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The development of novel antimicrobial peptides and various strategies to improve their activity and… Kumar, Prashant Sandeep 2018

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  THE DEVELOPMENT OF NOVEL ANTIMICROBIAL PEPTIDES AND VARIOUS STRATEGIES TO IMPROVE THEIR ACTIVITY AND BIOCOMPATIBILITY  by  Prashant Sandeep Kumar  B.Sc., The University of British Columbia, 2011  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Chemistry)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)   July 2018  © Prashant Sandeep Kumar, 2018  ii  The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: The Development Of Novel Antimicrobial Peptides And Various Strategies To Improve Their Activity And Biocompatibility  submitted by Prashant S. Kumar  in partial fulfillment of the requirements the degree of Doctor Of Philosophy in Chemistry   Examining Committee: Suzana K. Straus, Chemistry  Co-supervisor Jayachandran N. Kizhakkedathu, Pathology and Laboratory Medicine  Co-supervisor  Katherine S. Ryan, Chemistry  Supervisory Committee Member Russ Algar, Chemistry  University Examiner Dirk Lange, Medicine  University Examiner  Additional Supervisory Committee Members: Hongbin Li, Chemistry Supervisory Committee Member iii  Abstract With the advent of antibiotic resistance and crisis, it is crucial to find substitutes to conventional antibiotics. Antimicrobial peptides (AMPs) are considered to be viable alternatives, because they are broad spectrum and bacteria develop little or no resistance towards AMPs. Interestingly, only few AMPs are used as therapeutics, due to problems such as host toxicity, protease cleavage and short half-life. Therefore, there is a need to improve the efficacy of AMPs by the use of D-peptides and/or delivery vehicles. The introduction of the thesis describes the diversity and various mechanisms of action (MOA) of AMPs. The issues and ways to improve the efficacy of AMPs, which forms the foundation of this thesis, are also discussed. Recently, hyperbranched polyglycerol (HPG) has gained attention due to its excellent biocompatibility, multifunctionality and long blood circulation time. The body of the thesis describes a methodology to covalently attach aurein 2.2 and its mutants to HPG and study the influence of the molecular weight on the antimicrobial activity. A peptide array was used to design tryptophan and arginine mutants of aurein 2.2. Mutant peptide 77 had significantly superior antimicrobial and antibiofilm activity compared to aurein 2.2 but was more toxic. We found that HPG can be used as a general scaffold to alleviate the toxicity of the peptides, however the antimicrobial activity of the peptides decrease as the molecular weight of HPG increases.  The conjugates/peptides were tested in an in vivo mice skin infection (abscess) model. Surprisingly, peptide 73 and aurein 2.2 has similar efficacy in vivo indicating both the antimicrobial activity and toxicity, i.e. therapeutic index, are important. The conjugates (HPG-73c) were not active in mice abscess model, whereas 73c and D-73 encapsulated in micelles composed of DSPE-PEG2000 had excellent activity suggesting the release of the peptide from iv  the delivery vehicle is necessary for in vivo activity. Without encapsulation D-73 was too toxic. A bacterial expression system was used to produce isotopically (15N) labeled aurein 2.2 and its interaction with whole bacterial cells was examined by nuclear magnetic resonance (NMR) and scanning electron microscopy (SEM) confirming the MOA. Finally, the results presented will be discussed in the broad context of designing AMPs for therapeutics and understanding their MOA.    v  Lay Summary Antibiotic resistance has become a major public health concern. Indeed, some studies report that ABR could lead to 10 million deaths per year by 2050, making it crucial to find timely alternatives to currently used antibiotics. Antimicrobial peptides (AMPs) have shown great promise, because bacteria develop no or low resistance to AMPs. However, only few antimicrobial peptides are commercially available for use. This is due to problems with toxicity, short half-life, and rapid kidney clearance. This thesis focuses on developing strategies to improve the antimicrobial activity and properties of AMPs, by developing more active peptides and using delivery systems, such as biocompatible polymers and encapsulating agents. The activity of the peptides was tested using resistant bacteria, as well as in a mouse skin infection (abscess) model. Finally, a new method was developed to study how these peptides interact with whole bacterial cells, in order to understand how they function. Overall, the findings presented in this thesis lay the foundation for the future development of biocompatible AMPs, an essential tool to combat ABR.   vi  Preface Ethics approval was received from UBC for studies conducted at the Centre for Blood Research (UBC Ethics approval no: H10-01896). Animal experiments were performed in accordance with The Canadian Council on Animal Care (CCAC) guidelines and were approved by the University of British Columbia Animal Care Committee (certificate number A14-0363). This research project was conducted under the supervision of Dr. Suzana K. Straus (Department of Chemistry, UBC, Vancouver) and Dr. Jayachandran N. Kizhakkedathu (Centre for Blood Research, UBC, Vancouver). A version of Chapter 1 has been published as a first authored, invited review paper in the journal Biomolecules (DOI: 10.3390/biom8010004). I was responsible for conducting the literature review and writing the review paper which was edited by my supervisors.   Chapter 2 is based on the study published in the journal ACS Biomacromolecules (DOI: 10.1021/bm5018244) and describes the effect of conjugation of aurein peptides to hyperbranched polyglycerol (HPG) on antimicrobial activity, biocompatibility and structure. I was responsible for writing the paper which was edited by my supervisors. I was responsible for designing the experiments with my supervisors, synthesizing the peptides and conjugates and running most of the experiments except the following: Rajesh A. Shenoi synthesized the 44,000 Dalton HPG; Michael Nguyen synthesized the aurein 2.2 peptide under my supervision; and Benjamin Lai ran the complement activation and cell toxicity studies.    Chapter 3 is based on the study published in the journal ACS Applied Material and Interfaces (DOI: 10.1021/acsami.7b09471). This work examines the influence of the peptide sequence and HPG molecular weight on the antimicrobial activity and biocompatibility.  I was responsible for writing the paper which was edited by my supervisors. I designed the peptide vii  array with Suzana K. Straus, synthesising the polymers, peptides and conjugates. Srinivas Abbina synthesized the 100,000 Dalton HPG. Allen Takayesu synthesized peptide 73 and 77 under my supervision. Manu Thomas Kalathottukaren performed the blood coagulation studies (aPTT and PT) and Usama Abbasi ran the cell toxicity studies.   Chapter 4 is based on the study conducted in collaboration with Dr. Bob Hancock’s laboratory at the Centre for Microbial Diseases and Immunity Research, UBC, Vancouver. Studies in this chapter investigated the antibiofilm activity and efficacy of the peptides in mice abscess model. I was responsible for designing and synthesizing all the peptides and conjugates and running the experiments except for all the in vivo mice studies which were conducted by Daniel Pletzer and all the antibiofilm experiments which were done by John Cheng.   Chapter 5 is based on the overexpression, purification and esterification of aurein 2.2. The expressed isotopically labelled peptide was then utilized to probe the interaction of aurein 2.2 with whole bacterial cells by nuclear magnetic resonance (NMR) and scanning electron microscopy (SEM). The plasmid used for expression in this chapter was kindly provided by Dr. Hans J. Vogel from the University of Calgary. I was responsible for designing the experiments with my supervisors. All the experiments were also conducted by me with help from Suzana K. Straus for the NMR experiment. Allen Takayesu conducted some of the expression experiments under my supervision.  Publications 1) Kumar, P.; Kizhakkedathu, J.; Straus, S. Antimicrobial Peptides: Diversity, Mechanism of Action and Strategies to Improve the Activity and Biocompatibility In Vivo. Biomolecules 2018, 8, 4, doi:10.3390/biom8010004.  2) Kumar, P.; Takayesu, A.; Abbasi, U.; Kalathottukaren, M. T.; Abbina, S.; Kizhakkedathu, J. N.; Straus, S. K. Antimicrobial Peptide–Polymer Conjugates with High Activity: Influence of Polymer Molecular Weight and Peptide Sequence on Antimicrobial viii  Activity, Proteolysis, and Biocompatibility. ACS Appl. Mater. Interfaces 2017, acsami.7b09471, doi:10.1021/acsami.7b09471.  3) Kumar, P.; Shenoi, R. A.; Lai, B. F. L.; Nguyen, M.; Kizhakkedathu, J. N.; Straus, S. K. Conjugation of Aurein 2.2 to HPG Yields an Antimicrobial with Better Properties. Biomacromolecules 2015, 16, 913–923, doi:10.1021/bm5018244.  4) Abbina, S.; Vappala, S.; Kumar, P.; Siren, E. M. J.; La, C. C.; Abbasi, U.; Brooks, D. E.; Kizhakkedathu, J. N. Hyperbranched polyglycerols: recent advances in synthesis, biocompatibility and biomedical applications. J. Mater. Chem. B 2017, 5, 9249–9277, doi:10.1039/C7TB02515G.  5) Wenzel, M.; Senges, C. H. R.; Zhang, J.; Suleman, S.; Nguyen, M.; Kumar, P.; Chiriac, A. I.; Stepanek, J. J.; Raatschen, N.; May, C.; Krämer, U.; Sahl, H.-G.; Straus, S. K.; Bandow, J. E. Antimicrobial Peptides from the Aurein Family Form Ion-Selective Pores in Bacillus subtilis. ChemBioChem 2015, 16, 1101–1108, doi:10.1002/cbic.201500020.  6)  Sang, Y.; Tait, A. R.; Scott, W. R. P.; Creagh, A. L.; Kumar, P.; Haynes, C. A.; Straus, S. K. Probing the Interaction between U24 and the SH3 Domain of Fyn Tyrosine Kinase. Biochemistry 2014, 53, 6092–6102, doi:10.1021/bi500945x.  ix  Table of Contents Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ......................................................................................................................... ix List of Tables ............................................................................................................................. xvii List of Figures ........................................................................................................................... xviii List of Symbols ......................................................................................................................... xxiii List of Abbreviations ............................................................................................................... xxiv Acknowledgements ................................................................................................................. xxvii Dedication ................................................................................................................................. xxix Chapter 1: Introduction ................................................................................................................1 1.1  Antibiotics ....................................................................................................................... 1 1.1.1  History of antibiotics ................................................................................................... 1 1.1.2  Structure of Bacterial Cell and Membrane ................................................................. 2 1.1.3  Categories of antibiotics ............................................................................................. 3 1.1.4  Antibiotic resistance .................................................................................................... 4 1.2  Antimicrobial peptides .................................................................................................... 5 1.2.1  Antimicrobial peptides: history and diversity ............................................................. 5 1.2.2  Categories of antimicrobial peptides .......................................................................... 9 1.2.3  Common properties of antimicrobial peptides. ......................................................... 12 1.2.4  Mechanism of antimicrobial peptide action .............................................................. 14 1.2.4.1  Direct killing: membrane permeabilizing mechanism of action ....................... 15 x  1.2.4.2  Direct killing: non membrane targeting mechanisms of action ........................ 20 1.2.4.3  Immune modulation mechanism of action ........................................................ 22 1.2.5  Aurein peptides ......................................................................................................... 23 1.2.6  Challenges with antimicrobial peptides .................................................................... 24 1.3  Strategies to improve antimicrobial peptides ................................................................ 25 1.3.1  Chemical modification of AMPs .............................................................................. 25 1.3.2  Delivery systems for AMPs ...................................................................................... 28 1.4  Hyperbranched polyglycerol ......................................................................................... 31 1.5  Thesis rationale, hypotheses and aims .......................................................................... 34 1.5.1  Development of novel antimicrobial conjugates ...................................................... 34 1.5.2  Development of potent novel antimicrobial peptides and conjugates ...................... 35 1.5.3  In vivo efficacy of the peptides and the conjugates in mice models ......................... 36 1.5.4  Interaction of aurein 2.2 with whole bacterial cells by NMR. .................................. 37 1.6  Summary ....................................................................................................................... 39 Chapter 2: Conjugation of aurein 2.2 to HPG yields an antimicrobial with better properties. .....................................................................................................................................40 2.1  Synopsis ........................................................................................................................ 40 2.2  Background ................................................................................................................... 41 2.3  Methods......................................................................................................................... 43 2.3.1  Peptide Synthesis and Purification............................................................................ 43 2.3.2  Synthesis of HPG ...................................................................................................... 44 2.3.3  Amine Modification of HPG .................................................................................... 44 2.3.4  Conjugation of Peptide with amine-modified HPG .................................................. 45 xi  2.3.5  Characterization of HPG Peptide Conjugates ........................................................... 45 2.3.6  MIC Determination ................................................................................................... 46 2.3.7 Blood Compatibility Analysis...................................................................................... 47 2.3.7.1 Platelet Activation Analysis: Flow Cytometry ......................................................... 47 2.3.7.2 Platelet Aggregation Studies ..................................................................................... 48 2.3.7.3 Red Blood Cell Lysis Studies ................................................................................... 48 2.3.7.4  Complement Activation Analysis: CH50 Assay................................................... 49 2.3.7.5  Blood Coagulation by Activated Partial Thromboplastin Time (aPTT) and Partial Thromboplastin Time (PT) Analysis .................................................................................... 50 2.3.8  Cell Toxicity analysis ........................................................................................... 51 2.3.9  Biomembrane Interaction by CD Spectroscopy ................................................... 52 2.4  Results and Discussion ................................................................................................. 53 2.4.1  Synthesis of Conjugates ............................................................................................ 53 2.4.2  Antimicrobial Activity of the Bioconjugates ............................................................ 55 2.4.3  Peptide Secondary Structure on Bioconjugates ........................................................ 56 2.4.4  Blood Compatibility of the Peptide and Peptide Conjugates.................................... 58 2.4.5  Cell Compatibility of the Peptide and Conjugates .................................................... 65 2.5  Summary ....................................................................................................................... 66 Chapter 3: Antimicrobial peptide-polymer conjugates with high activity: Influence of polymer molecular weight and peptide sequence on antimicrobial activity, proteolysis and biocompatibility ...........................................................................................................................68 3.1  Synopsis ........................................................................................................................ 68 3.2  Background ................................................................................................................... 69 xii  3.3  Methods......................................................................................................................... 70 3.3.1  Peptide array ............................................................................................................. 70 3.3.2  Peptide synthesis and purification ............................................................................ 71 3.3.3  Synthesis of HPG ...................................................................................................... 71 3.3.4  Amine Modification of HPG .................................................................................... 72 3.3.5  Conjugation of peptide with HPG-amine ................................................................. 73 3.3.6  Synthesis of PEG 77c conjugate ............................................................................... 74 3.3.7  Antimicrobial activity measurements (minimum inhibitory concentration or MIC) 74 3.3.8  MIC determination after tryptic degradation ............................................................ 74 3.3.9  Biocompatibility analysis .......................................................................................... 75 3.3.10  Biomembrane interaction: CD spectroscopy ........................................................ 75 3.4  Results and Discussion ................................................................................................. 75 3.4.1  Discovery of potent peptides using peptide array based on aurein peptide .............. 75 3.4.2  Synthesis of aurein 2.2∆3-cys and peptide 77c conjugates ...................................... 77 3.4.3  Antimicrobial activity of the conjugates ................................................................... 79 3.4.4  Resistance to proteolysis: tryptic degradation .......................................................... 82 3.4.5  Peptide secondary structure on bioconjugates .......................................................... 84 3.4.6  Cell compatibility of the peptides and conjugates .................................................... 85 3.4.7  Blood compatibility of the peptide and peptide conjugates ...................................... 89 3.5  Summary ....................................................................................................................... 97 Chapter 4: The in vitro biofilm activity and the in vivo efficacy of the novel peptides and the polymer conjugates. .....................................................................................................................98 4.1  Synopsis ........................................................................................................................ 98 xiii  4.2  Background ................................................................................................................... 98 4.3  Materials and methods ................................................................................................ 101 4.3.1  Peptide synthesis and purification .......................................................................... 101 4.3.2  Synthesis of the bioconjugate ................................................................................. 101 4.3.3  Antimicrobial activity measurements: MIC determination .................................... 101 4.3.4  Biofilm studies ........................................................................................................ 102 4.3.5  Red blood cell lysis ................................................................................................. 102 4.3.6  Mouse skin infection model .................................................................................... 103 Bacterial strains and growth conditions for animal studies ................................................ 103 Ethics statement .............................................................................................................. 103 Cutaneous mouse infection model .................................................................................. 103 Statistical analysis ........................................................................................................... 104 4.4  Results and Discussion ............................................................................................... 104 4.4.1  Antibacterial activity of the novel peptides in vitro ................................................ 104 4.4.2  Efficacy of the peptides against Staphylococcus aureus biofilms .......................... 107 4.4.3  Tolerance of the peptides ........................................................................................ 109 4.4.4  In vitro therapeutic index of the peptides ............................................................... 110 4.4.5  Efficacy of peptides in a mouse abscess model ...................................................... 111 4.4.6  Peptide aggregation ................................................................................................. 116 4.4.7  Efficacy of the HPG conjugates in the mouse abscess model ................................ 117 4.4.8  Peptide design paradigms ....................................................................................... 118 4.5  Summary ..................................................................................................................... 119 xiv  Chapter 5: Overexpression, purification and esterification of aurein 2.2 utilized to probe the interaction of aurein 2.2 with whole bacterial cells by NMR. .........................................121 5.1  Synopsis ...................................................................................................................... 121 5.2  Background ................................................................................................................. 121 5.3  Methods and materials ................................................................................................ 124 5.3.1  Expression of Calmodulin-aurein fusion protein in E.coli ..................................... 124 5.3.2  SDS PAGE .............................................................................................................. 125 5.3.3  Purification of Calmodulin-aurein fusion protein in E.coli .................................... 125 5.3.4  Digestion of Calmodulin-aurein fusion protein ...................................................... 126 5.3.5  MALDI-TOF........................................................................................................... 126 5.3.6  Peptide purification by RP-HPLC .......................................................................... 127 5.3.7  Peptide Esterification .............................................................................................. 127 5.3.8  MIC Assays ............................................................................................................. 127 5.3.9  2D NMR.................................................................................................................. 127 5.3.10  Scanning electron microscope studies ................................................................ 128 5.4  Results And Discussion .............................................................................................. 128 5.4.1  Expression and purification of CaM-aurein 2.2 ...................................................... 128 5.4.2  Cleavage of CaM-aurein 2.2 and purification of aurein 2.2 ................................... 130 5.4.3  Chemical esterification of aurein 2.2 retains antimicrobial activity. ...................... 132 5.4.4  Whole cell NMR and SEM ..................................................................................... 134 5.5  Summary ..................................................................................................................... 137 Chapter 6: Conclusions and Future work ...............................................................................139 6.1  Conclusions ................................................................................................................. 139 xv  6.2  Future work ................................................................................................................. 143 Bibliography ...............................................................................................................................148 Appendix A ............................................................................................................................. 168 A.1  HPLC trace of Aurein 2.2∆3-cys ............................................................................ 168 A.2  Proton NMR of amine functionalized HPG ............................................................ 168 A.3  Proton NMR of HPG-bifunctional linker ............................................................... 169 A.4  UV spectra of the HPG-bifunctional linker ............................................................ 169 A.5  HPLC purification of the conjugate ........................................................................ 170 A.6  Proton NMR of HPG-Aurein 2.2∆3-cys ................................................................. 170 A.7  Size exclusion chromatography of conjugates ........................................................ 171 A.8  CD spectroscopy of Aurein 2.2∆3-cys .................................................................... 171 A.9  CD spectroscopy of the conjugates ......................................................................... 172 A.10  Platelet aggregation by the bioconjugates ............................................................... 172 Appendix B ............................................................................................................................. 174 B.1  Proton NMR of HPG-phthalimide .......................................................................... 174 B.2  Proton NMR of HPG-Amine .................................................................................. 174 B.3  Proton NMR of HPG maleimide ............................................................................ 175 B.4  Proton NMR of HPG 77c ........................................................................................ 175 B.5  Proton NMR of PEG 77c ........................................................................................ 176 B.6  Bacteria killing and RBC lysis in whole blood/bacteria mixture ........................... 176 B.7  Bacteria killing in serum/bacteria mixture .............................................................. 178 B.8  Maldi of Matrix ....................................................................................................... 178 B.9  aPTT analaysis of 77c and the conjugates .............................................................. 179 xvi  B.10  Thromboelastometry of conjugates and peptides ................................................... 179 B.11  aPTT analaysis of 77c and the conjugates .............................................................. 180 Appendix C ............................................................................................................................. 181 C.1  Toxicity and precipitation of D-peptides at various concentrations in saline. ........ 181 C.2  Toxicity and precipitation of D-peptides at various concentrations in DSPE-PEG2000 ............................................................................................................................. 182 C.3  Average hydrodynamic size of peptide 73c in water and DSPE-PEG2000. .......... 183 Appendix D ............................................................................................................................. 184 D.1  MS/MS spectra of aurein 2.2  and diesterified aurein 2.2 ...................................... 184  xvii  List of Tables  Table 1.1: Mechanism of antibiotic action and acquired resistance for different of antibiotic classes. ............................................................................................................................................ 6 Table 1.2: Classes of antimicrobial peptides based on structure .................................................. 10 Table 1.3: Antimicrobial peptide in clinical trials ........................................................................ 25 Table 2.1: Sequence and molecular weight of the peptides and the bioconjugates made .……...54 Table 2.2: Secondary structure of Aurein 2.2∆3-cys and HPG-Aurein 2.2∆3-cys in various environments determined by CD spectroscopy (representative spectra shown in Figure 2.3). .... 58 Table 3.1: Antimicrobial activity (MICs) of selected peptides identified in the peptide array….71 Table 3.2: Characteristics of the peptides and the HPG conjugates used in this study@. ............. 80 Table 3.3: Alpha helical content (%) of the various conjugates studied here, as determined from fits from the CD spectra (Figure 3.4). ........................................................................................... 87 Table 4.1: Various properties of some of the peptides used in this studies…………………….105 Table 4.2: Antimicrobial activity of the peptides. ...................................................................... 105 Table 4.3: Anti-biofilm activity of the peptides. ......................................................................... 108 Table 4.4: Therapeutic index of the peptides .............................................................................. 112 Table 4.5: Average hydrodynamic size of peptide 73c ............................................................... 116 Table 5.1: Antimicrobial activity of expressed and synthetic aurein 2.2……………………….133    xviii  List of Figures  Figure 1.1: Cell envelope of Gram postive and Gram negative bacteria.. ...................................... 2 Figure 1.2: The β lactam ring of the generalized penicillin molecule.. .......................................... 4 Figure 1.3: Sources of antimicrobial peptides (total 2818) as of September 2017 from the antimicrobial peptide database. ....................................................................................................... 7 Figure 1.4: Structural diversity of AMPs.. .................................................................................... 11 Figure 1.5: Various mechanism of action of antimicrobial peptides.. .......................................... 15 Figure 1.6: Initial interaction of cationic AMPs with the multicellular animal (left) or bacterial (right) membrane. RBC: red blood cell.. ...................................................................................... 17 Figure 1.7: Proposed mechanism of action for AMPs in bacteria.. .............................................. 19 Figure 1.8: Structure of lipid II molecules with pyrophosphate sugar moiety circled in red. ...... 21 Figure 1.9: Chemical modifications AMPs. ................................................................................. 28 Figure 1.10: Various polymers used for AMP conjugation. ......................................................... 30 Figure 1.11: Generic structure of HPG. ........................................................................................ 32 Figure 2.1: Synthesis of HPG.. ..................................................................................................... 42 Figure 2.2: Synthetic route for the conjugation of the aurein peptide with hyperbranched polyglycerol (HPG).. ..................................................................................................................... 53 Figure 2.3: CD spectra of the HPG-Aurein 2.2∆3-cys 5% in PBS buffer and lipids (1:1 POPC/POPG) and the peptide under the same conditions............................................................ 57 Figure 2.4: Influence of free aurein peptide and HPG peptide conjugates concentration on red blood cell lysis. ............................................................................................................................. 60 xix  Figure 2.5: Influence of free aurein peptide and HPG peptide conjugates concentration on complement activation.. ................................................................................................................ 62 Figure 2.6: Influence of free aurein peptide and HPG peptide conjugates concentration on platelet activation. ......................................................................................................................... 63 Figure 2.7: Influence of free aurein peptide and HPG peptide conjugates concentration on intrinsic pathway of blood coagulation. ........................................................................................ 65 Figure 2.8: Cell biocompatibility of the peptides and the conjugates against. ............................. 66 Figure 3.1: Helical wheels for some of the peptides generated in the array……………………..77 Figure 3.2: Synthetic scheme for the conjugation of the peptides with HPG.. ............................. 78 Figure 3.3: MALDI-TOF mass spectra of 77c, 22k 77c and trypsin after 3 hours of digestion ... 83 Figure 3.4: CD Spectra of the conjugates in phosphate buffer and POPC/PG (1:35). ................. 86 Figure 3.5: Fibroblast cell viability upon exposure to free peptides and HPG peptide conjugates....................................................................................................................................................... 88 Figure 3.6: Red blood cell lysis upon exposure to free peptides and HPG peptide conjugates. ... 90 Figure 3.7: Platelet activation upon exposure to free peptides and HPG peptide conjugates ...... 92 Figure 3.8: Complement activation upon exposure to free peptide and HPG peptide conjugates........................................................................................................................................................ 94 Figure 3.9: Prothrombin time upon exposure to free aurein peptide and HPG peptide conjugates....................................................................................................................................................... 96 Figure 4.1: Red blood cell lysis upon exposure to free peptides and HPG peptide conjugates...110 Figure 4.2: RBC lysis vs log of peptide concentration curves to extrapolate the concentration at which the peptides cause 50% RBC lysis (LD50). ...................................................................... 111 xx  Figure 4.3: Infection and therapeutic treatment of mouse cutaneous abscesses. All mice were infected with MRSA USA300 and treated with saline solution, aurein and peptide 73 (dose = 5 mg/kg) 1 h post-bacterial infection.. ........................................................................................... 113 Figure 4.4: Therapeutic treatment of mouse cutaneous abscesses with D and L peptides. ........ 115 Figure 4.5: Therapeutic treatment of mouse cutaneous abscesses with HPG-73c conjugates. .. 118 Figure 5.1: Calmodulin (CaM)-aurein 2.2 fusion construct with TEV protease cut site……….123 Figure 5.2: Expression and purification of CaM-aurein 2.2. ...................................................... 129 Figure 5.3: MALDI-TOF of the cleavage mixture.. ................................................................... 131 Figure 5.4: HPLC purification of the cleaved CaM-aurein 2.2.. ................................................ 131 Figure 5.5: MALDI-TOF of the diesterified aurein 2.2.. ............................................................ 133 Figure 5.6: 1H,15N-HSQC spectra of whole S. aureus cells with the aurein peptides. ............... 135 Figure 5.7: CD spectra of expressed peptides.. ........................................................................... 136 Figure 5.8: SEM of S. aureus after incubating for 1 h with the expressed peptides ................... 137 Figure 6.1: Design of HPG-73 conjugate that would lead to the release of peptide 73 at the site of infection. ……………….………………………………………………………………………146 Figure A.1: HPLC purification of the Aurein 2.2∆3cys. Peptide elutes between 55-57min…...168 Figure A.2: Proton NMR (in D2O) of amine modified HPG. ..................................................... 168 Figure A.3: Proton NMR (in deuterated DMSO) of HPG-bifunctional Linker. ......................... 169 Figure A.4: UV-Vis of HPG-Bifunctional linker. ...................................................................... 169 Figure A.5: Size exclusion HPLC purification of the bioconjugates .......................................... 170 Figure A.6: Proton NMR (in D2O) of HPG-Aurein 2.2∆3-cys................................................... 170 Figure A.7: Size exclusion chromatographs of HPG-Aurein 2.2∆3-cys  and HPG-Bifunctional linker. .......................................................................................................................................... 171 xxi  Figure A.8: CD spectra of the Aurein 2.2∆3-cys in and trifluroethanol (TFE) and lipids(POPC/POPG). ................................................................................................................... 171 Figure A.9: CD spectra of the HPG-Aurein 2.2∆3-cys in PBS buffer, Trifluroethanol (TFE) and lipids(POPC/POPG).. .................................................................................................................. 172 Figure A.10: Platelet aggregation by the bioconjugates.. ........................................................... 172 Figure B.1: 1H NMR (in 100% DMSO-d6) of phthalimide modified 22k HPG……………….174 Figure B. 2: 1H NMR (in 100% DMSO-d6) of 22k HPG-NH2 after deprotection of the phthalimide. ................................................................................................................................ 174 Figure B.3: 1H NMR (in 100% DMSO-d6) of 22k HPG maleimide. ......................................... 175 Figure B.4: 1H NMR (in 100% DMSO- d6) of 22k HPG 77c. ................................................... 175 Figure B. 5: 1H NMR (in 100% D2O) of 5k mPEG 77c ............................................................. 176 Figure B.6a: Bacterial colony forming units per milliliters (CFU/mL) in whole blood in presence of peptide/conjugates. ................................................................................................................. 176 Figure B.6b: Percent RBC lysis in the whole blood in presence of bacteria and peptide/conjugate...................................................................................................................................................... 177 Figure B.7: The ability for the peptide and conjugates to kill bacteria in serum.. ...................... 178 Figure B.8: Appendix B.8: MALDI of matrix. ........................................................................... 178 Figure B.9: Influence of free peptide and HPG peptide conjugates on activated Partial Thromboplastin Time (aPTT) ..................................................................................................... 179 Figure B.10: Typical thromboelastometry of the HEPES-buffered saline (HBS), 22K 77c  and 77c. .............................................................................................................................................. 180 Figure B.11: Whole blood clotting time by Thromboelastometry of the 22k 77c and 77c ........ 180 Figure C. 1: CD-1 mice were injected with 50 ul of different peptide concentrations on the left xxii  and right side of the dorsum. The skin inspected three days post injection……………..181 Figure C.2: CD-1 mice were injected with 50 ul of different peptide concentrations on the left and right side of the dorsum. The skin inspected three days post injection. No visible peptide/conjugate precipitation at various concentration in DSPE-PEG2000 ............................ 182 Figure C.3: Average hydrodynamic size and the intensity percent of peptide 73c in water and DSPE-PEG2000. ......................................................................................................................... 183 Figure D.1: MS/MS spectra of aurein 2.2 and diesterified aurein 2.2………………………….184    xxiii  List of Symbols °C Degree Celsius g  Gram h  Hour Hz  Hertz K  Kelvin kg  Kilogram mg  Milligram MHz  Megahertz (106 Hertz) mL  Milliliter mM  Millimolar µg  Microgram µL  Microliter nm  Nanometer nM  Nanomolar s  Second        xxiv  List of Abbreviations 1H NMR   Proton Nuclear Magnetic Resonance 31P NMR   Phosphorus-31 Nuclear Magnetic Resonance ABR   Antibiotic resistance  AD   Anno domini AMP    Antimicrobial peptides  ANOVA   Analysis of variance aPTT    Activated partial thromboplastin time BCA   Bicinchoninic acid CA   California  Ca   Community acquired  CD    Circular Dichroism CHCl3    Chloroform D2O    Deuterated Water DD   D-alanyl-D-alanine DHPC    1,2-dihexanoyl-sn-glycero-3-phosphocholine DLS    Dynamic light scattering DMF   Dimethylformamide DMPC   1,2-dimyristoyl-sn-glycero-3-phosphocholine DMPG   1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] DMSO   Dimethyl sulfoxide DNA   Deoxyribonucleic acid  DPC    Dodecylphosphocholine DSC    Differential Scanning Calorimetry DSPC   1,2-Distearoyl-sn-glycero-3-phosphocholine DSPE   1,2-Distearoyl-sn-glycero-3-phosphoethanolamine DSPG   1,2-Distearoyl-sn-glycero-3-phosphoglycerol DSPE-PEG2000 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]  EDTA    Ethylenediaminetetraacetic acid xxv  ELISA   Enzyme linked immunosorbent assay FBS   Fetal bovine serum  FDA    Food and Drug Administration HDP    Host Defense Peptide HEPES  N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid HIT    Heparin-induced thrombocytopenia HPG    Hyperbranched polyglycerol HPLC    High Performance Liquid Chromatography HUVEC  Human Umbilical Vein Endothelial Cells IDR   Innate defense regulator peptide ITC    Isothermal titration calorimetry IU    International unit kDa    Kilo Dalton MALDI-TOF  Matrix-Assisted Laser Desorption/Ionization-Time of flight spectrometry  MBEC   Minimal biofilm eradication concentration MBIC   Minimal biofilm inhibitory concentration MCP    Monocyte Chemoattractant Protein MDR   Multidrug resistance  MIC    Minimal inhibitory Concentration MOA   Mechanism of action  MOPS   4-morpholinepropanesulfonic acid mPEG    Methoxypolyethylene glycol MRSA   Methicillin-resistant Staphylococcus aureus MS/MS  Tandem mass spectrometry MW    Molecular weight OCD    Oriented Circular Dichroism OD    Optical Density PBMC   Peripheral Blood Mononuclear Cell PBS    Phosphate-buffered saline PC   Phosphatidylcholine xxvi  PDI   Polydispersity index PE   Phosphatidylethanolamine PEG    Polyethylene glycol PG   Phosphatidylglycerol POPC    1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine POPE    1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine POPG    1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] ppm    Parts per million PPP   Platelet-poor Plasma PRP    Platelet-rich Plasma PS   Phosphatidylserine PT   Prothrombin time RBC    Red blood cell REDOR   Rotational Echo Double Resonance Rh    Hydrodynamic radius RI   Retro-inverso  RNA   Ribonucleic acid  ROMBP  Ring opening multibranching polymerization   RP HPLC   Reverse-Phase High Performance Liquid Chromatography RT    Room temperature SDS    Sodium Dodecylsulfate SUV    Small Unilamellar Vesicle TEG    Thromboelastography TFA   Trifluoroacetic acid TFE    2,2,2-trifluoroethanol TFE-d3   Deuterated 2,2,2-trifluoroethanol TI   Therapeutic index UBC   University of British Columbia US/USA  United States of America UV   Ultraviolet xxvii  Acknowledgements I “give thanks to the Lord, for he is good and his love endures forever” (Psalm 107:1). I trust in you, God.  This research thesis would not have been possible without the help, advice and support from numerous individuals. I would like to express my sincere gratitude to these people here. First of all, I would like to thank my supervisor Dr. Suzana K Straus and co-supervisor Dr. Jayachandran N. Kizhakkedathu, for giving me an opportunity to learn, do research and teach in their laboratory. Thank you for having confidence in me and always being there for me. A million thanks for your patience in our weekly meetings, and for constantly providing guidance and motivation. I would like to thank my committee members, Dr. Hongbin Li, Dr. Derek P Gates, Dr. Katherine S. Ryan, for their valuable suggestions on this project, and in exam and committee meetings. Thank you Dr. Li and Dr. Ryan in particular for reading through this thesis. Dr. Donald E. Brooks, thank you very much for fruitful discussions and suggestions.  Special thanks to Dr. Rajesh A. Shenoi and Dr. Srinivas Abbina for being my mentors in the lab and sharing your knowledge. You both are an inspiration for me and other students in the Kizhakkedathu laboratory. I would like to thank Benjamin Lai, Dr. Manu Thomas Kalathottukaren, Dr. Frederico Rosell, Dr. Evan Haney, Irina Chafeeva, Iren Constantinescu, and Derrick Horne, for providing me excellent training on various techniques used for this project. I am grateful to Dr. Daniel Pletzer, Dr. John Cheng and Dr. Bob Hancock who provided invaluable guidance with interpretation of research findings through our collaboration. Thank you Dr. Hans J. Vogel for providing the plasmid for the studies in Chapter 5.  xxviii  I would also like to thank my students, Allen Takayesu, Michael Nguyen, Sayak Subhra Panda, José Ausencio Álvarez Huerta, Marty Yue, Waleed Aljehani, Aparna Mallaredy for their contribution in various projects and giving me a chance to mentor them and improve my leadership skills.  My sincerest gratitude to Usama Abbasi, Dr. Imran Ul-Haq, Dr. Jasmine Hamilton, , Dr. Anilkumar Parambath, Dr. Mahsa Alizadeh, Dr. Johan Janzen, Dr. Andrew Tait, Dr. Yurou Sang, Kevin Cooley, Matthew Drayton, Chantal Mustoe, Nigare Raheem, Sonja Horte, Dr. David Yang, Vincent Leung, Dr. Kai Yu, Yan Mei, Dr. Nima Khademmohtaram, Dr. Narges Hadjesfandiari, Erika Siren, Chanel La, Sreeparna Vappala, Na Li (April), Erika Das, Dr. Rafi Chapnian, Dr. Madhab Bagpai, Joseph Lee, Jin Zhang, Rui Zhang, Htet Ei Bo, Angela Dodd, Ruqaiba Desmond, Jung Hwan Bae (David), Selina Suleman, Marie Weinhart, Ainge Chang, Kyle Scoten, Kelly Hutchinson, Minnie Jiang, Cian Zybutz, Ethan Lee, Celebi Wan, and all past lab members of Brooks, Kizhakkedathu and Straus laboratories, for providing support and creating a fun-filled and positive  work environment. I would also like to thank all my family and friends, here in Canada and also in Fiji, for their love and support. Special thanks to my friends and members from the Acts 29 church. My sincere gratitude to the biology and chemistry department of Langara College, especially Dr. Shirley Wacowich-Sgarbi who provided the platform for my first ever research experience.  I am most grateful to my parents, especially my mother, Mohini Kumar, and my younger brother, for their unconditional love and prayers.  xxix  Dedication   To my Lord and savior, Jesus Christ, for his everlasting reckless love.   To my mother, Mohini Kumar;  for every sacrifice you have made for us.    For God so loved the world that He gave His one and only Son, that whoever believes in Him shall not perish but have eternal life. (John 3:16)                1  Chapter 1: Introductiona 1.1 Antibiotics 1.1.1 History of antibiotics Antibiotics are defined as therapeutic agents that are used to treat and prevent bacterial infections. The antibiotic can either kill bacteria and/or prevent bacterial growth. Although the first antibiotic was discovered in the 19th century, the use of antibiotics as therapeutic agents dates back to AD 350 by the Nubians who unknowingly used tetracycline based compounds1. Interestingly ancient Chinese and Greeks also used herbal medicine to combat bacterial infections. Egyptians would apply moldy bread to infected wounds2. In 1907 arsphenamine was synthesized by Alfred Berthelm in Paul Ehrlich’s lab. Ehrlich hypothesized that screening many compounds would eventually lead to the discovery of a ‘magic bullet’ that would kill the microbe but not the patient 3. Arsphenamine was eventually used to treat syphilis patients.   Many microorganisms also produce antibiotics to protect their environment niche and inhibit bacterial infection. In 1928 Sir Alexander Fleming noticed that the zone immediately around the Penicillium mold (fungi) inhibited Staphylococcus growth. He hypothesized the mold secreted an active compound to inhibit bacterial growth. The active compound secreted by the mold was penicillin which was further developed and purified by Ernst Chain and Howard Florey  and initially used in 1942 during World War II (WWII)4. The antibiotics field experienced a golden era as many families    aA version of Chapter 1 has been published. Kumar, P.; Kizhakkedathu, J.; Straus, S. Antimicrobial Peptides: Diversity, Mechanism of Action and Strategies to Improve the Activity and Biocompatibility In Vivo. Biomolecules 2018, 8, 4, doi:10.3390/biom8010004. 2  of antibiotics such as aminoglycosides, sulfonamides, cephalosporins and fluoroquinolones were discovered in the next 20 years. These new discoveries lead to extensive therapeutic research and the commercial industry against bacterial infections. Interestingly, various families of the antibiotics target different components of the bacteria which will be described in detail in section 1.1.3. 1.1.2 Structure of Bacterial Cell and Membrane  It is important to understand the basic structure and components of the bacterial cell before looking into the mechanism of action of certain antibiotics. Bacterial cells are divided into two broad classes i.e. Gram positive and Gram negative. Gram negative bacteria are generally characterized by their cell envelope which consists of a cell membrane, thin peptidoglycan (cell wall) and a bacterial outer membrane (Figure 1.1a)5. The area between the cell and outer membrane is known as the periplasmic space where many lipoproteins are located. Gram negative bacteria are also characterized by the presence of lipopolysacchride (LPS) on the outer membrane.   Figure 1.1: Cell envelope of Gram postive and Gram negative bacteria. Figure used with permission from Brown et. al. Extracellular Vesicles in Gram-Positive Bacteria, Mycobacteria and Fungi. Nat. Rev. Microbiol. 13 (10), 620–630 (2015). Cytoplasm  Cytoplasm  3  On the other hand, the cell envelope of Gram positive bacteria is comprised of an inner cell membrane and a thick peptidoglycan (cell wall) layer. The cell wall is composed of the peptidoglycan and different types of teichoic acid. Intracellular structures in Gram positive and Gram negative bacteria are very similar. Most bacteria consist of closed circular chromosomal DNA floating in the cytoplasm and ribosomes to facilitate protein synthesis. Common extracellular structures include the capsule and flagella6,7.  1.1.3 Categories of antibiotics  Antibiotics can be categorized according to their mechanism of action. There are hundreds of antibiotics8 which can be natural, semi-synthetic or synthetic making classification important. Antibiotics usually kill or inhibit the growth of the bacteria by interfering with a major pathway or by interacting with a specific structural component. The mechanism of action of the most common classes of antibiotics is summarized in Table 1.1.  Penicillin is the name given to a group of antibiotics which include penicillin G, penicillin V, ampicillin. The structural similarity is the β lactam ring (Figure 1.2) which is necessary to inhibit the cell wall synthesis in bacteria. The β lactam moiety binds to DD (D-alanyl-D-alanine)-transpeptidase, an enzyme that normally facilitates the peptidoglycan cross-linking. This binding interaction leads to weakening of the cell wall, leading to bacterial cell lysis due to the osmotic pressure9. Interestingly, tetracyclin-based antibiotics such as methacyclin work synergistically with penicillin as penicillin weakens the peptidoglycan and facilitates the entry of methacyclin into the bacteria cells to inhibit protein synthesis 10. 4   Figure 1.2: The β lactam ring of the generalized penicillin molecule. The R group moiety is different for Penicillin G, penicillin V, ampicillin and other penicillins. Image created using ChemDraw software.   1.1.4 Antibiotic resistance  Antibiotic resistance (ABR) is a natural phenomenon and was observed as early as the 1940s. For instance, penicillin was introduced as a therapeutic agent in 1942. Well before this, however, the β lactamase enzyme that destroys penicillin was identified in 194011. All ABR is genetically encoded and can be classified into two types: intrinsic or acquired resistance12. In intrinsic resistance, the bacteria have an innate ability to resist the antibiotic. For example, Gram negative bacteria show resistance to vancomycin because the antibiotic is too large to permeate the outer membrane. Some bacterial species have chromosomal DNA that also encodes for a efflux pump that enables them to remove the antibiotic from the cell13. In contrast, in acquired resistance (Table 1.1), the bacteria modify themselves such that they can now resist the antibiotic to which they were susceptible to earlier. Acquired resistance is mediated by mutations in the chromosomal DNA or horizontal gene transfer14. Non lethal mutations in the chromosomal DNA can arise by mistakes made by the DNA polymerase during DNA replication which might be beneficial for the bacteria if the mutation modifies the antibiotic target (e.g. rifampin and fluoroquinolones (ciprofloxanin)) (Table 1.1).  In one of the horizontal gene transfer mechanisms (conjugation), a specific gene coding ABR (e.g β lactamases genes (enzyme that cleaves the β 5  lactam ring)) is located on a small circular DNA, known as a plasmid, which can be transferred from one bacterium to another. This allows the bacteria that have just received the new plasmid to become resistant. Interestingly, most of the multidrug resistance (MDR) efflux pumps that were previously on chromosomal DNA are now found on plasmids15.   ABR is one of the greatest threats to human health in the future as it has been estimated that by 2050 ten million people may die per year due to ABR16. In the European Union, the current cost of treating ABR bacterial infections is 1.5 billion Euros per year 13. In addition to the ABR crisis, there were only a few new antibiotics developed in the past decade17. On average only one antibiotic has been developed per year in the past 15 years. Multiple drug resistant bacteria have become a global concern leading to an urgent need to discover and develop novel antibiotics.  1.2 Antimicrobial peptides  1.2.1 Antimicrobial peptides: history and diversity Antimicrobial peptides (AMPs), also known as host defense peptides, are virtually found in all forms of life. Antimicrobial peptides are produced by all organisms ranging from bacteria to plants, vertebrates and invertebrates (Figure 1.3). In bacteria the AMPs benefit individual bacterial species by killing other bacterial species that may compete for nutrients and the same environmental niche. Bacterial AMPs synthesized by ribosomes are known as bacteriocins and can be classified into two categories: lantibiotics and non-lantibiotics18,19. Lantibiotics are AMPs containing the non-natural amino acid lanthionine. Nisin, a lantibiotic, was one of the first AMPs isolated and characterized from Lactococcus lactis in 194720. It is active against a variety of Gram positive bacteria with a minimum inhibitory concentration (MIC) in the nanomolar range and has been used as a food preservative for 50 years with no significant development of  6   Table 1.1: Mechanism of antibiotic action and acquired resistance for different antibiotic classes.  Mechanism of action  Antibiotic class (examples) Mechanism of acquired resistance   Interfering of cell wall synthesis21 1. β lactams          a) Penicillin (penicillin, ampicillin )         b) Cephalosporins (cefotaxine)         c) Carbapenems (imipenem)         d) Monobactams (aztreonam) 2. Glycopeptides (vancomycin)  β lactamases produced by bacteria that cleave the β lactam ring  Modified or low affinity DD-transpeptidase  Removal by efflux pump    Inhibition of protein synthesis12,13 1. Tetracyclins (methacyclin) 2. Aminoglycosides (streptomycin) 3. Chloramphenicol  4. Lincosamide (clindamycin) 5. Macrolides (erythromycin) 6. Streptogramins (virginiamycin)  Removal by efflux pump  Enzymatic inactivation/modification  of the antibiotics  Modification of target such as ribosomes   Altered membrane permeability  Inhibition of folic acid  synthesis22 Sulfonamides (sulfamethoxazole)   New enzyme via acquisition of foreign genes  Inhibition of DNA replication22   Fluoroquinolones (ciprofloxanin)  Modification of the target enzymes involved in DNA replication  Inhibition of RNA synthesis12 Rifampin  Point mutation in the gene encoding for RNA polymerase  Disrupting of cell membrane structure or ion gradient22 1. Polypeptides (polymyxin, actinomycin) 2. Ionophores (gramicidin)   Altered membrane structure   Enzymatic degradation    7   resistance23. Other bacteriocins such as mersacidin have also been studied for their possible use against antibiotic resistant Gram positive bacteria24.  Figure 1.3: Sources of antimicrobial peptides (total 2818) as of September 2017 from the antimicrobial peptide database. Image created using GraphPad Prism software from numbers obtained from http://aps.unmc.edu/AP/ on September 20, 2017   Most AMPs reported to date are from eukaryotic origins such as plants, animals, and fungi (Figure 1.3). Since 1885, fluids such as blood, sweat, saliva plasma, white blood cell secretions and granule extracts have been prized for their antimicrobial properties25. However, it was not till 1981 that Hans Boman reported that the hemolymph (plasma and blood) of silk moth (Hyalophora cecropia) contained AMPs known as cecropins26. These peptides are cationic, amphipathic and have broad spectrum activity (i.e. are active against multiple types of microorganisms such a Gram positive and Gram negative bacteria and fungi). The field grew further when Rober Leher, Shunji Natori and Michael Zasloff isolated and described defensins27 (mammalian macrophages), sacrotoxins28 (fly Larvae) and magainins29 (skin’s of frogs (Xenopus Laevis)), respectively.  In eukaryotes, AMPs play an important role in innate immunity (first line of defense). Plants lack adaptive immunity (B cell and T cell mediated immunity) and hence AMPs play a  8   fundamental role in their protection against infection by bacteria and fungi. The presence of genes encoding for plant AMPs can be found in variety of plant species. Interestingly all plant AMPs are cysteine rich and contain many disulphide bonds30. The best studied groups of plant AMPs include the thionins31, plant defensins32 and cyclotides33. Plant AMPs can be found in leaves, flowers, seeds and tubers23.  Similar to plants, invertebrates lack an adaptive immune system and hence are completely dependent on the innate immune system for protection against infection. AMPs have been found in all invertebrates examined to date, which mostly includes insects and marine invertebrates34. AMPs can be found in the hemolymph, hemocytes (blood cells), phagocytes (white blood cells) and epithelial cells of these creatures23. As mentioned earlier, the first AMP (cecropins) from eukaryotes was discovered in silk moth, which is also found in fruit flies (Drosophila). Many other marine invertebrates such as shrimp, oysters and horseshoe crabs express AMPs constitutively35,36 (i.e. a gene is transcribed and translated continually to make a protein or peptides). Tachyplesin and polyphemusin are two potent AMPs produced by horseshoe crabs which possess antibacterial and antifungal activity at low micromolar range37. Interestingly, like some other AMPs, polyphemusin also shows antiviral activity against human immunodeficiency virus (HIV)38.  Although vertebrate immunity consists of both the innate and adaptive immune systems, AMPs have been isolated and characterized from a variety of vertebrates such as fish, mammals and amphibians, indicating the crucial role of AMPs in innate immunity. Vertebrate AMPs can be isolated from a variety of cells, such as granules of white blood cells (phagocytes, neutrophils, macrophages, natural killer cells), epithelial tissue situated in the mouth, lungs, or skin, and bodily fluids39–41. Interestingly, amphibian skin glands have been a rich source of AMPs, with  9   more than 500 AMPs reported to date23. Most vertebrate AMPs show direct antimicrobial activity at high concentration such as in the granules of white blood cells. However, some vertebrate AMPs have also shown to perform critical functions in immune modulation and controlling inflammation42–47. The two most prominent groups of AMPs in vertebrates are cathelicidins and defensins, which will be discussed further in the next section.   1.2.2 Categories of antimicrobial peptides Antimicrobial peptides are a distinct and diverse class of molecules. With over 2800 peptides sequences reported to date, it is important to categorize AMPs. AMPs can be categorized in many different ways, which can be based on the structure, sequence, or mechanism of action. As the activity of the peptides is dependent on the structure and the sequence, it is important to take both of these properties into account while categorizing AMPs. In this thesis, we will be focusing on eukaryotic cationic AMPs, as not many eukaryotic AMPs are anionic.  The first subgroup contains AMPs that adopt an alpha helical structure and which are predominantly found in the extracellular matrix of frogs and insects. Most of these peptides are unstructured in aqueous solution but become structured when in contact with trifluoroethanol, lipids, micelles and liposomes (Figure 1.4). An extensively studied human AMP which is a member of cathelicidins is LL-37 (Table 1.2). Cathelicidins are one of the most diverse AMPs of vertebrates, mainly found in mammals such as humans, mice, sheep, goat, horses and bovines48. Cathelicidin AMPs range from 12-80 amino acids and can adopt a variety of other structures (Table 1.2).  In addition to their antimicrobial activity, cathelicidins such as LL-37 play an important role in immunomodulatory and inflammation responses44.   10   Table 1.2: Classes of antimicrobial peptides based on structure Categorya  Peptides Unique structural/sequence feature Source        α helical peptides Aurein 1-249,50 Mellitin44, Brevinin 151 Maculatins52 Citropin53 Buforin II54 Amidated C-terminus Amidated C-terminus - Amidated C-terminus Amidated C-terminus - Frogs Bees Frogs Frogs Frogs Toad Cathelicidins48  LL-37  BMAP-27,28,34  Magainins   Cecropin   Amidated C-terminus - - Amidated C-terminus   Humans Bovine Frogs Insect      β sheet peptides  Cathelicidins48   Protegrins  Bactenecin    Cysteine rich Disulphide forming loop/Arginine rich   Pigs Bovine Defensinsb,41,55–57   α defensins   β defensins   θ defensins    Three disulphide bonds Three disulphide bonds Three disulphide bonds and cyclic  Mammals Mammals Gorilla Tachyplesins37 and  Polyphemusin58 Cysteine/arginine rich and amidated C-terminus Horse shoe crabs   Extended/flexible Cathelicidins48   PR-39  Tritrpticin  Indolicidin  Crotalicidin 15-34   Proline and arginine rich Tryptophan and arginine rich Tryptophan and amidated C-terminus Lysine rich  Pigs Pigs Bovine Snakes Histatins59 Histidine rich and amidated C-terminus Humans a Classification is based on the predominant structure, some peptides might have mixed alpha           helix and β sheet  b Analogues of defensins are also found in insects, plants and fungi and not restricted to mammals.  11   Another good example are the α helical magainins (Table 1.2), which were originally isolated from the African clawed frog Xenopus laevis and are active against Gram positive and Gram negative bacteria, fungi, yeast and viruses60. The structure and function relationship of the magainins has been well studied61,62. These AMPs were the first to be tested in the clinic, but ultimately failed23. However, the magainin analogue pexiganan is currently in clinical trials63. Most of the alpha helical peptides require amidation at the C-terminus for higher antimicrobial activity (Table 1.2). The amidation of the C-terminus enhances the electrostatic interaction between the positively charged peptide and the negatively charged bacterial membrane. This interaction stabilizes the helical structure at the membrane interface64. The second subclass of AMPs predominantly adopts a β-sheet structure (Figure 1.4). This class includes AMPs such as protegrins (from the cathelicidin family), defensins and tachyplesins. Interestingly nearly all β-sheet AMPs contain cysteine residues that are conserved and form disulphide bonds. In defensins, the disulfide bonds provide structural stability and minimize protease degradation65. β sheet AMPs are more structured in solution and do not    Figure 1.4: Structural diversity of AMPs. (a) the α-helical magainin, (b) β-sheet human defensin 5 and (c) extended coil indolicidin. Positively charged residues are colored blue whereas hydrophobic residues are red. The N and C termini are indicated. The figure was generated using CHIMERA66–69 and PowerPoint.   12    undergo major structural changes when going from an aqueous environment to a membrane environment70. Defensins are the largest group of AMPs that are further categorized in sub-families on the basis and location of the disulphide bond (Table 1.2). Defensins are also involved in antibacterial, antifungal, antiviral, immune and inflammation responses44. Knockout and transgenic mice experiments have indicated that α defensins are critical for protection against Escherichia coli71 and Salmonella enteric72. Tachyplesins and polyphemusin (arginine rich, ~30% by sequence) are other β-sheet AMPs that were isolated from hemocytes of horseshoe crabs51,58.   The third and last subclass of AMPs has a unique extended coil structure. Most of the AMPs in this category are from the cathelicidin family and consist of 2 or more proline residues, which are known to break secondary structural elements such as α-helices or β-sheets73. Indolicidin is a tryptophan rich AMP isolated from bovine neutrophils and consists of only 13 amino acids74. Nuclear magnetic resonance (NMR) and circular dichroism (CD) studies reveal that indolicidin forms a unique membrane-associated peptide structure with well defined extended structure in the presence of micelles74,75 (Figure 1.4c). In a more recent study, Falcao et al76 dissected the crotalicidin peptide and discovered that the C-terminus fragment (crotalicidin 15-34) showed activity against Gram negative bacteria  and tumors. NMR studies of the active crotalicidin 15-34 revealed that the AMP adopts mostly an extended structure (83%) and was only 17% α-helical. The N-terminus (crotalicidin 1-14) was fully α- helical but inactive.  1.2.3 Common properties of antimicrobial peptides.  Although AMPs are a diverse group of molecules in terms of sequence, structure and sources, there are several properties that are common to almost all AMPs. Firstly, most AMPs  13   display a net positive charge (cationic) ranging from +2 to +13 and may contain a specific cationic domain. The cationic nature can be attributed to the presence of lysine and arginine (and sometimes histidine) residues. Many studies have demonstrated the correlation between charge and antimicrobial activity of AMPs77–82. Increasing the charge of magainin 2 from +3 to +5 improved the antibacterial activity against both Gram positive and Gram negative bacteria, but an increase to +6 or +7 lead to increased hemolytic activity and loss of antimicrobial activity78. The loss of antimicrobial activity may be due to the fact that an extremely strong interaction between the peptide and the phospholipid head group would prevent translocation of the peptide into the inner leaflet of the membrane83.    Secondly, hydrophobicity is a key feature for all AMPs and is defined as the percent of hydrophobic residues such as valine, leucine, isoleucine, alanine, methionine, phenylalanine, tyrosine and tryptophan in the peptide sequence (typically 50% for AMPs).  Hydrophobicity governs the extent to which the water-soluble AMPs will be able to partition into the membrane lipid bilayer. It is required for membrane permeabilization; however, excessive levels of hydrophobicity can lead to mammalian cell toxicity and loss of antimicrobial selectivity83–85. Chen et al. examined the influence of hydrophobicity in a synthetic α helical AMP (V13KL) on the antimicrobial activity and hemolysis of human red blood cells (RBCs)85. The results suggest that there is an optimal hydrophobicity needed for good antimicrobial activity. Sequences with hydrophobicities below and very much above this threshold made the peptides inactive85. The decrease in activity when the hydrophobicity is high may be due to the increased likelihood of dimerization, thereby preventing access of the peptide to the bacterial membrane. Additionally, increasing the hydrophobicity of the non polar face of the amphipathic α helix also enhances the  14   lysis of RBCs. This may be due to the membrane discrimination mechanism as peptides with higher hydrophobicity penetrate deeper into the hydrophobic core of the RBC membrane85.  Another feature shared by all antimicrobial peptides is amphipathicity. Amphipathicity refers to the relative abundance of hydrophilic and hydrophobic residues or domains within the AMPs. It can be thought of as the balance between the cationic and hydrophobic residues, not just at the primary sequence level, but also in terms of the 2D or 3D structure of the AMPs. Amphipathicity can be achieved by a number of peptide conformations such as the ones listed in Table 1.2, but the most elegant example is the α  helix. The α helix allows the peptide to form two “faces”, namely the polar and nonpolar face referring to the arrangement of the hydrophobic and hydrophilic side chains of the residues in the helix. Amphipathicity of AMPs can be reflected by calculating the hydrophobic moment which is the vector sum of individual amino acid hydrophobicity, standardized to an ideal helix83 (calculated using many websites such as http://www.bioinformatics.nl/emboss-explorer/). Interestingly, for α-helical AMPs it was previously thought that disruption of the amphipathicity leads to an increase in antimicrobial activity and reduction in RBC lysis59,86–89, however, a recent study by Zhang et al. on melittin related peptides demonstrated that increased amphipathicity also leads to a decrease in RBC lysis90 suggesting a complicated relationship between amphipathicity, hydrophobicity and net charge. It would seem rather that the different parameters play a unique role, depending on the peptide sequence.   1.2.4 Mechanism of antimicrobial peptide action  Antimicrobial peptides are unique molecules and their mechanism of action (MOA) has been studied extensively since they were discovered. It is important to understand the MOA of these AMPs to facilitate further development as therapeutic agents. It was originally thought that  15   membrane targeting was the only mode of action, but there is increasing evidence now that AMPs have other MOA56 (Figure 1.5). The MOA can be divided into two major classes: direct killing and immune modulation. The direct killing mechanism of action can be further divided into membrane targeting and non-membrane targeting, which will be the focal point of the following sections.   Figure 1.5: Various mechanisms of action of antimicrobial peptides. Figure adapted with permission from Ulm, H et. al. Antimicrobial Host Defensins – Specific Antibiotic Activities and Innate Defense Modulation. Front. Immunol. 3, 249 (2012).  1.2.4.1 Direct killing: membrane permeabilizing mechanism of action  The membrane targeting AMPs can have receptor mediated or non-receptor mediated interactions. The receptor mediated pathway mostly includes AMPs produced by bacteria and which are active in vitro in the nanomolar range, such as nisin91. The nisin peptide has two domains: the first domain binds with high affinity to the lipid II molecule, a membrane anchored cell wall precursor. The second region is the membrane-embedded pore forming domain. Mesentericin is another example of a receptor mediated membrane targeting AMP92. AMP  16    Most vertebrate and invertebrate AMPs target the membrane without specifically interacting with receptors. These AMPs are typically active in vitro against microbes at micromolar concentrations93 and function by interacting with the components of the membrane. The outer surface (Figure 1.1) of Gram positive bacteria and Gram negative bacteria contains teichoic and lipopolysaccharide, each conferring net negative charge on the surface allowing the initial electrostatic attraction with cationic AMPs83,93. More importantly, AMPs target a fundamental difference in design between bacterial membrane and membrane of multicellular animals. The outer monolayer (leaflet) (Figure 1.6) of the lipid bilayer in bacterial membranes is mostly made up of lipids with negatively charged head groups such as phosphatidylglycerol (PG) and cardiolipin94 whereas the outer leaflet of the animal membranes are made up of zwitterionic phospholipids such as phosphatidylcholine (PC), sphingomyelin and other neutral components such as cholesterol95. Most of the lipids with negatively charged head groups are in the inner leaflet facing the cytoplasm in animal membranes95,96. The positively charged AMP has strong electrostatic interaction with the negatively charged phospholipids on the outer leaflet of the bacterial membrane (Figure 1.6).  Moreover, some AMPs are even sensitive to other properties of the lipids and not just the charge 97,98. Magainins can induce leakage more effectively in liposomes made of PG, an anionic phospholipid found predominantly in bacterial membrane compared to liposomes composed of negatively charged phosphatidylserine (PS), a major phospholipid of animal membranes97. Lipids have different shapes depending on the size of the head group and hydrophobic tails. PS   17     Figure 1.6: Initial interaction of cationic AMPs with the multicellular animal (left) or bacterial (right) membrane. RBC: red blood cell. Figure created using PowerPoint and Chimera.  and PG have molecular shape similar to a cone and cylinder respectively and hence display different membrane curvature properties99. The specific interactions are not only limited to anionic lipids as AMPs, such as plant cyclotides, can also bind specifically to zwitterionic lipid such as phosphatidylethanolamine (PE), which is abundantly present at the surface of bacterial membranes and also found in great quantity in the inner cytoplasmic leaflet of the animal membranes100,101. These studies suggest that membrane charge is not the only factor that is important for the initial interaction but other properties such as for example membrane curvature may also play an important role97,102,103.   After the initial electrostatic and hydrophobic interactions, the AMPs accumulate at the surface and self assemble on the bacterial membrane after reaching a certain concentration104,105. At this stage various models have been used to describe the action of AMPs. The models can be  18   classified under two broad categories: transmembrane pore and non-pore models. The transmembrane pore models can be further subdivided into the barrel-stave pore and toroidal pore models. In the barrel stave model, the AMPs are initially oriented parallel to the membrane but eventually insert perpendicularly in the lipid bilayer106(Figure 1.7a). This promotes lateral peptide-peptide interactions, in a manner similar to that of membrane protein ion channels. Peptide amphipathic structure (α and/or β sheet) is essential in this pore formation mechanism as the hydrophobic regions interact with the membrane lipids and hydrophilic residues form the lumen of the channels107,108 (ring like a barrel pore).  A unique property associated with AMPs in this category is a minimum length of ~22 residues (α helical) or ~8 residues (β sheet) to span the lipid bilayer. Only a few AMPs, such as alamethicin109, pardaxin110,111 and protegrins107, have been shown to form barrel stave channels. Furthermore, in the toroidal pore model, the peptides also insert perpendicularly in the lipid bilayer but specific peptide-peptide interactions are not present109. Instead the peptides induce a local curvature of the lipid bilayer with the pores partly formed by peptides and partly by the phospholipid head group (Figure 1.7b). The dynamic and transient lipid-peptide supramolecule is known as the “toroidal pore”. The distinguishing feature of this model as compared to the barrel-stave pore is the net arrangement of the bilayer: in the barrel-stave pore, the hydrophobic and hydrophilic arrangement of the lipids is maintained, whereas in toroidal pores model the hydrophobic and hydrophilic arrangement of the bilayer is disrupted. This provides alternate surfaces for the lipid tail and the lipid head group to interact with. As the pores are transient upon disintegration, some peptides translocate to the inner cytoplasmic leaflet entering the cytoplasm and potentially targeting intracellular components112. Other features of the toroidal pore include ion selectivity and discrete size83. A number of AMPs such as magainin  19   270, lacticin Q70, aurein 2.2113 and melittin70,109 have been shown to form toroidal pores. Both pore forming models (toroidal pore and barrel stave) lead to membrane depolarization and eventually cell death.   Figure 1.7: Proposed mechanisms of action for AMPs in bacteria. This figure was generated using Microsoft PowerPoint and Chimera.  AMPs can also act without forming specific pores in the membrane. One of these models is designated as the carpet model70,93,83. In this case, the AMPs adsorb parallel to the lipid bilayer and reach a threshold concentration to cover the surface of the membrane, thereby forming a “carpet” (Figure 1.7c). This leads to unfavorable interactions on the membrane surface. Consequently, the membrane integrity is lost, producing a detergent-like effect, which eventually disintegrates the membrane by forming micelles. The final collapse of the membrane bilayer  20   structure into micelles is also known as the detergent-like model (Figure 1.7d). The carpet model does not require specific peptide-peptide interactions of the membrane-bound peptide monomers; it also does not require the peptide to insert into the hydrophobic core to form transmembrane channels or specific peptide structures83. Many peptides act as antimicrobial agents despite their specific amino acid composition or the length of the sequence. Such AMPs typically act using the carpet model93, and do so at high concentrations because of their amphiphilic nature23. Examples of AMPs acting by the carpet model are cecropin114, indolicidin53 , aurein 1.2115, and LL-3793 .  Overall, there are a number of models to describe the MOA of AMPs. In addition to those given above, there are other related models such the Shai-Huang-Matsazuki model, the interfacial activity model, and the electroporation model70. Some models do not make the specific distinctions shown in Figure 1.7. For example, it has been suggested that the carpet-like mechanism is a prerequisite step for the toroidal pore model70. Most studies to elucidate the MOA of AMPs involve the use of model membranes. The mode of action of only a few AMPs has been investigated with whole bacterial cells using imaging techniques116,117. It is possible that different results may be obtained using different membrane models or assay conditions, i.e. that the models described here may or may not translate directly to what is occurring in bacteria. 1.2.4.2 Direct killing: non membrane targeting mechanisms of action  The non-membrane targeting AMPs can be divided into two broad categories: those that target the bacterial cell wall and those that have intracellular targets (Figure 1.5). Similarly to conventional antibiotics like penicillin, AMPs can also inhibit cell wall synthesis. Although most conventional antibiotics bind to specific proteins/enzymes involved in the synthesis of the cell wall components, AMPs often interact with various precursor molecules that are required for cell  21   wall synthesis. One molecule that is a prime target is the highly conserved lipid II, a precursor molecule in the synthesis of the cell wall118. For instance, AMPs such as defensins bind to the negatively charged pyrophosphate sugar moiety of the lipid II molecule119 (Figure 1.8). The binding event can further promote formation of pores and membrane disruption118. AMPs such as the human β defensin 3119 and α defensin 1120 rely on selective binding to lipid II to confer bactericidal activity.  Figure 1.8: Structure of lipid II molecules with pyrophosphate sugar moiety circled in red. This figure was generated using Chemdraw.   When AMPs were first discovered, it was thought that they could not have intracellular targets. Studies with the original α helical peptides such as magainin, cecropin and melittin showed that an all D-amino acid version of these peptides was equipotent compared to the natural all L-amino acid  peptides121, supporting the idea that stereospecific targets such as  22   proteins or DNA/RNA were not required for antibacterial activity, further confirming that AMPs target the membrane98. However, subsequent studies revealed that other AMPs with all-D or all-L amino acids did not have equal activity122. Now it is well established that several AMPs have intracellular targets as some AMPs do not cause membrane permeabilization at the minimal effective concentration, but still cause bacterial death107.  Mechanistically, these AMPs interact with the cytoplasmic membrane first and then accumulate intracellularly, where they can block critical cellular processes. Many novel mechanisms involving intracellular targets, such as inhibition of protein/nucleic acid synthesis and disruption of enzymatic/protein activity, have been discovered107. For example, buforin II, an histone derived AMP from frogs, translocates through the bacterial membrane without permeabilization and binds to the DNA and RNA of E.coli54. Human α defensin 5 also translocates into the cytoplasm of E.coli and accumulates at the cell division plate and at opposite poles suggesting part of the antibacterial activity might be due to the targets in the cytoplasm. Indolicidin123, human β defensin 4124, human α defensin 157 and PR-39125 have also been shown to target intracellular components in the bacterial cell.  1.2.4.3 Immune modulation mechanism of action  In addition to direct killing of microbes, AMPs can also recruit and activate immune cells (Figure 1.5), resulting in enhanced microbial killing and/or control of inflammation42,126,127. As AMPs are produced by many immune cells such as neutrophils and macrophages, they are one of the first molecules that encounter invading microbes23. In an infection, it is important to produce an immune response to attract other immune cells and also control inflammation. Interestingly some AMPs can produce a variety of immune responses, such as activation, attraction, and differentiation of white blood cells, stimulation of angiogenesis (formation of new blood  23   vessels), reduction of inflammation by lowering the expression of proinflammatory chemokines, and controlling the expression of chemokines and reactive oxygen/nitrogen species46,42,126,128,129.  The human AMPs such LL-37 and β defensins have the ability to attract (chemoattract) many immune cells such as mast cells130, leukocytes131 and dentritic cells132. Innate defense regulators (IDR) which are synthetic versions of natural AMPs, such IDR-1 and IDR-1018, also suppress pro-inflammatory cytokines in mice infection models133,134. IDR-1018 has also shown promise in reducing the inflammation response in severe malaria, without having direct anti-malaria activity. Mice treated with a combination of anti-malaria agents and IDR-1018 demonstrated a reduction in the harmful neural inflammation which otherwise leads to death. Although most AMPs have been shown to interact with innate immune system components such as neutrophils and macrophages, there is evidence that they are also involved in modulation of the adaptive immune system, i.e. the T and B cells. The exact mechanisms are not well understood42, however, some studies show that AMPs may act as vaccine adjuvants129,135. Interestingly, all these studies show that AMPs work in many independent or co-operative “multi-hit”136 mechanisms of action, making AMPs ideal candidates for future development. 1.2.5 Aurein peptides Aurein peptides are secreted from the granular dorsal glands of the Australian Green and Golden Bell Frog Litoria aurea and the Southern Bell Frog L. raniformis. There are more than 30 aurein peptides from five different families, ranging from the short active aurein peptides (aurein 1-3) to the longer peptides such aurein 4.1 and 5.1 which are not active50. Most aurein peptides are active against Gram positive bacteria such as Staphylococcus aureus and Staphylococcus epidermidis. Some peptides, such as aurein 1.2, 3.2 and 3.3, display their strongest activity against 30-50 different types of cancer49. Most of the active aurein peptides are  24   amidated at the C-terminus and usually adopt an α-helical structure when in contact with phospholipids or a membrane-mimetic environment50,64,113,115,137.   The mechanism of action and structure of aurein 2.2 has been extensively studied in recent years. Earlier studies with aurein 2.2 have shown that it is important to study the bilayer perturbation in membrane models made from PC and PG rather than PC alone, indicating that electrostatic interactions are important in the lipid–peptide interaction137. In a POPC/POPG (1:1) membrane model, the peptides induce toroidal pores. In contrast, in a DMPC/DMPG (1:1) membrane model, the peptides work in a detergent like model indicating the importance of the hydrophobic thickness of the lipid bilayer and the membrane composition113,138. The truncation of the N-terminus leads to the loss of antimicrobial activity but makes the peptide immunomodulatory139. In a more recent study, Wenzel et al. showed that aurein 2.2 forms ion selective pores, permitting the translocation of ions such as potassium and magnesium. In addition aurein 2.2 also causes membrane permeabilization, which disrupts the membrane potential and decreases the energy supply of the cells leading to cell death140.  1.2.6 Challenges with antimicrobial peptides  Although many eukaryotic AMPs have been identified and characterized, not many have made it to clinical trials (Table 1.3) and only a few have been approved by the US Food and Drug Administration (FDA). Most AMPs in clinical trials are analogues of natural AMPs, but there are some that are completely synthetic (e.g. IMX942). The majority of AMPs in clinical trials are limited to topical applications, due to the systemic toxicity, the susceptibility of the peptides to protease degradation and the rapid kidney clearance23,105,141 of these peptides if they are ingested. Oral administration of AMPs can lead to proteolytic digestion by enzymes in the digestive tract such as trypsin and pepsin. Systemic administration results in short half lives in  25   vivo, protease degradation and cytotoxic profiles in blood43.  Many strategies have been investigated to circumvent these issues and to improve the efficacy of AMPs. These include chemical modification of AMPs142 and the use of delivery vehicles143. These strategies will be discussed in more detail below.  Table 1.3: Antimicrobial peptide in clinical trials43,126,144,63 Peptide Progress Application AMP analogue (host) Pexiganan Phase III Topical application for diabetic foot ulcers Magainin (frogs) OP145 Phase I/II Bacterial ear infection LL-37 (humans) Omiganan Phase III Topical cream for prevention of cathether infection, severe acne, rosacea, atopic dermatitis Indolicidin (bovine) PAC 113 Phase II Mouth wash for fungal/yeast infection Histatin (humans) Iseganan Phase III Treatment of inflammation and ulceration of digestive system mucous membrane Protegin-1  (pigs) IMX942 Phase II Intravenous administration against hospital-acquired bacterial infections Synthetic  analogue of IDR-1  1.3 Strategies to improve antimicrobial peptides  1.3.1 Chemical modification of AMPs        Various chemical modifications of AMPs have been utilized to improve the stability of peptides against proteolytic digestion including the use of D-amino acids, cyclization, acetylation and peptidomimetics. Incorporation of non natural D-amino acids into AMP sequences reverses the stereochemistry of the peptide and hence prevents protease degradation, as enzymes are stereospecific.   26   In a recent study, Zhao et al. isolated a lysine rich AMP from the venom of the social wasp (Polybia paulista), MPI, which was active against Gram positive and Gram negative bacteria and also fungi145. In order to prevent proteolytic digestion by trypsin the authors designed two peptides, one with all the amino acids replaced with D-amino acids, D-MPI, and the other peptide with only the lysine residues substituted with D-amino acid (Figure 1.9a), D-lys-MPI, because trypsin cleaves after positively charged amino acids such as lysine145. Interestingly, both the peptides, D-MPI and D-lys-MPI were resistant to trypsin digestion, however only D-MPI was equipotent in terms of activity when compared to MPI. D-lys-MPI was inactive because the secondary structure was destabilized upon introduction of single D-amino acids. D-MPI adopted a right handed α-helical conformation, whereas the D-lys-MPI did not adopt any regular structure.  In a similar study, D-BMAP28, a peptide from bovine myeloid, was made proteolytically stable by replacing all amino acids by the D- counterparts. D-BMAP28 remained equipotent in terms of both its antimicrobial and immunomodulatory activities when compared to BMAP38146. Moreover, Falciani et al. reported that another AMP, D-M33, was more active against biofilms formed by Gram positive bacteria, as compared to M33147. Overall, the use of D-amino acids in AMPs leads to retention of the antimicrobial activity, while preventing proteolysis. This confirms that these AMPs interact with the bacterial membrane without making use of specific receptors43, since the stereochemistry of the amino acids has no impact on membrane binding. Finally, it should be emphasized that the synthesis of peptides containing D-amino acids is very costly148. Alternative strategies are thus important to reduce the economic impact.  These alternative approaches are many and variable. For instance, the substitution of positively-charged arginine in a sequence with other charged non natural amino acids, such as L- 27   orthothine and L-homoarginine (Figure 1.9b), also increases proteolysis stability of AMPs149. Moreover, acetylation of the N-terminus also increases the proteolytic stability of peptides as it blocks the activity of aminopeptidases; however, this leads to removal of a positive charge which in most cases decreases the antimicrobial activity150,151. Cyclization of peptides by different methods also prevents protease degradation (Figure 1.9c). Cyclization by joining the backbone N and C termini or by disulfide bridges similar to human defensins are common strategies used to increase serum stability of AMPs152. In a recent study, click chemistry was developed for specific cyclization of certain amino acids153. The results suggested that the α helical structure was critical for activity as the i, i+4 cyclization (1st and 4th amino acid cyclized) retained the structure and activity compared to i, i+6 cyclization, which was not structured and inactive.  Further strategies include the use of peptidomimetics: peptide-like polymers made from a backbone that is altered when compared to a peptide154–156. The main concept in peptidomimetics is to maintain the activity of the peptidomimetics by conserving the 2D and 3D spatial arrangement of the side chains, but modify the backbone to prevent proteolysis degradation. Some examples of peptidomimetics include peptoids, ceragenins, oligoacyllysines and β-peptides155,156. Peptoids are isomers of peptides in which the side chain is bonded to the backbone nitrogen instead of the alpha carbon making them resistant to protease degradation157 (Figure 1.9d). Peptoids derived from pexiganan have been shown not only to mimic the 1D structure but also mimic the 2D structure, function and mechanism of action of pexiganan158. CD studies confirmed that peptoids adopt α-helical structure in the presence of phospholipids, whereas X-ray reflectivity showed peptoids bind to the membrane and are membrane active158. Cyclization of peptoids also enhances the membrane permeation properties leading to better antimicrobials159.   28    Figure 1.9: Chemical modifications of AMPs. (a) Use of D amino acids such as lysine, (b) Use of non natural amino acids such as L-homoarginine, (c) Various cyclization strategies. (d) 1-dimensional structural difference between a peptide and peptoid. This figure was generated using ChemDraw.   1.3.2 Delivery systems for AMPs   Another important strategy to improve the properties of AMPs is to use delivery systems, i.e. systems ranging from inorganic and polymer materials, surfactant/lipid self assembly systems to peptide self assembly systems143,160 which can be used to improve the stability, toxicity, half-life and release profile of AMPs. The AMPs can be covalently attached to the delivery system or non-covalently encapsulated by these different types of systems. From their mechanism of action, it is well known that AMPs interact with lipids and hence lipid-based delivery systems of peptides are well studied161. The encapsulation of LL37 within liposomes composed of DSPC/DSPE-PEG/cholesterol ensured enhanced bioactivity and reduced toxicity towards cell  29   cultures162. In a similar study, DSPC/DSPG liposomes were found to encapsulate nisin and protect it from extreme alkaline/acidic conditions and elevated temperature163. Other nanoparticles for encapsulation of AMPs include DNA cages, metal particles, polymeric systems, carbon nanotubes and mesoporous silica particles160,164.  As mentioned above, in addition to encapsulation, AMPs can be covalently attached to various molecules, e.g. PEG (Figure 1.10a). PEGylation is a process by which a polyethylene glycol chain is added to a biomolecule. PEGylation is one of the most extensively investigated strategies to improve the performance of proteins, peptides and other biomolecules. The advantages of PEGylation include reduced non-specific uptake in tissue, reduced cell toxicity, increased blood half-life and reduced proteolytic degradation160,165. Many AMPs such as tachyplesin I166, magainin 2167, and nisin168 have been PEGylated, leading to improved properties. However, this improvement is often at the expense of the antimicrobial activity. For instance, PEGylation of the cyclic peptide tachyplesin and of magainin reduced toxicity towards CHO-K1 cells, but also decreased antimicrobial activity towards E. coli and S. epidermidis. Interestingly, PEGylation of nisin via the amine group of lysine lead to a conjugate that was inactive. It was hypothesized that the amino group of the lysine residue is needed to bind to the pyrophosphate of the lipid II molecule, hence the loss of activity. In other words, the site and nature of the conjugation chemistry168 has an impact on the properties of the resulting compounds. Finally, PEGylation of KYE28 revealed that increasing the length of PEG lead to a partial loss in antimicrobial activity, but also to a strong decrease in hemolysis and to improved selectivity in blood and bacteria mixtures169. Important drawbacks of linear PEG is its relatively large hydrodynamic size, high intrinsic viscosity in aqueous conditions which increases with  30   increase in molecular weight (and limits the generation of concentrated peptide formulations) and the lack of multiple functionality 40.  Alternatively, AMPs can also be conjugated to biopolymers such as chitosan and hyaluronic acid, which have multiple functional groups for the attachment of peptides (Figure 1.10b and c). Chitosan is a linear biocompatible, biodegradable carbohydrate polymer with some antimicrobial activity170. Conjugation of the short and moderately active anolin to chitosan increased the antimicrobial activity of the conjugate and abolished the hemolytic activity171. In general, the antimicrobial activity increased in proportion to the number of peptides attached to the chitosan polymer. Recently, click chemistry has been used as an interesting approach to attach AMPs to chitosan at specific sites172.    Figure 1.10: Various polymers used for AMP conjugation. (a) polyethylene glycol, (b) chitosan, (c) hyaluronic acid. Image created using ChemDraw.  Another biocompatible, biodegradable linear carbohydrate polymer utilized for AMP conjugation is hyaluronic acid. In contrast to PEGylation, conjugation of nisin to hyaluronic acid results in a conjugate which maintains antimicrobial activity173. The hyaluronic-nisin conjugates  31   were mostly active against Gram positive bacteria, and in the presence of EDTA the antimicrobial activity extended towards Gram negative bacteria. The EDTA binds to divalent cations such as Mg2+ and disturbs the lipopolysacchride-divalent cation interaction, disintegrating the outer membrane of the Gram negative bacteria, making it more susceptible to the hyaluronic-nisin conjugate.  1.4 Hyperbranched polyglycerol  Recently hyperbranched polyglycerol (HPG), a synthetic polymer, has gained a lot of attention in the research community due to its excellent biological properties174. HPG was originally synthesized by cationic ring opening multi branching polymerization (ROMPB) but due to various limitations, such as multiple side reactions, anionic ROMBP with slow monomer addition has been utilized thereafter175–177. The synthesis of high molecular weight HPG (>100,000 Da) usually requires an emulsifying agent such as 1,4 dioxane as a solvent178 due to the poor mixing that arises because the viscosity of the solution increases as polymerization proceeds177. HPG has been used in many biomedical applications due to the ease in its synthesis, globular structure, and various hydroxyl groups available for functionalization, low viscosity, high solubility and excellent biocompatibility.  HPG has excellent blood compatibility compared to PEG. Studies by the Brooks and Kizhakkedathu groups demonstrated that HPG had no effect on red blood cell aggregation and lysis, platelet activation and aggregation, complement activation, and blood coagulation179–181. In contrast, similar molecular weight PEG aggregate red blood cells, activate platelets and the complement system and alter blood coagulation181. Additionally, high and low molecular weight HPG are also compatible with many different types of cells in cell culture models such as fibroblasts182, human umbilical vein endothelial  cells (HUVEC)181, human peritoneal  32   mesothelial182 cells and H9e2 cells183. More importantly, the in vivo profile of HPG looks promising in different models: for instance, hydrophobically modified184, cationic185 or drug conjugated186,187 HPGs were all well tolerated in mice and found to be non toxic.  Figure 1.11: Generic structure of HPG. Image created using ChemDraw.   Furthermore, the pharmacokinetic properties of HPG and modified HPG have been well studied. The circulation half-life for 100kDa HPG is much longer (~40 hrs) compared to similar molecular weight PEG and linear polyglycerols (LPGs)181. The circulation half-life can be fine-tuned by varying the molecular weight181,188 and/or by incorporating ketal linkages189 in the HPG backbone. The repeated injection of the HPG modified red blood cells in mice models suggest that mice do not generate an immune response to the HPG molecule, unlike what was observed for PEG190. Although HPG has excellent in vivo biocompatibility, high molecular weight HPG is known to accumulate in liver and other organs187; however, the accumulation is lower compared to other polymers of similar molecular weight191.   33    Moreover, due to its excellent cell tolerance, blood and in vivo biocompatibility,  HPG has been utilized for many biomedical applications such as the development of long circulating drug conjugates186,187, anticoagulant neutralizing agents185,192, modification of red blood cell surface193,194, as an osmotic agent in peritoneal dialysis195 and for cell/organ preservation196. There are only a few examples of HPG conjugated peptides used for biomedical applications. For instance, Freund and co-workers utilized the multivalency effect of HPG by conjugating many peptides to the HPG scaffold197. The proline rich peptide used is a binding partner to FBP21, a protein involved in splicing of pre-mRNA in the nucleus of eukaryotic cells. The binding affinity was enhanced by nearly 9-fold once the peptides were conjugated to HPG compared to free peptides197. In another example, Zhang et al. conjugated a very short peptide, denoted RGD (arginine-glycine-aspartic acid), to HPG polymer for its use in antiplatelet therapy198. RGD is known to bind to platelet integrin GPIIbIIIa and interrupts platelet-fibrinogen binding and platelet aggregation during thrombosis198. Conjugation of the RGD peptides to high molecular weight HPG increased the platelet inhibitory function by 2-3 orders of magnitude. Moderate levels of conjugated peptides could lead to suitable candidates for the development of an antithrombotic drug.  Additionally, Haag and co-workers conjugated a tolerogenic peptide to HPG to increase the bioavailability and efficacy in vaccination strategies. Tolerogenic peptides have the ability to provide immunological tolerance199. The tolerogenic peptide pOVA is presented via the major histocompatibility complex II (MHCII) of the antigen presenting cell (APC) and recognized by T-cells. The uptake, storage and stability of the HPG-pOVA conjugates were dependent on the linkage chemistry. The degradable ester linked conjugates had superior properties compared to amide linked polymers. The authors also speculated that the linker structure might additionally  34   target the conjugates to specific APC and favor a tolerogenic T-cell response. These conjugates are promising novel candidates for vaccines that could be used for the treatment of autoimmune diseases and allergies199. 1.5 Thesis rationale, hypotheses and aims The emergence of antibiotic resistant microbes (Table 1.1)  and the limited discovery and development of new antibiotics is one of the greatest threats to human health16. Although antimicrobial peptides can be used as alternatives or synergistically with antibiotics to treat antibiotic resistant microbes, there are several limitations associated with the use of AMPs. These limitations include systemic toxicity, proteolytic degradation and short circulation half-lives23,105,141, for example. Although aurein 2.2 shows good activity against Gram positive bacteria in vitro, it is however, toxic towards peripheral blood mononuclear cells (PMBCs)139.  The main objectives of this thesis are hence: 1) to develop ways to increase the biocompatibility of aurein 2.2 while maintaining sufficient antimicrobial activity via polymer conjugation approaches, specifically conjugation to hyperbranched polyglycerols (HPG); 2) to investigate the influence of polymer molecular weight and peptide sequence on the antimicrobial activity; 3) to determine the efficacy of the optimal peptide and polymer peptide conjugate in mice models of infection, and 4) to establish Nuclear Magnetic Resonance (NMR) methodology that can be used in the future to study the mechanism of action of the peptides. These will be described briefly below. 1.5.1 Development of novel antimicrobial conjugates   In recent years different polymer systems have been developed to alleviate the limitations associated with AMPs but lead to a significant decrease in the activity of peptides169,171,173170. Our laboratory has pioneered the synthesis and characterization of HPG, and has shown that it is  35   a highly biocompatible polymer185,186,200. Studies reported in Chapter 2 uncover the molecular design of the HPG-aurein 2.2 conjugates, and the influence of peptide density and structure on their antimicrobial activity. We hypothesize that conjugation of multiple aurein 2.2 peptides to HPG would decrease the toxicity of the peptides but will maintain the antimicrobial activity due to the multivalent presentation and compact nature of HPG. To test this hypothesis, the specific aims are to:  1. Develop a methodology to synthesize the HPG-aurein 2.2 conjugate with different number of peptides per polymer. 2. Compare the antimicrobial activity of the HPG-aurein 2.2 conjugates. 3. Measure the toxicity of the HPG-aurein 2.2 conjugates against various blood components and mammalian cells. 4. Correlate the activity of the HPG-aurein 2.2 conjugates to the peptide structure using biomembrane mimicking phospholipid vesicles. 1.5.2 Development of potent novel antimicrobial peptides and conjugates Many studies have designed mutants of natural AMPs to increase the antimicrobial activity of peptides. Increasing the charge of the peptide is a common strategy used to enhance antimicrobial activity87. Although making single amino acid mutations in a peptide sequence and purifying them is a laborious task, it has been used for decades to design AMP analogues139. A more efficient method to generate peptide analogues is SPOT synthesis of peptides on cellulose membrane which enables the screening of hundreds or even thousands of peptides at once201–203. It has been shown previously that polymer scaffold molecular weight could influence the activity of peptides conjugated due to the increased molecular volume and changes in the peptide  36   conjugation density204. Moreover, the molecular weight of HPG can be fine tuned for various biomedical applications.  Studies reported in Chapter 3 provide insight into the influence of peptide sequence and polymers molecular weight on antimicrobial activity, blood and cell toxicity, and stability against proteolytic degradation of the conjugates. It is hypothesized that the tryptophan and arginine mutation of the aurein 2.2, the parent peptide, would lead to novel peptides with potent antimicrobial activity and conjugation of the peptide to the polymers will result in conjugates with excellent antimicrobial activity and biocompatibility. The specific aims for this chapter are to:  1. Design and screen an antimicrobial peptide array containing arginine and tryptophan mutants.  2. Synthesize various molecular weights of HPG and conjugate them with novel peptides.  3. Evaluate the antimicrobial activity and toxicity of the conjugates and the novel peptides. 4. Determine the proteolytic susceptibility and structure-function relationship of the best candidate conjugate. 1.5.3 In vivo efficacy of the peptides and the conjugates in mice models  An abscess is a common skin condition caused by bacterial infection and is characterized by the accumulation of pus in the skin which normally appears red, swollen and tender and may cause severe pain205. In 2005, approximately 3.2 million people with abscesses were treated in hospital emergency departments in the US alone206. Abscesses are treated by surgically draining the pus and treating with antibiotics to prevent spreading and reoccurrence207. Antibiotics do not work within the abscess due to limited penetration, high bacterial loads and low pH208. Methicillin resistant Staphylococcus aureus (MRSA) is an example of a bacterial species that  37   causes difficult to treat abscesses. Mansour et al. have shown that MRSA in abscess and biofilm conditions produce similar stress response which can be targeted in mice by antimicrobial peptide DJK-5207. In another study, D-amino acid AMPs eradicate biofilms formed by multi-resistant Pseudomonas aeruginosa in invertebrate (Caenorhabditis elegans and Galleria mellonella) survival models209.  The novel peptides developed in Chapter 3 showed enhanced in vitro antimicrobial activity against S. aureus compared to the aurein peptides. Studies reported in Chapter 4 investigate the in vivo efficacy of the peptides and the conjugates in mice abscess models. Our hypothesis is that the novel peptides and their conjugates will be effective in preventing abscesses in mice models, and the D-amino acid version of the peptides will further enhance their in vivo efficacy. The specific aims to test this hypothesis are to:  1. Evaluate the efficacy of the various novel peptide analogues in a mouse abscess model. 2. Conjugate the most active peptide to HPG and evaluate in mouse abscess model. 3. Design the D-amino acid version of the most active peptide and evaluate its in vivo efficacy. 1.5.4 Interaction of aurein 2.2 with whole bacterial cells by NMR. The mechanism of action (MOA) of aurein 2.2 has been well studied and many techniques such as NMR,  CD spectroscopy and leakage assays117,139 have been developed to understand the bacterial killing action of AMPs. Although the MOA of some AMPs is well understood, most studies use model membrane systems such as supported bilayers, liposomes, vesicles and micelles showing that AMPs damage the membrane or form membrane pores via various mechanisms such as toroidal pore, barrel stave or detergent models as described in section 1.2.4.2. Hence, the main killing action of AMPs is thought to target the bacterial cell  38   membrane, but as mentioned earlier, AMPs may also target the cell wall or other intracellular components160. To date only a few studies have explored the membrane/cell wall-peptide or peptide-intracellular component interactions in living cells116. Therefore, it is important to investigate the detailed structural interaction between AMPs and their targets in living cells to further understand their MOA.  NMR is one of the most powerful techniques that can used to elucidate the detailed structural  interactions between AMPs with potential molecular targets such as membranes, proteins and nucleotides210.  To date only a few whole cell NMR experiments have been reported to determine the interactions of AMPs with different bacterial components. Solid state NMR studies (cross-polarization magic-angle spinning, CPMAS) of whole planktonic cells were able to detect the total 13C and 15N composition of intact S. aureus cells and pinpoint the general MOA211. On the other hand, isotopic labeling of peptides instead of the bacterial cells can be used to determine the specific interaction of AMPs with live bacterial cells. Chemical isotopic labeling of the AMPs is not practical due to high cost; however, recombinant expression of AMPs in bacteria has been recently well developed by the Hans J. Vogel lab, using a calmodulin fusion construct 210. A common disadvantage with AMPs expressed in bacteria is their free C-terminus whereas most AMPs, including aurein 2.2, require an amidated C-terminus to display potent antimicrobial activity.   Studies reported in Chapter 5, describe a methodology to express and purify aurein 2.2 in a bacterial expression system and chemically modify the carboxyl C-terminus to restore the activity which was then used to probe the interaction of aurein 2.2 with whole bacterial cells by NMR. The specific aims are to:  39   1. Express, purify and cleave the isotopically (15N) labeled calmodulin-aurein fusion protein. 2. Esterify the carboxyl C-terminus of expressed aurein 2.2 and measure the antimicrobial activity. 3. Develop a preliminary 2D NMR methodology to study the interaction of 15N labeled aurein 2.2 with whole bacterial cells.  1.6 Summary  Overall, these studies will provide fundamental information of AMP-polymer conjugates which can be used to design novel drug candidates with better antimicrobial activity, and can be further developed into antibiotics that can combat the ABR crisis. It also provides a useful tool to understand the mechanism of other AMPs and conjugates in the future.    40   Chapter 2: Conjugation of aurein 2.2 to HPG yields an antimicrobial with better propertiesb 2.1 Synopsis  Aurein 2.2 is an antimicrobial peptide (AMP) whose mechanism of action is quite well understood and with good activity against Gram positive bacteria. It is, however, cytotoxic. Recently, hyperbranched polyglycerol (HPG) has been gaining a lot of attention as an alternative to PEG due to its excellent biocompatibility. In this Chapter, I report the synthesis of HPG conjugates of antimicrobial peptides. Aurein 2.2 peptide was conjugated to high molecular weight HPG with varying number of peptides per polymer and their biocompatibility and antimicrobial activities were investigated. The antimicrobial activity of the peptide and its conjugates were determined by measuring the minimal inhibition concentration (MIC) against S. aureus and S. epidermidis. The interaction of aurein 2.2 peptide and the conjugates with model bacterial biomembranes was investigated using CD spectroscopy to understand the mode of action of the conjugates. The biocompatibility of the AMP-polymer conjugates was investigated by measuring the red cell lysis, platelet activation and aggregation, complement activation, blood coagulation, and cell toxicity. Results presented in this chapter show that the size of the conjugates and the peptide density influence the biocompatibility of the antimicrobial conjugates. These results will help to further define the properties of HPG-AMP conjugates and set the stage for development of better therapeutic agents.   bA version of Chapter 2 has been published. Kumar, P.; Shenoi, R. A.; Lai, B. F. L.; Nguyen, M.; Kizhakkedathu, J. N.; Straus, S. K. Conjugation of Aurein 2.2 to HPG Yields an Antimicrobial with Better Properties. Biomacromolecules 2015, 16, 913–923, doi:10.1021/bm5018244.  41   2.2 Background As outlined in Chapter 1, it has become increasingly important to find substitutes to conventionally used antibiotics. Antimicrobial peptides (AMP) are considered to be viable alternatives because they have a broad antimicrobial spectrum and are unique, since bacteria develop little or no resistance towards AMPs212–214. Aurein 2.2 (GLFDIVKKVVGALGSL-CONH2) has also been extensively studied113,137,138,215. It has been shown that the N-terminus is essential for activity; however, the truncation of the C-terminus by three residues (GLFDIVKKVVGAL-CONH2) has no effect on structure and function.  Although AMPs are promising therapeutic agents, only a few are used for systemic therapy. Examples of AMPs in clinical trials are arenicin and the lantibiotics MU1140 and NVB302, used to treat infections related to Gram-positive bacteria63. The small number of AMPs currently being used is due to problems such as unknown toxicity against host cells, short circulation half-life due to protease digestion and rapid kidney clearance167,216. In order to circumvent these issues, researchers are investigating ways to conjugate AMPs to biocompatible polymers to alter their residence time in the body and their tissue distribution.  A number of studies in the literature have demonstrated how polyethyleneglycol (PEG) can be used as a polymer to conjugate peptides and proteins (PEGylation) in order to improve efficacy and tissue distribution in vivo167,217. A polymer that has recently gained a lot of attention is hyperbranched polyglycerol (HPG). HPGs have been used for the conjugation of biomolecules due to their ease of one-pot synthesis (Figure 2.1),  excellent biocompatibility (equal or better than PEG) and long blood circulation, which is molecular-weight dependent181,218,219. Other beneficial characteristics of HPG are described in detail in section 1.3.   42    Figure 2.1: Synthesis of HPG. Anionic ring opening polymerization of the 1,1,1-Tris(hydroxymethyl) propane initiator. Figure generated using ChemDraw.  In this chapter, we investigated the utility of HPG in the generation of antimicrobial peptide conjugates. We hypothesized that conjugation of AMPs to HPG would enhance the biocompatibility of the antimicrobial peptides without considerably diminishing their antimicrobial properties. The multifunctionality of the HPG would allow the conjugation of multiple peptides per scaffold. The C-terminus of aurein 2.2 was conjugated to HPG (HPGylation). The antimicrobial activity of conjugates was measured against S. aureus and S. epidermidis in comparison to the unconjugated peptides. Solution CD spectroscopy was used to examine the interaction of the conjugates with model biomembranes to understand their mode of action. Biocompatibility of the conjugates was investigated using hemocompatibility measurements including red blood cell lysis, platelet activation and aggregation, complement activation and blood coagulation, as well as cell toxicity tests.   43   2.3 Methods All chemicals were purchased from Sigma Aldrich Canada Ltd. (Oakville, ON) and used without further purification, unless indicated otherwise. Glycidol was purified by vacuum distillation and stored at 4 ºC. Cellulose ester dialysis membranes were obtained from Spectra/Por Biotech (Rancho Dominguez, CA, US). Pierce BCA protein assay kit was purchased from Thermo Fisher Scientific Inc. (Rockford, IL, US). POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, purity > 99%) and POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol, purity > 99%) were purchased from Avanti Polar Lipids (Alabaster, AL, US) and used without further purification. 2.3.1  Peptide Synthesis and Purification Aurein 2.2∆3 (GLFDIVKKVVGAL-CONH2) and aurein 2.2∆3-cys (GLFDIVKKVVGALC-CONH2) were synthesized (using Fmoc chemistry) using a solid phase peptide synthesizer from CS Bio Co. (Menlo Park, CA, US), as previously described113,137,138,215. The first residue at the C-terminus was double-coupled to the Rink resin to produce C-terminally amidated peptides. The crude products were purified by preparative reverse phase high performance liquid chromatography on a Waters 600 system (Mississauga, Ontario, Canada) monitored using a UV detector at 229 nm and using a Phenomenex (Torrance, CA, US) C4 preparative column (20.0 µm, 2.1 cm x 25.0 cm). The mobile phase was composed of two buffers (A and B) with gradient flow. Buffer A was composed of 90% ddH2O, 10% acetonitrile, 0.1% TFA and buffer B was made up of 10% ddH2O, 90% acetonitrile, 0.1% TFA. The peptides were purified to > 95% purity (Appendix A.1). The identity of the peptides was confirmed by MALDI-time of flight mass spectrometry, for aurein 2.2∆3 (theoretical mass: 1357.70 Da;  44   experimental: 1357.67 Da) and aurein 2.2∆3-cys (theoretical mass: 1460.83 Da; experimental: 1460.49 Da).   2.3.2 Synthesis of HPG Synthesis of hyperbranched polyglycerol (HPG) was carried out as described in a previous report220. Briefly (Figure 2.1), the reaction was carried out in a three-necked glass reaction flask with a mechanical stirrer, an automatic dosage pump and temperature control under argon. 1,1,1-tris(hydroxymethyl) propane (0.24 g) was partially (10%) deprotonated with potassium methoxide (25 wt% in methanol, 0.22 mL). Methanol was removed under vacuum for 5 h. The flask was heated to 95ºC and glycidol (18 mL) was added dropwise for 12 h and stirred for another 4 h. Polymer was dissolved in methanol and precipitated three times from acetone. The polymer was dissolved in water and purified by dialysis against water by using a regenerated cellulose membrane (MWCO 1000) and characterized by gel permeation chromatography (GPC) using DAWN-EOS multi-angle light scattering (MALLS) (Wyatt technology Inc., Santa Barbara CA, USA) in conjunction with an optilab RI detector. The mobile phase used was 1.0 M NaNO3 with a dn/dc value of 0.12 mL/g used for molecular weight calculation. 2.3.3 Amine Modification of HPG HPG (44000 g/mol, 0.5 g) was dissolved in N-methyl pyrrolidinone (NMP) and reacted with 0.004 g of sodium hydride (NaH) in a glove box for 4 h at room temperature (2.5% of the hydroxyl groups are deprotonated to get 15 amine groups eventually). The azido-epoxide compound was added to the solution and stirred for 15 h at 70 ºC. The polymer solution was then cooled to room temperature (22 ºC) and precipitated three times from cold diethyl ether and centrifuged to yield the azide functionalized HPG. HPG-azide was reacted with triphenyl phosphine (0.060 g) and water (0.025 mL) in tetrahydrofuran for 15 h at room temperature to  45   reduce the azide group and generate the amine-functionalized HPG. To obtain the 5% amine modified polymer twice the molar ratio of reagents was used when compared to the 2.5% modification described above. The products were characterized by proton NMR. 2.3.4 Conjugation of Peptide with amine-modified HPG The amine-functionalized HPG (2.5%) (0.01 g, 44000 g/mol) was reacted with activated ester of iodoacetic acid (iodoacetic acid N-hydroxysuccinimide ester) (0.001 g) in dimethylformamide (DMF) at room temperature (22 ºC) for 15 h. The resulting solution was dialyzed (MWCO 1000) for 24 h to remove the unreacted reagents. The product was characterized using proton NMR, UV-Vis and GPC.The iodoacetamide modified HPG (0.01 g. 44000 g/mol) was further reacted with sulfhydryl containing aurein 2.2∆3-cys (GLFDIVKKVVGALC-CONH2) (0.005 g, 1460.49 g/mol) at pH 8.0 in PBS (phosphate buffered saline):acetonitrile mixture 50:50 (V:V). To obtain the HPG-Aurein 2.2∆3-cys 5% conjugate, twice the molar ratio of iodoacetamide modified HPG and aurein 2.2∆3-cys were used when compared to 2.5%. The HPG conjugated peptide was purified by size exclusion chromatography using a Waters 600 system (Mississauga, ON, Canada) with a 229 nm UV detector and CATSEC column (CATSEC 300, Eprogen) (Appendix A.6). The mobile phase used was 0.2 M NaCl in 20% ethanol/water mixture. The product was further characterized by proton NMR and GPC. 2.3.5  Characterization of HPG Peptide Conjugates Absolute molecular weights of the conjugates were determined by GPC on a Waters 2690 separation module fitted with a DAWN-EOS multi-angle light scattering (MALLS) (Wyatt technology Inc., Santa Barbara CA, USA) in conjunction with an optilab RI detector181.  The mobile phase used was 1.0 M NaNO3 with a dn/dc value of 0.12 mL/g used for the molecular weight calculation. NMR spectra were recorded on a Bruker 300 and 500 MHz NMR  46   spectrometers by using deuterated solvents (Cambridge Isotope Laboratories, 99.8 %D) with the solvent peak used as a reference. The peptide concentration was determined with the Pierce BCA protein assay kit.  2.3.6  MIC Determination The MICs for the peptides and the conjugates were determined based on previously described methodology with minor modification113,137,138,215. Five milliliters of Mueller Hilton Broth (MHB) was inoculated with the bacterial strain (S. aureus C622 and S. epidermidis C621) and incubated overnight at 37 ºC. The day of the experiment, serial dilutions of the peptides at 2× the required concentration in sterile water in a polypropylene 96-well plate (Thermo Fisher Scientific Inc. Rockford, US) was made. First, the peptides/conjugates were diluted in equal volumes of sterile water to get 2× the required maximal concentration. Secondly, serial doubling dilutions were made in sterile water to get the peptides at 2× the required test concentrations, e.g. 1000, 500, 250….16.5 µg/ml. With a multichannel pipettor, 50 µL/well of the 2x peptide dilutions were added to columns 2 to 11 of the 96-well plates. For each strain, the OD600 (absorbance of the bacterial cells at 600 nm)  was measured and a 1:50 dilution of the overnight cultures were made to get 2-7×105 CFU/ml knowing that C622 has 7.3×108 CFU/OD and C621 has 3×108 CFU/OD. Colony forming units (CFU) is a rough estimate of the number of viable bacterial cells. With a multichannel pipette, 50 µL of the diluted bacteria (diluted in 2× MHB) was added to columns 2 to 12 of the plates containing 50 µL/well of the 2× peptide dilutions. Column 12 was the bacterial growth control. MHB was added to column 1 as a media control. The plates were incubated at 37 ºC overnight and the MIC was checked after 18-24 h. The MIC was determined visually as the lowest concentration where no growth is observed.  47   2.3.7 Blood Compatibility Analysis Blood was withdrawn from consenting unmedicated donors into a 3.8% sodium citrated tube with a blood/coagulant ratio of 9:1 or serum tube at the Centre for Blood Research, University of British Columbia. Serum was prepared by centrifuging whole blood samples in serum tubes at 1200g for 30 min in an Allegra X-22R centrifuge (Beckman Coulter, Canada). Platelet rich plasma (PRP) and platelet poor plasma (PPP) were prepared by centrifuging citrated whole blood samples at 150g for 20 min and 1200g for 30 min, respectively. Red blood cell (RBC) suspension was prepared by washing packed RBC with PBS three times to yield 80% hematocrit. The polymer/peptide stock solutions were made in PBS (with final concentrations as indicated in figure captions). 2.3.7.1 Platelet Activation Analysis: Flow Cytometry The level of platelet activation was quantified by flow cytometry194. Ninety microliters of PRP was incubated at 37 C with 10 μL of stock polymer/peptide samples (final concentration indicated in figure captions). After 1 h, aliquots of the incubation mixtures were removed for assessment of the platelet activation state. Five microliters of post-incubation platelet/polymer mixture, diluted in 45 μL PBS buffer, was incubated for 20 minutes in the dark with 5 μL of monoclonal anti-CD62-PE (Immunotech, Marseille, France). The reaction was then stopped with 0.3 mL of phosphate-buffered saline solution.  The level of platelet activation was analyzed in a BD FACS Canto II flow cytometer (Becton Dickinson, ON, Canada) by gating platelet-specific events based on their light scattering profile. Activation of platelets was expressed as the percentage of platelet activation marker CD62P-PE (phycoerythrin) fluorescence detected in the 10,000 total events counted. Duplicate measurements were done; the mean of which was  48   reported. One U/mL of human thrombin (SigmaAldrich, Oakville, ON, Canada) was used as a positive control for the flow cytometric analysis. 2.3.7.2 Platelet Aggregation Studies To evaluate the influence of the conjugates on platelets, a microplate platelet aggregation assay was performed. Platelet concentration in platelet-rich plasma (PRP) was made to 2.5 × 108 cells/mL. For all other samples, aggregation was quantified by mixing 10 µL of HPG-peptide conjugates with 90 µL of PRP.  The absorbance was then measured at 595 nm at an interval of 15 seconds for 20 minutes with shaking at 37 °C using a Spectramax PLUS plate reader (Molecular devices, Sunnyvale, CA, US). Platelet aggregation was induced by adding 5 µM adenosine diphosphate (positive control). The full experimental procedure and analysis were previously published221. The percentage of aggregation was calculated using the formula: % of aggregation = Abs PRP - Abs sample/ Abs PRP –Abs PPP where PRP= Platelet rich plasma, PPP= Platelet poor plasma, and Abs= Absorbance. 2.3.7.3 Red Blood Cell Lysis Studies Ten microliters of each of the stock polymer conjugate/peptide concentrations were mixed with 90 μL of 10% hematocrit RBC suspension and incubated for 1 h at 37 C. The complete lysis of red blood cells by dH2O is used to define the 100% lysis point and phosphate-buffered saline mixed with the hematocrit acts as the normal control (minimal lysis). The percent of red blood cell lysis was measured by using the Drabkin method222. Five microliters of the washed blood cells/polymer conjugate/peptide solution was added to 1 mL of Drabkin’s solution. After centrifugation, 50 μL of supernatant of the blood/polymer solution was also subjected to 1 mL of Drabkin’s solution. The difference in optical density (OD) was measured using a light  49   spectrophotometer at 540 nm. The percent of red blood cell lysis in the sample was the OD of the supernatant divided by the OD of blood/polymer solution222. The selectivity of the conjugates was also tested by using a combined blood and bacteria experiment169,223. Bacterial suspensions (S. aureus C622 and S. epidermidis C621) were diluted to 2x106 CFU/mL in MHB. Forty five microliters of the diluted bacterial suspension were added to 45 μL of 50% citrated blood (1:1 whole blood:PBS). Ten microliters of peptide conjugate (125 μg/mL final concentration) was added to the mixture and incubated for 1 h at 37 C. Ten microliters of the incubation mixture was serial diluted in MHB and plated on Mueller Hilton agar, followed by incubation at 37 C for 18-24 h for cfu determination. Percent lysis was also analyzed for the mixture by Drabkin's method as described above.   2.3.7.4 Complement Activation Analysis: CH50 Assay A hemolytic assay was performed to analyze the level of complement activation by the polymers, peptides and the conjugates. Two incubation steps were utilized. First, ten microliters of stock polymer conjugate/peptide solutions (final concentration of the conjugate in μg/mL is indicated in the figure captions) were incubated and reacted with 90 µL of GVB2+ (Gelatin veronal buffer) diluted human serum, at 37 °C. After 1 h incubation, the polymer conjugated-treated serum (60 μL) was diluted with 120 μL of GVB2+ buffer. The diluted serum was then incubated in equal volume with the antibody-sensitized sheep RBC (EA cells, Comptech, TX, US) for 1 h at 37 °C, in order to measure the amount of complement activity remaining. Heat-aggregated IgG (final concentration 5 mg/mL) and PBS were also incubated with GVB2+-diluted human serum for 1 h at 37 C, as the positive and negative controls, respectively. All reactions were stopped by the addition of 0.3 mL of GVB-EDTA. Control tubes containing equal volumes of EA cells and GVB2+ buffer were subject to either GVB-EDTA (blank control) or dH2O (100%  50   lysis control). Intact EA cells were spun down at 8000 rpm for 3 min and the supernatants were sampled.  Percentage EA lysis was calculated using average absorbance values as follows: %EA lysis = (OD540, test sample – OD540, blank)/(OD540, 100% lysis – OD540, blank) X 100. Percentage of complement activated (consumed) by the polymer/conjugate samples was expressed as: 100 %- % EA lysis224. 2.3.7.5 Blood Coagulation by Activated Partial Thromboplastin Time (aPTT) and Partial Thromboplastin Time (PT) Analysis aPTT and PT assays were performed as previously described224. Sodium citrate anticoagulated PPP was used for aPTT and PT analysis. The influence of peptide and peptide conjugate on the coagulation cascade was examined by mixing PPP with the polymer conjugate/peptide solution (9:1 V/V; varying final concentration depends on stock concentration used) at 37 °C. Control experiments were performed with phosphate-buffered saline solution with PPP. For the aPTT and PT analyses, the coagulation reagents actin FSL and Innovin thromboplastin (Siemens Healthcare, Marburg, Germany) were used, respectively. Each experiment was repeated in triplicates on the STart®4 coagulometer (Diagnostica Stago, France). The average values of the clotting time were obtained from at least three different donors. For aPTT analysis, 180 µL of PPP was added to 20 µL of stock polymer/peptide solution (final concentration indicated in figure captions) at room temperature. Actin FSL (200 µL) was added and 100 µL of the polymer conjugate/peptide-PPP-reagent mixture was then added into a cuvette-strip and warmed to 37 °C for 180 s. Fifty microliters of pre-warmed 0.025 M CaCl2 was then added to the cuvette and the time for clot formation was measured and recorded.  51   For PT analysis, 180 µL of PPP and 20 µL of stock polymer solution were mixed at room temperature. Fifty microliters of the mixture was then added into a cuvette-strip and warmed up at 37 °C for 60 s. One hundred microliters of Innovin thromboplastin (pre-warmed at 37°C) was then added to the cuvette and the time for clot formation was measured and recorded. 2.3.8 Cell Toxicity analysis Human umbilical vascular endothelial cells (HUVEC), and fibroblasts (3T3) were used to examine the effect of bioconjugates on cell viability. All cell lines were cultured in their respective growth media at 37 C under a humidified 95%:5% (V/V) mixture of air and CO2.  HUVEC cell line is cultured in endothelial cell growth medium (EGM-2 BulletKit, Lonza), and fibroblast is cultured in Dulbecco’s modified eagle medium (DMEM, Lonza) with 10% fetal bovine serum (FBS, Lonza). Before the addition of peptide/bioconjugate samples to the cells, each cell line was plated into separate tissue culture-treated 96 well assay plates (approximately 3000 cells/well) overnight to allow for cell attachment to the well surface. The polymers were dissolved in respective growth media for the targeting cell lines and were incubated with the cells for 48 h. Starting with an initial peptide/bioconjugates concentration of 250 g/mL, serial dilutions of the polymer solutions with media was performed to examine the effect of concentration  on cells. Fifty percent DMSO was used as a positive control for toxicity measurements. After a 48 h incubation period, cell viability was measured by adding 20 μL of the 3-[[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium] (MTS) reagent (CellTiter 96® AQueous One Solution Cell Proliferation Assay, Promega) to each of the wells. The colorimetric absorbance was measured at two wavelengths, 490 nm and 600 nm. Cells only treated with basic growth media were used as the baseline and was expressed  52   as 100% viable. The viability of HUVEC and fibroblast after exposure to the bioconjugates or 50% DMSO was expressed as the percentage of the viable cells compared to medium-treated cells. 2.3.9  Biomembrane Interaction by CD Spectroscopy Circular dichroism (CD) experiments were conducted using a JASCO J-810 spectropolarimeter at 25 ºC, as previously described113,137,138,215. For the samples prepared with lipids, the procedure outlined in113,137,138,215 was used. Briefly, samples with a constant peptide concentration of 75 μM were prepared in different peptide to lipid (P/L) molar ratios of 1:35 and 1:70, using the 1:1 lipid mixtures of POPC/POPG. Appropriate amounts of lipids in chloroform were dried using a stream of nitrogen gas to remove most of the chloroform and vacuum dried overnight in a 5.0 ml glass vial. After adding 450.0 μL of ddH2O and 50 μL of peptide stock solution in ddH2O to the dried lipids, the mixture was sonicated in a water bath for a minimum of 30 min (until the solution was no longer turbid) to ensure lipid vesicle formation. Two hundred microliters of sample was placed in a 1 mm path length for the CD measurements. The spectra were obtained over a wavelength range of 190 nm - 280 nm. Continuous scanning mode with a response time of 1 second with 0.5 nm steps, bandwidth of 1.5 nm and a scan speed of 200 nm/min were used. Spectra were corrected by subtracting the background (example PBS spectrum) from the sample spectrum. To improve the signal-to-noise ratio, an average of 10 scans was obtained. The helical content was calculated using three fitting programs (CDSSTR225, CONTINLL226, and SELCON3227) and an average was reported.  53   2.4 Results and Discussion  2.4.1 Synthesis of Conjugates For the synthesis of the conjugates, a C-terminus truncated peptide with an additional cysteine at the C terminus (aurein 2.2∆3-cys (GLFDIVKKVVGALC-CONH2)) was used. The C-terminus was chosen for the cysteine addition and truncation because it was previously shown that the N-terminus is critical for activity of aurein peptides, whereas the C-terminus is not215. The cysteine residue is incorporated in order to facilitate the conjugation chemistry (Figure 2.2). The insertion of the cysteine residue and the truncation of the C-terminus did not affect the antimicrobial activity against the tested strains (Table 2.1) as the minimum inhibitory concentrations (MIC) are similar to previously reported values113,138,215.  Figure 2.2: Synthetic route for the conjugation of the aurein peptide with hyperbranched polyglycerol (HPG). Only some of hydroxyl groups of HPG are represented explicitly for clarity. Figure created using ChemDraw.  54   Table 2.1: Sequence and molecular weight of the peptides and the bioconjugates made in this study.  Sequence Molecular weight PDI (Mw/Mn) MICc S.aureus  (µg/mL) MICc S. epidermidis (µg/mL) MIC (S.aureus)/5% Hemolysisd Aurein 2.2∆3 GLFDIVKKVVGAL 1358a - 16 32 - Aurein 2.2∆3-cys GLFDIVKKVVGALC 1461a - 16 16 0.50 HPG-Bifunctional linker - 44000b 1.1 >1000 >1000 - HPG-Aurein2.2∆3-cys(2.5%) - 56000b (7- 8 peptides) 1.3 125 150 <0.50 HPG-Aurein2.2∆3-cys (5%) - 69000b (17-18 peptides) 1.4 110 120 0.88  aMolecular weight determined by MALDI-time of flight mass spectrometry. bMolecular weight determined by  GPC analysis. cMICs reported are the most frequent values observed (n = 3 or more). dMIC (S. aureus)/ Concentration at which 5% hemolysis is observed (from Figure 2.4).  A smaller number indicates higher activity with respect to hemolysis.  All peptides used in this chapter had an amidated C-terminus.  HPG (44 kDa) was synthesized using anionic polymerization (Figure 2.1). Some of the hydroxyl groups were further modified to an azide functionality and finally reduced to an amine containing polymer (Figure 2.2), which showed characteristic proton NMR signals (Appendix A.2). The amine modified HPG was then reacted with a bifunctional linker (iodoacetic acid N-hydroxysuccinimide ester) yielding the HPG-bifunctional linker species. HPG-bifunctional linker showed characteristic amide peaks (Appendix A.3) and absorbed light in the UV region (Appendix A.4) due to the addition of the iodoacetamide group.  The HPG-bifunctional linker was finally conjugated to aurein 2.2∆3-cys to yield the peptide conjugate. To ensure that all the unreacted peptides are removed, the bioconjugate was  55   purified using size exclusion HPLC (Appendix A.5) and characterized by proton NMR (Appendix A.6).  After conjugation, the molecular weight of the parent was increased from 44kDa to 56kDa as determined by GPC analysis (Appendix A.7) upon peptide conjugation, which indicated approximately 7-8 peptides molecules per HPG molecule (Table 2.1). The PDI of HPG-Aurein 2.2∆3-cys (2.5%) conjugate did not change considerably (PDI= 1.3) in comparison to HPG-bifunctional linker (PDI= 1.1) indicating that there is a uniform distribution of peptides on the polymer molecules (Table 2.1). For HPG-Aurein 2.2∆3-cys (5%) conjugate, the molecular weight increased from 44kDa to 69kDa indicating 17-18 peptides per HPG molecule. The PDI of this conjugate was 1.4.  2.4.2 Antimicrobial Activity of the Bioconjugates  It is crucial to determine the peptide concentration of the HPG-conjugate to compare the bioconjugate and free peptide activity. The peptide concentration was determined using the BCA protein assay kit. Table 2.1 summarizes the MIC values for the free aurein 2.2∆3-cys peptide and the HPG-Aurein 2.2∆3-cys bioconjugates against clinical Gram positive bacteria (S. aureus (C621) and S. epidermidis (C622)). The HPGylated peptide (both 2.5% and 5%) showed approximately 7 fold weaker antimicrobial activity compared to the free peptide against S. aureus. In the case of S. epidermidis, it was in the range 3 to 7-fold weaker for conjugates. This was expected as polymer conjugation to AMPs is known to decrease the antimicrobial activity166–168. Interestingly, the decrease in activity is similar to magainin (5-10 fold), another alpha helical antimicrobial167. The decrease in activity could be due to various reasons such as interaction of the peptides with the polymer chain or modification of peptide folding, solubility and hydrophobicity. The decrease in antimicrobial activity of the HPGylated peptides is less  56   prominent when compared to PEGylated tachyplesin 1 (32-64 fold decrease in activity relative to free AMP) and nisin (inactive)166,168. However, important differences should be noted as in these examples the molecular weight of PEG was 5 kDa and a mono-PEGylated species was used, in contrast to HPG (44 kDa with 7-8 peptides or 17-18 peptides). Interestingly, the 2.5% and 5% conjugates showed similar antimicrobial activity, suggesting that a threshold number of peptide per HPG molecule is needed for antimicrobial activity. We anticipate that the optimization of the polymer molecular weight and structure of the polymer would further modulate the antimicrobial activity of the conjugates.  2.4.3 Peptide Secondary Structure on Bioconjugates  To further understand and correlate the antimicrobial activity of the HPG-conjugates, we investigated the secondary structure of the aurein peptide conjugated to HPG using CD spectroscopy (Figure 2.3). Cationic AMPs are unfolded in an aqueous environment but adopt secondary structure upon interaction with a lipid membrane. Electrostatic and hydrophobic interactions are important for this to occur. After initial electrostatic interaction with the negatively charged head groups of the outer leaflet of the membrane phospholipids, the peptides insert into the membrane. The thermodynamically favorable hydrophobic-hydrophobic interaction with the interfacial membrane lipids leads to the alpha helical conformation of many AMPs212–214. Aurein 2.2 adopts an alpha-helical structure when in contact with lipids113,138,215. CD data (Figure 2.3; Appendix A.8) confirmed that aurein 2.2∆3-cys also adopts alpha helical structure in the presence of TFE and POPC/POPG (1:1) lipid vesicles. Peptide to lipid (P/L) ratios of 1:35 and 1:70 (mol/mol) were tested. The percentage of alpha helical content (Table 2.2) is similar to the parent  peptide aurein 2.2, within the error inherent in CD113,138,215.   57    Figure 2.3: CD spectra of the HPG-Aurein 2.2∆3-cys 5% in PBS buffer and lipids (1:1 POPC/POPG and the peptide under the same conditions. In the presence of lipids, the peptides and peptide conjugates adopt an alpha helical structure. Figure generated using Graphpad prism.  Aurein 2.2∆3-cys conjugated to HPG was still able to form alpha helices (Figure 2.3; Appendix A.9) but to a much lesser extent (Table 2.2). For the 2.5% conjugate, the helicity in the presence of TFE, POPC/POPG (P/L=1:35), and POPC/POPG (P/L=1:70) was found to be 35%, 31% and 19%, respectively. The 5% conjugate (Appendix A.8 and A.9) was slightly more structured in TFE and POPC/POPG lipids (Table 2.2). Overall for both the conjugates, the percent helical content is at least 3 fold lower when compared to peptide only (Table 2.2). This indicates that HPGylation of aurein 2.2∆3-cys peptide alters or destabilizes the secondary structure of the peptide, which is consistent with the literature data on PEGylation of the magainin 2 analogue167. A decrease in alpha helical content may correlate with the decrease in antimicrobial activity, as demonstrated for peptides conjugated to polymer brushes228.     58   Table 2.2: Secondary structure of Aurein 2.2∆3-cys and HPG-Aurein 2.2∆3-cys in various environments determined by CD spectroscopy (representative spectra shown in Figure 2.3).  Environment α helical content (%)a Aurein 2.2∆3-cys   PBS Trifluoroethanol POPC/POPG (1:35)b POPC/POPG (1:70)b 15±5 98±5 96±5 84±8 HPG-Aurein2.2∆3-cys (2.5%) PBS Trifluoroethanol POPC/POPG (1:35)b POPC/POPG (1:70)b 10±5 35±8 31±13 19±13 HPG-Aurein2.2∆3-cys (5%) PBS Trifluoroethanol POPC/POPG (1:35)b POPC/POPG (1:70)b 12±5 42±8 36±13 28±13 aaverage from three software programs: CDSSTR225, CONTINLL226, and SELCON3227.  bP/L ratios  2.4.4 Blood Compatibility of the Peptide and Peptide Conjugates   Since HPG-aurein conjugates are designed as agents to be administered intravenously, and will interact with blood immediately upon administration, the determination of their interaction with blood components will provide important information on the biocompatibility of the conjugates. There are limited studies available in the literature on the interaction of different blood components with cationic AMPs or its conjugates with polymers. It is also important to understand whether the decoration of cationic AMPs on HPG would alter their blood interaction. Thus we tested the interaction of HPG conjugates with various blood components by measuring the red cell lysis, platelet aggregation and activation, complement activation and blood coagulation.  Previous studies have demonstrated that the toxicity of AMPs can be correlated with their ability to lyse red blood cells.  Recently, Zhao et al. used a computational approach to develop   59   peptides with improved antimicrobial activity while minimizing red blood cell lysis229. To study the interaction of conjugates with RBCs, washed RBCs were incubated with the conjugates at 37 °C and the percent lysis was measured. Aurein 2.2∆3-cys causes RBC lysis at concentrations as low as 62.5 µg/mL, whereas HPG-Aurein 2.2∆3-cys (2.5%) was not cytotoxic at this concentration (Figure 2.4a). Even at twice the new MIC value (250 µg/mL) of this conjugate, HPG-Aurein 2.2∆3-cys (2.5%) shows no change in RBC lysis (Figure 2.4a) compared to control buffer, demonstrating the improvement in biocompatibility. However, as more peptide groups are attached, as in HPG-Aurein 2.2∆3-cys (5%), an increase in lysis is seen (Figure 2.4a) indicating more peptide-like characteristics. These results suggest an optimal number of peptides are needed to enhance biocompatibility and retain antimicrobial activity. The charge and hydrophobic masking effect of HPG may contribute to the diminished toxicity of the conjugates towards RBCs. It is also possible that the peptides cannot permeate the RBC membrane due to the steric hindrance introduced by the large HPG moiety. A similar trend has been observed upon polymer conjugation of the synthetic AMP CaLL230.  To mimic the situation in a systemic infection situation and to demonstrate whether HPGylated aurein peptides show selectivity towards bacterial cells in the presence of whole blood, hemolysis and antimicrobial activity were monitored in the same sample. In the combined (blood and bacteria) experiments, there was a further decrease in hemolysis as a general trend, with the conjugated peptides (both 2.5% and 5%) now showing hemolysis comparable PBS (Figure 2.4b). Even at this high concentration (125 μg/mL) the conjugates showed selectivity towards bacterial cells (Figure 2.4c) with no significant hemolysis (Figure 2.4b). Bacterial counts were not detectable in these experiments. These findings are similar to those seen for PEGylated   60      Figure 2.4: (a) Influence of free aurein peptide and HPG peptide conjugates concentration on red blood cell lysis. Hemolysis of free peptides and peptides conjugated to HPG at various concentrations after incubation for 1 h with washed red blood cells at 37 °C. Positive control was water and PBS buffer was used as normal control. *P=0.0316; **P=0.0011; ***P=0.0073. (b) Combined RBC lysis in the presence of bacterial cells. Peptide/conjugates (125 μg/mL) were added to the 50% citrated RBC/bacterial mixture (1:1). (c) Bacterial Colony forming unit per milliliters (CFU/mL) of the same samples were also observed. No bacterial counts were detected for the HPGylated and free peptides. Figure generated using Graphpad prism.  PBS 15.6 31.2 62.5 125 25005101520g/mL% RBC lysisAurein 2.23-cysHPG-Aurein 2.23-cys 2.5%HPG-Aurein 2.23-cys 5%******(a) 61   KYE28 (peptide derived from Heparin cofactor II), which also showed an improved hemolysis profile in the presence of whole blood169. The complement system is part of the innate immune system and is involved in the first line of defense. It is comprised of as many as 30 different proteins circulating in the blood. Upon activation by foreign molecules, these proteins are cleaved and release anaphylatoxins which in turn can cause inflammation. To investigate whether the HPGylated peptide activated the complement system, the amount of complement activation was measured by a complement consumption analysis using a modified antibody-sensitized sheep erythrocyte complement lysis (CH50) assay231. The bioconjugates were incubated with human serum followed by the addition of antibody-sensitized sheep erythrocytes. Lysis of the antibody-sensitized sheep erythrocyte which was measured by hemoglobin release indicated the percent complement consumption. HPGylated and free peptides do not activate complement at the tested concentrations (Figure 2.5) indicating that these conjugates are not interacting with the complement system. Unmodified HPG also showed no complement activation181.  Platelets are involved in blood clotting. Activation of platelets can lead to thrombotic complications i.e. formation of blood clots inside blood vessels. It has been shown that cationic polymers and peptides activate platelets185. Thus we investigated whether HPG-aurein conjugate can induce platelet activation and aggregation in human PRP (platelet rich plasma). Platelet activation was measured from the expression of a glycoprotein P-selectin CD62 on the surface of platelets. Monoclonal anti-CD62P-FITC (fluorescently labelled) antibody based flow cytometry analysis was used for this purpose.  62    Figure 2.5: Influence of free aurein peptide and HPG peptide conjugates concentration on complement activation. Complement activation in human serum was measured by a complement consumption assay (CH50) using antibody-sensitized sheep red blood cells. The percentage of complement proteins consumed is reported. Heat-aggregated Immunoglobulin G (IgG) (final concentration 5 mg/mL) was used a positive control and PBS buffer as a normal control. Figure generated using Graphpad prism. Results are courtesy of Benjamin Lai.  The influence of the concentration of HPG conjugates and the peptide on platelet activation (% expression of CD62P) is given in Figure 2.6a. Results show that the extent of platelet activation by HPGylated and free peptides was very similar to buffer control (Figure 2.6a), indicating that the bioconjugates did not induce platelet activation at the tested concentrations. A similar observation was seen for the platelet aggregation analysis (Figure 2.6b). To test the extent of aggregation by the bioconjugates, adjusted PRP was incubated with peptides or conjugates and the absorbance was measured for 20 minutes. As the aggregation increases more light can pass through and the measured absorbance decreases (Appendix A.10).   63   Quantification of the percent platelet aggregation in the last 5 minutes (plateau phase) is shown in Figure 2.6b. Results show that the free peptides and the conjugates did not cause platelet aggregation, as the values were comparable to the buffer control.      Figure 2.6: (a) Influence of free aurein peptide and HPG peptide conjugates concentration on platelet activation. Platelet activation was measured in human platelet rich plasma by counting platelets (flow cytometry) that expressed the activation marker CD62P using the monoclonal anti-CD62-PE antibody. Human thrombin and PBS buffer were used as positive and negative controls, respectively. (b) Influence of free aurein peptide and HPG peptide conjugates concentration on platelet aggregation. The peptides and bioconjugates were incubated with adjusted platelet rich plasma (PRP) for 20 minutes. Absorbance at the plateau phase was used to calculate percent aggregation (see Appendix A.10 ). ADP (5 µM) was used as a positive control and PBS buffer as normal control. Figure generated GraphPad prism.  Blood coagulation in the presence of free peptide and conjugates were studied by measuring the blood clotting time using a clinical coagulation assay224. This test provides important information on the pro- or anti-coagulant activities of the AMP conjugates. Cationic  64   peptides and polymers were shown to influence the blood coagulation by, in most cases, delaying the onset of coagulation185,220. The conjugates/peptides were incubated with human PPP (platelet poor plasma) and the clotting time was measured by aPTT (activated partial thromboplastin time), which measures the intrinsic pathway of blood coagulation. Clotting time is the time it takes for a fibrin clot to form once thromboplastin reagent and calcium chloride is added. The peptide and the conjugates did not significantly affect the aPTT values when compared to PBS control (Figure 2.7a) at all the concentrations tested. The extrinsic pathway of blood coagulation in the presence of peptide conjugates and free peptides was probed by measuring the prothrombin time (PT).  Aurein 2.2∆3-cys prolonged PT at concentrations as low as 125 µg/mL, whereas HPG-Aurein 2.2∆3-cys (both 2.5% and 5%) did not significantly change the PT at this concentration (Figure 2.7b). At 250 µg/mL (twice the new MIC), HPG-Aurein 2.2∆3-cys (2.5%) did not alter clotting time as significantly as the free peptides. However, upon increase in the peptide density, the coagulation time increased at the highest concentration tested (Figure 2.7b) in comparison to the control buffer, suggesting the importance of peptide density on the conjugates. This also suggests a way to further improve the properties of the conjugates. The mechanism by which the free peptide and the conjugate influence the extrinsic pathway of coagulation needs to be further investigated. A possible explanation for the observations made here could be that the hydrophobic and electrostatic interactions between the peptides and clotting factors result in delayed coagulation. However, in the conjugate, the hydrophobic masking effect of HPG may contribute to the diminished interaction with clotting factors, leading to the observed unchanged clotting time. More experiments are needed, however, to confirm this hypothesis.    65    Figure 2.7: (a) Influence of free aurein peptide and HPG peptide conjugates concentration on intrinsic pathway of blood coagulation. Intrinsic pathway blood coagulation was monitored by measuring the activated partial thromboplastin time (aPTT) in human plasma at 37 ºC. PBS buffer was used as a negative control. (b) Influence of free aurein peptide and HPG peptide conjugates concentration on extrinsic pathway of blood coagulation. Prothrombin Time (PT) was measured by incubating peptide and conjugates with human plasma at 37 ºC. *P=0.0395; **P=0.0018; ***P=0.0198. Figure generated using Graphpad prism.  2.4.5 Cell Compatibility of the Peptide and Conjugates   The cell compatibility of the peptides and the conjugates was assessed by incubating HUVECs and fibroblasts with various peptide/conjugate concentrations for 48 h and measuring the viable cells by the MTS assay (Figure 2.8). Aurein 2.2∆3-cys caused a decrease in cell viability at concentrations as low as 31.2 µg/mL (HUVEC) and 125 µg/mL (fibroblasts), whereas HPG-Aurein 2.2∆3-cys (2.5% and 5%) were nontoxic at twice the new MIC concentration (250 µg/mL). An explanation for these observations is that the HPGylated peptides could not permeate the cell walls effectively because of the steric hindrance introduced by the   66   large HPG moiety, similar to that observed for PEGylated tachyplesin and magainin166,168. Another synthetic AMP CaLL  also showed enhanced cell compatibility230  Figure 2.8: Cell biocompatibility of the peptides and the conjugates against (a) human umbilical vein endothelial cells *P<0.0001; **P=0.1372; ***P=0.6501 and (b) fibroblasts *P=0.0070; **P=0.9725; ***P=0.8895 measured by the MTS assay after 48 h of incubation. Figure generated using GraphPad prism. Results are courtesy of Benjamin Lai.   2.5 Summary In this chapter, I describe the first design of a multifunctional HPG-peptide conjugate based on the alpha helical AMP aurein 2.2 and the examination of the effect of HPGylation on the antimicrobial activity, interaction with blood components and structure of the peptide. The conjugates were well characterized for composition and molecular weight. The HPGylation of aurein 2.2∆3-cys decreased the antimicrobial activity (~3-7-fold) and the alpha helical content (~3 fold). The biomembrane interaction of the HPG conjugate depends on the amount of peptide substitution; the higher the peptide density the higher the interaction. On the other hand, the HPGylation of the aurein 2.2 decreased the interaction with blood components especially to red  67   blood cells and the blood coagulation system; the peptide density on the HPG is an important contributing factor. HPGylated and free aurein 2.2 did not aggregate or activate platelets nor did they activate the complement system. Finally, the HPGylated peptides were also non-toxic to HUVECs and fibroblasts. The results presented here also demonstrate the importance of optimizing the peptide density on the HPG scaffold to provide optimal antimicrobial activity without generating toxicity. Future work will examine the interdependence of the peptide and HPG scaffold properties by examining the mechanism of action of the conjugates, as was done for the parent peptide alone113,138,215 (Chapter 5). Parameters to vary include the HPG molecular weight. PEG analogs will also be made for direct comparison. Finally, the results presented here demonstrate the potential of using HPG-peptide conjugates in the design of a variety of important biomolecules, such as e.g. conjugates with anti-inflammatory activities derived from peptides with immunomodulating properties230.    68   Chapter 3: Antimicrobial peptide-polymer conjugates with high activity: Influence of polymer molecular weight and peptide sequence on antimicrobial activity, proteolysis and biocompatibilityc  3.1 Synopsis  In this chapter we report the synthesis, characterization, activity and biocompatibility of a novel series of antimicrobial peptide-polymer conjugates. Using the parent peptide aurein 2.2, we designed a peptide array (~100 peptides) with single and multiple W and R mutations and identified antimicrobial peptides (AMPs) with potent activity against S. aureus. These novel AMPs were conjugated to hyperbranched polyglycerol (HPG) of different molecular weights and number of peptides to improve their antimicrobial activity and toxicity. The cell and blood compatibility studies of these conjugates demonstrated better properties than the AMP alone.  However, conjugates showed lower antimicrobial activity in comparison to peptides, as determined from minimal inhibition concentrations (MICs) against Staphylococcus aureus but considerably better than the available polymer-AMP conjugates in the literature. In addition to measuring MICs and characterizing the biocompatibility, CD spectroscopy was used to investigate the interaction of the novel conjugates with model bacterial biomembranes. Moreover, the novel conjugates were exposed to trypsin to evaluate their stability. It was found that the conjugates resist proteolysis in comparison to unprotected peptides. The peptide-conjugates were active in serum and whole blood. Overall, the results show that combining a  cA version of Chapter 3 has been published. Kumar, P.; Takayesu, A.; Abbasi, U.; Kalathottukaren, M. T.; Abbina, S.; Kizhakkedathu, J. N.; Straus, S. K. Antimicrobial Peptide–Polymer Conjugates with High Activity: Influence of Polymer Molecular Weight and Peptide Sequence on Antimicrobial Activity, Proteolysis, and Biocompatibility. ACS Appl. Mater. Interfaces 2017, acsami.7b09471, doi:10.1021/acsami.7b09471.  69   highly active AMP and low molecular weight HPG yields bioconjugates with excellent biocompatibility, MICs below 100 μg/ml, and proteolytic stability which could potentially improve its utility for in vivo applications. 3.2 Background In Chapter 2, antimicrobial peptide aurein 2.2 was covalently attached to a 44 kDa HPG scaffold. It was found that peptide density on the conjugate is important for determining biocompatibility and efficacy. Although the antimicrobial activity of aurein 2.2 is improved compared to PEG conjugates, there was a 7-8-fold decrease (16 μg/ml to 110-120 μg/ml) in MIC.  Thus further improvements in the activity are desired.   In this chapter, we therefore investigate strategies to improve the activity of the AMP-HPG bioconjugates, while preserving biocompatibility. We initially used a peptide array, where tryptophan (W) and arginine (R) mutations were introduced, to create more active mutants of aurein 2.2. These more active AMPs were then conjugated to HPGs of different molecular weights (22 kDa to 105 kDa). The antimicrobial activities of the newly identified and most active peptide and its HPG conjugates were measured against S. aureus and compared to the unconjugated peptides. In addition to the biocompatibility measurements, we investigated the resistance against tryptic degradation of the HPG conjugates as compared to the free peptides. Solution CD spectroscopy was used to examine the peptide secondary structure when bioconjugates interacted with model biomembranes in order to understand their mode of action. Overall, these studies aimed to devise improved bioconjugates and to further our understanding of the parameters that are important for obtaining optimal properties.  70   3.3 Methods All chemicals were sourced from Sigma Aldrich Canada Ltd. (Oakville, ON) and used without further purification, except for glycidol, which was purified by vacuum distillation. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) were purchased from Avanti Polar Lipids (Alabaster, AL). Pierce BCA protein assay kit was bought from Thermo Fisher Scientific Inc. (Rockford, IL). Dialysis membrane was obtained from Spectra/Por Biotech (Rancho Dominguez, CA). 3.3.1 Peptide array Considering the importance of charge, hydrophobicity and amphipathicity a peptide array with tryptophan (W) and arginine (R) mutations was designed using the aurein 2.2∆3 peptide as the framework. Tryptophan and arginine were chosen because they have special characteristics that allow them to interact with each other and the bacterial membrane232. The helical wheel projection was used as a guide to ensure that the amphipathic nature of the peptides was preserved. We started with a single mutation by replacing the lysine (K) residues with R or hydrophobic residues with W, followed by multiple R and W mutations. Quantitative structure-activity relationship (QSAR) have been used previously to design AMPs with better antimicrobial activities. This method requires a large dataset from a systematic peptide array with thousands of peptides 233 so a more targeted approach was used here.  Arrays of peptides were SPOT-synthesized on cellulose membranes by Kinexus Inc. (Vancouver, BC, Canada), as described previously234,202. Each cellulose spot was assumed to have a peptide density of 150 nmol based on Pierce BCA protein assay kit and a purity of ~70%. Peptides were dissolved in 200 μL of autoclaved water and the solution was incubated at room  71   temperature for ~4 h with gentle shaking to solubilize the peptide from the cellulose membrane. The stock peptide solutions were serially diluted and used for the antimicrobial screens described below. From the array, some of the most active peptides (Table 3.1) were synthesized using solid phase peptide synthesis and purified (95% purity), as described in Chapter 2.3.1. The antimicrobial activity of the peptides synthesized in house was verified and found to be consistent with those found for the peptides in the array (Table 3.1).  Table 3.1: Antimicrobial activity (MICs) of selected peptides identified in the peptide array.@  Peptide MIC S. aureus (μg/mL) 1. GLFDIVKKVVGAL (parent  peptide) 32 24. GLWDIWKKWWGWL 16 34. GLFDIVRRVVRAL 16 77. RLWDIVRRVWGWL 4 91. GLWDIWRRWWRWL 8 @ The parent peptide, aurein 2.2, was used as a template. The other peptides in the library were obtained by incorporating arginine (R) and tryptophan (W) residues at various locations. All peptides used in this chapter had an amidated C-terminus.  3.3.2 Peptide synthesis and purification Solid phase peptide synthesis (Fmoc) was used to synthesize aurein 2.2∆3-cys and its RW mutants (Table 3.1), as previously described113-215 with details given in Chapter 2.3.1.    3.3.3 Synthesis of HPG Synthesis and characterization of hyperbranched polyglycerol (HPG) with different molecular weights was carried out as previously reported235,220 and in Chapter 2. Briefly, For 22  72   kDa HPG, 1,1,1-tris(hydroxymethyl) propane (0.240 g) was partially (10%) deprotonated with CH3OK (25 wt% in methanol, 0.22mL). Methanol removed under vacuum for 5 h. The flask was heated to 95 ºC and glycidol (9mL) was added dropwise for 12 h and stirred for another 4 h. For 44 kDa HPG, glycidol (20mL) was added dropwise for 15 h. For 105 kDa HPG, glycidol (50mL) was also added dropwise for 15 h. The workup protocol, absolute molecular weight and polydispersity of the HPGs were determined using gel permeation chromatography (GPC) coupled with multi angle light scattering (MALS) as described in Chapter 2.3.2  3.3.4 Amine Modification of HPG HPG was vacuum dried for overnight at 70 °C prior to the reaction.  A flame dried round bottom flask was loaded with dried HPG (22 kDa, 200 mg) in anhydrous DMF (20 mL) under Ar. To this solution, NaH (3 eq, w.r.t. targeted -OH groups; 15 mg) was added slowly and stirred at RT for 2 h. To the obtained white cloudy solution, N-glycidylphthalimide (1.5 eq; 54 mg) was added and stirred for another 20 h at 70 °C. The reaction mixture was quenched with dilute HCl (pH ~7) and the resulting reaction mixture was concentrated approximately 3 times the initial concentration. The polymer was purified by precipitation from acetone, followed by dialysis against water (MWCO – 10000 Da, 24 h). The polymer was lyophilized and the number of phthalimide groups on HPG was determined by 1H NMR (Appendix B.1). The HPG-phthalimide was deprotected to HPG amine by refluxing it with hydrazine (20 eq, 35 wt. % in H2O; 383 mg) in methanol (20 mL) for 24 h. The reduction of HPG-phthalimide was confirmed by 1H NMR (Appendix B.2) and the resulting HPG amine was purified by dialysis and lyophilized. The number of amine groups was equivalent to phthalimide groups on HPG. For 44 kDa (200 mg), 3 eq of NaH (13 mg), 1.5 eq of N-glycidylphthalimide (55 mg) of and 20 eq, 35 wt.% in H2O  73   hydrazine (335 mg). For 105 kDa (200 mg), 3 eq of NaH (9 mg), 1.5 eq of N glycidylphthalimide (34 mg) of and 20 eq, 35 wt. % in H2O hydrazine (210 mg).  3.3.5 Conjugation of peptide with HPG-amine  The HPG-amine (0.014 g, 22000 g/mol) was reacted with 3-(maleimido)propionic acid N-hydroxysuccinimide ester (0.009 g) in dimethylformamide (DMF) at room temperature (22 ºC) for 24 h. The reaction mixture was dialyzed in DMF (MWCO 1000) for 24 h to remove the unreacted 3-(maleimido)propionic acid N-hydroxysuccinimide ester. 1H NMR was used to characterize the product (Appendix B.3). The maleimide modified HPG (0.010g, 22000 g/mol) was reacted with sulfhydryl containing aurein2.2∆3-cys (GLFDIVKKVVGALC-CONH2) (0.007g, 1460.49 g/mol) or its RW mutant  (peptide 77c, 0.009 g, 1857.34 g/mol) in DMF for 24 hours. The resulting conjugate was purified by size exclusion chromatography (Waters 600 system Mississauga, Ontario, Canada) using a CATSEC column (CATSEC 300, Eprogen)181 and a UV detector (229 nm) with a mobile phase composed of 0.5M NaCl in 20% ethanol/water mixture. The product was further characterized by GPC and 1H NMR (Appendix B.4). The maleimide to thiol-maleimide conversion for 22k 77c and 22k aurein 2.2∆3-cys was 70% and 80%, respectively. The unreacted maleimide was quenched with ethanethiol. For 44k 77c, 44k HPG amine (10 mg), 2 eq of 3-(maleimido)propionic acid N-hydroxysuccinimide ester (5 mg) was added, followed by 1.2 eq of peptide 77c (9 mg). The maleimide to thiol-maleimide conversion was 57%. For 44k aurein 2.2∆3-cys, 1.2 eq of aurein 2.2∆3-cys peptide (8 mg) was added. In this case, the maleimide to thiol-maleimide conversion was between 79%.   For 105k 77c, 105k HPG amine (10 mg), 2 eq of 3-(maleimido)propionic acid N-hydroxysuccinimide ester (2 mg) was added, followed by 1.2 eq of peptide 77c (10 mg). The  74   maleimide to thiol-maleimide conversion was 51%. For 105k aurein 2.2∆3-cys, 1.2 eq of aurein 2.2∆3-cys peptide (8 mg) was added. Here, the maleimide to thiol-maleimide conversion was 67%. The conjugates were characterized for their molecular weights by GPC-MALS, conjugation efficiency by 1H NMR and bicinchoninic acid (BCA) protein assay as described in Chapter 2.3.4 and 2.3.5 3.3.6 Synthesis of PEG 77c conjugate The mPEG-maleimide (5k) was purchased from advanced polymer materials Inc. (Montreal, Canada). The mPEG maleimide (0.01 g) was dissolved in 1 mL of acetonitrile and added to sulfhydryl containing peptide 77c (0.004 g, 1857.34 g/mol) dissolved in 1 mL of DMF and stirred for 24 h. The PEG conjugated peptide was purified the same way as the HPG conjugated peptide (described above) using a size exclusion CATSEC 100 column and characterized by 1H NMR (Appendix B.5). 3.3.7 Antimicrobial activity measurements (minimum inhibitory concentration or MIC) The MICs of the peptides/conjugates against S. aureus were measured based on previously described methodology235;113,138,215 and Chapter 2.3.6. 3.3.8 MIC determination after tryptic degradation To determine whether conjugation of the AMP with HPG prevents or alters degradation of the peptides by trypsin, we incubated the peptides/conjugates with trypsin. Trypsin (3 µg) was added to peptide 77c (200 µg/mL solution) and the 22K-77c conjugate (200 µg/mL solution) and incubated at 37 C for 3 h. After 3 h, the MALDI spectra of the two solutions were acquired. The trypsin-digested samples without purification were used for determination of the MIC of the peptides/conjugates, as described in Chapter 2.3.6.  75   3.3.9 Biocompatibility analysis The biocompatibility of conjugates and peptides were evaluated by measuring the cell viability against mouse embryonic fibroblasts (3T3-L1) by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium) assay. The blood compatibility of the conjugates were determined by measuring their effect on blood coagulation, effects on platelets, complement activation and red blood cell lysis using human blood. The blood was obtained from unmedicated consenting donors; the protocol was approved by the clinical ethical committee of the University of British Columbia. Detailed protocols are described in sections 2.3.7 to 2.3.12.  3.3.10 Biomembrane interaction: CD spectroscopy As previously described113,138,215, a JASCO J-810 spectropolarimeter was used to conduct circular dichroism experiments at 25 ºC. A 1mm cuvette was filled with 200 µL of sample and the spectra were obtained over a wavelength range of 190 nm - 250 nm. A continuous scanning mode was employed with a response time of 1 second, bandwidth of 1.5 nm, 0.5 nm steps, and a scan speed of 200 nm/min. An average of 5 scans was obtained to improve the signal to noise ratio. The spectra were corrected by subtracting the background (e.g. phospholipid spectrum) from the sample spectrum. An average alpha helical content was reported by calculating the output %helix from three fitting programs (CDSSTR, CONTINLL, and SELCON3)236 and averaging these three numbers.  3.4 Results and Discussion 3.4.1 Discovery of potent peptides using peptide array based on aurein peptide Trytophan and arginine have unique properties that allow them to interact with each other and the bacterial membrane232. The cation-π interactions between the residues allow maximum  76   hydrogen bonding for the arginine side chain and allow the peptide to penetrate deeper into the bacterial membrane232. Using the parent peptide aurein 2.2, with three residues removed from the C-terminus, we designed a peptide array (~100 peptides) with single and multiple W and R mutations. Previous work had shown that truncation of aurein 2.2 by three residues at the C-terminus has minimal impact on its activity138. In order to determine how the mutations impact antimicrobial activity, the sequences were plotted as helical wheels237 (Figure 3.1). Interestingly, changing single hydrophobic residues, e.g. F or V, for W does not improve activity (Figure 3.1b). However, adding a W near the interface of the hydrophobic/hydrophilic boundary results in a peptide that is 2x more active. Positioning the tryptophan at this interface may help the peptide anchor into the membrane better, as W is known to snorkel238. Mutation of the lysine residues for arginine (Figure 3.1c) at single positions (K7 or K8) has a surprising negative impact on activity. The mutation of G11R is most likely beneficial as this introduces a positive charge in close proximity to the negatively charged D4. Active AMPs typically have multiple positive charges239. From the peptide array, we identified peptide 77 (Figure 3.1d) that was 8 fold more active (MIC of 4 μg/mL) than the parent peptide (Table 3.1). A comparison of peptides with multiple mutations (Figures 3.1d, 3.1e, 3.1f) shows that clear trends are difficult to extract. Peptides 77 and 91 have the same number of R residues (and hence the same overall charge), yet peptide 91 is less active (Table 3.1). The addition of more W residues in peptide 91 does not appear to be beneficial, when comparing to peptide 77. Finally, although peptides 87 and 77 have the same number of mutations, their difference in activity is dramatically different. Overall, an examination of the sequence indicates that both the position and number of R and W residues play a critical role with respect to the activity of AMPs.   77    Figure 3.1: Helical wheels for some of the peptides generated in the array from a) the parent peptide aurein 2.2-∆3. The helical wheel for aurein 2.2-∆3 was generated using the script available at http://rzlab.ucr.edu/scripts/wheel/wheel.cgi. In b), single mutations of W are shown, along with the fold increase/decrease in MIC, as indicated in the legend. In c), single mutations in R are shown. In d)-f), some of the more active peptides are illustrated: d) peptide 77, which is 8x more active than the parent peptide; e) peptide 91, which is 4x more active; and f) peptide 87, which is just as active as aurein 2.2-∆3. Figure created by Suzana K. Straus and used here with her permission.  3.4.2 Synthesis of aurein 2.2∆3-cys and peptide 77c conjugates To develop the HPG conjugates, peptides with an additional cysteine at the C terminus were synthesized to facilitate the conjugation (Figure 3.2). Since the N-terminus is critical for activity of aurein peptides138,215, the C-terminus was chosen for the cysteine addition. The  78   addition of the cysteine residue had minimal to no impact on the antimicrobial activity of the peptides (Table 3.2).   Figure 3.2: Synthetic scheme for the conjugation of the peptides with HPG. Only a small number of hydroxyl groups of HPG are shown to ensure clarity. A new amine modification protocol and bifunctional linker was used for accurate quantification of the amine and maleimide groups, respectively. This also ensured water free reactions as peptide 77c was not as readily soluble in water as aurein 2.2∆3-cys. Figure generated using ChemDraw.  A general scheme for the HPG-AMP conjugate synthesis is shown in Figure 3.2. HPGs (22, 44 and 105 k) were synthesized by polymerization of glycidol via anionic ring opening and were modified to generate primary amine functionality240. This was further modified with a bifunctional linker (3-(maleimido) propionic acid N-hydroxysuccinimide ester) to generate maleimide groups on HPG. Aurein 2.2∆3-cys and peptide 77c were each conjugated to this  79   scaffold via Michael type addition of the thiol group of the peptide and maleimide groups on HPG. The number of maleimide groups on the polymer was varied for the generation of conjugates with different number of peptides (Table 3.2). The bioconjugate was purified using size exclusion chromatography and characterized by proton NMR. The NMR data for the precursors and the final conjugates are shown in Appendix B.3 and B.4. The molecular weight of the HPG conjugates increased compared to the parent polymer (Table 3.2) with a molecular weight distribution (PDI) in the range of 1.2 to 1.5. The PDI of the conjugates did not increase considerably compared to the parent HPG suggesting a uniform distribution of the peptides on the polymer molecule. The number of peptides on the conjugates was determined by determining the peptide concentration obtained from a BCA protein kit and the conjugate molecular weight (Table 3.2). For a given molecular weight scaffold, the number of aurein 2.2∆3-cys and peptide 77c remained similar, and thus comparisons were made for their activity and biocompatibility (see below).  3.4.3 Antimicrobial activity of the conjugates Table 3.2 summarizes the minimal inhibitory concentration (MIC) values for the free peptides and their bioconjugates against clinical Gram positive strain S. aureus (C622). The MIC value of the conjugates is with respect to peptide concentration. The MIC value obtained for 44k aurein 2.2∆3-cys of 150 μg/ml is close to the previously obtained value for the HPG- aurein2.2∆3-cys (5%) of 110 μg/ml235. These two samples contain a similar number of peptides. In terms of fold decrease in activity relative to the free peptide (calculated using the 95% confidence interval values241 in Table 3.2), this translates into values of 4-5 for the conjugate reported here versus 5-8 for the conjugate in Chapter 2. This suggests that the difference in conjugation chemistry used here results in a slightly better conjugate, but the more striking result of this work is that we were  80   able to synthesize a conjugate (22k 77c) with an MIC of 50 μg/mL, which is one of the most active polymer-AMP conjugates reported to date in literature166,167,235. Table 3.2: Characteristics of the peptides and the HPG conjugates used in this study@.  @ The number average molecular weights (Mn) HPGs used in this study are 22k (PDI 1.1), 44k (PDI 1.2), and 105k (PDI 1.2). For comparison, a mono-substituted PEG-peptide 77c construct was also synthesized. The number of peptides attached is indicated in brackets in the first column, as determined by GPC analysis. The sequence for aurein 2.2∆3-cys and peptide 77c was GLFDIVKKVVGALC and RLWDIVRRVWGWLC, respectively.  aMolecular weight determined by MALDI-TOF. bMolecular weight determined by  GPC-MALLS. cMICs are the most frequent values observed out of three repeats.  d 95% confidence intervals  Peptide/conjugates  Number of peptide groups per polymer Molecular weight PDI MIC S.aureus c (95% CId) μg/mL Fold decrease in activity Number of peptides/k Da         GPC NMR aurein 2.2∆3-cys - - 1 461a - 32 (26-38) 1  peptide 77c - - 1 858a - 8 (5-11) 1  22k aurein 2.2∆3-cys  8 ± 2 8 ± 1 33 000b 1.2 85 (74-95) 3 0.24 22k 77c  8 ± 1 7 ± 1 36 000b 1.3 50 (39-61) 6 0.22 44k aurein 2.2∆3-cys  16 ± 2 15 ± 2 68 000b 1.4 150 (133-167) 5 0.24 44k 77c 13 ± 1 11 ± 3 67 000b 1.3 100 (83-117) 13 0.19 105k aurein 2.2∆3-cys 30 ± 3 26 ± 5 148 000b 1.4 475 (390-560) 15 0.20 105k 77c  25 ± 4 20 ± 5 151 000b 1.4 325 (268-382) 41 0.16 PEG 5k-77c - 1  1.2 125 (102-148) 16 0.20  81   Looking at the entries in Table 3.2 in general, the HPGylated peptides showed weaker antimicrobial activity compared to the free peptides. This was expected as polymer conjugation to AMPs is known to decrease their antimicrobial activity166–168,235. A few important observations were made based on the MIC values of the conjugates. For both peptides, with an increase in the molecular weight of the polymer scaffold, the antimicrobial activity of the conjugates decreased (Table 3.2). The activity decrease was slightly more pronounced in the case of peptide 77c conjugates than aurein 2.2∆3-cys. This suggests that the peptide sequence plays a crucial role in determining the activity of the conjugates. However, it is important to point out that since peptide 77c is more potent than aurein 2.2∆3-cys its conjugates had overall lower MICs. In practical terms, both the molecular weight and peptide sequence are critical factors that impact the antimicrobial activity of the conjugates. In comparison to PEG 5K 77c, the 22k 77c and 44k 77c are more active (Table 3.2).    The increase in MIC of the conjugates with increasing molecular weights of conjugates could be related to differences in the interaction of the peptides with the polymer chain, to modifications of the secondary structure of the peptide, to its hydrophobicity and solubility, or to changes in the interaction of conjugates with bacterial membrane. Based on the MIC values and peptide density (last column in Table 3.2), we can state that PEG lowers the activity of the peptide more than HPG upon conjugation. This could be possibly due to the compact structure of HPG compared to PEG181. Our current data as well as literature support this observation. For instance, the decrease in antimicrobial activity of 22k 77c is less prominent when compared to PEGylated AMPs like tachyplesin 1 which showed a 32-64 fold decrease in activity or nisin which became inactive upon PEGylation166,168. It is important to note when comparing the activities of HPGylated and  82   the PEGylated peptide 77c (Table 3.2) that not only is there a difference in the molecular weight of the polymer, but there is also a difference in the number of peptides attached.  We also investigated the activity of peptide and conjugates in serum and whole blood (Appendix B.6 and Appendix B.7). At the concentrations studied, both the peptide 77c and 22K 77c were effective at clearing the bacteria.   3.4.4 Resistance to proteolysis: tryptic degradation Many strategies have been employed to limit enzymatic degradation of antimicrobial peptides for their systemic use. These include chemical modification of the peptide (side chain groups, N and C-termini,) incorporation of D amino acids, and cyclization239,242–244. Traditionally polymer conjugation to peptides has increased the stability of peptides against proteolytic degradation 245,246. In the current work, we determined whether the conjugation of peptide 77c to HPG prevents the peptides from protease degradation. We incubated peptide 77c and 22K 77c with trypsin for 3 h and characterized the peptide fragments generated by MALDI-TOF mass spectrometry. We have seen that the conjugated peptide (22K 77c) did not show characteristic cleavage of the naked peptide 77c (characteristic m/z 1114, 988) when incubated with trypsin (Figure 3.3), suggesting that the conjugated peptide 77c on HPG is resistant to tryptic degradation. The additional peaks present in the MALDI spectra were from the matrix, as can be seen from the MS spectrum in Appendix B.8.  83      Figure 3.3: MALDI-TOF mass spectra of a) 77c, b) 22k 77c and c) trypsin after 3 hours of digestion. Trypsin was added to the peptides/conjugates and incubated at 37 C for 3 hours, followed by MALDI characterization. Trypsin cleaves after positively charged residues such as arginine as shown in a). The peptide fragments detected for peptide 77c are highlighted in the red box in a). Figure generated using Data explorer and Adobe.   84   In order to validate that the degradation of the peptides leads to loss in antimicrobial activity, the MIC of the samples treated with trypsin were determined. Peptide 77c was inactive  once treated with trypsin for 3 hours, whereas 22K 77c retained the same MIC (50 µg/mL) before and after trypsin treatment. This further indicates that the peptide is protected from protease degradation once conjugated to HPG and maintains activity. Trypsin alone did not possess any antimicrobial activity. 3.4.5 Peptide secondary structure on bioconjugates   To further investigate the mechanism of action of HPG-conjugates, we determined the secondary structure of the conjugates using CD spectroscopy. Typically, AMPs do not adopt secondary structure in an aqueous environment but fold upon interaction with a lipid membrane. Initially, positively charged residues interact with the negatively charged head groups found in the outer leaflet of the membrane bilayer, followed by thermodynamically favorable hydrophobic-hydrophobic interactions between the AMP and the interfacial membrane lipids, leading to the formation of a specific conformation212–214. In our study, both free peptides are random coil in phosphate buffer (Figure 3.4a). Interestingly, peptide 77 adopts an alpha helical conformation once conjugated to HPG in phosphate buffer without the presence of phospholipids or mimics such as trifluoroethanol (TFE). All molecular weight conjugates (22k 77c, 44k 77c and 105k 77c) of 77c adopt alpha helical structure in phosphate buffer, which indicates it is an intrinsic property of the peptide-polymer conjugate. In the presence of phospholipids, the percent helicity of the 77c conjugates increases indicating a more favorable conformation upon   85   interaction with a membrane mimetic (Table 3.3, Figure 3.4). However, the increase in helicity is much smaller for the conjugates than the free peptide in the presence of POPC/POPG (1:1) relative to PBS.  On the other hand, once HPGylated with various molecular weight HPG, aurein 2.2∆3-cys remains as a random coil. It only adopts an alpha helical structure in the presence of phospholipids. Compared to the free peptide, the percent helicity for the HPGylated aurein 2.2∆3-cys is much lower than the free aurein 2.2∆3-cys, which is similar to previous studies235. HPGylation of aurein 2.2∆3-cys peptide does not affect the structure of the peptide, whereas HPGylation of the peptide 77c enhances the secondary structure of the peptide in buffer. However, HPGylation of both peptides leads to a decrease in percent helicity in a membrane-like environment when compared to the respective free peptides (Table 3.3). Finally, the data in Table 3.3 clearly shows that the helicity for a given peptide conjugate decreases with increasing HPG molecular weight. In this case, the alpha helical content correlates well with the activity (Table 3.2). 3.4.6 Cell compatibility of the peptides and conjugates  The biocompatibility of the antimicrobial peptides and their conjugates is crucial for their use in various applications247–249. Therefore, the cell viability was assessed against human fibroblasts by the MTT assay. It is known from previous studies that aurein 2.2∆3-cys is cytotoxic to fibroblasts235. It causes a decrease in cell viability at a concentration of 62.5μg/mL (Figure 3.5a). However, once conjugated to HPG (22k, 44K or 105k), its cell tolerance is increased irrespective of the molecular weight of the HPG used (Figure 3.5a).    86      Figure 3.4: CD Spectra of the conjugates in a) phosphate buffer and b) POPC/POPG (1:35). The peptides and conjugates have a higher helical content in the presence of lipids. Interestingly, conjugation of peptide 77c to HPG results in more structured peptides. Figure generated using Graphpad prism.    87   Table 3.3: Alpha helical content (%) of the various conjugates studied here, as determined from fits from the CD spectra (Figure 3.4).   PBS POPC/POPG (1:1) Peptide/Lipid (1:35) aurein 2.2∆3-cys 7±5 97±5 22k aurein 2.2∆3-cys  8±5 47±6 44k aurein 2.2∆3-cys  4±2 40±5 105k aurein 2.2∆3-cys  5±2 30±8 peptide 77c 8±5 73±6 22k 77c  40±5 65±6 44k 77c  31±6 52±7 105k 77c  23±4 41±8 Average values from three software programs: CDSSTR225, CONTINLL226, and SELCON3227  Peptide 77c was also toxic towards fibroblast at 2x the MIC concentrations (15.62 μg/mL). However, once HPGylated, peptide 77c (22k 77c) showed better tolerance even at concentrations of 5x the MIC (250 μg/mL). All the HPGylated peptides showed the same trend irrespective of the molecular weight of the scaffold. It is possible that the large HPG moiety prevents the HPGylated peptides from permeating through the extracellular matrix effectively, because of steric hindrance. This phenomenon has been previously observed for PEGylated magainin, tachyplesin, and a synthetic AMP CaLL166,167,250. Our biomembrane interaction data (Table 3.3 and Figure 3.4) support this statement; there was a significant decrease in the secondary structure formation upon conjugation of the peptides to HPG.   88     Figure 3.5: Fibroblast cell viability upon exposure to free peptides and HPG peptide conjugates. a) aurein 2.2∆3-cys *P=0.0056; **P=0.2121. b) peptide 77c *P=0.0047; **P=0.2007. Viability of the cell after incubation with free peptides and peptides conjugated to HPG at various concentrations for 48 h at 37 °C. Figure generated using Graphpad prism. Results are courtesy of Usama Abbasi.    89   3.4.7 Blood compatibility of the peptide and peptide conjugates    Although the conjugates are compatible with cultured cells, it is important to determine the interaction with blood, as these conjugates will be potentially used in systemic administration. There are only a few reports in the literature on the interaction of cationic AMPs and/or conjugates169,251 with various blood components. Moreover, researchers are actively seeking to develop peptides that minimize red blood cell lysis, as described in a recent study that used computational methods229. Given this importance, we sought to understand whether the attachment of two different (in terms of activity) cationic AMPs on HPG (with different molecular weight) would alter their blood interaction. Platelet activation, red cell lysis, complement activation and blood coagulation were used to test the interaction of HPG conjugates with different blood components. For all the experiments volume ratio of blood/plasma to peptide/conjugate was kept at 9:1 v/v.   Washed RBCs were incubated with the peptide/conjugates and the percent lysis was measured in order to investigate the interaction of conjugates with RBCs. Aurein 2.2∆3-cys causes about 10% RBC lysis at 2x the MIC concentration (62.5 µg/mL), whereas 22k HPG-aurein 2.2∆3-cys (MIC 85 µg/mL) was not cytotoxic at nearly 3x its new MIC concentration (250 µg/mL, Figure 3.6). Peptide 77c caused similar RBC lysis compared to aurein 2.2∆3-cys at 2x the MIC.  At 16 µg/mL (2x the MIC), peptide 77c also caused about 10% RBC lysis, however once conjugated to 22k polymer (22K 77c, MIC 50 µg/mL), the conjugate cause 10% lysis at 5x the new MIC (250 µg/mL).  90     Figure 3.6: Red blood cell lysis upon exposure to free peptides and HPG peptide conjugates: a) aurein 2.2∆3-cys *P=0.0075; **P=0.5469. b) peptide 77c *P=0.0004; **P=0.0122. Hemolysis of free peptides and HPG conjugates was measured at various concentrations, after 1h incubation at 37 °C with washed red blood cells. PBS buffer was used as normal control. Figure generated using Graphpad prism.   91   The data suggests that neither the activity of the peptide nor the polymer molecular weight influence the RBC lysis. The hydrophobic masking effect and charge of HPG work universally, contributing to the diminished toxicity of all the conjugates towards RBCs. A very similar observation was made upon polymer conjugation of other AMPs, such as KYE28169 and CaLL250.  Platelets are important players in the blood clotting cascade. Many thrombotic complications, such as the formation of blood clots inside blood vessels, can arise when platelets are activated. Cationic peptides/polymers have been shown to activate platelets185. Thus we examined whether the peptides/conjugates induce platelet activation in human PRP. Expressed glycoprotein P-selectin CD62 on the surface of platelets by fluorescently labeled monoclonal anti-CD62P-FITC antibody and flow cytometry was used to measure platelet activation. Aurein 2.2∆3-cys and all of its various molecular weight conjugates do not activate platelets at the concentrations indicated (Figure 3.7a). Interestingly, peptide 77c on its own activates platelets at 250 µg/mL (30% activation), but none of the HPGylated 77c conjugates activate platelets (Figure 3.7a). Again, this suggests that HPG has the ability to mask the negative effects of the peptides, independent of HPG molecular weight.    92     Figure 3.7: Platelet activation upon exposure to free peptides and HPG peptide conjugates: a) aurein 2.2∆3-cys; b) peptide 77c. *P=0.0094; **P=0.5944. Human platelet rich plasma was used to measure platelet activation. Platelets that expressed the activation marker CD62P using the monoclonal anti-CD62-PE antibody were counted (flow cytometry). PBS buffer was used as a negative control. Figure generated using Graphpad prism. 01020304050 15.62 µg/mL31.25 µg/mL125 µg/mL62.5 µg/mL250 µg/mL(a) 93    The complement system is an important part of innate immunity. As many as 30 different proteins are part of this system. Complement activation upon interaction with the peptides and/or the conjugates is an indication of inflammatory potential such as histamine release and suggests blood incompatibility. Complement activation was determined by a complement consumption analysis using a antibody-sensitized sheep erythrocyte complement lysis (CH50) assay using human serum231 to determine whether the free and HPGylated peptides had an effect on the complement system. Neither aurein 2.2∆3-cys, peptide 77, nor the HPGylated peptides activate complement (Figure 3.8), indicating that they are all blood compatible.  Moreover, clinical coagulation assays were used to study the blood coagulation profile of the peptides and conjugates224. This test provides important information on the pro- or anti-coagulant activities of the peptides/conjugates. Blood coagulation was measured by clinical coagulation assays activated partial thromboplastin time (aPTT) and partial thromboplastin time (PT) in human plasma. In aPTT measurements, clotting time is taken as the time it takes for fibrin clot to form once thromboplastin reagent and calcium chloride is added. Aurein peptides were shown to influence the blood coagulation by delaying the onset of coagulation235. Both peptides and the conjugates did not significantly affect the aPTT values when compared to the HEPES-buffered saline (HBS) control (Appendix B.9).   94     Figure 3.8: Complement activation upon exposure to free peptide and HPG peptide conjugates: a) aurein 2.2∆3-cys; b) peptide 77c. A complement consumption assay (CH50) was used to determine complement activation, using antibody-sensitized sheep red blood cells and human serum. The percentage of consumed complement proteins is shown. PBS buffer was used as a normal control. Figure generated using Graphpad prism.  95    Prothrombin time (PT) was measured to characterize the extrinsic pathway of blood coagulation upon addition of free peptides and conjugates. Cationic peptides and polymers have been shown to delay prothrombin time185,220. Interestingly, peptide 77c has no impact on PT whereas aurein 2.2∆3-cys prolonged PT at concentrations as low as 125 µg/mL, suggesting that peptide 77c is not changing the blood coagulation profile compared to aurein 2.2∆3-cys. None of the conjugates showed any changes in the PT (Figure 3.9). In the conjugates, HPG may mask the hydrophobic nature of the peptides and prevent non-specific interactions with blood coagulation proteins, which leads to normal prothrombin time.  To further investigate the influence of our peptide and its conjugates on whole blood clotting profile, we used rotational thromboelastometry (ROTEM) measurements in human whole blood. Representative ROTEM traces are shown in Appendix B.10 and clotting time is shown in Appendix B.11. Both the peptide and conjugate (peptide 77c and 22K 77c) showed similar clotting profile as the buffer control suggesting that the conjugate or peptide is not altering the blood coagulation profile. The clotting time of the peptide and conjugates is also not significantly different from the buffer control under these conditions.     96     Figure 3.9: Prothrombin time upon exposure to free aurein peptide and HPG peptide conjugates: a) aurein 2.2∆3-cys *P=0.0067; **P=0.2803. b) peptide 77c. Prothrombin time (PT) was measured by incubating peptide and conjugates with platelet poor plasma at 37 °C. Figure generated using Graphpad prism. Results are courtesy of Dr. Manu Thomas Kalathottukaren.  97   3.5 Summary  In this chapter, we examined how the parameters such as peptide sequence and size of HPG impact the antimicrobial activity and blood compatibility of AMP/HPG bioconjugates. Specifically, we designed a R and W mutant peptide library derived from the previously studied aurein 2.2 peptide and obtained peptide 77 that was 4-8 fold more active compared to the parent peptide. We conjugated both aurein 2.2 and the new peptide to HPGs of varied molecular weight and examined the effect of HPGylation on the antimicrobial activity, structure and interaction with blood components. Overall, HPGylation of both peptides decreased the antimicrobial activity, with the best molecular weight being 22k, where a factor of 3-6 in the increase in MICs was observed. More importantly, we obtained a bioconjugate with the best MIC value reported in literature to date, namely 50 μg/ml. This bioconjuate (22K 77c) also showed resistance to tryptic digestion and maintained antimicrobial activity compared to the free peptide. HPGylation decreased the alpha helical content of both peptides in a membrane environment; however, in buffer HPGylated peptide 77c adopted an alpha helical structure compared to HPGylated aurein 2.2∆3-cys which was random coil. Even though the activity and structure of the peptides are different, HPGylation reduced the interaction with red blood cells and platelets. All HPGylated peptides were biocompatible with fibroblast cells, demonstrating their non-toxic behavior compared to the free peptides. The results presented here demonstrate that it is possible to optimize peptide sequence and HPG molecular weight to obtain bioconjugates with excellent properties. The results suggest that conjugating highly active peptides with low molecular weight HPG is the best strategy.   98   Chapter 4: The in vitro biofilm activity and the in vivo efficacy of the novel peptides and the polymer conjugates.  4.1 Synopsis In Chapter 3, we developed novel peptides and conjugates with good antimicrobial activity. In order to determine their usefulness as potential therapeutic agents and more importantly, to determine whether HPG-conjugation could be part of a broader strategy to help develop novel antimicrobial agents, it is important to test the activity of these peptides and bioconjugates developed in Chapter 3 in biofilm inhibition and eradication, and in wound healing. To this end, we describe the evaluation of antimicrobial, anti-biofilm, toxicity, and in vivo activity in a mice abscess model in this chapter. As discussed in Chapter 1, other strategies to increase the efficacy of AMPs include the use of D-amino acids (Chapter 1.3.1). Hence, we also conducted activity tests on peptides where the sequence was preserved but the L-amino acids were replaced by their D- counterparts. Likewise, we investigated peptides where both the order of sequence and the use of D-amino acids were combined.  4.2 Background As described in Chapter 1, antimicrobial peptides are sometimes also referred to as host defense peptides (HPDs) in order to reflect their multi-faceted activities. Up to this point, we have examined the activity of aurein peptides, their derivatives and the HPG conjugates in terms of their ability to act against planktonic bacteria. However, many AMPs also display activity against bacteria in other forms such as in biofilm. A biofilm is a dense assembly of surface adhered bacteria localized in an extracellular polymeric substance (EPS) made from polysaccharides, DNA and proteins252. The EPS typically blocks the transport of the antibiotics  99   through the biofilm making biofilms particularly antibiotic resistant252 and consequently difficult to treat. It has been reported previously that some AMPs are also effective in clearing bacteria and enhance the wound healing in different skin infections. For instance, AMPs show potential for all of these activities, e.g. D-M33 posses good antibiofilm activity (Chapter 1.3.1), whereas DJK-5 displays both antibiofilm activity and wound healing properties in skin infection models207 (Chapter 1.3). Since antibiofilm activities of AMPs were already presented in Chapter 1, we will not discuss them again here but rather focus on wound healing. Cutaneous abscesses are one of the most common skin conditions that are caused by bacterial infection and are characterized by the accumulation of pus in the skin, leading to lesions that normally appear red and swollen and that may cause severe pain205. Although both Gram positive and Gram negative bacteria cause abscesses, community associated methicillin resistant Staphylococcus aureus (Ca MRSA) is the most common agent253. A Ca-MRSA strain that has been frequently recovered is USA 300254. In 2005, approximately 3.2 million people with abscesses were treated in hospital emergency departments in the USA alone206. Abscesses are treated by surgically draining the pus and treating with antibiotics to prevent spreading and reoccurrence207. Common antibiotics used to treat MRSA abscesses are trimethoprim-sulfamethoxazole and tetracyclines, however, antibiotics do not work within the abscess due to limited penetration, high bacterial loads and low pH208. As many bacterial strains are also acquiring antibiotic resistance, there is an urgent need for alternatives and adjuncts.  More recently, AMPs  have been utilized to treat skin infections caused by multidrug resistant pathogens255,256. Interestingly most AMPs in clinical trials to date have been used as topical antibacterial agents for skin related infections. AMPs have many advantages over  100   traditional antibiotics:  they display broad-spectrum antimicrobial and immunomodulatory activity; they can neutralize virulence factors; and finally, they show low or no resistance to the treatment. Interestingly, Mansour et al. have shown that abscess and biofilm caused by MRSA (USA 300) produces a similar stress response which can be targeted in mice by antimicrobial peptide DJK-5207. The DJK-5 peptide was initially developed for a screen of anti-biofilm peptides. Moreover, in a similar study, AMPs such as WR12 and IK8 showed good anti-biofilm activity and were used effectively as topical antibacterial agents for the treatment of MRSA associated skin infections in mice models255. Interestingly D-IK8 peptide showed the best activity against intracellular MRSA. Finally, in another study, D-amino acid AMPs eradicate biofilms formed by multi-resistant Pseudomonas aeruginosa in invertebrate (C. elegans and G. mellonell) survival models209.  Although AMPs have various activities, not many have been FDA approved due to their toxicity, the susceptibility of peptides to protease degradation and the rapid kidney clearance23,105,141. Many strategies have been investigated to circumvent these issues and to improve the efficacy of AMPs. These include chemical modification of AMPs142 and the use of delivery vehicles143 as discussed in Chapter 1.3. The use of D amino acids prevents protease degradation. On the other hand toxicity can be mitigated by either conjugation of AMPs to polymers such as HPG (Chapter 3) or encapsulation of the peptides using delivery vehicles such as liposomes or micelles. The encapsulation of LL37 within liposomes composed of DSPC/DSPE-PEG/cholesterol ensured enhanced bioactivity and reduced toxicity in cell cultures162. In Chapter 3, we developed peptide 77 and its HPG conjugates that showed enhanced in vitro antimicrobial activity against S. aureus compared to the aurein peptides. In this Chapter, we investigate the in vitro anti-biofilm and in vivo activity of peptide 73 and other peptides that  101   are closely related to 77 but more active in vivo when compared to 77. Our hypothesis is that novel peptides will be effective in healing abscesses in mouse models, and their conjugates will mitigate the toxicity of the AMPs in vivo. The D-amino acid version of peptide 73 (D-73) and retro-inverso (RI-73, D amino acid with the sequence reversed) may further enhance its in vivo efficacy. We also examined the role of encapsulation of the novel peptides using the abscess mouse model. Finally, the resulting data will allow us to determine the relationship between the in vitro and in vivo activity of the peptides/conjugates. Such information is important in the design of functional AMPs, with potential clinical applications. 4.3  Materials and methods All chemicals were purchased from Sigma Aldrich Canada Ltd. (Oakville, ON) and used without further purification except for glycidol, which was purified by vacuum distillation. Dialysis membrane was obtained from Spectra/Por Biotech (Rancho Dominguez, CA). 4.3.1 Peptide synthesis and purification Solid phase peptide synthesis (Fmoc) was used to synthesize aurein 2.2, aurein 2.2∆3-cys and its RW mutants (Table 4.1), as previously described113,215,257 with details given in Chapter 2.3.1 and 3.3.2. 4.3.2 Synthesis of the bioconjugate Synthesis and characterization of HPG-73c was carried out as described in Chapter 3.3.3 to 3.3.5. 4.3.3 Antimicrobial activity measurements: MIC determination The MICs of the peptides/conjugates against S. aureus were measured based on previously described methodology235;113,138,215 found in Chapter 2.3.6.  102   4.3.4 Biofilm studies  The bioluminescence- and biofluorescence-based static microtiter plate assays were established in a similar fashion as previously described 258. Briefly, an overnight culture was diluted to an OD = 0.01 in tryptic soy broth supplemented with 1% glucose (w/v) or in BM2 supplemented with 0.4% glucose (w/v), 0.5 mM Mg2+, and, for USA300-lac::lux/USA300 GFP or PAO1 lux/PAO1 GFP strains, respectively, and then 90 µL was added to each well of a 96-well Costar polypropylene plate (Corning Inc., Corning, NY) containing 10 µL of peptide diluted in water or water alone.  Each peptide was evaluated for antibiofilm activity at concentrations of 64, 32, 16, 8, 4, 2, 1 and 0.5 µg/mL.  After overnight growth, the planktonic cells were washed away with deionized water and the remaining adhered biomass was resuspended in 150 µL of 10% LB/ 90% PBS (v/v) solution by pipetting up-and-down (withdrawing the liquid up and squirting it back down).  Total biofilm mass was quantified by measuring luminescence or fluorescence (eGFP, ex = 488 nm, em = 530 nm) using a microtiter plate reader (Bio-Tek Instruments Inc., Winooski, VT).  The percent biofilm inhibition was calculated in relation to the amount of biofilm grown in the absence of peptide (defined as 100%) and the media sterility control (defined as 0% growth).  Results from three separate biological replicates were averaged. 4.3.5 Red blood cell lysis  Blood was withdrawn from consenting unmedicated donors into a 3.8% sodium citrated tube with a blood/coagulant ratio of 9:1 or serum tube at the Centre for Blood Research, University of British Columbia. Red blood cell (RBC) suspension was prepared by washing packed RBC with PBS three times to yield 80% hematocrit. Detailed protocols of the red blood lysis studies can be found in Chapter 2.3.9.  103   4.3.6 Mouse skin infection model Bacterial strains and growth conditions for animal studies     Bacterial strains used in this study were the Pseudomonas aeruginosa laboratory wild type strain PAO1259, the P. aeruginosa Liverpool Epidemic Strain isolate LESB58260, and the Staphylococcus aureus LAC (USA300) strain261. All organisms were cultured in double Yeast Tryptone (dYT), shaking at 250 rpm, at 37 °C. Bacterial growth was monitored using a spectrophotometer at the optical density of 600 nm (OD600).  Ethics statement     Animal experiments were performed in accordance with The Canadian Council on Animal Care (CCAC) guidelines and were approved by the University of British Columbia Animal Care Committee (certificate number A14-0363). Mice used in this study were female outbred CD-1. All animals were purchased from Charles River Laboratories (Wilmington, MA), were 7 weeks of age, and weighed about 25 ± 3 g at the time of the experiments. 1 to 3% isoflurane was used to anesthetize the mice. Mice were euthanized with carbon dioxide. Cutaneous mouse infection model     The abscess infection model was described earlier262. Briefly, all bacterial strains were grown to an OD600 of 1.0 in dYT broth and subsequently washed twice with sterile PBS and adjusted to 1  107 CFU/ml for P. aeruginosa PAO1 and 5  107 CFU/ml for P. aeruginosa LESB58 and S. aureus LAC, respectively. The fur on the backs of the mice was removed by shaving and application of chemical depilatories. A 50 L bacterial suspension was injected into the right side of the dorsum. All utilized peptides were tested for skin toxicity prior efficacy testing. Concentrations used were 5 mg/kg for L-73, D-73 and RI-73. Peptides or saline (50 L) were directly injected subcutaneously into the infected area [intra-abscess (IA) injection] at 1 h  104   post infection. The progression of the disease/infection was monitored daily and abscess lesion sizes (visible dermonecrosis) on day three measured using a caliper. Swelling/inflammation was not considered in the measurements. Skin abscesses were excised (including all accumulated pus), homogenized in sterile PBS using a Mini-Beadbeater-96 (Biospec products) for 5 min and bacterial counts determined by serial dilution. Experiments were performed at least 3 times independently with 3 to 4 animals per group.  Statistical analysis     Statistical evaluations were performed using GraphPad Prism 7.0 (GraphPad Software, La Jolla, CA, USA). P-values were calculated using one-way ANOVA, Kruskal-Wallis multiple-comparison test followed by the Dunn procedure. Data was considered significant when p-values were below 0.05 or 0.01 as indicated. 4.4 Results and Discussion 4.4.1 Antibacterial activity of the novel peptides in vitro We explored the antimicrobial activity of a range of peptides derived from aurein 2.2: aurein 2.2, aurein 2.2Δ3 (where the last 3 residues at the C-terminus are removed from aurein 2.2), peptide 77 and 77c (which was discussed in Chapter 3) and the new peptide 73, a D-amino acid version (D-73) and a retro-inverso version (RI-73). The sequences of some of these peptides and their hydrophobicity are given in Table 4.1. The peptides listed above were tested against a standard S. aureus strains, MRSA USA 300 strain and P. aeruginosa. MICs are listed in Table 4.2.  As can be seen from the activities, the novel peptides 73 and 77 are 4 times more active against C622 strain of S. aureus (MIC, 4 g/mL) compared to the parent aurein 2.2 peptide  105   (MIC, 16 g/mL) and 8 times more active than the peptides it was derived from (aurein 2.2∆3, MIC, 32 g/mL). These peptides both have increased charge (+2 to +3) and hydrophobicity (Table 4.1), suggesting that this may be a contributing factor to increased activity. A similar trend was seen in magainin 2 where increasing the charge of magainin 2 from +3 to +5 improved its antibacterial activity against Gram positive bacteria78. As hydrophobicity governs the extent to which the water-soluble AMPs will be able to partition into the membrane lipid bilayer, it is key to maintain a threshold hydrophobicity as sequences with hydrophobicity below and very much above this threshold made the peptides inactive and toxic respectively85. Interestingly peptides like 73 and 77 are composed of tryptophan and arginine residues that have unique properties that allow them to interact with each other and the bacterial membrane which may also explain the increase in activity of the peptides263,232.   Table 4.1: Various properties of some of the peptides used in this studies a Calculated from heliquest http://heliquest.ipmc.cnrs.fr/  All peptides used in this chapter had an amidated C-terminus.   Peptide Molecular weight  Net charge Hydrophobicitya Hydrophobicity momenta Aurein 2.2∆3 (GLFDIVKKVVGAL) 1357.68 +2 0.632 0.73 Aurein 2.2  (GLFDIVKKVVGALGSL) 1614.97 +2 0.617 0.603 73 (RLWDIVRRWVGWL) 1755.10 +3 0.815 0.753 77 (RLWDIVRRVWGWL) 1755.10 +3 0.815 0.721 73c (RLWDIVRRWVGWLC) 1857.24 +3 0.866 0.670  106   Table 4.2: Antimicrobial activity of the peptides. * MIC results are courtesy of Dr. John Cheng from the Hancock lab  Moreover, the D and retro-inverso (RI) versions of peptide 73 (Table 4.2) had similar activity compared to 73 suggesting that the use of D amino acids does not affect the antimicrobial activity of these peptides similar to lysine rich D-MPI peptide145. This indicates that peptide 73 interacts with the bacteria without making use of specific receptors since the stereochemistry of the amino acids has no impact on the antimicrobial activity43. Peptide D-73 also showed the best antimicrobial activity against P. aeruginosa with an MIC of 16 g/mL (Table 4.2). The addition of a cysteine to the C-terminus of the peptide 73 decreases its antimicrobial activity drastically by 4 fold (MIC from 4 to 16 g/mL, Table 4.2). Addition of a cysteine residue might promote   Peptide MIC   S. aureus C622  (μg/mL) MIC* Ca-MRSA (USA300-lac::lux) (μg/mL) MIC* P. aeruginosa (PA01 lux) (μg/mL) Aurein  2.2 (GLFDIVKKVVGALGSL) 16 32 >64 Aurein 2.2∆3 (GLFDIVKKVVGAL) 32 64 >64 73 (RLWDIVRRWVGWL) 4 4 64 73c (RLWDIVRRWVGWLC) 16 16 >64 77 (RLWDIVRRVWGWL) 4 4 64 D-73 (rlwdivrrwvGwl) 4 4 16 RI-73 (lwGvwrrvidwlr) 4 4 64 22k -73c (7-8 peptides) 50 - -  107   disulfide bridges that affect the antimicrobial activity of the peptide. Cathelicidin-derived peptides, E6 and Tet20 also show a decrease in antimicrobial activity when a cysteine is added at the C-terminus264.   4.4.2 Efficacy of the peptides against Staphylococcus aureus biofilms Biofilms are one of the most complex and dense assembly of cells that display emergent properties such as antibiotic resistance that are not present in free-living bacteria265. The formation of a biofilm is a major virulence factor of S.aureus. Biofilm is made of a complex matrix which protects the deeply positioned bacteria from antibiotics and host immune defense266. This eventually leads to the release of the bacteria in the host which can cause recurring and chronic infections255. Given the serious consequences of biofilms and the fact that it has been previously reported that some AMPs can lead to the inhibition and/or eradication of biofilms, we wanted to assess if our peptides are active against S.aureus biofilms.   Firstly, we evaluated our peptides as an agent to inhibit S. aureus biofilm by measuring the minimum biofilm inhibitory concentration (MBIC). Peptides 73 and 77 were both more effective at inhibiting 85% of the biofilm formation with an MBIC of 2 g/mL compared to the aurein peptides (Table 4.3). The addition of a cysteine to the C-terminus of peptide 73 also decreased its ability to inhibit biofilm formation, in analogy to the MIC data presented in the previous section.  In addition, the D-peptides (D-73 and RI-73) also maintain the inhibitory properties (MBIC, 2 and 4 g/mL, respectively). Inhibition of bacterial growth is important but it is also critical that the peptides can eradicate biofilms too. In general the trend for biofilm eradication is similar to that observed for MBICs,  108   but higher concentrations of the peptide are generally needed to eradicate biofilm. For example peptide 73 has a MBIC of 2 g/mL and MIC of  µg/mL but a MBEC of 16 g/mL (Table 4.3). Unlike peptide 73, aurein 2.2 does not possess a strong anti-biofilm activity.  Table 4.3: Anti-biofilm activity of the peptides. *All MBIC85 (Peptide concentration when 85% of the biofilm is inhibited) and MBEC80 (Peptide concentration when 80% of the biofilm is eradicated) results are courtesy of Dr. John Cheng from the Hancock lab  Generally, the MBECs reported in Table 4.3 are much higher than the MIC: for example peptide, 73 has a MBEC of 16 g/mL which is 8x the MIC, indicating it is more difficult to eradicate Peptide MBIC85 Ca-MRSA USA300 GFP (μg/mL) MBIC85 P. aeruginosa (PA01 lux) (μg/mL) MBEC80  (μg/mL) Ca-MRSA USA300 GFP (μg/mL) Aurein  2.2 (GLFDIVKKVVGALGSL) 16 >64 >64 Aurein 2.2∆3 (GLFDIVKKVVGAL) 32 >64 >64 73 (RLWDIVRRWVGWL) 2 >64 16 73c (RLWDIVRRWVGWLC) 8 64 - 77 (RLWDIVRRVWGWL) 2 >64 - D-73 (rlwdivrrwvGwl) 2 32 32 RI-73 (lwGvwrrvidwlr) 4 16 32 22k -73c (7-8 peptides) 50 - -  109   than to inhibit biofilms. In a similar study, peptide WR12 which is also a W and R rich peptide needed 8x the MIC to eradicate 80% of the S.aureus biofilm255.  4.4.3 Tolerance of the peptides  Host toxicity is one of the major limitations of AMPs that has prevented advancement of these drugs into clinical trials. Similar to studies done in Chapters 2 and 3, we evaluated the toxicity of the peptides by measuring the red blood cell lysis at 37 °C for 1 hour.  None of the peptides caused any significant RBC lysis near the MIC and MBIC. For example, aurein 2.2 and 73 are not lytic (similar lysis to PBS buffer <5%) at 25 g/mL and 12.5 g/mL respectively. However, at higher concentrations (100 g/mL) peptides 73, 73c, and 77 are more toxic compared to the original aurein peptide (Figure 4.1). Interestingly, D-73 and RI-73 have similar antimicrobial activity compared to peptide 73, but they are both more toxic towards RBCs (Figure 4.1). RI-73 is the most toxic peptide causing approximately 65% lysis of RBCs at 100 g/mL. Such a tendency has been previously observed in the literature: for instance, the RI version of certain cell penetrating peptides also caused severe cell toxicity267, whereas the original peptide did not. Our collaborators also tested toxicity of these peptides in different cells lines (data not shown). A similar toxicity trend to these seen in Figure 4.1 was observed using the LDH release assay with PBMCs (peripheral blood mononuclear cells).  110    Figure 4.1: Red blood cell lysis upon exposure to free peptides and HPG peptide conjugates: Hemolysis of the free peptides and the HPG conjugates was measured at various concentrations, after 1h incubation at 37 °C with washed red blood cells. PBS buffer was used as normal control. The experiment was repeated three times. Figure generated using Graphpad prism.  4.4.4 In vitro therapeutic index of the peptides  The therapeutic index (TI) which is a ratio of the concentration at which the peptide causes 50% toxicity (RBC lysis or cell viability) and the MIC is a parameter used to represent the specificity of the peptides for bacterial versus mammalian cells268. A plot of Log [peptide concentration] versus percent RBC lysis with a non linear regression (Graphpad Prism) was used to determine the 50% lysis concentration (also known as LD50) (Figure 4.2). Aurein 2.2 and peptide 73 have the best therapeutic index (42 and 55 respectively) in vitro compared to the other peptides. The therapeutic index for some peptides in DSPE-PEG2000 was also examined. The  111   DSPE-PEG2000 encapsulated peptides were less toxic in vitro but the antimicrobial activity also decreased and a clear trend was not visible (Table 4.4).   Figure 4.2: RBC lysis vs log of peptide concentration curves to extrapolate the concentration at which the peptides cause 50% RBC lysis (LD50). Figure generated using Graphpad prism with a hyperbolic non linear fit, % RBC lysis =100/(1+10^((LogLD50-X))), where X is the Log [peptide].   4.4.5 Efficacy of peptides in a mouse abscess model  As these novel peptides displayed good antimicrobial and antibiofilm activity and no toxicity at MIC, we conducted in vivo studies in a mouse abscess model207,262. Briefly, mice were injected with community acquired USA 300 MRSA strain and treated with a single subcutaneous [intra-abscess (IA)] injection of peptides or saline 1 hour post infection. The abscess area (visible dermonecrosis) and the intra abscess bacterial count were determined 3 days post infection to 1.0 1.5 2.0 2.5 3.0020406080Log [peptide]% RBC lysis7 373cD-73RI-73Aurein 2.2Aurein 2.23-cys7 7 112   evaluate the efficacy of the peptides. Peptides 73 and 77 have the same hydrophobicity, charge and in vitro antimicrobial activity but only peptide 73 significantly decreased the lesion size by Table 4.4: Therapeutic index of the peptides *MIC against S. aureus C622  approximately 3 fold compared to saline control in the mouse model (Figure 4.3a). Moreover, mice treated with peptide 73 also showed a 10-fold reduction in bacteria recovered from the abscess site (Figure 4.3b). The in vivo difference in activity between peptide 73 and 77 might be due to the fact that peptide 77 is more toxic in vitro (Figure 4.1) and also in vivo (Appendix C), Peptide MIC CA-MRSA  (μg/mL) 50% RBC lysis  (μg/mL) Therapeutic index [50% RBC lysis]/MIC Aurein  2.2 (GLFDIVKKVVGALGSL) 32 1350 42 Aurein 2.2∆3 (GLFDIVKKVVGAL) 64 1577 25 73 (RLWDIVRRWVGWL) 4 221 55 73c (RLWDIVRRWVGWLC) 16 139 9 77 (RLWDIVRRVWGWL) 4 107 27 D-73 (rlwdivrrwvGwl) 4 97 24 RI-73 (lwGvwrrvidwlr) 4 80 20 22k -73c (7-8 peptides) 50* >1500 >30 73 in DSPE-PEG2000 40 941 23 73c in DSPE-PEG2000 625 850 1.36 D-73 in DSPE-PEG2000 30 795 27  113   which might lead to inactivation or clearance of the peptide in vivo. The TI (Table 4.3) for peptide 73 (TI=55) is 2-fold better compared to 77 (TI=27), correlating with the in vivo activity and indicating the fine balance needed in terms of the in vitro toxicity and antimicrobial activity for optimum in vivo activity. Interestingly, aurein 2.2 which is the natural parent peptide isolated from Australian frog (skin) has similar activity as peptide 73 in reducing lesion size and viable bacteria counts (Figure 4.3). Aurein 2.2 has both lower antibacterial activity and toxicity in vitro and has a TI of 42 which is similar to peptide 73 (TI=55), again correlating well with the in vivo activity.   Figure 4.3: Infection and therapeutic treatment of mouse cutaneous abscesses. All mice were infected with MRSA USA300 and treated with saline solution, aurein and peptide 73 (dose = 5 mg/kg) 1 h post-bacterial infection. Lesion sizes and CFU counts were determined 3 days postinfection. The experiment was repeated at least twice. Results are courtesy of Dr. Daniel Pletzer from the Hancock lab.    114   In addition to peptide 73, the two versions of the peptide containing D-amino acids, namely D-73 and RI-73, were also tested in the mouse model. Unfortunately both the peptide D-73 and RI-7 were toxic and precipitated at 5 mg/kg in saline solution under the skin (Appendix C.1). This is consistent to the in vitro toxicity profile, as both peptide D-73 and RI-73 are more toxic compared to the L versions (Figure 4.1). Many stragegies have been used to alleviate the toxicity of AMPs but the easiest way is by encapsulation. DSPE-PEG2000 has been previously utilized to encapsulate LL-37 by the formation of liposomes to increase the bicompatability162. This encapsulation strategy was attempted here, but instead of making liposomes, we dissolved D-73 and RI-73 in DSPE-PEG2000 and evaluated the toxicity and precipitation in vivo. Interestingly dissolving these peptides in DSPE-PEG2000 prevented any precipitation and toxicity at various concentrations (Appendix C.2) due to the formation of miclles. The activity of various peptides dissolved in DSPE-PEG2000 in the mouse abcess model is summarized in Figure 4.4. Of the two D peptides, D-73 significantly decreased the lesion size and bacterial count in the abscess, whereas RI-73 was similar to the DSPE-PEG2000 control. D-73 also perfomed better than peptide 73 suggesting the benefit of using D-peptides as long as the toxicity is alleviated. Aurein 2.2∆3, from which peptide 73 is derived, was not effective in the mouse model. Peptide 73c, which had solubility issues in saline but good solublilty in DSPE-PEG2000, was as effective as D-73 in reducing lesion size and viable bacterial counts.  115    Figure 4.4: Therapeutic treatment of mouse cutaneous abscesses with D and L peptides. All mice were infected with MRSA USA300 and treated with DSPE-PEG2000 solution and various peptides dissolves in DSPE-PEG2000 solution (5 mg/kg) 1 h post-bacterial infection. Lesion sizes and CFU counts were determined 3 days postinfection. The experiment was repeated at least two times. Results are courtesy of Dr. Daniel Pletzer from the Hancock lab (UBC).   116   4.4.6 Peptide aggregation  Tryptophan and arginine containing AMPs have been well known to aggregate which may relate to its toxicity269. Peptide 73c was one of the most active peptide in the mice abscess model when dissolved in PEG but had poor solubility in water and saline solution. To investigate the aggregation behavior of peptide 73c the average hydrodynamic diameter (Hd) was measured in water and DSPE-PEG2000 at 3 mg/mL (concentration injected in abscesses). From Table 4.5 (and also Appendix C.3), it can be concluded that peptide 73c forms aggregates of various sizes with an average Hd of 329 nm and a large standard deviation (100 nm). Upon encapsulation with DSPE-PEG2000, most (74%) of the peptide 73c forms micelles with a Hd of 11.9 nm, whereas 26% still exists in the aggregate state. In another study, DSPE-PEG2000 encapuslated cabozantinib, an anticancer drug, also had an Hd of 11 nm270. The formation of micelles may account for the decreased toxicity and increased solubility of peptide 73c and hence better efficacy in vivo. However, in the future it would be beneficial to increase the concentration of DSPE-PEG2000 or use a better encapsulationg agent to increase the loading efficiency.   Table 4.5: Average hydrodynamic size of peptide 73c   Average hydrodynamic  size (nm) Standard deviation (nm) % intensity 73c in deionised water 329 100 90 73c in  DSPE-PEG2000 11.9 1.6 74 254 30  26  117   4.4.7 Efficacy of the HPG conjugates in the mouse abscess model  Results from Chapter 2 and 3 suggest that the HPG peptide conjugates were non toxic and showed moderate antimicrobial activity against S. aureus. These conjugates made use of aurein 2.2∆3 or peptide 77, which the mouse abscess model shows to be less effective than some of the other peptides tested (Figure 4.2 and 4.3). Since peptide 73c showed good activity upon encapsulation, a conjugate consisting of peptide 73c and 22K HPG was synthesized and the in vivo efficacy was evaluated. HPG-73c was not toxic and did not precipitate under the skin at concentrations as high as 30 mg/kg (9 mg/kg peptide 73c equivalent) in saline, but the solution was cloudy indicating that solubility is an issue. HPG-73c was not effective in reducing the lesion size or bacterial counts in the mouse abscess model (Figure 4.5). This may be due to the fact, as observed in Chapter 3, that conjugation has an impact on antimicrobial activity (recall the 6-7 fold increase in MIC). Compared to the free peptides shown in Figure 4.4, the HPG conjugate was already tested at a higher concentration (30 mg/kg) to try to account for the lower MIC of the bioconjugate. However, this higher concentration is most likely still low (i.e. when comparing dosage of 5 mg/kg for an MIC of 4 µg/mL (free peptide) versus 30 mg/kg for an MIC of 50 µg/mL for conjugate). As mentioned above, going to higher concentrations is problematic, firstly because the solubility is poor, but also in terms of the high amount of material required. It is difficult to speculate whether a really high dose would lead to a statistically significant change in the abscess size and bacterial count.  118    Figure 4.5: Therapeutic treatment of mouse cutaneous abscesses with HPG-73c conjugates. All mice were infected with MRSA USA300 and treated with HPG solution and HPG73c (30 mg/kg) 1 h post-bacterial infection. Lesion sizes and CFU counts were determined 3 days postinfection. The experiment was repeated at least two times. Results are courtesy of Dr. Daniel Pletzer from the Hancock lab (UBC).  4.4.8  Peptide design paradigms  Determining the MIC of the peptides is one of the most prominent parameters used in the screening of AMPs. Although it is a good starting point, the peptide with the best MIC does not always guarantee that the peptide would be more effective in different animal models such as the mouse abscess model. For example, peptide 73 has a better MIC than aurein 2.2 but has similar activity in the abscess model. The data presented in this chapter strongly suggests that it is important to test the peptides in animal models to better understand the efficacy of the peptides in vivo before further development of the AMPs. It is also worth screening the toxicity of the HPGHPG73-v2020406080100 Abscess area (mm2)HPGHPG73-v21001011071081091010 CFU/abscess   119   peptides and determining the therapeutic index in vitro, as it correlates well with the activity in vivo for our mice abscess model.   As discussed in Chapter 1.3 there are many strategies to improve the activity of AMPs. One of the easiest and most frequent strategies used to improve the activity of AMPs is the use of D-peptides to prevent proteolytic degradation. Peptide D-73 and RI-73 have similar MICs compared to 73 but could not be administered in vivo because of the toxicity issues. The toxicity of the D-73 peptides was mitigated by the use of DSPE-PEG2000 as a non covalent delivery vehicle which enabled the peptides to be released and effectively treat the abscess; however RI-73 was still ineffective in mice. Interestingly, the use of D-peptides also improved the in vivo efficacy of other peptides such as DJK-5207 and D-IK8255.  Another strategy used to improve the efficacy of AMPs is the use of covalent attachment to polymer molecules (conjugation). Similarly to the results presented in Chapter 3, the conjugation of peptide 73c to HPG alleviated the toxicity but the conjugates were not effective in the abscess model at the concentrations tested. One approach would be to increase the dosage but the solubility of the conjugate might limit this option. A better strategy would require the peptides to be released at the site of infection either by enzymatic cleavage or a degradable linker, similar to the DSPE-PEG2000 formulation used for 73c and D-73. Recently, an immunomodulatory peptide pOVA when conjugated to HPG via a cleavable ester linkage had superior properties compared to the non cleavable amide linkage199.  4.5 Summary In this chapter, we successfully demonstrated the antibacterial and antibiofilm activity of various novel peptides and conjugates. The therapeutic index of the peptides in vitro correlated  120   well with the in vivo activity in a mouse abscess model indicating the fine balance needed between antimicrobial activity and toxicity. This was particularly evident in the case of peptide 73, with its superior antimicrobial activity but higher toxicity, as compared to aurein 2.2. Peptide 73c and D-73 were the most active peptides in vivo when used in a formulation with DSPE-PEG2000 which aided in alleviating the toxicity of both the peptides. Although conjugation of peptide 73c to HPG also mitigated the toxicity of the peptide, the conjugate was not effective in vivo indicating that the release of the peptide may be an important step for an in vivo active formulation.   121   Chapter 5: Overexpression, purification and esterification of aurein 2.2 utilized to probe the interaction of aurein 2.2 with whole bacterial cells by NMR.  5.1 Synopsis The antibiotic crisis has lead to a pressing need for alternatives such as antimicrobial peptides. Better understanding the structure and mechanism of action (MOA) of AMPs would lead to the discovery of more potent and less toxic AMPs.  Many models have been utilized to describe the MOA but only a few studies have probed the interaction of AMPs with live bacterial cells. In this chapter, we use a calmodulin-aurein 2.2 (CaM-aurein 2.2) fusion construct to obtain isotopically 15N labeled aurein 2.2 using a bacterial expression system. Expression of AMPs yield peptides with carboxylic acid at the C-terminus which typically renders the peptide inactive; consequently, blocking the free carboxyl at the C-terminus via esterification was required to restore the activity of aurein 2.2. The interaction of the esterified and 15N labeled aurein 2.2 with live Staphylococcus aureus was probed using NMR (1H,15N-HSQC) and scanning electron microscopy (SEM). This system can be further developed and utilised to study the interaction of other AMPs and polymer conjugates with live bacteria in the future.   5.2 Background Antimicrobial peptides such as aurein 2.2 and 73 have good antibacterial activity both in vivo and in vitro. The mechanism of action (MOA) of AMPs like aurein 2.2 has been well studied and many techniques such as NMR,  CD spectroscopy and leakage assays117,139 have been utilized to understand the bacterial killing action of AMPs. Although the MOA of some AMPs is well understood, most studies use model membrane systems such as supported bilayers,  122   liposomes, vesicles and micelles showing that AMPs damage the membrane or form membrane pores via various mechanisms such as toroidal pore, barrel stave or detergent models as described in Chapter 1.2.4. Hence, the main killing action of AMPs is thought to target the bacterial cell membrane, but AMPs may also target the cell wall or other intracellular components160. To date only a few studies have explored the membrane/cell wall-peptide or peptide-intracellular component interactions in living cells116. Therefore, it is important to investigate the detailed structural interaction between AMPs and its targets in living cells to further understand their MOA.  NMR is one of the most powerful techniques that can used to elucidate the detailed structural  interactions between AMPs with potential molecular targets such as membranes, proteins and nucleotides210.  To date only a few whole cell NMR experiments have been reported to determine the interactions of AMPs with different bacterial components. Solid state NMR studies (cross-polarization magic-angle spinning, CPMAS) of whole planktonic cells were able to detect the total 13C and 15N composition of intact S. aureus cells211. The general mode of antibiotic action could be identified as whole cells treated with fosfomycin had lower peptidoglycan composition compared to cells treated with chloramphenicol which contained a higher percentage of peptidoglycan but a reduction in the cytoplasmic protein content211. In a more recent study, 13C and 15N CPMAS NMR was used to detect ribosomes in intact whole cells which could possibly be used in the future to determine the interaction of other ribosome binding compounds271.  Most whole cell NMR studies to date utilize isotopic labeling of the bacterial cells rather than the antimicrobial compound such as AMPs. AMPs are synthesized by solid phase peptide synthesis and chemical isotopic labeling of the AMPs is not practical due to the high cost. Few  123   recombinant expression systems in bacteria have been utilized to produce isotopic labeled AMPs210,272. Vidovic et al. used a construct in which AMP LAH4 was fused to the histone fold domain of the human transcription factor TAF12272. Interestingly, this fusion construct was expressed within insoluble inclusion bodies and did not require any purification and was cleaved chemically by formic acid due to the unique Asp-Pro bond. Moreover, recombinant expression of AMPs has also been developed by Ishida et al. using a calmodulin fusion construct (Figure 5.1), with a Tobacco Etch Virus (TEV) cleavage site210. With the development of this unique system, it is now possible to isotopically label AMPs and to study the interaction of AMPs with whole bacterial cells by NMR. NMR spectroscopy also becomes more complex when polymer conjugates are to be studied due to the signals from the polymer leading to indistinguishable overlapping signals. Isotopic labeling of the AMPs in this case will enable us to specifically observe the AMP signal only.   Figure 5.1: Calmodulin (CaM)-aurein 2.2 fusion construct with TEV protease cut site (between glutamine and glycine) and the restriction enzyme site. Figure modified from Ishida, H et. al210.   Calmodulin is a ubiquitous calcium sensor protein found in eukaryotes. Its negative surface charge allows it to bind to many peptide sequences with positive charge and hydrophobic residues273–275. As most AMPs consist of a high proportion of such amino acids, having an AMP-calmodulin fusion is advantageous as this construct will prevent proteolytic degradation and toxic effects arising from AMPs during expression210. Ishida et al have shown that many AMPs such as indolicidin and magainin can be expressed using the calmodulin fusion construct and the  124   AMPs bind to calmodulin in the micromolar range210. The resulting AMPs are produced in milligram quantities with a carboxy C-terminus215.   In this chapter, we used the recombinant expression system developed by Ishida et al. to express aurein 2.2 for the first time.  This yielded a peptide that could still adopt an alpha helical secondary structure but did not display any antimicrobial activity due to the free carboxyl at the C-terminus. To restore the activity of aurein 2.2, which in its natural form has an amidated C terminus, the free carboxyls (C-terminus and aspartic acid) were esterified using trimethylsilyl chloride (TMSCl). Isotopically (15N) labeled aurein 2.2 was expressed and esterified to yield a peptide with good antimicrobial activity. NMR studies (2-dimensional 15N-1H heteronuclear single quantum coherence (HSQC experiment)) with intact whole bacterial cells were performed to verify the mechanism of action and as a proof of concept that will allow us to study the detailed structure and interactions of other AMPS/conjugates with whole bacteria cell in the future.   5.3 Methods and materials  All chemicals were purchased from Sigma Aldrich Canada Ltd (Oakville, ON) unless mentioned otherwise and used without further purification. Dialysis membrane was obtained from Spectra/Por Biotech (Rancho Dominguez, CA). 5.3.1 Expression of Calmodulin-aurein fusion protein in E.coli The pET15b plasmid containing the pCaM-Aurein2.2 construct was provided by the Hans Vogel lab (University of Calgary, Canada). The pCaM-TEV site-Aurein2.2 construct was prepared as previously described210. E. coli cells transformed with the pCaM-TEV site-Aurein2.2 construct were grown in Lysogeny broth (LB) or minimal media (M9) at 37 oC with 100 g/L ampicillin. For the preparation of  15N-labelled aurein2.2 peptide, M9 media was prepared using  125   15NH4Cl. Cultures were grown to an OD600 of 0.5-0.7 and induced for 4 hours using 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, see section 5.3.2 for details) was used to confirm if the induced expression was successful. Cells were harvested using centrifugation at 10,000 rpm for 10 min before being resuspended in 25 mL of 20 mM Tris-NaCl buffer (20 mM Trisaminomethane; 500 mM NaCl; pH 7.0). Two hundred microliters of 250 μM phenylmethylsulfonyl fluoride (PMSF), 25 μL DNAse and a few flakes of lysozyme were added to the resuspension mixture before French pressing three times. The supernatant was collected after centrifugation (15,000 rpm, 55 min, 4 °C) and filtered through 0.25 μm filters (Millipore). 5.3.2 SDS PAGE One milliliter samples before and after IPTG induction were collected, centrifuged (13000 rpm, 10 min, 25 oC) and resuspended in 100 μL dH2O. One hundred microliters of protein sample was mixed with 25 μL of 5X SDS sample loading buffer (Biorad) and then heated at 80 °C for 10 min. Ten microliters of each boiled sample solution as well as 10 μL of PageRuler™ protein ladder were then loaded into a premade 12% acrylamide Tris-Bis gel. SDS-PAGE sample solutions collected from the Ni-NTA washes and elutions were directly loaded onto the gel. The gel was run at a constant 120 mV for 2 hrs and stained using 50 mL of Coomassie R-250 solution for 40 min on a rocker. Gels were destained using 50 mL of premade destaining solution (30% methanol, 10% acetic acid in water) for 2 h. 5.3.3 Purification of Calmodulin-aurein fusion protein in E.coli The supernatant described in section 5.3.1 was applied to a Ni-NTA (His)6-tag protein binding column (GE Healthcare). Prior to the application of the supernatant, the column was cleaned with 10 mL 50 mM EDTA solution (3x), and 10 mL 0.5M NaOH (3x) before recharging  126   the column using 1ml of 0.1M NiCl2. After loading the supernatant on to the Ni-NTA column, the column was washed with 10 column volumes of Tris-NaCl buffer until the OD280 of the washes were within 0.030 of the Tris-NaCl buffer blank. The His-tagged fusion protein was eluted with Tris-NaCl buffer, 400 mM imidazole, pH 7.5. The purity of the fusion protein was confirmed by SDS-PAGE. The Ni-NTA elutions were dialyzed overnight against 20 mM Tris-NaCl buffer at 4 °C (8k dialysis bag) to remove the excess imidazole.  5.3.4 Digestion of Calmodulin-aurein fusion protein  Tobacco Etch Virus (TEV) protease was prepared using the same expression protocol as described previously276. Prior to Tobacco Etch Virus (TEV) digestion of the eluted fusion protein, the dialyzed elutions were concentrated using 10k cut-off Amicon centrifuge tubes (4000 x rpm, 15 min, 4 oC) to a total final volume of 4-5 mL. Two hundred and fifty microliters of TEV protease was added for OD280 of 0.5 of the elutions. To this mixture EDTA and DTT were added, for a final concentration of 5 mM EDTA and 2 mM respectively. This solution was placed on a rocker for 24-48 h at 4 °C. MALDI-TOF was used to confirm the presence of the cleaved peptide and monitor the completion of the cleavage reaction. The peptide was purified using RP-HPLC. 5.3.5 MALDI-TOF One microliter of the sample mixture was mixed with 1 μL of 5 mg/mL α-Cyano-4-hydroxycinnamic acid (CHCA) matrix solution (70% acetonitrile, 30% dH2O, 0.1% TFA). The cleavage mixture was diluted (32-128 fold) in the α-Cyano-4-hydroxycinnamic acid solution to prevent formation of salt crystals. One microliter of each prepared sample solution was spotted onto a plate and dried completely. MALDI-TOF MS spectra were acquired using a Voyager-DE™ STR MALDI-TOF mass spectrometer.   127   5.3.6 Peptide purification by RP-HPLC RP-HPLC purification of expressed aurein 2.2 was carried out using a similar protocol given in Chapter 2.3.1. Fractions were collected for each corresponding peak seen in the HPLC trace and confirmed using MALDI-TOF. 5.3.7 Peptide Esterification Purified aurein 2.2 was esterified at the C-terminus and the aspartaic acid residue carboxylic acid moiety using the esterification protocol described previously277. Lyophilized peptide (1 mg) was dissolved in 2 mL of anhydrous methanol and sonicated for 10 min. 4 equivalents (450 μL) of TMSCl was added to the reaction mixture and stirred for 4 h. The progress of the reaction was monitored every 1 h using MALDI-TOF. After completion of the reaction, the solution was quenched with 6 mL dH2O and immediately lyophilized for a second esterification reaction to ensure the peptide was diesterified. The final diesterified product was purified using RP-HPLC and characterized by MS/MS. 5.3.8 MIC Assays The MICs of the esterified peptides against S. aureus (C622) were measured based on previously described methodology113,138,215,235 and Chapter 2.3.6. 5.3.9 2D NMR  NMR spectra for solution state peptide samples were acquired on a Bruker 500 MHz instrument (Bruker Biospin, Milton, ON), operating at a 1H frequency of 499.4 MHz. 1H-15N NMR spectra were collected using a Heteronuclear Single Quantum Coherence (HSQC) pulse sequence278–280 at 298 K. The NMR sample was prepared to yield 0.5 mM 15N labelled aurein peptide in 25% deuterated TFE and 10% D2O (by volume), for a total sample volume of 600 μL.  128   For the intact whole bacterial cell studies, an overnight culture of S. aureus was grown in lysogeny broth (LB) and diluted to an OD600 of 0.8 which corresponds to approximately 106 CFU/mL. Two hundred fifty microliters of the bacterial sample were spun down and resuspended in 250 μL of 10 mM phosphate buffer and 50 µL was transferred to a NMR tube containing 0.35 mM (~500 µg/mL) 15N aurein 2.2 (ester or carboxyl version dissolved in 10 mM phosphate) and 10% D2O, for a total volume of 600 uL. 5.3.10 Scanning electron microscope studies The morphology of the S. aureus cells in the presence of the aurein peptides was determined with SEM. Twenty microliters of expressed aurein 2.2 or diesterified aurein 2.2 (0.35 mM final concentration) were incubated with 20 µL of S. aureus (108 CFU/mL resuspended in 10 mM phosphate buffer) for 1 hour at 37 °C. Incubation mixtures were fixed using 2.5% glutaraldehyde and repeatedly washed with 0.1 M sodium cacodylate (pH=7.4), then subjected to post-fixation with 1% v/v osmium tetroxide. The fixed samples were washed three times with distilled water and dehydrated with a gradient series of ethanol (20-95% v/v). Cells were then dried with CO2 in a Tousimis Autosamdri 815B critical-point dryer, mounted onto stubs, and gold sputter-coated for SEM using a Hitachi S-4700 field emission scanning electron microscope at various magnifications. Multiple images from different areas were captured. 5.4 Results And Discussion 5.4.1 Expression and purification of CaM-aurein 2.2 The expression of AMPs in bacteria has been previously studied using several fusion partners such as ketosteroid isomerase (KSI)281 and small ubiquitin-related modifier (SUMO)282.  The expression of KSI-melittin leads to inhibition of culture growth due to the toxic peptides whereas the expression of  SUMO-melittin fusion results in poor culture growth after IPTG  129   induction210. On the other hand, the CaM fusion construct has been developed to express a handful of  AMPs such as melittin, tritrpticin, fowlicidin, indolicidin, magainin II, human-β-defensin 2 and lactoferrampin B210. For the first time, we were able to express CaM-aurein 2.2 in LB and minimal media (M9). There was no “leaky” expression (expression before adding the inducer (IPTG)) of CaM-aurein 2.2 before IPTG expression as seen from the gel and a maximum protein expression level was reached between 3 to 4 h (Figure 5.2).        Figure 5.2: Expression and purification of CaM-aurein 2.2. SDS-PAGE  (left) of samples from the E. coli expression cells taken at different times before and after IPTG induction (PL: protein ladder, Cal: commercial calmodulin, M: before IPTG  induction). SDS-PAGE (right) of Ni-NTA column purification. After the sample was loaded, washes (W1 to W5) of the Ni-NTA resin were collected until A280 was similar to the buffer only (~0.03). Elution samples (E1 and E2) were collected using 400 mM elution buffer. Figure generated using PowerPoint.   Moreover, the presence of a polyhistidine (His)6- tag at the N-terminus of the CaM-aurein 2.2 allowed for the purification directly via Nickel-NTA affinity chromatography (Figure 5.2).  The washing with Tris-NaCl buffer removed most of the impurities. However, some high 17000- 26000- 34000- 43000- 55000- 72000- 95000- Da  130   molecular weight impurities coeluted with CaM-aurein 2.2 during the imidazole elution in the first 10 mL (E1), but the second 10 mL elution did not have any impurities. The impurities may be due to the non-specific interaction a bacterial protein containing adjacent histidine residues with the nickel-NTA or non polar interactions with the nickel-NTA resin283. A very high purity of the CaM-aurein 2.2 fusion protein is not necessary as the CaM-aurein 2.2 has to be cleaved and purified. As in the case for melittin, tritrpticin, fowlicidin, indolicidin, magainin II, human-β-defensin 2 and lactoferrampin, the CaM fusion system was able to mask the antimicrobial activity of aurein 2.2 and protect it from degradation during expression.  5.4.2 Cleavage of CaM-aurein 2.2 and purification of aurein 2.2   After purification, the CaM-aurein 2.2 fusion protein was dialyzed overnight in digestion buffer and subjected to TEV protease. The cleavage was monitored by MALDI-TOF and after 48 h the cleavage was complete. After optimization of the digestion buffer, complete cleavage of the fusion protein lead to the appearance of two distinct MALDI-TOF peaks, namely the CaM at 20,193 Daltons (Figure 5.3) and that for aurein 2.2 peak at 1618 Daltons (Figure 5.5). Incomplete cleavage causes the appearance of CaM-aurein 2.2 at 21,782 Daltons (Figure 5.3). Upon complete cleavage, the reaction mixture was filtered and HPLC purified. HPLC peaks were identified by MALDI-TOF and the aurein 2.2 was successfully purified (Figure 5.4). Aurein 2.2 eluted after cleaved CaM, as would be expected for a more hydrophobic peptide. The hydrophilic water soluble CaM allows the largely hydrophobic aurein 2.2 to be well solubilized during the expression and purification process. The final yield of aurein 2.2 was approximately 1-2 mg per 1 L culture (similar yields in M9 media and LB).  131    Figure 5.3: MALDI-TOF of the cleavage mixture. The peaks with maxima at m/z of 21782.15 and 20193.67 correspond to the CaM-aurein 2.2 and CaM, respectively. Figure generated using data explorer and Adobe.     Figure 5.4: HPLC purification of the cleaved CaM-aurein 2.2. CaM elutes between 48-60 minutes, where as the more hydrophobic aurein 2.2 elutes between 56-57 minutes. Figure generated using Adobe.      132   5.4.3 Chemical esterification of aurein 2.2 retains antimicrobial activity.  One of the major disadvantages of expressing AMPs in most bacterial expression system is the inability to express peptides in the active amidated form. As mentioned in Chapter 1, most AMPs such as aurein 2.2 require the C-terminus to be amidated to exhibit antimicrobial activity113,138,139,215. Expressed aurein 2.2 consists of a carboxylic acid at the C-terminus (aurein 2.2-COOH) and was inactive against S. aureus (Table 5.1) with an MIC greater than 250 g/mL compared to the synthetic amidated aurein (MIC of 32 g/mL). Many enzymatic methods such as transacylation284 and amidation285 have been utilized to neutralize the negative charge of the carboxylic acid at the C-terminus of expressed peptides. These methods are usually time consuming and expensive. Using a previous reported277 TMSCl/methanol protocol to esterify various amino acids, we were able to rapidly and selectively esterify the C-terminus and the aspartic acid of the expressed aurein 2.2 (diesterification, 2 CH3 group, ~ +30 Daltons ) (Figure 5.5) within an hour of the reaction, however monoesterified  (~ +15 Daltons) products were also obtained.  Hence, to completely diesterify aurein 2.2, the reaction mixture was freeze dried and esterified with TMSCl/methanol again. The diesterification product was purified by HPLC and the antimicrobial activity was determined. The MS/MS (Appendix D.1) analysis of the purified diesterfied aurein 2.2 confirmed the addition of a CH3 group at the aspartic acid and the C-terminus. Diesterification of aurein 2.2 partially rescued the antimicrobial activity of the expressed peptide with an MIC of 64 g/mL compared to the synthetic amidated aurein 2.2 (32 µg/mL). Esterification of the aspartic acid could affect the structure and mechanism of action of aurein 2.2 as it has been recently suggested that aurein 2.2 forms ion selective pores in the membrane of Bacillus subtilis and that the aspartic acid may be involved in binding select  133   cations140. Future work should involve the selective removal of the ester from the aspartic acid or specific esterification or amidation of the C-terminus.   Table 5.1: Antimicrobial activity of expressed and synthetic aurein 2.2        Figure 5.5: MALDI-TOF of aurein 2.2 (left) and diesterified aurein 2.2 (right). The 1618.08 (right spectra) peak corresponds to aurein 2.2-COOH. The 1647.99 peak, correspond to the diesterified aurein 2.2 and its sodium adduct (~+23) respectively. Monoesterified product is at 1632.32. Figure generated using Data explorer and Adobe.   Peptide MIC   S. aureus C622 (μg/mL) Aurein  2.2 (synthetic) (GLFDIVKKVVGALGSL-CONH2) 32 Aurein  2.2 (expressed) (GLFDIVKKVVGALGSL-COOH) >250 Aurein  2.2 (expressed and diesterified) (GLFD*IVKKVVGALGSL-COOCH3) 64  134   5.4.4 Whole cell NMR and SEM  Recently a handful of studies have been reported to investigate the interaction of AMPs with living bacterial cells. Some techniques used include live cell imaging116, CD286 and NMR211,271 spectroscopy. To determine the interaction of aurein 2.2 with living S. aureus, we incubated 15N-labelled aurein 2.2 with S. aureus and acquired 1H,15N-HSQC NMR spectra at different time points. Comparing the 1H,15N-HSQC spectra of expressed aurein 2.2 and diesterified aurein 2.2, we can see clear differences between at the different time points (Figure 5.6). Expressed aurein 2.2 (aurein 2.2-COOH), which is inactive against S. aureus, does not show any differences after 1, 2 and 3 h of incubation, whereas in the spectra of diesterified aurein 2.2 the peak intensity decreased drastically with respect to time (Figure 5.6).  Amidated aurein 2.2 targets the bacterial membrane by inserting and forming ion selective pores which is consistent with the toroidal pore model140. The drastic decrease in peak intensity could be explained by the fact that diesterified aurein 2.2 inserts into the membrane of S. aureus which leads to slower tumbling of the peptide in solution and faster relaxation of transverse magnetization (short T2) resulting in line broadeing287. The sharp peaks seen at the end of 3 hours when all the bacteria were killed could be from unbound peptide, freely tumbling in solution. In contrast, the aurein 2.2-COOH spectra does not show any significant decrease in peak intensity indicating the peptides do not bind and insert into the membrane and hence are inactive against S. aureus. Aurein 2.3-COOH, another inactive AMP against S. aureus does not bind to PC/PG model membranes as effectively as its amidated counterpart, despite still forming an alpha helical structure139. Expressed aurein 2.2-COOH shows a similar behavior as it is   135     Figure 5.6: 1H,15N-HSQC spectra of whole S. aureus cells with the aurein peptides. Aurein 2.2-COOH (top) and diesterified aurein 2.2 (bottom) were mixed with S. aureus cells and the spectra were acquired during 1, 2 and 3 hours of incubation. The start contour levels, number of levels and the level multiplier for aurein 2.2-COOH was 2x107, 16 and 1.2 respectively, for all the time points whereas for diesterified aurein 2.2 they were set to 8.5x107, 16 and 1.2 respectively.    1 hr 2 hr 3 hr 1 hr 2 hr 3 hr  136   inactive but can form alpha helical structures, whereas diesterified aurein 2.2 is active and can form an alpha helical structure (Figure 5.7). It will be interesting in the future to express an AMP that is not membrane active but has intracellular targets and study the interaction with live S. aureus cells via 1H,15N-HSQC as intracellular protein/peptides that are more freely tumbling are easier to visualize by NMR288,289. The expression system could potentially also be used to determine the structure of bioconjugates of AMPs and their interaction with live bacterial cells.   Figure 5.7: CD spectra of expressed peptides. Both aurein 2.2-COOH and diesterified aurein 2.2 adopt an alpha helical structure in trifluoroethanol (TFE). Figure generated using data Graphpad prism.  In order to visualize the S. aureus during the NMR experiment, we collected SEM images post 1 hour incubation with the peptides (Figure 5.8). In both the samples, the S. aureus seems to aggregate but clear differences can be seen. In the case of aurein 2.2-COOH, the S. aureus are round and intact whereas the S. aureus cells are smaller and lysed when treated with diesterified  137   aurein 2.2 (Figure 5.8). There is also more lipid and cell debris in the latter case from the ruptured cells. Similar distortions have been observed when S. aureus is treated with PGla, a α-helical peptide from the magainin family that can destroy bacteria by interacting with the lipid membrane290.     Figure 5.8: SEM of S. aureus after incubating for 1 h with aurein 2.2-COOH (left) and diesterified aurein 2.2 (right). The cells look smaller and compact when treated with diesterified aurein 2.2. Figure generated using PowerPoint.  5.5 Summary In this chapter, for the first time we report a protocol for overexpression and purification of aurein 2.2 using a bacterial expression system. The aurein peptide obtained was inactive against S. aureus because of the free carboxylic acid at the C-terminus. Diesterification of the C-terminus and the aspartic acid carboxylic acids was confirmed by MALDI-TOF and MS/MS, which rescued the antimicrobial activity of the peptide against S. aureus. The interaction of diesterified aurein 2.2 and aurein 2.2-COOH with live S. aureus was probed using 1H,15N-HSQC  138   NMR spectroscopy and it was confirmed that diesterified aurein 2,2 binds to bacterial membranes, whereas aurein 2.2-COOH does not interact with the bacterial membrane, in agreement with previously reported results. SEM studies further confirmed that diesterified aurein 2.2 causes significant morphological changes of the bacterial membrane, whereas aurein 2.2-COOH does not. This system will not only allow us to just study the structure of the AMPs and bioconjugates but collect dynamic NMR data with living cells in the future.    139   Chapter 6: Conclusions and Future work  6.1  Conclusions  Antimicrobial peptides are unique molecules that are promising candidates to treat multidrug resistant organisms291. Interestingly, AMPs have many mechanisms of action and can also display antiviral51 and anticancer activities292. Although many AMPs have been identified and characterized, not many were able make it to clinical trials and only a few have been approved by the US Food and Drug Administration (FDA) due to issues with toxicity, protease cleavage and rapid kidney clearance23,105,141. Over the last decade, different strategies have been utilized to resolve these issues to improve the efficacy of AMPs to push forward their development as therapeutic agents. This thesis examined various approaches from designing new AMPs, using delivery systems for AMPs to decrease the toxicity and studying the mechanism of action of AMPs.   The conjugation of AMPs to polymers like PEG leads to improved characteristics such as lower toxicity, prevention of proteolytic cleavage and increased blood half-life160.  We hypothesized that conjugation of aurein 2.2 would have similar benefits. In Chapter 2, we developed the chemistry and for the first time conjugated aurein 2.2 to hyperbranched polyglycerol (HPG), a biocompatible polymer. We were able to demonstrate that the conjugation lead to decreased toxicity of AMPs toward mammalian cells such as fibroblasts and to blood components such as red blood cells, complement system and platelets. The neutral charge and hydrophobic masking effect of HPG likely contributes to the diminished toxicity of the conjugates. Although the antimicrobial activity of the aurein 2.2 conjugates was decreased, it was not as severe as PEG conjugates reported in the literature166–168. This may be due to the fact that multiple peptides were attached to HPG compared to PEG which typically has one or two  140   peptides per polymer molecule; hence a polymer with multiple functional groups is advantageous over a polymer with limited functionalities.  Furthermore in Chapter 3, we hypothesized that the strategic design of a peptide array would yield at least a few peptides with significantly better antimicrobial activity. From the array we got two hits, peptide 73 and 77 which had specific arginine and tryptophan substitutions and were 4 times more active than aurein 2.2. Although arginine and tryptophan substitutions generally lead to an increase in activity232, the specific correlation between activity and the location of these substitutions are more complicated to predict. Hence SPOT synthesis arrays were a good strategy as they permit the synthesis and testing of many peptides. Indeed, many of the peptides in the array were not as active as the parent peptide. Software programs such as quantitative structure-activity relationship (QSAR), could have been used to design AMPs with better antimicrobial activities but an initial dataset, which comes from the systematic peptide array, is required233. This chapter also described the successful conjugation of a more active peptide such as 77c to HPG and showed that this leads to a conjugate with higher antimicrobial activity and alleviation of the toxicity, as was the case in Chapter 2. The conjugates were also resistant to proteolytic degradation. This suggests that HPG could be used as a generic polymer scaffold for various AMPs.    AMPs have been conjugated to various polymers such as PEG, chitosan, hyaluronic acid293 but the influence on polymer molecular weight on activity and biocompatibility of the AMPs has not been investigated to date. The remainder of the studies in Chapter 3 showed that a decrease in molecular weight of HPG leads to increase in activity, but does not have any significant effect on the biocompatibility, again suggesting the usefulness of HPG (22K HPG-77  141   being the most active). Finally, through our search for more active HPG conjugates, we have also made AMPs (peptide 73 and 77) which have diverse activities. In Chapter 4, we explored the activities of peptide 73 and 77. As mentioned in Chapter 1, AMPs can have other activities such as antibiofilm and wound healing44. Aurein 2.2 has no antibiofilm activity, but peptide 73 and 77 were both found to be highly active against S. aureus biofilms. This indicates that peptides can have different activities and that the mechanism of action to kill bacteria in planktonic state and in a biofilm may be different. As the peptides and conjugates had good in vitro activity against S. aureus, we investigated the in vivo activity in mice abscess model. Interestingly, the in vivo data suggested that aurein 2.2 and peptide 73 were equally effective in mice even though their antimicrobial activity in vitro is very different. However, if one considers the balance between activity and toxicity, i.e. the therapeutic index, both aurein 2.2 and peptide 73 were very similar. This clearly indicates that one needs to consider the in vivo toxicity of AMPs and not just their antimicrobial activity during the design of these peptides233. It is also extremely critical to test parent peptides in an in vivo model before designing peptide arrays, as the parent peptide might be highly active in vivo (as in the case of aurein 2.2 compared to 73). In the case of aurein 2.2, further design of the peptide lead to peptide 73, which has additional antibioflim activity (which aurein 2.2 does not have). An in vivo antibiofilm model would be useful to determine whether this improvement is significant.  Chemical modification of AMPs such as the use of non natural149, D-amino acids146, and cyclization152 has been used to prevent proteolytic cleavage and this typically leads to the improved activity of AMPs. The development of D-73 (a peptide with all D-amino acids) resulted in a peptide with similar antimicrobial activity as the parent peptide, but increased toxicity in vitro. The toxicity of D-73 required micellar encapsulation of the peptide within  142   DSPE-PEG2000 to exhibit any activity in vivo. The encapsulation of peptide within the micelle masks the toxicity of the D-peptides. As the encapsulation formulation for D-73 is as effective as peptide 73 without a delivery system, one could conclude that the synthesis or purchase of expensive D-peptides does not necessarily yield a better drug candidate. On the other hand, the micellar encapsulation was very effective in general as it does not only improve the toxicity, but also increases the solubility of the peptides (e.g. as for peptide 73c). In other words, a careful choice of amino acid sequence and formulation needs to be taken into account when developing an optimal antimicrobial with good activity and low toxicity.  In recent years, many covalent delivery systems have been designed to further the pursuit of developing AMPs160. Although the attachment of AMPs to polymers also improves various in vitro properties such as toxicity, solubility and protease cleavage, as seen in Chapter 2 and 3, the best in vitro conjugate (22k HPG-73c) was ineffective in the mouse abscess model. However, the non covalent micellar encapsulation delivery systems discussed above were very effective. The data suggests that the better strategy requires peptides to be released from the delivery system to effectively kill bacteria and heal abscesses. Again it is important to investigate the effectiveness of the conjugates both in an in vivo and in vitro model, as the activities can differ drastically.  Finally, as described in Chapter 1.2, it is important to study the mechanism of action of AMPs in order to understand how they function and to help in the design of better AMPs. As a proof of concept, we showed in Chapter 5 how AMPs can be expressed and how the antimicrobial activity of the expressed aurein 2.2 can be rescued by simple esterification. This approach could be extended to a range of expression systems developed to produce AMPs284,285. We demonstrated that obtaining active isotopically labeled AMPs is feasible. This opens the door  143   for various whole cell NMR experiments. With whole cell 1H,15N-HSQC NMR experiments, we were able to confirm the mechanism of action of aurein 2.2, which binds to the bacterial membrane. Future whole cell experiments may provide new insight into the differences in mechanism of action of various peptides and conjugates, in particular in the case where the mechanism of action of the AMPs involves intracellular targets. 6.2 Future work  Studies in this thesis have revealed significant insight into the in vitro and in vivo activities of AMPs, which has in turn opened many avenues to explore. In Chapter 3, we developed peptide 73 on the basis of the in vitro antimicrobial activities alone, however, in Chapter 4 the in vivo data indicated that the toxicity profiles also need consideration. Future development of AMPs (peptide array) should consider both the toxicity such as RBC lysis and the antimicrobial activity, i.e. the therapeutic index, so that peptides with moderate antimicrobial activities are not overlooked294,295. For example, the aurein 2.2 parent peptide had moderate in vitro activity as compared to peptide 73, but their therapeutic indices were similar and could be used to account for their similar in vivo activity. The peptide with the best therapeutic index should also be used as the parent peptide for further development of the AMPs (peptide arrays). In addition, as most QSAR programs used to design novel AMPs only consider the antimicrobial activity of the peptides233, it will be useful if the method could be expanded and the therapeutic indices were also considered for a more efficient design.  In this thesis, the mechanism of action (MOA) of the peptides 73 and 77 was not investigated. Many arginine and tryptophan rich peptide target the bacterial membrane232 but recent oriented CD results (data not shown) from our lab suggest that peptide 73 and 77 do not insert into model membranes as effectively as aurein 2.2. Membrane leakage data also indicates  144   that peptide 73 and 77 cause less leakage as compared to aurein 2.2. It is hence possible that peptide 73 and 77 target other bacterial components, such as the cell wall or intracellular components. The expression system utilized in Chapter 5 could be used to produce isotopically labeled 73 and 77, in order to probe the interaction with live bacteria and compare it to the results on aurein 2.2 presented in Chapter 5. Moreover, this labelling approach could be used in conjunction with experiments that rely on isotopic labelling of the bacterial cells. For instance, recent studies examined the changes in bacterial cell wall composition using solid state 13C-CPMAS NMR experiments. The general MOA could be elucidated using whole cells treated with fosfomycin, a cell wall biosynthesis inhibitor, as the spectra shown a clear reduction in peptidoglycan composition. This was in contrast to cells treated with chloramphenicol, a protein synthesis inhibitor, which contained a higher percentage of peptidoglycan but a reduction in the cytoplasmic protein content211. Finally, for further pinpointing the MOA of peptides 73 and 77, additional methods could be used. If the peptides inhibit protein synthesis then 13C and 15N CPMAS NMR can be used to detect intact whole cell NMR of ribosomes and possibly the interaction of ribosomes with peptide 73 and 77271. AMPs such as indolicidin, an arginine and tryptophan rich peptide, target other intracellular components such DNA filamentation in E. coli cells and has the ability to bind DNA and membranes296,297. DNA binding experiments of peptide 73 and 77 could be performed by isothermal titration calorimetry (ITC) or NMR spectroscopy to determine the binding affinity. The expression system developed in Chapter 5 could be used to produce isotopically labeled 73 and NMR experiments could be used, for instance, to measure the distance between the 15N in the peptide and 31P in the DNA (e.g. using REDOR) and thus provide structural insight into the interactions involved. Such methods would work if the binding interactions were strong211,298,299.  145   Preliminary results in Chapter 4 show that peptide 73 and 77 also display superior antibiofilm activity compared to aurein 2.2. Bacteria in biofilms regulate stress response pathways which include signaling molecules such as Guanosine 5’-diphosphate 3’-diphosphate (ppGpp) and Guanosine 5’-triphosphate 3’-diphosphate (pppGpp). Peptide 73 and 77 have similar antibiofilm activity when compared to AMPs such as DJK-5 and IDR-1018 that have been shown to bind and degrade these signaling molecules207,300. It would be interesting to investigate the binding interaction of peptide 73 and 77 with ppGpp and/or pppGpp and with other nucleotide signaling molecules which are involved in the cells energy transformation such as cyclic adenine monophosphate (cAMP)301.   Furthermore, in vivo studies in Chapter 4 show that HPG-73c was not effective in the mouse abscess model, whereas the DSPE-PEG2000 encapsulated 73c showed excellent activity. These results indicate that the peptide needs to be released from the delivery vehicle to exhibit any antimicrobial activity in vivo. The development of an acid cleavable functional group such as an ester or specific enzymatic cleavable linker between peptide 73 and HPG would allow for release of the peptide at the site of infection (Figure 6.1). Many bacterial and host enzymes are released at the site of infection: for example, bacteria typically release gelatinase that are involved in tissue damage in host302,303. Host cells (including immune cells) also release enzymes such as the matrix metalloproteases (MMPs) which play an important role in various host responses such as combating bacterial infections, inflammatory response and tissue remodeling304.    146    Figure 6.1: Design of HPG-73 conjugate that would lead to the release of peptide 73 at the site of infection. The cleavage site could be an acid cleavable functional group such as an ester or specific enzyme cleavable. Figure generated using PowerPoint.     Recently, Qi et al. developed chitosan-AMP-PEG conjugates which assembled into nanoparticles and were cleaved by bacterial (S. aureus) gelatinase, MMP-2 and MMP-9 to expose the peptide on the chitosan to exhibit antimicrobial activity in vivo and in vitro305. Although the AMP was not released in this example, a similar strategy could be used to design HPG-73 conjugates that would lead to the release of the peptides at the site of infection. An enzymatically cleavable linker would require incorporation of additional amino acids in the peptide sequence which can be easily designed during the peptide synthesis, however, it should be noted that peptide 73 was derived from aurein 2.2 which requires the N-terminus for activity113,138,139,215, therefore, additional amino acids should most likely be incorporated at the  C-terminus.   Finally, the data from Chapter 4 indicated that only 60-70% of peptide 73c was encapsulated in DSPE-PEG2000 micelles. Future studies should ensure that the DSPE-PEG2000 concentration in increased or a different micelle forming agent could be used that will allow better encapsulation efficiency (close to 100% of the peptide being encapsulated) before the in vivo experiments. The PEG in DSPE-PEG comes in various molecular weights (2000, 3000 and 5000) and consequently different critical micellar concentrations306. The effect of the PEG chain length on encapsulation efficiency of the peptide should be investigated. Moreover, DSPE-PEG2000 has been FDA approved for the delivery of Doxil® (Doxorubicin), an anticancer drug307, however the formulation was composed of liposomes instead of micelles. Liposomal  147   (DSPE-PEG based) encapsulation of LL37 has also lead to improved toxicity of the peptides in vitro162; therefore it will be useful to encapsulate peptide 73c or D-73 using DSPE-PEG based  liposomes in the future to determine whether this would increase the in vivo efficacy.  Overall, the in vitro studies suggested above will further our understanding on the antimicrobial and antibiofilm mechanism of action of peptide 73 and 77, whereas the in vivo studies will give insights into the factors that are important for development of AMPs as drugs. 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Pharmaceutics 2017, 9 (4), 12.                                           168   Appendix A   A.1 HPLC trace of Aurein 2.2∆3-cys  Figure A.1: HPLC purification of the Aurein 2.2∆3cys. Peptide elutes between 55-57mins.   A.2 Proton NMR of amine functionalized HPG         Figure A.2: Proton NMR (in D2O) of amine modified HPG. Protons from the CH2 group next to the amine show around 3.0ppm (A).  HPG-119K-amine-260212.001.001.1r.esp5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 169   A.3 Proton NMR of HPG-bifunctional linker PK-HPG-linker.001.001.1r.esp7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0  Figure A.3: Proton NMR (in deuterated DMSO) of HPG-bifunctional Linker. The Amide (NH) peak at 6.5ppm indicates the formation of an amide bond. In deuterated DMSO the protons from the hydoxyl groups of HPG can also be observed between 4.5-5.0ppm  A.4 UV spectra of the HPG-bifunctional linker  Figure A.4: UV-Vis of HPG-Bifunctional linker. Amine modified HPG does not absorb light in the UV-Vis region. However, once conjugated to the bifunctional linker it absorbs between 250-280 nm. Concentration of both the compounds was constant at 1 mg/mL.  170   A.5 HPLC purification of the conjugate  Figure A.5: Size exclusion HPLC purification of the bioconjugates. HPH-Aurein 2.2∆3-cys elutes between 10-12min whereas the unreacted  peptide elutes around 18-20min.  A.6 Proton NMR of HPG-Aurein 2.2∆3-cys     Figure A.6: Proton NMR (in D2O) of HPG-Aurein 2.2∆3-cys. The peak that eluted around 10-12minutes in the HPLC trace (figure A.5) was used for Proton NMR experiment. Additional peaks from the peptides can be observed between 0-5.0 ppm.   Amut2Cys_HPG.014.001.1r.esp5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 171   A.7 Size exclusion chromatography of conjugates  Figure A.7: Size exclusion chromatographs of HPG-Aurein 2.2∆3-cys (Mn = 56,000) and HPG-Bifunctional linker (Mn = 44,000) at flow rate 0.8 mL/min at 25ºC.  A.8 CD spectroscopy of Aurein 2.2∆3-cys  Figure A.8: CD spectra of the Aurein 2.2∆3-cys in and trifluroethanol (TFE) and lipids (POPC/POPG).    172   A.9 CD spectroscopy of the conjugates  Figure A.9: CD spectra of the HPG-Aurein 2.2∆3-cys in PBS buffer, Trifluroethanol (TFE) and lipids (POPC/POPG). In the presence of TFE and lipids the peptides conjugated to the polymer is still able to adopt a alpha helical structure.   A.10 Platelet aggregation by the bioconjugates  Figure A.10: Platelet aggregation by the bioconjugates. Aggregation was measured by measuring absorbance at 595 nm. Adjusted Platelet rich plasma (PRP) was incubated with peptides or conjugates and the absorbance was measured for 20 minutes. As aggregation increases the absorbance decreases as more light can pass through. The rate of aggregation  173   can be measured by calculating the slope during the first 5 minutes. The end point (total amount of platelet aggregated) can be calculated by plateau phase (last 5 minutes). 5 µM ADP was used as a positive control and PBS buffer as normal control. The absorbance of adjusted PRP and PPP is used as theoretical 100% aggregation and no aggregation respectively for calculation of percent aggregation.                      174   Appendix B   B.1 Proton NMR of HPG-phthalimide   Figure B.1: 1H NMR (in 100% DMSO-d6) of phthalimide modified 22k HPG. Protons from the phthalimide group appear around 7.5-8 ppm.   B.2 Proton NMR of HPG-Amine  Figure B. 2: 1H NMR (in 100% DMSO-d6) of 22k HPG-NH2 after deprotection of the phthalimide.    175   B.3 Proton NMR of HPG maleimide  Figure B.3: 1H NMR (in 100% DMSO-d6) of 22k HPG maleimide.   B.4 Proton NMR of HPG 77c  Figure B.4: 1H NMR (in 100% DMSO- d6) of 22k HPG 77c.   PK_HPG_MA_2.001 analysed.esp8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5Chemical Shift (ppm)0.0050.0100.0150.020Normalized IntensityD M S OW ater7.007.017.9422K-77C.001.esp10 9 8 7 6 5 4 3 2 1 0Chemical Shift (ppm)-0.0006-0.0005-0.0004-0.0003-0.0002-0.000100.00010.00020.00030.00040.00050.00060.00070.0008Normalized Intensity 176   B.5 Proton NMR of PEG 77c 5k PEG 77c.001.esp6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0Chemical Shift (ppm)0.00050.00100.00150.00200.00250.00300.00350.00400.00450.00500.00550.00600.00650.0070Normalized Intensity Figure B. 5: 1H NMR (in 100% D2O) of 5k mPEG 77c  B.6 Bacteria killing and RBC lysis in whole blood/bacteria mixture  Figure B.6a: Bacterial colony forming units per milliliters (CFU/mL) in whole blood in presence of peptide/conjugates. No bacterial counts were detected for the 77c and 22k 77c (125 µg/mL). In the combined blood and bacteria experiment, bacteria (S. aureus C622) was diluted to 2x106 CFU/mL in LB. Forty five microliters of 50% citrated blood (1:1  177   whole blood:PBS) was added and to 45 μL of the diluted bacterial suspension followed by 10 μL of peptide 77c or 22k 77c (125 µg/mL final concentration) and  incubated at 37 C for 1 h. The incubation mixture (5 μL) was serial diluted in LB and plated on LB agar, followed by incubation for 18-24 hours at 37 C for CFU determination. Percent lysis for the mixture was measured by the Drabkin's method as described in the main text.      Figure B.6b: Percent RBC lysis in the whole blood in presence of bacteria and peptide/conjugate. Combined RBC lysis in the presence of bacterial cells. Peptide/conjugates (125 ug/mL) were added to the 50% citrated RBC/bacterial mixture (1:1). Lysis was determined by Drabkin's method.   178   B.7 Bacteria killing in serum/bacteria mixture  Figure B.7: The ability for the peptide and conjugates to kill bacteria in serum. Both 77c (125 µg/mL) and 22K 77c (125 µg/mL) cleared bacteria in serum efficiently. Bacteria killing assay was performed the same way as the combined blood lysis study. Instead of adding whole blood, 45 μL of serum was added to forty five microliters of the diluted bacteria. Mixture was incubated for 1 h at 37 C and five microliters of the incubation mixture was serial diluted in LB and plated on LB agar to determine the CFU count.  B.8 Maldi of Matrix  Figure B.8: MALDI of matrix.  179    B.9 aPTT analaysis of 77c and the conjugates  Figure B.9: Influence of free peptide and HPG peptide conjugates on activated Partial Thromboplastin Time (aPTT). Results are courtesy of Dr. Manu Thomas Kalathottukaren.  B.10 Thromboelastometry of conjugates and peptides   180   Figure B.10: Typical thromboelastometry of the HEPES-buffered saline (HBS) (red), 22K 77c (purple) and 77c (green), all at 125 µg/mL.  B.11 aPTT analaysis of 77c and the conjugates   Figure B.11: Whole blood clotting time by Thromboelastometry of the 22k 77c and 77c, all at 125 μg/mL (n=3). No significant (ns) difference was observed in the clotting time of 22k 77c and 77c compared to buffer.              181    Appendix C   C.1 Toxicity and precipitation of D-peptides at various concentrations in saline.   Figure C. 1: CD-1 mice were injected with 50 ul of different peptide concentrations on the left and right side of the dorsum. The skin inspected three days post injection. Upper panel: The backside of the mice. Lower panel: Skin flap to show the injection side under the skin. The red arrows point to peptide precipitation. Results are courtesy of Dr. Daniel Pletzer from the Hancock lab (UBC).          182     C.2 Toxicity and precipitation of D-peptides at various concentrations in DSPE-PEG2000  Figure C.2: CD-1 mice were injected with 50 ul of different peptide concentrations on the left and right side of the dorsum. The skin inspected three days post injection. No visible peptide/conjugate precipitation at various concentration in DSPE-PEG2000. Results are courtesy of Dr. Daniel Pletzer from the Hancock lab (UBC).   183   C.3 Average hydrodynamic size of peptide 73c in water and DSPE-PEG2000.  Figure C.3: Average hydrodynamic size and the intensity percent of peptide 73c in water and DSPE-PEG2000.               02040600.1 1 10 100 1000 10000Intensity (Percent)Size (d.nm)Size Distribution by IntensityRecord 5: 73C 3MG/ML IN DESPE PEG 1 Record 14: 73C 3MG/ML IN w ater 1Record 49: peg 184   Appendix D   D.1 MS/MS spectra of aurein 2.2  and diesterified aurein 2.2    Figure D.1: MS/MS spectra of aurein 2.2 (top) and diesterified aurein 2.2 (bottom). The corresponding b ions for the C-terminus (L16) and aspartic acid (D4) residues are denoted and with an asterisk (*) to indicate an increase of +15 Da. 

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