Pharmaceutical Sciences, Faculty of
DSpace
UBCV
Bergamo, Marta
Doctor of Philosophy - PhD
University of British Columbia
Macroaggregated albumin microparticles (MAA) are particles routinely used in the clinic as imaging agents for the diagnosis of pulmonary embolism (PE) due to their ability to passively target the lungs. Since albumin is biocompatible and biodegradable, MAA represents an attractive carrier for the delivery of drugs to the lungs. Particularly, there is still a great need for lung targeting delivery systems of fibrinolysis-enhancing drugs for the treatment of PE. While these drugs are effective in resolving blood clots entrapped in the pulmonary vasculature, they are administered intravenously resulting in severe side effects limiting their clinical use. Therefore, it is critical to develop formulations for their targeted delivery to reduce systemic exposure, thus improving their safety profile. The aim of this project was to formulate the anticoagulant tiplaxtinin with albumin microparticles for delivery to the lungs. MAA loaded with tiplaxtinin (MATi particles) were prepared by one-pot synthesis of the drug with bovine serum albumin (BSA) at controlled conditions. Particles were characterized for their size, drug loading, stability, and cytotoxicity. Acute toxicity of the particles was investigated in vivo by intravenous administration of MATi particles prepared with radiolabeled BSA (¹¹¹In-MATi) to healthy mice. Dual radiolabeling of the MATi particles was obtained by labeling tiplaxtinin with an additional radioisotope (³H). This allowed us to investigate the biodistribution of the albumin particles and the drug simultaneously and compare it to the unformulated tiplaxtinin following intravenous administration into healthy mice. MATi particles had a size distribution comparable to clinically used MAA, averaged a drug loading of 26%, showed good stability, and signs of toxicity observed were affected by the drug itself. In vivo, 60% of injected dose accumulated in the lungs upon injection and showed no overt signs of toxicity. When delivered with the particles, up to a 12-fold increase of tiplaxtinin was observed in the lungs compared to the unformulated drug. MATi particles allowed to overcome the solubility issue of tiplaxtinin and thereby could be administered at a higher concentration compared to unformulated drug. Future work involves the investigations of MATi particles in a disease model.
MACROAGGREGATED ALBUMIN PARTICLES AS INTRAVENOUS DRUG FORMULATION FOR LUNG DELIVERY by Marta Bergamo M.Sc., The University of Copenhagen, 2018 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Pharmaceutical Sciences) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2024 © Marta Bergamo, 2024 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Macroaggregated albumin particles as intravenous drug formulation for lung delivery submitted by Marta Bergamo in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Pharmaceutical Sciences Examining Committee: Prof. Urs Häfeli, Faculty of Pharmaceutical Sciences, UBC Supervisor Prof. Marcel Bally, Faculty of Medicine, UBCch University Examiner Prof. Charles Haynes, Michael Smith Laboratories, UBC University Examiner Prof. Marianna Foldvari, School of Pharmacy, University of Waterloo External Examiner Additional Supervisory Committee Members: Prof. Abby Collier, Faculty of Pharmaceutical Sciences, UBC Supervisory Committee Member Prof. Hanne Mørck Nielsen, Department of Pharmacy, University of Copenhagen Supervisory Committee Member Prof. Christian Kastrup, Michael Smith Laboratories, UBC Supervisory Committee Member iii Abstract Macroaggregated albumin microparticles (MAA) are particles routinely used in the clinic as imaging agents for the diagnosis of pulmonary embolism (PE) due to their ability to passively target the lungs. Since albumin is biocompatible and biodegradable, MAA represents an attractive carrier for the delivery of drugs to the lungs. Particularly, there is still a great need for lung targeting delivery systems of fibrinolysis-enhancing drugs for the treatment of PE. While these drugs are effective in resolving blood clots entrapped in the pulmonary vasculature, they are administered intravenously resulting in severe side effects limiting their clinical use. Therefore, it is critical to develop formulations for their targeted delivery to reduce systemic exposure, thus improving their safety profile. The aim of this project was to formulate the anticoagulant tiplaxtinin with albumin microparticles for delivery to the lungs. MAA loaded with tiplaxtinin (MATi particles) were prepared by one-pot synthesis of the drug with bovine serum albumin (BSA) at controlled conditions. Particles were characterized for their size, drug loading, stability, and cytotoxicity. Acute toxicity of the particles was investigated in vivo by intravenous administration of MATi particles prepared with radiolabeled BSA (111In-MATi) to healthy mice. Dual radiolabeling of the MATi particles was obtained by labeling tiplaxtinin with an additional radioisotope (3H). This allowed us to investigate the biodistribution of the albumin particles and the drug simultaneously and compare it to the unformulated tiplaxtinin following intravenous administration into healthy mice. MATi particles had a size distribution comparable to clinically used MAA, averaged a drug loading of 26%, showed good stability, and signs of toxicity observed were affected by the drug itself. In vivo, 60% of injected dose accumulated in the lungs upon injection and showed no overt signs of toxicity. When delivered with the particles, up to a 12-fold increase of tiplaxtinin was iv observed in the lungs compared to the unformulated drug. MATi particles allowed to overcome the solubility issue of tiplaxtinin and thereby could be administered at a higher concentration compared to unformulated drug. Future work involves the investigations of MATi particles in a disease model. v Lay Summary Albumin represents the ideal drug carrier due to its lack of toxicity and ease of degradation. In hospitals, albumin aggregates in the micrometer size are routinely used for the diagnosis of pulmonary embolism due to their ability of passively accumulate in the lungs. In the more severe cases of pulmonary embolism, fibrinolytics, a class of drugs known as ‘clot busters’, are administered intravenously to patients to dissolve blood clots present in their lungs. However, this treatment can cause adverse effects, such as hemorrhage, due to the patient’s systemic exposure to the drug. In this thesis we investigated the use of albumin aggregates as drug carriers for the targeted delivery of an anticoagulant drug (tiplaxtinin) to the lungs. With this formulation, up to a 12-fold increase of tiplaxtinin was observed in the lungs compared to the free drug, as well as reducing delivery to off-target organs. vi Preface This thesis has been conducted at the University of British Columbia (Vancouver) in the research group of Dr. Urs Häfeli at the Faculty of Pharmaceutical Sciences. The research was made possible by a grant from the Novo Nordisk Foundation funding the Center for Biopharmaceuticals and Biobarriers in Drug Delivery (BioDelivery, Grand Challenge Program; NNF16OC0021948). The Lundbeck Foundation of Denmark (UBC-SUND Lundbeck Foundation Professorship to Dr. Urs Häfeli; project number 2014-4176) is also acknowledged. Part of the thesis is going to be published as a paper: Bergamo M., Esposito T., Morales A., Rodriguez-Rodriguez C., Bobak T., Saatchi K., Häfeli U. O. (2024). Tiplaxtinin-loaded macroaggregated albumin particles as lung-targeting drug delivery system for the treatment of pulmonary embolism (in preparation). Chapter 1: The idealization and writing of this chapter were conducted by myself and reviewed by Dr. Urs Häfeli. Chapter 2 and Supplementary Data in Appendix A: Conceptualization and experimental design were developed by myself in discussion with Drs. Urs Häfeli, Tullio Esposito, and Katayoun Saatchi. Experimental work was performed by myself, with the contribution of Thomas Bobak for the acquisition of SEM images, and Aurelio Morales for the FT-IR measurements and analysis. Data analysis and visualization was performed by myself. Chapter 3 and Supplementary Data in Appendix B: Conceptualization and methodology for the material in this chapter were prepared by myself in discussion with Drs. Urs Häfeli, Tullio Esposito, and Katayoun Saatchi. Experimental work was performed by myself with the contribution of Dr. Katayoun Saatchi for the radiolabeling of bovine serum albumin. Aurelio Morales cultured the cells and performed the cytotoxicity assays. Dr. Cristina Rodriguez-Rodriguez, Maryam Osooly, and I carried out the dose study in vivo. Data analysis and visualization was performed by myself. Chapter 4 and Supplementary Data in Appendix C: Conceptualization and experimental design were developed by myself in discussion with Drs. Urs Häfeli, Tullio Esposito, and Katayoun Saatchi. Experiments were carried out by myself with the assistance of Dr. Katayoun Saatchi for the radiolabeling of the MATi particles, and Dr. Cristina Rodriguez-Rodriguez, Maryam Osooly, and Aurelio Morales for the biodistribution study. Danielle Hanke and Petar Iliev assisted with the HPLC-MS experiment and analysis. Data analysis and visualization was performed by myself. vii Chapter 5: Writing was conducted by myself and reviewed by Dr. Urs Häfeli. Ethic Statement: The animal experiments conducted for this thesis were performed under the protocol A20-0132 approved by the Animal Care Committee (ACC) of the University of British Columbia. Human blood obtained for the experiments in this project was donated by healthy volunteers under the protocol H22-03777 approved by the Clinical Research Ethics Board (CREB) at the University of British Columbia. viii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ....................................................................................................................... viii List of Tables ............................................................................................................................... xii List of Figures ............................................................................................................................. xiii List of Abbreviations ...................................................................................................................xv Acknowledgements ................................................................................................................... xvii Dedication ................................................................................................................................... xix Chapter 1: Introduction ................................................................................................................1 1.1 The Importance of Drug Delivery Systems .................................................................... 1 1.2 Albumin – The Ideal Carrier ........................................................................................... 2 1.2.1 Albumin Nanoparticles ............................................................................................... 4 1.2.2 Albumin Microparticles .............................................................................................. 5 1.2.3 99mTc-MAA for Diagnostic Imaging of Pulmonary Embolism .................................. 7 1.3 Management of Pulmonary Embolism ........................................................................... 8 1.4 Project Overview and Significance ................................................................................. 9 Chapter 2: MATi Particles Preparation and Characterization ..............................................11 2.1 Synopsis ........................................................................................................................ 11 2.2 Background ................................................................................................................... 12 2.3 Results and Discussion ................................................................................................. 18 ix 2.3.1 MATi Particles Formulation ..................................................................................... 18 2.3.2 Drug Loading ............................................................................................................ 22 2.3.3 MATi Particles Stability ........................................................................................... 24 2.4 Chapter Summary ......................................................................................................... 27 2.5 Experimental Section .................................................................................................... 28 2.5.1 Materials and Reagents ............................................................................................. 28 2.5.2 MATi Particles Preparation ...................................................................................... 28 2.5.3 Microscopy and SEM images ................................................................................... 29 2.5.4 Drug Loading Assessment ........................................................................................ 29 2.5.5 FT-IR Spectra for Qualitative Drug Loading ............................................................ 30 2.5.6 MATi Particles Storage Stability .............................................................................. 31 2.5.7 MATi Particles Stability at 37° C ............................................................................. 31 2.5.8 Data Visualization and Statistical Analysis .............................................................. 32 Chapter 3: Toxicity Profile of MATi Particles ..........................................................................33 3.1 Synopsis ........................................................................................................................ 33 3.2 Background ................................................................................................................... 33 3.3 Results and Discussion ................................................................................................. 34 3.3.1 MATi Particles Cytotoxicity ..................................................................................... 34 3.3.2 In Vivo Dose Study ................................................................................................... 39 3.4 Chapter Summary ......................................................................................................... 43 3.5 Experimental Section .................................................................................................... 44 3.5.1 Materials and Reagents ............................................................................................. 44 3.5.2 MTT Assay ............................................................................................................... 44 x 3.5.3 Cell Death with YOYOTM-1 Dye .............................................................................. 45 3.5.4 111In-MATi Preparation and Characterization .......................................................... 46 3.5.5 Animals ..................................................................................................................... 48 3.5.6 Dose Study ................................................................................................................ 48 3.5.7 Data Visualization and Analysis ............................................................................... 49 Chapter 4: Biodistribution of the MATi Particles in a Healthy Murine Model ....................50 4.1 Synopsis ........................................................................................................................ 50 4.2 Background ................................................................................................................... 50 4.3 Results and Discussion ................................................................................................. 55 4.3.1 Dual Labeling of MATi Particles and Dose Assessment.......................................... 55 4.3.2 Biodistribution of 111In-MATi 3H-TPLN in a Healthy Murine Model ..................... 59 4.3.3 Activity of Tiplaxtinin for Blood Clot Resolution .................................................... 66 4.4 Chapter Summary ......................................................................................................... 69 4.5 Experimental Section .................................................................................................... 70 4.5.1 Materials and Reagents ............................................................................................. 70 4.5.2 Tiplaxtinin Solubility Assessment ............................................................................ 70 4.5.3 Radiolabeling Stability.............................................................................................. 71 4.5.4 Preparation of 111In-MATi [3H-TPLN] and Characterization ................................... 72 4.5.5 Animals ..................................................................................................................... 73 4.5.6 Biodistribution Study in Healthy Murine Model ...................................................... 73 4.5.7 Tiplaxtinin Thermostability ...................................................................................... 74 4.5.8 Tiplaxtinin Activity ................................................................................................... 75 4.5.9 Data Visualization and Statistical Analysis .............................................................. 76 xi Chapter 5: Closing Thoughts ......................................................................................................77 5.1 Conclusion .................................................................................................................... 77 5.2 Project Limitations ........................................................................................................ 78 5.3 Future Work and Outlook ............................................................................................. 79 References .....................................................................................................................................82 Appendices ....................................................................................................................................93 Appendix A Materials in Support of Chapter 2 ........................................................................ 93 A.1 MATi Particles Formulation ..................................................................................... 93 A.2 Drug Loading ............................................................................................................ 94 A.3 MATi Particles Stability ........................................................................................... 95 Appendix B Material in Support of Chapter 3 .......................................................................... 98 B.1 Dose Study ................................................................................................................ 98 Appendix C Material in Support of Chapter 4 .......................................................................... 98 C.1 Tiplaxtinin Solubility ................................................................................................ 98 C.2 Biodistribution Study ................................................................................................ 99 C.3 Thermostability of Tiplaxtinin ................................................................................ 101 xii List of Tables Table 2.1: Summary of FDA approved PAs ................................................................................. 13 Table 5.1: Dose study experimental details .................................................................................. 98 xiii List of Figures Chapter 1: Figure 1.1: Albumin as a drug carrier. ............................................................................................ 4 Chapter 2: Figure 2.1: Tiplaxtinin .................................................................................................................. 18 Figure 2.2: Morphology and shape of MATi particles and commercial MAA ............................ 20 Figure 2.3: Particles size distribution and inter-batch variability ................................................. 22 Figure 2.4: Quantitative and qualitative loading of tiplaxtinin. .................................................... 24 Figure 2.5: MATi particles stability at storage conditions ........................................................... 25 Figure 2.6: MATi particles stability at 37° C ............................................................................... 27 Chapter 3: Figure 3.1: Cytotoxicity study ...................................................................................................... 37 Figure 3.2: Effect of the drug and the carrier on the overall toxicity of MATi particles ............. 39 Figure 3.3: Radiolabeling of BSA and preparation of 111In-MATi particles ................................ 41 Figure 3.4: Biodistribution of MATi particles in healthy mice. ................................................... 43 Chapter 4: Figure 4.1: Scheme of radiolabeling methods .............................................................................. 52 Figure 4.2: Solubility of tiplaxtinin in aqueous-based media ....................................................... 57 Figure 4.3: Radiolabeling of tiplaxtinin and BSA ........................................................................ 58 Figure 4.4: Radiolabeling stability of 111In-DTPA complex ........................................................ 59 Figure 4.5: Experimental design of the biodistribution study....................................................... 60 Figure 4.6: Biodistribution of 111In-MATi[3H-TPLN] and free 3H-TPLN in lungs and liver ...... 62 Figure 4.7: Biodistribution of 111In-MATi[3H-TPLN] and free 3H-TPLN ................................... 64 xiv Figure 4.8: Distribution of the MATi particles in the left and right lung ..................................... 65 Figure 4.9: Thermostability of tiplaxtinin ..................................................................................... 67 Figure 4.10: Lytic effect of tiplaxtinin .......................................................................................... 69 Appendices: Supplementary Figure A.1.1: Particles size distribution .............................................................. 93 Supplementary Figure A.1.2: Particles size distribution and inter-batch variability .................... 93 Supplementary Figure A.2.1: MATi particles color change ......................................................... 94 Supplementary Figure A.2.2: Purification of tiplaxtinin from the MATi particles supernatant ... 94 Supplementary Figure A.2.3: FT-IR spectra of TPLN, blank MATi, and MATi particles .......... 95 Supplementary Figure A.3.1: MATi and MAA stability at storage conditions ............................ 95 Supplementary Figure A.3.2: MATi stability at 37° C ................................................................. 96 Supplementary Figure A.3.3: MAA stability at 37° C ................................................................. 97 Supplementary Figure C.1.1: Tiplaxtinin solubility in different media. ....................................... 98 Supplementary Figure C.2.1: Activity concentration (%ID/g) of free 3H-TPLN and 111In-MATi[3H-TPLN] in lungs and liver ............................................................................................. 99 Supplementary Figure C.2.2: Biodistribution of 111In-MATi (%ID/g)......................................... 99 Supplementary Figure C.2.3: Biodistribution of free 3H-TPLN (%ID/g) .................................. 100 Supplementary Figure C.2.4: Biodistribution of MATi[3H-TPLN] (%ID/g) ............................. 100 Supplementary Figure C.3.1: Mass spectrometry fragmentation of tiplaxtinin .......................... 101 xv List of Abbreviations ° C Degree (Celsius) %ID/g Percentage of injected dose per organ %ID/organ Percentage of injected dose per gram 111In Indium-111 3H Tritium 68Ga Gallium-68 99mTc Metastable Technetium-99 AUC Area under the curve BSA Bovine serum albumin MBq Mega-Becquerel CO2 Carbon dioxide COVID-19 Coronavirus disease 2019 Da Dalton DDSs Drug delivery systems DLS Dynamic light scattering DMSO Dimethyl sulfoxide DTPA Diethylenetriaminepentaacetic acid EDTA Ethylenediaminetetraacetic acid FBS Fetal bovine serum FT-IR Fourier-transform infrared spectroscopy GLP-1 Glucagon-like peptide 1 GSH Glutathione reduced HEPES 4-2(-Hydroxyethyl)-1-piperazineethanesulfonic acid HPLC-MS High performance liquid chromatography mass spectroscopy HUVEC Human umbilical vein endothelial cells ID Injected dose i.v. Intravenous k Kilo (103) keV Kiloelectron volt M Molar MAA Macroaggregated albumin MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide MW Molecular weight MWCO Molecular weight cut off NMP N-Methyl-2-pyrrolidone OD Optical density PAs Plasminogen activators PAGE Polyacrylamide gel electrophoresis PAI-1 Plasminogen activator inhibitor 1 PBS Phosphate-buffered saline PE Pulmonary embolism xvi PET Positron emission tomography p.i. Post injection RCC Radiochemical conversion RCY Radiochemical yield RES Reticuloendothelial system Rf Retention factor SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2 SDS Sodium dodecyl sulfate SEM Scanning electron microscope SPECT Single photon emission computed tomography t 1/2 Half-life TLC Thin layer chromatography TPLN Tiplaxtinin UV-Vis Ultraviolet-visible xvii Acknowledgements As John Donne famously wrote ‘no man is an island’, and I believe this holds very true in science as well where collaborations, contributions, and support are essential for the progression of knowledge. Therefore, there are quite few people that I would like to acknowledge for helping me during these years. Firstly, I would like to thank my supervisor, Dr. Urs Häfeli, for welcoming me into your lab not once but twice, for your support over all these years, for allowing me scientific creativity and let me pursue my scientific interests, I greatly appreciate it. I am also grateful to Drs. Abby Collier, Hanne Mørck Nielsen, and Ujendra Kumar for the continuous support and encouragement. I am equally grateful to the rest of my supervisory committee (Drs. Christian Kastrup and Fergal Donnellan) for their time and precious feedback over these years. I would like to also thank Dr. Thomas Chang for the incredible amount of support provided from the very beginning, and for always making time for a chat. In the same way I am very grateful to Dr. Katayoun Saatchi for having your door always open for me, for the numerous chats, for consistently checking in on me to make sure I was doing ok, and for all the help received both within and outside the lab. I would like to thank also Dr. Tullio Esposito for helping shaping this project, and the continuous feedback and mentoring. I initially got to know you as my co-supervisor, you slowly became a close friend that I could always count on. Special thanks to Aurelio Morales for the incredible help provided in this project and for our brainstorming sessions. Thank you also for all the chats, the fun times, adventures, and the hours spent discussing the most random topics. I would like to thank also Dr. Cristina Rodriguez-Rodriguez and Maryam Osooly for their assistance during the in vivo studies. I will be forever in awe with your organizational skills that make even the busiest days go smooth. It has been a great pleasure working with you, making even the stressful days fun. I would like to acknowledge Thomas Bobak as well for his help acquiring the SEM images, as well as the coffee breaks and the good times in Vancouver. Many thanks to Zeynab, Gokce, Cristina, and Tanya for all the laughs inside and outside the lab, the chats, and the endless support since my very first day in the Häfeli lab. Overall, I would like to thank all the past and present lab members for creating such a friendly and welcoming environment. Thanks to my officemates - Lukas, Sahithi, and Lenni - for making the crazy PhD days survivable, even fun, and always be ready to help each other out. I am also very grateful to all my other grad student fellows and Vancouver xviii friends – Danielle, Pepy, Lukas, Mike, Thomas, Roli, Riley, Jonas, and Ravi – for making the faculty lively and fun, for helping me in so many different ways, for the trips, the laughs, and memorable moments. I am also very thankful to all the Tavernians (Monica, Lennart, Naya, Adria, Michael, Alex, Danielle, Riley, and Lukas) for making our place a familiar and fun place to live in. Special thanks to Moni and Lenni, the original Tavernians, for making La Taverna home. Lenni, thank you for being my roommate, labmate, officemate, climbing buddy, fellow LotR lover, and great friend. Life in Vancouver would have not been the same without you. I would like to thank also the friends and family back home that kept supporting me from the other side of the world. In particular, Silvia, Giulia, and Gaia. There are no words to explain how much I cherish our friendship and the rare but perfect moments together. You inspire me constantly. Special thanks to Silvia for being there for me every step of the way and doing life together despite the distance, and Lucia for being by my side since we took our very first steps and continuing doing so even now that we have learnt to run, fall, jump, and do cartwheels. Last but surely not least, I am extremely grateful to my family. Thank you for supporting all my decisions without hesitation (except for the tattoo), even if it meant making sacrifices or not seeing me for years. Thank you for your never-ending support, encouragement, and love. Thank you for being my safe port. xix Dedication To my family. As we make mistakes, we create an archive of solutions. - Sara Lando (photographer)1 Chapter 1: Introduction 1.1 The Importance of Drug Delivery Systems Drug delivery systems (DDSs) are at the interface between the drug and the body’s physical and chemical barriers, thus contributing to the enhancement of the drug’s efficacy and safety by regulating its location and release in the body [1, 2]. The recent marketing of the COVID-19 vaccines from Pfizer-BioNTech and Moderna, reminded us of the essential role played by DDSs for the successful translation of therapies from bench to bed [3]. Indeed, over the years, drugs with good therapeutic potential have been developed, but often their less-than-ideal pharmacokinetics limited their use. In an interesting review from 2021, Vargason et al. identified DDSs as one of the three paradigms of drug delivery, together with drug and microenvironmental modifications [2]. These paradigms were assessed to be applicable to all classes of drugs, e.g., small molecules, proteins, and nucleic acids [2]. DDSs were described as the conjunction of the other two paradigms as they can integrate drug and microenvironment modifications in one system [2]. An example of this is represented by Rybelsus, the oral formulation of semaglutide by Novo Nordisk, which combines chemical modifications of the protein glucagon-like peptide 1 (GLP-1) to improve its proteolytic stability, with the presence of a permeation enhancer to improve its absorption [2, 4]. DDSs also allow to modulate drugs’ pharmacokinetics often by providing targeted delivery, improve half-life, or controlled release [2]. Therefore, it becomes apparent that DDSs play a crucial role in the development of successful therapies as they allow to repurpose and/or enhance drugs’ efficacy. DDSs are also critical for delivering drugs via less invasive routes of administration, e.g., transdermal, pulmonary, and oral [5]. This opens the doors to a wide range of possibilities that can lead to therapies with improve patient compliance and lower off-target toxicity. Therefore, it is not 2 surprising that over the last decades there has been a surge in the investigation of DDSs, which have been prepared with a wide array of materials ranging from polymers to liposomes and live cells, thus making the possible designs for DDSs innumerable [1, 5]. However, in this scenario of vast possibilities, I was struck by something Robert Langer, considered one of the giants in the drug formulation world, said during his keynote talk at a conference focused on nanomedicine. During his talk, he mentioned two important things: 1) formulations should be pharmacologically elegant, meaning that they should be simple and robust; 2) focus should be on platforms, i.e., formulations that are compatible with multiple drugs, thus can be employed for treatment of different diseases [6]. The initial idea for the formulation investigated here was based on these principles. For this reason, our starting point is a well-known drug carrier: albumin. 1.2 Albumin – The Ideal Carrier One of the cornerstones of drug delivery is represented by albumin. Albumin has a molecular weight of 66.4 kDa, it is mainly produced by the liver and has a biological half-life of ~19 days in humans [7]. In its structure there are multiple binding sites that allow the binding of various endogenous and exogenous molecules, e.g., bilirubin, ibuprofen [8, 9]. Furthermore, albumin is biodegradable and highly biocompatible, thus making it an ideal carrier for drug delivery. For this reason, different types of albumins, e.g., from human and bovine serum, have been broadly studied and employed to control drugs’ pharmacokinetics, prolong their half-lives, or provide targeted therapy [7, 10, 11]. The high versatility of albumin resulted in a wide array of drug delivery systems being investigated for different applications as well as routes of administration [8]. These albumin-based DDSs could be broadly categorized in: non-covalent association, 3 albumin conjugates, fusion proteins, nanoparticles, and microparticles (Fig. 1.1). In the first case, drugs can be engineered with a moiety that provides reversable binding to albumin, resulting in longer circulatory half-life and slower clearance of the drug [12]. A renowned example is represented by Novo Nordisk’s liraglutide, a GLP-1 analog designed with the addition of a fatty acid for non-covalent binding of albumin following administration, which resulted in a longer half-life of the drug, thus allowing for a once-a-day administration [13]. Concerning drug conjugates, they are prepared by exploiting the functional groups present on the side chains of the albumin’ amino acids, such as cysteine and lysine [7, 14]. Drugs are chemically linked to albumin via covalent bonds, and drug binding can be performed before administration or in situ, i.e., drugs bind to circulating albumin [12, 14]. Drug release can then be triggered by an external stimulus, e.g., by using linkers sensitive to environmental change [14]. For example, aldoxorubicin is a formulation that reached clinical trials and uses an acid labile linker to bind the anti-cancer drug doxorubicin with the free cysteine (Cys34) of circulating albumin [15]. The linker breaks and releases the drug at the tumor site due to the lower pH of the tumor environment. Conjugation of doxorubicin with albumin led to an improved safety profile of doxorubicin compared to the free drug [16]. Differently, the creation of fusion proteins requires DNA engineering to express the protein-based drugs together with albumin [7]. The drug is fused to either the N- or C-terminal of albumin, or to both ends creating a bivalent fusion protein [7]. Similarly to albumin conjugates, fusion proteins can be expressed with cleavable linkers for the release of the drug [11]. An advantage of fusion protein is the expression of the drug-albumin combination as a single identity [12]. However, careful consideration should be taken in its design as the drug might interfere with the correct folding of albumin [12]. A successful example of a fusion protein that reached the market is represented by Idelvion®, a recombinant 4 coagulation factor IX bound to recombinant human albumin for the treatment of hemophilia B. Expressing factor IX with albumin resulted in an increased half-life of the enzyme, which allowed for once-a-week administration regimen thus reducing the treatment burden compared to replacement therapy [17]. While these strategies proved valid for the control of drugs pharmacokinetics, for the scope of this thesis I will focus on albumin nano- and micro-particles. Figure 1.1: Albumin as a drug carrier Schematic representation of the five main categories of albumin-based drug delivery systems. Figure inspired from Rahimizadeh et al. (2020) [12] and prepared using BioRender.com. 1.2.1 Albumin Nanoparticles The great versatility of albumin translates to the preparation of nanoparticles as well, due to the protein’s high availability, low toxicity, biodegradability, and cost effectiveness [18]. Albumin nanoparticles can be prepared via different methods such as coacervation, nano-spray drying, and Nab-technology [10]. Due to the ability of 1) loading different kind of drugs and 2) modifying 5 the particles surface with targeting or therapeutic molecules, albumin nanoparticles have been investigated for several applications, ranging from cancer to bacterial infections and HIV-1 [10]. Employing albumin nanoparticles in various applications is possible also because of potential customization of the particles size, which can range between 10 to 200 nm [19]. The ability to prepare particles of different sizes allowed to study them for the administration via different routes as well [19]. So far, some of the most successful DDSs prepared with albumin nanoparticles, have the communal aspects of being able to improve solubility of hydrophobic drugs, which is often the case for small molecules, and the possibility of modifying the drugs pharmacokinetics to enhance their therapeutic effect. A famous example is represented by the successful albumin nanoparticles-based Abraxane®, approved by FDA in 2005 [10]. Prepared with the Nab-technology, Abraxane is composed by the hydrophobic chemotherapeutic drug paclitaxel bound to albumin nanoparticles with an average size of 130 nm [7]. Due to the low solubility of paclitaxel, the previous approved formulation - known as Taxol® - required to mix the drug with Cremophor EL (PEG-35 castor oil) and ethanol to dissolve it, and then dilute the solution with 0.9% sodium chloride (saline) or 5% dextrose solution for its administration [20]. However, the vehicle used causes nephron- and neuro-toxicity and it can cause hypersensitivity reactions, therefore pre-treatment with antihistamines and corticosteroids is performed [20]. In contrast, the formulation of paclitaxel with albumin resulted in good solubility in aqueous based solutions, avoiding the use of Cremophor EL and improving the overall safety profile of the drug. Higher doses can thus be administered and result in enhanced efficacy [10]. 1.2.2 Albumin Microparticles Similar to nanoparticles, albumin microparticles can provide a versatile platform for the formulation of various drugs, demonstrated by decades of research in the field. Indeed, already in 6 a review by Gupta et al. from 1989, the group presented an overview of albumin microparticles formulated with a multitude of drugs [21]. Their review stated that the first study performed in vivo to assess the biodistribution of drug loaded albumin microparticles dated back to 1977 [21]. Although research in this area is certainly not new, it was surprising to notice that it is very limited compared to the nanoparticles counterpart. Nevertheless, albumin microparticles still hold great potential due to the advantageous features of albumin described in the previous paragraphs. Albumin microspheres can range from 1-100 µm in size [22]. The average particle size determines their biodistribution in vivo following intravenous (i.v.) administration. Smaller particles (1- 3 µm) distribute in liver and spleen following clearance by the reticuloendothelial system (RES), and they can accumulate in solid tumors via the enhanced permeability and retention (EPR) effect due to the leaky vasculature of tumors [22]. The RES, as part of the innate immune system, physiologically serves to clear foreign bodies from the circulation to protect the organism, thus resulting in the clearance of nanoparticles and small microparticles as well [23]. Differently, bigger particles (> 15 µm) accumulate in the lungs due to mechanical filtering of the lungs’ capillary bed, thus blockade of the capillary vessels is solely due to the size of the microparticles [21, 22]. Albumin microparticles are commonly prepared as a water in oil emulsion, then the particles are stabilized either via chemical cross-linking or thermal aggregation [24, 25]. A wide array of albumin microparticles for therapeutic applications has been investigated. Similar to albumin nanoparticles, various microparticles loaded with anti-cancer drugs were prepared. For example, in 1977, Sugibayashi et al. prepared bovine serum albumin (BSA) microparticles loaded with 5-fluorouracil (0.4 – 1 µm size), which accumulated in the liver following i.v. administration, showing that the drug was retained for longer at the 7 targeted organ compared to the free drug [26]. In 1984, Fujimoto et al. investigated mitomycin C loaded BSA microparticles (average size 45 µm) for intra-arterial administration [27]. The bigger size of the microparticles caused the formation of a depot close to the tumor site, which provided sustained release of the drug [27]. Similarly, Goldberg et al. prepared doxorubicin-loaded albumin microparticles administered intra-arterially for the treatment of hepatic cancer [28]. More recent studies conducted by Chaurasiya et al. (2018) and Gülsu et al. (2022) investigated albumin microparticles loaded with paclitaxel and doxorubicin, respectively [29, 30]. Beyond cancer treatment, albumin microparticles were prepared for several other applications, including bacterial and fungal infections, as well as for various administration routes, such as inhalation and intramuscular injection [29, 31-35]. Even though a wide array of formulations for therapeutic applications have been studied over the last decades, the translation into marketed products was poor. Conversely, the diagnostic counterpart was surely more successful with macroaggregated albumin particles radiolabeled with 99mTc (99mTc-MAA) routinely used in the hospitals for diagnostic imaging [22]. 1.2.3 99mTc-MAA for Diagnostic Imaging of Pulmonary Embolism 99mTc-MAA are albumin microparticles commonly used for diagnostic imaging and are prepared by heat stabilization [36]. They are stored as lyophilized kits, and they can be easily prepared by addition of the radioisotope 99mTc [37, 38]. The latter is in a saline solution and once added to the kit allows the re-suspension of the MAA particles as well as a one-step labeling with the radioisotope. The 99mTc-MAA thus prepared have an average size between 10-90 µm [36]. Following intravenous administration, 99mTc-MAA distribute uniformly in the lungs capillary bed, where they become entrapped by mechanical filtration due to their size. This allows to assess the patients’ lung perfusion and evaluate whether the pulmonary vessels might be blocked 8 by an embolus, i.e., thrombus, often caused by a blood clot [39, 40]. When this condition occurs, the patient is diagnosed with pulmonary embolism (PE) [39]. These thrombi are typically formed in another part of the body, such as the deep veins in the legs, and once dislodged are carried to the lungs via the circulatory system where they are trapped causing symptoms like shortness of breath, chest pain, and tachycardia, or death in the worst cases [39, 41]. Furthermore, the recent global pandemic caused by the virus SARS-CoV-2 resulted in a steep increase of patients presenting signs of hypercoagulability, which leads to the formation of thrombi particularly in the pulmonary vasculature, therefore increasing the PE incidence in the population [42-46]. It was also observed that infection by SARS-CoV-2 can predispose to venous thromboembolism, a risk factor for PE [43, 46]. 1.3 Management of Pulmonary Embolism Once diagnosed, PE patients are stratified into massive, submassive and low-risk PE based on the severity of the disease [47]. In cases of low-risk PE, systemic administration of anticoagulants, e.g., heparins, is commonly performed; this treatment prevents the thrombus to increase in size, allowing the body’s natural fibrinolytic activity to dissolve the clot and reinstate the blood flow through the occluded vessel [41, 47]. Even though thrombotic events observed in COVID-19 patients have a different etiology compared to PE, there has been increasing evidence that prophylaxis and treatment with anticoagulants leads to increased survival, especially in critically ill patients [42, 45, 48]. For patients presenting with massive PE, a more aggressive therapy is needed. In these cases, patients are administered intravenously with thrombolytic agents, such as the recombinant enzyme tissue plasminogen activator (rtPA), which promote fibrinolysis, i.e., breakdown of the fibrin net that stabilizes the blood clot, and allows the blood to flow again [49, 50]. However, treatment with fibrinolytics can result in considerable adverse 9 effects, e.g., hemorrhage, therefore this therapy is performed in clinical settings with only a subset of strictly monitored patients [51-53]. Selected patients presenting submassive or massive PE with a high risk of bleeding that are not eligible for systemic thrombolysis, can be treated locally by either performing 1) catheter guided thrombolysis, where thrombolytic agents are delivered near the clot using a catheter, or 2) mechanical or surgical embolectomy, where the thrombus is surgically removed [50, 54]. However, these procedures are invasive, need to be performed in clinical settings, and require skilled surgeons. Therefore, it is critical to formulate thrombolytic agents for their local delivery, thus reducing systemic exposure of the patients to the drugs, and avoiding the need of interventional techniques such as surgery. 1.4 Project Overview and Significance As seen in the previous paragraphs, 99mTc-MAA particles are routinely used in the clinic as an imaging agent for the diagnosis of PE as they can passively target the lungs, thus showing uneven perfusion in relation to ventilation if emboli are present and obstruct the blood flow [55]. Furthermore, it was reported how DDSs based on albumin nanoparticles and microparticles allowed the loading of different kind of drugs, help overcome solubility problems, and decrease drugs’ toxicity by providing targeted treatment. Therefore, albumin microparticles could be exploited as a targeted drug delivery system for the potential treatment of pulmonary embolism, thus transforming a diagnostic agent into a therapeutic one. While drug loaded albumin microspheres have been widely studied over the last decades, they have been often investigated for 1) the treatment of other diseases involving lungs, e.g., cancer and bronchitis; 2) different applications, e.g., vaccines; or 3) different routes of administration, e.g., inhalation [56-58]. In this project, the MAA particles will be loaded with a fibrinolysis-enhancer (tiplaxtinin), for its delivery to the lungs following intravenous administration, 10 decreasing accumulation and toxicity in off-target organs. The parenteral route represents a more desirable route of administration compared to the more user-friendly inhalation, as it allows to deliver the drug directly into the blood vessel where the blood clot is present. Another drawback of inhalation is the mucus, which poses an additional physical barrier for the delivery of the drug to the site of action. The overarching aim of the project is to prepare a formulation that provides a safer profile than the current fibrinolytic agents, thus allowing to treat a larger cohort of patients. We hypothesized that formulation of tiplaxtinin with albumin microparticles (MATi particles) would provide targeted delivery to the lungs for the potential treatment of pulmonary embolism. The primary objective of this thesis is the preparation and characterization of MATi particles. This was achieved using a one-pot synthesis technique and stabilizing the particles via heat. MATi particles were then characterized for their size, morphology, drug loading, and stability (Chapter 2). Cytotoxicity profile of the MATi particles was then investigated in relevant cell lines using two different techniques: 1) MTT assay, which allowed us to evaluate the cell viability following incubation with the particles for 24 h; 2) YOYOTM-1 dye assay, which was used to monitor cell death up to 84 h (Chapter 3). Acute toxicity of the MATi particles was also investigated in vivo in a healthy murine model following intravenous administration of two different doses of MATi particles (Chapter 3). The secondary objective was to assess the in vivo biodistribution of the carrier (albumin microparticles) and the drug (tiplaxtinin) and compare it to the unformulated drug as control (Chapter 4). This was performed by labeling the carrier and the drug with two different radioisotopes (111In-MATi[3H-TPLN]), which allowed us to assess their biodistribution simultaneously by quantifying the two signals in the organs of interest. This also provided insights on the lungs’ exposure to the formulated drug vs unformulated tiplaxtinin. 11 Chapter 2: MATi Particles Preparation and Characterization 2.1 Synopsis Thrombolytic agents, also known as “clot busters”, are a class of drugs that can actively degrade blood clots either via direct or indirect mechanism. The first thrombolytic agent was approved by FDA in 1987. Since then, new fibrinolytic agents with improved characteristics, e.g., longer half-life and higher specificity, were approved and marketed within few years. However, since the 2000s, no new fibrinolytic reached the market and, even with an improved pharmacological profile, the last generation of fibrinolytic agents has a limited use in the clinic due to their considerable side effects. For this reason, over the last couple of decades, there has been a great effort to research new drugs with a more desirable pharmacological profile, or create formulations for their targeted delivery. In this context, nanoparticles saw an incredible surge due to their versatile nature in terms of formulating materials, large range of drugs that can be loaded, potential of surface modifications and release control. However, even with these appealing features and the high number of studies performed, the clinical translation has been slow. Interestingly, microparticles did not receive the same attention, even though they might hold great potential. While promising results have been achieved with microbubbles in association with ultrasound, the investigation of polymeric microparticles for the delivery of fibrinolytic agents has been very limited. As we believe microparticles have the potential to provide targeted delivery, our aim in this chapter was to formulate and characterize a thrombolytic drug (tiplaxtinin) with albumin microparticles as a lung-targeting drug delivery system for the potential treatment of pulmonary embolism. 12 2.2 Background Thrombosis occurs when a thrombus, i.e., a blood clot, obstructs a vessel limiting its blood flow. Thrombosis can be distinguished in arterial or venous depending on where the thrombus formed and by its composition, with venous thrombi being richer in red blood cells and fibrin compared to the arterial ones [59]. Current treatments for thrombosis, and patients presenting massive pulmonary embolism, can include systemic administration of thrombolytic agents either via bolus dose or perfusion [60-62]. Thrombolytics can be distinguished in two main categories of drugs: 1) drugs that can directly lyse the thrombus, e.g., administration of plasmin; 2) drugs that can indirectly cause lysis of the blood clot [63]; for the sake of this thesis, I will focus on the latter category. These drugs are also known as plasminogen activators (PAs), due to their ability to activate the enzyme plasminogen into plasmin, which can break down the fibrin network into soluble fragments, thus dissolving the blood clot [64]. The first PA discovered was streptokinase, which has microbial origins and was approved in 1987 [65, 66]. Together with urokinase, they were part of the first generation of PAs. However, the first generation had considerable limitations such as short plasma half-life, lack of specificity, and hemorrhagic risk [59]. Since then, new generations of PAs have been approved, with the third one seeing the market around the 2000s [65]. This generation includes reteplase and tenecteplase which are a recombinant and a genetically modified protein of the endogenous tissue-type plasminogen activator (tPA) [64]. Physiologically, tPA is a serine protease mainly secreted by endothelial cells in response to stimuli and it cleaves plasminogen into its active form plasmin [67]. These engineered versions of tPA have an improved half-life, resistance to inhibitors, and are selective for fibrin compared to the first generation of PAs [68]. However, they did not decrease the mortality rate or the 13 bleeding risk, and their use in the clinic is thus still limited [68]. Table 1 summarizes the main characteristics of FDA approved PAs. Table 2.1: Summary of FDA approved PAs Plasminogen activator Half-life (min) Fibrin specificity Year of approval Indication Drawbacks First generation Streptokinase 18 No 1987 AMI, PE, DVT, arterial thrombosis Immunogenic, non-specific Urokinase 6-18 No 1978/2002 PE Non-specific Second generation Alteplase 6 + + 1987 AMI, PE, acute ischemic stroke Short half-life, expensive Anistreplase* 90-105 No 1989 AMI Immunogenic, non-specific Third generation Reteplase 15 + 1996 AMI Low specificity Tenecteplase 10-24 + + + 2000 AMI Expensive *Anistreplase has been discontinued in US [64]. AMI, acute myocardial infraction; DVT, deep vein thrombosis; PE, pulmonary embolism; + low fibrin specificity, ++ moderate fibrin specificity, +++, high fibrin specificity. Table adapted with permission from Adivitiya and Y.P. Khasa (2017) [65]. The shortcomings of current treatments listed in Table 1 highlight the critical need to develop drug delivery systems that can safely release their payload directly at the site of action, i.e., in proximity of the thrombus. Systemic exposure to these drugs can thus be decreased, reducing 14 side effects. For this reason, over the last years, great attention has been given to the investigation of nanoparticles for the targeted treatment of thrombosis [63]. In the literature, there is a plethora of nanoformulations that have been developed to execute their thrombolytic function in proximity of the blood clot [59, 69]. This has been achieved in different ways, for example via surface modification to improve targeting, drug release at the site of action by stimuli activation, e.g., ultrasound, magnetic field, or by creating bioinspired nanoformulations [70, 71]. While these nanoparticles hold great potential and have shown promising results in vitro and preclinically, clinical translation has been low. This could be due to few factors such as 1) the complexity of synthesis of some formulations, which could hinder their scale-up and result in high production costs and 2) rapid accumulation and onset speed, which are critical parameters to consider in thrombolysis therapy, thus formulations that require external stimuli, e.g.¸ ultrasound, to exert their function might cause treatment delays as they have to be performed by trained personnel in clinical settings [72]. Other studies focused on the development of formulations in the micron range, by investigating the use of microbubbles and microparticles for the dissolution of thrombi. Microbubbles are formulations composed of a gaseous core and proteinaceous or lipidic shell and it was shown that they could increase the efficacy of sonothrombolysis [59]. This technique uses ultrasound to initiate lysis and it can be coupled with thrombolytic drugs and/or microbubbles to improve its efficacy [73, 74]. Even though sonothrombolysis techniques were tested in clinical trials, safety concerns or no significant benefits compared to standard procedures were observed [75, 76]. While sonothrombolysis still holds good potential, success of this technique can be limited by the size of the blood clot and the attenuation conditions for the ultrasound [77]. 15 Besides microbubbles, microparticles formulations have also been investigated for the treatment of thrombosis. An example of polymer-based microparticles is provided by the work of Gunawan et al., where microparticles were loaded with urokinase plasminogen activator (uPA) and the surface functionalized with an activated platelet targeting antibody [78]. The drug release of these particles is stimulus-activated as they can be degraded by thrombin, thus releasing the cargo in the thrombus microenvironment [78]. However, only a small difference was shown compared to the control when efficacy was tested in vitro, and the particle synthesis required multiple steps (over 5) [78]. Differently, Ye et al. proposed in 2018 a synthesis involving less steps, for the preparation of microcapsules loaded with nattokinase and surface-modified with fibrinogen [79]. The latter served as a targeting molecule, as fibrinogen has high affinity for active platelets. The particles showed promising results when efficacy was investigated in vitro [79]. Another interesting study performed by Korin et al. showed the preparation of a shear sensitive drug delivery system where the microparticles prepared are aggregates of multiple polymeric nanoparticles [80]. These microparticles are designed to break up into the smaller components when in an environment of higher shear stress, condition found in proximity of blood clots. The formulation would then exert its thrombolytic role at the site of action via tissue plasminogen activator (tPA), which was used to coat the nanoparticles [80]. This drug delivery system achieved promising results when investigated in vivo in disease models, including a pulmonary embolism model in mice [80]. Similarly to microbubbles, these microparticles formulations would typically have a dimeter size below around 6 µm. This is because the pulmonary capillaries have a dimeter of around 8 µm, therefore when the microparticles are administered intravenously, they are small enough that they can move into circulation and act on a systemic level by resolving clots that can be found outside the lungs [81]. 16 In all these drug formulations, albumin occupied a small place. Even though this protein has been widely used for delivery of different drugs, such as anti-cancers and antibiotics, very little research has been performed on using it as a carrier for anti-thrombotic therapies [21]. To the best of my knowledge only one study performed by Ibrahim et al. in 2017 used albumin microparticles as a targeted delivery system for pulmonary embolism [82]. In this work, the group prepared albumin particles loaded with enoxaparin [82]. It is important to note that enoxaparin is an anticoagulant, therefore it prevents the formation of thrombi and limits the growth of already existing ones by irreversibly inhibiting factor Xa via complexation with antithrombin III, and is generally more indicated for milder cases [83]. For this reason, anticoagulants are usually indicated for prophylaxis, treatment of less severe cases of PE, or in combination with thrombolytic agents [41]. Furthermore, the group prepared the formulation using a water in oil emulsion technique using glutaraldehyde as cross-linking agent for the stabilization of the particles [82]. However, in our project, we wanted to investigate particles that could be prepared without the use of cross-linking agents. Although they can prevent drug leakage, they can also slow the release of the drug [84, 85]. While this can be advantageous in certain circumstances, e.g., for sustained release, this could be problematic for the treatment of thrombosis, as rapid drug accumulation and onset at the site of action are crucial [72]. The aim of this chapter, therefore, is to formulate the fibrinolytic enhancer drug tiplaxtinin with albumin microparticles and characterize the formulation. In this project, tiplaxtinin was used as a model drug, as in the literature is a well characterized small molecule that can enhance the endogenous fibrinolytic process. This small molecule was chosen over an FDA approved thrombolytic agent, as the ones currently on the market are proteins: since our preparation method for the microparticles involves high temperatures, proteins were excluded as they lack 17 the thermostability to undergo this process and maintain their activity. Besides thermostability, tiplaxtinin was chosen for the presence of a functional group that could be exploited for conjugation in future development. Furthermore, tiplaxtinin (Fig. 2.1) is a hydrophobic small molecule (MW = 439.38 g/mol) and it was firstly described in 2004 as a selective inhibitor of plasminogen activator Inhibitor type-1 (PAI-1) [86]. Since PAI-1 has been shown to play an important role in the pathological development of venous thrombosis, among other diseases, it was considered an attractive molecular target for the treatment of cardiovascular diseases [87-89]. This is due to its inhibitory role of urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA), which are enzymes that can convert plasminogen into the active form plasmin. Therefore, when there is a homeostasis imbalance, with elevated levels of PAI-1 present, it can lead to the development of thrombosis [90]. In a study by Gorlatova et al. it was shown that tiplaxtinin can reversibly bind to the hydrophobic region of PAI-1 in its active form [91]. It was therefore speculated that the small molecule hinders the conformational change needed by PAI-1 to bind and inhibit the plasminogen activators, thus inhibiting it [91]. The IC50 of the small molecule was firstly assessed to be 2.7 µM in vitro [86]. Thrombolytic efficacy of tiplaxtinin was also proved both in rodent and non-rodent animal models. In disease models, the small molecule increased time to vessel occlusion and decreased blood clot weight compared to the controls following oral administration [86]. Indeed, the drug showed good oral bioavailability in rats (around 93%) and dogs (around 43%) [92, 93]. 18 Figure 2.1: Tiplaxtinin Chemical structure of the PAI-1 inhibitor tiplaxtinin. In this chapter, we formulated tiplaxtinin using bovine serum albumin (BSA) to create microparticles for its delivery to the lungs. Microparticles were prepared with a thermal aggregation method, avoiding the use of cross-linking agents, and then characterized for their size distribution, morphology, drug loading, and stability. 2.3 Results and Discussion 2.3.1 MATi Particles Formulation MATi particles were prepared using a synthesis method based on a protocol previously established in the lab, which employs pH, addition of metals, and heat to induce albumin aggregation, thus forming the particles. In the formulation, a pH of 5.5 was used. When the solution is heated and the pH is closer to the isoelectric point of BSA (pI ~ 4.8), conformational changes of the protein’s tertiary structure are predominant, and are mediated by non-specific interactions [94, 95]. Heat induced aggregation is a rather well-known technique, where a temperature increase causes 1) denaturation of alpha helices and formation of beta strands, and 2) exposure of hydrophobic regions and thiol groups that can form new intermolecular interactions [96, 97]. Studies reported that above 65° C, a conformational change on the secondary structure of BSA was observed, with alpha helices denaturing, and forming a structure 19 closer to beta sheets [97, 98]. It was also shown that at higher temperatures, BSA undergoes conformational changes in its tertiary structure, and it can form new intermolecular bonds during the aggregation process [97]. To prepare the MATi particles, the solution containing albumin was heated at 80° C for 9 min, based on a study by Borzova et al. which reported that at this temperature, only 1.5 min were needed for half-conversion of BSA to its denatured state [99]. Due to the structural rearrangement undergone by BSA at elevated temperatures, Holm et al. observed formation of fibrils when they investigated the morphology of BSA aggregates [100]. This result was confirmed by Borzova et al. when the group analyzed BSA following incubation at 80° C [99]. These observations could explain the presence of some filaments visible in SEM images of MATi, whereas areas showing amorphous structure could be due to the use of a more acidic pH closer to the BSA isoelectric point, as reported by Vetri et al. [95] (Fig. 2.2 A). Interestingly, images of a commercial kit of MAA showed a more globular morphology (Fig. 2.2 B). This was consistent to the one previously reported by other groups, while Canziani et al., showed the co-existence of a smoother structure [101-103]. These differences, both compared to MATi particles and other MAA kit, could be due to the different nature of the albumin (bovine vs human) and the parameters used for their preparation [94]. As expected, MATi particles did not present a spherical shape when observed at the microscope, as they are formed by protein denaturation and aggregation (Fig. 2.2 C). This was consistent with the shape observed for commercial MAA, as well as with what was reported in the literature [104, 105] (Fig. 2.2 D). 20 Figure 2.2: Morphology and shape of MATi particles and commercial MAA SEM images of dried (A) MATi particle showing a mixture of amorphous and more filament-like structures, formed during the thermal aggregation process due to the formation of new beta-strands; and (B) MAA from a commercial kit, showing a more globular morphology. Microscopy images of resuspended (C) MATi particles and (D) MAA (commercial kit) showing the characteristic non-spherical shape due to the aggregation of albumin. Lastly, FeCl2 was added to the formulation as it was shown that metals can promote albumin aggregation and increase the average size of the aggregates [105, 106]. While the method used allows to play with the parameters described to obtain particles of a certain size, the resulting size distribution is still rather wide. However, this was observed also with clinically used MAA. In the U.S. Pharmacopeia, it is stated that 90% of 99mTc-MAA 21 particles should be in a size range within 10 to 90 µm, and no microparticles should be bigger than 150 µm [107]. These parameters are indicated for the following reasons: 1) microparticles smaller than 10 µm are filtered through the pulmonary capillaries and do not accumulate in the lungs; 2) microparticles bigger than 150 µm can block the bigger pulmonary arteries, thus posing health risks to the patients [108]. Furthermore, the commercial monograph of 99mTc-MAA DraxImage® indicates the average size between 20 to 40 µm [37]. MATi particles showed an average size of 25 ± 5 µm (N = 15), and for all the batches tested over 83% of the particles were within the 10-90 µm range; 60% of which were over 90%. When measured by laser diffraction, MATi particles showed a comparable size distribution to that of a 99mTc-MAA commercial kit (Fig. 2.3 A). Blank MATi particles, i.e., empty MATi particles, were also prepared and their size distribution investigated as they were used in downstream experiments as control (vehicle). Their size distribution was similar to the other types of particles tested and they had an average size of 23 ± 3 µm (N = 4) (Fig. 2.3 A). Microscopy images of MATi particles showed the presence of smaller particles, which was confirmed by size distribution analysis expressed as number (%). However, commercial MAA showed a similar profile (Supplementary Fig. A.1.1). While this synthesis method uses temperature and pH to produce microparticles within a desired range, inter-batch variability was observed. Fig. 2.3 B highlights the differences in size distribution of representative MATi particles prepared on different days. Nevertheless, the average particle size was consistently within the recommended range. 22 Figure 2.3: Particles size distribution and inter-batch variability A) Blank MATi (control) and MATi particles, and MAA from a commercial kit showing a similar size distribution with the majority of the particles within the 10-90 µm range, and no particles bigger than 150 µm. B) Representative size distributions of three batches of MATi particles prepared on different days, showing the variability within the 10-90 µm range. Measurements are shown as average (n = 3) without standard deviation for visual clarity (see Supplementary Fig. A.1.2 for graphs including SD). 2.3.2 Drug Loading Drug loading of tiplaxtinin was assessed both quantitatively and qualitatively. At a first visual analysis, it was interesting to notice that MATi particles showcased a characteristic yellow/orange color, whereas the empty MATi particles remained white (Supplementary Fig. A.2.1), thus potentially indicating drug loading, as tiplaxtinin is a yellow powder. To analyze drug loading quantitatively, the amount of drug present in the MATi particles was assessed indirectly by measuring tiplaxtinin in the supernatant by UV-Vis. The amount of drug loaded for each batch was then calculated by subtracting from the initial amount of drug added. Since the supernatant contained a mixture of buffer, organic solvent, as well as unformulated tiplaxtinin and BSA, the small molecule needed to be purified from the other components of the solution. This was performed by means of a PD MiniTrap G25, a size exclusion column with an exclusion limit of Mr = 5000. Due to the considerable size difference between BSA (66.4 kDa) and tiplaxtinin (439 Da), it was possible to separate them: BSA, which is bigger than the column 23 pores, would directly pass through the column, thus eluting in the first fractions. Tiplaxtinin, however, by being smaller than the cutoff, would get trapped within the pores of the column bed, thus eluting in later fractions (example of elution profile, Supplementary Fig. A.2.2). Separation of the two components was also confirmed by analysis of the eluted fractions both by UV-Vis and gamma counter when BSA was labelled with the radioisotope 111In (Chapter 3). As shown in Fig. 2.4 A, higher activity is present in the initial fractions when BSA elutes, while is not present in the following fractions corresponding to the tiplaxtinin peak. The amount present in the supernatant was then measured by UV-Vis and calculated using a calibration curve. Average drug loading was assessed to be 26 ± 13 % (N = 8). We hypothesized that the standard deviation assessed could be due to the inter-batch variability observed in the previous experiment and shown in paragraph 2.3.1. Taking into consideration the standard deviation, drug loading was quantified for each batch and batches were not mixed before experiments, so one batch was used per experiment. Nevertheless, direct measurements were possible when particles were prepared with radiolabeled 3H-TPLN (Chapter 4). Qualitative assessment of the drug loading was performed by FT-IR using a Spotlight 400 FT-IR Imaging System. Initially, reference spectra of tiplaxtinin and aggregated BSA, i.e., empty MATi, were acquired and characteristic peaks were assessed to be 1260 cm-1 and 1641 cm-1, respectively (Supplementary Fig. A.2.3). The former band represents the aryl ether group of tiplaxtinin, while the latter one corresponds to the N-H bend [109]. We then proceeded to analyze a sample containing a mix of blank and loaded MATi particles. The instrument allowed us to obtain an initial survey image of the sample using the microscope. Afterwards, the area surveyed was analyzed by IR and from the full spectrum obtained, it was possible to extract single wavelength images: one in blue for albumin and one in red for tiplaxtinin. Once the 24 images were merged, it was possible to localize MATi particles as the ones expressing both signals (white arrows, Fig. 2.4 B), among the blank ones. Figure 2.4: Quantitative and qualitative loading of tiplaxtinin. A) Elution profile of tiplaxtinin (black) measured by UV-Vis at 314 nm, and 111In-BSA (pink) measured by gamma counter, showing the separation of the two molecules. B) Images overlay of a mix of blank MATi and MATi particles acquired by IR single wavelength extraction obtained for the characteristic band of aggregated BSA (blue) and tiplaxtinin (red). White arrows indicate some of the particles showing overlapping signal (purple), thus identifying loaded particles. UV-Vis data is presented as mean ± SD (n = 3, error bars covered by the symbols). Gamma counter measurement is n = 1. 2.3.3 MATi Particles Stability MATi particles were firstly tested for their stability at storage conditions and compared to a commercial kit of MAA as control. A fresh batch of particles was prepared and their size distribution assessed using a Mastersizer (day 0). The particles were then stored at 4° C in saline solution and changes in size were monitored for signs of degradation, i.e., presence of smaller size particles, over a period of two weeks. As shown in Fig. 2.5 A, the particles proved to be very stable at the conditions tested. Minor signs of degradation were observed as the mean diameter of the MATi particles decreased from 22 ± 1 µm to 20 ± 0.5 µm (n = 3) over 14 days, 25 whereas the mean diameter of MAA from a commercial kit was 17 ± 0.3 µm on day 0 and 18 ± 0.3 µm (n = 3) on day 14 (Fig. 2.5 B). Nevertheless, the difference between mean diameters was found non-significant for both type of particles. Differently, when MATi particles were stored long term for up to six months, clear signs of degradation were visible as the size distribution shifted towards smaller particles. The average diameter of the particles significantly decreased from 31 ± 2 µm to 11 ± 1 µm (p = 0.015) (Fig. 2.5 C). Even though MATi particles proved to be stable for 14 days, batches were used within 24 h from their preparation for our experiments. Figure 2.5: MATi particles stability at storage conditions A) Size distribution of MATi particles measured over two weeks shows good stability while stored in saline solution at 4° C. B) Size distribution of MAA from a commercial kit showing very good stability in storage conditions over 14 day. C) MATi particles shows signs of degradation at 6 months timepoint, as a higher number of smaller particles are present compared to the day they were prepared (day 0). Measurements are shown as average (n = 3) without standard deviation for visual clarity (see Supplementary Fig. A.3.1 for graphs including SD). Stability of MATi particles was also assessed at 37° C when incubated with different media and compared to the stability of a commercial MAA kit. Particles were added to a 96 well plate and incubated at 37° C in 1x PBS, 5 mM glutathione (GSH), or 50% plasma. Potential degradation of the particles was monitored over time using a live-cell imager and then quantified using the instrument’s software. Stability of the particles is expressed as confluency, i.e., area occupied by the particles over the total area of the well. Therefore, an increase in confluency indicates particles degradation as the particles break up and cover more surface. 26 While MATi and MAA followed similar patterns, differences between the two types of particles can be noticed. MAA particles proved to be very stable in PBS, while MATi particles showed minor signs of degradation over three days (Fig. 2.6 A). Particle stability was also assessed following incubation with glutathione (GSH). GSH acts as a reductant and is widely distributed in our organism, including the blood at concentrations of around 5 µmol/liter [110, 111]. Therefore, GSH was used to assess potential degradation of the particles by reduction of the disulfide bridges formed during the thermal aggregation. MAA particles proved to be very stable over the period tested. Following a minor initial increase in confluency, MATi particles also showed very good stability for the period tested (Fig. 2.6 B). This result is comparable with the observations of Esposito et al., where heat-denatured macroaggregated albumin microparticles showed to be stable when incubated with GSH for less than 40 h [112]. MATi particles showed a similar trend when incubated with PBS and GSH, with a steeper confluency increase within the first 6 h, then they remained rather stable over time. It was hypothesized that the initial increase might be due to smaller MATi particles settling at to the bottom of the well, as shown by the microscope images (Supplementary Figure A.3.2), whereas this is not the case for MAA (Supplementary Figure A.3.3). Interestingly, degradation was observed for both MAA and MATi particles when incubated with 50% human plasma, with MATi showing a faster degradation kinetic compared to MAA (Fig. 2.6 C). 27 Figure 2.6: MATi particles stability at 37° C Stability of MATi particles and MAA from a commercial kit was assessed at 37° C. A) When incubated with PBS, MATi particles showed signs of degradation as the confluency increased, while MAA were very stable over 72 h. B) MATi particles showed an initial increase in confluency follow by a plateau, indicating overall good stability when incubated with the reducing agent GSH (5 µM). C) Both MATi and MAA particles degraded over time when incubated with 50% plasma. However, MATi showed a faster degradation rate compared to MAA. N = 2, line represents the mean value. Dashed line representing 0.5 normalized confluency. 2.4 Chapter Summary In this chapter we were able to formulate the thrombolytic drug tiplaxtinin with albumin microparticles for its delivery to the lungs’ capillary bed. The MATi particles were prepared using a one-pot synthesis method, using temperature and pH to obtain particles with an average size of 25 µm. Even though some inter-batch variability was observed, more than 80% of the particles were within the 10 to 90 µm range, and drug loading was assessed to be around 26%. MATi particles proved to be very stable at storage conditions (4° C in saline solution) over two weeks, while degradation was observed following six months storage. When stability was investigated at 37° C over three days, MATi particles showed a similar pattern to MAA from a commercial kit. Both particles were generally stable in PBS or when incubated with the reducing agent GSH. However, degradation was observed for when they were incubated with 50% plasma, with MATi particles showing a faster rate of degradation compared to MAA. 28 2.5 Experimental Section 2.5.1 Materials and Reagents Tiplaxtinin was purchased from Cayman Chemicals (Ann Arbor, MI, USA) at a purity of ≥ 98%, while BSA (IgG-free and protease-free) was purchased from Jackson ImmunoResearch Laboratories Inc (West Grove, PA, USA). The commercial kits of MAA used was DraxImage® MAA, provided by Jubilant Radiopharma (Montreal, QC, Canada), here referred as MAA. 1-Methyl-2-pyrrolidone (NMP) was obtained from MilliporeSigma (Oakville, ON, Canada). FeCl2 (98%) and L-Glutathione reduced (≥ 98%) were purchased from Sigma-Aldrich, Inc (St. Louis, MO, USA). PD MiniTrapTM G-25 columns for supernatant purification were purchased from GE HealthCare Technologies, Inc (Chicago, IL, USA). PBS (1x) used in the experiments was purchased by ThermoFisher Scientific (Burnaby, BC, Canada). Falcon® 96-well plates were obtained from Corning (Corning, NY, USA). Saline was prepared in-house by dissolving NaCl (0.9% w/v) in Type 1 ultrapure water. Type 1 ultrapure water was used for all the experiments and was obtained from a Milli-Q® filtration system (MilliporeSigma). 2.5.2 MATi Particles Preparation FeCl2 was dissolved using NMP for a final concentration of 50 mg/mL, then 3 µL were added to 72 µL of tiplaxtinin in NMP (25 mg/mL). The total volume was transferred to a 10 mL round bottom flask containing a PTFE stir bar set at 150 rpm. BSA (2% w/v) in 0.5 M phosphate buffer pH 5.5, 75 µL) was added to the flask and the solution kept under continuous stirring at room temperature for 1 min, while 100 µL MilliQ water and 100 µL NMP were added to the flask. Afterwards, the flask was stoppered and lowered into a water bath at 80° C for 9 min. The flask was then cooled down to room temperature before removing the solution containing the particles, and place it into an Eppendorf tube®. Particles were centrifuge at 1000 rpm for 5 min, the 29 supernatant was collected and particles were washed thrice with 150 µL saline by centrifuge at 1000 rpm for 5 min. 2.5.3 Microscopy and SEM images MATi particles and commercial MAA were fixed by chemical cross-linkage. Glutaraldehyde (12.5%, 300 µL) was added to the particles suspended in MilliQ water for a final concentration of 8%. Samples were placed on an end-to-end rotator and mixed gently for 24 h. After the incubation, the samples were centrifuged at 1500 rpm for 5 min, and the supernatant discarded. Particles were then washed thrice with MilliQ water previously filtered with a PVDF filter with pores size of 0.22 µm. Afterwards, few µL of the samples were spotted on a sample stud covered with carbon tape and samples were left to air dry overnight in a fume hood. The following day, the samples were sputter coated with a 10 nm layer of iridium using a Leica EM MED020 coating system (Wetzlar, Germany). Particles morphology was investigated using a Hitachi SU3500 (Tokyo, Japan) at 1.5 kV. 2.5.4 Drug Loading Assessment Tiplaxtinin present in the supernatant collected during the MATi particles preparation was purified using a PD MiniTrapTM G-25 column containing a crossed-linked dextran matrix (exclusion limit, Mr: 5000). The storage solution contained in the column was eluted, and the column equilibrated with at least 6 mL of MilliQ water. MATi particles supernatant (20 µL) was loaded onto the column. Sodium phosphate buffer (0.05 M) containing 150 mM NaCl pH 7 was added (1 mL aliquot, 2 mL total) to elute BSA and allow tiplaxtinin to be absorbed into the column. Additional fractions (500 µL each) were collected using the same buffer. Fractions containing tiplaxtinin were eluted using 40% EtOH (500 µL each). All fractions were analyzed by UV-Vis using a NanoDrop 2000 (ThermoFisher Scientific, 30 USA) by measuring the absorbance at 280 nm (BSA peak) and 314 nm (TPLN peak). Fractions containing tiplaxtinin were collected and the amount of drug present in the supernatant was calculated using a calibration curve of tiplaxtinin prepared in 40% EtOH. Drug loading was calculated using the following equation: 𝐷𝑟𝑢𝑔 𝑙𝑜𝑎𝑑𝑖𝑛𝑔 (%) = (𝑇𝑜𝑡𝑎𝑙 𝑑𝑟𝑢𝑔 𝑎𝑑𝑑𝑒𝑑 − 𝐷𝑟𝑢𝑔 𝑖𝑛 𝑠𝑢𝑝𝑒𝑟𝑛𝑎𝑡𝑎𝑛𝑡)𝑇𝑜𝑡𝑎𝑙 𝑑𝑟𝑢𝑔 𝑎𝑑𝑑𝑒𝑑∗ 100 2.5.5 FT-IR Spectra for Qualitative Drug Loading Drug loading was assessed qualitatively using a Spotlight 400 FT-IR Imaging System (PerkinElmer, USA). Initially, few mg of tiplaxtinin in powder (as received from the seller), blank MATi, and MATi particles (both in MilliQ water) were added onto a gold-plated slide and left to air dry in the fume hood. Afterwards, the slide was placed on the microscope holder and a visual survey (5% illumination) of the slide was performed to spatially locate the samples. Following, an IR background scan was performed on a sample-free area using a scanning range of 4000 – 744 cm-1, IR resolution of 16 cm-1, and pixel size resolution of 6.25 x 6.25 µm. Areas containing each sample were then scanned in the same fashion, thus obtaining a full IR spectrum for tiplaxtinin, blank MATi, and MATi particles. Characteristic bands for tiplaxtinin and aggregated BSA (based on blank MATi spectrum) were identified at 1260 cm-1 and 1641 cm-1, respectively. New blank MATi and MATi particles were then mixed in MilliQ water, and the sample was prepared on the gold-plated slide in the same manner. A spectral map of the sample was then acquired with the same parameters, but at a higher IR resolution of 4 cm-1. For each characteristic band, a single wavelength intensity image was extracted by performing a background subtraction from an adjacent baseline IR region of the sample corresponding to 1290 cm-1 for tiplaxtinin and 1714 cm-1 for aggregated BSA. The two images were merged to show 31 both presence of the drug and the protein. Areas of the images showing both signals were indicative of the presence of MATi particles. 2.5.6 MATi Particles Storage Stability Storage stability of the MATi particles was tested by monitoring their size distribution over two weeks. MAA from a commercial kit was tested for the same time period as control. A fresh batch of MATi particles was prepared as explained in paragraph 2.4.2. Size distribution of the freshly prepared particles (day 0) was assessed using a Mastersizer 2000E Particle Size Analyzer equipped with a Hydro 2000SM Dispersion Unit (Malvern Instruments, Inc., Malvern, UK). The samples were added to the dispersion unit with a stirring speed set at 2000 rpm and measured using water as dispersant. The particles were stored in saline at 4° C, over the period tested. Size distribution of the MATi particles was measured in the same manner at 1, 2, 4, 7, and 14 days. Long term storage of MATi particles was tested over a time period of six months. 2.5.7 MATi Particles Stability at 37° C Stability of MATi particles was assessed at 37° C when incubated with 1x PBS, 50% human plasma (supplemented with 1 U/mL penicillin and 1 µg/mL streptomycin), and 5 µM glutathione (GSH). Plasma was prepared from whole blood collected from healthy volunteers using BD Vacutainer® collection tube (Dutscher, Bernolsheim, France) with 9 NC sodium citrate 0.109M to prevent coagulation. If not processed right after collection, blood was kept at 4°C and processed within 2 h. Whole blood was centrifuged at 2500 g for 20 min at 4°C, and plasma was collected as the supernatant and stored at -80° C for a maximum of six months. Blank MATi and MATi particles were prepared in aseptic conditions, while MAA (DraxImage MAA) were reconstituted using 1 mL of sterile saline solution. Particles were added to a 96 well plate with the abovementioned media (Vf = 100 µL) and placed in an incubator at 37° C and 5% CO2 for 72 32 h. Every 6 hours, phase contrast images of each well (4 images per well) were acquired using an Incucyte® SX5 live-cell imager (Sartorius, Göttingen, Germany), equipped with a 10x objective. Each image was then analyzed by overlaying a confluency mask using the Incucyte 2022B software. Each value obtained per well was normalized to its 0 h timepoint and graphed against time. 2.5.8 Data Visualization and Statistical Analysis All figures were prepared using Prism 10 software (GraphPad, USA) and are presented as mean ± standard deviation, with n = 3 unless stated otherwise. ChemDraw version 19.1 (PerkinElmer, USA) was used for the representation of chemical structures. Paired t-test (two-tailed) analysis were performed with Prism10. Statistical significance was set to p < 0.05. 33 Chapter 3: Toxicity Profile of MATi Particles 3.1 Synopsis When designing a drug delivery system, it is important to consider the potential toxicity effect of each component. For this reason, albumin was chosen for the preparation of MATi particles as it has advantageous qualities such as biocompatibility, low immunogenicity, and biodegradability that makes it a very attractive drug carrier. We were interested in evaluating the cytotoxicity of MATi particles in relevant cell lines, and compared them to unformulated tiplaxtinin and empty particles. Another important aspect that we had to consider when preparing this formulation, was the number of MATi particles that could be safely administered. Indeed, when particles accumulate in the lungs through mechanical filtering, they cause microembolization in the capillaries and arterioles. Therefore, it is important to ensure that the pulmonary vasculature is not completely blocked causing a lethal embolism. As the maximum number of particles that can be administered limits the final drug dose in a formulation, it is particularly important to know how many particles can be safely administered. In this chapter, our aim was to define the toxicological profile of MATi particles in vitro and in vivo. 3.2 Background Albumin has been a long time player in the world of drug delivery and human, recombinant, and bovine serum albumin have been extensively investigated as drug carriers [10]. The main reason is that albumin has several advantageous features, including biodegradability and biocompatibility, which makes it a very appealing molecule for the preparation of drug delivery systems [113]. Particularly, various studies reported good cell compatibility of bovine serum albumin-based formulations in several cell lines [114-117]. This is a key characteristic as drug 34 delivery systems should not cause additional toxicity. Here, we were interested in investigating the cytotoxicity of MATi particles in pulmonary and endothelial cell lines. Another important aspect to account for when passively targeting the lungs is the number of particles that can be administered: when particles are injected intravenously and they reach the pulmonary bed, they obstruct the blood flow, thus causing microembolization. For this reason, it is also very important that the particles prepared do not contain particles with a size bigger than 150 µm, as they could lodge in the bigger pulmonary arteries, thus posing patients at great risk by blocking the blood flow and impairing the efficient exchange of gases causing hypoxemia [108]. Guidelines recommend doses of 99mTc-MAA, for example, so that less than 1 in 1000 capillaries are blocked, thus ensuring patient safety [36, 39]. The total number of particles that can be administered without being hazardous, could constitute a limiting factor when it comes to drug formulation as a therapeutic dose has to be achieved at the target organ. Therefore, we performed an in vivo dose study in a murine model by administering two different doses of MATi particles to ensure their safety profile and their ability to concentrate in the lungs. In summary, the aim of this chapter was to investigate the cytotoxicity as well as the in vivo acute toxicity of MATi particles. This study also allowed us to confirm the correct biodistribution of the particles and their accumulation in the lungs in preparation for the biodistribution study (Chapter 4). 3.3 Results and Discussion 3.3.1 MATi Particles Cytotoxicity Potential toxicity of the MATi particles was investigated both in vitro and in vivo. As the particles can passively target the lungs via mechanical filtration and lodge in the lung capillaries and arterioles, cytotoxicity was evaluated in a pulmonary epithelial cell line (A549) and an 35 endothelial cell line (HUVEC) [118, 119]. Viability of the cells in presence of MATi particles, blank MATi, or free tiplaxtinin was investigated via MTT assay at a 24 h timepoint, whereas cell death was studied via YOYOTM -1 dye over 84 h. The former assay evaluates cells’ metabolic activity via their ability to reduce MTT into blue-violet formazan crystals, thus allowing to indirectly assess cell viability: higher presence of metabolically active cells can reduce more MTT, therefore the color obtained when dissolving the formazan crystals will be more intense, thus resulting in higher optical density readings [120]. Cell viability is then calculated as a percentage by setting the readings for untreated cells, i.e., incubated with only media, as 100% viability. Differently, YOYOTM-1 dye allows to directly measure cell death as its fluorescence is substantially enhanced upon binding to the cells’ DNA [121]. Cell membrane is normally impermeable to YOYOTM-1, but once cells die their membrane integrity is lost, thus allowing the dye to permeate and bind to the DNA [122]. By means of a live-cell imager, it was possible to monitor and quantify the fluorescence over time. Overall, in both A594 and HUVEC cells, an increased in cell death was observed at higher concentrations of free tiplaxtinin and MATi particles tested (Fig. 3.1A, B), which aligns with the results from the MTT assay. Cell viability of A549 cells showed a decrease in a concentration dependent manner when cells were incubated with free tiplaxtinin and MATi particles (Fig. 3.1C). In a study from 2020 conducted by Krämer et al., no signs of toxicity were observed when A549 cells were incubated with tiplaxtinin up to 20 µM [123]. This is in agreement with our results as 84% cell viability was observed when cells were incubated with 16 µM free tiplaxtinin. A steep decrease in cell viability was recorded when cells were incubated with 32 µM or higher concentrations of free tiplaxtinin (53%). Interestingly, Feng et al. showed increasing apoptosis when A549 cells where incubated with increasing concentrations of tiplaxtinin up to 50 µM [124]. The group hypothesized the pro-tumorigenic 36 role of PAI-1, thus inferring that the pharmacological inhibition of PAI-1 led to the apoptosis observed. Due to the high biocompatibility and low immunogenicity of BSA, cytotoxicity was not expected for blank MATi particles. Interestingly, a decrease in cell viability was observed as the number of particles increased, although to a lesser extent compared to MATi particles and free tiplaxtinin (Fig. 3.1C). This finding is in contrast with results reported by other groups, where no signs of cytotoxicity were observed for BSA nanoparticles tested via MTT assay in multiple cell lines [125-127]. Our hypothesis is that the toxicity observed might be due to mechanical stress that the particles exert on the cells. Indeed, the particles tend to settle at the bottom of the wells due to their size. This might hinder the cells’ ability to proliferate as both A549 and HUVEC cells are adherent cell lines. 37 Figure 3.1: Cytotoxicity study Analysis of cell death when MATi, blank MATi particles or free tiplaxtinin were incubated with A) A549 or B) HUVEC cells up to 84 h. Increased cell mortality was observed for MATi particles and tiplaxtinin at the higher concentrations tested. Data presented as mean (n = 3). C) Cell viability of A549 at 24 h when incubated with MATi, blank MATi, or free tiplaxtinin. Formulated and unformulated drug showed a concentration dependent toxicity, while blank MATi showed higher biocompatibility (n = 3). Dashed line represents 50% viability. Cytotoxicity was observed for both the drug and the carrier, although to different extent. To investigate the effect that each of them played on the overall toxicity observed for the MATi particles, the two components were analyzed separately based on the data acquired via MTT assay with HUVEC cells. Firstly, dose-response curves were obtained for increasing concentrations of free tiplaxtinin and number of blank MATi particles (Fig. 3.2A, B). Free tiplaxtinin exhibited the typical sigmoidal curve and was fitted with a nonlinear regression line, while blank MATi’s graph was fitted with a linear regression line (R2 = 0.78). Using these 38 models, cell viability was calculated for the concentration of tiplaxtinin and number of blank MATi particles needed. The values obtained were normalized to the cell viability measured for the MATi particles at the same concentration of tiplaxtinin or number of particles. As shown in Fig. 3.2C, at lower concentrations of the drug the MATi/TPLN cell viability ratio is below 1 and it increases as the drug concentration increases, thus indicating that at higher concentrations the toxicity observed is mainly caused by tiplaxtinin. On the other hand, the MATi/blank MATi cell viability ratio decreases as the number of particles increases, which corresponds to higher amount of tiplaxtinin. The decreased ratio signifies that for the same number of particles, cells incubated with blank MATi particles showed higher cell viability, thus confirming that the cell toxicity observed is mainly due to tiplaxtinin (Fig. 3.2D). While there is no consensus in the literature concerning PAI-1 role in angiogenesis at the moment, some studies showed the involvement of the protein in the process and maintenance of vascular integrity [128, 129]. Therefore, the toxicity observed in HUVEC cells could be caused by the pharmacological inhibition of PAI-1. The IC50 calculated for free tiplaxtinin was 31.9 µM and 27.5 µM for A549 and HUVEC cells, respectively. These values are an order of magnitude higher than the IC50 for the inhibition of PAI-1 activity reported in the literature (2.7 µM) [86]. This was encouraging as it demonstrates that a therapeutic effect could be achieved at a concentration far below the toxic one. 39 Figure 3.2: Effect of the drug and the carrier on the overall toxicity of MATi particles A) Cell viability of HUVEC cells incubated with increasing concentrations of free tiplaxtinin (control) for 24 h, fitted using a non-linear regression function. N = 3. B) Cell viability (%) of HUVEC cells incubated with increasing amounts of blank MATi for 24 h, fitted with a linear regression function. Each data point represents n = 3, data shown from three independent experiments (N = 3). C) Cell viability ratio of MATi particles over free tiplaxtinin. The ratio increases as the concentration of drug increases, thus suggesting that tiplaxtinin is the main source of toxicity at high concentrations. N = 2, line represents mean. D) Cell viability ratio of MATi particles over blank MATi. The decreasing ratio as the number of particles increases indicates that at high number of particles cytotoxicity of MATi particles is not influenced by the carrier. Each data point represents n = 3, data shown from two independent experiments (N = 2). Data presented as mean ± SD. Dashed line represents 50% viability. 3.3.2 In Vivo Dose Study For the in vivo dose study radioactive labelled MATi particles (111In-MATi) were prepared, which allowed us to also quantify the fraction of particles reaching the organs of interest and 40 confirm particles accumulation in the lungs upon intravenous administration. To label the particles with the radioisotope 111In, BSA was modified with the chelator DTPA (DTPA-BSA) via a stable thiourea bond [112]. DTPA was the chelator of choice for 111In, as it was reported to have higher affinity for the radiometal compared to endogenous molecules such as transferrin, thus minimizing potential transchelation of 111In in vivo [130]. When analyzed via gel electrophoresis, native BSA, DTPA-BSA, and labeled 111In-DTPA-BSA showed similar molecular size with a main protein band slightly higher than 50 kDa (Fig. 3.3A). The signal provided by 111In represented by the heat map, and its corresponding radiotrace, was present at the same molecular weight of BSA confirming the successful radiolabeling of the protein (Fig. 3.3A). To prepare 111In-MATi particles, unmodified BSA was spiked with 10 % 111In-DTPA-BSA, afterwards the particles were prepared in the same manner as described in Chapter 2. Laser diffraction measurements showed that particles prepared with 10% DTPA-BSA and radiolabeled particles, remained within the same size distribution range of unmodified MATi particles (Fig. 3.3B). Following 111In-MATi preparation, particles were washed with a saline solution as per protocol, which served also as purification step from 111In that became unbound during the synthesis process. This was confirmed by radio-instant thin layer chromatography (ITLC), which showed 95% purity for the 111In-MATi particles (Fig. 3.3C). This ITLC system uses EDTA as mobile phase as it can chelate free 111In and transport it to the solvent front (retention factor, Rf = 1), whereas 111In bound to the particles remains at the origin (Rf = 0). 41 Figure 3.3: Radiolabeling of BSA and preparation of 111In-MATi particles A) Gel electrophoresis of the modified DTPA-BSA, and 111In-DTPA-BSA showing similar molecular weight to the native BSA. 111In signal is represented by the heat map and the corresponding radiotrace, at the same molecular weight protein band, confirming the radiolabeling of BSA. B) Cold MATi particles, MATi particles spiked with 10% DTPA-BSA, and radiolabeled 111In-MATi showed comparable size distributions. Graph is shown as average (n = 3) without SD for visual clarity. C) Representative radiotraces of ITLCs analysis of 111In-MATi, which remained at Rf = 0 proving successful purification of the particles, and a sample of free 111InCl3, which travelled to the front with the mobile phase (Rf = 1). Once the particles were prepared, they could be administered into healthy mice. The human dose for 99mTc-MAA ranges from 200,000 to 700,000 particles, with the a recommended one of around 300,000 particles [36, 37]. For the in vivo study, 111In-MATi particles were administered intravenously to healthy mice in two different doses containing either 500,000 or 700,000 42 particles (experimental details in Table 5.1). Mice were then sacrificed at 15 min post-injection, the organs of interest were collected, and activity in each of them quantified. After administration of the two doses, mice did not show overt signs of acute toxicity. Following the injections, mice were placed into their cages and monitored for physical activity and behavior. During the monitoring period, mice were active, drank water, and showed no signs of distress, e.g., hunched posture or ruffled fur. As particles of smaller size were noticed when characterizing them by laser diffraction and microscopy (Chapter 2.3.1), we also investigated during this study the biodistribution of the MATi particles upon injection, to ensure that high accumulation in the lungs was reached. Indeed, particles that are smaller than ∼ 8 µm can readily pass through the capillaries, being picked up by the RES, and being transported to off target organs such as liver and spleen [81, 131]. For both doses, MATi particles highly accumulated in the lungs 15 min post-administration with an injected dose per organ (%ID/organ) between 60 to 80% (Fig. 3.4A). The dose reaching the liver at the same timepoint was less than 20%, while activity in the spleen was negligible (around 1%). As lungs weigh around 6 times less than liver, when activity was analyzed as percentage of injected dose per gram of tissue (%ID/g), very high concentration of activity was present in the target organ, while considerably lower amounts were found in the excretion organs (Fig. 3.4B). Ratio of the activity/gram in the lungs over other organs showed that lungs had around 25 times higher activity than the liver, nearly 40 times higher activity than the kidneys and spleen, and over 100 times higher activity then the blood at 15 min post-injection (Fig. 3.4C). 43 Figure 3.4: Biodistribution of MATi particles in healthy mice. Biodistribution analysis of MATi particles following intravenous administration of 500,000 or 700,000 particles expressed as A) injected dose per organ and B) injected dose per gram. Graphs show high accumulation of the MATi particles 15 min post-injection. C) Ratio of the amount of activity in the lungs compared to other organs, show similar ratios for both doses administered. Data presented as mean + SD, n = 3. This study proved that up to 700,000 MATi particles could be administered to healthy mice without obvious signs of toxicity and that the majority of them reached the lungs upon intravenous administration. 3.4 Chapter Summary MATi particles were studied in vitro and in vivo. Signs of cytotoxicity were observed for the particles at higher concentrations for both A549 and HUVEC cells, showing a similar pattern to free tiplaxtinin. By singularly analyzing the effect of the carrier and the drug on the overall cytotoxicity of MATi particles, it was found that at higher concentrations the decrease in cell viability was mainly influenced by tiplaxtinin. In preparation for the in vivo dose study, BSA was successfully modified and labeled with the radioisotope 111In. The radioactive particles 111In-MATi thus obtained retained the right size for lung targeting. When administered intravenously to healthy mice, doses of 500,000 and 700,000 MATi particles were well tolerated. Post-sacrifice 44 analysis showed high accumulation of the MATi particles in the lungs. Minor activity was found in off target organs such as liver and spleen, thus confirming that MATi particles could effectively target the capillary bed of lungs. 3.5 Experimental Section 3.5.1 Materials and Reagents Consumables for cell culturing and in vitro experiments using A549 cells were acquired from ThermoFisher Scientific (Burnaby, BC, Canada) and Corning (Corning, NY, USA), while consumables for the HUVEC cells were purchased from Lonza (Basel, Switzerland). MATi particles and blank MATi used for the cytotoxicity assays were prepared under aseptic conditions. The number of particles was measured using a CytoFLEX LX flow cytometer (Beckman Coulter, IN, USA), as previously performed in the lab [112, 132]. 111InCl3 in 0.05 N HCl was purchased from BWXT Isotope Technology Group (Vancouver, BC, Canada). Materials and reagents purchased from Bio-Rad Laboratories (Hercules, CA, USA) were used to prepare, run, and stain the SDS polyacrylamide gel. Saline was prepared in-house by dissolving NaCl (0.9% w/v) in Type 1 ultrapure water. For all experiments type 1 ultrapure water was used and was obtained from a Milli-Q® filtration system (MilliporeSigma). 3.5.2 MTT Assay A549 cells were cultured in F12K media supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin (100 U/mL and 100 µg/mL, respectively), while HUVEC cells were cultured in endothelial cell basal growth medium-2 (EBM-2) supplemented with EGMTM-2 MV microvascular endothelial SingleQuotsTM kit, prepared as suggested by the vendor. Cells were seeded on a 96 well plate at a density of 5000 cells/well (A549) or 2000 cells/well (HUVEC) and incubated at 37° C and 5% CO2 for 24 h to let them adhere to the bottom of the 45 wells. Following incubation, MATi particles or free tiplaxtinin in fresh media were added to the plates in final concentrations ranging from 2.5 to 256 μM, whereas MATi particles and blank MATi were added in amounts from 5,116 to 413,000 and from 1860 to 952,380, respectively. Only media was added to the untreated cells, used as negative control. Afterwards, cells were incubated again at 37° C, 5% CO2 for an additional 24 h. Following incubation, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) solution (20 μL, 5 mg/mL) was added to each well and cells were incubated in the same fashion for additional 2 h. Media was then slowly removed and 150 μL dimethylsulfoxide DMSO added to each well to solubilize the formazan crystals formed. Absorbance was measured at 540 nm (formazan absorption wavelength) and 670 nm (baseline) using the BioTek Synergy MX microplate reader. Cell viability was calculated using the following equation: 𝐶𝑒𝑙𝑙 𝑣𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦 (%) = 𝐴 𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑐𝑒𝑙𝑙𝑠 𝑎𝑡 540 − 𝐴 𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑐𝑒𝑙𝑙𝑠 𝑎𝑡 670𝐴 𝑢𝑛𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑐𝑒𝑙𝑙𝑠 𝑎𝑡 540 − 𝐴 𝑢𝑛𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑐𝑒𝑙𝑙𝑠 𝑎𝑡 670 × 100 where A treated cells at 540 and A treated cells at 670 are the absorbances measured at 540 nm and 670 nm of cells incubated with MATi particles, blank MATi, or free Tiplaxtinin, whereas A untreated cells at 540 and A untreated cells at 670 are the absorbance measured at 540 and 670 nm of cells incubated with only media. 3.5.3 Cell Death with YOYOTM-1 Dye For this assays, A549 and HUVEC cells were cultured and seeded in the same manner as described per the MTT assay. Cells were then incubated for 24 h at 37° C and 5% CO2 to let them adhere to the bottom of the wells. Afterwards, a 2-fold serial dilution of MATi, blank MATi particles, and free tiplaxtinin was prepared in fresh media (final drug concentrations ranging from 2.5 to 127 µM and particles from 5,116 to 512,680) and 25 µL of each sample was 46 added to the cells. Only media was added to the untreated cells as negative control. For each well, 25 µL of media containing YOYO-1 dye (1 µL per 10 mL of media) were added. The plates were then placed in the ZOOM Incucyte live cell imager (Essen Bioscience, Ann Arbor, MI, US) or Incucyte® SX5 Live-Cell Analysis System (Sartorius, Göttingen, Germany) and phase contrast images at 20x or 10x were acquired every 6 h for a maximum of 84 h. At the same timepoints, the plates were also scanned using the green fluorescence channel and images showing fluorescence of the YOYOTM-1 dye were obtained. Images were then processed using the correspondent instrument software, Incucyte ZOOM 2016a or 2022B Rev2 and number of green fluorescence objects per well were obtained at each time point, which were then graphed against time. When analyzing the data with the 2022b Rev2 software, a threshold of 5 GFU was set to filter out background fluorescence. 3.5.4 111In-MATi Preparation and Characterization Radiolabeling of BSA for the in vivo dosage study was performed as stated elsewhere [112]. Briefly, BSA (11.7 mg, 0.17 µmol) was dissolved in 0.1 M NaHCO3. Following addition of p-SCN-Bn-DTPA (20 µL, 0.6 µmol) to the BSA solution, the sample was stirred at room temperature for 5 h. The sample was purified from unbound chelator with MilliQ water using an Amicon Ultra 0.5 mL centrifugal filter (30 kDa MWCO) membrane (MilliporeSigma). DTPA–BSA was then resuspended in 0.1 M (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES) buffer and lyophilized in aliquots using a FreeZone freeze dryer (Labconco; Kansas City, MO). Aliquots were stored at −20 °C as ready-to-radiolabel kits and used within 12-month. For the radiolabeling, one DTPA-BSA kit (~2 mg in 100 µL HEPES buffer) was thawed at room temperature and dissolved in MilliQ water and diluted 1:1 (v/v) with 0.1 M HEPES. 111InCl3 in 0.05 N HCl (17 MBq) was then added to the sample and incubated for 30 min at 700 rpm and 47 21° C. After incubation, a Tec-Control 150-771 strip (BioDex, Shirley, NY) and 0.1 M EDTA as mobile phase were used to investigate the radiochemical conversion (RCC) of DTPA-BSA in 111In-DTPA-BSA. For this, 1 µL of reaction solution was spotted at retention factor (Rf) = 0 on the strip, which was then developed using the mobile phase. The radioactive signal present on the strip was visualized and by using phosphor plate developed with a Cyclone phosphor imager, and quantified using the OptiQuant software (Packard Instruments, Downers Grove, IL, USA). Buffer exchange was then performed by centrifuging the sample twice with 0.5 M phosphate buffer pH 5.5 using an Amicon Ultra 0.5 mL centrifugal filter with a 30 kDa MWCO. Final RCC of purified 111In-DTPA-BSA was assessed using the same Tec-Control 150-771 strip system. To further confirm the radiolabeling of BSA and evaluate potential degradation of the protein due to the process, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was employed. Gels were prepared with a 12% resolving phase and a 4% stacking phase. Samples of native, DTPA-, and 111In-DTPA- modified BSA were mixed 1:1 (v/v) with 2x Laemmli buffer and loaded onto the wells of the gel (Cf = ~1 mg/mL, Vf = 10 µL). The buffer used to run the gel contained 25 mM Tris, 192 mM glycine, and 0.1% (w/v) SDS, pH 8.3. Gel staining was performed with Coomassie Brilliant Blue mix with 45% (v/v) methanol, 10% (v/v) glacial acetic acid in water. Once destained with water, the gels were scanned with an Odyssey CLx Imager (LI-COR Biosciences, Lincoln, NE, US) and analyzed with the correspondent software Image Studio (LI-COR). Radioactivity in the gel was analyzed via phosphorimaging as described for the ITLCs. To prepare radiolabeled MATi and blank MATi particles, cold BSA was spiked with 10% 111In-DTPA-BSA (0.1 mg). Particles were then prepared as stated in Chapter 2.4.2 and the final products 111In-MATi and blank 111In-MATi were obtained. The radioactive particles were then washed thrice by centrifuge (1000 rpm, 5 min) and resuspended in saline. RCC was then 48 assessed in the same manner as previously stated. Particles size distribution was measured with a Mastersizer 2000E Particle Size Analyzer equipped with a Hydro 2000SM Dispersion Unit (Malvern Instruments, Inc., Malvern, UK). 3.5.5 Animals Healthy female C57Bl/6 mice aged 4-6 weeks and with an average weight of 18 g were purchased from Charles Rivers Laboratories (Wilmington, MA, USA). Animals were acclimatized previous to the experiment and were housed in cages with unrestricted access to food and water. The mice were housed at least two per cage, and sources of enrichment and nesting materials were provided. 3.5.6 Dose Study 111In-MATi were diluted in saline to prepare injections containing 500,000 or 700,000 particles in 100 µL. C57Bl/6 mice were administered with the radiolabeled particles intravenously sine anesthesia and they were then placed back into their cages. After 15 min post-injection, the mice were firstly anesthetized using isoflurane (5% in O2), and then sacrificed via CO2 inhalation and cardiac puncture. Afterwards, the organs and tissue of interest, i.e., blood, lungs, liver, kidneys, spleen, pancreas, heart, bladder, urine, were collected in pre-weighted vials. Weight of each organ was measured and activity quantified using a Cobra II 5010 gamma counter (Packard Instruments). Percentage of injected dose per organ (%ID/organ) was calculated as per the following equation: %𝐼𝐷𝑜𝑟𝑔𝑎𝑛=𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 𝑜𝑟𝑔𝑎𝑛𝐼𝑛𝑗𝑒𝑐𝑡𝑒𝑑 𝑑𝑜𝑠𝑒× 100 Where the organ activity was decay corrected for the injection time. Percentage of injected dose per gram (% ID/g) was obtained by dividing % ID/organ by the weight of the corresponding 49 organ. As it was not always possible to fully remove the blood, a sample was measured by gamma counting and then standardized organ weight was used to calculate the % ID/organ. 3.5.7 Data Visualization and Analysis Figures were prepared with Prism 10 software (GraphPad, USA) and are presented as mean ± SD, n = 3 unless stated otherwise. Regression analysis, and calculation of IC50 were performed with Prism10. Radiotraces were prepared with OriginPro 2017 (OriginLab, USA). 50 Chapter 4: Biodistribution of the MATi Particles in a Healthy Murine Model 4.1 Synopsis Over the last few decades, and in particular following the roll out of the Pfizer and Moderna Covid-19 vaccines, DDSs proved their critical role in improving the drugs’ properties and offering targeted delivery. However, when preparing a new formulation, it is pivotal to understand the in vivo biodistribution of both carrier and drug. Indeed, this information provides useful insights for the refinement of the formulation design and the translational process from bench to bed. Nuclear medicine offers a valuable tool for the simultaneous assessment of the main components of a DDS in preclinical trials, for example via dual radiolabeling. We therefore used this technique to investigate the biodistribution of our carrier and drug simultaneously in a healthy murine model and assess the lungs’ exposure to tiplaxtinin over 24 h. 4.2 Background Drug delivery systems are critical for the manipulation of a drug’s pharmacokinetics and targeting to provide successful therapies [2]. For example, DDSs have been employed for the improvement of drugs solubility, half-lives, or targeted delivery leading to reduced off-target toxicity [133-135]. Some of these formulations were successfully translated into the clinic, such as Abraxane, where formulation of the anti-cancer drug paclitaxel with albumin nanoparticles improved its accumulation at tumor site and decreased toxicity effects, or the more recent Covid-19 vaccine, where lipid nanoparticles were used to improve stability of the loaded mRNA [136, 137]. However, like drugs, DDSs are also affected by biological interactions and/or physical barriers in the organism [2]. For example, nanoparticles in the circulatory system often accumulate in the liver and spleen due to reticuloendothelial system clearance [138]. Therefore, when designing new formulations, it becomes critical to investigate the biodistribution of the 51 major components via in vivo studies to understand their biodistribution and pharmacokinetics [139]. This kind of data is pivotal for further optimization and development of the DDSs. In this framework, nuclear imagining plays a remarkable role as it allows to non-invasively monitor a radiotracer, like DDSs, in vivo and provides fully quantitative analysis [139]. The term radiotracer has a rather broad definition and it includes any compound that is labeled with a radioisotope, such as protein, peptides, or small molecules [140]. Nuclear imaging includes techniques like positron emission tomography (PET) and single photon emission computed tomography (SPECT) [141]. Both PET and SPECT can detect gamma rays, which are ionizing photons emitted by nuclides when they decay to a more stable isotope [142]. PET imaging is based on the detection of photons pairs released in opposite directions (180° angle) at a discreet energy of 511 KeV, which is the result of radionuclides undergoing positron decay [143]. On the other hand, SPECT imaging is based on radionuclides that decay by electron capture or isomeric transition leading to the emission of single-photons with different energy peaks, which are specific for each radionuclide [144]. Other important characteristics of the radionuclides that should be considered when designing a radiotracer are their physical half-life, decay mode, and production method [145, 146]. Evaluating all these attributes is critical in deciding the most suited radiolabeling method as the final product should retain the characteristics of the native molecule [145]. Radiolabeling can be performed via two main techniques 1) direct, or 2) indirect (Fig. 4.1) [146]. With the first method, the radionuclide is bound directly to the molecule, whereas in the second case a chelator or spacer is required for the binding [146, 147]. Examples of direct labeling include 99mTc-MAA. When 99mTc in saline is added to the MAA kit, stannous chloride contained in the kit reduces 99mTc to a lower oxidation number allowing its conjugation to the particles [148]. Strikingly, the exact mechanism by which 99mTc binds to MAA has not 52 been fully elucidated yet, even though it was suggested that 99mTc might interact with the thiol groups of albumin [103]. On the other end, indirect labeling requires a chelator, which can coordinate with a radiometal and is bound to the molecule of interest via a linker or spacer [146]. The complex thus formed should prove to be stable to be used in vivo [149]. The very recently FDA approved 68Ga-PSMA-11 is a nuclear imaging agent for prostate cancer, which uses the chelator N,N′-bis-[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N′-diacetic acid (HBED-CC) for the stable binding of the PET radionuclide 68Ga [150]. Figure 4.1: Scheme of radiolabeling methods Schematic representation of direct and indirect radiolabeling methods. Figure inspired by Varani et al. (2022) [146] and Kozlovskaya et al. (2023) [151]. The figure was prepared with BioRender.com. The wide range of radionuclides as well as radiolabeling methods available makes nuclear imaging a rather versatile technique that can be applied in different settings, i.e., pre-clinical and clinical, diagnostic, theranostic, and therapeutics [145]. Furthermore, due to the different energy peaks of photons, two radionuclides can be used simultaneously to perform dual imaging [152]. Indeed, the difference in energies allows to distinguish the two signals and identify the in vivo distribution of the single radionuclide [152]. In the area of drug formulation, dual imaging 53 becomes a powerful tool as it allows to obtain information about a carrier and its drug at the same time [153-156]. This is advantageous as it results in less imaging time, reduced number of animals per study, and decreased data variability as distribution of the formulation can be evaluated in the same animal. Although dual imaging is a very appealing technique, its use is limited by the combinations of radionuclides that can be imaged simultaneously. This is because the photons emitted by the two radionuclides should have energy peaks different enough that the two signals can be separated or they can be corrected for photons crosstalk, i.e., when the two signals overlap [152, 154]. However, this is not always possible as the radio-tag should not change the physico-chemical properties of the native molecule, thus further restricting the combinations available [145]. This was the case for this project: while we were interested in monitoring both carrier and drug simultaneously in vivo, tiplaxtinin limited considerably our options. Indeed, due to its low molecular weight (439.38 g/mol) addition of a chelator would have caused a considerable change in its mass, whereas the absence of functional groups, such as phenols, hindered our ability to perform common direct labeling methods like radioiodination [147]. For this reason, we had to unfortunately renounce the possibility of performing imaging to investigate our formulation in vivo, but it was possible to evaluate it following mice sacrifice. To obtain two distinct signals, radionuclides undergoing different decay types were chosen: 1) 3H, a beta-particles emitter with a half-life of 12 years, and 2) 111In, a gamma-ray emitter with a half-life of 2.8 days. 3H decays solely by emission of low energy beta-particles produced by β- decay [157]. 111In, instead, emits gamma rays at peak energies of 171 and 245 keV upon electron capture [149]. Due to the very different decay method between 3H and 111In, their signals can be detected by different instrumentation: 111In gamma rays can be detected by solid and liquid scintillators, whereas β- -particles from 3H can be detected only by liquid scintillators. Therefore, 54 the two signals can be measured by: 1) gamma counter for 111In; 2) beta scintillator for 3H. Gamma counters contain inorganic or solid scintillators most commonly made of thallium-doped sodium iodide ((NaI(Tl)) crystals [158]. Gamma rays interact with the atoms in the crystals, exciting them. These atoms then release the energy as light as they go through de-excitation, and the light emitted is translated into electrical signal by a photomultiplier [158]. Contrarily, organic scintillators are usually in liquid form in a scintillation cocktail to which the radioactive sample is added [158]. The scintillation cocktail comprises few elements with the most important being the primary solute, i.e., scintillator, which shifts to an excited state as the decay energy is absorbed, and releases it as light when returning to rest state [158]. Other components of the cocktail are the organic solvent in which the primary solute and the sample are dissolved in and which allows the energy transfer from the radionuclide to the scintillator, and the secondary solute, which might be needed to shift the light emitted from the scintillator to a longer wavelength thus optimizing the detection by the photomultiplier [158]. Although the mechanism is different, the main principle behind both instruments is the same: the light produced by the scintillator is proportional to the energy deposited by interaction with gamma rays or beta particles [158]. Since gamma rays can be detected by organic scintillators as well, for this study radionuclides with widely different physical half-lives were chosen. Due to the shorter half-life of 67 h, 111In decays faster than 3H, which has a half-life of 12.4 years [159]. Therefore 3H signal can still be measured after 111In decay, ensuring no interferences from the gamma rays. With this method we were able to study the biodistribution of the main components of the MATi particles in vivo. The main aim of the chapter was to prepare dual labeled 111In-MATi[3H-MATi] particles and investigate the biodistribution of the drug and the carrier simultaneously in a healthy murine 55 model, as well as assess the exposure of the lungs to tiplaxtinin over 24 h and compare it to the unformulated drug. The secondary aim was to evaluate whether the concentration of tiplaxtinin that we were able to deliver with the MATi particles would be sufficient to elicit an anticoagulatory response. 4.3 Results and Discussion 4.3.1 Dual Labeling of MATi Particles and Dose Assessment For the biodistribution study, the formulated tiplaxtinin was compared to the free drug as control. Both MATi particles and tiplaxtinin were administered intravenously, therefore a compatible vehicle was needed, e.g., saline. However, due to the hydrophobic nature of tiplaxtinin its solubility in aqueous-based solutions is rather low, thus precipitation could occur. In the literature, studies administering tiplaxtinin in vivo used the oral route as it showed good bioavailability in initial pharmacokinetics assessments with 93% availability in rodents and 32% in a canine model [86, 92]. Tiplaxtinin was administered orally after easily dissolving it in oil, manually administering it or adding it to the animals’ diet [128, 160, 161]. However, in our study the aim was to compare the biodistribution of the drug via the same route of administration of the MATi particles, as well as demonstrate whether our formulation could overcome solubility problems. Therefore, we were interested in investigating the concentration of free tiplaxtinin that could be administered with an i.v. friendly vehicle, e.g., saline, without the addition of harmful solvent such as DMSO that could cause toxicity. Thus, serial dilution of tiplaxtinin in isotonic solutions, i.e., saline and 1x PBS, were investigated for signs of precipitation. As the structure of BSA contains pockets that can bind hydrophobic drugs, it is often employed to improve their solubility for intravenous administration [162]. For this reason, a solution of 1% (w/v) BSA in saline was used as control. Serial dilutions were therefore prepared in 1x PBS, saline, or 1% 56 BSA in saline for final concentrations ranging from 0.2 μM to 25 μM. The samples were added to a 96 well plate and optical density (OD) measured at 405 nm [163]. An increase in OD indicates presence of precipitates, suggesting poor drug solubility. Interestingly, when tiplaxtinin was dissolved in saline, OD increased considerably as the drug concentration increased, with OD being above background even at the lowest concentration tested (Fig. 7). This confirmed that saline was not a suitable media for the drug. Although 1x PBS seemed to improve the solubility of tiplaxtinin compared to saline, OD measurements showed values above background for all the concentrations tested. As expected, no signs of precipitation were observed when tiplaxtinin was mixed with 1% BSA in saline, thus indicating binding of the drug to the protein and proving BSA to be a compatible drug carrier (Fig. 4.2). These observations were further confirmed by dynamic light scattering (DLS) analysis of the samples (Supplementary Fig. C.1.1). Due to the improved solubility of the drug in PBS compared to saline, the latter was chosen as the injection media for the in vivo study. However, as aggregation signs were observed at the lower concentrations of tiplaxtinin, it was decided to administer only tracer doses of the drug, i.e., in the nM range, to avoid precipitation while still being able to quantify the activity from 3H. 57 Figure 4.2: Solubility of tiplaxtinin in aqueous-based media Serial dilutions of tiplaxtinin in saline, 1x PBS, or 1% BSA in saline were measured at 405 nm. An increase in optical density indicates presence of precipitates. As BSA can bind hydrophobic drugs improving their solubility, it was used as control. PBS showed better compatibility with tiplaxtinin compared to saline. n = 3. To investigate the biodistribution of MATi particles in a murine model, dual radiolabeling was performed. The latter technique allowed us to monitor the drug and its carrier simultaneously. To prepare the dual labeled MATi particles (111In-MATi [3H-TPLN]), the drug was labeled directly via 3H substitution (Fig. 4.3A), whereas the carrier, i.e., BSA, was labeled indirectly with 111In via DTPA chelation, in the same fashion as described in Chapter 3.5.4 (Fig. 4.3B). 0 5 10 15 20 250.0000.0150.0300.0450.060Concentration (µM)Absorbance (405 nm)TPLN in PBSTPLN in SalineTPLN + 1% BSA58 Figure 4.3: Radiolabeling of tiplaxtinin and BSA A) Tiplaxtinin was radiolabeled with the beta-particles emitter 3H. Tiplaxtinin was labeled with 3H via substitution on the aryl group. B) BSA was radiolabeled with the gamma emitter 111In via chelation with DTPA. Taking advantage of the amines present on the BSA structure, DTPA was bound to the protein via a thiourea link. Figure B reproduced with permission from Esposito et al. (2021) [112]. To evaluate the radiolabeling stability of the particles, the 111In-DTPA complex was challenged with the competitive chelator ethylenediaminetetraacetic acid (EDTA). Free 111In-DTPA-BSA was used as control. 111In-MATi particles showed good radiolabeling stability for the first 2 h, while it decreased at the later timepoints, with 57% bound activity after 24 h, compared to 94% of the control 111In-DTPA-BSA (Fig. 4.4A). However, when 111In-MATi particles were tested in physiologically relevant media, i.e., 50% human plasma, at 37°C, the particles proved to be very stable with 96% bound activity at 24 h, which matched the value obtained for 111In-DTPA-BSA (96%) (Fig. 4.4B). The plasma stability observed was very similar, although slightly higher, to results reported in the literature. For example, Loob-de Hooge et al. observed a 7% decreased of bound 111In per day when incubating 111In-DTPA-Trastuzumab in human serum at 37° C [164]. Similarly, Alirezapour et al. reported a 96 ± 0.91% bound activity when evaluating the stability of 111In-DTPA-mAb using the same conditions in a recent study from 2022 [165]. Differently, Palmowski et al. observed that MAA labeled with 111In-DTPA had a plasma stability of 95% 59 activity bound up to 3 days [166]. However, the temperature at which the 111In-DTPA-MAA were incubated was not specified. Figure 4.4: Radiolabeling stability of 111In-DTPA complex A) Radiolabeling stability of 111In-MATi was tested in presence of the competitive chelator EDTA. 111In-DTPA-BSA was used as control. The radiolabeled particles showed good stability up to 2 h, while at 24 h 56% of 111In was bound to the particles. N = 3 ± SD. B) When 111In-MATi were incubated at 37° C in 50% human plasma they showed very good stability with 96% activity bound to the particles at 24 h. N = 3. The labeling of tiplaxtinin with 3H also allowed us to directly assessed the drug loading by measuring activity in the particles, which resulted in 12 ± 3 % (N = 4). Although in the lower end, this result was found not significantly different from the drug loading assessed via indirect method using UV-Vis. The difference observed in the drug loading averages measured, directly and indirectly, could be due to the diverse methods employed for their assessment. 4.3.2 Biodistribution of 111In-MATi 3H-TPLN in a Healthy Murine Model For this study, healthy C57Bl/6 were administered intravenously with the dual labeled 111In-MATi [3H-TPLN] particles or free 3H-TPLN as control. At set timepoints up to 24 h, mice were sacrificed, organs of interest collected, and 111In and 3H signals quantified for each organ. A scheme of the study design is shown in Fig. 4.5 below. 60 Figure 4.5: Experimental design of the biodistribution study To assess the biodistribution of the dual labeled 111In-MATi[3H-TPLN] particles in a healthy murine model, 250000 particles were administered intravenously, while free 3H-TPLN was administered as control. Animals were then sacrificed at set timepoints (n = 3 per timepoint) and activity in each organ quantified. As expected, the albumin microparticles highly accumulated in the lungs of the mice upon injection. Consistently to the previous study, lungs had 60.4% of injected dose per organ (ID/organ) at 0.1 h post injection (Fig. 4.6A) and minimal accumulation in the liver and spleen was observed at the same timepoint, with only 9.03% and 0.88% ID/organ, respectively (Fig. 4.6B, 4.7C). The difference in organ accumulation was even more dramatic when the activity measured was standardized by the organ weight and expressed as %ID/g of tissue (Supplementary Fig. C.2.1A, B). As the particles break down and enter the systemic circulation, the activity in the lungs decreases while activity in the liver increases (Fig. 4.6A, B). Particles degradation in the lungs could be mainly caused by mechanical deterioration due to the shear force of blood in the vessels, and to a lesser extent by enzymatic degradation [112]. The half-life of the 111In-MATi particles in the lungs was 2.3 h. While an initial study of 99mTc-MAA biodistribution in humans performed by Chandra et al., reported the particles half-life in the lungs to be nearly 4 h, our value aligns with the one described for the commercial 99mTc-MAA 61 kit by DraxImage (2-3 h) [37, 167]. The half-life observed for free and formulated tiplaxtinin in the lungs was very similar (0.43 and 0.4 h, respectively). Nearly all of the free 3H-tiplaxtinin (control) administered reached the liver upon injection (87.7% ID/organ), it was then rapidly excreted via the hepatobiliary system within the first hour (where the liver concentration decreased below 20% ID/organ) until reaching only 1% ID/organ at 24 h post-injection (Fig. 4.6B). Following administration, only 0.54% ID/organ of free 3H-tiplaxtinin was able to reach the lungs (Fig. 4.6A). However, once the drug was formulated with the particles, significantly higher amount of tiplaxtinin was delivered to the lungs over the first 6 h, with an initial 2.7% ID/organ at 0.1 h (Fig. 4.6A). When looking at %ID/g, formulated tiplaxtinin reached 21.5% compared to 4.7% of the unformulated one (Supplementary Fig. C.1.2A). However, within 24 h p.i. virtually all of the free drug and formulation were excreted. Furthermore, only 37% ID/organ of the formulated tiplaxtinin was found in the liver at 0.1 h, more than two-fold decrease compared to the free drug (Fig. 4.6B). Optimization of the formulation might reduce even further the amount of tiplaxtinin reaching the liver. Nevertheless, MATi particles were able to decrease the amount of drug in the liver by more than half. Overall, with the MATi particles we were able to deliver up to a 12-fold increase of tiplaxtinin to the lungs (Fig. 4.6C). Fig. 4.6D shows the higher exposure of the lungs to drug when formulated with the MATi particles. The area under the curve (AUC) for formulated tiplaxtinin was five times higher than the unformulated one (21.3, and 4.2 %ID × h/organ, respectively). 62 Figure 4.6: Biodistribution of 111In-MATi[3H-TPLN] and free 3H-TPLN in lungs and liver A) Activity of dual labeled 111In-MATi[3H-TPLN] and free 3H-TPLN in the lungs over 24 h. When formulated, significantly higher amount of tiplaxtinin was delivered to the lungs. n = 3. B) Activity of dual labeled 111In-MATi[3H-TPLN] and free 3H-TPLN in the liver over 24 h. Most of the unformulated tiplaxtinin (87.7% ID/organ) reached the liver upon administration, whereas it decreases to 37% once formulated. n = 3. C) Ratio of formulated over unformulated tiplaxtinin in the lungs over time shows up to a 12-fold increase of tiplaxtinin in the targeted organ when formulated with the MATi particles. D) Exposure of the lungs to the free 3H-TPLN (black, AUC = 4.2) and to MATi[3H-TPLN] (magenta, AUC = 21.3). Statistical analysis performed by two-way ANOVA with Holm-Sidak correction (*, p < 0.05; ***, p< 0.005; ****, p<0.0001). %ID, percentage of injected dose; TPLN, tiplaxtinin. Activity present in all the other organs and tissues analyzed was below 15% ID/organ in both groups for all the timepoints tested. For the 3H signal measured in both free and formulated tiplaxtinin, higher activity was found in the kidneys at 0.1 h p.i. which decreased steeply within the first hour (Fig. 4.7A, B). Although the activity was only 3.5% and 2.5% for the free and 63 formulated drug, respectively, it might be due to a small fraction of 3H being quickly excreted through the renal pathway. Differently, the 111In signal provided by the carrier, activity in the kidneys increased over time (1.6% 0.1 h p.i. to 11.7% 24 h p.i.) as the particles are degraded and BSA enters the circulation and it is excreted (Fig. 4.7C). As a result, activity in the urine was detectable up to 6 h p.i. (Supplementary Fig. C.2.2). A similar pattern was observed for the spleen (0.88% at 0.1 h p.i. to 4.99% at 24 h p.i.) as the particles break into smaller parts and are carried to the spleen and liver by the RES. Analysis of %ID/g showed that activity concentrated more in the spleen then the liver, with 28.4% and 56.3% at 24 h, respectively (Supplementary Fig. C.2.1B and C.2.2). As tiplaxtinin reaches the liver upon administration, both in the unformulated and formulated form, and is excreted through the hepatobiliary system, activity increased in the small intestine (Fig. 4.7A, B) within the first hour to then decrease over time as the drug is excreted through the feces (Supplementary Fig. C.2.3 and C.2.4). 64 Figure 4.7: Biodistribution of 111In-MATi[3H-TPLN] and free 3H-TPLN A) Biodistribution of free 3H-TPLN over 24 h. At 0.1 h post-injection (p.i.), higher activity was observed in the blood and kidneys, while within the first hour activity increases in the small intestine as the drug is excreted via the hepatobiliary pathway and feces over 6 h p.i. B) Biodistribution of formulated 3H-TPLN followed a similar pattern as the unformulated drug. However, higher activity was measured in the blood at 0.1 h p.i., and decreased activity was in the kidney for the same timepoint. C) Biodistribution of 111In-MATi over 24 h. Activity increased over time in the kidneys and spleen as the particles are broken down and dislodge from the lungs. Activity increased in the urine as well as it is excreted via renal pathway. %ID, percentage of injected dose; TPLN, tiplaxtinin. n =3. As the particles should distribute uniformly in the lungs, activity provided by 111In bound to the carrier was analyzed separately in the right and left lungs of mice to evaluate potential differences. Anatomically, the left lung of mice is smaller than the right one as it is composed by 65 one lobe only, whereas the right one is formed by four lobes [168]. Therefore, the average weight of the left lung is generally half the weight of the right. For this reason, when comparing the ID/organ between the two sides a difference is noticeable with 39.6% in the right lung compared to 20.7% of the left lung at 0.1 h p.i. (Fig. 4.8A). However, when the value was standardized by the weight of the organ, obtaining the percentage of ID per gram of organ (%ID/g), the activity present in the right and left lung was very similar (465% and 509%, respectively) and no significant difference was found (Fig. 4.8B). This indicates that the MATi particles distributed uniformly in the lungs’ capillaries and arterioles. Figure 4.8: Distribution of the MATi particles in the left and right lung A) Percentage of injected dose measured for the carrier (111In-MATi) in the left and right lung of mice over 24 h showing higher accumulation of 111In-MATi in the right lung as its weight is roughly double the weight of the left lung. B) When the activity measured was standardized by the organ weight (%ID/g), no significant difference was found between the left and right lungs, indicating uniform distribution of the particles. Statistical analysis performed with two-way ANOVA with Holm-Sidak correction. ID, injected dose; L, left; R, right. n = 3. Dual radiolabeling provided us important insights on the MATi particles proving that we were able to achieve higher accumulation of the drug in the organ of interest, i.e., lungs. However, optimization is needed to further reduce the drug reaching off-target organs, i.e., the liver. 66 4.3.3 Activity of Tiplaxtinin for Blood Clot Resolution Since MATi particles were prepared via thermal aggregation, tiplaxtinin was chosen over marketed PAs due to its higher thermostability. Indeed, loss of their tertiary and/or secondary structure during the heating process would likely result in lack of fibrinolytic activity [169]. On the other hand, tiplaxtinin is as a small molecule, thus should endure the process and retain its properties. To test this hypothesis, tiplaxtinin purified from the MATi particles supernatant was analyzed by high performance liquid chromatography coupled with mass spectrometry (HPLC-MS). Tiplaxtinin purchased and stored at -20° C, as per seller indications, was used as control. Chromatogram at 254 nm of the control tiplaxtinin showed one major peak at 8 min representing the drug (Fig. 4.9A). Purity of the sample was assessed at 97.8% by integration of the area under the curve. Similarly, tiplaxtinin purified from the supernatant showed the same major peak and purity was assessed at 95.7%, thus indicating good stability of the molecule throughout the process of the MATi particles (Fig. 4.9B). Mass spectra of the control and purified tiplaxtinin confirmed that the peak observed in the chromatogram was tiplaxtinin, as a peak corresponding to its molecular weight (439.38 g/mol) was visible. However, in both mass spectra, another peak showed the presence of a molecule with molecular weight of 296 g/mol at the tiplaxtinin’s eluting time. As the peak was present also in the control, the hypothesis of it being a degradation product was discarded. Instead, it was hypothesized that the molecule observed is a mass spectroscopy fragmentation of tiplaxtinin. By studying different fragmentation patterns typical of mass spectroscopy, it was found that a tiplaxtinin fragment with molecular weight of 296 g/mol could be obtained following a proposed fragmentation of the aryl ether, loss of the hydrogen from the primary amine, and alpha cleavage (for details see Appendix C.3.1). 67 Figure 4.9: Thermostability of tiplaxtinin A) Chromatogram and correspondent mass spectrum of tiplaxtinin as received from the provider. The drug showed nearly 98% purity and a peak at the correct molecular weight was present (m/z = 440.1). B) Chromatogram and correspondent mass spectrum of tiplaxtinin following MATi particles preparation. The drug proved thermostable with a main peak on the chromatogram showing nearly 96% purity, and a peak at corresponding to tiplaxtinin molecular weight on the mass spectrum. In both mass spectra another peak at a m/z value of 296.2 was visible, which was assessed to be result of tiplaxtinin fragmentation due to mass spectroscopy. Overall, tiplaxtinin was considered thermostable and could withstand the MATi particles preparation process, indicating that the drug released by the MATi particles at the site of action would be in its active form. We were therefore interested in studying whether the concentration of tiplaxtinin that we were able to deliver to the lungs at 0.1 h p.i., would be sufficient to provide 68 a thrombolytic effect. Firstly, the drug concentration in lungs over time was estimated based on the percentage of injected dose reaching the lungs and the lungs’ blood volume of mice [170]. It was calculated that when tiplaxtinin was formulated with the MATi particles the initial concentration in the lungs was 6.9 µM, which is higher than the established drug IC50 (2.7 µM) (Fig. 4.10A) [86]. As the drug is released, enters the circulation and is excreted, the concentration in the lungs decreases over time until reaching 0.02 µM at 24 h. However, when the free drug was administered, the drug concentration in the lungs did not exceed 0.5 nM at any timepoint. The lower concentration of the unformulated tiplaxtinin was most likely due to two main reasons 1) poor solubility of the drug, which allowed us to only administer nanograms of 3H-TPLN; 2) lack of targeting, which did not allow the drug to accumulate in the lungs. Therefore, MATi particles allowed us to overcome the drug solubility problems and administered a considerable higher dose, which resulted in a concentration of 2 orders of magnitude higher in the targeted organ upon injection. To assess the potential of the drug to inhibit the target PAI-1 in the lungs, an in vitro clot lysis assay was performed. The assay utilizes the euglobulin fraction of the blood, which contains various proteins involved in blood coagulation and fibrinolysis including PAI-1 and plasminogen [171]. This assay allows to monitor blood clot formation and lysis by measuring absorbance at 405 nm over time: as the blood clot is formed, the absorbance increases and, consequentially, as the clot undergoes lysis, absorbance decreases. Therefore, a decrease in the AUC indicates an increase in clot lysis. As shown in Fig. 4.10B, the AUCs obtained from the clot lysis assay decreased as the concentration of tiplaxtinin increased, meaning a faster lysis. From the graph, it is visible that the tiplaxtinin concentration of 6.9 μM (dashed line) obtained in the mice lungs at 69 0.1 h p.i. using the MATi microparticles, can accelerate clot lysis compared to free tiplaxtinin (dashed line). Figure 4.10: Lytic effect of tiplaxtinin A) Estimated concentration of tiplaxtinin over 24 h, showing a 4 order of magnitude increase when the drug was delivered with the MATi particles n = 3. B) A decrease in AUC as the concentration of tiplaxtinin increases indicates the lytic effect of the drug in vitro. n = 1. The clot lysis assay indicates that the estimated concentration of tiplaxtinin delivered to the lungs by the MATi particles could have lytic effects on the blood clots present. However, further investigations in in vivo models are needed for conclusive results. 4.4 Chapter Summary In this chapter we showed the utility of dual radiolabeling as a tool for the fully quantitative analysis of drug delivery systems in a murine model. The dual labeling and biodistribution analysis allowed us to characterize the drug and the carrier simultaneously while reducing inter-mice variability as well as the overall number of mice needed for the study. Biodistribution analysis showed that with the MATi particles it was possible to deliver a significantly higher amount of tiplaxtinin to the lungs within the first 6 h from administration compared to the free 70 drug, with a maximum of 12-fold increase at 3 h p.i. With the MATi particles it was also possible to reduce by more than half the amount of tiplaxtinin reaching the liver at 0.1 h post-injection. However, optimization of the MATi particles could lead to a further decrease of the amount of tiplaxtinin reaching the off-target organ. Lastly, it was shown that tiplaxtinin is thermostable and can withstand the particles’ preparation process thus retaining its activity. With an in vitro clot lysis assay, it was shown that even at low concentrations, tiplaxtinin could induce a lytic response which helped to complement what was observed in vivo. 4.5 Experimental Section 4.5.1 Materials and Reagents 111InCl3 in 0.05 N HCl was purchased from BWXT Isotope Technology Group (Vancouver, BC, Canada), while 3H-tiplaxtinin was custom prepared by ViTrax radiopharmaceuticals using a substitution method (Placentia, CA, USA). Acetonitrile (HPLC grade, ≥ 99.9%) was obtained from Sigma Aldrich (Merck KGgA, Darmstadt, Germany). PBS (1x) used in the experiments was purchased by ThermoFisher Scientific (Burnaby, BC, Canada). Falcon® 96-well plates were obtained from Corning (Corning, NY, USA). Saline was prepared in-house by dissolving NaCl (0.9% w/v) in Type 1 ultrapure water. For all experiments type 1 ultrapure water was used and was obtained from an in-house Milli-Q® filtration system (MilliporeSigma). 4.5.2 Tiplaxtinin Solubility Assessment Serial dilutions (2-fold) of tiplaxtinin were prepared in acetonitrile to fully dissolve the drug. Each sample was then diluted in 1x PBS, saline, or 1% BSA in saline for final concentrations ranging from 0.2 to 25 μM. The samples (100 µL) were then added to a 96-plate (Corning®), and optical density measured at 405 nm using a BioTek® SynergyTM HT microplate reader (Winooski, VT, USA). Samples (40 µL) at the same concentrations were added to disposable 71 cuvettes and measured by dynamic light scattering using a Zetasizer Nano ZS (Malvern Panalytical, UK). 4.5.3 Radiolabeling Stability Radiolabeling stability was investigated by challenging the 111In-DTPA complex with chelator EDTA. Blank 111In-MATi (50 µL, ~0.19 MBq) were incubated with 5 µL 0.01M EDTA at 37° C and 700 rpm using a thermomixer (Canadawide Scientific, Ottawa, ON, Canada). 111In-DTPA-BSA (50 µL, 0.85 MBq) was used as control. At 0, 0.5, 1, 2, 16, and 24 h samples were gently resuspended and 1 µL was spotted at Rf = 0 on a Tec-Control 150-771 strip (BioDex, Shirley, NY) and developed with 0.1 M EDTA as mobile phase. Once all the samples were collected, the strips were developed with a phosphor plate and presence of free 111In quantified using the Cyclone Phosphorimager and OptiQuant software (Packard Instruments, Downers Grove, IL, USA). Bound activity of the samples was normalized to their 0 h timepoint and graphed over time. To assess plasma stability, whole blood obtained from donors was collected in BD Vacutainer® collection tube (Dutscher, Bernolsheim, France) with 9 NC sodium citrate 0.109M to prevent coagulation. Blood was maintained at 4° C until processed (within 2 h from of drawing). To obtain plasma, the whole blood was centrifuged at 2000 g for 15 min at 4° C and the supernatant collected and aliquoted. Plasma was kept at -80° C and used within 3 months. Plasma stability of the particles was tested by pre-warming at 37° C 50% of human plasma in 1x PBS containing 100 U/mL penicillin and 100 µg/mL streptomycin pl. Blank 111In-MATi (50 µL, 0.19 MBq) and 111In-DTPA-BSA (50 µL, 0.85 MBq) were added to the plasma and maintained at 37° C, 700 rpm throughout the whole experiment. At 0, 0.5, 1, 2, 16, and 24 h, 10 µL samples were collected and placed on dry ice. Following the last timepoint, samples were thawed at room 72 temperature and mixed with 2x Laemmli buffer in 1:1 (v/v) ratio and run on a 12% SDS PAGE gel. Gels were stained using Coomassie Blue to visualize the proteins, while radioactivity was identified by using the Cyclone Phosphor imager and analyzed with the OptiQuant software (Packard Instruments, Downers Grove, IL, USA). Radioactive bands at ~60 kDa (BSA MW) were quantified and normalized to their 0 h timepoint and graphed against time. 4.5.4 Preparation of 111In-MATi [3H-TPLN] and Characterization 3H-TPLN was shipped in acetonitrile and store at -20° C as per provider recommendations. As the radiolabeled drug was shipped very diluted in organic solvent, for the preparation of dual labeled MATi particles it was necessary to concentrate it and change the solvent to NMP to prepare the particles as per protocol. 3H-TPLN (600 µL, 4 µmol) in acetonitrile was added to a glass vial and the solvent evaporated using a vacuum pump. 3H-TPLN was then reconcentrated in 32 µL of NMP. The concentrated 3H-TPLN was used to spike cold tiplaxtinin for a final amount of 1.8 mg in a total of 72 µL NMP. The spiked TPLN was mixed with 3 µL of FeCl2 (50 mg/mL) in a 10 mL round bottom flask containing a PTFE stirrer set at 150 rpm. Afterwards, BSA spiked with 10% 111In-DTPA-BSA (2% w/v, 75 µL) in 0.5 M phosphate buffer pH 5.5 was added to the flask and the solution stirred for 1 min. In the meantime, 100 µL MQ water and 100 µL NMP were added to the flask. Under continuous stirring, the flask was stoppered and lowered in a water bath at 80° C for 9 min. Afterwards, the solution was left to cool at room temperature, then the particles were collected and washed thrice with 150 µL 1x PBS. Particles were diluted accordingly with 1x PBS to obtain 250,000 111In-MATi [3H-TPLN] particles/injection. For the group receiving free 3H-TPLN, 60 µL (0.67 MBq, 0.8 µmol) of the radiolabeled drug were diluted in 2.28 mL 1x PBS and 130 µL were used per injection. The particles size distribution was assessed with a Mastersizer 2000E Particle Size Analyzer equipped with a Hydro 2000SM 73 Dispersion Unit (Malvern Instruments, Inc., Malvern, UK). Drug loading was assessed directly by mixing 10 µL of the particles prepared with 5 mL CytoScintTM ES scintillation fluid (MP Biomedicals; Irvine, CA, USA) and measured with a LS 6500 multi-purpose scintillation counter (Beckman CoulterTM ; Brea, CA, USA) following 111In decay. 4.5.5 Animals Healthy female C57Bl/6 mice age 4-6 weeks and an average weight of 18 g were purchased from Charles Rivers Laboratories (Wilmington, MA, USA). This study was designed for the biodistribution assessment of the particles and healthy animals were used, therefore the sex of the animals is not critical. However, sex should be carefully considered when designing in vivo efficacy studies. Animals were acclimatized previous to the experiment and were housed in cages with unrestricted access to food and water. The mice were housed at least two per cage, and sources of enrichment and nesting materials were provided. 4.5.6 Biodistribution Study in Healthy Murine Model For this study, healthy C57Bl/6 mice were randomly divided into two groups (n = 3 per timepoint) and were administered intravenously sine anesthesia with either 1) 250,000 111In-MATi[3H-TPLN] particles in 1x PBS (~ 0.15 MBq/injection 111In, and ~ 0.074 MBq/injection 3H, ~ 20 µg of tiplaxtinin per injection) or 2) free 3H-TPLN in 1x PBS (around 0.01 MBq/injection, ~ 6 ng tiplaxtinin/injection). Based on the dose study in Chapter 2, we knew that the number of particles administered was safe, as well as activity administered for both 111In and 3H would allow for quantification. Syringes were weighed before and after injection to calculate the volume administered. Based on the volume administered, it was possible to calculate the injected activity for both 111In and 3H using the following equation: 74 𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 𝑎𝑑𝑚𝑖𝑛𝑖𝑠𝑡𝑒𝑟𝑒𝑑 =𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑖𝑛𝑗𝑒𝑐𝑡𝑖𝑜𝑛 (𝑐𝑝𝑚µ𝐿 )𝑉𝑜𝑙𝑢𝑚𝑒 𝑖𝑛𝑗𝑒𝑐𝑡𝑒𝑑 (µ𝐿) At 0, 1, 3, 6, and 24 h mice were firstly anesthetized with isoflurane (5% in O2), then sacrificed by CO2 inhalation and cardiac puncture. Following sacrifice, organs were collected and placed in pre-weighted tubes. Tubes were then weighted again to obtain the total organ weight, and 111In activity measured with a Cobra II autogamma counter (Packard Instruments, USA). Organs were then kept in the freezer for at least a month to let 111In decay. For the digestion of the organ and quantification of 3H signal, the method used was based on PerkinElmer’s protocols [172, 173]. Briefly, organs (either whole or a piece) were transferred into glass scintillation vials (20 mL), thawed at room temperature, and digested by incubating them with 1 mL of alkaline solution (Solvable, Perkin Elmer) at 50-60° C until dissolution. H2O2 (0.2 – 0.3 mL) was then added in aliquots of 100 µL at time to decolorize the samples and the sample further incubated for 30 min at 50-60° C. Samples were then left to cool at room temperature before adding 10 mL of liquid scintillation cocktail (Ultima Gold, Perkin Elmer). Samples were temperature and light adapted for at least 1 h before measuring them on a LS 6500 multi-purpose scintillation counter (Beckman CoulterTM ; Brea, CA, USA). Percentage of injected dose per organ and gram was calculated in the same manner as described in Chapter 3.5.6. Urine samples for free 3H-TPLN and MATi[3H-TPLN] were not analyzed as the volume was too little for transfer into the scintillation vials. Each organ was assessed in triplicate except for blood at 6 h in the MATi[3H] group, where n = 2 due to a processing mistake of one sample. 4.5.7 Tiplaxtinin Thermostability To evaluate potential degradation of tiplaxtinin during the MATi particles preparation process, mass spectrometry was performed. Tiplaxtinin received by the seller was dissolved in 75 acetonitrile (0.1 mM) and used as control. A fresh batch of MATi particles was prepared as per protocol (Chapter 2) and tiplaxtinin in the supernatant was purified with a PD MiniTrapTM G-25 column containing a crossed-linked dextran matrix (exclusion limit, Mr: 5000) in the same fashion as described in Chapter 2. The purified tiplaxtinin was diluted with 40% ethanol to a concentration of 0.1 mM. Both samples were filtered with a Millex®-GV (PVDF, 0.22 µm pore size) filter. Control and purified tiplaxtinin were then analyzed with an Agilent HPLC column Zorbax SB-C18 and streamlined to a single quadrupole LC/MSD (Agilent InfinityLab G6125B). Using 95% water + 0.1% formic acid (FA) as solvent A, and 100% acetonitrile (MeCn) + 0.1% formic acid (FA) as solvent B, a solvent gradient was run over 14 min: 0-3 min 90% solvent A; 3-6 min 90% solvent B; 6-9 min 99% solvent B; 9-12 min maintained 99% solvent B; 12-12.01 min decreased to 10% solvent B; 12.01-14 min maintained 10% solvent B; flow 0.5 mL/min. UV absorbance was measured at 254 nm and used for purification analysis by integrating the area under the curve (AUC). Mass spectra were acquired by API-ES ion source. 4.5.8 Tiplaxtinin Activity The euglobulin lysis assay was used to assess tiplaxtinin activity [171]. Citrated whole blood obtained from a donor was centrifuge at 2500 g at 4° C for 20 min. The plasma (supernatant) was collected and centrifuged again at 2500 g at 4°C for 20 min. Acetic acid (0.016%) was added (1:18 v/v) to the blood fraction obtained, and placed on ice for 10 min. The sample was then centrifuged at 2000 g for 5 min at room temperature. The supernatant was discarded and the pellet (euglobulin fraction) gently resuspended in the same volume as the initial plasma using 154 mM NaCl 2.6 mM sodium borate, pH 9.0. The sample was placed in a water bath at 37° C for 90 s, the tube was then gently inverted several times and incubated for an additional 90 s. In a 96 well plate, tiplaxtinin (60 µL) was added to the wells in final 76 concentrations of 2.5 - 20 μM. 1x PBS was used as negative control. Afterwards, 120 μL of 25 mM CaCl2 were added to the wells, followed by 120 μL of euglobulin fraction. As soon as the latter was added to the plate, optical density at 405 nm was measured every 3 min for 720 min with a BioTek Synergy MX microplate reader. 4.5.9 Data Visualization and Statistical Analysis Figures and statistical analysis were performed using Prism10 (GraphPad, USA). Data are presented as mean ± SD, n = 3 unless otherwise stated. Statistical tests were performed using Prism10, namely unpaired t-test and two-way ANOVA with Holm-Sidak correction. Statistical significance was set at p < 0.05. Chemical structures were prepared with ChemDraw 19.1 (PerkinElmer, USA). Illustrations were created with BioRender.com. 77 Chapter 5: Closing Thoughts 5.1 Conclusion Currently FDA approved fibrinolytic agents are very effective in resolving blood clots and reinstate the blood flood in the obstructed vessels. The main hurdle with this class of drugs resides in their severe side effects, which limit their use in the clinic, e.g., patients presenting with submassive PE are not recommended to receive treatment with PA due to the risks involved, even though it could benefit them [174]. Therefore, the literature highlights the need for targeted delivery of these drugs. Although great investigational work has been performed on DDSs for fibrinolytics agents in the nanometers range, studies evaluating micro-based formulations are rather low. Furthermore, considering the expansive body of work in the drug-loaded albumin microparticles area, it was surprising to find that studies investigating albumin microparticles as potential DDS for fibrinolytic agents are very scarce. In this thesis we exploited the passive lung targeting ability of macroaggregated albumin microparticles to prepare a formulation for the potential local treatment of pulmonary embolism, thus exploring the possibility of converting a diagnostic tool into a therapeutic one. We were able to formulate the PAI-1 inhibitor tiplaxtinin with albumin particles using a one-pot synthesis and heat for particles stabilization. The particles formed were within the size range for mechanical filtration (10-90 µm) of the pulmonary capillaries and showed good stability at storage conditions as well as when incubated with PBS and GSH at 37°C. Increasing signs of degradation were observed over time when the particles were incubated with plasma, which is preferable for a fast release of the drug at the site of action. Signs of cytotoxicity in endothelial and pulmonary epithelial cell lines were observed when incubated with large numbers of particles, and analysis showed that the toxic effect was mainly due to tiplaxtinin itself. Therefore, 78 the carrier appears to not exacerbate the toxicity, which is a desirable characteristic for a carrier. When administered in vivo, MATi particles were well tolerated for doses containing up to 700,000 particles and at least 60% of the injected dose was found in the lungs 15 min p.i. Lastly, we were able to investigate the biodistribution of the carrier and the drug simultaneously in a fully quantitative manner in a murine model. This was possible by labeling the two components with radioisotopes having different physical properties, 111In and 3H, allowing us to distinguish and measure each signal within the same mouse, thus reducing inter-mice variability as well as the total number of mice needed for the study Following intravenous administration, we obtained a significantly higher concentration of tiplaxtinin in the lungs for the first six hours compared to the unformulated drug as well as ~ 5 times higher exposure of the lungs to the drug. Furthermore, our formulation allowed us to overcome solubility problems due to the hydrophobic nature of tiplaxtinin. This will allow to administer higher a concentration in future therapeutic investigations. 5.2 Project Limitations For passive targeting, size of the microparticles is critical for ensuring mechanical filtration and entrapment in the capillary bed. For diagnostic imaging with 99mTc-MAA, high accumulation of the particles in the lungs is desirable in order to increase the signal to noise ratio and obtain images with improved quality. However, in this case, the size distribution of the particles can be wider as long as particles are big enough (> 10 µm) to be entrapped in the capillaries, but small enough (< 100 µm) to not block the bigger branches of the pulmonary arteries. Differently, for drug delivery systems, the particles should be as monodisperse as possible because 1) uniformity of the formulation, hence uniform drug encapsulation, allows for a more precise dosing; 2) drug release can be affected by the size of the particles [175]. While the MATi particles here prepared 79 had a consistent average size and were within the size distribution that allowed us to passively target the lungs and deliver higher concentration of drug, optimization of the formulation might yield to an even narrower size distribution. This could be achieved, for example, by heating and cooling the particles more than once or stir them for a longer period of time. However, caution should be taken as these parameters can affect the final average size of the particles. Another option could be via mechanical filtration by passing the particles through a sizing screen. Furthermore, formulation parameters like buffer pH, heating time, and stirring speed, were optimized to obtain particles within the desired size range. However, this was only possible in small volume batches. Therefore, it would be important to assess the scale up of the formulation. As the MATi particles showed good stability at storage conditions, being able to prepare bigger batches would decrease inter-experimental variability and be more time effective. Finally, by scaling up the formulation it would be possible to investigate the stability of the MATi particles following lyophilization and storage at room temperature. This would allow to potentially prepare kits similar to 99mTc-MAA ones, that are easy to store and can be reconstituted via simple addition of saline solution previous to administration. 5.3 Future Work and Outlook With this project we showed the feasibility of using albumin microparticles for local drug delivery to the lungs. This was a first step that we hope would prompt further research on albumin microparticles as targeting carriers for fibrinolytic drugs. In a second step, it would be interesting to show increased delivery of tiplaxtinin using the MATi particles not only into the lungs of healthy mice, but also in a disease model, and investigate their efficacy in vivo as well. Pulmonary embolism can be caused in animals by administering microemboli prepared ex vivo either by coagulation of whole blood or by mixing fibrinogen with 80 thrombin [176, 177]. Once prepared, the microemboli are administered either via tail vein or jugular vein [176]. Due to physiological presence of anticoagulant molecules in the organism, autolysis of the exogenous blood clots has been observed in vivo. However, the time for this process may vary [176-178]. Therefore, it would be critical to firstly establish important parameters of the model, such as time to autolysis and distribution of the microemboli. This would be a predictive model as it would allow to also assess the influence of high shear stress on our formulation. Indeed, local shear stress increases when the vessels are obstructed, which might hinder the delivery of the drug [80]. Lastly, on a grand scale, MATi particles were originally thought of as a potential platform DDS, hence the decision to investigate an easy one-pot synthesis method and to avoid the use of chemical cross-linking. Glutaraldehyde is a cross-linking agent commonly used in albumin formulations for particle stabilization. However, it can cause toxicity as well as limit the drugs that can potentially be loaded onto the MATi particles. Indeed, glutaraldehyde uses amines for the cross-linking process that might result in a decreased activity for drugs containing this functional group. Using the MATi particles for the treatment of pulmonary embolism was the first application we designed this formulation for, as the particles would accumulate directly at the site of action, i.e., in proximity of the blood clots within the pulmonary vessels. Furthermore, perspective patients that could particularly benefit from MATi particles are COVID-19 patients as it was found that the hypercoagulability state observed is due to activation of the thrombosis process as well as upregulation of PAI-1 due to release of IL-6 by the innate immune system [179]. Initial clinical trials have been performed to assess the safety and efficacy of PAI-1 inhibitors in COVID-19 patients [180]. 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Rusu, A., et al., Overview of Side-Effects of Antibacterial Fluoroquinolones: New Drugs versus Old Drugs, a Step Forward in the Safety Profile? Pharmaceutics, 2023. 15(3). 182. FDA. FDA approves safety labeling changes for fluoroquinolones. 2016; Available from: https://www.fda.gov/drugs/information-drug-class/fda-approves-safety-labeling-changes-fluoroquinolones. 92 183. EMA. Disabling and potentially permanent side effects lead to suspension or restrictions of quinolone and fluoroquinolone antibiotics. 2018; Available from: https://www.ema.europa.eu/en/news/disabling-potentially-permanent-side-effects-lead-suspension-or-restrictions-quinolone-fluoroquinolone-antibiotics. 93 Appendices Appendix A Materials in Support of Chapter 2 A.1 MATi Particles Formulation Supplementary Figure A.1.1: Particles size distribution Size distribution of MATi particles (magenta) and MAA from a commercial kit (blank) expressed as number (%) showing the presence of particles of smaller sizes. Supplementary Figure A.1.2: Particles size distribution and inter-batch variability On the left, size distribution of blank and MATi particles compared to a MAA commercial kit. On the right, size distribution of three different batches of MATi particles prepared on three different days. 0.1 1 10 100 10000510152025Number %Size (µm)Number (%)MAAMATi94 A.2 Drug Loading Supplementary Figure A.2.1: MATi particles color change On the left, blank MATi particles showing the typical white color. On the right, MATi particles showing the characteristic yellow color. Supplementary Figure A.2.2: Purification of tiplaxtinin from the MATi particles supernatant Example of an elution profile obtained by UV-Vis analysis of the fractions collected from a PD MiniTrap G25 column. The peak indicate presence of tiplaxtinin and those fractions were used for drug loading analysis. 95 Supplementary Figure A.2.3: FT-IR spectra of TPLN, blank MATi, and MATi particles FT-IR spectra shown as percentage transmittance (%T) of blank MATi (blue), MATi particles (pink), and tiplaxtinin (green) A.3 MATi Particles Stability Supplementary Figure A.3.1: MATi and MAA stability at storage conditions Particles were stored in saline at 4° C and stability was investigated for A) MATi particles over 14 days, B) MAA from a commercial kit over 14 days, and C) MATi particles over 6 months. 96 Supplementary Figure A.3.2: MATi stability at 37° C Phase contrast images of MATi particles at 0 h, 6 h, 72 h incubated with 1x PBS, 5 µM GSH, or 50% human plasma at 37° C 97 Supplementary Figure A.3.3: MAA stability at 37° C Phase contrast images of MATi particles at 0 h, 6 h, and 72 h incubated with 1x PBS, 5 µM GSH, or 50% human plasma at 37° C 98 Appendix B Material in Support of Chapter 3 B.1 Dose Study Table 5.1: Dose study experimental details Number of particles Radioactive dose administered (MBq) Number of mice (n) Sacrifice time (h post-injection) 500,000 0.097 ± 0.02 3 0.25 700,000 0.143 ± 0.004 3 0.25 Appendix C Material in Support of Chapter 4 C.1 Tiplaxtinin Solubility Supplementary Figure C.1.1: Tiplaxtinin solubility in different media Two-fold dilution (0.2 – 25 µM) of tiplaxtinin in saline, 1x PBS, or 1% BSA measured by DLS. Controls are the corresponding medium without tiplaxtinin. 99 C.2 Biodistribution Study Supplementary Figure C.2.1: Activity concentration (%ID/g) of free 3H-TPLN and 111In-MATi[3H-TPLN] in lungs and liver Activity of dual labeled 111In-MATi[3H-TPLN] and free 3H-TPLN expressed as percentage of injected dose per gram (%ID/g) over 24 h in A) the lungs and B) liver. (**, p < 0.01; ***, p < 0.001; ****, p < 0.0001). %ID, percentage of injected dose; TPLN, tiplaxtinin. Supplementary Figure C.2.2: Biodistribution of 111In-MATi (%ID/g) Biodistribution of free 111In-MATi over 24 h expressed as percentage of injected dose per gram (%ID/g). Activity increased over time in the kidneys and spleen as the particles are broken down and dislodge from the lungs. Activity was mainly concentrated in the urine for the first 6 h p.i.,, whereas negligible activity was measured in the feces. %ID, percentage of injected dose; TPLN, tiplaxtinin. 100 Supplementary Figure C.2.3: Biodistribution of free 3H-TPLN (%ID/g) Biodistribution of free 3H-TPLN over 24 h. At 0.1 h post-injection (p.i.), higher activity was observed in the kidneys, while within the first hour activity increases in the small intestine as the drug is excreted via the hepatobiliary pathway and feces over 6 h p.i. Surprisingly, high activity was measured in the bladder at 24 h p.i. %ID, percentage of injected dose; TPLN, tiplaxtinin. Supplementary Figure C.2.4: Biodistribution of MATi[3H-TPLN] (%ID/g) Biodistribution of MATi[3H-TPLN] over 24 h. At 0.1 h post-injection (p.i.), higher activity was observed in the blood, heart, and kidneys, while within the first hour activity increases in the small intestine as the drug is excreted via the hepatobiliary pathway and feces over 6 h p.i. %ID, percentage of injected dose; TPLN, tiplaxtinin 101 C.3 Thermostability of Tiplaxtinin Supplementary Figure C.3.1: Mass spectrometry fragmentation of tiplaxtinin Scheme of potential fragmentation pattern resulting in a molecule with a molecular weight of 296 g/mol.
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Häfeli, Urs
Macroaggregated albumin particles as intravenous drug formulation for lung delivery
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