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The development and characterization of nanocomposite films for the controlled release and localized… Chakraborti, Michelle 2011

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THE DEVELOPMENT AND CHARACTERIZATION OF NANOCOMPOSITE FILMS FOR THE CONTROLLED RELEASE AND LOCALIZED DELIVERY OF ALENDRONATE AND TETRACYCLINE FOR PERIODONTAL APPLICATION  `  by  Michelle Chakraborti B.Pharm., Panjab University, 2008  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  Master of Science in The Faculty of Graduate Studies  (Pharmaceutical Sciences)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  May 2011  © Michelle Chakraborti, 2011  ABSTRACT  It has been proposed that localized and controlled delivery of alendronate and tetracycline to periodontal pocket fluids via guided tissue regeneration (GTR) membranes may be a valuable adjunctive treatment for advanced periodontitis. The objectives of this work were to develop a co-loaded, controlled release tetracycline and alendronate nanocomposite plasticized poly(lacticco-glycolic acid) (PLGA) film that would form a suitable matrix supporting osteoblast proliferation and differentiation. Alendronate release was successfully controlled, with complete suppression of the burst phase of release by intercalation of alendronate anions in magnesium/aluminum layered double hydroxide (LDH) clay nanoparticles and dispersed in the PLGA film matrix. Tetracycline, loaded as free drug into the film together with alendronate-LDH clay complex released more rapidly than alendronate, but showed evidence of intercalation in the LDH clay particles. The dual drug loaded nanocomposite films were biocompatible with osteoblasts and after five week incubations, significant increase in alkaline phosphatase activity and bone nodule formation were observed.  ii  PREFACE This thesis is comprised of one manuscript (Chapter 2) that has been submitted for publication, of which I am the principal author. Details of the specific nature of the experiments and the scope of the thesis work were engineered in discussions between Dr. Helen Burt, John Jackson and myself. During the study, I received training in the handling of various equipment from John Jackson. I performed all the experiments, recorded and analyzed the data. I wrote the thesis and manuscript under the guidance of my supervisor Dr Helen Burt.  iii  TABLE OF CONTENTS  ABSTRACT.............................................................................................................................. ii PREFACE ............................................................................................................................... iii TABLE OF CONTENTS ........................................................................................................ iv LIST OF TABLES ................................................................................................................. vii LIST OF FIGURES .............................................................................................................. viii LIST OF ABBREVIATIONS ................................................................................................ xii ACKNOWLEDGEMENTS .................................................................................................. xiii DEDICATION .......................................................................................................................xiv 1. INTRODUCTION .................................................................................................................1 1.1 Periodontal disease ..............................................................................................................2 1.1.1 Introduction to disease characteristics ..........................................................................2 1.1.2 Pathogenesis ................................................................................................................3 1.1.3 Regenerative therapy using GTR .................................................................................5 1.2 Drugs used in GTR film formulation..................................................................................7 1.2.1 Alendronate .................................................................................................................7 1.2.1.1 Chemistry and physicochemical properties ............................................................7 1.2.1.2 Pharmacology and pharmacokinetics .................................................................. 10 1.2.1.3 Controlled release alendronate delivery systems for periodontal treatment .......... 13 1.2.2 Tetracycline hydrochloride ........................................................................................ 14 1.2.2.1 Chemistry and physicochemical properties .......................................................... 14 1.2.2.2 Pharmacology and pharmacokinetics .................................................................. 16 1.2.2.3 Controlled release tetracycline delivery systems for periodontal application ........ 18 1.3 Biodegradable polymeric drug delivery systems .............................................................. 18 1.3.1 Polymer structure ...................................................................................................... 19 1.3.2 Polymer morphology ................................................................................................. 20 1.3.2.1 Thermal transitions in polymers .......................................................................... 20 1.3.3 Polymer molecular weight and distribution ................................................................ 22 1.3.4 Controlled release drug delivery systems ................................................................... 23 1.3.4.1 Mechanisms of drug release from polymers ........................................................ 26 1.3.4.1.1 Diffusion controlled drug release .................................................................. 26 1.3.4.1.2 Degradation controlled drug release .............................................................. 26 iv  1.3.4.2. Factors affecting drug release ............................................................................. 26 1.4 Polymers used in film manufacture .................................................................................. 28 1.4.1 Poly(DL-lactic-co-glycolic acid) (PLGA) .................................................................. 28 1.4.2 Methoxypoly(ethylene glycol) (MePEG) ................................................................... 29 1.5 Nanoparticulate layered double hydroxide (LDH) clay ................................................... 30 1.5.1 Chemistry and physicochemical properties ................................................................ 31 1.5.2 Drug loading and release characteristics .................................................................... 31 1.6 Goal, Hypotheses and objectives of study......................................................................... 33 2. DRUG INTERCALATION IN LAYERED DOUBLE HYDROXIDE CLAY: APPLICATION IN THE DEVELOPMENT OF A NANOCOMPOSITE FILM FOR GUIDED TISSUE REGENERATION ................................................................................... 35 2.1. Introduction ...................................................................................................................... 35 2.2. Material and Methods ...................................................................................................... 41 2.2.1 Size analysis and elemental analysis of layered double hydroxide clay ...................... 41 2.2.2. Drug binding to layered double hydroxide clay......................................................... 41 2.2.3 Characterization of the alendronate-LDH clay complex ............................................. 42 2.2.3.1 X-ray powder diffraction ..................................................................................... 42 2.2.3.2 Differential scanning calorimetry (DSC) ............................................................. 42 2.2.3.3 Alendronate release from LDH-clay complex ..................................................... 42 2.2.4 Preparation of the dual-drug loaded nanocomposite film formulations ....................... 43 2.2.5 Characterization of the nanocomposite film formulations .......................................... 43 2.2.5.1 Drug release studies ............................................................................................ 43 2.2.5.1.1 Analysis of alendronate ................................................................................ 43 2.2.5.1.2 Analysis of tetracycline hydrochloride .......................................................... 44 2.2.6 Osteoblast culture ...................................................................................................... 45 2.2.7 Osteoblast viability .................................................................................................... 45 2.2.8 Alkaline phosphatase activity .................................................................................... 46 2.2.9 Bone nodule formation .............................................................................................. 47 2.2.10 Statistical analysis ................................................................................................... 48 2.3. Results ............................................................................................................................... 48 2.3.1 Size and composition of LDH clay ............................................................................ 48 2.3.2. Adsorption isotherm and characterization of alendronate-LDH clay complex ........... 48 2.3.3 Characterization of the nanocomposite film formulation ............................................ 51 2.3.3.1 Tetracycline and alendronate release from film formulations ............................... 52 2.3.3.2 Stress-strain determination by thermal mechanical analysis (TMA)..................... 54 v  2.3.4 Osteoblast viability .................................................................................................... 56 2.3.5 Alkaline phosphatase activity (ALP) and bone nodule formation ............................... 57 2.4. Discussion ......................................................................................................................... 60 3. CONCLUSION.................................................................................................................... 66 3.1 Suggestions for future work .............................................................................................. 66 BIBLIOGRAPHY ................................................................................................................... 68 APPENDIX: ASSAY DETAILS FOR DRUG ANALYSIS ................................................... 83  vi  LIST OF TABLES  Table 2-1: Target properties and formulation strategies of a co-loaded antibacterial/bisphosphonate guided tissue regeneration (GTR) membrane for application in periodontal therapy ............................................................................................................. 38  vii  LIST OF FIGURES  Figure 1-1: Pathogenesis of periodontal disease. Comparison of tooth structure in a healthy and diseased state: Periodontal disease is accompanied by tooth pocket formation and plaque deposition [Figure adapted from American Academy of Periodontology, www.perio.org] ...........5  Figure 1-2: Chemical structure of (A) Pyrophosphate (B) Bisphosphonate (C) Alendronate sodium. .......................................................................................................................................8  Figure 1-3: Acid-base chemistry of alendronate ..........................................................................9  Figure 1-4: Chemical structure of tetracycline hydrochloride. ................................................... 14  Figure 1-5: Acid-base chemistry of tetracycline ........................................................................ 15  Figure 1-6: Degradation of tetracycline under strong acidic conditions. .................................... 16  Figure 1-7: Representation of the fringed micelle model of polymer crystallinity. The diagram shows the presence of the amorphous and crystalline regions in the polymer [Figure adapted from Odian, 1991] ..................................................................................................................... 21  Figure 1-8: Representation of the chain folded model of polymer crystallinity. [Figure adapted from Odian, 1991]. ................................................................................................................................................. 22  viii  Figure 1-9: Representation of controlled drug release systems: A) Reservoir system and B) Matrix system. The arrows indicate drug release from the polymeric system into the external environment. ............................................................................................................................. 25  Figure 1-10: Chemical structure of Poly(D,L-lactic-co-glycolic acid) ....................................... 29  Figure 1-11: Chemical structure of methoxypoly(ethylene glycol) ............................................ 29  Figure 2-1: Graphical representation of the structure of layered double hydroxide clay. The figure depicts the presence of carbonate anions in the interlayer space....................................... 40  Figure 2-2: Adsorption isotherm: alendronate bound to layered double hydroxide clay at room temperature. Data expressed as mean±S.D; (n=3). ..................................................................... 49  Figure 2-3: X-ray diffraction patterns of: (a) layered double hydroxide clay (b) alendronatelayered double hydroxide clay complex. The arrows indicate the d-spacing at 11° and 7° 2θ respectively. .............................................................................................................................. 50  Figure 2-4: Cumulative alendronate release from alendronate-layered double hydroxide clay complex in PBS (pH 7.4) at 37° C under gentle agitation. Data expressed as mean±S.D; (n=3).. 51  Figure 2-5: Cumulative release of tetracycline from films composed of a polymeric blend of PLGA (85:15) and MePEG (15% w/w) loaded with (% w/w): (∆) 5% tetracycline (□) 5% tetracycline and 4.2% alendronate-LDH clay complex [at 37°C; in PBS (0.1 mM)]. Data expressed as mean±S.D; (n=5) .................................................................................................. 53  ix  Figure 2-6: Cumulative release of alendronate from films composed of a polymeric blend of PLGA (85:15) and MePEG (15% w/w) loaded with (% w/w): (o) 4.2% alendronate (□) 5% tetracycline and 4.2% alendronate-LDH clay complex [at 37°C; in PBS (0.1 mM)]. Data expressed as mean±S.D; (n= 5). ................................................................................................ 54  Figure 2-7: Variation of Young’s modulus with an increase in incubation time in aqueous media (0.1 mM PBS; 37° C) on films composed of a polymeric blend of PLGA (85:15) and MePEG (15% w/w) loaded with (% w/w): (▲) 5% tetracycline and 4.2% alendronate (□) 5% tetracycline and 4.2% alendronate-LDH clay complex. Data expressed as mean±S.D; (n=3). ....................... 55  Figure 2-8: Osteoblast viability after a two day incubation period on films composed of a blend of PLGA (85:15) and MePEG (15% w/w) loaded with 0.03, 0.1, 0.75 and 4.2 (% w/w): a) alendronate and b) alendronate-LDH clay complex. Data expressed as mean±S.D; (n=6). **P< 0.05. Films composed of 0.03% alendronate were the control group. (Separate viability studies containing groups composed of cells (no films), films composed of PLGA and MePEG and PLGA, MePEG and LDH clay were performed. No significant difference was observed in the osteoblast viability (%) among the groups.) .............................................................................. 57  Figure 2-9: Alkaline phosphatase activity of osteoblasts grown for 5 weeks on (A) no films; (B) films composed of a blend of PLGA (85:15) and MePEG (15% w/w) loaded with (% w/w) a) 5% tetracycline b) 5% tetracycline and 4.2% alendronate and c) 5% tetracycline and 4.2% alendronate-LDH clay complex. Data expressed as mean±S.D; (n=12).  ***  P< 0.05. TET and AL:  abbreviations used for tetracycline and alendronate respectively. .............................................. 58  x  Figure 2-10: Bone nodule formation. Osteoblasts grown for 5 weeks on (A) no films (B) films composed of a blend of PLGA (85:15) and MePEG (15% w/w) loaded with (% w/w) a) 5% tetracycline b) 5% tetracycline and 4.2% alendronate and c) 5% tetracycline and 4.2% alendronate-LDH clay complex. Data expressed as mean±S.D; (n=12). ***P< 0.05. TET and AL: abbreviations used for tetracycline and alendronate respectively. .............................................. 59  Figure A-1: Chromatogram of alendronate in phosphate buffered saline. Method: HPLC with fluorescence detector. Mobile phase: EDTA (1 mM)/ methanol (97:3 v/v) mixture (pH 6.5); flow rate of 1 mL/min (Alltech nucleosil 100 C18 column). ............................................................... 83  Figure A-2: Standard curve for alendronate sodium standard solutions using HPLC with fluorescence detection. R2= 0.9977............................................................................................ 84  Figure A-3: Chromatogram of tetracycline hydrochloride showing the presence of two peaks on both days 1 and 6 respectively. Peak 2 indicates the standard tetracycline peak. The area under the curve of the first peak (degradative tetracycline) increased from day 1 to day 6. The mobile phase was composed of 12% v/v acetonitrile in 1.3 g/L oxalate solution (pH 2.1), having a flow rate of 1mL/min (reverse phase C18 Novapak column). ................................................................................................................................................. 86  Figure A-4: Standard curve for tetracycline hydrochloride standard solutions using HPLC with UV-vis detection. R2= 1. ........................................................................................................... 87  xi  LIST OF ABBREVIATIONS 2θ  2 theta, X-ray diffraction angle  α-MEM  Minimal essential medium  °  C  Degrees celsius  DCM  Dichloromethane  DSC  Differential scanning calorimetry  EDTA  Ethylenediamine tetraacetic acid  GTR  Guided tissue regeneration  HPLC  High performance liquid chromatography  ‫גּ‬em  Emission wavelength  ‫גּ‬ex  Excitation wavelength  LDH clay  Layered double hydroxide clay  MePEG  Methoxy(polyethylene glycol)  Mn  Number average molecular weight  MTS  3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium  MW  Molecular weight  Mw  Weight average molecular weight  nm  Nanometer  PBS  Phosphate buffered saline  PLGA  Poly(lactic-co-glycolic acid)  SEM  Scanning electron microscope  Tg  Glass transition temperature  Tm  Melting transition temperature  w/v  Weight per volume  w/w  Weight per weight  XRD  X-ray powder diffraction  xii  ACKNOWLEDGEMENTS  I would like to thank my supervisor, Dr. Helen Burt, for her support, guidance and patience over the course of my M.Sc. studies. I thank my committee members: Drs. Urs Häfeli, Kishor Wasan and Adam Frankel for their guidance and direction on my research project. Thank you to Drs. Lucy Marzban and Judy Wong for chairing my committee. I am grateful to Dr. Donald Brunette (Faculty of Dentistry, UBC) for providing osteoblast cell cultures and Dr. David Plackett (Risø National Laboratory for Sustainable Energy, Denmark) for providing the supply of layered double hydroxide clay during the course of this work. I would also like to thank Ms. Anita Lam (UBC Chemistry) for help in the X-ray diffraction analysis of the samples.  Thank you to John Jackson for advice and direction on my work. I would like to thank my colleagues in the lab for their help and encouragement over the past two and a half years: Dr. Kevin Letchford, Clement Mugabe, Sam Gilchrist, Antonia Tsallas, Leon Wan, Dr. Lucy Ye, Ben Wasserman, Ana Schteinman, Rakhi Pandey and Arvin Mazhary. Thanks to Wes Wong, Barb Conway, Suzana Topic, Rachel Wu and Jamal Kurtu for their kind help whenever needed. A special thanks to Sam Gilchrist for the illustration in Figure 2-1 and Maryam Zamiri for drawing the chemical structures.  Finally, I would like to thank my parents Drs. Anuradha and Pradip Chakraborti and my brother Monideep for their continuous encouragement and support throughout the period.  xiii  DEDICATION I would like to dedicate this work to my parents. Thank you for always being there and believing in me, especially during the hard times in life.  xiv  1. INTRODUCTION Periodontal disease is an inflammatory disease, which affects the tissues surrounding and supporting the teeth. It is caused by periodontal pathogens that initiate damage by triggering host mediated responses. Towards the later phase of the disease, the supporting collagen of the periodontium degenerates, followed by the resorption of alveolar bone, ultimately leading to tooth loss (Pihlstrom et al., 2005). The current method used for treatment is ‘guided tissue regeneration’ (GTR). In this method, a barrier film is inserted under the gum and over gingival tissue to guide bone tissue regeneration. However, studies indicate a large variability in the surgical outcome of GTR procedures. One of the main causes for variability comes from bacterial colonization of the membrane, following its placement into the periodontal pocket which results in limited bone growth (Lynch, 1999). We believe that the localized co-delivery of the antibiotic tetracycline, and bone resorption inhibitory agent alendronate, in a biodegradable polymeric film may solve this problem. Previous studies in our lab in which alendronate was loaded in polymeric films and incubated with osteoblasts, showed an initial burst release of alendronate that was toxic to the osteoblasts (Long et al., 2009). We hypothesized that the initial burst release of alendronate could be controlled or eliminated by binding the drug to nanoparticulate clays called layered double hydroxide (LDH), followed by incorporation of the drug loaded clay into a polymeric matrix. The layered double hydroxides are layered solids having interlayer charge compensating anions. These nanoclays have the potential to intercalate anionic molecules and control their release through interlayer anion exchange (Ambrogi et al., 2001; Choy et al., 2007).  1  The goal of the project was to develop, characterize and determine osteoblast biocompatibility of a biodegradable controlled release nanocomposite film formulation for the localized delivery of alendronate and tetracycline over a period of 2-4 weeks as a suitable guided tissue regeneration film.  1.1 Periodontal disease 1.1.1 Introduction to disease characteristics Periodontal disease is an inflammatory disorder that affects the supporting tissue structures of the teeth. It is one of the most prevalent chronic oral diseases affecting about 90% of the world population (Pihlstrom et al., 2005). According to the Canadian Health Measures Survey (oral health component) conducted from 2007-2009, 21% of the Canadian adult population has, or is likely to have been, affected by the disease (Cooney, 2010). Recent evidence suggests that periodontal disease may be linked with cardiovascular, diabetes and respiratory disease (Pihlstrom et al., 2005). Therefore maintenance of oral hygiene and proper management of disease is essential in order to prevent further progression of periodontitis. Periodontal disease includes both gingivitis and periodontitis. Gingivitis is the mildest form of the disease where inflammation is confined to the gingiva. It is readily reversible with effective oral hygiene. The spread of inflammation into the deeper tissues leads to degeneration of the periodontium and alveolar bone resorption, ultimately causing loosening and loss of tooth attachment. This condition, known as periodontitis is irreversible in nature and is characterized by chronic gingival inflammation, deep pocket formation and ultimately leads to loss of tooth attachment (Jain et al., 2008; Pihlstrom et al., 2005).  2  1.1.2 Pathogenesis Periodontal disease is a pathological condition characterized by inflammation of the periodontium, caused by pathogenic microflora present in the plaque biofilm which develop on the surface of teeth (Figure 1-1). A variety of aerobic and anaerobic microbial species like Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans and spirochaete Treponema denticola reside in the oral cavity (Pihlstrom et al., 2005). They are responsible for plaque biofilm formation on the teeth surface/gingiva. The biofilm formation triggers a variety of inflammatory responses but it is the host response that is responsible for tissue destruction. The bacterial action on the teeth surface/gingival epithelium leads to the release of various chemotactic factors, such as cytokines that attract the polymorphonuclear leucocytes (neutrophils) into the inflammed periodontal cavity. They are responsible for phagocytosis and removal of bacteria. However, the presence of a high microbial load in the tissues, leads to the ‘overloading’ of neutrophils, thereby resulting in cell lysis and release of various enzymes like matrix metalloproteinases and collagenases that cause tissue destruction. This condition may be reversed with proper oral hygiene when limited to the gingival epithelial tissue. However if the immune-inflammatory conditions persist, it may lead to further apical migration of the gingival epithelium along the root surface, leading to the formation of deep periodontal pockets (Jain et al., 2008; Kinane, 2001; Pihlstrom et al., 2005; Sbordone and Bortolaia, 2003) The development of periodontal pockets is a characteristic feature of periodontal disease. They are chronic inflammatory lesions that are able to undergo repair. However, complete healing does not occur due to continuous bacterial activity which stimulates inflammatory 3  responses leading to degeneration of the newly formed tissue (Sbordone and Bortolaia, 2003). The periodontal pocket contains debris of microorganisms and their metabolic products, gingival fluid, food remnants, salivary mucin, epithelial cells and leukocytes. Pocket formation starts due to destruction of the periodontium and progressive deepening leads to further tissue damage, ultimately causing loosening and loss of tooth attachment (Kinane, 2001; Pihlstrom et al., 2005; Sbordone and Bortolaia, 2003).  4  Figure 1-1: Pathogenesis of periodontal disease. Comparison of tooth structure in a healthy and diseased state: Periodontal disease is accompanied by tooth pocket formation and plaque deposition [Figure used with permission from American Academy of Periodontology, www.perio.org] 1.1.3 Regenerative therapy using GTR Periodontal therapy depends upon severity or stage of the disease. Initial treatment, or the non surgical phase, involves the mechanical removal of calculus and root planing, followed by systemic or local antimicrobial therapy. However, if the disease persists, the treatment shifts to phase two therapy, known as the surgical phase. This involves the placement of implants in the periodontal pocket for regeneration of the lost periodontal tissues. The remarkable healing 5  capacity of periodontal tissues has made periodontal regenerative therapy an important part of treatment. Currently, guided tissue regeneration is an important technique used in regenerative therapy (Newman 2006). Based upon a literature review conducted by Needleman et al, there was a significant improvement in treatment outcomes, such as an increase in attachment level and probing depth level, provided by GTR therapy, compared to conventional therapy (debridement surgery) (Needleman et al., 2006). Guided tissue regeneration is a regenerative technique which involves the placement of a membrane between the tooth and gingival connective tissue. The membrane acts as a physical barrier that prevents the migration of the gingival connective tissue into the wound area, thereby maintaining a selective space for the inward migration of periodontal progenitor cells derived from the periodontal ligament and alveolar bone from the exposed root surface, promoting formation of new attachment in the traumatised area (Gottlow et al., 1992; Lynch, 1999; Villar and Cochran, 2010). Along with favouring selective repopulation of the wound area, the barrier protects the blood clot during the initial healing phase and ensures space maintenance for new periodontal tissue (Villar and Cochran, 2010) A large number of materials have been used for the GTR film manufacture. They may be classified based upon being either non-resorbable or bioabsorbable. Non-resorbable or nondegradable membranes require an additional surgical procedure for removal from the periodontal pocket, which is a major disadvantage in comparison to bioabsorbable barriers. Furthermore, early exposure to the oral environment and subsequent bacterial colonization of the membrane, leads to infection of the site, resulting in bone loss. Poly(tetrafluoroethylene) (PTFE) membranes are the most commonly used non-resorbable membranes. Some examples are: 6  Cytoplast®Regentex GBR-200 membranes (composed of high-density poly(tetrafluoroethylene)(d-PTFE) and TefGen-FD® membranes, composed of nano-porous poly(tetrafluoroethylene) (nPTFE) (Kasaj et al., 2008; Teparat et al., 1998). The advantage of bioabsorbable membranes is their biodegradability. Hence they do not require any further surgical removal. Some of the major disadvantages of these membranes include the lack of stiffness resulting in collapse in the defect area with epithelial down growth and early loss of the material (Newman 2006). Some examples are: Resodont® membranes (made from equine type I collagen) and BioGide® membranes (made from porcine type I and III collagen). Hyaluronic acid, poly (lactide) and poly(lactide-co-glycolide) are also materials that have been used to make bioabsorbable membranes (Kasaj et al., 2008; Villar and Cochran, 2010). In in vitro studies carried out by Kasaj et al, bioabsorbable membranes were shown to be more compatible for stimulation of cell proliferation in comparison to the non-resorbable membranes (Kasaj et al., 2008). 1.2 Drugs used in GTR film formulation 1.2.1 Alendronate Alendronate is a potent inhibitor of bone resorption, commonly used for the treatment of bone disorders such as Paget’s disease, osteoporosis, osteolytic bone metastases and periodontal disease (Russell et al., 2008). 1.2.1.1 Chemistry and physicochemical properties Alendronate belongs to the group of bisphosphonate drugs: amino-bisphosphonates. Bisphosphonates are stable synthetic analogues of pyrophosphate (Figure 1-2). The P-O-P backbone in the pyrophosphate structure is replaced by the P-C-P backbone in bisphosphonates, 7  thereby rendering them stable to enzyme and chemical hydrolysis (Hirabayashi and Fujisaki, 2003). The presence of both phosphonate groups is vital for bone mineral affinity and antiresorptive activity (Luckman et al., 1998; Russell et al., 2008). The side chains (R1 and R2) present on the carbon atom further enhance these properties. As in the alendronate structure, it has been observed that the presence of a hydroxyl group at the R1 position and a nitrogen derivative at the R2 position further enhances the bone mineral affinity and anti-resorptive activity respectively (Reszka and Rodan, 2004).  A  B  C  Figure 1-2: Chemical structure of (A) Pyrophosphate (B) Bisphosphonate (C) Alendronate sodium.  8  Alendronate is a negatively charged drug molecule that possesses 5 pKa values: 0.8, 2.2, 6.3, 10.9 and 12.2 (Lin et al., 1994) (Figure 1-3). At the intestinal pH of 6-8, it is entirely ionized. Alendronate has an octanol/water partition coefficient of 0.00117 that is independent of the pH range from 2-11. This indicates low lipophilicity of the molecule and may be a reason for the poor intestinal absorption (Lin et al., 1994).  Figure 1-3: Acid-base chemistry of alendronate.  Alendronate is used in the form of its sodium salt. The various crystalline hydrates have different water solubilities: alendronate monosodium trihydrate and alendronate monosodium anhydrous have solubilities of 40 mg/mL and 300 mg/mL respectively (Ezra, 2000). However 9  the calcium salt of alendronate has been observed to be highly insoluble and more toxic than the free acid form (Twiss et al., 1999). 1.2.1.2 Pharmacology and pharmacokinetics Bone is composed of 65% mineral (hydroxyapatite) and 35% matrix (mainly collagen). Due to affinity for hydroxyapatite, alendronate selectively accumulates in bone (Russell et al., 2008). The principle effect of alendronate is the inhibition of bone resorption. Osteoclasts bind to bone through their ruffled border surface. During bone resorption, the area beneath the osteoclast is acidified by the vacuolar-type proton pumps present in the ruffled border of the osteoclast. The prevailing acidic environment causes dissolution of the bone mineral, followed by uptake of alendronate into the osteoclasts where it further triggers various biochemical events. At the molecular level, it acts on the mevalonate pathway by inhibiting the enzyme farnesyl pyrophosphate synthase, thereby, preventing the formation of farnesyl pyrophosphate. Farnesyl pyrophosphate is involved in the prenylation of small GTPase signaling proteins. Prenylation involves the transfer of the isoprenoid goup from farnesyl pyrophosphate onto specific target proteins, forming farnesylated proteins such as Ras Rho, Rac, Cdc42, and Rab families that are responsible for the regulation of various osteoclast cell processes like membrane ruffling, cytoskeletal arrangement and trafficking of intracellular vesicles. Hence, inhibition of the mevalonate pathway may result in loss of osteoclast function, leading to apoptosis of the osteoclasts (Rogers, 2004). Furthermore, at the cellular level, a decrease in osteoclast recruitment is observed. (Rodan, 1998; Russell et al., 1999; Russell et al., 2007; Tenenbaum et al., 2002). The loss of the ruffled border of the osteoclast inactivates it from further resorption and a corresponding decrease in depth of the resorption site is observed. 10  Apart from direct involvement with osteoclasts, alendronate has also been shown to cause an increase in osteoblast cell proliferation and maturation (Im et al., 2004; Kim et al., 2008; von Knoch et al., 2005). Clinical studies have demonstrated that alendronate is a relatively safe and well tolerated drug (Greenspan et al., 2002; Watts et al., 1999; Yanık et al., 2007). However, there are some adverse reactions that have been reported with drug administration, such as oesophageal ulcers and gastrointestinal events (Marshall, 2002; Woo et al., 2010), osteonecrosis of the jaw (Perrotta et al., 2010; Yarom et al., 2007), hepatotoxicity (Yanık et al., 2007), auditory hallucinations and visual disturbances (Coleman et al., 2004). Osteonecrosis of the jaw (ONJ) is a relatively recently discovered potential adverse effect of bisphosphonates. This condition was first reported in 2003 by Marx in relation to the use of the bisphosphonates pamidronate or zoledronate for the treatment of various bone disorders (Marx, 2003). Osteonecrosis of the jaw is characterized by the presence of exposed necrotic bone in the oral cavity (mandible, maxilla or palate) for more than six weeks. However the pathobiology of the adverse effect is still not clearly understood. Bisphosphonate induced low bone turnover may be proposed as a reason for osteonecrosis of the jaw as it leads to bone cell necrosis, apoptosis and decreased blood flow (Rizzoli et al., 2008). As no diagnostic tests are available, clinical manifestations are the basis for identification. Most of the cases reported (95%) have been associated with the intravenous administration of pamidronate or zoledronate used for treatment of patients suffering from metastatic bone disease undergoing dental procedures (Bilezikian, 2005; Migliorati et al., 2011). Fewer cases have been reported in patients on oral alendronate therapy as the dose is considerably lower compared to intravenous administration. However, in 11  cases where bisphosphonates have been used in periodontal therapy, no cases of ONJ have been reported to date. However, it should be noted that the patients were followed for a period of 3 years, which may be insufficient considering the long half life of alendronate (Jeffcoat, 2006). Therefore, more evidence is required to further clarify the magnitude and risk of ONJ with bisphosphonate therapy. Alendronate is poorly absorbed via the oral route. The oral bioavailability is 0.7% in humans (Lin, 1996). The poor absorption may be attributed to the presence of bulky and negatively charged phosphonate groups which results in the poor lipophilicity of the molecule (Reszka and Rodan, 2004). Absorption is further hindered by food. Therefore, patients are counselled to take the drug 30 minutes prior to food intake. Absorption of alendronate from the gastrointestinal tract into the blood stream occurs by paracellular transport through the tight junctions between the epithelial cells (Lin et al., 1994; Porras et al., 1999). Following absorption, the drug molecules bind to the albumin proteins in the plasma (78%) and are then rapidly distributed in the body either by uptake into the bone (50-60%) or are eliminated through renal clearance. The drug distribution is higher in the areas of rapid bone turnover (Sparidans et al., 1998). Alendronate does not undergo metabolism as the P-C-P backbone is resistant to enzymatic attack by enzymes like alkaline phosphatase and pyrophosphatase. The elimination of alendronate by urinary excretion consists of two phases: in the first phase, 90% of the alendronate (not retained on the bone surface) is excreted within the first 24 hours, while the second phase, the half life is about 10 days. Once the drug is localized onto the resorptive bone surface and embedded into the bone surface, it is released back slowly as a part of normal bone turnover. The average terminal  12  elimination half-life from the skeleton is about 10 years, (as estimated by urinary excretion in an 18 month follow-up study) (Reszka and Rodan, 2004). 1.2.1.3 Controlled release alendronate delivery systems for periodontal treatment In periodontitis, inflammation surrounding the tooth may further penetrate into the deeper tissues like the alveolar bone, thereby causing bone resorption. The use of alendronate as an adjunct to non-surgical treatment in periodontitis has shown promising results in the clinical outcomes of therapy. In a randomized, placebo controlled clinical study conducted over a year, the bisphosphonate treatment group showed significant improvement in the clinical outcomes of therapy i.e. increase in the attachment level and probing depth compared to the control patient group (Lane et al., 2005). Local application of alendronate has also been shown to inhibit bone resorption. The application of an alendronate soaked pellet to the exposed bone surface caused a significant reduction in bone resorption in rats (Nafea et al., 2007). Similar studies carried out with alendronate encapsulated gelatin sponge pellets in rat models have also shown a reduction in bone loss (Binderman et al., 2000). Along with local application, the encapsulation of alendronate in controlled release delivery systems may provide further advantages, such as better patient compliance as the frequency of administration would be reduced. A Carbopol® gel loaded with alendronate and placed in periodontal pockets, significantly improved clinical parameters like clinical attachment level, gingival index, pocket depth and there was an increase in new bone formation (Reddy et al., 2005). In previous work carried out in our laboratory, alendronate was encapsulated in biodegradable polymeric films and in vitro studies using osteoblasts cultured on these films showed an increase in the alkaline phosphatase activity and bone nodule formation 13  (Long et al., 2009). Alendronate has been encapsulated in polymeric microspheres and was suggested to have potential application in periodontal treatment. In vitro studies showed that the drug was completely released over two weeks (Nafea et al., 2007). 1.2.2 Tetracycline hydrochloride Tetracycline hydrochloride possesses both antibacterial activity and the ability to promote fibroblast and connective tissue attachment to tooth surfaces. This may help in the regeneration of periodontal tissues lost in disease (Wikesjo et al., 1986). 1.2.2.1 Chemistry and physicochemical properties Tetracycline hydrochloride belongs to a family of broad spectrum antibiotics possessing a highly functionalized naphthacene ring structure (Figure 1-4). Tetracycline is commercially used in the form of the hydrochloride salt which renders the molecule water soluble (Lemke, 2008)..  Figure 1-4: Chemical structure of tetracycline hydrochloride.  Tetracycline hydrochloride is an amphoteric drug molecule, having pKa values of 3.3, 7.8 and 9.5 (Day, 1978) (Figure 1-5). Therefore, tetracycline contains localized charges across all pH values, achieving an overall neutral state as zwitterions (Anderson et al., 2005; Wu and Fassihi,  14  2005). As they form complexes with divalent metal ions, the chelated complexes may interfere with their activity and absorption in the body (Lemke, 2008).  Figure 1-5: Acid-base chemistry of tetracycline.  Tetracycline hydrochloride undergoes photodegradation in solution darkening from yellow to brown (Lemke, 2008). The stability of the molecule is poor in strong alkaline and acidic medium. It may undergo epimerization (which is reversible) forming 4-epitetracycline under weak acidic conditions and anhydro-tetracycline under strong acidic conditions (Lemke, 2008). On further epimerization of anhydrotetracycline and dehydration of epitetracycline, another toxic product called epianhydrotetracycline is formed. (Pena et al., 1998; Wu and Fassihi, 2005) (Figure 1-6).  15  Tetracycline  4-epitetracycline (inactive)  Anhydrotetracycline (inactive)  4-epianhydrotetracycline (inactive)  Figure 1-6: Degradation of tetracycline under strong acidic conditions.  1.2.2.2 Pharmacology and pharmacokinetics Tetracycline exerts its antimicrobial action by inhibiting protein synthesis in microorganisms. The uptake of the tetracycline molecule through the organism cell wall occurs by a combination of passive diffusion through the outer membrane pores and an active transport process through the inner membrane. On entering the cell, the molecule binds reversibly to the 30S ribosomal subunit, preventing the binding of aminoacyl transfer RNA, thereby inhibiting protein synthesis and ultimately cell growth (Katzung, 2004; Seymour, 1995). Tetracycline is bacteriostatic in nature and is active against gram positive and gram negative bacteria as well as some anaerobes, chlamydia, protozoa, mycoplasma and rickettsiae (Katzung, 2004). 16  Tetracycline also inhibits host collagenolytic enzymes (Golub et al., 1998; Golub et al., 1991; Rifkin et al., 1993) such as matrix metalloproteinases (MMP) responsible for collagen breakdown in the bone (Weinberg and Bral, 1998). Some of the adverse events associated with tetracycline include nausea, vomiting and diarrhoea (Katzung, 2004). Tetracycline may alter the intestinal microflora by suppressing the activity of some organisms, thereby leading to an overgrowth of other organisms such as pseudomonas or staphylococci and may lead to various intestinal disturbances (Katzung, 2004). Tetracycline is known to chelate with calcium deposited in the bone or teeth especially in children, which may cause growth deformity or discoloration of teeth. Other effects may include photosensitivity to sunlight, renal dysfunction and impairment in liver function especially during pregnancy (Katzung, 2004; Weinberg and Bral, 1998). Following oral administration, about 75% of the tetracycline dose is absorbed from the stomach and upper duodenum. The absorption is hindered in the presence of food, dairy products and substances containing divalent cations such as antacids (Lemke, 2008). About 40-80% is bound to serum proteins. Following absorption, tetracycline is widely distributed in the body except for the cerebrospinal fluid. Due to affinity for calcium present in bone, it selectively distributes into the region of newly formed bone. Tetracycline is excreted in the urine and bile with some of the drug excreted in the bile being reabsorbed from the intestine through enterohepatic circulation. Renal clearance occurs through glomerular filtration. About 40-70% the dose of tetracycline is excreted in the urine (Katzung, 2004). Tetracycline hydrochloride has a half life of 6-8 hours in healthy adults (Katzung, 2004; Weinberg and Bral, 1998).  17  1.2.2.3 Controlled release tetracycline delivery systems for periodontal application Tetracycline is one of the most widely used antibiotics for periodontal application and can concentrate in the gingival crevicular fluid of the periodontal pocket, at concentrations 5-10 times greater than the serum levels (Golub et al., 1991). Intra-pocket devices containing the drug are beneficial for periodontal application with enhanced efficacy, decreased side effects and greater patient compliance. Tetracycline has been successfully encapsulated in microspheres, gels, fibres and films for use as intra-pocket controlled release delivery systems in periodontal treatment (Agarwal et al., 1993; Jain et al., 2008; Webber et al., 1998). Ethyl vinyl acetate (EVA) fibres containing 25% tetracycline hydrochloride and wound around the base of the tooth showed a reduction in pocket depth and gain in tooth attachment in periodontal disease patients (Tonetti et al., 1990). Furthermore, the use of tetracycline fibres as an adjunct to scaling and root planing treatment, has been shown to significantly decrease the recurrence of periodontal disease (4%) in comparison with scaling and root planing alone (9%) (Michalowicz et al., 1995). Owen et al developed a tetracycline loaded (5% and 10%) plasticized PLGA membrane that may have potential application as a guided tissue regeneration membrane. The drug was released in a controlled release manner (a fast initial burst followed by a slower controlled release) and the membrane was observed to be biocompatible and had optimal stiffness to act as a barrier membrane (Owen et al., 2010). 1.3 Biodegradable polymeric drug delivery systems A biomaterial may be defined as material that is in contact with the biological system for the purpose of evaluation, treatment, augmentation or replacement of any tissue or organ function in the body (Williams, 1999). Biomaterials are divided into four classes: polymers, metals, 18  ceramics and plant or animal derived materials (Ratner, 2004). Polymers represent the largest class of biomaterials. They may occur naturally as polysaccharides, proteins and polynucleotides or are synthetic in origin such as polyesters and polyanhydrides. Biodegradable polymers have been used extensively as delivery vehicles for a broad range of drugs and drug classes and the field has been reviewed by many groups (Amsden, 2010; Heller, 1984; Kumari et al., 2010; Ulbrich et al., 1997; Yu et al., 2010). Biodegradable polymers are also used as implants or devices such as guided tissue regeneration membranes, vascular stents, bone screws, bone plates, staples etc (Nair and Laurencin, 2007). Biodegradable polymers undergo degradation in the body via enzymatic and hydrolytic processes. 1.3.1 Polymer structure Polymers are macromolecules composed of repeating monomer units, which are bonded to each other forming linear, branched or cross-linked structures. The properties of polymers are defined by the constitution, configuration and conformation of the atoms forming the polymer. Constitution refers to the atoms that make up the polymer, configuration determines the arrangement of the different atoms about the polymer backbone while conformation describes how the configuration of the polymer chain leads to a three dimensional structure in space (Rosen, 1993). Polymers composed of one type of monomer are called homopolymers. Poly(lactic acid) and poly(glycolic acid) are examples of homopolymers. Polymers containing two or more types of repeat units are termed copolymers. Depending upon the arrangement of the repeat units, the copolymer may further be classified as: random copolymer (arrangement of the repeat units is random), alternating copolymer (alternate arrangement of the repeat units along the chain), block 19  copolymer (polymer chain contains a sequence or block of each monomer and depending upon the number of blocks, they may be diblock or triblock) and graft copolymers (main polymer chain is formed of one type of repeat units, from which a series of another type of repeat units branch out) (Rosen, 1993). Network polymers are formed when branches from the main polymer chain are connected to the other polymer chains through cross-linking. 1.3.2 Polymer morphology 1.3.2.1 Thermal transitions in polymers Polymers are rarely observed to be totally crystalline. They generally have the properties of both crystalline solids and highly viscous liquids, that is, they are semi crystalline. They consist of crystalline regions termed crystallites interspersed among the amorphous regions. This has been observed in the X-ray diffraction patterns of polymer samples, where the sharp peaks, indicative of long range order of molecules, represent the crystallites and the typical amorphous halo represents the amorphous phase (Odian, 1991). A model developed to explain the polymer morphology is the fringed micelle model. According to the model, the crystallites or fringed micelles are composed of regularly aligned portions of the polymer chains within randomly arranged chains of the amorphous areas. The observation of the growth of single crystals of polymers led to the second model termed the folded chain lamella model (Odian, 1991) (Figure1-7). Single crystal growth, in the form of thin plate like crystallites termed lamellae were obtained from dilute polymer solutions. However, from the X-ray diffraction pattern of these crystallites, it was observed that the polymer chains were aligned perpendicular to the flat surfaces of the crystallite even though the extended length  20  of the polymer chain was greater than the crystal thickness. Thus, it was presumed that the chain folded back on itself (Grulke, 1994) (Figure 1-8). Semicrystalline polymers are characterized by two types of transition temperatures: the crystalline melting temperature (Tm), which is the melting temperature of the crystalline portion of the polymer and the glass transition temperature (Tg), that is characteristic of the amorphous region. The glass transition temperature may be defined as a narrow range of temperature, above which the material is rubbery and below which it exists as a glass. Amorphous polymers exhibit two types of mechanical behaviour: they can be a glassy, hard and rigid material material below Tg or a rubbery, flexible, soft material above Tg. (Odian, 1991).  Amorphous region  Crystalline region  Figure 1-7: Representation of the fringed micelle model of polymer crystallinity. The diagram shows the presence of the amorphous and crystalline regions in the polymer [Figure adapted from Odian, 1991].  21  Figure 1-8: Representation of the chain folded model of polymer crystallinity. [Figure adapted from Odian, 1991]. 1.3.3 Polymer molecular weight and distribution Polymers are macromolecules, composed of mixtures of molecules having different molecular weights. This property distinguishes polymers from low molecular weight compounds. Therefore, the molecular weight of the polymer is measured in terms of an average molecular weight (Odian, 1991). Polymer molecular weight can be defined by several different average molecular weights like number average molecular weight (Mn) and weight average molecular weight (Mw). These average molecular weights are obtained by different analytical methods. As the properties measured by these methods are biased towards different sized polymer molecules in a sample, the values of the average molecular weights are different. For most purposes, Mn and Mw are used to characterize the molecular weight of polymers (Odian, 1991).  22  The number average molecular weight (Mn) is defined as the total weight (w) of the molecules present in the polymer sample divided by the total number of moles (N x).  Analytical techniques based upon the number of moles present in a sample, such as methods measuring colligative properties are used to determine M n. The weight average molecular weight (Mw) is the molecular weight based upon the weight of molecules present at each size level. It is defined as  Where, wx is the weight of x-mer molecules in the sample having molecular weight Mx. The ratio of Mw to Mn is termed the polydispersity index of the polymer.. The ratio of M w to Mn is generally greater than unity and increases with an increase in polydispersity of the polymer sample. The closer the value to unity, narrower is the distribution of the molecular weights. 1.3.4 Controlled release drug delivery systems Controlled drug release systems ideally deliver the drug at a pre-determined rate for a prolonged period of time. The drug release rate is determined by the design of the system and is nearly independent of environmental conditions (Langer, 1990). The primary advantage of such systems is that the drug concentration is maintained within its therapeutic window at the target site or in the blood for prolonged time periods. Controlled release systems are divided into two types: the matrix and reservoir system (Figure 1-9). In a matrix system, the drug is uniformly  23  distributed in the polymer and depending upon miscibility characteristics, is present as the molecular form and/or solid drug particles. A matrix system, in which the drug is uniformly dissolved or dispersed in the polymer, is termed as monolithic solution or dispersion. In the reservoir system, the drug phase is surrounded by an inert rate limiting polymer coating or membrane (Sinko, 1993).  24  Figure 1-9: Representation of controlled drug release systems: A) Reservoir system and B) Matrix system. The arrows indicate drug release from the polymeric system into the external environment. 25  1.3.4.1 Mechanisms of drug release from polymers 1.3.4.1.1 Diffusion controlled drug release In diffusion controlled systems, the drug is released into the surrounding medium through diffusion. In reservoir systems, the rate of drug release is controlled by the diffusion of the drug through the inert rate limiting membrane (Baker, 1987) while in matrix systems such as a monolithic solution or dispersion, the drug diffuses out of the system as a result of the concentration gradient. The rate of drug release is described by Fick’s law.  where J is the flux (diffusion per unit area), D is the diffusion coefficient between the matrix and the surrounding medium. 1.3.4.1.2 Degradation controlled drug release The rate of drug release from a biodegradable polymer matrix is also controlled by the rate of polymer degradation. Bulk or homogeneous degradation involves the degradation of the whole matrix, as in polyesters and in surface or heterogeneous degradation, only the surface of the polymer undergoes degradation, thereby retaining the structural integrity of the matrix (Sinko, 1993). Poly(ortho)esters and polyanhydrides are examples of polymers that undergo surface degradation (Sinko, 1993) 1.3.4.2. Factors affecting drug release The rate of drug release from biodegradable polymers is influenced by factors such as polymer composition and morphology, nature of the drug molecule, drug loading, geometry, addition of other agents in polymers like plasticizers and biodegradation of the polymer (Alexis, 2005). 26  In monolithic dispersion systems, where the drug is dispersed in the polymeric matrix, drug release is related to drug loading, solubility and diffusivity of the drug. At low drug loadings (05%), drug release involves the dissolution of the drug in the polymer matrix, followed by diffusion to the surface of the device. This system is called as a simple monolithic dispersion. However, at drug loadings of 5-10%, the dissolution of drug particles in the matrix leaves behind cavities, which are eventually filled with fluid from the external environment. The cavities are pathways for the release of the remaining drug from the polymer. These systems are called complex monolithic dispersions. At higher drug loadings (usually greater than 20%), the dissolved particles cause cavities that interconnect with one another and form a continuous fluid filled channel through which the drug is released (Baker, 1987). As water penetrates through the polymeric matrix, the drug molecule with higher water solubility dissolves rapidly in the ‘external fluid’ and partitions out from the polymer in comparison to a more hydrophobic drug. Water penetrates hydrophobic polymers and the crystallite regions of semicrystalline polymers less readily and degradation rate is slower than for hydrophilic polymers and the amorphous regions of polymers. Hence slower polymer degradation rates will result in decreased drug release rates (Chasin, 1990) The addition of plasticizers to the polymer matrix may alter the rate of drug release. If the plasticizer is more water soluble than the polymer, it dissolves out of the matrix on contact with aqueous media, thereby creating pores and channels for further water penetration, which in turn accelerates the drug release from the system (Alexis, 2005). For example, the addition of water soluble methoxypoly(ethylene glycol) to poly(lactic-co-glycolic acid) polymers causes an increase in the rate of drug release (Jackson et al., 2004; Owen et al., 2010). 27  1.4 Polymers used in film manufacture 1.4.1 Poly(DL-lactic-co-glycolic acid) (PLGA) Poly(D,L-lactic-co-glycolic acid) is an aliphatic polyester which is synthesized by the ring opening polymerization reaction of lactide and glycolide. It is a copolymer which is identified by the ratio of monomers used (Figure 1-10) (Vert et al., 1992). The copolymer lactic acid: glycolic acid ratio selected for the study was in the weight ratio of 85:15. Poly(lactic acid) is more hydrophobic than poly(glycolic acid) and copolymers containing a higher proportion of lactic acid absorb less water and hence degrade more slowly (Vert et al., 1992). PLGA is typically amorphous at most ratios of lactic acid to glycolic acid, with a Tg of about 47°C for 85:15 PLGA (Jackson et al., 2004). It is the most commonly used synthetic biodegradable polymer with applications as films, nanospheres, microspheres, fibers, implants and grafts. The polymer degrades via a three phase process: random chain scission, matrix solubilisation and matrix erosion and clearance. Biodegradation is initiated through random hydrolysis of the ester bonds and chain scission into smaller chain oligomers. When the oligomers decrease in molecular weight to a point where they become water soluble, mass loss and erosion of the polymer matrix takes place. Ultimately, lactic acid and glycolic acid is formed and metabolized and cleared via the cellular tricarboxylic acid cycle (TCA) or renal clearance (Vert et al., 1992). The hydrolysis of ester bonds depends upon accessibility of the aqueous fluid to the ester linkage. Water penetration is dependent on molecular weight, hydrophobicity, copolymer composition and crystallinity of the polymer (Grizzi et al., 1995; Park, 1995; Vert et al., 1992).  28  n= number of lactic acid units; m= number of glycolic acid units  Figure 1-10: Chemical structure of Poly(D,L-lactic-co-glycolic acid)  1.4.2 Methoxypoly(ethylene glycol) (MePEG) Individual films were prepared containing blends of PLGA and MePEG. MePEG is a hydrophilic polyether (Figure 1-11). It is used extensively as a biomaterial for drug delivery applications due to its water solubility and biocompatibility MePEG has been shown to function as a plasticizer in PLGA films as it causes a decrease in the glass transition temperature (Jackson et al., 2004).  n= number of MePEG repeat units  Figure 1-11: Chemical structure of methoxypoly(ethylene glycol)  29  1.5 Nanoparticulate layered double hydroxide (LDH) clay Clay minerals have been utilized for their medicinal properties (Choy et al., 2007). They have been used as both excipients and active principle ingredients in pharmaceutical formulations. For example, as excipients they have been used as emulsifying agents and lubricants in formulations. They are also used as the active principle ingredient in laxatives and anti-diarrhoeals (Aguzzi et al., 2007). These minerals possess properties including high adsorption capacity, chemical inertness and low toxicity that makes them favourable for use in pharmaceutical formulations (Choy et al., 2007; Del Hoyo, 2007; Delhoyo, 2007). There has been a recent interest in the use of clay materials as drug carriers, controlled release agents, stabilizers and solubility enhancers (Del Hoyo, 2007). Layered double hydroxides are synthetic hydrotalcite-like clays which have been used for a wide range of applications as catalysts, pharmaceuticals and cosmetics (Choy et al., 2007). Particularly in the area of pharmaceutical research, they have potential application as nanocarriers for various drug molecules like antibiotics (Trikeriotis and Ghanotakis, 2007), antiinflammatory agents (Ambrogi et al., 2001; Ambrogi et al., 2002; Khan et al., 2009; Li et al., 2004), antihypertensive drugs (Xia et al., 2008) anticoagulants (Gu et al., 2010) and anticancer agents (Choy et al., 2004; Oh et al., 2006; Qin et al., 2010). The drug loaded layered double hydroxide clay intercalated hybrids may be used for the purpose of controlled release, stabilization, protection and solubility enhancement of various drug molecules (Choy et al., 2007; Oh et al., 2009).  30  1.5.1 Chemistry and physicochemical properties Layered double hydroxide clay (LDH), also known as anionic clays, are layered nano-materials having the general formula MII1−xMIIIx(OH)2 Yn-x/n. m H2O where MII and MIII represent the divalent and trivalent metal cations and Yn- represents the hydrated exchangeable anions present in the interlayer space (Parello et al., 2010). The molecular formula of the parent molecule is Mg6 Al2 (OH)16 CO3·4H2O. They possess a brucite-like [Mg(OH)2] layered structure which has a thickness of 0.48 nm (Ambrogi et al., 2001). Some of the divalent cations in the brucite structure are replaced by the trivalent cations, thereby imparting an excess positive charge to the structure. This excess positive charge is balanced by the anions present in the interlayer space, thereby rendering the structure electrically neutral. The layered structure is stabilized by hydrogen bonding among the water molecules, anions and hydroxide layers (Gasser, 2009). The variation of the divalent and trivalent cations, interlayer anions and stoichiometric coefficient (x), imparts flexibility to the structure, forming a large variety of isostructural compounds (Evans and Duan, 2006). Layered double hydroxide clay is basic in nature (Evans and Duan, 2006). It possesses a high specific surface area and also a high layer charge density (2-5 mEq/g), that accounts for the strong electrostatic forces between the brucite layers and anions (Del Hoyo, 2007). From the biological point of view, they are stable, inert and biocompatible systems (Oh et al., 2009). 1.5.2 Drug loading and release characteristics  The incorporation of the drug molecules (guest) into the interlayer space of layered double hydroxide clay (host) by ion exchange mechanism, reconstruction or co-precipitation methods is  31  termed hybridization or intercalation (Aisawa, 2001; Choy et al., 2007; Nakayama, 2004). The molecules incorporated in the interlayer space may be organic anions such as fenbufen (Li et al., 2004) and ibuprofen (Ambrogi et al., 2001) or zwitterions (exist as neutral molecules at pH 7) as in the case of some amino acids like phenylalanine (Aisawa, 2001). Drug molecules may be adsorbed from solution into the interlayer space of the layered double hydroxide clay particles. The adsorption process has been shown to follow the Langmuir model of adsorption (monolayer adsorption) (Gasser, 2009; Nakayama et al., 2003). The Langmuir equation is represented as: (Gasser, 2009) where Ce= Concentration of solution at equilibrium qe= Amount adsorbed at equilibrium Q0= Langmuir constant (related to monolayer adsorption capacity) b= Constant (related to enthalpy of adsorption) The adsorption or intercalation of the anions is influenced by the electrostatic interactions between the layered double hydroxide clay and intercalated drug molecule (Lee et al., 2008). The dimension of the interlayer space is affected by the size and nature of the functional groups of the drug molecule, the orientation of the drug molecule and the electrostatic interactions between the layered double hydroxide clay and drug molecule (Khan and O'Hare, 2002). The release or de-intercalation of the drug molecule from the layered double hydroxide clay interlayer space may occur through ion exchange or displacement reactions (Evans and Duan, 2006). The release profile of the drug molecule from the ‘hybrid’ is characterized by its biphasic nature i.e. an initial burst phase release is observed, followed by a slow sustained release (Khan 32  et al., 2001; Kong et al., 2010; Nakayama et al., 2003). The release mechanism suggests that the fast burst phase of release may occur due to surface diffusion i.e. the diffusion of the anions present on the external surface of clay into the release medium through anion exchange. This is followed by a slow sustained release of the drug that may occur due to diffusion of the anions from the interlayer space onto the external surface of the clay, followed by release into the medium via ion exchange (Kong et al., 2010). The release of the guest molecule (e.g. anionic drug molecule) from the ‘hybrid’ is affected by the strength of the electrostatic interactions between the LDH clay and drug anion. It has been observed that by increasing the electrostatic attraction, the release rate may be decreased and vice-versa (Choy et al., 2007). The rigidity of the layers and the diffusion path length also affect the drug release from the interlayer space (Ambrogi et al., 2001; Evans and Duan, 2006). As described above, the initial large burst phase of release due to surface diffusion of the drug near the external regions of the clay particles results in a decrease in the interlayer distance in these regions. This is followed by the diffusion of the drug anions out of more inner regions of the clay particles, and since the diffusional path length and tortuosity increases, the release becomes slower and more controlled (Ambrogi et al., 2001). 1.6 Goal, hypotheses and objectives of study The overall goal of the project was to develop and characterize a controlled release, dual alendronate and tetracycline loaded plasticized nanocomposite film formulation that may have potential application as a guided tissue regeneration film to treat periodontal disease. The target characteristics of the nanocomposite film formulation proposed are provided in Table 2.1.  33  We hypothesized that: (1) The controlled release and elimination of the burst phase of alendronate release may be achieved by binding alendronate to nanoparticulate clay, followed by addition to the polymer blend. (2) That tetracycline-alendronate loaded nanocomposite film may form a biocompatible surface to provide osteoblast cell proliferation. The objectives of the project were:   To determine the binding of alendronate to clay particles and the physicochemical properties of alendronate loaded LDH clay.    To evaluate the loading, chemical stability and release of tetracycline from PLGA membranes.    To prepare and characterize dual drug loaded nanocomposite membranes.    To evaluate the proliferation of osteoblasts on drug loaded nanocomposite membranes.  34  2. DRUG INTERCALATION IN LAYERED DOUBLE HYDROXIDE CLAY: APPLICATION IN THE DEVELOPMENT OF A NANOCOMPOSITE FILM FOR GUIDED TISSUE REGENERATION 2.1. Introduction Periodontal diseases such as gingivitis and periodontitis, are inflammatory disorders that affect the tissues supporting the teeth (Pihlstrom et al., 2005) and are caused by bacterial infections in the tissues adjacent to the teeth. As the inflammation progresses, periodontal pockets or crevices, form between the gingival tissues and the tooth root, causing degeneration of the periodontium and resorption of alveolar bone, which can lead to tooth loosening and eventually, tooth loss (Haffajee and Socransky, 1986). Significant bacterial loads and a broad range of microflora are commonly found in periodontal pockets (Pihlstrom et al., 2005). The first-line, non-surgical approach to treating periodontal disease includes removal of dental plaque and calculus and the adjunctive localized delivery of antibiotics to the gingival crevicular fluid (GCF) within the periodontal pocket (Pihlstrom et al., 2005), via irrigation solutions or controlled release gels, fibres and implants (Agarwal et al., 1993; Webber et al., 1998). For more advanced disease, surgical strategies include guided tissue regeneration (GTR), a method in which a barrier film is surgically placed between the tooth and gingival connective tissue, allowing the detached root surface to be repopulated with regenerating cells such as osteoblasts and periodontal ligament cells (Gottlow et al., 1986; Nyman et al., 1982). The film acts as a mechanical barrier allowing undisturbed and guided bone tissue regeneration (Gottlow et al., 1986; Nyman et al., 1982; Wang et al., 2002). This chapter has been submitted for publication.  35  Bioabsorbable membranes composed of collagen, calcium sulphate or synthetic polyesters may promote periodontal regeneration, by providing a protected space for inward migration of regenerating cells, and the field has recently been reviewed by Villar et al (Villar and Cochran, 2010). Retention of the physical integrity of the membrane was suggested to be about 6 weeks for the healing process, after which biodegradation and resorption would be optimal. However, it was noted that these biodegradable GTR membranes possess only a limited clinical efficacy since they have no biological effects on cellular proliferation or differentiation (Villar and Cochran, 2010). Furthermore, significant variability in the surgical outcomes of GTR procedures has been observed (Gottlow et al., 1986) frequently due to bacterial colonization of the membrane following its placement into the periodontal pocket, which may limit the proliferation of the regenerating cells like osteoblasts (Tempro and Nalbandian, 1993). Our group has developed biodegradable, plasticized poly(lactic-co-glycolic acid) (PLGA) films loaded with either tetracycline or alendronate for potential application as GTR membranes (Long et al., 2009; Owen et al., 2010). Tetracycline and other similar broad spectrum antibiotics, including minocycline and doxycycline, have been used extensively in the treatment of periodontal disease. Tetracycline not only eliminates or reduces microbial load in the GCF, but it also inhibits metalloproteinases (collagenases) that breakdown collagen and exacerbate inflammation (Golub et al., 1998; Golub et al., 1991; Rifkin et al., 1993; Weinberg and Bral, 1998). Alendronate, an amino-bisphosphonate, is a potent inhibitor of osteoclastic resorption and may trigger the proliferation of osteoblasts (Fleisch, 1998; Im et al., 2004; Reinholz et al., 2000; von Knoch et al., 2005). Commonly, the drug is used for the treatment of bone disorders such as osteoporosis, Paget’s disease and osteolytic bone metastases (Fleisch, 2007) and is proposed for 36  use in the treatment of periodontal disease (Shinoda and Takeyama, 2006; Tenenbaum et al., 2002). Studies have shown that the local delivery of alendronate may improve the bone growth around dental implants (Yaffe et al., 1997; Yaffe et al., 2003). In our previous work, we demonstrated that, although controlled release of alendronate could be achieved from 0.1% and 0.25% alendronate loaded PLGA films over 30 days, there was only a marginal increase in osteoblast cell numbers and a higher alendronate loading (0.5%) in the film produced evidence of osteoblast toxicity, thought to be due to the very large burst phase of alendronate release (Long et al., 2009). We hypothesized that a combination drug loaded GTR film formulation of tetracycline and alendronate, that would also eliminate the burst phase of alendronate release, would be effective in promoting osteoblast viability and proliferation. Our goal was to develop co-drug loaded films with target properties as summarized in Table 2-1. The use of anionic clay nanoparticles to form an intercalation compound with alendronate was explored to provide an additional controlled release strategy for alendronate.  37  Table 2-1: Target properties and formulation strategies of a co-loaded antibacterial/bisphosphonate guided tissue regeneration (GTR) membrane for application in periodontal therapy  Feature of GTR membrane  Target properties and characteristics  Placement  Between tooth and gingival connective tissue  Tissue-membrane interface  Biocompatible for tissue integration  Lifetime of GTR membrane  Biodegradable PLGA-based film to avoid surgical removal Biodegradation lifetime > 4-6 weeks for periodontal attachment Initial handling: flexible, with some elasticity to conform and stretch around tooth. MePEG350 used as a plasticizer. After placement: stiffening to protect defect space. MePEG diffuses out of film in aqueous fluids.  Mechanical properties  Drug loading and approx release lifetime Antibacterial:  Tetracycline: protect from bacterial invasion (release over ~2weeks)  Bisphosphonate:  Alendronate: enhance bone growth (release over ~4-6 weeks)  Reduction in alendronate burst phase of release  Alendronate bound to nano-clay particles and dispersed in polymer matrix  Other  Sterile Chemical stability maintained  38  Layered double hydroxides have the general formula: MII1−xMIIIx(OH)2 Yn-x/n. m H2O where MII and MIII represent the divalent and trivalent metal cations and Yn- represents the hydrated exchangeable anions present in the interlayer space (Parello et al., 2010). They possess a basic structure of brucite [Mg(OH)2]. The brucite - like sheets are positively charged due to the partial substitution of divalent metal cations by trivalent ions. The positive charge is balanced by interlayer anions like carbonates along with water molecules, therefore rendering the structure electrically neutral. The layered structure is stabilized by hydrogen bonding among the water molecules, anions and hydroxide layers (Gasser, 2009) and a schematic representation is shown in Figure 2-1. Layered double hydroxides possess a high specific surface area and high layer charge density (2-5 mEq/g), which results in strong electrostatic forces between the brucite sheets and anions. They also possess anion exchange properties (Choy et al., 2007; Del Hoyo, 2007). Recent studies have shown that LDH clay can bind with, and retain different drugs and modulate and/or delay their release (Aguzzi et al., 2007; Ambrogi et al., 2002; Evans and Duan, 2006; Li et al., 2004; Mohanambe and Vasudevan, 2005; Pihlstrom et al., 2005; Trikeriotis and Ghanotakis, 2007; Zhang et al., 2006; Zhang, 2004). The objectives of this work were to develop a co-loaded, controlled release tetracycline and alendronate nanocomposite plasticized PLGA film, with the target characteristics appropriate for a GTR membrane (Table 2-1), eliminating the burst phase of alendronate release and forming a suitable matrix supporting osteoblast proliferation and differentiation.  39  Figure 2-1: Graphical representation of the structure of layered double hydroxide clay. The figure depicts the presence of carbonate anions in the interlayer space.  40  2.2. Material and Methods Poly(D,L lactic-co-glycolic acid) (PLGA), with a weight percentage ratio of 85/15 (intrinsic viscosity of 0.61 dL/g) was obtained from Birmingham Polymers (Birmingham, AL). Methoxy poly(ethylene glycol) (MePEG) (molecular weight 350 g/mol) was obtained from Union Carbide (Danbury, CT). Alendronate sodium trihydrate and tetracycline hydrochloride were obtained from Sigma-Aldrich (St Louis, MO). Aluminium/magnesium carbonate layered double hydroxide clay (LDH clay) was kindly provided by Dr. David Plackett (Risø DTU National Laboratory, Copenhagen). All solvents used were of HPLC grade and obtained from Fisher Scientific (Fairlawn, NJ). 2.2.1 Size analysis and elemental analysis of layered double hydroxide clay LDH clay samples contained particle aggregates and were therefore de-aggregated by ultrasonicating a suspension of LDH clay in ethanol before size analysis was performed using the Malvern Zetasizer Nano ZS (Malvern Zetasizer Ltd, UK) and elemental analysis using the Hitachi S-3000N scanning electron microscope- energy-dispersive X-ray spectroscopy analysis (SEM-EDX) (Tokyo, Japan). Samples were coated with a 60:40 alloy of gold:palladium using a Denton Vacuum Desk II sputtercoater (Moorestown, NJ) at 50 torr. 2.2.2. Drug binding to layered double hydroxide clay Increasing concentrations of alendronate in deionized water (15.625 µg/mL to 4 mg/mL) were added to LDH clay (2 mg). The dispersion was vortexed for 2 minutes and incubated for 1 hour in a rotary shaker (37° C), followed by centrifugation for 5 minutes (18000× g). The supernatant (representing the unbound concentration of alendronate) was analyzed using HPLC with  41  fluorescence detection (Long et al., 2009) as described below. The amount of alendronate bound was calculated by subtracting the unbound amount from the total amount of drug added. A binding study was carried out using tetracycline, as described above. Supernatants were analyzed using HPLC with UV detection (Owen et al., 2010) (see below) and the amount of tetracycline bound by clay determined. 2.2.3 Characterization of the alendronate-LDH clay complex 2.2.3.1 X-ray powder diffraction X-ray powder diffraction patterns of alendronate loaded clay and control clay samples were obtained using a Bruker D8 advanced diffractometer with a Cu source at 25 ° C. Approximately 100 mg of sample was packed into the sample holder and scanned from 2 - 60° 2θ using a step size of 0.020⁰ and step time of 1second/step. 2.2.3.2 Differential scanning calorimetry (DSC) Samples (about 5 mg) of LDH clay or alendronate-LDH clay complex were placed in a crimped aluminum DSC pan and heated between 30 - 300° C at a heating rate of 10° C/min under nitrogen flow. 2.2.3.3 Alendronate release from LDH-clay complex In vitro release studies were carried out in phosphate buffered saline (PBS) (0.1 mM; pH 7.4) at 37° C. Alendronate was bound to LDH clay using the procedure described above. This was followed by the incubation of the complex with PBS (1mL) for pre-determined time intervals, after which it was centrifuged (18000× g) for 10 minutes. The supernatant was withdrawn for analysis and replaced by PBS (1mL). 42  2.2.4 Preparation of the dual-drug loaded nanocomposite film formulations Film casting solutions were prepared by dissolving PLGA in dichloromethane at a concentration of 20% (w/v) of polymer solution, followed by the addition of MePEG (15% w/w) to the polymeric solution. Tetracycline hydrochloride (5% w/w) was pre-dissolved in a minimal volume of dimethylsulphoxide and added to the above prepared film casting solution. Alendronate (5% w/w) was bound to LDH clay as described above. The alendronate-LDH clay complex did not disperse readily in the polymer solution, so that 100 µL polysorbate 20 (2% v/v) was added to aid dispersion of the complex in the polymeric solution. The dispersion formed was added to the film casting solution and mixed vigorously by ultrasonication and vortexing until the complex was homogeneously suspended, following which, a 100 µL aliquot of the dispersion was cast on 1cm × 1cm teflon templates applied to glass slides. 2.2.5 Characterization of the nanocomposite film formulations 2.2.5.1 Drug release studies The films were weighed individually and placed in 20 mL glass vials. To the vials, were added 5 mL PBS (pH 7.4, 0.1 mM) and the samples were incubated in a rotary shaker at 37 ° C. At predetermined intervals, all the PBS was withdrawn and replaced with fresh PBS. The samples were analyzed using HPLC analysis methods for alendronate and tetracycline. 2.2.5.1.1 Analysis of alendronate Samples were assayed for alendronate using HPLC with fluorescence detection using previously described methods (Long et al., 2009). Briefly, samples were prepared by adding 100 µL of the drug release sample to 900 µL of 0.13 M EDTA buffer (pH 10) and 500 µL of fluorescamine dissolved in acetonitrile (2 mg/mL). The samples were then gently agitated till a yellow colored 43  solution was formed (approximately 10 seconds), 1 mL dichloromethane was added and the samples were shaken vigorously. The solution separated into two layers. The yellow upper layer was withdrawn for HPLC analysis with fluorescence detection (‫גּ‬ex= 395 nm, ‫גּ‬em= 480nm). Analysis of alendronate was performed on a Waters HPLC system (Milford, MA, USA) consisting of a model 717 plus autosampler, 1525 binary HPLC pump and 2475 multi ‫גּ‬ fluorescence detector. The mobile phase was composed of EDTA (1 mM)/ methanol (97:3 v/v) mixture (pH 6.5) with a flow rate of 1 mL/min (Alltech nucleosil 100 C 18 column). (Further details on HPLC analysis of alendronate are provided in the appendix) 2.2.5.1.2 Analysis of tetracycline hydrochloride The samples were assayed for tetracycline hydrochloride using HPLC with UV absorbance detection at 358 nm (Owen et al., 2010). Analysis was performed on a Waters HPLC system (Milford, MA, USA) consisting of a model 717 plus autosampler, 1525 binary HPLC pump and 2487 dual ‫ גּ‬absorbance detector. The mobile phase was composed of 12% v/v acetonitrile in 1.3 g/L oxalate solution (pH 2.1), having a flow rate of 1mL/min (reverse phase C 18 Novapak column). (Further details on HPLC analysis of tetracycline hydrochloride are provided in the appendix) 2.2.5.2 Stress-strain determination Stress-strain experiments were carried out at room temperature using a thermal mechanical analyzer (TMA) (TA instruments, New Castle, Delaware) as previously described (Jackson et al., 2004). The rectangular films were measured using digital calipers (Mitutoyo, Japan). The film was subjected to increased force per unit length and the film extension was measured. Film recovery was observed before adding more weight. Measurements were discontinued when the 44  membrane ceased to recover. Film extension was measured after incubation in PBS (pH 7.4) for predetermined time intervals. Film stiffness was calculated in terms of Young’s modulus (stress/strain). 2.2.6 Osteoblast culture Calvarial osteoblasts (from newborn Sprague Dawley rats) were kindly provided by Dr. Donald Brunette (Department of Oral, Biological and Medical Sciences, Faculty of Dentistry, UBC, Vancouver, Canada). The cells were subcultured by trypsinization as described previously (Long et al., 2009) and maintained in minimal essential medium (α-MEM) (Stem Cell Technologies, Vancouver, Canada) that was composed of fetal calf serum (15%; Cansera International, Rexdale, ON, Canada), amphotericin B (Fungizone®) (3 µg/mL; Gibco, Grand Island, NY, USA), penicillin G (100 µg/mL) and gentamycin (50 µg/mL) (Sigma-Aldrich, St Louis, MO, USA) in a humidified atmosphere with CO2 (5%) at 37° C. The experiments were performed in 24 or 48 well Falcon™ tissue culture treated plates (BD Bioscience). The polymeric films were sterilized by glow discharge using the plasma sterilizer (PDC 32-G connected to a high performance vacuum pump SPX, Owatonna, MN, USA). The osteoblasts used were between a 510 cell passage number. 2.2.7 Osteoblast viability Cell viability was determined by the MTS assay kit (CellTiter 96 ® AQueous One Solution Cell Proliferation Assay; Promega, WI, USA). The films used in the study were prepared as described above and were composed of the following groups: free alendronate and alendronate bound to LDH clay in the concentrations,  45  0.03%, 0.1%, 0.75% and 4.2% (w/w). The films were placed in the individual wells of the plate (n=6), followed by the addition of media (500 µL) and osteoblast cells (1× 10 4) into each well. The plates were incubated for 2 days after which the media was removed from the wells and washed with PBS. Finally, the MTS reagent (200 µL) was added to each well and the plate was incubated for 3 hours. The absorbance was measured at 492 nm (subtracting the blank reading at 600 nm) using the Labsystems Multiskan Ascent photometric plate reader (Labsystems, Helsinki, Finland). 2.2.8 Alkaline phosphatase activity Bone specific alkaline phosphatase (ALP) is a membrane bound exoenzyme produced by osteoblasts. The presence of the enzyme indicates the onset of osteoblastic cell differentiation, thus it is used as a marker for bone formation (Long et al., 2009). Osteoblasts were grown for a period of 5 weeks in wells of tissue culture plates. Control wells had no films. Sterilized films, placed in individual wells, were composed of tetracycline, tetracycline and free alendronate, and tetracycline and alendronate-LDH clay complex. As described previously, the films in each well were seeded with 1× 104 cells per well and reseeded after two days with another 1× 104 cells in 500 µL media, supplemented with 10 mM sodium beta glycerophosphate and ascorbic acid (50 µg/µL). After 5 weeks of culture, the media was removed and each well was washed three times with PBS. The osteoblast cell extract was prepared by lysing the cells with a probe sonicator for 20 seconds in 1 mL Tris buffer (0.1 M, pH 7.2) containing 0.1% Triton-X 100 at 4° C. The sonicated cell lysates were then centrifuged at 1500× g (for 10 min), at 4 ° C and the supernatant obtained was used in the assay for analysis of alkaline phosphatase activity. Alkaline phosphatase activity of the osteoblasts was determined spectrophotometrically at 405 nm by 46  quantifying the conversion of p-nitro phenyl phosphate to p-nitro phenol, on the addition of 50 µL cell extract to 150 µL alkaline phosphatase reagent (Promega, WI, USA). ALP activity was normalized to the total protein content of the cells. Protein content was determined spectrophotometrically using a Micro BCA protein assay kit (Thermo Scientific, IL, USA). 2.2.9 Bone nodule formation Bone nodules deposited by osteoblasts, were observed by alizarin red dye staining (SigmaAldrich St Louis, MO) of cells after 5 weeks of cell growth in wells of tissue culture plates. Control wells had no films. Sterilized films, placed in individual wells, were composed of tetracycline, tetracycline and free alendronate, and tetracycline and alendronate-LDH clay complex as described previously for the ALP assay (Long et al., 2009). Briefly, the osteoblast cells present were fixed with 1 mL paraformaldehyde solution (4% w/v, Sigma) for 24 hours, following which the fixative was removed and each well was washed three times with PBS. To each well, 500 µL alizarin dye in PBS (20 mg/mL) were added for 5 minutes, after which the dye was removed and each well was washed three times with PBS in order to remove excess stain. The dye from the stained nodules was extracted using cetyl pyridinium chloride (10% w/v in PBS; Sigma-Aldrich) as previously described (Stanford et al., 1995). Briefly, cetyl pyridinium chloride (1 mL) was added to each well. After the dye was extracted, the solution was transferred into the 96-well plates, and its absorbance was measured at 540 nm using the Labsystems Multiskan Ascent photometric plate reader (Labsystems, Helsinki, Finland).  47  2.2.10 Statistical analysis Statistical analyses were performed using GraphPad Prism® version 5 (GraphPad Software, San Diego, CA). Significant differences among the groups was determined using a one way ANOVA followed by Turkey- Kramer’s post hoc test with a significance level p<0.05. 2.3. Results 2.3.1 Size and composition of LDH clay The LDH clay particles possessed an average diameter of 557± 95 nm. Surface X-ray analysis showed the following elements and their amounts by weight %: carbon (18.24), oxygen (59.59), sodium (0.35), magnesium (15.68) and aluminium (6.13), indicating the clay sample was a magnesium aluminum hydroxycarbonate. 2.3.2. Adsorption isotherm and characterization of alendronate-LDH clay complex Alendronate bound to LDH clay particles in a concentration dependent manner. Saturation binding was observed at an alendronate concentration of approximately 2 mg/mL, where about 600 µg alendronate was bound per mg LDH clay (~ 32% w/w drug bound) (Figure 2-2). The X-ray diffraction patterns for LDH clay and alendronate-LDH clay complex samples are shown in Figure 3. LDH clay samples showed a strong X-ray diffraction peak at 11° (2θ) which corresponded to a d-spacing of 0.77 nm. Alendronate-LDH clay complex sample showed a smaller peak at 11° and an additional peak at 7° (2θ) corresponding to a d-spacing of 1.28 nm (Figure 2-3). DSC scans for both LDH clay and alendronate-LDH clay complex showed very similar broad endotherms beginning at about 70° C, with the peaks occurring at 127° C (data not shown) indicative of loss of adsorbed and intercalated water from the clay particles. These LDH type 48  clays, with and without intercalated drug, are well known to exhibit this distinctive decrease in water content between 70 – 200o C (Gasser, 2009). Figure 2-4 shows the alendronate release profile from alendronate-LDH clay complex. There was a very large initial burst release of alendronate (~45%) from the LDH clay particles over the first 6 hours, followed by a period of slower sustained release, with approximately 80% drug released in 6 days.  Amount of alendronate bound/amount of LDH clay (µg/mg)  700  600  500  400  300  200  100  0 0  500  1000  1500  2000  2500  3000  3500  4000  4500  Alendronate concentration (µg/mL)  Figure 2-2: Adsorption isotherm: alendronate bound to layered double hydroxide clay at room temperature. Data expressed as mean±S.D; (n=3).  49  d= 0.77 nm  Intensity (cps)  200  100  (a) LDH clay 0  (b) alendronate-LDH clay complex  d= 1.28 nm  2  10  20  30  40  50  60  2θ  Figure 2-3: X-ray diffraction patterns of: (a) layered double hydroxide clay (b) alendronatelayered double hydroxide clay complex. The arrows indicate the d-spacing at 11° and 7° 2θ respectively.  50  100  cumulative alendronate release (%)  90 80 70 60 50 40 30 20 10 0 0  2  4  6  8  10  Time (days)  Figure 2-4: Cumulative alendronate release from alendronate-layered double hydroxide clay complex in PBS (pH 7.4) at 37° C under gentle agitation. Data expressed as mean±S.D; (n=3).  2.3.3 Characterization of the nanocomposite film formulation The film matrix used in these studies was a miscible blend of 15% MePEG in PLGA. Previous studies in our lab have shown that the addition of MePEG to PLGA causes a concentration dependent decrease in the Tg. The MePEG was added as a plasticizing agent and produced a film with a glass transition temperature (T g) of 11° C (Jackson et al., 2004). The films were prepared by the solvent evaporation method, with co-loading of the drugs accomplished by addition of free tetracycline and alendronate as the alendronate-LDH clay complex.  51  2.3.3.1 Tetracycline and alendronate release from film formulations  The release profiles for tetracycline from films loaded either with free tetracycline or nanocomposite films co-loaded with free tetracycline and alendronate-LDH clay complex are shown in Figure 2-5. Films loaded with free tetracycline showed a burst phase of release of about 65% in the first 6 hours and almost 80% drug released over the next 3 days. However, the tetracycline/alendronate co-loaded nanocomposite film produced a significantly reduced tetracycline burst phase down to about 10% with 50% of tetracycline released over 7 days (Figure 2-5). On completion of the release studies, the films were dissolved in order to determine the recovery of intact tetracycline by HPLC. About 80% (w/w) tetracycline was released from tetracycline loaded films and upon dissolution of the films, no additional tetracycline was detected. From the dual drug loaded nanocomposite films 50% (w/w) tetracycline was released and on dissolving these films, about 28% (w/w) of intact tetracycline was recovered, indicating that about 80% (w/w) of the total drug present could be accounted for. Given the greatly reduced tetracycline release from nanocomposite films compared to tetracycline when loaded as the free form in the films, it was possible that tetracycline was adsorbed or bound by the LDH clay particles loaded in the nanocomposite films. Tetracycline was shown to bind to LDH clay particles with a binding curve very similar to that of alendronate (Figure 2-2), with maximal uptake of about 600 µg tetracycline bound per mg of clay at a concentration of 4 mg/mL tetracycline (data not shown). Alendronate release profiles from free alendronate loaded films, showed 35% burst phase with a slower release phase of up to 80% after 10 days (Figure 2-6) whereas nanocomposite 52  films co-loaded with free tetracycline and alendronate-LDH clay complex produced no burst phase and a slow controlled release of alendronate of about 15% over 10 days.  90  cumulative tetracycline release (%)  80 70 tetracycline loaded film 60  50 40 tetracycline and alendronate - LDH clay complex loaded film  30 20 10 0 0  2  4  6  8  10  12  Time (days)  Figure 2-5: Cumulative release of tetracycline from films composed of a polymeric blend of PLGA (85:15) and MePEG (15% w/w) loaded with (% w/w): (∆) 5% tetracycline (□) 5% tetracycline and 4.2% alendronate-LDH clay complex [at 37°C; in PBS (0.1 mM)]. Data expressed as mean±S.D; (n=5).  53  120  cumulative alendronate release (%)  100  80  alendronate loaded film  60  40 tetracycline and alendronate - LDH clay complex loaded film 20  0 0  2  4  6  8  10  12  Time (days)  Figure 2-6: Cumulative release of alendronate from films composed of a polymeric blend of PLGA (85:15) and MePEG (15% w/w) loaded with (% w/w): (o) 4.2% alendronate (□) 5% tetracycline and 4.2% alendronate-LDH clay complex [at 37°C; in PBS (0.1 mM)]. Data expressed as mean±S.D; (n= 5).  2.3.3.2 Stress-strain determination by thermal mechanical analysis (TMA) PLGA films plasticized with 15% MePEG have excellent mechanical properties (Table 1) being flexible, with some elasticity. Previous work in our laboratory has shown that following incubation of MePEG/PLGA films in PBS, the water soluble MePEG is rapidly released from the film (Owen et al., 2010), producing a film that, when handled, appears stiffer with less flexibility. Young’s modulus (Y) defined as the ratio of stress (force per unit area) to strain (change in length per unit length) is a measure of the material stiffness. Films incubated for  54  different time periods in PBS (Figure 2-7) showed an increase in Young’s modulus over 2 hours, indicating enhanced stiffness of the films.  500 450 tetracycline and alendronate LDH clay complex loaded film  Young's modulus (MPa)  400 350 300  tetracycline and alendronate loaded film  250  200 150 100 50 0  -20  0  20  40  60  80  100  120  140  Time (minutes)  Figure 2-7: Variation of Young’s modulus with an increase in incubation time in aqueous media (0.1 mM PBS; 37° C) on films composed of a polymeric blend of PLGA (85:15) and MePEG (15% w/w) loaded with (% w/w): (▲) 5% tetracycline and 4.2% alendronate (□) 5% tetracycline and 4.2% alendronate-LDH clay complex. Data expressed as mean±S.D; (n=3).  55  2.3.4 Osteoblast viability Osteoblast viability experiments were performed on the following control groups: no films (only cells growing in the well) and films composed of PLGA and MePEG, PLGA, MePEG and LDH clay. No significant difference was observed in cell viability among these groups (data not shown). Osteoblast viability was then evaluated in free alendronate loaded films and alendronate-LDH clay complex loaded films. In this study, the osteoblast viability (%) of the film groups were compared to the control films composed of alendronate 0.03%. There was no change in osteoblast viability at low alendronate concentrations (up to 0.1% w/w) for the free or bound alendronate films (Figure 2-8). However, when the alendronate concentration was increased to 0.75%and 4.2%, a significant decrease in cell viability was observed in films containing free alendronate but cell viability was unchanged in the films containing the same concentration of alendronate bound to LDH clay (1:1 ratio).  56  Figure 2-8: Osteoblast viability after a two day incubation period on films composed of a blend of PLGA (85:15) and MePEG (15% w/w) loaded with 0.03, 0.1, 0.75 and 4.2 (% w/w): a) alendronate and b) alendronate-LDH clay complex. Data expressed as mean±S.D; (n=6). **P< 0.05. Films composed of 0.03% alendronate were the control group. (Separate viability studies containing groups composed of cells (no films), films composed of PLGA and MePEG and PLGA, MePEG and LDH clay were performed. No significant difference was observed in the osteoblast viability (%) among those groups in comparison to the control).  2.3.5 Alkaline phosphatase activity (ALP) and bone nodule formation The effect of alendronate released from film formulations on the ALP activity (Figure 2-9) and bone nodule formation of osteoblasts (Figure 2-10) was evaluated. There was no significant difference in the ALP activity and bone nodule formation in the plates containing no film controls, free tetracycline loaded films and free tetracycline plus free alendronate loaded films. However, a significant increase in both the ALP activity and bone nodule formation was 57  observed for the free tetracycline and alendronate-LDH clay complex nanocomposite film compared to the free drug loaded film formulations.  Figure 2-9: Alkaline phosphatase activity of osteoblasts grown for 5 weeks on no films; and films composed of a blend of PLGA (85:15) and MePEG (15% w/w) loaded with (% w/w) a) 5% tetracycline b) 5% tetracycline and 4.2% alendronate and c) 5% tetracycline and 4.2% alendronate-LDH clay complex. Data expressed as mean±S.D; (n=12).  ***  P< 0.05. TET and AL:  abbreviations used for tetracycline and alendronate respectively.  58  Figure 2-10: Bone nodule formation. Osteoblasts grown for 5 weeks on no films and films composed of a blend of PLGA (85:15) and MePEG (15% w/w) loaded with (% w/w) a) 5% tetracycline b) 5% tetracycline and 4.2% alendronate and c) 5% tetracycline and 4.2% alendronate-LDH clay complex. Data expressed as mean±S.D; (n=12).  ***  P< 0.05. TET and AL:  abbreviations used for tetracycline and alendronate respectively.  59  2.4. Discussion Untreated periodontal disease is characterized by advanced bacterial infection that leads to the spread of inflammation into the deeper tissues and alveolar bone resorption. Therefore, the objective of our study was to co-deliver both an antibacterial agent, tetracycline, and a bone resorption inhibitory agent, alendronate, into the periodontal pocket using a biocompatible and biodegradable film with a proposed degradation/drug release lifetime of about 4-6 weeks, suitable mechanical properties and, importantly, suppression of the burst phase of release of the water soluble alendronate from the film to avoid osteoblast toxicity issues (Table 2-1). Extensive binding of alendronate to the LDH clay particles of around 32% w/w was observed, similar to other reports of binding of anionic drugs to LDH clays, including captopril (Zhang et al., 2006), non-steroidal anti-inflammatory compounds (Ambrogi et al., 2002; Mohanambe and Vasudevan, 2005) and antibiotics (Trikeriotis and Ghanotakis, 2007). The binding has been shown to be due to the anion exchange properties of the LDH clay and replacement of the interlayer anions with drug anions. Ambrogi et al (Ambrogi et al., 2001) showed the binding of ibuprofen to a Mg/Al-based LDH clay composed of exchangeable chloride ions to be 0.5 g ibuprofen per 1 g of LDH-ibuprofen intercalated product. The drug anions were incorporated until all the clay chloride ions had been exchanged by ibuprofen. These guest-host intercalation structures can be analyzed by X-ray diffraction, as shown in Figure 2-3. LDH clay showed three relatively intense peaks at low 2θ, indicative of a crystallized form of LDH clay. The interlayer distance of the LDH host particles is represented by the peak at 2θ of 11° with a d-spacing of 0.77 nm. As a consequence of intercalation of  60  alendronate ions, the interlayer distance increased to a d-spacing value of 1.28 nm. The thickness of the brucite layer, shown schematically in Figure 2- 1 has been reported by several groups as 0.48 nm (Ambrogi et al., 2001; Zhang et al., 2006). Overall, an expansion of about 0.5 nm was observed upon alendronate intercalation which is lower than for ibuprofen intercalation into a nitrate exchanging anionic clay (Ambrogi et al., 2001) but likely reflects easier accommodation of the alendronate anion into the interlayer space and less requirement for lattice expansion. The peak at 11° 2θ did not disappear completely for the alendronate-LDH clay complex, indicating that not all interlayer sites exchanged with alendronate. Ambrogi et al also showed that the original anionic clay (with exchangeable chloride ions) showing a d-spacing of 0.78 nm was retained in the X-ray diffraction pattern following intercalation of diclofenac into the clay (Ambrogi et al., 2002). The release profile of the alendronate-LDH clay complex in PBS is characterized by an initial burst phase release followed by a slower sustained release of the remaining drug over 4-6 days (Figure 2-4). Alendronate release from the LDH clay occurred as phosphate and/or chloride ions in the PBS buffer exchanged with alendronate and allowed alendronate anions to be released from the clay structure into the release medium. Transport of alendronate ions out of the matrix is thought to be controlled primarily via diffusion out of the clay matrix and is related to the rigidity of the layers and the diffusion path length (Ambrogi et al., 2001). It has been suggested that the initial large burst phase of release is due to anion exchange near the external regions of the clay particles, with concurrent decrease in interlayer distance in these regions. Release then becomes slower and more controlled as a result of the diffusional path length and tortuosity becoming increased for diffusion of the larger drug anions out of more internal regions of the 61  clay particles (Ambrogi et al., 2001). Interestingly, most reports of intercalated drug release from anionic LDH clays show release to be very rapid and complete within minutes (Ambrogi et al., 2001; Li et al., 2004; Trikeriotis and Ghanotakis, 2007; Zhang et al., 2006) to several hours (Ambrogi et al., 2002). Gasser (Gasser, 2009) also noted that, whereas most drugs are shown to release rapidly via ion exchange from the clays, the release of vitamin C from intercalated LDH clays was not completed within 120 min. Gasser suggested that the much slower release of vitamin C from the LDH clays was due to drug molecules being deeply embedded in the LDH host structure (Gasser, 2009). We have suggested that the targeted mechanical properties of the GTR film require a flexible, elastic film for initial placement, which was achieved by the blending of 15% MePEG into the PLGA (Table 2-1). The Young’s modulus values for these plasticized films loaded with either free tetracycline/alendronate or tetracycline/alendronate-LDH clay complex were between 7 MPa to 12 MPa. Following exposure of the film to aqueous fluids, the Young’s modulus of both films increased dramatically up to 2 hours (Figure 2-7), corresponding to a time dependent loss of the water soluble MePEG from the film matrix (Jackson et al., 2004). This increase in Young’s modulus of the film was considered to be an important feature of the GTR film, to impart stiffness to create a seal around the tooth, protect the defect space and prevent gingival cells from populating the void space during healing. Furthermore, increased membrane stiffness could prevent collapse of the GTR film in the void space, which is often observed as the films are not able to bear the pressure of the growing cells (Owen et al., 2010). Although the Young’s modulus for the LDH-containing films was higher than the films with no clay following incubation, there were no differences in the films in the dry state (before incubation). It is 62  possible that MePEG was released faster from the LDH clay-containing films because of either greater water uptake or increased porosity of films containing clay nanoparticles. Alendronate release profiles from PLGA films (with 15% MePEG) loaded with either free alendronate or alendronate-LDH clay complex particles (4.2 % w/w loading) are compared in Figure 2-6. Tetracycline was loaded in both groups of films at 5% w/w. The data demonstrates that alendronate release is dramatically influenced by the combination effect of the anion exchanging clay and the polymer matrix, such that the burst phase of alendronate release is essentially eliminated and release rates significantly lowered. Tetracycline release profiles from the plasticized films described above, loaded with either free tetracycline or free tetracycline/alendronate-LDH clay complex are compared in Figure 2-5. Interestingly, although tetracycline was loaded in the free state into all films, tetracycline release rate was markedly decreased by the presence of the alendronate-LDH clay complex. Further binding studies showed that there was significant tetracycline binding by the LDH clay nanoparticles. The binding is suggested to be due to intercalation within the clay particles, given that it has been reported that in addition to anionic compounds, neutral and zwitterionic species may also undergo intercalation (Choy et al., 2007). Hence, the decreased tetracycline release rate from the dual drug loaded nanocomposite films was likely caused by the uptake of PBS buffer into the film, followed by diffusion of tetracycline onto available sites in the interlayer spacing of the LDH clay nanoparticles. Dissolution of films on completion of release studies and recovery of tetracycline in the film, showed significant levels of intact drug (28% of the initial loading) remaining, suggesting that tetracycline was intercalated in the clay particles and retained in the film. 63  Nanocomposite film formulations loaded with alendronate were evaluated for biocompatibility and ability to provide a surface for maintaining viability of osteoblast cells (Figure 2-8). Tetracycline was not included in the films for the viability studies as tetracycline interfered with the experimental conditions. Initial studies with co-loaded films, showed that as significant amounts of tetracycline were released from the films within the first 2 days, yellow precipitates were observed over the cell layers and cells could not be visualized. The plasticized PLGA films could be loaded with alendronate-LDH clay complex nanoparticles up to 4.2% without any decrease in cell viability, whereas free alendronate in the films showed evidence of osteoblast toxicity at a loading level of 0.75% and higher. The viability studies were followed by long term studies, where the effects of the nanocomposite formulations were observed on ALP and bone nodule formation over a period of five weeks. Bone specific alkaline phosphatase (ALP) is a membrane bound exoenzyme produced by osteoblasts. It is required during bone formation for osteoid formation and matrix mineralization (Mohamadnia et al., 2007). An increase in ALP levels signifies an increase in osteoblastic activity. Following cell differentiation, mineralization of the matrix takes place and calcium deposits are laid down in the extracellular matrix. The deposits of calcium in the matrix are referred to as bone nodules and they are an indication of early cell maturation (Long et al., 2009). Figures 2-9 and 2-10 demonstrate that either free tetracycline or free alendronate loaded into the films had no effect on ALP activity and bone nodule formation in the cell cultures. Although alendronate has been shown to increase proliferation and enhance osteoblastic differentiation at submicromolar concentrations (Long et al., 2009), we believe that for the films containing free co-loaded drugs, the burst phase of alendronate release was toxic to osteoblasts, 64  thus inhibiting osteoblast proliferation and differentiation. However, after the initial burst phase of release was complete, the reduced amounts of alendronate released may have been favorable for cell growth, allowing the cells to recover and proliferate. Tetracycline was released rapidly from the nanocomposite films and showed a large burst phase and this is suggested to be optimal for maintaining high concentrations of the antibiotic as wound healing occurs (Owen et al., 2010).  65  3. CONCLUSION A plasticized biodegradable PLGA film formulation based on dual drug loading of free tetracycline and alendronate intercalated within LDH clay nanoparticles was developed with the target properties suitable for potential application as a GTR membrane. The alendronate-LDH clay complex in the polymer film matrix successfully controlled the release of alendronate from the film and suppressed the burst phase of release. The long term increase in osteoblastic activity observed may be favourable in a periodontal setting as the amount of bone that regenerates in presently used GTR membranes (along with bone grafts) has not been sufficient to bring about total healing (Lynch, 1999).  3.1 Suggestions for future work  The film release studies were carried out in phosphate buffered saline (pH 7.4, 0.1mM). However, in the periodontal cavity, as the guided tissue regeneration membrane is in contact with the gingival crevicular fluid (inflammatory fluid in periodontitis), release studies in gingival fluid simulated medium would provide more relevant in vitro data. Alendronate is known to cause both osteoblast proliferation as well as osteoclast resorption. The experiments conducted in the study were carried out on osteoblasts. Therefore, in future studies, the effect of alendronate release on osteoclasts, in addition to osteoblasts should be determined (Boanini et al., 2008). 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This method involves the derivatization of the amino group of the alendronate molecule with a fluorescent tag molecule- fluorescamine, resulting in a fluorescent fluorophore that is detected by HPLC with fluorescence detection ( ex= 395 nm,  em=  480nm). From the chromatogram, the  characteristic alendronate peak was observed around 3 minutes and the unreacted fluorescamine around 10 minutes (Figure A-1).  Figure A-1: Chromatogram of alendronate in phosphate buffered saline. Method: HPLC with fluorescence detector. Mobile phase: EDTA (1 mM)/ methanol (97:3 v/v) mixture (pH 6.5); flow rate of 1 mL/min (Alltech nucleosil 100 C18 column) 83  From the standard curve, the linearity was observed to exist over a broad range (15.625-2000 μg/mL). The limit of quantitation calculated was 0.5 μg/mL while the limit of detection was 0.2 μg/mL (Figure A-2).  Figure A-2: Standard curve for alendronate sodium standard solutions using HPLC with fluorescence detection. R2= 0.9977.  Tetracycline assay details: Tetracycline may be analysed using variety of methods like UV-vis, fluorescence, HPLC etc. In this project, tetracycline was analysed by HPLC with UV-vis detection. This method has been observed to be suitable for detection of tetracycline in the presence of PBS and has previously 84  been used in our lab for tetracycline detection in release studies (Owen et al., 2010). From the chromatogram, the presence of two significant peaks were observed: peak 1 at 7 minutes and the main tetracycline peak around 9 minutes (Figure A-3). The first peak was also observed in the tetracycline standards. If tetracycline solutions were left at room temperature over several days, the area under peak 1 was observed to increase with time, while the area under the second peak decreased, indicative of tetracycline degradation. Therefore, at the end of the release study, the films were dissolved in order to determine the recovery of intact tetracycline by HPLC.  85  9.230  Peak 2  0.80  Day 1  AU  0.60  1.248 1.550 1.881  0.20  7.037  0.40  0.00 2.00  4.00  6.00  8.00  Peak 1  10.00 Minutes  12.00  14.00  16.00  18.00  20.00  18.00  20.00  9.643  Peak 2  Day 6  0.40  7.058  AU  0.60  5.216  1.994  1.308 1.524  0.20  0.00 2.00  4.00  6.00  8.00  Peak 1  10.00 Minutes  12.00  14.00  16.00  Figure A-3: Chromatogram of tetracycline hydrochloride showing the presence of two peaks on both days 1 and 6 respectively. Peak 2 indicates the standard tetracycline peak. The area under the curve of the first peak (degradative tetracycline) increased from day 1 to day 6. The mobile phase was composed of 12% v/v acetonitrile in 1.3 g/L oxalate solution (pH 2.1), having a flow rate of 1mL/min (reverse phase C18 Novapak column).  86  From the standard curve, the linearity was observed to exist over a broad range (15.625-2000 µg/mL). The limit of quantitation calculated was 0.7 µg/mL while the limit of detection was 0.5 µg/mL (Figure A-4).  80000000 Peak area (Absorbance units)  70000000 60000000 50000000  40000000 30000000 20000000 10000000 0 0  500  1000  1500  2000  2500  Tetracycline concentration (µg/mL)  Figure A-4: Standard curve for tetracycline hydrochloride solution in PBS using HPLC with UV-vis detection. R2= 1.  87  

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