Antimicrobial Bioadhesive Polymer Complexes for the Oral Cavity
Total Page：16
File Type：pdf, Size：1020Kb
ANTIMICROBIAL BIOADHESIVE POLYMER COMPLEXES FOR THE ORAL CAVITY GEMMA KEEGAN A thesis submitted in partial fulfilment of the requirements of the University of Brighton for the degree of Doctor of Philosophy March 2007 The University of Brighton in collaboration with GlaxoSmithKline. -2- ABSTRACT Due to the problems associated with local antimicrobial delivery to the oral cavity, such as poor retention times, the use ofbioadhesive polymers within oral healthcare products may significantly improve therapeutic efficacy. In the current study, bioadhesive antimicrobial-polymer complexes were investigated as a formulation strategy to improve the substantivity of antimicrobial compounds within the oral cavity. Interactions between Carbopol'P' polymers and antimicrobial metals were investigated using a dialysis technique. Carbopol 971P-zinc complexes exhibited ideal properties, with zinc retained by the polymer in deionised water and displaced in the presence of sodium chloride and calcium chloride at a rate determined by the pH of the solution. Carbopol 971P-silver and Carbopol 971P-copper complexes were both retained by the polymer in deionised water but did not demonstrate ideal displacement in the presence of competing ions. Both Carbopol 971P-zinc and Carbopol 971P-silver complexes exhibited bioadhesive properties comparable to the polymer alone when assessed using an in vitro staining technique and texture probe analysis, however Carbopol 971P- copper adhesion was significantly reduced. Interactions between chitosan and fluoride did not prevent fluoride release in deionised water when using the dialysis technique, therefore microparticles were formulated using a water-in-oil solvent evaporation technique and by spray-drying. Spray-dried fluoride microparticles exhibited a smaller size distribution, sustained fluoride release, improved fluoride loading and bioadhesion to oesophageal epithelium when compared to particles prepared from a water-in-oil solvent evaporation technique. Scanning electron microscopy revealed the presence of crystals on the surface of particles prepared by water-in-oil solvent evaporation, which were absent in particles prepared by spray- drying. Higher starting concentrations of chitosan were found to improve the fluoride loading of spray-dried microparticles; however the addition of glutaraldehyde had no significant effect on the parameters measured. Antimicrobial activity was initially assessed usmg a broth microdilution technique against a range of planktonic oral bacteria. Optimised chitosan-fluoride microparticles -3- demonstrated some activity at the highest concentration tested but was not effective against all species tested. Antimicrobial assessment of Carbopol 971P-zinc complexes suggested a lack of zinc bioavailability, possibly due to the lack of displacement Subsequent assessment of metal salts alone established the antimicrobial activity of zinc and silver, with copper demonstrating the lowest efficacy. The use of a bioavailability model developed at GlaxoSmithKline found no improvement in zinc or silver efficacy when formulated with Carbopol 971P in preventing the formation of a biofilm on hydroxyapatite. Despite the lack of significant antimicrobial effects, the potential for bioadhesive polymers in the improvement of antimicrobial retention within the oral cavity has been demonstrated through the characteristics of the complexes formed. -4- TABLE OF CONTENTS ABSTRACT 2 TABLE OF CONTENTS 4 ACKNOWLEGDEMENTS 9 AUTHOR'S DECLARATION 10 CHAPTER 1: 11 GENERAL INTRODUCTION 11 1.1 THE ORAL CAVITY 12 1.1.10VERVIEW 12 1.1.2 THE TEETH 12 1.1.3 THE ORALMUCOSA 13 1.1.4 SALIVA 15 1.1.5 MICROBIALECOLOGYOFTHEORALCAVITY 17 1.1.5.1 Dental plaque 20 1.2 DENTAL DISEASES 21 1.2.1 DENTALCARIES 21 1.2.2 PERIODONTALDISEASE 22 1.2.2.1 Gingivitis 23 1.2.2.2 Periodontitis 24 1.3 ORAL HEALTHCARE 2S 1.3.1 ORAL HEALTHCAREPRODUCTS 25 1.3.2 ANTIMICROBIALSIN ORALHEALTHCARE 27 1.3.3 CONSIDERATIONSANDLIMITAnONS IN ANTIMICROBIALORALHEALTHCARE. 29 1.3.3.1 Human oral retention and clearance 29 1.3.3.2 Emerging microbial resistance 30 1.3.3.3 In vitro vs. in vivo 31 1.3.3.4 Formulation 32 1.4 BIOADHESION 33 1.4.1 THE THEORIESOFADHESION 33 1.4.1.1 The electronic theory 34 1.4.1.2 The adsorption theory 34 1.4.1.3 The diffusion theory 34 1.4.1.4 The wetting theory 35 1.4.1.5 The fracture theory 35 1.4.2 MUCOADHESION 36 1.4.2.1 The contact stage 36 1.4.2.2 The consolidation stage 37 1.4.3 FACTORSIMPORTANTTOMUCOADHESION 39 1.4.4 METHODSTODETERMINEMUCOADHES[ON 40 1.4.4.1In vitro test methods 40 1.4.4.2In vivo test methods 42 1.4.4 MUCOADHESIVEPOLYMERS 43 - 5 - 1.4.4.1 Poly(acrylic acid)-based polymers 44 1.4.4.1a Carbopol 44 1.4.4.1 b Polycarbophil 45 1.4.4.2 Chitosan-based polymers 45 1.4.4.3 Other mucoadhesive polymers 46 1.4.4.3a Cellulose-based polymers 46 1.4.4.3b Natural polysaccharides 46 1.4.4.3c Other synthetic polymers 48 1.4.5 MUCOADHESIVEDRUGANDANTIMICROBIALDELIVERYIN THE ORALCAVITY 48 1.4.5.1 Solid dosage forms 48 1.4.5.2 Semi-solid dosage forms 51 1.4.5.3 Liquid dosage forms 53 1.4.5.4 Particulate systems 54 1.4.6 A NEW DEVELOPMENTINANTIMICROBIALTHERAPYWITHINTHE ORALCAVITY 55 1.5 AIMS AND OBJECTIVES 57 CHAPTER2 58 IN VITRO ASSESSMENT OF BIOADHESIVE POLYMERS 58 2.1. INTRODUCTION 59 2.Ll BACKGROUND 59 2.1.2 DETECTINGPOLYMERONCELL SURFACES:HISTOLOGICALSTAININGAND DYES 60 2.1.3 MEASURINGADHESIVEBONDSTRENGTH:TEXTUREPROBEANALYSIS 61 2.2 MATERIALS ANDMETHODS 65 2.2.1 MATERIALS 65 2.2.2 METHODS 65 2.2.2.1 In vitro direct staining technique 65 2.2.2.1 a Preparation of aqueous polymer dispersions 65 2.2.2.1 b In vitro mucoadhesion of polymer dispersions to human buccal cells in sucrose 66 2.2.2.1 c In vitro mucoadhesion of polymer dispersions to human buccal cells in artificial saliva 66 2.2.2.ld Cell staining and analysis 67 2.2.2.2 Texture probe analysis 68 2.2.2.2a Preparation of polymer samples 68 2.2.2.2b Preparation of porcine oesophageal tissue 68 2.2.2.2c Texture probe analysis 68 2.2.3 STATISTICALANALYSIS 69 2.3 RESULTS AND DISCUSSION 70 2.3.1 IN VITRO STAlNlNGTECHNIQUE 70 2.3.1.1 Microscopy of treated cells 72 2.3.1.2 Stain intensity 75 2.3.2 TEXTURE PROBEANALYSIS 76 2.3.2.1 Poly(acrylic acid) based polymers 78 2.3.2.2 Chitosan polymers 81 2.4 CONCLUSIONS 87 CHAPTER3 88 DEVELOPMENT OF A POLY(ACRYLIC ACID)-METAL ION ORAL DELIVERY SYSTEM 88 -6- 3.1 INTRODUCTION 89 3.1.1 BACKGROUND 89 3.1.2 DIALYSIS: A METHODOF EVALUATINGTHERATEOF ACTIVEAGENTRELEASE. 90 3.1.3 METAL IONDETECTIONINSOLUTION- ATOMIC ABSORPTIONSPECTROSCOPY 92 3.2 MATERIALS ANDMETHODS 94 3.2.1 MATERIALS 94 3.2.2 METHODS 94 3.2.2.1 Preparation and characterisation of metal ion-polymer complexes 94 3.2.2.1 a Preparation of aqueous polymer dispersions and metal ion solutions 94 3.2.2.1b Development of Carbo pol 971P-metal salt solution 95 3.2.2.1c Stability of metal-polymer complex 95 3.2.2.1d Displacement of metal from the polymer complex 96 3.2.2.1e Atomic absorption spectroscopy 97 3.2.2.2 In vitro assessment ofbioadhesion I: Direct staining technique 98 3.2.2.2a In vitro mucoadhesion of polymer dispersions to human buccal cells in sucrose 98 3.2.2.2b In vitro mucoadhesion of polymer dispersions to human buccal cells in artificial saliva 98 3.2.2.2c Cell staining and analysis 99 3.2.2.3 In vitro assessment ofbioadhesion II: Texture probe analysis 99 3.2.2.3a Preparation of texture probe analysis samples 99 3.2.2.3b Porcine oesophageal tissue 99 3.2.2.3c Texture probe analysis 99 3.2.3 STATISTICALANALYSIS 100 3.3 RESULTS AND DISCUSSION 101 3.3.1 DEVELOPMENTOF CARBOPOL971P-METAL SALTSOLUTIONS 101 3.3.2 STABILITYOF CARBOPOL971P-METAL SALTCOMPLEX 103 3.3.3 DISPLACEMENTFROMTHE METAL-POLYMERCOMPLEX 108 3.3.4 IN VITROASSESSMENTOFBIOADHESIONI: DIRECT STAININGTECHNIQUE. 114 3.3.4.1 Bioadhesion in sucrose 114 3.3.4.2 Bioadhesion in artificial saliva 117 3.3.5 IN VITROASSESSMENTOFBIOADHESIONII: TEXTUREPROBEANALYSIS 120 3.4 CONCLUSIONS 124 CHAPTER 4 125 DEVELOPMENT OF A CHITOSAN-FLUORIDE ORAL DELIVERY SYSTEM 125 4.1 INTRODUCTION 126 4.1.1 BACKGROUND 126 4.1.2 DETECTINGFREEFLUORIDEUSINGPOTENTIOMETRY-FLUORIDEIONSELECTIVE ELECTRODES 127 4.1.3 MANUFACTURINGMICROPARTICLESBYSPRAYDRYING 129 4.1.4 PARTICLE SIZINGUSINGLASERDIFFRACTION 131 4.1.5 INVESTIGATINGMICROPARTICLEMORPHOLOGY-SCANNINGELECTRONMICROSCOPY 132 4.2 MATERIALS ANDMETHODS 135 4.2.1 MATERIALS 135 4.2.2 METHODS 135 4.2.2.1 Preparation and characterisation of chitosan-fluoride complexes 135 4.2.2.1a Preparation of polymer dispersions and fluoride solutions 135 4.2.2.1 b Stability of chitosan-fluoride complexes 136 4.2.2.1c Displacement of fluoride from the chitosan complex 137 4.2.2.2 Analysis of fluoride samples using an ion selective electrode 137 4.2.2.2a Preparation of ionic strength (IS) adjustment buffers 137 -7- 4.2.2.2b Preparation of calibration standards 138 4.2.2.2c Calibration of the ion selective electrode 138 4.2.2.2d Analysis of fluoride samples 139 4.2.2.3 Preparation of chitosan-fluoride microparticles 140 4.2.2.3a Preparation of aqueous phase 140 4.2.2.3b Preparation of micro particles using spray drying 140 4.2.2.3c Preparation of micro particles using water in oil (W/O) emulsion solvent evaporation technique 141 4.2.2.4 Characterisation of chitosan-fluoride microparticles 142 4.2.2.4a Particle size 142 4.2.2.4b Fluoride loading 142 4.2.2.4c In vitro fluoride release 143 4.2.2.4d Texture probe analysis 144 4.2.2.4e Scanning electron microscopy 144 4.2.3 STATISTICALANALYSIS 144 4.3 RESULTS AND DISCUSSION 146 4.3.1 DEVELOPMENTANDSTABILITYOFAQUEOUSCHITOSAN-FLUORIDECOMPLEXES 146 4.3.2 DISPLACEMENTOFSODIUMMONOFLUOROPHOSPHATEFROMCHITOSANINISOTONIC