A study of alginate matrices as anti microbial wound dressing

A STUDY OF ALGINATE MATRICES AS ANTI-MICROBIAL WOUND DRESSING GOH CHEONG HIAN (B.Sc.(Pharm.)(Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACY NATIONAL UNIVERSITY OF SINGAPORE 2009 ACKNOWLEDGEMENTS I would like to thank my supervisors, Associate Professor Chan Lai Wah and Associate Professor Paul Heng Wan Sia, for their support and faith in me during my higher degree Their encouragement and guidance had allowed me to enjoy research and fulfil my potential I wish to thank the National University of Singapore for providing me the research scholarship and use of the research facilities in the Department of Pharmacy and GEA-NUS Pharmaceutical Processing Research Laboratory I also wish to express my appreciation to the helpful staff members in the Department of Pharmacy for their technical assistance My gratitude to the lecturers who had taught me, my friends and colleagues in the Department of Pharmacy and GEA-NUS PPRL who had provided me with valuable advice and support when needed Special thanks to Associate Professor Phan and Anan for their assistance in carrying out the cytotoxicity studies Personal thanks to Chye Khoon who helped me from time to time The continual support from my family and loved one had been very important in keeping my spirits up in the face of challenges and difficulties encountered during the course of work Last but not least, I would like to thank them for their understanding and patience in being there for me throughout my research years Goh Cheong Hian 2009 i TABLE OF CONTENTS ACKNOWLEDGEMENTS i TABLE OF CONTENTS ii SUMMARY vi LIST OF TABLES viii LIST OF FIGURES x I INTRODUCTION A Skin Physiology A1 Structure of the skin A2 Wound and wound healing A3 Factors affecting wound healing A3.1 Nutritional status, mineral and trace elements A3.2 Wound infection A3.2.1 A3.2.2 B Chronic wound infections A3.2.3 A3.3 Skin pathogens Burn infections Wound environment 10 Wound Management 11 B1 Wound dressings 11 B1.1 B1.2 B2 Properties of an ideal wound dressing 12 Types of wound dressings 13 Review on alginates 19 B2.1 B2.2 Selective binding of alginates and applications 21 B2.3 Advantages of alginates in wound management 23 B2.4 B3 Structure of alginates 20 Immunological and cytotoxic effects of alginates 25 Management of wound infections 26 B3.1 Topical antiseptics 26 ii B3.2 B4 Topical antibiotics 29 Research and development in wound management products 34 B4.1 Product development 34 B4.2 Critical parameters in the development of alginate dressing 38 II HYPOTHESIS AND OBJECTIVES 43 III EXPERIMENTAL 45 A Materials 45 A1 A2 Anti-microbials 45 A3 Test bacteria and culture medium 45 A4 Chemicals used in cross-linking 45 A5 Reagents used in digestion 48 A6 Reagents used in media preparation 48 A7 Skin cells and tissue culture materials 48 A8 B Sodium alginate 45 Additives 49 Methods 50 B1 Preparation of test inoculum 50 B2 Minimum Inhibitory Concentration (MIC) determination 50 B3 Interaction between cross-linking cation and anti-microbial agent 51 B4 Preparation of alginate pellets 54 B5 Physical characterization of alginate pellets 55 B6 Determination of the anti-microbial activities of blank and SDSloaded alginate pellets 55 B7 Preparation of sodium alginate films 56 B8 Preparation of cross-linked alginate films 56 B9 Determination of film thickness 56 B10 Weight of the alginate film before and after cross-linking 57 iii B11 Determination of fluid uptake and degradation of alginate films 57 B12 Determination of mechanical properties of alginate films 58 B13 Determination of specific cation content and release profiles of alginate films 59 B14 Preparation of cell culture for cytotoxicity assay 60 B15 Preparation of test solutions of cations and additives for in vitro cytotoxicity assay 60 B16 Determination of in vitro cytotoxicity of cations and additives 61 B17 Preparation of alginate fibres 62 B18 Examination of fibre morphology 62 B19 Determination of fibre strength 63 B20 Contents of Na+ and Cu2+ in alginate fibres and the release profiles of Cu2+ in acidic condition 64 B21 B22 Determination of in vitro cytotoxicity of alginate fibres 65 B23 IV Determination of fluid uptake and degradation of alginate fibres 64 Statistical analysis 65 RESULTS AND DISCUSSION 66 Part Anti-microbial activities of cross-linking cations and anti-microbial agents when used alone, in combination and when incorporated into alginate matrices 66 A Compatibility between cross-linking cations and anti-microbial agents 66 B Pellet morphology 71 C Feasibility of incorporating anti-microbial agents in alginate dressing 74 Part Influence of cations on the functionality of alginates as wound dressings 88 A Effect of cations on extent of cross-linking and film characteristics 88 B Effect of cations on mechanical properties of films 97 iv C Effect of cations on fluid absorbency and degradation profiles of alginate films 102 D Release of cations from alginate films 113 Part Cytotoxic effects of cross-linking cations, additives and alginate on skin cells 118 Part Effects of additives and cross-linking cations on the properties of alginate fibres and viability of skin fibroblasts 128 A Effect of Cu2+ concentration on the fabrication of alginate fibres 128 B Effect of additives on alginate fibre morphology 129 C Effects of additives and Cu2+ concentration on extent of crosslinking with alginates 134 D Effect of additives on fibre strength 136 E Release of cations from alginate fibres in acidic condition 138 F Effect of additives on fluid absorbency and degradation profiles of alginate fibres 141 G In vitro cytotoxicity of soluble extracts from alginate fibres 145 V CONCLUSIONS 149 VI FUTURE DIRECTIONS 152 VII REFERENCES 153 VIII PUBLICATIONS/ PRESENTATIONS ARISING FROM THIS STUDY 184 v SUMMARY This study investigated the effects of cross-linking cations and additives in the development of alginate matrices as anti-microbial wound dressings Al3+, Ca2+, Cu2+ and Zn2+ were used as cross-linking cations and PEG 1500, PEG 6000, PVP C15 and PVP S630 as additives with cross-linked alginate pellets, films and fibres as test models The cross-linking cations exerted a strong influence on the morphology and anti-microbial properties of the cross-linked pellets produced Generally, pellets cross-linked by Ca2+ or Cu2+ were more spherical The cross-linking cations were generally compatible with the common topical anti-microbial agents However, antagonism was observed for the combination of Ca2+ and gentamicin against Staphylococcus aureus The cross-linked alginate pellets exhibited different activities against the skin pathogens, Staphylococcus aureus and Pseudomonas aeruginosa, with higher anti-microbial activities against the Gram-negative bacterium and increased activities with higher blank pellet loads Alginate pellets that were crosslinked with Cu2+ exhibited good anti-microbial activities against both test pathogens The incorporation of Zn2+ and sulphadiazine sodium into alginate pellets resulted in synergistic activities against Pseudomonas aeruginosa The cross-linking cations and presence of added Na+ during cross-linking affected the functional properties of the alginate films produced Greater extent of cross-linking was observed for films cross-linked in the absence of added Na+ The added Na+ did not improve the tensile strength of the films cross-linked by Cu2+ or Zn2+ Films cross-linked by combinations of cations exhibited greater extent of crosslinking but little improvement in tensile strength The cross-linked films exhibited similar trend in fluid uptake and corresponding degradation in all the test media, and were generally less affected by pH Alginate films cross-linked with added Na+ exhibited significant increase in fluid uptake capacity but lower extent of degradation than those cross-linked in the absence of Na+ The added Na+ also influenced the vi release of cations from alginate matrices to different extents in pH 4.0 and 8.0 The percentage of cations released from the films was lowest for Cu2+ but relatively high for Ca2+ and Zn2+ The cross-linking cations and additives exhibited different toxicities to skin fibroblasts, with greater toxicity experienced at a higher concentration or over a longer exposure time The alginate, Keltone® LVCR, did not cause any significant toxicity The additives modified the functional properties and cytotoxicity of the copper alginate fibres developed Polyvinylpyrrolidone (PVP) S630 was found to have the least harmful effect among the additives studied Polyethylene glycol (PEG) and PVP C15 exhibited insignificant or negative effects on the extent of cross-linking in the fibres In contrast, the effects of PVP S630 on the extent of cross-linking varied with the concentrations used The strength of fibres generally decreased when additives were incorporated The different concentrations and types of additives affected the release of Cu2+, fluid uptake and degradation of the fibres as well as their cytotoxicity to skin fibroblasts in different ways and to different extents Therefore, by careful choice of the type and proportion of additives, copper alginate fibres with suitable characteristics for use in wound dressings can be achieved vii LIST OF TABLES Table Types of commercial primary dressings (Adapted with modifications from Degreef and Flour, 1994; Erwin-Toth and Hocevar, 1995) 14-16 Table Properties of common topical anti-microbial agents and their associated products used for wound management 27-28 Table Research and development of wound management products 35-36 Table Factors affecting the fabrication of alginate fibres 41 Table Types of alginate pellets prepared 54 Table Types of alginate films prepared 57 Table Types of additive solutions prepared for cytotoxicity assay 60 Table Types of alginate fibres prepared 63 Table Types of soluble extract medium tested 65 Table 10 Anti-microbial activities of cross-linking cations and antimicrobial agents, from highest to lowest 67 Table 11 FICmean and categorical interpretation of cross-linking cationanti-microbial combinations tested against P aeruginosa and S aureus 68 Table 12 Physical properties of blank alginate pellets 73 Table 13 Time-kill equations for low loads (~0.5 g) of alginate pellets against S aureus 76 Table 14 Comparison of anti-microbial activities of varying loads of blank and SDS-loaded alginate pellets 78 Table 15 Time-kill equations for medium loads (~1.0 g) of alginate pellets against S aureus 80 Table 16 Time-kill equations for high loads (~2.0 g) of alginate pellets against S aureus 82 Table 17 Influence of pellet load on anti-microbial activities 83 Table 18 Time-kill equations for low loads (~0.5 g) of alginate pellets against P aeruginosa 86 Table 19 Test for synergism in anti-microbial activities between Zn2+ and SDS in alginate pellets against P aeruginosa 86 Table 20 Proportion of cross-linking cations to Na+ in the various 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Properties of common topical anti- microbial agents and their associated products used for wound management Class Anti- microbial agent Products (Manufacturer) Mechanism of action Quaternary ammonium... (functional capability) and nature of dressing (safety and quality) The design of a wound dressing can affect the overall outcome of wound management For instance, the ease of application improves patient