Local administration in osteomyelitis by drug encapsulated polymer beads

LOCAL ADMINISTRATION IN OSTEOMYELITIS BY DRUG ENCAPSULATED POLYMER BEADS PAVAN KUMAR NARAHARISETTI NATIONAL UNIVERSITY OF SINGAPORE 2003 LOCAL ADMINISTRATION IN OSTEOMYELITIS BY DRUG ENCAPSULATED POLYMER BEADS PAVAN KUMAR NARAHARISETTI (B.Tech, RECW-KAKATIYA UNIVERSITY, 2001) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF ENGINEERING DEPARTMENT OF CHEMICAL AND ENVIRONMENTAL ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2003 ACKNOWLEDGEMENTS I would like to thank my supervisor Prof Chi-Hwa Wang who with his constant guidance, moral support, comments and suggestions has helped me overcome many difficulties in research and beyond I would like to thank The Department of Chemical and Environmental Engineering, National University of Singapore for providing a generous research scholarship; advanced facilities and study leave for research attachment I thank Prof Si-Shen Feng for permitting me to use certain facilities in his lab I would like to thank Prof Duu-Jung Lee at The Department of Chemical Engineering, National Taiwan University for giving me an opportunity to visit Taiwan for a research attachment and I am indebted to Dr YiChih Fu and Ms Po Ya Chang for support that they have provided me in research and in personal matters during the stay I would like to thank Dr Madhusudana Rao Suryadevara, Dr Rensheng Deng, Dr.Kewu Zhu and all the colleagues and friends for the moral support that they have given me without which I wouldn’t have been able to work comfortably during the period Dr Jianjun Wang, Mr Fangjing Wang and Ms Khin Yin Win have been a constant source of inspiration and are the people whom I used to look upon for guidance apart from my supervisor I enjoyed working with the under-graduate students Mr Bing Fai Kwong, Mr Herman Chian Guan Lee and Ms Magdeleine Duan Ning Lew i Acknowledgements I am grateful to all the technical support staff and administrative staff especially Mdm Li Yoong Khoo, Ms Chai Keng Lee, Mr.Boey Kok Hong, Mdm Susan Chia Yuit Ching, Mdm Xiang Li, Ms Siew Ping Goh for the support in technical and administrative work ii TABLE OF CONTENTS ACKNOWLEDGEMENTS i TABLE OF CONTENTS iii SUMMARY iv NOMENCLATURE ix LIST OF TABLES xi LIST OF FIGURES xii CHAPTER INTRODUCTION 1.1 Motivation and Objectives 1.2 Organization of thesis CHAPTER BACKGROUND AND LITERATURE REVIEW 2.1 Materials for Drug Delivery System 2.1.1 Polymeric materials 2.1.2 Materials in Orthopedic Implants 14 2.1.3 Micro organisms in Polymeric Implants 16 2.2 Types of drug delivery systems 16 2.3 Composition, healing and regeneration of bone 2.3.1 Composition 17 2.3.2 Healing of bone 21 2.3.3 Bone regeneration 22 iii Table of Contents 2.4 Analysis of Gentamicin 23 CHAPTER MATERIALS AN METHODS 3.1 Materials 31 3.2 Preparation of micro particles and discs 31 3.3 Encapsulation efficiency and Analysis of Gentamicin 32 3.4 Morphology, particle size distribution and degradation study 34 3.5 In vitro release 34 3.6 In vivo study on rabbit model 34 CHAPTER RESULTS AND DISCUSSIONS 4.1 Modification of porosity of discs 37 4.2 The usage of Gentamicin as a solid 55 4.3 Rabbit models for in vivo study 60 CHAPTER CONCLUSIONS AND RECOMMENDATIONS 5.1 Conclusions 67 5.2 Recommendations for Future work 68 REFERENCES 72 APPENDIX A Conference papers 82 “30th Annual meeting and exposition of the society of controlled release society, 19-23 July 2003, Glasgow, United Kingdom” iv Table of Contents A.1.1 Double walled microspheres for the encapsulation of a hydrophilic drug Pavan Kumar Naraharisetti, Magdeleine Duan Ning Lew, Chi-Hwa Wang, YinChih Fu A.1.2 In Vitro Release of Gentamicin from Implantable Discs and Modified Discs Pavan Kumar Naraharisetti, Chi-Hwa Wang, Yin-Chih Fu A.3 Double walled microspheres 87 A.4 Thin flims 90 v SUMMARY Osteomyelitis is an infection of the bone and successful treatment involves the removal of the effected bone and the tissue by a surgical procedure and implantation of a composition of Gentamicin that gives sustained release of the drug for a period of about 6-8 weeks Systemic administration of Gentamicin has shown that high dosages are required in order to achieve sufficient concentration at the local area Local administration and sustained release is being currently achieved by Gentamicin-loaded poly methyl methacrylate (PMMA) beads However, PMMA is not biodegradable and a second surgical procedure is required in order to remove the PMMA beads Also, it was observed that the PMMA beads did not release all of the Gentamicin The current study aims to develop a biodegradable/biocompatible composition that gives sustained release and hence reducing the need for a second surgery We have started working on microspheres made by spray drying a mixture of poly lactide-co-glycolide solution and Gentamicin solution and the results have shown that all of the Gentamicin was released out in less than one day Also, there is not much effect of the type of polymer in terms of copolymer ratios of PLA and PGA on the release characteristics We have then worked on the possibility of compressing the microspheres into a disc and studied the release characteristics The release from the discs has given a biphasic behavior with an initial release followed by a lag phase and a second release The initial release corresponds to the release of the drug that was present in the microspheres, which are present near the surface when the disc was compressed vi Summary Depending on the type of polymer the period of the lag phase varied The second release starts when the polymer starts to degrade With this result, we have directed our effort in order to modify the release profile by eliminating the lag phase Hence a hydrophilic and biocompatible polymer poly ethylene glycol (PEG) was introduced before spray drying with an intension that PEG may migrate out of the discs as it is hydrophilic and thus making the discs porous and giving way for the drug to come out easily Methylcellulose was tried for modifying the porosity of the discs Also, pure microspheres made of PLGA were blended with hydroxylapatite and β-tricalcium phosphate and discs were made and the in vitro release was studied In all the cases it was observed that only the initial release was increased with increased addition of another material to PLGA The lag phase was reduced in time, but still the profile followed a biphasic behavior In an effort to get a composition that does not give a biphasic behavior, discs with solid Gentamicin were prepared Gentamicin as solid was spray dried with the polymer and the resultant microparticles were compressed to obtain a disc This procedure does not give uniformity of Gentamicin on the molecular level, but still can give sufficient uniformity in distribution that allows for the results to be reproduced It was observed that 10% drug loading was optimum for Gentamicin to be used as solid in spray drying The results showed that about 60 % of the drug is released in about 5-6 days and the remaining drug is released in about 30 days in total Since 60% initial release might be large, a more hydrophobic PLGA (PLGA 85:15) was introduced anticipating lesser initial release vii Summary However, all samples followed a similar profile and the hydrophobic-hydrophilic nature of the polymer did not play a major role when Gentamicin is used as solid Samples, which gave up to 30 days release, were then used for in vivo studies on rabbit models Male New Zealand rabbits were used in the study and the discs were implanted in the bone after drilling the bone Study involved the blood sampling from the bone and the ear to check the local area concentration and systemic concentration respectively It was observed that the local area concentration was relatively very large when compared to the systemic concentration and also the systemic concentration was negligible after one week Another batch of rabbits were operated and PMMA beads were implanted These beads were removed after a two-week period and them the biodegradable discs were implanted and similar study was performed In the thesis the results and discussions pertaining to the above studies are presented in detail Future work related to the use of discs in bacteria infected rabbits to further establish the efficacy of the composition and the possibility of extending the project to tissue and bone regeneration are presented viii References _ Lenz R W Biodegradable polymers in advances in polymer science Biopolymers (eds NA Peppas and RS Langer) Springer Verlag, Berlin 1-40, 1993 Leikweg W G, Greenfiled L J Vascular prosthetic infections, collected, experience and results of treatment surgery Surgery 81, 335-342, 1977 Lewis D H In Biodegradable Polymers as drug delivery systems Chasin M Langer R, Eds Marcel Dekker, New York 1-42, 1990 Mauduit J, Bukh, N, Vert M, Gentamicin poly lactic acid blends aimed at sustained release local antibiotic therapy administered per operatively I the case of Gentamicin base and Gentamicin sulfate in poly dl lactic acid oligomers, J Controlled Release, 23, 209-220, 1993 Mauduit J, Bukh, N, Vert M, Gentamicin poly lactic acid blends aimed at sustained rlease local antibiotic therapy administered per operatively II The case of Gentamicin sulfate in high molecular weight poly dl lactic acid and poly l lactic acid J Controlled Release, 23, 221-230, 1993 Mauduit J, Bukh, N, Vert M, Gentamicin poly lactic acid blends aimed at sustained rlease local antibiotic therapy administered per operatively.III The case of Gentamicin sulfate 76 References _ in films prepared from high and low molecular weight poly dl lactic acid J Controlled Release, 25, 43-49, 1993 Medline plus health information A service of the US national library of medicine and the National institutes of health Mills S N, Davis S S In ‘Polymers in controlled drug delivery; IllumL, Davis SS, Eds: IOP Publishing, Bristol, UK, pp1-14, 1987 Moreno M A, Frutos P, Ballesteros M P Extraction and liquid chromatographic detection of 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I, perera A, Evora C In vivo-in vitro study of biodegradable and osteointegrable Gentamicin bone implants Eu J Pharma and Biopharma 51, 151158, 2001 Schaal K P, Pulverer G Epidermilogic, etiologic, diagnostic and the therapeutic aspects of endogenous Actinomycetes infections, in Biological, Biochemical and Biomedical aspects of Actinomyecetes (eds L.Ortiz-ortiz, LF Bojalil and Y.Yakoleft)Academic Press New York 13,32,1984 Seth N K, Franson T R, Rose H D, Buckmire F L A, Sohnle P G Colonization of bacteria on poly vinyl chloride and Teflon intravascular catheters in hospitalized patients J.Clin.Microb 18, 1060-1063, 1983 79 References _ Seth N K, Rose H D, Franson T R, Buckmire F L A and Sohnle P G In vitro bacterial adherence to intrvascular catheters J.Sur.Res 34, 213-218, 1983 Soriano I, Evora C, Formulation of calcium phosphates/ poly dl lactide blends containing Gentamicin for bone implantation, J Controlled Release, 68, 121-134, 2000 Sriramkamal J, Dennis HR, A bioresorbable, polylactide reservoir for diffusional and osmotically controlled drug delivery, AAPS PharmSciTech, 1, 4, article 29, 2000 Stead D A, Richards R M Sensitive high performance liquid chromatographic assay of aminoglycosides in biological matrices enables the direct estimation of bacterial drug uptake Journal of chromatography B Biomedical Science applications 2, 415-421, 1997 Sujata V B Biomaterials Alpha Science International Pangbourne England, 2002 Toyo’oka T Modern derivatization methods for separation sciences Wiley 1999 Tuli S M Allogenic decalbone implants for repair of bone defect in Biomechanics(eds KB Sahay, RK Saxxena) Wiley Eastern India, 1989 Wang F M Engg Thesis, Department of Chemical and Environmental Engineering, National University of Singapore, 2001 80 References _ Warren J W Infections associated with urological devices, in Infections associated with prosthetic devices (eds, B Sugarman, EJ Young)CRC Press Boca Raton 23-56, 1984 Wei G, Kotoura Y, Oka M, yamamura T, Wada 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encapsulation of a hydrophilic drug Pavan Kumar Naraharisetti, Magdeleine Duan Ning Lew, Chi-Hwa Wang, Yin-Chih Fu A.1.2 In Vitro Release of Gentamicin from Implantable Discs and Modified Discs Pavan Kumar Naraharisetti, Chi-Hwa Wang, Yin-Chih Fu 82 Double walled microspheres for the encapsulation of a hydrophilic drug Pavan Kumar Naraharisetti1, Magdeleine Duan Ning Lew1, Chi-Hwa Wang1,2*, Yin-Chih Fu3 Department of Chemical and Environmental Engineering, National University of Singapore, Engineering Drive 4, Singapore 117576, Singapore Molecular Engineering of Biological and Chemical Systems, Singapore-MIT Alliance, Singapore 117576 *chewch@nus.edu.sg Department of Orthopaedic Surgery, Kaohsiung Medical University Hospital, 100 Shith-Chuan 1st Road Kaohsiung, Taiwan 807 Abstract Doubled wall microspheres are required especially when a better encapsulation efficiency and controlled release is necessary In this study, we will present the fabrication and characterization of double walled microspheres by exploring the solubility and insolubility of polymers in different solvents Introduction Poly lactide-co-glycolide (PLGA50:50) is soluble in both dichloromethane(DCM) and ethyl acetate (EA) whereas poly lactide (PLLA) is soluble only in DCM This property of PLLA is utilized in order to fabricate doubled walled microspheres When PLLA solution in DCM is poured into PLGA solution in EA, then a PLLA inner core and PLGA outer cover can be prepared The fabrication and characterization will be presented Experimental Method A solvent evaporation method is used for the fabrication of double walled microspheres Briefly, 100mg of PLLA is dissolved in DCM and 12mg of Gentamicin as solid is suspended in it by ultrasonication 200mg of PLGA is dissolved in EA (see Table for composition) Three compositions as described in the table are fabricated PLLA-GS solution is poured into ethyl acetate containing PLGA and ultrasonicated again This mixture is poured into 500ml of 2% poly vinyl alcohol (PVA) and stirred at 400rpm for hours The microspheres formed were filtered washed and dried The composition of core and shell polymer is analyzed using Fourier Transform Infrared Spectroscopy (FTIR)microscope Cross sectioning of the microspheres is done using a freeze microtome and SEM is used to get the micrographs Gentamicin is analyzed using a high-pressure liquid chromatography (HPLC) after derivatization with o-phthalaldehyde reagent Results and Discussion When higher concentration of PLLA (consider as A) is poured into a lower concentration of PLGA (consider as B), then drops of A will be formed and around which B will be distributed During the hardening of micro spheres, it was observed that, at the instant the drop is formed, the drop of B is still not hardened and is soft Then DCM in it tries to migrate radially outside and hardening first occurs at the interface of PLLA (inside) and PLGA (outside) Now, at the interface, PLLA is totally solid and PLGA is in dissolved state in EA Still DCM tries to come out radially in the outward direction and EA tries to go inside Along the path which EA travels, PLLA will solidify from outside to inside Along the path of DCM, PLLA tries to go outside from inside A stage reaches where the core has only EA, for concentration balance with outside EA and DCM diffused mostly into outside EA At this stage, PLLA is entirely solidified inside and PLGA is in dissolved state on the outside When this is poured into water phase both EA and DCM will diffuse out leaving a crater inside Since this explanation might be an ideal situation, we might see PLLA diffusing a bit into outer cover, and PLGA diffusing a bit into inner core and hence overlapping of FTIR results It can be seen in SEM of A (Fig 1) that the surface has a largely porous structure This may be possible due to the rapid escape of DCM/EA from inside to the outside of the microsphere SEM of B (Fig 2) shows a relatively smooth surface, however a few pores can be observed and this also can be explained by the diffusion of solvent from inside to the outside Surface SEM of C also looks similar and hence it is not shown Cross-section SEM of C(Fig 3) shows a hole inside with a visible change in smoothness from inside to the outside The FTIR results of samples B and C (Fig and 5) against a blank PLGA and PLLA have shown the formation of a double wall structure However, certain amount of PLGA can be seen in the core region and certain amount of PLLA can be seen in the shell region This along with the FTIR results it is found that PLLA comes on the inside and PLGA on the outside Conclusions We were able to prepare double wall microspheres and have observed that the encapsulation efficiencies can be achieved However, further studies need to be carried out in order to increase the encapsulation efficiency In vitro analysis should also be compared with single wall microspheres Sample A B %PLGA (w/v) %PLLA (w/v) 10 E.E (%)