A surface-eroding antibiotic delivery system based on poly-(trimethylene carbonate)Otto S. Kluina, Henny C. van der Meia, Henk J. Busschera, Danie lle Neuta, b,*aDepartment of Biomedical Engineering, University Medical Center Groningen, and University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The NetherlandsbDepartment of Orthopedic Surgery, University Medical Center Groningen, and University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlandsarti cle i nfoArticle history:Received 17 March 2009Accepted 10 May 2009Available online 4 June 2009Keywords:Poly(trimethylene carbonate)In vitro degradationEnzymatic surface erosionAntibiotic releaseabstractBiodegradable delivery systems that do not produce acidic compounds during degradation are preferredfor local antibiotic delivery in bone infections in order to avoid adverse bone reactions. Poly(trimethylenecarbonate) (PTMC) has good biocompatibility, and is such a polymer. The objective of this in vitro studywas to explore the suitability of PTMC as an antibiotic releasing polymer for the local treatment of boneinfections. Degradationbehaviourandcorrespondingreleaseprolesof gentamicinandvancomycinfrom slowly degrading PTMC168 and faster degrading PTMC339 discs were compared in the absence andpresence of a lipase solution. Gentamicin release in the absence of lipase was diffusion-controlled, whilevancomycin release was limited. Surface erosion of PTMC only occurred in the presence of lipase. Bothantibiotics were released in high concentrations from PTMC in the presence of lipase through a combi-nationofsurfaceerosionanddiffusion. Thisillustratesthemajoradvantageofsurface-erodingbiode-gradable polymers, allowing release of larger antibiotic molecules like vancomycin. 2009 Elsevier Ltd. All rights reserved.1. IntroductionBoneandsoft tissueinfections, suchasosteomyelitisor theinfected diabetic foot are difcult to treat [1,2]. Usually blood owispoor under these pathological conditions, and systemically appliedantibiotics are unable to achieve the local concentrations needed toeradicate the infecting bacteria, which are, in addition, protected bytheir biolm mode of growth [3]. In order to treat these infections,local drug delivery systems are needed to achieve effective levels ofantibiotics. Nowadays, research on antibiotic delivering carriers forthe local treatment of infections is concentrated on biodegradablepolymers [47]. Biodegradable carriers not only supply a high localdrug concentration for a xed amount of time, but can also releasetheir entire antibiotic content [4]. Moreover, there is no necessityfor a second surgical intervention to remove the carrier. The mostfrequently investigated biodegradable antibiotic carriers consist ofpoly(lactic acid) (PLA) and/or poly(glycolic acid) (PGA) [4].However, the use of PLA and PGA implants in bone is controversial:bone resorption can develop as a result of a decrease in pH whenthepolymerdegrades[6]. Therefore, thesepolymersmaynotbeideal forthelocaltreatment ofbone infections. For this purpose,carriers that do not produce acidic degradation products are highlypreferable. Thisrequirement canbe fullledby the useofbiode-gradable and biocompatible poly(trimethylene carbonate) (PTMC)[8,9], which degrades in water, CO2 and propanediol.Enzymesplay animportantroleintheinvivodegradationofPTMC, which can be simulated in vitro by the use of a lipase solution[10]. In fact, in the absence of lipase, PTMC hardly degrades in vitro,and this sharply contrast with the high degradation rates observedinvivo [10]. Degradation of PTMC is characterised by surfaceerosion, which occurs faster for PTMC of higher molecular weight[9,10]. Drug release kinetics from surface-eroding polymers showa close relationshiptothe polymer degradationrate [11], butwhetherdrugreleasekineticsfromPTMCareinuencedbyitsmolecular weight needs to be determined.Antibiotics incorporated in local delivery systems ideallydemonstratebroadandsufcient antibacterial efcacy, markedwatersolubilityfacilitatingitsrelease, andchemicalandthermalstability [12]. Aminoglycosides in general and gentamicin inparticular are preferred antibiotics, both from a bacteriological andphysico-chemical point of view. However, the increasing resistanceto gentamicin ofstaphylococciin bone infections [13,14]necessi-tates the use of different antibiotics. Vancomycin is the antibiotic oflast resort used for the treatment of clinically resistant bacteria, likemethicillin-resistantStaphylococcusaureus(MRSA). Nevertheless,the use of vancomycin in non-biodegradable drug carriers like bone*Corresponding author. Department of Biomedical Engineering (Sector F),UniversityMedical CenterGroningen, andUniversityof Groningen, POBox196,9700 AD Groningen, The Netherlands. Fax: 31 50 3633159.E-mail address: d.neut@med.umcg.nl (D. Neut).Contents lists available at ScienceDirectBiomaterialsj ournal homepage: www. el sevi er. com/ l ocat e/ bi omat eri al s0142-9612/$ see front matter 2009 Elsevier Ltd. All rights reserved.doi:10.1016/j.biomaterials.2009.05.012Biomaterials 30 (2009) 47384742cement is limited because it demonstrates poor release [15], due toits high molecular weight, impeding diffusion through the polymermatrix [7]. PTMC, however, is not expected to hamper the release ofhigh molecular weight drugs because of its degradation by surfaceerosion.The aim of this study is to explore the suitability of biodegrad-able PTMC of different molecular weights as an antibiotic deliverysystem, focusingonthedegradationprocessandcorrespondingantibiotic release of gentamicin and vancomycin.2. Materials and methods2.1. MaterialsHigh purity, polymerization grade 1,3-trimethylene carbonate (TMC) waspurchased fromBoehringer Ingelheim(Ingelheim, Germany). The antibioticsgentamicin sulphate (molar mass 723 g/mol) and vancomycin hydrochloride (molarmass 1486 g/mol) were purchased from SigmaAldrich (St. Louis, MO, USA). LipasefromThermomyceslanuginosus(EC3.1.1.3, minimum100.000 units/g, theaqueoussolution contains 2 wt% enzyme concentrate, 0.5 wt% CaCl2 and 25 wt% propyleneglycol) was purchasedfromSigma(St. Louis, MO, USA) andusedas received.Tetrahydrofuran(THF), anhydroushexane, andanalytical gradechloroformwerepurchased from Merck (Darmstadt, Germany), and used as received.2.2. Antibiotic loading of the polymersTwotypesof PTMCwithdifferent molecularweightswereusedtoprepareantibiotic-loaded polymer lms: low molecular weight PTMC (PTMC168,Mw168 103g/mol, Mw/Mn 1.15, intrinsic viscosityat 25 Cinchloroform2.2 dl/g)andhighmolecularweight PTMC(PTMC339,Mw339 103g/mol, Mw/Mn 1.17, intrinsicviscosityat25 Cinchloroform4.3 dl/g). Thepolymersweresynthesized, puried, and characterized as previously described [16]. Briey, PTMCsolutions were prepared by dissolving 2 g of polymer in 75 ml THF. To these solu-tions, 0.20 g of antibiotic (gentamicin sulphate or vancomycin hydrochloride) wasadded, and after 5 min of ultrasonic mixing followed by magnetic stirring for 24 h,the solutions containing the antibiotic dispersion were precipitated into an excess ofanhydrous hexane. The composite was then collected and dried under vacuum atroom temperature.PTMC lms with dispersed antibiotic particles incorporated were prepared bycompression moulding at 70 C, using stainless steel moulds and a laboratory press(Carver Inc., Wabash, IN, USA). Of each polymer, three separate lms (approximatethickness500 mm)containingeithergentamicinsulphateorvancomycinchloridewereprepared. Fromtheselms, discswithadiameterof5 mmwerepunched.Films without antibiotics were prepared as well.The initial antibiotic contents of the discs were determined in triplicate usingauorescencepolarizationimmuno-assay(AxSYM, Abbott Laboratories, AbbottPark, IL, USA) after appropriate dilution. To this end, the discs were dissolved in 3 mlchloroformandextractedvetimes with3 ml water tominimizetheresidualantibiotic in the chloroform to less than 5%. The aqueous fractions were collected,and their gentamicin or vancomycin content measured.2.3. Degradation experimentsTo investigate the degradationbehaviour, gentamicin-loaded PTMC168andPTMC339discs were incubated in 5 ml phosphate buffered saline (PBS) or in a lipase-containingsolutionat37 C. Themass, thicknessanddiameterofthespecimenswere determined before incubation and at predetermined time points during thedegradation process. After one day, the discs were blotted dry on ltration paper,analyzed and re-immersed in the degradation media. This procedure was repeatedafter 2, 3, 7, 10, 14and28days. Degradationexperiments wereperformedintriplicate.Molecular weights were determined after 0, 2, and 7 days of incubation usingnon-loaded PTMC168 and PTMC339 discs. Weight average (Mw) and number average(Mn) molecular weights, molecular weight distributions(Mw/Mn) andintrinsicviscosities of the polymers were determined by gel permeation chromatography, aspreviouslydescribed[9]. Changesinthesurfacestructureof gentamicin-loadedPTMC discs upon incubation in PBS and lipase solutions were visualized by scanningelectron microscopy (SEM). After 0, 2, 7 and 14 days, the polymer specimens wererinsed with demineralised water and dried using ltration paper. The samples weresputter-coatedwitha3 nmthickgold/palladiumlayerforexaminationat2.0 kVusing a JEOL 630 1F eld emission SEM.2.4. Antibiotic releasePTMC168and PTMC339discs loaded with gentamicinor vancomycinwereimmersed in 5 ml PBS or in lipase solutions and incubated for 4 weeks at 37 C. After1, 2, 3, 7, 10, 14 and 28 days of incubation, 150 ml aliquots of the media were taken,and the antibiotic concentrations were indirectly determined by measuring bacte-rial inhibition zones [17]. A gentamicin-sensitive S. aureus A20734 (minimal inhib-itory concentration for gentamicin 0.50 mg/ml) was used to determine theconcentrations of gentamicin released, while a vancomycin-sensitive S. aureus 5298(minimal inhibitory concentration for vancomycin 0.064 mg/ml) was used for van-comycin concentration measurements. Inhibition zone measurements were chosenfor determining theantibioticrelease, inorder toensurethattheantibioticshadretained their activity throughout the entire process of PTMC preparation.Staphylococci wereculturedaerobicallyfromcryopreservativebeads(ProtectTechnical Service Consultants Ltd., United Kingdom) onto blood agar plates at 37 Covernight. A18 hpreculture ofbothbacterialstrainswas madein TryptoneSoyaBroth (TSB). These precultures were diluted to a concentration of approximately 108bacteria per ml in order to inoculate TSB agar plates with a sterile cotton swab. Thethickness of the agar was about 5 mm. Ten minutes after inoculation, 15 ml of PBS orlipasealiquotsweretransferredtothecentreof eachplate, andtheplatewassubsequently incubated aerobically at 37 C. After overnight incubation, clear areasaround the position of the sample droplet indicated the absence of bacterial growth.Thediameters of theseinhibitionzones weremeasuredintwoperpendiculardirections and related with the diameters of inhibition zones around droplets of PBSor lipase solutions with known antibiotic concentrations, in order to calculate theantibiotic concentration in the aliquots.2.5. Statistical analysisData are presented as mean values with standard deviations. The Student t-testforindependent sampleswasused, anda95%(p