Acta of Bioengineering and Biomechanics Original paperVol. 17, No. 3, 2015 DOI: 10.5277/ABB-00188-2014-02

Gentamicin loaded PLGA nanoparticlesas local drug delivery system for the osteomyelitis treatment

URSZULA POSADOWSKA1, MONIKA BRZYCHCZY-WŁOCH2, ELŻBIETA PAMUŁA1*

1 AGH University of Science and Technology, Faculty of Materials Science and Ceramics,Department of Biomaterials, Kraków, Poland.

2 Jagiellonian University, Medical College, Department of Microbiology, Kraków, Poland.

Since there are more and more cases of multiresistance among microorganisms, rational use of antibiotics (especially their systemicvs. local application) is of great importance. Here we propose polymeric nanoparticles as locally applied gentamicin delivery systemuseful in osteomyelitis therapy. Gentamicin sulphate (GS) was encapsulated in the poly(lactide-co-glycolide) (PLGA 85:15) nanoparti-cles by double emulsification (water/oil/water, W1/O/W2). The nanoparticles were characterized by dynamic light scattering, laser elec-trophoresis and atomic force microscopy. UV-vis spectroscopy (O-phthaldialdehyde assay, OPA) and Kirby-Bauer tests were used toevaluate drug release and antimicrobial activity, respectively. Physicochemical characterization showed that size, shape and drug solubi-lization of the nanoparticles mainly depended on GS content and concentration of surface stabilizer (polyvinyl alcohol, PVA). Laserelectrophoresis demonstrated negative value of zeta potential of the nanoparticles attributed to PLGA carboxyl end group presence. Drugrelease studies showed initial burst release followed by prolonged 35-day sustained gentamicin delivery. Agar-diffusion tests performedwith pathogens causing osteomyelitis (Staphylococcus aureus and Staphylococcus epidermidis, both reference strains and clinical iso-lates) showed antibacterial activity of GS loaded nanoparticles (GS-NPs). It can be concluded that GS-NPs are a promising form ofbiomaterials useful in osteomyelitis therapy.

Key words: poly(lactide-co-glycolide), nanoparticles, gentamicin sulphate, ostemyelitis, Staphylococcus aureus, Staphylococcusepidermidis

List of abbreviations

AFM – atomic force microscopyDLS – dynamic light scatteringGS – gentamicin sulphateGS-NPs – gentamicin sulphate loaded nanoparticlesMIC – minimal inhibitory concentrationOPA – O-phthaldialdehydePBS – phosphate buffered salinePLGA – poly(lactide-co-glycolide)PVA – polyvinyl alcoholUHQ-water – ultra high quality waterW1/O/W2 – water-oil-water emulsificationZr(acac)4 – zirconium acetylacetonate%EE – encapsulation efficiency%LE – loading efficiency

1. Introduction

Surgical actions are accompanied by the risk ofmicrobial infections, independently of the tissueaffected. In the case of bone, such infections oftenresult in serious skeletal tissue damage and fur-thermore in the spreading of the microbes to theadjacent tissues and organs [16]. As a result of mi-crobes abundance a chronic inflammation arisesthat utmost concerns tibia, femur, clavicle [24]. Thedeveloping onset of symptoms is called osteo-myelitis [24]. The consequence of osteomyelitisis chronic inflammation, abscesses formation, sinus

______________________________

* Corresponding author: Elżbieta Pamuła, AGH University of Science and Technology, Faculty of Materials Science and Ceramics,Department of Biomaterials, al. A. Mickiewicza 30, 30-059 Kraków, Poland. Tel. +48 12 617 44 48, fax ++48 12 617 33 71, e-mail:epamula@agh.edu.pl

Received: September 19th, 2014Accepted for publication: November 18th, 2014

U. POSADOWSKA et al.42

appearance, severe pain, and finally tissue necrosis[24].

One of the possible strategies to treat osteomyelitisis aminoglycoside antibiotic administration. Gentami-cin sulphate (GS) is here utmost used agent that dealswith severe infections caused by Gram-positive (e.g.,Staphylococcus spp.) and also by Gram-negative bacte-ria. GS, acts by irreversible hindering bacteria 30S ribo-somal subunit and thus causes misreading of bacterial t-RNA molecule. Contact with antibiotic makes the mi-croorganisms incapable to synthesize proteins essentialto their growth and thus prevents showing up or spread-ing of the infection [25]. Recently, there are two mainprocedures applied in order to struggle with osteomye-litis. The first approach implies intravenous injections byan indwelling catheter, applied three times a day [15],whilst the second is based on locally placed bone cementor collagen sponges saturated with GS [22]. Since suc-cess of the antyosteomyelitic therapy firmly depends onthe proper doses of antibiotic availability, administeredclosely at the infected site that should last at least up to6 weeks, a very promising solution seems to be encap-sulation of antimicrobial drug into slow degradingbiomaterial which is thought to, after being placed di-rectly inside the bone, delay gentamicin release to ap-propriate rate, dose and time [4]. Poly(lactide-co-glycolide) (PLGA) was chosen to encapsulate the drug,because this FDA-approved resorbable copolymer hasa tunable degradation rate (i.e., by molar ratio of lactideto glycolide, chain structure, molecular weight) [15].PLGA has already been used for the production ofsutures, osteosynthesis devices, scaffolds for bone tissueregeneration and drug delivery systems [15], [19].

Thus, the main objective of our study was to pre-pare and optimize in terms of size and solubilizationa new drug delivery system containing gentamicin.The system proposed was based on PLGA 85:15 andwas produced by the double emulsification technique.After preparation, the detailed physicochemical char-acterization of the nanoparticles was performed, andthe most highly loaded drug delivery systems weevaluated in context of GS release kinetics and theirantimicrobial properties.

2. Materials and methods

2.1. Materials

Gentamicin sulphate (Gentamicini sulfas, C21H43N5O7·H2SO4) was obtained from Galpharm, Poland while

polyvinyl alcohol (PVA, Mowiol 4-88, Mn = 31,000 Da),phosphate buffered saline (PBS) were obtained fromSigma-Aldrich, Poland; dichloromethane, methanol,mercaptoethanol, isopropanol, orthophthaldialdehyde(OPA) were all of analytical grade and were pur-chased from POCh, Poland. Ultra high quality water(UHQ-water) was produced in UHQ-PS purificationsystem (Elga, UK). Poly(lactide-co-glycolide) (PLGA,85:15, Mn = 80 kDa, d = 1.9) was synthesized in theCentre of Polymer and Carbon Materials of the PolishAcademy of Sciences in Zabrze, Poland by a ring-opening polymerization in bulk at 100 °C with zirco-nium acetylacetonate (Zr(acac)4) initiator [20], [21].

2.2. Preparationof the nanoparticles

Nanoparticles loaded with gentamicin sulphate(GS-NPs) were prepared by means of a water-oil-water (W1/O/W2) emulsification solvent evaporationmethod. Firstly, an oily phase (O) was prepared bydissolving 100 mg of PLGA in 6 ml of dichlo-romethane (1.67% w/v). Secondly, internal W1/Oemulsion was prepared by dispersing GS solution(2, 10 or 20 mg in 100 µl of UHQ-water and 45 µl of4% w/v PVA) within an oily phase during ultrasoni-cation (3 min, Sonics VibraCellTM, USA, 40% of thecycle) on ice. In order to form double emulsionW1/O/W2, obtained earlier W1/O emulsion was addeddrop by drop to 20 ml of 1%–4% (w/v) PVA solution(external water phase, W2) in conditions as men-tioned above. Formed double emulsion W1/O/W2was left overnight on mechanical agitator (1000 rpm)in order to allow dichloromethane evaporation. For-matted colloidal dispersion of loaded carriers wasfurther centrifuged (14,000 rpm, 4 °C, 20 min, MPW351R, Medical Instruments, Poland) and three timesflushed with UHQ-water. Afterwards, a portion ofnanoparticles was freeze-dried and then stored at4 °C. Before further assessments such as size, surfacecharge, morphology the particles were redispersedin UHQ-water using ultrasonic bath (POLSONIC®SONIC-3, 10 min).

2.3. Physicochemical analysisof nanoparticles

– size and zeta potential

The size as well as polydispersity index of obtainednanoparticles were determined with the use of dy-

Gentamicin loaded PLGA nanoparticles as local drug delivery system for the osteomyelitis treatment 43

namic light scattering method (DLS) on ZetasizerNano ZS (Malvern Instruments) equipped with He-Nelaser 632.8 nm. To perform the measurements thescattering angle 173° was set and correlator ALV 500was engaged. Size values were determined accordingto Stokes-Einstein equation. Zeta potential was meas-ured by laser Doppler electrophoresis method on thesame apparatus and calculated using the Smoluchow-ski equation out of electrophoretic mobility data per-formed in triplicate for every sample.

2.4. Morphologyof the nanoparticles

Morphological evaluation of nanoparticles wasperformed on atomic force microscope (AFM). Nano-particles suspension was placed on microscopic glassand water was allowed to evaporate for 12 h at roomtemperature. Topographic images were recorded in airusing Si3N4 tips with a nominal radius of curvature of30 nm (NanoProbeTM tips) in contact mode on AFM(Explorer, Thermomicroscopes, Veeco). A feedbackmechanism was employed to adjust the tip-to-sampledistance to maintain a constant force of 0.1 nN be-tween the tip and the sample with proportional, inte-gral and derivative parameters of 1, 0.3 and 0, respec-tively. All the images were flattened and treated usingthe software SPMLab6.02.

2.5. Solubilizationof the antibiotic

In order to assess an amount of GS that actuallyremained enclosed within the nanoparticle structure,supernatant out of particles centrifugation was col-lected. A GS content was examined spectropho-tometrically with the use of an OPA test [1] in thespectrophotometer (UV-VIS Cecil CE 2502, CorstonUK) at 332 nm. A standard calibration curve was plot-ted in appropriate PVA concentration within range 5–400 µg/ml of the drug. Parameters referring encapsula-tion efficiency (%EE) and loading efficiency (%LE)were calculated according to the formulas

%,100

%

⋅=systemtheinGSofmassinitial

lesnanoparticinGSofmassEfficiencyionEncapsulat

(1)

%.100

%

⋅=lesnanoparticofmass

lesnanoparticinGSofmassEfficiencyLoading

(2)

All experiments were performed in triplicate andthe results were expressed as mean ± standard error ofthe mean (S.E.M.).

2.6. Release study

The release profile of GS from GS-NPs suspendedin PBS buffer was analyzed. End-sealed dialysis bags(ZelluTransRoth, MWCO 12 kDa) were filled with1 ml of the suspension (10 mg of the GS-NPs pre-pared in the following conditions: 4% w/v PVA and20 mg of GS per 100 mg PLGA which in total con-tained 1 mg of drug) and immediately immersed in thevials with 20 ml of PBS at pH 7.2. Then the vials wereplaced on mechanical stirrer (50 rpm) at 37 °C. Atpredetermined time intervals 2 ml of PBS that con-tained released antibiotic were collected and analyzedin terms of the drug content (the aliquots that werewithdrawn from the system were replaced with 2 mlof fresh PBS). To determine antibiotic amount anOPA assay according to the method described by An-halt et al. [1] was used. The results were expressed asamount of released antibiotic with respect to timepassage.

2.7. Antimicrobial activity

Kirby-Bauer method (Agar diffusion test) was ap-plied in order to determine antimicrobial activity ofGS-NPs. 30 mg of the GS-NPs (produced in the follow-ing conditions: 4% w/v PVA, 20 mg of GS/100 mgPLGA) were mixed with 0.5 ml of PBS buffer pH 7.4and shaken overnight on vortex at room temperature.After centrifugation the aliquots were gathered andzone inhibition test against two main bone-relatedGram-positive pathogens Staphylococcus aureus andStaphylococcus epidermidis was performed. The ref-erence strains used in the study were S. aureus DSM24167 (Deutsche Sammlung von Mikroorganismenund Zellkulturen) and S. epidermidis ATCC 700296(American Type Culture Collection). The clinicalisolates of S. aureus (SA1-KCR) and S. epidermidis(SE1-KCR) were isolated from patients with jointinfection hospitalized at the Kraków Centre of Rehabili-tation and Orthopedics in 2012. The used strains wereincubated in 5 mL of Bacto™ Tryptic Soy Broth (Bec-ton Dickinson) for 16 h at 37 °C and prepared in con-centration 0.5 in McFarland scale (1.5 × 108 CFU/ml)in physiological saline solution. Then they were seededon Mueller-Hinton agar (Difco) plates in which 3 mm

U. POSADOWSKA et al.44

wells were cut and in these wells 50 μl of aliquotswere placed. As a reference 10 µg gentamicin stan-dards (Oxoid, UK) were used. The plates were thenincubated at 37 °C for 18 h and the zone of microbesgrowth inhibition was measured using CalibratingViewer [mm]. The experiment was performed in trip-licate.

3. Results

3.1. Physicochemical propertiesof nanoparticles

The results of size, zeta potential, polydispersityindex and solubilization parameters of GS-NPs areshown in Table 1. PLGA GS-NPs were formulatedwith an average size of 200–400 nm. Nanoparticleswere also of a low polydispersity, ca.0.3, which corre-sponded to a uniform size distribution. The zeta po-tential was in the range –7.2 mV to –1.1 mV. Thesolubilization efficiency was in the range 2.7–52.2%,and was the highest for the highest GS amount (drugto polymer ratio 20:100) as well as the surfactant con-centration (4%). Under these conditions loading effi-ciency was also highest (up to 10.3%).

3.2. Morphology of the nanoparticles

AFM images demonstrated that the nanoparticleswere spherical and uniform in shape (Fig. 1). The sizeof the particles decreased with increase in surfactantconcentration (Fig. 1a vs. b). The nanoparticles loaded

with the highest GS amount (drug to polymer ratio20:100, PVA 4%) were the biggest (Fig. 1c).

3.3. Drug release studyof the nanoparticles

GS release profile from nanoparticles suspended inPBS buffer and images of such nanoparticles in thetest-tubes are shown in Figs. 2a and 2b, respectively.

Table 1. Size, zeta potential, polydispersity indices and solubilization parameters of GS-NPsfor different preparation conditions

Composition DH Pdl ζ Solubilization parameters

GS concentration[mg/100 mgof PLGA]

PVAconcentration

[%][nm] [–] [mV] % EE % LE

2 mg 1% 307 ± 8 0.38 ± 0.04 –7.2 ± 1.0 2.7 ± 1.8 0.06 ± 0.052 mg 2% 219 ± 11 0.27 ± 0.03 –1.1 ± 0.1 11.2 ± 1.5 0.34 ± 0.022 mg 3% 214 ± 4 0.24 ± 0.01 –5.1 ± 0.5 21.3 ± 1.8 0.31 ± 0.012 mg 4% 222 ± 37 0.30 ± 0.02 –3.6 ± 0.2 15.6 ± 0.9 0.37 ± 0.01

10 mg 4% 234 ± 11 0.21 ± 0.01 –5.2 ± 0.5 45.7 ± 1.0 7.25 ± 0.0620 mg 4% 391 ± 23 0.346 ± 0.002 –3.5 ± 0.3 52.4 ± 0.5 10.28 ± 0.04

DH – hydrodynamic diameter, Pdl – polydisperisty index, ζ – Zeta potential, %EE – encapsulationefficiency, %LE – loading efficiency.

Fig. 1. AFM images of GS-NPs: drug to polymer ratio 2:100 mgand PVA 1% (a), drug to polymer ratio 2:100 mg

and PVA 4% (b); drug to polymer ratio 20:100 and PVA 4% (c);contact mode, 2 µm × 2 µm

a) 2:100 mg

b) 2:100 mg

c) 2:100 mg

Gentamicin loaded PLGA nanoparticles as local drug delivery system for the osteomyelitis treatment 45

The sample was liquid, turbid and milk-white. Theburst release of the drug was observed. It was foundthat 25% of the loaded drug was released in the first12 h. Next, a sustained drug release phase was ob-served that continued up to day 35, when the entireamount of encapsulated drug (1 mg) had been re-leased.

3.4. Antimicrobial activity

In Fig. 3, the plates with the tested strains ofstaphylococci grown for 18 h with GS-NPs aliquots

Fig. 3. Antimicrobial activity of GS-NPs aliquots.Inhibition zone of: the reference strains of S. aureus DSM 24167 (a),

the clinical isolate of S. aureus SA1-KCR (b), the reference strainsof S. epidermidis ATCC 700296 (c) and the clinical isolate

of S. epidermidis SE1-KCR (d)

are shown. Around wells that contained GS-NPs ali-quots the zone of growth inhibition was observed,with diameters in the range 32–39 mm depending onthe pathogen type; precise values of the diameters ofthe growth inhibition zones are shown in Fig. 4. For10 µg standards of gentamicin the zones of growthinhibition were 27 mm (for S. aureus DSM 24167 andS. epidermidis SE1-KCR) and 28 mm (for S. epider-midis ATCC 700296 and S. aureus SA1-KCR) (datanot presented).

4. Discussion

4.1. Characteristics ofgentamicin-loaded nanoparticles

In this study, GS-NPs with diameter of 200–400 nmwere produced. It was achieved by utilization ofPLGA solution of low concentration (1.67%) in W1/Ophase. Such a concentration resulted in low viscosityof the W1/O, which enabled it to be dispersed betterduring the second emulsification stage (W2) as shownpreviously [18]. Surfactant amount, acting in the ex-periment as an interphase stabilizer, also had a signifi-cant impact on the size of developing particles. Whenhigher concentrations of PVA (2%, 3% or 4% w/v)were used, the size of the particles decreased. It alsoresulted in a decrease in polydispersity index as previ-ously reported by others [10]. The AFM analysis con-firmed the sphericity and narrow size distribution ofthe nanoparticles. Determination of size by DLSmeasurements and AFM visualization yielded similarresults, although the light scattering technique pro-vides the values derived from hydrodynamic meas-

Fig. 2. Drug release study from gentamicin loaded nanoparticles (GS-NPs) (a)and morphology of the sample in the test tube (b)

a) b)

a b

c d

U. POSADOWSKA et al.46

urements, while AFM provides data for the driedsamples. It can be anticipated that the obtained nano-particles with such a size will remain within the extra-cellular space of bone tissue. It was reported so farthat too small carriers (500 nm) undergophagocytic uptake [23].

The nanoparticles demonstrated negative zeta po-tential values (–7.2 mV to –1.1 mV), which can beattributed to the presence of the ionized carboxylgroup of PLGA on the particles’ surface. Interest-ingly, pure PLGA showed a much lower zeta potential(–30 mV to –50 mV) [3], which suggests that PVAmolecules adsorb on the surface of the nanoparticles.A similar size and zeta potential values were also re-ported by Grabowski et al. who studied PLGA nano-particles obtained with the use of PVA and attributedsuch values of zeta potential to the remnants of sur-factant [12].

Regarding solubilization, it was observed that bothencapsulation and loading efficiencies significantlyincreased with the surfactant concentration. This canbe explained by the fact that smaller droplets devel-oped when the PVA concentration was higher andsmaller particles exposed a higher relative surfacearea. As a consequence such particles solidified fasterbecause the solvent evaporated more rapidly. Hencethe escape of the phase containing the drug (W1) tothe external water phase W2 was prevented. Similarenhancement of solubilization with increased PVAconcentration has already been described in otherstudies [6].

Additionally, both solubilization parameters wereincreased by higher drug concentration in the W1phase. It can be concluded that changes in surfactantconcentration and the drug amount are the factors thatfinely tune the solubilization parameters as well asparticle size during formation of GS-NPs.

4.2. Drug release kinetics

Systemic drug infusions of GS (applied as generalway of treating osteomyelitis) are known to provokenephro- and ototoxicity [11]. On the other hand localtreatment of osteomyelitis with the use of GS loadedcements or collagen sponges requires traumatic surgi-cal procedure [7]. Thus recently great attention is paidon local tissue-directed ways of antibiotics admini-stration via injections. Effective intra bone osteomye-litis treatment requires maintenance a therapeutic an-tibiotic concentration for at least 3–4 weeks [20]. Inour drug release studies from GS-NPs we observed

immediate drug release phase accounting for ca. 25%of the whole GS dose embedded in the nanoparti-cles followed by prolonged drug release phase up to35 days. Such shape of release curve is typical ofdiffusion – erosion controlled mechanism [17]. Ac-cording to Jain et al. [13], immediate release phasecan be ascribed to release of surface-associatedamount of antibiotic driven by diffusion. Next stage ofdrug release originated from hydrolytic bulk degrada-tion of the polymer which caused diffusion of oli-gomers out of the nanoparticle that created micro-cavities in the particles and facilitated drug migration[13]. Initial burst of drug dose followed by its sus-tained release is in compliance with recommendationsof gentamicin use [11].

4.3. Antimicrobial activityagainst staphylococci

The antimicrobial effect of the GS-NPs was evalu-ated by observation of growth inhibition zones of S.aureus and S. epidermidis strains in contact with ali-quots containing GS-NPs. Such pathogens were cho-sen because they are mainly responsible for intra boneinfection development leading to osteomyelitis: 34% and32% of reported cases (totally 2/3 of the whole cases) arecaused by S. aureus or by S. epidermidis, respec-tively [5]. In our studies (see Fig. 3) we observed thatby application of GS-NPs suspension we succeededwith stopping growth of staphylococci species. Thediameters of the inhibitions zones after 18 h of incuba-tion were between 32–39 mm and 39–37 mm (seeFig. 4) for S. aureus and S. epidermidis, respectively. Ingeneral for the S. epidermidis type higher diametersrange were obtained than for S. aureus. The observations

Fig. 4. Diameters of growth inhibition zonesof staphylococci strains in contact with GS-NPs;

the reference strain of S. aureus DSM 24167, the clinical isolateof S. aureus SA1-KCR, the reference strain of S. epidermidis

ATCC 700296 and the clinical isolate of S. epidermidis SE1-KCR

Zone

dia

met

er [m

m]

Gentamicin loaded PLGA nanoparticles as local drug delivery system for the osteomyelitis treatment 47

correspond to minimal inhibitory concentrationvalues (MIC) of GS for both species: for S. epider-midis MIC (0.064 μg/ml) is lower than for S. aureus(0.500 μg/ml) [14].

Resistance to antibiotics is nowadays one of themayor concerns in the antimicrobial therapy. Oftenthe reference strains of microorganisms are more sus-ceptible to antibiotics whereas for clinical isolates theresponse is reduced [5]. Clinical staphylococci speciescan be methicillin/oxacillin, aminoglycosides, macro-lides, lincosamides, tetracyclines, trimethoprim orsulfonamides resistant [2]. In conducted studies bothtypes of microorganisms, e.g. reference strains andclinical isolates were analyzed. For all tested condi-tions significant inhibition zones were noted whichoverall proved broad antibacterial activity of GS-NPs.According to the European Committee on AntimicrobialSusceptibility Testing from 01.01.2014 (EUCAST) zonediameter breakpoint for gentamicin [10 µg] forS. aureus is 18 mm, and for coagulase-negativestaphylococci eg. S. epidermidis is 22 mm [9]. Basedon these recommendations it can be concluded that theprocedure of GS-NPs manufacturing did not affectantibiotic activity of the encapsulated drug and GS-NPs seem to be promising drug delivery system in thetreatment of osteomyelitis.

5. Conclusion

Based on the results obtained we conclude that byencapsulation of GS in PLGA nanoparticles it waspossible to fabricate prospective drug delivery sys-tems for bone infection treatment. By changing manu-facturing parameters (GS content in relation to PLGAand PVA concentration) it was possible to adjust sizeand solubilization parameters of GS-NPs. Short burstrelease followed by long sustained release of antibi-otic was obtained up to 5 weeks. Drug released fromGS-NPs demonstrated antimicrobial activity againstreference strains and clinical isolates of S. aureus andS. epidermidis, which are responsible for majority ofosteomyelitis cases.

Acknowledgments

The authors would like to thank Professor Piotr Dobrzyński(Center of Polymer and Carbon Materials, Polish Academy of Sci-ences) for providing us with degradable copolymer and Dr. ŁukaszZych (Department of Ceramics and Refractories, Faculty of Mate-rials Science and Ceramics) for the access to the Zetasizer. PolishNational Science Centre (Grant no: 012/05/B/ST8/00129) pro-vided financial support to this project.

References

[1] ANHALT J.P., Assay of gentamicin in serum by high-pres-sure liquid chromatography, Antimicrob. Agents. Ch, 1997,Vol. 11(4), 651–655.

[2] BAQUERO F., Gram-positive resistance: challenge for thedevelopment of new antibiotics, J. Antimicrob. Chemother.,1997, Vol. 39, 1–6.

[3] BROC-RYCKEWAERT L., CARPENTIER R., LIPKA E., DAHER S.,VACCHER C., BETBEDER D. FURMAN C., Development of in-novative paclitaxel-loaded small PLGA nanoparticles: Studyof their antiproliferative activity and their molecular inter-actions on prostatic cancer cells, Intern. J. Pharm., 2013,Vol. 454(2), 712–719.

[4] CALHOUN J., MANRING M.M., SHIRTLIFF M., Osteomyelitis ofthe long bones, Semin. Plast. Surg., 2009, Vol. 23(2), 59–72.

[5] CAMPOCCIA D., MANTANARO L., ARCIOLA C.R., The significanceof infection related to orthopedic devices and issues of antibioticresistance, Biomaterials, 2006, Vol. 27(11), 2331–2339.

[6] CAPAN Y., JIANG G., GIOVAGNOLI S., NA K.H., DE LUCA P.P.,Preparation and characterization of poly(d,l-lactide-co-glycolide) microspheres for controlled release of human growthhormone, AAPS Pharm. Sci. Tech., 2003, Vol. 4(2), 147–156.

[7] COSTERTON J.W., Biofilm theory guide the treatment of de-vice-related orthopaedic infections, Clin. Orthop. Relat. Res.,2005, Vol. 437, 7–11.

[8] DOBRZYNSKI P., KASPERCZYK J., JANECZEK H., Synthesis ofbiodegradable copolymers with the use of low toxic zirco-nium compounds. 1. Copolymerization of glycolide withL-Lactide initiated by Zr(Acac)4, Macromolecules, 2001,Vol. 34, 5090–5099.

[9] European Committee on Antimicrobial SusceptibilityTesting. Breakpoint tables for interpretation of MICs andzone diameters. Version 4.0, valid from 2014-01-01.http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Breakpoint_tables/Breakpoint_table_v_4.0.pdf. Accessed7 November 2014.

[10] FECZKÓ T., TÓTH J., DÓSA G., GYENIS J., Optimization ofprotein encapsulation in PLGA nanoparticles, Chem. Eng.Process, 2011, Vol. 50(8), 757–765.

[11] FLOCZYKOWSKI B., STORER A., Gentamicin Dosing andMonitoring Challenges in End-Stage Renal Disease, Adv.Pharmacoepidem. Drug. Safety, 2013, Vol. 2(3), DOI:0.4172/2167-1052.1000135.

[12] GRABOWSKI N., HILLAIREAU H., VERGNAUD J., SANTIAGO L.A.,KERDINE-ROMER S., PALLARDY M., TSAPIS N., FATTAL E.,Toxicity of surface-modified PLGA nanoparticles toward lungalveolar epithelial cells, Intern. J. Pharm., 2013, Vol. 454(2),686–694.

[13] JAIN G.K., PATHAN S.A., AKHTER S., AHMAD N., JAIN N.,TALEGAONKAR S., KHAR R.K., AHMAD F.J., Mechanisticstudy of hydrolytic erosion and drug release behaviour ofPLGA nanoparticles: Influence of chitosan, Polym. Degrad.Stab., 2010, Vol. 95, 2360–2366.

[14] KAYA E.G., OZBILGE H., ALBAYRAK S., Determination of theeffect of gentamicin against Staphylococcus aureus by usingmicrobroth kinetic system, ANKEM Derg., 2009, Vol. 23(3),110–114.

[15] KLOSE D., SIEPMANN F., ELKHARRAZ K., SIEPMANN J.,PLGA-based drug delivery systems: importance of the type ofdrug and device geometry, Int. J. Pharm., 2008, Vol. 354(1–2),95–103.

U. POSADOWSKA et al.48

[16] LEE D.W., YUN Y.P., PARK K., KIM S.E., Gentamicin andbone morphogenic protein-2 (BMP-2)-delivering hepa-rinized-titanium implant with enhanced antibacterialactivity and osteointegration, Bone, 2012, Vol. 50(4),974–982.

[17] LIN C.C., METTERS A.T., Hydrogels in controlled releaseformulations: network design and mathematical modeling,Adv. Drug. Deliv. Rev., 2006, Vol. 58, 1379–1408.

[18] MORENO D., ZALBA S., NAVARRO I., TROS DE ILARDUYA C.,GARRIDO M., Pharmacodynamics of cisplatin-loaded PLGAnanoparticles administered to tumor-bearing mice, Eur. J.Pharm. Biopharm., 2010, Vol. 74(2), 265–274.

[19] PAMULA E., FILOVÁ E., BACÁKOVÁ L., LISÁ V., ADAMCZYK D.,Resorbable polymeric scaffolds for bone tissue engineering:the influence of their microstructure on the growth of humanosteoblast-like MG 63 cells, J. Biomed. Mater. Res. A, 2009,Vol. 89(2), 432–443.

[20] RAPP R.P., KENT M.E., SMITH K.E., Antibiotic Beads andOsteomyelitis: Here Today, What's Coming Tomorrow? Or-thopedics, 2006, Vol. 29(7), 345–349.

[21] RUMIAN L., WOJAK I., SCHARNWEBER D., PAMULA E., Re-sorbable scaffolds modified with collagen type I or hydroxy-apatite: in vitro studies on human mesenchymal stem cells,Acta Bioeng. Biomech., 2013, Vol. 15(1), 61–67.

[22] SASAKI T., ISHIBASHI Y., KATANO H., NAGUMO A., TOH S., Invitro elution of vancomycin from calcium phosphate cement,J. Arthroplast., 2005, Vol. 20(8), 1055–1059.

[23] SIVARAMAN B., RAMAMURTHI A., Multifunctional nanoparti-cles for doxycycline delivery towards localized elastic matrixstabilization and regenerative repair, Acta Biomater., 2013,Vol. 9(5), 6511–6525.

[24] SMITH I.M., AUSTIN O.M.B., BATCHELOR A.G., The treat-ment of chronic osteomyelitis: A 10 year audit, Journal ofPlastic, Reconstructive & Aesthetic Surgery, 2006, Vol. 59(1),11–15.

[25] TSAI A., UEMURA S., JOHANSSON M., PUGLISI E.V.,MARSHALL R.A., AITKEN C.E., KORLACH J., EHRENBERG M.,PUGLISI J.D., The impact of aminoglycosides on the dynamicsof translation elongation, Cell. Rep., 2013; Vol. 3(2), DOI:10.1016/j.celrep.2013.01.027.

/ColorImageDict > /JPEG2000ColorACSImageDict > /JPEG2000ColorImageDict > /AntiAliasGrayImages false /CropGrayImages true /GrayImageMinResolution 300 /GrayImageMinResolutionPolicy /OK /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 300 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict > /GrayImageDict > /JPEG2000GrayACSImageDict > /JPEG2000GrayImageDict > /AntiAliasMonoImages false /CropMonoImages true /MonoImageMinResolution 1200 /MonoImageMinResolutionPolicy /OK /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 1200 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict > /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile () /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False

/CreateJDFFile false /Description > /Namespace [ (Adobe) (Common) (1.0) ] /OtherNamespaces [ > /FormElements false /GenerateStructure false /IncludeBookmarks false /IncludeHyperlinks false /IncludeInteractive false /IncludeLayers false /IncludeProfiles false /MultimediaHandling /UseObjectSettings /Namespace [ (Adobe) (CreativeSuite) (2.0) ] /PDFXOutputIntentProfileSelector /DocumentCMYK /PreserveEditing true /UntaggedCMYKHandling /LeaveUntagged /UntaggedRGBHandling /UseDocumentProfile /UseDocumentBleed false >> ]>> setdistillerparams> setpagedevice