Gentamicin-eluting bioresorbable composite fibers for wound healing applications

Gentamicin-eluting bioresorbable composite fibers forwound healing applications

Meital Zilberman,1 Esty Golerkansky,1 Jonathan J. Elsner,1 Israela Berdicevsky21Department of Biomedical Engineering, Faculty of Engineering, Tel-Aviv University, Tel-Aviv 69978, Israel2Department of Microbiology, Faculty of Medicine, Technion–Israel Institute of Technology, Haifa 32000, Israel

Received 12 August 2007; revised 19 December 2007; accepted 2 January 2008Published online 28 April 2008 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.32013

Abstract: New gentamicin-eluting bioresorbable core/shell fiber structures were developed and studied. Thesestructures were composed of a polyglyconate core and aporous poly(DL-lactic-co-glycolic acid) (PDLGA) shellloaded with the antibiotic agent gentamicin, preparedusing freeze drying of inverted emulsions. These uniquefibers are designed to be used as basic elements of biore-sorbable burn and ulcer dressings. The investigationfocused on the effects of the emulsion’s composition (for-mulation) on the shell’s microstructure, on the drug releaseprofile from the fibers, and on bacterial inhibition. Therelease profiles generally exhibited an initial burst effectaccompanied by a decrease in release rates with time. Al-bumin was found to be the most effective surfactant forstabilizing the inverted emulsions. All three formulation

parameters had a significant effect on gentamicin’s releaseprofile. An increase in the polymer and organic:aqueousphase ratio or a decrease in the drug content resulted in alower burst release and a more moderate release profile.The released gentamicin also resulted in a significantdecrease in bacterial viability and practically no bacteriasurvived after 2 days when using bacterial concentrationsof 1 3 107 CFU/mL. Thus, our new fiber structures areeffective against the relevant bacterial strains and can beused as basic elements of bioresorbable drug-elutingwound dressings. � 2008 Wiley Periodicals, Inc. J BiomedMater Res 89A: 654–666, 2009

Key words: composite fibers; wound dressings; poly(DL-lac-tic-co-glycolic acid); gentamicin; controlled drug delivery

INTRODUCTION

Organ or tissue failure or loss is one of the mostfrequent and devastating problems in human health-care. The skin is the largest organ of the body andhas many different functions. Wounds with tissueloss include burn wounds, wounds caused as aresult of trauma, diabetic ulcers, and pressure sores.The regeneration of damaged skin includes repara-tive cell adhesion, migration, proliferation, and dif-ferentiation that results in skin reconstruction. Themoist, warm, and nutritious environment providedby wounds, together with a diminished immunefunction secondary to inadequate wound perfusion,may enable the build-up of physical factors such asdevitalized, ischemic, hypoxic, or necrotic tissue andforeign material, all of which provide an ideal envi-

ronment for bacterial growth. In burns, infection isthe major complication after the initial period ofshock. It is now estimated that about 75% of themortality following burn injuries is related to infec-tions rather than to osmotic shock and hypovolemia.1

Several types of chemical agents may be used asgermicidal compounds, for example silver ions, io-dine, chlorhexidine and antibiotics. These agents candiffuse from the dressing into the wound bed,thereby inhibiting microbial growth.2 Acticoat1

(Smith and Nephew), Silverlon1 (Argentum), andSilvaSorbTM are silver-impregnated dressings de-signed to provide controlled release of silver througha slow but sustained release mechanism, whichhelps avoid toxicity yet ensures delivery of a thera-peutic dose of silver to the wound.3 Acticoat1 is a 3-ply gauze dressing consisting of an absorbent rayonpolyester core, with upper and lower layers of nano-crystalline silver-coated high-density polyethylenemesh.4 It is applied wet and is then moistened withwater several times a day to allow the release of sil-ver, which provides an antimicrobial effect for3 days. The fabrication of a more elaborate bilayerchitosan membrane consisting of a dense upper layerand sponge-like chitosan lower layer able to deliver

This article is based on Esty Golerkansky’s MSc thesis.Correspondence to: M. Zilberman; e-mail: meitalz@eng.

tau.ac.ilContract grant sponsors: RAMOT (Horowitz) and Ela

Kodesz Foundations, Tel-Aviv University

� 2008 Wiley Periodicals, Inc.

sulfazidime has been reported.5 This dressing exhib-ited a two-phase release of sulfadiazine, with an ini-tial burst effect followed by a slow release. Themajor drawback associated with most dressing solu-tions presented to date is the need to remove thedressing, consequently causing pain and possiblyharming the vulnerable underlying skin. Further-more, concerns have been raised by cliniciansregarding safety issues related to the silver ions thatare included in most of these products. In the cur-rent study, we developed and investigated biore-sorbable fibers which contain the antibacterial agentgentamicin. These fibers are designed to be used asbasic elements of drug-eluting wound dressings.These burn and ulcer dressings will be bioresorb-able, thus eliminating the need for their removal.Furthermore, gentamicin, which is effective against abroad spectrum of Gram-positive and Gram-negativebacteria, will be released in a controlled manner.

Few controlled-release fiber systems based on poly-mers have been investigated to date.6–13 The twobasic types of drug-loaded fibers that have beenreported are monolithic fibers and reservoir fibers.In systems that use monolithic fibers, the drug is dis-solved or dispersed throughout the polymer fiber.For example, curcumin, paclitaxel, and dexametha-sone have been melt spun with poly(L-lactic acid)(PLLA) to generate drug-loaded fibers6 and aqueousdrugs have been solution spun with PLLA.7 Varioussteroid-loaded fiber systems have demonstrated theexpected first-order release kinetics.8–10 In systemsthat use hollow reservoir fibers, drugs such as dexa-methasone and methotrexane have been added tothe internal section of the fiber.11–13 The advantagesof drug-loaded fibers include ease of fabrication,high surface area for controlled release, and local-ized delivery of bioactive agents to their target. Dis-advantages include poor mechanical properties dueto drug incorporation and limitations in drug load-ing. Furthermore, many drugs and all proteins donot tolerate melt processing and organic solvents.

In one of our recent studies, we presented a newconcept of core/shell fiber structures, which success-fully meet these challenges.14 These composite fiberscombine a dense polymer core fiber and a drug/pro-tein-loaded porous shell structure, that is, the drugor protein is located in a separate compartment (a‘‘shell’’) around a melt spun ‘‘core’’ fiber. The shell isprepared using freeze drying of inverted emulsionswith mild processing conditions. This results ingood mechanical properties as well as in the desireddrug release profile.14 Our new fibers are ideal forforming thin, delicate, biomedically important struc-tures for various applications, such as fiber-basedendovascular stents that mechanically support bloodvessels while delivering drugs for preventing reste-nosis directly to the blood vessel wall, or drug deliv-

ery systems for cancer treatment. The antiprolifera-tive drugs used for these applications are water-in-soluble, and therefore diffuse slowly out of thefibers,15 whereas gentamicin is a water-soluble anti-biotic. It is therefore of interest to investigate itseffect on the emulsion’s stability and on the drugrelease profile from these composite fiber structures.

Since our fiber’s shell preparation is based oninverted emulsions, we will first describe this typeof emulsion. Emulsions are metastable colloids madeof two immiscible fluids, where one fluid is dis-persed in the other in the presence of a surface-active agent (surfactant).16 Inverted emulsions arecomposed of water droplets dispersed in a continu-ous oil (organic) phase. Emulsions are obtained byshearing two immiscible fluids, leading to the frag-mentation of one phase into the other. Their life-timemay vary considerably, depending on the tempera-ture and their composition. The instability is due tothe large interface area, and therefore a large surfaceenergy which is associated with finely dispersed sys-tems.17 The stability of emulsions is highly importantfor their use as drug delivery systems. Separating atwo-phase system produces a large surface area perdrop, leading to a high excess Gibbs energy perdrop and thus to a tendency in the direction ofdecreasing the Gibbs energy.16,17 The destabilizationof a two-phase emulsion goes through several con-secutive and parallel steps before reaching the finalstage of separated layers. Several types of interactionpatterns may arise when two particles driven by ran-dom Brownian motion approach each other, includ-ing flocculation, coalescence, Ostwald ripening, andcreaming.16,18,19 The microstructure of the freezedried emulsion can serve as a measure for the emul-sion’s stability.

The present study focuses on composite core/shellfiber structures loaded with gentamicin. The porousshell (drug-containing section) was prepared usingthe technique of freeze drying an inverted emulsion.The effects of the emulsion’s formulation (com-ponents) on the shell’s microstructure and on thedrug release profile and bacterial inhibition aredescribed.

MATERIALS AND METHODS

Materials

MaxonTM polyglyconate monofilament sutures, with adiameter of 0.20–0.25 mm, United States Surgical, wereused as core fibers.

Bioresorbable porous structures (the shell coating) weremade of 75/25 poly(DL-lactic-co-glycolic acid) (PDLGA), in-herent viscosity (i.v.) 5 0.65 dL/g (in CHCl3 at 308C,

GENTAMICIN-ELUTING COMPOSITE FIBERS 655

Journal of Biomedical Materials Research Part A

approximately 97,100 g/mole), Absorbable Polymer Tech-nologies, Inc., USA.

Drug: gentamicin sulfate (cell-culture tested), 590 lggentamicin base per milligram, Sigma (G-1264).

Surface active agents

1. Bovine serum albumin (BSA), molecular weight 566,000 Da, Sigma (A-4503).

2. Poly(vinyl alcohol) (PVA), 87–89% hydrolyzed, molec-ular weight5 13,000–23,000 Da, Aldrich (36,317-0).

3. Horseradish peroxidase (HRP) with an initial enzy-matic activity of 560 U/mg, Fluka (77,335).

Reagents

Isopropyl alcohol (propanol) was purchased from Fru-tarom, Israel.

O-phthaldialdehyde (P0657), sodium borate 0.04 M(B0127), and 2-hydroxyethylmercaptan (63,690) were pur-chased from Sigma.

1,1,1,3,3,3-Hexafluoro-2-propanol (H1008) was pur-chased from Spectrum Chemical Manufacturing Corp.

Microorganisms

Clinically isolated strains of Staphylococcus aureus, Staph-ylococcus epidermidis, and Pseudomonas aeruginosa were usedin this study. The strains were kindly provided by the Mi-crobiology Department of the Rambam Medical Center,Haifa, Israel.

Preparation of core/shell fiber structures

Fiber surface treatment

The sutures were surface-treated so as to dispose of theoriginal fiber’s coating and to enhance adhesion betweenthe core fiber and the coating. The Maxon fibers wereslightly stretched on special holders and dipped in1,1,1,3,3,3 hexafluoro-2-propanol for 45 s. The fibers werethen washed with 70% ethanol and dried.

Emulsion formation

A known amount of PDLGA was dissolved in chloro-form to form an organic solution. A known amount of

gentamicin was dissolved in double-distilled water andthen poured into the organic phase (in a test tube) andhomogenization of the emulsion was performed using aKinematica PT-3100 Polytron homogenizer operating at15,000 rpm (medium rate, which was found to be optimal)for 3 min. An emulsion formulation containing 20% w/vpolymer in the organic solution, 20% w/w gentamicin (rel-ative to the polymer load), and an organic to aqueous(O:A) phase ratio of 6:1 v/v was used as the reference for-mulation. Other formulations included 17.8% and 26.7%w/v polymer, 0, 5, and 10% w/w gentamicin, and O:Aphase ratios of 4:1, 8:1 and 12:1. Bovine serum albumin(BSA) (5% w/v relative to the water quantity) was addedto most of the emulsions as a surface active agent for sta-bilization. The effect of HRP and PVA (also 5% w/v rela-tive to the water quantity) as surfactants was also studied.All formulations are presented in Tables I and II.

Core/shell fiber structure formation

The treated core Maxon fibers were dip-coated (whileplaced on holders) in fresh emulsions and then frozen im-mediately in a liquid nitrogen bath. The holders plus sam-ples were then placed in a precooled (21058C) freeze dryer(Virtis 101 equipped with a nitrogen trap) capable of work-ing with organic solvents (freezing temperature of the con-denser was approximately 21058C) and freeze dried inorder to preserve the microstructure of the emulsion-basedcore/shell fiber structures. Drying was performed in twostages:

1. The freeze dryer chamber pressure was reduced to100 mTorr, while the temperature remained at21008C.

2. The condenser was turned off and its plate tempera-ture slowly increased to room temperature, while thepressure was monitored between 100 and 700 mTorr.During this step, the liquid nitrogen trap condensedthe excess water and solvent vapors.

The samples were stored in desiccators until use.

In vitro drug release studies

The composite core/shell fiber structures (15 cm eachsample) were immersed in phosphate buffered saline(PBS) at 378C for 112 days to determine gentamicin’srelease kinetics from these structures. The release studieswere conducted in closed glass vessels containing 2 mL

TABLE IThe Effect of Surfactants on the Shell’s Structural Characteristics

Surfactant Type(5% w/v)

Mean PoreSize (lm) Porosity

CoatingThickness (lm)

EncapsulationEfficiency (%)

No surfactant 4.88 6 0.35 54.5 25.1 6 1.01 20.8Albumin 1.01 6 0.03 44.5 28.6 6 0.36 64.0HRP 3.21 6 0.25 57.4 20.6 6 0.37 60.9PVA 11.45 6 2.5 25.3 51.2 6 2.6 52.9

656 ZILBERMAN ET AL.


PBS medium, using a horizontal bath shaker operated at aconstant rate of 130 rpm. The medium was removed (com-pletely) periodically, at each sampling time (6 h, 1, 2, 7, 14,21, 28, 35, 42, 56, 70, 84, 98, and 112 d), and fresh mediumwas introduced.

The gentamicin content in each sample was determinedwith a UV–vis scanning spectrophotometer (Anthos, Zen-yth 200rt). Sampath and Robinson’s procedure20 for ana-lyzing gentamicin was followed with a slight modification.O-phthaldialdehyde reagent was prepared by adding 2.5 go-phthaldialdehyde, 62.5 mL methanol, and 3 mL 2-hydroxyethylmercaptan to 560 mL 0.04M sodium borate indistilled water. Two-hundred microliter of gentamicin so-lution, 200 lL isopropanol (to prevent sedimentation), and200 lL o-phthaldialdehyde reagent were reacted for45 min at room temperature. The absorbance, which cor-responds to the gentamicin concentration, was thenmeasured at 333 nm. The gentamicin working range was10–140 lg/mL. A calibration curve was prepared for eachset of measurements. The calibration lines were the samewithin the experimental error for at least 1000 h (correla-tion coefficient >0.99, slope: 0.0018), indicating that theformulated o-phthaldialdehyde reagent was stable. The cu-mulative release profiles were determined relative to theinitial amount of gentamicin in the composite fibers (quan-tity released during the incubation period plus the residueremaining in the fibers). All experiments were performedin triplicate. Results are presented as means 6 standarderrors. The effects of the emulsion’s composition on therelease profile were studied by examining the followingparameters: polymer content in the organic phase (%w/v),drug content (relative to polymer content, %w/w), organi-c:aqueous (O:A) phase ratio, and surfactant type.

Residual drug recovery from composite fibers

Residual gentamicin recovery from the composite fiberswas measured as follows: the fibers were placed in 1 mLmethylene chloride (to dissolve the remaining PDLGAcoating) and 2 mL water were added (to dissolve the gen-

tamicin residues). Methylene chloride evaporation wasperformed under a nitrogen stream for 60 min, until aclear aqueous solution was achieved. The solution wasthen filtered in order to dispose of polymer particles. Thegentamicin concentration was estimated using a UV/visscanning spectrophotometer (Anthos, Zenyth 200rt).

The encapsulation efficiency was determined as theactual gentamicin concentration encapsulated in the fibersdivided by the theoretical value (the quantity that wasadded to the emulsion when it was created).

A similar process of drug recovery (from fresh fibersnot used for the drug release study) was used to evaluatethe encapsulation efficiency and to elucidate the effect ofthe emulsion formulation on the encapsulation efficiency.The experiments were performed in triplicate.

Morphological characterization

The morphology of the composite core/shell fiber struc-tures (cryogenically fractured surfaces) was observed usinga Jeol JSM-6300 scanning electron microscope (SEM) at anaccelerating voltage of 5 kV. The SEM samples were Ausputtered before observation. The mean pore diameter andporosity of the observed morphologies was analyzed usingSigma Scan Pro software and statistics were drawn usingSPSS 10 software. Statistical significance was determinedusing the ANOVA (Tukey-Kramer) method. For each SEMfractograph, the area occupied by the pores was calculatedusing the Sigma Scan Pro software to evaluate the porosityof the samples. The porosity was determined as the areaoccupied by the pores divided by the total area.

Microbiological study

Preparation of bacteria

All strains were grown overnight on Muller-Hinton(Difco) agar plates21 at 378C. The bacterial cells were col-

TABLE IIThe Examined Specimens and Their Shell’s Structural Characteristics

Process Parameters AmountMean PoreSize (lm) Porosity

CoatingThickness (lm)

EncapsulationEfficiency (%)

Polymer content (% w/v) 17.8 1.56 6 0.06 53.3 21.1 6 0.48 26.620.0 1.10 6 0.03 43.5 28.0 6 0.12 54.226.7 1.01 6 0.03 44.5 28.6 6 0.36 64.0

Gentamicin content (% w/w) 0 1.11 6 0.03 41.3 38.9 6 0.77 –5 1.56 6 0.07 52.1 26.6 6 0.52 36.7

10 1.16 6 0.03 47.7 22.0 6 0.54 40.820 1.10 6 0.03 43.5 28.0 6 0.12 54.2

Organic to aqueous phase ratio (v/v) 4:1 1.13 6 0.03 67.8 27.7 6 0.30 36.46:1 1.10 6 0.03 43.5 28.0 6 0.12 54.28:1 0.90 6 0.03 11.0 28.9 6 0.18 50.3

12:1 1.10 6 0.03 8.1 37.8 6 1.88 55.2Organic to aqueous phase ratio

(without albumin as surfactant) (v/v)4:1 5.77 6 0.31 67.4 35.3 6 0.63 42.66:1 4.98 6 0.26 67.6 33.2 6 1.41 20.88:1 2.22 6 0.20 39.4 34.5 6 0.27 18.3

12:1 2.16 6 0.08 30.4 37.2 6 0.32 36.4



lected and resuspended in saline, and adjusted to 1 3 107

CFU/mL and 1 3 108 CFU/mL by visual comparison witha 0.5 McFarland standard.

Evaluation of residual bacteria

Samples of 10 lL or 100 lL were collected, at the appro-priate time, and spread on agar plates containing Muller-Hinton. Colony forming unit per milliliter were countedafter 24 h incubation at 378C.

MIC

The minimal inhibitory concentrations (MICs) of genta-micin against all tested microorganisms were determinedusing the twofold tube dilution method in Muller-Hintonbroth and are presented in Table III.

Microbiological experiment design

A release study from selected fibers was performed inthe presence of bacteria, to study the effect of drug releaseon the kinetics of residual bacteria. This experiment simu-lates a situation in which a wound is created and thereforethe skin in this area cannot function as a barrier againstbacteria. The bacterial strains were added at the beginningof the release study at a concentration of 1 3 107 CFU/mLor 1 3 108 CFU/mL, and the effect of the gentamicinreleased from the fibers was tested. The gentamicin con-centration in each tube was estimated by the ‘‘o-ophthalde-hyde’’ method. The samples were collected at time 0 andafter 6 h, 24 h, 2 d, 7 d, and 14 d, and viable counts wereperformed. Microorganisms in the presence of PBS onlyserved as the control. Bacterial growth was counted onMuller-Hinton agar plates.

The final calculation established the residual number ofbacteria in the presence of the released gentamicin.

RESULTS AND DISCUSSION

Composite fiber structures with a core diameter ofapproximately 200 lm and a shell thickness of 20–50 lm were produced and studied. The freeze dry-ing technique is unique in being able to preserve theliquid structure in solids. We used this technique toproduce the shell from inverted emulsions in whichthe continuous phase contained polymer dissolved

in a solvent, with water and drug dissolved in it asthe dispersed phase. A SEM fractograph showingthe bulk morphology of the reference specimen ispresented in [Fig. 1(a)]. The quality of the interfacebetween the fiber and the porous coating is high[Fig. 1(b)], that is the surface treatment enabled goodadhesion between the core and the shell. The shell’sporous structure contains round-shaped pores witha diameter of 1.0–11.5 lm and a porosity of 8–68%(Tables I and II). It can be seen that the shell’s micro-structure is uniform, probably due to rapid quench-ing of the emulsion, which enabled the preservationof its microstructure.

The shell’s microstructure affects the drug releaseprofile and can also serve as a good measure of theemulsion’s stability. At the early stages of this study,we found that the basic inverted emulsions (without

TABLE IIIMIC Values

Microorganism MIC (mg/mL)

Pseudomonas aeruginosa 2.5Staphylococcus epidermidis 5Staphylococcus aureus 6.3

Figure 1. SEM fractographs of a gentamicin-loaded com-posite fiber demonstrating the concept of core/shell fiberstructures.



drug) are stable. However, when the water-solublegentamicin was incorporated into the aqueous phaseof the emulsion, the interfacial tension increased andthe emulsion became less stable. Therefore, a prelim-inary study focused on the effect of surfactants onthe emulsion’s stability and on the resulting shellmicrostructure. Three types of surfactants wereadded (5% w/v) to emulsions containing 26.7% w/vpolymer, 20% w/w gentamicin, and an O:A phaseratio of 6:1. HRP and PVA were chosen as surfac-tants because we found them effective in stabilizinginverted emulsions with even more extreme O:Aphase ratios, such as 16:1.14 PVA is a well-knownemulsion stabilizer, and we therefore decided to tryit in our study. The effects of these surfactants onthe shell structure are presented in Figure 2 andTable I. Our results show that both albumin andHRP act as surfactants, that is their incorporationinto the emulsion resulted in a smaller pore size andthey increased the encapsulation efficiency from 20.8to 64 and 60.9%, respectively. In contradistinction,PVA exhibited an adverse effect on the emulsion’sstability, which resulted in an increased pore size,with relatively thick polymer domains betweenpores and reduced porosity. The effect of these threesurfactants on the gentamicin release profile fromthe composite fibers was also investigated. Theresults are presented in Figure 3. Composite fibersloaded with albumin as surfactant demonstrated arelatively small burst release of 33%, followed by amoderate release profile, which enabled release of

most of the loaded gentamicin within two weeks.The HRP and PVA loaded fibers exhibited a burstrelease of 55 and 58%, respectively, and most of theloaded gentamicin was released within 1 week. Al-bumin was shown to be very effective in our sys-tems. As a surfactant, it is located at the interfacebetween the aqueous phase and the organic phase,reduces the interfacial tension between the twophases, and therefore significantly decreases thepore size. It also enables a high encapsulation effi-ciency and relatively low burst release and drug

Figure 2. Effect of the addition of (5% w/v) surfactant on the shell microstructure of samples containing 20% w/w drug,26.7% w/v polymer and O:A phase ratio of 6:1: (a) no surfactant, (b) albumin, (c) HRP, and (d) PVA.

Figure 3. Effect of the addition of (5% w/v) surfactant onthe release profile of gentamicin from core/shell fiberstructures containing 20% w/w gentamicin, 26.7% w/vpolymer and O:A phase ratio of 6:1: green circle,– albumin;red square, HRP; and blue triangle PVA. [Color figure canbe viewed in the online issue, which is available atwww.interscience.wiley.com.]



release rate, probably due to its ability to bind thegentamicin through specific interactions. The abilityof albumin to bind drugs is well-known.22,23 Albu-min can interact with acidic or basic drugs by vander Waals dispersion forces, hydrogen bonds, andionic interactions. We chose albumin as the preferredsurfactant in our systems, based on these results.

We continued this study with composite fibersbased on shell formulations, which contained 5% w/valbumin. The effects of the emulsion’s formulation,that is polymer and drug contents and O:A phase ratio,on the shell microstructure (reflects the emulsion’s sta-bility) and on the resulting gentamicin release profilewere studied and are described below in detail. Inaddition, selected samples were chosen to study thekinetics of bacterial inhibition.

The shell microstructure

A relatively narrow polymer content range of17.8–26.7% w/v was used in this study. Polymercontents below 17.8% w/v produced a nonhomoge-nous structure reflected by a partially porous struc-ture, due to the emulsion’s instability, whereas earlyphase separation of the emulsion occurred at poly-mer contents above 26.7% w/v, since the organicphase is rapidly rejected by the water. An optimalpolymer content range of 17.8–26.7% w/v was there-fore used, in which a relatively stable and homoge-nous porous structure was achieved. The effects of

polymer content on the morphological characteristicsof the shell in this range are presented in Figure 4and Table II. An increase in polymer contentresulted in a small decrease in the shell’s porosityand mean pore size. According to the theory ofemulsion stability, polymer content affects an emul-sion’s stability either by increasing its viscosity orpromoting surface stability.16 The viscosity of the or-ganic phase is exponentially dependent on the den-sity of the organic polymer solution. A higher viscos-ity reduces the tendency of droplets to move, whichleads to breaking of the emulsion. A higher viscosityalso reduces the diffusion of water through the or-ganic phase and therefore the rate of creation oflarger drops at the expense of smaller drops isreduced (Ostwald ripening).16–19 High viscositiestherefore produce structures with a smaller meanpore diameter. Increasing the polymer content maytherefore reduce flocculation and coalescence. Poly-mer content would thus be expected to affect theshell’s pore size. However, in the present study, theviscosity probably did not change so much due tothe chosen specific range of polymer contents. It isalso important to note that the fibers with 26.7%polymer showed two size populations: pores with adiameter of 1–2 lm as well as very small nanopores.These two pore populations probably result from adegree of emulsion instability. If a more stable emul-sion were created, we would probably have obtainedonly the very small pore population.

Figure 4. SEM fractographs of composite fibers showing the effect of polymer content on the shell microstructure of sam-ples containing 20% w/w gentamicin, 5% w/v albumin and O:A phase ratio of 6:1. Polymer contents used: (a) 17.8% w/v,(b) 20% w/v, (c) and (d) 26.7% w/v.



The gentamicin content did not exhibit any signifi-cant effect on the shell’s structural characteristics(Table II). Gentamicin is a water-soluble drug (5 g/100 mL) and its presence in the emulsion’s aqueousphase therefore changes the emulsion’s hydropho-bic–hydrophilic balance. Higher gentamicin contentsare thus expected to increase the interfacial tensionbetween the water and the organic phase, and resultin lower emulsion stability. However, such an effectwas not obtained in our systems because the emul-sions were stabilized with a relatively high albumincontent of 5% w/v.

The effects of the O:A phase ratio on the shell’sstructural characteristics, derived from emulsionswith and without albumin, are presented in Figure 5and Table II. When the emulsion did not contain al-bumin, the size of the pores and the porosity exhib-ited a decreasing trend with the decrease in theaqueous phase content (or increase in the O:A phaseratio), and as a result the wall thickness (betweenpores) increased, as expected. When the emulsioncontained albumin as surfactant, the O:A phase ratiodid not show any significant effect on the pore size,due to the stabilizing effect of the surfactant. The po-rosity decreased with the increase in the O:A phaseratio, as expected. As stated above, gentamicin is awater-soluble agent which tends to diffuse outquickly from a porous structure. Relatively high O:Aratios can therefore be beneficial for the release of

gentamicin, because thick polymer walls betweenpores may result in a lower diffusion rate of the gen-tamicin molecules.

In vitro gentamicin release from core/shellfiber structures

The effects of the emulsion’s formulation (compo-sition) on the gentamicin release from the porousshell are presented in Figures 6–8. The emulsion pa-rameters include polymer content, drug content, O:Aphase ratio, and the presence of albumin. Most albu-min-loaded fiber structures generally exhibited drugrelease profiles of an initial burst effect accompaniedby a decreased release rate with time. Furthermore,at least 60% of the gentamicin was released withinthe first month, and the remaining drug wasreleased within the second month.

An increase in the emulsion’s polymer contentresulted in a dramatic decrease in the burst releaseand a more moderate overall profile (Fig. 6). Burstrelease values of 83, 51, and 33% were obtained forshell structures with polymer contents of 17.8, 20,and 26.7%, respectively. This behavior probablyresults from the general small decrease in pore sizeand pore density (porosity) (Fig. 4 and Table II),which affects the diffusion of the drug molecules.Unexpectedly, the release rate from the 26.7% poly-

Figure 5. SEM fractographs of composite fibers showing the effect of the O:A phase ratio on the shell microstructure ofsamples containing 20% w/v polymer and 20% w/w gentamicin: (a) 6:1, (b) 6:1 and 5% w/v albumin, (c) 12:1, (d) 12:1and 5% w/v albumin.



mer sample was higher than the release rateobtained from the 20% polymer sample (Fig. 6). Thisphenomenon probably results from changes in themicrostructure. Both shell structures exhibited a sim-ilar mean pore size, but the 26.7% polymer emulsionwas less stable and resulted in a structure of twopore size populations (Fig. 4). Such a microstructureis more effective for the diffusion of a bioactiveagent than a uniform structure. Furthermore, sincesome of the 26.7% emulsion was destroyed duringthe process step, most of the drug was located nearthe surface and was therefore released at a fasterrate.

The emulsion, which contained 26.7% polymer,enabled a relatively low burst release (33%, Fig. 6)and the effect of the gentamicin content on therelease profile was therefore studied on samples con-taining this polymer content. The burst releaseincreased with the increase in drug content (Fig. 7),because of a higher driving force for diffusion, asexpected. Samples with 5% gentamicin exhibited aburst release of 14% and samples with 20% gentami-cin exhibited a burst release of 33%. The 5% genta-micin sample released 60% of the drug within thefirst 3 days due to diffusion, and then exhibited asecond phase of release only after 3 weeks, probablydue to polymer degradation. In contradistinction, the20% gentamicin sample exhibited a decreased rate ofrelease after the initial burst, and 90% of the loadeddrug was released within 3 weeks.

The effect of the O:A phase ratio on the releaseprofile of gentamicin from composite fibers based onan emulsion with 20% w/v polymer is presented inFigure 8. Samples which contained albumin as thesurfactant exhibited a release pattern of three phases:burst release during the first 2 days, then drug

release at a nearly constant rate for 4 weeks, and athird phase of release with an increased rate attrib-uted to degradation of the host polymer [Fig. 8(a)].An increase in the O:A phase ratio (or decrease inthe aqueous phase content) resulted in a decrease inthe burst release and a small increase in the rate ofrelease during the second phase of drug release.These changes are obtained due to changes in theshell microstructure. Although the mean pore sizealmost did not change with the O:A phase ratio,samples with a high O:A phase ratio, such as 12:1,demonstrated relatively large polymeric domainsbetween pores which create a barrier to the diffusionof drug molecules. A similar effect of the O:A phaseratio on the burst release was obtained in samples,which did not contain albumin as surfactant. How-ever, in these samples, the rate of release during thesecond phase was very low [Fig. 8(b)]. The effect ofalbumin as a surfactant on the shell microstructureand on the resulting gentamicin release profile fromthe composite fibers can be summarized as follows:In all studied O:A phase ratios, samples which con-tained albumin exhibited a smaller mean pore sizeand smaller porosity than samples which did notcontain albumin. As a result, incorporation of albu-min enabled a lower burst release through a finermicrostructure, thus resulting in a more moderaterelease profile.

Microbiological evaluation of the effectof gentamicin release on bacterial viability

The purpose of these experiments was to monitorthe effectiveness of various concentrations of the an-

Figure 6. Effect of polymer content on the release profileof gentamicin from core/shell fiber structures containing20% w/w gentamicin, 5% w/v albumin, and O:A phaseratio of 6:1. Polymer contents used: blue triangle, 17.8%w/v; red square, 20% w/v; green circle, 26.7% w/v. [Colorfigure can be viewed in the online issue, which is availableat www.interscience.wiley.com.]

Figure 7. Effect of the gentamicin content on its releaseprofile from core/shell fiber structures containing 26.7%w/v polymer, 5% w/v albumin and O:A phase ratio of6:1. Gentamicin contents used: green diagonal, 5% w/wand blue triangle, 20% w/w. [Color figure can be viewedin the online issue, which is available at www.interscience.wiley.com.]



tibiotic released from the fibers in terms of the resid-ual bacteria compared with the initial number ofbacteria. Bacteria present in PBS only served as thecontrol. We chose the following three types of fiberswith different release profiles:

1. Fibers with a shell based on 26.7% w/v poly-mer, 20% w/v drug, 6:1 O:A phase ratio andalbumin as surfactant. These fibers demon-strated a moderate burst release of 32% fol-lowed by a moderate release profile.

2. Fibers with a shell based on 20% w/v polymer,20% w/v drug, 6:1 O:A. These fibers demon-strated a high burst release of approximately60%.

3. Fibers with a shell based on 26.7% w/v poly-mer, 5% w/v drug, 6:1 O:A phase ratio and al-bumin as surfactant. These fibers demonstrateda low burst release of 13% during the first dayand 60% within 3 days. After 3 days the releasepattern was similar to that of sample II.

Figure 9 presents all three profiles, for compari-son. The bacterial strains used in this study wereStaphylococcus aureus, Staphylococcus epidermidis, andPseudomonas aeruginosa. Their MIC values are pre-sented in Table III. All three strains were clinicallyisolated. These strains were chosen since they areprevalent in wound infections, especially Staphylo-coccus aureus and Pseudomonas aeruginosa. The thirdstrain, Staphylococcus epidermidis, usually comprisesthe normal flora of the skin. However, under graveconditions, it can cause wound infections. More-over, these bacteria can produce biofilms, whichprevent antibiotics from reaching the target, there-fore causing resistance. The bacteria were added atthe beginning of the fibers’ release, to simulate con-tamination at the time of implantation. Two initialbacterial concentrations were used: 1 3 107 CFU/mL and 1 3 108 CFU/mL. The results for all threebacterial types are presented in Figures 10 and 11,respectively. These specific bacterial concentrationsare relatively high. Most infections involve lowerconcentrations. We therefore assume that if our gen-tamicin-eluting fibers are effective against such con-centrations, they will also be effective against seri-ous infections.

Most bacteria did not survive after 2 days when abacterial concentration of 1 3 107 CFU/mL wasused, compared with the control where all the bacte-ria survived even after 14 days (Fig. 10). All fibersexhibited marked gentamicin release, which was re-sponsible for the dramatic decrease in bacterial sur-vival. Moreover, the shell’s preparation did notaffect gentamicin’s activity as an antimicrobial agent.

Figure 9. Gentamicin’s release profile from fiber sampleswhich were used for microbiological evaluation: yellowsquare, sample I (26.7% w/v polymer, 20% w/v drug, 6:1O:A phase ratio, and 5% w/v albumin); black circle, sam-ple II (20% w/v polymer, 20% w/v drug, 6:1 O:A); andred triangle, sample III (26.7% w/v polymer, 5% w/vdrug, 6:1 O:A phase ratio and 5% w/v albumin). [Colorfigure can be viewed in the online issue, which is availableat www.interscience.wiley.com.]

Figure 8. Effect of the emulsion’s O:A phase ratio on thegentamicin release profile from core/shell fiber structurescontaining 20% w/v polymer and 20% w/w gentamicin.(a) Samples which contained 5% w/v albumin, and (b)samples without albumin. O:A phase ratio used: greencircle, 6:1; blue triangle, 8:1; and red square, 12:1. [Colorfigure can be viewed in the online issue, which is availableat www.interscience.wiley.com.]



Although all three fiber samples exhibited a differentburst release during the first 6 hours, they released60–75% of their gentamicin contents after 2 days.When the initial drug contents and encapsulationefficiencies (Table II) are considered, it appears thatfibers I and II release similar drug quantities duringthe first 2 days, while fiber III which contained only5% w/v gentamicin released a smaller quantity.Indeed, after 2 days of gentamicin release, only Pseu-domonas aeruginosa remained while being exposed tothe gentamicin released from fiber III. When sampleII with a high burst release was used in the presenceof an extremely high concentration of the starter(1 3 108 CFU/mL), which simulates a serious infec-

tion, all three bacteria survived for less than 7 days.When fiber samples I and III were used, Pseudomonasaeruginosa survived for less than 7 days but smallquantities of Staphylococcus aureus and Staphylococcusepidermidis survived for more than 7 days (Fig. 11).Since the MIC values of these two bacteria arehigher that that of the former, the fibers with a rela-tively low burst release could totally eradicate themonly after more than 7 days.

These results indicate that bacterial survival isdose-dependent and that the MIC also plays an im-portant role. Drug quantities higher than the MICvalues should be released in order to eradicate allbacteria within a few days and prevent infection. Infact, a release profile with a medium burst effect, fol-lowed by continued moderate release is preferable.

Figure 10. Number of colony forming units (CFU) versustime, when an initial bacterial concentration of 1 3 107

CFU/mL was used: (a) Staphylococcus aureus, (b) Staphylo-coccus epidermidis, (c) Pseudomonas aeruginosa. The releasingfibers are: Yellow square, sample I (26.7% w/v polymer,20% w/v drug, 6:1 O:A phase ratio and 5% w/v albumin);black square, sample II (20% w/v polymer, 20% w/vdrug, 6:1 O:A); red square, sample III (26.7% w/v poly-mer, 5% w/v drug, 6:1 O:A phase ratio and 5% w/v albu-min); control––reference fiber without gentamicin. [Colorfigure can be viewed in the online issue, which is availableat www.interscience.wiley.com.]

Figure 11. Number of colony forming units (CFU) versustime, when an initial bacterial concentration of 1 3 108

CFU/mL was used: (a) Staphylococcus aureus, (b) Staphylo-coccus epidermidis, (c) Pseudomonas aeruginosa. The releasingfibers are: yellow square, sample I (26.7% w/v polymer,20% w/v drug, 6:1 O:A phase ratio, and 5% w/v albu-min); black square, sample II (20% w/v polymer, 20% w/vdrug, 6:1 O:A); red square, sample III (26.7% w/v poly-mer, 5% w/v drug, 6:1 O:A phase ratio, and 5% w/v albu-min); control––reference fiber without gentamicin. [Colorfigure can be viewed in the online issue, which is availableat www.interscience.wiley.com.]



This study demonstrates that our new fiber struc-tures are effective against the relevant bacterialstrains and can therefore be used as basic elementsof bioresorbable drug-eluting wound dressings. Anappropriate concentration of the ‘‘bioactive’’ (genta-micin-eluting) fibers in the wound dressings shouldbe chosen, to achieve effectiveness against the rele-vant bacterial strains without exceeding the thera-peutic window (4–12 lg/mL).24

SUMMARY AND CONCLUSIONS

New gentamicin-eluting bioresorbable core/shellfiber structures were developed and studied. Thesestructures were composed of a polyglyconate coreand a porous PDLGA shell loaded with the antibi-otic agent gentamicin, prepared using freeze dryingof inverted emulsions. These unique fibers aredesigned to be used as basic elements of bioresorb-able burn and ulcer dressings. Their investigationfocused on the effects of the emulsion’s composition(formulation) on the shell microstructure, on thedrug release profile from the fibers, and on theresulting bacterial inhibition.

In general, porous ‘‘shell’’ structures (mean poros-ity of 8–68% and mean pore size of 1–11 lm) wereobtained with good adhesion to the core fiber. Thefollowing emulsion parameters were chosen toobtain a stable emulsion that will result in a homo-geneous porous shell structure and a feasible releaseprofile of the water-soluble drug: 17.8–26.7% w/vpolymer content in the organic phase, 5–20% w/wgentamicin (relative to the polymer) and organic:a-queous (O:A) phase ratio in the 4:1–12:1 range. Therelease profiles generally exhibited an initial bursteffect accompanied by a decrease in release rateswith time. Albumin was found to be the most effec-tive surfactant for stabilizing the inverted emulsions.Its incorporation into the aqueous phase resulted ina lower burst release through a finer microstructureand therefore a more moderate release profile.

Most of the study focused on samples which werestabilized with albumin. In these structures, the O:Aphase ratio strongly affects the shell structure. Thepolymer content demonstrated some effect on themicrostructure, whereas the drug content had practi-cally no effect on the microstructure. All three for-mulation parameters had a significant effect on gen-tamicin’s release profile: an increase in the polymerand O:A phase ratio or a decrease in the drug con-tent resulted in a lower burst release and a moremoderate release profile. The released gentamicinalso resulted in a significant decrease in the bacterialviability and practically no bacteria survived after2 days when a bacterial concentration of 1 3 107

CFU/mL was used. Even at higher bacterial concen-

trations (1 3 108 CFU/mL), the three types of bacte-ria survived less than 7 days in most release profiles.The fiber preparation did not affect gentamicin’s po-tency as an antibacterial agent. Our new fiber struc-tures are thus effective against the relevant bacterialstrains and can be used as basic elements of biore-sorbable drug-eluting wound dressings.

The authors are grateful to the RAMOT (Horowitz) andto the Ela Kodesz foundations, Tel-Aviv University, forsupporting this research.

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Gentamicin-eluting bioresorbable composite fibers for wound healing applications

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