Biomedical coatings based on chitin soluble extract for inhibition of fungal adhesion to polymeric surfaces

Biomedical coatings based on chitin soluble extract forinhibition of fungal adhesion to polymeric surfaces

Meital Zilberman,1 Alon Navon,1 Hana Sandovsky-Losica,2 Esther Segal21Department of Biomedical Engineering, Tel Aviv University, Tel Aviv 69978, Israel2Department of Human Microbiology, Tel Aviv University, Tel Aviv 69978, Israel

Received 5 March 2006; revised 15 June 2006; accepted 10 August 2006Published online 21 November 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.31048

Abstract: Indwelling medical devices made of polymericmaterials, such as intravenous (IV) catheters, are known riskfactors for development of fungal infection, particularly sys-temic candidiasis, that are a significant cause of morbidityand lethality in compromised patients. Candida can form abiofilm on the polymeric surface, serving as a nidus for sys-temic, difficult to eradicate infection. The current researchfocuses on development and study of a chitin soluble extract(CSE) coating on polyurethane (PU), in order to reduce thelevel of adherence of C. albicans to the PU surface. The immo-bilization of CSE onto the PU surface was performed usingboth, chemical binding and physical adsorption. Our resultsindicate that CSE develops a unique tertiary structure in

which basic elements in the range of 100 nm build ‘‘fingers’’and these ‘‘fingers’’ are arranged in a concentric structurearound a center. The CSE coated films showed 75% inhibitionof C. albicans adhesion for the chemical binding and 83% forthe physical adsorption coating and even after 11 weeks theinhibitory effect on adhesion is still significant. Hence, ournew coatings may lead to a new generation of medical devi-ces with surfaces that can prevent fungal adhesion. � 2006Wiley Periodicals, Inc. J Biomed Mater Res 81A: 392–398, 2007

Key words: chitin soluble extract (CSE); intravenous (IV)catheters; fungal adhesion; biofilm prevention; biomedicalcoatings

INTRODUCTION

Candidiasis encompasses a wide range of infectionscaused by the fungi of the genus Candida. In additionto causing mucosal and cutaneous infections, these op-portunist pathogens can cause acute or chronic deep-seated infection in immunocompromised or debilitatedindividuals. Candidiasis is the most common systemicfungal infection.

Today’s hospitals rely on intravenous (IV) cathetersas essential tools to deliver IV medications,1 bloodproducts, and nutritional fluids to patients. Approxi-mately, 90% of all patients entering the hospital envi-ronment for care have some form of IV therapy duringtheir hospital stay. Most IV catheters are made of poly-urethane (PU). The medical consequences of catheter-related infections can be disastrous; they includepotentially life-threatening systemic infections and ex-pensive care. It is known that central venous cathetersinfections is the most frequent factor limiting theirprolonged use2,3 and in most cases demand catheterremoval.4,5 The reasons for the increase in incidence ofinvasive fungal infections in patients are: intensity of

chemotherapy employed in cancer patients, the pro-longed use of immunosuppressive agents in bonemarrow transplant patients, the use of broad-spectrumantibiotics, and long term use of central venous cathe-ters.6,7 Complications due to infections are frequentevents for all types of central venous catheters.8,9

Treating the infection presents various problems. Inmany instances the use of antifungal drugs incites sideeffects, which present a severe problem to the alreadysensitive population, which is primarily at risk of deepinfections. It is also possible that the treatment resultsin the appearance of resistant strains. It may thereforebe desired to prevent the infection in the first place.Although the number and types of treatment beingadministered by infusion therapy are becoming in-creasingly complex7,10,11 the routes by which infectioncan develop remain relatively constant.12 In short-termcatheters (up to 8 days), catheter colonization mostcommonly results from skin microorganisms (75–90%), followed by the catheter hub\lumen (10–50%),the bloodstream (3–10% up to 50% in ICU patients),and infusion material (2–3%).13 For long-term cathe-ters (more then 8 days), the source of colonization ismost commonly the hub\lumen (66%) followed by theskin (26%).

Clinical studies have shown that catheter-relatedinfection is reduced significantly by use of central ve-nous catheters impregnated with chlorhexidine and

Correspondence to: M. Zilberman; e-mail: [email protected]

' 2006 Wiley Periodicals, Inc.

silver sulfadiazine.14–16 These catheters are impreg-nated with antiseptics on their outer surface only.The lack of antimicrobial protection in the luminalsurfaces may account for some reports that show thatthey were not effective in preventing catheter-relatedbloodstream infections when placed for more then5 days.17–20 However, a recent in vitro and in vivostudy21 which examined the efficacy of cathetersimpregnated with antiseptics and antibiotics foundthat antibiotics catheters were not effective againstCandida species.

Chitin is the second most abundant polysaccharidein nature (after cellulose). At least 10 giga-tons of chi-tin are synthesized and degraded each year in thebiosphere. Chitin consists mainly of the aminosugarN-acetylglucosamine (NAG), which is partially deace-tylated. Chitin is present in nature usually as a com-plex with other polysaccharides and with proteins.Chitin is a renewable resource and is isolated fromcrab and shrimp waste. A procedure for preparationof chitin soluble extract (CSE) was developed in ourLab.22 We previously studied the inhibition of Candidaadherence to dentures and to soft contact lenses madeof methylacrylate, using CSE.23–25 The adherence ofC. albicans to dentures23 and to soft contact lenses aswell as that of Aspergillus spp.24,25 was inhibited. Thestudy revealed that addition of CSE to commercialcontact lenses maintenance solutions had a synergis-tic effect.24

In the present study, we have developed a CSE-based coating to PU surfaces in order to reduce theadherence of C. albicans to the PU. An attempt toachieve chemical binding of CSE to PU surfacesthrough a spacer was based on covalent binding theamine groups in the CSE molecules to the PU surface.The effectiveness of this coating (chemical binding)was compared to that achieved using physicaladsorption of CSE to the PU. In addition, the effect of‘‘shelf life’’ time on the coating stability was studiedfor both types of coatings.

MATERIALS AND METHODS

Materials

PU, medical grade Walopur 4201 AU, 25 mm film pro-duced by Bayer, was contributed by MTRE advanced Tech-nologies (Or-Akiva, Israel). Chitin (Poly(N-acetyl-1,4-b-D-glucopyranosamine) was purchased from Fluka. WaterSoluble Carbodiimide (WSC), (N-Ethyl-N0-(3-dimethylami-nopropyl)carbodiimide), 155.24 g/mol, produced by Sigma-Aldrich, was used as a spacer molecule. Trypsin (from pigspancreas), produced by Kibutz ‘‘Bet-Haemek’’, Israel, wasused to remove the adherent microorganisms from the PUspecimens.

Solvents and reagents

Buffer: Morpholino Ethane Sulphonic Acid (MES), (2-(N-Morpholino) ethanesulfonic Acid), molecular weight: 195.25,produced by: Nacalai Tesque, Kyoto, Japan. Dimethylforma-mide (DMF), molecular weight: 73.09, produced by: Sigma-Aldrich, was used as solvent. Citric Acid, molecular weight:192.12, produced by Frutarom, Israel. Sodium hydroxide(NaOH), molecular weight: 40, produced by Frutarom,Israel. Methyl Alcohol (Methanol), molecular weight: 32.04,produced by Sigma-Aldrich. Calcofluor, white (C40H44N12O10S2),a product of Sigma-Aldrich). sabouraud dextrose agar (SDA)with 0.05% chloramophenicol, produced by Difco Laborato-ries, was used for culturing Candida and enumeration of col-ony forming units (CFU). Bovine serum albumin (CAS no.9048-46-8), produced by Amresco solon, Ohio, was used tocover the wells of the microtiter plates.

Preparation of PU films

The raw PU thin films (25 mm) were converted to thickerfilms of *1 mm thickness that can be easily handled, by a3-step solution processing method as follows:

a. Thin film dissolution in DMF at room temperatureuntil polymer dissolution.

b. Solution casting in petri dishes and slow solventevaporation at a rate of approximately 7 mL/day, atroom temperature.

c. Isothermal heat treatment (508C, 500 mbar) for1 h in avacuum oven in order to dispose off solvent residues.

Disks of 10 mm diameter were cut out from the films,and etched by NaOH solution (4M), in order to introducecarboxyl groups on their surfaces.

CSE preparation

CSE was produced from commercially obtained chitin asdescribed previously,22 namely, chitin was suspended insterile distilled water to a concentration of 20% w/v. Thesuspension was shaken for 6 h and filtered. The supernatantwas frozen at �208C and freeze dried, using a freeze dryer(Virtis CT4KZL-105, SP Industries Company, NY). Thedried CSE was dissolved in ddH2O to get a concentrationof 25 mg/mL.

CSE chemical binding to PU

We chose to use a method, developed by Kishida et al.26

in order to chemically attach proteins to PU surfaces. Usingthis technique, CSE was bound to hydrolyzed PU using N-Ethyl-N0-(3-dimethylaminopropyl) carbodiimide (water solu-ble carbodiimide; WSC) as spacer. In order to do so, PU diskswere immersed in a mixture of 10% aqueous citric acid solu-tion and methanol (1:3 V/V) for 3 h at 258C and then washedwith double distilled water (ddH2O). The citric acid-treatedsamples were immersed in a 10% aqueous solution of WSC

BIOMEDICAL COATINGS 393

Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a

at 48C for 30 min (pH 4.75, buffered with 2-(N-Morpholino)ethanesulfonic acid, washed with ddH2O and then immersedin an aqueous solution of CSE (25 mg/mL) for 3 h at 258C.These treated samples were finally washed with ddH2O anddried using a vacuum oven (200 mbar, 408C). A schematicrepresentation of the chemical binding of CSE to PU surfacethrough WSC spacer is presented in Figure 1.

CSE physical binding to PU (direct binding)

PU disks (10 mm diameter, 1 mm thickness) wereimmersed directly in a solution of CSE (25 mg/mL) for 3 hat 258C, and then washed with ddH2O and dried using avacuum oven (200 mbar, 408C). These samples presentphysical binding of CSE to PU surfaces.

Candida growth conditions

Adherence to PU in vitro was tested with C. albicans CBS562, the major pathogenic Candida species. The organismswere grown for 69–120 h at 288C on SDA. For the adherenceassay the Candida yeasts were subcultured from slants intoyeast extract (YE) broth and grown under constant shakingat 288C for 18 h, harvested by centrifugation, washed withPBS, and resuspended to 2 � 107 cells/mL.

In vitro adherence assay

Tissue culture microtiter plates (Corning) were coveredwith 5% BSA dissolved in PBS (1 mL/well) in order to re-duce nonspecific adherence of Candida to the surface of thewell. The microtiter plates were incubated at 378C for 1 h,traces of albumin removed and dried.

One milliliter of yeast suspension (2 � 107 cells/mL) wasadded to each well (16 mm diameter) and then the 10 mm PUspecimens were placed in the wells, following which theplates were incubated at 378C for 1 h under rotational shaking(170 rpm) to prevent passive sedimentation of Candida.23,24

Quantitative evaluation of Candida adherence

Adherence of C. albicans to PU was evaluated by remov-ing adherent microorganisms from the PU specimens byincubation in 0.25% Trypsin solution (pH ¼ 7) for 30 min at378C (Trypsin a proteolytic enzyme, probably affects thebinding to the PU), and plating the suspension on SDAplates for determination of Candida CFU.

Microstructure

The surfaces of the treated and neat PU films were ob-served using High Resolution Scanning Electron Microscopy(HRSEM, Jeol JSM 6700) at accelerating voltage of 1 kV. Thechanges in surface morphology, due to surface treatments,were observed in order to characterize the coating’s struc-ture as well as adhesion to PU.

Statistical analysis

Mean values and standard errors were calculated for all re-sults. The results were evaluated using Excel t-test and ‘‘p’’values less than 0.01 were considered as statistically signifi-cant.

RESULTS

The surface structure of PU films

HRSEM was performed in order to characterize thesurface structure of the treated PU films, the structureof the coating, its dispersion and binding to PU. Themorphology of the neat PU films and WSC (spacer)treated PU films is presented in Figure 2, and themorphology of CSE-treated PU films is presented inFigure 3. Neat solution cast PU film exhibit relativelyrough surface structure, with holes of 10–20 mm diameter[Fig. 2(a)]. A relatively high magnification shows addi-tional roughness in the range of 0.1–1 mm [Fig. 2(b)]. PUfilms treated with WSC spacer molecules exhibited sur-face structure characteristics similar to those observedfor neat PU, but with holes and cracks that were probablycreated during the preliminary treatment [Fig. 2(c)].

In contrast, HRSEM observations of films treatedwith CSE—chemical binding (PU/WSC/CSE) indicatethat CSE molecules create interesting structures on thesurface of the treated PU. These round bright structuresare spread all over the sample [Fig. 3(a)]. Higher mag-nifications indicate that these concentric structures of5–20 mm diameter typically consist of a central regionand peripheral region. The latter is a ‘‘finger-like’’ struc-ture of ‘‘fingers’’ of 1–2 mm length and 0.5–1 mm thick-ness [Fig. 3(b)]. A very high magnification of theseunique structures indicates that they are actually com-posed of basic elements of very fine structure—typi-

Figure 1. A schematic representation showing CSE chemi-cal binding to polyurethane via spacer (WSC) molecule.[Color figure can be viewed in the online issue, which isavailable at www.interscience.wiley.com.]

394 ZILBERMAN ET AL.


cally 100 nm [Fig. 3(c)]. Hence, CSE develops a uniquetertiary structure in which basic elements in the rangeof 100 nm build ‘‘fingers’’ and these ‘‘fingers’’ are arrangedin a concentric structure around a center. Direct depo-sition (physical binding) of CSE on the surface of PUfilms (PU/CSE) resulted in similar structure to thatobserved for PU/WSC/CSE films [Fig. 3(d,e)]. How-ever, in this case the number of the CSE structuresseems to be lower [Fig. 3(d)].

Calcofluor is a fluorescent dye that has affinity tochitin and to some extent to glucan. Fungi can bestained by Calcofluor, as they contain chitin/glucanin their cell walls. We used Calcofluor to demonstrate

presence of CSE (a chitin derivative) on the coatedPU films. The results presented in Figure 4 show thatthe coated specimens are bright, suggesting the pres-ence of CSE on the PU surface.

Inhibition of C. albicans attachment to PU films

After preparation of coated PU specimens, quantita-tive evaluation of Candida adherence was performed.Based on the experience gained in our laboratory, theassays were set up to initial Candida concentration of2 � 107 cells per 1 mL. The percentage of adherence

Figure 2. HRSEM micrographs of PU film surfaces: (a) uncoated PU (�250), (b) uncoated PU (�10,000), (c) WSC-treatedPU (�250), (d) WSC-treated PU (�10,000).

Figure 3. HRSEM micrographs of PU film surfaces coated with CSE: (a) PU/WSC/CSE (�250), (b) PU/WSC/CSE(�10,000), (c) PU/WSC/CSE (�35,000), (d) PU/CSE (�250), (e) PU/ CSE (�10,000).



refers to the number of CFU that adhered relative tothe initial concentration of Candida.

The results (Fig. 5) show that the mean adherenceof C. albicans to neat PU was 3.85%, to PU/WSC/CSE(chemical binding through a spacer)—0.95% and toPU/CSE (physical binding)—0.63%. The analysis ofthe results demonstrate that CSE attached to PU sur-face inhibits fungal adherence in both cases, by 75%in comparison to untreated controls when boundchemically through a spacer and by 83% when physi-cally adsorbed.

‘‘Shelf life’’ of coated PU

As it is important to evaluate the effect of time onthe stability of the CSE coating and learn if it is stillactive and inhibits fungal adherence also after some‘‘shelf life,’’ we conducted such assays for 11 weeks.Two batches of specimens were prepared at the sameday. Twenty specimens of each type, PU/WSC/CSE(binding through spacer) and PU/CSE (direct physi-

cal binding), were used. The results are presented inFigure 6. It can be noted that PU/WSC/CSE specimensshowed a constant decrease in inhibition of fungal ad-herence with time. In contrast, the PU/CSE specimensshowed improved ability between week 2 and week 5,and then decrease up to week 11. However, even after11 weeks there was still significant inhibitory activity.

DISCUSSION

HRSEM observations indicate that CSE develops aunique tertiary structure in which basic elements inthe range of 100 nm build ‘‘fingers’’ and these ‘‘fingers’’are arranged in a concentric structure around a center(Fig. 3). Hence, it is suggested that although the WSCmolecules are probably spread uniformly on the sur-face of the PU film and are chemically bound to PUmolecules, the CSE tends to stay in relatively big or-dered shapes. This means that only small part of theWSC actually acts as spacer (binder) which connectsbetween WSC and the PU surface at a molecular level.Another possibility is that only a very small quantityof WSC was successfully bound to the PU film. Thestrong tendency of CSE molecules to aggregate andform relatively large shapes on the surface of the PUfilm, rather than spread uniformly, probably resultsfrom the significant difference in the nature (surfacetension, mainly) of these materials. While PU is a hy-drophobic polymer with relatively low surface tension,

Figure 4. A photograph of PU-based films treated with cal-cofluor (�10): right specimen—uncoated PU film, left speci-men—PU/WSC/CSE film. [Color figure can be viewed in theonline issue, which is available at www.interscience.wiley.com.]

Figure 5. Mean adherence of C. albicans to coated PU sur-faces. [Color figure can be viewed in the online issue, whichis available at www.interscience.wiley.com.]

Figure 6. Effect of time (‘‘shelf life’’ test) on the activityof the coatings: n—PU/WSC/CSE (chemical binding), l—PU/CSE (physical binding), ~—uncoated PU. [Color figurecan be viewed in the online issue, which is available atwww.interscience.wiley.com.]



the CSE is composed of polysaccharides and proteins,which are more hydrophilic and therefore of highersurface tension. Direct deposition of CSE on the surfaceof PU films [Fig. 3(d,e)] resulted in similar structure tothat obtained when we tried to bind CSE at molecularlevel through the WSC spacer [Fig. 3(a–c)].

As presented in Figure 5, the mean adherence rateof C. albicans onto neat PU specimens was 3.85%, ascompared to 0.95% adherence rate of C. albicans toPU/WSC/CSE and 0.63% to PU/CSE (physical bind-ing) specimens, thus attachment of C. albicans wasreduced by 75 or 83%, respectively. In order to evalu-ate the effect of time on the CSE stability and see if itis still active and inhibits fungal adherence, a shelflife test was conducted. The results are presented inFigure 6. After taking into account the microstructureof the coated specimens and the results from the ad-herence assays, it is clear that CSE ‘‘prefers itself’’ onbinding to the PU surface. Figure 6 shows a constantdecrease in inhibition ability for the PU/WSC/CSEspecimens. In contrast, the PU/CSE specimens showedimproved ability between week 2 and week 5, andthen decrease up to week 11. A possible reason for theunstable activity of the direct conjugation specimensmay be the relatively weak binding achieved by direct(physical) adsorption. As established previously,23–25

presence of CSE inhibits Candida adhesion. Hence,even when CSE molecules are not strongly bound tothe PU surface, during the few first weeks of the testthey will still remain on the PU surface and inhibit fun-gal adhesion.

Our original thought was to bind the protein mole-cules of the CSE to the PU surface through the WSCspacer, as previously done with other proteins.26

However, this chemical binding process is probablynot effective enough in our system, due to the morecomplex nature of the CSE compared to simple pro-teins such as albumin. Our shelf life test showed thatafter 8 weeks the increasing positive performance gapbetween the PU/WSC/CSE samples and PU/CSEsamples indicates that some chemical binding hasprobably occurred in the PU/WSC/CSE samples.However, in both cases the CSE is probably locatedon the PU surface, with some physical binding ratherthan chemical one. Accordingly, we can also assumethat if higher CSE concentrations are used, the effec-tiveness against fungal adhesion would increase, forboth types of coatings.

SUMMARY AND CONCLUSIONS

Novel coatings based on CSE for inhibition of fun-gal adhesion to PU surfaces were developed andstudied. Two methods for CSE attachment to the PUsurfaces were used: chemical binding through spacer

and physical binding. This study focuses on the micro-structure of the CSE coating and on its ability to re-duce the adherence of C. albicans to the PU surfaces.

CSE molecules showed strong tendency to aggre-gate and develop a unique tertiary structure, in whichbasic elements in the range of 100 nm build ‘‘fingers’’and these ‘‘fingers’’ are arranged in a concentric struc-ture around a center.

The CSE-coated films showed 75% inhibition ofC. albicans adherence for the chemical binding and83% for the physical adsorption coating, and even after11 weeks the adhesion prevention effect is still signifi-cant. It is therefore suggested that these new coatingsmay lead to a new generation of medical devices withsurface that can prevent fungal adhesion.

The authors thank Dr. Eddie Sionov and Mr. Ben Schin-dler (Department of Human Microbiology, Tel Aviv Uni-versity, Tel Aviv, Israel) for their generous assistance withthe microbiological tests.

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Biomedical coatings based on chitin soluble extract for inhibition of fungal adhesion to polymeric surfaces