Antifungal Activity and Cytotoxicity of Zinc, Calcium,or Copper Alginate Fibers

Ying Gong & Guangting Han & Yuanming Zhang &

Ying Pan & Xianbo Li & Yanzhi Xia & Yan Wu

Received: 6 December 2011 /Accepted: 7 March 2012 /Published online: 20 March 2012# Springer Science+Business Media, LLC 2012

Abstract The antifungal properties and cytotoxicity of algi-nate fibers were investigated to widen their application intissue engineering. Calcium, zinc, and copper alginate fiberswere separately prepared by replacing Na+ with Ca2+, Zn2+, orCu2+. The antifungal properties of the three alginate fiberswere studied after coming into contact withCandida albicans.Then, the fungal inhibitory rates were measured using theplate-count method following shake-flask test. Moreover, aninhibition-zone test and observation by scanning electronmicroscopy were carried out. The inhibitory rate of the calci-um, copper, and zinc alginate fibers were, respectively, 49.1,68.6, and 92.2 %. The results from inhibition-zone test andshake-flask test show that zinc alginate fibers have the mostsignificant antifungal action and that copper alginate fibershave obvious inhibitory action, but the calcium alginate fibershave weak inhibitory effects. The scanning electron micro-graphs similarly illustrate that the fungal surfaces show mostscraggly after the interaction between C. albicans and zincalginate fibers. Moreover, the relative growth rates of zinc orcalcium alginate fibers in human embryonic kidney cells andhuman fibroblast cells were more than 100 %. No significantresults were obtained (P>0.05). The calcium alginate fibers inhuman fibroblast cells were not much different from thenegative control group (P>0.05). However, zinc alginatefibers had a significant change (P<0.05). Therefore, the ex-cellent antifungal property of zinc alginate fibers demonstratespotential application in skin tissue engineering comparingwith calcium or copper alginate fibers.

Keywords Cytotoxicity . Alginate fibers . Antifungalproperty . Biomaterials

Introduction

Biomaterial-centered infection (BCI), indicated by micro-bial biofilm formation, is the key factor restraining theutilization of biomaterials [1]. Despite the use of anti-biotics and sterile conditions, many patients still sufferfrom BCI, and the microbial biofilm formation on im-plant surfaces is a common reason for the failure of somemedical equipment [2]. Thus, numerous efforts havebeen taken to reduce pathogen growth on implant surfa-ces. The adhesion of microorganisms to medical equip-ment is the first step for biofilm formation, which isrelevant to the physicochemical properties of its surfaces.Therefore, the careful design of the biomaterials candecrease incidence rate of BCI and avoid implant removal,as well as surgeries [3].

Calcium alginate fibers are firstly invented in 1944 [4].Calcium alginate fibers are extensively used in wound man-agement in recent years because they have advantages overtraditional cotton gauzes, including softness, high absorben-cy, and the ease of fabricating different products [5]. Severalbacteria and fungi cause wound infections, such as Escher-ichia coli, Staphylococcus aureus, Pseudomonas aerugi-nosa, and Candida albicans (C. albicans), because mostwounds offer a favorable environment for both fungi andbacteria [6–8]. In addition, infections in traumatic and sur-gical wounds cause a serious antibiotic-resistance problem ifa bacterial biofilm forms [9]. However, calcium alginatefibers do not control bacteria reproduction efficiently. Pre-vious researches have shown that zinc or copper alginatefibers possess good antibacterial activation [10]. In addition,

Y. Gong :G. Han (*) :Y. Zhang :Y. Pan :X. Li :Y. Xia :Y. WuShandong Provincial Key Laboratory of New Fiber Materialsand Modern Textile, the Growing Base for State Key Laboratory,Qingdao University,Qingdao 266071, People’s Republic of Chinae-mail: kychgt@qdu.edu.cn

Biol Trace Elem Res (2012) 148:415–419DOI 10.1007/s12011-012-9388-7

both zinc and copper are vital microelements required by thebody, and zinc is known to promote wound healing [11].Some researchers have modified alginate fibers to kill fungiand bacteria [12]; however, such modifications may alter theproperty of fibers. The calcium, zinc, or copper alginatefibers in this paper were prepared using ion exchange duringthe wet spinning course [13].

Tissue engineering (TE) involves the formation of porif-erous materials with an open and interconnected pore canal,which is beneficial to cell migration, nutrient delivery, andwaste excretion [14]. As a natural polymer biomaterial,alginate is widely used in TE and wound treatment due toits good biocompatibility and low toxicity [15, 16]. Amongthe alginate fibers, calcium fibers are commonly used inpreparing surgical dressings, gauze, etc. Many studies havereported that calcium alginate dressings can induce hemostaticaction without cytotoxicity, thus accelerating the healing pro-cess of wound [17].

However, few researchers have explored the cytotox-icity of zinc alginate fibers on human embryonic kidneycells (293 cells) and human fibroblast cells by comparingwith calcium alginate fibers. Not much is known aboutthe impact of zinc alginate fibers on kidney and skin[18]. This paper examined the cytotoxicity of zinc algi-nate fibers using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-nyltetrazolium bromide (MTT) assay and compared theresults with those obtained from calcium alginate fibers.The current study investigated the properties of alginatefibers as suitable biomaterials for reducing bacterial biofilmformation and enhancing the wound healing rate in skintissue engineering.

Materials and Methods

Antifungal Properties Assay

C. albicans (ATCC 10231) was transferred twice, and anaxenic culture colony was grown in Sabouraud dextrose broth(Beijing Land Bridge Technology Co., Ltd.) at 37°C for 24 h.Calcium, zinc, and copper alginate fibers were separately per-formed by replacing Na+ with Ca2+, Zn2+, or Cu2+ in coagu-lating bath. The three kinds of alginate fibers were sterilizedthrough 2 h of UV irradiation before the experiments (each sidefor an hour). The reagents weremostly analytical reagent grade.

First, 0.1 ml of culture solution was seeded into sterilizedSabouraud dextrose agar. Then, 0.1 g of the fibers wasplaced on plates and incubated at 37°C for 24 h, and thediameter of the resulting inhibition zones caused by thethree alginate fibers was analyzed.

The culture solution was diluted to 105 CFU/ml with 0.9 %NaCl. Then, 1 ml of diluent was added to the flasks andincubated at 37°C for 24 h. The fibers were respectively addedto the flasks and shaken at 37°C for 24 h. After that, 0.1 ml ofsolution from the flasks was diluted to 1:10 and 1:100, and0.1 ml of diluent was added into Petri dishes, pouring Sabo-uraud dextrose agar. The number of fungi was counted after24 h. The inhibitory rate was calculated using the followingequation:

Inhibitory rate ¼ A� Bð Þ=A½ � � 100%

where A was the number of fungal colony on blank controlplates and B was that on test plates.

Then, 0.5 ml of diluent after incubation with the fiberswas fixed in the centrifuge tube using 2.5 % glutaraldehyde(HPLC grade). The specimens were dehydrated, gold-coated through direct current sputtering, and photographedunder a JEOL JSM-6390LV microscope.

Morphology Assay

Using the scanning electron microscope (SEM, JSM-6390LV,Japan), the fiber morphology of the longitudinal surface andcross-section images of calcium or zinc alginate fibers wasexamined. The average fiber diameter was calculated usingImage-Pro Plus 6.0 software.

Fig. 1 Inhibition zones ofalginate fibers against C.albicans. a Calcium alginatefibers. b Copper alginate fibers.c Zinc alginate fibers

Fig. 2 Inhibitory rates of the three alginate fibers against C. albicans

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Cytotoxicity Assay

Cytotoxicity tests were performed by placing these fibersinto cells. A total of nine experimental groups were preparedaccording to the following weights of calcium and zincalginate fibers: 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.08,0.10, and 0.15 g. Next, 1-cm lengths of fibers were cutand sterilized with UV irradiation for 1 h.

All fibers were introduced into human embryonic kidneycells (293 cells) and human fibroblast cells. These cells werein their exponential growth phase in the 20 ml RPMI-1640cell growth medium (GIBCO) and contained 10 % fetalbovine serum (GIBCO), streptomycin 0.1 mg/ml, and peni-cillin 100 U/ml. There were no fibers added in the negativecontrol group.

Cells were incubated at 37°C for 48 h. All cells wereobserved through the inverted microscope at×200 magnifi-cations. After digesting 0.25 % trypsin, the 200 μl of cells

per well (n03/group) was inoculated in 96-well plates. Allplates were incubated for 4 h and then combined with 50 μlof MTT solution.

Afterwards, 150 μl of dimethylsulfoxide was added to thewells and left to be absorbed for 10 min. The amount ofabsorption (A) was read through a spectrophotometer at450 nm. The following equation was used to calculate relativegrowth rate (RGR):

RGR %ð Þ ¼ Atest=Anegative controlÞ � 100%

Statistical Analysis

The experimental data were analyzed by using the softwareof SPSS 11.5. Some data are presented as arithmetic meanvalues and standard deviation. The differences between themean values of RGR of zinc alginate fibers and negativecontrol group were analyzed for statistical significance by

Fig. 3 SEM micrographs of C.albicans morphology aftercontact with the alginate fibers.a Normal negative controlgroup. b Zinc alginate fibersgroup. c Copper alginate fibersgroup. d Calcium alginatefibers group

Fig. 4 a Micrographs of thelongitudinal surface of the zincalginate fibers. b Cross sectionof the zinc alginate fibers. cLongitudinal surface of thecalcium alginate fibers. d Crosssection of the calcium alginatefibers

Antifungal Activity and Cytotoxicity of Alginate Fibers 417

ANOVA and post hoc tests. The statistical analysis of cal-cium alginate fibers was similar. Probability level values atP<0.05 were significant.

Results and Discussion

Antifungal Properties Assay

The inhibition-zone test is a qualitative approach to test theantimicrobial activity. Figure 1 shows that the culture plateswith zinc or copper alginate fibers had evident inhibitionzone compared with calcium alginate fibers and that thefibers contacted with agar were gelatinous because alginatefibers dissolved after 48 h.

The three alginate fibers were obtained using the ion-exchange method; the metal ions Ca2+, Zn2+, or Cu2+ easilyexchanged with sodium ion in the agar after the fibers touchedthe agar. The positively charged zinc and copper ions are likelyabsorbed on the surface of negatively charged fungi, and then,the ions destroy the cell membrane, thereby restraining thegrowth of fungi. Therefore, the three alginate fibers weredissoluble and antifungal. Figure 2 shows the result of theinhibitory rate of the three kinds of alginate fibers. The meanantifungal rates were 49.1, 68.6, and 92.2 %, respectively.

A shake-flask test is suitable for both insoluble anddissoluble fibers, which is a quantitative method for testingthe antifungal activity of fibers. Zinc alginate fibers have thebest antifungal property against C. albicans. Similarly, thebacterial activity against E. coli for the zinc or copperalginate fibers is stronger than that of cotton fibers [11].

Changes in the appearance of C. albicans after incubationwith alginate fibers were investigated under scanning elec-tron microscopy. Figure 3d shows that most unbroken fungiare present on the calcium alginate fibers; the surface offungi is uneven on the copper alginate fibers (Fig. 3c);however, many fungal hyphae are cracked on zinc alginatefibers (Fig. 3b).

The three alginate fibers had different effects on C. albi-cans, which was associated with the metal ions used. Themetal ions from the calcium, copper, and zinc alginate fibersreplaced sodium ions in the agar, and sodium ions came intothe interior of the fibers while the metal ions dispersed in theagar and fungi. The metal ions adhered to the fungi, thenpenetrated into them and damaged the cell membrane orcombined with the protein, which induced cell death. Cop-per or zinc ions could penetrate the cell wall more easilythan calcium ions, damage cell membranes, and result inprotein denaturation to restrain fungal growth and even killthem. The results indicate that zinc alginate fibers had abetter effect against C. albicans than calcium or copperalginate fibers.

Morphology Analysis

The diameters of zinc alginate fibers were 19.16±3.36 μm(Fig. 4), which were noticeably bigger than those of calciumalginate fibers (12.03±0.87 μm, P<0.05). The morpholo-gies of both zinc alginate fibers (Fig. 4a, b) and calciumalginate fibers (Fig. 4c, d) were similar. A few longitudinalgrooves without a skin-core structure could be seen on thesurface of the two fibers, which might be caused by the

Fig. 5 Cytotoxicities ofcalcium or zinc alginate fiberson 293 cells. No significantdifference is observedcompared with the negativecontrol group (P>0.05 byANOVA)

Fig. 6 Cytotoxicities ofcalcium or zinc alginate fiberson human fibroblast cells. Theresults are significantlydifferent from the negativecontrol group (*P<0.05 byANOVA)

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solvent. Therefore, moisture absorption of the two fibersmay be enhanced by extending the surface-specific areas.

Cytotoxicity Tests

In this study, according to ISO 10993-5:1999, the materialsdemonstrate no cytotoxicity when RGR is more than 75 %(Figs. 5 and 6). Therefore, the calcium alginate fibers andzinc alginate fibers attain the biosafety level standard ofbiomaterials. Moreover, there was a significant differencebetween the RGR of zinc alginate fibers groups and thenegative control group in human fibroblast cells (P<0.05)(Fig. 6), which means zinc alginate fibers (>0.05 g) not onlyhad no cytotoxicity but also could promote human fibroblastcells growth.

The biomaterial used for tissue engineering should pro-mote cell and tissue growth while maintaining its nontox-icity, nonsensitization, and nonrejection reaction. Usingbiomaterials from an animal may present potential compli-cations because these may contain carrying various hazard-ous components, including bacteria and viruses that causediseases.

Alginates that come from plants are used as cell scaffoldfor islet cells and chondrocytes. According to a previouswork, antibiotic calcium alginate dressings have no obvioustoxicity reaction to fibroblasts cells, and calcium alginatemay augment the proliferation of fibroblasts cells; however,because they release calcium ions, these may decrease fibro-blast motility [19].

Zinc acts as a cofactor in plentiful transcription factorsand enzyme systems involving zinc-dependent matrix met-alloproteinases that increase autodebridement and keratino-cyte migration for wound repair. Topical zinc therapy isnonrecognized although clinical evidence underlines its sig-nificance in autodebridement, anti-infective action, and pro-motion of epithelialization [20].

Conclusions

Zinc alginate fibers have superior antifungal propertiesagainst C. albicans compared with calcium or copperalginate fibers; zinc alginate fibers have far implicationsand enable significant advances in skin tissue engineer-ing for regenerative medicine. In addition, the incidenceof BCI would be dramatically reduced with the use ofzinc alginate fibers, and fungal resistance might beprevented.

Calcium alginate fibers are not harmful to cells, but zincalginate fibers can enhance cell growth. The mechanism ofcell proliferation is caused by the release of zinc ions fromfibers. Therefore, the zinc alginate fibers are better thancalcium alginate fibers in promoting cell growth.

Acknowledgments This study was funded by a grant from the NationalHigh Technology Research and Development Program of China(2008AA03Z509), the National Basic Research Program of China(2011CB612308), and Sci-Tech Plan Project of Qingdao (10-3-4-3-6-jch).

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