Indian Journal of Fibre & Textile Research Vol. 3 1 , December 2006, pp. 543-547

Drug release and antimicrobial studies on chitosan-coated cotton yarns

o L Shanmugasundaram, V R Giri Dev", R Neelakandan & M Madhusoothanan

Department of Texti le Technology, A C College of Technology, Anna University, Chennai 600 025, Tamilnadu, India

and

G Suseela Rajkumar

Department of Bacteriology, Central Leather Research Insti tute, Chennai 600 020, Tamil nadu, India

Received 4 August 2005; revised received and accepted 2 December 2005

Cotton yarns coated with chitosan have been prepared by the oxidation of cotton yarns with sodium periodate at 60°C in water and subsequent treatment w ith a solution of chitosan in aqueous acetic acid. Infrared spectra of the chitosan-coated cotton yarn show the formation of Schiffs base between the chitosan and the oxidized cellulose. The chitosan-coated yarns have been further immobi l ized with tetracyc l ine drug and the effect of drug concentration and treatment time on drug release characteristics and antimicrobial activity studied. The study shows good drug release characteristics and antimicrobial activity against E.co/i and S. aureus.

Keywords: Chitosan , Coated yarn, Cotton, Drug release, Sodium periodate, Tetracycline hydrochloride IPC Code: Int. CI.8 A6 1 L l 5/00, D0683/00

1 Introduction Textile materials are used for the appl ications in

medical field as sutures and as wound dressings. Nowadays, wound closure has become a critical part in surgery and postoperative procedures. The surgeon ' s skill and the patien t ' s condit ion are not the only factors, which ensure successful outcome; i t also depends on the qual i ty of wound closing material because there is every possibi l i ty of bacterial invasion at the i nfected si te. A wound dressing material which is effective against bacteria will be an ideal material for these kinds of appl ications wi thout losing their i nherent textile characterist ics. The textile substrates used as wound closing materials should act as reservoir of antimicrobial agents and should release them gradually at the affected s ite for prolonged period of t ime.

Efforts have been put by researchers to provide such kind of textile substrates by incorporation of antimicrobial agents while producing the fibre l -3, coating with antimicrobial agents4-6 and by incorporating them US111 g surface modification techniques.7-JO

Chitosan, a polysaccharide obtained by alkaline de­acetylation of chit in from skeletons of crustaceans and arthropods, has attracted considerable interests due to

"To whom all the correspondence should be addressed. E-mai l : vrgiridev@yahoo.com

i ts biological properties such as antimicrobial, anti tumour and i mmunological effects. Chitosan, being non-toxic and biodegradable, finds application _

i n waste water treatment, textile and other medical appl ications. I I

In texti le applications, chitosan i s used as biodegradable sutures, antimicrobial agent, dye-fixing agent and as a finishing chemical. 1 2- 1 6 The usage of chitosan 111 biomedical application has drawn considerable attention due to i ts antimicrobial properties. Chitosan inhibits the growth of bacteria by inducing the leakage of i ntra- cellular components due to i ts poly-cationic nature. 1 7

The present investigation i s aimed at evaluating the drug loaded chitosan cotton yarns for their drug release characteristics and antimicrobial acti vity against E.coli and S.aureus. Cotton yarns coated with chitosan fol lowed by i mmobilization of drug have been used aiming at two layers of defense at the i nfected si te.

2 Materials and Methods

2.1 Materials

Scoured cotton yarns ( 1 1 12 Ne) were used for the study. Chitosan of N-degree of deacetylation of 0.82 (T]=300 cps), obtained from Central Institute of Fisheries Technology, was used as such for coating of the yarns. Commercial ly available tetracycline

544 INDIAN J. FIBRE TEXT. RES. , DECEMBER 2006

hydrochloride drug used for periodontal d iseases was purchased from the market and used without further purifi cation.

2.2 Methods

2.2.1 Oxidatioll of Cotton Yam With SodiulII Periodate

Scoured cotton yarn was immersed i n a solution of sodium periodate in deionized water (400 ml) at concentration of 1 .0 mg/m!. The solution was then stirred for I h at 60°C. The cotton yarn was taken out, washed with deionized water several t imes to remove the oxidant, and finally soaked in deionized water (400ml) at an ambient temperature. This oxidized material was used for the next reaction without drying as suggested earlier.6

2.2.2 Treatmellt with Chitosall

The chi tosan solution was prepared by stlrnng a d ispersion of chitosan (2.0 g) in 2 .0% (v/v) aqueous acetic acid solution ( 100 ml) for 1 h at 60°C .The above-mentioned oxidized cotton yarn was i mmersed in the chitosan solution with constant st irring for 2 h at 60°C, washed with deionized water several times and dried at 60°C for 25 min to produce the drug carrier cotton yarn as suggested earli er.6

2.2.3 1mmobilizatioll of Tetracyclille Hydrochloride Drug

The cotton samples (0.3 g) were immersed separately i n 5% and 1 0% (w/v) aqueous solutions of tetracycl ine hydrochloride. The solutions were than allowed to remain stand still for 1 2 h for one set of samples and 24 h for another set of samples. Finally, the samples were taken out, washed with dist i l led water and dried at room temperature for two days. The drug add-on (%) on the sample was calculated using the following relationship:

where WI is the weight of drug immobil ized cotton yarn; and W2, the weight of unimmobilized cotton yarn. The drug add-on of various samples taken for the study are given i n Table 1 .

2.2.4 Drug Release Studies

The drug-loaded samples (0.3g with respective drug add-on) were taken as such and suspended in 1 0 ml normal water for 24 h at room temperature. After 24 h, the supernatant from the sample tubes was tested for drug concentration using H i tach i UV­visible spectrophotometer (U32 1 O Japan) . The sample

Table 1- Drug add-on i n cotton yarn samples

Sample Drug Treatment Drug add-Oil concentration t ime %

% h Cotton yarn (C) 5 1 2 73

5 24 89 1 0 1 2 87 1 0 24 98

Chitosan-coated 5 1 2 1 25 cotton yarn (CC) 5 24 1 47

i O 1 2 155 1 0 24 1 66

tubes were replenished wi th fresh water, which was tested for drug concentration the next day. The entire procedure was continued for days t i l l measurable release of the drug continued. Standard calibration plots were drawn to measure the amount of drug released during each day.

2.2.5 Antimicrobial Test

The chitosan-coated cotton yarn, drug-i mmobi l ized yarn and drug-released yarn were tested for antimicrobial activities against E.coli and S.aureliS according to 'agar diffusion test' and 'shake flask test ' . Immobil ized ant imicrobial agents, such as surface-bonded materials, are not free to diffuse into their environment under normal conditions of use. Thi s dynamic shake flask test was developed for routine qual i ty control and screening tests in order to overcome difficulties when usmg classical antimicrobial test methods to evaluate substrate­bound antimicrobials . 1 8

This standard method was used t o measure the reduction rate in the number of colonies formed and provided quantitative data, which could then be converted to the average colony forming units per mil l i l i ter (CFU/ml) of buffer solution in the flask. The reduction rate in the number of colonies was calculated using the following formula:

Reduction rate (%) = [(B-A)/BJ x 1 00

where A i s the CFU/ml for the flask containing the treated substrate after 3 h and 6 h contact time; and B, the CFU/ml for the flask at t ime zero prior to the addition of the treated substrate.

The test tube was shaken at 37°C for 6 h on a rotary shaker and 1 00 III of the serially di lute test solution was added to the agar plate which was incubated at 37°C for 24 h. The number of colonies in the agar plate was counted. A sample photograph of agar plates used for the study i s shown Fig. 1 .

SHANMUGASUNDARAM ef 01. : ANTIMICROBIAL STUDIES ON C HITOSAN-COATED COTION YARNS 545

Fig. I - (a) In i t ia l samples showing the growth of bacteria and (b) agar plates showing 1 00% reduction in bacterial growth

2.2.6 FTlR and SEM Studies

Fourier transform infrared (FTIR) spectroscopy studies on samples were carried out on Horiba FT-2 1 0 system; the spectra were recorded i n the transmission mode. The surface characteristics of the uncoated and coated samples were studied using scanning electron microscope (Hitachi S-2400). A thick layer of s ilver coating was provided for conduction.

3 Results and Discussion

3.1 Chitosan-coated Cotton Yarn

Chitosan is the second most abundant biopolymer available on the earth next to cellulose. Chitosan has excellent biological properties and is being used profoundly in medical field because of its biodegradabi l i ty , biocompatibil ity, antimicrobial activity, non-toxic i ty and versat i le chemical and physical properties. I n this study, chitosan was applied for it to be second l ine attack against microbes if the drug immobil i zed is exhausted (Fig. 2) . It has been shown by various researchers that the chitosan can be bonded to the polymer substrate by oxi dation using sodium per iodate.6 According to the procedure as described earlier6, the cotton yarn was first oxidized by sodium periodate to c leave the 2, 3-vicinal diol of the cellulose glucose uni ts ( I ) , g iv ing the dialdehyde cellulose ( I I ) (Fig. 3). The resulting aldehyde group on the cellulose fibre would possess the abi l i ty to couple wi th an amino group of chitosan. Treatment of the cotton yarn with a chitosan solution in acetic acid would result in the formation of an Schiffs base.

This was further confirmed by infrared studies. Fig. 4 shows the spectra of cellulose, cellulose oxidized with sodium periodate and chitosan-coated cotton yarn. The characteristic absorption band of the oxidized cellulose clearly appeared at 1 738 cm' l due

Cotton yarn

Even i f Drug Exhausts ­chitosan polymer in the yarn acts as second l i ne of defense ( antimicrobial i n nature)

Chitosan Coated Cotton

Yarn

Infected Site

Fig. 2 - Mechanism of action of modified yarn at the infected site

I I

I I I

Fig. 3 - Chemical reaction between oxidized cellulose and chi tosan6 [ I - cel lulose g lucose unit; I I-dialdehyde cellu lose and I I I- chitosan-coated cotton 1

97·UL_--'-_L...---'-_-'-_.L---L_.....L..._-'----L_� 1800 1760 1720 1680 1640 lEO)

Fig. 4 - I nfrared spectra of (a) uncoated , (b) oxidized and (c) chitosan-coated cotton yarns

546 I NDIAN J. FIBRE TEXT. RES., DECEMBER 2006

to the stretching vibration of C=O (double bond of the aldehyde group). After treatment with chitosan, the characteristic absorption band shifted to 1 725- 1 720 cm' l region, suggesting the formation of Schiffs base (C=N double bond) between the aldehyde group and the amino group of chitosan. The SEM photographs of the uncoated and chitosan-coated cotton yarns are presented i n Fig .5 . The coated yarn shows the presence of chitosan on the substrate whereas i n case of uncoated yarn the surface of the substrate i s smooth. The deposition of chitosan on the surface i ndicates that the surface characteristics such as friction are bound to vary and further studies has to be carried out to confirm thi s argument.

3.2 Drug Release Studies

Tetracycline hydrochloride is primarily bacteriostatic and it exerts its antimicrobial effect by the inhibition of protein synthesis. It is active against a wide range of gram posit ive and gram negative organi sms and hence chosen as drug for this study. The drug was immobil ized on the chitosan-coated yarn as mentioned earlier and the drug release behaviour was studied.

The amount of drug released from the chitosan­coated cotton yarn is given in Figs 6 and 7. It can be observed from the figures that the drug imbibed on the surface of the cotton yarn is released qui cker initially and at the end of seventh day of the trials, the yarn is left with residual drug and chi tosan. This indicates that there will be antimicrobial activity shown by the yarn at the infected site even after seven days. This makes the yarn for its usage in a number of wound healing applications as sutures and drug delivery medium in case of oral diseases. Moreover, it can be seen from the figures that with the i ncrease i n treatment t ime from 1 2h to 24 h, more amount o f drug gets imbibed on the surface, but the release trend in

Fig. 5 - SEM photographs o f (a) uncoated, and (b) chitosan-coated cotton yarns

both the cases is s imilar. When compared with uncoated yarns, the amount of drug absorbed on chitosan-coated yarn is more due to the polycationic nature of chitosan.

3.3 Antimicrobial Activity

As such, chitosan-coated cotton yarn, drug-loaded yarn and drug-released yarn (after seven days) were taken for antimicrobial effect against E.coli and S.aureus bacteria by agar diffusion test and shake flask test for varying concentration of drug and treatment time. It can be seen from Table 2 that for agar diffusion method, the zone of inhibition is greater than 2 cm (initial chitosan-coated sample) for all samples. The high zone diameter indicates that the drug is easily released on contact with the nutrient broth.

The samples were also tested for their antimicrobial activity using shake flask method and the results are g iven i n Table 2. 1 00% reduction in bacterial population is observed even after seven days for al l concentrations and treatment t imes for chitosan­coated samples. However, uncoated yarns immobilized with drug show negligible activity against bacteria after seven days.

-0 :\l

200 '"'

-+- C(73%) " " 150 -- CC( 1 25%) 0:: ;n� -+- C(87%) � t;l) 100 -- CC ( l 55%) -o E ,... � 0 C 50 " 0 E -< 0

II 1II IV V VI VII

Days

Fig. 6-Amount of drug released (C - cotton yarn treated with 73% & 87% drug add-on, and CC - chitosan-coated yarn treated with 1 25% & 1 55% drug add-on) for a period of 1 2 h

250 -+- C(89%) --- CC(l47%) -+- C(98%) -- CC( l66%)

II III IV V VI VII Days

Fig. 7 - Amount of drug released (C - cotton yarn treated with 89% & 98% drug add-on, and CC - chitosan-coated yarn treated with 1 47% & 1 66 % drug add-on) for a period of 24h

SHANMUGASUNDARAM et al.: ANTIM ICROBIAL STUDIES ON CHITOSAN-COATED COTTON YARNS 547

Table 2-Antimicrobial testing by agar d iffusion method and shake flask method

SI.No Sample description" Zone of i nhibition, cm Reduction in bacterial population , % (Al:lar diffusion method) (Shake flask method) E. coli S. aurellS E. coli S. aurellS

C(5%, 1 2h, 73%) 2. 1 2 .3 1 00 100 2 C (5%, 24h, 89%) 2 .5 2 .7 1 00 1 00 3 C ( l 0%, 1 2h, 87%) 2.7 3.0 1 00 100 4 C ( l0%,24h ,98%) 2 .4 2.5 1 00 100 5 CC(5%, 1 2h , 1 25%) 2.7 3.0 1 00 1 00 6 CC(5%,24h , 1 47%) 2 .6 2 .5 1 00 1 00 7 CC( l 0% , 1 2h, 1 55%) 2.6 2.6 1 00 1 00

8 CC( 1 0%,24h , 1 66%) 2.7 2.5 1 00 1 00 9 C (5%, 1 2h, 73%) Nil N i l 8 1 0

1 0 C (5%, 24h, 89%) Ni l N i l 7 9 I I C ( 10%, 1 2h, 87%) N i l Ni l 10 1 2 1 2 C ( 1 0%, 24h, 98%) N i l N i l 14 I S

1 3 CC(5%, 1 2h, 1 25%) 2.7 2.5 1 00 1 00 1 4 CC(5%, 24h, 1 47%) 2 .6 2 .6 1 00 1 00 1 5 CC( 1 O%, 1 2h, 1 55%) 2.7 2.5 1 00 1 00

1 6 CC( 1 0%,24h, 1 66%) 2 .6 2.6 1 00 1 00

1 7 Chitosan-coated yarn 2.0 2.0 1 00 1 00

1 8 Cotton yarn Ni l N i l N i l N i l

"Values in parentheses i ndicate the concentration of drug i n aqueous solution, treatment t ime and drug add-on. Serial nos 1 -8 show antimicrobial activity of samples taken i nit ial ly. Serial nos 9 - 1 6 show antimicrobial activity of samples taken after 7 days.

Most of the surgical yarns and wound dress ing materials are kept at the infected site for 6-8 days. A wound dressing material which offers resistance for growth i n this period can be considered for usage i n thi s kind of appl ications. The study shows that the cotton material is l eft with res idual drug and chitosan coating. Even if the drug on the surface of the substrate is exhausted chitosan coating on the surface offers the second l ine of defense against bacteria (chitosan i s known for wound heal i ng and ant imicrobial nature), thereby offering the medical personnel to use for substrates which needs to be antimicrobial nature for prolonged time period.

4 Conclusions Drug release studies show that there is a residual

drug left on the substrate even after seven days. The cotton yarns show 1 00% antimicrobial activity against bacteria E.coli and S.aureus even after seven days. The yarns developed may find appl ications where textile substrates are being used as medium for drug release such as in case of periodontal d iseases. Further cl inical i nvestigations have to be carried out to study

the usage of the developed substrate for i ts oral application.

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