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 MadhusoothananDepartment of Textile Technology, A C College of Technology, Anna University, Chennai 600 025, Tamilnadu, India and
G Suseela Rajkumar
Department of Bacteriology, Central Leather Research Institute, Chennai 600 020, Tamilnadu, 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 with 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 lized with tetracycline 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 applications 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 patient's condition are not the only factors, which ensure successful outcome; it also depends on the qual i ty of wound closing material because there is every possibility of bacterial invasion at the i nfected site. A wound dressing material which is effective against bacteria will be an ideal material for these kinds of applications without losing their i nherent textile characteristics. 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 time.
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 de
acetylation of chitin from skeletons of crustaceans and arthropods, has attracted considerable interests due to
"To whom all the correspondence should be addressed.
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 applications. I I
In textile applications, chitosan i s used as biodegradable sutures, antimicrobial agent, dye-fixing agent and as a finishing chemical. 12-16 The usage of chitosan 111 biomedical application has drawn considerable attention due to its antimicrobial properties. Chitosan inhibits the growth of bacteria by inducing the leakage of i ntra- cellular components due to its poly-cationic nature. 17
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
andS.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 site.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. Commercially available tetracycline
544 INDIAN J. FIBRE TEXT. RES., DECEMBER 2006
hydrochloride drug used for periodontal diseases 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 i m mersed 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 times 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 chitosan solution was prepared by stlrnng a dispersion of chitosan (2.0 g) in 2.0% (v/v) aqueous acetic acid solution ( 1 00 ml) for 1 h at 60°C .The above-mentioned oxidized cotton yarn was i mmersed in the chitosan solution with constant stirring 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 earlier.6
2.2.3 1mmobilizatioll of Tetracyclille Hydrochloride Drug
The cotton samples (0.3 g) were i m mersed separately i n 5% and 1 0% (w/v) aqueous solutions of tetracycline 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 distilled 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 immobilized 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 UV
visible spectrophotometer (U32 1 O Japan). The sample
Table 1- Drug add-on in cotton yarn samples
Sample Drug Treatment Drug add-Oil
concentration time %
% 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 with fresh water, which was tested for drug concentration the next day. The entire procedure was continued for days till 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 mmobilized yarn and drug-released yarn were tested for antimicrobial activities against
E.coli
andS.aureliS
according to 'agar diffusion test' and 'shake flask test' . Immobilized antimicrobial 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 quality control and screening tests in order to overcome difficulties when usmg classical antimicrobial test methods to evaluate substrate
bound 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 milliliter (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 time 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 dilute 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) Initial 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 silver 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 biodegradability, biocompatibility, antimicrobial activity, non-toxicity and versatile chemical and physical properties. I n this study, chitosan was applied for it to be second line attack against microbes if the drug immobilized 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 cleave the 2, 3-vicinal diol of the cellulose glucose uni ts (I), giving the dialdehyde cellulose (II) (Fig. 3). The resulting aldehyde group on the cellulose fibre would possess the ability to couple with 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
Cotton yarn
Even if Drug Exhausts chitosan polymer in the yarn acts as second line of defense ( antimicrobial in 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 chitosan6 [ I - cellulose glucose unit; I I-dialdehyde cellulose and I I I- chitosan-coated cotton 1
97·UL_--'-_L...---'-_-'-_.L---L_...L..._-'----L_�
1800 1760 1720 1680 1640 lEO)
Fig. 4 -Infrared spectra of (a) uncoated , (b) oxidized and
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 o f chitosan o n 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 anti microbial effect by the inhibition of protein synthesis. It is active against a wide range of gram positive and gram negative organi sms and hence chosen as drug for this study.
The drug was i m mobilized on the chitosan-coated yarn as mentioned earlier and the drug release behaviour was studied.
The amount of drug released from the chitosan
coated 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 time from 1 2h to 24 h, more amount o f drug gets imbibed on the surface, but the release trend in
Fig. 5 - S E M photographs o f (a) uncoated, and (b) chitosan-coated cotton yarns
both the cases is similar. 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
andS.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 given i n Table 2. 1 00% reduction in bacterial population is observed even after seven days for all concentrations and treatment times for chitosan
coated samples. However, uncoated yarns immobilized with drug show negligible activity against bacteria after seven days.
-0 :\l 200
'"' -+- C(73%)
"
" 1 5 0 -- 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 diffusion 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 1 00
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 1 00
4 C ( l0%,24h ,98%) 2.4 2.5 1 00 1 00
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 Nil 8 1 0
1 0 C (5%, 24h, 89%) Nil Nil 7 9
I I C ( 10%, 1 2h, 87%) Nil Nil 10 1 2
1 2 C ( 1 0%, 24h, 98%) Nil Nil 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 Nil Nil Nil N i l
"Values in parentheses i ndicate the concentration o f drug i n aqueous solution, treatment time and drug add-on.
Serial nos 1 -8 show antimicrobial activity of samples taken i nitially.
Serial nos 9 - 1 6 show antimicrobial activity of samples taken after 7 days.
Most of the surgical yarns and wound dressing 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 applications. The study shows that the cotton material is l eft with residual drug and chitosan coating. Even if the drug on the surface of the substrate is exhausted chitosan coating on the surface offers the second line of defense against bacteria (chitosan i s known for wound heali ng and antimicrobial 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
andS.aureus
even after seven days.The yarns developed may find applications where textile substrates are being used as medium for drug release such as in case of periodontal diseases. Further clinical i nvestigations have to be carried out to study
the usage of the developed substrate for its oral application.
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