Approach To Fabricate a Compact Cotton Patch without Weaving: A

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A Novel Approach to Fabricate Compact Cotton Patch Without Weaving: A Smart Bandage Material Achyut Konwar, Raghuram Kandimalla, Sanjeeb Kalita, and Devasish Chowdhury ACS Sustainable Chem. Eng., Just Accepted Manuscript • Publication Date (Web): 05 Apr 2018 Downloaded from http://pubs.acs.org on April 5, 2018

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ACS Sustainable Chemistry & Engineering

A Novel Approach to Fabricate Compact Cotton Patch Without Weaving: A Smart Bandage Material Achyut Konwara, Raghuram Kandimallab, Sanjeeb Kalitab and Devasish Chowdhurya* a

Material Nanochemistry Laboratory, Physical Sciences Division and

b

Drug Discovery

Laboratory, Life Sciences Division, Institute of Advanced Study in Science and Technology, Paschim Boragaon, Garchuk, Guwahati, 781035, India. E-mail: [email protected]; Fax: +91 361 2279909; Tel: +91 361 2912073 KEYWORDS: cotton, compact absorbent patch, smart bandage material, graphene oxide, curcumin.

ABSTRACT: In this present work, we are reporting a novel approach for fabrication of a kind of highly absorbent, highly stable, flexible and compact bio-polymeric hydrogel bounded cotton patch. This novel technique not only exempts the spinning of cotton yarn and use of loom or other kinds of weaving machines but also makes it possible to get a soft but mechanically robust textile material. The mechanical strength, softness as well as the flexibility of such patches can be tuned just by varying the concentration of the bio-polymer. Incorporation of graphene oxide in such patches not only improved the mechanical strength, but also added the antimicrobial property to the patches. These patches are even stable in presence of aqueous medium. Most importantly the polymeric matrix hydrogel part, present over the surface of cotton fibers creates

ACS Paragon Plus Environment

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the possibility of loading different kinds of drugs, micro or nanomaterials, different chemicals etc. for numerous applications. With this hydrogel coated cotton patch we finally came out with a bandage material of complete package with the minimal number of ingredients viz. graphene oxide and curcumin, specially designed to work on infected wounds.

INTRODUCTION Minute observation of natural phenomena’s can sometimes become a great source of ideas and inspiration for the development of wonderful technologies. Many advanced technologies of human civilization are the outcome of mimicking the nature. This mimicking nature has also been reflected by the scientific reports coming out from the research and developments going on worldwide in the past decades1-5. In this context, we are going to introduce a new idea to develop a kind of highly absorbent, stable, robust cotton patch following the mechanism by which a silk-worm makes its cocoon. The cocoon is composed of the fibroin protein fibers coated with another kind of protein called sericin, which also acts as an antimicrobial adhesive matrix to make a compact structure of macroscopic protein fibers6. The present novel approach leads to the fabrication of a highly absorbent compact cotton patch by binding the fibers of a piece of well-stacked cotton wool with a chitosan-based soft hydrogel system which has the interesting self-healing ability7,8. This novel technique not only exempts the spinning of cotton yarn and use of loom or other kinds of weaving machines but also makes it possible to get a mechanically robust textile material with high absorption capability. Another most interesting advantage of such a patch is that any drug, chemical or nanomaterial can be loaded in it during the time of its fabrication or after fabrication as well. The absorption capability of the hydrogel matrix coating on the cotton fiber surface facilitates the post fabrication loading of chemicals, drugs or nanomaterials. Cotton is a soft, absorbent and


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breathable natural fibre, making it the perfect fibre for clothing and undergarments worn close to the skin and contains cellulose amounts to as much as 96% of the dry weight of the fibres. Cellulose is a poly-disperse polymer of high molecular weight, typically 6 x 105-1.5 x 106, comprising long chains of α-D-glucose units joined by β-1,4-glycosidic links9. Chitosan, the cationic (1-4)-2-amino-2-deoxy--D-glucan is industrially obtained from partly acetylated chitin. It is the only pseudo-natural polymer which can be applied as solution, film, fiber and even in hydrogel form and also widely used in biomedical applications7. Thus prepared hydrogel bound cotton patches were further incorporated with Graphene oxide (GO), a well-known nano-filler for improvement of mechanical property along with the added advantage of having antimicrobial activity. Moreover, graphene oxide (GO), chemically exfoliated from oxidized graphite is considered as a promising material for biological applications

owing

to

its

excellent

aqueous

processability,

amphiphilicity,

surface

functionalization ability, surface enhanced Raman scattering (SERS) property and fluorescence quenching ability. GO is dispersible in an aqueous medium and proven to be non-cytotoxic at low concentration although it has some adverse effect at high concentration. These antibacterial GO and other graphite based materials act through physical damages upon direct contact with bacterial membranes by sharp edges of graphene sheets8,10-13. At last, with the aim to come out with an effective bandage material specially for the infected wounds we introduced curcumin. Curcumin (Diferuloylmethane) is a naturally occurring phytochemical polyphenol derived from the rhizome of turmeric (Curcuma longa), a traditional medicine widely and popularly used in different parts of the world from the ancient days for treatment of several kinds of diseases including wounds and burns. Curcumin is considered to be beneficial against oxidative damage, due to its antioxidant properties, and is


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already been proven to be helpful to the better healing of the wound. There are several reports with in vitro and in vivo studies which demonstrate the effectiveness of curcumin in decreasing the release of inflammatory cytokines like interleukin-8 and tumor necrosis factor-α from monocytes and macrophages. Moreover, it has been shown to inhibit enzymes associated with inflammation, such as cyclooxygenase and lipoxygenase. Along with the anti-inflammatory activity, curcumin has the ability to scavenge free radicals, which is the major cause of inflammation during wound healing activity. From the experimental data using animal models, recently it has been established that treatment of curcumin may assist wound healing by increasing

the

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neovascularization. Curcumin has the extra advantage with its proven antimicrobial activity14-19. In short, this work is an endeavour to establish a method of fabrication of a soft, highly absorbent, mechanically stable compact patch from raw cotton using an interesting biopolymeric hydrogel system having the self-healing ability. This work also studies the tuning of mechanical, structural and absorption properties of the patches introducing graphene oxide (GO) in to the patches. Introduction of GO in to the patches makes them antimicrobial in nature. Finally loading of curcumin makes such patches a very effective bandage material with excellent wound healing ability which has been studied in detail.

EXPERIMENTAL Materials used. Bio-polymer Chitosan (M.W. ~ 1,93,400) was purchased from SRL, India. We used absorbent cotton wool obtained from ‘Perfect Surgicare’, India. Graphite nanopowder was obtained from SRL, India. Other chemicals viz., glycerol, acetic acid, sodium hydroxide, nitric acid and sulphuric acid were purchased from Merck India and used as obtained.


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Preparation of graphene oxide. Graphene oxide was synthesized by oxidation and exfoliation of commercially available graphite nano powder, following an already reported method8. In this simple and single step method, 0.1 g graphite nanopowder was dispersed in 40 mL of 3:1 acid mixture of concentrated H2SO4 (98 wt%) and HNO3 (16 M). This mixture was then sonicated in a bath sonicator for 24 h followed by dilution with Milli-Q water. The graphene oxides formed were collected by centrifugation and washed with Milli-Q water for several times to remove all the acids. Then, these graphene oxide nano platelets were again dispersed in the required amount of Milli-Q water and sonicated with the help of an ultra-probe sonicator. Fabrication of compact chitosan hydrogel bound cotton patch. The bio-polymer chitosan was dissolved in a 0.1 M acetic acid solution containing 20% glycerol by volume. A slice of nicely packed cotton wool of weight 1.5 g (of size ⁓ (6x6) cm2) was then soaked in 30 mL of that solution. The volume of the solution was adjusted in such a way, which was just the maximum volume level-the slice of cotton could absorb inside it. Then the piece of cotton wool wetted with the chitosan solution was dried in a vacuum oven at 70 oC. The dried piece of cotton slice was soaked in a crosslinking bath containing 1N NaOH solution for 2 hours. The chitosan hydrogel coated piece of cotton wool was taken out from the crosslinking bath and washed for several times with distilled water. This was followed by the compression with the help of a mini lab scale compression moulding machine for 5 minutes applying a pressure of 10 tons. The compressed hydrogel bound cotton patch was then washed with distilled water and allowed to dry again at 70 oC. Here we fabricated cotton patches with 2, 5 and 20 % of chitosan with respect to the weight of the cotton and toward this end 0.1, 0.25 and 1% (W/V) chitosan solutions were used.


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Fabrication of compact chitosan-graphene oxide nanocomposite hydrogel bound cotton patch. Chitosan-gaphene oxide nanocomposite hydrogel bound cotton patch was fabricated by following the similar procedure described for compact chitosan hydrogel bound cotton patch. First the required amount of graphene oxide (GO) dispersion was added to a solution of chitosan dissolved in 0.1 M acetic acid solution containing 20% glycerol by volume. The required volume of that solution was then allowed to absorb by the piece of cotton wool and dried it in hot air oven at 70 oC. The dried piece of cotton slice was then cross-linked by soaking in the 1N NaOH solution for 2 hours followed by washing and compression for 5 minutes at pressure 10 tons. The compressed chitosan-graphene oxide nanocomposite hydrogel bound cotton patch was then washed and dried again at 70 oC. Loading of curcumin in the hydrogel bound cotton patch. 5 mg of curcumin dissolved in minimal amount of ethanol was allowed to absorb by the cotton patches of 2 cm diameter each. The absorption of the curcumin solution in to the cotton patches was done by repeated absorption and then drying process until the whole curcumin solution get absorbed by the patch. We followed this procedure to load curcumin so that we can get maximum curcumin loading on the outer surface of the patches. So curcumin is mostly loaded in the chitosan hydrogel coating part on the surface of the cotton fibers. CHARACTERIZATION The nano material and the cotton patches were characterized by Fourier transform infrared spectrometer (Nicolet 6700 FT-IR) to investigate different types of interactions and functional groups present. Scanning electron microscope (SEM) images were collected on a ‘Carl Zeiss ∑igma VP’ instrument. To study the mechanical properties of the fabricated compact


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hydrogel bound patches we used a universal testing machine (‘Tinius Olsen 5ST’ model) with a load cell of 2.5 KN capacity. The speed of the testing was maintained at 5 mm/min. Antimicrobial property study. Evaluation of the antimicrobial activity of the patches, was done by direct contact based method and also monitored the killing kinetics. Evaluation of the antibacterial activity was performed against Staphyllococcus aureus (S. aureus) (ATCC 25923) and Escherichia coli (E. coli) (MTCC 40). Direct contact method. UV-sterilized bandage materials were subjected to antibacterial activity analysis. The test materials (sample size 1 cm2) were incubated in separate wells (24 well microtiter plate) having 2 mL of nutrient broth containing S. aureus and E. coli at concentration of 107 CFU/mL. After, 48 hrs of incubation at 37 oC, 200 µL aliquots were collected and inoculated in to the nutrient agar plates. After the stipulated incubation period, the culture plates were photographed for enumeration of bacterial colony forming units. To ensure validity of result, tests were conducted in triplicate. Time kill assay. To determine the rate at which the fabricated bandages materials killed the tested bacteria, kill test was performed. Overnight grown cultures of the tested bacteria were washed twice in PBS (centrifugation speed 12000×g); maintained a final concentration of approximately 2 × 105 CFU/mL in nutrient broth and then incubated along with the bandage materials (1 cm2) at 37 oC with shaking. 0, 2, 4 and 6 hour of post treatment, samples were withdrawn and analyzed to enumerate the number of viable bacteria. The CFU of both the test bacteria were compared and plotted as the CFU/mL over time post treatment. Independent experiments were performed in triplicates.


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In-vivo wound healing study in animals. Male wistar rats weighing between 180-200g were procured from animal house at Institute of Advanced Study in Science and Technology (IASST), Guwahati, Assam. All the animals were housed for minimum of five days for acclimatization to laboratory environment before the initiation of the experiment in polypropylene cages. All the animals were fed with standard rodent pellet diet obtained from Provimi Animal Nutrition Pvt. Ltd., India and water ad libitum throughout the experimental period. The experimental room was maintained as per the standard guidelines (Temperature: 22 ± 2 0C; Relative humidity: 60–70%; 12 h–12 h light–dark cycle). All the experimental were carried during 09:00 and 17:00 h. The experimental protocol was approved (IASST/IAEC/2015-16/754) by institutional animal ethical committee (IAEC) of IASST, Guwahati which have approval from Committee for Control and Supervision of Experimentation on Animals (CPCSEA). Wound induction. Rats were anaesthetized with ketamine hydrochloride 80 mg/kg and xylazine 10 mg/kg cocktail by intraperitoneal (i.p.) injection. The dorsal region of the rats was shaved to remove the fur and sterilized the surgical area with povidone iodine solution. Further, 1.5 cm x 1.5 cm diameter wounds were created with serial scalpel and covered with sterile gauge. Wound infection and treatment. After 24 h of surgery, wound of the animals were infected with Staphylococcus aureus. Briefly-30 µL suspension of S. aureus containing 5 x 108 CFU/mL inoculated on the wounded area of mildly anesthetized (Ether) animals. Grouping. After three days of observation period, animals were divided randomly into five groups (n=6) for the treatment. Group-I. Rats with wound + No infection and treated with “chitosan hydrogel coated cotton bandage”..


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Group-II. Rats with wound + S. aureus infection and treated with “chitosan hydrogel coated cotton bandage”. Group-III. Rats with wound + S. aureus infection and treated with “chitosan-GO nanocomposite hydrogel coated cotton bandage”. Group-IV. Rats with wound + S. aureus infection and treated with “chitosan hydrogel coated cotton bandage with curcumin loading”. Group-V. Rats with wound + S. aureus infection and treated with “chitosan-GO nanocomposite hydrogel coated cotton bandage with curcumin loading”. CFU count determination. Efficacy of the prepared bandage material on the S. aureus infected wounds was evaluated on the basis of CFU count determination on various time intervals. Briefly-on 3rd, 7th, 14th & 21st day of treatment period, wound samples were excised and homogenized in sterile phosphate buffer saline. 100 fold tissue homogenate dilutions were plated on nutrient agar plates to enumerate the CFU count after 24 h of incubation at 37 ± 2 oC. Based on the excised tissue weight all the results were normalized. Blood and tissue harvesting. Blood was collected from the animals at 7th, 14th & 21st day of post treatment through retro orbital route for the measurement of cytokine levels. At the end of the study i.e. 21st day, all the animals from each group were sacrificed and wounded area from two animals was collected in 10% buffered formaldehyde to proceed for histopathology analysis. Wounded area from remaining four animals were collected in liquid nitrogen for the biochemical estimation.


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Measurement of serum inflammatory cytokine levels. Blood samples were homogenized at 1500 rpm for 10 min at 4 oC and the supernatant was collected in separate tubes. Inflammatory cytokines like TNF-α and IL-1β levels were measured by using ELISA kits from R&D systems, USA as per the manufacturer’s instructions. All the samples were performed in the duplicate and results were expressed in Pg/ml. Biochemical estimation-measurement of hydroxyproline. Collagen content in the wound tissue was estimated by measuring the hydroxyproline levels in the wound homogenates. Wound tissues from the different groups were dried in a hot air oven (60 oC) and hydrolyzed in 6N hydro chloric acid for 4h at 130 oC in air tight tubes. The pH of the hydrolysates was neutralized to 7.0 and subjected to Chloramine-T oxidation for 20 min. The reaction was terminated by the addition of 0.4 M perchloric acid. The colour was developed by the addition of Ehrlich reagent at 60 oC and hydroxyproline content was measured spectrophotometrically at 557 nm using UVvisible spectrophotometer (Shimadzu, Japan). Measurement of hexosamine. The wounded tissues were hydrolyzed with 6 N HCL for 8 h at 98 oC and the resulting mixture is neutralized with 4 N NaOH, diluted with Milli-Q water. The diluted solution was heated at 96 oC for 40 min by adding acetyl acetone solution. The reaction mixture was cooled and 96 % ethanol followed by r-dimethylamino-benzaldehyde solution (Ehrlich's reagent) was added. Further the resulting solution was mixed thoroughly and kept at 27 oC for 1 h. The absorbance was measured at 530 nm by using UV-Visible spectrophotometer (Shimadzu, Japan). Hexosamine content was measured by spectral matching with standard curve and expressed as mg/g dry tissue weight.


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Histopathology of wound. Wounded tissue collected in the 10% buffered formaldehyde was preserved at least for 24 h and dehydrated gradually with ethanol from 70 to 100 %. The tissues were further cleared in the xylene and embedded in paraffin. Thin sections (4-5 µm) were prepared by using microtome and stained with hematoxylin-eosin to observe the pathological changes under light microscope at 10x magnification.

RESULTS AND DISCUSSION Cotton fibers were successfully coated by first absorption of a mixture of chitosan and glycerol in a 0.1 M acetic acid medium by a piece of cotton wool followed by cross-linking of this polymeric system on the surface of the cotton fibers by neutralization with NaOH. Figure 1 shows a schematic presentation of the protocol followed for the fabrication of such patches. Chitosan is not soluble in neutral aqueous medium. It dissolves in mild acidic condition and acquires a positive charge. In our case, the zeta


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Figure 1: Schematic representation showing the protocol for fabrication of compact biopolymeric hydrogel coated cotton patches. potential value of a chitosan solution dissolved in 0.1M acetic acid solution is found to be +37.5 mV (Figure S1, in the ESI). Literature says that the NH2 group of chitosan gets protonated to NH3+ group in acidic medium. This positive charge on chitosan is responsible for the solubility of the polymer in acidic medium20,21. The positive charge on each polymer molecules generates an electrostatic repulsive force which makes the polymer chains to go into the solution. It is a well-known fact that any polymer molecule when it goes into solution, disentanglement occurs and become aligned while they may remain in coiled state in solid form22. So, when we again neutralize a chitosan solution initially at lower pH, the chitosan molecules lose their positive charge due to the


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a

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Figure 2: (a) FTIR spectra of cotton (CT), chitosan (CH), chitosan hydrogel coated (CTCH2) and chitosan-GO (CTCH2GO2) nanocomposite hydrogel coated patches (b) SEM images of coated and uncoated cotton fibers and different patches with varying chitosan as well as GO concentration. deprotonation of the NH3+ to NH2. In this state there is no electrostatic repulsive force among the polymer chain molecules and they can easily come close to each other. The molecules when come sufficiently close to one another, they can form different types of secondary interactions like hydrogen bonding, polar-polar interaction etc. But since the polymer chain molecules are in an aligned state in the chitosan solution so they can form a three dimensional network structure with the help of secondary interactions to one another including the glycerol molecules and entrapping water molecules inside the gel structure. The glycerol molecules inside the hydrogel


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matrix helps to form a more uniform hydrogel product compared to the chitosan hydrogel in absence of glycerol (Figure S2, ESI). We have synthesized the chitosan hydrogel film in absence of the cotton wool and the FTIR spectrum of this dried gel film was collected. This FTIR spectrum of the hydrogel (Figure S3, ESI) was compared with that of the dry chitosan. The peak appearing at 3380 cm-1 due to the –OH stretching frequency in the FTIR spectrum for chitosan broadens up in case of the hydrogel indicating extensive hydrogen bonding interaction in the chitosan hydrogel system. FTIR spectrum (Figure 2a) shows a broadening of the peak for –OH stretching (3374 cm-1) in case of the hydrogel coated cotton patch compared to that of bare cotton (3345 cm-1) and chitosan (3380 cm-1) indicating increase in the hydrogen bonding interaction in the coated one. May be because of this same reason, the peak responsible for the C-O-C bond in the cotton fiber (1060 cm-1) shifted to 1048 cm-1 in the hydrogel coated cotton patch. Moreover, appearance of a peak at 1599 cm-1, which is for the N-H stretching indicates the presence of chitosan hydrogel coating on the surface of cotton fibers. The coating of the cotton fibers by the hydrogel matrix system and graphene oxide loaded hydrogel system is vividly perceptible from the SEM images (Figure 2b). Surface morphology of the cotton fibers changes after coating. Just after the cross-linking process of the hydrogel system on the surface of the cotton fibers, the cotton wool was compressed with a compression moulding machine by applying pressure (as shown in scheme, Figure 1). This compression process leads to the formation of a compact structure. After drying, this particular hydrogel system acts as an adhesive matrix due to its self-healing property and holds the cotton fibers close to one another and thus provides mechanical strength to the whole patch. Thus we have fabricated cotton patches with 2, 5 and 20% chitosan concentration with respect to that of weight of the cotton fibers.


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With the aim to improve the mechanical properties of the cotton patches, we incorporated graphene oxide (GO) which have the added advantage of antimicrobial property in the biopolymeric hydrogel matrix. The cotton fibers were coated with chitosan-graphene oxide nanocomposite hydrogel with three different concentration of GO i.e. 0.5, 1 and 2% with respect to the weight of cotton fibers. For convenience of reporting we have coded the patch samples. Thus the chitosan hydrogel coated patch without GO is coded as CTCHx, where x represents the percentage of chitosan; patch samples with GO is coded as CTCHxGOy, where y represents the percentage of GO and x represents the percentage of chitosan. Hence for example, patches with 2% chitosan and having GO concentration 0, 0.5, 1 and 2% will have the codes as CTCH2 (patch without GO), CTCH2GO0.5, CTCH2GO1 and CTCH2GO2. Realising the potential application of such a bio-polymeric hydrogel coated patch as a smart bandage material, we endeavoured to come out with a compact bandage patch specially designed to work effectively on infected wounds. With this aim, ‘curcumin’ was chosen and loaded in the patch of our choice. We want to add that in this work curcumin was taken as model compound for antimicrobial agent. However, as already mentioned any antimicrobial, antifungal or antibacterial molecules can be loaded. For the use as bandage material in animal model we loaded 5mg of curcumin solubilized in ethanol in the hydrogel coated patches of diameter 2 cm2 by the process of repetitive absorption and drying process. The curcumin loaded patches have been coded as CTCHx+Cur (bandage without GO) or CTCHxGOy+Cur (bandage with GO). Mechanical properties of the patches. The mechanical strength in to the patches come because of the hydrogel coating. In their fabrication process, the chitosan hydrogel coated patches are compressed as a result of which the hydrogel coating part on the surface of cotton fibers come in contact with each other. As we already have mentioned that the chitosan hydrogel


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has self-healing ability, so after drying, the coated fibers remains in contact with each other with the help of strong hydrogen bonding8. Thus the hydrogel coating part on the cotton fibers binds the fibers with one another which

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CTCH5

CTCH5GO0.5 CTCH5GO1

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Figure 3: (a) Different states of the patch under stretch untill break experiment in an UTM. Stress-strain profile of the cotton patches with (b) different chitosan concentration, (c) 2% chitosan and varying the GO concentration. Bar diagram showing comparison of tensile strength of different patches with 2% chitosan and different GO concentration (d), 5% chitosan and different GO concentration (e), 20% chitosan and different GO concentration (f) in the dry state as well as in the wet state after soaking in water for 24 hours. eliminates the necessity of weaving and provides the patches their mechanical strength. It was observed that by varying the chitosan percentage, it is possible to tune the tensile strength, flexibility and soft ness of such patches. So, different patches with varying chitosan


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concentration viz., 2%, 5% and 20% possessed an average tensile strength of about 6, 12.5 and 19 MPa respectively. Here it is noteworthy to mention that the thickness of the fabricated patches was within 0.5 to 1 mm. With the increase in chitosan concentration, the thickness of the layer of hydrogel coating on the surface of cotton fibers increases which in turn increases the compactness of the patches as depicted by the SEM images (Figure 2b). Presence of higher amount of hydrogel matrix can fill the gap between the cotton fibers, connects and binds each and every fiber in a more effective way which helps in improvement of the tensile strength of the patches. But on the other hand with the increase in the chitosan concentration flexibility of the patches decreases. After incorporation of GOs in the hydrogel matrix, the tensile strength values seemed to be improved further irrespective of the chitosan concentration present in the patches (Figure 3). It was also observed that 1% is the optimum concentration of GOs, which when loaded in the patches showed the maximum tensile strength value. After incorporation of 1% GO in the hydrogel matrix coating, the maximum average tensile strength values reached to ⁓10, 19 and 27.5 MPa for the patches with 2, 5 and 20% chitosan respectively. GO with its aromatic structure acts as an effective filler for improvement of mechanical property in polymeric nanocomposite systems. Moreover, the presence of polar groups on the surface of GO facilitates good compatibility with the chitosan hydrogel matrix and increases the crosslinking density of the hydrogel matrix system which as a whole enhances the compactness of the patches. But at higher concentration of GOs, the tensile strength value decreases due to the overloading of GO nanomaterials7, 8, 23, 24. The nature of the stress strain curve clearly shows the tough nature of the patches. Since the patches are totally fibrous inside, so they do not possess any sharp breaking point. So we collected the stress-strain profile of the patches up to a minimum force of 15N for each sample. This fibrous system is actually responsible for the tough and flexible nature of such


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patches as a result of which they can be easily bent like any elastic material as per application requirement. The mechanical strength of the patches is seemed to be comparable or even better compared to some already reported polymeric systems for bandage application25,26,27,28. But these patches have the advantages over polymeric systems, because most of the bio-polymeric materials loses their mechanical strength when they come in contact with water because of their solubility in aqueous medium. We also studied the retention of mechanical strength of such patches in presence of water and our observation revealed that almost all the patches retained above 60% of their original strength in the wet condition and even after dipping into the water for 24 hours. This retention of mechanical strength is because of the use of a water insoluble hydrogel matrix system for binding the cotton fibers. Thermostability of the patches. Cotton fiber shows good thermostability in the thermogravimetric analysis (Figure 4). The chemical structure of cotton fibers starts to degrade at around 280 oC. In case of the Chitosan hydrogel coated cotton patches, the first phase of degradation starts at around 170 oC. This phase of degradation is because of the structural breaking of glycerol and side chains of chitosan present in the hydrogel coating part. The second phase of degradation starts at around 270 oC which is maily because of the breakdown of the cotton fibers. The third phase of degradation in all the


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Figure 4: TGA thermogram of the raw cotton fibers as well as the patches with different chitosan as well as graphene oxide concentrations.

patches as well as in the cotton fibers is due the degradation of the aromatic structures7. Evaluation of antimicrobial activity of the patches. Cotton based patches with GO and curcumin exhibited significant antibacterial activity against both pathogenic gram positive and gram negative bacteria, viz., Staphylococcus aureus and Escherichia coli. We carried out the detail antimicrobial activity evaluation of the patches by two different methods. In the first method, after incubating the bandage materials with 107 CFU of test bacteria, the aliquots from the respective culture tubes, were cultivated in nutrient agar plates to tally the number of viable bacterial cell count. Figure 5(a) represents the typical photographs of re-cultivated (107 CFU/mL) E. coli and S. aureus colonies after treatment against four different types of patches (viz. CTCH2, CTCH2GO2, CTCH2+Cur and CTCH2GO2+Cur). Both CTCH2GO2 and CTCH2+Cur patches were seemed to exhibit antimicrobial activity by reducing the bacterial


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colonies on the re-cultivated agar plates after treatment. The study of the time killing assay also shows reduction of CFU count with time for both the pathogenic microorganism (Figure 5(b)). The antimicrobial activity of GO and its mode of action has already been well established by several research group

a

b

8

Medium CTCH2 CTCH2GO2 CTCH2+Cur CTCH2GO2+Cur

CFU/mL

7 6 5 4 3 2 0h

2h

7

4h

6h

Time

8

CFU/mL

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 39

Medium CTCH2 CTCH2GO2 CTCH2+Cur CTCH2GO2+Cur

6 5 4 3 0h

2h

4h

6h

Time

Figure 5: (a) Typical photographs of re-cultivated microorganism colonies for Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) on nutrient agar culture plates, where the concentrations of bacteria seeded onto the patches were 107 CFU/mL (b) CFU count of E. coli and S. aureus at different intervals on treatment with different patch samples.

as well as by our team in our previous report8. Upon direct contact with the microbial cell, the sharp edges of GO nano-sheet results in membrane stress and subsequent superoxide anion independent oxidation, which in turn, led to the oxidation of proteins, lipids, and nucleic acids and ultimately resulted in membrane damage and cell death. On the other-hand curcumin is also a well-known antimicrobial agent and as per experimental report it can induce membrane


20

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permeabilization of bacterial cell and leads to membrane damage and leakage which intern kill the bacteria29. CTCH2GO2+Cur treatment resulted in absence or minimal growth of microorganism colonies; which depicts significant antibacterial activity of the same due to the combined action of both the antimicrobial agents. Evaluation of biocompatibility and cytotoxicity of the patches. We also endeavoured to study the biocompatibility and cytotoxicity of the patches that are going to be used as wound dressing material. This was done by two different methods, viz. haemolytic activity study and MTT essay study. Haemolytic activity study. In biosafety evaluation studies of various biomedical materials, human RBCs have been widely used which constitutes 40-50% (v/v) of human whole blood. In this study we evaluated the effect of the prepared bandage materials on the lysis and morphology of the RBCs. Upon treatment for 1 h, we found that the tested bandage materials (CTCH2, CTCH2GO2, CTCH2+Cur & CTCH2GO2+Cur) did not induced significant hemolysis. The hemolysis percentage of all the tested materials is far below the permeable limit (5%)30 which suggests that the bandage materials are hemocompatible (Figure 6(b)). Particulate state, particle size, surface charge/oxygen content and the extent of exfoliation of GO play a crucial role in hemolysis. Smaller the particles the greater are the hemolysis, whereas GO sheets in aggregated form demonstrate mild hemolysis. In this present study electrostatically adsorbed chitosan


21


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Figure 6: (a) Scanning electron microscopy (SEM) of human blood cells incubating with distilled water (DW), saline water (SW) and different bandage materials viz.; CTCH2, CTCH2GO2, CTCH2+Cur and CTCH2GO2+Cur (b) Percent hemolysis of human blood cell by different types of bandage materials (c) MTT assay study result. covers the GO sheets and abolish the hemolytic potential31. To confirm further, RBCs after treatment with bandage materials were subjected to FE-SEM analysis. Naturally, the shape of


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mature RBCs is like biconcave disk and they are very sensitive to interactions with membraneactive substances. Interaction with CTCH2 & CTCH2GO2 did not distorted the morphology of RBCs, whereas CTCH2+Cur & CTCH2GO2+Cur treatments resulted in slight alteration in shape (Figure 6(a)). These alterations are due to presence of curcumin which is a well-known anti-cancer agent. MTT assay. MTT assay was also performed to explore the cytotoxicity of the prepared bandage materials. CTCH2 and CTCH2GO2 exhibited negligible cytotoxic effects on the mitochondrial activity of mouse L929 fibroblastic cell line. CTCH2+Cur & CTCH2GO2+Cur demonstrated mild effects on the cell viability compared to that of the former two, which might be due to presence of curcumin in both samples (Figure 5(c)). More than 80 % cell viability (⁓92%, 86%, 84% and 82% for CTCH2, CTCH2GO2, CTCH2+Cur and CTCH2GO2+Cur respectively) was found for all the samples after treatment for 72 hours. There are only a few studies could be found on investigations of in vitro cytotoxicity of GO materials. GO is reported to have negligible toxicity towards human alveolar epithelial A547 cells, A549 cells and mouse fibroblast L929 cells8,32. It is also reported that GO substrates are highly biocompatible and possess enhanced genetrans fection efficacy in NIH-3T3 fibroblast33. Though GO is reported as an antimicrobial nano materials it does not induces mammalian cell toxicity. Size, surface charge, surface functional groups, residual precursors and particulate state are the possible parameters to be considered in regard to GO cytotoxicity. Curcumin, one of the components of the prepared bandage materials is a well-known chemotherapeutic anticancer agent and possesses significant wound healing property. It was reported that, unlike other anticancer agent curcumin does not induce any toxicity towards normal cells34. In this present study, mouse fibroblast cells were found to be non-sensitive to CTCH2+Cur and CTCH2GO2+Cur as


23


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concluded from MTT results. The minimal cell death shown by CTCH2+Cur and CTCH2GO2+Cur is may be due to disturbance created by the bandage materials in the physiological environment of incubated cells. Absorption properties of the patches. The patches were found to possess excellent absorption capability when we carried out a time dependent absorption study in water. We calculated the swelling ratio of the patches with the help of the formula given below7,8. Swelling ratio (%) = [(W s - W d)/ W d] x 100% ………………………1 where, Ws and Wd are the weights of the swollen and dried samples, respectively. The time dependant swelling ratio of the samples are shown in Figure 7. The absorption property of the patches is also influenced by the amount of chitosan present in the patches. The patch CTCH2 (i.e. the patch with 2% chitosan) shows the maximum water absorption ability with more than 200% of its original weight (S.R ⁓236%) within 5 sec and after 2 hour, it reaches a saturation absorption with S.R. ⁓280%. The patch CTCH5 and CTCH20 absorbs ⁓180% and 147% within 5 sec with saturation absorption of 195% and 193% after 2 hours respectively. The high absorption capability of the patches is mainly because their porous structure although the hydrogel matrix coating part also has the capability to absorb water to some extent7,8. That is why, the patches with less chitosan content shows higher absorption capacity because of their less compactness compared to the higher chitosan containing patches which is vividly perceptible from the SEM images (Figure 2(b)). Although the absorption property of the patches can be understood from the measurement of swelling ratio (Figure 6), but to provide a direct quantitative idea,


24

Page 25 of 39

300

a

Swelling ratio (%)

Swelling ratio (%)

300 250 200 150 100

CTCH2 CTCH5 CTCH20

50 0

b

250 200 150 CTCH2 CTCH2GO0.5 CTCH2GO1 CTCH2GO2

100 50 0

0S

5s 30s 1m 5m 10m 30m 1h

2h

--

0S

300

c

Swelling ratio (%)

Swelling ratio (%)

300 250 200 150 100

CTCH5 CTCH5GO0.5 CTCH5GO1 CTCH5GO2

50 0 0S

2h

--

5s 30s 1m 5m 10m 30m 1h

d

250 200 150 100 CTCH20 CTCH20GO0.5 CTCH20GO1 CTCH20GO2

50 0

2h

--

0S

Time

5s 30s 1m 5m 10m 30m 1h

2h

--

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300

300

e Swelling ratio (%)

250

5s 30s 1m 5m 10m 30m 1h

Time

Time

Swelling ratio (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


200 150

CTCH2+cur CTCH2GO2+cur

100 50 0

250

f

200 150

CTCH2GO0 CTCH2GO2 CTCH2GO0+cur CTCH2GO2+cur

100 50 0

0S

5s 30s 1m 5m 10m 30m 1h

2h

--

0S

Time

5s 30s 1m 5m 10m 30m 1h

2h

-

Time

Figure 7: Graphical representation of the comparative study of absorption properties of different patches in water with varying chitosan and GO concentration (a, b, c, d) and after loading of curcumin (e). Absorption properties of different patches with 2% chitosan concentration and varying GO concentration in blood plasma (f).


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we want to mention here that a small patch of size (4 × 4 cm2) and thickness ⁓1mm can easily absorb ⁓2 mL (by CTCH2 and CTCH2+Cur) or ⁓ 1.7 mL of water (by CTCH2GO2 and CTCH2GO2+Cur) instantly. This absorption capability is again directly proportionalto the volume of the patches. Thus we can easily increase the absorption capability of the patches by increasing their thickness, without changing the area. The thickness of the patches can be increased just by increasing the thickness of the cotton wool used for their fabrication. As the compactness of the patches increases after incorporation of GO, hence the absorption capability of the GO incorporated patches decreased to some extent, but still they possess very good absorption capability. To test the effectiveness of the patches as bandage material we tried to mimic the real environment of the wound and absorption of the patches were tested against blood plasma. The patches showed very satisfactory absorptivity against blood plasma, although the SR value is only just a little less than water which may be due to the presence of high protein content in blood plasma increasing the viscosity of the whole liquid substance. Wound-healing ability of the patches. We carried out an in-vivo wound healing study for a period of 21 days using four different types of bandage materials, viz., CTCH2, CTCH2GO2, CTCH2+Cur and CTCH2GO2+Cur on S. aureus infected excised wounds in wistar rats. Among the cotton patches, we preferred the patches with chitosan concentration 2% for wound dressing application and test in animal model because of their sufficient flexibility, softness and higher absorption rate. Photographic images of the wounds treated with different bandage materials and their healing with time within the period of observation are shown in Figure 8. A non-infected wound covered with CTCH2 bandage patch is also shown by the images. Interestingly, the infected wounds treated with the CTCH2GO2+Cur bandage patch showed faster and satisfactory wound healing rate along with the hair growth in the wounded area within the days of


26

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observation. This effective wound healing ability of such bandage patches come from GO, a highly active antimicrobial agent and curcumin, the active part of the most popular drug of Ayurveda for wound treatment i.e. ‘Turmeric’. Antimicrobial activity of GO and curcumin along with its faster and effective wound healing ability has already been well established18,19. The combined effect of GO and curcumin makes the CTCH2GO2+Cur bandage patch an excellent smart wound dressing material for the wounds infected by microbes. We confirmed the release of curcumin from the bandage patch in a neutral aqueous medium

b

a

CTCH2

CTCH2

NORMAL

INFECTED

CTCH2GO2

INFECTED

CTCH2+Cur CTCH2GO2+Cur

INFECTED

INFECTED

Day 3

Day 7

Day 14

Day 21

Figure 8: (a) Observation of post-operative wound healing of animal wounds treated with different bandage treatments at various time intervals (b) microscopic images showing the histopathology of the wounds after completion of the period of observation.


27


with the help UV-vis-spectroscopy (Figure S7). Along with fastening the wound healing, such bandage materials also take care of the pus material ejaculated from the degraded wounds with microbial infection by their property of high absorptivity. In addition, such patches with their porous nature (as evident from the SEM images) provide ventilation to the wound which is one of the significant advantages of such patches. It is noteworthy to mention here that although few reports have been found on wound healing ability of curcumin and graphene based bandage materials, but within the limit of our knowledge this is the first report to study the ability of wound healing by a bandage material with curcumin, graphene oxide and the combination of the two on an infected wound. To monitor the healing of the wounds treated with bandages, loaded with different agents, we studied different aspects that indicates the curing of the wounded area.

a 14 12

b

c

Inf. wound+ CTCH2 Inf. wound+ CTCH2GO2 Inf. wound+ CTCH2+Cur Inf. wound+ CTCH2GO2+Cur

10

CFU/mL

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 39

8 6 4 2 0 Day-3

Day-7

Day-14

Day-21

Time

Figure 9: Colony forming unit (of infected S. aureus) data from the excised wound treated with different bandage treatments at various time intervals (a). Effect of surgery and different bandage treatments on (b) plasma pro inflammatory cytokine Tumor necrosis factor α levels and (c) plasma pro inflammatory cytokine Interleukin 1β levels and at different time intervals.


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Sl. no 1 2 3 4 5

Treatments CTCH2 @Normal wound CTCH2 @Infected wound CTCH2GO2 @Infected wound CTCH2+Cur @Infected wound CTCH2GO2+Cur @Infected wound

Hexosamine (mg/100mg of tissue) 7th day 14th day 21st day 0.52 ± 0.06 0.69 ± 0.11 0.82 ± 0.09

Hydroxyproline (mg/g tissue) 7th day 14th day 21st day 19.5 ± 2.26 38.2 ± 3.44 67.2 ± 4.82

0.12 ± 0.04

0.16 ± 0.03

0.18 ± 0.05

9.5 ± 1.12

13.4 ± 1.36

17.4 ± 2.36

0.32 ± 0.08

0.53 ± 0.07

0.66 ± 0.08

15.4 ± 1.93

28.4 ± 2.82

48.2 ± 3.35

0.39 ± 0.1

0.61 ± 0.11

0.73 ± 0.09

17.6 ± 2.62

34.5 ± 3.16

55.1 ± 4.14

0.58 ± 0.07

0.77± 0.08

0.89 ± 0.05

24.6 ± 2.17

42.3 ± 3.35

74.4 ± 5.28

Table 1: Hexosamine and hydroxyproline content of granulation on different days of healing after treating the wound with different bandage treatments.

Effect on S. aureus load. S. aureus load on the infected excised wounds were measured in terms of CFU count in respective post-operative days (3rd, 7th, 14th & 21st day). Both CTCH2GO2 & CTCH2+Cur treated group of wounds demonstrated significant decrease in the S. aureus CFU burden compared to the CTCH2 treated wounds.

Both CTCH2GO2 and

CTCH2+Cur treated wounds showed almost equal results while in case of the CTCH2GO2+Cur treated wounds S. aureus concentration decreased significantly (Figure 9). Effect on connective tissue markers. Collagen being a major component of connective tissue provides a frame work for structural strength and helps in tissue regeneration. Augmented levels of hydroxyproline and hexosamine are regarded as one of the crucial parameters for the wound healing process as increase in both the markers is an index of neo collagen synthesis. Treatment with CTCH2GO2 & CTCH2+Cur bandage materials resulted in significant increase in both hydroxyproline and hexosamine content than CTCH2 treated infected wound


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areas at different time intervals (7th, 14th & 21st day). Whereas, CTCH2GO2+Cur treatment resulted in significant acceleration in both the marker’s level as compared to remaining treatment groups (Table 1). Repair and healing of injured tissues involves hemostasis, inflammation, proliferation and remodeling. Microorganisms, sequestered at the skin surface gain access to the underlying tissues through injured skin which leads to delayed wound healing. Wound healing follows a cascade of complex processes for eradication of invaded microbial pathogens from the effected tissue which ultimately leads to remodeling of the same. GO containing bandage materials significantly ameliorate wound healing process through antibacterial action. Curcumin is well known for its wound healing and antimicrobial potential. Curcumin improves healing of injured tissues through modulating collagen synthesis and decrease in ROS generation35. Effect on inflammatory cytokine levels. In this present study, CTCH2 treated infected wounds showed higher levels of both inflammatory cytokine (IL-1β & TNF-α). CTCH2GO2, CTCH2+Cur and CTCH2GO2+Cur treatment significantly decreased both the cytokines where, CTCH2GO2+Cur showed better results among all treatments. TNF-α and IL-1β alters the matrix metalloproteinase synthesis resulted in increased collagenase activity36. Infections resulted pronounced inflammatory response which is governed by an elevated production and release of inflammatory cytokines.

GO reduced the bacterial load at the wound site which leads to

decrease in the levels of inflammatory markers and fasten the wound healing process. Curcumin has also been found to inhibit the release of proinflammatory cytokine (IL-1β & TNF-α) from monocytes and macrophages. It is also reported that curcumin possesses the ability to inhibit the inflammatory enzymes such as cyclooxygenase and lipoxygenase levels and helps in fasten the wound healing process37,38.


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Histopathology of wounds. Histopathology analysis of the wounded area was preformed after 21 days post treatment with different bandages to confirm the wound healing process. In the sample collected from CTCH2 treated infected wounds dead cells with chronic cell infiltrate was observed indicating no sign of healing. Treatment with CTCH2GO2, CTCH2+Cur and CTCH2GO2+Cur where the apparent wound healing was visible also showed the neovascularization, epithelialization, collagenase tissue with all the adnexal structures like sweat glands and hair follicles. Complete wound healing was observed for the infected wound on treatment with CTCH2GO2+Cur bandage along with the normal wound with almost nil inflammatory cell infiltrate. CONCLUSION To summarise, a type of bio-polymeric hydrogel bound cotton based compact patch were fabricated by a compression technique. The structural design of such patches allows to load different types of chemicals, drugs, nano or micro structured materials to modify their properties. Mechanical properties of the patches could be tuned by varying the percentage of the biopolymer used. Such patches retain their mechanical strength even in the wet state and soaking in water for 24 hours. Incorporation of GO not only provided the antimicrobial activity but it also improves the mechanical strength of the patches. After loading of curcumin, the antimicrobial activity of the GO incorporated patches further enhanced. All the patches possess good absorption capability. Patches are non-cytotoxic in nature as proven from the hemolysis and MTT assay test. Use of this patches as bandage material in animal model depicted the efficiency of such patches as the wound dressing material. The loading of curcumin in the GO incorporated patches showed the maximum wound healing rate compared to the other bandage patches.


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ASSOCIATED CONTENT Supporting Information. Supporting information part contains the details of materials used, characterization, SEM image, FTIR spectra, XRD spectra of GO, XRD spectra of the patches, pictures showing the synthesis of chitosan hydrogel both in presence and absence of Glycerol, Zeta potential graph chitosan solution, FTIR spectra of Chitosan and dry Chitosan-hydrogel film and stress-strain profile of the cotton patches with 5% and 20% chitosan concentration with varying GO percentage in dry as well as in wet state. Time dependent release study of curcumin from the patches in neutral aqueous medium is also given. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Fax: +91 361 2279909. Tel.: +91 361 2912073. Author Contributions Product design, material fabrication along with the physical and mechanical testing were done by A. Konwar and D. Chowdhury. Biological testings (both in-vitro and in-vivo) were done by R. Kandimalla and S. Kalita and both contributed equally in their part. ACKNOWLEDGMENT The authors thank SERB, New Delhi, Grant No. SB/S1/PC-69/2012 and BRNS, Mumbai, Grant No. 34/14/20/2014-BRNS. AK wants to thank IASST for fellowship. REFERENCES 1. Salsi, E.; Bayden, A. S.; Spyrakis, F.; Amadasi, A.; Campanini, B.; Bettati, S.; Dodatko, T.; Cozzini, P.; Kellogg, G. E.; Cook, P. F.; Roderick, S. L.; Mozzarelli, A. Design of O-


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Table of Content (TOC) Synopsis: A bio-polymeric hydrogel coated cotton based compact hybrid patch is fabricated. Any drug can be loaded into such patches.


Approach To Fabricate a Compact Cotton Patch without Weaving: A

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