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Synthesis and characterization of chitosan-PVP-nanocellulose composites for in-vitro wound dressing application
Accepted Manuscript Title: Synthesis and characterization of Chitosan–PVP–nanocellulose composites for In-vitro Wound dressing application Authors: R. Poonguzhali, S. Khaleel Basha, V. Sugantha Kumari PII: DOI: Reference:
S0141-8130(17)31037-1 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.07.006 BIOMAC 7813
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
21-3-2017 21-6-2017 2-7-2017
Please cite this article as: R.Poonguzhali, S.Khaleel Basha, V.Sugantha Kumari, Synthesis and characterization of Chitosan–PVP–nanocellulose composites for In-vitro Wound dressing application, International Journal of Biological Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.07.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Synthesis and characterization of Chitosan–PVP–nanocellulose composites for In-vitro Wound dressing application R. Poonguzhali a, S. Khaleel Basha b, V. Sugantha Kumari a*, a
Department of Chemistry, Auxilium College, Vellore 632 006, India
b
Department of Chemistry, C. Abdul Hakeem College, Melvisharam 632 509, India
Corresponding author [email protected] Contact number: 9894029585
Highlights
We synthesized Chitosan-PVP composites with different Nanocellulose contents.
Addition of nanocellulose increased swelling ratio of CPN composite.
The prepared nanocomposites were themally stable.
The bionanocomposites studied by hemolysis and cytocompatibility.
Influence of nanocellulose in the Chitosan/PVP matrix on in vitro wound dressing application was significant.
Abstract Biocompatible Chitosan/Poly (vinyl pyrrolidone) /Nanocellulose (CPN) composites were successfully prepared by solution casting method. The prepared bionanocomposites were characterized by Transmission electron microscopy (TEM), Thermo gravimetric analysis (TGA), X-ray diffraction (XRD) and Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) spectra. TEM images revealed the average particle size of the nanocellulose is 6.1 nm. Thermogravimetric analysis indicated that the thermal stability of the composites was decreased with increasing concentration of nanocellulose. The CPN composites were characterized for physical properties like Thickness, Barrier properties and 1
mechanical testing. Water vapor and oxygen permeability evaluations indicated that CPN composite could maintain a moist environment over wound bed. The nanocomposite showed enhanced swelling, blood compatibility and antibacterial activity. Cytotoxicity of the composite has been analyzed in normal mouse embryonic fibroblast cells. The results have shown the CPN3% composite shows a high level of antibacterial property when compared to the other composites. The biological study suggests that CPN3% composite may be a potential candidate as a wound healing material for biomedical application. Keywords: Chitosan; Poly (vinyl pyrrolidone); Nanocellulose; Cytotoxicity; Wound healing
1.
Introduction Skin injuries represent an important health problem that needs to be managed properly in
order to avoid serious consequences in terms of morbidity, disability and life quality. In vitro wound healing assays have been used for decades to study the major signal transduction pathways in wound healing [1-3]. Wound healing generally follows a well-defined, yet a complex cascade of processes commonly divided into four main stages; coagulation, inflammation, cell proliferation with repair of the matrix, and epithelialization with remodeling of the scarred tissue. Various formulations such as ointments and wound dressings have been developed for the treatment of severe wounds. However, when ointments or creams are used, their frequent reapplication and washing of the wound region often lead to pain or burden on the patient. In order to avoid this discomfort, we are introducing a novel biocomposite based nanocellulose incorporated Chitosan-Poly (vinyl pyrrolidone) for wounds. In recent years, biomaterial-based wound dressings have been widely used, such as natural abundant Chitosan [4], collagen [5], alginate [6] because of its nontoxic, biocompatible, biodegradable, analgesic, moisture retentive and readily available properties. Different forms of chitosan as wound dressings, such as hydrogels, membranes, scaffolds, and sponges were reported. Film forming ability is another especial aspect of chitosan in comparison with other biopolymers. Chitosan (CS) is a natural biopolymer that is derived from chitin, a major component of crustacean outer skeletons, has been widely investigated as an antimicrobial agent for preventing and treating infections owing to its intrinsic antimicrobial properties [7]. Chitosan is known in the wound management field for its hemostatic properties, biological activities and affects 2
macrophage function that helps in faster wound healing [8]. It also has an aptitude to stimulate cell proliferation and histoarchitectural tissue organization. The biological properties including bacteriostatic and fungistatic properties are particularly useful for wound treatment [9]. Poly (vinyl pyrolidone) (PVP) is a synthetic linear, non-toxic, biocompatible polymer, frequently used in controlled drug release [10], tissue engineering [11] and wound dressings [12]. It is easily soluble in water and in many organic solvents. PVP hydrogels, which has PEG as a plasticizer, were successfully prepared by gamma-radiation technique for pressure ulcer dressing applications [13]. Aytimur et al., [14] have prepared a nanofiber mat by electrospinning method and their basic composition is Poly (vinyl pyrrolidone) (PVP), Poly (vinyl alcohol) (PVA) and Poly (acrylic acid) (PAA). Based on clinical and in vivo studies, it has been proved that PVP is effective in the treatment of wound healing. In recent years there has been increasing interest in the use of nanocellulose in the biomedical field. Nanocellulose has many of the properties required for an ideal wound-care dressing (e.g. High water-holding capacity, high elasticity and conformability, and high mechanical strength) [15]. Prevention of microbial infection is one of the most valuable properties for wound dressings. Nanocellulose has little potential antimicrobial activity against pathogenic microorganisms [16]. Nanocellulose has a high surface to volume ratio, which can enhance cell attachment, cell migration and cell proliferation [17]. The network effect of nanocellulose/matrix and reinforcing effect of nanocellulose has been proved by Yvonne Aitomaki et al., [18]. Composites based on chitosan and nanocellulose are attracting significant interest because of the structural similarity of these two components. Danial Dehnad et al., prepared nanocomposites based on chitosan and nanocellulose composites functionalized with glycerol [19]. Meriem Fardioui et al., prepared bionanocomposite materials based on chitosan reinforced with nanocrystalline cellulose and Organo-Modified Montmorillonite for the packaging applications [20]. Jaya Sundaram et al., have prepared biodegradable composite membranes with antimicrobial properties consisting of nanocellulose fibrils, chitosan, and S-Nitroso-N-acetylpenicillamine tested for food packaging applications [21]. Narges Naseri developed Porous electrospun nanocomposite mats based on chitosancellulose nanocrystals and the mats showed permeability to oxygen and CO2 as well as compatibility towards adipose derived stem cells, thus, they are a potential candidate for use as a wound dressing material [22]. Archana et al prepared chitosan wound dressing [23] composed 3
of chitosan, PVP with nanosilver oxide [24] and TiO2 nanocomposites [25]. Recently, CSbentonite nanocomposite films can potentially be a promising candidate for wound dressing application [26]. In this study, Chitosan and Poly (vinyl pyrrolidone) (PVP) were used as matrix materials for the development of nanocomposites filled with different amount of cellulose nanoparticles. The bionanocomposites have been characterized through a variety of techniques to obtain information about their crystalline nature, thermal stability, cytotoxicity, blood compatibility, barrier properties and antibacterial property. The effects of the nanocellulose content on the mechanical properties, morphology and water solubility of bionanocomposite were investigated. These novel biomaterials offers great potential used in biomedical applications, particularly as wound dressings. The CPN is expected to protect the wound from further infections and provide a better healing environment. 2. Experimental 2.1. Materials The Hibiscus Cannabinus used as raw material was obtained from the local farming community. Chitosan (MW = 190,000-300,000 g/mol with 80 % deacetylation), was purchased from Sisco Research Laboratories Pvt. Ltd. Mumbai, India. PVP (approximate MW= 40,000g/mol), Acetic acid, Sodium Hydroxide, Sodium hypochlorite, Oxalic acid, deionised Water and other analytical grade reagents were purchased from Spectrochemicals Pvt. Ltd. Mumbai, India. 2.2. Preparation of cellulose nanocrystals from Hibiscus cannabinus Alkali treatment The alkali treatment was performed to purify the cellulose by removing lignin and hemicellulose from Hibiscus cannabinus fibers. The fiber was cut into small piece, and then treated with an alkali solution (2 weight% NaOH). The mixture was transferred into a flask and treatment was performed in an autoclave with a pressure of 15 lbs and at a temperature of 120°C for a period of 1h. The alkali treated fiber was then filtered and washed several times using distilled water. Bleaching process
4
Following alkali treatment, the bleaching process was completed by adding a buffer solution of acetic acid, aqueous chlorite (2 %) for an hour and repeated for several times followed by washing with water till the smell of bleaching agent was removed. Acid hydrolysis The acid hydrolysis treatment was conducted on the fibers after alkali treatment and bleaching using oxalic acid for 5h in an autoclave in the pressure of 20 lbs. The fiber was neutralized and the suspension was diluted with water, kept for stirring in a magnetic stirrer for 8h. The pH of the suspension was around 6.7. The nanocellulose was obtained from the suspension by drying it at room temperature. The schematic presentation of whole process is shown in Fig. 1.
2.3. Chitosan/ Poly(vinyl pyrrolidone)/Nanocellulose blending and Nanocomposite preparation The Solution casting method was used to prepare the bionanocomposite of chitosan/PVP/nanocellulose (CPN). Scheme-1 shows the flow chart of preparation of CPN composite. Chitosan solution (1% w/v) was prepared in acetic acid (1 % v/v) stirring was conducted at room temperature for 2h using Homogenizer. PVP solution (1% v/v) was prepared in deionized water. The obtained solution of PVP was added into the chitosan solution, followed by mechanical stirring of 4 h to form CS/PVP (50:50) blend solution. To this blend solution of CS/PVP aqueous dispersion of nanocellulose of different compositions (1wt%, 3wt%, 5wt % and 10% wt) were added with constant stirring for 8 hours at room temperature to get a homogenous solution. The resulting mixture was deformed under rest for 1h at room temperature then it was cast in a Teflon mold and placed in a 37°C oven to evaporate water. The dried composites were peeled from the Teflon plate and kept in a desiccator at room temperature until further use. The membranes were coded as CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5%, CPN10%.
2.4. Characterization techniques 2.4.1. Transmission electron microscopy (TEM) Transmission electron microscopy (model Philips CM 200) was used to determine the dimensions of the nanocellulose obtained from the Hibiscus Cannabinus fibers and CPN nanocomposites. A drop of a diluted suspension of nanocellulose and its nanocomposites was
5
deposited on the surface of a clean copper grid and coated with a thin carbon film and dried at room temperature. 2.4.2. Thickness of the composite Thickness of the different composite was determined by a hand-held digimatic micrometer Mitutoyo Corporation, Japan. Model-MDC-1”SB. For each sample, 5 random positions around the composite were measured and mean value was taken as thickness of composite. 2.4.3. Barrier properties The moisture permeability of the composite was carried out by measuring the water vapor transmission rate (WVTR) according to the ASTM standard method E 96-95. To measure WVTR, the composites were mounted at the top of the vial (19 mm diameter) containing 10 mLs of water. Then, the vials were placed in a desiccator with a saturated solution to ammonium sulfate at 37°C. The assembly was weighed at regular intervals of time and a weight loss vs. Time plot was constructed. From the slope of the plot, WVTR was calculated as follows: WVTR = slope × 24⁄A
g
[m2 /day]
(1)
Where‘A’represents the permeation area of the sample (m2). Experiments were done in triplicate. Oxygen permeability (Dk) was evaluated at 25 °C on composite equilibrated at 54% RH by measuring the oxygen transference rate (OTR) with a gas permeability tester (Ox-Tran 1/50 system), following the ASTM D3985-05 standard. Dk was calculated following the expression: Dk = (OTR × l)/ΔP
(2)
Where ‘l’ is the composite thickness and ‘ΔP’ is the difference between oxygen partial pressure across the composite. Four replicates per composite were made. 2.4.4. Swelling parameter The swelling parameters like swelling ratio and swelling coefficient were evaluated to assess the extent of swelling behaviour of nanocomposites in PBS solution. The swelling ratio was measured by immersing a preweighed dry sample in appropriate swelling medium of phosphate buffer saline solution (PBS) to pH 7.4 at a given time interval (1h, 2h, 3h, 4h, 5h, 6h, 7h, and 8h) at 37°C. Excess surface water was blotted out with filter paper before weighing. Highly swollen samples were placed like sandwich between two sieves and then blotted with filter paper. Percentage swelling of the composite at equilibrium was calculated from the formula. 6
Swelling % = Ww − Wd /Wd × 100 %
(3)
Where Ww and Wd are weights of wet and dry sample respectively. The swelling behavior of the composites can also be analyzed from the swelling coefficient values. It is an index of the ability with which the sample swells and this was determined using the equation. Swelling coefficient, α = {As/m} × [1/d]
(4)
Where, As is the weight of the solvent sorbed at equilibrium swelling, ‘m’ the mass of the sample before swelling and‘d’ the density of the solvent used. 2.4.5. Mechanical studies Tensile testing was carried out using MTS Criterion 5 kN testing Machine according to ASTM D-638-2010 with a crosshead speed of 50 mm/min. The measurements were done at 25 ºC. Sample dimensions were 4mm X 10mm. At least five sample specimens for each set were tested to get the average value. 2.4.6. Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) spectra The chemical structure of the prepared membranes was characterized using an attenuated total reflectance Fourier transform (ATR-FTIR) spectrophotometer (Shimadzu IR affinity-1S). Each spectrum was accrued of transmittance mode on a Quest ATR ZnSe crystal cell by accumulation of 250 scans with a resolution of 4 cm-1 and a wave number range of 4000-400 cm-1. 2.4.7. Thermogravimetric analysis (TGA) The thermal behaviour of the sample were performed with a NETZSCH STA 449F3 thermal gravimetric analyzer under a nitrogen atmosphere and at a heating rate of 10°C /min in the temperature range of 25-600°C. 2.4.8. X-ray diffraction analysis (XRD) X-ray diffraction patterns of membranes were analyzed using a X-ray diffractometer (XRD-SchimadzuXD-D1) with Nickel-filtered Cu- Kα radiation at voltage 30kV and current of 45 mA. The samples were scanned from 10º to 60 º 2θ at a scanning rate of 3º min−1. 2.4.9. Antibacterial Activity The antibacterial activity of CPN composite was tested by an inhibition zone method. Staphylococcus aureus MTCC1688 and Pseudomonas aeruginosa MTCC3615 were used as examples of gram-positive and gram-negative bacteria. Zone inhibition test was carried out with a modified agar diffusion assay. CPN composite was placed on nutrient agar in petri dishes 7
which had been seeded with 20 µl of bacterial cell suspensions. The petri dishes were examined for zone of inhibition after 24 h incubation at 37 ºC. The presence of any clear zone that formed around the film disc on the plate medium was recorded as an indication of inhibition against the bacterial species. The area of the whole zone was calculated and then subtracted from the disc area, and the difference in area was reported as the zone of inhibition. 2.4.10. Hemocompatibility In this experiment, a dry composite was equilibrated in saline water (0.8 % NaCl solution) for 24 hrs at 37 ± 0.5 ºC. Fresh goat blood (0.25ml) was added onto the wet composite. After 20 minutes 2 mL of saline water was added onto the composite to stop hemolysis and the test sample was incubated for 60 min at 37 ± 0.5ºC. Positive and negative controls were obtained by adding 0.25 ml of fresh goat blood and saline solution respectively into 2.0 ml distilled water. Incubated test samples were centrifuged at1000 rpm for 45 min and hemoglobin released was measured by the optical densities (OD) of the supernatant at 540nm using an UV-Visible Spectrophotometer (Shimadzu UV Visible Spectrophotometer, UVmini-1240). The percentage of hemolysis was calculated according to the following formula. Hemolysis (%) = (ODs – OD (-)) / (OD (+) – OD (-)) × 100
(5)
Where ODS is the optical density of the sample, OD (-) is the optical density of negative control, OD (+) is the optical density of positive control. 2.4.11. Cytotoxicity Cytotoxicity assay of CPN composites was performed on the basis of NIH3T3 cell viability using MTT (3-(4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide) assay (Sigma, St Louis, USA). NIH3T3 cells were mouse embryonic fibroblast cell line cultured in Dulbecco’s minimal essential medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), antibiotics (100 U/mL penicillin and 100 g/mL streptomycin) at37◦C and 5% CO2. The composites were immersed in DMEM culture media (0.2 g dry composite per mL media) for 24 h. Then, the extract was added over cultured NIH3T3 cells in 96-well plate for 1, 3, 5 days. The viability of cells in each well was evaluated by MTT method (Liu et al., 2010), and DMSO solution with a concentration of 5.0 % was used as a control. 2.5. Statistical analysis All the experiments were performed in triplicate and student’s t-test was performed to determine the statistical significance p<0.05 was considered statistically significance. 8
3. Results and discussion 3.1. Transmission electron microscopy (TEM) The morphology of synthesized acid hydrolysed nanocellulose and nanocomposites reinforced with 3 wt% and 5 wt% of nanocellulose was examined using TEM at the nanoscale. The shape of the nanocellulose in Fig.2 (a) is observed as needle like shape and the size distribution histogram (Fig.2 (b)) obtained through particle size analysis tests for nanocellulose also revealed mean diameter of 6.1 ± 5 nm. The corresponding Selected Area Electron Diffraction (SAED) Fig.2 (c) shows the crystallinity of the nanocellulose. A visible diffraction pattern as bright spots proves crystallinity of the nanocellulose. In the TEM image of Fig.3 (a, b) shows the shape of the prepared nanocomposites is spherical and the interface of nanocellulose with CS/PVP matrix appears to be efficiently bonded. In other words, there is no trace of polymer aggregation visible on to the surface of the nanocellulose. This is a very important aspect for nanocomposites in order to get maximum advantage of incorporating nano-fillers in polymer matrices and particles are fairly monodispersed with respect to their size, which is approximately 2-10 nm. The narrow size distribution and absence of agglomerates prove that CS/PVP matrix prevents agglomeration of the nanocellulose. Chitosan and PVP have oxygen and nitrogen bearing groups that can stabilize the nanoparticle surface. The strongest interactions with nanocellulose likely involve with oxygen and hydrogen. 3.2. Thickness of the composite Thickness of CPN nanocomposite was varied from 32.82 ± 2.4 to 46.18 ± 7.5 µm, higher than normal thickness of chitosan/PVP membrane (Table 1). The biomaterial for wound dressing should ideally be thinner than the human dermis, whose thickness varies from 30-50 µm depending on age, sex and body region where the biomaterial is to be applied. The use of scaffolds with thickness lower than 1 mm has been described in the literature for the regeneration of human skin [27], which indicates that the composite produced can be considered adequate for healing purpose. Chitosan is slowly biodegradable and positively charged so that a thinner chitosan composite can act as a dermis substitute. Therefore, we considered that 30-50 µm dry thickness of the nanocomposite was enough for cell attachment in vitro and for in vivo biodegradation in a certain period of skin tissue healing. 3.3. Barrier properties 9
The ideal wound dressing should has several characters, such as ability to control gases diffusion, maintains a moist environment around the wound, prevent further inflammation, simple and easy to use with little or no pain from the wound and cosmetically acceptable. The wound dressing material has to be permeable to control the moisture and gases in order to help in the wound healing process [28]. Fig.4. shows the water vapour transmission rate and oxygen permeability of composites. The WVTR of CPN0% composite was 2128.0 g/m2 day, and it decreased significantly when nanocellulose was incorporated. The WVTR decreased linearly down to 1742.9 g/m2 day when the nanocellulose was added up to 3 wt%, and then it increased slightly with increase in the concentration of nanocellulose. The nanocellulose in the composite act as an impermeable barrier against water vapor which increased the tortuous path for water vapor to diffuse through the membrane and resulted in the decreased WVTR of the composite [29]. The reduction in WVTR is probably due to surface densification and the partial closure of the surface pores by the nanocellulose. Water molecules might be strongly adsorbed to the surface or of the nanocellulose which could result in lower water vapor transmission through the composite. This is mainly due to the high affinity between water and the composite. Providing better cellular growth, oxygen is required at almost every step of wound healing process and to reduce the growth of anaerobic bacteria. So evaluation of oxygen permeability of nanocomposite is important for its application as wound dressing. In the present case, nanocomposites have shown considerable permeability to oxygen. The results showed the nanocomposite which can allow oxygen molecules to penetrate through them and reach the wound site to help in the wound healing process. From Fig.4 it can be observed that the Dk of the nanocomposite rises upon increasing nanocellulose concentration. The increased number of polar -OH groups with increasing nanofiller content leads to an enhancement of the oxygen solubility in the nanocomposite.
3.4. Swelling parameter Swelling behaviour and structural stability of nanocomposite are critical for their practical use in wound dressing application. Most natural polymers, including chitosan, swell readily in biological fluids. There are several parameters affecting the swelling ratio, hydrophilicity, stiffness and pore structure of a nanocomposite. The sample with the highest 10
degree of swelling will have the highest surface area/volume ratio. The swelling parameter of CPN composite is presented in Fig.5 (a, b). Incorporation of nanocellulose significantly increases the swelling percentage of CPN, with the most significant increase at 5 wt % nanocellulose. The main reason for higher swelling rate of nanocomposite is due to the presence of hydrogen bonding of water molecules to the free -OH and -COOH groups present in cellulose molecules [30]. The hydrophilic nature of chitosan and also PVP may be a major factor that influences the extent of swelling of the CS / PVP matrix. The swelling coefficient values of the nanocomposite were decreased upon increasing nanocellulose loading, but the decrease is not regular. It is clear that as nanocellulose increases, the equilibrium solvent uptake decreases. This is due to the increased hindrance exerted by the crystals at higher loadings. Nanocomposite with 3 wt % nanocellulose content shows lower values for swelling coefficient than that with 5 wt% nanocellulose content. 3.5. Mechanical studies The mechanical properties of the composites depend on the orientation of the filler, content and size as well as the interaction between filler and polymer matrix [31]. The mechanical properties of the CPN composites are shown in Table 2. The remarkable increase in tensile strength and modulus of these composites indicates the presence of some interaction between nanocellulose and chitosan molecules in the composite. Increasing NC level incorporated into CS/PVP matrix could increase Young’s Modulus by 1743-2190 MPa. This could be relevant to two factors: (a) proper connections between polymer-nanocrystals (b) efficient transfer of involved stress through polymer-NC layers. In fact, improvement of tensile properties for the composite is due to a suitable stress transfer throughout polymer, even distribution of entered stress and minimizing the areas of stress concentrations. With the addition of nanofiller, Elongation at break dropped from 53.2% for CPN0% to 50.5% for CPN10%. This decrease in the Elongation at Break value indicated that motion of the CS/PVP matrix was restricted because of its interactions with nanocellulose. The mechanical test result suggests that there is an optimum concentration of the filler to induce the maximum increment of the strength of the nanocomposite. 3.6. Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) spectra To analyze the nanocomposites structure and to identify the molecular groups in chemical interaction with nanocellulose, the prepared bio-nanocomposite will be investigated by 11
Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR). Fig.6. represents the overall ATR- FTIR spectra of the nanocomposite membrane with varying nanocellulose concentration. The CPN0% showed distinct -C=O group absorption spectra at 1647 cm-1 and -OH absorption for chitosan in the blend at 3084 cm-1. In case of chitosan/PVP/nanocellulose composite membrane, additional bands were observed in addition to the bands of cellulose polymer. These additional bands are attributed to the nanocellulose and PVP in the composite membrane. The vibrational peak at 1430 cm−1 corresponds to -CH2 bending vibration that attributed to the crystalline peak of cellulose, however, the vibrational peak at 896 cm−1 is attributed to the C-O-C stretching vibration of amorphous band of cellulose. Finally, the band observed in the 1028–1161 cm-1 range was attributed to C–O stretching and C– H rocking vibrations of the pyranose ring skeleton [32]. It can be clearly seen that the resulting nanocellulose band around 1000 and 3400 cm−1 due to the isolated cellulose component. These bands could be associated with glucose units (with three additional hydroxyl groups) exposed on the cellulose backbone structure. However the most prominent band at 1553 cm-1 assigned to amino group in pure chitosan film shifts to a higher wavenumber (1557 cm-1) in the presence of nanocellulose. It is quite evident that the primary amino groups are in interaction with the cellulose surface (Scheme-2). The amino groups act as capping sites for the cellulose nanoparticles stabilization.
3.7. Thermogravimetric analysis (TGA) The thermal stability of the CPN composites of different ratios was studied by means integral (TGA) and derivative (DTG) thermogravimetric curves and the results are shown in Fig.7 (a, b). The results from the TGA show residual weight vs temperature for prepared nanocomposite. The TGA curves of chitosan, nanocellulose was almost similar and chitosan, PVP, nanocellulose shows single weight loss region ranging from 250°C-310°C, 410°C-470°C and 260°C-330°C respectively. The TGA curves of all composite shows three weight loss regions. The first region in the range of 70-150°C is due to the evaporation of weakly bound water with a weight loss of around 10%. The second transition region for CPN0% was around 150-320°C, due to structural degradation of PVP with weight loss of 30% and for CPN0.5%, CPN1% were around 150-320°C and 150-360°C due to structural degradation and burning out of nanocellulose and CPN3%, CPN5% and CPN10% were in the region of 150-390°C. The third 12
stage of weight loss occurred for CPN0% was 320-480°C due to cleave of backbone of chitosan where the weight loss around 85% and increasing gradually to 100%. The third stage loss of CPN0.5% and CPN1% were in the region of 320-480°C and 360-480°C. The TG curves of CPN3%, CPN5% and CPN10% were quite similar to each other, and their degradation started much earlier as compared to CPN0%, CPN0.5% and CPN1%. From the Fig.7 it can be concluded that the thermal stability decreases with increases nanocellulose content.
3.8. X-ray diffraction analysis (XRD) X-ray diffraction was used to determine information regarding the crystalline structure of the nanocomposite. The CPN composites obtained with various nanocellulose concentrations had almost same XRD patterns and the results were shown in Fig.8. The CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5%, CPN10% exhibited its characteristic semicrystalline peaks around 2θ = 22.0, 21.9, 21.7, 21.5, 21.4 and 21.2 respectively. Even though the nanocellulose prepared by acid hydrolysis possess enough crystallinity observed by TEM the synergetic amorphous effect of CS/PVP matrix diminishes the crystallinity of nanocellulose. 3.9. Antibacterial Activity CPN composite was tested against microorganisms to determine antibacterial activity. Table 3 shows a typical antibacterial test result of chitosan-based nanocomposite against Staphylococcus aureus MTCC1688 and Pseudomonas aeruginosa MTCC3615 determined by the zone inhibition method. The diameter of the growth inhibition zone was dependent on the composite used. As shown in Fig.9. the largest inhibition zone were observed for the Gramnegative bacteria Pseudomonas aeruginosa, while the smallest one were observed for the gram positive Staphylococcus aureus bacteria for CPN1%, CPN3% and CPN5%. The reason could be explained in two aspects. On the one hand, chitosan exhibited bactericidal activity through contact killing or dissolving from the nanocomposite. Interaction between positively charged chitosan molecules and negatively charged microbial cell membranes leads to the leakage of bacteria by disrupting the inner organelles or disturbing the metabolism of bacterial strains and another is the extremely high surface area of nanocellulose facilitated the adsorption of target bacteria, which accelerated the rate of antibacterial reaction [33]. According to Zivanovic and Draughon [34], the proposed mechanism of antibacterial activity of bionanocomposites are their attack on the phospholipid cell membrane, which causes 13
increased permeability and leakage of cytoplasm, or in their interaction with enzymes located on the cell wall. Thus, the resistance of Gram-negative bacteria to the composite likely lies in the protective role of their cell wall lipopolysaccharides or outer membrane proteins. 3.10. Hemocompatibility Hemolysis assay was carried out to study the hemolytic effect of composite on blood. The basic observation was to find out the release of haemoglobin into plasma due to damage of the erythrocyte membrane. The hemolysis of CPN composite was measured against positive and negative control. The % hemolysis of CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5%, CPN10% were 0.67±0.08, 0.74±0.2, 0.89±0.5, 1.94±0.26, 5.1±0.6 and 6.4±0.2 % respectively. As shown in Fig. 10, all the composites were showed very low hemolysis ratios at the low concentration of nanocellulose. When the concentration was increased to 5 and 10%, the CPN composite showed a relatively higher hemolysis of 5.1±0.6 and 6.4±0.2 % respectively, while the other composite still exhibited low hemolysis ratios. Generally, smaller the hemolysis ratio value, the better the blood compatibility of the biomaterial [35]. The prepared nanocomposite could be highly hemocompatible since the percentage of hemolysis for the membranes were <2%. 3.11. Cytotoxicity Cytotoxicity is one of the most important properties for material used in for wound healing applications. The fibroblasts (NIH3T3) stand for a typical embryonic cell and were used to evaluate the cytotoxicity of wound dressing material. Bionanocomposite with cell viability larger than 80% can be considered as noncytotoxic defined by the ISO standard [36]. As shown in Fig.11, the cell viability data of control (5% DMSO) did not show any toxicity at 1, 3 and 5 days in contact with NIH3T3 cells. All bionanocomposites showed 40-70% viability after 1 day of incubation and 60-80 % viability after 3 and 5 days of incubation. Cell viability of CPN3% and CPN5% was increased up to 90% at 5 days of incubation. The cell viability increased due to the multiplication of NIH3T3 cells. Hence the above results suggest that CPN3% and CPN5% had more cell viability and good compatibility. 4. Conclusion High performance novel polymer bio-nanocomposite films were prepared by solution casting of chitosan, Poly (vinyl pyrrolidone) (PVP) and nanocellulose. The structural characterizations showed that the chitosan and PVP are perfectly compatible and miscible polymers by the hydrogen bond interactions between the carbonyl groups of PVP and amino and 14
hydroxyl groups of chitosan, resulting in the formation of new biocompatible homogeneous blend matrix for bionanocomposite development with nanocellulose. When the nanocellulose has been added to CS/PVP blend, some additional hydrogen bonding can occurs between the hydroxyl groups in nanocellulose and amino and hydroxyl groups of CS and between the carbonyl groups of PVP. Therefore, an interconnected structure is assumed to be formed in CS/PVP strengthened by nanocellulose. Since that the special two-dimensional morphology of the nanocellulose as well as its functionalized surface provides well homogeneous dispersion, which leads to a high contact area in the CS/PVP matrix. Owing to these strong interfacial interactions, large improvements of certain properties, such as barrier properties and mechanical properties were achieved for CS/PVP-based nanocomposite. With the incorporation of 5% wt of nanocellulose, the swelling property is increased to a greater extent. In addition, tensile strength of CPN3%, CPN5%, CPN10% was increased by 35.6 ± 5.8, 38.4 ± 2.7 and 39.7 ± 6.9 respectively. Bionanocomposites of CPN3%, CPN5% exhibits superior antibacterial activity against gram positive and gram negative bacteria pathogens and good blood compatible property and cytotoxicity. Therefore, the prepared bio-nanocomposite membrane with features of good tensile, good thermal and high swelling property will have potential applications as an eventual wound dressing material.
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Figure legends Fig.1. Schematic representation of Isolation of Nanocellulose from Hibiscus cannabinus Fig.2. Transmission electron micrograph of a) Nanocellulose b) Histogram of nanocellulose c) SAED pattern of nanocellulose. Fig.3. Transmission electron micrograph of a) CPN3% and b) CPN5%. Fig.4. Water vapour transmission rate (WVTR) and Oxygen permeability (OP) of CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5% and CPN10% bionanocomposites. Fig.5. a) Swelling Ratio and b) Swelling Coefficient of CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5% and CPN10% bionanocomposites. Fig.6. ATR-FTIR Spectra of CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5% and CPN10% bionanocomposites. Fig.7. a) TGA thermogram and b) DTG curves of CS, PVP, Nanocellulose, CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5% and CPN10% bionanocomposites. Fig.8. XRD patterns of CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5% and CPN10% bionanocomposites. Fig.9. Inhibitory effect of CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5% and CPN10% bionanocomposites against a) Staphylococcus aureus and b) Pseudomonas aeruginosa. Fig.10. Hemolysis of CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5% and CPN10% bionanocomposites. Fig.11. Cytotoxicity of CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5% and CPN10% bionanocomposites expressed as a percentage of cell viability for incubation time of 1, 3 and 5 days.
18
Fig.1. Schematic representation of Isolation of Nanocellulose from Hibiscus cannabinus
19
Fig.2. Transmission electron micrograph of a) Nanocellulose b) Histogram of nanocellulose c) SAED pattern of nanocellulose.
Fig.3. Transmission electron micrograph of a) CPN3% b) CPN5%.
20
Fig.4. Water vapour transmission rate (WVTR) and Oxygen permeability (OP) of CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5% and CPN10% bionanocomposites.
21
Fig.5. a) Swelling Ratio and b) Swelling Coefficient of CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5% and CPN10% bionanocomposites.
22
Fig.6. ATR-FTIR Spectra of CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5% and CPN10% bionanocomposites.
Fig.7. a) TGA thermogram and b) DTG curves of CS, PVP, Nanocellulose, CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5% and CPN10% bionanocomposites.
23
Fig.8. XRD patterns of CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5% and CPN10% bionanocomposites.
24
Fig.9. Inhibitory effect of CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5% and CPN10% bionanocomposites against a) Staphylococcus aureus and b) Pseudomonas aeruginosa.
Fig.10. Hemolysis of CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5% and CPN10% bionanocomposites.
25
Fig.11. Cytotoxicity of CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5% and CPN10% bionanocomposites expressed as a percentage of cell viability, for incubation time of 1, 3 and 5 days.
26
Scheme-1. Flow diagram of the Synthesis of Chitosan/ Poly (vinyl pyrrolidone)/Nanocellulose composite.
Scheme-2. Intermolecular Hydrogen bond in CPN bionanocomposite.
27
Table 1 Thickness of the CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5% and CPN10% bionanocomposites. Sample
Nanocomposites
Thickness(µm)
a
CPN0%
32.82 ± 2.4
b
CPN0.5%
34.61 ± 1.9
c
CPN1%
36.83 ± 4.9
d
CPN3%
39.04 ± 5.7
e
CPN5%
43.96 ± 3.3
f
CPN10%
46.18 ± 7.5
Table 2 Mechanical properties of CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5% and CPN10% bionanocomposites. Data are presented as the mean ± SD (n = 4, P<0.05). Sample
Tensile strength
Elongation at break
Young’s Modulus
(MPa)
(%)
(MPa)
CPN0%
27.1 ± 9.5
53.2 ± 7.2
1743.2 ± 2.8
CPN0.5%
31.7 ± 7.3
52.9 ± 4.5
1842.8 ± 7.1
CPN1%
34.0 ± 8.3
51.9 ± 5.4
1895.3 ± 5.8
CPN3%
35.6 ± 5.8
51.5 ± 4.1
1902.4 ± 3.0
CPN5%
38.4 ± 2.7
50.9 ± 3.8
2127.5 ± 7.9
CPN10%
39.7 ± 6.9
50.5 ± 1.3
2190.4 ± 3.8
28
Table 3 Antibacterial activity of CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5% and CPN10% bionanocomposites. Sample code
Zone of Inhibition(mm) S. aureus MTCC 1688
P. aeruginosa MTCC3615
CPN0%
10.9 ± 0.7
10.9 ± 0.3
CPN0.5%
11.5 ± 0.3
11.2 ± 0.0
CPN1%
11.3 ± 0.8
11.9 ± 0.6
CPN3%
11.8 ± 0.1
12.3 ± 0.2
CPN5%
12.6 ± 0.2
12.8 ± 0.9
CPN10%
12.5 ± 0.4
12.5 ± 0.3
29
S0141-8130(17)31037-1 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.07.006 BIOMAC 7813
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
21-3-2017 21-6-2017 2-7-2017
Please cite this article as: R.Poonguzhali, S.Khaleel Basha, V.Sugantha Kumari, Synthesis and characterization of Chitosan–PVP–nanocellulose composites for In-vitro Wound dressing application, International Journal of Biological Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.07.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Synthesis and characterization of Chitosan–PVP–nanocellulose composites for In-vitro Wound dressing application R. Poonguzhali a, S. Khaleel Basha b, V. Sugantha Kumari a*, a
Department of Chemistry, Auxilium College, Vellore 632 006, India
b
Department of Chemistry, C. Abdul Hakeem College, Melvisharam 632 509, India
Corresponding author [email protected] Contact number: 9894029585
Highlights
We synthesized Chitosan-PVP composites with different Nanocellulose contents.
Addition of nanocellulose increased swelling ratio of CPN composite.
The prepared nanocomposites were themally stable.
The bionanocomposites studied by hemolysis and cytocompatibility.
Influence of nanocellulose in the Chitosan/PVP matrix on in vitro wound dressing application was significant.
Abstract Biocompatible Chitosan/Poly (vinyl pyrrolidone) /Nanocellulose (CPN) composites were successfully prepared by solution casting method. The prepared bionanocomposites were characterized by Transmission electron microscopy (TEM), Thermo gravimetric analysis (TGA), X-ray diffraction (XRD) and Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) spectra. TEM images revealed the average particle size of the nanocellulose is 6.1 nm. Thermogravimetric analysis indicated that the thermal stability of the composites was decreased with increasing concentration of nanocellulose. The CPN composites were characterized for physical properties like Thickness, Barrier properties and 1
mechanical testing. Water vapor and oxygen permeability evaluations indicated that CPN composite could maintain a moist environment over wound bed. The nanocomposite showed enhanced swelling, blood compatibility and antibacterial activity. Cytotoxicity of the composite has been analyzed in normal mouse embryonic fibroblast cells. The results have shown the CPN3% composite shows a high level of antibacterial property when compared to the other composites. The biological study suggests that CPN3% composite may be a potential candidate as a wound healing material for biomedical application. Keywords: Chitosan; Poly (vinyl pyrrolidone); Nanocellulose; Cytotoxicity; Wound healing
1.
Introduction Skin injuries represent an important health problem that needs to be managed properly in
order to avoid serious consequences in terms of morbidity, disability and life quality. In vitro wound healing assays have been used for decades to study the major signal transduction pathways in wound healing [1-3]. Wound healing generally follows a well-defined, yet a complex cascade of processes commonly divided into four main stages; coagulation, inflammation, cell proliferation with repair of the matrix, and epithelialization with remodeling of the scarred tissue. Various formulations such as ointments and wound dressings have been developed for the treatment of severe wounds. However, when ointments or creams are used, their frequent reapplication and washing of the wound region often lead to pain or burden on the patient. In order to avoid this discomfort, we are introducing a novel biocomposite based nanocellulose incorporated Chitosan-Poly (vinyl pyrrolidone) for wounds. In recent years, biomaterial-based wound dressings have been widely used, such as natural abundant Chitosan [4], collagen [5], alginate [6] because of its nontoxic, biocompatible, biodegradable, analgesic, moisture retentive and readily available properties. Different forms of chitosan as wound dressings, such as hydrogels, membranes, scaffolds, and sponges were reported. Film forming ability is another especial aspect of chitosan in comparison with other biopolymers. Chitosan (CS) is a natural biopolymer that is derived from chitin, a major component of crustacean outer skeletons, has been widely investigated as an antimicrobial agent for preventing and treating infections owing to its intrinsic antimicrobial properties [7]. Chitosan is known in the wound management field for its hemostatic properties, biological activities and affects 2
macrophage function that helps in faster wound healing [8]. It also has an aptitude to stimulate cell proliferation and histoarchitectural tissue organization. The biological properties including bacteriostatic and fungistatic properties are particularly useful for wound treatment [9]. Poly (vinyl pyrolidone) (PVP) is a synthetic linear, non-toxic, biocompatible polymer, frequently used in controlled drug release [10], tissue engineering [11] and wound dressings [12]. It is easily soluble in water and in many organic solvents. PVP hydrogels, which has PEG as a plasticizer, were successfully prepared by gamma-radiation technique for pressure ulcer dressing applications [13]. Aytimur et al., [14] have prepared a nanofiber mat by electrospinning method and their basic composition is Poly (vinyl pyrrolidone) (PVP), Poly (vinyl alcohol) (PVA) and Poly (acrylic acid) (PAA). Based on clinical and in vivo studies, it has been proved that PVP is effective in the treatment of wound healing. In recent years there has been increasing interest in the use of nanocellulose in the biomedical field. Nanocellulose has many of the properties required for an ideal wound-care dressing (e.g. High water-holding capacity, high elasticity and conformability, and high mechanical strength) [15]. Prevention of microbial infection is one of the most valuable properties for wound dressings. Nanocellulose has little potential antimicrobial activity against pathogenic microorganisms [16]. Nanocellulose has a high surface to volume ratio, which can enhance cell attachment, cell migration and cell proliferation [17]. The network effect of nanocellulose/matrix and reinforcing effect of nanocellulose has been proved by Yvonne Aitomaki et al., [18]. Composites based on chitosan and nanocellulose are attracting significant interest because of the structural similarity of these two components. Danial Dehnad et al., prepared nanocomposites based on chitosan and nanocellulose composites functionalized with glycerol [19]. Meriem Fardioui et al., prepared bionanocomposite materials based on chitosan reinforced with nanocrystalline cellulose and Organo-Modified Montmorillonite for the packaging applications [20]. Jaya Sundaram et al., have prepared biodegradable composite membranes with antimicrobial properties consisting of nanocellulose fibrils, chitosan, and S-Nitroso-N-acetylpenicillamine tested for food packaging applications [21]. Narges Naseri developed Porous electrospun nanocomposite mats based on chitosancellulose nanocrystals and the mats showed permeability to oxygen and CO2 as well as compatibility towards adipose derived stem cells, thus, they are a potential candidate for use as a wound dressing material [22]. Archana et al prepared chitosan wound dressing [23] composed 3
of chitosan, PVP with nanosilver oxide [24] and TiO2 nanocomposites [25]. Recently, CSbentonite nanocomposite films can potentially be a promising candidate for wound dressing application [26]. In this study, Chitosan and Poly (vinyl pyrrolidone) (PVP) were used as matrix materials for the development of nanocomposites filled with different amount of cellulose nanoparticles. The bionanocomposites have been characterized through a variety of techniques to obtain information about their crystalline nature, thermal stability, cytotoxicity, blood compatibility, barrier properties and antibacterial property. The effects of the nanocellulose content on the mechanical properties, morphology and water solubility of bionanocomposite were investigated. These novel biomaterials offers great potential used in biomedical applications, particularly as wound dressings. The CPN is expected to protect the wound from further infections and provide a better healing environment. 2. Experimental 2.1. Materials The Hibiscus Cannabinus used as raw material was obtained from the local farming community. Chitosan (MW = 190,000-300,000 g/mol with 80 % deacetylation), was purchased from Sisco Research Laboratories Pvt. Ltd. Mumbai, India. PVP (approximate MW= 40,000g/mol), Acetic acid, Sodium Hydroxide, Sodium hypochlorite, Oxalic acid, deionised Water and other analytical grade reagents were purchased from Spectrochemicals Pvt. Ltd. Mumbai, India. 2.2. Preparation of cellulose nanocrystals from Hibiscus cannabinus Alkali treatment The alkali treatment was performed to purify the cellulose by removing lignin and hemicellulose from Hibiscus cannabinus fibers. The fiber was cut into small piece, and then treated with an alkali solution (2 weight% NaOH). The mixture was transferred into a flask and treatment was performed in an autoclave with a pressure of 15 lbs and at a temperature of 120°C for a period of 1h. The alkali treated fiber was then filtered and washed several times using distilled water. Bleaching process
4
Following alkali treatment, the bleaching process was completed by adding a buffer solution of acetic acid, aqueous chlorite (2 %) for an hour and repeated for several times followed by washing with water till the smell of bleaching agent was removed. Acid hydrolysis The acid hydrolysis treatment was conducted on the fibers after alkali treatment and bleaching using oxalic acid for 5h in an autoclave in the pressure of 20 lbs. The fiber was neutralized and the suspension was diluted with water, kept for stirring in a magnetic stirrer for 8h. The pH of the suspension was around 6.7. The nanocellulose was obtained from the suspension by drying it at room temperature. The schematic presentation of whole process is shown in Fig. 1.
2.3. Chitosan/ Poly(vinyl pyrrolidone)/Nanocellulose blending and Nanocomposite preparation The Solution casting method was used to prepare the bionanocomposite of chitosan/PVP/nanocellulose (CPN). Scheme-1 shows the flow chart of preparation of CPN composite. Chitosan solution (1% w/v) was prepared in acetic acid (1 % v/v) stirring was conducted at room temperature for 2h using Homogenizer. PVP solution (1% v/v) was prepared in deionized water. The obtained solution of PVP was added into the chitosan solution, followed by mechanical stirring of 4 h to form CS/PVP (50:50) blend solution. To this blend solution of CS/PVP aqueous dispersion of nanocellulose of different compositions (1wt%, 3wt%, 5wt % and 10% wt) were added with constant stirring for 8 hours at room temperature to get a homogenous solution. The resulting mixture was deformed under rest for 1h at room temperature then it was cast in a Teflon mold and placed in a 37°C oven to evaporate water. The dried composites were peeled from the Teflon plate and kept in a desiccator at room temperature until further use. The membranes were coded as CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5%, CPN10%.
2.4. Characterization techniques 2.4.1. Transmission electron microscopy (TEM) Transmission electron microscopy (model Philips CM 200) was used to determine the dimensions of the nanocellulose obtained from the Hibiscus Cannabinus fibers and CPN nanocomposites. A drop of a diluted suspension of nanocellulose and its nanocomposites was
5
deposited on the surface of a clean copper grid and coated with a thin carbon film and dried at room temperature. 2.4.2. Thickness of the composite Thickness of the different composite was determined by a hand-held digimatic micrometer Mitutoyo Corporation, Japan. Model-MDC-1”SB. For each sample, 5 random positions around the composite were measured and mean value was taken as thickness of composite. 2.4.3. Barrier properties The moisture permeability of the composite was carried out by measuring the water vapor transmission rate (WVTR) according to the ASTM standard method E 96-95. To measure WVTR, the composites were mounted at the top of the vial (19 mm diameter) containing 10 mLs of water. Then, the vials were placed in a desiccator with a saturated solution to ammonium sulfate at 37°C. The assembly was weighed at regular intervals of time and a weight loss vs. Time plot was constructed. From the slope of the plot, WVTR was calculated as follows: WVTR = slope × 24⁄A
g
[m2 /day]
(1)
Where‘A’represents the permeation area of the sample (m2). Experiments were done in triplicate. Oxygen permeability (Dk) was evaluated at 25 °C on composite equilibrated at 54% RH by measuring the oxygen transference rate (OTR) with a gas permeability tester (Ox-Tran 1/50 system), following the ASTM D3985-05 standard. Dk was calculated following the expression: Dk = (OTR × l)/ΔP
(2)
Where ‘l’ is the composite thickness and ‘ΔP’ is the difference between oxygen partial pressure across the composite. Four replicates per composite were made. 2.4.4. Swelling parameter The swelling parameters like swelling ratio and swelling coefficient were evaluated to assess the extent of swelling behaviour of nanocomposites in PBS solution. The swelling ratio was measured by immersing a preweighed dry sample in appropriate swelling medium of phosphate buffer saline solution (PBS) to pH 7.4 at a given time interval (1h, 2h, 3h, 4h, 5h, 6h, 7h, and 8h) at 37°C. Excess surface water was blotted out with filter paper before weighing. Highly swollen samples were placed like sandwich between two sieves and then blotted with filter paper. Percentage swelling of the composite at equilibrium was calculated from the formula. 6
Swelling % = Ww − Wd /Wd × 100 %
(3)
Where Ww and Wd are weights of wet and dry sample respectively. The swelling behavior of the composites can also be analyzed from the swelling coefficient values. It is an index of the ability with which the sample swells and this was determined using the equation. Swelling coefficient, α = {As/m} × [1/d]
(4)
Where, As is the weight of the solvent sorbed at equilibrium swelling, ‘m’ the mass of the sample before swelling and‘d’ the density of the solvent used. 2.4.5. Mechanical studies Tensile testing was carried out using MTS Criterion 5 kN testing Machine according to ASTM D-638-2010 with a crosshead speed of 50 mm/min. The measurements were done at 25 ºC. Sample dimensions were 4mm X 10mm. At least five sample specimens for each set were tested to get the average value. 2.4.6. Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) spectra The chemical structure of the prepared membranes was characterized using an attenuated total reflectance Fourier transform (ATR-FTIR) spectrophotometer (Shimadzu IR affinity-1S). Each spectrum was accrued of transmittance mode on a Quest ATR ZnSe crystal cell by accumulation of 250 scans with a resolution of 4 cm-1 and a wave number range of 4000-400 cm-1. 2.4.7. Thermogravimetric analysis (TGA) The thermal behaviour of the sample were performed with a NETZSCH STA 449F3 thermal gravimetric analyzer under a nitrogen atmosphere and at a heating rate of 10°C /min in the temperature range of 25-600°C. 2.4.8. X-ray diffraction analysis (XRD) X-ray diffraction patterns of membranes were analyzed using a X-ray diffractometer (XRD-SchimadzuXD-D1) with Nickel-filtered Cu- Kα radiation at voltage 30kV and current of 45 mA. The samples were scanned from 10º to 60 º 2θ at a scanning rate of 3º min−1. 2.4.9. Antibacterial Activity The antibacterial activity of CPN composite was tested by an inhibition zone method. Staphylococcus aureus MTCC1688 and Pseudomonas aeruginosa MTCC3615 were used as examples of gram-positive and gram-negative bacteria. Zone inhibition test was carried out with a modified agar diffusion assay. CPN composite was placed on nutrient agar in petri dishes 7
which had been seeded with 20 µl of bacterial cell suspensions. The petri dishes were examined for zone of inhibition after 24 h incubation at 37 ºC. The presence of any clear zone that formed around the film disc on the plate medium was recorded as an indication of inhibition against the bacterial species. The area of the whole zone was calculated and then subtracted from the disc area, and the difference in area was reported as the zone of inhibition. 2.4.10. Hemocompatibility In this experiment, a dry composite was equilibrated in saline water (0.8 % NaCl solution) for 24 hrs at 37 ± 0.5 ºC. Fresh goat blood (0.25ml) was added onto the wet composite. After 20 minutes 2 mL of saline water was added onto the composite to stop hemolysis and the test sample was incubated for 60 min at 37 ± 0.5ºC. Positive and negative controls were obtained by adding 0.25 ml of fresh goat blood and saline solution respectively into 2.0 ml distilled water. Incubated test samples were centrifuged at1000 rpm for 45 min and hemoglobin released was measured by the optical densities (OD) of the supernatant at 540nm using an UV-Visible Spectrophotometer (Shimadzu UV Visible Spectrophotometer, UVmini-1240). The percentage of hemolysis was calculated according to the following formula. Hemolysis (%) = (ODs – OD (-)) / (OD (+) – OD (-)) × 100
(5)
Where ODS is the optical density of the sample, OD (-) is the optical density of negative control, OD (+) is the optical density of positive control. 2.4.11. Cytotoxicity Cytotoxicity assay of CPN composites was performed on the basis of NIH3T3 cell viability using MTT (3-(4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide) assay (Sigma, St Louis, USA). NIH3T3 cells were mouse embryonic fibroblast cell line cultured in Dulbecco’s minimal essential medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), antibiotics (100 U/mL penicillin and 100 g/mL streptomycin) at37◦C and 5% CO2. The composites were immersed in DMEM culture media (0.2 g dry composite per mL media) for 24 h. Then, the extract was added over cultured NIH3T3 cells in 96-well plate for 1, 3, 5 days. The viability of cells in each well was evaluated by MTT method (Liu et al., 2010), and DMSO solution with a concentration of 5.0 % was used as a control. 2.5. Statistical analysis All the experiments were performed in triplicate and student’s t-test was performed to determine the statistical significance p<0.05 was considered statistically significance. 8
3. Results and discussion 3.1. Transmission electron microscopy (TEM) The morphology of synthesized acid hydrolysed nanocellulose and nanocomposites reinforced with 3 wt% and 5 wt% of nanocellulose was examined using TEM at the nanoscale. The shape of the nanocellulose in Fig.2 (a) is observed as needle like shape and the size distribution histogram (Fig.2 (b)) obtained through particle size analysis tests for nanocellulose also revealed mean diameter of 6.1 ± 5 nm. The corresponding Selected Area Electron Diffraction (SAED) Fig.2 (c) shows the crystallinity of the nanocellulose. A visible diffraction pattern as bright spots proves crystallinity of the nanocellulose. In the TEM image of Fig.3 (a, b) shows the shape of the prepared nanocomposites is spherical and the interface of nanocellulose with CS/PVP matrix appears to be efficiently bonded. In other words, there is no trace of polymer aggregation visible on to the surface of the nanocellulose. This is a very important aspect for nanocomposites in order to get maximum advantage of incorporating nano-fillers in polymer matrices and particles are fairly monodispersed with respect to their size, which is approximately 2-10 nm. The narrow size distribution and absence of agglomerates prove that CS/PVP matrix prevents agglomeration of the nanocellulose. Chitosan and PVP have oxygen and nitrogen bearing groups that can stabilize the nanoparticle surface. The strongest interactions with nanocellulose likely involve with oxygen and hydrogen. 3.2. Thickness of the composite Thickness of CPN nanocomposite was varied from 32.82 ± 2.4 to 46.18 ± 7.5 µm, higher than normal thickness of chitosan/PVP membrane (Table 1). The biomaterial for wound dressing should ideally be thinner than the human dermis, whose thickness varies from 30-50 µm depending on age, sex and body region where the biomaterial is to be applied. The use of scaffolds with thickness lower than 1 mm has been described in the literature for the regeneration of human skin [27], which indicates that the composite produced can be considered adequate for healing purpose. Chitosan is slowly biodegradable and positively charged so that a thinner chitosan composite can act as a dermis substitute. Therefore, we considered that 30-50 µm dry thickness of the nanocomposite was enough for cell attachment in vitro and for in vivo biodegradation in a certain period of skin tissue healing. 3.3. Barrier properties 9
The ideal wound dressing should has several characters, such as ability to control gases diffusion, maintains a moist environment around the wound, prevent further inflammation, simple and easy to use with little or no pain from the wound and cosmetically acceptable. The wound dressing material has to be permeable to control the moisture and gases in order to help in the wound healing process [28]. Fig.4. shows the water vapour transmission rate and oxygen permeability of composites. The WVTR of CPN0% composite was 2128.0 g/m2 day, and it decreased significantly when nanocellulose was incorporated. The WVTR decreased linearly down to 1742.9 g/m2 day when the nanocellulose was added up to 3 wt%, and then it increased slightly with increase in the concentration of nanocellulose. The nanocellulose in the composite act as an impermeable barrier against water vapor which increased the tortuous path for water vapor to diffuse through the membrane and resulted in the decreased WVTR of the composite [29]. The reduction in WVTR is probably due to surface densification and the partial closure of the surface pores by the nanocellulose. Water molecules might be strongly adsorbed to the surface or of the nanocellulose which could result in lower water vapor transmission through the composite. This is mainly due to the high affinity between water and the composite. Providing better cellular growth, oxygen is required at almost every step of wound healing process and to reduce the growth of anaerobic bacteria. So evaluation of oxygen permeability of nanocomposite is important for its application as wound dressing. In the present case, nanocomposites have shown considerable permeability to oxygen. The results showed the nanocomposite which can allow oxygen molecules to penetrate through them and reach the wound site to help in the wound healing process. From Fig.4 it can be observed that the Dk of the nanocomposite rises upon increasing nanocellulose concentration. The increased number of polar -OH groups with increasing nanofiller content leads to an enhancement of the oxygen solubility in the nanocomposite.
3.4. Swelling parameter Swelling behaviour and structural stability of nanocomposite are critical for their practical use in wound dressing application. Most natural polymers, including chitosan, swell readily in biological fluids. There are several parameters affecting the swelling ratio, hydrophilicity, stiffness and pore structure of a nanocomposite. The sample with the highest 10
degree of swelling will have the highest surface area/volume ratio. The swelling parameter of CPN composite is presented in Fig.5 (a, b). Incorporation of nanocellulose significantly increases the swelling percentage of CPN, with the most significant increase at 5 wt % nanocellulose. The main reason for higher swelling rate of nanocomposite is due to the presence of hydrogen bonding of water molecules to the free -OH and -COOH groups present in cellulose molecules [30]. The hydrophilic nature of chitosan and also PVP may be a major factor that influences the extent of swelling of the CS / PVP matrix. The swelling coefficient values of the nanocomposite were decreased upon increasing nanocellulose loading, but the decrease is not regular. It is clear that as nanocellulose increases, the equilibrium solvent uptake decreases. This is due to the increased hindrance exerted by the crystals at higher loadings. Nanocomposite with 3 wt % nanocellulose content shows lower values for swelling coefficient than that with 5 wt% nanocellulose content. 3.5. Mechanical studies The mechanical properties of the composites depend on the orientation of the filler, content and size as well as the interaction between filler and polymer matrix [31]. The mechanical properties of the CPN composites are shown in Table 2. The remarkable increase in tensile strength and modulus of these composites indicates the presence of some interaction between nanocellulose and chitosan molecules in the composite. Increasing NC level incorporated into CS/PVP matrix could increase Young’s Modulus by 1743-2190 MPa. This could be relevant to two factors: (a) proper connections between polymer-nanocrystals (b) efficient transfer of involved stress through polymer-NC layers. In fact, improvement of tensile properties for the composite is due to a suitable stress transfer throughout polymer, even distribution of entered stress and minimizing the areas of stress concentrations. With the addition of nanofiller, Elongation at break dropped from 53.2% for CPN0% to 50.5% for CPN10%. This decrease in the Elongation at Break value indicated that motion of the CS/PVP matrix was restricted because of its interactions with nanocellulose. The mechanical test result suggests that there is an optimum concentration of the filler to induce the maximum increment of the strength of the nanocomposite. 3.6. Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) spectra To analyze the nanocomposites structure and to identify the molecular groups in chemical interaction with nanocellulose, the prepared bio-nanocomposite will be investigated by 11
Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR). Fig.6. represents the overall ATR- FTIR spectra of the nanocomposite membrane with varying nanocellulose concentration. The CPN0% showed distinct -C=O group absorption spectra at 1647 cm-1 and -OH absorption for chitosan in the blend at 3084 cm-1. In case of chitosan/PVP/nanocellulose composite membrane, additional bands were observed in addition to the bands of cellulose polymer. These additional bands are attributed to the nanocellulose and PVP in the composite membrane. The vibrational peak at 1430 cm−1 corresponds to -CH2 bending vibration that attributed to the crystalline peak of cellulose, however, the vibrational peak at 896 cm−1 is attributed to the C-O-C stretching vibration of amorphous band of cellulose. Finally, the band observed in the 1028–1161 cm-1 range was attributed to C–O stretching and C– H rocking vibrations of the pyranose ring skeleton [32]. It can be clearly seen that the resulting nanocellulose band around 1000 and 3400 cm−1 due to the isolated cellulose component. These bands could be associated with glucose units (with three additional hydroxyl groups) exposed on the cellulose backbone structure. However the most prominent band at 1553 cm-1 assigned to amino group in pure chitosan film shifts to a higher wavenumber (1557 cm-1) in the presence of nanocellulose. It is quite evident that the primary amino groups are in interaction with the cellulose surface (Scheme-2). The amino groups act as capping sites for the cellulose nanoparticles stabilization.
3.7. Thermogravimetric analysis (TGA) The thermal stability of the CPN composites of different ratios was studied by means integral (TGA) and derivative (DTG) thermogravimetric curves and the results are shown in Fig.7 (a, b). The results from the TGA show residual weight vs temperature for prepared nanocomposite. The TGA curves of chitosan, nanocellulose was almost similar and chitosan, PVP, nanocellulose shows single weight loss region ranging from 250°C-310°C, 410°C-470°C and 260°C-330°C respectively. The TGA curves of all composite shows three weight loss regions. The first region in the range of 70-150°C is due to the evaporation of weakly bound water with a weight loss of around 10%. The second transition region for CPN0% was around 150-320°C, due to structural degradation of PVP with weight loss of 30% and for CPN0.5%, CPN1% were around 150-320°C and 150-360°C due to structural degradation and burning out of nanocellulose and CPN3%, CPN5% and CPN10% were in the region of 150-390°C. The third 12
stage of weight loss occurred for CPN0% was 320-480°C due to cleave of backbone of chitosan where the weight loss around 85% and increasing gradually to 100%. The third stage loss of CPN0.5% and CPN1% were in the region of 320-480°C and 360-480°C. The TG curves of CPN3%, CPN5% and CPN10% were quite similar to each other, and their degradation started much earlier as compared to CPN0%, CPN0.5% and CPN1%. From the Fig.7 it can be concluded that the thermal stability decreases with increases nanocellulose content.
3.8. X-ray diffraction analysis (XRD) X-ray diffraction was used to determine information regarding the crystalline structure of the nanocomposite. The CPN composites obtained with various nanocellulose concentrations had almost same XRD patterns and the results were shown in Fig.8. The CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5%, CPN10% exhibited its characteristic semicrystalline peaks around 2θ = 22.0, 21.9, 21.7, 21.5, 21.4 and 21.2 respectively. Even though the nanocellulose prepared by acid hydrolysis possess enough crystallinity observed by TEM the synergetic amorphous effect of CS/PVP matrix diminishes the crystallinity of nanocellulose. 3.9. Antibacterial Activity CPN composite was tested against microorganisms to determine antibacterial activity. Table 3 shows a typical antibacterial test result of chitosan-based nanocomposite against Staphylococcus aureus MTCC1688 and Pseudomonas aeruginosa MTCC3615 determined by the zone inhibition method. The diameter of the growth inhibition zone was dependent on the composite used. As shown in Fig.9. the largest inhibition zone were observed for the Gramnegative bacteria Pseudomonas aeruginosa, while the smallest one were observed for the gram positive Staphylococcus aureus bacteria for CPN1%, CPN3% and CPN5%. The reason could be explained in two aspects. On the one hand, chitosan exhibited bactericidal activity through contact killing or dissolving from the nanocomposite. Interaction between positively charged chitosan molecules and negatively charged microbial cell membranes leads to the leakage of bacteria by disrupting the inner organelles or disturbing the metabolism of bacterial strains and another is the extremely high surface area of nanocellulose facilitated the adsorption of target bacteria, which accelerated the rate of antibacterial reaction [33]. According to Zivanovic and Draughon [34], the proposed mechanism of antibacterial activity of bionanocomposites are their attack on the phospholipid cell membrane, which causes 13
increased permeability and leakage of cytoplasm, or in their interaction with enzymes located on the cell wall. Thus, the resistance of Gram-negative bacteria to the composite likely lies in the protective role of their cell wall lipopolysaccharides or outer membrane proteins. 3.10. Hemocompatibility Hemolysis assay was carried out to study the hemolytic effect of composite on blood. The basic observation was to find out the release of haemoglobin into plasma due to damage of the erythrocyte membrane. The hemolysis of CPN composite was measured against positive and negative control. The % hemolysis of CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5%, CPN10% were 0.67±0.08, 0.74±0.2, 0.89±0.5, 1.94±0.26, 5.1±0.6 and 6.4±0.2 % respectively. As shown in Fig. 10, all the composites were showed very low hemolysis ratios at the low concentration of nanocellulose. When the concentration was increased to 5 and 10%, the CPN composite showed a relatively higher hemolysis of 5.1±0.6 and 6.4±0.2 % respectively, while the other composite still exhibited low hemolysis ratios. Generally, smaller the hemolysis ratio value, the better the blood compatibility of the biomaterial [35]. The prepared nanocomposite could be highly hemocompatible since the percentage of hemolysis for the membranes were <2%. 3.11. Cytotoxicity Cytotoxicity is one of the most important properties for material used in for wound healing applications. The fibroblasts (NIH3T3) stand for a typical embryonic cell and were used to evaluate the cytotoxicity of wound dressing material. Bionanocomposite with cell viability larger than 80% can be considered as noncytotoxic defined by the ISO standard [36]. As shown in Fig.11, the cell viability data of control (5% DMSO) did not show any toxicity at 1, 3 and 5 days in contact with NIH3T3 cells. All bionanocomposites showed 40-70% viability after 1 day of incubation and 60-80 % viability after 3 and 5 days of incubation. Cell viability of CPN3% and CPN5% was increased up to 90% at 5 days of incubation. The cell viability increased due to the multiplication of NIH3T3 cells. Hence the above results suggest that CPN3% and CPN5% had more cell viability and good compatibility. 4. Conclusion High performance novel polymer bio-nanocomposite films were prepared by solution casting of chitosan, Poly (vinyl pyrrolidone) (PVP) and nanocellulose. The structural characterizations showed that the chitosan and PVP are perfectly compatible and miscible polymers by the hydrogen bond interactions between the carbonyl groups of PVP and amino and 14
hydroxyl groups of chitosan, resulting in the formation of new biocompatible homogeneous blend matrix for bionanocomposite development with nanocellulose. When the nanocellulose has been added to CS/PVP blend, some additional hydrogen bonding can occurs between the hydroxyl groups in nanocellulose and amino and hydroxyl groups of CS and between the carbonyl groups of PVP. Therefore, an interconnected structure is assumed to be formed in CS/PVP strengthened by nanocellulose. Since that the special two-dimensional morphology of the nanocellulose as well as its functionalized surface provides well homogeneous dispersion, which leads to a high contact area in the CS/PVP matrix. Owing to these strong interfacial interactions, large improvements of certain properties, such as barrier properties and mechanical properties were achieved for CS/PVP-based nanocomposite. With the incorporation of 5% wt of nanocellulose, the swelling property is increased to a greater extent. In addition, tensile strength of CPN3%, CPN5%, CPN10% was increased by 35.6 ± 5.8, 38.4 ± 2.7 and 39.7 ± 6.9 respectively. Bionanocomposites of CPN3%, CPN5% exhibits superior antibacterial activity against gram positive and gram negative bacteria pathogens and good blood compatible property and cytotoxicity. Therefore, the prepared bio-nanocomposite membrane with features of good tensile, good thermal and high swelling property will have potential applications as an eventual wound dressing material.
15
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Figure legends Fig.1. Schematic representation of Isolation of Nanocellulose from Hibiscus cannabinus Fig.2. Transmission electron micrograph of a) Nanocellulose b) Histogram of nanocellulose c) SAED pattern of nanocellulose. Fig.3. Transmission electron micrograph of a) CPN3% and b) CPN5%. Fig.4. Water vapour transmission rate (WVTR) and Oxygen permeability (OP) of CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5% and CPN10% bionanocomposites. Fig.5. a) Swelling Ratio and b) Swelling Coefficient of CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5% and CPN10% bionanocomposites. Fig.6. ATR-FTIR Spectra of CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5% and CPN10% bionanocomposites. Fig.7. a) TGA thermogram and b) DTG curves of CS, PVP, Nanocellulose, CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5% and CPN10% bionanocomposites. Fig.8. XRD patterns of CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5% and CPN10% bionanocomposites. Fig.9. Inhibitory effect of CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5% and CPN10% bionanocomposites against a) Staphylococcus aureus and b) Pseudomonas aeruginosa. Fig.10. Hemolysis of CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5% and CPN10% bionanocomposites. Fig.11. Cytotoxicity of CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5% and CPN10% bionanocomposites expressed as a percentage of cell viability for incubation time of 1, 3 and 5 days.
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Fig.1. Schematic representation of Isolation of Nanocellulose from Hibiscus cannabinus
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Fig.2. Transmission electron micrograph of a) Nanocellulose b) Histogram of nanocellulose c) SAED pattern of nanocellulose.
Fig.3. Transmission electron micrograph of a) CPN3% b) CPN5%.
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Fig.4. Water vapour transmission rate (WVTR) and Oxygen permeability (OP) of CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5% and CPN10% bionanocomposites.
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Fig.5. a) Swelling Ratio and b) Swelling Coefficient of CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5% and CPN10% bionanocomposites.
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Fig.6. ATR-FTIR Spectra of CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5% and CPN10% bionanocomposites.
Fig.7. a) TGA thermogram and b) DTG curves of CS, PVP, Nanocellulose, CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5% and CPN10% bionanocomposites.
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Fig.8. XRD patterns of CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5% and CPN10% bionanocomposites.
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Fig.9. Inhibitory effect of CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5% and CPN10% bionanocomposites against a) Staphylococcus aureus and b) Pseudomonas aeruginosa.
Fig.10. Hemolysis of CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5% and CPN10% bionanocomposites.
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Fig.11. Cytotoxicity of CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5% and CPN10% bionanocomposites expressed as a percentage of cell viability, for incubation time of 1, 3 and 5 days.
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Scheme-1. Flow diagram of the Synthesis of Chitosan/ Poly (vinyl pyrrolidone)/Nanocellulose composite.
Scheme-2. Intermolecular Hydrogen bond in CPN bionanocomposite.
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Table 1 Thickness of the CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5% and CPN10% bionanocomposites. Sample
Nanocomposites
Thickness(µm)
a
CPN0%
32.82 ± 2.4
b
CPN0.5%
34.61 ± 1.9
c
CPN1%
36.83 ± 4.9
d
CPN3%
39.04 ± 5.7
e
CPN5%
43.96 ± 3.3
f
CPN10%
46.18 ± 7.5
Table 2 Mechanical properties of CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5% and CPN10% bionanocomposites. Data are presented as the mean ± SD (n = 4, P<0.05). Sample
Tensile strength
Elongation at break
Young’s Modulus
(MPa)
(%)
(MPa)
CPN0%
27.1 ± 9.5
53.2 ± 7.2
1743.2 ± 2.8
CPN0.5%
31.7 ± 7.3
52.9 ± 4.5
1842.8 ± 7.1
CPN1%
34.0 ± 8.3
51.9 ± 5.4
1895.3 ± 5.8
CPN3%
35.6 ± 5.8
51.5 ± 4.1
1902.4 ± 3.0
CPN5%
38.4 ± 2.7
50.9 ± 3.8
2127.5 ± 7.9
CPN10%
39.7 ± 6.9
50.5 ± 1.3
2190.4 ± 3.8
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Table 3 Antibacterial activity of CPN0%, CPN0.5%, CPN1%, CPN3%, CPN5% and CPN10% bionanocomposites. Sample code
Zone of Inhibition(mm) S. aureus MTCC 1688
P. aeruginosa MTCC3615
CPN0%
10.9 ± 0.7
10.9 ± 0.3
CPN0.5%
11.5 ± 0.3
11.2 ± 0.0
CPN1%
11.3 ± 0.8
11.9 ± 0.6
CPN3%
11.8 ± 0.1
12.3 ± 0.2
CPN5%
12.6 ± 0.2
12.8 ± 0.9
CPN10%
12.5 ± 0.4
12.5 ± 0.3
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