Functionalization of carbopol with NVP for designing antibiotic drug loaded hydrogel dressings for better wound management

1. Introduction

The concept of wound treatment always focuses on reducing the risk caused by wound itself, minimizing potential environmental complications and enhancing proper regeneration and reestablishment of the wounded skin tissue. [1] Infected skin that is left untreated may delay healing and even lead to death.[1, 2] Recently, a wide range of wound dressings are available in market, but hydrogel is a material of choice for designing dressings for better wound management.Hydrogel dressings are more successful in clinical results as compared to the conventional wound dressings. Hydrogel wound dressings containing antibiotic drugs have been used against wound infections.[3]In order to maximise the effectiveness of antibiotics for longer periods, slow and sustained release of antibiotics is very important. These drug loaded hydrogel dressings have been found to be instrumental in achieving successful and long lasting healing effects from antibiotic loaded wound dressings.[4]

Hydrogels are three-dimensional, hydrophilic, polymeric networks capable of absorbing large amounts of biological fluids. These are closely related to the natural living tissue, more than any other class of synthetic biomaterials due to their high water content, porosity and soft consistency. [5]They can be made from a wide range of biocompatible materials. They can provide moist healing environment, and slow drug delivery at wound site.[6] Hydrogels are capable of promoting the autolytic debridement of necrotic tissues and are usually more efficient at drying wounds with few exudates.[ 7 ] A great advantage of hydrogel dressings is that they are usually applied and removed without interfering with the wound bed. In addition, these dressings are flexible, nonantigenic, and permeable to water, oxygen and metabolites.[ 8]

In view of the above, it is reasonable to assume that a antibiotic drug (moxifloxacin) loaded hydrogel dressings composed of carbopol-poly (NVP) would provide the slow release of antibiotic drug locally in a controlled manner besideproviding moistwound for improving wound healing. Poly (NVP) is a water solublehydrophilic biocompatiblepolymer.[ 9, 10 ] Its hydrogel films have been applied as local dressings.[ 11 ] These hydrogel dressings can act as a barrier against bacterial penetration.[ 12] It is one of the most frequently used material in biomedical applications due to their biocompatibility with living tissues and extremely low cytotoxicity.[ 13, 14]Inclusion of poly(NVP) in the composite hydrogels can improve swelling and other biomedical properties.[ 9, 15] On the other hand, carbopol is a hydrophilic, non-toxic, biocompatible, bioadhesive and non-irritant polymer.[ 16, 17]It has been used in drug delivery systems for buccal, transdermal,[ 18] ocular, rectal, vaginal,[ 19 ] nasal[20] and wound dressing applications.[ 21]

2. Experimental

2.1 Materials used

1-Vinyl-2-pyrollidone (NVP) [MerckSpecialities Pvt. Ltd. Mumbai-India], carbopol (CP)[Loba Chemie Pvt. Ltd., Mumbai-India], N,N-methylenebisacrylamide (NN-MBA) [Acros organics, New Jersey-USA], ammonium persulphate (APS) [Qualigens Fine Chemicals, Mumbai-India], glycerol, [S.D. Fine Chemical Ltd., Mumbai, India[, and moxifloxacin HCl [Lifestar Pharma Pvt. Ltd., New Delhi, India] were used as received.

2.2 Synthesis of CP-cl-poly(NVP) polymers

Synthesis of the polymers was carried out by chemically induced free radical copolymerization method using APS as initiator and NN-MBA as cross-linker. Polymer reaction was carried out with solution of definite concentration of CP [2.0% (w/v)], taken in beaker. This solution was prepared by stirring at 100 rpm for definite time after 12 hours hydration. To this homogenous content, solutions of a definite concentration of [NVP] = 28.15×10² mol/L, [NN-MBA] = 8.10×10³ mol/L, glycerol = 0.27 mol/L and [APS] = 5.48×10³ mol/L were added, and the reaction mixture was again stirred. The reaction content was stirred for total 12 hours at 25 °C. The reaction mixture was then transferred to the petri dish which was placed in hot air oven maintained at 55 °C to get the cross-linked polymer. The polymer films were formed by solution casting method. The cross-linked polymer formed was washed with distilled water and ethanol to remove the soluble fractions left in the polymer. Then it was dried in an oven maintained at 40 °C till constant weight was obtained. This cross-linked polymer was named as CP-cl-poly(NVP) polymer.

2.3 Characterization

The polymers were characterized by scanning electronmicrography (SEMs), energy dispersion X-ray analysis (EDX), atomic force microscopy (AFM), Fourier transform infrared (FTIR) spectroscopy, ¹³C nuclear magnetic resonance (NMR) spectroscopy, X-ray diffraction study (XRD), thermo gravimetric analysis (TGA), differential scanning calorimetry (DSC) and swelling studies.SEM-EDX analysis was done on FEI Quanta 200F (The Netherlands). AFM (NTEGRA, NT-MDT, Russia) of polymer film was done in 5m² areas, in semicontact mode. FTIR of polymer was recorded on Perkin Elmer-Spectrum RX-I (USA) The solid state ¹³C NMR of polymer was carried out using JEOL RESONANCE ECX 400 solid state NMR spectrometer operating at a magnetic field of 9.3 T and at a frequency of 100 MHz for carbons. X-ray diffractogram were obtained using a Bruker D8 Advance diffractometer (USA), operating at 40 kV with Cu-K radiation.TGA of polymer was done using EXSTAR TG/DTA 6300 thermal analyzer (Japan), at a scan rate of 10 °C/min.DSC thermogram of polymer was recorded with NETZSCH DSC 204 (USA) in temperature range 25-500 °C at a scan rate of 10 °C/min.

2.4 Swelling Studies

Swelling studies of the polymers were carried out in different mediums for 24 hours, by gravimetric method.[ 22]

2.5 Drug release studies

Initially the drug loading into hydrogels was carried out by the swelling equilibrium method.[ 22] Release profile of moxifloxacin HCl from drug loaded polymer samples was carried out in simulated wound fluid (SWF) and release was determined from the standard curve made on Cary 100 Bio, Varian, UV visible spectrophotometer at λ_max (288nm). Drug release mechanism was evaluated by using power law expression given by Ritger and Peppas.[ 23, 24] Different kinetic models (i.e. zero order, first order, Higuchi square root law, Korsmeyer-Peppas and Hixson-Crowell cube root model) were applied to drug release profileto determine the best fit model for the release of drug from the drug loaded polymers.[25, 26]

2.6 Blood compatibility

As a preliminary investigation, the in vitro biocompatibility was determined on the basis of thrombogenicity and hemolysis assessment. Thrombogenicity of CP-clpoly(NVP) polymer was carried out using a gravimetric method.[ 27] The positive and negative control was taken as glass beaker with absence of sample and glass beaker with absence of sample and blood respectively.[ 28 ] The thrombose percentage is calculated as follow:

The haemoglobin released by haemolysis was measured by the optical densities (OD) of the supernatants at 540 nm using a UV-visible spectrophotometer. The percentage of haemolyis (%) was calculated as follows:

Baljit Singh, et al. Functionalization of carbopol with NVP for designing antibiotic drug loaded hydrogel dressings for better wound management3

2.7 Mucoadhesion studies

Mucoadhesive property of the polymer was investigated by using texture analyzer (TA-XT, Stable Micro Systems, UK) equipped with a 5 kg load cell and a mucoadhesive holder. The maximum force required to separate the probe from the goat mucosa (i.e. maximum detachment force, F_max) and distance travelled by film before detachment could be directly recorded in the instrument.

2.8 Oxygen permeability

Permeability of synthesized polymer towards O₂ has been evaluated by using Winkler's method.[ 29] The test flasks were placed in an open environment for 24 h.[ 3] The collected water samples were then analyzed for dissolved oxygen according to Winkler's method.[ 29]

2.9 Water vapour transmission rate

WVTR of polymer film was determined gravimetrically using desiccant method.[30] All the vials were then placed in desiccator, containing saturated solution of NaCl at 37 °C (Relative humidity 75%).[ 31] These vials were weighed after every 24 hours up to eight days and WVTR (g/m²/day) was calculated using following equation.[31]

In this equation ( w/t) is the slope of the plot ‘w’ vs ‘t’, where ‘w’ is the weight gain (g) along the specified time period, ‘t’ (h) and A is the effective transfer area (m²).

2.10 Microbial penetration

In the present study, microbial penetration test was performed for polymer film to check their capacity, to prevent secondary infection. The polymeric film (thickness = 1mm) was placed on top of the test tubes (test area: 1.3±0.03 cm²) containing 5ml of sterile nutrient broth (2.5% w/v) (Merck Specilities Pvt. Ltd. Mumbai-India) and was sealed to test microbial penetration through them. Before test, polymeric films, nutrient broth and glass test tubes were sterilized in autoclave at 121 °C, 15lbs pressure for 20 minutes. The negative control was the closed test tube with an airtight cap preventing any kind of microbial penetration, while the positive control was the open test tube having no barrier for microbes. The test tubes were placed in an open environment and observed for 30 days period. Permeability for micro-organisms was evaluated by observing microbial growth (in term of turbidity) in test tubes. Any visual turbidity of the nutrient broth was considered as sign of microbial contamination.

2.11 Mechanical properties

In order to access the mechanical strength of the polymer film, some important mechanical tests such as, tensile strength, burst strength, resilience, relaxation, and folding endurance have been performed.

2.11.1 Tensile Strength

Tensile strength of polymeric film was studied using texture analyzer (TA-XT, Stable Micro Systems, UK) equipped with 50 kg load cell.

2.11.2 Bursting strength

Bursting strength of polymeric film was studied using texture analyzer (TA-XT, Stable Micro Systems, UK) equipped with 50 kg load cell. The force (N) and distance (mm) at break point were recorded.

2.11.3 Resilience

Resilience is measurement of how well a sample recovers from deformation. Resilience of polymeric film was studied using texture analyzer (TA-XT, Stable Micro Systems, UK) equipped with 50 kg load cell. Resilience was calculated by using following equation.

where A₂ is the work returned by the sample as compressive strain is removed (known as recoverable work) andA1 is work required for compression.

2.11.4 Stress relaxation test

Stress relaxation of polymeric film was studied using texture analyzer (TA-XT, Stable Micro Systems, UK) equipped with 50 kg load cell. Stress relaxation was evaluated in terms of retained force (%) which was calculated by the following equation.

2.11.5 Folding endurance

The flexibility of the film was accessed from the determination of the folding endurance of film. During folding endurance test, polymer film of thickness 1 mm, length 30 mm and breath 30 mm was repeatedly folded and de-folded at the same place until it breaks. The number of repeated folding and de-folding at the same place without breaking or cracking gives the value of folding endurance.[ 32]

3. Result and discussion

3.1 Characterization

A photograph of CP-cl-poly(NVP) polymer film was showed transparent nature of the wound dressing (Figure 1(a)) which helps in monitoring of the wound healing progress wound exudates accumulation without removal of wound dressing.[ 33] Generally transparent wound dressings are preferred for partial-thickness wounds with moderate exudation. SEM image ofCP-clpoly(NVP)polymer film is given Figure 1(b)). This image showed less degree of heterogeneity in the polymer surface which indicating the high degree of miscibility of polymeric contents. Further, the average surface roughness (S_a) was 14.26 nm and root mean square roughness (S_q) was 18.59 nm from the AFM image of CP-cl-poly(NVP) polymer film (Figure 1(c))). Recently, AFM has been implemented as a surface characterization technique in biomaterial research and it is very helpful for the determination and verification of morphological features of polymer film. AFM particularly permits non-destructive imaging of surfaces on a molecular scale.[34] Being more hydrophilic poly(NVP) is more miscible in CP and it increases surface hydrophilicity of the crosslinked polymer. A more hydrophilic surface and high water content in the hydrogel leads to less surface roughness.[ 35] It has also been reported that grafting of NVP on polymers can make the surface smoother.[36, 37] In another research Kennedy and co-workers (2009) have reported that polymer grafted with NVP give smooth surface in comparison to acrylic acid grafted polymers.

(a) (b) (c)

Figure 1 (a) Photograph, (b) SEM image, and (c) AFM image of CP-cl-poly(NVP) polymer film

Results of EDX analysis of CP and CP-cl-poly(NVP) polymers indicating the incorporation of poly(NVP) in the crosslinked polymer in the form of additional peak of nitrogen which is correlated to the presence of poly(NVP) and cross linker NNMBA in the polymer film (Figure 2).

(a) (b)

Figure 2 Elemental analysis results of (a) CP and (b) CP-cl-poly(NVP) polymer

FTIR spectra of CP and CP-cl-poly(NVP) polymer are presented in Figure 3. In case of CP, a broad band at 3116.4 cm^-1 (due to O-H stretching), a band at 2961.4 cm^-1 (due to C-H stretching), a sharp intense band at 1714.1 cm^-1 (due to C=O stretching), at 1454.4 cm^-1 (due to C-H bending ), at 1413.5 cm^-1 (due to O-H in plane bending), and at 1247.4 (due to C-O stretching) have been observed. Beside, absorption band at 1171.4 cm^-1 due to C-O-C stretching was observed which has proved ethereal crosslonking in CP. Some less intense bands at 1114.6 and 1048.6 cm^-1 (due to O-H deformations), along with a sharp band at 801.5 cm^-1 due to =C-H out of plane bending vibrations have been observed. This band at 801.5 cm^-1 is characteristic of CP 940, which appears due to the presence of crosslinker (allyl ether of pentaerythrol) in carpool 940.[38–40] Baljit Singh, et al. In case of CP-cl-poly(NVP) polymera broad band around 3432.33 cm^-1 (due to O-H), a band at 2926.37 cm^-1 (due to C-H stretching), at 1737.32 cm^-1 (C=O stretching of CP), at 1636.36 cm^-1 (C=O stretching of polyNVP), at 1457.37 cm^-1 (ring CH2 wagging, ring C-N stretching of polyNVP), at 1162.36 (C-O-C stretching in CP and ring CH₂ twist of polyNVP). Some less intense bands at 1113.37 and 1051.36 cm^-1 (due to O-H deformations) have been observed.[39–41]

(a) (b)

Figure 3 FTIR spectra of (a) CP and (b) CP-cl-poly(NVP) polymer

¹³CNMR spectra of CP and CP-cl-poly(NVP) polymer are presented in Figure 4. In case of CPan intense splitted peak at 183.49, 179.90 ppm [due to carbon ofcarbonyl groups] and another intense but broad peak at 42.57 ppm [due to methylene (-CH₂-CH-) and methine (-CH₂-CH-) groups of acrylic chains] has been observed.[38, 42] The signal due to methylene and methine carbons overlapped to give a single broad peak at 42.57 ppm. In case of CP-cl-poly(NVP) polymer, the chemical shift peak at 178.44 ppm [due to carbonyl (C=O) carbons of poly(NVP) and CP] was observed. Broad peak along with some adjacent smaller peaks was observed, due to different environments of carbonyl carbon in the major constituents. Some peaks at 43.43 ppm [due to CP main chain carbons (-CH-), vinylic carbons (-CH-N<) and ring carbons (-CH2-N) of poly (NVP)].This peak represents the polymerization of N-vinyl 2-pyrrolidone to poly (N-vinyl 2-pyrrolidone). The absence of characteristic peak of NVP around =94.0 and 129.0 ppm for sp² hybridized carbon atoms (CH₂=CH-) indicate the involvement of double bond in grafting and cross-linking during polymerization reaction. Chemical shift peaks at 31.97 ppm [due to ring carbons of NVP (O=C-CH₂-CH₂-) and18.90 ppm [due to ring carbons of NVP -CH₂-CH₂-CH₂)] were also observed.[38, 43, 44] Besides, sharp peaks at 73.50 ppm and 63.94 ppm can be attributed to formation of some new cross links during polymerization reaction.

(a) (b)

Figure 4 ¹³CNMR spectra of (a) CP and (b) CP-cl-poly(NVP) polymer

XRD spectra of CP and CP-cl-poly(NVP) polymer are presented in Figure 5. In case of CP there is no crystalline region in its X-ray diffractogram, indicating completely amorphous nature of CP.[45] XRD spectra of CPcl-poly(NVP) polymer also showed no crystalline region in its polymer sample. Process of cross-linking and grafting may have hindered formation of any regular pattern in polymeric samples.

(a) (b)

Figure 5 XRD of (a) CP and (b) CP-cl-poly(NVP) polymer

TGA, DTA and DTG of CP and CP-cl-poly(NVP) polymer are presented in Figure 6. In each case weight loss due to entrapped moisture has been ignored and initial decomposition temperature (IDT) has been taken as the temperature where actual degradation of material started. In case of CP, initial 7.59% weight loss occurred up to 100 °C, which indicates that CP has 7.59% bounded water. Kanis and co-workers (2000) have observed about 5% weight loss upto 150 °C due to bounded water. Ignoring the initial weight loss due to free and bounded water, two stages decomposition was observed in CP. In the present case, first stage started at 198.99 °C (residue left = 88.78%) and second stage started at 396.86 °C (residue left = 31.92%). IDT and final decomposition temperature (FDT) have been observed 199 °C and 462 °C (residue left = 5.29%) respectively. Early degradation after 199 °C can be attributed to the formation of formation of cyclic structures (anhydrides) associated with loss of water loss of water. Final decomposition of CP after 396.86 °C onwards occuredat a very high rate, due to decarboxylation, formation of unsaturated structures, depolymerisation of the residual polymer and and complete degradation of polymer forming gaseous products.[ 46–48]In case of CP-cl-poly(NVP)initial 4.30% weight loss occurred upto 100 °C, which indicates that CP-cl-poly (NVP) polymer has 4.30% bounded water. Three stage decomposition observed in the case of crosslinked polymer. In the present case, first stage started at 152.95 °C (residue left = 91.25%), second stage started at 315.64 °C (residue left = 71.29%) and third stage started at 439.24 °C (residue left = 26.17%). IDT and FDT have been observed 157 °C and 527 °C (residue left = 0.05%) respectively.

(a) (b)

Figure 6 TGA/DTA/DTG curves of (a) CP and (b) CP-cl-poly(NVP) polymer

TGA curves show that thermal degradation of crosslinked hydrogel was slow in comparison to CP, indicating more interactions in cross-linked polymer. 50% weight loss occurred at 325 °C and 390 °C for CP and CP-cl-poly(NVP) polymer respectively, indicating that crosslinked polymeric filmsto be more thermally stable than CP. Thermal degradation of the hydrogels was slow and gradual in comparison to CP, as no sudden weight loss has been observed. Increase in thermal stability of cross-linked hydrogels can be attributed to crosslinking and grafting reactions.[49] Besides the strong interaction between carboxyl of CP and carbonyl group of polyNVP may be one factor for enhanced thermal stability.[ 50]

DTG analysis was studied as a function of rate of weight loss ( g/min) with increase in temperature. In case of CP, two peaks at 251 °C (590 μg/min) and 424 °C (2810 μg/min) have been observed. In case of CP-cl-poly(NVP) polymer three stage degradation has been observed, where first stage is associated with very slow degradation rate with a peak at 190 °C (211 μg/min), second stage degradation showed DTG peak at 400 °C (870 μg/min), third peak at 493 °C (1030 μg/min). Results of DTG support the TGA, as relatively slow degradation rate was observed in cross-linked polymer matrices in comparison to CP. It is important to mention here that temperature at which maximum degradation rate was observed shifted from 424 °C to 493 °C on going from CP to cross-linked polymer. The results of thermal degradation thus confirm the conclusion that cross linking and grafting has occurred which has improved thermal stability of CP-cl-poly(NVP) polymer in comparison to CP.

DSC curves of CP and CP-cl-poly(NVP) polymer are presented in Figure 7. In case of CP two endothermic peaks, at 61.0 and 228.7 °C, were observed, with a heat of fusion 122.6 and 248.7 J/g respectively. The initial endotherm can be due to the evaporation of unbound water in the polymer and the latter can be attributed to the loss of water due to formation of anhydrides in CP.[ 51] Glass transition temperature (T_g) for CP has been observed in the temperature range 128.5-135.1 °C ( Cp = 0.574 J/g K). Gómez-Carracedo et al. (2004) have reported that various grades of CPs show Tg in the range 130-140 C. CP-cl-poly(NVP) polymer showed quite different DSC thermo gram in comparison to CP. Two endothermic peaks have been observed at 89.3 °C and 221.9 °C. The first endotherm with a heat of fusion 89.74 J/g, can be attributed to the evaporation of unbound water in the cross-linked polymer and second endotherm (108.6 J/g) can be due to anhydride formation in the polymer.[ 52]

(a) (b)

Figure 7 DSC curves of (a) CP and (b) CP-cl-poly(NVP) polymer

3.2 Swelling studies

The swelling polymers was determined in simulated wound fluid (pH=8), phosphate buffer saline and pH 2.2 buffer to evaluate the effect of nature of swelling medium on network structure of hydrogels (Table 1). Swelling was observed more in SWF (1137.35±31.21%) as compared to swelling in pH 2.2 buffer (287.49±23.47%) and PBS (1100.04±12.25%). pH sensitivity of the hydrogel can be attributed to the presence of CP in the polymer matrix and in alkaline solution, partially ionization of carboxylic groups develop internal ion osmotic pressure due to electrostatic repulsion and swelling of hydrogels.[ 53 ] On the other hand, at lower pH solution, the hydrogen-bonding interactions leading to generation of additional physical crosslinking. So, the electrostatic repulsion due to COOgroups is restricted, and the polymeric network tends to shrink.[54] The swelling of polymers was observed less in 0.9% NaCl solution (355.04±5.88%) as compared to distilled water (1198.78±34.30%). Increase in salt concentration shielded the electrostatic repulsion between charged groups, causing decrease of hydrogel swelling.[ 45]Swelling of hydrogels increased with increase in temperature of swelling medium. At higher temperature the chain mobility increases which facilitates the network expansion and leads to increase in swelling of hydrogel [55](Table 1).

Table 1 Results of swelling for CP-cl-poly(NVP) hydrogels

Overall, these hydrogel wound dressing showed high wound fluid absorption capacity and one gram of hydrogel film has taken 11.37±0.31 g of simulated wound fluid. Moist wound bed has been widely accepted as the most ideal environment for effective wound healing.[ 56] The accumulation of wound exudates often causes maceration and bacterial overgrowth in the wound site.

3.3 Drug release profile

Release profile of moxifloxacin from drug loaded CP-cl-poly(NVP) hydrogels was evaluated in simulated wound fluid (SWF) and results are shown in Figure 8 and Table 2. The release profile of entrapped drug showed slow and sustained release in SWF. No significant burst release was observed and considerable amount of drug release was observed after every half an hour, which can help in maintaining constant drug concentration at the wound site for a long time. The high surface area and porous structure of the hydrogels enabled the drugs to diffuse into the aqueous medium. Penetration of solvent led to formation of micro-cavities which cause the drug migration into solvent.[57] Diffusion exponent and various diffusion coefficients for the release of drug from the drug loaded polymers have been calculated and results have been presented in Table 2. The release of moxifloxacin from hydrogels occurred through Case-II diffusion mechanism. Case II diffusion mechanism occurs when rate of diffusion is very rapid as compared to the rate of relaxation of polymeric chains (relaxation controlled transport).The values of initial and late time diffusion coefficients were found comparable. The in vitro drug release data from CP-cl-poly(NVP) hydrogels in different releasing mediums were evaluated kinetically using various important mathematical models like zero order, first order, Higuchi, KorsmeyerPeppas, and Hixson-Crowell models. When the respective R² were compared, it was found that it followed best by Hixson-Crowell's model with highest values of regression coefficient. Hixson-Crowell's model applies to such pharmaceutical dosages, where the dissolution occurs in planes that are parallel to the drug surface if the dosage dimensions diminish proportionally, in such a manner that the initial geometrical form keeps constant all the time.[58]

Figure 8 Release profile of moxifloxacin hydrochloride from drug loaded CP-cl-poly(NVP) hydrogels, in simulated wound fluid at 37 °C

Table 2Results of diffusion exponent ‘n’, gel characteristic constant ‘k’, various diffusion coefficients, and correlation coefficients of different models for release profile of moxifloxacin hydrochloride from drug loaded CP-cl-poly(NVP) hydrogels, in simulated wound fluid at 37 °C

3.4 Blood compatibility

Hemolysis, defined as the release of hemoglobin into plasma due to damage of erythrocytes membranes, is directly related to blood compatibility of material. Direct contact hemolytic assay is considered to be reliable method for measuring blood biocompatibility of biomaterials. Results of hemolysis test for CP-cl-poly(NVP) polymer have shown (3.97±0.72)% haemolysis classifying it as non-haemolytic material. These hydrogels can be regarded as safe for wound applications. Li, et al. have reported increase in blood compatibility on grafting with NVP.[59] Moreover, Blood compatibility of these crosslinked polymer can be attributed to its constituting materials poly(NVP) and CP, which are highly hydrophilic and biocompatible in nature.[ 13, 60] These results revealed the hemocompatibility of hydrogel dressings, which indicates material will not alter the integrity of the blood during in-vivo application.During thrombogenicity test, weight of the clot formed with hydrogels was slightly less (0.31±0.02 g) in comparison to blood without hydrogels (0.34±0.05 g). These results indicate that 91.39±7.44% thrombogenicity was observed in the presence of CP-cl-poly(NVP) polymer, classifying the material as non-thrombogenic (Table 3).

3.5 Mucoadhesion studies

The results of mucoadhesion studies for CP-clpoly(NVP) polymer are presented in Table 3. Maximum detachment force (F_max) required for detachment of hydrogels from mucosal surface was 79.98±8.16 mN. Biadhesive properties in the present case can be attributed to synergistic mucoadhesive effect of poly(NVP) and CP. CP and poly (NVP) are known to improve mucoadhesive properties of the biomaterials.[ 61, 62] Carboxylic groups of CP are capable of forming strong hydrogen bonds with oligosaccharide chains present in mucins leading to adhesion.[ 62] Mucoadhesion is desirable for a topical wound dressing as it will help the dressing to retain its position with less chances of dislocation from the wound site. Mucoadhesive polymers are widely explored for developing novel drug delivery systems. The close contact between a delivery system and the absorbing cell layer will improve both efficiency and effectiveness of the mucoadhesive system. Interaction of a polymeric system with these mucus glycoproteins is the major reason for the mucoadhesion phenomenon. Inter-penetration and inter-diffusion of polymeric chains with the mucus lining is one among the important steps in determining the bioadhesive interactions. Specific non-covalent interactions between the functional groups in the polymers and the mucus glycoproteins further reinforce these interactions.[ 63 ]

3.6 Oxygen permeability

The results of oxygen permeability are presented in Table 3. In the present case, the airtight flask (negative control) and opened flask (positive control) had dissolved oxygen values of 4.95±0.07mg/L and 7.4±0.0mg/L, respectively, whereas flasks covered CPcl-poly(NVP)polymer film had dissolved oxygen values of 6.43±0.05 mg/L. The results show that CP-clpoly(NVP) polymer film has shown considerable permeability to oxygen. Similar results have been reported in literature.[ 64] These results can be attributed to hydrophilicity of hydrogel films as observed in swelling studies. Hydrophilicity of the material results in water plasticization effect that will lead to polymer chain relaxation, increase in pore size and hence improved gaseous permeability.[ 65, 66] Gaseous permeability especially for oxygen is a crucial factor for proper wound healing. On one side oxygen is necessary for regeneration of damaged tissue, while on the other side wound hypoxia leads to impaired wound healing. All result suggested that sufficient oxygen was able to penetrate through the polymeric network of crosslinked films. Oxygen availability is suitable for tissue homoeostasis, energy production, cell membrane maintenance, mitochondrial function, and cellular repair.[ 64]

Table 3Results of biomedical properties of ofCP-cl-poly(NVP) hydrogels

3.7 Water vapour transmission rate

Parallel to oxygen permeability, evaluation of water vapour transmittance through the films is also necessary. WVTR for CP-cl-poly(NVP) films was 400.00±13.90 g/m²/day, and for open vial it was 4192.38±106.29 g/m²/day. However in case of sealed vial no water vapor transmittance was observed indicating CP-cl-poly(NVP) films were permeable to water vapors, but to a very less extent in comparison to open vial. Excess evaporation from the wound leads to wound dehydration and complete sealing leads to collection of wound exudates, both results in delayed wound healing. Intermediate WVTR for CP-cl-poly(NVP) hydrogels will help to maintain moist wound healing environment without collection of wound exudates.

3.8 Microbial penetration

Microbial penetration test was carried out for a month period and it was found that no microbial contamination occurred in open environment in case of nutrient broth sealed with CP-cl-poly(NVP) hydrogel films, while high turbidity in the open test tube was observed as a result of microbial contamination (Table 3). This test highlights that these hydrogel films could be considered as a good barrier against the microbes to prevent any secondary infection during wound healing. Similar results have been reported in literature.[ 56] The goal of wound therapy is to keep the wound microorganism content as low as possible in order to prevent infection and accordingly to stimulate the repair process. The hydrogel has the potential to mimic the extracellular matrix, which may lead to the tissue regeneration necessary for wound healing. In designing a matrix which promotes wound healing, it is important to provide an adequate antimicrobial protection. Hydrogel dressings are considered as a good barrier against microbes and this is important, especially for protecting the wound from further infection and accelerating the healing of the wound.[ 56]

3.9 Mechanical Properties

The results of mechanical properties (viz. tensile strength, burst strength, resilience, stress relaxation and folding endurance) of the CP-cl-poly(NVP) polymer film are given in Table 3. The polymers showed tensile strength (0.65±0.12 Nmm^-2) and elongation at break (119.38±14.26%). Tensile strength represented to the maximum tension that a material can withstand without tearing, while elongation at break was used to evaluate the malleability of materials. A crosslinked network imparts greater tensile strength and a more malleability to the hydrogels.[ 3] The considerable tensile strength are the results of long polymeric chains and interpenetrating network present in the polymeric film. Results of burst strength test revealed that 8.25±0.31 N force was required to burst the polymer film with a ball probe (P/5s) and distance at burst (15.30±1.65 mm) was shown by the polymer, indicating high mechanical strength. In stress relaxation test, when a constant strain (2mm) was applied for 30 second (F_max=0.54±0.09 N) on 1mm thick polymeric films, 0.23±0.02 N force was relaxed/retained by the polymer.

So, stress relaxation test shows 43.56±2.69% force was retained by the polymer sample indicting viscoelastic behaviour of the polymer film. The folding endurance was determined by repeatedly folding one film at the same place till it broke or folded up to 300 times manually, which was considered satisfactory to reveal good film properties. These polymer films did not show any cracks even after folding for more than 300 times. Folding Endurance test results reveal that these hydrogels were flexible and suitable for wound dressing applications. High flexibility can be attributed to high hydrophilicity of the hydrogels and glycerol, the biocompatible plasticizer.[ 65 ] Resilience is evaluated from of work returned by the sample when it is strained upto a distance (2 mm in present case). 25.49±0.72% resilience of the polymers was observed, indicating slight elastic character of the polymer film. Resilience and elongation values are measure for elasticity of a material, and in the present case the polymer film has shown high values for elongation and was resilient upto an extent, displaying elastic nature of the material.[ 66] Hence, the mechanical parameters obtained for the CP-cl-poly(NVP) polymer film indicate that it meets the requirements for a potential wound dressing material. Compromised mechanical properties of hydrogels often limit the scope of their applications.

4. Conclusion

From the forgone discussion it can be concluded that modification of CP with poly(NVP) was successfully carried out by free radical polymerization. The polymer was obtained in the form of a transparent flexible film. SEM and AFM studies demonstrate small degree of heterogeneity of the surface of the polymer film. The hydrogels showed high simulated wound fluid absorption and hydrogel dressings were found non-hemolytic, non-thrombogenic, oxygen permeable and water vapour permeable. In addition these hydrogels were transparent which will allow monitoring wound healing progress. Moxifloxacin released was observed in controlled manner from drug loaded hydrogels. Drug release from hydrogels occurred through Case-II diffusion mechanism and best fitted in HixsonCrowells model. Since the polymers were transparent, real time monitoring of wound healing is possible. These polymer films displayed high mechanical strength and flexibility. Overall the preliminary investigation suggested that CP-cl-poly(NVP) hydrogel films can act as potential candidate for wound dressing applications.