International Journal of Biological Macromolecules 79 (2015) 440–448

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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Investigation of curcumin release from chitosan/cellulose micro crystals (CMC) antimicrobial films S.K. Bajpai a,∗ , Navin Chand b , Sonam Ahuja a a b

Polymer Research Laboratory, Department of Chemistry, Govt. Model Science College, Jabalpur, M.P. 482001, India Advanced Materials and Processes Research Institute (CSIR), Bhopal, M.P., India

a r t i c l e

i n f o

Article history: Received 14 March 2015 Received in revised form 1 May 2015 Accepted 13 May 2015 Available online 21 May 2015 Keywords: Phase inversion Chitosan Cellulose Curcumin complex Wound dressings

a b s t r a c t Following the novel ‘vapor induced phase inversion’ (VIPI) method, we have prepared curcumin loaded chitosan/cellulose micro crystals composite films and characterized them by thermo gravimetric analysis (TGA), X-ray diffraction (XRD) and scanning electron microscopy (SEM). The equilibrium moisture absorption behavior of these films was investigated under different relative humidity (RH) environments and the data obtained was interpreted by the GAB isotherm model successfully. The films were also studied for their curcumin release behavior in the physiological fluid (PF) at 37 ◦ C and the kinetic data obtained was best interpreted by Higuchi model. Finally, the films showed fair antimicrobial action against bacteria and fungi. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Since ancient time, Turmeric has been used as a natural healing and antimicrobial agent against wounds [1]. The main biologically active ingredient of Turmeric is curcumin which is a poly (phenolic) compound and actually responsible for antimicrobial properties. The Food and Drug Administration Department of USA has recognized it as a safe compound [2]. There have been some reports which claim that curcumin has excellent anticancer properties [3]. Indeed, a large amount of research has been carried out to prove antimicrobial and wound healing properties of curcumin [4–6]. Chitosan is a biopolymer and it is obtained from the de-acetylation of its native polymer chitin [7]. The de-acetylation is carried out under strong alkaline conditions and degree of de-acetylation varies with the experimental conditions[8]. Chitosan is a biocompatible and biodegradable polymer and has a number of biomedical applications which include wound dressings [9], drug delivery [10], porous scaffolds for cells re-generation [11,12], etc. Because of low cost, abundant availability and excellent film forming capability [13], it is also used in food packaging [14]. Most recently [15], we reported a unique approach, i.e. ‘vapor induced phase inversion (VIPI)’ to prepare cellulose crystals-loaded chitosan films with controllable water absorption and moisture permeation properties. Following

∗ Corresponding author. Tel.: +91 9993220651. E-mail address: [email protected] (S.K. Bajpai). http://dx.doi.org/10.1016/j.ijbiomac.2015.05.012 0141-8130/© 2015 Elsevier B.V. All rights reserved.

the same VIPI approach, we hereby report some physico-chemical properties and moisture absorption behavior of curcumin loaded chitosan/cellulose composite films. The curcumin release behavior and antimicrobial activity of these films is also studied.

2. Materials and methods 2.1. Materials Chitosan with a degree of deacetylation (DD) of 93% and viscosity average molecular weight of 1463 kDa was obtained from Research Lab, Mumbai, India and was used with no further treatment. Cellulose micro crystals (CMC), liquid ammonia, various salts to produce required relative humidity (RH) were obtained from Hi Media Chemicals, Mumbai, India and were analytical grade. Turmeric was purchased from the local market. The double distilled water was used throughout the investigations.

2.2. Extraction of CC from turmeric Curcumin (Cur) was obtained from turmeric following the method proposed elsewhere [16]. In brief, 15 g of fine turmeric powder was suspended in 150 ml of acetone under moderate stirring for 72 h at 30 ◦ C. The mixture was filtered and the filtrate was poured into a Petri plate and the solvent was evaporated under vacuum to obtain semi-dry oily mass. The oily mass was weighed and

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dissolved in 50 ml of dimethyl sulfoxide (DMSO) to give a reddishbrown curcumin solution. 2.3. Preparation of Cur-loaded Ch/CMC films by VIPI approach In order to prepare the Cur-loaded films, 2% chitosan solution was prepared in 2% solution of acetic acid, followed by addition of pre-calculated quantity of CMC under vigorous stirring for 1 h to ensure complete missing. Now, a pre-calculated volume of curcumin dispersion was also mixed thoroughly into the above film forming solution and the solution was transferred into Petri plates, followed by their immediate exposure to NH3 gas (for detailed procedure see our ref.[15]). In all four films were prepared, each one containing same amount of curcumin, i.e. 450 ␮g per g of film. These films were designated as Ch/CMC (0)450 , Ch/CMC(10)450 , Ch/CMC(20)450 , and Ch/CMC(30)450 , where the number in subscript is the amount of curcumin in ␮g present in 1 g of the film. 2.4. Characterization of films The Cur-loaded films were characterized by SEM, XRD, TGA and mechanical analysis. The X-ray diffraction (XRD) method was used to measure the crystalline nature of Ch/CMC(20) and Cur-loaded Ch/CMC(20)450 films. These measurements were carried out on a Rikagu Diffractometer (Cu radiation = 0.1546 nm) running at 40 kV and 40 mA. The diffractogram was recorded in the 2 range of 5–60◦ at the rate of 2◦ min−1 . The surface morphology of the plain and Cur loaded films was investigated using a JEOL 6400F microscope operated with an accelerating voltage of 2 kV and a working distance of 4.4 mm. 50 ␮l sediment suspension (0.01 wt%) was dropped onto clean silicon wafers followed by air-drying for 24 h and then sputtered with an approximately 6 nm layer of gold/palladium. The mechanical properties of the films were determined according to the procedure reported elsewhere [17]. Film samples, with the dimensions of 39 mm × 5.8 mm, were equilibrated under the RH of 50% at 23 ◦ C for a period of 24 h and their tensile strength (TS) and percent elongation at break (PE) were measured by using an Instron Universal Testing Instrument (Model 1011). The initial grip separation and crosshead speed were set to 40 and 200 mm per min, respectively. All the determinations were made in triplicate. The TS (MPa) and % elongation at break (PE) were determined using the following expressions: breaking force(N) tensile strength(MPa) = cross-sectional area of sample(mm2 ) PE =

increase in length at the time of break × 100 initial length

2.5. Determination of moisture sorption isotherm A gravimetric static method was employed to determine moisture absorption isotherms [18]. Saturated solutions of various salts were prepared in polypropylene chambers to create environments with desired relative humidity (RH) (see Table 1). The chambers Table 1 The water activities (aw ) of saturated salt solutions 30 ◦ C. Salt

aw at 30 ◦ C

KOH CH3 COOK K2 CO3 Mg(NO3 )2 NaCl KCl K2 SO4

0.0738 0.2161 0.4317 0.5140 0.7509 0.8362 0.9700

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were kept in an incubator (Sanyo MIR 152) at 30 ◦ C. Now, preweighed film samples were put in small crucibles of aluminum foils and placed in air-tight polypropylene chambers. The film samples took almost three days to attain the equilibrium. The equilibrium moisture content (EMC) of the film samples was determined using the vacuum oven method [19] and expressed as g/g dry solids. All the moisture absorption experiments were carried out in triplicate and the average values were used to plot the isotherms. The EMC was determined using following formula: EMC =

(final weight − initial weight) (g/g) initial weight

(1)

2.6. Curcumin release studies The pre-weighed Cur loaded film was placed in 25 ml of physiological fluid (PF) at 37 ◦ C. The amounts of curcumin released at different time intervals were determined spectrophotometrically (Shimadzu, Genesis 10-S) at 526 nm. After each measurement, the film was transferred in to 25 ml of fresh physiological fluid. Calibration curve, prepared for the curcumin solutions of known concentrations in the appropriate range, was used to determine the amount of curcumin in the release media. 2.7. Blood compatibility A representative sample Ch/CMC(20)450 was used to determine the total protein adsorption using the procedure given in a report from the International Standard Organization (ISO) [20]. 2.7.1. Protein and albumin adsorption study A piece of the film sample Ch/CMC(20)450 ,with surface area of 1 cm2 , was incubated in 0.9% saline solution at 37 ◦ C for a period of 24 h, followed by its transfer into 10 ml of pure frozen plasma (PFP) serum for 3 h under normal stirring. The film was taken out after 3 h and the protein contents was measured using the (Bicinchoninic acid) BCA reagent. This method is based on the fact that protein reduces Cu2+ to Cu1+ in the alkaline medium. The BCA combines with Cu1+ to create a purple-colored product with a maximum absorbance at 546 nm. The protein adsorption was calculated using the following formula: total protein concentration (g/dl) =

absorbance of test sample × 60 absorbance of standard

The albumin content was determined using Bromocresol green (BCG) test. The Bromocresol green test is based on the fact that at acidic pH (approximately 4.0), albumin act as a cation and binds to the anionic dye Bromocresol Green (BCG), forming a green colored complex. The absorbance of soluble complex is measured at 630 nm (Shimadzu, Genesis 10-S). The color intensity of the complex is proportional to albumin concentration in the sample. acidic pH

albumin + BGC −→ green coloured complex albumin concentration (g/dl) =

absorbance of test sample × 36 absorbance of standard

2.8. Antibacterial test by the ‘zone inhibition method’ The antibacterial tests were carried out with curcumin loaded film sample Ch/CMC (20)450 by ‘zone of inhibition’ method. In brief, the culture medium was prepared by mixing nutrient agar (2.8 g) and agar powder (1 g) in 100 ml of distilled water in a conical flask and autoclaved for 30 min. The medium was transferred into sterilized Petri plates in a laminar air flow. After solidification of media, Escherichia coli culture was streaked on its surface. Now, the film

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sample Ch/CMC (20)450 was placed in center of the Petri plate and the Petri plate was incubated for 2 days at 37 ◦ C in the incubator [21]. 2.9. Antifungal activity We also carried out the antifungal activity of the film sample Ch/CMC (20)450 against Candida albicans, and Candida parapsilosis. As even to fourteen days old culture was obtained from Fungal Disease Diagnostic Center, Jabalpur (India). For disc diffusion test, films were cut into disc shape with a diameter of 5 mm, then sterilized by autoclaving for 30 min at 120 ◦ C, and finally placed on different cultured agar plates. The plates were incubated for 1 day at 37 ◦ C in an incubation chamber. 3. Results and discussion 3.1. Characterization of films The surface morphology of the film samples Ch/CMC (20) and Ch/CMC(20)450 was determined using scanning electron microscopy. The results are shown in Fig. 1. The SEM images of film samples Ch/CMC(20) and Ch/CMC(20)450 with 100× magnifications are shown in Fig. 1(a) and (b), respectively. It is observed that surface texture of film Ch/CMC(20) is quite rough due to the presence of well dispersed cellulose micro crystals throughout the matrix. Overall, a uniform distribution of CMC

is visible (although with a few agglomerations). However, the surface texture of Cur loaded film sample Ch/CMC (20)450 exhibits different texture. It may be noticed that the whole surface have a relatively smoother texture and the curcumin molecules seems to have superimposed on the cellulose crystals and the grooves visible in the plain film are not so pronounced now. In other words, the pronounced appearance of the cellulose crystals in the film sample Ch/CMC(20) is depressed greatly due to presence of curcumin which must have formed a layer on the cellulose crystals throughout the film matrix. The SEM images with 200 times magnifications are also shown in Fig. 1(c) and (d), respectively. These images further support our observations. In order to investigate the crystalline nature of the films, XRD analysis of film samples Ch/CMC (20) and Ch/CMC (20)450 was carried out. The diffractograms of films Ch/CMC (20) and Ch/CMC (20)450 are shown in Fig. 2(a) and (b), respectively. In the diffractogram (a), two characteristic peaks of chitosan are observed at 10◦ and 23◦ [22]. It is also seen that three moderate peaks, corresponding to (1 0 1), (1 0 1) planes of cellulose-I and (1 1 0) plane of cellulose-II, also appear at 14.6◦ , 16.3◦ and 20◦ , respectively [23]. The most intense peak, occurring at 22.5◦ , corresponds to the (0 0 2) lattice plane of cellulose-I. In fact, the two peaks, namely one at 22.5◦ for cellulose-I and the other one at 23◦ for chitosan superimpose and cannot be distinguished separately. The diffraction pattern of sample Ch/CMC (20)450 , is shown in Fig. 2(b). It contains a number of peaks, though not prominently visible, in the 2 range of 10–30◦ . These peaks indicate the presence of highly crystalline curcumin in the film matrix [24]. The

Fig. 1. SEM images of (a) plain Ch/CMC(0) and (b) Ch/CMC(20)450 film samples at 100× magnifications., (c) plain Ch/CMC(0) and (d) Ch/CMC(20)450 film samples at 200× magnifications.

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Fig. 2. XRD of (a) Ch/CMC(20) and (b) Ch/CMC(20)450 film samples with XRD of curcumin in inset.

XRD of pure curcumin is also shown in the inset for comparison. It is noteworthy here that we did not get such prominent peaks in the diffractogram of Ch/CMC (20)450 , probably be due to the very low concentration of curcumin in the film. 3.2. Thermal stability of films The thermogravimetric analysis of native chitosan powder, cellulose crystals and Ch/CMC (20)450 film is shown in Fig. 3.

It can be seen that cellulose powder suffers a drastic weight loss of nearly 76% in the temperature range of 200–380 ◦ C where all cellulose is pyrolyzed. The drastic weight loss is attributable to the liberation of volatile hydrocarbon from rapid thermal decomposition of cellulose chains. However, beyond 400 ◦ C there is gradual weigh loss probably due to the steady decomposition of the remaining heavy components mainly from lignin. The thermogram of chitosan powder showed a two stage degradation behavior. There is weight loss of 46% in the first phase in the temperature

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Fig. 3. TGA of Ch, CMC and Ch/CMC(20)450 with curcumin TGA in inset.

range of ambient to 390 ◦ C. In the second phase, the weight loss observed was almost 37% in the temperature range of 400–800 ◦ C. Such a two stage degradation of chitosan has also been reported previously [25]. The thermogram of Ch/CMC(20)450 shows an intermediate degradation behavior. The degradation is a little faster than that of chitosan but is slow enough as compared to that of cellulose crystals. More interestingly, the thermogram of Ch/CMC (20)450 is more similar to that of chitosan in nature, probably due to the relatively much higher content of chitosan in the film as compared to cellulose content which is only 20% to that of chitosan. Finally, it is also noteworthy that presence of curcumin did not affect the thermal stability of the film because of very negligible content of curcumin as compared to the total mass of polymer (see TGA of curcumin in inset as obtained from the literature) [26]. According to the report, curcumin does not show any moisture loss up to 200 ◦ C because of its poor hydrophilic nature and a slower mass loss is observed at 240 ◦ C due to thermal degradation and it becomes prominent on reaching at 500 ◦ C. The presence of curcumin does not make any influence on the stability of the film because the Cur content is only 450 ␮g, as compared to the total mass of 1 × 106 ␮g of the film.

3.3. Tensile strength measurement A wound dressing hydrogel film must be able to withstand the normal stress encountered during its handling and application in the wound. The tensile strength (TS) is the maximum stress sustained by the film during the tension test. Tensile strength (TS) is one of the significant parameters which need to be evaluated to test the accessibility of a wound dressing film. In addition, percent elongation at break (PE) is also an important parameter which indicates stretch ability of the film. This is also required to be quite fair during the application of the film on the affected area of wound [27]. The results of mechanical analysis are shown in Fig. 4. It can be seen that the TS of films increases from 5.7 to 13.8 MPa with the increase in cellulose content from 0% to 20%. However, for the sample Ch/CMC (30)450 the TS decreases to 8.1. The observed increase in TS with cellulose content may be attributable to the fact that cellulose crystals, being self-reinforced, possess fair stiffness[28] and as their content in the film increases, the TS also increases. In addition, with the increase in cellulose content, the interfacial interactions between polar groups of cellulose micro crystals and chitosan chains become prominent [29]. These

interactions help in transfer of applied stress from the matrix to the cellulose micro crystals, and therefore enhance the tensile strength of resulting films. In a recent work, Zimmermann et al. [30] have reported a threefold increase in the tensile strength of PVA film due to addition of 20% (by weight) of cellulose fibers. In the present work, we have also observed an almost three fold increase in the TS of the chitosan film due to addition of cellulose crystals (20% of chitosan content by weight). Moreover, in a study by Atefa et al. [31], the TS of agar-films increased with addition of nano cellulose up to 2.5%, but later on the TS started to decrease with further increase in the cellulose content. In the present work, the observed decrease in TS with increase in CMC content beyond 20% (by weight) may probably be due to the over exceeded stiffness that could bring internal cracks within the film matrix, making the film brittle and so reducing its mechanical strength. In addition, the heterogeneous size distribution and agglomeration of CMC (as was also visible in SEM images) could be another reason for the observed decrease in TS of the film Ch/CMC (30)450 . Similar results have also been reported by Piyada et al. [32] for the rice starch films, reinforced with rice starch nanocrystals. A close look at the percent elongation (PE) values reveals an almost opposite trend with the exception of the film sample Ch/CMC (30)450 . The observed decrease in PE with CMC content may probably be due to the increased nature of nanofillers. These dispersed cellulose crystals restrict the segmental motion of polymeric chains, thus reducing their elongation tendency. However, the sample Ch/CMC (30)450 has the highest

Fig. 4. Tensile strength and % elongation of various Cur-loaded film samples.

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Fig. 5. (a) Equilibrium moisture contents at different relative humidity for various film samples and (b) evaluation of GAB parameters.

cellulose content and there may be heterogeneous distribution of cellulose crystals with a few agglomerations with inter and intra molecular crosslinks among cellulose crystals. This provides more freedom to chitosan molecules for movement under applied stress, thus increasing PE of the film. 3.4. Equilibrium moisture uptake studies The tendency of a wound dressing film to absorb moisture helps to maintain a moist environment at the wound site, which ultimately makes the healing process faster [33]. In the presence of a moist environment, the desiccated tissues are dehydrated, thus promoting autolysis, epidermal migration and connective tissue synthesis [34]. The equilibrium moisture uptake data for the samples Ch/CMC (0)450 , Ch/CMC (10)450 , Ch/CMC (20)450 and Ch/CMC(30)450 are shown in Fig. 5(a). All the curves obtained are sigmoid in shape, exhibiting type-II characteristic isotherms. Such curves are typical for most of the biopolymers like starch [35], chitosan [36], etc. The curves may be divided into three zones, i.e. zone-I, zone-II and zone-III with water activity ranges of 0.0–0.2, 0.2–0.7 and 0.7–1.0, respectively. The variation in moisture uptake with water activities for all the films may be explained as follows. In the water activity (aw ) range of 0–0.2 (zone-I), the equilibrium moisture content (EMC) increases with aw and the film sample Ch/CMC (30)450 shows the maximum moisture absorption. This is attributable to the fact that in this zone, water vapor molecules are readily absorbed on the active polar groups (i.e. –OH groups) present on the surface of the film. Here, the dispersed cellulose crystals with their surface hydroxyls also contribute towards moisture uptake. In other words, the density of the polar –OH groups on the film surface increases with CMC content in the film. As the aw increases beyond 0.2 and enters into the zone II with aw range of 0.2–0.7, the EMC increases with relatively slower rate. The reason is that moisture uptake occurs at less active sites. It is reported that, in this region unfolding or relaxation of polymer chains opens up new sites for uptake [37]. Finally, there is sharp increase in the EMC with aw , in the range of 0.7–1.0. Here, the moisture molecules diffuse into the voids and capillaries within the film matrix. In addition, a cross over can be clearly observed. The film Ch/CMC (0)450 shows maximum moisture absorption whiles the film Ch/CMC(30)450 absorbs minimum moisture. The relatively faster moisture uptake in this zone is simply because of the capillary action. When water vapor molecules enter into the film matrix, the –OH groups of cellulose crystals in the bulk of the films do not interact with invading water vapor molecules. The reason is that there are strong inter and intra

H-bonding interactions among the –OH groups of cellulosic chains. Therefore, these –OH groups do not bind with the incoming water vapor molecules. Therefore, moisture uptake of the films decreases with increase in their cellulose content. The equilibrium moisture sorption data obtained for various film samples was analyzed using the GAB model. The GAB (Guggenheim-Anderson-de Boer) is a three parameters model which is a theoretically derived isotherm model [38], and is given as: M=

M0 CKaw (1 − Kaw )(1 − Kaw + cKaw )

(2)

where M0 is the monolayer moisture content, C a constant related to the first layer heat of sorption and K is a factor related to the heat of sorption of the multilayer. In order to determine the parameters of GAB isotherm model, GAB equation was re-arranged into a second degree polynomial equation. aw = ˛a2w + ˇaw +  M

(3)

where

 1 

˛=

k M0

ˇ=

1 2 1− M0 C



C −1

(4)

 (5)

and =

1 M0 Ck

(6)

A non-linear regression analysis of aw /M versus aw yielded a polynomial of second-order as shown in Fig. 5(b) for the samples Ch/CMC (0)450 , Ch/CMC (10)450 , Ch/CMC (20)450 and Ch/CMC (30)450 , respectively. The coefficients ˛, ˇ and , obtained from the polynomial equation, were substituted in Eqs. (4)–(6) to obtain GAB constants. These values are given in Table 2. As stated earlier, GAB isotherm model has a sound theoretical basis and therefore its parameters need Table 2 Parameters associated with GAB isotherm model. Sample code

Ch/CMC(0)450 Ch/CMC(10)450 Ch/CMC(20)450 Ch/CMC(30)450

GAB isotherm parameters M0 (g/g)

C

K

0.092 0.048 0.032 0.023

4.99 3.84 7.41 15.13

0.94 0.98 0.96 0.53

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Fig. 7. Higuchi model plots for various curcumin loaded film samples.

Fig. 6. Curcumin release profiles for various Cur-loaded film samples in the physiological fluid at 37 ◦ C.

preclinical and clinical studies in mice, rats and humans have also revealed a low bioavailability of curcumin [40].

to be discussed here. The monolayer moisture content (M0 ) represents the amount of water strongly bound to the active sites on the film surface. Therefore, M0 is the maximum moisture content with which the film can exhibit fairly longer stability without any deterioration. Below this EMC level, extent of deteriorative reactions is minimum. The values of monolayer content M0 , as shown in Table 2, indicate that the moisture uptake of films decreases with the increase in their cellulose contents. This may simply be attributed to the presence of cellulose micro crystals which, due to strong interand intra-molecular bonding, do not interact with incoming polar water vapor molecules. As their concentration in the film increases, the overall moisture absorption tendency of films decreases. The GAB constant C is indicative of adsorbent adsorbate interactions and it also decides the shape of the isotherm: for C > 2, the GAB model should yield a sigmoidal shaped curve with point of inflection (type II of Brunauers (1943) classification): and for 0 < C < 2 the isotherm is of the type III only (isotherm without point of inflection). In this study, the value of C was greater than 2 for all the four film samples studied and the isotherm curves obtained were also sigmoid, thus supporting above predictions. Finally, the constant K measures the interactions between the water molecules in multilayer with the substrate and tends to fall between the energy values of the molecules in the monolayer and that of bulk water. The values of K, for all the samples, lie between 0 and 1 as has been prescribed.

3.5.1. Modeling of the release data The curcumin release data was applied on the Higuchi model [41], which was initially developed for the planar systems, but later on extended to systems with different geometries. It is based on the following assumptions: (1) initial drug concentration in the matrix is much higher than its solubility, (2) thickness of drug particles is much smaller than the thickness of the matrix and (3) drug diffusivity is constant and (4) perfect sink conditions are always maintained. The most simplified form of this model is given as:

3.5. Curcumin release study Cellulose crystals, present within the chitosan films, serve as diffusion barrier and they are expected to retard the release of curcumin from the films. The release profiles of Cur-loaded film samples Ch/CMC (0)450 , Ch/CMC(10)450 , Ch/CMC(20)450 , and Ch/CMC(30)450 are shown in Fig. 6. It can be seen that the film sample Ch/CMC (0)450 exhibits maximum release while minimum release of curcumin was obtained for the film sample Ch/CMC (30)450 . This indicates that amount of Cur released decreases with the increase in the cellulose content of the films. The amounts of Cur released from the samples Ch/CMC (0)450 , Ch/CMC(10)450 , Ch/CMC(20)450 , and Ch/CMC(30)450 in 36 h was 365, 300, 270 and 140 ␮g, respectively. Here, it is also noteworthy that the total loading of curcumin in each film was around 450 ␮g. Such a lower curcumin release from these films is attributable to the fact that Cur has very poor solubility in water. It has been reported that Cur has a solubility of around 2.67 ␮g/ml at pH 7.3 [39]. Many

F = KH t 1/2

(7)

where F may be taken as the fractional release and KH is the Higuchi constant. The major benefits of this equation include the possibility to: (i) facilitate device optimization, and (ii) to better understand the underlying drug release mechanisms. The equation can also be applied to other types of drug delivery systems than thin ointment films, e.g., controlled release transdermal patches or films for oral controlled drug delivery. The dynamic release data, shown in Fig. 6, was used to draw plots between F and t1/2 , which were linear with fair regressions as shown in Fig. 7. It can be seen that all the linear plots pass through origin as demanded by the Higuchi model. The regressions R2 for the release of Cur from the film samples Ch/CMC (0)450 , Ch/CMC(10)450 , Ch/CMC(20)450 , and Ch/CMC(30)450 were 0.894, 0.970,0.988 and 0.950, respectively. It appears that the plain sample Ch/CMC(0)450 does not exhibit a fair regression, thus indicating that plain film does not demonstrate an excellent diffusion-controlled release of curcumin. This may be due to the fact that the sample Ch/CMC (0)450 does not contain cellulose crystals within the chitosan matrix. Therefore, when solvent enters into the film matrix, Cur is released along with the swelling of the chitosan film. As the film is not crosslinked, there is appreciable relaxation of chitosan segments along with segmental motion within the matrix. In this way, the curcumin release is not totally diffusion controlled but it is partly contributed by chain relaxation process also. However, the situation is different with the other three film samples which contain 10%, 20% and 30% (by weight) cellulose micro crystals. The cellulose crystals act as a diffusion barrier against the release of Cur molecules from the samples. In addition, there may probably be physical crosslinks between the surface hydroxyls of cellulose crystals and –OH groups of chitosan molecules as shown in Fig. 8. The presence of H-bonding interactions within the film matrix restricts the relaxation and segmental motion of chitosan chains,

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Fig. 8. Physical crosslinks between the surface hydroxyls of cellulose micro crystals and –OH groups of chitosan molecules.

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cells [42]. Therefore, protein adsorption activity of a film is also related to its cell adhesion properties. The protein adsorption capacity of the representative film sample Ch/CMC(20)450 was determined using human pure frozen plasma (PFP) serum as a standard protein. The protein adsorption of each sample was measured using the Bicinchoninic acid (BCA) reagent, and compared against initial protein content of serum, i.e. 60 ␮g/␮l. The amount of protein adsorbed by the film sample Ch/CMC(20)450 was 25.21 ␮g/␮l/cm2 which is an appreciable quantity. This shows that the film sample Ch/CMC(20)450 has fair protein adsorption capacity[43]. Finally, the film sample Ch/CMC(20)450 showed albumin adsorption of10.9 ␮g/␮l/cm2 , while the initial albumin concentration was 36 ␮g/␮l, thus indicating that the film showed fair albumin adsorption capacity. 3.7. Antibacterial activity

thus rendering an almost diffusion-controlled release behavior to these film samples. The values of Higuchi constants for the samples Ch/CMC (0)450 , Ch/CMC (10)450 , Ch/CMC(20)450 , and Ch/CMC(30)450 were 11.5, 8.6, 8.0 and 4.1 × 10−2 h−1/2 , respectively.

The antibacterial action of sample curcumin loaded Ch/CMC(20)450 was investigated qualitatively by using zone inhibition method. The results of antibacterial tests are shown in Fig. 9. The radius of zone developed was found to be 4 cm [44].

3.6. Protein and albumin adsorption test

3.8. Antifungal activity

Owing to the bacterial and fungal infections, an appreciable amount of proteins is found in wound exudates. Therefore, proteins adsorption is necessary for adhesion of cells like fibroblasts

The use of representative sample Ch/CMC(20)450 as functional wound dressings was also assessed by observing their antifungal against C. albicans and C. parapsilosis. Fig. 10(a) and (b) shows

Fig. 9. Antibactarial activity of film samples (a) Ch/CMC(20) and (b) Ch/CMC(20)450 against E. coli.

Fig. 10. Antifungal activity of film samples (a) Ch/CMC(20)450 against Candida albicans and (b) Ch/CMC(20)450 against Candida parapslosis.

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the typical antifungal test results of films by the disc method. It was found that the curcumin loaded film sample Ch/CMC(20)450 demonstrated inhibition zones of nearly 3 and 3.5 cm for C. albicans, and C. parapsilosis, respectively. It is noteworthy here that the plain sample Ch/CMC(20) did not show any antifungal action, but a dense population of colonies was observed(data not shown). Although chitosan has fair reputation as an antimicrobial compound [45], but in the presence study we did not observe its antimicrobial action. This may probably be due to the fact that the chitosan used in the study had a higher molar mass of around 1463 kDa and so it did not show biocidal action. 4. Conclusion This study concludes that curcumin loaded Ch/CMC films demonstrate controlled release of Cur, extended over a time period of 36 h. The amount of curcumin released shows a negative dependence on the concentration of cellulose crystals dispersed within the chitosan matrix. The dispersed CMC produce additional physical crosslinks and retard the release rate. The films show fair antimicrobial activities against bacteria and fungi. Conflict of interest All the authors hereby declare that they do not have any conflict of interest about this manuscript. Acknowledgment The authors are thankful to IIITDM, Jabalpur India for providing facilities for SEM analysis. References [1] A. Kumar, A. Ahuja, J. Ali, S. Baboota, Crit. Rev. Drug Carrier Syst. 27 (4) (2010) 279–312. [2] U.S. Food and Drug Administration Food additive status list, Revised as of April 1 [Accessed on July 17, 2012.]. [3] S.S. Chung, J.V. Vadgama, Anticancer Res. 35 (1) (2015) 39–46. [4] A.E. Krausz, B.L. Adler, V. Cabral, M. Navati, J. Doerner, R.A. Charafeddine, D. Chandra, H. Liang, L. Gunther, A. Clendaniel, S. Harper, J.M. Friedman, J.D. Nosanchuk, A. Friedman, J. Nano medicine 11 (1) (2015) 195–206. [5] D. Mehrabani, M. Farjam, B. Geramizadeh, N. Tanideh, M. Amini, M.R. Panjehshahin, World J. Plast. Surg. 4 (1) (2015) 29–35. [6] S.Z. Fu, X.H. Meng, J. Fan, L.L. Yang, Q.L. Wen, S.J. Ye, S. Lin, B.Q. Wang, L.L. Chen, J.B. Wu, Y. Chen, J.M. Fan, Z. Li, J. Biomed. Mater. Res. B Appl. Biomater. 102 (3) (2014) 533–542. [7] M. Rinaudo, Prog. Polym. Sci. 31 (7) (2006) 603–632.

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cellulose micro crystals (CMC) antimicrobial films.

Following the novel 'vapor induced phase inversion' (VIPI) method, we have prepared curcumin loaded chitosan/cellulose micro crystals composite films ...
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