International Journal of Biological Macromolecules 79 (2015) 76–85

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

Minocycline-loaded cellulose nano whiskers/poly(sodium acrylate) composite hydrogel films as wound dressing S.K. Bajpai a , V. Pathak b , Bhawna Soni a,∗ a b

Polymer Research Laboratory, Department of Chemistry, Govt. Model Science College, Jabalpur 482001, MP, India Department of Physical Science, Mahatma Gandhi Gramodaya University, Chitrakoot, MP, India

a r t i c l e

i n f o

Article history: Received 7 February 2015 Received in revised form 31 March 2015 Accepted 20 April 2015 Available online 2 May 2015 Keywords: Cellulose nano whiskers Hydrogel film Drug release Wound dressing Blood compatibility test

a b s t r a c t In this work, antibiotic drug Minocycline (Mic) loaded cellulose nano-whiskers (CNWs)/poly(sodium acrylate) hydrogel films were prepared and investigated for their drug releasing capacity in physiological buffer solution (PBS) at 37 ◦ C. The (CNWs)/poly(sodium acrylate) film, containing 9.7% (w/w) of CNWs, demonstrated Mic release of 2500 ␮g/g while the plain poly(acrylate) film showed 3100 ␮g/g of drug release. In addition, with the increase in the concentration of cross-linker N,N -methylene bisacrylamide (MB) from to, the drug release from the resulting films decreased from 507 to 191 ␮g/g. The release exponent ‘n’ for films with different compositions was found in the range of 0.45 to 0.89, thus indicating non-Fickian release mechanism. The Schott model was employed to interpret the kinetic drug release data successfully. The film samples poly(SA) and CNWs/poly(SA) (both not containing drug) showed thrombus formation of 0.010 ± 0.001 g and 0.007 ± 0.001 g, respectively, thus showing the non-thrombogenic behavior. In percent Hemolysis, both of the film samples of 1.136 ± 0.012 and 0.5 ± 0.020, respectively, thus indicating non-hemolytic behavior. In addition, both of the film samples demonstrated protein adsorption of 49.02 ± 0.59 ␮g/␮L and 51.20 ± 0.51 ␮g/␮L per cm2 , thus revealing a fair degree of protein adsorption. Finally, the Mic-loaded films showed fair anti-fungal and antibacterial properties. © 2015 Elsevier B.V. All rights reserved.

1. Introduction The replacement or regeneration of damaged epidermal tissues by new ones may be referred to as wound healing process [1]. However, with new biopolymers and fabrication techniques, a wound dressing material is expected to have extraordinary properties which enhance the healing process of a wound. For an effective design of a functional wound dressing, characteristics of the wound type, wound healing time, physical, mechanical, and chemical properties of the dressing material must be taken into consideration. Ultimately, the main purpose is to achieve the highest rate of healing and the best esthetic repair of the wound [2]. The primary goals of wound care are rapid wound closure and leave minimal or esthetically acceptable scar. Wound management is important in providing optimum healing milieu for wound healing [3]. Depending on the severity of the wound, the desirable wound dressing may therefore serve among the purposes of (a) to provide moisture and occlusion, (b) protection from

∗ Corresponding author. Tel.: +91 9993220651. E-mail addresses: [email protected] (S.K. Bajpai), [email protected] (B. Soni). http://dx.doi.org/10.1016/j.ijbiomac.2015.04.060 0141-8130/© 2015 Elsevier B.V. All rights reserved.

infections and contamination, (c) debridement, and (d) easy application and removal avoiding dressing-related trauma [4,5]. In addition, it should be able to permeate the gases and keep the wound environment moist [6]. Finally, fluid balance, particularly in burn injury, is also very important since heavy loss of water from the body by exudation and evaporation may lead to a fall in body temperature and increase in the metabolic rate. Apart from the above mentioned requirements, dressing should also have certain other properties like ease of application and removal, and proper adherence. Occasionally, drug-loaded wound dressings are used to treat wound locally such as anti-infections due to secondary infection or for pain control, especially in chronic wounds [7]. Recent advances in the field of biomaterials and their medical applications indicate the significance and potential of various microbial polysaccharides in the development of novel classes of medical materials. Several of the microbial-derived polysaccharides possessing novel and interesting physical and biological properties already have been applied in biotechnology products or are presently being widely investigated (e.g. hyaluronic acid, dextran, alginate, scleroglucan, chitosan etc.) [8–12]. However, these biopolymers are usually crosslinked to regulate their physic-chemical properties by crosslinking them with glutaraldehyde, epichlorohydrin etc. which are reported to be toxic [13,14]. Indeed, synthetic polymers based

S.K. Bajpai et al. / International Journal of Biological Macromolecules 79 (2015) 76–85

hydrogels like poly(sodium acrylate), poly(acrylamide) etc. have also been frequently used for wound dressing and other biomedical applications. The hydrogels have put their strong candidature as wound dressings because their soft, rubbery, and flexible texture resembles the human tissues [15]. In addition, they provide excellent water absorption properties, and keep the wound environment moist [16]. In order to prepare wound dressing films with controllable drug release properties, we have previously reported synthesis and water absorption behavior of cellulose nano-whiskers (CNWs) loaded poly(SA) films [17,18]. It was observed that amount of CNWs, present in the film matrix, could easily control the water absorption and moisture permeation properties of composite films. It is believed that cellulose crystals possess a distinctive nanofibrillar structure that may become a perfect matrix as an optimal wound healing environment. As a biopolymer, cellulose is the most

DE =

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chamber at ambient temperature till they showed constant weight. The compositions of various films prepared are given in Table 1. 2.4. Determination of drug entrapment The drug entrapped per gram of the film was determined as described below: A pre-weighed piece of completely dry film was placed in 25 mL of aqueous drug solution containing definite quantity of drug Mic and equilibrated for a period of 24 h to ensure equilibrium loading. Now, the film sample was taken out carefully, hung for 2 min over the solution to let the loosely bound surface solution be dropped into the drug solution and then this remaining drug solution was again made up to 25 mL with distilled water and its absorbance was recorded at 390 nm (Shimadzu, Genesis 10-S). After transforming the absorbance into concentration, the amount of drug entrapped per gram of film was determined using the expression:

(Initial drug content in solution − Final drug content in solution) ␮g/g Weight of dry film

common biodegradable natural material and is present in a large amount of natural substances [19]. In continuation of our previous works, we hereby report drug release behavior and antimicrobial action of Minocycline loaded CNWs/poly(SA) hydrogel films. 2. Materials and methods 2.1. Materials The cellulose pulp, used to prepare cellulose nanocrystals (CNWs), was obtained from a paper mill (Amlai Paper Mill, Shahdol, M.P., and India) and was used without any further chemical or physical treatments. The monomer sodium acrylate (SA), cross-linker N,N methylenebis-acrylamide (MB), initiator potassium persulfate (KPS), catalyst N,N,N,N tetra methyl ethylenediamine (TEMED), calcium chloride, 36% formaldehyde, and sodium chloride were obtained from Hi Media Chemicals, Mumbai, India. Minocycline (molecular formula C23 H27 N3 O7 ·HCl; molar mass 493.95) was a gift from Ranbaxy Laboratories (Gurgaon, Haryana, India) and certified to contain 99.62% (w/w) on dry basis. Total protein and albumin assay kits were obtained from Hi Media Chemicals, Mumbai, India. The double distilled water was used throughout the investigations.

The drug contents (␮g/g) in the film samples A, B, C, D and E were found to be 5380, 4968, 3728, 2915 and 6952 ␮g/g of film, respectively. 2.5. Drug release study The pre-weighed drug loaded film was placed in 25 mL of physiological fluid (PF) at 37 ◦ C. The amount of drug released at different time intervals was studied spectrophotometrically (Shimadzu, Genesis 10-S) at 390 nm. After each measurement, the hydrogel was put in to 25 mL of fresh physiological fluid. Calibration curve, prepared for the drug solutions of known concentrations in the appropriate range, was used to determine the amount of drug released. 2.6. In vitro cytotoxicity test In vitro cytotoxicity of film was evaluated by an extract method, based on a protocol that was adapted from ISO 10993-5, 2009 Standard Test Method (STM). The test was carried out in source of cell ATCC strain L-929 is an established and well characterized mammalian cell line that has demonstrated reproducible results and minimum essential medium supplemented with fetal bovine serum. The test sample was sterilized by steam at 121 ◦ C for 20 min. Preparation of test samples by these steps.

2.2. Method of preparation and characterization of CNWs In order to obtain cellulose nano-whiskers (CNWs), the controlled acid hydrolysis of de-watered cellulose pulp (DCP) was carried out. The synthesis conditions were as follows: concentration of sulphuric acid = 64 (wt%), hydrolysis temperature = 45 ◦ C, and reaction time = 75 min. A detailed characterization of CNWs has been reported in our previous research document [17].

(1) Positive control was prepared by diluting phenol stock solution (13 mg/mL) to 1.3 mg/mL with culture medium containing serum. (2) Negative control was prepared by 1.25 cm2 ultra high molecular weight poly ethylene containing 1 mL of physiological saline at 50 ± 2 ◦ C for 72 ± 2 h. (3) Extract was prepared by incubating 0.2 g of test sample in 1 mL of physiological saline at 50 ± 2 ◦ C for 72 ± 2 h.

2.3. Preparation of drug loaded CNWs/poly(SA) hydrogel films The CNWs loaded poly(SA) films were prepared by aqueous free radical polymerization of monomer SA in the presence of dispersed cellulose nano-whiskers, using MB as cross linker and TEMED as catalyst [18]. In order to prepare drug loaded films, method of equilibration was followed. A pre-determined quantity of drug was dissolved in definite volume of water and the CNWs/poly(SA) films were equilibrated for a period of 12 h. After loading, films were taken out, washed superficially with distilled water to remove the drug which was loosely bound on the surface so that ‘burst effect’ could be eliminated. The drug-loaded films were dried in a dust free

2.6.1. Procedure of in vitro cytotoxicity test An in vitro cytotoxic study test using Test on Extract method was performed with test sample based on ISO 10993-5, 2009. Test sample was soaked in 5 mL physiological saline prior to extraction. After soaking completely with saline, additional 1mLsaline was added before dilution of extract. The extract was mixed with MEM2X (1 part of extract and 1 part of MEM2X) medium to get 50% extract. Physiological saline without test material processed similar to test sample was considered as reagent control. Different dilution of test sample extract, positive control and 100% extracts of negative control, reagent control in triplicate were placed on sub

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Table 1 Compositions of various CNWs/poly(SA) film samples synthesized* . Sample code

SA (mmol)

MB (␮mol)

KPS (␮mol)

TEMED (␮mol)

CNWs (wt%)

Minocycline drug (␮mol)

A* B* A B C D E

21.28 21.28 21.28 21.28 21.28 21.28 21.28

195 195 195 195 351 507 195

223 223 223 223 223 223 223

60 60 60 60 60 60 60

0 9.7 0 9.7 9.7 9.7 9.7

0 0 40.49 40.49 40.49 40.49 80.98

*

The total volume of the reaction mixture was 10.0 mL.

Table 2 Cells were examined microscopically and cellular responses were scored. Grade

Reactivity

Condition of all cultures

0 1

None Slight

2

Mild

3

Moderate

4

Severe

Discrete intra-cytoplasmatic granules, no cell lysis, no reduction of cell growth Not more than 20% of the cells are round, loosely attached and without intra-cytoplasmatic granules, or show changes in morphology; occasional lysed cells are present; only slight growth inhibition observable Not more than 20% of the cells are round, devoid of intra-cytoplasmatic granules, no extensive cell lysis; not more than 50% growth inhibition observable Not more than 70% of the cell layers contain rounded cells or are lysed; cell layers not completely destroyed, but more than 50% growth inhibition observable Nearly complete or complete destruction of the cell layers

confluent monolayer of L-929 cells. After incubation of cells with extract of test sample and control at 37 ± 1 ◦ C for 24 to 26 h, cell culture was examined microscopically for cellular response. Cells were examined microscopically and cellular responses were scored as 0, 1, 2, 3 and 4 based on Table 2. 2.7. Blood compatibility The blood compatibility of samples was determined in the terms of thrombogenicity, haemolytic potential and total protein adsorption following the procedure given in a report from the International Standard Organization (ISO) [20]. All tests were carried out at pH 7.4 [21]. 2.7.1. Thrombus formation test The thrombus formation on polymeric film surface was evaluated by gravimetric method. Prior to the test, the films (1 cm2 ) were kept in 0.9% saline solution for 24 h at 37 ◦ C. Thereafter, films were removed from saline solution and then 0.05 mL of acid citrate dextrose (ACD) blood was poured over the films, followed by addition of 0.03 mL of 0.1 M CaCl2 and leaving the films for 10 min [21]. After 45 min, 4 mL of distilled water was added to each film to stop the clotting process. A 36% formaldehyde solution (2 mL) was used to fix the clot. The clot was dried and weighed [22]. The positive control test was carried out by taking same amount of ACD blood without sample and negative control test was carried out by saline and distilled water without sample and blood, respectively [23]. The thrombus percentage was calculated as follow: Thrombose (%) =

 Weight of test sample − Weight of (−) control  Weight of (+)control − Weight of (−) control

× 100%

2.7.2. Hemolysis test The film samples were analyzed for its hemolytic behavior using the method described in American Society for Testing and Materials (ASTM) ASTM F 756-00 (2000). The sample, with surface area of 1 cm2 was incubated in 7 mL of 0.9% saline at 37 ◦ C for a period of 24 h. Now the saline was removed and test tube was filled with 0.05 mL of acid citrate dextrose (ACD) blood. After 15 min 10 mL of 0.9% saline solution was added on the film surface and incubated

for 3 h at 37 ◦ C. Positive and negative controls were prepared by adding the same amount of ACD blood to 10 mL of distilled water and 0.9% saline, respectively. To ensure the contact of film surface with blood, each tube was gently inverted. After incubation, each tube was centrifuged at 104 rpm for 15 min. The optical density (OD) of the supernatant was measured with a UV–visible spectrophotometer (Shimadzu) at 545 nm to determine the amount of hemoglobin released. The percentage of Hemolysis was calculated as follows [24]: Heamolysis(%) =

 OD of test sample − OD of(−)control  OD of(+)control − OD of(−)control

× 100%

As per ASTM F 756-00, materials can be classified in to three categories on the basis of their % Hemolysis (hemolytic index), i.e. with >5% Hemolysis are hemolytic; while 2% to 5% are slightly hemolytic and