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International Journal of Biological Macromolecules 75 (2015) 437–446

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

j ourna l h o mepa ge: www.elsev ier .com/ locate / i jb iomac

otential use of curcumin loaded carboxymethylated guar gumrafted gelatin film for biomedical applications

iyali Jana Mannaa,1, Tapas Mitraa,∗∗,1, Nilkamal Pramanika, V. Kavithab,. Gnanamanib, P.P. Kundua,∗

Department of Polymer Science & Technology, University of Calcutta, University College of Science & Technology, 92, A.P.C road,olkata 700009, West Bengal IndiaMicrobiology Division, CSIR-Central Leather Research Institute, Adyar, Chennai 600020, Tamil Nadu India

r t i c l e i n f o

rticle history:eceived 10 October 2014eceived in revised form2 December 2014ccepted 14 January 2015vailable online 4 February 2015

a b s t r a c t

Present study describes the synthesis of carboxylmethyl guar gum (CMGG) from the native guar gum(GG). Further, the prepared CMGG is grafted with gelatin to form CMGG-g-gelatin and then mixed withcurcumin to prepare a biomaterial. The resultant biomaterial is subjected to the analysis of 1H NMR, ATR-FTIR, TGA, SEM and XRD ensure the carboxymethylation and grafting. The results reveal that 45% of theamine groups of gelatin have been reacted with the COOH group of CMGG and 90–95% of curcumin is

eywords:elatinuar gumurcumin

released from CMGG-g-gelatin after 96 h of incubation in the phosphate buffer at physiological pH. In vitrocell line studies reveal the biocompatibility of the biomaterial and the antimicrobial studies display thegrowth inhibition against gram +ve and gram −ve organisms at a considerable level. Overall, the studyindicates that the incorporation of curcumin into CMGG-g-gelatin can improve the functional propertyof guar gum as well as gelatin.

© 2015 Published by Elsevier B.V.

. Introduction

Gelatin is a hydrolyzed product of a native collagen, an abundanttructural protein found in the various parts of the animal body,uch as skin, tendon, cartilage and bone [1]. Gelatin has been widelysed in the pharmaceutical and medical fields as sealants for vas-ular prostheses [2–4], drug delivery vehicle material [5–7], woundressing material [8,9], etc. However, at a temperature above 35 ◦C,he secondary bonding structure of gelatin is completely brokennd thereby destroys the physical network and imparts poor ther-al and mechanical properties. Therefore, the use of stabilizers

o increase the stability of gelatin by cross-linking is a commonractice. The modification of biopolymer (collagen, gelatin, etc.)sing an exogenous cross-linker has been studied extensively for aumber of biomedical applications. Several physical and chemical

ethods have been reported for cross-linking of collagenous mate-

ials. Physical methods include dehydro-thermal treatment andV irradiation [10,11]; however, they are generally less efficient.

∗ Corresponding author. Tel.: +91 9609121901; fax: +91 3323525106.∗∗ Corresponding author.

E-mail addresses: tapasbiochem1@gmail.com (T. Mitra), ppk923@yahoo.comP.P. Kundu).

1 Both authors contributed equally to this work.

ttp://dx.doi.org/10.1016/j.ijbiomac.2015.01.047141-8130/© 2015 Published by Elsevier B.V.

Many chemicals such as carbodiimide [12], formaldehyde [13], glu-taraldehyde [14,15] and oxidized polysaccharides such as dextran[16,17], chondroitin sulfate [18,19], starch [20] and genipin havebeen used to modify the gelatin for biomedical applications [21].Though the stability of the biopolymer is achieved by cross-linkingagents, the low mechanical strength restricts its application. Inaddition, the biocompatibility of the resultant biopolymer is ques-tionable because of the release of toxic components from some ofthe cross-linkers during its usage.

Thus, in order to obviate the problems associated with mechan-ical property and biocompatibility, an attempt is made in thepresent study by grafting the gelatin molecule with a modified nat-ural polysaccharide, carboxy methylated guargum (CMGG). Guargum is a polygalactomannan derived from the seeds of a legumi-nacea plant, Cyamopsis tetragonolobus, and its molecular structureshows a backbone of �-d-mannopyranoses linked at 1→4 positionto which, on the average, every alternate mannose and �-d-galactose is linked to 1→6 position [22]. GG is a non-toxic andbiodegradable polymer and it has found various applications suchas emulsifier, suspending and bioadhesive agent [23]. It is wellknown that curcumin is a natural pigment derived from Curcuma

longa and has well been explored for its antimicrobial [24], antiviral[25], anticancer [26] and wound healing activities [27]. However,curcumin is sparingly soluble in water; this hydrophobic natureof curcumin inhibits its vascular and oral administration. These

438 P.J. Manna et al. / International Journal of Biological Macromolecules 75 (2015) 437–446

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roperties of curcumin can be utilized by incorporating in the filmf carboxymethylated guargum grafted gelatin (CMGG-g-gelatin)or a possible application as an antimicrobial patch for wound heal-ng. Wound healing is a complex and multifunctional process thatelps in the contraction and closure of the wound and restoration ofissue integrity [28]. Infections at the wound site are cited as one ofhe reason for delayed wound healing [29]. Currently, the demandor new wound healing materials with inherent antimicrobial prop-rties is on the rise. Guar and xanthan gums were already patenteds bioabsorbable materials for wound dressing [30]. Therefore, guarum has been selected for the present study to protect gelatin fromaster degradation by the gelatinase enzyme at the wound environ-

ent. Hence, the durability of the CMGG-g-gelatin material will bencreased and it can withstand at the wound site for a prolongedime for sustained release of curcumin.

The major objective of the present study is to prepare a polysac-haride (from plant) and protein (from animal) based graftediomaterial with inherent antimicrobial property as an efficientound healing material. As far as we know, this is the first kind

f report for the preparation of a biomaterial by using a modi-ed plant based material (CMGG) and an animal based materialgelatin), for the delivery of another natural product, curcumin forhe application of an effective wound healing material.

. Experimental details

.1. Materials

Guargum, chloro acetic acid and gelatin were obtained fromerck (India) and picrylsulfonic acid [2,4,6-trinitrobenzene sul-

onic acid (TNBS)], curcumin were obtained from Sigma–AldrichUSA). 3-[4,5-Dimethylthiazol-2-yl]-2,5-dephenyltetrazolium bro-

ide (MTT), 1-ethyl-3(3-dimethyl aminopropyl) carbodiimideEDC), N-hydroxy succinamide (NHS) and 2-(N-morpholino)thanesulfonic acid (MES buffer) were obtained from HiMediaIndia). All of the other reagents were of analytical reagent gradend used without further purification.

.2. GG purification procedure

The commercial guar gum was purified according to the proce-ure as described in the literature [31] with some modifications.

rude guar gum (10 g) was stirred in 250 ml of cold distilled water

or 24 h at room temperature. The clear supernatant was obtainedy centrifugation and ethanol was added to the clear supernatanto precipitate out the carbohydrate. The material was washed again

lation of guar gum.

with ethanol, followed by distilled water and subsequently freeze-dried.

2.3. Preparation of carboxymethyl guargum (CMGG)

GG was derivatized to sodium carboxymethyl guargum fol-lowing the method as reported previously [32]. In brief, forcarboxymethylation, 6 g of purified guar gum was dispersed in120 ml of isopropanol and water mixture (8:2, v/v ratio) taken ina 250 ml round bottom flask connected to an oil bath, and fittedout with a magnetic stirrer. Then, about 18 g of NaOH was addedand continued the stirring for an hour at 50 ◦C. An aliquot of 10 mlchloro-acetic acid of specified weight (6 g) was then added to thereaction mixture, over a period of 30 min. The reaction mixture washeated to a specified temperature (50 ◦C) with continuous stirringfor 4 h, to drive the reaction process to completion (Scheme 1).The reaction product was repeatedly extracted with ethanol andseparated by centrifugation. After the third extraction, the pH wasadjusted to 7 with several drops of glacial acetic acid. Finally, theprecipitate was washed with water and 80% ethanol, and subse-quently vacuum-dried. The modification of GG to CMGG is shownin Scheme 1.

2.4. Determination of degree of substitution (DS) of CMGG

The degree of substitution of CMGG was estimated using atitrimetric method reported elsewhere [33]. 1.5 g of CMGG wasdispersed in 50 ml of 2 M HCl solution using 70% methanol as sol-vent, and the suspension was stirred continuously for 2 h. Duringthis process, the sodium form of CMGG (Na-CMGG) was convertedto its hydrogen form (H-CMGG). The product was then washedwith 95% (v/v) ethanol to remove the chlorine and the dispersionwas filtered. The filtrate was then dried in a vacuum oven at 60 ◦Cfor 2 h. 0.5 g of the dried H-CMGG was dissolved in 50 ml of 0.1 MNaOH solution and stirred for 2 h and the excess of NaOH wasback-titrated with 0.1 M HCl solution using phenolphthalein as anindicator. The DS was calculated using the following equation:

WA = (CNaOHVNaOH − CHClVHCl)m

DS = 162WA

(5900 − 58WA)

where CNaOH and CHCl are the molar concentration of standardNaOH and HCl solutions, WA is the mass fraction of CH2COOH,VNaOH is the volume of NaOH and VHCl is the volume of HCl and mis the weight (g) of polymer taken.

P.J. Manna et al. / International Journal of Biological Macromolecules 75 (2015) 437–446 439

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Scheme 2. Schematic representation on the ch

.5. Preparation of carboxymethylated guargum grafted gelatinCMGG-g-gelatin)

Covalent interaction between CMGG and gelatin was performedsing 1-ethyl-3(3-dimethyl aminopropyl) carbodiimide (EDC) and-hydroxy succinamide (NHS) using the coupling chemistry. Atrst, 3 g of CMGG was dissolved in 50 ml of 0.1 M 2-(N-morpholino)thanesulfonic acid (MES buffer) to maintain the pH of the solutiont 6.5. Then EDC and NHS were added to the solution in the moleatio of 1:2:2 [34] to activate the carboxyl group of CMGG. The car-oxyl group was activated for 24 h at room temperature. 3.0 g ofelatin was dissolved in 50 ml of 1% acetic acid solution and it wasdded to the activated CMGG solution and allowed the reactiono proceed further for another 48 h at room temperature to formhe final product of CMGG grafted gelatin (Scheme 2). The resul-ant solution was purified by exhaustive dialysis (MWCO-10 kDa)gainst double distilled water for 48 h, and then the solution wasried as a film.

.6. Preparation of curcumin loaded CMGG-g-gelatin film

Curcumin loaded CMGG-g-gelatin was prepared by a simple,on covalent interaction method. Curcumin was weighed accu-ately to get a final concentration of 1 mg/ml in acetone and wasixed with 60 ml of a freshly prepared solution of CMGG-g-gelatinith constant stirring for 24 h, at room temperature. The suspen-

ion was then squeezed through a muslin cloth to remove anyrecipitate formed during the process. The solution was then placed

n a polypropylene plate for drying in a vacuum oven at 50 ◦CScheme 3).

ry behind the preparation of CMGG-g-gelatin.

2.7. Texture and morphology of the biopolymers

The physical texture and the morphology of the GG, CMGG,gelatin and CMGG-g-gelatin were assessed using the physicaltouch, followed by observation under a scanning electron micro-scope (SEM) (ZEISS EVO-MA 10 Scanning Electron Microscope) at ahigh voltage of 15 kV.

2.8. Analysis of functional groups by FT-IR spectroscopy

The analysis of the functional groups present in GG, CMGG,gelatin and CMGG-g-gelatin were made by a Fourier transforminfrared spectrophotometer (Alpha, Bruker, Germany). All spec-tra were recorded at the resolution of 4 cm−1 in the range of400–4000 cm−1.

2.9. X-ray diffraction analysis

The X-ray diffraction of the polymer film was performedby a wide angle X-ray scattering diffractometer (PanalyticalX-ray Diffractometer, model-X’pert Pro) with Cuk� radiation(� = 1.54060) in the range of 5–69◦ (2�) at 40 kV and 30 mA.

2.10. NMR analysis

The 1H nuclear magnetic resonance (1H NMR) spectrum was

determined by a Bruker AV 3000 Supercon NMR system (Germany)at 300 MHz using D2O/DCl and D2O as solvent. Chemical shift (ı)was reported in ppm using tetramethylsilane (TMS) as an internalreference.

440 P.J. Manna et al. / International Journal of Biological Macromolecules 75 (2015) 437–446

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Scheme 3. Interaction of curcumin with CMGG-g

.11. Estimation of interacted amine group of CMGG-g-gelatin byNBS assay

The analysis of the interacted amine groups was quantifiedsing TNBS assay according to the procedure summarized by Mitrat al. [35]. In brief, native and grafted (CMGG and CMGG-g-gelatin)iopolymers were cut into small pieces of size 4.5 mm. Six mil-

igrams of cut pieces were immersed in a 2 ml of solution consistedf 1 ml of 4% (w/v) di-sodium hydrogen orthophosphate and 1 mlf 0.5% (v/v) TNBS, and incubated at 40 ◦C for 2 h. Termination ofeaction was done with the addition of 3 ml of 6 M (v/v) HCl solu-ion and the incubation was continued at 60 ◦C for 90 min. Thebsorbance of the resulting solution was measured at 345 nm andhe interacted amine group was calculated from the difference inhe absorbance divided by the absorbance of the native materialnd then multiplied with 100. The absorbance of the resulting mix-ure was measured at A345 nm using UV–vis spectrophotometer.

.12. Analysis of mechanical properties

Mechanical properties, viz., Young’s modulus, tensile strengthnd the maximum stretching the length of the dried film wereeasured using a Universal Testing Machine (LLOYD model no

R10k Plus) at a crosshead speed of 5 mm min−1 at 25 ◦C and at5% relative humidity. Length and width of the dumbbell shapedest samples were maintained at 20 and 5 mm respectively. All ofhe mechanical tests were performed with dried samples and werexamined in triplicate.

.13. Thermo gravimetric analysis (TGA)

Thermal decomposition analysis of GG, CMGG, gelatin andMGG-g-gelatin was carried out under nitrogen flow (40 and0 ml min−1) with ramp 20 ◦C min−1 using a TGA Q 50 (V20.6 build1) instrument.

.14. In vitro curcumin release

A known amount of curcumin loaded CMGG-g-gelatin film

50 mg) was immersed in 2 ml of phosphate buffer solution, pH 7.4t 37 ◦C, followed by the transfer into 7 centrifuge tubes and wasept in a shaker. Free curcumin was not soluble in water; there-ore, at predetermined time intervals, the solution was centrifuged

in through non covalent interaction (H-bonding).

at 5000 RPM for 7 min to separate the released curcumin from theCMGG-g-gelatin film. The released curcumin was redissolved in3 ml of ethanol and determined its concentration by UV–vis spec-trometer at 425 nm. The concentration of releasing curcumin wasthen calculated using a standard curve of curcumin in ethanol. Thepercentage of curcumin released was determined from the follow-ing equation: curcumin release (%) = 100 × curcumin released ontime (t)/total curcumin loaded in CMGG-g-gelatin film.

2.15. Evaluation of antimicrobial activity

The antimicrobial activity of the prepared curcumin loaded/notloaded CMGG-g-gelatin film was tested by the disk diffusionmethod against gram-negative bacteria namely Escherichia coli,Enterobacter aerogenes, Vibrio vulnificus and Pseudomonasaeruginosa and gram-positive bacteria, namely Bacillus cereus,Bacillus subtilis, Lysinibacillus and Staphylococcus aureus. The filmswere cut into a disk shape with 5 mm diameter followed bysterilization under UV light for 10 min and placed on agar plateswith various cultures. The plates were incubated for 24 h at 37 ◦Cin an incubator and the zone of inhibition was then calculated. Thezone of inhibition was not observed for native CMGG-g-gelatinfilm (not loaded with curcumin). This experiment was performedin triplicate with each organism and an average diameter of zoneof inhibition was noted.

2.16. Hemolytic assay

Blood compatibility was examined by hemolytic assay. Freshhuman blood was used in this study. RBC lysis investigates the pro-tein denaturing effect of curcumin incorporated CMGG-g-gelatinusing a biological material (RBC) as a substrate. Freshly isolatedRBCs were incubated in phosphate buffer saline. An irritatingsubstance will cause lysis of the RBCs, leading to the release ofhemoglobin in the sample. The cell debris and intact cells wereseparated by centrifugation and the amount of hemoglobin, whichcorresponded to the number of cells lysed by the curcumin loadedCMGG-g-gelatin, was assayed by spectrophotometry. At first, dif-ferent volume of samples in tubes (10, 20, 30, 50, 75, 100 �l) was

taken and made up to 950 �l with PBS, followed by the additionand mixing of 50 �l of RBC sample. The samples were incubated inthe dark for 10 min and then centrifuged for 10 min at 6000 RPM.The OD value of the supernatant was measured at 540 nm using

P.J. Manna et al. / International Journal of Biological Macromolecules 75 (2015) 437–446 441

Fig. 1. (a) Digital images of CMGG, gelatin, CMGG-g-gelatin and curcumin loaded CMGG-g-gelatin biopolymers. (b) SEM Micrographs of CMGG, gelatin and CMGG-g-gelatinbiopolymers. (Arrow mark indicates the smooth surface of CMGG, fibers in gelatin and network structure in CMGG-gelatin film).

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spectrophotometer. The obtained results were compared withhe positive control (50 �l RBC + 950 �l H2O) and the negative con-

rol (50 �l RBC + 950 �l PBS). Each concentration was evaluated inriplicate. The % of lysis calculated by the formula; % lysis = (ODf sample − OD of positive control) × 100/(OD of negative con-rol − OD of positive control) [36].

2.17. In vitro assessment of cell compatibility of the GG, CMGG,CMGG-g-gelatin and curcumin loaded CMGG-g-gelatin

Biocompatibility in terms of cytotoxicity and cell proliferation ofthe prepared biomaterials were analyzed using NIH 3T3 fibroblastcell line [37].

4 Biological Macromolecules 75 (2015) 437–446

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42 P.J. Manna et al. / International Journal of

.18. Cell proliferation study (MTT assay)

Cells were grown in DMEM (Dulbecco’s modified Eagle’sedium) supplemented with 10% (v/v) fetal bovine serum and

% antibiotic and were incubated at 37 ◦C in 5% CO2 humidifiedtmosphere. Polystyrene 96 well culture plates (Tarson, India)ere coated with GG, CMGG, CMGG-g-gelatin and curcumin loadedMGG-g-gelatin biomaterials. The plates were dried in a laminar airow hood followed by the UV sterilization. The cells were seededt the density of 0.5 × 106 per well and incubated at 37 ◦C in aumidified atmosphere containing 5% CO2. After 12 h of incuba-ion, the supernatant of each well was replaced with MTT dilutedn serum-free medium and the plates were incubated at 37 ◦C for

h. After removing the MTT solution, a mixture of acid and iso-ropanol (0.04 N HCl in isopropanol) was added to each well andipette up and down to dissolve all of the dark blue crystals andhen left at room temperature for a few minutes to ensure all crys-als were dissolved. Finally, absorbance was measured at 570 nmsing a UV spectrophotometer. Each experiment was performed at

east three times. The sets of three wells for the MTT assay weresed for each experimental variable.

. Results and discussion

The CMGG is prepared from GG by the consecutive two-stepeaction. In step one, a strong base such as sodium hydroxide depro-onates the free hydroxyl groups from CH2OH group of GG toorm alkoxides, thereby increases their nucleophilicity. In the sec-nd step, the prepared guar alkoxide reacts with chloroacetic acid,s a consequence of which carboxymethyl groups are formed on theG backbone. The overall reaction is briefly shown in Scheme 1.

Fig. 1a and b demonstrates the digital images and morpholog-cal features of the CMGG, gelatin, CMGG-g-gelatin and curcuminoaded CMGG-g-gelatin film. Thin film of CMGG is very smooth,

hereas the native gelatin has fiber like structure. The CMGG-g-elatin film displays a network like structure; this may be attributedo the grafting of gelatin into the CMGG backbone. This high net-ork like structure of the CMGG-g-gelatin suggests the suitability

f this biopolymer for biomedical applications, including serving as delivery vehicle of various drug molecules.

FT-IR studies are conducted to ensure the chemical modifica-ions in GG and gelatin structures. Fig. 2 illustrates the FT-IR spectraletails of GG, CMGG, gelatin, CMGG-g-gelatin. As per the spectrumf the native GG, the absorption band at around 3279 cm−1 is dueo the O-H stretching vibration and another band at 2918 cm−1 isssigned as a C H stretching vibration band. The band at 1140 cm−1

or C O stretching, vibration indicates the presence of alcoholicroup in GG. The absorption band at around 1007 cm−1 is due tohe glycosidic linkage of pyranose ring of GG [38]. A slight mod-fication is observed in the spectrum of CMGG. Two new bandsre appearing at 1555, and 1320 cm−1, corresponding to the COO−

symmetric and symmetric stretching vibration, respectively. Thisndicates the presence of COOH group in the CMGG. The intensityf the peak found in GG at 3279 cm−1 is reduced in CMGG; this isue to the successful carboxymethylation in the hydroxyl group ofG [39]. The sharp peak at 2886 cm−1 is due to the C H stretchingnd the peak at 1014 cm−1 is due to the bending of CH2 O CH240]. The FT-IR spectra of gelatin show strong absorption bandst 3277, 1632, 1522 and 1236 cm−1 due to the N H stretching,

O stretching (amide I), amide II and C N stretching, respec-ively [41]. FT-IR spectrum of CMGG-g-gelatin displays significant

hanges compared to the parent molecules due to the covalentnteraction between NH2 group of gelatin and COOH group ofMGG, leading to the formation of amide linkages. In the nativeelatin spectrum, the sharp, intense amide-I and II bands which

Fig. 2. FT-IR spectrum of GG, CMGG, gelatin and CMGG-g-gelatin biopolymers.

are observed at around 1632 and 1522 cm−1 are reduced (amide Iand II and free primary amines); this may be due to the reductionof free NH2 group in gelatin. Results from FT-IR analysis reflectthat gelatin is grafted with CMGG through the formation of amidelinkages [41].

The wide angle X-ray diffractogram of native GG, CMGG, gelatinand CMGG-g-gelatin are represented in Fig. 3. From the obtainedresult, it is observed that the native GG exhibits low crystallinity.Similar kind of observation has been reported by Pal et al. [42] fornative GG. After carboxymethylation, a typical reduction in crys-tallinity is observed in CMGG. This loss in crystallinity may bedue to the carboxymethylation of the hydroxyl groups of nativeGG. The intermolecular hydrogen bonding in GG is responsiblefor the crystallinity in GG; when the interaction is disrupted, itleads to the reduction in the crystallinity of CMGG. Gelatin has asmall peak at 2� = 7◦ and a large broad, amorphous peak at 2� = 22◦,whereas in CMGG-g-gelatin, the intensity of this peak is decreased.The decrease in the degree of crystallinity is due to the increasein covalent interaction between CMGG and gelatin, leading to thegrafting of gelatin with CMGG through the formation amide link-ages [43,44].

Fig. 4 describes the 1H NMR spectra wherein the native GG spec-trum shows two peaks at ı 4.7 and at ı 3.5–4.2 due to the anomericprotons and sugar protons, respectively [40]. On the other hand,the CMGG spectrum reveals the occurrence of new proton peaks

at ı = 3.8, 3.9 and 4.08 ppm, attributed to the methylene protons inthe carboxymethoxy substituent at the position of C-6 of the �-d-galactose unit and at the position of C-3 of the �-d-mannose unit.The peak at ı = 4.6 and 4.95 is due to the anomeric proton of CMGG

P.J. Manna et al. / International Journal of Biological Macromolecules 75 (2015) 437–446 443

Fig. 3. XRD patterns of GG, CMGG, gelatin and CMGG-g-gelatin biopolymers.

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Table 2Thermal analysis of GG, CMGG, gelatin and CMGG-gelatin under N2 air atmosphere.

Temperature (◦C) % of weight loss (heating rate 20 ◦C min−1)

GG CMGG Gelatin CMGG-gelatin

100 4 10 7 5200 18 15 15 13300 60 49 32 24

45% for CMGG-g-gelatin film.With regard to the mechanical property, CMGG-g-gelatin bio-

material shows tensile strength of 41.64 MPa, where the tensilestrength of CMGG and native gelatin strength is only 26.07 and

Table 3

Fig. 4. 1H NMR spectra of native guar gum and 1H NMR spectra of CMGG.

verlay by the solvent peak (D2O-d), and the other peaks located at

round ı = 3–4.1 is due to sugar protons of CMGG [40]. The resultsbtained from proton NMR spectra confirm the polysaccharide car-oxymethylation reaction. Partial degree of substitution of CMGG

s calculated to be 0.45 from the calculation procedure described in

able 1ssessment of mechanical properties of GG, CMGG, gelatin and CMGG-gelatin.

Sample Name Strain atmaximum load

Tensilestrength (MPa)

Young’smodulus (GPa)

GG 0.045 1.36 2.70607CMGG 0.049 26.07 116.342Gelatin 0.108 3.35 111.18792CMGG-gelatin 0.261 41.64 358.53377

400 73 66 76 58500 79 72 88 67600 80 73 93 70

Section 2. The percentage of the interacted amine group is observed

Antimicrobial activity of the curcumin loaded CMGG-gelatin film against gram-negative and gram-positive bacteria.

Microorganisms Average diameter of zone ofinhibition (mm) (mean ± SD)

Gram-negativeE. coli 16 ± 1.0E. aerogenes 16 ± 0.5V. vulnificus 16 ± 1.0P. aeruginosa 12 ± 0.5

Gram-positiveB. cereus 16 ± 1.0B. subtilis 17 ± 1.0Lysinibacillus 15 ± 2.0S. aureus 15 ± 1.0

444 P.J. Manna et al. / International Journal of Biological Macromolecules 75 (2015) 437–446

Fig. 5. Thermo gravimetric analysis of GG, CMGG, gelatin and CMGG-g-gelatinbiopolymers.

Fig. 6. Blood compatibility studies of curcumin loaded CMGG-g-gelatin where (1)Positive control (50 �l RBC + 950 �l H2O); (2) Negative control (50 �l RBC + 950 �l

Fig. 7. The microscopic NIH 3T3 cell morphology of (a) CMGG, (b) gelatin, (c) CMGG-g-gelatin, CMGG-g-gelatin, CMGG-g-gelatin-curcumin and control after 12 h of incubation.

PBS); and (3–8) 10, 20, 30, 50, 75 and 100 �l of CMGG-g-gelatin sample make up to950 �l with PBS and then 50 �l of RBC sample was added and mix.

3.35 MPa, respectively. Nearly two fold increases in tensile strengthis observed for CMGG-g-gelatin compared to CMGG, whereas12–13 fold increase in tensile strength is observed for CMGG-g-gelatin in comparison to the native gelatin. The strain at maximumload of CMGG-g-gelatin is observed much higher than that of the

native GG, CMGG and gelatin; this may be due to the grafting ofgelatin with CMGG. The ultimate tensile strength (MPa), strain atmaximum load and Young’s modulus (GPa) of GG, CMGG, gelatinand CMGG-g-gelatin are represented in Table 1.

gelatin, (d) CMGG-g-gelatin-curcumin, (e) control and (f) MTT analysis of CMGG,

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P.J. Manna et al. / International Journal of

Thermo gravimetric analyses of GG, CMGG, gelatin and CMGG--gelatin are represented in Fig. 5 and the corresponding thermalegradation values are reported in Table 2. From the results, it

s observed that the incorporation of gelatin to CMGG tends tohift the thermal region of higher temperature and such a shifts attributed to an increase in the thermal stability. The thermaltability also influences on the durability of the biopolymers.

The release of curcumin from CMGG-g-gelatin film is conductedn phosphate buffer solution to evaluate its sustained delivery. Theverage concentration of curcumin release from curcumin loadedMGG-g-gelatin is approximately 14.8, 17.5, 23.7, 28.3, 37.6, 49.9,9.3, 81.7 and 93.5% in the respective duration of 1, 3, 6, 9, 12, 24, 48,2 and 96 h. Van Cuong Nguyen et al., Mei Dai et al. [45,46] observed

similar kind of release pattern of curcumin from curcumin-loadedhitosan/gelatin composite sponge and chitosan-alginate spongend they observed 80–90% of curcumin release after 50–96 h ofncubation in PBS. Comparing all of these results, it is confirmed thathe curcumin loaded CMGG-g-gelatin film prepared in the presenttudy has a better sustained release property. The material with thebove said property will be very helpful as a wound dressing mate-ial because the curcumin will release on the wound site at a slowerate and this will restrict the bacterial growth for a prolonged time,eading to quick subsequent wound healing.

The results of the antibacterial activity studies of the preparedurcumin loaded CMGG-g-gelatin film against the identified bacte-ia (details given in Section 2) has been listed in Table 3. Thenhibition zones are obtained in the range between 16 and 17 mmor both gram-negative and gram-positive bacteria. The results sug-est that the prepared curcumin loaded film has a similar kindf antibacterial efficiency against both gram-negative and gram-ositive bacteria. Due to this antibacterial property, it can bepplied as an efficient wound healing material because the inherentntimicrobial property of the material will not allow the microbialrowth on the wound site, leading to quick healing of the wound.

The hemolysis assay is a significant index of the material for thepplication in the biomedical field because the material is usuallyxposed to blood environment and damaged the erythrocytes in aertain degree. In the present study, the assay is carried out to eval-ate the blood compatibility of curcumin loaded CMGG-g-gelatin.he results in Fig. 6 shows that there is no damage on the erythro-ytes by the curcumin loaded CMGG-g-gelatin. This reveals that therepared biomaterial has high blood compatibility.

With reference to the cytotoxicity of the resulting biomateri-ls, cell proliferation assays are carried out. MTT assay is done toheck the toxicity of the prepared biomaterials (CMGG, gelatin,MGG-g-gelatin and curcumin loaded CMGG-g-gelatin). Only cellshat are metabolically normal can turn the tetrazolium salts intourple crystals. Compared with the native gelatin, CMGG, CMGG-g-elatin and curcumin loaded CMGG-g-gelatin exhibit no significantifferences in the absorbance (Fig. 7). This indicates that biomate-ials in direct contact with fibroblast do not lead to apoptosis orecrosis. MTT results clearly indicate that NIH 3T3 cells proliferateell on the surface of the CMGG-g-gelatin and curcumin loadedMGG-g-gelatin film.

. Conclusion

In the present study, we have synthesized carboxymethylateduar gum from native guar gum with the DS value of 0.45 andxpounded the possibility of grafting of gelatin with CMGG. Further,he study has also explored for the enhancement of the mechanical

roperty and thermal stability of the CMGG-g-gelatin film com-ared to the native CMGG and gelatin film. To the best of ournowledge, this is the first kind of report of grafting between annimal based product, i.e. gelatin and a plant based product, i.e.

[

[

ical Macromolecules 75 (2015) 437–446 445

guar gum through its modification (CMGG). The COOH group isinserted into GG to increase its functionality which makes it capableto react with the free NH2 group of gelatin. To make the CMGG-g-gelatin film as an efficient wound healing material, curcumin(very familiar for its antibacterial activity) is loaded to incorpo-rate its antibacterial activity. The obtained results from the diskdiffusion test against various microbes indicate that the curcuminloaded CMGG-g-gelatin film has appreciable antibacterial activ-ity. In vitro cell line result and hemocompatibility assay stronglysuggest about the biocompatibility and blood compatibility of theCMGG-g-gelatin film.

Acknowledgments

One of the authors Dr. Tapas Mitra acknowledges UGC, NewDelhi for financial assistance provided in the form of Dr. D. S. KothariPost Doctoral Fellowship.

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