Carboxymethylation of Locust Bean Gum: Application in

Article pubs.acs.org/IECR

Carboxymethylation of Locust Bean Gum: Application in Interpenetrating Polymer Network Microspheres for Controlled Drug Delivery Santanu Kaity and Animesh Ghosh* Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, Ranchi 835215, Jharkhand, India S Supporting Information *

ABSTRACT: For hydrophilic modification, the sodium carboxymethyl ether of locust bean gum was developed by Williamson synthesis using monochloroacetic acid as the etherifying agent. The modification reaction was optimized in terms of concentration of monochloroacetic acid and sodium hydroxide. The modified gum was evaluated for its degree of substitution, elemental analysis, viscosity, swelling, and contact angle. The etherification of locust bean gum was further confirmed by FTIR, 13 C NMR, DSC, and XRD techniques. Acute oral toxicity and biodegradability studies showed that the modified gum was safe enough for internal use. This carboxymethylated gum with poly(vinyl alcohol) was utilized to prepare the interpenetrating polymer network microspheres of buflomedil hydrochloride for controlled drug delivery. The microspheres were evaluated for their drug entrapment efficiency, swelling, and particle size. The microspheres were further characterized by FTIR, 13C NMR, and XRD techniques. The in vitro release study of microspheres showed retarded drug release up to 12 h. has a molecular weight range of 3.0 × 106−3.6 × 106 g/mol. The mannose:galactose (M:G) ratio of LBG from Ceratonia siliqua is 3.5:1.13 LBG is a nongelling neutral polysaccharide which is highly viscous and relatively stable against variations in pH, salinity, and temperature. Dissolved LBG adopts a disordered, fluctuating, random coil conformation.14 Galactomannans exist as poorly soluble “random coils”, which need high temperature and vigorous agitation for complete dissolution in water. Carboxylated, hydroxylated, and phosphate derivatives of galactomannans have been prepared15 to achieve the best water-binding capacity and to increase the water solubility. An interpenetrating polymer network (IPN) is a combination of two polymers exhibiting different characteristics. Whenever an IPN hydrogel is formed from two polymers at a given temperature, physical phase separation between the component polymers would be almost impossible because of the infinite zero viscosity of the gel. IPN is also attractive in producing synergistic properties from the component polymers. For example, when a hydrophilic gelling polymer is interpenetrated with a relatively hydrophobic gelling polymer, the resultant IPN hydrogel is expected to have an improved capability of immobilizing a drug. This would open up new avenues to use IPN in designing novel drug release systems.16 Therefore, an IPN of highly water soluble carboxymethyl locust bean gum (CMLBG) and poly(vinyl alcohol) (PVA) may be beneficial for controlling the release rate of highly water soluble drugs such as buflomedil hydrochloride (BH).

1. INTRODUCTION The field of natural polymer modification is a rapidly developing area because of its wide range of applicability in drug delivery, biomaterials development, food and beverages, water purification, paper, textiles, fuel cells, etc. Modification of natural polymers is sometimes required due to their uncontrolled rates of hydration, thickening, and drops in viscosity on storage, and because of the possibility of microbial contamination.1 Natural polysaccharides are unique raw materials because of their wide availability, renewability, stability, and hydrophilic and modifiable natures. They offer tremendous potential for the development of alternate drug delivery, biomaterial, and water purification systems.2−6 Natural gums are polysaccharides consisting of multiple sugar units linked together to create large molecules. Gums are frequently produced by higher plants as a result of their protection mechanisms following injury.1 Among various polymeric modifications of natural gums, carboxymethylation leads to formation of polyelectrolyte which increases the solubility of the polysaccharide. Other advantages of carboxymethylation are its cost effectiveness and the nontoxicity of the products.7 The reaction is based on the Williamson synthesis, whereby the polysaccharide alkoxide reacts with monochloroacetic acid (MCA) and the primary and secondary alcohol groups are substituted by carboxyl groups.8 Many natural gums such as gellan,9 tamarind kernel powder,10 Cassia tora gum,11 and guar gum12 were investigated using the carboxymethylation process. Locust bean gum (LBG) is composed of galactomannans, which are a series of natural polysaccharides from various sources sharing a similar chemical structure of a β-1−4-Dmannose backbone substituted to varying degrees at the 6 position with single α-linked D-galactose residues. The extent of galactose substitution is a function of the source species. LBG © 2013 American Chemical Society

Received: Revised: Accepted: Published: 10033

February 7, 2013 July 1, 2013 July 3, 2013 July 3, 2013 dx.doi.org/10.1021/ie400445h | Ind. Eng. Chem. Res. 2013, 52, 10033−10045

Industrial & Engineering Chemistry Research

Article

neutralized by glacial acetic acid. The modified polymer was dialyzed against water (48 h) and vacuum-dried at 45 °C. 2.3. Characterization of Carboxymethylated Gum. 2.3.1. Determination of Degree of Carboxymethyl Substitution (DS). DS was determined by potentiometric backtitration. The degree of O-carboxymethyl group substitution was calculated from the equation20

Buflomedil is readily absorbed in the gastrointestinal tract and has a plasma half-life of approximately 2−3 h. Pharmacologically, buflomedil increases perfusion to impaired vascular beds of the microcirculation. A nonspecific α receptor blocking activity appears to be involved, at least in part, in this pharmacologic effect.17 The usual oral dose of BH is 300−600 mg/day. A number of studies have demonstrated that buflomedil increases erythrocyte deformability, associated with increases in erythrocyte adenosine triphosphate (ATP) and cyclic adenosine monophosphate (cAMP) and a decrease in erythrocyte 2,3-diphosphoglycerate.18 Long-term use of BH in high doses can produce drug accumulation and drug related toxicity. Therefore, a controlled release approach such as IPN microspheres of BH can reduce the dose and related side effects. We herein present work on carboxymethylation of locust bean gum (LBG) using various amounts of MCA and sodium hydroxide, detailed characterization of CMLBG, and development of an interpenetrating polymer network with PVA for controlled oral delivery of BH. The carboxymethyl locust bean gum (CMLBG) was characterized in terms of degree of substitution (DS), viscosity, molecular weight determination, elemental analysis, swelling study, contact angle study, scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, 13C nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), X-ray diffraction study (XRD), and biocompatibility study. The IPN particles were characterized by SEM, particle size, FTIR, NMR, and XRD. An in vitro drug release study was performed in phosphate buffer (pH 6.8) and acidic media (pH 1.2). The drug release mechanism was also evaluated using different empirical equations.

DS = 0.162A /(1 − 0.058A)

(1)

where A is the milliequivalents of NaOH required per gram of sample (Supporting Information, section 2.3.1). The method of successive approximation was applied to obtain the final DS.21 2.3.2. Measurement of Viscosity. The viscosity of LBG and various grades of CMLBG (0.1% w/v) were determined by a programmable Brookfield viscometer (Model DV-II+ Pro, Brookfield Engineering Laboratories, Inc. Middleboro, MA) at 32.7 °C. Locust bean gum was dissolved in hot water and conditioned at 32.7 °C, and the various batches of CMLBG were dissolved in normal water and conditioned at the same temperature. The spindle (spindle no. CPE 41) was rotated at 2.0 rpm. 2.3.3. Determination of Weight Average Molecular Weight. The weight average molecular weight was determined by static light scattering (SLS) analysis. The weight average molecular weight (Mw) of LBG and different grades of CMLBG were determined by static light scattering (SLS) analysis using a light scattering spectrophotometer (Model Nano ZS, Malvern, U.K.). 2.3.4. Contact Angle and Surface Energy Measurement. Wettability determinations of native LBG and CMLBG were performed by contact angle measurements of samples using a video-based contact angle meter OCA 20 (DataPhysics, Germany) attached to a camera. Before measurement the samples were prepared by dissolution in water (1% w/v solution), casting on glass slides, and vacuum-drying at 45 °C. The wetting liquid used was Millipore grade distilled water (liquid surface tension (γ1) = 72.8 mJ/m2). The value is the average of five samples for each experiment. Surface energy was calculated using an equation of state, Schultz Method-2, using Data Physics SCA20 software (version 2.01). 2.3.5. Elemental Analysis. The elemental analysis of native gum and different grades of CMLBG was done with an elemental analyzer (Model Vario EL III, Elementer, Hanau, Germany). The estimation of three elements, that is, carbon, hydrogen, and oxygen, was undertaken. 2.3.6. Differential Scanning Calorimetry. DSC thermograms of pristine gum and modified gums were obtained by using a DSC-60 (Shimadzu, Japan). Each sample (3−7 mg) was accurately weighed into a 40 μL aluminum pan in a hermetically sealed condition. The measurements were performed in an atmosphere of nitrogen between 20 and 250 °C at a heating rate of 10 °C/min. 2.3.7. Acute Oral Toxicity Study. An acute oral toxicity study of CMLBG was performed as per the Organization of Economic Co-operation and Development (OECD) guideline for the test of chemicals 425, adopted Dec 17, 2001. Five nulliparous and nonpregnant 5 week old female mice (Swiss albino strain) were taken for this study. The study protocol was prior approved by the animal ethics committee (CPCSEA Approval No. 621/02/ac/CPCSEA) of Birla Institute of Technology, Ranchi, India. Mice were housed in a polycarbonate cage with sufficient food, and deionized reverse osmosis water was available to them ad libitum at 20−25 °C and 40−

2. MATERIALS AND METHODS 2.1. Materials. Locust bean gum was purchased from HiMedia Laboratories Private Ltd. (Mumbai, India; MW 3.1 × 106). Monochloroacetic acid (MCA), sodium hydroxide, methanol, and hydrochloric acid were of laboratory reagent grade (SD Fine-Chem. Ltd., Mumbai, India). Poly(vinyl alcohol) (PVA; 98% hydrolyzed, average molecular weight 125 000) and light liquid paraffin (LLP; viscosity 25−80 mPa at 20 °C) were procured from HiMedia Laboratories Private Ltd. (Mumbai, India). Span 80 was procured from Pioneer InOrganics (Delhi, India). Hydrochloric acid (HCl; 30%, ultrapure) was obtained from HiMedia Laboratories Private Ltd. (Mumbai, India). Glycine and glutaraldehyde (GA; 25%, v/v) were supplied by Merck Ltd. (Mumbai, India). Acetone was procured from Qualigens fine chemicals (Mumbai, India). All the reagents were used without further purification. Water used was of Milli-Q grade. 2.2. Synthesis of Sodium Carboxymethyl Locust Bean Gum (CMLBG). Locust bean gum was tailored to sodium carboxymethyl locust bean gum with slight modification of the method reported earlier.19 Briefly, 10 g of locust bean gum was kneaded with different amounts of ice-cold NaOH solution (10 M) over a period of 45 min. Different amounts of monochloroacetic acid were dissolved in 10 mL of water and added slowly to the dough for a period of 1 h at 15 °C. The mixture was heated to 65 °C and stirred for at least another 1 h. To study the effect of temperature, some batches were prepared at 35 and 95 °C. The wet semisolid mass was washed with 80% (v/v) methanol/water mixture (50 mL × 3) for 15 min and 10034

dx.doi.org/10.1021/ie400445h | Ind. Eng. Chem. Res. 2013, 52, 10033−10045


Article

Table 1. Different Batches of CMLBG with Their Codes, Conditions of Preparation, DS Values, Viscosities, and Weight Average Molecular Weights batch

vol of 10 M NaOH (mL)

MCA (g)

temp (°C)

molar ratio NaOH/MCA/LBG

DS

viscosity (cP)

F1 F2 F3 F4 F5 F6 F7 F8 F9 LBG

2.8 5.6 8.4 11.2 14.0 11.2 11.2 11.2 11.2 0.0

2.64 2.64 2.64 2.64 2.64 5.28 7.92 5.28 5.28 0.00

65 65 65 65 65 65 65 95 35 0

1:1:1 2:1:1 3:1:1 4:1:1 5:1:1 4:2:1 4:3:1 4:2:1 4:2:1 0:0:0

0.244 0.317 0.332 0.582 0.485 0.653 0.646 0.618 0.427 0.000

73.7 75.2 75.8 78.1 77.6 71.9 71.8 71.3 77.2 122.0

Mw (g/mol) 2.07 2.33 2.41 2.72 2.55 1.83 1.72 1.68 2.49 3.13

× × × × × × × × × ×

106 106 106 106 106 106 106 106 106 106

For the preparation of blank microspheres, a similar method was adopted except for the addition of drug into the system. 2.4.1. Drug Entrapment Efficiency (DEE) Study. Accurately weighed, 10 mg of dried microspheres was crushed with the help of a mortar and pestle, transferred into 50 mL of pH 6.8 phosphate buffer solution, and heated at 50 °C to extract the drug. After 24 h, the suspension was filtered and centrifuged to remove the polymeric debris. Then, the supernatant was taken and analyzed with a spectrophotometer (UV-1800, Shimadzu, Japan) at λmax = 282 nm. All samples were analyzed in triplicate. The drug entrapment efficiency (%) was calculated by using the following equation:26

70% relative humidity in a 12 h light on/light off cycle. A single dose of 2000 mg/kg body weight of CMLBG was administered by gavage using a stomach tube to the first animal. The same dose was administered to the remaining four animals after survival of the first animal. The animals were kept under continuous observation up to 4 h after dosing. The observation was continued up to 14 days. The mortality rate was evaluated by visible observation and reported accordingly.22 Serum biochemical studies were performed in different time intervals. The animals were sacrificed on the 15th day, and histological (liver, kidney, lung, and stomach) studies were performed.23,24 2.3.8. Biodegradability Study. Sample films of CMLBG (5% gum solution was cast on a Petri dish and dried) was inoculated with Aspergillus niger on a medium and incubated at the surrounding temperature (25−37 °C) for 21 days. Samples were cut (2.5 cm × 2.5 cm) and faced on the surface of mineral salt agar in a Petri dish containing no additional carbon source. Before placing the samples, agar surfaces were cultivated with A. niger from tapioca slices. Thereafter, the films were examined for evidence of colony growth.22 2.4. Preparation of IPN Hydrogel Microspheres. Microspheres composed of optimized CMLBG (F6, Table 1) and PVA containing buflomedil hydrochloride (BH) were prepared by a water-in-oil (w/o) emulsion-cross-linking method.25 Briefly, 20 mL of 2% (w/v) aqueous polymeric solution (total polymer amount was kept constant) was prepared by dissolving varying amounts of CMLBG and PVA. First, PVA was dissolved in water (80 °C) by continuous stirring until a transparent solution was obtained. It was then kept for cooling at room temperature. CMLBG was then dispersed in PVA solution with continuous stirring. The required amount of drug was added in the polymeric dispersion and stirred (3 h) with the help of a magnetic stirrer to obtain a homogeneous, bubble-free drug−polymer mixture. The drug− polymer mixture was slowly added to light liquid paraffin (100 g, w/w) containing 1% (w/w) Tween-80 under constant mechanical stirring at 500 rpm for 10 min. A milky white emulsion (w/o) was formed. To this emulsion, GA containing 0.5 mL of 1 N HCl was added slowly and stirring was continued for 3 h in order to produce hardened microspheres. The microspheres were then filtered and washed with acetone, 0.1 M glycine solution, and water to remove excess amount of liquid paraffin, unreacted GA, and surfactant, respectively. Complete removal of unreacted GA was tested by treating the filtrate with Fehling’s reagent. A negative result was obtained, which confirmed the absence of unreacted GA. Hardened microspheres were vacuum-dried at 40 °C for 24 h and stored in a desiccator until further use.

entrapment efficiency (%) = (actual drug content/theoretical drug content)· 100 (2)

2.4.2. Swelling Study. Equilibrium swelling measurements of both LBG and CMLBG were done in various buffers. A small, previously weighed piece of the material (W1) was immersed in 50 mL of buffer (pH 1.2 and 6.8) and left to swell for 2 h. Then, the swollen piece was recovered and the excess water was removed carefully with tissue paper and reweighed (W2) to an accuracy of ±0.01 mg on an electronic microbalance (Mettler, Model AT120, Greifensee, Switzerland). The pH-dependent equilibrium water uptake of the blank microspheres was measured by a similar method. A 10 mg sample of IPN particles was immersed in 50 mL of buffer (pH 1.2 and 6.8) for 24 h at 37 °C. The swelling index was calculated by the following formula:27 swelling index = [(W2 − W1)/ W1]·100

(3)

where W2 and W1 are the swollen and dry weights of the gum/ microspheres, respectively. 2.4.3. Particle Size Analysis. Particle size analysis was done with a Mastersizer 2000, version 5.40 (Malvern Instruments Ltd., U.K.) which allows sample measurement in the range 0.020−2000 mm. The particle size was measured by the laser light scattering technique. The polydispersity index was determined according to the following equation: polydispersity index = [d(0.9) − d(0.1)]/d(0.5)

(4)

where d(0.9) corresponds to the particle size by volume immediately above 90% of the sample, d(0.5) corresponds to the particle size immediately above 50% of the sample, and d(0.1) corresponds to the particle size immediately above 10% of the sample.26 10035



Article

gum was prepared by the Williamson synthesis.29 The schematic representation of the reaction process and the physical appearance of the native and modified LBG aqueous solutions are given in Scheme S1 and Figure S1 (Supporting Information). The carboxymethyl groups are formed by an SN2 reaction between the gum alkoxide and MCA. The main reaction is given by

2.4.4. Fourier Transform Infrared (FTIR) Spectroscopy. The FTIR spectra of LBG, various grades of CMLBGs, buflomedil hydrochloride, drug−polymer physical mixture, blank microspheres, and drug loaded microspheres were carried out by an FTIR-8400S (Shimadzu, Japan) to confirm the formation of CMLBG and the compatibility of different ingredients of the IPN formulation. A small amount of each material was mixed with KBr (1 wt % sample content), taken into a sample holder, and scanned in the range 600−4000 cm−1. 2.4.5. Solid State 13C NMR Spectroscopy. Solid state 13C NMR of native gum, CMLBG, and glutaraldehyde cross-linked blank formulation was studied to confirm the formation of the CMLBG and glutaraldehyde cross-linked IPN network. For solid state 13C NMR, approximately 300 mg of sample was inserted into the ceramic rotor on a Bruker AMX 400 spectrometer. The spectrum was recorded at 75.5 MHz. 2.4.6. Scanning Electron Microscopy. The surface morphology and topography of LBG, CMLBG, and IPN microspheres were investigated by a scanning electron microscope (JSM6390LV, Jeol, Japan). Prior to examination, the samples were mounted onto stubs using double-sided dried carbon tape and vacuum coated with gold−palladium film (thickness 2 nm) by a sputter coater (Edward S-150, U.K.) to render them electrically conductive. 2.4.7. Qualitative X-ray Diffractometry. Ground samples of pristine gum, carboxymethylated gum, BH, and blank and drug loaded IPN microspheres were scanned from 10 to 60° 2θ, using an X-ray diffractometer (Bruker AXS D8 Advance, Germany; configuration vertical, θ/2θ geometry; X-ray Cu, wavelength 1.5406 Å, detector Si (Li) PSD). The diffractometer was run at a scanning speed of 2 deg/min and a chart speed of 2 deg/2 cm per 2θ, and the angular range fixed was from 10 to 60°. 2.4.8. In Vitro Release Study. Drug release from the IPN microspheres containing different drug loadings, IPN compositions, and extents of cross-linking were investigated at pH 1.2 for the initial 2 h, followed by the release in phosphate buffer of pH 6.8. This experiment was performed in triplicate in a tablet dissolution tester (Electro Lab, TDT-08L, India) equipped with eight baskets (glass jars) at a stirring speed of 50 rpm. A weighed quantity of each sample (equivalent to 100 mg of drug) was placed in 900 mL of dissolution medium maintained at 37.5 °C. At regular intervals of time, sample aliquots were withdrawn and analyzed using a UV spectrophotometer (UV1800, Shimadzu, Japan) at fixed λmax = 282 nm. 2.4.9. Drug Release Kinetics. The drug release data were fitted to various empirical equations such as zero order (Qt = Kt), first order (ln Qt = ln Q0 − Kt), Higuchi kinetics (Qt = Ktn), Hixson−Crowell (Q01/3 − Qt1/3 = Kt), and the power-law equation Mt/M∞ = ktn, where Mt/M∞ is the fraction of drug release at time t, k is the release rate constant, and n is the diffusion exponent that denotes the drug release mechanism.28 A least squares regression method was used to determine the values of n and k. Values of n = 0.43 or less indicate Fickian transport, whereas values of n = 0.43−0.85 indicate anomalous or non-Fickian drug transport from spherical matrixes. Exponent values of n greater than 0.85 signify the super case II transport mechanism. Q0 and Qt are amounts of drug released at times 0 and t. K is the release rate constant.

LBG − OH + NaOH → LBG − ONa + H 2O

(5)

LBG − ONa + ClCH 2COONa → LBG − OCH 2COONa + NaCl

(6)

The side reaction takes place in both the liquid bulk and the gum phase,30 and results in the formation of sodium glycolate from MCA and sodium hydroxide. NaOH + ClCH 2COONa → HOCH 2COONa + NaCl (7)

Processing variables optimization is highly required to get the highest possible carboxymethylated locust bean gum. The levels of formulation variables used for the optimization are shown in Table 1. 3.2. Effects of Reaction Parameters on Degree of Substitution (DS). The extent of carboxymethylation of carbohydrates is expressed in terms of degree of substitution, which is the average number of substituted carboxymethyl groups per anhydro sugar unit. DS values of different batches of CMLBG are given in Table 1. The average degree of carboxymethyl substitution was found to be 0.244−0.653 (Table 1). 3.2.1. Effect of Sodium Hydroxide. The DS of LBG is highly dependent on sodium hydroxide as seen from Table 1 and Figure S2a (Supporting Information). As the amount of sodium hydroxide was increased from 2.8 to 11.2 mL, the DS value was also increased; the DS value decreased when the volume of NaOH increased to 14 mL. This observation can be explained by considering the carboxymethylation process, where two competitive reactions take place simultaneously. The first involves reaction of the LBG hydroxyl with sodium monochloroacetate in the presence of sodium hydroxide to give CMLBG. When 14 mL of NaOH was used, the glycolate formation predominated over the first reaction and hence the DS value was decreased (0.485). Similar observations have been reported for sago waste.31 3.2.2. Effect of Monochloroactic Acid (MCA). The effect of MCA is shown in Figure S2b (Supporting Information) and Table 1. At an optimized concentration of NaOH, when the amount of MCA was increased from 2.64 to 5.28 g, the DS value was increased, but above that, the DS value decreased. Since the amount of LBG and the temperature were fixed, the value of the DS might have decreased due to the formation of alkali glycolate. Excess amount of MCA may accelerate the process of glycolate formation. A similar phenomenon was reported for Cassia tora gum.11 3.2.3. Effect of Temperature. As shown in Figure S2c (Supporting Information) and Table 1, the DS value of CMLBG was dependent on the reaction temperature. A maximum DS of 0.653 was obtained at 65 °C. When the reaction temperature was increased to 95 °C, the DS value decreased to 0.618. This may be due to chain degradation of LBG, where chemical elimination of water from LBG originates primarily from an intramolecular elimination leading to C2, C3

3. RESULTS AND DISCUSSION 3.1. Synthesis of Carboxymethyl Locust Bean Gum (CMLBG). In the present study carboxymethylated locust bean 10036



Article

Table 2. Various Batches of CMLBG, Elemental Analysis, Equilibrium Swelling, Contact Angle, and Surface Energy elem analysis (%)

equilib swelling (%)

batch

C

H

O

pH 1.2

pH 6.8

contact angle (deg)

surf. energy (mN/m)

F1 F2 F3 F4 F5 F6 F7 F8 F9 LBG

35.40 34.47 34.26 30.61 32.45 28.05 28.60 30.03 32.74 38.91

6.211 5.979 6.187 6.447 5.961 6.299 5.791 5.458 6.242 7.216

58.04 58.62 59.21 62.70 61.32 65.40 65.23 64.05 60.79 52.76

144.26 147.98 153.12 173.19 165.72 211.53 198.18 187.42 158.13 139.33

251.23 253.36 261.51 281.55 272.58 325.76 310.51 293.84 266.49 141.21

61.78 58.47 47.66 29.43 30.45 21.66 24.23 25.01 46.96 65.28

46.66 48.76 55.10 64.65 64.23 67.96 66.09 65.70 55.43 44.62

study is shown in Table 2 and Figure 1. The carboxymethylated form showed a sharp increase (for the best optimized CMLBG,

unsaturation or a ketone group on C2. Concurrently, there may be intermolecular elimination between hydroxyl groups of neighboring chains giving rise to cross-linking by ether linkages, thus decreasing the sites of −OH groups for carboxymethylation.31 When the temperature was reduced to 35 °C, the DS value was markedly reduced (0.427). This may be due to the lack of swellability of LBG and less intimacy of reactants with the polymer chains. Therefore, 65 °C was found to be the optimum temperature for the carboxymethylation of LBG. 3.3. Viscosity Study. It can be said from Table 1 that the viscosity of the native LBG (122 cP) is much higher than that of CMLBG. Since Williamson’s etherification reaction was carried out invariably in aqueous medium using concentrated NaOH and MCA at elevated temperature, a nonspecific degradation by β-elimination and/or peeling reaction may initiate at the reducing sugar unit, which may reduce the viscosity of the derivatized material. It is also reported that polysaccharides such as guar gum, even in oxygen-free alkaline solution, generate saccharinic acids32 which can also reduce the viscosity of the system since it is has higher water solubility. In the present study, when the DS of CMLBG was the lowest (0.244), the viscosity was 73.7 cP, and when the DS increased to a value of 0.582, then the viscosity was observed to be highest (78.1 cP). This increase in viscosity may be due to the introduction of carboxymethyl groups. However, the derivatives having higher DS values (0.618−0.653) showed a marked decrease in viscosity. This may be due to steric hindrance of carboxymethyl groups and decreased intermolecular forces,10 or there may be reduction of the molecular weight due to polymer chain degradation during the alkali kneading of the carboxymethylation process. 3.4. Weight Average Molecular Weight. The result of the weight average molecular weight study is shown in Table 1. In the case of pristine gum the molecular weight was found to be 3.13 × 106 g/mol, whereas in the case of optimized CMLBG it was 2.29 × 106 g/mol. This decrease in molecular weight may be due to the degradation of polymer chains in the high alkali environment of the carboxymethylation process. Among the carboxymethylated batches, the molecular weight initially increased with the increment of the DS value. The increase in molecular weight is due to the increase in hydrodynamic volume by the carboxymethyl group. After a certain DS value (0.582), the molecular weight started to decrease. This was due to polymer chain disruption in highly alkaline medium during the carboxymethylation process. 3.5. Contact Angle and Surface Energy Measurement. Here we performed this study to identify the change in the wetting ability of the etherified form of LBG. The result of this

Figure 1. Water contact angle of native LBG and different batches of CMLBG.

i.e., F6, the contact angle is 21.66° and the surface energy is 67.96 mN/m) in the wetting ability in water compared with that of native LBG (contact angle 65.28° and surface energy 44.62 mN/m). The water contact angle was decreased and the surface energy was increased gradually with the DS (Table 1 and 2). When CMLBG comes into contact with water molecules, it easily breaks the cohesive force of water molecules due to the presence of highly polar groups on the surface. The higher substituted batches had smaller contact angles because of their high affinity toward water molecules. The high attraction of CMLBG molecules toward water can also be 10037



Article

due to the presence of a high amount of surface −COO− groups. 3.6. Elemental Analysis. The result of the elemental analysis is given in Table 2. It can be said from the observed data that there was a significant increase in oxygen content in all the modified batches compared with the native gum (52.76%). This may be due to attachment of the −CH2COONa group in all three replaceable −OH groups. However, the carbon content was reduced in all the modified samples and this may be due to polymer chain distortion during the vigorous stirring of the carboxymethylation process. The hydrogen content of the modified gum (6.299% for F6) was less than that of native gum (7.216%); this may be due to formation of water molecules (eq 5) during the carboxymethylation process. Thus, from the elemental analysis, it can be said that LBG was successfully modified to CMLBG. 3.7. Differential Scanning Calorimetry. Differential scanning calorimetry analysis gives an account of thermal transitions during heating under an inert atmosphere. DSC thermograms of native LBG and different batches of CMLBG are presented in Figure 2. The thermogram of native LBG

peak temperature (for the optimized batch it is 93.29 °C) of the native gum. The endothermic transitions of polysaccharides depend upon the thermal history of the material and also on their structural features. The decomposition temperature of CMLBG batches is lower than that of native gum, due to the carboxymethyl etherification. Moreover, due to the breaking of intermolecular and intramolecular hydrogen bonds during carboxymethylation, movement of chain segments may occur, which accelerates the degradation of modified polymers in lower temperature.33 This result supports the result obtained from XRD analysis (data not shown). 3.8. Acute Oral Toxicity Study. From the oral toxicity study it was found that there was no mortality within the observation period of 14 days after dosing. The different hematological and blood chemistry data were collected at different time points of the study period and are presented in Table S1 (Supporting Information). No abnormalities were found in all hematological and blood chemistry parameters. Moreover, the histological sections of lung, kidney, liver, and stomach (Figure S3, Supporting Information) did not show any abnormality on 15th day of toxicity study. As per the Organization of Economic Co-operation and Development (OECD) guideline for the test of chemicals 425, adopted Dec 17, 2001, Annexure-4, the LD50 value is greater than the 2000 mg/kg dose of CMLBG, so recognizing the need to protect animal welfare, testing in animals is discouraged. As per the globally harmonized system (GHS), if the LD50 value is greater than the 2000 mg/kg dose, then the test product will fall under “category 5” and the toxicity rating will be “zero”. Therefore, CMLBG is under “category 5” and its toxicity rating is “zero”. 3.9. Biodegradability Study. The biodegradability study shows the fungal growth (Figure S4, Supporting Information) for various batches of CMLBG films after 21 days. The microscopic study revealed apparent fungi growth on 0, 3, 7, 14, and 21 days in all the Petri dishes. The apparent growth of fungi in mineral salt agar medium (no carbon source) proves that the carbon present in the CMLBG had been utilized by the fungi for its growth. Thus it can be easily concluded that CMLBG is biodegradable in nature. 3.10. Formation of Microspheres. In the present study, we have prepared the interpenetrating polymer network hydrogel microspheres of CMLBG and PVA by an emulsioncross-linking method. The carboxymethylated form of LBG is highly swellable and pH sensitive. However, this swelling behavior is abrupt. To control this abrupt swelling property, we have blended PVA with CMLBG and cross-linked them by GA.

Figure 2. DSC thermograms of pristine LBG and different batches of CMLBG.

showed a broad endothermic peak at 104.40 °C. Most of the CMLBG samples showed their endothermic peaks below the

Table 3. Formulation Variables, Particle Size, Polydispersity Index, % Drug Entrapment Efficiency (DEE), and Equilibrium Water Uptake at pH 1.2 and 6.8 equilib water uptake (%) formuln

CMLBG:PVA

BH loading (%)

GA (mL)

part. size [d(0.5)] (μm)

PDI

B1 B2 B3 B4 B5 B6 B7 B8 B9 B10

1:1 1:1.5 1:2 1:2 1:2 1:2 1:2 0:1 1.5:1 2:1

50 50 50 25 75 50 50 50 50 50

3 3 3 3 3 1 5 3 3 3

834.11 781.73 732.34 719.52 755.42 949.25 496.17 378.61 868.29 961.64

1.41 1.34 1.17 1.57 1.19 1.22 1.70 1.94 1.18 1.32

10038

DEE% (±SD, n = 3)

pH 1.2

pH 6.8

± ± ± ± ± ± ± ± ± ±

172.33 166.42 121.65 119.26 159.14 128.79 113.51 106.27 179.12 186.57

244.91 236.38 224.7 212.81 229.17 232.53 162.15 144.65 267.36 278.44

37.33 48.52 62.61 59.73 65.11 41.83 63.26 45.31 51.72 55.42

1.18 2.44 1.27 1.62 1.85 2.51 2.41 2.73 1.39 1.71



Article

effect on the swelling behavior. Swelling was slightly increased with increased drug loading (B4 and B5, from 212.81 to 229.17%). This was due to the hydrophilic nature of BH. 3.13. Particle Size Analysis. Results of the particle size analysis of IPN microspheres are presented in Table 3. Results show that the arithmetic mean diameter varied from 378.61 to 961.64 μm. Particle size varied depending on the amount crosslinker, IPN blend ratio, and amount of drug loading. The results showed that, in a fixed IPN blend composition, when the amount of cross-linker was increased from 2.5 (B6) to 7.5 mL (B7), the diameter was reduced from 949.25 to 496.17 μm (Figure S7, Supporting Information). This can be explained by the fact that, as the amount of cross-linker was increased, it squeezed the polymer matrix to a greater extent and reduced the internal void space. As the amount of CMLBG was increased in the formulation (from B3 to B9 and B10), the diameter of the particle also increased from 732.34 μm to 868.29 μm and 961.64 μm. This can be explained on the basis of the hydrodynamic viscosity concept; i.e., as the amount of CMLBG increased, the interfacial viscosity of the polymer droplets in the emulsion also increased, leading to formation of larger particles. An increase in drug loading (B4, B3, and B5) also resulted in the formation of larger particles. This was due to the fact that drug molecules might have occupied the free volume spaces within the matrix, thereby hindering the inward shrinkage of the polymer matrix. The polydispersity index of the particles was calculated, and it showed a moderately narrow distribution range of IPN particles. 3.14. Fourier Transform Infrared (FTIR) Spectroscopy. The FTIR spectra of pristine gum and the CMLBGs are shown in Figure S6 (Supporting Information). A strong O−H stretching peak of the hydroxyl group was observed in LBG near 3300 cm−1,35 and it was due to hydrogen bonding involving the hydroxyl groups on the gum molecules. In the case of CMLBGs this −OH stretching peak was broadened. This might be an indication of the utilization of hydroxyl groups in the carboxymethylation reaction. The C−H stretching vibrations were observed at 2950 cm−1, and additional characteristic bands of LBG appeared at 1400 and 1050 cm−1, attributed to C−H and O−H bending vibrations, respectively. The broad peak at about 1000−1100 cm−1 was mainly attributed to C−O−H stretching/bending, and was also identified in all modified batches without marked changes. The presence of carboxymethylated groups in CMLBG was confirmed by the peaks at about 900 cm−1, representing symmetric C−O−C stretching. In addition, the C−O stretching of ether and the CO stretching of acid were observed in derivatized gum, respectively, at 1450 and 1600 cm−1; the same were absent in LBG. A substantial increase in the intensity of this band was observed after carboxymethylation (spectrum F6 of Figure S6, Supporting Information), caused by the introduction of new carboxylic groups per macromolecule. As more groups were introduced (higher DS), the peak at 1600 cm−1 became more intense. The peak intensity at 1450 cm−1 did not increase markedly, indicating that CMLBG samples were predominantly in acidic form.21 Hence, it can be said that LBG was successfully modified to CMLBG by this process and all the processing variables had significant effects on the DS of CMLBG. The FTIR spectral analysis of buflomedil hydrochloride, PVA, physical mixture, placebo microspheres, and drug loaded formulation (Figure 3) was done to examine the compatibility

The cross-linker, GA, is a bifunctional cross-linker and helps to form the network structure. During the formation of microspheres an acetal ring was formed between the hydroxyl groups of CMLBG−PVA polymer blend and aldehyde groups of GA to produce the network structure (Scheme S2, Supporting Information). It made the matrix network rigid and insoluble. Conditions for the preparation of different batches of microspheres are shown in Table 3. 3.11. Drug Entrapment Efficiency. The result of the DEE study (Table 3) shows that the values depend on formulation variables, i.e., drug/polymer ratio, polymer composition, and extent of cross-linking. An increase in the amount of PVA in the blend results in higher DEE (for B1, B2, and B3, DEE = 37.33, 48.52, and 62.61%, respectively) and the opposite phenomenon was observed in higher CMLBG containing formulations (for B9 and B10, DEE = 51.72 and 55.42%, respectively). This might be due to the fact that, with increasing amount of CMLBG, the number of −COOH groups increased, which would be responsible for increased hydrophilicity, resulting in low DEE values. However, with increasing concentration of cross-linking agent (for B6, B3, and B7, DEE = 41.83, 62.61, and 63.26%, respectively), DEE also increased due to the rigid network which retained more drug particles and prevented drug leaching. The percent DEE results were also dependent on the percent drug loading. Formulations containing high drug loading exhibit slightly higher DEE values (B4, B3, and B5) due to the accumulation of more drug particles inside the free void spaces of the matrixes. 3.12. Swelling Study. As shown in Table 2, the modified form of LBG showed a pH dependent swelling property. Native LBG showed less swelling in both acidic and basic media (139.33 and 141.21%) than modified forms did. CMLBG showed higher swelling in pH 6.8 than in pH 1.2. The swelling of CMLBG batches was increased with the DS (Tables 1 and 2). The optimized batch (F6) showed the highest swelling in both acidic and basic media (211.53 and 325.76%). Being nonionic, LBG is not affected by pH or ionic strength. The pH dependent swelling of CMLBG was due to the presence of carboxymethyl groups. The pendant carboxymethyl groups ionize if the pH of the environment is above the pKa of the carboxylic group (3.4−3.7). Thus, at low pH (1.2), the carboxyl groups were protonated to promote the formation of intramolecular hydrogen bonds, thereby reducing the overall swelling ratio. At higher pH (pH 6.8), the carboxyl groups were deprotonated and a higher swelling ratio was observed due to the collective electrostatic repulsion forces between the ionized acid groups. A similar pH dependent swelling behavior has been observed in polymers containing pendant carboxylic acid groups such as carboxymethyl κ-carrageenan.34 For similar reasons the IPN particles showed pH dependent swelling behavior. Results are shown in Table 3. The swelling of particles was very much dependent upon the cross-linking density. At lower cross-link density (B6) swelling increased (232.53%) because of the loose network structure. This resulted in the creation of a large hydrodynamic free volume, which accommodated more solvent molecules, resulting in higher swelling. The swelling of particles was also dependent upon the blend composition. For instance, the 100% PVA containing formulation (B8) showed the least swelling (144.65%) compared to other formulations containing CMLBG. This was because of the increased hydrophilic nature of the matrix due to the presence of −COOH groups of CMLBG. However, the amount of drug loading showed less 10039



Article

Figure 3. FTIR spectra of (a) buflomedil hydrochloride, (b) PVA, (c) drug−polymer physical mixture, (d) placebo microspheres, and (e) BH loaded optimized formulation.

spectra without significant changes, indicating the compatibility of the ingredients. However, peak intensities were reduced, which might be due to the dilution effect by other components of the system. In the case of the placebo formulation (Figure 3d), a considerable reduction of the PVA hydroxyl peak intensity was observed, indicating a possible formation of acetal bridges.37 Moreover, a small absorption peak at about 1246 cm−1 was observed in the spectrum. This might be due to ether linkage (C−O−C stretching) formed between the −OH group of PVA and CMLBG by the help of glutaraldehyde.38 Peaks near 1724 and 1649 cm−1 may be due to the CO groups of PVA acetate and CMLBG, respectively. This significant peak shift of the CO group (in pristine PVA it was at 1741 cm−1 and in pristine CMLBG it was at 1600 cm−1) indicated the formation of the IPN network structure. In the drug loaded microsphere spectrum (Figure 3e), peaks were observed that were additional to those of the placebo microspheres. This was due to the incorporation of BH in the

of drug in the formulation and to confirm the formation of the IPN matrix structure. Buflomedil hydrochloride (Figure 3a) showed its characteristic peaks near 2968 cm−1 for methoxy CH stretching (CH3−O−). A small peak at about 1340 cm−1 was observed for aromatic C−N stretching. The absorption band at 1696 cm−1 was of the ketonic carbonyl group. In the case of PVA (Figure 3b), all major peaks related to hydroxyl and acetate groups were observed. The large bands observed between 3600 and 3250 cm−1 were linked to the stretching of O−H from the intermolecular and intramolecular hydrogen bonds. The vibrational band observed between 2854 and 2924 cm−1 referred to the stretching C−H from alkyl groups, and the peaks between 1741 and 1735 cm−1 were due to the stretching of CO and C−O from the acetate group of PVA. The sharp peak obtained near 1462 cm−1 indicated the bending vibration of CH2 groups. The stretching intensity of 1741−1735 cm−1 indicated that PVA was less hydrolyzed.36 In the physical mixture (Figure 3c), peaks of all the components (BH, PVA, and CMLBG) were present in the 10040



Article

absorption peak at δ = 62 ppm is for the C-6 carbon of the mannan unit. The absorption peak at δ = 70 ppm broadened at 80 ppm because the signals of C-2, C-3, and C-5, had merged into it. Some notches on that peak give strong evidence of this fact. The small hump at δ = 81 ppm is for the C-4 mannan carbon atom. The sharp absorption peak at δ = 102.7 ppm is due to the C-1 mannan carbon.39 In the case of CMLBG, it can be seen in Figure 4b that new signals at δ = 170.0, 178.5, and 179.7 ppm emerged in the spectrum, which represent the carbonyl atom of the carboxymethyl groups at the 2-O-, 3-O-, and 6-O-positions.40,41 The CO resonance signal was observed at 167.6 ppm. Moreover, a new peak in the spectrum of CMLBG at δ = 71.7 was detected, which corresponded to methylene groups at the 2-O-, 3-O-, and 6-O- positions. On the basis of these observations, it can be concluded that LBG was carboxymethylated at all three positions. Yang et al.42 reported the NMR spectra of PVA, where a sharp peak at δ = 43 was for the CH2 carbon of PVA and three split peaks at about δ = 60−80 ppm were for the resonance of CH carbons of PVA. The 13C NMR spectrum of placebo IPN microspheres is shown in Figure 4c. The absorption peak at δ = 21.60 ppm was due to the aliphatic carbon of −CH2 present in PVA. Absorption peaks in the range 37−42 ppm corresponded to aliphatic carbons of glutaraldehyde. The absorption peak at δ = 63.33 ppm is for the C-6 carbon atom of the mannan residue of CMLBG. The broad absorption peak at δ = 72.09 is for the C2, C-3, and C-5 carbons of the LBG mannan unit. The CH resonance peaks of PVA might be merged into this broad peak and hence were not visible distinctly. The peak at δ = 100.99 ppm was due to the acetal carbon atom43 which formed between the CMLBG and PVA hydroxyl group due to crosslinking by glutaraldehyde, proving the formation of IPN. The C-1 mannan carbon of CMLBG might be overlapped with the peak of acetal carbon atom. The peak at δ = 170.45 ppm was for the carbonyl carbon atom of CMLBG. Hence, the solid state NMR spectra of the IPN particles showed strong evidence of formation of the glutaraldehyde cross-linked IPN matrix. 3.16. Scanning Electron Microscopy. The scanning electron microscopy of LBG and CMLBG was done to investigate the surface morphologies of the native gum and the modified gum (Figure S5, Supporting Information). Studies revealed that the native gum was of oval, globular, and ellipsoid shapes (Figure S5a,b, Supporting Information). The modified gum showed some sharp edge breaking points (Figure S5c,d, Supporting Information). This can be due to an increase in the amorphus nature of gum after modification. The alkaline environment during the carboxymethylation process accounted for the structural changes. SEM images of the IPN particles are shown in Figure 5. Figure 5a represents a group of microspheres, while Figure 5b represents an individual microsphere. Figure 5c,d shows the surface of the microsphere, Figure 5e shows the surface before dissolution, and Figure 5f shows the surface after dissolution; the images were taken at magnifications of 50×, 150×, 300×, and 1000×. The microspheres were spherical in nature with rough surfaces, exhibiting pores on the surface, which might be created during the particle hardening stage. Figure 5d shows evidence of the network structure. From Figure 5f it can be said that the drug release from the microspheres followed an erosion controlled release mechanism.

IPN matrix. No extra peak due to drug−polymer interaction was identified, indicating that the materials and the process of preparation were compatible to produce this type of delivery system. 3.15. Solid State 13C NMR Spectroscopy. The solid state 13 C NMR spectra of native LBG and optimized CMLBG are shown in Figure 4. From the NMR spectrum of LBG (Figure 4a), it can be said that it has three distinct peaks. The

Figure 4. Solid state 13C NMR spectra of (a) LBG, (b) CMLBG, and (c) CMLBG−PVA IPN. 10041



Article

Figure 5. SEM images of (a) group of microspheres, (b) individual microsphere, (c) surface of microsphere, (d) network structure on surface, (e) surface of particle before dissolution, and (f) surface of particle after dissolution.

3.18.1. Effect of Cross-Linker. The case of the formulation having a higher cross-linker in a fixed blend ratio and drug loading (B7, B3, and B6) showed less drug release (75, 82, and 86%, respectively) (Figure 7a). This was due to fact that higher cross-linker concentration increased the rigidity of the network, which in turn prevented the imbibition of buffer into the polymer matrix. Therefore, there was a reduction in network erosion and loosening leading to reduction in drug release. 3.18.2. Effect of Blend Ratio. In a fixed cross-linker concentration and drug loading when the IPN blend ratio was changed from 1:1 to 1:2 (CMLBG:PVA), the particles showed less drug release (82% for B3 and 86% for B1) (Figure 7b). However, when the amount of CMLBG was increased in the IPN (B9 and B10), the CPR was greater (91 and 95%, respectively). Particles composed of only PVA (B8) showed the

3.17. Qualitative X-ray Diffractometry. From the diffractogram of BH (Figure 6) it can be said that BH is highly crystalline in nature. BH shows its characteristic peaks at 2θ of 13−50°. Moreover, a sharp, intense peak at about 2θ of 25° proves its crystalline nature. However, these peaks disappeared in BH loaded microspheres (Figure 6), confirming that the drug was molecularly dispersed in the polymer matrix. The peak intensity at 2θ of 25° was higher in drug loaded formulation than in the placebo, which confirmed the presence of BH in the IPN matrix. 3.18. In Vitro Drug Release and Release Kinetics. The cumulative percentage release (CPR) vs time plot of the release data shows that the drug release was dependent on the crosslinker concentration, blend ratio, and drug loading. 10042



Article

least drug release (72%). This might be due to the hydrophilic nature of gum. Since CMLBG has a high number of −COOH groups, due to its hydrophilic nature, higher gum containing formulations showed higher interactions with media and accelerated the drug release process. 3.18.3. Effect of Drug Loading. Formulations having a fixed blend ratio and cross-linker concentration but higher drug loading (B5) showed higher drug release (88%) (Figure 7c). This can be explained by the fact that the concentration gradient of drug in the case of high drug containing formulations was higher than that in less drug containing formulations. This accelerated the dissolution of the drug particle and leaching out from the matrix more easily. Moreover, as less amount of drug was dispersed in almost the same volume of polymer matrix, drug molecules would experience higher obstacles to coming out from the matrix of less drug loaded formulations. In a recent study we have reported IPN hydrogel microspheres of LBG−PVA for controlled delivery of BH.44 Here, in this study we tried to explore the effect of carboxymethylation of LBG and its incorporation in the IPN system with PVA. The comparison of the dissolution profile of best optimized formulations (for CMLBG−PVA IPN B3 and LBG−PVA IPN F12)44 showed that the difference in the release profile at pH 1.2 (up to 2 h) for both cases was not significant (P value > 0.05). The release profile in phosphate buffer, pH 6.8, also did not show any significant difference up to 7 h (P value > 0.05). However, from 8 h there was a highly significant difference between both release profiles (P value < 0.001). This was due to the presence of the −COOH group in CMLBG, which swelled and eroded to a much higher extent than in LBG and helped in a higher amount of drug release in the same time points. 3.18.4. Release Kinetics. From the analysis of in vitro release data using different empirical equations, the fit was best with the zero order model (Table S2, Supporting Information), suggesting that the release was not dependent on the amount of drug present in the microspheres. The drug release data were also fitted with the power law equation and the values of n varied from 0.97 to 1.229, indicating the super case II transport mechanism.

Figure 6. XRD patterns of pure drug, blank formulation, and drug loaded formulation.

4. CONCLUSION In the present study locust bean gum was successfully modified to its carboxymethylated derivative by Williamson synthesis. The carboxymethylation process was optimized, and it was observed that, at 65 °C, the native gum produced the best substituted product (F6, DS = 0.653) in the NaOH/MCA/ LBG molar ratio of 4:2:1. The modified gum showed a marked decrease in its viscosity and molecular weight compared with its native form. The carboxymethylated derivative showed a pH dependent swelling property. There was a sharp reduction of the water contact angle of the modified gum, indicating its hydrophilic nature. Results of FTIR and solid state 13C NMR studies revealed that the native LBG was successfully modified to its carboxymethylated form. The DSC study also supported the fact that the native gum was modified to its carboxymethylated form. Toxicity and biodegradability studies showed that CMLBG was safe enough for internal use. The glutaraldehyde cross-linked IPN microspheres were prepared by using optimized CMLBG and PVA for controlled oral delivery of BH. Particles showed pH dependent swelling behavior. Depending upon different formulation variables, the particles

Figure 7. Effect of formulation variables on in vitro drug release: (a) effect of cross-linker, (b) effect of blend ratio, and (c) effect of drug loading. 10043



Article

were optimized. The particles were in the size range 378.61− 961.64 μm with low PDIs. The particles showed moderate drug entrapment efficiency. The FTIR and NMR data confirmed the formation of a glutaraldehyde cross-linked network structure. The FTIR and XRD data further confirmed the formation of IPN microspheres of BH. Drug release from IPN particles showed a dependency upon the cross-linker amount, blend ratio, and percentage drug loading. From the kinetic modeling of the release data, it was observed that particles followed zero order and super case II release kinetics. Hence, it can be concluded that the CMLBG−PVA IPN system can be a better approach for controlled oral drug delivery.

■

■

REFERENCES

(1) Rana, V.; Raja, P.; Tiwary, A. K.; Singh, R. S.; Kennedy, J. F.; Knill, C. J. Modified Gums: Approaches and Applications in Drug Delivery. Carbohydr. Polym. 2011, 83, 1031. (2) Chauhan, G. S.; Dhiman, S. K.; Guleria, L. K.; Kaur, I. Polymers from Renewable Resources: Kinetics Studies of the Radiochemical Graft Copolymerization of Styrene onto Cellulose Extracted from Pine Needles and the Effect of Some Additives on the Grafting Parameters in an Aqueous Medium. J. Appl. Polym. Sci. 2002, 83, 1490. (3) Okieimen, F. E. Grafting Ethyl Methacrylate onto Partially Hydrolysed Starch Using Ceric Ion as Initiator. Indian J. Chem. Technol. 2003, 10, 197. (4) Singh, V.; Tiwari, A.; Tripathi, D. N. T.; Sanghi, R. Microwave Assisted Synthesis of Guar-g-Polyacrylamide. Carbohydr. Polym. 2004, 58, 1. (5) Chan, A. W.; Whitney, R. A.; Neufeld, R. J. Semisynthesis of a Controlled Stimuli-Responsive Alginate Hydrogel. Biomacromolecules 2009, 10, 609. (6) Božanić, D. K.; Trandafilović, L. V.; Luyt, A. S.; Djoković, V. ‘Green’ Synthesis and Optical Properties of Silver-Chitosan Complexes and Nanocomposites. React. Funct. Polym. 2010, 70, 869. (7) Verraest, D. L.; Peters, J. A.; Kuzee, H. C.; Batelaan, J. G.; Van Bekkum, H. Carboxymethylation of Inulin. Carbohydr. Res. 1995, 271, 101. (8) Ege, S. N. Organic Chemistry; D. C. Heath and Co., Lexington, MA, 1989. (9) Miyamoto, K.; Tsuji, K.; Nakamura, T.; Tokita, M.; Komai, T. Preparation of Carboxymethyl-Gellan. Carbohydr. Polym. 1996, 30, 161. (10) Goyal, P.; Kumar, V.; Sharma, P. Carboxymethylation of Tamarind Kernel Powder. Carbohydr. Polym. 2007, 69, 251. (11) Sharma, B. R.; Kumar, V.; Soni, P. L. Carbamoyl Ethylation of Cassia Tora Gum. Carbohydr. Polym. 2003, 54, 143. (12) Sharma, B. R.; Kumar, V.; Soni, P. L. Carbamoyl Ethylation of Guar Gum. Carbohydr. Polym. 2004, 58, 449. (13) Mannion, R. O.; Melia, C. D.; Launay, B.; Cuvelier, G.; Hill, S. E.; Harding, S. E.; Mitchell, J. R. Xanthan/Locust Bean Gum Interactions at Room Temperature. Carbohydr. Polym. 1992, 19, 91. (14) Lundin, L.; Hermansson, A. M. Supermolecular Aspects of Xanthan-Locust Bean Gum Gels based on Rheology and Electron Microscopy. Carbohydr. Polym. 1995, 26, 129. (15) Garcia-Ochoa, F.; Casas, J. A. Viscosity of Locust Bean (Ceratonia siliqua) Gum Solutions. J. Sci. Food Agric. 1992, 59, 97. (16) Clissold, S. P.; Lynch, S.; Sorkin, E. M. Buflomedil. A Review of its Pharmacodynamic and Pharmacokinetic Properties, and Therapeutic Efficacy in Peripheral and Cerebral Vascular Diseases. Drugs 1987, 33, 430. (17) Rokhade, A. P.; Agnihotri, S. A.; Patil, S. A.; Mallikarjuna, N. N.; Kulkarni, P. V.; Aminabhavi, T. M. Semi-Interpenetrating Polymer Network Microspheres of Gelatin and Sodium Carboxymethyl Cellulose for Controlled Release of Ketorolac Tromethamine. Carbohydr. Polym. 2006, 65, 243. (18) Dubourg, A.; Scamuffa, R. F. An Experimental Overview of a New Vasoactive Drug: Buflomedil HCl. Angiology 1981, 32, 663. (19) Ray, R.; Maity, S.; Mandal, S.; Chatterjee, T. K.; Sa, B. Development and Evaluation of a New Interpenetrating Network Bead of Sodium Carboxymethyl Xanthan and Sodium Alginate for Ibuprofen Release. Pharmacol. Pharm. 2010, 1, 9. (20) Green, J. W. Carboxymethyl cellulose. In Methods in Carbohydrate Chemistry; Whistler, R. L., Ed.; Academic Press: New York, 1963.

ASSOCIATED CONTENT

* Supporting Information S

Detailed process of determination of DS, mechanism of synthesis of CMLBG, different hematological and blood chemistry data during toxicity study, kinetic model correlation coefficient of invitro release data, schemes of Preparation of carboxymethyl locust bean gum by Williamson ether synthesis, Probable mechanism of network formation by glutaraldehyde cross-linking, Solution characteristics of locust bean gum and carboxymethyl locust bean gum, effect of NaOH, MCA and temperature on DS, histopathological study, Evidence of biodegradation, SEM images of LBG and CMLBG, FTIR spectrum of LBG and different batches of CMLBG, Particle size distribution of IPN microspheres. This material is available free of charge via the Internet at http://pubs.acs.org.

■

SEM = scanning electron microscopy KBr = potassium bromide C = carbon N = nitrogen O = oxygen

AUTHOR INFORMATION

Corresponding Author

*Tel.: +91-9470339587. Fax: +91-6512275290. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS S.K. is thankful to CSIR, New Delhi, for financial support as CSIR-SRF [Grant 09/554(0029)/2011; EMR-I]. The authors are thankful to GCTS, Asansol (India), for viscosity study; CIF, BITMesra (India), for SEM; STIC, Cochin University (India), for PXRD analysis; NMR study centre, IISc-Bangalore (India), for solid state NMR; and DRL-DRDO, Tezpur, for providing other instrumental facilities under the grant [DRLTP1-2011/TASK-46]. The authors are also thankful to the authorities at BITMesra for providing necessary facilities for this work. The authors alone are responsible for the content and the writing of this article.

■

ABBREVIATIONS LBG = locust bean gum CMLBG = carboxymethyl locust bean gum MCA = monochloroacetic acid NaOH = sodium hydroxide PVA = poly(vinyl alcohol) IPN = interpenetrating polymer network BH = buflomedil hydrochloride FTIR = Fourier transform infrared NMR = nuclear magnetic resonance PDI = polydispersity index XRD = X-ray diffraction DSC = differential scanning calorimetry 10044



Article

(21) Silva, D. A.; de Paula, R. C. M.; Feitosa, J. P. A.; de Brito, A. C. F.; Maciel, J. S.; Paula, H. C. B. Carboxymethylation of Cashew Tree Exudate Polysaccharide. Carbohydr. Polym. 2004, 58, 163. (22) Vijan, V.; Kaity, S.; Biswas, S.; Isaac, J.; Ghosh, A. Microwave Assisted Synthesis and Characterization of Acrylamide Grafted Gellan, Application in Drug Delivery. Carbohydr. Polym. 2012, 90, 496. (23) Perry, L. M. Medicinal Plants of East and Southeast Asia: Attributed Properties and Uses; MIT Press: Cambridge, MA, 1980. (24) Rosidah, Y. M. F.; Sadikun, A.; Asmawi, M. Z. Antioxidant Potential of G. procumbens. Pharm. Biol. 2008, 46, 616. (25) Angadi, C. S.; Manjeshwar, S. L.; Aminabhai, T. M. Stearic AcidCoated Chitosan-Based Interpenetrating Polymer Network Microspheres: Controlled Release Characteristics. Ind. Eng. Chem. Res. 2011, 50, 4504. (26) Maiti, S.; Kaity, S.; Ray, S.; Sa, B. Development and Evaluation of Xanthan Gum Facilitated Ethyl Cellulose Microsponges for Controlled Percutaneous Delivery of Diclofenac Sodium. Acta Pharm. 2011, 61, 257. (27) Bhattacharya, S. S.; Sen, K. K.; Sen, S. O.; Banerjee, S.; Kaity, S.; Ghosh, A. K.; Ghosh, A. Synthesis and Characterization of Poly(acrylic acid)/Modified Bentonite Superabsorbent Polymer. Int. J. Polym. Mater. 2011, 60, 1015. (28) Korsemeyer, R. W.; Gunny, R.; Peppas, N. A. Mechanism of Solute Release From Hydrophilic Polymers. Int. J. Pharm. 1983, 15, 25. (29) Williamson, A. Theory of Aetherification. Philos. Mag. 1850, 37, 350 (from A Source Book in Chemistry, 1400−1900; Leicester, H. M., Klickstein, H. S., Eds.; Harvard: Cambridge, MA, 1952). (30) Tijsen, C. J.; Scherpenkate, H. J.; Stamhuis, E. J.; Beenackers, A. A. C. M. Optimisation of the Process Conditions for the Modification of Starch. Chem. Eng. Sci. 1999, 54, 2765. (31) Pushpamalar, V.; Langford, S. J.; Ahmad, M.; Lim, Y. Y. Optimization of Reaction Conditions for Preparing Carboxymethyl Cellulose from Sago Waste. Carbohydr. Polym. 2006, 64, 312. (32) Whistler, R. L.; BeMiller, J. N. Alkaline Degradation of Polysaccharides. Adv. Carbohydr Chem. 1958, 13, 289. (33) Gong, H.; Liu, M.; Chen, J.; Han, F.; Gao, C.; Zhang, B. Synthesis and Characterization of Carboxymethyl Guar Gum and Rheological Properties of its Solutions. Carbohydr. Polym. 2012, 88, 1015. (34) Leong, K. H.; Chung, L. Y.; Noordin, M. I.; Mohamad, K.; Nishikawa, M.; Onuki, Y.; Morishita, M.; Takayama, K. Carboxymethylation of Kappa-Carrageenan for Intestinal-Targeted Delivery of Bioactive Macromolecules. Carbohydr. Polym. 2011, 83, 1507. (35) Parvathy, K. S.; Susheelamma, N. S.; Tharanathan, R. N.; Gaonkar, A. K. A Simple Non-Aqueous Method for Carboxymethylation of Galactomannans. Carbohydr. Polym. 2005, 62, 137. (36) Andrade, G.; Barbosa-Stancioli, E. F.; Mansur, A. A. P.; Vasconcelos, W. L.; Mansur, H. S. Small-Angle X-ray Scattering and FTIR Characterization of Nanostructured Poly(vinyl alcohol)/Silicate Hybrids for Immunoassay Applications. J. Mater. Sci. 2008, 43, 450. (37) Mansur, H. S.; Sadahira, C. M.; Souza, A. N.; Mansur, A. A. P. FTIR Spectroscopy Characterization of Poly(Vinyl Alcohol) Hydrogel with Different Hydrolysis Degree and Chemically Crosslinked with Glutaraldehyde. Mater. Sci. Eng., C 2008, 28, 539. (38) Kajjari, P. B.; Manjeshwar, L. S.; Aminabhavi, T. M. Novel Interpenetrating Polymer Network Hydrogel Microspheres of Chitosan and Poly(acrylamide)-grafted-Guar Gum for Controlled Release of Ciprofloxacin. Ind. Eng. Chem. Res. 2011, 50, 13280. (39) Newman, R. H.; Hemmingson, J. A. Interactions between Locust Bean Gum and Cellulose Characterized by 13C NMR Spectroscopy. Carbohydr. Polym. 1998, 36, 167. (40) Peschel, D.; Zhang, K.; Aggarwal, N.; Brendler, E.; Fischer, S.; Groth, T. Synthesis of Novel Celluloses Derivatives and Investigation of their Mitogenic Activity in the Presence and Absence of FGF2. Acta Biomater. 2010, 6, 2116. (41) Kurita, Y.; Isogai, A. N-Alkylations of Chitosan Promoted with Sodium Hydrogen Carbonate under Aqueous Conditions. Int. J. Biol. Macromol. 2012, 50, 741.

(42) Yang, H.; Hu, S.; Horii, F.; Endo, R.; Hayashi, T. CP/MAS 13C NMR Analysis of the Structure and Hydrogen Bonding of MeltCrystallized Poly(Vinyl Alcohol) Films. Polym. J. 2006, 47, 1995. (43) Webster, E. A.; Chudek, J. A.; Hopkins, D. W. Carbon Transformations during Decomposition of Different Components of Plant Leaves in Soil. Soil Biol. Biochem. 2000, 32, 301. (44) Kaity, S.; Isaac, J.; Ghosh, A. Interpenetrating Polymer Network of Locust Bean Gum-Poly(Vinyl Alcohol) for Controlled Release Drug Delivery. Carbohydr. Polym. 2013, 94, 456.

10045


Carboxymethylation of Locust Bean Gum: Application in

Recommend Documents