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Preparation and Characterization of Crosslinked Gum Acacia Microspheres by Single Step Emulsion In-Situ Polymer Crosslinking Method: A Potential Vehicle for Controlled Drug Delivery

Parul K Patel1* and Pandya SS2

Department of Pharmaceutical Sciences, Jodhpur National University, Jodhpur, Rajasthan, India.

B. Pharmacy College, Rampura, Po. Kankanpur, Taluka –Godhra, Gujarat, India.

*Corresponding Author:
Parul K Patel
Babaria Institute of Pharmacy BITS Edu campus Vadodara Mumbai NH#8, Varnama Vadodara , Gujarat.

Received date: 21/02/2013 Accepted date: 11/03/2013 Revised date: 28/02/2013

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Because of its biodegradability several natural polymers, such as plant polysaccharides, have been proposed as appropriate excipients for the development of controlled drug delivery systems for oral administration. In this work microspheres of crossinked gum acacia were prepared by single step emulsion crosslinking method using glutaraldehyde (GL) as the crosslinking agent and Hydrochloric acid (HCl) as the catalyst.. Aqueous gum acacia solution (10% w/v) was emulsified with castor oil using span 80 as emulsifier (3%) and reacted with GL (25% solution) at different temperature. After a certain time of reaction the mixture was cooled and the oily phase decanted. The microspheres were washed with isopropyl alcohol (IPA) and dried at 40OC to constant weight. The FTIR spectra and the swelling properties of obtained microspheres showed that gum acacia could be crosslinked using glutaraldehyde. Degree of swelling and % weight loss decreased with increase in amount of GL and severity of reaction conditions (temperature and time). Crosslinking with higher amount of glutaraldehyde produced microspheres with lower swelling degree. These results lead to the conclusion that crosslinked GA presents good perspectives for its use in modified release pharmaceutical formulations.

Key words

Gum acacia, microspheres, crosslinking, swelling degree


Controlled release of active ingredients in specified regions of the digestive tract is a most challenging area in the field of development of the controlled drug delivery systems. Site-specific drug therapy based on polymer matrix and coating systems is a growing field of research and application technology. Several natural polymers, such as those found in the diet, have recently been proposed as appropriate excipients for the development of controlled drug delivery devices for oral administration based on their microbial biodegradability [1, 2]. A large number of these polysaccharides and oligosaccharides may form the basis for a suitable biodegradable carrier. The administration of drugs directly to the colon, particularly to the proximal portion of the large intestine, has been evaluated as a site for local colonic pathologies but also for systemic drug delivery. Colon targeting still remains one of the most challenging systems to drug delivery. Many diseases of the colon, such as bowel disease, constipation, carcinomas and infections could be benefit from colon specific delivery.[1-3] In these cases, local administration of drugs is advantageous as it promotes reduced exposure of the organism to the drug, as a result of smaller doses employed and reduced systemic absorption, which minimizes the occurrence of side effects related to another use for colon-specific release is shown in the case of drugs degraded on upper portions of the GIT (stomach and small intestine), such as proteins and peptide hormones, [3] successful oral administration of these agents is conditioned to the protection of the pharmaceutical form against gastric and duodenal enzyme attack. Various options for colon-specific delivery have been proposed, including the use of pro-drugs, which release the active drug on the colon, and the production of hydrogels, matrices and coated solid forms, employing biodegradable polymers such as polysaccharides [2,3,4].

The drug release from the system activated by colonic microflora appears to be more suitable with regard to selectivity. Also due to the toxicity associated with the synthetic polymeric systems, a wide variety of natural polymers are being used for the design and development of colon-targeted delivery systems. Most of these systems are based on the knowledge that anaerobic bacteria in the colon are able to recognize the various substrates and degrade them with the enzymes. The application of biodegradable natural polymers, which are resistant to degradation in the upper GIT (above the colon), has gained tremendous importance in pre-biotic food systems. Most of the recent research includes natural polysaccharides, especially from plant origin, being applied to create degradable colon-specific substrates. A number of colon-targeted delivery systems based both on polysaccharides and synthetic polymers, such as acrylics, have been designed and developed by various research groups [5,6,7]. Polysaccharides with a large number of derivatizable groups, a wide range of molecular weight, varying chemical composition and above all being stable, safe and biodegradable, offer properties preferable over all the other approaches. Exploiting the use of these naturally occurring dietary polysaccharides for colonic drug carrier means that issues of safety, toxicity and availability are simplified. An important pre-requisite for a colon-specific drug carrier made of natural and modified polysaccharide hydrogel, is its ability to hydrate and resultant swelling which creates a diffusion barrier at the surface of the solid dosage form during its passage through the GI tract. These hydrated layers of polymers allow the penetration of colonic enzymes/bacteria which leads to the degradation of the polysaccharide barrier, hence releasing the drug at the target site [1,3].

Most of the natural gums are safe enough for oral consumption in the form of food additives or drug carriers. Among the advantages of natural gums over their synthetic counterparts are their biocompatibility, low cost, low toxicity (ecofriendliness) and relative widespread availability. Gums are metabolized by the intestinal microflora and ultimately degraded to their individual component sugars. In addition, enzymes available in the intestine can cleave the gums at specific sites. For example, α-galactosidase can hydrolyze terminal non reducing galactose residues to produce free α-d-galactose. However, there are certain problems associated with the use of gums. These include uncontrolled rates of hydration, pH dependent solubility, thickening, drop in viscosity on storage, and the possibility of microbial contamination. Chemical modification of gums not only minimizes these drawbacks but also enables their use for specific drug delivery purposes. Crosslinked or derivatives of gums are widely being investigated for the design of new delivery systems with tailor-made drug release profiles. An additional advantage of biodegradability confers the property of complete drug release to the dosage form due to the degradation of gums by colonic bacteria and enzymes present in the distal portion of the gastro-intestinal tract.

Gum Acacia; Indian Gum is the air-hardened, gummy exudation from the stem and branches of Acacia nilotica (Linn.) Del. subsp. Indica (Benth.) Brenan (syn. A. arabica Willd. var. indica Benth.) (Fam. Leguminosae), or other species of Acacia. It is available as pieces (tears) or in the form of a powder acacia. The powder form is white or yellowish-white ; odourless; on treatment with water it dissolves to give a mucilaginous liquid which is colourless or yellowish, dense, viscous, adhesive and translucent [8]. Chemically GA consists of a mixture of high molecular weight polysaccharide (major component) and hydroxyproline rich glycoprotein (minor component). The basic structural units of the gum are L-arabinose (45-65%), D-galactose (23-36%), L-rhamnose (2-3%) and uronic acid (8-14%) (Fig 1&2) [9]. GA enjoys a wide range of applications in industries such as paper, textile, food and pharmaceutical, due to its water binding capacity and high thickening efficiency. GA is hydrophilic and susceptible to easy biodegradation [10]. In the pharmaceutical industry, GA is used in pharmaceutical preparations and as a carrier of drugs since it is considered a physiologically harmless substance. Additionally, recent studies have highlighted GA antioxidant properties [11, 12], anticancer properties [13], its role in the metabolism of lipids [14] and its positive results when being used in treatments for several degenerative diseases such as kidney failure [15,16], cardiovascular [17] and gastrointestinal [18]. GA has high water solubility and a relatively low viscosity compared with other gums. Most gums cannot dissolve in water in concentrations above 5% due to their high viscosity. Instead, GA can get dissolved in water in a concentration of 50% w/v, forming a fluid solution with acidic properties (pH ~ 4.5). GA has been extensively tested for its properties as non-digestible polysaccharide which can reach the large intestine without digestion; in the small intestine. GA is slowly fermented by the bacterial flora of the large intestine producing short chain fatty acids [19]. However, since natural GA is highly water-soluble it becomes impossible to use it as carrier for controlled release oral dosage form, because the polysaccharide would promptly dissolve in the aqueous content of the digestive tube, completely releasing the drug. In order to overcome this disability of the natural gum, modification of the polymer solubility through crosslinking reaction can be employed. This kind of reaction reduces the amount of hydroxyl groups on the polysaccharide chain bonding the chains together, hence decreasing its affinity by water. Chemical modifications applied to polysaccharides can produce new compounds that can be used for novel drug delivery system. However, these structural modifications should maintain the potential biodegradability of the polymer by colonic micro flora at a specific portion of the GIT. Aldehyde derivatives such as formaldehyde, glutaraldehyde or other bifunctional reactants have been used to produce insoluble biodegradable microspheres [20]. Glutaraldehyde is used as a crosslinking agent to obtain rigid microspheres. This method has been widely studied in various formulations by different researchers [21, 22]. In this method, it is important to remove excess oil by washing the particles with solvents such as isopropyl alcohol (IPA). Otherwise, the oil retained in the microspheres may cause aggregation and alter the morphological properties of the microspheres. This washing procedure is also said to remove excess of the cross-linking agent [27]. IPA used to remove excess glutaraldehyde, extracts the water content of microspheres to obtain hardened microspheres that are easily filtered and dried.

Crosslinked Gum acacia microspheres were prepared by single step emulsion in-situ crosslinking technique in which gluataraldehyde 25% solution was used as crosslinking agent. The obtained crosslinked GA microspheres were analyzed through FTIR which allows us to identify certain characteristic bonds on the compounds that indicate the formation of new products. In addition, equilibrium swelling study of microspheres was carried out to study the effect of variables (temperature, amount of GL and reaction time).


Gum Acacia IP was purchased from Loba Chemie Pvt Ltd (Lot No.: SL 12981107), Mumbai, Glutaraldehyde was purchased from S.D. Fine chemicals, Mumbai. All other chemicals used in the study were of analytical grade

Method of Preparation of Crosslinked Gum Acacia Microspheres

The chemical crosslinking method for preparation of gum acacia microspheres involves emulsification followed by crosslinking with a suitable crosslinking agent (glutaraldehyde). An aqueous solution of gum acacia was prepared in distilled water The aqueous phase was dispersed in castor oil containing 3% span 80 (emulsifying agent), using overhead stirrer with three blade paddle. The biphasic system was stirred with heating (45°C, 50°C, 55°C, 60°C, 70°C), at 2000 rpm. The pH of the emulsion was adjusted to 3 with hydrochloric acid. Glutaraldehdye solution (25%w/v) was added as crosslinking agent to the above emulsion. Fifteen batches were prepared by varying the amount of crosslinking agent, temperature and reaction time, one at a time. The oily phase was decanted and the product was filtered, washed with distilled water followed by isopropyl alcohol. The microspheres were dried at 400C. Upon drying, an off white colored free flowing, fine powder was obtained. The product was kept in desiccators till further evaluation.

Fourier Transform Infrared Spectroscopy

The spectra were recorded for product using FTIR spectrophotometer (Jasco FTIR-410). Samples were prepared by KBr disk method and scanned over the range of 400-4000 cm-1; the resolution was 2/cm. The spectra were recorded for Gum acacia and crosslinked gum acacia microspheres to identify certain characteristic bonds on the compounds that indicate the formation of new products

Differential Scanning Calorimetry

The thermal behavior of gum acacia before and after crosslinking was examined with Perkin Elmer, Model: Pyris 1, DSC. Indium std was used for calibration and done every once in a week. Approximately 2.5 mg of sample was weighed in an aluminium pan and crimped with aluminium lid by using crimper. Sample was placed in a cell against a reference cell which having empty aluminium pan. DSC curves were recorded at a temperature range of 60-3000 C. with heating rate of 200C per min.

Shape and Surface Morphology

Surface and shape characteristics of microspheres were evaluated by means of scanning electron microscopy. The scanning electron microscopy samples were prepared by lightly sprinkling the microsphere powder on a double adhesive tape, which stuck to an aluminum stub. The stubs were then coated with gold to a thickness of ~300 Å using a sputter coater, and the photographs of samples were taken. Also Photomicrographs of crosslinked gum acacia microspheres obtained from various batches were taken using a digital trinocular microscope (Axioplan microscope, MPM-400 with image analyzer, Zeiss, Oberkochen, Germany). A small volume of ethereal suspension of microspheres was taken on a clean slide and was allowed to air-dry. The slide containing the dry film of microspheres was mounted on the stage of the microscope for observation.

Percentage Yield

The yield was calculated as the weight of the microspheres recovered from each batch divided by total weight of drug and polymer used to prepare that batch multiplied by 100.

Equilibrium Swelling Study and % Weight Loss

To evaluate the effectiveness of in-situ chemical crosslinking equilibrium swelling and % weight loss of treated GA was studied.

Equilibrium Swelling

The equilibrium swelling ratio which signifies the expanding and retracting forces between crosslink’s at equilibrium, was determined by water uptake measurement. The terms ‘swelling ratio’, [25] ‘equilibrium degree of swelling’ (EDS) [26] or ‘degree of swelling’ [27] has been used for more or less similar measurements. A pre-weighed amount (100 mg) of microspheres was placed in PBS (pH 7.4) and allowed to swell for 24 hrs, which is sufficient to reach the equilibrium state. The weight of swollen samples was measured after blotting excessive water gently with filter paper. The degree of swelling (α) was then calculated from the following formula [28]

α = W-W0/ W0

Where W0 is the initial weight of the microspheres and W is the weight of the microspheres at equilibrium swelling in the medium.

% Weight Loss

To evaluate the effectiveness of in-situ chemical crosslinking % weight loss of the product in Phosphate buffer saline (Ph 7.4) was studied. The weight (W1) of a 25mm glass fibre paper (pore size 2 micron) was determined following drying in an oven at 105oC for 1 hour and subsequently cooled in a desiccators containing silica gel. Dispersion of pre-weighed (S) sample from each batch was prepared in Phosphate buffer saline (Ph 7.4) followed by overnight hydration at room temperature. The hydrated dispersion was then centrifuged for 2-5 minutes at 2500 rpm prior to filtration. Drying of the filter paper with retained amount was carried out in an oven at 105oC followed by cooling to a constant weight (W3).

Amount retained (R) = W3-W1

Weight loss = S-R

% Weight loss = (S-R/S) X100

Results and Discussion


The evaluation of the FTIR spectra and DSC curves of products compared to natural Gum Acacia as reference, strongly suggests the occurrence of the proposed crosslinking reaction.

The FTIR spectrum of both Gum acacia and crosslinked gum acacia (Fig 4) depict a characteristic absorption band at 3420 cm-1 representing the presence of a hydrogen bonded OH group. The amino group which shows a characteristic absorption band in the region of 3400 - 3500 cm-1 must have been masked by an O-H group absorption band. The polymers also showed the characteristic bands of amine stretch (NH bend) around 1650 cm-1. The ether linkage is manifested as a characteristic band at 1074 cm-1. The intense absorption band at 1746.33 cm-1 in IR spectra of microspheres expresses the carbonyl group which could be due formation of new acetal groups. This new peak indicates that glutaraldehyde had been reacted with hydroxyl of gum acacia. According to previous authors [29, 30, 31, 32] in crosslinking reaction, the aldehyde groups from glutaraldehyde reacts with hydroxyl group from polymer under acidic condition, and then forms acetal bridges. In the present work, spectra IR indicated that microspheres based on glutaraldehyde–crosslinked gum acacia were prepared successfully. Broader peak at 1263 cm-1 and 1145 in spectra of crosslinked gum acacia microspheres as compared to that of gum acacia indicates ether linkages (cyclic ether large ring – C-O- stretch), reflective of the glutaraldehyde-crosslinked gum acacia. The mechanism of cross linking reaction was predicted as in Fig. 3.

DSC thermal profiles for gum acacia (Fig 5) with low water content (<15%) showed an endothermic event at about 87.440C. This can be related to water evaporation. No degradation DSC was observed for gum acacia, meanwhile, microspheres of gum acacia cross-linked with glutaraldehyde showed a different pattern of the DSC thermogram (fig 6) with a broad endothermic peak between 80 and 120OC and a new endothermic peak at 160.81° C reflective of formation of new compound.

Evaluation of Gum Acacia Microspheres

Percentage yield

The percentage yield obtained for in various batches is between 55.0- 92.16%. The product obtained at temperature lower than 60OC was mucilaginous in nature could not be separated from the oily phase. At 60OC and above granular product was obtained with phase separation which could be filtered and dried after adequate washing with IPA. Minimum time taken for complete evaporation of aqueous phase with appearance of granular product was found to be 5 hours at 600C. Further increasing the time, while maintaining constant temperature slightly decreased the product yield. The product yield was found to increase with increase in the concentration of gum acacia in aqueous phase (P15-P17) as well as with increase in amount of glutaraldehyde (P1-P5) suggesting increase crosslink density and therefore increase in water insoluble fraction in the product.

Equilibrium Swelling Study

Gum Acacia is known to swell in aqueous environments due to hydration. As a new polymeric structure is formed by introducing bridges between polymeric chains during the cross-linking procedure, the extent of the swelling process depends on the degree of cross-linking. Therefore, the denser the cross-linking bridges between the molecules, the more packed is the structure. Such a structure can be characterized by lower and slower penetration of the solvent through the chain structure of the polymer, suggesting that the swelling ratio and hence the release characteristics of the microsphere can be controlled by varying the content of the cross-linking agent used during the manufacturing process. Since glutaraldehyde is responsible for the formation of crosslinks, increasing the amount of glutaraldehyde and the crosslinking time increased the polymer density, resulting in reduction of the macromolecular chains mobility, and the formation of more stable and rigid spheres that showed a lower tendency to swell. However increase in the severity of the treatment conditions (i.e. increasing treatment temperature or time) also resulted in product exhibiting a darker color.

% Weight loss

Upon crosslinking, GA should not be dissolved completely in aqueous solvent. Table 2 shows the effects of crosslinkng agent (GL), temperature and time of treatment on weight loss and swelling behavior of the treated GA after submersion in Phosphate Buffer Saline at room temperature for 24 hrs. Weight loss and degree of swelling was highest in batch treated without GL (P-1) which subsequently decreased with increase in amount of GL and finally reached a plateau. Weight loss, for a fixed treatment temperature (P10-P15), decreased appreciably with initial increase in the treatment time and finally reached a plateau value at longer treatment times. With increasing treatment temperature, the weight loss was found to decrease.

Shape and Surface Morphology

Scanning electron micrograph and images of Trinocular microscope (Figure 7&8) indicate that cross-linked gum acacia microspheres possessed a fairly smooth surface and spherical shape.


Gums are abundantly found in nature. Among the advantages of natural gums over their synthetic counterparts are their biocompatibility, low cost, low toxicity (ecofriendliness) and relative widespread availability. GA has been extensively tested for its properties as non-digestible polysaccharide which can reach the large intestine without digestion; in the small intestine. GA is slowly fermented by the bacterial flora of the large intestine producing short chain fatty acids. However, the highly swellable nature of their putative form often restricts their use for delivering drugs to distal parts of the gastrointestinal tract. Swelling properties are strongly influenced by the crosslinking reaction parameters, like amount of crosslinking agent and reaction time. The FTIR spectra of obtained crosslinked gum acacia microspheres showed the new peak that indicated the formation of crosslinked structure. Crosslinking reduced the solubility of the natural gum which is indicated by decrease in weight loss on increasing temperature and reaction time. The increase in the severity of the treatment conditions (i.e. increasing treatment temperature and time) also resulted in product exhibiting a darker color. The increasing amount of glutaraldehyde will increase the crosslinked density, indicated based on the reduction of swelling ability and % weight loss of obtained gum acacia microspheres. Crosslinked gum acacia microspheres can be a potential carrier for colon specific drug delivery since reduced hydrophilicity due to crosslinking can prevent premature drug release. The drug release can be controlled by controlling the density of crosslinking by varying the amount of GL, temperature and time of reaction, and hence the degree of swelling.


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