Assam Bora Rice Starch Based Biocompatible Mucoadhesive

Sep 20, 2012 - Nanomedicine Research Lab, Department of Pharmaceutics, Faculty of Pharmacy, Hamdard University, New Delhi, India 110062. ABSTRACT: ...
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Assam Bora Rice Starch Based Biocompatible Mucoadhesive Microsphere for Targeted Delivery of 5‑Fluorouracil in Colorectal Cancer Mohammad Zaki Ahmad,† Sohail Akhter,‡ Mohammed Anwar,‡ and Farhan Jalees Ahmad*,‡ †

Dreamz College of Pharmacy, Khilra-Meramesit, Sundernagar, Mandi, Himachal Pradesh, India 175036 Nanomedicine Research Lab, Department of Pharmaceutics, Faculty of Pharmacy, Hamdard University, New Delhi, India 110062



ABSTRACT: The aim of this study was to develop novel colon targeted mucoadhesive microspheres (MAMs) for site specific delivery of 5-fluorouracil (5-FU) to colon without the drug being released in the stomach or small intestine. MAMs were prepared using Assam Bora rice starch, a natural mucoadhesive polymer, by a double emulsion solvent evaporation method. The microspheres were characterized for their shape, size, surface morphology, size distribution, incorporation efficiency, and in vitro and in vivo drug release studies. The release study confirmed the insignificant release of 5-FU in physiological condition of stomach and small intestine and major drug release in the cecal content. In vivo release study of the optimized MAMs was compared with immediate release (IR) 5-FU. 5-FU was distributed predominantly in the upper GI tract from the IR, whereas 5-FU was distributed primarily to the lower part of the GI tract from the MAM formulation. Enhanced levels of liver enzymes were found in animals given IR 5-FU as well as augmented levels of serum albumin, creatinine, leucocytopenia and thrombocytopenia was also observed. Thus to sum up, it can be appropriately established that the 5-FU release pattern from MAMs exhibits slow and extended release over longer periods of time with reduced systemic side effects. KEYWORDS: 5-fluorouracil, colon-specific, Assam Bora rice starch, mucoadhesion, drug absorption and organ distribution



INTRODUCTION The colon and rectum are parts of the digestive system of human beings. Cancer affecting either of these organs may be called colorectal cancer (CRC). CRC is a leading cause of death due to cancer in the world, and more than 66,000 cases of CRC are reported in the Indian subcontinent every year.1 The main treatments for colorectal cancer are radiation therapy, surgery, and/or chemotherapy. From several decades 5-fluorouracil (5FU) remains the sole utmost effective chemotherapeutic agent for the treatment CRC.2,3 Conventional cancer chemotherapy is not very effective for treatment of colorectal cancer, as the drug molecule does not reach the target site at therapeutic concentration. On the other hand, intravenous administration of 5-FU produces severe systemic, cytotoxic, and other toxic effects such as diarrhea, ECG changes, and an increase in cardiac enzymes, discoloration along the vein through which medication is given, low blood counts, low platelets, and bone marrow depression.4 The cytotoxic effects of 5-FU have been well characterized, and include the following: inhibition of the target enzyme thymidylate synthase by the 5-FU metabolite 5fluoro-2′-deoxyuridine-5′-monophosphate; incorporation of 5FU into RNA, with subsequent alterations in RNA processing and function; and incorporation of 5-FU into DNA, with resultant DNA damage.5−9 The oral bioavailability of 5-FU is © 2012 American Chemical Society

erratic, so the drug is usually administered by the intravenous route.10 However, it produces systemic side effects, which reduce patient compliance, and a high failure rate due to interactions with nontargeted sites.11,12 In terms of drug delivery, site-specific delivery of 5-FU to receptor sites has the potential to reduce the systemic side effects and to increase the pharmacological response.13−16 This problem can be circumvented by reduction of the carrier size and the development of a multiparticulate colon-specific drug-delivery system,13 since size-dependent GI retention has been reported in earlier studies.17 The colonic region is inhabited by more than 400 species of bacteria, which mostly ferment the polymers which are not digested by enzymes present in the human gastrointestinal (GI) tract.18 Among the different approaches to achieve colon specific drug delivery, the use of polymers specifically biodegraded by colonic bacterial enzymes holds assurance.19,20 The important bacteria present in the colon such as Bacteroides, Bif idobacterium, Eubacterium, Peptococcus, Lactobacillus, and Received: Revised: Accepted: Published: 2986

October 6, 2011 August 17, 2012 September 20, 2012 September 20, 2012 dx.doi.org/10.1021/mp300289y | Mol. Pharmaceutics 2012, 9, 2986−2994

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Table 1. Composition and Characterization of Developed MAM Formulationa

a

formulations

Assam Bora rice starch (mg)

F1 F2 F3 F4 F5

1000 1500 2000 2500 3000

mean particle sizeb (μm) 210 234 278 305 367

± ± ± ± ±

0.08 0.01 0.01 0.07 0.09

entrapment efficiencyb (%) 92.49 91.27 92.78 89.75 89.67

± ± ± ± ±

0.22 0.27 0.38 0.03 0.09

angle of reposeb 20.18 21.20 24.11 22.10 23.42

± ± ± ± ±

compressibility indexb

0.30 0.01 0.07 0.01 0.08

13.02 13.02 13.67 13.29 12.45

± ± ± ± ±

0.02 0.01 0.03 0.01 0.07

In each case 1000 mg of 5-fluorouracil was used. bEach value of the characterization parameters represents the mean ± SD of six determinations.

Clostridium secrete a wide range of reductive and hydrolytic enzymes such as β-glucuronidase, β-xylosidase, β-galactosidase, α-arabinosidase, nitroreductase, azoreductase, deaminase, and urea hydroxylase.21 Upon reaching the colon, polymer undergoes assimilation by microorganisms, degradation by enzymes, or breakdown of the polymer backbone leading to a subsequent reduction in their molecular weight and, hence, loss of mechanical strength. They are then unable to hold the drug entity to further extent.22 This concept has been exploited to trigger drug release and its delivery to the colon.23,24 The present investigation is aimed at using a natural, safe, inexpensive, and abundantly available Assam Bora rice starch for colon targeted microsphere formulation of 5-FU. Assam Bora rice locally known as Bora Chaval was introduced in Assam from Thailand or Myanmar by Thai-Ahoms25,26 and is now widely distributed throughout the upper Assam. Obtained starch from this rice (Assam Bora rice starch) is characterized by high amylopectin content (>95% of amylopectin) with branched waxy polymer, having physical stability and resistance toward enzymatic action.26−28 Assam Bora rice starch hydrates and swells in cold water forming viscous colloidal dispersion or sols, responsible for its mucoadhesive nature. Moreover, it is totally degraded by colonic bacteria but undigested in the upper GI tract. So, on account of its mucoadhesive nature, drug release retarding property, and susceptibility to microbial degradation in the large intestine,28,29 we have utilized Assam Bora rice starch in our study to deliver 5-FU to target colon in the form of mucoadhesive microspheres (MAMs).

formulation code are given in Table 1. A Silverson homogenizer fitted with 6 blade butterfly propeller was used for rapid mixing for 60 min. For the preparation of the second emulsion, 60 mL of the first emulsion was added dropwise (using 20 gauge hypodermic needle) to 300 mL of light liquid paraffin containing 1.5% (v/v) span 80. The resulting double emulsion was stirred at 1000 rpm for 1 h with heating at 50 °C to promote the evaporation of water. Solid MAMs were subsequently separated from the oil by centrifugation at 10000 rpm, washed in hexane, freeze-dried overnight (Hetro Drywinner, Birkerod, Denmark), and kept in an airtight container for further studies. For each polymer to drug ratio, 6 batches of MAMs were prepared for the purpose of assessing the reproducibility of drug loading. The composition of selected MAMs is given Table 1. Particle Size, Size Distribution, and Morphology. The particle size distribution of the prepared microspheres was measured by the quasi elastic light scattering technique (Photocor FC with manual goniometer, software 288 channel, Photocor Instruments Inc., College Park, MD). Weighed microspheres (100 mg) were suspended in double distilled water (4 mL) and vortexed before measurement. The obtained homogeneous dispersion was examined to determine the particle size distribution. The morphology of the microspheres was studied by scanning electron micrographs (SEM) taken with a scanning electron microscope (Hitachi S-300N, Germany). The samples were gold coated (at about 100 Å) on metal stabs with the aid of double-sided adhesive tape in KSE24 M high vacuum evaporator. Selected regions that were scanned depicting distinct morphological feature were photographed. Determination of Flow Properties. The flow properties of the prepared microspheres were investigated by the angle of repose, bulk density, and tapped density measurements. The angle of repose was determined by the fixed-base cone method. Bulk and tapped densities were measured in 100 mL of a graduated cylinder. The sample contained in the cylinder was tapped mechanically by means of a constant-velocity rotating cam. The tapped volume was noted down when it showed no change in its value, and bulk density and tapped density were calculated. The result obtained from bulk density and tapped density was used to calculate the compressibility index.



MATERIALS AND METHODS Assam Bora rice was procured from local villagers of Dibrugarh district of upper Assam. All other chemicals used in the experiment were of analytical reagent grade and used without further purification. Starch from Assam Bora rice was isolated by following the general procedure of Ahmad et al. with slight modification.26−30 The drug, 5-FU (98- 99% purity), was obtained as gift sample from Taj Pharmaceutical Limited, Valsad, Gujarat, India. Assam Bora rice starch was isolated and further processed in our laboratory. Preparation of Microspheres. MAMs were prepared by a double emulsion solvent evaporation method as described earlier by Sandra et al., 2005, with slight modification31 Aqueous polymer dispersion (A) was prepared by dispersing the weighed amount of Assam Bora rice starch (Table1) in 100 mL of distilled water and further stirred at 1000 rpm for 8 h. The solution (A) was stored in a sealed container at 40 °C for 36 h prior to use. Drug solution (B) was prepared by dissolving 1000 mg of 5-FU and 75 mg of Mg-stearate (Anti Adherent) in 40 mL of methylene chloride and stirred at 1000 rpm for 1 h. The first emulsion was prepared by emulsifying solution B into 100 mL of solution A with further addition of 0.25 mL of Tween 80 dropwise to enhance the process of emulsification. The compositions of different formulations along with

compressibility index = 100 ×

ρtapped − ρbulk ρtapped

(1)

where ρtapped is tapped density and ρbulk is bulk density. Entrapment Efficiency. Unentrapped drug was separated by centrifugation, and the drug which remained entrapped in the MAMs was determined after complete disruption by crushing 100 mg of MAMs in glass mortar, dispersing in 100 mL of phosphate buffer (pH 7.4), and further analyzing the resultant solution for 5-FU using the high performance liquid 2987

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blood sample, 1.0 mL of blood was taken with 2.0 mL of methanol, vortexed, and centrifuged for 15 min at 10,000 rpm. The supernatant was taken and evaporated to dryness, and the residue was reconstituted with mobile phase. Aliquots of 20 μL from each sample were injected using the manual injector. All samples were filtered through a 0.45 μm pore size membrane filter before injection. In Vitro Drug Release Study. The in vitro drug release study was carried out in 0.1 M HCl (2 h) and phosphate buffer (3 h) to investigate the ability of 5-FU MAMs to remain intact with respect to pH conditions prevailing in stomach and small intestine. These studies were carried out using USP34-NF29 dissolution rate test apparatus (dissolution apparatus II at 100 rpm and 37 ± 0.5 °C). The ability of the 5-FU MAMs to release 5-FU in the physiological environment of colon was assessed by continuing the drug release studies in goat cecal content. Release of 5-FU from the microspheres was studied for the initial 2 h in acidic medium, followed by phosphate buffer (pH 7.4) for 3 h, and further the medium was replaced by goat cecal content and the dissolution was carried out for the next 19 h. The anaerobic environment of the cecal content was maintained by continuous CO2 bubbling into the beaker containing cecal content and the formulation. At regular intervals of time, 10 mL of the sample was withdrawn and replaced with 10 mL of fresh medium to maintain the sink conditions in each case. Withdrawn samples were filtered through a syringe filter (0.22 μm) and analyzed for 5-FU by HPLC as described previously. A control study (without goat cecal content) was also performed with all the formulations. The drug release studies were performed in triplicate in every case. The prepared microspheres were observed under different magnifications to analyze the surface and morphology of the microsphere before and after dissolution. Release Kinetics Study. To investigate the mechanism of drug release from the microsphere, the in vitro release profile was analyzed by applying various kinetic models (Higuchi, Korsmeyer−Peppas, zero order, and first order). The release constants were calculated from the slope of respective plots in different models. The best-fit solution was identified by R2 value approaching 1. In Vivo Study. In Vivo Drug Absorption Study. Formulation F3 was selected on the basis of in vitro release performance for further in vivo study. The animal protocol to carry out in vivo study was approved by animal ethical committee, and their guidelines were followed for the studies. Male albino rats, 7−9 weeks old and weighing 200−250 g, were used for the study. The animals were kept under standard laboratory conditions (temperature, 25 ± 2 °C; relative humidity, 55 ± 5%), housed in polypropylene cages, with free access to standard laboratory diet (Lipton feed, Mumbai, India) and water ad libitum. For the study, the animals were housed in five groups, and each group contained 18 rats. The first group served as a control. The second and third groups received IR formulation of 8.05 mg of 5-FU (IR formulation was prepared as per the method reported by Rahman et al.13). Animals of the fourth and fifth groups were given MAMs (F3) containing the equivalent amount of 5-FU (8.05 mg). The formulation was orally administered in suspension form by oral gavages followed by sufficient volume of drinking water. The blood samples (1 mL) of both control and test groups (second and fourth) were collected at predetermined time intervals (0, 2, 3, 6, 12, and 24 h) through the tail vein of rat in vacutainer

chromatography (HPLC, Shimazdu, Japan) method as established by Rahman et al. with slight modification.13 entrapment efficiency (EF) actual drug content = initial amount of the drug added in the formulation × 100

(2)

Mucoadhesion Test. Whole GIT of goat was commercially collected from the slaughter house immediately after sacrification of goat and was stored in physiological solution (previously bubbled with CO2) at −20 °C before use. GI tissues from different parts of goat GI tract (i.e., stomach, small intestinal, and large intestinal tissues) were washed with deionized water to remove nondigested food from lumen and then placed in normal saline solution at 4 °C and used within 2 h. The underlying connective tissues were subsequently removed to isolate the mucosal membrane. The mucoadhesion test of MAM disks was carried out using a texture analyzer (TA.XTplus, Stable Micro Systems, U.K.) with 50 N load cell equipped with mucoadhesive holder.32 All the MAM disks individually were attached to the cylindrical probe (10 mm in diameter) by double-sided adhesive tape. The GI tissues (about 20 × 10 mm) were equilibrated for 15 min at 37.0 ± 0.5 °C before being placed onto the holder stage of the mucoadhesive holder and maintained at 37 °C during the test in 200 mL of the medium. MAMs were hydrated in each medium (0.1 N HCl and phosphate buffer pH 7.4 and cecal content) for 5 min prior to contact with GI mucosa at a force of 0.05 N for 120 s. Probe withdrawal speed was 0.5 mm/s. Using Texture Exponent 32-bit software, the maximum force required to separate the probe from the tissue (i.e., maximum detachment force; Fmax) could be directly calculated. Swelling Index. The swelling studies on microspheres were performed with water, 0.1 N HCl, and phosphate buffer pH 7.4 as method described by Jain et al., 2004.33 In brief, 100 mg of MAMs, accurately weighed, was immersed in a slight excess of double distilled water, phosphate buffer (pH 7.4), and 0.1 N HCl, respectively, and kept for 24 h. The swelling index (SI) was calculated by using the following formula: ⎞ ⎛ W − Ws SI = ⎜ i × 100⎟ ⎠ ⎝ Ws

where SI = percentage swelling of microsphere, Wi = initial weight of microsphere, and Ws = weight of microspheres after swelling. HPLC Analysis of 5-Fluorouracil. 5-FU was analyzed by reverse phase HPLC column (Supelco column 516, C18, 5 μm, 250 mm × 4.6 mm). The HPLC system (SCL-10 AVP, Shimadzu, Japan) consisted of a binary pump (LC-10 ATVP, Shimadzu, Japan) and a UV−vis detector (SPD-10 AVP, Shimadzu, Japan). The mobile phase was composed of HPLC grade acetonitrile/acetate buffer (pH 4.0) in the ratio of 20:80 with a flow rate of 1 mL/min. The detection wavelength was set at 260 nm, and the retention time was 7.2 min. The assay was linear (r2 = 0.9995) in the concentration range of 0.01−40 μg/ mL with a lowest detection limit at 0.005 μg/mL. The percentage recoveries (average) ranged from 97.0% to 99.9%. No formulation component showed any sort of interference. For the analysis of samples from dissolution solution, aliquots of 20 μL from each sample were injected via the manual injector into a HPLC system, and in the case of analysis of 2988

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Figure 1. Scanning electron photomicrograph of MAMs (F3).

undispersed, aggregated mass of MAMs. Furthermore, at stirring speed above 1000 rpm, small size and dispersed MAMs were formed due to the development of high shear and turbulence. However, the turbulence produced causes the adhesion of MAMs to the wall of container and the blade surface. So, the optimized stirring speed was kept at 800−1000 rpm resulting in spherical and dispersed microsphere formation. Particle Size and Particle and Size Distribution. Microspheres formed were sufficiently rigid, and a SEM image showed a spherical shape with slight rough surface (Figure 1). The mean particle size for the formulations (F1− F5) was 210 ± 0.08 μm to 367 ± 0.09 μm (Table 1). Here in this case, it was seen that with the increase of Bora rice starch concentration, the size of MAMs increased. This may be due to the increase in viscosity of the first emulsion, which in turn increased the droplet size during the second emulsification process. Additionally, accumulation of starch polymer under the influence of chemical interaction like H-bonding may also be a contributing factor. Furthermore, entrapment of drug also affected the size but did not show linear relation with the particle size. Flow Properties. Angle of repose and compressibility index, the simple, faster and popular means of predicting flow properties, were chosen for flow characterization. The angle of repose and compressibility index of the formulated microspheres were in the range of 20.18 ± 0.30 to 24.11 ± 0.07, 12.45 ± 0.07 to 13.67 ± 0.03 respectively (Table 1). Values indicate good flow property (USP34-NF29) and suggest that MAMs can easily be encapsulated and handled during processing. Entrapment Efficiency. Entrapment efficiency (%) of selected formulations for 5-FU is summarized in the Table 1. 5FU, being sparingly soluble in water have a low tendency to diffuse out of the MAM core, and therefore high drug loading was achieved with Bora rice starch. Furthermore, high entrapment may also be due to inclusion of drug into hollow hilum of the Assam Bora rice starch. Mucoadhesion Test. The mucoadhesion test was carried out using goat stomach mucosa with 0.1 N HCl small intestinal (duodenal section) with phosphate buffer of pH 7.4 and large intestinal mucosa cecal content. The Fmax for all the formulations (F1−F5) are shown in Figure 2. Swelling Index. Swelling property is directly related to the mucoadhesive capability of the formulation as it has been reported earlier that the adhesive nature and the cohesiveness of the polymers are affected by swelling behavior.33,38 MAMs are anticipated to take up water from the underlying mucosal tissues by absorption, swelling, and capillary action leading to

tubes. The blood samples were then mixed with 2.0 mL of methanol, vortexed, and centrifuged for 15 min at 10,000 rpm. The supernatant was taken and evaporated to dryness, and the residue was reconstituted with mobile phase to quantify the drug using HPLC. Pharmacokinetic data were analyzed by fitting to a noncompartmental model using WinNonlin software, version 5.3 (Pharsight Corporation, Mountain View, CA, USA). Organ Distribution Study. Three animals from the third and fifth groups were killed by deep ether anesthesia at 0, 2, 4, 6, and 10 h after formulation administration, the entire GI tract was removed, and mesenteric and fatty tissues were separated. The GI tract was segmented into stomach, small intestine, cecum, and colon. These organs were homogenized by a micro tissue homogenizer (Remi Ltd., Mumbai) with a small amount of phosphate buffer pH 7.4 premixed with 1.5 mL of acetonitrile and kept for 45 min. The samples were centrifuged, supernatants collected were appropriately diluted with mobile phase, and the drug content was determined by the HPLC method as described in the section HPLC Analysis of 5Fluorouracil. Biochemical Studies. The carcass was opened by bilateral thoracotomy immediately after death, and blood (5 mL) was withdrawn by cardiac puncture. Sodium-EDTA was added to 2 mL of blood for determination of white blood cell (WBC) and platelet counts. The remaining blood was centrifuged at 3000 rpm for 15 min to separate the serum and was stored at −20 °C for biochemical estimation to be conducted later. WBC and platelets were determined by a cell counter (Sysmex-K100, Transasia Biomedicals Ltd., Mumbai, India). SGOT and SGPT, alkaline phosphatase, creatinine, and albumin concentrations were estimated by a UV kinetic method,34 p-nitrophenyl phosphate (PNPP) method,35 alkaline picric acid method,36 and BCG (Bromocresol green) method,37 respectively. Levels of SGOT, SGPT, albumin, creatinine, and alkaline phosphatase in the serum were determined using a clinical chemistry analyzer (ERBA Chem-5 Plus Transasia Biomedicals Ltd., Mumbai, India). Statistical Analysis. The mean percentage of 5-FU released from MAMs from various polymer ratios in the dissolution medium (with and without goat cecal content) for 24 h was compared. GraphPad Instat software was used for statistical analysis (GraphPad Software Inc., San Diego, CA, USA). Data are expressed as mean ± SD. Data were analyzed by ANOVA. Differences were considered significant at P < 0.05.



RESULTS During the optimization step of MAM synthesis, it was found that a stirring speed ≤800 rpm results in the formation of an 2989

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release was not due to mechanical erosion which is likely to occur because of bowel movements. The scanning electron micrographs taken before and after dissolution in the three different dissolution media depict the effect of release environment. SEM photographs after the dissolution in 0.1 M HCl and phosphate buffer do not show any change on the microsphere surface (Figure 4). However the SEM photographs of the microsphere after the dissolution into cecal content show the presence of holes on the surface of the microsphere due to breakdown of polymer backbone owing to microbial degradation (Figure 4). Release Kinetics Study. Values of regression coefficient (R2) for release profile of selected MAMs (F1−F5) in different kinetic models are given in Table 3. Results showed that, with the application of the Higuchi model, every formulation value of R2 was approaching 1. Overall curve fitting showed that drug release from MAMs follows the Higuchi model, indicating the controlled release nature of the prepared formulation. In vivo drug absorption and organ distribution study of optimized formulation was performed in order to establish its targeting potential to the colon. In Vivo Study. In Vivo Drug Absorption Study. The pharmacokinetic parameters are shown in Table 4. The two formulations produced markedly different serum drug concentration profiles. The mean peak plasma concentration (Cmax) was distinctly different for the two formulations: 10.56 ± 2.43 μg/mL versus 93.23 ± 4.34 μg/mL for the MAMs and IR formulation, respectively. Time to reach Cmax, i.e., Tmax, was 18.60 ± 1.23 h for MAMs, which was significantly higher than for IR formulation, 1.5 ± 0.813. The mean residence time (MRT) was 16.54 ± 2.34 h for MAMs, which was 10.60 times higher than that for the IR formulation (1.56 ± 0.23 h). All of the differences in pharmacokinetic parameters (Cmax, Tmax, and MRT) for the two formulations were significant (P < 0.001). Organ Distribution Study. The results of organ distribution indicated that maximum concentration 67 ± 5.4% of 5-FU was observed after 2 h in stomach following oral administration of 5-FU from IR formulation and in the subsequent hour; much less drug reached the small intestine, and afterward no drug was found in the colon. Assam Bora rice starch based MAMs were observed and found intact in the upper part of the GIT. Approximately 2.5−4.5% of total drug loaded was released during its transit through the upper GIT. After 8−10 h, the maximum percentage of drug was observed in the colon (92 ± 0.4%), and a very insignificant amount of drug was found in the stomach and small intestine (Figure 5). The release pattern of 5-FU from MAMs can be understood by the protective nature of Assam Bora rice starch. The drug 5-FU was released from MAMs only after reaching the colon owing to microbial degradation with microbial flora residing in the colon.

Figure 2. Maximum detachment force on different parts of GIT with MAMs.

additive adhesion.39,40 Table 2 shows the percentage swelling of different microsphere formulations at different time intervals, indicating variable swelling index of microspheres in water and acidic and alkaline medium with high swelling in phosphate buffer followed by water and 0.1 N HCl. It may be due to the greater gelling behavior of polymer in alkaline medium, followed by water and least in acidic medium. In Vitro Drug Release Study. Drug release performance of selected MAMs in different media (0.1 N HCl, phosphate buffer pH 7.4, and goat cecal content) at regular intervals of time has been depicted in Figure 3a. The results indicate that minimal amount of drug is released from the Assam Bora rice starch based MAMs in the physiological environment of stomach and intestine. There was no measurable drug release up to 2 h in simulated gastric fluid (pH 1.2), while at pH 7.4, the 5-FU release was quite insignificant (