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Drug delivery systems (DDS) that can precisely control the release rates of drugs have an enormous impact on the healthcare system (1). The controlled release DDS are mainly aimed at controlling the rate of drug delivery and sustaining the duration of therapeutic activity. Drug release from these systems should be at a desired rate, predictable and reproducible. Amongst the various approaches for oral controlled DDS, microspheres have generated much interest among researchers around the world (2,3). However, the success of these microspheres is limited owing to their short residence time at the site of absorption. It would, therefore, be advantageous to have a means of providing an intimate contact of the DDS with the absorbing membrane. This can be achieved by coupling mucoadhesive characteristics to microspheres and developing mucoadhesive microspheres. Mucoadhesion is a topic of current interest in the design of controlled release drug delivery systems to prolong the residence time of the dosage form at the site of application or absorption and to improve and enhance the bioavailability of drugs (4-6).
Pectin, a natural ionic polysaccharide, mainly consists of linearly connected α-(1-4)- D-galacturonic acid residues. The degree of esterification (DE), which is expressed as a percentage of carboxyl groups (esterified) is an important means to classify pectin (7). Low methoxyl pectin (< 50% Degree of esterification) is mainly used for preparation of microspheres by orifice-ionic gelation or emulsification ionic gelation technique. Microspheres are formed by intermolecular cross-linking between divalent calcium ions and the negatively charged carboxyl group of LMP. Divalent metals establish direct polyanion-cation-polyanion interaction between pairs of carboxylic groups on neighboring helices producing an 'egg-box model' (8). LMP is hydrophilic polymer containing a large number of H-bonding groups (e.g. carboxyl groups) which are possible to form H-bond with functional groups in mucus. This has been proposed as mechanism in mucoadhesion process.
Glimepiride is a second generation sulfonylurea, mainly used in management of type-2 diabetes mellitus. Glimepiride has a short biological half life of 5 hrs, which necessitates its administration in 2 or 3 doses of 1 mg to 2 mg per day (9). Thus, the development of controlled release dosage form would clearly be advantageous.
The present work was designed with an aim of formulating mucoadhesive microspheres of glimepiride using LMP as a main polymer and to further evaluate the effect of co-polymers such as HPMC, NaCMC, MC and carbopol 934P on the mucoadhesive properties and the release of glimepiride from the resulting microspheres. It is noteworthy that there are very few literature available describing microspheres with LMP and the effect of co-polymer on its properties.
MATERIALS AND METHODS
Glimepiride was obtained as a gift sample from Apex Formulations Pvt. Ltd, Ahmedabad. LMP was obtained as a gift sample from Krishna Pectins Pvt. Ltd, Mumbai. HPMC K15M was obtained from Colorcon Industries, India. NaCMC, MC, Carbopol 934P and calcium chloride were procured from S.D. Fine Chem. Ltd, India. All other chemicals used were of analytical grade.
Preparation of mucoadhesive microspheres
Microspheres were prepared by orifice-ionic gelation method (10-12). Drug and polymers were passed through sieve #80 before use. Polymeric solutions (3% w/v) of LMP alone and in combination with HPMC, NaCMC, MC and carbopol 934P was prepared in three different ratios viz. 9:1, 8:2 and 7:3 (Table I), by dissolving 600 mg of polymer mixtures in 20 ml of distilled water with constant stirring for 1 to 2 h. Glimepiride (25% w/w of dry polymer weight) was dispersed in the polymeric solution under constant stirring on magnetic stirrer to form a smooth viscous dispersion with a core to coat ratio of 1:4. This dispersion was added dropwise using 24G syringe needle into 5% w/v calcium chloride solution maintained under gentle agitation. The added droplets were retained in calcium chloride solution for 15 minutes to complete the curing reaction, to produce spherical rigid microspheres. The microspheres were collected by filtration and washed twice with distilled water and dried at room temperature for 24 h followed by 40°C for 2 h.
Characterization of prepared mucoadhesive microspheres
Particle Size Determination
The particle size of prepared microspheres as determined with an optical microscope fitted with a calibrated eye piece micrometer. The mean of 100 microspheres was noted as particle size. All the studies were carried out in triplicate.
Scanning Electron Microscopy (SEM)
Morphological examination of the surface and internal structure of microspheres was performed using a scanning electron microscope (JSM-840A, Jeol, Japan). The sample was sputtered with gold and the observations were made under vacuum.
100 mg of microspheres was crushed in a glass mortar and suspended in minimal amount of distilled water for dissolving the coat shell of microspheres. The suspension was suitably diluted with methanol in 100 ml graduated flask and filtered to separate the shell fragments. Drug content was analyzed after suitable dilution by UV-spectrophotometer at a wavelength of 228 nm (UV-1601, Shimadzu, Japan) against suitable blank. All the studies were carried out in triplicate.
The percentage drug encapsulation efficiency (EE) of each formulation was calculated using the following equation:
Actual Drug Content
EE (%) = X 100
Theoretical Drug Content
Swelling index of microspheres was determined in 0.1 N HCl (pH 1.2) and phosphate buffer (pH 7.4). 100 mg of microspheres were taken in a wire mesh basket and immersed in the medium. At different time intervals the weight of the swollen microspheres was recorded after wiping the excess of liquid with tissue paper. The swelling index was calculated by using the following equation.
(Wt - Wo)
Swelling Index =
Where, Wo is the initial weight of microspheres and Wt is the weight of the microspheres at time't'.
In vitro Drug Release
In vitro drug release studies were performed using USP dissolution test apparatus I (basket type). The dissolution studies were performed in 900 ml dissolution medium (0.1 N HCl - pH 1.2 / phosphate buffer - pH 7.4), at 50 rpm maintained at 37 ± 0.5°C. 0.5% of SLS was mixed in the buffer to maintain sink condition. At a predetermined time intervals an aliquot of 5 ml was withdrawn and replenished with fresh medium. Amount of drug in each aliquot was assayed on a UV-spectrophotometer (UV-1601, Shimadzu, Japan) at 228 nm using a suitable blank. All trials were conducted in triplicate and the average (± S.D) reading was noted.
Release Kinetics andMechanism
For analyzing the drug release kinetics, in vitro release data was fitted to; Zero order equation, Qt = Kot; First order equation, Qt = Qoe-Kt ; Higuchi's square root model, (13,14) Qt = KH√t, where, ´Qt´is amount of drug released at a time t, Qo is the initial amount of drug in the dissolution medium. K, Ko and KH are release constants. The mechanism of drug release was further analyzed using the Korsemeyer-Peppas empirical power law equation, (15) Mt/M∞ = Ktn, where, Mt/M∞ is the fraction of drug released at a time 't', K is the structural and geometrical constant and 'n' is the release exponent.
Mucoadhesion Testing by In Vitro wash-off Test
Freshly excised pieces of intestinal mucosa (2 x 2 cm2) from sheep were mounted on glass slides (3 x 1 inches) with cyanoacrylate glue. Approximately 50 microspheres were spread onto each wet rinsed tissue specimen, and immediately the slide was hung onto the arm of USP tablet disintegrating test apparatus. The tissue specimen was given a slow, regular up and down movement in the beaker containing 0.1N HCl (pH 1.2) / phosphate buffer (pH 7.4) maintained at 37 ± 0.2°C. At predetermined time intervals the machine was stopped and the number of microspheres still adhering to the tissue was counted (10-12).
RESULTS AND DISCUSSION
Glimepiride loaded LMP microspheres containing 3% w/v LMP produced satisfactory microspheres and this was selected as the maximum LMP concentration, above which the solution was too viscous to drop through a syringe with a needle of 24 gauze size.
Particle Size Determination
Microspheres were found to be large, discrete and free flowing. The particle size of microspheres increased with the increase in concentration of mucoadhesive polymers. The mean particle size was found to be in the range of 791.90±4.58 to 960.88±4.61 μm (Table I).
The SEM photographs of formulation N (Figure 1) indicated that the microspheres were almost spherical with rough surface due to higher concentration of drug uniformly dispersed at molecular level in the calcium-pectinate matrices. Surface morphology also revealed presence of cracks and deposits of the fine drug crystals on the surface.
Drug Content and Encapsulation Efficiency
Drug content for all the formulations was found to be within the range of 15.74±0.12 to 16.92 ± 0.37 mg. The EE of the preliminary batches of microspheres prepared using LMP alone was found to be decreasing with increasing the calcium chloride concentration above 5%, which may be attributed to the weakening of surface gel strength due to an excess of calcium ions. The calcium concentration versus gel strength curve reaches a point where further increase in calcium concentration does not increase the gel strength and this is indicated as calcium saturation. As the calcium concentration continues to increase beyond this point, the gel strength decreases. This is caused by pre-gelation, a rapid reaction between pectin molecules and calcium ions, resulting in non-homogeneous gel structure (8). All the formulations showed good EE within the range of 78.69±0.59% to 85.84±0.78% (Table I). The EE of microspheres was found to be decreasing with increase in concentration of HPMC, NaCMC and MC which may be attributed to the lesser availability of LMP to encapsulate the drug (8). Whereas in case of carbopol 934P, the EE was found to be increasing with increase in concentration of carbopol which may be due to the formation of denser network structure of polymers. Formulation N12 showed the highest EE of 85.84±0.78%.
The release of a drug from a polymeric matrix is controlled by the swelling behavior of the polymer. To study the effect of swelling of LMP microspheres on drug release, swelling ratio of microspheres was measured in terms of water uptake at selected time intervals. Swelling ratio was determined in two different media and varied with pH of the media used. Amongst the two media used, swelling ratio was high at pH 7.4 in comparison to pH 1.2 which is similar to the earlier report by Chung et al (16). At pH 1.2, the swelling of microspheres was found to be increasing with increase in the concentration of HPMC, NaCMC, MC and carbopol 934P as compared to LMP alone, but to the limited extent, which may be due to rehydration. At pH 1.2, the microspheres reached to their maximum swelling ratio within 3 to 4 h and then gradually decreased towards their equilibrium state (Figure 2 and 3). This initial swelling may be attributed to the highly hydrophilic nature of the polymers used which gets hydrated when comes in the contact with the fluid. Formulation N showed lowest swelling because, in solutions of low pH, the ionization of carboxylate groups on LMP might be repressed and there is less water incorporation into interchain entanglements due to closer network structure of hydrogel (7) i.e. less swellable microspheres.
Whereas in case of pH 7.4 totally opposite result were obtained, the swelling of microspheres was increased considerably. As the concentration of co-polymers such as HPMC, NaCMC and MC was increased in microspheres the swelling ratio was decreased and erosion was observed within 7 h (Figure 4 and 5). Formulation N containing only LMP showed highest swelling in pH 7.4, which may be because of ionization of carboxylate groups on LMP was favoured and LMP contains ester, hydroxyl and carboxyl groups that can easily form hydrogen bonds with water molecules upon hydration leading to a more swellable network structure and therefore the microspheres swelled to a great extent (16). The swelling ratio of microspheres reached to a maximum within 7 h and then decreased due to the slow and gradual erosion of the hydrogel network. Phosphate buffer (pH 7.4) contains a chelating agent such as phosphate ions which can weaken the calcium cross linking leading to the weakening of microspheres structure and erosion of the hydrogel matrix (16). The enhanced erosion rate was observed with the increase in concentration of HPMC in the formulations N1, N2 and N3 i.e. 6 h, 5 h and 4 h respectively. As the microspheres swells, the matrix experiences intra-matrix swelling force which promotes erosion and leaching of the drug leaving behind a highly porous matrix. Water influx weakens the network integrity of the polymer, thus influencing structural resistance of the swollen microspheres, which in turn results in pronounced erosion of the loose gel layer (17). For formulations N4, N5 and N6 with the increase in concentration of NaCMC, erosion of microspheres resulted at 7 h, 6 h and 5 h respectively. Similar results were obtained in case of formulations N7, N8 and N9 containing increasing amount of MC. The water soluble hydrophilic polymers like NaCMC and MC dissolve rapidly and introduce porosity. The void volume is thus expected to be occupied by the external solvent which diffuses into the microspheres and thereby accelerate the erosion of the gel layer (17). Whereas, in case of the microspheres N10, N11 and N12 containing carbopol 934P as a co-polymer, with the increase in concentration of co-polymer the swelling ratio was also increased, due to the ionization of carboxyl groups present in carbopol 934P (18) and the microspheres remained intact i.e. no erosion was observed even after 8 h, indicating that the swelling ratio has not reached to its maximum.
In Vitro Release Studies
The release of glimepiride from microspheres was studied in two different media viz. 0.1 N HCl - pH 1.2 (Figure 6 and 7) and phosphate buffer - pH 7.4 (Figure 8 and 9). In pH 1.2, glimepiride release from all the formulations N to N12 was found to be more controlled, with the maximum of 35.58±0.7% in 8 h (N3) and their dissolution profile reached to almost a steady state within 4 to 5 h. Formulation N showed highest control on drug release i.e. 14.78±0.19% in 8 h, because the calcium ions in calcium-pectinate microspheres were totally discharged in acidic environment and the carboxyl groups were shifted to an unionized form, indicating the acid resistant nature of calcium-pectinate matrix. It was found that the use of calcium chloride as a cross-linking agent lead to LMP microspheres stronger in the upper GIT at pH 1.2, due to a higher binding ability with a higher affinity and a higher pectinate gel strength. These properties resulted to a decrease of swelling of microspheres in pH 1.2 and consequently a decrease of drug release (19). The order of releasing rate observed with various microspheres was LMP<LMP-carbopol 934P<LMP-NaCMC<LMP-MC<LMP-HPMC.
Microspheres showed faster drug release in pH 7.4 as compared to pH 1.2. This is believed to be due to the presence of a calcium pectinate matrix, which altered the drug release profile in pH 7.4 from pH 1.2. At pH 7.4, the ionization of the carboxyl groups on LMP is favoured leading to the increased swelling of microspheres and decreased mechanical rigidity of the calcium pectinate matrix due to erosion, which accelerates the drug release (16). Moreover, for poorly water soluble drugs like glimepiride, erosion of the polymer gel layer is believed to be the predominant mechanism for drug release. Microspheres prepared using LMP alone i.e. formulation N showed better control over the release of glimepiride i.e. 84.30±2.82% in 8 h as compared to the microspheres prepared using HPMC, NaCMC and MC as co-polymers. The possible reason may be due to their faster rates of erosion as compared to microspheres containing LMP alone. In case of formulations N1, N2 and N3, with the increase in concentration of HPMC, the erosion of microspheres occurred at faster rate leading to increased release of drug i.e. 97.70±2.27% in 8 h, 98.48±2.03% in 7 h and 98.77±2.13% in 6 h respectively. HPMC as a co-polymer, exhibited low release retardant effect due to the presence of extensive amount of hydroxyl groups, which leads to highly swollen matrix and loose gel layer formation, followed by erosion. In the formulations N4, N5 and N6, with the increase in the concentration of NaCMC the release of glimepiride from the microspheres was also found to be increasing i.e. 87.64±2.67% in 8 h, 95.42±1.57% in 8 h and 96.14±2.51% in 7 h respectively. During dissolution, formulations containing NaCMC swelled forming a gel layer on the exposed surface of microspheres. The loosely bound polymer molecules in these microspheres were readily eroded, allowing the easy and faster release of glimepiride (17). Formulation N7, N8 and N9 containing MC as a co-polymer showed very low control over the release of glimepiride from the microspheres i.e. 91.65±3.59% in 8 h, 98.50±2.09% in 8 h and 98.86±2.02% in 7 h respectively. The reason may to attributed to the highly hydrophilic nature of the MC, acting as an channeling agent and causing the formation of micro-cavities in the matrix, which leads to the leaching of the drug from the porous matrix, resulting in increased drug release rates. Whereas in case of formulations N10, N11 and N12, it was found that an increase in carbopol 934P concentration resulted in better control over the release of glimepiride i.e. 78.23±2.13%, 72.53±2.29% and 66.86±1.70% in 8 h respectively. Formulation N12 showed the highest control over the drug release i.e. 66.86±1.70% in 8 h, which may be due to some physical and chemical interactions that might have taken place during the calcium-pectinate and carbopol 934P gel formation leading to the formation of cross linked network and decreasing the release of drug. The order of releasing rate observed with various microbeads was LMP-carbopol 934P<LMP<LMP-NaCMC<LMP-MC<LMP-HPMC.
The effect of progressive change in pH, on the drug release pattern of glimepiride microspheres was further evaluated on the formulations N and N12, by performing the in vitro release study following a pH progression method i.e. pH 1.2 for first 2 h and pH 7.4 for the rest of study. As expected very low drug release was observed in pH 1.2 whereas, faster drug release was observed in pH 7.4 for both the formulations which corroborates with the results of in vitro release studies in pH 1.2 and 7.4. This pH-responsive release was believed to be due to the carboxyl groups of LMP which were shifted from unionized form in pH 1.2 to the ionized form in pH 7.4, leading to the faster drug release. On changing the pH from lower to higher level, the drug release slowed to some extent (Figure 10). At the end of 8 h, 76.60±1.17% of drug was released from N in comparison to 84.30±2.82% in 8 h at pH 7.4. Similar pattern was observed in case of N12 at the end of 8 h, 59.13±1.12% of drug was release in comparison to 66.86±1.70% in 8 h at pH 7.4.
Drug Release Kinetics and Mechanism
Analysis of release datas according to different kinetic models is shown in Table II. All the prepared formulations followed the first order kinetics. The r2 values of Higuchi's plot indicated that the drug release from all the formulation followed diffusion mechanism. Further the data treatment using Korsemeyer-Peppas equation indicated that formulation either followed Fickian diffusion or Anomalous mechanism based on the formulations. The release exponent 'n' was less than 0.5 for all the formulations from N to N12 in pH 1.2 indicating Fickian diffusion as release mechanism, when the polymer dissociation was almost negligible in the dissolution medium. Whereas in pH 7.4, the release exponent 'n' was less than 0.5 for the formulations N1, N2, N3, N6, N7, N8 and N9 indicating Fickian diffusion as release mechanism. However for the formulations N, N4, N5, N10, N11, and N12 the 'n' value was > 0.5 which indicates that the drug was released by a combination of drug diffusion as well as polymeric chain relaxation.
Mucoadhesion Testing by In Vitro wash-off Test
Microspheres prepared using LMP in combination with co-polymers showed fairly good mucoadhesive properties in the in-vitro washoff test. The wash-off was faster at gastric pH (pH 1.2) i.e. within 5hr (Table III) as compared to intestinal pH (pH 7.4) i.e. within 8 hr (Table IV) which is in line with the earlier report by Thirawong et al (20). The possible explanation is that, pectin and mucin both possesses positive charge in pH 1.2 due to unionization of the molecules since the environmental pH was lower than their pKa; pKa of pectin and mucin were about 3-4 and 2.6, respectively. In other words, gelation of pectin was reduced, leading to the separation of the pectin and mucin molecules from each others in acidic environment. In contrast, both pectin and mucin are ionized at pH 7.4 which may establish the bond formation between them. Pectin and mucin both possesses negative charge in pH 7.4. The negative charges of pectin and mucin were due to the ionization of carboxyl groups in pectin and sialic acid in mucin since the environmental pH was higher than their pKa. Moreover, the uncharged segments in pectin molecules could interact with mucin via Hydrogen bond formation showing stronger mucoadhesive properties of pectin at this pH (21).
It was found that as the concentration of mucoadhesive polymers HPMC, NaCMC, MC and carbopol 934P was increased the microspheres exhibited good mucoadhesive properties at pH 1.2 as well as pH 7.4. However, better mucoadhesion was achieved at pH 7.4 with the order of LMP<LMP-MC<LMP-HPMC<LMP-NaCMC <LMP-carbopol 934P. The results showed that formulation N12 containing carbopol 934P as a co-polymer showed greater mucoadhesion as compared to that of HPMC, MC and NaCMC in both pH 1.2 and 7.4 i.e. upto 5 h and 8 h respectively, which is similar to the observations drawn in the earlier research by Chowdary et al (10). Explanation similar to that of pectin can be given for carbopol 934P because the interaction between pectin and mucin is similar to that of carbopol 934P and mucin, because of the same negative charges which are present on carbopol 934P and mucin due to the ionization of carboxyl groups in carbopol 934P and sialic acid in mucin, which has an axial carboxyl group.
The results of the studies concluded that choice of combination of polymers instead of single polymer may be an effective strategy for the designing and development of glimepiride loaded mucoadhesive microspheres for easy, reproducible and cost effective method to prove its potential for safe and effective controlled for oral drug delivery. The calcium pectinate matrix alone was able to control the release of drug in gastric pH, whereas LMP in combination with carbopol 934P showed more efficient controlled release as compared to other polymers in intestinal pH. LMP alone was not able to exhibit good mucoadhesive properties. However, the combination of LMP with other mucoadhesive polymers (HPMC, NaCMC, MC and carbopol 934P) exhibited good mucoadhesive properties. Our findings indicate that an inherent paradox for mucoadhesive microspheres exist, with the factors leading to an increased mucoadhesion on one hand, inevitably leading to a faster drug release on the other in case of co-polymers such as HPMC, NaCMC and MC . It may be concluded that the glimepiride microspheres prepared using the combination LMP and carbopol 934P in ratio 7:3 (N12) showed good EE, swelling, promising controlled release and mucoadhesive properties, thus seems to be a potential candidate for the development of mucoadhesive microspheres for therapeutic use. An in vivo study needs to be designed and executed to substantiate further in vitro - in vivo correlation.
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