In-vitro Evaluation of Glimepiride Mucoadhesive Microspheres to Improve the Sustain Drug Delivery

The oral controlled release drug delivery have recently been of increasing interest in pharmaceutical field to achieve improved therapeutic advantages such as ease of doing administration, patient compliance and flexibility in formulation (1). Over the past few decades, several oral control release drug delivery approaches being designed and developed, including floating systems that causes buoyancy in gastric fluid help to improve the pharmacotherapy (2). Many approaches have been reported in the literature for improved gastroretention for oral sustained release dosage forms viz. floatation (3), bio- or mucoadhesion (2), sedimentation (1), unfoldable, expandable, or swellable systems (2), super porous hydrogel systems (1), magnetic systems (3). Furthermore the gastroretentive system can act locally within the stomach and prolong the intimate contact with the absorbing membrane thus increasing its efficacy. The detailed literature on classification of gastroretentive systems has been well reviewed elsewhere. The most common approach was gastroretention based on floating system (4). The disadvantage of floating devices administered in a single-unit form such as hydrodynamically balanced systems (HBS) are unreliable in prolonging the GRT owing. Bioadhesive drug delivery systems are used to localize a delivery device within the human to enhance the drug absorption in a site-specific manner (5). In this approach, various bioadhesive polymers are used and they can adhere to the epithelial surface in the stomach. Thus, they increase GRT of the dosage forms. The basis of microadhesion in that a dosage form can stick to the mucosal surface by different mechanism. This mechanism is (1, 3, 5):-

i. The wetting theory, which is based on the ability of bioadhesive polymers to spread and develop intimate contact with the mucous layers.

ii. The diffusion theory, which proposes physical entanglement of mucin strands the flexible polymer chains, or an interpenetration of mucin strands into the porous structure of the polymer substrate.

iii. The absorption theory, suggests that bioadhesion is due to secondary forces such as Vander Waal forces and hydrogen bonding.

iv. The electron theory, which proposes attractive electrostatic forces between the glycoprotein mucin net work and the bio adhesive material.

Gastric mucoadhesion does not tend to be strong enough to impart to dosage forms the ability to resist the strong population forces of the stomach wall. The continuous production of mucous by the gastric mucosa to replace that is lost through peristaltic contractions and the dilution of the stomach content also seems to limit the potential of mucoadhesion as a gastroretentive force (6). The major challenge for bioadhesive drug delivery systems is the high turnover rate of the gastric mucus in the GIT and resulting limited retention times. Furthermore, it is very difficult to target specifically the gastric mucus with bioadhesive polymers. Materials commonly used for bioadhesion are poly acrylic acid, polylactic acids, cholestyramine, HPMC, sodium CMC, chitosan, sodium alginate, sucralfate, tragacanth, dextrin, gliadin, lectin etc (4).

Microspheres are spherical particles that range in diameter from 10 μm to 1000 μm. Microspheres are essential for improving the way conventional drugs are absorbed and lessening their side effects. Mucoadhesive microspheres include microparticles and microcapsules of 1 to 1000 μm in diameter consisting either entirely of mucoadhesive polymer or having an outer coating with adhesive property (5). Microspheres have the potential to be used for controlled as well as spatial drug delivery. Incorporating mucoadhesivenes to microspheres leads to efficient absorption and enhanced bioavailability of drug. Specific targeting of drug to the absorption site is achieved by using homing devices (ligand) like plant lactin, bacterial adhesion etc. on the surface of the microspheres. Mucoadhesive microspheres can be tailored to adhere to mucosal linings of GIT, thus offering the possibilities of localized as well as systemic absorption of drug in controlled manner. Reduction in size leads to an increase in surface area and can boost the strength of the poorly soluble substance (6, 7). Maintaining a constant level of medication in the body to enhance patient compliance. Polymer-based drug packaging keeps the medication from undergoing enzymatic cleavage while allowing it to be used with a drug delivery system. Shorter dosage intervals increase patient compliance (8).

The properties of mucoadhesive microspheres, e.g., their surface characteristics, force of mucoadhesion, release pattern of the drug, and clearance, are influenced by the type of polymers used to prepare them. Polymer microspheres can be used to deliver drug in a rate controlled manner and sometimes in targeted manner (9). The polymers that are commonly employed in the manufacture of mucoadhesive drug delivery platforms that adhere to mucin-epithelial surfaces may be conveniently divided into three broad categories as defined by Park and Robinson. Polymers are typically utilized as microspheres. First-generation mucoadhesive polymers may be divided into three main sub-categories, namely: Anionic polymers, Cationic polymers and non-ionic polymers. Of these, anionic and cationic polymers have been shown to exhibit the greatest mucoadhesive strength. Consequently, such charged polymeric systems will now be examined in more detail. The major disadvantage of using first generation mucoadhesive systems is that adhesion may occur at sites other than those intended (10). A scenario that is particularly true for platforms designed to adhere to a distal target such as those hypothesized in targeted mucoadhesion within the GI tract. Unlike first-generation non-specific platforms, certain second-generation polymer platforms are less susceptible to mucus turnover rates, with some species binding directly to mucosal surfaces; more accurately termed ‘‘cytoadhesives” (11).

Furthermore as surface carbohydrate and protein composition at potential target sites vary regionally, more accurate drug delivery may be achievable. They fall into two categories including Synthetic polymers and Natural polymers. Synthetic polymers are divided into two types including Non-biodegradable polymers having Poly methyl methacrylate (PMMA), Acrolein, Glycidyl methacrylate, Epoxy polymers, and Biodegradable polymers having Lactides, Glycosides & their co polymers Poly alkyl cyano Acrylates Poly anhydrides. Natural polymers obtained from different sources like Proteins, carbohydrates and chemically modified Carbohydrates, Proteins, Albumin, Gelatin, Collagen, Carbohydrates, Agarose, Carrageenan, Chitosan, and Starch. Chemically modified carbohydrates Poly dextran , Poly starch5,6. Thiolated polymers (thiomers) are a type of second-generation mucoadhesive derived from hydrophilic polymers such as polyacrylates, chitosan or deacetylated gellan gum (8, 9, 10, 11). The presence of thiol groups allows the formation of covalent bonds with cysteine- rich sub domains of the mucus gel layer, leading to increased residence time and improved bioavailability. In this respect thiomers mimic the natural mechanism of secreted mucus glycoproteins that are also covalently anchored in the mucus layer by the formation of disulphide bonds (9). Whilst first-generation mucoadhesive platforms are facilitated via non-covalent secondary interactions, the covalent bonding mechanisms involved in second generation systems lead to interactions that are less susceptible to changes in ionic strength and/or the pH. Mucoadhesive microspheres can be prepared using one of the following methods (5, 7, 8, 9, 12):

1. Emulsion cross-linking method/chemical denaturation

2. Emulsification and ionotropic Gelation

3. Solvent evaporation

4. Spray drying

5. Hot melt microencapsulation

6. Solvent removal

7. Ionic gelation (hydrogel microspheres)

8. Phase inversion microencapsulation

In the present work, glimepiride (Glimepiride is widely used oral antidiabetic drug acts as a model drug in this research work belonging to the class of sulfonylurea in BCS-II category. It is commonly prescribed for the management of type 2 diabetes mellitus by stimulating insulin release from pancreatic beta cells. However, glimepiride has a relatively moderate to short biological half-life and its rapid metabolism and elimination can lead to the need for frequent dosing) mucoadhesive microsphere made of low molecular mass chitosan and ethyl cellulose was developed. These glimepiride microsphere were evaluated their particle size, zeta potential, % of yield value, drug entrapment efficiency, drug loading, stability. In addition, in vitro drug release was performed.

MATERIALS AND METHODS

Glimepiride was a gift sample from Ajanta Pharma. Pvt. Ltd., India

Other materials were purchased from different sources like

Low molecular mass chitosan, ethyl cellulose: Sigma Aldrich, USA.

Glutaraldehyde, liquid paraffin and tween 80: Loba Chemie Pvt. Ltd., India

All other chemicals and reagents used were of analytical grade.

Formulation of mucoadhesive microspheres of glimepiride

Glimepiride mucoadhesive microspheres were prepared using different ratios of low molecular mass chitosan and ethyl cellulose by addition of crosslinking agent glutaraldehyde, using emulsifying crosslinking method. Briefly, required quantity of low molecular mass chitosan and ethyl cellulose solution was prepared by dissolving in double-distilled deionised water and stirring it continuously until the attainment of a homogeneous dispersion. The drug was dispersed in the above polymer solution, which was added slowly to light liquid paraffin (100 g, w/w) containing 2% (w/w) tween-80 under constant stirring at 600 rpm speed for about 15 min.  To this w/o emulsion, required quantity of glutaraldehyde as a crosslinking agent were added slowly and stirred for 3 h. The hardened mucoadhesive microspheres were separated by filtration, washed repeatedly with distilled water to remove the unreacted glutaraldehyde. Solid mucoadhessive microspheres obtained were vacuum dried at 40?C for 24 h and stored in desiccators until further use (12, 19, 20). Totally, five formulations were prepared as per the formulation codes assigned in Table 1.

Table 1: Formula of different ingredients used to prepare mucoadhesive microsphere

Evaluation of particle size and zeta potential

The mean particle size of the mucoadhesive microspheres was determined by Photon Correlation Spectroscopy (PCS) on a submicron particle size analyzer (Malvern particle size analyser) at a scattering angle of 90°. A sample (0.5mg) of the microsphere suspended in 5 ml of distilled water was used for the measurement. The zeta potential of the drug-loaded blend microspheres was measured on a zetasizer (Malvern particle size analyser) by determining the electrophoresis mobility in a micro electrophoresis flow cell. All the samples were measured in water at 25°C in triplicate (21).

Evaluation % of yield value

The prepared formulation were collected and weighed for each formulation code. The percentage yield (%) was calculated using formula given below (22):

% Yield = Actual weight of product/Total weight of drug and polymer???? 100

Evaluation of drug entrapment and drug loading efficiency

Amount of Glimepiride in each formulation was calculated according to procedure given below: Equivalent to 10mg of chitosan mucoadhesive microspheres from each batch were accurately weighed. The powder of chitosan blend microspheres were dissolved in 10 ml phosphate buffer and centrifuged at 1000 rpm. This supernatant solution is then filtered through whatmann filter paper No. 44. After filtration, from this solution 0.1 ml was taken out and diluted up to 10 ml phosphate buffer solution. The supernant was analyzed for drug content by measuring the absorbance at 244nm (23, 24).

Evaluation of in-vitro drug release study

The prepared mucoadhesive microspheres were evaluated for in vitro drug release. The drug release studies were carried out using USP I Basket type dissolution test apparatus. The dissolution study was carried out in 900 ml dissolution medium which was stirred at 100 rpm maintained at 37±0.5°C. A weighed quantity of formulation (equivalent to 10mg) was filled in capsule and kept in basket of dissolution apparatus with 900 ml of phosphate buffer (pH-7.4) at 37±0.5°C. Samples were withdrawn at different time interval and compensated with same amount of fresh dissolution medium. Volume of sample withdrawn was made up to 5ml by media. The samples withdrawn were assayed spectrophotometrically at 234 nm for percent of release from mucoadhesive microspheres using UV visible spectrophotometer. The release of mucoadhesive microsphere was calculated with the help of Standard curve of Glimepiride (25, 26).

 Statistical analysis

All determined data are expressed as mean ± standard deviation (Here n = 3).

RESULTS AND DISCUSSION

Preparation of mucoadhesive microspheres of glimepiride

In generally mucoadhesive was more reliable, economical, and easy to handling dosage system as compare to another system of those drugs having large extent absorbed in small intestine. Previously many similar works reported in this area, (12, 14, 27-30) Here this microspheres was designed by addition of polymer low molecular mass chitosan and ethyl cellulose with the help of emulsifying crosslinking method which achieving good adhesion to the intestinal wall and release the drug for a long periods of time due to suitable composition of polymer enhancer to developed the system. 

Particle size and zeta potential

The brief data of particle size and zeta potential is shown in Table 2. The particle sizes of all formulation range of 1.05 ± 0.34 to 1.43 ± 0.13 mm. The results projected the GM3 formulation having average particle size 1.05 ± 0.34 due to suitable composition of polymeric concentration help to easily mucoadhesive and sustain release the drug. The zeta potential of the drug-loaded blend microspheres was measured on a zetasizer (Malvern Instruments) by determining the electrophoretic mobility in a micro electrophoresis flow cell. All the samples were measured in water at 25°C in triplicate. Results of zeta potential of optimized formulation FM3 blend microspheres were found to be -35.45 mV which provide the greater stability.

Table 2: Particle size and zeta potential of glimepiride mucoadhesive microspheres

% of yield

It is important to increase amount of production of mucoadhesive microspheres of glimepiride. Therefore % of yield value was found to be satisfactory between 76.05 ± 0.34 to 82.03 ± 0.17 which demonstrates that microspheres were prepared by suitable composition of polymer and drug. although the value of % of yield in GM3 was high as compare to other. The brief data of % of yield value is shown in Table 2.

Table 3: % o yield of glimepiride mucoadhesive microspheres

Drug entrapment and loading efficiency

Here the formulation GM3 showed highest % EE i.e. 78.32 ± 1.98% due to suitable composition of two polymer. When the amount of polymers was increased or decreases in their ratio then this value was fluctuated. The value of drug entrapment efficiency and drug loading efficiency was shown within the value of 74.15 ± 1.18 to 78.32± 1.98 and 79.63 ± 0.42 to 87.03 ± 0.57 respectfully.

Table 4: Drug entrapment and loading uniformity of glimepiride mucoadhesive microspheres

In vitro drug release

In vitro release of glimepiride from mucoadhesive microspheres has been given on Figure 2. All the formulation performs good release profile prepared through suitable composite polymer low molecular mass chitosan and ethyl cellulose with their proper ratio by the addition of suitable crosslinking agent. These emulsifying crosslinking  method help to release the drug more than 12 hours because of sutable composition of polymer. Here all the formulation specially GM3 have better sustain release the drug because the quantity of low molecular mass chitosan and ethyl cellulose are equally 15 mg as compare to GB1.

Figure 2: In vitro release of drug (glimepiride) from these mucoadhesive microspheres