INTRODUCTION:

The goal of any drug delivery system is to provide a therapeutic amount of drug to the proper site in the body promptly and then maintain the desired drug concentration in the body over an entire period of treatment. This is possible through administration of conventional dosage form in a particular dose and particular frequency to provide a prompt release of drug. Therefore to achieve and maintain the concentration within the therapeutically effective range needs repeated administration in a day. This results in a significant fluctuation in a plasma drug level leading to several undesirable toxic effects and poor patient compliance^1,2.

The development of a drug delivery system faces several challenges: reaching the target site, which is often, far away from the administration site (drug targeting), remaining at the target site to deliver the drug, preferably in a time controlled manner, limiting the drug’s adverse effects and ensuring biocompatibility.

Controlled drug delivery systems have acquired a centre stage in the area of pharmaceutical R and D sector. Such systems offer temporal and/or spatial control over the release of drug and grant a new lease of life to a drug molecule in terms of controlled drug delivery systems for obvious advantages of oral route of drug administration.

Recently, dosage forms that can precisely control the release rates and target drugs to a specific body site have made an enormous impact in the formulation and development of novel drug delivery systems. Microspheres form an important part of such novel drug delivery systems. The success of these microspheres is limited due to the short residence time at the site of absorption. It would therefore advantageous to have means for providing an intimate contact of the drug delivery system with the absorbing membranes. This can be achieved by coupling bioadhesion characteristics to microspheres and developing bioadhesive microspheres.^3,4

Stavudine (2',3'-didehydro-3'-deoxythymidine), is a nucleoside analogue of thymidine used in the treatment of HIV⁵. The usual dose of stavudine is 40 mg which is taken twice daily and has a shorter half-life of 0.8-1.5 hours. To overcome inherent drawbacks associated with conventional dosage forms of stavudine, an attempt is being made to develop an alternative drug delivery system in the form of mucoadhesive microspheres.

MATERIALS AND METHODS:

Materials:

Stavudine was obtained as gift sample from Hetero labs limited, Baddi and Carbopol 934, Chitosan and Sodium alginate were of analytical grade.

Preparation of Mucoadhesive Microspheres: Ionotropic Gellation Method^(6-9):

Accurately weighed amount of sodium alginate and mucoadhesive polymer were dissolved in purified water (10 ml) separately (Table-1). Then both the solutions were mixed to form homogeneous polymer solution. Then the drug was added to the polymer solution and mixed thoroughly with the help of mechanical stirrer to form viscous dispersion. The resulting dispersion was added drop wise into 10% w/v calcium chloride solution (100 ml) through a syringe with needle of 21 size. This should be done with continuous stirring at 500 rpm. The added droplets were retained in the calcium chloride solution for 15 minutes to produce spherical rigid microspheres. Finally the microspheres were collected by decantation and the product thus separated was washed repeatedly with water and dried at 45° C for 12 hours and stored in desiccators.

Table 1: Formulation Design of Mucoadhesive Microspheres

Formulation	Drug (mg)	Sodium Alginate (mg)	Carbopol 934 (mg)	Chitosan (mg)
F₁	60	375	125	-
F₂	60	750	250	-
F₃	60	1125	375	-
F₄	60	375	-	125
F₅	60	750	-	250
F₆	60	1125	-	375

Evaluation of Mucoadhesive Microspheres:¹⁰

1. Particle Size:^11,12

Determination of average particle size of mucoadhesive microspheres loaded with stavudine was carried out by using optical microscopy. A minute quantity of microspheres was spread on a clean glass slide and average size of 300 microspheres was determined in each batch.

2. Percentage Yield:^13-16

The measured weight was divided by total amount of all non-volatile components which were used for the preparation of microsphere. Percentage yield can be calculated using the formula

% Yield = [Total weight of excipient and drug / Actual weight of product] x 100

3. Encapsulation Efficiency and Drug Loading: ^{17, 18}

To determine the amount of drug encapsulated in mucoadhesive microspheres, a weighed amount (50 mg) of microspheres was suspended into 50 ml of ethanol and sonicated for 15 min in order to extract the entrapped drug completely. The solution was filtered through Whatman filter paper. 1 ml of this solution was withdrawn and diluted to 50 ml with pH 7.2 phosphate buffer solution. This solution was assayed for drug content by UV spectrophotometer at 266 nm. Calculating this concentration with the dilution factor we get the percentage drug content.

a. Encapsulation Efficiency was calculated as:¹⁹

EE (%) = [Actual Drug Content / Theoretical Drug Content] X 100

b. Drug Loading was calculated as:²⁰

DL (%) = [Actual Drug Content / Weight of Powdered icrospheres] X 100

4. Degree of Swelling: ^20,21

The swell ability of mucoadhesive microspheres in physiological media was determined by swelling them in the Phosphate Buffer Solution of pH 7.2. Accurately weighed 100 mg of microspheres were immersed in little excess of phosphate buffer of pH 7.2 for 12 hours and washed. The degree of swelling was calculated using following formula:

α = (W_s-W_o) / W_o

Where, α is the degree of swelling; W_o is the weight of microspheres before swelling; W_s is the weight of microspheres after swelling.

5. In-vitro Mucoadhesion Studies: ^22-24

A small portion of the sheep intestinal mucosa was mounted on a glass slide and accurately weighed microspheres were sprinkled on the mucosa. This glass slide was kept in desiccator for 15 min to allow the polymer to interact with the membrane and finally placed in the cell that was attached to the outer assembly at an angle of 45º. Phosphate buffer solution pH 7.2, previously warmed to 37 ± 5ºC was circulated all over the microspheres and membrane at the rate of 1 ml/min. Washings were collected at different time intervals and microspheres were collected by centrifugation followed by drying at 50ºC. The weight of washed out microspheres was determined and percentage mucoadhesion was calculated by following formula:

% Mucoadhesion = (W_a-W_l) X 100 / W_a

Where, W_a = weight of microspheres applied; W_l = weight of microspheres leached out.

6. Scanning Electron Microscopy:²⁵

Dry microspheres are kept in a brass stub coated with gold in an ion sputter. Then picture of microspheres were taken by random scanning of the stub. The SEM analysis of the mucoadhesive microspheres was carried out by using JEOL–6360A analytical scanning electron microscope.

7. In-vitro Dissolution Study:²⁶

Mucoadhesive microspheres equivalent to 100 mg of stavudine was loaded into the basket of the dissolution apparatus. Dissolution study carried out for 12 hrs in phosphate buffer of pH 7.2. 1 ml of the sample was withdrawn from the dissolution media at suitable time intervals and diluted to 10 ml using pH 7.2 phosphate buffer and the same amount was replaced with fresh buffer. The absorbance was measured at 266 nm by using Shimadzu 1700 UV spectrophotometer, against a blank solution.

8. Stability Study:^27-29

From the six batches of mucoadhesive microspheres, formulation F₃ and F₆were tested for stability studies. These two formulations were divided into 3 sample sets and stored at 4 ± 1^○C; 25± 2^○C and 60 ± 5% RH; 37± 2^○C and 65 ± 5% RH. After 30 days, the drug release of selected formulations was determined by the method discussed previously in vitro drug release.

RESULTS AND DISCUSSION:

In the current research, mucoadhesive microspheres loaded with stavudine were developed and evaluated.

IR Studies:

The physical mixture showed identical spectrum with respect to the spectrum of the pure drug, indicating there is no chemical interaction between the drug molecule and polymers used. (Fig 1, 2 and 3)

Fig 1: FTIR spectrum of pure stavudine

Fig 2: FTIR spectrum of physical mixture of Drug + Carbopol

Fig 3: FTIR spectrum of physical mixture of Drug + Chitosan

Particle Size:

With increase in polymer concentration, the mean particle size of the microspheres significantly increased and was range was between 806.19 μm to 842.37 μm. (Table 2)

Percentage Yield:

Percentage yield of the formulations were carried out and was found to be within the range of 89.46 to 95.56 % (Table 3).

Percent Encapsulation Efficiency and Percent Drug Loading:

Percent Encapsulation Efficiency and Percent Drug Loading of the formulations were found to be within the range of 89.46 to 95.56% and 18.21 to 21.28%. (Table 2 and Fig 4)

Degree of Swelling and Percent Mucoadhesion:

Degree of swelling and percentage mucoadhesion of the formulations were carried out and were found to be within the range between 1.16 to 1.48 and 90.83 to 98.51% (Table 2 and Fig 4).

Table 2: Particle Size, Percentage Yield, % Encapsulation, % Drug Loading, Degree of Swelling and % Mucoadhesion

Formulation	Particle Size (µm)	Percentage Yield	%Drug Loading	%Encapsulation Efficiency	Degree of Swelling	% Mucoadhesion
F₁	820.12	91.90	18.21	96.92	1.38	94.12
F₂	829.83	93.29	20.79	96.81	1.42	96.84
F₃	842.37	95.56	21.28	98.16	1.48	98.51
F₄	806.19	89.46	18.76	98.12	1.16	90.83
F₅	813.64	92.69	19.16	98.43	1.21	94.65
F₆	827.51	94.18	19.22	98.65	1.32	96.79

Scanning Electron Microscopy:

Scanning electron microscopy (SEM) confirms the outer surface of F₃ formulation was smooth and dense, while the internal surface was porous (Fig 5).

Fig 5: SEM Photograph of Mucoadhesive Microspheres (F₃)

In-vitro release studies^30-34

The In vitro release studies of mucoadhesive microspheres were carried out in phosphate buffer of pH 7.2 as a dissolution medium for a period of 12 hours. The release showed a biphasic release with an initial burst effect. At the end of first 30 minutes drug release was found to be 15.48%, 17.55%, 21.6%, 13.59%, 15.12% and 16.74% for F₁to F₆ respectively. The percentage drug release for F₁, F₂, F₃, F₄, F₅ and F₆were found to be 96.3%, 97.38%, 97.95%, 94.57%, 95.50% and 96.06% at the end of 12^th hour. (Table 3 and Fig 6)

Table 3: In vitro release of mucoadhesive microspheres in phosphate buffer

Formulation	% Drug release at 12^th hour
F₁	96.30
F₂	97.38
F₃	97.95
F₄	94.57
F₅	95.50
F₆	96.06

Stability Studies:^35,36

These studies revealed that, the formulations F₃ and F₆ maintained at 4±1°C showed 97.19% and 95.84% of drug release respectively. Formulations maintained at 25±2°C and 60±5% relative humidity (RH) showed 98.26% and 97.41% and formulations stored at 37±2°C 65± 5% RH showed 99.71% and 97.98% of drug release after 12 hours for F₃ and F₆ respectively. These results indicate that the drug release from the formulations maintained at 4±1°C was lowest followed by formulation maintained at 25±2°C; 60±5% RH and 37±2°C; 65±5% RH. (Table 4)

Table 4: Stability Studies – Percentage Drug Release

Formulation code	Percentage Drug Release
Formulation code	4°C± 1	25 ± 2°C and 60 ± 5% RH	37 ± 2°C and 65 ± 5% RH
F₃	97.19	98.26	99.71
F₆	95.84	97.41	97.98

On comparing this data with the previous release data of F₃ and F₆, it was observed that there was no much difference in the drug release of formulation maintained at 4±1°C. There was a slight increase in drug release for formulation maintained at 25±2°C and 60±5% RH and 37±2°C and 65±5% RH. These results may be attributed to erosion of polymer matrix to some extent during storage.

Fig 6: In-vitro dissolution profile of mucoadhesive microspheres of stavudine in pH 7.2 buffer

CONCLUSION:

By studying all the experimental results mucoahesive microspheres loaded with hydrophilic macromolecular bioadhesive polymers can be successfully formulated by Ionotropic Gellation method. All the formulations showed optimum results of which formulation containing carbopol showed the best results in all the evaluated parameters. Thus F₃with highest concentration of Carbopol can be concluded as the ideal batch of formulation.

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Received on 22.11.2013 Modified on 15.01.2014

Res. J. Pharm. Dosage Form. and Tech. 6(2):April- June 2014; Page 99-104