1. INTRODUCTION:

Microencapsulation is a technology to entrapping solids, or gases inside one or more polymeric coating.¹Microencapsulation helps to separate a core material from its environment until it is released. It protects the unstable core from its environment thereby improving its stability, extends the core’s shelf life and provides a release.^2-3

There are various approaches in delivering a therapeutic substance to the target site in a controlled release fashion. One such approach is using microspheres are characteristically free flowing powders consisting of proteins or synthetic polymers which are biodegradable in nature and ideally having a particle size less than 200µm.⁴

Microencapsulation is a well-known method that is used to modify and delay drug release from pharmaceutical dosage forms. A great number of Microencapsulation techniques are available for the formation of sustain release micro particulates drug delivery system. One of the popular methods for the encapsulation of drugs within water insoluble polymers is the emulsion solvent Evaporation method.

Ø Preparation of microspheres should satisfy following criteria.

· The ability to incorporate reasonably high concentration of the drug.

· Stability of the preparation after synthesis with a clinically acceptable shelf life.

· Controlled particle size and dispersability in aqueous vehicles for injection.

· Release of active reagents with a good control over a wide time scale.

· Biocompatibility with a controllable biodegradability and susceptibility to chemical modification.

Ø Advantages of Microspheres -

· Reliable means to deliver the drug to the target site with specificity, if modified, and to maintain the desired concentration at the site of interest without untoward effects.

· Solid biodegradable microspheres have the potential throughout the particle matrix for the controlled release of drug.

· Microspheres received much attention not only for prolonged release, but also for targeting of anticancer drug to the tumors.⁵

· The size, surface charge and surface hydrophilicity of microspheres have been found to be important in determining the fate of particles in vivo.

· Studies on the macrophage uptake of microspheres have demonstrated their potential in targeting drug to pathogens residing intracellularly.

· Blood flow determination. Relatively microspheres (10-15µm in diameter) are useful for regional blood flow studies in tissues and organs. This type of study has been carried out using radio labeled Microspheres; however fluorescent microspheres have been shown to be superior in chronic flow measurements.^6-10

2. Materials:

Zidovudine was obtained as a gift sample from Cadila pharma Ltd, Indore. Chitosan was obtained as a gift sample from Cadila Pharmaceutical Ltd Indore, Glutareldehyde solution-25% was purchased from Cadila Pharmaceutical Ltd Indore, Light liquid paraffin, Glutaraldehyde, Span 80, N-hexane and Acetone were procured from Central Drug House Pvt. Ltd. Mumbai. Methanol, Distilled water and other reagents were of analytical grade.

3. PREFORMULATION STUDY OF DRUG ZIDOVUDINE:

3.1 Preparation of Stock solutions

Standard Zidovudine 100 mg was weighed and dissolved in 50 mL of methanol in a 100 mL volumetric flask. The flask was shaken and volume was made up to the mark with methanol to give a solution containing 1000 μg / mL (stock solution I). From the stock solution I, 10mL was taken and placed into 100 mL volumetric flask. The volume was made up to mark with distilled water to give a stock solution containing 100 μg / mL (stock solution II).

3.2 Calibration curve for the Zidovudine (2 – 20 μg / ml)

Appropriate volume of aliquots from standard Zidovudine stock solution II were transferred to different volumetric flasks of 10 mL capacity. The volume was adjusted to the mark with distilled water to obtain concentrations of 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 μg / mL. Absorbance spectra of each solution against distilled water as blank were measured at 266 nm and the graphs of absorbance against concentration were plotted and shown in Figure 2. The regression equation and coefficient of determination was determined.

Figure 1 UV Spectra of Zidovudine at 266 nm

Table 1 Results of calibration curve at 266 nm for Zidovudine by UV spectroscopy

Sl. No.	Concentration (μg/ml)	Absorbance at 266 nm
1	2	0.099
2	4	0.190
3	6	0.278
4	8	0.374
5	10	0.465
6	12	0.553
7	14	0.641
8	16	0.714
9	18	0.791
10	20	0.886

Figure 2 Linearity plot or calibration curve for Zidovudine at 266 nm by UV Spectroscopy at 266 Zidovudine

4. Method of Preparation of chitosan Microspheres:

4.1 Plain microsphere (Without Drug) Chitosan microspheres were prepared by simple emulsification technique based on glutaraldehyde crosslinking as reported by Thanoo et al., 1992. Chitosan was used as a polymer and glutaraldehyde was used as cross-linking agent. 400mg of chitosan was dissolved in 10ml 0.1% w/v solution of acetic acid solution. Then take light and heavy liquid paraffin (1:1) in 250 ml beaker and stirred for 30 minutes by using a mechanical stirrer at 2000 RPM. Then add 10 drops of surfactant Span 80 was added and then the aqueous phase containing chitosan was added and stirred further for 30 minutes which resulted in formation of the O/W emulsion. Glutaraldehyde previously saturated with toluene was added drop wise to O/W emulsion and stirred at 2000 RPM for 4 hrs. The upper layer of the emulsion was discarded and the prepared microspheres where washed three time with acetone and N-hexane. The prepared microspheres where dried in air.

4.2 Drug loaded Microspheres: For the preparation of drug loaded microspheres same procedure as reported in section 4.1 was followed except that drug was added to liquid paraffin solution (Fig.3).

4.3 OPTIMIZATION OF DRUG LOADED MICROSPHERES

Preparation of chitosan microspheres involves various process variables out of which the following were selected for the optimization of formulation:

(A) Effect of varying polymer concentration (Chitosan).

(B) Effect of varying drug concentration (Methotrexate).

(D) Effect of varying cross linking agent concentration (Glutaraldehyde).

(E) Effect of varying stirring rate.

A) Effect of Polymer conc. on particle size and drug entrapment efficiency

Particle size of the chitosan microspheres varied from 177.62 to 190.67 mm. On increasing the concentration of chitosan from 200mg to 500 mg. The average particle size of microspheres increased with increasing amount of polymer solution, which can be attributed due to greater quantity of polymer available for formation of microspheres. The drug entrapment efficiency increases from 66.38 ± 0.62 % to 78.08 ± 0.51 % (Table 2, Figure 4 and 5), on increasing the concentration of chitosan from 200 mg to 400 mg which may be due to increase in viscosity of the solution which prevent the drug crystals from leaving the droplets. However on further increasing the concentration of polymer the entrapment efficiency was found to be decreased as highly viscous solution were prepared, which were difficult to process.

Table 2: Effect of varying polymer concentration on particle size and drug entrapment efficiency of chitosan microspheres

Formulation Code	Polymer Concentration (mg)	Particle Size (µm)*	*% Drug Entrapment Efficiency**
ZC₁	200	177.62±2.28	66.38 ±0.62
ZC₂	300	188.14±1.22	73.13±1.51
ZC₃	400	187.16±2.35	78.08±0.51
ZC₄	500	190.67±3.45	55.31±1.58

Fig 4.Effect of varying polymer concentration on Particle size of chitosan microspheres

Fig 5 Effect of varying polymer concentration on drug entrapment efficiency of chitosan microspheres

B) Effect of Drug conc. on particle size and drug entrapment efficiency

The effect of variation of drug content was studied with an increase in drug concentration the particle size of chitosan microspheres was found to increase from 184.14±2.19μm to 185.79±2.73 μm (Table 3 and Figure 6). However, on further increasing the drug concentration above 15 mg does not affect the particle size. Increase in particle size may be because of increase in viscosity of the droplets present in the internal phase caused by the increase in drug concentration.

The drug entrapment efficiency increased from 59.37±2.23 % to 74.98±0.76 (Table 3 and Figure 7) with increase in drug concentration may be due to greater free space available in the microspheres for accommodating the drug. However, on further increase in drug concentration particle size remained constant as no free space may be available for accommodating the free drug.

Table 3: Effect of varying drug concentration on particle size and drug entrapment efficiency of chitosan microspheres

Formulation Code	Drug Concentration (mg)	Particle Size (µm)*	*% Drug Entrapment Efficiency**
ZL₁	10	184.14±2.19	68.24±2.34
ZL₂	15	185.65 ±3.23	74.98±0.76
ZL₃	20	186.25±4.12	75.06±0.23
ZL₄	25	186.79±2.73	74.88±0.32

* Value represent mean ± SD (n=3)

Fig 6 Effect of varying drug concentration on particle size of chitosan microspheres

Fig 7 Effect of varying drug concentration on drug entrapment efficiency of chitosan microspheres

C) Effect of varying emulsifier concentration.

On increasing the emulsifier concentration from 0.5 to 1.25% the mean particle size of the microsphere span 80 increased to stabilization of the emulsion droplets avoiding their coalescence resulting in formation of small sized Microspheres. The drug loading efficiency varying emulsifier concentration from 0.5 to 1.25. (Table 4, Fig 8 and 9)

Table 4: Effect of varying emulsifier concentration on particle size and drug entrapment efficiency of chitosan microspheres

Formulation Code	Emulsifier Concentration w/v	Particle Size (µm)*	*%Drug Entrapment Efficiency**
ZS₁	0.50%	189.18±3.28	70.32±0.93
ZS₂	0.75%	175.76±1.39	72.41±0.52
ZS₃	1.0%	172.37±2.73	71.01±0.79
ZS₄	1.25%	174.00±4.32	73.92±1.12

* Value represent mean ± SD (n=3)

Fig 8 Effect of varying emulsifier concentration on particle size of chitosan microspheres

Fig 9 Effect of varying emulsifier concentration on drug entrapment efficiency of chitosan microspheres

D) Effect of varying Crosslinking agents concentration

Chitosan loaded microspheres were also characterized to evaluate the effect of the varying concentration of glutaraldehyde on mean particle size, size distribution. Particle size of the chitosan microspheres increased from 190.49±1.32 μm to 192.29±1.21 μm with increasing concentration of glutaraldehyde from 0.8 ml to 1.0 ml (Table 5 and Figure 10). The average particle size of microspheres increases with increasing concentration after which it remains constant. The increase in particle size with an increase in cross linking agent may be due to formation of chitosan microspheres. However, after further increase in cross linking agent concentration no change in particle size was observed as any free molecules of chitosan were available for cross linking. The drug entrapment efficiency was found to be increasing with an increase in concentration of cross linking agent from 65.57±1.52 to 74.76±0.55 (Table 5 and Figure 11) after which it remained constant. This may be attributed to formation of rigid surface of the microspheres with increasing cross linking agent concentration, which prevented the leakage of the drug from the microspheres surface.

Table 5: Effect of varying cross linking agent concentration on particle size and drug entrapment efficiency of chitosan microspheres

Formulation Code	Cross linking agent (ml)	Particle Size (µm)*	*% Drug Entrapment Efficiency**
ZA₁	0.8	190.49±1.32	65.57±1.52
ZA₂	1.0	192.29±1.21	74.76±0.55
ZA₃	1.2	191.48±0.76	74.76±1.22
ZA₄	1.4	192.01±0.83	73.23±0.78

* Value represent mean ± SD (n=3)

Fig 10 Effect of varying cross linking agent on particle size of chitosan microspheres

Fig 11 Effect of varying cross linking agent on drug entrapment efficiency of chitosan microspheres

(E) Effect of varying stirring rate.

The particle size of chitosan microspheres decreases with an increase in stirring speed (Table 6 and Figure 12) from 191.32±0.72 mm to 184.63±0.33 mm which may be due to the production of small sized droplets which undergo cross linking in presence of glutaraldehyde thus producing small microspheres. Results suggested that there was a stirring rate limit for a particular polymer concentration. Higher stirring rate did not result in further reduction in mean diameter significantly. The drug entrapment efficiency increases from 68.65±1.11 % to 76.64±0.27 %( Table 6 and Figure 13). The stirring speed of 2000RPM was found to be optimum as 74.34±0.44 of drug was loaded at this speed. High stirring speed produced irregular shape microspheres, but a slight increase in entrapment efficiency was observed (Table 6)

Table 6: Effect of varying stirring rate on particle size and drug entrapment efficiency of chitosan microspheres

Formulation Code	Stirring rate	Particle Size (µm)*	% Drug* Entrapment Efficiency
SR₁	1500	191.32±0.72	68.65±1.11
SR₂	2000	189.67±0.34	74.34±0.44
SR₃	2500	185.32±0.12	75.43±1.78
SR₄	3000	184.63±0.33	76.64±0.27

* Value represent mean ± SD (n=3)

Fig 12 Effect of varying stirring rate on particle size of chitosan microspheres

Fig 13 Effect of varying stirring rate on drug entrapment efficiency of chitosan microspheres

4.4 Scanning Electron Microscopy

SEM was used to investigate the morphology as well as particle size of microspheres. As showed in Photomicrograph. 14 (a) and (b), microspheres displayed a spherical shape with a smooth surface and no aggregation was observed. No difference was observed in the morphological properties of microspheres due to presence of the drug.

(a) uncoated chitosan Microspheres,

(b) Coated chitosan microspheres

Fig 14 SEM Photomicrographs of Microspheres

4.5 In Vitro Drug Release Studies

The drug release of chitosan microspheres was studied which revel that as the time of duration increases the release of drug content also increases i.e. 4% Cumulative Drug Release at the 1 hour and 72% Cumulative Drug Release after 8 hours. (Shown in Table 7 and Fig 15)

Fig 15 Cumulative % drug release from chitosan microsphere in simulated gastric fluid

Table 7: Cumulative % drug release from chitosan microspheres in simulated gastric fluid

S.No.	Time (hrs)	*% Cumulative Drug Release (PC₁)**
1	1	4.02±0.37
2	2	12.03±0.50
3	3	18.30±0.43
4	4	32.84±0.12
5	5	43.50±1.32
6	6	54.65±0.52
7	7	61.32±0.69
8	8	72.04±0.73

5 RESULTS:

In the present study a satisfactory attempt was made to develop microparticulate drug delivery system of zidovudine with improved bioavailability, efficient targeting and dose reduction. From the experimental results demonstrated that Chitosan polymer is a suitable macromolecule for the preparation of microspheres of ZidovudineParticle size analysis revealed that the microspheres were in the range (172 to 192µm) and all the formulations showed ideal surface methodology. Formulation SR4 showed maximum percent drug release. Present study shows that the targeting efficiency of drug loaded microspheres over free drug was, higher, which may provide increased therapeutic efficacy.

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Received on 23.10.2013 Accepted on 25.11.2013

Asian J. Res. Pharm. Sci. 2013; Vol. 3: Issue 4, Pg 200-205