INTRODUCTION:

A well planned controlled drug delivery system can defeat some of the harms of conventional therapy and enhance of a given drug. To obtain maximum therapeutic efficacy, it becomes essential to carry the agent to target tissue in the most favorable amount in the right period of time there by causing modest toxicity and minimal side effects. These are various approaches in delivering a beneficial substance to the target site in a sustained controlled release fashion.

The approach facilities the correct delivery of small quantity of the effective drugs, reduced drugs concentration at the site other than the target site and the defense of the liable compound before and after the administration and prior to appearance at the site of action. One such approach is using microspheres as carries of drugs^[1]. Microspheres are defined a “ monolithic sphere or remedial agent distributed throughout the matrix either as a molecular dispersion of particular dispersion of particles” they can also defined as a structure made up of continuous phase of one or more miscible polymers in which drug particles are dispersed at the molecular or macroscopic level^[2-3]. Microspheres are characteristically free flowing powders consisting of proteins or synthetic polymers having a particle size ranging from 1-1000 μm^[4]. Lamivudine (2′,3′-dideoxy-3′-thiacytidine) is an antiretroviral medicine used to avoid and treat HIV/AIDS and used to treat perpetual hepatitis B. It is of the nucleoside simple opposite transcriptase inhibitor (NRTI) class^[5]. It can hinder both sorts (1 and 2) of HIV reverse transcriptase furthermore the converse transcriptase of hepatitis B infection. It is phosphorylated to dynamic metabolites that go after fuse into viral DNA. They hinder the HIV reverse transcriptase protein aggressively and go about as a chain eliminator of DNA blend^[6]. The absence of a 3'- OH bunch in the joined nucleoside simple keeps the arrangement of the 5' to 3' phosphodiester linkage crucial for DNA chain stretching, and in this manner, the viral DNA development is ended^[7]. Fundamental purpose behind determination of this medication is low biological half-life, less protein binding, reduce the harmful impacts, diminish the measurements and expansion the patient consistence.

MATERIALS AND METHODS:

Materials:

Lamivudine was a gift sample from Karnataka Anti-biotics ,Bangalore. HPMC, Di‐sodium hydrogen phosphate, potassium dihydrogen phosphate, diethyl ether, tween‐60, span‐60 were purchased from A.R. Loba Chemical Pvt. Ltd, Mumbai. Sunflower oil purchased from Keerthi Agencies Koti, Hyderabad. All other chemicals used were of L.R. grade.

Methods:

Table 1. Formulation design forLamivudine Microspheres

S. No	Ingredients	LM 1	LM 2
1	Lamuvidine	300 mg	300 mg
2	HPMC K4M	300 mg	600 mg
3	Egg albumin	5 ml	5 ml
4	Sunflower oil	100 ml	100 ml
5	Diethyl ether	5 ml	5 ml
6	Surfactants	0.1-1%	0.1-1%

Preparation of microspheres of Lamivudine by emulsification heat stabilizing method:

300 mg Lamivudine of and polymer (HPMC) were disintegrated in 20 ml of deionized water and included 5 ml of egg albumin, 0.1% of Tween‐80, blending it for 30 min. The readied arrangement was utilized as aqueous phase. The oil stage was set up by blending 20 ml of sunflower oil and 5ml of diethyl ether with 1% span‐80 (as emulsifier) and mixed it for 20 mins at 800‐1000 rpm on an magnetic stirrer^[8]. The essential emulsion was set up by including the oil stage drop shrewd to the watery stage mixed it for 30 mins at 800‐1000 rpm. The readied essential emulsion was added to pre‐heated (65 to 70oC) sunflower oil (80 ml) by utilizing 21 No. needle and mixed it 1000‐1200 rpm for 2 hrs till the hardening of microspheres shaped. The suspension was then permitted to cool to room temperature with persistent blending utilizing an magnetic stirrer. On cooling, 100 ml of anhydrous ether was included. The suspension containing the micro‐spheres was centrifuged for 15 min and the settled microspheres were washed three times with ether to evacuate hints of oil on microspheres surfaces^[9]. The acquired microspheres were then vacuum dried in a desiccator overnight and put away at 4o c in dark.

CHARACTERIZATION:

Fourier Transform Infra-Red spectroscopy analysis:

IR spectral analysis of pure drug, and polymers was carried out. The samples were crushed with KBr to get pellets by applying pressure on 600 Kg/cm² and scanned in (Shimadzu, 8400 Series, Tokyo, Japan) from 400 -4000cm^-1[10].

Determination of % yield of microspheres:^[^11]

The dried microspheres were collected and weighed accurately. The percentage yield was then calculated using formula given below.

	Mass of micro‐spheres obtained
% Yield =		X 100

	Total weight of drug and polymer

Determination of drug content:^[^12]

Lamuvudine content in the micro‐spheres was evaluated by an UV spectrophotometer strategy in light of the estimation of absorbance at 254 nm in phosphate buffer of pH 7.4. The technique was approved for linearity, exactness and accuracy. The technique complied with Beer's law in the focus scope of 5‐50 µg/ml.

Weight of drug in microspheres

% Loading = ----------------------------------------- x 100

Weight of microspheres

Zeta potential Analysis:

The zetapotential was measured utilizing the (Beckman Coulter Delsa Nano C, Brea, USA. Instrument). The specimen was weakened with distilled water and taken in the cuvettes and temperature kept up at 25°^C[13].

Scanning Electron Microscopy:

Scanning electron microscopy (JEOL 5400, Tokyo, Japan) was utilized to decide shape, surface geography and composition and look at the morphology of broke or separated surface. Sample spreads on the little square plate and covered with a gold particle for 5-6 mins^[14]. The readied test was kept inside the chamber and pictures caught with various amplifications.

Invitro release studies Shaking flask method:^[^15,16]

Drug loaded microspheres equal to 100 mg of drug were weighed and transferred into a 100 ml conical flask. To this 100ml of pH 7.4 phosphate buffer saline was added, then the flasks were kept in a metabolic shaker and the shaker was adjusted to 50 horizontal shakes per minutes at 37 ± 0.5^oC. One ml of the drug releasing media was withdrawn at various time interval of 30 min, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 hours and replaced by the same volume of phosphate buffer saline. These samples were filtered through 0.45 μm membrane filter. The filtrate was diluted suitably. The drug was estimate in each batch by UV‐Visible Spectrophotometer at 254 nm.

RESULTS AND DISCUSSION:

In the present study an attempt was made to formulate Lamivudine as micro particulate drug delivery system in order to localize drug at the absorption site, enhance its bioavailability, reduce dose, thereby improving patient compliance. Micro particulate system of Lamivudine was formulated using HPMC as carrier by emulsification heat stabilizing method. Prior to formulation, Preformulation studies were carried out in order to establish compatibility between drug and polymer by IR spectroscopy(Figure1). The formulations (LM1 and LM2) were prepared by varying the ratio of drug and polymer. Preformulation studies revealed that the drug Lamivudine and HPMC were satisfactorily compatible, without any significant changes in the chemical nature of the drug. These formulations were subjected to various evaluation parameters like % practical yield, drug entrapment efficiency, particle size distribution, invitro release studies. The results of all parameters are tabulated (Table 2 and Table 3) and depicted graphically.

Percentage practical yield was found to be maximum in formulation LM2. Particle size of the drug loaded microspheres revealed that the particles were in micron range. Drug entrapment efficiency was found to be maximum in LM2. It was observed that drug entrapment efficiency increased with increase in concentration of drug added in consecutive formulations. Microspheres were examined for various evaluation parameters.

The drug entrapment for LM 1 showed in the range of 68.52±0.75and in LM 2 79.84±0.26 and % practical yield was 73..84±1.54 for LM 1 and 85.24±1.37 for LM 2. Invitro release study was analyzed using various mathematical models % drug releases with respect to time were found to be highest for formulation LM1 and lowest for formulation LM2. The drug release of prepared Microspheres with different combination of polymers HPMC and Egg albumin of different ratios showed slower release when there was an increase in the concentration. It was also observed that drug entrapment and drug loading also affect the drug release from microspheres. Formulation LM 1 showed 84.20±0.16 release of the drug for a period of 10 h faster release when compared to other formulation LM 2 showed drug release of 79.37±0.20% for a period of 10 h (Figure.2).

Table 3: In vitro cumulative drug release data ofLamivudine loaded microspheres

S. no	Time in hrs	Cumulative % drug release (LM 1)	Cumulative % drug release (LM 2)
1	0.5	7.85±0.24	6.25±0.87
2	1	14.61±0.58	12.46±0.95
3	2	21.14±0.67	19.32±0.14
4	3	29.25±0.31	26.95±0.76
5	4	37.47±0.47	35.74±0.68
6	5	42.58±0.98	40.82±0.35
7	6	51.35±0.72	49.21±0.59
8	7	60.84±0.83	58.78±0.41
9	8	69.92±0.38	67.91±0.72
10	9	75.39±0.74	72.68±0.94
11	10	84.20±0.16	79.37±0.20

Figure 2. In vitro cumulative drug release data of Lamivudine loaded Microspheres

Scanning electron microscopic (SEM) photographs showed microspheres of spherical, discrete nature, and distinct size and a nearly smooth surface (Figure 3). Zeta potential is used to conclude the electrophoretic mobility of particles. The scale of the zeta potential gives a signal of the possible stability of the colloidal system. It was determined for the optimized formulation LM 2 and was found negative with 18.40±1.2 mV(Figure 4).

Figure 3.SEM of (LM 2) 750x & 500x

Figure 4. Zetapotential of LM 2 microspheres

CONCLUSION:

In the present study a satisfactory attempt was made to develop micro particulate drug delivery system of the Lamivudine with improved bioavailability. From the experiment results it can be concluded that HPMC polymer is a suitable for the preparation of microspheres of Lamivudine. Particle size analysis reveals that the microspheres were in the range and all the formulation showed surface characters. LM 2 formulation shows sustained release and may be reduce the adverse effect of Lamivudine. Therefore increasing the patient compliance.

ACKNOWLEDGEMENTS:

The authors are grateful to the Jyothismathi college of pharmacy, Thurkapally (v), Sharmeerpet (M), R.R-Dist. For providing research facilities.

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Received on 16.10.2016 Accepted on 29.11.2016

Asian J. Res. Pharm. Sci. 2016; 6(4): 235-239.

DOI: 10.5958/2231-5659.2016.00033.3

Formulation code	Drug:polymer	% practical yield	% Encapsulation Efficiency
LM 1	1:1	73..84±1.54	68.52±0.75
LM 2	1:2	85.24±1.37	79.84±0.26

ABSTRACT: