INTRODUCTION:

Mono or multi nuclear materials embedded in spherical coating matrix are called microspheres.Microspheres are solid, approximately spherical particles ranging in size from 1µm to 1000µm. These are made of polymeric, waxy or other protective materials that are biodegradable synthetic polymers and modified natural products such as starches, gums, proteins fats, and waxes.

Microspheres are small and have large surface to volume ratios. At the lower end of their size range they have colloidal properties¹. The interfacial properties of microspheres are extremely important, often dictating their activity. In fact principle of microsphere manufacture depends on the creation of an interfacial area, involving polymeric materials that will form an interfacial boundary and a method of cross-linking to impart permanency. The method of manufacturing described later are by no means comprehensive and the reader should bear in mind that if the aforementioned criteria are adhered to, the only limitation to the manufacture of microspheres are the researchers imagination².

Preparation of microspheres needs mainly two ingredients. They are Core material – drug and Coating material – polymer. The important physiochemical characters that have to be considered before microspheres preparation are Particle size, Polymer and it’s molecular weight, Drug and polymer ratio, Total mass of drug and polymer³.

Ideal properties of microspheres: Longer duration of action, Control of content release, Increase of therapeutic efficiency, Protection of drug, Reduction of toxicity, Biocompatibility, Sterilizability, Relative stability, Water solubility and dispersibility, Bio resorbability, Targetability, Polyvalent^4,5.

Criteria for the preparation of Microspheres:

Ability to incorporate reasonably high concentration of drug, Stability of the preparation with a clinically acceptable shelf life, Controllable particle size, Dispersability in aqueous vehicle for injection, Release of active agent with good control over wide time scale, Biocompatibility with biodegradability, Susceptible to chemical modification^6-10.

Advantages:

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^11-14, Microspheres received much attention not only for prolonged release, but also for targeting of anticancer drugs to the tumor, 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 drugs to pathogens residing intracellularly^15-20.

Types of microspheres:

Albumin microspheres, Gelatin microspheres, Starch microspheres, Dextran microspheres, PLGA microspheres, Poly phosphazene microspheres, Poly anhydride microspheres, Chitosan microspheres, Carrageenan microspheres, Alginate microspheres, Poly (alkyl cyanoacrylate) microsphere, Poly acrolein microspheres^20-24.

Methods:

Different types of methods are employed for the preparation of microspheres^25-27. They include, Solvent evaporation, Single emulsion method, Double emulsion method, Polymerization technique (Conventional polymerization, Interfacial polymerization), Phase separation co-acervation technique, Spray drying and spray congealing, Solvent removal, Solvent extraction, Freeze drying, Chemical and thermal cross linking, Precipitation, Hot melt microencapsulation method^28-30.

MATERIALS AND METHODS:

Materials:

Candesartan provided by Provided by Chandra labs, hyderabad, Sodium alginate, carbopol 934, HPMC K4M, guar gum and calcium chloride is obtained from sree srinivas private limited, hyd.

Method of preparation:

Ionotropic gelation method:

Batches of microspheres were prepared by ionotropic gelation method which involved reaction between sodium alginate and polycationic ions like calcium to produce a hydrogel network of calcium alginate. Sodium alginate and the mucoadhesive polymer were dispersed in purified water (10 ml) to form a homogeneous polymer mixture. The API, Candesartan were added to the polymer premix and mixed thoroughly with a stirrer to form a viscous dispersion. The resulting dispersion was then added through a 22G needle into calcium chloride (4% w/v) aqueous solution. The addition was done with continuous stirring at 200rpm. The added droplets were retained in the calcium chloride solution for 30 minutes to complete the curing reaction and to produce rigid spherical microspheres. The microspheres were collected by decantation, and the product thus separated was washed repeatedly with purified water to remove excess calcium impurity deposited on the surface of microspheres and then air-dried and the formulation chart is mentioned in the table no1.

Table no1: Prepared formulation of Bioadhesive Microspheres

Formulation code	Drug: polymer	Sodium alginate: Carbopol
F1	1:1	3:1
F2	1:2	3:1
F3	1:3	3:1

Formulation code	Drug: polymer	Sodium alginate: Guar gum
F4	1:1	3:1
F5	1:2	3:1
F6	1:3	3:1

Formulation code	Drug: polymer	Sodium alginate: HPMC K4M
F7	1:1	3:1
F8	1:2	3:1
F9	1:3	3:1

RESULTS AND DISCUSSION:

Physical appearance:

Physical appearance of the drug was examined by organoleptic properties and results were obtained as follows:

· Color: White or almost white

· Odor: Odorless

· State: Fine powder

EVALUATION AND CHARACTERISATION OF MICROSPHERES

FTIR studies

Figure No: 1 FTIR Spectra of Candesartan and optimised formulation

The FTIR studies of pure drug and the optimised formulation were performed as shown in figure1. The result was found to be there is no interaction with the pure drug and other excipients and showns compatability with each other.

PERCENTAGE YIELD:

It was observed that as the polymer ratio in the formulation increases, the product yield also increases. The low percentage yield in some formulations may be due to blocking of needle and wastage of the drug- polymer solution, adhesion of polymer solution to the magnetic bead and microspheres lost during the washing process. The percentage yield was found to be in the range of 80 to 85% for microspheres containing sodium alginate along with carbopol 934 as copolymer, 78.22 to 88% for microspheres containing sodium alginate along with Guargum as copolymer and 80 to 87% for microspheres containing sodium alginate along with HPMC K4M as copolymer. The percentage yield of the prepared microspheres is recorded in Table 2 and displayed in Figures 2.

DRUG ENTRAPMENT EFFICIENCY:

Percentage Drug entrapment efficiency of Candesartan ranged from 72.66 to 86.66% for microspheres containing sodium alginate along with carbopol 934 as copolymer, 68.66 to 83% for microspheres containing sodium alginate along with Guargum as copolymer and 72.66 to 88.73 % for microspheres containing sodium alginate along with HPMC K4M as copolymer. The drug entrapment efficiency of the prepared microspheres increased progressively with an increase in proportion of the respective polymers. Increase in the polymer concentration increases the viscosity of the dispersed phase. The particle size increases exponentially with viscosity. The higher viscosity of the polymer solution at the highest polymer concentration would be expected to decrease the diffusion of the drug into the external phase which would result in higher entrapment efficiency. The % drug entrapment efficiency of the prepared microspheres is displayed in Table 2, and displayed in Figure 2.

Table No: 2 Percentage yield and percentage drug entrapment efficiency of the prepared microspheres

S.No.	Formulation code	% yield	% Drug entrapment efficiency
1	F1	80	77.66
2	F2	83.33	78.4
3	F3	85	86.66
4	F4	88	68.66
5	F5	78.22	75.2
6	F6	80	83
7	F7	80	72.86
8	F8	87	80.66
9	F9	80	88.73

Figure No :2 Graphical representation of percentage yield and drug entrapment efficiency of formulations F1-F9

PARTICLE SIZE ANALYSIS :

The mean size increased with increasing polymer concentration which is due to a significant increase in the viscosity, thus leading to an increased droplet size and finally a higher microspheres size. Microspheres containing sodium alginate along with carbopol 934 as copolymer had a size range of 512µm to 711µm, microspheres containing sodium alginate along with Guargum as copolymer exhibited a size range between 517µm to 792µm and microspheres containing sodium alginate along with HPMC K4M as copolymer had a size range of 664µm to 834µm. The particle size data is presented in Tables 3 and displayed in Figure 3. The effect of drug to polymer ratio on particle size is displayed in Figure 3. The particle size as well as % drug entrapment efficiency of the microspheres increased with increase in the polymer concentration.

SWELLING STUDY:

The swelling ratio is expressed as the percentage of water in the hydrogel at any instant during swelling. Swell ability is an important characteristic as it affects mucoadhesion as well as drug release profiles of polymeric drug delivery systems. Swell ability is an indicative parameter for rapid availability of drug solution for diffusion with greater flux. Swell ability data revealed that amount of polymer plays an important role in solvent transfer. It can be concluded from the data shown in Table no 3 that with an increase in polymer concentration, the percentage of swelling also increases. Thus we can say that amount of polymer directly affects the swelling ratio. As the polymer to drug ratio increased, the percentage of swelling increased from 35.21to 39.86. The percentage swelling of the prepared microspheres. The effect of drug to polymer ratio on percentage swelling is displayed in Figure 3.

Table No :3 Average Particle Size analysis and Percentage Swelling for formulation F1-F9

Formulation code	Average particle size(µm)	PERCENTAGE SWELLING
F1	512	36.32
F2	617	36.86
F3	711	39.86
F4	517	35.21
F5	642	36.02
F6	792	37.78
F7	834	35.75
F8	664	37.21
F9	774	38.54

Figure No :3 Graphical representation of average particle size and Percentage swelling index for formulations F1-F9.

IN-VITRO DRUG RELEASE STUDIES:

Dissolution studies of all the formulations were carried out using dissolution apparatus USP type I. The dissolution studies were conducted by using dissolution media,7.4 pH phosphate buffer.

The results of the in-vitro dissolution studies of formulations F₁ toF₉and shown in table no 4. Figure 4 shows the comparison of % CDR for formulations F₁ to F₃, figure 5 for formulations F₄ to F₆ and figure 6 for F7 to F_9.

The formulations F₁, F₂, F₃ containing Sodium alginate along with carbopol as copolymer showed a maximum release of 86.64% at 12 hours for F3 formulation. The formulations F₄, F₅, F₆ containing Sodium alginate along with Guargum showed a maximum release of 93.33% at 12 hours respectively for F6 formulation. The formulations F₇, F₈, F₉containing Sodium alginate along with HPMC K4M showed a maximum release of 98.55 % at 12 hours respectively for F9 formulation. The formulations F₉ Sodium alginate along with HPMC K4M showed a maximum release of 98.55 % at 12 hours. And it is the best formulation. This shows that more sustained release was observed with the increase in percentage of polymers. As the polymer to drug ratio increases the rate of drug release also increases. The release of drug has been controlled by swelling control release mechanism and due to larger particle size with restricted total surface area.

Table No :4 In-Vitro drug release data of all formulations Candesartan microspheres.

TIME (hr)	Cumulative Percent Of Drug Released
TIME (hr)	F1	F2	F3	F4	F5	F6	F7	F8	F9
0	0	0	0	0	0	0	0	0	0
1	21.11	18.66	15.88	27.77	22.44	18.44	25.77	21.55	18.66
2	31.55	25.11	24.22	36.44	32.22	29.33	35.33	31.77	26.55
3	39.77	35.44	32.60	37.22	40.88	39.55	39.50	40.44	36.55
4	47.77	40.66	39.33	40.81	48.60	45.55	43.55	48.44	43.66
5	56.66	52.00	44.55	43.85	53.11	49.01	49.45	53.01	54.55
6	58.89	57.33	50.77	46.01	57.55	53.44	53.61	57.11	62.33
7	61.90	60.00	59.77	50.91	60.77	57.33	54	60.33	67.68
8	64.00	63.11	65.35	54.77	63.55	65.33	63.55	63.11	73.55
9	67.44	69.01	71.55	59.66	66.62	77.55	75.33	70.22	78.55
10	70.55	73.56	76.55	64.01	70.44	85.56	78.45	76.00	83
11	75.33	75.33	80.46	75.77	76.55	88.55	80.01	83.11	90
12	82.26	83.66	86.64	76.90	82.55	93.33	83.00	86.33	98.55

Figure No: 4 of Comparison In-Vitro drug release profile of Candesartan microspheres containing sodium alginate along with Carbopol 934 as copolymer (F1-F3).

Figure No: 5 -Comparison of In-Vitro drug release profile of Candesartan microspheres containing sodium alginate along with Guargum as copolymer (F4-F6).

Figure No: 6 Comparison of In-Vitro drug release profile of Candesartan microspheres containing sodium alginate along with HPMC K4M as copolymer (F7-F9)

COMPARISION STUDIES:

Drug release profile of pure drug, optimized formulation and the marketed tablet was compared and the drug release of the marketed formulation was found to be 73.80% due to its hepatic first pass metabolism as shown in figure no 7. And the drug release of the optimized formulation was found to be 98.55%. The marketed tablets administration is in the form of oral route and its shows hepatic first pass metabolism. The marketed formulations also shows problem when the dose exceed 100mg daily so mostly the marketed oral formulation are modified and formulated in to the microspheres which shows advantages over oral marketed formulation.

Figure No:7 Comparison of In-Vitro drug release profile between pure drug, marketed drug (atacand 32mg) and best formulation (F9)

STABILITY STUDIES:

Stability studies are conducted for optimized formulation at 5⁰, 25⁰, 60% RH, 30⁰C/65% RH for a period of three months . Samples were withdrawn at 15 days time intervals and evaluated for physical appearance, drug content, drug release. Stability studies shown no changes in the product after 3 months, it is considered as a stable product as shown in table no5.

Table no 5: Stability studies

S.NO	OBSERVATIONS	BEFORE STABILITY TESTING	AFTER 3 MONTHS
1	Appearance	White or almost white	White or almost white
4	Percentage yield	80	80
3	In vitro studies	98.55%	98.00%

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Received on 09.12.2018 Modified on 29.12.2018

Res. J. Pharma. Dosage Forms and Tech.2019; 11(2):81-86.

DOI: 10.5958/0975-4377.2019.00013.2