INTRODUCTION:

Microencapsulation has been used in the pharmaceutical industry for the conversion of liquids to solids, taste masking of bitter drugs, acquiring prolonged or sustained release, reducing gastric irritation and environmental protection of labile moieties¹. Microcapsules having core material and coating material. Core material is the drug substance which is to be coated by a coating material generally polymers are used. An important class of polymer mediated drug delivery systems that are applied for controlled drug delivery is the microcapsules^2,3. Microcapsules continue to be of much interest in controlled release based on relative ease of design and formulation and partly on the advantages of microparticulate system. Ethyl cellulose is a non biodegradable and biocompatible polymer used as encapsulating materials for the controlled release of pharmaceuticals.

Nicardipine Hydrochloride used as a calcium channel blocker⁴. Due to its considerable first pass metabolism and a high clearance, thus necessitating frequent administration of large doses.

Microencapsulation provides the prolonged release of drug in a single dose. So to reduce the dose frequency, the purpose of the present work was to prepare and evaluate microcapsules of Nicardipine Hydrochloride by Coacervation phase separation induced by temperature change method.

MATERIALS AND METHODS:

Nicardipine hydrochloride was a gift sample from Cipla Ltd, Goa. Cyclohexane and Ethyl cellulose were purchased from Merck Pvt. Ltd, Mumbai. All other reagents used were of analytical grade.

Methods for preparation of microcapsules:

Microcapsules were prepared by Coacervation phase separation induced by temperature change method. Thermal induced Coacervation technique developed by Miller et al. Ethyl cellulose was dispersed in 50 ml of Cyclohexane. The mixture was heated to boiling to form homogeneous polymer solution. Core material (1.6 g) was then dispersed in the solution with stirring. The mixture was allowed to cool with continuous stirring to effect phase separation of ethyl cellulose and encapsulation of the core material. The mixture was allowed to cool to room temperature and filtered by membrane filter. The encapsulated product was then air dried to obtain discrete microcapsules. Now repeat the same experiment with same amount of solvent and core material but the amount of coating material was different i.e. 400mg, 800mg, and 1200mg. The rotation speed of mechanical stirrer is also a variable i.e. 200 rpm, 400 rpm and 600 rpm. The results are shown in Table 01.

Table 01: Formulation of Nicardipine hydrochloride microcapsules

Phase separation induced by temperature change
Batch code	Rotation (RPM)	Core:coat ratio
FI	200	4 :1
F2	200	4 :2
F3	200	4 :3
F4	400	4 :1
F5	400	4 :2
F6	400	4 :3
F7	600	4 :1
F8	600	4:2
F9	600	4 :3

Drug polymer compatibility study:⁵

Compatibilities among the drug-polymers can be confirmed by carrying out infrared light absorption scanning spectroscopy (IR) studies. Infra red spectra of pure drug and mixture of formulations were recorded by dispersion of drug and mixture of formulations in suitable solvent (KBr) using Fourier Transform Infrared Spectrophotometer (FTIR). A base line correction was made using dried potassium bromide and then the spectra of the dried mixture of drug, formulation mixture and potassium bromide and then the spectra of the dried mixture of drug, formulation mixture and potassium bromide were recorded .The FTIR studies were conducted at National Institute of Technology (NIT), Rourkela, Odisha.

Solubility determination:⁶

The solubility was studies in various aqueous and non aqueous solvents.

A cleaned and dried graduated test tube of 10 ml was taken and 10 ml of 0.1 N HCL was poured into it, then unknown quantity of Nicardipine was added to it and dissolved properly by shaking. The shaking was continued until the drug went into solution that means until a clear solution was obtained. If the drug was undisclosed in the solution even after shaking with hand then the test tube containing the drug with solvent was subjected for shaking in a mechanical shaker for 12 hrs. The above solution was then filtered, dilutions were made and absorbance was noted in UV Spectrophotometer at 238nm. Likewise, solubility was determined in phosphate buffer of pH 6.8, and 7.2.

Evaluation of microcapsules:

Particle size measurement study:⁷

Particle size analysis was done by sieving method using Indian standard sieves ≠ 10, 12, 16, 20, 22, 40 and 44. Average particle size was calculated using the formula

d_avg =∑dn/∑n

Where n is frequency weight and d is the mean diameter

Rheology properties:⁸

Angle of repose, Carr’s index, Bulk density and Hausner’s ratio were determined to assess the flow ability of the prepared microcapsules.

Drug content estimation:⁹

Drug content of the different coat:core ratio of microcapsules were estimated. From each batch of the microcapsules four sample of 100 mg each were taken and analyzed for the drug content. 100 mg of microcapsule were weighed and transfer into mortar and pastel and triturate properly. Remove this powder into 100 ml volumetric flask with the help of 5 ml of methanol and the volume was made up to the mark with 0.1 N HCL, kept for 24 hours and filtered. The drug content was analyzed using UV- Visible double beam spectrophotometer at 238 nm. Percentage of drug content was calculated by the formula

Percentage drug content = weight of pure drug present in microcapsules / weight of microcapsules x 100

Drug Entrapment Study:¹⁰

The Drug Entrapment Efficiency (DEE) was calculated by the equation

EE = (Pc / Tc) X 100

Pc : Practical content.

Tc : Theoretical content.

Loose surface crystals study:¹¹

Microcapsules of Nicardipine were evaluated by loose surface crystal study to observe the excess drug present on the surface of microcapsules. From each batch, 100mg equivalent of microcapsules was shaken in 20 ml of double distilled water for 5 minute and then filtered through Whattman filter paper 41. The amount of drug lost in filtrate was determined spectroscopically and percentage of total drugwas calculated.

In-vitro drug release study:¹²

Dissolution studies were conducted for microcapsules of Nicardipine by using USP paddle type apparatus at 37 ± 0.5ºC at 50 rpm. 900 ml of 0.1 N HCl was used as dissolution medium. At various time intervals, 5 ml of sample was withdrawn from a fixed position of the vessel and replaced with fresh dissolution medium. The absorbance of filtered sample was analyzed by using UV-Visible double beam spectrophotometer at 238 nm. The percentage drug released at various time intervals was calculated.

In- vitro drug release kinetic study:¹³

In order to study the exact mechanism of drug release from Nicardipine microcapsules, drug release data was analyzed according to Zero order, First order, Higuchi Equationand Korsemeyer-Peppas model.

Scanning Electron Microscopy (SEM):¹⁴

Scanning Electron Microscopy (Stereo scan S250 MK III, Cambridge, UK) was carried out to study the morphological characteristics of Nicardipine hydrochloride microcapsules. The dried microcapsules were coated with gold (100 A^°) under an argon atmosphere in a gold coating unit and Scanning electron micrographs of both higher and lower resolutions were observed. The scanning electron microscopy was held at Birbal Sahini Institute of Palaeobotany, Lucknow (U.P.)

RESULTS AND DISCUSSION:

Drug-polymer compatibility study:

The FT-IR (Shimadzu IR Spectrophotometer, model 840, Japan) was used for these IR analyses in the frequency range between 4000 and 600 cm^-1and at 1 cm^-1 resolution. The results indicate that there was no drug interaction and compexation occur during the manufacturing process. The results are shown in Figs 01 to 03.

Figure 01: FTIR study of Nicardipine Hydrochloride

Figure 02. FTIR study of Ethyl cellulose

Figure 03: FTIR study of drug and polymer

Solubility determination:

Solubility of Nicardipine Hydrochloride was varied in different pH environment. Nicardipine hydrochloride is more soluble in acidic pH and it was found to be 15.357 mg/ml .The results are shown in Table 02.

Table 02: Solubility values of Nicardipine in different pH ranges

pH	Solubility(mg/ml)
1.2	15.357
6.8	5.252
7.2	4.269

Particle size measurement of microcapsules:

The average particle size was found to be in the range of 222 µm to 389 µm. The average particle size of microcapsule increases as the concentration of the polymer increases whereas particle size decreases by increases of rotation speed of stirrer. Particles sizes of different formulations are mentioned in Table 03.

Table 03: Particle size of microcapsules

Batch code	Particle size (micrometer)
F1	312
F2	345
F3	389
F4	290
F5	298
F6	305
F7	222
F8	234
F9	248

Rheological determination of microcapsules:

The rheological properties of the microcapsules are presented in Table 04. Particle size of the microcapsules increases as the drug: polymer ratio decreases. Low value of angle of repose indicate that the microcapsules have better flow properties and the better flow properties indicated that non aggregated of microcapsules.

Table 04: Rheological properties of microcapsules

Batch code	Carr’s index	Hausner‘s ratio	Angle of repose (in degree)
F1	9.34	1.03	25.1
F2	9.17	1.04	28.7
F3	11.67	1.02	24.9
F4	4.84	1.09	23.1
F5	9.34	1.26	22.5
F6	9.18	1.04	21.7
F7	10.22	1.01	27.7
F8	9.78	1.06	22.8
F9	11.80	1.32	29.8

Drug content of prepared microcapsules:

The minimum percentage of drug content was found to be 54.3% in formulation F9 in the drug : polymer ratio (4:3). Similarly maximum percentage of drug content was found to be 85.4 % in formulation F1 in the drug: polymer ratio (4:1).It seems that value of percentage of drug content decreases with the increase of coat:core ratio probably due to its thick layer of coat formed by increase of coat: core ratio. Percentage drug contents of different formulations of microcapsules are mentioned in Table 05.

Table 05: Percentage drug content of microcapsules

Batch code	Drug: polymer	% drug content
F1	4 : 1	85.4
F2	4 : 2	67.9
F3	4 : 3	61.1
F4	4 : 1	79.8
F5	4 : 2	66.5
F6	4 : 3	58.4
F7	4 : 1	82.7
F8	4 : 2	60.0
F9	4 : 3	54.3

Entrapment Efficiency:

Entrapment Efficiency was found to be in the range of 82 % to 106 %. Entrapment Efficiency decreases with increase of rotation speed of stirrer, probably it is due to smaller microcapsules formed by increase of rotation speed of stirrer. The results are shown in Table 06.

Table 06: Entrapment efficiency of the microcapsules

Batch code	Drug : polymer ratio	Theoretical Drug content (%)	Estimated drug Content (%)	Entrapment efficiency (%)
F1	4 : 1	80.00	80.8	101
F2	4 : 2	66.60	67.9	102
F3	4 : 3	57.14	61.1	106
F4	4 : 1	80.00	79.8	99
F5	4 : 2	66.66	66.5	99
F6	4 : 3	57.14	58.2	102
F7	4 :1	80.00	65.7	82
F8	4 : 2	66.66	60.0	90
F9	4 : 3	57.14	54.3	95

Loose surface crystal studies:

Loose surface crystal studies were observed to estimate the excess amount of drug attached on the surface of microcapsules after a successful drug entrapment. Percent of loose drug on surface were found to be in the range of 4.54% to 8.76%.The results obtained are mentioned in the Table 07.

Table 07: Loose surface crystal study of microcapsules

Batch code	Percentage drug on surface
F1	4. 4
F2	5.65
F3	6.54
F4	6.65
F5	7.43
F6	5.76
F7	4.87
F8	8.76
F9	6.87

In-vitro drug release study:

The in-vitro drug release study of the microcapsules was performed in 0.1N HCL. All the formulations were found to release Nicardipine hydrochloride in a controlled manner for prolonged periods over 12 hours. The cumulative percent of drug release from the formulations F1 to F9 were found to be 6.9% to 18.4% drug release in first hour and 55.9 % to 98.8 % drug release at the end of 12 hrs. Among the nine formulations, Formulation F3 shows minimum drug release i.e.55.9%, whereas formulation F7 shows maximum drug release i.e. 98.8%. at the end of 12 hrs. It seems that drug release from formulations decreases with increase in polymer : drug ratio and increases with increase in rotation speed of stirrer. The results are mentioned in Fig. 04.

Figure 04: Comparative cumulative drug release profile of formulations F1 to F9

In- vitro drug release kinetic study.

The in-vitro drug release data of all the formulations were fit into Zero order, First order, Higuchi Equationand Korsemeyer-Peppas model. The results are shown in Table 08.

Among the zero order and first order equations, the Zero order Regression co-efficient (R²) value was found to be more than the First order. So all the formulations F1 to F9 followed Zero order drug release. Similarly, Regression co-efficient (R²) value between Higuchi Equationand Korsemeyer-Peppas model, the Korsemeyer-Peppas model (R²) value was found be more and also ‘n’ values are greater than 0.5 i.e. all the formulations followed Non-Fickian diffusion mechanism. Hence all the formulations followed the Zero order drug release with Non-Fickian diffusion mechanism.

Scanning Electron Microscopy (SEM)

The microcapsules prepared were found to be spherical to near spherical and without aggregation, (as revealed in SEM studies), discrete and free flowing. SEM of microcapsules are shown in Fig;05. The scanning electron microscopy was held at Birbal Sahini Institute of Palaeobotany, Lucknow (U.P.)

Figure 05: Microcapsules prepared by Coacervation phase separation method

Batch code	Zero order R²	First order R²	Higuchi R²	Korsemeyer- Peppas R²	Diffusion exponent(n)
F1	0.982	0.898	0.911	0.988	0.716
F2	0.976	0.812	0.963	0.995	0.754
F3	0.988	0.821	0.941	0.991	0.652
F4	0.997	0.791	0.885	0.992	0.784
F5	0.981	0.858	0.927	0.989	0.567
F6	0.99.1	0.875	0.946	0.987	0.765
F7	0.984	0.894	0.934	0.997	0.795
F8	0.983	0.868	0.972	0.989	0.843
F9	0.991	0.843	0.891	0.988	0.876