FORMULATION AND INVITRO EVALUATION OF 5-FLUOROURACIL MICROCAPSULES BY USING DIFFERENT METHODS OF MICROENCAPSULATION

ABOUT AUTHORS:
S.Shanmugam¹, J. Srikanth Reddy*¹, T. Vetrichelvan²
Adhiparasakthi College of Pharmacy,
Melmaruvathur, 603 319, Tamilnadu, India.
*srikanthjeedipelly@gmail.com

ABSTRCT:
The present study in formulation and evaluation of 5-fluorouracil microcapsules. 5-fluorouracil which is used as a anti cancer drug to treat cancer. The capsules were prepared by coacervation phase separation and emulsion solvent evaporation by using gelatin, sodium alginate and ethyl cellulose. The prepared microcapsules were evaluated with various evaluation methods such as drug content, in-vitro drug release studies, kinetic studies and stability studies as per ICH guidelines were performed. The formulated extended release microcapsules were prepared by powder layering technique. In these formulations containing 150mg of 5-fluorouracil was loaded in it. The particle size and dissolution study of  F9 formulation was concluded as the best formulation among other formulations, which showing the most desired drug release. It will be considered as optimized formulation. No significant change was observed in the drug content physical properties and dissolution rate of these micro pellets after the storage period of three months at 40±2ºc and 75±5%RH.

INTRODUCTION
Microcapsules are small particles that contain an active agent or core material surrounded by a coating or shell. (Commercial microcapsules typically have a diameter between 3 & 800 micrometer and 10-90% core).

Microspheres:
Microspheres are solid, spherical particles containing dispersed drug molecules, either in solution or crystalline form, among the polymer molecule.
Microcapsules have an either spherical geometry with a continuous core region surrounded by    a continuous shell or have an irregular geometry and contain a number of small droplets or particles of core.

Reasons for Encapsulation:
There are several reasons why substances may be encapsulated
1. To protect reactive substances from the environment
2. To convert liquid active components into a dry solid system
3. To separate incompatible components for functional reasons
4. To mask undesired properties of the active components
5. To protect the immediate environment of the microcapsules from the active components

1.2 METHOD OF MICROCAPSULE PREPARATION:

(1) Coacervation – phase separation        

(2) Interfacial polymerization      

(3) In-Situ polymerization                                    

(4) Solvent evaporation                

(5) Solvent extraction

(6) Spray drying

(7) Fluidized Bed Coating

(8) MultiorificeCentrifugal process

(9) Pan coating

1. Coacervation – Phase Separation^:  (Nitika Agnihotri, et al. 2012)
This process of microencapsulation is generally referred to The National Cash Register (NCR) Corporation and the patents of B.K. Green.

This process consists of three Steps-

• Formation of three immiscible phases; a liquid manufacturing phase, a core material phase and a coating material phase
• Deposition of the liquid polymer coating on the core material
• Rigidizing of the coating material

Step-1: The first step of coacervation phase separation involves the formation of three immiscible chemical phases: a liquid vehicle phase, a coating material phase and a core material phase. The three phases are formed by dispersing the core material in a solution of coating polymer, the vehicle phase is used as a solvent for polymer. The coating material phase consists of a polymer in a liquid phase, is formed by using one of the of phase separation- coacervation method, i.e. .by changing the temperature of the polymer solution, by adding a solution, or by inducing a polymer- polymer interaction.

Step-2: It involves the deposition of the liquid polymer coating upon the core material. This is done by controlled mixing of liquid coating material and the core material in the manufacturing vehicle. Step-3: In the last step rigidizing of the coating material done by the thermal, cross linkingdesolvationtechniques.

Fig 1.4: Coacervation process: (a) Core material dispersion in solution of shell polymer; (b) Separation of coacervate from solution; (c) Coating of core material by micro droplets of coacervate; (d) Coalescence of coacervate to form continuous shell around core particles.

4. Solvent-Evaporation Method^: (Hammad Umar, et al. 2011)
(Emulsification- Evaporation Method)
This technique is based on the evaporation of the internal phase of an emulsion by agitation. Initially, the coating polymeric material is dissolved in a volatile organic solvent. The core to be encapsulated is then dispersed in the coating polymer solution to form a suspension or emulsion.

In the next step, this organic solution is emulsified under agitation in dispersing phase, which is immiscible with the organic solvent, which contains the emulsifier. Once the emulsion is stabilized, agitation is maintained and the solvent evaporates after diffusing through the continuous phase. This results in the formation of microcapsule. On the completion of the process, the microcapsules held in suspension in the continuous phase are recovered by filtration or centrifugation and are washed and dried.

MATERIALS AND METHODS

MATERIALS
5-fluorouracil was received as a gift sample from Bindu Pharma Pvt.Ltd., Andhrapradesh. Gelatin, Sodium alginate and Ethyl cellulose were obtained from natco pharma Ltd., andrapradesh.

PREPARATION OF MEBEVERINE MICROCAPSULES
The microcapsules of 5-fluorouracil were prepared by coacervation phase separation by change in pH method and emulsion solvent evaporation. The formulation of sustained release microcapsules of 5-fluorouracil were done by using polymers gelatin, sodium alginate and ethyl cellulose. (Table 1)

EVALUATION OF MICROCAPSULES

APPEARANCE
The microcapsules were visually observed for physical appearance of microcapsules.

PARTICLE SIZE
Particle size distribution of microcapsules was determined by using phase contraction microscopy.

DRUG CONTENT
50 mg capsules were weighed and powdered and was transferred to a 100 ml volumetric flask and 15 ml pH 7.0 is added.  The drug is extracted in pH 7.0 by vigorously shaking the Stoppard flask for 2 hrs.  Then the volume is adjusted to the mark with distilled water and the liquid is filtered.  The drug content was determined by measuring the absorbance at 266 nm after appropriate dilution. The drug content was calculated using the standard calibration curve.  The mean percent drug content was calculated.

SCANNING ELECTRONMICROSCOPY
Morphological examination of the surface and internal structure of the dried beads was performed by using a scanning electron microscope (SEM). Microcapsules before dissolution only subjected to SEM study since, after dissolution the capsules become swollen palpable mass. Photographs were taken within the range of 50-500 magnification.

IN-VITRO DRUG RELEASE STUDIES
The release of drug from the developed formulation in the GIT was determined using USP dissolution type II apparatus. The drug release studies were carried out in pH 1.2 for 2 hrs, in pH 7.4 for next 10 hrs at 37± 0.50ºC and 100 rpm. At regular time interval, 5 ml of sample was withdrawn from the dissolution medium and replaced with equal volume of fresh medium. After filtration and appropriate dilution, the samples were analyzed at 266nm for 5-fluorouracil in HCl against blank using UV-Visible spectrophotometer. The amount of drug present in the samples was calculated using standard curve.⁸

RELEASE DRUG DATA MODEL FITTING
The suitability of several equation that are reported in the literature to identify the mechanisms for the release of drug was tested with respect to the release data up to the first 50% drug release. The data were evaluated according to the following equations: ⁶

Zero order model

Mt = M0+ K0t

Higuchi model11

Mt = M0 +KH t 0.5

Korsmeyer-Peppas model12

Mt = M0 + KKtn

Where Mt is the amount of drug dissolved in time t. M0 is the initial amount of the drug. K0 is the Zero order release constant, KH is the Higuchi rate constant, K is a release constant and n is the release exponent that characterizes the mechanism of drug release.

STABILITY STUDIES
Stability studies were carried out at 40°C/ 75% RH for the optimized formulation for 3 months. The microcapsules were stored at 40°C/75% RH as per ICH guidelines and various parameters (drug content and drug release profile) were monitored periodically for 3 months.⁹

RESULTS AND DICUSSION
The microcapsules were prepared by coacervation phase seperation by change in pH method and emulsion solvent evoperation by using different polymers shows significant results during their evaluation.

The appearance shows the pellets being spherical in shape and showing smooth surface of capsules. Results shown in (Table 2) (Figure 1).

The size of micro capsules found to be in the range of 4.31 µm to 515.74 µm and it was observed that increase in concentration of coating polymer particle size of the micro pellets significantly increased. The average particle size is highest for F9. Theparticle size distribution is uniform and narrow. Results shown in (Table 3).

Drug content was found to be uniform among different batches of microcapsules and ranged from 36.76 to 75.08 %. These results showed that the all formulations having percentage drug content within the specified limits as per IP. Results shown in (Table 3).

The scanning electron microscope shows the pellets being spheroid in shape. Surface depression was noted at the point of contact on the drying paper. (Figure 3)

The in vitro drug release data of all the formulations were fitted in zero order, first order and peppas model and the rate constant (k), correlation coefficient (R²) and n values were compared to know the mechanism of drug release from the microcapsules. Comparing the R² values of all formulations, it is evident that F1-F9 formulations following Higuchi release, The formulation F9 showing high cumulative % drug release after 12 hrs was found to be 94.63%, which contains ethyl cellulose. (Table 5 and 6)

The selected formulations F9 were subjected to stability studies as per ICH guidelines. There were no change in drug content and cumulative percentage drug release at 40°C/ 75% RH. All the parameters were within the limit after 90 days. Results shown in (Figure 4 and Figure 5).

Table 1: Composition of Microcapsules of 5-fluorouracil

Formulation	5-fluorouracil	Na.alginate (1%)	Gelatine (1%)	Ethylcellulose (1%)	Dil.HCl	Chloroform	Na cmc (1%)
F1	150mg	50ml	50ml	_	Q.s	_	_
F2	150mg	100ml	50ml	_	Q.s	_	_
F3	150mg	200ml	50ml	_	Q.s	_	_
F4	150mg	300ml	50ml	_	Q.s	_	_
F5	150mg	500ml	50ml	_	Q.s	_	_
F6	150mg	_	_	50ml	_	25ml	100ml
F7	150mg	_	_	100ml	_	25ml	100ml
F8	150mg	_	_	200ml	_	25ml	100ml
F9	150mg	_	_	300ml	_	25ml	100ml

Table 2: General appearance study of microcapsules

Parameters	F1-F5	F6-F9
Composition	Gelatin and Sodim alginate	Ethyl cellulose
Shape	Spherical	Spherical
Size by visualization	Large	Small
Colour	Creamish white	More white than control
Stickiness	None	None
Odour	No	No

Table 3: particle size for various formulations of microcapsules

Formulations Code	Particle size (µm ± S.D)	Drug Content* (%)
F1	205.97±0.41	53.35±0.94
F2	207.64±0.375	56.81±1.31
F3	168.98±0.452	63.61±1.71
F4	469.72±0.271	36.76±1.59
F5	515.74±0.376	38.09±1.57
F6	14.56±0.166	46.15±1.50
F7	10.99±0.336	45.01±1.36
F8	5.60±0.150	47.92±1.81
F9	4.31±0.240	51.08±1.25

All the values are expressed as a mean ±SD., n = 3

Table 4: In-vitro drug release data of Formulation F1-F9

S. No.	Medium	Time (hrs)	Cumu lative drug Release (%) F1	Cumu lative drug Release (%) F2	Cumu lative drug Release (%) F3	Cumu lative drug Release (%) F4	Cumu lative drug Release (%) F5	Cumu lative drug Release (%) F6	Cumu lative drug Release (%) F7	Cumu lative drug Release (%) F8	Cumu lative drug Release (%) F9
1	0.1N HCl	0	0	0	0	0	0	0	0	0	0
2		1	5.90	5.90	5.71	5.91	5.19	5.76	5.76	6.36	6.05
3	pH 7.4 phosphate buffer	2	12.8	12.36	12.03	12.53	11.31	12.57	12.57	14.42	13.06
4		3	16.83	15.97	15.49	15.98	14.85	17.92	17.92	18.01	17.7
5		4	23.29	22.9	21.83	22.61	21.25	25.41	25.41	24.79	25.2
6		5	26.82	26.35	25.74	26.94	25	30.02	30.02	28.69	29.82
7		6	57.22	56.48	60.9	55.76	51.68	61.89	61.89	60.66	61.52
8		7	77.35	77.53	78.06	76.23	74.31	80.48	80.48	80.42	83.26
9		8	80.18	81.13	85.57	78.53	78.35	82.65	82.65	83.62	85.24
10		10	81.58	82.32	88.62	81.95	79.35	84.25	84.25	85.94	88.89
11		12	82.79	81.79	94.3	85.1	80.37	86.69	86.61	87.93	94.63

Table 6: Different Kinetic models for Formulations F1-F9

Table 6: Different Kinetic models for Formulations F1-F9

Code	Zero order		First order		Higuchi		Peppas		Best fitting model
	R2	K0	R2	K1	R2	K	R2	n
F1	0.7199	0.0182	0.7203	0.0002	0.9710	0.0524	0.9554	0.3914	Higuchi
F2	0.7284	0.0182	0.7288	0.0002	0.9709	0.0524	0.9538	0.3950	Higuchi
F3	0.7815	0.7610	0.7540	0.0542	0.8934	0.0549	0.7133	0.2578	Higuchi
F4	0.8484	0.6789	0.8928	0.0549	0.9395	0.0764	0.9411	0.3533	Higuchi
F5	0.7248	0.0179	0.7252	0.0002	0.9716	0.0516	0.9558	0.3912	Higuchi
F6	0.7423	0.0849	0.8414	0.0088	0.9441	0.2727	0.9416	0.3975	Higuchi
F7	0.7336	0.0186	0.7340	0.0002	0.9744	0.0535	0.9621	0.4040	Higuchi
F8	0.7371	0.0186	0.7375	0.0002	0.9730	0.0535	0.9556	0.0643	Higuchi
F9	0.7650	0.0189	0.7653	0.0002	0.9765	0.0543	0.9593	0.3914	Higuchi

ACKNOWLEDGEMENT:
 We express our sincere thanks to our management, Adhiparasakthi College of Pharmacy, melmaruvathur, kanchipuram for their support to providing the facilities to complete the work. We also extend our thanks to Bindu pharmaceuticals Pvt. Ltd., Andhrapradesh, Richer Healthcare for providing us gift samples of drug and polymers.
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