Microspheres for Targeting an Alkaloidal Anticancer Drug in Colon Cancer
Dheeraj Ahirwar1, Bharti Ahirwar2, Anish Chandy1
1School of Pharmacy, Chouksey Engg. College, Bilaspur, CG
2Department of Pharmacy, Gurughasidas Vishvavidyalaya, Bilaspur, CG
*Corresponding Author E-mail: email@example.com
The aim of the present study is to develop a multiparticulate system containing pectin microspheres for the colon targeted delivery of Vincristine Sulphate for the treatment of irritable bowel syndrome. This work combines pH-dependent solubility of shellac polymers and microbial degradability of pectin polymers. The yield of preparation and the encapsulation efficiencies were high for all pectin microspheres. Shellac coating of pectin microspheres was performed by oil-in-oil solvent evaporation method using coat: core ratio (5:1). Microspheres were evaluated for surface morphology particle size and size distribution, percentage drug entrapment, and in vitro drug release in simulated gastrointestinal fluids (SGF). The release profile of Vincristine Sulphate from shellac-coated pectin microspheres was pH dependent. In acidic medium, the release rate was much slower; however the drug was released quickly at pH 7.4. It is concluded from the present investigation that Shellac-coated pectin microspheres are promising controlled release carriers for colon-targeted delivery of Vincristine Sulphate.
Colon Cancer is the 2nd most lethal cancer causing mortality in America. It occurs at the lower part of the colon and spread to rectal region 1,2. It is a metastatic cancer and absence of proper treatment can cause its spread to other organs. Vincristine shows, promising result in different types of cancers such as Colon, Hodgkin, Lymphoma, Blastoma and willims tumour 3. It is white to slightly yellow, amorphous, light sensitive, odorless powder, having pH (0.1% solution) 3.5 - 4.5. It is poorly absorbed orally. The clearance of the drug after intravenous injection follows a triphasic decay pattern: a very rapid steep descent (alpha phase); a narrow-middle region (beta-phase) and a much longer region (gamma phase). The terminal phase half-life of the drug varies from 15-155 hours3. Both in vivo and in vitro laboratory tests have failed to demonstrate conclusively that this product is mutagenic. No evidence of carcinogenicity was found following intraperitoneal administration in rats and mice, although those study were limited. 4
Vincristine blocks mutosis by arresting cell in metaphase and may also interfere with amino acid metabolism. It is cell cycle specific for the M-phase of the cell division.4The drug is having good solubility in water and other solvents which is a barrier to prevent its release from sites other than colon in GIT. Thus bio-degradable pectin microparticles were designed as a drug delivery system which offers decent approach for sustain release of drug and have potential for colon specific drug delivery.5,6 In this type of drug delivery system pectin a reteropolysaccharides derived from the cell wall of the plants and used a carrier agent to entrap drug and prevent its release in other part of GIT7. The formulation is designed to swell then allow drug to slowly release drug from by degradation of pectin by pectinolytic enzyme secreted by microorganisams found in the colon.
MATERIALS AND METHODS:
Preparation of Microparticle
The drug vincristine sulphate was obtained as gift sample from Dr. Reddys Lab, Mumbai. Pectin and shellac Acetone, n-Hexane, and light liquid paraffin Span 80 were purchased from CDH chemicals Mumbai. All other chemicals used were of analytical grade and were used as received.
Pectin microspheres were prepared by emulsion cross-linking method. Pectin dissolved in 20 ml of distilled water and uniform solution was prepared.5 Dispersion of vincristin sulphate was added to the uniform polymeric solution with stirring. Aqueous polymeric solution containing drug molecules was dispersed in 40 ml of light liquid paraffin containing span 80 (1.25% w/v) and stirred at 1000 rpm continuously to obtain stable w/o emulsion. The solution was rapidly cooled to 5°C by placing the beaker in an ice bath. After 20 min of stirring 10 ml of 3% w/v CaCl2 was added gradually to the system and stirred for 1 hr (allows the time for cross-microspheres were prepared by using different ratios of vincristine sulphate. There were more microparticles prepared to obtain optimum preparation by using varying concentration of Pectin (1:2, 1:3, 1:4 and 1:5), surfactant (75%, 1%, 1.25% and 2%) and by varying stirring speed (500, 1000, 1500 and 2000).
Microencapsulation of pectin microspheres:
The vincristine sulphate loaded Pectin microspheres were microencapsulated by emulsion-solvent evaporation technique.8 The Pectin micrcspheres (100 mg) were suspended in 20 ml of coating solution prepared by dissolution of shellac (500 mg) in ethanol-acetone mixture and then emulsified into 40 ml of light liquid paraffin containing span 80. The emulsification process was carried out for 2 h at 1000 rpm with mechanical stirrer. The Shellac coated microspheres were collected and rinsed with n-hexane and dried.
Particle size analysis:
Particle size distribution of the microspheres was determined by optical microscopy using calibrated ocular eyepiece and by scanning electron microscopy. Product dispersed in light liquid paraffin and a smear of the dispersion was observed under compound microscope- The size of 100 microspheres was measured in each case against a calibrated eyepiece in micrometer.
Determination of shape:
Morphological appearance and surface characteristics of the microspheres were studied by dispersing the microspheres in liquid paraffin and observed under light microscope.
Scanning Electron Microscopy
The shape and surface morphology of pectin microspheres and shellac-coated pectin microspheres were investigated using scanning electron microscopy (SEM). The samples for SEM study were prepared by lightly sprinkling the formulation on a double adhesive tape stuck to an aluminum stub. The stubs were zero with gold to a thickness of ~300 A under an argon atmosphere using a gold sputter module in a high-vacuum-evaporator. The coated samples were then scanned and photomicrographs were taken with scanning electron microscope.6
Determination of % Drug Entrapment
Efficiency of drug entrapment for each batch was calculated in term of percentage drug entrapment. Drug analysis of Vincristine Sulphate was done by dispersing 10 mg of Vincristine Sulphate microparticles in 10 ml phosphate-buffered saline (PBS; pH 7.4) under of solution constant stirring at 200 rpm/min at 37°C. The samples were centrifuged and the amount of free Vincristine Sulphate in the supernatant was determined at predefined time intervals using a UV spectrophotometer Elico -159 at 256nm. This analysis was performed three times for sample.
In Vitro Drug Release Study
The drug dissolution test of microspheres was carried out using USP rotating basket method in stimulated gastrointestinal fluid. Microspheres (10 mg) were weighed accurately and placed in the dissolution medium. The content was rotated at 50 rpm at 37°C ± 0.5 °C. Perfect sink conditions while GI transit condition was simulated by altering the pH of dissolution medium at fixed time intervals. For First 2 hrs pH was kept at 1.2 pH using 0.1 N HCl. Then 4 gm mixture of KH2PO4 and Na2HPO4.2H2O in ratio of 7:9 was added to elevate the pH to 6.8 and after 5 hrs 1M NaOH, and 4 % w/v pectinase enzyme was added to obtain 7.4 pH upto 24 hours. The samples were withdrawn from the dissolution medium by pipette fitted with microfilter (0.45µm), The rate of drug release was analyzed at 256nm using Elico -159 UV Spectroscope. The concentration of drug in the samples was calculated based on average of triplicate samples.
In view of the potential utility of optimized formulation for targeting of Vincristin Sulphate to colon, stability studies were carried out at 40°C / 75% RH for 3 months to assess their long term stability.
RESULT AND DISCUSSION:
Pectin microspheres of Vincristin Sulphate were successfully by emulsion cross linking method. Uniform, surface cross-linked and almost spherical microspheres were obtained as shown in SEM photomicrographs (Fig. 1A). The pectin microspheres coated with shellac by emulsion solvent evaporation method, using coat: core 5:1. The coated microspheres were found to be of spherical shape as observed in SEM photomicrographs (Fig. 1 B). The method was optimized using different drug polymer ratio, stirring rate and emulsifier concentration to produce microspheres of small size and narrow size distribution, high drug loading efficiency and controlled drug release at the colonic pH.
Figure 1: scanning electron photomicrographs of (A) shellac coated pectin microsphere SP2 (B) pectin microsphere P2
Particle size and shape analysis:
The Scanning electron microscopy is used to identify the shape and particle size of the micropsheres. Stirring rate, concentration of surfactant and Drug-polymer ratio has specific effect on the size of the microspheres. The mean diameter of microspheres varied from 30 µm to 34 µm with varying pectin concentration from 2% wt/vol to 5% wt/vol. The mean diameter of microspheres was found to vary from 35 um to 28 µm on varying emulsifier concentration (Span 80) from 1 % wt/vol to 2% wt/vol. Increasing Span 80 concentration from 1% to 2 % wt/vol led to stablization of the emulsion droplets avoiding their coalescence, resulting in smaller microspheres. The speed of stirrer was varied in at 500 rpm, 1000 rpm, 1500 rpm and 2000 rpm. The mean diameter of microspheres decreased from 36 µm to 30 µm with increasing agitation speed of the mechanical stirrer from 500 rpm to 2000 rpm. This result was expected because high stirring rates provide the shearing force needed to separate the oil phase into smaller globules.6 Fig 2 shows the effect of stirring speed, Surfactant Concentration and drug polymer ratio on particle size of microspheres.
Figure 2 Effect of Drug polymer Ratio, Surfactant concentration and Stirring Speed on Particle Size of Microspheres
Determination of % Drug Entrapment
The percentage drug entrapment varied from 82% to73% with varying drug polymer ratio from 2% to 5%. The highest drug loading efficiency was found with 2% pectin. A higher concentration of polymer gave larger microspheres with defective texture7. Figure 2 shows the effect of drug-polymer ratio on particle size, % drug entrapment and % yield. The entrapment efficiency varied from 77% to 81% with varying emulsifier concentration from 1% to 2% during preparation of pectin microspheres5. Fig 3 shows the effect of surfactant concentration on particle size, % drug entrapment and % yield. The stirring speed of 1000 rpm was found to be optimum for pectin microspheres, as the drug loading efficiency was 78%. High stirring speed produced an irregular shape of microspheres but a slightly increased entrapment efficacy was found9.
Figure 3 Percentage cumulative in vitro Vincristine Sulphate release from, pectin microspheres in simulated gastrointestinal fluids. Values are average of 3 readings ± standard deviation.
Figure 4. Percentage cumilative in vitro Vincristine Sulphate release from Shellac-coated pectin microspheres in stimulated gastrointestinal fluids of different pH. Values are average of readings ± standard deviation.
in-Vitro Drug Release Studies in Simulated Gastrointestinal Fluids:
In vitro drug release study of Pectin microspheres and Shellac-coated pectin microspheres was performed in pH progression medium at 37°C ± 0.5°C. The plot of cumulative drug release vs. time in simulated gastrointestinal conditions is shown in Figure 3 and 4, respectively. The drug release patterns shown in Figure 3 indicate that the rate of release of Vincristin Sulphate from pectin microspheres was mainly influenced by polymer concentration. When drug to polymer ratio increases from 1:2 to 1:5 a decrease in release rate was observed. The in vitro drug release studies of P1-P4 formulations in simulated gastro intestinal fluids showed a burst release pattern in the initial hour. The burst release may be due to solubility of pectin in the acidic pH. In order to prevent the drug release in stomach and small intestine these pectin microspheres were encapsulated with shellac, which shows solubility at a pH >7. The cumulative percentage drug release from Shellac-coated pectin microspheres showed the desired rate, as there was no measurable drug release observed up to 2 hours in SGF (pH 1.2), while in SIF (Simulated Intestinal Fluid) (pH 6.8), the drug release was quite insignificant (>1%) up to 5 hours. But in colonic fluid maximum drug release was observed due to dissolution of the shellac coat at pH 7.4 and the pectin microspheres were degraded on exposure to the colonic fluid and results in higher percentage of drug release. Significant release Vincristin Sulphate release from Shellac- coated pectin microspheres in SGF followed the order S-P1 > S-P2 > S-P3> S-P4 (Figure 4).The release of drug from microspheres decreased as the polymer concentration increased, suggesting that drug release could be controlled by varying the polymer concentration.10 This could be attributed to an increase in the density of the polymer matrix and the diffusional path length that the drug has to traverse.
The optimized formulation SP2 was kept at 40°C / 75% RH for 3 months to assess their long term stability. There is no appreciable change in drug content and dissolution profile of optimized formulation after storage at 40°C / 75% RH for 3 months, which was 98.45 ± 0,22% and 96.23 ±0.14% shown in Figure no 5.
Figure 5 Percentage drug release of optimized formulation subjected for stability testing Stability study
The particle size and entrapment efficiency were found to be varied by changing various formulation parameters like surfactant concentration, Drug polymer ratio and stirring speed. Scanning electron microscopy indicates that the microspheres have the rough and porous surface due to arising as a trace of solvent evaporation during the process. The release profile of Vincristine Sulphate from Shellac coated pectin microspheres was pH dependent. In acidic medium, the release rate was negligible; however, the drug was released quickly at pH 7.4. It is concluded from the present investigation that Shellac coated pectin microspheres are promising as a carrier for colon targeted delivery of Vincristine Sulphate.
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Received on 18.03.2013 Modified on 12.04.2013
Accepted on 28.04.2013 © RJPT All right reserved