INTRODUCTION:

Viruses are the ultimate expression of parasitism: they not only take nutrition from the host cell but also direct its metabolic machinery to synthesize new virus particles. Viral chemotherapy, therefore, is difficult, as it would require interference with cellular metabolism in the host¹. The most convenient and commonly employed route of drug delivery has historically been by oral ingestion². Drugs that are easily absorbed from the gastrointestinal tract and having a short half life are eliminated quickly from the blood circulation. However, the intestinal epithelium may constitute a permeability barrier for the absorption of orally administered drugs. This problem stimulated a search for new strategies to overcome mucosal barriers. Among various approaches, the ability of colloidal systems (liposomes, nanoparticles and polymeric micelles) to cross the intestinal mucosa has been investigated extensively³. Also the enhancements of electrostatic interaction between the mucosal surfaces and drug have a marked effect on their uptake and overall bioavailability. Nanoparticles are defined as solid, submicron-sized drug carrier that may or may not be biodegradable. The term nanoparticle is a collective name for both nanospheres have a matrix type of structure or encapsulated within the particle⁴. Nanoparticles composed of physiologically tolerated lipid components, at room temperature the particles are in the solid state.

Received on 09.12.2014 Modified on 30.12.2014

Research J. Pharm. and Tech. 8(1): Jan. 2015; Page 35-37

DOI: 10.5958/0974-360X.2015.00007.4

The purpose of the present investigation is to develop acyclovir loaded nanoparticles using chitosan polymer through ionic gelation method and to improve oral bioavailability acyclovir drug by incorporating into nanoparticles⁵.

MATERIALS AND METHODS:

Acyclovir was received as gift sample from Indoco Remedies Rabale, Navi Mumbai. Chitosan was purchased from India Sea Foods, Cochin. Acetic acid and Tween 80 were purchased from SD Fine chemical Ltd, Mumbai. Methanol and ethanol were purchased from Qualigens fine chemicals, Mumbai. Potassium Chloride and Hydrochloric acid were purchased from Ranbaxy Fine Chemicals Ltd., New Delhi. Sorbitol solution, 70% non crystalline, Glycerol, Dispersible cellulose, Methyl parahydroxy benzoate, Propyl parahydroxy benzoate free samples were supplied by Indoco Remedies Rabale. And all other chemicals were of reagent grade and used without further modification.

Methods:

Preparation of Acyclovir nanoparticles using chitosan⁶

Drug loaded chitosan nanoparticles were prepared by the method reported by Calvo et al. (1997b) with some modifications based on the ionic gelation of acyclovir with TPP anions. Chitosan was dissolved in acetic aqueous solution (6 % v/v) at various concentrations such as 1.0, 2.0, 3.0, 4.0 and 5.0 mg/ml. 10 mg of drug (acyclovir) was dissolved in 5 ml of 2 % w/v tween 80 solution, which was added to the chitosan solution. Under magnetic stirring at room temperature, 5 ml of 0.25 % sodium tripolyphosphate (TPP) aqueous solution was added drop wise into drug and polymeric mixture, respectively. The stirring was continued for about 20 – 25 min. The obtained nanoparticle suspension was centrifuged at 12000x rpm for 30 min using C24 centrifuge. The formation of the particle was a result of the interaction between the negative groups of the TPP and the positively charged amino groups of chitosan (ionic gelation).

Formulation plan for Acyclovir nanoparticles using chitosan

Batch	Formula
Batch	Drug (Acyclovir) (mg)	0.25 % TPP solution (ml)	10 ml Polymer (Chitosan) solution (%)
FM – 1	10	5	0.1
FM – 2	10	5	0.2
FM – 3	10	5	0.3
FM – 4	10	5	0.4
FM – 5	10	5	0.5

Formulation of Acyclovir nanoparticles suspension:

Charge approximately 5% of the final batch volume of Purified water at 20±3ºC to a suitable vessel equipped with a mixer propeller. Add dispersible cellulose to the above purified water and mix until dissolved. Then re-circulate the mixture in microfluidizer. After that add 7% of the final batch volume of purified water to above solution and mix for 5 minutes. Then add acyclovir loaded nanoparticles to the vessel with constant mixing. Continue mixing until it is fully dispersed. Add methyl parahydroxy benzoate and propyl parahydroxy benzoate to it and mix for approximately 5 minutes. Add sorbitol solution slowly with constant mixing. Continue to mix after addition of the sorbitol solution. Then add glycerin with constant mixing and mix for 5 minutes. Eventually add the flavors and mix for approximately five minutes. Add purified water and make up to the final volume and then mix until a uniform suspension is attained.

Manufacturing formula of Batch No. R&D/05/13

Sr.No	Ingredient	Qty Per Dosage (mg/ml)
1.	Acyclovir loaded nanoparticles	Equivalent to 40 mg of Acyclovir
2.	Sorbitol solution, 70% non crystalline	350.000
3.	Glycerol	100.000
4.	Dispersible cellulose	25.000
5.	Methyl parahydroxy benzoate	0.004
6.	Propyl parahydroxy benzoate	0.002
7.	Flavour banana	0.005
8.	Vanillin	0.005
9.	Purified Water	Q.s. to add 1 ml

Final batch was charged for Stability study up to 6 months as per ICH guidelines.

Evaluation of Acyclovir loaded nanoparticles suspension:

a. Particle size:

Particle size analysis was done by scanning electron microscopy (SEM). SEM is the most commonly used method for characterizing drug delivery system, due to simplicity in sample preparation and ease of operation. Three dimensional information about macro (0.1-10 mm), meso (1-100 µm) and nanostructure (10-1,000 nm), is often found within the same micrograph. SEM has been used to determine particle size, distribution, surface topography, texture and to examine the morphology of fractured or sectioned surface.

Particle size analysis was done by using JEOL JSM-T330A scanning microscope. Cleaned brass specimen stud was used for mounting the samples. Solvent paint was applied on these stud and while the paint was wet, the pellets were placed on each stud and allowed to dry, then the sample was observed in scanning electron microscopy and photographs were taken.

b. pH:

pH of final suspension was measured on thermolab digital pH meter at room temperature.

c. Sedimentation volume:

Physical stability is defined as the condition in which the particles remain uniformly distributed throughout the dispersion without any signs of sedimentation. Therefore the extent of sedimentation and ease of redispersibility evaluated by sedimentation volume method.

It is defined as ratio of ultimate volume of the sediment to initial volume of the suspension. Normally sedimentation value is between 0 to1. The higher the value, the better is the physical stability.

d. Viscosity:

The viscosity of the optimized nanosuspension was determined by using Brookfield DV-E Rheometer (Brookfield Engineering, Middleboro, MA, USA) using a spindle no.00 in triplicate at 25°C. The speed of the spindle was adjusted to 100 rpm. Wait time for the operation was 50 min. Shear rate applied was 413 per min and diameter of the spindle used was 50 mm (R. Parveen et al. 2011, V. Bali et al. 2010).

In vivo Biodistribution Studies of Final batch:

All animal experiments were approved and performed in accordance with the guidelines of by Institutional Animal Ethics Committee (Ref No. METIP/IAEC/2011-2012/25). Male Sprague Dawley rats weighing 250–270 g were selected for the biodistribution studies which were divided into two group, one for oral administration of Test sample and another for oral administration of marketed sample. To Group I, 5 ml of the formulation (200 mg/5ml DRUG loaded nanosuspesion) were given with the help of micropipette attached with LDPE tubing, having 0.1 mm internal diameter. The rats were held from the back in slanted position during administration. The blood was collected using syringe. Blood samples were anticoagulated with heparin and centrifuged at 3000 rpm for 10 min to obtain plasma. At each time point, 6 rats were taken for measurements. All plasma samples were stored for up to 48 hr in a deep freezer (−70⁰ C) until HPLC analysis (Zhang Q. et al., 2004).

A. Processing of samples:

The whole procedure was carried out at room temperature. To 200 µl plasma samples 25 µl of the Internal Standard (40 µg /ml) was spiked and vortex mixed for 30 s. Then 0.5 ml of acetonitrile was added and vortex-mixed for 1 min. The sample was centrifuged at 8000 rpm for 5 min in a microcentrifuge. The supernatant layer (0.75 ml) was transferred to a 15 ml glass test tube, and then 4.5 ml of extraction solvent, methyl t-butyl ether– n-hexane (9:1) were added. The sample was vortex-mixed for 3 min using a multi-tube vortexer. The organic layer (4 ml) was quantitatively transferred to a 6 ml glass tube and evaporated to dryness using an evaporator at 40 ⁰C under a stream of nitrogen. Then the dried extract was reconstituted in 100 µl of water–methanol (50:50, v/v; diluent) and a 20 µl aliquot was injected into chromatographic system (Mudigonda, K et. al., 2006).

B. Chromatographic conditions:

The chromatographic separation was performed at ambient temperature with a reversed-phase, 150 X 4 mm base specific column packed with 5µm C18 silica reversed-phase particles (Lichrospher 60 Select B). The mobile phase was a mixture of 10 mm ammonium acetate buffer–acetonitrile (45:55, v/v) pumped at a flow-rate of 1.0 mL/min. Detection was performed at a wavelength of 240 nm.

C. Calibration curves of Drug in plasma:

Calibration curves of DRUG were prepared with plasma spiked with known amounts of the drug utilizing its HPLC peak area ratio to the internal standard. The linear range of DRUG was 500–5000 ng/ml, 500–5000 ng/g for plasma samples. The detection limits were 100 ng/ml (or 100 ng/g).

D. Data analysis:

All data are reported as mean ± S.D and the difference between the groups were tested using Student’s t-test at the level of P < 0.05. All concentration data were dose- and weight- normalized. Pharmacokinetic parameters for drug formulations were calculated using Kinetica 5.0 software. The C_max and T_max values of the oral administration were read directly from the concentration–time profile. The area under the concentration–time curve (AUC_{0 →t}) was calculated by the trapezoidal rule. The absolute oral bioavailability of drug from nanosuapension was calculated.

RESULTS AND DISCUSSION:

Following table describes the results of test samples and marketed samples for each test performed during the production of the Suspension.

Sr. No	Test	Results for test samples	Results for marketed sample
1.	Particle size	250-300 nm	10000 – 15000 nm
2.	pH	6.1	5-7
3.	Viscosity	64 cp	65 cP
4.	Sedimentation volume	0.8-1.0	0.8-1.0

Stability results up to 6 months for accelerated, intermediate and long term conditions was found to be satisfactory.

In vivo study results:

The results of biodistribution studies showed the time profile of Drug concentration in plasma higher for nanosuspension compare to marketed sample. The profiles of Acyclovir level in plasma displayed an initial absorption phase and maximum concentration achieved after about 4.5±1 hrs in plasma for Nanosuspension while 2.5±1 hrs in marketed sample.

Pharmacokinetic parameters	Sample	Nano-suspension	Marketed sample
C_max (ng/ml)	Blood Plasma	300.66±47.0	205.20±39.0
T_max (hrs)	Blood Plasma	4.5±1	2.0±1
AUC_{0-12 hrs} (ng/ml*min)	Blood Plasma	9457±843	5124±712

CONCLUSION:

The present investigation was aimed at developing and characterizing oral suspension of chitosan-acyclovir nanoparticles and to improve oral bioavailability. Physical characterization and stability data showed that final formulation was stable and drug is in nano form. In vivo study results confirmed increase in extent of absorption of Acyclovir in rats. From the above studies, it is revealed that the present work was a satisfactory preliminary study of improving bioavailability using chitosan nanoparticles. Clinical study in humans can be done using this formulation for the further study.

ACKNOWLEDGEMENTS:

Kishore Uttam Kothule would like to thanks IIT, Powai for providing facilities to perform SEM studies. The authors would also like to acknowledge Indoco Remedies Rabale, Navi Mumbai India, for providing the gift samples of Acyclovir and other excipeints. The authors are again grateful to Indoco Remedies Rabale, Navi Mumbai and MET’s college for extending facilities to perform various experimental and analytical studies.

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