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International Journal
of Pharmaceutical Sciences and Drug Research ISSN: 0975-248X | DOI Prefix: 10.25004 | CODEN (USA): IJPSPP

Volume No.: 9 (2017) Issue No: 3

International Journal of Pharmaceutical Sciences and Drug Research 2017; 9(3): 118-125 http://dx.doi.org/10.25004/IJPSDR.2017.090304
Research Article

Formulation and in vivo Evaluation of Acyclovir Loaded Chitosan Nanoparticles for Ocular Delivery

S. Selvaraj1*, V. Niraimathi2

1Department of Pharmaceutics, JKK Munirajah Institute of Health Sciences, College of Pharmacy, Gobichettipalayam- 638506, Tamilnadu, India

2Department of Pharmaceutical Chemistry, College of Pharmacy, Madurai Medical College, Madurai- 625020, Tamilnadu, India

*Corresponding author: Dr. S. Selvaraj

 

Address: Department of Pharmaceutics, JKK Munirajah Institute of Health Sciences, College of Pharmacy, Gobichettipalayam- 638506, Tamilnadu, India

 

Tel.: +91-9994756130; +91-4285-260755E-mail *: selvasangari@gmail.com

Relevant conflicts of interest/financial disclosures: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Received: 14 April, 2017; Revised: 25 April, 2017; Accepted: 30 April 2017; Published: 29 May 2017Copyright © 2017 S. Selvaraj et al. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms. 

ABSTRACT

The present study was aimed to formulate and evaluate chitosan nanoparticles containing acyclovir as potential ophthalmic drug delivery system. The topical application of acyclovir as eye ointment remains a concern for effective management of various ocular viral diseases owing to poor ocular drug bioavailability. The acyclovir loaded chitosan nanoparticles were prepared by ionic gelation of chitosan. Differential scanning calorimetry and fourier transform infra red spectroscopy measurements were carried out on the prepared nanoparticles, pure acyclovir and chitosan polymer. Fifteen different formulations were prepared and evaluated for particle size, Zeta potential, scanning electron microscopy, entrapment and loading capacity and in-vitro drug release profile. All the prepared formulations resulted in nano size in 377.9 to 720.6 nm and displayed spherical shape with zeta potential of +33.2 to +42.8 mV. The encapsulation efficiency and loading capacity were 70.7% - 90.9% and 25% - 50.8% respectively. The acyclovir loaded chitosan nanoparticles displayed crystallinity than acyclovir. The in-vitro release profile of acyclovir from the nanoparticles showed a sustained release of the drug over a prolonged period of 24 hours and fit best with Higuchi model with zero order and non- Fickian diffusion was superior phenomenon. The in vivo results reveal that ocular viral infections can be inhibited by the nanoparticles more significantly than the drug in conventional dosage forms. No appreciable difference was observed during 90 days in which nanoparticles were stored at various temperatures. Thus the results suggest that acyclovir loaded chitosan nanoparticle suspension appears promising for effective management of ocular viral infections. 

Keywords: Acyclovir, Chitosan nanoparticles, Ocular delivery, Ionic gelation method.

 

INTRODUCTION

 

Herpes simplex virus’s infection is very common in humans. It has been estimated that one third of the world population have recurrent infection. Keratitis caused by herpes simplex viruses is the most common cause of cornea-derived blindness in developed nations. [1-2] Acyclovir is an antiviral drug with a significant and highly specific activity against herpes viruses and is widely used in the treatment of various ocular viral diseases. [3-6] The drug possess 10-30% of bioavailability with short plasma half-life of 2–3 hours, therefore necessitates 4–5 times application when administered as an ophthalmic ointment. These conventional preparations are ill-accepted on account of their short pre-corneal retention time, greasiness and vision-blurring effects. [7]

Many attempts have been made to improve the ocular bioavailability and the therapeutic effectiveness of acyclovir, e.g., chemical modification of the drug and its incorporation into colloidal systems such as liposomes or nanoparticles. [8-9] Nanoparticles have been used as ophthalmic delivery systems because they are able to penetrate into the corneal or conjunctival tissue by an endocytotic mechanism. [10] Further nanoparticles owing to their polymeric nature present some important advantages such as high storage stability, controlled release of the encapsulated drug and a prolonged residence time in the precorneal area, particularly in the case of ocular inflammation and /or infection. [11] Chitosan (Poly [-(1, 4)-2-amino-2-deoxy-D-glucopyranose)]) is a natural carbohydrate polymer prepared by the partial N-deacetylation of chitin. Among the mucoadhesive polymers investigated until now, the cationic polymer chitosan has attracted a great deal of attention because of its unique properties, such as acceptable biocompatibility, biodegradability and ability to enhance the paracelluar transport of drugs. [12] Besides, the cornea and conjunctiva have a negative charge; use of the cationic polymer chitosan will interact intimately with these extra ocular structures, which would increase the concentration and residence time of the associated drug. Moreover, chitosan has recently been proposed as a material with a good potential for ocular drug delivery. Among the various methods developed for preparation of nanoparticles, ionic gelation method is simple to operate and also to optimize the required particle size of the drug that can penetrate the ocular surface and hence this method was followed in the study. However, literature search indicates that the role of chitosan concentration on nanoparticles has not been studied in detail and hence the present study was attempted to demonstrate the influence of chitosan concentration on the physicochemical characteristics and release profile of the chitosan nanoparticles.

 

MATERIALS & METHODS

 Materials

 

Acyclovir was obtained as a gift sample from Micro labs (Hosur, India). Chitosan (degree of deacetylation of 85%; intrinsic viscosity,1390 ml/g in 0.30 M acetic acid/0.2 M sodium acetate solution; and viscometric molecular weight, 4.08 × 105 Da) was obtained as gift sample from Central Institute of Fisheries Technology (Cochin, India). Sodium tripolyphosphate (TPP) was purchased from S.D. Fine Chemicals Ltd (Mumbai, India) and Tween-80 was supplied by Loba Chemie Pvt Ltd (Mumbai, India). Ultra pure water was purchased from Himedia Ltd (Mumbai, India). All other reagents and solvents used were of analytical grade.

 

 

Methods

 

Compatibility studies

Before formulation of drug substances into a dosage form, it is essential that the drug and polymer should be chemically and physically characterized. Compatibility studies give the information needed to define the nature of the drug substance and provide a framework for the drug combination with pharmaceutical excipient in the fabrication of a dosage form. [13-14]

Fourier Transform Infra Red Spectroscopy

 

Compatibility study of pure acyclovir, chitosan polymer, acyclovir with chitosan polymer and acyclovir loaded chitosan nanoparticles (F-9) were determined by FTIR Spectroscopy using Perkin Elmer RX1. The pellets were prepared by gently mixing sample with potassium bromide at high pressure. The scanning range used is 450 to 4000 cm -1. The pellets thus prepared were examined and the spectra of drug and the polymer in the formulations were compared with that of pure drug and polymer spectra.

 

Differential Scanning Calorimetry

 

Differential scanning calorimetric curve of pure acyclovir, chitosan polymer, acyclovir with chitosan polymer and acyclovir loaded chitosan nanoparticles (F-9) were carried out by using thermal analysis instrument equipped with liquid nitrogen sub ambient accessory; 2-6mg samples were accurately weighed in aluminum pans hermetically sealed and heated at a rate of 10ºC per min-1 under nitrogen flow of 40 mL/ min.

 

 

Preparation of acyclovir loaded chitosan nanoparticles

 

Chitosan nanoparticles were prepared based on the ionic gelation of chitosan with sodium tripolyphosphate anions. [15] Chitosan nanoparticles were prepared by ionic gelation of chitosan solution with sodium tripolyphosphate prepared in the presence of Tween 80 as a re-suspending agent to prevent aggregation, at ambient temperature. 350 mg acyclovir and various concentrations of chitosan dissolved in acetic acid in aqueous solution under magnetic stirring at room temperature for 45 min in the presence of Tween 80; 10 mL of sodium tripolyphosphate aqueous solution was added into 10 mL chitosan-acyclovir solution and the mixture was subjected to different sonication times. The nanosuspensions were cold centrifuged at 12000 g in a glucose bed for 30 min using Hitachi centrifuge. The supernatant liquid was analyzed by spectrophotometer to calculate the drug loading and percentage drug entrapment. The final suspensions were frozen and lyophilized by using freeze drier (Lab Conico, USA) at 0.4 mbar and -40ºC for 5 hours using glucose and lactose. The lyophilized nanoparticles were stored in a desiccator at 4ºC. The concentrations and amounts applied are summarized in Table 1.

 

 

 

 

 

Table 1: Composition of Acyclovir Loaded Chitosan Nanoparticles

Formulation Code

Drug

(mg)

Polymer

(mg)

Tween 80

(0.5%)

STPP

(0.25%)

Sonication Time (min)

F-1

 

350

 

150

 

0.5

 

0.25

 

0

 

F-2

 

350

 

150

 

0.5

 

0.25

 

5

 

F-3

 

350

 

150

 

0.5

 

0.25

 

10

 

F-4

 

350

 

250

 

0.5

 

0.25

 

0

 

F-5

 

350

 

250

 

0.5

 

0.25

 

5

 

F-6

 

350

 

250

 

0.5

 

0.25

 

10

 

F-7

 

350

 

350

 

0.5

 

0.25

 

0

 

F-8

 

350

 

350

 

0.5

 

0.25

 

5

 

F-9

 

350

 

350

 

0.5

 

0.25

 

10

 

F-10

 

350

 

450

 

0.5

 

0.25

 

0

 

F-11

 

350

 

450

 

0.5

 

0.25

 

5

 

F-12

 

350

 

450

 

0.5

 

0.25

 

10

 

F-13

 

350

 

550

 

0.5

 

0.25

 

0

 

F-14

 

350

 

550

 

0.5

 

0.25

 

5

 

F-15

 

350

 

550

 

0.5

 

0.25

 

10

 

 

 

Evaluation of acyclovir loaded chitosan nanoparticles

 

Evaluation of pH

 

pH is one of the most important factors involved in the formulation process. The pH of ophthalmic formulation should be such that the formulation will be stable at that pH and at the same time there would be no irritation to the patient upon administration of the formulation. The pH of the prepared formulations was checked by using pH meter (Hanna instruments, Italy H198).

 

 

Particle size and Zeta potential

 

The prepared acyclovir nanoparticles were evaluated for their particle size and zeta potential by zeta potential analyzer Zetasizer 3000 HS (Malvern instrument, UK). The formulations were diluted to 1:1000 with the aqueous phase of the formulation to get a suitable kilo counts per second. Analysis was carried out at 25°C with an angle of detection of 90 degrees.

 

 

Surface morphology by Scanning Electron Microscopy

 

The morphology of the acyclovir nanoparticles were analyzed by scanning electron microscope. The instrument used for this determination was JEOL MODEL JSM 6400 scanning electron microscope. The nanoparticles were mounted directly on the SEM stub, using double sided, sticking tape and coated with platinum and scanned in a high vacuum chamber with a focused electron beam. Secondary electrons, emitted from the samples were detected and the image formed.

 

Acyclovir Encapsulation efficiency and loading capacity

 

 

 

The Encapsulation efficiency and loading capacity of the nanoparticles were determined by the separation of nanoparticles from the aqueous medium containing non associated acyclovir by cold centrifugation at 12000 g for 30 minutes. The amount of free acyclovir in the supernatant was measured by UV method at 253 nm. [11] The acyclovir encapsulation efficiency (EE) and loading capacity (LC) of the nanoparticles was calculated as follows:

 

 

In-vitro release studies

The acyclovir loaded chitosan nanoparticles were separated from the aqueous suspension medium through ultracentrifugation. Nanoparticles of acyclovir were redispersed in 10 mL of (pH 7.4) phosphate buffer solution and placed in a dialysis membrane bag with a molecular cut-off of 5 kDa which acts as a donor compartment. The bag was tied and placed into 10 mL of phosphate buffer solution in a beaker which acts as a receptor compartment. The entire system was kept at 37ºC with continuous magnetic stirring; at appropriate time intervals 1 mL of the release medium was removed and 1mL fresh phosphate buffer solution was added into the system. The amount of acyclovir in the release medium was estimated by UV-Visible spectrophotometer at 253 nm.

 

Kinetics of drug release

 

In order to understand the mechanism and kinetic of drug release, the drug release data of the in-vitro diffusion study were analyzed with various kinetic models like zero order, first order, Higuchi’s and Peppas. Coefficient of correlation (r) values were calculated for the linear curves by regression analysis. [16-17]

 

In-vivo drug release studies

Six healthy albino rabbits (1.5–2.2 kg) were selected for the study with no signs of ocular inflammation. As per CPCSEA guidelines the procedures were followed. The experimental protocols have been approved by the Institutional Animal Ethics Committee (Nandha College of Pharmacy, 688/2/C-CPCSEA dated 21-02-2015). Out of 15 formulations F-9 was taken for in-vivo study on the basis of in-vitro drug release study. The marketed formulation of acyclovir (Acivir Eye 3% w/w ointment) was used as control. The left eyes of rabbits were used for control preparation and the right eye of the rabbits for prepared nano suspension. Aqueous humor acyclovir levels were monitored at 1, 4, 8, 16, 20 and 24 hours after instillation of nanosuspension and control into the conjunctival sac. During the time interval the rabbits were anaesthesized by i.v. injection of ketamine (25 mg/kg) and 150μL aqueous humor aqueous was withdrawn from the limbus region of rabbits with a heparin-rinsed glass syringe connected to a 27-gauge needle. Samples were denatured by the addition of an equivalent volume of 2% zinc sulphate solution centrifuged and the supernatant liquid was filtered through a 0.2-μm millipore membrane. A similar experiment was carried out on the second group of rabbits with marketed formulation of acyclovir. Samples were analyzed by high performance liquid chromatography at a wavelength of 253 nm [18-20]

 

The HPLC system was operated in a binary mode with a photodiode array detector, auto injector and column oven. The analysis was performed at 253 nm on a reversed phase chemisorb C-18 cartridge column (250 mm×4.6 mm, 5 μm) maintained at 25ºC. The mobile phase comprised of 0.02M potassium dihydrogen phosphate - acetonitrile in the ratio of 99:1 with a flow rate of 1 mL/ min. Acyclovir concentration in aqueous humor was estimated using linearity chart. [21-22]

 

Stability Studies

The short term stability study was carried out as per ICH guidelines using the optimized formulation F-9. Formulation F-9 was divided into 3 sets of samples and stored at 4°C in refrigerator, room temperature 37°C and at ambient temperature. In vitro release study of formulation F-9 was carried out after 90 days of storage. [23]

RESULT & DISCUSSION

 The results of the present investigation demonstrated the potential use of chitosan nanoparticles for effective delivery of acyclovir for treating various ocular viral diseases. Drug delivery system for the ocular surface must overcome important physical barriers to reach the target cells. Different colloidal systems have been developed to solve these problems. [24] Among them chitosan based systems are acknowledged more suitable for ocular pathway, based on the favorable biological characteristics of chitosan. [25-26] Several studies have shown that nanoparticles can transport across epithelia more readily than microparticles. [27] Moreover chitosan nanoparticles can be easily prepared under mild conditions, and can be incorporated in macromolecular bioactive compounds. This characteristic feature is extremely beneficial for drugs, proteins, genes or hydrophobic molecules that are poorly transported across epithelia. [25-26]

Compatibility studies

Fourier Transform Infra Red Spectroscopy (FTIR)

There was no appearance or disappearance of any characteristics peaks of pure drug or of polymer in the physical mixture and acyclovir loaded nanoparticles, thus indicating absence of any physical interaction between the drug and polymer. The results show that the incorporation of the drug in to the polymer did not change the characteristics of the drug. Observations of compatibility studies of infrared spectral data for the pure drug, the mixture of acyclovir with chitosan and acyclovir loaded chitosan nanoparticle (F-9) are shown in Table 2.

 

Differential Scanning Calorimetry (DSC)

The results of the DSC study of acyclovir, chitosan, acyclovir with chitosan and acyclovir loaded chitosan nanoparticle (F-9) are shown in Figs. 1A, 1B, 1C and 1D. The DSC curve of acyclovir showed characteristic peaks at 120 .61ºC, 150.48ºC and 254.07ºC. The DSC curve of chitosan showed characteristic broader peak at 102.81ºC. The thermogram of acyclovir chitosan mixture exhibited same characteristic peaks of acyclovir at 121.06ºC, 150.48ºC and 254.07ºC. The results of the thermogram suggested that there was no chemical interaction between acyclovir and chitosan.

 

From the IR spectral analysis and DSC study, it was found that IR spectrum and thermogram of pure acyclovir and combination of pure drug with polymer like chitosan and prepared nanoparticles showed all the characteristic peaks of acyclovir confirming the physical and chemical compatibility of the pure drug and polymer.

Table 2: FT-IR spectral data

Molecular

Vibration

Wave number in cm-1

Pure acyclovir

Acyclovir with polymer

Acyclovir loaded nanoparticles

NH stretch in NH2

3184.14

 

3184.26

 

3232.77

 

C = N stretch

 

1633.12

 

1633.14

 

1694.84

 

C = O stretch

 

1716.69

 

1717.96

 

1694.84

 

CH2 stretch

 

2927.96

 

2928.61

 

2873.04

 

OH stretch

 

3442.06

 

3442.48

 

3444.97

 

Fig. 1A: Thermogram of acyclovir

Fig. 1B: Thermogram of chitosan

Fig. 1C: Thermogram of acyclovir and chitosan

Fig. 1D: Thermogram of formulation F-9

Evaluation of acyclovir loaded chitosan nanoparticles

pH

pH values for all the formulations are within acceptable range 6.6-7.4 and hence would not cause any irritation upon administration of the formulation. It was also observed that increase in chitosan polymer causes a slight increase in pH for formulations

 

Particle size and Zeta potential

The mean particle size and zeta potential of acyclovir loaded chitosan nanoparticles (F1– F15) are shown in Table 3 and Fig. 2. The mean particle size of the acyclovir loaded chitosan nanoparticles ranged between 377.9 to720.6 nm. The sizes of nanoparticles of all the formulations are given in nanometer. The maximum size of nanoparticles was observed in F-15 as compared to other formulations and the least size was seen in F-1. Thus the results indicated that the size of the nanoparticles varied with the polymer concentration.

 

The presence of a nonionic surfactant is very important for the so-called “long-term” stability of the nanosphere colloidal suspension, which is determined by the adsorption of hydrophilic macromolecules on the nanosphere surface, thus increasing the steric repulsion between particles. The presence of hydrophilic macromolecules on the surface of nanosphere leads to a change of the surface properties (zeta potential) of the colloidal carrier. Considering these factors the non-ionic surfactant Tween 80 (0.05%) was used to stabilize the formulation.