INTRODUCTION:

Drugs that are easily absorbed from the gastrointestinal tract (GIT) and have a short half life are eliminated quickly from the blood circulation, require frequent dosing. To avoid this problem, the peroral controlled release (CR) formulations have been developed in attempt to release the drug slowly into the GIT and maintain a constant drug concentration in serum for longer period of time. Such oral drug delivery devices have a restriction due to the gastric retention time (GRT), release of drug in a region where it is poorly absorbed. Therefore for maximum absorption of drug it is important to release the drug in appropriate region of GIT.

Thus the real issue in the development of oral controlled release dosage form is not just to prolong the delivery of the drug, but to prolong the presence of the dosage forms in the stomach and in the small intestine until all the drug is released for the desired period of time. It may improve bioavailability and reduce drug waste.

Acyclovir is deoxiguanosine analogue antiviral drug which shows its action by inhibiting nucleic acid synthesis. It was chosen as a model drug since it has a very short half life (2.5-3.3 hrs) and low bioavailability (22 ± 8 %). The objective of the present study was to prepare nanoparticles of Acyclovir in order to sustained release in Intestine, which may result in enhanced absorption and thereby improved bioavailability.

MATERIAL AND METHODS:

Materials:

Acyclovir drug supplied as a gift sample by Wockhardt Pvt. Ltd. Aurangabad (India), chitosan polymer purchased from Central Institute of Fisheries Technology, Cochin, sodium tripolyphoshate purchased from Poonmani Labs, Coimbature (India).All chemical were of analytical reagent grade and were used as received.

Preparation of Chitosan Nanoparticles^{(4, 5)}:

Chitosan nanoparticles were prepared according to the procedure first reported by Calvo et al. (1997b) based on the ionic gelation of chitosan with sodium tripolyphosphate (TPP) anions. Nanoparticles were prepared by using different drug to polymer ratio. Quantity of drug in all formulations is kept constant i.e. 200 mg. The different ratio of drug and polymer is as shown in table 8.

RESULTS:

Characterization of nanoparticles:

SEM photomicrograph of acyclovir and chitosan

Figure No. 01

Figure No. 02

In vitro drug release study

Graph No.01

Chitosan was dissolved in acetic aqueous solution (3 mg/ml). Calculated quantity of drug was added to the chitosan solution and sonicated for uniform distribution. 0.5 %w/v of Tween 80 was added to the chitosan solution as a suspending agent, to prevent particle aggregation while stirring at 25⁰C. 4 ml TPP aqueous solution with various concentrations (0.2, 0.4, 0.6, 0.8, 1.0 mg/ml) was added into 30 ml chitosan/drug solution, respectively.

Three kinds of phenomena were observed: solution, aggregates and opalescent suspension. The zone of opalescent suspension was further examined as nanoparticles. Nanoparticles were collected by centrifugation at 8,000 rpm for 30 min. Then supernatants were discarded and the concentrated nanoparticles solution was freeze dried.

CHARACTERIZATION OF NANOPARTICLES:

Characterization of the morphology of the nanoparticles:

The surface morphology (roundness, smoothness and formation of aggregates) and the size of nanoparticles formulations were studied by scanning electron microscope (SEM). The data obtained after the observation were analyzed accordingly.

Percentage Yield^(6):

The percentage yield of different formulations was determined by weighing the nanoparticles after freeze drying. The percentage yield was calculated as follows

Each determination was made in triplicate.

Drug Entrapment Efficiency ^{(6, 7)}

The various formulations of the nanoparticles were subjected for drug content analysis. Suspension of the various formulations was prepared by suspending nanoparticles (equivalent to 50 mg of pure acyclovir) in aqueous solution. Each suspension was centrifuged at 15,000 rpm for 40 min at 24 ^◦C to separate the free drug in the supernatant from the drug incorporated in the nanoparticles. Concentrations of acyclovir in the supernatant were determined by UV-visible spectrometry at 254 nm after suitable dilution. The amount of the drug incorporated in nanoparticles was calculated from the difference in drug concentrations between the supernatant and the original given concentrations. The entrapment efficiency was calculated according to the following equation:

Each determination was made in triplicate

Fourier Transform Infra-red Spectroscopy (FT-IR) Analysis^(8):

The Fourier transform infra-red analysis was conducted for the analysis of drug polymer interaction and stability of drug during formulation process.

Table No.01: Drug Entrapment Efficiency of Different Formulation:

Sr. No.	Formulation	*Entrapment efficiency (% w/w)**
1	FN1	37.42 + 0.05
2	FN2	43.15+ 0.07
3	FN3	66.24 + 0.03
4	FN4	57.14 + 0.02
5	FN5	54.88 + 0.06
6	FN6	40.67 + 0.07
7	FN7	49.43 + 0.04
8	FN8	61.54 + 0.08
9	FN9	57.11 + 0.02
10	FN10	56.33 + 0.01

*Average of three preparation + S.D, FN: Formulation number.

Graph No.02

Table No.02: Percentage Yield of Different Formulation

Sr. No.	Formulation code	*Percentage yield (%)**
1	FN1	52.51 + 0.8
2	FN2	63.67 + 0.05
3	FN3	71.48 + 0.03
4	FN4	69.24 + 0.05
5	FN5	62.09 + 0.04
6	FN6	58.47 + 0.04
7	FN7	67.04 + 0.07
8	FN8	70.97 + 0.05
9	FN9	63.33 + 0.03
10	FN10	62.12 + 0.02

*Average of three preparation + S.D, FN: Formulation number.

Fourier transform infra-red spectrum of pure acyclovir and formulated nanoparticles were recorded. The formulation was kept for stability study before going for the FT-IR study. After the completion of the stability study formulation is used for the FT-IR study and the peaks of acyclovir were observed. Infrared absorption spectra of acyclovir and nanoparticles in the wavelength region of 450cm^-1 to 3600cm^-1 were recorded using FT-IR (SHIMADZU, JAPAN). Resolution used in the scans was 4 cm^-1 and the spectra were averaged over 20 scans.

In-vitro Release Studies⁽⁹⁾:

In vitro release pattern of nanoparticle suspension was carried out by dialysis bag method. An amount of nanoparticles equivalent to 50 mg pure acyclovir was weighed and filled in dialysis bag (Hi media). In the acid stage, dialysis bag was placed in a round bottomed cylindrical vessel containing 75 ml of 0.1 N HCl. The vessel was placed over magnetic stirrer (50 rpm) and the temperature was maintained at 37 + 0.5⁰C. Aliquots were withdrawn at predetermined time intervals and immediately replaced with the fresh medium equilibrated at 37⁰C. After two h, 25 ml of 0.2 M tribasic sodium phosphate was added to change the pH of test medium to 7.4, and the test was continued for a further 8 h. The sink condition was maintained throughout the experiment. The withdrawn samples were diluted and analyzed for drug content using U.V. spectrophotometer at 254 nm keeping phosphate buffer pH 7.4 as blank. All the determinations were made in triplicate.

Stability Study:

Graph No.03

Graph No.04

Kinetic modeling (^{14, 15)}:

1. Zero order kinetics:

Drug dissolution from pharmaceutical dosage forms that do not disaggregate and release the drug slowly, assuming that the area does not change and no equilibrium conditions are obtained can be represented by the following equation –

Q _t= Q _o + K _ot

Where Q _t= amount of drug dissolved in time t, Q _o = initial amount of drug in the solution and K _o= zero order release constant.

3. Higuchi model:

Higuchi developed several theoretical models to study the release of water-soluble and low soluble drugs incorporated in semisolids and or solid matrices. Mathematical expressions were obtained for drug particles dispersed in a uniform matrix behaving as the diffusion media. And the equation is

Q _{t =} K_H. t ^1/2

Where Q _t
= Amount of drug released in time t, K _H = Higuchi dissolution constant.

Table No.02: Kinetic modeling study:

Formulations	Zero order		Higuchi’s		Peppa’s Plot
Formulations	(Ko )	Correlation (r2 )	( K_H)	Correlation (r2)	Slope ( n )	Correlation (r2)
FN*1	10.9412	0.9986	26.9752	0.9865	1.5440	0.9317
FN2	9.5861	0.9916	26.2620	0.9489	1.4231	0.9268
FN3	8.3421	0.9965	23.9612	0.9912	1.2990	0.9694
FN4	9.3501	0.9902	25.6138	0.9158	1.3241	0.9351
FN5	8.8413	0.9912	24.0358	0.9286	1.3088	0.9661
FN6	9.0304	0.9847	23.3320	0.9161	1.4013	0.9355
FN7	8.8635	0.9903	24.2015	0.9421	1.3291	0.9334
FN8	8.5012	0.9901	24.2061	0.9210	1.3084	0.9528
FN9	9.1021	0.9914	24.9181	0.9031	1.3469	0.9274
FN10	8.6831	0.9857	24.9281	0.9277	1.3095	0.9613

FN*: Formulation number

Table No. 04: In vivo absorption study:

Sr. no	Time(hour)	Concentration of Acyclovir nanoparitcles in plasma	Concentration of pure Acyclovir solution in plasma (µg/ml)
1	0	0	0
2	2	0.889	2.724667
3	4	2.867	7.494667
4	6	6.962333	5.797667
5	8	4.845667	0
6	12	1.05	0

4. Krosmeyer and Peppas release model:

To study this model the release rate data are fitted to the following equation

M_t / M _¥ = K.t ⁿ

Where M_t / M _¥is the fraction of drug release, K is the release constant, t is the release time and n is the Diffusional exponent for the drug release that is dependent on the shape of the matrix dosage form.

If the exponent n = 0.5 or near, then the drug release mechanism is Fickian diffusion, and if n have value near 1.0 then it is non-Fickian diffusion.

Stability Study⁽¹⁶⁾

Samples from each batch were withdrawn after the definite time intervals and the residual amount of drug in the vesicles was determined. Stability data of three formulations were further analyzed for significant difference by paired t-test.

All the batches of acyclovir nanoparticles were tested for stability. The preparations were divided into 3 sets and were stored at 5-8⁰C (refrigerator) 27⁰C and at 40⁰C. After 15, 30 and 60 days drug content of all the formulations was determined by the method discussed previously in entrapment efficiency section.

Zeta potential ⁽¹⁷⁾:

The zeta potential value of the sample was measured by a zeta potential probe model DT-300. A nanoparticle which gives best results (FN₃) between particle size, entrapment efficiency, % yield and drug release, was determined for zeta potential. Zeta potential can help you to understand the characteristics of a suspension by understanding how individual colloids interact with one another. At times we may want to maximize the repulsive forces between them in order to keep each particle discrete and prevent them from gathering into larger, faster settling agglomerates. Each

colloid carries a “like” electrical charge which produces a force of mutual electrostatic repulsion between adjacent particles. If the charge is high enough, the colloids will remain discrete, disperse and in suspension.

In-vivo Mucoadhesion Study^{(14, 15)}:

The in vivo gastric mucoadhesive property of chitosan nanoparticles was evaluated by using fluoresein isothiocyanate (FITC) entrapped nanoparticles of formulation FN₃ in albino rat stomach. Albino rats (250-300 g) of either sex were fasted for 24 h before the experiments but were allowed free access to water. FITC-entrapped nanoparticles (10 mg) were placed in a polyethylene tube that had one end covered with hydroxypropyl cellulose film. Approximately 10 mg of nanoparticle was orally administered through the polyethylene tube attached to a gastric sonde with 2 ml of water. The gastric sonde was attached to a microsyringe containing 0.2 ml of water. The nanoparticles and water were pushed out through the polyethylene tube and orally administered to conscious rats. After 2.5 h, all the nanoparticles remaining in the stomach were collected after 3 washings with PBS (pH 7.4) and then centrifuged at 10,000 rpm for 10 minutes.

The resulting residues were dispersed in 5ml solution 1%NaOH. Samples were treated for 2 h by ultrasonication and left overnight at room temperature until mucus and nanoparticles were completely dissolved. The residues were then filtered and the fluorescence intensity of the filtrate was determined in a fluorescence spectrophotometer (systronics-photofluorimeter 151) at an emission wavelength of 495 nm and excitation wavelength of 520 nm. The amount of FITC-entrapped acyclovir nanoparticles remaining in the stomach was calculated from a calibration curve. The gastric mucoadhesion was expressed as the percentage of nanoparticles remaining in the stomach after perfusion.

In-vivo study:^{17, 18}

Acyclovir is well known anti-viral agent, which found to be most effective against many viral infection. Hence here also an attempt made to determine its antiviral activity with the help of best formulation (FN₃) in comparison to pure drug solution. The study protocol was approved by institutional Animal Ethics committee for the use of animal in research (proposal no.NCP/IAEC/PG/05/2008-2009) The in vivo study carried on male albino Wistar rats weighing 200 to 250 g. The animals were fasted overnight prior to the experiment but had free access to water. The Acyclovir nanoparticles of acyclovir were administered by oral snode in an equivalent dose of 19 mg/kg of acyclovir to first group of rat. The pure Acyclovir solution was administered in the same manner to the second group of rats. The blood samples (approximately 300-400 μL) were collected from the retro-orbital vein using a heparinized needle (18- 20 size) at 0, 1, 2, 4, 6, and 12 hours after administration. The blood samples were collected into a heparinized microcentrifuge tube. Then the samples were subjected to centrifugation on a laboratory centrifuge (Sigma, 3K30) at 10 000 rpm for 10 minutes at 0⁰C, and supernatant plasma was collected into another microcentrifuge tube and kept at –20⁰ C until analysis.

The concentration of acyclovir in plasma samples was determined by HPLC analysis. The HPLC system consisted of Hewlett-Packard (Agilent) 1100 series components, including a quaternary pump, auto sampler, and variable wavelength UV detector (Palo Alto, CA). The acyclovir was detected at 252 nm. Chromatographic separations were achieved using an Inertsil ODS-3V column (250 _ 4.6 mm, 5 μm) (GL Science, Tokyo, Japan). The mobile phase used for the plasma sample was 20mM ammonium acetate with 5mM octane sulfonic acid sodium salt in water (pH adjusted to 3 by acetic acid)-methanol (98:2, v/v). Filtration of the buffer was done using 0.2μm nylon 6.6 membrane filters, and it was degassed by sonication. Exactly 200 µL of the thawed plasma samples was mixed with 100 μL of water by vortexing for 1 minute using a Vibromixer (SPINIX, Mumbai, India). To this was added 20 μL of 35% perchloric acid, and the solution was mixed for 1 minute for protein precipitation. Then this mixture was centrifuged at 10 000 rpm for 10 minutes using a Biofuge Pico Micro centrifuge (Heraeus Instruments, Hanau, Germany). After centrifugation, 50 µL of supernatant solution was injected into the HPLC system. The linearity of the method was found suitable in the range of 2 to 20 μg/mL.

DISCUSSION:

The percentage yield of different formulation was determined by weighing the nanoparticles after drying. The percentage yield of different formulations was in the range of 52.51 + 0.08% to 71.04 + 0.03 % as shown in table and percentage yield for all formulation was almost similar. The percentage yield of the formulation mainly depends on the ionic gelation process. If the ratio of the TPP to chitosan is not maintained properly it may result in less gelation, which ultimately leads to less percentage yield.

The drug entrapment efficiency of different formulations was in the range of 37.42 + 0.05 to 66.24 + 0.03 %w/w. Drug entrapment efficiency increases with increasing to polymer ratio and concentration of TPP in most of the formulations. Sonication of the solution after addition of drug in polymer solution also plays important role in drug entrapment efficiency, as sonication leads to uniform distribution of the drug. Uniform distribution of drug will also give consistent entrapment efficiency of the same batch with less deviation. The entrapment efficiency increases with the increase in polymer: TPP ratio and is highest when the concentration of TPP used is 0.6 mg/ml.

The various kinetic models were applied to in vitro release data for prediction of the drug release kinetic mechanism. The release constants were calculated from the slope of appropriate plots, and the regression coefficient (r²) was determined. It was found that the in vitro drug release of nanoparticles was best explained by zero order kinetics as the plots shows highest linearity. The correlation coefficient (r²) was in the range of 0.9857 to 0.9986 for various formulations as shown in Table 26. For formulation FN₃ correlation coefficient (r²) is found to be 0.9965, indicating that the drug release was nearly independent of concentration, followed by Higuchi’s (r²= 0.9489 with K_H = 26.2620).

In the current study, drug release kinetic according to korsmeyer-peppa’s model is also followed. The values of release rate exponent (n), calculated as per the equation proposed by peppa’s, and all the slope values ranges from 1.3084 to 1.5440 revealed the fact that the drug release follows a super case II transport.

Stability study is the important part of the study for any pharmaceutical formulation. There are procedures given for the stability study in ICH guidelines. In response to that stability study was carried out for the formulation FN₃ by exposing it to temperature 5-8^oC, 270C and 40⁰C for 2 months. The sample was analyzed for drug content at regular interval of two months and it was evident that there was no remarkable change in the drug content of nanoparticles. Results show that formulation FN₃ was stable at mentioned temperatures. As the drug used, Acyclovir, is recommended to be stored below 25⁰C, stability study has not been performed above ambient temperature.

Zeta potential of chitosan nanoparticles (FN₃) was determined and it was found +33.19 ± 0.34 mV and it is due to presence of positively charged amino groups on the polymer backbone had a positive zeta potential. The positive values indicate that the chitosan nanoparticles were stabilized by electrostatic repulsion forces.

The FITC labeled Acyclovir nanoparitcles were removed from the stomach after 2.5 hrs and examined under fluorescence spectrophotometer at an emission wavelength of 495 nm and excitation wavelength of 520 nm. From the standard graph it was found that the 69.41 % of the nanoparticle were present in stomach. Amino acid group on the polymer backbone can promote hydrogen bonding interactions with the mucosa. As the Acyclovir is best absorbed from stomach, it could give uniform release of the drug from formulation.

The in vivo study carried out to determine the absorption study chitosan nanoparitcles of acyclovir in comparison to pure drug solution. The plasma sample from rat was collected after administration on specific interval of hour (0, 1, 2, 4,6,12, hour). HPLC analysis was done to find out the acyclovir nanoparticles are more absorbed than the pure acyclovir and above observation find that increase the bioavaibility of acyclovir.

From past few years nanoparticles have been studied by many workers as a choice of novel drug delivery system to provide a better drug bioavailability considering, high penetration property of the nanoparticles encapsulated agents through biological membrane and the stability of them.

The present formulation study on Acyclovir is an attempt to prepare nanoparticles drug delivery system and evaluate its performance both, In-vitro and In-vivo. The formulations were prepared, varying the ratios of polymer and TPP, by ionic gelation method.

An ideal or best formulation of nanoparticles is the one which gives high entrapment efficiency along with good stability and drug release profile. In the present study entrapment efficiency is found to be drug and polymer ratio dependent. The release rate is found to be depended on both polymer concentration and TPP concentration.

In the present study entrapment efficiency is found to be dependent on drug and polymer ratio. The drug entrapment efficiency of different formulation is in the range of 21.19 + 0.06 to 68.96 + 0.04 % w/w. The formulation FN₃, which showed higher entrapment efficiency (68.96 + 0.04 % w/w) provides desired drug release rate.

Zeta potential study proved that the above formulation have an excellent stability. The positive value (+33.19 ± 0.34 mV) indicates that the chitosan nanoparticles were stabilized by electrostatic repulsion forces.

The in-vivo results of Nanoparticles have emerged as an efficient means of enhancing the bioavailability and sustained delivery of Acyclovir. In-vivo mucoadhesion study with FITC loaded nanoparticles has elucidated that the 67.56 % of nanoparticles adheres to the stomach.

By these facts, study can be concluded by saying that nanoparticles prepared from chitosan and TPP using concentrations of 2 mg/ml and 1.0 mg/ml, respectively, is a promising approach to enhancing the bioavailability of Acyclovir.

ACKNOWLEDGEMENTS:

Authors are thankful to Nandha College of Pharmacy for their financial support throughout the project work.

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Received on 01.08.2009 Modified on 23.09.2009

Research J. Pharm. and Tech. 3(1): Jan. - Mar. 2010; Page 121-126