Formulation and in vitro evaluation of antineoplastic drug loaded nanoparticles as drug delivery system

The main aim of the present work was to formulate anti-neoplastic drug loaded polymeric nanoparticles using biodegradable polymers (Chitosan and Eudragit RS 100) by emulsion droplet coalescence method. The model drug used here is 5-fluorouracil which is a pyrimidine analogue that is mainly used to treat colonic carcinoma, under the category of anti-neoplastic drugs. Tween 20 was used as emulsifier and colloidal stabilizer. The prepared nanoparticles were evaluated for particle size, surface morphology by TEM, surface charge, drug loading and entrapment efficiency, and for drug release by diffusion. Results show that the prepared nanoparticles are in nanosize, below 1000 nm, having appropriate zeta potential values with better entrapment of drug and controlled release of drug for a period of 12 h. From the obtained formulations, EF5 was selected as best with high entrapment efficiency, optimum zeta potential, and showing more controlled release of drug.


INTRODUCTION
Drug delivery research is clearly moving from the microto the nano-size scale.Nanotechnology is therefore emerging as a field in medicine that is expected to elicit significant therapeutic benefits.The development of effective nano delivery systems capable of carrying a drug specifically and safely to a desired site of action is one of the most challenging tasks of pharmaceutical formulation investigators.They are attempting to reformlate and add new indications to the existing blockbuster drugs to maintain positive scientific outcomes and therapeutic breakthroughs (Yashwant and Deepak, 2009).
5-Flourouracil (5-FU or 5-fluoro-2,4-pyrimidinedione) is an antimetabolite of pyrimidine analogue type, with a broad spectrum activity against solid tumors (of gastrointestinal tract, pancreas, ovary, brain, breast, etc).Due to its structure, 5-fluorouracil interferes with nucleoside metabolism and can incorporate into RNA and DNA, leading to cytotoxicity and cell death (Zhang et al., 2008;Arias et al., 2008).Limitations are short biological half-life due to rapid metabolism, incomplete and non uniform oral absorption by dihydropyramidine dehydrogenase and nonselective action against healthy cells.To prolong the circulation time of 5-fluorouracil and increase its efficacy, its delivery has to be modified by incorporation into nanoparticulate carriers to reduce the 5-fluorouracil associated side effects and thereby improve its therapeutic index (Li et al., 2008).Polymer systems can be to physically trap an antitumor agent and release it in a sustain form directly at the tomor site (Simeonova et al., 2003;Elvire et al., 2004).Chitosan is a linear polysaccharide of [ (Yashwant and Deepak, 2009;Li et al., 2008) linked 2-acetamido-2-deoxy-dglucopyranose, is a natural polysaccharide derived from chitin by deacetylation (Figure 1).
Chitosan is a cationic polymer, and regarded as  biocompatible, biodegradable, and nontoxic (Dang and Leong, 2006;Thanou et al., 2001).Eudragit RS 100 is a copolymer of ethyl acrylate, methyl methacrylate and a low content of methacrylic acid ester with quaternary ammonium groups (Figure 2).It shows unique dissolution behavior above pH 7.0.So, it has been used as pHsensitive polymers in various applications including enteric coating materials (Sand drug delivery vehicles).In the present work, nanoparticles of 5-Fluorouracil were prepared using chitosan, Eudragit RS 100, to deliver the drug to disease site and achieve gradual drug release (Onishi et al., 2005).This is based on the concept of localization at the disease site by nanoparticles and control release by ester hydrolysis.Secondary coating of Eudragit over 5-fluorouracil loaded chitosan nanoparticles (Ch-5-FU-NP) was use full to protect the drug from acidic environment, that is, pH below 6 and shows a unique drug release above pH 6.8.By the combination of these two polymers, that is, chitosan, Eudragit targeting specific sites in the body simplifies drug administration procedures, reduce the quantity of drug require to reach therapeutic levels, decrease the drug concentration at non target sites.
The objective of this work as to deliver the 5fluorouracil drug loaded nanoparticles as oral delivery that offers certain advantages over the current regimen of chemotherapy by injection or infusion.Oral site specific rate-controlled expect to reduce systemic side-effects and also to provide an effective and safe therapy for colon cancer with reduce dose and duration of therapy.

MATERIALS AND METHODS
5 Fluorouracil (5Fu) was obtained from Celone Pharmaceuticals Pvt. Ltd., India.Eudragit RS 100, from R ohm Pharma (Darmstadt, Germany).Chitosan (CS, degree of deacetylation was 95.3% and was a gift sample from Indian Sea foods, Cochin).Liquid paraffin, Tween 20, and sodium chloride were obtained from SD Fine chemicals, Mumbai, India.Sodium hydroxide and sodium dihydrogen phosphate were procured from Loba chemicals Mumbai, India.All other chemicals were of analytical grade and were used without further purification.

Preformulation studies
FTIR studies: Drug identification was carried out by FTIR spectroscopy.The spectrum was recorded for 5-fluorouracil using spectrum BX (Perkin Elmer) infrared spectrophotometer.Samples were prepared in KBr disk (2 mg sample in 200 mg KBr) with an hydrostatic press at a force of 40 psi for 4 min.The scanning range was 400 to 4000cm -1 and the resolution was 4 cm -1 (Sharma, 2011; Aslam et al., 2004).
Differential scanning calorimetric (DSC) studies: DSC analysis of the drug 5-fluorouracil was carried out using DSC 200F3 Maia equipped with computer analyzer.Samples (3 to 7 mg) were heated under nitrogen atmosphere on an aluminum pan at a heating rate of 10°C min -1 over the temperature range of 0 to 500°C.(Babu et al., 2006).
X-ray diffraction (XRD) studies: XRD patterns were traced for the 5-fluorouracil employing X-ray diffract meter (Philips PW 1729, Analytical XRD, Holland) using filtered CuK(α) radiation (intensity ratio (α1/α2): 0.500), a voltage of 40 KV, a current of 30 m and receiving slit of 0.2 inches.The samples were analyzed over 2θ range of 5.010 to 39.990 with scanning step size of 0.020 (2q) and scan step time of 1 s (Denizli et al., 1988).

Preparation of drug-loaded nanoparticles
Drug-loaded nanoparticles were prepared using emulsion droplet coalescence method (Conti et al., 1998).Chitosan (CS) was dissolved in 1% acetic acid and 50 mg of 5-flourouracil in phosphate buffer saline.This solution was added to 10 ml of liquid paraffin containing 0.5% v/v Tween 20.This mixture was stirred using a homogenizer for 3 min to form water in oil (w/o) emulsion.Similarly, another w/o emulsion consisting of 1% Eudragit RS 100 in 3 M sodium hydroxide solution was prepared.Then, these two emulsions were stirred using homogenizer.As a result of coalescence of droplets, chitosan was solidified to produce nanoparticles.Eudragit RS 100 produces secondary coating over chitosan nanoparticles.The obtained 5-flourouracil drug loaded nanoparticles were centrifuged at 3000 rpm for 60 min using Remi centrifuge and washed using ethanol and water, repeatedly to remove the remaining surfactant and liquid paraffin.Later, they were dried in air for 3 h and kept in hot air oven at 50°C for 4 h and stored in a desiccator.

Transmission electron microscope (TEM)
The particle shape and morphology of the prepared 5-fluorouracil nanoparticles were determined by TEM analysis.The nanoparticles were viewed using Philips TEM model CM200 as shown in Figure 13a, b, c, d, e and f for morphological examination.
The sample can be mounted on carbon/formvar coated copper grid or can be made of disc type with a thinned (electron transparency) central area of size 3 mm.Operating voltages are 20 to 200 kv and the resolution was 2.4 A° (Carlos, 2005).

Zeta potential measurement of the nanoparticles
Zeta potential of the nanoparticles was determined by laser Doppler anemometry using a Malvern Zetasizer also called Doppler electrophoretic light scatter analyzer.It is used to measure velocities and thereby zeta potential of colloid particles (Sanyogitta, 2007).

Percentage yield
The nanoparticles were prepared by emulsion droplet coalescence method from the study of [16] and percentage yield was calculated by dividing the weight of obtained nanoparticles by the weight of calculated ingredients of nanoparticles and expressed in terms of percentage.

Loading efficiency
The loading efficiency was determined by centrifuging the drugloaded nanoparticles at 5000 rpm for 30 min and separate the supernatant, and collected particles were washed with water and then subjected to another cycle of centrifugation.The amount of free 5-fluorouracil in the supernatant was assayed by UV spectrophotometer (UV-1700 Lab india) at 266 nm.The loading efficiency was calculated by using the formula (Leroux et al., 1995;Ganta et al., 2009):

Entrapment efficiency
The entrapment efficiency was determined using the following formula: Zeta potential and polydispersity Zeta potential and particle size are two important characteristics of nanoparticles that were determined by Zetasizer 3000 analyser system (Malvern instrument, UK) (24-25)

In vitro drug release study
The in vitro drug release profile of 5-FU-Ch-NP was determined using dialysis membrane bag.5-FU-Ch-NP (20 mg) was placed in to dialysis bag (with a molecular cut-off of 5 kDa).5-FU-Ch-NP loaded dialysis bag was incubated in 70 ml phosphate buffer (pH 7.4).The system was maintained at 37±0.5°C with mild magnetic stirring.At appropriate time interval, 4 ml of the release media was taken and equivalent volume of fresh phosphate buffered saline (PBS) solution was supplemented in order to keep the volume of the system identical.The sample was assayed at 266 nm by UV-Spectrophotometer (Lab India) and the cumulative percentage of drug release was calculated (Wan et al., 2009)

In vitro release kinetics study
In order to analyze the drug release mechanism, in vitro release data were fitted into a zero-order, first order, Higuchi, and Korsmeyer-peppas model.

Zero order kinetics
The zero order rate equation describes the systems where the drug release rate is independent of its concentration.Q1 = Q0 + K0 t

First order kinetics
The first order equation describes the release from a system where the release rate is concentration dependent.Kinetic equation for the first order release is as follows: Log Qt = log Q0 + K1 t/2.303

Higuchi model
Higuchi describes drug release as a diffusion process based in the Fick's law, square root time dependent.

Korsmeyer-Peppas model
To find out the drug release mechanism, first 60% drug release data can be fitted in Korsmeyer-Peppas model which is often used to describe the drug release behavior from polymeric systems when the mechanism is not well-known or when more than one type of release phenomena is involved.

RESULTS AND DISCUSSION
The identification of drug was studied by FTIR, DSC and XRD analysis.The FTIR spectra for pure drug 5fluorouracil are as shown in Figure 3    DSC thermogram of 5-fluorouracil is as shown in Figure 8.This reveals that the drug shows good thermal stability up to its melting point.The onset melting peak is about 273.47°C.It suggests that the drug is stable up to 280.13°C and undergoes degradation above that temperature.
DSC thermograms for chitosan and Eudragit RS 100 are as shown in Figures 9 and 10 and it was found that the onset of melting peak is 294.8°C and it is stable up to 321°C.The other peak observed may be due to presence of moisture.The thermogram of Eudragit RS 100 onset melting peak was observed at 376.08°C and it shows good thermal stability up to 389.6°C.The other peak observed at 412.6°C may be due to the presence of moisture.DSC thermogram for physical mixture is as shown in Figure 11  Figure 12.Sharp peak between 28.385 and 28.525° was the characteristic of 5-fluorouracil.The most intensive peaks of 5-fluorouracil were observed at 2 of 17, 29, and 32° suggesting the crystalline nature of drug.5-Fluorouracil loaded polymeric nanoparticles were successfully prepared by emulsion droplet coalescence method using biodegradable polymers chitosan and Eudragit RS 100.All the prepared drug loaded nanoparticles are in white and powdery appearance.The prepared nanoparticles were studied for various evaluation parameters.
Particle size and morphological characteristics for some of the prepared nanoparticles were studied by TEM analysis using Philips TEM model CM200.The corresponding images are as shown in Figure 13.They reveal that the particles of all formulations are in submicron sizes of peculiar shapes having rough or irregular surfaces; some aggregates are also observed which can be attributed to the gelling property of chitosan leading to particle-particle aggregation.
Percentage yield or particle recovery of prepared nanoparticles was calculated and the resulted values are summarized in the Table 1.The percentage yield of prepared nanoparticles was in the range of 64.76 to 87.14%.The variations in the percentage yield may be attributed to process parameters.Drug loading and entrapment efficiency of each formulation was calculated and the results were summarized in the Table 2. From the Table 2, it was found that the loading efficiency of CF1 to CF6 formulations was in range of 13.17 to 19.84% and for EF1 to EF5 formulations it was found between 13.55 and 20.16%.Subsequently, the entrapment efficiency of CF1 to CF6 was found to be in range of 65.5 to 75.6% and for EF1 to EF5 formulations it was found between 64.6 and 81.6%.From these results, it was evident that the drug loading efficiency of formulation increases with the increased chitosan and Eudragit RS 100 concentration.
Average zeta potential, average diameter and polydispersity index (PDI) values of the analysed formulations were summarized in Table 2 from which it was observed that the zeta potential values were in range of ±2.46 to ±48.4 indicating the increasing colloidal stability of nanoparticles with increased polymer concentration.The positive zeta potential values are representative to quaternary ammonium group present in Eudragit RS 100.The PDI values were within the range of 0.14 to 0.687.This indicates that the particles are in uniform size distribution.From the Table 2, it was observed that as polymer concentration increases, the particle size also increased.The size distribution of nanoparticles was found to be in desired range, that is, between 21 to 778.2 nm.The zeta potential and size distribution for optimized formulations are given in the Figure 14a The in vitro drug release study was performed by diffusion method for a period of 12 h using modified permeation apparatus.The percent cumulative drug released for each formulation was recorded and given in Table 3.The graphical representations of time versus percent cumulative drug release (CDR) plots for formulations CF1 to CF6 are as shown in Figure 16, and for EF1 to EF5 in Figure 17.From the graphs, it has been shown that percent cumulative drug release of CF1 to CF6 formulations was in range of 68.28 to 80.58% and for EF1 to EF5 formulations between 61.82 and 80.94%.
The drug release study shows that the drug release from the prepared nanoparticles was found to be more controlled as the polymer concentration increases.It is evident that from the results, the drug entrapment efficiency and the particle size have a direct effect on the drug release profile of formulations.From the results, EF5 formulation having high entrapment efficiency (81.6%) and particle size of 778.2 nm was found to have more controlled drug release as it is having high concentration of Eudragit RS 100 as secondary coating.The percent cumulative drug released for EF5 after 12 h was found to be 61.82%.
The mechanism of 5-fluorouracil release and the kinetic order of drug release from the nanoparticles were studied by fitting the in vitro drug release data of formulations into different kinetic models: zero order, first order, Higuchi and Korsmeyer-Peppas models.As shown in Table 4, it is evident that R 2 values for zero order plots of all formulations (CF1 to EF5) were ranging from 0.939 to 0.991; for first order plots, 0.805 to 0.953; for Higuchi

Conclusion
The emulsion droplet coalescence method procedure
and the peaks observed from the graph is evident that the absorption bands at1661.51,1449.89,3136.40,1430.70 and 1246.87 cm -1 indicate the presence of C=O, C=C, N-H, C-F and C-N stretching vibrations corresponding to 5fluorouracil, the peak at 1349.35 cm -1 refers to vibration of pyrimidine compound confirming 5-fluorouracil.Drug-polymer compatibility studies were performed by using FTIR and DSC techniques.The FTIR spectra for 5fluorouracil and mixture of 5-fluorouracil with different polymers are as shown in Figures 4, 5, 6 and 7, respectively and the characteristic peaks observed all the characteristic absorption bands of 5-fluorouracil were
,b and Figure 15a,b respectively.

Table 2 .
Evaluation parameters of 5-fluorouracil nanoparticles..766 to 0.904; and for Peppas equation, 0.931 to 0.998.The diffusion exponent values of Peppas plots were in range of 0.435 to 0.705.This data reveals that drug release from nanoparticles follows zero order release kinetics with non-Fickian diffusion for all formulations except CF2 and EF3 formulations which are following zero order release with Fickian diffusion.