DEVELOPMENT AND IN VITRO EVALUATION OF 5-FLUOROURACIL NANOPARTICLES BY SALTING OUT TECHNIQUE

Objectives: At the current miserable state of the prevalence of cancers, there is a need for the development of simple technologies to prepare formulations of anticancer drugs with less economy and investment. Hence, the aim of the present work is to prepare nanoparticles of 5-fluorouracil (5-FU) by simple technique, such as salting out method. Methods: Nanoparticles containing 10 mg of 5-FU were prepared by salting out method using Eudragit-100 as polymer. The prepared nanoparticles were evaluated by particle size, zeta potential, in vitro drug release studies, and drug-excipient interaction studies. Results: Nanoparticles prepared by salting out methods showed higher dissolution rate for formulation F3 and F5 revealed high percentage release of 98.6±0.24 in 60 min and 86.5±0.39% in 120 min. Fourier transform infrared (FTIR) spectra revealed no interaction between drug and excipients used for preparation. Conclusion: 5-FU nanoparticles can be produced successfully by salting out method using drug to polymer (Eudragit S-100) ratio of 1:3 to possess ideal drug release characteristics and average particle size of 205.1 nm. were taken in 20 ml of distilled water and mixed well to dissolve completely (aqueous phase). Organic phase is suddenly poured in to aqueous part under stirring. Stirring is continued for about 3 h under mechanical stirring for about 1500 rpm. After stirring, small quantity of water was added to the dispersion, mixed well, and subjected for vacuum filtration. The filtrate was dried in Salting out is based on separation of water miscible solvent from aqueous solutions through salting out effect. Polymer and drug are initially dissolved in a solvent which is emulsified into aqueous gel containing salting out agent. This o/w emulsion is diluted with sufficient quantity of water to enhance diffusion of solvent into aqueous phase. Thus inducing the formation of nanospheres. In this method, several manufacturing parameters can be varied including stirring rate, internal/external ratio, concentration of polymers in the organic phase, type of electrolyte, and type of stabilizer in aqueous phase [10]. ABSTRACT


INTRODUCTION
At present, cancer is the leading cause of mortality and one of the most difficult global health hazards to tackle [1]. Due to high prevalence of various types of cancers right now, there is a large demand for therapeutics. The prevalence did not spare the people based on their economic and social status. India is ranked one with oral cancer, especially in low-class public. According to the WHO, cancer is the second leading cause of human deaths. Hence, there is a need and demand for the production of formulations of anticancer drugs by economical and simple techniques with optimum quality.
Pancreatic cancer is the ninth more prevalent and challenging to treat. Several formulations are now available for the treatment of pancreatic cancer and 5-fluorouracil (5-FU) is one of the most widely used drugs to treat pancreatic cancer [2,3]. It is a sparingly soluble drug with less cellular uptake [4]. Hence, the conversion of the dug in the form of nanoparticles may solve these defects to enhance solubility, leading enhanced cellular uptake and permeability. At this context, formulations of 5-FU can develop using nanotechnology. Especially, nanoparticles as a tool for targeted therapy in cancer treatment have an attention due to higher intracellular uptake compared to microparticles (>1 um) [5,6]. The effective delivery of the drug in the epithelium of the blood in human tumors has been according to pore size, which varies from 200 to 600 nm and the particles should be <200 nm [7][8][9].
Hence, the aim of the present work is the design of nanoparticles of 5-FU by simple method such as salting out technique as this particular method is not so far reported for the production of nanoparticles of 5-FU. This technique may become a promising, simple, and economical process for the production of nanoparticles possessing optimum solubility and drug release characteristics.

Materials
5-FU was obtained as a gift sample from Aurobindo Pharma Ltd., Eudragit S-100 and sodium carboxymethyl cellulose (CMC) were purchased from Merck Chemical Company (Mumbai, India), zinc sulfate and ethanol were obtained from Sigma-Aldrich (Mumbai, India), and all other chemicals used in the study are of analytical grade.

Preparation of nanoparticles of 5-FU
Nanoparticles of 5-FU were prepared by salting out method [11]. Various trials containing 10 mg of 5-FU were tried and five number of formulations (F1-F5) produced with clear yield are presented in Table 1. During the preparation, 5-FU and Eudragit S-100 were dissolved in ethanol (organic phase). In another beaker, 2 g of sodium CMC and 4 g of zinc sulfate were taken in 20 ml of distilled water and mixed well to dissolve completely (aqueous phase). Organic phase is suddenly poured in to aqueous part under stirring. Stirring is continued for about 3 h under mechanical stirring for about 1500 rpm. After stirring, small quantity of water was added to the dispersion, mixed well, and subjected for vacuum filtration. The filtrate was dried in Salting out is based on separation of water miscible solvent from aqueous solutions through salting out effect. Polymer and drug are initially dissolved in a solvent which is emulsified into aqueous gel containing salting out agent. This o/w emulsion is diluted with sufficient quantity of water to enhance diffusion of solvent into aqueous phase. Thus inducing the formation of nanospheres. In this method, several manufacturing parameters can be varied including stirring rate, internal/external ratio, concentration of polymers in the organic phase, type of electrolyte, and type of stabilizer in aqueous phase [10].

Sailaja and Jyothi
lyophilizer (Lyodel, JAPAN) for 24 h and the product was subjected to in vitro evaluation.

In vitro evaluation of nanoparticles of 5-FU
Prepared formulations, F1-F5, are subjected to in vitro evaluation by particle size determination, zeta potential measurement using Zetasizer, scanning electron microscopy, entrapment efficiency, in vitro dissolution studies, and drug-excipient interaction studies by Fourier transform infrared (FTIR).

Drug content estimation
Nanoparticles equivalent to containing 10 mg of 5-FU were weighed and taken in 100 ml beaker containing 50 ml of ethanol. The solution was stirred at 1000 rpm for 4 h in magnetic stirrer. The resultant solution was filtered and estimated for drug content using ultraviolet (UV)--visible spectrophotometer at λ max at 265 nm.

In vitro dissolution studies
The drug release studies were carried out using USP XX1 dissolution testing type-II apparatus (Electrolab, INDIA) for prepared formulations F1-F5 and for pure drug. The dissolution medium was pH 6.8 phosphate buffer maintained at temperature of 37±1°C with rotating speed of 100 rpm. At predetermined time intervals, aliquot samples were withdrawn and diluted wherever necessary and analyzed for drug content by UV spectrophotometer (Systronics, INDIA) λ max at 265 nm. The volume withdrawn was replaced with fresh dissolution medium maintained at same temperature.

Particle size determination and zeta potential measurement
Particle size and zeta potential of formulations F3 and F5 with enhanced % drug release values among all prepared formulations were determined using Zetasizer (Horiba).

FTIR analysis
The FTIR spectra of the samples were recorded for formulation F3 and for pure 5-FU using a FTIR spectrometer (Bruker, JAPAN). A small quantity of nanoparticles was mixed with 200 mg of KBr and compressed to form pellets. These pellets were scanned in transmission mode in the spectral region 4000-400 cm −1 using a resolution of 4 cm −1 and 32 coadded scans.

RESULTS AND DISCUSSION
In the present work, five formulations, F1-F5, were tried to produce nanoparticles of 5-FU by salting out method. This method is based on the separation of water miscible solvent from aqueous solution through salting out effect. The concentration of zinc sulfate will prevent the miscibility of ethanol into aqueous medium on mechanical agitation of this system, emulsion is formed. The formed emulation droplet size controlled by salting out agent. The increasing concentration of salting agent reduced the size of particles.

Drug content
The results of percentage drug content of formulations are presented in Table 2. It was observed that as the drug to polymer concentration increases from F3 to F5, drug content was found to be acceptable with the range of 96.5%±0.61-99.7%±0.55.

In vitro drug release studies
Cellular uptake of anticancer drug delivery systems plays a crucial role to elicit effective action against the cancerous tissues. Hence, as an indirect assessment, the prepared formulations were assessed for in vitro drug release studies. The results of drug releasing studies of F1-F5 and pure 5-FU are presented in Table 3 and Fig. 1. From the data, it is observed that there is enhanced % drug release from all the prepared formulations compared to pure drug release. Among all, formulations, F3 (98.5%) and F5 (88.07%), evidenced high % drug release values.

Particle size and zeta potential
The particle size of prepared nanoparticles of formulations F3 and F4 is determined as the drug release is high from them. The scan copies indicating particle and zeta potential obtained from Zetasizer are presented in Figs. 2 and 3 and the values are shown in Table 4. The mean particle size of formulation F3 is 205.1 nm and formulation F5 is 231.6 nm. This indicates that the present method used for preparing nanoparticles is successful in producing the yield in nanosize range. Zeta potential influences the stability of nanoparticles. Extremely negative values of zeta potential indicate large repulsive forces showing the stability of prepared nanoparticles.       there is no interaction between 5-FU and the excipients used to prepare nanoparticles.

Drug release kinetics
Drug release kinetics of promising formulation F3 was assessed by zero-order, first-order, Higuchi, and Korsmeyer-Peppas (k-p) mechanisms and the relevant plots are shown in Fig. 6 and the data of copies given plots. It is evident that regression values for zero order were more linear (0.976) compared to the first order (0.879), indicating that the release of drug is dose independent. Korsmeyer-Peppas plot (0.903) with n = 0.862 which falling in between 0.45 CONCLUSION 5-FU nanoparticles were successfully produced by salting out method using drug-to-polymer (Eudragit S-100) ratio of 1:3 to possess ideal drug release characteristics of 72.6% in 90 min and 98.5% in 120 min with average particle size 205.1 nm.
Scope 5-FU can be produced as nanoparticles by salting out method which on large scale after pilot plant scale-up studies which may become a promising economical method on the part of the industrial production.