Design and Evaluation of Sorafenib Tosylate Nanoparticles Including Assessment of IC50 Values using PC Cell Lines

Introduction: Sorafenib tosylate is an anticancer drug used for treatment of pancreatic cancer. In the present research work, Sorafenib tosylate is converted to nanoparticles with an aim to assess its anticancer activity with reduced concentration expecting less side effects of the parent drug. Objective: The aim of the present research work is preparation of nanoparticles of Sorafenib tosylate, evaluation at in-vitro level and to possess ideal drug release characteristics. IC50 values of nanoparticles of sorafenib tosylate are reasonably reduced compared to pure drugs indicating very chances of reduced side effects with nanoparticles to treat pancreatic cancer effectively with reduced side effects.


INTRODUCTION
Anti-cancer drugs prepared in the form of nanoparticles possess many therapeutic advantages including greater site-specific effect, high efficacy with less dose, less side effects to treat tumour cells [1,2].
Sorafenib tosylate (SRB) was approved by USFDA in December 2005, and received European commission marketing authorization in July 2006 for the use in the treatment of hepatocellular carcinoma that cannot be removed by surgery, renal cell carcinoma. It is one of the most preferred kinase inhibitor drug that formerly approved for therapy for primary kidney cancer (advanced renal cell carcinoma) [3].
It is a poorly water-soluble drug and commercially available as film coated tablets. The conversion to nanoparticles can be a promising approach to develop formulation suitable for oral or IV formulation due to expected rapid solubility, dissolution and bioavailability to treat pancreatic cancer [4]. Especially with the conversion of sorafenib tosylste to nanoparticles is expected to exhibit cytotoxic effect with much reduced concentration of active drug hence cause less side effects that are commonly possessed by chemotherapeutics.
Hence the main aim of the present research work is development and evaluation of nanoparticles of Sorafenib tosylate and to assess their activity for treatment of pancreatic cancer at in-vitro level by MTT Assay method using PC cell lines and to compare IC50 values of sorafenib tosylste nanoparticles and pure drug [5,6].

Preparation of nanoparticles of sorafenib tosylate by salting out method
Nanoparticles of sorafenib tosylate containing 200 mg of drug were prepared by salting out method. Various formulations were tried by changing composition and 8 no. of formulations (F1 to F8) produced with clear yield are presented in Table 1. During the preparation, Sorafenib tosylate and eudragit L-100 were dissolved in ethanol (organic phase). In another beaker, 2 gm of sodium CMC and 4gm of zinc sulphate 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 3h under mechanical stirring for about 1500 rpm. After stirring a small quantity of water was added to the dispersion, mixed well and subjected for vacuum filtration. The filtrate was dried in Lyophillizer (Lyodel, JAPAN) for 24 hrs and the product was subjected to in-vitro evaluation [7,8].

In-vitro evaluation of nanoparticles of sorafenib tosylate
Prepared formulations, F1 to F8 are subjected to in-vitro evaluation by particle size determination, zeta potential measurement by using Zeta sizer, scanning electron microscopy, entrapment efficiency, in-vitro dissolution studies and drugexcipient interaction studies by FT-IR. The dissolution medium was a pH 6.8 phosphate buffer that was kept at 37±1º C with a 100 rpm rotation speed. At predetermined time intervals aliquot samples were withdrawn and diluted wherever necessary and analysed 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 F6 and F8 with enhanced % drug release values among all prepared formulations was determined by using zeta sizer (Horriba).

FTIR analysis
The IR spectra of the samples were recorded for nanoparticles of SRB, F6 and for pure Sorafenib tosylate using a Fourier transform infrared spectrometer (Bruker, JAPAN). A small quantity of nanoparticles was mixed with 200mg 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 4cm-1 and 32-co-added scans [9,10].

MTT assay principle
MTT Assay is a colorimetric assay that measures the reduction of yellow 3-(4,5-dimethythiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase. The assay is based on the quantity of cells present as well as the premise that tetrazolium is not reduced by dead cells or their products.
MTT enters the cells and travels to the mitochondria, where it is converted to insoluble dark purple formazan crystals. The cells are subsequently dissolved in DMSO, and the solubilized formazan reagent is spectrophotometrically quantified at 570 nm.

Cell Line and maintenance
The Cancer cell lines were purchased from NCCS, Pune and the cells were maintained in MEM supplemented with 10 % FBS and the antibiotics penicillin/streptomycin (0.5 mL-1), in atmosphere of 5% CO 2 /95% air at 37 0 C.

Preparation of test compound
For MTT assay, each test compound was weighed separately and dissolved in DMSO.
Final concentration was made with medium and the cells were treated with series of concentrations from 1 to 5 µg/ ml.

Procedure
Cell viability was evaluated by the MTT Assay with three independent experiments with six concentrations of compounds in triplicates. Cells were trypsinized and performed trypan blue assay to know viable cells in cell suspension. Cells were counted by hemocytometer and seeded at density of 5.0 X 10 3 cells / well in 100 μl media in 96 well plate culture medium and incubated overnight at 37 0 C. After incubation, take off the old media and add fresh media 100 µl with different concentrations of test compound in represented wells in 96 plates [11,12].
After 48 hrs., Discarded the drug solution and added the fresh medium with MTT solution (0.5 mg / mL-1) was added to each well and plates were incubated at 37 0 C for 3 hrs. At the end of incubation time, precipitates are formed as a result of the reduction of the MTT salt to chromophoreformazan crystals by the cells with metabolically active mitochondria. The optical density of solubilized crystals in DMSO was measured at 570 nm on a microplate reader. The percentage growth inhibition was calculated following The concentration of the test compounds used to kill 50% of the of the growth of the cell lines, IC50 value was determined by using linear regression equation i.e. y=mx+c. Here, Y = 50, M and C values were derived from the viability graph.

RESULTS AND DISCUSSION
In this study, eight formulations (F1 to F8) were used to create Sorafenib tosylate nanoparticles using the salting out method. This approach relies on the salting out action to separate water miscible solvent from aqueous solution. On mechanical agitation of this system, the concentration of Zinc sulphate will impede the miscibility of ethanol in the aqueous medium, resulting in the formation of an emulsion. Salting out agent controls the size of the produced emulation droplet. The size of the particles decreased as the concentration of salting agent increased.

Drug Content
The results percent drug content of formulations are presented in Table 2. It was observed that as the drug to polymer concentration increases from F6to F8, Drug content were found to be acceptable with the range of 96.5% ±0.61 to 99.7%±0.55.

In-vitro drug release studies
The absorption of anticancer drug delivery systems by cells is critical for successful action against malignant tissues. As a result, the produced formulations were evaluated in-vitro drug release studies as an indirect assessment. The results of drug releasing studies of F4 to F8 and pure Sorafenib tosylate 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, formulation F3 evidenced high % drug release of 90.2% in 120 min and 82.5% in 90 min.

Particle size and zeta potential
The particle size of prepared nanoparticles of formulations F1 and F3 is determined as the drug release is high from them. The Scan copies indicating particle and zeta potential obtained from zeta sizer are presented in Fig. 3 and Fig. 4 and the values are shown in Table 4. As shown in table and zeata sizer data. the mean particle size of formulation, F6 is 205.1nm and formulation F8 is 231.6nm. These results evidence that method used for preparing nanoparticles is successful in producing the yield in nano size range. Extremely negative values of zeta potential -4.3mv and -2.2mv indicates large repulsive forces showing the stability of prepared nanoparticles.

Drug-excipient interaction studies by FT-IR
The FI-IR spectra of formulation F2 and pure Sorafenib tosylate are given in Fig. 5 and 6 and the absorption peaks are shown in Table 5 . All these peaks are also present in spectrum of prepared nanoparticles formulation with slight change. Hence it is considered that there is no interaction between Sorafenib tosylate and the excipients used to prepare nano particles.

Assessment of anticancer activity by MTT assay technique
The results of evaluation of anti-cancer activity of promising nanoparticles of sorafenib tosylate as well as pure durgs are shown in Figs 9 and 10 and respectively. The figures show much reduced staining in case of nanoparticles in Fig.9 compared to pure drug in Fig 10. The data showing the % inhibition and % viability of cancer cells against the pure drugs and nano particles are shown Tables 7 to 9. As presented, the IC50 value of Nano particles of sorafenib tosylate is 0.848 ± 0.217 and pure drug is 1.92±0.140. These values indicated reasonable reduction in IC50 values. This reduction in IC50 value for nanoparticles show the anticancer effect with much less reduced side effects possessed by psorafenib tosylate.

CONCLUSION
Sorafenib tosylate nanoparticles were successfully produced by salting out method using drug to polymer (Sorafenib tosylate: Eudragit L-100) ratio of 1:3 by salting out method to possess ideal drug release characteristics of 82.5% in 90min and 90.2% in 120min with average particle size 205.1nm. IC50 values of nanoparticles of sorafenib tosylate are reasonably reduced compared to pure drugs indicating very chances of reduced side effects with nanoparticles produced by simple technique of salting out method and hence effective in treating pancreatic cancer.

DISCLAIMER
The research was not funded by the any funding agencies and is purely by personal efforts of the authors.

CONSENT
It is not applicable.