Multifunctional nanoparticles containing hydrophilic and hydrophobic drugs for anticancer drug delivery

Combination therapy is routinely used in anticancer therapy for maximizing efficacy and minimizing systemic toxicity. The nanoparticle drug delivery of the hydrophobic paclitaxel with hydrophilic gemcitabine offers the advantages of a combination therapy with the inherent advantage of a nanoparticle system. The objective of the present study was to develop and characterize PLGA nanoparticles containing both hydrophilic and hydrophobic anticancer agents and to evaluate the effect of drying techniques that includes lyophilization and spray drying, on physicochemical characteristics of these nanoparticles. Paclitaxel and gemcitabine were successfully entrapped into PLGA matrix using spontaneous emulsification-solvent diffusion method using PVA (2%) as a stabilizer. The nano-sized emulsion was obtained by using sonication followed by high pressure homogenization which was further subjected to lyophilization and spray drying. The drug entrapment efficiency and drug loading was determined by biphasic extraction. An HPLC method was developed and validated for simultaneous determination of paclitaxel and gemcitabine. The stability of both the formulations was evaluated over a period of 60 days. The surface morphology was analyzed using Atomic force microscopy (AFM). The physical state of the drug in the particles was analyzed using differential scanning calorimeter (DSC) and X-ray diffractometer (XRD). Moisture content and weight loss on heating were measured using Karl Fischer titrimetry and thermogravimetric analysis, respectively. The in vitro release of the drugs was evaluated at 37°C in pH 7.4 phosphate buffer containing 0.5% (w/v) Tween 80 by using HPLC analysis. The cytotoxicity of the formulation was tested in cancer cell lines (A549, CL66, MDA-MB-231) and normal cell lines (WI-26 A4, MDCK). Cellular uptake of nanoparticles and subcellular localization studies using rhodamine 6G loaded nanoparticles were performed in MDA-MB-231 and MDCK cells. The safety of lyophilized nanoparticle on injection was tested using hemolysis assay on red blood cells. A sensitive HPLC method for detection of paclitaxel and gemcitabine concentrations as low as 0.3mug/mL, was developed and validated for linearity, precision and accuracy. The spontaneous emulsification solvent diffusion method gave nanosized particles with higher entrapment of hydrophobic drug. Use of high pressure homogenization significantly reduced the particle size of the nanoemulsion as compared to sonication. Particle size of blank and drug loaded lyophilized nanoparticles was 175.8+/-0.63nm and 199.5+/-3.6nm respectively. The particle size of blank and drug loaded spray dried nanoparticles was 318.4+/-51.3nm and 657.3+/-11.7nm respectively. The lyophilized nanoparticles were stable over a period of 60 days at room temperature. The surface morphology observed in AFM showed spherical particles and differences in particle size of lyophilized and spray dried nanoparticles. Both the drugs were found to be present in amorphous or dissolved state in lyophilized and spray dried nanoparticles. The nanoparticle formulations showed a sustained release for paclitaxel and gemcitabine. The overall uptake of paclitaxel and gemcitabine from the nanoparticles was higher in cancer cells than normal cells. The confocal microscopic images confirmed the internalization of these nanoparticles into the lysosomes of the cells. The paclitaxel-gemcitabine nanoparticles were cytotoxic in the cell lines tested. The lyophilized nanoparticles, intended for injectable use, produced no significant hemolysis of red blood cells. The particle size, morphology, moisture content, release characteristics and cellular uptake were found to change with method of drying. However, zeta potential and physical state of the drug were seen to be unaffected by drying technique used.