Abstract
The relapse of cancer after first line therapy with anticancer agents is a common occurrence. This recurrence is generally accepted to be due to the presence of a small subpopulation of cells called cancer stem cells. Thus, it is essential to use a combination therapy consisting of at least one antitumor agent to which the cancer stem cells are susceptible. The objective of the present study was to develop and characterize nanoparticles containing two anticancer agents having different mechanisms of action that would target both differentiated as well as cancer stem cells.|The two hydrophobic antitumor agents (cyclopamine and paclitaxel) were entrapped in glyceryl monooleate (GMO)-chitosan solid lipid nanoparticles using Poloxamer 407 as a stabilizer. The nanoemulsion was prepared by double emulsion method using ultrasonication. This emulsion was freeze dried. A sensitive UPLC method for simultaneous detection and quantification of both the drugs was successfully developed and validated. The particle size (PS) and zeta potential (ZP) was determined using the Zetameter. The surface morphology was analyzed using the Atomic Force Microscopy (AFM). The drug loaded nanoparticles were monitored for their physical and chemical stability over a period of 60 days. Differential scanning calorimetry (DSC) was used to help analyze the physical state of drugs in the nanoparticles. Percentage of weight loss on heating and moisture content of the nanoparticles was assessed using thermogravimetric analysis (TGA) and Karl Fisher titrimetry respectively. The in vitro release of cyclopamine and paclitaxel was evaluated in phosphate buffer containing 0.5% w/v Tween 80 at 37ºCover 7 days. The in vitro cytotoxicity was tested on prostate cancer cells (DU145 and DU145 TXR) and normal cells (Wi26 A4) whereas the cellular uptake of the nanoparticles was studied using the cancer cells. The nanoparticles were also tested for their hemolytic potential on red blood cells. The cytotoxicity studied showed that the blank GMO-Chitosan nanoparticles were as cytotoxic as the drug loaded ones. This led to the development of an alternative nanoparticle system for drug delivery. The new system chosen for the study was the Poly (D, L)- Lactic-co-Glycolic Acid (PLGA) system. Blank and drug loaded PLGA nanoparticles were prepared by single emulsion followed by solvent evaporation technique. The PLGA nanoparticles were characterized using the same tests that were used to characterize GMO-Chitosan nanoparticles. |The UPLC method developed for the detection of drugs was validated for specificity, linearity, precision and accuracy. This method was able to detect the drug concentrations to as low as 3.1 µg/mL. The GMO-Chitosan nanoparticles obtained after freeze drying showed high entrapment efficiencies for both the drugs. The particle size of drug loaded GMO-Chitosan and PLGA nanoparticles was found to be 278.4±16.4 nm and 234.5±6.8 nm respectively. The GMO-Chitosan nanoparticles showed a positive zeta potential whereas the PLGA ones showed a negative zeta potential. The GMO-Chitosan nanoparticles were found to be physically stable; however, they showed a decrease in the cyclopamine content (approximately 59% w/w) over a period of 2 months. The AFM images showed that both GMO-Chitosan as well as PLGA nanoparticles were spherical in shape. Both drugs were found to be in non-crystalline state in both nanoparticles. A sustained release for both the drugs was observed in GMO-Chitosan and PLGA nanoparticles. Cytotoxicity studies revealed that blank GMO-Chitosan nanoparticles were as cytotoxic as the drug loaded ones whereas PLGA blank particles showed no cytotoxicity in all the cell lines tested. Similar results were replicated in the cellular uptake studies. Confocal microscopic studies revealed that PLGA nanoparticles were completely internalized into the cells tested after 5 minutes of treatment in the cancer cells. GMO-Chitosan and PLGA nanoparticles showed no hemolysis which confirmed their suitability to be used as parenteral formulations.