Abstract
The current investigation involves preparation and characterization of a nanoparticulate drug delivery system which contains a model hydrophilic drug. The drug chosen for the purposes of these studies is methylglyoxal, which has shown significant anticancer properties. A rapid, sensitive and efficient Ultra Performance Liquid Chromatography (UPLC) method was developed and validated for the purpose of detection and quantification of methylglyoxal. The drug was first derivitized, and then analyzed by the UPLC. The derivitization process involved a reaction between methylglyoxal and ophenylenediamine to form its quinoxaine derivative. The reaction was carried out for 4 hours at room temperature, in the dark. The derivative was filtered through a 0.2 μm syringe filter and injected on to the UPLC. The mobile phase consisted of 70% (v/v) water and 30% (v/v) acetonitrile at a flow rate of 0.6 ml/min. The chromatographic separation was achieved on Waters Acquity UPLC system using an Acquity UPLC BEH C18 (50 x 2.1 mm), 1.7 μm column. The column effluents were monitored at 312 nm using a PDA detector. The method validation parameters include within day and day to day precision, linearity, accuracy and sensitivity. The retention time of the quinoxaline derivative of methylglyoxal was found to be 0.54 minutes. No interfering peaks from the mobile phase and the derivatized blank samples were detected with the drug peak. The standard curve was linear over a concentration range of 0.15-35.1 μg/ml. The RSD values for the within day and day to day precision ranged from 0.01 to 1.65% and 1.17 to 4.35%, respectively. The accuracy of this method ranged from 94 to 97.5% with a RSD value of 1.33 to 8.49%. GMO-Chitosan nanoparticles were prepared using multiple emulsion solvent evaporation technique. Two types of nanoparticles were prepared – crosslinked (with sodium TPP) and non-crosllinked (without sodium TPP) and were characterized. The surface morphology was studied using images obtained from a scanning electron microscope (SEM). The particle size and the surface charge were measured using a zetameter. The loss of weight on heating and moisture content were acquired using the thermogravimetric assay (TGA) and Karl Fisher Titrimetry (KFT) respectively. Differential scanning calorimetry was used to study the physical state of the drug in the formulation. The drug loading, entrapment efficiency and in vitro release pattern of the drug from the formulation were also studied. The samples for these studies were analyzed using the previously described UPLC method. The cytotoxicity profile of the nanoparticulate system and the free drug solution were observed and compared in normal (MDCK and WI26) and cancerous (A549, Cl66, HeLa and Caco-2) cell lines using the MTT cytotoxcity assay. The cellular uptake of the formulations and the free drug solution were studied in normal (MDCK and WI26) and cancerous (A549, Cl66 and HeLa) cell lines by cell lysis method. The SEM images revealed that both; the crosslinked and noncrosslinked nanoparticles were agglomerated. The surface of the crosslinked particles appeared to be more porous than that of the non-crosslinked one. The particle size and zeta potential of nanoparticles with TPP were 207.26±8.39 nm and -18.38±4.88 mV and that of nanoparticles without TPP were 240.2±23.14 nm and -13.37±3.31 mV, respectively. The drug load for nanoparticles without and with TPP was found to be 13.5±0.15% and 8.3±0.03% with entrapment efficiencies of 62.3±0.7% and 38±0.1% respectively. The in vitro release studies revealed that 93.8±2.6% and 98.6±2.3% of the drug was released from nanoparticles with TPP and without TPP within 8 hours. WI26 cells showed higher survival when treated with crosslinked nanoparticles compared to drug solution in 24 and 72 hours. However, a reverse trend was observed with noncrosslinked nanoparticles. On the other hand, MDCK, A549, Cl66, HeLa and Caco-2 cells showed lower survival when treated with nanoparticles, compared to drug solution in 24 and 72 hours. The cellular uptake of drug solution was significantly greater than nanoparticles in WI26 and HeLa cells. The cellular uptake of drug solution and nanoparticles were approximately same in MDCK, A549 and Cl66 cells. A sensitive UPLC method for the determination of methylglyoxal in aqueous solutions was developed and validated. A hydrophilic drug (methylglyoxal) was successfully entrapped in GMO-Chitosan system, with and without crosslinker. The entrapment efficiency was found to decrease with the addition of crosslinker. The crosslinker had no significant effect on the particle size, zeta potential and the drug release of nanoparticles, but generally showeda significant effect on its cytoxocity and cellular uptake in normal cells. No significant difference was seen between the uptake of drug solution and nanoparticles by the cancer cells in general.