Polymeric nanoparticles for the controlled and specific delivery of drugs and genes

Abstract

In this research, the process of preparation of biodegradable polymeric nanoparticles was evaluated. The emulsification and nanoprecipitation techniques were used to prepare drug and gene loaded nanoparticles of poly-dl-lactic-co-glycolic acid (PLGA). A comparison of the main parameters controlling the preparation of nanoparticles by both techniques was completed. These parameters included: the polymer concentration; surfactant concentration; organic to aqueous rate; sonication amplitude or injection speed; and the agitation speed during solvent evaporation. The centrifugation speeds during purification of nanoparticles and the use of cryoprotectants in the emulsification and nanoprecipitation techniques were also investigated. Polymeric nanoparticles of PLGA (PNP) loaded with methylene blue (MB) were prepared by a combination of the single and double emulsification techniques, and compared with the individual techniques. PNP loaded with MB (MB-PNP) sizes obtained from the combined technique are similar to the single emulsion technique, while the diameter of particles prepared by double emulsion increased in proportion to the mass of MB used in the preparation. Experimental release of MB from MB-PNP nanoparticles was evaluated obtaining a monophasic release profile. A mathematical model of release that simultaneously combines the mechanisms of initial burst and drug diffusion was used to successfully describe the experimental release. This drug release mathematical analysis could be extended to other drugs with partial solubility. The relationship between ROS produced by photoactivation of MB and BEAS-2B cell survival was evaluated. Increments of ROS produced by MB photoactivation are directly related to BEAS-2B cell survival. Even though BEAS-2B cells presented oxidative stress when exposed to MB alone, results suggest that BEAS-2B cells are more resistant to ROS than some cancerous cell lines reported in literature. Therefore, in lung cancer photodynamic treatments using MB, selective cell damage could be achieved. PLGA was modified with polyethylene glycol (PEG) and functionalized with folic acid (FA) following basic carbodiimide chemistry to obtain the copolymer PLGA-PEG-FA. This synthesis was verified by FT-IR and spectrophotometry methods. Nanoparticles of PLGA-PEG-FA loaded with pVAX1-NH36 (pDNA-PNP) were prepared by using the double emulsification-solvent evaporation technique. Plasmid pVAX1-NH36 was replicated in E. coli cell cultures and purified using a commercial kit. Experimental drug release presented a multiphase release profile for the duration of more than 30-days. Plasmid release was effectively analyzed with a mathematical model that considers a simultaneous contribution of initial burst and the degradation-relaxation of nanoparticle. This mathematical analysis presents a novel approach to describe and predict the release of plasmid DNA from biodegradable nanoparticles. A plasmid containing green fluorescent protein (pGFP) was encapsulated into PLGARhodamine nanoparticles (PLGA-Rh) to study plasmid expression and cellular uptake. PLGA-Rh nanoparticles loaded with pGFP (pGFP-PNP) were prepared by using double emulsification-solvent evaporation technique. PLGA-Rh synthesis was verified by FTIR. The difference in size and zeta potential between blank nanoparticles and pGFP-PNP suggest the successful encapsulation of the pGFP and the in vitro release studies showed a single-stage release profile with 10-days of duration. Cellular uptake and plasmid expression were confirmed by fluorescence microscopy visualization of the nanoparticles and the GFP protein on H441 cells.