Effect of different parameters on the pDNA-loaded microspheres prepared by double emulsion solvent evaporation method

Biodegradable poly(D,L-lactide-co-glycolide) (PLGA) microspheres is a promising gene carrier system to deliver the gene in gene therapy. This study describes fabrication and characterization of plasmid DNA-loaded PLGA microspheres using a versatile double-emulsion (w/o/w) solvent-evaporation method. For microsphere fabrication, plasmid DNA in TE buffer was added to PLGA solution, previously dissolved in DCM, and was homogenized to form the primary water-in-oil (w/o) emulsion. This w/o emulsion was immediately injected into 1% aqueous PVA and homogenized to form the secondary water-in-oil-in-water (w/o/w) emulsion. The w/o/w emulsion was then transferred to a continuously stirred hardening tank of 1% aqueous PVA and the stirring was continued for 2 hours to allow complete evaporation of DCM. The hardened microspheres were collected by centrifugation, washed and freeze-dried. Several parameters have been investigated including PVA concentration, different types of surfactants and surfactant blends and PLGA molecular weight. Resultant microsoheres were characterized for size distribution and external morphology by laser and scanning electron microscopy, respectively. Encapsulation efficiency was also calculated and the DNA was quantified by UV absorbance (NanoQuant). it was found that, increasing the PVA concentration from 1% w/v to 5% w/v reduced that mean particle size from 11.39um to 3.74um. These size were also evident by the microimages that depicted smooth surfaces of microspheres yielded for the range of PVA concentration. This research is still ongoing the future aims include transfection on neuro cell line to see feasibility of using this carrier system to deliver relevant gene to treat neurodegenerative diseases at molecular level