Synthesis of Polymeric Nanoparticles for Plasmid DNA Delivery
Dugas, A.C1. , R.K. Cooper2, F.M. Enright2, and C. M. Sabliov1
1 BAE Department, LSU AgCenter
2 Veterinary Science, LSU AgCenter
ABSTRACT
The goal of the research proposed is to design and synthesize a nanoparticle delivery system for plasmid DNA that will efficiently deliver genetic information to targeted cells in vitro and in vivo. A surface cationic, plasmid-docking polymeric nanoparticle system will be developed to entrap plasmid DNA. In the plasmid-docking nanoparticle system, surface-cationic particles are synthesized without the encapsulation of the pDNA. Prior to delivery, pDNA is incorporated and surrounded by multiple nanoparticles for protection. It is hypothesized that the cationic particle system will neutralize the negatively charged DNA by the surface binding which will, in turn, assist in cell and nuclear membrane penetration when compared to naked pDNA. The plasmid-docking system is also thought to exhibit a slightly positive surface charge upon degradation which may assist in cellular endo-lysosomal release. The following characteristics will be considered when classifying the particle system as successful: biodegradability and biocompatibility, size, particle stability in storage and during sterilization, high plasmid DNA entrapment efficiency, plasmid DNA protection in vitro and in vivo, and delivery of “active” plasmid DNA to nuclei of targeted cells more efficiently than with naked DNA injections.
Experimental Design and Multivariate Analysis for Optimizing Poly (D,L-lactide-co-glycolide) (PLGA) Nanoparticle Synthesis Using Molecular Micelles
Gabriela M. Ganea a, Cristina M. Sabliov b, [1], Abiodun O. Ishola c, Sayo O. Fakayode a, Isiah M. Warner a
a Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803, USA
b Department of Biological and Agricultural Engineering,
Louisiana State University Agricultural Center, Baton Rouge, LA 70803, USA
c College of Basic Sciences, Louisiana State University, Baton Rouge, LA 70803, USA
Abstract
The utility of polymeric nanoparticles as drug delivery systems depends on effective control of synthetic parameters with a significant impact on their physico-chemical characteristics. In this study, a chemometric central composite experimental design (CCD) was used to optimize the synthesis of poly (D,L lactide-co-glycolide) (PLGA) nanoparticles by emulsification solvent evaporation using anionic molecular micelles, such as poly (sodium N-undecylenic sulfate) (poly-SUS), poly (sodium N-undecanoyl-glycinate) (poly-SUG) and poly (sodium N-undecanoyl-L-leucyl-valinate) (poly-L-SULV) as well as conventional emulsifiers, such as anionic sodium dodecyl sulfate (SDS) and non-ionic poly (vinyl alcohol) (PVA). The individual and combined effects of PLGA concentration, emulsifier concentration, homogenization speed, and sonication time (design variables) on particle size and polydispersity index (responses) were investigated using multivariate analysis. The most significant design variables influencing the nanoparticle size and size distribution were PLGA concentration and emulsifier concentration (p < 0.05) in comparison to the other design variables. The quadratic model demonstrated the highest predictive ability when the molecular micelles were used as emulsifiers. The PLGA nanoparticles optimally synthesized according to the CCD were further purified by dialysis and then freeze dried. Dried nanoparticles synthesized with molecular micelles and PVA were readily re-suspended in water, as compared with SDS for which nanoparticle aggregation occurred. The size of PLGA nanoparticles synthesized using molecular micelles increased after freeze drying, but remained below 100 nm when poly-L-SULV was used as emulsifier. The PDI values indicated monodisperse nanoparticle suspensions after purification and freeze drying for all investigated molecular micelles (PDI < 0.100). The nanoparticle suspensions synthesized using molecular micelles were the most stable after dialysis and freeze drying, having low negative zeta potential values ranging from - 54 ± 1.6 mV for poly-L-SULV to - 63.2 ± 0.4 mV for poly-SUS. Transmission electron microscopy (TEM) micrographs showed spherical shape and smooth surface for the PLGA nanoparticles synthesized using molecular micelles.