Gene delivery nanoparticles fabricated by supercritical fluid extraction of emulsions

Abstract

Non-viral polymeric gene delivery systems offer increased protection from nuclease degradation, enhanced plasmid DNA (pDNA) uptake, and controlled dosing to sustain the duration of pDNA action. Such gene delivery systems can be formulated from biocompatible and biodegradable polymers such as poly(D,L-lactic-co-glycolic) acid (PLGA). Experimental loading of hydrophilic macromolecules such as pDNA is low in polymeric particles. The study purpose was to develop a supercritical fluid extraction of emulsions (SFEE) process based on CO(2) for preparing pEGFP-PLGA nanoparticles with high plasmid loading and loading efficiency. Another objective was to determine the efficacy of pFlt23k, an anti-angiogenic pDNA capable of inhibiting vascular endothelial growth factor (VEGF) secretion, following nanoparticle formation using the SFEE process. Results indicated that the SFEE process allows high actual loading of pDNA (19.7%, w/w), high loading efficiency (>98%), and low residual solvents (<50 ppm), due to rapid particle formation from efficient solvent removal provided by the SFEE process. pFlt23K-PLGA nanoparticles were capable of in vitro transfection, significantly reducing secreted VEGF from human lung alveolar epithelial cells (A549) under normoxic and hypoxic conditions. pFlt23K-PLGA nanoparticles did not exhibit cytotoxicity and are of potential value in treating neovascular disorders wherein VEGF levels are elevated.

Figure 1

SFEE piping and instrumentation diagram of semi-continuous process.

Figure 2

Transmission electron micrographs of: A) SFEE-PLGA 85:15- placebo nanoparticles (69,000×), B) pFlt23K-PLGA (2% w/w loaded) nanoparticles (82,000×), and C) pEGFP-PLGA (20% w/w loaded) nanoparticles (54800×).

Figure 3

In vitro cumulative amount released (μg) of plasmid DNA from pFlt23K-PLGA (2% w/w loaded) and pEGFP-PLGA (20% w/w loaded) nanoparticles. Data represented as mean ± SD (n = 3).

Figure 4

VEGF secreted from A549 cells in response to various treatment groups. pFlt23K concentration used = 5 μg/well. The data is expressed as mean ± SD for n = 8; *denotes significant difference from normoxia control, and ^†denotes significant difference from hypoxia control (p ≤ 0.05).

Figure 5

MTT assay assessing the cytotoxicty of the various treatments in VEGF ELISA experiments. The data is expressed as mean ± SD for n = 5.

Figure 6

Mechanism (proposed by Chattopdhyay et al. modified for plasmid DNA incorporation) of mass transfer in solvent extraction during SFEE processing. N_Transfer:

Direct mass transfer of solvent into SC CO₂

N_Diffusion:

Diffusion of solvent into emulsion continuous phase, subsequent mass transfer from this mixture into SC CO₂.

SC CO₂ flux:

SC CO₂ movement into the emulsion droplet leading to expansion of the organic phase.