Encapsulating Polyethyleneimine-DNA Nanoplexes into PEGylated Biodegradable Microparticles Increases Transgene Expression In Vitro and Reduces Inflammatory Responses In Vivo

Abstract

Encapsulating genetic material into biocompatible polymeric microparticles is a means to improving gene transfection while simultaneously decreasing the tendency for inflammatory responses; and can be advantageous in terms of delivering material directly to the lungs via aerosolization for applications such as vaccinations. In this study, we investigated the advantages of using polymeric microparticles carrying the luciferase reporter gene in increasing transfection efficiency in the readily transfectable HEK293 cell line and the difficult to transfect RAW264.7 cell line. The results indicated that there was a limit to the ratio of nitrogen in polyethylenimine (PEI) to phosphate in DNA (N/P ratio) beyond which further increases in transgene expression no longer, or only marginally, occurred. Microparticles encapsulating PEI:DNA nanoplexes induced cellular toxicity in a dose-dependent manner. PEGylation increased transgene expression, likely related to enhanced degradation of particles. Furthermore, intra-tracheal instillation in rats allowed us to investigate the inflammatory response in the lung as a function of PEGylation, porosity, and size. Porosity did not influence cell counts in bronchoalveolar lavage fluid in the absence of PEG, but in particles containing PEG, non-porous particles recruited fewer inflammatory cells than their porous counterparts. Finally, both 1 μm and 10 μm porous PLA-PEG particles recruited more neutrophils than 4 μm particles. Thus, we have shown that PEGylation and lack of porosity are advantageous for faster release of genetic cargo from microparticles and a reduced inflammatory response, respectively.

Keywords: Gene transfection; PEGylation; PLA-PEG; PLGA; Porosity.

Fig. 1

Relative cell viability as a percentage of the control (untreated cells) for HEK293 cells determined from the MTT assay. Cells were incubated with blank porous PLA-PEG microparticles. All reported values are the mean and standard deviation for n = 3

Fig. 2

Relative cell viability as the percentage of the control (untreated cells) for HEK293 (a) and RAW264.7 (b) cells incubated with PEI:pDNA loaded porous PLA-PEG microparticles with N/P = 15 and DNA concentration of 0.08 mg/mL. All reported values are the mean and standard deviation for n = 3

Fig. 3

Luciferase expression in HEK293 (black bars) and RAW264.7 (gray bars) cells for free PEI:pDNA nanoplexes (i.e., no microparticle encapsulation) after 48 h of transfection. All reported values are the mean and standard deviation for n = 3. Statistical significance was calculated using ordinary two-way ANOVA, comparing each value to the control (N/P 0) data set

Fig. 4

Luciferase expression in HEK293 (a) and RAW264.7 (b) cells following transfection with PEI:pDNA loaded porous microparticles for both PLA (black bars) and PLA-PEG (white bars). Microparticles were added at a concentration of 3 mg/mL (1 μg DNA). All reported values are the mean and standard deviation for n = 3 trials. Statistical analysis was performed using a two-way ANOVA with comparisons between PLA and PLA-PEG at each time point. Significant differences are denoted by ****p < 0.00001 or n.s. for no significance

Fig. 5

Number of macrophages, neutrophils, and total cells in BAL fluid of exposed and control animals. All values are the mean and standard deviation for n = 3