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. 2020 Sep 21;3(9):6263-6272.
doi: 10.1021/acsabm.0c00761. Epub 2020 Aug 4.

Serum-Independent Nonviral Gene Delivery to Innate and Adaptive Immune Cells Using Immunoplexes

Atanu Chakraborty  1 , Jackline Joy Martín Lasola  2 , Nhu Truong  1 , Ryan M Pearson  1   2   3
Affiliations

Serum-Independent Nonviral Gene Delivery to Innate and Adaptive Immune Cells Using Immunoplexes

Atanu Chakraborty et al. ACS Appl Bio Mater. .

Abstract

Genetic engineering of innate and adaptive immune cells represents a potential solution to treat numerous immune-mediated pathologies. Current immune engineering methods to introduce nucleic acids into cells with high efficiency rely on physical mechanisms such as electroporation, viral vectors, or other chemical methods. Gene delivery using non-viral nanoparticles offers significant flexibility in biomaterial design to tune critical parameters such as nano-bio interactions, transfection efficiency, and toxicity profiles. However, their clinical utility has been limited due to complex synthetic procedures, high toxicity at increased polymer (nitrogen, N) to DNA ratios (phosphate, P) (N/P ratios), poor transfection efficiency and nanoparticle stability in the presence of serum, and short-term gene expression. Here, we describe the development of a simple, polymer-based non-viral gene delivery platform based on simple modifications of polyethylenimine (PEI) that displays potent and serum-independent transfection of innate and adaptive immune cells. Cationic acetylated PEI (Ac-PEI) was synthesized and complexed with plasmid DNA (pDNA) followed by enveloping with an anionic polyelectrolyte layer of poly(ethylene-alt-maleic acid) (PEMA) to form immunoplexes (IPs). Cellular interactions and gene expression could be precisely controlled in murine RAW 264.7 macrophages, murine DC2.4 dendritic cells, and human Jurkat T cells by altering the levels of PEMA envelopment, thus providing a strategy to engineer specific cell targeting into the IP platform. Optimally formulated IPs for immune cell transfection in the presence of serum utilized high N/P ratios to enable high stability, displayed reduced toxicity, high gene expression, and a lengthened duration of gene expression (>3 days) compared to non-enveloped controls. These results demonstrate the potential of engineered IPs to serve as simple, modular, targetable, and efficient non-viral gene delivery platform to efficiently alter gene expression within cells of the immune system.

Keywords: Gene delivery; Immune cells; Nanoparticle; Polyethylenimine; Serum independent.

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Figures

Figure 1.
Figure 1.. Synthesis and characterization of acetylated PEI (Ac-PEI).
A) Synthesis scheme for acetylated PEI. B) Representative 1H-NMR spectra for PEI and Ac-PEI in D2O. The peak at 1.7–1.8 ppm is due to acetylated primary amines whereas the peak at 1.8–1.9 ppm is from acetylated secondary amines of PEI. C) Quantification of PEI degree of acetylation and molecular weight determined by 1H-NMR for various modified PEI polymers.
Figure 2.
Figure 2.. Ac-PEI polyplex formation and characterization.
A) Schematic representation of polyplex formation using acetylated PEI (Ac-PEI) and plasmid DNA (pDNA). B) Agarose gel electrophoresis showing complex formation of various Ac-PEI polymers with GFP plasmid at different N/P ratios. C) GFP expression in RAW 264.7 macrophages using AC-PEI/GFP polyplexes prepared at N/P ratio 20. Ac20-PEI shows higher GFP signals compared to Ac40-PEI and Ac60-PEI. Representative images of at least n=3 experiments.
Figure 3.
Figure 3.. Serum-dependent transfection efficiency and toxicity of Ac-PEI polyplexes.
Transfection efficiency of Ac20-PEI (Ac-PEI) polyplexes at various N/P ratios in RAW 264.7 (A) and Jurkat cells (B) determined by flow cytometry. Median Fluorescence Intensity of GFP expression for RAW 264.7 (C) and Jurkat cells (D). Cell viability determined by MTS assay for RAW 264.7 (E) and Jurkat cells (F). Cells were incubated in serum-free or serum-containing medium for 4 hrs with polyplexes, washed, and incubated for 24 hrs prior to analysis. Data are representative of n=3 experiments. Statistical differences between groups were determined by performing a one-way ANOVA and Tukey’s post hoc test (p < 0.05). Error bars represent SD.
Figure 4.
Figure 4.. Preparation, characterization, and stability assessment of immunoplexes (IPs).
A) Schematic representation of enveloping Ac-PEI/GFP polyplexes with various wt.% of PEMA to form IPs. B, C) Hydrodynamic size and zeta potential of IPs, respectively. Agarose gel electrophoresis to assess the stability of Ac-PEI/GFP polyplexes following PEMA enveloping at N/P 30 in the absence (D) or presence (E) of physiologically-relevant concentration serum (55% v/v). IPs are stable after enveloping with PEMA and in presence of serum as no DNA band is observed. PEMA (poly(ethylene-alt-maleic acid)). n=3 for each experiment. Statistical differences between groups were determined by performing a one-way ANOVA and Tukey’s post hoc test (p < 0.05). All bars in panel B are significantly different from each other. Error bars represent SD.
Figure 5.
Figure 5.. Serum- and PEMA enveloping-dependent transfection and toxicity of IPs.
Transfection efficiency of IPs prepared with various wt.% of PEMA (10% to 50%) determined by flow cytometry in RAW 264.7 (A) and Jurkat cells (B). Median Fluorescence Intensity of GFP expression for RAW 264.7 (C) and Jurkat cells (D). Cell viability determined by MTS assay for RAW 264.7 (E) and Jurkat cells (F). Cells were incubated in serum-free or serum-containing medium for 4 hrs with polyplexes, washed, and incubated for 24 hrs prior to analysis. Data are representative of n=3 experiments. Statistical differences between groups were determined by performing a one-way ANOVA and Tukey’s post hoc test (p < 0.05). Error bars represent SD.
Figure 6.
Figure 6.. Persistence of GFP expression in RAW 264.7 macrophages.
A) Fluorescence micrographs of RAW 264.7 macrophages transfected in serum-containing medium with various IPs enveloped with various wt.% of PEMA. Images are representative of at least n=3 experiments. Cells were incubated in serum-containing medium for 4 hrs with IPs, excess IPs were washed away, and images were acquired at various time points. B) Transfection efficiency of IPs, 24 hr post cell treatment. C) Fold-change enhancements in transfection efficiency relative to Ac-PEI30 for IPs. IP30-10 displayed significantly longer and higher levels of transfection compared to Ac-PEI30 polyplexes. Data are representative of n=3 experiments and as mean +/- SD.
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