Rational design, fabrication, characterization and in vitro testing of biodegradable microparticles that generate targeted and sustained transgene expression in HepG2 liver cells

Abstract

Poly(lactide-co-glycolide) (PLGA) microparticles have significant potential for sustained delivery of plasmid DNA (pDNA). However, unmodified PLGA microparticles have poor transfection efficiencies. In this study, we use several approaches to enhance the transfection efficiencies of PLGA microparticles in a HepG2 liver cell line. Polyethylenimine (PEI) is used to condense the pDNA prior to loading into the PLGA microparticles. This provides enhanced loading efficiencies and greater protection to the pDNA during the entrapment process. In addition, the pDNA used (ApoE) incorporates a hybrid liver-specific murine albumin enhancer/α1 antitrypsin promoter (AlbE/hAAT) to enhance transgene expression in human liver (HepG2) cells. The percentage of surfactant used in the preparation of the microparticles, the polymer composition of the PLGA, the ratio of the PEI to pDNA (N/P), the structure of the PEI and the potential utility of a galactose targeting ligand were then investigated to further optimize the efficacy of the cationic microparticle non-viral delivery system in transfecting HepG2 cells. For each PLGA PEI-pDNA microparticle formulation prepared, we evaluated particle size, ζ-potential, loading of pDNA, cytotoxicity, and transgene expression in HepG2 cells and control human embryonic kidney (HEK293) and monkey African green kidney fibroblast-like (COS7) cells. Loading PLGA particles with PEI-ApoE pDNA complexes resulted in a significant reduction in particle size when compared to PLGA microparticles loaded with ApoE pDNA alone. Scanning electron microscopy images showed that all the particle formulations were smooth and spherical in appearance. Incorporation of the cationic PEI in the PLGA particles changed the ζ-potential from negative to positive. Complexing PEI with ApoE pDNA increased the loading efficiency of the ApoE pDNA into the PLGA microparticles. The cytotoxicity of PLGA particles loaded with PEI-ApoE pDNA complexes was similar to PLGA particles loaded with ApoE pDNA alone. The transfection efficiency of all particle formulations prepared with ApoE pDNA was significantly higher in HepG2 cells when compared to HEK293 and COS7 cell lines. The release of PEI-pDNA complexes from particles prepared with different PLGA polymer compositions including PLGA 50-50, PLGA 75-25, and PLGA 85-15 was sustained in all cases but the release profile was dependent on the polymer composition. Transmission electron microscopy images showed that PEI-pDNA complexes remained structurally intact after release. The optimum formulation for PLGA particles loaded with PEI-ApoE pDNA complexes was prepared using 2% polyvinyl alcohol, 50-50 PLGA compositions and N/P ratios of 5-10. Strong sustained transgene expression in HepG2 cells was generated by PLGA PEI-ApoE pDNA particles up to the full 13 days tested.

Figure 1

Chemical structure of (a) linear polyethylenimine (b) branched polyethylenimine and (c) poly (lactide-co-glycolide).

Figure 2

Schematic of the preparation of (a) PLGA particles loaded with ApoE pDNA and (b) PLGA particles loaded with PEI-ApoE pDNA complexes.

Figure 3

Scanning electron micrographs of PLGA particles loaded with PEI-ApoE pDNA complexes (formulation 1 to 11) and PLGA particles loaded with ApoE pDNA.

Figure 3

Scanning electron micrographs of PLGA particles loaded with PEI-ApoE pDNA complexes (formulation 1 to 11) and PLGA particles loaded with ApoE pDNA.

Figure 4

Cumulative (%) release of PEI-ApoE pDNA complexes from PLGA particles loaded with PEI-ApoE pDNA complexes prepared using PLGA 50-50, PLGA 75-25 and PLGA 85-15. Experiments were carried out in PBS buffer pH 7.4 at 37ºC. Data is presented as means ± SD and are representative of three independent experiments.

Figure 5

Transmission electron micrographs (TEM) of PEI-ApoE pDNA complexes (a) before encapsulation in PLGA particles and (b) 5 days after release from PLGA particles. The bar represents 100 nm.

Figure 6

Luciferase activity of (a) HepG2, (b) HEK293, (c) COS-7 cells that have been treated with unmodified PLGA particles loaded with ApoE pDNA (PLGA/ApoE), PLGA particles loaded with PEI-ApoE pDNA complexes prepared using varying concentrations of PVA solution (0.5%, 1.0%, 1.5%, 2.0% and 5% (w/v)). Transfection was performed by incubating these formulations with cells for 4 hours (reporter gene: ApoE pDNA; pDNA: 1 μg/well). Data is presented as the mean ± standard deviation (n = 3). ND represents naked DNA.

Figure 6

Luciferase activity of (a) HepG2, (b) HEK293, (c) COS-7 cells that have been treated with unmodified PLGA particles loaded with ApoE pDNA (PLGA/ApoE), PLGA particles loaded with PEI-ApoE pDNA complexes prepared using varying concentrations of PVA solution (0.5%, 1.0%, 1.5%, 2.0% and 5% (w/v)). Transfection was performed by incubating these formulations with cells for 4 hours (reporter gene: ApoE pDNA; pDNA: 1 μg/well). Data is presented as the mean ± standard deviation (n = 3). ND represents naked DNA.

Figure 7

Luciferase activity from (a) HepG2, (b) HEK293, (c) COS-7 cells that have been treated with unmodified PLGA particles loaded with ApoE pDNA (PLGA/ApoE), PLGA particles loaded with PEI-ApoE pDNA complexes prepared with varying polymer compositions of PLGA polymer (PLGA 50-50, PLGA 75-25 and PLGA 85-15). Transfection experiments were performed by incubating these formulations with cells for 4 h (reporter gene: ApoE pDNA; pDNA: 1 μg/well). Data is presented as the mean ± standard deviation (n = 3). ND represents naked DNA.

Figure 7

Luciferase activity from (a) HepG2, (b) HEK293, (c) COS-7 cells that have been treated with unmodified PLGA particles loaded with ApoE pDNA (PLGA/ApoE), PLGA particles loaded with PEI-ApoE pDNA complexes prepared with varying polymer compositions of PLGA polymer (PLGA 50-50, PLGA 75-25 and PLGA 85-15). Transfection experiments were performed by incubating these formulations with cells for 4 h (reporter gene: ApoE pDNA; pDNA: 1 μg/well). Data is presented as the mean ± standard deviation (n = 3). ND represents naked DNA.

Figure 8

Luciferase activity from (a) HepG2, (b) HEK293, (c) COS-7 cells that have been treated with unmodified PLGA particles loaded with ApoE pDNA (PLGA/ApoE), PLGA particles loaded with PEI-ApoE pDNA complexes prepared with varying types of PEI polymer (branched PEI (BPEI), linear PEI (LPEI) and galactosylated PEI (GPEI)). Transfection experiments were performed by incubating these formulations with cells for 4 h (reporter gene: ApoE pDNA; pDNA: 1 μg/well). Data is presented as the mean ± standard deviation (n = 3). ND represents naked DNA.

Figure 8

Luciferase activity from (a) HepG2, (b) HEK293, (c) COS-7 cells that have been treated with unmodified PLGA particles loaded with ApoE pDNA (PLGA/ApoE), PLGA particles loaded with PEI-ApoE pDNA complexes prepared with varying types of PEI polymer (branched PEI (BPEI), linear PEI (LPEI) and galactosylated PEI (GPEI)). Transfection experiments were performed by incubating these formulations with cells for 4 h (reporter gene: ApoE pDNA; pDNA: 1 μg/well). Data is presented as the mean ± standard deviation (n = 3). ND represents naked DNA.

Figure 9

Luciferase activity from (a) HepG2, (b) HEK293, (c) COS-7 cells that have been treated with unmodified PLGA particles loaded with ApoE pDNA (PLGA/ApoE), PLGA particles loaded with PEI-ApoE pDNA complexes prepared with varying N/P ratio of PEI-ApoE pDNA complexes (2, 5 and 10). Transfection experiments were performed by incubating these particles with cells for 4 h (reporter gene: ApoE pDNA; pDNA: 1 μg/well). Data is presented as the mean ± standard deviation (n = 3). ND represents naked DNA.

Figure 9

Luciferase activity from (a) HepG2, (b) HEK293, (c) COS-7 cells that have been treated with unmodified PLGA particles loaded with ApoE pDNA (PLGA/ApoE), PLGA particles loaded with PEI-ApoE pDNA complexes prepared with varying N/P ratio of PEI-ApoE pDNA complexes (2, 5 and 10). Transfection experiments were performed by incubating these particles with cells for 4 h (reporter gene: ApoE pDNA; pDNA: 1 μg/well). Data is presented as the mean ± standard deviation (n = 3). ND represents naked DNA.

Figure 10

Luciferase activity from HepG2 cells that have been treated with naked ApoE pDNA, BPEI-ApoE pDNA complexes at N/P ratio 5 and PLGA particles loaded with PEI-ApoE pDNA complexes prepared with varying concentrations of PVA solution (0.5%, 1.5%, 2.0% and 5% (w/v)). Transfection was performed by incubating these particles with cells for 4 hours, 1 day, 3 days, 5 days, 7 days, 11 days and 13 days (reporter gene: ApoE pDNA; pDNA: 1 μg/well). Data is presented as the mean ± standard deviation (n = 3).

Figure 11

Luciferase activity of HepG2 cells that have been treated with naked ApoE pDNA, BPEI-ApoE pDNA complexes at N/P ratio 5 and PLGA particles loaded with PEI-ApoE pDNA complexes prepared with varying polymer compositions of PLGA polymer (PLGA 50-50, PLGA 75-25 and PLGA 85-15). Transfection was performed by incubating these particles with cells for 4 hours, 1 day, 3 days, 5 days, 7 days, 11 days and 13 days (reporter gene: ApoE pDNA; pDNA: 1 μg/well). Data is presented as the mean ± standard deviation (n = 3).