Hyaluronic acid and fibrin hydrogels with concentrated DNA/PEI polyplexes for local gene delivery

Abstract

Local delivery of DNA through a hydrogel scaffold would increase the applicability of gene therapy in tissue regeneration and cancer therapy. However, the delivery of DNA/cationic polymer nanoparticles (polyplexes) using hydrogels is challenging due to the aggregation and inactivation of polyplexes during their incorporation into hydrogel scaffolds. We developed a novel process (termed caged nanoparticle encapsulation or CnE) to load concentrated and unaggregated non-viral gene delivery nanoparticles into various hydrogels. Previously, we showed that PEG hydrogels loaded with DNA/PEI polyplexes through this process were able to deliver genes both in vitro and in vivo. In this study, we found that hyaluronic acid and fibrin hydrogels with concentrated and unaggregated polyplexes loaded through CnE were able to deliver genes in vivo as well, demonstrating the universality of the process.

Figure 1

HA or fibrin induced aggregation of polyplexes. (A, C) DNA/PEI polyplexes containing 5 or 50 μg DNA at N/P = 15 were prepared in 100 μL water or 1% HA or 5 mg/mL fibrinogen solution. The size distributions of the formed polyplexes were measured with DLS. (B, D) DNA/L-PEI polyplexes containing 100 μg DNA at N/P = 15 were encapsulated in 100 μL HA (B) or fibrin (D) hydrogels. The DNA was stained with ethidium bromide and visualized with a fluorecence microscope. Scale bar: 100 μm.

Figure 2

Polyplex distribution in HA and fibrin hydrogels. DNA/PEI polyplexes containing 0 to 300 μg DNA at N/P = 15 were encapsulated in 100 μL HA (A–F) or fibrin (5 or 15mg/mL) (G–L) hydrogels through CnE. The DNA was stained with ethidium bromide and visualized with a fluorescence (A–C, G–I) or confocal microscope (D–F, J–L). Scale bar: 50 μm for (A–C, G–I) and 1 μm for (D–F, J–L).

Figure 3

DNA release from HA and fibrin hydrogels. (A) polyplexes containing 100 μg DNA at N/P = 15 were encapsulated into 100 μL HA hydrogel. The hydrogel was incubated in PBS (pH 7.4) at 37 °C. (B) polyplexes containing 50 μg DNA at N/P = 15 were encapsulated into 100 μL 5 or 15 mg/mL fibrin hydrogel. The hydrogels were incubated in PBS (pH 7.4) at 37 °C. The released DNA was quantified with HOECHST.

Figure 4

DNA loaded HA hydrogels resulted in gene transfer in CAM model. A HA hydrogel with polyplexes was placed on top of CAM for 3 days. The gel with CAM was cut, fixed and stained with x-gal solution for 48 hrs. Positive β-galactosidase expression resulted in blue color. 100 μg pVEGF or pβgal at N/P = 15 was loaded into 100 μL gel. The dashed line highlights the edge of the hydrogel and “G” indicates the gel area

Figure 5

pVEGF loaded HA hydrogels resulted in enhanced angiogenesis in a CAM model. A HA hydrogel with polyplexes containing pVEGF was placed on top of the CAM for 3 days. pVEGF was transferred to cells and produced a high concentration of VEGF at the gel area, which resulted in hyperbranced neovessels (arrow). VEGF diffusing out the gel created a decreasing VEGF gradient around the gel and led to radial neovessels around the gel (arrowheads). (A–D) Gross pictures on the gel edge were recorded before the CAM was infused with FITC-dextran for fluorescent imaging (E–H) at the gel area. Induced neovessels were found both around the gel (C–D, arrow heads) and at the gel area (G–H, arrows) with pVEGF, which were not found in the negative control (No DNA). Polyplexes at N/P = 15 were used. The dashed line highlights the edge of the hydrogel and “G” indicates the gel area. Scale bar: E–H: 200 μm

Figure 6

DNA loaded fibrin hydrogels resulted in gene transfer in the CAM model. A fibrin hydrogel with polyplexes was placed on top of CAM for 3 days. The gel with CAM was cut, fixed and stained with x-gal solution for 48 hrs. Positive β-galactosidase expression resulted in blue color. (A–C) and (D–F) showed the low and high magnifications, respectively. 50 μg pβgal at N/P = 15 was loaded to 100μL 5 or 15 mg/mL fibrin gels. The dashed line highlights the edge of the hydrogel and “G” indicates the gel area. The white arrows in (E and F) point out some of the stained nucleus.

Figure 7

pVEGF loaded fibrin hydrogels resulted in enhanced angiogenesis in a CAM model. A fibrin hydrogel with polyplexes containing pVEGF was placed on top of the CAM for 3 days. pVEGF was transferred to cells and produced a high concentration of VEGF at the gel area, which resulted in hyperbranced neovessels (arrow). VEGF diffusing out the gel created a decreasing VEGF gradient around the gel and led to radial neovessels around the gel (arrowheads). (A–C) Gross pictures on the gel edge were recorded before the CAM was infused with FITC-dextran for fluorescent imaging (D–F) at the gel area. Induced neovessels were found both around the gel (B–C, arrow heads) and at the gel area (E–F, arrows) with pVEGF, which were not found in the negative control (No DNA). 50 μg pVEGF at N/P = 15 were used for both the 5 and 15 mg/mL gels. The dashed line outlines the edge of the hydrogel and “G” indicates the gel area. Scale bar: D–F: 200μm.