DNA delivery from hyaluronic acid-collagen hydrogels via a substrate-mediated approach

Abstract

Efficient and controlled gene delivery from biodegradable materials can be employed to stimulate cellular processes that lead to tissue regeneration. In this report, a substrate-mediated approach was developed to deliver DNA from hyaluronic acid-collagen hydrogels. The hydrogels were formed by crosslinking HA with poly(ethylene glycol) diglycidyl ether. Poly(ethylene imine)(PEI)/DNA complexes were immobilized to the substrate using either biotin/neutravidin or non-specific adsorption. Complexes were formed in the presence or absence of salt to regulate complex size, and resulted in complexes with z-average diameters of 1221.7 +/- 152.3 and 139.4 +/- 1.3 nm, respectively. During 48-h incubation in PBS or hyaluronidase, DNA was released slowly from the hydrogel substrate (<30% of immobilized DNA), which was enhanced by incubation with conditioned media (approximately 50% of immobilized DNA). Transgene expression mediated by immobilized, large diameter complexes was 3 to 7-fold greater than for small diameter complexes. However, the percentage of cells expressing the transgene was greater for small diameter complexes (48.7%) than for large diameter complexes (22.3%). Spatially controlled gene transfer was achieved by topographically patterning the hydrogel to pattern cell adhesion. Biomaterial-based gene delivery can be applicable to numerous tissue engineering applications, or as a tool to examine tissue formation.

Fig. 1

(A) z-average diameter of DNA/PEI complexes at N/P ratios of 2.5, 5, 10 and 20 for complexes formed in 150 mm NaCl or water. (B) Zeta potential of DNA/PEI complexes at N/P ratios of 2.5, 5, 10 and 20 for complexes formed in 150 mm NaCl. Plotted data is an average of triplicate conditions ± SD.

Fig. 2

DNA/PEI complex aggregation. Complexes were formed in water at different N/P ratios and complex size was monitored following addition of salt to the complexes. Plotted data is an average of triplicate conditions ± SD.

Fig. 3

Surface densities of DNA/PEI complexes immobilized to HA-collagen- NA hydrogels for complexes formed in 15 0mm NaCl (B) or water (C). Plotted data is an average of triplicate conditions±SD. Fluorescence photomicrographs of immobilized biotinylated DNA/PEI complexes (N/P=5) to HA-collagen-NA hydrogels. Biotinylated complexes formed in water (B) or 150mm NaCl (C) were incubated on the substrate for 120 min. Images were captured after washing the substrate with PBS buffer.

Fig. 4

Spatially controlled gene transfer of NIH/3T3 cells plated on topographically patterned HA-collagen-NA hydrogels with immobilized DNA/PEI complexes. Overlay of GFP positive cells and rhodamine phalloidin staining are shown. Magnifications correspond to 100 × (A) and 200 × (B).

Fig. 5

Transgene expression in NIH/3T3 cells mediated by immobilized complexes to HA-collagen-NA substrates. % biotinylated PEI indicate the percent biotinylation of the complexes. The symbols * and ** represents a statistically significant level of p<0.05 and <0.01; respectively, for single comparisons between biotinylated complexes and non-biotinylated complexes. Complexes were formed at an N/P ratio of 5. Plotted data is an average of triplicate conditions ± SD.

Fig. 6

GFP transgene expression in NIH/3T3 cells mediated by immobilized complexes to HA-collagen-NA substrates. The cell nuclei are stained with HOESCHT dye for visualization (A). The percentage of transfected cells was calculated by dividing the number of GFP positive cells by the total number of nuclei (B). The symbol *** represents a statistically significant level of p<0.0001 for a single comparison between large and small complexes. Complexes were formed at an N/P ratio of 5. Plotted data is an average of triplicate conditions ± SD.

Fig. 7

Release kinetics of biotinylated and non-biotinylated complexes immobilized to HA-collagen-NA hydrogels against PBS (A), conditioned media (B) or hyaluronidase (C). Complexes containing 0% and 25% biotinylated PEI were immobilized to the hydrogel substrates. The symbols * and *** represents a statistically significant level of p<0.05 and p<0.001; respectively, for single comparisons between biotinylated complexes and non-biotinylated complexes. Plotted data is an average of triplicate conditions ± SD.

Fig. 8

Luciferase transgene expression in NIH/3T3 cells mediated by immobilized complexes to HA-collagen-NA substrates for cells directly attached to the hydrogel (open diamonds) or not in contact with the hydrogel (closed diamonds). % biotinylated PEI indicate the percent biotinylation of the complexes. All complexes were formed at an N/P ratio of 5. Plotted data are an average of triplicate conditions ± SD.

Fig. 9

Luciferase transgene expression in NIH/3T3 cells mediated by immobilized complexes to HA-collagen-NA substrates. % RGD-PEI indicates the percent RGD modification of the complexes. DNA/PEI complexes were formed (N/P=5) with mixtures of three types of PEI: biotinylated, RGD-modified and unmodified. The percentage of biotinylated PEI was kept constant at 25% and the percentage of RGD-PEI was varied from 0% to 75% to obtain different quantities of RGD in the complex. Plotted data is an average of triplicate conditions ± SD.