Local gene delivery from ECM-coated poly(lactide-co-glycolide) multiple channel bridges after spinal cord injury

Abstract

Tissue engineering scaffolds with complex geometries can provide an architecture that directs tissue formation. Drug delivery from these scaffolds to promote regeneration is often challenging due to the complex fabrication processes. Surface-mediated DNA delivery from multiple channel bridges was applied to deliver lipoplexes in vivo to the injured spinal cord. The surface properties of the polymer, DNA deposition with or without drying, and the presence of ECM components were investigated. In vitro studies revealed that fibronectin produced greater expression levels and immobilization efficiencies compared with collagen, laminin, and no coating. In addition, lipoplex incubation on ECM-coated PLG increased expression relative to either of the drying methods. Additionally, the incubation method had more homogeneously distributed lipoplexes and a higher number of transfected cells relative to the dried conditions. Translation to three-dimensional bridges led to high levels of transgene expression in vitro. In vivo, lipoplexes immobilized to the bridge produced transgene expression levels in a rat spinal cord hemisection model that were 2-fold greater than naked plasmid. Additionally, expression with lipoplexes persisted for at least three weeks. Surface-mediated delivery can be applied to scaffolds with complex geometries to promote transgene expression in vivo.

Figure 1

Transgene expression for surface treatment, and adsorption efficiency and expression levels for multiple deposition methods. (A) Transgene expression for PLG surfaces with and without pre-hydrolyzing treatment. DNA complexes were mixed with ECM (25 μg) and subsequently dried on PLG disks. Significant differences based on a t-test between surfaces with and without pre-hydrolyzing treatment are denoted by an asterisk (p<0.05). (B) Immobilization efficiency of DNA complexes onto ECM (25 μg) coated PLG disks using i) incubation, ii) 2-step drying, and iii) 1-step drying. (C) Transgene expression in vitro using the three deposition methods. Significant differences based on a Tukey multiple comparisons analysis are denoted by different letters (p<0.05).

Figure 2

Distribution of transfected cells and immobilized complexes. Transfected cells (blue: A,C,E) and immobilized lipoplexes (red: B,D,F) on fibronectin (25 μg) coated disks. Complexes were deposited by incubation (A,B), 2-step drying (C,D), or 1-step drying (E,F). Transfected cells: 10X magnification, scale bar: 200 μm; fluorescently labeled DNA: scale bar: 400 μm.

Figure 3

Fibronectin density and transfection. (A) Cell proliferation on PLG surfaces coated with multiple fibronectin densities. (B) Transgene expression on PLG disks coated with varied fibronectin density. Significant differences based on a Tukey multiple comparisons analysis are denoted by different letters (p<0.05).

Figure 4

Transgene expression on fibronectin coated PLG disks incubated with multiple doses of DNA complexes. Significant differences based on a Tukey multiple comparisons analysis are denoted by different letters (p<0.05).

Figure 5

ECM and complex deposition and transfection on bridges. (A) Sirius red stain of fibronectin coated multiple channel bridges. From left to right: top view and cross section of bridge coated with 24 μg fibronectin, and top view and cross-section of uncoated bridge. Scale bar: 150 μm. (B) Bridge coated with 24 μg fibronectin and incubated with 1 μg fluorescently tagged DNA (red). From left to right: top view and cross section of bridge imaged with fluorescence microscopy, and cross section bridge imaged with phase contrast microscopy, Scale bar: 200 μm. (C) Release of uncomplexed and complexed DNA. Significant differences based on a t-test are denoted by an asterisk (p<0.05). (D) Transgene expression in vitro for different doses of DNA incubated with fibronectin coated bridges.

Figure 6

In vivo expression with spinal cord bridges. (A) Transgene expression in rat spinal cord hemisection model after 1 week using either 16 μg complexed (n=8) or naked plasmid (n=4) incubated with fibronectin coated bridges. Significant differences based on an ANOVA with posthoc Wilcoxon 1-way test are denoted by an asterisk (p<0.05). (B) Transgene expression at multiple time points after implantation of fibronectin coated bridges incubated with 16 μg lipoplexes: 3 days (n=10), 1 week (n=8), 2 weeks (n=10), 3 weeks (n=4).