Chitosan Particles Complexed with CA5-HIF-1α Plasmids Increase Angiogenesis and Improve Wound Healing

Abstract

Wound therapies involving gene delivery to the skin have significant potential due to the advantage and ease of local treatment. However, choosing the appropriate vector to enable successful gene expression while also ensuring that the treatment's immediate material components are conducive to healing itself is critical. In this study, we utilized a particulate formulation of the polymer chitosan (chitosan particles, CPs) as a non-viral vector for the delivery of a plasmid encoding human CA5-HIF-1α, a degradation resistant form of HIF-1α, to enhance wound healing. We also compared the angiogenic potential of our treatment (HIF/CPs) to that of chitosan particles containing only the plasmid backbone (bb/CPs) and the chitosan particle vector alone (CPs). Our results indicate that chitosan particles exert angiogenic effects that are enhanced with the human CA5-HIF-1α-encoded plasmid. Moreover, HIF/CPs enhanced wound healing in diabetic db/db mice (p < 0.01), and healed tissue was found to contain a significantly increased number of blood vessels compared to bb/CPs (p < 0.01), CPs (p < 0.05) and no-treatment groups (p < 0.01). Thus, this study represents a method of gene delivery to the skin that utilizes an inherently pro-wound-healing polymer as a vector for plasmid DNA that has broad application for the expression of other therapeutic genes.

Keywords: hypoxia-inducible factor-1α; nanoparticles; non-viral gene therapy; wound healing.

Figure 1

Characterization of chitosan particles (CPs) and chitosan particle/CA5-HIF-1α plasmid complexes (HIF/CP). Scanning electron microscopy images at (A) low magnification and (B) high magnification of particles. (C) Nanoparticle tracking analysis showing particle size distribution. (D) Dynamic light-scattering analysis of CPs showing z-average, number mean, volume mean, and intensity mean diameters. (E) Polydispersity index of CPs. (F) Concentration of CPs as determined by nanotracking analysis. (G) Zeta potential of CPs showing particles have a positive charge. (H) Gel electrophoresis of CA5-HIF-1α plasmids alone along with 50% CPs in solution with CA5-HIF-1α plasmid, and 50% CPs alone. (I) Atomic force microscopy images of CA5-HIF-1α plasmids alone and complexed with 50% CPs in solution (green arrows: plasmid DNA; red arrows: chitosan particles). For all panels, n = 3.

Figure 2

In vitro assessment of HIF/CPs’ angiogenic activity. HUVECs were treated with growth media only (EGM; positive control), basal media only (EBM; negative control), 100 μg/mL CA5-HIF-1α plasmids (HIF) in basal media, 50% chitosan particle solution (CPs) in basal media, or 100 μg/mL CA5-HIF-1α plasmids with 50% chitosan particles in solution (HIF/CPs) in basal media. Brightfield images were taken at 0 h and 12 h post-wounding of the cell layer. Representative images for each group and time point are displayed, and the gap area is outlined with a white line. Statistical significance was calculated using one-way ANOVA with Holm–Sidak’s multiple comparison test (* p < 0.05, *** p < 0.001, and **** p < 0.0001) (n = 3).

Figure 3

Luciferase expression over time when using chitosan particles for plasmid transfection. Verification of transfection efficiency of luciferase plasmids (Luc) using chitosan particles (CPs) in an in vivo reporter assay wound model. Statistical significance was calculated using two-way ANOVA with Holm–Sidak’s multiple comparison test (* p < 0.05) (n = 3).

Figure 4

HIF/CPs induce expression of HIF-1α among hair follicles on wound edge in Sprague Dawley rats. Immunohistochemistry of HIF-1α in skin surrounding the wound with no treatment, 50 μg (based on plasmid content) of 50% chitosan particle/CA5-HIF-1α plasmid complexes (HIF/CPs), 50 μg (based on plasmid content) of 50% chitosan particles with plasmid backbone (bb/CPs), or 50% chitosan particles (CPs). Treatments were injected into the dermis of skin surrounding the wound on day 3 post-wounding, and samples were collected on day 5 for analysis via immunohistochemistry (hair follicular regions expressing HIF-1α shown with white arrows).

Figure 5

Western blots of skin treated with HIF/CPs in Sprague Dawley rats for HIF-1α and VEGFA. Western blots for (A) HIF1α and (B) VEGFA of skin with no treatment or treatment with 50 μg (based on plasmid content) of 50% chitosan particle/CA5-HIF-1α plasmid complexes (HIF/CPs), 50 μg (based on plasmid content) of 50% chitosan particles with plasmid backbone (bb/CPs), or 50% chitosan particles (CPs). Treatments were injected into the dermis of skin surrounding the wound on day 3 post-wounding, and samples were collected on day 5 for analysis via Western blot. Relative expression of HIF1α and VEGFA from each sample was determined via normalization to β-actin from its respective lane. Statistical significance was calculated using one-way ANOVA with Holm–Sidak’s multiple comparison test (* p < 0.05, *** p < 0.001) (n = 6).

Figure 6

HIF/CPs improve wound healing in an excisional db/db mouse model. (A) Representative images of 8 mm wounds with no treatment or treatment with HIF/CPs, bb/CPs, or CPs. (B) Wound closure over time and (C) individual wound sizes on each day of measurements as assessed using digital planimetry. (D) Representative H&E histology of healed tissue from day 21. White arrows indicate blood vessels within the tissue. In the no-treatment group, one mouse perished by day 7 and another was sacrificed on day 14 due to wound infection. # indicates statistical significance (p < 0.01) of improved overall healing, assessed using a two-way ANOVA of HIF/CPs compared to both CPs and no treatment. Statistical significance was calculated using (B) two-way and (C) one-way ANOVA with Holm–Sidak’s multiple comparison test (* p < 0.05) (n = 8).

Figure 7

HIF/CPs induce angiogenesis in healed tissue of db/db mice. CD31+ immunohistochemistry in day 21 tissue from db/db mice. The number of positive structures was counted and tissue in field of view was measured in vessels/mm². The tissue of all 8 mice per group was analyzed, except for the 2 mice in the no-treatment group, which had either perished or were sacrificed prematurely due to wound infection. Statistical significance was calculated using one-way ANOVA with Holm–Sidak’s multiple comparison test (* p < 0.05, ** p < 0.01; n = 8).