Local Silencing of Connective Tissue Growth Factor by siRNA/Peptide Improves Dermal Collagen Arrangements

Abstract

Collagen organization within tissues has a critical role in wound regeneration. Collagen fibril diameter, arrangements and maturity between connective tissue growth factor (CTGF) small interfering RNA (siRNA) and mismatch scrambled siRNA-treated wound were compared to evaluate the efficacy of CTGF siRNA as a future implement for scar preventive medicine. Nanocomplexes of CTGF small interfering RNA (CTGF siRNA) with cell penetrating peptides (KALA and MPG∆NLS) were formulated and their effects on CTGF downregulation, collagen fibril diameter and arrangement were investigated. Various ratios of CTGF siRNA and peptide complexes were prepared and down-regulation were evaluated by immunoblot analysis. Control and CTGF siRNA modified cells-populated collagen lattices were prepared and rates of contraction measured. Collagen organization in rabbit ear 8mm biopsy punch wound at 1day to 8wks post injury time were investigated by transmission electron microscopy and histology was investigated with Olympus System and TS-Auto software. CTGF expression was down-regulated to 40% of control by CTGF siRNA/KALA (1:24) complexes (p < 0.01) and collagen lattice contraction was inhibited. However, down-regulated of CTGF by CTGF siRNA/MPG∆NLS complexes was not statistically significant. CTGF KALA-treated wound appeared with well formed-basket weave pattern of collagen fibrils with mean diameter of 128 ± 22nm (n = 821). Mismatch siRNA/KALA-treated wound showed a high frequency of parallel small diameter fibrils (mean 90 ± 20nm, n = 563). Controlling over-expression of CTGF by peptide-mediated siRNA delivery could improve the collagen orientation and tissue remodeling in full thickness rabbit ear wound.