Cutaneous chronic wounds are characterized by the absence of healing six weeks after the injury. The classic treatment is the debridement of the wound bed followed by a compression method. When this technique is not effective enough, the application of wound dressings is required. Several hulman trials with protein therapy have failed because of the rapid diffusion and short half-life of the cytokines in the wound[1]. Therefore gene therapy represents an interesting alternative. Local delivery of nucleic acids through a hydrogel extends the application of gene therapy for the treatment of chronic wounds. However, the inactivation and fast release of encapsulated DNA strongly compromise their efficacy. Nanoscale delivery systems such as silica nanoparticles (SiNPs) hold great promise for gene therapy owing to their high cellular uptake, DNA protection and possible cell targeting[2].
In this study, nanocomposites consisting of plasmid DNA-PEI-SiNP complexes, collagen hydrogel and 3T3 mouse fibroblasts have been evaluated as gene delivery systems to obtain a local and sustained release of biomolecules. Plasmid DNA encoding for Interleukin 10 (IL-10) was chosen as this cytokine is able to modulate inflammation of chronic wounds.
SiNPs were modified with PEI (polyethyleneimine) of different molecular weights (1.8 kDa, 10 kDa and 25 kDa) by electrostatic absorption. SiNP-PEI complexes were associated with plasmids encoding for Luciferase or human IL-10 at a weight ratio of 30:1. Then, complexes were encapsulated within collagen hydrogel with 3T3 mouse fibroblasts. Cell transfection was assessed by measuring the luciferase activity or the IL-10 production. Cell viability was evaluated by Alamar Blue Assay. Subsequently, the effect of nanocomposites to modulate inflammation was evaluated on activated mouse RAW 264.7 macrophages. The ability of IL-10 doses released from nanocomposites to downregulate the TNF-α expression in macrophages was analyzed over one week.
Successful cell transfection within the collagen/silica nanocomposites was detected by sustained release of luciferase over one week. The best transfection efficiency was obtained with PEI 10KDa (Figure 1). When compared with the soluble form of PEI 25 KDa, the transfection was lower. However, the incubation with increasing quantities of soluble PEI or complexed with SINP revealed a cytotoxicity of PEI 25 KDa and 10 KDa from 10 µg/mL (Figure 1). In contrast, PEI was not toxic until the 100 µg/mL when associated with silica nanoparticles. Interestingly, SiNP-PEI complexes were confined within the collagen network as no transfection was possible out of the nanocomposites. Finally, SiNP-PEI complexes were confined within the collagen network as no transfection was possible out of the nanocomposites. Tested on activated macrophages, nanocomposites permitted the sustained release of IL-10 (500 pg/mL) which could inhibit the expression of TNF-α by 50% in macrophages (Figure 2).
These results show that nanocomposites obtained by entrapping SiNP-PEI-DNA together with cells inside a collagen hydrogel can work as a reservoir for local and controlled gene delivery. In addition, it is possible to create cell factories producing high doses of IL-10 which modulate inflammation. These biomaterials are promising for the local treatment of cutaneous chronic wounds.
Bernard Haye; Corinne Illoul
References:
[1] Menke,N.; Ward, K.; Witten, T.; Bonchev, D.; Diegelmann, R. Impaired wound healing. Clin Dermatol. 2007, 25, 19–25.
[2] Guo, X.; Huang, L. Recent Advances in Non viral Vectors for Gene Delivery. Acc. Chem. Res. 2012, 45, 971-979.