Nonviral Transfection Strategies for Keratinocytes, Fibroblasts, and Endothelial Progenitor Cells for Ex Vivo Gene Transfer to Skin Wounds

Abstract

In a search for the optimal nonviral gene transfer technique in epidermal and dermal supportive extracellular matrix studies, we investigated the efficiency of late generation liposomal transfection reagents and nucleofection of fibroblasts (FBs), endothelial progenitor cells (EPCs), and keratinocytes (KCs) as essential indicators of healing skin wounds. FBs, KCs, and EPCs were grown under serum-reduced conditions and manipulated according to optimized in vitro manufacturer protocols. Fugene HD, Effectene, PEI, and Lipofectin were compared to Amaxa Nucleofection. A green fluorescent protein (GFP)-encoded reporter gene plasmid was transfected, and transfection efficiencies were determined by green-fluorescence-activated cell sorting. Normal cell morphologies were observed after either transfection or nucleofection. For KC cell cultures, Fugene HD resulted in the highest transfection efficiency in human (41%) and porcine (42%) KCs. For EPCs, Effectene was optimal for human-derived cells (42%), whereas nucleofection was optimal (32%) for porcine cells. For FBs, however, nucleofection resulted in the highest transfection rates in human (46%) and porcine (60%) FBs. For specific epidermal cell studies, Fugene HD was the preferred gene transfer method, whereas Effectene appeared to be the optimal reagent for pro-angiogenic studies. Nucleofection in combination with FBs is the best combination to achieve the highest overall transfection rate and is thus the optimal combination for use in ex vivo gene transfer strategies of wound healing or skin tissue engineering.

Topical delivery of siRNA-based spherical nucleic acid nanoparticle conjugates for gene regulation

“…To evaluate the potential for targeted gene suppression by siRNA-based SNA-NCs through noninvasive topical delivery, we first investigated their uptake in primary normal human keratinocytes (hKCs), which are typically difficult to transfect (25). hKCs were incubated with Cy3 fluorophore-labeled SNA-NCs (Table S1), and entry was studied by confocal fluorescence microscopy.…”

Gene Therapy for Pyoderma Gangrenosum: Optimal Transfection Conditions and Effect of Drugs on Gene Delivery in the HaCaT Cell Line Using Cationic Liposomes

“…However, as most patients with PG and other skin conditions already use multiple topical and systemic treatments to control the painful lesions, the impact of this is likely to be reduced. In addition, wound healing is a complex process involving many interacting cell types and growth factors, which is time limited, and therefore transgene expression which is only temporary is not unfavourable [54]. PG is a non-malignant disease, so a transfection rate of 100% is not required; the relatively low transfection efficiency of liposomes [15] is not necessarily problematic.…”

Nanomagnetic Activation as a Way to Control the Efficacy of Nucleic Acid Delivery

“…Complexes of nTMag particles associated with pDNA under an oscillating magnetic field with a frequency of 2 Hz yielded 25% transgene-expressing cells compared with 22% with Lipofectamine 2000 (8). The best reported results for the transfection of porcine primary fibroblasts have been achieved with optimized nucleofection protocols (6,(23)(24)(25). Richter et al (25) reported a rate of 53% eGFP-positive PFF cells with low cytotoxicity.…”