Cationic lipid and polymer-based gene delivery to human pancreatic islets

Abstract

Transplantation of pancreatic islets has great potential for treating Type I diabetes. Ex vivo gene therapy may promote re-vascularization or inhibit apoptosis of the islets and promote graft. In this study, we investigated the feasibility of non-viral gene delivery using Enhanced Green Fluorescent Protein (EGFP) and human Vascular Endothelial Growth Factor (hVEGF(165)) expression plasmids as model reporter and therapeutic genes. LipofectAMINE/pDNA and Superfect/pDNA complexes showed high transfection efficiency in rapidly dividing Jurkat cells, but low transfection in non-dividing human islets. LipofectAMINE/pCAGGS-hVEGF transfected islets showed relatively higher levels of hVEGF than in those transfected with LipofectAMINE/pCMS-EGFP complexes or 5% glucose. To exclude endogenously secreted hVEGF, real time RT-PCR experiment was repeated using pCAGGS vector-specific forward primer and hVEGF gene-specific reverse primer. In this case, both non-transfected islets and the islets transfected with LipofectAMINE/pCMS-EGFP complexes showed negligible amplification of hVEGF. On glucose challenge, insulin release from LipofectAMINE/pCAGGS-hVEGF transfected human islets increased from 10.78 +/- 4.56 to 65 +/- 5 ng/ml, suggesting little adverse effect on islet beta cell response to glucose challenge. The low transfection efficiency is due to the islets being a cluster of approximately 1000 non-dividing cells. This underscores the importance of experimentation with the actual human islets.

Adenovirus-based vascular endothelial growth factor gene delivery to human pancreatic islets

“…46 The islet samples were used for glucose-stimulated insulin release. 19 There was no increase in NO release in the presence of these cytokines. Similarly, the presence of 500 Units/ml of TNF-a and IFN-g had little effect on insulin release, as in all the transfected islet samples there was increase in insulin with increase in glucose concentration from 60 to 300 mg/dl (data not shown).…”

“…19 Non-transfected islets were used as controls. Transfection media was removed by centrifugation at 400 g and the islets were sequentially incubated at 371C for 1 h with MEM media containing low (0.6 mg/ml, basal), high (3 mg/ml, stimulated) and glucose plus theophylline (1.8 mg/ml), and again back at the basal glucose levels for 1 h each at 371C in 5% CO 2 incubator.…”

“…17,18 Therefore, we recently investigated the possible use of plasmid DNA-based gene delivery approaches for transient expression of human VEGF (hVEGF) to improve neovascularization. 19 We recently constructed a bicistronic plasmid DNA encoding GFP and hVEGF and tested for transfection efficiency and revascularization after transplantation under the kidney capsules of the NOD-SCID mice. 20 At this stage, it is not yet clear whether high or low levels of hVEGF gene expression are required for revascularization of human islets to improve islet engraftment after transplantation.…”

“…Since islet is a compact cluster of about 1000 non-dividing cells, it might be difficult to transfect all cells by the available non-viral methods. 19,20 There are also reports of low transfection of lipofectin/pDNA, lipofectAMINE/pDNA, DOSPA:-DOPE liposome/pDNA and polyethylenimine (PEI)/ pDNA complexes into intact mouse, rat and porcine islets. [21][22][23] In contrast, adenoviral vectors are known to efficiently transfect non-proliferating cells, such as pancreatic islets.…”

Combined treatment of human MCF-7 breast carcinoma with antibody, cationic lipid and hyaluronic acid using ex vivo assays

“…It has already been shown that Lipofectamine (LipA) used in vitro transfers the transfected material into the cell through lipid membrane [10]. This process results in the direct delivery of the protein to the target without interfering with cell membrane and influences several cancer cells' activities, including growth and adhesion.…”