Folate Receptor Mediated Gene Delivery to Oral Cancer Cells

Objectives: The minimal constitutive expression of the folate receptor in normal tissues and its over-expression in many types of cancer render it a promising target for tumor-specific gene delivery. We tested the hypothesis that inclusion of folate-conjugated lipids in cationic lipid-DNA complexes (“lipoplexes”) used as non-viral vectors will enhance gene delivery to oral squamous cell carcinoma cells compared to non-targeted lipoplexes.

Methods: Folate-poly(ethylene glycol)(PEG)-conjugated lipids or control PEG-lipids were included in cationic liposomes composed of DOTAP/cholesterol (1:1) at concentrations ranging from 1 mol% to 0.1 mol%. A plasmid expressing luciferase (pCMV.luc) was complexed with liposomes to form “lipoplexes”. The lipoplexes were incubated for 4 h with the human oral epidermoid carcinoma cell line KB and non-tumor-derived GMSM-K oral epithelial cells, and the murine SCCVII (oral squamous cell carcinoma) and the non-tumorigenic NIH-3T3 embryonic fibroblasts for. The cells were washed and incubated for an additional 48 h and luciferase activity in cell lysates was measured using a Promega kit. To assess the level of folate receptors on cells the folate-PEG- and PEG-lipid-containing liposomes were labeled with rhodamine-phosphatidylethanolamine, and binding of these liposomes to cells was observed by fluorescence microscopy.

Results: Fluorescently labeled Folate-PEG-lipid containing liposomes bound at a higher level to cancer cells than to normal human and murine cells, indicating the presence of folate receptors on the cancer cells. Folate-PEG-lipid lipoplexes resulted in a 5-6 fold increase in gene delivery over non-targeted control mPEG-lipid lipoplexes in both human and murine tumor cells, when used at a 0.2 mol% concentration. Non-tumor cells showed no significant increase with folate over control PEG. However, the PEG-lipid itself was inhibitory to transfection when used at concentrations over 0.1 mol%, compared to plain DOTAP/cholesterol liposomes.

Conclusion: Folate receptors may be a significant target for future gene therapy applications.