Intracellular Delivery of Functional Proteins with DNA−Protein Nanogels−Lipids Complex
Résumé
Using functional proteins for therapeutic purposes due to their high selectivity and/or catalytic properties can enable the control of various cellular processes; however, the transport of active proteins inside living cells remains a major challenge. In contrast, intracellular delivery of nucleic acids has become a routine method for a number of applications in gene therapy, genome editing, or immunization. Here we report a functionalizable platform constituting of DNA–protein nanogel carriers cross-linked through streptavidin–biotin or streptactin–biotin interactions and demonstrate its applicability for intracellular delivery of active proteins. We show that the nanogels can be loaded with proteins bearing either biotin, streptavidin, or strep-tag, and the resulting functionalized nanogels can be delivered into living cells after complexation with cationic lipid vectors. We use this approach for delivery of alkaline phosphatase enzyme, which is shown to keep its catalytic activity after internalization by mouse melanoma B16 cells, as demonstrated by the DDAO–phosphate assay. The resulting functionalized nanogels have dimensions on the order of 100 nm, contain around 100 enzyme molecules, and are shown to be transfectable at low lipid concentrations (charge ratio R± = 0.75). This ensures the low toxicity of our system, which in combination with high local enzyme concentration (∼100 μM) underlines potential interest of this nanoplatform for biomedical applications.
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