Intracellular Delivery of mRNA in Adherent and Suspension Cells by Vapor Nanobubble Photoporation

Efficient and safe cell engineering by transfection of nucleic acids remains one of the long-standing hurdles for fundamental biomedical research and many new therapeutic applications, such as CAR T cell-based therapies. mRNA has recently gained increasing attention as a more safe and versatile alternative tool over viral- or DNA transposon-based approaches for the generation of adoptive T cells. However, limitations associated with existing nonviral mRNA delivery approaches hamper progress on genetic engineering of these hard-to-transfect immune cells. In this study, we demonstrate that gold nanoparticle-mediated vapor nanobubble (VNB) photoporation is a promising upcoming physical transfection method capable of delivering mRNA in both adherent and suspension cells. Initial transfection experiments on HeLa cells showed the importance of transfection buffer and cargo concentration, while the technology was furthermore shown to be effective for mRNA delivery in Jurkat T cells with transfection efficiencies up to 45%. Importantly, compared to electroporation, which is the reference technology for nonviral transfection of T cells, a fivefold increase in the number of transfected viable Jurkat T cells was observed. Altogether, our results point toward the use of VNB photoporation as a more gentle and efficient technology for intracellular mRNA delivery in adherent and suspension cells, with promising potential for the future engineering of cells in therapeutic and fundamental research applications.

Transient Low-Temperature Effects on Propidium Iodide Uptake in Lance Array Nanoinjected HeLa Cells

Understanding environmental factors relative to transfection protocols is key for improving genetic engineering outcomes. In the following work, the effects of temperature on a nonviral transfection procedure previously described as lance array nanoinjection are examined in context of molecular delivery of propidium iodide (PI), a cell membrane impermeable nucleic acid dye, to HeLa 229 cells. For treatment samples, variables include varying the temperature of the injection solution (3C and 23C) and the magnitude of the pulsed voltage used during lance insertion into the cells (+5 V and +7 V). Results indicate that PI is delivered at levels significantly higher for samples injected at 3C as opposed to 23C at four different postinjection intervals (t = 0, 3, 6, 9 mins; p-value ≤ 0.005), reaching a maximum value of 8.3 times the positive control for 3 C/7 V pulsed samples. Suggested in this work is that between 3 and 6 mins postinjection, a large number of induced pores from the injection event close. While residual levels of PI still continue to enter the treatment samples after 6 mins, it occurs at decreased levels, suggesting from a physiological perspective that many lance array nanoinjection (LAN) induced pores have closed, some are still present.