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.

Long-term live-cell microscopy with labeled nanobodies delivered by laser-induced photoporation

Fluorescence microscopy is the method of choice for studying intracellular dynamics.However,its success depends on the.availability of specific and stable markers.A prominent example of markers that are rapidly gaining interest are nanobodies(Nbs.-15 kDa),which can be functionalized with bright and photostable organic fluorophores.Due to their relatively small size and high specificity,Nbs offer great potential for high-quality long-term subcellular imaging,but suffer from the fact that they cannot spontaneously cross the plasma membrane of live cells.We have recently discovered that laser-induced photoporation is well suited to deliver extrinsic labels to living cells without compromising their viability.Being a laser-based technology,it is readily compatible with light microscopy and the typical cell recipients used for that.Spurred by these promising initial results,we demonstrate here for the first time successful long-term imaging of specific subcellular structures with labeled nanobodies in living cells.We illustrate this using Nbs that target GFP/YFP-protein constructs accessible in the cytoplasm,actin-bundling protein Fascin,and the histone H2A/H2B heterodimers.With an efficiency of more than 80%labeled cells and minimal toxicity(-2%),photoporation proved to be an excellent intracellular delivery method for Nbs.Time-lapse microscopy revealed that cell division rate and migration remained unaffected,confirming excellent cell viability and functionality.We conclude that laser-induced photoporation labeled Nbs can be easily delivered into living cells,laying the foundation for further development of a broad range of Nbs with intracellular targets as a toolbox for long-term live-cell microscopy.