Nanoparticle-sensitized photoporation is an upcoming approach for intracellular delivery of biologics, combining high efficiency and throughput with excellent cell viability. However, as it relies on close contact between nanoparticles and cells, its translation towards clinical applications is hampered by safety and regulatory concerns. Here, we show that light-sensitive iron oxide nanoparticles (IONPs) embedded in biocompatible electrospun nanofibers induce membrane permeabilization by photothermal effects without direct cellular contact with IONPs. The photothermal nanofibers are successfully used to deliver effector molecules, including CRISPR/Cas9 ribonucleoprotein complexes and siRNA, in adherent and suspension cells, including embryonic stem cells and hard-to-transfect T-cells without affecting cell proliferation or phenotype. In vivo experiments furthermore demonstrate successful tumor regression in mice treated with CAR-T cells in which expression of PD1 is downregulated after nanofiber photoporation with siPD1. In conclusion, cell membrane permeabilization with photothermal nanofibers is a promising concept towards the safe and more efficient production of engineered cells for therapeutic applications, including stem cell or adoptive T cell therapy.

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.

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The modification of CD4+ T cells with exogenous nucleic acids or proteins is a critical step in several research and therapeutic applications, such as HIV studies and cancer immunotherapies. However, efficient cell transfections are not always easily achieved when working with these primary hard-to-transfect cells. While the modification of T cells is typically performed by viral transduction or electroporation, their use is associated with safety issues or cytotoxicity. Vapor nanobubble (VNB) photoporation with sensitizing gold nanoparticles (AuNPs) has recently emerged as a new technology for safe and flexible cell transfections. In this work, we evaluated the potential of VNB photoporation as a novel technique for the intracellular delivery of macromolecules in primary human CD4+ T cells using fluorescent dextrans as model molecules. Our results show that VNB photoporation enables efficient delivery of fluorescent dextrans of 10 kDa in Jurkat (>60% FD10+ cells) as well as in primary human CD4+ T cells (±40% FD10+ cells), with limited cell toxicity (>70% cell viability). We also demonstrated that the technique allows the delivery of dextrans that are up to 500 kDa in Jurkat cells, suggesting its applicability for the delivery of biological macromolecules with a wide range of molecular weights. Altogether, VNB photoporation represents a promising technique for the universal delivery of macromolecules in view of engineering CD4+ T cells for use in a wide variety of research and therapeutic applications.