Photothermal nanofibers enable macromolecule delivery in unstimulated human T cells

Abstract: Cell therapies such as adoptive T cell transfer require ex vivo modification of cells with exogenous cargo to modulate their phenotype (e.g., to express a synthetic antigen receptor) for optimal therapeutic efficacy upon reinfusion in a patient. Several studies have shown superior anti-tumor activity of minimally differentiated T cell subsets over their activated counterparts. Therefore, developing techniques for safe and efficient manipulation of these quiescent cells is important for both clinical applications and fundamental studies of T cell biology. Photoporation with photothermal electrospun nanofibers (PEN) is an efficient and minimally perturbing nonviral intracellular delivery technique for activated and expanded T cells. However, the technique has not yet been applied to unstimulated T cells. Here, we investigated the potential of PEN photoporation for delivery of macromolecules into these cells. First, we confirmed with inductively coupled plasma tandem mass spectrometry that there was no significant iron release from fibers after laser activation of PEN substrates for laser fluences up to 0.36 J/cm2. Next, we demonstrated successful intracellular delivery of 150 kDa FITC-dextran as model macromolecule in resting and pre-activated lymphocytes with 55-60 % delivery efficiency. By analyzing metabolic activity, activation surface marker presentation and extracellular cytokine release, we found that PEN treatment had no effect on cell proliferation and limited impact on T cell activation propensity for all tested irradiation energies. Thus, our findings show that PEN photoporation holds promise as a safe and efficient delivery strategy, paving the way for its use in genetic modification of minimally differentiated T cells.