Photothermal nanofibres enable safe engineering of therapeutic cells

Abstract

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.

The cellular response to plasma membrane disruption for nanomaterial delivery

“…Moreover, it can do so in both adherent and suspension cells with high efficiency and low cytotoxicity [ 3 , 66 , 69 , 73 ] and it has the precision to target organelle membranes such as the nuclear envelope [ 74 ]. Instead of adding photothermal NPs to cells, it is also possible to integrate photothermal features into substrates onto which cells can be cultured [ 75 – 77 ]. This has the advantage that the nanosensitizers will not be ingested by the cell, thereby eliminating a potential safety issue for clinical translation.…”

“…VNB photoporation of Jurkat T cells using nanosecond pulsed irradiation of 60 nm spherical AuNPs showed unaltered proliferation rates up to 5 days post-transfection compared to untreated cells [ 3 ]. Primary human T cells and human embryonic stem cells (hESCs) treated with photothermal electrospun nanofibers (PEN photoporation) also did not suffer from a proliferative slowdown [ 6 ].…”

“…Moreover, it was found that T-cells showed undiminished effector responses and homing capabilities after cell squeezing, indicating this technique does not affect T-cell functionality [ 52 ]. The impact of nanoparticle sensitized photoporation was found to be modest as well [ 149 ], and photoporation with photothermal nanofibers was shown to leave the differentiation potential of hESCs intact, as well as preserving T cell homeostasis and functionality in vitro and in vivo [ 6 ].…”

“…In the past decade, the field of membrane disruption methods for intracellular delivery has grown exponentially owing to advances in nanotechnology, including the emergence of various nanomaterials and nanofabrication methods [ 1 ]. One noteworthy example is the combination of nanosensitizers with laser irradiation, which has gained significant interest due to its ability to permeabilize the PM of both adherent and suspension cells with high throughput and spatiotemporal selectivity [ 2 – 6 ]. While most studies on intracellular delivery technologies focus on achieving high delivery efficiencies with minimal acute cytotoxicity, recent studies show that surviving cells can suffer from more subtle effects, such as alterations in morphology and functionality.…”

Designing Functionalized Polyelectrolyte Microcapsules for Cancer Treatment

“…The MCs can be functionalized with high-molecular-weight compounds, including capture molecules for the specific recognition of target tumor cells (e.g., monoclonal anti-HER2, anti-EGFR antibodies, bispecific anti-EGFR antibodies [4,10,29]), short interfering RNA recognizing programmed cell death protein 1 mRNA [30], and low-molecularweight antitumor agents (e.g., gemcitabine, 5-fluorouracil, phthalocyanines, and doxorubicin [6,15,31,32]). Doxorubicin (DOX) is the most commonly used first-line drug for a number of malignant tumors, such as ovarian, breast, and prostate cancers, as well as lymphomas and leukemias [33].…”