Intracellular Photothermal Delivery for Suspension Cells Using Sharp Nanoscale Tips in Microwells.

Efficient intracellular delivery of biomolecules into cells that grow in suspension is of great interest for biomedical research, such as for applications in cancer immunotherapy. Although tremendous effort has been expended, it remains challenging for existing transfer platforms to deliver materials efficiently into suspension cells. Here, we demonstrate a high-efficiency photothermal delivery approach for suspension cells using sharp nanoscale metal-coated tips positioned at the edge of microwells, which provide controllable membrane disruption for each cell in an array. Self-aligned microfabrication generates a uniform microwell array with three-dimensional nanoscale metallic sharp tip structures. Suspension cells self-position by gravity within each microwell in direct contact with eight sharp tips, where laser-induced cavitation bubbles generate transient pores in the cell membrane to facilitate intracellular delivery of extracellular cargo. A range of cargo sizes were tested on this platform using Ramos suspension B cells with an efficiency of >84% for Calcein green (0.6 kDa) and >45% for FITC-dextran (2000 kDa), with retained viability of >96% and a throughput of >100 000 cells delivered per minute. The bacterial enzyme ß-lactamase (29 kDa) was delivered into Ramos B cells and retained its biological activity, whereas a green fluorescence protein expression plasmid was delivered into Ramos B cells with a transfection efficiency of >58%, and a viability of >89% achieved.

Magnetic Force-driven in Situ Selective Intracellular Delivery.

Intracellular delivery of functional materials holds great promise in biologic research and therapeutic applications but poses challenges to existing techniques, including the reliance on exogenous vectors and lack of selectivity. To address these problems, we propose a vector-free approach that utilizes millimeter-sized iron rods or spheres driven by magnetic forces to selectively deform targeted cells, which in turn generates transient disruption in cell membranes and enables the delivery of foreign materials into cytosols. A range of functional materials with the size from a few nanometers to hundreds of nanometers have been successfully delivered into various types of mammalian cells in situ with high efficiency and viability and minimal undesired effects. Mechanistically, material delivery is mediated by force-induced transient membrane disruption and restoration, which depend on actin cytoskeleton and calcium signaling. When used for siRNA delivery, CXCR4 is effectively silenced and cell migration and proliferation are significantly inhibited. Remarkably, cell patterns with various complexities are generated, demonstrating the unique ability of our approach in selectively delivering materials into targeted cells in situ. In summary, we have developed a magnetic force-driven intracellular delivery method with in situ selectivity, which may have tremendous applications in biology and medicine.

Microfluidic platform for probing cancer cells migration property under periodic mechanical confinement.

Cancer cell migration and invasion, which are involved in tumour metastasis, are hard to predict and control. Numerous studies have demonstrated that physical cues influence cancer cell migration and affect tumour metastasis. In this study, we proposed the use of a microchannel chip equipped with a number of vertical constrictions to produce periodic compression forces on cells passing through narrow channels. The chip with repeated vertical confinement was applied on adherent MHCC-97L liver cancer cells and suspended OCI-AML leukaemia cells to determine the migration ability of these cancer cells. Given the stimulation of the periodic mechanical confinement on-chip, the migration ability of cancer cells was promoted. Moreover, the migration speed increased as the stimulation was enhanced. Both AFM nanoindentation and optical stretching tests on cancer cells were performed to measure their mechanical property. After confinement stimulation, the cancer cells possessed higher deformability and lower stiffness than non-stimulating cells. The confinement stimulation altered the cell cytoskeleton, which governs the migration speed. This phenomenon was determined through gene expression analysis. The proposed on-chip cell migration assays will help characterise the migration property of cancer cells and benefit the development of new therapeutic strategies for metastasis.