UCLA

Intracellular delivery of diverse biomolecules, such as protein, nucleic acids, nano-devices, has been of great importance and interest in biomedical fields like cancer therapy, gene editing and intracellular environment probing. Although tremendous effort has been expended, it remains challenging for existing transfer platforms to meet the emerging requirements of the cutting-edge research. In this thesis, I focused on three major hurdles in the current intracellular delivery, which are suspension cell delivery, complexity of incorporating nanotechnology, and large cargo delivery.

Photothermal mechanism is the underlying physics throughout all the work to be introduced here. It utilizes the light energy and transforms it into thermal energy and then into mechanical energy, serving for different functions in delivery. Nanosecond laser was chosen as the original power tool due to its high energy density, remote operation capability, and selective absorption. The combination of laser and micro/nano structure has been extensively explored to develop various delivery capabilities.

The first problem tackled in this thesis is to deliver materials into suspension cells with high efficiency, viability, and throughput. Suspension cells, especially lymphocytes, which represent 25-30% of immune cells, are of great interest in cancer immunotherapies and known as hard-to-transfect cells. To achieve effective delivery, the microwell structure with metallic sharp tips were designed to provide both cell anchoring and controllable membrane disruption on each cell. 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 β-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.

The second problem raised from the notice of the huge potential of nanostructures, especially combined with photothermal mechanism, in contrast with their current limited applications in this field. Nanostructures, such as nanoneedle array, have been adopted in the intracellular delivery field due to its unique scale advantages, including minimal damage of the cell membrane and large cargo loading capacity from high surface-to-volume ratio. However, nanotechnologies have suffered from its complexity of high-precision fabrication and are limited to small area. Thus, we demonstrate the fabrication of large-area plasmonic gold (Au) nanodisk arrays that enable photothermal intracellular delivery of biomolecular cargo at high efficiency. The Au nanodisks (350�nm in diameter) were fabricated using chemical lift-off lithography (CLL), a high-throughput and low-cost for nanoscale chemical patterning. This technique is applied to produce Au nanostructures on a variety of substrates (e.g., silicon, glass, and plastic), which facilitate in situ intracellular delivery in laboratory cell culture environments, enabling integration with existing medical devices. Nanosecond laser pulses were used to excite the plasmonic nanostructures, thereby generating transient pores at the outer membranes of targeted cells that enable the delivery of biomolecules via diffusion. We studied nanodisks of various sizes and found that an increase in delivery efficiency correlated with decreasing disk radius, which we attribute to higher density of pores per cell. Delivery efficiencies of >98% were achieved with 1-μm Au plasmonic disk arrays, using the cell impermeable dye Calcein (0.6�kDa) as a model payload, while maintaining cell viabilities at >98%. The highly efficient intracellular delivery approach demonstrated in this work will facilitate translational studies targeting molecular screening and drug testing that bridge laboratory and clinical investigations.

Despite that major problems were nicely solved in the prior two projects, an apparent drawback appears, as the delivery efficiency drops significantly when cargo size increases. Photothermal energy was adopted, in both projects, to generate bubble explosion near the adjacent cell membrane so as to disrupt the membrane. Cargoes had to passively diffuse into the membrane, which posed the hardship to large cargoes. Thus, in the third project, the integration of membrane disruption and active pumping was studied to facilitate large cargo delivery with precise control and large-area uniformity. We utilized the high initial pressure of the laser-induced bubbles as the pump source for high-speed fluidic jet, which cuts the cell membrane and delivers cargos into the cytosol and nucleus. The fabrication processes of the devices are designed to be conventional and simple with large-area uniformity. The penetration was demonstrated by injecting 140 nm polystyrene beads into Agarose hydrogel which was prepared to have similar Young’s Modulus as cells. With delicate device designs, we achieved penetration depths from tens of microns to a hundred microns, indicating the capability of three-dimensional tissue delivery and epidermal in vivo delivery, besides intracellular delivery into single layer of cells.