(266d) Engineering High-Throughput Gold Nanoshell-Liposomes for Effective mRNA Delivery

Altering immune system cells such as T-cells or NK-cells with specific protein targeting moieties is a promising route for cancer and infectious disease treatment¹. Non-viral vector delivery of mRNA to code for these proteins is advantageous due to the relative ease of mRNA manufacture and complete absence of genomic integration². mRNA only needs to reach the cell cytoplasm to catalyze the production of exogenous proteins. mRNA is transiently active and does not integrate into the genome, so cell modifications are not permanent or replicated, which makes it safer than DNA delivered by viral vectors³. However, efficient intracellular delivery of mRNA to the cell cytosol in vitro or in vivo continues to be a major hurdle. The large molecular weight (10⁵–10⁶ Da) and high negative charge of mRNA prevent mRNA from crossing cellular membranes⁴. Moreover, mRNA is an inherently unstable molecule, which is highly prone to degradation by 5’ exonucleases, 3’ exonucleases, and endonucleases⁵. Consequently, development of high throughput and efficient delivery systems are imperative for manufacturing protein-modified immune cells ex vivo. Different strategies have been investigated to improve RNA delivery, such as condensation of mRNA in lipid nanoparticles as in the Moderna and Pfizer vaccines or electroporation, both of which are inefficient and often toxic. There are few available viral vectors for mRNA, and all have drawbacks associated with genome integration, and possible host rejection (immunogenicity and cytotoxicity)⁶.

To create a high throughput, high viability ex-vivo mRNA delivery we have encapsulated mRNA in neutral liposomes that are then coupled to plasmon-resonant hollow gold nanoshells (HGN). The HGN plasmon resonance can be tuned to absorb physiologically friendly near infra-red light of 650 – 1040 nm wavelength⁷. After mixing the HGN-liposomes with the cells to be transfected, the mixture can be irradiated with picosecond pulsed near infra-red light that induce vapor nanobubbles around the HGN. These nanobubbles grow and collapse, thereby rupturing both the cell and liposome membrane, thereby releasing the mRNA to the cell cytoplasm. Once delivered, the mRNA can produce the proteins of interest (Fig 1). Here we present an extensive analysis into HGN synthesis, sterilization and liposome coupling to minimize mRNA degradation, greatly improving the transfection efficiency. We show high efficiency mRNA-induced green fluorescent protein production in Jurkat (non-adherent) and HEK-273 (adherent) cells.

Synthesis and characterization: HGN were synthesized using the galvanic replacement reaction from silver nanoparticle templates in aqueous solution. The silver template size and the gold salt to silver ratio determines the size and shell thickness of the HGN, which in turn determines the plasmon resonance wavelength. The HGN were characterized using UV-Vis, and Nanosight particle counting and TEM imaging and yielded an average size of 30 nm and shell thicknesses of 2- 5 nm giving plasmon resonances of 650 -1040 nm, depending on the shell diameter to thickness ratio. Dipalmitoylphosphatidylcholine (DPPC) with small mole fraction of thiol terminated distearoylphosphatidylethanolamine- polyethylene glycol formed liposomes using the thin film hydration method, followed by manual extrusion to 200 nm. These liposomes were characterized by Nanosight and TEM. Different batches of liposomes were made containing carboxyfluorescin (CF), quantum dots or eGFP-mRNA. The liposomes were, thereafter, coated with gold nanoparticles by mixing the HGN with the liposomes; the HGN reacted with the thiol terminated PEG such that 1-3 HGN were bound to each liposome.

In-vitro testing: Transfection was achieved by suspension of the HGN-liposomal mixture in cell media at different concentrations. The HGN in cells were triggered by picosecond pulses of near-infrared light to release dye and/or mRNA from liposomes to cell. Cytotoxicity was measured using an MTT assay kit. The uptake of HGN-liposome was confirmed and quantified using flow cytometry and fluorescent microscope images. Transfection efficiency of the system was measured for all the GFP-positive formulations.

Results: Our results showcase successful HGN-liposome synthesis and characterization. The HGN are tethered to the liposomes as shown by TEM. Intracellular uptake of the HGN-Liposome contents was successful demonstrated by fluorescent microscopy and flow cytometry of 6-carboxyfluorescein (small molecule), quantum dots (2 - 5 nm in diameter) and expression of eGFP. The liposome-HGN showed little to no toxicity to both adherent and non-adherent cells. NIR pico-second light mediated release showed efficient uptake of carboxyfluorescein, quantum dots and eGFP mRNA. The HGN/mRNA mixture had successful cell transfection with high efficiency of eGPF expression with no minimal effects on cell viability post transfection. We plan to improve the throughput rate by increasing the flow rate of the cells through the laser light pulses to achieve a highly efficient method of ex-vivo cell modification.

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