Methods and systems for biomolecule delivery

Abstract

The invention relates to a multiwell plate, an electrotransfection system comprising the multiwell plate, and a corresponding method for delivering a biologically relevant cargo into immune cells, wherein the multiwell plate comprising a plurality of wells, wherein a plurality of hollow nanostraws project from a bottom of each well to a height of between 1.5 μm and 2 μm, and have an inner channel with a diameter of between 200 nm and 400 nm. In one embodiment, the immune cells are CD4+ T cells, CD8+ T cells, natural killer (NK) cells, regulatory T cells (Treg), yδ T cells, macrophages, dendritic cells (DCs), or neutrophils. In one embodiment, the cargo is a polynucleotide, a polypeptide, a nucleoprotein, a polysaccharide, or a combination thereof. In another embodiment, the cargo is a plasmid, an mRNA, a miRNA, a polypeptide, a ribonucleoprotein, or a combination thereof.

Description

[0001] Methods and Systems for Biomolecule Delivery Technical field

[0002] The present invention relates, in general terms, to devices for electrotransfection, and more specifically to nanostraw electrotransfection devices.

[0003] Background

[0004] The delivery of biomolecules into mammalian cells for cell therapy still suffers from various technical and practical limitations. Immune cells are particularly difficult to transfect, and conventional delivery methods have struggled to attain a sufficiently high transfection efficiency and cell viability necessary for high-throughput cell manufacturing. Viral vectorbased delivery systems, while efficient, are time-consuming to manufacture, limited in terms of the cargo capacity and the type of cargo that can be delivered, and present risks associated with insertional mutagenesis. Bulk electroporation often requires high concentrations of cargo, and even then transfection efficiency can be variable. Furthermore, the high-voltage electrical pulses used in bulk electroporation can compromise cell viability and induce perturbations that affect cell function.

[0005] There thus remains a need for delivery platforms that can provide efficient, high-throughput transfection of diverse cargo types into immune cells, while preserving cell viability and function. It would be desirable to overcome or alleviate at least one of the above-described problems, or at least to provide a useful alternative.

[0006] Summary

[0007] Disclosed herein is a method for delivering a biologically relevant cargo into cells, the method comprising: a) applying a force to drive a suspension of cells into contact with a plurality of nanostraws, wherein the nanostraws project from a substrate to a height of between about 1.5 pm and about 2 pm, and wherein the nanostraws have an inner channel with a diameter of between about 200 nm and about 400 nm; and b) applying a pulsed electrical field to drive the cargo through the inner channel of the nanostraws into an intracellular volume of the immune cells.Disclosed herein is an electrotransfection system for delivering a biologically relevant cargo into cells, the system comprising: a multiwell plate having a plurality of wells, wherein a plurality of hollow nanostraws project from a bottom of each well to a height of between about 1.5 pm and about 2 pm, and wherein the nanostraws have an inner channel with a diameter of between about 200 nm and about 400 nm; a cargo assembly comprising a frame coupled to a base electrode forming a reservoir for holding the cargo, the cargo assembly configured to receive the multiwell plate such that the nanostraws in each well are in fluid communication with the reservoir; and a cover for the multiwell plate, the cover having a plurality of top electrodes arranged thereon, each top electrode configured to extend into a well of the multiwell plate when the cover is positioned over the multiwell plate

[0008] Disclosed herein is the use of the electrotransfection system as defined herein for delivering a biologically relevant cargo into immune cells.

[0009] Disclosed herein is a multiwell plate for an electrotransfection system as defined herein, the multiwell plate having a plurality of wells, wherein a plurality of hollow nanostraws project from a bottom of each well to a height of between about 1.5 pm and about 2 pm, and wherein the nano straws have an inner channel with a diameter of between about 200 nm and about 400 nm.

[0010] Disclosed herein is a multiwell plate for an electrotransfection system as defined herein, the multiwell plate having a plurality of wells, wherein a plurality of hollow nanostraws project from a bottom of each well to a height of between about 1.5 pm and about 2 pm, and wherein the nano straws have an inner channel with a diameter of between about 200 nm and about 400 nm.

[0011] Disclosed herein is a kit for an electrotransfection system as defined herein, the kit comprising: a cargo assembly comprising a frame configured for coupling to a base electrode to form a reservoir for holding a cargo, the cargo assembly configured to receive a multiwell plate; and a cover having a plurality of top electrodes arranged thereon, each top electrode configured to extend into a well of the multiwell plate when the cover is positioned over the multiwell plate.Brief description of the drawings

[0012] Embodiments of the present invention will now be described, by way of non-limiting example, with reference to the drawings in which:

[0013] Figure 1 shows fabrication of high aspect-ratio nanostraws, (a) Schematic showing fabrication workflow for high aspect-ratio nanostraws. Field emission-scanning electron microscopy (FE-SEM) images captured during the different nano-fabrication steps at (i) 25,000x magnification and (ii) 65,000x magnification respectively (Scale: 1 pm), (b) Aluminum oxide (alumina) thickness measurements obtained from ellipsometer at different ALD deposition cycles, indicating an increase in coating thickness with deposition cycles. ***p <0.001 (c) Alumina thickness measurements obtained using atomic force microscopy between three different batch repetitions (A, B, and C) of 120 ALD deposition cycles, indicating manufacturing consistency, (d) Heat map plot showing the influence of plasma treatment time on the height of nano straws, (c) Heat map showing the influence of oxygen gas flow in reactive ion etch (RIE) chamber on the height of nanostraws (RIE: 650 W ICP, 20 W RF, 60°C, 90 sec), (f) Heat map showing the effect of reactive ion plasma composition in the RIE chamber (40 sccm O2) and plasma etch durations on height of nanostraws (RIE: 50 W ICP, 20 W RF, 25°C).

[0014] Figure 2 shows NExT transfection system enables intracellular delivery to diverse primary human immune cell types, (a) Schematic of NExT transfection system (b) FE-SEM image showing close adherence of CD8+ T cells around high aspect-ratio nanostraws after centrifugation and incubation for 2 hours, (c) Transfection efficiency of FITC-Bovine Serum Albumin (BSA) into CD4+ and CD8+ cells (n = 3). (d) Transfection efficiency of GFP-Cas9 into CD4+ and CD8+ T cells demonstrate donor-to-donor differences (n = 5 donors), (e) Transfection efficiency of mCherry mRNA with > 80% in CD8+ T cells after 24 hours, (f) eGFP mRNA delivery with > 50% is observed in CD4+ T cells after 24 hours (n = 3). (g) Transfection efficiency of eGFP mRNA of up to 60% efficiency in NK cells (cell selection with CD56+ marker) after 24 hours (n = 4). (h) Delivery of eGFP mRNA in CD4+CD25+ Tregcells was observed in over 30% cells with 2 min electric pulse after 24 hours, (n = 3). (i) Transfection of eGFP mRNA into human dendritic cells was observed up to 45% efficiency with 2 min electric pulse delivered at 50V (n = 3). (j) The transfection efficiency of eGFP mRNA resulted in delivery efficiency of up to 50% in y8-T cells within 24 hours, (n = 3 donors), (k) Transfection efficiency of THP-l-derived Ml macrophages with GPC3CAR constructs evaluated 24 hours after transfection by measuring the GFP expression. (1) NExT could achieve up to 90% delivery efficiency of 6-FAM-labelled siRNA into primary bone marrow derived mouse neutrophils (n = 3). Statistical significance was determined by one-way ANOVA test with post-tests performed with Tukey’s multiple comparisons test * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

[0015] Figure 3 shows NExT transfection of RNP induced CRISPR / Cas9 mediated genome editing in primary human CD8+ T cells. ‘NExT’ refers to NExT transfected cells and ‘CTRL’ refers to non-transfected cells (a) Agarose gels showing the T7E1 cleaved bands, (b) Western blots showing CXCR4 knockout, (c) Knockout of TRAC locus as determined using flow cytometry. Statistical significance was determined by one-way ANOVA test with post-tests performed with Tukey’s multiple comparisons test, ns - not significant, * p < 0.05.

[0016] Figure 4 shows that NExT is minimally perturbative to primary human immune cells. “NExT” refers to NExT transfected cells and “CTRL” refers to non-transfected cells, (a) Proliferation of CD4+T and CD8+ T cells observed up to 14 days post-NExT transfection showed that NExT transfection had minimal adverse effect on cell proliferation (n = 3). (b-c) Intracellular cell staining of cytotoxic granule proteins Perforin and Granzyme B analyzed at 4 days and 7 days after NExT transfection respectively demonstrating insignificant changes between NExT transfected cells and non-transfected cells (n = 4). (d) The expression of activation markers CD25 and (c) HLA-DR on the surface of CD4+ and CD8+ T cells was monitored up to 7 days following NExT transfection. Flow cytometry analyses conducted at 4- and 7-days post-transfection revealed minimal perturbations in activation marker expression on both CD4+ and CD8+ T cells as a result of NExT transfection (n > 4). (f) IP- 10 chemokine induced Transwell migration of CD4+ and (g) CD8+ T cells were evaluated with flow cytometry 7- and 14-days after NExT transfection respectively. The data indicated that NExT had minimal impact on the migratory properties of transfected cells, with the exception of one CD4+ and CD8+ T cell donor at 7 days. By day 14, the migratory properties returned to normal, (n = 4). (h) Differentiation marker expression on surface of CD8+ and CD4+ T cells measured using flow cytometry 4 days and 7 days after NExT transfection (n > 5). NExT provided gentle transfection and did not perturb T-cell differentiation. Using CD45RA and CCR7 surface marker expression, T cells can be categorized into Naive (CD45RA+CCR7+), Central Memory (CD45RA-CCR7+), Effector Memory (CD45RA-CCR7+) and Effector / EMRA (CD45RA+CCR7-) subsets, (i-j) Intracellular Cell Staining for cytokine expression and surface exhaustion marker analysis respectively were performed with NExT transfected CD8+ T cells and non-transfected cellsup to 14 days after transfection. There were minimal changes in the expression of cytokines such as IL-2, TNF-a, IFN-y and IL-6 and minimal changes in exhaustion marker expression of PD-1, LAG-3 and TIM-3. Statistical significance was determined by one-way ANOVA test with post-tests performed with Tukey’s multiple comparisons test: ns - not significant; * p < 0.05; **** p < 0.0001.

[0017] Figure 5 shows that the multiwell NExT platform enhances transfection throughput and exhibits comparable efficiencies to single well NExT system, (a) Schematic of high-throughput 96-multiwell NExT system, (b-d) Transfection efficiency of BSA-FITC protein, Dextran-FITC, and eGFP mRNA into various cell types. Transfection efficiency was evaluated 24 hours after transfection using flow cytometry.

[0018] Figure 6. (A) Longer exposure time to reactive ion plasma etching (RIE) increases the track-etched PETE membrane pore. RIE was performed with 5 sccm O2 + 45 sccm Ar gases in the etch chamber. The average membrane pore size was increased to -300 nm with a 4 min plasma etch time. (B) Effect of plasma gas composition and RF Power on membrane pore size. Membrane pore size was observed to increase up to -400 nm with a 4 min plasma etch time with modified RF Power and plasma gas composition. (C, D) Heat map depicting the height of nanostraw etching effected by increasing plasma etch durations with 30 sccm O2, and 30 sccm O2 + 3 sccm Ar reactive ion plasma, respectively in RIE chamber (RIE: 650 W ICP, 20W RF, 25°C).

[0019] Figure 7 shows NExT transfection of Dextran (70 kDa) polysaccharide into primary CD8+ T cells. (A) Transfection efficiency measured 24 hours after transfection (n = 2 donors) using flow cytometry. (B) Cell viability measured after 24 hours.

[0020] Figure 8 shows effect of NExT transfection on immune cell viability 24 hours after transfection. (A, B) Transfection of BSA-FITC into CD8+ and CD4+ T cells respectively. (C, D) Transfection of mRNA into CD8+ and CD4+ T cells respectively. (E) Transfection of eGFP mRNA into y5-T cells. (F) Transfection of GPC3-CAR plasmid constructs into THP-1 derived Ml macrophages. (G) Transfection of 6-FAM-labelled siRNA into bone marrow derived mouse neutrophils. (H) Transfection of eGFP mRNA into CD4+CD25+Trccells. (I) Transfection of eGFP mRNA into CD56+NK cells. Statistical significance was determined by one-way ANOVA test with post-tests performed with Tukey’s multiple comparisons test: * p < 0.05; **** p < 0.0001; ns - not significant.

[0021] Figure 9 shows Western blot 4 days after NExT transfection of CXCR4-targeting Cas9 RNPs show into human CD8+ T cells. “NExT” refers to NExT transfected cells and “CTRL” refers to non-transfected cells.Figure 10 shows CD3 expression in CD8+ T cells following NExT transfection of Cas9 mRNA and gRNA targeting the TRAC gene locus. Knockout of the TRAC gene eliminates CD3 expression. “NExT” refers to NExT transfected cells and “CTRL” refers to nontransfected cells.

[0022] Figure 11 shows secretion of perforin and granzyme B from CD4+ T cells or CD8+ T cells following NExT transfection. NExT” refers to NExT transfected cells and “CTRL” refers to non-transfected cells. Median Fluorescence Intensities (MFI) of perforin and granzyme B expression was measured 4 days and 7 days after NExT transfection.

[0023] Figure 12 shows expression of activation marker CD25 and HLA-DR in either CD4+ and CD8+ T cells following NExT transfection. “NExT” refers to NExT transfected cells and “CTRL” refers to non-transfected cells. Intracellular Median Fluorescence Intensities (MFI) of the markers was measured 4 days and 7 days after NExT transfection.

[0024] Figure 13 shows expression of the cell exhaustion markers PD-1, TIM-3, and LAG-3, expressed as Median Fluorescence Intensities (MFI) and measured 4 days and 7 days after NExT transfection (n = 3 or 5). “NExT” refers to NExT transfected cells and “CTRL” refers to non-transfected cells. Statistical significance was determined by one-way ANOVA test with post-tests performed with Tukey’s multiple comparisons test: ns - not significant. Figure 14 shows an embodiment of a high-throughput 96-well NExT transfection system. (A) Photos of the embodiment showing the multi-well plate, cargo assembly and plate lid connected to an electrical pulse generator and oscilloscope. (B-C) Top and side views of the transfection system.

[0025] Figure 15 shows top and side plan and perspective views of a cargo assembly of one embodiment of the electrotransfection system.

[0026] Figure 16 shows top and side plan and perspective views of a cargo assembly of another embodiment of the electrotransfection system.

[0027] Figure 17 shows top and side plan and perspective views of a cargo assembly of a third embodiment of the electrotransfection system. Dimensions are shown in millimetres. Figure 18 shows a top plan view of an insert for attachment to a base electrode to provide channels in the cargo reservoir.

[0028] Figure 19 shows optimi sation of parameters for nanostra w-medi ated transfection of mRN A into NK cells, (a) Schematic showing cells on nanostraws in a well of the transfection device, (b) SEM images of nanostraw array (left) and primary NK cell on the nanostraws (right), (c- f) Radar charts showing effect of voltage, number of pulses, days of NK cell activation following transfection and nanostraw density on: (i) transfection efficiency, (ii) NK cellviability, (iii) cargo uptake measured based on change in Median Fluorescence Index (MFI), (iv) activation index (NKG2D expression), and (v) proliferation index (Ki67 expression), (g-h) Effect of media osmolarity and CAR mRNA concentration on transfection efficiency. The surface CAR expression was captured using a CD19-PE protein with flow cytometry 24 hours after transfection, (i) Orthogonal sections of a representative NK cell showing nanostraws (red) penetrating through the nucleus (blue) and cytoplasm (green). Measurement of the height of nanowire penetration inside a primary human NK cell. Data represents 10 randomly selected cells with at least 5 nanostraw height measurements per cell. Data are presented as mean ± s.d. (j) Orthogonal projection of Fig. 20(1) showing nanostraws (red), nucleus (blue), and cytoplasm (green). Measurement of the number of nanostraws penetrating each cell based on a total of 15 cells analysed across different nanostraw samples. Data are presented as mean ± s.d.

[0029] Figure 20 shows that NExT delivery of CD 19 CAR mRNA into human NK cells do not compromise cell phenotype and function, (a) Effect of delivery voltage on transfection efficiency, (b-d) Killing of Raji (CD19+), Nalm-6 (CD19+) and K562 (CD19-) target cells by CD19 CAR-NK effector cells, assessed with Delfia EuTDA Cytotoxicity assay. Effector: Target ratios of 0.25:1 to 10:1 were tested, (e-g) Expression of CD107a degranulation marker, Granzyme-B and Perforin of transfected CAR-NK cells. Significant increase in CD107a expression following transfection indicated that NK cells were activated following CAR mRNA delivery, (h) Expression of TNF-a and IFN-y cytokines in a representative NK cell donor is demonstrated, (i-k) Surface expression of PD-1, NKG2D and CD57 in transfected CAR NK cells. (1) Proliferation of NK cells and CD 19 CAR-NK cells cultured with Raji tumor cells were monitored for 2 weeks after CAR mRNA delivery using NExT.

[0030] Figure 21 shows NExT delivery of fluorescein-tagged miR-186-5p (FL-miR-186) into human NK cells, (a) Effect of voltage and pulse duration on transfection efficiency, (b) Effect of cargo concentration on transfection efficiency, (c) Effect of centrifugation speed and nanostraw density on transfection efficiency, (d) Effect of nanostraw height on transfection efficiency.

[0031] Figure 22 shows simultaneous delivery of mRNA and miRNA into CAR-NK cells using NExT. (a-c) NExT delivery of miR-186-5p into human NK and CD 19 CAR-NK cells improved the expression of IFNy and the activation markers NKG2D and NKG2A. (d-c) NExT delivery of miR-186-5p into human NK cells and CD 19 CAR-NK cells improved cell proliferation and migratory changes (n = 6). (f) Western blot showing expression of proteinsin the TGF p signalling pathway in NK cells with or without miR-186-5p transfection in the presence of TGF. g) Benchmarking of nanostraw delivery of fluorescein-tagged miRNA-186 to conventional delivery methods, including the electroporation programs CM137, DN100, EO115, and the lipid-based methods Lipofectamine 3000 and Lipofectamine RNAiMax. Cell fluorescence was measured 24 hours after miRNA delivery, (h) Cell viability measured 24 hours following the benchmarking experiments, (i-j) Short-term tumour cell killing (6 hour incubation) performed using Raji cell spheroids and NK cells prepared using different conditions (2:1 effector: target ratio). Annexin+Pl- indicates early apoptotic cells and Annexin+PI+ indicates late apoptotic cells, (k) Effect of effector Target ratio on cell killing, (n) Representative fluorescence images from spheroid cultures with different effectortarget ratios.

[0032] Detailed description

[0033] The inventors have engineered a nanostraw electrotransfection system capable of highly efficient delivery of a variety of biomolecular cargo into primary cells, in particular immune cells, without compromising the viability or critical biological functioning of transfected cells. The system comprises a multiwell plate with wells containing high aspect ratio nanostraws projecting from the base of each well, together with a holding frame for the multiwell plate which contains a reservoir for the cargo, and an electrode-fitted cover. By applying gentle electric pulses, liquid cargo in the reservoir may be drawn through the nanostraws into cells which are in contact with the tip of the nanostraws. The inventors show that a variety of clinically relevant biomolecules, including proteins, polysaccharides, nucleic acids (such as RNA and plasmid DNA), and ribonucleoproteins (RNPs) can be precisely and efficiently delivered into adherent and non-adherent cells using the system. The system, also referred to as the Nanostraw Electro-actuated Transfection (NExT) system herein, improves scalability of transfection processes and can be easily integrated into automated workflows. Up to about 1.5x107cells may be simultaneously transfected in a 96-well plate (~150,000 cells / well) using the system of this disclosure.

[0034] Unlike conventional methods, the NExT system leverages physical forces and localised electric fields for intracellular delivery, eliminating the need for complex cargo packaging or the reliance on specialised biological mechanisms like endocytosis and receptor-mediated transport. This approach makes the NExT system a robust, cargo-agnostic and cell type-independent transfection tool. Furthermore, it can be seen in this disclosure that transfection using the device does not negatively impact essential biological functions, such as cell migration, proliferation, activation, differentiation, exhaustion, cytokine expression, or cytotoxic granule release, ensuring that transfected cells retain their therapeutic potential.

[0035] While recent studies have demonstrated the success of nanostraw-assisted electroporation for transfecting some cell types, such as primary neurons, stem cells and cardiomyocytes, the use of high aspect-ratio nanostructures for the transfection and reprogramming of primary human immune cells like CD3+ T cells is still underexplored. The inventors show that the NExT system can transfect a range of primary immune cells beyond CD8+ T and CD4+ T cells, including y5 T cells, Tregcells, natural killer (NK) cells, dendritic cells (DCs), macrophages, and neutrophils, all of which can be alternatives to CD8+ T cells for cell-based therapies.

[0036] Safe and effective delivery of gene editing components into primary immune cells remains a significant challenge. The inventors show that the NExT system can achieve CRISPR / Cas9-mediated gene editing in primary human CD8+ T cells, at efficiencies comparable to or exceeding the industry-standard Lonza 4D Nucleofector. Furthermore, cargoes of different sizes, such as mRNA and microRNA (miRNA), can be simultaneously introduced into immune cells using the NExT system, to regulate multiple biological pathways.

[0037] Accordingly, this disclosure provides systems and methods for electrotransfecting cells and in particular immune cells.

[0038] Disclosed herein is an electrotransfection system for delivering biologically relevant cargo into cells, the system comprising: a multiwell plate having a plurality of wells, wherein a plurality of hollow nanostraws project from a bottom of each well to a height of between about 1.5 pm and about 2 pm, and wherein the nanostraws have an inner channel with a diameter of between about 200 nm and about 400 nm; a cargo assembly comprising a frame coupled to a base electrode forming a reservoir for holding liquid cargo, the frame configured to hold the multiwell plate such that the nanostraws are in fluid communication with the reservoir; and a cover having a plurality of top electrodes arranged thereon, the cover configured for positioning in overlying relation to the multiwell plate when the plate is heldin the cargo assembly frame such that each top electrode extends into a well of the plate.

[0039] Disclosed herein is a method for delivering a biologically relevant cargo into cells, the method comprising: a) applying a force to drive a suspension of cells into contact with a plurality of nanostraws, wherein the nanostraws project from a substrate to a height of between about 1.5 pm and about 2 pm, and wherein the nanostraws have an inner channel with diameter of between about 200 nm and about 400 nm; and b) applying a pulsed electrical field to drive a liquid cargo through the inner channel of the nanostraws into an intracellular volume of the immune cells.

[0040] Electrotransfection system

[0041] Fig. 5a shows an exemplary electrotransfection system 100 of this disclosure. The system comprises a multiwell culture plate 102 (shown in the figure is a 96-well plate) fitted with hollow nanostraws 110 for high-throughput electrotransfection, a cargo assembly 112 for holding the plate and a cargo of interest, and a cover 128 for the plate. The cargo assembly and plate cover contain base and top electrodes 116, 128 respectively, arranged such that a voltage applied between the base and top electrodes drives the cargo through the inner channels of the nanostraws into cells that are in contact with the nanostraws in each well.

[0042] The nanostraws 110 project from the base of each well to a height of between about 1.5 pm and about 2 pm. The inner channel of each nanostraw has a diameter of between about 200 nm and about 400 nm. The inventors have found that these dimensions are optimal for nanostraw penetration into immune cells for cargo delivery.

[0043] Suitable materials for fabricating the nanostraws include but are not limited to aluminium oxide (alumina), silicon dioxide (silica) and hafnium dioxide (hafnia). In one embodiment, the nanostraws are aluminium oxide (alumina) nanostraws. The inventors have found that alumina is particularly advantageous as it is biocompatible and exhibits strong chemical resistance across a wide range of pH conditions compared to silica. Alumina also possesses good dielectric properties (e.g., high breakdown voltage and low leakage currents), which resists breakage at the nanostraw tips during electrotransfection (where the electrical pulses are focused).A nanoporous membrane 108 may be used as a template for fabricating the nanostraws 110, with the nanostraws projecting from the pores of the membrane such that the inner channels of the nanostraws are continuous with the membrane pores. The membrane 108 may be a track-etched polymeric membrane with a thickness of between 5 pm to 20 pm. The polymer material of the membrane is chosen based on compatibility with cell culture and with the deposition and etching process conditions for fabricating of the nanostraws, as discussed below. Non-limiting examples of suitable polymeric membranes include polycarbonate and polyester (e.g., polyethylene terephthalate) track-etched membranes, which have good mechanical strength and chemical resistance.

[0044] The fabrication of the nanostraw membrane typically follows a four-step process as shown in Fig. 1A. First, the membranes arc exposed to oxygen plasma to remove surface debris. Next, atomic layer deposition (ALD) is used to deposit a thin layer of metal oxide (e.g., aluminium oxide) about 20 nm thick on the membrane and on the inner surface of the pores. The metal oxide layer is then anisotropically etched using reactive ion etching (RTE) to expose the polymer membrane. Finally, an oxygen plasma is used for anisotropic etching of the polymer membrane to expose nanostraws of a particular height.

[0045] The inventors have found that fluorine-based etch chemistry (e.g., a SF6-based etching mixture) is advantageous for etching the metal oxide compared to chlorine-based etching mixtures (e.g., BCl3+ Cl2), as fluorine-based mixtures produce high and consistent etch rates while being relatively easy to control and generating fewer toxic by-products.

[0046] The pore density of the membrane determines the nanostraw density, while the pore diameter determines the outer diameter of the nanostraws. In some embodiments, the nanostraw density is about 107cm-2to about 108cm'2in each well. The thickness of the metal oxide coating influences the thickness of the nanostraw walls and thus the mechanical strength of the nanostraws. An alumina coating of between about 15 nm to about 25 nm thick (preferably about 20 nm) is found to produce nanostraws with reasonable mechanical strength.

[0047] The nanostraw membrane 108 may be attached to a bottomless multiwell plate 104 using clinical-grade adhesives known in the art. The nanostraws 110 may be fabricated on islands on the membrane corresponding to the positions of the wells on the multiwell plate 104 using a suitable mask, as shown in Fig. 5a. A piece of double-sided tape 106 with cut-outscorresponding to the positions of the wells is then used to attach the nanostraw membrane 108 to the bottom of the plate 104 such that the nanostraw-containing portions of the membrane form the bottom of each well. Alternatively, well-sized cut-outs may be punched out from the nanostraw membrane and attached individually to the base of each well of the bottomless multiwell plate 104.

[0048] The multiwell plate 104 may be fabricated from any material suitable for cell culture, nonlimiting examples of which include polystyrene, polyvinyl chloride, polypropylene, polyethylene terephthalate, and high- and low-density polyethylene.

[0049] A cell-adhesive coating may be provided on the bottom of each well. Advantageously, this allows the cells to be retained close to the nanostraws to enhance transfection. In some embodiments, the cell-adhesive coating comprises fibronectin and / or poly-L-lysine. In some embodiments, the cell-adhesive coating comprises an antibody or antigen-binding fragment thereof which binds to an immune cell, such as to a surface component of an immune cell. In one embodiment, the cell-adhesive coating comprises an antibody or antigen-binding fragment thereof which specifically binds to CD45 or CD3 on an immune cell. The celladhesive coating may be applied by physical adsorption or bioconjugation che istry known to the skilled person.

[0050] The cargo assembly 112 comprises a holding frame 114 that is coupled to a base electrode 116 to form a cargo reservoir 118. The frame 114 is configured to receive and securely hold the multiwell plate 102. For example, the frame may include a stage formed around an interior perimeter of the frame that is vertically offset from the base electrode 116. The stage protrudes horizontally inward into the cargo reservoir 118 and forms a ledge on which the multiwell plate 102 sits when positioned in the cargo assembly 112. When the plate 102 is properly seated in the frame 114, the base ends of the nanostraws 110 are in fluid communication with the cargo reservoir 118. The vertical offset between the stage and the upper surface of the base electrode determines the volume of cargo solution required to fill the reservoir to a level sufficient to contact the base ends of the nanotubes. To reduce the volume of cargo solution needed, the vertical offset may be arranged to be 2 mm or less.

[0051] The base electrode 116 forms the base of the cargo assembly and is arranged such that the upper surface of the electrode is in electrical communication with cargo held in the reservoir.The base electrode 116 may be fixedly or detachably coupled to the cargo assembly frame 114. The frame 114 may comprise a slot or fittings for receiving the base electrode. The coupling between frame and base electrode is suitably watertight to prevent leakage of the cargo from the reservoir. The frame may comprise an electrical contact in electrical communication with the base electrode. Alternatively, the electrical contact may be on the base electrode.

[0052] In some embodiments, the base electrode 116 is a plate electrode. The base electrode may comprise an indium tin oxide (ITO) coating, at least on its upper surface. ITO coatings can be easily deposited onto a variety of substrates, including glass and polymer sheets, using methods known in the ait, e.g., vacuum deposition. ITO coatings have high electrical conductivity while being water resistant and resistant to physical damage such as delamination, flaking, cracking and crazing, thus conferring reusability to the base electrode.

[0053] The system may include different frames each sized to accommodate a multiwell plate of a certain configuration. Figs. 15 to 17 show cargo assembly frames with different heights for use with multiwell plates of different well depths. In addition, the frame may include cutouts or slots to accommodate corresponding features on the multiwell plate. For example, vertical grooves may be added to the frame to accommodate corresponding ridges on the multiwell plate.

[0054] The cargo assembly may comprise a plurality of channels 122 extending across a surface of the base electrode 116 in contact with the cargo reservoir 118. Each channel is closed at one end 126 and has an opening at the other end 124 for receiving the cargo. The channels 122 are bounded by channel walls that extend substantially vertically from the supper surface of the base electrode 116. The height of these walls is selected such that when the multiwell plate is seated in the frame, the top of the walls contact the bottom of a multiwell plate 102 to form a fluid-tight seal with the bottom of the plate. An insert 120 containing the channel walls may be attached to the base electrode to form the channels 122. The attachment may be through various means including adhesive bonding or mechanical clamping.

[0055] Referring to Fig. 5a, each channel may underlie a row of wells when the multiwell plate is held in the cargo assembly. The channels may have a width that is approximately the diameter of each well, so that substantially all of the nanostraws in each well are in fluidcommunication with a channel. Alternatively, the channels may have a width that is smaller than the diameter of each well, so that only a portion of the nanostraws in each well are in fluid communication with a channel. In one embodiment, the channels have a width that is between about 1 mm to about 2 mm. The inclusion of the channels can reduce the volume of cargo needed for effective transfection compared to having an open reservoir or having individual cargo reservoirs for each well.

[0056] Suitable materials for the frame and channels of the cargo assembly include but are not limited to acrylics and plastics such as polypropylene, polycarbonate, polyethylene terephthalate and polyethylene.

[0057] In some embodiments, the cargo assembly comprises one or more inlets for receiving the cargo, the one or more inlets in fluid communication with the reservoir. The inlets may be positioned to allow the cargo to be introduced into the reservoir with the multiwell plate already seated in the cargo assembly. For example, the inlets may be provided in the side of the frame or on a top surface of the frame away from where the multiwell plate is to be held. Where channels are present in the cargo assembly, a separate inlet may be provided for each channel. This allows independent filling of each channel, such as with different cargo solutions. Alternatively, a single inlet may communicate with multiple channels, as shown in Fig. 18.

[0058] The cover 128 is configured to be positioned over the multiwell plate such that each top electrode 130 extends into a well of the plate. The cover 128 may be further configured to be securable to the plate for longer term cultures, such as using a friction fit, a snap fit, a screw fit, etc. The cover 128 may comprise one or more electrical contacts in electrical communication with the plurality of top electrodes. In some embodiments, the cover 128 is provided with an inlet 132 configured to be in fluid communication with the cargo reservoir when the cover is positioned over a multi well plate that is installed in the holding frame. The cover inlet 132 connects to a channel formed within the cover. When the cover is positioned over the plate, thus channel aligns with a correspondingly positioned channel provided in the plate, thereby establishing a continuous pathway from cover inlet through the plate and into the cargo reservoir below. This configuration allows reservoir filling even when cells arc actively being cultured in the wells of the plate on the cargo assembly.The top electrodes 130 may be configured as wires, pins, plates or other structures. In some embodiments, the plurality of top electrodes 130 are wire or stylus electrodes. The top electrodes may be formed from any suitable conductive material, including carbon, aluminium, silver, gold and platinum. In one embodiment, the plurality of top electrodes 130 are platinum electrodes. The top electrodes are preferably substantially identical so that the same voltage is produced in each well.

[0059] Each top electrode 130 may extend into a well to a distance of about 0.5 cm to about 1.25 cm from the bottom of the well when the cover is positioned over with the plate 102. In one embodiment, each top electrode extends into a well to a distance of about 0.5 cm from the bottom of the well. In use, the wells arc filled with culture medium to a sufficient volume to allow immersion of the top electrodes in the culture medium when the cover is placed over the plate.

[0060] The multiwell plate, plate cover and / or channels of the cargo assembly may be transparent or include transparent regions to allow visual inspection of the wells during operation.

[0061] The system 100 includes or is configured to connect to a power supply 134 via one or more electrical contacts provided on the frame 114, base electrode 116 or cover 128. The power supply may include programmable controls to deliver specific electrical protocols.

[0062] The electrotransfection system of this disclosure can be sold as a kit. For example, the cover and cargo assembly (with or without the base electrode) may be provided in a kit as reusable components. The kit may contain cargo assemblies with different frame heights and / or covers with different top electrode lengths or electrode arrangement for accommodating multiwell plates of different well depths or different number of wells. The kit may further contain one or more multiwell plates as single-use components.

[0063] Disclosed herein is a kit for an electrotransfection system as defined herein, the kit comprising: a cargo assembly comprising a frame configured for coupling to a base electrode to form a reservoir for holding a cargo, the cargo assembly configured to receive a multiwell plate; and a cover having a plurality of top electrodes arranged thereon, each top electrode configured to extend into a well of the multiwell plate when the cover is positioned over themultiwell plate.

[0064] In some embodiments, the kit further comprises a base electrode detachably attachable to the cargo assembly frame. In some embodiments, the kit further comprises one or more multiwell plates as defined herein.

[0065] Also disclosed is a multiwell plate for an electrotransfection system as defined herein, the multiwell plate having a plurality of wells, wherein a plurality of hollow nanostraws project from a bottom of each well to a height of between about 1.5 pm and about 2 pm, and wherein the nano straws have an inner channel with a diameter of between about 200 nm and about 400 nm.

[0066] The electrotransfection system may be used to deliver a biologically relevant cargo into immune cells using methods as described below.

[0067] Methods for electrotransfection

[0068] Methods herein may be used to deliver biologically relevant cargo into any cell, including adherent and non-adherent cells. The cells may be cell lines or primary cells. The methods are particularly suitable for delivering cargo to immune cells, including primary immune cells and immune cell lines.

[0069] In some embodiments, methods herein are used to deliver biological cargo into primary immune cells. In one embodiment, the immune cells are selected from CD4+ T cells, CD8+ T cells, natural killer (NK) cells, regulatory T cells (Treg), y5 T cells, macrophages, dendritic cells (DCs), and neutrophils.

[0070] A suspension of the immune cells is added to a culture well containing the nanostraws. The nanostraws project from the bottom of the well (such as from a membrane spanning the well bottom) to a height of between about 1.5 pm and about 2 pm. Each nano straw has an inner channel with a diameter of between about 200 nm and about 400 nm. The cells may be allowed to settle onto the nanostraws over a period of time. Preferably, a force is applied to drive the cell suspension into contact with the nanostraws, so that the nanostraws penetrate the cells. For example, a centrifugal force may be applied using a centrifuge to drive thecells into contact with the nanostraws.

[0071] In preferred embodiments, the cells form a single layer on the nano straws to reduce overcrowding and improve electrotransfection efficiency. This may be achieved by selecting an appropriate seeding density for the cell suspension. The seeding density may vary between cell types due to differences in their sizes. The inventors have found that a seeding density of between about 27,000 cells / cm3to about 450,000 cells / cm3is suitable for immune cells. This corresponds a cell density of about 22,000 cells / cm2to about 700,000 cells / cm2on the nanostraw substrate for transfection. The cells may be suspended in a medium with an osmolarity of between about 290 mOsm / L to about 310 mOstn / L. The media osmolarity may be adjusted by adding sterile water or NaCl solution.

[0072] The cargo is added to the cargo reservoir on the side of the nanostraw substrate or membrane opposite to the side that is in contact with the cells. The cargo reservoir is filled to a level that contacts the base ends of the nanostraws. Once contact is made, the liquid is drawn into the hollow interior of the nanotubes by capillary action.

[0073] The cargo is typically provided in a liquid preparation such as an aqueous solution and suspension. In some embodiments, the cargo preparation has an osmolarity not exceeding about 0.05 mOsm / L. For example, the cargo may be dissolved or suspended in distilled water or O. OOlx PBS to provide a very low osmolarity preparation. Advantageously, the low osmolarity can minimise the voltage drop at the nanostraw-cell interface to attain better transfection outcomes. Cargo media with higher salt content (e.g., lx PBS or 0. lx PBS) may lead to an abrupt loss of cell viability or poor transfection outcomes due to insufficient cell permeabilisation.

[0074] The cargo may be a polynucleotide, polypeptide, nucleoprotein, polysaccharide, or a combination thereof. The polynucleotide may be, for example, single- or double -stranded DNA or RNA or a combination thereof of any length. The polynucleotide may be linear or circular. The polypeptide may be a peptide, protein or protein fragment of any size. The nucleoprotein may be a ribonucleoprotein, such as a complex of a Cas protein and a guide RNA (gRNA). The polysaccharide may be neutral or charged (e.g., polycationic or poly anionic). The cargo may be delivered with a carrier, for example, a lipid or polymeric carrier to improve the stability and / or solubility of the cargo. In some embodiments, thecargo is selected from a plasmid, an mRNA, a miRNA, a polypeptide, a ribonucleoprotein, or a combination thereof.

[0075] The cargo introduction step may occur before the multiwell plate is placed in the cargo assembly frame (i.c., the reservoir is pre-filled), or after the plate is in position. The inlet on the frame accommodates both approaches.

[0076] Preferably, a DC voltage is used to provide the pulsed electrical field. The voltage is applied through electrodes positioned on the two sides of the nanostraw substrate / membrane, i.e., through one or more top electrodes positioned on the cell side and one or more base electrodes positioned on the cargo side of the nanostraw substrate / membrane. The polarity of the electrodes may be selected based on the cargo to be delivered. For example, to deliver polynucleotides, the electrotransfection system may be configured to have the top electrode be the anode / positive electrode and the bottom electrode be the cathode / negative electrode, to provide electro-driven flow of the negatively-charged polynucleotides from the cargo side through the nano straws into the cells.

[0077] The applied voltage, pulse width, pulse frequency, and total duration of stimulation may be varied according to the cargo, cargo medium, cell type, and cell culture medium used so as to optimise transfection efficiency. For transfecting immune cells, the inventors have found that ideal voltages are between about 25 V to about 40 V, suitable pulse widths are between about 100 ps to about 300 ps, and suitable pulse frequencies are between about 20 Hz to about 40 Hz. Due to factors such as circuit resistance, electrode conductivity, and salt concentration in the cargo and culture media, the voltage across the electrotransfection system may be lower than the applied voltage. A voltage offset may be applied where necessary to account for this voltage drop.

[0078] The electrical field may be applied as a single pulse or in a train of multiple pulses. The inventors have found that multiple pulses can improve transfection efficiency into some cells (e.g., 76 T cells), but may negatively impact cell viability and / or overall health of other cells.

[0079] For transfecting immune cells, the pulsed electrical field may be applied once for a duration of about 30 s to about 4 min. Alternatively, the pulsed electrical field may be applied between 1 and 5 times, each time for a duration of about 40 s. hr one embodiment, the pulsed electricalfield is applied for a duration of about 30 s to about 2 min to transfect a polynucleotide into an immune cell. In one embodiment, the pulsed electrical field is applied for a duration of about 30 s to about 4 min to transfect a polypeptide or a polysaccharide into an immune cell.

[0080] In some embodiments, the immune cells may be cultured in the culture chamber with the nanostraws prior to the application of the pulsed electrical field to, for example, allow the cells to settle on and / or adhere to the substrate. Alternatively or additionally, the immune cells may be cultured in the culture chamber after electroporation to, for example, allow for expansion of the cell population.

[0081] Terms used to describe the spatial relationships between different components or features, such as “above”, “below”, “top”, “bottom”, “upper”, “lower”, “front”, “back”, and similar terms, are used for descriptive purposes only to facilitate understanding of the written description, and are not meant to imply or limit the device or apparatus to any particular orientation or configuration of the described elements, unless explicitly stated.

[0082] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0083] As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an agent” includes a plurality of agents, including mixtures thereof.

[0084] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0085] Throughout this specification and the claims which follow, unless the context requires otherwise, the phrase "consisting essentially of", and variations such as "consists essentially of' will be understood to indicate that the recited element(s) is / are essential i.e. necessary elements of the invention. The phrase allows for the presence of other non-rccitcd elements which do not materially affect the characteristics of the invention but excludes additionalunspecified elements which would affect the basic and novel characteristics of the method defined.

[0086] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

[0087] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications, which fall within the spirit and scope. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

[0088] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the ail to which this invention belongs.

[0089] Certain embodiments of the invention will now be described with reference to the following examples which are intended for the purpose of illustration only and are not intended to limit the scope of the generality hereinbefore described.

[0090] EXAMPLES

[0091] 1: Nanostraw Electro-actuated Transfection

[0092]

[0093] Methods

[0094] System fabrication

[0095] The nanostraw membrane platform was fabricated on a thin 12 pm thick polyethylene terephthalate membrane with track etched pores purchased from GVS Pore Technologies, USA (Catalog: 1221385) and ipCELLCULTURE membrane filters from it4ip S. A. (AR Brown Co., Ltd.). These track-etched polyethylene terephthalate (PETE) membranes have apore density of 1×107- 3×108cm-2and a mean pore size of 220 nm. The membranes underwent surface cleaning with oxygen plasma at 100W RF Power for 30 sec to remove debris from the surface. For experiments involving pore size expansion, reactive ion plasma etching (RIE) was performed up to 4 min at 25°C using Inductively Coupled Plasma-Reactive Ion Etching (ICP-RIE) (Oxford Plasmalab 100, ICP-RIE) and the following gas composition and plasma etch conditions (5 seem O2 + 45 seem Ar, 100-300W RF Power), unless otherwise described. Using Atomic Layer Deposition, a thin 20 nm layer of aluminum oxide / alumina was uniformly deposited over the PETE surface (Savannah S 100, Cambridge Nanotech). The alumina deposition was performed using an exposure mode with 25 ms pulse time of precursor gases tri-methyl aluminum (TMA) and water (H2O) with intermediate purging using 45 sec N2 gas flow. The process was repeated for 120 cycles at 100°C to deposit a 20 nm thick aluminum oxide coating. The nanostraws were then developed following a top-down fashion in two steps. First, the aluminum oxide deposited over the surface of PETE was selectively and anisotropically etched in plasma containing sulfur hexafluoride (SFe), oxygen (O2) and argon (Ar) using Inductively Coupled Plasma-Rcactivc Ion Etching (ICP-RIE) (Oxford Plasmalab 100, ICP-RIE: 10 sccm SF6+ 10 sccm O2+ 10 sccm Ar, 650W ICP Power, 200W RF Power, 25°C, 30 sec). Second, the PETE membrane was selectively etched using O2 plasma to expose alumina nanostraws of 1.5-2 pm height (Oxford Plasmalab 100, ICP-RIE: 30 seem O2, 650W ICP Power, 20W RF Power, 25°C, 4p8 min).

[0096] Field Emission- Scanning Electron Microscopy (FE-SEM)

[0097] Characterization of the nanopores and nanostraws was performed with FE-SEM imaging. To minimize charging phenomenon, nanostraw membrane samples were coated with a 15-25 nm conductive layer of gold and imaged with 30–45° tilted stage, field emission HV gun at 2-10 kV (Verios FESEM 460). Membrane pore size and nanostraw height measurements were performed with the help of xT Microscope Control Verios software v5.0.2.

[0098] Ellipsometer measurements

[0099] Ellipsometry was used to measure the thickness of aluminum oxide coating after ALD deposition. Ellipsometer readings were captured using the Accurion ellipsometer and analyzed using EP4 Model software by comparing against previously established models of aluminum oxide as ‘layer stack’, while PETE membrane and crystalline silicon are added asthe ‘substrate’ within the software. Crystalline silicon wafers were used here as a support to hold the thin PETE membranes in place.

[0100] Atomic force microscopy (AFM) imaging

[0101] Surface roughness measurements were taken using Scanning Probe Microscope using Bruker Dimension Icon AFM. Imaging was performed with AC tapping mode using ScanAsyst Imaging platform. Each image was recorded at 0.6 Hz with a scan size of 20 pm. Step height measurement was performed using the Nanoscope Analysis software vl.8 to measure the difference in aluminum oxide thickness between coated and uncoated Step Regions Of Interest (ROI) and calculate the coating thickness of aluminum oxide.

[0102] Results

[0103] The NExT platform comprises millions of high aspect ratio nanostraws distributed across a thin polyester membrane, and the use of short voltage pulses to permeabilize cell membrane and electrophoretically deliver biomolecules from the nanostraws into the cytosol.

[0104] The nanostraw membrane was fabricated using a four-step protocol. Track-etched polyethylene terephthalate (PETE) was selected as the membrane substrate due to its superior mechanical strength. Figure la shows the fabrication process, starting with surface cleaning and culminating in the creation of nanostraws with a final height of -2 pm and diameters ranging from 200-400 nm. Nanostraw density may be varied between 107cm-2to 3×108cm-2. The thickness of the alumina coating, which impacts the wall thickness and durability of the nanostraws during cell transfection, was a critical parameter. Figure Ib-c show that measurements of alumina coating thickness using ellipsometry and atomic force microscope are in excellent agreement, with mean thicknesses of 19.3 nm and 19.9 nm respectively.

[0105] To avoid production of toxic by-products associated with chlorine -based etching mixtures (BCl3+ Cl2), which are commonly used for etching aluminium oxide, a SF6-based etching mixture containing SFe, Ar and O2 was used. SF6allows a high etch rate for aluminium oxide, while Ar and O2 maintain the etch rate and anisotropy, and remove etch residuals from the RIE chamber. The RTE step was optimized to remove the -20 nm aluminium oxide layer anisotropically from the surface of the polymer membrane. A plasma gas mixturecontaining 10 sccm SF6, 10 sccm O2, and 25 sccm Ar maintained at 5 mTorr pressure, with etching at 650 W ICP Power and 200 W Bias for 30 sec, was found to give consistent etching.

[0106] A comprehensive analysis of the effect of plasma etching on nanostraw height was conducted. As shown in Figure 6, different etching factors such as plasma gas composition and etching durations affected the track-etched nanopore sizes and nanostraw heights. It was found that there was an increase in nanostraw height up to 2 pm with longer plasma treatment (RIE: 650 W ICP, 20 W RF, 60°C, 30 sccm O2) (Figure Id). Furthermore, higher oxygen gas flow also resulted in taller nanostraws (Figure le) by increasing the oxygen plasma concentration and the etch rate. Figure If summarizes the relationship between nanostraw height, 40 seem (standard cubic centimeters per minute) O2 flow rate, and duration of plasma treatment. An 8 min plasma treatment was selected for fabrication as it consistently produced nanostraws with a height of 2 pm without any structural artifacts or membrane moisture loss.

[0107] To ensure that equal volumes of cargo can flow to all the wells of the device, a multichannel cargo flow reservoir was fabricated which holds cargo beneath the multiwell nanostraw membrane. The reservoir was made out of acrylic blocks of ~1 cm thickness, and the channels run across individual rows of wells. To promote automation in cell manufacturing and processing, cargo flow into the multichannel reservoir can be performed with the help of a syringe pump.

[0108] The multiwell plate was fabricated by attaching the nanostraw membrane sheet to a bottomless 96-well plate (GBO Biosciences) using a clinical grade biocompatible adhesive tape. This reduces manufacturing time and costs.

[0109] The cover for the plate contains platinum (Pt) wire electrodes which protrude vertically into the nanostraw wells. To maintain equal voltage in all the wells, the Pt wires were cut to equal lengths and optimally spaced 0.5 cm-1.25 cm from the bottom of the nanostraw membranes (a typical height is 0.5 cm).

[0110] Due to factors such as circuit resistance, bottom electrode conductivity, salt concentration in the media and osmolality of cargo solution, it was found that there was a notable voltage drop across the multiwell plate (ranging between cell types and applications from 1.0 V-35.0 V). To mitigate this issue, the pulse generator amplitude was offset by about +5 V for most applications.

[0111] Example 2: Transfection of immune cells using nanostraw electrotransfection device Methods

[0112] Cell culture

[0113] Human peripheral blood collected from healthy donors were used to isolate Peripheral Blood Mononuclear Cells (PBMCs) using Ficoll-paque density gradient lymphocyte separating medium (Cytiva Life Sciences, Catalog: 17144003) and Sepmate inserts (STEMCELL Technologies). CD4+ T cells and CD8+ T cells were then negatively isolated from PBMCs using Dynabeads Untouched cell isolation kits (Catalogs: 11352D, 11348D). Human T cells were cultured in RPMI 1640 (Gibco, Life Technologies, Catalog: 11875093) supplemented with 10% fetal bovine serum (FBS-Heat Inactivated, Life Technologies, Catalog: 16140071) and 1% penicillin- streptomycin (PenStrep, Gibco, Catalog: 15140122) (referred to as complete RPMI) in a 37°C / 5% CO2 incubator. T cells are activated with Dynabeads T cell activator CD3 / CD28 paramagnetic beads (Life Technologies, Catalog: 11141D) at 1:1 bead to cell ratio and stimulated with 100 International Unit (IU) of IL-2 / mL (Merck Millipore, Singapore, Catalog: HIL2-RO) of culture media renewed every 2-3 days. After 3 days of activation, Dynabeads were removed from CD4+ or CD8+ T cells, and the cells were prepared for NExT transfection in Opti-MEM media.

[0114] Human NK cells were sorted using Fluorescent Activated Cell Sorting (FACS) from healthy donor PBMCs by excluding B cells and T cells. For FACS analyses, fluorochrome conjugated antibodies against CD3, CD56, CD20 were used for staining PBMCs. The sorted NK cells were rested overnight in complete RPMI 30 lU / mL IL -2. Next day, the media was replaced with Opti-MEM and the cells were ready for transfection.

[0115] Human Treg cells used for this research were sorted out using Fluorescent Activated Cell Sorting (FACS) from healthy donor PBMCs by cell selection of CD4+CD25+ cells. For FACS analyses, fluorochrome conjugated antibodies against CD4, CD25, CD 127 were used for staining PBMCs. Sorted Treg cells were rested overnight in complete RPMI supplemented with 100 lU / mL IL-2. Next day, the cells were activated with Dynabeads CD3 / CD28Activation beads at a 4: 1 bead to cell ratio in complete RPMI supplemented with 300 lU / mL IL-2. After culturing for 3 days, the Dynabeads were removed, and the cells were prepared for NExT transfection.

[0116] The mononuclear cell (MNC) fraction from cord blood (CB) from full-term infants was separated using Ficoll-Paque PLUS (GE Healthcare, Uppsala, Sweden) through centrifugation and y8 T cells were enriched from CB MNCs with the EasySep Human y6 T cell isolation kit (STEMCELL Technologies) following the manufacturer’s protocol. The enriched CB-GDT cells w'ere cultured in a feeder cell-based expansion system for two weeks and then used for transfection during their expansion phase.

[0117] Monocytes were isolated from human PBMCs obtained from healthy donors using the CD14 MicroBeads kit (Miltenyi Biotec, Catalog: 130-050-201), following the manufacturer’s protocol for positive selection. Monocytes were maintained in dendritic cell (DC) media (CellGenix, Catalog: 20801-0500), supplemented with 1% PenStrep solution (Thermo Fisher, Catalog: 15140122), 1% human serum (SigmaAldrich, Catalog: H3667-100 mL), supplemented with 800 to 1600 lU / mL Recombinant Human GM-CSF (R& D Systems, Catalog: 215-GM-CF) and 10 ng / mL Recombinant Human IL-4 (PeproTech, Cat. No. 200-04). Subsequently, cells were mature in medium containing 100 lU / mL IFN-y (R& D Systems, Catalog: 285-IF-100) and 10 ng / mL lipopolysaccharide (LPS) (Sigma Aldrich, Catalog: L4516-1MG). The cells were replated and allowed to mature for 16 to 20 hours before being harvested for transfection.

[0118] The THP1 monocyte cells are seeded into the NExT wells and incubated with 150 nM phorbol 12-myristate 13-acetate (PMA) purchased from StemCell Technologies (Catalog: 74042) in complete RPMI for 24 hours to differentiate into M0 macrophages, followed by a 24 hour rest in complete RPMI. The next day, the M0 macrophages were polarised to Ml macrophages by addition of 20 ng / mL IFN-y and 10 ng / mL LPS and incubating for another 24 hours. After M1 polarisation, the RPMI is removed from the NExT wells and filled with Opti-MEM medium prepared for NExT transfection.

[0119] Bone marrow-derived mouse neutrophils were isolated using the following procedure. Wild type C57BL / 6 mice were euthanized via carbon dioxide inhalation in a chamber with rapid air displacement with compressed carbon dioxide. Animals were disinfected using 70%ethanol. Skin and muscles were removed from the lower limbs. Left and right femurs were dislocated from the hip joints carefully to avoid the fracture of femur heads. Tibia and fibula were separated from the femur. The bones were disinfected with 70% ethanol and washed with an ice-cold sterile PBS. To obtain the bone marrow, epiphyses of the bones were cut off and the medullary cavity was flushed with 3 mL of ice-cold sterile Phosphate buffered saline (PBS) using a 30-gauge needle. Bone marrow was treated with red blood cell lysis buffer for 3 minutes. The cells were centrifuged at 500 g for 3 minutes, resuspended in Percoll solution (1.04 g / mL) and overlaid on top of denser Percoll solution (1.08 g / mL). The reagent was centrifuged at 1500 g for 10 minutes without brake. The interphase layer, which consisted of enriched bone marrow-derived neutrophils were retrieved and washed twice with PBS. After washing, the cells were resuspended in plain RPMI (Gibco, Texas, USA, Catalog: 11875093) with lx PenStrep and later in lx Opti-MEM medium for NExT transfection.

[0120] Field Emission- Scanning Electron Microscopy (FE-SEM)

[0121] CD8+ T cell samples were seeded onto the NExT wells (pre-coated with fibronectin) and centrifuged at 600 g for 3 minutes, followed by a 2 hour incubation in complete RPMI to let the cells adhere close to the membrane surface. The wells were rinsed with PBS three times, and fixed with 2.5% glutaraldehyde (Sigma-Aldrich, Catalog: G6257) in PBS at room temperature for 1 hour. Glutaraldehyde solution was removed, and the NExT sample wells were rinsed with PBS three times with 10 minutes incubation each. Post-fixation was performed with freshly prepared 1% aqueous osmium tetroxide (OsCU) and incubated for 1 hour at room temperature. Following OsO incubation, the wells were rinsed with three rounds of deionised water for 10 minutes incubation each. Samples were subjected to gradual ethanol dehydration with a series of washes in increasing concentrations of ethanol: 25%, 50%, 75%, 95% (one round of incubation for 15 mins), 100% (three rounds of incubation for 10 mins each). The samples were then run through critical point dryer (Leica EM CPD300) and coated with 25 nm gold using Leica EM ACE 200 sputter coater. FE-SEM images were captured using FEI Quanta 650 FEG SEM.

[0122] Transfection using NExT device

[0123] To assemble the NExT wells, fabricated nanostraw membranes were cut into circular shapes and adhered to a cylindrical transparent plastic tube of 6.5 mm inner diameter and 9 mm height using AS-110 medical-grade biocompatible double-sided tape (AR Global, China;Catalog: ARcare® 90445Q). The assembled NExT wells were sterilized using 70% ethanol for 15 minutes followed by two times rinse with deionized water, once with lx PBS and UV sterilized for 1 hour. The cell culture sterile NExT wells were then coated with fibronectin using previously established protocols (Merck Millipore, Singapore, Catalog: F0895). Excess fibronectin was removed and the wells were rinsed twice with lx PBS and once with complete RPMI or cell culture medium. Primary T cells were plated at a density of 150,000 cells per well and centrifuged at 600 g for 5 minutes to maintain close adherence of cells with the nanostraws. Seeding density for other cell types were chosen to minimise overcrowding on the nanostraw membrane. For instance, the seeding density varied from 50,000 to 300,000 cells per well for different cell types. Adherent human DCs and Ml macrophages were cultured for prolonged duration (2 hours to overnight) to allow adhesion to the nanostraw membrane.

[0124] For transfection, a drop of transfection cargo (< 10 pl) was placed on top of Indium Tin Oxide (ITO) coated glass electrode (cathode) and the NExT transfection well was placed on top of the cargo. A platinum (Pt) electrode (anode) was dipped into the NExT well in contact w'ith the culture media, and a pulsed electric field (40 V, 40 Hz, 200 ps, 2 min) was applied between the electrodes using a high voltage electric generator (A-M Systems). The delivered electric pulses were confirmed using an oscilloscope (Agilent Technologies).

[0125] For all experiments involving the single-w'ell NExT system seeded with primary immune cells, each experiment was repeated using cells isolated from at least three independent healthy donors. Experiments with immortalized suspension and adherent cells were obtained with at least three biological replicates. In all cases, the control group consisted of cells seeded into NExT wells w'ith cargo loaded beneath the wells but without the delivery of NExT electric pulses. This control group served as a baseline to assess spontaneous cell penetration or endocytosis, which was consistently minimal without NExT electric pulses. For all experiments involving the multi- well NExT system, primary immune cells and immortalized cells were seeded in columns and were isolated from at least three independent donors and had three biological replicates respectively.

[0126] The cells were returned to a 37°C / 5% CO2 incubator immediately after transfection. Transfection efficiency was evaluated using flow cytometry (based on expression of eGFP or mCherry), and cell viability was evaluated based on Trypan blue exclusion (LifeTechnologies, Catalog: T8154) 24 hours after transfection. The transfected cells can be removed from the wells for downstream assays or cultured in the NExT wells. To assess immune cell functionalities, the primary T cells were incubated up to 14 days after transfection in the 37°C / 5% CO2 incubator seeded into appropriate cell culture vessels.

[0127] CAR construct and virus packaging

[0128] The CAR construct used consists of a CD8 signal peptide sequence, GPC3 scFv extracellular domain, CD8 hinge and CD8 transmembrane domain, followed by a 4- IBB co-stimulatory domain and CD3z intracellular signaling domain, linked to EGFP through a P2A sequence, expressed under the control of an EFla promoter.

[0129] 1×107HEK 293T (Cat. No. CRL-3216, ATCC, Manassas, Virginia, USA) were seeded in a 10 cm diameter tissue culture dish to achieve 70-75% confluence. Transfection mix was prepared by mixing packaging plasmid PsPAX, PMD2G and CAR plasmid at a DNA: PEI molar ratio of 1:1 in OptiMEM medium (Cat. No. 31985070, Gibco, Waltham, Massachusetts, United States). Medium were changed to DMEM (Cat. No. 11995065, Life Technologies, Carlsbad, California, United States) containing 5% FBS and 1% PS after 6 hours of transfection. Supernatant containing virus particles were harvested 48 hours and 96 hours of post-transfection. The supernatant was filtered with 0.45 pm filter and then centrifuged at a high speed at 15,000 g for 2 hours.

[0130] THP-1 culture and polarization

[0131] THP-1 were cultured in complete RPMI medium (Cat No. A1049101 Gibco, Waltham, Massachusetts, United States) containing 10% Fetal Bovine Serum (FBS) (Cat. No.

[0132] 10270106, Life Technologies, Carlsbad, California, United States) and lx Penicillin-Streptomycin (PS) (Cat. No.15140- 122, Gibco, Waltham, Massachusetts, United States) supplemented with 2-Mercaptoethanol (Cat. No. 21985023, Life Technologies, Carlsbad, California, United States). Cells were sub-cultured when the density reached 8xl05cells / mL.

[0133] To differentiate THP-1 monocytes into macrophages (M0), cells were incubated with 150 nM phorbol 12-myristate 13-acetate (PMA) (Cat. No. P8139-1MG, Sigma-Aldrich, St. Louis, Missouri, United States) for 24 hours, followed by 24 hours in RPMI medium without PMA. To polarize M0 macrophages into Ml, 20 ng / mL of interferon-gamma (IFN-y) (Cat. No. 285-IF-100, R& D Systems, Minneapolis, Minnesota, United States) and 10 ng / mL oflipopolysaccharide (LPS) (Cat. No. L2630-100MG, Sigma- Aldrich, St. Louis, Missouri, United States) were added and incubated for 24 hours.

[0134] THP-1 transfection with CAR

[0135] 0.267 pg of plasmid was transfected into 105THP-1-derived Ml macrophages using Neon Transfection System 10 pL Kit (Cat. No. MPK1025, Thermofisher Scientific, Waltham, Massachusetts, United States) with voltage of 1700 V, pulse width of 20 ms, pulse number of 1. GFP expression was analyzed by FACS, 24 hours after transfection.

[0136] THP-1 virus transduction with CAR

[0137] CAR lentivirus was added to 105THP-1 derived Ml macrophages with a MOI of 1. GFP expression was analyzed by FACS after 24 hours.

[0138] Flow cytometry

[0139] Transfection efficiency was evaluated using flow cytometry to measure the fluorescence emitted by transfected cells 24 hours after NExT. Cell samples recovered from the NExT wells were spun down at 300 g for 5 min and washed twice with lx PBS. T cells were stained with Live / Dead™ cell viability stains: Far Red, Violet (Life Technologies). After washing the cells to remove excess viability stain, the cells were stained with FACS buffer (2% FBS in PBS buffer) containing anti-human antibodies for the detection of surface marker expression. The cell samples were incubated for 30 min at 4°C in the dark, washed twice, and resuspended in FACS buffer. The transfection efficiency was calculated using the fluorescence captured using either the mCherry or FITC / GFP channel. The fluorochrome conjugated antibodies were purchased from BioLegend (USA), Invitrogen / Life Technologies, and BD Biosciences (USA). The cells were analyzed using CytoFLEX LX flow cytometer (Beckman Coulter) and the Flow data was analyzed using CytExpert and FlowJo (vlO) software and the artwork done in Inkscape 1.3. All the antibodies used in the research work are listed in Table 1.

[0140] CXCR4 sgRNA preparation

[0141] The 20 nt target sequence for the CXCR4 gene locus was retrieved from Schumann et al. (2015). To prepare the CXCR4 sgRNA, an overlapping PCR was performed with the DNA template encoding for a T7 promoter, the 20-nt target sequence and sgRNA scaffold sequence (Integrated DNA Technologies, Inc.) and PRIMESTAR Max DNA Polymerase2X Premix (Takara Bio USA, Catalog: R045). The PCR product estimated size ~ 120 bp) was separated using a 2% (w / v) agarose gel and the product band was excised and cleaned up using GeneJET Genomic DNA Purification Kit (ThermoFisher Scientific, Catalog: K0721) (as shown in Figure 16A). This dsDNA served as the DNA template for in vitro transcription of the CXCR4 sgRNA. In vitro transcription of sgRNA was performed for 16 hours at 37°C using HiScribe Quick T7 High Yield RNA Synthesis Kit following the manufacturer’s protocol (New England Biolabs, Catalog: E2050). The iVT synthesized sgRNA was cleaned up using Monarch RNA Cleanup Kit (New England Biolabs, Catalog: T2050L). RNA concentrations were measured using Nanodrop Onecspectrophotometer (ThermoFisher Scientific) and RNA aliquots were stored at -80°C. Before using the sgRNA for cell experiments, the in vitro digestion of DNA was tested with Cas9 nuclease following manufacturer’s protocol (New England Biolabs) ensuring in vitro DNA cleavage activity of the CXCR4 sgRNA (Figure 16B).

[0142] CXCR4 Cas9 RNP assembly and NExT transfection

[0143] Cas9 RNPs were prepared by incubating EnGen™ Cas9 NLS protein (20 pM) with CXCR4 sgRNA (40pM) at a 1:2 molar ratio in Cas9 nuclease reaction buffer. The mixture was incubated at 37°C for 15 min. After the incubation, the Cas9 RNPs were immediately transfected into the CD8+ T cells seeded onto the wells using NExT technique (40 V, 30 s, 40 Hz, 200 ps). Each NExT transfection well contained 60-80 pmol of Cas9 RNPs with a buffer volume of 5 pl in the transfection reservoir. After transfection, the cells were recovered and cultured for at least 4 days in complete RPMI supplemented with 100 lU / mL IL -2, IL-7 (PeproTech, Catalog: 200-07) and IL-15 (PeproTech, Catalog: 200-15) (2 ng / mL each) before CXCR4 knockout analysis was performed by T7E1 assay and CXCR4 protein expression quantified with Western blotting.

[0144] CXCR4 Cas9 RNP assembly and Lonza 4D nucleofection

[0145] Cas9 RNPs were prepared by incubating EnGen™ Cas9 NLS protein (20 pM) (New England Biolabs, Catalog: M0646M) with CXCR4 sgRNA (40 pM) at a 1:2 molar ratio in Cas9 nuclease reaction buffer. The mixture was incubated at 37°C for 15 mins. After incubation, the Cas9 RNPs were immediately transfected into the CD8+ T cells using 4D nucleofector (Lonza Biosciences) and P3 solution kit using the program code EH115. 106activated CD8+ T cells were washed twice in Opti-MEM medium before suspending in 20 pl Nucleofection buffer with the Cas9 RNPs. Each 20 pl Nucleofection reaction well wasloaded with 106cells in a buffer volume of 20 pl ± 5 pl to accommodate the volume of Cas9 RNPs (80 pmol).

[0146] T7 Endonuclease I (T7 El) assay

[0147] Gene edited CD8+ T cells expanded for at least 3 days after transfection were collected and washed twice with ice cold lx PBS. Genomic DNA was isolated from the cell pellets using EZNA Tissue DNA Kit (Omega Biotek) and the dsDNA concentration was measured using Nanodrop Onccspectrophotometer (ThermoFisher Scientific). 100 ng of genomic DNA was used to amplify CXCR4 gene using gene-specific PCR with 2X PrimeSTAR PCR Master Mix (Takara Bio USA), forward 5’-AGAGGAGTTAGCCAAGATGTGACTTTGAAACC-3’ (SEQ ID NO: 1) and reverse 5’- GGACAGGATGACAATACCAGGCAGGATAAGGCC-3’ (SEQ ID NO: 2) primers. The thermocycler setting for PCR includes one denaturation cycle of 98°C for 5 min, 35 cycles of 98°C for 10 s, 60°C for 30 s, and 72°C for 30s, and one extension cycle of 72°C for 10 min. The PCR product (938 bp) was separated using a 2% (w / v) agarose gel and the CXCR4 band was excised and cleaned-up using the GeneJET Genomic DNA Purification Kit. 200 ng of the cleaned-up dsDNA PCR product was used to set up the T7 El assay. The PCR product was first denatured at 95°C for 5 mins, then annealed at the following ramp rates (95°C-85°C at -2°C / sec and 85°C-25°C at -0.1°C / sec) and held at 4°C in a thermocycler. The annealed products were added with T7E1 enzyme and incubated at 37°C for 15 min. The reaction is halted by adding 0.25 M EDTA to the mixture, and the DNA fragments are visualized by separating them on a 2% (w / v) agarose gel. Densitometry analysis was performed with the use of ImageJ software (National Institute of Health, USA) by calculating the mean gray values. The total editing efficiency (%) was calculated using the following formula, % gene modification = 100 x (1 - (1 - fraction cleaved)1 / 2), where fraction cleaved is the ratio between the intensity of the cleaved bands and the sum of intensities of cleaved and parent bands.

[0148] Western blotting

[0149] Gene edited CD8+ T cells expanded for at least 4 days after transfection were collected and washed twice with ice cold lx PBS. The cell pellets were resuspended in RIPA buffer (Catalog: 89900) supplemented with lx Halt™ Protease Inhibitor Cocktail (ThermoFisher Scientific, Catalog: 87786) and incubated on ice for 30 mins. Cell lysates were centrifuged at 14,000 g for 15 minutes at 4°C and the supernatants were collected and stored at -80°C.The protein concentration was quantified by BCA assay (ThermoFisher Scientific, Catalog: 23225), following manufacturer’ s protocol and the cell lysates were separated by 10% Tris-Glycine SDS-PAGE (Bio-Rad). Proteins were wet transferred onto a 0.22 µm Immun-Blot PVDF Membrane (Bio-Rad). The membrane was blocked in 5% BSA for 1 hour at room temperature, washed thrice for 10 minutes with lx TBST and incubated overnight at 40°C with the CXCR4 primary antibody in lx TBST at recommended concentrations. After 3x washes with lx TBST for 10 mins each, secondary antibody was added and incubated for 1 hour. After three washes with lx TBST for 10 mins each, Pierce ECL substrate (ThermoFisher Scientific, Catalog: 32106) was used and chemiluminescence images were captured using ImageQuant™ TL chemiluminescence imager (Cytiva Life Sciences). The images were quantified by calculating the mean gray values using ImageJ software (National Institute of Health, USA).

[0150] TRAC Cas9 RNP assembly, transfection and analysis

[0151] TRAC crRNA and tracrRNA were mixed at 1:1 ratio and annealed in a conventional thermocycler to assemble the TRAC guide RNA (gRNA) with the following steps, 95°C for 30 s, 95°C-85°C at -2°C / sec ramp rate and 85°C-25°C at -0.1°C / sec ramp rate. Cas9 RNPs were prepared by mixing the Cas9 protein (Concentration: 40 pM, Source: qb3 lab, UC Berkeley) with the annealed TRAC gRNA at a 1:2 molar ratio similar to the steps mentioned above, and immediately used for transfection with NExT technique (40 V, 30s, 40Hz, 200µs) and Lonza 4D nucleofection. Two Cas9 RNPs transfection mixture were prepared for NExT transfection: (1) 60 pmol RNPs and (2) 80 pmol RNPs added with Cas9 buffer to maintain a volume of 5µl in the transfection reservoir, while Lonza nucleofection was performed with 120 pmol RNPs in the cuvette (Program Code: EH115, P3 buffer) following manufacturer’s protocol. After transfection, the cells were recovered and cultured for at least 4 days in complete RPMI supplemented with 100 lU / mL IL-2, IL-7 (PeproTech, Catalog: 200-07) and IL-15 (PeproTech, Catalog: 200-15) (2ng / mL each) before TRAC knockout analysis was performed by evaluating CD3 surface protein expression. CD3 is only expressed on the cell surface as a complex with the TCR. Cell staining for flow cytometry followed the aforementioned steps. Live CD8+ T cells were gated for CD3 surface protein expression with flow cytometry to assess the TRAC knockout efficiency between the two transfection techniques.

[0152] TRAC guide RNA (gRNA) assembly, transfection with Cas9 mRNA and analysisTRAC gRNA annealing was performed following the aforementioned steps. Cas9 transfection mixtures were prepared by mixing Cas9 mRNA with the annealed TRAC gRNA and immediately used for transfection with NExT technique (40 V, 30 s, 40 Hz, 200 ps) and Lonza 4D nucleofection. Two Cas9 transfection mixtures were prepared for NExT transfection: (1) 1.12 pg mRNA + 120 pmol TRAC gRNA, and (2) 1.5 pg mRNA + 160 pmol TRAC gRNA added with Cas9 buffer to maintain a volume of 5 pl in the transfection reservoir, while Lonza nucleofection was performed with 3.0 pg mRNA + 320 pmol TRAC gRNA in the cuvette (Program Code: EH115, P3 buffer) following manufacturer’s protocol. After transfection, the cells were recovered and cultured for at least 4 days in complete RPMI supplemented with 100 lU / mL IL-2, IL-7 (PeproTech, Catalog: 200-07) and IL-15 (PeproTech, Catalog: 200-15) (2 ng / mL each) before TRAC knockout analysis was performed by evaluating CD3 surface protein expression similar to the previous methods.

[0153] Proliferation assay

[0154] Proliferation assay was performed by culturing NExT transfected T cells (NExT) and nontransfected T cells (CTRL) for up to 14 days post-transfection. Cells recovered from the transfected and non-transfected NExT wells were counted using Countess 3 Automated Cell Counter prior to seeding for experiment. The two experimental conditions denoted by CTRL and NExT arc seeded in equal numbers in a 48 well plate and arc cultured in complete RPMI medium supplemented with 10% FBS, 1% PenStrep and 100 lU / mL IL-2 and subsequently expanded in appropriate culture vessels. Cell count was performed on 2, 4, 7, 10, 14 days post transfection. Cell counts were calculated using CountBright™ Plus Absolute Counting Beads and flow cytometry data was captured using Attune flow cytometer (Life Technologies, Catalog: C36995).

[0155] Marker expression, cytokine release and granule secretion

[0156] Cells collected from the NExT transfected culture wells and non-transfected wells were cultured in appropriate cell culture plates in complete RPMI medium supplemented with 10% FBS, 1% PenStrep and 100 TU / mL IL-2. Activation and differentiation marker expression was assessed 4 days and 7 days after NExT transfection (NExT samples) and compared against CTRL cells to investigate the effect of NExT transfection on immune cell activation and differentiation into T-cell subsets. Cells are stained for T-cell activation markers (CD25, HLA-DR), differentiation markers (CD45RA, CCR7) and exhaustionmarkers (PD-1, TTM-3, and LAG-3) with the fluorochrome conjugated antibodies denoted in Table 1.

[0157] Cytokine granules are vesicles containing cytotoxic glycoproteins such as perforin and granzyme B secreted by cytotoxic T cells at the tumor site. Cytokine expression was investigated with expression of IL-2, IFN-y, TNF- a, IL-6. The ability of NExT transfected T cells to secrete cytotoxic granules and cytokines were analyzed by staining the intracellular proteins using Intracellular Cell Staining (ICS) flow cytometry. ICS was performed with CTRL and NExT samples on 4 days and 7 days post transfection The cells were prepared using eBioscience™ Intracellular Fixation & Permeabilization Buffer Set (Life Technologies) following manufacturer’s protocol. Following fixation and permeabilization, the cells were stained with respective fluorochrome antibodies (Table 1), washed, and resuspended in FACS buffer. Flow cytometry data was captured using CytoFlex LX flow cytometer (Beckmann Coulter) and the data was analyzed using CytExpert software.

[0158] T cell migration assay

[0159] Chemokine-induced transwell migration was performed by seeding 100,000 live NExT transfected cells and non-transfected control cells in the upper Boyden chamber (Coming, 5 pm pore size) and adding chemokine IP-10 (Sigma Aldrich) at 25 ng / mL concentration in the bottom chamber. The cells were allowed to migrate to the lower chamber overnight in a standard 37°C / 5% CO2 incubator. The absolute number of migrated cells were counted using Attune flow cytometer (Life Technologies) with CountBright™ Plus Absolute Counting Beads (Figure 13). The experiment was performed with NExT cells 4 days and 7 days after transfection and compared against non-transfected CTRL samples.

[0160] Nanostraw penetration into cells

[0161] APTES-functionalised nanostraws were loaded with sulfo-Cy5 (red fluorescence). Primary human NK cells were seeded on the nanostraws, and the well was centrifuged at 600g for 5 min, following which the cells were cultured overnight. The cells were fixed and stained with Phalloidin-FITC (actin stain, green fluorescence) and DAPI (nuclear stain, blue fluoresence). 3D z-stacked confocal images were obtained to capture nanowire penetration into the NK cells. The extent of penetration and number of nanostraw associated with each cell were measured.Orthogonal sections of NK cells showed that the nanostraws penetrated both the cell cytoplasm and nucleus. Measurement of the height of nanowire penetration inside a primary human NK cell. Data represents 10 randomly selected cells with at least 5 nanostraw height measurements per cell. Data are presented as mean ± s.d.

[0162] NK cell killing assays

[0163] Raji cells (4000 cells) were cultured for 3 days using low attachment culture plates to form spheroids. Raji cells were stained with CellTrace Far Red. Short-term tumour cell killing (6 hour incubation) was performed with Raji cell spheroids and NK cells prepared under different conditions: No Transfection Control (CTRL), miR-186-5p transfected and cotransfection of CD19 CAR mRNA and miR-186-5p pre-treated with or without TGF p. NK cells were stained with CD56 marker or Cell Tracker CMFDA and added at different effector: target ratios (0.5:1 to 5:1).

[0164] Cell killing was evaluated in NK cell-spheroid cultures after 1 day using Annexin V FITC and PI dye staining. Staining was evaluated using flow cytometry performed on 10,000 target cells. Flow cytometry graphs were distinguished as early apoptotic cells based on Annexin+PI- expression or late apoptotic cells based on Annexin+PI+ expression. Spheroid images were captured using an epifluorescence microscope and spheroid diameter was measured.

[0165] Table 1. List of fluorochrome antibodies used.

[0166] Marker (anti-human) Fluorochrome conjugate Manufacturer (Catalog No,) CD3 APC / Fire™ 750 BioLegend (317351)

[0167] PerCP / Cyanine5.5 BioLegend (300327) FITC BioLegend (317306) PerCP / Cyanine5.5 Invitrogen (45-0037-42) APC BioLegend (300412) V500 BD Horizon™ (561416) CD4 PerCP / Cyanine5.5 BioLegend (317427 / 8)

[0168] APC / Cyanine7 BioLegend (344615 / 6) CD8 PE BioLegend (344705)

[0169] Brilliant Violet™ 510 BioLegend (344731 / 2)

[0170]

[0171] CD20 PE / Cyanine5 BioLegend (302308) CD25 Brilliant Violet™ 605 BioLegend (302631)

[0172] PE BioLegend (302605 / 6) CD45RA Brilliant Violet™ 421 BioLegend (304129) CD56 (NCAM) PE BioLegend (362507)

[0173] Brilliant Violet™ 711 BioLegend (318335) APC BD Pharmingen™ (555518) CD 127 (IL-7Ra) FITC BioLegend (351312)

[0174] CD 184 (CXCR4) PE / Cyanine5 BioLegend 306507 CD223 (LAG-3) PerCP / Cyanine5.5 BioLegend (369311)

[0175] CD 197 (CCR7) APC BioLegend (353214)

[0176] PE / Cyanine7 BioLegend (353226) CD279 (PD-1) PE / Cyanine7 BioLegend (329917) CD366 (TIM-3) APC BioLegend (345011) Granzyme-B PE BioLegend (372207) HLA-DR FITC BioLegend (307603)

[0177] IFN-y APC / Fire™ 750 BioLegend (502547)

[0178] IL-2 APC BioLegend (500309)

[0179] IL-6 PE / Cyanine7 BioLegend (501119) Perforin FITC BioLegend (308103)

[0180] TCR-y5 PE BD Pharmingen™ (561994) TNF-a PerCP / Cyanine5.5 BioLegend (502925)

[0181]

[0182] Results

[0183] A key challenge for cell manufacturing is ability to deliver various biomolecular cargo into a variety of cell types in a manner that is minimally perturbative, high-throughput and scalable. To address this challenge, a 96-well high-throughput NExT device was designed which is capable of transfecting over 14 million cells in a single run. Each well in the device can hold at least 150,000 cells, and the system allows the simultaneous transfection of cells from multiple donors (Figure 5a). A schematic and photos illustrating the components of the 96-well NExT system are provided in Figure 15.Figure 2a shows the general setup of the NExT platform for transfection. The magnified image exhibits a single NExT well, comprising a plastic cylinder with nanostraw membrane adhered at the bottom, facilitating delivery of biomolecule cargoes at the bottom and cells overlaid on the top. The circuit is closed by placing the Pt electrode anode dipped into the media and the bottom ITO glass electrode serving as the cathode. To enhance membrane permeabilization, nanostraw wells were pre-coated with fibronectin before seeding and centrifuging cells at 600 g for 5 mins (Figure 2a).

[0184] For transfection, 5-10 pl of cargo was placed on an Indium Tin Oxide (ITO)-coated glass electrode, with the NExT well positioned on top. The second platinum (Pt) electrode was immersed in the cell culture media inside the NExT wells to complete the circuit. The electrode polarity was determined by the charge of the payload being transfected. A negative cathode polarity applied at the ITO glass to electrophoretically transport negatively charged nucleic acids into the cells. By applying 200 ps square-waved DC pulses at 20-40 Hz for 30 sec to 2 min, transient pores formed in the cell membrane allowed cargo delivery into the cytosol. Suspension cells were recovered by pipetting and cultured in an incubator for downstream experiments, while adherent cell types, such as DC and macrophages, were gently detached using a mild detachment solution. Unlike other transfection technologies, NExT does not require specialized electroporation buffers and can transfect cells in standard culture media such as RPMI or reduced-serum Opti-MEM. General transfection protocols recommend using serum- free or reduced- serum media to prevent RNA degradation by RNases. Field emission-scanning electron microscopy (FE-SEM) images showed CD8+ T cells enveloping the nanostraws after centrifugation and 2 hours of incubation, a critical step for transient membrane permeabilization at the cell-nano straw interface necessary for effective transfection (Figure 2b).

[0185] The multiwell device facilitates the screening of allogeneic donors, transfection conditions and cargo compatibility for various cell types. Using the multiwell NExT device, simultaneous transfection of six different cell types (human CD4+ T cells, human CD8+ T cells, Raji cells, Jurkat cells, MDA-MB-231 cells and HEK-293T cells) with diverse cargo, including BSA-F1TC (Figure 5b), Dextran-FITC (Figure 5c) and eGFP mRNA (Figure 5d) was shown. Transfection efficiency was evaluated 24 hours post-transfection using flow cytometry'. A linear regression analysis comparing transfection efficiency using the 96-well NExT device and a single NExT well show that transfection efficiencies between the singlewell and multiwell NExT platforms were comparable, demonstrating the scalability and effectiveness of the high-throughput NExT system.

[0186] (a) Delivery of protein, polysaccharide and nucleic acids into immune cells Highly efficient delivery of small proteins, such as FITC-Bovine Serum Albumin (BSA), into CD4+ and CD8+ T cells could be achieved using the nanostraw device with a 2 min train of voltage pulses. Figure 2c shows more than 80% delivery of FITC-BSA into CD4+ and CD8+ T cells (n = 3 donors). Larger protein complexes, such as GFP-Cas9, could also be delivered into primary CD4+ and CD8+ T cells obtained from healthy donors. Significant donor-to-donor variability in transfection efficiency was observed, likely due to differences in cell-nanostraw interactions and donor health status. Despite these variations, consistent transfection efficiencies of 25-50% was achieved in CD8+ T cells and 50-90% in CD4+ T cells across the five donors for Cas9-GFP delivery (Figure 2d).

[0187] It was shown that high molecular weight polysaccharides, such as Dextran-FITC (70 kDa), could also be delivered into immune cells. Post-transfection 24 hours, Dextran-70 could be detected in up to 91.6 ± 0.32% of primary' CD8+ T cells, with minimal impact on cell viability (Figure 7). It was further demonstrated that the device could deliver larger and more complex biological cargo, including mRNA coding for mChcrry and cGFP (~1 kb), achieving up to 83% mCherry expression in CD8+ T cells (Figure 2e) and up to 54% eGFP transfection efficiency for CD4+ T cells (Figure 2f).

[0188] (b) NExT device delivers cargo into a variety of immune cells

[0189] Beyond CD3+ T cells, the ability of the NExT device to transfect a wide range of primary immune cell types was demonstrated with nucleic acid cargo used for CAR-immune cell therapy. Using a 2 min duration of 40 V pulses, mRNA was successfully transfected into human CD56+ NK cells (Figure 2g, mean transfection efficiency = 59.8 ± 2.55%), CD4+CD25+ Tregcells (Figure 2h, mean transfection efficiency = 34.07 ± 9.19%), and human gamma-delta (yS) T cells (Fig. 2j, mean transfection efficiency = 50.9%). 50 V pulses w'ere used to successfully transfect eGFP mRNA into human DCs (Figure 2i, mean efficiency = 46.98%). Moreover, it was shown that GPC3 -targeting CAR-GFP DNA plasmid could be transfected into Ml macrophages derived from THP-1 cells with an efficiency of 23.08 ± 4.15%, which is higher than the gold-standard techniques of viral transduction (8.75 ± 2.26%) and bulk electroporation (17 ± 1.09%) (Figure 2k). Neutrophilsare notoriously difficult to transfect due to their short lifespan and rapid turnover. It was shown that the NExT device could efficiently deliver siRNA (Accell Green non-targeting Control labelled with 6-FAM) into primary bone-marrow derived mouse neutrophils with a transfection efficiency of 92.59% as measured 6 hours post-delivery (Figure 21).

[0190] Maintaining cell viability post-transfection is critical, as it directly impacts cell yield for therapeutic application. Cell viability analyses were conducted using Trypan blue staining and flow cytometry post-transfection. In Figure 8, it can be seen that NExT transfection did not significantly alter cell viability in various immune cell types transfected with BSA-FITC (Figures 8A, B) or mRNA (Figures 8C-H). The exception was primary NK cells (Figure 81), where a loss of viability was observed, consistent with the challenges of maintaining NK cells in vitro post- transfection for extended periods.

[0191] (c) NExT transfection enables gene editing in primary immune cells

[0192] Knockout (KO) of the CXCR4 and TRAC gene loci are known to improve the efficacy of CAR-T treatment in clinical settings. CXCR4 KO in CAR-T cells has been shown to counteract the immunosuppressive effects of the CXCL12-mediated tumor microenvironment, thereby improving treatment outcomes. Similarly, knocking out the endogenous TCRa-chain constant region locus {TRAC has been associated with reduced T cell exhaustion and enhanced CAR expression in T cells. To demonstrate CRISPR-based gene editing of immune cells using the NExT device, CRISPR / Cas9 RNPs and guide RNAs targeting CXCR4 and TRAC were transfected into CD8+ T cells.

[0193] A 54.18% KO of the CXCR4 gene locus was achieved, as measured using the T7E1 assay 3 days after transfection (Figure 3 a). The T7E1 editing efficiency was calculated by comparing the intensities of the cleavage products to the parent DNA band. Figure 3a shows the three bands at 544 bp, 394 bp (representing the cleaved products) and 938 bp (representing the uncleaved parent band). The CXCR4 KO resulted in -60% reduction in CXCR4 protein expression in CD8+ T cells as measured 4 days after transfection (Figure 3b), with some donors showing up to an 80% decrease in CXCR4 expression relative to untreated samples (Figure 9).

[0194] The TRAC locus was also successfully knocked out in most of the transfected cells, as demonstrated by a downregulation of cell surface CD3 protein expression of up to 81.9%following the delivery of Cas9 RNPs targeting the TRAC (CD3 is only expressed on the cell surface in the presence of the TCR) (Figure 3c). NExT achieved KO efficiencies comparable to or better than bulk electroporation (up to 88.6% CD3 protein KO) with a significantly lower dosage of RNPs (80 pmol for NExT compared to 120 pmol for bulk electroporation). While previous studies have reported gene editing with nanochannels in HEK293T and Jurkat cell lines, this is the first report of gene editing in primary CD8+ T cells using high aspect-ratio nanostructures.

[0195] Gene editing could also be performed by transfecting Cas9 mRNA and a suitable gRNA using the NExT device. Up to 15% TRAC gene KO in CD8+ T cells was achieved (Figure 10). The KO rate was lower than transfecting Cas9 RNPs possibly due to factors such as nuclear localization, Cas9 protein expression levels or instability of the duplexed gRNA. Generally, duplexed gRNA can be stabilized by RNPs and less so by Cas9 mRNA. Using single-strand gRNA together with Cas9 mRNA could potentially enhance this knockout efficiency. Nevertheless, NExT demonstrated the ability to co-deliver multiple clinically relevant biomolecule cargoes simultaneously, showcasing its potential as a robust and versatile tool for gene editing in primary immune cells.

[0196] (d) NExT transfection does not perturb critical biological attributes of primary immune cells

[0197] For CAR-T cell therapies to be effective, maintaining critical biological attributes such as proliferation rate, tumor trafficking, activation and subtype differentiation upon tumor antigen exposure, pro-inflammatory cytokine secretion, and resistance to antigen-induced exhaustion are essential. These factors are hallmarks of CAR-T cell quality and their cytotoxic efficacy against tumor cells. While achieving high transfection efficiency is important, emerging transfection systems must also ensure minimal perturbation of these crucial biological properties. Given the diverse roles that immune cells play in tumor elimination, comprehensive assays were conducted to determine whether these essential traits were preserved following NExT transfection of PBS (mimicking cargo solution).

[0198] Figure 4a shows minimal differences in cell counts between NExT-transfected and nontransfected controls for CD4+ and CD8+ T cells cultured up to 2 weeks post-transfection, indicating that NExT does not adversely affect T cell proliferation. Production of cytotoxic granules, specifically perforin and granzyme B, was assessed, since this is critical for T cell-mediated killing of tumor cells. Using Intracellular Cell Staining (ICS), It was found that NExT did not significantly alter the secretion of perforin or granzyme B (Figure 4b-c) in CD4+ and CD8+ T cells up to 7 days post-transfection. T cell activation is a key mechanism in their response to tumor cells, typically marked by the expression of surface markers such as HLA-DR and CD25. Although these markers are not immediate indicators of T cell activation, their sustained expression on the T cell surface for over 2 weeks suggest an ongoing activated state. The results showed no significant difference in the expression of CD25 and HLA-DR on CD4+ and CD8+ T cells following NExT transfection compared to non-transfected controls (Figure 4d-e). Additionally, median fluorescence intensities (MFI) analyses of these markers revealed minimal changes between NExT -transfected cells and controls (Figure 11-12).

[0199] To investigate whether NExT affects T cell migratory behaviors, a migration assay was established using a Boyden chamber with an IP- 10 chemoattractant gradient. The number of cells migrating to the bottom chamber after overnight incubation was counted. While one CD4+ and one CD8+ sample exhibited significant differences in migration at day 7 post-NExT transfection, this was resolved by day 14 (Figure 4f-g). Differentiation of T cells into various effector or memory subtypes is crucial for the effectiveness of adoptively transferred T cells. Clinical studies have shown that less differentiated subtypes such as naive (TN), central memory (TCM), and stem-like memory (TSCM) T cells, exhibit better in vivo persistence and anti-tumor activity compared to terminally differentiated effector T cells (TEff). It was found that NExT transfection did not significantly affect T cell differentiation up to 7 days post-transfection, suggesting minimal perturbation (Figure 4h).

[0200] CD8+ T cells produce cytokines such as IL-2, TNF, IFN-y, that aid their cytotoxic activity against tumor cells, but over-production of IL-6 is associated with cytokine release syndrome and neurotoxicity. As shown in Figure 4i-j, NExT transfection minimally alters expression of key cytokines (IL-2, TNF, IFN-y and IL-6) and exhaustion markers (PD-1, TIM-3 and LAG-3) 14 days post-transfection in CD8+ T cells co-cultured with Raji tumor cells (1:1). This demonstrates that NExT has little to no adverse impact on cytokine expression and T cell exhaustion, both of which are critical for CD8+ T cell antitumor potency. Figure 14 shows that NExT transfection induced minimal changes to the MFI values of exhaustion marker expression PD-1, TIM-3 and LAG-3 in CD8+ T cells.(e) NExT is capable of simultaneous delivery of different cargoes into cells Exposure to TGF-P reduces NK cell proliferation, hampers surface expression of CXCR3 on NK cells, and reduces cell migration towards the chemoattractant IP10. TGF upregulates expression of TGF-P R2 and p-SMAD2 / 3. Conversely, the miRNA miR-186-5p reduces upregulation of TGF-P R2 and p-SMAD2 / 3, thereby contributing to immune rescue of NK cells. Simultaneous delivery of CD19 CAR mRNA and miR-186-5p can attenuate NK cell exhaustion and reverse TGF-P-induced NK cell immune suppression.

[0201] (f) Discussion

[0202] Previous studies have reported substantial immunogenicity and global gene perturbations associated with viral and bulk electroporation transfection methods. For example, bulk electroporation can trigger significant calcium ion influx, causing cell stress and global gene alterations. These issues often impair CAR-T cell quality during cell manufacturing, leading to T cell exhaustion, terminal differentiation, and ultimately reducing therapeutic efficacy.

[0203] The NExT device provides a cargo delivery solution with high transfection efficiencies without compromising the viability and critical biological functioning of transfected primary human immune cells. By applying gentle electric pulses through nanostraws, a variety of clinically relevant biomolecules, including proteins, polysaccharides, nucleic acids (such as RNA and plasmid DNA), and CRISPR / Cas9 RNPs can be precisely and efficiently delivered into primary human immune cells using the device. Unlike conventional methods, NExT leverages physical forces and localized electric fields for intracellular delivery, eliminating the need for complex cargo packaging or the reliance on specialized biological mechanisms like endocytosis and receptor-mediated transport. This approach makes the NExT device a robust, cargo-agnostic and cell type-independent transfection tool. Furthermore, transfection using the device does not negatively impact essential biological functions, such as cell migration, proliferation, activation, differentiation, exhaustion, cytokine expression, or cytotoxic granule release, ensuring that transfected cells retain their therapeutic potential.

[0204] While recent studies have demonstrated the success of nanostraw-assisted electroporation for transfecting some cell types, such as primary neurons, stem cells and cardiomyocytes, the use of high aspect-ratio nanostructures for the transfection and reprogramming of primary human immune cells like CD3+ T cells is still underexplored. This study shows that the NExT device can transfect a range of primary human immune cells beyond CD8+ T andCD4+ T cells, including yS-T cells, Tregcells, NK cells, DCs, macrophages, and neutrophils, cell types that are actively being developed as alternatives to CD8+ T cell-based therapies.

[0205] CRISPR-Cas9 technology has revolutionized translational biomedical research by emerging as a powerful means for precise gene editing. However, the safe and effective delivery of gene editing components into hard-to-transfect primary immune cells remains a significant challenge. Here, it was demonstrated that NExT transfection can achieve CRISPR / Cas9-mediated gene editing of the CXCR4 gene in up to 54.18% of primary human CD8+ T cells, and TRAC gene knockout in human CD8+ T cells with an efficiency of up to 81.9%, comparable to, or exceeding the industry-standard Lonza 4D Nucleofector.

[0206] The multiwcll NExT device is capable of processing -14.4 million cells in a single run. Given that each CAR-T dose requires > 106cells per kg of patient weight, the high-throughput NExT system increases the yield of transfected cells, reducing cell expansion time and overall manufacturing costs. The multiwell device is particularly useful for screening compatible cargos for genetically engineering immune cells from different allogeneic sources. In the context of clinical T-cell manufacturing, viral vectors and bulk electroporation are widely used for their high efficiency. However, their drawbacks — including safety and immunogenicity concerns, the need for stringent regulatory approvals, limited cargo capacity and variety, cell toxicity, diminished T-cell functionality, high manufacturing costs and long processing time, make them less appealing in the long run. This work with the multiwell NExT device shows great promise in overcoming these limitations, significantly advancing CAR-immune cell manufacturing and therapy. The device also improves scalability and integration with automated workflows.

[0207] It will be appreciated that many further modifications and permutations of various aspects of the described embodiments are possible. Accordingly, the described aspects are intended to embrace all such alterations, modifications, and variations that fall within the spirit and scope of the appended claims.

Claims

CLAIMS1. A method for delivering a biologically relevant cargo into immune cells, the method comprising:a) applying a force to drive a suspension of immune cells into contact with a plurality of nanostraws, wherein the nanostraws project from a substrate to a height of between about 1.5 pm and about 2 pm, and wherein the nanostraws have an inner channel with a diameter of between about 200 nm and about 400 nm; and b) applying a pulsed electrical field to drive the cargo through the inner channel of the nanostraws into an intracellular volume of the immune cells.

2. The method of claim 1, wherein the immune cells are primary immune cells.

3. The method of claim 2, wherein the primary immune cells are selected from CD4+ T cells, CD8+ T cells, yd T cells, Tregcells, natural killer (NK) cells, macrophages, dendritic cells (DCs), and neutrophils.

4. The method of any one of claims 1 to 3, wherein the nanostraws are present at a density of about 107cm-2to about 108cm-2on the substrate.

5. The method of any one of claims 1 to 4, wherein the cells are present at a density of about 27,000 to about 450,000 cells / cm3.

6. The method of any one of claims 1 to 5, wherein the pulsed electrical field is applied at a voltage of about 25 V to about 40 V.

7. The method of any one of claims 1 to 6, wherein the pulsed electrical field is applied at a pulse width of about 100 ps to about 300 ps.

8. The method of any one of claims 1 to 7, wherein the pulsed electrical field is applied at a pulse frequency of about 20 Hz to about 40 Hz.

9. The method of any one of claims 1 to 8, wherein the pulsed electrical field is applied once for a duration of about 30 s to about 4 min.

10. The method of any one of claims 1 to 8, wherein the pulsed electrical field is applied between 1 and 5 times, each time for a duration of about 40 s.

11. The method of any one of claims 1 to 10, wherein the cargo is selected from a polynucleotide, a polypeptide, a nucleoprotein, a polysaccharide, or a combination thereof.

12. The method of claim 11, wherein the cargo is selected from a plasmid, an mRNA, a miRNA, a polypeptide, a ribonucleoprotein, or a combination thereof.

13. The method of any one of claims 1 to 12, wherein the cargo is provided in a liquid preparation with an osmolarity not exceeding about 0.05 mOsm / L.

14. The method of any one of claims 1 to 13, wherein the cells are suspended in a medium with an osmolarity of between about 290 mOsm / L to about 310 mOsm / L.

15. The method of any one of claims 1 to 14, wherein the method further comprises culturing the electropulsed immune cells for at least 4 days.

16. An electrotransfection system for delivering a biologically relevant cargo into cells, the system comprising:a multiwell plate having a plurality of wells, wherein a plurality of hollow nanostraws project from a bottom of each well to a height of between about 1.5 pm and about 2 pm, and wherein the nanostraws have an inner channel with a diameter of between about 200 nm and about 400 nm;a cargo assembly comprising a frame coupled to a base electrode forming a reservoir for holding the cargo, the cargo assembly configured to receive the multiwell plate such that the nanostraws in each well are in fluid communication with the reservoir; and a cover for the multiwell plate, the cover having a plurality of top electrodes arranged thereon, each top electrode configured to extend into a well of the multiwell plate when the cover is positioned over the multiwell plate.

17. The electrotransfection system of claim 16, wherein the base electrode comprises aplurality of channels extending across a surface thereof for holding the cargo.

18. The electrotransfection system of claim 17, wherein each channel is configured to underlie a row of wells of a multiwell plate held in the cargo assembly.

19. The electrotransfection system of claim 17 or 18, wherein each channel is bounded by channel walls extending vertically from the surface of the base electrode.

20. The electrotransfection system of claim 19, wherein the channel walls are configured to contact a bottom surface of a multiwell plate held in the cargo assembly to form a fluid seal between each channel and the bottom surface of the plate.

21. The electrotransfection system of claim 19 or 20, wherein the channel walls are formed by an insert that is coupled to the surface of the base electrode.

22. The electrotransfection system of any one of claims 16 to 21, wherein the nanostraws project from pores of a nanoporous membrane that spans the bottom of each well.

23. The electrotransfection system of any one of claims 16 to 22, wherein the nanostraw density is about 107cm-2to about 108cm-2in each well.

24. The electrotransfection system of any one of claims 16 to 23, wherein the nanostraws are aluminium oxide (alumina) nano straws.

25. The electrotransfection system of any one of claims 16 to 24, further comprising a cell- adhesive coating on the bottom of each well.

26. The electrotransfection system of claim 25, wherein the cell-adhesive coating comprises an antibody or antigen-binding fragment thereof which binds to an immune cell.

27. The electrotransfection system of claim 26, wherein the cell-adhesive coating comprises an antibody or antigen-binding fragment thereof which binds specifically to CD45 or28. The electrotransfection system of any one of claims 16 to 27, wherein the cargo assembly comprises at least one inlet for receiving the cargo, the at least one inlet in fluid communication with the reservoir.

29. The electrotransfection system of any one of claims 16 to 28, wherein the base electrode is detachably attached to the cargo assembly frame.

30. The electrotransfection system of any one of claims 16 to 29, wherein the cargo assembly frame comprises a slot in a side wall thereof for receiving the base electrode.

31. The electrotransfection system of any one of claims 16 to 30, wherein the base electrode is a plate electrode.

32. The electrotransfection system of any one of claims 16 to 31, wherein the base electrode comprises an indium tin oxide (ITO) coating.

33. The electrotransfection system of any one of claims 16 to 32, wherein the plurality of top electrodes are wire electrodes.

34. The electrotransfection system of any one of claims 16 to 33, wherein the plurality of top electrodes are platinum electrodes.

35. The electrotransfection system of any one of claims 16 to 34, wherein each top electrode is configured to extend into a well of the multiwell plate to a distance of about 0.5 cm to about 1.25 cm from the bottom of the well when the cover is positioned over the multiwell plate.

36. Use of the electrotransfection system of any one of claims 16 to 35 for delivering a biologically relevant cargo into immune cells.

37. A multiwell plate for an electrotransfection system of any one of claims 16 to 35, the multiwell plate having a plurality of wells, wherein a plurality of hollow nanostraws project from a bottom of each well to a height of between about 1.5 pm and about 2 pm, and wherein the nanostraws have an inner channel with a diameter of between about200 nm and about 400 nm.

38. A kit for an electrotransfection system of any one of claims 16 to 35, the kit comprising:a cargo assembly comprising a frame configured for coupling to a base electrode to form a reservoir for holding a cargo, the cargo assembly configured to receive a multiwell plate; anda cover having a plurality of top electrodes arranged thereon, each top electrode configured to extend into a well of the multi well plate when the cover is positioned over the multiwell plate.

39. The kit of claim 38, further comprising a base electrode detachably attachable to the cargo assembly frame.

40. The kit of claim 38 or 39, further comprising a multiwell plate of claim 37.

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