NANOWIRE MEDIATED DELIVERY SYSTEM AND METHODS

COMPRISING THE SAME

TECHNICAL FIELD

The present invention relates generally to a molecular delivery system and methods of using the molecular delivery system to deliver molecules into cells. The molecular delivery system comprises a nanowire array (NW) comprising a substrate and a plurality of nanowires (NWs). BACKGROUND

Intracellular delivery of bioactive molecules into cells is critical for a plethora of progressive therapeutic, clinical and research applications. Intracellular delivery of molecules (for example, nucleic acids) can be achieved using multiple approaches, including viral (for example, adenoviruses, retroviruses, adeno-associated viruses, herpes simplex viruses and vaccinia viruses), chemical (for example, lipofection, calcium-phosphate, DEA-dextran) and physical (for example, electroporation, bombardment, microinjection) methods.

However, a high-throughput, scalable, efficient, flexible and non-destructive platform is yet to be developed for delivering exogenous molecules (for example, plasmid DNAs, siRNAs, peptides and proteins) across diverse cell types, especially into hard-to-transfect primary immune cells. Current techniques for transfecting live immune cells typically yield low delivery efficiencies, activate the immune response, induce non specific inflammation, require a lengthy viral transfection process, suffer from biological safety issues, suffer from cargo specificity, and/or require harsh conditions that result in widespread apoptosis.

Thus, despite advances in the field, new methods are required to deliver genes into diverse cell types effectively and with high yields, and in particular primary cells.

SUMMARY OF THE INVENTION

The present inventors have developed a method for delivering molecules into cells

(e.g. mammalian cells), including difficult to transfect primary cells. Advantageously, in some embodiments, the present inventors have shown that this method results in high delivery (or transfection) efficiency with relatively low transfection-related toxicity.

The present disclosure relates to a method of delivering a molecule into a cell using a nanowire (NW) array. The NW array comprises a substrate having a surface and a plurality of NWs attached to the surface. The plurality of NWs may extend from the surface of the substrate, for example, the NWs are upstanding from the substrate, and extend away from the attachment point on the substrate.

In one aspect, the present disclosure relates to a method of delivering a molecule into a cell, the method comprising the steps of:

a) obtaining a nanowire (NW) array comprising a substrate having a surface and a plurality of nanowires (NWs) attached to the surface;

b) applying a mixture comprising the molecule and a linker onto the NWs;

c) incubating the NWs with the molecule and the linker to form functionalised NWs, wherein the molecules are attached to the functionalised NWs via the linker; d) adding cells onto the functionalised NWs;

e) incubating the cells with the functionalised NWs to deliver the molecule into at least some of the cells.

In one embodiment, the method of delivering a molecule into a cell is a non-viral delivery method.

The present inventors have shown that applying a mixture comprising the molecule and a linker onto the NWs to form functionalised NWs may result in a higher transfection efficiency than stepwise addition of the linker then the molecule (i.e. layer by layer) according to at least some embodiments or examples described herein.

The linker may be, for example, selected from the group consisting of a silane , a cleavable linker, poly-lysine, a synthetic polymer, a plasma polymer, collagen, fibronectin and laminin. For example, the linker may be poly-lysine such as poly-D- lysine. In another example, the mixture comprises poly-D lysine and plasmid.

The method may comprise adding a first molecule to be delivered to the cells. The method may further comprise adding a second different molecule to be delivered to the cells. The method may further comprise adding one or more further different molecules to be delivered or co-delivered to the cells.

Similarly, the method may comprise adding one or more different linkers.

In one embodiment, step c) comprises incubating the NWs with the molecule and the linker for about 1 to 6 h. In another or further embodiment, step c) comprises incubating the NWs with the molecule and the linker at a temperature of about 1 to 10°C.

In one embodiment, the method further comprises the step of removing unbound linker and/or unbound molecule. For example, the unbound linker and/or unbound molecule may be removed via aspiration. One or more wash steps may be performed.

In a further embodiment, the NWs are dried after the unbound linker and/or unbound molecule is removed. In one embodiment, the method further comprises the step of centrifuging the cells and functionalised NWs during the incubation step e). Advantageously, this centrifugation step can provide for a reduced incubation time and/or higher % uptake of molecules into the cells.

Advantageously, the method can be used to mediate high efficiency transfection with short incubation times. Therefore, in one embodiment, step e) comprises incubating the cells with the functionalised NWs for about 6 to 12 h.

In another or further embodiment, step e) comprises incubating the cells with the functionalised NWs at a temperature of about 30 to 40°C.

The present inventors have also shown that the cells can be recovered from the NWs without impacting significantly on apoptosis and proliferative capacity of the cells. Accordingly, in one embodiment, the method further comprises the step of detaching the cells from the functionalised NWs after step e). The method may further comprise the step of re-culturing the detached cells.

In some embodiments, the molecule is a nucleic acid, a protein, a polysaccharide, or a small molecule. For example, the molecule is a DNA, RNA, antisense, or siRNA molecule. In one example, the molecule is a non-viral molecule. In a further example, the nucleic acid is a vector or a plasmid. The nucleic acid may encode a chimeric antigen receptor (CAR).

In some embodiments, the cell is an immune cell, immortalised cell, neuron, fibroblast, or stem cell. The present inventors have also shown that the method can be used to deliver molecules to transfect cells that are difficult to transfect, such as primary cells. Accordingly, in one embodiment, the cell is a primary cell such as a primary immune cell or stem cell. The primary immune cell may be a T-cell.

In some embodiments, the majority of the NWs attached to the surface may extend along a uniform direction, such as a substantially vertical projections (i.e., 60 to 90°) protruding from a solid base or platform.

In some embodiments, i) the average density of the NWs is about 0.1 to about 1.0 NWs per pm ² ; ii) the average length of the NWs is at least about 3 pm; and/or iii) the average diameter of the NWs is less than about 500 nm. In some embodiments, i) the average density of the NWs is about 0.1 to about 1.0 NWs per pm ² ; ii) the average length of the NWs is at least about 3 pm; and iii) the average diameter of the NWs is less than about 500 nm. In one embodiment, the average length of the NWs is about 3 pm to about 3.5 pm. In one embodiment, the average length of the NWs is about 3.2pm.

In one embodiment, the NWs may be silicon NWs, polymeric NWs, or a combination thereof. In a preferred embodiment, the NWs are silicon NWs. In another embodiment, the NWs are polymeric NWs, for example ORMOCOMP NWs, SU8 NWs, or polystyrene NWs.

In another embodiment, the NWs are a mixture of silicon NWs and polymeric

NWs.

In one embodiment, the NWs are silicon NWs.

In some embodiments embodiment, i) the average density of the silicon NWs is about 0.2 to about 0.4 NWs per pm ² ; ii) the average length of the silicon NWs is at least about 3 pm; and/or iii) the average diameter of the silicon NWs is less than about 150 nm. In some embodiments, i) the average density of the silicon NWs is about 0.2 to about 0.4 NWs per pm ² ; ii) the average length of the silicon NWs is at least about 3 pm; and iii) the average diameter of the silicon NWs is less than about 150 nm.

In one embodiment, the average density of the silicon NWs is about 0.3 NWs per pm ² . In another or further embodiment, the average length of the silicon NWs is about 3 to about 4 pm. In another or further embodiment, the average diameter of the silicon NWs is about 100 nm. In one embodiment, the average density of the silicon NWs is about 0.3 NWs per pm ² ; the average length of the silicon NWs is about 3 to about 4 pm; and the average diameter of the silicon NWs is about 100 nm. In one embodiment, the average length of the silicon NWs is about 3 pm to about 3.5 pm. In one embodiment, the average length of the silicon NWs is about 3.2pm.

In one embodiment, the average density of the silicon NWs is about 0.3 NWs per pm ² , the average length of the silicon NWs is about 3.2 pm, and the average diameter of the silicon NWs is about 100 nm.

In one embodiment, the NWs of step a) are attached to the surface along a substantially vertical direction to the surface.

It will be appreciated that embodiments as described herein in relation to the method of delivering a molecule to a cell according to the first aspect, including embodiments relating to the NWs, linker, molecule, and cells, can also provide embodiments for the molecule delivery system according to the second aspect, the kit according to the third aspect, the nanowire array according to the fourth aspect, the method of functionalising a plurality of NWs according to the fifth aspect, the population of cells according to the sixth aspect, the method of treatment/prevention according to the seventh aspect, and/or the method of providing a T-cell response according to the eighth aspect, as defined below, and vice versa.

In a second aspect, the present disclosure relates to a molecular delivery system comprising: a) a nanowire (NW) array comprising substrate having a surface and plurality of nanowires (NWs) attached to the surface, wherein NWs comprise one or more of the following features:

i) an average density of about 0.1 to about 1.0 NWs per pm ² ; ii) an average length of at least about 3 pm; and/or

iii) an average diameter of less than about 500 nm;,

b) a linker and

c) a molecule.

In one embodiment, the molecular delivery system is a non-viral delivery system.

In one embodiment, the NWs have i) an average density of about 0.1 to about 1.0 NWs per pm ² ; ii) an average length of at least about 3 pm; and iii) an average diameter of less than about 500 nm.

In a third aspect, the present disclosure relates to a kit for delivering a molecule to a cell, the kit comprising:

a) a nanowire (NW) array comprising a substrate having a surface, and plurality of nanowires (NWs) attached to the surface, wherein NWs comprise one or more of the following features:

i) an average density of about 0.1 to about 1.0 NWs per pm ² ; ii) an average length of at least about 3 pm; and/or

iii) an average diameter of less than about 500 nm.

In one embodiment, the kit further comprises b) a linker.

In one embodiment, the NWs comprise i) an average density of about 0.1 to about 1.0 NWs per pm ² ; ii) an average length of at least about 3 pm; and iii) an average diameter of less than about 500 nm.

In a further embodiment, the linker in the kit is a poly-lysine linker. For example, the linker is a poly-D-lysine linker. In another example, the poly-D-lysine is provided as an aqueous solution at a concentration of about 140 to 200 pg.mL ¹ .

In a further embodiment, the kit further comprises c) a molecule.

In a fourth aspect, there is provided a nanowire array, comprising:

a) a nanowire (NW) array comprising a substrate having a surface, and plurality of nanowires (NWs) attached to the surface, wherein NWs comprise one or more of the following features:

i) an average density of about 0.1 to about 1.0 NWs per pm ² ; ii) an average length of at least about 3 pm; and/or

iii) an average diameter of less than about 500 nm. In one embodiment, the NWs comprise i) an average density of about 0.1 to about 1.0 NWs per pm ² ; ii) an average length of at least about 3 pm; and iii) an average diameter of less than about 500 nm.

In a fifth aspect, there is provided method of functionalising a plurality of nanowires (NWs) with a linker and a molecule, the method comprising:

a) applying a mixture comprising the molecule and linker onto the NWs, and b) incubating the NWs with the molecule and the linker to form functionalised NWs, wherein the molecules are attached to the functionalised NWs via the linker.

In a sixth aspect, the present disclosure relates to population of cells transfected with a molecule by NWs. The cells may be transiently or stably transfected with the molecule, by application and incubation of the cells on nanowire (NWs) arrays.

In one embodiment, the population of cells are primary immune cells. For example, the population of cells comprises chimeric antigen receptor (CAR) such as CAR ⁺ T cells.

In another or further embodiment, the population of cells were not activated prior to transfection with the molecule.

In a seventh aspect, the present disclosure also relates to a method of treating and/or preventing a disease or condition in a subject, the method comprising administering to the subject a population of cells transfected with a molecule by NW arrays.

Any disease that involves the specific or enhanced expression of a particular antigen can be treated by targeting CAR ⁺ cells to the antigen. For example, autoimmune diseases, infections, and cancers can be treated. These include cancers, such as primary, metastatic, recurrent, sensitive-to-therapy, refractory -to-therapy cancers (e.g., chemo- refractory cancer). The cancer may be of the blood, lung, brain, colon, prostate, breast, liver, kidney, stomach, cervix, ovary, testes, pituitary gland, esophagus, spleen, skin, bone, and so forth (e.g., B-cell lymphomas or a melanomas). In the case of cancer treatment, CAR ⁺ cells typically target a cancer cell antigen (also known as a tumour- associated antigen (TAA)).

In an eighth aspect, the present disclosure relates to a method of providing a T- cell response in a subject having a disease comprising obtaining cells from the subject (comprising T-cells or T-cell progenitors); transfecting the cells with a nucleic acid encoding CAR construct according to the disclosure and administering an effective amount of the transgenic cells to the subject to provide a T-cell response. For example, the cells from the subject may be obtained from peripheral blood or umbilical cord blood. The cells may be collected by for example, apheresis or venipuncture. In some embodiments, the method comprises culturing the population of transgenic CAR ⁺ cells ex vivo in a medium that selectively enhances proliferation of CAR-expressing T-cells. For example, the transgenic CAR ⁺ cells are cultured ex vivo for less than 21 days, such as for less than 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 days or less. In certain embodiments, the CAR ⁺ cells are cultured ex vivo no more that 3 to 5 days. In some embodiments, the CAR ⁺ cells are co-cultured with antigen presenting cells such as dendritic cells, activating and propagating cells (AaPCs) or inactivated (e.g., irradiated) artificial antigen presenting cells (aAPCs) ex vivo for a limited period of time in order to expand the CAR ⁺ cell population according to methods known in the art.

The disclosed delivery system can be utilised to manufacture CAR ⁺ T cells for various tumor antigens (e.g., CD19, ROR1, CD56, EGFR, CD123, c-met, GD2). CAR ⁺ T cells generated using this technology can be used to treat patients with leukemias (AML, ALL, CML), infections and/or solid tumors. For example, methods of the disclosure can be used to treat cell proliferative diseases, fungal, viral, bacterial or parasitic infections. Pathogens that may be targeted include, with limitation, Plasmodium , trypanosome, Aspergillus, Candida , HSV, RSV, EBV, CMV, JC virus, BK virus, or Ebola pathogens. Further examples of antigens that can be targeted by CAR cells include, without limitation, CD 19, CD20, carcinoembryonic antigen, alphafetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, melanoma-associated antigen, mutated p53, mutated ras, HER2/Neu, ERBB2, folate binding protein, HIV-1 envelope glycoprotein gpl20, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD23, CD30, CD56, c-Met, meothelin, GD3, HERV-K, IL-l lRalpha, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII, or VEGFR2. In still further embodiments, a CAR can be conjugated or fused with a cytokine, such as IL-2, IL-7, IL-15, IL-21 or a combination thereof.

General terms

Throughout this disclosure, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (e.g. one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter. Thus, as used herein, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. For example, reference to "a" includes a single as well as two or more; reference to "an" includes a single as well as two or more; reference to "the" includes a single as well as two or more and so forth.

As used herein, the term“about”, unless stated to the contrary, refers to +/- 10%, more preferably +/- 5%, more preferably +/- 1%, of the designated value.

Those skilled in the art will appreciate that the disclosure herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure 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 or any two or more of said steps or features.

Each example, aspect and embodiment of the present disclosure described herein is to be applied mutatis mutandis to each and every other example, aspect or embodiment unless specifically stated otherwise. The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the disclosure as described herein.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

The term "consists of, or variations such as "consisting of, refers to the inclusion of any stated element, integer or step, or group of elements, integers or steps, that are recited in context with this term, and excludes any other element, integer or step, or group of elements, integers or steps, that are not recited in context with this term.

The term“NW” is an abbreviation of the term nanowire. These terms are used interchangeably throughout the disclosure.

The term“NW” or“NWs” or nanowire or nanowires refers to a material in the shape of a wire or rod having a diameter in the range of 1 nm to 1 pm.

The term“Si” refers to silicon. These terms are used interchangeably throughout the disclosure.

The term“SiNW” or“SiNWs” refers to silicon NWs. These terms are used interchangeably throughout the disclosure.

The term“pNW” or“pNWs” refers to polymeric NWs. These terms are used interchangeably throughout the disclosure.

The term“molecule” is understood to mean any substance or matter that can be delivered to the cell. The term molecule also includes two or more molecules. For example, the term molecule includes, but is not limited to, a protein, a nucleic acid, a nanoparticle, a dye, a toxin, a virus, and a polymer particle. The molecule may be a viral or a non-viral molecule.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF DRAWINGS

Embodiments are described in further detail below, by way of example, with reference to the accompanying drawings briefly described below:

FIGURE 1A: Schematic summarising of a method of delivering a molecule to a cell comprising a substrate and a plurality of silicon NWs;

FIGURE IB: A representative cross-sectional SEM image of silicon NW arrays of the present invention;

FIGURE 2: An enlargement of the circled area 200 of the molecule delivery system in Figure 1 depicting a single NW with molecules (X and Y) attached to the surface via linkers (A and B);

FIGURE 3: A schematic depicting the adding of cells onto the NWs in a cell plate using an embodiment of the invention, where 300 is the cell plate and 301 is a well of that cell plate;

FIGURE 4: Cell viability of cells harvested from flat Si and SiNWs. a) Live/dead staining of GPE86, LI.2 and Jurkat cells on flat Si (i, iii, and v, respectively) and SiNWs (ii, iv, and vi, respectively), after 6 h incubation. FDA (green) indicates live cells and PI (red) indicates dead cells. Scale bars, 100 pm. b-d) Quantification of the viability of GPE86 cells (b), LI.2 cells (c) and Jurkat cells (d) on flat Si (red dots) and SiNWs (blue dots) analysed from (a). Each symbol in (b-d) represents an individual substrate. Data are representative of three independent experiments with at least three substrates per group.

FIGURE 5: Penetration, cellular deformations and endocytosis induced by SiNWs. a-c) SEM images after FIB milling at 90° demonstrating the interface of GPE86 (a), LI.2 (b) and Jurkat (c) cells with SiNWs. d) SEM images after FIB milling at 45° revealing the SiNW (indicated by yellow arrows) penetration into the nucleus and cytoplasm of a LI .2 cell (i) as well as the engulfment of SiNWs by the cell membrane of a Jurkat cell (ii). e) Representative confocal images showing the localisation of nucleus (blue), caveolin-1 (Cav-1, green) and clathrin heavy chain (CHC, magenta) within GPE86 cells cultured on flat Si (i) and SiNWs (ii), which were coated with Cy3-gWiz- GFP (Cy3-gWiz, red) plasmids. The bottom right images in i, ii are enlargements (x2) of the areas outlined in‘Merge’, showing Cav-1 and Cy3-gWiz channels. Original images (a-d) are black- white inverted. Scale bars, 2 pm (a-d) and 10 pm (e).

FIGURE 6: Cell proliferation and apoptosis after detachment from SiNW substrates a) Expression of CTV in GPE86, LI .2 and Jurkat cells on Day 2 and 3 after detachment from 6 h-incubation on flat Si (magenta curve) and SiNWs (blue curve). Unstained cells (solid gray) and CTV freshly stained cells (solid red) served as negative and positive controls, respectively b) Quantification of the proliferation rate of cells harvested from flat Si (magenta dots) and SiNWs (blue triangles) as shown in (a) c) Flow cytometry of Jurkat cells harvested from flat Si and SiNWs at different incubation times (2, 6, 12, and 24 h), and stained with Annexin V-FITC/PI Apoptosis Kit. Gating strategy showing 4 populations (Annexin V /PL, Annexin V ⁺ /PL, Annexin V ⁺ /PI ⁺ and Annexin V /PI ⁺ ). d) Graph plotting of the percentages of the four populations (Annexin V /PL (black), Annexin V ⁺ /PL (green), Annexin V ⁺ /PI ⁺ (red) and Annexin V ^_/ PI ⁺ (blue)) at different incubation times as in (c). e) Statistical analysis of the percentages of the four populations (annexin V-/PI-, annexin V+/PI-, annexin V+/PI+, and annexin V-/PI+) after detachment from flat Si and SiNWs at different incubation times as in (a). Data are representative of three independent experiments (a,b) or two experiments (c,d) with at least two substrates per group

FIGURE 7: Effect of external centrifugal force on plasmid insertion and cell transfection by flat silicon substrates and SiNWs. a) Flow cytometry of LI .2 cells harvested after 6 h incubation on flat Si (with spin), SiNWs (without spin) and SiNWs (with spin), coated with Cy3-gWiz-GFP plasmids at different concentrations (10, 50, and 100 ng pL ^_1 ). Numbers adjacent to outlined areas indicate the percentage of Cy3 ⁺ cells (indicative of plasmid insertion) b) Percentages of Cy3 ⁺ LI .2 cells as shown in (a). Black, orange and blue dots indicated cells harvested from flat Si (spin, black dots), SiNWs (non-spin, orange dots) and SiNWs (spin, blue dots), respectively. *P = 0.0104, **P = 0.003, ****p < 0.0001 (Two-way ANOVA). c) Flow cytometry of GPE86, LI .2, and Jurkat cells 48 h after harvesting from plasmid-coated flat Si and SiNWs (with 6 h incubation). Numbers adjacent to outlined areas indicate the percentage of GFP ⁺ cells (indicative of positive transfection) d) Percentage of GFP ⁺ cells harvested from flat Si (red dots) and SiNWs (blue dots) as in (c). ***P = 0.0002, ****P < 0.0001 (Mann- Whitney’s U-tests). Each symbol (b,d) represents an individual substrate. Data are representative of two (a,b) and three independent experiments (c,d) with at least three substrates per group.

FIGURE 8: Comparison of the transfection efficiency on primary T cells via stepwise and mixed addition of PDL and Cy3-plasmids. a) Flow cytometry detection of Cy3-plasmids within primary T cells after 6 h culture on flat Si (i) and SiNWs (ii), coated stepwise with PDL and then Cy3-plasmids (PDL ¹ + Plas ² ), and SiNWs (iii) coated with a mixture of PDL and Cy-Plasmids (PDL-Plas Mix) b) Quantification of Cy3 ⁺ % within each group of T cells as shown in a. c) Flow cytometry detection of Cy3 and GFP expression within T cells 48 h after detaching from flat Si (i) and SiNWs (ii), coated with PDL ¹ + Plas ² , and SiNWs (iii) coated with PDL-Plas Mix. d) Quantification of GFP ⁺ Cy3 ⁺ % within each group of T cells as shown in c. ***P = 0.0005, ****P < 0.0001 (Two-way ANOVA).

FIGURE 9: Transfection of Cy3-gWiz-GFP plasmids into inactivated primary mouse T cells a) Flow cytometry detection of Cy3 expression within primary mouse T cells, detached from flat Si and SiNWs coated with Cy3-gWiz-GFP plasmids, after 6 h incubation b) Quantification of the percentage of Cy3 ⁺ cells harvested from flat Si and SiNWs as in (a) c) Flow cytometry detection of Cy3 and GFP expression within primary mouse T cells 48 h after re-culture in fresh media d) Quantification of the percentage of GFP ⁺ Cy3 ⁺ cells harvested from flat Si and SiNWs as in (c). ***P = 0.0002, ****P < 0.0001 (Mann-Whitney’s U-tests). Each symbol (b,d) represents an individual substrate. Data are representative of two independent experiments with at least three substrates per group e) T cell activation study. Heat map showing the mean expression level of key T cell activation markers within inactivated T cells after 6 h interfacing with flat Si and SiNWs, and within T cells pre-activated with anti-CD3, anti-CD28, and IL-2 for 48 h (activated T). Freshly isolated inactivated T cells served as the negative control (neg Ctrl). **P = 0.0066, ****p < 0.0001 (Dunnetf s multiple comparisons test).

FIGURE 10: Transfection of Cy3-tagged CAR construct encoding human CD19 receptor on primary mouse T cells via SiNWs. a) Flow cytometry detection of the expression of Cy3 and human CD 19 within primary mouse T cells that were re-cultured in fresh media for 48 h following incubation for 6h on functionalised flat Si (left) and SiNWs (right). Gating in circle indicates Cy3 ⁺ CD19 ⁺ double positive T cell population, with the adjacent number showing its percentage b) Quantification of the percentage of Cy3 ⁺ CD19 ⁺ double positive T cell population from flat Si and SiNW substrates. ***P = 0.0003 (Mann-Whitney’s U-tests). Each symbol (b) represents an individual substrate. FIGURE 11 A: SEM images of SiNW arrays, of other SiNW arrays prepared according to the present invention.

FIGURE 11B: SEM images of polymeric NWs. SEM images of SU8, PS, PDMS, and OROMOCOMP NW arrays prepared according to the present invention.

FIGURE 11C: Cell interface with polymeric NWs. Adherent and non-adherent cells on polymeric NWs. (a-b) Zoom-out (a) and Zoom-in (b) SEM images of GPE86 fibroblast cells on SU8 NWs. (c-d) Zoom-out (c) and Zoom-in (d) SEM images of Jurkat cells on the surface of ORMOCOMP NWs.

FIGURE 12: Viability of Jurkat cells on flat Si and SiNWs at different incubation times a) Live/dead staining of Jurkat cells after 2, 6, 12, and 24 h incubation on flat Si (i, ii, iii, iv, respectively) and SiNWs (v, vi, vii, viii, respectively). FDA (green) indicates live cells and PI (red) indicates dead cells. Scale bars, 100 pm. b) Quantification of the viability of Jurkat cells on flat Si (blue dots) and SiNWs (red dots) analysed from (a). Each symbol represents an individual substrate. Data are representative of two independent experiments with at least two substrates per group.

FIGURE 13A: NW-mediated siRNA transfection into primary immune B cells. Cell count of Ramos (human B cell line) (a,b) and primary human B cells (c) transfected with siOTP Control (blue dots) or siTOX siRNAs (red dots) coated on flat Si and SiNWs at 4 h, 24 h, and 48 h post transfection d) Quantification of siTOX transfection efficiency normalised to control siOTP after 4 h. Each symbol represents an individual substrate. *P = 0.0417, ****P < 0.0001 (Two-way ANOVA). Data are representative of two independent experiments with at least three substrates per group.

FIGURE 13B: Comparison of loading efficiencies. Loading efficiencies of plasmid onto Flat Si substrate compared to silicon nanowires.

FIGURE 14: Cell-SiNW interface a-c) Confocal imaging of fluorescence stained GPE86 (a), LI .2 (b), and Jurkat (c) cells after centrifugation and 6 h incubation on SiNWs. Cells were fixed and stained with phalloidin to reveal the F-actin (red), and Hoechst to reveal the nuclei (blue). White circles in the merged images indicate the positions of SiNWs inside the cells. Inset (merge column, a) is an enlargement (x3) of the area outlined in the main image. Scale bars, 10 pm (a) and 5 pm (b,c). d) SEM imaging showing zoom-out and zoom-in tilted (45°) views of GPE86 (i, ii), LI .2 (iii, iv) and Jurkat (v, vi) cells on SiNWs after 6 h incubation. Scale bars, 50 pm (i, iii, v), 5 pm (ii, iv, vi) and 1 pm for the insets.

FIGURE 15: Cell focal adhesion formation and migration on SiNWs. a-c) Confocal imaging of fluorescence stained GPE86 (a), LI .2 (b) and Jurkat (c) cells after 6 h incubation on SiNWs. Cells were stained with Hoechst (blue) to reveal the nuclei, and vinculin (green), phalloidin (red), and ?-integrin (magenta) to reveal the cytoskeleton and focal adhesion points on the substrates. Scale bars, 10 pm. d-f) Polar plots showing the migration trajectory of GPE86 (d), LI .2 (e), Jurkat (f) cells on SiNWs over 60 h. Sequential confocal images were taken every 10 min. Each curve in color represents the trajectory, including heading and length, of a single cell migrating from their origin g) Graph plotting of the migration lengths (mean shown in red) of all three cell types. Each symbol represents the migration length of one single cell (N > 200).

FIGURE 16: Membrane perturbation and nuclear deformation induced by SiNWs. 3D (a) and slice view (b) of confocal imaging of GPE86 cells on FITC (green)- labelled SiNWs after 6 h incubation. Cells were fixed and stained with phalloidin (red) and Hoechst (blue). SiNWs are depicted in white in (b) to represent their interaction with cell membrane and the nucleus. Scale bar, 10 pm.

FIGURE 17: pGFP transfection into HEK293 cells via SiNWs with different geometry, (a) Flow cytometry analysis of GFP expression within HEK293 cells 48 h after detachment from pGFP-coated flat Si and SiNWs (#88, 89, 90, 91, 92, and 94). (b) Histogram of CTV expression within HEK293 cells Confocal images 48 h after detachment from the 6 types of SiNWs as shown in (a). Freshly CTV-stained and non- stained cells served as positive and negative controls, respectively (c) Quantification of the percentage of GFP ⁺ cells in (a) (d) Quantification of CTV GMFI of cells in (b). (e) Geometric parameters of the 6 types of SiNWs used for (a-d). (f-h) Linear regression showing the relationship between pGFP transfection efficiency in HEK293 cells with parameters of SiNW arrays, including density (f), tip diameter (g), and height (h).

FIGURE 18: Scanning laser microscopy image of PDMS NWs. (a) Top-view profile of PS NWs with 200 x magnification (b) Determination of the geometry of individual NWs based on the profile, showing that the NWs have a height of 3.5 pm and a pitch of 3 pm. (c) PS NWs mapped in 3D after the scanning laser procedure (d) Tilted view of the PS NWs with 800 x magnification.

FIGURE 19: Viability of Jurkut cells on polymeric NWs. (a-d) Confocal images of Jurkat cells on 4 types of polymeric NWs with PDL coating after 6 h culture. Cells were stained with PI (left), and Hoechst 33342 (middle), FDA (right), which stand for dead cells, total cell nucleus, and viable cells, respectively (a) The confocal images demonstrated high density of live cells without any dead cells on the PS NWs. (b) SU8 NWs and (c) ORMOCOMP NWs showed similar results to the PS NW samples. Cells also attached to the (d) PDMS NWs. (e) Quantification of Jurkat cell viability on 4 types of polymeric flat and NWs with PDL coating after 6 h culture. Cells on both flat and VA- NWs, made from ORMOCOMP, SU8, and PS, showed high viability close to 100 % after 6 h culture. Cell viability on PDMS substrates including flat and NW samples, with 50 % and 76 %, respectively.

FIGURE 20: Delivery of ssDNA-FAM into L1.2 cells by polymeric NWs. (a)

FACS analysis of ssDNA-FAM insertion into LI .2 cells by 3 types (ORMOCOMP, PS, and SU8) of polymeric flat and NW substrates. The control (Ctrl) sample showed LI .2 cells without ssDNA transfection, setting the baseline for FACS analysis (b) Quantification of ssDNA insertion into LI .2 cells via 3 types of polymeric substrates as shown in (a) after 6 h culture.

FIGURE 21: Cy5-GFP-mRNA delivery into L1.2 cells via SiNWs. Confocal images showing signals of Cy5 (magenta) and GFP (green) expression inside LI .2 cells on flat Si (a) and SiNWs (b) with magnification of 60 ^c (a, b i) and 180 ^c (b ii). Cells were stained with Hoechst 33342 (blue).

FIGURE 22: Cy5-GFP-mRNA delivery into L1.2 cells via polymeric SU8

NWs. Confocal images showing signals of Cy5 (magenta) and GFP (green) expression inside LI .2 cells on flat SU8 (a) and SU8 NWs (b) with magnification of 60 ^c (a, b i) and 180 x (b ii). Cells were stained with Hoechst 33342 (blue).

FIGURE 23: Cy5-GFP-mRNA delivery efficiency by polymeric substrates and Si substrates to Jurkat cells, (a) FACS analysis of Jurkat cells cultured on mRNA- coated PDMS, ORMOCOMP, type I PS (PS1), type II PS (PS2), SU8, and Si flat and NW substrates. The control (Ctrl) sample showed LI .2 cells without ssDNA transfection, setting the baseline for FACS analysis (b) Quantification of the percentage of Cy5 ⁺ population within Jurkat cells, as gated in (a).

DETATT/ED DESCRIPTION

The present disclosure relates to a NW based molecular delivery system and methods of using the system to deliver exogenous molecules into cells. One advantage of the disclosed delivery system and methods is that exogenous molecules (e.g., RNAs, peptides, and proteins) can be delivered into cells, including difficult to transfect primary cells, with unexpectedly high efficiency and relatively low transfection-related toxicity. The disclosed delivery system can therefore be used, for example, to generate clinical grade CAR ⁺ T cells. Advantageously, the disclosed delivery system allows for high- throughput delivery of molecules into cells for therapeutic interventions. In one embodiment, the delivery system is a non-viral delivery system. In some embodiments, the non-viral approach disclosed has significant advantages to viral mediated transduction. In some embodiments, the transfected cells do not include added viral sequences. In some embodiments, the disclosed methods are generally cheaper, quicker, highly scalable, and/or avoid T cell activation, and/or avoid genotoxicity due to virus- mediated transduction and/or immunogenicity due to use of virus. The present disclosure may also offer additional advantages over other chemical based methods of gene delivery which whilst typically cheaper usually achieve lower transfection efficiency, particularly of primary cells as well as suspension cell lines, and result in cell toxicity, especially in the case of primary cells.

Molecular delivery system

Referring to Figure 1 A, an example of a molecule delivery system and method of delivering molecules into cells is shown (100). A cell (101) is brought into contact with a NW array comprising a substrate having a surface (102) and a plurality of NWs (103) adhered (i.e. attached) to the surface of the substrate. The terms adhered and attached are used interchangeably. The geometry of the NWs in the NW array (e.g. pitch/density, height, diameter etc.) can vary and is described herein.

Referring to Figure 2, the NWs (200) can be functionalised to have molecules (X and/or Y) attached to the NW surface which can be delivered into cells. Suitable molecules that can be delivered into cells are defined herein. The molecules can be attached to the NW surface via a linker molecule (A and/or B). Suitable linker molecules are defined herein. In Figure 1 A, the functionalised NWs (103) can penetrate into cells (101) which have been added onto the NWs. Following incubation, at least some of the molecules X and/or Y may be delivered or co-delivered into at least some of the cells. In some embodiments, the linker molecule can also be delivered into the cell. The introduction of the molecules X and/or Y into the cell may alter the cellular function of the cell which is indicated by the solid black line (104) around the cell, for example, result in a transformed immune cell (e.g. a CAR ⁺ T cell), an edited genome (e.g. by delivering a programmable nuclease, such as a ribonucleoprotein comprising a programmable nuclease, for example Cas9 RNP ) or gene silencing (e.g. by RNAi).

Nanowire arrays

The molecular delivery system comprises a NW array. The NW array comprises a substrate (e.g. a base), and a plurality of NWs attached to a surface of the substrate and are extending from the substrate.

In one embodiment, the NWs are protruding (e.g. extending) from a solid base or platform. In other words, the plurality of NWs extend from the solid base.

In some embodiments, the NWs are attached to the substrate surface along a substantially vertical direction to the surface of the substrate. The NWs may be described as a plurality of vertical NWs. In this embodiment, a person skilled in the art would understand the NWs as being“vertically aligned” or“vertically aligned NWs”. In some embodiments, the NWs (103) may form at an angle with respect to the substrate (102), for example an angle of about 60° to about 90°, about 70° to about 90°, about 80° to about 90°, about 85°, or about 90°. In some embodiments, the NWs average angle with respect to the substrate is about 90°. It will be appreciated that the alignment of the NWs is not limited by the material forming the NWs.

The NWs are attached to the substrate (102). The substrate may also be called a base. The substrate (102) is in direct communication with the ends of the plurality of the NWs.

The substrate (102) may be a solid base or platform. Any reference to“solid” in relation to the substrate/base of the NW array refers to the base of the NW array being substantially solid, for example to essentially exclude a base with a degree of porosity which allows fluid to pass through the substrate.

The substrate (102) may be formed of the same or different materials as the NWs. For example, the substrate may comprise silicon, silicon oxide, glass and/or organic polymers (e.g. polystyrene (PS), polydimethylsiloxane (PDMS), SU8 and OROMOCOMP). In other embodiments, the substrate can be selected from one or more of the suitable materials listed for the NWs defined below. In one embodiment, the substrate comprises or consists of silicon. In another embodiment, the substrate comprises or consists of glass. In yet another embodiment, the substrate comprises or consists of a polymer (e.g. polystyrene).

A top-down process that involves removing predefined structures from a supporting substrate may be used to obtain the plurality of NWs (103) attached (i.e. adhered) to the surface of a substrate. As a non-limiting example, the NW array may be formed by a combination of direct electron beam lithography (EBL) and deep reactive ion etching (DRIE) which can yield NW arrays with controlled geometry at predefined locations on the surface of the base. In some embodiments, the sites where NWs are to be formed may be patterned into a soft mask and subsequently etched to develop the patterned sites into three-dimensional NWs. Methods for patterning the soft mask include, but are not limited to, photolithography, electron beam lithography, and nanosphere lithography casting. For example, a suitable resist may be spin coated onto a substrate and subsequently loaded into an electron beam lithography (EBL) system to form a patterned soft-mask to define the NW cross-section morphology and diameter. Deep reactive ion etching (DRIE or RIE) may then be used to obtain a plurality of silicon NWs which uses alternate cycles of passivation (e.g. O2 passivation) and etching steps (e.g. SFe etching) of the patterned soft mask substrate in order to obtain a plurality of NWs on the surface of the substrate. Here, the non-masked area of the substrate is etched away, and the masked area of the substrate is preserved thus forming the plurality of NWs. In some embodiments, this integrated approach provides efficient control over the NW etching site locations where the spacing, height and density of the NWs can be independently controlled. In this example, using EBL and DRIE, the sites where the NWs are defined by a patterned mask which is subsequently etched to develop the patterned sites into three-dimensional NWs. An example of suitable EBL and DRIE conditions to prepare the NW array are outlined in the examples, for example in relation to silicon NWs.

In one embodiment, the substrate and the NWs may be integral with one and other. For example, the plurality of NWs and the substrate (e.g. base) may be provided as a single phase (e.g. the substrate is formed in-situ as a result of the formation of the NWs via reaction ion etching(RIE). In one embodiment, it will be appreciated that where RIE is used to prepare the NW array (e.g. a silicon NW array), the substrate and NWs (e.g. silicon NWs) are integral with each other. In some embodiments, by varying the masking, passivation and etching steps of the RIE process used to prepare the NW array, the diameter, density, length, and the tip sharpness of the silicon NWs can be varied..

Alternatively, the NWs may be obtained by growing the NWs from a precursor material. As a non-limiting example, to prepare silicon NWs, chemical vapour deposition (CVD) may be used to grow silicon NWs by placing or patterning noble catalyst or seed particles (typically with a diameter of 1 nm to a few hundred nm) atop the substrate and adding a silane gas precursor to the catalyst or seed particles. When the particles become saturated with the silane precursor at the specific eutectic point, silicon NWs can begin to grow in a shape that minimizes the system's energy. By varying the substrate crystallinity, catalyst/seed particles (e.g., type, size, density, and deposition method on the substrate), and growth conditions, the diameter, density and length of the silicon NWs can be varied. The NW array may also be created via integrating and combining bottom- up and top-down approaches - such as colloidal lithography, metal-assisted chemical etching, and chemical vapour deposition. The NWs may also be prepared separately and subsequently attached to the surface of the base.

Another top-down process that may be used to prepare the NW arrays may comprise nanoimprint lithography, for example to prepare polymeric NWs. As a non limiting example, where nanoimprint lithography is used to prepare polymeric NWs, a negative master mould may be used as a template (for example based on one or more silicon NW arrays as described herein) to define the NWs morphology and dimension. The negative master template may be stamped onto a polymer layer that has been applied to the surface of a substrate (e.g. glass), and is subsequently cured (e.g. via heat and/or pressure or UV) within the template to form the NW array. Examples of suitable nanoimprint lithography conditions to prepare the NW array are outlined in the examples, for example in relation to polymeric NWs.

Alternatively, the substrate and NWs may be provided as separate, non-integral components, for example, where the base is of different material to that of the NWs. In this example, the plurality of NWs may be prepared separately (for example by solution phase methods) and subsequently attached to the surface of the substrate.

The NW array may also be a microwell plate as described herein, wherein the plurality of NWs extend from the substrate of one of one or more of the wells of the microwell plate. For example, the array may be prepared by EBL and RIE to define a microwell plate comprising a plurality of NWs extending from the base of one or more wells of the microwell plate. In this example, the NW array is integral with the microwell plate. In this example, the microwell plate may comprise a plurality of NW arrays, for example each well of the microwell plate houses a NW array. In some embodiments, the microwell may form part of a kit as described herein.

In one embodiment, the surface of the NWs may have a native oxide layer. For example, referring to Figure 2, a NW may have a native oxide layer (201). By way of example, where the NWs are silicon NWs, the native oxide layer may comprising a Si- O layer. Alternatively, the oxygen atoms in the native oxide layer can be replaced with hydrogen creating hydride terminated NWs. Other possible terminations include alkyl (e.g. methyl, ethyl, propyl) and amine or epoxy, and also include other metal species, such as gold nanoparticles. It will be appreciated that, when present, the nature of the native oxide layer (201) will depend on the composition of NW. For example, if the NWs are silicon, then the native oxide layer (201), if present, will be a silicon oxide (Si-O) layer.

Referring to Figure 1 A, the NWs (103) may form an array on the substrate (102). In some embodiments, the NW array comprises a plurality of NWs that are regularly spaced apart from one and other, as seen in Figure IB by way of example for SiNWs. Alternatively, the NW array comprises a plurality of NWs that are irregularly spaced on the substrate e.g. stochastic positioning. It will be appreciated that the NW array as described herein, is not limited to any specific spatial arrangement of the NWs. The NW density, length and/or diameter (e.g. geometry) may be varied depending on the type of cell and/or molecule being delivered (for example, see Figure 11 in relation to SiNWs). In some embodiments, the density of the NWs can be tailored to accommodate various cell sizes. Alternatively or additionally, in some embodiments, the height of the NWs can be tailored to increase the transfection efficiency of molecules into cells.

The nanowires (103) may be solid. Any reference to“solid” in relation to the NWs refers to the NWs being substantially solid, for example to essentially NWs with a degree of porosity or openings which allows fluid to pass through the interior of the NWs (in contrast to a hollow morphology such as a nanostraws which are often configured to be in fluid communication with an external reservoir housing a fluidic medium which can be pumped through the nanostraws). In some embodiments, owing to the solid nature of the NWs, the delivery system described herein is not configured to be integrated with an external pump, such as microfluidic pump, which is often used to deliver a fluidic medium through hollow nanostraws.

The NW array has a density. In some embodiments, the average density of the NWs is at least about 0.01, 0.02, 0.03, 0.05, 0.07, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 7 or 10 NWs per pm ² . In some embodiments, the average density of the NWs is less than about 10, 7, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5 ,0.4, 0.3, 0.2, 0.1, 0.07, 0.05, 0.03, 0.02, or 0.01 NWs per pm ² . Combinations of these average density values are also possible, e.g., the average density of the NWs is about 0.1 to about 1.0 NWs per pm ² , about 0.2 to about 0.4 NWs per pm ² , or about 0.5 to about 1 NWs per pm ² . In some embodiments, the average density of the NWs is about 0.6 to about 0.9 NWs per pm ² , for example about 0.75 NWs per pm ² . In some preferred embodiments, the NWs have a low density, for example of about 0.05 to about 1 NWs per pm ² , preferably about 0.1 to about 0.5 NWs per pm ² , and more preferably about 0.2 to about 0.4 NWs per pm ² . In one preferred embodiment, the average density is about 0.3 NWs per pm ² . In other embodiments, the NWs have a density of about 0.5 to about 1 NWs per pm ² , preferably about 0.75 NWs per pm ² .

The density of the NWs may be selected depending on the type of cell being transfected. For example, the NWs may have a density where, upon addition to the NW array, cells do not settle between adjacent NWs. Reducing such settling can result in an increased number of cells being transfected in a given population. Alternatively or additionally, the NWs may have a density to provide multiple NW contact points with a given cell (e.g. on average at least about 10, 50, 100, 200, 500, or 1 x 10 ³ NWs penetrating a single cell). By increasing the number of contact points (e.g. penetrations) per cell, a higher payload of molecules may be delivered into the cell. For example, suspension cells such as primary lymphocytes (e.g. B or T cells) are much smaller in size compared with larger adherent cells (e.g. fibroblast cells) and in some embodiments require the use of a higher density NW array. For example, if the cells comprise lymphocytes (e.g. B or T cells) which may have an average cell size of between about 5 mih to 12 mih, in some embodiments, the NWs may have a density of between about 0.2 to 1.0 NWs per mih ² , for example about 0.2 to 0.4 NWs per pm ² , e.g. about 0.3 NWs per pm ² . Alternatively, for larger adherent cells (e.g. fibroblasts) which may have an average cell size of between about 10 pm to about 15 pm, in some embodiments, the density of the NWs may be between about 0.15 to 0.2 NWs per pm ² . It will be appreciated that the other NW array densities are also possible depending on the size of the cells being transfected.

In some embodiments, the NWs may be of any suitable length which is measured as the distance from the substrate to the tip of the NW. The terms length and height can also be used interchangeably. The NWs may have substantially the same lengths or different lengths. In some embodiments, the average length of the NWs is at least about 0.1 pm, 0.2 pm, 0.5 pm, 1 pm, 1.5 pm, 2 pm, 2.5 pm, 3 pm, 3.5 pm, 4 pm, 4.5 pm, 5 pm, 5.5 pm, 6 pm, 6.5 pm, 7 pm, 7.5 pm, 8 pm, 8.5 pm, 9 pm, 9.5 pm, or 10 pm. In some embodiments, the average length of the NWs is less than about 10 pm, 9.5 pm, 9 pm, 8.5 pm, 8 pm, 7.5 pm, 7 pm, 6.5 pm, 6 pm, 5.5 pm, 5 pm, 4.5 pm, 4 pm, 3.5 pm, 3 pm, 2.5 pm, 2 pm, 1.5 pm, 1 pm, 0.5 pm, or 0.2 pm. Combinations of these average length values are also possible, e.g., the average length of the NWs is from about 2 pm and about 8 pm. In some preferred embodiments, the average length of the NWs is at least about 3 pm, for example about 3 pm to about 4 pm, for example from about 3 pm to about 3.5pm, for example about 3 pm, e.g. about 3.2 pm. Further advantages may be provided by using shorter NWs to deliver molecules into cells according to at least some embodiments or examples described herein, such as higher transfection efficiency (see for example Figure 17).

The cross-section of the NWs may have any arbitrary shape, including, but not limited to, circular, square, rectangular, elliptical and tubular. Regular and irregular shapes are included. The NWs may also have any suitable diameter, or narrowest cross- section dimension if the NWs are not cylindrical in shape (e.g. spike morphology). For example, the NWs may be conically-shaped (see Figure IB). The term“conically- shaped” or“conical” refers to a three-dimensional geometric shape that tapers from a first diameter to an apex or vertex having a narrower diameter (e.g. a cone). If the NWs are conically-shaped, the diameter is taken to be the narrowest cross-section dimension (e.g. at the tip/vertex of the conically shaped nanowires). It will be appreciated that conically shaped NWs are distinctly different to needle-shaped, rod, or pillar NWs, all of which have substantially the same diameter along the length of the NWs (e.g. not conical). In some embodiments, the inventors have identified that conically shaped NWs are mechanically stable due to the larger diameter at the base compared to the tip, which may provide further advantages such as increased resilience (e.g. harder to break and/or bend) compared with needle, rod or pillar shaped NWs. The inventors have also identified that owing to the tapered geometry of conically shaped NWs, cells often interface with only the narrower tip area of the NWs, which can minimise the degree of penetration (e.g. reduces the risk of piercing through the entire cell and nucleus), which in some embodiments can lead to improved cell viability. It will be appreciated that the NWs depicted in the schematic diagrams represented in Figures 1 to 3 are not intended to limit the NWs to any morphology, but rather to demonstrate an embodiment of the delivery system described herein.

The NWs may be have substantially the same diameters, or may have different diameters. In some embodiments, the average diameter of the NWs is at least about 10 nm, 20 nm, 30 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 120 nm, 140 nm, 160 nm, 180 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, or 500 nm. In some embodiments, the average diameter of the NWs is less than about 500 nm, 450 nm, 400 nm, 350 nm, 300 nm, 250 nm, 200 nm, 180 nm, 160 nm, 140 nm, 120 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 30 nm, or 20 nm. Combinations of these average diameter values are also possible, e.g. the average diameter of the NWs may be from about 50 nm to about 200 nm, from about 60 nm to about 150 nm, or from about 80 nm to about 120 nm. In one embodiment, the average diameter of the NWs less than about 150 nm, preferably about 100 nm. In another embodiment, the average diameter of the NWs is about 300 nm to about 500 nm, preferably about 400 nm.

The NW density, length, and diameter may be varied depending on the type of cell and/or molecule being delivered. In other words, the geometry of the NWs are programmable depending on the cell type and/or molecule. For example, the density and/or height of the NWs can be varied depending on the type of cell, or to increase transfection efficiency, according to at least some embodiments or examples as described herein.

It will be appreciated that the NWs may have a combination of any one of the lengths, diameters, and densities described herein. For example, i) the average density of the NWs is about 0.1 to about 1.0 NWs per pm ² ; ii) the average length of the NWs is at least about 3 pm; and/or iii) the average diameter of the NWs is less than about 500 nm. In some embodiments, i) the average density of the NWs is about 0.2 to about 0.4 NWs per pm ² ; ii) the average length of the NWs is at least about 3 pm; and/or iii) the average diameter of the NWs is less than about 150 nm. In one embodiment, i) the average density of the NWs is about 0.2 to about 0.4 NWs per pm ² ; ii) the average length of the NWs is at about 3 pm to 4 pm; and/or iii) the average diameter of the NWs is less than about 150 nm. In one embodiment, the average density of the NWs is about 0.3 NWs per pm ² , the average length of the NWs is about 3.2 pm, and the average diameter of the NWs is about 100 nm. Other combinations are also possible.

Nanowire material

The NWs may be made of a suitable material. For example, the NWs may be formed from materials with low cytotoxicity. Suitable materials include, but are not limited to, silicon, silicon oxide, silicon nitride, silicon carbide, iron oxide, aluminium oxide, iridium oxide, tungsten, stainless steel, silver, platinum and gold. Other suitable materials include aluminium, copper, molybdenum, tantalum, titanium, nickel, tungsten, chromium or palladium. In some embodiments, the NW comprises or consists essentially of a semiconductor. Typically, a semiconductor is an element having semiconductive or semi-metallic properties (i.e. between metallic and non-metallic properties. An example of a semiconductor is silicon. Other non-limiting examples include elemental semiconductors, such as gallium, germanium, diamond (carbon), tin selenium, tellurium, boron or phosphorous. More than one element may be present in the NWs as the semiconductor, for example gallium nitride.

In one embodiment, the plurality of NWs are silicon NWs, polymeric NWs, or a combination thereof. In one embodiment, the NWs do not comprise or consist of alumina (AI2O3) or diamond. In a preferred embodiment, the plurality of NWs are silicon NWs. The silicon NWs can be prepared according to processes defined herein. The average density, length and diameter of the silicon NWs can vary and can be selected from any one of the density, length and diameter values described herein.

In one embodiment, the plurality of NWs are silicon NWs. The silicon NWs can be prepared according to processes defined herein. The average density, length and diameter of the silicon NWs can vary and can be selected from any one of the density, length and diameter values described herein.

In some embodiments, the average density of the silicon NWs may be at least about 0.01, 0.02, 0.03, 0.05, 0.07, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 7 or 10 NWs per pm ² . In some embodiments, the average density of the silicon NWs is less than about 10, 7, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5 ,0.4, 0.3, 0.2, 0.1, 0.07, 0.05, 0.03, 0.02, or 0.01 NWs per pm ² . Combinations of these average density values are also possible, e.g., the average density of the silicon NWs is about 0.1 to about 1.0 NWs per pm ² , about 0.2 to about 0.4 NWs per pm ² , or about 0.5 to about 1 NWs per pm ² . In some preferred embodiments, the silicon NWs have a low density, for example of about 0.05 to about 1 NWs per pm ² , preferably about 0.1 to about 0.5 NWs per pm ² , and more preferably about 0.2 to about 0.4 NWs per pm ² . In one preferred embodiment, the average density of the silicon NWs is about 0.3 NWs per pm ² .

In some embodiments, the silicon NWs may be of any suitable length (e.g. height) which is measured as the distance from the substrate to the tip of the NW. The NWs may have substantially the same lengths or different lengths. In some embodiments, the average length of the silicon NWs is at least about 0.1 pm, 0.2 pm, 0.5 pm, 1 pm, 1.5 pm, 2 pm, 2.5 pm, 3 pm, 3.5 pm, 4 pm, 4.5 pm, 5 pm, 5.5 pm, 6 pm, 6.5 pm, 7 pm, 7.5 pm, 8 pm, 8.5 pm, 9 pm, 9.5 pm, or 10 pm. In some embodiments, the average length of the silicon NWs is less than about 10 pm, 9.5 pm, 9 pm, 8.5 pm, 8 pm, 7.5 pm, 7 pm, 6.5 pm, 6 pm, 5.5 pm, 5 pm, 4.5 pm, 4 pm, 3.5 pm, 3 pm, 2.5 pm, 2 pm, 1.5 pm, 1 pm, 0.5 pm, or 0.2 pm. Combinations of these average length values are also possible, e.g., the average length of the silicon NWs is from about 2 pm and about 8 pm. In some preferred embodiments, the average length of the silicon NWs is at least about 3 pm, for example about 3 pm to about 4 pm, for example about 3 pm to about 3.5 pm, for example about 3 pm, e.g. about 3.2 pm. Further advantages may be provided by using shorter Si NWs to deliver molecules into cells according to at least some embodiments or examples described herein, such as higher transfection efficiency.

The cross-section of the silicon NWs may have any arbitrary shape, including, but not limited to, circular, square, rectangular, elliptical and tubular. Regular and irregular shapes are included. The silicon NWs may also have any suitable diameter, or narrowest cross-section dimension if the silicon NWs are not cylindrical in shape (e.g. spike morphology). In some embodiments, the Si NWs may be conically-shaped (see Figure IB). Further advantages may be provided by conically-shaped Si NWs according to at least some embodiments or examples described herein, such as improved cell viability following penetration by one or more conically-shaped Si NWs.

The silicon NWs may be have substantially the same diameters, or may have different diameters. In some embodiments, the average diameter of the silicon NWs is at least about 10 nm, 20 nm, 30 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 120 nm, 140 nm, 160 nm, 180 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, or 500 nm. In some embodiments, the average diameter of the silicon NWs is less than about 500 nm, 450 nm, 400 nm, 350 nm, 300 nm, 250 nm, 200 nm, 180 nm, 160 nm, 140 nm, 120 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 30 nm, or 20 nm. Combinations of these average diameter values are also possible, e.g. the average diameter of the silicon NWs may be from about 50 nm to about 200 nm, from about 60 nm to about 150 nm, or from about 80 nm to about 120 nm. In one embodiment, the average diameter of the silicon NWs less than about 150 nm, preferably about 100 nm or less. It will be appreciated that the silicon NWs may have a combination of any one of the lengths, diameters, and densities described herein. For example, i) the average density of the silicon NWs is about 0.1 to about 1.0 NWs per pm ² ; ii) the average length of the silicon NWs is at least about 3 pm; and/or iii) the average diameter of the silicon NWs is less than about 500 nm. In some embodiments, i) the average density of the silicon NWs is about 0.2 to about 0.4 NWs per pm ² ; ii) the average length of the silicon NWs is at least about 3 pm; and/or iii) the average diameter of the silicon NWs is less than about 150 nm. In one embodiment, i) the average density of the silicon NWs is about 0.2 to about 0.4 NWs per pm ² ; ii) the average length of the silicon NWs is at about 3 pm to 4 pm; and/or iii) the average diameter of the silicon NWs is less than about 150 nm. In one embodiment, the average density of the silicon NWs is about 0.3 NWs per pm ² , the average length of the silicon NWs is about 3.2 pm, and the average diameter of the silicon NWs is about 100 nm. Other combinations are also possible.

In another embodiment, the plurality of NWs are polymeric NWs. The average density, length and diameter of the polymeric NWs can vary and can be selected from any one of the density, length and diameter values described herein, including any combinations thereof.

The polymeric NWs can be prepared according to processes defined herein. The polymeric NWs may be formed from one or more polymers. For example, the polymeric NWs may be formed from two or more different types of polymers, such as 3 or more different type of polymers, such as 4 or more different types of polymers and including 5 or more different types of polymers. Any suitable polymer may be used to prepare the polymeric NWs. For example, suitable polymeric NWs may include, but are not limited to, polyester NWs, polydimethylsiloxane (PDMS), polycarbonate NWs, polypropylene NWs, polystyrene NWs, polyvinyl NWs (such as polyvinyl chloride (PVC) NWs), polyurethane NWs, polyether NWs, polyamide NWs, polyimide NWs, or copolymer NWs such as PETG NWs (glycol-modified polyethylene terephthalate), methacrylate NWs, polythiophene NWs, polyacetylene NWs, poly aniline NWs, and polypyrrole NWs. Polyester NWs of interest may be aliphatic polyester NWs such as polyglycolide (PGA) NWs, polylactide (PLA) NWs, polyethylene adipate (PEA) NWs, polyhydroxyalkanoate (PHA) NWs, polycaprolactone (PCL) NWs, polyhydroxybutyrate (PHB) NWs, poly (3 - hydroxybutyrate-co-3 -hydroxy valerate) (PHBV) NWs or aromatic polyesters NWs such as polyethylene terephthalate (PET) NWs, polybutylene terephthalate (PBT) NWs, polytrimethylene terephthalate (PTT) NWs, polytrimethylene terephthalate (PTT) NWs and polyethylene naphthalate (PEN) NWs, among other polyester polymeric NWs, and combinations thereof. In one embodiment, the polymeric NWs are polypyrrole NWs. In another embodiment, the polymeric NWs are polystyrene (PS) NWs. In another embodiment, the polymeric NWs are polydimethylsiloxane (PDMS) NWs.

In another embodiment, the polymeric NWs are epoxy based photoresist polymeric NWs. For example, the polymeric NWs are SU-8 NWs. SU-8 derives its name from the presence of 8 epoxy groups, being the statistical average per moiety. An example of the structure of the SU-8 monomer is provided as follows:

In another embodiment, the polymeric NWs are organically modified ceramic polymeric (ORMOCER) NWs. For example, the ORMOCER NWs are made from the commercially available ORMOCOMP, manufactured by Microresist Technology GmbH, Berlin, Germany. In another embodiment, the polymeric NWs are Thermanox™ NWs. Thermanox™ is commercially available from ThermoFisher Scientific, and has good cell compatibility and low cytotoxicity and is used extensively in cell cultures.

In one embodiment, the polymeric NWs are polydimethylsiloxane (PDMS) NWs. The PDMS may be hard PDMS (h-PDMS) or soft PDMS (s-PDMS).

In some embodiments, depending on the type of polymer, the molecular weight of the polymeric NWs will vary ranging from 5 kDa to 500 kDa, such as from 10 kDa to 400 kDa, such as from 15 kDa to 300 kDa, such as from 20 kDa to 200 kDa, such as from 25 kDa to 150 kDa and including from 50 kDa to 100 kDa.

In some embodiments, the average density of the polymeric NWs may be at least about 0.01, 0.02, 0.03, 0.05, 0.07, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 7 or 10 NWs per pm ² . In some embodiments, the average density of the polymeric NWs is less than about 10, 7, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5 ,0.4, 0.3, 0.2, 0.1, 0.07, 0.05, 0.03, 0.02, or 0.01 NWs per pm ² . Combinations of these average density values are also possible, e.g., the average density of the polymeric NWs is about 0.1 to about 1.0 NWs per pm ² , about 0.2 to about 0.4 NWs per pm ² , or about 0.5 to about 1 NWs per pm ² . In some embodiments, the average density of the polymeric NWs is about 0.6 to about 0.9 NWs per pm ² , for example about 0.75 NWs per pm ² . In some preferred embodiments, the polymeric NWs have a low density, for example of about 0.05 to about 1 NWs per pm ² , preferably about 0.1 to about 0.5 NWs per pm ² , and more preferably about 0.2 to about 0.4 NWs per pm ² . In one preferred embodiment, the average density of the polymeric NWs is about 0.5 to about 1 NWs per pm ² , preferably about 0.75 NWs per pm ² .

In some embodiments, the polymeric NWs may be of any suitable length which is measured as the distance from the substrate to the tip of the NW. The NWs may have substantially the same lengths or different lengths. In some embodiments, the average length of the polymeric NWs is at least about 0.1 pm, 0.2 pm, 0.5 pm, 1 pm, 1.5 pm, 2 pm, 2.5 pm, 3 pm, 3.5 pm, 4 pm, 4.5 pm, 5 pm, 5.5 pm, 6 pm, 6.5 pm, 7 pm, 7.5 pm, 8 pm, 8.5 pm, 9 pm, 9.5 pm, or 10 pm. In some embodiments, the average length of the polymeric NWs is less than about 10 pm, 9.5 pm, 9 pm, 8.5 pm, 8 pm, 7.5 pm, 7 pm, 6.5 pm, 6 pm, 5.5 pm, 5 pm, 4.5 pm, 4 pm, 3.5 pm, 3 pm, 2.5 pm, 2 pm, 1.5 pm, 1 pm, 0.5 pm, or 0.2 pm. Combinations of these average length values are also possible, e.g., the average length of the polymeric NWs is from about 2 pm and about 8 pm. In some preferred embodiments, the average length of the polymeric NWs is at least about 3 pm, preferably about 3 pm to about 4 pm, for example about 3 pm, e.g. about 3.2 pm. Further advantages may be provided by using shorter polymeric NWs to deliver molecules into cells according to at least some embodiments or examples described herein, such as higher transfection efficiency.

The cross-section of the polymeric NWs may have any arbitrary shape, including, but not limited to, circular, square, rectangular, elliptical and tubular. Regular and irregular shapes are included. The polymeric NWs may also have any suitable diameter, or narrowest cross-section dimension if the polymeric NWs are not cylindrical in shape (e.g. conically-shaped/spike morphology). In some embodiments, the polymeric NWs may be conically-shaped. Further advantages may be provided by conically-shaped polymeric NWs according to at least some embodiments or examples described herein, such as improved cell viability following penetration by one or more conically-shaped polymeric NWs. The polymeric NWs may be have substantially the same diameters, or may have different diameters. In some embodiments, the average diameter of the polymeric NWs is at least about 10 nm, 20 nm, 30 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 120 nm, 140 nm, 160 nm, 180 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, or 500 nm. In some embodiments, the average diameter of the polymeric NWs is less than about 500 nm, 450 nm, 400 nm, 350 nm, 300 nm, 250 nm, 200 nm, 180 nm, 160 nm, 140 nm, 120 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 30 nm, or 20 nm. Combinations of these average diameter values are also possible, e.g. the average diameter of the polymeric NWs may be from about 50 nm to about 200 nm, from about 60 nm to about 150 nm, or from about 80 nm to about 120 nm. In one embodiment, the average diameter of the polymeric NWs less than about 350 nm, 250 nm, or 150 nm, preferably about 100 nm or less. In another embodiment, the average diameter of the polymeric NWs is about 300 nm to about 500 nm, preferably about 400 nm.

It will be appreciated that the silicon NWs may have a combination of any one of the lengths, diameters, and densities described herein.

The skilled person will appreciate that the molecular delivery efficiency to different cell types can be manipulated by varying the design parameters of NW arrays (range of diameters, heights, and densities, and more complex and hierarchical architectural designs). For example, silicon NWs with an average density of about 0.2 to about 0.4 NWs per pm ² , average length (e.g. height) of at least about 3 pm, and average diameter of less than about 150 nm show unexpectedly high delivery efficiency to human primary immune cells. In one embodiment, the average density of the silicon NWs is about 0.3 NWs per pm ² , the average length of the silicon NWs about 3.2 pm, and the average diameter of the silicon NWs is about 100 nm, as seen in Figure IB. According to at least some embodiments and examples described herein, silicon NWs having a length (e.g. height) of between about 3 to about 3.5 pm, exhibit higher transfection efficiency than comparably longer silicon NWs.

In one embodiment, the plurality of NWs may comprise both silicon NWs and polymeric NWs. Where the plurality of NWs comprise both silicon NWs and polymeric NWs, the average density, length and diameter for each of the silicon NWs and polymeric NWs can vary and can be selected from the density, length and diameter values described herein.

Linker and molecules

The molecule delivery system may include linkers and molecules. In some embodiments, the NWs may have one or molecules attached to its surface which can subsequently be delivered to a cell. For example, referring to Figure 2, each NW may have one or more molecules (e.g. X and/or Y) attached to its surface, for example at least 1 molecule, 2 molecules, 3 molecules, 5 molecules, 10 molecules, 15 molecules, or at least 25 molecules attached the NW surface. Where more than one molecule is attached to the NW, the molecules may be the same or different. The molecules X and/or Y may be attached to the surface of the NW via a linker A and/or B. With different surfaces on the NWs (e.g. Si-0 termination or hydride termination), the attachment between the linker and the NW can be covalent, electrostatic, photosensitive, or hydrolysable (e.g. with or without an enzyme), labelled as 203 or 204, in Figure 2. For example, the linker may be attached via an electrostatic interaction. In another example, the linker may be attached via a covalent bond. By way of example only, a poly-lysine molecule may be applied to the NW surface forming an electrostatic interaction attaching the linker to the NW.

In some embodiments, the linker may be attached to the NW surface via a functional moiety. The functional moiety may be selected from— OH,— CHO,— COOH,— SOrH,— CN,— NH ₂ ,— SH,—COSH, and— COOR. Where applicable, these moieties may be protonated or deprotonated to form a positively or negatively charged moiety which can attach to the NW surface.

In another example, the linker may be attached to the NW surface via the native oxide layer of the NW.

In another example, the linker may be a silane molecule which is attached to the NW surface resulting in a covalent Si-0 bond.

In some embodiments, the linker may be a silane, an epoxy silane, poly-lysine, a cleavable linker, a plasma polymer, a synthetic polymer, collagen, fibronectin or laminin. In one preferred embodiment, the linker is poly-lysine.

In some embodiments, the linker may have an average molecular weight of at least about 10 kDa, 20 kDa, 50 kDa, 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 400 kDa, or 500 kDa. In other embodiments, the poly-lysine linker may have an average molecular weight of less than about 500 kDa, 400 kDa, 300 kDa, 250 kDa, 200 kDa, 150 kDa, 100 kDa, 50 kDa, 20 kDa, or 10 kDa. Combinations of these average molecular weight values are also possible. In one embodiment, the average molecular weight of the may be from about 10 kDa to about 500 kDa.

In a preferred embodiment, the linker is poly-lysine. The poly-lysine may be poly- L-lysine or poly-D-lysine. The poly-D-lysine may be polymerised at the a position or e position. In a preferred embodiment, the poly-D-lysine is polymerised at the a position forming a-poly-D-lysine (PDL), which may allow for a greater number of poly-D-lysine molecules to be attached to the silicon NW surface. In one preferred embodiment, the linker is poly-D-lysine.

In other embodiments, the linker may be a silane. For example, the linker molecule may be an aminosilane such as (3- aminopropyl)-trimethoxy silane (APTMS), (3 -aminopropyl)-tri ethoxy silane, 3 -(2- aminoethylamino)propyl- dimethoxymethylsilane, (3 -aminopropyl)-di ethoxy- methylsilane, [3-(2- aminoethylamino)propyl]trimethoxysilane, bis[3- (trimethoxysilyl)propyl] amine, and (l l-aminoundecyl)-triethoxysilane. Other examples include glycidoxysilanes such as 3- glycidoxypropyldimethylethoxy silane and 3- glycidyloxypropyl)trimethoxy silane; mercaptosilanes such as (3-mercaptopropyl)- trimethoxysilane and (11- mercaptoundecyl)-trimethoxysilane, and other silanes such as trimethoxy(octyl) silane, trichloro(propyl)silane, trimethoxyphenylsilane, trimethoxy(2- phenylethyl) silane, allyltriethoxysilane, allyltrimethoxysilane, 3-[bis(2- hydroxyethyl) aminojpropyl- tri ethoxy dilane, 3-(trichlorosilyl)propyl methacrylate, and (3-bromopropyl) trimethoxysilane. In one embodiment, the linker is (3- aminopropyl)-trimethoxysilane (APTMS).

In other embodiments, the linker may be a biomolecular entity including, but not limited to, amino acids, proteins, sugars, DNA, antibodies, antigens, and enzymes; synthetic and organic polymers including, but not limited to, polyamide, polyester, polyimide, polyacrylic polymers, and plasma polymers. The linker may also be a cleavable i.e. a cleavable linker.

In some embodiments, each NW is attached to more than one linker molecule. It will be appreciated that depending on the type of linker molecule, the number of linker molecules attached to the surface of the NW will vary. For example, in some embodiments the linker is poly-lysine (e.g. poly-D-lysine) which is a synthetic polymer comprising repeating unit molecules of lysine. Without wishing to be bound by theory, depending on the number of repeating units, the number of poly-D-lysine linkers attached to the NW surface may vary due to steric hindrance and/or site of attachment (e.g. via interaction with amino or carboxyl groups).

In other embodiments, each linker may attach to more than one molecule. For example, where the linker is poly-lysine (e.g. poly-D-lysine), the positively charged amino terminated side chains may interact with a negatively charged molecule (e.g. a DNA plasmid). Depending on the degree of polymerisation of poly-D-lysine, each poly- D-lysine linker may be attached to more than one molecule.

The interaction/attachment between the linker (A or B) and the molecule to be delivered (X or Y) can be covalent, electrostatic, photosensitive, or hydrolysable, labelled as 202 or 205 in Figure 2. In one embodiment, the molecule may be attached to the linker via an electrostatic interaction, such that the molecule can be detached from the NWs and released into the cytoplasm of the cell following penetration.

In some embodiments, the molecule may be attached to the linker via a functional moiety. The functional moiety may be selected from— OH,— CHO,— COOH,— SO3H, — CN,— NH ₂ ,— SH,— COSH, and— COOR. Where applicable, these moieties may be protonated or deprotonated to form a positively or negatively charged moiety which can attach to the molecule.

In one embodiment, the linker molecule is poly-lysine which carries positive charges due to terminal amino groups and the molecule is a nucleic acid which carries negative charges which form an electrostatic interaction allowing for the release of the nucleic acid into the cytoplasm of the cell following penetration. For example, the linker may be poly-lysine (e.g. poly-D-lysine) which attaches to the molecules via electrostatic interactions thus allowing for molecular delivery with high efficiency.

The molecules may be delivered to the outer surface of the cell or may be delivered into the cell, for example via endocytosis following penetration of one or more NWs into the cell. As used herein,“endocytosis” refers to the cellular process in which substances, such as molecules, are brought into the cell. The molecule to be internalized is surrounded by an area of cell membrane, which then buds off inside the cell to form a vesicle containing the ingested material. Endocytosis includes pinocytosis and phagocytosis. The present inventors have surprisingly identified that, in some embodiments, the NW array can efficiently deliver molecules to cells via endocytosis whilst maintaining good cell viability. In some embodiments, the molecule is delivered to the cells via endocytosis. In one embodiment, the molecule is delivered to the cell while the cell is in contact with at least one of the NWs.

The molecule may be, but is not limited to, a small molecule, a protein, a nucleic acid, nanoparticles, dyes, a virus, a viral particle or polymeric molecules, or combinations thereof.

The small molecule may be any molecule with a molecular weight below 1000 Da. Non-limiting examples of molecules that may be considered to be small molecules include synthetic compounds, drug molecules, oligosaccharides, oligonucleotides, and peptides.

The protein may be a natural protein, fusion/chimeric protein or a fragment thereof, an antibody, an enzyme, or a transmember protein. The protein may be a chimeric protein, for example a chimeric antigen receptor (CAR). The enzyme may be a programmable nuclease such as Cas9. The protein fragments may a peptide, including immunogenic peptide for use in a vaccine, or an antigen binding fragment of an antibody (e.g., dAb, Fv, scFv, dimeric scFv, diabody, triabody, tetrabody, Fab, Fab’, F(ab’)2, or Fc fusions thereof). The protein may be a tagged protein, for example a fluorescence- tagged protein, such as fluorescence tagged IgGs (e.g. IgG-AF647 and IgG-AF488). The antibody may be any suitable monoclonal antibody. The dye can be any suitable dye, for example betalains e.g. betacyanins and betaxanthins. The dye may be an intracellular dye. The polymer or polymeric molecules may be a polysaccharide (e.g. a carbohydrate). The nanoparticles may be metallic nanoparticles (e.g. gold nanoparticles) or semiconductor nanoparticles (e.g. quantum dots).

The nucleic acid may be DNA, including linear and plasmid DNAs; RNA, including mRNA, siRNA, guide RNA for gene editing, dsRNA, and microRNA, PNA, CRISPR, and microRNA.

In some embodiments, two or more molecules as described herein can be delivered (e.g. co-delivered) to cells.

In some embodiments the molecule may be, but is not limited to, a small molecule, a protein (e.g., a natural protein or a fusion protein, a programmable nuclease such as Cas9, a chimeric antigen receptor (CAR), an enzyme, an antibody (e.g., monoclonal Ab)) or protein fragments such as such as an immunogenic peptide for use in a vaccine or an antigen binding fragment of an antibody (e.g., dAb, Fv, scFv, dimeric scFv, diabody, triabody, tetrabody, Fab, Fab’, F(ab’)2, or Fc fusions thereof), a nucleic acid (e.g., DNA, including linear and plasmid DNAs; RNA, including mRNA, siRNA, guide RNA for gene editing, dsRNA, and microRNA, PNA, CRISPR, and microRNA), nanoparticles, dyes (such as betalains e.g. betacyanins and betaxanthins), a virus, a viral particle, and polymers such as a polysaccharide (e.g. a carbohydrate). The molecule may also be a fluorescence-tagged protein, such as fluorescence tagged IgGs (e.g. IgG-AF647 and IgG-AF488). In some embodiments, the molecule may be a DNA, RNA, siRNA, a protein. Two or more molecules may also be delivered (e.g. co-delivered) to cells.

In an embodiment, the molecule is not a virus (e.g. the delivery method is a non- viral delivery method), nor is it contained within a virus (such as a genetically modified viral genome). In this instance, the invention has significant advantages to viral mediated delivery of a molecule to a cell. For example, methods of delivering non-viral molecules to cells using the NT array disclosed herein are generally cheaper, quicker, highly scalable, and/or avoid T cell activation, and/or avoid genotoxicity which can arise from virus-mediated transduction and/or immunogenicity due to use of virus.

The deliberate delivery or introduction of nucleic acids into eukaryotic cells is typically referred to as“transfection”. Transfection may also refer to other methods and cell types, although other terms are often preferred. Transformation is typically used to describe non-viral DNA transfer in bacteria and non-animal eukaryotic cells, including plant cells. Genetic material (such as supercoiled plasmid DNA or siRNA constructs), or even proteins such as antibodies, may be“transfected”. As used herein,“transfection” relates generally to delivery of a molecule into a cell including bacterial and non-animal eukaryotic cells.

The present invention includes use of vectors for manipulation or transfer of genetic constructs. A vector is a nucleic acid molecule, preferably a DNA molecule, that can be used to artificially carry foreign genetic material; into another cell, where it can be replicated or expressed. A vector containing foreign DNA is referred to as a “recombinant vector”. Examples of vectors include, but are not limited to, plasmids, viral vectors, cosmids, extrachromosomal elements, minichromosomes.

The vector is generally a DNA sequence that consists of an insert (transgene) and a larger sequence that serves as the "backbone" of the vector. The purpose of a vector which transfers genetic information to another cell is typically to isolate, multiply, or express the insert in the target cell. Vectors designed specifically for the expression of the transgene in the target cell are called“expression vectors”, and generally have a promoter sequence that drives expression of the transgene. Selection of appropriate vectors is within the knowledge of those having skill in the art.

As used herein, the term "promoter" is to be taken in its broadest context and includes the transcriptional regulatory sequences of a genomic gene, including the TATA box or initiator element, which is required for accurate transcription initiation, with or without additional regulatory elements (e.g., upstream activating sequences, transcription factor binding sites, enhancers and silencers) that alter expression of a nucleic acid, for example, in response to a developmental and/or external stimulus, or in a tissue specific manner. In the present context, the term "promoter" is also used to describe a recombinant, synthetic or fusion nucleic acid, or derivative which confers, activates or enhances the expression of a nucleic acid to which it is operably linked. Exemplary promoters can contain additional copies of one or more specific regulatory elements to further enhance expression and/or alter the spatial expression and/or temporal expression of said nucleic acid.

As used herein, the term "operably linked to" means positioning a promoter relative to a nucleic acid such that expression of the nucleic acid is controlled by the promoter. A promoter can be operably linked to numerous nucleic acids, for example, through an internal ribosome entry site.

The molecule may be able to modulate the expression or activity of a cellular target. The term "cellular target" refers to any component of a cell. Non-limiting examples of cellular targets include DNA, RNA, a protein, an organelle, a lipid, or the cytoskeleton of a cell. Other examples include the lysosome, mitochondria, ribosome, nucleus, or the cell membrane. In some embodiments, the molecule is siRNA. siRNA, or“Small Interfering RNA,” in general is a class of double-stranded RNA molecules, typically 20-25 base pairs in length. siRNA plays a role in the RNA interference (RNAi) pathway, where it interferes with the expression of specific genes with complementary nucleotide sequence. Thus, for example, siRNA may have a sequence that is antisense to a sequence within a target gene. siRNA also acts in RNAi-related pathways in some cases, e.g., as an antiviral mechanism or in shaping the chromatin structure of a genome. The siRNAs typically have a structure comprising a short (usually 21 -bp) double- stranded RNA (dsRNA) with phosphorylated 5' ends and hydroxylated 3' ends with two overhanging nucleotides. siRNAs are typically produced by the Dicer enzyme reacting with various precursor RNAs. Those of ordinary skill in the art will be able to identify siRNAs, many of which have been catalogued in publically accessible databases.

In some embodiments, the NW array described herein can be used for ex vivo gene editing. In some embodiments, the molecule is a programmable nuclease. As used herein, the term“programmable nuclease” relates to nucleases that can be“targeted” (“programmed”) to recognize and edit a pre-determined site in a genome.

In an embodiment, the programmable nuclease can induce site specific DNA cleavage at the pre-determined sequence or nucleic acid site. In an embodiment, the programmable nuclease may be programmed to recognize a genomic location with a DNA binding protein domain, or combination of DNA binding protein domains. In an embodiment, the programmable nuclease may be programmed to recognize a genomic location by a combination of DNA-binding zinc-finger protein (ZFP) domains. ZFPs recognize a specific 3 -bp in a DNA sequence, a combination of ZFPs can be used to recognize a specific a specific genomic location. In an embodiment, the programmable nuclease may be programmed to recognize a genomic location by transcription activator like effectors (TALEs) DNA binding domains. In an alternate embodiment, the programmable nuclease may be programmed to recognize a genomic location by one or more RNA sequences. In an alternate embodiment, the programmable nuclease may be programmed by one or more DNA sequences. In an alternate embodiment, the programmable nuclease may be programmed by one or more hybrid DNA/RNA sequences. In an alternate embodiment, the programmable nuclease may be programmed by one or more of an RNA sequence, a DNA sequence or a hybrid DNA/RNA sequence.

Programmable nucleases that can be used in accordance with the present disclosure include, but are not limited to, RNA-guided engineered nuclease (RGEN) derived from the bacterial clustered regularly interspaced short palindromic repeat (CRISPR)-cas (CRISPR-associated) system, zinc-finger nuclease (ZFN), transcription activator-like nuclease (TALEN), and argonautes.

In an embodiment, the programmable nuclease is a Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) nuclease (Barrangou, 2012). CRISPR is a microbial nuclease system involved in defence against invading phages and plasmids. CRISPR loci in microbial hosts contain a combination of CRISPR-associated (Cas) genes as well as non-coding RNA elements capable of programming the specificity of the CRISPR-mediated nucleic acid cleavage. Three types (I-III) of CRISPR systems have been identified across a wide range of bacterial hosts. One key feature of each CRISPR locus is the presence of an array of repetitive sequences (direct repeats) interspaced by short stretches of non-repetitive sequences (spacers). The non-coding CRISPR array is transcribed and cleaved within direct repeats into short crRNAs containing individual spacer sequences, which direct Cas nucleases to the target site (protospacer).

The Type II CRISPR carries out targeted DNA double-strand break in four sequential steps (for example, see Cong et ah, 2013). First, two non-coding RNA, the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the repeat regions of the pre-crRNA and mediates the processing of pre-crRNA into mature crRNAs containing individual spacer sequences. Third, the mature crRNA:tracrRNA complex directs Cas9 to the target DNA via Wastson-Crick base-pairing between the spacer on the crRNA and the protospacer on the target DNA next to the protospacer adjacent motif (PAM), an additional requirement for target recognition. Finally, Cas9 mediates cleavage of target DNA to create a double-stranded break within the protospacer. The CRISPR system can also be used to generate single- stranded breaks in the genome. Thus the CRISPR system can be used for RNA guided (or RNA programmed) site specific genome editing.

In an embodiment, the nuclease is a RNA-guided engineered nuclease (RGEN). In an embodiment, when the nuclease is an RGEN a guide for the nuclease, such as an RNA guide, is co-delivered to the cell with the nuclease such as in the form of a ribonucleoprotein (RNP). In an embodiment, the RGEN is from an archaeal genome or is a recombinant version thereof. In an embodiment, the RGEN is from a bacterial genome or is a recombinant version thereof. In an embodiment, the RGEN is from a Type I (CRISPR)-cas (CRISPR-associated) system. In an embodiment, the RGEN is from a Type II (CRISPR)-cas (CRISPR-associated) system. In an embodiment, the RGEN is from a Type III (CRISPR)-cas (CRISPR-associated) system. In an embodiment, the nuclease is a class I RGEN. In an embodiment, the nuclease is a class II RGEN. In an embodiment, the RGEN is a multi-component enzyme. In an embodiment, the RGEN is a single component enzyme. In an embodiment, the RGEN is CAS3. In an embodiment, the RGEN is CASIO. In an embodiment, the RGEN is CAS9. In an embodiment, the RGEN is Cpfl. In an embodiment, the RGEN is a dCAS9 or a CAS9 nickase. In a further preferred embodiment, the RGEN is a base editing enzyme or a deaminase. In a further preferred embodiment the RGEN is CAS9 coupled with a second enzyme, such as a reverse transcriptase. In an embodiment, the RGEN is targeted by a single RNA or DNA. In an embodiment, the RGEN is targeted by more than one RNA and/or DNA. In an embodiment, the RGEN is a recombinant and/or a high fidelity nuclease.

In an embodiment, the programmable nuclease may be a transcription activator like effector (TALE) nuclease (see, e.g., Zhang et al., 2011). TALEs are transcription factors from the plant pathogen Xanthomonas that can be readily engineered to bind new DNA targets. TALEs or truncated versions thereof may be linked to the catalytic domain of endonucleases such as Fokl to create targeting endonuclease called TALE nucleases or TALENs.

In an embodiment, the programmable nuclease is a zinc-finger nuclease (ZFN). In one embodiment, each monomer of the ZFN comprises 3 or more zinc finger-based DNA binding domains, wherein each zinc finger-based DNA binding domain binds to a 3 bp subsite. In other embodiments, the ZFN is a chimeric protein comprising a zinc finger-based DNA binding domain operably linked to an independent nuclease. In one embodiment, the independent endonuclease is a Fokl endonuclease. In one embodiment, the nuclease agent comprises a first ZFN and a second ZFN, wherein each of the first ZFN and the second ZFN is operably linked to a Fokl nuclease, wherein the first and the second ZFN recognize two contiguous target DNA sequences in each strand of the target DNA sequence separated by about 6 bp to about 40 bp cleavage site or about a 5 bp to about 6 bp cleavage site, and wherein the Fokl nucleases dimerize and make a double strand break (see, for example, US20060246567, US20080182332, US20020081614, US20030021776, WO/2002/057308, US20130123484, US20100291048 and

WO/2011/017293).

In an embodiment, the programmable nuclease may be a DNA programmed argonaute (WO 14/189628). Prokaryotic and eukaryotic argonautes are enzymes involved in RNA interference pathways. An argonaute can bind and cleave a target nucleic acid by forming a complex with a designed nucleic acid-targeting acid. Cleavage can introduce double stranded breaks in the target nucleic acid which can be repaired by non-homologous end joining machinery. A DNA“guided” or“programmed” argonaute can be directed to introducing double stranded DNA breaks in predetermined locations in DNA.

Functionalisation of the NWs

In some embodiments, the methods of the present disclosure require applying a mixture comprising the molecule and the linker onto NWs and incubating the NWs with the molecule and the linker to form functionalised NWs, wherein the molecules are attached to the functionalised NWs via the linker.

Accordingly, the present disclosure also provides a method of functionalising a plurality of nanowires (NWs) with a linker and a molecule, the method comprising: a) applying a mixture comprising the molecule and linker onto the NWs, and b) incubating the NWs with the molecule and the linker to form functionalised NWs, wherein the molecules are attached to the functionalised NWs via the linker.

The NWs may be part of a NW array. The NWs/NW array may be selected from any one or more of the NWs/NW array described herein.

The molecule and linker in the mixture may be selected from any one or more of the linkers and any one or more of the molecules described herein.

The present inventors have shown that applying a mixture comprising the molecule and a linker onto the NWs to form functionalised NWs results in a higher transfection efficiency than stepwise addition of the linker then the molecule. In some embodiments, the linker and molecule in the mixture may form a complex at the time of application to the surface of the NWs. For example, the linker and molecule may interact (e.g. couple) to form a single molecule or complex comprising both the linker and the molecule prior to application to the NW surface. In one embodiment, the mixture comprises a polyplex formed by the linker and the molecule. As used herein, the term “polyplex” refers to a positively charged complex as a result of condensation by the electrostatic interactions between the linker (e.g. poly-D-lysine) and the molecule (e.g. plasmid).

In one embodiment, the mixture comprises poly-D-lysine as a linker, and a plasmid as the molecule. In another embodiment, the mixture may comprise poly-D- lysine as a linker, and siRNA as the molecule. In yet another embodiment, the mixture may comprise poly-D-lysine as a linker, and a nucleic acid as the molecule, for example a nucleic acid encoding a chimeric antigen receptor (CAR). Other molecule and linker mixtures are also contemplated.

In some embodiments, the concentration of molecule in the molecule and linker mixture is at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, or 500 pg.mL ¹ . In other embodiments, the concentration of molecule in the molecule and linker mixture is less than about 500, 400, 300, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or 5 pg.mL ¹ . Combinations of these concentrations are also possible, e.g. the concentration of molecule in the molecule and linker mixture may be about 50 pg.mL ¹ to about 200 pg.mL ¹ , about 60 pg.mL ¹ to about 100 pg.mL ¹ , or about 70 pg.mL ¹ to about 90 pg.mL ¹ . In one embodiment, the concentration of molecule in the molecule and linker mixture is at least about 80 pg.mL ¹ .

In some embodiments, the concentration of linker in the molecule and linker mixture is at least about 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, or 500 pg.mL ¹ . In other embodiments, the concentration of linker in the molecule and linker mixture is less than about 500, 400, 300, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, or 5 pg.mL ¹ . Combinations of these concentrations are also possible, e.g. the concentration of linker in the molecule and linker mixture may be about 10 pg.mL ¹ to about 200 pg.mL ¹ , or about 50 pg.mL ¹ to about 150 pg.mL ¹ . In one embodiment, the concentration of molecule in the molecule and linker mixture is at about 40 pg.mL ¹ to about 60 pg.mL ¹ , for example about 50 pg.mL ¹ . In another embodiment, the concentration of molecule in the molecule and linker mixture is at about 80 pg.mL ¹ to about 120 pg.mL ¹ , for example about 100 pg.mL ¹ .

In another embodiment, the mass ratio of molecule to linker in the mixture may be about 1 : 100 to about 100: 1. For example, the mass ratio of molecule to linker in the mixture may be about 1 :50 to about 50: 1, about 1 : 10 to about 10: 1, about 1 :5 to about 5: 1, or about 1 :2 to about 2: 1.

In some embodiments, the NWs are incubated with the molecule and the linker for at least about 1 h, 2 h, 3 h, 4 h, 5 h, 6 h ,7 h, 8 h, 9 h, or 10 h. In other embodiments, the NWs are incubated with the molecule and the linker for less than about 10 h, 9 h, 8 h, 7 h, 6 h, 5 h, 4 h, 3 h, 2 h or 1 h. Combinations of these NW, linker and molecule incubation time are also possible, e.g. the NWs are incubated with the molecule and the linker for about 1 h to about 10 h, about 2 h to 8 h, or about 5 h to 6 h.

In some embodiments, the NWs are incubated with the molecule and the linker at a temperature of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or 25°C. In other embodiments, the NWs are incubated with the molecule with the molecule and the linker at a temperature of less than 25, 20, 28, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 °C. Combinations of these NW, linker and molecule incubation temperatures are also possible, e.g., the NWs are incubated with the molecule and the linker at a temperature of about 1 to 10°C, about 2 to 7°C, preferably about 4°C. In a preferred embodiment, the NWs are incubated with the linker and the molecule at for about 1 to 6 h and/or at a temperature of about 1 to 10°C, more preferably for about 1 to 6 h and at a temperature of about 1 to 10°C, even more preferably for about 2 h and at a temperature of about 4°C.

In some embodiments, the method further comprises the step of removing any linker and/or molecule not attached to the NWs. Various methods of removing unbound linkers and/or molecules are known in the art and include aspiration, washing, etc. In another embodiment, the NWs are dried after any unbound linker and/or unbound molecule are removed prior to adding the cells.

The number of molecules attached to the NWs can be defined in terms of a loading efficiency. The loading efficiency (%LE) is calculated according to the following formula: %LE = [(total molecules added to the NWs- free molecules not attached to the NWs)/total molecules added to the NWs] x 100%. In some embodiments, the molecules can be loaded onto the NWs with a loading efficiency of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%. Combinations of these loading efficiency values are also possible. For example, in some embodiments, the molecules can be loaded onto the NWs with a loading efficiency of about 40% to about 95%, about 60% to about 90%, or about 75% to about 85%.

In another embodiment, the linker and molecule may be added onto the NWs via stepwise addition. For example, the linker may be first applied onto the NWs, and then incubated with the NWs to form linker functionalised NWs. The molecule may then subsequently be applied onto the linker functionalised NWs, and incubated with the linker functionalised NWs to form functionalised NWs, wherein the molecules are attached to the functionalised NWs via the linker.

It will be appreciated that the embodiments described above in relation to the mixture addition of both the linker and molecule onto the NWs, can also provide embodiments for the stepwise addition of the linker and molecule.

Cells

Cells can be combined with the NW array (e.g. cells are added onto the NWs). The present inventors have identified the required design parameters of NW arrays (range of diameters, heights, and densities) to allow for, in some embodiments, high-throughput delivery of molecules into cells including difficult to transfect primary cells.

The cells may be prokaryotic cells or eukaryotic cells. The cells may be from a single-celled organism or a multi-celled organism. In some cases, the cells are genetically engineered, e.g., the cells may be chimeric cells. The cells may be bacterial, fungi, plant, or animal cells, etc. The cells may be from a human or a non-human animal or mammal. For instance, if the cell is from an animal, the cell may be a cardiac cell, a fibroblast, a keratinocyte, a hepatocyte, a chondrocyte, a neural cell, an osteocyte, an osteoblast, a muscle cell, a blood cell, an endothelial cell, a stem cell, an immune cell (e.g., a T-cell, a B-cell, a macrophage, a neutrophil, a basophil, a mast cell, an eosinophil), etc. In some cases, the cell is a cancer cell. The cells may be non-adherent cell lines (e.g. LI .2, mouse immune B; Jurkat, human CD4 ⁺ T; Ramos, human immune B) or adherent cell lines (GPE86, mouse embryonic fibroblast), as well as primary cells. In a preferred embodiment, the cell is a primary cell such as a primary immune cell or stem cell. In one embodiment, the cell is a primary immune cell, for example, a T cell. In one embodiment, the cell is an immortalised cell. The immortalised or primary cells may be an immune cell, neuron, endothelial cell, epithelial cell, or fibroblast, or mammalian cell.

In an embodiment, the cell has been modified to remove the cell wall, such as from a plant cell, e.g. a plant protoplast. Techniques for removing or permeating the wall of a cell without effecting viability are well known in the art.

As used herein, the term“immortalised cells” refers to a population of cells from a multicellular organism which would normally not proliferate indefinitely but, due to mutation, have evaded normal cellular senescence and instead can keep undergoing division.

As used herein, the term“immune cells” refer to cells of the immune system, which defend the body against disease and foreign materials. Non-limiting examples of immune cells include dendritic cells, such as bone marrow-derived dendritic cells; lymphocytes, such as B cells, T cells, and natural killer cells; and macrophages. The immune cells may, in some embodiments, be derived from bone marrow, spleen, or blood from a suitable subject. For example, the immune cells may arise from a human or a non human mammal, such as a monkey, ape, cow, sheep, goat, horse, donkey, llama, rabbit, pig, mouse, rat, guinea pig, hamster, dog, cat, etc. In a preferred embodiment, the immune cell is a T-cell, preferably a human T-cell.

As used herein, the term“stem cells” refers to clonogenic cells capable of both self-renewal and multilineage differentiation. Based on their origin, stem cells are categorised either as embryonic stem cells (ESCs) or as postnatal stem cells/somatic stem cells/adult stem cells (ASCs).

Embryonic stem cells (ESCs) can be derived from embryos that are 2-11 days old called blastocysts. They are totipotent - capable of differentiating into any type of cell including germ cells. ESCs are considered immortal as they can be propagated and maintained in an undifferentiated state indefinitely. Adult stem cells (ASCs) are found in most adult tissues. They are multipotent - capable of differentiating into more than one cell type but not all cell types. Depending on their origin, AASCs can be further classified as hemopoetic stem cells (HSCs) and mesenchymal stem cells (MSCs). HSCs can be obtained either from cord blood or peripheral blood. MSCs are those that originate from the mesoderm layer of the fetus and in the adult reside in a variety of tissues such as the bone marrow stem cells (BMSCc), limbal stem cells, hepatic stem cells, dermal stem cells, etc. The stem cells may be induced pluripotent stem cells.

Stem cells have also been isolated from orofacial tissues which include adult tooth pulp tissue, pulp tissue of deciduous teeth, periodontal ligament, apical papilla, and buccal mucosa.

HSCs can be divided into a long-term subset, capable of indefinite self-renewal, and a short-term subset that self-renew for a defined interval. HSCs give rise to nonself- renewing oligolineage progenitors, which in turn give rise to progeny that are more restricted in their differentiation potential, and finally to functionally mature cells including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, dendritic cells), erythroid (erythrocytes), megakaryocytic (platelets) and lymphoid lineages (T-cells, B-cells, NK-cells).

The cells can be added onto the functionalised NWs in any manner. Unless otherwise stated, it will be understood that the cells can be added by any means onto the NWs, and provided the cells interact with the surface of the NWs, they will be considered to have been“added onto” the NWs. For example, the NW array may be introduced into a medium comprising the cells, or the cells may be dropped onto the NW array (e.g. via a pipette), both of which results in the cells being added onto the NWs. In other words, the cells are combined with the NW array. The cells may first be harvested and/or resuspended in a suitable medium, for example, culture medium, at a given concentration. In one embodiment, the cells are added onto the functionalised NWs using, for example, a pipette, a multichannel pipette, or an automated cell plating system. In one embodiment, the cells are added to a well plate wherein one or more wells comprises one or more substrates comprising the plurality of NWs as described above. For example, referring to Figure 3, one or more substrates comprising the plurality of NWs are placed within the wells of a multi well plate, for example a 6, 24, 48 or 96-well plate, and the cells are subsequently added onto the NWs within the wells of the plate.

In some embodiments, the number of cells per well may be at least about 10, 20, 40, 50, 70, 100, 120, or 150 x 10 ³ cells/well. In other embodiments, the number of cells per well may be less than about 150, 120, 100, 70, 50, 40, 20, or 10 x 10 ³ cells/well. For example, 100 x 10 ³ cells/well of cells (e.g., primary B or T cells) can be seeded onto linker and molecule coated s NWs in a 48-well plate.

Following addition of the cells onto the NWs, one or more of the cells may be penetrated by at least one of the plurality of NWs. In some cases, merely placing or plating the cells on the NWs is sufficient to cause at least some of the NWs to be mechanically inserted into the cells (i.e. penetration). For example, a population of cells suspended in medium may be added to the surface of the substrate containing the NWs, and as the cells settle from being suspended in the medium to the surface of the substrate, at least some of the cells may encounter NWs, which may (at least in some cases) become inserted into the cells. It will be appreciated that NW insertion/penetration may occur as the cells naturally sink onto the plurality of NWs (e.g. by gravity). The NWs may penetrate partially or completely into the cells, depending on the size or dimensions of the NW and/or the size and shape of the cell. For example, the NWs may be inserted into the cytosol of a cell, or into an organelle within the cell, such as into a mitochondria, a lysosome, the nucleus, or a vacuole. In some embodiments, in a population of cells added onto the NWs, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the cells may have at least one NW penetrating the cells. In other embodiments, in a population of cells added onto the NWs, less than about 90%, 85% 80%, 75%, 70%, 65%, 60%, 55% or 50% of the cells may have at least one NW penetrating the cells. Combinations of these penetration % values are also possible, e.g., in a population of cells added onto the NWs, about 80% to about 90% of the cells may have at least one NW penetrating the cells. In some embodiments, there may be more than one NW penetrating each cell, for example on average at least about 5, 10, 50, 100, 200, 500, or 1 x 10 ³ NWs. By increasing the number of NWs contacting/penetrating a single cell, in some embodiments, the transfection efficiency may increase due to a larger payload of molecules being delivered (e.g. endocytosis) into a single cell owing to the multiple penetration sites. The number of NWs penetrating a single cell can be controlled by the density. For example, a NW array having a density of about 0.2 to about 0.4 NWs per pm ² may result in at least 5 NWs penetrating a single cell following addition of the cells onto the NWs.

In some embodiments, the surface of the NWs may be further treated in any fashion prior to adding the cells that allows binding of cells to occur thereto. For example, the NW surface may be ionised and/or coated with any of a wide variety of hydrophilic and/or cytophilic materials, for example, materials having exposed carboxylic acid, alcohol, and/or amino groups. In other embodiments, the surface of the NWs may be reacted in such a manner as to produce carboxylic acid, alcohol, and/or amino groups on the surface which can bind to the cells. In some cases, the surface of the substrate may be coated with a biological material that promotes adhesion or binding of cells, for example, materials such as fibronectin, laminin, vitronectin, albumin, collagen, or peptides or proteins containing RGD sequences. Alternatively, the linker molecule present on the NWs may also promote adhesion or binding of cells. For example, the linker poly-D4ysine allows for the attachment of molecules for delivering into cells and also promotes cell adhesion with the NWs prior to penetration.

The cells are incubated with the functionalised NWs to allow for release of the molecules into the penetrated cells from the NWs. The cells and NWs may be incubated for at least about 1 h, 4 h, 6 h, 12 h, 1 day, 2 days, 3 days, 4 days, or 5 days. The cells and NWs may be incubated for less than about 5 days, 4, days, 3 days, 2 days, 1 day, 12 h, 6 h, 4 h or 1 h. Combinations of these cell and NW incubation times are also possible, e.g. the cells and NWs are incubated for about 6 to 12 h. In some embodiments, the cells and NWs are incubated at a temperature of about 30 to 40°C, preferably approximately 37°C, or other temperatures suitable for the cell type. In some embodiments, the cells and NWs are incubated in a humidified 5% CO2 atmosphere.

In addition, it should be noted that in some embodiments, the cells may be cultured using any suitable cell culturing technique, e.g., before or after insertion of the NWs. For example, mammalian cells may be cultured at 37°C in a humidified 5% CO2 atmosphere in appropriate cell medium.

In some embodiments, following incubation, the percentage of cells being transfected (e.g. successfully having molecules delivered into them) may be at least 30%, 35%, 40%, 45 %, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the total number of cells added to the NWs. Combinations of these transfection percentages are also possible, e.g., the percentage of cells being transfected (e.g. successfully having molecules delivered into them) may be about 30% to about 40% of the total number of cells added to the NWs, about 40% to about 80% of the total number of cells added to the NWs, or about 70% to about 80% of the total number of cells added to the NWs. In the context of the present disclosure,“high transfection efficiency” and“high delivery efficiency” is understood to mean that the percentage of cells being transfected (e.g. successfully having molecules delivered into them) may be about at least about 30% of the total number of cells added to the NWs.

In one embodiment, the method further comprises the step of centrifuging the cells and the NWs. The inventors have surprisingly identified that centrifuging the cells and the NWs not only facilitates the interaction between the cells and the NWs, but also provides an external force which allows for enhanced penetration of the cells through the cell membrane and even the nucleus. In addition, the inventors have surprisingly identified no significant difference in cell viability before and after centrifugation. In some embodiments, this enhanced penetration via centrifugation may result in enhanced intracellular delivery of molecules through direct insertion of the molecules attached to the NWs and/or endocytosis triggered by the cell membrane curvature. Further advantages may be provided by centrifuging the NWs and cells compared to natural sinking (non-centrifuged) including a reduced incubation time required to deliver the molecules into cells and/or higher % uptake of molecules in cells without significant impact on cell viability.

In some embodiments, the cells and NWs are centrifuged for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, or 30 minutes. In other embodiments, the cells and NWs are centrifuged for less than about 30, 25, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 minute. Combinations of these centrifuge times are also possible, e.g., the cells and NWs are centrifuged for about 10 minutes to about 20 minutes. The cells and NWs may be centrifuged at a temperature of at least 30°C, preferably about 32°C, or other temperatures suitable for the cell type. The cells and NWs may be centrifuged at a speed of about 200 x gravity (200 g = about 3.92 N). In other embodiments, the cells and NWs may be centrifuged at a speed of at least about 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 RPM. In one embodiment, the centrifuge speed is about 1000 RPM.

The skilled person will appreciate that the molecular delivery efficiency to different cell types can be manipulated by varying the centrifugation time and/or speed. For example, a centrifugation time of about 15 minutes at 200 x g shows unexpectedly high delivery efficiency to human primary immune cells.

The present inventors have shown that the cells can be unexpectedly detached from the NWs while maintaining high cell viability and proliferative capacity as compared to the viability and proliferation of cells transfected on flat surfaces (for example flat Si surfaces). Such detachment processes are known in the art, and include trypsinisation or gentle pipetting. For example, the cells can be detached and harvested from the NWs after 2 h, 6 h, 12 h and 24 h of incubation.

In some embodiments, the detached and harvested cells can be further cultured to expand the transfected population. For example, the cells may be cultured for 1, 2 or 3 days.

In an embodiment, the molecule is a polynucleotide which becomes integrated into the genome of the cell. In an alternate embodiment, the molecule modifies the genome of the cell such as when using a programmable nuclease. In some embodiments, the cells are transfected with foreign genetic material. In some embodiments the cells are transiently transfected and express the foreign gene but do not integrate it into their genome. Thus the new gene will not be replicated. These cells express the transiently transfected gene for a finite period of time, usually several days, after which the foreign gene is lost through cell division or other factors. In other embodiments, stably transfected cells are generated. To accomplish this, a marker gene is typically co-transfected, which gives the cell some selectable advantage, such as resistance towards a certain toxin. Some (very few) of the transfected cells will, by chance, have integrated the foreign genetic material into their genome. If the toxin is then added to the cell culture, only those few cells with the marker gene integrated into their genomes will be able to proliferate, while other cells will die. After applying this selective stress (selection pressure) for some time, only the cells with a stable transfection remain and can be cultivated further.

Contemplated uses of the disclosed system and methods include, for example, the production of cells for therapeutic use, production of cells that may produce a recombinant product which may be a therapeutic, nutraceutical or food, for assaying the effect (intra and/or intercellular effect) of a molecule (e.g., candidate therapeutic), for assaying dose-response of a molecule, etc. The skilled person will appreciate that the intended use of the cells determines whether the cells need to be detached from the NWs.

CAR ⁺ T cells

To express specific cancer-targeting proteins on T cell surface, virus-mediated DNA transfer is typically used in the prior art. However, a lengthy viral transfection may suffer from immunogenicity, low transfection efficiency in quiescent cells, high toxicity, and biological safety issues.

The system and delivery methods of this disclosure can be used to generate antigen-specific CAR ⁺ T cells. In certain embodiments, the system disclosed herein is used to mediate gene transfer of CARs into primary immune T cells. A wide range of CAR constructs and expression vectors for the same are known in the art. Using the NW array and methods disclosed herein, T cells can be genetically engineered to produce CARs on their surface. Advantageously, the T cells do not need to be first activated, for example, using low-level activation by phytohemagglutinin (PHA) prior to transfection. Further, short term culture (6-12 h) is sufficient for cells to uptake the CAR gene with high efficiency (see Figure 10).

CARs encode for transmembrane chimeric molecules with dual function: (a) immune recognition of tumor antigens expressed on the surface of tumor cells; (b) active promotion and propagation of signaling events controlling the activation of the lytic machine. CARs comprise an extracellular domain with a tumor-binding moiety, typically a single-chain variable fragment (scFv), followed by a hinge/spacer of varying length and flexibility, a transmembrane (TM) region, and one or more signaling domains associated with the T-cell signaling. The first generation CARs are equipped with the stimulatory domain of the z-chain; in the second generation CARs, the presence of costimulatory domains (CD28) provides additional signals to ensure full activation; in the third generation CARs an additional transducer domain (CD27, 41-BB or 0X40) is added to the z-chain and CD28 to maximize strength, potency, and duration of the delivered signals; the fourth generation CARs include armored CARs, engineered to synthetize and deliver interleukins. Armored CARs combine the CAR functional activities with the secretion of IL-2 or IL-12 expressed as an independent gene in the same CAR vector.

A chimeric antigen receptor (CAR) recognizes cell-surface tumor-associated antigen independent of human leukocyte antigen (HLA) and employs one or more signaling molecules to activate genetically modified T cells for killing, proliferation, and/or cytokine production. Adoptive transfer of T cells expressing CAR has shown promise in multiple clinical trials.

In one embodiment, cells are removed from a patient’s body (for example, blood is removed from the patient to obtain T cells) and genetically modified so that they can recognize the patient’s cancer cells (for example, transfected with gene encoding a CAR using the system and delivery methods of the present disclosure) and the modified T cells reintroduced into the patient. The modified T cells, when reintroduced into the patient’s body, multiply and attack cancer cells. In some embodiments, the modified T cells are cultured ex vivo prior to being reintroduced into the patient. Preferably, the T cells are not activated prior to introduction of the gene encoding the CAR.

Kits and nanowire arrays

In another embodiment, a kit may be provided for delivering a molecule to a cell. The kit may include a molecular delivery system as described above. The kit may further comprise a linker.

For example, the kit may comprise a nanowire (NW) array, comprising a) a substrate having a surface, and plurality of NWs attached to the surface, wherein the NWs comprise one or more of the following features:

i) an average density of about 0.1 to about 1.0 NWs per pm ² ; ii) an average length of at least about 3 pm; and/or iii) an average diameter of less than about 500 nm.

In one embodiment, the NWs comprise i) an average density of about 0.1 to about 1.0 NWs per pm ² ; ii) an average length of at least about 3 pm; and iii) an average diameter of less than about 500 nm.

In one embodiment, the kit may further comprise a linker. In a further embodiment, if present, the linker of kit may be a silane, poly-lysine , a cleavable linker, a plasma polymer, a synthetic polymer, collagen, fibronectin or laminin, for example poly-lysine, preferably poly-D-lysine. In a further embodiment, the linker is provided as an aqueous solution at a concentration of about 140 to 200 pg.mL ¹ .

In a further or another embodiment, the kit may further comprise a molecule. In other embodiments, the kit may further comprise cells.

It will be appreciated that embodiments described for the NWs, linker and molecules earlier in relation to the molecule delivery system and method of delivery a molecule to a cell can also provide embodiments for the NWs, linker and molecules that are part of the kit.

In some embodiments, the kit may also include written information on how to prepare a mixture of the linker with a molecule for applying to the surface of the NWs as described above in relation to the embodiments for the molecule delivery system and method of delivery a molecule to a cell.

In another embodiment, the kit comprises a multiwell plate comprising the NWs as discussed above. The multiwell plates may be of any size. However, in certain embodiments of the kit, the multi well plate has the dimensions of a microwell plate, e.g., having standard dimensions (about 5 inches x about 3.33 inches, or about 128 mm x 86 mm) and/or standard numbers of wells therein. For example, there may be 6, 24, 48, 96, 384, 1536 or 3456 wells present in the multiwell plate. The multiwell plates may be fabricated from any suitable material, e.g., polystyrene, polypropylene, polycarbonate, cyclo-olefins, or the like. Microwell plates can be made by injection molding, casting, machining, laser cutting, or vacuum sheet forming one or more resins, and can be made from transparent or opaque materials. Many such microwell plates are commercially available.

In some embodiments, the multiwell plate is prepared by immobilising a bottomless multiwell plate with a substrate comprising a plurality of NWs as discussed above. For example, the bottomless multiwell plate may be a commercially available bottomless microwell plate, e.g., a bottomless 384-well microwell plate. The substrate and NWs form a base of the bottomless microwell plate. In some embodiments, the multiwell plate and the substrate may be immobilised with respect to each other by the use of a suitable adhesive. Non-limiting examples of adhesives include acrylic adhesives, pressure- sensitive adhesives, silicone adhesives (e.g., UV curable silicones or RTV silicones), biocompatible adhesives, epoxies, or the like. Non-limiting examples of biocompatible glues include, but are not limited to, Master Bond EP42HT -2ND-2MED BLACK and Master Bond EP42HT-2 CLEAR (Master Bond). The adhesive, in some cases, may be a permanent adhesive. Many such adhesives can be obtained commercially from companies such as 3M, Loctite, or Adhesives Research.

The multiwell plate and the substrate may be directly immobilised to each other, and/or there may be other materials positioned between the multiwell plate and the substrate, for example, one or more gaskets (e.g., comprising silicone, rubber, neoprene, nitrile rubber, fiberglass, polytetrafluoroethylene, etc.). In some cases, these materials may be dimensioned and arranged to be in the same pattern as the wells (or a subset thereof) of the multiwell plate to which they are being attached.

The present disclosure also provides nanowire array comprising a plurality of nanowires (NW s).

In one embodiment, the nanowire array comprises:

a) a substrate having a surface, and plurality of nanowires (NWs) attached to the surface, wherein NWs comprise one or more of the following features:

i) an average density of about 0.1 to about 1.0 NWs per pm ² ; ii) an average length of at least about 3 pm; and/or

iii) an average diameter of less than about 500 nm.

In some embodiments, the NWs are silicon NWs, polymeric NWs, or a combination thereof. In one embodiment, the NWs are silicon NWs.

It will be appreciated that embodiments described for the NWs earlier in relation to the molecule delivery system, method of delivery a molecule to a cell, and kit, can also provide embodiments for the NWs of the nanowire array.

EXAMPLES

In order that the invention may be more clearly understood, particular embodiments of the invention are described in further detail below by reference to the following non-limiting experimental materials, methodologies and examples.

Assays and tests used to assess the application of this invention.

FITC Labeling of SiNWs Substrates were first ozone-cleaned (10 min), and then incubated in 2% (v/v) 3- aminopropyltrimethoxysilane (APTMS, Sigma-Aldrich) in dry toluene (Sigma-Aldrich) in an orbital shaker for 10 min, RT. Substrates were rinsed three times with toluene and dried at 110°C on a hot plate for 10 min. Silanised substrates were then immersed to freshly prepared solution of FITC (ImM in DPBS, Gibco). After 10 minutes incubation at RT, substrates were washed three times with DPBS and left to dry at RT.

Cell Viability Assay

The viability of cells on substrates was assayed by live-dead staining using a final concentration of 15 pg mL ^_1 FDA (Sigma-Aldrich) and 5 pg mL ^_1 PI (Sigma-Aldrich) in media for 5 minutes at 37°C. After staining, samples were rinsed with DPBS before being observed under an inverted Ti-S fluorescence microscope (Nikon) using standard filters for FITC (495 nm excitation/517 nm emission) for FDA and tetramethylrhodamine (TRITC, 538 nm excitation/619 nm emission nm) for PI. Observations were conducted at five different locations on the surface of each sample at the magnification of 10 ^c objective lens. All experiments were repeated at least three times.

Cell Fixation and Fluorescence Staining

Cells grown on the substrates were washed with DPBS and then fixed in a solution of 4% paraformaldehyde (Electron Microscopy Sciences) for 10 min, followed by permeabilisation with 0.1% Triton X-100 (Sigma-Aldrich) in DPBS for 5 minutes at RT and washing three times with DPBS. Cells were blocked with 1% (w/v) bovine serum albumin (BSA, Sigma-Aldrich) solution for 1 h at RT and washed three times with DPBS. For primary antibody staining, cells were stained with Hoechst (Hoechst 33342, Sigma-Aldrich), Alexa Fluor 568 Phalloidin (Life Technologies), anti-vinculin monoclonal antibody (rabbit, Sigma-Aldrich), anti- ?-integrin monoclonal antibody (mouse, Invitrogen), anti-caveolin-1 polyclonal antibody (rabbit, Abeam), and anti- clathrin heavy chain monoclonal antibody (mouse, Life Technologies), for 45 min, RT. After washing three times with DPBS, cells were then further stained with secondary antibodies, Alexa Fluor 488 chicken anti-rabbit IgG (Life Technologies) and Alexa Fluor 647 goat anti-mouse IgG (Life Technologies), for 30 min, RT. Cells were washed three times with DPBS and left in DPBS for confocal imaging.

Confocal Laser Scanning Microscopy

A Nikon AIR confocal laser scanning microscope system was used for fluorescence imaging. Hoechst, Alexa Fluor 488 chicken anti-rabbit IgG (indicating vinculin), Alexa Fluor 568 Phalloidin, and Alexa Fluor 647 goat anti-mouse IgG (indicating /-integrin) were excited at 340 nm, 488 nm, 578 nm, 647 nm, with emission at 510 nm, 520 nm, 600 nm and 670 nm, respectively. Observations were conducted at more than ten different locations on the surface of each sample at the magnification of 60 x water immersed objective lens. Images were analysed using the Nikon NIS EI ements Advanced Research software provided by the manufacturer.

Live Cell Microscopy for Migration Study

20,000 pR-GFP GPE86 cells (constitutively expressing GFP) were seeded onto substrates in 48-well plate, followed by centrifugation at 200 g, 32°C, 15 min. After centrifugation, substrates carrying cells were transferred to a Nunc glass bottom dish (Thermo Fisher Scientific) with fresh complete DMEM and placed in the portable incubation chamber provided by the manufacturer, with temperature at 37°C and 5% CO2. GFP was excited using the 488 nm laser with low intensity, to avoid phototoxicity that might induce cell death, and detected at 513 nm. Observation was conducted at the magnification of 10 x objective lens. Confocal images were taken every 10 minutes over 60 h. Live cell images were analysed using Nikon NIS-A Advanced 2D Tracking Module.

Sample Preparation for SEM Imaging

Cells grown on the substrates were rinsed with 0.1 M sodium cacodylate buffer (Electron Microscopy Sciences) and fixed with 2.5% glutaraldehyde (Electron Microscopy Sciences) in 0.1 M sodium cacodylate at 4°C overnight. Following this, substrates were washed (3 x 5 min) with chilled 0.1 M sodium cacodylate buffer and post-fixed with 1% osmium tetroxide (Electron Microscopy Sciences) in 0.1 M sodium cacodylate at RT for 1 h. After repeating the washing step, substrates were gradually dehydrated with increasing concentrations of ethanol: 50%, 70%, 90% (1 x 10 min) and 100% (2 x 10 min) at RT, and finally critical point dried (CPD 030 Critical Point Dryer, BAL-TEC). Substrates were then mounted on SEM stubs and sputter-coated with a thin layer of either gold or platinum in order to increase their conductivity.

Staining of Intracellular Compartments

The sample preparation combined heavy metal staining with resin embedding. In particular, samples were rinsed with 0.1 M sodium cacodylate buffer and fixed with 2.5% glutaraldehyde in the same buffer at 4°C overnight (4 h in the case of LI .2 and Jurkat cells). Following this, samples were washed (3 x 5 min) with chilled 0.1 M sodium cacodylate buffer and quenched with chilled 20 mM glycine solution (Sigma-Aldrich) in the same buffer for 20 min. After repeating the washing step samples were post-fixed by combining equal volumes of 4% aqueous osmium tetroxide with 2% potassium ferrocyanide (UNIVAR) in 0.2 M sodium cacodylate buffer on ice for 1 h. Samples were then washed again (3 x 5 min) with chilled buffer and incubated with 1% tannic acid (BDH) in water at RT for 20 min. After rinsing with buffer (2 x 5 min) samples were further incubated with 2% aqueous osmium tetroxide at RT for 30 min. Following this, samples were washed (2 x 5 min) with distilled water and incubated with syringe-filtered 4% aqueous uranyl acetate (UNIVAR) at 4°C overnight. Samples were then washed (3 x 5 min) with chilled distilled water and gradually dehydrated with increasing concentrations of ethanol: 10%, 30%, 50%, 70%, 90% and 100% (1 x 7 min) at RT. An epoxy -based resin (Epon 812) 20 mL solution was prepared by initially mixing 12.2 g of DDSA (Dodecenyl Succinic Anhydride Specially Distilled, Electron Microscopy Sciences), 4.4 g of Araldite (GY 502, Electron Microscopy Sciences) and 6.2 g of Procure 812 (EMBED 812 RESIN) using a mechanical stirrer. Once the solution was uniformly mixed, 0.8 ml of BDMA (N-benzyldimethylamine, Electron Microscopy Sciences) was added to it while stirring. Samples were then infiltrated with increasing concentrations of the freshly prepared resin solution in 100% ethanol at RT and in a sealed container using the following ratios: 1 :3 (3 h), 1 :2 (3 h), 1 : 1 (overnight), 2: 1 (3 h), 3 : 1 (3 h). Following this, samples were finally infiltrated with 100% resin solution overnight. Prior to polymerisation at 60°C, the excess resin was drained away by mounting the samples vertically for 1 h.

SUM Imaging

SEM imaging of both bare SiNWs and SiNW substrates with cells was performed on a Nova NanoSEM 430 (FEI). The images were taken at tilted (45°) or top views with an electron beam acceleration voltage of 5 kV and a current of 0.11 nA, while using a secondary electron detector.

FIB Sectioning and Imaging

FIB sectioning of SiNW substrates with cells was performed using a Thermo Fischer Helios G4 UX FIB-SEM vertically and at 45° to the sample surface The stage is tilted at 7°C in order for the milling to be performed at 45° to the sample surface. Prior to FIB sectioning, the region of interest was protected from ion beam (i-beam) damage using i-beam assisted deposition of -0.5 pm thick platinum layer. The coating was carried out at 30 kV using i-beam current of 0.26-0.44 nA, depending on the area size. Following this, rough milling was performed at acceleration voltage of 30 kV and a current of 20 nA. The resulting cross sections were then polished with a voltage of 30 kV and a current ranging between 1.2-2.4 nA. Images were taken using an electron beam at acceleration voltage of 3 kV and a current of 200 pA, while using immersion mode, TLD detector operating in back-scattered (BS) electron collection mode, at a dwell time of 5 ps and 6144 x 4096 pixel ² resolution. During sequential sectioning, images were taken every 50 nm using previously mentioned e-beam conditions.

Flow Cytometry

A LSRIIb flow cytometer (BD) was used to investigate the proliferation, apoptosis and transfection efficiency of cells harvested from the substrates.

Flow Cytometry - Proliferation Assay

On Day 1, 20,000 cells/well of GPE86, and 40,000 cells/well of LI .2 and Jurkat cells were stained with 5 mM CellTrace Violet (CTV) reagent (Invitrogen), seeded onto substrates in 48-well plate and centrifugated at 200 g, 32°C, 15 min. Substrates carrying cells were then transferred to a new plate with fresh media. After 6 h incubation at 37°C with 5% CO2, cells were harvested from the substrates as described above and placed back in fresh media culture. On Day 2 and Day 3, cells were resuspended in fluorescence- activated cell sorting (FACS) buffer (1 x DPBS containing 1% BSA, 2 mM EDTA, and 0.1% sodium azide) and cell proliferation was determined by measuring the fluorescence intensity of CTV using the LSRIIb. Flow cytometry analysis was performed using 405 nm excitation and a 460 nm bandpass emission filter. Unstained cells and CTV-stained cells fixed in FACS buffer after 6 h incubation served as negative and positive controls, respectively.

Flow Cytometry - Apoptosis Assay

The FITC Annexin V/Dead Cell Apoptosis Kit (Thermo Fisher Scientific) with FITC annexin V and PI for flow cytometry was used for a rapid and convenient apoptosis assay. 40,000 cells/well of the T cells, Jurkat cells were seeded onto PDL-coated substrates (linker being polylysine, PDL) in 48-well plate, followed by centrifugation at 200 g, 32°C, 15 min. Substrates carrying cells were then transferred to a new plate with fresh media. At time points of 2 h, 6 h, 12 h and 24 h, cells were harvested from the substrates as described above and stained with the FITC annexin V/Dead Cell Apoptosis Kit following the manufacture protocol. Stained cells were analysed on LSRIIb. FITC Annexin V and PI were excited using 488 nm laser and detected using 530 nm and 670 nm filters, respectively.

Flow Cytometry - Detection of Plasmid Insertion and GFP Expression

Cells were harvested from substrates loaded with plasmids after 6 h incubation. For detection of Cy3 tagged-plasmid insertion, harvested cells were stained with Hoechst (to distinguish from non-living particles) and PI (to exclude dead cells) and cells were immediately run through flow cytometry analysis. To investigate the transfection efficiency, GFP expression was measured 48 h after harvesting. The excitation/emission wavelengths for Cy3, Hoechst, PI and GFP on LSRIIb were 561/580 nm, 355/495 nm, 488/670 nm, and 488/540 nm, respectively. Flow cytometry analysis was performed with proper negative and positive controls. Compensation was done to avoid fluorescence leakage between different channels.

Data Processing and Statistical Analysis

Fluorescence microscopy and SEM images were processed and analysed by NIS EI ement (Nikon), Image J, Microscopy Image Browser (MIB, University of Helsinki) and Amira (Thermo Fisher Scientific). The contrast and the brightness were not varied from the original pictures. Flow cytometry data were analysed with FlowJo. All statistical analysis were performed using Prism GraphPad 7. Non-parametric two-sided Mann- Whitney U-tests were performed for comparison between two groups. A one-way ANOVA was used to calculate univariate data set with more than two groups. Two-way ANOVA tests were used to calculate multivariate data set. Values are represented as mean and mean ± standard deviation.

Example 1: Preparation of Si Nanowires

Preparation of Si Wafer Substrates

Before polystyrene microsphere (PSMS, Polysciences, Inc.) deposition, flat silicon wafers (3", p-type, 3-6 Ge , <100>, Siltronix, France) were cut into quarter pieces and cleaned by sonication in 1 : 1 solution of ethanol: acetone for 5 minutes and then sonicated again in MilliQ water for 5 min. This was followed by dipping the wafer pieces into boiling Piranha solution (3 : 1 HiSCUHiCk v/v, 75°C, Avantor Performance Materials) for 1 h to remove any organic contaminants, then washing with water and drying under a nitrogen jet. Convective Assembly Deposition

Hexagonally close-packed PSMS monolayers were deposited over quarter pieces of a 3" Si wafer by convective assembly. The apparatus included a mounted microscope slide that was used as a blade for the PSMS depositions, a 50 mm motorised translation stage (MTS50-Z8, Thorlabs, Inc.) and a Compact Sub-Hertz Pendulum (CSP) vibration isolation system (TMC). The blade was adjusted to leave a small space between the bottom edge of the blade and the Si substrate. 25 pL of a suspension of PSMS (Polybead microspheres solutions, various diameters, 2.5% w/v in water) was injected into the space between the blade and the sample using a micropipette, forming a meniscus between the pinned substrate and the bottom edge of the blade. This resulted in a continuous contact line of PSMS suspension on the Si substrate. To deposit PSMS in a uniform monolayer, operating parameters such as stage velocity and PS suspension concentration were adjusted.

Oxygen Plasma Reactive Ion Etching of Polystyrene Microspheres

Samples prepared by convective assembly were inserted into PlasmalablOO ICP380 deep reactive ion etcher (Oxford Instruments), where oxygen plasma treatment was performed in order to reduce the size of the PSMS. During the etching step a flow rate of 100 seem O2 was used with inductively coupled plasma (ICP) power of 100 W and bias power of 50 W. The APC valve position was set at 35 and He pressure was set at 10 Torr. The reduced size PSMS served as a mask for the subsequent silicon etching in two steps.

Deep Reactive Ion Etching of Silicon

1. Bosch process: Silicon etching was performed by alternate cycles of passivation and etching steps in order to obtain SiNWs with a cylindrical profile. During the passivation step (6 s), a flow rate of 150 seem C4F8 and 3 seem SF6 was used with ICP power of 1500 W and Bias power of 5 W. During the etching step (8 s), a flow rate of 150 seem SFe and 3 seem C4F8 was used with ICP power of 2000 W and bias power of 20 W. The APC valve position was set at 70 and He pressure was set at 10 Torr in both steps. The etching depth of Si was controlled by the number of cycles. Following the Bosch process, PSMS were removed either by sonication of the samples in MilliQ water for 2 minutes or by performing oxygen plasma RIE with high power. In the case of sonication samples were then dried under a nitrogen jet and inserted back to the deep reactive ion etcher. 2. Pseudo Bosch process: Silicon etching was performed in a simultaneous flow of 100 seem SFe and 40 seem C4F8 at a pressure of 10 mTorr with ICP power of 1500 W and Bias power of 50 W to achieve a conically-shaped profile of the SiNWs. He pressure was set at 10 Torr. The diameter of the tip as well as the final length of the SiNWs was controlled by the etching time.

Functionalisation of SiNW’s with Linker and Molecules

1. Stepwise addition: Substrates were immersed in 70% ethanol and allowed to dry at room temperature (RT) for 2 h in a laminar flow cabinet. Following this, substrates were coated with 10 pL of the linker, PDL (Sigma- Aldrich), at the concentration of 167 pg mL ^_1 in H2O. After 4 h incubation at 4°C, excessive PDL solution was removed by aspiration before applying of a molecule, in this case nucleic acid loading. 10 pL of gWiz-GFP or Cy3-gWiz-GFP plasmid DNAs (10, 50 or 100 ng pL ^-1 in H2O; Aldevron), or siRNAs (siOTP (20 nM in H2O); siTOX (20 nM in H2O); Dharmacon) were placed on the PDL-coated substrates and allowed to stand overnight, 4 °C. Cargo-loaded substrates were briefly dried just before cell seeding.

2. Mixture addition: Preparation of linker and molecule mixture. Loading of linker and molecule mixture onto SiNWs: Substrates were immersed in 70% ethanol and allowed to dry at RT for 2 h in a laminar flow cabinet. 5 pL of PDL (Sigma- Aldrich) at the concentration of 167 pg mL ^_1 in H2O was mixed with 5 pL of gWiz-GFP or Cy3- gWiz-GFP plasmid DNAs (100 or 200 ng pL ^-1 in H2O; Aldevron), or siRNAs (siOTP (40 nM in H2O); siTOX (40 nM in H2O); Dharmacon) and allow to stand for 10 min, RT. Following this, substrates were coated with 10 pL of PDL-cargo mixture and incubate for 2 h at RT or overnight, 4 °C. Mixture-loaded substrates were briefly dried just before cell seeding.

Example 2: Preparation of molecules

Preparation of Fluorescentlv Tassed Plasmids

The Label IT Tracker™ Intracellular Nucleic Acid Localisation Kit (Mirus, Japan) was used to add a fluorescence tag to the molecule, that being plasmids. 5 pg of gWiz-GFP plasmids were mixed with 2.5 pL of Cy3, 5 pL of Labeling Buffer A (provided in the Kit), and 37.5 pL of water to make a final volume of 50 pL. The mixture solution was then incubated at 37 °C for 1 h. Cy3 -tagged plasmids were then purified using ethanol precipitation. Figure 13B shows that the loading efficiencies of these plasmids were 79.4±22.5% for SiNWs which is comparable to the efficiencies of plasmids loaded onto flat Si substrates ( 81.2 ± 17.8%) Example 3: Preparation of cell cultures

Four types of cell lines, GPE86 (ATCC, mouse embryonic fibroblasts), LI .2 (ATCC, mouse B cells), Jurkat (ATCC, human CD4 ⁺ T cells), Ramos (ATCC, human B cells), primary human B cells (isolated from human tonsils, approved by the Australian National University’s Human Research Ethics Committee and the University Hospitals Institutional Review Board), and primary mouse T cells (isolated from mouse lymph nodes, approved by the Australian National University’s Animal Experimentation Ethics Committee) were investigated in this study. GPE86 cells were grown and maintained in complete Dulbecco’s modified Eagle’s medium (DMEM (Gibco), supplemented with 10% fetal bovine serum (FBS, Gibco), 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U mL ^_1 penicillin, and 100 pg mL ^_1 streptomycin (Gibco). LI .2, Jurkat, and Ramos cells were grown and maintained in complete RPMI (RPMI-1640 (Gibco), consisting of 10% FBS, 10 mM HEPES, U non-essential amino acids solution (Gibco), 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U mL ^_1 penicillin, 100 pg mL ^_1 streptomycin, and 55 pM 2-mercaptoethanol (Gibco)). Primary human B cells were grown and maintained in complete RPMI supplemented with lipopolysaccharide (LPS; 1 pg mL ^_1 ), anti-CD40 (5 pg mL ^_1 ) and interleukin (IL)-4 (20 ng mL ^_1 ). Primary mouse T cells were grown and maintained in complete RPMI supplemented with anti-mouse CD3 (5 pg mL ^_1 ), anti mouse CD28 (5 pg mL ^_1 ) and IL-2 (20 ng mL ^_1 ). All cells were incubated at 37 °C with 5% CO2.

Example 4: SiNW-mediated Cell Transfection

20,000 cells/well of GPE86 and HEK293, 40,000 cells/well of LI .2, Jurkat, and Ramos, and 100,000 cells/well of primary B and T cells were seeded onto PDL-coated and cargo-loaded substrates described above in 48-well plate, in 250 pL Opti-MEM (Gibco), followed by centrifugation at 200 g, 32°C, 15 min. After centrifugation and 6 h incubation at 37°C in a humidified 5% CO2 atmosphere, substrates carrying cells were gently rinsed, and transferred to new plates. The transfected cells were then detached from the substrates using 0.25% Trypsin/EDTA (Gibco) (adherent cells) or by gently pipetting (suspension cells), and cultured with fresh DMEM or RPMI media (complete DMEM for GPE86 cells, and complete RPMI for LI .2, Jurkat, Ramos, primary B and T cells) for 24-48 h. Example 5: Silicon NW Arrays

A library of silicon NW arrays were prepared using a combination of nanosphere lithography or electron-beam lithography with deep reactive ion etching (DRIE) as described above.

Referring to Figure IB, a silicon NW array, a representative cross-sectional SEM image of a silicon NW array. Scale bar, 2 pm. Average dimensions of SiNWs used in this study: tip diameter 100 nm, length 3.2 pm, and density 0.3 NWs pm ^-2 . Referring to Figure 11 A, cross sectional SEM images of various other SiNW arrays fabricated by nanosphere lithography and DRIE are provided. SiNW dimensions: (i) diameter: 95 nm, length: 3 pm and density: 2.5 NWs pm ^-2 ; (ii) diameter: 60 nm, length: 2.9 pm and density: 0.3 NWs pm ^-2 ; (iii) diameter: 165 nm, length: 2.6 pm and density: 0.3 NWs pm ² ; (iv) diameter: 195 nm, length: 3.2 pm and density: 0.3 NWs pm ^-2 ; (v) diameter: 530 nm, length: 10 pm and density: 0.15 NWs pm ^-2 ; (vi) diameter: 130 nm, length: 10.3 pm and density: 0.15 NWs pm ^-2 .

Unless otherwise stated, the silicon NWs used in the following cell viability and transfection studies had the following average dimensions: tip diameter 100 nm, length 3.2 pm, and density 0.3 NWs pm ^-2 .

Example 6: Preparation of Polymeric NWs

Referring to Figure 1 IB, the SU8 (an epoxy based photoresist polymer, Micro Chem) and OROMOCOMP (hybrid polymer made by micro-resist technology) and polystyrene (PS; Petri dishes, Falcon, and tissue culture well plate, Eppendorf) NWs were fabricated by nanoimprint of SU8, ORMOCOMP, or PS with a h-PDMS/s-PDMS composite (Gelest, Inc.; Sylgard 184, Sigma Aldrich) negative master mold fabricated from the silicon NWs template. PDMS nanowires were fabricated by casting of PDMS (Sylgard 184, Sigma Aldrich) utilised the same negative master mold.

SU8 (i) NW dimension: diameter: <250 nm, length: 3 pm and density: 0.75 NWs pm ^-2 . PS (ii) NW dimension: diameter: <250 nm, length: 3 pm and density: 0.75 NWs pm ^-2 . PDMS (iii) NW dimension: diameter: <350 nm, length: 3 pm and density: 0.75 NWs pm ^-2 . ORMOCOMP (iv) diameter: <250 nm, length: 3 pm and density: 0.75 NWs pm ^-2 .

Fabrication of Negative Master Mold

Silicon NWs template was cleaned by piranha solution and (3 : 1 H2SO4 : H2O2 v/v, 75 °C, Avantor Performance Materials) for 15 min to remove any organic contaminants, then washing with water and drying under a nitrogen jet. The template was placed in air plasma cleaner (Harrick Plasma) for 5 min and silanised by 15 pL of Trichloro(lH, 1H, 2H, 2H - perfluoro-octly)silane (Sigma Aldrich) in vapour phase for 15 min. h-PDMS (Gelest, Inc.) was spin coated on silicon nanowires template with 500 rpm for 3 s, then 3000 rpm for 1 min. It was left to cure in 60 °C oven for 30 min. This was followed by depositing a s-PDMS (Sylgard 184, Sigma Aldrich) layer on top of h-PDMS and further cured in 60 °C oven for 2 h. At this point, the negative master mold can be removed from the silicon template. The negative master mold was silanised in preparation for replicating polymeric nanowires.

Casting and Nanoimyrint of the SU8 and ORMOCOMP Nanowires

Glass substrate was rinsed by acetone, isopropanol (Sigma-aldrich) and de ionised water, then dried under nitrogen jet and placed in air plasma cleaner for 5 min. 5pL of SU8 (MicroChem, SU8 class 2002) or ORMOCOMP (Micro-resist technology) was cast on the glass substrate, followed by the PDMS negative master mold. Nanoimprint was performed with NX B-200 (Nanonex). 3 min pumping time with 75 °C pre-imprint temperature and 70 PSI were used to pre-condition the imprint. 95 °C and 70 PSI were used for nanoimprint with processing time of 5 min followed by 2 min of UV exposure time. The sample was cooled to 60 °C before releasing from the system. SU8 or ORMOCOMP nanowires can be removed from the template with the nanowires pattern successfully transferred.

Nanoinwrint/Hot Embossing of PS Nanowires

PS was rinsed by isopropanol (Sigma-aldrich) and de-ionised water, then dried under nitrogen jet and placed in air plasma cleaner for 5 min. PS was then sandwiched between the PDMS negative master mold and glass substrate to create a flat base for the imprint. Nanoimprint was performed with NX B-200 (Nanonex). 3 min pumping time with 170 °C pre-imprint temperature and 85 PSI were used to pre-condition the imprint. 220 °C and 85 PSI were used for nanoimprint with processing time of 5 min. The sample was cooled to 70 °C before releasing from the system. PS nanowires can be removed from the template.

Casting of PDMS Nanowires

PDMS solution (1 part of curing agent with 10 parts of PDMS base solution) was used to cast onto the prepared negative master mold with glass substrate. The sample was placed in vacuum to cure overnight to ensure maximum solution infiltration into the negative master mold. The sample was then cured in 60 °C oven for 30 min and can be removed from the negative master mold, obtaining PDMS nanowires on glass substrate.

Sharpening of the SU8, ORMOCOMP , and PS Nanowires

The as prepared SU8, ORMOCOMP, and PS nanowires can be further sharpened by applying oxygen plasma (Plasmalab system 100, OXFORD Instruments) with lOOsccm oxygen flow rate and 50W RF power under 14.5mTorr chamber pressure. The sharpening of different types of polymeric nanowires is controlled by the etching time.

Example 7: Cell viability after Detachment from Silicon NWs

The viability of cells cultured on SiNWs were followed and their proliferation was tracked up to three days after detaching from SiNWs. Neglectable difference was observed in the viability of GPE86, LI.2, and Jurkat cells, after 6 h culture on flat Si and SiNWs (Figure 4). Briefly, when compared to a control flat silicon substrate (i.e. no NWs), negligible difference was observed in the viability of cells after incubation for 6 h on silicon NWs having a density of 0.3 NWs per pm ² , length 3.2 pm, diameter 100 nm) (see Figure 1A and 4). Maintaining cell viability was surprising especially as the cells were penetrated by one or more of the silicon NWs, breaching the cell membrane and in some cases penetrating the nucleus, as seen in Figure 5.

Example 8: Cell Proliferation after Detachment from Silicon NWs

To investigate the proliferative capacity post NW-culture, GPE86, LI.2, and Jurkat cells were first labeled with a proliferation tracking dye, CellTrace Violet (CTV), before seeding onto SiNWs. After 6 h incubation, the cells were detached from SiNWs either by trypsinisation (GPE86) or gentle pipetting (LI.2 and Jurkat). Harvested cells were then cultured back in fresh media and analysed by flow cytometry on Days 2 and 3. Because CTV covalently binds to intracellular proteins in the cytoplasm and nucleus, and halves in amount with each round of cell division, the proliferation rate of cells is negatively correlated with CTV fluorescence intensity. CTV unstained cells (CTV-) and CTV stained cells (CTV ⁺ , fixed in FACS buffer after 6 h incubation) worked as negative and positive controls, respectively.

Among the three cell types, LI.2 cells were most prolific on Days 2 and 3, indicated by their lower CTV fluorescence intensity (Figure 6a). On Day 3, CTV intensity in LI.2 cells was reduced almost to the level of the negative control. Although reduction of LI.2 cells harvested from SiNWs lagged slightly compared with that from control flat Si, as measured by CTV intensity, the percentage of cells undertaking proliferation was very similar for both substrates (99.5 ± 0.1%; Figure 6b). On Day 2, GPE86 cells demonstrated less CTV intensity and therefore reduced proliferation (56.4 ± 2.8%) compared with LI .2 cells. But CTV intensity dropped continuously from Days 2 to 3, indicating ongoing proliferation (reaching 80.5 ± 3.6% on Day 3). No significant difference was observed in CTV intensity and proliferation rate between GPE86 cells harvested from SiNWs and flat Si. Jurkat cells showed the lowest proliferation rate. But similar to GPE86 cells, the CTV intensity of Jurkat cells continued to decrease from Days 2 to 3, with the proliferation percentage increasing from 23.0 ± 5.8% to 64.6 ± 0.4%.

These results confirm that cells harvested from the NWs demonstrated ongoing proliferation, confirming that cells can be successfully detached and thus harvested from the silicon NWs whilst maintaining their proliferative capacity. The proliferation rate also reflects the ability of the three cell types to recover from the culture on SiNWs. GPE86 and particularly Jurkat cells required longer time to recover than LI .2 cells after culturing on SiNWs, suggesting a higher sensitivity of Jurkat cells towards cellular deformations caused by SiNWs.

To test whether SiNWs induce any unexpected damage, Jurkat cells were harvested at different incubation times (2, 6, 12, and 24 h) and then stained with the annexin V-FITC/propidium iodide (PI), to probe apoptosis/necrosis pathways. Flow cytometry results (Figure 6c and d) showed that over 95% of Jurkat cells maintained viability (annexin V/PE) after 2, 6, and 12 h culture on both SiNWs and flat Si, with less than 5% becoming apoptotic (annexin V ⁺ /PE); this was consistent with the viability result achieved by live/dead (FDA/PI) staining in situ on SiNWs (Figure 12). After 24 h incubation, the rate of apoptosis (annexin V ⁺ /PI ) increased to up to 10% for cells on SiNWs, more than double of that on flat Si. The population of dead/necrotic cells (annexin V ⁺ /PI ⁺ ) remained negligible, 0.83 ± 0.1% and 0.74 ± 0.2% on flat Si and SiNWs respectively, indicating that SiNWs did not induce necrotic cell death of Jurkat cells. Overall, SiNWs induced minimal necrosis and negligible apoptosis for up to 12 h.

Example 9: Silicon NW Mediated Transfection of Plasmids into Immortalised Cell Lines

Having demonstrated that SiNWs induce membrane disruption and nuclear perturbation, while preserving cell viability and proliferative capacity after detaching and harvesting from the SiNWs, the delivery of plasmids into various cell lines was tested. Flow cytometry techniques were used to detect the fluorescence in the transfected and then detached cells. Cy3-tagged plasmids (Cy3-gWiz-GFP) were added onto flat Si and SiNWs at three distinct concentrations: 10, 50, and 100 ng pL ^_1 . LI .2 cells were seeded onto the substrates, with or without the application of centrifugal external force. Cells were incubated for 6 h before detaching from the substrates by gentle pipetting. Harvested cells were stained with Hoechst and PI, and analysed by means of flow cytometry to measure the Cy3 intensity within viable (Hoechst ⁺ /PL) population (Figure 7a and b).

The results showed no Cy3 ⁺ population in LI .2 cells cultured on flat Si, demonstrating no plasmid insertion at all three tested concentrations. Similar results were obtained for the other cell types. However, for LI .2 cells cultured on SiNWs coated with plasmids at 10 and 50 ng pL ^_1 , an observed increase in Cy3 ⁺ population from 0.3 ± 0.2 to 5.3 ± 1.8%. When exposed to further increased plasmid concentration (from 50 to 100 ng pL ^_1 ), no further enhancement in plasmid uptake was observed in LI .2 cells settled on SiNWs by gravity alone (Figure 7a center and 7b). By contrast, when applying external centrifugal force to strengthen the cell-SiNW interaction, the percentage of LI .2 cells gaining Cy3-tagged plasmids rose more than three times from 6.9 ± 3.1% for 50 ng pL ¹ to 22.3 ± 7.0% for 100 ng pL ^_1 (Figure 7a right, and 6b).

Also observed in Figure 7, when applying external centrifugal force to enhance NW penetration of the cells (e.g. with spin), the percentage of LI .2 cells uptaking Cy3- tagged plasmids increased compared to cells that were incubated on NWs with no centrifugal force applied (e.g. without spin).

After validating the insertion of plasmids into LI .2 cells mediated by SiNWs, the efficiency of reporter gene expression among all three cell types was investigated by measuring the GFP intensity. GPE86, LI .2, and Jurkat cells were cultured on flat Si and SiNWs coated with untagged plasmids (gWiz-GFP). After centrifugation and 6 h incubation, cells were harvested from the SiNWs, returned to fresh media, and cultured for another 48 h to allow for the expression of GFP. Flow cytometry analysis was performed to determine the fluorescence intensity of GFP within these cells. Corresponding to the null plasmid insertion described above, control flat Si rendered no GFP ⁺ population for any of the three cell types (Figure 7c and 7d). For cells harvested from SiNW-mediated transfection, LI .2 cells exhibited the highest transfection efficiency, with 25.4 ± 5.3% GFP ⁺ population, followed by GPE86 cells, with 21.4 ± 3.3% and Jurkat cells, with 5.2 ± 0.7%.

Example 10: Silicon NW Mediated Delivery of Nucleic Acids into Primary Immune Cells and Generation of CAR ⁺ Cells ex Vivo The efficiency of transfecting nucleic acids into hard-to-transfect primary immune cells was investigated. The transfection of‘TOX’ siRNA (siTOX) - a highly toxic RNA sequence that induces cell death - using SiNWs led to 75.8 ± 9.2% and 90.7 ± 2.8% cell death of Ramos and primary human B cells, respectively (Figure 13 A), when normalised to those transfected with negative control ΌTR’ siRNA (siOTP). These results are in line with previous studies that have shown high transfection efficiency of small siRNAs into both primary B cells and T cells via SiNWs.

Example 11: Silicon NW Mediated Delivery of Plasmids into Primary Immune Cells and Generation of CAR ⁺ Cells ex Vivo

The delivery of larger gene constructs (e.g. plasmids) into primary immune cells, in particular T cells, was investigated. Such delivery may induce and modulate the T cells specific anti-tumour responses, such as in chimeric antigen receptor T cell immunotherapy. Inactivated T cells isolated from wildtype mice were transfected with Cy3-gWiz-GFP plasmids coated on flat Si and SiNWs. By detecting the Cy3 expression through flow cytometry, it was determined that 32.8 ± 0.4% of primary mouse T cells obtained Cy3-gWiz-GFP plasmids from SiNWs after 6 h incubation, whereas only less than 1% of Cy3 ⁺ T cells were observed from flat Si (Figure 9a and 9b). Additionally, the 6 h interfacing period caused almost no alteration to the expression level of key surface activation markers and major inflammatory cytokines within T cells harvested from SiNWs. This indicates that the majority of T cells remained at naive stage after SiNWmediated transfection (see Figure 9e).

To further investigate the transfection efficiency in terms of GFP expression, the T cells were detached from flat Si and SiNWs and cultured in fresh media for another 48 h. The result showed that 30.7 ± 2.2% of T cells harvested from SiNWs became Cy3 ⁺ GFP ⁺ , indicating the preservation of Cy3-gWiz-GFP plasmids within the transfected T cells and their expression of GFP reporter gene after 48 h (Figure 9c and 9d).

Example 12: Silicon NW Mediated Delivery of a CAR Construct into Inactivated Primary T-cells

Transfection of inactivated primary mouse T cells with a Cy3-tagged CAR construct, which encodes human CD 19 receptor (Cy3 -CD 19-CAR) was investigated. After 6 h incubation on Cy3-CD19-CAR-coated flat Si and SiNWs, T cells were detached and put back in in fresh media for another 48 h (see Figure 10). By staining with FITC- conjugated human CD19 antibody and analysing via flow cytometry, we detected 5.7 ± 1.1% and 15.8 ± 1.0% Cy3 ⁺ CD19 ⁺ T cells from flat Si and SiNWs, respectively, demonstrating the significantly higher transfection efficiency and expression of Cy3- CD19-CAR construct within T cells via SiNWs.

This data validates the use of SiNWs as a promising platform for transfecting siRNAs into primary immune B cells, and plasmids including CAR constructs, into inactivated primary T cells.

Overall, high transfection efficiency and good cell viability has been achieved in small primary immune cells using the SiNW’s of the present invention (diameter 100 nm, length 3.2 pm, and density 0.3 NWs pm ^-2 ) . Shorter NWs (e.g. less than 3.5 pm) are often used to maintain cell viability and/or provide improved transfection. The current results represent a surprising advantage.

Example 13: Functionalisation of Silicon NWs using Linker and Molecule Mixture

Referring to Figure 8, higher transfection efficiency was achieved on primary T cells incubated for 6 h on silicon NWs functionalised via the addition of a poly-D-lysine and Cy3-plasmid (PDL-Plas) mixture compared with cells incubated for 6 h on NWs functionalised via the stepwise addition of poly-D-lysine followed by the Cy3-plasmid. For example, referring to Figure 8a and b, the percentage of primary T cells gaining Cy3- tagged plasmids after 6 h incubation on silicon NWs functionalised with a linker- molecule mixture (e.g. PDL-Plas mix) was about 30%, more than three times that for cells incubated on silicon NWs coated stepwise with linker then molecule (PDL ¹ + Plasmid ² ).

Without wishing to be bound by theory, it is believed that the linker and molecule are coupling spontaneously in the mixture, e.g. to for example to form a polyplex, namely a positively charged complex as a result of condensation by the electrostatic interactions between the linker (e.g. PDL) and the molecule (e.g. plasmid), which upon addition to the NWs results in a higher number of molecules attached to the NW surface.. For example, owing to the linker size, the positive amino side chains of poly-D-lysine may couple to numerous negatively charged plasmid, thus coupling multiple plasmid molecules per linker molecule, for example in the form of a polyplex. Adding this mixture to the NW surface may therefore result in a higher % loading efficiency of plasmid molecules compared with stepwise addition, where the linker attaches to the NW surface first via various possible points (e.g. via amine or carboxylic acid), thus reducing the number of possible linking sites available for molecules to attach.

Example 14A: Cell-Silicon NW Interface To study the interfacial interactions of cells with SiNWs, three cell types were examined: GPE86, LI .2, and Jurkat cells (see Figure 14). Substrates were coated with positively charged poly-D-lysine (PDL). Such adhesion-mediating molecule coating serves a twofold purpose: to promote cell adhesion and to bind plasmid DNA (. All three cell types were seeded onto PDL-treated substrates. To achieve forcible interfacing and tight intracellular access, a controlled external force via centrifugation-induced gravity (200 g, -3.92 nN, 32°C, 15 min) was applied. After 6 h incubation, all three cell types were fixed and stained with fluorescence markers (phalloidin for F-actin and Hoechst for the nuclei) to assess the cell-SiNW interface via confocal florescence microscopy (Figure 14a-c) and scanning electron microscope (SEM) (Figure 14d).

Cellular adhesion is considered a crucial process for development and maintenance of a functional cell-SiNW interface, which is known to promote cell penetration. Figure 14a-c are representative fluorescence confocal images demonstrating the forcible interfacing achieved between SiNWs and GPE86 (a), LI .2 (b), and Jurkat (c) cells. The individual SiNW elements appear as black dots within all three cell types (marked as white circles in merged channel), indirectly suggesting the assisted impalement of SiNWs into the cell body (nucleus and cytoplasm) by applied centrifugal force.

Multiple SiNWs were noted to interact with each cell type (Figure 14a-c, merged channel). In parallel, selected tilt-view SEM images illustrate the cellular morphology and adhesion of the plated cells on the SiNWs (Figure 14d). Adherent GPE86 cells displayed mainly a flattened and more elongated morphology, generating long lamellipodia with filopodial protrusions anchored on the tips of SiNWs (Figure 14d ii). Short lamellipodia with filopodial protrusions were also observed on suspension-type Jurkat cells (Figure 14d vi), whereas LI .2 cells largely maintained their globular shape (Figure 14d iv).

To better understand the cell proteins and complexes that participate in focal adhesion on SiNWs, the distribution of F-actin (actin filament), ?-integrin (transmembrane receptor), and vinculin (membrane-cytoskeletal protein) was examined, which are reported to play important roles in mediating cell adhesion to the extracellular matrix (ECM). Figure 15a-c display the fluorescence staining of GPE86, LI .2, and Jurkat cells cultured on SiNWs. Interestingly, GPE86 cells formed ring-shaped adhesion complexes, assembled mainly by vinculins rather than F-actin or ?-integrin (Figure 15a). Increased vinculin clustering indicates the potential for creating strong focal adhesion of GPE86 cells on the SiNWs substrate. The observation of long lamellipodia with filopodial protrusions along SiNWs in SEM (Figure 15d ii) supports this view. For LI .2 cells, vinculins were observed to be colocalised with the multilobed nucleus. While F- actin was found across the whole cell, it was mainly the transmembrane receptor b- integrin that accumulated along the short filopodial protrusions (Figure 15b), facilitating the adhesion of LI .2 cells on SiNWs. Similar small‘feet’ made by ?-integrins were also found on the cell surface of Jurkat cells, whereas vinculins were mainly distributed in the perinuclear region and F-actin accumulated at the membrane site (Figure 15c). Formation of small Lintegrin protrusions on LI .2 and Jurkat cell surfaces, instead of ring-shaped vinculin complexes around the SiNWs, at least partially explains why suspension cells largely maintain globular shape and are less embedded onto SiNWs compared with their adherent counterparts.

To further investigate the alterations of cell focal adhesions over an extended period and their influence on cell motility on SiNWs, confocal live cell imaging to record the migration trajectory of all three cell types on SiNW substrates was performed (Figure 15d to f) over 60 h. The mean migration lengths of GPE86, LI .2, and Jurkat cells were 142.9, 295.5, and 251.9 pm, respectively (Figure 15g). For the larger adherent GPE86 cells, though some nuclei performed little movement, their cell membrane displayed high fluidity and plasticity as evidenced by the creation of long and diverse protrusions at different orientations along the SiNWs.

Taken together, a functional cell-SiNW interface forms using the silicon NW arrays, where the cells can form focal adhesions on SiNWs within 6 h after plating and exhibit cell motility over 60 h.

Example 14B: Cell-Polymer NW Interface

To study the interfacial interactions of cells with polymeric NWs, two cell types were examined: adherent GPE86 fibroblast cells and non-adherent Jurkat cells. Polymeric NW substrates were placed in air plasma for 5 min and immersed in 70% ethanol and allowed to dry at room temperature for 2 h in a laminar flow cabinet. Next, the substrates were coated with 10 pL of PDL (Sigma- Aldrich) at the concentration of 167 pg mL ¹ in FLO under vacuum for 5 min. The substrates were then transferred to 4 °C environment and incubated for 1 h. Excessive PDL solution was aspirated before cell seeding following protocols outlined in Example 4 for SiNWs.

Multiple pNWs were noted to interact with each cell type (Figure 11C a-d). Similar to the cell interaction observed on SiNWs (see Example 4; Figure 14d), adherent GPE86 cells displayed mainly a flattened and more elongated morphology, generating long lamellipodia with filopodial protrusions anchored on the tips of pNWs (Figure 11C b). Short lamellipodia with filopodial protrusions were also observed on suspension-type Jurkat cells (Figure 11C d).

Example 15: Cellular Deformations Induced by Silicon NWs

To reveal the actual types of interaction at the cell-SiNW interface, two complementary characterisation techniques were used: confocal microscopy and focus ion beam (FIB)-SEM imaging.

The SiNWs were labelled with a fluorescent dye (fluorescein isocyanate; FITC), making the NWs visible under the confocal microscope. GPE86, LI .2, and Jurkat cells were seeded onto the FITC-labeled SiNWs. After short-term centrifugation and 6 h incubation, cells were fixed and stained with Hoechst and phalloidin, representing the nucleus and F-actin, respectively. Figure 16 is 3D and 2D slice views of the compiled z- stack confocal images; they show that some SiNWs can breach the cell membrane of a GPE86 cell, entering the cytoplasm and further perturbing the nucleus (this is revealed by the absence of phalloidin and Hoechst staining in the corresponding positions of SiNWs that are illustrated in white). Similar results were obtained for the LI .2 and Jurkat cells.

FIB-SEM imaging was used as a complementary method to unveil more details of the cell-SiNW interface at a higher nanoscale resolution. Samples were prepared through heavy-metal staining followed by resin embedding, to enhance the contrast between SiNWs, cell membrane, and intracellular compartments under the FIB-SEM. FIB milling was performed either at 90° or at 45° to the sample surface to investigate the cell-SiNW interface from different perspectives. In Figure 5, multiple interacting routes were observed even within the same single cell-SiNW interface. Figure 5a presents two distinct patterns by which two SiNWs interact with a GPE86 cell. One SiNW is shown to clearly breach the cell membrane and further perturb the nucleus, while the second SiNW is engulfed by a continuous intact plasma membrane. Figure 5b manifests SiNW insertion into the cytoplasm of a LI .2 cell; Figure 5c displays a SiNW perturbing the cell membrane and nucleus of a Jurkat cell. Sequential SEM imaging of the FIB sectioning at 45° provides additional insights into the cell-SiNW interactions. Figure 5d i show clear evidence that the SiNWs (yellow arrows) directly penetrate into both the cytoplasm and nucleus of the LI .2 cell. By contrast, Figure 5d ii demonstrates that most SiNWs were engulfed by the intact cell membrane of the Jurkat cell. The softness of Jurkat cells, attributed to a smaller size of actin meshwork and less stress fibers, may act to enhance membrane invagination rather than penetration by SiNWs. The observation by confocal and FIB-SEM suggests that direct penetration through membrane and nucleus might not be the prevalent, at least not the only, mechanism behind SiNW-mediated intracellular delivery. To understand whether other mechanisms, such as endocytosis, are involved, the role of two endocytotic markers was investigated: clathrin heavy chain (CHC), a clathrin coat protein involved in CME; and caveolin-1 (CAV-1), a key protein for CavME. Plasmids tagged with a Cy3 fluorescence dye (Cy3-gWiz-GFP) were coated onto flat Si and SiNWs. Confocal microscopy images in Figure 5e demonstrate the distribution of CAV-1 and CHC within GPE86 cells after 6 h interfacing with plasmid-coated SiNWs. Compared to the random localisation of CAV- 1 in all three cell types on flat Si, CAV-1 highly colocalised with Cy3-gWiz-GFP plasmids coated on SiNWs (Figure 5e). But the clustering of CHC around SiNWs was less obvious than that of CAV-1, indicating caveolae are the key elements that promote endocytosis during cell-SiNW interaction.

These results demonstrate that SiNWs induce cellular deformations, which lead to direct membrane penetration as well as accumulation of functional endocytotic vesicles, particularly caveolae, which play a crucial role in CavME. The synergy of both mechanisms may contribute to the efficient intracellular delivery through SiNWs.

Example 16: Correlation between Transfection Efficiency with SiNW Geometry

The transfection efficiency is correlated to the SiNW geometry (e.g. length/height). Figure 17a demonstrates flow cytometry analysis of GFP expression within HEK293 cells 48 h after detachment from pGFP-coated flat Si and SiNW arrays with varying geometrical characteristics (e.g. varying the SiNW arrays diameter, length (i.e. height) and density) as summarized in Figure 17e.

Quantification of the percentage of GFP ⁺ cells in Figure 17a is depicted in Figure 17c. For example, referring to SiNW arrays #92 and #88, SiNWs that have the same density and diameter but are shorter in length/height (e.g. #92: 3.2 pm length) exhibit higher transfection efficiency than corresponding longer SiNWs (e.g. #88: 3.5 pm length). Correlation plots between SiNW geometrical characteristics (density, diameter, and height) and transfection efficiency are included in Figure 17f-h. Referring to Figure 17h, in some embodiments, the transfection efficiency strongly depends on SiNW height. As seen in Figures 17b,d. HEK293 cells demonstrated proliferation capacity 2 days after being harvested from all 6 SiNW samples. Example 17: Characterisation of Polymeric NWs by Laser Scanning Confocal Microscopy

Laser scanning confocal microscopy was used to characterise and control the quality of fabricated polymeric NWs. Referring to Figure 18, uniform polystyrene (PS) NWs ( were prepared having a length (height) of 3.5 pm and a pitch of 3 pm.

Example 18: Cell Viability on Polymeric NWs

The viability of Jurkat cells cultured on both flat and NW SU8, ORMOCOMP, and PS NWs were close to 100%, as seen in Figure 19 a-c, e.

Example 19: Delivery of ssDNAs into L1.2 Cells by Polymeric NWs

Transfection experiments were performed by delivering FAM-tagged ssDNAs (FAM-ssDNAs) into LI .2 cells using PDL-coated ORMOCOMP, SU8, and PS NWs. The first experiment was to investigate the insertion of ssDNAs into cells after a 6 h interfacing period. Cells were spun down by 250 g-force in centrifuge after seeding onto the pNWs. A control sample consisted of cells without ssDNA transfection was included to set the baseline for FACS analysis. Referring to Figure 20a, flow cytometry analysis showed the ssDNA insertion into LI .2 cells after 6 h incubation. The insertion percentage for ORMOCOMP, PS, and SU8 samples were 9%, 4%, and 20%, respectively, demonstrating successful transfection across each pNW array.

Example 20: Delivery of mRNAs into Suspension Cells by SiNWs and Polymeric NWs

The confocal microscopy images of LI .2 cells on SiNW substrates showed significant signals of both Cy5 and GFP after 6 h of interfacing (Figure 21), indicating the insertion of Cy5-tagged GFP-encoding mRNA (Cy5-mRNA-GFP) into LI .2 cells and that they started expressing GFP within 6 h. Lower signals of Cy5 and GFP signals were observed on SU8 NW samples (Figure 22).

The Cy5-GFP-mRNA delivery experiment was repeated on Jurkat cells, with the inclusion of 5 polymeric (PDMS, ORMOCOMP, 2 types of PS: PS1 and PS2, and SU8) substrates and Si substrates (flat vs NWs). All samples were coated with PDL under vacuum before the loading of mRNAs. Results of the FACS analysis are shown in Figure 23a. Cy5 insertion efficiency ranging from 5.5 % to 18.7 % was observed among samples, with no GFP expression in LI .2 cells. PS 2 and PDMS substrates demonstrated high Cy5 insertion efficiency (18.7 % and 15.6 % respectively) (Figure 23b). It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

The present application claims priority from AU 2019902302 filed 28 June 2019, the entire contents of which are incorporated herein by reference.

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