Transfection of cells on three-dimensional cell constructs with surface-capped protein nanoparticles

Abstract

A method of manipulating cell genetics includes transferring a genetic payload from at least one non-viral nanoparticle capable of penetrating tissue to at least one cell growing and supported on a three-dimensional construct by contacting the nanoparticle with the at least one cell. The transferring occurs without disassociating or breaking up the three-dimensional construct. Also provided is a genetic cell manipulation system, including a three-dimensional cell construct configured to support and promote three-dimensional growth of cells. Also, at least one non-viral nanoparticle is provided that is configured to penetrate tissue. The nanoparticle defines a core region comprising a water-soluble protein and a genetic payload and has a peripheral surface comprising a positively-charged capping agent disposed thereon. The nanoparticle is configured to deliver the genetic payload to at least a portion of the cells disposed on the three-dimensional cell construct, without disassociating or breaking up the three-dimensional construct.

Description

Attorney Docket No. 2115-008429-WO-POATRANSFECTION OF CELLS ON THREE-DIMENSIONAL CELL CONSTRUCTS WITH SURFACE-CAPPED PROTEIN NANOPARTICLESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 727,835, filed on December 4, 2024. The entire disclosure of the above application is incorporated herein by reference.FIELD

[0002] The present disclosure relates to methods of manipulating cell genetics and systems for genetic manipulation of cells proliferating on three-dimensional constructs with at least one non- viral nanoparticle, which may be a surface-capped protein-based nanoparticle.BACKGROUND

[0003] This section provides background information related to the present disclosure which is not necessarily prior art.

[0004] Autologous cell therapy is a rapidly growing therapeutic modality that includes immuno-oncology and regenerative medicine applications. Non-viral vectors offer a more affordable, simpler, and effective alternative to viral transmission. However, one significant issue in non-viral autologous cell therapy is the number of viable dosed cells, which is low using current approaches. For example, electroporation is commonly used for genetic manipulation of cells, such as DNA transfection, in autologous cell therapy research.

[0005] Electroporation generally involves an external electrical field being applied, for example, as a pulse of energy, to the cells to be treated to create openings in the cell membrane that temporarily increases permeability. In this manner, genetic manipulation agents, such as DNA, can be transferred into the cell via the permeable cell membrane. While electroporation typically has a high transfection efficiency, is easy to apply, and has a short duration for the procedure, it also has significant disadvantages. For example, electroporation presents a significant risk to cell viability, resulting in a very low survival rate post-procedure, even when the transfection efficiency exceeds 90%. This poses a significant technical challenge in autologous cell therapy, where the number of viable dosed cells is crucial. Moreover, where cells are grown on three-dimensional tissue constructs, electroporation cannot achieve a high transfection efficiency, because of closely packed structure of the cells growing. Thus, where electroporation is used to transfect DNA into cells supported and growing on a three-dimensional cell construct,Attorney Docket No. 2115-008429-WO-POA the cells must be detached using chemical or biological reagents, which can disassociate, disintegrate, or break-up the three-dimensional cell construct while also leading to gene retention problems in the transfected cells. It would be desirable to manipulate cell genetics with a high efficiency rate, while increasing viability of cells that further can remain intact and growing on a three-dimensional cell construct.SUMMARY

[0006] This section provides a general summary of the disclosure, and is not a comprehensive disclosure of its full scope or all of its features.

[0007] In certain aspects, the present disclosure relates to a method of manipulating cell genetics. The method optionally comprises transferring a genetic payload from at least one non- viral nanoparticle capable of penetrating tissue to at least one cell growing and supported on a three-dimensional construct. The transferring occurs by contacting the at least one non-viral nanoparticle with the at least one cell. Further, the transferring occurs without disassociating or breaking up the three-dimensional construct.

[0008] In one aspect, the at least one non-viral nanoparticle is selected from the group consisting of: a lipid nanoparticle, a protein nanoparticle, a polymeric nanoparticle, and combinations thereof.

[0009] In one aspect, the at least one non-viral nanoparticle comprises a core region comprising a water-soluble protein and the genetic payload. The core region defines a peripheral surface that comprises a positively-charged capping agent disposed thereon. The transferring further comprises delivering the genetic payload from the core region to an interior of the at least one cell, while the cell remains disposed within the cellular support system.

[0010] In one further aspect, the positively-charged capping agent is selected from the group consisting of: polyethylene imine (PEI), polylysine, polyarginine, and combinations thereof.

[0011] In one further aspect, the water-soluble protein is selected from the group consisting of albumin, ovalbumin, mucin, transferrin, insulin, lysozyme, hemoglobin, collagen, catalase, horseradish peroxidase, glucose oxidase, and combinations thereof.

[0012] In one further aspect, the water-soluble protein comprises albumin.

[0013] In one further aspect, the at least one non-viral nanoparticle is pH responsive, so that the at least one non-viral nanoparticle is stable outside the cell, but after passing through a cell membrane of the cell, at least partially degrades at pH inside the cell to release the polynucleotide agent.Attorney Docket No. 2115-008429-WO-POA

[0014] In one aspect, the at least one non- viral nanoparticle is not crosslinked and is free of added crosslinking agents.

[0015] In one aspect, the nanoparticle is formed by electrohydrodynamic jetting.

[0016] In one aspect, the genetic payload comprises a polynucleotide agent selected from the group consisting of: DNA, RNA, plasmids, plasmid DNA (pDNA), short interfering sequence of double stranded RNA (siRNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, small nuclear RNA, single stranded DNA, microDNA, guide RNA, CRISPR CAS-9, aptamers, and combinations thereof.

[0017] In one aspect, the three-dimensional cell construct is selected from the group consisting of: a tissue explant, an organoid, a three-dimensional printed cell structure, and combinations thereof.

[0018] In one aspect, the three-dimensional construct is a three-dimensional cell scaffold comprising a plurality of voids. The non- viral nanoparticle has a particle size permitting passage through the plurality of voids. The genetic payload comprises a polynucleotide agent, where the at least one nanoparticle comprises a core region comprising a water-soluble protein and the polynucleotide agent. The core region defines a peripheral surface comprising a positively- charged capping agent disposed thereon. The transferring further comprises delivering the polynucleotide agent from the core region to an interior of the cell, while the cell remains disposed within the three-dimensional cell scaffold.

[0019] In one aspect, the three-dimensional cell scaffold further comprises at least one protein-based fiber spanning each void of the plurality of voids, wherein the protein-based fiber comprises an extracellular matrix protein selected from the group consisting of: laminin, fibronectin, and a combination thereof.

[0020] In one aspect, the transferring occurs without any viral vectors or electroporation.

[0021] In one aspect, the at least one cell comprises a plurality of cells supported and growing on the three-dimensional construct and the at least one non-viral nanoparticle comprises a plurality of non-viral nanoparticles. Thus, the transferring of the genetic payload occurs in bulk from at least a portion of the plurality of non-viral nanoparticles to at least a portion of the plurality of cells.

[0022] In certain other aspects, the present disclosure relates to a cell genetic manipulation system. The cell genetic manipulation system may comprise a three-dimensional cell construct configured to support and promote three-dimensional growth of cells. Further, at least one non- viral nanoparticle is configured to penetrate tissue. The at least one non-viral nanoparticle defines a core region comprising a water-soluble protein and a genetic payload. The core region defines aAttorney Docket No. 2115-008429-WO-POA peripheral surface comprising a positively-charged capping agent disposed thereon. The at least one non-viral nanoparticle is configured to deliver the genetic payload to at least a portion of the cells disposed on the three-dimensional cell construct, without disassociating or breaking up the three-dimensional construct.

[0023] In one aspect, the at least one non-viral nanoparticle is configured to deliver the genetic payload from the core region to an interior of a respective cell of the portion of cells, while the cells remain disposed on the three-dimensional cell construct.

[0024] In one aspect, the at least one non-viral nanoparticle is pH responsive, so that the at least one non-viral nanoparticle is stable outside the respective cell, but after passing through a cell membrane of the cell, at least partially degrades at pH inside the respective cell to release the genetic payload.

[0025] In one aspect, the positively-charge capping agent is selected from the group consisting of: polyethylene imine (PEI), polylysine, polyarginine, and combinations thereof.

[0026] In one aspect, the water-soluble protein is selected from the group consisting of albumin, ovalbumin, mucin, transferrin, insulin, lysozyme, hemoglobin, collagen, catalase, horseradish peroxidase, glucose oxidase, and combinations thereof.

[0027] In one aspect, the water-soluble protein comprises albumin.

[0028] In one aspect, the at least one non-viral nanoparticle is not crosslinked and is free of crosslinking agents.

[0029] In one aspect, the nanoparticle is formed by electrohydrodynamic jetting.

[0030] In one aspect, the genetic payload comprises a polynucleotide agent selected from the group consisting of: DNA, RNA, plasmids, plasmid DNA (pDNA), short interfering sequence of double stranded RNA (siRNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, small nuclear RNA, single stranded DNA, microDNA, guide RNA, CRISPR CAS-9, aptamers, and combinations thereof.

[0031] In one aspect, the three-dimensional cell construct is selected from the group consisting of: a tissue explant, an organoid, a three-dimensional printed cell structure, and combinations thereof.

[0032] In one aspect, the three-dimensional construct is a three-dimensional cell scaffold comprising a plurality of voids. The non-viral nanoparticle has a particle size configured to permit passage through the plurality of voids. The genetic payload comprises a polynucleotide agent, such that the polynucleotide agent is transferred from the core region to an interior of the cell, while the cell remains disposed within the three-dimensional cell scaffold.Attorney Docket No. 2115-008429-WO-POA

[0033] In one aspect, the three-dimensional cell scaffold further comprises at least one protein-based fiber spanning each void of the plurality of voids, wherein the protein-based fiber comprises an extracellular matrix protein selected from the group consisting of: laminin, fibronectin, and a combination thereof.

[0034] In one aspect, the genetic cell manipulation system is free of any viral vectors or electroporation.

[0035] In one aspect, the at least one non-viral nanoparticle comprises a plurality of non- viral nanoparticles. The transferring of the genetic payload occurs in bulk from at least a portion of the plurality of non-viral nanoparticles to at least a portion of the cells disposed on the three-dimensional cell construct.

[0036] Further areas of applicability will become apparent from the description provided herein. The description and specific examples in this summary are intended for purposes of illustration only and are not intended to limit the scope of the present disclosure.DRAWINGS

[0037] The drawings described herein are for illustrative purposes only of selected embodiments and not all possible implementations, and are not intended to limit the scope of the present disclosure.

[0038] FIG. 1 shows a genetic cell manipulation system for transferring a genetic payload from non-viral nanoparticles to cells proliferating on a three-dimensional construct according to certain aspects of the present disclosure.

[0039] FIGS. 2A-2E. FIG. 2 A shows a schematic diagram of fabrication of non-viral nanoparticles comprising a protein-based core having a genetic payload and surface-capped with a positively-charged molecule (scPNP) formed by electrohydrodynamic (EHD-jetting) in accordance with certain aspects of the present disclosure. FIG. 2B shows an SEM image of scPNPs formed by the process in FIG. 2 A. FIG. 2C shows a size histogram of scPNPs in the SEM image of FIG. 2B. FIG. 2D shows a DLS spectrum of scPNPs, where I and N represent intensity and number respectively. FIG. 2E shows a zeta view spectrum of scPNPs.

[0040] FIGS. 3A-3F show morphologies of cells before a conventional electroporation process (FIGS. 3A, 3D), after electroporation (FIGS. 3B, 3E), and after 2 days of recovery (FIGS. 3C, 3F). FIGS. 3A, 3B, and 3C represent cells treated with 7.5 pg / ml of DNA, while FIGS. 3D, 3E, and 3F represent cells treated with 100 pg / ml of DNA.

[0041] FIGS. 4A-4H show dependency of cell transfection on the concentration of seeding cells and the dosing amount of nanoparticles prepared in accordance with certain aspectsAttorney Docket No. 2115-008429-WO-POA of the present disclosure (surface-capped protein nanoparticles (scPNPs)). FIG. 4A shows uptake of scPNPs as a function of the concentration of scPNPs. FIG. 4B shows transfection efficiency as a function of the concentration of scPNPs, measured via flow cytometry analysis of green fluorescent protein (GFP) signal. FIGS. 4C-4F show flow cytometry analysis of GFP signal in cells demonstrating transfection efficiency with varying doses of scPNPs. FIG. 4E shows an untreated control. FIG. 4F shows a concentration of 5.55 x 106scPNPs / microliters. FIG. 4G shows 11.11 x 106scPNPs / microliters. FIG. 4H shows 16.66 x 106scPNPs / microliters. FIG. 4G shows transfection efficiency of scPNP-treated cells as a function of seeding cell density, determined by flow cytometry analysis. Data represent the mean transfection efficiency ± SEM (n=3 independent experiments). FIG. 4H shows an event count of GFP-positive signals as a function of seeding cell density assessed by flow cytometry.

[0042] FIGS. 5A-5F show transfection efficiency and uptake of nanoparticles prepared in accordance with certain aspects of the present disclosure (surface-capped protein nanoparticles (scPNPs)) as a function of repetitive addition using 3x scPNP solution. FIG. 5A shows transfection efficiency of scPNP-treated cells as a function of the number of repetitive injections, measured by flow cytometry analysis of GFP signal. Data are presented as mean ± SEM (n=3 independent experiments). FIG. 5B shows flow cytometry determined the uptake of scPNPs with varying numbers of repetitive injections. FIGS. 5C-5F shows flow cytometry analysis of GFP signal in cells demonstrating transfection efficiency with repetitive injections of scPNPs (3x). FIG. 5C shows a non-treated control, FIG. 5D shows a one-time injection of scPNPs, FIG. 5E shows two- times injection of scPNPs, and FIG. 5F shows three-times injection of scPNPs.

[0043] FIGS. 6A-6H show transfection efficiency and uptake of nanoparticles prepared in accordance with certain aspects of the present disclosure (surface-capped protein nanoparticles (scPNPs)) as a function of DNA content. FIG. 6A shows transfection efficiency of scPNPs with varying DNA content, determined by flow cytometry analysis of GFP signal. Data are presented as mean ± SEM (n=3 independent experiments). FIG. 6B shows uptake of scPNPs with varying DNA content, assessed by flow cytometry. FIGS. 6C-6D show confocal microscopy images showing: FIG. 5C shows non-treated control cells, while FIG. 6D shows scPNP-treated cells. DAPI stains cell nuclei (blue), and green fluorescence indicates GFP signal. Insets show low- magnification images of each sample; scale bars: 50 micrometers (pm - main images) and 500 pm (insets). FIGS. 6E-6G show flow cytometry analysis of transfection efficiency of scPNPs with varying DNA content. FIG. 6E shows a non-treated control, while FIG. 6F shows 10 wt. % DNA content; (g) 20 wt. % DNA content. FIG. 6H shows cell viability as a function of DNA content,Attorney Docket No. 2115-008429-WO-POA determined by CCK-8. Data are presented as mean ± SEM (n=3 independent experiments). Statistical significance is indicated as determined by one-way ANOVA(p < 0.05).

[0044] FIGS. 7A-7E show scalability of transfection on scaffolds using nanoparticles prepared in accordance with certain aspects of the present disclosure (surface-capped protein nanoparticles (scPNPs)). 324 mm2-sized 3D EECM scaffolds were used, and the scPNPs were treated with the same concentration of DNA. FIG. 7A shows fluorescence microscope image of non-treated control scaffold. FIG. 7B shows fluorescence microscope image of the scPNP-treated scaffold, with a magnified region designated by the square. Green fluorescence indicates the GFP signal. FIG. 7C shows flow cytometry data of non-treated control cells. FIG. 7D shows flow cytometry data of the scPNP-treated cells. FIG. 7E shows viability of cells post-treatment, measured by Euna cell counter with trypan blue exclusion method.

[0045] FIG. 8 is a table showing a comparison of electroporation transfection versus on-scaffold transfection via nanoparticles prepared in accordance with certain aspects of the present disclosure (surface-capped protein nanoparticles (scPNPs)).

[0046] Corresponding reference numerals indicate corresponding parts throughout the several views of the drawings.DETAILED DESCRIPTION

[0047] Example embodiments are provided so that this disclosure will be thorough, and will fully convey the scope to those who are skilled in the art. Numerous specific details are set forth such as examples of specific compositions, components, devices, and methods, to provide a thorough understanding of embodiments of the present disclosure. It will be apparent to those skilled in the art that specific details need not be employed, that example embodiments may be embodied in many different forms and that neither should be construed to limit the scope of the disclosure. In some example embodiments, well-known processes, well-known device structures, and well-known technologies are not described in detail.

[0048] The terminology used herein is for the purpose of describing particular example embodiments only and is not intended to be limiting. As used herein, the singular forms “a,” “an,” and “the” may be intended to include the plural forms as well, unless the context clearly indicates otherwise. The terms “comprises,” “comprising,” “including,” and “having,” are inclusive and therefore specify the presence of stated features, elements, compositions, steps, integers, operations, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. Although the open-ended term “comprising,” is to be understood as a non-restrictive term used to describe andAttorney Docket No. 2115-008429-WO-POA claim various embodiments set forth herein, in certain aspects, the term may alternatively be understood to instead be a more limiting and restrictive term, such as “consisting of’ or “consisting essentially of.” Thus, for any given embodiment reciting compositions, materials, components, elements, features, integers, operations, and / or process steps, the present disclosure also specifically includes embodiments consisting of, or consisting essentially of, such recited compositions, materials, components, elements, features, integers, operations, and / or process steps. In the case of “consisting of,” the alternative embodiment excludes any additional compositions, materials, components, elements, features, integers, operations, and / or process steps, while in the case of “consisting essentially of,” any additional compositions, materials, components, elements, features, integers, operations, and / or process steps that materially affect the basic and novel characteristics are excluded from such an embodiment, but any compositions, materials, components, elements, features, integers, operations, and / or process steps that do not materially affect the basic and novel characteristics can be included in the embodiment.

[0049] Any method steps, processes, and operations described herein are not to be construed as necessarily requiring their performance in the particular order discussed or illustrated, unless specifically identified as an order of performance. It is also to be understood that additional or alternative steps may be employed, unless otherwise indicated.

[0050] When a component, element, or layer is referred to as being “on,” “engaged to,” “connected to,” or “coupled to” another element or layer, it may be directly on, engaged, connected or coupled to the other component, element, or layer, or intervening elements or layers may be present. In contrast, when an element is referred to as being “directly on,” “directly engaged to,” “directly connected to,” or “directly coupled to” another element or layer, there may be no intervening elements or layers present. Other words used to describe the relationship between elements should be interpreted in a like fashion (e.g., “between” versus “directly between,” “adjacent” versus “directly adjacent,” etc.). As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items.

[0051] Although the terms first, second, third, etc. may be used herein to describe various steps, elements, components, regions, layers and / or sections, these steps, elements, components, regions, layers and / or sections should not be limited by these terms, unless otherwise indicated. These terms may be only used to distinguish one step, element, component, region, layer or section from another step, element, component, region, layer or section. Terms such as “first,” “second,” and other numerical terms when used herein do not imply a sequence or order unless clearly indicated by the context. Thus, a first step, element, component, region, layer or section discussedAttorney Docket No. 2115-008429-WO-POA below could be termed a second step, element, component, region, layer or section without departing from the teachings of the example embodiments.

[0052] Spatially or temporally relative terms, such as “before,” “after,” “inner,” “outer,” “beneath,” “below,” “lower,” “above,” “upper,” and the like, may be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the figures. Spatially or temporally relative terms may be intended to encompass different orientations of the device or system in use or operation in addition to the orientation depicted in the figures.

[0053] Throughout this disclosure, the numerical values represent approximate measures or limits to ranges to encompass minor deviations from the given values and embodiments having about the value mentioned as well as those having exactly the value mentioned. Other than in the working examples provided at the end of the detailed description, all numerical values of parameters (e.g., of quantities or conditions) in this specification, including the appended claims, are to be understood as being modified in all instances by the term “about” whether or not “about” actually appears before the numerical value. “About” indicates that the stated numerical value allows some slight imprecision (with some approach to exactness in the value; approximately or reasonably close to the value; nearly). If the imprecision provided by “about” is not otherwise understood in the art with this ordinary meaning, then “about” as used herein indicates at least variations that may arise from ordinary methods of measuring and using such parameters. For example, “about” may comprise a variation of less than or equal to 5%, optionally less than or equal to 4%, optionally less than or equal to 3%, optionally less than or equal to 2%, optionally less than or equal to 1%, optionally less than or equal to 0.5%, and in certain aspects, optionally less than or equal to 0.1%.

[0054] In addition, disclosure of ranges includes disclosure of all values and further divided ranges within the entire range, including endpoints and sub-ranges given for the ranges.

[0055] Example embodiments will now be described more fully with reference to the accompanying drawings.

[0056] The present disclosure contemplates methods of manipulating cell genetics and cell genetic manipulation systems. The manipulating of genetics of one or more cells may include transfection, which refers to the introduction of a polynucleotide agent, such as nucleic acids, delivered into eukaryotic cells, for example, into animal cells. In the past, the term transfection was used to denote the uptake of viral nucleic acid from a prokaryote-infecting virus or bacteriophage, which would result in an infection and production of mature virus particles.Attorney Docket No. 2115-008429-WO-POAHowever, transfection in the present context encompasses various processes where foreign polynucleotides, such as nucleic acid, are artificially introduced into a cell.

[0057] In certain aspects, the present disclosure contemplates methods of manipulating cell genetics, for example, in a transfection process, where a genetic payload is transferred from at least one non- viral nanoparticle capable of penetrating tissue to at least one cell growing and supported on a three-dimensional construct. The transferring occurs of the genetic payload by contacting the at least one non- viral nanoparticle with the at least one cell. As will be described further below, at least a portion of the nanoparticle may be transferred into and enter the cell, where the genetic payload is released. The nanoparticle is desirably capable of penetrating tissue, which includes a collection of cells having a common structure or function, meaning that the nanoparticles can contact and penetrate at least portions of the tissue to access one or more cells and thus release its genetic payload to one or more cells. As discussed above, the collection of cells may be growing and supported on the three-dimensional cell construct. In accordance with various aspects of the present disclosure, the transferring occurs without disassociating, disintegrating, destroying, or breaking up the three-dimensional cell construct, in other words, the three-dimensional construct remains largely structurally intact. In this manner, the cells that are genetically manipulated may remain on or near the three-dimensional construct to continue growing and proliferating after the transfer process.

[0058] FIG. 1 shows an example of a cell genetic manipulation system, where a genetic payload is transferred from non-viral nanoparticles to cells proliferating on a three-dimensional construct according to certain aspects of the present disclosure. One example of a three- dimensional cell construct is shown. The three-dimensional cell construct is a structure configured to support and promote cell growth, especially three-dimensional growth, of cells. “Promoting” cell growth, cell proliferation, cell differentiation, cell repair, or cell regeneration means that a detectable increase occurs in either a rate or a measurable outcome of such processes when the cellular support system is present as compared to a cell or organism’s process in the absence of the cellular support system, for example, conducting such processes naturally. By way of example, as appreciated by those of skill in the art, promoting cell growth in the cellular support system may increase a growth rate of target cells or increase a total cell count of the target cells, when compared to cell growth or cell count of the target cells in the absence of such a cellular support system. By “supporting” cell growth, cell proliferation, cell differentiation, cell repair, or cell regeneration, it is meant that the cellular support system provides a physical substrate for one or more target cells that enhances target cell growth, vitality, proliferation, differentiation, repair, or regeneration, by way of non-limiting example. As appreciated by those of skill in the art, theAttorney Docket No. 2115-008429-WO-POA cellular support system may both support and promote the growth, proliferation, vitality, differentiation, repair, and / or regeneration processes of one or more target cells in vitro, ex vivo, or in vivo, for example. The cellular support system thus can serve a role as a cellular scaffold structure that supports and / or promotes target cell growth, target cell proliferation, target cell differentiation, target cell repair, and / or target cell regeneration in three-dimensions, in contrast to the support and growth on conventional two-dimensional planar or two-dimensional scaffold surfaces.

[0059] By way of non-limiting example, such a three-dimensional cell construct may include structures or scaffolds, for example, a tissue explant, an organoid, and / or a manufactured cellular support structure, such as a three-dimensional printed or additively manufactured cellular support structure. In certain aspects, the three-dimensional scaffold structure may be used for genetic manipulation of cells or tissue comprising cells ex vivo or in vitro. In other aspects, the three-dimensional construct or scaffold structure may be an implantable device that is used in vivo and thus introduced into a subject, such as a human. In certain variations, a three-dimensional construct may be a three-dimensional scaffold structure comprising a plurality of openings or voids. In certain variations, a plurality of protein-based fibers span each void. Various three- dimensional scaffold structures configured to support and promote three-dimensional growth of cells and are described in commonly owned U.S. Patent No. 11,479,753 to Ramcharan, et al., U.S. Publication No. 2023 / 0357710 to Lahann, et al., and WO 2024 / 238935 to Kim, et al. the relevant portions of each of which are incorporated herein by reference.

[0060] For example, as shown in FIG. 1, a cellular support system includes a three- dimensional scaffold structure comprising a plurality of voids, which is labeled as a three- dimensional engineered extracellular matrix (3D EECM). The cellular support system may further include a suspended protein bridge spanning across the at least one void in the three-dimensional scaffold structure. Voids may include pores, surface features, holes, openings, roughness, or topography, by way of example. The shape of the voids or pores is not limited, but may be any number of shapes including those having a cross-sectional shape of a square, a circle, a rectangle, an oval, a parallelogram, a triangle, or any other regular or irregular cross-sectional or three- dimensional shape. The overall shape of the three-dimensional construct or scaffold structure may be of any shape, including customized shapes and sizes, and is not correlated with the shape of the void(s).

[0061] The suspended protein bridge is capable of supporting cells and promotes three- dimensional cellular growth. The combination of these two components forms a solid, three-dimensional protein matrix that mimics the extracellular matrix as deposited by cells in vivo,Attorney Docket No. 2115-008429-WO-POA hence referred to as the three-dimensional engineered extracellular matrix (3D EECM). The 3D EECM scaffold structure has a plurality of interior voids that have a representative rectangular shape in a regular repeating three-dimensional mesh pattern; however, as noted above the voids are not limited to these shapes or positions and may have different shapes and arrangements within the scaffold structure. The scaffold structure includes walls that define and surround each void. Each void has at least one suspended protein bridge spanning from and anchored to a first interior surface to a second interior surface across each void. As shown, each void has multiple suspended protein bridges within each void, for example, each void may have one bridge to thousands of bridges per void, for example, in certain variations, from greater than or equal to about 10 bridges to less than or equal to about 1,000 bridges. In certain aspects, the suspended protein bridges may be a plurality of suspended bridge structures numerous enough to form a suspended protein mesh across the void.

[0062] In various aspects, the three-dimensional scaffold structure may be formed of a biocompatible material. By “biocompatible,” it is meant that a material or combination of materials can be contacted with cells and tissue in vitro, ex vivo, or in vivo, or used with mammals or other organisms and has acceptable toxicological properties for contact and / or beneficial use with such cells, tissue, and / or animals. For instance, in certain aspects, a biocompatible material may be one that is suitable for implantation into a subject without adverse consequences, for example, without substantial toxicity or acute or chronic inflammatory response and / or acute rejection of the material by the immune system, for instance, via a T-cell response. It will be recognized, of course, that “biocompatibility” is a relative term, and some degree of inflammatory and / or immune response is to be expected even for materials that are highly compatible with living tissue. However, non-biocompatible materials are typically those materials that are highly toxic, inflammatory and / or are acutely rejected by the immune system, e.g., a non-biocompatible material implanted into a subject may provoke an immune response in the subject that is severe enough such that the rejection of the material by the immune system cannot be adequately controlled, in some cases even with the use of immunosuppressant drugs, and often can be of a degree such that the material must be removed from the subject. In certain aspects, biocompatible materials are those that are approved for use in humans by an appropriate regulatory agency, such as the Federal Drug Administration (FDA) in the United States; the European Commission (EC) / European Medicines Agency (EMEA) in Europe; or Health Products and Food Branch (HPFB) in Canada.

[0063] In certain variations, the scaffold structure is formed of a biodegradable material, while in other variations; the scaffold structure is formed of a non-biodegradable material. AAttorney Docket No. 2115-008429-WO-POA biodegradable material may dissolve or disintegrate ex vivo or in vivo. “Dissolving” refers to physical disintegration, erosion, disruption and / or dissolution of a material and may include the resorption of a material by a living organism. Dissolution or erosion occurs when the material is exposed to a solvent comprising a high concentration of water, such as growth or culture media, serum, blood, saliva, bodily fluids, and the like. As noted above, the three-dimensional tissue scaffold is preferably formed of a material that does not dissolve during the process of transferring the genetic materials to the cells on the tissue scaffold, but may dissolve or degrade on a longer time scale. In certain aspects, the three-dimensional scaffold structure optionally comprises a combination of biocompatible materials, like a combination of polymer materials.

[0064] The material forming the scaffold structure may include a biofunctional active ingredient that is released as the biodegradable material dissolves or disintegrates. Biofunctional active ingredients or agents may include pharmaceutical active ingredients, proteins, peptides, growth factors, biofactors, imaging agents by way of non-limiting example. The biofunctional active ingredient may be dispersed within the material that forms the scaffold. Inclusion of a biofunctional active ingredient or agent may be preferred where the scaffold material is biodegradable thus allowing the release of various compounds of interest such as pharmaceuticals or imaging agents.

[0065] The three-dimensional scaffold structure can be made of a wide variety of materials, including inorganic and organic biocompatible materials. The three-dimensional scaffold structure may be formed from a material selected from the group consisting of: a polymeric material, a composite material (having a polymeric material and a reinforcement material), a metal material, a ceramic material, a glass material, a ceramic material, a biologically- derived material (a material derived from a biological source, such as cellulose or paper), and combinations thereof and need not be uniform or homogeneous throughout the scaffold structure (e.g., there may be distinct regions of the scaffold with different compositions). Certain polymeric and composite materials may be biodegradable, while other polymeric, composite, and metal materials are not biodegradable. Specifically, biocompatible polymer materials, such as biodegradable or non-biodegradable polymers, synthetic or natural polymers can be used.

[0066] By way of example, suitable polymers include polyethers, such as a polyethylene oxide (PEO), polyoxyethylene glycol or polyethylene glycol (PEG), biodegradable polymers such polyesters like polylactic acid, polycaprolactone, polyglycolic acid, poly(lactide-co-glycolide polymer (PLGA), poly(lactide-co-caprolactone), and copolymers, derivatives, and combinations thereof. Suitable water-soluble and / or hydrophilic polymers, which are biocompatible, include cellulose ether polymers like hydroxypropyl methyl cellulose (HPMC), hydroxypropyl celluloseAttorney Docket No. 2115-008429-WO-POA(HPC), hydroxyethyl cellulose (HEC), methyl cellulose (MC), carboxymethyl cellulose (CMC), and combinations thereof. Various polysaccharides include starches such as maltodextrin, amylose, com starch, potato starch, rice starch, tapioca starch, pea starch, sweet potato starch, barley starch, wheat starch, modified starch (e.g., hydroxypropylated high amylose starch), and the like.

[0067] In certain variations, the three-dimensional scaffold structure comprises a polymer and is formed from a polymeric precursor or is a polymer selected from the group consisting of: polylactic acid, polyglycolide, polycaprolactone, poly(lactide-co-glycolide), poly(lactide-co- caprolactone), polyethylene glycol, polydimethylsiloxane, polyurethanes, polyolefins, polyamides, celluloses, lignins, starches, biodegradable polyesters, polystyrene, and combinations thereof.

[0068] Other water-soluble polymers among those useful herein include, without limitation, sodium alginate, carrageenan, xanthan gum, gum acacia, Arabic gum, guar gum, pullulan, agar, chitin, chitosan, pectin, karaya gum, locust bean gum, various polysaccharides; starches such as maltodextrin, amylose, com starch, potato starch, rice starch, tapioca starch, pea starch, sweet potato starch, barley starch, wheat starch, modified starch (e.g., hydroxypropylated high amylose starch), dextrin, levan, elsinan and gluten; and proteins such as collagen, whey protein isolate, casein, milk protein, soy protein, keratin, and gelatin.

[0069] The choice of the scaffold structure material can vary based upon the intended application of the cell culture system. When used to facilitate three-dimensional culture of adherent cells, polymeric materials may be advantageous by providing a softer mechanical environment. These include, without limitation, polylactic acid, polyglycolide, polyethylene glycol, polycaprolactone, starches, biodegradable polyesters, co-polymers such as poly(lactide- co-glycolide) and poly(lactide-co-caprolactone), and non-degradable materials such as polystyrene. If intended for long-term use, or as an implant with intention of surgical removal, a metal scaffold may be preferred for rigidity, stability, and lack of degradability.

[0070] In certain aspects, the three-dimensional scaffold structure may be custom-made or commercially available. Scaffolds can be custom made with desired opening sizes and shape. For polymeric scaffolds, fabrication options include without limitation, solid free form fabrication, additive manufacturing (e.g., 3D printing, 3D jet writing, direct writing), and extrusion. Metal scaffolds, again without limitation, can be milled, machined, photochemically etched, or formed via additive manufacturing (e.g., direct metal laser sintering). In certain aspects, three-dimensional scaffold structures may be formed via photolithography from a suitable commercially available resist SU-8 sold by MicroChem, which is a negative tone photoresist high contrast epoxy basedAttorney Docket No. 2115-008429-WO-POA material. In other aspects, the three-dimensional scaffold structure may be formed from nonwoven electrospun polymeric microfiber mats. In another aspect, three-dimensional scaffold structure may include an array of micropillars. Another suitable commercially available three- dimensional scaffold structure further includes a mesh filter screen commercially available from Component Supply. In other certain variations, the three-dimensional scaffold structure may include a non-woven fiber mat, a woven fiber mat, or paper substrate. Other suitable three- dimensional scaffold structures include a porous hydrogel or Matrigel™ natural ECM-based hydrogel.

[0071] As shown in FIG. 1, the cellular support system also includes at least one suspended protein bridge comprising at least one protein spanning across the at least one void in the three-dimensional scaffold structure. A protein, as used herein, is a polypeptide chain comprising bonded amino acids, where the polypeptide chain has undergone folding (including primary, secondary, and tertiary folding) to form the complex folded molecule. In certain aspects, the protein has greater than 50 amino acids. The protein for forming the suspended bridge within the variation shown in FIG. 1 may be initially dissolved as a solubilized protein in a liquid. The dissolved protein may be transformed into an insoluble protein bridge, notably without the need for use of cells to do so. The final product of the suspended protein bridge may comprise an insoluble molecule formed into a complex folded shape. The complexly folded molecule is insoluble in water and aqueous solutions, preferably is fibrillar, and is also biologically active to cells in the environment.

[0072] The proteins selected for use in the cellular support system are those proteins that can form suspended protein bridges across or within the voids / pores of the aforementioned three- dimensional scaffold structure. Different proteins can be combined to form suspended protein supports of varying composition. Importantly, proteins that do not form suspended protein supports can be combined with / co-assembled with proteins that do form suspensions to create bridge structures containing multiple proteins. Thus, the composition forming the protein bridge may comprise a plurality of distinct proteins.

[0073] In certain aspects, the suspended protein bridge comprises one or more extracellular proteins, such as one or more extracellular matrix proteins. An extracellular matrix protein is one or more of the large structural fibrillar proteins often found physiologically in the extracellular matrix (ECM) of animals or plants. In certain variations, the suspended protein bridge comprises one or more proteins selected from the group consisting of: fibronectins, collagens, laminins, tenascins, elastin, vitronectin, periostin, and combinations thereof. In other variations, the suspended protein bridge comprises one or more proteins selected from the groupAttorney Docket No. 2115-008429-WO-POA consisting of: fibronectins, collagens, laminins, and combinations thereof. In certain aspects, the at least one extracellular matrix protein comprises fibronectin. The suspended fibril may further comprise one or more proteins in addition to fibronectin, for example, those selected from the group consisting of: collagens, laminins, tenascins, elastin, vitronectin, periostin, and combinations thereof. In certain variations, the suspended fibril further comprise collagen, such as Type I collagen.

[0074] As noted above, the suspended protein bridges can include mixtures of such proteins, each at different relative ratios. The protein material forming the suspended protein bridges may also form a surface coating on one or more regions of the interior surfaces of the scaffold structure voids / pores. The coating may serve to structurally and mechanically anchor the suspended protein bridge to an interior surface of the void of the scaffold structure. In this manner, the suspended protein bridge is capable of spanning regions of the void while supporting the additional weight of cells that adhere to it.

[0075] The suspended protein bridge thus provides cells with an ECM-like network of protein which is susceptible to remodeling by the cells, allowing for cell migration, proliferation, and metastasis. When cells secrete insoluble proteins to form their microenvironment, they are also revealing biologically active cryptic binding sites on the protein that are otherwise inaccessible to cells when the protein is solubilized. In this variation, because the proteins present in the protein bridge are insoluble, the cryptic binding sites can be revealed despite being fully- defined and cell-free.

[0076] In certain other variations, the suspended protein bridges may further comprise other components, such as a glycan. The glycan may be associated or conjugated with the extracellular matrix protein within the fibril (suspended protein bridge), for example, associating via weak forces or bonded together (referred to herein as “decorating” the extracellular matrix protein with a glycan). For example, in certain variations, a controlled disulfide conjugation strategy occurs at fibronectin (Fnlll) with minimally modified (e.g., thiol functionalized) hyaluronic acid (HA). In certain aspects, a fibronectin fibril assembly can be induced with hydrodynamic fibrillogenesis that is not believed to be reliant on domain interactions or disulfide bonds involving Fnlll. In this manner, the at least one suspended fibril or bridge is created that supports cells and promotes three-dimensional cellular growth.

[0077] The at least one glycan may comprise a glycosaminoglycan. In certain variations, the glycosaminoglycan comprises hyaluronic acid (HA). It may be functionalized, for example, with a thiol-reactive group prior to forming the fibrillary network on the three-dimensional support, as will be described further below.Attorney Docket No. 2115-008429-WO-POA

[0078] With renewed reference to FIG. 1, the three-dimensional construct or tissue scaffold structure (3D EECM) may be seeded with cells, by way of non-limiting example, with CCL-247 cells. After the cells have grown on the three-dimensional tissue scaffold to a sufficient extent and are proliferating and capable of uptake, the genetic manipulation process may be initiated. Thus, one or more non-viral nanoparticles comprising a genetic payload and capable of penetrating tissue (including a plurality of cells typically having the same structures and / or functions) may be introduced to the cells growing on the three-dimensional tissue scaffold with suspended protein bridges.

[0079] In various aspects, the methods of the present disclosure contemplate using at least one non-viral nanoparticle having a genetic payload to be delivered to a cell on the three- dimensional cell construct, while the cell remains intact on or within the three-dimensional cell construct. Such a nano-particle may be selected from the group consisting of: a lipid nanoparticle, a protein nanoparticle, a polymeric nanoparticle, and combinations thereof. In certain variations, like the one shown in FIG. 1, the nanoparticle is protein-based and defines a core region comprising a water-soluble protein and the genetic payload, where the core region further defines a peripheral surface comprising a positively-charged or cationic capping agent disposed thereon. The positively-charged capping agent disposed around the peripheral surface of the core region (for example, defining a continuous or discontinuous “shell” region) is selected from the group consisting of: polyethylene imine (PEI), polylysine, polyarginine, and combinations thereof. In various aspects, the water-soluble protein is selected from the group consisting of albumin, ovalbumin, mucin, transferrin, insulin, lysozyme, hemoglobin, collagen, catalase, horseradish peroxidase, glucose oxidase, and combinations thereof. In certain variations, the water-soluble protein comprises albumin, such as human serum albumin (HSA). Notably, the positively-charged capping agent serves to stabilize the core region and nanoparticle and further can render the nanoparticles insoluble in aqueous solutions.

[0080] In certain aspects, the positively-charged capping agent renders a surface of the nanoparticle cationic, for example, having a zeta (Q potential value at a physiological neutral pH (e.g., a pH of between about 7 to about 7.5) that is greater than or equal to about 0 to less than or equal to about +50 mV, optionally greater than or equal to about +2 mV to less than or equal to about +30 mV, and in other variations greater than or equal to about +5 mV to less than or equal to about +25 mV. In other aspects, the nanoparticles or components may have a net positive charge of less than or equal to about 30 mV or optionally less than or equal to about 25 mV. In certain variations, the nanoparticle may have a net positive charge and / or a zeta potentialAttorney Docket No. 2115-008429-WO-POA corresponding to any of the values specified above. In one variation, a zeta potential may be about +20 mV at a pH of about 7.

[0081] The genetic payload included in the non- viral nanoparticle is a genetic engineering agent that is delivered to a cell and can modify one or more genes or the expression of one or more genes in at least one cell growing or supported on the three-dimensional cell construct. For example, the genetic payload may add, remove, or modify nucleic acids (e.g., DNA) of genes to result in a genetic modification to the cells. The genetic payload may comprise a polynucleotide agent that when transferred and delivered to the cell achieves genetic modification or manipulation of the cell. By way of example, polynucleotide agents may be selected from the group consisting of: DNA, RNA, plasmids, plasmid DNA (pDNA), short interfering sequence of double stranded RNA (siRNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, small nuclear RNA, single stranded DNA, microDNA, guide RNA, CRISPR CAS-9, aptamers, and combinations thereof. In certain variations, the cell DNA may be modified, for example, by using CRISPR CAS-9, whereas in other cases, a polynucleotide agent, like plasmid DNA (pDNA), can encode for generating a protein. In certain aspects, the present technology is particular advantageous for use in autologous cell therapy, which uses a patient’s own cells to mitigate immune system rejection and enhance therapeutic outcomes. Such cell therapy may be used for cancer treatment, among other treatments.

[0082] In various aspects, a non-viral nanoparticle having such components may have a diameter or major dimension that is in the nanoscale range. The non-viral nanoparticle desirably has an average particle size permitting passage through the plurality of voids in a three- dimensional cell construct and optionally of a size that nanoparticles can pass between cells in the scaffold. Thus, a nanoparticle is a material that has a variety of shapes or morphologies, however, generally has at least one spatial dimension that is less than about 1 micrometer (i.e., 1,000 nm), optionally less than about 0.75 micrometers (i.e., 750 nm), optionally less than about 0.5 micrometers (i.e., 500 nm), and in certain aspects, less than about 0.25 micrometers (i.e., 250 nm). In some instances, the nanoparticle has a least one spatial dimension that is greater than or equal to about 10 nm to less than about 300 nm (e.g., mean diameter of about 10 nm to about 300 nm or median diameter of about 10 nm to about 300 nm), optionally a diameter of greater than or equal to about 100 nm to less than or equal to about 300 nm, optionally greater than or equal to about 100 nm to less than or equal to about 200 nm, optionally greater than or equal to about 100 nm to less than or equal to about 150 nm. For example, suitable nanoparticle diameters may be about 150 nm, about 200 nm, about 250 nm, or about 300 nm. In some instances, the nanoparticle has at least one spatial dimension that is less than about 100 nm (e.g., diameter of less than 100 nmAttorney Docket No. 2115-008429-WO-POA(e.g., mean diameter of less than 100 nm or median diameter of less than 100 nm), e.g., diameter between 10 nm and 100 nm e.g., mean diameter between 10 nm and 100 nm or median diameter between 10 nm and 100 nm), e.g., about 95 nm, about 90 nm, about 85 nm, about 80 nm, about 75 nm, about 70 nm, about 65 nm, about 60 nm, about 55 nm, about 50 nm, or about 40 nm.

[0083] The nanoparticle may have a variety of geometries or morphologies, including, by way of non-limiting example, substantially round shapes, like spheres and ellipsoids / ovals, rectangles, polygons, discoids / discs, ellipsoids, toroids, cones, pyramids, rods / cylinders, and the like. In certain aspects, the nanoparticle may have a substantially round shape, such as spheres, ellipsoids, hemispheres, and the like.

[0084] In certain variations, the nanoparticle comprises a water-soluble protein that is not cross-linked and is free of any externally added cross-linking agents. The water-soluble protein may have an average molecular weight of greater than or equal to about 8 kDa and less than or equal to about 700 kDa, so long as it is water soluble. The protein may optionally have an average molecular weight of greater than or equal to about 10 kDa to less than or equal to about 400 kDa. For example, human serum albumin (HSA) has an average molecular weight of about 67 kDa.

[0085] The protein-based nanoparticle may be formed by electrohydrodynamic jetting, for example, as shown in FIG. 2A. By way of non-limiting example, electrohydrodynamic jetting enables formation of protein-based nanoparticles, including not only the protein (shown as the serum albumin), but also a genetic payload for modifying cell genetics, such as a polynucleotide agent (shown as plasmid DNA (pDNA) that is delivered to a cell and encodes for a protein, for example, green fluorescent protein (GFP)), and the positively charged surface-capping component (here the polyethylene imine (PEI). In certain aspects, the water-soluble protein is present in the core of the nanoparticle at greater than or equal to about 50% by weight to less than or equal to about 95% by weight (e.g., about 50% by weight, about 55% by weight, about 60% by weight, about 65% by weight, about 70% by weight, about 75% by weight, about 80% by weight, about 85% by weight, about 90% by weight, or about 95% by weight (e.g., dry weight) of the core of the nanoparticle), while the polynucleotide agent or other genetic payload agent is present at greater than or equal to about 5% by weight to less than or equal to about 50% by weight (e.g., about 5% by weight, about 10% by weight, about 15% by weight, about 20% by weight, about 25% by weight, about 30% by weight, about 35% by weight, about 40% by weight, about 45% by weight, or about 50% by weight (e.g., dry weight) of the core of the nanoparticle).

[0086] While a large variety of proteins can be used, in certain aspects, the protein is not cross-linked and is water-soluble. Thus, the protein may be dissolved in water or carriers that are aqueous solutions that may comprise predominantly water. In certain aspects, proteins that areAttorney Docket No. 2115-008429-WO-POA excluded from suitable proteins include transmembrane proteins, polytopic proteins that aggregate and precipitate in water, and proteins with a very high molecular weight, e.g., a molecular weight greater than 700 kDa, greater than or equal to about 750 kDa, or greater than or equal to about 800 kDa. In some instances, the protein of the nanoparticle is not laminin. In other instances, the protein is not fibronectin. In yet other instances, the protein is not laminin or fibronectin. In some instances, the protein of the nanoparticle is not a native matrix protein (e.g., not a naturally occurring extracellular matrix protein). Further, in certain aspects, small proteins with molecular weights less than 8 kDa may be avoided, such as hirudin, which is only made up of 65 amino acids and has a molecular weight of about 6.7 kDa.

[0087] In certain aspects, the water-soluble protein having the desired molecular weight is selected from the group consisting of: albumin, ovalbumin, mucin, transferrin, insulin, lysozyme, hemoglobin, collagen, and combinations thereof. It should be noted that some of these proteins have varying molecular weights, so the protein selected desirably has a molecular weight in the range discussed above to ensure capability to be electrohydrodynamically jetted in an aqueous liquid, as will be described further below.

[0088] FIG. 2A generally shows an electrified jetting process used to develop liquid jets having a nanometer- sized diameter, using electro-hydrodynamic forces. An electrohydrodynamic jetting system includes at least one source of a liquid contained in a channel that is fed to a nozzle. A syringe pump (not shown) may be used to drive the liquids into the nozzle. At the nozzle, a pendant droplet is formed of conducting liquid. The nozzle(s) are in electrical communication with a power supply that can be applied during the jetting operation. As shown, there is also an electrically conductive and grounded plate disposed below and spaced apart from the nozzle. The power supply is also in electrical communication with the plate. Thus, the droplet from the nozzle is exposed to an electric potential of a few kilovolts generated by the power supply, where the force balance between electric field and surface tension causes the meniscus of the pendent droplet to develop a conical shape, the so-called Taylor cone (not shown). Above a critical point, a highly charged liquid jet or ejected stream is ejected from an apex of the cone.

[0089] In one variation, the electric field is generated by the potential difference between nozzle(s) and plate. Typically, an electric field is formed by applying a potential difference between at least two electrodes from about 0.1 kV to about 25 kV. Various configurations of plates and geometries may be used to generate the electric field as known to those of skill in the art and are contemplated by the present disclosure. In the variation shown in FIG. 2A, the ejected stream is fragmented due to instabilities generated by the electric field, thereby forming a spray ofAttorney Docket No. 2115-008429-WO-POA droplets that form the protein-based nanoparticles. The solvents in the liquid in the ejected stream are rapidly removed (e.g., volatilized or evaporated) from the stream during the jetting process.

[0090] Morphological control can be achieved with the exemplary electric jetting formation methods described herein. Therefore, the ejected stream exiting the first nozzle as a pendant droplet can be fragmented to small droplets, instead of sustained and elongated jetting that leads to a continuous fiber. The size of the droplets can also be controlled. Such control is attained by changing either the material properties of jetting liquids or the working parameters of electrified jetting that breaks-up the jet stream. It should be appreciated, however, that the final morphology of the ejected stream is not always the same as those of the solid nanoparticle products collected on the substrates. The shape of final nanoparticles can also be controlled by a sol-gel transition process or by subsequent processing after formation by electric jetting.

[0091] Since the electrified jetting methods are related to electrohydrodynamic processes, the properties of the jetting liquid and operating parameters are interrelated. Moreover, when the jetting liquids are not one-component systems (i.e., mixtures of two or more compounds), the jetting liquid is a solution having properties governed by several parameters of the solvent and solutes. It should be appreciated that liquid properties, solution parameters, and operating parameters are related, as recognized by those of skill in the art. Relevant material properties include viscosity, surface tension, volatility, thermal and electrical conductivity, dielectric permittivity, and density. Relevant solution properties include concentrations, molecular weight, solvent mixtures, surfactants, doping agent, and crosslinking agents. Finally, relevant operating parameters include flow rate of the liquid streams, electric potential, temperature, humidity, and ambient pressure. With regard to the operating parameters, the average size and size distributions of the droplets in electrospraying with cone-jet mode also are believed to be dependent on the flow rate (e.g., pumping rate of the jetting liquid). At a fixed flow rate, one or several relatively monodisperse classes of nanocomponent diameters are formed. At minimum flow rate, the modality of the distributions and diameter of the droplet itself also show their minima. When the flow rate is changed, the electric field can be adjusted by changing either distance or electric potential between the electrodes in order to sustain a stable cone-jet mode. Higher flow rates may be accompanied by a higher electrical field applied for mass balance of jetting liquids. When the diameter of droplets is larger than desired, solvent evaporation does not fully occur before the droplets reach the collecting substrate, so the resulting droplets may be wet and flat.

[0092] As shown in FIG. 2A, a first liquid stream can be jetted through a first nozzle. The first liquid stream may comprise the water-soluble protein (e.g., serum albumin) and the polynucleotide agent (plasmid DNA (pDNA) for encoding the eGFP gene. The first liquid streamAttorney Docket No. 2115-008429-WO-POA may include water and optionally aqueous solvents. The liquid may include components that adjust pH, such as acids (e.g., acetic acid) or bases, one or more solutes, e.g., buffers. The first liquid stream can be exposed to an electric field sufficient to solidify the first liquid and form the nanoparticles. Thus, the methods provided herein may be considered to be electrified jetting, such as that disclosed by Roh et al. in “Biphasic Janus Particles With Nanoscale Anisotropy,” Nature Materials, Vol. 4, pp. 759-763 (October, 2005), as well as in U.S. Patent No. 7,767,017 to Lahann et al. The contents of each of these respective references are hereby incorporated by reference in their respective entireties. However, it should be noted that the techniques described in the Roh and Lahann et al. references pertain to polymers rather than proteins, as described herein.

[0093] After formation of the nanoparticles by using the first liquid stream, a second liquid stream can be applied through a second nozzle to stabilize the nanoparticles, thus making them insoluble in aqueous solutions. The second liquid stream may comprise the molecule e.g., polyethylene imine (PEI)) that form a surface-cap over an exposed surface of the core region that comprises the water-soluble protein and polynucleotide agent. The second liquid stream may further include water or other components, such as buffers. In this manner, the second liquid stream may be directed towards the jetted nanoparticles (formed via the electrohydrodynamic jetting of the first liquid stream) on the plate or collector. In certain aspects, mild physical agitation of the plate or collector may be conducted to remove the nanoparticles.

[0094] In various aspects, such a protein-based nanoparticle has a water-soluble protein disposed in the core that is free of any crosslinking (including self-cross-linking) and does not contain any added crosslinking agents. The positively-charged capping agent serves to stabilize the nanoparticles as they are delivered to cell. In certain aspects, the nanoparticles having the positively-charged capping agent are insoluble in aqueous systems. However, the nanoparticle may be pH responsive, so that the nanoparticle is stable outside the cell, but may at least partially degrade at pH inside a cell. For example, the nanoparticle may be capable of passing through a cell membrane of a target cell. After passing through a cell membrane and into the cell, the nanoparticle including the core region may at least partially degrade at the pH inside the cell to release the genetic payload / polynucleotide agent.

[0095] For example, in FIG. 1, at least one non- viral nanoparticle comprises a core region comprising a water soluble protein and the genetic payload, marked as purple. In accordance with certain aspects of the present disclosure, the core region defines a peripheral surface comprising a positively-charged capping agent shown in yellow disposed thereon. This is referred to as a surface-capped protein nanoparticle (ScPNP). The ScPNPs contact the cells in the three- dimensional scaffold structure and transfer the genetic payload into the cell. For example, theAttorney Docket No. 2115-008429-WO-POA transferring may include delivering the genetic payload from the core region of the nanoparticle to an interior of the at least one cell. This process occurs while the cells remain intact and disposed within the cellular support system, without any need to remove them from the three-dimensional structure, for example, by dissolving or disintegrating it, as would be necessary in traditional genetic manipulation techniques like electroporation. In this manner, the intact cells are transfected with the genetic payload, so that the genetic payload can manipulate cell genetics while the cells remain growing on the three-dimensional cell construct.

[0096] In various aspects, the present disclosure contemplates methods of modifying or manipulating cell genetics. The method may comprise delivering or transferring a genetic payload from at least one non-viral nanoparticle capable of penetrating tissue to at least one cell growing and supported on a three-dimensional construct. The transferring may occur by contacting the at least one non-viral nanoparticle with the at least one cell. Further, in accordance with various aspects of the present disclosure, the transferring occurs without disassociating with or breaking up the three-dimensional construct.

[0097] The at least one non-viral nanoparticle may be selected from the group consisting of: a lipid nanoparticle, a protein nanoparticle, a polymeric nanoparticle, and combinations thereof. In one variation, the at least one non-viral nanoparticle comprises a core region comprising a water-soluble protein and the genetic payload and the core region defines a peripheral surface comprising a positively-charged capping agent disposed thereon. The transferring further comprises delivering the genetic payload from the core region to an interior of the at least one cell, while the cell remains disposed within the cellular support system. The positively-charged capping agent is optionally selected from the group consisting of: polyethylene imine (PEI), polylysine, polyarginine, and combinations thereof. The water-soluble protein is optionally selected from the group consisting of albumin, ovalbumin, mucin, transferrin, insulin, lysozyme, hemoglobin, collagen, catalase, horseradish peroxidase, glucose oxidase, and combinations thereof. In certain aspects, the water-soluble protein comprises albumin. In one variation, the at least one non-viral nanoparticle is pH responsive, so that the at least one non-viral nanoparticle is stable outside the cell. The nanoparticle may be capable of penetrating through a cell membrane, such that after passing through a cell membrane of the cell, the nanoparticle at least partially degrades at pH inside the cell to release the polynucleotide agent. Moreover, as discussed above, the at least one non-viral nanoparticle is not crosslinked and is free of any added crosslinking agents. The nanoparticle may be formed by electrohydrodynamic jetting, as described above.Attorney Docket No. 2115-008429-WO-POA

[0098] The genetic payload may comprise a polynucleotide agent selected from the group consisting of: DNA, RNA, plasmids, plasmid DNA (pDNA), short interfering sequence of double stranded RNA (siRNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, small nuclear RNA, single stranded DNA, microDNA, guide RNA, CRISPR CAS-9, aptamers, and combinations thereof.

[0099] The three-dimensional cell construct may be selected from the group consisting of: a tissue explant, an organoid, a three-dimensional printed cell structure, and combinations thereof. In one variation, the three-dimensional construct is a three-dimensional cell scaffold comprising a plurality of voids. The non-viral nanoparticle has a particle size permitting passage through the plurality of voids. The genetic payload comprises a polynucleotide agent, wherein the at least one nanoparticle comprises a core region comprising a water-soluble protein and the polynucleotide agent and the core region defines a peripheral surface comprising a positively-charged capping agent disposed thereon. The transferring further comprises delivering the polynucleotide agent from the core region to an interior of the cell, while the cell remains disposed within the three- dimensional cell scaffold.

[0100] The three-dimensional cell scaffold may further comprise at least one protein-based fiber spanning each void of the plurality of voids, where the protein-based fiber comprises an extracellular matrix protein selected from the group consisting of: laminin, fibronectin, and a combination thereof. In accordance with various aspects of the present disclosure, the transferring of the genetic payload occurs without any viral vectors or electroporation.

[0101] The at least one cell comprises a plurality of cells supported and growing on the three-dimensional construct and the at least one non-viral nanoparticle comprises a plurality of non-viral nanoparticles. The transferring of the genetic payload occurs in bulk form to at least a portion of the plurality of non-viral nanoparticles to at least a portion of the plurality of cells. Stated in another way, the transfer of the genetic payload is a bulk transfer process, where multiple nanoparticles transfer genetic payload, like nucleotide agents, into multiple cells while the cells are intact on the three-dimensional scaffold. Thus, the present disclosure contemplates methods of using non-viral, tissue-penetrating nanoparticles, such as surface-capped protein nanoparticles (scPNPs) within a three-dimensional-engineered extracellular matrix enabling high yield gene transfer while preserving cell viability and integrity. The 3D EECM offers a natural-like environment that supports cell proliferation and reduces the need for cellular disruption, making this approach a significant advancement in gene delivery technology for cancer treatment, among others treatments and applications.Attorney Docket No. 2115-008429-WO-POA

[0102] In certain aspects, a transfection efficiency, reflecting a percentage of cells present having delivered DNA after the transfer / transfection process conducted in accordance with certain aspects of the present disclosure may be greater than or equal to about 30%, optionally greater than or equal to about 35%, optionally greater than or equal to about 40%, optionally greater than or equal to about 45%, optionally greater than or equal to about 50%, optionally greater than or equal to about 55%, optionally greater than or equal to about 60%, optionally greater than or equal to about 65%, optionally greater than or equal to about 70%, optionally greater than or equal to about 75%, optionally greater than or equal to about 80%, optionally greater than or equal to about 85%, optionally greater than or equal to about 90%, and in certain variations, optionally greater than or equal to about 95%.

[0103] In certain aspects, a cell viability, reflecting a percentage of cells still alive viable after having delivered DNA via the transfer / transfection process conducted in accordance with certain aspects of the present disclosure may be greater than or equal to about 60%, optionally greater than or equal to about 65%, optionally greater than or equal to about 70%, optionally greater than or equal to about 75%, optionally greater than or equal to about 80%, optionally greater than or equal to about 85%, optionally greater than or equal to about 90%, optionally greater than or equal to about 95%, optionally greater than or equal to about 97%, optionally greater than or equal to about 98%, and in certain variations, optionally greater than or equal to about 99%.

[0104] In yet other aspects, a yield of the process, reflecting the cell viability multiplied by the transfection efficiency, where DNA is modified in the cells via the transfer / transfection process conducted in accordance with certain aspects of the present disclosure, may be greater than or equal to about 50%, optionally greater than or equal to about 55%, optionally greater than or equal to about 60%, optionally greater than or equal to about 65%, optionally greater than or equal to about 70%, optionally greater than or equal to about 75%, optionally greater than or equal to about 80%, optionally greater than or equal to about 85%, optionally greater than or equal to about 90%, and in certain variations, optionally greater than or equal to about 95%.

[0105] By way of further example, as shown in FIGS. 1 and 2A-2E, non-viral, tissuepenetrating nanoparticles, namely surface-capped protein nanoparticles (scPNPs) are used for transfection of cells growing and supported on a 3D EECM. These scPNPs are created through electrohydrodynamic (EHD) jetting of human serum albumin (HSA), which are then capped and collected using polyethyleneimine (PEI). The serum albumin was used as the primary structural component and pDNA encoded the eGFP gene. The EDH-jetting process applied high voltage to an aqueous protein solution, forming nanodroplets that dried into protein nanoparticles on aAttorney Docket No. 2115-008429-WO-POA conductive collector. Post-jetting, PEI treatment stabilized the nanoparticles, making them insoluble in aqueous solutions. SEM imaging showed uniform, spherical particles about 100 ± 10 nm in diameter, as conformed by DLS and NTA analysis, which also indicated particle hydration. FIG. 2B shows an SEM image of scPNPs formed by the process in FIG. 2A. FIG. 2C shows a size histogram of scPNPs in the SEM image of FIG. 2B. FIG. 2D shows a DES spectrum of scPNPs, where I and N represent intensity and number respectively. FIG. 2E shows a zeta view spectrum of scPNPs. The scPNPs had a positive surface charge of about ± 20 mV.

[0106] Cells are seeded onto the 3D EECM and they are allowed to grow for one day. The cells are then treated on the scaffold with nanoparticles carrying a GFP plasmid (pDNA). As the cells proliferate, they take up the scPNPs. Preliminary results show that transfection on 3D EECM with scPNPs yielded GFP signal intensities comparable to those achieved with electroporation, while maintaining higher cell viability. For example, in certain examples, the cell manipulation system provided up to about 80% of transfection efficiency (with 10,000 seeding cells and 3 times scPNPs with 10 wt.% of pDNA). In another example, a system having 50,000 seeding cells and 3x scPNPs with 20 wt.% of pDNA), provided a transfection efficiency of greater than or equal to about 67%.

[0107] As such, on- scaffold transfection is an efficient and less harmful alternative to electroporation for DNA transfection in cell therapy applications. For example, higher numbers of cells may be transfected on the scaffold.

[0108] By way of further example, FIGS. 3A-3F show cell morphologies before, during, and after a conventional electroporation process, where FIGS. 3A-3C represent cells treated with 7.5 pg / ml of DNA and FIGS. 3D-3F represent cells treated with 100 pg / ml of DNA. FIGS. 3A and 3D show cells prior to electroporation, FIGS. 3B and 3E show after electroporation, while FIGS. 3C-3F show the cells after 2 days of recovery. As can be seen, electroporation impacts cell morphology substantially and reduces a number of viable cells.

[0109] A dependency of cell transfection on the concentration of seeding cells and the dosing amount of nanoparticles prepared in accordance with certain aspects of the present disclosure (surface-capped protein nanoparticles (scPNPs)) is explored in FIGS. 4A-4H. FIG. 4A shows uptake of scPNPs as a function of the concentration of scPNPs. FIG. 4B shows transfection efficiency as a function of the concentration of scPNPs, measured via flow cytometry analysis of green fluorescent protein (GFP) signal. FIGS. 4C-4F show flow cytometry analysis of GFP signal in cells demonstrating transfection efficiency with varying doses of scPNPs. FIG. 4E shows an untreated control. FIG. 4F shows a concentration of 5.55 x 106scPNPs / pE. FIG. 4G shows 11.11 x 106scPNPs / pE. FIG. 4H shows 16.66 x 106scPNPs / pE. FIG. 4G shows transfection efficiencyAttorney Docket No. 2115-008429-WO-POA of scPNP-treated cells as a function of seeding cell density, determined by flow cytometry analysis. Data represent the mean transfection efficiency ± SEM (n=3 independent experiments). FIG. 4H shows an event count of GFP-positive signals as a function of seeding cell density assessed by flow cytometry. A ratio of cells to delivered genes is related to treatment efficiency. To optimize conditions, experiments varied cell-to-scPNP ratios and DNA doses within a fixed scPNP and cell number. Initially, 50,000 cells were cultured and treated with difference scPNP quantities (lx, 2x, 3x). Cellular uptake increased with scPNP quantity, reaching up to about 99.16%. Higher scPNP quantities (2x and 3x) yielded transfection efficiencies of about 45.68% and 49.93%, respectively. Eower cell seeding (10,000 cells with 3x scPNPs) resulted in higher transfection efficiency (about 84.8%) compared to 50,000 cells, but the latter yielded a larger total number of transfected cells, overall.

[0110] Enhanced transfection efficiency and uptake occurs via multiple rounds of (surface-capped protein nanoparticles (scPNPs)) treatment of cells on a three-dimensional scaffold (3D EECM) as shown in FIGS. 5A-5F as a function of repetitive addition using 3x scPNP solution. FIG. 5A shows transfection efficiency of scPNP-treated cells as a function of the number of repetitive injections, measured by flow cytometry analysis of GFP signal. Data are presented as mean ± SEM (n=3 independent experiments). FIG. 5B shows flow cytometry determined the uptake of scPNPs with varying numbers of repetitive injections. FIGS. 5C-5F shows flow cytometry analysis of GFP signal in cells demonstrating transfection efficiency with repetitive injections of scPNPs (3x). FIG. 5C shows a non-treated control, FIG. 5D shows a one-time injection of scPNPs, FIG. 5E shows two-times injection of scPNPs, and FIG. 5F shows three- times injection of scPNPs.

[0111] This experiment explores ways to increase the scPNP-to-cell ratio within volume and concentration constraints by applying multiple rounds of scPNP treatments to the cells on the scaffold. Single, double, and triple dosing of scPNPs yielded transfection efficiencies of about 50.63%, about 61.7%, and 63.96%, respectively. While the increase was significant between singe and double treatments, It was less pronounced between double and triple. This could be due to limited time for GFP expression after the third dose. Nevertheless, trip dosing still improved efficiency, showcasing the benefit of sequential treatments. Despite the increase in transfection efficiency, uptake was near 100% with just a single dose, consistent with previous observations. Population analysis revealed that the highest GFP expression levels with triple scPNP treatments, indicating the greatest transfection occurred with multiple doses, demonstrating that repetitive scPNP treatments leveraging their stability can significantly enhance transfection outcomes.Attorney Docket No. 2115-008429-WO-POA

[0112] FIGS. 6A-6H show enhanced transfection efficiency and uptake of nanoparticles prepared in accordance with certain aspects of the present disclosure (surface-capped protein nanoparticles (scPNPs)) as a function of DNA content in the nanoparticles. FIG. 6A shows transfection efficiency of scPNPs with varying DNA content, determined by flow cytometry analysis of GFP signal. Data are presented as mean ± SEM (n=3 independent experiments). FIG. 6B shows uptake of scPNPs with varying DNA content, assessed by flow cytometry. FIGS. 6C- 6D show confocal microscopy images showing: FIG. 5C shows non-treated control cells, while FIG. 6D shows scPNP-treated cells. DAPI stains cell nuclei (blue), and green fluorescence indicates GFP signal. Insets show low-magnification images of each sample; scale bars: 50 pm (main images) and 500 pm (insets). FIGS. 6E-6G show flow cytometry analysis of transfection efficiency of scPNPs with varying DNA content. FIG. 6E shows a non-treated control, while FIG. 6F shows 10 wt. % DNA content; (g) 20 wt. % DNA content. FIG. 6H shows cell viability as a function of DNA content, determined by CCK-8. Data are presented as mean ± SEM (n=3 independent experiments). Statistical significance is indicated as determined by one-way ANOVA (p < 0.05). Transfection efficiency can be adjusted by both the number of scPNPs and the genetic payload dose during fabrication. The pDNA content was varied from 10 wt. % to 20 wt. % relative to serum albumin, maintaining a constant scPNP concentration (3x) and seeding with 50,000 cells. Cellular uptake of scPNPs stayed above 90%, regardless of pDNA amount, indicating that surface charge and uptake mechanism were unaffected by pDNA quantity. Transfection efficiency increased from about 50.36% to about 68.8% as pDNA content rose from 10 wt. % to 20 wt. % and confocal microscopy confirmed GFP expression in cells treated with higher pDNA scPNPs. Additionally, a CCK-8 assay showed that relative metabolic activity decreased slightly with scPNP treatment but remained higher than with electroporation indicating a milder impact on cellular health.

[0113] Transfection efficiency is also explored in a large-scale organoid as reflected in FIGS. 7A-7E, which shows scalability of transfection on scaffolds using nanoparticles prepared in accordance with certain aspects of the present disclosure (surface-capped protein nanoparticles (scPNPs)). 324 mm2-sized 3D EECM scaffolds were used, and the scPNPs were treated with the same concentration of DNA. FIG. 7A shows fluorescence microscope image of non-treated control scaffold. FIG. 7B shows fluorescence microscope image of the scPNP-treated scaffold. Green fluorescence indicates the GFP signal. FIG. 7C shows flow cytometry data of non-treated control cells. FIG. 7D shows flow cytometry data of the scPNP-treated cells. FIG. 7E shows viability of cells post-treatment, measured by Luna cell counter with trypan blue exclusion method.Attorney Docket No. 2115-008429-WO-POA

[0114] Given the simplicity of the scPNP treatment, the process can be easily scaled up. The 3D EECM scaffold size was increased from a 96-well plate (25 mm2) to 12-well plate (324 mm2), and increased the amount of scPNPs tenfold while keeping the pDNA concentration at 7.5 pg / microliters. Larger fluorescent microscope images of treated and untreated scaffolds showed extensive green fluorescence, indicating effective GFP distribution comparable to smaller-scali experiments. Flow cytometry revealed a transfection efficiency of about 64.46% similar to the smaller system. Post-collagenase-I detachment cell viability was over 90%, confirming that scPNP treatment did not result in significant cell death.

[0115] FIG. 8 is a table showing a comparison on electroporation and on-scaffold transfection via nanoparticles prepared in accordance with certain aspects of the present disclosure (surface-capped protein nanoparticles (scPNPs)). A higher DNA concentration typically used with electroporation of about 100 micro grams / mL is used for a first example of cell electroporation, as well as a lower DNA concentration equivalent to the DNA concentration used to test the transfection of cells on the scaffold (7.5 micro grams / mL). In the direct comparison of the same DNA concentration, viability of cells in electroporation was only about 5.88% and, for the example according to certain aspects of the present disclosure, was about 90.45%. Comparatively, the conventional concentration of about 100 micrograms / liter was about 19.1%, still well below the 90.45% viability of the inventive example. Transfection efficiency was similar albeit lower in the comparative 100 micrograms / liter electroporation (about 63.92% versus 68.8% in the inventive example). However, the comparative 7.5 micro gram / mL transfection efficiency was only about 18.69%. Thus, a yield (viability x transfection efficiency) for the inventive example was about 62.22% in this example, as compared to the conventional 100 micrograms / liter (about 12.22% yield) and the comparative 7.5 micrograms / liter (about 1.09% yield).

[0116] In summary, these experimental results help demonstrate the cell manipulation / transfection systems according to certain variations of the present disclosure (that use three-dimensional cell constructs, such as the 3D EECM scaffolds, and surface-capped protein nanoparticles (scPNPs) and having a genetic payload of the pDNA that expresses GFP in genetically modified cells) is a superior alternative to electroporation for gene transfection in 3D cell culture environments, offering enhanced transfection efficiency, higher cell viability, and the potential for scalable gene delivery. The present disclosure envisions more effective gene therapy applications, emphasizing the importance of optimizing both scPNP-to-cell ratios and pDNA content with scPNPs to maximize therapeutic outcomes. This technology uses a nanoparticlebased approach for DNA transfection in autologous cell therapy research that improves cell viability and transfection efficiency. On-scaffold transfection methods provided herein haveAttorney Docket No. 2115-008429-WO-POA potential applications not only in autologous cell therapy but also in applications where scaffold implantation is necessary.

[0117] Moreover, in accordance with various aspects of the present disclosure, there is no need for harmful chemical or biological treatments (serving to detach cells from the 3D EECM) prior to transferring the genetic payload. The inventive technology can eliminate any requirement for expensive machinery, such as electroporation devices. The present disclosure also contemplates a smaller, comprehensive kit capable of handling both cell proliferation and transfection, thus making the technique more user-friendly and compact. Specifically, the technology uses surface-capped protein nanoparticles (scPNPs) for transfection on 3D engineered extracellular matrix (EECM).

[0118] The foregoing description of the embodiments has been provided for purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosure. Individual elements or features of a particular embodiment are generally not limited to that particular embodiment, but, where applicable, are interchangeable and can be used in a selected embodiment, even if not specifically shown or described. The same may also be varied in many ways. Such variations are not to be regarded as a departure from the disclosure, and all such modifications are intended to be included within the scope of the disclosure.

Claims

Attorney Docket No. 2115-008429-WO-POACLAIMSWhat is claimed is:

1. A method of manipulating cell genetics, the method comprising: transferring a genetic payload from at least one non-viral nanoparticle capable of penetrating tissue to at least one cell growing and supported on a three-dimensional construct by contacting the at least one non-viral nanoparticle with the at least one cell, wherein the transferring occurs without disassociating or breaking up the three-dimensional construct.

2. The method of claim 1, wherein the at least one non-viral nanoparticle is selected from the group consisting of: a lipid nanoparticle, a protein nanoparticle, a polymeric nanoparticle, and combinations thereof.

3. The method of claim 1, wherein the at least one non-viral nanoparticle comprises a core region comprising a water-soluble protein and the genetic payload and the core region defines a peripheral surface comprising a positively-charged capping agent disposed thereon; and the transferring further comprises delivering the genetic payload from the core region to an interior of the at least one cell, while the cell remains disposed within the cellular support system.

4. The method of claim 3, wherein the positively-charged capping agent is selected from the group consisting of: polyethylene imine (PEI), poly lysine, poly arginine, and combinations thereof.

5. The method of claim 3, wherein the water-soluble protein is selected from the group consisting of albumin, ovalbumin, mucin, transferrin, insulin, lysozyme, hemoglobin, collagen, catalase, horseradish peroxidase, glucose oxidase, and combinations thereof.

6. The method of claim 3, wherein the water-soluble protein comprises albumin.

7. The method of claim 3, wherein the at least one non-viral nanoparticle is pH responsive, so that the at least one non-viral nanoparticle is stable outside the cell, but after passing through a cell membrane of the cell, at least partially degrades at pH inside the cell to release the polynucleotide agent.

8. The method of claim 1, wherein the at least one non-viral nanoparticle is not crosslinked and is free of added crosslinking agents.

9. The method of claim 1, wherein the nanoparticle is formed by electrohydrodynamic jetting.

10. The method of claim 1, wherein the genetic payload comprises a polynucleotide agent selected from the group consisting of: DNA, RNA, plasmids, plasmid DNA (pDNA), shortAttorney Docket No. 2115-008429-WO-POA interfering sequence of double stranded RNA (siRNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, small nuclear RNA, single stranded DNA, microDNA, guide RNA, CRISPR CAS-9, aptamers, and combinations thereof.

11. The method of claim 1, wherein the three-dimensional cell construct is selected from the group consisting of: a tissue explant, an organoid, a three-dimensional printed cell structure, and combinations thereof.

12. The method of claim 1, wherein the three-dimensional construct is a three-dimensional cell scaffold comprising a plurality of voids, wherein the non- viral nanoparticle has a particle size permitting passage through the plurality of voids, the genetic payload comprises a polynucleotide agent, wherein the at least one nanoparticle comprises a core region comprising a water-soluble protein and the polynucleotide agent and the core region defines a peripheral surface comprising a positively-charged capping agent disposed thereon; and the transferring further comprises delivering the polynucleotide agent from the core region to an interior of the cell, while the cell remains disposed within the three-dimensional cell scaffold.

13. The method of claim 12, wherein the three-dimensional cell scaffold further comprises at least one protein-based fiber spanning each void of the plurality of voids, wherein the protein-based fiber comprises an extracellular matrix protein selected from the group consisting of: laminin, fibronectin, and a combination thereof.

14. The method of claim 1, wherein the transferring occurs without any viral vectors or electroporation.

15. The method of claim 1, wherein the at least one cell comprises a plurality of cells supported and growing on the three-dimensional construct and the at least one non- viral nanoparticle comprises a plurality of non-viral nanoparticles, wherein the transferring of the genetic payload occurs in bulk from at least a portion of the plurality of non-viral nanoparticles to at least a portion of the plurality of cells.

16. A cell genetic manipulation system comprising: a three-dimensional cell construct configured to support and promote three- dimensional growth of cells; and at least one non-viral nanoparticle configured to penetrate tissue and defining a core region comprising a water-soluble protein and a genetic payload, wherein the core region defines a peripheral surface comprising a positively-charged capping agent disposed thereon and the at least one non-viral nanoparticle is configured to deliver the genetic payload to at least aAttorney Docket No. 2115-008429-WO-POA portion of the cells disposed on the three-dimensional cell construct, without disassociating or breaking up the three-dimensional construct.

17. The cell genetic manipulation system of claim 16, wherein the at least one non- viral nanoparticle is configured to deliver the genetic payload from the core region to an interior of a respective cell of the portion of cells, while the cells remain disposed on the three-dimensional cell construct.

18. The cell genetic manipulation system of claim 17 wherein the at least one non- viral nanoparticle is pH responsive, so that the at least one non-viral nanoparticle is stable outside the respective cell, but after passing through a cell membrane of the cell, at least partially degrades at pH inside the respective cell to release the genetic payload.

19. The cell genetic manipulation system of claim 16, wherein the positively-charge capping agent is selected from the group consisting of: polyethylene imine (PEI), polylysine, polyarginine, and combinations thereof.

20. The cell genetic manipulation system of claim 16, wherein the water-soluble protein is selected from the group consisting of albumin, ovalbumin, mucin, transferrin, insulin, lysozyme, hemoglobin, collagen, catalase, horseradish peroxidase, glucose oxidase, and combinations thereof.

21. The cell genetic manipulation system of claim 16, wherein the water-soluble protein comprises albumin.

22. The cell genetic manipulation system of claim 16, wherein the at least one non- viral nanoparticle is not crosslinked and is free of added crosslinking agents.

23. The cell genetic manipulation system of claim 16, wherein the nanoparticle is formed by electrohydrodynamic jetting.

24. The cell genetic manipulation system of claim 16, wherein the genetic payload comprises a polynucleotide agent selected from the group consisting of: DNA, RNA, plasmids, plasmid DNA (pDNA), short interfering sequence of double stranded RNA (siRNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, small nuclear RNA, single stranded DNA, microDNA, guide RNA, CRISPR CAS-9, aptamers, and combinations thereof.

25. The cell genetic manipulation system of claim 16, wherein the three-dimensional cell construct is selected from the group consisting of: a tissue explant, an organoid, a three- dimensional printed cell structure, and combinations thereof.

26. The cell genetic manipulation system of claim 16, wherein the three-dimensional construct is a three-dimensional cell scaffold comprising a plurality of voids, wherein the non- viral nanoparticle has a particle size configured to permit passage through the plurality of voids,Attorney Docket No. 2115-008429-WO-POA the genetic payload comprises a polynucleotide agent, and the polynucleotide agent is transferred from the core region to an interior of the cell, while the cell remains disposed within the three- dimensional cell scaffold.

27. The cell genetic manipulation system of claim 16, wherein the three-dimensional cell scaffold further comprises at least one protein-based fiber spanning each void of the plurality of voids, wherein the protein-based fiber comprises an extracellular matrix protein selected from the group consisting of: laminin, fibronectin, and a combination thereof.

28. The cell genetic manipulation system of claim 16, wherein the genetic cell manipulation system is free of any viral vectors or electroporation.

29. The cell genetic manipulation system of claim 16, wherein the at least one non- viral nanoparticle comprises a plurality of non- viral nanoparticles, wherein the transferring of the genetic payload occurs in bulk from at least a portion of the plurality of non- viral nanoparticles to at least a portion of the cells disposed on the three dimensional cell construct.

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