Systems, devices, and methods for controlled poration of and substance delivery into the cellular nucleus

Abstract

Disclosed are systems, devices, and methods for delivering substances from the cytoplasm into the nucleus of cell by selectively disrupting the nuclear membrane. In some aspects, a method for delivering substances into a nucleus of a cell includes creating one or more pores in the nucleus of the cell by imposing one or more nanoscale geometric and / or physical constraints on the cell. In some embodiments of the method, the method includes enabling entry of substances into the nucleus through the created one or more pores.

Description

SYSTEMS, DEVICES, AND METHODS FOR CONTROLLED PORATION OF AND SUBSTANCE DELIVERY INTO THE CELLULAR NUCLEUSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefits of U.S. Provisional Application No. 63 / 677,950, filed on July 31, 2024. The entire contents of the aforementioned patent application are incorporated by reference as part of the disclosure of this document.TECHNICAL FIELD

[0002] This patent document relates to nanotechnology, and in particular to delivering substances from the cytoplasm into the nucleus of cell by selectively disrupting the nuclear membrane.BACKGROUND

[0003] Nanotechnology encompasses techniques or processes for fabricating structures, devices, and systems with features at the molecular or atomic scale, typically ranging from one to several hundred nanometers. For example, nanoscale devices can be configured to sizes similar to some large molecules, e.g., biomolecules such as enzymes. At these dimensions, materials can exhibit unique physical, chemical, and biological properties. Such properties enable the development of nanoscale devices and systems for a variety of biomedical applications.SUMMARY

[0004] Most existing chemical-free delivery techniques are limited to disrupting the plasma membrane of a cell, but cannot achieve direct delivery to the cell nucleus. Nonchemical approaches for breaking the cell membrane, such as microfluidic platforms that mechanically deform cells by forcing them through constricted channels, primarily compromise the integrity of the plasma membrane rather than the nuclear envelope. Direct delivery of substances into the nucleus typically relies on bulk electroporation, which involves the application of a large electric field to permeabilize cellular membranes. However, this method often results in significant cell damage, reduced cell viability, and extremely low transfection efficiencies. Consequently, there remains a need for improved delivery methods that can selectively disrupt the nuclear membrane to facilitate efficient and viable nuclear transfection without adversely affecting the plasma membrane.

[0005] The technology disclosed in this patent document relates to systems, devices, and methods for controllably porating a cell’s nucleus and delivering substances from the cytoplasm into the nucleus of the cell. Example substances that can be delivered by the disclosed systems, devices, and methods include, but are not limited to, biomolecules, nanoparticles, and genetic materials, among other deliverable materials. In some examples, a substance for delivery into the porated nucleus can include a viral vector, such as adeno- associated viral (AAV) vectors. In some examples, a substance for delivery into the porated nucleus can include plasmid DNA, mRNA, or genome editing tools like CRISPR / Cas9. In some examples, a substance for delivery into the porated nucleus can include a biologic or pharmaceutical drug or prodrug. In some examples, a substance for delivery into the porated nucleus can include a nanoparticle carrying a payload, such as a biologic or pharmaceutical drug.

[0006] In some aspects, a method for porating and delivering substances into a cellular nucleus, in accordance with the present technology, includes receiving, on a nucleoporation device comprising a substrate and a plurality of nanostructures on the substrate, a cell on the plurality of nanostructures; inducing, by the nucleoporation device, formation of transient pores in a nuclear membrane of the cell, wherein the plurality of nanostructures do not significantly puncture or penetrate a plasma membrane of the cell; and facilitating, through the transient pores in the nuclear membrane of the cell induced by the nucleoporation device, an unregulated entry of one or more substances from a cytoplasm of the cell into the nucleus.

[0007] In some aspects, a nucleoporation device for disrupting a nuclear membrane of a cell, in accordance with the present technology, includes a substrate; and a plurality of nanostructures, wherein the nanostructures are configured to induce formation of transient pores in the nuclear membrane of the cell without significantly puncturing or penetrating a plasma membrane of the cell.

[0008] In some aspects, a system for porating and delivering substances into a cellular nucleus, in accordance with the present technology, includes a nucleoporation device comprising a substrate, and a plurality of nanostructures, wherein the nanostructures are configured to induce formation of transient pores in a nuclear membrane of a cell without significantly puncturing or penetrating a plasma membrane of the cell; a cell imaging device interfaced with the nucleoporation device, the cell imaging device including a camera and a cell positioning device, wherein the camera is configured to acquire images of the cell on the nucleoporation device, and wherein the cell positioning device is configured to move the nucleoporation device with respect to the camera; and a data processing device in datacommunication with the cell imaging device, wherein the data processing device is configured to control the camera to acquire the images and the cell positioning device to move the nucleoporation device.

[0009] In some aspects, a method in accordance with the present technology includes delivering substances into the nucleus of cells. The entry of substances into the cell nucleus is a highly regulated process. Implementations of the method can bypass this process and for a short period of time create pores in the nucleus that enable entry of substances into the nucleus. For example, imposing very small (nanoscale) geometric / physical constraints on the cell creates pores in their nuclei. Therefore, by putting cells on nano-patterned surfaces, the disclosed method can induce these pores and deliver substances to the nucleus.

[0010] In some aspects, a method in accordance with the present technology includes:(1) selective disruption of the nuclear membrane without disruption of the plasma membrane;(2) high-throughput and scalability, using a nanofabrication technique that patterns thousands of nanopillars to induce nuclear poration for delivery; (3) compatibility with many cell types; (4) transient membrane disruption, such that cells remain viable and continue to perform regular processes; and (5) repair of the pores after a period of time. The nanopillars are engineered to create Gaussian-like curvature of the cells on the nanopillars to induce poration of the cellular nucleus while not significantly, if at all, disrupting the cellular membrane of the cells, and which the cellular nucleus transiently self-repairs. In some aspects, the method involves adding cells on top of nano-patterns of modulated geometries that induce nuclear disruption, i.e. poration. Agents may be delivered to the cytoplasm by different means, and, because of the nuclear poration, they directly translocate to the nucleus.

[0011] In some aspects, a method for delivering substances into a nucleus of a cell includes creating one or more pores in the nucleus of the cell by imposing one or more nanoscale geometric and / or physical constraints on the cell; and enabling controlled entry of substances into the nucleus, wherein the one or more nanoscale geometric and / or physical constraints on the cell do not cause significant puncture of or penetration through a cellular membrane of the cell that would threaten cellular viability.

[0012] The above and other aspects and implementations of the disclosed technology are described in more detail in the drawings, the detailed description, and the claims.BRIEF DESCRIPTION OF DRAWINGS

[0013] FIG. 1A shows a diagram depicting an example embodiment of a system for nucleoporation in accordance with the present technology.

[0014] FIGS. 1B-1F show diagrams, images, and data plots depicting an example embodiment of a nucleoporation device with engineered nanotopographies and method for breaching the nuclear membrane of a cell without penetrating the cell membrane, in accordance with the present technology.

[0015] FIG. 1G shows a diagram depicting an example embodiment of a method for delivering molecules into a nucleus of a cell, in accordance with the present technology.

[0016] FIG. 1H shows a scanning electron microscopy image depicting an example nanoring structures for a nucleoporation device in accordance with the present technology.

[0017] FIG. II shows a scanning electron microscopy image of an example embodiment of a nucleoporation device in accordance with the present technology featuring multiple groups of nanostructures.

[0018] FIGS. 2A-2C show data plots depicting an example analysis of nanotopography- induced nuclear envelope breach across various cell types.

[0019] FIGS. 3A-3O show diagrams, images, and data plots depicting example temporal and size-dependent effects of nanotopography-induced nuclear membrane openings.

[0020] FIGS. 4A-4E show diagrams, images, and data plots depicting an example nuclear envelope repair on nanotopography.

[0021] FIG. 4F shows data images and data plots from an example implementation of a nucleoporation method depicting rapid cellular nucleoporation and self-repair induced by an example embodiment of a nucleoporation device in accordance with the present technology.

[0022] FIGS. 5A-5C show diagrams, images, and data plots depicting example implementation results of cells on exemplary nanopillar structures, in accordance with the present technology.

[0023] FIGS. 6A-6C show diagrams, images, and data plots depicting example implementation results of nanopillar topography modulated nuclear envelope rupture.

[0024] FIGS. 7A-7E shows data plots depicting example results, including comparative analyses of ruptured and non-ruptured cells on various nanopillar substrates, as well as quantification of nuclear deformation and Ku-80 localization within the cell nucleus.

[0025] FIGS. 8A and 8B show images depicting an example implementation of human stem cells that underwent attempted gene editing using CAS9, where CAS9 was only in cell cytosol (FIG. 8 A) due to delivery by conventional non-viral delivery method (lipof ection), and where CAS9 was in cellular nucleus (FIG. 8B) due to delivery by implementing a nucleoporation method in accordance with the present technology.

[0026] FIGS. 9 A and 9B show diagrams depicting existing methods and an example implementation for delivering large plasmid DNA to the nucleus, in accordance with the present technology.

[0027] FIG. 10 shows a diagram depicting direct nuclear delivery of plasmids using a nanopatterned platform during a curvature induced breach.

[0028] FIGS. 11 A-l IF show diagrams, images, and data plots depicting example temporal and size-dependent effects of nuclear membrane openings induced by nanotopography.

[0029] FIGS. 12A-12D show images and data plots depicting example segmentation and analysis of nuclear breach using artificial intelligence.

[0030] FIGS. 13A-13B show images and data plots depicting an example demonstration of plasmid DNA nuclear delivery on nanopillars, in accordance with the present technology.

[0031] FIGS. 14A-14M show diagrams, images, and data plots depicting example characterization results of cell-nanopillar interaction using example embodiments of a nucleoporation device, in accordance with the present technology.

[0032] FIG. 15A-15H shows example data plots and images depicting the role of Lamin A / C on nanotopography-induced nuclear rupture.

[0033] FIGS. 16A-16J show images and data plots depicting the effects of actin contraction and polymerization on nuclear envelope rupture.

[0034] FIGS. 17A-17C show images and data plots depicting 3D SRS imaging of lipid composition of U2OS cells cultured on example nanopillars.

[0035] FIG. 18 shows a schematic diagram of an example embodiment of a method for fabricating nucleoporation devices, in accordance with the present technology.DETAILED DESCRIPTION

[0036] Efficient delivery of molecules, such as nucleic acids, proteins, or therapeutic agents, into the nucleus of a cell, referred to as nuclear delivery, is critical for applications in gene therapy, genome editing, and molecular biology research. Conventional transfection methods often rely on disrupting the plasma membrane (also referred to as cell membrane) to facilitate molecular entry into the cell. However, these approaches can result in low transfection efficiencies and unintended cellular damage. Therefore, there remains a need for improved systems, devices, and methods that enable the selective delivery of molecules directly into the nucleus without compromising the integrity of the plasma membrane.

[0037] Disclosed are systems, devices, and methods for delivering substances from the cytoplasm into the nucleus of a cell by selectively disrupting the nuclear membrane (alsoreferred to as nuclear envelope). The disclosed systems, devices, and methods control nuclear membrane disruption by a process referred to herein as “nucleoporation.” Nucleoporation is a nondamaging, non-deleterious process that controllably and transiently disrupts the integrity of the nuclear membrane of a cell through nanoscale-imposed changes of cellular morphology to induce an internal reshaping of the cell’s inner composition (e.g., cytoskeleton) that causes formation of openings or gaps of the nuclear membrane. The pores in the transiently permeabilized nuclear envelope are self-repaired by the cell. Such nanoscale-imposed changes are achieved through engineered nanostructures, e.g., with engineered nano-scale surface topographies, produced on a nano-biomedical device, platform, and / or system in accordance with the present technology.

[0038] Nanostructures, including but not limited to nanopillars, nanoneedles, nanorings, nanocrowns, and nanowires, can be biomimetically engineered to mimic natural structures. These nanostructures can be fabricated from a variety of materials, such as glass, silicon, polymers, and metals, allowing for tailored physical, chemical, and biological properties._One example includes nanostructures designed to function like viral spike proteins, which, when interfaced with a cell, can enable the nanostructures to bypass certain biological barriers, like the plasma membrane of a cell. Such biomimetic properties have led to applications in nanoelectronics for intracellular sensing and ex vivo and in vivo drug delivery platforms. Notable examples include planar drug delivery patches with nanopillars and nanoneedles, nanopillar electrodes for neuron and cardiac electrophysiology, and spiky nanoparticles that enhance cellular uptake and modulate drug release. While studies have shown that some engineered nanostructures can induce plasma membrane remodeling and increased endocytosis, a cost is incurred due to their structural characteristics that cause irreparable damage to the cell membranes and / or other harmful interactions with other organelles.

[0039] The nuclear membrane serves as the main barrier regulating selective molecular exchange between the cytoplasm and nucleoplasm. This barrier is primarily mediated by the nuclear pore complex (NPC), which strictly controls the transport of substances into and out of the nucleus. Overcoming this barrier is crucial for delivery of cargo into the nucleus of cells and for applications in intra-nuclear sensing. To overcome the NPC-mediated barrier, nanotopographies can be employed to induce nucleoporation through mechanical stretching of the NPC, thereby facilitating the entry of cargo into the nucleus. Here it is shown that nano topography can temporarily breach the nuclear membrane of many cell types. It is further demonstrated that by tuning their geometries and duration of interaction with cells, the nanotopographic features can control the incidence and size of nuclear membrane breaches.

[0040] Other example implementations can concentrate on the direct delivery of various biomolecules into the nucleus of cells employing nano topography. With capabilities to directly observe nuclear opening and repair events, these occurrences can be linked to nuclear delivery events. This enabled rational design of nanotopographic features to achieve nuclear delivery in various cell types. The example results presented in this disclosure increase confidence that the method can be applied in diverse cell types for various therapeutic applications.

[0041] In some embodiments in accordance with the disclosed technology, a method for delivering one or more substances into a nucleus of a cell includes placing the cell on a substrate comprising a surface having a plurality of nanostructures, and inducing formation of one or more transient pores in a nuclear membrane of the cell based on nanostructures imposing a physical effect on the cell’s internal structures (e.g., cytoskeleton) while conformed on the surface of the substrate (without significantly puncturing or penetrating the cells’ plasma membrane), thereby permitting unregulated entry of the one or more molecules from a cytoplasm of the cell into the nucleus.

[0042] The disclosed technology provides a cellular nucleoporation device that includes a plurality of nanostructures configured to receive a cell (e.g., the cell is placed on the plurality of nanostructures), and the cellular nucleoporation device induces formation of one or more transient pores in a nuclear membrane of the cell without damage to the cell (e.g., the plurality of nanostructures do not significantly puncture or penetrate the plasma membrane of the cell), thereby permitting unregulated entry of the one or more molecules from a cytoplasm of the cell into the nucleus.

[0043] Nuclear delivery is a critical aspect of biomedical research. One of the primary targets for delivered genes is the nucleus, where gene expression occurs. Efficient nuclear delivery is particularly important for gene delivery and genome editing applications, as these processes require the direct introduction of genetic material — such as plasmid DNA, mRNA, or genome editing tools like CRISPR / Cas9 — into the nucleus to achieve desired genetic modifications. Nuclear delivery enables precise manipulation of genetic material, including silencing or overexpressing endogenous genes and introducing exogenous genes, which is essential for elucidating cellular functions, developing therapies, and investigating disease mechanisms. Advances in nuclear delivery strategies significantly enhance the efficiency and specificity of gene delivery and genome editing, paving the way for new and improved therapeutic approaches and improved understanding of genetic regulation.

[0044] While various methods have been developed for the delivery of cargo to the cytoplasm, efficient delivery into the nucleus remains a significant challenge. For instance,DNA is rapidly degraded in the cytoplasm by enzymes within a few hours, significantly decreasing transfection efficiency. Existing nuclear delivery techniques are generally low throughput and predominantly depend on slow, internal cellular processes such as mitosis or transport through the nuclear pore complex (NPC). These internal processes impose significant limitations on both the size of the cargo that can be delivered and the range of cell types that can be effectively targeted. For example, adeno-associated virus (AAV) and other viral vectors have been explored for nuclear delivery; however, their use is often limited by immunogenicity, genotoxicity, restricted cargo capacity, and high production costs. Furthermore, transfection of non-dividing cells remains a significant challenge as conventional techniques reliant on cell division are ineffective.

[0045] Two principal methods enable cargo to reach the cell nucleus: transport through the NPC and pore formation in the nuclear envelope via physical disruption. NPC-mediated transport occurs either by passive diffusion or active targeting. Passive diffusion is limited to molecules up to approximately 9 nm in size and is dependent on concentration. Stimuli- responsive systems take advantage of the acidic environment of the cytosol, endosomes, or lysosomes, using near infrared (NIR) irradiation, or using ultrasound to release smaller nanoparticles from larger nanoparticles to enter the nucleus through the NPC. Active targeting strategies overcome the size limit of particles entering the nucleus by employing nuclear localization signal (NLS) peptides — such as SV40 T antigen, adenoviral, and TAT peptides — to achieve better kinetics in delivering cargo up to 30 nm, and in some examples up to 50 nm. Despite these advances, nuclear delivery remains limited by cargo size, material compatibility, and the efficiency of NLS conjugation, resulting in low transport efficiency for most macromolecular nanoparticles.

[0046] Methods for nuclear membrane disruption and nuclear delivery are less sensitive to cell and cargo properties. Some techniques include light-induced membrane peroxidation, plasmonic nanobubble generation, and image-guided photoporation. For example, light induced disruption through nucleus membrane peroxidation has led to 50 nm particles delivered to the cell nucleus. Short-lived plasmonic vapor nanobubbles have been able to burst through the nuclear membrane, which these ablative nanobubbles avoided irreversible damage to the membrane. And, image guided photoporation used laser beam to deliver 30 nm cargo to the cell, the photoporation was able to permeabilize the nuclear envelope. Other strategies, including micro-acoustofluidic sonoporation, carbon nanofiber-mediated perforation, and microtube array-based microinjection, have also been explored. However, these methods suffer from low delivery efficiency, limited controllability, complex fabrication requirements,and can compromise cell viability due to heat, DNA damage, or mechanical stress. Moreover, microfluidic and inertial microfluidic platforms that mechanically disrupt membranes or use electric fields can facilitate nuclear entry, but are limited by device complexity, cell heterogeneity, and potential adverse effects on cell function and viability. Other physical approaches, such as nanosecond pulsed electric fields and micro-orifice electroporation, can permeabilize the nuclear envelope but are limited by cell viability concerns, electric field nonuniformity, and technical challenges in cell handling.

[0047] Thus, current existing methods remain constrained by issues of efficiency, throughput, versatility, and safety. Therefore, there remains a need for a chemical-free, rapid, and high-throughput nuclear delivery platform that is effective across diverse cell types. An ideal method should enable rapid delivery to minimize biomolecule degradation, support high- throughput transfection, be versatile for various cell types — including non-dividing cells — and be minimally- or non-invasive to preserve cell viability. Although current state-of-the-art methods offer some advantages, none can achieve all the above objectives.

[0048] Moreover, for gene editing therapies, currently more than 70% of therapy costs and complexities are due to the delivery of the genes into the cellular nucleus. For gene editing to be effective, the editing machinery (e.g., viral vectors or non-viral vectors, like lipid nanoparticles) must enter the nucleus for gene correction to be conducted. Viral vectors are expensive and hard to scale; and non-viral vector particles have slow and poor uptake, typically getting stuck in the cytosol of the cell.

[0049] The disclosed technology includes systems, devices, and methods to address and solve the aforementioned problems and challenges. Some embodiments, in accordance with the present technology, provides a high-throughput biophysical platform to overcome the nucleo-cytoplasmic barrier controllably and transiently for rapid delivery of genes from the cytoplasm to the nucleus. Nuclear curvature induced by engineered material structures with nanoscale surface topographies are configured to transiently breach the nuclear membrane without damage to the cellular nucleus or the cellular membrane. The disclosed methods, systems, and devices are able to deliver genes, drugs, and other substances to the nucleus during this breach at a high throughput with minimal cell damage.

[0050] Example substances that can be delivered by the disclosed systems, devices, and methods upon nucleoporation of a cell’s nucleus include, but are not limited to, biomolecules, nanoparticles, and genetic materials, among other deliverable materials. In some examples, a substance for delivery into the porated nucleus can include a viral vector, such as adeno- associated viral (AAV) vectors. In some examples, a substance for delivery into the poratednucleus can include plasmid DNA, mRNA, or genome editing tools like CRISPR / Cas9. In some examples, a substance for delivery into the porated nucleus can include a biologic or pharmaceutical drug or prodrug. In some examples, a substance for delivery into the porated nucleus can include a nanoparticle carrying a payload, such as a biologic or pharmaceutical drug.

[0051] FIG. 1A shows a diagram depicting an example embodiment of a system 100 for nucleoporation in accordance with the present technology. The system 100 includes a nucleoporation device 110. The example embodiment of the nucleoporation device 1 10 shown in FIG. 1A includes a substrate 111 and a plurality of nanostructures 112 disposed on the substrate 111. The plurality of nanostructures 112 are configured to provide one or more nanoscale surface topographies engineered to induce nanoscale morphological changes to the nuclear membrane (of a cell attached to the nucleoporation device 110) that cause a breach (e.g., openings) of the nuclear membrane that can thereby allow for molecular transfer for the cell nucleus.

[0052] In some embodiments, the system 100 (optionally) can include a cell handling and / or imaging system 120, which can be interfaced with the nucleoporation device 110 (e.g., one or a plurality of nucleoporation device(s) 110). In some embodiments, the (optional) cell handling and / or imaging system 120 can include one or more imaging devices, including but not limited to, e.g., a microscope, stage positioner, a camera, and a computing system for controlling the imaging of cells on one or a plurality of nucleoporation device(s) 110 and storing of images acquired. In various embodiments, the microscope can include, but is not limited to, an inverted or upright light or brightfield microscope and / or fluorescent microscope. In some embodiments, the microscope can include a confocal microscope. In various embodiments, the stage positioner can include a micro- and / or nano-stepper to cause a stage, on which the one or plurality of nucleoporation device(s) 110 are positioned, to move with controlled spatial resolution (e.g., millimeter, micrometer, and / or nanometer resolution). In various implementations, the computing system of the cell handling and / or imaging system 120 can include a laptop computer, a desktop computer, or a mobile computing device (e.g., smartphone, smartwatch, or other) that stores and executes software to control the one or more of the camera to capture images, the stage positioner to move the stage, and / or the microscope to change magnification and focus of imaging (e.g., change and / or focuses lenses for some embodiments of the microscope). Further, in some embodiments of the (optional) cell handling and / or imaging system 120 can include one or more imaging devices can include a cell seeding device or system to deposit cells on the one or plurality of nucleoporation device(s) 110. Insome example embodiments, the (optional) cell handling and / or imaging system 120 comprises a microfluidic device configured to automate cell seeding, guidance onto and off of nanopillarbased nucleoporation substrates, mixing and delivery of biological media for cell maintenance, and / or fluidic interfacing with a holding apparatus compatible with real-time imaging systems. Whereas, in some example embodiments, the cell seeding device can include a cell solution deposition device, such as an automated or semi-automated pipetting system, or 3D bio-printer, that is configured to deposit cells in solution or in a gel on the one or plurality of nucleoporation device(s) 1 10 (e.g., which can be positioned for imaging by the imaging device or system). Various embodiments of the cell seeding device can include single-cell seeding or deposition techniques or multi-cell seeding or deposition techniques.

[0053] In some embodiments, the (optional) cell handing and / or imaging system 120 can include one or more well plates comprising one or more wells within which one or a plurality of the nucleoporation device(s) 110 can be contained and cell solution can be immersed thereon. In some implementations, for example, a single well plate can be used, e.g., such as a petri dish. In some implementations, for example, a multi-well plate can be used. The multi-well plate can be configured as a standard or a customized cell / tissue culture vessels with single or a plural number of wells / chambers (e.g. 1, 2, 4, 6, 8, 12, 24, 48, 96, 384, 1,536, etc.). In some embodiments, for example, the one or more well plates can have a modified bottom base wall and / or side wall(s) of the wells to provide physical features and / or chemical layers that can reduce or eliminate a meniscus formed between the liquid solution surface and the wall(s) of the well plate.

[0054] In some embodiments, the system 100 (optionally) can include a data processing system 130, which can be interfaced with the nucleoporation device 110 and / or the (optional) cell handling and / or imaging system 120. In some embodiments, for example, the data processing system 130 can include a remote user computer to obtain, analyze, and / or output data associated with the nucleoporation device 110 (and / or the cell handling and / or imaging system 120) implementing a nuclear membrane disruption application and / or substance delivery application. For example, in some embodiments, the data processing system 130 can include a personal computer such as a desktop or laptop computer, a mobile computing device such as a smartphone, tablet, smartwatch, etc., or other computing device; and additionally or alternatively, in some embodiments, for example, the data processing system 130 can include one or more computing devices in a computer system or communication network accessible via the Internet (referred to as “the cloud”), e.g., including servers and / or databases in the cloud. In some embodiments, for example, the data processing system 130 includes a softwareapplication (“app”) that is stored and operated on one or multiple computing devices of the data processing system 130 (using one or more processors and one or more memories of the one or multiple computing devices) to process the data received from the nucleoporation device 110 and / or the cell handling and / or imaging system 120.

[0055] In various implementations in accordance with the disclosed technology, the system 100 can be combined with any one or multiple of a variety of cytoplasmic (cell membrane) delivery devices, systems, and techniques, including but not limited to lipid nanoparticles, electroporation, optoporation, or mechanoporation, which can be used to aid an example nuclear delivery application by following an initial cytoplasmic delivery or be in parallel with cytoplasmic delivery technique.

[0056] FIGS. 1B-1F shows diagrams, images, and data plots depicting an example implementation of an example embodiment of the nucleoporation device 110 (labeled 110B in FIG. IB), demonstrating how its engineered nanostructures 112B controllably induced breaching of the nuclear membrane (e.g., pores) without penetrating or damaging the cell membrane. For instance, as illustrated in FIG. IB, the nucleoporation device HOB includes an array of nanopillars 112B disposed on a substrate 1 1 IB arrays, which, when a cell is placed on the device 110B, induces the nuclear membrane to morph at the nanometer scale and thereby produce openings within the nuclear membrane. The lower inset box 109 of FIG. IB shows a cross-sectional confocal fluorescent image of the cell, demonstrating the presence of Ku-80- FITC in both the nucleus and cytosol, which indicates successful transport of the labeled protein across the compromised nuclear envelope.

[0057] FIG. 1C shows an illustrative diagram of an example embodiment of a method for nucleoporation of a cell, in accordance with the present technology, labeled 190 in FIG. 1C, implemented on the exemplary device 110B. As shown in FIG. 1C, the method includes a process 191 to seed a cell on the nucleoporation device HOB. In some implementations of the process 191, for example, the cell can be seeded by a variety of cell seeding techniques, including by microfluidic cell placement, chemo-passive cell adhesion, mechanical cell placement (e.g., via a cell seeding device), or other. Also, in some implementations, the process 191 includes seeding a plurality of cells at portions of the nucleoporation device HOB, e.g., where one or more cells are seeded at a first region of the nucleoporation device 110B, another one or more cells are seeded at a second region of the nucleoporation device HOB, etc. Whereas, in some implementations, the process 191 includes seeding a single cell at one or more portions of the nucleoporation device 110B (like illustrated in the diagram of FIG. 1C).

[0058] The method 190 includes a process 192 to induce cell deformation of the seeded cell(s) on the nucleoporation device HOB. For example, cell deformation refers to overall cellular morphology conforming to the engineered topologies of the array of nanostructures 1 12B of the nucleoporation device HOB. The method 190 includes a process 193 to induce nuclear membrane deformation, where the shape of the nuclear membrane undergoes curvatures at the nanoscale based on the cellular deformation (discussed in detail later). The method 190 includes a process 194 to facilitate the breach the nuclear membrane based on the nuclear membrane deformation. The method 190 includes a process 195 to facilitate the repair of the nuclear membrane. Example data demonstrating results of an example implementation of the method 190 is shown in FIGS. 1C-1F, including, for example, side-view confocal microscopy images of nanopillars with U2OS cells stained with Ku- 80, in which these representative images of cells demonstrate the ability of nanostructures 112B (e.g., nanopillars) for inducing nanoscale curvature to the nucleus of the cells.

[0059] FIG. ID shows fluorescent microscopy images depicting cells on a flat substrate (top image) versus an engineered nanopillar substrate of an example nucleoporation device 110B (bottom image), which the cells are immunostained with Ku-80 antibody, and where the images demonstrate the diffusion of Ku-80 to the cytoplasm on nanopillar areas due to the nuclear envelope breaching.

[0060] FIG. IE shows a panel of immunofluorescent images comparing HeLa cells on a flat substrate (top row) and an array of engineered nanopillar surfaces (bottom row). In the example implementation, the HeLa cells were stained with DAPI (blue), Lamin A / C (magenta), Ku-80 (green), and Actin-phalloidin (red). The images of FIG. IE, for example, demonstrate nuclear deformation and altered Ku-80 distribution on nanopillar interfaces. Notably, 30- degree tilted angle SEM images were captured (shown in bottom-left portion of FIG. IE) that depict the distinctive nanopillar arrays (e.g., pitch=3.55± 0.06 m, height= 3.1+ 0.1|im, Diameter= 0.88+ 0.03 pm, Mean+ S.D).

[0061] FIG. IF shows a data plot depicting a quantification of the cytoplasmic-to-nuclear Ku-80 ratio, which indicates higher cytoplasmic Ku-80 presence on the exemplary nanopillar substrates (e.g., 0.129 +.018, n>58) compared to flat controls (e.g., 0.023+0.002, n=23).

[0062] Example embodiments and implementations of the disclosed nucleoporation technology are described herein that provide the first instance of a cellular nucleoporation device with engineered nanotopographic material structures that can breach the nucleo- cytoplasmic barrier by inducing transient openings in nuclear membranes without penetrating cells (that is, insignificantly puncturing or otherwise damaging the cell’s plasmic membrane),thereby leading to temporary and controlled breaches that allow for free exchange of molecules across the nuclear membranes. These openings are induced by the nanoscale curvature on the nuclear membrane, which can be controlled by altering the dimensions of nanopillars and the duration of their interaction with cells. And, cell viability is sustained throughout implementation of the nucleoporation technique, where the nuclear membrane transiently selfrepairs after the technique and the cell membrane does not undergo significant puncture or disruption while the cell is positioned on the engineered nanostructures, where, for example, an insignificant puncture refers to no puncture or de minimus puncture of the cell membrane that does not risk cell viability and is capable of plasma membrane repair or recovery.

[0063] As disclosed herein, different nanotopography geometries can induce varying degrees of curvature in the nuclear membrane, influencing the likelihood of breach incidence, the extent of nuclear envelope disruption, and the time required for membrane repair. These factors can critically affect the efficacy and efficiency of nuclear transfection. Additionally, the nuclear membranes of various cell types can be breached using nano-scale surface topographies configured by engineering material structures to induce and control nuclear membrane disruption, e.g., nucleoporation. Such control by the engineered nanostructures over the integrity of the nuclear membrane can be both spatial and temporal. Spatial control includes the ability to precisely localize nuclear membrane breaches to specific regions of the cell, which is determined by the size and geometry of the engineered nanostructures used — for example, nanostructures (such as nanopillars) with diameters ranging from 300 nm to 6 pm, heights from 500 nm to 5 pm, and nanostructure spacings ranging in the microns, e.g., such as 1 pm to 10 pm spacing, or particularly at substantially 6 pm (± 1 pm) spacing or at substantially 3 pm (± 0.5 pm) spacing. Temporal control includes the ability to regulate the duration of these breaches, which can last from as short as 1 hour to as long as 4 to 5 days, e.g., depending on the interaction parameters between the nanostructures and the cells. Moreover, these breaches of the nuclear membrane through such engineered nanostructures are temporary and can be repaired through endosomal sorting complexes required for transport (ES CRT) -mediated mechanisms.

[0064] The embodiments described herein demonstrate improved delivery, efficiency, and safety of therapeutic agents to the nucleus, leading to more effective and targeted treatments for a variety of genetic disorders and diseases. These discoveries pave the way for innovative methods of direct nuclear sensing and delivery using nanotopographic materials. By controlling the timing and duration of nuclear membrane openings and repairs using rationallydesigned nanotopographic materials, molecules can be delivered precisely and rapidly — within one hour — directly to cell nuclei, potentially revolutionizing targeted therapeutic interventions.

[0065] The interface between cells and the nanotopographic surfaces of the exemplary nucleoporation devices of the disclosed technology, which result in nanotopography-induced nuclear envelope (NE) rupture, have been examined as to how various cellular properties — including nucleoskeletal elements, cytoskeletal dynamics, and lipid composition — impact cellular and nuclear behavior and stability. Example experimental implementations of the disclosed nucleoporation device have produced results showing that a reduction in Lamin A / C levels within the nucleus leads to a loss of nuclear stiffness, thereby increasing nuclear deformability and susceptibility to rupture when exposed to nanotopographies. Additionally, the example implementations include investigations into the role of cytoskeletal elements, such as actomyosin contractility and actin polymerization, which have demonstrated that actin polymerization plays a predominant role in curvature-induced NE rupture. Furthermore, analysis of lipid composition has shown that higher levels of saturated fatty acids, which render the nuclear membrane more rigid, correspondingly increase susceptibility to rupture. Collectively, these findings demonstrate that interactions between cellular components and nanotopography can influence cellular behavior, thereby evidencing the capabilities of the disclosed nucleoporation devices, systems, and methods, which can be used in a variety of applications to study and modulate cellular and nuclear function. Example results of the example implementations are discussed later in this disclosure, in connection with FIGS. 14A- 17C.

[0066] FIG. 1G shows a diagram depicting an example embodiment of a method 150 for delivering molecules into a nucleus of a cell, in accordance with the present technology. The method 150 includes a process 151 to create one or more pores (e.g. nanometer scale pores, up to 30 nm or up to 50 nm) in the nucleus of the cell by imposing one or more nanoscale geometric and / or physical constraints on the cell, where the one or more nanoscale geometric and / or physical constraints on the cell do not cause significant puncture of or penetration through a cellular membrane of the cell. For example, the process 151 can control (e.g. , provide direct influence over) the pore size formed and duration of the formed pores in the nuclear membrane through the engineered nanostructures 11 1. As an example, the process 151 can control the pore size to be in a range of 10 nm, if not smaller, to 500 nm, if not larger, which is shown by exemplary data from example implementations where small substances like Ku80 dimer (e.g., -10 nm) have passed through pores to large substances like plasmid nucleic acid with thousands of base pairs (e.g., -540 nm in diameter as circular DNA) have passed throughnucleoporated pores of the nuclear envelope. The method 150 includes a process 155 to enable controlled entry of molecules into the nucleus through the one or more pores created by the imposition of the one or more nanoscale geometric and / or physical constraints on the cell.

[0067] In some embodiments of the process 151 of the method 150, the imposing one or more nanoscale geometric and / or physical constraints on the cell includes putting the cell on a nanopatterned surface comprising one or more nanostructures. In some embodiments of the method 150, the process 151 includes inducing transient openings in a nuclear membrane of the nucleus of the cell by breaching a nucleo-cytoplasmic barrier, which can allow for molecular exchange across the nuclear membrane. In some embodiments of the process 151, the openings are induced, at least temporarily, by at least one of an indention depth of or a nanoscale curvature of the one or more nanostructures on the nanopatterned surface. For instance, the nanostructures are engineered to create Gaussian-like curvature of the nucleus (e.g., a plurality of ‘bell curves’ for the cell’s morphology) on the nanostructures to induce nucleoporation of the cellular nucleus while not significantly, if at all, disrupting the cellular membrane, such that the induced openings (nucleopores) are transient, whereby the nuclear membrane is able to self-repair.

[0068] In some embodiments of the method 150, the method 150 may further include a process 157 to facilitate repair of the openings through at least one ESCRT-mediated mechanism (ESCRT: endosomal sorting complexes required for transport). In certain embodiments of the process 155 of the method 150, the enabling the unregulated entry of molecules into the nucleus occurs in a period of time prior to mitosis of the cell. In some embodiments of the method 150, the molecules entering the nucleus comprise nucleic acid, protein, peptide, or gene editing component. In some embodiments of the process 155 the method 150, the molecules are deposited in a solution surrounding the cell. In some embodiments of the method 150, the method 150 includes delivering molecules into a plurality of nuclei of a plurality of cells, respectively, by putting the plurality of cells on an array of nanopatterned surfaces. In some embodiments, the method 150 is used for direct nuclear delivery of therapeutic agents, gene editing reagents, or biosensors. In some embodiments, the method 150 includes controlling a degree of nuclear membrane disruption, where, for example, the controlling the degree of nuclear membrane disruption can be performed at least partially based on controlling at least one of an indention depth of or a nanoscale curvature of one or more nanostructures that impose the one or more nanoscale geometric and / or physical constraints on the cell.

[0069] In some embodiments of the nucleoporation device 150, the nanostructures 112 include nanopillars that can be configured to have a diameter in a range of 200 nm to 5 pm (e.g., including in a range of 200 nm to 700 nm). In some embodiments, the exemplary nanopillars can be configured to have a height in a range of 1 pm to 5 pm (e.g., including in a range of 2 pm to 3 pm). In some embodiments, the exemplary nanopillars can be configured to have a spacing on the substrate 111 in a range of 2 pm to 10 pm (e.g., including in a range of 2.5 pm to 5 pm). For example, the nanopillars can be structured to span vertically from the base substrate 111 and have a cylindrical geometry, a cone-like geometry, or a combination of a cylindrical and cone geometry, where the base of the pillar is cylindrical to a region that spans with a cone geometry that terminates at a tip. Nanopillars can be configured to be solid, e.g., typically rigid, and provide vertical topographic cues with defined spacing and aspect ratio. As such, for example, in some embodiments, exemplary nanopillars can be configured to have a tip angle in a range of 9 to 16 degrees (e.g., including in a range of 9° to 13°).

[0070] In some embodiments of the nucleoporation device 150, the nanostructures 112 include nanorings that can be configured to have a diameter in a range of 500 nm to 5 pm with a ring / wall thickness of 10nm-200nm. In some embodiments, the exemplary nanorings can be configured to have a height in a range of 1 pm to 5 pm (e.g., including in a range of 2 pm to 3 pm). In some embodiments, the exemplary nanorings can be configured to have a spacing on the substrate 111 in a range of 2 pm to 10 pm (e.g., including in a range of 2.5 pm to 5 pm). For example, the nanorings can be structured with a cylindrical and / or conical geometry that has an outer wall than spans from the base substrate and a hollow or at least partially-hollow interior section, such that the outer wall forms a perimeter at the top of the nanostructure. Nanorings are structured to be (at least partially) hollow, ring-like structures with an open center and raised circular perimeter. For example, compared to nanopillars, example engineered nanorings offer a lateral confinement geometry with reduced vertical protrusion in the center, which can affect nuclear membrane curvature differently.

[0071] In some embodiments of the nucleoporation device 150, the nanostructures 112 include nanocrowns that can be configured to have a diameter, height, and / or spacing similar to that of the exemplary nanorings. For example, the nancrowns can be configured to have a diameter in a range of 500 nm to 5 pm with a ring / wall thickness of 10nm-200nm, a height in a range of 1 pm to 5 pm (e.g., including in a range of 2 pm to 3 pm), and a spacing on the substrate 111 in a range of 2 pm to 10 pm (e.g., including in a range of 2.5 pm to 5 pm). For example, the nanocrowns can be structured similar to the nanorings, e.g., including acylindrical and / or conical geometry that has an outer wall than spans from the base substrate and a hollow or at least partially-hollow interior section, such that the outer wall forms a perimeter at the top of the nanostructure. Yet, the outer wall can include indentations from the peak height down at least a portion of the outer wall (e.g., including down to the base substrate in some embodiments), thereby providing crenelation-like structures of the exemplary nanocrown nanostructure. For example, nanocrowns can be configured as modified nanorings with a recessed central region surrounded by a raised outer rim — resembling a crown; where, for example, this structure presents both vertical and circumferential curvature, potentially enhancing membrane wrapping or cell anchoring at the rim.

[0072] In some embodiments of the nucleoporation device 150, the nanostructures 112 include nanoneedles that can be configured to have a diameter in a range of 100 nm to 1 pm, a height in a range of 1 pm to 5 pm, and a spacing on the substrate 1 1 1 in a range of 2 pm to 10 pm (e.g., including in a range of 2.5 pm to 5 pm). For example, nanoneedles are high- aspect-ratio, sharp-tipped vertical structures (e.g., with height-to-diameter aspect ratios in a range of 10:1 or greater, e.g., up to 50:1), which are engineered for membrane penetration or close contact. Compared to nanopillars, they are narrower and sharper, increasing local mechanical stress and enabling applications like intracellular delivery.

[0073] In some embodiments of the nucleoporation device 150, the nanostructures 112 include nanowires that can be configured to have a diameter in a range of 50 nm to 5 pm, a height in a range of 500 nm to 5 pm, and a spacing on the substrate 111 in a range of 1 pm to 10 pm (e.g., including in a range of 2.5 pm to 5 pm). For example, nanowires are elongated, flexible or rigid structures that are longer in one horizontal axis. Example embodiments of the engineered nanowires can differ from nanopillars by their anisotropy and horizontal extension, offering directional cues and enabling electrical interfacing or axon guidance.

[0074] FIG. 1H shows a scanning electron microscopy (SEM) image depicting an example engineered nanotopography ring structure, e.g., exemplary nanoring structures. The SEM image depicts an array of SiCh nanorings with 100 nm to 200 nm wall thickness.

[0075] FIG. II shows a scanning electron microscopy (SEM) image of an example embodiment of the nucleoporation device 150 having a first group of nanostructures 112 (left side) having 6 pm spacing and a second group of nanostructures 112 (right side) having 3 pm spacing. The example nanostructures 112 in the two groups include a base diameter of 1 pm, a tip diameter of 300 nm, and a height of 2 pm.

[0076] In some embodiments, the nanostructures 112 (e.g., nanopillars, nanoneedles, nanorings, nanocrowns, nanowires) are formed from a material that includes one or more of silicon dioxide, silicon, metals, and / or one or more polymers. In some embodiments, the nanostructures 112 are configured to have a particular shape and / or geometry that includes, but is not limited to, cylindrical, conical, annular (e.g., ring-like), rectangular prisms, and / or pyramidal. In some embodiments, the nanostructures are arranged in a pattern that includes at least one of a regular array, a random array, and / or concentric rings. Such configurations of the nanostructures 1 12 of example embodiments of the nucleoporation device 110 can facilitate the adhesion, spreading, and conformation of the cells on the device 110 and cause nuclear membrane breach / rupture without any significant puncture / piercing / breach of the cell membrane.

[0077] In some (optional) embodiments, for example, the nanostructures 112 may comprise a coating to promote cell adhesion. In some embodiments, for example, the (optional) coating can include one or more of poly-L-lysine, gelatin, and / or fibronectin. Additionally or alternatively, in some embodiments, the coating may include extracellular matrix (ECM) proteins such as laminin, vitronectin, collagen, matrigel, or combinations thereof, to further enhance cellular attachment and compatibility with a variety of cell types.

[0078] Example embodiments of the nucleoporation device 150 comprising the nanostructures 112 configured as nanopillars were implemented to demonstrate nucleoporation. For example, nanopillars with dimensions of 500 nm in diameter, 2.5 pm spacing, and 3 m height demonstrated that up to 78% of cells exhibit nuclear membrane breach under these conditions (e.g., shown in FIG. 9B). These exemplary nanopillars can be fabricated in any suitable manner, including, for example, on transparent fused silica substrates using a two-step dry and wet etching technique, as described herein, such as in connection with FIG. 18. The disclosed technology includes a variety of nanotopographies (e.g., nanoscale protrusions from the surface of a material) that can induce these passive ruptures of the nuclear membrane while preserving the integrity of the cellular membrane.

[0079] The nanostructures 112 are configured to induce nanoscale curvature upon the plasma and nuclear membranes, e.g., as illustrated in FIGS. IB and 1C. Fluorescent imaging reveals that cells grown on the nanopillar platforms exhibit both positive and negative curvature in their nuclear membranes (FIG. IE). In contrast with the disclosed technology, nanoscale indentation of the nuclear membrane by atomic force microscopy (AFM) has been shown to rupture cell nuclei, but at the detriment to the viability of the cell. Whereas, using the disclosed nucleoporation technology, the curvature induced by nanotopography during cell adhesion andspreading can lead to breaching of nuclear membranes, resulting in device-controlled breaches for substance exchange between the nucleus and cytoplasm, as illustrated schematically in FIG. 1C.

[0080] To assess this, cells were stained with a nuclear rupture reporter, Ku-80; a protein typically localized to the nucleus in most cell types that diffuses into the cytoplasm upon a breach in the nuclear membranes. Several cells on nanopillars exhibited leakage of ku-80 into the cytoplasm, indicating nuclear membrane openings (FIG. ID). However, the plasma membranes of these cells remain intact, as evidenced by the absence of Ku-80 diffusion outside the cytoplasm. Notably, no such leakage was observed on the flat regions of the same platform that lacked nanopillars (FIG. ID). To quantify nuclear opening events, immunostaining was performed to delineate nuclear and cytoplasmic boundaries using DAPI and Actin, respectively (FIG. IE), and the cytoplasmic-to-nuclear ratio of Ku-80 was calculated. A significant increase in this ratio was identified as a nuclear opening event, as described in the image analysis of methods section. Analysis revealed that HeLa cells on nanopillar substrates exhibited a significant increase and a much broader range in the cytoplasmic-to-nuclear ratio of Ku-80 (0.129 ± 0.018) compared to those on flat substrates (0.023 + 0.002), indicating a high incidence of nuclear membrane breaches on nanopillars (FIG. IF).

[0081] These example results demonstrate that forces arising from cell interaction with nano-topography can induce openings in the nuclear membrane without requiring any other external stimulus. To further assess whether cells with diverse structural properties would experience such nuclear membrane breaches on nanopillars, a variety of cell types were subsequently cultured on them.

[0082] To explore the versatility of the engineered nanotopography surfaces of exemplary nucleoporation devices 110 in inducing nuclear membrane openings, a range of cell types with diverse structural and morphological properties were investigated, including epithelial-like cells (U2OS, HeLa, HEK-293), stem cell-derived cardiomyocytes (siLMNA iPSC-CM), and NIH-3T3 fibroblast cells (shown in FIG. 2A-2C).

[0083] FIGS. 2A-2C show images and data plots depicting an example analysis of nanotopography-induced nuclear envelope breach across various cell types. In FIG. 2A, the top panel shows a fluorescent image of nuclei of epithelial cells (U2OS, HeLa, HEK293) with a Ku-80 stain inside the nucleus (intact nucleus), and the bottom portion of each panel shows cells with breached nuclear membranes as indicated by Ku-80 mislocalization to the cytoplasm, with each panel including a data plot showing the intensity profile of Ku-80. FIG. 2B shows images and data plots for stem cell-derived-like cells (siLMNA iPSC-CM) displaying intact(top) and breached (bottom) nuclear membranes on nanopillars. FIG. 2C shows images and data plots for fibroblast cells (NIH-3T3), which demonstrate distinct Ku-80 translocation in the cytoplasm in cells (top), and, upon Nanopillar-induced breach of the nuclear membranes, Ku- 80 translocated to the nucleus, as depicted by intensity profiling (bottom).

[0084] As these example results showed, all tested cell types exhibited curvature-induced nuclear membrane openings on the nanopillar platforms. In epithelial cells and iPSC-CMs, nuclear membrane openings were evident from the mislocalization of Ku-80 from the nucleus to the cytoplasm (FIGS. 2A and 2B). Conversely, in NIH-3T3 cells it was found that Ku-80 was predominantly located in the cytoplasm under non-breached conditions, consistent with previous studies. (FIG. 2C). Upon nuclear membrane breaching induced by nanopillars some Ku-80 localized to the nucleus. These results not only indicate the versatility of the platform to induce nuclear membrane openings in various cell types, but also highlights bidirectional transfer of proteins between the nucleus and the cytoplasm during such breaches.

[0085] Having shown the versatility of the platform in inducing nuclear membrane openings in various cell types, the temporal dynamics of nuclear membrane openings on nanopillars for one cell type (U2OS) were investigate. In separate experiments, the number of breached nuclear membranes on nanopillars at 1, 5, and 8 hours after seeding were quantified. See FIG. 5.

[0086] FIGS. 3A-3O show diagrams, images, and data plots depicting an example embodiment of temporal and size-dependent effects of nanotopography-induced nuclear membrane openings.

[0087] FIG. 3A shows a schematic and quantitative analysis highlighting a significant increase in the cytoplasmic to nuclear Ku- 80 ratio from 1 to 5 hours. A cytoplasmic / nuclear Ku-80 ratio threshold of 0.16% is established to identify nuclear membrane openings. Data indicate that 6.5% of cells exhibit nuclear membrane breaches at the 1-hr point, with the proportion of breached cells increasing to 20.5% and 19.3% at 5 and 8 hours, respectively. (n>92). FIG. 3B shows a side-view immunofluorescent image of Lamin A / C on nanopillars, showing the curvature and indentation depth of the nucleus due to the nanopillars. FIG. 3C shows a data plot depicting the indentation depth of the nuclear envelope at 1, 5, and 8 hours post seeding illustrates a significant increase from 1.57+ 0.19 pm to 2.53+ 0.24 pm and 2.41+0.39 (e.g., n > 8 cells per condition; and each value represents the mean values of indentation induces by each nanopillar). FIG. 3D shows a data plot depicting the normalized maximum curvature of the nuclear envelope, exhibiting an upward trend from 1 hour to 8 hrs. (n>8 cells per condition. Each value represents the mean values curvature induces by eachnanopillar). FIG. 3E shows a data plot depicting that cells have higher area from 5 to 8 hrs., indicating the cellular spreading, while the breach incidence remains unchanged between 5 and 8 hrs., and indicating that breaching likely is not dependent on cellular area. FIG. 3F shows that cell circularity measurements suggest an initial round morphology at 1 hr., which decreases over time, exhibiting higher cell deformation. FIG. 3G demonstrates that the nucleus area parameter shows a full spread of the nucleus by the 5 -hr time point and remains consistent through to the 8-hr time point. FIG. 3H shows that the nuclear circularity increases by 5 hours and then stabilizes up to the 8-hr time point. The nuclear circularity is higher after cells spread suggesting the nuclear deformation while cells are in early adhesion and spreading phase. For example, 1 hr shows lowest circularity and highest deformability of nucleus. FIG. 31 shows tilted 30-degree SEM images of flat, large (height= 4.70±0.02pm, Diameter= 1.30±0.01pm) and small nanopillars (height= 3.18±O.O3pm, Diameter= 0.51+0.03pm), schematic of isotropic etching of nanopillars and cytoplasmic / nuclear Ku- 80 ratio, showing notably higher presence of Ku-80 in the cytoplasm on small nanopillars compared to large nanopillars and flat substrate. Small nanopillars exhibit the highest incidence of nuclear envelope breach at 78.1%, while large nanopillars demonstrate a significantly lower breach rate of 12.1%. (n>98). FIG. 3J illustrates the indentation depth of nuclear envelope associated with large and small pillars, deeper indentation for larger pillars 3.41+ 0.34 pm compared to small pillars 2.58+ 0.24 pm (n>8). FIG. 3K illustrates the normalized maximum curvature of nuclear envelope where the small nanopillars induce higher curvature 3.15+ 0.73 pm compared to large pillars 2.24+ 0.90pm. (n>8). FIG. 3L shows that analysis of cell area suggests that cells on both large and small nanopillars exhibit a reduced spreading compared to flat surfaces. FIG. 3M shows that cell circularity measurements indicate greater cell deformation on large nanopillars when compared to flat and small nanopillar substrates. FIG. 3N shows that nuclear area analysis detects no significant difference in nuclear spreading across different substrate topographies. FIG. 30 demonstrates that the nucleus circularity shows a significant nucleus deformation on small pillars where it is higher than flat substrate as well as large nanopillars.

[0088] As these example results showed, a small percentage of cells exhibit openings as early as 1-hour of cell-nanopillar interactions, with breached nuclear membranes increasing from 6.5% to 20.5% during the first 5 hours. No significant changes were observed in this percentage between 5 and 8 hours. (FIG. 3A). To understand the mechanisms, confocal microscopy was performed to quantify the indentation depth and maximum curvature of the nuclear membrane at various durations of cell-nanopillar interactions. (FIG. 3B). A significant increase in the indentation depth from 1.57+ 0. 19 pm at 1 hour increasing sharply to 2.53+ 0.24pm at 5 hours was observed. Interestingly no significant increase was observed in the indentation depth between 5 and 8 hours, similar to non-significant breach incident difference between 5 and 8 hrs. (FIG. 3B). These results suggest that deeper indentation correlates with an increased likelihood of nuclear membrane opening. (FIG. 3C). This is further evidenced by the lack of any no significant increase in the percentage of cells with nuclear membrane breaches between 5 and 8 hours, as no significant increase is observed in their indentation depths. (FIG. 3C). Additionally, the normalized maximum curvature values show a consistent increase from 0.65+0.15 pm at 1 hr to 1.59+0.41 and 2.34+0.84 pm at 8 hours, suggesting that prolonged exposure to nanopillars increases the curvature and deformation of nuclear membrane on nanopillars which leads to more frequent nuclear membrane breaches. (FIG. 3D.)

[0089] Furthermore, it was determined that the spreading of cells and nuclei on nanopillars leads to increased nuclear membrane breaches. To explore this, cell and nuclear morphologies, specifically spreading (area) and shape (circularity), were quantified at 1, 5, and 8 hrs. (FIGS. 3E-H). Although cell area remained largely unchanged from 1 to 5 hours (FIG. 3E), cell shape changed significantly (FIG. 3F), indicating cell deformation around the nanopillars during this period. More importantly, significant changes in nuclear area (FIG. 3G) and shape (FIG. 3H) were observed between 1 and 5 hrs., aligning with the rise in nuclear membrane breaches. These metrics remained unchanged from 5 to 8 hrs. (FIGS. 3G and 3H), underscoring that nuclear spreading and deformation is key to nuclear membrane breach on nanopillars. Interestingly, although cell spreading area and shape continued to change significantly from 5 to 8 hrs. (FIGS. 3E and 3F), these alterations did not correlate with any further increase in nuclear membrane breaching. This finding indicates that changes in nuclear dimensions and shape, rather than those of the cell, are the primary drivers behind the nuclear membrane breaching on nanopillars.

[0090] Together these results show that the incidence of nuclear membrane breaching can be controlled by adjusting the duration of interactions between the nanopillars and cells.

[0091] To investigate whether the size of nanopillars could influence the frequency of nuclear membrane breaches, isotropic wet etching to reduce nanopillar dimensions were employed, keeping other factors constant. (FIG. 31). The percentage of cells with nuclear membrane openings on large (height= 4.7+0.02, Diameter= 1.30+0.01) and small pillars (height= 3.18+0.033, Diameter= 0.515+0.03) were compared. See FIGS. 6A-C.

[0092] Interestingly, for example, an increase in the percentage of cells with breached nuclear membranes was found, e.g., from 12% to 78% with nanopillar size reduction, while smooth surfaces showed none (FIG. 31). Also, for example, it was found that, although smallernanopillars induce a lower indentation depth (2.58± 0.24) compared to larger nanopillars (3.41+ 0.34), the normalized maximum curvature of the nuclear membrane is much higher on smaller nanopillars (3.15+ 0.73pm) compared with larger ones (2.243+ 0.90pm) (FIGS. 3J and 3K). The higher curvature associated with smaller nanopillars appears to be the critical factor in the increased frequency of nuclear envelope breaches. Despite the larger pillars causing deeper indentation, their relatively lower curvature contributes to a reduced breaching of the nuclear membrane suggesting that while indentation depth contributes to the mechanical disruption of the nuclear envelope, it is the degree of curvature that more significantly dictates the likelihood of formation of membrane openings.

[0093] To assess whether changes in cell and nuclear morphology, specifically in area and circularity, contribute to the higher incidence of nuclear membrane breaches on smaller nanopillars, these aspects of cell and nuclear structure were compared. The analysis showed that cells on nanopillars had a smaller spreading area than those on flat surfaces, regardless of the nanopillar size, with no notable difference in cell area between small and large nanopillars. (FIG. 3L). Additionally, it was found that cell and nuclear circularity, rather than spreading area, varies between small and large nanopillars. (FIGS. 3L-O). This suggests that the distinct cell and nuclear circularity imposed by the size of the nanopillars may play a role in nuclear membrane breaching. See FIGS. 7A-E.

[0094] Nuclear envelope repair can be initiated through the recruitment of ESCRT-III complex proteins, including CHMP4B, which can be guided by LEM-domain proteins. DNA- binding proteins including Ku70 / 80 and sensors such as cGAS localize to exposed chromatin, initiating DNA damage response and promoting nuclear integrity restoration.

[0095] FIGS. 4A-E show diagrams, images, and data plots depicting an example nuclear envelope repair on nanotopography. FIG. 4A illustrates time-lapse imaging of U2OS cells expressing NLS-GFP undergoing nuclear membrane breach and repair. Notably, after 90 minutes, cells display signs of breaching with subsequent repair mechanisms relocalizing NLS- GFP to the nucleus after 2.5 hrs. U2OS cells. FIG. 4B shows the cytoplasmic and nuclear NLS-GFP signal ratio, illustrating the decreasing ratio after the rupture incident, which suggests activation of the repair mechanism. FIG. 4C presents an overall schematic and timelapse images of U2OS cells expressing NLS-GFP and cGAS-mCherry, showing membrane breach and repair. The cGAS-mCherry binds to the DNA exposed to the cytoplasm upon breaching. Cells exhibit breaching after 5 hrs., as indicated by mislocalization of NLS to the cytoplasm and the appearance of cGAS signals. Additionally, cells repair after 7 hours, while the cGAS remains in the breach sites as indicated by the profile analysis of cGAS and NLSover the yellow line. Representative images of U2OS cells transfected with NLS-GP and cGAS-mChery show the site of the membrane breach on nanopillars induced by the curvature. FIG. 4D displays the profile intensity of the NLS and cGAS, showing an increased cytoplasmic to nuclear NLS ratio followed by a sudden increase in the cGAS signals, which confirms the presence of both cGAs and NLS upon membrane opening. Additionally, after 7 hours, Ku-80 is retained to the while the cGAS signal is still present. FIG. 4E provides immunofluorescence microscopy images of U2OS cells, viewed both from the top and the side. The nuclei are stained with blue DAPI stain; Ku-80, which signals nuclear envelope breach, is in green; and CHMP4B, the red-stained ESCRT-III protein, is implicated in nuclear membrane repair. The highlighted yellow region within the images shows the area of nuclear envelope disruption — characterized by gaps in the DAPI and Ku- 80 where CHMP4B is localized — suggesting active repair processes. Side-view images further emphasize CHMP4B's presence at the sites of nuclear breach and repair on the nanopillars.

[0096] Whether nuclear membrane breaches induced by nanopillars are transient were also investigated. By conducting live-cell imaging of cells expressing NLS-GFP as a nuclear rupture reporter, it was observed that the nuclear membrane repairs itself within approximately 1 .5 hr after the breach, as shown in FIGS. 4A and 4B. This repair process is evident from the gradual decrease in the cytoplasmic to nuclear ratio of NLS-GFP following breach, followed by its complete relocalization to the nucleus after 1.5 hrs., indicating a successful repair mechanism. Additionally, the initiation of a rupture event coincided with the localized expression of a cytoplasmic DNA-binding protein fused to mCherry, Cyclic GMP-AMP synthase (cGAS), which marks the opening site and indicates that some DNA is exposed to the cytosol at these locations (FIGS. 4C and 4D). Furthermore, cGAS remained at the breach site even after the nuclear membrane was fully repaired and NLS-GFP had relocalized to the nucleus, corroborating previous studies that have demonstrated the persistent presence of cGAS at nuclear membrane rupture sites during cancer cell migration. Finally, through two- photon microscopy, it was shown that ESCRT-III repair machinery, specifically CHMP4B are recruited to nuclear membrane openings and aid in repairing the nuclear membrane (FIG.4E). Altogether, these results suggest that nanotopography-induced nuclear membrane openings are repairable and transient.

[0097] FIG. 4F shows data images and data plots from another example implementation of the nucleoporation method depicting the cytoplasmic and nuclear NLS-GFP signal over time prior to, at, and after nucleoporation induced by an example embodiment of the nucleoporation device. The example data demonstrates a rapid nucleoporation period (e.g., 15 min) andimmediate rapid self-repair (e.g., less than 60 minutes) of the cell on an example nucleoporation device that induced nucleoporation.

[0098] FIGS. 5A-C show diagrams, images, and data plots depicting example implementation results of cells on exemplary nanopillar structures. FIG. 5 A presents a 1-hr study of the U2OS cells on the nanopillar followed by SEM and immunofluorescence images. These images reveal that cells retain a round shape and remain non-ruptured, as confirmed by the profile intensity plot where DAPI and Ku-80 signals overlap, demonstrating the non-rupture status of cells. FIG. 5B shows results from a 5hr study of the U2OS cells on nanopillars, demonstrating that the cells spread over the nanopillars and exhibit nuclear deformation leading to nuclear rupture, as evidenced by the diffusion of Ku-80 into the cytoplasm, as confirmed by intensity profiles along the yellow line. FIG. 5C illustrates observations at the 8-hour time point, where further cell spreading is noted, and Ku-80 persists in the cytoplasm but at reduced concentrations, indicating the initiation of nuclear envelope repair mechanisms.

[0099] FIGS. 6A-C show diagrams, images, and data plots depicting example implementation results of nanopillar topography modulates nuclear envelope rupture. FIG. 6A presents fluorescent microscopy and SEM images of cells on flat surfaces, showing the spreading of U2-OS cells and intact nuclear envelopes with Ku-80 localized within the nucleus, suggesting no envelope rupture. FIG. 6B shows that cells cultured on low aspect ratio nanopillars have an effective attachment and spreading; however, the Ku-80 remains within the nuclear confines. FIG. 6C demonstrates that high AR nanopillars lead to significant cell and nuclear deformation and result in nuclear envelope rupture, as evidenced by the translocation of Ku- 80 from the nucleus to the cytoplasm.

[0100] FIGS. 7A-E show data plots depicting example results. FIG. 7A-7D show a comparison of ruptured and non-ruptured cells on small nanopillars, which do not show significant changes in cell spreading, and deformation and nuclear area while the nuclear deformation is significantly different in low aspect ratio and high aspect ratio pillars with ruptures cells indicating higher deformation. FIG. 7E shows a comparison of ruptured and non-ruptured Ku-80 in the cell nucleus revealing a significant difference in Ku-80 present in the non-ruptured cell nucleus compared to ruptured cell nucleus.

[0101] FIGS. 8A and 8B show images depicting an example implementation of human stem cells that underwent attempted gene editing using CAS9, where CAS9 was only in cell cytosol (FIG. 8A) due to delivery by conventional non- viral delivery method (lipof ection), and where CAS9 was in cellular nucleus (FIG. 8B) due to delivery by implementing a nucleoporation method in accordance with the present technology.

[0102] FIGS. 9A and 9B show methods for delivery of large plasmid DNA to the nucleus. FIG. 9A shows that initially, the plasmid is delivered to the cytoplasm using a carrier, such as a liposome. Subsequently, the plasmid translocates to the nucleus over several hours either during cell division (mitosis) or through active transport via the nuclear pore complex (NPC). This active transport is facilitated by binding to importin, a protein that recognizes a nuclear localization signal (NLS) often attached to the plasmid. FIG. 9B shows a novel method for nuclear delivery by inducing nanoscale curvature on the nuclear membrane through external nanotopography. This method results in a brief breach of the nuclear membrane, allowing the delivery of cargo directly into the nucleus.

[0103] FIG. 10A shows the direct nuclear delivery of plasmids using a nanopatterned platform during a curvature induced breach. Notably, the cytoplasm remains intact during this breach.

[0104] FIG. 11A shows platforms developed with various geometries by altering the diameter, spacing, and height of the nanopatterns. FIG. 11B shows that the platforms can induce varying degrees of curvature and indentation on the nuclear membrane. FIG. 11C shows that nanopillars can be fabricated by wet etching large pillars to obtain smaller pillars. FIG. HD demonstrates that smaller nanopillars induce higher curvature on nuclear membranes. FIG. HE demonstrates that despite their smaller size, these nanopillars cause smaller indentations. FIG. 1 IF shows a time-lapse image displaying the breach of the nuclear membrane and its repair within approximately one hour.

[0105] FIG. 12A shows that predefined algorithms, such as Biodock, can be used to accurately segment cells and their corresponding nuclei. FIG. 12B shows an optimized Line Drawing: For each cell, the optimal line is drawn based on three criteria: passing through the nucleus, being the longest possible line within the cell, and being the longest within the nucleus. This optimized line captures key structural features of both the cell and the nucleus. Pixel Profile Analysis: Normalized pixel intensity profiles were obtained for both the nuclear (DAPI) channel and the KU80 channel along the optimized lines. FIG. 12C shows Ruptured Nuclei: Pixel intensity profiles for KU80 show a more uniform distribution in ruptured nuclei, indicating a compromised nuclear envelope. FIG. 12D shows Non-Ruptured Nuclei: In nonruptured nuclei, pixel intensity profiles for KU80 and DAPI follow similar trends, suggesting an intact nuclear envelope. These analyses enable the automated distinction between ruptured and non-ruptured nuclei based on pixel intensity profiles.

[0106] FIG. 13A shows nuclear delivery of plasmid DNA and expression during mitosis (Mitotic). In cases on the flat / smooth surface, the expression coincided with a cell division(mitosis) event. During mitosis the nuclear membrane disassembles and the plasmid can therefore reach the nucleus and be expressed. FIG. 13B shows nuclear delivery of plasmid DNA and expression during interphase (Non-mitotic). On nanopillars, most expressions did not involve a mitosis event indicating a different pathway for nuclear entry on nanopillars.Exemplary Implementations of Systems, Devices, and Methods for Nucleoporation

[0107] Nanopillar Fabrication: Example implementations included the following example embodiments of methods in accordance with the present technology. In some examples, nanopillar chips fabricated on a 4-inch fused quartz wafer (Wafer Pro) and underwent RCA cleaning using both SCI and SC3 solutions, followed by a spin rinse and drying using a Spin Rinse Dryer (MEI). AZ 1512 photoresist (EMD Performance Materials). Spin coated at 4000 rpm for 45 seconds, accelerating at 12000 rpm during a three-step procedure, achieving a photoresist thickness of ~ 1.2 pm. Example custom design file (.gds) was used to pattern the wafers, which were then exposed using the Heidelberg MLA system at a wavelength of 375 nm and a dosage of 300. The substrate developed using AZ400 developer (AZ Electronic Materials) for 30 seconds. Chromium (Cr) with a purity of 99.998% was deposited onto the patterned substrates via a Temescal Ebeam evaporator. A lift-off process was carried out using RR41, acetone, and isopropyl alcohol (IPA) in sequence. The substrates dry etched for 50 minutes in an Oxford Plasmalab 80 using Reactive Ion Etching (RIE) system using argon (Ar) at 35 seem and chlorotrifluoromethane (ChF3) at 25 seem, under conditions of 50 millitorr and 200 watts.

[0108] Wafer wet etched for 10 minutes using a chromium etchant (Transene Company Inc.) followed by wet etching in buffered oxide etch (BOE) 20: 1 to remove the exposed quartz. The final step involved dicing the wafers into chips measuring 1 cm x 1 cm for further functionalization.

[0109] Nanopillar Preparation: For Gelatin NP chips were sterilized by rinsing with 70% ethanol, followed by deionized water, and air-dried. The chips UVO treated for 10 minutes and incubated with 100 pg / mL poly-L-lysine (PLL) solution. PLL was applied by inverting the chip onto a 50 pL droplet on parafilm, with the nanopillar side contacting the droplet, and incubated at room temperature for 30 minutes. The chip was then washed 3 times with PBS. 0.5% glutaraldehyde solution for added for 30 minutes to crosslink the proteins and washed with PBS. Example proteins can include various extracellular matrix (ECM) proteins or peptiedes that promote and / or facilitate cells to adhere to the surface on which the proteins are coated. Examples include but are not limited to fibronectin, vitronectin, osteopontin, laminin,collagen I, collagen IV, gelatin, elastin, Matrigel (basement-membrane extract), Geltrex, hyaluronic acid, heparan sulfate, silk fibroin, elastin-like polypeptides, RGD peptide, poly-L- lysine (PEL), poly-D-lysine (PDL), poly-L-omithine (PLO), polydopamine. Finally, a prewarmed 0.1% gelatin solution was applied, and the chip was incubated for another 30 minutes at room temperature.

[0110] To coat the nanopillar chips with FN for iPSC-CM and NIH-3T3 cells, the chips were sterilized and rinsed with 70% ethanol followed by deionized water, allowing them to air dry after each rinse. 50 pL of 50 pg / mL FN was applied and the chips were incubated in an incubator at 37°C for at least 2 hours or alternatively or overnight at 4°C.

[0111] Cell Culture and seeding: U2OS, NIH-3T3, iPSC-CM, HeLa, and HEK-293 cells were obtained. U2OS cells were cultured in McCoy's 5A medium (ACC) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and 1% penicillin-streptomycin (PenStrep, ThermoFisher Scientific). HeLa cells were maintained in Eagle's Minimum Essential Medium (EMEM, ATCC) with the same supplements. HEK-293 cells were grown in Dulbecco's Modified Eagle Medium (DMEM, ThermoFisher Scientific) with 1% PenStrep, and NIH-3T3 cells were cultured in DMEM supplemented with 10% FBS and 1% PenStrep. All cell lines were cultured under humidified conditions at 37°C and 5% CO2.

[0112] To seed cells onto NP chips, cells were first detached using TrypLE ™ Express Enzyme (Gibco) and a 100 pL suspension containing 50,000 cells was prepared. The NP chips were placed in a 24- well plate and rinsed three times with PBS. The droplet of cells was added and mixed thoroughly to ensure uniform distribution of cells. After 10 minutes of initial adhesion, 1 mL of growth media was added and the cells were incubated at 37°C for further analysis.

[0113] Plasmid transduction: Plasmid was expanded according to the Addgene agar stab protocol. Briefly, a bacterial colony was collected from the stab and streaked onto an LB agar plate (vendor).

[0114] All plasmids were streaked from provided bacterial stabs onto agarose plates containing the respective antibiotic and incubated overnight at 37°C. Individual colonies for each plasmid were selected and grown overnight in lOmL of LB Broth incubated with the appropriate antibiotic, at 37°C with shaking. After overnight growth, plasmid DNA was extracted by miniprep with the QIAprep Spin Miniprep Kit (ID 27104) from Qiagen. Miniprep procedure was followed as recommended by the manufacturer. Purified DNA plasmids were eluted in ultrapure H 2 O and DNA concentration and purity was validated through A260 ratios on a NanoDrop Lite Spectrophotometer from ThermoFisher.

[0115] Cell lines and transfections: U2OS cells were transfected using to express nuclear rupture indicator NLS-GFP (pCMV-PV-NLS-GFP) and cGAS-mCherry (pCDH-cGAS- E225A D227A-mCherry2-EFl-Puro for live imaging. The cells were starved in serum-free minimal medium (OPTI-MEM) for 30 minutes. A complex of 1 pg of plasmid was mixed with 3 pl of Viafect (Promega) in OPTI-MEM and mixed and incubated at room temperature for 20 minutes. The media exchanged with serum-free McCoy 5a, and complex added to the cells. After 1 day transfection medium was replaced with growth media and cells recovered before live imaging.

[0116] Immunostaining: Cells fixed using 4% paraformaldehyde (Electron Microscopy Sciences, USA), for a duration of 10 minutes at room temperature and washed with PBS to remove excess fixative. Cells were permeabilized with a 1% solution of Triton™ X-100 (Sigma-Aldrich, USA) for 10 minutes and blocked using a 2% w / v solution of Bovine Serum Albumin (BSA) (Thermo Scientific™, USA) for one hour.

[0117] Antibodies used for immunofluorescence included: Rabbit anti-Ku-80 (1 :200, Cell Signaling), Mouse anti-Lamin A / C (1 :400, Biolegend), and Rabbit anti-CHMP4B (1:100, Novus Biologicals). Secondary antibodies conjugated to Alexa Fluor 488, 594, or 647 (Thermo Fisher Scientific) were used at 1: 1,000. Samples were washed in PBS and stained with 4', 6- diamidino-2-phenylindole (DAPI) (Thermo Scientific™, USA) for 5 minutes and Alexa 594- phalloidin (Invitrogen™, USA) for 20 minutes in the dark to stain the nucleus and F-actin.

[0118] Time lapse imaging: For live imaging, nanopillar chips are placed on a glass bottom well plate. Echo Revolution System is used with 20x Olympus objective with stage top incubator (Live Cell Instrument) for the duration of the experiment. Cells are maintained at 37 °C and 5% CO2 throughout the experiment.

[0119] SEM imaging: Scanning Electron Microscopy (SEM) was performed using a FEI Apreo FESEM system. Imaging was conducted under low vacuum conditions and operated at 5 kV voltage. To enhance the visibility of the nanopillars' height, images were captured at a 30-45-degree tilt.

[0120] Cell samples were fixed and dehydrated for SEM imaging. Briefly, 2.5% glutaraldehyde added and incubate overnight at 4 degree and rinsed with PBS at least 3 times. Samples dehydrated through a series of ethanol washes, increasing the concentration from 30% to 100%, with each step lasting 10 minutes. Finally, sample covered with Hexamethyldisilazane (HMDS) for 15 minutes and HDMS removed and air dried overnight before SEM imaging.

[0121] Fluorescence and confocal microscopy: Images were collected with Echo Revolution microscope with following objectives. 20x PLAN Fluorite LWD CC Phase Phi NA 0.45, 40x PLAN Fluorite LWD CC Phase Ph2 NA 0.60, 60x PLAN Fluorite Water Dipping NA 1.00.

[0122] An upright laser-scanning microscope (DIY multiphoton, Olympus) with a 25x water objective (XLPLN, WMP2, 1.05 NA, Olympus) was applied for near-IR throughput. The setup included a synchronization of pulsed pump and stokes beams through a picoEmerald system (Applied Physics & Electronics), with the pump beam tunable between 780 and 990 nm, a pulse width of 5-6 picoseconds, and a repetition rate of 80 MHz, while the Stokes beam was fixed at 1031 nm with identical pulse width and repetition rate. Both beams were directed into the microscope and the transmission was collected via a high numerical aperture (NA) oil condenser (1.4NA). For the two-photon fluorescence (TPF) imaging, 781 nm and 810 nm pump beams were employed for capturing DAPI and FITC stained images, respectively. Each pixel was exposed for 12.5 microseconds, and images were averaged over three frames. The backscattered light was separated using a dual filter cube with passbands at 460 nm and 515 nm. All images were captured at a resolution of 512 x 512 pixels, and a z-step size of 1.

[0123] Image analysis: ImageJ 1.53 (NIH, US) used for image analysis to quantify nuclear envelope rupture via the cytoplasmic to nuclear ratio of Ku- 80 protein intensity. Images were processed to select the nuclear and cytoplasmic area using immunofluorescence staining; Actin staining provided the cellular outline, while DAPI staining defined nuclear regions. To calculate the cytoplasmic / nuclear ratio of Ku-80, intensity measurements were performed separately on cytoplasmic and nucleic regions. Initially, the DAPI channel was used to threshold and define nuclear areas, followed by measurement of Ku-80 intensity within these boundaries. Subsequently, the Actin channel outlined the entire cell for assessing total cellular Ku-80 intensity. The area and circularity of cells and nucleus calculated using analyze particle command as follow:Circularitywhere A shows the area and P is the indicator of perimeter.

[0124] To quantify the curvature characteristics of an ellipse relative to a circular geometry with an equivalent area, the images are resliced from confocal imaging and reconstructed the side view of the images for fitting the ellipse, the normalized maximum curvature (KnOrm) is calculated using image! for calculating the major axis (a) and minor axis (b) of ellipse.

[0125] The maximum curvature of the ellipse is defined as below, where it is assumed that a > b > 0:

[0126] To normalize the ellipse's curvature, the curvature of a circle whose area is equal to that of the ellipse is used. The curvature of a circle with the same area as the ellipse defined by:

[0127] The normalized maximum curvature is then calculated by dividing the maximum curvature of the ellipse by the curvature of the equivalent circle:

[0128] To differentiate between ruptured and non-ruptured cells based on Ku-80 intensity ratios, Gaussian Mixture Models (GMM) is utilized. This statistical method models data as a combination of Gaussian distributions, each representing a different cluster within the dataset. In the analysis, GMM was applied to identify two clusters: one for ruptured cells and another for non-ruptured cells. The intersection of these distributions provided a data-driven threshold, optimizing the distinction between the two states. The GMM determined a threshold value of 0.16, which effectively separated cells with ruptured nuclei from those intact, highlighting its utility in refining the analysis without relying on arbitrary preset values.

[0129] Statistical analysis: Unless otherwise noted, all experimental results were taken from at least three independent experiments. For comparisons, student's t-tests (comparing two groups) or one-way ANOVA (for experiments with more than two groups) with post-hoc tests were used. All tests were performed using GraphPad Prism., * denotes p <0.05, ** denotes p <0.01, and *** denotes p <0.001. Unless otherwise indicated, error bars represent the standard error of the mean (SEM).Exemplary Implementations of Studying the Mechanism of Nucleoporation

[0130] Example implementations were conducted for studying the mechanism of nuclear membrane disruption and breach induced by the disclosed nucleoporation devices, systems, and methods.

[0131] As discussed above, the nuclear envelope (NE) serves as a critical barrier separating and regulating the materials exchange between the cytoplasm and nucleus maintaining cellular integrity and function. In pathological conditions, increased levels of NE rupture can be linked to several degenerative diseases, such as laminopathies including premature ageing syndromes, muscular dystrophies, and dilated cardiomyopathies. Furthermore, cancer cells can experience NE rupture as they migrate through tight constrictions to invade neighboring tissue, potentially driving genome instability and enabling tumor progression. Under physiological conditions, NE rupture can occur at sites of high cytoskeletal stress and curvature for many cell types.

[0132] Yet, in all these pathophysiological scenarios, despite the significant disruption of NE, cells maintain their ability to function after NE rupture suggesting the existence of evolutionarily conserved mechanisms for NE repair. Therefore, modulating NE rupture and repair is an aim of the disclosed technology for creating capabilities to model disease and develop new therapies, which the disclosed technology can provide with high throughput to induce NE rupture and repair mechanisms for various cell types. NE rupture can provide access to the nucleus which paves the way for the direct intranuclear delivery of the cargo.

[0133] The example implementations of example embodiments of the nucleoporation device 110, discussed below, demonstrate that nuclear curvatures induced by nanoscale surface topography can cause NE rupture and recruit NE repair machinery. This provides a simple, high-throughput, and versatile platform for examining NE rupture and repair dynamics under various conditions. The example implementations further investigate the mechanisms of NE rupture on nanotopography, focusing on the role of key regulators of NE integrity, including nucleoskeletal and cytoskeletal elements, as well as lipid composition. The example results show that decreased Lamin A / C levels significantly increase the incidence of nanotopography- induced NE rupture by altering the shape of nuclei. Additionally, the example findings show acto-myosin contractility actin polymerization plays significant roles in NE rupture on nanotopographies; however, actin polymerization has a more pronounce impact on inducing NE rupture on nanotopographies. Furthermore, through advanced Stimulated Raman Scattering (SRS) imaging of lipids, the example results indicate that ruptured cells exhibit lower levels of unsaturated lipids, suggesting new roles for lipid metabolism in NE rupture.

[0134] The example implementations were carried out as follows. For example, example embodiments of the nucleoporation device 110 that included an array of nanopillars as the nanostructures 112 (referred to below as “nanopillar chips”) were produced on a 4-inch fused quartz wafer. Initially, the wafers were subjected to RCA cleaning using SCI and SC3 solutions. Post-cleaning, a spin rinse dryer was used for spin rinsing and drying. The AZ 1512 photoresist was applied using a spin coating process set at 4000 rpm for 45 seconds with an acceleration phase reaching 12000 rpm, resulting in a photoresist layer approximately 1.2 pm thick. The patterning of the wafers was carried out using a custom design file (.gds) and the Heidelberg MLA system, which exposed the photoresist at a 375 nm wavelength and a dosage of 300. After exposure, the wafers were developed in AZ400 developer for 30 seconds, for example. Subsequently, a Temescal Ebeam evaporator was used to deposit a 99.998% pure chromium layer onto the patterned substrates. A lift-off process followed, utilizing RR41, acetone, and isopropyl alcohol (IP A) sequentially to remove excess material. The substrates then underwent dry etching for 50 minutes in a Reactive Ion Etching (RIE) system. For example, this process involved using argon (Ar) at a flow rate of 35 seem and chlorotrifluoromethane (ChF3) at 25 seem, under a pressure of 50 millitorr and a power setting of 200 watts. For the final steps, the wafers were wet etched for 10 minutes using a chromium etchant from Transene Company Inc., followed by a buffered oxide etch (BOE) 20: 1 to remove the exposed quartz areas. The completed wafers were then diced into 1 cm x 1 cm chips, ready for further functionalization. Scanning Electron Microscopy (SEM) images revealed the precise dimensions and alignment of the nanopillars, with a height of 3.24+0.25 m a pitch of 3.49+0.06 pm and a diameter of 0.90+0.016pm. To prepare for cell culture, chips were sterilized with 70% ethanol, UVO treated for 10 minutes and incubated with a 100 pg / mL poly- L-lysine (PLL) solution for 30 minutes. After washing three times with PBS, a 0.5% glutaraldehyde solution was added for 30 minutes to crosslink the proteins, followed by another PBS wash. Finally, a pre-warmed 0.1% gelatin solution was applied, and the chips were incubated for 30 minutes at room temperature.

[0135] Cell Culture. U2OS cells (were cultured in McCoy’s 5 A Medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, and maintained at 37 °C with 5% CO2. Cells at passages 2-8 were detached using TrypLE™ Express Enzyme (IX). After centrifugation, the supernatant was removed to obtain a cell pellet. The pellet was resuspended in McCoy’s 5 A Medium. A droplet of cell suspension with 50k cell was added to each nanopillar chip and incubated at 37 °C for 8 hours for full attachment and spread.

[0136] Drug Treatment. For the example myosin II inhibition experiments, U2OS cells were treated with 10 pM blebbistatin (Blebb, EMD Millipore). Cells were incubated at 37°C for 8 hours before fixation. For actin polymerization inhibition experiments, U2OS cells were treated with 1 pg / mL cytochalasin D (CytoD). CytoD was reconstituted in DMSO to a concentration of 1 mg / mL andl pL of the CytoD solution was added to 1 mL of media, resulting in a final concentration of 1 pg / mL.

[0137] Cell transfections. The following were used: pCMV-PV-NLS-GFP (Addgene plasmid # 17300; http: / / n2t.net / addgene: 17300 ; RRID: Addgene_ 17300); and pCDH-cGAS- E225A D227A-mCherry2-EFl-Puro (Addgene plasmid # 132771; http: / / n2t.net / addgene: 132771; RRID:Addgene_132771). U2OS cells were transfected to express the nuclear rupture indicator NLS-GFP (pCMV-PV-NLS-GFP) and cGAS-mCherry (pCDH-cGAS-E225A D227A-mCherry2-EFl-Puro) for live imaging. The cells were first starved in serum-free minimal medium (OPTI-MEM) for 30 minutes. To prepare the transfection complex, 1 pg of plasmid DNA was mixed with 3 pL of Viafect in OPTI-MEM, then incubated at room temperature for 20 minutes. The culture medium was then replaced with serum-free McCoy’s 5 A, and the transfection complex was added to the cells. After 24 hours, the transfection medium was replaced with growth media, and the cells were allowed to recover before live imaging. For Lamin A / C silencing experiments, U2OS cells were transfected with Lamin A / C siRNA (sc-35776) using the siRNA Transfection Reagent (sc- 29528) and siRNA Transfection Medium (sc-36868). The siRNA was reconstituted in RNAse- free water to a final concentration of 10 pM. Cells were treated with the siRNA following the manufacturer’s protocol, and the efficiency of Lamin A / C knockdown was confirmed by immunofluorescence staining using Lamin A / C antibodies.

[0138] Immunostaining. Cells were initially fixed with 4% paraformaldehyde at room temperature for 10 minutes. After fixation, cells were rinsed with PBS to eliminate any residual fixative. Permeabilization was performed using a 1% Triton™ X-100 solution, e.g., for 10 minutes. Subsequently, cells were blocked with a 2% w / v Bovine Serum Albumin (BSA) solution, e.g., for one hour. For immunofluorescence staining, Rabbit anti-Ku-80 (1 :200) and Mouse anti-Lamin A / C (1 :400) were used as primary antibodies. The secondary antibodies, conjugated to Alexa Fluor 488, 594, or 647, were applied at a dilution of 1:1,000. Following antibody incubation, samples were washed with PBS. To visualize the nucleus and F-actin, cells were stained with 4’,6-diamidino-2-phenylindole (DAPI) for 5 minutes and Alexa 594- phalloidin for 20 minutes, both in the dark.

[0139] Fluorescence microscopy. Images were captured using an Echo Revolution microscope, equipped with several objectives: a 20x PLAN Fluorite LWD CC Phase Phi with a numerical aperture (NA) of 0.45, a 40x PLAN Fluorite LWD CC Phase Ph2 with a NA of 0.60, and a 60x PLAN Fluorite Water Dipping objective with a NA of 1.00.

[0140] Multimodal optical imaging system. An upright laser-scanning microscope (DIY multiphoton, Olympus) with a 25x water objective (XLPLN, WMP2, 1.05 NA, Olympus) was used for near-IR imaging. The setup included synchronization of pulsed pump and Stokes beams through a picoEmerald system (Applied Physics & Electronics), with the pump beam tunable between 780 and 990 nm, a pulse width of 5-6 picoseconds, and a repetition rate of 80 MHz. The Stokes beam was fixed at 1031 nm with the same pulse width and repetition rate. Both beams were directed into the microscope, and transmission was collected via a high numerical aperture (NA) oil condenser (1.4 NA). For two-photon fluorescence (TPF) imaging, 781 nm and 810 nm pump beams were used to capture DAPI and FITC stained images, respectively. Each pixel was exposed for 12.5 microseconds, and images were averaged over three frames. The backscattered light was separated using a dual filter cube with passbands at 460 nm and 515 nm.

[0141] In stimulated Raman scattering (SRS) imaging mode, a high optical density (O.D.) shortpass filter from Thorlabs, with a cutoff at 950 nm, was used to block the Stokes beam while transmitting the pump beam to a silicon photodiode. This configuration was set up for the detection of the stimulated Raman loss signal. The signal from the photodiode was processed through a filter and demodulated by a lock-in amplifier operating at 20 MHz. The resultant demodulated current was integrated into Olympus' FV3000 software module FV-OSR to produce images during the scanning process. All 3D images were captured at a resolution of 512 x 512 pixels, with a z-step size of 1 pm.

[0142] Scanning Electron Microscopy imaging. The nanopillar structures were analyzed using a FEI Apreo Field Emission Scanning Electron Microscope (FESEM). The imaging process was carried out under low vacuum conditions with an operating voltage of 5 kV. To clearly display the height of the nanopillars, the samples were tilted between 30 and 45 degrees during imaging.

[0143] Image analysis. Image analysis was performed using ImageJ 1.53 (NIH, US). For quantifying nuclear envelope rupture via the cytoplasmic to nuclear ratio of Ku- 80 protein intensity, immunofluorescence staining was employed to identify nuclear and cytoplasmic areas. Actin staining outlined the entire cell, while DAPI staining defined nuclear regions. The DAPI channel was used to threshold and delineate nuclear areas, followed by measuring Ku-80 intensity within these nuclear boundaries. Subsequently, the Actin channel was used to outline the entire cell, allowing for the assessment of total cellular Ku-80 intensity. Analysis of the cytoplasmic Ku-80 images was conducted by subtracting the background intensity from every pixel in the Ku- 80 image and removing all signal within the nuclear region, defined by the bounds of Hoechst staining.

[0144] Rupture determination. The ratio of a protein’s total cytoplasmic intensity to its total nuclear intensity (cytoplasmic / nuclear ratio) in immunofluorescence images of fixed cells is determined using anti-KU80. This ratio can vary for nonruptured nuclei in images. Based on fluorescent images of anti-KU80, nonruptured nuclei have a specific cytoplasmic / nuclear ratio. This ratio reflects changes in protein localization caused by rupture, making it a reliable method for detecting nuclear envelope rupture. To confirm and validate this method, we used Gaussian Mixture Models (GMM). This statistical approach models data as a combination of Gaussian distributions, each representing a different cluster. In our analysis, GMM identified two clusters: one for ruptured cells and another for non-ruptured cells. The point where these distributions intersected provided a data-driven threshold, optimizing the distinction between the two states. The GMM determined a threshold value of 0.16, effectively separating cells with ruptured nuclei from those with intact nuclei.

[0145] H2AX foci determination. H2AX foci analysis was performed using ImageJ. Z- stack images were converted to maximum intensity projections (MIP) using the “Z Project” function. Deconvolution was performed with a PSF generator plugin considering microscope specifications (NA=1.4 for a 60X objective, refractive index 1.3). Deconvolved images were analyzed for clarity and foci definition improvement. The LUT was adjusted to red with intensity levels set between 0-100 or 0-200. Nuclei were segmented based on DAPI staining and converted to binary for foci counting using the "Threshold" function. Foci were counted using “Analyze Particles” with a size range of 10-100 pixels, ensuring no duplicates. Data on foci counts and sizes were compiled for statistical analysis to compare different conditions.

[0146] Statistical analysis. Unless otherwise noted, the example experimental results were taken from at least three independents experiments. For comparisons, we used student’s t-tests (comparing two groups) or one-way ANOVA (for experiments with more than two groups) with post-hoc tests. All tests were performed using GraphPad Prism., * denotes p <0.05, ** denotes p <0.01, and *** denotes p <0.001. Unless otherwise indicated, error bars represent the standard deviation (SD).

[0147] The example results of the example implementations included characterization of cell-nanopillar interactions leading to nuclear envelope rupture.

[0148] FIGS. 14A-14M show diagrams, images, and data plots depicting example characterization results of cell-nanopillar interaction using an example embodiment of the nucleoporation device 1 10. FIG. 14A shows a graphical illustration of current mechanical methods for inducing nuclear envelope rupture including Atomic Force Microscopy (AFM), Vapor nanobubbles (VNB) and microfluidic constriction, which, in contrast with the disclosed technology, are ineffective at controllably disrupting the integrity of the nuclear membrane without causing cell damage. FIG. 14B shows a high-resolution confocal fluorescence image of the nucleus of a U2OS cell indented by the nanopillars on an exemplary nanopillar chip. The image shows the indentation and curvature of the nucleus in side-view and 3D (scale bar: 5 pm). FIG. 14C shows schematic illustrations of cell interactions with nanopillars. The normal condition illustrates the nuclear deformation without rupture where the Ku-80 (Green) signal overlaps with the nucleus (D API-blue). FIG. 14D shows schematic illustrations of cell interactions with nanopillars where in the ruptured state Ku-80 mislocalized to the cytoplasm (scale bars: 10 pm). FIG. 14E shows immunofluorescence microscopy images of U2OS cells cultured on nanopillar substrates, DAPI (cyan), Ku-80 (green), F-Actin (red) and Lamin A / C (magenta) (scale bars: 20 pm). FIG. 14F shows a data plot depicting the quantification of cytoplasmic to nuclear intensity of Ku-80, which demonstrates significant increase in the Ku- 80 ratio on nanopillars associated with higher rupture incidence. FIG. 14G shows a data plot depicting the quantification of cytoplasmic cGAS, which indicates 9.3 % of cells on flat and 61.9% cells on nanopillars showing cGAS accumulation as a rupture indicator. FIG. 14H shows fluorescence microscopy images showing the presence of cGAS (red) and DNA (cyan) in cells cultured on nanopillar substrates, highlighting nuclear rupture sites (indicated by arrows) (scale bars: 5 pm). FIG. 141 shows a data plot depicting the quantification of yH2AX foci in cells cultured on flat and nanopillar substrates. Cells on nanopillar substrates show increased DNA damage, indicated by a higher number of yH2AX foci that can be associated with higher number of NER (scale bars: 5 pm). FIG. 14J shows representative images of nucleus stained with yH2AX shows higher number of foci.

[0149] To determine whether nanotopography can induce NE rupture, an example embodiment of the nucleoporation device 110 was used, which included arrays of glass nanopillars and which were interfaced with U2OS cells. 3D reconstruction of the nucleus on the exemplary nanopillar chip platform using confocal microscopy revealed nanoscale indentations of the nucleus and presence of highly curved surfaces on the NE, indicating that the cell nucleus adapts to the nanotopography of the nanopillars, e.g., shown in FIG. 14B. The example data of FIGS. 14B-14H show nanotopographies can induce transient openings in thenuclear membrane, and these topographical features cause significant NE rupture, accompanied by nuclear deformation and the activation of repair machinery.

[0150] To detect NE rupture caused by these induced curvatures, cells were fixed and immunostained for a NE rupture reporter, Ku-80, which exclusively localized to the nucleus when the NE was intact (FIG. 14C) and diffused to the cytoplasm during NE rupture (FIG. 14D). To quantify NE rupture incidence as well as cell and nuclear morphologies, we used a DAPI and Lamin A / C stains to identify nuclear boundaries, and actin to identify the cell boundaries (FIG. 14E). A comparison of cytoplasmic to nuclear ratios of Ku-80 on flat surfaces vs the exemplary nanopillar chip platform indicated high incidence of NE rupture on nanotopography (FIG. 14F). Additionally, cells and their nuclei exhibited significant morphological changes on nanopillars. Specifically, cell areas increased while nuclear areas decreased on nanopillars, as shown in FIG. 14K. Furthermore, nuclear circularity was lower on nanopillars suggesting a higher degree of deformation of the nuclei on nanopillars, consistent with observations in other studies, as demonstrated in the plots of FIG. 14K.

[0151] It is understood that NE rupture can result in the exposure of double- stranded DNA to the cytoplasm, DNA damage, and the activation of repair response. The example experimental implementations addressed this issue whether similar deleterious processes would occur on nanotopograpy-induced NE rupture. Exposure of double- stranded DNA to the cytoplasm is indicated by the accumulation of cyclic GMP-AMP synthase (cGAS) at the NE. cGAS is a cytosolic DNA sensor that detects the presence of double- stranded DNA in the cytoplasm. Upon detecting cytoplasmic DNA, cGAS synthesizes cyclic GMP-AMP (cGAMP), which then activates the stimulator of interferon genes (STING) pathway, leading to the production of type I interferons and other inflammatory cytokines. This signaling cascade is crucial for initiating an immune response to DNA damage and facilitating repair processes.

[0152] The example results of the implemented experiments showed that cGAS accumulates at the NE upon nanotopography-induced NE rupture and remains present even after NE repair, as demonstrated in FIG. 14L. Quantitative analysis and representative fluorescent images of the cells on flat and nanopillar showed cGAS accumulation in 61.9% of cells on nanopillars, while only 9.3% of cells on flat showed cGAS accumulation (FIG. 14G and 14H). These example results suggest that DNA is exposed to the cytoplasm during nanotopography-induced NE rupture and that the STING pathway may be increasingly activated on nanopillars.

[0153] DNA damage can be identified through the presence of vH2AX foci in the nucleus. Histone variant H2AX is rapidly phosphorylated to form yH2AX at sites of DNA double-strandbreaks, serving as a marker for DNA damage. Quantification of yH2AX foci, as shown in FIG. 141, demonstrates a significant increase in the number of foci in cells cultured on nanopillars compared to those on flat surfaces. This significant increase demonstrates higher DNA damage and repair activity due to mechanical stress induced by the curvature. Overlay images of yH2AX and DAPI highlighted the presence of DNA damage foci within the nucleus (FIG. 14J).

[0154] Activation of NE repair machinery, namely ESCRT-III proteins has been observed upon NE rupture. It was found that ESCRT-III protein CHMP4B accumulates as sites of nanotopography-induced NE rupture. Additionally, we performed SRS imaging on these samples to investigate the localization of ESCRT-III protein, CHMP4B with newly protein synthesis. Interestingly, it was observed in FIG. 14M that the co-localization of CHMP4B protein stain with sites of new protein synthesis, indicating its active involvement in the NE repair process.

[0155] These example results collectively indicate that external nanoscale surface topography can induce NE rupture, cause DNA damage and activate ESCRT-III mediated repair machinery offering a versatile and simple platform for studying curvature induced NE rupture and repair and its downstream consequences.

[0156] To better understand the mechanisms of nanotopography-induced NE rupture, the role of key components involved in the maintenance of NE integrity was also investigated in the example implementations.

[0157] Lower Lamin A / C levels increase incidence of nanotopogrpahy-induced NE rupture. One of the major contributors to NE integrity is Lamin A / C, which provides structural support to the NE by forming a dense network under the inner nuclear membrane. Decreased levels of Lamin A / C lower nuclear stiffness and increase local NE deformations. Thus, the example implementations investigated whether nuclear deformations around the nanopillars leading to NE rupture is dependent on lamin A / C levels which impact nuclear stiffness. To test this, Lamin A / C was silenced using small interfering RNA (siRNA) targeting the LMNA gene (siLMNA).

[0158] FIG. 15 A- 15H shows example data plots and images depicting the role of Lamin A / C on nanotopography-induced nuclear rupture. FIG. 15A shows a data plot depicting quantitative analysis of the Lamin A / C intensity in control U2OS cells and Lamin A / C silenced (siLMNA) cells showing significant decrease on Lamin A / C intensity on siLMNA cells, e.g., indicating successful Lamin silencing of cells. FIG. 15B shows a data plot depicting the correlation of Lamin A / C intensity with cytoplasmic to nuclear of ku-80 ratio which is anindicator of NER. Higher Lamin A / C is associated with lower Ku-80 cyto / nuc ratio demonstrating the impact if Lamin A / C on nanoropography -induced rupture incidence. FIG. 15C shows immunofluorescent images of control and siLMNA-treated cells stained for DAPI (blue), Ku-80 (green), and Lamin A / C (magenta), showing the nuclear envelope deformation and Ku-80 mislocalization in siLMNA-treated cells, e.g., indicating nuclear envelope rupture. FIG. 15D shows a data plot depicting quantification of the Ku-80 cytoplasmic-to-nuclear ratio, demonstrating higher ratios in siLMNA-treated cells, e.g., indicating increased nuclear envelope rupture incidence of 54% compared to 23% of control sample. FIG. 15E and FIG. 15F show data plots depicting the quantitative analysis of cellular area and circularity shows similar area and circularity of siLMNA and control cells. FIG. 15E and FIG. 15F show data plots depicting the nuclear area and circularity demonstrates higher nuclear area in siLMNA sample compared to the control sample while the circularity remains similar which can be related to the higher adaption of nucleus on siLMNA cells to the nanopillar resulting in the higher surface area.

[0159] It was observed that a significant decrease in Lamin A / C intensity in siLMNA- treated cells, confirming successful silencing of siLMNA genes (FIG. 15A). Lower Lamin A / C levels were associated with higher Ku-80 cytoplasmic / nuclear ratios (FIG. 15B, 15C) indicating that depletion of Lamin A / C increases the probability of nanotopography-induced NE rupture. Interestingly the ruptured cells in the siLMNA sample (shown by arrows) indicated lower Lamin A / C levels. Further quantification of the Ku-80 cytoplasmic-to-nuclear ratio showed that 54% of siLMNA-treated cells exhibit nanotopography-induced NE rupture compared to 23% of control cells (FIG. 15D). When comparing cellular and nuclear morphologies, no change was observed in cell area and circularity between siLMNA-treated and control cells (FIGS. 15E, 15F). However, the nuclear area significantly increased in siLMNA-treated cells suggesting an increased deformation of the nuclei around the nanopillars leading to the flattening of the nucleus (FIGS. 15G, 15H). These example results suggest that reduced Lamin A / C levels increase NE rupture incidence suggesting that nuclear stiffness plays an important role in nanotopography-induced NE rupture.

[0160] The role of cytoskeletal dynamics in nanotopogrpahy-induced NE rupture was also studied by the example implementations.

[0161] Actin polymerization but not contraction is required for nanotopogrpahy-induced NE rupture. Following the study of the role of Lamin A / C in maintaining NE integrity, the contribution of cytoskeletal dynamics to nanotopogrpahy-induced NE rupture was explored. Actin dynamics, involving both polymerization and acto-myosin contraction, are anothercrucial factor for maintaining cellular and nuclear architecture. Actin filaments form a dynamic cytoskeletal network that interacts with the NE, influencing its mechanical properties and stability.

[0162] FIGS. 16A-16J show images and data plots depicting the effects of actin contraction and polymerization on nuclear envelope rupture. FIG. 16A shows representative immunofluorescent images of U2OS cells under DMSO, Blebb, and CytoD treatments. Staining includes DAPI (blue) for nuclei, Ku- 80 (green) for nuclear envelope rupture, and Actin (red) for cytoskeleton visualization. The white arrow in the DMSO-treated cell image indicates a nuclear envelope rupture cells. FIG. 16B shows a data plot depicting the cytoplasmic-to-nuclear Ku-80 ratio for cells treated with DMSO (control), Blebbistatin (Blebb), and Cytochalasin D (CytoD). The line indicates the threshold for nuclear envelope rupture. Control group showing a higher percentage of ruptured cells (44%) compared to Blebb (18%) and CytoD (2%) samples, demonstrating the importance of actin-myosin contraction and actin polymerization on the NER. FIGS. 16C-16F show data plots depicting the morphological analysis of the cells on nanopillars treated with Blebb and CytoD, which show CytoD significantly decreases the cell and nuclear area while increase the cell and nuclear circularity due to inhibiting the actin polymerization, while the area and circularity of cells and nucleus remains similar on sample treated with Blebb sample. FIGS. 16G-16J show data plots depicting the quantitative comparison of cellular morphologies between control (DMSO) and Blebbistatin (Blebb) treated cells for ruptured and non-ruptured cells, and nuclear area and circularity of ruptured cells significant differences on non-ruptured cells on control sample and sample treated with Blebb. Cells that experienced NER showed lower cellular and nuclear area compared to the control sample experiencing rupture while the nuclear circularity remains similar indicating nuclear deformation can be major contributor to the nuclear envelope rupture.

[0163] To investigate the role of actin dynamics in nanotopogrpahy-induced NE rupture, cells were treated with Blebbistatin (Blebb) to inhibit acto-myosin contraction and Cytochalasin D (CytoD) to inhibit actin polymerization (FIG. 16A). Although inhibiting actomyosin contraction reduced nanotopography-induced NE rupture incidence by over 50%, it did not completely eliminate rupture, indicating that acto-myosin contraction is important, but not essential for rupture (FIG. 16B). On the other hand, inhibiting actin polymerization decreased nanotopography-induced NE rupture incidence by over 95% suggesting its crucial role in rupture incidence (FIG. 16B).

[0164] Through analyzing cell and nuclear morphologies, it was shown that inhibiting actin polymerization on nanopillars significantly decreased cell and nuclear area whileincreasing circularity, suggesting a reduction in cell spreading and adhesion to the nanopillars in these cells, potentially reducing local NE deformations at the nanopillars (FIGS. 16C-16F). In contrast, inhibiting acto-myosin contraction resulted in no overall change in cell or nuclear area or circularity (FIGS. 16C-16F). However, without acto-myosin contraction, cells exhibited NE rupture at lower cell and nuclear areas, when compared to control NE ruptured cells (FIGS. 16G-16I). These example results suggesting that the dynamics of cell-nanopillar interactions and the local deformations at the cell / nanopillar interface are altered with inhibition of acto-myosin contraction.

[0165] Collectively these findings indicate that actin polymerization is crucial for nanotopogrpahy-induced NE rupture, as evidenced by significant morphological changes and reduced rupture in CytoD-treated cells. Inhibiting actin-myosin contraction with Blebb had a less pronounced effect on nanotopogrpahy-induced NE rupture.

[0166] The role of lipid saturation and unsaturation in nanotopogrpahy-induced NE rupture was also studied by the example implementations.

[0167] Building upon the findings on the roles of Lamin A / C and actin dynamics in maintaining nuclear envelope (NE) integrity, the impact of lipid composition on nuclear stability was also studied. The integrity and functionality of cell membranes, including the nuclear envelope, are heavily influenced by their lipid compositions. Lipids are important in cell signaling and membrane dynamics, which can influence the flexibility, fluidity, and stability of cell membrane. Specifically, the balance between saturated fatty acids (SFAs) and unsaturated fatty acids (USFAs) plays a vital role in modulating membrane rigidity. Therefore, Stimulated Raman Scattering (SRS) imaging was employed, providing a cutting-edge label- free technology with high spatial resolution that enabled us to visualize and quantify the lipid compositions of U2OS cells cultured on nanopillar arrays in 3D.

[0168] FIGS. 17A-17C show images and data plots depicting 3D SRS imaging of lipid composition of U2OS cells cultured on example nanopillars. FIG. 17A shows an image panel from 3D imaging of normal cell and ruptured cell on nanopillars showing various biomolecules. FIG. 17B shows an image panel of maximum projection of 3D images from FIG. 17A that highlight the total SFAs and USFAs from normal and ruptured cell. The ratiometric images of USFAs I SFAs show the spatial distribution of lipid unsaturation metabolism within cells. FIG. 17C shows a data plot depicting the quantitative analysis of the lipid unsaturation between normal and ruptured cells. Statistical significance was determined by using two-way t-test. *, p < 0.05. The scale bar in FIGS. 17 A and 17B is 20 pm.

[0169] As observed in FIG. 17A, the nuclear region exhibited significant amount of SFAs and USFAs. To further explore the role of lipid metabolism in rupture state of the cells, the ratiometric images of USFAs / SFAs were used as the lipid unsaturation index in normal and ruptured cells, which correlated with the rigidity of cell membrane (FIG. 17B). The example findings revealed that normal cells showed higher lipid unsaturation index compared to the ruptured cells (FIG. 17C). The example results demonstrated the increased rigidity associated with SFAs likely contributes to the observed NE rupture in cells on nanopillars.

[0170] These example implementations reveal how cells experience nuclear envelope rupture and repair, e.g., identifying and examining the role of critical factors such as nucleoskeletal elements, cytoskeletal dynamics, and lipid composition in rupture event. These example results offer insights into the cellular properties that can impact the rupture and repair event. These example findings not only can shape an understanding of nuclear mechanics but also how cells behavior can be modulated by the external cues, e.g., such as the engineered surface topographies and biomaterials in accordance with the present technology.

[0171] The example findings highlight the critical role of Lamin A / C in maintaining nuclear integrity, where reduced levels lead to increased rupture. Similarly, the role of actin polymerization and actomyosin contractility investigated that showed critical role of actin polymerization. Additionally, the lipid composition of the nuclear envelope proved to be a crucial factor in nuclear stability. The example results shows that cells undergo NE event have lower unsaturation of fatty acid correlating to membrane rigidity. This is particularly important in diseases modeling, e.g., such as cancer where NE integrity can be critical for tumor progression. Additionally, the example implementations show that biomaterials can be tuned to interact with cellular structures to mimic the tissue environment.Exemplary Method for Fabrication of Nucleoporation Devices

[0172] In some aspects, the disclosed technology includes a method of fabricating various example embodiments of the nucleoporation device 110.

[0173] In some embodiments, the fabrication method can include a process to form a plurality of nanostructures (e.g., nanostructures 112) on a surface of a substrate (e.g., substrate 111) to have a first structural configuration comprising a first diameter or length, a first height, and a first spacing of at least one nanostructure of the plurality of nanostructures from another nanostructure of the plurality of nanostructures. The fabrication method can include a process to size the formed nanostructures to remove structural aspects of the first structural configuration of each nanostructure by a deleterious etching process to produce a plurality offinal nanostructures on the substrate. For instance, the plurality of final nanostructures on the substrate include defined surface curvatures and are configured to impose nanoscale geometric and / or physical constraints on a cell based on contact of the cell with the final nanostructures that cause the cell to mechanically deform or reshape at least a portion of a cell body and / or its nucleus.

[0174] FIG. 18 shows a schematic diagram of an example embodiment of at least a portion of the fabrication method. For example, the fabrication method shown in FIG. 18, labeled method 1800, includes a pre-processing protocol that includes a process 1801 to provide a substrate (e.g., wafer including silicon and / or silicon dioxide); a process 1802 to apply a photoresist layer (e.g. , AZ 1512) on the substrate, e.g. , by spin coating. The fabrication method includes the process to form the nanostructures on the substrate that includes a process 1803 to remove portions of the photoresist layer through laser exposure that exposes portions of the substrate (e.g., based on a pattern); a process 1804 to deposit a chromium layer on the photoresist layer and exposed substrate; a process 1805 to remove the photoresist, e.g., through lift off process, thereby resulting in the patterned chromium on the previously-exposed substrate; and a process 1806 to create nanopillar structures by etching, e.g., dry reactive ion etching, the nanopillar structures from the substrate not protected by the deposited chromium layer, and a process 1807 to remove the chromium layer (e.g., wet etching).Examples

[0175] In some embodiments in accordance with the present technology (example Al), a method for delivering molecules into a nucleus of a cell includes creating one or more pores in the nucleus of the cell by imposing one or more nanoscale geometric and / or physical constraints on the cell; and enabling unregulated entry of molecules into the nucleus, wherein the one or more nanoscale geometric and / or physical constraints on the cell do not cause puncture of or penetration through a cellular membrane of the cell.

[0176] Example A2 includes the method of example Al or any of examples Al -A 14, wherein the imposing one or more nanoscale geometric and / or physical constraints on the cell includes putting the cell on a nanopatterned surface comprising one or more nanostructures.

[0177] Example A3 includes the method of example A2 or any of examples A1-A14, comprising inducing transient openings in a nuclear membrane of the nucleus of the cell by breaching a nucleo-cytoplasmic barrier and allowing molecular exchange across the nuclear membrane.

[0178] Example A4 includes the method of example A3 or any of examples A1-A14, wherein the openings are induced, at least temporarily, by at least one of an indention depth of or a nanoscale curvature of the one or more nanostructures on the nanopattemed surface.

[0179] Example A5 includes the method of examples A3 or A4 or any of examples Al- A14, further comprising repairing the openings through at least one endosomal sorting complexes required for transport (ES CRT) -mediated mechanism.

[0180] Example A6 includes the method of example Al or any of examples A1-A14, wherein the enabling the unregulated entry of molecules into the nucleus occurs in a period of time prior to mitosis of the cell.

[0181] Example A7 includes the method of example Al or any of examples A1-A14, wherein the molecules are deposited in a solution surrounding the cell.

[0182] Example A8 includes the method of any of examples A1-A7 or any of examples A1-A14, wherein the method includes delivering molecules into a plurality of nuclei of a plurality of cells, respectively, by putting the plurality of cells on an array of nanopatterned surfaces.

[0183] Example A9 includes the method of any of examples A1-A8 or any of examples Al -Al 4, comprising controlling a degree of nuclear membrane disruption.

[0184] Example A10 includes the method of example A9 or any of examples A1-A14, wherein the controlling the degree of nuclear membrane disruption is based on controlling at least one of an indention depth of or a nanoscale curvature of one or more nanostructures that impose the one or more nanoscale geometric and / or physical constraints on the cell.

[0185] Example Al 1 includes the method of any of examples A1-A10 or any of examples A1-A14, wherein the method is implemented for direct molecular delivery into a nucleus.

[0186] Example A12 includes the method of any of examples Al-Al 1 or any of examples A1-A14, wherein the direct molecular delivery includes delivery of one or more substances to the nucleus.

[0187] Example A13 includes the method of example A12 or any of examples A1-A14, wherein the one or more substances includes a gene.

[0188] Example A14 includes the method of any of examples A1-A13, wherein the method is implemented for direct nuclear sensing.

[0189] In some embodiments in accordance with the present technology (example Bl), a method for porating and delivering substances into a cellular nucleus includes receiving, on a nucleoporation device comprising a substrate and a plurality of nanostructures on the substrate, a cell on the plurality of nanostructures; inducing, by the nucleoporation device, formation oftransient pores in a nuclear membrane of the cell, wherein the plurality of nanostructures do not significantly puncture or penetrate a plasma membrane of the cell; and facilitating, through the transient pores in the nuclear membrane of the cell induced by the nucleoporation device, an unregulated entry of one or more substances from a cytoplasm of the cell into the nucleus.

[0190] Example B2 includes the method of example Bl or any of examples Bl -Bl 7, wherein the inducing the formation of the transient pores in the nuclear membrane of the cell comprises imposing multiple nanoscale geometric and / or physical constraints on the cell by contacting the cell with the nanostructures that mechanically deform or reshape a morphology of at least a portion of a body of the cell and the cell’s nucleus.

[0191] Example B3 includes the method of example B2 or any of examples Bl -Bl 7, comprising controlling a duration of opening, a pore size magnitude, or an amount of the transient pores based on the nanostructures having a defined shape, size, and / or surface curvature that mechanically deform or reshape the morphology of at least a portion of a body of the cell and the cell’s nucleus.

[0192] Example B4 includes the method of example B3 or any of examples B1-B17, wherein the formation of the transient pores is controlled by adjusting at least one of a geometry, a size, or an arrangement of the nanostructures.

[0193] Example B5 includes the method of example B3 or any of examples B1-B17, wherein the formation of the transient pores is controlled by adjusting the duration of contact between the cell and the nanostructures.

[0194] Example B6 includes the method of example Bl or any of examples Bl -Bl 7, wherein the formation of the transient pores are induced, at least temporarily, based on at least one of an indention depth or a nanoscale curvature of the nanostructures.

[0195] Example B7 includes the method of example B l or any of examples B l -Bl 7, wherein the transient pores formed in the nuclear membrane are capable of self-repair based on a defined shape, size, and / or surface curvature of the nanostructures that does not threaten viability of the cell.

[0196] Example B8 includes the method of example Bl or any of examples B1-B17, wherein the transient pores formed in the nuclear membrane are capable of self-repair further facilitated by an ESCRT-mediated mechanism.

[0197] Example B9 includes the method of example Bl or any of examples Bl -Bl 7, further comprising: breaching the cellular membrane of the cell on the nucleoporation device by one or more of electroporation, optoporation, or mechanoporation.

[0198] Example BIO includes the method of example Bl or any of examples B1-B17, further comprising: supplying the one or more substances into cytoplasm of the cell while the cell is on the nucleoporation device by providing lipid particles encapsulating the one or more one or more substances therein.

[0199] Example Bl l includes the method of example Bl or any of examples B1-B17, wherein the facilitating the unregulated entry of the one or more substances from cytoplasm of the cell into the nucleus through the transient pores formed in the nuclear membrane comprises depositing a solution of the one or more substances in a cell solution on the nucleoporation device.

[0200] Example B12 includes the method of any of examples Bl -Bl l or any of examples B1-B17, wherein the method is used for direct nuclear delivery of therapeutic agents, gene editing reagents, or biosensors.

[0201] Example Bl 3 includes the method of any of examples Bl -Bl 2 or any of examples B1-B17, wherein the unregulated entry of the one or more substances into the nucleus occurs in a period of time prior to mitosis of the cell.

[0202] Example B 14 includes the method of any of examples B 1 -B 13 or any of examples B1-B17, wherein the method is implemented for direct nuclear sensing.

[0203] Example Bl 5 includes the method of any of examples Bl -Bl 4 or any of examples B1-B17, wherein the method is implemented for direct substance delivery into a nucleus.

[0204] Example B 16 includes the method of any of examples B 1 -B 15 or any of examples B 1-B 17, wherein the plurality of nanostructures comprises nanopillars, nanorings, nanoneedles, nanowires, nanocrowns, or a combination thereof.

[0205] Example B17 includes the method of any of examples Bl -Bl 6, wherein the one or more substances comprise a nucleic acid, protein, peptide, or gene editing component.

[0206] In some embodiments in accordance with the present technology (example B 18), a method for porating and delivering substances into a cellular nucleus includes receiving, on a nucleoporation device comprising a substrate and a plurality of nanostructures on the substrate, a cell on the plurality of nanostructures; and inducing, by the nucleoporation device, formation of one or more transient pores in a nuclear membrane of the cell, wherein said plurality of nanostructures do not significantly puncture or penetrate a plasma membrane of the cell.

[0207] Example B 18B includes the method of example B 18, wherein the method includes one or more features of the method of any of examples B 1-B 17.

[0208] In some embodiments in accordance with the present technology (example B 19), a method for delivering one or more substances into a nucleus of a cell includes placing the cellon a substrate comprising a surface having a plurality of nanostructures, wherein the plurality of nanostructures induce formation of one or more transient pores in a nuclear membrane of the cell, thereby permitting unregulated entry of the one or more substances from a cytoplasm of the cell into the nucleus, and wherein said plurality of nanostructures do not significantly puncture or penetrate a plasma membrane of the cell.

[0209] Example B 19B includes the method of example B 19, wherein the method includes one or more features of the method of any of examples B1-B17.

[0210] Tn some embodiments in accordance with the present technology (example B20), a method for delivering one or more substances into a cellular nucleus includes providing a cellular nucleoporation device that comprises a plurality of nanostructures configured to receive a cell, wherein when the cell is placed on the plurality of nanostructures, the cellular nucleoporation device induces formation of one or more transient pores in a nuclear membrane of the cell, thereby permitting unregulated entry of the one or more substances from a cytoplasm of the cell into a nucleus of the cell, and wherein said plurality of nanostructures do not significantly puncture or penetrate a plasma membrane of the cell.

[0211] Example B20B includes the method of example B20, wherein the method includes one or more features of the method of any of examples B1-B17.

[0212] In some embodiments in accordance with the present technology (example B21), a nucleoporation device for disrupting a nuclear membrane of a cell includes a substrate; and a plurality of nanostructures, wherein the nanostructures are configured to induce formation of transient pores in the nuclear membrane of the cell without significantly puncturing or penetrating a plasma membrane of the cell.

[0213] Example B22 includes the device of example B21 or any of examples B21-B40, wherein the plurality of nanostructures is configured to impose nanoscale geometric and / or physical constraints on the cell based on contact of the cell with the nanostructures having defined shapes, sizes, or surface curvatures that mechanically deform or reshape at least a portion of the cell or its nucleus.

[0214] Example B23 includes the device of example B21 or any of examples B21-B40, wherein the nanostructures comprise nanopillars, nanorings, nanoneedles, nanowires, nanocrowns, or a combination thereof.

[0215] Example B24 includes the device of example B21 or any of examples B21-B40, wherein the plurality of nanostructures is arranged in an array of the nanostructures on the substrate.

[0216] Example B25 includes the device of example B24 or any of examples B21-B40, wherein the array of the nanostructures includes a first array of a first group of nanostructures having a first spacing between the nanostructures of the first group, and wherein the array of nanostructures includes a second array of a second group of nanostructures having a second spacing between the nanostructures of the second group.

[0217] Example B26 includes the device of example B25 or any of examples B21-B40, wherein the first array includes 6 pm spacing, and wherein the second group includes substantially 3 pm spacing.

[0218] Example B27 includes the device of example B24 or any of examples B21-B40, wherein the array of the nanostructures is configured to facilitate high-throughput processing of a plurality of cells deposited on the substrate of the nucleoporation device.

[0219] Example B28 includes the device of example B24 or any of examples B21-B40, wherein the array of the nanostructures is arranged in a pattern selected from the group consisting of a regular or repeating arrangement of the nanostructures, a random arrangement of the nanostructures, and a concentric arrangement of the nanostructures.

[0220] Example B29 includes the device of example B21 or any of examples B21-B40, wherein the nanostructures have a diameter ranging from 200 nm to 2 pm.

[0221] Example B30 includes the device of example B21 or any of examples B21-B40, wherein the nanostructures have a height ranging from 1 pm to 5 pm.

[0222] Example B31 includes the device of example B21 or any of examples B21-B40, wherein the nanostructures are spaced apart by a distance ranging from 2 pm to 10 pm.

[0223] Example B32 includes the device of example B21 or any of examples B21-B40, wherein the nanostructures comprise a material including one or more of silicon dioxide, silicon, one or more metals, or a polymer.

[0224] Example B33 includes the device of example B21 or any of examples B21-B40, further comprising: a coating covering at least a portion of at least some of the nanostructures on the substrate, the coating including a layer of one or more of a protein or a peptide configured to promote cell adhesion.

[0225] Example B34 includes the device of example B33 or any of examples B21-B40, wherein the coating includes at least one of poly-L-lysine, gelatin, fibronectin, vitronectin, laminin, collagen, matrigel, or a combination thereof.

[0226] Example B35 includes the device of example B21 or any of examples B21-B40, further comprising: a packaging article or apparatus configured to contain the nucleoporation device during a nucleoporation implementation of the cell on the nucleoporation device.

[0227] Example B36 includes the device of example B35 or any of examples B21-B40, wherein the packing article or apparatus includes a single well plate or petri dish or a multiwell plate.

[0228] Example B37 includes the device of example B35 or any of examples B21-B40, wherein the packing article or apparatus includes a standard multi- well plate comprising 1 , 2, 4, 6, 8, 12, 24, 48, 96, 384, or 1,536 wells.

[0229] Example B38 includes the device of example B21 or any of examples B21-B40, wherein the device is configured to controllably porate and deliver one or more substances into a nucleus of the cell by: receiving the cell on the plurality of nanostructures; inducing formation of the transient pores in the nuclear membrane of the cell, wherein the plurality of nanostructures do not significantly puncture or penetrate a plasma membrane of the cell; and facilitating, through the transient pores in the nuclear membrane of the cell induced by the nucleoporation device, an unregulated entry of the one or more substances from a cytoplasm of the cell into the nucleus.

[0230] Example B39 includes the device of example B38 or any of examples B21-B40, wherein the device is configured for use in gene therapy, drug delivery, or biosensing applications.

[0231] Example B40 includes the device of example B38 or any of examples B21-B39, wherein the one or more substances comprise a nucleic acid, protein, peptide, or gene editing component.

[0232] In some embodiments in accordance with the present technology (example B41), a system for porating and delivering substances into a cellular nucleus includes a nucleoporation device comprising a substrate, and a plurality of nanostructures, wherein the nanostructures are configured to induce formation of transient pores in a nuclear membrane of a cell without significantly puncturing or penetrating a plasma membrane of the cell; a cell imaging device interfaced with the nucleoporation device, the cell imaging device including a camera and a cell positioning device, wherein the camera is configured to acquire images of the cell on the nucleoporation device, and wherein the cell positioning device is configured to move the nucleoporation device with respect to the camera; and a data processing device in data communication with the cell imaging device, wherein the data processing device is configured to control the camera to acquire the images and the cell positioning device to move the nucleoporation device.

[0233] Example B42 includes the system of example B41 or any of examples B41-B47, wherein the plurality of nanostructures of the nucleoporation device is configured to imposenanoscale geometric and / or physical constraints on the cell based on contact of the cell with the nanostructures having defined shapes, sizes, or surface curvatures that mechanically deform or reshape at least a portion of the cell or its nucleus.

[0234] Example B43 includes the system of example B41 or any of examples B41-B47, wherein the nanostructures of the nucleoporation device comprise nanopillars, nanorings, nanoneedles, nanowires, nanocrowns, or a combination thereof.

[0235] Example B44 includes the system of example B41 or any of examples B41-B47, wherein the cell imaging device includes a microscope comprising one or more lenses and a stage and interfaced with the cell positioning device and the camera.

[0236] Example B45 includes the system of example B41 or any of examples B41-B47, wherein the cell positioning device includes a stepper device configured to move the nucleoporation device by one or more nanometers or one or more micrometers with respect to the camera.

[0237] Example B46 includes the system of example B41 or any of examples B41-B47, wherein the data processing device includes a laptop computer, a desktop computer, or a mobile computing device including a smartphone, a tablet, a smartwatch, or a smartglasses.

[0238] Example B47 includes the system of example B41 or any of examples B41-B46, wherein the data processing device is in data communication with one or more computing devices in a computer communication network, wherein the data processing device includes a software application that is stored and operated on one or both of the data processing device and the one or more computing devices in the computer communication network to process data received from at least one of the nucleoporation device or the cell imaging device.

[0239] In some embodiments in accordance with the present technology (example B48), a method of fabricating a nucleoporation device includes forming a plurality of nanostructures on a surface of a substrate to have a first structural configuration comprising a first diameter or length, a first height, and a first spacing of at least one nanostructure of the plurality of nanostructures from another nanostructure of the plurality of nanostructures; and sizing the formed nanostructures to remove structural aspects of the first structural configuration of each nanostructure by a deleterious etching process to produce a plurality of final nanostructures on the substrate, wherein the plurality of final nanostructures on the substrate include defined surface curvatures and are configured to impose nanoscale geometric and / or physical constraints on a cell based on contact of the cell with the final nanostructures that cause the cell to mechanically deform or reshape at least a portion of a cell body and / or its nucleus.

[0240] Example B49 includes the method of example B48 or any of examples B48-B56, wherein the forming includes using one or more photolithographic, chemical, or physical vapor deposition, or etching techniques to create the plurality of nanostructures to have the first structural configuration.

[0241] Example B50 includes the method of example B48 or any of examples B48-B56, wherein the deleterious etching process includes wet etching.

[0242] Example B51 includes the method of example B50 or any of examples B48-B56, wherein the formed nanostructures include silicon oxide, and wherein the wet etching includes using a chromium etchant followed by a buffered oxide etch (BOE) to remove exposed silicon oxide.

[0243] Example B52 includes the method of example B48 or any of examples B48-B56, wherein the final nanostructures on the substrate comprise one or more of nanopillars, nanorings, nanoneedles, nanowires, nanocrowns, or combinations thereof.

[0244] Example B53 includes the method of example B48 or any of examples B48-B56, wherein the nanostructures are formed by photolithography followed by chemical etching and material deposition.

[0245] Example B54 includes the method of example B48 or any of examples B48-B56, wherein the method does not include an electron beam-based fabrication step.

[0246] Example B55 includes the method of example B48 or any of examples B48-B56, wherein the nanostructures have one or both of a spacing and a size dimension including a height or a diameter that is effective to interact with the cell and induce nanoscale deformation of a nuclear membrane of the cell.

[0247] Example B56 includes the method of example B48 or any of examples B48-B55, further comprising functionalizing one or both of the substrate and at least some of the plurality of final nanostructures with a coating to promote cell adhesion.Conclusion

[0248] Implementations of the subject matter and the functional operations described in this patent document may be implemented using various systems, digital electronic circuitry, and / or in computer software, firmware, or hardware, including the structures disclosed in this specification and their structural equivalents, or in combinations of one or more of them. Implementations of subject matter described in this specification may be implemented using one or more computer program products, i.e., one or more modules of computer program instructions encoded on a tangible and non-transitory computer readable medium for executionby, or to control the operation of, data processing apparatus. The computer readable medium can be a machine-readable storage device, a machine-readable storage substrate, a memory device, a composition of matter effecting a machine-readable propagated signal, or a combination of one or more of them. The terms “data processing unit” or “data processing apparatus” or “data processor” or the like encompass all apparatuses, devices, and machines for processing data, including by way of example a programmable processor, a computer, or multiple processors or computers. An apparatus can include, in addition to hardware, code that creates an execution environment for the computer program in question, e.g., code that constitutes processor firmware, a protocol stack, a database management system, an operating system, or a combination of one or more of them.

[0249] A computer program (also known as a program, software, software application, script, or code) can be written in any form of programming language, including compiled or interpreted languages, and it can be deployed in any form, including as a stand-alone program or as a module, component, subroutine, or other unit suitable for use in a computing environment. A computer program does not necessarily correspond to a file in a file system. A program can be stored in a portion of a file that holds other programs or data (e.g., one or more scripts stored in a markup language document), in a single file dedicated to the program in question, or in multiple coordinated files (e.g., files that store one or more modules, sub programs, or portions of code). A computer program can be deployed to be executed on one computer or on multiple computers that are located at one site or distributed across multiple sites and interconnected by a communication network.

[0250] The processes and logic flows described in this specification can be performed by one or more programmable processors executing one or more computer programs to perform functions by operating on input data and generating output. The processes and logic flows can also be performed by, and apparatus can also be implemented as, special purpose logic circuitry, e.g., an FPGA (field programmable gate array) or an ASIC (application specific integrated circuit) or other circuitry.

[0251] Processors suitable for the execution of a computer program include, by way of example, both general and special purpose microprocessors, and any one or more processors of any kind of digital computer. Generally, a processor will receive instructions and data from a read only memory or a random access memory or both. The essential elements of a computer are a processor for performing instructions and one or more memory devices for storing instructions and data. Generally, a computer will also include, or be operatively coupled to receive data from or transfer data to, or both, one or more mass storage devices for storing data,e.g., magnetic, magneto optical disks, or optical disks. However, a computer need not have such devices. Computer readable media suitable for storing computer program instructions and data include all forms of nonvolatile memory, media and memory devices, including by way of example semiconductor memory devices, e.g., EPROM, EEPROM, and flash memory devices. The processor and the memory can be supplemented by, or incorporated in, special purpose logic circuitry.

[0252] While this patent document contains many specifics, these should not be construed as limitations on the scope of any invention or of what may be claimed, but rather as descriptions of features that may be specific to particular embodiments of particular inventions. Certain features that are described in this patent document in the context of separate embodiments can also be implemented in combination in a single embodiment. Conversely, various features that are described in the context of a single embodiment can also be implemented in multiple embodiments separately or in any suitable subcombination. Moreover, although features may be described above as acting in certain combinations and even initially claimed as such, one or more features from a claimed combination can in some cases be excised from the combination, and the claimed combination may be directed to a subcombination or variation of a subcombination.

[0253] Similarly, while operations are depicted in the drawings in a particular order, this should not be understood as requiring that such operations be performed in the particular order shown or in sequential order, or that all illustrated operations be performed, to achieve desirable results. Moreover, the separation of various system components in the embodiments described in this patent document should not be understood as requiring such separation in all embodiments.

[0254] Only a few implementations and examples are described and other implementations, enhancements and variations can be made based on what is described and illustrated in this patent document.

Claims

CLAIMSWhat is claimed is:

1. A method for porating and delivering substances into a cellular nucleus, comprising: receiving, on a nucleoporation device comprising a substrate and a plurality of nanostructures on the substrate, a cell on the plurality of nanostructures; inducing, by the nucleoporation device, formation of transient pores in a nuclear membrane of the cell, wherein the plurality of nanostructures do not significantly puncture or penetrate a plasma membrane of the cell; and facilitating, through the transient pores in the nuclear membrane of the cell induced by the nucleoporation device, an unregulated entry of one or more substances from a cytoplasm of the cell into the nucleus.

2. The method of claim 1, wherein the inducing the formation of the transient pores in the nuclear membrane of the cell comprises imposing multiple nanoscale geometric and / or physical constraints on the cell by contacting the cell with the nanostructures that mechanically deform or reshape a morphology of at least a portion of a body of the cell and the cell’s nucleus.

3. The method of claim 2, comprising: controlling a duration of opening, a pore size magnitude, or an amount of the transient pores based on the nanostructures having a defined shape, size, and / or surface curvature that mechanically deform or reshape the morphology of at least a portion of a body of the cell and the cell’s nucleus.

4. The method of claim 3, wherein the formation of the transient pores is controlled by adjusting at least one of a geometry, a size, or an arrangement of the nanostructures.

5. The method of claim 3, wherein the formation of the transient pores is controlled by adjusting the duration of contact between the cell and the nanostructures.

6. The method of claim 1, wherein the formation of the transient pores are induced, at least temporarily, based on at least one of an indention depth or a nanoscale curvature of the nanostructures.

7. The method of claim 1, wherein the transient pores formed in the nuclear membrane are capable of self-repair based on a defined shape, size, and / or surface curvature of the nanostructures that does not threaten viability of the cell.

8. The method of claim 1, wherein the transient pores formed in the nuclear membrane are capable of self-repair further facilitated by an ESCRT-mediated mechanism.

9. The method of claim 1, further comprising: breaching the cellular membrane of the cell on the nucleoporation device by one or more of electroporation, optoporation, or mechanoporation.

10. The method of claim 1, further comprising: supplying the one or more substances into cytoplasm of the cell while the cell is on the nucleoporation device by providing lipid particles encapsulating the one or more one or more substances therein.

11. The method of claim 1 , wherein the facilitating the unregulated entry of the one or more substances from cytoplasm of the cell into the nucleus through the transient pores formed in the nuclear membrane comprises depositing a solution of the one or more substances in a cell solution on the nucleoporation device.

12. The method of any one of claims 1-11, wherein the method is used for direct nuclear delivery of therapeutic agents, gene editing reagents, or biosensors.

13. The method of any one of claims 1-12, wherein the unregulated entry of the one or more substances into the nucleus occurs in a period of time prior to mitosis of the cell.

14. The method of any one of claims 1-13, wherein the method is implemented for direct nuclear sensing.

15. The method of any one of claims 1-14, wherein the method is implemented for direct substance delivery into a nucleus.

16. The method of any one of claims 1-15, wherein the plurality of nanostructures comprises nanopillars, nanorings, nanoneedles, nano wires, nanocrowns, or a combination thereof.

17. The method of any one of claims 1-16, wherein the one or more substances comprise a nucleic acid, protein, peptide, or gene editing component.

18. A method for porating and delivering substances into a cellular nucleus, comprising: receiving, on a nucleoporation device comprising a substrate and a plurality of nanostructures on the substrate, a cell on the plurality of nanostructures; and inducing, by the nucleoporation device, formation of one or more transient pores in a nuclear membrane of the cell, wherein said plurality of nanostructures do not significantly puncture or penetrate a plasma membrane of the cell.

19. A method for delivering one or more substances into a nucleus of a cell, comprising: placing the cell on a substrate comprising a surface having a plurality of nanostructures, wherein the plurality of nanostructures induce formation of one or more transient pores in a nuclear membrane of the cell, thereby permitting unregulated entry of the one or more substances from a cytoplasm of the cell into the nucleus, and wherein said plurality of nanostructures do not significantly puncture or penetrate a plasma membrane of the cell.

20. A method for delivering one or more substances into a cellular nucleus, comprising: providing a cellular nucleoporation device that comprises a plurality of nanostructures configured to receive a cell, wherein when the cell is placed on the plurality of nanostructures, the cellular nucleoporation device induces formation of one or more transient pores in a nuclear membrane of the cell, thereby permitting unregulated entry of the one or more substances from a cytoplasm of the cell into a nucleus of the cell, and wherein said plurality of nanostructures do not significantly puncture or penetrate a plasma membrane of the cell.

21. A nucleoporation device for disrupting a nuclear membrane of a cell, comprising: a substrate; and a plurality of nanostructures, wherein the nanostructures are configured to induce formation of transient pores in the nuclear membrane of the cell without significantly puncturing or penetrating a plasma membrane of the cell.

22. The device of claim 21, wherein the plurality of nanostructures is configured to impose nanoscale geometric and / or physical constraints on the cell based on contact of the cell with the nanostructures having defined shapes, sizes, or surface curvatures that mechanically deform or reshape at least a portion of the cell or its nucleus.

23. The device of claim 21, wherein the nanostructures comprise nanopillars, nanorings, nanoneedles, nanowires, nanocrowns, or a combination thereof.

24. The device of claim 21, wherein the plurality of nanostructures is arranged in an array of the nanostructures on the substrate.

25. The device of claim 24, wherein the array of the nanostructures includes a first array of a first group of nanostructures having a first spacing between the nanostructures of the first group, and wherein the array of nanostructures includes a second array of a second group of nanostructures having a second spacing between the nanostructures of the second group.

26. The device of claim 25, wherein the first array includes 6 pm spacing, and wherein the second group includes substantially 3 pm spacing.

27. The device of claim 24, wherein the array of the nanostructures is configured to facilitate high-throughput processing of a plurality of cells deposited on the substrate of the nucleoporation device.

28. The device of claim 24, wherein the array of the nanostructures is arranged in a pattern selected from the group consisting of a regular or repeating arrangement of the nanostructures, a random arrangement of the nanostructures, and a concentric arrangement of the nanostructures.

29. The device of claim 21 , wherein the nanostructures have a diameter ranging from 200 nm to 2 pm.

30. The device of claim 21 , wherein the nanostructures have a height ranging from 1 pm to 5 pm.

31. The device of claim 21, wherein the nanostructures are spaced apart by a distance ranging from 2 pm to 10 pm.

32. The device of claim 21, wherein the nanostructures comprise a material including one or more of silicon dioxide, silicon, one or more metals, or a polymer.

33. The device of claim 21 , further comprising: a coating covering at least a portion of at least some of the nanostructures on the substrate, the coating including a layer of one or more of a protein or a peptide configured to promote cell adhesion.

34. The device of claim 33, wherein the coating includes at least one of poly-L-lysine, gelatin, fibronectin, vitronectin, laminin, collagen, matrigel, or a combination thereof.

35. The device of claim 21 , further comprising: a packaging article or apparatus configured to contain the nucleoporation device during a nucleoporation implementation of the cell on the nucleoporation device.

36. The device of claim 35, wherein the packing article or apparatus includes a single well plate or petri dish or a multi- well plate.

37. The device of claim 35, wherein the packing article or apparatus includes a standard multi-well plate comprising 1, 2, 4, 6, 8, 12, 24, 48, 96, 384, or 1,536 wells.

38. The device of claim 21, wherein the device is configured to controllably porate and deliver one or more substances into a nucleus of the cell by : receiving the cell on the plurality of nanostructures; inducing formation of the transient pores in the nuclear membrane of the cell, wherein the plurality of nanostructures do not significantly puncture or penetrate a plasma membrane of the cell; and facilitating, through the transient pores in the nuclear membrane of the cell induced by the nucleoporation device, an unregulated entry of the one or more substances from a cytoplasm of the cell into the nucleus.

39. The device of claim 38, wherein the device is configured for use in gene therapy, drug delivery, or biosensing applications.

40. The device of claim 38, wherein the one or more substances comprise a nucleic acid, protein, peptide, or gene editing component.

41. A system for porating and delivering substances into a cellular nucleus, comprising: a nucleoporation device comprising a substrate, and a plurality of nanostructures, wherein the nanostructures are configured to induce formation of transient pores in a nuclear membrane of a cell without significantly puncturing or penetrating a plasma membrane of the cell; a cell imaging device interfaced with the nucleoporation device, the cell imaging device including a camera and a cell positioning device, wherein the camera is configured to acquire images of the cell on the nucleoporation device, and wherein the cell positioning device is configured to move the nucleoporation device with respect to the camera; and a data processing device in data communication with the cell imaging device, wherein the data processing device is configured to control the camera to acquire the images and the cell positioning device to move the nucleoporation device.

42. The system of claim 41 , wherein the plurality of nanostructures of the nucleoporation device is configured to impose nanoscale geometric and / or physical constraints on the cell based on contact of the cell with the nanostructures having defined shapes, sizes, or surface curvatures that mechanically deform or reshape at least a portion of the cell or its nucleus.

43. The system of claim 41 , wherein the nanostructures of the nucleoporation device comprise nanopillars, nanorings, nanoneedles, nanowires, nanocrowns, or a combination thereof.

44. The system of claim 41 , wherein the cell imaging device includes a microscope comprising one or more lenses and a stage and interfaced with the cell positioning device and the camera.

45. The system of claim 41, wherein the cell positioning device includes a stepper device configured to move the nucleoporation device by one or more nanometers or one or more micrometers with respect to the camera.

46. The system of claim 41 , wherein the data processing device includes a laptop computer, a desktop computer, or a mobile computing device including a smartphone, a tablet, a smartwatch, or a smartglasses.

47. The system of claim 41 , wherein the data processing device is in data communication with one or more computing devices in a computer communication network, wherein the dataprocessing device includes a software application that is stored and operated on one or both of the data processing device and the one or more computing devices in the computer communication network to process data received from at least one of the nucleoporation device or the cell imaging device.

48. A method of fabricating a nucleoporation device comprising: forming a plurality of nanostructures on a surface of a substrate to have a first structural configuration comprising a first diameter or length, a first height, and a first spacing of at least one nanostructure of the plurality of nanostructures from another nanostructure of the plurality of nanostructures; and sizing the formed nanostructures to remove structural aspects of the first structural configuration of each nanostructure by a deleterious etching process to produce a plurality of final nanostructures on the substrate, wherein the plurality of final nanostructures on the substrate include defined surface curvatures and are configured to impose nanoscale geometric and / or physical constraints on a cell based on contact of the cell with the final nanostructures that cause the cell to mechanically deform or reshape at least a portion of a cell body and / or its nucleus.

49. The method of claim 48, wherein the forming includes using one or more photolithographic, chemical, or physical vapor deposition, or etching techniques to create the plurality of nanostructures to have the first structural configuration.

50. The method of claim 48, wherein the deleterious etching process includes wet etching.

51. The method of claim 50, wherein the formed nanostructures include silicon oxide, and wherein the wet etching includes using a chromium etchant followed by a buffered oxide etch (BOE) to remove exposed silicon oxide.

52. The method of claim 48, wherein the final nanostructures on the substrate comprise one or more of nanopillars, nanorings, nanoneedles, nanowires, nanocrowns, or combinations thereof.

53. The method of claim 48, wherein the nanostructures are formed by photolithography followed by chemical etching and material deposition.

54. The method of claim 48, wherein the method does not include an electron beam-based fabrication step.

55. The method of claim 48, wherein the nanostructures have one or both of a spacing and a size dimension including a height or a diameter that is effective to interact with the cell and induce nanoscale deformation of a nuclear membrane of the cell.

56. The method of claim 48, further comprising: functionalizing one or both of the substrate and at least some of the plurality of final nanostructures with a coating to promote cell adhesion.

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