Cellular interaction assays involving delivery of one or more components by mechanoporation

Abstract

In some aspects, provided herein are methods for detecting an interaction in a live cell comprising an intact cell membrane, the methods comprising delivering one or more components useful therefor using a mechanical deformation delivery technique. In other aspects, the disclosure is directed to systems, kits, and compositions useful for the methods taught herein.

Description

Attorney Docket No. 285192001040CELLULAR INTERACTION ASSAYS INVOLVING DELIVERY OF ONE OR MORE CELL IMPERMEABLE COMPONENTSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims priority to U. S. Provisional Application No. 63 / 778,054 filed on March 26, 2025, and U. S. Provisional Application No. 63 / 888,844, filed on September 26, 2025, the content of each of which is hereby incorporated herein by reference in their entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The content of the electronic sequence listing (285192001040seqlist.xml; Size: 4,689 bytes; and Date of Creation: March 16, 2026) is herein incorporated by reference in its entirety.FIELD OF THE INVENTION

[0003] The present invention, in some aspects, relates to methods for detecting an interaction in a live cell comprising an intact cell membrane, the methods comprising delivering one or more components useful therefor using a mechanical deformation delivery technique. In other aspects, the disclosure is directed to systems, kits, and compositions useful for the methods taught herein.BACKGROUND OF THE INVENTION

[0004] Understanding how aspects within cells (such as polypeptides) behave and interact with other aspects therein, including exogenous materials (such as therapeutic drug candidates), provides valuable insights into cellular processes and mechanisms. Many cellular assays have been developed to provide such insights, however, such assays are generally performed, at least in part, using overly modulated cells which do not reflect accurate biological states and / or as a terminal assay (z.e., one time point is captured only once the cell is not living and / or does not have an intact cell membrane). Accordingly, such assays do not reflect an accurate biological1MF-367693737Attorney Docket No. 285192001040 state of the cells and / or do not allow for the measure of cellular kinetics across a plurality of time points from the same sample.BRIEF SUMMARY OF THE INVENTION

[0005] In certain aspects, provided herein is a method of detecting an interaction in a live cell comprising an intact cell membrane, the method comprising: delivering a first component, or a precursor thereof, to the live cell using a mechanical deformation delivery technique, wherein the first component is configured to interact with a second component in the live cell, and wherein a detectable signal is produced or producible when the first component interacts with the second component; and measuring the detectable signal to detect the interaction in the live cell comprising the intact cell membrane.

[0006] In some embodiments, the first component and the second component form a luminescent enzyme. In some embodiments, the method further comprises delivering a substrate of the luminescent enzyme to the live cell comprising an intact cell membrane.

[0007] In some embodiments, the first component and the second component exhibit resonance energy transfer to produce the detectable signal.

[0008] In some embodiments, the first component comprises a non-deoxyribonucleic acid component. In some embodiments, the first component comprises a polypeptide.

[0009] In some embodiments, the first component comprises a synthetic component. In some embodiments, the synthetic component is a synthetic fluorophore or a detection particle.

[0010] In some embodiments, the method further comprises delivering the second component, or a precursor thereof, to the live cell.

[0011] In some embodiments, the second component, or a precursor thereof, is co-delivered with the first component using the mechanical deformation delivery technique.

[0012] In certain aspects, provided herein is a method of detecting an interaction in a live cell comprising an intact cell membrane, the method comprising: delivering a component comprising a luminescent enzyme, or an aspect thereof, or a precursor thereof, to the live cell using a mechanical deformation delivery technique, wherein the component comprising the luminescent enzyme, or the aspect thereof, associates or is associated with a target in the live cell; and2MF-367693737Attorney Docket No. 285192001040 measuring a luminescence output of the luminescent enzyme in the live cell comprising the intact cell membrane to detect the interaction in the live cell comprising the intact cell membrane. In some embodiments, the method further comprises delivering a substrate of the luminescent enzyme to the live cell. In some embodiments, the substrate is co-delivered to the live cell along with the component comprising the luminescent enzyme, or the aspect thereof, or the precursor thereof, using the mechanical deformation delivery technique. In some embodiments, the component comprising the luminescent enzyme, or the aspect thereof, or the precursor thereof, comprises the target or a precursor thereof. In some embodiments, the target is a polypeptide. In some embodiments, the target is a nucleic acid. In some embodiments, the target is a subcellular structure. In some embodiments, the luminescent enzyme, or the aspect thereof, delivered to the live cell via the mechanical deformation delivery technique is delivered in polypeptide form. In some embodiments, the luminescent enzyme, or the aspect thereof, delivered to the live cell via the mechanical deformation delivery technique is delivered in a non-deoxyribonucleic acid form. In some embodiments, the luminescent enzyme is composed of a complex comprising two or more aspects of the luminescent enzyme, and wherein upon complex formation in the live cell the luminescent enzyme is catalytically active. In some embodiments, the live cell comprises another aspect of the luminescent enzyme associated with a second target, and wherein the aspect of the luminescent enzyme associated with the target and the other aspect of the luminescent enzyme form a catalytically active enzyme. In some embodiments, the live cell comprises another aspect of the luminescent enzyme, and wherein the aspect of the luminescent enzyme associated with the target and the other aspect of the luminescent enzyme form a catalytically active enzyme. In some embodiments, the luminescent enzyme comprises LgBiT (SEQ ID NO:1) and SmBiT (SEQ ID NO:2). In some embodiments, the luminescent enzyme comprises LgBiT (SEQ ID NO:1) and HiBiT (SEQ ID NO:3). In some embodiments, the luminescent enzyme comprises LgBiT (SEQ ID NO:1) and natural peptide (NP: SEQ ID NO:4). In some embodiments, the live cell expresses the target fused to the aspect of the luminescent enzyme, wherein the delivering the component comprising the luminescent enzyme, or the aspect thereof, or the precursor thereof, to the live cell using the mechanical deformation delivery technique comprises delivering another aspect of the luminescent enzyme, or a precursor thereof, and wherein the aspect of the luminescent enzyme fused to the target and the other aspect of the luminescent enzyme associate to become catalytically active. In some embodiments, the luminescent enzyme, or the aspect thereof, or the precursor thereof, is3MF-367693737Attorney Docket No. 285192001040 substantially cell membrane impermeable. In some embodiments, the substrate of the luminescent enzyme is cell permeable. In some embodiments, the substrate of the luminescent enzyme is a luciferin. In some embodiments, the substrate of the luminescent enzyme is substantially cell impermeable.

[0013] In certain aspects, provided herein is a method of detecting an interaction in a live cell comprising an intact cell membrane, the method comprising: delivering a first component comprising an acceptor chromophore, or a precursor thereof, and / or a second component comprising a donor chromophore, or a precursor thereof, to the live cell using a mechanical deformation delivery technique, wherein the first component comprising the acceptor chromophore and / or the second component comprising the donor chromophore associates or is associated with a target in the live cell; subjecting the live cell to an excitation light to excite the chromophore donor; and measuring a fluorescence output in the live cell comprising the intact cell membrane to detect the interaction in the live cell. In some embodiments, the delivering comprises delivering the first component comprising the acceptor chromophore, or the precursor thereof. In some embodiments, the delivering comprises delivering the second component comprising the donor chromophore, or the precursor thereof. In some embodiments, the delivering comprises delivering the first component comprising the acceptor chromophore, or the precursor thereof, and the second component comprising the donor chromophore, or the precursor thereof. In some embodiments, the donor chromophore is a natural molecule. In some embodiments, the donor chromophore is an autofluorescent polypeptide. In some embodiments, the autofluorescent polypeptide is a green fluorescent protein (GFP). In some embodiments, the second component comprising the donor chromophore, or the precursor thereof, is a nucleic acid encoding the donor chromophore. In some embodiments, the nucleic acid encoding the donor chromophore also encodes the target. In some embodiments, the donor chromophore is a synthetic molecule. In some embodiments, the second component comprising the donor chromophore, or the precursor thereof, is substantially cell impermeable. In some embodiments, the first component comprising the acceptor chromophore, or the precursor thereof, is substantially cell impermeable. In some embodiments, the acceptor chromophore is synthetic. In some embodiments, the second component comprising the acceptor chromophore comprises a small molecule. In some embodiments, the second component comprising the acceptor chromophore is a small molecule fluorophore. In some embodiments, the detecting the interaction in the live cell comprising the intact cell membrane is based on a FRET assay. In 4MF-367693737Attorney Docket No. 285192001040 some embodiments, the detecting the interaction in the live cell comprising the intact cell membrane is based on a time-resolved FRET assay. In some embodiments, the detecting the interaction in the live cell comprising the intact cell membrane is based on a homogeneous time-resolved fluorescence assay. In some embodiments, the donor chromophore is a cryptate. In some embodiments, the donor chromophore is europium cryptate or terbium cryptate. In some embodiments, the acceptor chromophore is XL665, fluorescein, GFP, or d2.

[0014] In certain aspects, provided herein is a method of detecting an interaction in a live cell comprising an intact cell membrane, the method comprising: delivering: (a) a first component comprising an acceptor chromophore, or a precursor thereof, and a second component comprising a donor chromophore, or an aspect thereof, or a precursor thereof, to the live cell using a mechanical deformation delivery technique, or (b) the first component comprising the acceptor chromophore, or the precursor thereof, to the live cell using the mechanical deformation delivery technique, wherein the live cell comprises the donor chromophore, or (c) the second component comprising the donor chromophore, or the aspect thereof, or the precursor thereof, to the live cell using the mechanical deformation delivery technique, wherein the live cell comprises the acceptor chromophore, wherein the first component comprising the acceptor chromophore and / or the second component comprising the donor chromophore associates or is associated with a target in the live cell; and measuring a fluorescence output in the live cell comprising the intact cell membrane to detect the interaction in the live cell.

[0015] In some embodiments, the donor chromophore is a luminescent enzyme. In some embodiments, the donor chromophore is a luciferase. In some embodiments, the luciferase is composed of a complex comprising two or more portions of the luminescent enzyme, and wherein upon complex formation the luminescent enzyme is catalytically active. In some embodiments, the first component comprising the acceptor chromophore associates or is associated with the target. In some embodiments, the second component comprising the donor chromophore associates or is associated with a second target. In some embodiments, the second component comprising the donor chromophore associates or is associated with the target. In some embodiments, the first component comprising the acceptor chromophore associates or is associated with a second target. In some embodiments, the first component comprising the acceptor chromophore associates or is associated with a test moiety. In some embodiments, the second component comprising the donor chromophore associates or is associated with a test moiety. In some embodiments, the test moiety associates with the target. In some embodiments,5MF-367693737Attorney Docket No. 285192001040 the test moiety comprises a small molecule. In some embodiments, the test moiety comprises a polypeptide.

[0016] In certain aspects, provided herein is a method of detecting an interaction in a live cell comprising an intact cell membrane, the method comprising: delivering a first component comprising an acceptor bead and a second component comprising a donor bead to the live cell using a mechanical deformation delivery technique, wherein the first component comprising the acceptor bead is configured to associate with a first target and the second component comprising the donor bead is configured to associate with a second target; and measuring a fluorescence output in the live cell comprising an intact cell membrane to detect the interaction in the live cell. In some embodiments, the first target and second target are on the same macromolecule. In some embodiments, the first target and second targe are on different macromolecules.

[0017] In some embodiments, the live cell further comprises a PROTAC configured to associate with the target in the cell. In some embodiments, the cell further comprises a PROTAC configured to associate with a macromolecule comprising the first target or a macromolecule comprising the second target. In some embodiments, the method further comprises delivering the PROTAC to the cell. In some embodiments, the measuring comprises measuring at two or more time points. In some embodiments, the measuring is performed via fluorescence microscopy. In some embodiments, the measuring is performed via a plate reader. In some embodiments, the mechanical deformation delivery technique comprising passing the cell through a constriction-based structure to form pores in the membrane of the cell. In some embodiments, the constriction-based structure comprises a filter or a microfluidic channel. In some embodiments, the live cell is selected from the group consisting of a primary cell, iPSC, HeLa, cardiomyocyte, and HEK293.

[0018] In certain aspects, provided herein is an engineered live cell comprising an intact cell membrane and a component useful for any method described herein. In some embodiments, at least one of the components is delivered using a mechanical deformation delivery technique.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1A shows a bar chart displaying the percentage of live cells with intact cell membranes having 3 kDa dextran delivered thereto using the mechanical deformation delivery6MF-367693737Attorney Docket No. 285192001040 technique. FIG. IB shows a bar chart displaying the percentage of live cells with intact cell membranes having PROTAC delivered thereto using the mechanical deformation delivery technique. FIG. 1C shows histograms displaying the population of live cells with intact membranes over a continuous plot of Cascade Blue fluorescence levels (left; 3 kDa dextran) and Texas Red fluorescence levels (right; PROTAC) following delivery to cells via the mechanical deformation delivery technique.

[0020] FIG. 2 shows a bar chart displaying luminescence levels of live cells with intact cell membranes.

[0021] FIG. 3 shows a bar chart displaying the BRET Ratio (mBu) of live NanoLuc-Src cells with intact cell membranes subjected to mechanical deformation delivery of a kinase tracer.

[0022] FIG. 4 shows a bar chart displaying luminescence levels from Lumit Immunoassay reagents configured for detection of pBTK.

[0023] FIG. 5A and 5B show bar charts displaying luminescence levels from a titration of Lumit Immunoassay reagents configured for detection of pBTK. FIG. 5B is an expanded view of certain lower reagent amounts.

[0024] FIG. 6 shows a bar chart displaying luminescence levels from Lumit Immunoassay reagents configured for detection of BTK.

[0025] FIG. 7 A shows a bar chart displaying the BRET Ratio (mBu) of live cells with intact cell membranes that were treated with bosutinib and subjected to mechanical deformation delivery of a kinase tracer. FIG. 7B shows a bar chart displaying the BRET Ratio (mBu) of live cells with intact cell membranes that were treated with ponatinib and subjected to mechanical deformation delivery of kinase tracer. FIG. 7C shows a bar chart displaying the BRET Ratio (mBu) of live cells with intact cell membranes that were treated with dasatinib and subjected to mechanical deformation delivery of kinase tracer.

[0026] FIG. 8A shows a bar chart displaying the viability of HeLa cells after mechanical deformation delivery of cereblon. FIG. 8B shows a bar chart displaying the percentage of Cy5-positive HeLa cells following delivery of Cy5-labeled cereblon via the mechanical deformation delivery technique.MF-367693737Attorney Docket No. 285192001040

[0027] FIG. 9A shows a bar chart displaying raw luminescence levels over time of live cells with intact cell membranes subjected to the mechanical deformation delivery technique. FIG.9B shows a bar chart displaying raw luminescence levels over time of lysate samples.

[0028] FIG. 10A shows a bar chart displaying luminescence levels of activated B cell lysates compared to activated B cells subjected to the mechanical deformation delivery technique. FIG.10B shows a bar chart displaying the difference in raw luminescence between activated B cell lysates versus activated B cells subjected to the mechanical deformation delivery technique.DETAILED DESCRIPTION OF THE INVENTION

[0029] Provided in the present application, in certain aspects, are methods of detecting an interaction in a live cell comprising an intact cell membrane, the methods comprising delivering one or more components to the live cell using a mechanical deformation delivery technique, such as passing the cell through a filter comprising pores therethrough. Following delivery, interaction of said one or more components produces a detectable signal. In some embodiments, the interaction is an interaction of reagents delivered to a cell, such as split luciferase system. For example, said reagents may be configured to specifically detect one of more target polypeptide, such as a specific target protein. In some embodiments, the methods taught herein may be used to detect and / or quantify the presence of a target in a cell, such as a polypeptide, and characteristics thereof, such as location, distribution, interaction(s), and degradation. In some embodiments, the detectable signal can be measured from the live cell comprising an intact cell membrane, such as via light produced or a change in fluorescence. In some embodiments, the detectable signal is further evaluated following manipulation of the live cell, such as following cell lysis. As described herein, in some embodiments, the detected interaction in a live cell comprising an intact cell membrane is indicative of an interaction between two or more components, such as reagents useful for a bioluminescence-based, FRET-based, BRET-based, HTRF-based, or AlphaLISA-based interaction assay. In some embodiments, said methodology is configured to detect the presence (including quantified amount or absence) of a target in the live cell comprising the intact cell membrane. In some embodiments, said methodology is configured to detect an interaction between two or more aspects in a live cell comprising an intact cell membrane, e.g., the interaction between two proteins or the interaction between a small molecule and a protein. As described herein, the one8MF-367693737Attorney Docket No. 285192001040 or more components (e.g., reagents) useful for performing the interaction assay may be a cell membrane impermeable. The description provided herein is based, at least in part, on the inventors’ unique perspectives and findings regarding the discovery of new and useful interaction assay formats in live cells comprising an intact cell membrane involving the mechanical deformation delivery of components useful thereof. Said assay formats provide measurements in cells exhibiting a more biological relevant state as compared to conventional assays, as well as allow for the measurement of cellular kinetics following delivery of said components. Surprisingly, the inventor’s found that complex detection systems can be delivered to live cells using a mechanical deformation delivery technique, wherein the reagents of the detection systems are delivered in sufficient quantity and are able to interact with one another so as to enable detection of aspects e.g., a target protein) within a live cell with an intact cell membrane. Moreover, the teachings provided herein can be applied to primary cells, as demonstrated in the Examples, which are known to be more difficult to manipulate than immortalized cells. Such findings represent a significant advancement in the field.

[0030] Thus, provided herein, in some aspects, is a method of detecting an interaction in a live cell comprising an intact cell membrane, the method comprising: delivering a first component, or a precursor thereof, to the live cell using a mechanical deformation delivery technique, wherein the first component is configured to interact with a second component in the live cell, and wherein a detectable signal is produced or producible when the first component interacts with the second component; and measuring the detectable signal to detect the interaction in the live cell comprising the intact cell membrane.

[0031] In other aspects, provided herein is a method of detecting an interaction in a live cell comprising an intact cell membrane, the method comprising: delivering a component comprising a luminescent enzyme, or an aspect thereof, or a precursor thereof, to the live cell using a mechanical deformation delivery technique, wherein the component comprising the luminescent enzyme, or the aspect thereof, associates or is associated with a target in the live cell; and measuring a luminescence output of the luminescent enzyme in the live cell comprising the intact cell membrane to detect the interaction in the live cell comprising the intact cell membrane.

[0032] In other aspects, provided herein is a method of detecting an interaction in a live cell comprising an intact cell membrane, the method comprising: delivering a first component9MF-367693737Attorney Docket No. 285192001040 comprising an acceptor chromophore, or a precursor thereof, and / or a second component comprising a donor chromophore, or a precursor thereof, to the live cell using a mechanical deformation delivery technique, wherein the first component comprising the acceptor chromophore and / or the second component comprising the donor chromophore associates or is associated with a target in the live cell; subjecting the live cell to an excitation light to excite the chromophore donor; and measuring a fluorescence output in the live cell comprising the intact cell membrane to detect the interaction in the live cell.

[0033] In other aspects, provided herein is a method of detecting an interaction in a live cell comprising an intact cell membrane, the method comprising: delivering (a)a first component comprising an acceptor chromophore, or a precursor thereof, and a second component comprising a donor chromophore, or an aspect thereof, or a precursor thereof, to the live cell using a mechanical deformation delivery technique, or (b) the first component comprising the acceptor chromophore, or the precursor thereof, to the live cell using the mechanical deformation delivery technique, wherein the live cell comprises the donor chromophore, or (c) the second component comprising the donor chromophore, or the aspect thereof, or the precursor thereof, to the live cell using the mechanical deformation delivery technique, wherein the live cell comprises the acceptor chromophore, wherein the first component comprising the acceptor chromophore and / or the second component comprising the donor chromophore associates or is associated with a target in the live cell; and measuring a fluorescence output in the live cell comprising the intact cell membrane to detect the interaction in the live cell.

[0034] In other aspects, provided herein is a method of detecting an interaction in a live cell comprising an intact cell membrane, the method comprising: delivering a first component comprising an acceptor bead and a second component comprising a donor bead to the live cell using a mechanical deformation delivery technique, wherein the first component comprising the acceptor bead is configured to associate with a first target and the second component comprising the donor bead is configured to associate with a second target; and measuring a fluorescence output in the live cell comprising an intact cell membrane to detect the interaction in the live cell.

[0035] In other aspects, provided herein are engineered live cells comprising an intact cell membrane, wherein the live cells comprise one or more components described herein as useful for an interaction assay taught herein.10MF-367693737Attorney Docket No. 285192001040

[0036] In other aspects, provided herein are kits comprising one or more aspects taught herein as useful for generating the engineered live cells describe herein.

[0037] The disclosures of all publications, patents, patent applications and published patent applications referred to herein are hereby incorporated herein by reference in their entirety.I. Definitions

[0038] In general, terms used in the claims and the specification are intended to be construed as having the plain meaning understood by a person of ordinary skill in the art. Certain terms are defined below to provide additional clarity. In case of conflict between the plain meaning and the provided definitions, the provided definitions are to be used.

[0039] As used herein, the term “individual” refers to a mammal and includes, but is not limited to, human, bovine, horse, feline, canine, rodent, rat, mouse, dog, or primate (such as a nonhuman primate). In some embodiments, the individual is a human individual.

[0040] The terms “polypeptide” and “protein,” as used herein, may be used interchangeably to refer to a polymer comprising amino acid residues, and are not limited to a minimum length. Such polymers may contain natural or non-natural amino acid residues, or combinations thereof, and include, but are not limited to, peptides, polypeptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Full-length polypeptides or proteins, and fragments thereof, are encompassed by this definition. The terms also include modified species thereof, e.g., post-translational modifications of one or more residues, for example, methylation, phosphorylation glycosylation, sialylation, or acetylation.

[0041] The term “antibody” includes full-length antibodies and antigen-binding fragments thereof. A full-length antibody comprises two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable region in both chains generally contain three highly variable loops called the complementarity determining regions (CDRs) (light chain (LC) CDRs including LC-CDR1, LC-CDR2, and LC-CDR3, heavy chain (HC) CDRs including HC-CDR1, HC-CDR2, and HC-CDR3). CDR boundaries for the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDRs of the heavy or11MF-367693737Attorney Docket No. 285192001040 light chains are interposed between flanking stretches known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several of the major antibody classes are divided into subclasses such as IgGl (yl heavy chain), lgG2 (y2 heavy chain), lgG3 (y3 heavy chain), lgG4 (y4 heavy chain), IgAl (al heavy chain), or lgA2 (a2 heavy chain).

[0042] The term “antigen-binding fragment” as used herein refers to an antibody fragment including, for example, a diabody, a Fab, a Fab', a F(ab')2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide stabilized diabody (ds diabody), a single-chain antibody molecule (scFv), an scFv dimer (bivalent diabody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a camelized single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not comprise a complete antibody structure. An antigen-binding fragment is capable of binding to the same antigen to which the parent antibody or a parent antibody fragment (e.g., a parent scFv) binds. In some embodiments, an antigen-binding fragment may comprise one or more CDRs from a particular human antibody grafted to a framework region from one or more different human antibodies.

[0043] The terms “comprising,” “having,” “containing,” and “including,” and other similar forms, and grammatical equivalents thereof, as used herein, are intended to be equivalent in meaning and to be open ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. For example, an article “comprising” components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components. As such, it is intended and understood that “comprises” and similar forms thereof, and grammatical equivalents thereof, include disclosure of embodiments of “consisting essentially of’ or “consisting of.”

[0044] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for12MF-367693737Attorney Docket No. 285192001040 convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For instance, where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictate otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure. In some embodiments, two opposing and open ended ranges are provided for a feature, and in such description it is envisioned that combinations of those two ranges are provided herein. For example, in some embodiments, it is described that a feature is greater than about 10 units, and it is described (such as in another sentence) that the feature is less than about 20 units, and thus, the range of about 10 units to about 20 units is described herein.

[0045] As used herein, the term “about” a number refers to that number plus or minus 10% of that number. The term “about” a range refers to that range minus 10% of its lowest value and plus 10% of its greatest value. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.

[0046] As used herein, including in the appended claims, the singular forms “a,” “or,” and “the” include plural referents unless the context clearly dictates otherwise.

[0047] Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of the present disclosure. The following description illustrates the disclosure and, of course, should not be construed in any way as limiting the scope of the inventions described herein.II. Interaction assay methods

[0048] Provided herein, in certain aspects, are methods of detecting an interaction in a live cell comprising an intact cell membrane, the methods comprising delivering one or more components useful therefor using a mechanical deformation delivery technique, such as by passing the live cell through a filter comprising pore therethrough.13MF-367693737Attorney Docket No. 285192001040

[0049] In some embodiments, provided is a method of detecting an interaction in a live cell comprising an intact cell membrane, the method comprising: delivering a first component, or a precursor thereof, to the live cell using a mechanical deformation delivery technique, wherein the first component is configured to interact with a second component in the live cell, and wherein a detectable signal is produced or producible when the first component interacts with the second component; and measuring the detectable signal to detect the interaction in the live cell comprising the intact cell membrane. For example, in some embodiments, the first component is an aspect of a luminescent enzyme, and the second component is a second aspect of the luminescent enzyme, wherein the first component and the second component form an active luminescent enzyme, and wherein the second component is associated (e.g., fused) with a target in the cell, such as a protein, nucleic acid, or subcellular structure. In some embodiments, the first component is associated with a second target in the cell, wherein the target and second target are the same or different (e.g., to assess intramolecular versus intermolecular interactions). In some embodiments, the target is exogenous to the cell and is delivered by mechanical deformation delivery technique. In some embodiments, the target is endogenous to the cell, however, the method comprises delivering an additional amount of the target or increasing expression of the target in said cell. In some embodiments, the method comprises delivering a labeled version of a target, such as a target labeled with a moiety useful for the methods described herein, e.g., a luminescent enzyme or an aspect thereof or a component that fluoresces or is designed to fluoresce following completion of a reaction. In some embodiments, the precursor thereof comprises a nucleic acid encoding the first component. In some embodiments, the nucleic acid comprises a DNA or an RNA. In some embodiments, the first component comprises a non-deoxyribonucleic acid component. In some embodiments, the first component comprises a polypeptide. In some embodiments, the first component comprises a synthetic component. In some embodiments, the synthetic component is a synthetic fluorophore or a detection particle. In some embodiments, the method further comprises delivering the second component, or a precursor thereof, to the live cell. In some embodiments, the precursor thereof, comprises a nucleic acid encoding the second component. In some embodiments, the nucleic acid comprises a DNA or an RNA. In some embodiments, the second component, or a precursor thereof, is co-delivered with the first component using the mechanical deformation delivery technique. In some embodiments, the second component, or a precursor thereof, is delivered sequentially after the first component using the mechanical deformation delivery technique. In14MF-367693737Attorney Docket No. 285192001040 some embodiments, the first component, or a precursor thereof, is delivered sequentially after the second component using the mechanical deformation delivery technique. In some embodiments, the mechanical deformation delivery technique comprises passing the live cell through a filter comprising pores therethrough, wherein the component(s) to be delivered to the live cell are in the presence of the live cell as it passes through the filter. In some embodiments, the mechanical deformation delivery technique comprises passing the live cell through a filter comprising pores therethrough, wherein the component(s) to be delivered to the live cell are not in the presence of the live cell as it passes through the filter and the component(s) are later place in the presence of the live cell while the live cell has open pores through the cell membrane. In some embodiments, the detectable signal indicates the interaction in the live cell comprises a direct interaction between the first component and the second component. In some embodiments, the detectable signal indicates the interaction in the live cell comprises a minimal distance between the first component and the second component need to produce the detectable signal. In some embodiments, the live cell is not genetically modified to express the first component and / or the second component. In some embodiments, the live cell does not comprise a nucleic acid encoding the first component and / or the second component.

[0050] In some embodiments, the first component and the second component form a luminescent enzyme (e.g., two-part luminescent enzymes such as LgBiT and SmBiT or LgBiT and HiBiT). In some embodiments, the method further comprises delivering a substrate of the luminescent enzyme to the live cell comprising the intact cell membrane. As certain substrates are cell membrane permeable, said method do not necessarily require delivery using a mechanical deformation delivery technique. In some embodiment, the substrate can be codelivered during delivery of one or more of the taught components. In some embodiments, the formed luminescent enzyme is catalytically active such that the detection event can occur.

[0051] In some embodiments, the first component and the second component exhibit resonance energy transfer to produce the detectable signal, such as useful for FRET, BRET, or AlphaLISA assay formats. For example, in some embodiments, the first component comprises a fluorescent donor and the second component comprises a fluorescent acceptor.

[0052] In some embodiments, the first component comprises a colorimetric enzyme, or an aspect thereof. In some embodiments, the colorimetric enzymes include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (ALP), glucose oxidase (GOx), lactate15MF-367693737Attorney Docket No. 285192001040 dehydrogenase (LDH), P-glucuronidase, and tyrosinase. In some embodiments, the associated substrates include, but are not limited to, 3,3'-diaminobenzidine (DAB), p-nitrophenyl phosphate (pNPP), glucose, lactate, glucuronide conjugates, and L-tyrosine. In some embodiments, the method comprises delivering a substrate of the colorimetric enzyme useful for producing the detectable signal. In some embodiments, the substrate can be co-delivered during delivery of one or more of the taught components. In some embodiments, the substrate can be separately delivered, such as using a mechanical deformation delivery technique or by admixing with the cell if the substrate is cell membrane permeable.

[0053] In some embodiments, the first component is configured to interact with the second component in the live cell, wherein the interaction results in a covalent bond(s) being formed between the first component and the second component. In some embodiments, the first component is configured to interact with the second component in the live cell such that a click chemistry reaction occurs covalently connecting the first component and the second component. In some embodiments, the first component comprises an acceptor chromophore and the second component comprises a donor chromophore, wherein a detectable signal is produced or producible when the first component interacts with the second component such that one or more covalent bonds are formed. In some embodiments, the formation of a covalent bond between a first component and a second component results in a detectable signal, such as a change in a fluorescent property associated with at least one of the components. In some embodiments, the interaction between the first component and the second component are further detectable due to a covalent modification, e.g., detectable based on a change in a target and / or component characteristic such as molecular weight. In some embodiments, the method comprises further detecting said signal, such as via immunoprecipitation, immunoblotting, and / or mass spectrometry analysis. Thus, in some embodiments, the methods provided herein enable detection of a signal in a live cell comprising an intact cell membrane as well as further analysis of an aspect from said live cell. For example, detection of a compound and / or molecule identity following cell lysis and / or extraction.

[0054] In some embodiments, wherein a first component is configured to interact with a second component in the live cell to enable formation of a detectable signal, the methods described herein encompass when said first component is a multi-part system, e.g., a primary and secondary antibody. In some embodiments, the second component is a multi-part system, e.g., a primary and second antibody. Molecules providing specific affinity, e.g., antibodies, can be used 16MF-367693737Attorney Docket No. 285192001040 in many assay formats taught herein, e.g., to bring a specific component in proximity, such as bound to, a target or to serve as a binding point for another molecule providing specific affinity, e.g., a secondary antibody. Antibody conjugates are well known in the art and antibodies conjugated to components described herein that are useful for the methods taught herein are encompassed in the instant application. The methods taught herein may comprise a variety of combinations of the components described herein, and may be arranged in a diverse array of configurations. In some instances, such as provided in section below, components and configurations are described in a modular fashion, and such description is not intended to limit the scope of the claimed subject matter encompassed herein. Specific interaction assay formats are described in more detail below.A. Bioluminescence-based interaction assays

[0055] In certain aspects, the method of detecting an interaction in a live cell comprising an intact cell membrane involves the use of a bioluminescent enzyme, such as a luciferase.Luciferase encompasses a group of enzymes that can produce bioluminescence through oxidative decarboxylation of a luciferase substrate, resulting in the release of energy in the form of light. The interaction assays taught herein are configured such that the bioluminescent light output is a measure of the biological activity being investigated, e.g., the presence of a specific target protein and / or the interaction between a protein and another protein or a small molecule. In some embodiments, the luciferase used in the methods taught herein is a two-subunit luciferase system. When the aspects of the luciferase come together an active luciferase is produced such that a substrate can then be used to generate a measurable signal. It is also envisioned that other multipart luciferase systems can be used in the methods described herein. In some embodiments, the two subunits, or complementation partners, utilized may be Large BiT (LgBiT; SEQ ID NO:1), a 17.6 kDa protein, and Small BiT (SmBiT; 1.3 kDa; SEQ ID NO:2), which is an 11-amino-acids polypeptide tag. LgBiT can bind to surface-exposed or secreted proteins tagged with SmBiT or HiBiT. Because LgBiT and SmBiT weakly associates (KD = 190 pM), the resulting luminescence signal is reversible. Alternatively, the tag may be HiBiT (SEQ ID NO:3), another 11-amino-acids polypeptide tag that has higher affinity to LgBiT (KD = 0.7 nM). Due to the higher affinity between LgBiT and HiBiT, the resulting luminescence signal is more permanent as compared to when SmBiT is used. Accordingly, HiBiT can be used17MF-367693737Attorney Docket No. 285192001040 to detect low-abundance proteins at endogenous levels. LgBiT, SmBiT, and HiBiT are cell-impermeable and conventional techniques require the subunits to be stably expressed or transfected as DNA.

[0056] In some embodiments, there is provided a method of detecting an interaction in a live cell comprising an intact cell membrane, the method comprising delivering a component comprising a first aspect of a split luciferase system, or a precursor thereof, and a component comprising a second aspect of the split luciferase system, or a precursor thereof, to the live cell using a mechanical deformation delivery technique, and measuring a luminescence output of the split luciferase system in the live cell comprising the intact cell membrane to detect the interaction in the live cell comprising the intact cell membrane. Examples of split luciferase systems can be found in, e.g., Lang et al., Curr Protoc Toxicol, 82, 2019, which is hereby incorporated herein by reference in its entirety. In some embodiments, the component comprising the first aspect of the split luciferase system comprises NLuc (amino acids 1-416 of Flue). In some embodiments, the component comprising the first aspect of the split luciferase system comprises CLuc (amino acids 398-550 of Flue).

[0057] In some embodiments, there is provided a method of detecting an interaction in a live cell comprising an intact cell membrane, the method comprising: delivering a component comprising a luminescent enzyme, or an aspect thereof, or a precursor thereof, to the live cell using a mechanical deformation delivery technique, wherein the component comprising the luminescent enzyme, or the aspect thereof, associates or is associated with a target in the live cell; and measuring a luminescence output of the luminescent enzyme in the live cell comprising the intact cell membrane to detect the interaction in the live cell comprising the intact cell membrane. In some embodiments, the method further comprises delivering a substrate of the luminescent enzyme to the live cell (such as either co-delivered with one or more components taught herein or delivered at another stage). In some embodiments, the substrate is co-delivered to the live cell along with the component comprising the luminescent enzyme, or the aspect thereof, or the precursor thereof, using the mechanical deformation delivery technique. In some embodiments, the live cell is not genetically modified to express the component comprising a luminescent enzyme, or an aspect thereof, or a precursor thereof and / or the substrate of the luminescent enzyme. In some embodiments, the live cell does not comprise a nucleic acid encoding the luminescent enzyme, or an aspect thereof, or a precursor thereof and / or a nucleic acid encoding the substrate of the luminescent enzyme. In some embodiments, the live cell is 18MF-367693737Attorney Docket No. 285192001040 selected from the group consisting of iPSC, HeLa, cardiomyocytes, and HEK293. In some embodiments, the live cell is a primary cell.

[0058] In some embodiments, the component comprising the luminescent enzyme, or the aspect thereof, or the precursor thereof, comprises the target or a precursor thereof. In some embodiments, the target is a polypeptide. In some embodiments, the target is a nucleic acid. In some embodiments, the target is a subcellular structure. The luminescent enzyme, or the aspect thereof, may be fused to the target or may associate with the target (such as via binding). In some embodiments, the live cell is not genetically modified to express the luminescent enzyme, or the aspect thereof fused or associated with the target. In some embodiments, the live cell does not comprise a nucleic acid encoding the luminescent enzyme, or the aspect thereof fused with the target.

[0059] In some embodiments, the luminescent enzyme, or the aspect thereof, delivered to the live cell via the mechanical deformation delivery technique is delivered in polypeptide form. In some embodiments, the luminescent enzyme, or the aspect thereof, delivered to the live cell via the mechanical deformation delivery technique is delivered in a non-deoxyribonucleic acid form (e.g., RNA). In some embodiments, the luminescent enzyme is composed of a complex comprising two or more aspects of the luminescent enzyme, and wherein upon complex formation in the live cell the luminescent enzyme is catalytically active. In some embodiments, the live cell comprises another aspect of the luminescent enzyme associated with a second target, and wherein the aspect of the luminescent enzyme associated with the target and the other aspect of the luminescent enzyme form a catalytically active enzyme. In some embodiments, the live cell comprises another aspect of the luminescent enzyme, and wherein the aspect of the luminescent enzyme associated with the target and the other aspect of the luminescent enzyme form a catalytically active enzyme. In some embodiments, the luminescent enzyme comprises LgBiT (SEQ ID NO:1) and SmBiT (SEQ ID NO:2). In some embodiments, the luminescent enzyme comprises LgBiT (SEQ ID NO:1) and HiBiT (SEQ ID NO:3). In some embodiments, the luminescent enzyme comprises LgBiT (SEQ ID NO:1) and natural peptide (NP: SEQ ID NO:4). In some embodiments, the live cell expresses the target fused to the aspect of the luminescent enzyme (such as SmBiT or HiBiT), wherein the delivering the component comprising the luminescent enzyme, or the aspect thereof, or the precursor thereof, to the live cell using the mechanical deformation delivery technique comprises delivering another aspect of the luminescent enzyme, or a precursor thereof (such as LgBiT), and wherein the aspect of the 19MF-367693737Attorney Docket No. 285192001040 luminescent enzyme fused to the target and the other aspect of the luminescent enzyme associate to become catalytically active. In some embodiments, the luminescent enzyme, or the aspect thereof, or the precursor thereof, is substantially cell membrane impermeable.

[0060] In some embodiments, the substrate of the luminescent enzyme is cell permeable. In some embodiments, the substrate of the luminescent enzyme is a luciferin. In some embodiments, the luciferin is selected from the group consisting of D-luciferin, bacterial luciferin, Diplocardia luciferin, Fridericia luciferin, coelenterazine luciferin, fungal luciferin, Cypridina luciferin, Latia luciferin, dinoflagellate luciferin, and Odontosyllis luciferin. In some embodiments, the substrate is selected from the group consisting of D-luciferin, coelenterazine, furimazine, reduced riboflavin phosphate, luciferin, and akalumine. In some embodiments, the luciferase is selected from the group consisting of firefly luciferase, bacterial luciferase (e.g., Vibrio harveyi), Diplocardia longa luciferase, Fridericia luciferase, gaussia luciferase, Metridia luciferase, fungal luciferase, Cypridina luciferase, dinoflagellate luciferase, e.g., Gonycaulax, Odontosyllis luciferin, click beetle luciferase, copepod (e.g., Gaussia princepds), ostracod luciferase (e.g., Cypridina noctiluca), jellyfish luciferase (e.g., Aequorea victori), and renilla luciferase (e.g., Renilla reniformis). See, e.g., Bose, TheScientist, Luciferase: A Powerful Bioluminescent Research Tool, 2024, which is hereby incorporated herein by reference in its entirety.

[0061] In some embodiments, the substrate of the luminescent enzyme is substantially cell impermeable.

[0062] In some embodiments, the live cell comprises one aspect of the luminescent enzyme associated with the target and a second aspect of the luminescent enzyme forming a catalytically active enzyme. In some embodiments, the one aspect of the luminescent enzyme associated with the target comprises SmBiT (SEQ ID NO:2) and the second aspect of the luminescent enzyme forming a catalytically active enzyme comprises LgBiT (SEQ ID NO:1). In some embodiments, the one aspect that is delivered includes the target associated with the aspect of the luminescent enzyme. In some embodiments, the one aspect (including, in some embodiments, the target associated with the aspect of the luminescent enzyme) and the second aspect are co-delivered via the mechanical deformation delivery technique to the live cell comprising the intact cell membrane. In some embodiments, the one aspect (including, in some embodiments, the target associated with the aspect of the luminescent enzyme) and the second aspect are separately20MF-367693737Attorney Docket No. 285192001040 delivered. In some embodiments, the one aspect is delivered via the mechanical deformation delivery technique to the live cell comprising the intact cell membrane and the second aspect is sequentially delivered via the mechanical deformation delivery technique to the live cell comprising the intact cell membrane. In some embodiments, the second aspect is delivered via the mechanical deformation delivery technique to the live cell comprising the intact cell membrane and the one aspect is sequentially delivered via the mechanical deformation delivery technique to the live cell comprising the intact cell membrane. In some embodiments, upon complex formation in the live cell comprising the intact cell membrane the luminescent enzyme is catalytically active. In some embodiments, the live cell is selected from the group consisting of iPSC, HeLa, cardiomyocytes, and HEK293. In some embodiments, the live cell is a primary cell.

[0063] In some embodiments, the live cell comprises one aspect of the luminescent enzyme associated with the target and a second aspect of the luminescent enzyme forming a catalytically active enzyme. In some embodiments, the one aspect of the luminescent enzyme associated with the target comprises HiBiT (SEQ ID NO:3) and the second aspect of the luminescent enzyme forming a catalytically active enzyme comprises LgBiT (SEQ ID NO:1). In some embodiments, the one aspect that is delivered includes the target associated with the aspect of the luminescent enzyme. In some embodiments, the one aspect (including, in some embodiments, the target associated with the aspect of the luminescent enzyme) and the second aspect are co-delivered via the mechanical deformation delivery technique to the live cell comprising the intact cell membrane. In some embodiments, the one aspect (including, in some embodiments, the target associated with the aspect of the luminescent enzyme) is delivered via the mechanical deformation delivery technique to the live cell comprising the intact cell membrane and the second aspect is sequentially delivered via the mechanical deformation delivery technique to the live cell comprising the intact cell membrane. In some embodiments, the second aspect is delivered via the mechanical deformation delivery technique to the live cell comprising the intact cell membrane and the one aspect is sequentially delivered via the mechanical deformation delivery technique to the live cell comprising the intact cell membrane. In some embodiments, upon complex formation in the live cell comprising the intact cell membrane the luminescent enzyme is catalytically active. In some embodiments, the live cell is selected from the group consisting of iPSC, HeLa, cardiomyocytes, and HEK293. In some embodiments, the live cell is a primary cell.21MF-367693737Attorney Docket No. 285192001040

[0064] In some embodiments, the live cell comprises one aspect of the luminescent enzyme associated with the target and a second aspect of the luminescent enzyme forming a catalytically active enzyme. In some embodiments, the one aspect of the luminescent enzyme associated with the target comprises SmBiT (SEQ ID NO:2). In some embodiments, the LgBiT (SEQ ID NO:1) has affinity for the second aspect of the luminescent enzyme. In some embodiments, the LgBiT (SEQ ID NO:1) is delivered via the mechanical deformation delivery technique to the live cell comprising the intact cell membrane and associates with the second aspect of the luminescent enzyme forming a catalytically active enzyme. In some embodiments, upon complex formation in the live cell comprising the intact cell membrane the luminescent enzyme is catalytically active. In some embodiments, the live cell is selected from the group consisting of iPSC, HeLa, cardiomyocytes, and HEK293. In some embodiments, the live cell is a primary cell.

[0065] In some embodiments, the live cell comprises one aspect of the luminescent enzyme associated with the target and a second aspect of the luminescent enzyme forming a catalytically active enzyme. In some embodiments, the one aspect of the luminescent enzyme associated with the target comprises HiBiT (SEQ ID NO:3). In some embodiments, the LgBiT (SEQ ID NO:1) has affinity for the second aspect of the luminescent enzyme. In some embodiments, the LgBiT (SEQ ID NO:1) is delivered via the mechanical deformation delivery technique to the live cell comprising the intact cell membrane and associates with the second aspect of the luminescent enzyme forming a catalytically active enzyme. In some embodiments, upon complex formation in the live cell comprising the intact cell membrane the luminescent enzyme is catalytically active. In some embodiments, the live cell is selected from the group consisting of iPSC, HeLa, cardiomyocytes, and HEK293. In some embodiments, the live cell is a primary cell.

[0066] In some embodiments, the LgBiT, SmBiT, and / or HiBiT are fused to an affinity moiety, wherein the affinity moiety binds to the target.

[0067] In some embodiments, the live cell comprises one aspect of the luminescent enzyme associated with the target and a second aspect of the luminescent enzyme forming a catalytically active enzyme. In some embodiments, the one aspect of the luminescent enzyme associated with the target comprises natural peptide (NP: SEQ ID NO:4) and the second aspect of the luminescent enzyme forming a catalytically active enzyme comprises LgBiT (SEQ ID NO:1). In some embodiments, the second aspect of the luminescent enzyme has affinity for the one aspect of the luminescent enzyme associated with the target comprising natural peptide (NP: SEQ ID22MF-367693737Attorney Docket No. 285192001040 NO:4). In some embodiments, the one aspect and the second aspect are co-delivered via the mechanical deformation delivery technique to the live cell comprising the intact cell membrane. In some embodiments, the one aspect is delivered via the mechanical deformation delivery technique to the live cell comprising the intact cell membrane and the second aspect is sequentially delivered via the mechanical deformation delivery technique to the live cell comprising the intact cell membrane. In some embodiments, the second aspect is delivered via the mechanical deformation delivery technique to the live cell comprising the intact cell membrane and the one aspect is sequentially delivered via the mechanical deformation delivery technique to the live cell comprising the intact cell membrane. In some embodiments, upon complex formation in the live cell comprising the intact cell membrane the luminescent enzyme is catalytically active. In some embodiments, the live cell is selected from the group consisting of iPSC, HeLa, cardiomyocytes, and HEK293. In some embodiments, the live cell is a primary cell.

[0068] In some embodiments, the bioluminescence-based methodology is configured to detect the presence (including the quantification or absence) or an aspect of in a live cell comprising an intact cell membrane, e.g., a target including a target protein. In some embodiments, the method comprises a component comprising a luminescent enzyme and a substrate of the luminescent enzyme, wherein the component comprising the luminescent enzyme associates with the target, wherein association of the luminescent enzyme and the substrate indicates the presence (including the quantification) or an aspect thereof in a live cell comprising an intact cell membrane, e.g., a target polypeptide in the live cell. In some embodiments, the method comprises a component comprising a luminescent enzyme and a substrate of the luminescent enzyme, wherein the component comprising the luminescent enzyme does not associate with the target, wherein lack of an association of the luminescent enzyme and the substrate indicates the absence (including the quantification) or an aspect of in a live cell comprising an intact cell membrane.B Fluorescence Resonance Energy Transfer (FRET) assays

[0069] In certain aspects, the method of detecting an interaction in a live cell comprising an intact cell membrane involves the use of FRET components. Fluorescence resonance energy transfer (FRET) is a technique the measures the transfer of energy between a donor molecule23MF-367693737Attorney Docket No. 285192001040 (e.g., dye or chromophore) and an acceptor molecule (e.g., chromophore). Unlike bioluminescence resonance energy transfer (BRET), FRET relies on an external light source, and thus in certain aspects provided herein, the methods comprise subjecting the live cell comprising an intact cell membrane with a light source configured to excite the donor molecule. The donor molecule initially absorbs the energy from the external light source, and the energy is then transferred to the acceptor molecule. FRET occurs when the distance between the donor and acceptor is approximately 1-10 nm, and thus FRET-based interaction assays described herein are useful for detecting interactions approximately within such distances.

[0070] In some embodiments, there is provided a method of detecting an interaction in a live cell comprising an intact cell membrane, the method comprising: delivering a first component comprising an acceptor chromophore, or a precursor thereof, and / or a second component comprising a donor chromophore, or a precursor thereof, to the live cell using a mechanical deformation delivery technique, wherein the first component comprising the acceptor chromophore and / or the second component comprising the donor chromophore associates or is associated with a target in the live cell; subjecting the live cell to an excitation light to excite the chromophore donor; and measuring a fluorescence output in the live cell comprising the intact cell membrane to detect the interaction in the live cell. In some embodiments, the delivering comprises delivering the first component comprising the acceptor chromophore, or the precursor thereof. In some embodiments, the delivering comprises delivering the second component comprising the donor chromophore, or the precursor thereof. In some embodiments, the delivering comprises delivering the first component comprising the acceptor chromophore, or the precursor thereof, and the second component comprising the donor chromophore, or the precursor thereof. In some embodiments, the live cell is not genetically modified to express the first component comprising the acceptor chromophore, or a precursor thereof, the second component comprising a donor chromophore, or a precursor thereof, the first component comprising the acceptor chromophore associated with a target in the live cell, and / or the second component comprising the donor chromophore associated with a target in the live cell. In some embodiments, the live cell does not comprise a nucleic acid encoding the first component comprising the acceptor chromophore, or a precursor thereof, a nucleic acid encoding the second component comprising a donor chromophore, or a precursor thereof, a nucleic acid encoding a first component comprising the acceptor chromophore fused with a target in the live cell, and / or a nucleic acid encoding a second component comprising the donor chromophore fused with a 24MF-367693737Attorney Docket No. 285192001040 target in the live cell. In some embodiments, the live cell is selected from the group consisting of iPSC, HeLa, cardiomyocytes, and HEK293. In some embodiments, the live cell is a primary cell.

[0071] In some embodiments, the donor chromophore is a natural molecule. In some embodiments, the donor chromophore is an autofluorescent polypeptide. In some embodiments, the autofluorescent polypeptide is a green fluorescent protein (GFP). In some embodiments, the second component comprising the donor chromophore, or the precursor thereof (e.g., DNA or RNA), is a nucleic acid encoding the donor chromophore. In some embodiments, the donor chromophore also encodes the target such that a fusion product comprising the target and the donor chromophore is produced (likewise, the fusion product is what can be delivered via the mechanical deformation delivery technique). In some embodiments, the donor chromophore is a synthetic molecule. In some embodiments, the second component comprising the donor chromophore, or the precursor thereof, is substantially cell impermeable. In some embodiments, the first component comprising the acceptor chromophore, or the precursor thereof, is substantially cell impermeable. In some embodiments, the acceptor chromophore is synthetic. In some embodiments, the second component comprising the acceptor chromophore comprises a small molecule. In some embodiments, the second component comprising the acceptor chromophore is a small molecule fluorophore.

[0072] In some embodiments, the first component comprising the acceptor chromophore associates or is associated with the target. In some embodiments, the second component comprising the donor chromophore associates or is associated with a second target. In some embodiments, the second component comprising the donor chromophore associates or is associated with the target. In some embodiments, the first component comprising the acceptor chromophore associates or is associated with a second target. In some embodiments, the first component comprising the acceptor chromophore associates or is associated with a test moiety. In some embodiments, the second component comprising the donor chromophore associates or is associated with a test moiety. In some embodiments, the test moiety associates with the target. In some embodiments, the test moiety comprises a small molecule. In some embodiments, the test moiety comprises a polypeptide.

[0073] In some embodiments, the donor chromophores include, but are not limited to, GFP (green fluorescent protein), CFP (cyan fluorescent protein), Clover, mClover3, mNeonGreen, Europium chelate, Terbium chelate, ATTO 390, ATTO 425, and ATTO 488.25MF-367693737Attorney Docket No. 285192001040

[0074] In some embodiments, the acceptor chromophores include, but are not limited to, YFP (yellow fluorescent protein), Lumio Green, Lumio Red, ULight, Surelight APC, AlexaFluor 647, XL665 / d2, HiLyte647, Far-red dye, fluorescein, GFP, and ATTO 490LS.

[0075] In some embodiments, the fluorescent proteins include, but are not limited to, Aquamarine, ECFP, mTurquoise2, mCerulean3, LUMP, mTFPl, EYFP, mVenus, sEYFP, mCitrine, YPet, EGFP, NowGFP, Clover, mClover3, mNeonGreen, mRuby2, mRuby3, mCherry, mPlum, eqFP650, mCardinal, IFP1.4, iRFP, mAmetrine, LSS-mOrange, tdTomato, mKate2, ShadowG, REAChl, REACh2, sREACh, rsTagRFP, PA-GFP, Phanta, T-Sapphire, mTagBFP, sfGFP, CyOFPl, mOrange2, mKOK, TagRFP, and DsRed.

[0076] In some embodiments, the live cell comprises a second component comprising a donor chromophore, or a precursor thereof. In some embodiments, a first component comprising an acceptor chromophore is delivered via the mechanical deformation delivery technique to the live cell comprising the intact cell membrane and associates with a target in the live cell comprising the intact cell membrane.

[0077] In some embodiments, the live cell comprises a first component comprising an acceptor chromophore, or a precursor thereof. In some embodiments, a second component comprising a donor chromophore, or a precursor thereof is delivered via the mechanical deformation delivery technique to the live cell comprising the intact cell membrane and associates with a target in the live cell comprising the intact cell membrane.

[0078] In some embodiments, a first component comprising an acceptor chromophore, or a precursor thereof, and a second component comprising a donor chromophore, or a precursor thereof are co-delivered via the mechanical deformation delivery technique to the live cell comprising the intact cell membrane and the first component and the second component associate with their respective targets in the live cell comprising the intact cell membrane. In some embodiments, a first component comprising an acceptor chromophore, or a precursor thereof, and a second component comprising a donor chromophore, or a precursor thereof are sequentially delivered via the mechanical deformation delivery technique to the live cell comprising the intact cell membrane and the first component and the second component associate with their respective targets in the live cell comprising the intact cell membrane.

[0079] In some embodiments, the methods provided herein comprise an acceptor chromophore fused to an affinity moiety, such as an antibody or a portion thereof that specifically binds to a 26MF-367693737Attorney Docket No. 285192001040 target. In some embodiments, the methods provided herein comprise a donor chromophore fused to an affinity moiety, such as an antibody or a portion thereof that specifically binds to a target. In some embodiments, the methods provided herein comprise an acceptor chromophore fused to an affinity moiety, such as an antibody or a portion thereof that specifically binds to a target, and a donor acceptor chromophore fused to an affinity moiety, such as an antibody or a portion thereof that specifically binds to a target. In some embodiments, the affinity moiety of an acceptor chromophore binds to a different epitope than the affinity moiety of the donor chromophore (e.g., different portions of a single protein or different proteins). In some embodiments, such examples of acceptor chromophores and / or donor chromophores are delivered to a cell in a polypeptide form or a nucleic acid form that is subsequently expressed in the cell. In some embodiments, the FRET-based methodology is configured to detect the presence (including the quantification or absence) or an aspect of in a live cell comprising an intact cell membrane, e.g., a target including a target protein. In some embodiments, the method comprises a first component comprising an acceptor chromophore and a second component comprising the donor chromophore, wherein the second component comprising the donor chromophore associates or is associated with a target in the live cell, wherein a fluorescence output indicates the presence (including the quantification) thereof in a live cell comprising an intact cell membrane, e.g., a target including a target protein. In some embodiments, the method comprises a first component comprising an acceptor chromophore and a second component comprising the donor chromophore, wherein the second component comprising the donor chromophore associates or is associated with a target in the live cell, wherein a lack of fluorescence output (such as at a specific wavelength) indicates the absence thereof, e.g., a target including a target protein.C. Homogeneous Time-Resolved Fluorescence (HTRF) assays

[0080] In certain aspects, the method of detecting an interaction in a live cell comprising an intact cell membrane involves the use of HTRF components. Homogeneous Time Resolved Fluorescence (HTRF) is an immunoassay that combines FRET with time-resolved (TR) fluorescent detection. TR-FRET introduces a time delay between the initial light excitation and fluorescence detection, which eliminates the non-specific, short-lived background fluorescence, by using long-lived fluorophores (e.g., lanthanides) as donors. Four specific fluorophores are27MF-367693737Attorney Docket No. 285192001040 used in HTRF to form different TR-FRET couples: Europium cryptate (Eu3+ cryptate; donor), Lumi4-Tb (Tb2+ cryptate; donor), XL665 (acceptor), and d2 (acceptor). The cryptate donors are typically excited at 337 nm while the acceptor and donor emit at 620 nm and 665 nm, respectively. Emission at 665 nm indicates energy transfer. A ratiometric measurement is taken for a specific time window on microplate readers.

[0081] In some embodiments, there is provided a method of detecting an interaction in a live cell comprising an intact cell membrane, the method comprising: delivering a first component comprising an acceptor chromophore, or a precursor thereof, and / or a second component comprising a donor chromophore, or a precursor thereof, to the live cell using a mechanical deformation delivery technique, wherein the first component comprising the acceptor chromophore and / or the second component comprising the donor chromophore associates or is associated with a target in the live cell; subjecting the live cell to an excitation light to excite the chromophore donor; and measuring a fluorescence output in the live cell comprising the intact cell membrane to detect the interaction in the live cell. In some embodiments, the delivering comprises delivering the first component comprising the acceptor chromophore, or the precursor thereof. In some embodiments, the delivering comprises delivering the second component comprising the donor chromophore, or the precursor thereof. In some embodiments, the delivering comprises delivering the first component comprising the acceptor chromophore, or the precursor thereof, and the second component comprising the donor chromophore, or the precursor thereof. In some embodiments, the live cell is selected from the group consisting of iPSC, HeLa, cardiomyocytes, and HEK293. In some embodiments, the live cell is a primary cell.

[0082] In some embodiments, the donor chromophore is a natural molecule. In some embodiments, the donor chromophore is an autofluorescent polypeptide. In some embodiments, the autofluorescent polypeptide is a green fluorescent protein (GFP). In some embodiments, the second component comprising the donor chromophore, or the precursor thereof (e.g., DNA or RNA), is a nucleic acid encoding the donor chromophore. In some embodiments, the donor chromophore also encodes the target and the donor chromophore is produced (likewise, the fusion product is what can be delivered via the mechanical deformation delivery technique). In some embodiments, the donor chromophore is a synthetic molecule. In some embodiments, the second component comprising the donor chromophore, or the precursor thereof, is substantially cell impermeable. In some embodiments, the first component comprising the acceptor chromophore, or the precursor thereof, is substantially cell impermeable. In some embodiments,28MF-367693737Attorney Docket No. 285192001040 the acceptor chromophore is synthetic. In some embodiments, the second component comprising the acceptor chromophore comprises a small molecule. In some embodiments, the second component comprising the acceptor chromophore is a small molecule fluorophore.

[0083] In some embodiments, the detecting the interaction in the live cell comprising the intact cell membrane is based on a FRET assay. In some embodiments, the detecting the interaction in the live cell comprising the intact cell membrane is based on a time-resolved FRET assay. In some embodiments, the detecting the interaction in the live cell comprising the intact cell membrane is based on a homogeneous time-resolved fluorescence assay. In some embodiments, the donor chromophore is a cryptate or terbium cryptate. In some embodiments, the acceptor chromophore is XL665 or d2.

[0084] In some embodiments, the first component comprising the acceptor chromophore associates or is associated with the target. In some embodiments, the second component comprising the donor chromophore associates or is associated with a second target. In some embodiments, the second component comprising the donor chromophore associates or is associated with the target. In some embodiments, the first component comprising the acceptor chromophore associates or is associated with a second target. In some embodiments, the first component comprising the acceptor chromophore associates or is associated with a test moiety. In some embodiments, the second component comprising the donor chromophore associates or is associated with a test moiety. In some embodiments, the test moiety associates with the target. In some embodiments, the test moiety comprises a small molecule. In some embodiments, the test moiety comprises a polypeptide. In some embodiments, the live cell is not genetically modified to express the first component comprising the acceptor chromophore, or a precursor thereof, the second component comprising a donor chromophore, or a precursor thereof, the first component comprising the acceptor chromophore associated with a target in the live cell, and / or the second component comprising the donor chromophore associated with a second target in the live cell. In some embodiments, the live cell does not comprise a nucleic acid encoding the first component comprising the acceptor chromophore, or a precursor thereof, a nucleic acid encoding the second component comprising a donor chromophore, or a precursor thereof, a nucleic acid encoding a first component comprising the acceptor chromophore fused with a target in the live cell, and / or a nucleic acid encoding a second component comprising the donor chromophore fused with a second target in the live cell.29MF-367693737Attorney Docket No. 285192001040

[0085] In some embodiments, the methods provided herein comprise an acceptor chromophore fused to an affinity moiety, such as an antibody or a portion thereof that specifically binds to a target. In some embodiments, the methods provided herein comprise a donor chromophore fused to an affinity moiety, such as an antibody or a portion thereof that specifically binds to a target. In some embodiments, the methods provided herein comprise an acceptor chromophore fused to an affinity moiety, such as an antibody or a portion thereof that specifically binds to a target, and a donor acceptor chromophore fused to an affinity moiety, such as an antibody or a portion thereof that specifically binds to a target. In some embodiments, the affinity moiety of an acceptor chromophore binds to a different epitope than the affinity moiety of the donor chromophore (e.g., different portions of a single protein or different proteins). In some embodiments, such examples of acceptor chromophores and / or donor chromophores are delivered to a cell in a polypeptide form or a nucleic acid form that is subsequently expressed in the cell.

[0086] In some embodiments, the HTRF-based methodology is configured to detect the presence (including the quantification or absence) of an aspect of in a live cell comprising an intact cell membrane, e.g., a target including a target protein. In some embodiments, the method comprises a first component comprising an acceptor chromophore and a second component comprising the donor chromophore, wherein the second component comprising the donor chromophore associates or is associated with a target in the live cell, wherein a fluorescence output indicates the presence (including the quantification) of an aspect of in a live cell comprising an intact cell membrane, e.g., a target including a target protein. In some embodiments, the method comprises a first component comprising an acceptor chromophore and a second component comprising the donor chromophore, wherein the second component comprising the donor chromophore associates or is associated with a target in the live cell, wherein a lack of fluorescence output indicates the absence of an aspect of in a live cell comprising an intact cell membrane, e.g., a target including a target protein.D. Bioluminescence Resonance Energy Transfer (BRET) assays

[0087] In certain aspects, the method of detecting an interaction in a live cell comprising an intact cell membrane involves the use of BRET components. Bioluminescence resonance energy transfer (BRET) is a technique that measures the transfer of energy between a luminescence30MF-367693737Attorney Docket No. 285192001040 donor (e.g., luciferase) and a fluorescence acceptor (e.g., GFP) without external illumination. In BRET, a protein of interest is fused to a bioluminescent energy donor while the other protein is fused to a fluorescent energy acceptor. BRET occurs when the distance between the donor and acceptor is less than 10 nm.

[0088] In some embodiments, there is provided a method of detecting an interaction in a live cell comprising an intact cell membrane, the method comprising: delivering: (a) a first component comprising an acceptor chromophore, or a precursor thereof, and a second component comprising a donor chromophore, or an aspect thereof, or a precursor thereof, to the live cell using a mechanical deformation delivery technique, or (b) the first component comprising the acceptor chromophore, or the precursor thereof, to the live cell using the mechanical deformation delivery technique, wherein the live cell comprises the donor chromophore, or (c) the second component comprising the donor chromophore, or the aspect thereof, or the precursor thereof, to the live cell using the mechanical deformation delivery technique, wherein the live cell comprises the acceptor chromophore, wherein the first component comprising the acceptor chromophore and / or the second component comprising the donor chromophore associates or is associated with a target in the live cell; and measuring a fluorescence output in the live cell comprising the intact cell membrane to detect the interaction in the live cell. In some embodiments, the live cell is not genetically modified to express the first component comprising the acceptor chromophore, or a precursor thereof, the second component comprising a donor chromophore, or a precursor thereof, the first component comprising the acceptor chromophore associated with a target in the live cell, and / or the second component comprising the donor chromophore associated with a target in the live cell. In some embodiments, the live cell does not comprise a nucleic acid encoding the first component comprising the acceptor chromophore, or a precursor thereof, a nucleic acid encoding the second component comprising a donor chromophore, or a precursor thereof, a nucleic acid encoding a first component comprising the acceptor chromophore fused with a target in the live cell, and / or a nucleic acid encoding a second component comprising the donor chromophore fused with a target in the live cell. In some embodiments, the live cell is selected from the group consisting of iPSC, HeLa, cardiomyocytes, and HEK293. In some embodiments, the live cell is a primary cell.

[0089] In some embodiments, the donor chromophore is a luminescent enzyme, such as described above including multipart luminescent enzymes such as LgBiT and SmBiT. In some embodiments, the donor chromophore is a luciferase. In some embodiments, the luciferase is 31MF-367693737Attorney Docket No. 285192001040 composed of a complex comprising two or more portions of the luminescent enzyme, and wherein upon complex formation the luminescent enzyme is catalytically active.

[0090] In some embodiments, the donor chromophore is a luminescent enzyme. In some embodiments, the luminescent enzyme is NanoLuc® luciferase. In some embodiments, the second component comprises a NanoLuc® luciferase. In some embodiments, the first component comprising the acceptor chromophore, or the precursor thereof is delivered via the mechanical deformation delivery technique and the second component comprising a NanoLuc® luciferase is sequentially delivered via the mechanical deformation delivery technique to the live cell comprising the intact cell membrane. In some embodiments, the second component comprising a NanoLuc® luciferase is delivered via the mechanical deformation delivery technique and first component comprising the acceptor chromophore, or the precursor thereof is sequentially delivered via the mechanical deformation delivery technique to the live cell comprising the intact cell membrane. In some embodiments, the first component comprising the acceptor chromophore, or the precursor thereof, and the second component comprises a NanoLuc® luciferase are co-delivered via the mechanical deformation delivery technique to the live cell comprising the intact cell membrane. In some embodiments, similarly, the luciferase can be delivered in two or more component parts, such as describe herein using LgBiT and SmBiT or LgBiT and HiBiT.

[0091] In some embodiments, the first component comprising the acceptor chromophore associates or is associated with the target. In some embodiments, the second component comprising the donor chromophore associates or is associated with a second target. In some embodiments, the second component comprising the donor chromophore associates or is associated with the target. In some embodiments, the first component comprising the acceptor chromophore associates or is associated with a second target. In some embodiments, the first component comprising the acceptor chromophore associates or is associated with a test moiety. In some embodiments, the second component comprising the donor chromophore associates or is associated with a test moiety. In some embodiments, the test moiety associates with the target. In some embodiments, the test moiety comprises a small molecule. In some embodiments, the test moiety comprises a polypeptide.

[0092] In some embodiments, the BRET-based methodology is configured to detect the presence (including the quantification or absence) of an aspect of in a live cell comprising an32MF-367693737Attorney Docket No. 285192001040 intact cell membrane, e.g., a target including a target protein. In some embodiments, the method comprises a first component comprising an acceptor chromophore and a second component comprising the donor chromophore, wherein the second component comprising the donor chromophore associates or is associated with a target in the live cell, wherein a fluorescence output indicates the presence (including the quantification) of an aspect of in a live cell comprising an intact cell membrane, e.g., a target including a target protein. In some embodiments, the method comprises a first component comprising an acceptor chromophore and a second component comprising the donor chromophore, wherein the second component comprising the donor chromophore associates or is associated with a target in the live cell, wherein a lack of fluorescence output indicates the absence of an aspect of in a live cell comprising an intact cell membrane, e.g., a target including a target proteinE. AlphaLISA assays

[0093] In certain aspects, the method of detecting an interaction in a live cell comprising an intact cell membrane involves the use of AlphaLISA components. AlphaLISA is a bead-based immunoassay for analyte detection in biological samples, e.g., Beaudet et al., Nature Methods, 5, 2008, which is incorporated herein by reference in its entirety. In certain aspects, AlphaLISA can may be configured as a sandwich or competition immunoassay. In certain embodiments, the sandwich AlphaLISA immunoassay uses two types of beads: 1) a biotinylated antibody bound to a streptavidin-coated donor beads and 2) a second antibody conjugated to AlphaLISA acceptor beads. Both antibodies recognize the same analyte. Binding of both antibodies to the analyte brings the donor and acceptor beads into proximity. In some embodiments, in the AlphaLISA competition immunoassay, the streptavidin-coated donor beads are conjugated to a biotinylated analyte, rather than a biotinylated antibody. The donor beads are used with an antibody conjugated to AlphaLISA acceptor beads. Excitation of the donor beads at 680 nm generates a flow of singlet oxygen that trigger a cascade of chemical reactions in the acceptor beads, leading to chemiluminescent emission at 615 nm.

[0094] In some embodiments, there is provided a method of detecting an interaction in a live cell comprising an intact cell membrane, the method comprising: delivering a first component comprising an acceptor bead and a second component comprising a donor bead to the live cell using a mechanical deformation delivery technique, wherein the first component comprising the33MF-367693737Attorney Docket No. 285192001040 acceptor bead is configured to associate with a first target and the second component comprising the donor bead is configured to associate with a second target; and measuring a fluorescence output in the live cell comprising an intact cell membrane to detect the interaction in the live cell. In some embodiments, the first target and second target are on the same macromolecule. In some embodiments, the first target and second targe are on different macromolecules. In some embodiments, the live cell is not genetically modified to express the first component comprising the acceptor bead, the second component comprising the donor bead, the first component comprising the acceptor bead associated with a first target in the live cell, and / or the second component comprising the donor bead associated with a second target in the live cell. In some embodiments, the live cell does not comprise a nucleic acid encoding the first component comprising the acceptor bead, a nucleic acid encoding the second component comprising the donor bead, a nucleic acid encoding a first component comprising the acceptor bead fused with a first target, and / or a nucleic acid encoding a second component comprising the donor bead fused with a second target. In some embodiments, the live cell is selected from the group consisting of iPSC, HeLa, cardiomyocytes, and HEK293. In some embodiments, the live cell is a primary cell.

[0095] In some embodiments, the AlphaLISA-based methodology is configured to detect the presence (including the quantification or absence) or an aspect of in a live cell comprising an intact cell membrane, e.g., a target including a target protein. In some embodiments, the method comprises a first component comprising an acceptor bead and a second component comprising a donor bead, wherein the acceptor bead and the donor bead associate with the same target, such as by binding specifically thereto via non- overlapping epitopes.F. Lumit Immunoassays

[0096] In certain aspects, the methods described herein for detecting an interaction in a live cell comprising an intact cell membrane involves the use of Lumit Immunoassay reagents. Lumit Immunoassays are configured with primary and secondary antibodies sets configured to detect a target, e.g., a protein or protein complex, when two secondary antibodies are brought in proximity with one another. For example, in some embodiments, the Lumit Immunoassay reagents comprise: a first primary antibody that specifically binds to a first epitope of a target polypeptide; a second primary antibody that specifically binds to a second epitope of the target polypeptide; a first secondary antibody that specifically binds to the first primary antibody; and a34MF-367693737Attorney Docket No. 285192001040 second secondary antibody that specifically binds to the second primary antibody, wherein the first secondary antibody comprises a first aspect of a luciferase (or another split enzyme allowing for generation of a detectable signal), wherein the second secondary antibody comprises a second aspect of the luciferase (or another split enzyme allow for generation of a detectable signal), and wherein the first and second aspect of the luciferase form a functional luciferase. In some embodiments, a first primary antibody and a second primary antibody are derived from a different organism such that they can be distinguished using anti-species specific antibodies. In certain aspects, provided herein is a method of a method of detecting an interaction in a live cell comprising an intact cell membrane, the method comprising: delivering, using a mechanical deformation delivery technique, (a) a first binding agent (e.g., a primary antibody) that specifically binds a cellular target (e.g., a protein), (b) a second binding agent (e.g., a primary antibody) that specifically binds the cellular target (e.g., a protein), wherein the first binding agent and the second binding agent bind to different aspects of the target (e.g., different epitopes), (c) a first secondary binding agent that specifically binds the first binding agent, wherein the first secondary binding agent comprises a first aspect of a split luciferase system, and (d) a second secondary binding agent that specifically binds the second binding agent, wherein the second secondary binding agent comprises a second aspect of a split luciferase system, wherein the first aspect and the second aspect of the split luciferase system form an active luciferase; and measuring a fluorescence output in the live cell comprising an intact cell membrane to detect the interaction in the live cell. As described herein, the components delivered to the live cell may be delivered in various forms, such as a polypeptide or a nucleic acid encoding a polypeptide. Examples of split luciferase systems can be found in, e.g., Lang et al., Curr Protoc Toxicol, 82, 2019, which is hereby incorporated herein by reference in its entirety. In some embodiments, the luciferase system used in the methods taught herein is a two-subunit luciferase system. When the aspects of the luciferase come together (i.e., interact) an active luciferase is produced such that a substrate can then be used to generate a measurable signal. It is also envisioned that other multipart luciferase systems can be used in the methods described herein. In some embodiments, the two subunits, or complementation partners, utilized may be Large BiT (LgBiT; SEQ ID NO:1), a 17.6 kDa protein, and Small BiT (SmBiT; 1.3 kDa; SEQ ID NO:2), which is an 11-amino-acids polypeptide tag. LgBiT can bind to surface-exposed or secreted proteins tagged with SmBiT or HiBiT. Because LgBiT and SmBiT weakly associates (KD = 190 pM), the resulting luminescence signal is reversible. Alternatively, the tag35MF-367693737Attorney Docket No. 285192001040 may be HiBiT (SEQ ID NO:3), another 11-amino-acids polypeptide tag that has higher affinity to LgBiT (KD = 0.7 nM). Due to the higher affinity between LgBiT and HiBiT, the resulting luminescence signal is more permanent as compared to when SmBiT is used. Accordingly, HiBiT can be used to detect low-abundance proteins at endogenous levels. LgBiT, SmBiT, and HiBiT are cell-impermeable and conventional techniques require the subunits to be stably expressed or transfected as DNA. In some embodiments, the component comprising the first aspect of the split luciferase system comprises NLuc (amino acids 1-416 of Flue). In some embodiments, the component comprising the first aspect of the split luciferase system comprises CLuc (amino acids 398-550 of Flue).

[0097] In some embodiments, the method further comprises delivering a substrate of the luminescent enzyme to the live cell (such as either co-delivered with one or more components taught herein or delivered at another stage). In some embodiments, the substrate is co-delivered to the live cell along with the component comprising the luminescent enzyme, or the aspect thereof, using the mechanical deformation delivery technique. In some embodiments, the substrate is incubated with the live cell after delivery of components thereto, wherein the substrate is cell membrane permeable. In some embodiments, the live cell is not genetically modified to express the component comprising a luminescent enzyme, or an aspect thereof, and / or the substrate of the luminescent enzyme. In some embodiments, the live cell does not comprise a nucleic acid encoding the luminescent enzyme, or an aspect thereof, and / or a nucleic acid encoding the substrate of the luminescent enzyme. In some embodiments, the live cell is selected from the group consisting of iPSC, HeLa, cardiomyocytes, Ramos cells, and HEK293. In some embodiments, the live cell is a primary cell.G. PROTAC

[0098] In certain aspects, the methods described herein may further comprise the use of one or more proteolysis targeting chimera (PROTAC) components. PROTACs may be configured to target the degradation of one or more specific polypeptides. Accordingly, as the assays configured herein may be used to study cellular kinetics, PROTACs may be used to assess the cell when there is a degradation of a polypeptide over time. In some embodiments, the PROTAC degrades the polypeptide that is the target(s) of the interaction assay.36MF-367693737Attorney Docket No. 285192001040

[0099] In some embodiments, the live cell further comprises a PROTAC configured to associate with the target in the cell. In some embodiments, the cell further comprises a PROTAC configured to associate with a macromolecule comprising the first target or a macromolecule comprising the second target. In some embodiments, the method further comprises delivering the PROTAC to the cell. In some embodiments, the PROTAC degrades the first target or the second target. In some embodiments, the PROTAC degrades a component associated with the first target or a component associated with the second target. In some embodiments, the live cell is not genetically modified to express the PROTAC, the PROTAC associated with a target in the live cell, the PROTAC associated with a macromolecule comprising the first target, and / or the PROTAC associated with a macromolecule comprising the second target.II. Cellular kinetics and measurement techniques

[0100] The interaction assays taught herein may be performed to obtain information regarding cellular kinetics. As discussed herein, the delivery techniques encompassed by the methods described herein result in live cell comprising intact cell membranes, and accordingly, in certain aspects, the interaction assay formats allow for non-terminal measurements (measurements that can be taken while the cell is alive). In some embodiments, the measuring comprises measuring at two or more time points. The methods described herein can be used with a diverse array of cell numbers. In some embodiments, the number of cells used for the methods described herein is at least about 1,000 cells to about 10,000,000 cells (including for example about any of 1,000 cells, 10,000 cells, 100,000 cells, 1,000,000 cells, 10,000,000 cells, or any ranges within any of the numerical values).

[0101] In some embodiments, the methods taught herein comprise delivery of an impermeable degrader and / or polypeptide via mechanical deformation to assess cellular activity following delivery. In some embodiments, the methods taught herein comprises delivery of a nucleic acid, e.g., mRNA, encoding a polypeptide of interest comprising a component useful for an assay taught herein and another component useful for said assay, e.g., LgBiT and a polypeptide of interest comprising SmBiT. In some embodiments, the delivery is to a primary cell, wherein the method comprises measuring a detectable signal to detect the interaction in the live primary cell comprising the intact cell membrane.37MF-367693737Attorney Docket No. 285192001040

[0102] The methods described herein comprise measuring a detectable signal, and such measuring may be performed on any suitable device. One of ordinary skill in the art will readily appreciate that the measuring device may be selected based on characteristics of the detectable signal. In some embodiments, the measuring is performed via fluorescence microscopy. In some embodiments, the measuring is performed via a plate reader.I. Example assay methods

[0103] In certain aspects, the method described herein is a FRET-based assay performed in a live cell comprising an intact cell membrane, the method comprising: delivering a first component comprising an acceptor chromophore, or a precursor thereof, and / or a second component comprising a donor chromophore, or a precursor thereof, to the live cell using a mechanical deformation delivery technique, wherein the first component comprising the acceptor chromophore and / or the second component comprising the donor chromophore associates or is associated with a target in the live cell; subjecting the live cell to an excitation light to excite the chromophore donor; and measuring a fluorescence output in the live cell comprising the intact cell membrane to detect the interaction in the live cell. In some embodiments, the first component comprises a binding agent, such as an antibody. In some embodiments, the second component comprises a binding agent, such as an antibody. In some embodiments, the first component and / or second component associate with the target based on secondary (or more) interactions (e.g., one or more primary binding agents bind to the target and the first component and the second component associate with a target via the primary binding agent(s)). In some embodiments, the interaction detected in the live cell is the interaction of the acceptor chromophore and the donor chromophore in the live cell. In some embodiments, the first component comprises a binding agent, such as an antibody, that specifically binds the target (endogenous or exogenous), such as a polypeptide. In some embodiments, the second component comprises a binding agent, such as an antibody, that specifically binds the target (endogenous or exogenous), such as a polypeptide. In some embodiments, the target associated with the first component and the target associated with the second component are the same target. In some embodiments, the target associated with the first component and the target associated with the second component are the different targets, wherein the different targets38MF-367693737Attorney Docket No. 285192001040 include, e.g., dimers or multimers of a complex or interact with one another. In some embodiments, the interaction detected in the live cell is in the cytoplasm.

[0104] In certain aspects, the method described herein is a Lumit-based assay performed in a live cell comprising an intact cell membrane, the method comprising: delivering a component comprising a luminescent enzyme, or an aspect thereof, or a precursor thereof, to the live cell using a mechanical deformation delivery technique, wherein the component comprising the luminescent enzyme, or the aspect thereof, associates or is associated with a target in the live cell; and measuring a luminescence output of the luminescent enzyme in the live cell comprising the intact cell membrane to detect the interaction in the live cell comprising the intact cell membrane. In some embodiments, the component comprising the luminescent enzyme, or an aspect thereof, or a precursor thereof, comprises LgBiT. In some embodiments, the method further comprises delivering a second component comprising a luminescent enzyme, or an aspect thereof, or a precursor thereof, to the live cell using a mechanical deformation delivery technique (including separate delivery or co-delivery with the component. In some embodiments, the second component comprising a luminescent enzyme, or an aspect thereof, or a precursor thereof, comprises SmBiT. In some embodiments, the component comprises a binding agent, such as an antibody. In some embodiments, the second component comprises a binding agent, such as an antibody. In some embodiments, the component and / or second component associate with the target based on secondary (or more) interactions e.g., one or more primary binding agents bind to the target and the component and the second component associate with a target via the primary binding agent(s)). In some embodiments, the interaction detected in the live cell is the interaction of LgBiT and SmBiT in the live cell. In some embodiments, the component comprises a binding agent, such as an antibody, that specifically binds the target (endogenous or exogenous), such as a polypeptide. In some embodiments, the second component comprises a binding agent, such as an antibody, that specifically binds the target (endogenous or exogenous), such as a polypeptide. In some embodiments, the target associated with the component and the target associated with the second component are the same target. In some embodiments, the target associated with the component and the target associated with the second component are the different targets, wherein the different targets include, e.g., dimers or multimers of a complex or interact with one another. In some embodiments, the precursor of the component or second component is a nucleic acid (e.g., mRNA) encoding a polypeptide. In some embodiments, the substrate of the luminescent assay is cell-membrane 39MF-367693737Attorney Docket No. 285192001040 permeable. In some embodiments, the substrate of the luminescent assay is co-delivered with the component and / or second component. In some embodiments, the substrate of the luminescent assay is delivered using a mechanical deformation delivery technique. In some embodiments, the interaction detected in the live cell is in the cytoplasm.

[0105] In certain aspects, the method described herein is a HiBiT-based assay performed in a live cell comprising an intact cell membrane, the method comprising: delivering a component comprising a luminescent enzyme, or an aspect thereof, or a precursor thereof, to the live cell using a mechanical deformation delivery technique, wherein the component comprising the luminescent enzyme, or the aspect thereof, associates or is associated with a target in the live cell; and measuring a luminescence output of the luminescent enzyme in the live cell comprising the intact cell membrane to detect the interaction in the live cell comprising the intact cell membrane. In some embodiments, the component comprising the luminescent enzyme, or an aspect thereof, or a precursor thereof, comprises LgBiT. In some embodiments, the method further comprises delivering a second component comprising a luminescent enzyme, or an aspect thereof, or a precursor thereof, to the live cell using a mechanical deformation delivery technique (including separate delivery or co-delivery with the component. In some embodiments, the second component comprising a luminescent enzyme, or an aspect thereof, or a precursor thereof, comprises HiBiT. In some embodiments, the component comprises a binding agent, such as an antibody. In some embodiments, the second component comprises a binding agent, such as an antibody. In some embodiments, the component and / or second component associate with the target based on secondary (or more) interactions (e.g., one or more primary binding agents bind to the target and the component and the second component associate with a target via the primary binding agent(s)). In some embodiments, the interaction detected in the live cell is the interaction of LgBiT and HiBiT in the live cell. In some embodiments, the component comprises a binding agent, such as an antibody, that specifically binds the target (endogenous or exogenous), such as a polypeptide. In some embodiments, the second component comprises a binding agent, such as an antibody, that specifically binds the target (endogenous or exogenous), such as a polypeptide. In some embodiments, the target associated with the component and the target associated with the second component are the same target. In some embodiments, the target associated with the component and the target associated with the second component are the different targets, wherein the different targets include, e.g., dimers or multimers of a complex or interact with one another. In some embodiments, the 40MF-367693737Attorney Docket No. 285192001040 precursor of the component or second component is a nucleic acid (e.g., mRNA) encoding a polypeptide. In some embodiments, the substrate of the luminescent assay is cell-membrane permeable. In some embodiments, the substrate of the luminescent assay is co-delivered with the component and / or second component. In some embodiments, the substrate of the luminescent assay is delivered using a mechanical deformation delivery technique. In some embodiments, the interaction detected in the live cell is in the cytoplasm.

[0106] In certain aspects, the method described herein is a target engagement assay comprising use of a cell impermeable tracer according to the methods taught herein. In some embodiments, the cell impermeable tracer comprises a chemical compound, such a small molecule therapeutic candidate e.g., having regulatory approval or an application submitted thereof, under research for use in treating a human condition, and / or characterized by one or more of Lipinski’s rule of 5). In certain aspects, the target engagement assay is a FRET-based assay, wherein the method comprises: delivering a first component comprising an acceptor chromophore, or a precursor thereof, and / or a second component comprising a donor chromophore, or a precursor thereof, to the live cell using a mechanical deformation delivery technique, wherein the first component comprising the acceptor chromophore and / or the second component comprising the donor chromophore associates or is associated with a target in the live cell; subjecting the live cell to an excitation light to excite the chromophore donor; and measuring a fluorescence output in the live cell comprising the intact cell membrane to detect the interaction in the live cell. In some embodiments, the first component comprises the chemical compound, wherein second component comprises a binding target of the chemical compound. In some embodiments, the second component comprises the chemical compound, wherein first component comprises a binding target of the chemical compound. In certain aspects, the target engagement assay is a BRET-based assay, wherein the method comprises: delivering a first component comprising an acceptor chromophore, or a precursor thereof, and / or a second component comprising a donor chromophore, or a precursor thereof, to the live cell using a mechanical deformation delivery technique, wherein the first component comprising the acceptor chromophore and / or the second component comprising the donor chromophore associates or is associated with a target in the live cell; and measuring a fluorescence output in the live cell comprising the intact cell membrane to detect the interaction in the live cell. In some embodiments, the first component comprises the chemical compound, wherein second component comprises a binding target of the chemical compound and the chromophore donor, such as luciferase.41MF-367693737Attorney Docket No. 285192001040III. Cells

[0107] The methods provided herein are suitable to a diverse array of cells. A live cell according to the present disclosure may be any suitable cell known in the art, such as bacterial, mammalian, insect, fungal, or plant cells. The live cells may be cell lines, primary cells, stem cells, differentiated cells derived or produced from such stem cells, hematopoietic stem cells, induced pluripotent stem cells, immortalized cells, and the like. In some embodiments, the live cell is a human cell. In some embodiments, the live cell is selected from the group consisting of iPSC, HeLa, cardiomyocytes, and HEK293.

[0108] In some embodiments, the live cell is a somatic cell. In some embodiments, the live cells are derived from skin or other organs, e.g., heart, brain or spinal cord, liver, lung, kidney, pancreas, bladder, bone marrow, spleen, intestine, or stomach. The live cells can be from humans or other mammals e.g., rodent, non-human primate, bovine, or porcine cells).

[0109] In some embodiments, the live cell is a mammalian cell, for example a primary cell or a cell line, selected from the group consisting of: islet cells, beta islet cells, pancreatic islet cells, immune cells, B cells, T cells, natural killer (NK) cells, natural killer T (NKT) cells, macrophage cells, endothelial cells, muscle cells, cardiac muscle cells, smooth muscle cells, skeletal muscle cells, dopaminergic neurons, retinal pigmented epithelium cells, optic cells, hepatocytes, thyroid cells, skin cells, glial progenitor cells, neural cells, cardiac cells, stem cells, hematopoietic stem cells, induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), pluripotent stem cell (PSCs), blood cells, endothelial stem cells, epithelial stem cells, adipose stem or progenitor cells, germline stem cells, lung stem or progenitor cells, mammary stem cells, olfactory adult stem cells, hair follicle stem cells, multipotent stem cells, amniotic stem cells, cord blood stem cells, neural stem or progenitor cells, CHO cells, BHK cells, MDCK cells, C3H 10T1 / 2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, HEK293 cells, HEK293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, NS0, PerC6, Sp2 / 0, BHK, C127, and 211 A cells. In some embodiments, the live cell is a B lymphocyte. In some embodiments, the live cell is a Ramos cell.42MF-367693737Attorney Docket No. 285192001040

[0110] In some embodiments, the live cell is an immune cell, such as, for example, an immune cell selected from the group consisting of: a natural killer (NK) cell, a natural killer T (NKT) cell, a T cell (e.g., CTL), a CD 14+ cell, a dendritic cell, a PBMC cell, and any combination thereof. In some embodiments, the cell is an NK cell, a T cell (e.g., CTL), or a PBMC.

[0111] In some embodiments, the live cell is a stem cell or progenitor cell (e.g., iPSC, embryonic stem cell, hematopoietic stem cell, mesenchymal stem cell, endothelial stem cell, epithelial stem cell, adipose stem or progenitor cells, germline stem cells, lung stem or progenitor cells, mammary stem cells, olfactory adult stem cells, hair follicle stem cells, multipotent stem cells, amniotic stem cells, cord blood stem cells, or neural stem or progenitor cells). In some embodiments, the stem cells are adult stem cells (e.g., somatic stem cells or tissue specific stem cells). In some embodiments, the stem or progenitor cell is capable of being differentiated (e.g., the stem cell is totipotent, pluripotent, or multipotent). In some embodiments, the live cell is isolated from embryonic or neonatal tissue. In some embodiments, the live cell is a fibroblast, monocytic precursor, B cell, exocrine cell, pancreatic progenitor, endocrine progenitor, hepatoblast, myoblast, preadipocyte, progenitor cell, hepatocyte, chondrocyte, smooth muscle cell, K562 human erythroid leukemia cell line, bone cell, synovial cell, tendon cell, ligament cell, meniscus cell, adipose cell, dendritic cells, or natural killer cell. In some embodiments, the cell is manipulated (e.g., converted or differentiated) into a muscle cell, erythroid-megakaryocytic cell, eosinophil, iPSC, macrophage, T cell, islet beta-cell, neuron, cardiomyocyte, blood cell, endocrine progenitor, exocrine progenitor, ductal cell, acinar cell, alpha cell, beta cell, delta cell, PP cell, hepatocyte, cholangiocyte, or brown adipocyte. In some embodiments, the live cell is a muscle cell (e.g., skeletal, smooth, or cardiac muscle cell), erythroid-megakaryocytic cell, eosinophil, iPSC, macrophage, T cell, islet beta-cell, neuron, cardiomyocyte, blood cell (e.g., red blood cell, white blood cell, or platelet), endocrine progenitor, exocrine progenitor, ductal cell, acinar cell, alpha cell, beta cell, delta cell, PP cell, hepatocyte, cholangiocyte, or white or brown adipocyte. In some embodiments, the live cell is a hormone-secreting cell (e.g., a cell that secretes insulin, oxytocin, endorphin, vasopressin, serotonin, somatostatin, gastrin, secretin, glucagon, thyroid hormone, bombesin, cholecystokinin, testosterone, estrogen, or progesterone, renin, ghrelin, amylin, or pancreatic polypeptide), an epidermal keratinocyte, an epithelial cell (e.g., an exocrine secretory epithelial cell, a thyroid epithelial cell, a keratinizing epithelial cell, a gall bladder epithelial cell, or a surface epithelial cell of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra, or 43MF-367693737Attorney Docket No. 285192001040 vagina), a kidney cell, a germ cell, a skeletal joint synovium cell, a periosteum cell, a bone cell (e.g., osteoclast, osteocyte, or osteoblast), a perichondrium cell (e.g., a chondroblast or chondrocyte), a cartilage cell (e.g., chondrocyte), a fibroblast, an endothelial cell, a pericardium cell, a meningeal cell, a keratinocyte precursor cell, a keratinocyte stem cell, a pericyte, a glial cell, an ependymal cell, a cell isolated from an amniotic or placental membrane, or a serosal cell (e.g., a serosal cell lining body cavities).

[0112] In certain aspects, provided herein are engineered live cells comprising intact cell membranes and comprising one or more components taught useful for the described interaction assays. In some embodiments, provided herein are live cells comprising intact cell membranes and comprising one or more components taught herein useful for the described interaction assays, wherein the live cell is produced by a mechanical deformation delivery technique of at least one of the one or more components.IV. Mechanical deformation delivery techniques

[0113] The methods provided herein comprise delivering one or more components useful for the described interaction assays using a mechanical deformation delivery technique. In some embodiments, the mechanical deformation delivery technique comprising passing the cell through a constriction-based structure to form pores in the membrane of the cell. In some embodiments, the constriction-based structure comprises a filter or a microfluidic channel. In some embodiments, the mechanical deformation delivery technique comprises passing the cell through a microfluidic channel, such as described in US Pat. No. 10,696,944, which is incorporated herein by reference in its entirety.

[0114] In certain aspects, provided herein are methods of detecting an interaction in a live cell, wherein the methods comprise subjecting a cell suspension to a mechanical deformation, such as by passing the cell suspension through a material having pores therethrough, e.g., a filter.

[0115] In some embodiments, the method comprises subjecting a cell, such as in a cell suspension, to a mechanical deformation. In some embodiments, the method comprises passing a cell, such as in a cell solution, through a material having pores therethrough. In some embodiments, the cell is subjected to a mechanical deformation in the presence of a cargo, such as one or more components useful for the interaction assays described herein. For example, in44MF-367693737Attorney Docket No. 285192001040 some embodiments, the method is performed by subjecting a cell suspension comprising a cell and a cargo, such as a first component, or a precursor thereof, and / or a second component, or a precursor thereof, to a mechanical deformation, such as passing the cell suspension through a material comprising pores therethrough. In some embodiments, the cell is subjected to a mechanical deformation in the absence of a cargo, such as a first component, or a precursor thereof, and / or a second component, or a precursor thereof, wherein following deformation the cell is admixed with the cargo (such as providing the cargo during a period when pores still exist in a cell following a mechanical deformation). For example, in some embodiments, the method is performed by subjecting a cell suspension comprising a cell to a mechanical deformation, such as passing the cell suspension through a material comprising pores therethrough, followed by mixing the processed cell suspension with a cargo, such as a first component, or a precursor thereof, and / or a second component, or a precursor thereof.

[0116] The methods disclosed herein may comprise a variety of different combinations of the component features described herein. In some instances, such methods and component features are described in a modular fashion. The modular discussion of such component features does not limit the scope of the inventions provided herein and one of ordinary skill in the art will readily appreciate how certain features from the sections below can be combined to perform the cellular delivery methods provided herein.A. Mechanical deformation delivery and components thereof

[0117] In certain aspects, provided herein are mechanical deformation processes for use in the methods of detecting an interaction in a live cell taught herein. Such mechanical deformation processes are suitable for deforming a cell such that pores exist in the cell for a short period of time (e.g., 30 seconds) after deformation allowing for cargo delivery to the inside of the cell, and after said period of time the pores close enclosing the delivered cargo within the cell. As described herein, a cell mechanoporation component used in a mechanical deformation delivery may comprise one or more constrictions, or narrowings, that deform the cells that pass through the component. The use of the term cell mechanoporation component in this disclosure can be understood to encompass mechanoporation devices known to one of ordinary skill in the art, such as a cell perturbation filters (e.g., filters), microfluidic chips containing one or more channels with constrictions or confinements in parallel or series, post configurations to induce45MF-367693737Attorney Docket No. 285192001040 perturbations, constrictions created through depth changes in fluidic channels, a microneedle or microneedle array, etc. In the instance a filter or microfluidic chip is referred to throughout this disclosure, it is to be understood that the other cell mechanoporation component can be reasonably incorporated in its place.

[0118] In some embodiments, the cargo to be delivered is present with the cell when passing through one or more constrictions for mechanical deformation. In some embodiments, the cargo to be delivered is admixed with the cell after passing through one or more constrictions for mechanical deformation. In some embodiments, a first cargo is to be delivered is present with the cell when passing through one or more constrictions for mechanical deformation, and a second cargo to be delivered is admixed with the cell after passing through the one or more constrictions. In some embodiments, the first cargo and the second cargo are different, e.g., the first cargo comprises a reagent to be delivered to all cells and the second cargo comprises a specific test compound, such as a small molecule candidate therapeutic. In some embodiments, the methods taught herein are performed in a high-throughput manner, e.g., based on configurations described in US 2025 / 0186995, which is hereby incorporated herein by reference in its entirety. For example, in some embodiments, the methods taught herein are used to assess 10, 100, 1,000, or 10,000 or more different conditions within a set about of time, e.g., 72 hours or less, 48 hours or less, 36 hours or less, or 24 hours or less.

[0119] As described herein, the cell mechanoporation component can be a microfluidic chip comprising one or more channels. Alternatively, the cell mechanoporation component can be a micro- sieved filter or a track-etched filter.

[0120] In some embodiments, the mechanical deformation comprises passing a cell suspension, or a portion thereof, through a material having pores therethrough, e.g., a filter, a microfluidic device comprising a plurality of constrictions or confinements, or a microneedle or microneedle array, or subjecting the cell suspension, or a portion thereof, to high shear forces with or without the presence of an electric field. Examples of certain mechanical deformation processes, including associated devices, are found in US Pat. No. 11,613,759 and Chakrabarty et al., Materials Today Bio, 13, 2022, the entire contents of each of which are incorporated by reference herein in their entirety.

[0121] As discussed in more detail below, in some embodiments, the method comprises passing a cell suspension, or a portion thereof including a cell, through a material having pores46MF-367693737Attorney Docket No. 285192001040 therethrough, e.g., a filter. In some embodiments, the cell is deformed by passing through a pore of the material having pores therethrough, and the processed cell can be collected in a filtrate after passing through the material.1. Materials having pores therethrough and method steps and conditions associated therewith

[0122] In certain aspects, the methods of the present disclosure are directed to delivering one or more components useful for the interaction assays taught herein into a cell by passing the cell through a material having pores therethrough, wherein the pores deform the cell, causing a perturbation of the cell and allowing delivery of the cargos, such as by having the cargos in the presence of the cell before, during, and / or after the cell is perturbed. In some embodiments, the material having pores therethrough is a filter.

[0123] The material having pores therethrough as disclosed herein can be made of any one of a number of materials, and combinations thereof, and take any one of a number of forms. In some embodiments, the material having pores therethrough is a filter (also referred to herein and in the art as a membrane). In some embodiments, the material having pores therethrough is a STERLITECH™ polycarbonate filter (PCT8013100). In some embodiments, the material having pores therethrough is a tangential flow filter. In some embodiments, the material having pores therethrough is a sponge or sponge-like matrix. In some embodiments, the material having pores therethrough is a matrix. In some embodiments, the material having pores therethrough is a microsieve. In some embodiments, the material having pores therethrough is not a net.

[0124] In some embodiments, the material having pores therethrough comprises a silicon-based material, such as silicon dioxide (also known as silica or SiCh). In some embodiments, the material having pores therethrough comprises a silicon dioxide (also known as silica or SiCh). In some embodiments, the material having pores therethrough comprises silicon nitride. In some embodiments, the material having pores therethrough comprises glass (also referred to as silicon oxide). In some embodiments, the material having pores therethrough comprises a polymer-based material, such as a polycarbonate. In some embodiments, the polymer-based material is a track-etched filter. In some embodiments, the material having pores therethrough comprises a metal-based material.MF-367693737Attorney Docket No. 285192001040

[0125] In some embodiments, the material having pores therethrough comprises a material having tortuous paths. In some embodiments, the material having tortuous paths comprises cellulose acetate. In some embodiments, the material having pores therethrough comprises a material selected from, without limitation, synthetic or natural polymers, polycarbonate, silicon, glass, metal, alloy, cellulose nitrate, silver, cellulose acetate, nylon, polyester, polyethersulfone, polyacrylonitrile (PAN), polypropylene, PVDF, polytetrafluorethylene, mixed cellulose ester, porcelain, graphite, and ceramic, or a mixture thereof.

[0126] The material having pores therethrough may be manufactured using any technique known in the art, including, without limitation, etching, track-etching, lithography, laser ablation, injection molding, stamping, micro-hole punching, polymeric- sponge, direct foaming, extrusion, and hot embossing. Etching involves the process of using a chemical, such as a strong acid, to cut into a material, such as a metal, in a desired pattern. Track-etched materials are formed by bombarding a solid film with particles that form tracks of damaged material through the film. The film is then subjected to a chemical agent that selectively etches the damaged tracks to create perforations through the film. The cross-sectional widths of the pores can be controlled by the incubation time of the etchant on the film. Lithography can include microlithography, nanolithography, and x-ray lithography. In lithography, a lithographic apparatus applies a desired pattern onto a target portion of the substrate. For example, in x-ray lithography, x-rays are used to transfer a pattern from a mask to a light-sensitive chemical photoresist on the desired substrate. In photolithography, light is used to transfer the desired pattern from a photomask to a light-sensitive photoresist on the substrate. Subsequent chemical application is used to engrave the pattern into the substrate material beneath the photoresist. Laser ablation is the process of removing material from a solid surface by irradiating with a laser beam, while injection molding involves injecting a material into a desired mold, where it then cools and hardens into the desired structure. Stamping and micro-hole punching methods utilize tools to cut or imprint desired forms into the substrate material. To produce ceramic filter membranes, the polymeric- sponge method involves saturating a polymeric sponge with a ceramic slurry, which is then burned out to leave a porous ceramic. In the direct foaming method, a chemical mixture containing the desired ceramic component and organic materials is treated to evolve a gas. Bubbles are then produced in the material, causing it to foam. The resulting porous ceramic material is then dried and fired. To produce a honeycomb or cellular structure, a plastic-forming method called extrusion is used, where a mixture of ceramic powder 48MF-367693737Attorney Docket No. 285192001040 plus additives is forced through a shaped die. Hot embossing involves the stamping of a pattern into a polymer softened by raising the temperature of the polymer just above its glass transition temperature.

[0127] In some embodiments, the material having pores therethrough is a nanostructure membrane. In some embodiments, phase change is used to produce nano structured membranes. Phase change methods include, without limitation, precipitation from the vapor phase, dry-wet phase inversion, and thermally induced phase separation. In the precipitation from the vapor phase method, a cast polymer solution composed of polymer and solvent is introduced into a nonsolvent vapor environment saturated with solvent vapor. The saturated solvent vapor suppresses the evaporation of solvent from the film. The nonsolvent molecules subsequently diffuse into the film, leading to polymer setting. In the dry-wet phase inversion method, a polymer solution composed of polymer and solvent is prepared. The solution is cast on a suitable surface and after partial evaporation of the solvent, the cast film is immersed in a gelatin. Then, the nonsolvent diffuses into the polymer solution film through the thin solid layer while the solvent diffuses out, creating a porous membrane. In the thermally induced phase separation method, a polymer is mixed with a solvent at a high temperature and the polymer solution is cast into a film. When the solution is cooled, it enters into an immiscible area due to the loss of solvency.

[0128] In some embodiments, the material having pores therethrough comprise a block copolymer. Block copolymers are composed of two or more blocks of different polymerized monomers linked by covalent bonds. Block copolymer components can microphase separate to form periodic nanostructures. In this process, due to incompatibility between the blocks, block copopolymers undergo phase separation, creating nanometer-sized structures.

[0129] In some embodiments, the material having pores therethrough comprises a microsieve. In some examples, microsieves are used in cell separation, CTC isolation or droplet emulsification techniques. In some embodiments, pores of a microsieve are generated by photolithography on a ceramic substrate with a silicon support. In some embodiments, the microsieve is made of polycarbonate. Microsieves are available from, e.g., Aquamarijn. In some embodiments, the microsieve has a pore size ranging from about 2 pm to about 10 pm, such as about 4 pm to about 10 pm. In some embodiments, the microsieve has a porosity of greater than about any of 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%.49MF-367693737Attorney Docket No. 285192001040

[0130] The material having pores therethrough may be characterized by a surface, such as a top surface or a surface across a cross-section of a material, upon which a cell suspension first contacts the material when the cell suspension is passed through the material having pores therethrough. In some embodiments, the surface is substantially perpendicular to the general direction of flow a cell suspension will take through a material having pores therethrough when said cell suspension is passed through the material. The 2-dimensional shape of the surface can be any shape, such as, without limitation, circular, elliptical, round, square, star- shaped, triangular, polygonal, pentagonal, hexagonal, heptagonal, or octagonal. In some embodiments, the surface is round in shape. In some embodiments, the surface of the material having pores therethrough has a largest cross-sectional width of about 1 mm to about 3 m, such as any of about 1 mm to about 750 mm, about 1 mm to about 500 mm, about 1 mm to about 250 mm, about 1 mm to about 100 mm, about 1 mm to about 50 mm, about 1 mm to about 25 mm, about 1 mm to about 10 mm, about 1 mm to about 5 mm, or about 1 mm to about 2.5mm.

[0131] The surface of a material having pores therethrough may be substantially flat (such as having a surface variance as measured by two parallel planes of 1 mm or less). In some embodiments, the surface of a material having pores therethrough, or a portion thereof, has a designed topography, including having a surface variance as measured by two parallel planes of more than 1 mm.

[0132] The material having pores therethrough may be characterized by a thickness. In some embodiments, the thickness of a material is substantially parallel to the general direction of flow a cell suspension will take through a material having pores therethrough when said cell suspension is passed through the material. In some embodiments, the thickness of a material having pores therethrough is uniform. In some embodiments, the thickness of a material having pores therethrough is variable. For example, in some embodiments, portions of a material having pores therethrough are thicker or thinner than other portions of the material having pores therethrough. In some embodiments, the surface thickness varies by about 1-90% as compared to the smallest thickness. In some embodiments, the material having pores therethrough has a thickness of about 0.01 pm to about 20 mm, such as any of about 0.01 pm to about 5 mm, about 0.01 pm to about 2.5 mm, about 0.01 pm to about 1 mm, about 0.01 pm to about 750 pm, about 0.01 pm to about 500 pm, about 0.01 pm to about 250 pm, about 0.01 pm to about 100 pm, about 0.01 pm to about 90 pm, about 0.01 pm to about 80 pm, about 0.01 pm to about 70 pm, about 0.01 pm to about 60 pm, about 0.01 pm to about 50 pm, about 0.01 pm to about 40 pm,50MF-367693737Attorney Docket No. 285192001040 about 0.01 pm to about 30 |am, about 0.01 |am to about 20 |am, 1 |jm to about 20 |am, about 0.01 |am to about 10 |am, about 0.01 |am to about 5 |am, about 0.01 |am to about 1 |am, about 0.01 |am to about 0.5 |am, about 0.01 |am to about 0.1 |am, about 0.01 |am to about 0.05 |am, about 0.05 |am to about 5 mm, about 0.1 |am to about 5 mm, about 0.5 |am to about 5 mm, about 1 |am to about 5 mm, about 5 |am to about 5 mm, about 10 |am to about 5 mm, about 20 |am to about 5 mm, about 30 |am to about 5 mm, about 40 |am to about 5 mm, about 50 |am to about 5 mm, about 60 |am to about 5 mm, about 70 |am to about 5 mm, about 80 |am to about 5 mm, about 90 |am to about 5 mm, about 100 |am to about 5 mm, about 250 |am to about 5 mm, about 500 |am to about 5 mm, about 750 |am to about 5 mm, about 1 mm to about 5 mm, or about 2.5 mm to about 5 mm. In some embodiments, the material having pores therethrough has a thickness of about 1 pm to about 20 pm, such as about any of 1 pm, 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, 7 pm, 8 pm, 9 pm, 10 pm, 11 pm, 12 pm, 13 pm, 14 pm, 15 pm, 16 pm, 17 pm, 18 pm, 19 pm, or 20 pm.

[0133] In some embodiments, the material having pores therethrough comprises a surface, such as a top surface or a cross-sectional surface, having a largest cross-sectional width of about 1 mm to about 3 m, including any sub-ranges (such as disclosed herein) and a thickness of about 0.01 pm to about 20 mm, including any sub-ranges (such as disclosed herein).

[0134] The material having pores therethrough can be formed in a variety of different shapes, including 3-dimensional shapes. In some embodiments, the 3-dimensional shape is cylindrical, conical, or cuboidal.

[0135] The material having pores therethrough can be characterized by the size(s) of the pores. In some embodiments, the pore size described herein reflects the smallest cross-sectional width across a pore. In some embodiments, the pore size described herein reflects the average cross-sectional width across a pore, such as evaluated for a single pore across the thickness of a material having pores therethrough and / or for a population of pores. In some embodiments, the pore size reflects an average of a population of pores of a material having pores therethrough. In some embodiments, the pores are characterized by a cross-sectional shape of the pores or an aspect (such as observed for a thickness of the material) thereof. In some embodiments, the pores have a cross-sectional shape that is circular or roughly circular. In some embodiments, the pores have a cross-sectional shape that is polygonal (e.g., triangular, square, rectangular, pentagonal, or hexagonal). In some embodiments, the cross-sectional shape of a pore is circular,51MF-367693737Attorney Docket No. 285192001040 elliptical, square, rectangular, star-shaped, triangular, polygonal, pentagonal, hexagonal, heptagonal, or octagonal.

[0136] In some embodiments, the material having pores therethrough comprises pores having a size, such as a cross-sectional width, selected based on the cell being passed therethrough. For example, in some embodiments, the smallest cross-sectional width across a pore is about 99% or less, such as about any of 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, or 20% or less, the average size of a cell (such as measured by the diameter or cross-sectional distance of the cell). In some embodiments, the cell has a cross-sectional width (e.g., diameter) in the cell suspension of between about 5-15 pm, e.g., dendritic cells are 7-8 pm in diameter. In another example, the cross-sectional width e.g., diameter) of a human ovum is about 120 pm. In some embodiments, the cross-sectional width of pores of a material described herein is about 0.01 pm to about 300 pm, such as about 0.01 pm to about 250 pm, about 0.01 pm to about 200 pm, about 0.01 pm to about 150 pm, about 0.01 pm to about 100 pm, about 0.01 pm to about 50 pm, about 0.01 pm to about 25 pm, about 25 pm to about 300 pm, about 50 pm to about 300 pm, about 100 pm to about 300 pm, about 150 pm to about 300 pm, about 200 pm to about 300 pm, or about 250 pm to about 300 pm. In some embodiments, the pore cross-sectional width is about any of 10 pm or less, 9.5 pm or less, 9.0 pm or less, 8.5 pm or less, 8.0 pm or less, 7.5 pm or less, 7.0 pm or less, 6.5 pm or less, 6.0 pm or less, 5.5 pm or less, 5.0 pm or less, 4.5 pm or less, 4.0 pm or less, 3.5 pm or less, 3.0 pm or less, 2.5 pm or less, 2.0 pm or less, 1.5 pm or less, 1.0 pm or less, or 0.5 pm or less. In some embodiments, the pores are heterogeneous in cross-sectional width. In some embodiments, the pores are substantially homogenous (such as within about 20% or less variance) in cross-sectional width.

[0137] In some embodiments, the material having pores therethrough comprises pores having an average size of about 0.1 pm to about 300 pm, such as about any of 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, 7 pm, 8 pm, 9 pm, 10 pm, 11 pm, 12 pm, 13 pm, 14 pm, 15 pm, 16 pm, 17 pm, 18 pm, 19 pm, or 20 pm. In some embodiments, the pore has a uniform cross-sectional dimension along the length of the pore passage, e.g., through the thickness of the material having pores therethrough. In some embodiments, the pore has a variable cross-sectional dimension along the length of the pore passage, e.g., increasing or decreasing.52MF-367693737Attorney Docket No. 285192001040

[0138] In some embodiments, the entrances and exits of a pore through a material having pores therethrough may have a variety of angles. In some embodiments, the pore angle can be selected to minimize clogging of the pore while cells are passing through. For example, the angle of the entrance or exit portion can be between about 0 and about 90 degrees. In some embodiments, the pores have identical entrance and exit angles. In some embodiments, the pores have different entrance and exit angles. In some embodiments, the pore edge is smooth, e.g., rounded or curved. A smooth pore edge has a continuous, flat, and even surface without bumps, ridges, or uneven parts. In some embodiments, the pore edge is sharp. A sharp pore edge has a thin edge that is pointed or at an acute angle. In some embodiments, the pore passage is straight. A straight pore passage does not contain curves, bends, angles, or other irregularities. In some embodiments, the pore passage is curved. A curved pore passage is bent or deviates from a straight line. In some embodiments, the pore passage has multiple curves, e.g., about 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more. In some embodiments, the pore has a fluted entrance and exit shape. In some embodiments, the pore shape is homogenous among other pores within a given material or a portion thereof. In some embodiments, the pore shape is heterogeneous (z.e., mixed or varied) among pores within a given material or a portion thereof.

[0139] The material having pores therethrough described herein may have a range of total pore numbers and / or area sizes having said pores. In some embodiments, the pores encompass about 10-80% of the total surface area of a material having pores therethrough. In some embodiments, the material having pores therethrough comprises about l. OxlO3to about l. OxlO30pores, including, e.g., at least about any of l. OxlO3pores, l. OxlO4pores, l. OxlO5pores, l. OxlO6pores, l. OxlO7pores, l. OxlO8pores, l. OxlO9pores, l. OxlO10pores, l. OxlO11pores, l. OxlO12pores, l. OxlO13pores, l. OxlO14pores, l. OxlO15pores, l. OxlO16pores, l. OxlO17pores, l. OxlO18pores, l. OxlO19pores, l. OxlO20pores, l. OxlO21pores, l. OxlO22pores, l. OxlO23pores, l. OxlO24pores, l. OxlO25pores, l. OxlO26pores, l. OxlO27pores, l. OxlO28pores, l. OxlO29pores, or l. OxlO30pores. In some embodiments, the material having pores therethrough comprises about 10 to about l. OxlO15pores per mm2.

[0140] The pores can be distributed in numerous ways within a given material having pores therethrough. In some embodiments, the pores are distributed in grid as viewed from a surface of the material. In one such example, the pores are distributed side-by-side as viewed from a surface of the material, including having substantially equal distancing from one another. In 53MF-367693737Attorney Docket No. 285192001040 some embodiments, the pore distribution is ordered or homogeneous as viewed from a surface of the material. In some embodiments, the pores are distributed in a regular, systematic pattern as viewed from a surface of the material. In some embodiments, the pore distribution is random or heterogeneous as viewed from a surface of the material. In some embodiments, the pores are distributed in an irregular, disordered pattern or are different distances apart as viewed from a surface of the material. In some embodiments, the pores are arranged in an irregular pattern as viewed from a surface of the material. In some embodiments, the pores are heterogeneous in size, such as pore size. In some embodiments, the pores are heterogeneous in shape, such as cross-sectional shape.

[0141] In some embodiments, the material having pores therethrough comprises more than one separate piece, such as a first material piece and a second material piece. In some embodiments, the pieces of material constituting the material having pores therethrough are arranged in series. In some embodiments, the pieces of a material having pores therethrough are arranged such that a cell solution travels through each piece in series. In some embodiments, at least two of the pieces of a material constituting the material having pores therethrough are the same. In some embodiments, at least two of the pieces of a material constituting the material having pores therethrough are different.

[0142] In some embodiments, the surface of a material having pores therethrough comprises a coating. Such coating can be selected from any material known in the art, including, without limitation, Teflon, an adhesive coating, surfactants, anticoagulants such as heparin, EDTA, citrate, and oxalate, proteins, adhesion molecules, antibodies, factors that modulate cellular function, nucleic acids, lipids, carbohydrates, complexes such as lipid-carbohydrate complexes, or transmembrane proteins. In some embodiments, the coating comprises, including is, polyvinylpyrrolidone. In some embodiments, the coating is covalently attached to a surface. In some embodiments, the coating is non-covalently attached to a surface. In some embodiments, the coating is released, or removed, as the cells pass through the pores.

[0143] In some embodiments, the material having pores therethrough comprises a modified mechanical or chemical property, e.g., exposed aspects thereof (such as a surface and / or pore) are modified to have a desired property such as hydrophilicity, hydrophobicity, charge (positive, negative, neutral, or zwitter ionic), or smoothness.54MF-367693737Attorney Docket No. 285192001040

[0144] In some embodiments, the material having pores therethrough is configured with a directionality, e.g., configured such that a cell suspension is intended to enter and exit specific portions of a material having pores therethrough.

[0145] As described herein, the delivery methods comprise subjecting a cell suspension to a mechanical deformation. Such mechanical deformation may be performed under certain conditions, such as based on pressure and / or flow rate of the cell suspension.

[0146] In some embodiments, the cell suspension is passed through a material having pores therethrough at about 1 psi (pounds per square inch) to about 100 psi, such as any of about 1 psi to about 10 psi, about 1 psi to about 20 psi, about 5 psi to about 12 psi, or about (5 psi to about 10 psi). In some embodiments, the cell suspension is passed through a material having pores therethrough at about any of 1 psi, 2 psi, 3 psi, 4 psi, 5 psi, 6 psi, 7 psi, 8 psi, 9 psi, 10 psi, 11 psi, 12 psi, 13 psi, 14 psi, 15 psi, 16 psi, 17 psi, 18 psi, 19 psi, 20 psi, 21 psi, 22 psi, 23 psi, 24 psi, 25 psi, 26 psi, 27 psi, 28 psi, 29 psi, 30 psi, 31 psi, 32 psi, 33 psi, 34 psi, 35 psi, 36 psi, 37 psi, 38 psi, 39 psi, 40 psi, 41 psi, 42 psi, 43 psi, 44 psi, 45 psi, 46 psi, 47 psi, 48 psi, 49 psi, 50 psi, 51 psi, 52 psi, 53 psi, 54 psi, 55 psi, 56 psi, 57 psi, 58 psi, 59 psi, 60 psi, 61 psi, 62 psi, 63 psi, 64 psi, 65 psi, 66 psi, 67 psi, 68 psi, 69 psi, 70 psi, 71 psi, 72 psi, 73 psi, 74 psi, 75 psi, 76 psi, 77 psi, 78 psi, 79 psi, 80 psi, 81 psi, 82 psi, 83 psi, 84 psi, 85 psi, 86 psi, 87 psi, 88 psi, 89 psi, 90 psi, 91 psi, 92 psi, 93 psi, 94 psi, 95 psi, 96 psi, 97 psi, 98 psi, 99 psi, or 100 psi. In some embodiments, the pressure is as measured upstream of a material having pores therethrough, e.g., in a cell suspension prior to passing through the material having pores therethrough. In some embodiments, the pressure is held within + / - about 20% variance of a desired pressure during the period of time that a cell suspension is passed through a material having pores therethrough. In some embodiments, two or more pressures are used to pass a cell suspension through a material having pores therethrough. In some embodiments, the pressure for passing a cell suspension through a material having pores therethrough is a positive pressure, such as created by a pump or syringe pushing a cell suspension through a material having pores therethrough. In some embodiments, the pressure for passing a cell suspension through a material having pores therethrough is a negative pressure, such as created by a vacuum source pulling a cell suspension through a material having pores therethrough. Techniques for passing a cell suspension through a material having pores therethrough are understood by one of skill in the art and include, e.g., a peristaltic pump, a centrifugal pump, a syringe pump, a piston pump,55MF-367693737Attorney Docket No. 285192001040 rotary vane pump, diaphragm pump, turbomolecular pump, or a scroll pump, or any combination or hybrid thereof.

[0147] In some embodiments, the cell suspension is passed through the material having pores therethrough at a cell speed of about 0.1 mm / second to about 5 m / second, such as about any of 0.1 mm / second to about 1 mm / second, about 1 mm / second to about 1 cm / second, about 1 cm / second to about 10 cm / second, about 10 cm / second to about 25 cm / second, about 25 cm / second to about 50 cm / second or about 50 cm / second to about 100 cm / second, about 100 cm / second to about 500 cm / second, about 500 cm / second to about 1 m / second, or about 1 m / second to about 5 m / second. In some embodiments, the flow rate of a cell suspension through a material having pores therethrough is between about 0.001 mb / cm / second to about 100 L / cm2 / second, such as any of about 0.001 mL / cm2 / second to about 75 L / cm2 / second, about 0.001 mb / cm2 / second to about 50 L / cm2 / second, about 0.001 mL / cm2 / second to about 25 L / cm2 / second, about 0.001 mL / cm / second to about 10 L / cm2 / second, about 0.001 mL / cm2 / second to about 7.5 L / cm / second, about 0.001 mL / cm2 / second to about 5.0 L / cm2 / second, about 0.001 mL / cm2 / second to about 2.5 L / cm2 / second, about 0.001 mL / cm2 / second to about 1 L / cm / second, about 0.001 mL / cm2 / second to about 0.1 L / cm2 / second, about 0.001 mL / cm2 / second to about 75 mL / cm2 / second, about 0.001 mL / cm2 / second to about 50 mL / cm2 / second, about 0.001 mL / cm2 / second to about 25 mL / cm2 / second, about 0.001 mL / cm2 / second to about 10 mL / cm2 / second, about 0.001 mL / cm2 / second to about 1 mL / cm2 / second, about 0.001 mL / cm2 / second to about 0.1 mL / cm2 / second, about 0.001 mL / cm2 / second to about 0.01 mL / cm2 / second, about 0.001 mL / cm2 / second to about 100 L / cm2 / second, about 0.01 mL / cm2 / second to about 100 L / cm2 / second, about 0.1 mL / cm2 / second to about 100 L / cm2 / second, about 1 mL / cm2 / second to about 100 L / cm2 / second, about 10 mL / cm2 / second to about 100 L / cm2 / second, about 50 mL / cm2 / second to about 100 L / cm2 / second, about 0.1 L / cm2 / second to about 100 L / cm2 / second, about 0.5 L / cm2 / second to about 100 L / cm2 / second, about 1 L / cm2 / second to about 100 L / cm2 / second, about 2.5 L / cm2 / second to about 100 L / cm2 / second, about 5 L / cm2 / second to about 100 L / cm2 / second, about 7.5 L / cm2 / second to about 100 L / cm2 / second, about 10 L / cm2 / second to about 100 L / cm2 / second, about 25 L / cm2 / second to about 100 L / cm2 / second, about 50 L / cm2 / second to about 100 L / cm2 / second, or about 75 L / cm2 / second to about 100 L / cm2 / second.

[0148] In some embodiments, the porous surface or channels of the cell mechanop oration component includes uniformly sized pores (and / or channels). In some embodiments, the porous 56MF-367693737Attorney Docket No. 285192001040 surface of the cell constricting component includes varying pore sizes throughout the surface and / or may comprise channels of varying sizes. The pores / channels (hereinafter referred to collectively as pores for simplicity, unless stated otherwise) of the cell mechanoporation component may comprise one or more cross-sectional shapes including but not limited to circular, rectangular (e.g., square), elliptical, triangular, or another polygonal shape. In the instance the cross-section of the pores comprises a circular shape, the diameter of each of the one or more pores may be between 2 pm and 20 pm. For example, the diameter of a given pore may be about 0.1 pm, 0.5 pm, 1 pm, 2 pm, 5 pm, 10 pm, 15 pm, or 20 pm. The diameter of each of the pores of the cell mechanoporation component may be based on the diameter of the cells in the cell mixture. For example, the diameter of each of the pores may be a smaller diameter than a cell of a cell mixture passing through the cell mechanoporation component, such that forcing the cell through the pore due to centripetal force causes a perturbation in (or deformation of) the membrane of the cell as the cell is constricted by the pore. The pore diameter may be between 10% and 99% of the diameter of the cells in a cell suspension, such as about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the diameter of the cells. Each of the aforementioned characteristics of pores of a porous surface are understood to be attributable to, e.g., channels of a microfluidic chip. It is to be understood that reference to a diameter of a cell in the cell suspension means the diameter of the cell in the cell mixture prior to being passed through the filter, e.g., as the cell approaches the filter, unless otherwise specified.

[0149] In the instance the cell mechanoporation component comprises a filter (e.g., a microsieved filter or track-etched filter), the porous surface of the filter may comprise a range of total pores. For example, the pores may cover between about 0.1% and about 60% of the total surface area of the filter. In some embodiments, the porous surface may comprise between about l. OxlO1to about l. OxlO10pores per millimeter of surface area. The porous surface may in some embodiments comprise between about l. OxlO1to about 1.0 xlO15pores per millimeter of surface area. The filter may have a cross-sectional shape comprising a circular, triangular, rectangular, elliptical, or other polygonal shape.

[0150] The filter (e.g., a micro-sieved filter or track-etched filter) may have a thickness, or length, defined by the axis by which the mixture travels along as it passes through the cell mechanoporation component. The thickness of the filter be between about 0.1 pm and 100 pm. For example, the thickness of the filter may be less than or equal to about 0.1 pm, 0.5 pm, 1 pm, 5 pm, 10 pm, 50 pm, 100 pm, 500 pm, or 1 mm. In some embodiments, the thickness of the 57MF-367693737Attorney Docket No. 285192001040 filter may be greater than or equal to about 0.1 qm, 0.5 qm, 1 qm, 5 qm, 10 qm, 50 qm, 100 qm, 500 qm, or 1 mm. Thus, given the thickness of the filter the pores of a filter may comprise a 3-dimensional shape (e.g., like a channel). In the instance a pore comprises a circular cross-section with a uniform diameter along the thickness of the filter, the 3-dimensional shape of the pore may comprise a cylinder. In the instance the diameter of the pore varies (e.g., increases or decreases) along the thickness of the filter, the 3-dimensional shape of the pore may be conical.B. Co-delivery

[0151] The methods provided herein further enable the co-delivery of more than one cargo, including, e.g., a polypeptide, a nucleic acid molecule, PROTAC, or elements thereof, or a chemical compound such as a small molecule (e.g., 2 kDa or less in size). In some embodiments, the co-delivery is performed in a unified step (e.g., the first component, or the precursor thereof, and / or the second component, or the precursor thereof, e.g., via a single mechanical deformation process), such as using a cell suspension comprising the first component, or the precursor thereof, and / or the second component, or the precursor thereof. In some embodiments, the codelivery is performed by sequentially or concurrently exposing a cell to a first component, or a precursor thereof, and / or a second component, or a precursor thereof and another cargo, e.g., such as passing a cell suspension comprising the first component, or the precursor thereof, and / or the second component, or the precursor thereof through a material having pores therethrough and then subsequently adding the other cargo while the cells still contain pores from the mechanical deformation. In some embodiments, provided herein are methods comprising two or more steps of subjecting a cell suspension to a mechanical deformation, e.g., subjected a cell suspension to a mechanical deformation to deliver a first component, or a precursor thereof, and / or a second component, or a precursor thereof, wherein the mechanical deformation is configured such that at least two of the first component, or the precursor thereof, and / or the second component, or the precursor thereof enters the cell, and subjecting the cell suspension to a mechanical deformation to deliver one or more other cargos, including one or more gene editing complexes, a nucleic acid, such as RNA, e.g., mRNA or siRNA, a sugar polymer, or other labeling agent. In some embodiments, the methods allow for the passage of time between mechanical deformation steps, such as to expand cells following a delivery step. For example, in some embodiments, the methods comprise at least about 1 day, such as at least58MF-367693737Attorney Docket No. 285192001040 about any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 21, 28, 35, or 42 days between a first mechanical deformation step and a second mechanical deformation step. One of ordinary skill in the art will readily recognize that the cell suspension may change between the mechanical deformation steps, such as accomplished by changing cellular media, buffer exchange, or cell replication and / or death and / or change (e.g., differentiation and / or activation), however use of this identifier in subsequent steps is to convey the lineage between multiple rounds of mechanical deformation. In some embodiments, the methods provided herein comprise subjecting a cell suspension to a mechanical deformation to deliver a first component, or a precursor thereof, and / or a second component, or a precursor thereof, and subjecting the cell suspension to a mechanical deformation to deliver a nucleic acid, such as RNA, e.g., mRNA. In some embodiments, the methods provided herein comprise subjecting a cell suspension to a mechanical deformation to deliver a first component, or a precursor thereof, and / or a second component, or a precursor thereof, and subjecting the cell suspension to a mechanical deformation to deliver a nucleic acid, such as RNA, e.g., mRNA. In some embodiments, the co-delivered cargo is a polypeptide, a nucleic acid (such as RNA, e.g., mRNA, or DNA), or a complex, such as a complex comprising a polypeptide and a nucleic acid. In some embodiments, the polypeptide is a peptide. In some embodiments, the polypeptide is a protein.

[0152] In some embodiments, the methods described herein further enable the sequential delivery of cargo, including, e.g., a polypeptide or a nucleic acid molecule (such as a first component, or a precursor thereof, and / or a second component, or a precursor thereof). In some embodiments, the method comprises a first step comprising processing a cell such that a first cargo enters the cell followed by a second step comprising processing the cell such a second cargo enters the cell. In some embodiments, the first and second steps are separated by a rest period. In some embodiments, the first step comprising processing the cell such that the first cargo enters the cell comprises a step of delivering a first component, or a precursor thereof. In some embodiments, the rest period comprises between about 0 to about 24 hours. In some embodiments, the rest period comprises between about 0 hours to about 4 hours, between about 4 hours to about 8 hours, between about 8 hours to about 12, between about 12 to about 16, between about 16 to about 20 or between about 20 to about 24 hours. In some embodiments, the rest period comprises between about 1 to about 14 days. In some embodiments, the rest period comprises about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days,59MF-367693737Attorney Docket No. 285192001040 about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, or about 14 days. In some embodiments, one or more cargos are delivered at each sequential delivery step. In some embodiments, the first step comprising processing a cell such that a first cargo enters the cell is performed prior to cell expansion. In some embodiments, the first step comprising processing a cell such that a first cargo enters the cell is performed prior to cell differentiation or activation.C. Cell suspensions and cells

[0153] The method provided herein comprise subjecting a cell to a mechanical deformation, such as passing a cell through a pore of a material comprising pores therethrough. To perform such methodology, cells are generally processed in a cell suspension.

[0154] In some embodiments, the cell suspension has a volume of about 10 pL to about 5 L, such as any of 100 pL, 500 pL, 1 mL, 5 mL, 10 mL, 50 mL, 100 mL, 200 mL, 300 mL, 400 mL, 500 mL, 600 mL, 700 mL, 800 mL, 900 mL, 1 L, 2 L, 3 L, 4 L, or 5 L. In some embodiments, the cell suspension has a volume of at least about 10 pL, such as at least about any of 100 pL, 500 pL, 1 mL, 5 mL, 10 mL, 50 mL, 100 mL, 200 mL, 300 mL, 400 mL, 500 mL, 600 mL, 700 mL, 800 mL, 900 mL, 1 L, 2 L, 3 L, 4 L, or 5 L. Accordingly, in some embodiments, the methods comprise selecting a mechanical deformation technique, or component thereof, suitable for processing such volume.

[0155] It is conceived that, in some embodiments, the cargo to be delivered is a limited resource, such as based on availability and / or cost, and thus the volume of the cell suspension can be modulated accordingly to ensure a certain concentration of the cargo is present in the cell suspension for delivery. In some embodiments, the concentration of the cargo present in the cell suspension for delivery is between about 0.01 mg / mL to about 10 mg / mL, such as any of about 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, or 10 mg / mL. In some embodiments, the concentration of the cargo present in the cell suspension for delivery is between about 0.05 mg / mL to about 2 mg / mL. In some embodiments, the concentration of the cargo present in the cell suspension for delivery is at least about 0.01 mg / mL, such as at least about 0.01 mg / mL, 0.02 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, or 10 mg / mL. In some embodiments, the concentration of the cargo present in the cell suspension for delivery is between about 0.01 mg / mL to about 10 mg / mL, such as any of about 1 mg / mL, 2 mg / mL, 360MF-367693737Attorney Docket No. 285192001040 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, or 10 mg / mL, and the cell density is about 20 million cells / mL to about 500 million cells / mL.

[0156] In some embodiments, the cell suspension has a cell density of at least about 100 cells / mL, such as at least about 200 cells / mL, 300 cells / mL, 400 cells / mL, 500 cells / mL, 600 cells / mL, 700 cells / mL, 800 cells / mL, 900 cells / mL, 1,000 cells / mL, 1,250 cells / mL, 1,500 cells / mL, 1,750 cells / mL, 2,000 cells / mL, 2,250 cells / mL, 2,500 cells / mL, 2,750 cells / mL, 3,000 cells / mL, 3,250 cells / mL, 3,500 cells / mL, 3,750 cells / mL, 4,000 cells / mL, 4,250 cells / mL, 4,500 cells / mL, 4,750 cells / mL, 5,000 cells / mL, 10,000 cells / mL, 50,000 cell / mL, 100,000 cell / mL, 1,000,000 cell / mL, 5,000,000 cells / mL, 10,000,000 cells / mL, 25,000,000 cells / mL, 50,000,000 cells / mL, 100,000,000 cells / mL, 150,000,000 cells / mL, or 200,000,000 cells / mL. In some embodiments, the cell suspension has a cell density of about 1,000,000 cells / mL to about 200,000,000 cells / mL. In some embodiments, the cell suspension has a cell density of about 20,000,000 cells / mL to about 100,000,000 cells / mL.

[0157] In some embodiments, the cell is a cell obtained from, or derived from, an individual. In some embodiments, the individual is a mouse. In some embodiments, the individual is a nonhuman primate. In some embodiments, the individual is a human. In some embodiments, the individual is a mouse, dog, cat, horse, rat, goat or rabbit.

[0158] In some embodiments, the cell suspension substantially contains a single cell type (e.g., at least about 90% of the cells in a cell suspension are of a single cell type, e.g., T cells). In some embodiments, the cell suspension comprises a plurality of cell types, e.g., as found in cells from whole blood, lymph, and / or peripheral blood mononuclear cells (PBMCs). In some embodiments, the cell suspension comprises a purified cell population.

[0159] In some embodiments, the cell suspension comprises a buffer. Such buffer may, in certain aspects, serve as a vehicle to enable processing of cells as described herein. As the buffer will contain cells, buffers envisioned herein are compatible with the cell being processed, such as, e.g., to avoid lysis and / or undesirable alterations of said cell. For example, in some embodiments, the buffer has a desired osmolarity, salt concentration, serum content, cell concentration, and pH. In some embodiments, the buffer is a physiological saline solution or a physiological medium other than blood.

[0160] In some embodiments, the cell suspension comprises an aqueous solution. In some embodiments, the aqueous solution comprises cell culture medium, PBS, salts, sugars, growth 61MF-367693737Attorney Docket No. 285192001040 factors, animal derived products, bulking materials, surfactants, lubricants, vitamins, proteins, chelators, and / or an agent that impacts actin polymerization. In some embodiments, the cell culture medium is DMEM, OptiMEM, EVIDM, RPMI, or X-VIVO. Additionally, solution buffer can include one or more lubricants (pluronics or other surfactants) that can be designed to reduce or eliminate clogging of the surface and improve cell viability. Exemplary surfactants include, without limitation, poloxamer, polysorbates, sugars such as mannitol, animal derived serum, and albumin protein.

[0161] In some embodiments, the cells can be incubated in one or more solutions that aid in the delivery of the cargo to the interior of the cell. In some embodiments, the aqueous solution comprises an agent that impacts actin polymerization. In some embodiments, the agent that impacts actin polymerization is Latrunculin A, Cytochalasin, and / or Colchicine.I). Efficacy of delivery, co-delivery, and cell viability

[0162] The methods of delivery described herein, provide, in certain aspects, a degree of delivery efficacy and / or cell viability. For example, the methods described herein provide a desired yield of engineered cells that are viable for downstream uses.

[0163] In some embodiments, following the subjecting a cell suspension to a mechanical deformation, such as passing cells through a material having pores therethrough, at least about 25%, such as at least about any of 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, of the cells receive at least one, including two or more or all, of components useful for the interaction assays taught herein.

[0164] In some embodiments, following the subjecting a cell suspension to a mechanical deformation, such as passing cells through a material having pores therethrough, at least about 25%, such as at least about any of 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, of the cells are edited according to the design of at least one of components useful for the interaction assays taught herein.

[0165] In some embodiments, following the subjecting a cell suspension to a mechanical deformation, such as passing cells through a material having pores therethrough, at least about 25%, such as at least about any of 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, of cells are viable.62MF-367693737Attorney Docket No. 285192001040E. Additional aspects of the methods provided herein

[0166] The present disclosure further encompasses both upstream and downstream processes useful or associated with the delivery methods described herein. For example, in some embodiments, the method further comprises obtaining a cell or cells, such as from an individual. In some embodiments, the method further comprises obtaining a cell suspension comprising a cell or cells. In some embodiments, the method further comprises preparing a mechanical deformation technique, or component thereof, such as a material having pores therethrough, for a method of delivery as described herein (such as washing said material). In some embodiments, the method further incubating a cell or cells processed with a mechanical deformation such that the cell or cells close the pores made therein. In some embodiments, the method further comprises storing a cell suspension or cells subjected to the delivery methods described herein. In some embodiments, the method further comprises validating delivery of cargo to a cell or cells following performing a delivery method described herein.V. Systems, Kits, and Components Useful for the Methods Taught Herein

[0167] In other aspects, the present disclosure provides systems, kits, components, and compositions (such as consumables) useful for the methods described herein. For example, provided herein is a system comprising a device for subjecting the cell suspension to a mechanical deformation delivery technique, such as a material having pores therethrough, wherein the subjecting the cell suspension to the process for mechanical deformation allows at least one of the components useful for the interaction assays taught herein to enter the cell, and a cell suspension comprising a cell. In some embodiments, the kit, or another composition of a system provided herein, further comprises one or more components useful for the interaction assays taught herein. For example, in some embodiments, the kit comprises a first component, such as a component useful for an assay taught herein. In some embodiments, the kit comprises an engineered cell comprising an intact cell membrane and comprising one or more components taught useful for the described interaction assays as taught herein. In some embodiments, the63MF-367693737Attorney Docket No. 285192001040 components of the systems or kits may come in separate containers. In some embodiments, the system or kit comprises instructions for use according to the methods described herein.

[0168] The kits of the present application are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Kits may optionally provide additional components such as buffers and interpretative information.VII. Sequences

[0169] Provided herein are sequences useful for the present disclosure.LgBiT - SEQ ID NO: 1 MVFTLEDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPIQRIVRSGENALKIDIH VIIPYEGLSADQMAQIEEVFKVVYPVDDHHFKVILPYGTLVIDGVTPNMLNYFGRPYEGI AVFDGKKITVTGTLWNGNKIIDERLITPDGSMLFRVTINSSmBiT - SEQ ID NO: 2VTGYRLFEEILHiBiT - SEQ ID NO: 3VSGWRLFKKISNatural Peptide (NP) - SEQ ID NO: 4GVTGWRLCERILAEXAMPLES

[0170] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they64MF-367693737Attorney Docket No. 285192001040 intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. The examples below are intended to be purely exemplary of the application and should therefore not be considered to limit the application in any way.Example 1

[0171] This example demonstrates the co-delivery of 3kDa dextran and a PROTAC to HEK293 cells using a mechanical deformation delivery technique. Specifically, the 3kDa dextran was fluorescently labeled with cascade blue and the PROTAC was fluorescently labeled with Texas red. The cells were not genetically modified to express the 3kDa dextran and PROTAC.

[0172] HEK293 cells were lifted into a single cell suspension with 0.25% Trypsin / EDTA, then quenched with DMEM+GlutaMAX containing 10% FBS and 1% P / S (complete DIO). Cells were centrifuged and resuspended in complete DIO before being passed through a 40 pm mesh strainer, then spun resuspended at 10 M / mL in OptiMEM. Cells were combined with 3kDa dextran fluorescently labeled with Cascade blue (0.1 mg / mL) and a PROTAC fluorescently labeled with Texas Red (10 pM) (cargo). 100 pl of the cell and cargo solution was pipetted into a cartridge containing a silicon membrane with 10 pm pores. The solution was mechanoporated into a 1.5 mL tube at 12 PSI. Complete D10 was added to the collection tube 30s after mechanoporation. Intracellular delivery of 3 kDa dextran and PROTAC into live cells was measured with flow cytometry within 30 minutes of mechanical deformation.

[0173] As shown in FIGS. 1A and IB, co-delivery of 3 kDa dextran and the PROTAC was observed in live cells with intact cell membranes, thus demonstrating the ability of the mechanical deformation delivery technique to deliver the PROTAC along with a co-payload. Flow cytometry measurements confirm delivery of 3kDa dextran and the PROTAC (FIG. 1C).Example 2

[0174] This example demonstrates the delivery of a LgBiT protein to cells expressing KRAS G12C fused to HiBiT using a mechanical deformation delivery technique. The luciferase LgBiT / HiBiT system enables intracellular measurements of amounts of KRAS G12C. The mechanical 65MF-367693737Attorney Docket No. 285192001040 deformation delivery technique allowed for delivery of LgBiT to engineered live cells having intact cell membranes. The cells were not genetically modified to express LgBiT.

[0175] MiaPaCa-2 HiBiT-KRAS(G12C)-KI cells were plated in 6-well plates and treated with 0, 1, 5, or 10 pM of the KRAS-targeting PROTAC LC-2 for 24 hours. After 24 hours, cells were lifted into single cell suspension and combined with 3 kDa Cascade blue dextran (0.1 mg / mL) and LgBiT protein (% reaction volume) in a 100 pl reaction volume at 3 M / mL in OptiMEM. Cells were mechanically deformed through a cartridge containing a silicon membrane with 12 pm diameter pores at 9 PSI and collected in a 1.5 mL tube. 1 mL of complete growth media (DMEM+GlutaMAX with 10% FBS and 1% Pen / Strep) was added to the cell and cargo mixture. Delivery of dextran into live cells was measured using flow cytometry. Cells were washed with CO2 independent media before plating in a 1:100 dilution of NanoGio Endurazine (Promega) in CO2 independent media containing LC-2 (0, 1, 5, or 10 pM). Cells were incubated for 2 hours at 37 °C before reading out luminescence on a plate reader.

[0176] As shown in FIG. 2, luminescence measured 2 hours after delivery decreased with increasing concentrations of LC-2, suggesting that increased amounts of LC-2 degraded more KRAS G12C.Example 3

[0177] This example demonstrates an assay performed according to the methodology described herein wherein a NanoBRET system was delivered to live cells resulting in an engineered live cell with an intact cell membrane. The cells were not genetically modified to express the NanoBRET system.

[0178] The NanoBRET system is an interaction-based assay designed to assess protein-protein interactions by measuring energy transfer from a bioluminescent protein donor to a fluorescent protein acceptor. Generally, a NanoLuc fusion protein serves as the energy donor while a fluorescent fusion protein serves as the energy acceptor. When the protein fused to NanoLuc is in proximity to the protein fused to a fluorescent molecule, energy transfer occurs, and the BRET ratio is calculated.

[0179] HeLa cells were transfected with a Src-NanoLucif erase fusion vector using Lipofectamine 3000 in OptiMEM. After 20 hours, transfected cells were lifted into a single cell66MF-367693737Attorney Docket No. 285192001040 suspension using 0.25% Trypsin / EDTA and quenched with DMEM+GlutaMAX containing 10% FBS and 1% Pen / Strep (complete DMEM). Cells were passed through a 40 pm mesh strainer before resuspending at 10 M / mL in OptiMEM. Cells were mixed in a 100 pl reaction volume containing 3 kDa dextran fluorescently labeled with Cascade Blue (0.1 mg / ml) and a cell impermeable kinase tracer (Kinase Tracer-01) at 0, 0.5, 1, or 5 pM. The cell and cargo mixture was pipetted into a cartridge containing a silicon membrane with 11 pm pores and mechanically deformed at 10 PSI before collecting in a 1.5 mL tube. Cells mixed with the cargo but not mechanically deformed were used as control samples. Complete DMEM was added to samples following mechanical deformation. Cells were washed twice with OptiMEM and intracellular delivery of dextran to live cells was measured with flow cytometry. Cells were plated in 100 pl of OptiMEM in wells of a white-walled 96 well plate and incubated for 2 hours at 37°C before adding 50 pl of a solution containing 1:166 NanoGio substrate (Promega) and 1:500 NanoLuc extracellular inhibitor (Promega). After 3 minutes, Bioluminescence Resonance Energy Transfer (BRET) was measured on a plate reader using 450 BP (bandpass) to measure donor and 600 LP (long pass) to measure acceptor. BRET ratio was calculated using the ratio of acceptor to donor luminescence and subtracting the BRET ratio for a control sample in which no kinase tracer was added.

[0180] In this example, a NanoLuc fusion protein, namely, a NanoLuc fused to Src kinase, and fluorescent fusion protein, namely, a Src kinase tracer (which are both cell membrane impermeable) were delivered into cells by a mechanical deformation delivery technique. As shown in FIG. 3, the BRET ratio increased with increasing concentrations of the Src kinase tracer delivered into the cell (mechanical deformation delivery condition), representing a successful dose response using this delivery technique. In contrast, substantially no signal was measured when both NanoLuc and the Src kinase tracer were mixed with the live cells but not subjected to the mechanical deformation delivery technique.

[0181] Pre-treatment of HeLa cells expressing NanoLuc-tagged Src kinase with cell-permeable Src kinase inhibitors (e.g., ponatinib, bosutinib, or dasatinib) resulted in a dose-dependent decrease of the BRET ratio following the introduction of a Src kinase tracer to Hela cells via the mechanical deformation delivery technique. No BRET ratio was observed for the no mechanical deformation delivery conditions since the Src kinase tracer is cell membrane impermeable (FIGs. 7A-7C). For HeLa cells that were pre-treated with ponatinib or bosutinib, the BRET ratio progressively decreased relative to increasing concentrations of the Src kinase inhibitors,67MF-367693737Attorney Docket No. 285192001040 compared to the no Src kinase inhibitor control condition (FIGs. 7A and 7B). For HeLa cells that were pre-treated with dasatinib, a large decrease in the BRET ratio was observed at 1.1 pM dasatinib pre-treatment and the BRET ratio remained low at higher concentrations of dasatinib pre-treatment, compared to the no Src kinase inhibitor control condition (FIG. 7C).

[0182] Taken together, this example shows that the NanoLuc fusion protein (e.g., NanoLuc fused to Src kinase and a Src kinase tracer) that enters HeLa cells via the mechanical deformation delivery technique is functional. In the absence of Src kinase inhibitors, an increasing BRET ratio is observed relative to increasing concentrations of the Src kinase tracer delivered into the cell. However, in the presence of Src kinase inhibitors, a decreasting BRET ratio was observed relative to increasing concentrations of the Src kinase inhibitor.Example 4

[0183] This example demonstrates an assay performed according to the methodology described herein, wherein a Lumit Immunoassay system was delivered to live cells resulting in an engineered live cell with an intact cell membrane having reagents therein for the detection of cellular components, namely, pBTK and BTK.

[0184] A Lumit Immunoassay system was configured for detecting the phosphorylated form of BTK (pBTK). To detect pBTK, the system used a first primary rabbit antibody that specifically bound to pBTK, and a second primary mouse antibody that bound to another portion of BTK (it is envisioned that the species could be adjusted as long as secondary antibodies specific to two different species could be utilized to distinguish to two primary antibodies). Secondary antibodies labeled with LgBiT and SmBiT, respectively, could then be used to detect pBTK. Ramos cells were harvested and resuspended to a concentration of 5.14M / mL in RPMI with no supplements. After 3 hours, the cells were split into 2 equal groups, one treated with ImM of pervanadate solution (designed to increase phosphorylation levels of BTK) consisting of 16 mM Na3VO4, 0.03% H2O2, and water. The other group was treated with an equal volume of RPMI. After 40 minutes, cells were harvested and passed through a 40pm mesh strainer before resuspending at 25M / mL in OptiMEM. Treated and untreated cells were mixed in a 200uL reaction volume containing 30uL of each Phospho-BTK mAB (Rabbit), BTK mAB (Mouse), Anti-Mouse Ab-LgBiT, Anti-Rabbit Ab-SmBiT. The cell and cargo mixture were pipetted into a cartridge containing a silicon membrane with 7 pm pores and mechanically deformed at 10 PSI 68MF-367693737Attorney Docket No. 285192001040 before being collected in a 1.5 mL tube. RPMI was added to samples following mechanical deformation. Cells were washed twice with ImL of OptiMEM, then 100 pl of cells were plated in a white- walled 96 well plate and incubated for 20 minutes at room temperature. After incubation, 25uL of Lumit Detection Reagent diluted 1:12.5 in Immunoassay Reaction Buffer was added to each well. The plate was shaken at 400rpm for 2 minutes and read using the preset Lumit Immunoassay settings on the Promega GloMax. Raw luminescence was recorded and compared within the treated and untreated cell groups.

[0185] A control was also prepared wherein no cargo (no primary or second antibodies) was admixed with the cells during the preparation noted above. A second control was prepared using cell lysates per manufacturer’s instructions of a conventional Lumit Immunoassay (such samples were not passed through a membrane for mechanical deformation delivery).

[0186] The results are provided in FIG. 4, showing that the methods described herein can be utilized to deliver the Lumit Immunoassay system such that targets, namely, pBTK in this example, can be detected in live cells with an intact cell membrane.

[0187] Using the method described above, a titration of antibodies concentrations was performed. Specifically, 5 pL, 10 pL, 15 pL, and 30 pL of the antibodies solutions described above were used. As shown in FIGS. 5A and 5B, it was demonstrated that lower concentrations of antibodies can also confidently detect the present of pBTK in live cells having an intact cell membrane.

[0188] A Lumit Immunoassay system was configured for detecting BTK. Ramos cells were harvested and resuspended to a concentration of 1.5M / mL in RPMI with 10% FBS and 1% Pen / Strep (complete RPMI) and split between 2 cultures. A 50nM solution of MT-802 (a BTK PROTAC degrader) was added to one group of cells and the other received equal volume of complete RPMI. After 5 hours, cells were harvested and passed through a 40pm mesh strainer before resuspending at 7M / mL in OptiMEM. Treated and untreated cells were mixed in a 200uL reaction volume containing 30uL of each BTK mAB (Rabbit), BTK mAB (Mouse), Anti-Mouse Ab-LgBiT, Anti-Rabbit Ab-SmBiT. The cell and cargo mixture was pipetted into a cartridge containing a silicon membrane with 7 pm pores and mechanically deformed at 10 PSI before collecting in a 1.5 mL tube. Complete RPMI was added to samples following mechanical deformation. Cells were washed twice with ImL of OptiMEM, then 100 pl of cells were plated in a white- walled 96 well plate and incubated for 20 minutes at room temperature. After69MF-367693737Attorney Docket No. 285192001040 incubation, 25uL of Lumit Detection Reagent diluted 1:12.5 in Immunoassay Reaction Buffer was added to each well. The plate was shaken at 400rpm for 2 minutes and read using the preset Lumit Immunoassay settings on the Promega GloMax. Raw luminescence was recorded and compared within the treated and untreated cell groups.

[0189] A second control was prepared using cell lysates per manufacturer’s instructions of a conventional Lumit Immunoassay (such samples were not passed through a membrane for mechanical deformation delivery).

[0190] The results are provided in FIG. 6, showing that the methods described herein can be utilized to deliver the Lumit Immunoassay system such that targets, namely, BTK in this example, can be detected in live cells with an intact cell membrane. The methods described herein show comparable detection of BTK degradation as in the lysate assay.

[0191] Viability studies were performed to confirm Ramos cells remained viable following mechanical deformation delivery described above (with and without cargo delivery described above) for MT-802 and untreated cell samples. Additionally, it was confirmed that 3 kDa dextran delivery using the mechanical deformation delivery described above (with and without cargo delivery described above) was consistent for MT-802 and untreated cell samples. Data not provided.Example 5

[0192] This example demonstrates an assay performed according to the methodology described herein wherein a fluorescently tagged cereblon protein was delivered to live cells (HeLa cells) resulting in an engineered live cell with an intact cell membrane. The genome of the HeLa cells was not genetically modified to express cereblon, the substrate-recognition subunit of an E3 ligase complex.

[0193] HeLa cells were lifted into a single cell suspension with 0.25% Trypsin / EDTA, then quenched with DMEM+GlutaMAX containing 10% FBS and 1% P / S (complete D10). Cells were centrifuged and resuspended in complete D10 before being passed through a 40 pm mesh strainer, then spun resuspended at 50 M / mL in OptiMEM. Cells were combined with 3kDa dextran fluorescently labeled with Cascade blue (0.1 mg / mL) and a cereblon protein fluorescently labeled with Cy5 (1 pM) (cargo). 100 pl of the cell and cargo solution was pipetted70MF-367693737Attorney Docket No. 285192001040 into a cartridge containing a silicon membrane with 11 |jm pores. The solution was mechanoporated into a 1.5 mL tube at 10 PSI. Complete D10 was added to the collection tube 30s after mechanoporation. Intracellular delivery of 3 kDa dextran and cereblon into live cells was measured with flow cytometry within 1 hour of mechanical deformation.

[0194] As shown in FIG. 8A, the viability of HeLa cells that were subjected to the mechanical deformation delivery technique was comparable to that of HeLa cells that were not subjected to the delivery technique. Further, 80% of live HeLa cells were positive for Cy5 compared to control, indicating that the delivery of cereblon was highly effective (FIG. 8B). This example demonstrates that cereblon can be tagged and tracked in degradation-related assays in live cells with an intact cell membrane, which can provide useful insights into underlying drug candidate mechanism and kinetics.Example 6

[0195] This example demonstrates an assay performed according to the methodology described herein wherein a Lumit Immunoassay system comprising two different primary antibodies and two different secondary antibodies was delivered to live cells resulting in an engineered live cell with an intact cell membrane. The genome of the cells was not genetically modified to express the Lumit Immunoassay system.

[0196] A Lumit Immunoassay system was configured for detecting the phosphorylated form of BTK (pBTK). To detect pBTK, the system used a first primary rabbit antibody that specifically bound to pBTK, and a second primary mouse antibody that bound to another portion of BTK (it is envisioned that the species could be adjusted as long as secondary antibodies specific to two different species could be utilized to distinguish to two primary antibodies). Secondary antibodies labeled with LgBiT and SmBiT, respectively, could then be used to detect pBTK. Ramos cells were harvested and resuspended to a concentration of 5.14M / mL in RPMI with no supplements. After 3 hours, the cells were split into 2 equal groups, one treated with ImM of pervanadate solution (designed to increase phosphorylation levels of BTK) consisting of 16 mM Na3VO4, 0.03% H2O2, and water. The other group was treated with an equal volume of RPMI. After 40 minutes, cells were harvested and passed through a 40pm mesh strainer before resuspending at 25M / mL in OptiMEM. Treated and untreated cells were mixed in a 200uL reaction volume containing 30uL of each Phospho-BTK mAB (Rabbit), BTK mAB (Mouse),71MF-367693737Attorney Docket No. 285192001040 Anti-Mouse Ab-LgBiT, Anti-Rabbit Ab-SmBiT. The cell and cargo mixture were pipetted into a cartridge containing a silicon membrane with 7 pm pores and mechanically deformed at 10 PSI before being collected in a 1.5 mL tube. RPMI was added to samples following mechanical deformation. Cells were washed twice with ImL of OptiMEM, then 100 pl of cells were plated in a white- walled 96 well plate for each timepoint readout (0, 30, 60, 90, and 120 minutes). After a 20-minute incubation, 25uL of Lumit Detection Reagent diluted 1:12.5 in Immunoassay Reaction Buffer was added to wells for time 0 readout. The plate was shaken at 400rpm for 2 minutes and read using the preset Lumit Immunoassay setting. The plate was then returned to the incubator and Lumit Detection Reagent was added to the remaining wells at timepoints of 30, 60, 90, and 120 minutes.

[0197] A Lumit Immunoassay system comprising two different primary antibodies and two different secondary antibodies was delivered to live cells to detect differences in pBTK levels in untreated versus in pervanadate-treated cells. The raw luminescence of samples (untreated controls and pervanadate stimulated) subjected to a mechanical deformation delivery technique (FIG. 9A) and lysate samples (the Lumit Immunoassay system was admixed with a cell lysate without being subjected to mechanical deformation delivery) was measured (FIG. 9B). The raw luminescence of untreated controls and pervanadate stimulated samples showed a similar trend between live cells and lysate samples, with highest raw luminescence at 0 minute with a slow decrease over time (FIGs. 9A-9B).

[0198] By demonstrating that multiple antibodies can be delivered intracellularly and remain functional, this opens up new applications in using antibodies for intracellular staining in live cells. This is particularly useful as it does not require the protein of interest to be genetically tagged or pre-tagged in any other way and can directly detect native protein levels in a way that provides useful mechanistic and kinetic information.Example 7

[0199] This example demonstrates the delivery of a Lumit Immunoassay system to primary cells (activated B cells) resulting in an engineered primary live cell with an intact cell membrane having reagents therein for the detection of cellular components, namely, pBTK.72MF-367693737Attorney Docket No. 285192001040

[0200] Bulk PBMCs were cultured in Immunocult B Cell Expansion Media at a cell concentration of 1 M / mL. PBMCs were stimulated with CpG oligodeoxynucleotides (CpG ODN) at a 1 ug / mL for 72 hours. After activation, the cell suspension was washed and cultured at a cell concentration 1 M / mL with 100 ng / mL of IL-7 added to the culturing media for 4 more days. After 7 days in culture, the activated B cells were used for the Lumit experimental following the same workflow as Example 4.

[0201] As shown in FIG. 10A, phosphorylated BTK as detected by luminescence was higher in B cells stimulated with pervanadate, as compared to unstimulated B cells, in both B cell lysate samples and B cells subjected to mechanical deformation delivery technique described herein. For the comparison between unstimulated controls and pervanadate stimulated, the raw luminescence for B cell lysate samples and live B cells subjected to mechanical deformation delivery technique was comparable (FIG. 10B). This example shows that the mechanical deformation delivery technique can surprisingly deliver impermeable systems into primary cells, which is notoriously difficult to accomplish.MF-367693737

Claims

Attorney Docket No. 285192001040CLAIMS1. A method of detecting an interaction in a live cell comprising an intact cell membrane, the method comprising:delivering a first component, or a precursor thereof, to the live cell using a mechanical deformation delivery technique,wherein the first component is configured to interact with a second component in the live cell, andwherein a detectable signal is produced or producible when the first component interacts with the second component; andmeasuring the detectable signal to detect the interaction in the live cell comprising the intact cell membrane.

2. The method of claim 1, wherein the first component and the second component form a luminescent enzyme.

3. The method of claim 2, further comprising delivering a substrate of the luminescent enzyme to the live cell comprising an intact cell membrane.

4. The method of claim 1, wherein the first component and the second component exhibit resonance energy transfer to produce the detectable signal.

5. The method of any one of claims 1-4, wherein the first component comprises a non- deoxyribonucleic acid component.

6. The method of any one of claims 1-5, wherein the first component comprises a polypeptide, e.g., antibody conjugate.

7. The method of any one of claims 1-5, wherein the first component comprises a synthetic component.

8. The method of claim 7, wherein the synthetic component is a synthetic fluorophore or a detection particle.

9. The method of any one of claims 1-8, further comprising delivering the second component, or a precursor thereof, to the live cell.74MF-367693737Attorney Docket No. 285192001040 10. The method of claim 9, wherein the second component, or a precursor thereof, is codelivered with the first component using the mechanical deformation delivery technique.

11. A method of detecting an interaction in a live cell comprising an intact cell membrane, the method comprising:delivering a component comprising a luminescent enzyme, or an aspect thereof, or a precursor thereof, to the live cell using a mechanical deformation delivery technique,wherein the component comprising the luminescent enzyme, or the aspect thereof, associates or is associated with a target in the live cell; and measuring a luminescence output of the luminescent enzyme in the live cell comprising the intact cell membrane to detect the interaction in the live cell comprising the intact cell membrane.

12. The method of claim 11, further comprising delivering a substrate of the luminescent enzyme to the live cell.

13. The method of claim 12, wherein the substrate is co-delivered to the live cell along with the component comprising the luminescent enzyme, or the aspect thereof, or the precursor thereof, using the mechanical deformation delivery technique.

14. The method of any one of claims 1-13, wherein the component comprising the luminescent enzyme, or the aspect thereof, or the precursor thereof, comprises the target or a precursor thereof.

15. The method of any one of claims 1-14, wherein the target is a polypeptide.

16. The method of any one of claims 1-14, wherein the target is a nucleic acid.

17. The method of any one of claims 1-14, wherein the target is a subcellular structure.

18. The method of any one of claims 1-17, wherein the luminescent enzyme, or the aspect thereof, delivered to the live cell via the mechanical deformation delivery technique is delivered in polypeptide form, e.g., antibody conjugate.75MF-367693737Attorney Docket No. 285192001040 19. The method of any one of claims 1-18, wherein the luminescent enzyme, or the aspect thereof, delivered to the live cell via the mechanical deformation delivery technique is delivered in a non-deoxyribonucleic acid form.

20. The method of any one of claims 1-19, wherein the luminescent enzyme is composed of a complex comprising two or more aspects of the luminescent enzyme, and wherein upon complex formation in the live cell the luminescent enzyme is catalytically active.

21. The method of claim 20, wherein the live cell comprises another aspect of the luminescent enzyme associated with a second target, and wherein the aspect of the luminescent enzyme associated with the target and the other aspect of the luminescent enzyme form a catalytically active enzyme.

22. The method of claim 10, wherein the live cell comprises another aspect of the luminescent enzyme, and wherein the aspect of the luminescent enzyme associated with the target and the other aspect of the luminescent enzyme form a catalytically active enzyme.

23. The method of any one of claims 1-22, wherein the luminescent enzyme comprises LgBiT (SEQ ID NO:1) and SmBiT (SEQ ID NO:2).

24. The method of any one of claims 1-22, wherein the luminescent enzyme comprises LgBiT (SEQ ID NO:1) and HiBiT (SEQ ID NO:3).

25. The method of any one of claims 1-22, wherein the luminescent enzyme comprises LgBiT (SEQ ID NO:1) and natural peptide (NP: SEQ ID NO:4).

26. The method of any one of claims 1-25, wherein the live cell expresses the target fused to the aspect of the luminescent enzyme, wherein the delivering the component comprising the luminescent enzyme, or the aspect thereof, or the precursor thereof, to the live cell using the mechanical deformation delivery technique comprises delivering another aspect of the luminescent enzyme, or a precursor thereof, and wherein the aspect of the luminescent enzyme fused to the target and the other aspect of the luminescent enzyme associate to become catalytically active.76MF-367693737Attorney Docket No. 285192001040 27. The method of any one of claims 1-26, wherein the luminescent enzyme, or the aspect thereof, or the precursor thereof, is substantially cell membrane impermeable.

28. The method of any one of claims 1-27, wherein the substrate of the luminescent enzyme is cell permeable.

29. The method of any one of claims 1-28, wherein the substrate of the luminescent enzyme is a luciferin.

30. The method of any one of claims 1-27, wherein the substrate of the luminescent enzyme is substantially cell impermeable.

31. A method of detecting an interaction in a live cell comprising an intact cell membrane, the method comprising:delivering a first component comprising an acceptor chromophore, or a precursor thereof, and / or a second component comprising a donor chromophore, or a precursor thereof, to the live cell using a mechanical deformation delivery technique,wherein the first component comprising the acceptor chromophore and / or the second component comprising the donor chromophore associates or is associated with a target in the live cell;subjecting the live cell to an excitation light to excite the chromophore donor; and measuring a fluorescence output in the live cell comprising the intact cell membrane to detect the interaction in the live cell.

32. The method of claim 31, wherein the delivering comprises delivering the first component comprising the acceptor chromophore, or the precursor thereof.

33. The method of claim 31, wherein the delivering comprises delivering the second component comprising the donor chromophore, or the precursor thereof.

34. The method of claim 31, wherein the delivering comprises delivering the first component comprising the acceptor chromophore, or the precursor thereof, and the second component comprising the donor chromophore, or the precursor thereof.77MF-367693737Attorney Docket No. 285192001040 35. The method of any one of claims 31-34, wherein the donor chromophore is a natural molecule.

36. The method of any one of claims 31-35, wherein the donor chromophore is an autofluorescent polypeptide.

37. The method of claim 36, wherein the autofluorescent polypeptide is a green fluorescent protein (GFP).

38. The method of any one of claims 31-37, wherein the second component comprising the donor chromophore, or the precursor thereof, is a nucleic acid encoding the donor chromophore.

39. The method of claim 38, wherein the nucleic acid encoding the donor chromophore also encodes the target.

40. The method of any one of claims 31-34, wherein the donor chromophore is a synthetic molecule.

41. The method of any one of claims 31-40, wherein the second component comprising the donor chromophore, or the precursor thereof, is substantially cell impermeable.

42. The method of any one of claims 31-41, wherein the first component comprising the acceptor chromophore, or the precursor thereof, is substantially cell impermeable.

43. The method of any one of claims 31-42, wherein the acceptor chromophore is synthetic.

44. The method of any one of claims 31-43, wherein the second component comprising the acceptor chromophore comprises a small molecule.

45. The method of any one of claims 31-44, wherein the second component comprising the acceptor chromophore is a small molecule fluorophore.

46. The method of any one of claims 31-45, wherein the detecting the interaction in the live cell comprising the intact cell membrane is based on a FRET assay.78MF-367693737Attorney Docket No. 285192001040 47. The method of any one of claims 31-45, wherein the detecting the interaction in the live cell comprising the intact cell membrane is based on a time-resolved FRET assay.

48. The method of any one of claims 31-45, wherein the detecting the interaction in the live cell comprising the intact cell membrane is based on a homogeneous time- resolved fluorescence assay.

49. The method of claim 48, wherein the donor chromophore is a cryptate.

50. The method of claim 49, wherein the donor chromophore is europium cryptate or terbium cryptate.

51. The method of claim 48 or 49, wherein the acceptor chromophore is XL665, fluorescein, GFP, or d2.

52. A method of detecting an interaction in a live cell comprising an intact cell membrane, the method comprising:delivering:(a) a first component comprising an acceptor chromophore, or a precursor thereof, and a second component comprising a donor chromophore, or an aspect thereof, or a precursor thereof, to the live cell using a mechanical deformation delivery technique, or(b) the first component comprising the acceptor chromophore, or the precursor thereof, to the live cell using the mechanical deformation delivery technique, wherein the live cell comprises the donor chromophore, or (c) the second component comprising the donor chromophore, or the aspect thereof, or the precursor thereof, to the live cell using the mechanical deformation delivery technique, wherein the live cell comprises the acceptor chromophore,wherein the first component comprising the acceptor chromophore and / or the second component comprising the donor chromophore associates or is associated with a target in the live cell; and measuring a fluorescence output in the live cell comprising the intact cell membrane to detect the interaction in the live cell.79MF-367693737Attorney Docket No. 285192001040 53. The method of claim 52, wherein the donor chromophore is a luminescent enzyme.

54. The method of claim 52 or 53, wherein the donor chromophore is a luciferase.

55. The method of claim 54, wherein the luciferase is composed of a complex comprising two or more portions of the luminescent enzyme, and wherein upon complex formation the luminescent enzyme is catalytically active.

56. The method of any one of claims 41-55, wherein the first component comprising the acceptor chromophore associates or is associated with the target.

57. The method of claim 56, wherein the second component comprising the donor chromophore associates or is associated with a second target.

58. The method of any one of claims 41-55, wherein the second component comprising the donor chromophore associates or is associated with the target.

59. The method of claim 58, wherein the first component comprising the acceptor chromophore associates or is associated with a second target.

60. The method of any one of claims 31-55, wherein the first component comprising the acceptor chromophore associates or is associated with a test moiety.

61. The method of any one of claims 31-55, wherein the second component comprising the donor chromophore associates or is associated with a test moiety.

62. The method of claim 60 or 61, wherein the test moiety associates with the target.

63. The method of any one of claims 60-62, wherein the test moiety comprises a small molecule.

64. The method of any one of claims 60-62, wherein the test moiety comprises a polypeptide.

65. A method of detecting an interaction in a live cell comprising an intact cell membrane, the method comprising:80MF-367693737Attorney Docket No. 285192001040 delivering a first component comprising an acceptor bead and a second component comprising a donor bead to the live cell using a mechanical deformation delivery technique,wherein the first component comprising the acceptor bead is configured to associate with a first target and the second component comprising the donor bead is configured to associate with a second target; and measuring a fluorescence output in the live cell comprising an intact cell membrane to detect the interaction in the live cell.

66. The method of claim 65, wherein the first target and second target are on the same macromolecule.

67. The method of claim 65, wherein the first target and second targe are on different macromolecules.

68. The method of any one of claims 1-67, wherein the live cell further comprises a PROTAC configured to associate with the target in the cell.

69. The method of any one of claims 65-67, wherein the cell further comprises a PROTAC configured to associate with a macromolecule comprising the first target or a macromolecule comprising the second target.

70. The method of claim 68 or 69, further comprising delivering the PROTAC to the cell.

71. The method of any one of claims 1-70, wherein the measuring comprises measuring at two or more time points.

72. The method of any one of claims 1-71, wherein the measuring is performed via fluorescence microscopy.

73. The method of any one of claims 1-71, wherein the measuring is performed via a plate reader.

74. The method of any one of claims 1-73, wherein the mechanical deformation delivery technique comprising passing the cell through a constriction-based structure to form pores in the membrane of the cell.81MF-367693737Attorney Docket No. 285192001040 75. The method of claim 74, wherein the constriction-based structure comprises a filter or a microfluidic channel.

76. The method any one of claims 1-75, wherein the live cell is selected from the group consisting of a primary cell, iPSC, HeLa, cardiomyocyte, and HEK293.

77. An engineered live cell comprising an intact cell membrane and a component useful for the methods of claims 1-73.

78. The engineered live cell of claim 77, wherein at least one of the components is delivered using a mechanical deformation delivery technique.MF-367693737

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