Method of analyzing ligand-receptor complexes

Abstract

Disclosed herein are methods of and compositions for identifying components of a molecular pathway, wherein the molecular pathway comprises a cell surface receptor. The method comprises use of ligand-coated nanoparticles and cells that internalize the ligand-coated nanoparticles. The methods allow analysis of a wide range of ligand- receptor complexes.

Description

TITLE: METHOD OF ANALYZING LIGAND-RECEPTOR COMPLEXESCROSS REFERENCE:

[0001] This PCT application claims the benefit to U.S. Provisional Application No.63 / 710,403, filed October 22, 2024, U.S. Provisional Application No. 63 / 767,898, filed March 6, 2025, the contents of which are incorporated herein in their entirety by reference.FIELD:

[0002] The present disclosure generally relates to methods of analyzing ligandreceptor complexes, more specifically, with live cell affinity receptor chromatography (LARC) coupled with the use of ligand-coated nanoparticles.BACKGROUND:

[0003] Receptors on the cell membrane recruit a large complex of receptor enzymes to the inner membrane surface at the site of activation by voltage and phosphorylation events that may be stabilized by the presence of the membrane and / or cytoskeleton and that require cellular energy and organization to assemble. Ligand receptor complexes cannot be reconstituted after homogenization. Ligand receptor complexes on the surface of live cells may be captured by affixing the ligand to chromatography beads and interacting the ligand with the receptor on the surface of live cells to assemble the receptor complex prior to homogenization and isolation of the ligandreceptor complex on the chromatography supports termed live cell affinity receptor chromatography (LARC) that has been shown to work well for phagocytic ligands such as IgG.

[0004] US20080124310A1 discloses a method for analyzing receptor signaling complexes from live cells using microbeads coated with receptor ligands. The ligand-coated beads are allowed to bind receptors on live cells, causing phagocytosis. The receptor-ligand-bead complexes are separated from the cells by, for example, mechanical disruption. The microbeads can then be collected and the bound complexes can be analyzed, for example, by mass spectrometry.

[0005] Many cell surface receptors are not phagocytic receptors. There remains a need for methods that allows analysis of a wide range of receptor complexes.SUMMARY:

[0006] In one aspect, provided herein is a method of identifying one or more components of a molecular pathway, the molecular pathway comprising a cell surface receptor, the method comprising: contacting a ligand-coated nanoparticle comprising at least one ligand and a nanoparticle with a cell comprising the cell surface receptor capable of binding the at least one ligand of the ligand-coated nanoparticle, thereby forming a ligand-coated nanoparticle complex which is internalized by the cell via endocytosis; disrupting the cell; isolating the ligand-coated nanoparticle complex; and analyzing the ligand-coated nanoparticle complex; thereby identifying one or more components of the molecular pathway.

[0007] In one aspect, provided herein is a method of identifying one or more components of a molecular pathway, the molecular pathway comprising a cell surface receptor, the method comprising: contacting a ligand-coated nanoparticle comprising at least one ligand and a nanoparticle with a cell comprising the cell surface receptor capable of binding the at least one ligand of the ligand-coated nanoparticle, thereby forming a ligand-coated nanoparticle complex; disrupting the cell; isolating the ligand-coated nanoparticle complex; and analyzing the ligand-coated nanoparticle complex; thereby identifying one or more components of the molecular pathway.

[0008] In some embodiments, the ligand-coated nanoparticle complex is a cell surface complex. In some embodiments, the ligand-coated nanoparticle complex is an internalized complex.

[0009] In some embodiments, the at least one ligand is immobilized on the nanoparticle via an anchor. In some embodiments, the anchor is selected from: (i) a phospholipid; and (ii) a protein comprising a thio, a disulfide, a carboxyl, or any combination thereof. In some embodiments, the anchor is an amphiphilic phospholipid. In some embodiments, the anchor is adsorbed to the nanoparticle. In some embodiments, the anchor is covalently attached to the nanoparticle. In some embodiments, the at least one ligand is covalently attached to the anchor.

[0010] In some embodiments, the at least one ligand comprises a protein, a nucleic acid, a lipid, or derivative thereof.

[0011] In some embodiments, the at least one ligand is associated with regulation of cell differentiation and / or metabolism.

[0012] In some embodiments, the nanoparticle has a diameter of less than about 1 pm, less than about 800nm, less than about 600nm, less than about 400nm, or less than about 200nm.

[0013] In some embodiments, the nanoparticle comprises silica, polystyrene, polyacetate, polyacrylate, alginate, cellulose and / or PVDF.

[0014] In some embodiments, the ligand-coated nanoparticle complex comprises a receptor and a ligand-coated nanoparticle, wherein the ligand-coated nanoparticle comprises a nanoparticle and a plurality of ligands.

[0015] In some embodiments, the ligand-coated nanoparticle complex comprises at least one additional component.

[0016] In some embodiments, the at least one additional component is a cytoplasmic molecule.

[0017] In some embodiments, the ligand is capable of binding to a cell surface receptor.

[0018] In some embodiments, the ligand is associated with regulation of cellular differentiation and / or metabolism.

[0019] In some embodiments, the ligand is a hormone, or a fragment or variant thereof.

[0020] In some embodiments, the ligand is a growth factor, or a fragment or variant thereof.

[0021] In some embodiments, the ligand is a protein of the immunoglobulin superfamily, or a fragment or variant thereof.

[0022] In some embodiments, the ligand is an immunoglobulin, or a fragment or variant thereof.

[0023] In some embodiments, the ligand is an IgG, or a fragment or variant thereof.

[0024] In some embodiments, the ligand is an antigen-binding fragment.

[0025] In some embodiments, the ligand is insulin, or a fragment or variant thereof.

[0026] In some embodiments, the ligand is an insulin-like growth factor, or a fragment or variant thereof.

[0027] In some embodiments, the fragment or variant thereof is a functional fragment or variant.

[0028] In some embodiments, the nanoparticle has a diameter of less than about 1 pm, less than about 800nm, less than about 600nm, less than about 400nm, or less than about 200nm.

[0029] In some embodiments, the nanoparticle comprises silica, polystyrene, polyacetate, polyacrylate, alginate, cellulose and / or PVDF.

[0030] In some embodiments, the ligand-coated nanoparticle complex is derived from an actin-independent event.

[0031] In some embodiments, the ligand-coated nanoparticle complex is derived from a clathrin-dependent event.

[0032] In some embodiments, the ligand-coated nanoparticle complex is derived from a dynamin-dependent event.

[0033] In some embodiments, the ligand-coated nanoparticle complex is derived from an endocytic event.

[0034] In some embodiments, disrupting the cell does not disrupt the ligand-coated nanoparticle complex.

[0035] In some embodiments, disrupting the cell comprises use of a French press.

[0036] In some embodiments, isolating the ligand-coated nanoparticle complex comprises centrifugation.

[0037] In some embodiments, isolating the ligand-coated nanoparticle complex comprises ultracentrifugation.

[0038] In some embodiments, ultracentrifugation comprises ultracentrifugation over a sucrose gradient.

[0039] In some embodiments, analyzing the ligand-coated nanoparticle complex comprises separating the one or more components of the molecular pathway.

[0040] In some embodiments, the separation is by liquid chromatography, optionally normal phase chromatography or reverse phase liquid chromatography.

[0041] In some embodiments, the liquid chromatography is high-performance liquid chromatography (HPLC).

[0042] In some embodiments, the HPLC is nanoflow liquid chromatography.

[0043] In some embodiments, analyzing the ligand-coated nanoparticle complex comprises digest of the one or more components of the molecular pathway.

[0044] In some embodiments, analyzing the ligand-coated nanoparticle complex comprises performing mass spectrometry, optionally electrospray ionization or nano electrospray tandem MS (ESI-MS / MS) or matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF).

[0045] In some embodiments, analyzing the ligand-coated nanoparticle complex comprises electrospray or nano electrospray ionization (ESI), MALDI, chemical ionization, electron impact, laser desorption, electrical ionization, or heat ionization.

[0046] In some embodiments, analyzing the ligand-coated nanoparticle complex comprises recording product ion intensity by single ion monitoring (SIM) and / or product ion parent to fragment transition by single reagent monitoring (SRM).

[0047] In some embodiments, analyzing the ligand-coated nanoparticle complex comprises de novo sequencing.

[0048] In some embodiments, analyzing the ligand-coated nanoparticle complex comprises fitting of observed MS / MS spectra to a predicted library.

[0049] In some embodiments, the fitting of observed MS / MS spectra to the predicted library comprises 64 bit computation.

[0050] In some embodiments, the 64 bit computation comprises use of cross correlation, XTANDEM, SEQUEST, regression, goodness of fit, heuristic algorithms, or combination thereof.

[0051] In another aspect, provided herein is a non-naturally occurring cell, comprising an endosome comprising a ligand-coated nanoparticle complex.

[0052] In another aspect, provided herein is a method of identifying one or more components of a molecular pathway, the method comprising: providing the non-naturally occurring cell disclosed herein; disrupting the non-naturally occurring cell; isolating the ligand-coated nanoparticle complex, wherein the ligand-coated nanoparticle complex comprises the one or more components of the molecular pathway; analyzing the isolated ligand-coated nanoparticle complex; thereby identifying the one or more components of the molecular pathway.

[0053] Other features and advantages of the present disclosure will become apparent from the following detailed description. The detailed description and the specific examples while indicating embodiments of the disclosure are given by way of illustration only. These detailed description and specific examples are provided for the purposes of explanation, and not limitation, of the disclosure.BRIEF DESCRIPTION OF THE DRAWINGS:

[0054] FIGs. 1A-1E are images showing internalization of human insulin coated PCM 0.75 urn nanobeads in INSR siRNA transfected RAW 264.7 cells. (A) Overlay. (B) Actin stained by Phalloidin-i405 (405 nm). (C) External human insulin coated nanobeads (488 nm). (D) Cy3 -tagged INSR siRNAs (550nm). (E) External and internalized human insulin coated nanobeads (647 nm).

[0055] FIGs. 2A-2E are images showing internalization of human insulin coated PCM 0.75 urn nanobeads in Universal Negative Control siRNA transfected RAW 264.7 cells (control). (A) Overlay. (B) Actin stained by Phalloidin-i405 (405 nm). (C) External human insulin coated nanobeads (488 nm). (D) Cy3 -tagged Universal Negative control siRNAs (550nm). (E) External and internalized human insulin coated nanobeads (647 nm).

[0056] Fig. 3 is a graph showing average number of human insulin coated PCM 0.75 urn nanobeads internalized by RAW 264.7 cells transfected with universal negative control siRNA or various insulin receptor siRNAs. Significantly reduced nanobeadinternalization was observed in RAW264.7 cells transfected with INSR siRNA 857 & 859. (* : P < 0.05).

[0057] FIGs. 4A-4D are images showing insulin receptor knockdown in RAW264.7 cells by transient transfection (INSR siRNA- 857). (A) Overlay. Arrow indicates a nontransfected cell next to INSR siRNA transfected RAW264.7 cells. (B) Actin stained by Phalloidin-i405 (405 nm). (C) Insulin receptor 488 nm (extra bright dots are cy3-labelled siRNAs bleeding into the 488 nm channel). Arrow indicates a non-transfected cell next to INSR siRNA transfected RAW264.7 cells. (D) Cy3-labelled INSR siRNA 857 550nm channel.

[0058] FIGs. 5A-5D are images showing internalization of human insulin coated PCM 0.75 urn nanobeads by RAW 264.7 cells treated with AG1024 (120 uM). (A) Overlay. (B) Actin (405 nm). (C) External human insulin coated nanobeads (488nm). (D) External and internalized human insulin coated nanobeads (647nm).

[0059] FIGs. 6A-6D are images showing internalization of human insulin coated PCM 0.75 urn nanobeads by RAW 264.7 cells treated with DMSO (control). (A) Overlay. (B) Actin (405 nm). (C) External human insulin coated nanobeads (488nm). (D) External and internalized human insulin coated nanobeads (647nm).

[0060] FIG. 7 is a graph that shows AG1024 (120 uM) treatment significantly reduced the internalization of human insulin coated PCM 0.75 urn nanobeads by RAW 264.7 cells. (* P-value < 0.05).

[0061] FIGs. 8A-8D are images showing internalization of human insulin coated PCM 0.75 urn nanobeads by RAW 264.7 cells treated with ceritinib (13.4 uM). (A) Overlay. (B) Actin (405 nm). (C) External human insulin coated nanobeads (488nm). (D) Internalized and external human insulin coated nanobeads (647 nm).

[0062] FIGs. 9A-9D are images showing internalization of human insulin coated PCM 0.75 urn nanobeads by RAW 264.7 cells treated with DMSO (control). (A) Overlay. (B) Actin (405 nm). (C) External human insulin coated nanobeads (488nm). (D) Internalized and external human insulin coated nanobeads (647 nm).

[0063] FIG. 10 is a graph showing ceritinib (13.4 uM) treatment significantly reduced the internalization of human insulin coated PCM 0.75 urn nanobeads by RAW 264.7 cells. (* P-value < 0.01 ).

[0064] FIGs. 11 A-11 D are images showing internalization of human insulin coated PCM 0.75 urn nanobeads by RAW 264.7 cells treated with dynasore hydrate 80 uM. (A) Overlay. (B) Actin stained with Phalloidin 405nm. (C) External human insulin coated nanobeads (488 nm). (D) External and internalized nanobeads 647 nm.

[0065] FIGs. 12A-12D are images showing internalization of human insulin coated PCM 0.75 urn nanobeads by RAW 264.7 cells treated with DMSO (control). (A) Overlay. (B) Actin stained with Phalloidin 405nm. (C) External human insulin coated nanobeads (488 nm). (D) External and internalized nanobeads 647 nm.

[0066] FIG. 13 is a graph showing dynasore hydrate treatment (80 uM) significantly reduces the uptake of human insulin coated PCM 0.75 urn nanobeads by RAW 264.7 cells. (* P-value < 0.05).

[0067] FIGs. 14A-14C are images showing aggregation of clathrin around human insulin coated nanobeads during internalization in RAW264.7 cells. (A) Overlay. (B) Clathrin 488nm. (C) Human insulin coated nanobeads 647 nm.

[0068] FIGs. 15A-15D are images showing internalization of human insulin coated PCM 0.75 urn nanobeads by RAW 264.7 cells treated with DMSO (control). (A) Overlay. (B) Actin stained with Phalloidin 405nm. (C) External human insulin coated nanobeads (488 nm). (D) External and internalized nanobeads 647 nm.

[0069] FIGs. 16A-16D are images showing internalization of human insulin coated PCM 0.75 urn nanobeads by RAW 264.7 cells treated with Pitstop2 (3.75 uM). (A) Overlay. (B) Actin stained with Phalloidin 405nm. (C) External human insulin coated nanobeads (488 nm). (D) External and internalized nanobeads 647 nm.

[0070] FIG. 17 is a graph showing treating RAW 264.7 cells with Pitstop2 (3.75 uM) significantly reduced the internalization of human insulin coated nanobeads. (* P value < 0.05).

[0071] FIGs. 18A-18D are images showing RAW264.7 cells transfected with Empty-GFP plasmid- human insulin coated PCM 0.75 urn. (A) Overlay. (B) Actin (405nm). (C) Empty-GFP (488nm). (D) External and internalized human insulin coated beads (647nm).

[0072] FIGs. 19A-19D are images showing RAW264.7 cells transfected with IR-GFP plasmid- human insulin coated PCM 0.75 urn. (A) Overlay. (B) Actin (405 nm). (C) IR-GFP (488nm). (D) External and internalized human insulin coated beads (647nm).

[0073] FIGs. 20A-20D are images showing presence of IR-GFP (Arrow) around the internalized human insulin coated nanobead in RAW 264.7 cells. (A) Overlay. (B) Actin (405 nm). (C) IR-GFP (488nm). (D) External and internalized human insulin coated beads (647nm).

[0074] FIG. 21 is a graph showing internalization of human insulin coated PCM 0.75 urn nanobeads was significantly reduced in IR-GFP transfected RAW264.7 cells when compared to empty-GFP transfected RAW 264.7 cells, * P value < 0.05.

[0075] FIGs. 22A-22D are images showing internalization of human insulin coated PCM 0.75 urn nanobeads by RAW 264.7 cells treated with DMSO alone. (A) Overlay. (B) Actin (405 nm). (C) External human insulin coated beads (488nm). (D) External and internalized human insulin coated beads (647nm).

[0076] FIGs. 23A-23D are images showing internalization of human insulin coated PCM 0.75 urn nanobeads by RAW 264.7 cells treated with wortmannin (1uM). (A) Overlay. (B) Actin (405 nm). (C) External human insulin coated beads (488nm). (D) External and internalized human insulin coated beads (647nm).

[0077] FIGs. 24A-24D are images showing internalization of human insulin coated PCM 0.75 urn nanobeads by RAW 264.7 cells treated with wortmannin (50 nM). (A) Overlay. (B) Actin (405 nm). (C) External human insulin coated beads (488nm). (D) External and internalized human insulin coated beads (647nm).

[0078] FIG. 25 is a graph showing wortmannin treatment did not significantly alter the internalization of human insulin coated PCM 0.75 urn nanobeads by RAW 264.7 cells.

[0079] FIG. 26 is a STRING protein-protein interaction network based on the human insulin coated Nanobead-LARC mass spec data.

[0080] FIGs. 27A-27D are images showing internalization of IGF-1 coated nanobeads in RAW 264.7 cells treated with DMSO (control). (A) Overlay. (B) Actin stainedwith Phalloidin-i405 at 405 nm channel. (C) External IGF-1 coated PCM 0.75 um nanobeads at 488 nm channel. (D) Internalized and external IGF-1 coated PCM 0.75 um nanobeads at 647 nm channel.

[0081] FIGs. 28A-28D are images showing internalization of IGF-1 coated nanobeads in RAW 264.7 cells treated with AG1024 (80 uM). (A) Overlay. (B) Actin stained with Phalloidin-i405 at 405 nm channel. (C) External IGF-1 coated PCM 0.75 um nanobeads at 488 nm channel. (D) Internalized and external IGF-1 coated PCM 0.75 um nanobeads at 647 nm channel.

[0082] FIGs. 29A-29D are images showing internalization of IGF-1 coated nanobeads in RAW 264.7 cells treated with Ceritinib (8.96 uM). (A) Overlay. (B) Actin stained with Phalloidin-i405 at 405 nm channel. (C) External IGF-1 coated PCM 0.75 um nanobeads at 488 nm channel. (D) Internalized and external IGF-1 coated PCM 0.75 um nanobeads at 647 nm channel.

[0083] FIG. 30 is a graph showing the effect of ceritinib and AG1024 on the internalization of IGF-1 coated PCM 0.75um nanobeads in. RAW. 264.7 cells. (* P value < 0.05).

[0084] FIGs. 31A-31D are images showing internalization of IgG coated nanobeads in RAW 264.7 cells treated with DMSO (control). (A) Overlay. (B) Actin stained with Phalloidin-i405 at 405 nm channel. (C) External IgG coated PCM 0.75 um nanobeads at 488 nm channel. (D) Internalized and external IgG coated PCM 0.75 um nanobeads at 647 nm channel.

[0085] FIGs. 32A-32D are images showing internalization of IgG coated nanobeads in RAW 264.7 cells treated with pitstop2 (30 uM). (A) Overlay. (B) Actin stained with Phalloidin-i405 at 405 nm channel. (C) External IgG coated PCM 0.75 um nanobeads at 488 nm channel. (D) Internalized and external IgG coated PCM 0.75 um nanobeads at 647 nm channel.

[0086] FIGs. 33A-33D are images showing internalization of IgG coated nanobeads in RAW 264.7 cells treated with Ceritinib (13.4 uM). (A) Overlay. (B) Actin stained with Phalloidin-i405 at 405 nm channel. (C) External IgG coated PCM 0.75 um nanobeads at 488 nm channel. (D) Internalized and external IgG coated PCM 0.75 um nanobeads at 647 nm channel.

[0087] FIG. 34 is a graph showing the effect of ceritinib (13.4 uM) and pitstop2 (30 uM) on the internalization of IgG coated PCM 0.75um nanobeads in RAW 264.7 cells.

[0088] FIG. 35 is a graph comparing human insulin coating methods of PCM 0.75 urn nanobeads based on the total amount of protein on the beads as determined by BCA assay.

[0089] FIG. 36 is an image showing internalization of IGF-1 coated nanobeads in L6 myoblast cells treated with DMSO (control). The image is an overlay of actin stained with Phalloidin-i405 at 405 nm channel, external IGF-1 coated PCM 0.75 urn nanobeads at 488 nm channel, and internalized and external IGF-1 coated PCM 0.75 urn nanobeads at 647 nm channel.

[0090] FIG. 37 is an image showing internalization of IGF-1 coated nanobeads in L6 myoblast cells treated with linsitinib (47.45 uM). The image is an overlay of actin stained with Phalloidin-i405 at 405 nm channel, external IGF-1 coated PCM 0.75 urn nanobeads at 488 nm channel, and internalized and external IGF-1 coated PCM 0.75 urn nanobeads at 647 nm channel.

[0091] FIG. 38 is an image showing internalization of IGF-1 coated nanobeads in L6 myoblast cells treated with ceritinib (4.48 uM). The image is an overlay of actin stained with Phalloidin-i405 at 405 nm channel, external IGF-1 coated PCM 0.75 urn nanobeads at 488 nm channel, and internalized and external IGF-1 coated PCM 0.75 urn nanobeads at 647 nm channel.

[0092] FIG. 39 is an image showing internalization of IGF-1 coated nanobeads in L6 myoblast cells treated with AG1024 (40 uM). The image is an overlay of actin stained with Phalloidin-i405 at 405 nm channel, external IGF-1 coated PCM 0.75 urn nanobeads at 488 nm channel, and internalized and external IGF-1 coated PCM 0.75 urn nanobeads at 647 nm channel.

[0093] FIG. 40 is an image showing internalization of IGF-1 coated nanobeads in L6 myoblast cells treated with Pitstop2 (15 uM). The image is an overlay of actin stained with Phalloidin-i405 at 405 nm channel, external IGF-1 coated PCM 0.75 urn nanobeads at 488 nm channel, and internalized and external IGF-1 coated PCM 0.75 urn nanobeads at 647 nm channel.

[0094] FIG. 41 is a graph showing the effect of Ag1024, ceritinib, linsitinib, and pitstop2 on the internalization of IGF-1 coated PCM 0.75um nanobeads by L6 myoblast cells. N = 3, * P value < 0.05.

[0095] FIG. 42 is an image showing internalization of IGF-1 coated nanobeads in COS-7 (fibroblast) cells treated with DMSO (control). The image is an overlay of actin stained with Phalloidin-i405 at 405 nm channel, external IGF-1 coated PCM 0.75 urn nanobeads at 488 nm channel, and internalized and external IGF-1 coated PCM 0.75 urn nanobeads at 647 nm channel.

[0096] FIG. 43 is an image showing internalization of IGF-1 coated nanobeads in COS-7 (fibroblast) cells treated with Pitstop2 (15 uM). The image is an overlay of actin stained with Phalloidin-i405 at 405 nm channel, external IGF-1 coated PCM 0.75 urn nanobeads at 488 nm channel, and internalized and external IGF-1 coated PCM 0.75 urn nanobeads at 647 nm channel.

[0097] FIG. 44 is a graph showing the effect of Pitstop2 on the internalization of IGF-1 coated PCM 0.75um nanobeads by COS-7 fibroblast cells.

[0098] FIG. 45 is an illustration of the uptake of ligand-coated nanoparticles.

[0099] FIG. 46A is a graph showing a comparison of internalization of different types of beads by endocytosis according to some embodiments.

[0100] FIG. 46B is a graph showing a comparison internalization of different types of beads by phagocytosis according to some embodiments.

[0101] FIGs. 46C-46D are images showing internalization of 0.2 urn beads in cells pretreated with DMSO (FIG. 46C) or wortmannin (FIG. 46D).

[0102] FIG. 47 depicts assays where nanobeads were coated with human IgG and FcGR1 and incubated with RAW 264.7 cells.

[0103] FIG. 48 depicts assays where nanobeads were coated with human IgG and FcGR1 and incubated with RAW 264.7 cells and fibrolasts.

[0104] FIG. 49 depicts assays where nanobeads were coated with human IgG and incubated with RAW 264.7 cells. Images were taken at 0 min, 5 min, 10 min, and 20 min.

[0105] FIG. 50 depicts assays where nanobeads were coated with human IgG and incubated with RAW 264.7 cells. Images were taken at 0 min, 5 min, 20 min, and 30 min.DETAILED DESCRIPTION OF THE DISCLOSURE:

[0106] The following is a detailed description provided to aid those skilled in the art in practicing the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the disclosure. All publications, patent applications, patents, figures and other references mentioned herein are expressly incorporated by reference in their entirety.

[0107] All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.I. Definitions

[0108] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by those of ordinary skill in the art to which the disclosure pertains.

[0109] As used herein, the singular forms “a,” “an,” and “the” include plural references, unless indicated otherwise. For example, a reference to “a ligand” can be a reference to more than one ligand.

[0110] The term “or” is used to mean “and / or”, unless it is indicated explicitly to refer to alternatives only. It should also be noted that the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise. These terms can convey that any combination is specifically contemplated. Solely for illustrative purposes, the expression “A, B, and / or C” can mean A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C.

[0111] The term “comprise”, “comprising”, or the like means additional elements or components other than those recited may be present. Other terms such as “include”, “contain”, “have” and the like have similar meaning.

[0112] The term “consist of”, “consisting of” or the like means no additional component is present.

[0113] The term “about” or “approximately” when used in reference to a particular recited value, means the value may vary from the recited value by no more than 10%, 5%, 2% or 1%. Where a particular value is recited, it can be understood that the value is modified by the term “about” or “approximately”, unless indicated otherwise.

[0114] The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g. 1 to 5 includes 1 , 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about."

[0115] The term “nucleic acid” as used herein means a polymer of at least two nucleotides, and encompass analogs and derivatives of natural nucleotides and modifications. A nucleic acid may be single-stranded or double-stranded, or may contain portions of both. A nucleic acid may be DNA, RNA, or a hybrid.

[0116] The terms “polypeptide”, “peptide” and “protein” are used interchangeably and refer to a polymer of at least two amino acids. The amino acids may be natural amino acids, or analogues or derivatives thereof. The term encompasses modifications, such as modifications to the backbone and modifications to side chains.

[0117] The term “variant” in the context of a protein or polypeptide means a protein or polypeptide that share a certain percentage amino acid sequence identity with a reference upon alignment of the amino acid sequences.

[0118] The definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art.

[0119] For example, in the following passages, different aspects of the disclosure are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary.II. Methods

[0120] It is demonstrated herein that an insulin-coated nanoparticle can be internalized by a cell via endocytosis, and analysis of the complexes formed resulted in the identification of previously un-identified protein-protein interactions. Internalization of the insulin-coated nanoparticle can be significantly reduced when the receptor of insulin is inhibited or when the level of the receptor is knocked-down by siRNA.

[0121] Accordingly, provided herein is a method. The method can be for identifying one or more components of a molecular pathway. The molecular pathway can comprise a cell surface receptor. The method can comprise contacting a ligand-coated nanoparticle with a cell. The ligand-coated nanoparticle can comprise at least one ligand. The ligand-coated nanoparticle can comprise a nanoparticle. The cell can comprise a cell surface receptor. The cell surface receptor can be capable of binding the at least one ligand of the ligand-coated nanoparticle. Contacting the ligand-coated nanoparticle with the cell can result in forming a ligand-coated nanoparticle complex. The ligand-coated nanoparticle complex can be a cell surface complex. The ligand-coated nanoparticle complex can be internalized by the cell. The internalization by the cell can occur via endocytosis. The method can comprise disrupting the cell. The method can comprise isolating the ligand-coated nanoparticle complex. The method can comprise analyzing the ligand-coated nanoparticle complex. The method thereby identifies one or more components of the molecular pathway.

[0122] Provided herein is a method of identifying one or more components of a molecular pathway, the molecular pathway comprising a cell surface receptor, the method comprising: contacting a ligand-coated nanoparticle comprising at least one ligand and a nanoparticle with a cell comprising the cell surface receptor capable of binding the at least one ligand of the ligand-coated nanoparticle, thereby forming a ligand-coated nanoparticle complex which is internalized by the cell via endocytosis; disrupting the cell; isolating the ligand-coated nanoparticle complex; and analyzing the ligand-coated nanoparticle complex; thereby identifying one or more components of the molecular pathway.

[0123] Also provided herein is a method of identifying one or more components of a molecular pathway, the molecular pathway comprising a cell surface receptor, the method comprising: contacting a ligand-coated nanoparticle comprising at least oneligand and a nanoparticle with a cell comprising the cell surface receptor capable of binding the at least one ligand of the ligand-coated nanoparticle, thereby forming a ligand-coated nanoparticle complex; disrupting the cell; isolating the ligand-coated nanoparticle complex; and analyzing the ligand-coated nanoparticle complex; thereby identifying one or more components of the molecular pathway.

[0124] In some embodiments, the ligand-coated nanoparticle complex is a cell surface complex. In some embodiments, the ligand-coated nanoparticle complex is an internalized complex.

[0125] The cell surface receptor can be any cell surface receptor. Examples of cell surface receptors include but not limited to G-protein-coupled receptors, ion channels, enzyme linked receptors, hormone receptors, immunoglobulin receptors, etc.

[0126] Also provided herein is a method of identifying one or more components of a molecular pathway, the method comprising: providing the non-naturally occurring cell disclosed herein; disrupting the non-naturally occurring cell; isolating the ligand-coated nanoparticle complex, wherein the ligand-coated nanoparticle complex comprises the one or more components of the molecular pathway; analyzing the isolated ligand-coated nanoparticle complex; thereby identifying the one or more components of the molecular pathway.

[0127] Also provided is a method of identifying one or more components associated with a ligand-receptor complex. The ligand-receptor complex can be a cell surface complex. The ligand-receptor complex can be an internalized complex.

[0128] Also provided is a method of identifying one or more components associated with a cell surface ligand-receptor complex. Also provided is a method of identifying one or more components associated with a ligand-receptor complex that has been internalized.

[0129] Also provided herein is a method of identifying one or more components associated with a ligand-receptor complex that has been internalized by endocytosis.

[0130] Also provided herein is a screening method.

[0131] Also provided herein is a method of screening drug candidates.Molecular pathway

[0132] As used herein, the term “molecular pathway” refers to a series of events at the molecular level that can generate a biological effect. The events can be extracellular, on a cell surface, and / or intracellular. For example, a molecular pathway can initiate outside a cell, such as on a cell surface, and the series of events can continue within the cell.

[0133] Where a molecular pathway initiates outside of a cell through binding of a ligand to a cell surface receptor, the resulting ligand-receptor complex can be internalized. Internalization of the ligand-receptor complex can occur through, for example, pinching in of a patch of the cell membrane, forming an intracellular vesicle, such as an endosome.

[0134] The molecular pathway can be a signaling pathway. The molecular pathway can be associated with cell processes such as cell differentiation and cell metabolism. The molecular pathway can relate to for example, normal state, abnormal state, disease state, and / or pre-disease state of the cell.Ligand-coated nanoparticle

[0135] The term “ligand-coated nanoparticle” as used herein refers to a nanoparticle with at least one ligand associated with it in such a way that the ligand is available for binding with another component, such as a receptor.

[0136] In some embodiments, the ligand-coated nanoparticle comprises a nanoparticle and at least one ligand. In some embodiments, the ligand-coated nanoparticle comprises a nanoparticle and a plurality of ligands.

[0137] The ligand of the ligand-coated nanoparticle can be any molecule, such as a peptide, a nucleic acid, a lipid, or a derivative thereof, that is capable of binding another component, such as a receptor. The ligand can be naturally occurring or synthetic. For example, the ligand can be an IgG, a hormone such as insulin, or a DNA oligomer. A nanoparticle can be coated with different ligands. For example, a nanoparticle can be coated with two or more monoclonal antibodies that bind different targets. When the ligand is a polyclonal antibody, the nanoparticle may be coated with a plurality of ligands comprising antibodies with different binding domains. A nanoparticle can be coated with ligands of different types, such as an antibody and a hormone. A library of molecules may be used to coat a nanoparticle, which may result in the nanoparticle being coated with different ligands.

[0138] The nanoparticle can be a solid phase. The ligand can be immobilized onto the solid phase.

[0139] The nanoparticle can be any suitable material that allows association of the ligand, such as silica, polystyrene, polyacetate, polyacrylate, alginate, cellulose, polyvinylidene difluoride (PVDF) or other polymers, and derivatives thereof.

[0140] The nanoparticle can have a diameter of less than 1 pm.

[0141] In some embodiments, the nanoparticle has a diameter of less than about 1 pm. In some embodiments, the nanoparticle has a diameter of less than about 500 nm. In some embodiments, the nanoparticle has a diameter of less than about 300 nm. In some embodiments, the nanoparticle has a diameter of less than about 200 nm. In some embodiments, the nanoparticle has a diameter of about 100 nm.

[0142] The ligand can be associated with the nanoparticles using any suitable method. For example, if the ligand comprises a peptide, it can be immobilized on the nanoparticles by crosslinking to carboxyl or amine groups of the peptide. Such crosslinking can be performed with for example, NHS or EDC. The ligand can also associate with the nanoparticles non-covalently, for example, by adsorption.

[0143] In some embodiments, the ligand-coated nanoparticle is generated by a method that comprises crosslinking. In some embodiments, the ligand-coated nanoparticle is generated by a method that comprises adsorption.

[0144] In some embodiments, the ligand can be associated with the nanoparticle via streptavidin. For example, streptavidin can be attached to the nanoparticle, and the ligand, such as an antibody, can be conjugated to biotin, allowing it to associate with the nanoparticle. Different ligands can associate with the same nanoparticle this way, by conjugating biotin with different ligands.

[0145] The ligand can also associate with the nanoparticles through a linker, optionally a cleavable linker.Attaching the at least one ligand to the nanoparticle via an anchor

[0146] Anchor, which can be proteins and / or phospholipids, can be used in combination with a ligand where the anchor is coprinted with the ligand or where the ligand is attached for example covalently to the anchor.

[0147] The term “phospholipid” as used herein lipids containing phosphoric acid as mono- or di-esters, and includes phospholipids that comprise saturated or unsaturated C12, C14, C16, C18, C20 fatty acid chains, for example including phosphatidic acids and phosphoglycerides, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl glycerol (diacyl glycerol), phosphatidyl inositol, cardiolipin, galactolipids, sphingosine, ceramide, sphingomyelin, glycolipid and combinations thereof.

[0148] For example, the phospholipid can be covalently attached to the nanoparticle and / or or adsorbed to the nanoparticle. In some embodiments, the phospholipid is not covalently attached to the nanoparticle. For example, the phospholipid optionally phosphatidyl serine or phosphatidyl ethanolamine, can be adsorbed to a nanoparticle and a PEG bifunctional crosslinker can be attached to its primary amine. The at least one ligand can then be covalently attached to the crosslinker. In other embodiments, for example involving phosphatidyl choline and nucleic acid ligands, the phosphatidylcholine and nucleic acid, optionally DNA can be coprinted.

[0149] Also in some embodiments, the anchor is adsorbed to the nanoparticle via C12, C14, C16, C18, C20 phospholipid alkyl tail.

[0150] The anchor can be a protein, for example, albumin or other protein that is reduced and / or denatured. The 17 disulphide bonds present in albumin can be reduced and / or the protein can be denatured with for example DTT, urea, detergent or guanidine to expose covalent attachment sights for attaching the ligand.

[0151] The anchor can be albumin or another protein such as ovalbumin, myosin, actin or Ribulose-1 ,5-bisphosphate carboxylase-oxygenase (RuBisCo). Where the anchor is a protein, it can be a protein with at least 10, 12, 14, 16, 17, 18 or more disulphide bonds.

[0152] The anchor, or the anchor and the ligand, can be microspotted on the nanoparticle. Where the nanoparticle comprises PVDF, the PVDF can be prewet by microspotting with a polar solvent optionally methanol, for example as described in US provisional Application No. No. 63 / 338,324, filed May 4, 2022. The microspot prewetting may also minimize the amount of reagent and / or sample that may be necessary.

[0153] The PVDF may be briefly dried between the application of the methanol or other polar solvent and the ligand.

[0154] Various crosslinkers can be used to crosslink ligand with the anchor or with the nanoparticle.

[0155] For nucleic acids, hydrophilic crosslinkers may be used in combination with the anchor. This may ensure that the nucleic acid strand is positioned such that its hybridizable surface is accessible to the sample.

[0156] Examples of suitable crosslinkers for linking ligand to an anchor include PEGylated, long-chain SMCC crosslinkers. SM(PEG)n cross linkers are amine-to-sulfhydryl crosslinkers that differ in length from 17.6 to 95.2 angstroms as a result of polyethylene glycol spacer arms containing n equals 2 to 24 ethylene glycol units. SM(PEG)n NHS- and maleimide-activated PEG compounds can be used for crosslinking between primary amines (NH2) and sulfhydryl (SH) groups in proteins and other molecules. The number of ethylene glycol units can for example be n = 2, 4, 5, 6, 8, 12 or 24. The N-hydroxysuccinimide ester (NHS) group reacts specifically and efficiently with lysine and N-terminal amino groups to form stable amide bonds. The maleimide group reacts with reduced sulfhydryls to form stable thioether bonds. Other linkers can also be used, for example, EDO.

[0157] In some embodiments, the at least one ligand is immobilized on the nanoparticle via an anchor. In some embodiments, the anchor comprises a phospholipid. In some embodiments, the phospholipid comprises an amphiphilic phospholipid. In some embodiments, the phospholipid comprises phosphatidylcholine. In some embodiments, the anchor comprises a protein. In some embodiments, the protein comprises a thio, a disulfide, a carboxyl, or any combination thereof.

[0158] In some embodiments, the anchor is adsorbed to the nanoparticle. In some embodiments, the at least one ligand is adsorbed to the nanoparticle. In some embodiments, the anchor is covalently attached to the nanoparticle. In some embodiments, the at least one ligand is covalently attached to the anchor.Ligand-coated nanoparticle complex

[0159] As used herein, the term “ligand-coated nanoparticle complex” refers to a complex of the ligand-coated nanoparticle and at least one component that binds the ligand of the ligand-coated nanoparticle, such as a cell-surface receptor that binds theligand, or an intracellular molecule that binds or associates with the ligand-receptor complex.

[0160] In some embodiments, the at least one component comprises a cell surface receptor.

[0161] When the ligand-coated nanoparticle binds a cell surface receptor, the cell can internalize the ligand-coated nanoparticle complex, forming an intracellular vesicle comprising the ligand-coated nanoparticle complex.

[0162] As demonstrated herein, internalization of the ligand-coated nanoparticle can be significantly reduced when the receptor of the ligand is inhibited or when the level of the receptor is knocked-down by siRNA.

[0163] In some embodiments, internalization of the ligand-coated nanoparticle occurs upon binding of the ligand-coated nanoparticle with a receptor on the cell surface.

[0164] In some embodiments, the ligand-coated nanoparticle complex comprises a ligand-coated nanoparticle and a receptor.

[0165] As used herein, the term “intracellular vesicle” refers to a membrane-bound vesicle inside a cell. An intracellular vesicle can be formed, for example, through pinching in of a patch of the cell membrane. Extracellular content can become internalized through such process.

[0166] It is demonstrated herein that internalization of the ligand-coated nanoparticle complex can occur through endocytosis, which comprises an actin-independent, clathrin-dependent, and dynamin-dependent mechanism.

[0167] In some embodiments, the intracellular vesicle is formed by an actin-independent mechanism. In some embodiments, the intracellular vesicle is formed by a clathrin-dependent mechanism. In some embodiments, the intracellular vesicle is formed by a dynamin-dependent mechanism. In some embodiments, the intracellular vesicle is formed by a clathrin- and dynamin-dependent mechanism.

[0168] In some embodiments, the intracellular vesicle is formed by endocytosis.

[0169] In some embodiments, the intracellular vesicle comprises an endosome.

[0170] The receptor can be a cell surface receptor and can comprise at least one of: an ectodomain, which is outside of the cell prior to internalization; a transmembrane domain; and an endodomain, which is inside of the cell (i.e. on the cytoplasmic side of the cell) prior to internalization.

[0171] In some embodiments, the receptor is bound to a membrane of the endosome.

[0172] In some embodiments, the receptor is bound to the ligand-coated nanoparticle.

[0173] In some embodiments, the ligand-coated nanoparticle is inside the endosome.

[0174] Binding of the ligand-coated nanoparticle and the receptor can result in recruitment of other components. This can be caused by for example activation of the receptor and / or internalization of the ligand-coated nanoparticle complex. The other components can be part of a molecular pathway that comprises the ligand and the receptor.

[0175] In some embodiments, the ligand-coated nanoparticle complex comprises an activated receptor.

[0176] In some embodiments, the ligand-coated nanoparticle complex further comprises at least one additional component. In some embodiments, the at least one additional component comprises a cytoplasmic molecule. In some embodiments, the at least one additional component is bound to the receptor at a domain external to the endosome.Ligand

[0177] The ligand of the ligand-coated nanoparticle can be any molecule, such as a peptide, a nucleic acid, a lipid, or a derivative thereof, that is capable of binding another component, such as a receptor. The ligand can be naturally occurring or synthetic. The ligand can be part of a molecular pathway, such as one that regulates cellular differentiation and / or metabolism. More than one ligand may be associated with the nanoparticle.

[0178] In some embodiments, the ligand of the ligand-coated nanoparticle is capable of binding a cell surface receptor.

[0179] In some embodiments, the ligand is associated with regulation of cellular differentiation and / or metabolism.

[0180] In some embodiments, the ligand is hormone, or fragment or variant thereof. In some embodiments, the ligand is growth factor, or fragment or variant thereof. In some embodiments, the ligand is insulin, or fragment or variant thereof. In some embodiments, the ligand is an insulin-like growth factor, or fragment or variant thereof. In some embodiments, the ligand is a protein of the immunoglobulin superfamily, or fragment or variant thereof. In some embodiments, the ligand is an immunoglobulin, or fragment or variant thereof. For example, an immunoglobulin fragment can be an antigen-binding domain of the immunoglobulin. The antigen-binding domain may be a product of V(D)J recombination. In some embodiments, the ligand is an IgG, or fragment or variant thereof.

[0181] As used herein, the term “fragment or variant” when referring to a ligand means a portion of the ligand or a version of the ligand that retains at least some level of a function of the full length or wildtype ligand. The full length or wildtype ligand can have more than one function, and a fragment or variant of the ligand can retain for example a reduced level of a first function, while the other function or functions of the full length or wildtype ligand is / are abolished.

[0182] In some embodiments, the fragment or variant thereof is a functional fragment or variant.

[0183] As used herein, the term “functional” when referring to a ligand fragment or variant, means the ligand fragment or variant retains some ability to bind a receptor that enables the resulting complex to be internalized.

[0184] The ligand can be a library of binding agents, such as an aptamer library. If the ligand is provided as a library of binding agents, the method can be used, for example, to interrogate multiple molecular pathways concurrently, study all signaling pathways capable of being activated, screen for drug candidates etc.Contacting the ligand-coated nanoparticle with the cell

[0185] Contacting the ligand-coated nanoparticle with the cell can comprise any means to allow the ligand-coated nanoparticle and the cell to come into contact. For example, the ligand-coated nanoparticle and the cell can be incubated together in a medium.

[0186] Crosslinking can be performed to fix the ligand-receptor complex using for example formaldehyde or glutaraldehyde.

[0187] It is disclosed herein that the affinity of protein-protein interactions may have kd values as low as E-9 to E-13 and these natural affinities can be sufficient to capture the ligand and receptor interactions.

[0188] Accordingly, in some embodiments, the method does not comprise crosslinking the ligand-coated nanoparticle complex.

[0189] After contacting the ligand-coated nanoparticle with the cell, unbound ligand-coated nanoparticles can be removed by any suitable method.Isolating and analyzing the ligand-coated nanoparticle complex

[0190] The method disclosed herein can further comprise disrupting the cell after formation of the ligand-coated nanoparticle complex. Disrupting the cell can release internalized ligand-coated nanoparticle complex. Disrupting the cell can also release complexes on the cell surface that have not been internalized, or that cannot be internalized. Disruption of the cell can be achieved by any suitable method that would not result in dissociation of the ligand-coated nanoparticle complex. Such method can include, for example, the use of a French press.

[0191] Disrupting the cell can occur a period of time after contacting of the ligand-coated nanoparticle with the cell. Alternatively, or in addition, the ligand-coated nanoparticle and the cell can be in contact for a period of time before removal of any unbound ligand-coated nanoparticle. Such time period(s) can allow internalization of the ligand-coated nanoparticle complex and / or association of additional component(s).

[0192] After disruption of the cell, the ligand-coated nanoparticle complex can be isolated, for example, by centrifugation. Centrifugation includes ultracentrifugation over sucrose gradients. Other methods can be used. For example, if the nanoparticles are magnetic nanoparticles, then a magnetic field can be used.

[0193] The proteins that are associated with the ligand-coated nanoparticle complex can be recovered from the nanoparticles by any suitable method, and subject to chemical digestion or enzyme digestion.Analyzing the ligand-coated nanoparticle complex

[0194] The digested product can be analyzed by tandem mass spectrometry, optionally electrospray ionization or nano electrospray tandem MS (ESI-MS / MS) or matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF).

[0195] The MS can comprise electrospray or nano electrospray ionization (ESI), MALDI, chemical ionization, electron impact, laser desorption, electrical ionization, or heat ionization.

[0196] Analysis can comprise recording product ion intensity by single ion monitoring (SIM) and / or product ion parent to fragment transition by single reagent monitoring (SRM).

[0197] The amino acid sequence of the digested products can be derived from the MS / MS spectra by de novo sequencing, and / or by fitting of observed MS / MS spectra to a predicted library.

[0198] The fitting of observed MS / MS spectra to a predicted library can comprise 64 bit computation using cross correlation, XTANDEM, SEQUEST, regression, goodness of fit, heuristic algorithms, or combination thereof.

[0199] The one or more components of the molecular pathway identified by the method disclosed herein may be validated by any suitable method. For example, the gene encoding the identified component can be deleted or disrupted in a cell, the expression of the identified component can be knocked down by treatments, e.g. by siRNA or CRISPR or oligonucleotides or aptamers or morpholinos, or expression of mutants, activity of the identified component may be inhibited with a drug or an inhibitor, or ligands or agonist or antagonist or etc. Receptor function can then be assayed. For example, internalization of ligand-receptor complexes can be measured by confocal microscopy before and after siRNA or drug or other treatments, and downstream events may be measured by biochemical assays.

[0200] The method disclosed herein can be used in a variety of studies, for example, to identify new components of a molecular pathway that are downstream of binding of a ligand with a cell surface receptor. The method disclosed herein can be used in screening studies. For example, drug candidates can be identified by screening a library of molecules and identify candidates that can disrupt or enhance receptor activation and / or function, for example, by measuring internalization of ligand-coated beads by confocal microscopy. The library of molecules can comprise small molecules, peptides, antigen-binding fragments, nucleic acids, lipids, ligands, receptors, agonists, antagonists, antibodies or fragments thereof, single variable domains, conjugated variable domains, drug-conjugated molecules, and other types of molecules. Further, a library of ligands can be used to generate the ligand-coated nanoparticles. Different cells may be used, for example, normal cells compared to diseased or abnormal cells, which can also allow identification of new drug targets.

[0201] The method disclosed herein can be used with any suitable cell type. The cell can be a cell derived from tissues / organs such as muscle, adipose, nerve, connective tissue, epidermis, dermis, blood, eye, brain, heart, liver, intestine, breast, ovary, gonad, testicle, colon, lung, stomach, kidney, pancreas, spleen, tumor, connective, muscle-skeletal, bone, skin, vein, artery, an embryo or endoderm, mesoderm or ectoderm, or any other tissues / organs. The cell can be a cell of the digestive system, neurological system, respiratory system, circulatory system, lymphatic system, or any other systems. The cell can be a normal cell or a diseased / abnormal cell. The cell can be a stem cell, a germ cell, an eosinophil, a basophil, an astrocyte, an oligodendrocyte, a microglia, an ependymal cell, a Schwann cell, an adipocyte, a chondrocyte, a hepatocyte, a pancreatic islet cell, an alveolar cell, an enterocyte, a photoreceptor cell, a leukocyte, a lymphocyte, a plasma cell, or any other cell. The cell can for example be an immune cell, such as a macrophage, a neutrophil, a natural killer cell, a dendritic cell, a B cell, a mast cell, a T cell, a regulatory T cell, a helper T-cell. The cell can be a non-immune cell, such as a fibroblast cell. The cell can be a primary cell, an immortalized cell, a cultured cell, a cell line, a cell that has been selected based on specific biomarker(s), etc.

[0202] In yet another aspect, provided herein is a method of identifying a molecule capable of modifying a function of a cell surface receptor, comprising: providing a library comprising the molecule; contacting the library with a cell comprising the cell surfacereceptor; contacting a ligand-coated nanoparticle comprising a ligand capable of binding the cell surface receptor with the cell; assaying for the function of the cell surface receptor; thereby identifying the molecule.

[0203] In some embodiments, modifying the function of the cell surface receptor comprises enhancing receptor activation. In some embodiments, the modifying the function of the cell surface receptor comprises reducing receptor activation. Some embodiments, the modifying the function of the cell surface receptor comprises abolishing receptor activation.

[0204] In some embodiments, assaying for the function of the cell surface receptor comprises assaying for internalization of the cell surface receptor. In some embodiments, assaying for the function of the cell surface receptor comprises performing microscopy, optionally confocal microscopy. In some embodiments, assaying for the function of the cell surface receptor comprises performing a biochemical assay.II. Non-naturally occurring cells

[0205] As demonstrated herein, a ligand-coated nanoparticle can be internalized by a cell via an actin-independent mechanism and internalization is significantly reduced by inhibiting a receptor of the ligand or a receptor complex of the ligand. Without wishing to be bound by a theory, the ligand-coated nanoparticle can bind a cell surface receptor, forming a ligand-coated nanoparticle complex, triggering endocytosis, and internalization of the ligand-coated nanoparticle complex. Upon internalization, an endosome is formed with the ligand-coated nanoparticle encapsulated. The ligand-coated nanoparticle complex can remain intact during formation of the endosome. Upon binding of the ligand-coated nanoparticle to the cell surface receptor, the membrane-bound receptor can associate with other components inside the cell. Said other components can be part of a molecular pathway.

[0206] Accordingly, provided herein is a non-naturally occurring cell. As used herein, the term “non-naturally occurring cell” refers to a cell not found in nature. In some embodiments, the non-naturally occurring cell comprises an endosome. In some embodiments, the endosome comprises a ligand-coated nanoparticle complex.

[0207] In some embodiments, provided herein is a non-naturally occurring cell, comprising an endosome comprising a ligand-coated nanoparticle complex.

[0208] In some embodiments, the ligand-coated nanoparticle complex comprises a receptor and a ligand-coated nanoparticle, wherein the ligand-coated nanoparticle comprises a nanoparticle and a plurality of ligands.

[0209] In some embodiments, the receptor is bound to a membrane of the endosome. In some embodiments, the receptor is bound to the ligand-coated nanoparticle. In some embodiments, the ligand-coated nanoparticle is inside the endosome. In some embodiments, the receptor comprises a transmembrane domain, a domain internal to the endosome, and a domain external of the endosome.

[0210] In some embodiments, the ligand-coated nanoparticle complex comprises at least one additional component. In some embodiments, the at least one additional component is bound to the receptor at the domain external to the endosome. In some embodiments, the at least one additional component is a cytoplasmic molecule. In some embodiments, the ligand is capable of binding to a cell surface receptor. In some embodiments, the ligand is associated with regulation of cellular differentiation and / or metabolism.

[0211] In some embodiments, the ligand is a protein, a nucleic acid, a lipid, or derivative thereof. In some embodiments, the ligand is a hormone, or a fragment or variant thereof. In some embodiments, the ligand is a growth factor, or a fragment or variant thereof. In some embodiments, the ligand is insulin, or a fragment or variant thereof. In some embodiments, the ligand is an insulin-like growth factor, or a fragment or variant thereof. In some embodiments, the ligand is a protein of the immunoglobulin superfamily, or a fragment or variant thereof. In some embodiments, the ligand is an immunoglobulin, or a fragment or variant thereof. In some embodiments, the ligand is an IgG, or a fragment or variant thereof. In some embodiments, the fragment or variant thereof is a functional fragment or variant.

[0212] In some embodiments, the nanoparticle has a diameter of less than about 1 pm, less than about 800nm, less than about 600nm, less than about 400nm, or less than about 200nm. In some embodiments, the nanoparticle comprises silica, polystyrene, polyacetate, polyacrylate, alginate, cellulose and / or PVDF. In some embodiments, the ligand-coated nanoparticle complex is derived from an actin-independent event. In some embodiments, the ligand-coated nanoparticle complex is derived from a clathrin-dependent event. In some embodiments, the ligand-coated nanoparticle complex is derived from a dynamin-dependent event. In some embodiments, the ligand-coated nanoparticle complex is derived from an endocytic event.

[0213] In another aspect, provided herein is a method of identifying one or more components of a molecular pathway, the method comprising: providing the non-naturally occurring cell disclosed herein; disrupting the non-naturally occurring cell; isolating the ligand-coated nanoparticle complex, wherein the ligand-coated nanoparticle complex comprises the one or more components of the molecular pathway; analyzing the isolated ligand-coated nanoparticle complex; thereby identifying the one or more components of the molecular pathway.

[0214] Although any compositions and methods similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, examples of compositions and methods are now described.EXAMPLESExample 1Materials and methods

[0215] Reagents

[0216] Insulin, Human Recombinant was purchased from Sigma-Aldrich. Insulin (C27C9) Rabbit mAb (Alexa Fluor ® 647 Conjugate) and Insulin (C27C9) Rabbit mAb (Alexa Fluor ® 488 Conjugate) were purchased from Cell Signaling. Insulin Receptor p (E9L5V) XP® Rabbit mAb #23413 was purchased from Cell Signaling. Clathrin Heavy Chain (D3C6) XP® Rabbit mAb was purchased from Cell Signaling. Insulin mouse monoclonal Antibody (2D11-H5) Alexa Fluor® 647 was purchased from Santa Cruz biotechnology. Alexa Fluor® 488-conjugated AffiniPure Fab Fragment Donkey AntiRabbit IgG (H+L) was purchased from Jackson Immunoresearch. Wortmannin, Pitstop2, dynasore hydrate and AG 1024 (insulin receptor / IGF1 R. inhibitor) were purchased from Sigma-Aldrich. GFP-Standard format (empty GFP) and hIR-GFP plasmids were obtained from Addgene. Phalloidin-iFluor 405 Reagent (ab176752) was purchased from Abeam. Raw 264.7 macrophage-like cells were obtained from American Type Tissue Collection (ATCC, Manassas, VA, USA). Polybead® Carboxylate Microspheres 0.75pm (07759-15)were purchased from Polysciences. Labelled. Universal negative control cy3-labelled siRNA and cy3-labelled INSR siRNAs: SASI_Mm01_00090856, SASI_Mm01_00090857, SASI_Mm01_00090859, SASI_Mm01_00090860 were purchased from Millipore Sigma.

[0217] Coating PCM 0.75 urn (polybead carboxylate microspheres 0.75 urn, “PCM 0.75um”) Nanobeads with human insulin

[0218] Human insulin recombinant (Sigma) 5 mg was dissolved in 0.5 ml Modified Krebs-Henseliet buffer and was mixed with PCM 0.75um (5mg) resuspended in surface binding buffer (200 ul) on a rocking incubator overnight at room temperature. To remove the unbound human Insulin, eppendorf tubes were centrifuged at 16000 g for 10 min, the supernatant was removed, and the bead pellet was resuspended in 0.5 ml surface binding buffer (repeated 3X). After the third wash, the bead pellet was resuspended in 250 ul PBS 1X.

[0219] Investigating the effect of Wortmannin on the uptake of human insulin coated PCM 0.75um nanobeads by RAW264.7 cells

[0220] In each well of the 6-well plates was placed a circular cover slip and 2 ml DMEM (FBS 5%) was added to each well. 1x 10A5 RAW 264.7 cells were seeded in each well and the plate was incubated for 24 hours in a cell culture incubator (37 C, CO25%). After 48 hrs DMEM media was removed, and each well was washed with PBS 1X twice after which 2 ml RPMI media was added. 1 ul of wortmannin stock (2 mM) was added to the corresponding wells (to have a final concentration of 1 uM), 1 ul of wortmannin stock (100 uM) was added to the corresponding wells (to have a final concentration of 50 nM. In the control wells 1 ul of DMSO was added. The plate was left at 37 Celsius in the incubator for 60 min. To each well then was added 30 ul of human insulin coated nanobead suspension (10 mg / ml) and the plates were incubated at 37 Celsius. (CO25%) for 30 minutes. After the 30-min incubation, the media was removed, and wells were washed with 2 ml ice-cold PBS 1x twice. Cells were fixed with 2 ml of 4 % paraformaldehyde (in PBS 1x) over 60 minutes at room temperature. The fixation was stopped by washing the wells with 2ml ice cold PBS 1x three times, quenched with 2 ml 5% glycine (5 min, RT) and washed 3x with 2 ml PBS 1x (5 min, RT). BSA 4% (IgG free) in PBS 1x was used as the blocking buffer, 50 ul of the blocking buffer was placed on a parafilm and cover slips were placed on top followed by incubation for 30 min at RT. Tostain the external human insulin coated nanobeads, Insulin (C27C9) Rabbit mAb (Alexa Fluor® 488 Conjugate) at 1 :25 ratio in the blocking buffer was used at RT for 1hr. Afterwards, Cover slips were washed with PBS1X three times, and the cells were permeabilized by Triton X-1000.1% for 30 minutes at RT. Both internalized and external insulin coated nanobeads were stained with Insulin (C27C9) Rabbit mAb (Alexa Fluor® 647 Conjugate) at 1 :25 ratio in BSA 4%. Actin-filaments were stained with Phalloidin-iFluor 405 reagent (ab176752) at 1 :50 ratio. Cover slips were mounted on slides by DAKO fluorescent mounting medium. Slides were imaged by Z-stacking method using a ZEISS LSM900 confocal laser scanning microscope.

[0221] Investigating the effect of Dynasore hydrate on the uptake of human insulin coated PCM 0.75um nanobeads by RAW264.7 cells

[0222] In each well of the 6-well plates was placed a circular cover slip and 2 ml DMEM (FBS 5%) was added to each well. 1x 10A5 RAW 264.7 cells were seeded in each well and the plate was incubated for 24 hours in a cell culture incubator (37 C, CO25%). After 48 hrs DMEM media was removed, and each well was washed with PBS 1X twice after which 2 ml RPMI media was added, 2 ul Dynasore hydrate (80 mM in DMSO) was added to the corresponding wells (to have a final concentration of 80 uM). In the control wells 2 ul of DMSO was added. The plate was left at 37 Celsius in the incubator for 30 min. To each well then was added 40 ul of human insulin coated nanobead suspension (10 mg / ml) and the plates were incubated at 37 Celsius. (CO25%) for 45 minutes. After the 45-min incubation, the media was removed, and wells were washed with 2 ml ice-cold PBS 1x twice. Cells were fixed with 2 ml of 4 % paraformaldehyde (in PBS 1 x) over 60 minutes at room temperature. The fixation was stopped by washing the wells with 2ml ice cold PBS 1x three times, quenched with 2 ml 5% glycine (5 min, RT) and washed 3x with 2 ml PBS 1x (5 min, RT). BSA 4% (IgG free) in PBS 1x was used as the blocking buffer, 50 ul of the blocking buffer was placed on a parafilm and cover slips were placed on top followed by incubation for 30 min at RT. To stain the external human insulin coated nanobeads, Insulin (C27C9) Rabbit mAb (Alexa Fluor® 488 Conjugate) at 1 :25 ratio in the blocking buffer was used at RT for 1 hr. Afterwards, Cover slips were washed with PBS1 X three times, and the cells were permeabilized by T riton X-1000.1 % for 30 minutes at RT. Both internalized and external insulin coated nanobeads were stained with Insulin (C27C9) Rabbit mAb (Alexa Fluor® 647 Conjugate) at 1 :25 ratio in BSA 4%. Actin-filaments were stained with Phalloidin-iFluor 405 reagent (ab176752) at 1 :50 ratio. Cover slips were mounted on slides by DAKO fluorescent mounting medium. Slides were imaged by Z-stacking method using a ZEISS LSM900 confocal laser scanning microscope.

[0223] Investigating the effect of Pitstop2 on the uptake of human insulin coated PCM 0.75um nanobeads by RAW264.7 cells

[0224] In each well of the 6-well plates was placed a circular cover slip and 2 ml DMEM (FBS 5%) was added to each well. 1x 10A5 RAW 264.7 cells were seeded in each well and the plate was incubated for 48 hours in a cell culture incubator (37 C, CO25%). After 48 hrs DMEM media was removed, and each well was washed with PBS 1X twice after which 2 ml RPMI media was added. 6 ul Pitstop2 (10 mM in DMSO) was added to the corresponding wells (to have a final concentration of 30 uM). In the control wells 6 ul of DMSO was added. The plate was left at 37 Celsius in the incubator for 30 min. To each well then was added 40 ul of human insulin coated nanobead suspension (10 mg / ml) and the plates were incubated at 37 Celsius. (CO2 5%) for 45 minutes. After the 45-min incubation, the media was removed, and wells were washed with 2 ml ice-cold PBS 1x twice. Cells were fixed with 2 ml of 4 % paraformaldehyde (in PBS 1 x) over 60 minutes at room temperature. The fixation was stopped by washing the wells with 2ml ice cold PBS 1 x three times, quenched with 2 ml 5% glycine (5 min, RT) and washed 3x with 2 ml PBS 1x (5 min, RT). BSA4% (IgG free) in PBS 1x was used as the blocking buffer, 50 ul of the blocking buffer was placed on a parafilm and cover slips were placed on top followed by incubation for 30 min at RT. To stain the external human insulin coated nanobeads, Insulin (C27C9) Rabbit mAb (Alexa Fluor® 488 Conjugate) at 1 :25 ratio in the blocking buffer was used at RT for 1 hr. Afterwards, Cover slips were washed with PBS1X three times, and the cells were permeabilized by Triton X-1000.1% for 30 minutes at RT. Both internalized and external insulin coated nanobeads were stained with Insulin (C27C9) Rabbit mAb (Alexa Fluor® 647 Conjugate) at 1 :25 ratio in BSA 4%. Actin-filaments were stained with Phalloidin-iFluor 405 reagent (ab176752) at 1 :50 ratio. Cover slips were mounted on slides by DAKO fluorescent mounting medium. Slides were imaged by Z-stacking method using a ZEISS LSM900 confocal laser scanning microscope. Clathrin staining was done in separate experiments with similar setup where Rabbit monoclonal anti-Clathrin primaryantibody was used at 1 :25 ratio in BSA 4% and secondary antibody Donkey anti Rabbit 488 nm conjugated at 1 :100 in BSA 4%.

[0225] Investigating the effect of Insulin receptor siRNA transfection on the uptake of human insulin coated PCM 0.75um nanobeads by RAW264.7 cells

[0226] In each well of the 6-well plates was placed a circular cover slip and 2 ml DMEM (FBS 5%) was added to each well. 1x 10A5 RAW 264.7 cells were seeded in each well and the plate was incubated for 24 hours in a cell culture incubator (37 C, CO25%). After 24 hrs DMEM media was removed, and each well was washed with PBS 1X twice after which 1 ml Opti-MEM media was added. RAW264.7 cells in the corresponding wells were transfected with cy3-labelled INSR siRNAs (856, 857, 859, 860) or cy3-labelled universal negative control siRNA using X-treme gene siRNA transfection reagent (Sigma) according to manufacturer’s protocol. The plates were incubated for 24 hours at 37 Celsius. (CO2 5%). After 24 hrs Opti-MEM media was removed, and each well was washed with PBS 1X twice after which 2 ml RPMI media was added. To each well then was added 40 ul of human insulin coated nanobead suspension (10 mg / ml) and the plates were incubated at 37 Celsius. (CO2 5%) for 45 minutes. After the 45-min incubation, the media was removed, and wells were washed with 2 ml ice-cold PBS 1x twice. Cells were fixed with 2 ml of 4 % paraformaldehyde (in PBS 1 x) over 60 minutes at room temperature. The fixation was stopped by washing the wells with 2ml ice cold PBS 1x three times, quenched with 2 ml 5% glycine (5 min, RT) and washed 3x with 2 ml PBS 1x (5 min, RT). BSA 4% (IgG free) in PBS 1x was used as the blocking buffer, 50 ul of the blocking buffer was placed on a parafilm and cover slips were placed on top followed by incubation for 30 min at RT. To stain the external human insulin coated nanobeads, Insulin (C27C9) Rabbit mAb (Alexa Fluor® 488 Conjugate) at 1 :25 ratio in the blocking buffer was used at RT for 1 hr. Afterwards, Cover slips were washed with PBS1X three times, and the cells were permeabilized by Triton X-100 0.1% for 30 minutes at RT. Both internalized and external insulin coated nanobeads were stained with Insulin (C27C9) Rabbit mAb (Alexa Fluor® 647 Conjugate) at 1 :25 ratio in BSA 4%. Actin-filaments were stained with Phalloidin-iFluor 405 reagent (ab176752) at 1 :50 ratio. Cover slips were mounted on slides by DAKO fluorescent mounting medium. Slides were imaged by Z-stacking method using a ZEISS LSM900 confocal laser scanning microscope.

[0227] Transfecting RAW264.7 cells with IR-GFP and empty GFP plasmids and investigating the effect on the uptake of human insulin coated PCM 0.75um nanobeads

[0228] In each well of the 6-well plates was placed a circular cover slip and 2 ml DMEM (FBS 5%) was added to each well. 1x 10A5 RAW 264.7 cells were seeded in each well and the plate was incubated for 24 hours in a cell culture incubator (37 C, CO25%). After 24 hrs DMEM media was removed, and each well was washed with PBS 1X twice after which 1 ml Opti-MEM media was added. RAW264.7 cells in the corresponding wells were transfected with IR-GFP or empty GFP plasmid (info) by FuGene 6 transfection reagent (promega) according to the manufacturer’s protocol. The plates were incubated for 24 hours at 37 Celsius. (CO2 5%). After 24 hrs Opti-MEM media was removed, and each well was washed with PBS 1X twice after which 2 ml RPMI media was added. To each well then was added 40 ul of human insulin coated nanobead suspension (10 mg / ml) and the plates were incubated at 37 Celsius. (CO25%) for 45 minutes. After the 45-min incubation, the media was removed, and wells were washed with 2 ml ice-cold PBS 1x twice. Cells were fixed with 2 ml of 4 % paraformaldehyde (in PBS 1 x) over 60 minutes at room temperature. The fixation was stopped by washing the wells with 2ml ice cold PBS 1 x three times, quenched with 2 ml 5% glycine (5 min, RT) and washed 3x with 2 ml PBS 1x (5 min, RT). The cells were permeabilized by Triton X-1000.1% for 30 minutes at RT. The wells were washed 3 times with PBS1X. BSA 4% (IgG free) in PBS 1x was used as the blocking buffer, 50 ul of the blocking buffer was placed on a parafilm and cover slips were placed on top followed by incubation for 30 min at RT. Both internalized and external insulin coated nanobeads were stained with Insulin (C27C9) Rabbit mAb (Alexa Fluor® 647 Conjugate) at 1 :25 ratio in BSA 4%. Actin-filaments were stained with Phalloidin-iFluor 405 reagent (ab176752) at 1 :50 ratio. Cover slips were mounted on slides by DAKO fluorescent mounting medium. Slides were imaged by Z-stacking method using a ZEISS LSM900 confocal laser scanning microscope.

[0229] Investigating the effect of AG 1024 on the uptake of human insulin coated PCM 0.75um nanobeads by RAW264.7 cells

[0230] In each well of the 6-well plates was placed a circular cover slip and 2 ml DMEM (FBS 5%) was added to each well. 1x 10A5 RAW 264.7 cells were seeded in each well and the plate was incubated for 48 hours in a cell culture incubator (37 C, CO25%). After 48 hrs DMEM media was removed, and each well was washed with PBS 1X twiceafter which 2 ml RPMI media was added. 6 ul AG1024 (40 mM in DMSO) was added to the corresponding wells (to have a final concentration of 120 uM). In the control wells 6 ul of DMSO was added. The plate was left at 37 Celsius in the incubator for 60 min. To each well then was added 40 ul of human insulin coated nanobead suspension (10 mg / ml) and the plates were incubated at 37 Celsius. (CO2 5%) for 45 minutes. After the 45-min incubation, the media was removed, and wells were washed with 2 ml ice-cold PBS 1x twice. Cells were fixed with 2 ml of 4 % paraformaldehyde (in PBS 1 x) over 60 minutes at room temperature. The fixation was stopped by washing the wells with 2ml ice cold PBS 1 x three times, quenched with 2 ml 5% glycine (5 min, RT) and washed 3x with 2 ml PBS 1x (5 min, RT). BSA4% (IgG free) in PBS 1x was used as the blocking buffer, 50 ul of the blocking buffer was placed on a parafilm and cover slips were placed on top followed by incubation for 30 min at RT. To stain the external human insulin coated nanobeads, Insulin (C27C9) Rabbit mAb (Alexa Fluor® 488 Conjugate) at 1 :25 ratio in the blocking buffer was used at RT for 1 hr. Afterwards, Cover slips were washed with PBS1X three times, and the cells were permeabilized by Triton X-1000.1% for 30 minutes at RT. Both internalized and external insulin coated nanobeads were stained with Insulin (C27C9) Rabbit mAb (Alexa Fluor® 647 Conjugate) at 1 :25 ratio in BSA 4%. Actin-filaments were stained with Phalloidin-iFluor 405 reagent (ab176752) at 1 :50 ratio. Cover slips were mounted on slides by DAKO fluorescent mounting medium. Slides were imaged by Z-stacking method using a ZEISS LSM900 confocal laser scanning microscope.

[0231] Human insulin coated Nanobeads LARC protocol

[0232] PCM 0.75 urn (10 mg) were washed in PBS1X and coupling buffer (10 mM sodium phosphate pH 7.2) mixture (1 :1 , total volume 500 ul) followed by centrifugation at 16000 g for 10 min three times. After the last wash nanobeads were resuspended in 300 ul coupling buffer. Human insulin (2 mg, dissolved in 200 ul modified Krebs-Henseleit buffer) was added to the bead suspension followed by incubation on a rocking platform for 16 hrs. For the negative control (uncoated nanobeads) only modified Krebs-Henseleit buffer (200 ul) was added. To remove the unbound human insulin, nanobeads were washed in PBS1X and coupling buffer (10 mM sodium phosphate pH 7.2) mixture (1 :1, total volume 500 ul) followed by centrifugation at 16000 g for 10 min three times after which the beads were resuspended in 500 ul PBS1X. RAW 264.7 cells were cultured in 75 cm2 flasks using DMEM media (FBS 5%) till -70% confluency. Each was washed with5 ml PBS1X 5 times after which 10 ml RPMI 1640 were added. To each flask was added 100 ul of the ligand coated nanobead suspension (or uncoated nanobeads in the case of the negative control) followed by incubation at 37 degrees Celsius (5% CO2) for 15, 30, 45 or 60 minutes. After the incubation period, flasks were washed with ice cold PBS1X (1mM MgCI2) five times followed by the addition of the homogenization buffer (10 ml). Cells were scraped from the surface and were applied to a French press twice at 1500 PSI. (For crude extract the homogenized RAW 264.7 cells were mixed with human insulin coated pern 0.75 urn nanobeads on ice for 45 minutes). Samples were centrifuged and the pellet (containing the nanobeads) was resuspended in PBS1X (200 u) and was applied on top of 60% sucrose gradient centrifuge tubes followed by centrifugation at 40,000 g for 1 hr. at 4 degrees Celsius. Nanobead pellet was collected, and the attached proteins were eluted in salt / acetonitrile gradient solutions. Each fraction was digested with trypsin and then lyophilized before being applied to LC-MS / MS.

[0233] Comparison of human insulin coating methods of PCM 0.75 urn nanobeads

[0234] To 4 eppendorf tubes, 2.5 mg PCM 0.75 urn nanobeads were added. Two Human insulin stocks were prepared as follows: in the first 5mg / ml in PBS1 x (HCI pH 2.5) and in the second 5 mg / ml in modified Krebs-Henseleit buffer (pH 7.4). To one vial (#1) containing 2.5 mg PCM 0.75 urn nanobeads was added 2.5 mg of Human insulin dissolved in PBS1x (HCI pH 2.5). To the second vial (#2) containing 2.5 mg PCM 0.75 urn nanobeads was added 2.5 mg of Human insulin dissolved in modified Krebs-Henseleit buffer.

[0235] To Vial #3, 500 ul of 2mM EDC + 5mM NHS solution in 0.1 M MES buffer + 0.5 M NaCI, pH6.0 was added and the eppendorf tube was placed on rocking platform for 15min at RT. Eppendorf tube was centrifuged at 16000 g for 3 min. bead pellet was resuspended in PBS1X 1ml followed by centrifugation. Supernatant was removed and beads were resuspended in 500 ul 0.2M sodium bicarbonate buffer, pH 8.3. Afterwards, 2.5 mg Human insulin dissolved in PBS1x (HCI pH 2.5) was added. To the fourth vial only PBS1x (HCI pH 2.5) was added. All eppendorf tubes were placed on a rocking platform for 2h at RT. Eppendorf tubes were then washed in 200 ul PBS1X followed by centrifugation at 16000 g three times.

[0236] After the final wash the bead pellet was resuspended in 100 ul PBS 1X. Concentration of protein in each vial was calculated by BCA assay according to the manufacturer’s protocol.

[0237] Coating PCM 0.75 urn nanobeads with human IGF-1

[0238] IGF-1 (200 ug) was dissolved in 0.1 ml ddH2O (nuclease / protease free) to have a final concentration of 2 mg / ml. PCM 0.75 urn nanobeads (5mg) were washed with PBS 1X three times followed by centrifugation at 16000 g for 3 minutes. After the last wash nanobeads were resuspended in 200 ul PBS 1X. IGF-1 (200 ug) was added to the nanobead suspension followed by incubation on a rocking platform for 4 hrs. To remove the unbound human IGF-1, eppendorf tubes were centrifuged at 16000 g for 3 min, the supernatant was removed, and the bead pellet was resuspended in 0.5 ml sterile PBS1X (repeated 3X). After the third wash, the bead pellet was resuspended in 600 ul PBS 1X.

[0239] Investigating the effect of AG 1024 and centinib on the internalization of IGF- 1 coated PCM 0.75um nanobeads by RAW264.7 cells.

[0240] In each well of the 6-well plates was placed a circular cover slip and 2 ml DMEM (FBS 5%) was added to each well. 1x 10A5 RAW 264.7 cells were seeded in each well and the plate was incubated for 48 hours in a cell culture incubator (37 C, CO25%). After 48 hrs DMEM media was removed, and each well was washed with PBS 1X twice after which 1 ml RPMI media was added. 2 ul AG1024 (40 mM in DMSO) was added to the corresponding wells (to have a final concentration of 80 uM). Ceritinib (4.48 mM in DMSO, 2 ul) was added to the corresponding wells to have a final concentration of 8.96 uM. In the control wells 2 ul of DMSO was added. The plate was left at 37 Celsius in the incubator for 3 hrs. To each well then was added 100 ul of human IGF-1 coated nanobead suspension and the plates were incubated at 37 Celsius (CO25%) for 45 minutes. After the 45-min incubation, the media was removed, and wells were washed with 2 ml ice-cold PBS 1x twice. Cells were fixed with 2 ml of 4 % paraformaldehyde (in PBS 1x) over 60 minutes at room temperature. The fixation was stopped by washing the wells with 2ml ice cold PBS 1x three times, quenched with 2 ml 5% glycine (5 min, RT) and washed 3x with 2 ml PBS 1x (5 min, RT). BSA 4% (IgG free) in PBS 1x was used as the blocking buffer, 50 ul of the blocking buffer was placed on a parafilm and cover slips were placed on top followed by incubation for 30 min at RT. To stain the external human IGF-1 coatednanobeads, Anti- IG F1 antibody (ab9572, Abeam) Rabbit polyclonal Ab at 1 :25 ratio in the blocking buffer was used at RT for 1 hr. Afterwards, Cover slips were washed with PBS1 X three times, followed by staining with the secondary antibody (Alexa Fluor® 488 AffiniPure™ Donkey Anti-Rabbit IgG (H+L), Jackson ImmunoResearch) at 1 :25 in BSA 4% for 1 hr. Cover slips were washed with PBS1X three times and the cells were permeabilized by Triton X-1000.1% for 30 minutes at RT. Both internalized and external insulin coated nanobeads were stained with I Anti-IGF1 antibody (ab9572, Abeam) Rabbit polyclonal Ab at 1 :25 ratio at in BSA 4%. Actin-filaments were stained with Phalloidin-iFluor 405 reagent (ab176752, Abeam) at 1 :50 ratio for 1 hr at RT. Cover slips were washed in PBS1X 3 times and were mounted on slides by DAKO fluorescent mounting medium. Slides were imaged by Z-stacking method using a ZEISS LSM900 confocal laser scanning microscope.

[0241] Coating PCM 0.75 urn nanobeads with human IgG

[0242] IgG (200 ug) was dissolved in 0.1 ml PBS1X (sterile) to have a final concentration of 2 mg / ml. PCM 0.75 urn nanobeads (5mg) were washed with PBS 1X three times followed by centrifugation at 16000 g for 3 minutes. After the last wash nanobeads were resuspended in 200 ul PBS 1 X. IgG (200 ug) was added to the nanobead suspension followed by incubation on a rocking platform for 2 hrs. To remove the unbound human IgG, eppendorf tubes were centrifuged at 16000 g for 3 min, the supernatant was removed, and the bead pellet was resuspended in 0.5 ml sterile PBS1X (repeated 3X). After the third wash, the bead pellet was resuspended in 600 ul PBS 1X.

[0243] Investigating the effect of Pitstop2 and ceritinib on the internalization of IgG coated PCM 0.75um nanobeads by RAW264.7 cells

[0244] In each well of the 6-well plates was placed a circular cover slip and 2 ml DMEM (FBS 5%) was added to each well. 1x 10A5 RAW 264.7 cells were seeded in each well and the plate was incubated for 48 hours in a cell culture incubator (37 C, CO25%). After 48 hrs DMEM media was removed, and each well was washed with PBS 1X twice after which 2 ml RPMI media was added. Pitstop2 (10 mM in DMSO, 6 ul) was added to the corresponding wells (to have a final concentration of 30 uM). Ceritinib (4.48 mM in DMSO, 6 ul) was added to the corresponding wells to have a final concentration of 13.44 uM. In the control wells 6 ul of DMSO was added. The plate was left at 37 Celsius in theincubator for 3 hrs. To each well then was added 100 ul of human IgG coated nanobead suspension and the plates were incubated at 37 Celsius (CO25%) for 45 minutes. After the 45-min incubation, the media was removed, and wells were washed with 2 ml ice-cold PBS 1x twice. Cells were fixed with 2 ml of 4 % paraformaldehyde (in PBS 1x) over 60 minutes at room temperature. The fixation was stopped by washing the wells with 2 ml ice cold PBS 1x three times, quenched with 2 ml 5% glycine (5 min, RT) and washed 3x with 2 ml PBS 1x (5 min, RT). BSA 4% (IgG free) in PBS 1x was used as the blocking buffer, 50 ul of the blocking buffer was placed on a parafilm and cover slips were placed on top followed by incubation for 30 min at RT. To stain the external human IgG coated nanobeads, Donkey anti-Human Ab (Alexa Fluor® 488 AffiniPure™ Donkey Anti-Human IgG (H+L), Jackson ImmunoResearch) at 1 :25 ratio in the blocking buffer was used at RT for 1 hr. Afterwards, Cover slips were washed with PBS1X three times, and the cells were permeabilized by Triton X-1000.1% for 30 minutes at RT. Both internalized and external insulin coated nanobeads were stained with Alexa Fluor® 647 AffiniPure™ Donkey AntiHuman IgG (H+L) (Jackson ImmunoResearch ) at 1 :25 ratio in BSA 4%. Actin-filaments were stained with Phalloidin-iFluor 405 reagent (ab176752, Abeam) at 1 :50 ratio for 1 hr at RT. Cover slips were washed in PBS1X 3 times and were mounted on slides by DAKO fluorescent mounting medium. Slides were imaged by Z-stacking method using a ZEISS LSM900 confocal laser scanning microscope.

[0245] Investigating the effect of AG 1024, ceritinib, pitstop2 and linsitinib on the internalization of IGF-1 coated PCM 0.75um nanobeads by L6 myoblasts

[0246] In each well of the 6-well plates was placed a circular cover slip and 2 ml MEM alpha (FBS 10%) was added to each well. 1x 10A5 L6 myoblast cells were seeded in each well and the plate was incubated for 48 hours in a cell culture incubator (37 C, CO25%). After 48 hrs media was removed, and each well was washed with PBS 1X twice after which 1 ml RPMI media was added. 1 ul AG1024 (40 mM in DMSO) was added to the corresponding wells (to have a final concentration of 40 uM). Ceritinib (4.48 mM in DMSO, 1 ul) was added to the corresponding wells to have a final concentration of 4.48 uM. In the control wells 1 ul of DMSO was added. Linsitinib (47.45 mM in DMSO, 1 ul) and piststop2 (15 mM, 1 ul) were added to the corresponding wells to have a final concentration of 47.45 uM and 15 uM respectively. The plate was left at 37 Celsius in the incubator for 1 hr. To each well then was added 100 ul of human IGF-1 coated nanobead suspensionand the plates were incubated at 37 Celsius (C02 5%) for 45 minutes. After the 45-min incubation, the media was removed, and wells were washed with 2 ml ice-cold PBS 1x twice. Cells were fixed with 2 ml of 4 % paraformaldehyde (in PBS 1 x) over 60 minutes at room temperature. The fixation was stopped by washing the wells with 2ml ice cold PBS 1 x three times, quenched with 2 ml 5% glycine (5 min, RT) and washed 3x with 2 ml PBS 1x (5 min, RT). BSA4% (IgG free) in PBS 1x was used as the blocking buffer, 50 ul of the blocking buffer was placed on a parafilm and cover slips were placed on top followed by incubation for 30 min at RT. To stain the external human IGF-1 coated nanobeads, Anti-IGF1 antibody (ab9572, Abeam) Rabbit polyclonal Ab at 1 :25 ratio in the blocking buffer was used at RT for 1 hr. Afterwards, Cover slips were washed with PBS1X three times, followed by staining with the secondary antibody (Alexa Fluor® 488 Aff ini Pure™ Donkey Anti-Rabbit IgG (H+L), Jackson ImmunoResearch) at 1 :25 in BSA 4% for 1 hr. Cover slips were washed with PBS1X three times and the cells were permeabilized by Triton X-100 0.1% for 30 minutes at RT. Both internalized and external insulin coated nanobeads were stained with I Anti-IGF1 antibody (ab9572, Abeam) Rabbit polyclonal Ab at 1 :25 ratio at in BSA 4%. Actin-filaments were stained with Phalloidin-iFluor 405 reagent (ab176752, Abeam) at 1 :50 ratio for 1 hr at RT. Cover slips were washed in PBS1 X 3 times and were mounted on slides by DAKO fluorescent mounting medium. Slides were imaged by Z-stacking method using a ZEISS LSM900 confocal laser scanning microscope.

[0247] Investigating the effect of pitstop2 on the internalization of IGF- 1 coated PCM 0.75um nanobeads by COS-7 fibroblast cells

[0248] In each well of the 6-well plates was placed a circular cover slip and 2 ml MEM alpha (FBS 10%) was added to each well. 1x 10A5 COS-7 cells were seeded in each well and the plate was incubated for 48 hours in a cell culture incubator (37 C, CO2 5%). After 48 hrs media was removed, and each well was washed with PBS 1X twice after which 1 ml RPMI media was added. Piststop2 (15 mM, 1 ul) was added to the corresponding wells to have a final concentration of 15 uM. The plate was incubated at 37 Celsius (5% CO2) for 1 hr. To each well then was added 100 ul of human IGF-1 coated nanobead suspension and the plates were incubated at 37 Celsius (CO2 5%) for 45 minutes. After the 45-min incubation, the media was removed, and wells were washed with 2 ml ice-cold PBS 1x twice. Cells were fixed with 2 ml of 4 % paraformaldehyde (in PBS 1x) over 60 minutes at room temperature. The fixation was stopped by washing thewells with 2ml ice cold PBS 1x three times, quenched with 2 ml 5% glycine (5 min, RT) and washed 3x with 2 ml PBS 1x (5 min, RT). BSA 4% (IgG free) in PBS 1x was used as the blocking buffer, 50 ul of the blocking buffer was placed on a parafilm and cover slips were placed on top followed by incubation for 30 min at RT. To stain the external human IGF-1 coated nanobeads, Anti-IGF1 antibody (ab9572, Abeam) Rabbit polyclonal Ab at 1 :25 ratio in the blocking buffer was used at RT for 1 hr. Afterwards, Cover slips were washed with PBS1X three times, followed by staining with the secondary antibody (Alexa Fluor® 488 AffiniPure™ Donkey Anti-Rabbit IgG (H+L), Jackson ImmunoResearch) at 1 :25 in BSA 4% for 1 hr. Cover slips were washed with PBS1X three times and the cells were permeabilized by Triton X-100 0.1% for 30 minutes at RT. Both internalized and external insulin coated nanobeads were stained with I Anti-IGF1 antibody (ab9572, Abeam) Rabbit polyclonal Ab at 1 :25 ratio at in BSA 4%. Actin-filaments were stained with Phalloidin-iFluor 405 reagent (ab176752, Abeam) at 1 :50 ratio for 1 hr at RT. Cover slips were washed in PBS1X 3 times and were mounted on slides by DAKO fluorescent mounting medium. Slides were imaged by Z-stacking method using a ZEISS LSM900 confocal laser scanning microscope.Example 2

[0249] Knock down of INSR by labelled siRNA INSR transfection reduces the uptake of human insulin coated nanobeads by RAW 264.7 cells

[0250] To investigate whether insulin receptor (IR) is involved in the process by which the human insulin coated beads are internalized in RAW264.7 cells, the cells were transfected with cy3-labelled INSR siRNAs or non-targeting siRNA (universal negative control) for 24 hours. The internalization of the insulin-coated beads was then compared between the cells that had taken up the labelled INSR siRNAs to those containing labelled non-targeting siRNAs (as confirmed by confocal laser scanning microscopy). RAW264.7 cells transfected with two of the INSR siRNAs showed significantly reduced internalization of the human insulin coated nanobeads compared to the cells transfected with nontargeting siRNA (FIG. 3). This suggests that IR is probably involved in the mechanism by which human insulin coated nanobeads are internalized in RAW 264.7 cells.

[0251] The knockdown of insulin receptor by INSR siRNAs was confirmed in the transfected RAW264.7 cells. As can be seen in FIGs. 4A-4D in which green fluorescence(488nm) indicates the presence of insulin receptors, the non-transfected cell (indicated by white arrow in FIGs. 4A and 4C) shows higher expression of INSR compared to the adjacent transfected cells.Example 3

[0252] Biochemical inhibitor of INSR and IGF1R, AG1024 reduced the internalization of human insulin coated nanobeads in RAW 264.7 cells

[0253] The effect of IR inhibitor AG 1024 on the internalization of coated nanobeads was investigated. AG1024 is a cell permeable, reversible, and specific inhibitor of insulin receptor and inulin-like growth factor 1 receptor tyrosine kinase activity. RAW264.7 cells were pretreated with AG1024 at 120 uM (FIGs. 5A-5D) or DMSO (control) (FIGs. 6A-6D) for one hour. Cells were then treated with human insulin coated nanobeads for 45 minutes and the number of internalized beads in each treatment was determined. AG1024 pretreatment significantly reduced the internalization of the human insulin coated nanobeads suggesting that Insulin receptor activity required for the internalization of the nanobeads (FIG. 7).Example 4

[0254] Ceritinib, a receptor tyrosine kinase inhibitor significantly reduced the internalization of human insulin coated (and IGF-1 coated) nanobeads

[0255] The effect of multiple tyrosine kinase receptor inhibitor and antineoplastic drug ceritinib on the internalization of human insulin coated nanobeads was investigated. Ceritinib is a small molecule, ATP-competitive, and potent inhibitor of several tyrosine kinase receptors including insulin receptor, insulin-like growth factor 1 receptor and fms related receptor tyrosine kinase (FLT3). RAW264.7 cells were pretreated with ceritinib at 13.4 uM or DMSO (control) for 24 hours. Cells were then treated with human insulin coated nanobeads for 45 minutes and the number of internalized beads in each treatment was determined. Ceritinib pretreatment significantly reduced the internalization of the human insulin coated nanobeads suggesting that insulin receptor tyrosine kinase activation is required for the internalization of the nanobeads (Fig 8-10).Example 5

[0256] Inhibitors of endocytosis significantly reduce the uptake of coated nanobeads by macrophages

[0257] To investigate whether endocytosis is involved in the internalization of the human insulin coated nanobeads, two inhibitors of endocytosis Pitstop 2 and dynasore hydrate were used. Pitstop 2, a clathrin-dependent endocytosis inhibitor, inhibits the interaction of amphyphysin with the N-terminal domain of clathrin. Dynasore hydrate is an inhibitor of GTPase activity of dynamins which is required for the fission of budding vesicles during endocytosis. Both compounds are cell permeable. RAW 264.7 cells were pretreated with dynasore hydrate (80uM), pitstop2 (3.75uM) or DMSO alone (control) in RPMI medium prior to the addition of the human insulin coated nanobeads. The internalization of the human insulin coated nanobeads in RAW 264.7 cells was significantly reduced as a result of either treatment when compared to the control condition (DMSO alone) (FIGs 11-17). Pitstop2 at higher concentrations (up to 30 mM) is known to exert a stronger inhibitory effect on endocytosis, however higher concentrations of pitstop2 (7.5. uM, 15 uM, 30 uM) caused high levels of background fluorescence and disturbed high contrast imaging of human insulin coated nanobeads stained with the corresponding Alexa-fluor conjugated antibodies. The results acquired, together with the results obtained from the wortmannin treatment experiment, suggests that endocytosis (and not pinocytosis or phagocytosis) is most likely the mechanism involved in the internalization of the insulin coated nanobeads.Example 6

[0258] IR-GFP showed a dominant negative effect on the internalization of human insulin coated PCM 0.75 um nanobeads

[0259] Human insulin coated PCM 0.75 um beads were presented to IR-GFP transfected RAW 264.7, as well as empty-GFP transfected cells (control), both cultured as monolayers on top of 25 mm diameter circular glass cover slips in RPMI media. After fixing the cells, the internalization of the coated beads was investigated by confocal laser scanning microscopy. In IR-GFP transfected RAW 264.7 cells, the green fluorescence (488 nm) is more intense in the cell membrane where IR-GFPs are present in higher density (Fig. 3). The green fluorescence (488 nm channel) around the internalized beads indicates the presence of IR-GFP, which suggests that the human insulin coated beadscaptured some of the IR-GFPs (initially present in the cell membrane) during their internalization (Fig. 3). However, there is no such pattern of fluorescence around the internalized nanobeads in empty-GFP transfected RAW264.7 cells (Fig. 1). Also, IR-GFP transfected RAW 264.7 cells showed significantly reduced internalization of the human insulin coated nanobeads when compared to empty-GFP transfected cells suggesting the presence of a dominant negative effect.Example 7

[0260] Phagocytosis inhibitors do not affect the internalization of beads

[0261] Wortmannin does not inhibit human insulin coated PCM 0.75 urn nanobeads internalization

[0262] The effect of wortmannin, a specific inhibitor of the phosphatidylinositol 3-kinase (PI3K) which is reported to inhibit phagocytosis in a concentration dependent manner, at 50 nM and 1 uM was investigated on the uptake of human insulin coated PCM 0.75 urn nanobeads by RAW 264.7 cells. Cells were pre-treated with wortmannin (FIGs.23A-23D, FIGs. 24A-24D) or DMSO (FIGs 22A-22D) for 60 minutes after which human insulin coated beads were added to the media. The total number of beads internalized after 30 minutes was compared to the control condition (DMSO alone). No significant difference between the two treatments on the number of internalized nanobeads was observed suggesting that the internalization of the insulin coated beads is not affected by wortmannin (FIG. 25).

[0263] Conclusion

[0264] The mechanisms of the uptake of human insulin coated nanobeads (polybead carboxylate microspheres 0.75 urn, “PCM 0.75um”) by RAW 264.7 cells were investigated. Treating the cells with wortmannin did not have a significant effect on the uptake of the beads however dynasore hydrate (an inhibitor of endocytosis, and Pitstop2) significantly reduced the uptake of the beads. Insulin receptors knock down also significantly reduced the uptake of insulin coated beads. In addition, IR GFP transfected RAW264.7 cells showed significantly reduced uptake of the human insulin coated beads when compared to Empty GFP transfected cells suggesting a dominant negative effect. The results suggested that the uptake of the insulin coated nanobeads occurs through insulin receptor dependent endocytosis.Example 8

[0265] Nano-LARC reveals previously un-identified protein-protein interactions

[0266] Network analysis was performed on the human insulin coated Nanobead-LARC mass spec data using the STRING (Protein-Protein Interaction Networks Functional Enrichment Analysis) (FIG. 26). Nano-LARC reveals previously un-identified protein-protein interactions where the low PPI-p value 1E-16 indicates a low probability that the network is a random assemblage of proteins that is consistent with the capture of the whole nano receptor supramolecular complex.Example 9

[0267] AG1024 and ceritinib reduced the internalization of IGF-1 coated nanobeads in RAW 264.7 cells

[0268] The effect of AG1024 on the internalization of IGF-1 coated nanobeads was investigated. RAW264.7 cells were pretreated with AG1024 at 80 uM (FIGs. 28A-28D) or DMSO (control) (FIGs. 27A-27D) for 3 hrs. Cells were then treated with IGF-1 coated nanobeads for 45 minutes and the number of internalized beads in each treatment was determined. AG1024 pretreatment significantly reduced the internalization of the human insulin coated nanobeads suggesting that Insulin receptor activity required for the internalization of the nanobeads (FIG. 30).

[0269] The effect of ceritinib on the internalization of IGF-1 coated nanobeads was investigated. RAW264.7 cells were pretreated with ceritinib at 8.96 uM (FIGs. 29A-29D) or DMSO (control) (FIGs. 27A-27D) for 3 hours. Cells were then treated with IGF-1 coated nanobeads for 45 minutes and the number of internalized beads in each treatment was determined. Ceritinib pretreatment significantly reduced the internalization of the IGF-1 coated nanobeads (FIG. 30).Example 10

[0270] Pitstop2 and ceritinib reduced the internalization of IgG coated nanobeads in RAW 264.7 cells

[0271] The effect of Pitstop2 on the internalization of IGF-1 coated nanobeads was investigated. RAW264.7 cells were pretreated with Pitstop2 at 30 uM (FIGs. 32A-32D) orDMSO (control) (FIGs. 31A-31D) for 3 hours. Cells were then treated with IGF-1 coated nanobeads for 45 minutes and the number of internalized beads in each treatment was determined. Ceritinib pretreatment significantly reduced the internalization of the IGF-1 coated nanobeads (FIG. 34).

[0272] The effect of ceritinib on the internalization of IGF-1 coated nanobeads was investigated. RAW264.7 cells were pretreated with ceritinib at 13.44 uM (FIGs. 33A-33D) or DMSO (control) (FIGs. 31A-31D) for 3 hrs. Cells were then treated with IGF-1 coated nanobeads for 45 minutes and the number of internalized beads in each treatment was determined. AG1024 pretreatment significantly reduced the internalization of the human insulin coated nanobeads suggesting that Insulin receptor activity required for the internalization of the nanobeads (FIG. 34).Example 11

[0273] Comparison of human insulin coating methods of PCM 0.75 um nanobeads

[0274] Three different methods of coating PCM 0.75 um nanobeads with human insulin were compared.

[0275] Human insulin was either dissolved in PBS1X (pH 2.5 with HCI) or modified krebs-Henseleit buffer (pH 7.4) and afterwards was mixed with PCM 0.75 um suspension to be coated by adsorption. In the third method PCM 0.75 um nanobeads were first treated with EDC+NHS and then were mixed with Human insulin dissolved in PBS1 X (pH 2.5 with HCI). After the washing steps to remove the unbound insulin, the amount of human insulin attached to PCM 0.75 um nanobeads was determined by BCA assay. The adsorption method using modified Krebs-Henseleit buffer resulted in the highest amount of human insulin coating of the nanobeads (FIG. 35). The other advantage of this method is that the pH is within the physiological pH range and therefore may be less likely to cause protein denaturation. Therefore, this method was chosen for coating the nanobeads with human insulin.Example 12

[0276] Ceritinib, linsitinib and Pitstop 2 significantly reduced the internalization of IGF-1 coated nanobeads in L6 myoblast cells

[0277] The effects of AG1024, ceritinib, pitstop2 and linsitinib on the internalization of IGF-1 coated nanobeads by L6 myoblasts were investigated. L6 myoblast cells were pretreated with AG1024 (40 uM), ceritinib (4.48 uM), linsitinib (47.45 uM), Pitstop2 (15 uM) or DMSO (control) (FIGs. 36-40) for 1 hour. Cells were then treated with IGF-1 coated nanobeads for 45 minutes and the number of internalized beads in each treatment was determined. Pretreatment with ceritinib linsitinib or Pitstop 2 significantly reduced the internalization of the IGF-1 coated nanobeads by L6 myoblast cells (FIG. 41).Example 13

[0278] Pitstop 2 reduced the internalization of IGF-1 coated nanobeads in COS-7 fibroblast cells

[0279] The effect of Pitstop2 on the internalization of IGF-1 coated nanobeads by COS-7 fibroblast cells was investigated. COS-7 fibroblast cells were pretreated with Pitstop2 (15 uM) or DMSO (control) (FIGs. 42-43) for 1 hour. Cells were then treated with IGF-1 coated nanobeads for 45 minutes and the number of internalized beads in each treatment was determined. Pretreatment with Pitstop 2 reduced the internalization of the IGF-1 coated nanobeads by COS-7 fibroblast cells (FIG. 44).Example 14

[0280] In this example, internalization by phagocytosis or endocytosis of different types of beads with different sizes coated with IgG was examined.

[0281] 0.75 urn Polybead Carboxylate Microsphere (PCM) and 0.8 urn polystyrene beads were coated with IgG. RAW264.7 cells were pretreated with DMSO or wortmannin (1 uM) for 30 minutes, followed by internalization assay for 30 minutes.

[0282] Results are shown in FIG. 46A.

[0283] 2.0 urn Polybead Carboxylate Microsphere (PCM) and 2.0 urn polystyrene beads were coated with IgG. RAW264.7 cells were pretreated with DMSO or wortmannin (1 uM) for 30 minutes, followed by internalization assay for 30 minutes.

[0284] Results are shown in FIG. 46B.

[0285] 0.2 urn Polybead Carboxylate Microsphere (PCM) and 0.2 urn polystyrene beads were coated with IgG. RAW264.7 cells were pretreated with DMSO or wortmannin (1 uM) for 30 minutes, followed by internalization assay for 30 minutes.

[0286] Results are shown in FIGs. 46C (DMSO) and 46D (wortmannin).Example 15

[0287] In this example, nanobeads coated with multiple ligands were used in internalization assays.

[0288] 800 nm nanobeads were coated with human IgG and FCGR1 , and applied to RAW264.7 cells. Cells were incubated on ice for 5 minutes. Results are shown in FIG.47.

[0289] 800 nm nanobeads were coated with human IgG and FCGR1 , and applied to RAW264.7 cells and fibroblast cells. Cells were incubated on ice for 5 minutes. Results are shown in FIG. 48.Example 16

[0290] This example shows time course experiments:

[0291] 0.2 urn PCM nanobeads coated by IgG were incubated with RAW264.7 cells for 0, 5, 10, or 20 minutes. Results are shown in FIG. 49.

[0292] .2 urn PCM nanobeads coated by IgG were incubated with RAW264.7 cells for 0, 5, 10, or 20 minutes. Results are shown in FIG. 50.

[0293] While the present application has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the application is not limited to the disclosed examples. To the contrary, the application is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[0294] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[0295] The scope of the claims should not be limited by the embodiments and examples, but should be given the broadest interpretation consistent with the description as a whole.

Claims

CLAIMS:1 . A method of identifying one or more components of a molecular pathway, the molecular pathway comprising a cell surface receptor, the method comprising: contacting a ligand-coated nanoparticle comprising at least one ligand and a nanoparticle with a cell comprising the cell surface receptor capable of binding the at least one ligand of the ligand-coated nanoparticle, thereby forming a ligand- coated nanoparticle complex which is internalized by the cell via endocytosis; disrupting the cell;isolating the ligand-coated nanoparticle complex; andanalyzing the ligand-coated nanoparticle complex;thereby identifying one or more components of the molecular pathway.

2. A method of identifying one or more components of a molecular pathway, the molecular pathway comprising a cell surface receptor, the method comprising: contacting a ligand-coated nanoparticle comprising at least one ligand and a nanoparticle with a cell comprising the cell surface receptor capable of binding the at least one ligand of the ligand-coated nanoparticle, thereby forming a ligand- coated nanoparticle complex;disrupting the cell;isolating the ligand-coated nanoparticle complex; andanalyzing the ligand-coated nanoparticle complex;thereby identifying one or more components of the molecular pathway.

3. The method of any of the preceding claims, wherein the ligand-coated nanoparticle complex is a cell surface complex.

4. The method of any of the preceding claims, wherein the ligand-coated nanoparticle complex is an internalized complex.

5. The method of any of the preceding claims, wherein the at least one ligand is immobilized on the nanoparticle via an anchor.

6. The method of any of the preceding claims, wherein the anchor is selected from: (i) a phospholipid; and (ii) a protein comprising a thio, a disulfide, a carboxyl, or any combination thereof.

7. The method of any of the preceding claims, wherein the anchor is an amphiphilic phospholipid.

8. The method of any of the preceding claims, wherein the anchor is adsorbed to the nanoparticle.

9. The method of any of the preceding claims, wherein the anchor is covalently attached to the nanoparticle.

10. The method of any of the preceding claims, wherein the at least one ligand is covalently attached to the anchor.

11. The method of any of the preceding claims, wherein the at least one ligand comprises a protein, a nucleic acid, a lipid, or derivative thereof.

12. The method of any of the preceding claims, wherein the at least one ligand is associated with regulation of cell differentiation and / or metabolism.

13. The method of any of the preceding claims, wherein the at least one ligand comprises a hormone, or a fragment or variant thereof.

14. The method of any of the preceding claims, wherein the at least one ligand comprises a growth factor, or a fragment or variant thereof.

15. The method of any of the preceding claims, wherein the at least one ligand comprises insulin, or a fragment or variant thereof.

16. The method of any of the preceding claims, wherein the at least one ligand comprises an insulin-like growth factor, or a fragment or variant thereof.

17. The method of any of the preceding claims, wherein the at least one ligand comprises a protein of the immunoglobulin superfamily, or a fragment or variant thereof.

18. The method of any of the preceding claims, wherein the at least one ligand comprises an immunoglobulin, or a fragment or variant thereof.

19. The method of any of the preceding claims, wherein the at least one ligand comprises an IgG, or a fragment or variant thereof.

20. The method of any of the preceding claims, wherein the fragment or variant thereof is a functional fragment or variant.

21. The method of any of the preceding claims, wherein the nanoparticle has a diameter of less than about 1 pm, less than about 800nm, less than about 600nm, less than about 400nm, or less than about 200nm.

22. The method of any of the preceding claims, wherein the nanoparticle comprises silica, polystyrene, polyacetate, polyacrylate, alginate, cellulose and / or PVDF.

23. The method of any of the preceding claims, wherein disrupting the live cell does not disrupt the ligand-coated nanoparticle complex.

24. The method of any of the preceding claims, wherein disrupting the non-naturally occurring cell comprises use of a French press.

25. The method of any of the preceding claims, wherein isolating the ligand-coated nanoparticle complex comprises centrifugation.

26. The method of any of the preceding claims, wherein isolating the ligand-coated nanoparticle complex comprises ultracentrifugation.

27. The method of any of the preceding claims, wherein ultracentrifugation comprises ultracentrifugation over a sucrose gradient.

28. The method of any of the preceding claims, wherein analyzing the ligand-coated nanoparticle complex comprises separating the one or more components of the molecular pathway.

29. The method of any of the preceding claims, wherein the separation is by liquid chromatography, optionally normal phase chromatography or reverse phase liquid chromatography.

30. The method of any of the preceding claims, wherein the liquid chromatography is high-performance liquid chromatography (HPLC).

31. The method of any of the preceding claims, wherein the HPLC is nanoflow liquid chromatography.

32. The method of any of the preceding claims, wherein analyzing the ligand-coated nanoparticle complex comprises digest of the one or more components of the molecular pathway.

33. The method of any of the preceding claims, wherein analyzing the ligand-coated nanoparticle complex comprises performing mass spectrometry, optionally electrospray ionization or nano electrospray tandem MS (ESI-MSZMS)or matrix- assisted laser desorption / ionization time-of-flight (MALDI-TOF).

34. The method of any of the preceding claims, wherein analyzing the ligand-coated nanoparticle complex comprises electrospray or nano electrospray ionization (ESI), MALDI, chemical ionization, electron impact, laser desorption, electrical ionization, or heat ionization.

35. The method of any of the preceding claims, wherein analyzing the ligand-coated nanoparticle complex comprises recording product ion intensity by single ion monitoring (SIM) and / or product ion parent to fragment transition by single reagent monitoring (SRM).

36. The method of any of the preceding claims, wherein analyzing the ligand-coated nanoparticle complex comprises de novo sequencing.

37. The method of any of the preceding claims, wherein analyzing the ligand-coated nanoparticle complex comprises fitting of observed MS / MS spectra to a predicted library.

38. The method of any of the preceding claims, wherein the fitting of observed MS / MS spectra to the predicted library comprises 64 bit computation.

39. The method of any of the preceding claims, wherein the 64 bit computation comprises use of cross correlation, XTANDEM, SEQUEST, regression, goodness of fit, heuristic algorithms, or combination thereof.

40. The method of any of the preceding claims, further comprising validating the identified one or more components of the molecular pathway.

41. A method of identifying a molecule capable of modifying a function of a cell surface receptor, comprising:providing a library comprising the molecule;contacting the library with a cell comprising the cell surface receptor; contacting a ligand-coated nanoparticle comprising a ligand capable of binding the cell surface receptor with the cell;assaying for the function of the cell surface receptor,thereby identifying the molecule.

42. A non-naturally occurring cell, comprising an endosome comprising a ligand- coated nanoparticle complex.

43. The non-naturally occurring cell of any of the preceding claims, wherein the ligand- coated nanoparticle complex comprises a receptor and a ligand-coated nanoparticle, wherein the ligand-coated nanoparticle comprises a nanoparticle and a plurality of ligands.

44. The non-naturally occurring cell of any of the preceding claims, wherein the receptor is bound to a membrane of the endosome.

45. The non-naturally occurring cell of any of the preceding claims, wherein the receptor is bound to the ligand-coated nanoparticle.

46. The non-naturally occurring cell of any of the preceding claims, wherein the ligand- coated nanoparticle is inside the endosome.

47. The non-naturally occurring cell of any of the preceding claims, wherein the receptor comprises a transmembrane domain, a domain internal to the endosome, and a domain external of the endosome.

48. The non-naturally occurring cell of any of the preceding claims, wherein the ligand- coated nanoparticle complex comprises at least one additional component.

49. The non-naturally occurring cell of any of the preceding claims, wherein the at least one additional component is bound to the receptor at the domain external to the endosome.

50. The non-naturally occurring cell of any of the preceding claims, wherein the at least one additional component is a cytoplasmic molecule.

51. The non-naturally occurring cell of any of the preceding claims, wherein the ligand is capable of binding to a cell surface receptor.

52. The non-naturally occurring cell of any of the preceding claims, wherein the ligand is associated with regulation of cellular differentiation and / or metabolism.

53. The non-naturally occurring cell of any of the preceding claims, wherein the ligand is a protein, a nucleic acid, a lipid, or derivative thereof.

54. The non-naturally occurring cell of any of the preceding claims, wherein the ligand is a hormone, or a fragment or variant thereof.

55. The non-naturally occurring cell of any of the preceding claims, wherein the ligand is a growth factor, or a fragment or variant thereof.

56. The non-naturally occurring cell of any of the preceding claims, wherein the ligand is insulin, or a fragment or variant thereof.

57. The non-naturally occurring cell of any of the preceding claims, wherein the ligand is an insulin-like growth factor, or a fragment or variant thereof.

58. The non-naturally occurring cell of any of the preceding claims, wherein the ligand is a protein of the immunoglobulin superfamily, or a fragment or variant thereof.

59. non-naturally occurring cell of any of the preceding claims, wherein the ligand is an immunoglobulin, or a fragment or variant thereof.

60. The non-naturally occurring cell of any of the preceding claims, wherein the ligand is an IgG, or a fragment or variant thereof.

61. The non-naturally occurring cell of any of the preceding claims, wherein the fragment or variant thereof is a functional fragment or variant.

62. The non-naturally occurring cell of any of the preceding claims, wherein the nanoparticle has a diameter of less than about 1pm, less than about 800nm, less than about 600nm, less than about 400nm, or less than about 200nm.

63. The non-naturally occurring cell of any of the preceding claims, wherein the nanoparticle comprises silica, polystyrene, polyacetate, polyacrylate, alginate, cellulose and / or PVDF.

64. The non-naturally occurring cell of any of the preceding claims, wherein the ligand- coated nanoparticle complex is derived from an actin-independent event.

65. The non-naturally occurring cell of any of the preceding claims, wherein the ligand- coated nanoparticle complex is derived from a clathrin-dependent event.

66. The non-naturally occurring cell of any of the preceding claims, wherein the ligand- coated nanoparticle complex is derived from a dynamin-dependent event.

67. The non-naturally occurring cell of any of the preceding claims, wherein the ligand- coated nanoparticle complex is derived from an endocytic event.

68. A method of identifying one or more components of a molecular pathway, the method comprising:providing the non-naturally occurring cell of any of the preceding claims; disrupting the non-naturally occurring cell;isolating the ligand-coated nanoparticle complex, wherein the ligand-coated nanoparticle complex comprises the one or more components of the molecular pathway;analyzing the isolated ligand-coated nanoparticle complex;thereby identifying the one or more components of the molecular pathway.

69. The method of any of the preceding claims, wherein disrupting the non-naturally occurring cell does not disrupt the ligand-coated nanoparticle complex.

70. The method of any of the preceding claims, wherein disrupting the non-naturally occurring cell comprises use of a French press.

71. The method of any of the preceding claims, wherein isolating the ligand-coated nanoparticle complex comprises centrifugation.

72. The method of any of the preceding claims, wherein isolating the ligand-coated nanoparticle complex comprises ultracentrifugation.

73. The method of any of the preceding claims, wherein ultracentrifugation comprises ultracentrifugation over a sucrose gradient.

74. The method of any of the preceding claims, wherein analyzing the ligand-coated nanoparticle complex comprises digest of the one or more components of the molecular pathway.

75. The method of any of the preceding claims, wherein analyzing the ligand-coated nanoparticle complex comprises performing mass spectrometry, top down mass spectrometry, electrospray or nano electrospray ionization mass spectrometry, MALDI mass spectrometry, chemical ionization mass spectrometry, electron impact ionization electron impact ionization, or liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS / MS).

76. The method of any of the preceding claims, wherein analyzing the ligand-coated nanoparticle complex comprises de novo sequencing.

77. The method of any of the preceding claims, wherein analyzing the ligand-coated nanoparticle complex comprises fitting of observed MS / MS spectra to a predicted library.

78. The method of any of the preceding claims, wherein the fitting of observed MS / MS spectra to the predicted library comprises 64 bit computation.

79. The method of any of the preceding claims, wherein the 64 bit computation comprises use of cross correlation, XTANDEM, SEQUEST, regression, goodness of fit, heuristic algorithms, or combination thereof.

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