Endocytic agents

Abstract

Disclosed herein are compounds comprising an agent or a probe connected with a chemical arm via a cleavable or non-cleavable chemical bond or linker unit where the compounds has a binding affinity to an endocytosis-mediating membrane component. Also disclosed are methods of using and making the same.

Description

[0001] ENDOCYTIC AGENTS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 420,326 filed on October 28, 2022, the contents of which are incorporated by reference in their entireties.

[0004] BACKGROUND OF THE INVENTION

[0005] According to traditional guidelines, such as “the rule of 5’ (Ro5)” and later extended ones (extended Ro5; eRo5 and beyond Ro5; bRo5), majority of therapeutic agents suffer from poor drug-likeness due to unfavorable stability, aqueous solubility, and / or membrane permeability, directly resulting in the high rate of attrition in drug development. Among these factors, poor permeability has been recognized as the most important issue since drugs must cross numerous membrane barriers to reach to the drug acting sites after administration. Based on the passive permeation, stability, aqueous solubility, and membrane permeability are highly associated with each other and sensitive to the structure changes and structural modifications for adjusting one nature will inevitably affect one or more other properties. To increase drug passive permeability, the common method is to enhance the lipophilicity of compounds via structural optimizations on a case-by-case basis, making it readily dissolved in membrane phospholipids and rapidly diffused across cellular membranes, but at the expense of reduced solubility (due to reduced hydrophilicity), stability, and / or toxicity. For most therapeutic agents, favorable stability and aqueous solubility could be achieved via structural modification and salting technologies, leaving how to improve therapeutic agents’ membrane permeability without sacrificing solubility and stability as an ultimate question to be solved in traditional drug discovery and development campaigns. Evidence shows that as molecular size increases, passive permeability of drugs drops off drastically. Unfortunately, the bioactivities of eRo5 and bRo5 molecules such as chimeric molecules have been credited from the passive permeability and the optimization of eRo5 and bRo5 molecules has been based on the passive diffusion, although according to the passive diffusion theory, the modification of large molecules, such as PROTAC molecules, to improve membrane passive permeability balanced with favorable metabolic stability and solubility are challenging or even impossible. Hence, there is a need in the art for a general method which can be used to speed-up the therapeutics development and widen the therapeutic applications of agents via enhancing permeability without sacrificing solubility and stability. BRIEF SUMMARY OF THE INVENTION

[0006] Disclosed herein are compounds comprising an agent or a probe connected with a chemical arm via a cleavable or non-cleavable chemical bond or linker unit where the compounds has a binding affinity to an endocytosis-mediating membrane component. The compound may have a binding affinity KD to the endocytosis-mediating membrane component of less than 20.0 mM. In some embodiments, the the compound is represented by a Formula (I): f1, wherein m, n, and p represent integer from 0 to

[0007] 100. An exemplary compound may have m, n, and p equal to 1. The compounds may comprise an agent that is a degrader, stabilizer, inhibitor, modulator, or activator. In some embodiments, the agent is a protein binder. In some embodiments, the compound comprises a probe that is a diagnostic agent. The chemical arm may be an atom, an agent, a probe, or a portion of an agent or a binder or a probe. In some instances, he chemical arm has a binding affinity Kp to the endocytosis-mediating membrane component of less than 20.0 mM. Suitably, the endocytosis- mediating membrane component is a cell membrane lipid, carbohydrate, or protein. The endocytosis-mediating membrane component may be a glycolipid, glycoprotein, phospholipid, ceramides, and cholesterol. In some embodiments, the endocytosis-mediating membrane component is a glycolipid, or a glycoprotein comprises 2-100 straight or branched monosaccharide units. The endocytosis-mediating membrane component may be an integral membrane protein, peripheral membrane protein, lipid-anchored protein, globular protein, or glycoprotein.

[0008] Also disclosed are nanostructures comprising liquid or cytoplasm enclosed by a lipid bilayer, an endocytosis-meditating membrane component, and any of the compounds described herein. In some instances, the nanostructure has a binding affinity Ko to the endocytosis-mediating membrane component of less than 20.0 mM. Exemplary nanostructures include extracellular vesicles, microvesicles, endocytic agents-vesicle complexes, or exocytic vesicles.

[0009] Methods of preparing nanostructures are also provided. The method may comprise contacting any of the compounds disclosed herein with a cell or vesicle comprising the endocytosis-meditating membrane component. The nanostructure may be prepared in vivo, in vitro, or ex vivo.

[0010] Method for internalizing any of the compounds disclosed herein within a cell are also provided. The method may comprise contacting any of the compounds disclosed herein with a cell comprising the endocytosis-mediating membrane component. In some embodiments, a nanostructure comprising the compound is contacted with the cell.

[0011] A method for isolating the compound from a nanostructure is also provided. The method may comprise lysing the nanostructure and separating the compound from a lysate.

[0012] A method of determining a qualitative or quantitative presence of a compound or nanostructure in a cell, body, solution, or medium is also provided. The method may comprise centrifuging a sample comprising the cell, body, solution, or medium and detecting for the compound or nanostructure.

[0013] A method for isolating the compound or nanostructure from a cell, body, solution, or medium is also provided. The method may comprise centrifuging a sample comprising the cell, body, solution, or medium.

[0014] A method for treating a subject is also provided. The method may comprise administering a compound or nanostructure to a subject in need thereof.

[0015] A method for identifying an endocytic agent is also provided. The method may comprise contacting a compound with a first cell and a second cell, wherein the presence of an endocytosis- mediating membrane component in the second cell has been modulated relative to the first cell, and comparing the activity of the compound in contact with the first cell to the activity of the compound in contact with the second cell to determine the compound is the endocytic agent.

[0016] A method for identifying an endocytosis-mediating membrane component is also provided. The method may comprise contacting a cell with the compound or nanostructure, wherein the compound comprises a detectable label and wherein the endocytosis-mediating membrane component in a cell can be identified by determining an interaction between endocytosis-mediating membrane component and detectable label.

[0017] A method of identifying an endocytosis-mediating membrane component is also provided. The method may comprise comparing the sensitivity of a first cell and a second cell to the treatment of a compound or nanostructure and comparing genomic expression or protein abundance of a membrane component in the first cell and the second cell, wherein increased sensitivity of the first cell or the second cell to the compound or nanostructure identifies the endocytosis-mediating membrane component.

[0018] A method for selecting a subject for treatment with a compound or nanostructure having a binding affinity to an endocytosis-mediating membrane component is also provide. The method may comprise determining a qualitative or quantitative presence of the endocytosis-mediating membrane component in a sample obtained from a prospective subject and administering the compound or nanostructure to the prospective subject when the endocytosis-mediating membrane component is present in the sample.

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention.

[0021] Figures 1A-1E illustrate endocytic agents and their utilization.

[0022] Figure 1A depicts the mechanisms for cellular pick up and utilities of endocytic agents and the methods to generate endocytic agents or polypharmacological endocytic agent with increased binding affinity and / or valent with endocytosis-mediating membrane component(s) for enhanced endocytosis efficacy and / or efficiency.

[0023] Figure IB depicts the mechanisms for endocytic agents’ absorption into blood after oral, inhalation, or topical administration. The endocytic agent can be enclosed in intracellular vesicles via multiple paths and released into blood stream in forms of free endocytic agent and / or exocytic vesicles. Cytoplasmic proteins include but are not limited to fatty acid binding proteins (FABPs).

[0024] Figure 1C depicts the mechanisms for endocytic agents’ absorption by crossing blood brain barrier into brain tissue from blood stream. The endocytic agent can be enclosed in intracellular vesicles via multiple paths and released into blood stream in forms of free endocytic agent and / or exocytic vesicles.

[0025] Figure ID depicts the mechanisms for cellular pick up, release, and utilization of endocytic agents and exocytic vesicles for biological functions.

[0026] Figure IE depicts the relative permeability of agents with a range of molecular weights via passive diffusion (top curve at low molecular weight) or endocytosis (bottom curve at low molecular weight).

[0027] Figures 2A-2E show endocytic agents bind with human CD36 protein. Figure 2A depicts the immunoblotting assay that demonstrates the expressions of CD36 protein in LNCaP PCa cells’ membrane and cytosolic compartment.

[0028] Figure 2B depicts the pull-down assay that demonstrates CD36 protein in LNCaP cell membrane is one of the targets of polypharm cological endocytic agents as exemplified by Example 1. Mass spectrometry (MS) determines the binding of Example 1 with bromodomain and extra- terminal (BET) family of proteins, CD36, and proteins associated with endocytic vesicles formation, tethering, fusion, trafficking, and membrane component recycling, such as CLH1, UBR4, AP1B1, RAB5A, BIG1, AP2B1, AP3D1, ARP2, and AP3B1.

[0029] Figure 2C shows that endocytic agents as exemplified by Example 1, Example 3, or Example 4 bind with hCD36 proteins. Immunoblot for CD36 after treatment of purified his-tagged hCD36 proteins with biotin control or endocytic agent examples.

[0030] Figure 2D shows that polypharmcological endocytic agent as exemplified by Example 6 or Example 7 binds with hCD36 proteins. Immunoblot for CD36 after treatment of purified his- tagged hCD36 proteins with biotin control or endocytic agent examples.

[0031] Figure 2E shows that representative endocytic agent Example 8 binds with human CD36 protein as determined by ITC assay. The titrations consisted of injections of Example 8 solution in injection syringe into the human CD36 protein (hCD36) solution in sample cell at a rate of 0.5 pl / s at 150 s time intervals. K / j value was determined using a sequential binding site model.

[0032] Figures 3A-3O show that endocytosis is the dominant path for agent uptake.

[0033] Figure 3A shows the CD36 protein levels in LNCaP PCa cells transfected with shRNA with Luc control or CD36 targeting sequence.

[0034] Figure 3B shows the EEA1 protein levels in LNCaP cells transfected with shRNA with Luc control or EEA1 targeting sequence.

[0035] Figure 3C shows the Rab5 protein levels in LNCaP cells transfected with shRNA with Luc control or Rab5 targeting sequence.

[0036] Figure 3D shows the CD36 protein levels in 22Rvl PCa cells transfected with siRNA with scrambled sequence or CD36 targeting sequence.

[0037] Figure 3E shows that EEA1 / Rab5 proteins are required for endocytosis and facilitate uptake of transferrin. Representative confocal images of morphology by DAPI and transferrin in LNCaP cells with stable shLuc, shEEAl, or shRab5 treated for indicated durations. Scale bars: 100 pm. Figure 3F shows that colocalization assay demonstrates the dependence of CD36- mediated endocytosis for the uptake of polypharmacological endocytic agent as exemplified by Example 10 in LNCaP cells. The fluorescence of endocytic agent Example 10 and DAPI from cells were imaged on a fluorescent microscopy after treatment of LNCaP cells with Example 10 for indicated durations. Scale bars: 200 pm.

[0038] Figure 3G shows that colocalization assay demonstrates the dependence of CD36- mediated endocytosis for the uptake of trivalent polypharmacological endocytic agent as exemplified by Example 12 in LNCaP cells. The fluorescence of endocytic agent Example 12 and DAPI from cells were imaged on a fluorescent microscopy after treatment of LNCaP cells with Example 12 for indicated durations. Scale bars: 100 pm.

[0039] Figure 3H shows that colocalization assay demonstrates the dependence of CD36- mediated endocytosis for the uptake of trivalent polypharmacological endocytic agent as exemplified by Example 8 in LNCaP cells. The fluorescence of endocytic agent Example 8 and DAPI from cells were imaged on a fluorescent microscopy after treatment of LNCaP cells with Example 8 for indicated durations. Scale bars: 100 pm.

[0040] Figure 31 shows that colocalization assay demonstrates the dependence of CD36-mediated endocytosis for the uptake of trivalent polypharmacological endocytic agent as exemplified by Example 8 in 22Rvl cells. The fluorescence of endocytic agent Example 8, CD36 protein, EEA1 protein, and DAPI from cells were imaged on a fluorescent microscopy after treatment of 22Rvl cells with Example 8 at 500 nM for 0.5 hour.

[0041] Figure 3 J shows that colocalization assay demonstrates the dependence of CD36-mediated endocytosis for the uptake of polypharmacological endocytic agent as exemplified by Example 13 in LNCaP cells. The fluorescence of endocytic agent Example 13 and DAPI from cells were imaged on a fluorescent microscopy after treatment of LNCaP cells with Example 13 for indicated durations. Scale bars: 200 pm.

[0042] Figure 3K shows that colocalization assay demonstrates the dependence of CD36- mediated endocytosis for the uptake of bivalent polypharmacological endocytic agent as exemplified by Example 14 in LNCaP cells. The fluorescence of endocytic agent Example 14 and DAPI from cells were imaged on a fluorescent microscopy after treatment of LNCaP cells with Example 14 for indicated durations. Scale bars: 100 pm. Figure 3L shows that colocalization assay demonstrates the dependence of CD36- mediated endocytosis for the uptake of bivalent polypharmacological endocytic agent as exemplified by Example 15 in LNCaP cells. The fluorescence of endocytic agent Example 15 and DAPI from cells were imaged on a fluorescent microscopy after treatment of LNCaP cells with Example 15 for indicated durations. Scale bars: 100 pm.

[0043] Figure 3M shows that colocalization assay demonstrates the dependence of CD36- mediated endocytosis for the uptake of bivalent polypharmacological endocytic agent as exemplified by Example 15 in 22Rvl cells. The fluorescence of endocytic agent Example 15, CD36 protein, EEA1 protein, and DAPI from cells were imaged on a fluorescent microscopy after treatment of 22Rvl cells with Example 15 at 500 nM for 0.5 hour. Scale bar: 100 pm.

[0044] Figure 3N shows that colocalization assay demonstrates the dependence of CD36- mediated endocytosis for the uptake of bivalent polypharmacological endocytic agent as exemplified by Example 16 in 22Rvl cells. The fluorescence of endocytic agent Example 16 and DAPI from cells were imaged on a fluorescent microscopy after treatment of 22Rvl cells with Example 16 for indicated durations. Scale bars: 100 pm.

[0045] Figure 30 shows that trivalent polypharmacological endocytic agent Example 8 or Example 12 and bivalent polypharmacological endocytic agent Example 14 or Example 15 has little or no visible toxicity on LNCaP or 22Rvl cells viability, (n = 3)

[0046] Figures 4A-4F show that CD36-mediated endocytosis determines the uptake and biological function of drugs.

[0047] Figure 4A shows that polypharmacological endocytic agent Trivalent-BETDl (Example 17) induces BRD4 degradation in LNCap or 22Rvl PCa cells. Protein levels of BRD4 in LNCaP or 22Rvl cells treated with Example 17 and with or without the pretreatment of MG132 were determined by western blotting assay.

[0048] Figure 4B shows that shCD36 in LNCaP or 22Rvl PCa cells reverses the BRD4 degradation by polypharmacological endocytic agent Trivalent-BETDl (Example 17). LNCaP or 22Rvl cells stably transfected with shLuc or shCD36 were treated with Example 17 then the protein levels of BRD4 and CD36 were determined by western blotting assay.

[0049] Figure 4C shows that polypharmacological bivalent endocytic agent ARV110 (Example 19) induces AR degradation in LNCaP or 22Rvl PCa cells. Protein levels of AR in LNCaP or 22Rvl cells treated with Example 19 and with or without the pretreatment of MG132 were determined by western blotting assay.

[0050] Figure 4D shows that shCD36 in LNCaP or 22Rvl PCa cells reverses the AR degradation by polypharmacological bivalent endocytic agent ARV110 (Example 19). LNCaP or 22Rvl cells stably transfected with shLuc or shCD36 were treated with Example 19 then the protein levels of AR and CD36 were determined by western blotting assay.

[0051] Figures 4E and 4F show shCD36 antagonists the anti-tumor effects of polypharmacological endocytic agent ARV110 (Example 19) in LNCaP -xenografted mice model, (n = 5). In Figure 4E, the top curve represents shCD36#2+ARV110; the second from top curve represents shCD36#2; the third from top curve represents shLuc+vehicle; the fourth from top curve represents shCD36#l+ARV110; the fifth from top curve represents shCD36#l; and the bottom curve represents shLuc+ARVl 10.

[0052] Figures 5A-5D show that conjugating an agent with a chemical arm enhances cellular uptake via CD36-mediated endocytosis.

[0053] Figure 5A shows that conjugating an agent (Example 20) with a dicarboxylic acid chemical arm enhances and speeds-up cellular uptake of agent. Green fluorescence of endocytic agent Bivalent-BDP-FLl (Example 20) or Trivalent BDP-FLl-C12Na (Example 21) from cells were imaged on a fluorescent microscopy after treatment of HCC1806 cells with Example 20 or Example 21 at 500 nM for indicated durations. Scale bar: 100 pm.

[0054] Figure 5B shows that colocalization assay demonstrates CD36-mediated endocytosis is the dominant path for endocytic agent Bivalent-BDP-FLl (Example 20) Trivalent-BDP-FLl- C12Na (Example 21), or Trivalent-BDP-FLl-C14Na (Example 22) uptake in HCC1806 cells. Fluorescence of endocytic agent Example 20, Example 21, or Example 22 , CD36 protein, EEA1 protein, and DAPI from cells were imaged on a fluorescent microscopy after treatment of HCC1806 cells with Example 20 or Example 21 at 500 nM for 0.5 hour. Examples 21 and 22 with higher binding affinity and more binding arms (valent) than 20 enhance and speed-up cellular uptakes. Scale bar: 100 pm.

[0055] Figure 5C shows that colocalization assay demonstrates CD36-mediated endocytosis is the dominant path for endocytic agent Bivalent-BDP-FLl (Example 20), Trivalent-BDP-FLl- C12Na (Example 21), or Trivalent-BDP-FLl-C14Na (Example 22) uptake in shCD36 HCC1806 cells. Fluorescence of endocytic agent Example 20, Example 21 , or Example 22 , CD36 protein, EEA1 protein, and DAPI from cells were imaged on a fluorescent microscopy after treatment of shCD36 HCC1806 cells with Example 20 or Example 21 at 500 nM for 0.5 hour. Scale bar: 100 pm.

[0056] Figure 5D shows that Bivalent-BDP-FLl (Example 20), Trivalent-BDP-FLl-C12Na (Example 21), or Trivalent-BDP-FLl-C14Na (Example 22) has little cytotoxicity in HCC1806 cells. Meanwhile, trivalent endocytic agents Example 28 and 29 exerted more potent cytotoxicity than bivalent endocytic agent Example 23 in HCC1806 TNBC cells, (n = 2)

[0057] Figures 6A-6D show that CACO2 cells uptake endocytic agents via endocytosis and release free endocytic agents and exocytic vesicles.

[0058] Figure 6A depicts a representative method in present disclosure for isolating and / or detecting endocytic agents and / or exocytic vesicles in medium with cells after treating with endocytic agents.

[0059] Figure 6B depicts a representative method in present disclosure for isolating and / or detecting endocytic agents and / or exocytic vesicles in animal blood and / or tissue after treating animal with endocytic agents via oral administration.

[0060] Figure 6C shows the detection of polypharmacological bivalent endocytic agent ARV110 (Example 19) in supernatants or exosome lysates isolated from CACO2 cells-containing culture medium after Example 19 treatment. Compared with the exosome lysates samples, no or little Example 19 is detectable in supernatants samples. Endocytic agents in all samples were detected by LC-mass spectrum.

[0061] Figure 6D shows the CD36, CD9, HSP70, and TSG101 protein levels in exocytic vesicles released by CACO2 cells after treatment with DMSO control or representative polypharmacological bivalent endocytic agent ARV110 (Example 19). Immunoblot for CD36, CD9, HSP70, TGS101 proteins in exosomes isolated from CACO2 cells after treatment of CACO2 cells with DMSO vehicle control or Example 19. Compared with the control vehicle, Example 19 treatment enhances the expression of CD36 in exocytic vehicles.

[0062] Figure 6E shows the CD36, CD9, HSP70, and TSG101 protein levels in exosomes isolated from plasma of rats after treatment of rats with vehicle or representative polypharmacological bivalent endocytic agent ARV110 (Example 19) at indicated dosage via oral administration. Compared with the control vehicle, Example 19 treatment enhances the expression of CD36 in exocytic vehicles in vivo. Meanwhile, compared with the supernatant’s samples in plasma from rats after treatment with ARV110, higher abundances of Example 19 were detected in all the corresponding exosome lysates samples by LC-mass spectrum.

[0063] Figures 7A-7K show that increasing binding affinity (see Table 2) and / or binding valent (see Table 1) with CD36 results in augmented agent uptake and therapeutic consequences.

[0064] Figure 7A shows that some of the trivalent endocytic agents (Examples 24 to 31) with higher binding affinity and more binding arms (valent) induce deeper BRD4 degradation than bivalent endocytic agents (Example 23) in HCC1806 TNBC or 22Rvl PCa cells under same conditions. Immunoblot for BRD4 after treatment of HCC 1806 or 22Rvl cells with DMSO vehicle control or endocytic agents.

[0065] Figure 7B shows that trivalent endocytic agent Example 29 with higher binding affinity and more binding arms (valent) induces deeper BRD4 degradation than bivalent endocytic agents (Example 23) in HCC 1806 TNBC cells under same conditions. BRD4 protein was examined by immunoblotting and BRD4 protein level was quantified by densitometry and normalized to the corresponding density of P-tubulin protein.

[0066] Figure 7C shows that trivalent endocytic agent Example 29 with higher binding affinity and more binding arms (valent) induces faster BRD4 degradation than bivalent endocytic agents (Example 23) in HCC1806 TNBC cells under same conditions. Immunoblot for BRD4 after treatment of HCC 1806 cells with DMSO vehicle control or endocytic agents for indicated durations.

[0067] Figure 7D shows that pretreatment of MG132 reverses the BRD4 degradation by endocytic agent Example 23 or Example 29 in HCC1806 cells. Protein levels of BRD4 in HCC1806 cells treated with endocytic agent examples with or without the pretreatment of MG132 at indicated concentrations were determined by western blotting assay.

[0068] Figure 7E shows that shCD36 reverses the BRD4 degradation by endocytic agent Example 23 or Example 29 in HCC 1806 cells. HCC 1806 cells stably transfected with or without shCD36 were treated with endocytic agent examples at indicated concentrations, then the protein levels of BRD4 and CD36 were determined by western blotting assay.

[0069] Figure 7F shows that pretreatment of SYK inhibitor, which was known for blocking endocytosis reverses the BRD4 degradation by endocytic agent Example 23 or Example 29 in HCC 1806 cells. Protein levels of BRD4 in HCC 1806 cells treated with endocytic agent examples with or without the pretreatment of entospletinib at indicated concentrations were determined by western blotting assay.

[0070] Figure 7G shows that some of the trivalent endocytic agents (Examples 32 to 37) with higher binding affinity and more binding arms (valent) induce deeper BRD4 degradation than bivalent endocytic agents (Example 23) in HCC1806 TNBC or 22Rvl PCa cells under same conditions. Immunoblot for BRD4 after treatment of HCC 1806 or 22Rvl cells with DMSO vehicle control or endocytic agents.

[0071] Figure 7H shows that trivalent endocytic agent Example 35 with higher binding affinity and more binding arms (valent) induces deeper BRD4 degradation than bivalent endocytic agents (Example 23) in 22Rvl PCa cells under same conditions. BRD4 protein was examined by immunoblotting and BRD4 protein level was quantified by densitometry and normalized to the corresponding density of P-tubulin protein.

[0072] Figure 71 shows that trivalent endocytic agent Example 35 with higher binding affinity and more binding arms (valent) induces faster BRD4 degradation than bivalent endocytic agents (Example 23) in 22Rvl PCa cells. Immunoblot for BRD4 after treatment of 22Rvl cells with DMSO vehicle control or endocytic agents for indicated durations.

[0073] Figure 7 J shows that pretreatment of MG132 reverses the BRD4 degradation by endocytic agent Example 23, Example 35, or Example 36 in 22Rvl PCa cells. Protein levels of BRD4 in 22Rvl cells treated with endocytic agent examples with or without the pretreatment of MG132 at indicated concentrations were determined by western blotting assay.

[0074] Figure 7K shows that shCD36 reverses the BRD4 degradation by endocytic agent Example 23 or Example 35 in 22Rvl PCa cells. 22Rvl cells stably transfected with or without shCD36 were treated with endocytic agent examples at indicated concentrations, then the protein levels of BRD4 were determined by western blotting assay.

[0075] Figures 8A-8H show that introducing a chemical arm via cleavable bond or moiety augments agent uptake and therapeutic consequences.

[0076] Figure 8A shows that some of the endocytic agents conjugated with a chemical arm via cleavable bond (Examples 44 to 51) induce deeper AR protein degradation than endocytic agent ARV1 10 (Example 19) in MDA-MB-453 TNBC cells under same conditions. Different from the data in MDA-MB-453 cells, Examples 44 to 51 are incapable of inducing AR degradation in C4- 2 PCa cells, indicating the isoform and / or conformational diversity of membrane proteins exist in different cancer cell lines. Immunoblot for AR after treatment of MDA-MB-453 or C4-2 cells with DMSO vehicle control, enzalutamide or Example 19, or Examples 44 to 51

[0077] Figure 8B shows that some of the endocytic agents conjugated with diverse chemical arms via cleavable bond (Examples 46-4, 46, 52, or 53) induce deeper AR protein degradation than endocytic agent ARV110 (Example 19) in MDA-MB-453 TNBC cells under same conditions. Immunoblot for AR after treatment of MDA-MB-453 cells with DMSO vehicle control, enzalutamide, or endocytic agent examples.

[0078] Figure 8C shows that endocytic agent conjugated with a chemical arm via cleavable bond (Example 46) induces a deeper AR degradation than endocytic agent ARV110 (Example 19) in MDA-MB-453 TNBC cells under same conditions. AR protein was examined by immunoblotting and AR protein level was quantified by densitometry and normalized to the corresponding density of GAPDH protein.

[0079] Figure 8D shows that endocytic agent conjugated with a chemical arm via cleavable bond (Example 46) induces a faster AR degradation than endocytic agent ARV110 (Example 19) in MDA-MB-453 TNBC cells under same conditions. Immunoblot for AR after treatment of MDA- MB-453 cells with DMSO vehicle control, Example 19, or Example 46 for indicated durations.

[0080] Figure 8E shows that shCD36 partially reverses the AR degradation by endocytic agent Example 19 or Example 46 in MDA-MB-453 TNBC cells, suggesting the involvement of CD36 isoforms or other endocytosis-mediating membrane component(s) for endocytic agent pick up. MDA-MB-453 cells transfected with or without shCD36 were treated with endocytic agent examples at indicated concentrations, then the protein levels of AR and CD36 were determined by western blotting assay.

[0081] Figure 8F shows that pretreatment of MG132 reverses the AR degradation by endocytic agent Example 19 or Example 46 in MDA-MB-453 TNBC cells. Protein levels of AR in MDA- MB-453 cells treated with endocytic agent examples and with or without the pretreatment of MG132 at indicated concentrations were determined by western blotting assay.

[0082] Figure 8G shows that pretreatment of SYK inhibitor reverses the AR degradation by Example 19 or Example 46 in MDA-MB-453 TNBC cells. Protein levels of AR in MDA-MB-453 cells treated with endocytic agent examples and with or without the pretreatment of entospletinib at indicated concentrations were determined by western blotting assay. Figure 8H shows that some of the endocytic agents conjugated with a chemical arm via cleavable bond (Examples 54 to 61) induce deeper BRD4 protein degradation than bivalent endocytic agent Examples 23 in HCC1806 TNBC or 22Rvl PCa cells under same conditions. Examples 58 and 59 with an additional binding arm but without enhanced binding affinity (see Table 2) induce deeper BRD4 degradation. Some of the endocytic agents showed different potencies in triggering BRD4 degradation in HCC1806 and 22Rvl cells indicating the isoform and / or conformational diversity of membrane proteins exist in different cancer cell lines. Immunoblot for BRD4 after treatment of 22Rvl cells with DMSO vehicle control, Examples 23, or Examples 54 to 61.

[0083] Figures 9A-9G show that introducing a chemical arm via relatively stable bond or moiety augments agent uptake and therapeutic consequences.

[0084] Figure 9A shows that some of the polypharmacological trivalent endocytic agents Examples 63 to 70 induce deeper BRD4 protein degradation than polypharmacological bivalent endocytic agent Examples 62 in HCC1806 TNBC or 22Rvl PCa cells under same conditions. Some of the endocytic agents showed different potencies in triggering BRD4 degradation in HCC1806 and 22Rvl cells indicating the isoform and / or conformational diversity of membrane proteins exist in different cancer cell lines. Immunoblot for BRD4 after treatment of HCC1806 or 22Rvl cells with DMSO vehicle control or Examples 62 to 70.

[0085] Figure 9B shows that some of the polypharmacological trivalent endocytic agents Examples 71 to 77 with higher binding affinity and more binding arms (valent) induce deeper BRD4 protein degradation than polypharmacological bivalent endocytic agent Examples 62 in HCC1806 TNBC, 22Rvl, orLNCaP PCa cells under same conditions. Immunoblot for BRD4 after treatment of HCC1806, 22Rvl, or LNCaP cells with DMSO vehicle control, Examples 62, or Examples 71 to 77.

[0086] Figure 9C shows that some of the polypharmacological trivalent endocytic agents Examples 78 to 85 induce deeper BRD4 protein degradation than polypharmacological bivalent endocytic agent Examples 62 in 22Rvl PCa cells under same conditions. Immunoblot for BRD4 after treatment of 22Rvl cells with DMSO vehicle control, Examples 62, or Examples 78 to 85.

[0087] Figure 9D shows that polypharmacological trivalent endocytic agent Example 71 with higher binding affinity and more binding arms (valent) induces a deeper BRD4 degradation than polypharmacological bivalent endocytic agent Examples 62 in 22Rvl cells under same conditions. BRD4 protein was examined by immunoblotting and BRD4 protein level was quantified by densitometry and normalized to the corresponding density of p-tubulin protein.

[0088] Figure 9E shows that polypharmacological trivalent endocytic agent Example 71 induces a faster BRD4 degradation than polypharmacological bivalent endocytic agent Examples 62 in 22Rvl cells under same conditions. Immunoblot for BRD4 after treatment of 22Rvl cells with DMSO vehicle control, Example 62, or Example 71 at indicated concentrations.

[0089] Figure 9F shows that pretreatment of MG132 reverses the BRD4 degradation by polypharmacological endocytic agent Example 62, Example 71, or Example 79 in 22Rvl cells. Protein levels of BRD4 in 22Rvl cells treated with polypharmacological endocytic agent examples and with or without the pretreatment of MG132 at indicated concentrations were determined by western blotting assay.

[0090] Figure 9G shows that shCD36 in 22Rvl cells reverses the BRD4 degradation by polypharmacological endocytic agent Example 62 or Example 79 in 22Rvl cells. 22Rvl cells stably transfected with or without shCD36 were treated with polypharmacological endocytic agent examples at indicated concentrations, then the protein levels of BRD4 and CD36 were determined by western blotting assay.

[0091] Figures 10A-10D show that introducing a chemical arm augments agent uptake and enlarge therapeutic efficacy and safety window for agents.

[0092] Figure 10A shows that polypharmacological trivalent endocytic agent Example 28 or 29 demonstrates greater anti-tumor efficacy than polypharmacological bivalent endocytic agent Example 23 in breast cancer HCC1806-xenografted mice model. In the graph, the top curve represents vehicle; the second from top curve represents Example 23; the third from top curve represents Example 28; and the bottom curve represents Example 29.

[0093] Figure 10B shows that polypharmacological trivalent endocytic agent Example 28 or 29 has no visible toxicity in HCC 1806-xenografted mice model. The top curve represents vehicle; the second from top curve represents Example 23; the third from top curve represents Example 28; and the bottom curve represents Example 29.

[0094] Figure IOC shows that polypharmacological trivalent endocytic agent Example 28 or 29 demonstrates higher potency than polypharmacological bivalent endocytic agent Example 23 in reducing BRD4 protein levels in breast cancer HCC 1806 xenografts. Immunohistochemistry for BRD4 (brown to black) of a representative tumor of a mouse treated with vehicle, Example 23, Example 28, or Example 29. Scale bars: 20 pm.

[0095] Figure 10D shows that polypharmacological trivalent endocytic agent Example 36 demonstrates greater anti-tumor efficacy than polypharmacological bivalent endocytic agent Example 23 in prostate cancer 22Rvl-xenografted mice model. Meanwhile, polypharmacological trivalent endocytic agent Example 71 or 79 demonstrates greater anti-tumor efficacy than polypharmacological bivalent endocytic agent Example 62 in prostate cancer 22Rvl -xenograft mice model. In the graph, the top curve represents vehicle; the second from top curve represents Example 23; the third from top curve represents Example 62; the forth from top curve represents Example 36; the fifth from top curve represents Example 79; and the bottom curve represents Example 71.

[0096] Figure 10E shows that polypharmacological trivalent endocytic agent Example 23, 36, 62 or 71 has no visible toxicity in 22Rvl-xenografted mice model.

[0097] Figure 11 shows that endocytic agent conjugated with a chemical arm (Example 29) demonstrates higher solubility than endocytic agent Example 23 in water. After standing still for 24 hours, no precipitation was observed in Example 29 water solution and LC-mass analysis confirmed the stability of Example 29 in water solution.

[0098] DETAILED DESCRIPTION OF THE INVENTION

[0099] Disclosed herein are compounds comprising an endocytic agent or probe connected / or fused with an additional membrane binding moiety. The additional membrane binding moiety is selected to enhance the endocytic efficacy and / or efficiency via an endocytosis-mediating membrane component. Connection or fusion of an additional membrane binding moiety to the endocytic agent or probe allows for greater uptake of the endocytic agents across a membrane than the original agent or probe.

[0100] Disclosed herein are also the methods to screen and identify the endocytic agents including known chemicals with unknown endocytic mechanisms for cellular penetration.

[0101] A benefit of the present technology is that it provides mechanistic basis and methods of an alternative and simplified drug discovery and development process based on chemical endocytosis compared to the classical drug discovery and development based on passive diffusion. Endocytosis is a biological process by which all cells absorb large-sized and large volume fraction external materials, such as nutrients, viruses, and bacteria, by engulfing them with the cell membrane. This process is initiated with recruitment of substances (such as protein, fatty acids, and exosome particles) that bind with endocytosis-mediating membrane component(s), such as membrane receptors including transporters. Binding to the endocytosis-mediating membrane component(s) forms an endocytic pit or invagination of plasma membrane. Membrane internalization is completed by a scission process to release the early endosome inside the cell. After internalization, materials and endosome contents can be released intracellularly and endocytosis-mediating membrane component(s) may be recycled to cell surface for another rounds of delivery. Additionally, the substances and endocytosis-mediating membrane components can be released by cells to extracellular compartment in forms of extracellular vesicles (EVs) such as exosomes by exocytic progresses. Compared to passive diffusion driven by concentration gradient from high to low until reaching plateau where equilibrium is reached, higher efficacy and efficiency of endocytic processes is proven by over 95% some nutrients in our daily diet are trafficked through endocytosis and enter blood circulation (Deanna M. Minich, et al., Journal of Lipid Research, 38, 1709-1721, 1997). For example, as a multi-functional scavenger receptor with multiple ligands, CD36 abundantly expressed in the intestine on the luminal surface of enterocytes, facilitating CD36-mediated endocytosis as the dominant path for highly efficient absorption of a large scale of fatty acids in food (Vincenza Cifarelli, et al., Comprehensive Physiology, (8)2, 493-507, 2018; Helene Poirier, et al., European Journal of Biochemistry, 238(2), 368-373, 1996). Binding of fatty acids to CD36 activates its downstream kinases and initiate endocytosis, efficiently facilitating the uptake of supply through membrane barriers (Jian-Wei Hao, et al, Nature Communications, 11(1), 4765, 2020; Richard F. Collins, et al., Journal of Biological Chemistry, 284(44), 30288-30297, 2009). As revealed by the crystallization studies, a distinctive feature of CD36 is a large hydrophobic groove (residue 127-279) that spans most of the length of the protein ectodomain, allowing the binding and accommodation of fatty acids, such as stearic acid (Cl 8:0) and kink- shaped docosahexaenoic acid (C22:6) (Fu-Lien Hsieh, et al., Nature Communications, 7, 12837, 2016; Zineb Tarhda, et al., Bioinformatics and Biology Insights, 7, 369-373, 2013). Additionally, endocytosis not only plays many vital roles in normal cell physiology but also play significant roles in pathology, such as recycling membrane receptors for dysregulated signaling transduction and providing steady-increased influx of exogenous nutrient supplies for constitutive anabolism of cancer cells. For example, to thrive under stress, cancer cells upregulate the expression of CD36 for increased production of lipid mass and the generation of oncogenic signaling lipid in cancer cells. The increased expression of CD36 correlates with a poor prognosis in various cancer cells, such as lung squamous cells, glioblastoma, leukemia, prostate, bladder, and breast carcinomas (Gloria Pascual, et al., Nature, 541, 41-45, 2017; Tian Zhang, et al., Blood Cancer Discovery, 1(2), 198-213, 2020; Matthew J. Watt, et al., Science Translational Medicine, 11(478), eaau5758, 2019; AritroNath, et al., Scientific Reports, 5, 14752, 2015; James S. Hale, et al., Stem Cell, 32(7), 1746- 1758, 2014). However, for the cellular endocytosis of chemicals by targeting endocytosis- mediating membrane component(s) for effective and efficient uptake of therapeutic agents was unexploited. The chemical endocytic approach speeds-up the therapeutics development and expands the scope of therapeutic agents that can be effectively utilized for study, diagnosis, prevention, and treatment of subjects. Broadly, comparing with the current method that seeks the balance between contradictory parameters (stability, aqueous solubility, and membrane permeability) of an agent, medicinal chemistry methods based on chemical endocytosis in the present technology can simplify and revolutionize the process of drug discovery and development merely by improving the permeability via enhancing the efficacy and / or efficiency of endocytosis and adjusting metabolic stability of agents with retaining biological activity, as the solubility can be relatively easier improved by the salting or charge formation techniques in the art. In addition, a precision health care method based on the differential expressions of endocytosis-mediating membrane component(s) can be used for patient stratification for the personalized selection of agents and agents’ administration route.

[0102] A benefit of the present technology is that endocytosis allows for the uptake of agents across a membrane. Particularly, the present technology is useful in allowing for or increasing uptake of large and / or polar agents that can be effectively utilized for study, diagnosis, and treatment of subjects. As predicted by “the rule of 5’ (Ro5)” (Christopher A. Lipinski, et al., Advanced Drug Delivery Reviews, 46, 3-26, 2001), the most influential framework correlating the physicochemical properties of a given compound with its membrane permeability, drugs with smaller size (< 500 Da), lower polarity (topological polar surface area (tPSA) 140 A), and higher lipophilicity (but LogP should be 5) are more readily to across the cell membrane via passive diffusion. As the increasing of molecule size (especially M.W. > 700 Da), drugs presenting higher polarity (higher tPSA values) or lower lipophilicity (lower LogP values) could face a sharp decrease in passive diffusion through the cell membrane (Par Matsson, et al., Journal of Medicinal Chemistry, 60(5), 1662-1664, 2017; Cameron R. Pye, et al., Journal of Medicinal Chemistry, 60(5), 1665-1672, 2017). As disclosed in present disclosure, endocytic agents which 1) have increased binding affinity and / or binding valent with endocytosis-mediating membrane component(s), 2) allow for or increase dimerization or clustering of endocytosis-mediating membrane components (Laura Salavessa, et al., Proceedings of the National Academy of Sciences of the United States of America, 118(37), e2024893118, 2021; John Maringa Githaka, et al., Journal of Cell Science, 129(22), 4175-4189, 2016), or 3) allow for or increase conformational changes of endocytosis- mediating membrane component(s), can be better absorbed into cells via endocytosis.

[0103] As used in present technology, “clustering" or “cluster” refers to the process that endocytosis mediating membrane components assemble into nano- and micro-scale domains to govern biological processes, including but not limited to, cell adhesion, endocytosis, and immune responses. For example, upon binding with ligand(s), clustering of endocytosis mediating membrane proteins results in local protein crowding, and / or membrane conformational change(s), promoting membrane curvature and initiation of endocytic processes. It is understood that the “clustering" or “cluster” of endocytosis mediating membrane component) includes the dimerization, oligomerization, and multimerization of membrane components. The “clustering" or “cluster” of endocytosis-mediating membrane components can be homogeneous, heterogeneous, or in combination of homogeneous and heterogeneous mixtures at any ratio. It is also understood that the conformational changes of endocytosis-mediating membrane component(s) can be independent with and / or in associated with the “clustering" or “cluster” of endocytosis mediating membrane components.

[0104] Upon receiving input signals, such as ligand binding, chemical modification, or change in environment, membrane proteins undergo conformational changes and proteins with different conformations can exert diverse or distinctive biological functions. For example, after receiving binding stimulus, membrane protein can switch conformations and affect clathrin lattice growth, curvature, and endocytosis in cells (Kazuki Obashi, et al., 14, 732, Nature Communication, 2023). Conformational change of a membrane protein by ligand binding is dependent on factors including binding affinity and binding domain (Anna Vangone, et al., eLife, 4, e07454, 2015). Increasing binding valent for picking up additional binding domain even without enhancing binding affinity can trigger conformational change and further enhance endocytosis. A benefit of the present technology is that it allows for or enhances conformational changes of endocytosis-mediating membrane component(s) via structural modification of endocytic agents for enhanced endocytosis efficacy and / or efficiency, wherein the structural modification of endocytic agents include, but are not limited to, making charge molecules or salting techniques in the art, by forming multivalent endocytic agent via connecting charged, chargeable and other hydrophilic chemical arm(s), or by structural modification using medicinal chemistry strategies in the art including introducing reversible or non-reversible covalent bond(s) or moiety(moieties). Conformation changes of endocytosis-mediating membrane component(s) for enhanced endocytic efficiency and / or efficacy can be achieved via 1) increasing the binding affinity and / or binding valent of endocytic agents with endocytosis-mediating membrane component(s) and / or 2) increasing dimerization or clustering of endocytosis-mediating membrane component, through structural modification of endocytic agents. Also, the conformation changes of endocytosis-mediating membrane component(s) for enhanced endocytic efficiency and / or efficacy can be achieved via accommodating 1) endocytosis-mediating membrane component(s) environmental factors, such as pH values, salinity, oxygen gradient, carbon dioxide gradient, H2O2 gradient, nutrient gradient, and therapeutic compound gradient, 2) interaction(s) with membrane cofactor proteins, and / or 3) status of post translational modification of endocytosis-mediating membrane component s) and membrane cofactor proteins, such as ubiquitination, phosphorylation, palmitoylation, glycosylation, acetylation, and lipidation, through the utilization or structural modification of endocytic agents.

[0105] Another benefit of the present technology is that it allows for or increase agent’s permeability without sacrificing solubility and stability. Based on passive diffusion theory, common methods to allow for or increase agents’ permeability are to enhance the lipophilicity of compounds via structural optimizations on a case-by-case basis, making it readily dissolved in membrane phospholipids and rapidly diffused across cellular membranes, but at the expense of reduced solubility (due to reduced hydrophilicity), stability, and / or intrinsic biological activities. However, evidence shows structural optimizations to bRo5 agents to allow for or increase membrane passive permeability balanced with favorable metabolic stability and solubility are challenging or even impossible (Par Matsson, et al., Journal of Medicinal Chemistry, 60(5), 1662- 1664, 2017; Victoria G. Klein, etal., Journal ofMedicinal Chemistry, 64(24), 18082-18101, 2021). Comparing with the current methods which seek balance between contradictory parameters (stability, aqueous solubility, and membrane permeability) of a therapeutic agent, the present technology simplifies and revolutionizes the process of drug discovery and development merely by improving the metabolic stability and endocytic efficiency of chemical molecules, as aqueous solubility could be relatively easier achieved via such as salting technologies. The salt or charge formation can not only improve the solubility, but also can increase the binding affinity and / or binding valent to endocytosis mediating membrane component(s), to increase the cellular uptake spontaneously. In the meantime, increasing aqueous solubility decreases the first path metabolism and lowers the liability of stability. The present technology allows for improved solubility and stability profiles by increasing permeability via enhancing the endocytic efficiency / and or efficacy.

[0106] Another benefit of the present technology is that it allows for increasing solubility and / or binding affinity and / or binding valent to endocytosis mediating membrane component(s), dimerization or clustering of endocytosis-mediating membrane components, and / or conformational changes of endocytosis-mediating membrane component(s) for enhanced agent’s endocytic efficacy and / or efficiency by making charge molecules or salting techniques in the art, by forming multivalent endocytic agent via connecting charged, chargeable and other hydrophilic chemical arm(s), or by structural modification using medicinal chemistry strategies in the art including introducing reversible or non-reversible covalent bond(s) or moiety(moieties). The resulting endocytic agent can be present in cation or anion form in aqueous solution at the certain pH value and has increased both solubility and binding affinity and / or binding valent of an endocytic agent for picking-up the salt bridging, hydrogen binding interactions or reversible / non- reversible covalent bond formation with endocytosis-mediating membrane component(s) for enhanced endocytic efficacy and / or efficiency. Chargeable or hydrogen bond-forming moieties, such as lysine, arginine, cysteine, serine, threonine, lipid, and carbohydrates, are frequently found in the membrane components, such as transmembrane proteins, are more prone to bind with ionic agents or agents possessing ionic bond(s) via salt bridge(s), hydrogen bonding interactions or reversible covalent bond formation (Ondrej Kuda, et al., Journal of Biological Chemistry, 288(22), 15547-15555, 2013; Dante Necukai, et al., Nature, 504(7478), 172-176, 2013; Joanna S. G. Slusky, et al., Bioinformatics, 29(17), 2122-2128, 2013; Yibo Wang, et al., Journal of Physical Chemistry B, 125(8), 2124-2133, 2021; Anupam Bandyopadhyay, et al., Current Opinion in Chemistry Biology, 34, 110-116, 2016). Thus, for example, by structural modification using reversible covalent chemistry strategies in the art, including attaching or replacing any chemical bond(s) or moiety(moieties) within an agent with chemical bond(s) or moiety(moieties) that can form reversible covalent bond(s) with any nucleophiles in biology (including lysine, arginine, cysteine, serine, threonine, and carbohydrates) (Anupam Bandy opadhyay, et al., Current Opinion in Chemistry Biology, 34, 110-116, 2016), the resulting endocytic agents can have increased binding affinity and / or binding valent with endocytosis-mediating membrane component(s), allow for or increase dimerization or clustering of endocytosis-mediating membrane components, and / or allow for or increase conformational changes of endocytosis-mediating membrane component(s) for enhanced endocytic efficacy and / or efficiency. Alternatively, by structural modification including attaching or replacing any chemical bond(s) or moiety(moieties) within an agent with chemical bond(s) or moiety(moieties) that can form reversible or irreversible covalent bond(s) with any nucleophiles in biology, in combination with strategies including attaching or replacing any chemical bond(s) or moiety(moieties) within an agent with cleavable chemical bond(s) or moi ety(moi eties), the resulting endocytic agents can have increased binding affinity and / or binding valent with endocytosis-mediating membrane component s), allow for or increase dimerization or clustering of endocytosis-mediating membrane components, and / or allow for or increase conformational changes of endocytosis-mediating membrane component(s) for enhanced endocytic efficacy and / or efficiency, wherein cleavable chemical bond(s) or moiety(moieties) in resulting endocytic agents can be cleaved in cells or bodies, resulting the release of biological active endocytic agents from endocytosis-mediating membrane component(s) to exert any biological functions.. Hence, the present technology allows for endocytic agents to possess increased solubility and / or permeability spontaneously via endocytosis for disease diagnosis and treatment.

[0107] Another benefit of the present technology is that it allows for adjusting metabolic stability of an endocytic agent. To meet agent stability requirement for treatment and diagnosis of subjects, the methodology of current disclosure, include but not limit to, adding or removing substituents, fragment replacement, cyclization, scaffold-hopping, bioisosterism, linkerology (linker-activity relationship studies), prodrug, or lowing the overall LogP values. In certain embodiments, the method refers to deuterium and / or fluorine replacement of any hydrogen atoms on endocytic agent. In certain embodiments, the method refers to introducing substitutes to reduce metabolism at the soft spots in the agent. These methods allow for balancing between endocytic agent’s intended functionality, binding affinity to endocytosis-mediating membrane components and metabolic stability for disease treatment. In the classical drug discovery and development campaigns, polar or hydrophilic compounds were determined poorly permeable, as high polarity or hydrophilicity forge compounds hardly dissolved in membrane lipid bilayers and diffused across cellular membranes. As the polarity increases, the permeability of polar or hydrophilic compounds drops off drastically. However, protein, especially membrane protein surface generally contains hydrophilic exterior facing amino acids and some of post-translation modifications (PTMs) moieties, such as glycan groups, tend to be located on the surface of membrane proteins capable to bind with polar compounds via salt bridge or hydrogen bond formations, including but not limited to, inorganic compounds, chelation, metal-based compounds, and / or polar organic compounds such as phosphatase inhibitors, peptide-based compounds, whilst the membrane protein often has a small molecular binding region. Thus, a benefit of the present technology is that endocytosis is a general pathway for the uptake of endocytic agents with no limitations on endocytic agent’s molecular weight, polarity and lipophilicity. Particularly, a benefit of the present technology is that endocytosis can facilitate the cellular uptake of hydrophilic agents, including but are not limited to, inorganic compounds, chelation, metal-based compounds, and / or polar organic compounds such as phosphatase inhibitors and peptide-based compounds. For example, by appropriately linking a small molecule membrane protein binder with a phosphatase inhibitor or insulin, the salt bridge(s) or hydrogen bind(s) can be formed between the phosphatase inhibitor or insulin with the surface amino acids of membrane protein for inducing the conformational change in the endocytic process. With the technology, polar compounds can be absorbed and can be used via oral administration. By introducing a facilitated diffusion glucose transporter (GLUT) binding moiety onto a phosphatase inhibitor with a cleavable or non-cleavable bond or moiety, the resulting endocytic phosphatase inhibitor agent can be used via oral administration, due to the increased binding affinity and / or binding valent to endocytosis mediating membrane component(s), and / or enhanced dimerization or clustering of endocytosis-mediating membrane components, and / or conformational changes of endocytosis-mediating membrane component(s) for endocytosis. By introducing a CD36 binding moiety onto insulin molecule with a cleavable or non-cleavable bond or moiety, the resulting endocytic insulin agent can be used via oral administration, due to the increased binding affinity and / or binding valent to endocytosis mediating membrane component(s), and / or enhanced dimerization or clustering of endocytosis-mediating membrane components, and / or conformational changes of endocytosis-mediating membrane component(s) for endocytosis. Insulin stability can be elevated from the exosome formation after endocytosis and endosomal escape.

[0108] As the driving force for the endocytic uptake of agents is the binding between endocytic agents and endocytosis-mediating membrane component(s) and endocytosis is a process that can be independent from passive diffusion, a benefit of the present technology is that it is useful in allowing endocytic agents across cellular membranes and / or body barriers via endocytosis alone or in combination with other mechanisms, including but not limited to, passive diffusion, facilitated diffusion, transporter-mediated influx and / or efflux, and paracellular transport.

[0109] A benefit of the present technology is that it expands the current concept of polypharmacology from seeking combination or synergistic effects of an agent’s intrinsic activities on multiple disease-related targets or pathways (Rajan Chaudhari, et al., Expert Opinion on Drug Discovery, 15(9), 1025-1044, 2020) to the combination or synergistic effects on disease-related targets or pathways and binding properties with endocytosis-mediating membrane component(s). The synergistic or additive effects on disease-related targets or pathways and endocytosis- mediating membrane component(s) can lower the requirements of endocytic agent’s dose for therapeutic purposes; as increased binding affinity and / or binding valent of endocytic agent’s with endocytosis-mediating membrane component(s), dimerization or clustering of endocytosis mediating membrane components, and / or conformational changes of endocytosis-mediating membrane component s) can result in enhanced endocytic efficacy and / or efficiency and be better absorbed via endocytosis, which can make up the insufficient intrinsic activities of some endocytic agent on disease-related targets or pathways for disease diagnosis and treatment.

[0110] As the binding nature of an endocytic agent with endocytosis-mediating membrane component(s) can be independent from its binding ability with intrinsic pharmacological target(s), it allows for an endocytic agent to be a polypharmacological compound which spontaneously possesses binding affinities to both endocytosis-mediating membrane component(s) for endocytosis and pharmacological target(s) for intrinsic pharmacological activities. Hence another benefit of the present technology are methods to identify or generate novel endocytic agents via polypharmacologically endocytic and functional targeting spontaneously. One method is to make structural modification directly on an agent, making it readily bind with both endocytosis- mediating membrane component(s) for endocytosis and pharmacological target(s) for intrinsic pharmacological activities. Wherein the process to identify or generate novel polypharmacologically endocytic and functional targeting agents, classical medicinal chemistry in the art is used by monitoring the activities for both endocytic and biological target modulation efficiencies spontaneously. For example, to develop an endocytic BCL-2 inhibitor, an assay to measure CD36 binding affinity by SPR and an assay to measure BCL-2 targeting efficiency by SPR can be used in the classical medicinal chemistry optimization process in the art to identify or generate endocytic and BCL-2 dual targeting agents. Multiple chemical arms in ligands aid in improving binding affinity of both CD36 and BCL2, and / or efficient up-take. Also, multivalent agents or agents with increased binding valent can enhance the dimerization or clustering of membrane components and / or induce the conformational changes of membrane components to activate the endocytic cascade. Additionally, as revealed by crystallization studies and molecular modeling, bell-shaped ligands or macro ligands containing multiple side chains could give good alignments with the binding pocket of CD36 (residue 127-279). Hence, another method in present disclosure to identify or generate novel endocytic agents with enhanced polypharmacological natures, including endocytic efficacy and / or efficiency and targeting biological functions, is merely by connecting or fusing additional chemical arm(s) with binding affinity to the endocytosis- mediating membrane component(s) and / or target protein(s), via either surface or within pocket interaction, onto endocytic agent via cleavable or non-cleavable chemical bond or linker unit(s) to make it as a multivalent compound, the resulting multivalent endocytic agent can 1) have increased affinity and / or binding valent with endocytosis-mediating membrane component(s), and / or increased activity on biological target(s), 2) allow for or increase of dimerization or clustering of endocytosis-mediating membrane components, or 3) allow for or increase conformational changes of endocytosis-mediating membrane component(s) for enhanced endocytic efficacy and / or efficiency and be better absorbed via endocytosis and still have retained or increased targeting biological functions. The chemical arms with affinity to CD36 and biological targets can be built up by the library synthesis and follow-up by screening. Comparing with the current methods in medicinal chemistry which adjust one intrinsic nature of agents via structural modifications but inevitably affects other(s), the present technology allows for the increased permeability of an endocytic agent without sacrificing or even with enhancing solubility, stability and its intrinsic pharmacological activities for disease study, diagnosis, prevention, and treatment.

[0111] As the results of endocytosis, foreign materials can be picked up by cells and released from the (donor) cells in forms of extracellular vesicles (EVs), including but not limited to exosomes, apoptotic bodies, and microvesicles (MVs) or ectosomes, via exocytosis or membrane fusion, wherein the foreign materials can exist at any sites of extracellular vesicles. The endogenous extracellular vesicles can be up-taken by any (acceptor) cells via endocytosis, membrane fusion, or transcytosis resulting transport of the foreign materials across more than one cell layers (Ravi Shah, et al., TheNew England Journal ofMedicine, 8(379), 958-966, 2018; Oscar P. B. Wiklander, et al., Science Translational Medicine, 11(492), eaav8521, 2019; Raghu Kalluri, et al., Science, 367(6478), eaau6977, 2020). Hence, another benefit of the present technology are the methods to transport endocytic agents across more than one cell layers in body, wherein the endocytic agents can be up-taken by cells via endocytosis and can be released in forms of free endocytic agent molecules and / or endocytic agents-vesicle complexes (namely, exocytic vesicles), which comprise endocytic agents and lipid bilayer vesicles including extracellular vesicles, and the exocytic vesicles can be up-taken by any (acceptor) cells for the action via endocytosis or membrane fusion for any purpose of applications. Particularly, an additional benefit of the present technology is a “one-step” method to generate and use of exocytic vesicles in human and animals, wherein, without further processes including agents or vesicles isolation, cells bodies pick up endocytic agents via endocytosis and sequentially secrete out exocytic vesicles via exocytosis, and the resulting endogenous exocytic vesicles can be directly distributed into any tissue(s) and used by bodies including brain for any purposes.

[0112] Another benefit of the present technology are the methods of making, isolating, and using exocytic vesicles in vitro and / or in vivo for any purposes. Particularly, one method to generate exocytic vesicles is that cells in an animal body can up-take endocytic agents via endocytosis and then secrete out exocytic vesicles via exocytosis spontaneously. Another method to generate functional exocytic vesicles is that endocytic agent molecules can be linked with extracellular vesicles and / or exocytic vesicles via covalent or non-covalent bond(s) in situ. Another method to generate exocytic vesicles is to load endocytic agent into exocytic vesicles in vitro by any technology in the art. Another method is to isolate the exocytic vesicles from cells, body fluids, tissues, organs, products, or cultural mediums by any extracellular vesicles’ isolation techniques in the art.

[0113] As extracellular vesicles (EVs) carry soluble materials, such as soluble cytokines, allowing for adequate solubility of EVs in aqueous solution (Ana Paula Ramos, et al., Journal of Extracellular Biology, 1(1), e34, 2022). Hence, another benefit of the present technology are the methods of making and using exocytic vesicles in vitro or in vivo for increased aqueous solubility of endocytic agents for any purposes. For example, to have aqueous solubility in animal bodies, a method to increase the aqueous solubility of agents is to allow for or increase the uptake of endocytic agents via above mentioned methods, resulting in the increased loading of endocytic agents into exocytic vesicles and / or releasing of exocytic vesicles via cell endocy tic / exocytic process. The endocytic agents can be dissolved in aqueous solution in forms of exocytic vesicles.

[0114] Another benefit of the present technology is methods to adjust the endocytic agents’ absorption, distribution, metabolism, and excretion (ADME) properties in animal bodies. For example, to extend the residency time and / or half-lives of given endocytic agents in animal bodies, a method of present technology is to allow for or increase the uptake of endocytic agents by endocytosis via above mentioned methods to allow for or increase the loading of endocytic agents into exocytic vesicles via cell endocytosis and the releasing of exocytic vesicles, for reducing or preventing endocytic agents from the first pass metabolism and / or excretion. To improve the brain penetration of agents, a method of present technology is to allow for or increase the uptake of endocytic agents by endocytosis via above mentioned methods to allow for or increase the loading of endocytic agents into exocytic vesicles via cell endocytosis and the releasing of exocytic vesicles, for reducing or preventing endocytic agents from the blood-brain barrier efflux transport.

[0115] Another benefit of the present technology is methods to evaluate and / or determine endocytic agents’ ADME properties in the drug discovery and development process comprising a process or step for isolating and / or lysing exocytic vesicles from a sample. For examples, in certain embodiments, the endocytic agent concentration in blood or tissues after endocytic agent administration includes the free endocytic agent molecules in blood or tissues and the endocytic agent molecules entrapped in exocytic vesicles, such as exosomes, in blood or tissues. To release the endocytic agent from exocytic vesicles for endocytic agent detections, any technology and stills in the art for isolating and / or lysing EVs is applicable to isolate and / or lyse exocytic vesicles in present disclosure.

[0116] Another benefit of the present technology are the methods to reduce the toxicity of agents / drugs by making and using exocytic vesicles with endocytic agents in vitro or in vivo. Hematological toxicities represent the main toxicity of the cytotoxic agents (Etienne Chatelut, et al., Investigational New Drugs, 21, 141-148, 2003). For example, most of the PARE inhibitors in clinic are observed with hematological toxicities, including neutropenia, anemia, thrombocytopenia, fatigue, and bleeding, due to toxicity of PARP inhibitors to platelets, white blood cells, and red blood cells (Yamin Shu, et al., Cancer Medicine, 12(3), 3365-3375, 2023). As disclosed in present disclosure, endocytic agents can be up-taken by and released from the cells in forms of exocytic vesicles, such as endocytic agents-loading exosomes, wherein the endocytic agents can exist in exosomes, preventing or reducing the direct contact between free endocytic agent molecules and blood cells. Additionally, as the natural particles, exosomes have unique properties, such as innate stability and low immunogenicity(Raghu Kalluri, et al., Science, 367, eaau6977, 2020). Hence, another benefit of the present technology is the methods of using endocytosis-mediating membrane component in cells and tissues and exocytosis process to release exocytic vesicles to lower the toxicity of drugs by preventing or reducing the toxicity of agents / drugs. For example, after endocytic agents’ oral administration, the cells in gastrointestinal tract can pick up endocytic agents via endocytosis and sequentially secrete out exocytic vesicles into circulatory system, and the resulting exocytic vesicles reduce hematological toxicities comparing with the free endocytic agent molecules (Ashish K. Agrawal, et al., Nanomedicine, 13, 1627-1636, 2017). Particularly, an additional benefit of the present technology is the methods to lower toxicity of agent by allowing for endocytosis or adjusting endocytic efficacy and / or efficiency via any structural modification methods disclosed in present technology to adjust the endocytic efficacy and / or efficiency, loading of endocytic agents into exocytic vesicles, and / or releasing of exocytic vesicles in different cells and tissues.

[0117] Another benefit of the present technology is that the different expression of endocytosis- mediating membrane component(s) in cells and tissues can be used for increasing potency and reducing toxicity of endocytic agents. Alteration of metabolic activities has been shown to support the malignant properties of cancer cells (Ralph J. DeBeradinis, et al., Science Advances, 2(5), el600200, 2016). For example, recently studies demonstrated that high CD36 expression plays a critical role in tumor initiation, development, invasion, and metastasis by fueling tumor cells with increased nutrients supply, such as fatty acids and lipids (Gloria Pascual, et al., Nature, 541, 41- 45, 2017; Matthew J. Watt, et al., Science Translational Medicine, 11(478), eaau5758, 2019). Hence, targeted delivery of agents by employing the over-expression of endocytosis-mediating membrane component on cancer cells or upregulated nutrient uptake via endocytosis in cancerous cells can minimize potential toxicity on normal cells and enlarge therapeutic window for agents. While examples in this disclosure demonstrate that CD36-mediated endocytosis mediates endocytic agents’ absorption into cells, other endocytosis-mediating membrane component(s) described in, but not limited to (Sara Sigismund, et al., Nature Review Molecular Cell Biology, 22, 625-643, 2021), for examples scavenger receptors (SRs), endothelial cell protein C receptor (EPCR), fatty acid binding protein (FABPpm), fatty acid transport proteins (FATPs), free fatty acid receptor 1 (GPR40) and epidermal growth factor receptor (EGFR), facilitated glucose transporters (GLUTs), including all variants, mutations, splice variants, indels and fusions can be employed for agents’ uptake via endocytosis. Also, other chemical modifications on endocytic agents can give stronger binding affinities or more binding valent with endocytosis-mediating membrane component(s), allow for or increase dimerization or clustering of endocytosis- mediating membrane components, and / or allow for or increase conformational change of endocytosis-mediating membrane component(s) for enhanced endocytic efficacy and / or efficiency can serve as the platform(s) for up-takes of especially eRo5 and bRo5 drugs with enhanced efficacy and / or efficiency. Thus, the different expression of endocytic membrane proteins can be used for patient stratification for endocytic drugs.

[0118] Another benefit of the present technology is that the different expression of endocytosis- mediating membrane component(s) in cells and tissues can be utilized for a special delivery system such as topical, inhaled, intraperitoneal, intravenous, microinjection, and oral delivery. For example, owing to the abundant expression of endocytosis-mediating membrane component s), such as CD36 or GLUTs, in the intestine on luminal surface of enterocytes (Fatiha Nassir, et al., Journal of Biological Chemistry, 282(27), 19493-19501, 2007), with appropriate optimization for the affinity to endocytosis-mediating membrane component(s) and metabolic stability, endocytosis agents, especially those endocytosis agents lie in eRo5 and bRo5 chemical spaces, will bear acceptable oral bioavailability. Another benefit of the present technology are administrations of endocytic agents by any appropriate routes, including by oral (including buccal or sublingual), topical (including buccal, sublingual, transdermal, or eye drop), inhalation, parenteral (including subcutaneous, intramuscular, intravenous, or intradermal) route, intraperitoneal injection, and microneedle patches.

[0119] Another benefit of the present technology is that the conformation diversity of endocytosis- mediating membrane component(s) can be used for structure design and modification of endocytic agents. Membrane proteins possesses various conformation states in different cells or under different conditions, including but are not limited to cell size and volume, cellular microenvironment, and posttranslational modification status (Raghavendar Reddy Sanganna Gari, et al., Nature Communications, 12, 4363, 2021; Diego del Alamo, et al., eLife, 11, e75751, 2022; Alex R Terry, et al., Cell Metabolism, 35, 1-7, 2023). Different conformations of membrane protein exhibit distinctive sensitivities to ligands (Dante Necukai, et al., Nature, 504(7478), 172- 176, 2013; Fu-Lien Hsieh, et al., Nature Communications, 7, 12837, 2016). Hence, a benefit of the present technology is design or structural modifications of endocytic agents using medicinal chemistry strategies in the art or utilization of endocytic agents to target specific conformations(s) of endocytosis-mediating membrane component(s) for any purposes. For example, CD36 protein locating on the membranes of normal cells and cancer cells can adopt different conformations. It is reasonable to take advantage of the conformation diversity of CD36 on normal cells and cancer cells for the structural design or modification of endocytic agents using medicinal chemistry strategies in the art or selection of endocytic agents to target the specific conformation (s) of CD36 on cancer cells for enhanced anti-tumor efficacy and reduced toxicity. As protein conformational switches alter their shape and biological functions upon receiving an input signal, such as ligand binding, chemical modification, or change in environment (Matin Dutertre, et al., Perspective in Pharmacology, 295(2), 431-437, 2000), a benefit of the present technology is to take advantage of any approaches in the art, such as application of mechanical, electrical, heat, cold, light, or radiation stimulation and / or the presence of one endocytic agent, to change conformations of endocytosis-mediating membrane component(s), resulting in the alterations of biological events in cells and / or sensitivity of the endocytosis-mediating membrane component(s) to endocytic agent.

[0120] Another benefit of the present technology is that the differential expressions and / or conformations of endocytosis-mediating membrane component(s) can be used for precision health care for individuals using present endocytic agent. Meanwhile, the feedback of endocytic agents’ treatments in clinic can be used to adjust the route for administrations of endocytic agents and / or improve outcomes of endocytic agents’ treatments. Studies evidence the existences of differential expressions of membrane component(s) among individuals, cells and tissues in one individual, and different disease stage in one individual (Roy L. Silverstein, et al., Science Signaling, 2(72), re3, 2009; Aritro Nath, et al., Scientific Reports, 4(6), 18669, 2016; Matthew J. Watt, et al., Science Translational Medicine, 11(478), eaau5758, 2019). In addition, membrane protein possesses various conformational states and / or posttranslational statues on different cells or under different conditions, resulting in distinctive sensitivities of membrane proteins to ligands. The present disclosure demonstrates that endocytosis is a pathway for cellular up-take of endocytic agents, and endocytosis-mediating membrane component(s), such as CD36, has different expressions and / or structural conformations in cells and / or tissues in different patients (Vincenza Cifarelli, et al., Comprehensive Physiology, (8)2, 493-507, 2018; Helene Poirier, et al., European Journal of Biochemistry, 238(2), 368-373, 1996). Hence, a benefit of the present technology is to stratify patients for the personalized selection of endocytic agents and endocytic agents’ administration route, wherein the endocytosis-mediating membrane component(s) can be used as biomarker(s) in cells and / or tissues and the different expressions and / or conformations of endocytosis-mediating membrane component(s) in patient’s cells and / or tissues are used for selection and / or administration of endocytic agents to treat a subject from patient stratification, dosage route and dosage selection. The diversity of genetic profiling widely exists among different cells, tissues, and bodies. It is reasonable to take advantage of differences of genetic profiling among cells, tissues, and bodies to move biological active agents within drug discovery and development value chain. As the first PROTAC targeting androgen receptor (AR) into clinical trials, although ARV- 110 was able to completely degrade AR and most of its point mutants in the preclinical studies, patients with tumors harboring AR T878 or H875 point mutations were found to be particularly sensitive to the ARV110 treatment in clinical studies (Xin Gao, et al., Journal of Clinical Oncology, 40(6,) suppl. 017, 2022). As we demonstrated all these agents / drugs / vesicles as endocytic agents, including but not limited to agents disclosed here in examples (Table 1), the precise selection of patents with high expression and / or ligand-sensitive conformation of endocytosis-mediating membrane component(s), such as CD36 in disease tissues and / or absorption tissues such as intestine, can increase the clinical benefit of endocytic agents for patients.

[0121] Another benefit of the present technology is the endocytic agents can be used for the study, diagnosis, prevention, and treatment of any subjects, including but not limited to, aging and age- related diseases and conditions, body weight management, cancers, central nervous systems (CNS) diseases and conditions, cardiovascular diseases (CVDs), diabetes mellitus, eye diseases and conditions, hypertension, immune system diseases, infections, inflammation and inflammation- associated diseases and conditions, obesity and obesity-associated diseases and conditions, respiratory diseases and conditions, and skin diseases and conditions. Due to the barriers in human body against foreign materials, drug discovery for the treatment of subjects, especially CNS or skin diseases and conditions, has been challenging (Sung Min Pyo, et al., Skin Pharmacology and Physiology, 32, 283-293, 2019; William M Pardridge, NeuroRX, 2(1), 3-14, 2005). After endocytosis and exocytosis results in the release of the extracellular vesicles (EVs) and free nutrients by exocytosis process (Marcel Grapp, et al., Nature Communications, 4, 2123, 2013; Raghu Kalluri, et al., Science, 367(640), 2020), which facilitates the penetration of nutrients in free forms and / or EVs through membrane barriers, such as blood-retinal barrier (Monica Diaz- Coranguez, et al., Vision Research, 139, 123-137, 2017), lung endothelial and epithelial barrier (Mikihisa Takano, et al., Expert Opinion on Drug Delivery, 12(5), 813-825, 2015; Julia Voigt, et al., Proceedings of the National Academy of Sciences, 111(8), 2942-2947, 2014), skin barrier (Noriaki Nagai, et al., International Journal of Molecular Sciences, 19(7), 2138, 2018) and brain- blood-barrier (Mathew W. Smith, et al., Journal of Drug Targeting, 14(4), 191-214, 2006). For example, by binding with receptors or proteins expressed in the brain-blood-barrier, such as scavenger receptors (e.g., CD36), major facilitator superfamily domain-containing protein 2 (Mfsd2a), flotillin-1, flotillin-2, glucose transporter 1 (GLUT1), glutathione transporter, amino acids transporters (e.g., L-type amino acid transporter 1, LAT1), transferrin receptor, lactoferrin receptor, low density lipoprotein receptor, nicotinic acetylcholine receptor, insulin receptor, insulin-like growth factor receptor, integrin (e.g., aVP3 integrin), and / or CD13 / APN receptor, endocytic agents can be absorbed into brain endothelial cells, be transported through brain-blood- barrier via endocytosis, and reach the brain parenchyma in forms of free endocytic agents, exocytic vesicles, or the mixture of free endocytic agents and exocytic vesicle via endocytosis / exocytosis route. Additionally, due to endocytosis, the endocytic agents are absorbed into cells and located in intracellular organelles and / or vesicles, such as endosomes and multivesicular bodies, reducing or preventing pumping endocytic agents out of the cells by efflux transporters, such as P-glycoprotein (P-gp), breast cancer resistant protein (BCRP) and the multidrug resistance-associated proteins MRP1, MRP3, MRP4 and MRP6. It is understood that taking advantage of the expression of endocytosis-mediating membrane component(s) in membrane barriers and / or any structural modifications on agent in present disclosure can result in enhanced efficacy and / or efficiency of endocytosis of endocytic agents and transport of endocytic agents across membrane barriers for any purposes. Hence, a particular benefit of the present technology is that, endocytic agents can be used for the study, diagnosis, prevention, and treatment of eye, respiratory, skin, and CNS conditions and diseases, as after membrane component-mediated endocytosis, endocytic agents can be transported through membrane barriers in forms of free endocytic agents, exocytic vesicles, or the mixture of free endocytic agents and exocytic vesicle at any ratio via endocytosis / exocytosis route.

[0122] Another benefit of the present technology is a method to identify literature-known bioactive compounds that can be up-taken by cells via endocytosis. Wherein the method includes adjusting (such as, increasing, decreasing, or deletion) the expression of endocytosis-mediating membrane component(s) in cells, tissues, and / or bodies by endocytosis-mediating membrane component(s) gene editing, knockdown, or silencing, followed by comparing the activities of bioactive compounds in cells, tissues, and / or bodies with and without endocytosis-mediating membrane component(s) gene editing, knockdown, or silencing. Comparing with the activities of compounds in cells, tissues, and / or bodies without endocytosis-mediating membrane component(s) gene editing, knockdown, or silencing, the compounds which are up-taken via endocytosis have significantly increased or decreased bioactivity in cells, tissues, and / or bodies with endocytosis- mediating membrane component(s) editing, knockdown, or silencing.

[0123] Another benefit of the present technology are methods of using identified endocytic agents as probes to identify the biological target(s) in cells and bodies by any technology and stills in the art. For example, the identified endocytic agent which possess biotin, fluorescent, Halo-tag ligand(s), SNAP -tag ligand(s), CLIP -tag ligand(s), or chemical bond(s) or moiety(moieties) that can have covalent bond(s) formation with any biological target(s) are particularly suitable for identifying biological target(s) of endocytic agent and derivatives, in combination with technology and stills including microscope imaging, immunophenotyping, immunoprecipitation, flow cytometry, fluorescent-activated cell sorting (FACS), fluorescence resonance energy transfer (FRET), and / or omics (including genomics, epigenomics, transcriptomics, proteomics, and metabolomics) analysis. Particularly, a benefit of the present technology is methods to identify the endocytosis-mediating membrane target(s). A method to identify the endocytosis-mediating membrane target(s) may use a labeled endocytic agent followed by agent administration. Detecting and identification techniques include microscope imaging, immunoprecipitation, immunophenotyping, flow cytometry, fluorescent-activated cell sorting (FACS), fluorescence resonance energy transfer (FRET), omics (including genomics, epigenomics, transcriptomics, proteomics, and metabolomics) analysis, and / or positron emission tomography (PET) scan using computed tomography (CT) or magnetic resonance imaging (MRI). For example, connecting a labeling probe, such as biotin, onto an endocytic agent at any appropriate site results in a biotin- labeled endocytic agent. Subsequently, the endocytosis targeting the endocytosis-mediating membrane component(s) of the endocytic agent can be identified by culturing cell with biotin- labeled endocytic agent, isolating membrane proteins, followed by immunoprecipitation, FACS, omics analysis, western-blot confirmation. Another method is to use any genomic scanning technologies in the art to identify the endocytosis-mediating membrane target(s). For example, comparisons of gene expression between cells, tissue, or bodies with different sensitivities to certain endocytic agents can be used to identify the endocytosis-mediating membrane target(s). Another method is to use any gene-editing technology in the arts to identify the endocytosis- mediating membrane target(s), wherein the gene-edited cells, tissues, or bodies can be more sensitive or resistant to endocytic agent treatment. For example, cells with gene expression inhibition and activation, through CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa), respectively, act differently or complementarily to certain endocytic agent treatments, which can be used to identify the endocytosis-mediating membrane target(s).

[0124] As used herein, “gene editing" refers to the process of artificially introducing a genetic modification. Genetic engineering can be performed at the DNA, RNA, or epigenetic level. Genetic modifications include: (i) deletion of an endogenous gene; (ii) introduction of a recombinant nucleic acid encoding a wild-type or mutant form of an endogenous or exogenous protein; (iii) introduction of an RNA molecule (e.g., small-interfering RNA (siRNA), short hairpin RNA (shRNA), anti-sense RNA, and micro RNA (miRNA)) that interferes with the functional expression of a protein; or (iv) altering the promoter or enhancer elements (i . e. , regulatory elements) of one or more endogenous genes. It is understood that item (ii) includes replacement of an endogenous gene (e.g., by homologous recombination) with a gene encoding an altered or entirely different protein, and that item (iv) includes modification or manipulation of the regulatory regions of a target gene or of any region that is contiguous with a target gene (e.g., up to 5 KB on either side of the target sequence). Genetic engineering also includes altering an endogenous gene to produce a protein having additions (e.g., a heterologous sequence), deletions, or substitutions (e.g., mutations such as point mutations; conservative or non-conservative mutations). Mutations can be introduced specifically (e.g., by site-directed mutagenesis or homologous recombination) or can be introduced randomly (e.g., chemically mutagenized). Thus, genetic modifications may modulate a gene in several ways, such as increased-expression, increased function, reduced- expression, reduced function, or gene knockout. Exemplary methods include, but are not limited to, RNA-based RNA interference including small interfering RNA (siRNA) and short hairpin RNA (shRNA), DNA-based RNA interference including antisense oligonucleotides, and CRISP- mediated gene genome editing techniques.

[0125] Another benefit of the present technology is that different microenvironmental factors of cells and tissues can be used as modulator(s) when using or selecting present endocytic agent for any purposes, wherein the microenvironment of cells and tissues include but are not limited to, pH values, salinity, oxygen gradient, carbon dioxide gradient, H2O2 gradient, nutrient gradient, and therapeutic compound gradient. For example, due to the acidic environment of cancer cells, endocytic agents with basic group(s) can be used for tumor-targeted delivery for enhanced therapeutic functions and lower toxicities. Similarly, endocytic agents with basic amino group(s) can be used for enhanced BBB (blood brain barrier) localization for higher therapeutic functions and lower toxicities.

[0126] Another benefit of the present technology is that the expressions of endocytosis-mediating membrane component(s) with diverse conformations, isoforms (or variants), and / or post- translational modifications can be used as modulator(s) when using or selecting present endocytic agent for any purposes. Another benefit of the present technology is that the expressions of cofactors that form complex with endocytosis-mediating membrane component can be used as modulator(s) when using or selecting present endocytic agent for any purposes. Membrane proteins have diverse conformations under different environments and protein with diverse conformations exhibits various affinities to ligand(s) or other protein(s) and biological functions. Membrane proteins isoforms can have unique expression difference among cells and individuals and / or functions. Additionally, the diversity of glycosylation status in membrane protein can produce complex pleiotropy, where unique modifications on one glycosylation site may alter function or recognition within a specific cellular context but may cause other effects or be functionally silent in other contexts. Thus, for example, by targeting the specific conformations, isoforms, glycosylation, palmitoylation, or phosphorylation status of endocytosis-mediating membrane component or cofactors in cancer cells, endocytic agents can be used for enhanced therapeutic functions and lower toxicities in cancer treatments.

[0127] Another benefit of the present technology is that the endocytic agent binds with endocytosis-mediating membrane component(s) and triggers the internalization of membrane via endosome / lysosome pathway for digestion. The present technology is useful in utilizing endocytic agent(s) for delivering disease-related endocytosis-mediating membrane component(s) or extracellular materials into cells via endocytosis and destruct or degrade the disease-related endocytosis-mediating membrane component(s)or extracellular materials via endosome / lysosome system. The disease related endocytosis-mediating membrane component(s)or extracellular materials include but not limit to membrane proteins or extracellular proteins, carbohydrates, lipids, pathogens, particles, virus, fungi, protozoa, bacteria, vectors, cell debris, and another cell.

[0128] Another benefit of the present technique is that it allows for activating the endocytic process or increasing expression of endocytosis-mediated membrane component(s) via regulating input signals, such as the binding of endogenous or exogenous substances with a membrane protein, for enhanced endocytic absorption of endocytic agents. For example, the binding of insulin with insulin receptor on the membrane activate the endocytic cycling of endocytosis-mediating membrane component(s) and / or increase the expression / or relocation of endocytosis-mediating membrane component(s) such as GLUTs, resulting in the enhanced absorption of endocytic agents via endocytosis. It can be understood that endocytic agents can bind with endocytosis-mediating membrane component(s) and non-endocytosis-mediating membrane component(s) simultaneously for activating the endocytic process or increasing expression of endocytosis-mediated membrane component(s) and enhancing absorption via endocytosis.

[0129] As shown in Figure 1A, disclosed herein are endocytic agents, or salts thereof, which can bind with endocytosis-mediating membrane component s) and absorbed into cells via endocytosis. Also disclosed in Figure 1 A are methods in drug discovery to allow for or adjust an agent’s binding affinity and / or valent with endocytosis-mediating membrane component(s) for enhanced endocytic efficacy and / or efficiency.

[0130] In Figure IB, disclosed are the absorption mechanisms of endocytic agents in different routes of administration (including oral, topical and inhalation) and sequential distribution, metabolism, and excretion (ADME). Also disclosed are methods to evaluate or determine the endocytic agents’ ADME properties in the drug discovery and development process.

[0131] As shown in Figure 1C, disclosed are methods to administer endocytic agents or exocytic vesicles via oral or intravenous route for the study, diagnosis, prevention, and treatment of any subjects, especially for CNS conditions and diseases. Also disclosed are methods to utilize endocytic agents in combination with extracellular vesicles (EVs) for the study, diagnosis, prevention, and treatment of any diseases. As shown in Figure ID, disclosed are the mechanisms of action of endocytic agents. Either endocytic agents or extracellular vesicle after absorption, or exocytic vesicles comprising endocytic agents, can be absorbed into cells for the action via endocytosis or membrane fusion for any purpose of applications.

[0132] Also disclosed are methods to use an “one-step” method to generate and use of exocytic vesicles in human or animals, wherein, without further processes including agents or vesicles isolation, cells in body up-take endocytic agents via endocytosis and sequentially secrete out exocytic vesicles, and the resulting endogenous exocytic vesicles can be directly used by bodies for any purposes. Also disclosed are methods to isolate exocytic vesicles. Wherein a method to make exocytic vesicles is that cells can up-take endocytic agents via endocytosis and then secrete out exocytic vesicles via exocytosis. Wherein another method to form functional exocytic vesicles is that endocytic agent molecules can be linked with extracellular vesicle and / or exocytic vesicles via covalent or non-covalent bond(s) in situ. Disclosed are methods of making and using the same. Endocytic Agents

[0133] Endocytic agent, or agent, refers to any compound or a portion or a conjugated agent thereof that can bind with endocytosis-mediating membrane component(s) and be absorbed by cells via endocytosis. In some embodiments, the agent is a therapeutic agent, a diagnostic agent, a binding agent, a conjugate, a nanoparticle, or a vesicle. A therapeutic agent is any compound that is useful in the treatment of a subject. A diagnostic agent is any compound that is useful in providing qualitative or quantitative information about a biomolecular target or a biological environment of interest. A binding agent is any compound which bind with endocytosis-mediating membrane component(s) or extracellular materials, including membrane proteins or extracellular proteins, carbohydrates, lipids, pathogens, particles, virus, bacteria, fungi, protozoa, vectors, cell debris, and cells. A conjugate is a compound composed of one or more than one agent(s) conjugated with one or more than one chemical arm(s) via cleavable or non-cleavable chemical bond or linker unit(s). A vesicle is a structure within or outside a cell, consisting of liquid or cytoplasm enclosed by a lipid bilayer.

[0134] The endocytic agents of the present disclosure as discussed below include, but not limit to, neutral compound, the free base or acid, their salts, solvates, and prodrugs and can include oxidized sulfur atoms or quaternized nitrogen atoms in their structures, although not explicitly stated or shown, particularly the pharmaceutically acceptable forms thereof. Such forms, particularly the pharmaceutically acceptable forms, are intended to be embraced by the appended claims.

[0135] In some embodiments, the endocytic agent has a binding affinity with an endocytosis- mediating membrane component with binding affinity Ko value lower than 20.0 mM.

[0136] In some embodiments, the endocytic agent has a molecular weight greater than 200 Da. In certain embodiments, the endocytic agent has a molecular weight greater than 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000 Da. In some embodiments, the endocytic agent may have a molecular weight between 200 and 10000 Da.

[0137] In some embodiments, the endocytic agent is a therapeutic agent. Exemplary therapeutic agent includes, without limitation, a drug, a protein inhibitor or antagonist, a protein activator or agonist, a protein modulator, a molecular glue that induce or stabilize protein-protein interactions, a protein degrader, or a multivalent agent, a protein binder, a diagnostic agent or chemical probe, or a vesicle, including their isotopomers, such as deuterium / or fluorine substituted derivatives, analogs and chelates, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and pharmaceutically acceptable carrier. A therapeutic agent is also a protein, DNA, or RNA modifier.

[0138] In certain embodiments, the therapeutic agent is a drug which can bind with membrane receptors that are involved in the endocytic process of cells. Exemplary drugs include, without limitation, any of the following compounds: 68Ga-DOTA-F API-46, 68Ga-DOTA-2P9FAPI)2, ABT-737, acarbose, acetaminosalol, actinomycin D, adenosine Ai receptor (AiR) agonists, aliskiren, AMG-131, argatroban, ascomycin, asukamycin, asunaprevir, atazanavir, atorvastatin, AZD5153, azithromycin, B-cell lymphoma 2 (BCL-2family proteins inhibitors such as venetoclax (ABT-119), navitoclax (ABT-263), APG-2575 (Lisaftoclax), UBX1325 (CAS No: 2271269-01- 1), APG-1252 (Pelcitoclax), APG-2575 (Lisaftoclax), AMG176, AMG397, AZD4606 (CAS: 2241039-81-4), AZD5991, AZD4747 (CAS: 2489226-14-2), BGJ-398, birinapant, bis(7)-tacrine, BLU-945, BMS-777607, BMS-791325, BMS-986165 (CAS: 1609392-27-9), Brigatinib (CAS: 1197953-54-0), Brigimadlin, camdronate, camptothecin, candoxatril, capreomycin, ceritinib, CHF-6366, clarithromycin, cobimetinib, combretatropone, Compound 28 (Fedor Romanov- Michailidis, et al., Journal of Medicinal Chemistry, 66, 6122-6148, 2023), cosmomycin D, CPT- Gly-PEG-Folate, CUDC-101, CUDC-907, cyclosporine A, daclatasvir, dactinomycin, dalfopristin, danamide F, danaprevir, Danoprevir (CAS: 850876-88-9), Danuglipron (CAS: 2230198-02-2), DHP1808, diaspirine, digoxin, diprovocim-X, divarisib (CAS: 2417987-45-0), diamino allose phosphates (DAPs), DNL343, doxorubicin, dasatinib, DU1301, ECPU-0001, EDO-S101, EML981, epcoritamab (CAS: 2134641-34-0), eptifibatide, EPZ-5676, ergotamine, eritoran, erythromycin A, erythronolides, etoposide, erythromycin A, estramustine, ethacraplatin, everolimus, EZN-2208 (CAS: 946062-05-1), FAPI-46, FAPI-dimer, Fenebrutinib (CAS: 1434048-34-6), fedratinib, Fosinopril (CAS: 98048-97-6), fostamatinib, G protein-coupled receptors (GPCRs) inhibitors, G protein-coupled receptors (GPCRs) agonists, gartisertib, Glecaprevir (CAS: 1365970-03-1), himeic acid A, histone deacetylase (HDAC) inhibitors, homoharringtonine, inhibitor of apopotosis (IAP) protein inhibitors such as AZD5582 (CAS: 1258392-53-8), SM-164 (CAS No.: 957135-43-2) and xevinapant, indoleamine 2, 3 -dioxygenase (IDO) inhibitors, indobufen, IR820-SS-CPT, IT-101, itraconazole, ivermectin, JNJ78394355, JS230, KX2-361, ladostigil, lapatinib, leucomycin, Lipitor (atorvastatin), lonafarnib, Lumakras (CAS: 2296729-00-3), LUNA18 (CAS: 2676177-63-0), LY3502970 (CAS: 2212020-52-3), mammalian target of rapamycin (mTOR) and / or FK506-binding proteins (FKBP) proteins inhibitors, medoxomil, Milademetan (CAS: 1398568-47-2), MIP-1404, MK-1468, MK-8768 (CAS: 1432729-22-0), mometasone furoate, motixafortide (CAS: 664334-36-5), MPI8 (CAS: 856242-63-2), MRT-2359 (CAS: 2803881-11-8), MRTX-849 (CAS: 2326521-71-3), Muvalaplin (CAS: 2565656-70-2), NDI-034858 (CAS: 2272904-53-5), nilotinib, nintedanib, NOSH-aspirin (NBS-1120), NKTR-102 (CAS: 1193151-09-5), NKTR-105, nirmatrelvir, ODDA-PTX, omavel oxoIone, anobinost, ortataxel, ouabain, PAANIB-1, paclitaxel, Pacritinib (CAS: 937272- 79-2), pelabresib, Pevonedistat (CAS: 905579-51-3), PF-03715455, phakelli statins, pictilisib, PiflufolastatF-18 injection, Pluvicto (CAS No.: 1703749-62-5), peroxisome proliferator-activated receptors (PPARs) agnoists, PRMT inhibitors, protein phosphatase inhibitors, protein arginine methyltransferases (PRMTs) inhibitors, pralsetinib, pseudomonic acid A, PSMA-11, PSMA-617, PSMA-1007, PSMA I&S, PSMA I&T, PSMA SMOL-TTC monomer, PSMA SMOL-TTC dimer, PSMA SMOL-TTC trimer, PSMA SMOL-TTC tetramer, PU-H71, pyrilutamide, quizartinib (CAS: 950769-58-1), quinupristin, rapamycin, Rapalink-1, REC-3599, rifampicin, rifapentine, rifabutin, rifaximin, Rilzabrutinib (CAS: 1575596-29-0), ritonavir, rivaroxaban, RMC-4998 (CAS: 2642037-07-6), RMC-6291 (CAS: 2641998-63-0), RMC-6236 (CAS: 2765081-21-6), RMC- 4998 (CAS: 2642037-07-6), roxithromycin, RPT193 (CAS: 2366152-15-8), sanguinamide A, S63845, S64315, saquinavir, scavenge receptors (SRs) inhibitor, activator, or binders, endothelial cell protein C receptor (EPCR) inhibitor, activator, or binders, setileuton, SHP-1971, simeprevir, sirolimus, solute carriers (SLC) transporters inhibitor, activator, or binders, Simeprevir (CAS: 923604-59-5), sparsentan (CAS: 254740-64-2), spiramycin, staurosporine, stimulator of interferon genes (STING) agonists, Sug-HisVal-CPT, tacrolimus, taladegib, Tapotoclax (CAS: 1883727-34- 1), Tat-P4-(C5)2 (doi.org / 10. 15252 / emmm.201911248), TNG348, tryptophan 2,3- dioxygenase (TDO) inhibitors, telithromycin, thiosptrepton, tinostamustine, Tirbanibulin (KX2- 391), trioxaquine, trypdronate, tubacin, tubocurarine, VCP746 (CAS No.: 1582751-84-5), volasertib, vazegepant (CAS: 1337918-83-8), VX-548 (CAS: 2649467-58-1), zatebradine, zotarolimus, deuterium substituted derivatives, analogs and chelates, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and pharmaceutically acceptable carrier.

[0139] In certain embodiments, the therapeutic agent is a protein inhibitor or antagonist, including their isotopomers, such as deuterium substituted derivatives.

[0140] The protein inhibitor or antagonist may target any suitable protein including all variants, mutations, splice variants, indels and fusions of these target proteins listed. Examples include, without limitation, 5HT2c receptor, on A- AR, a-2 adrenergic receptor, a-synuclein, AAK1, ATP- binding cassette (ABC) transporters such as MDR1 / 2 / 3 / 4 / 5 and ABCG2, ABL, cholesterol acyltransferases (ACATs), ACE protein, ACK, ACTR2, ACTR2B, ADCK1 / 2 / 3 / 4 / 5, protein kinase B (AKT), AlphaKl / 2 / 3, anaplastic lymphoma kinase (ALK), ALIX, Amnionless, AMPA receptors (AMPARs), AMP -related protein kinase (AMPK), amyloid beta, Angiotensin II receptor, ANKRD3, Apaf-1, apolipoprotein E receptor (ApoER), amyloid precursor protein (APP), androgen receptor (AR), ARAF, ARG, AR-V7, ileal apical sodium / bile acid co-transporter (ASBT), V-type proton ATPase 6 (ATP6V), ATP6V1H, avidin, amino acids transporters, alanine serine cysteine transporters (ASCTs), ASGPR, ASK1 / 2, Ataxin-1, ataxia-telangiectasia mutated protein (ATM), ATM and Rad3 -related protein (ATR), Aurora kinases, AXL, P-site amyloid precursor protein-cleaving enzyme 1 (BACE1), 02-adrenergic receptors, BAD, BARK1 / 2, Bax, BCKDK, B-cell leukemia / lymphoma (BCLs) family proteins such as BCL2, BCL-XL, and MCL- 1, BCR- ABL, bromodomain and extraterminal domain family proteins (BETs) such as BRD2 / 3 / 4 / T, beta-catenin, BIKE / BMP2K, BLK, BMPR1A, BMPR1B, BMPR2, BMX / ETX, brother of CDO (Boc), BRD9, BMI1, BRAF, BRAFV600E, brassinosteriod insensitive 1 (BRI1), BRK, BRM, BRSK1, BRSK2, Bruton tyrosine kinase (BTK), BUB1, C3G, calreticulin, cell adhesion molecule (CAM) receptors, CAMK I a / p / 7 / 6, CAMK2a / p / y / d, CAMK4, caMLCK, cannabinoid- 1 receptor, coxsackievirus-adenovirus receptor (CAR), CBL-B, CRISPR associated proteins (Cas), CASK, Caspase-3, Caspase-6, Caspase-7, Caspase-9, CBF0, CBL, CBP, chemokine receptors such as CC chemokine receptors (CCRs) and CXC chemokine receptors (CXCRs) such as CXCR2, CXCR4, and CXCR7, CCK4 / PTK7, CCR2, CCR9, CCRK, cluster of differentiations (CDs) such as CD4, CD9, CD13 / APN receptor, CD25, CD26(DPP4), CD30, CD33, CD36, CD38, CD46, CD56, , CD63, CD71, CD74, CD80, CD81, CD82, CD83, CD86, CD123, CD138, CD147, CD152, CD152 (CTLA4), CD166, CD174, CD197, CD205, CD227, CD228, CD269, CD276, and CD326, CDC7, CDC20, CDC25, CDC37,

[0141] CDK1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12 / 13 / 14 / 15 / 16 / 17 / 18, CDKL1 / 2 / 3 / 4 / 5, CDON, CEACAM5, cholesteryl ester transfer protein (CETP), c-Fos, cystic fibrosis transmembrane conductance regulator (CFTR), cyclic GMP-AMP synthase (eGas), Chakl / 2, CHK1 / 2, CI-M6PR, CKla, CKla2, CK18, CKle, CKlyl / 2 / 3, CK2al / 2, CLIK1, CLIK1L, Clip, CLK1 / 2 / 3 / 4, human collectin placenta 1 (CL-P1), CMYC, cone opsins, COT / TPL2, cell-penetrating peptide (CPP), connexins, coronavirus protease, CRABP, C-RAF, cereblon (CRBN), CREB, CRK, CROC, CRIPTO, CSF2R, CSF1R / FMS, CSK, C-TAK1, CTK, cubilin, cyclin D, cyclin E, CytoC, DAPK1 / 2 / 3, DCAMKL1 / 2 / 3, DDR1 / 2, diglyceride acyltransferase (DGAT), DLK, DMPK1 / 2, DNA-PK, DP- 1, DPP -4, DRAK1 / 2, deubiquitylating enzymes (DUBs), DYRK1A / 1B / / 2 / 3 / 4, E2F, excitatory amino acid carrier 1 (EAAC1), E-cadherin, endothelin-converting enzyme (ECE), ED-B, EED, EF2K, eEF2K, EIF2A3, EIF4E, EIF4G, ELK, ENL, endothelin B receptor, epithelial cell adhesion molecule (EpCAM), endothelial cell protein C receptor (EPCR), Ephrin receptors (EphRs), estrogen receptor (ER), ER81, ErbB2, ErbB3, ETS transcription factor (ERG), ERK1 / 2 / 3 / 4 / 5 / 7 / 8, ERRa, EZH2, fatty acid binding proteins (FABPs), FADD, FAK, FAP, fatty acid transport proteins (FATPs), neonatal Fc receptor (FCRN), FER, FKBPs, FLIP, folate receptors such as reduced folate carrier, FOLT1 / 2 / 3 and proton-coupled folate transporter (PCFT), F0XM1, FOXO1 / 2 / 3 / 4, FRK, Frizzled4, fructose-l,6-bisphosphatase, FYN, y-aminobutyric acid type A receptors (GAB AAR), GAK, Gap-1, growth arrest specific 1 (Gasl), GCC, GCK, GCN2, GD2, ghrelin receptor (ghrelinR), glucose-dependent insulinotropic polypeptide (GIP), Globo H, glucagon receptor, glucagon-like peptide 1 (GLP-1), glutamate transporters (GLTs), glucokinase, Ionotropic AMP A glutamate receptors (GluRs), glucose-6-phosphatase, glucose transporters (GLUTs), glutathione transporters, glycogen phosphorylase, glycogen synthase kinase, glycoproteins such as gp 18, gp31 , and gp60, G protein-coupled receptors (GPCRs) such as GPR20, GPR20, free fatty acid receptor 1 (GPR40), GPR119, and GPR120, GPNMB, GPRK4 / 5 / 6 / 7, GRB2, GSK-3, GSPT1, Haspin kinase, HCK, hepatitis C virus (HCV) NS3 / 4A, histone deacetylases (HDACs), HDL receptor (HDLR), HectH9, HH498, HIF1, HIPK1 / 2 / 3 / 4, HMGN1, HMG-CoA reductase, hnRNPs, H-PGDS, HPGCR, hematopoietic progenitor kinase 1 (HPK1), HRAS, HRI, hRpnl3Pru, HSD-110 (11 P-hydroxysteriod dehydrogenase), heat shock proteins (HSPs), huntingtin protein (HTT), HUNK, ICK, intermediate-density lipoprotein receptor (IDLR), IDO-1, IFN-yR, IGF-1R, IGF-2R, IKK-alpha, IKK-beta, IKK-gamma, IKK-epsilon, Ikaros (IKZF1), Helios (IKZF2), Aiolos (IKZF3), inhibitor of apoptosis (IAP) proteins such as cIAPs and XIAP, IKZF4, IL-4R, IL-10R, ILK, integrins such as aVP3, a4pi and a5pi integrins, insulin receptors (IRs), insulin-like growth factor receptor, INSR, IRE1 / 2, IRR, ITKIMP, integrin receptors (IRs), IRA2, IRAK1 / 2 / 3 / 4, IRE1, IRR, Janus tyrosine kinase (JAKs), c-Jun N-terminal kinases (JNKs), potassium-chloride cotransporter 2 (KCC2), KDM5C, KHS1 / 2, inward rectifier potassium channel (Kir2.3), KIS, KIT, Kelch domain-containing protein 2 (KLHDC2), KLF4, KRAS, KRASG12C, KSR1 / 2, Lamin, lysosome associated membrane proteins (Lamps), LAMT0R2, LANA, lactoferrin receptor, L-amino acid transporters (LATs), LATS1 / 2, LCK, low- density lipoprotein receptor (LDLR), LEF, LIMK1 / 2, LKB1, LMR1 / AATK, LMR2 / 3, LOK, low- density lipoprotein receptor related proteins (LRPs), LRRK1 / 2, LTK, LXR-0, LYN, LZK, LZTFL1, m5C, m6A, MAK, MALTs, MAP3K4 / 8, MAP4K3, MAP4K5, MAPK, MAPKlb, MAPKAPK2 / 3 / 5, MARK1 / 2 / 3 / 4, MAST1 / 2 / 3 / 4 / L, MC4 receptor, MCAK, MCH-1 receptor, monocarboxylic acid transporters (MCTs), mouse double minute 2 homolog (MDM2), MDMx, megalin, mitogen-activated protein kinase (MEKs), MEKK 1 / 2 / 3, MEL, MELK, MER, MERTK, mesothelin, MET, Mfsd2a, metabotropic glutamate receptors (mGluls), major histocompatibility complex class I proteins (MHC-I), MHC-II, MIF, MINCLE, MINK1, MISR2, MITF, MKK1 / 2 / 3 / 4 / 6 / 7, MKP3, MLK1 / 2 / 3 / 4, MLKL, MLN1 / 3, MNK1 / 2, MOK, MOS, MPK, MPSK1, MRCKa / p, multidrug resistance proteins (MRPs), MSK1 / 2, MSSK1, MST1 / 2 / 3 / 4, mammalian target of rapamycin (mTOR), MUCL, MUSK, Myc proteins, MYO3A / 3B, MYT1, NAMPT, N- cadherin, K+-dependent Na+ / Ca2+exchanger 2 (NCKX2), NDC80, NDR1 / 2, NEK1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11, NF-kappa B, Nicastrin, nicotinic acetylcholine receptor, NIK, NEK, NOTCH receptors, niemann-Pick Cl-like 1 (NPC1L1), N-methyl D-aspartate receptors (NRs), NRAS, NRBP1 / 2, NRF2, NSD2, NSD3, nucleoside transporters (NTs), sodium / taurocholate co- transporting peptide (NTCP), NuaKl / 2, NUAK1, organic anion transporters (OATs), organic anion transporting polypeptides (OATPs; 0ATP1B1, 0ATP2B1, OATP4C1, etc.), Obscurin, organic cation transporters (OCTs), OSR1, organic solute transporter (OST), otoferlin, P2X purinoceptor 4 receptor (P2x4R), purinergic receptor P2Y12 (P2Y12), pl6INK4A, pl8INK4, pl9INK4D, p21, p27kipl, p38a, p38p, p38y, p386, p53, p65 / RELA, p70S6K, p70S6Kb, p90, p300 protein, PAK1 / 2 / 4 / 5 / 6, pan-coronavirus antiviral, protease-activated receptors (PARs), PARPs, PASK, PAX, PBRM1, PCSK9, PD-1, phosphodiesterases (PDEs), PDE4, PDE8, PDGFRa / 0, pdhkl / 2 / 3 / 4, PDK1, PD-L1, PEA-15, PEK, peptide transporters (PEPTs), p-glycoprotein (P-gp), PHKyl / 2, phosphoenol pyruvate carboxykinase, phosphoinositide 3-kinase (PI3K), PIM1 / 2 / 3, PINK1, PITSLRE, PKA, PKACa / p / y, PKBa / 0, PKC, PKD1 / 2 / 3, PKG1 / 2, PKN1 / 2 / 3, PKR, PLC, PLDL, PLK1 / 2 / 3 / 4, PMEL17, PRAK, peroxisome proliferator-activated receptors (PPARs), PRC2, PRK2, PRKX, PRKY, PRMT5, protein phosphatase, protein tyrosine phosphatase (PTP), PRP4, PRPK, PSKH1 / 2, prostate-specific membrane antigen (PSMA), receptor patched 1 (PTCHI), PTEN, protein tyrosine kinases (PTKs), PVRL4, pyruvate dehydrokinase, PYK2, QIK, QSK, R1P1, RACK-1, Rac proteins, RAF protein, RAP1, Raptor, RAR, Ras protein family (RAS), Rb, retinol binding proteins (RBPs), RET, riboflavin transporter proteins (RFVTs), RFX1, RHAU, RHODK, RI0K1 / 2, receptor interacting protein kinases (RIPKs), rhodopsin, ribonuclease K (RNASEK), RNAseL, ring finger proteins (RNFs), R0CK1 / 2, renal outer medullary potassium channel (ROMK), RON, R0R1 / 2, ROS, RPN11, RPN13, ribosomal s6 kinases, receptor serine / threonine kinases (RSKs), such as transforming growth factor 0 (TGF-0) receptor and proteins as listed in Aristidis Moustakas, et al, Receptor Serine / Threonine Kinases (ISBN: 978-3- 540-44244-8), receptor tyrosine kinases (RTKs), such as fibroblast growth factor receptors (FGFR), FLT1 / 2 / 3 / 4, FGFR1 / 2 / 3 / 4, RET, tropomyosin-related kinases (TRKs), epidermal growth factor receptor (EGFR), human epidermal growth factor receptors (HERs), vascular endothelial growth factor receptors (VEGFRs), TYRO3 and other proteins as listed in publication Mark A. Lemmon and Joseph Schlessinger, Cell, 141(7) 1117-1134, 2010, RUNX1 / 2 / 3, RYK, spindle assembly checkpoint (SAC), Sanpodo / Notch, SAP, SARS-CoV-2, SBK, solute carriers (SCs), SCF, SCYL1 / 2 / 3, SF3B1, serine / threonine-protein kinases (SGKs), SGLTs, SHC, SHP2, SIK2 / 3, SIRT2, SIX1, skMLCK, S-phase kinase associated protein 1 (SKP1), S-phase kinase associated protein 2 (SKP2), solute carriers (SLC, such as SCL19A1) transporters, such as hMATEl and proteins listed in publication Enrico Girardi, et al., Nature Chemical Biology, 16, 469-478, 2020, SLK, SLOB, Smac, Smad proteins, SMARCAs, sodium-coupled monocarboxylate transporters (SMCTs), SMG1, smMLCK, sodium dependent multivitamin transporter (SMVT), synaptosomal- associated protein (SNAP), SNRK, sortilin-related CNS expressed la (SorCSla), SorCSlc, protein son of sevenless (SOS), SOS1, SOX2, secreted protein acidic and rich in cysteine (SPARC), SPEG, speckle type BTB / POZ protein (SPOP), SPRED, SPRY, SPv-NKIR, scavenger receptors (SRs) such as CD36, LAMP1, andLAMP2, Src protein, SRF, SRM, SRPK1 / 2, SSTK, StaO, signal transducer and activator of transcription proteins (STATs), STING, serine / threonine kinases (STKs), STLK3 / 5 / 6, syntaxins (STXs), SuRTK106, SUV39HI, SUZ12, sodium-vitamin C co- transporters (SVCTs), synaptotagmins (SYTs), triiodothyronine (T3), TAB, TAK1, TAL, TAO1 / 2 / 3, Tau protein, TBCK, TBK1, t-cell factor / lymphoid enhancer-binding factors (TCF / LEF family), TEC, TESK1 / 2, TFAM, transcription factor EB (TFEB), transferrin receptor (TfR), TGF- P, trans-Golgi network (TGN38), thiamine transporters (THTRs), TIE1, TIE2 / TEK, TIFla / p / y, Titin / TTN, TLK1 / 2, toll-like receptors (TLRs) such as TLR4, TNK1, TOPK, TPL2 / COT, TPNlp, TRAD, TRAF proteins, Trb 1 / 2 / 3, tripartite motif family proteins (TRIMs), Trio, TRRAP, TSC2, TSG101, TSSK1 / 2 / 3 / 4, TTBK1 / 2, TTK, TXK, TYK2, tyrosinase, tubulin, UBF, UCHL5, ULK1 / 2 / 3 / 4, ubiquitin specific peptidase (USP) such as USP7, USP11, and USP14, VACAMKL protein, vesicle associated membrane proteins (VAMPs), vitamin D receptors (VDR), vesicular GABA transporter (VGAT), vesicular glutamate transporters (VGLUTs), very low-density lipoprotein receptor (VLDLR), urate transporter 1 (URAT1), VRK1 / 2 / 3, WAVE-2, WDR5, WEE- 1, WEE-1B, WNK1 / 2 / 3 / 4, Wnt, XBP1, YANK1 / 2 / 3, YBX1, YES, YSK1, ZAK, ZAP70, ZC1 / 2 / 3 / 4, ZFP91, and zinc ring finger proteins (ZRNFs), including all variants, mutations, splice variants, indels and fusions of these target proteins listed.

[0142] In certain embodiments, the protein inhibitors or antagonists are molecules targeting B-cell lymphoma 2 (BCL-2) family proteins. In certain embodiments, the protein inhibitor or antagonist targeting BCL-2 family proteins is selected from ABT737 (CAS: 852808-04-9), ABT263 (CAS: 923564-51-6), ABT199 (CAS: 1257044-40-8), GX15-070 (CAS: 803712-79-0), UBX1325 (Lisaftoclax), APG-1252 (pelcitoclax), APG-2575 (Lisaftoclax), or ©-(-)-gossypol (CAS: 90141- 22-3), any deuterium substituted derivatives, analogs and chelates, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and pharmaceutically acceptable carrier. In certain embodiments, the protein inhibitors or antagonists are molecules targeting proprotein convertase subtilisin / kexin type 9 (PCSK9). In certain embodiments, the protein inhibitor or antagonist targeting PCSK9 protein is selected from CVI-LM001, PF-06815345, MK- 0616 or enlicitide chloride (CAS: 2407527-16-4), NN6434, 13PCSK9i, and any molecules listed in publication Shakir Ahamad, et al., Journal ofMedicinal Chemistry, 65(23), 15513-15539, 2022, any deuterium substituted derivatives, analogs and chelates, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and pharmaceutically acceptable carrier.

[0143] In certain embodiments, the protein inhibitors or antagonists are molecules targeting protein phosphatase. In certain embodiments, the protein inhibitor or antagonist targeting protein phosphatase is selected from TNO155 (CAS: 1801765-04-7), RMC-4630, RMC-4550 (CAS: 2172651-73-7), IFB-088 (CAS: 951441-04-6), SHP1 inhibitor (CAS: 56932-43-5), SHP009 (CAS: 1801747-42-1), DPM-1001 (1471172-27-6), AKB-9778 (CAS: 1008510-37-9), LB-100 (CAS: 1026680-07-8), any deuterium substituted derivatives, analogs and chelates, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and pharmaceutically acceptable carrier.

[0144] In certain embodiments, the protein inhibitors or antagonists are molecules targeting histone deacetylase (HDACs). In certain embodiments, the HDAC inhibitor or antagonist is selected from, but not limited to, vorinostat, romidepsin, belinostat (PXD101), Panobinostat (LBH589), valproic acid, entinostat (MS275), butyric acid, trichostatin A, givinostat (ITF2357), citarinostat (ACY-241), mocetinostat (MGCD0103), pracinostat (SB 939), resminostat, RGFP966, CUDC-101, abexinostat (PCI-24781), nocetinostat, phenylbutyrate, tacedinaline, tubacin, tubastatin A, R306464, SE-7552, MPT0B451, dacinostat (LAQ824), HDAC10-IN-1, HDAC10- IN-2, AR-42, GSK3117391, MC1568, quisinostat (JNJ-26481585), PCI-34051, droxinostat, RGFP966, ricolinostat (ACY-1215), tacedinaline (CI994), fimepinostat (CUDC-907), M344, RG2833 (RGFP109), scriptaid, TMP269, TMP195, santacruzamate A (CAY10683), SKLB-23bb, ACY-775, BRD73954, CXD101, suberohydroxamic acid, BRD3308, HPOB, LMK-235, nexturastat A, BML-210 (CAY10433), KA2507, TC-H 106, Tucidinostat (Chidamide), SIS17, WT161, CAY10603, ACY-738, tinostamustine(EDO-S101), domatinostat (4SC-202), BG45, any deuterium substituted derivatives, analogs and chelates, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and pharmaceutically acceptable carrier. In certain embodiments, the protein inhibitors or antagonists are molecules targeting mammalian target of rapamycin (mTOR) and / or FK506-binding proteins (FKBP) proteins. In certain embodiments, the mTOR and / or FKBP (or 4EBO1) inhibitor or antagonist is rapamycin, Rapalink-1 (CAS: 1887095-82-0), orRMC-5552 (CAS: 2382768-62-7), any deuterium substituted derivatives, analogs and chelates, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and pharmaceutically acceptable carrier.

[0145] In certain embodiments, the protein inhibitors or antagonists are molecules targeting protein methyl transferases such as arginine methyltransferases (PRMTs). In certain embodiments, the PRMTs inhibitor or antagonist is selected from, but not limited to, S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), AMI-1, sinefungin, homosinefungin, GSK3326595, JNJ-63619178, GSK3368715, EML108, EPZ004777, and EML981, any deuterium substituted derivatives, analogs and chelates, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and pharmaceutically acceptable carrier.

[0146] In certain embodiments, the protein inhibitors or antagonists are molecules targeting signal transducer and activator of transcription proteins (STATs).

[0147] In certain embodiments, the therapeutic agent is a protein activator or agonist and modulator, including their isotopomers, such as deuterium substituted derivatives. The protein activator or agonist may target any suitable protein. Examples include, without limitation, adenosine Ai receptor (AiR), A2A receptor, A ?B receptor, A3 receptor, AMPK, cGAS, chemokine receptors, FXR, GCK, glucagon-like peptide 1 (GLP-1), G protein-coupled receptors (GPCRs) such as GPR20, GPR20, free fatty acid receptor 1 (GPR40), GPR119, and GPR120, integrins, peroxisome proliferator-activated receptors (PPARs), ring finger proteins (RNFs), stimulator of interferon genes (STING), Toll-like receptors (TLRs) such as TLR4, zinc ring finger proteins (ZRNFs), including all variants, mutations, splice variants, indels and fusions of these target proteins listed.

[0148] In some embodiments, the activators or agonists are molecules targeting adenosine Ai receptor (AiR). In certain embodiments, the AiR activator or agonist is selected from, but not limited to, CPA (CAS: 41552-82-3), BnOCPA, LUF6258 and VCP746 (CAS: 1582751-84-5), any deuterium substituted derivatives, analogs and chelates, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and pharmaceutically acceptable carrier. In certain embodiments, the activators or agonists are molecules targeting glucagon-like peptide 1 (GLP-1). In certain embodiments, the GLP1 activator or agonist is selected from, but not limited to, Lixisenatide / AVEOOlO / ZPIO / Lyxumia, Exenatide / Exendin-4 / Byetta / Bydureon / ITCA 650 / AC-2993, Liraglutide / Victoza, Tirzepatide, Semaglutide, Taspoglutide, Syncria / Albiglutide, Dulaglutide, rExendin-4, CJC-1134-PC, PB-1023, TTP-054, Efpeglenatide / HM-11260C, CM-3, GLP-1 Eligen, ORMD-0901, NN-9924, NN-9926, NN-9927, Nodexen, Viador-GLP-1, CVX-096, ZYOG-1, ZYD-1, GSK-2374697, DA-3091, MAR-701, MAR709, ZP-2929, ZP-3022, ZP-DI-70, TT-401, MK-8521, MED10382, BHM-034, HM12525A, MOD-6030, CAM-2036, DA-15864, ARI-2651, ARI-2255, LY3298176, NN1177, Exenatide-XTEN and Glucagon-XTEN, NN9030, and any deuterium substituted derivatives, analogs and chelates, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and pharmaceutically acceptable carrier.

[0149] In certain embodiments, the activators or agonists and modulators are molecules targeting integrins. In certain embodiments, the integrin activator or agonist and modulators is selected from, but not limited to, SAR-1118, BMS-587101, l,2,3,4-tetrahydroquinoline-6-carboxylicacid, HC- 0303, Compactin, AJM-300, HMR-1031, Firategrast, Tirofiban, Eptifibatide, MK-0429, ATN-161, JSM-6427, and any deuterium substituted derivatives, analogs and chelates, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and pharmaceutically acceptable carrier.

[0150] In certain embodiments, the activators or agonists are molecules targeting peroxisome proliferator-activated receptors (PPARs). In certain embodiments, the PPAR activator or agonist is selected from, but not limited to, GW0742, L-165041, MA-0211, KD-3010, CER-002, SAR351034, Oxeglitazar, LY518674, ZYH7, Ki l l, Macuneos, Efatutazone, CHS-131, OMS-405, GED 0507-34-Levo, T2D 959, Lanifibranor, Gemfibrozil, Rosiglitazone, Ciprofibrate, Piolitazone, Bezafibrate, Lobeglitazone, Fenofibrate, Saroglitazar, Pemafibrate, any deuterium substituted derivatives, analogs and chelates, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and pharmaceutically acceptable carrier.

[0151] In certain embodiments, the activators or agonists are molecules targeting stimulator of interferon genes (STING) protein. In certain embodiments, the STING activator or agonist is selected from, but not limited to, c(di-GMP), 3’,3’-cGAMP, 2’,3’-cGAMP, ML-RR-S2-cGAMP, ADU-S100, ML-RR-S2-CDG, DMXAA, aminobenzimidazoles, ExoSTING, MV-626, SB11285, STACT-TREX1, SYN-STING (SYNB1891), E7766, GSK3745417, MK-1454, MK-2118, BMS- 986301, SB-11285, IMSA-101, BI-1387446, TAK676, SNX281, HG-381, DN-015089, any deuterium substituted derivatives, analogs and chelates, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and pharmaceutically acceptable carrier.

[0152] In certain embodiment, the therapeutic agent is a pyrophosphate- or bisphosphonate- containing agent that can bind to bone mineral, are taken up by bone cells via membrane receptors such as SCL37A3. In certain embodiment, the pyrophosphate- or bisphosphonate-containing agent is selected from, but not limited to, pamidronic acid, risedronic acid, alendronic acid, zoledronic acid, ibandronic acid, minodronic acid, compounds in publication Jaeok Park, et al., Frontiers in Chemistry, 8, 612728, 2021 and Zhou Yu, et al., eLife, e36620, 2018, any deuterium substituted derivatives, analogs and chelates, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and pharmaceutically acceptable carrier.

[0153] In certain embodiments, the therapeutic agent is a molecular glue or degrader, including their isotopomers, such as deuterium substituted derivatives. A molecular glue can be any compound that can stabilize the interaction between two or more than two proteins. A degrader can be any compound that can bind with protein of interest (POI) and induce the degradation of POI via direct modulation of POI, such as modification of POI’s surface topology.

[0154] In some embodiments, the therapeutic agent is a multivalent endocytic agent. A multivalent endocytic agent herein, composes of agent(s) or probe(s) conjugated with chemical arm(s) in any equivalent and sequences via cleavable or non-cleavable chemical bond or linker unit(s) , wherein, the agent can be any compounds with biological activities and the probes can be any diagnostic agent or chemical probe. The multivalent endocytic agent can bind with endocytosis-mediating membrane component(s) and be absorbed via endocytosis. The multivalent compound may have a binding affinity Kn to endocytosis-meditating membrane component(s) less than 20.0 mM.

[0155] In certain embodiments, the multivalent endocytic agent includes, without limitation, structures as present by general Formula (I): wherein m, n, and p represent integer from 0 to 100. Each of m, n, and p may be independently, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, the multivalent endocytic agent itself can exert biological functions in bacteria, viruses, fungi, protozoa, vectors, cells, tissues, and animal bodies. In certain embodiment, the chemical bond or linker unit(s) B / L within multivalent endocytic agent can be cleaved by enzymes (or protein) in bacteria, viruses, fungi, protozoa, vectors, cells, tissues and animal bodies and a portion of the multivalent endocytic agent is released to exert any biological functions. The components and extracellular materials in internalized membrane or, such as proteins, carbohydrates, lipids, pathogens, particles, viruses, bacteria, fungi, protozoa, vectors, cell debris, and another cell, can be also degraded by lysosomes.

[0156] The agent and chemical arm are covalently conjugated by chemical bond or linker unit(s)

[0157] B / L In certain embodiments, the chemical bond or linker unit(s) B / L can be attached at any sites of the agent(s) or probe(s) probe and chemical arm(s) L.Qhemical arm

[0158] In certain embodiments, the site of conjugate between the agent(s) or probe(s) and chemical arm(s) may be selected to allow for, improve, or enhance the intended functionality of the agent, meanwhile, allow for, improve, or enhance the binding affinity to an endocytosis-meditating membrane component for enhanced endocytic efficacy and / or efficiency. For example, conjugation between the agent(s) or probe(s) and chemical arm(s) result in multivalent endocytic agent(s).

[0159] The site of conjugate between the agent(s) or probe(s) and chemical arm(s) may be selected to impair, diminish, or remove the intended functionality of the agent.

[0160] In certain embodiments, the chemical bond or linker unit(s) B / L may be a multivalent polyethylene glycol, alkoxy, alkyl, alkylene, alkyne, cycloalkyl, cycloalkyene, cycloalkyne, heterocyclyl, aryl, heteroaryl, which may terminate (at either or both termini) in at least one of a - H, -D, -O(R), =0, -S(R), =S, =NR, =N(R), -N(R)(R’), -Se(R), =Se, -F, -Cl, -Br, -I, -F, -18F, - C(R)(R’)(R”), =C(R)(R’), =C(R), -Si(R)(R’)(R”), =Si(R)(R’), = Si(R), -OS(O)2R, -OS(O)R, -

[0161] S(O)R, -S(O)2R, -P(0)(R)(R’), -P(R)(R’), -P(0)(0H)0-, -S-, -N(R')-, -C(0)-, -C(0)0-, -0C(0)-, - 0 C(0) 0 -,-C(S)-, -C(S)O-, -OC(S)-, -OC(S)O-, -C(N0R)-, -C(0)N(R')-, -C(0)N(R)C(0)-, - C(0)N(R)C(0)N(R')-, -N(R)C(0)-, -N(R')C(0)N(R)-, -N(R)C(0)0-, -0C(0)N(R)-, -C(NR')-, - N(R)C(NR')-, -C(NR')N(R)-, -N(R)C(NR')N(R)-, -S(0) 2- , -0S(0)-, -S(0)0-, -S(0)-, -0S(0)2- -S(O)20-, -N(R)S(0)2- , -S(0)2N(R)-, -N(R')S(0)-, -S(0)N(R)-, -N(R)S(0)2N(R')-, - N(R')S(0)N(R')-, -S-S-, -0-Si(R)(R’)-0-,)-, -C(=N)N(R)(R’)-, -C(R)=C(R’)-, -C = C-, C3-12 cycloalkane, C3-12 cycloalkene, C3-12 cycloalkyne, 3- to l2-membered heterocycles, 5- to l2- membered aryl, 5- to l2-membered heteroaryl, any deuterium substituted derivatives, or any combination thereof, wherein R, R’, or R’’ is H, D, 1-100 polyethylene glycol, C1-C100 alkoxy, C1-C100 alkyl, C2-C100 alkylene, C2-C100 alkyne, C3-C100 cycloalkyl, C3-C100 cycloalkyene, C3-C100 cycloalkyne, C3-C100 heterocyclyl, C6-C100 aryl, or C1-C100 heteroaryl, wherein the one or both terminating groups may be the same or different. In certain embodiments, the chemical bond(s) or linker unit(s) can be multivalent chain unit(s) , which contain one or more than one core attached with one or more chemical bond or linker unit(s) in any equivalent and sequences, wherein m, and n represent integer from . In certain embodiments, the core includes, without limitation, an atom H, C, Si, N, P, B, O, S, Se, 1-100 polyethylene glycol, C100 alkoxy, C1-C100 alkyl, C2-C100 alkylene, C2- C100 alkyne, C3-C100 cycloalkyl, C3-C100 cycloalkyene, C3-C100 cycloalkyne, C3-C100 heterocyclyl, C6-C100 aryl, or C1-C100 heteroaryl, any deuterium substituted derivatives, analogs and chelates, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and pharmaceutically acceptable carrier. In certain embodiments, the chemical bond or linker unit(s) may be a bivalent or trivalent polyethylene glycol, alkoxy, alkyl, alkylene, alk ycloalkyl, cycloalkyene, cycloalkyne, heterocyclyl, aryl, heteroaryl, which may terminate (at either or both termini) in at least one of a -H, -D, -O(R), =O, -S(R), =S, =NR, =N(R), -N(R)(R’), -Se(R), =Se, -F, -Cl, -Br, -I, -F, -18F, -C(R)(R’)(R’’), =C(R)(R’), ≡C(R), -Si(R)(R’)(R’’), =Si(R)(R’), ≡Si(R), -OS(O)2R, - OS(O)R, -S(O)R, -S(O)2R, -P(O)(R)(R’), -P(R)(R’), -P(O)(OH)O-, -S-, -N(R')-, -C(O)-, -C(O)O-, -OC(O)-, - O C(O) O -,-C(S)-, -C(S)O-, -OC(S)-, -OC(S)O-, -C(NOR)-, -C(O)N(R')-, - C(O)N(R)C(O)-, -C(O)N(R)C(O)N(R')-, -N(R)C(O)-, -N(R')C(O)N(R)-, -N(R)C(O)O-, - OC(O)N(R)-, -C(NR')-, -N(R)C(NR')-, -C(NR')N(R)-, -N(R)C(NR')N(R)-, -S(O) 2- , -OS(O)-, - S(O)O-, -S(O)-, -OS(O)2- -S(O)20-, -N(R)S(O)2- , -S(O)2N(R)-, -N(R')S(O)-, -S(O)N(R)-, - N(R)S(O)2N(R')-, -N(R')S(O)N(R')-, -S-S-, -O-Si(R)(R’)-O-,)-, -C(=N)N(R)(R’)-, -C(R)=C(R’)-, - C ≡ C-, C3-12 cycloalkane, C3-12 cycloalkene, C3-12 cycloalkyne, 3- to l2-membered heterocycles, 5- to l2-membered aryl, 5- to l2-membered heteroaryl, any deuterium substituted derivatives, or any combination thereof, wherein R, R’, or R’’ is H, D, 1-100 polyethylene glycol, C1-C100 alkoxy, C1-C100 alkyl, C2-C100 alkylene, C2-C100 alkyne, C3-C100 cycloalkyl, C3- C100 cycloalkyene, C3-C100 cycloalkyne, C3-C100 heterocyclyl, C6-C100 aryl, or C1-C100 heteroaryl, wherein the one or both terminating groups may be the same or different. In some embodiment, m or n is integral from 0 to 50. In certain embodiments, the linker is a cleavable bond. Cleavable bonds include, without limitation, phosphate ester, amide, ester, dialkyl or diaryl dialkoxysilane, cyanoethyl group, sulfone, ethylene, glycolyl disuccinate, cyclic acetal, 2-N-acyl nitrobenzenesulfonamide, a- thiophenyl ester, unsaturated vinyl sulfide, sulfonamide, malondialdehyde (MDA)-indole derivative, levulinoyl ester, hydrazone, oxime, imine, acylhydrazone, alkyl thioester, thioester, disulfide bridges, azo compounds, 2-nitrobenzyl derivatives, phenacyl ester, 8-quinolinyl benzenesulfonate, coumarin, bis-arylhydrazone, bimane bi-thiopropionic acid derivatives, paramethoxybenzyl derivative, tert-butylcarbamate analogue, orthoester, acetal, aconityl, silyl ether, b-thiopropionate, phosphoramidate, disulfide, vinyl ether, polyketal, allyl ester, picolinate ester, vicinal diols, and selenium compounds. In certain embodiments, the agent(s) or probe(s) is a portion of a drug or therapeutic agent or diagnostic agent or chemical probe substituted derivatives, analogs and chelates, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and pharmaceutically acceptable carrier. In certain embodiments, the chemical arm(s) is an atom or an atom group, a compound, or a portion of compound which can bind with membrane component(s) or extracellular materials, including membrane proteins, extracellular proteins, carbohydrates, lipids, pathogens, particles, virus, bacteria, fungi, protozoa, vectors, cell debris, and another cell. Hence, the multivalent endocytic agent can be used to internalize cell membrane to form endosome, and subsequently traffic multivalent endocytic agent itself, membrane proteins, extracellular proteins, carbohydrates, lipids, pathogens, particles, bacteria, viruses, fungi, protozoa, vectors, cell debris, and another cell into cell or cell lysosomes. In certain embodiments, the chemical arm(s) is a binding moiety which can bind to an endocytosis-meditating membrane component(s), wherein the chemical arm is selected from, without limitation, an atom, a chemic uding reversible or irreversible covalent bond, a charged or chargeable (at the certain pH value) chemical moiety, a hydrophilic moiety, a lipophilic moiety, a reversible or irreversible covalent bond-containing chemical moiety, an agent, a portion of agent, membrane binding chemical fragments (MBCFs), substituted mono- or di-carboxylic acid derivatives (SMDAs), lipid and derivatives (LAs), substituted phosphoric acid derivatives, glyceride and derivatives (GAs), phospholipid and derivatives (PPAs), steroid, vitamin and derivatives (VtAs) , amino acid and peptides and derivatives (AAPs), mono-sugars, saccharide and derivatives (SCAs), nucleobase and nucleoside and nucleotide derivatives (NNNAs), reported membrane proteins binders and derivatives (RMPBs), any deuterium substituted derivatives, analogs and chelates, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and pharmaceutically acceptable carrier.

[0162] In certain embodiments for membrane binding chemical fragments, substituted mono- or di-carboxylic acid derivatives (SMDAs) includes, without limitation, saturated or unsaturated alkyl or heteroalkyl chain also include chains with 1—50 terminal carbolic acid moieties. Without limitation, the alkyl or heteroalkyl with terminal carbolic acid moieties are represented by propionic acid, butyric acid, valeric acid, hexanoic acid, enanthic acid, octanoic acid, nonanoic acid, decanoic acid, undecylenic acid, lauric acid, tridecanoic acid, myristic acid, palmitic acid, palmitic acid, hexadecanoic acid, heptadecanoic acid, stearic acid, nonadecanoic acid, arachidic acid, heneicosanoic acid, docosanoic acid, tricosanoic acid, tetracosanoic acid, pentacosanoic acid, hexacosanoic acid, tetradecenoic acid, pentadecanoic acid, palmitoleic acid, oleic acid, eicosenoic acid, docosenoic acid, tetracosanoic acid, eicosapentaenoic acid, docosatrienoic acid, docosahexaenoic acid, octadecadienoic acid, octadecatrienoic acid, eicosadienoic acid, eicosatrienoic acid, eicosatetraenoic acid, mevalonate acid, carotenoic acid, retinoic acid, dihydroretinoic acid, fenofibric acid, any deuterium substituted derivatives, analogs and chelates, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and pharmaceutically acceptable carrier.

[0163] In certain embodiments for membrane binding chemical fragments, the terminal carbolic acid moieties can be replaced, without limitation, with hydroxamic acid, hydroxamic acid ester, hydroxamic acid amide, carbonic acid, carbonic ester, carbonic amide, sulfonic acid, sulfonic ester, sulfonic amide, sulfurous acid, sulfurous ester, sulfurous amide, nitric acid, nitric ester, nitric amide, nitrous acid, nitrous ester, nitrous amide, boronic acid, boronic ester, boronic amide, phosphoric acid, phosphoric ester, phosphoric amide, phosphorous acid, phosphorous ester, phosphorous amide, phosphinic acid, phosphinic ester, phosphinic amide, pyrophosphoric acid, pyrophosphoric ester, pyrophosphoric amide, alcohol, aldehyde, anime, any deuterium substituted derivatives, analogs and chelates, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and pharmaceutically acceptable carrier.

[0164] In certain embodiments for membrane binding chemical fragments, the lipid and derivatives (LAs) include, without limitation, saturated or unsaturated C4-C100 alkyl chain optionally substituted with 0-6 R1groups, saturated or unsaturated C4-C100 heteroalkyl chain optionally substituted with 0-6 groups, glyceride, phospholipid, ceramides, any deuterium substituted derivatives, analogs and chelates, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and pharmaceutically acceptable carrier.

[0165] In certain embodiments for membrane binding chemical fragments, the saturated or unsaturated alkyl or heteroalkyl chain includes, without limitation, phytoene, phytofluene, neurosporene, lycopene, didehydrolycopene, apolycopene, aponeurosporene, diaponeurosporene, cryptoxanthin, lutein, zeaxanthin, phytoene, carotenal, retinal, squalene, squalane, squalene 2,3- oxide, squalene 2,3:22,23 -dioxide, polypodatetraene, isodammara-20(21), 24-diene, isodammara- 12,24-diene, dammara- 13 (17), 24-diene, eupha-7, 24-diene, dammara-20(21), 24-diene, oxidosqualene, farnesol, farnesyl acetate, l l-hydroxy-10,l l-dihydrofarnesyl acetate, 10-bromo- 11 -hydroxy- 10, 11 -dihydrofamesol, 10, 11 -epoxy farnesyl acetate, 10,11 -epoxyfarnesol, any deuterium substituted derivatives, analogs and chelates, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and pharmaceutically acceptable carrier.

[0166] In certain embodiments for membrane binding chemical fragments, the glyceride and derivatives (GAs) includes, without limitation, monoerucin, monolaurin, monomyristin, monopalmitin, monostearin, l,3-dioleoyl-2-palmitoyl-glycerol, 1,3-dipalmitolein, 1,2-diolein, 1,3-diarachidonin, 1,3 -dipalmitin, 1,2-dipalmitin, 1,3 -di stearin, tripalmitolein, trielaidin, tripetroselaidin, trilinolein, trimyristin, tripalmitin, tristearin, 1,3-dipalmitelaidin, 2-acetyl-l,3- dicaffeoyl glycerol, 2-acetyl-l-caffeoyl-3-coumaroyl glycerol, 2-acetyl-l-feruloyl-3-caffeoyl glycerol, 2-acetyl-l-feruloyl-3-coumaroyl glycerol, 2-acetyl-l,3-diferuloyl glycerol, 2-acetyl-l- caffeoyl -3 -cinnamoyl glycerol, 2-acetyl-l,3-dicoumaroyl glycerol, 2-acetyl-l-coumaroyl-3- feruloylglycerol, acetyl coumaroyl glycerol, coumaroyl glycerol, 1,3-dicoumaroyl glycerol, 1- coumaroyl-3-caffeoyl glycerol, caffeoyl glycerol, tricoumaroyl glycerol, coumaroyl feruloyl glycerol, dicaffeoyl coumaroyl glycerol, dicaffeoyl feruloyl glycerol, any deuterium substituted derivatives, analogs and chelates, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and pharmaceutically acceptable carrier.

[0167] In certain embodiments for membrane binding chemical fragments, the phospholipid and derivatives (PPAs) includes, without limitation, phosphatidic acid, cardiolipin (CL), lysobisphosphatidic acid (LBPA), lysophosphatidic acid (LPA), phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylserine (PtSer), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidyserine (PS), phosphatidylinositol (PI), phosphatidylinositol, sphingomyelin, any deuterium substituted derivatives, analogs and chelates, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and pharmaceutically acceptable carrier.

[0168] In certain embodiments for membrane binding chemical fragments, the steroid includes, without limitation, cholesterol, ergosterol, lithocholic acid, 7-dehydrocholesterol, 22,23- duhydroergosterol, 7-dehydrositosterol, 7-dehydrostigmasterol, 7-dehydrocamperterol, pregnenolone, 17a-hydroxypregnenolone, 16a-hydroxypregnenolone, 20a-dihydropregnenolone, dehydroepiandrosterone (DHEA), 7a-hydroxy-DHEA, 7-oxo-DHEA, 7P-hydroxy-DHEA, 5- androstene-30, 17|3-diol, 5-androstene-3p,7a,17p-triol, 5-androstene-3p,7p,17p-triol, 5- androstene-3p,16a,17p-triol, progesterone, 17a-hydroxyprogesterone, 17a,20a-dihydroxy-4- pregnene-3-one, 16a-hydroxyprogesterone, 20a-dihydroprogesterone, androstenedione, testosterone, 16a-hydroxytestosterone, 5a-dihydrotestosterone, estrone, estradiol, estriol, 5a- dihydroprogesterone, allopregnanolone, isopregnanolone, 5P-dihydroprogesterone, pregnanolone, epipregnanolone, 5a,20a-tetrahydroprogesterone, 5a-pregnane-3a,20a-diol, 5a-pregnane-3p,20a- diol, 5p,20a-tetrahydroprogesterone, 5P-pregnane-3a,20a-diol, 5P-pregnane-3p,20a-diol, 17a- hydroxyallopregnanolone, 17a-hydroxypregnanolone, 5a-pregnane-3a,17a,20a-triol, 5a- pregnane-3p,17a,20a-triol, 5p-pregnane-3a,17a,20a-triol, 5a-androstane-3, 17-dione, androsterone, epiandrosterone, etiochol anol one, 5a-androstane-3a,17P-diol, 5a-androstane- 3p,17p-diol, 5a-androstane-3a,17p-diol, cortisol, cortisone, corticosterone, 21 -deoxycortisol, 11- deoxy corticosterone, 3a,5a-tetrahydrocorticosterone, 3a,5P-tetrahydrocorticosterone, l ip- hydroxyandrostenedione, tetrahymanol, l ip-hydroxyandrosterone, l ip-hydroxy epiandrosterone, l ip-hydroxyetiocholanolone, hopene, hopanol, hop-22(29)-ene, hopan-22-ol, diplopterol, tetrahymanol, bacteriohopaneteirol, aminobacteriohopanetriol, lanosterol, 24,25-oxidolanosterol, 24,25-epoxycholesterol, pregnenolone sulfate, 17a-hydroxypregnenolone sulfate, 20a- dihydropregnenolone sulfate, DHEA sulfate, androstenediol sulfate, 5-androstene-30,16a,170- triol sulfate, conjugated 17a,20a-dihydroxy-4-pregnen-3-one, conjugated 20a- dihydroprogesterone, conjugated testosterone, conjugated epitestosterone, estrone sulfate, estradiol sulfate, estriol sulfate, allopregnanolone sulfate, isopregnanolone sulfate, conjugated pregnanolone, conjugated epipregnanolone, conjugated 5a,20a-tetrahydroprogesterone, conjugated 5a-pregnane-3a,20a-diol, conjugated 5a-pregnane-30,2Oa-diol, conjugated 50,20a- tetrahydroprogesterone, conjugated 50-pregnane-3a,2Oa-diol, conjugated 50-pregnane-30,2Oa- diol, 17a-hydroxyallopregnanolone sulfate, conjugated 17a-hydroxy pregnanolone, 5a-pregnane- 3a,17a,20a-triol, 5a-pregnane-30,17a,2Oa-triol, 50-pregnane-3a,17a,2Oa-triol, androsterone sulfate, epiandrosterone sulfate, etiocholanolone sulfate, epietiocholanolone sulfate, conjugated 5a-androstane-3a,170-diol, conjugated 5a-androstane-30,170-diol, conjugated 50-androstane- 3a,170-diol, conjugated 50-androstane-30,170-diol, conjugated 3a,5a-tetrahydrocorticosterone, conjugated 3a,50-tetrahydrocorticosterone, 110-hydroxyandrosterone sulfate, 110- hydroxy epiandrosterone sulfate, 110-hydroxy etiocholanolone sulfate, any deuterium substituted derivatives, analogs and chelates, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and pharmaceutically acceptable carrier.

[0169] In certain embodiments for membrane binding chemical fragments, the vitamin and derivatives (VtAs) includes, without limitation, coenzyme Q10, vitamin A, vitamin B 1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenate), vitamin B6 (pyridoxine), vitamin B7 (biotin), vitamin B8 (inositol), vitamin B9 (folic acid), vitamin B12 (cobalamins or methylcobalamin), vitamin C, vitamin D2 (ergocalciferol), vitamin D3 (cholecalciferol), vitamin D4 (22-dihydroergocalciferol), vitamin D5 (sitocalciferol), vitamin D6 (calciferol), vitamin D7, vitamin E, vitamin KI (phylloquinone), vitamin KiO (phylloquinone epoxide), vitamin K2 (menaquinones), vitamin K3 (menadione), any deuterium substituted derivatives, analogs and chelates, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and pharmaceutically acceptable carrier.

[0170] In certain embodiments for membrane binding chemical fragments, the amino acid and peptides and derivatives (AAPs) include, without limitation, glutamic acid (Glu), glutamine (Gin), glycine (Gly), alanine (Ala), valine (Vai), leucine (Leu), isoleucine (He), phenylalanine (Phe), tryptophan (Trp), tyrosine (Tyr), aspartic acid (Asp), histidine (His), asparagine (Asn), lysine (Lys), methionine (Met), arginine (Arg), serine (Ser), threonine (Thr), cysteine (Cys), proline (Pro), 2-60 amino acids containing peptides, any deuterium substituted derivatives, analogs and chelates, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and pharmaceutically acceptable carrier.

[0171] In certain embodiments for membrane binding chemical fragments, the saccharide and derivatives (SCAs) include, without limitation, monosaccharides, oligosaccharide, polysaccharides, glycosides, glycoproteins, glycolipids.

[0172] In certain embodiments for membrane binding chemical fragments, the saccharide and derivatives (SCAs)includes, without limitation, D-ribose, Z-ribose, D-arabinose, Z-arabinose, D- xylose, Z-xylose, ZMyxose, Z-lyxose, D-allose, Z-allose, D-altrose, Z-altrose, D-glucose, L- glucose, D-mannose, Z-mannose, D-gulose, Z-gulose, D-ldose, Z-ldose, D-galactose, Z-galactose, D-talose, Z-talose, D-fucose, Z-fucose, A-acetylneuraminic acid, A-acetyl-D-glucosamine, N- acetyl-D-galactosamine, trehalose, maltose, sucrose, cellobiose, kestoses, raffinoses, nystose, fructosyl-nistose, glucan, arabinoxylan, apigenin-7-O-glucoside, quercetin-3-O-glucoside, isorhamnetin-3-O-rutinoside, kaempferol -p-coum aroyl rhamnoside, quercetin-3-O-rutinoside, quercetin-3-O-glucuronide, kaempferol-3-O-rutinoside, isorhamnetin-O-pentoside, quercetin-3- O-rhamnoside, isorhamnetin-O-glucuronide, kaempferol -methyl ether-Oglucoside, isorhamnetin- O-acetylrutinoside, rhamnetin-O-glucuronide, quercetin-dimethyl ether-O-rutinoside, quercetin- dimethyl ether-O-glucuronide, kaempferol-O- / ?-coumaroyl rhamnoside, quercetin-7-O-glycoside, luteolin 7-O-glucoside, naringenin 7-rhamnoglucoside, cerebrosides, gangliosides, glucosylcerebrosides, lactosylceramides, hopane glycolipid, oligosaccharide or polysaccharides containing 2-100 units of monosaccharide and / or derivatives and can be straight or branched, any deuterium substituted derivatives, analogs and chelates, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and pharmaceutically acceptable carrier.

[0173] In certain embodiments for membrane binding chemical fragments, the nucleobase and nucleoside and nucleotide derivatives (NNNAs) include, without limitation, adenine, guanine, thymine, cytosine, uracil, hypoxanthine, xanthine, epiguanine, dihydrouracil, adenosine, guanosine, thymidine, cytidine, uridine, inosine, xanthosine, 7-methylguanosine, dihydrouridine, cAMP, pppGpp, NADP, FAD, ATP, ADP, AMP, GTP, GDP, GMP, UTP, UDP, UMP, CTP, CDP, CMP, TTP, TDP, TMP, any deuterium substituted derivatives, analogs and chelates, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and pharmaceutically acceptable carrier.

[0174] In certain embodiments, the reported membrane proteins binders and derivatives (MPBDs) include, without limitation, an atom, drug, a portion of a drug, or derivative and chelates, any deuterium substituted derivatives, a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, that targets endocytosis-mediating membrane components.

[0175] In certain embodiments, the chelating atom includes, but is not limited to, Ag+, Cu+, Au+, Hg2+, Pb2+, Cu2+, Cd2+, Zn2+, Se2', Se4+, Se6+, and Sb3+.

[0176] In certain embodiments, the selected drug, a portion of a drug, or drug derivative and chelates (ADDs), any deuterium substituted derivatives, a pharmaceutically acceptable salt or stereoisomer, or any combination thereof are reported ones targeting proteins. In certain embodiments, the target protein includes, but not limited to, target proteins listed in patent US20210002296 Al, including all variants, mutations, splice variants, indels and fusions of these target proteins listed. Examples include, without limitation, 5HT2c receptor, OUA-AR, a-2 adrenergic receptor, a-synuclein, AAK1, ATP -binding cassette (ABC) transporters such as MDR1 / 2 / 3 / 4 / 5 and ABCG2, ABL, cholesterol acyltransferases (ACATs), ACE protein, ACK, ACTR2, ACTR2B, ADCK1 / 2 / 3 / 4 / 5, protein kinase B (AKT), AlphaKl / 2 / 3, amino acids transporters, ALIX, anaplastic lymphoma kinase (ALK), Amnionless, AMPA receptors (AMPARs), AMP-related protein kinase (AMPK), amyloid beta, Angiotensin II receptor, ANKRD3, Apaf-1, apolipoprotein E receptor (ApoER), amyloid precursor protein (APP), androgen receptor (AR), ARAF, ARG, AR-V7, ileal apical sodium / bile acid co-transporter (ASBT), V-type proton ATPase 6 (ATP6V), ATP6V1H, avidin, anine serine cysteine transporters (ASCTs), ASGPR, ASK1 / 2, Ataxin-1, ataxia-telangiectasia mutated protein (ATM), ATM and Rad3 -related protein (ATR), Aurora kinases, AXL, p-site amyloid precursor protein-cleaving enzyme 1 (BACE1), p2-adrenergic receptors, BAD, BARK1 / 2, Bax, BCKDK, B-cell leukemia / lymphoma (BCLs) family proteins such as BCL2, BCL-XL, and MCL-1, BCR-ABL, bromodomain and extra-terminal domain family proteins (BETs) such as BRD2 / 3 / 4 / T, beta- catenin, BIKE / BMP2K, BLK, BMPR1A, BMPR1B, BMPR2, BMX / ETX, brother of CDO (Boc), BRD9, BMI1, BRAF, BRAFV600E, brassinosteriod insensitive 1 (BRI1), BRK, BRM, BRSK1, BRSK2, Bruton tyrosine kinase (BTK), BUB1, C3G, calreticulin, cell adhesion molecule (CAM) receptors, CAMKla / p / y / 5, CAMK2a / p / y / 5, CAMK4, caMLCK, cannabinoid- 1 receptor, coxsackievirus-adenovirus receptor (CAR), CRISPR associated proteins (Cas), CASK, Caspase- 3, Caspase-6, Caspase-7, Caspase-9, CBF0, CBL-B, CBP, chemokine receptors such as CC chemokine receptors (CCRs) and CXC chemokine receptors (CXCRs) such as CXCR2, CXCR4, and CXCR7, CCK4 / PTK7, CCR2, CCR9, CCRK, cluster of differentiations (CDs) such as CD4, CD9, CD13 / APN receptor, CD25, CD26 (DPP4), CD30, CD33, CD36, CD38, CD46, CD56, , CD63, CD71, CD74, CD80, CD81, CD82, CD86, CD123, CD138, CD147, CD152, CD152 (CTLA4), CD166, CD174, CD197, CD205, CD227, CD228, CD269, CD276, and CD326, CDC7, CDC20, CDC25, CDC37, CDK1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12 / 13 / 14 / 15 / 16 / 17 / 18, CDKL 1 / 2 / 3 / 4 / 5, CDON, CEACAM5, cholesteryl ester transfer protein (CETP), c-Fos, cystic fibrosis transmembrane conductance regulator (CFTR), cyclic GMP-AMP synthase (eGas), Chakl / 2, CHK1 / 2, CI-M6PR, CKla, CKla2, CK15, CKle, CKlyl / 2 / 3, CK2al / 2, CLIK1, CLIK1L, Clip, CLK1 / 2 / 3 / 4, human collectin placenta 1 (CL-P1), CMYC, cone opsins, COT / TPL2, cell- penetrating peptide (CPP), connexins, coronavirus protease, CRABP, C-RAF, cereblon (CRBN), CREB, CRK, CRIK, CRIPTO, CSF2R, CSF1R / FMS, CSK, C-TAK1, CTK, cubilin, cyclin D, cyclin E, CytoC, DAPK1 / 2 / 3, DCAMKL1 / 2 / 3, DDR1 / 2, diglyceride acyltransferase (DGAT), DLK, DMPK1 / 2, DNA-PK, DP-1, DPP-4, DRAK1 / 2, deubiquitylating enzymes (DUBs), DYRK1A / 1B / / 2 / 3 / 4, E2F, excitatory amino acid carrier 1 (EAAC1), E-cadherin, endothelin- converting enzyme (ECE), ED-B, EED, EF2K, eEF2K, EIF2A3, EIF4E, EIF4G, ELK, ENL, endothelin B receptor, epithelial cell adhesion molecule (EpCAM), endothelial cell protein C receptor (EPCR), Ephrin receptors (EphRs), estrogen receptor (ER), ER81, ErbB2, ErbB3, ETS transcription factor (ERG), ERK 1 / 2 / 3 / 4 / 5 / 7 / 8, ERRa, EZH2, fatty acid binding proteins (FABPs), FADD, FAK, FAP, fatty acid transport proteins (FATPs), neonatal Fc receptor (FCRN), FER, FKBPs, FLIP, Flotillin-1, Flotillin-2, folate receptors such as reduced folate carrier, FOLT1 / 2 / 3 and proton-coupled folate transporter (PCFT), F0XM1, FOXO1 / 2 / 3 / 4, FRK, Frizzled4, fructose- 1,6-bisphosphatase, FYN, y-aminobutyric acid type A receptors (GAB AAR), GAK, Gap-1, growth arrest specific 1 (Gasl), GCC, GCK, GCN2, GD2, ghrelin receptor (ghrelinR), glucose- dependent insulinotropic polypeptide (GIP), Globo H, glucagon receptor, glucagon-like peptide 1 (GLP-1), glutamate transporters (GLTs), glucokinase, Ionotropic AMPA glutamate receptors (GluRs), glucose-6-phosphatase, glucose transporters (GLUTs), glutathione transporter, glycogen phosphorylase, glycogen synthase kinase, glycoproteins such as gpl 8, gp31, and gp60, G protein- coupled receptors (GPCRs) such as GPR20, GPR20, free fatty acid receptor 1 (GPR40), GPR119, and GPR120, GPNMB, GPRK4 / 5 / 6 / 7, GRB2, GSK-3, GSPT1, Haspin kinase, HCK, hepatitis C virus (HCV) NS3 / 4A, histone deacetylases (HDACs), HDL receptor (HDLR), HectH9, HH498, FHF1, FHPK1 / 2 / 3 / 4, HMGN1, HMG-CoA reductase, hnRNPs, H-PGDS, HPGCR, hematopoietic progenitor kinase 1 (HPK1), HRAS, HRI, hRpnl3Pru, HSD-110 (11 P-hydroxysteriod dehydrogease), heat shock proteins (HSPs), huntingtin protein (HTT), HUNK, inhibitor of apopotosis (IAP) proteins such as cIAPs and XIAP, ICK, intermediate-density lipoprotein receptor (IDLR), IDO-1, IFN-yR, IGF-1R, IGF-2r, IKK-alpha, IKK -beta, IKK -gamma, IKK- epsilon, Ikaros (IKZF1), Helios (IKZF2), Aiolos (IKZF3), IKZF4, IL-4R, IL-10R, ILK, integrins such as aVp3, a4pl and a5p l integrins, insulin receptors (IRs), insulin-like growth factor receptor, INSR, IRE1 / 2, IRR, ITKIMP, integrin receptors (IRs), IRA2, IRAK1 / 2 / 3 / 4, IRE1, IRR, Janus tyrosine kinase (JAKs), c-Jun N-terminal kinases (JNKs), potassium-chloride cotransporter 2 (KCC2), KDM5C, KHS1 / 2, inward rectifier potassium channel (Kir2.3), KIS, KIT, Kelch domain- containing protein 2 (KLHDC2), KLF4, KRAS, KRASG12C, KSR1 / 2, Lamin, lysosome associated membrane proteins (Lamps), LAMT0R2, LANA, lactoferrin receptor, L-amino acid transporters (LATs), LATS1 / 2, LCK, low-density lipoprotein receptor (LDLR), LEF, LIMK1 / 2, LKB1, LMR1 / AATK, LMR2 / 3, LOK, low-density lipoprotein receptor related proteins (LRPs), LRRK1 / 2, LTK, LXR-£, LYN, LZK, LZTFL1, m5C, m6A, MAK, MALTs, MAP3K4 / 8, MAP4K3, MAP4K5, MAPK, MAPKlb, MAPKAPK2 / 3 / 5, MARK1 / 2 / 3 / 4, MAST1 / 2 / 3 / 4 / L, MC4 receptor, MCAK, MCH-1 receptor, monocarboxylic acid transporters (MCTs), mouse double minute 2 homolog (MDM2), MDMx, megalin, mitogen-activated protein kinase (MEKs), MEKK1 / 2 / 3, MEL, MELK, MER, MERTK, mesothelin, MET, Mfsd2a, metabotropic glutamate receptors (mGluls), major histocompatibility complex class I proteins (MHC-I), MHC-II, MIF, MINCLE, MINK1, MISR2, MITF, MKK1 / 2 / 3 / 4 / 6 / 7, MKP3, MLK1 / 2 / 3 / 4, MLKL, MLN1 / 3, MNK1 / 2, MOK, MOS, MPK, MPSK1, MRCKa / p, multidrug resistance proteins (MRPs), MSK1 / 2, MSSK1, MST1 / 2 / 3 / 4, mammalian target of rapamycin (mTOR), MUCL, MUSK, Myc proteins, MYO3A / 3B, MYT1, NAMPT, N-cadherin, K+-dependent Na+ / Ca2+exchanger 2 (NCKX2), NDC80, NDR1 / 2, NEK1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11, NF-kappa B, Nicastrin, nicotinic acetylcholine receptor, NIK, NLK, NOTCH receptors, niemann-Pick Cl -like 1 (NPC1L1), N- methyl D-aspartate receptors (NRs), NRAS, NRBP1 / 2, NRF2, NSD2, NSD3, nucleoside transporters (NTs, such as hENTl, hCNTl-3, etc.), sodium / taurocholate co-transporting peptide (NTCP), NuaKl / 2, NUAK1, organic anion transporters (OATs), organic anion transporting polypeptides (OATPs, 0ATP1B1, 0ATP2B1, OATP4C1, etc.), Obscurin, organic cation transporters (OCTs), OSR1, organic solute transporter (OST), otoferlin, P2X purinoceptor 4 receptor (P2x4R), purinergic receptor P2Y12 (P2Y12), pl6INK4A, pl8INK4, pl9INK4D, p21, p27kipl, p38a, p380, p38y, p388, p53, p65 / RELA, p70S6K, p70S6Kb, p90, p300 protein, PAK1 / 2 / 4 / 5 / 6, pan-coronavirus antiviral, protease-activated receptors (PARs), PARPs, PASK, PAX, PBRM1, PCSK9, PD-1, phosphodiesterases (PDEs), PDE4, PDE5, PDGFRa / 0, pdhkl / 2 / 3 / 4, PDK1, PD-L1, PEA-15, PEIZO1, PEIZ02, PEK, peptide transporters (PEPTs), p-glycoprotein (P- gp), PHKyl / 2, phosphoenol pyruvate carboxykinase, phosphoinositide 3-kinase (PI3K), PIM1 / 2 / 3, PINK1, PITSLRE, PKA, PKACa / 0 / y, PKBa / 0, PKC, PKD1 / 2 / 3, PKG1 / 2, PKN1 / 2 / 3, PKR, PLC, PLDL, PLK1 / 2 / 3 / 4, PMEL17, PRAK, peroxisome proliferator-activated receptors (PPARs), PRC2, PRK2, PRKX, PRKY, PRMT5, protein phosphatase, protein tyrosine phosphatase (PTP), PRP4, PRPK, PSKH1 / 2, prostate-specific membrane antigen (PSMA), receptor patched 1 (PTCHI), PTEN, protein tyrosine kinases (PTKs), PVRL4, pyruvate dehydrokinase, PYK2, QIK, QSK, R1P1, RACK-1, Rac proteins, RAF protein, RAP1, Raptor, RAR, Ras protein family (RAS), Rb, retinol binding proteins (RBPs), RET, riboflavin transporter proteins (RFVTs), RFX1, RHAU, RHODK, RI0K1 / 2, receptor interacting protein kinases (RIPKs), rhodopsin, ribonuclease K (RNASEK), RNAseL, ring finger proteins (RNFs), R0CK1 / 2, renal outer medullary potassium channel (ROMK), RON, R0R1 / 2, ROS, RPN11, RPN13, ribosomal s6 kinases, receptor serine / threonine kinases (RSKs), such as transforming growth factor 0 (TGF-0) receptor and proteins as listed in Aristidis Moustakas, et al, Receptor Serine / Threonine Kinases (ISBN: 978-3- 540-44244-8), receptor tyrosine kinases (RTKs), such as fibroblast growth factor receptors (FGFR), FLT1 / 2 / 3 / 4, FGFR1 / 2 / 3 / 4, RET, tropomyosin-related kinases (TRKs), epidermal growth factor receptor (EGFR), human epidermal growth factor receptors (HERs), vascular endothelial growth factor receptors (VEGFRs), TYRO3 and other proteins as listed in publication Mark A. Lemmon and Joseph Schlessinger, Cell, 141(7) 1117-1134, 2010, RUNX1 / 2 / 3, RYK, spindle assembly checkpoint (SAC), Sanpodo / Notch, SAP, SARS-CoV-2, SBK, solute carriers (SCs), SCF, SCYL1 / 2 / 3, SF3B1, serine / threonine-protein kinases (SGKs), SGLTs, SHC, SHP2, SIK2 / 3, SIRT2, SIX1, skMLCK, S-phase kinase associated protein 1 (SKP1), S-phase kinase associated protein 2 (SKP2), solute carriers (SLC, such as SCL19A1) transporters, such as hMATEl and proteins listed in publication Enrico Girardi, et al., Nature Chemical Biology, 16, 469-478, 2020, SLK, SLOB, Smac, Smad proteins, SMARCAs, sodium-coupled monocarboxylate transporters (SMCTs), SMG1, smMLCK, sodium dependent multivitamin transporter (SMVT), synaptosomal- associated protein (SNAP), SNRK, sortilin-related CNS expressed la (SorCSla), SorCSlc, protein son of sevenless (SOS), SOS1, SOX2, secreted protein acidic and rich in cysteine (SPARC), SPEG, speckle type BTB / POZ protein (SPOP), SPRED, SPRY, SPv-NKIR, scavenger receptors (SRs) such as CD36, LAMP1, andLAMP2, Src protein, SRF, SRM, SRPK1 / 2, SSTK, StaO, signal transducer and activator of transcription proteins (STATs), STING, serine / threonine kinases (STKs), STLK3 / 5 / 6, syntaxins (STXs), SuRTK106, SUV39HI, SUZ12, sodium-vitamin C co- transporters (SVCTs), synaptotagmins (SYTs), triiodothyronine (T3), TAB, TAK1, TAL, TAO1 / 2 / 3, Tau protein, TBCK, TBK1, t-cell factor / lymphoid enhancer-binding factors (TCF / LEF family), TEC, TESK1 / 2, TFAM, transcription factor EB (TFEB), transferrin receptor (TfR), TGF- P, trans-Golgi network (TGN38), thiamine transporters (THTRs), TIE1, TIE2 / TEK, TIFla / p / y, Titin / TTN, TLK1 / 2, toll-like receptors (TLRs) such as TLR4, TNK1, TOPK, TPL2 / COT, TPNlp, TRAD, TRAF proteins, Trb 1 / 2 / 3, tripartite motif family proteins (TRIMs), Trio, TRRAP, TSC2, TSG101, TSSK1 / 2 / 3 / 4, TTBK1 / 2, TTK, TXK, TYK2, tyrosinase, tubulin, UBF, UCHL5, ULK1 / 2 / 3 / 4, ubiquitin specific peptidase (USP) such as USP7, USP11, and USP14, VACAMKL protein, vesicle associated membrane proteins (VAMPs), vitamin D receptors (VDR), vesicular GABA transporter (VGAT), vesicular glutamate transporters (VGLUTs), very low-density lipoprotein receptor (VLDLR), urate transporter 1 (URAT1), VRK1 / 2 / 3, WAVE-2, WDR5, WEE- 1, WEE-1B, WNK1 / 2 / 3 / 4, Wnt, XBP1, YANK1 / 2 / 3, YBX1, YES, YSK1, ZAK, ZAP70, ZC1 / 2 / 3 / 4, ZFP91, and zinc ring finger proteins (ZRNFs), including all variants, mutations, splice variants, indels and fusions of these target proteins listed.

[0177] In certain embodiments, the multivalent endocytic agent is a bivalent or multiple valent agent for protein inhibition and chemical modification of protein, DNA, or RNA including not limited to protein ubiquitination, protein de-ubiquitination, protein phosphorylation, protein methylation, protein acetylation, protein folding or unfolding, and DNA / RNA degradation.

[0178] In certain embodiment, the bivalent or multiple valent agents are a protein degrader, including not limited to proteolysis-targeting chimeras (PROTACs). A PROTAC degrader of proteins after protein ubiquitination is compound that physically degrades a target protein by cleaving one or more bonds of the target protein by proteolysis. PROTAC also includes all PROTAC variances such as photocaged PROTAC, tag-based PROTACs, etc. Exemplary protein degraders with other mechanisms include, without limitation, protein molecular degraders, autophagosome-tethering compounds (ATTECs), autophagy-targeting chimeras (AUTACs), chaperone-mediated protein degraders (CHAMPs), BacPROTAC, ASGPR targeting chimeras (ATACs), lysosome-targeting chimeras (LYTACs), mitochondrial protease targeting chimera (MtPTAC). The bivalent or multiple valent agent also include deubiquitinase-targeting chimeras (DUBTACs), RESTORAC, enhancement-targeting chimera (ENTAC), and phosphorylation- inducing chimeric small molecules (PHICSs), ribonuclease targeting chimeras (RIBOTACs), phosphatase recruiting chimera (PhoRC), dephosphorylation-targeting chimera (DEPTAC), phosphorylation-targeting chimeras (PhosTAC), phosphorylation-inducing chimeric small molecules (PHICS), acetylation tagging molecule (AceTAG), regulated induced proximity targeting chimeras (RIPTAC), transcriptional / epigenetic chemical inducers of proximity (TOP), caspase cleavage targeting chimeras (CACTACs), their deuterium substituted derivatives, their analogs, or combinations thereof.

[0179] In certain embodiments, functional moieties in bivalent or multiple valent agents or bivalent or multiple valent agents themself are represented by any of the following compounds: 3- aminophthalic acid (CAS: 5434-20-8), 4-aminoisoindoline-l, 3-dione (CAS: 2518-24-3), 4- aminoisobenzofuran-1, 3-dione (CAS: 17395-99-2), 5-aminoisoindoline-l, 3-dione (CAS: 3676- 85-5), 5-aminoisobenzofuran-l, 3-dione (CAS: 17011-53-9), 4-aminophthalic acid (CAS: 5434- 21-9), 753b, A7, A16, A031, A1874 (CAS: 2064292-12-0), ABBV-101, Ab-PROTAC 3, ACBI1 (CAS: 2375564-55-7), ACBI3, AC-0682, AC-0716, AC-0676, ACBI2 (CAS: 2913161-19-8), AGB1, AM-A3, AMG232 (CAS: 1352066-68-2), AP-01-104, APG-265, APR-Cy3, AR-VHL- SF2, AR2-VHL-SF2, ARCC-4 (CAS: 1973403-00-7), ARD-69 (CAS: 2316837-10-0), ARD-61 (CAS: 2316837-08-6), ARD-69, ARD-2128 (CAS: 2222111-87-5), ARD1676, GT20029, ARD- 266, ARD-2051 (CAS: 2632305-17-8), ARD-2585 (CAS: 2757422-79-8), AR-ARL, AR-LDD, ARV110 (CAS: 2222112-77-6), ARV-766 (CAS: 2750830-09-0), ARV-471 (CAS: 2229711-68- 4), ARV-771 (CAS: 1949837-12-0), ARV-825 (1818885-28-7), ARV-763, ASP-3082, AT-1 (CAS: 2098836-45-2), ATTEC, AU-15330, AUTAC4, Avadomide, AZD9496-based PROTAC (CN112979747A), Azo-PROTAC-4C, 0-NF-ATRA, BC5P, BCPyr, BETd-260 (CAS: 2093388- 62-4), B03, BGB-16673, BI-3663 (CAS: 2341740-84-7), BI3802 (CAS: 2166387-65-9), BP3, BRD4-SF2, BSJ-4-116 (CAS: 2519823-34-6), BT1, BTX-1188, BTX-9341, BWA-522, C004019, C13 (Jingyu Zhang, et al., Journal of Medicinal Chemistry, 65, 13, 9096-9125, 2022), CC885 (CAS: 1010100-07-8), CC-90009 (CAS : 1860875-51-9), CC-92480 (CAS: 2259648-80-9) (Joshua D. Hansen, et al., Journal of Medicinal Chemistry, 63, 6648-6676, 2020), CC-94676, CC- 99282, CCR9-PROTAC, CCT369260, CCW 28-3, CDDO-JQ1, CDDO-Me, CFT7455 (NCT04756726), CFT8634 (CAS: 2704617-96-7), CFT8919, CFT1946 (CAS: 2882165-79-7), CG416, CG428, CG001419, CL1-YL2, CLL1-5, CM11, Compound 3 (Mingming Wei, et al., European Journal of Medicinal Chemistry, 209, 112903, 2021), Compound 6 (Archana Bhumireddy, et al., Bioorganic & Medicinal Chemistry Letter, 55, 128448, 2022), Compound 6c (Lijie Peng, et al., ACS Medicinal Chemistry Letters, 10, 767-772, 2019), Compound 9 (Joao Nunes, et al., ACS Medicinal Chemistry Letters, 10, 1081-1085, 2019), Compound 21b (Guoshun Luo, et al., Acta Pharmaceutica Sinica B, 11(5), 1300-1314, 2021), Compound 955 (Jing Pei, et al., Cell Chemical Biology, 30, 203-213, 2023), CP5V (CAS: 2509359-75-3), CP-10 (CAS: 2366268-80-4), CPD-1224 (CAS: 2891620-68-9), CPR3, CPR4, CPS2, CRBN-6-5-VHL, D15 (Pengyun Li, et al., Acta Pharmaceutica Sinica B, 13(6), 2715-2735, 2023), d4E-4, d4E-6, d9A-2, dBETl (CAS: 1799711-21-9), dBET6 (CAS: 1950634-92-0), DBt-10 (Martin Schroder, et al., bioRxiv preprint, 2023, DOI: 10.1101 / 2023.04.09.536153), dCBP-1 (CAS: 2484739-25-3), DCY- 09-192, DD-03-171 (CAS: 2366132-45-6), DD-04-015, dFKBP-1 (CAS: 1799711-22-0), DGY- 04-035, DGY-06-177, DGY-06-177-pkl, DGY-06-177-pk2, DGY-09-192, DKY709 (NCT03891953), dMCLl-2 (CAS: 2351218-88-5), DPI, DP-C-1, DP-C-4, DP-V-4, DS-3032 (CAS: 1398568-47-2), DT2216 (CAS: 2365172-42-3), dTRIM24 (CAS: 2170695-14-2), ERD- 148, ERD-308 (CAS: 2320561-35-9), ERD-3111 (CAS: 2832865-25-3), ERG OP-CI, ER PROTAC (ES2717436T3), FA-S2-POMA, FA-S2-MS4048, FHD-609 (CAS: 2676211-64-4), Folate-ARV-771, Folate-MS432, Folate-MS99, Fulvestrant, G4-PROTAC, GBD-9, GBM-475, GMB-475 (CAS: 2490599-18-1), GNE-987, GSK215, GT20029, GT19630, GT19715, GW3965- PEG5-VH032, HaloPROTAC-3, HBL-4, HC-X029, HC-X035, HD-TAC7, HJM-561, HER2-14, HP14, HP17, HP518, HP568, HPB-143, HRS-1358, HRS-5041, HSK29116, 1-6, 1-685, Iberdomide, INY-03-041, ITRI-90, ITRI-125 and ITRL126 (Chiu-Lien Hung, et al., eBioMedicine, 90, 104500, 2023), JB170, JH-XI-10-02 (CAS: 2209085-22-1), JP-1, JP-2, JP-3, JP-4, JP-5, JP-6, JPX-0802, JPX-1185, JPX1188, JNJ-1013, JMKX000623, KB02-JQ-1 (CAS: 2384184-44-3), KB02-SLF (CAS: 2384184-40-9), KP-14, KRAS PROTACs in patent WO2022173032 including KRAS G12D inhibitor 17 (CAS: 2821793-99-9), KT-333, KT-413, KF-253, KT-474 (CAS: 2432994-31-3), KTX-335, KTX-652, KTX-959, KTX-978, KTX-214, KYH1872, L18i, LC-2, LC-BM12, LEF1 OP-V1, LC-MB12 (CAS: 828438-38-4), lenalidomide, LG1188, LT-002, macroPROTAC-1, MK-8242 (CAS: 147-94-4), MD13, MD-222 (CAS: 2136246-72-3), MD-224 (CAS: 2136247-12-4), MDEG-541, MEK PR0TAC3 (Stefan Vollmer, et al., Journal of Medicinal Chemistry, 63, 157-162, 2020), MG-277 (CAS: 2411085-89-5), MM-02-08, MM-03-75, and MM- 04-09 (Margot Meyers, et al., bioRxiv Preprint, 2023, DOI: 10.1101 / 2023.08.11.553046), MS-170 (CAS: 2376136-61-5), MR837 (CAS: 1210906-48-1), MS28 (CAS: 2093386-22-0), MS33, MS39, MS67, MS83, MS98, MS154, MS170, MS4077 (CAS: 2230077-10-6), MS432, MS910, MS928, MS934, MS1943, MS4332, MS9715, MT-802 (CAS: 2231744-29-7), MTX-23 (CAS: 2488296- 74-6), MZ1, N3-NF-KB-ODN, dNF-KB #15, dNF-KB #16, N3-E2F-ODN, dE2F #16, dE2F #17, NH2, NJH-04-086, NJH-04-087, NJH-04-098, NR-6a, NR-7h, Nutlin 3a, Nutlin 3, NVP-CGM097 (CAS: 1313363-54-0), NRX-0492 (CAS: 2416130-57-7), NX-0479, NX-2127 (CAS: 2416131- 46-7), NX-5948 (CAS: 2649400-34-8), olutasidenib, ORM-5029, ovalicin, P3, P4B, P19As, P19P, P22, P22A, P22D, PAP508, pc-PROTACl, PG21, pc-PROTAC3, P13i (CAS: 2360561-66-4), PF15, pomalidomide, PP-C8, Pre-PROTAC, PROTAC-8, PROTAC ERRa (CAS: 1801547-15- 8), PROTAC RIPK2 (Daniel P. Bondeson, et al., Nature Chemical Biology, 11, 611-617, 2015), RIPK2 PROTACs (Anh-Tuan Pham, et al., Frontiers in Pharmacology, 14, 1127722, 2023), PRE3789, PROTAC MDM2 degrader- 1 (CAS: 2249944-98-5), PROTAC BRD9 degrader- 1 (CAS: 2097971-01-0), PROTAC BET degrader 23, PROTAC-D, PROTAC ER degrader-3 (CAS: 2158322-29-1), PROTAC-FCPF, PROTAC-O412, PROTAC(HPGDS)-1, PROTAC(HPGDS)-7, PRTC, PZ703b, PZ15527, QCA570 (CAS: 2207569-08-0), R1-5C, RBN012811, RC-1, RC-3, RG7112 (CAS: 939981-39-2), RG7388 (CAS: 1229705-06-9), RNK05047, Rucaparib-AP6, SAR405838 (CAS: 1303607-60-4), SARD279, SD-36 (CAS: 2429877-44-9), SD-91, SHP2-D26 (CAS: 2458219-65-1), SIAIS001, SIAIS056, SIAIS091, SIAIS117, SIAIS164018, SIAIS117 (CAS: 2353494-84-3), SIAIS125, SIAIS126, SIAIS178 (2376047-73-1), SIAIS629048, SIAIS629049, SIAIS629050, SIAIS629051, SIM1, SJ995973 (CAS: 2882065-25-8), SJF620 (CAS: 2376187-16-3), SJF-0628, SJF-0661, SJH1-62B, SK-575, SNIPER(ER)-3, SNIPER(ER)- 87 (CAS: 2222354-91-6), SNIPER-BRD4-1, SNIPER- ABL-62, SP4 (CAS: 2624181-69-5), SPB5208, SMD-3040, SR-1114, STEAPl-5a, STEAPl-13a, stimuli-responsive PROTACs (sr- PROTACs), T1101 tosylate (CAS: 2250404-95-4), TD-004 (Chung Hyo Kang, et al., Biochemical and Biophysical Research Communications, 502(2), 542-547, 2018.), TD-9, TD-165, TD-428 (CAS: 2334525-50-5), TD-802, thalidomide, TAI-1 (CAS: 1334921-03-7), TM-P4-Thal, TMX- 2172, UBX-382, UI-EP002, UNC6852 (CAS: 2688842-08-0), UNC7700, UNC7698, VHLL— X- BCN#15, VHLL— X-BCN#16, VHLL— X-BCN&17, Versortrexate (VSTX), VZ185, WB214, WWL0245, xStAx-VHLL, XD2-149, XY028-140 (CAS: 2229974-83-6), XY-06-007, XH2, XL01126, XL5-VHL-2, XY-4-88, XY-07-035, XY-07-096, XY-07-093, XY-07-143, XY-07-189, XZ739 (CAS: 2365172-19-4), XZ424, XZ9002, YF135, YKL-04-085, YM181, YUM70 (CAS: 423145-35-1), YX-2-107, YX-02-030 (Clare M. Adams, et al., Cancer Discovery, 13(5), 1210- 1229, 2023), YZ167, YZ268, ZB-S-29, ZCY-PROTAC, ZNL-02-096, ZXH-4-130, and ZXH-4- 137, any deuterium substituted derivatives, analogs and chelates, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and pharmaceutically acceptable carrier.

[0180] In certain embodiments, the bivalent or multiple valent agents are a PROTAC molecule. PROTACs are heterobifunctional molecules consisting of one or more than one warhead that bind proteins of interest (POI), a linker, and one or more than one ligand that recruit E3 ubiquitin ligases. By simultaneously binding to POI and E3 ligase, PROTACs can bring two and more proteins into proximity and facilitate ubiquitination for sequential proteasomal degradation of POI. In contrast to the protein inhibition, the event-driven pharmacological mechanism of PROTACs is catalytic in nature. Lower than effective inhibition drug concentration would give sufficient and prolonged target degradation and avoid off-target toxicity caused by high-dose drugs. Unfortunately, PROTACs suffer from poor durability, due to high molecular weight which is often associated with metabolic instability, poor solubility, and permeability. In theory, metabolic vulnerability and insolubility can be completely resolved by blocking metabolic hot spots and forming the salt on a basic or acidic functional group, respectively. In addition, formulation technologies can be employed to argument aqueous solubility. However, as proven by numerous published evidence, PROTACs are found to be hard or prohibitive for passive permeability, and the structural modifications of PROTACs for a better cell permeability via classical medicinal chemistry is significantly limited or even impossible, due to the large molecular size of PROTAC agents.

[0181] The warhead of the PROTAC may target any suitable protein of interest (POI). In certain embodiments, the target protein includes, but not limited to, target proteins listed in patent US20210002296A1, including all variants, mutations, splice variants, indels and fusions of these target proteins listed. Examples include, without limitation, 5HT2c receptor, OUA-AR, a-2 adrenergic receptor, a-synuclein, AAK1, ATP -binding cassette (ABC) transporters such as MDR1 / 2 / 3 / 4 / 5 and ABCG2, ABL, cholesterol acyltransferases (ACATs), ACE protein, ACK, ACTR2, ACTR2B, ADCK1 / 2 / 3 / 4 / 5, protein kinase B (AKT), AlphaKl / 2 / 3, amino acids transporters, anaplastic lymphoma kinase (ALK), ALIX, Amnionless, AMPA receptors (AMPARs), AMP-related protein kinase (AMPK), amyloid beta, Angiotensin II receptor, ANKRD3, Apaf-1, apolipoprotein E receptor (ApoER), amyloid precursor protein (APP), androgen receptor (AR), ARAF, ARG, AR-V7, ileal apical sodium / bile acid co-transporter (ASBT), V-type proton ATPase 6 (ATP6V), ATP6V1H, avidin, anine serine cysteine transporters (ASCTs), ASGPR, ASK1 / 2, Ataxin-1, ataxia-telangiectasia mutated protein (ATM), ATM and Rad3 -related protein (ATR), Aurora kinases, AXL, P-site amyloid precursor protein-cleaving enzyme 1 (BACE1), p2-adrenergic receptors, BAD, BARK1 / 2, Bax, BCKDK, B-cell leukemia / lymphoma (BCLs) family proteins such as BCL2, BCL-XL, and MCL-1, BCR-ABL, bromodomain and extraterminal domain family proteins (BETs) such as BRD2 / 3 / 4 / T, beta-catenin, BIKE / BMP2K, BLK, BMPR1A, BMPR1B, BMPR2, BMX / ETX, brother of CDO (Boc), BRD9, BMI1, BRAF, BRAFV600E, brassinosteriod insensitive 1 (BRI1), BRK, BRM, BRSK1, BRSK2, Bruton tyrosine kinase (BTK), BUB1, C3G, calreticulin, cell adhesion molecule (CAM) receptors, CAMK Ia / p / y / o, CAMK2a / p / y / 5, CAMK4, caMLCK, cannabinoid-1 receptor, coxsackievirus- adenovirus receptor (CAR), CRISPR associated proteins (Cas), CASK, Caspase-3, Caspase-6, Caspase-7, Caspase-9, CBFp, CBL-B, CBP, chemokine receptors such as CC chemokine receptors (CCRs) and CXC chemokine receptors (CXCRs) such as CXCR2, CXCR4, and CXCR7, CCK4 / PTK7, CCR2, CCR9, CCRK, cluster of differentiations (CDs) such as CD4, CD9, CD13 / APN receptor, CD25, CD26 (DPP4), CD30, CD33, CD36, CD38, CD46, CD56, CD63, CD71, CD74, CD80, CD81, CD83, CD86, CD 123, CD 138, CD 147, CD 152, CD 152 (CTLA4), CD166, CD174, CD197, CD205, CD227, CD228, CD269, CD276, and CD326, CDC7, CDC20, CDC25, CDC37, CDK1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12 / 13 / 14 / 15 / 16 / 17 / 18, CDKL 1 / 2 / 3 / 4 / 5, CDON, CEACAM5, cholesteryl ester transfer protein (CETP), c-Fos, cystic fibrosis transmembrane conductance regulator (CFTR), cyclic GMP-AMP synthase (eGas), Chakl / 2, CHK1 / 2, CLM6PR, CKla, CKla2, CK15, CKls, CKlyl / 2 / 3, CK2al / 2, CLIK1, CLIK1L, Clip, CLK1 / 2 / 3 / 4, human collectin placenta 1 (CL-P1), CMYC, cone opsins, COT / TPL2, cell-penetrating peptide (CPP), connexins, coronavirus protease, CRABP, C-RAF, cereblon (CRBN), CREB, CRK, CRIK, CRIPTO, CSF2R, CSF1R / FMS, CSK, C-TAK1, CTK, cubilin, cyclin D, cyclin E, CytoC, DAPK1 / 2 / 3, DCAMKL1 / 2 / 3, DDR1 / 2, diglyceride acyltransferase (DGAT), DLK, DMPK1 / 2, DNA-PK, DP-1, DPP-4, DRAK1 / 2, deubiquitylating enzymes (DUBs), DYRK1A / 1B / / 2 / 3 / 4, E2F, excitatory amino acid carrier 1 (EAAC1), E-cadherin, endothelin-converting enzyme (ECE), ED- B, EED, EF2K, eEF2K, EIF2A3, EIF4E, EIF4G, ELK, ENL, endothelin B receptor, epithelial cell adhesion molecule (EpCAM), endothelial cell protein C receptor (EPCR), Ephrin receptors (EphRs), estrogen receptor (ER), ER81, ErbB2, ErbB3, ETS transcription factor (ERG), ERK1 / 2 / 3 / 4 / 5 / 7 / 8, ERRa, EZH2, fatty acid binding proteins (FABPs), FADD, FAK, FAP, fatty acid transport proteins (FATPs), neonatal Fc receptor (FCRN), FER, FKBPs, FLIP, Flotillin-1, Flotillin-2, folate receptors such as reduced folate carrier, FOLT1 / 2 / 3 and proton-coupled folate transporter (PCFT), F0XM1, FOXO1 / 2 / 3 / 4, FRK, Frizzled4, fructose-l,6-bisphosphatase, FYN, y-aminobutyric acid type A receptors (GABAAR), GAK, Gap-1, growth arrest specific 1 (Gasl), GCC, GCK, GCN2, GD2, ghrelin receptor (ghrelinR), glucose-dependent insulinotropic polypeptide (GIP), Globo H, glucagon receptor, glucagon -like peptide 1 (GLP-1), glutamate transporters (GLTs), glucokinase, Ionotropic AMPA glutamate receptors (GluRs), glucose-6- phosphatase, glucose transporters (GLUTs), glutathione transporter, glycogen phosphorylase, glycogen synthase kinase, glycoproteins such as gpl8, gp31, and gp60, G protein-coupled receptors (GPCRs) such as GPR20, GPR20, free fatty acid receptor 1 (GPR40), GPR119, and GPR120, GPNMB, GPRK4 / 5 / 6 / 7, GRB2, GSK-3, GSPT1, Haspin kinase, HCK, hepatitis C virus (HCV) NS3 / 4A, histone deacetylases (HDACs), HDL receptor (HDLR), HectH9, HH498, HIF1, FHPK1 / 2 / 3 / 4, HMGN1, HMG-CoA reductase, hnRNPs, H-PGDS, HPGCR, hematopoietic progenitor kinase 1 (HPK1), HRAS, HRI, hRpnl3Pru, HSD-110 (11 P-hydroxysteriod dehydrogease), heat shock proteins (HSPs), huntingtin protein (HTT), HUNK, inhibitor of apopotosis (IAP) proteins such as cIAPs and XIAP, ICK, intermediate-density lipoprotein receptor (IDLR), IDO-1, IFN-yR, IGF-1R, IGF-2r, IKK-alpha, IKK-beta, IKK-gamma, IKK-epsilon, Ikaros (IKZF1), Helios (IKZF2), Aiolos (IKZF3), IKZF4, IL-4R, IL-10R, ILK, integrins such as aVp3, a4pi and a5pi integrins, insulin receptors (IRs), INSR, IRE1 / 2, IRR, ITKIMP, integrin receptors (IRs), insulin-like growth factor receptor, IRA2, IRAK1 / 2 / 3 / 4, IRE1, IRR, Janus tyrosine kinase (JAKs), c-Jun N-terminal kinases (JNKs), potassium-chloride cotransporter 2 (KCC2), KDM5C, KHS1 / 2, inward rectifier potassium channel (Kir2.3), KIS, KIT, Kelch domain- containing protein 2 (KLHDC2), KLF4, KRAS, KRASG12C, KSR1 / 2, Lamin, lysosome associated membrane proteins (Lamps), LAMT0R2, LANA, lactoferrin receptor, L-amino acid transporters (LATs), LATS1 / 2, LCK, low-density lipoprotein receptor (LDLR), LEF, LIMK1 / 2, LKB1, LMR1 / AATK, LMR2 / 3, LOK, low-density lipoprotein receptor related proteins (LRPs), LRRK1 / 2, LTK, LXR-0, LYN, LZK, LZTFL1, m5C, m6A, MAK, MALTs, MAP3K4 / 8, MAP4K3, MAP4K5, MAPK, MAPKlb, MAPKAPK2 / 3 / 5, MARK1 / 2 / 3 / 4, MAST1 / 2 / 3 / 4 / L, MC4 receptor, MCAK, MCH-1 receptor, monocarboxylic acid transporters (MCTs), mouse double minute 2 homolog (MDM2), MDMx, megalin, mitogen-activated protein kinase kinase (MEKs), MEKK1 / 2 / 3, MEL, MELK, MER, MERTK, mesothelin, MET, metabotropic glutamate receptors (mGluls), Mfsd2a, major histocompatibility complex class I proteins (MHC-I), MHC-II, MIF, MINCLE, MINK1, MISR2, MITF, MKK1 / 2 / 3 / 4 / 6 / 7, MKP3, MLK1 / 2 / 3 / 4, MLKL, MLN1 / 3, MNK1 / 2, MOK, MOS, MPK, MPSK1, MRCKa / p, multidrug resistance proteins (MRPs), MSK1 / 2, MSSK1, MST1 / 2 / 3 / 4, mammalian target of rapamycin (mTOR), MUCL, MUSK, Myc proteins, MYO3A / 3B, MYT1, NAMPT, N-cadherin, K+-dependent Na+ / Ca2+exchanger 2 (NCKX2), NDC80, NDR1 / 2, NEK1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11, NF-kappa B, Nicastrin, nicotinic acetylcholine receptor, NIK, NLK, NOTCH receptors, niemann-Pick Cl -like 1 (NPC1L1), N- methyl D-aspartate receptors (NRs), NRAS, NRBP1 / 2, NRF2, NSD2, NSD3, nucleoside transporters (NTs), sodium / taurocholate co-transporting peptide (NTCP), NuaKl / 2, NUAK1, organic anion transporters (OATs), organic anion transporting polypeptides (OATPs), Obscurin, organic cation transporters (OCTs), OSR1, organic solute transporter (OST), otoferlin, P2X purinoceptor 4 receptor (P2x4R), purinergic receptor P2Y12 (P2Y12), pl6INK4A, pl8INK4, pl9INK4D, p21, p27kipl, p38a, p38p, p38y, p386, p53, p65 / RELA, p70S6K, p70S6Kb, p90, p300 protein, PAK1 / 2 / 4 / 5 / 6, pan-coronavirus antiviral, protease-activated receptors (PARs), PARPs, PASK, PAX, PBRM1, PCSK9, PD-1, phosphodiesterases (PDEs), PDE4, PDE8, PDGFRa / p, pdhkl / 2 / 3 / 4, PDK1, PD-L1, PEA-15, PEK, peptide transporters (PEPTs), p-glycoprotein (P-gp), PHKyl / 2, phosphoenol pyruvate carboxykinase, phosphoinositide 3-kinase (PI3K), PIM1 / 2 / 3, PINK1, PITSLRE, PKA, PKACa / p / y, PKBa / 0, PKC, PKD1 / 2 / 3, PKG1 / 2, PKN1 / 2 / 3, PKR, PLC, PLDL, PLK1 / 2 / 3 / 4, PMEL17, PRAK, peroxisome proliferator-activated receptors (PPARs), PRC2, PRK2, PRKX, PRKY, PRMT5, protein phosphatase, protein tyrosine phosphatase (PTP), PRP4, PRPK, PSKH1 / 2, prostate-specific membrane antigen (PSMA), receptor patched 1 (PTCHI), PTEN, protein tyrosine kinases (PTKs), PVRL4, pyruvate dehydrokinase, PYK2, QIK, QSK, R1P1, RACK-1, Rac proteins, RAF protein, RAP1, Raptor, RAR, Ras protein family (RAS), Rb, retinol binding proteins (RBPs), RET, riboflavin transporter proteins (RFVTs), RFX1, RHAU, RHODK, RI0K1 / 2, receptor interacting protein kinases (RIPKs), rhodopsin, ribonuclease K (RNASEK), RNAseL, ring finger proteins (RNFs), R0CK1 / 2, renal outer medullary potassium channel (ROMK), RON, R0R1 / 2, ROS, RPN11, RPN13, ribosomal s6 kinases, receptor serine / threonine kinases (RSKs), such as transforming growth factor P (TGF-P) receptor and proteins as listed in Aristidis Moustakas, et al, Receptor Serine / Threonine Kinases (ISBN: 978-3- 540-44244-8), receptor tyrosine kinases (RTKs), such as fibroblast growth factor receptors (FGFR), FLT1 / 2 / 3 / 4, FGFR1 / 2 / 3 / 4, RET, tropomyosin-related kinases (TRKs), epidermal growth factor receptor (EGFR), human epidermal growth factor receptors (HERs), vascular endothelial growth factor receptors (VEGFRs), TYRO3, and other proteins as listed in publication Mark A. Lemmon and Joseph Schlessinger, Cell, 141(7) 1117-1134, 2010, RUNX1 / 2 / 3, RYK, spindle assembly checkpoint (SAC), Sanpodo / Notch, SAP, SARS-CoV-2, SBK, solute carriers (SCs), SCF, SCYL1 / 2 / 3, SF3B1, serine / threonine-protein kinases (SGKs), SGLTs, SHC, SHP2, SIK2 / 3, SIRT2, SIX1, skMLCK, S-phase kinase associated protein 1 (SKP1), S-phase kinase associated protein 2 (SKP2), solute carriers (SLC) transporters, such as hMATEl and proteins listed in publication Enrico Girardi, et al., Nature Chemical Biology, 16, 469-478, 2020, SLK, SLOB, Smac, Smad proteins, SMARCAs, sodium -coupled monocarboxylate transporters (SMCTs), SMG1, smMLCK, sodium dependent multivitamin transporter (SMVT), synaptosomal-associated protein (SNAP), SNRK, sortilin-related CNS expressed la (SorCSla), SorCSlc, protein son of sevenless (SOS), SOS1, SOX2, secreted protein acidic and rich in cysteine (SPARC), SPEG, speckle type BTB / POZ protein (SPOP), SPRED, SPRY, SPv-NKIR, scavenger receptors (SRs) such as CD36, LAMP1, and LAMP2, SR-Bs, Src protein, SRF, SRM, SRPK1 / 2, SSTK, StaO, signal transducer and activator of transcription proteins (STATs), STING, serine / threonine kinases (STKs), STLK3 / 5 / 6, syntaxins (STXs), SuRTK106, SUV39HI, SUZ12, sodium-vitamin C co-transporters (SVCTs), synaptotagmins (SYTs), triiodothyronine (T3), TAB, TAK1, TAL, TAO1 / 2 / 3, Tau protein, TBCK, TBK1, t-cell factor / lymphoid enhancer-binding factors (TCF / LEF family), TEC, TESK1 / 2, TFAM, transcription factor EB (TFEB), transferrin receptor (TfR), TGF-P, trans-Golgi network (TGN38), thiamine transporters (THTRs), TIE1, TIE2 / TEK, TIF Ia / p / y, Titin / TTN, TLK1 / 2, toll-like receptors (TLRs) such as TLR4, TNK1, TOPK, TPL2 / COT, TPNlp, TRAD, TRAF proteins, Trbl / 2 / 3, tripartite motif family proteins (TRIMs), Trio, TRRAP, TSC2, TSG101, TSSK1 / 2 / 3 / 4, TTBK1 / 2, TTK, TXK, TYK2, tyrosinase, tubulin, UBF, UCHL5, ULK1 / 2 / 3 / 4, ubiquitin specific peptidase (USP) such as USP7, USP11, and USP14, VACAMKL protein, vesicle associated membrane proteins (VAMPs), vitamin D receptors (VDR), vesicular GABA transporter (VGAT), vesicular glutamate transporters (VGLUTs), very low-density lipoprotein receptor (VLDLR), urate transporter 1 (URAT1), VRK1 / 2 / 3, WAVE-2, WDR5, WEE-1, WEE- IB, WNK1 / 2 / 3 / 4, Wnt, XBP1, YANK1 / 2 / 3, YBX1, YES, YSK1, ZAK, ZAP70, ZC1 / 2 / 3 / 4, ZFP91, and zinc ring finger proteins (ZRNFs), including all variants, mutations, splice variants, indels and fusions of these target proteins listed.

[0182] In certain embodiments, the E3 ligase is selected from following proteins, without limitation: inhibitor of apoptosis (IAP) proteins, X-linked inhibitor of apoptosis protein (XIAP), aryl hydrocarbon receptor (AhR), ring finger protein 4 (RNF4), ring finger protein 114 (RNF114), Fem-1 homolog B (FEM1B) protein, von Hippel-Lindau (VHL), casitas B-lineage lymphoma proto-oncogene-B (CBL-B) protein, cereblon (CRBN), damage specific DNA binding protein 1 (DDB1), mouse double minute 2 homolog (MDM2) protein, Kelch-like ECH-associated protein 1 (KEAP1), Kelch domain-containing protein 2 (KLHDC2), S-phase kinase associated protein 1 (SKP1), S-phase kinase associated protein 2 (SKP2), ubiquitin-protein ligase N-recognin 5 (UBR5), DDB1 and CUL4 associated factor 1 (DCAF1) protein, DDB1 and CUL4 associated factor 11 (DCAF11) protein, DDB1 and CUL4 associated factor 15 (DCAF15) protein, and DDB1 and CUL4 associated factor 16 (DCAF16) including all variants, mutations, splice variants, indels and fusions of these target proteins listed.

[0183] The PROTAC may comprise one or more than one E3 ligase ligand targeting IAP, VHL,

[0184] CBL-B, CRBN, DDB1, MDM2, KEAP1, KLHDC2, SKP1, SKP2, DCAF1, DCAF15, DCAF16,

[0185] UBR5, or any combination thereof.

[0186] The POI binding ligand and the E3 ligase ligand may be chemically linked or coupled via chemical bond or linker unit(s) B / L The chemical bond or linker unit(s) can be attached at any sites of the POI binding ligand and the E3 ligase ligand. The linker should allow for appropriate formation of a target protein-ligase complex. The linker group may comprise one or more structural units Bl / Ll

[0187] In certain embodiments, the chemical bond or linker unit(s) Lg / LJ can be a multivalent polyethylene glycol, alkoxy, alkyl, alkylene, alkyne, cycloalkyl, cycloalkyene, cycloalkyne, heterocyclyl, aryl, heteroaryl, which may terminate (at either or both termini) in at least one of a - H, -D, -O(R), =0, -S(R), =S, =NR, =N(R), -N(R)(R’), -Se(R), =Se, -F, -Cl, -Br, -I, -F, -18F, - C(R)(R’)(R”), =C(R)(R’), = C(R), -Si(R)(R’)(R”), =Si(R)(R’), = Si(R), -OS(O)2R, -OS(O)R, -

[0188] S(O)R, -S(O)2R, -P(0)(R)(R’), -P(R)(R’), -P(0)(0H)0-, -S-, -N(R')-, -C(0)-, -C(0)0-, -0C(0)-, - 0 C(0) 0 -,-C(S)-, -C(S)O-, -OC(S)-, -OC(S)O-, -C(N0R)-, -C(0)N(R')-, -C(0)N(R)C(0)-, - C(O)N(R)C(O)N(R')-, -N(R)C(O)-, -N(R')C(O)N(R)-, -N(R)C(O)O-, -OC(O)N(R)-, -C(NR')-, - N(R)C(NR')-, -C(NR')N(R)-, -N(R)C(NR')N(R)-, -S(O) 2- , -OS(O)-, -S(O)O-, -S(O)-, -OS(O)2- -S(O)20-, -N(R)S(O)2- , -S(O)2N(R)-, -N(R)S(O)-, -S(O)N(R)-, -N(R)S(O)2N(R')-, - N(R')S(O)N(R')-, -S-S-, -O-Si(R)(R’)-O-,)-, -C(=N)N(R)(R’)-, -C(R)=C(R’)-, -C = C-, C3-12 cycloalkane, C3-12 cycloalkene, C3-12 cycloalkyne, 3- to 12-membered heterocycles, 5- to 12- membered aryl, 5- to 12-membered heteroaryl, any deuterium substituted derivatives, or any combination thereof, wherein R, R’, or R” is H, D, 1-100 polyethylene glycol, C1-C100 alkoxy,

[0189] Cl -Cl 00 alkyl, C2-C100 alkylene, C2-C100 alkyne, C3-C100 cycloalkyl, C3-C100 cycloalky ene, C3-C100 cycloalkyne, C3-C100 heterocyclyl, C6-C100 aryl, or Cl -Cl 00 heteroaryl, wherein the one or both terminating groups may be the same or different.

[0190] In certain embodiments, the chemical bond or linker unit(s) can be multivalent chain unit(s) which contain one or more than one core with one or more chemical bond or linker unit(s) ILLUJ in any equivalent and sequences, wherein m, and n represent integer from 0 to 100.

[0191] In certain embodiments, the core includes, without limitation, an atom H, C, Si, N, P,

[0192] B, O, S, Se, 1-100 polyethylene glycol, Cl -Cl 00 alkoxy, Cl -Cl 00 alkyl, C2-C100 alkylene, C2- C100 alkyne, C3-C100 cycloalkyl, C3-C100 cycloalkyene, C3-C100 cycloalkyne, C3-C100 heterocyclyl, C6-C100 aryl, or C1-C100 heteroaryl, any deuterium substituted derivatives, analogs and chelates, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and pharmaceutically acceptable carrier.

[0193] In certain embodiments, the chemical bond or linker unit(s) B1ZL1 may be a bivalent or trivalent polyethylene glycol, alkoxy, alkyl, alkylene, alkyne, cycloalkyl, cycloalkyene, cycloalkyne, heterocyclyl, aryl, heteroaryl, which may terminate (at either or both termini) in at least one of a -H, -D, -O(R), =0, -S(R), =S, =NR, =N(R), -N(R)(R’), -Se(R), =Se, -F, -Cl, -Br, -I, -F, -18F, -C(R)(R’)(R”), =C(R)(R’), =C(R), -Si(R)(R’)(R”), =Si(R)(R’), = Si(R), -OS(O)2R, -

[0194] OS(O)R, -S(O)R, -S(O)2R, -P(O)(R)(R’), -P(R)(R’), -P(O)(OH)O-, -S-, -N(R')-, -C(O)-, -C(O)O-, -OC(O)-, - O C(O) O -,-C(S)-, -C(S)O-, -OC(S)-, -OC(S)O-, -C(NOR)-, -C(O)N(R')-, - C(O)N(R)C(O)-, -C(O)N(R)C(O)N(R')-, -N(R)C(O)-, -N(R')C(O)N(R)-, -N(R)C(O)O-, - OC(O)N(R)-, -C(NR')-, -N(R)C(NR')-, -C(NR')N(R)-, -N(R)C(NR')N(R)-, -S(O) 2- , -OS(O)-, - S(0)0-, -S(0)-, -0S(0)2- -S(O)20-, -N(R)S(O)2- , -S(O)2N(R)-, -N(R')S(O)-, -S(O)N(R)-, - N(R)S(O)2N(R')-, -N(R')S(O)N(R')-, -S-S-, -O-Si(R)(R’)-O-,)-, -C(=N)N(R)(R’)-, -C(R)=C(R’)-, - C = C-, C3-12 cycloalkane, C3-12 cycloalkene, C3-12 cycloalkyne, 3- to 12-membered heterocycles, 5- to 12-membered aryl, 5- to 12-membered heteroaryl, any deuterium substituted derivatives, or any combination thereof, wherein R, R’, or R” is H, D, 1-100 polyethylene glycol, Cl -Cl 00 alkoxy, Cl -Cl 00 alkyl, C2-C100 alkylene, C2-C100 alkyne, C3-C100 cycloalkyl, C3- C100 cycloalky ene, C3-C100 cycloalkyne, C3-C100 heterocyclyl, C6-C100 aryl, or Cl -Cl 00 heteroaryl, wherein the one or both terminating groups may be the same or different. In some embodiment, m or n is integral from 0 to 50.

[0195] In certain embodiments, the PROTAC targets androgen receptor (AR), B-cell lymphoma- 2 (BCL-2) family proteins, bromodomain and extrateminal (BET) family proteins, bromodomain containing 9 (BRD9) protein, Bruton’s tyrosine kinase (BTK), CREB-binding protein (CBP) and / or p300 proteins, cyclin-dependent kinases (CDKs), epidermal growth factor receptor (EGFR), estrogen receptor (ER), interleukin- 1 receptor-associated kinases (IRAKs), Janus kinase (JAK) family proteins, Myc proteins, RAF proteins, RAS proteins, SMARCA proteins, signal transducer and activator of transcription proteins (STATs), Tau proteins, tropomyosin receptor kinases (TRKs), or any combination thereof.

[0196] In certain embodiments, the bivalent or multiple valent agents are an AT AC or a LYTAC. ATAC or LYTAC is a heterobifunctional molecule consisting of a POI ligand, an endocytic agent, and a linker between the two moieties. An ATAC or a LYTAC can trigger lysosomal degradation of membrane POIs or extracellular POIs. In certain embodiments, the ATACs or LYTACs, or structural moieties in LYTACs in herein, are agents that can bind with endocytosis-mediating membrane component(s), such as CD36 or GLUTs, and be up-taken into cells via endocytosis.

[0197] In certain embodiments, the bivalent or multiple valent agents are a MtPTAC. MtPTAC refers to a heterobifunctional molecule consisting of a POI ligand, a mitochondrial caseinolytic protease P (ClpP) ligand, and a linker between the two moieties. MtPTAC activates the hydrolase activity of ClpP while simultaneously bringing POI into proximity with ClpP for degradation. In some embodiments, the MtPTAC is selected from all compounds in publication Dachi Wang, et al., Journal of American Chemistry Society, 145(23), 12861-12869, 2023, any deuterium substituted derivatives, analogs and chelates, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and pharmaceutically acceptable carrier. In certain embodiments, the bivalent or multiple valent agents are an AUTAC or AUTOTAC. An AUTAC is a heterobifunctional molecule consisting of a POI ligand, an autophagosome recruiting motif, and a linker between the two moieties. By binding with POIs, AUTAC molecules can trigger the degradation of POIs by recruiting autophagosomes. The POI can be any suitable protein of interest. An AUTOTAC is composed of an autophagy-targeting ligand, a POI ligand, and a linker. AUTOTA can directly tether receptor p62 to the POI and induce autophagy of POI.

[0198] In certain embodiments, the AUTACs and AUTOTACs are represented by any of the following compounds: AUTAC1, AUTAC2, AUTAC3, AUTAC4, PHTPP-1304, VinclozolinM2- 2204, Fumagilin-105, PBA-1105, PBA-1106, Anlel38b-F105, and PBA-1105b, any deuterium substituted derivatives, analogs and chelates, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and pharmaceutically acceptable carrier.

[0199] In certain embodiments, the bivalent or multiple valent agents are a nucleic acid degrader. Examples include, without limitation, ribonuclease-targeting chimeras (RIBOTACs), proximity- induced nucleic acid degrader (PINAD), or RNA-degrading chimeras. A RIBOTAC is a heterobifunctional molecule consisting of an RNA-targeting ligand, an RNase recruiter or binder, and a linker between the two moieties. RIBOTACs function by recruiting an endogenous RNase to a specific RNA, activating the RNase, and inducing cleavage of the target RNA. The RNases includes, without limitation, RNase A, RNase H, RNase III, RNase L, RNase P, RNase PhyM, RNase Tl, RNase T2, RNaseU2, RNase V, RNase E, RNase G, polynucleotide phosphorylase (PNPase), RNase PH, RNase R, RNase D, RNase T, oligoribonuclease, exoribonuclease I, and exoribonuclease II, including all variants, mutations, splice variants, indels and fusions of these proteins listed.

[0200] In certain embodiments, the RIBOTACs are represented by any of the following compounds: Compound 2 and Compound 5 in publication Matthew G. Costales, et al., Proceedings of the National Academy of Sciences, 117(5), 2406-2411, 2020, C5-RIBOTAC in publication Hafeez S. Haniff, et al., ACS Central Science, 6(10), 1713-1721, 2020, any deuterium substituted derivatives, analogs and chelates, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and pharmaceutically acceptable carrier.

[0201] In certain embodiments, the bivalent or multiple valent agents are a deubiquitinase- targeting chimera (DUBTAC), RESTORAC, or ENTAC. A DUBTAC, RESTORAC, or ENTAC is a heterobifunctional molecule consisting of a deubiquitinating enzyme or deubiquitinases (DUBs) recruiter or binder linked to a POI ligand via a linker, to stabilize the levels of POI degraded in a ubiquitin-dependent manner. The POI can be any suitable protein of interest. The DUBs include, without limitation, ubiquitin-specific protease (USP / UBP) superfamily proteins, such as USP1, USP2, USP3, USP4, USP5, USP6, USP7, USP8, USP9X, USP9Y, USP10, USP11, USP12, USP13, USP14, USP15, USP16, USP17, USP17L2, USP17L3, USP17L4, USP17L5, USP17L7, USP17L8, USP18, USP19, USP20, USP21, USP22, USP23, USP24, USP25, USP26, USP27X, USP28, USP29, USP30, USP31, USP32, USP33, USP34, USP35, USP36, USP37, USP38, USP39, USP40, USP41, USP42, USP43, USP44, USP45, USP46; the ovarian tumor (OTU) superfamily proteins, such as 0TUB1, 0TUB2; the Machado- Josephin domain (MJD) superfamily proteins, such as ATXN3, ATXN3L;the ubiquitin C-terminal hydrolase (UCH) superfamily proteins, such as BAP1, UCHL1, UCHL3, UCHL5; the MINDY family of K48- specific deubiquitinases, such as MINDY1, MINDY2, MINDY3, MINDY4; and the ZUFSP family proteins such as ZUP, including all variants, mutations, splice variants, indels and fusions of these proteins listed.

[0202] In certain embodiments, the DUBTACs are represented by any of the following compounds: 05IB9, 11JQ15, 11JQ16, NJH-2-075 (CAS: 2858812-70-9), NJH-2-056 (CAS: 2858812-69-6), NJH-2-057 (CAS: 2858812-70-9), LEB-03-153 (CAS: 2858812-88-9), LEB-03-144 (CAS: 2858812-89-0), LEB-03-145 (CAS: 2858812-90-3), LEB-03-146 (CAS: 2858812-91-4), FOXO- DUBTAC #6, p53-DUBTAC #6, p53-DUBTAC #7, and IRF-DUBTAC #7 in publication Jing Liu, et al., Journal of American Chemistry Society, 144, 12934-12941, 2022, any deuterium substituted derivatives, analogs and chelates, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and pharmaceutically acceptable carrier.

[0203] In certain embodiments, the bivalent or multiple valent agents are a phosphatase recruiting chimera (PhoRC), dephosphorylation-targeting chimera (DEPTAC), or phosphorylation -targeting chimeras (PhosTAC). A PhoRC, DEPTAC or PhosTAC is a heterobifunctional molecule consisting of a phosphatase recruiter or binder linked to a POI ligand via a linker. PhoRCs, DEPTACs or PhosTACs can trigger the dephosphorylation of POIs by the function of protein phosphatases (PPs). The POI can be any suitable protein of interest. The PPs includes, without limitation, tyrosine-specific phosphatases, serine-Zthreonine-specific phosphatases, dual specificity phosphatases, and histidine phosphatase, including all variants, mutations, splice variants, indels and fusions of these target proteins listed.

[0204] In certain embodiments, the PhoRC, DEPTAC or PhosTAC are represented by any of the following compounds: compound 1, compound 3, compound 4a, compound 5a, and compound 7 in publication Sayumi Yamazoe, et al., Journal of Medicinal Chemistry, 63, 2807-2813, 2020, DDO-3709R8, DDO-3710, and DDO-3711 in publication Zhang Qiuyue, et al., Journal of the America Chemistry Society, 145, 1118-1128, 2023, PhosTAC 7 in publication Chen P. H, et al., ACS Chemical Biology, 16, 2808-2815, 2021, any deuterium substituted derivatives, analogs and chelates, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and pharmaceutically acceptable carrier.

[0205] In certain embodiments, the bivalent or multiple valent agents are a regulated induced proximity targeting chimeras (RIPTAC). ARIPTAC is a heterobifunctional small molecule, which elicits a stable ternary complex between a target protein selectively expressed in cancer tissue and a pan-expressed protein essential for cell survival. The resulting cooperative protein-protein interaction (PPI) abrogates the function of the essential protein, thus leading to cell death selectively in cells expressing the target protein.

[0206] In certain embodiments, the RIPTACs are represented by any of the following compounds listed in publication Kanak Raina, et al., bioRxiv preprint, 2023, https: / / doi.org / 10.1101 / 2023.01.01.522436, in publication Zonghui Ma, et al., Drug Discovery Today, 2023, https: / / doi.Org / 10.1016 / j.drudis.2023.103774, and in patent WO2023059581 Al, any deuterium substituted derivatives, analogs and chelates, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and pharmaceutically acceptable carrier.

[0207] In certain embodiments, proximity-inducing modalities are represented by any compounds listed in Chem. Soc. Rev. 52, 5485-5515, 2023, any deuterium substituted derivatives, analogs and chelates, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and pharmaceutically acceptable carrier.

[0208] In certain embodiments, the bivalent or multiple valent agents are a transcriptional / epigenetic chemical inducers of proximity (TOP). A TCIP is a heterobifunctional small molecule, which recruit the endogenous cancer driver, or a downstream transcription factor, to the promoters of cell death genes thereby activating their expression. In certain embodiments, the TCIPs are represented by any of the following compounds listed in publication Sai Gourisankar, et al., Nature, 620, 417-425, 2023 and in patent WO2022098989A1, any deuterium substituted derivatives, analogs and chelates, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and pharmaceutically acceptable carrier.

[0209] In certain embodiments, the bivalent or multiple valent agents are a phosphorylation- inducing chimeric small molecules (PHICS). APHICS is a heterobifunctional molecule consisting of a kinase activator, POI binder, and a linker that recruits kinases to phosphorylate POIs by inducing the translocation of substrates to bring them into proximity. The POI can be any suitable protein of interest. The kinase includes, without limitation, AMPK and PKC, including all variants, mutations, splice variants, indels and fusions of these target proteins listed.

[0210] In certain embodiments, the PHICS are represented by any of the following compounds: PHICS1, PHICS2, and PHICS3 in publication Sachini U. Siriwardena, et al., Journal of the American Chemistry Society, 142, 14052-14057, 2020, any deuterium substituted derivatives, analogs and chelates, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and pharmaceutically acceptable carrier.

[0211] In certain embodiments, the bivalent or multiple valent agents are an acetylation tagging molecule (AceTAG). An AceTAG heterobifunctional molecule consisting of a lysine acetyltransferase, a POI binding ligand, and a linker. By binding with lysine acetyltransferase and POI, an AceTAG can modulate the distance between lysine acetyltransferase and POI, and thus inducing the POI acetylation. The POI can be any suitable protein of interest.

[0212] In certain embodiments, the AceTAGs are represented by AceTAG- 1 in publication Wesley W. Wang, et al., Journal of the American Chemistry Society, 143, 16700-16708, 2021, any deuterium substituted derivatives, analogs and chelates, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and pharmaceutically acceptable carrier.

[0213] In certain embodiments, the bivalent or multiple valent agents are chaperone-mediated protein degraders (CHAMPs). A CHAMP heterobifunctional molecule consisting of a chaperone protein binding ligand, a POI binding ligand, and a linker. By binding with chaperone protein and POI, a CHAMP can modulate the distance between chaperone protein and POI, and thus inducing the POI degradation. The POI can be any suitable protein of interest. In certain embodiments, the bivalent or multiple valent agents are BacPROTACs as endocytic agents via bacterial cell membrane protein (s) like CD36 etc.. A BacPROTAC heterobifunctional molecule consisting of a bacterial CIpCP protease binding ligand, a POI binding ligand, and a linker. By binding with bacterial CIpCP protease and POI, a BacPROTAC can modulate the distance between CIpCP protease and POI, and thus inducing the POI degradation. The POI can be any suitable protein of interest.

[0214] In certain embodiments, the bivalent or multiple valent agents are caspase cleavage targeting chimeras (CACTACs) as endocytic agents. A CACTAC heterobifunctional molecule consisting of a binding ligand to caspase, a POI binding ligand, and a linker. By binding with caspase and POI, a CACTAC can modulate the distance between caspase and POI, and thus inducing the cleavage of the POI. The POI can be any suitable protein of interest.

[0215] In some embodiments, the therapeutic agent is a binding agent. A binding agent is any compound which bind with membrane or extracellular materials, including membrane proteins or extracellular proteins, carbohydrates, lipids, pathogens, particles, virus, bacteria, fungi, protozoa, vectors, cell debris, and another cell. Hence, the binding agent can be used to internalize cell membrane to form endosome, and subsequently traffic membrane proteins, extracellular proteins, carbohydrates, lipids, pathogens, particles, virus, bacteria, fungi, protozoa, vectors, cell debris, and another cell into (recipient) cells via endocytosis. The component in internalized membrane or extracellular materials, such as proteins, carbohydrates, lipids, pathogens, particles, virus, bacteria, fungi, protozoa, vectors, cell debris, and another cell, can be eventually degraded by lysosome of (recipient) cells.

[0216] In some embodiments, the therapeutic agent is a DNA groove binder or RNA binder. DNA minor groove binders are crescent-shaped molecules that selectively bind non-covalently to the groove of DNA, the furrows in the DNA helix (Sayantan Bhaduri, et al., Beilstein Journal of Organic Chemistry, 14, 1051-1086, 2017). RNA binders are molecules that can specifically bind with RNA (Jessica L. Childs-Disney, et al., Nature Reviews Drug Discovery, 21, 736-762, 2022). Binding to DNA or RNA with specific sequences usually takes place by a combination of directed hydrogen bonding to base pair edges. By binding with DNA or RNA, the molecules can affect the functions of DNA or RNA.

[0217] In some embodiments, the therapeutic agent is a diagnostic agent or chemical probe. Diagnostic agent or chemical probe comprise a detectable label. Detectable labels include, but are not limited to, a binding label, a chromophore, an enzyme label, a bioluminescent label, a fluorescent label, a quencher, a radiolabel, or any other label suitable for a means of detection. Binding labels provide for a detectable signal via a binding event. In some embodiments, a binding label may be biotin, compounds used in HaloTag, CLIP-Tag, or SNAP-Tag technology (Jonas Wilhelm, et al., Biochemistry, 60(3), 2560-2575, 2021), an antibody, an antigen, or any other label capable of providing a detectable signal via a binding event. Chromophores provide a detectable signal via the absorbance and emission of photons. In some embodiments, the chromophore is a fluorophore, a phosphor, a dye, a quantum dot, or any other chromophore capable of absorbing and emitting detectable photons. Enzyme labels provide a detectable signal via a reaction with a substrate. Bioluminescent labels provide a detectable signed via the emission of light from a protein. Quenchers or fluorescent labels provide a detectable signal via the modulation of the photon emission from a chromophore. Radiolabels provided for a detectable signal via radioactive decay. As demonstrated in the Examples, conjugated diagnostic agents comprising a fluorophore successfully crossed membrane, but other detectable labels may be incorporated into the cargo. Examples of diagnostic agents or chemical probes include , but are not limited to : Alexa Fluor dyes (Alexa Fluor 350, Alexa Fluor 488 , Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680, AMCA, AMCA-S, BODIPY dyes (BODIPY FL, BODIPY ROG, BODIPY TMR, BODIPY TR, BODIPY 530 / 550, BODIPY 558 / 568 , BODIPY 564 / 570, BODIPY 576 / 589 , BODIPY 581 / 591 , BODIPY 630 / 650 , BODIPY 650 / 665) , Carboxy rhodamine 6G, carboxy-X-rhodamine (ROX), Cascade Blue, Cascade Yellow, Coumarin 343, Cyanine dyes (Cy3 , Cy5 , Cy3.5 , Cy5.5) , Dansyl, Dapoxyl, Dialkylamino coumarin, 4',5-Dichloro-2', 7'-dimethoxy-fluorescein, DM-NeRF, Eosin, Erythrosin, Fluorescein, FAM , Hydroxycoumarin, IRDyes (IRD40 , IRD 700 , IRD 800), JOE, Lissamine rhodamine B, Marina Blue, Methoxycoumarin, Naphthofluorescein, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, PyMPO, Pyrene, Rhodamine B, Rhodamine 6G, Rhodamine Green, Rhodamine Red, Rhodol Green, 2',4,5',7'-Tetra-bromosulfone-fluorescein, Tetramethyl- rhodamine (TMR), Carboxytetramethylrhodamine (TAMRA), Texas Red, Texas Red-X.

[0218] In certain embodiments, diagnostic or tracking agent is multivalent compound which contains one or more chemical fragments with fluorescence as the detectable label. The emission and excitation wavelengths of diagnostic agent are, but not limited to, from 200 nm to 900 nm. The diagnostic or tracking agent can be used for imaging in vitro and in vivo. In certain embodiments, the endocytic agent is an exocytic vesicle (or endocytic agents- vesicle complex). An exocytic vesicle refers to a vesicle or vesicle composition comprising vesicle forming components or particles, and one or more than one endocytic agent(s) associated with the vesicle forming components or particles, wherein the vesicle forming components includes any cytoplasm and endocytic-mediating membrane components of cells, including nucleic acids, proteins, lipids, carbohydrates, and metabolites. The endocytic agents can be located at any sites of the vesicle, including encapsulated or entrapped in said vesicle forming components formed of lipids, attached onto the membrane components (inward and / or outward), inserted into or across the membrane components. The endocytic agents can bind with any components of the vesicle via covalent or non-covalent bond(s) with binding affinity K / j less than 20.0 mM.

[0219] The exocytic vesicle has a diameter lower than 10000 nm. In certain embodiments, the exocytic vesicles have a diameter between 10 and 10000 nm. In certain embodiments, the exocytic vesicle has a diameter between 10 and 1000 nm.

[0220] In certain embodiments, the exocytic vesicle can be an extracellular vesicle (EV) which contains or binds with endocytic agent(s) in any equivalent. The EV includes, but is not limited to, exosomes, microvesicles, microparticles, apoptosis body, oncosomes, ectosomes, synaptic vesicles, prostasome, and matrix vesicles. The endocytic agent(s) can be selected from any endocytic agent(s) as defined in present disclosure. In said exocytic vesicles, the endocytic agent(s) can exist at any sites of the EVs and / or can bind with any components of the EVs via covalent or non-covalent bond(s).

[0221] In certain embodiments, the exocytic vesicles can be generated by any cells and can be secreted from the cells into extracellular space through exocytosis, transcytosis, and / or membrane fusion. In certain embodiments, the exocytic vesicles can be generated in vitro and / or in vivo by mixing vesicle forming components or particles (including EVs) with endocytic agent(s) in any equivalents, in any suitable solution, medium, or body fluids, wherein the endocytic agent(s) can form covalent or non-covalent bond(s) with vesicle forming components or particles (including EVs).

[0222] Based on the facts that foreign materials can be up-taken by cells and released from the (donor) cells in forms of extracellular vesicles (EVs), and extracellular vesicles can be up-taken by any the same cells or another cells via exocytosis or membrane fusion (Ravi Shah, et al., The New England Journal of Medicine, 8(379), 958-966, 2018; Oscar P. B. Wiklander, et al., Science Translational Medicine, 11(492), eaav8521, 2019; Raghu Kalluri, et al., Science, 367(6478), eaau6977, 2020), the present exocytic vesicle can be used to transport any endocytic agent(s) as defined in present disclosure into cells. In addition, the present exocytic vesicle can be used to across membrane barriers (including blood-retinal barrier, lung endothelial and epithelial barrier, skin barrier and brain-blood-barrier) and / or delivery any endocytic agent(s) as defined in present disclosure into any target tissues and organs in animal bodies. Particularly, the present exocytic vesicles are suitable for the delivery of endocytic agent(s) for studying, diagnosing, preventing, treating eye, respiratory, skin, and CNS conditions and diseases.

[0223] In present disclose, exocytic vesicle is termed as one kind of endocytic agents. All benefits and methods associated with endocytic agents are applicable to exocytic vesicles.

[0224] Endocytosis-mediating Membrane Component

[0225] Endocytosis-mediating membrane component includes proteins, lipids, and carbohydrates, which are components of a membrane, including cellular membrane, that mediates an endocytic process or the binding of which allows for increased uptake via endocytosis.

[0226] In some embodiments, the lipid includes glycolipid, phospholipids, ceramides, and cholesterol.

[0227] In some embodiments, the carbohydrates are monosaccharide or molecules consisting of 2-100 units of monosaccharide and / or derivatives and can be straight or branched, which are attached to proteins, forming glycoprotein, or lipids, forming glycolipids.

[0228] In certain embodiments, the carbohydrates include, without limitation, N- acetylgalactosamine (GalNAc), chondroitin sulfate (CS), dermatan sulfate (DS), heparin sulfate (HS), keratan sulfate (KS), hyaluronic acid, and sialic acid (SA).

[0229] In some embodiments, the proteins include, without limitation, integral membrane proteins, peripheral membrane proteins, lipid-anchored proteins, globular proteins, and glycoproteins, including all variants, mutations, splice variants, substituted, and fusions.

[0230] In certain embodiments, the proteins include, without limitation, ATP -binding cassette (ABC) transporters such as MDR1 / 2 / 3 / 4 / 5 and ABCG2, anaplastic lymphoma kinase (ALK), Amnionless, AMPA receptors (AMPARs) (Maria Fiuza, et al., Journal of Cell Biology, 216(10), 3323-3338, 2017), apolipoprotein E receptor (ApoER), amino acids transporters, amyloid precursor protein (APP), ileal apical sodium / bile acid co-transporter (ASBT), anine serine cysteine transporters (ASCTs), asialogycoprotein receptors (ASPGPR), V-type proton ATPase 6 (ATP6V), ATP6V1H, avidin, £-site amyloid precursor protein-cleaving enzyme 1 (BACE1), p2-adrenergic receptors, brother of CDO (Boc), brassinosteriod insensitive-1 (BRI1), cell adhesion molecule (CAM) receptors, coxsackievirus-adenovirus receptor (CAR), chemokine receptors such as CC chemokine receptors (CCRs) and CXC chemokine receptors (CXCRs) such as CXCR2, CXCR4, and CXCR7, cluster of differentiations (CDs), such as CD4, CD9, CD13 / APN receptor, CD25, CD26 (DPP4), CD30, CD33, CD36, CD38, CD46, CD56, , CD63, CD71, CD74, CD80, CD81, CD82, CD86, CD 123, CD 138, CD 147, CD 152, CD 152 (CTLA4), CD 166, CD 174, CD 197, CD205, CD227, CD228, CD269, CD276, and CD326, cation-dependent MPR (CD-MPR), Cdon, cystic fibrosis transmembrane conductance regulator (CFTR), cation-independent MPR / insulin- like growth factor-II (IGF-II) receptor (CI-MPR), IGF-2R, chemokine receptors, human collectin placenta 1 (CL-P1), cone opsins, connexins, cell-penetrating peptide (CPP), CSF2R, CSF1R / FM, excitatory amino acid carrier 1 (EAAC1), E-cadherin, early endosomal autoantigen 1 (EEA1), epithelial cell adhesion molecule (EpCAM), erythropoietin-producing hepatocellular carcinoma receptors (EphRs), endothelial cell protein C receptor (EPCR), fatty acid binding proteins (FABPs), fatty acid transport proteins (FATPs), Major facilitator superfamily domain-containing protein 2 (mfsd2), aneonatal Fc receptor (FCRN), free fatty acid receptors (FFARs), Flotillin- 1 , Flotillin-2, folate receptors such as reduced folate carrier, FOLT1 / 2 / 3 and proton-coupled folate transporter (PCFT), Frizzled4, y-aminobutyric acid type A receptors (GABAAR), growth arrest specific 1 (Gasl), glutamate transporters (GLT), glucose transporters (GLUTs), glutathione transporter, ionotropic AMPA glutamate receptors (GluRs), GLP1, glycoproteins such as gpl8, gp31, and gp60, glucokinase, G protein-coupled receptors (GPCRs) such as GPR20, GPR20, free fatty acid receptor 1 (GPR40), GPR119, and GPR120, HDLR, IFN-yR, intermediate-density lipoprotein receptor (IDLR), IL-10R, IL-4R, integrins such as aV03, a4|31 and a5|31 integrins, insulin receptors (IRs), insulin-like growth factor receptor, potassium-chloride cotransporter 2 (KCC2), inward rectifier potassium channel (Kir2.3), lactoferrin receptor, L-amino acid transporters (LATs), lysosome-associated membrane proteins (LAMPs), low-density lipoprotein receptor (LDLR), low-density lipoprotein receptor related proteins (LRPs), major facilitator superfamily domain-containing protein 2 (Mfsd2a), monocarboxylic acid transporters (MCTs), multi drug resistance proteins (MRPs), MET, metabotropic glutamate receptors (mGluls), major histocompatibility complex class II molecules (MHC-II), MHC-I, MINCLE, multidrug resistance proteins (MRPs), A-cadherin, K+-dependent Na+ / Ca2+exchanger 2 (NCKX2), Nicastrin, nicotinic acetylcholine receptor, NOTCH receptors, niemann-Pick Cl-like 1 (NPC1L1), / V-m ethyl D- aspartate receptors (NRs), nucleoside transporters (NTs), sodium / taurocholate co-transporting peptide (NTCP), organic anion transporters (OATs), organic anion transporting polypeptides (OATPs), organic cation transporters (OCTs), organic solute transporter (OST), otoferlin, P2X purinoceptor 4 receptor (P2x4R), purinergic receptor P2Y12 (P2Y12), protease-activated receptors (PARs), PEIZO1, PEIZO2, peptide transporters (PEPTs), p-gly coprotein (P-gp), peroxisome proliferator-activated receptors (PPARs), prostate-specific membrane antigen (PSMA), receptor patched 1 (PTCHI), RAS, retinol binding proteins (RBPs), riboflavin transporter proteins (RFVTs), rhodopsin, ribonuclease K (RNASEK), ring finger proteins (RNFs), renal outer medullary potassium channel (ROMK), receptor serine / threonine kinases (RSKs), such as transforming growth factor 0 (TGF-0) receptor and proteins as listed in Aristidis Moustakas, et al, Receptor Serine / Threonine Kinases (ISBN: 978-3-540-44244-8), receptor tyrosine kinases (RTKs), such as fibroblast growth factor receptors (FGFR), FLT1 / 2 / 3 / 4, FGFR1 / 2 / 3 / 4, RET, tropomyosin-related kinases (TRKs), epidermal growth factor receptor (EGFR), human epidermal growth factor receptors (HERs), vascular endothelial growth factor receptors (VEGFRs), TYRO3, and other proteins as listed in publication Mark A. Lemmon and Joseph Schlessinger, Cell, 141(7) 1117-1134, 2010, Sanpodo / Notch, solute carriers (SLC) transporters, such as hMATEl and proteins listed in publication Enrico Girardi, et al., Nature Chemical Biology, 16, 469-478, 2020, solute carrier family 37 member A3 (SLC37A3), SGLTs, sodium -coupled monocarboxylate transporters (SMCTs), sodium dependent multivitamin transporter (SMVT), synaptosomal- associated protein (SNAP), sortilin-related CNS expressed la (SorCSla), SorCSlcsecreted protein acidic and rich in cysteine (SPARC), SPv-NKIR, SRC, scavenger receptors (SRs) such as CD36, LAMP1, and LAMP2, syntaxins (STXs), sodium-vitamin C co-transporters (SVCTs), synaptotagmins (SYTs), triiodothyronine (T3), transferrin receptor (TfR), transforming growth factor 0 (TGF-0), trans-Golgi network (TGN38), thiamine transporters (THTRs), toll-like receptors (TLRs) such as TLR4, TPNlp, vesicle associated membrane proteins (VAMPs), vitamin D receptors (VDR), vesicular GABA transporter (VGAT), vesicular glutamate transporters (VGLUTs), very low-density lipoprotein receptor (VLDLR), urate transporter 1 (URAT1), and zinc ring finger proteins (ZRNFs), including all variants, mutations, splice variants, indels and fusions of these target proteins listed. Similar transporters or receptors expressed on microorganism cell membranes are also applied for such as BacPROTACs. Chemistries and Terms

[0231] As used herein, represents a binding site;

[0232] ^is a single bond or a double bond;

[0233] R, R’, and R” are, each independently, H, D, 0, =0, S, =S, S-S, =NH, =N(0H), N(0H), N(H)0, CH2F, CHF2, CF3, 1-100 polyethylene glycol, Cl -Cl 00 alkoxy, Cl -Cl 00 alkyl, C2-C100 alkylene, C2-C100 alkyne, C3-C100 cycloalkyl, C3-C100 cycloalky ene, C3-C100 cycloalkyne, C3-C100 heterocyclyl, C6-C100 aryl, or Cl -Cl 00 heteroaryl, 1-100 polyethylene glycol-Ra, Cl- C100 alkyl-Ra, C2-C100 alkylene-Ra, C2-C100 alkyne-Ra, C3-C100 cycloalkyl-Ra, C3-C100 cycloalkyene-Ra, C3-C100 cycloalkyne-Ra, C3-C100 heterocyclyl -Ra, C6-C100 aryl-Ra, or Cl- C100 heteroaryl -Ra, B(ORa2), Si(ORa3), C = C, C = CRa, CH=CH2, CH=CHRaCH=C(Ra)2,

[0234] CRa=CHRaCRa=CRaC(O), CRa=C(Ra)2, CORa, CONH2, C(O)ORa, OC(O)Ra, OC(O)ORa, OC(O)N(Ra)2, C0NHRa, CON(Ra)2, NH2, N(H)Ra, N(Ra)2, N+(Ra)3, NHNH2, NHN(H)Ra, - NHN(Ra)2, NHC(O)RaNHC(0)0Ra, NHC(0)NH2, NHC(0)NHRa, NHC(0)N(Ra)2, NRaC(O)NH2, NRaC(0)NHRa, NRaC(O)N(Ra)2,NRaC(S)N(Ra)2, NHC(NH)NH2, NHC(NH)NHRa, NHC(NH)N(Ra)2, NRaC(NH)NH2, NRaC(NH)NHRa, NRaC(NH)N(Ra)2, NRaC(NRa)N(Ra)2, NHS(O)Ra, NHS(O)NH2, NHS(O)NHRa, NHS(O)N(Ra)2, NRaS(O)NH2, NRaS(O)NHRa, NRaS(O)N(Ra)2, NHS(O)2Ra, NHS(O)2NH2, NHS(O)2N(H)Ra, NHS(O)2N(Ra)2, NRaS(O)2NH2, NRaS(O)2NHRa, NRaS(O)2N(Ra)2, OH, ORa, OS(O)2OH, OS(O)2ORa, OS(O)OH, OS(O)ORa, OS(O)2NH2, OS(O)2N(H)Ra, OS(O)2N(Ra)2, OS(O)NH2, OS(O)N(H)Ra, OS(O)N(Ra)2, SH, SRa, S(O)Ra, S(O)2NH2, S(O)2NHRa, S(O)2N(Ra)2, S(O)N(H)Ra, S(O)N(Ra)2, SON(Ra)3, SO2H, SO2Ra, P(O)(OH)2, P(O)(OH)(ORa), P(O)(ORa)2, P(O)(OH)OP(O)(OH)2, P(O)(OH)OP(O)(OH)(ORa), P(O)(OH)OP(O)(ORa)2, P(O)(OH)OP(O)(OH)OP(O)(OH)2,

[0235] P(O)(OH)OP(O)(OH)OP(O)(OH)(ORa), P(O)(OH)OP(O)(OH)OP(O)(ORa)2, OP(O)(ORa), OP(O)OP(O)(OH)2, OP(O)OP(O)(OH)(ORa), OP(O)OP(O)(ORa)2,

[0236] OP(O)OP(O)(OH)OP(O)(OH)2, OP(O)OP(O)(OH)OP(O)(OH)(ORa),

[0237]

[0238] MBCFs, SMDAs, LAs, GAs, PPAs, VtAs, AAPs, SCAs, NNNAs, RMPBs, ADDs, an agent, or a portion of an agent, any deuterium substituted derivatives, or any combination thereof, wherein the interrupting and the one or both terminating groups may be the same or different;

[0239] U, V, U’, V’, V”, V’”, W, W’, X’, Ra, Ra, Rb, Rb, Rc, Re, RdRd , Re , Rf , Rg, and Rhare independently selected from -H, -D, -F, -Cl, -18F, -CH3, -CF3, -CDH2, -CD2H, -CD3F, Cl, Br, CF3, CHF2, CH2F, OH, SH, NH2, NHNH2, COOH, CONH2, S0NH2, SO2NH2, B(OH2), Si(OH3), NHC(NH)NH2, C(O)(NH)OH, P(0)(0H)2, P(O)(OH)OP(O)(OH)2,

[0240] P(O)(OH)OP(O)(OH)OP(O)(OH)2,R‘F, R;C1, R‘Br, RjCF3, RiCHF2, RiCH2F, R*OH, R'SH, R‘NH2, R‘NHNH2, R'COOH, C(O)OR‘, OC(O)R‘, R‘CONH2, R‘SONH2, R‘SO2NH2, R‘B(OH)2, R‘Si(OH)3, R‘NHC(NH)NH2, RiC(O)(NH)OH, RiP(O)(OH)2, RiP(O)(OH)OP(O)(OH)2, R‘P(O)(OH)OP(O)(OH)OP(O)(OH)2, an agent, or a portion of an agent, any deuterium substituted derivatives, or any combination thereof, wherein the interrupting and the one or both terminating groups may be the same or different, wherein Racan be at any site of 1-100 polyethylene glycol, Cl -Cl 00 alkoxy, Cl -Cl 00 alkyl, C2-C100 alkylene, C2-C100 alkyne, C3-C100 cycloalkyl, C3- C100 cycloalky ene, C3-C100 cycloalkyne, C3-C100 heterocyclyl, C6-C100 aryl, or Cl -Cl 00 heteroaryl in the 1-100 polyethylene gly col -Ra, C1-C100 alkyl-Ra, C2-C100 alkylene-Ra, C2-C100 alkyne-Ra, C3-C100 cycloalkyl-Ra, C3-C100 cycloalkyene-Ra, C3-C100 cycloalkyne-Ra, C3- C100 heterocyclyl-Ra, C6-C100 aryl-Ra, or Cl -Cl 00 heteroaryl -Ra;

[0241] R' is 1-100 polyethylene glycol, C1-C100 alkoxy, C1-C100 alkyl, C2-C100 alkylene, C2- C100 alkyne, C3-C100 cycloalkyl, C3-C100 cycloalkyene, C3-C100 cycloalkyne, C3-C100 heterocyclyl, C6-C100 aryl, or Cl -Cl 00 heteroaryl, linker, any deuterium substituted derivatives, or any combination thereof, wherein the interrupting and the one or both terminating groups may be the same or different.

[0242] The chemical bond or linker unit(s) l^lZUcan share the same structure with chemical bond or linker unit(s) B / L and binds with any sites of ligand, E3 ligase ligand, agent, chemical arm,

[0243] SMDAs, LAs, Gas, PPAs, VtAs, AAPs, SCAs, NNNAs, RMPBs, or ADDs; m, n, and q are independently selected from 0 to 100, included any number in between.

[0244] The term “substituted” means that the specified group or moiety bears one or more suitable substituents wherein the substituents may connect to the specified group or moiety at one or more positions. For example, an aryl substituted with a cycloalkyl may indicate that the cycloalkyl connects to one atom of the aryl with a bond or by fusing with the aryl and sharing two or more common atoms.

[0245] Unless otherwise specifically defined, “aryl” means a cyclic, aromatic hydrocarbon group having 1 to 100 aromatic rings, including monocyclic or bicyclic groups such as phenyl, biphenyl, or naphthyl. When containing two aromatic rings (e.g., bicyclic), the aromatic rings of the aryl group are optionally joined at a single point (e.g., biphenyl), or fused (e g., naphthyl). The aryl group is optionally substituted by one or more substituents, e.g., 1 to 100 substituents, at any point of attachment. Exemplary substituents include, but are not limited to , -H , -halogen , -CN , -O(C1- C100)alkyl, -(Cl-ClOO)alkyl, -O(C2-C100)alkenyl, -O(C2-C100)alkynyl, -(C2-C100)alkenyl, - (C2-C100)alkynyl, -OH, -OP(O)(OH)2, -OC(O)(Cl-C100)alkyl, -C(O)(Cl-C100)alkyl, - OC(O)O(Cl-C100)alkyl, -NH2, -NH((Cl-C100)alkyl), -N(Cl-C100)alkyl)2, -S(O)2-, -(Cl- C100)alkyl, -S(O)NH(Cl-C100)alkyl, and -S(O)N((Cl-C100)alkyl)2. The substituents are themselves optionally substituted. Furthermore, when containing two fused rings, the aryl groups optionally have an unsaturated or partially saturated ring fused with a fully saturated ring. Exemplary ring systems of these aryl groups include, but are not limited to, phenyl, biphenyl, naphthyl, anthracenyl, phenalenyl, phenanthrenyl, indanyl, indenyl, tetrahydronaphthalenyl, tetrahydrobenzoannulenyl, and the like. Unless otherwise specifically defined, "heteroaryl" means a monovalent monocyclic aromatic radical of 3 to 100 ring atoms or a polycyclic aromatic radical, containing one or more ring heteroatoms selected from N, 0, or S, the remaining ring atoms being C. Heteroaryl as herein defined also means a bicyclic heteroaromatic group wherein the heteroatom is selected from N, 0, or S. The aromatic radical is optionally substituted independently with one or more substituents described herein. Examples include, but are not limited to, furyl, thienyl, pyrrolyl, pyridyl, pyrazolyl,pyrimidinyl, imidazolyl, isoxazolyl, oxazolyl, oxadiazolyl, pyrazinyl, indolyl, thiophen-2-yl, quinolyl, benzopyranyl, isothiazolyl, thiazolyl, thiadiazole, indazole, benzimidazolyl, thieno[3,2-b]thiophene, triazolyl, triazinyl, imidazo[l,2-b]pyrazolyl, furo[2,3- c]pyridinyl, imidazo[l,2-a]pyridinyl, indazolyl, pyrrolo [2,3 -c]pyridinyl, pyrrolo [3,2- cjpyridinyl, pyrazolo[3,4-c]pyridinyl, thieno[3,2-c]pyridinyl, thieno[2, 3 -cjpyridinyl, thieno[2,3-b]pyridinyl, benzothiazolyl, indolyl, indolinyl, indolinonyl, dihydrobenzothiophenyl, dihydrobenzofuranyl, benzofuran, chromanyl, thiochromanyl,tetrahydroquinolinyl, dihydrobenzothiazine, dihydrobenzoxanyl, quinolinyl, isoquinolinyl, 1,6-naphthyridinyl, benzo[de]isoquinolinyl, pyrido[4,3-b][l,6]naphthyridinyl, thieno[2,3-b]pyrazinyl, quinazolinyl, tetrazolo [1,5 -a]pyridinyl, [l,2,4]triazolo[4,3-a]pyridinyl, isoindolyl, pyrrolo[2,3-b]pyridinyl, pyrrolo[ 3,4 -b ]pyridinyl, pyrrolo[3,2-b]pyridinyl, imidazo[5,4-b]pyridinyl, pyrrolo[l,2-a]pyrimidinyl, tetrahydropyrrolo[l,2-a]pyrimidinyl, 3,4-dihydro-2H-lA2-pyrrolo[2,l-b]pyrimidine, dibenzo[b,d]thiophene, pyridin-2-one, furo[3,2-c]pyridinyl, furo[2,3-c]pyridinyl, lH-pyrido[3,4- b][l,4]thiazinyl, benzooxazolyl, benzoisoxazolyl, furo[2,3-b]pyridinyl, benzothiophenyl, 1,5- naphthyridinyl, furo[3,2-b]pyridine, [l,2,4]triazolo[l,5-a]pyridinyl, benzo[l,2,3]triazolyl, imidazo[l,2-a]pyrimidinyl, [l,2,4]triazolo[4,3-b]pyridazinyl, benzo[c][l,2,5]thiadiazolyl, benzo[c][l,2,5]oxadiazole, l,3-dihydro-2H-benzo[d]imidazol-2-one, 3,4-dihydro-2H- pyrazolo[l,5-b][l,2]oxazinyl, 4,5,6,7-tetrahydropyrazolo[l,5-a]pyridinyl, thiazolo[5,4- d]thiazolyl, imidazo[2,l-b][l,3,4]thiadiazolyl, thieno[2,3-b]pyrrolyl, 3H-indolyl,and derivatives thereof. Furthermore, when containing two fused rings the aryl groups herein defined may have an unsaturated or partially saturated ring fused with a fully saturated ring. Exemplary ring systems of these heteroaryl groups include indolinyl, indolinonyl, dihydrobenzothiophenyl, dihydrobenzofuran, chromanyl, thiochromanyl, tetrahydroquinolinyl, dihydrobenzothiazine, 3,4 dihydro-lH-isoquinolinyl, 2,3 -dihydrobenzofuran, indolinyl, indolyl, and dihydrobenzoxanyl.

[0246] Halogen or “halo” means fluorine, chlorine, bromine, iodine, and their isotopomers. “Alkyl” means a straight or branched chain saturated hydrocarbon containing 1-100 carbon atoms. Examples of a (Cl -Cl 00) alkyl group include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, neopentyl, and isohexyl.

[0247] “Alkoxy” means a straight or branched chain saturated hydrocarbon containing 1-100 carbon atoms containing a terminal “O” in the chain, e.g., O(alkyl). Examples of alkoxy groups include, without limitation, methoxy, ethoxy, propoxy, butoxy, t-butoxy, or pentoxy groups.

[0248] “Alkyene” means a straight or branched chain unsaturated hydrocarbon containing 2-100 carbon atoms. The “Akyene” group contains at least one double bond in the chain. The double bond of an alkenyl group can be unconjugated or conjugated to another unsaturated group. Examples of alkenyl groups include ethenyl, propenyl, n-butenyl, isobutenyl, pentenyl, or hexenyl. An alkenyl group can be unsubstituted or substituted and may be straight or branched.

[0249] “Alkyne” means a straight or branched chain unsaturated hydrocarbon containing 2-100 carbon atoms. The “Alkyne” group contains at least one triple bond in the chain. Examples of alkenyl groups include ethynyl, propargyl, n-butynyl, isobutynyl, pentynyl, or hexynyl. An alkynyl group can be unsubstituted or substituted.

[0250] "Cycloalkyl" or "carbocyclyl” means a monocyclic or polycyclic saturated carbon ring containing 3-100 carbon atoms. Examples of cycloalkyl groups include, without limitations, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptanyl, cyclooctanyl, norboranyl, norborenyl, bicyclo[2.2.2]octanyl, or bicyclo [2.2.2]octenyl and derivatives thereof. A (C3-C100) cycloalkyl is a cycloalkyl group containing between 3 and 100 carbon atoms. A cycloalkyl group can be fused (e.g., decalin) or bridged (e.g., norbomane).

[0251] " Cycloalky ene" means a monocyclic or polycyclic carbon ring containing 3-100 carbon atoms. The "Cycloalky ene" group contains at least one double bond in the chain. The double bond of a cycloalkyene group can be unconjugated or conjugated to another unsaturated group. Examples of cycloalkyene groups include cyclopropenyl, cyclobutenyl, butadienyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, cycloheptenyl, cycloheptadienyl, cycloheptatrienyl, cyclooctenyl, cyclooctadienyl, or cyclooctatetraenyl.

[0252] "Cycloalkyne" means a monocyclic or polycyclic carbon ring containing 3-100 carbon atoms. The " Cycloalkyne " group contains at least one triple bond in the chain. The triple bond of a cycloalkyne group can be unconjugated or conjugated to another unsaturated group. Examples of cycloalkyne groups include cyclooctynyl. Heterocyclyl” or "heterocycloalkyl” means saturated or partially saturated monocyclic or polycyclic ring containing carbon and at least one heteroatom selected from oxygen, nitrogen, or sulfur (0, N, or S) and wherein there is not delocalized n electrons (aromaticity) shared among the ring carbon or heteroatoms. The heterocycloalkyl ring structure may be substituted by one or more substituents. The substituents can themselves be optionally substituted. Examples of heterocyclyl rings include , but are not limited to oxetanyl, azetadinyl, tetrahydrofuranyl, tetrahydropyranyl, pyrrolidinyl, oxazolinyl, oxazolidinyl, thiazolinyl, thiazolidinyl, pyranyl, thiopyranyl, tetrahydropyranyl, dioxalinyl, piperidinyl,morpholinyl, thiomorpholinyl, thiomorpholinyl S-oxide, thiomorpholinyl S-dioxide, piperazinyl, azepinyl, oxepinyl, diazepinyl, tropanyl, oxazolidinonyl, 1,4-dioxanyl, dihydrofuranyl, 1,3-dioxolanyl, imidazolidinyl, imidazolinyl, dithiolanyl, and homotropanyl.

[0253] “Haloalkyl” means an alkyl group substituted with one or more halogens. Examples of haloalkyl groups include, but are not limited to, trifluoromethyl, difluoromethyl, pentafluoroethyl, or tri chloromethyl.

[0254] “Haloalkoxy” means an alkoxy group substituted with one or more halogens. Examples of haloalkyl groups include, but are not limited to, trifluoromethoxy, difluoromethoxy, pentafluoroethoxy, or trichloromethoxy.

[0255] “Cyano” means a substituent having a carbon atom joined to a nitrogen atom by a triple bond, e.g., C = N.

[0256] “Amino” means a substituent containing at least one nitrogen atom (e.g., -NH2).

[0257] “Isomers” means compounds having the same number and kind of atoms, and hence the same molecular weight, but differing with respect to the arrangement or configuration of the atoms in space. The term includes stereoisomers and geometric isomers.

[0258] “Stereoisomer” or “optical isomer” mean a stable isomer that has at least one chiral atom or restricted rotation giving rise to perpendicular dissymmetric planes (e.g., certain biphenyls, allenes, and spiro compounds) and can rotate the plane of plane-polarized light. Because asymmetric centers and other chemical structures exist in the compounds of the disclosure, which may give rise to stereoisomerism, the disclosure contemplates stereoisomers and mixtures thereof. The compounds of the disclosure and their salts include asymmetric carbon atoms and may therefore exist as single stereoisomers, racemates, and as mixtures of enantiomers and diastereomers. Typically, such compounds will be prepared as a racemic mixture. If desired, however, such compounds can be prepared or isolated as pure stereoisomers, i.e., as individual enantiomers or diastereomers, or as stereoisomer enriched mixtures. As discussed in more detail below , individual stereoisomers of compounds are prepared by synthesis from optically active starting materials containing the desired chiral centers or by preparation of mixtures of enantiomeric products followed by separation or resolution, such as conversion to a mixture of diastereomers followed by separation or recrystallization, chromatographic techniques, use of chiral resolving agents, or direct separation of the enantiomers on chiral chromatographic columns. Starting compounds with particular stereochemistry are either commercially available or are made by the methods described below and resolved by techniques well-known in the art.

[0259] “Enantiomers” means a pair of stereoisomers that are non - superimposable mirror images of each other.

[0260] "Diastereoisomers” or “diastereomers” mean optical isomers, which are not mirror images of each other.

[0261] “Racemic mixture” or “racemate” mean a mixture containing equal parts of individual enantiomers.

[0262] "Non-racemic mixture” means a mixture containing unequal parts of individual enantiomers.

[0263] “Geometrical isomer” means a stable isomer which results from restricted freedom of rotation about double bonds (e.g., c / .s-2-butene and trans 2-butene) or in a cyclic structure (e.g., cis-l,3-dichlorocyclobutane and trans- 1,3 -di chlorocyclobutane). Because carbon-carbon double (olefinic) bonds, C=N double bonds, cyclic structures, and the like may be present in the compounds of the disclosure, the disclosure contemplates each of the various stable geometric isomers and mixtures thereof resulting from the arrangement of substituents around these double bonds and in these cyclic structures. The substituents and the isomers are designated using the cis I trans convention or using the E or Z system, wherein the term “E” means higher order substituents on opposite sides of the double bond, and the term “Z” means higher order substituents on the same side of the double bond. A thorough discussion of E and Z isomerism is provided in J. March, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 4th ed., John Wiley & Sons, 1992, which is hereby incorporated by reference in its entirety.

[0264] Some of the compounds of the disclosure can exist in more than one tautomeric form. As mentioned above, the compounds of the disclosure include all such tautomer. It is well-known in the art that the biological and pharmacological activity of a compound is sensitive to the stereochemistry of the compound. Thus, for example, enantiomers often exhibit strikingly different biological activity including differences in pharmacokinetic properties, metabolism, solubility, protein binding, and the like, and pharmacological properties, including the type of activity displayed, the degree of activity, toxicity, and the like. Thus, one skilled in the art will appreciate that one enantiomer may be more active or may exhibit beneficial effects when enriched relative to the other enantiomer or when separated from the other enantiomer. Additionally, one skilled in the art would know how to separate, enrich, or selectively prepare the enantiomers of the compounds of the disclosure from this disclosure and the knowledge of the prior art.

[0265] Thus, although the racemic form of drug may be used, it is often less effective than administering an equal amount of enantiomerically pure drug. Thus, if one enantiomer is pharmacologically more active, less toxic, or has a preferred disposition in the body than the other enantiomer, it would be therapeutically more beneficial to administer that enantiomer preferentially.

[0266] Preparation of pure enantiomers or mixtures of desired enantiomeric excess (ee) or enantiomeric purity are accomplished by one or more of the many methods of (a) separation or resolution of enantiomers, or (b) enantioselective synthesis known to those of skill in the art, or a combination thereof. These resolution methods generally rely on chiral recognition and Such methods are disclosed generally in Chiral Separation Techniques: A Practical Approach (2ndEd.), G. Subramanian (ed.), Wiley-VCH, 2000; T. E. Beasley andR.P.W. Scott, Chiral Chromatography, John Wiley & Sons, 1999; and Satinder Ahuja, Chiral Separations by Chromatography, Am. Chem. Soc., 2000.

[0267] In general, all tautomeric forms and isomeric forms and mixtures, whether individual geometric isomers or stereoisomers or racemic or non-racemic mixtures, of a chemical structure or compound is intended, unless the specific stereochemistry or isomeric form is specifically indicated in the compound name or structure.

[0268] “Charged” refers to ionic forms of an atom or a group of atoms where the number of electrons is not equal to the number of protons. The charged atom or a group of atoms can be anion (negative charge or negative charged) and cation (positive charge or positive charged) at certain pH values. “Chargeable” or “chemically chargeable” refers to the capability of being ionized of an atom or a group of atoms in aqueous solutions at certain pH values.

[0269] The “chemical bond(s) or moi ety(moi eties) that can form reversible or irreversible covalent bond(s) with any nucleophiles in biology” refers to any chemical bonds or moieties that can reversibly or irreversibly crosslink the endocytic agents and any nucleophiles in biology (including lysine, arginine, cysteine, serine, threonine, and carbohydrates) via covalent bonds. Specially, wherein the “chemical bond(s) or moiety(moieties) that can form reversible covalent bond(s) with any nucleophiles in biology” refers to any chemical bonds or moieties that can crosslink the endocytic agents and any nucleophiles in biology via covalent bonds, but the dissociation of said covalent bond(s) can be broken and / or reformed in any conditions. In certain embodiments, the “chemical bond(s) or moiety(moieties) that can form reversible or irreversible covalent bond(s) with any nucleophiles in biology” can be selected from any chemical bonds, linkages, or moieties as listed in patents WO2020252397A1 and WO2011018611A1, and in publication Anupam Bandyopadhyay, et al., Current Opinion Chemical Biology, 34, 110-116, 2016, Hannah Kiely- Collins, et al., Cell Chemical Biology, 28(7), 952-968, 2021, Fandi Sutanto, et al., RSC Medicinal Chemistry, 11, 876-884, 2020, and Sijie Wang, et al., ChemRxiv preprint, 2022 (DOI: 10.26434 / chemrxiv-2022-tvgnl). In certain embodiment, the “chemical bond(s) or moiety(moieties) that can form reversible or irreversible covalent bond(s) with any nucleophiles in biology” includes, but not limited to, substituted or non- substituted Michael acceptors (including acrylamides, acrylates), cyclic imines, thiols, a-cyanoacrylamides (or acrylates), a- substituted acrylonitriles, a.p-diketoamide with arginine (Ziyang Zhang, et al., Journal of the American Chemistry Society, 144(35), 15916-15921, 2022), P-lactone derivatives (Ziyang Zhang, et al., Nature Chemical Biology, 18, 1177-1183, 2022), benzylidine rhodanine derivatives, disulfide bond, boronic acids, a-ketoamides, nitrile moieties, methyl esters, ketones, ortho- phenoxide moieties, aromatic aldehydes, coumarin 3-aldehydes, ortAo-boronic acid substituent benzaldehyde (or acetophenone), and any deuterium substituted derivatives, or any combination thereof, wherein the interrupting and the one or both terminating groups may be the same or different.

[0270] Methods and Pharmaceutical Administration

[0271] An aspect of the technology provides for methods that affect cellular uptake of agents via endocytosis. In some embodiments, the method refers to employ structural modification strategies used in the art to allow for or adjust an agent’s binding affinity and / or binding valent with endocytosis- mediating membrane component(s), the dimerization or clustering of endocytosis-mediating membrane components, and / or the conformational changes of endocytosis-mediating membrane component(s) for enhanced endocytic efficacy and / or efficiency. The structural modification strategies include but are not limited to adding or removing substituents, fragment replacement, cyclization, scaffold-hopping, bioisosterism, linkerology (linker-activity relationship studies), and prodrug. This method allows for enhancing the intended biological activities of the agent and agent’s binding affinity to endocytosis-mediating membrane component / s) for enhanced endocytic efficacy and or efficiency for disease diagnosis and treatment. In some embodiments, the method refers to covalently conjugating chemical arm(s) onto an agent via cleavable or non-cleavable chemical bond(s) or linker / s) to generate a multivalent endocytic agent. The multivalent endocytic agent can have a better absorption via the enhanced endocytosis for disease diagnosis and treatment. In certain embodiments, the method refers to covalently conjugating chemical arm(s) onto an agent via chemical bond(s) or linker / s) to generate a multivalent endocytic agent, wherein the chemical arm(s) is selected to allow for or increase the resulting multivalent endocytic agent’s binding affinity and / or binding valent to endocytosis-mediating membrane component / s), the dimerization or clustering of endocytosis-mediating membrane components, and / or the conformational change of endocytosis-mediating membrane component(s) for enhanced endocytic efficacy and / or efficiency without sacrificing the intrinsic pharmacological activities of the agent. In certain embodiments, the method refers to covalently conjugating chemical arm(s) onto a therapeutic agent via cleavable bond(s) or linker(s), wherein the chemical arm / s) is selected to impair, diminish, or remove the intended functionality of the therapeutic agent until to the chemical arm(s) is cleaved from the therapeutic agent. This method allows for enhanced targeted delivery into cells or tissues via endocytosis.

[0272] In some embodiments, the method refers to employ structural modification strategies used to allow for or enhance conformational changes of endocytosis-mediating membrane component / s) by structural modifications of endocytic agents for any purposes, wherein the structural modification strategies include, but are not limited to, making charge molecules or salting techniques in the art, by forming multivalent endocytic agent via connecting charged, chargeable and other hydrophilic chemical arm / s), or by structural modification using medicinal chemistry strategies in the art including introducing reversible or non-reversible covalent bond(s) or moiety(moieties). In certain embodiments, a method to allow for or enhance conformational changes of endocytosis-mediating membrane component(s) for enhanced endocytic efficiency and / or efficacy of endocytic agent is to increase the binding affinity and / or binding valent of endocytic agents with endocytosis-mediating membrane component(s). In certain embodiments, a method to allow for or enhance conformational changes of endocytosis-mediating membrane component(s) for enhanced endocytic efficiency and / or efficacy of endocytic agent is to increase the dimerization or clustering of endocytosis-mediating membrane component. In certain embodiments, a method to allow for or enhance conformational changes of endocytosis-mediating membrane component(s) for enhanced endocytic efficiency and / or efficacy of endocytic agent is to adjust endocytosis-mediating membrane component(s) environmental factors, interaction(s) with membrane cofactor proteins, and / or status of post translational modification of endocytosis- mediating membrane component(s) and membrane cofactor proteins by utilization or structural modification strategies used in the art of endocytic agents.

[0273] An aspect of the technology provides for methods to allow for or increase the binding affinity and / or binding valent of an endocytic agent with endocytosis-mediating membrane component(s), the dimerization or clustering of endocytosis-mediating membrane components, and / or the conformational change of endocytosis-mediating membrane component s) for agent’s enhanced endocytic efficacy and / or efficiency. In some embodiments, the methods refer to form multivalent endocytic agent or use medicinal chemistry strategies in the art, such as introducing reversible or irreversible covalent bond(s) or moiety(moieties) at any appropriate site of the endocytic agent in any equivalent. In certain embodiments, the strategies refer to conjugating chemical arm(s) containing reversible or irreversible covalent bond(s) or moi ety(moi eties) onto an agent via cleavable or non-cleavable chemical bond(s) or linker(s), or the replacement of any bonds or moi eties on endocytic agent with reversible covalent bond(s) or moiety(moieties). The resulting endocytic agent can form reversible or irreversible covalent interactions with endocytosis- mediating membrane component(s), allow for or increase dimerization or clustering of endocytosis-mediating membrane components, and / or allow for or increase conformational change of endocytosis-mediating membrane component(s) for enhanced endocytic efficacy and / or efficiency. An aspect of the technology provides for methods to increase an endocytic agent’s solubility and binding affinity and / or binding valent with endocytosis-mediating membrane component(s) for enhanced endocytic efficacy and / or efficiency. In some embodiments, the method refers employ salting techniques in the art, structural modification on an agent using medicinal chemistry strategies in the art, and / or forming multivalent endocytic agent via conjugating with chargeable or charged chemical arm(s) to allow for increasing an endocytic agent’s solubility and binding affinity and / or binding valent with endocytosis-mediating membrane component(s) for enhanced endocytic efficacy and / or efficiency. The resulting endocytic agent can be present in cation or anion form in aqueous solution at a certain pH value and has increased solubility. This method allows for endocytic agents to possess enhanced solubility and permeability via endocytosis spontaneously for disease diagnosis and treatment. In some embodiments, the method refers to forming exocytic vesicles in vitro or in vivo to allow for increasing an endocytic agent’s stability and solubility.

[0274] An aspect of the technology provides for methods to allow for the endocytic uptake of agents with no limitations on endocytic agent’s molecular weight, polarity, and lipophilicity. In certain embodiment, the present technology provides for methods to facilitate the endocytic uptake of hydrophilic agents, including but are not limited to, inorganic compounds, chelation, metal- based compounds, and / or polar organic compounds such as peptide-based compounds. For example, by binding with endocytosis-mediating membrane component via salt bridges and / or hydrogen bond formations, highly polar compounds can be up-taken by cells via endocytosis. Another aspect of the technology provides for methods to increase binding affinity and / or binding valent to endocytosis mediating membrane component(s), and / or dimerization or clustering of endocytosis-mediating membrane components, and / or conformational changes of endocytosis- mediating membrane component(s) for enhanced polar or hydrophilic agent’s endocytic efficacy and / or efficiency by making charge molecules or salting techniques in the art, by forming multivalent endocytic agent via connecting chemical arm(s), or by structural modification using medicinal chemistry strategies in the art including introducing reversible or non-reversible covalent bond(s) or moiety(moieties). In certain embodiments, by introducing a moiety targeting the hydrophobic core of CD36 onto, for example, a polar phosphatase inhibitor, the resulting endocytic phosphatase inhibitor agent can have increased binding affinity and / or binding valent to endocytosis mediating membrane component(s), dimerization, and / or clustering of endocytosis- mediating membrane components, and / or conformational changes of endocytosis-mediating membrane component(s) for enhanced endocytic efficacy and / or efficiency. In certain embodiments, by introducing a CD36 binding moiety onto insulin molecule with cleavable or non- cleavable bonds or moieties, the resulting endocytic insulin agent can be used via oral administration, due to the increased binding affinity and / or binding valent to endocytosis mediating membrane component(s), dimerization or clustering of endocytosis-mediating membrane components, and / or conformational changes of endocytosis-mediating membrane component(s) for enhanced endocytic efficacy and / or efficiency. The encapsulation of insulin in exosomes after endocytosis and exocytosis enhances stability. Another benefit of the technology provides for methods to allow for endocytic agents across cellular membranes and / or body barriers via endocytosis alone or in combination with other mechanisms, including but not limited to, passive diffusion, facilitated diffusion, transporter-mediated influx and / or efflux, and paracellular transport.

[0275] An aspect of the technology provides for methods to affect the metabolic stability of endocytic agents. To meet agent stability requirement for treatment and diagnosis of subjects, the methodology of current disclosure, include but not limit to, adding or removing substituents, fragment replacement, cyclization, scaffold-hopping, bioisosterism, linkerology (linker-activity relationship studies), prodrug, lowing the overall LogP values, or forming exocytic vesicles in vitro or in vivo. In certain embodiments, the method refers to deuterium replacement of any hydrogen atoms on endocytic agent. In certain embodiments, the method refers to introducing substitutes to reduce metabolism at the soft spots in the agent.

[0276] An aspect of the technology provides methods to make and / or use (polypharmacological) endocytic agents for any purposes, wherein at least one target of the (polypharmacological) endocytic agents is endocytosis-mediating membrane component(s). As the binding nature of an endocytic agent with endocytosis-mediating membrane component s) can be independent with its binding ability with intrinsic pharmacological target(s), it allows for an endocytic agent to be a (polypharmacological) compound which spontaneously possesses binding affinities to both endocytosis-mediating membrane component s) for endocytosis and pharmacological target(s) for intrinsic pharmacological activities without balancing. In some embodiments, a method to identify or generate (polypharmacological) endocytic agents is to make structural modification directly on an agent, making it readily bind with both endocytosis-mediating membrane component(s) for endocytosis and pharmacological target(s) for intrinsic pharmacological activities. Wherein the process to identify or generate novel (polypharmacological) endocytic and functional targeting agents, classical medicinal chemistry in art is to monitor the activities for both endocytic and biological target modulation efficiencies spontaneously. The structural modification includes, but is not limited to, structural modification on an agent using medicinal chemistry strategies in the art, forming multivalent endocytic agent via conjugating with chargeable or charged chemical arm(s), and forming exocytic vesicles.

[0277] An aspect of the technology provides for methods to make and / or use (polypharmacological) endocytic agents and exocytic vesicles to transport (polypharmacological) endocytic agents and exocytic vesicles across barriers in human body against foreign materials for any purposes, wherein, the barriers are blood-retinal barrier, lung endothelial and epithelial barrier, skin barrier, brain-blood-barrier. In some embodiment, a method to make and / or use (polypharmacological) endocytic agents and exocytic vesicles is endocytic agents and exocytic vesicles bind with receptors or proteins expressed in cells in barriers, such as scavenger receptors, Mfsd2a, GLUT1, Flotillin-1, Flotillin-2, glutathione transporter, amino acids transporters, transferrin receptor, lactoferrin receptor, low density lipoprotein receptor, nicotinic acetylcholine receptor, insulin receptor, insulin-like growth factor receptor, integrin, and / or CD13 / APN receptor, and are transported through barriers via endocytosis / exocytosis route in forms of free endocytic agents, exocytic vesicles, or the mixture of free endocytic agents and exocytic vesicles at any ratio. In some embodiment, a method to make and / or use (polypharmacological) endocytic agents and exocytic vesicles is the endocytic agents are absorbed into cells via endocytosis and wrapped in intracellular organelles or vesicles, such as endosomes and multivesicular bodies, reducing or preventing pumping endocytic agents out of the cells by efflux transporters, such as P-gp, BCRP and the multidrug resistance-associated proteins MRP1, MRP3, MRP4 and MRP6. In some embodiment, a method of using expression of endocytosis-mediating membrane component s) in membrane barriers and / or any structural modifications on agent in present disclosure to enhance efficacy and / or efficiency of endocytosis of endocytic agents and transport of endocytic agents across membrane barriers for any purposes. Particularly, a method of using (polypharmacological) endocytic agents and exocytic vesicles is to administrate (polypharmacological) endocytic agents and exocytic vesicles by any routes for the treatment of eye, respiratory, skin, and CNS conditions and diseases, wherein the (polypharmacological) endocytic agents and exocytic vesicles can be used alone or in combination at any ratio.

[0278] An aspect of the technology provides for methods to transport endocytic agents across more than one cell layers in body for any purposes, wherein the endocytic agents can be up-taken by cells via endocytosis and can be released in forms of free endocytic agent molecules and / or endocytic agents-vesicle complexes (namely, exocytic vesicles), which comprise endocytic agents and lipid bilayer vesicles including extracellular vesicles, and the free endocytic agent molecules and / or exocytic vesicles can be up-taken by any (acceptor) cells again for the action via endocytosis or membrane fusion for any purpose of applications.

[0279] An aspect of the technology provides for methods to make exocytic vesicles in vitro and / or in vivo for any purposes. Particularly, an aspect of the technology provides for a “one-step” method to generate and use of exocytic vesicles in animal bodies, wherein, without further processes including agents or vesicles isolation, cells bodies up-take endocytic agents via endocytosis and sequentially secrete out exocytic vesicles, and the resulting endogenous exocytic vesicles can be directly used by bodies for any purposes. In certain embodiments, the “body” or “animal body” refers to a human body. In another embodiment, a method to generate exocytic vesicles is that free endocytic agent molecules bind with extracellular vesicle and / or exocytic vesicles via covalent or non-covalent bond(s)in situ. In another embodiment, a method to generate exocytic vesicles is to load endocytic agent into exocytic vesicles in vitro by any technology in the art. In another embodiment, a method to isolate the exocytic vesicles from cells, body fluids, tissues, organs, products, or cultural mediums by any extracellular vesicles’ isolation techniques in the art.

[0280] An aspect of the technology provides for methods to adjust the endocytic agents’ absorption, distribution, metabolism, and excretion (ADME) properties in animal bodies. In certain embodiment, to extend the residency time and / or half-lives of given endocytic agents in animal bodies, a method is to enhance endocytic agents’ endocytic efficacy and / or efficiency via above mentioned methods, to increase the loading of endocytic agents into exocytic vesicles via cell endocytosis or the releasing of exocytic vesicles, which can reduce or prevent endocytic agents from fast metabolism and / or excretion.

[0281] An aspect of the technology provides for methods to evaluate and / or determine endocytic agents’ ADME properties in the drug discovery and development process, wherein a process or step for exocytic vesicles isolation and / or lysis by any technology and stills in the art is involved. In certain embodiment, the technology, skill, and agents can be used for exocytic vesicles isolation process includes, but not limited to, any suitable methods for extracellular vesicles (EVs) or cells isolation as listed in publications Brennan K., et al., Scientific Reports, 10, 1039, 2020 and Thanaporn Liangsupree, et al., 1636, 461773, 2021. In certain embodiment, the technology, skill, and agents can be used for exocytic vesicles lysing process includes, but not limited to, any suitable methods or agents for lysing extracellular vesicles (EVs) or cells or causing EV or cell membranes to rupture, including freeze-thaw cycle process, and any methods or agents as listed in publication Prabal Subedi, et al., Analytical Biochemistry, 584, 113390, 2019.

[0282] In some embodiments, the method refers to reduce the toxicity of agents / drugs by making and using exocytic vesicles from endocytic agents in vitro or in vivo. As endocytic agents can be up-taken by and released from cells in forms of exocytic vesicles, such as endocytic agents- exosomes complexes, wherein the endocytic agents can exist in exosomes, preventing or reducing the direct contact between free endocytic agent molecules and cells in body, such as blood cells. In certain embodiment, a method refers to administrate endocytic agents orally, the cells within human body, such as cells in gastrointestinal tract, liver, or tumors, uptake endocytic agents via endocytosis and secrete out exocytic vesicles, and the resulting exocytic vesicles reduce toxicity comparing with the free endocytic agent molecules. In certain embodiment, a method to reduced toxicity is adjusting agents’ endocytosis efficacy and / or efficiency by any above methods, loading of endocytic agents into exocytic vesicles, and / or releasing of exocytic vesicles. For example, by connecting or fusing additional chemical arm(s) with binding affinity to the endocytosis-mediating membrane component(s) onto endocytic agent via cleavable or non-cleavable chemical bond or linker unit(s) to make is as a multivalent compound, the resulting multivalent endocytic agent can have better affinity with endocytosis-mediating membrane component(s) for enhanced endocytic efficacy and / or efficiency, enhanced loading of endocytic agent into exocytic vesicles, and / or increased releasing of exocytic vesicles, and eventually reduced toxicity, as the formation of exocytic vesicles prevent or reduce the direct contact between free endocytic agent molecules and cells such as blood cells.

[0283] An aspect of the technology provides for methods to design or structural modifications of endocytic agents using medicinal chemistry strategies in the art or utilization of endocytic agents to target specific confirmation(s) of endocytosis-mediating membrane component(s) for any purposes. In certain embodiment, the present technology provides for methods to take advantage of the conformation diversity of CD36 on normal cells and cancer cells for the structural design or modification of endocytic agents using medicinal chemistry strategies in the art or selection of endocytic agents to target the specific conformation(s) of CD36 on cancer cells for enhanced anti- tumor efficacy and reduced toxicity. Another benefit of the present technology provides for methods to take advantage of any approaches in the art, such as application of mechanical, electrical, heat, cold, light, or radiation stimulation and / or the presence of one endocytic agent, to change conformations of endocytosis-mediating membrane component(s), resulting in the alterations of biological events in cells and / or sensitivity of the endocytosis-mediating membrane component(s) to endocytic agent.

[0284] An aspect of the technology provides for general methods for improved clinical outcomes. In some embodiments, the method refers to use the expression difference of endocytosis-mediating membrane component(s) for patient stratification, dosage route and dosage selection. Meanwhile, the feedback of endocytic agents’ treatments in clinic can be used to adjust the route for administrations of endocytic agents and / or improve outcomes of endocytic agents’ treatments. In certain embodiments, the method refers to the treatment of a subject in need of an endocytic agent. In certain embodiment, the method refers to the treatment of a subject in need of an endocytic agent via a special delivery system such as topical, inhaled, intraperitoneal, intravenous, and oral delivery by employing different expression of endocytosis-mediating membrane component(s) in cells and tissues. For example, CD36-mediated endocytosis is a dominant pathway for cellular uptake of nutrients and macromolecules (Itirgen Pohl, et al., Molecular Biology of the Cell, 16(1), 24-31, 2005; Nanxia Zhao, et al., Advanced NanoBiomed Research, 2(6), 2100120, 2022; Vincenza Cifarelli, et al., Comprehensive Physiology, 8(2), 493-507, 2018; Youchun Zeng, et al., Journal of Biological Chemistry, 278(46), 45931-45936, 2003). CD36 is highly expression in intestine, cancer cells, metabolism related cells, and upregulated in patients’ central nervous system with pathological changes (Vincenza Cifarelli, et al., Comprehensive Physiology, 8(2), 493-507, 2018; Shunjie Bai, et al., Translational Psychiatry, 11(16), 2021; Octavian loghen, et al., European Journal of Neuroscience, 53, 2500-2510, 2021). Besides, receptor-mediated endocytosis and exocytosis route is a major path to transport nutrients, such as folates and analogues, into brain parenchyma in forms of free nutrient compounds and nutrient compounds-loaded extracellular vesicles (Marcel Grapp, et al., Nature Communications, 4, 2123, 2013; Andong Qiu, et al., Cell, 127, 917-928, 2006). Thus, present technology particularly provides general methods for increasing potency and reducing toxicity of much broader endocytic agents into central nervous system after oral or iv delivery for therapeutic and prophylactic administration.

[0285] An aspect of the technology provides for methods to identify literature-known bioactive compounds that can be up-taken by cells via endocytosis. Wherein the method includes adjusting the expression of endocytosis-mediating membrane component s) in cells, tissues, and / or bodies by endocytosis-mediating membrane component(s) gene editing, followed by comparing the activities of bioactive compounds in cells, tissues, and / or bodies with and without endocytosis- mediating membrane component(s) gene editing. Comparing with the activities of compounds in cells, tissues, and / or bodies without endocytosis-mediating membrane component s) gene editing, the compounds which are up-taken via endocytosis have significantly increased or decreased bioactivity in cells, tissues, and / or bodies with endocytosis-mediating membrane component(s) editing.

[0286] An aspect of the technology provides for methods of using present endocytic agent as probes to identify the biological target(s) in cells and bodies by any technology and stills in the art. For example, the present endocytic agent which possess biotin, fluorescent, Halo-tag ligand(s), SNAP -tag ligand(s), CLIP -tag ligand(s), or chemical bond(s) or moiety(moieties) that can have covalent bond(s) formation with any biological target(s) are particularly suitable for identifying biological target(s) of endocytic agent and derivatives, in combination with technology and stills including microscope imaging, immunophenotyping, immunoprecipitation, flow cytometry, fluorescent-activated cell sorting (FACS), fluorescence resonance energy transfer (FRET), and / or omics (including genomics, epigenomics, transcriptomics, proteomics, and metabolomics) analysis. Particularly, an aspect of the technology provides for methods to identify the endocytosis mediating membrane target(s). In a certain embodiment, the method to identify the endocytosis- mediating membrane target(s) refers to use a labeled endocytic agent followed by agent administration and any detecting and identification techniques in the art. For example, connecting a labeling probe, such as biotin, onto an endocytic agent at any sites results in a biotin-labeled endocytic agent. Subsequently, the endocytosis targeting endocytosis-mediating membrane component(s) of the endocytic agent can be identified by culturing cell with biotin-labeled endocytic agent, isolating membrane proteins, followed by immunoprecipitation, FACS, omics analysis, western-blot confirmation. In another embodiment, the method to identify the endocytosis-mediating membrane target(s) refers to using any genomic scanning technologies in the art to identify the endocytosis-mediating membrane target(s). For example, comparisons of gene expression between cells, tissue, or bodies with different sensitivities to certain endocytic agents can be used to identify the endocytosis-mediating membrane target(s). In another embodiment, the method to identify the endocytosis-mediating membrane target(s) refers to use any gene-editing technology in the arts to identify the endocytosis-mediating membrane target(s), wherein the gene-edited cells, tissues, or bodies can be more sensitive or resistant to endocytic agent treatment. For example, cells with gene expression inhibition and activation, through CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa), respectively, act differently or complementarily to certain endocytic agent treatments, which can be used to identify the endocytosis-mediating membrane target(s).

[0287] An aspect of the technology provides for methods to use and select present endocytic agents basing on the different microenvironmental factors of cells and tissues for any purposes, wherein the microenvironment of cells and tissues include but are not limited to, pH values, salinity, oxygen gradient, carbon dioxide gradient, H2O2 gradient, nutrient gradient, and therapeutic compound gradient. For example, due to the acidic environment of cancer cells, endocytic agents with basic group(s) can be used for tumor-targeted delivery for enhanced therapeutic functions and lower toxicities.

[0288] An aspect of the technology provides for methods to use and select present endocytic agents basing on the expressions of endocytosis-mediating membrane component s) with diverse conformations, isoforms (or variants), and / or posttranslational modification such as glycosylation status for any purposes. Another aspect of the technology provides for methods to use and select present endocytic agents based on the expressions of cofactors that form complex with endocytosis-mediating membrane component for any purposes. For example, by targeting the specific conformations, isoforms, or glycosylation of endocytosis-mediating membrane component or cofactors in cancer cells, endocytic agents can be used for enhanced therapeutic functions and lower toxicities in cancer treatments.

[0289] An aspect of the technology provides for methods to activate the endocytic process or increase expression of endocytosis-mediated membrane component(s) for enhanced endocytic absorption of endocytic agents via regulating input signals, such as the binding of endogenous or exogenous substances with a membrane protein. In certain embodiments, the method refers to the utilization of the binding of an endogenous substance (e.g., insulin and derivatives) against a membrane protein (e.g., insulin receptor) to active the endocytic cycling of endocytosis-mediating membrane component(s) such as GLUT4 and / or increase the expression of endocytosis-mediating membrane component s), resulting in the enhanced absorption of endocytic agents via endocytosis. In certain embodiments, the method refers to the utilization of the binding of endocytic agents with endocytosis-mediating membrane component(s) and non-endocytosis-mediating membrane component(s) simultaneously for activating the endocytic process or increasing expression of endocytosis-mediated membrane component(s) and enhancing absorption via endocytosis.

[0290] An aspect of the technology provides for methods to deliver any disease-related endocytosis-mediating membrane component(s) or extracellular materials into cells via endocytosis and destruct or degrade the disease-related endocytosis-mediating membrane component(s) or extracellular materials via endosome / lysosome system. In certain embodiment, the disease related endocytosis-mediating membrane component(s) or extracellular materials include but not limit to membrane proteins or extracellular proteins, carbohydrates, lipids, pathogens, particles, virus, fungi, protozoa, bacteria, vectors, cell debris, and another cell.

[0291] An aspect of the technology provides for methods for treating subjects in need of any of the endocytic agent described herein. Suitably, the method comprises administering an effective amount of the agent to the subject. As used herein, the terms “treating” or “to treat” each mean to alleviate symptoms, eliminate the causation of resultant symptoms either on a temporary or permanent basis, and / or to prevent or slow the appearance or to reverse the progression or severity of resultant symptoms of the named disease or disorder. As such, the methods disclosed herein encompass both therapeutic and prophylactic administration.

[0292] An aspect of the technology provides for a method for diagnosing of subject in need of any of the endocytic agent described herein. Suitably, the method comprises administering an effective amount of the agent to the subject. As used herein, the terms “diagnosing” or “to diagnose” each mean to present or image the site of pathology, alleviate symptoms, eliminate the causation of resultant symptoms either on a temporary or permanent basis, and / or to prevent or slow the appearance or to reverse the progression or severity of resultant symptoms of the named disease or disorder. As such, the methods disclosed herein encompass both therapeutic and prophylactic administration.

[0293] A “subject” can be interchangeable with “patient” or “individual” and means an animal, which can be a human or non-human animal, in need of treatment. A “subject in need of treatment” includes a subject having a disease, disorder, or condition that is responsive to therapy with the endocytic agent disclosed herein, either alone or in combination with another agent. Preferably, a subject in need of a treatment includes, without limitations, aging and age-related diseases and conditions, body weight management, cancers, central nervous systems (CNS) diseases and conditions, cardiovascular diseases (CVDs) , diabetes mellitus, eye diseases and conditions, hypertension, immune system diseases, infections, inflammation and inflammation-associated diseases and conditions, obesity and obesity-associated diseases and conditions, respiratory diseases and conditions, or skin diseases and conditions.

[0294] In certain embodiments, a subject in need of treatment includes a subject in need of prevention, treatment, and study for aging and age-related diseases and conditions. “Aging is a physiological process mediated by biological and genetic pathways, which are directly linked to lifespan and are a driving force for age-related diseases. Aging mechanisms have been identified, including but not limited to genomic instability, telomere shortening, and cellular senescence. For example, aging-related diseases include, but not limited to, cardiovascular diseases, cancer, diabetes mellitus, immune system disorders, hearing loss, macular degeneration, and musculoskeletal disorders, such as osteoarthritis.

[0295] In certain embodiments, a subject in need of treatment includes a subject in need of prevention, treatment, and study for cancer. “Cancer” refers to the pathological process that results in the formation and growth of a cancerous or malignant neoplasm, i.e., abnormal tissue that grows by cellular proliferation, often more rapidly than normal and continues to grow after the stimuli that initiated the new growth cease. For example, cancers include, but are not limited to, mesothelioma, leukemias, and lymphomas such as cutaneous T-cell lymphomas (CTCL), non- cutaneous peripheral T-cell lymphomas, lymphomas associated with human T-cell lymphotropic virus (HTLV) such as adult T-cell leukemia / lymphoma (ATLL), B-cell lymphoma, acute nonlymphocytic leukemias, chronic lymphocytic leukemia, chronic myelogenous leukemia , acute myelogenous leukemia, lymphomas, and multiple myeloma, non-Hodgkin lymphoma, acute lymphatic leukemia (ALL), chronic lymphatic leukemia (CLL), Hodgkin’s lymphoma, Burkitt lymphoma, adult T-cell leukemia lymphoma, acute-myeloid leukemia (AML), chronic myeloid leukemia (CML), or hepatocellular carcinoma. Further examples include myelodysplastic syndrome, childhood solid tumors such as brain tumors, neuroblastoma, retinoblastoma, Wilms’ tumor, bone tumors, and soft-tissue sarcomas, common solid tumors of adults such as head and neck cancers (e.g., oral, laryngeal, nasopharyngeal, and esophageal), genitourinary cancers (e.g., prostate, bladder, renal, uterine, ovarian , testicular), lung cancer (e.g., small cell and non-small cell), breast cancer (e.g., triple-negative breast cancer (TNBC)), pancreatic cancer, melanoma, and other skin cancers, stomach cancer, brain tumors, tumors related to Gorlin’s syndrome (e.g., medulloblastoma or meningioma), or liver cancer.

[0296] Additional exemplary forms of cancer which can be prevented or treated by the endocytic agents include, but are not limited to, cancer of skeletal or smooth muscle, stomach cancer, cancer of the small intestine, rectum carcinoma, cancer of the salivary gland, endometrial cancer, adrenal cancer, anal cancer, rectal cancer, parathyroid cancer, pituitary cancer, colon carcinoma, Familiarly adenomatous polyposis carcinoma, and hereditary non-polyposis colorectal cancer, labial carcinoma, larynx carcinoma, carcinoma, tongue carcinoma, salivary gland carcinoma, gastric carcinoma, , thyroid cancer (medullary and papillary thyroid carcinoma), renal carcinoma, kidney parenchyma carcinoma, kidney carcinoma, cervix carcinoma, uterine corpus carcinoma, endometrium carcinoma, chorion carcinoma, testis carcinoma, urinary carcinoma, brain tumors such as glioblastoma, astrocytoma, meningioma, medulloblastoma and peripheral neuroectodermal tumors, gallbladder carcinoma, bronchial carcinoma, basalioma, teratoma, retinoblastoma, seminoma, rhabdomyosarcoma, craniopharyngioma, osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma, Ewing’s sarcoma, plasmacytoma, melanoma, nasopharyngeal cancer (NPC), microsatellite stable colorectal cancer (mssCRC), thymoma, carcinoid, acute myelogenous leukemia, and gastrointestinal stromal tumor (GIST), breast cancer, triple-negative breast cancer (TNBC), small cell lung cancer, non-small cell lung cancer, prostate cancer, castration-resistant prostate cancer (CRPC), or metastatic castration- resistant prostate cancer (mCRPC).

[0297] In certain embodiments, a subject in need of treatment includes a subject in need of prevention, treatment, and study for cardiovascular disease. “Cardiovascular disease” refers to a group of disorders of the heart and blood vessels. For example, cardiovascular disease includes, but not limit to, coronary heart disease, cerebrovascular disease, peripheral arterial disease, rheumatic heart disease, congenital heart disease, deep vein thrombosis, or pulmonary embolism.

[0298] In certain embodiments, a subject in need of treatment includes a subject in need of prevention, treatment, and study for central nervous system (CNS) diseases and conditions. “CNS diseases and conditions” refers to a wide group of neurological disorders that affect the structure or function of the brain or spinal cord, which collectively form the central nervous system. For example, CNS diseases include, but not limit to, brain tumors, neurodegenerative diseases, such as Alzheimer disease, frontotemporal dementias, Pick disease, progressive supranuclear palsy, corticobasal degeneration, vascular dementia, Parkinson disease, dementia with Lewy bodies, Huntington disease, spinocerebellar ataxia, Friedrich ataxia, ataxia telangiectasia, amyotrophic lateral sclerosis (ALS), bulbospinal atrophy, or spinal muscular atrophy.

[0299] In certain embodiments, a subject in need of treatment includes a subject in need of prevention, treatment, and study for diabetes mellitus. “Diabetes mellitus” refers to a group of diseases in which the body’s ability to produce or respond to insulin is impaired, resulting in the abnormal metabolism of carbohydrates and elevated levels of glucose in the blood and urine. For example, diabetes mellitus includes, but not limited to, type 2 diabetes, gestational diabetes, prediabetes, monogenic diabetes, cystic fibrosis-related diabetes, or drug or chemical-induced diabetes.

[0300] In certain embodiments, a subject in need of treatment includes a subject in need of prevention, treatment, and study for eye diseases and conditions. “Eye diseases and conditions” refers to any diseases or disorders that affect the human eye. For example, eye diseases include, but not limited to, age-related macular degeneration (AMD), amblyopia, anophthalmia and microphthalmia, astigmatism, Behcet's disease, Bietti's crystalline dystrophy, blepharitis, blepharospasm, cataracts, central retinal vein occlusion (CRVO), cerebral visual impairment (CVI), coloboma, color blindness, convergence insufficiency, corneal conditions, diabetic retinopathy, dry eye, eye cancer, farsightedness (Hyperopia), floaters, glaucoma, Graves’ eye disease, idiopathic intracranial hypertension, low vision, macular edema, macular hole, macular pucker, nearsightedness (Myopia), ocular histoplasmosis syndrome (OHS), pink Eye, presbyopia, rare diseases, refractive errors, retinal detachment, retinitis pigmentosa, retinoblastoma, retinopathy of prematurity, Stargardt disease, Usher syndrome, uveitis, or vitreous detachment.

[0301] In certain embodiments, a subject in need of treatment includes a subject in need of prevention, treatment, and study for hypertension. “Hypertension” refers to medical condition in which the blood pressure in the arteries is persistently elevated. For most adults, hypertension is present if the resting blood pressure is persistently at or above 120 / 80, 130 / 80 or 140 / 90 mmHg. For example, hypertension includes, but is not limited to, primary hypertension, or secondary hypertension. In certain embodiments, a subject in need of treatment includes a subject in need of prevention, treatment, and study for immune system diseases and conditions. “Immune system diseases and conditions” refers to immunodeficiency and autoimmune disorders. Wherein, “autoimmune diseases” refers to conditions arising from an abnormal immune response to a functioning body part. The functioning body part refers to any body part of an animal. For examples, autoimmune disease includes, but not limited to rheumatoid arthritis (RA), psoriasis / psoriatic arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), inflammatory bowel disease, Addison’s disease, Graves’ disease, Sjogren’s syndrome, Hashimoto’s thyroiditis, Myasthenia gravis, autoimmune vasculitis, pernicious anemia, or celiac disease.

[0302] In certain embodiments, a subject in need of treatment includes a subject in need of prevention, treatment, and study for inflammation and inflammation-associated diseases and conditions. “Inflammation” is when the immune system attacks the body’s own tissues, resulting in inflammation. Many conditions can be associated with inflammation, especially chronic inflammation. For example, inflammation-associated diseases include, but not limit to, Alzheimer’s disease, asthma, cancer, CDVs, rheumatoid arthritis (RA), ankylosing spondylitis (AS), or stroke.

[0303] In certain embodiments, a subject in need of treatment includes a subject in need of prevention, treatment, and study for body weight management, obesity, and obesity-associated diseases and conditions. “Obesity” refers to body conditions having a body mass index (BMI) of 30.0 or more. Obesity-associated diseases include, but are not limited to, type 2 diabetes, CDVs, fatty liver, or cancer. Body weight management refers to the techniques and physiological processes that contribute to a person’s ability to attain and maintain a certain weight.

[0304] In certain embodiment, the endocytic agents described herein can be used for preventing, treating, and studying respiratory diseases and conditions. “Respiratory diseases” refers to diseases that affect the lung and other parts of the respiratory system. For example, respiratory diseases include, but not limited to, asthma, chronic obstructive pulmonary disease (COPD), pulmonary, pneumonia, idiopathic pulmonary fibrosis, or lung cancer.

[0305] In certain embodiments, a subject in need of treatment includes a subject in need of prevention, treatment, and study for skin diseases and conditions. “Skin diseases and conditions” refers to any conditions that affect skin. For example, skin diseases and conditions include, but not limited to, acanthosis nigricans, acne, acne keloidalis nuchae, acne scars, actinic keratosis, alopecia areata, athlete's foot, atopic dermatitis, basal cell carcinoma, bed bugs, birthmarks, boils and styes, botulinum toxin, bullous pemphigoid, cellulitis, central centrifugal cicatricial alopecia (CCCA), chemical peels, chickenpox, cold sores, contact dermatitis, cradle cap, cutaneous T-cell lymphoma, dandruff, dermatofibrosarcoma protuberan (DFSP), diabetes-related skin conditions, diaper rash, dry skin, dyshidrotic eczema, epidermolysis bullosa, female pattern hair loss, folliculitis, frontal fibrosing alopecia, genital herpes, genital warts, granuloma annulare, hair loss, hand-foot-and- mouth disease, head lice, heart disease-related skin conditions, herpes simplex, hidradenitis suppurativa, hives, hyperhidrosis, ichthyosis vulgaris, imiquimod, impetigo, isotretinoin, keloid scars, keratosis pilaris, kidney disease-related skin conditions, lasers-related skin conditions, leprosy, lichen planus, lupus, lyme disease, melanoma, melasma, merkel cell carcinoma, moles, molluscum contagiosum, monkeypox rash, nail fungus, neurodermatitis, nickel allergy, nummular dermatitis, ocular rosacea, pemphigus, perioral dermatitis, pityriasis rosea, poison ivy, oak, and sumac-related skin conditions, prurigo nodularis, psoriasis, psoriatic arthritis, rashes, ringworm, rosacea, sarcoidosis, scabies, scalp psoriasis, scars, scleroderma, sebaceous carcinoma, seborrheic dermatitis, seborrheic keratoses, shingles, skin biopsy, skin cancer, squamous cell carcinoma, stasis dermatitis, stretch marks, syphilis, thyroid disease-related skin conditions, tinea versicolor, vitiligo, warts, wound, xeroderma or pigmentosum.

[0306] In some embodiments, a subject in need of a treatment includes a subject in need sensitization to a bioactive agent, such as anti-aging, anti-aging-related diseases, anti-cancer, anti- cardiovascular diseases, anti-diabetes, anti-eye diseases, anti-hypertension, anti-immune system diseases, anti-infections, anti-inflammation, anti-inflammation-associated diseases, anti-CNS diseases, body weight management, anti-obesity, anti-obesity-associated diseases, anti -respiratory diseases, anti-skin diseases and conditions. The terms "sensitize”, and "sensitizing" refer to making, through the administration of a first agent, a subject or a cell more susceptible, or more responsive, to the biological effects (e.g., promotion or retardation of an aspect of cellular function including, but not limited to, cell secretion, cell division, cell growth, proliferation, invasion, angiogenesis, necrosis, or apoptosis) of a second agent (e.g., the bioactive agent). The sensitizing effect of a first agent on a target cell can be measured as the difference in the intended biological effect (e.g., promotion or retardation of an aspect of cellular function including, but not limited to, cell growth, proliferation, invasion, angiogenesis, or apoptosis) observed upon the administration of a second agent with and without administration of the first agent. In some embodiments, the endocytic agents described herein can be administered with one or more bioactive agents, including anti-aging, anti-aging-related diseases, anti-cancer, anti- cardiovascular diseases, anti-diabetes, anti-eye diseases, anti-hypertension, anti-immune system diseases, anti-infections, anti-inflammation, anti-inflammation-associated diseases, anti-CNS diseases, anti -obesity, anti-obesity-associated diseases, anti -respiratory diseases, anti-skin diseases and conditions agents. The term “bioactive agent” is used to describe an agent with biological activity to assist in effecting an intended therapy, inhibition and / or prevent! on / prophyl axis for which the present compounds are used. Exemplary bioactive agents include anti -cancer agents. An "anti-cancer agent" means a compound or composition that can be combined with an endocytic agent to treat cancer, inhibit the growth or proliferation of a cancer cell, or kill a cancer cell. Suitably, endocytic agents can be administered before, during, or after administration of the bioactive agent. The endocytic agents can sensitize the subject to the bioactive agent. This allows for improved therapeutic efficacy of the bioactive agent, reduction in an effective amount of bioactive agent needed to achieve a desired effect or reduce the duration of treatment with the bioactive agent.

[0307] Although the present disclosure demonstrates the utility of the technology with various probes or therapeutic endocytic agents, the technology is not limited to those probes or therapeutic endocytic agents. In some embodiments, the endocytic agents include, but are not limited to, immunoconjugates, drugs, prodrugs, cytotoxic agents, pro-apoptotic agents, toxins, nucleases (including DNAses and RNAses), hormones, vitamins, immunomodulators, chelators, boron compounds, photoactive agents, radionuclides, oligonucleotides, interference, DNA, RNA, siRNA, RNAi, anti -angiogenic agents, protein inhibitors, protein activators, molecular glues, degraders, chemotherapeutic agents, cytokines, chemokines, amino acids, peptides, their deuterium substituted derivatives, or combinations thereof.

[0308] As used herein the term “effective amount” refers to the amount or dose of the compound, such as upon single or multiple dose administration to the subject, which provides the desired effect. With respect to sensitization, an effective amount will refer to the amount of a therapeutic agent that results in sensitization of the subject or cell as described above.

[0309] As used herein the term “certain weight”, with respect to the body weight management, refers to the body weight of a person that retains and decreases the desirable weight of a person. An effective amount can be determined by the attending diagnostician, as one skilled in the art, using known techniques and by observing results obtained under analogous circumstances.

[0310] In some embodiments, the endocytic agents utilized in the methods disclosed herein can be formulated as pharmaceutical compositions that include: (a) a therapeutically effective amount of one or more endocytic agents as described herein and (b) one or more pharmaceutically acceptable carriers, excipients, or diluents. Such compositions take any physical form, which is pharmaceutically acceptable; illustratively, they can be orally administered pharmaceutical compositions. Such pharmaceutical compositions contain an effective amount of a disclosed endocytic agent, which effective amount is related to the daily dose of the agent to be administered. Each dosage unit contains the daily dose of a given endocytic agent or each dosage unit contains a fraction of the daily dose, such as one-half or one-third of the dose. The amount of each endocytic agent to be contained in each dosage unit can depend, in part, on the identity of the endocytic agent chosen for the therapy and other factors, such as the indication for which it is given. The pharmaceutical compositions disclosed herein can be formulated to provide quick, sustained, or delayed release of the endocytic agent after administration to the patient by employing well known procedures. The endocytic agents for use according to the methods disclosed herein can be administered as a single compound or a combination of compounds. For example, an endocytic agent that anti-cancer activity can be administered as a single compound or in combination with another compound that promotes also promotes anti-cancer activity or that has a different pharmacological activity.

[0311] In certain embodiment, the endocytic agents utilized in the methods disclosed herein can be loaded with extracellular vesicles (EVs) to form endocytic agents-loaded EVs in vitro for any purposes, wherein the extracellular vesicles include, but are not limited to, exosomes, microvesicles, ectosomes, oncosomes, prostasome, and apoptotic bodies. It can be understood that the endocytic agents-loaded EVs can be obtained by any in vitro technologies in the art for drug- loaded EVs’ generation, isolation, and modification, which includes but is not limited to the methods as shown in publication Shuang Du, et al., loumal of Nanobiotechnology, 21, 231, 2023 and in publication Inge Katrin Herrmann, et al., Nature Nanotechnology, 16, 748-759, 2021. In some embodiments, the endocytic agent utilized in the methods disclosed herein can be loaded within exosomes by microfluidic droplet-based electroporation (pDES) as nanoparticles, which can result in enhanced stability, biocompatibility, transportability, and / or targeting ability over free endocytic agents.

[0312] In certain embodiments, the endocytic agents, including exocytic vesicles, utilized in the methods disclosed herein can be formulated with human serum albumin in vitro or in vivo. Exemplary formulation with human serum albumin is complex at a 7:1, 6: 1, 5:1, 4: 1, 3:1, 2: 1 or 1: 1 conjugate: albumin mol ratio. In some embodiments, the endocytic agent utilized in the methods disclosed herein can be formulated with albumin as nanoparticles, microparticles, albumin-coated liposomes, albumin microbubbles, and albumin nanocapsules. Exemplary nanoparticles range between 1 to 100 nanometers in size. In further embodiments, the endocytic agent-albumin complex can be used in combination with any nanoparticle technology thereof. Exemplary methods for formulated with human serum albumin include, but not limited to, the methods as shown in publications Ella N. Hoogenboezem, et al., Advanced Drug Delivery Reviews, 130, 73-89, 2018; CassandraE. Callmann, et al., loumal of American Chemistry Society, 141(30) 11765-11769, 2019; and Shrawani Lamichhane, et al., Archives of Pharmacal Research, 43, 118-133, 2020. Comparing with the free endocytic agents, the formulation of endocytic agent with albumin in vitro or in vivo can 1) extend the extend the residency time and / or half-lives of given endocytic agents or exocytic vesicles in animal bodies, 2) increase the aqueous solubility, 3) increase the penetration of endocytic agent across cell membranes and membrane barriers, such as blood-retinal barrier, lung endothelial and epithelial barrier, skin barrier, and brain-blood-barrier, 4) be used for delivery of endocytic agent to specific organ systems, such as liver and brain, 5) be used for delivery of endocytic agent to specific body tissue, such as tumor(s), and / or 6) mitigate the toxicity of endocytic agents in animal bodies.

[0313] In certain embodiments, the endocytic agents, including exocytic vesicles, utilized in the methods disclosed herein can be formulated as a pharmaceutical composition. The term “pharmaceutical composition” refers to an agent of the disclosure, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, together with at least one pharmaceutically acceptable carrier, in a form suitable for oral or parenteral administration.

[0314] In certain embodiments, the endocytic agents utilized in the methods disclosed herein can be formulated as a pharmaceutical composition that includes a carrier. For example, the carrier can be selected from the group consisting of proteins, carbohydrates, sugar, talc, magnesium stearate, cellulose, calcium carbonate, and starch-gelatin paste. In certain embodiments, the endocytic agents in the methods disclosed herein can be formulated as a pharmaceutical composition that includes one or more binding agents, diluents, filling agents, lubricating agents, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, and effervescent agents.

[0315] Suitable diluents include pharmaceutically acceptable inert fillers, such as microcrystalline cellulose, lactose, dibasic calcium phosphate, saccharides, and mixtures of any of the foregoing.

[0316] Suitable disintegrants include lightly crosslinked polyvinyl pyrrolidone, corn starch, potato starch, maize starch, and modified starches, croscarmellose sodium, cross-povidone, sodium starch glycolate, and mixtures thereof.

[0317] Examples of effervescent agents are effervescent couples such as an organic acid and a carbonate or bicarbonate. Alternatively, only the sodium bicarbonate component of the effervescent couple can be present.

[0318] In some embodiments, the endocytic agents utilized in the methods disclosed herein can be used alone or can be formulated as a pharmaceutical composition for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual, or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, or intradermal) route, intraperitoneal injection, microneedle patches, and topic via eye drop. Such formulations can be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier(s) or excipient(s). Preferably, the endocytic agents or compositions are administered topically, orally, intraperitoneally, intravenously, via inhalation, or via microinjection.

[0319] In certain embodiments, the endocytic agents utilized in the methods disclosed herein can be administered in conventional dosage forms prepared by combining the endocytic agents with standard pharmaceutical carriers or diluents according to conventional procedures well known in the art. These procedures involve mixing, granulating, and compressing or dissolving the ingredients as appropriate to the desired preparation.

[0320] In certain embodiments, the endocytic agents utilized in the methods disclosed herein can be formulated as a pharmaceutical composition in solid dosage form, although any pharmaceutically acceptable dosage form can be utilized. Exemplary solid dosage forms include, but are not limited to, tablets, capsules, sachets, lozenges, powders, pills, or granules, and the solid dosage form can be, for example, a fast melt dosage form, controlled release dosage form, lyophilized dosage form, delayed release dosage form, extended-release dosage form, pulsatile release dosage form, mixed immediate release and controlled release dosage form, or a combination thereof.

[0321] In certain embodiments, the endocytic agents utilized in the methods disclosed herein can be practiced using solvate forms of the compounds or salts, prodrugs, esters, and / or amides, thereof. Solvate forms include ethanol solvates, hydrates, and the like. “Hydrate” means a solvate wherein the solvent molecule(s) is / are water.

[0322] The formulations can be presented in unit-dose or multi-dose containers.

[0323] “Salt” means an ionic form of the parent compound or the product of the reaction between the parent compound with a suitable acid or base to make the acid salt or base salt of the parent compound. Generally, the salts are prepared by reacting with the free base or acid parent compound with stoichiometric amounts or with an excess of the desired salt forming inorganic or organic acid or base in a suitable solvent or various combinations of solvents.

[0324] The counter-ion forming a part of any salt of endocytic agents disclosed herein may not be critical to the activity of the compound, so long as the salt is pharmacologically acceptable and as long as the counter-ion does not contribute undesired qualities to the salt as a whole. Undesired qualities include undesirably solubility or toxicity.

[0325] “Prodrug” or “prodrug derivative” means a covalently bonded derivative or carrier of the parent compound or active drug substance which undergoes at least some biotransformation prior to exhibiting its pharmacological effect(s). More detailed definition and clinical advantages for Prodrugs and their preparations are known in the art, such as those described in A Textbook of Drug Design and Development, Krogsgaard-Larsen and H. Bundgaard (eds.), Gordon & Breach , 1991, particularly Chapter 5 : “ Design and Applications of Prodrugs” ; Design of Prodrugs, H. Bundgaard (ed.), Elsevier, 1985; Prodrugs: Topical and Ocular Drug Delivery, K. B. Sloan (ed .), Marcel Dekker, 1998; Methods in Enzymology, K. Widder et al. (eds.), Vol. 42 , Academic Press, 1985, particularly pp . 309-396; Burger's Medicinal Chemistry and Drug Discovery, 5th Ed., M. Wolff (ed.), John Wiley & Sons, 1995, particularly Vol. 1 and pp. 172-178 and pp. 949-982; Pro- Drugs as Novel Delivery Systems, T. Higuchi and V. Stella (eds.), Am. Chem. Soc., 1975; Bioreversible Carriers in Drug Design, E. B. Roche (ed.), Elsevier, 1987, each of which is incorporated herein by reference in their entireties. One example of pharmaceutically acceptable prodrugs is the ester of the endocytic agents which can be employed in the compositions and methods.

[0326] To meet drug stability requirement and / or for diagnostic applications, in some embodiment, the methodology of current disclosure can be applied to replace any atoms in endocytic agents with isotopically labelled atoms for achieving stability and / or for diagnostic applications of endocytic agents for disease treatment or diagnosis. The present disclosure encompasses any of the compound of the present disclosure being isotopically labelled, i.e., radiolabeled, by having one or more atoms replaced by an atom having a different atomic mass or mass number. Examples of isotopes that can be incorporated into compound of the present disclosure include isotopes of hydrogen, carbon, nitrogen, sulfur, oxygen, fluorine, and chlorine, such as2H (or deuterium (D)),3H,nC,13C,14C,15N,18O,17O,35S,18F, and36C1, e.g.,2H,3H, and13C. In one embodiment, a portion of the atoms at a position within a compound of the present disclosure are replaced, i.e., the compound of the present disclosure is enriched at a position with an atom having a different atomic mass or mass number. In one embodiment, multiple atoms can be replaced by isotopes, especially by deuterium isotopically labeled compounds of the disclosure can be prepared by methods known in the art.

[0327] “Endocytosis” or “Endocytic process” refers to any biological processes by which all cells absorb large-sized and large volume fraction external materials, such as nutrients, viruses, and bacteria, by engulfing them with the cell membrane. This process is initiated with recruitment of substances (such as protein, fatty acids, and exosome particles) that bind with endocytosis- mediating membrane component(s), such as membrane receptors including transporters, and involves the formation of intracellular vesicles. In present disclosure, the “endocytosis” or “endocytic process” include, but not limited to, phagocytosis, pinocytosis, macropinocytosis, receptor-mediated endocytosis, clathrin-dependent endocytosis, caveolin-dependent endocytosis, clathrin- and ca...

Claims

CLAIMS We claim:

1. A compound comprising an agent or a probconnected with a chemical arm via cleavable or non-cleavable chemical bond or linker unit , wherein the compound has a binding affinity to an endocytosis-mediating membrane component.

2. The compound of claim 1, wherein the compound has a binding affinity KDto the endocytosis-mediating membrane component of less than 20.0 mM.

3. The compound of any one of claims 1-2, wherein the compound is represented by a Formula ent integer f4. The compound of claim 3, wherein m, n, and p are 1. . The compound of any one of claims 1-4, wherein the compound comprises an agent that is a degrader, stabilizer, inhibitor, modulator, or activator.

6. The compound of claim 5, wherein the degrader is a PROTAC.

7. The compound of any one of claims 1-4, wherein the compound comprises an agent that is a protein binder.

8. The compound of claim 7, wherein the protein binder has a binding affinity KDto a protein of less than 20.0 mM.

9. The compound of claim 7-8, wherein the protein is an extracellular protein, intracellular protein, integral membrane protein, peripheral membrane protein, lipid-anchored protein, globular protein, or glycoprotein.

10. The compound of any one of claims 1-4, wherein the compound comprises a probe that is a diagnostic agent.

11. The compound of claim 10, wherein the diagnostic agent comprises a detectable label.

12. The compound of any one of claims 1-11, wherein the chemical arm is an atom, an agent, a probe, or a portion of an agent or a binder or a probe.

13. The compound of claim 12, wherein the chemical arm has a binding affinity KDto the endocytosis-mediating membrane component of less than 20.0 mM.

14. The compound of any one of claims 1-13, wherein the endocytosis-mediating membrane component is a cell membrane lipid, carbohydrate, or protein.

15. The compound of any one of claims 1-13, wherein the endocytosis-mediating membrane component is a glycolipid, glycoprotein, phospholipid, ceramides, and cholesterol.

16. The compound of any one of claims 1-13, wherein the endocytosis-mediating membrane component is a glycolipid, or a glycoprotein comprises 2-100 straight or branched monosaccharide units.

17. The compound of any one of claims 1-13, wherein the endocytosis-mediating membrane component is an integral membrane protein, peripheral membrane protein, lipid-anchored protein, globular protein, or glycoprotein.

18. A pharmaceutical composition comprising the compound according to any one of claims 1-17 and a pharmaceutically acceptable excipient, carrier, or diluent.

19. A nanostructure comprising liquid or cytoplasm enclosed by a lipid bilayer, an endocytosis- meditating membrane component, and the compound according to any one of claims 1-17.

20. The nanostructure of claim 19, wherein the nanostructure has a binding affinity KDto the endocytosis-mediating membrane component of less than 20.0 mM.

21. A method of preparing the nanostructure according to claim 19-20 comprising contacting the compound according to any one of claims 1-17 with a cell or vesicle comprising the endocytosis-meditating membrane component.

22. The method of claim 21, wherein the compound is contacted with a cell or vesicle comprising the endocytosis-meditating membrane component in vivo.

23. The method of claim 21, wherein the compound is contacted with a cell or vesicle comprising the endocytosis-meditating membrane component in vitro or ex vivo.

24. A method for internalizing a compound within a cell comprising contacting the compound according to any one of claims 1-17 with a cell comprising the endocytosis-mediating membrane component.

25. A method for internalizing a compound within a cell comprising contacting the nanostructure according to any one of claims 19-20 with a cell comprising the endocytosis- mediating membrane component.

26. A method for isolating the compound from nanostructure according to any one of claims 19-20 comprising lysing the nanostructure and separating the compound from a lysate.

27. A method of determining a qualitative or quantitative presence of a compound or nanostructure according to any one of claims 1-20 in a cell, body, solution, or medium comprising centrifuging a sample comprising the cell, body, solution, or medium and detecting for the compound or nanostructure.

28. A method for isolating the compound or nanostructure according to any one of claims 1- 20 from a cell, body, solution, or medium comprising centrifuging a sample comprising the cell, body, solution, or medium.

29. The method of any of claim 27-28, wherein the medium is cell culture medium, tissue culture medium, organ culture medium, body fluid, tissue, or organ.

30. A method for treating a subject comprising administering a compound or nanostructure according to any one of claims 1-20 to a subject in need thereof.

31. The method of claim 30, wherein an effective amount of the compound or nanostructure to treat the subject is lower than an effective amount of the agent or the probe absent the chemical arm to treat the subject.

32. The method of claim 30, wherein an effective amount of the compound or nanostructure to treat the subject is equal to an effective amount of the agent or the probe absent the chemical arm to treat the subject.

33. The method of claim 30, wherein an effective amount of the compound or nanostructure to treat the subject is higher than an effective amount of the agent or the probe absent the chemical arm to treat the subject.

34. A method for identifying an endocytic agent comprising contacting a compound with a first cell and a second cell, wherein the presence of an endocytosis-mediating membrane component in the second cell has been modulated relative to the first cell, and comparing the activity of the compound in contact with the first cell to the activity of the compound in contact with the second cell to determine the compound is the endocytic agent.

35. The method of claim 34, wherein the presence of the endocytosis-mediating membrane component of the second cell is less than the first cell.

36. The method of claim 35, wherein the presence of the endocytosis-mediating membrane component of the second cell has been lowered by gene editing, knockdown, or silencing.

37. The method of claim 34, wherein the presence of the endocytosis-mediating membrane component of the second cell is more than the first cell.

38. The method of claim 37, wherein the presence of the endocytosis-mediating membrane component of the second cell has been increased by gene editing or introduction of DNA or RNA encoding for the endocytosis-mediating membrane component.

39. The method of any one of claims 34-38, wherein the compound is contacted with the first cell and the second cell in vivo.

40. The method of any one of claims 34-38, wherein the compound is contacted with the first cell and the second cell in vitro or ex vivo.

41. The method of any one of claims 34-40 further comprising determining that the compound has a binding affinity KDto the endocytosis-mediating membrane component of less than 20.0 mM.

42. The method of any one of claims 34-41 further comprising administering a compound or nanostructure to a subject in need thereof.

43. A method for identifying an endocytosis-mediating membrane component comprising contacting a cell with the compound or nanostructure according to any one of claims 1-29, wherein the compound comprises a detectable label and wherein the endocytosis-mediating membrane component in a cell can be identified by determining an interaction between endocytosis-mediating membrane component and detectable label.

44. A method of identifying an endocytosis-mediating membrane component comprising comparing the sensitivity of a first cell and a second cell to the treatment of a compound or nanostructure according to any one of claims 1-20 and comparing genomic expression or protein abundance of a membrane component in the first cell and the second cell, wherein increased sensitivity of the first cell or the second cell to the compound or nanostructure identifies the endocytosis-mediating membrane component.

45. The method of claim 44, wherein the first cell with higher abundance of or the membrane component is more sensitive than the second cell with lower abundance or no membrane component.

46. The method of claim 44, wherein the first cell with higher expression of the membrane component is more sensitive than the second cell with lower or no expression of the membrane component.

47. The method of any one of claims 44-46 further comprising determining that the endocytosis-mediating membrane component has a binding affinity KDto the compound or nanostructure according to any one of claims 1-25 of less than 20.0 mM.

48. The method of 44-47, wherein sensitivity of the first cell and the second cell is determined by evaluating a change in a biological process when the first cell and the second cell are contacted with the compound or nanostructure.

49. A method for selecting a subject for treatment with a compound or nanostructure according to any one of claims 1-20 having a binding affinity to an endocytosis-mediating membrane component comprising determining a qualitative or quantitative presence of the endocytosis-mediating membrane component in a sample obtained from a prospective subject and administering the compound or nanostructure to the prospective subject when the endocytosis-mediating membrane component is present in the sample.

50. The method of claim 49, wherein the administration route or effective amount of the compound or nanostructure is determined from the qualitative or quantitative presence of an endocytosis-mediating membrane component in the sample.

51. The method of any one of claims 49-50, wherein the compound or nanostructure has a binding affinity to the endocytosis-mediating membrane component of less than 20 mM.

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