Method for delivery of an agent across biological barriers

Abstract

This disclosure provides a protein-vesicle conjugate comprising one or more secretory proteins or fragments thereof expressed on or bound to the surface of vesicles and, as appropriate, a pharmaceutical agent, and a method for delivering the pharmaceutical agent together with the protein-vesicle conjugate across biological barriers to cells and tissues containing the cells.

Description

Technical Field

[0001] This invention relates to the field of drug delivery to cells, and more particularly to the delivery of drugs together with protein-vesicle conjugates across biological barriers to cells. Prior Technology

[0002] The blood-brain barrier (BBB) ​​constitutes a specialized vascular system in the brain, maintaining central nervous system (CNS) homeostasis by precisely regulating the flow of specific nutrients and preventing the entry of harmful substances such as neurotoxic agents. However, the presence of the BBB poses a challenge to the treatment of CNS diseases, limiting the entry of certain drugs and macromolecular therapeutic agents (including biologics) into the brain. For example, after administration, the concentration of therapeutic antibodies in the brain is typically only 0.01% to 0.1% of that in plasma. While these low levels of antibodies can still produce pharmacological effects, achieving higher concentrations offers significant advantages, reducing the required dosage and improving the therapeutic window. Summary of the Invention

[0003] In one embodiment, this disclosure provides a method for delivering a drug across a biological barrier to cells and / or tissues containing such cells, comprising contacting a protein-vesicle conjugate or a pharmaceutical composition containing a protein-vesicle conjugate with the cells and / or tissues of an individual, wherein the protein-vesicle conjugate comprises: (a) one or more secretory proteins or fragments thereof expressed on or bound to the surface of a vesicle, and / or an antibody specific to one or more secretory proteins or fragments thereof and expressed on or bound to the surface of a vesicle; and (b) a drug embedded in a vesicle. In another embodiment, this disclosure provides a method for delivering a drug across a biological barrier to cells and / or tissues containing such cells, comprising contacting a protein-vesicle conjugate or a pharmaceutical composition containing a protein-vesicle conjugate with the cells and / or tissues of an individual, wherein the protein-vesicle conjugate comprises: (a) one or more secretory proteins or fragments thereof expressed on or bound to the surface of a vesicle, and (b) a drug embedded in a vesicle. In another embodiment, this disclosure provides a method for delivering a drug across a biological barrier to cells and / or tissues containing such cells, comprising contacting a protein-vesicle conjugate or a pharmaceutical composition containing a protein-vesicle conjugate with cells and / or tissues of an individual, wherein the protein-vesicle conjugate comprises: (a) an antibody that is specific to one or more secretory proteins or fragments thereof and is expressed on or bound to the surface of a vesicle; and (b) a drug encapsulated within a vesicle.

[0004] In some embodiments, the biological barriers described herein include, but are not limited to, the intestinal barrier, nasal barrier, lung barrier, blood-brain barrier, and skin barrier. In some embodiments, cells or tissues containing such cells may be present in the central nervous system of an individual. For example, cells or tissues containing such cells may be present in the brain of an individual. The biological barrier may be the blood-brain barrier.

[0005] In some embodiments, the cells described herein may be inflamed and / or damaged cells undergoing regulated cell death (apoptotic and non-apoptotic cell death). In some embodiments, the cells or tissues containing the cells are in the central nervous system of an individual. In some embodiments, the cells or tissues containing the cells are in the brain of an individual. In some embodiments, the cells may be neurons, microglia cells, or astrocytes.

[0006] In some embodiments, prior to the administration of the protein-vesicle conjugate, the method further comprises the step of inducing and / or administering the conjugate to inflamed and / or damaged cells within the blood-brain barrier. While not wishing to be bound by any particular theory, it is believed that the integrity of the blood-brain barrier may be compromised due to inflammation and / or damage in the brain, thus allowing the protein-vesicle conjugate to penetrate more easily.

[0007] The protein-vesicle conjugates described herein comprise one or more secretory proteins or fragments thereof expressed on or bound to the surface of vesicles, and, where appropriate, pharmaceutical agents.

[0008] Examples of one or more secreted proteins or fragments thereof described herein include, but are not limited to, P-selectin (P-sel), cation-independent mannose 6-phosphate receptor (CI-MPR), L-selectin (L-sel), E-selectin (E-sel), P-selectin glycoprotein ligand-1 (PSGL-1), CD22 (siglec2), galactoglobin-3 (Gal-3), clonoxin (Klotho), pentaxin 3 (PTX3), CD47, CD42b, dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin receptor (DC-SIGNR), intercellular adhesion molecule 1 (ICAM-1; CD54), and vascular cell adhesion molecule. VCAM), integrin α4β1, VE-caderin, annexin V, toll-like receptor 3 (TLR3), programmed cell death 1 ligand 1 (PD-L1), IL-1β, IL-1RA, TNF-α, etanercept (soluble TNF receptor), EGF, fibroblast growth factor (FGF), insulin, glial cell-line derived neurotrophic factor (GDNF), BDNF, β-NGF, NT-3, TGF-β1, activin A, BMP4, BMP6, BMP9, BMP-10, GDF-8, GDF-10, GDF-11 and their fragments. Examples of antibodies that are specific to one or more secreted proteins or fragments thereof described herein include, but are not limited to, anti-PSGL-1 immunoglobulin (anti-PSGL-1 Ig) and anti-PDL1 Ig.

[0009] In some embodiments, one or more secretory proteins or fragments thereof described herein are selected from P-selectin, b-NGF, NT3, TGF-b1, activin A, BMP4, BMP6, BMP9, BMP10 and fragments thereof.

[0010] In some embodiments, the cells described herein are neurons, and one or more secreted proteins or fragments thereof are selected from P-selectin, activin A, BMP9 and fragments thereof.

[0011] In some embodiments, the cells described herein are microglial cells, and one or more secreted proteins or fragments thereof are selected from P-selectin, β-NGF, NT3, activin A and fragments thereof.

[0012] In some embodiments, the cells described herein are astrocytes, and one or more secreted proteins or fragments thereof are selected from P-selectin, β-NGF, NT3, activin A, BMP6, BMP9 and fragments thereof.

[0013] In some embodiments, the protein-vesicle conjugates described herein comprise a combination of CI-MPR and clotrimazole, a combination of CI-MPR and P-selectin, or a combination of clotrimazole and P-selectin.

[0014] In some embodiments, the vesicles described herein are liposomes or microcells.

[0015] In some embodiments, the agents described herein are separated from secretory proteins or fragments thereof and encapsulated within vesicles or attached to the outer surface of vesicles.

[0016] In some embodiments, the agents described herein are diagnostic contrast agents, cell survival enhancers, cell survival inhibitors, cellular components, organelles, cells, cytotoxic agents, antitumor drugs, toxins or antibodies, lipids, proteins, DNA, RNA, therapeutic agents, or nanomaterials. Simple Explanation of the Diagram

[0017] Figures 1A to 1C present a quantitative analysis of relative fluorescence intensity in the mouse brain. Following a 20-hour restraint stress protocol, the ability of luciferin-loaded liposomes bound to different secreted proteins on their surface to cross the blood-brain barrier and stain the mouse brain was tested (Figures 1A and 1B, brain fluorescence intensity; Figure 1C, experimental overview and setup). Vehicle injection, along with injections of BSA- and IgG-Fc-bound liposomes, served as negative controls. Fluorescence intensity in the BSA-bound mouse brain was normalized to 1-fold (horizontal line). Single protein conjugates were used alone, with P-selectin (P-sel) and non-cation-dependent mannose-6-phosphate receptor (CI-MPR) used as positive controls. Relevant brain images are available in Figures 2A to 2F.

[0018] Figures 2A to 2F show representative images of the intensity of fluorescent dyes delivered to the mouse brain via a delivery system. The upper image shows the secreted proteins bound to liposomes, while the lower image presents the corresponding brain images. The corresponding quantitative analysis results are shown in Figures 1A to 1C.

[0019] Figures 3A to 3D illustrate the targeted rescue of inflamed and damaged brain cells using liposomes loaded with caspase-1 siRNA-binding secretory protein. Figure 3A provides an overview of the experiment. The markers used in flow cytometry analysis to label neurons, microglia, astrocytes, and pyroptosis signals were NeuN, F4 / 80, GFAP, and the activated form of caspase-1 (Figure 3A). Figures 3B, 3C, and 3D show the relative levels of pyroptosis (a regulated form of cell death) in neurons, microglia, and astrocytes in the mouse brain under activity-restricted stress conditions compared to the control group. # P < 0.05 relative to the BSA group indicates significant rescue. These findings are consistent with the luciferin delivery experiment (Figure 1), in which P-selectin, b-NGF, NT3, TGF-b1, activin A, BMP4, BMP6, BMP9, and BMP10 demonstrated effective drug delivery in neurons (B), microglial cells (C), and astrological glial cells (D) of the mouse brain after activity restriction stress. Furthermore, siRNA is an ideal carrier for this drug delivery system; as demonstrated in this experiment using apoptosis protease-1 siRNA, it effectively ameliorated pyroptosis and encephalitis induced by activity restriction stress in mice.

[0020] Figure 4 shows the direction of liposome binding to secreted proteins toward inflamed and damaged brain cells. Selected secreted proteins included the non-cation-dependent mannose-6-phosphate receptor (CI-MPR), clomiphene, and P-selectin (P-sel), while bovine serum albumin (BSA) served as a non-lectin negative control protein. The carrier indicates treatment using a solvent without fluorescent liposomes. The upper figure shows fluorescence images of the mouse brain after activity restriction stress, and the lower figure illustrates the treatments and corresponding relative fluorescence intensities (numerical). The combination of liposomes and effective secreted proteins can produce a synergistic effect in fluorescent dye delivery (lower figure; e.g., using a combination of CI-MPR, clomiphene, and P-sel).

[0021] Figures 5A to 5C illustrate the targeting of inflamed and damaged cells in the brain using liposomes loaded with an apoptosis protease-3 inhibitor and bound to secreted proteins. Figure 5A provides an overview of the experiment. Figures 5B and 5C show the assessment of relative IL-1 levels in the mouse brain under activity-restriction stress conditions compared to the control. Notably, intravenous administration of liposomes loaded with the apoptosis protease-3 inhibitor (z-DEVD-FMK) and bound to P-selectin [P-sel-lipo(C3in)], rather than liposomes bound to bovine serum albumin (BSA), significantly mitigated the increase in pro-inflammatory cytokine IL-1β levels induced by activity-restriction stress (* P < 0.05 compared to the untreated groups; # P < 0.05 for P-sel-lipo(C3in) compared to the carrier group) [P-sel-lipo(C3in): liposomes loaded with the apoptosis protease-3 inhibitor Z-DEVD-FMK and bound to P-selectin].

[0022] Figures 6A to 6C demonstrate that administration of liposomes loaded with an apoptosis protease-1 inhibitor and bound with P-selectin effectively improved LPS-induced anxiety and depressive tendencies. Image recordings of mouse trajectories during the open space test are shown in Figure 6A. Quantitative analysis of mouse behavior is shown in Figures 6B and 6C, revealing that intravenous treatment with liposomes loaded with the apoptosis protease-1 inhibitor z-WEHD-FMK and bound with P-selectin [P-sel-lipo (C1in)] significantly alleviated LPS-induced anxiety and depressive tendencies. Statistical significance of P < 0.05 compared to the untreated groups (UN) is indicated by *, and statistical significance of P < 0.05 when comparing liposomes loaded with the apoptosis protease-1 inhibitor and bound with P-selectin (P-sel-lipo (C1in)) to the vector group is indicated by #. [P-sel-lipo(C1in): a liposome loaded with the apoptosis protein-1 inhibitor z-WEHD-FMK and bound to P-selectin].

[0023] Figures 7A to 7K demonstrate significant improvements in activity-restriction stress-induced anxiety and depression-like behaviors following treatment with liposomes loaded with an apoptosis protein-1 inhibitor bound to P-selectin. Results of the outstretched arm test (Figures 7A to 7F) and the suspended tail test (Figures 7G to 7K) are presented. Representative video recordings of mouse trajectories in the outstretched arm test (Figures 7A to 7D) and the suspended tail test (Figures 7G to 7J) are shown. Quantitative analysis of mouse behavior (Figures 7E and 7F) confirms that intravenous treatment with liposomes [P-sel-lipo (Clin)] loaded with the apoptosis protein-1 inhibitor z-WEHD-FMK and bound to P-selectin significantly improves activity-restriction stress-induced anxiety and depression-like behaviors. Statistical significance of P < 0.05 compared to the individual untreated groups (UN) is indicated by *, and statistical significance of P < 0.05 compared to the vector group is indicated by #. [P-sel-lipo(C1in): liposomes loaded with the apoptosis protein-1 inhibitor z-WEHD-FMK and bound to P-selectin].

[0024] Figure 8 shows microscopic images of mouse brain slices obtained using fluorescence microscopy. In the absence of Cre recombinase expression, brain cells of transgenic mice [B6.129-(Gt(ROSA)26Sortm4(ACTB-tdTomato, EGFP)Luo / J] only expressed the red fluorescent protein tdTomato and not the enhanced green fluorescent protein (EGFP). However, when a functional Cre recombinase was introduced into these brain cells, it catalyzed the LoxP site in the transgenic gene, shutting down the red fluorescence expression of tdTomato and activating EGFP expression, resulting in the substitution of red fluorescence for green fluorescence. Our findings confirm that EGFP in transgenic mice [B6.129-(Gt(ROSA)26Sortm4(ACTB-tdTomato, EGFP)Luo / J] subjected to activity restriction stress... EGFP expression in brain cells (indicated by green spots). This EGFP expression was observed after delivery of the functional recombinant protein Cre recombinase using liposomes bound to the secretory protein P-selectin. These results indicate that this delivery system is well-suited for the efficient delivery of functional enzymes and proteins (such as those used in enzyme replacement therapy) to brain cells without significant lysosomal digestion.

[0025] Figures 9A to 9H demonstrate the significant improvement in LPS-induced anxiety-like and depressive-like behaviors after treatment with liposomes loaded with apoptosis protease-1 siRNA and bound to P-selectin. An overview of the experiment is presented in Figure 9A. Image recordings of mouse trajectories during the open space test are shown in Figures 9B to 9E. Quantitative analysis of mouse behavior is shown in Figures 9F to 9H, revealing a significant improvement in activity-restriction-induced anxiety-like and depressive-like behaviors after intravenous treatment with liposomes loaded with apoptosis protease-1 siRNA and bound to BSA compared to the control group. Statistical significance of P < 0.05 compared to the untreated groups (UN) is indicated by *, and statistical significance of P < 0.05 when comparing liposomes loaded with apoptosis protease-1 siRNA and bound to P-selectin (P-sel-lipo) to the vector group is indicated by #. (P-sel-lipo: liposomes loaded with apoptosis protease-1 siRNA and bound to P-selectin; BSA-lipo: liposomes loaded with apoptosis protease-1 siRNA and bound to BSA). Implementation

[0026] Unless specifically instructed otherwise, the applicant intends to use the following definitions when the definitions of terms deviate from their usual use.

[0027] As used in this specification and the accompanying claims, unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" include the plural references.

[0028] As used herein, unless otherwise stated, the use of "or" means "and / or". In the case of multiple items, the use of "or" is only to alternatively refer to more than one of the aforementioned independent items or items.

[0029] As used in this article, the term "one or more" is readily understood by those generally familiar with this technique, especially when read in the context in which it is used.

[0030] As used herein, the term "secretory protein" can refer to any protein secreted by cells. Secretory proteins can include hormones, enzymes, toxins, and antimicrobial peptides. In some embodiments, secretory proteins are lectins.

[0031] As used herein, the term "liposome" is a general term that encompasses a variety of monolayer and multilayer lipid carriers formed by creating a closed lipid bilayer or aggregate. Liposomes can be characterized as having a vesicle structure, having a bilayer membrane typically containing phospholipids and an internal medium typically containing an aqueous composition.

[0032] As used herein, the term "microcell" refers to an aggregate (or supramolecular assembly) of surfactant molecules dispersed in a liquid colloid. A typical microcell forms an aggregate in aqueous solution, in which the hydrophilic "head" region is in contact with the surrounding solvent, while the hydrophobic single-tailed region is isolated at the center of the microcell.

[0033] As used herein, the terms "medicine" or "therapeutic agent" refer to any medicine that can treat and / or improve a condition or disease.

[0034] As used interchangeably in this document, the terms “individual,” “subject,” “host,” and “patient” refer to mammals, including but not limited to rodents (rats, mice), non-human primates, humans, canines, felines, and ungulates (e.g., horses, cattle, sheep, pigs, goats).

[0035] As used herein, the term "therapeutic effective amount" or "effective amount" refers to the amount of vesicles that, when administered to a mammal or other individual for the purpose of treating a disease, are sufficient to achieve such treatment of the disease.

[0036] As used herein, the terms “treatment,” “treating,” and the like cover any treatment of a disease in mammals (specifically, humans), including: (a) preventing the development of a disease in an individual who may be susceptible to the disease but has not yet been diagnosed with it; (b) suppressing the disease, i.e., halting its development; and (c) alleviating the disease, i.e., resolving the disease.

[0037] As used in this article, the term "binding site" refers to the site where a covalent bond is formed between two macromolecules, mostly branched chains of the terminal and side chains, and occasionally linear bonds of the head and tail of the molecule.

[0038] This disclosure demonstrates that drug delivery across biological barriers can be achieved using vesicles that bind secretory proteins, as listed herein. Through the use of these vesicles, a wide variety of molecules, including small molecule drugs, fluorescent compounds, nanoparticles, proteins, nucleic acids, and combinations thereof, have been successfully delivered to cells. This highlights the potential of vesicles that bind secretory proteins as an effective tool for delivering a diverse range of substances to cells.

[0039] Apoptosis is controlled by the integration of multiple pro-apoptotic and anti-apoptotic signals. Drugs or therapeutic agents, delivered together with the vesicles of this invention, are delivered to apoptotic cell lines to target diseases associated with changes in apoptosis. Diseases associated with changes in apoptosis may include, but are not limited to, trauma, exposure to chemical and physical toxic factors, genetic diseases, age-related diseases, cardiovascular diseases, infectious diseases, neoplastic diseases, neurodegenerative diseases, metabolic diseases, aging, obesity, cancer, neurodegeneration induced by β-amyloid protein or α-synuclein or toxicity (Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis), muscular degenerative conditions or chronic pneumonia caused by cystic fibrosis, cardiovascular diseases (such as ischemia, heart failure, and infectious diseases), and autoimmune diseases (systemic lupus erythematosus, autoimmune lymphoproliferative syndrome, rheumatoid arthritis, and thyroiditis).

[0040] Autophagy is a lysosomal degradation pathway essential for survival, differentiation, development, and stability. Drugs or therapeutic agents are delivered to autophagic cell lines along with the vesicles of this invention to target diseases associated with autophagy dysregulation. Diseases associated with autophagy dysregulation include, but are not limited to, trauma, exposure to chemical and physical toxic factors, genetic diseases, age-related diseases, cardiovascular diseases, infectious diseases, neoplastic diseases, neurodegenerative diseases, metabolic diseases, aging (when ATG5 is overexpressed throughout the organism), obesity (when ATG7 or the pre-autophagy transcription factor EB [TFEB] is overexpressed in hepatocytes), cancer (when beclin 1 is expressed in KRAS-induced lung adenomas), neurodegeneration induced by β-amyloid or α-synuclein or toxicity (when TFEB or beclin 1 is overexpressed in the brain, or when cystin B (a lysosomal cysteine ​​protease inhibitor) is knocked out), muscle degenerative conditions (when TFEB or beclin 1 targets skeletal muscle), and chronic pneumonia caused by cystic fibrosis (when beclin 1 is expressed in the lungs).

[0041] Pyroptosis is a regulated form of cell death characterized by the activation of inflammatory apoptotic proteases, leading to cell lysis and the release of pro-inflammatory cytokines such as IL-1β and IL-18. This process is mediated by the formation of gasdermin pores in the cell membrane. In the central nervous system, pyroptosis is associated with various neurodegenerative and neuroinflammatory diseases. For example, in Alzheimer's disease, pyroptosis promotes neuronal death and the release of inflammatory mediators, exacerbating amyloid-β plaque formation and tau lesions. In multiple sclerosis, pyroptosis in microglial cells and astrocytes promotes demyelination and neuronal damage. Furthermore, in traumatic brain injury, pyroptosis-induced inflammation exacerbates neuronal damage and hinders recovery. These examples highlight the crucial role of pyroptosis in central nervous system diseases, making it a potential target for therapeutic interventions.

[0042] This disclosure provides a method for delivering a drug across a biological barrier to cells and / or tissues containing such cells, comprising contacting a protein-vesicle conjugate or a pharmaceutical composition containing a protein-vesicle conjugate with the cells and / or tissues of an individual, wherein the protein-vesicle conjugate comprises: (a) one or more secretory proteins or fragments thereof expressed on or bound to the surface of a vesicle, and / or one or more antibodies specific to one or more secretory proteins or fragments thereof and expressed on or bound to the surface of a vesicle; and (b) a drug encapsulated in a vesicle.

[0043] The liposomes described in this article include monolayer liposomes, multilayer liposomes, and multivesicular liposomes. The liposomes described in this article may contain positively charged, negatively charged, or neutral phospholipids.

[0044] The liposomes used in this invention can be provided by various known methods. For example, phospholipids, such as phospholipid dioleoylphosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), and / or EPC, can be dissolved in alcohols or other organic solvents and subsequently mixed with the components included in the lipid bilayer. The mixture may further include various cleaning agents. Typically, the lipid mixture is vortexed, frozen in a dry ice / acetone bath, and lyophilized overnight. The lyophilized formulation is stored long-term at -20°C or lower. The lyophilized liposomes are reconstituted when needed.

[0045] Alternatively, liposomes can be prepared by mixing lipids in a solvent in a container (e.g., a glass or pear-shaped flask). The volume of the container should be more than ten times larger than the volume of the intended liposome suspension. Using a rotary evaporator, the solvent is removed under negative pressure at approximately 40°C. Depending on the desired volume of liposomes, the solvent is typically removed over approximately 5 minutes to 2 hours. The composition can be further dried under vacuum in a desiccator. The dried lipids are usually discarded after about one week, as they tend to degrade over time.

[0046] The microcellular structure itself is largely determined by the type and composition of the polymer molecules used to form the microcells and the solvent environment of the microcells. In some embodiments, microcells are constructed using nonionic triblock copolymers composed of both hydrophilic and hydrophobic monomer units. In the embodiments disclosed herein, poloxamer, a poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer (PEO-PPO-PEO), is used. In some embodiments, the microcells disclosed herein can be prepared using PEG-PLA polymers with various block sizes (e.g., block sizes within a certain range mentioned) and various ratios (e.g., PEG:PLA from about 1:10 to about 10:1, or any integer ratio within that range).

[0047] In some embodiments, the protein-vesicle conjugate may be engineered or derived from cells.

[0048] The secretory proteins described in this article can be bound to vesicles using a shear-force-based approach by supplementing functionally labeled lipids (Yu B, Lee RJ, Lee LJ. Microfluidic methods for production of liposomes. Methods Enzymol. 2009;465:129-141; Jeong D, Jo W, Yoon J et al., Nanovesicles engineered from ES cells for enhanced cell proliferation. Biomaterials. 2014;35(34):9302-9310).

[0049] In some embodiments, the therapeutic agent is a cell survival enhancer (or cell death inhibitor) that can guide the rescue of drug-mediated tissue damage. In some embodiments, the agent is a cell survival inhibitor, cell death enhancer, or antitumor agent that can reduce the survival of target cells in tissues (such as tumors) containing naturally occurring autophagic and apoptotic cells, or reduce the survival of selected specific tissues in which autophagic and apoptotic cells have been artificially induced in a specific tissue using cytotoxic agents (such as drugs, toxins, or antibodies against tissue-specific proteins).

[0050] In one embodiment, the agent is a diagnostic agent or a therapeutic agent. In one embodiment, the agent is an autophagy or apoptosis drug. In some embodiments, examples of agents include, but are not limited to, antipathogenic drugs, autophagy inhibitors, enzyme inhibitors, cell signaling inhibitors, diagnostic contrast agents, cell survival enhancers (or cell death inhibitors), cell survival inhibitors (or cell death enhancers), cells (such as stem cells and precursor cells), cell components, organelles, cytotoxic agents, antitumor drugs, toxins or antibodies, lipids, proteins, DNA, RNA, therapeutic agents, and nanomaterials. In one embodiment, an antagonist of the aforementioned first or second protein (such as soluble forms, corresponding ligands, and neutralizing and blocking antibodies) can act as an antidote to reduce vesicle targeting autophagic and apoptotic cells and tissues containing autophagic and apoptotic cells. In some embodiments, the agent is bardoxolone methyl, chloroquine, quinine, hydrochloroquine, sorafenib, sunitinib, an Hsp90 inhibitor, metformin, or crizotinib. In some embodiments, the RNA may be one or more of the following: short interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), antisense RNA, guide RNA (gRNA), ribonuclease, and RNA aptamer.

[0051] In some embodiments, the agent is a stem cell or precursor cell. Delivery of stem cells and precursor cells can exert protective physiological functions and rescue tissues containing autophagic and apoptotic cells.

[0052] In some embodiments, the protein-vesicle conjugate or pharmaceutical composition of the present invention may be administered to a patient in various ways, including topically, non-enterically, intravenously, intradermally, subcutaneously, intramuscularly, via the colon, via the rectum, or intraperitoneally. Preferably, the pharmaceutical composition may be administered non-enterically, topically, intravenously, intramuscularly, subcutaneously, orally, or via the nose (e.g., by inhalation).

[0053] Protein-vesicle conjugates can be formulated into pharmaceutical compositions and can be formulated with one or more pharmaceutically acceptable carriers. Protein-vesicle conjugates can be formulated in a variety of different ways known to those generally skilled in the art. Pharmaceutically acceptable carriers are determined in part by the specific composition to which they are administered and the specific method used to administer the composition. Therefore, a wide variety of suitable formulations of the pharmaceutical compositions of the present invention exist (see, for example, Remington's Pharmaceutical Sciences, 20th Edition, Supplement, 2003, as described above). Effective formulations include oral and nasal formulations, formulations for non-enteral administration, and compositions formulated for long-term release.

[0054] For administration purposes, such as non-enteral administration, sterile aqueous solutions of water-soluble salts (e.g., NaCl) can be used. Other or alternative carriers may include sesame oil or peanut oil, and aqueous propylene glycol. The aqueous solution may be appropriately buffered as needed, and the liquid diluent may be first made isotonic with sufficient physiological saline or glucose. Such aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, intraperitoneal, and intratumoral (IT) injections.

[0055] The formulations suitable for oral administration may consist of: liquid solutions, such as an effective amount of the compound of the present invention suspended in a diluent (such as water, saline, or PEG 400); capsules, pouches, drug reservoirs, or tablets, each containing a predetermined amount of the active ingredient, in liquid, solid, granular, or gelatinous form; suitable suspensions in liquids; suitable emulsions; and patches. The described liquid solutions may be sterile solutions. Pharmaceutical forms may include one or more of the following: lactose, sucrose, mannitol, sorbitol, calcium phosphate, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silica, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffers, humectants, preservatives, flavorings, dyes, disintegrants, and pharmaceutically compatible carriers. Oral tablets may contain active ingredients in flavoring agents (such as sucrose) or tablets containing active ingredients in an inert matrix (such as gelatin and glycerin or sucrose and gum arabic latex, gel and the like), and in addition to the active ingredients, they may also contain carriers known in this art.

[0056] In some embodiments, the protein-vesicle conjugate may be administered together with one or more antagonists of one or more secretory proteins or fragments thereof. In some embodiments, the antagonists of one or more secretory proteins or fragments thereof are soluble forms of secretory proteins or fragments thereof, corresponding ligands of secretory proteins or fragments thereof, or antibodies against secretory proteins or fragments thereof.

[0057] In some embodiments, the method is directed at diseases associated with regulated cell death related to changes in brain cells, such as trauma, exposure to chemical and physical toxicities, genetic diseases, age-related diseases, cardiovascular diseases, infectious diseases, neoplastic diseases, neurodegenerative diseases, metabolic diseases, aging, obesity, cancer, inflammation, psychological stress, stroke, neurodegeneration induced by β-amyloid or α-synuclein or toxicity, or chronic pneumonia caused by cystic fibrosis, or autoimmune diseases. Neurodegenerative conditions may include brain fog, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, or stroke; brain injury-related cardiovascular conditions may include ischemia, heart failure, or infectious diseases; and autoimmune diseases may include systemic lupus erythematosus, autoimmune lymphoproliferative syndrome, rheumatoid arthritis, or thyroiditis.

[0058] Although the invention has been described with reference to preferred embodiments and examples, the scope of the invention is not limited to the embodiments described therein. It will be apparent to those skilled in the art that modifications and variations can be made to the described invention without departing from the spirit and scope of the invention as defined and limited by the appended claims. The following examples are provided to illustrate embodiments and advantages of the invention and are not intended to limit its scope. [, , ]

[0059] [Example] [1] Preparation of liposomes engineered from protein binders

[0060] [Preparation of Liposomes:] Liposomes were prepared using a liposome kit (Sigma-Aldrich Co.) and individual lipids. Surface proteins were incorporated into the lipids via a shear-force-based binding method (Yu, B.; Lee, RJ; Lee, LJ Microfluidic methods for production of liposomes. Methods Enzymol).

[2009] ,465, 129-141, doi:10.1016 / S0076-6879(09)65007-2;Jeong, D.; Jo, W.; Yoon, J.; Kim, J.; Gianchandani, S.; Gho, YS; Park, J. Nanovesicles engineered from ES cells for enhanced cell proliferation.Biomaterials

[2014] (35, 9302-9310, doi:10.1016 / j.biomaterials.2014.07.047). The protein binding procedure follows the method provided by the manufacturer.

[0061] [Synthesis and Binding of Liposomes:] The lipid system was synthesized using the previously described, well-established method (Chen, YL; Chen, YS; Chan, H.; Tseng, YH; Yang, SR; Tsai, HY; Liu, HY; Sun, DS; Chang, HH). The use of nanoscale visible light-responsive photocatalyst TiO2-Pt for the elimination of soil-borne pathogens. PLoS one

[2012] ,7, e31212, doi:10.1371 / journal.pone.0031212;Wu, CH; Kuo, YH; Hong, RL; Wu, HC alpha-Enolase-binding peptide enhances drug delivery efficiency and therapeutic efficacy against colorectal cancer.Sci Transl Med

[2015] ,7, 290ra291, doi:10.1126 / scitranslmed.aaa9391).

[0062] [Recombinant Proteins:] The recombinant proteins used in this study, such as recombinant MPR (non-cation-dependent mannose-6-phosphate receptor: CI-MPR; cation-dependent mannose-6-phosphate receptor: CD-MPR), were obtained from R and D Systems Co.

[0063] [Example] [2] [:] [Liposomes engineered with protein binders successfully delivered fluorescent dyes to the inflamed mouse brains after activity stress was restricted.] []

[0064] [Activity-Restriction Stress Mouse Model:] A mouse model of activity-restriction stress was used to induce psychological stress, encephalitis, and depressive and anxious behaviors. The activity-restriction stress procedure followed the previously described approach (Chu, X.; Zhou, Y.; Hu, Z.; Lou, J.; Song, W.; Li, J.; Liang, X.; Chen, C.; Wang, S.; Yang, B. et al., 24-hour-restraint stress induces long-term depressive-like phenotypes in mice. Sci Rep.).

[2016] ,6, 32935, doi:10.1038 / srep32935;Pethaperumal, S.; Hung, SC; Lien, TS; Sun, DS; Chang, HH P-Selectin is a Critical Factor for Platelet-Mediated Protection on Restraint Stress-Induced Gastrointestinal Injury in Mice.Int J Mol Sci

[2022] ,23, doi:10.3390 / ijms231911909;Chuang, DJ; Pethaperumal, S.; Siwakoti, B.; Chien, HJ; Cheng, CF; Hung, SC; Lien, TS; Sun, DS; Chang, HH Activating Transcription Factor 3 Protects against Restraint Stress-Induced Gastrointestinal Injury in Mice.Cells

[2021] (10, doi:10.3390 / cells10123530). During the stress period, mice in both the stressed and unstressed groups were deprived of food and water. For behavioral tests, the activity restriction stress lasted for 20 hours. After the activity restriction stress ended, mice in both the stressed and unstressed groups had a 2-hour period to obtain food and water to restore their energy.

[0065] Specialized liposomes engineered with protein-binding compounds were tested to selectively target inflamed and damaged cells in the brain. Targeting efficiency was visualized using a fluorescent imaging system (Thermo iBright FL1500) with liposomes labeled with a commercially available dye (calcium yellow-green pigment red; Sigma-Aldrich Co.). The acquired images confirmed that the fluorescent dye was successfully delivered to the inflamed mouse brain after activity-limiting stress (Figs. 2A–2F: brain images), demonstrating delivery at the cellular level.

[0066] The corresponding quantitative analysis results are shown in Figure 1 (Figure 1A and Figure 1B, brain fluorescence intensity; Figure 1C, experimental overview and setup). Groups with average fluorescence intensity higher than the BSA group (black horizontal line) were considered more effective in delivering these substances across the blood-brain barrier. These included P-sel, CI-MPR, L-selectin (L-sel), E-selectin (E-sel), P-selectin glycoprotein ligand-1 (PSGL-1), CD22 (siglec2), galactolectin-3 (Gal-3), clonoxin (Klotho), pentacyclic protein 3 (PTX3), CD47, CD42b, dendritic cell-specific intercellular adhesion molecule-3 binding to non-integrin (DC-SIGN) receptor (DC-SIGNR), intercellular adhesion molecule-1 (ICAM-1, CD54), vascular cell adhesion molecule (VCAM), integrin α4β1, VE-cadherin, annexin V, TLR-like receptor 3 (TLR3), planned cell death 1 ligand-1 (PD-L1), anti-PSGL-1 immunoglobulin (anti-PSGL-1 Ig), and anti-PDL1. Ig, IL-1β, IL-1RA, TNF-α, etanercept, EGF, fibroblast growth factor (FGF) 23, insulin, glial cell line-derived neurotrophic factor (GDNF), BDNF, β-NGF, NT-3, TGF-β1, activin A, BMP4, BMP6, BMP9, BMP-10, GDF-8, GDF-10, and GDF-11 were tested. In contrast, other proteins tested, such as BSA, IgG-Fc, mannose receptor (MMR, CD206), galactolectin-1 (Gal-1), CD31, CD36, CD44, CD300a, CLEC5, DC-SIGN, integrin αLβ2, TLR4, TSP1, anti-PD1 Ig, inhibin A, and GDF-15, did not demonstrate this role in delivering liposomes across the blood-brain barrier.

[0067] [Example] [3] [:] [Engineered liposomes that bind to secreted proteins can inhibit apoptosis proteases under activity-limiting stress.] [-1 siRNA] [Successfully delivered to the inflamed mouse brain] [,] [Thus effectively rescuing mouse brain cells] [(] [Including neurons, glial cells, and stellate glial cells] [)] [Pyroptosis of cells within the cell.] []

[0068] Engineered liposomes, bound to secreted proteins, were designed to selectively target inflamed and damaged cells in the brain. Targeting efficiency was visualized using a fluorescence imaging system (Thermo iBright FL1500) with liposomes labeled with a commercially available dye (calcein red; Sigma-Aldrich Co.). The acquired images confirmed successful delivery of the fluorescent dye to the inflamed mouse brain after activity-restricted stress. Figure 3A provides an overview of the experiment. The markers used in flow cytometry analysis to label neurons, microglia, astrocytes, and pyroptosis signals were NeuN, F4 / 80, GFAP, and the activated form of apoptosis protein-1, respectively (Figure 3A). Figures 3B, 3C, and 3D show the relative pyroptosis (a regulated form of cell death) in neurons, microglia, and astrocytes in the mouse brain under activity-restricted stress conditions compared to the control group. # P < 0.05 compared to the BSA group indicates significant rescue. These results are consistent with the luciferin delivery experiments (Figures 1 and 2), in which P-selectin, b-NGF, NT3, TGF-b1, activin A, BMP4, BMP6, BMP9, and BMP10 demonstrated effective drug delivery in neurons (B), microglial cells (C), and / or astrological glial cells (D) of the mouse brain after activity restriction stress. Furthermore, siRNA is an ideal carrier for this drug delivery system; as demonstrated in this experiment using apoptosis protease-1 siRNA, it effectively ameliorated pyroptosis and encephalitis induced by activity restriction stress in mice.

[0069] [Example] [4] [:] [Compared to using each secreted protein alone] [,] [Carries fluorescent markers and is associated with various secreted proteins] [(] [For example] [CI-MPR+KL] [、] [CI-MPR+P-] [Select elements and] [KL+P-] [Selection element] [)] [The bound liposomes exhibited significantly enhanced fluorescence.] []

[0070] Engineered liposomes, bound to secretory proteins, were designed to selectively target inflamed and damaged cells in the brain. Targeting efficiency was visualized using a fluorescence imaging system (Thermo iBright FL1500) with liposomes labeled with a commercially available dye (calcein red; Sigma-Aldrich Co.). The acquired images confirmed successful delivery of the fluorescent dye to the inflamed mouse brain after activity-limiting stress (Figure 4). The secretory proteins used in this experiment are listed in Figure 4, and corresponding quantifications of relative brain fluorescence intensity are provided in the same figure. This is consistent with the synergistic effect observed in the hepatitis rescue experiment shown in Figure 1. Compared to using each secretory protein alone, liposomes carrying fluorescent labels exhibited significantly enhanced fluorescence when bound to multiple secretory proteins (e.g., CI-MPR+KL, CI-MPR+P-selectin, and KL+P-selectin). Furthermore, these results indicate a synergistic effect when using this combination of secreted proteins in tandem with liposomes, even when used to deliver substances across the blood-brain barrier.

[0071] [Example] [5] [:] [and] [P-] Selectin-bound liposomes can promote the loading of apoptotic proteases. [-3] [Inhibitor] [(Z-DEVD-FMK)] [The liposomes penetrated through the mouse.] [blood-brain barrier] [。] []

[0072] These liposomes, bound to secreted proteins, were tested to specifically target inflamed and damaged cells in the brain. Activity-restricting stress induced inflammation in the mouse brain, as demonstrated by increased levels of the pro-inflammatory cytokine IL-1 (Fig. 5A: Experimental overview; Figs. 5B and 5C: IL-1 levels in the untreated group relative to the activity-restricting stress-carrier group). Notably, liposomes bound only to P-selectin facilitated the penetration of liposomes loaded with the apoptosis protein-3 inhibitor (z-DEVD-FMK) across the blood-brain barrier in mice, while liposomes bound to the control protein bovine serum albumin (BSA) did not exhibit this promoting effect.

[0073] [Example] [6] [:] [Combination] [P-] [Liposomes of Selectin significantly improve lipopolysaccharide in mice] [(LPS)] [Induced anxiety and depressive-like behaviors.] []

[0074] [LPS] [Induced Depressive Behavior:] LPS-induced depressive behavior was induced in mice using the previously described method (Hall, S.; Arora, D.; Anoopkumar-Dukie, S.; Grant, GD. Effect of Coffee in Lipopolysaccharide-Induced Indoleamine 2,3-Dioxygenase Activation and Depressive-like Behavior in Mice. J Agric Food Chem.).

[2016] , 64, 8745-8754, doi:10.1021 / acs.jafc.6b03568; O'Connor, JC; Lawson, MA; Andre, C.; Moreau, M.; Lestage, J.; Castanon, N.; Kelley, KW; Dantzer, R. Lipopolysaccharide-induced depressive-like behavior is mediated by indoleamine 2,3-dioxygenase activation in mice.Mol Psychiatry

[2009] ,14, 511-522, doi:10.1038 / sj.mp.4002148).

[0075] [Mouse Behavioral Testing:] The "Open Field Maze" was used to measure depressive and anxiety-like behaviors in mice. These tests followed a recognized protocol (Seibenhener, ML; Wooten, MC. Use of the Open Field Maze to measure locomotor and anxiety-like behavior in mice. J Vis Exp.

[2015] , 10.3791 / 52434, e52434, doi:10.3791 / 52434;Lopatina, O.; Yoshihara, T.; Nishimura, T.; Zhong, J.; Akther, S.; Fakhrul, AA; Liang, M.; Higashida, C.; Sumi, K.; Furuhara, K. et al. Anxiety- and depression-like behavior in mice lacking the CD157 / BST1 gene, a risk factor for Parkinson's disease. Front Behav Neurosci

[2014] , 8, 133, doi:10.3389 / fnbeh.2014.00133).

[0076] Based on the foregoing results, it has been established that activity-restricting stress has the ability to induce encephalitis. However, this encephalitis can be alleviated by delivering anti-cell death agents via liposomes that bind to secretory proteins and can cross the blood-brain barrier. Given these results, our aim was to further investigate whether treatment with liposomes loaded with an apoptosis protease-1 inhibitor and bound to secretory proteins could alleviate depressive behaviors exhibited in mice. Our results confirmed that liposomes bound to P-selectin significantly improved lipopolysaccharide (LPS)-induced anxiety-like and depressive-like behaviors in mice (Figure 6A: Recordings of open space tests in mice; Figures 6B and 6C: Quantitative analysis of mouse behavior). Notably, intravenous administration of liposomes loaded with the apoptosis protease-1 inhibitor z-WEHD-FMK and bound to P-selectin significantly alleviated LPS-induced depressive-like behaviors compared to the vector group (P-sel-lipo(C1in) vs. vector group, * P < 0.05). [P-sel-lipo(C1in): a liposome loaded with the apoptosis protein-1 inhibitor z-WEHD-FMK and bound to P-selectin].

[0077] [Example] [7] [:] [and] [P-] [Selectin-bound and loaded with apoptosis-protease] [-1] [The liposomes of the inhibitor significantly improved activity-limiting stress-induced anxiety and depressive-like behaviors.] []

[0078] [Mouse Behavioral Testing:] Mouse behavioral tests were conducted, including the arm extension test (Horii, Y.; McTaggart, I.; Kawaguchi, M. Testing Animal Anxiety in Rats: Effects of Open Arm Ledges and Closed Arm Wall Transparency in Elevated Plus Maze Test. J Vis Exp

[2018] (10.3791 / 56428, doi:10.3791 / 56428) and tail suspension test (Can, A.; Dao, DT; Terrillion, CE; Piantadosi, SC; Bhat, S.; Gould, TD The tail suspension test. J Vis Exp

[2012] , 10.3791 / 3769, e3769, doi:10.3791 / 3769), to measure depressive and anxiety-like behaviors in mice. These tests followed accepted protocols.

[0079] Our objective was to investigate whether drug-loaded secretory protein-liposomes could alleviate anxiety- and depression-like behaviors induced by activity restriction stress, based on a well-established model of activity restriction stress-induced depression. Previous studies have demonstrated that 20 hours of activity restriction stress can lead to a depression-like phenotype. Based on this model, additional analyses were performed using the Open Arm Test (OAT) and Tail Suspension Test (TST), recognized methods for assessing depression- and anxiety-like behaviors in mice. These tests were performed using methods previously described. Our results showed that liposomes bound only to P-selectin and loaded with an inhibitor of apoptosis protein-1 significantly improved activity restriction stress-induced anxiety- and depression-like behaviors, while liposomes bound to BSA did not show this improvement (Figures 7A to 7F), consistent with observations from the LPS model (Figure 6). Similarly, these results demonstrate that this secretory protein-based drug delivery system can deliver small molecules and peptide / short protein fragments to the brain and exert their medicinal effects.

[0080] [Example] [8] [:] This drug delivery system can deliver large functional enzymes. [(] [such as] [Cre DNA] [Recombinase] [)] [Successfully delivered across the blood-brain barrier to the mouse brain. This demonstrated gene editing and altered protein expression, confirmed by a shift from red fluorescent protein expression to green fluorescent protein expression.] []

[0081] Here, the Cre-Lox recombination system was used to demonstrate the efficient delivery of functional Cre recombinases to the mouse brain. Specifically, mice [B6.129-(Gt(ROSA)26Sor tm4(ACTB-tdTomato,-EGFP)Luo / J] were transfected with a vector designed with a CMV enhancer / chicken β-actin core promoter (pCA) to drive the expression of an optimized DsRed fluorescent protein variant (called tandem-dimer-Tomato, tdTomato) with an N-terminal membrane marker attached to the loxP site, followed by polyadenylation signaling. Adjacent to the second loxP site, an enhanced green fluorescent protein (EGFP) sequence with an N-terminal membrane marker was present, also followed by polyadenylation signaling. Additionally, a neomycin resistance gene cartridge (neo) attached to the frt site was also used. The cassette was located at the distal end of the expression vector. Red fluorescence was observed in all test tissues. When exogenous Cre recombinase was delivered to mouse brain cells, the regions containing loxP were excised to enable expression of the EGFP cassette. Thus, these cells receiving Cre recombinase became EGFP-labeled (Figure 8). These results indicate that this delivery system is well-suited for the efficient delivery of functional enzymes and proteins (such as those used in enzyme replacement therapy) to brain cells without significant lysosomal digestion.

[0082] [Example] [9] [:] [and] [P-] [Selectin-bound and loaded with apoptosis-protease] [-1 siRNA] [Liposomes significantly improve activity-limiting stress-induced] [Anxiety and depressive-like behaviors.] []

[0083] [Mouse Behavioral Testing:] An open-field maze (OFM) behavioral test was conducted in mice (Michael L. Seibenhener and Michael C. Wooten, Use of the Open Field Maze to Measure Locomotor and Anxiety-like Behavior in Mice. J Vis Exp. 2015; (96): 52434; doi: 10.3791 / 52434 PMCID: PMC4354627 PMID: 25742564) to measure depressive and anxiety-like behaviors in mice. This test followed a recognized protocol.

[0084] Our aim was to further investigate whether drug-loaded secretory protein-liposomes could alleviate anxiety-like and depressive-like behaviors induced by activity restriction stress, based on a model of depression induced by activity restriction stress. It has been reported that 24-hour activity restriction stress can induce a depressive-like phenotype. Based on this model, additional analysis was performed using the OFM test, a well-established method for measuring depressive and anxiety-like behaviors in mice. This test was performed using a previously described method. Our results showed that liposomes bound only to P-selectin and loaded with apoptosis protease-1 siRNA significantly improved activity restriction stress-induced anxiety-like and depressive-like behaviors, while liposomes bound to BSA did not show this improvement (Fig. 9A, Experimental Overview; Figs. 9B–9H). Similarly, these results demonstrate that this secretory protein-based drug delivery system can deliver small molecules and short nucleotide fragments to the brain and exert its pharmacological effects.

Claims

1. The use of a protein-vesicle conjugate in the preparation of a medicament for delivering a drug across a biological barrier to cells and / or tissues containing such cells, wherein the protein-vesicle conjugate is in contact with cells and / or tissues of an individual, wherein the protein-vesicle conjugate comprises: (a) one or more secretory proteins or fragments thereof expressed on or bound to the surface of a vesicle, and / or one or more antibodies expressed on or bound to the surface of a vesicle, wherein the one or more antibody systems are selected from the group consisting of: anti-PSGL-1 immunoglobulin (anti-PSGL-1 Ig) and anti-PDL1 Ig; and (b) a drug encapsulated in the vesicle.

2. As requested in claim 1, wherein the cell is an inflamed and / or damaged cell.

3. As claimed in claim 1, wherein the biological barrier is selected from the group consisting of: intestinal barrier, nasal barrier, lung barrier, blood-brain barrier and skin barrier.

4. As claimed in claim 3, wherein the biological barrier is a blood-brain barrier, and the individual contains induced and / or administered inflamed and / or damaged cells within the blood-brain barrier prior to administration of the protein-vesicle conjugate.

5. As claimed in claim 1, wherein the one or more secreted proteins or fragments thereof are selected from the group consisting of: P-selectin (P-sel), non-cation-dependent mannose-6-phosphate receptor (CI-MPR), L-selectin (L-sel), E-selectin (E-sel), P-selectin glycoprotein ligand-1 (PSGL-1), CD22 (siglec2), galactolectin-3 (Gal-3), clonoxin (Klotho), pentacyclic protein 3 (PTX3), CD47, CD42b, dendritic cell-specific intercellular adhesion molecule-3 binding non-integrin receptor (DC-SIGNR), intercellular adhesion molecule 1 (ICAM-1, CD54), vascular cell adhesion molecule (VCAM), integrin α4β1, VE-cadherin, annexin V, TLR-like receptor 3 (TLR3), planned cell death ligand 1. (PD-L1), IL-1β, IL-1RA, TNF-α, etanercept, EGF, fibroblast growth factor (FGF) 23, insulin, glial cell line-derived neurotrophic factor (GDNF), BDNF, β-NGF, NT-3, TGF-β1, activin A, BMP4, BMP6, BMP9, BMP-10, GDF-8, GDF-10, GDF-11 and their fragments.

6. As claimed in claim 1, wherein the cell is a neuron, and the one or more secreted proteins or fragments thereof are selected from the group consisting of P-selectin, activin A, BMP9 and fragments thereof.

7. As claimed in claim 1, wherein the cell is a microglial cell, and the one or more secreted proteins or fragments thereof are selected from the group consisting of: P-selectin, β-NGF, NT3, activin A and fragments thereof.

8. As claimed in claim 1, wherein the cell is an astrocyte, and the one or more secreted proteins or fragments thereof are selected from the group consisting of: P-selectin, β-NGF, NT3, activin A, BMP6, BMP9 and fragments thereof.

9. As claimed in claim 1, wherein the protein-vesicle conjugate comprises a combination of CI-MPR and clotrimazole, a combination of CI-MPR and P-selectin, or a combination of clotrimazole and P-selectin.

10. As claimed in claim 1, wherein the vesicle is a liposome or a microcell.

11. As claimed in claim 1, wherein the protein-vesicle conjugate is artificially engineered or cell-derived.

12. The use as claimed in claim 1, wherein the agent is separated from the one or more secretory proteins or fragments thereof and encapsulated within the vesicle or attached to the outer surface of the vesicle.

13. As claimed in claim 1, wherein the agent is a diagnostic contrast agent, a cell survival enhancer, a cell survival inhibitor, a cell component, an organelle, a cell, a cytotoxic agent, an antitumor drug, a toxin, an antibody, a lipid, a protein, DNA, RNA, a therapeutic agent, or a nanomaterial.

14. The use of any of the claims 1 to 13 is directed toward a disease related to regulated cell death associated with changes in brain cells.

15. As claimed in claim 14, wherein the disease associated with regulated cell death related to changes in brain cells is trauma, exposure to chemical and physical toxic factors, genetic diseases, age-related diseases, cardiovascular diseases associated with brain injury, infectious diseases, neoplastic diseases, neurodegenerative diseases, metabolic diseases, aging, obesity, cancer, inflammation, psychological stress, stroke, neurodegeneration induced by β-amyloid or α-synuclein or toxicity, muscle degenerative conditions or chronic pneumonia caused by cystic fibrosis, or autoimmune diseases.

16. As claimed in claim 15, wherein the neurodegenerative disease is brain fog, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, or stroke; the cardiovascular condition associated with brain injury is ischemia, heart failure, or an infectious disease; and the autoimmune disease is systemic lupus erythematosus, autoimmune lymphoproliferative syndrome, rheumatoid arthritis, or thyroiditis.

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