JP7253762B2 - Drug delivery to autophagic and apoptotic cells by vesicles with surface-expressed proteins - Google Patents

Description

The present invention relates to the field of drug delivery to cells. In particular, the present invention relates to the delivery of agents to cells and tissues undergoing autophagy and/or apoptosis via vesicles on which modified proteins are expressed or conjugated. .

Apoptosis is a well-studied pathway of programmed cell death. Non-apoptotic forms of cell exclusion include those with characteristics of necrosis and autophagy. Apoptosis plays a very important role in developing and maintaining the health of the body by eliminating old, unwanted and unhealthy cells. Too little or too much apoptosis may play a role in many diseases. When apoptosis does not work properly, cells that should be eliminated may remain and become immortal in cancer and leukemia, for example. Apoptosis is normal, but when overdone, it kills too many cells and causes significant tissue damage. This is the case in stroke and neurodegenerative disorders such as Alzheimer's disease, Huntington's disease and Parkinson's disease.

Autophagy, the process by which proteins and organelles are sequestered in double-membrane structures called autophagosomes and delivered to lysosomes for degradation, is of great importance in various diseases. In addition to cancer and neurodegeneration, modulation of autophagy has become a therapeutic strategy in a wide variety of additional diseases and disorders. For example, several liver, heart and muscle diseases have been linked to the accumulation of misfolded protein aggregates. In such diseases, agents that increase cellular autophagy may enhance clearance of disease-causing aggregates, thereby contributing to treatment and reducing disease severity. Additionally, inhibitors of autophagy may serve as therapeutic agents in the treatment of pancreatitis.

Therefore, there is a need to develop means to modulate apoptosis and autophagy in cells and thereby treat or ameliorate diseases associated with autophagy.

The present invention provides a protein conjugate vesicle comprising one or more lectins or fragments thereof expressed or conjugated on the surface of said vesicle and optionally a drug. Provide gated vesicles.

In one embodiment, the agent is enclosed inside the vesicle or attached to the outer surface of the vesicle. Specific embodiments of said vesicles include liposomes and micelles. The vesicles can be artificially modified or derived from cells.

Specific embodiments of said lectins or fragments include cation-dependent mannose-6-phosphate receptor (M6PR), P-selectin, E-selectin, L-selectin, P - selectin-ligand-1 (PSGL-1), CD22, CD206, galectin 3, annexin V, CD31, integrin αLβ2, VE-cadherin, CD44, CD300a, CD47, thrombospondin 1 (TSP1) and CD36 , but not limited to.

Specific embodiments of said lectins or fragments include CD300a, CD47, thrombospondin 1 (TSP1) and CD36, Toll-like receptor 4 (TLR4) or fragments thereof used as second proteins. but not limited to these.

Specific embodiments of said lectin or fragment include, but are not limited to, one or more of said first protein and one or more of said second protein.

In some embodiments, the vesicle comprises M6PR in combination with P-selectin, E-selectin, PSGL-1 or galectin-3. In some embodiments, the vesicle comprises Siglec2 in combination with P-selectin, galectin3 or CD31.

In some embodiments, the vesicle comprises P-selectin or M6PR in combination with TLR4, galectin 3, CLEC2, integrin αLβ2 or CD31.

Specific embodiments of said agents include diagnostic imaging agents, cell survival enhancing agents, cell survival inhibitors, cellular components, organelles, cells, cytotoxic agents, anti-tumor drugs, toxins or antibodies, lipids, proteins, Including but not limited to DNA, RNA, therapeutic agents or nanomaterials.

The invention also provides pharmaceutical compositions comprising the vesicles of the invention and a pharmaceutically acceptable carrier.

The invention also provides a method for targeting the delivery of agents to autophagic and/or apoptotic cells and tissues containing said cells, directed to the protein-conjugated vesicles of the invention. A method is provided comprising administering.

Liposome targeting to autophagy cells in vitro. ^* P<0.05 vs unconjugated group (n=3). Groups 1-15 showed different levels of cell binding of liposomes with or without protein conjugate. Groups: 1, unconjugated liposomes; 2, M6PR conjugated liposomes; 3, P-selectin conjugated liposomes; 4, E-selectin conjugated liposomes; 5, PSGL-1 conjugated liposomes; 7, CD206 conjugated liposomes; 8, Galectin 3 conjugated liposomes; 9, Annexin V conjugated liposomes; 10, Integrin αLβ2 conjugated liposomes; 11, VE-Cadherin conjugated liposomes; Conjugated liposomes; 14, conjugated liposomes with TSP1 and CD36. The unconjugated group was normalized to 100%.

Figure 3 shows liposome targeting to apoptotic cells in vitro. ^* P<0.05 vs absent group (n=3). Groups 1-15 showed different levels of cell binding of liposomes with or without protein conjugation. Groups: 1, unconjugated liposomes; 2, M6PR conjugated liposomes; 3, P-selectin conjugated liposomes; 4, E-selectin conjugated liposomes; 5, PSGL-1 conjugated liposomes; 7, CD206 conjugated liposomes; 8, Galectin 3 conjugated liposomes; 9, Annexin V conjugated liposomes; 10, Integrin αLβ2 conjugated liposomes; 11, VE-Cadherin conjugated liposomes; Conjugated liposomes; 14, conjugated liposomes with TSP1 and CD36. The unconjugated group was normalized to 100%.

An example of flow cytometric analysis and detection of autophagic cells (blue population) is shown.

An example of flow cytometric analysis and detection of apoptotic cells (purple population) is shown.

Targeting effect of recombinant protein-conjugated liposomes is demonstrated by in vivo imaging system (IVIS). Fluorescence intensity of recombinant M6PR conjugated liposomes (labeled fluorescent dye is calcein red).

Targeting effect of recombinant protein-conjugated liposomes on B16-F10 tumor cells is demonstrated by in vivo imaging system (IVIS). Fluorescently labeled liposomes were used. Fluorescence intensity of recombinant M6PR protein-conjugated liposomes (labeled fluorescent dye is calcein red). Absent: treated with unconjugated liposomes.

Figure 2 shows that an in vivo imaging system (IVIS) was used to confirm whether recombinant protein-conjugated fluorescent liposomes have targeting effects on the liver after injury. Relative fluorescence intensities of mouse organs including heart, lung, liver and spleen with and without TAA and liposome treatments were measured. A, Bright field images showed mouse organs including heart, lung, liver and spleen. BS, Fluorescence images, in these images the red areas in the pseudocolor images (B, D, F, H, J) are shown in C, E, G, I, K. Synergistic targeting: BC, M6PR + P-selectin; DE, M6PR + galectin3 and Siglec2; FG, M6PR + MMR and integrin αLβ2; HI, M6PR + CD31 and annexin V; JK, M6PR + CD44 and VE-cadherin . All protein-conjugated liposomes showed higher liver-preferential targeting properties compared to the control group treated with non-conjugated liposomes (vehicle). In addition, fluorescence levels are higher in such groups where two proteins are conjugated compared to groups where only one protein is conjugated.

Figure 2 shows that an in vivo imaging system (IVIS) is used to confirm whether recombinant protein-conjugated fluorescent liposomes have targeting effects on tumors. We found that fluorescence-loaded liposomes exhibited more efficient targeting to tumors formed from B16-F10 melanoma cells (M6PR group versus untreated and vehicle groups). , the group with M6PR + P-selectin, and the group with M6PR + galectin 3).

An in vivo imaging system (IVIS) was used to confirm whether recombinant protein-conjugated fluorescent liposomes have a targeting effect on injured white adipose tissue (pretreated with anti-adipose tissue antibodies). indicates that C57B1/6J mice were treated with or without rabbit anti-mouse adipocyte antibody. Mice were then treated with recombinant protein-conjugated fluorescent (calcein red) liposomes. Groups: 1, untreated; 2, unconjugated liposomes; 3, M6PR + galectin 3; 4, M6PR + P-selectin; 5, M6PR.

Mouse livers after injury contain autophagic and apoptotic cells. ^* P<0.05 compared to the normal group (n=4).

Solid tumors formed from mouse B16-F10 cells are shown to contain autophagic and apoptotic cells. ^* P<0.05 compared to the normal group (n=4).

Mouse adipose tissue treated with anti-fat antibodies contains autophagic and apoptotic cells. ^* P<0.05 compared to the normal group (n=4).

Blockage of liposome targets to apoptotic cells. Blocking antibodies and soluble M6PR recombinant proteins (panels 3 and 4, respectively) were able to block targeting of M6PR-conjugated liposomes/microvesicles (panel 2) targeting apoptotic cells, indicating antidote may work as Liposome preparations: Panel 1, unconjugated liposomes; Panel 2, M6PR + conjugated liposomes with P-selectin; Panel 3, M6PR + conjugated liposomes with P-selectin + blocking antibody; Panel 4, M6PR + conjugated liposomes with P-selectin. +Soluble M6PR recombinant protein.

Blockage of liposome targets to autophagy cells. Blocking antibodies and soluble M6PR recombinant proteins (panels 3 and 4, respectively) are able to block the targeting of M6PR+P-selectin conjugated liposomes/microvesicles (panel 2) to autophagic cells. can and may act as an antidote. Liposome preparations: Panel 1, unconjugated liposomes; Panel 2, M6PR + conjugated liposomes with P-selectin; Panel 3, M6PR + conjugated liposomes with P-selectin + blocking antibody; Panel 4, M6PR + conjugated liposomes with P-selectin. +Soluble M6PR recombinant protein.

Liposome targeting to apoptotic cells. Groups 1 to 4 represent the untreated group, the M6PR+P-selectin conjugate group, the M6PR+E-selectin conjugate group, and the M6PR+PSGL-1 conjugate group, respectively. ^* P<0.05 compared to the untreated (panel 1) group (n=4). These results demonstrate that specific blocking antibodies, specific soluble recombinant proteins and sialyl-Lewis x-oligosaccharides stimulated apoptotic cells of P-selectin-, E-selectin-, and PSGL-1-conjugated liposomes. suggested that it could serve as an antidote to prevent targeting to

Liposome targeting to autophagy cells. Groups 1 to 4 represent the untreated group, the M6PR+P-selectin conjugate group, the M6PR+E-selectin conjugate group, and the M6PR+PSGL-1 conjugate group, respectively. ^* P<0.05 compared to the untreated (panel 1) group (n=4). These results demonstrate that specific blocking antibodies, specific soluble recombinant proteins and sialyl-Lewis x-oligosaccharides inhibit autophagy of P-selectin-, E-selectin-, and PSGL-1-conjugated liposomes. suggested that it could act as an antidote to prevent targeting to cells.

Results for rescue of detached apoptotic B16-F10 cells in vitro of M6PR conjugated liposomes loaded with caspase-3 inhibitors are shown. ^* P<0.05 compared to the exfoliated group (n=4). These results suggested that M6PR-conjugated liposomes could specifically deliver the liposome-loaded caspase-3 inhibitor (BioVision) into apoptotic cells.

, Results for DNA-loaded M6PR-conjugated liposomes (white panels), RNA-loaded M6PR-conjugated liposomes (grey panels), and protein-loaded M6PR-conjugated liposomes (black panels) for delivery of DNA, RNA and protein to B16-F10 cells. indicates Relative fluorescence levels of B16-F10 cells with or without liposomes were analyzed using flow cytometry. ^* P<0.05, compared to the respective unconjugated group (n=4).

Results for rescuing liposomes/MV TAA-treated mice loaded with caspase-3 inhibitors conjugated with M6PR, M6PR + P-selectin, M6PR + E-selectin, and M6PR + PSGL-1 are shown. Plasma aspartate transaminase (AST) levels were analyzed. P-selectin: P-sel; E-selectin: E-sel; P-selectin glycoprotein ligand 1: PSGL-1. ○# P<0.05 compared to the unconjugated MV group. ^** P<0.01, compared to each recombinant protein conjugate group (n=6).

Results of rescue of M6PR, M6PR + P-selectin, M6PR + E-selectin, and M6PR + PSGL-1 conjugated Bcl-2 expression plasmid-loaded liposomes/MV TAA-treated mice are shown. Plasma aspartate transaminase (AST) levels were analyzed. P-selectin: P-sel; E-selectin: E-sel; P-selectin glycoprotein ligand 1: PSGL-1. # P<0.05 compared to the group without liposomes/MV. ^** P<0.01, compared to each recombinant protein conjugate group (n=6).

Results of rescue of M6PR, M6PR + P-selectin, M6PR + E-selectin, and M6PR + PSGL-1 conjugated caspase-3 siRNA-loaded liposomes/MV TAA-treated mice are shown. Plasma aspartate transaminase (AST) levels were analyzed. P-selectin: P-sel; E-selectin: E-sel; P-selectin glycoprotein ligand 1: PSGL-1. # P<0.05 compared to unconjugated MV group. ^** P<0.01, compared to each recombinant protein conjugate group (n=6).

Results of rescuing M6PR, M6PR + P-selectin, M6PR + E-selectin, and M6PR + PSGL-1 conjugated anti-apoptotic Bcl-xL-derived BH4 motif-loaded liposomes/MV TAA-treated mice are shown. Plasma aspartate transaminase (AST) levels were analyzed. P-selectin: P-sel; E-selectin: E-sel; P-selectin glycoprotein ligand 1: PSGL-1. ○# P<0.05 compared to the unconjugated MV group. ^** P<0.01, compared to each recombinant protein-conjugated group (loaded with anti-apoptotic Bcl-xL-derived BH4 motif) (n=6).

Synergistic effect of M6PR + galectin3, M6PR + P-selectin, Siglec2 + P-selectin, and Siglec2 + galectin3 conjugated caspase-3 inhibitor-loaded liposomes/MVs when rescuing TAA-treated mice. Platelet counts (PLT) and plasma alanine aminotransferase (ALT) levels were analyzed. P-selectin: P-sel; Galectin 3: Gal3. ## P<0.01 compared to the group without BSA. ^* P<0.05, ^** P<0.01, compared to each single recombinant protein conjugate group (loaded with caspase-3 inhibitor) (n=6).

IVIS analysis of M6PR and P-selectin (P-sel) conjugated liposomes/MVs for enhanced targeting of fluorescently labeled CD34+ stem cells to injured tissue. Shown here is the liver in a model of TAA-treated mice.

Results of rescue of M6PR, M6PR + P-selectin, M6PR + E-selectin, and M6PR + PSGL-1 conjugated liposomes/MV TAA-treated mice are shown. Plasma aspartate transaminase (AST) levels were analyzed. P-selectin: P-sel; E-selectin: E-sel; P-selectin glycoprotein ligand 1: PSGL-1. # P<0.05 compared to unconjugated MV group. ^** P<0.01, compared to each M6PR-conjugated liposome/MV group (n=6).

Effect of MP-loaded anti-cancer drugs on tumor growth rate: Figure 26A shows the tumor plots of each group. Vehicle: normal saline (control), MMC: mitomycin C (anti-cancer drug).

Effect of MP-loaded anti-cancer drugs on tumor growth rate: Figure 26B shows the weight and volume of tumors in each group. Vehicle: normal saline (control), MMC: mitomycin C (anti-cancer drug).

FIG. 27A shows that protein-conjugated modified liposomes (containing doxorubicin) can suppress tumor growth rate.

FIG. 27B shows that protein-conjugated modified liposomes (containing doxorubicin) can also reduce mortality in mice.

Effect of mitomycin C-loaded liposomes on adipose mice. Liposomes-drug: mitomycin C-loaded liposomes, CIg: control Ig.

FIG. 29A shows that protein-conjugated modified liposomes (containing doxorubicin) can reduce the rate of weight gain in mice.

Figure 29B shows that anti-fat antibodies or liposomes (containing doxorubicin) do not cause an increase in liver function indices.

Plasma MVs express relatively higher levels of surface P-selectin compared to serum MVs and cell (C6/36)-derived MVs. Analysis was performed by flow cytometry. ^* P<0.05, compared with the serum-derived MV group (n=4).

Results of liver injury rescue in the TAA mouse model of plasma-derived MP are shown (n=4).

Plasma levels of the liver enzyme AST, indicative of liver function in experimental mice, are shown (higher AST levels indicate lower liver function). ^* P<0.05, compared with the serum-derived MV group (n=4). These results suggested that plasma MVs could rescue injury in target tissues.

The present invention provides modified surface proteins expressed or vesicle-conjugated on the surface of vesicles for the specific targeting and delivery of drugs to autophagic and/or apoptotic cells. . In particular, the vesicles of the invention can achieve synergistic effects on targeting and drug delivery to autophagic and/or apoptotic cells and tissues containing autophagic and/or apoptotic cells.

Where the definition of a term departs from the commonly used meaning of that term, applicant intends to utilize the definitions provided below, except where specifically indicated.

As used in this specification and the appended claims, the singular forms (“a,” “an,” and “the”) include plural references unless the context clearly dictates otherwise. do.

As used herein, the use of "or (or alternatively)" means "and/or" unless stated otherwise. In the context of multiple dependent claims, the use of "or" refers only alternatively to two or more preceding independent or dependent claims.

As used herein, the term "one or more" is readily understood by those skilled in the art, especially when read within the context of their common usage.

As used herein, the term "liposome" is a generic term encompassing a variety of unilamellar and multilamellar lipid vesicles formed by the formation of encapsulated lipid bilayers or aggregates. be. Liposomes may be characterized as having a vesicular structure with a bilayer membrane that typically contains a phospholipid and an internal medium that typically contains an aqueous composition.

As used herein, the term "micelle" refers to an aggregate (or supramolecular assembly) of surfactant molecules dispersed in a liquid colloid. A typical micelle in aqueous solution is an aggregate in which the hydrophilic "head" region is in contact with the surrounding solvent, forming an aggregate that traps a single hydrophobic tail region in the center of the micelle.

As used herein, the term "drug" or the term "therapeutic agent" refers to an agent capable of effecting treatment and/or amelioration of a condition or disease.

As used interchangeably herein, the terms “individual,” “subject,” “host,” and “patient” refer to mammals, including murines (rats, mice, ), non-human primates, humans, dogs, cats, ungulates (eg, horses, cows, sheep, pigs, goats), and the like.

As used herein, the term "therapeutically effective amount" or the term "effective amount" refers to the therapeutically effective amount for a disease when administered to a mammal or other subject to treat the disease. The amount of the vesicles that is sufficient to effect such treatment is shown.

As used herein, the term "treatment," the term "treat," and like terms encompass the treatment of disease in mammals (especially humans), any treatment that includes (a ) preventing the disease from occurring in subjects who may be predisposed to the disease but have not yet been diagnosed with the disease; and (c) alleviating said disease, ie causing regression of said disease.

As used herein, the term "conjugation site" is the site of covalent bonding formed between two macromolecules, primarily end-to-side chain branched conjugations, and Sometimes it is a linear head-to-tail conjugation of molecules.

In one aspect, the invention provides protein-conjugated vesicles comprising one or more lectins or fragments thereof expressed or conjugated on the surface of the vesicle and, optionally, a drug. offer.

In one embodiment, the agent is enclosed inside the vesicle or attached to the outer surface of the vesicle.

In some embodiments, the vesicles are liposomes or micelles. The vesicles can be artificially modified or derived from cells.

In some embodiments, the lectin or fragment thereof is cation-gated mannose-6-phosphate receptor (M6PR), P-selectin, E-selectin, L-selectin, P-selectin-ligand-1 (PSGL -1), CD22, CD206, galectin 3, annexin V, CD31, integrin αLβ2, VE-cadherin, CD300a, CD47, thrombospondin 1 (TSP1) and CD36, or fragments thereof. In some embodiments, M6PR, P-selectin, E-selectin, P-selectin-ligand-1 (PSGL-1), CD22, CD206, galectin 3, annexin V, integrin αLβ2, VE-cadherin are alone; Sufficient for vesicle targeting to autophagic and/or apoptotic cells and tissues containing autophagic and/or apoptotic cells to act as the first protein (EP). In some further embodiments, the vesicle comprises M6PR in combination with P-selectin, E-selectin, PSGL-1 or galectin-3, or Siglec2 in combination with P-selectin or galectin-3. include.

In some embodiments, CD300a, CD47, thrombospondin 1 (TSP1) and CD36 can also serve as the second protein. Accordingly, the invention further provides M6PR, P-selectin, E-selectin, L-selectin, P-selectin-ligand-1 (PSGL-1), CD22, CD206, galectin 3, annexin V, CD31, integrins αLβ2 and VE. - one or more of the first proteins selected from the group consisting of cadherins and from the group consisting of CD300a, CD47, thrombospondin 1 (TSP1), Toll-like receptor 4 (TLR4) and CD36 and fragments thereof A vesicle is provided that includes one or more of the selected second proteins. In some embodiments, the vesicle comprises M6PR or P-selectin in combination with TLR4, galectin 3, CLEC2, integrin αLβ2 or CD31. The vesicles with combinations of protein labeling can achieve synergistic effects for targeting and drug delivery to autophagic and/or apoptotic cells and tissues containing autophagic and/or apoptotic cells. . A synergistic effect on targeting action can result in a lower effective dosage of the drug to be delivered, thus reducing the side effects of the drug.

In one embodiment, the agent is a diagnostic or therapeutic agent. In one embodiment, the agent is a drug that induces autophagy or apoptosis. In some embodiments, examples of such agents include antimalarial drugs, autophagy inhibitors, histone deacetylase (HDAC) inhibitors, antagonists of EP or AP described herein, diagnostic imaging. agents, cell survival-enhancing agents (or cell death inhibitors), cell survival inhibitors (or cell death-enhancing agents), cells (such as stem cells and progenitor cells), cell components, organelles, cytotoxic agents, antitumor agents Including, but not limited to, drugs, toxins or antibodies, lipids, proteins, DNA, RNA, therapeutic agents, and nanomaterials. In one embodiment, antagonists of the above-described first protein or second protein (e.g., soluble forms, corresponding ligands, neutralizing and blocking antibodies, etc.) are used in autophagic and apoptotic cells, as well as It can serve as an antidote to reduce vesicle targeting to tissues containing fuzzy and apoptotic cells. In some embodiments, the agent is bardoxolone methyl, chloroquine, quinine, hydrochloroquine, sorafenib, sunitinib, Hsp90 inhibitor, metformin or crizotinib.

Liposomes provided herein include unilamellar liposomes, multilamellar liposomes and multivesicular liposomes. Liposomes provided herein may be composed of positively charged, negatively charged, or neutral phospholipids.

Liposomes used in the present invention can be made by various methods known in the art. For example, phospholipids (such as the neutral phospholipids dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC) and/or EPC) are dissolved in alcohol or other organic solvents and then encapsulated in the lipid bilayer. Can be mixed with ingredients for This mixture may further contain various surfactants. Typically, the lipid mixture is vortexed, frozen in a dry ice/acetone bath, and subjected to lyophilization overnight. Freeze-dried preparations are stored at -20°C or below for extended periods of time. Lyophilized liposomes are reconstituted when needed.

Alternatively, liposomes can be prepared by mixing lipids with a solvent in a container (eg, a glass pear-shaped flask). The container should be 10 times larger in volume than the expected suspension volume of liposomes. Solvent is removed under negative pressure at approximately 40° C. using a rotary evaporator. The solvent is usually removed within about 5 minutes to 2 hours, depending on the desired volume of liposomes. The composition can be further dried in a desiccator under vacuum. Dried lipids generally tend to degrade over time and are discarded after about a week.

The micelle structure itself will be determined primarily by the type and composition of the polymer molecules used to form the micelle, as well as the micelle's solvent environment. In some embodiments, micelles are made using nonionic triblock copolymers composed of both hydrophilic and hydrophobic monomeric units. In embodiments of the present disclosure, poloxamers, triblock copolymers of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) are used. In some embodiments, micelles of the present disclosure use PEG-PLA polymers of varying block sizes (eg, block sizes within the range above) and varying ratios (eg, about 1 : PEG:PLA) in integer ratios from 10 to about 10:1, or any integer ratio within said ratios.

Conjugation of the proteins described herein into vesicles is accomplished through the recruitment of functional group-labeled lipids into the vesicles using shear force-based methods ( Ｙｕ Ｂ、Ｌｅｅ ＲＪ、Ｌｅｅ ＬＪ、Ｍｉｃｒｏｆｌｕｉｄｉｃ ｍｅｔｈｏｄｓ ｆｏｒ ｐｒｏｄｕｃｔｉｏｎ ｏｆ ｌｉｐｏｓｏｍｅｓ、Ｍｅｔｈｏｄｓ Ｅｎｚｙｍｏｌ．、２００９；４６５：１２９～１４１、および、Ｊｅｏｎｇ Ｄ、Ｊｏ Ｗ、Ｙｏｏｎ Ｊ他、Ｎａｎｏｖｅｓｉｃｌｅｓ ｅｎｇｉｎｅｅｒｅｄ ｆｒｏｍ ＥＳ ｃｅｌｌｓ ｆｏｒ ｅｎｈａｎｃｅｄ ｃｅｌｌ ｐｒｏｌｉｆｅｒａｔｉｏｎ、 Biomaterials, 2014;35(34):9302-9310).

In another aspect, the invention provides pharmaceutical compositions comprising a vesicle of the invention and a pharmaceutically acceptable carrier. The vesicles of the invention can be formulated in a variety of different ways known to those skilled in the art. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention (see, eg, Remington's Pharmaceutical Sciences, 20th Ed., 2003, supra). Effective formulations include oral and nasal formulations, formulations for parenteral administration, and compositions formulated for sustained release.

For administration, eg, for parenteral administration, sterile aqueous solutions of water-soluble salts (eg, NaCl) can be used. Additional or alternative carriers may include sesame or peanut oil, as well as aqueous propylene glycol. Aqueous solutions may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, intraperitoneal and intratumoral (IT) injection.

Formulations suitable for oral administration include (a) a liquid solution, such as an effective amount of a compound of the invention suspended in a diluent such as water, saline or PEG 400, b) capsules, sachets, depots or tablets, each containing a predetermined amount of the active ingredient as a liquid, solid, granules or gelatin, (c) a suspension in a suitable liquid, (d) a) a suitable emulsion; and (e) a patch. The liquid solutions described above can be sterile solutions. Pharmaceutical forms may contain lactose, sucrose, mannitol, sorbitol, calcium phosphate, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, coloring. agents, fillers, binders, diluents, buffering agents, wetting agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers. Lozenges can contain the active ingredient in a flavor (e.g., sucrose), similarly lozenges can contain the active ingredient in an inert base (e.g., gelatin and glycerin or sucrose and acacia emulsion, etc.) and gels. Agents and the like contain carriers known in the art in addition to the active ingredient.

In another aspect, the present invention provides delivery of an agent of interest to a subject, comprising administering a protein conjugate vesicle of the present invention to autophagic and/or apoptotic cells and tissue containing said cells. A method is provided for targeting against. In one embodiment, prior to administering the vesicle, the method further comprises administering an autophagy-inducing agent and/or an apoptosis-inducing agent to the target cell or target tissue. By using this step, autophagy or apoptosis occurs in the target cell or tissue, such that the vesicles of the invention are targeted to cells or tissues undergoing autophagy and/or apoptosis, and then , could deliver agents of interest to such cells or tissues. For example, an anti-obesity antibody is first administered to a subject such that adipocytes or adipose tissue undergoes autophagy and/or apoptosis, and then vesicles with the anti-obesity drug undergo autophagy and/or apoptosis. It is administered to target adipocytes or adipose tissue undergoing apoptosis so that these adipocytes or adipose tissue can be further damaged by said anti-obesity drug.

Autophagy is a lysosomal degradation pathway that is essential for survival, differentiation, development and homeostasis. Delivery of drugs or therapeutic agents with the vesicles of the invention to autophagic cells is directed to diseases associated with deregulation of autophagy. Diseases associated with dysregulation of autophagy include trauma, exposure to chemical and physical toxic agents, genetic diseases, aging-related diseases, cardiovascular diseases, infectious diseases, neoplastic diseases, neurodegenerative diseases, metabolic sexually transmitted disease, aging (when ATG5 is overexpressed throughout the organism), obesity (when ATG7 or autophagy-enhancing transcription factor EB [TFEB] is overexpressed in hepatocytes), cancer (beclin 1 is associated with KRAS-induced lung adenoma), β-amyloid or α-synuclein or toxicity-induced neurodegeneration (when TFEB or Beclin 1 is overexpressed in the brain, or cystatin B (an inhibitor of lysosomal cysteine proteases) knockout), myodegenerative conditions (when TFEB or Beclin 1 is targeted to skeletal muscle), and chronic pulmonary inflammation caused by cystic fibrosis (when Beclin 1 is expressed in the lung). including but not limited to:

Apoptosis is controlled by the integration of multiple pro-apoptotic and anti-apoptotic signals. Delivery of drugs or therapeutic agents to apoptotic cells using the vesicles of the invention targets diseases associated with altered apoptosis. Diseases associated with alterations in apoptosis include trauma, exposure to chemical and physical toxic agents, genetic diseases, aging-related diseases, age-related diseases, cardiovascular diseases, infectious diseases, neoplastic diseases, neurodegenerative diseases , metabolic disease, aging, obesity, cancer, β-amyloid or α-synuclein-induced neurodegeneration (Alzheimer's, Parkinson's, Huntington's, amyotrophic lateral sclerosis) or toxicity-induced neurodegeneration, muscle degeneration conditions, or chronic pulmonary inflammation caused by cystic fibrosis, cardiovascular disorders (such as ischemia, heart failure and infectious diseases) and autoimmune diseases (systemic lupus erythematosus, autoimmune lymphoproliferative syndrome, rheumatoid arthritis) and thyroiditis).

The vesicles of the invention can be used to treat or diagnose any disease requiring administration of a diagnostic or therapeutic agent. Any suitable drug or therapeutic agent can be used with the vesicles of the invention. Additionally, the vesicles of the present invention are useful for treating infections by various pathogens such as viruses, bacteria, fungi and parasites. Other diseases can be treated using the vesicles of the invention.

In some embodiments, the vesicles or pharmaceutical compositions of the invention are administered topically, parenterally, intravenously, intradermally, subcutaneously, intramuscularly, colonically, rectally or intraperitoneally. It can be administered to the patient in a variety of ways, including: Preferably, the pharmaceutical composition is administered by parenteral, topical, intravenous, intramuscular, subcutaneous, oral or nasal administration (eg, inhalation, etc.).

In certain embodiments, protein-conjugated vesicles can deliver lipids, proteins, DNA, RNA, therapeutic agents or nanomaterials. In some embodiments, the therapeutic agent is a cell survival-enhancing agent (or cell death inhibitor). Drug-mediated rescue of tissue injury can be achieved by delivering cell survival-enhancing agents (or cell death inhibitors) to the subject.

In some embodiments, the agent is a cell survival inhibitor, cell death enhancing agent, or an anti-tumor agent. Reducing the survival of target cells in such tissues (e.g., tumors, etc.) containing naturally occurring autophagic and apoptotic cells by delivery of cell survival inhibitors (cell death enhancing agents) or anti-tumor agents Alternatively, autophagic and apoptotic cells can be artificially induced in specific tissues using cytotoxic agents (e.g., drugs, toxins, or antibodies directed against tissue-specific proteins). specific tissue can be reduced.

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

Although the invention has been described with reference to preferred embodiments and examples thereof, the scope of the invention is not limited to only such described embodiments. As will be apparent to those skilled in the art, various modifications and adaptations to the above invention can be made without departing from the spirit and scope of the invention as defined and limited by the appended claims. . The following examples are provided for the purpose of illustrating various embodiments and advantages of the invention and are not intended to limit the scope of the invention.

Example 1 Liposome targeting to autophagy cells in vitro Preparation of liposomes

Liposomes were prepared by a liposome kit (Sigma-Aldrich Co.) and the respective lipids. Conjugation of surface proteins is accomplished via loading functional group-labeled lipids into liposomes using shear force-based methods (Yu B, Lee RJ, Lee LJ, Microfluidic methods for production of liposomes, Methods Enzymol., 2009;465:129-141, and Jeong D, Jo W, Yoon J et al., Nanovesicles engineered from ES cells for enhanced cell proliferation, Biomaterials, 31:33, 2034-9; . Protein conjugation of proteins (M6PR, P-selectin, E-selectin, PSGL-1, CD22, CD206, galectin 3, annexin V, integrin αLβ2, VE-cadherin, CD300a, CD47, TSP1 and CD36) to liposomes has been reported. , based on the methods provided by the product.

Measurement of relative association levels for autophagic and apoptotic cells

Mouse B16-F10 cells were suspended for 4 hours to induce autophagy and apoptosis. Autophagic and apoptotic cells were stained with the Cyto-ID Autophagy Detection Kit (Enzo Life Sciences) (see Figures 1 and 3) and the CaspGLOW™ Red Active Caspase-3 Staining Kit (BioVision) (Figures 2 and 3). 4), respectively, and labeled with green fluorescent dye (GFD). Populations containing autophagic and apoptotic cells were associated with various protein-conjugated liposomes labeled with the fluorescent dye calcein-red (CR). The percentage of the B16-F10 liposome-associated population (GFD and CR double positive population) was determined using flow cytometry. Levels of liposomes and fluorescent beads associated with unconjugated cells (“unconjugated” group) were normalized to 100% (see FIGS. 1 and 2).

Example 2 Liposomes Specifically Targeted to Damaged Tissue Via Various Protein Conjugates Targeting Modified Liposomes to Damaged Liver in a Mouse Model of Thioacetamide (TAA) Hepatitis

In a mouse model of thioacetamide (TAA) hepatitis, unconjugated liposomes (fluorescein-containing) and M6PR-conjugated modified liposomes (fluorescein-containing) were injected intravenously into experimental mice, respectively. After 24 hours, fluorescence levels were determined using the IVIS system (see Figure 5). These results suggested that M6PR-conjugated modified liposomes (containing fluorescein) could specifically deliver liposome-loaded fluorescein into the liver.

Synergy assays were performed according to the method described above. Results are shown in FIG. As shown in the figure, the fluorescence intensity in the liver of M6PR in combination with P-sel, gal-3, siglec2, MMR, αLβ2, CD31, annexin V, CD44 or VE-cadherin was significantly higher than M6PR alone. expensive. The above combinations exhibit a synergistic effect.

Solid tumor derived from mouse B16-F10 cell line

Mice were injected subcutaneously at the inguinal site with B16-F10 melanoma cells (1 ^* 10 ⁶ cells/mouse). On days 3 and 8, control (fluorescein-containing) and M6PR-conjugated modified liposomes were injected into the orbital sinus of mice, respectively. Mice were sacrificed on day 12. Tumor fluorescence intensity was monitored using IVIS® Spectrum. Results are shown in FIG. As shown in the figure, the fluorescence intensity of M6PR-conjugated modified liposomes in tumor was significantly higher than that in other organs, indicating that M6PR-conjugated modified liposomes can localize tumor sites. .

Synergy assays were performed according to the method described above. Control liposomes (containing fluorescein), M6PR conjugated liposomes, M6PR-P-sel conjugated liposomes and M6PR-Gal3 conjugated modified liposomes were injected into the orbital sinus of mice, respectively. Mice were sacrificed on day 12. Tumor fluorescence intensity was monitored using IVIS® Spectrum. Results are shown in FIG. As shown in the figure, the fluorescence intensity in tumor of M6PR-conjugated liposomes, M6PR-P-sel conjugated liposomes and M6PR-Gal3-conjugated modified liposomes is significantly higher than that in other organs. Moreover, M6PR-P-sel liposomes and M6PR-Gal3 conjugate modified liposomes show better synergistic potency than M6PR conjugate modified liposomes.

Adipose tissue after injection of anti-fat antibody

Mice on a high-fat diet were injected with control Ig or anti-fat antibody (75 μg/mouse) into the orbital sinus at 0 and 48 hours, respectively. (Fluorescein-containing) control liposomes, M6PR conjugated liposomes, M6PR-P-sel conjugated liposomes and M6PR-Gal3 conjugated liposomes were injected into the orbital sinus of mice at 6 h, 24 h, 54 h and 72 h, respectively. bottom. Mice were sacrificed 96 hours later and white adipose tissue was removed. Tumor fluorescence intensity was monitored using IVIS® Spectrum. Results are shown in FIG. As shown in the figure, fluorescence intensity in adipose tissue of M6PR conjugated liposomes, M6PR-P-sel conjugated liposomes and M6PR-Gal3 conjugated modified liposomes is significantly higher than control. Moreover, M6PR-P-sel liposomes and M6PR-Gal3 conjugate modified liposomes show a better synergistic effect than M6PR conjugate modified liposomes.

Injured tissue contains autophagic and apoptotic cells.

Thioacetamide (TAA) treated mouse liver

In a mouse model of thioacetamide (TAA) hepatitis, unconjugated liposomes (containing fluorescein) and M6PR-conjugated modified liposomes (containing fluorescein) were injected intravenously into the orbital sinus of experimental mice, respectively. Twenty-four hours later, autophagic and apoptotic liver cells were assayed for green fluorescence using the Cyto-ID Autophagy Detection Kit (Enzo Life Sciences) and the CaspGLOW™ Red Active Caspase-3 Staining Kit (BioVision), respectively. Labeled with a dye (GFD). A population containing autophagic and apoptotic liver cells was associated with M6PR-conjugated modified liposomes, which were labeled with the fluorescent dye calcein-red (CR). The percentage of liver cell-liposome associated population (GFD and CR double positive population) was determined using flow cytometry. Levels of liposomes and fluorescent beads associated with unconjugated cells (“unconjugated” group) were normalized to 100% (see FIG. 10).

Solid tumor formed by B16-F10 cells

Mice were injected subcutaneously at the inguinal site with B16-F10 melanoma cells (1 ^* 10 ⁶ cells/mouse). On days 3 and 8, control (fluorescein-containing) and M6PR-conjugated modified liposomes were injected into the orbital sinus of mice. Mice were sacrificed on day 12. Autophagic and apoptotic tumor cells were stained with green fluorescent dye (GFD) using the Cyto-ID Autophagy Detection Kit (Enzo Life Sciences) and the CaspGLOW™ Red Active Caspase-3 Staining Kit (BioVision), respectively. labeled. A population containing autophagic and apoptotic tumor cells was associated with M6PR-conjugated modified liposomes, which were labeled with the fluorescent dye calcein-red (CR). The percentage of liver cell-liposome associated population (GFD and CR double positive population) was determined using flow cytometry. Levels of liposomes and fluorescent beads associated with unconjugated cells (“unconjugated” group) were normalized to 100% (see FIG. 11).

Adipose tissue treated with anti-fat antibody

Mice on a high-fat diet were injected with control Ig or anti-fat antibody (75 μg/mouse) into the orbital sinus at 0 and 48 hours, respectively. (Fluorescein-containing) control liposomes, M6PR conjugated liposomes, M6PR-P-sel conjugated liposomes and M6PR-Gal3 conjugated modified liposomes were injected into the orbital sinus of mice at 6 h, 24 h, 54 h and 72 h, respectively. bottom. Mice were sacrificed 96 hours later and white adipose tissue was removed. Autophagic and apoptotic adipocytes were stained with green fluorescent dye (GFD) using the Cyto-ID Autophagy Detection Kit (Enzo Life Sciences) and the CaspGLOW™ Red Active Caspase-3 Staining Kit (BioVision), respectively. labeled. A population containing autophagic and apoptotic adipocytes was associated with M6PR-conjugated modified liposomes, which were labeled with the fluorescent dye calcein-red (CR). The percentage of liver cell-liposome associated population (GFD and CR double positive population) was determined using flow cytometry. Levels of liposomes and fluorescent beads associated with unconjugated cells (“unconjugated” group) were normalized to 100% (see FIG. 12).

Example 3 Treatment of blocking antibodies, soluble recombinant proteins and soluble M6P can block targeting of M6PR conjugated modified liposomes/microvesicles targeting autophagic and apoptotic cells, as an antidote Mouse B16-F10 cells were suspended for 4 hours and then treated with blocking antibody or soluble M6PR recombinant protein plus additional M6PR+P-selectin conjugated liposomes. Autophagic and apoptotic cells were labeled with green fluorescent dye (GFD) using the Cyto-ID Autophagy Detection Kit (Enzo Life Sciences) and the CaspGLOW™ Red Active Caspase-3 Staining Kit (BioVision), respectively. bottom. Populations containing autophagic and apoptotic cells were associated with M6PR+P-selectin-conjugated liposomes, which were labeled with the fluorescent dye calcein-red (CR). The percentage of the B16-F10-liposome-associated population (GFD and CR double positive population) was determined using flow cytometry. Levels of liposomes and fluorescent beads associated with unconjugated cells (“unconjugated” group) were normalized to 100% (see FIGS. 13 and 14).

Example 4 Liposome Loads (Lipids, DNA, RNA, Proteins, Drugs) Specifically Targeted to Autophagic and Apoptotic Cells in Vitro
Mouse B16-F10 cells were suspended for 4 hours to induce apoptosis, then treated with M6PR-conjugated liposomes loaded with a caspase-3 inhibitor and incubated with serum-free medium. After 24 hours, the percentage of apoptotic cells was determined using flow cytometry (see Figure 17).

表２．Ｍ６ＰＲおよび第２のタンパク質がコンジュゲートされたカスパーゼ－３阻害剤搭載リポソームを例として使用して、ＴＡＡ処置マウスの相乗的レスキューを、低下したＡＬＴレベルによる検出を介して分析した（＋ Ｐ＜０．０５、＋＋ Ｐ＜０．０１）。

Fluorescently labeled DNA, RNA and proteins were prepared using Molecular Probes® labeling chemistry (DNA, RNA and protein labeling kit; ThermoFisher Scientific Co.). Fluorescent DNA, fluorescent RNA and fluorescent protein (BH4 motif of Bcl-xL) were conjugated to or conjugated with a cell penetrating peptide (R811). Delivered to liposomes/MVs by gating (glutaraldehyde; Sigma-Aldrich Co.). These results indicate that M6PR-conjugated liposomes can achieve targeting of DNA-, RNA-, and protein-loaded liposomes to apoptotic cells (see Figure 18).
Table 1. Using single recombinant protein-conjugated caspase-3 inhibitor-loaded liposomes as an example, synergistic rescue of TAA-treated mice was analyzed via detection by reduced ALT levels (+P< 0.05, ++ P<0.01).

Table 2. Using M6PR and a second protein-conjugated caspase-3 inhibitor-loaded liposome as an example, synergistic rescue of TAA-treated mice was analyzed via detection by reduced ALT levels (+P<0 .05, ++ P<0.01).

Example 6 Demonstration of Modified Liposomes Specifically Targeted to Injured Tissue (IVIS) Via Various Protein Conjugates on the Liposome Surface In Vivo M6PR, M6PR + P-selectin in a Mouse Model of Thioacetamide (TAA) Hepatitis Caspase-3 inhibitor-loaded liposomes conjugated with conjugate, M6PR + E-selectin, and M6PR + PSGL-1 were injected intravenously into experimental mice. After 24 hours, plasma aspartate transaminase (AST) levels were analyzed (see Figure 19). These results suggested that selectin-conjugated liposomes could not only be targeted to tissues after injury, but could also carry drugs to treat the target tissue (see Figure 19). .

In a mouse model of thioacetamide (TAA) hepatitis, experimental mice were intravenously injected with M6PR, M6PR+P-selectin, M6PR+E-selectin, and Bcl-2-expressing plasmid-loaded liposomes conjugated with M6PR+PSGL-1. After 24 hours, plasma aspartate transaminase (AST) levels were analyzed (see Figure 20). These results suggested that selectin-conjugated liposomes could not only be targeted to post-injury tissue, but could also carry plasmid DNA to treat the target tissue.

In a mouse model of thioacetamide (TAA) hepatitis, experimental mice were intravenously injected with caspase-3 siRNA-loaded liposomes conjugated with M6PR, M6PR+P-selectin, M6PR+E-selectin, and M6PR+PSGL-1. After 24 hours, plasma alanine aminotransferase (ALT) levels were analyzed (see Figure 21). These results suggested that these protein-conjugated liposomes could not only be targeted to post-injury tissue, but could also deliver RNA to treat the target tissue (see FIG. 21). matter).

In a mouse model of thioacetamide (TAA) hepatitis, experimental mice were intravenously injected with anti-apoptotic Bcl-xL-derived BH4 motif-loaded liposomes conjugated with M6PR, M6PR + P-selectin, M6PR + E-selectin, and M6PR + PSGL-1. . After 24 hours, plasma alanine aminotransferase (ALT) levels were analyzed (see Figure 22).

In a mouse model of thioacetamide (TAA) hepatitis, experimental mice were intravenously injected with caspase-3 inhibitor-loaded liposomes conjugated with M6PR + galectin3, M6PR + P-selectin, Siglec2 + P-selectin, and Siglec2 + galectin3. After 24 hours, plasma alanine aminotransferase (ALT) levels were analyzed (see Figure 23).

Example 7 Targeting CD34+ Cells with Protein-Conjugated Liposomes of the Invention to Sites of Injury and Synergistic Effect of Protein-Conjugated Liposomes on CD34 ⁺ Cell-Mediated Rescue Fluorescent (calcein red) labeled mouse CD34 ⁺ stem cells (1 ×10 ⁷ cells/mouse) were injected intravenously into experimental mice along with M6PR-conjugated liposomes/MV and M6PR+P-sel conjugated liposomes/MV (2.5×10 ⁹ MVs per mouse). Fluorescence levels were determined using the IVIS system (see Figure 24).

In a mouse model of thioacetamide (TAA) hepatitis, mouse CD34 ⁺ stem cells (1×10 ⁷ cells/mouse) were transfected with liposomes/MVs conjugated with M6PR, M6PR + P-selectin, M6PR + E-selectin, and M6PR + PSGL-1 ( Experimental mice were injected intravenously with 2.5×10 ⁹ MVs per mouse). After 24 hours, plasma alanine aminotransferase (ALT) levels were analyzed (see Figure 25).

Example 8 Targeting Anticancer Drugs or Cytostatic Agents to Cancer Cells with Protein-Conjugated Liposomes of the Present Invention Mice were injected subcutaneously at the inguinal site with B16-F10 melanoma cells (1 ^* 10 ⁶ cells/mouse). On days 3 and 8, (MV (0.2 μg containing mitomycin C) and MV (2 μg containing cisplatin) were injected into the orbital sinus of mice, respectively. The size and weight of the tumor were measured (see Figures 26A and 26B).As shown in Figure 26, MVs delivered anti-cancer drugs to tumors and also delivered anti-cancer drugs to tumors. can be delivered into the body, resulting in inhibition or amelioration of tumor growth and reduction in tumor size.

According to the method described above, protein-conjugated modified liposomes (containing doxorubicin) were used as carriers to carry the anti-cancer drug. As shown in FIG. 27, protein-conjugate-modified liposomes (containing doxorubicin) can suppress tumor growth rate (see FIG. 27A) and can also reduce mouse mortality (see FIG. 27A). 27B).

Example 9 Targeting Drugs to Adipose Tissue with Protein-Conjugated Liposomes of the Present Invention Mice on a high-fat diet were injected with control Ig or anti-fat antibody (75 μg/mouse) into the orbital sinus at 0 and 48 hours, respectively. injected. At 6 hours, 24 hours, 54 hours and 72 hours, (MV (containing mitomycin C, 0.2 μg) and MV (containing cisplatin, 2 μg) were injected into the orbital sinus of mice, respectively. Mice were weighed. The results are shown in Figure 28. The results show that MVs (containing mitomycin C, 0.2 μg) can reduce weight gain in mice.

Protein-conjugated modified liposomes (containing doxorubicin) were used in the assay according to the method described above. As shown in Figure 29, protein-conjugated modified liposomes (containing doxorubicin) can reduce the rate of weight gain in mice (Figure 29A). In addition, anti-fat antibodies or (doxorubicin-containing) liposomes would not produce increases in liver function indices (FIG. 29B).

Example 10 Protein-Conjugated Liposomes of the Present Invention Only Mediate Rescue to Injured Tissues Without Loading Additional Drugs/Substances Plasma MVs compared to serum MVs and cell (C6/36)-derived MVs , express relatively higher levels of surface P-selectin than P-selectin. Analyzed by flow cytometry (see Figure 30).

In a mouse model of thioacetamide (TAA) hepatitis, experimental mice were intravenously injected with plasma MVs, serum MVs and cell (C6/36)-derived MVs (2.5×10 ⁹ MVs per mouse). After 24 hours, plasma alanine aminotransferase (ALT) levels were analyzed (see Figure 31).

In a mouse model of thioacetamide (TAA) hepatitis, murine CD34 ⁺ stem cells (1 x 10 ⁷ cells/mouse) were transfected into plasma MVs, serum MVs and cell (C6/36)-derived MVs (2.5 x 10 ⁹ per mouse). MV) were injected intravenously into experimental mice. After 24 hours, plasma alanine aminotransferase (ALT) levels were analyzed (see Figure 32).

Claims (18)

A vesicle conjugated with one or more lectins or fragments thereof, wherein:
said one or more lectins or fragments thereof conjugated to the surface of said vesicle;
said vesicles optionally containing a drug;
said one or more lectins or fragments thereof is mannose-6-phosphate receptor (M6PR), CD22, CD206, galectin 3, annexin V, integrin αLβ2, VE-cadherin, CD300a, thrombospondin 1 (TSP1) and CD36, and fragments thereof . one or more lectins or fragments thereof comprising a second protein selected from the group consisting of CD300a, thrombospondin 1 (TSP1), CD36 and Toll-like receptor 4 (TLR4) and fragments thereof
2. The vesicle of claim 1, wherein is further conjugated to the surface of said vesicle . 3. The vesicle of claim 1 or 2, wherein the agent is encapsulated within the vesicle or attached to the outer surface of the vesicle. A vesicle according to any one of claims 1 to 3, which is a liposome or micelle. A vesicle according to any one of claims 1 to 4 which is artificially modified or derived from cells. A vesicle conjugated with one or more lectins or fragments thereof, wherein:
said one or more lectins or fragments thereof conjugated to the surface of said vesicle;
A vesicle wherein said one or more lectins or fragments thereof is a combination of M6PR and P-selectin, E-selectin, PSGL-1 or galectin-3. A vesicle conjugated with one or more lectins or fragments thereof, wherein:
said one or more lectins or fragments thereof conjugated to the surface of said vesicle;
A vesicle wherein said one or more lectins or fragments thereof are a combination of CD22 and P-selectin, galectin 3 or CD31. A vesicle conjugated with one or more lectins or fragments thereof, wherein:
said one or more lectins or fragments thereof conjugated to the surface of said vesicle;
A vesicle wherein said one or more lectins or fragments thereof are P-selectin or M6PR in combination with TLR4, galectin 3, CLEC2, integrin αLβ2 or CD31. the agent is a diagnostic imaging agent, cell survival enhancer, cell survival inhibitor, cellular component, organelle, cell, cytotoxic agent, anti-tumor drug, toxin, antibody, lipid, protein, DNA, RNA, therapeutic agent or A vesicle according to any one of claims 1 to 8 , which is a nanomaterial. an antagonist of said first protein and said second protein selected from soluble forms, corresponding ligands, neutralizing antibodies and blocking antibodies;
6. Any of claims 2-5 , capable of serving as an antidote for reducing vesicle targeting to autophagic and/or apoptotic cells and tissues containing autophagic and/or apoptotic cells. The vesicle according to item 1. A pharmaceutical composition comprising a vesicle according to any one of claims 1-10 and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising autophagic and/or apoptotic cells and vesicles conjugated with one or more lectins or fragments thereof for targeted drug delivery to tissues containing said cells There is
said one or more lectins or fragments thereof conjugated to the surface of said vesicle;
said one or more lectins or fragments thereof is mannose-6-phosphate receptor (M6PR), P-selectin, E-selectin, L-selectin, P-selectin-ligand-1 (PSGL-1), CD22 , CD206, galectin 3, annexin V, integrin αLβ2, VE-cadherin, CD300a, CD31, CD44, CD47, thrombospondin 1 (TSP1) and CD36, and fragments thereof. an additional agent is administered prior to administering the pharmaceutical composition;
13. The pharmaceutical composition according to claim 12 , wherein said additional agent is an autophagy inducer and/or an apoptosis inducer. 14. The pharmaceutical composition according to claim 12 or 13 , which is used for treating or diagnosing a disease associated with autophagy deregulation. The diseases associated with deregulation of autophagy include trauma, exposure to chemical and physical virulence factors, genetic diseases, aging-related diseases, cardiovascular diseases, infectious diseases, neoplastic diseases, neurodegenerative diseases, metabolic diseases. sexually transmitted disease, aging, obesity, cancer, β-amyloid or α-synuclein or toxicity induced neurodegenerative, myodegenerative conditions, or chronic lung inflammation caused by cystic fibrosis. pharmaceutical composition of 14. The pharmaceutical composition according to claim 12 or 13 , which is used for treating or diagnosing a disease associated with altered apoptosis. The diseases associated with alterations in apoptosis include trauma, exposure to chemical and physical toxic factors, genetic diseases, aging-related diseases, cardiovascular diseases, infectious diseases, neoplastic diseases, neurodegenerative diseases, metabolic diseases. , aging, obesity, cancer, β-amyloid or α-synuclein or toxicity-induced neurodegenerative, muscle degenerative conditions, or chronic pulmonary inflammation caused by cystic fibrosis, or an autoimmune disease. 17. The pharmaceutical composition according to 16 . said neurodegenerative disease is Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis or stroke, said cardiovascular disorder is ischemia, heart failure or an infectious disease, and said autoimmune disease is systemic lupus erythematosus, autoimmune lymphoproliferative syndrome, rheumatoid arthritis or thyroiditis .

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