WO2014054588A1 - Nanogel/exosome complex and dds - Google Patents

Abstract

The present invention addresses the problem of providing a means for introducing an exosome into a cell. This complex, comprising a hydrophobic polysaccharide nanogel and an exosome, is provided as a means to solve such a problem.

Description

Nanogel / exosome complex and DDS

[Cross-reference of related applications]
This application claims priority based on Japanese Patent Application No. 2012-219155 filed on Oct. 1, 2012, the entire disclosure of which is incorporated herein by reference.

The present invention relates to a complex containing an exosome and a drug delivery system (DDS).

Exosomes are vesicles of about 50 to 200 nm derived from endosomes secreted by cells, and their existence has been known since the 1980s. In recent years, messenger RNA (mRNA) and microRNA (miRNA) are contained in this exosome, and it has been found to function as a carrier for carrying nucleic acids and proteins to other cells. It is becoming clear that exosomes containing cell membrane proteins and nucleic acids derived from specific cells are importantly involved in various life phenomena as long-distance intercellular communication pathways. It has been.

It is known that when cells are separated from each other in a living body, the cells exchange information via proteins, low molecular compounds, and the like. In recent years, RNA present in body fluids has attracted attention as a new means for intercellular communication. The presence of RNA in body fluids has been reported for a long time, but its role has been unknown for a long time. In addition, RNA was very unstable in body fluids, so it was not considered to be involved in long-distance cell-to-cell communication.

Recently, it has been reported that mRNA and miRNA exist in exosomes secreted from cells, and a new means of intercellular communication called secreted RNA is becoming clear. RNAs that are encapsulated and transported in exosomes are mainly mRNAs and miRNAs, and it has been discovered that mRNA encapsulated in exosomes is translated in cells that receive exosomes (Non-patent Document 1: Valadi H et al. Nat Cell Biol 2007, 9: 654-659). On the other hand, some reports have been made on the functional expression of exosome-derived miRNAs in recipient cells. Pegtel et al. Found that exosomes secreted from lymphocytes infected with EB virus (Epstein-Barr virus) contain miRNAs derived from EB virus, and mononuclear cells whose miRNAs are not infected with EB virus. It is clarified that it is carried into the derived dendritic cells and suppresses the expression of the target gene (Non-patent Document 2: Pegtel D M et al. Proc Natl Acad Sci USA 2010, 107: 6328-6333). Kosaka et al. Also confirmed that the target miRNA 含 ま was contained in the exosome derived from a kidney cell line that overexpressed a specific miRNA, and miRNA entered the recipient's cell and expressed the target gene. (Non-patent document 3: Kosaka 文献 N et al. J Biol Chem 2010, 285: 17442-17452).

There have been many studies on exosomes and immunity, and among them, many findings have been reported on exosome-mediated interactions between cancer cells and immune cells. An exosome secreted from a cancer cell contains a cancer cell-specific antigen, and induces an antigen-specific cytotoxic T cell via a dendritic cell (Non-patent Document 4: TheryTheC et al. Nat Rev Immunol 2009, 9: 581-593, Non-patent Document 5: Andre F et al. Lancet 2002, 360: 295-305). In particular, many cancer cell-specific membrane proteins and endosome-constituting proteins are contained as cancer antigens, and anti-tumor immunity is induced by incorporating such exosomes into antigen-presenting cells. It has also been clarified that cancer cell-derived exosomes exhibit not only an immunostimulatory effect but also an immunosuppressive effect. For example, T cell apoptosis induction and growth suppression by exosomes derived from cancer cells and body fluids of cancer patients (Non-patent Document 6: Andreola G et al. J Exp Med 2002, 195: 1303-1316), NK cells and T cell cells Inhibition of injury activity (Non-patent document 7: Clayton A et al. Cancer Res 2007, 67: 7458-7466, etc. has been reported, and exosomes secreted from cancer cells exhibit various immunosuppressive effects. It is also thought that it protects cancer cells from antitumor immunity.

As the immunoregulatory function by exosomes becomes clear, the development of new cancer immunotherapy using exosomes is being promoted. Many reports have shown that anti-tumor immunity can be induced by using exosomes secreted from dendritic cells sensitized to cancer antigens, and clinical trials using this type of exosomes have also begun (non-patent literature). 8: Tan A et al. Int J Nanomed 2010, 5: 889-900).

There are not many examples of applying exosomes as drug delivery carriers, but by appropriately selecting cells that secrete exosomes, ligands such as membrane proteins expressed specifically for diseases can also be used as endosomes and Attempts have been made to increase the target directivity by using it as a drug carrier by overexpressing it in the cell membrane and transferring it to the exosome. For example, Zhang et al. Succeeded in causing mouse lymphoma EL-4-derived exosome to contain curcumin, which suppresses the growth of cancer cells, and reaching mouse bone marrow cells (Non-Patent Document 9: Sun D et). al. Mol Ther 2010, 18: 1606-1614). It has been reported that exosomes are taken into recipient cells by various endocytosis and phagocytosis such as receptor / ligand interaction, macropinocytosis, etc. depending on the cell type (Non-Patent Document 10: Escrevent C et al. BMC cancer 2011 11: 108-118).

On the other hand, Erviti et al. Succeeded in sending an exosome loaded with siRNA into the mouse brain (Non-patent Document 11). In order to target the brain, dendritic cells expressing exosome membrane protein (Lamp2b) fused with neuron specific peptide (RVG peptide) were prepared using genetic engineering techniques. It has been reported that siRNA is encapsulated in exosomes collected from the cells using electroporation and administered to mice, and then siRNA is delivered to brain neurons, microglia, etc., and the target gene is knocked down. In addition, it has succeeded in knocking down BACE1, which is a target gene for Alzheimer's disease treatment, and is expected as a new therapeutic method.

On the other hand, we have previously found that hydrophobized polysaccharides in which hydrophobic groups are partially substituted with hydrophobic polysaccharides form aggregates (nanogels) of 20-30 nm in water. It has been found that nanogels have a chaperone function that assists in protein refolding, and that nanogels into which cationic groups have been introduced can introduce proteins and nucleic acids into cells. Cationic nanogels can spontaneously form nano-sized complexes (up to 50 nm) that are easily taken up by cells simply by mixing with the protein to be introduced, and also efficiently release proteins by chaperone function in the cells. This is a major feature not found in other carriers. Recently, siRNA (Non-patent document 12: Toita S et al. Chem. Lett 2009 38: 1114-1115), plasmid DNA (Non-patent document 13: Toita S et al. J Controlled Release 2011 155: 54-59) It has been reported that cationic nanogels are also useful for delivery. The application of nanogels to cancer vaccines is also underway.

Cancer immunotherapy with a cancer vaccine has features that are not available in other treatments, such as fewer side effects and the ability to suppress the growth, recurrence, and metastasis of cancer cells over a long period of time. In vaccine therapy, it is important to efficiently deliver antigen proteins specifically expressed by cancer cells and infected cells to macrophages and dendritic cells. The nanogel could easily encapsulate the antigen protein, and formed stable complex nanoparticles of 50 nm or less. For example, when CHP nanogel encapsulating erbB2 antigen protein as an oncogene product is administered subcutaneously to tumor-bearing mice, not only helper T cells that produce antibodies but also anti-tumor killer T cells are efficiently induced. (Non-Patent Document 14: Ikuta Y et al. Blood 2002 99: 3717-3724). Clinical trials have been conducted since 2004 and its effectiveness has been demonstrated (Non-patent document 15: Uenaka A et al. Cancer Immunity 2007 7: 9-19 Non-patent document 16: Kageyama S et al. Cancer Sci 2008 99: 601-607). Clinical usefulness of CHP nanogel encapsulating NY-ESO-1 protein of esophageal cancer antigen has been suggested, and it is proceeding to clinical trials. Recently, development of a mucosal vaccine utilizing the cell affinity of a cationic nanogel is also underway. Nasal vaccine is considered to be very effective as a preventive vaccine against respiratory infections such as influenza because an antigen-specific immune response can be induced not only in systemic tissues but also in mucosal tissues. On the other hand, the mucosal tissue is usually tightly covered with an epithelial layer, and in order to maximize the effect of nasal vaccine, the development of effective vaccine delivery technology for the upper respiratory tract mucosal immune system has to be developed. It has been considered essential. By embedding the vaccine antigen in a cationic nanogel and administering it intranasally, vaccine antigens such as Clostridium botulinum and tetanus that are highly lethal due to nerve paralysis when infected are effectively delivered to the upper respiratory tract mucosal immune system And succeeded in inducing a high level of protective immune response even in the absence of an adjuvant (Non-patent Document 17: Nochi T et al. Nat Mat 2010 9: 572-578). In addition, in order to confer cytophilicity on hydrophobic polysaccharides, we have developed an RGD-substituted nanogel incorporating an RGD peptide known as a cell adhesion signal and reported that it can be efficiently incorporated into cancer cells (non- Patent Document 18; A. Shimoda, S. Sawada, K. Akiyoshi, Cell specific peptide-conjugated polysaccharide nanogels for protein delivery, Macromol. Bioscience, 11, 882-888 (2011)).

It has also been reported that a hydrophobized polysaccharide nanogel obtained by introducing a hydrophobic molecule into a water-soluble polysaccharide interacts with a liposome composed of a molecular film on a phospholipid, and the surface of the liposome can be coated with the hydrophobized polysaccharide nanogel (Non-patent Document 19). : Kang EC et al. J. Bioact Compat Polym 1997 12: 14-26).

Usually, exosomes extracted and purified from specific cells have been shown to interact and be taken in by specific recipient cells, but do not interact with cells other than the recipient cells. Therefore, when exosomes are taken into any cell other than the original recipient cell, conventionally, the target cell is artificially expressed on the exosome surface by expressing a specific ligand for the cell membrane receptor expressed by the target cell. Strategies have been taken to realize the interaction of exosomes. As a technique for expressing a ligand on the exosome surface, most methods include introducing a fusion gene of a membrane protein and a ligand present on the exosome into an exosome-producing cell by genetic engineering. Due to the difficulty of the genetic engineering operation and the uncertain introduction of the ligand into the exosome inside the production cell, a method for incorporating the exosome into any cell has not been established yet.

Hydrophobized polysaccharide nanogel is described in Patent Document 1, for example.

WO00 / 12564

Valadi H et al. Nat Cell Biol 2007, 9: 654-659 Pegtel D M et al. Proc Natl Acad Sci USA 2010, 107: 6328-6333 Kosaka N et al. J Biol Chem 2010, 285: 17442-17452 Thery C et al. Nat Rev Immunol 2009, 9: 581-593 Andre F et al. Lancet 2002, 360: 295-305 Andreola G et al. J Exp Med 2002, 195: 1303-1316 Clayton A et al. Cancer Res 2007, 67: 7458-7466 Tan A et al. Int J Nanomed 2010, 5: 889-900 Sun D et al. Mol Ther 2010, 18: 1606-1614 Escrevent C et al. BMC cancer 2011 11: 108-118 Erviti L A, Seow Y, Yin H F, Betts C, Lakhal S et al .: Delivery of siRNA to the mouse brain by systemic injection of targeted exosome. Nat Biotechnol 2011, 29: 341-345 Toita S et al. Chem. Lett 2009 38: 1114-1115 S. Toita et al. J Controlled Release 2011 155: 54-59 Ikuta Y et al. Blood 2002 99: 3717-3724 Uenaka A et al. Cancer Immunity 2007 7: 9-19 Kageyama S et al. Cancer Sci 2008 99: 601-607 Nochi T et al. Nat Mat 2010 9: 572-578 A. Shimoda, S. Sawada, K. Akiyoshi, yoshiCell specific peptide-conjugated polysaccharide nanogels for protein delivery, Macromol. Bioscience, 11, 882-888 (2011) Kang EC et al. J. Bioact Compat Polym 1997 12: 14-26

An object of the present invention is to provide a method for incorporating exosomes into arbitrary cells.

The present invention relates to a complex for promoting uptake of exosomes into cells, a method for producing the same, and a method for introducing substances such as drugs and nucleic acids into cells.

Item 1. A complex composed of a hydrophobized polysaccharide nanogel and an exosome.

Item 2. The complex according to Item 1, wherein the hydrophobized polysaccharide nanogel has a polysaccharide portion and a hydrophobic portion.

Item 3. The polysaccharide part of the hydrophobized polysaccharide nanogel is at least one selected from the group consisting of pullulan, amylopectin, amylose, dextran, hydroxyethyl dextran, mannan, levan, inulin, chitin, chitosan, xyloglucan, and water-soluble cellulose. Item 3. The complex according to Item 2, wherein

Item 4. The complex according to Item 2 or 3, wherein the hydrophobic part is a hydrocarbon group or steryl group having 8 to 50 carbon atoms.

Item 5. The complex according to any one of Items 2 to 4, wherein the hydrophobic part is a cholesteryl group.

Item 6. The complex according to any one of Items 1 to 5, wherein the hydrophobized polysaccharide nanogel is cationic.

Item 7. The complex according to Item 6, wherein the hydrophobized polysaccharide nanogel is a cationic cholesterylated pullulan.

Item 8. The complex according to any one of Items 1 to 7, which has an amino group as a cationic group.

Item 9. The complex according to any one of Items 1 to 8, wherein the exosome is an extracellular secretory vesicle having a particle diameter of less than 200 nanometers.

Item 10. The complex according to any one of Items 1 to 8, wherein the exosome comprises a drug or siRNA.

Item 11. A substance-introducing carrier comprising the complex according to any one of Items 1 to 9.

Item 12. An agent for introducing a drug or siRNA comprising the complex according to Item 10.

Item 13. The method for producing a complex according to Item 10, wherein an exosome into which a drug or siRNA or a precursor thereof is introduced into a cell and a hydrophobic polysaccharide nanogel are mixed.

Item 14, The drug or siRNA or a precursor thereof is introduced into the cell, the cell is cultured to obtain an exosome, and the obtained exosome and the hydrophobized polysaccharide nanogel are mixed. A method for producing a composite.

Item 15. The method for producing a complex according to Item 13, wherein the drug or siRNA or a precursor thereof is introduced into the exosome by electroporation, and the obtained exosome and the hydrophobized polysaccharide nanogel are mixed.

According to the present invention, it is possible to impart an uptake function of exosomes to arbitrary cells, and to promote and improve uptake of various physiologically active substances such as nucleic acids, drugs and proteins into cells. In particular, it becomes possible to impart affinity to cells on the exosome surface by coating the exosome surface with a hydrophobic polysaccharide nanogel into which an affinity molecule for cells has been introduced.

According to the present invention, it is possible to introduce exosomes into cells that cannot inherently interact. As a method for imparting functions to exosome membranes, it enables reliable functionalization of the exosome membrane surface, which is impossible with genetic engineering techniques and probabilistic introduction of functionally modified membrane proteins into exosomes. The function to be imparted can be controlled by the functional molecule introduced into the nanogel.

CHP-NH ₂ Nanogel / exosome complex particle size measurement result of complex formation. Confocal laser microscope image of HeLa cells incorporating CHP-NH ₂ nanogel / CFSE-labeled exosome complex (a) CFSE-labeled exosome only (b) CHP-NH ₂ nanogel / CFSE-labeled exosome complex. The figure which reversed the color in the upper stage of FIG. 2A. Confocal laser microscope image of RAW264.7 cells incorporating CHP-NH ₂ nanogel / CFSE-labeled exosome complex (a) CFSE-labeled exosome only (b) CHP-NH ₂ nanogel / CFSE-labeled exosome complex. The figure which reversed the color in the upper stage of FIG. 3A. Intracellular introduction experiment of siRNA using CHP-NH ₂ nanogel / exosome complex (CT26 cells, CMS5 cells). (A) CHP-NH ₂ (+), exosome (+), siRNA (-). (B) CHP-NH ₂ (−), exosome (+), siRNA (+). (C) CHP-NH ₂ (+), exosome (+), siRNA (+) Intracellular introduction experiment of siRNA by CHP-NH ₂ nanogel / exosome complex (4T1 cells, K562 cells). (A) CHP-NH ₂ (+), exosome (+), siRNA (-). (B) CHP-NH ₂ (−), exosome (+), siRNA (+). (C) CHP-NH ₂ (+), exosome (+), siRNA (+) Intracellular introduction experiment of siRNA using CHP-NH ₂ nanogel / exosome complex (RAW264.7 cells). (A) CHP-NH ₂ (+), exosome (+), siRNA (-). (B) CHP-NH ₂ (−), exosome (+), siRNA (+). (C) CHP-NH ₂ (+), exosome (+), siRNA (+) An example of the hydrophobic nanogel of this invention is shown. 1: Cholesteryl group (1-5%), 2: Pullulan, 3: Hydrophobic nanogel (particle size ˜50 nm), p: Self-assembly. An example of the hydrophobic nanogel of this invention is shown. 1: cholesterol, 2: amino group, 3: pullulan, 4: cCHP nanogel (cationic nanogel), p: self-association. An example of the hydrophobic nanogel of this invention is shown. An example of the composite_body | complex of this invention is shown. 1: cationic nanogel, 2: exosome, 3: cationic nanogel / exosome complex, 4: endosome, 5: nucleus, 6: cytoplasm, p: introduction of a drug into the inner aqueous phase (for example, fluorescent dye as a model drug ), Q: complexation by electrostatic interaction, r: intracellular introduction by cationic nanogel, s: promotion of intracellular uptake of exosomes, t: membrane fusion of exosomes. Intracellular introduction of CHP-NH ₂ nanogel / exosome complex. 1: Exosome (eg, derived from K562) 2: CFSE-Exosome (CFSE-labeled exosome) 3: CHP-NH ₂ nanogel 4: CFSE-labeled exosome / CFSE-Exosome (CHP-NH ₂ nanogel / CFSE Labeled exosome complex), 5: cells (eg, HeLa, RAW264.7), p: CFSE staining (37 ° C., 4 h (4 hours)) and gel filtration purification, q: complexation (on ice) 30 min (30 minutes)), r: administration, s: 3 h (3 hours), t: 1 h (1 hour), i: Lysotracker (Red) added, ii: confocal microscopy.

4A, 4B, and 5, the horizontal axis indicates the fluorescence intensity of AF488 (Alexa-Fluor 488) and represents the amount of siRNA introduced. The vertical axis indicates the number of cells into which siRNA has been introduced. On the horizontal axis, the amount of siRNA introduced (fluorescence intensity) is small toward the left and increases toward the right. In the vertical axis, the number of cells is small toward the lower side and larger toward the upper side.

In one embodiment, the present invention provides a complex composed of a hydrophobized polysaccharide nanogel and an exosome.

The exosome in the present invention widely includes vesicles released from cells. The diameter of the exosome is about 30 to 200 nm, preferably about 30 to 100 nm, and includes phospholipids, lipids such as cholesterol, proteins, and the like. The exosome may be derived from any animal or plant species as long as it produces it. Examples of the animal species include vertebrates (eg, human, mouse, rat, monkey, dog, cat, cow, horse, pig, rat, mouse, hamster, rabbit, goat, chicken, salmon, tuna, etc.). However, preferably, when a target cell into which a physiologically active substance such as a drug or nucleic acid is introduced is derived from the same animal, and the target cell is an in vivo cell, a vesicle derived from the same animal as the target cell preferable. The cell type from which the exosome is derived is not particularly limited. For example, tumor cells, dendritic cells, macrophages, T cells, B cells, platelets, reticulocytes, epithelial cells, fibroblasts, stem cells, iPS cells, etc. Various types of cells can be mentioned. Exosomes can also be prepared from various body fluids such as blood, urine, ascites.

The hydrophobized polysaccharide nanogel used in the present invention is known and disclosed in Patent Document 1, for example.

The hydrophobized polysaccharide nanogel has a polysaccharide portion and a hydrophobic portion, and these are linked directly or via an appropriate linker group. The polysaccharide part should just be what combined 2 or more types of 1 type, or 2 or more types of monosaccharide molecules, such as glucose, galactose, mannose, and fructose. The polysaccharide moiety may have a modifying group such as hydroxymethyl, hydroxyethyl, hydroxypropyl, carboxymethyl. Specific examples of the polysaccharide moiety include pullulan, amylopectin, amylose, dextran, hydroxyethyl dextran, mannan, levan, inulin, chitin, chitosan, xyloglucan, water-soluble cellulose and the like. Among them, pullulan, amylopectin, amylose, dextran, hydroxyethyl dextran and the like which are homopolysaccharides composed of α-glucose are preferable, and pullulan is particularly preferable. The polysaccharide part may be a single polysaccharide or a combination of two or more polysaccharides.

Examples of the hydrophobic moiety include a hydrocarbon group or steryl group having 8 to 50 carbon atoms (may be 12 to 50 carbon atoms), preferably a steryl group, and particularly preferably a cholesteryl group. The linker group includes an ester bond (—COO— or —O—CO—), an ether group (—O—), an amide group (—CONH— or —NHCO—), a urethane bond (—NHCOO— or —OCONH). -), And a combination of one or more of them, and further combination of an alkylene group having 1 to 10 carbon atoms, an arylene group, and an aralkylene group (benzylene group, phenethylene group, etc.) as necessary. Good.

The hydrophobic part is about 0.1 to 20%, preferably about 0.3 to 15%, more preferably about 0.5 to 10%, particularly about 1 to 5% of the polysaccharide part by weight.

Hydrophobized polysaccharide nanogel can be manufactured according to the method of patent document 1, for example. Specifically, a hydroxyl group-containing hydrocarbon or sterol having 8 to 50 carbon atoms (or 12 to 50 carbon atoms) and OCN-R ¹ -NCO (where R ¹ is a hydrocarbon having 1 to 50 carbon atoms) A first reaction for producing an isocyanate group-containing hydrophobic compound in which one molecule of a hydroxyl group-containing hydrocarbon or cholesterol having 12 to 50 carbon atoms is reacted with a diisocyanate compound represented by the following formula: Further, the isocyanate group-containing hydrophobic compound obtained in the first step reaction is further reacted with a polysaccharide to produce a hydrocarbon group having 8 to 50 carbon atoms or a steryl group as the hydrophobic group. A method including a second reaction step may be mentioned. In this case, the reaction product of the second stage reaction can be purified with a ketone solvent to produce a highly pure hydrophobic group-containing polysaccharide (hydrophobic polysaccharide nanogel). (Hereafter, it may be described as “Method A”.)

The hydrophobized polysaccharide nanogel preferably has a cationic property. This is because cationic hydrophobic polysaccharides bind to exosomes that originally have anionic properties based on electrostatic interactions in addition to hydrophobic interactions. In order to make the hydrophobic polysaccharide nanogel cationic, a cationic amino acid (Lys, Arg, His) or a polymer thereof may be linked to the polysaccharide moiety, and an amino group, monoalkylamino group, dialkylamino group A cationic group such as an imino group, an ammonium group, a guanidino group, or an amidino group may be introduced into the hydrophobized polysaccharide nanogel. The introduction of the cationic group includes, for example, a cationic group via a C1-C10 alkylene group, arylene group, aralkylene group (benzylene group, phenethylene group, etc.), or a linker group as exemplified above. This can be done by introducing an appropriate functional group into the polysaccharide moiety. Alternatively, other cationic functional groups such as polyethyleneimine may be introduced.

Suitable hydrophobized polysaccharide nanogels are cholesterylated pullulan (CHP-NH ₂ , 1 to 10 cholesteryl groups, preferably 1 to several per 100 monosaccharides of pullulan having multiple cationic groups (preferably amino groups) 1 to 50, preferably 5 to 30 cationic groups (preferably amino groups) are introduced). The cholesteryl group is introduced into pullulan via a linker group (preferably a urethane bond).

As a method for introducing a cationic group (preferably an amino group) into the nanogel, the following method (hereinafter sometimes referred to as “Method B”) is preferably exemplified. Method B is described in the literature H. Ayame, N. Morimoto, motoand K. Akiyoshi, Self-assembled cationic nanogels for intracellular protein delivery system, Bioconjugate Chem., 19, 882-890 (2008).

1 g of cholesterol-dried pullulan (CHP) and 0.035 g of dimethylaminopyridine (DMAP) dried under reduced pressure were dissolved in 20 ml of a mixed solvent of dimethyl sulfoxide (DMSO) / pyridine (v: v = 1: 1). Slowly add 0.25 g / ml 4-nitrophenyl chloroformate / (DMSO / pyridine (v: v = 1: 1) mixed solvent) and stir for 4 hours. This is reprecipitated using a mixed solvent of ethanol / diethyl ether (v: v = 1: 1). The collected precipitate is dissolved in 300 ml of DMSO / pyridine (v: v = 1: 1) mixed solvent and slowly dissolved in 10 ml of 28.5% ethylenediamine / (DMSO / pyridine (v: v = 1: 1) mixed solvent). And stir for 4 days. This is reprecipitated using a mixed solvent of ethanol / diethyl ether (v: v = 1: 1). The precipitate is dried under vacuum, dissolved in 200 ml DMSO and dialyzed against distilled water. Further, dialyzed with 1N NaOH aqueous solution, neutralized with HCl, and further dialyzed with distilled water. This is lyophilized to give a milky white solid. NH ₂ Specific examples of the amino group are preferably exemplified. The number of substituents to be introduced can be appropriately changed. By changing the number of substituents to be introduced, it is possible to control the magnitude of the positive charge and to control the efficiency of incorporation of the complex into cells. Various cationic groups can be introduced by using other compounds having a cationic group instead of ethylenediamine.

The properties of the hydrophobized polysaccharide nanogel can be changed by changing the amount of hydrophobic substitution, such as cholesterol, depending on the size of the polysaccharide and the degree of hydrophobicity of the introduced hydrophobic portion. In order to control the hydrophobicity, it is also suitable to introduce an alkyl group having 10 to 30 carbon atoms, preferably about 12 to 20 carbon atoms, instead of the cholesteryl group or together with the cholesteryl group. The nanogel used in the present invention has an average particle diameter (diameter) of 5 to 200 nm, preferably 10 to 40 nm, more preferably 20 to 30 nm. Nanogels have already been widely marketed, and in the present invention, these commercially available products can be widely used.

In the present specification, the average particle diameter (diameter) can be measured by, for example, a dynamic light scattering method (DLS, Dynamic light scattering).

The ratio between the hydrophobized polysaccharide nanogel and the exosome particles is about 10: 1 to 1: 2. Hydrophobized polysaccharide nanogel and exosome form a complex by combining these particles at an appropriate ratio as shown in FIG. 7, for example.

The complex of the present invention can contain a physiologically active substance such as a drug or siRNA.

Since siRNA can be produced by chemical synthesis, in addition to natural siRNA, in order to improve intracellular stability (chemical and / or enzyme) and specific activity (affinity with target RNA) In addition, siRNA subjected to various chemical modifications can be included. For example, in order to prevent degradation by a hydrolase such as nuclease, a phosphate residue (phosphate) of each nucleotide constituting a nucleic acid is changed to a chemically modified phosphate such as phosphorothioate (PS), methylphosphonate, phosphorodithionate, etc. It can be substituted with a residue. In addition, the 2′-position hydroxyl group of each nucleotide sugar (ribose) is represented by —OR (R is, for example, CH ₃ (2′-O-Me), CH ₂ CH ₂ OCH ₃ (2′-O-MOE) CH ₂ CH ₂ NHC (NH) NH ₂ , CH ₂ CONHCH ₃ , CH ₂ CH ₂ CN and the like may be substituted). Furthermore, the base moiety (pyrimidine, purine) may be chemically modified, for example, introduction of a methyl group or a cationic functional group at the 5-position of the pyrimidine base, or substitution of the carbonyl group at the 2-position with thiocarbonyl. Is mentioned. Furthermore, although the phosphoric acid part and the hydroxyl part can be modified with biotin, amino group, lower alkylamine group, acetyl group, etc., it is not limited thereto.

The siRNA is a nucleotide sequence homologous to the nucleotide sequence of the target gene mRNA or the initial transcript or a partial sequence thereof (preferably within the coding region) (including an intron in the case of the initial transcript) and a complementary sequence thereof. This is a single-stranded oligo RNA. The length of a portion homologous to the target nucleotide sequence contained in siRNA is usually about 18 bases or more, for example, about 20 bases (typically about 21 to 23 bases) in length. Is not particularly limited as long as it can cause The total length of siRNA is usually about 18 bases or more, for example, about 20 bases (typically about 21 to 23 bases in length), but is not particularly limited as long as it can cause RNA interference. .

The relationship between the target nucleotide sequence and the sequence homologous to that contained in the siRNA may be 100% identical or may have base mutations (at least 70%, preferably 80%, more preferably 90%, most preferably within 95% identity range).

SiRNA may have an additional base at the 5 'or 3' end of 5 bases or less, preferably 2 bases, which does not form a base pair. The additional base may be DNA or RNA, but the use of DNA can improve the stability of siRNA. Examples of such additional base sequences include ug-3 ', uu-3', tg-3 ', tt-3', ggg-3 ', guuu-3', gttt-3 ', ttttt-3 Examples of the sequence include ', uuuuu-3', but are not limited thereto.

The siRNA may be directed to any target gene. However, when the nucleic acid introduction agent of the present invention is used as a prophylactic / therapeutic agent for diseases, siRNA encapsulated in exosomes has an enhanced expression of the target disease. It is preferable that the target is a gene involved in exacerbation, and more specifically, the antisense nucleic acid for the gene is a clinically advanced or preclinical stage gene or a newly known gene And the like.

SiRNA may be used alone or in combination of two or more.

The drug contained in the complex of the present invention is not particularly limited, and an antitumor agent is preferably exemplified. Antitumor agents include hormonal therapeutic agents (eg, phosfestol, diethylstilbestrol, chlorotrianiserin, medroxyprogesterone acetate, megestrol acetate, chlormadinone acetate, cyproterone acetate, danazol, allylestrenol, gestrinone. , Mepartricin, raloxifene, olmeloxifen, levormeloxifene, antiestrogens (eg, tamoxifen citrate, toremifene citrate, etc.), pill formulations, mepithiostane, testrolactone, aminoglutethimide, LH-RH agonists (eg, goserelin acetate) , Buserelin, leuprorelin, etc.), droloxifene, epithiostanol, ethinyl estradiol sulfonate, aromatase inhibitors (eg, fadrozole hydrochloride, anastrozo) , Letrozole, exemestane, borozole, formestane, etc.), antiandrogens (eg, flutamide, bicalutamide, nilutamide, etc.), 5α-reductase inhibitors (eg, finasteride, epristeride, etc.), corticosteroids (eg, dexamethasone) , Prednisolone, betamethasone, triamcinolone, etc.), androgen synthesis inhibitors (eg, abiraterone), retinoids and drugs that slow the metabolism of retinoids (eg, riarosol), among others, LH-RH agonists (eg, goserelin acetate) , Buserelin, leuprorelin, etc.)), alkylating agents (eg, nitrogen mustard, nitrogen mustard hydrochloride-N-oxide, chlorambutyl, cyclophosphamide, ifosfamide, Thiotepa, carbocon, improsulfan tosylate, busulfan, nimustine hydrochloride, mitobronitol, melphalan, dacarbazine, ranimustine, sodium estramsine phosphate, triethylenemelamine, carmustine, lomustine, streptozocin, pipobroman, etoglucid, carboplatin, cisplatin, Miboplatin, nedaplatin, oxaliplatin, altretamine, ambermustine, dibrospium hydrochloride, fotemustine, prednimustine, pumitepa, ribomustine, temozolomide, treosulphane, trophosphamide, dinostatin timamarer, carbocon, adzelesin, systemustin, biselecin) Antimetabolites (eg mercaptopurine, 6-mercaptopurine riboside, thioino , Methotrexate, enositabine, cytarabine, cytarabine ocphosphate, ancitabine hydrochloride, 5-FU drugs (eg, fluorouracil, tegafur, UFT, doxyfluridine, carmofur, garocitabine, emiteful, etc.), aminopterin, leucovorin calcium, tabloid Folinate calcium, levofolinate calcium, cladribine, emitefur, fludarabine, gemcitabine, hydroxycarbamide, pentostatin, piritrexim, idoxyuridine, mitoguazone, thiazofurin, ambmustine), anticancer antibiotics (eg, actinomycin D, actinomycin C, Mitomycin C, chromomycin A3, bleomycin hydrochloride, bleomycin sulfate, pepromy sulfate , Daunorubicin hydrochloride, doxorubicin hydrochloride, aclarubicin hydrochloride, pirarubicin hydrochloride, epirubicin hydrochloride, neocalcinostatin, misramicin, sarcomomycin, carcinophylline, mitotane, zorubicin hydrochloride, mitoxantrone hydrochloride, idarubicin hydrochloride), plant-derived anticancer agents (for example, , Etoposide, etoposide phosphate, vinblastine sulfate, vincristine sulfate, vindesine sulfate, teniposide, paclitaxel, docetaxel, vinorelbine, camptothecin, irinotecan hydrochloride, immunotherapeutic agent (BRM) (eg, picibanil, crestin, schizophyllan, fenibran, lentinone, ubenix Interleukin, macrophage colony stimulating factor, granulocyte colony stimulating factor, erythropoietin, lymphotoxin, BCG vaccine, Corynebacterium parvum, levamisole, polysaccharide K, procodazole), agents that inhibit the action of cell growth factors and their receptors (eg, trastuzumab (Herceptin ™; anti-HER2 antibody), ZD1839 (Iressa) And antibody drugs such as Gleevec). The types of cancer that are targeted by anti-tumor agents are colorectal cancer, liver cancer, kidney cancer, head and neck cancer, esophageal cancer, stomach cancer, biliary tract cancer, gallbladder / bile duct cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer. Cervical cancer, endometrial cancer, bladder cancer, prostate cancer, testicular tumor, bone / soft tissue sarcoma, leukemia, malignant lymphoma, multiple myeloma, skin cancer, brain tumor, etc., preferably colorectal cancer, stomach cancer , Head and neck cancer, lung cancer, breast cancer, pancreatic cancer, biliary tract cancer, liver cancer.

These drugs may be used alone or in combination of two or more.

By coating the exosome with a cell affinity molecule-introduced nanogel, strong cell affinity can be imparted to the exosome. Cytophilic molecules can be selected according to their use. When nanogels with cationic molecules are used, exosome-cell interactions due to electrostatic interactions with the cell membrane surface, regardless of cell type Can be promoted. It is also possible to prepare exosomes that interact specifically with target cells by introducing peptides, protein fragments, antibodies and the like into nanogels. For example, there is an RGD-substituted nanogel into which an RGD peptide known as a cell adhesion signal described above is introduced. A. Shimoda, S. Sawada, K. Akiyoshi, Cell specific peptide-conjugated polysaccharide nanogels for protein delivery, Macromol. Bioscience, 11, 882-888 (2011). In this method, basically, by mixing the exosome and the nanogel, the nanogel can be complexed and the interaction function with the cell can be imparted in a short time regardless of the origin of the exosome. Therefore, in the past, from the selection of cells to genetic modification and functional analysis of exosomes, it was possible not only to dramatically shorten the operation time, which took several months to confirm the promotion of interaction with cells, but also There may be cases where the modification itself is not necessary.

The complex of the present invention can be obtained by mixing exosome and hydrophobic polysaccharide nanogel in water. The obtained complex can be separated and recovered by centrifugation or the like. The exosome and the hydrophobized polysaccharide nanogel are mixed in such a ratio that part or all of the surface of the exosome is covered with the hydrophobized polysaccharide nanogel. When the hydrophobized polysaccharide nanogel is cationic, the charge of the complex is reduced. The ratio of both is adjusted so as to be anionic / cationic.

When the complex has a drug or siRNA or a precursor of the drug or siRNA, the complex of the present invention is formed by mixing the exosome introduced with the drug or siRNA or its precursor and the hydrophobic polysaccharide nanogel. be able to. An exosome into which a drug or siRNA or a precursor thereof has been introduced (that is, an exosome in which the drug or siRNA or a precursor thereof is encapsulated) is, for example, in one embodiment, a drug or siRNA or a ribosome in a cell that produces exosomes. After the precursor is introduced, exosomes in which these are encapsulated can be produced. The drug or siRNA or precursor thereof may be one that a cell producing exosome introduces itself into the cell (for example, synthesis, uptake from outside the cell, etc.). In another aspect, exosomes can be obtained by introducing drugs or siRNA or precursors thereof into exosomes by an appropriate method such as electroporation. Alternatively, the drug or siRNA or precursor thereof can be introduced after the complex is formed.

The complex of the present invention can be suitably used as a carrier for introducing a substance into cells. When the complex of the present invention contains a physiologically active substance such as a drug or siRNA, the complex of the present invention can be suitably used as an introduction agent for a drug or siRNA.

The target to which a drug, siRNA or the like is introduced may be a cell or a living body. Examples of the target cells include cells derived from humans or non-human animals such as mice, particularly cultured cells. Examples of living organisms to be used include humans; mammals such as monkeys, mice, rats, rabbits, cows, pigs, sheep and horses; birds such as chickens; and non-human animals such as fish.

When a target to which a drug, siRNA, or the like is to be introduced is a cell, it can be used as a carrier for introducing the substance into the cell or as an introducing agent by including an appropriate amount of the complex of the present invention in the medium.

When an object into which a drug, siRNA or the like is introduced is a living body such as a human, the introduction agent of the present invention is preferably used in a dosage form such as an injection, an eye drop, a nasal drop, an inhalant, a suppository. In this case, the introduction agent of the present invention is suitably provided as a pharmaceutical composition, and introduction agents of various dosage forms can be obtained by using a commonly used appropriate carrier. The daily dose of the introduction agent of the present invention for an adult can be appropriately set according to the drug to be introduced, siRNA and the like, but is about 0.1 to 1 mg / day, preferably about 0.5 to 1 mg / day. It can be selected from a range of about 500 mg.

The present invention also provides introduction of a physiologically active substance such as a drug or siRNA using the complex of the present invention into a cell.

Hereinafter, although the present invention will be described in more detail with reference to examples, it goes without saying that the present invention is not limited to the examples.

Example 1
<Experiment method>
1) Preparation of cationic nanogel solution In this experiment, cationic hydrophobized pullulan (CHP-) was introduced into pullulan, a linear water-soluble polysaccharide, by introducing 1.2 cholesterol per 100 monosaccharides and 15 amino groups per 100 monosaccharides. NH ₂ ) was dissolved in PBS and treated with a probe-type ultrasonic irradiator to prepare and use a CHP-NH ₂ nanogel solution. A hydrophobized pullulan was produced by the above method A, and an amino group was introduced into the hydrophobized pullulan by the above method B to obtain a cationic hydrophobized pullulan (CHP-NH ₂ ).

2) Exosome purification from cell culture supernatant K562 cells (human leukemia cell line) at a concentration of 1.0 to 2.0 × 10 ⁶ cells / mL, exosome-free medium (medium from which fetal bovine serum (FBS) -derived exosomes have been removed) After culturing for 15 hours, the cell suspension was recovered and centrifuged at 1000 rpm for 10 minutes, and then the supernatant was recovered and further centrifuged at 12000 g at 4 ° C. for 30 minutes. The supernatant was collected and centrifuged at 100,000 g at 4 ° C. for 2 hours. The supernatant was discarded, 5 mL of phosphate buffered saline (PBS) was added, and the mixture was centrifuged at 100,000 g at 4 ° C. for 2 hours. After centrifugation, the supernatant was discarded and the precipitate was resuspended with PBS to obtain a K562-derived exosome suspension.

RAW264.7 cells (mouse macrophage cell line) were suspended in 2 L of exosome-free medium at a concentration of 1 × 10 ⁶ cells / ml and cultured for 18-24 hours. The cell suspension was recovered, and 400 g, After centrifugation at 4 ° C for 10 minutes, the supernatant was recovered and further centrifuged at 10000 g and 4 ° C for 15 minutes. The supernatant was filtered through a filter having a pore size of 0.45 μm and a pore size of 0.22 μm, and then concentrated to 180 ml using an ultrafiltration membrane. The concentrated supernatant was passed through a filter having a pore size of 0.8 μm, and then centrifuged at 100000 g at 4 ° C. for 2 hours. The supernatant was removed by suction with an aspirator, and the precipitate containing exosomes was washed with PBS and centrifuged at 100,000 g for 2 hours at 4 ° C. After centrifugation, the supernatant was removed by suction with an aspirator, and the precipitate was resuspended in 200 μl of PBS to obtain a RAW264.7-derived exosome suspension. The exosome suspension was stored at 4 ° C. until use.

The protein concentration of the exosome was quantified using Micro BCA ^™ Protein Assay Kit manufactured by Thermo Scientific.

3) Binding reaction between latex beads and exosomes for flow cytometry (FCM) analysis After thoroughly mixing 4 μm diameter aldehyde / sulfate latex beads (Invitrogen), 1.4 mg of beads were collected, 3000 g, 4 ° C, The supernatant was removed after centrifugation for 20 minutes. The precipitated beads were suspended in 2- (N-Morpholino) ethanesulfonic acid (MES) buffer. This centrifugation and suspending operation was repeated three times to wash the beads, and then adjusted to a concentration of 20 mg / ml with MES buffer. The bead suspension was mixed well by vortexing and sonication, and then 0.2 mg of the bead suspension was collected, and 30 μg of RAW264.7-derived exosome suspension was gradually added in terms of protein amount. The reaction was allowed to proceed for 15 minutes at room temperature with stirring. MES buffer was added to a total volume of 1 ml, and the mixture was reacted at room temperature for 2 hours with stirring. The reaction was stopped by adding 300 μl of a 400 mM glycine solution and stirred for another 30 minutes. Centrifugation was performed at 3000 g and 4 ° C. for 20 minutes to precipitate the beads, and the supernatant was removed. The beads were suspended in PBS supplemented with 2% FCS (exosome-free). Centrifugation and suspension operations were repeated three times to wash the beads, and then stained with an antibody against a membrane protein expressed in an exome and analyzed by FCM.

4) Preparation of complex of exosome and cationic nanogel 30 μL of exosome suspension with a protein concentration of 100 μg / mL is mixed with 30 μL of CHP-NH ₂ nanogel solution (0 to 1.0 mg / mL) at 4 ° C. The mixture was allowed to stand for 30 minutes to form a composite, and the particle size was measured with a dynamic light scatterometer (DLS).

5) Intracellular evaluation of cationic nanogel / exosome complex 150,000 mL / mL of HeLa cell (human cervical cancer cell line) and RAW264.7 cell (mouse macrophage-like cell line) suspension was glass bottom dish. And precultured for 24 hours. 2 μL of 2 mg / mL fluorescent dye (CFSE (carboxyfluorescein diacetate succinimidyl ester) solution) was added to 100 μL of K562-derived exosome suspension with a protein concentration of 300 μg / mL, and the mixture was allowed to stand at 37 ° C. for 4 hours. Replace the buffer with PBS through PD SpinTrap G-25, mix 50 μL of the recovered CFSE-labeled exosome solution with 50 μL of cationic nanogel (CHP-NH ₂ ) solution (1 mg / mL), and combine at 4 ° C for 30 minutes Made it. Add 100 μL of the complexed solution to the cells, and after 3 hours, add 1 μL of intracellular endosome staining reagent (LysoTracker (registered trademark) Red DND-99) to the cells. Washed and observed with a confocal laser microscope. FIG. 8 schematically shows the experimental method.

10 μg of protein exosome and Alexa Fluor 488 (AF488) -labeled siRNA Qiagen, AllStars Negative Control siRNA (Alexa Fluor 488 modified), model number: 1027292) were added to 100 μl of PBS. SiRNA was introduced into RAW264.7-derived exosomes by adding to a 2-mm gap cuvette and electroporating with NEPA21 (Neppagene). Electroporation conditions are: poring pulse: voltage 100 V, pulse width 1 ms, pulse interval 50 ms, 2 times, attenuation 10%, polarity), transfer pulse: voltage 20 V, pulse width 50 ms, pulse interval 50 ms , 5 times, attenuation rate 40%, polarity +/-. The siRNA-introduced exosome was centrifuged at 100,000 g, 4 ° C. for 2 hours, precipitated, and then suspended in PBS. The exome was washed by repeating the operation of centrifuging and suspending twice in total, and suspended in 100 μl of PBS. The same volume of CHP-NH ₂ nanogel solution (50 μg / ml) was added to this, and complexed at 4 ° C. for 30 minutes. Then, 100 μl of the complexed solution is added to 150 μl of cell solution (1 × 10 ⁴ CT26 mouse colon cancer cell line, CMS5 mouse fibrosarcoma cell line, 4T1 mouse breast cancer cell line, K562 human leukemia cell line, or 2 × 10 ⁴ cells Incorporation of AF488-labeled siRNA into cells after 4 hours was examined by FCM analysis.

<Results and discussion>
It was obtained by mixing CHP-NH ₂ nanogel (particle size 38 nm) and K562-derived exosomes (particle size 145 nm) at various concentrations (“CHP-NH ₂ final concentration” in FIG. 1, 0 to 1.0 mg / mL). CHP-NH ₂ nanogel / exosomes results of particle size of the composite was measured by DLS, CHP-NH ₂ nanogel concentration particle diameter 0.05 mg / mL or more to form a CHP-NH ₂ nanogel / exosome complex of about 180nm (Fig. 1).

The CHP-NH ₂ nanogel / CFSE-labeled exosome complex prepared on the basis of the above results was added to the HeLa cell culture system and observed with a confocal laser microscope. Almost no exosome was observed (FIG. 2 (a), CFSE). On the other hand, under the condition where the CHP-NH ₂ nanogel / CFSE-labeled exosome complex was added, green fluorescence derived from CFSE-labeled exosome was observed in almost all cells (FIG. 2 (b), CFSE). In addition, when the intracellular localization was examined, exosomes existing in endosomes exhibiting yellow fluorescence were confirmed, but it was confirmed that most of the incorporated exosomes were present in the cytoplasmic part. It was shown that the incorporated exosome moves from the endosome to the cytoplasm. From these results, it was clarified that the cellular uptake of exosomes was promoted by complexing with CHP-NH ₂ nanogel. Next, when examining exosome uptake in RAW264.7 cells of different cell types, it was observed that even exosome alone was taken up into the cell, and no difference due to complexation with CHP-NH ₂ nanogel was confirmed. . This is RAW264.7 cells macrophage-like cells, phagocytosis is believed to be due to easily capture a strong extracellular material (FIG. 3 (a), exosomes only; (b) CHP-NH ₂ Nanogel / CFSE Labeled exosome complex).

CHP-NH ₂ nanogel / exosome complex containing AF488-labeled siRNA was added to CT26 cells, CMS5 cells, 4T1 cells or K562 cells, and uptake of siRNA into each cell after 4 hours was examined by FCM analysis. In all these cells, siRNA added using CHP-NH ₂ nanogel / exosome was taken up by the cell at a much higher rate than siRNA added using exosome alone (FIG. 4). On the other hand, in RAW264.7 cells, there was no difference in cellular uptake between siRNA added using CHP-NH ₂ nanogel / exosome and siRNA added using exosome alone (FIG. 5). This was thought to be because RAW264.7 cells are macrophage-like cells and have a strong phagocytosis and easily take up extracellular substances. The above results confirmed that exosomes complexed with CHP-NH ₂ nanogel deliver siRNA efficiently to cells in many cell types, especially cells with low phagocytosis.

The complex of the present invention can be used as a drug delivery system (DDS) because physiologically active substances such as drugs and nucleic acids can be introduced into cells. In addition, by introducing a labeling substance into the complex, it can be applied to bioimaging. Furthermore, it can also be used as a vaccine by introducing an antigen into the complex.

Claims (15)

A complex composed of hydrophobic polysaccharide nanogel and exosomes. The complex according to claim 1, wherein the hydrophobized polysaccharide nanogel has a polysaccharide portion and a hydrophobic portion. The polysaccharide part of the hydrophobized polysaccharide nanogel is at least one selected from the group consisting of pullulan, amylopectin, amylose, dextran, hydroxyethyldextran, mannan, levan, inulin, chitin, chitosan, xyloglucan, and water-soluble cellulose Item 3. The complex according to Item 2. The composite according to claim 2 or 3, wherein the hydrophobic part is a hydrocarbon group or a steryl group having 8 to 50 carbon atoms. The complex according to any one of claims 2 to 4, wherein the hydrophobic portion is a cholesteryl group. The complex according to any one of claims 1 to 5, wherein the hydrophobized polysaccharide nanogel is cationic. The complex according to claim 6, wherein the hydrophobized polysaccharide nanogel is a cationic cholesterylated pullulan. The complex according to any one of claims 1 to 7, which has an amino group as a cationic group. The complex according to any one of claims 1 to 8, wherein the exosome is an extracellular secretory vesicle having a particle diameter of less than 200 nanometers. The complex according to any one of claims 1 to 8, wherein the exosome comprises a drug or siRNA. A substance introduction carrier comprising the complex according to any one of claims 1 to 9. An agent for introducing a drug or siRNA comprising the complex according to claim 10. The method for producing a complex according to claim 10, wherein exosomes into which a drug or siRNA or a precursor thereof is introduced into a cell and a hydrophobic polysaccharide nanogel are mixed. 14. The complex according to claim 13, wherein a drug or siRNA or a precursor thereof is introduced into a cell, the cell is cultured to obtain an exosome, and the obtained exosome and the hydrophobized polysaccharide nanogel are mixed. Manufacturing method. 14. The method for producing a complex according to claim 13, wherein a drug or siRNA or a precursor thereof is introduced into the exosome by electroporation, and the obtained exosome and the hydrophobized polysaccharide nanogel are mixed.

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