Delivery system and preparation method and application thereof

Abstract

The present disclosure provides a delivery system for a pharmaceutically active ingredient, as well as a preparation method and use of the delivery system. The delivery system comprises lipid nanoparticles and extracellular vesicles, and the lipid nanoparticles and the extracellular vesicles form a chimeric structure through fusion polypeptide. The delivery system disclosed by the invention has the characteristics of high delivery efficiency, low immunogenicity, capability of efficiently loading mRNA and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and in particular relates to a delivery system for active pharmaceutical ingredients, and a preparation method and application of the delivery system. Background Art

[0002] The two key factors for the success of mRNA vaccines are breakthroughs in mRNA modification and vectors. mRNA needs to be packaged in a vector to avoid degradation by nucleases, and to pass through the cell membrane to release mRNA into the cytoplasm for translation. Currently, the carrier of mRNA vaccines is lipid nanoparticles (LNP). LNP is mainly composed of four lipids, including cholesterol, phospholipids, PEG derivatives and ionizable lipids.

[0003] mRNA shows great promise as a nucleic acid drug (such as in protein replacement therapy, cell therapy, and antibody therapy). The protein level that mRNA needs to produce as a nucleic acid drug may need to be 100-1000 times higher than that of mRNA vaccines, because vaccines can amplify immune signals through the body's immune response. In addition, mRNA nucleic acid drugs require repeated administration. However, LNP delivery efficiency is low, and only 1-2% of mRNA can be released into the cytoplasm for translation. Moreover, LNP has poor extrahepatic targeting, more than 80% of mRNA is enriched in the liver, and LNP has strong immunogenicity and toxic side effects, which are not suitable for repeated long-term administration. Therefore, it is necessary to develop a new mRNA delivery system with high delivery efficiency, low immunogenicity and small toxic side effects. Summary of the invention

[0004] In order to solve the technical problems of low delivery efficiency, high cytotoxicity and immunogenicity of existing LNP, the present invention uses fusion polypeptides to make the shell of lipid nanoparticles and the membrane of extracellular vesicles (EV) interlock, so as to form an interlocking structure between EV and LNP, and obtain a biomimetic EV-LNP delivery system. This system has the characteristics of high delivery efficiency, low immunogenicity, and efficient loading of active pharmaceutical ingredients.

[0005] In one aspect, the present disclosure provides a delivery system comprising lipid nanoparticles and extracellular vesicles, wherein the lipid nanoparticles and the extracellular vesicles form a chimeric structure by fusing polypeptides.

[0006] In some embodiments, the shell of the lipid nanoparticle partially forms a chimeric structure with the membrane of the extracellular vesicle.

[0007] In some embodiments, the fusion polypeptide is coupled to the outer surface of the lipid nanoparticle.

[0008] In some embodiments, the fusion polypeptide is linked to a polyethylene glycol (PEG) derivative on the outer surface of the lipid nanoparticle, such as DMG-PEG2000.

[0009] In some embodiments, the fusion polypeptide is an amphipathic polypeptide having 10-30 amino acids.

[0010] In some embodiments, the secondary structure of the fusion polypeptide has at least one of an alpha helix, a beta fold (beta sheet), a beta turn or a random coil, preferably an alpha helix.

[0011] In the present application, the fusion polypeptide is fixed on the surface of the nanoliposome by chemical coupling or the like, and then incubated with the extracellular vesicles. The fusion polypeptide is inserted into the membrane of the extracellular vesicle with its unique amphipathicity and spontaneous conformations such as α-helix without destroying the membrane structure of the extracellular vesicle. At the same time, the length of the fusion polypeptide is comparable to the membrane thickness of the extracellular vesicle, so that the outer shell of the lipid nanoparticle and the membrane of the extracellular vesicle are chimeric at the insertion point, but not fused. This delivery system with a special chimeric structure retains the basic morphology and function of the nanoliposome and the extracellular vesicle, and has the characteristics of high delivery efficiency, low immunogenicity, and the ability to efficiently load active drug ingredients.

[0012] The fusion polypeptide has amphipathicity, folds into an alpha helix or other spontaneous conformations, or has a polypeptide bend introduced by proline / glycine, and can promote fusion with the extracellular vesicle membrane or pore opening.

[0013] In some embodiments, the fusion polypeptide can spontaneously insert into the phospholipid bilayer structure in an α-helix or α-helix-bend-α-helix conformation and stably maintain the inserted form.

[0014] In some specific embodiments, the fusion polypeptide has an amino acid sequence shown in at least one of SEQ ID NO: 1 (LAKSWGRALKR), SEQ ID NO: 2 (VARALGRAIAKSIKR), SEQ ID NO: 3 (IARSLGKSEGVALKK), and SEQ ID NO: 4 (LAKALGKAEGVALSKVKR).

[0015] In some embodiments, the extracellular vesicles are extracellular vesicles obtained from at least one of pluripotent stem cells, mesenchymal stem cells, ectoderm-derived cells, mesoderm-derived cells, and endoderm-derived cells.

[0016] In some embodiments, the extracellular vesicles can be secreted into the culture medium during adherent / suspension cell culture, or sourced from body fluids such as blood and plasma, and further purified by centrifugation, chromatography, and the like.

[0017] In some embodiments, the lipid nanoparticles include steroids, neutral lipids, PEG derivatives, and ionizable lipids.

[0018] In some embodiments, based on the total molar amount of the lipid nanoparticles, the molar percentage of the steroidal compound is 20%-50%, the molar percentage of the neutral lipid is 10-40%, the molar percentage of the PEG derivative is 1-20%, and the molar percentage of the ionizable lipid is 20-65%.

[0019] In some embodiments, the steroidal compound includes but is not limited to at least one of cholesterol, coprosterol, non-sterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, brassicasterol, tomatine, tomatine, ursolic acid, α-tocopherol, corticosteroids and derivatives thereof.

[0020] In some embodiments, the neutral lipids include, but are not limited to, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DM ... At least one of 1-stearoyl-sn-glycero-3-phosphoethanolamine (DOPE), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin (SM), ceramide, sterols and their derivatives.

[0021] In some embodiments, the PEG derivatives include but are not limited to at least one of phosphatidylethanolamine modified with polyethylene glycol, phosphatidic acid modified with polyethylene glycol, ceramide modified with polyethylene glycol, dialkylamine modified with polyethylene glycol, diacylglycerol modified with polyethylene glycol, and dialkylglycerol modified with polyethylene glycol. Preferably, the PEG derivatives include at least one of DMG-PEG2000, ALC-0159 or DSPE-PEG2000.

[0022] In some embodiments, the ionizable lipid is a type of lipid that is almost uncharged under normal neutral physiological pH conditions but positively charged under acidic pH, and is roughly composed of an amine portion and a lipid portion, and the cationic amine portion and the polyanionic nucleic acid electrostatically interact to form a positively charged liposome or lipid membrane structure. In some embodiments, the ionizable lipid includes but is not limited to 4-dimethylaminobutyric acid dilinoleyl methyl ester (DLin-MC3-DMA), ALC0315, SM102, 1,1-((2-(4-(2-((2-(di(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazine-1-yl)ethyl)azanediyl)di(dodecan-2-ol) (C12-200) or The compounds shown in the figure, wherein: RCOO is selected from myristoyl, α-D-tocopheryl succinyl, linoleoyl and oleoyl; and X is selected from

[0023] In some embodiments, the lipid nanoparticles include cholesterol, phospholipids, PEG derivatives and ionizable lipids. In some specific embodiments, the phospholipids are selected from DOPE (1,2 dioleoyl-sn-glycero-3-phosphoethanolamine), the PEG derivative is DMG-PEG2000, and the ionizable lipid is ionizable lipid SM-102.

[0024] In another aspect, the present disclosure provides a method for preparing a delivery system, the method comprising:

[0025] (1) coupling the fusion polypeptide to the outer surface of lipid nanoparticles to obtain lipid nanoparticles coupled with the fusion polypeptide;

[0026] (2) Mixing the lipid nanoparticles coupled with the fusion polypeptide and the extracellular vesicles.

[0027] In some embodiments, in step (1), the fusion polypeptide is coupled to the PEG derivative of the lipid nanoparticle by chemical coupling.

[0028] In some embodiments, in step (1), a solution containing a PEG derivative and a fusion polypeptide is incubated, wherein the PEG derivative and the fusion polypeptide respectively have one of the chemical coupling linker pair members, and the polypeptide-PEG derivative is used as one of the raw materials for preparing lipid nanoparticles to obtain lipid nanoparticles containing the fusion polypeptide on the surface, and the ionizable lipids are contained inside the lipid nanoparticles.

[0029] In some embodiments, the molar ratio of the fusion polypeptide to the PEG derivative is (2-100): 1, preferably (2-10): 1. In some specific embodiments, the molar ratio of the fusion polypeptide to the PEG derivative can be 2: 1, 4: 1, 6: 1, 8: 1, 10: 1, 15: 1, 20: 1, 40: 1, 60: 1, 80: 1, 100: 1 or any value therebetween.

[0030] In some embodiments, the incubation temperature is 0-20° C., preferably 4-6° C. In some embodiments, the incubation temperature can be 0° C., 2° C., 4° C., 6° C., 8° C., 10° C., 15° C., 20° C. or any value therebetween.

[0031] In some embodiments, the incubation time is 2-20 hours, preferably 6-12 hours.

[0032] In some embodiments, in step (2), the molar ratio of the lipid nanoparticles coupled with the fusion polypeptide to the extracellular vesicles is 1:(1-10), preferably 1:(1-2). In some specific embodiments, the molar ratio of the lipid nanoparticles coupled with the fusion polypeptide to the extracellular vesicles can be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 or any value therebetween.

[0033] In some embodiments, the temperature for incubating the lipid nanoparticles coupled with the fusion polypeptide and the extracellular vesicles is 0-20° C., preferably 4-6° C. In some embodiments, the incubation temperature can be 0° C., 2° C., 4° C., 6° C., 8° C., 10° C., 15° C., 20° C. or any value therebetween.

[0034] In some embodiments, the lipid nanoparticles coupled with the fusion polypeptide and the extracellular vesicles are incubated for 0.5-5 h, preferably 0.5-1 h.

[0035] In some embodiments, the chemical coupling linker pairing members are paired chemical linkers, such as amine-reactive linkers, thiol-reactive linkers, or click chemistry linkers. Amine-reactive linkers can be linkers containing succinimidyl ester groups. Thiol-reactive linkers can be linkers containing maleimide groups. Click chemistry linkers can be linkers containing click chemistry functional groups, such as NHS-azide linkers, NHS-DBCO linkers, maleimide-azide linkers, or maleimide-DBCO linkers.

[0036] In some embodiments, step (1) further comprises mixing the lipid nanoparticles coupled with the fusion polypeptide with a solution containing a pharmaceutically active ingredient, and encapsulating the pharmaceutically active ingredient inside the lipid nanoparticles.

[0037] In yet another aspect, the present disclosure provides a pharmaceutical composition, comprising the above-mentioned delivery system or the delivery system prepared by the above-mentioned preparation method, and a pharmaceutically active ingredient.

[0038] In some embodiments, the pharmaceutically active ingredient is selected from nucleic acids, small molecule compounds, polypeptides and / or proteins.

[0039] In some embodiments, the nucleic acid is selected from the group consisting of DNA, RNA, mRNA, siRNA, rRNA, tRNA, snRNA, miRNA, plasmid, and combinations thereof.

[0040] In some embodiments, the nucleic acid is selected from mRNA.

[0041] In some embodiments, the pharmaceutically active ingredient is encapsulated inside the lipid nanoparticles.

[0042] In some embodiments, the method for preparing the pharmaceutical composition comprises the following steps:

[0043] (1) chemically coupling the fusion polypeptide into the PEG derivative of the lipid nanoparticle to obtain a lipid nanoparticle connected with the fusion polypeptide;

[0044] (2) connecting the active pharmaceutical ingredient to the lipid nanoparticles to obtain lipid nanoparticles connected with the active pharmaceutical ingredient;

[0045] (3) Mixing the extracellular vesicles with the lipid nanoparticles connected with the active pharmaceutical ingredient obtained in step (2).

[0046] In another aspect, the present disclosure provides use of the above-mentioned delivery system or the delivery system prepared by the above-mentioned preparation method in the delivery of active pharmaceutical ingredients.

[0047] In some embodiments, the pharmaceutically active ingredient is selected from nucleic acids, small molecule compounds, polypeptides and / or proteins.

[0048] In some embodiments, the nucleic acid is selected from DNA, RNA, mRNA, siRNA, rRNA, tRNA, snRNA, miRNA, plasmid and combinations thereof. In some embodiments, the pharmaceutically active ingredient is mRNA.

[0049] Compared with the prior art, the delivery system disclosed in the present invention has the following beneficial effects:

[0050] (1) The delivery efficiency is 1-2 orders of magnitude higher than that of existing LNP delivery systems;

[0051] (2) lower cytotoxicity and immunogenicity than existing LNP delivery systems;

[0052] (3) Easy to genetically engineer to target different tissues and organs. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 A schematic diagram of an embodiment of the present application for preparing an extracellular vesicle-lipid nanoparticle delivery system is shown, and lipid nanoparticles, extracellular vesicles, and a chimeric structure produced by the fusion of the two taken by a transmission electron microscope are also shown.

[0054] Figure 2 Nanoflow cytometric analysis of the labeling efficiency of extracellular vesicles with lipid nanoparticles is shown.

[0055] Figure 3 The fusion efficiency of lipid nanoparticles coupled with fusion polypeptides and lipid nanoparticles not coupled with fusion polypeptides with extracellular vesicles is shown.

[0056] Figure 4 The delivery efficiency of GFP-mRNA by different delivery systems is shown.

[0057] Figure 5 The chimeric delivery system obtained using different fusion polypeptides is shown to deliver the expression of GFP-mRNA in cells. DETAILED DESCRIPTION

[0058] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. The specific implementation methods described herein are only used to explain the present invention and are not intended to constitute any limitation to the present invention.

[0059] Extracellular vesicles (EV) are secreted autonomously by cells, can carry a variety of proteins and nucleic acids, and target the functions of target cells and tissues and organs. It has low immunogenicity, good tolerance for repeated administration, and is suitable as a small molecule or nucleic acid drug delivery vehicle. At present, there is a mature technology for mass production of extracellular vesicles, but drug delivery into vesicles still relies on traditional electroporation technology, ultrasound, extrusion or repeated freezing and thawing, which is inefficient and damages vesicle integrity. Therefore, the present disclosure provides a novel LNP and EV delivery system for mRNA vaccine and drug delivery. The delivery system disclosed in the present disclosure is 10-100 times more efficient than the existing LNP system in delivery efficiency, and has lower immunogenicity and cytotoxicity, and can efficiently load mRNA.

[0060] Specifically, see Figure 1 The present invention discloses a lipid nanoparticle-extracellular vesicle (LNP-EV) delivery system prepared by a method comprising the following steps:

[0061] (1) Screening a fusion polypeptide that can promote lipid fusion,

[0062] (2) The screened fusion polypeptide is chemically coupled into the PEG derivative of LNP,

[0063] (3) Synthesizing LNPs encapsulated with active pharmaceutical ingredients (e.g., mRNA),

[0064] (4) Isolation and extraction of extracellular vesicles,

[0065] (5) The extracellular vesicles of step (4) are mixed with the LNP of step (3) to form an LNP-EV delivery system.

[0066] The formed LNP-EV delivery system was observed by transmission electron microscopy, and it can be seen that the lipid nanoparticles and extracellular vesicles are fused to form a chimeric structure ( Figure 1 shown).

[0067] the term

[0068] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0069] The term "nucleic acid" includes one or more of the following types: polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base or a modified purine or pyrimidine base (including abasic sites). The term "nucleic acid", as used herein, also includes covalently bonded polymers of ribonucleosides or deoxyribonucleosides, the covalent bonding usually being through phosphodiester bonds between subunits, but in some cases through phosphorothioate, methylphosphonate, etc. "Nucleic acid" includes single-stranded and double-stranded DNA and single-stranded and double-stranded RNA. Exemplary nucleic acids include, but are not limited to, gDNA; hnRNA; mRNA; rRNA, tRNA, microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), and small sequential RNA (stRNA), etc., and any combination thereof.

[0070] The term "amphiphilic polypeptide" is a peptide molecule that has both a hydrophilic peptide sequence and a hydrophobic alkyl chain, also known as an amphiphilic polypeptide or a peptide amphiphile. An amphiphilic polypeptide generally comprises a hydrophilic peptide sequence and a hydrophobic alkyl chain attached to a lipid tail.

[0071] The secondary structure of a protein refers to the periodic structural conformation of the main chain atoms of a polypeptide chain arranged in one-dimensional direction under the action of hydrogen bonds. This is the local spatial conformation of the peptide chain and does not involve the side chain atoms of amino acids. There are many secondary structures of proteins, which can be mainly divided into α helix, β fold (β fold sheet), β turn, random coil, etc. α helix refers to the regular spiral rise of the main chain of the polypeptide chain around the central axis. Its structural characteristics are: ① The main chain skeleton forms a right-handed structure around the central axis; ② Each turn of the spiral is 3.6 amino acid residues, and the pitch is 0.54nm; ③ Hydrogen bonds are formed between adjacent spiral turns; ④ Side chain groups are located on the outside of the spiral. β fold: There are generally two forms of folding, one is parallel and the other is naturally antiparallel. Parallel β fold means that adjacent peptide chains are in the same direction, and antiparallel is in the opposite direction; its structural characteristics are: ① Several peptide chains or peptide segments are arranged in parallel or antiparallel sheets; ② All peptide bonds C=O and NH form interchain hydrogen bonds; ③ Side chain groups are alternately located above and below the sheet. β-turn often occurs at the corner when the polypeptide chain turns back 180°. It is usually composed of 4 amino acid residues, and forms a tight loop with the help of hydrogen bonds between the first and fourth residues. Random coil refers to the conformation formed by the irregular arrangement of the main chain skeleton, and also refers to those polypeptide regions that cannot be classified into a clear secondary structure. It often appears between α-helix and α-helix, α-helix and β-fold, and β-fold and β-fold.

[0072] The term "fusion polypeptide" relates to a protein comprising at least a first amino acid chain genetically linked to at least a second amino acid chain. Thus, a fusion polypeptide may comprise a multimer of peptides expressed as a single, linear polypeptide. It may comprise 1, 2, 3, 4 or even more peptides. The term "fusion" refers to components that are linked by peptide bonds (directly or through a peptide linker).

[0073] The term "chemical coupling" refers to coupling of a peptide to another substance (such as a second protein or non-protein moiety) by a chemical reaction method, including substitution (e.g., N-succinimidyl chemistry), addition or cycloaddition (e.g., maleimide chemistry or click chemistry) or oxidation chemistry (e.g., disulfide formation). These chemical reaction methods activate the carboxyl or amino group with a given compound and then covalently bond it to the amino or carboxyl group. In some embodiments, the fusion polypeptide is chemically coupled to the PEG derivative by covalent bond interactions (chemical linkers, such as amine-reactive linkers, thiol-reactive linkers, or click chemistry linkers). The amine-reactive linker can be a linker containing a succinimidyl ester group. The thiol-reactive linker can be a linker containing a maleimide group. The click chemistry linker can be a linker containing a click chemistry functional group, such as an NHS-azide linker, an NHS-DBCO linker, a maleimide-azide linker, or a maleimide-DBCO linker.

[0074] The term "chimeric structure" refers to the insertion of a fusion polypeptide into a phospholipid bilayer structure, so that the outer shell of the lipid nanoparticle and the membrane of the extracellular vesicle are chimeric at the insertion point but not fused, thereby forming an overall delivery system of the lipid nanoparticle and the extracellular vesicle, but at the same time retaining the basic morphology and function of the nanoliposome and the extracellular vesicle.

[0075] The term "delivery vector" can be used to prepare and deliver active ingredients using any method, including small molecule compounds and various nucleic acids to perform specific functions in vivo. For example, mRNA is delivered for in vivo production of proteins, such as antibodies, or antigens, or antibody fragments or antigen fragments, etc. In some embodiments, mRNA is encapsulated in a transfer vector, such as a nanoparticle. In addition, one purpose of this encapsulation is usually to protect nucleic acids from the effects of an environment that may contain enzymes or chemicals that degrade nucleic acids and / or cause rapid excretion of nucleic acids or receptors, and to promote cell uptake and expression of corresponding sequences. In some embodiments, the transport vector is a liposome vesicle or other means to promote nucleic acid transfer to target cells and tissues. Suitable transport vectors may include, but are not limited to, liposomes, lipid nanoparticles, lipid nanoparticles containing ceramide, proteoliposomes, nanoparticles, calcium phosphate-silicate nanoparticles, calcium phosphate nanoparticles, silica nanoparticles, nanocrystalline particles, semiconductor nanoparticles, poly (D-arginine), nano dendrimers, starch-based delivery systems, micelles, emulsions, vesicles, plasmids, viruses, calcium phosphate nucleotides, aptamers, peptides, and other carrier tags. Bioion capsules and other viral capsid protein assemblies are also contemplated as suitable transfer vehicles.

[0076] Extracellular vesicles (EVs) refer to vesicles with double-layer membrane structures that fall off from the cell membrane or are secreted by cells, with diameters ranging from 30nm to 1,000nm. Extracellular vesicles mainly include microvesicles (MVs) and exosomes (Exo). Microvesicles are small vesicles that fall off from the cell membrane after cell activation, damage or apoptosis, with a diameter of about 200 to 1000nm. Exosomes are released to the outside of the cell in the form of exocytosis after the fusion of multivesicular bodies (multivesicular bodies) in the cell with the cell membrane, with a diameter of about 30 to 200nm. Extracellular vesicles are widely present in cell culture supernatants and various body fluids (blood, lymph, saliva, urine, semen, and milk), carrying a variety of cell-derived proteins, lipids, DNA, mRNA, miRNA, etc., and are involved in processes such as intercellular communication, cell migration, angiogenesis, and immune regulation.

[0077] Extracellular vesicles have the characteristics of natural biocompatibility, high delivery efficiency, low toxicity and low immunogenicity, and are emerging drug delivery carriers. Engineered extracellular vesicles are usually designed by genetically modifying their donor cells. In some aspects, the extracellular vesicles described in the present disclosure are also loaded with a payload of interest, that is, the extracellular vesicles encapsulate substances such as proteins, nucleic acids, polypeptides, small molecules, etc. The payload is an exogenous or endogenous payload. The payload has the purpose of preventing and / or treating diseases. Specifically, the nucleic acid is selected from the group consisting of antisense oligonucleotides, siRNA, miRNA, mRNA, gRNA or plasmids. The polypeptides, proteins and small molecules are selected from drugs for preventing and / or treating multiple tissue and organ damage repair and regeneration, tumor diseases, or drugs or regulators for promoting embryonic development and / or stem cell proliferation.

[0078] In the present application, extracellular vesicles are derived from extracellular vesicles of cells of various properties. In some embodiments, extracellular vesicles are derived from stem cells, for example, derived from pluripotent stem cells, mesenchymal stem cells, ectoderm-derived cells, mesoderm-derived cells and endoderm-derived cells, etc. Pluripotent stem cells are cells that may have the ability to differentiate into various tissues in the body, particularly cells that can differentiate into all endoderm, mesoderm and ectoderm. Such cells include embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells). Mesenchymal stem cells are stem cells with the ability to mainly differentiate into mesoderm-derived tissues and part of ectoderm-derived tissues and endoderm-derived tissues. Cells of ectoderm origin include keratinized epithelial cells, stratified epithelial cells, inner hair cells of the organ of Corti, outer hair cells of the organ of Corti, basal cells of the olfactory epithelium, cold sensory neurons, heat sensory neurons, epidermal Merkel cells, olfactory sensory neurons, pain sensory neurons, retinal photoreceptor cells, deep sensory neurons, touch sensory neurons, type I carotid body cells, type II carotid body cells, vestibular type I hair cells, vestibular type II hair cells, type I taste bud cells, autonomic neurons, inner column cells of the organ of Corti, outer column cells of the organ of Corti, inner phalanx cells of the organ of Corti, outer phalanx cells of the organ of Corti, marginal cells of the organ of Corti, vestibular supporting cells, taste bud supporting cells, olfactory epithelium supporting cells, satellite cells, enteric glial cells, central nervous system neurons and glial cells, lens cells, etc.Mesoderm-derived cells include hepatocytes, adipocytes, Ito cells, pararenal cells, glomerular epithelial cells, proximal tubular brush border cells, thin segment cells of Henle's loop, distal tubular cells, collecting duct cells, type I alveolar epithelial cells, central acinar cells, smooth muscle duct cells, duct cells, intestinal brush border cells, exocrine gland striatal duct cells, gallbladder epithelial cells, efferent duct non-ciliated cells, epididymal chief cells, epididymal basal cells, ameloblasts, semilunar epithelial cells, corti interdental epithelial cells, loose connective tissue fibroblasts, corneal fibroblasts, tendon fibroblasts, bone marrow reticular connective tissue fibroblasts, other non-epithelial fibroblasts, pericytes, intervertebral disc nucleus pulposus cells, cementoblasts, odontoblasts, hyaline cartilage chondrocytes, fibrochondrocytes, elastic chondrocytes, osteoblasts, osteoprogenitor cells, Vitreous vitreous cells, ear astrocytes, pancreatic astrocytes, contractile cells, skeletal muscle cells, muscle satellite cells, cardiomyocytes, smooth muscle cells, iris myoepithelial cells, exocrine gland myoepithelial cells, megakaryocytes, monocytes, connective tissue macrophages, epithelial Langerhans cells, osteoclasts, dendritic cells, microglia, neutrophils, eosinophils, basophils, hybridoma cells, mast cells, Th2 cells, regulatory T cells, cytotoxic T cells, natural killer T cells, B cells, natural killer cells, reticulocytes, blood and immune cell system stem cells and progenitor cells, erythroblasts, bone marrow cells, oocytes, spermatocytes, spermatogonia, nurse cells, ovarian follicular cells, supporting cells, thymic epithelial cells, interstitial kidney cells and any cultured cells derived therefrom, etc. Cells of endoderm origin include salivary gland mucous cells, mammary gland cells, lacrimal gland cells, ear canal gland cells, exocrine glands, gland dark cells, exocrine clear cells, apocrine gland cells, ciliated gland cells, sebaceous gland cells, duodenal cells, seminal vesicle cells, prostate cells, glomerular urethral gland cells, urethral gland cells, endometrial cells, goblet cells, gastric mucosal cells, gastric chief cells, parietal cells, pancreatic acinar cells, small intestinal Paneth cells, lung type II pneumocytes, lung Clara cells, anterior pituitary cells, melanocyte stimulating hormone producing cells, large neurosecretory neurons, thyroid cells, parathyroid cells, adrenal cells, interstitial cells, internal capsule cells, corpus luteum cells, juxtaglomerular cells, macula densa cells, peripheral polar cells, glomerular mesangial cells, and any cells derived from these cultured cells. When obtaining extracellular vesicles from these cells, the cells are cultured in a normal culture medium generally used for cell culture for 1 to 30 days, the culture medium is collected, and if necessary, further purified by centrifugation, chromatography (e.g., gel filtration chromatography, column chromatography, size exclusion chromatography, phosphatidylserine affinity chromatography and other conventional column chromatography), flow cytometry and the like.

[0079] Lipid nanoparticle (LNP) has the structure that comprises single monolayer or double-layer lipid that compound is encapsulated in solid phase.Different from liposome, lipid nanoparticle does not have aqueous phase or other liquid phases in its inside, but directly and internal compound are compounded from the lipid of double-layer or single-layer shell, thereby it is encapsulated in solid core.Lipid nanoparticle is normally the spherical vesicle with relatively uniform shape and size distribution.It is generally believed that the diameter of lipid nanoparticle can be in the range of 10nm to 1000nm.

[0080] For lipid nanoparticle nucleic acid delivery systems, the lipid shell can be formulated to contain ionizable ionizable lipids that can be compounded and associated with the negatively charged backbone of the nucleic acid core. At physiological pH, the lipid nanoparticle can adopt a relatively neutral exterior, thereby significantly increasing the circulation half-life of the particle after intravenous administration. In the context of nucleic acid delivery, lipid nanoparticles have many advantages over other lipid-based nucleic acid delivery systems, including high nucleic acid encapsulation efficiency, potent transfection, improved penetration into tissues to deliver therapeutic agents, and low levels of cytotoxicity and immunogenicity.

[0081] The pharmaceutical composition can be formulated for administration via a specific route of administration. For example, the pharmaceutical composition can be formulated for intravenous, intratumoral, intraperitoneal, intradermal, subcutaneous, intranasal or other routes of administration.

[0082] As used herein, the term "delivery" encompasses both local delivery and systemic delivery. For example, the delivery of mRNA encompasses situations where mRNA is delivered to a target tissue and the encoded protein is expressed and retained in the target tissue (also referred to as "local distribution" or "local delivery"), and situations where mRNA is delivered to a target tissue and the encoded protein is expressed and secreted into the patient's circulatory system (e.g., serum), and systemically distributed and absorbed by other tissues (also referred to as "systemic delivery"). "Local delivery" refers to tissue-specific delivery or distribution. Typically, local delivery requires that a protein or peptide encoded by mRNA be translated and expressed in a cell or with limited secretion, which avoids entering the patient's circulatory system. The term "target tissue" refers to any tissue affected by the disease to be treated. In certain embodiments, the target tissue includes those tissues that display disease-related pathology, symptoms, or features.

[0083] The following are preferred embodiments of the present invention, and the present invention is not limited to the following preferred embodiments. It should be noted that for those skilled in the art, several modifications and improvements made on the basis of this invention and creative conception all belong to the protection scope of the present invention. The reagents used without indicating the manufacturer are all conventional products that can be obtained through commercial purchase.

[0084] Example 1

[0085] (1) Preparation of lipid nanoparticles (LNP)

[0086] Ionizable lipids can be protonated under acidic conditions to form ionizable lipids, which bind to negatively charged nucleic acids such as mRNA through electrostatic interaction to form lipid nanoparticles (LNPs) loaded with mRNA. In this experiment, a microfluidic mixing method was used to allow the lipid solution and the mRNA solution to fully and quickly form LNPs with uniform particle size in a micromixer. Since lipids are dissolved in ethanol and nucleic acids are dissolved in acidic buffers, dialysis or ultrafiltration is required to remove residual ethanol and replace the solution system with a suitable storage buffer (such as PBS).

[0087] Experimental materials (taking SM-102 ionizable lipid as an example):

[0088] Reagents: ionizable lipid SM-102, neutral auxiliary phospholipid DOPE, cholesterol (Chol), PEGylated lipid DMG-PEG2000, mRNA, citric acid, sodium citrate, diethyl pyrocarbonate (DEPC), anhydrous ethanol, ultrapure water, 75% alcohol.

[0089] Consumables: sterile syringes, sterile centrifuge tubes, sterile pipette tips, microfluidic chips, dispensing needles, Tygon hoses, stainless steel connectors, dialysis bags / boxes (3k / 10k / 20k), ultrafiltration tubes (100Kd).

[0090] Equipment: two syringe pumps, clean bench, magnetic stirrer, low-temperature centrifuge.

[0091] Experimental steps:

[0092] 1. Prepare lipid-ethanol solution, taking SM102-LNP as an example (refer to Table 1 below):

[0093] Table 1

[0094] SM-102 DOPE Chol DMG-PEG2000 Molecular weight 710.17 744.03 386.65 2509.2 Mole Percent 50％ 10％ 38.5％ 1.5％ 8mM system 4mM 0.8mM 3.08mM 0.12mM Mass concentration (mg / mL) 2.84 0.60 1.19 0.30 Stock solution (mg / mL) 50 10 10 30 To prepare 250μL, add (μL) 14.2 14.9 29.8 2.5

[0095] Note: If fluorescent labeling of LNP is required, lipophilic fluorescent dye (such as DiD, final concentration 10ug / mL) can be added to the lipid-ethanol solution.

[0096] 2. Prepare mRNA-acidic aqueous solution

[0097] In this example, green fluorescent protein mRNA (the mRNA is shown in SEQ ID NO: 7) was used as an example to prepare mRNA-LNPs samples.

[0098] The required mRNA mass was calculated based on N / P=6 and FRR=3 (the average molecular weight of mRNA nucleotides is 339, and each nucleotide contains one P atom. The ionizable lipid contains one N atom).

[0099] Use ultrapure water to prepare 50.0 mL of 100 mM citric acid and sodium citrate solutions respectively. Take 33.0 mL of citric acid solution and 17.0 mL of sodium citrate solution, mix and add diethyl pyrocarbonate (DEPC), let stand for 30 minutes and then sterilize by high pressure to remove DEPC. After sterilization, use DEPC-treated ultrapure water to make up to 100 mL to obtain 50 mM pH = 4 citric acid buffer. Dilute mRNA to the required concentration using citric acid buffer according to lipid concentration.

[0100] 3. Preparation of mRNA-LNPs samples using microfluidic chips

[0101] Assemble the microfluidic chip and dispensing needle, Tygon hose, and stainless steel connector. Rinse and sterilize with 75% alcohol and check for leakage.

[0102] The lipid-ethanol solution and mRNA-acidic aqueous solution were sucked into the syringes respectively, fixed to the syringe pump, and connected to the microfluidic chip. The lipid-ethanol phase flow rate was 125 μL / min, the mRNA-water phase flow rate was 375 μL / min, and the total flow rate was 500 μL / min. The uniformly mixed mRNA-LNPs sample was collected.

[0103] 4. Buffer exchange and sample preservation

[0104] The mRNA-LNP solution system is replaced into a suitable storage buffer (such as PBS) by dialysis or ultrafiltration.

[0105] (2) LNP with fusion peptide (LNP pep+ )

[0106] In this embodiment, the coupling method is the reaction of cysteine ​​and maleimide. On the basis of the fusion polypeptide (amino acid sequence as shown in SEQ ID NO: 1), an additional cysteine ​​is added to introduce a sulfhydryl group. A certain proportion of DMG-PEG2000-Mal (maleimide) is added to the lipid-ethanol solution, and the synthesis steps are the same as those in step 3 above to prepare mRNA-LNPs. The buffer of the synthesized mRNA-LNP-Mal is replaced with PBS (pH = 7.4), and a sulfhydryl polypeptide is added according to a molar ratio of polypeptide: DMG-PEG2000-Mal = 5: 1, and the reaction is carried out overnight at 4°C, and free polypeptides are removed by ultrafiltration.

[0107] (3) Preparation of extracellular vesicles (EVs)

[0108] Extracellular vesicles are a type of single-layer membrane-coated vesicles that are secreted by cells autonomously. Therefore, extracellular vesicles can be further separated and purified by collecting cell culture medium. Take the source of 293T cell line culture medium as an example:

[0109] 1. 293T cells were plated onto a 15 cm culture dish at a density of 60-80%, 12 mL of DMEM medium was added, and cultured in an incubator (37° C., 5% CO 2 ) for 3 days.

[0110] 2. Collect the culture medium into a 50 mL centrifuge tube, centrifuge at 12000 g for 15 minutes, and collect the supernatant.

[0111] 3. Place the supernatant into an SW41 ultracentrifuge tube and use a Beckman Coulter centrifuge to ultracentrifuge for 2 hours at 150,000 g. Discard the supernatant and resuspend the pellet in the centrifuge tube with 20 μL PBS buffer.

[0112] 4. The sample obtained in step 3 is further separated and purified by asymmetrical flow field-flow fractionation technology. The asymmetrical field-flow fractionation equipment is mainly composed of an isocratic pump from Agilent and an Eclipse device from Wyatt, and provides ultraviolet absorption, multi-angle laser and differential detection.

[0113] 5. The separated extracellular vesicle samples can be concentrated to the desired concentration by ultrafiltration.

[0114] (4) LNP coupled with fusion peptide (LNP pep+ ), LNPs not coupled with fusion peptides (LNPs pep- ) and the labeling efficiency of EVs expressing GFP fluorescent protein

[0115] DiD-labeled LNPs with and without fusion peptides and EVs expressing GFP fluorescent protein were prepared and analyzed by fluorescence nanoflow cytometry. The results showed that the DiD labeling efficiency of LNPs without fusion peptides was 75.0%, the DiD labeling efficiency of LNPs with fusion peptides was 84.7%, and the efficiency of EVs labeled with GFP was 36.8% ( Figure 2 ).

[0116] Example 2

[0117] (1) LNP pep+ - Preparation of EV delivery system

[0118] 1. Prepare the obtained LNP pep+The cells were replaced with EVs into a uniform buffer, in this example PBS buffer was used, and the particle concentration was determined by NTA (nanoparticle tracking analysis).

[0119] 2. According to the molar ratio LNP pep+ Mix the two in a ratio of EV=1:1.5 and incubate at 4°C in the dark for 1 hour to obtain LNP. pep+ -EV delivery system.

[0120] (2)LNP pep- - Preparation of EV delivery system

[0121] 1. Prepare the obtained LNP pep- The EVs were replaced with PBS buffer and the particle concentration was determined by NTA (Nanoparticle Tracking Analysis).

[0122] 2. According to the molar ratio LNP pep- Mix the two in a ratio of EV=1:1.5 and incubate at 4°C in the dark for 1 hour to obtain LNP. pep- -EV delivery system.

[0123] (3) Fusion efficiency verification

[0124] The uncoupled fusion peptide LNP (LNP pep- , control group) and coupled fusion peptide LNP (LNP pep+ , experimental group) were incubated with GFP-labeled EVs at a ratio of 1:1.5 to test the fusion efficiency of the two, and analyzed by fluorescence nanoflow cytometry. The results showed that the comprehensive fusion efficiency in the control group was 11.8%, and the comprehensive fusion efficiency in the experimental group was 36.8% ( Figure 3 ), it can be seen that the lipid nanoparticles coupled with the fusion polypeptide significantly improved the fusion efficiency with the extracellular blastocyst.

[0125] Example 3

[0126] In order to clarify the actual delivery efficiency of the delivery system, 293T cells were treated with mRNA-LNPs encapsulating green fluorescent protein GFP mRNA. The steps are as follows:

[0127] 1. Inoculate 293T cells in a 48-well culture plate at a cell count of 4.5w per well.

[0128] 2. Set up 4 groups according to 0.6 μg mRNA per well: Control group 1 (LNP pep- ), control group 2 (LNP pep- -EV)(LNP pep+ ), control group 3 (LNP pep- -EV) and experimental group (LNP pep+-EV), the preparation and fusion steps are as described in Examples 1 and 2 above.

[0129] 3. Add 300 μL of culture medium to each group product and mix well for cell culture.

[0130] 4. After 16 hours, photos of each group were collected using a fluorescence microscope, and the green fluorescence expression level was counted.

[0131] The results showed that the chimeric delivery system formed by LNP coupled with fusion peptide and EV can successfully deliver and promote the expression of GFP-mRNA in cells, and the delivery efficiency of this chimeric delivery system of lipid nanoparticles fused with extracellular vesicles is more than 70 times higher than that of lipid nanoparticles alone ( Figure 4 ).

[0132] Example 4

[0133] To verify the design principle of the polypeptide, four polypeptides (polypeptides 1-4) with different sequences were selected to couple lipid nanoparticles and fuse extracellular vesicles. The specific synthesis steps are referred to Example 1. The chimeric delivery system composed of polypeptides 1-4 was transferred to in vitro cultured cells, refer to Example 3. At the same time, negative control polypeptides (polypeptides 5-6) were designed and coupled to the surface of lipid nanoparticles to try to form a vesicle chimeric system. The polypeptide sequences are shown in Table 2 below.

[0134] Table 2

[0135] Peptide name sequence Sequence number Peptide 1 LAKSWGRALKR SEQ ID NO:1 Peptide 2 VARALGRAIAKSIKR SEQ ID NO:2 Peptide 3 IARSLGKSEGVALKK SEQ ID NO:3 Peptide 4 LAKALGKAEGVALSKVKR SEQ ID NO:4 Peptide 5 TPIGDGPVLLPDNH SEQ ID NO:5 Peptide 6 GEELFTGVVPILVE SEQ ID NO:6

[0136] The results showed that the delivery system composed of peptides 1-4 can significantly promote the expression of GFP-mRNA in lipid nanoparticles in cells, while peptides 5-6 cannot significantly increase the expression level of GFP-mRNA in lipid nanoparticles in cells cultured in vitro ( Figure 5 ).

Claims

1. A delivery system comprising lipid nanoparticles and extracellular vesicles, wherein the lipid nanoparticles and the extracellular vesicles form a chimeric structure by fusing polypeptides.

2. The delivery system according to claim 1, characterized in that A chimeric structure is partially formed between the shell of the lipid nanoparticle and the membrane of the extracellular vesicle.

3. The delivery system according to claim 1, characterized in that The fusion polypeptide is coupled to the outer surface of the lipid nanoparticle; Preferably, the fusion polypeptide is linked to a polyethylene glycol (PEG) derivative on the outer surface of the lipid nanoparticle.

4. The delivery system according to claim 1, characterized in that The fusion polypeptide is an amphipathic polypeptide having 10-30 amino acids; Preferably, the secondary structure of the fusion polypeptide has at least one of an α-helix, a β-fold (β-sheet), a β-turn or a random coil, preferably an α-helix; More preferably, the fusion polypeptide can spontaneously insert into the phospholipid bilayer structure in an α-helix or α-helix-bend-α-helix conformation. Further preferably, the fusion polypeptide has an amino acid sequence shown in at least one of SEQ ID NOs: 1-4.

5. The delivery system according to claim 1, characterized in that The extracellular vesicles are extracellular vesicles obtained from at least one of pluripotent stem cells, mesenchymal stem cells, ectoderm-derived cells, mesoderm-derived cells, and endoderm-derived cells.

6. The delivery system according to claim 1, characterized in that The lipid nanoparticles include steroids, neutral lipids, PEG derivatives and ionizable lipids; Preferably, the PEG derivative is selected from at least one of polyethylene glycol-modified phosphatidylethanolamine, polyethylene glycol-modified phosphatidic acid, polyethylene glycol-modified ceramide, polyethylene glycol-modified dialkylamine, polyethylene glycol-modified diacylglycerol, and polyethylene glycol-modified dialkylglycerol, preferably selected from at least one of DMG-PEG2000, ALC-0159 or DSPE-PEG2000.

7. A method for preparing a delivery system, the method comprising: (1) coupling the fusion polypeptide to the outer surface of lipid nanoparticles to obtain lipid nanoparticles coupled with the fusion polypeptide; (2) Mixing the lipid nanoparticles coupled with the fusion polypeptide and the extracellular vesicles.

8. The preparation method according to claim 7, characterized in that: In step (1), the fusion polypeptide is coupled to the PEG derivative of the lipid nanoparticle by chemical coupling; Preferably, in step (1), a solution containing a PEG derivative and a fusion polypeptide is incubated, wherein the PEG derivative and the fusion polypeptide respectively have one of the chemical coupling linker pairing members; More preferably, the molar ratio of the fusion polypeptide to the PEG derivative is (2-100):1, preferably (2-10):1; More preferably, the incubation temperature is 0-20°C, preferably 4-6°C, and the incubation time is 2-20h, preferably 6-12h.

9. The preparation method according to claim 7, characterized in that: In step (2), the molar ratio of the lipid nanoparticles coupled with the fusion polypeptide to the extracellular vesicles is 1:(1-10), preferably 1:(1-2); Preferably, the lipid nanoparticles coupled with the fusion polypeptide and the extracellular vesicles are incubated at a temperature of 0-20° C., preferably 4-6° C., for a time of 0.5-5 h, preferably 0.5-1 h.

10. The preparation method according to claim 7, characterized in that: Step (1) further comprises mixing the lipid nanoparticles coupled with the fusion polypeptide with a solution containing a pharmaceutically active ingredient, and encapsulating the pharmaceutically active ingredient inside the lipid nanoparticles.

11. A pharmaceutical composition comprising the delivery system according to any one of claims 1 to 6 or the delivery system prepared by the preparation method according to any one of claims 7 to 10, and a pharmaceutically active ingredient.

12. The pharmaceutical composition according to claim 11, characterized in that The active pharmaceutical ingredient is selected from nucleic acids, small molecule compounds, polypeptides and / or proteins; Preferably, the nucleic acid is selected from the group consisting of DNA, RNA, mRNA, siRNA, rRNA, tRNA, snRNA, miRNA, plasmid and combinations thereof, preferably mRNA.

13. The pharmaceutical composition according to claim 11 or 12, characterized in that The active pharmaceutical ingredient is encapsulated inside the lipid nanoparticles.

14. Use of the delivery system according to any one of claims 1 to 6 or the delivery system prepared by the preparation method according to any one of claims 7 to 10 in the delivery of active pharmaceutical ingredients, Preferably, the pharmaceutically active ingredient is selected from nucleic acids, small molecule compounds, polypeptides and / or proteins; Preferably, the nucleic acid is selected from the group consisting of DNA, RNA, mRNA, siRNA, rRNA, tRNA, snRNA, miRNA, plasmid and combinations thereof, more preferably mRNA.

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