Delivery systems, their preparation methods and applications

Abstract

This disclosure provides a delivery system for a pharmaceutical active ingredient, as well as a method for preparing the delivery system and its applications. The delivery system comprises lipid nanoparticles and extracellular vesicles, wherein the lipid nanoparticles and extracellular vesicles form a chimeric structure through fusion with a polypeptide. The delivery system of this disclosure features high delivery efficiency, low immunogenicity, and efficient loading of mRNA.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to a delivery system for active pharmaceutical ingredients, as well as the preparation method and application of the delivery system. Background Technology

[0002] Two key factors for the success of mRNA vaccines are breakthroughs in mRNA modification and vector technology. mRNA needs to be packaged in a vector to prevent degradation by nucleases and to cross the cell membrane to release it into the cytoplasm for translation. Currently, the vector for mRNA vaccines is lipid nanoparticles (LNPs). LNPs are mainly composed of four types of lipids, including cholesterol, phospholipids, PEG derivatives, and ionizable lipids.

[0003] mRNA shows great promise as a nucleic acid drug (e.g., in protein replacement therapy, cell therapy, and antibody therapy). However, mRNA as a nucleic acid drug may require protein levels 100-1000 times higher than mRNA vaccines, as vaccines amplify immune signals through the body's immune response. Furthermore, mRNA nucleic acid drugs require repeated administration. LNP, on the other hand, has low delivery efficiency, with only 1-2% of the mRNA released into the cytoplasm for translation. Moreover, LNP has poor extrahepatic targeting, with over 80% of the mRNA accumulating in the liver, and it exhibits strong immunogenicity and toxic side effects, making it unsuitable for repeated long-term administration. Therefore, there is a need to develop novel mRNA delivery systems with high delivery efficiency, low immunogenicity, and minimal toxicity. Summary of the Invention

[0004] To address the issues of low delivery efficiency, high cytotoxicity, and immunogenicity associated with existing lipid nanoparticles (LNPs), this disclosure discloses a biomimetic EV-LNP delivery system. This system utilizes a fusion peptide to create an intercalation between the shell of lipid nanoparticles and the membrane of extracellular vesicles (EVs), thereby forming a chimeric structure between the EVs and LNPs. This system features high delivery efficiency, low immunogenicity, and the ability to efficiently load active pharmaceutical ingredients.

[0005] In one aspect, this 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 peptides.

[0006] In some embodiments, the shell of the lipid nanoparticles partially forms an interlocking structure with the membrane of the extracellular vesicles.

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

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

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

[0010] In some embodiments, the secondary structure of the fusion polypeptide has at least one of α-helix, β-sheet (β-flask), β-turn or random coil, preferably α-helix.

[0011] In this application, the fusion peptide is immobilized on the surface of nanoliposomes through chemical coupling and other methods, and then incubated with extracellular vesicles. The fusion peptide, with its unique amphiphilicity and spontaneous conformation such as α-helix, inserts into the membrane of the extracellular vesicle without damaging the membrane structure of the extracellular vesicle. At the same time, the length of the fusion peptide is comparable to the membrane thickness of the extracellular vesicle, so that the shell of the lipid nanoparticle and the membrane of the extracellular vesicle intercalate at the insertion point, but do not fuse. This delivery system with a special intercalation structure retains the basic morphology and function of nanoliposomes and extracellular vesicles, and has the characteristics of high delivery efficiency, low immunogenicity, and efficient loading of active pharmaceutical ingredients.

[0012] The fusion polypeptide is amphiphilic, folds into an α-helix or other spontaneous conformation, or has polypeptide bending introduced by proline / glycine, which can promote fusion or opening of extracellular vesicle membranes.

[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 insertion morphology.

[0014] In some specific embodiments, the fusion polypeptide has at least one of the amino acid sequences shown in 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 derived from at least one of pluripotent stem cells, mesenchymal stem cells, ectoderm-derived cells, mesodermal-derived cells, and endoderm-derived cells.

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

[0017] In some embodiments, the lipid nanoparticles include steroidal compounds, 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 compounds include, but are not limited to, at least one of cholesterol, coccosterol, nonsterol, sitosterol, ergosterol, campesterol, stigmasterol, brassosterol, tomatine, ursolic acid, α-tocopherol, corticosteroids and their derivatives.

[0020] In some embodiments, the neutral lipids include, but are not limited to, 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), and 1,2-dioleoyl-sn-glycerol-3-phosphate choline. At least one of the following: 1-sn-glycerol-3-phosphate ethanolamine (DOPE), palmitoyl oleoyl phosphatidyl ethanolamine (POPE), distearate-phosphatidyl-ethanolamine (DSPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoyl phosphate ethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoyl ethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin (SM), ceramide, sterol and its derivatives.

[0021] In some embodiments, the PEG derivative includes, but is not limited to, 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, the PEG derivative comprises at least one of DMG-PEG2000, ALC-0159, or DSPE-PEG2000.

[0022] In some embodiments, ionizable lipids are a class of lipids that are almost uncharged under normal neutral physiological pH conditions but positively charged under acidic pH conditions. They are generally composed of an amine moiety and a lipid moiety, with the cationic amine moiety interacting electrostatically with the polyanionic nucleic acid to form a positively charged liposome or lipid membrane structure. In some embodiments, the ionizable lipids include, but are not limited to, dilinoleyl 4-dimethylaminobutyrate (DLin-MC3-DMA), ALC0315, SM102, 1,1-((2-(4-(2-((2-(di(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)di(dodecane-2-ol)(C12-200) or The compounds shown, etc., wherein: RCOO is selected from myristoyl, α-D-tocopherol succinoyl, 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-glycerol-3-phosphoethanolamine), the PEG derivatives are DMG-PEG2000, and the ionizable lipids are ionizable lipid SM-102.

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

[0025] (1) The fusion peptide is coupled to the outer surface of the lipid nanoparticle to obtain lipid nanoparticles coupled with the fusion peptide.

[0026] (2) The lipid nanoparticles coupled with the fusion peptide are mixed with extracellular vesicles.

[0027] In some embodiments, in step (1), the fusion peptide is chemically coupled to the PEG derivative of the lipid nanoparticles.

[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 each have one of the chemical coupling head pairing members, and the polypeptide-PEG derivative is used as one of the raw materials for preparing lipid nanoparticles to obtain lipid nanoparticles with the fusion polypeptide on the surface, wherein the ionizable lipid is contained inside the lipid nanoparticles.

[0029] In some embodiments, the molar ratio of the fusion peptide to the PEG derivative is (2-100):1, preferably (2-10):1. In some specific embodiments, the molar ratio of the fusion peptide 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 between them.

[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 between them.

[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 peptide 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 peptide 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 between them.

[0033] In some embodiments, the lipid nanoparticles coupled with the fusion peptide and the extracellular vesicles are incubated at a temperature of 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 between them.

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

[0035] In some embodiments, the chemical coupling connector pairing members are paired chemical connectors, such as amine reactive connectors, thiol reactive connectors, or click chemical connectors. Amine reactive connectors may be connectors containing succinimide ester groups. Thiol reactive connectors may be connectors containing maleimide groups. Click chemical connectors may be connectors containing click chemical functional groups, such as NHS-azide connectors, NHS-DBCO connectors, maleimide-azide connectors, or maleimide-DBCO connectors.

[0036] In some embodiments, step (1) further includes mixing the lipid nanoparticles coupled with the fusion peptide with a solution containing the active pharmaceutical ingredient, thereby encapsulating the active pharmaceutical ingredient inside the lipid nanoparticles.

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

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

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

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

[0041] In some embodiments, the active pharmaceutical ingredient is encapsulated within the lipid nanoparticles.

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

[0043] (1) The fusion peptide is chemically coupled into the PEG derivative of the lipid nanoparticle to obtain lipid nanoparticles with the fusion peptide.

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

[0045] (3) Mix extracellular vesicles with lipid nanoparticles with drug active ingredients obtained in step (2).

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

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

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

[0049] Compared with the prior art, the delivery system disclosed herein has the following advantages:

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

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

[0052] (3) It is easy to genetically engineer and thus target different tissues and organs. Attached Figure Description

[0053] Figure 1 This illustration shows a schematic diagram of a delivery system for preparing extracellular vesicle-lipid nanoparticles according to one embodiment of this application, along with images of lipid nanoparticles, extracellular vesicles, and the chimeric structure resulting from their fusion, captured by transmission electron microscopy.

[0054] Figure 2 The labeling efficiency of extracellular vesicles and lipid nanoparticles was demonstrated by nanoflow cytometry.

[0055] Figure 3 The fusion efficiency of lipid nanoparticles with coupled and uncoupled fused peptides to extracellular vesicles was shown.

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

[0057] Figure 5 The chimeric delivery system obtained using different fusion peptides demonstrates the delivery of GFP-mRNA expression in cells. Detailed Implementation

[0058] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention in any way.

[0059] Extracellular vesicles (EVs) are autonomously secreted by cells and can carry various proteins and nucleic acids to target and regulate the function of target cells and tissues. They exhibit low immunogenicity and good tolerability with repeated administration, making them suitable as delivery vehicles for small molecule or nucleic acid drugs. While mature technologies exist for the mass production of EVs, drug delivery into vesicles still relies on traditional methods such as electroporation, ultrasound, compression, or repeated freeze-thaw cycles, which are inefficient and damage vesicle integrity. Therefore, this disclosure provides a novel LNP and EV delivery system for mRNA vaccine and drug delivery. The delivery system disclosed herein is 10-100 times more efficient than existing LNP systems, exhibits lower immunogenicity and cytotoxicity, and can efficiently load mRNA.

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

[0061] (1) A fusion peptide that can promote lipid fusion was screened out.

[0062] (2) The selected fusion peptides were chemically coupled into the PEG derivative of LNP.

[0063] (3) Synthesize LNPs containing active pharmaceutical ingredients (e.g., mRNA).

[0064] (4) Isolate and extract extracellular vesicles.

[0065] (5) Mix the extracellular vesicles from step (4) with the LNPs from step (3) to form an LNP-EV delivery system.

[0066] Transmission electron microscopy revealed that the LNP-EV delivery system formed by the fusion of lipid nanoparticles and extracellular vesicles created a chimeric structure. Figure 1 (As shown).

[0067] the term

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

[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 a base-free site). The term "nucleic acid," as used herein, also includes covalently bonded polymers of ribonucleosides or deoxyribonucleosides, said covalent bond typically via a phosphodiester bond between subunits, but in some cases via phosphate thioesters, methylphosphonates, 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 hourly sequence RNA (stRNA), and any combination thereof.

[0070] The term "amphiphilic polypeptide" refers to a peptide molecule that combines a hydrophilic peptide sequence with a hydrophobic alkyl chain; it is also called an amphiphilic polypeptide or peptide amphiphile. Amphiphilic polypeptides typically consist of a hydrophilic peptide sequence attached to a lipid tail and a hydrophobic alkyl chain.

[0071] The secondary structure of a protein refers to the periodic structural conformation of the polypeptide backbone atoms arranged in a one-dimensional direction under the influence of hydrogen bonds. This is a local spatial conformation of the peptide chain and does not involve the amino acid side chain atoms. There are many types of protein secondary structures, mainly classified as α-helices, β-sheets (β-flaps), β-turns, random coils, etc. An α-helix refers to the polypeptide backbone ascending in a regular helical pattern around a central axis. Its structural characteristics are: ① The backbone backbone forms a right-handed structure around the central axis; ② Each turn of the helix consists of 3.6 amino acid residues, with a pitch of 0.54 nm; ③ Hydrogen bonds are formed between adjacent helical turns; ④ Side chain groups are located on the outside of the helix. β-sheets: There are generally two forms of β-sheets: parallel and antiparallel. Parallel β-sheets are when adjacent peptide chains are in the same direction, while antiparallel β-sheets are in opposite directions. Their structural characteristics are: ① Several peptide chains or segments are arranged in parallel or antiparallel sheets; ② All C=O and NH bonds in the peptide bonds form interchain hydrogen bonds; ③ Side chain groups are alternately located on the top and bottom of the sheet. β-turns typically occur at the 180° fold of a polypeptide chain, usually consisting of four amino acid residues. They form a tight loop through hydrogen bonds between the first and fourth residues. Random coils refer to conformations formed by the irregular arrangement of the main chain backbone, and also broadly refer to polypeptide regions that cannot be categorized into defined secondary structures. They commonly appear between α-helices, α-helices and β-sheets, and β-sheets.

[0072] The term "fusion polypeptide" refers to a protein comprising at least a first amino acid chain genetically linked to at least a second amino acid chain. Therefore, a fusion polypeptide can comprise a multimer of peptides expressed as a single, linear polypeptide. It can contain 1, 2, 3, 4, or even more peptides. The term "fusion" indicates components linked by peptide bonds (directly or via peptide linkers).

[0073] The term "chemical coupling" refers to the coupling of a peptide with other substances (such as a second protein or non-protein moiety) through chemical reactions, including substitution (e.g., N-succinimide chemistry), addition or cyclization (e.g., maleimide chemistry or click chemistry), or oxidation chemistry (e.g., disulfide formation). These chemical reactions activate a carboxyl or amino group with a given compound, which is then covalently bonded to the amino or carboxyl group. In some embodiments, the fusion peptide is chemically coupled to a PEG derivative via covalent interactions (chemical connectors, such as amine reactive connectors, thiol reactive connectors, or click chemistry connectors). Amine reactive connectors may be connectors containing succinimide ester groups. Thiol reactive connectors may be connectors containing maleimide groups. Click chemistry connectors may be connectors containing click chemistry functional groups, such as NHS-azide connectors, NHS-DBCO connectors, maleimide-azide connectors, or maleimide-DBCO connectors.

[0074] The term "chimerism" refers to the insertion of a fused polypeptide into a phospholipid bilayer structure, where the shell of the lipid nanoparticle and the membrane of the extracellular vesicle intercalate at the insertion point without fusing. This allows the lipid nanoparticle and the extracellular vesicle to form an integrated delivery system, while retaining the basic morphology and function of both the nanoliposomes and the extracellular vesicles.

[0075] The term "delivery carrier" refers to any method used to formulate and deliver active ingredients, including small molecule compounds and various nucleic acids, to perform a specific function in vivo. For example, delivering mRNA for the in vivo production of proteins, such as antibodies, antigens, or antibody fragments or antigen fragments. In some embodiments, mRNA is encapsulated in a transfer carrier, such as nanoparticles. In addition, one purpose of this encapsulation is generally to protect nucleic acids from environments containing enzymes or chemicals that may degrade nucleic acids and / or cause rapid excretion of nucleic acids, and to promote cellular uptake and expression of the corresponding sequence. In some embodiments, the transport carrier is a liposome vesicle, or other means of facilitating the transfer of nucleic acids to target cells and tissues. Suitable transport carriers may include, but are not limited to, liposomes, lipid nanoparticles, ceramide-containing lipid nanoparticles, protein liposomes, nanoparticles, calcium phosphate-silicate nanoparticles, calcium phosphate nanoparticles, silica nanoparticles, nanocrystals, semiconductor nanoparticles, poly(D-arginine), nanodendritic polymers, starch-based delivery systems, micelles, emulsions, vesicles, plasmids, viruses, calcium phosphate nucleotides, aptamers, peptides, and other carrier tags. We are also considering using bio-ion capsules and other viral capsid protein assemblies as suitable transfer vectors.

[0076] Extracellular vesicles (EVs) are small, double-membrane vesicles that detach from the cell membrane or are secreted by the cell, ranging in diameter from 30 nm to 1,000 nm. EVs mainly include microvesicles (MVs) and exosomes (Exosomes). Microvesicles are small vesicles that detach from the cell membrane after cell activation, damage, or apoptosis, with a diameter of approximately 200–1000 nm. Exosomes are released extracellularly in the form of secretions from multivesicular bodies fused to the cell membrane, with a diameter of approximately 30–200 nm. EVs are widely distributed in cell culture supernatants and various body fluids (blood, lymph, saliva, urine, semen, breast milk), carrying a variety of cell-derived proteins, lipids, DNA, mRNA, miRNA, etc., and participating in processes such as intercellular communication, cell migration, angiogenesis, and immune regulation.

[0077] Extracellular vesicles possess natural biocompatibility, high delivery efficiency, low toxicity, and low immunogenicity, making them an emerging drug delivery vector. Engineered extracellular vesicles are typically designed through genetic modification of donor cells. In some respects, the extracellular vesicles described in this disclosure also carry payloads of interest, i.e., extracellular vesicles encapsulate substances such as proteins, nucleic acids, peptides, and small molecules. The payloads can be exogenous or endogenous. The payloads have preventative and / or therapeutic uses for diseases. Specifically, the nucleic acids are selected from the group consisting of antisense oligonucleotides, siRNA, miRNA, mRNA, gRNA, or plasmids. The peptides, proteins, and small molecules are selected from drugs for the prevention and / or treatment of multi-organ damage repair and regeneration, tumor diseases, or drugs or regulators for promoting embryonic development and / or stem cell proliferation.

[0078] In this application, extracellular vesicles are derived from extracellular vesicles of various cell types. In some embodiments, extracellular vesicles are derived from stem cells, such as pluripotent stem cells, mesenchymal stem cells, ectoderm-derived cells, mesoderm-derived cells, and endoderm-derived cells. 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 that have the ability to primarily differentiate into mesodermal-derived tissues and partially into ectoderm-derived and endoderm-derived tissues. Ectodermal cells include keratinizing 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, tactile sensory neurons, type I carotid somatic cells, type II carotid somatic cells, type I vestibular hair cells, type II vestibular 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 epithelial supporting cells, satellite cells, intestinal glial cells, neurons and glial cells of the central nervous system, lens cells, etc.Mesodermal cells include hepatocytes, adipocytes, Ito cells, pararenal cells, glomerular epithelial cells, proximal renal tubule brush border cells, thin segment cells of the ring of Henry, distal renal tubule 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, and osteoprogenitor cells. Vitreous cells, auricular 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 cells, 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, stem cells and progenitor cells of the blood and immune cell system, erythroblasts, bone marrow cells, oocytes, sperm cells, spermatocytes, spermatogonia, spermatogonia, nurse cells, ovarian follicular cells, Sertoli cells, thymic epithelial cells, interstitial kidney cells, and any cultured cells derived therefrom. Endoderm-derived cells include salivary gland mucous cells, mammary gland cells, lacrimal gland cells, auditory gland cells, exocrine glands, glandular dark cells, exocrine clear cells, apocrine gland cells, ciliated gland cells, sebaceous gland cells, duodenal cells, seminal vesicle cells, prostate cells, bulbourethral gland cells, urethral gland cells, endometrial cells, goblet cells, gastric mucosal cells, gastric chief cells, parietal cells, pancreatic acinar cells, Panthen cells of the small intestine, type II lung cells, pulmonary Clara cells, anterior pituitary cells, melanocyte-stimulating hormone-producing cells, large neurosecreting neurons, thyroid cells, parathyroid cells, adrenal cells, interstitial cells, internal capsule cells, luteal cells, juxtaglomerular cells, macula densa cells, peripolar cells, glomerular mesangial cells, and any cells derived from these cultured cells. When obtaining extracellular vesicles from these cells, the cells are cultured in normal culture media typically used for cell culture for 1 to 30 days, the culture medium is collected, and if necessary, further purified by methods such as centrifugation, chromatography (e.g., gel filtration chromatography, column chromatography, size exclusion chromatography, phosphatidylserine affinity chromatography and other conventional column chromatography), and flow cytometry.

[0079] Lipid nanoparticles (LNPs) have a structure comprising a single monolayer or bilayer of lipids encapsulating a compound within a solid phase. Unlike liposomes, lipid nanoparticles do not have an aqueous or other liquid phase inside; instead, lipids from the monolayer or bilayer shell directly complex with the internal compound, thus encapsulating it within a solid core. Lipid nanoparticles are typically spherical vesicles with a relatively uniform shape and size distribution. The diameter of lipid nanoparticles is generally considered to range from 10 nm to 1000 nm.

[0080] For lipid nanoparticle nucleic acid delivery systems, the lipid shell can be formulated to contain ionizable lipids that can complex and associate with the negatively charged backbone of the nucleic acid core. At physiological pH, the lipid nanoparticles can utilize a relatively neutral outer shell, thereby significantly increasing the circulating half-life of the particles after intravenous administration. In the context of nucleic acid delivery, lipid nanoparticles offer numerous advantages over other lipid-based nucleic acid delivery systems, including high nucleic acid encapsulation efficiency, potent transfection, improved tissue penetration for therapeutic delivery, 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, mRNA delivery includes situations where mRNA is delivered to a target tissue and the encoded protein is expressed and retained within the target tissue (also known 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 distributed systemically and absorbed by other tissues (also known as “systemic delivery”). “Local delivery” refers to tissue-specific delivery or distribution. Typically, local delivery requires the protein or peptide encoded by the mRNA to be translated and expressed intracellularly or with limited secretion, avoiding entry into the patient’s circulatory system. The term “target tissue” refers to any tissue affected by the disease to be treated. In some embodiments, target tissue includes those tissues exhibiting disease-related pathology, symptoms, or features.

[0083] The following describes preferred embodiments of the present invention, but the scope of protection of the present invention is not limited to these preferred embodiments. It should be noted that any modifications and improvements made by those skilled in the art based on this inventive concept are within the scope of protection of the present invention. All reagents used, unless otherwise specified, are commercially available conventional products.

[0084] Example 1

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

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

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

[0088] Reagents: ionizable lipid SM-102, neutral cofactor 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 tubing, stainless steel connectors, dialysis bags / boxes (3k / 10k / 20k), ultrafiltration tubing (100kd).

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

[0091] Experimental steps:

[0092] 1. Prepare lipid-ethanol solutions, 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 percentage 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 a 250μL solution, add (μL) 14.2 14.9 29.8 2.5

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

[0096] 2. Prepare an acidic aqueous solution for mRNA.

[0097] In this embodiment, mRNA-LNPs samples were prepared using green fluorescent protein mRNA (the mRNA is shown in SEQ ID NO:7) as an example.

[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. Ionizable lipids contain one N atom).

[0099] Prepare 50.0 mL each of 100 mM citric acid and sodium citrate solutions using ultrapure water. Take 33.0 mL of the citric acid solution and 17.0 mL of the sodium citrate solution, mix them, add diethyl pyrocarbonate (DEPC), let stand for 30 minutes, then autoclave to remove DEPC. After sterilization, use DEPC-treated ultrapure water to bring the volume to 100 mL to obtain a 50 mM citrate buffer solution with pH = 4. Dilute the mRNA to the required concentration using the citrate buffer solution according to the lipid concentration.

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

[0101] Assemble the microfluidic chip and dispensing needle, Tygon tubing, and stainless steel connector. Flushing with 75% alcohol for sterilization and checking for leaks.

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

[0103] 4. Buffer replacement and sample preservation

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

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

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

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

[0108] Extracellular vesicles are a type of single-membrane vesicle that are autonomously secreted by cells and released into the extracellular space. Therefore, extracellular vesicles can be obtained by further isolation and purification from collected cell culture media. Taking the 293T cell line culture medium as an example:

[0109] 1. Spread 293T cells into a 15cm culture dish at a density of 60-80%, add 12mL of DMEM medium, and incubate in an incubator (37℃, 5% CO2) for 3 days.

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

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

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

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

[0114] (4) LNP of the conjugated fusion peptide (LNP) pep+ LNPs of uncoupled fusion peptides (LNPs) pep- and the labeling efficiency of EVs expressing GFP fluorescent protein.

[0115] DiD-labeled LNPs of conjugated and unconjugated fusion peptides, as well as EVs expressing GFP fluorescent protein, were prepared and analyzed by fluorescent nanoflow cytometry. The results showed that the DiD labeling efficiency of the unconjugated fusion peptide LNP was 75.0%, the DiD labeling efficiency of the conjugated fusion peptide was 84.7%, and the efficiency of GFP-labeled EVs 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 EVs were replaced with a uniform buffer solution, which in this example used PBS buffer, 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 1:1.5 ratio (EV = 1) 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 EV was 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 1:1.5 ratio (EV = 1) 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 the conjugated fusion peptide LNP (LNP pep+ The control group and experimental group were incubated with GFP-labeled EVs at a ratio of 1:1.5 to test the fusion efficiency, which was analyzed by fluorescent nanoflow cytometry. The results showed that the overall fusion efficiency in the control group was 11.8%, while the overall fusion efficiency in the experimental group was 36.8%. Figure 3 As can be seen, lipid nanoparticles coupled with fusion peptides significantly improved the fusion efficiency with extracellular blastocysts.

[0125] Example 3

[0126] To determine the actual delivery efficiency of the delivery system, 293T cells were treated with mRNA-LNPs containing mRNA encapsulating green fluorescent protein (GFP). The steps are as follows:

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

[0128] 2. Four groups were set up 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 each group of products to 300 μL of culture medium and mix well for cell culture.

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

[0131] The results showed that the delivery system, which combines LNPs and EVs with fusion peptides to form a chimeric structure, successfully delivered and promoted the expression of GFP-mRNA in cells. Furthermore, this chimeric delivery system, which combines lipid nanoparticles with extracellular vesicles, demonstrated a delivery efficiency more than 70 times higher than that of lipid nanoparticles alone. Figure 4 ).

[0132] Example 4

[0133] To verify the design principles of the peptides, four peptides with different sequences (peptides 1-4) were selected to couple lipid nanoparticles and fuse them with extracellular vesicles. The specific synthesis steps are described in Example 1. The chimeric delivery system composed of peptides 1-4 was transfected into in vitro cultured cells, as described in Example 3. Simultaneously, negative control peptides (peptides 5-6) were designed and coupled to the surface of lipid nanoparticles to attempt to form a vesicle chimeric system. The peptide sequences are shown in Table 2 below.

[0134] Table 2

[0135] polypeptide name sequence Serial Number Peptide 1 LAKSWGRALKR SEQ ID NO:1 Peptide 2 VARALGRAIAKSIKR SEQ ID NO:2 Polypeptide 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 systems composed of peptides 1-4 significantly promoted the expression of GFP-mRNA from lipid nanoparticles in cells, while peptides 5-6 did not significantly increase the expression level of GFP-mRNA from lipid nanoparticles in cultured cells in vitro. Figure 5 ).

Claims

1. A delivery system comprising lipid nanoparticles and extracellular vesicles, wherein the shell of the lipid nanoparticles and the membrane of the extracellular vesicles are partially intercalated by a fusion polypeptide having at least one amino acid sequence shown in SEQ ID NOs:1-4, the fusion polypeptide being chemically coupled to a polyethylene glycol (PEG) derivative on the outer surface of the lipid nanoparticles, the PEG derivative being 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.

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

3. The delivery system according to claim 1, characterized in that, The lipid nanoparticles include steroidal compounds, neutral lipids, the PEG derivatives, and ionizable lipids.

4. The delivery system according to claim 1, characterized in that, The PEG derivative is selected from at least one of DMG-PEG2000, ALC-0159 or DSPE-PEG2000.

5. A method for preparing a delivery system according to any one of claims 1-4, the method comprising: (1) The fusion peptide is coupled to the outer surface of the lipid nanoparticle to obtain lipid nanoparticles coupled with the fusion peptide. (2) Mix the lipid nanoparticles coupled with the fusion peptide with extracellular vesicles; In step (1), the fusion peptide is chemically coupled to the PEG derivative on the outer surface of the lipid nanoparticles.

6. The preparation method according to claim 5, characterized in that, In step (1), lipid nanoparticles containing PEG derivatives are incubated with a solution of fused peptides, wherein the PEG derivatives and the fused peptides each have one of the chemical coupling head pairing members.

7. The preparation method according to claim 5, characterized in that, The molar ratio of the fusion peptide to the PEG derivative is (2-100):

1.

8. The preparation method according to claim 5, characterized in that, The molar ratio of the fusion peptide to the PEG derivative is (2-10):

1.

9. The preparation method according to claim 6, characterized in that, The incubation temperature is 0-20℃, and the time is 2-20h.

10. The preparation method according to claim 6, characterized in that, The incubation temperature is 4-6℃, and the time is 6-12h.

11. The preparation method according to claim 5, 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).

12. The preparation method according to claim 5, characterized in that, In step (2), the molar ratio of the lipid nanoparticles coupled with the fusion polypeptide to the extracellular vesicles is 1:(1-2).

13. The preparation method according to claim 5, characterized in that, In step (2), the lipid nanoparticles coupled with the fusion peptide and the extracellular vesicles are mixed at a temperature of 0-20°C for a time of 0.5-5h.

14. The preparation method according to claim 5, characterized in that, In step (2), the lipid nanoparticles coupled with the fusion peptide and the extracellular vesicles are incubated at a temperature of 4-6°C for 0.5-1h.

15. The preparation method according to claim 5, characterized in that, Step (1) further includes mixing lipid nanoparticles coupled with fusion peptides with a solution containing a pharmaceutical active ingredient, thereby encapsulating the pharmaceutical active ingredient inside the lipid nanoparticles.

16. A pharmaceutical composition comprising a delivery system according to any one of claims 1-4 or a delivery system prepared by any one of claims 5-15, and a pharmaceutical active ingredient.

17. The pharmaceutical composition according to claim 16, characterized in that, The active pharmaceutical ingredient is selected from nucleic acids, plasmids, small molecule compounds, peptides and / or proteins.

18. The pharmaceutical composition according to claim 17, characterized in that, The nucleic acid is selected from DNA, RNA, and combinations thereof.

19. The pharmaceutical composition according to claim 17, characterized in that, The nucleic acid is selected from mRNA, siRNA, rRNA, tRNA, snRNA, miRNA, and combinations thereof.

20. The pharmaceutical composition according to any one of claims 16-19, characterized in that, The active pharmaceutical ingredient is encapsulated within the lipid nanoparticles.

21. The use of the delivery system according to any one of claims 1-4 or the delivery system prepared by the preparation method according to any one of claims 5-15 in the preparation of a drug active ingredient delivery system.

22. The application according to claim 21, characterized in that, The active pharmaceutical ingredient is selected from nucleic acids, plasmids, small molecule compounds, peptides and / or proteins.

23. The application according to claim 22, characterized in that, The nucleic acid is selected from DNA, RNA, and combinations thereof.

24. The application according to claim 22, characterized in that, The nucleic acid is selected from mRNA, siRNA, rRNA, tRNA, snRNA, miRNA, and combinations thereof.

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