WO2022152145A1 - 一种纳米复合物及其制备方法和用途 - Google Patents

一种纳米复合物及其制备方法和用途 Download PDF

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WO2022152145A1
WO2022152145A1 PCT/CN2022/071526 CN2022071526W WO2022152145A1 WO 2022152145 A1 WO2022152145 A1 WO 2022152145A1 CN 2022071526 W CN2022071526 W CN 2022071526W WO 2022152145 A1 WO2022152145 A1 WO 2022152145A1
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protein drug
protamine
hyaluronic acid
dosage
nanocomposite
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PCT/CN2022/071526
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English (en)
French (fr)
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高小玲
黄佳琳
江淦
宋清香
陈红专
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上海交通大学医学院
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Priority to JP2023542742A priority Critical patent/JP2023553518A/ja
Priority to EP22739023.4A priority patent/EP4279067A4/en
Priority to US18/271,947 priority patent/US20240115504A1/en
Publication of WO2022152145A1 publication Critical patent/WO2022152145A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1277—Processes for preparing; Proliposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/728—Hyaluronic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/38—Albumins
    • A61K38/385—Serum albumin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
    • C12Y111/01—Peroxidases (1.11.1)
    • C12Y111/01006—Catalase (1.11.1.6)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies

Definitions

  • the present invention relates to the field of biotechnology and chemical pharmacy, in particular to a nanocomposite for protein drug delivery, a preparation method thereof, and use as a protein drug delivery carrier.
  • Protein drug therapy is the introduction of normal protein drugs into specific tissues or cells of patients to correct dysfunctional or lost proteins for therapeutic purposes.
  • protein drugs Compared with traditional small molecule drugs, protein drugs have the advantages of high biological activity, strong specificity, and clear biological functions; compared with gene therapy, protein drugs are faster and safer, and can directly exert their biological functions at specific sites. function, and does not involve changes in gene sequence.
  • protein drugs have become the focus of the most attention in the field of medicine, ranking first in the world's top ten best-selling drugs for many years.
  • the marketed protein drugs are mainly limited to molecules that function extracellularly, such as antibodies, which greatly limits the large-scale development and application of protein drugs.
  • the first type of method is to directly covalently link or fuse the target protein with molecules that promote endocytosis, such as linking penetrating peptides, targeting molecules, etc. to promote the uptake of protein drugs by target cells.
  • This method can efficiently deliver proteins into cells, the proteins entering cells often lack the ability to escape from cell endosomes, resulting in the degradation of proteins by cell endosomes before their function can be exerted.
  • this method involves complex synthesis and purification processes. It is very likely that the structure and function of the protein are disrupted in the process.
  • the second class of methods utilizes carrier materials to deliver proteins through specific intermolecular recognition or cationic carrier materials.
  • protein delivery carriers are roughly divided into two categories. ), recognition between polyhistidine (His-tag) and metal ions, transcription factors and promoters) or chemical reactions (such as amino and azide, tetrazine (Tz) and cycloalkyne (TCO), etc. to bind proteins.
  • Such carrier materials have strong binding force to proteins, but the preparation process involves complex protein modification process and carrier material synthesis and modification process.
  • specific molecular markers of proteins and their covalent binding with carrier materials may damage the structure and structure of proteins. Function. Therefore, most of the current research focuses on the second type of carrier materials, namely cationic carrier materials, which directly bind target proteins through electrostatic and hydrophobic interactions.
  • cationic carriers can be directly used for binding through electrostatic or hydrophobic interactions. and delivery.
  • Cheng's research group directly constructed a library of cationic materials, grafted different fluorine-containing small molecular compounds onto polyethyleneimine, and obtained a library of fluorine-containing polymer materials, and further screened the protein delivery performance of the polymer materials in the library. Two high-performance fluorine-containing polymer materials were successfully obtained.
  • fluorine-containing polymers can efficiently deliver a variety of protein molecules including bovine serum albumin, ⁇ -galactosidase, saponin, etc. to different cells, and can maintain the biological activity of these proteins or small peptides
  • bovine serum albumin ⁇ -galactosidase
  • saponin saponin
  • Rotello's group inserted small polyglutamic acid peptides of different lengths into the N-terminus of Cas9 protein, so that the positively charged Cas9 protein has enough negatively charged regions, and co-incubated the protein with sgRNA to form a negatively charged complex. Electrostatic interaction co-incubated with arginine-modified gold nanoparticles to self-assemble to form nanocomplexes. It was found that the complexes could be efficiently taken up by HeLa cells through cholesterol-dependent membrane fusion, achieving high efficiency of AAVS1 and PTEN genes. edit.
  • Recombinant lipoprotein is a kind of nanoparticle constructed by simulating natural nanostructure - lipoprotein, which has been developed into a widely used nano-drug carrier. No immunogenicity; 2 long circulation time in vivo; 3 multimodal drug loading; 4 in vivo targeting properties. Recombinant lipoprotein nanocarriers have been successfully applied to the effective delivery of some hydrophobic drugs, amphiphilic drugs and a small amount of hydrophilic drugs such as polypeptides and nucleic acid drugs.
  • our research group achieved targeted delivery of neuroprotective peptides to the core lesions of Alzheimer's disease where ⁇ -amyloid aggregates by establishing ⁇ -helical peptide fusion technology (ACS Nano, 2015); proposed the development of hydrophobic co-precipitation technology, The calcium phosphate core was introduced into the recombinant lipoprotein core to achieve efficient nucleic acid drug delivery and targeted delivery to gliomas (Nat Commun, 2017 &2020; Adv Sci, 2020).
  • the technical problem to be solved by the present invention is to overcome the lack of a carrier method suitable for protein drugs in the prior art, so as to achieve its efficient intracellular and in vivo targeted delivery, and to provide a nanocomposite and its preparation method and use .
  • the present invention solves the above technical problems through the following technical solutions.
  • the first object of the present invention is to provide a nanocomposite comprising 0-60% protein drug, 0.03-15% hyaluronic acid, 0.1-20% protamine, 35-95% lipid Quality components and 2.5-40% "apolipoprotein and/or its mimetic peptide";
  • the lipid components include electrically neutral lipids and anionic lipids; the total dosage of the hyaluronic acid and the protamine is 0.03-15%;
  • the above percentages are the respective mass percentages of each component relative to the nanocomposite.
  • the nanocomposite when the protein drug content is 0%, the nanocomposite is an empty carrier without protein drug loaded. When the protein drug content is not 0, the nanocomposite is a protein drug-loaded nanocomposite.
  • the cationic lipid DOTAP is not included in the nanocomposite.
  • the protein drug can be a conventional protein drug in the art.
  • the molecular weight of the protein drug may be 10-255kDa, such as 10-16kDa, 15-40kDa, 30-50kDa, 60-80kDa, 140-180kDa, 200-255kDa or 210-255kDa, preferably 10-14kDa, 20-33kDa, 35-45kDa, 60-80kDa, 150-170kDa, 220-250kDa, 220-250kDa, for example 12.4kDa, 26kDa, 40kDa, 69.3kDa, 160kDa or 240kDa.
  • 10-255kDa such as 10-16kDa, 15-40kDa, 30-50kDa, 60-80kDa, 140-180kDa, 200-255kDa or 210-255kDa, preferably 10-14kDa, 20-33kDa, 35-45kDa, 60-80kDa, 150-170kDa, 220-250kDa, 220-
  • the isoelectric point of the protein drug may be 4-11, for example, 4-5.3, 3.7-5.7, 4.4-6.4, 6-8.5, 7-9, 8.3-10.3 or 9.3-11, preferably 4- 4.8, 4.2-5.5, 4.9-5.9, 6.5-8, 7.5-8.5, 8.8-9.8 or 9.8-10.8, and another example is 4.3, 4.7, 5.4, 7.2, 8, 9.3 or 10.3.
  • the isoelectric point of the protein drug may also be 4-6, 6-8 or 8-10.5.
  • the following protein drugs can be used:
  • HRP Horseradish peroxidase
  • IgG antibodies 8 160 Nerve Growth Factor ( ⁇ -NGF) 9.3
  • Cytochrome C (CC) 10.3 12.4
  • the electrically neutral lipids generally refer to lipids whose positive and negative ions are balanced in solution, and the overall solution does not exhibit charged properties outward, preferably including amphoteric lipids, nonionic lipids, cholesterol and derivatives thereof one or more of. More preferably, one or more of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and sphingomyelin are included.
  • the phosphatidylcholine is preferably dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine Choline (DOPC), Dilauroylphosphatidylcholine (DLPC), Dierucylphosphatidylcholine DEPC, 1-palmitoyl-2-oleoylphosphatidylcholine POPC, Egg Lecithin, Soy Lecithin, Hydrogenated Soybean One or more of lecithin HSPC and derivatives thereof, more preferably one or more of dimyristoyl phosphatidyl choline (DMPC), egg yolk lecithin and hydrogenated soybean lecithin HSPC, such as DMPC and Egg yolk lecithin, alternatively, DMPC and hydrogenated soy lecithin.
  • DMPC dimyristoylphosphatid
  • the phosphatidyl ethanolamine is preferably dimyristoyl phosphatidyl ethanolamine (DMPE), distearoyl phosphatidyl ethanolamine (DSPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), dioleoyl phosphatidyl ethanolamine (DOPE), dioleoyl phosphatidyl ethanolamine (DOPE).
  • DMPE dimyristoyl phosphatidyl ethanolamine
  • DSPE distearoyl phosphatidyl ethanolamine
  • DPPE dipalmitoyl phosphatidyl ethanolamine
  • DOPE dioleoyl phosphatidyl ethanolamine
  • DOPE dioleoyl phosphatidyl ethanolamine
  • Stearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 distearoyl phosphatidyl ethanolamine-polyethylene glycol 5000, dipalmitoyl phosphatidyl ethanolamine-polyethylene glycol 2000, dipalmitoyl phosphatidyl ethanolamine-polyethylene glycol One or more of Alcohol 5000 and its derivatives.
  • the phosphatidyl glycerol is preferably dimyristoyl phosphatidyl glycerol (DMPG), distearoyl phosphatidyl glycerol (DSPG), dipalmitoyl phosphatidyl glycerol (DPPG), dioleoyl phosphatidyl glycerol (DOPG), 1 - One or more of palmitoyl-2oleoylphosphatidylglycerol (POPG-Na), egg yolk phosphatidylglycerol (EPG) and derivatives thereof.
  • DMPG dimyristoyl phosphatidyl glycerol
  • DSPG distearoyl phosphatidyl glycerol
  • DPPG dipalmitoyl phosphatidyl glycerol
  • DOPG dioleoyl phosphatidyl glycerol
  • POPG-Na palmitoyl-2oleoy
  • the electrically neutral lipid is a mixture of dimyristoyl phosphatidyl choline (DMPC) and egg yolk lecithin
  • the mass ratio of dimyristoyl phosphatidyl choline (DMPC) and egg yolk lecithin may be 20: (8-12), for example 20:10.94.
  • the anionic lipid generally refers to the lipid whose negative charge is greater than the positive charge in the solution, and the whole solution exhibits a negative charge outward, preferably an anionic phospholipid.
  • the anionic phospholipid preferably includes one or more of phosphatidic acid, phosphatidylinositol, phosphatidylserine, cardiolipin, lysophospholipid and ganglioside, more preferably phosphatidic acid and/or ganglioside .
  • the phosphatidic acid is preferably dimyristoyl phosphatidic acid (DMPA), distearoyl phosphatidic acid (DSPA), dipalmitoyl phosphatidic acid (DPPA), dioleoyl phosphatidic acid (DOPA) and derivatives thereof
  • DMPA dimyristoyl phosphatidic acid
  • DSPA distearoyl phosphatidic acid
  • DPPA dipalmitoyl phosphatidic acid
  • DOPA dioleoyl phosphatidic acid
  • DMPA dimyristoyl phosphatidic acid
  • DSPA distearoyl phosphatidic acid
  • DPPA dipalmitoyl phosphatidic acid
  • DOPA dioleoyl phosphatidic acid
  • the phosphatidylserine is preferably dioleoylphosphatidylserine (DOPS) and/or dipalmitoylphosphatidylserine (DPPS).
  • DOPS dioleoylphosphatidylserine
  • DPPS dipalmitoylphosphatidylserine
  • the lysophospholipid is preferably one or more of stearoyl lysolecithin S-lysoPC, myristoyl lysolecithin M-LysoPC, palmitoyl lysolecithin P-LysoPC and derivatives thereof.
  • the ganglioside is preferably monosialotetrahexosylganglioside (GM1).
  • the mass ratio of ganglioside and phosphatidic acid may be 30.07:(3-7), for example, 30.07:5.
  • the "apolipoprotein and/or its mimetic peptide” may be ApoE and its mimetic peptide, ApoA-I, ApoA-II, ApoA-IV and its mimetic peptide, ApoC-I, ApoC-II, ApoC - One or more of III and its peptidomimetics, ApoB and its peptidomimetics, ApoJ and its peptidomimetics, such as ApoE or ApoA-I peptidomimetics.
  • the dosage of the protein drug may be 1-43%, such as 1.5%, 1.78%, 2.2%, 3%, 4%, 4.8%, 5%, 6%, 8%, 8.5%, 9% , 10%, 11%, 12.06%, 15%, 16%, 16.5%, 17%, 17.73%, 19%, 20%, 22%, 28.08%, 28.68%, 29.31%, 30%, 31.5%, 32 %, 30.9%, 33%, 33.07%, 34%, 34.5% or 41%.
  • the amount of the hyaluronic acid can be 0.03-5%, such as 0.04%, 0.05%, 0.06%, 0.08%, 0.09%, 0.1%, 0.13%, 0.14%, 0.15%, 0.2%, 0.24% %, 0.25%, 0.3%, 0.33%, 0.35%, 0.41%, 0.43%, 0.44%, 0.45%, 0.47%, 0.5%, 0.6%, 0.78%, 0.8%, 0.9%, 1.53%, 2.1%, 0.2%, 2.1%, 2.5% or 3%.
  • the amount of protamine can be 0.2-16%, such as 0.1%, 0.2%, 0.3%, 0.35%, 0.8%, 0.83%, 1%, 1.3%, 1.5%, 1.6%, 1.7% %, 2%, 2.34%, 2.39%, 2.44%, 3%, 3.26%, 3.3%, 3.5%, 3.7%, 4%, 4.3%, 4.5%, 4.85%, 5%, 5.1%, 5.5%, 5.53%, 6%, 6.5%, 7%, 13% or 14%.
  • the dosage of the "apolipoprotein and/or its mimetic peptide" may be 2.85% to 25%, such as 2.92%, 3%, 3.5%, 3.82%, 3.94%, 4%, 4.1%, 4.12% %, 4.43%, 4.5%, 4.65%, 4.68%, 4.78%, 4.83%, 4.89%, 5%, 5.4%, 5.8%, 6%, 6.35%, 6.52%, 7.21%, 10.35%, 16.5%, 16.37%, 19.29% or 20%.
  • the amount of the lipid component is preferably 35-90%.
  • the amount of the anionic lipid can be 14-40%, such as 16, 17.64%, 22%, 24%, 24.04%, 24.78%, 24.8%, 25%, 26%, 25.66%, 26.1% , 27%, 27.22%, 27.5%, 29.79%, 29%, 30%, 30.5%, 30.66%, 31%, 31.08%, 31.11%, 32%, 31.5%, 32%, 32.5%, 33%, 33.06 %, 33.12%, 33.6%, 34%, 34.44%, 35%, 35.07%, 36%, 37.22%, 37.72%, 37.8%, 38%, or 39%.
  • the amount of the electrically neutral lipid may be 22-60%, such as 23.5%, 24.36%, 30%, 30.94%, 32%, 33%, 34.2%, 34.22%, 35%, 36.09% , 37%, 37.44%, 37.5%, 38.25%, 39.08%, 39.91%, 41%, 41.21%, 42%, 42.34%, 42.92%, 43.5%, 44%, 45%, 45.61%, 46%, 46.4 %, 47%, 47.56%, 48.43%, 50%, 52.17%, 52.2%, 53%, 54%, 54.5%, 55.32%, 56%, or 57%.
  • the total dosage of the hyaluronic acid and the protamine may be 0.2-14%, such as 0.3%, 0.5%, 0.64%, 0.8%, 0.85%, 0.97%, 1%, 1.08%, 1.15%, 1.2%, 1.5%, 1.63%, 1.71%, 1.79%, 2%, 2.15%, 2.35%, 2.58%, 2.63%, 2.68%, 3.2%, 3.5%, 4%, 4.17%, 5% , 5.08%, 5.5%, 5.6%, 5.96%, 6.38%, 6.5%, 7%, 8% or 8.6%, 13.06% or 13.5%.
  • the particle size of the nanocomposite may be 10-1000 nm, preferably 10-100 nm.
  • 12 ⁇ 95nm another example is 20.30 ⁇ 5.89nm, 23.79 ⁇ 7.91nm, 25nm, 26.52 ⁇ 4.31nm, 27.31 ⁇ 10.84nm, 27.38 ⁇ 7.83nm, 27.55 ⁇ 6.99nm, 37.98 ⁇ 14.29nm, 28.96 ⁇ 8.74nm, 31.11 ⁇ 3.44nm, 36.30 ⁇ 6.41nm, 37.22 ⁇ 7.28nm, 37.55 ⁇ 13.73nm, 37.63 ⁇ 4.20nm, 37.68 ⁇ 2.20nm, 38nm, 39.12 ⁇ 4.84nm, 40.55 ⁇ 7.66nm, 55nm, 55.75 ⁇ 7.69nm, 57nm, 60nm, 63.62 ⁇ 1.97nm, 70nm, 74.20 ⁇ 14.23nm or 75.29 ⁇ 14.
  • the Zeta potential of the nanocomposite can be -70 ⁇ -15mV, such as -65, -64.87 ⁇ 3.30, -63.7 ⁇ 2.66, -58.87 ⁇ 4.90, -57.33 ⁇ 2.31, -56.33 ⁇ 3.26, - 55.20 ⁇ 10.74, -52.07 ⁇ 2.15, -50.10 ⁇ 3.18, -48.87 ⁇ 1.95, -45.20 ⁇ 2.15, -44.87 ⁇ 0.45mV, -43.93 ⁇ 14.03, -43.87 ⁇ 9.68, -43.20 ⁇ 2.75mV, -40.23 ⁇ 6.92 , -38.27 ⁇ 13.10, -36.83 ⁇ 2.71, -31.57 ⁇ 4.67, -30, -25, -20, -21.03 ⁇ 2.47 or -19.43 ⁇ 1.96mV.
  • the nanocomposite comprises 0-30% protein drug, 0.15-2.1% hyaluronic acid, 1-6.5% Protamine, 4.65-7% "apolipoprotein and/or its mimetic peptide", 30-53.5% electrically neutral phospholipid and 23.5-38.5% anionic phospholipid; wherein the hyaluronic acid and protamine The total amount is 1 to 9%.
  • the isoelectric point of the protein drug may be 4.2 ⁇ 5.5, for example, 4.7.
  • the molecular weight of the protein drug may be 60-80 kDa, for example, 69.3 kDa.
  • the protein drug can be bovine serum albumin.
  • the dosage of the protein drug may be 0%, 9%, 16%, 22%, 28.08%, 28.68%, 29.31% or 30%.
  • the amount of the hyaluronic acid can be 0.15%, 0.24%, 0.33%, 0.35%, 0.47% or 2.1%.
  • the amount of protamine may be 1%, 2%, 2.34%, 2.39%, 2.44%, 3.26%, 3.7% or 6.5%.
  • the dosage of the "apolipoprotein and/or its mimetic peptide” may be 4.65%, 4.68%, 4.78%, 4.83%, 4.89%, 5.4%, 6.35% or 6.52%.
  • the "apolipoprotein and/or its mimetic peptide” can be ApoE and its mimetic peptide, ApoA-I, ApoA-II, ApoA-IV and its mimetic peptide, ApoC-I, ApoC-II, ApoC-III and one or more of its peptidomimetics, such as ApoE.
  • the electrically neutral phospholipid can be one or more of dimyristoyl phosphatidylcholine DMPC, egg yolk lecithin and hydrogenated soybean lecithin HSPC, such as DMPC and egg yolk lecithin, or DMPC and hydrogenated soybean Lecithin.
  • the electrically neutral phospholipid is a mixture of dimyristoyl phosphatidyl choline DMPC and egg yolk lecithin
  • the mass ratio of dimyristoyl phosphatidyl choline DMPC and egg yolk lecithin may be 20:(8-12), for example 20:10.94.
  • the amount of the electrically neutral phospholipid may be 30.94%, 37%, 37.44%, 38.25%, 39.08%, 43.5%, 48.43% or 52.17%.
  • the anionic phospholipid can be phosphatidic acid and/or ganglioside.
  • the phosphatidic acid is preferably dimyristoyl phosphatidic acid (DMPA), distearoyl phosphatidic acid (DSPA), dipalmitoyl phosphatidic acid (DPPA) or dioleoyl phosphatidic acid (DOPA).
  • the ganglioside is preferably monosialotetrahexosylganglioside (GM1).
  • the mass ratio of monosialotetrahexosylganglioside GM1 and DOPA can be 30.07:(3 ⁇ 7), for example, 30.07:5 .
  • the amount of the anionic phospholipid can be 24.04%, 25.66%, 26%, 27.22%, 31.5%, 33.06%, 35.07% or 37.72%.
  • the total amount of the hyaluronic acid and the protamine may be 1.15%, 2.35%, 2.58%, 2.63%, 2.68%, 4.17% or 8.6%.
  • the particle size of the nanocomposite may be 20-95nm, such as 27.55 ⁇ 6.99nm, 36.30 ⁇ 6.41nm, 37.63 ⁇ 4.20nm, 37.68 ⁇ 2.20nm, 39.12 ⁇ 4.84nm, 40.55 ⁇ 7.66nm, 55.75 ⁇ 7.69 nm, or 75.29 ⁇ 14.53 nm.
  • the zeta potential of the nanocomposite may be -70 ⁇ -20mV, such as -63.7 ⁇ 2.66, -64.87 ⁇ 3.30, -52.07 ⁇ 2.15, -45.20 ⁇ 2.15, -44.87 ⁇ 0.45mV, -43.20 ⁇ 2.75mV , -40.23 ⁇ 6.92 or -38.27 ⁇ 13.10mV.
  • the nanocomposite comprises 4-17% protein drug, 0.05-0.25% hyaluronic acid, 1-3.3 % protamine, 4-16.5% "apolipoprotein and/or its mimetic peptide", 42-53% electrically neutral phospholipid and 31-38% anionic phospholipid; wherein, said hyaluronic acid and said protamine
  • the total dosage is 1 to 3.5%.
  • the isoelectric point of the protein drug may be 4-4.8, for example, 4.3.
  • the molecular weight of the protein drug may be 220-250kDa, for example, 240kDa.
  • the protein drug can be phycoerythrin.
  • the dosage of the protein drug may be 4.8%, 8%, 8.5%, 11% or 16%.
  • the amount of the hyaluronic acid can be 0.08%, 0.13%, 0.15% or 0.2%.
  • the amount of protamine may be 1%, 1.5% or 3%.
  • the dosage of the "apolipoprotein and/or its mimetic peptide” may be 4.12%, 5.8%, 10.35% or 16.37%.
  • the "apolipoprotein and/or its mimetic peptide” can be ApoE and its mimetic peptide, ApoA-I, ApoA-II, ApoA-IV and its mimetic peptide, ApoC-I, ApoC-II, ApoC-III and one or more of its peptidomimetics, such as ApoE.
  • the electrically neutral phospholipid can be phosphatidylcholine.
  • the phosphatidylcholine is preferably DMPC.
  • the amount of the electrically neutral phospholipid may be 42.92%, 43.5%, 46.4%, 47.56% or 52.2%.
  • the anionic phospholipid can be DOPA.
  • the amount of the anionic phospholipid may be 31.08%, 31.5%, 33.6%, 34.44% or 37.8%.
  • the total amount of the hyaluronic acid and the protamine may be 1.08%, 1.15%, 1.2%, 1.63% or 3.2%.
  • the particle size of the nanocomposite may be 12 ⁇ 60 nm, for example, 20.30 ⁇ 5.89 nm, 23.79 ⁇ 7.91 nm, 27.31 ⁇ 10.84 nm, 28.96 ⁇ 8.74 nm or 37.55 ⁇ 13.73 nm.
  • the zeta potential of the nanocomposite may be -65 ⁇ -30mV, such as -58.87 ⁇ 4.90, -56.33 ⁇ 3.26, -50.10 ⁇ 3.18, -43.93 ⁇ 14.03 or -36.83 ⁇ 2.71mV.
  • the nanocomposite comprises 31.5-34.5% protein drug, 0.25-0.45% hyaluronic acid, 0.1-0.35% % protamine, 3-5% "apolipoprotein and/or its mimetic peptide", 35-37.5% electrically neutral phospholipid and 25-27.5% anionic phospholipid; wherein said hyaluronic acid and said protamine
  • the total dosage is 0.5 to 0.85%.
  • the isoelectric point of the protein drug may be 9.8-10.8, for example, 10.3.
  • the molecular weight of the protein drug may be 10-14 kDa, for example, 12.4 kDa.
  • the protein drug can be cytochrome c.
  • the dosage of the protein drug may be 32-34%, for example, 33.07%.
  • the amount of the hyaluronic acid can be 0.3-0.5%, for example, 0.44%.
  • the amount of protamine may be 0.1-0.3%, for example, 0.2%.
  • the dosage of the "apolipoprotein and/or its mimetic peptide” may be 3.5-4.5%, for example, 4.1%.
  • the "apolipoprotein and/or its mimetic peptide” can be ApoE and its mimetic peptide, ApoA-I, ApoA-II, ApoA-IV and its mimetic peptide, ApoC-I, ApoC-II, ApoC-III and one or more of its peptidomimetics, such as ApoE.
  • the electrically neutral phospholipid can be phosphatidylcholine.
  • the phosphatidylcholine is preferably DMPC.
  • the amount of the electrically neutral phospholipid may be 35-37%, for example, 36.09%.
  • the anionic phospholipid can be DOPA.
  • the amount of the anionic phospholipid may be 25-27%, for example, 26.1%.
  • the total dosage of the hyaluronic acid and the protamine may be 0.5-0.8%. For example 0.64%.
  • the particle size of the nanocomposite may be 25 ⁇ 38 nm, for example, 31.11 ⁇ 3.44 nm.
  • the zeta potential of the nanocomposite may be -25 ⁇ -15mV, for example, -21.03 ⁇ 2.47mV.
  • the nanocomposite comprises 5-43% protein drug, 0.04-0.9% hyaluronic acid, 1-14 % protamine, 2.5-20% "apolipoprotein and/or its mimetic peptide", 23.5-46% electrically neutral phospholipid and 16-32% anionic phospholipid; wherein said hyaluronic acid and said protamine
  • the total dosage is 1 to 13.5%.
  • the isoelectric point of the protein drug may be 7.5-8.5, for example, 8.
  • the molecular weight of the protein drug may be 150-170 kDa, for example, 160 kDa.
  • the protein drug can be an IgG antibody.
  • the dosage of the protein drug may be 6%, 20%, 33% or 41%.
  • the amount of the hyaluronic acid can be 0.06%, 0.09%, 0.15%, 0.41% or 0.78%.
  • the amount of protamine may be 1.3%, 1.7%, 2%, 4.3% or 13%.
  • the dosage of the "apolipoprotein and/or its mimetic peptide" can be 2.85%, 2.92%, 3.94%, 7.21%, 19.29%.
  • the "apolipoprotein and/or its mimetic peptide” can be ApoE and its mimetic peptide, ApoA-I, ApoA-II, ApoA-IV and its mimetic peptide, ApoC-I, ApoC-II, ApoC-III and one or more of its peptidomimetics, such as ApoE or ApoA-I peptidomimetics.
  • the electrically neutral phospholipid can be phosphatidylcholine.
  • the phosphatidylcholine is preferably DMPC.
  • the amount of the electrically neutral phospholipid may be 24.36%, 34.22%, 41.21%, 42.34%, or 45%.
  • the anionic phospholipid can be DOPA.
  • the amount of the anionic phospholipid can be 17.64%, 24.78%, 29.79%, 30% or 30.66%.
  • the total amount of the hyaluronic acid and the protamine may be 1.71%, 1.79%, 2.15%, 5.08% or 13.06%.
  • the particle size of the nanocomposite may be 15-95 nm, for example, 26.52 ⁇ 4.31 nm, 27.38 ⁇ 7.83 nm, 37.98 ⁇ 14.29 nm or 74.20 ⁇ 14.23 nm.
  • the zeta potential of the nanocomposite may be -70 ⁇ -20mV, such as -57.33 ⁇ 2.31, -55.20 ⁇ 10.74, -43.87 ⁇ 9.68 or -31.57 ⁇ 4.67mV.
  • the nanocomposite comprises 1-3% protein drug, 0.2-0.6% hyaluronic acid, 4.5-6.5% % protamine, 2.5-5% "apolipoprotein and/or its mimetic peptide", 54-57% electrically neutral phospholipid and 32-35% anionic phospholipid; wherein, said hyaluronic acid and said protamine
  • the total dosage is 5 to 7%.
  • the isoelectric point of the protein drug may be 8.8 ⁇ 9.8, for example, 9.3.
  • the molecular weight of the protein drug may be 20-33 kDa, for example, 26 kDa.
  • the protein drug can be nerve growth factor ⁇ -NGF.
  • the dosage of the protein drug may be 1.5-2.2%, for example, 1.78%.
  • the amount of the hyaluronic acid can be 0.3-0.5%, for example, 0.43%.
  • the amount of protamine may be 5-6%, for example, 5.53%.
  • the dosage of the "apolipoprotein and/or its mimetic peptide” may be 3-4.5%, for example, 3.82%.
  • the "apolipoprotein and/or its mimetic peptide” can be ApoE and its mimetic peptide, ApoA-I, ApoA-II, ApoA-IV and its mimetic peptide, ApoC-I, ApoC-II, ApoC-III and one or more of its peptidomimetics, such as ApoE.
  • the electrically neutral phospholipid can be phosphatidylcholine.
  • the phosphatidylcholine is preferably DMPC.
  • the amount of the electrically neutral phospholipid may be 54.5-56%, for example, 55.32%.
  • the anionic phospholipid can be DOPA.
  • the amount of the anionic phospholipid can be 32.5-34%, for example, 33.12%.
  • the total amount of the hyaluronic acid and the protamine may be 5.5-6.5%, for example, 5.96%.
  • the nanocomposite comprises 15-20% protein drug, 0.05-0.8% hyaluronic acid, 0.8-1.6 % protamine, 3-6% "apolipoprotein and/or its mimetic peptide", 44-47% electrically neutral phospholipid and 29-33% anionic phospholipid; wherein, the hyaluronic acid and the protamine
  • the total dosage is 0.3 to 2%.
  • the isoelectric point of the protein drug may be 6.5-8, for example, 7.2.
  • the molecular weight of the protein drug may be 35-45 kDa, for example, 40 kDa.
  • the protein drug can be active enzyme protein drug HRP.
  • the dosage of the protein drug may be 16.5-19%, for example, 17.73%.
  • the amount of the hyaluronic acid can be 0.1-0.3%, for example, 0.14%.
  • the amount of protamine may be 0.8-1.5%, for example, 0.83%.
  • the dosage of the "apolipoprotein and/or its mimetic peptide” may be 4-5%, for example, 4.58%.
  • the "apolipoprotein and/or its mimetic peptide” can be ApoE and its mimetic peptide, ApoA-I, ApoA-II, ApoA-IV and its mimetic peptide, ApoC-I, ApoC-II, ApoC-III and one or more of its peptidomimetics, such as ApoE.
  • the electrically neutral phospholipid can be phosphatidylcholine.
  • the phosphatidylcholine is preferably DMPC.
  • the amount of the electrically neutral phospholipid may be 45-46%, for example, 45.61%.
  • the anionic phospholipid can be DOPA.
  • the amount of the anionic phospholipid may be 30.5-32%, for example, 31.11%.
  • the total amount of the hyaluronic acid and the protamine may be 0.5-1.5%, for example, 0.97%.
  • the particle size of the nanocomposite may be 20 ⁇ 57 nm, for example, 37.22 ⁇ 7.28 nm.
  • the zeta potential of the nanocomposite may be -55 ⁇ -15mV, for example, -48.87 ⁇ 1.95mV.
  • the nanocomposite comprises 10-33% protein drug, 0.2-3% hyaluronic acid, 3.5-7% % protamine, 3-6% "apolipoprotein and/or its mimetic peptide", 32-42% electrically neutral phospholipid and 22-39% anionic phospholipid; wherein, said hyaluronic acid and said protamine
  • the total dosage is 4 to 8%.
  • the isoelectric point of the protein drug may be 4.9-5.9, for example, 5.4.
  • the molecular weight of the protein drug may be 220-250kDa.
  • the protein drug can be catalase (CAT).
  • the dosage of the protein drug may be 11-32%, for example, 12.06 or 30.9%.
  • the amount of the hyaluronic acid may be 0.3-2.5%, for example, 0.5% or 1.53%.
  • the amount of protamine may be 4-5.5%, such as 4.85% or 5.1%.
  • the dosage of the "apolipoprotein and/or its mimetic peptide” may be 4-5%, for example, 4.43% or 4.5%.
  • the "apolipoprotein and/or its mimetic peptide” can be ApoE and its mimetic peptide, ApoA-I, ApoA-II, ApoA-IV and its mimetic peptide, ApoC-I, ApoC-II, ApoC-III and one or more of its peptidomimetics, such as ApoE.
  • the electrically neutral phospholipid can be phosphatidylcholine.
  • the phosphatidylcholine is preferably DMPC.
  • the amount of the electrically neutral phospholipid may be 33-41%, for example, 34.2% or 39.91%.
  • the anionic phospholipid can be DOPA.
  • the amount of the anionic phospholipid may be 24-38%, for example, 24.8% or 37.22%.
  • the total amount of the hyaluronic acid and the protamine may be 5-7%, for example, 5.6% or 6.38%.
  • the particle size of the nanocomposite may be 55 ⁇ 70 nm, for example, 63.62 ⁇ 1.97 nm.
  • the zeta potential of the nanocomposite may be -25 ⁇ -15mV, for example, -19.43 ⁇ 1.96mV.
  • the second object of the present invention is to provide a method for preparing the nanocomposite, which is prepared by the following method one or two:
  • the lipid composition prepares liposomes by conventional methods
  • the nanogel and the liposome are co-incubated to form a liposome containing the nanogel;
  • the mixture of the nanogel-containing liposome and "the apolipoprotein and/or its mimetic peptide” forms the nanocomplex by self-assembly;
  • the lipid composition is prepared by conventional methods for liposomes
  • the nanogel and the liposome are prepared by a microfluidic chip to prepare the liposome containing the nanogel, and after the solvent is removed by ultrafiltration, the liposome containing the protein drug is obtained;
  • the mixture of the protein-loaded liposome and "the apolipoprotein and/or its mimetic peptide” forms the nanocomplex by self-assembly.
  • the mixing operation and method can be conventional in the art.
  • the conventional method can be the preparation method of conventional liposomes in the art, such as a thin film hydration method, an injection method, a reconstitution method, a melting method or a freeze-drying method.
  • the liposome can be prepared by the following steps: the lipid component is dissolved in an organic solvent, and then the solvent is evaporated on a rotary evaporator to remove the solvent, so that the lipid component is in A film is formed on the wall of the vessel, then an appropriate amount of buffer solution is added, and it is fully hydrated and dispersed by shaking to obtain liposomes. Liposome particle size can be further reduced by sonication with a sonicator probe.
  • the liposome can be prepared by the following steps: dissolving the lipid component in ethanol to form a liposome.
  • Method 1 in S3 the operation and method of the incubation can be conventional in the art.
  • the operation and method of the self-assembly can be conventional in the field, for example, the mixture can be incubated on a shaking shaker at 120 rpm and 37° C. for 36 hours.
  • Method 2 S3, the method for preparing the nanogel-containing liposome with the nanogel and the liposome through a microfluidic chip can be a conventional method in the art.
  • the third object of the present invention is to provide an application of the nanocomposite in the delivery of the protein drug.
  • the nanocomposite can be used for intracellular delivery of protein drugs.
  • the nanocomposite can be used for in vivo delivery of protein drugs.
  • the nanocomposite can be used for intracerebral delivery of protein drugs.
  • the reagents and raw materials used in the present invention are all commercially available.
  • the nanocomposite of the present invention comprises a specific amount of protein drug, hyaluronic acid, protamine and recombinant lipoprotein, which constitutes a microenvironment suitable for protein drug through appropriate proportions of hyaluronic acid and protamine, and realizes the protein drug through recombinant lipoprotein. Efficient intracellular, in vivo and even intracerebral delivery of drugs.
  • the nanocomposite of the present invention provides a universal carrier for protein drugs with different physical and chemical properties (such as molecular weight 10-255KDa and PI 4-11), and realizes effective intracellular delivery and in vivo delivery.
  • the technical problem of insufficient in vivo and in vitro delivery of existing protein drugs including protein drugs whose molecular weight and PI exceed the examples).
  • Figure 1 shows the fluorescence distribution of the uptake of bovine serum albumin by Hela cells with different prescriptions.
  • Figure 2 shows the fluorescence distribution of phycoerythrin uptake by Hela cells with different prescriptions.
  • Figure 3 is a fluorescence distribution diagram of the uptake of cytochrome C by U87 cells with different prescriptions.
  • Figure 4 shows the fluorescence distribution of the uptake of Alexa Fluor488-IgG with different formulations by HT22 cells.
  • Figure 5 is a fluorescence distribution diagram of the uptake of Alexa Fluor488-IgG nanocomplexes loaded with different formulations by C6 cells.
  • Figure 6 shows that the nanocomplexes loaded with nerve growth factor (NGF) promote PC12 cell differentiation.
  • Figure 6A is the statistics of neurite lengths of different preparations promoting PC12 cell differentiation;
  • Figure 6B is the statistics of neurite branch points of different preparations promoting PC12 cell differentiation;
  • Figure 6C is a morphological diagram of different preparations promoting PC12 cell differentiation.
  • NGF nerve growth factor
  • Figure 7 is a graph showing the fluorescence distribution of the uptake of catalase by BV2 cells with different prescriptions.
  • Figure 8 shows that CAT-HA-PRTM-rHDL effectively increased intracellular catalase level (Figure 8A), and effectively reduced the increase in intracellular ROS level caused by H 2 O 2 ( Figure 8B).
  • Figure 9 shows that CAT-HA-PRTM-rHDL effectively inhibited cell membrane damage induced by H 2 O 2 (Figure 9A), and effectively increased cell viability (Figure 9B).
  • Figure 10 shows that in the cortical injury model of C57 mice, fluorescent dye DiR-labeled recombinant lipoprotein nanocomplexes (CAT-HA-PRTM-rHDL) and hyaluronan-loaded hyaluronic acid, protamine, and CAT were administered to the tail vein.
  • the liposomes of acid, protamine and CAT (CAT-HA-PRTM-LIPO) were taken 4 hours after administration, and the organs of mice were taken for in vivo imaging to evaluate the in vivo distribution and brain entry of the preparation.
  • Figure 11 shows the cortical injury model of CX3CR1-GFP mice. Different preparations labeled with fluorescent dye DiI were administered through the tail vein. After 4 hours of administration, the mouse brains were taken for frozen sections to evaluate the in vivo effects of CAT-loaded nanocomplexes on the brain. Microglia uptake.
  • Figure 12 shows the cortical injury model of C57 mice was constructed.
  • the Sham group was the sham injury control group. After modeling, the mice were given continuous administration for 1 week. Morris water maze test was used to investigate the latency and the number of times of crossing the platform of CCI model mice with different preparations loaded with CAT. and the effect of time in the target quadrant.
  • Figure 13 shows the use of SOD mouse model, continuous administration and simultaneous evaluation of mouse exercise ability.
  • Rod bar test, grasping bar test and hindlimb clenching test are used to investigate the effect of different preparations loaded with CAT on the exercise ability of SOD transgenic mice.
  • Figure 14 is a graph showing the fluorescence distribution of the uptake of the blank vector with or without protein loading by Hela cells ( Figure 14A) and the distribution of the blank vector with or without protein loading in the brain in a mouse cortical injury model ( Figure 14B). ).
  • lipids neutral phospholipid DMPC and/or lecithin lecithin, cationic phospholipid DOTAP, and anionic phospholipid DOPA and/or anionic sphingolipid monosialotetrahexosylganglioside Lipid GM1 and/or DMPA, DPPA, DSPA
  • lipids neutral phospholipid DMPC and/or lecithin lecithin, cationic phospholipid DOTAP, and anionic phospholipid DOPA and/or anionic sphingolipid monosialotetrahexosylganglioside Lipid GM1 and/or DMPA, DPPA, DSPA
  • the bovine serum albumin-loaded nanocomplexes were negatively stained with phosphotungstic acid, and the morphology was observed by transmission electron microscopy. After further sample preparation, cryo-electron microscopy was used to observe the structure. The particle size and surface potential were measured by laser particle size analyzer. The encapsulation efficiency and drug loading capacity of the protein drug by the nanocomplex loaded with bovine serum albumin were detected by a microplate reader.
  • control groups were: 1 free FITC-BSA protein; 2 hyaluronic acid, protamine, FITC-BSA complex (FITC-BSA-HA-PRTM), that is, according to the preparation method of step 1 of step (1)
  • the prepared protein-loaded complex 3.
  • BSA-loaded recombinant lipoprotein nanocomplex (FITC-BSA-rHDL) without hyaluronic acid and protamine.
  • Other preparation methods are the same as step (1); 4 Liposomes loaded with hyaluronic acid, protamine and FITC-BSA (FITC-BSA-HA-PRTM-LIPO), except that no apolipoprotein is added.
  • the preparation methods of liposomes are the same as steps (1) 12; 5FITC-BSA+ commercial protein transfection reagent (FITC-BSA-Pulsin); 6Containing bovine serum albumin with different anions (FITC-BSA) Recombinant lipoprotein nanocomplexes, the preparation method is the same as FITC-BSA-HA-PRTM-rHDL, except that DOPA is replaced by DMPA or DPPA or DSPA respectively to obtain the nanocomplexes loaded with bovine serum albumin, respectively FITC-BSA-HA- PRTM-rHDL-1, FITC-BSA-HA-PRTM-rHDL-2, FITC-BSA-HA-PRTM-rHDL-3.
  • the experimental group and the control group (administration concentration of 20 ⁇ g/mL, calculated by the amount of FITC-BSA protein) were incubated at 37 °C for 4 h. Then, the cells were fixed with 3.7% formaldehyde at 37°C for 10 min, Hoechest stained the nucleus for 10 min, washed three times with PBS, and then qualitatively observed by confocal imaging. Image J software was used for semi-quantitative analysis of the cellular uptake of FITC-BSA.
  • the optimized FITC-BSA-HA-PRTM-rHDL formulation delivers BSA more efficiently into cells than FITC-BSA, FITC-BSA-HA-PRTM, FITC-BSA-rHDL, FITC-BSA-HA-PRTM-LIPO (The average optical density value of cellular uptake is higher), and it is more evenly distributed in the cytoplasm (as shown in Table 1, Figure 1).
  • Table 1, Figure 1 The average optical density value of cellular uptake is higher
  • the commercially available protein transfection preparation group showed stronger cell-associated fluorescence intensity, most of the fluorescence signal was distributed extracellularly ( Figure 1).
  • the protein nanocomplexes formed by different anionic lipids DMPA, DPPA or DSPA can efficiently deliver proteins to the cell cytoplasm.
  • the prescription composition of FITC-BSA-HA-PRTM-rHDL in Table 1 is the prescription composition of Formula 6 in Table 2.
  • 2“Untested” means that no relevant testing has been done.
  • 3 Zeta potential represents the potential of the nanoparticle, which is determined by the material composing the nanocarrier. 4*** There is a significant difference between the FITC-BSA-HA-PRTM-rHDL group; ### and the FITC-BSA group have a significant difference.
  • Figure 1 shows the uptake of bovine serum albumin by Hela cells with different prescriptions.
  • Free green fluorescent protein-labeled bovine serum albumin FITC-BSA
  • FITC-BSA-HA-PRTM-rHDL recombinant lipoprotein nanocomplex
  • FITC-BSA-HA-PRTM-rHDL recombinant lipoprotein nanocomplex
  • FITC-BSA-HA-PRTM hyaluronic acid
  • FITC-BSA-HA-PRTM hyaluronic acid
  • BSA-loaded recombinant lipoprotein nanocomplex FITC-BSA-rHDL
  • Protamine, liposomes of FITC-BSA FITC-BSA-HA-PRTM-LIPO
  • FITC-BSA + commercial protein transfection reagent FITC-BSA-Pulsin
  • Formulas 1, 2, and 7 in Table 2 are control formulas, and formulas 3-6 are the formulas of the embodiments of the present invention.
  • the method for evaluating the stability of the preparation is: stand at 4°C for 12 hours.
  • the component EPC is cationic lipid.
  • Easy sedimentation in the stability of the formulation means that the sedimentation is visible to the naked eye when stored at 4°C for 12 h, and the particle size increases to more than 1000 nm.
  • 4 Formulas 3-6 have better stability. After standing at 4°C for 1 month, the particle size and potential are stable, and there is no change compared with before. 5
  • Statistical analysis Taking prescription 1 as the control, ***P ⁇ 0.001, there is a significant difference between prescription 1 and prescription 1. 6
  • lecithin refers to egg yolk lecithin, and the equivalent amount was replaced with hydrogenated soybean lecithin, and the effect was equivalent.
  • PE-HA-PRTM 1Incubate phycoerythrin (PE) with hyaluronic acid and protamine according to different mass ratios to form a carrier protein complex (PE-HA-PRTM);
  • PE-HA-PRTM-LIPO protein-loaded liposomes
  • PE-HA-PRTM-rHDL PE-loaded nanocomposite
  • the PE-loaded nanocomposites were negatively stained with phosphotungstic acid, and the morphology was observed by transmission electron microscopy. After further sample preparation, cryo-electron microscope was used to observe its structure. The particle size and surface potential were measured by laser particle size analyzer. The microplate reader detects the encapsulation efficiency and drug loading capacity of the protein drug by the PE-loaded nanocomposite.
  • PE-HA-PRTM-rHDL PE-loaded recombinant lipoprotein nanocomplex
  • Figure 2 shows the uptake of phycoerythrin by Hela cells with different prescriptions.
  • PE Phycoerythrin
  • PE-HA-PRTM-rHDL PE-loaded recombinant lipoprotein nanocomplex
  • PE-HA- PRTM PE-loaded recombinant lipoprotein nanocomplexes without hyaluronic acid, protamine
  • PE-rHDL PE-loaded liposomes
  • PE + commercial protein transfection reagent PE-Pulsin
  • formula 7 has the largest cell uptake average optical density value, indicating that its cell uptake efficiency is the highest and the effect is the best.
  • the cytochrome C-loaded nanocomplexes were negatively stained with phosphotungstic acid, and the morphology was observed by transmission electron microscopy. After further sample preparation, cryo-electron microscope was used to observe its structure. The particle size and surface potential were measured by laser particle size analyzer. The encapsulation efficiency and drug loading capacity of the protein drug by the cytochrome C-loaded nanocomposite were detected by a microplate reader.
  • the intracellular protein delivery of the cytochrome C-loaded nanocomplexes was observed by confocal laser microscopy.
  • the glioma cell line U87 was seeded in a confocal dish at a density of 50,000/well and cultured for 24h.
  • Aspirate the original culture solution add 500 ⁇ L of recombinant lipoprotein nanocomplex (FITC-CC-HA-PRTM-rHDL) loaded with green fluorescent protein-labeled cytochrome C (FITC-CC), and the free FITC-CC protein in the control group.
  • Hyaluronic acid, protamine, FITC-CC complex (FITC-CC-HA-PRTM); CC-loaded recombinant lipoprotein nanocomplex (FITC-CC-rHDL) without hyaluronic acid and protamine; Liposomes loaded with hyaluronic acid, protamine, FITC-CC (FITC-CC-HA-PRTM-LIPO) and FITC-CC + commercial protein transfection reagent (FITC-CC-Pulsin) (administration concentration of 20 ⁇ g /mL, calculated by the amount of FITC-CC protein), incubated at 37°C for 6h. Then, the cells were fixed with 3.7% formaldehyde at 37°C for 10 min, stained with Hoechest for 10 min, washed 3 times with PBS, and then qualitatively observed by confocal photography.
  • FITC-CC-HA-PRTM Hyaluronic acid, protamine, FITC-CC complex
  • FITC-CC-rHDL
  • FITC-CC-HA-PRTM-rHDL has better stability. After standing at 4°C for 1 month, the particle size and potential are stable, and there is no change compared with before. 3 Statistical analysis, with FITC-CC-HA-PRTM-rHDL as the control. ***P ⁇ 0.001 and FITC-CC-HA-PRTM-rHDL group have significant difference.
  • Figure 3 is a fluorescence distribution diagram of the uptake of cytochrome C by U87 cells with different prescriptions.
  • Green fluorescent protein-labeled cytochrome C FITC-CC
  • FITC-CC-HA-PRTM-rHDL recombinant lipoprotein nanocomplex
  • FITC-CC-HA-PRTM-rHDL recombinant lipoprotein nanocomplex
  • FITC-CC-HA-PRTM-rHDL loaded with hyaluronic acid, protamine, FITC-CC complex
  • FITC-CC-HA-PRTM FITC-CC-loaded recombinant lipoprotein nanocomplex
  • FITC-CC-rHDL without hyaluronic acid, protamine
  • hyaluronic acid-loaded Acid protamine, liposomes of FITC-CC complex
  • FITC-CC-HA-PRTM-LIPO FITC-CC + commercial protein transfection reagent
  • the nanocomplexes loaded with Alexa Fluor488-IgG antibody were negatively stained with phosphotungstic acid, and the morphology was observed by transmission electron microscope. After further sample preparation, cryo-electron microscope was used to observe its structure. The particle size and surface potential were measured by laser particle size analyzer. The encapsulation efficiency and drug loading capacity of protein drugs by nanocomplexes loaded with Alexa Fluor488-IgG antibody were detected by a microplate reader.
  • the cells were fixed with 3.7% formaldehyde at 37°C for 10 min, and the nucleus was stained with Hoechest for 10 min. After washing three times with PBS, the cells were qualitatively observed by confocal imaging, and the cellular uptake of Alexa Fluor488-IgG was semi-quantitatively analyzed by Image J software.
  • Alexa Fluor488-IgG-HA-PRTM Alexa Fluor488-IgG-HA-PRTM
  • Alexa Fluor488-IgG-rHDL Alexa Fluor488-IgG-HA-PRTM-rHDL formulation
  • Alexa Fluor488-IgG-HA-PRTM-rHDL formulation was more effective Alexa Fluor488-IgG was delivered into cells and distributed more evenly in the cytoplasm (Table 6, Figure 4).
  • the commercially available protein transfection preparation group showed stronger cell-associated fluorescence intensity, most of the fluorescence signal was distributed extracellularly.
  • Figure 4 shows the fluorescence distribution of the uptake of Alexa Fluor488-IgG with different formulations by HT22 cells.
  • Alexa Fluor488-IgG recombinant lipoprotein nanocomplexes loaded with hyaluronic acid, protamine, Alexa Fluor488-IgG (Alexa Fluor488-IgG-HA-PRTM-rHDL); hyaluronic acid, protamine, Alexa Fluor488-IgG Complex (Alexa Fluor488-IgG-HA-PRTM); Alexa Fluor488-IgG-loaded recombinant lipoprotein nanocomplex (Alexa Fluor488-IgG-rHDL) without hyaluronic acid, protamine; loaded with hyaluronic acid, protamine Protein, Alexa Fluor488-IgG liposome (Alexa Fluor488-IgG-HA-PRTM-LIPO) and Alexa Fluor488-IgG
  • formula 5 is a control group, and formulas 1-4 are all examples of the present invention. ***P ⁇ 0.001, ****P ⁇ 0.0001 was significantly different from formula 5.
  • Figure 5 is a fluorescence distribution diagram of the uptake of Alexa Fluor488-IgG nanocomplexes loaded with different formulations by C6 cells.
  • ⁇ -NGF was selected as the research model of neurotrophic protein drugs, and the preparation method was the same as that of Example 1, and the nanocomposite and the control group loaded with ⁇ -NGF were prepared respectively in the experimental group.
  • the prescriptions of the experimental group and the control group are shown in Table 8. .
  • Mouse primary neurons or PC12 cells were selected and inoculated in 96-well plates.
  • the concentration of ⁇ -NGF was 100ng/mL.
  • Different protein-carrying preparations were incubated with cells for 8 hours.
  • the incucyte real-time dynamic cell imaging technology was used to detect ⁇ -NGF.
  • the experimental results are shown in Figure 6.
  • the NGF-loaded nanocomposite quickly and efficiently promoted the differentiation of PC12 cells into neurons, and the neuron branch points and synapse lengths were significantly increased, and the morphology was similar to neurons.
  • Figure 6 shows that the nanocomplexes loaded with nerve growth factor (NGF) promote PC12 cell differentiation.
  • Figure 6A is the statistics of neurite lengths of different preparations promoting PC12 cell differentiation;
  • Figure 6B is the statistics of neurite branch points of different preparations promoting PC12 cell differentiation;
  • Figure 6C is a morphological diagram of different preparations promoting PC12 cell differentiation.
  • the recombinant lipoprotein nanocomplex (NGF-HA-PRTM-rHDL) loaded with hyaluronic acid, protamine and NGF was incubated with PC12 cells for 8 hours; the control was free NGF; NGF recombinant lipoprotein nanocomplex (NGF-rHDL); blank liposome (liposome) and culture medium blank control (Control).
  • NGF nerve growth factor
  • HRP was selected as the research model of active enzyme protein drugs, and the preparation method was the same as that of Example 1, and the HRP-loaded nanocomposite of the experimental group and the control group were prepared respectively.
  • the prescriptions of the experimental group and the control group are shown in Table 9.
  • Hela cells were selected and inoculated in a 96-well plate, the concentration of HRP was 10ug/mL, and the cells were incubated with different protein-loaded preparations for 6 hours, then the cells were rinsed 3 times with PBS, and 200ul of TMB chromogenic solution was added to each well, and incubated at room temperature in the dark. 3-30min until the color develops to the expected depth, and the absorbance value is measured at 650nm, which indicates the level of intracellular HRP enzyme activity.
  • the experimental results are shown in Table 9.
  • the intracellular level of horseradish peroxidase in the free HRP group is the lowest, and the liposomes loaded with hyaluronic acid, protamine and HRP can partially deliver the HRP enzyme to the cells to improve the intracellular enzymes.
  • the recombinant lipoprotein nanocomplex (HRP-HA-PRTM-rHDL) loaded with hyaluronic acid, protamine and HRP significantly increased the intracellular horseradish peroxidase activity.
  • the catalase-loaded nanocomplexes were negatively stained with phosphotungstic acid, and the morphology was observed by transmission electron microscopy. After further sample preparation, cryo-electron microscope was used to observe its structure. The particle size and surface potential were measured by laser particle size analyzer. The encapsulation efficiency and drug loading capacity of the protein drug by the catalase-loaded nanocomposite were detected by a microplate reader.
  • FITC-CAT-HA-PRTM hyaluronic acid, protamine, FITC-CAT complex
  • FITC-CAT-rHDL CAT-loaded recombinant lipoprotein nanocomplex
  • Liposomes loaded with hyaluronic acid, protamine, FITC-CAT FITC-CAT-HA-PRTM-LIPO
  • FITC-CAT + commercial protein transfection reagent FITC-CAT-Pulsin
  • FITC-CAT-HA-PRTM-rHDL formulation compared to FITC-CAT, FITC-CAT-HA-PRTM, FITC-CAT-rHDL, FITC-CAT-HA-PRTM-LIPO and commercially available protein transfection preparation groups CAT was more efficiently delivered into cells and more uniformly distributed in the cytoplasm (Table 10, Figure 7).
  • FIG. 7 is a graph showing the fluorescence distribution of the uptake of catalase by BV2 cells with different prescriptions.
  • Green fluorescent protein-labeled catalase FITC-CAT
  • FITC-CAT recombinant lipoprotein nanocomplexes loaded with hyaluronic acid, protamine, FITC-CAT
  • FITC-CAT-HA-PRTM-rHDL hyaluronic acid, fish Protamine, FITC-CAT complex
  • FITC-CAT-HA-PRTM FITC-CAT-loaded recombinant lipoprotein nanocomplex
  • FITC-CAT-rHDL without hyaluronic acid, protamine
  • hyaluronic acid-loaded protamine, liposomes of FITC-CAT
  • FITC-CAT-HA-PRTM-LIPO FITC-CAT + commercial protein transfection reagent
  • Example 8 Transfection ability, antioxidant ability and evaluation of cell viability of catalase (CAT)-loaded nanocomposite on HT22 cells
  • Catalase (CAT) was selected as the research model of the protein drug, and the preparation method was the same as that of Example 7, and the nanocomplexes carrying catalase (CAT) were prepared respectively.
  • the prescriptions are shown in Table 11.
  • the transfection ability of CAT-loaded nanocomplexes on HT22 cells was detected by catalase detection kit.
  • the reaction principle is that when hydrogen peroxide is relatively sufficient, catalase can catalyze the production of water and oxygen from hydrogen peroxide.
  • the residual hydrogen peroxide can oxidize the chromogenic substrate under the catalysis of peroxidase to produce a red product with a maximum absorption wavelength of 520 nm.
  • make a standard curve so that the catalase in the sample can calculate how much hydrogen peroxide catalyzes the conversion of hydrogen peroxide into water and oxygen per unit time and unit volume, so as to calculate the amount of hydrogen peroxide in the sample.
  • the enzymatic activity of catalase is that catalase can catalyze the production of water and oxygen from hydrogen peroxide.
  • the residual hydrogen peroxide can oxidize the chromogenic substrate under the catalysis of peroxidase to produce a red product with a maximum absorption wavelength of 520
  • the specific experimental plan is to seed HT22 cells in a 12-well plate, and after culturing in a carbon dioxide incubator for 24 hours, different preparations loaded with CAT protein, including recombinant lipoprotein nanocomplexes containing hyaluronic acid, protamine and CAT (CAT- HA-PRTM-rHDL) and the control group were the untreated group (Control), the free CAT protein group and the CAT+ commercial protein transfection reagent group (CAT-Pulsin) (the administration concentration was 20 ⁇ g/mL, according to the amount of CAT protein). calculation), incubated at 37°C for 6h. Cells were lysed and intracellular catalase levels were measured according to the protocol.
  • CAT protein including recombinant lipoprotein nanocomplexes containing hyaluronic acid, protamine and CAT (CAT- HA-PRTM-rHDL) and the control group were the untreated group (Control), the free CAT protein group and the CAT+ commercial protein trans
  • HT22 cells were seeded in a 96-well plate. When the confluence reached 70%, different CAT protein-loaded pharmaceutical preparations were given. After 4 hours of administration, the preparation solution was discarded and the fluorescent probe DCFH was added. -DA was incubated with the cells for 30 minutes, then the probe solution was discarded, and 400 ⁇ M of hydrogen peroxide was added to the cells for 15 minutes to induce oxidative damage, and quantitative analysis was performed by a microplate reader. The results are shown in Figure 10.
  • the control group was the completely untreated group with intracellular reactive oxygen species levels.
  • the intracellular ROS level surged after the induction of hydrogen peroxide alone, while the administration of CAT-HA-PRTM-rHDL significantly reduced the cellular ROS induced by hydrogen peroxide. level increases.
  • FIG 8. CAT-HA-PRTM - rHDL effectively increased intracellular catalase levels (Figure 8A) and effectively reduced H2O2 - induced increase in intracellular ROS levels (Figure 8B).
  • the lactate dehydrogenase cytotoxicity detection kit mainly evaluates the integrity of the cell membrane, which is also one of the indicators for evaluating the cell state.
  • the procedure of cell administration is the same as above. After 4 hours of administration, the preparation solution is discarded, and hydrogen peroxide is added to co-incubate with the cells to induce oxidative damage. , 1 hour before the scheduled detection time point, take out the cell culture plate from the cell incubator, and add the LDH release reagent provided by the kit to the "sample maximum enzyme activity control well", and the addition amount is 10% of the original culture medium volume . After adding LDH release reagent, mix by pipetting several times, and then continue to incubate in the cell culture incubator.
  • mice were anesthetized by intraperitoneal injection of 5% chloral hydrate, fixed in a brain stereotaxic apparatus, the scalp was cut under aseptic conditions, the right parietal bone was exposed, and a circular cranial window was opened between the right coronal suture and herringbone suture and beside the midline. , expose the dura mater, and set different striking parameters to simulate different degrees of cortical damage models. Strike parameters: speed 1.5m/s, depth 1mm, diameter of strike head 2mm, contact time 100ms to simulate moderate injury, used for routine brain injury experiments. The skull is closed after injury and the skin is sutured. In the sham-injured mice, only the scalp was incised to expose the right parietal bone, and no damage was done.
  • the DiR-labeled preparation was administered in the tail vein (calculated according to the dosage of phospholipid DMPC at a concentration of 20 mg/kg), and the heart, liver, spleen, lung, kidney and brain tissues were taken out 4 hours after administration. After rinsing with saline, they were placed in a small animal in vivo imager to collect images to observe the distribution of nanocomposites in the body and the dynamic changes of their transport to the brain. The experimental results are shown in Fig. 10.
  • FIG. 10 In the cortical injury model of C57 mice, fluorescent dye DiR-labeled recombinant lipoprotein nanocomplexes loaded with hyaluronic acid, protamine, CAT (CAT-HA-PRTM-rHDL) and hyaluronan-loaded hyaluronan were administered into the tail vein.
  • the liposomes of acid, protamine and CAT (CAT-HA-PRTM-LIPO) were taken 4 hours after administration, and the organs of mice were taken for in vivo imaging to evaluate the in vivo distribution and brain entry of the preparation.
  • Example 10 Evaluation of the distribution of catalase (CAT)-loaded nanocomposite (ie, the experimental group FITC-CAT-HA-PRTM-rHDL of Example 7) in the brain of mice
  • the present invention selects CX3CR1-GFP transgenic mice with microglia transformed into green fluorescent protein to construct a CCI model, and also administers DiI-labeled fluorescent preparations in the tail vein after CCI, and evaluates DiI-CAT-HA-PRTM- The distribution of rHDL preparation in the brain of CCI mice and its uptake by microglia. After 3 hours of administration to the tail vein, the mice were anesthetized and fixed for cardiac perfusion. Post-fixed in formaldehyde for 24h, rinsed with PBS, dehydrated in 15% and 30% sucrose solution to sink, then embedded in O.C.T.
  • FIG. 11 The cortical injury model of CX3CR1-GFP mice was constructed. Different preparations labeled with fluorescent dye DiI were administered through the tail vein. After 4 h of administration, the mouse brain was taken for frozen section to evaluate the in vivo effect of CAT-loaded nanocomplexes on the brain. Microglia uptake.
  • Example 11 The effect of catalase (CAT)-loaded nanocomposite (ie, the experimental group FITC-CAT-HA-PRTM-rHDL of Example 7) on spatial learning and memory ability of CCI mice
  • a mouse model of moderate CCI brain damage was constructed. Different preparations loaded with CAT were administered to the tail vein (the concentration calculated according to CAT was 13300 Units/kg), and the mice were subjected to behavioral training and testing using the Morris water maze after continuous administration for 7 days. .
  • the water maze consists of three parts: a circular pool, a platform and a recording system; the pool is 150cm in diameter and 50cm in height. The pool is divided into 4 quadrants (I, II, III and IV quadrants). The pool is filled with water to a depth of 30cm, and white food coloring is added to make the water. It became opaque white, so that the experimental mice could not directly see the platform and the bottom of the pool; the water temperature was maintained at about 25°C.
  • Spatial reference objects (doors, cameras, wall signs, etc.) were set around the pool, and the positions remained unchanged for mice to locate and memorize the location of the platform.
  • the cylindrical platform is 9cm in diameter and 29cm in height, wrapped with non-reflective black cloth, placed in the IV quadrant, and the plane is 1cm below the water surface.
  • a camera was placed above the center of the pool to automatically collect swimming images of animals, and the collected signals were directly input into the computer, and the Morris water maze video analysis system 2.0 was used to monitor and record the swimming trajectories of mice.
  • the positioning navigation experiment (Hidden platform test) started after the mice were continuously administered for 7 days and lasted for 5 days; the water entry points of each training were 4 quadrants arranged according to the random principle, and different mice were placed in the same position each time. The mice were placed in the water facing the pool wall, and the sequence of entering the water was different every two consecutive days. The computer monitored and recorded the route and the time (latency period) that the mice took from entering the water to find and climb the black platform. Each mouse is trained 4 times a day, and the incubation period set for each training is 60s. If the mouse does not find the platform within 60s, it needs to be led to the platform and stay for 10s. At this time, the incubation period is recorded as 60s.
  • the interval between two training sessions of the mice was 30 s; in the space exploration experiment (Probe trial), the platform was removed on the 6th day after the positioning navigation test on the 5th day, and the mice were placed in the water facing the pool wall from the water entry points of the II and III quadrants, respectively, and recorded. The percentage of time that the mouse was in the target quadrant (the quadrant where the platform was located) within 60 s and the trajectory of the mouse searching for the platform. The experimental results are shown in Figure 12.
  • mice gradually shortened, and CAT-HA-PRTM-rHDL significantly shortened the latency of mice to find platforms; The times of crossing the platform and the residence time on the target platform increased significantly in PRTM-rHDL mice, suggesting that CAT-HA-PRTM-rHDL can efficiently deliver CAT protein to the site of brain damage, relieve the increased level of oxidative stress caused by brain damage, and improve the space of mice Learning and memory ability.
  • FIG. 12 The cortical injury model of C57 mice was constructed.
  • the Sham group was the sham injury control group. After modeling, the drug was administered continuously for 1 week.
  • the Morris water maze test was used to investigate the latency and the number of crossing the platform of different preparations loaded with CAT on the CCI model mice. and the effect of time in the target quadrant.
  • Example 12 The effect of catalase (CAT)-loaded nanocomposite (ie, the experimental group FITC-CAT-HA-PRTM-rHDL of Example 7) on exercise capacity and survival time of SOD mice
  • mice were administered with SOD1-G93A (amyotrophic lateral sclerosis model) CAT-HA-PRTM-rHDL in the same manner as in Example 10, and the administration group Saline and free CAT protein were used as control groups, and the SOD mice started at 3 months of age. Dosing was administered continuously every day. At the same time of administration, mice exercise ability evaluation experiments were conducted, including rotarod test, grasping bar test and hindlimb gripping test. The specific steps of the rotarod experiment were as follows: in the first week, the mice were trained three times, and the rotational speed of the rotarod was 14 rpm. At this speed, the mice stayed on the rotarod for 180 seconds and were defined as asymptomatic.
  • mice When the mice stayed on the rotarod for less than 180 seconds Defined as the clinical onset time, the mice can be tested after training for one week; the grasping rod test is to record the time that the mouse relies on the upper limb to grasp the rod, the total detection time is 60 seconds, and the mouse falling latency within 60 seconds is recorded; the hind limb is clenched tightly. The experiment is to hold the tail of the mouse and hang the mouse upside down, record a 15-second video and record the hindlimb grip of the mouse within 15 seconds. ). The experimental results are shown in Figure 13. In the initial stage of the test, the mice in each group stayed on the rotarod for a similar time.
  • mice given CAT-HA-PRTM-rHDL As time went on (rest at 4°C overnight), the mice given CAT-HA-PRTM-rHDL The retention time on the rod was significantly prolonged, and the grasping time of the mice in the CAT-HA-PRTM-rHDL group was significantly longer than that in the control group. .
  • Example 13 Preparation and characterization of protein-free blank nanocomplexes and their cellular uptake efficiency and intracerebral distribution
  • HA-PRTM 1Incubate hyaluronic acid and protamine together according to different mass ratios to form a carrier complex
  • the nanocomplexes without protein drugs were negatively stained with phosphotungstic acid, and the morphology was observed by transmission electron microscopy. After further sample preparation, cryo-electron microscopy was used to observe the structure. The particle size and surface potential were measured by laser particle size analyzer.
  • Experimental group nanocomplexes without protein drugs (DiI-HA-PRTM-rHDL) labeled with red fluorescent probes.
  • Control group BSA protein drug-containing nanocomplex (DiI-BSA-HA-PRTM-rHDL) labeled with red fluorescent probe, the preparation method is the same as that of Example 1.
  • the experimental group and the control group (administration concentration of 10 ⁇ g/mL, calculated according to the mass of phospholipid DMPC) were incubated at 37°C for 4h respectively. Then, the cells were fixed in 3.7% formaldehyde at 37°C for 10 min, and the nucleus was stained with Hoechest for 10 min. After three washes with PBS, the cells were qualitatively observed by confocal imaging, and the cellular uptake of nanocomplexes was semi-quantitatively analyzed by Image J software.
  • mice were used to construct a CCI model.
  • DiI-labeled fluorescent preparations were also administered to the tail vein after CCI. After 3 hours of tail vein administration, the mice were anesthetized and fixed for cardiac perfusion. The intact brains of tumor-bearing mice were taken, placed in 4% paraformaldehyde, fixed for 24 hours, rinsed with PBS and then placed in 15% and 30% Dehydrated in sucrose solution to sink, then embedded in O.C.T.
  • FIG. 14 The cortical injury model of C57 mice was constructed. Different preparations labeled with fluorescent dye DiI were administered through the tail vein. After 4 hours of administration, the mouse brain was taken for frozen section, and the protein-loaded or unloaded nanocomplexes were evaluated in small cells. Distribution in the rat brain.

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Abstract

本发明公开了一种纳米复合物及其制备方法和用途。纳米复合物包括0～60％蛋白质药物、0.03～15％透明质酸和0.8～20％鱼精蛋白、35～95％脂质成分和2.5～40％载脂蛋白和/或其模拟肽；脂质成分包括电中性脂质和阴离子脂质；透明质酸和鱼精蛋白的总用量为0.03～15％。本发明纳米复合物为不同理化性质(例如分子量10-255KDa和PI 4-11)的蛋白质药物提供通用型载体，实现高效的细胞内、体内乃至脑内递送，所采用技术具备普适性，可有效解决现有蛋白质药物（包括分子量、PI超出实施例的蛋白质药物）体内外输送不足的技术问题。

Description

一种纳米复合物及其制备方法和用途
本申请要求申请日为2021/1/13的中国专利申请2021100403712的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及生物技术领域以及化学制药领域，具体涉及一种用于蛋白质药物递送的纳米复合物及其制备方法和作为蛋白质药物递送载体的用途。
背景技术
作为细胞功能的执行者，蛋白质在细胞代谢、基因调控、信号传导和免疫反应等病理生理过程中扮演着十分重要的角色。蛋白质药物治疗是将正常的蛋白质药物导入患者特定的组织或细胞中以纠正功能失调或丢失的蛋白质从而达到治疗的目的。相比于传统小分子药物，蛋白质药物有着生物活性高、特异性强、生物功能明确等优越性；与基因疗法相比，蛋白质药物则更为快速而安全，可在特定位点直接发挥其生物学功能，且不涉及基因序列的改变。近年来，蛋白质药物成为医药领域最受关注的焦点，连续多年占据全球前十大畅销药榜首。然而，由于缺乏有效的蛋白质药物递送手段，已上市的蛋白类药物主要局限在抗体等在胞外发挥作用的分子上，极大限制了蛋白质类药物的大规模开发应用。
目前，蛋白质递送的方式主要有两大类。第一类方法是直接将目的蛋白质共价连接或融合促进细胞内吞的分子，例如连接穿膜肽、靶向分子等促进靶细胞摄取蛋白质药物。该方法虽然可以将蛋白质高效递送到细胞内，但是进入细胞的蛋白质往往缺乏从细胞内涵体逃逸的能力，导致蛋白功能尚未发挥即被细胞内体降解；同时该方法涉及复杂的合成和纯化过程，蛋白质的结构和功能很有可能在此过程中被破坏。第二类方法是利用载体材料通过分子间的特异性识别或者阳离子载体材料递送蛋白质。根据载体材料与蛋白质之间结合方式的不同，蛋白质递送载体大致分为两大类，第一类载体材料一般是通过分子间存在的一些特异性识别(如生物素(biotin)和亲和素(avidin)、多聚组氨酸(His-tag)与金属离子、转录因子与启动子之间的识别)或化学反应(如氨基与叠氮、四嗪(Tz)与环炔(TCO)等反应)来结合蛋白质。此类载体材料与蛋白结合力较强，但制备过程涉及复杂的蛋白质修饰过程以及载体材料合成修饰过程，同时蛋白质的特异性分子标记及其与载体材料的共价结合均可能破坏蛋白质的结构和功能。因此目前大部分研究集中于第二大类载体材料，即阳离子载体材料，其通过静电、疏水相互作用直接结合目标蛋白 质。由于蛋白质分子具有复杂的三维结构，其表面电荷及亲疏水基团亦不是均匀分布的，大部分蛋白质表面有部分的疏水区和负电区，因此可以直接利用阳离子载体通过静电或疏水相互作用进行结合和递送。例如Cheng课题组直接构建阳离子材料库，将不同的含氟小分子化合物接枝到聚乙烯亚胺上，得到了一个含氟高分子材料库，进一步对库中高分子材料的蛋白质递送性能进行筛选，成功获得了两种高性能含氟高分子材料。这些含氟高分子可以将包括牛血清白蛋白、β-半乳糖苷酶、皂草素等在内的多种蛋白质分子高效递送到不同的细胞中，且能够维持这些蛋白质或小肽的生物活性，然而考虑到阳离子材料体内毒性问题，这类载体多仅限于体外蛋白转染。而对于荷正电的蛋白质，研究者可通过对蛋白质进行超负电荷化改造使其整体带负电，再与阳离子载体材料通过静电相互作用载带。例如Rotello课题组在Cas9蛋白N端插入了不同长度聚谷氨酸小肽，使正电的Cas9蛋白具有足够的负电荷区域，并将该蛋白与sgRNA共孵形成负电复合物，上述复合物通过静电作用与精氨酸修饰的金纳米颗粒共孵从而自组装形成纳米复合物，实验发现该复合物能够被HeLa细胞通过胆固醇依赖的膜融合方式高效摄取，实现对AAVS1和PTEN两种基因的高效编辑。然而，应用超负电荷化改造虽然可以实现对不同荷电特性蛋白质的递送，但同样需要对蛋白质进行比较复杂的生物或化学合成过程，在改造过程中蛋白质的结构和功能可能被破坏。可见，目前尚缺乏一种方便、普适的方法能够实现高效的蛋白质药物载带和体内递送。
重组脂蛋白是一种模拟天然纳米结构——脂蛋白构建的纳米颗粒，已被发展成为一种广泛应用的纳米药物载体，其具有以下优势：①生物相容好，可完全被机体代谢利用且无免疫原性；②体内循环时间长；③多模式载药；④体内靶向特性。重组脂蛋白纳米载体，已被成功应用于部分疏水性药物、两亲性药物和少量亲水性药物如多肽、核酸药物的有效地送。如本课题组通过建立α螺旋肽融合技术实现了神经保护肽脑内靶向递送至β淀粉样蛋白聚集的阿尔茨海默病核心病变部位(ACS Nano,2015)；提出发展疏水共沉淀技术，将磷酸钙内核引入重组脂蛋白核心，实现了高效的核酸药物载带和对脑胶质瘤的靶向递释(Nat Commun,2017&2020；Adv Sci,2020)。然而，由于蛋白质药物的活性高度依赖于其复杂的高级结构，现有疏水共沉淀“硬核”式的药物包载方法，只能实现将蛋白质与外界环境的物理阻隔，无法避免内核蛋白质在沉淀条件下发生结构破坏和活性丧失。因此，亟需发展适合蛋白质药物的载带方法，以实现其高效的靶向递送。
在以往研究中，Leaf Huang教授研究组发明了一种称为阳离子脂质体-聚阳离子-透明质酸纳米复合物，并通过在PEG化磷脂分子末端连接上靶向基团实现对肿瘤的特异性识别，用以靶向递送基因药物至肿瘤细胞。虽然透明质酸、鱼精蛋白复合物已被用于核酸 类药物递送，并可通过外壳包被含阳离子脂质、聚乙二醇化磷脂的脂质体，用于体内递送。然而，上述体系无法直接用于蛋白质药物递送，且存在非特异细胞毒性、诱发过敏反应等风险。
发明内容
本发明所要解决的技术问题是克服现有技术中缺乏一种适合蛋白质药物的载带方法，以实现其高效的细胞内和体内靶向递送，而提供一种纳米复合物及其制备方法和用途。
本发明通过以下技术方案解决上述技术问题。
本发明的第一个目的在于，提供一种纳米复合物，所述纳米复合物包括0～60％蛋白质药物、0.03～15％透明质酸和0.1～20％鱼精蛋白、35～95％脂质成分和2.5～40％“载脂蛋白和/或其模拟肽”；
所述脂质成分包括电中性脂质和阴离子脂质；所述透明质酸和所述鱼精蛋白的总用量为0.03～15％；
上述百分比为各组分分别相对于所述纳米复合物的质量百分比。
本发明中，当蛋白质药物含量为0％时，所述纳米复合物为未载有蛋白质药物的空载体。当蛋白质药物含量不为0时，所述纳米复合物为载蛋白质药物的纳米复合物。
本发明中，优选所述纳米复合物中不包括阳离子脂质DOTAP。
本发明中，所述蛋白质药物可为本领域常规的蛋白质药物。
本发明中，所述蛋白质药物的分子量可为10～255kDa，例如10～16kDa、15～40kDa、30～50kDa、60～80kDa 140～180kDa、200～255kDa或者210～255kDa，优选为10～14kDa、20～33kDa、35～45kDa、60～80kDa、150～170kDa、220～250kDa、220～250kDa，再例如12.4kDa、26kDa、40kDa、69.3kDa、160kDa或者240kDa。
本发明中，所述蛋白质药物的等电点可为4～11，例如4～5.3、3.7～5.7、4.4～6.4、6～8.5、7～9、8.3～10.3或者9.3～11，优选4～4.8、4.2～5.5、4.9～5.9、6.5～8、7.5～8.5、8.8～9.8或者9.8～10.8，再例如4.3、4.7、5.4、7.2、8、9.3或者10.3。
本发明中，所述蛋白质药物的等电点还可为4～6、6～8或者8～10.5。
本发明优选实施例中，可采用下述蛋白质药物：
蛋白 等电点 分子量(kDa)
藻红蛋白PE 4.3 240
牛血清白蛋白BSA 4.7 69.3
过氧化氢酶(CAT) 5.4 250
辣根过氧化物酶(HRP) 7.2 40
IgG抗体 8 160
神经生长因子(β-NGF) 9.3 26
细胞色素C(CC) 10.3 12.4
本发明中，所述电中性脂质一般是指在溶液中正负离子平衡，整体溶液向外不表现带电的属性的脂质，优选包括两性脂质、非离子型脂质、胆固醇及其衍生物中的一种或多种。更优选包括磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰甘油和鞘磷脂中的一种或多种。
其中，所述磷脂酰胆碱优选为二肉豆蔻酰磷脂酰胆碱(DMPC)、二棕榈酰磷脂酰胆碱(DPPC)、二硬脂酰基磷脂酰胆碱(DSPC)、二油酰基磷脂酰胆碱(DOPC)、二月桂酰基磷脂酰胆碱(DLPC)、二芥酰磷脂酰胆碱DEPC、1-棕榈酰基-2-油酰基磷脂酰胆碱POPC、蛋黄卵磷脂、大豆磷脂、氢化大豆卵磷脂HSPC及其衍生物中的一种或多种，更优选为二肉豆蔻酰磷脂酰胆碱(DMPC)、蛋黄卵磷脂和氢化大豆卵磷脂HSPC中的一种或多种，例如DMPC和蛋黄卵磷脂、或者，DMPC和氢化大豆卵磷脂。
所述磷脂酰乙醇胺优选为二肉豆蔻酰磷脂酰乙醇胺(DMPE)、二硬脂酰基磷脂酰乙醇胺(DSPE)、二棕榈酰磷脂酰乙醇胺(DPPE)、二油酰基磷脂酰乙醇胺(DOPE)、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000、二硬脂酰基磷脂酰乙醇胺-聚乙二醇5000、二棕榈酰磷脂酰乙醇胺-聚乙二醇2000、二棕榈酰磷脂酰乙醇胺-聚乙二醇5000及其衍生物中的一种或多种。
所述磷脂酰甘油优选为二肉豆蔻酰磷脂酰甘油(DMPG)、二硬脂酰基磷脂酰甘油(DSPG)、二棕榈酰磷脂酰甘油(DPPG)、二油酰基磷脂酰甘油(DOPG)、1-棕榈酰基-2油酰基磷脂酰甘油(POPG-Na)、蛋黄磷脂酰甘油(EPG)及其衍生物中的一种或多种。
优选，当所述电中性脂质为二肉豆蔻酰磷脂酰胆碱(DMPC)和蛋黄卵磷脂的混合物时，二肉豆蔻酰磷脂酰胆碱(DMPC)和蛋黄卵磷脂的质量比可为20：(8～12)，例如20:10.94。
本发明中，所述阴离子脂质一般是指在溶液中负电荷大于正电荷，整体溶液向外表现带负电荷属性的脂质，优选为阴离子磷脂。其中，所述阴离子磷脂优选包括磷脂酸、磷脂酰肌醇、磷脂酰丝氨酸、心磷脂、溶血磷脂和神经节苷脂中的一种或多种，更优选为磷脂酸和/或神经节苷脂。
其中，所述磷脂酸优选为二肉豆蔻酰磷脂酸(DMPA)、二硬脂酰磷脂酸(DSPA)、二棕榈酰磷脂酸(DPPA)、二油酰基磷脂酸(DOPA)及其衍生物中的一种或多种，更优选为二肉豆蔻酰磷脂酸(DMPA)、二硬脂酰磷脂酸(DSPA)、二棕榈酰磷脂酸(DPPA)或者二油酰基磷脂酸(DOPA)。
所述磷脂酰丝氨酸优选为二油酰基磷脂酰丝氨酸(DOPS)和/或二棕榈酰磷脂酰丝氨酸(DPPS)。
所述的溶血磷脂优选为硬脂酰溶血卵磷脂S-lysoPC、肉豆蔻酰溶血卵磷脂M-LysoPC、棕榈酰溶血卵磷脂P-LysoPC及其衍生物中的一种或多种。
所述的神经节苷脂优选单唾液酸四己糖神经节苷脂(GM1)。
优选，当阴离子磷脂为磷脂酸和神经节苷脂的混合物时，神经节苷脂和磷脂酸质量比可为30.07：(3～7)，例如30.07:5。
本发明中，所述“载脂蛋白和/或其模拟肽”可为ApoE及其模拟肽，ApoA-I、ApoA-II、ApoA-IV及其模拟肽，ApoC-I、ApoC-II、ApoC-III及其模拟肽，ApoB及其模拟肽，ApoJ及其模拟肽中的一种或多种，例如ApoE或者ApoA-I模拟肽。
本发明中，所述蛋白质药物的用量可为1～43％，例如1.5％、1.78％、2.2％、3％、4％、4.8％、5％、6％、8％、8.5％、9％、10％、11％、12.06％、15％、16％、16.5％、17％、17.73％、19％、20％、22％、28.08％、28.68％、29.31％、30％、31.5％、32％、30.9％、33％、33.07％、34％、34.5％或者41％。
本发明中，所述透明质酸的用量可为0.03～5％，例如0.04％、0.05％、0.06％、0.08％、0.09％、0.1％、0.13％、0.14％、0.15％、0.2％、0.24％、0.25％、0.3％、0.33％、0.35％、0.41％、0.43％、0.44％、0.45％、0.47％、0.5％、0.6％、0.78％、0.8％、0.9％、1.53％、2.1％、0.2％、2.1％、2.5％或者3％。
本发明中，所述鱼精蛋白的用量可为0.2～16％，例如0.1％、0.2％、0.3％、0.35％、0.8％、0.83％、1％、1.3％、1.5％、1.6％、1.7％、2％、2.34％、2.39％、2.44％、3％、3.26％、3.3％、3.5％、3.7％、4％、4.3％、4.5％、4.85％、5％、5.1％、5.5％、5.53％、6％、6.5％、7％、13％或者14％。
本发明中，所述“载脂蛋白和/或其模拟肽”的用量可为2.85％～25％，例如2.92％、3％、3.5％、3.82％、3.94％、4％、4.1％、4.12％、4.43％、4.5％、4.65％、4.68％、4.78％、4.83％、4.89％、5％、5.4％、5.8％、6％、6.35％、6.52％、7.21％、10.35％、16.5％、16.37％、19.29％或者20％。
本发明中，所述脂质成分的用量优选为35～90％。
本发明中，所述阴离子脂质的用量可为14～40％、例如16、17.64％、22％、24％、24.04％、24.78％、24.8％、25％、26％、25.66％、26.1％、27％、27.22％、27.5％、29.79％、29％、30％、30.5％、30.66％、31％、31.08％、31.11％、32％、31.5％、32％、32.5％、33％、33.06％、33.12％、33.6％、34％、34.44％、35％、35.07％、36％、37.22％、37.72％、37.8％、 38％或者39％。
本发明中，所述电中性脂质的用量可为22～60％，例如23.5％、24.36％、30％、30.94％、32％、33％、34.2％、34.22％、35％，36.09％、37％、37.44％、37.5％、38.25％、39.08％、39.91％、41％、41.21％、42％、42.34％、42.92％、43.5％、44％、45％、45.61％、46％、46.4％、47％、47.56％、48.43％、50％、52.17％、52.2％、53％、54％、54.5％、55.32％、56％或者57％。
本发明中，所述透明质酸和所述鱼精蛋白的总用量可为0.2～14％，例如0.3％、0.5％、0.64％、0.8％、0.85％、0.97％、1％、1.08％、1.15％、1.2％、1.5％、1.63％、1.71％、1.79％、2％、2.15％、2.35％、2.58％、2.63％、2.68％、3.2％、3.5％、4％、4.17％、5％、5.08％、5.5％、5.6％、5.96％、6.38％、6.5％、7％、8％或者8.6％、13.06％或者13.5％。
本发明中，所述纳米复合物的粒径可为10～1000nm，优选为10～100nm。例如12～95nm，再例如20.30±5.89nm、23.79±7.91nm、25nm、26.52±4.31nm、27.31±10.84nm、27.38±7.83nm、27.55±6.99nm、37.98±14.29nm、28.96±8.74nm、31.11±3.44nm、36.30±6.41nm、37.22±7.28nm、37.55±13.73nm、37.63±4.20nm、37.68±2.20nm、38nm、39.12±4.84nm、40.55±7.66nm、55nm、55.75±7.69nm、57nm、60nm、63.62±1.97nm、70nm、74.20±14.23nm或者75.29±14.53nm。
本发明中，所述纳米复合物的Zeta电位可为-70～-15mV，例如-65、-64.87±3.30、-63.7±2.66、-58.87±4.90、-57.33±2.31、-56.33±3.26、-55.20±10.74、-52.07±2.15、-50.10±3.18、-48.87±1.95、-45.20±2.15、-44.87±0.45mV、-43.93±14.03、-43.87±9.68、-43.20±2.75mV、-40.23±6.92、-38.27±13.10、-36.83±2.71、-31.57±4.67、-30、-25，-20、-21.03±2.47或者-19.43±1.96mV。
作为一个优选方案，当所述蛋白质药物的等电点4～5.7，分子量为60～80kDa时，所述纳米复合物包括0～30％蛋白质药物、0.15～2.1％透明质酸、1～6.5％鱼精蛋白、4.65～7％“载脂蛋白和/或其模拟肽”、30～53.5％电中性磷脂和23.5～38.5％阴离子磷脂；其中，所述透明质酸和所述鱼精蛋白的总用量为1～9％。
其中，所述蛋白质药物的等电点可为4.2～5.5，例如4.7。
其中，所述蛋白质药物的分子量可为60～80kDa，例如69.3kDa。
其中，所述蛋白质药物可为牛血清白蛋白。
其中，所述蛋白质药物的用量可为0％、9％、16％、22％、28.08％、28.68％、29.31％或者30％。
其中，所述透明质酸的用量可为0.15％、0.24％、0.33％、0.35％、0.47％或者2.1％。
其中，所述鱼精蛋白的用量可为1％、2％、2.34％、2.39％、2.44％、3.26％、3.7％或者6.5％。
其中，所述“载脂蛋白和/或其模拟肽”的用量可为4.65％、4.68％、4.78％、4.83％、4.89％、5.4％、6.35％或者6.52％。
其中，所述“载脂蛋白和/或其模拟肽”可为ApoE及其模拟肽，ApoA-I、ApoA-II、ApoA-IV及其模拟肽，ApoC-I、ApoC-II、ApoC-III及其模拟肽中的一种或多种，例如ApoE。
其中，所述电中性磷脂可为二肉豆蔻酰磷脂酰胆碱DMPC、蛋黄卵磷脂和氢化大豆卵磷脂HSPC中的一种或多种，例如DMPC和蛋黄卵磷脂，或者，DMPC和氢化大豆卵磷脂。
当电中性磷脂为二肉豆蔻酰磷脂酰胆碱DMPC和蛋黄卵磷脂的混合物时，二肉豆蔻酰磷脂酰胆碱DMPC和蛋黄卵磷脂的质量比可为20：(8～12)，例如20:10.94。
其中，所述电中性磷脂的用量可为30.94％、37％、37.44％、38.25％、39.08％、43.5％、48.43％或者52.17％。
其中，所述阴离子磷脂可为磷脂酸和/或神经节苷脂。所述磷脂酸优选为二肉豆蔻酰磷脂酸(DMPA)、二硬脂酰磷脂酸(DSPA)、二棕榈酰磷脂酸(DPPA)或者二油酰基磷脂酸(DOPA)。所述的神经节苷脂优选单唾液酸四己糖神经节苷脂(GM1)。
当阴离子磷脂为单唾液酸四己糖神经节苷脂GM1和DOPA的混合物时，单唾液酸四己糖神经节苷脂GM1和DOPA质量比可为30.07：(3～7)，例如30.07:5。
其中，所述阴离子磷脂的用量可为24.04％、25.66％、26％、27.22％、31.5％、33.06％、35.07％或者37.72％。
其中，所述透明质酸和所述鱼精蛋白的总用量可为1.15％、2.35％、2.58％、2.63％、2.68％、4.17％或者8.6％。
其中，所述纳米复合物的粒径可为20～95nm，例如27.55±6.99nm、36.30±6.41nm、37.63±4.20nm、37.68±2.20nm、39.12±4.84nm、40.55±7.66nm、55.75±7.69nm、或者75.29±14.53nm。
其中，所述纳米复合物的Zeta电位可为-70～-20mV，例如-63.7±2.66、-64.87±3.30、-52.07±2.15、-45.20±2.15、-44.87±0.45mV、-43.20±2.75mV、-40.23±6.92或者-38.27±13.10mV。
作为一个优选方案，当所述蛋白质药物的等电点为4～5.3、分子量为200～255kDa时，所述纳米复合物包括4～17％蛋白质药物、0.05～0.25％透明质酸、1～3.3％鱼精蛋白、4～16.5％“载脂蛋白和/或其模拟肽”、42～53％电中性磷脂和31～38％阴离子磷脂；其中，所 述透明质酸和所述鱼精蛋白的总用量为1～3.5％。
其中，所述蛋白质药物的等电点可为4～4.8，例如4.3。
其中，所述蛋白质药物的分子量可为220～250kDa，例如240kDa。
其中，所述蛋白质药物可为藻红蛋白。
其中，所述蛋白质药物的用量可为4.8％、8％、8.5％、11％或者16％。
其中，所述透明质酸的用量可为0.08％、0.13％、0.15％或者0.2％。
其中，所述鱼精蛋白的用量可为1％、1.5％或者3％。
其中，所述“载脂蛋白和/或其模拟肽”的用量可为4.12％、5.8％、10.35％或者16.37％。
其中，所述“载脂蛋白和/或其模拟肽”可为ApoE及其模拟肽，ApoA-I、ApoA-II、ApoA-IV及其模拟肽，ApoC-I、ApoC-II、ApoC-III及其模拟肽中的一种或多种，例如ApoE。
其中，所述电中性磷脂可为磷脂酰胆碱。所述磷脂酰胆碱优选为DMPC。
其中，所述电中性磷脂的用量可为42.92％、43.5％、46.4％、47.56％或者52.2％。
其中，所述阴离子磷脂可为DOPA。
其中，所述阴离子磷脂的用量可为31.08％、31.5％、33.6％、34.44％或者37.8％。
其中，所述透明质酸和所述鱼精蛋白的总用量可为1.08％、1.15％、1.2％、1.63％或者3.2％。
其中，所述纳米复合物的粒径可为12～60nm，例如20.30±5.89nm、23.79±7.91nm、27.31±10.84nm、28.96±8.74nm或者37.55±13.73nm。
其中，所述纳米复合物的Zeta电位可为-65～-30mV，例如-58.87±4.90、-56.33±3.26、-50.10±3.18、-43.93±14.03或者-36.83±2.71mV。
作为一个优选方案，当所述蛋白质药物为的等电点9.3～11，分子量为8～16kDa时，所述纳米复合物包括31.5～34.5％蛋白质药物、0.25～0.45％透明质酸、0.1～0.35％鱼精蛋白、3～5％“载脂蛋白和/或其模拟肽”、35～37.5％电中性磷脂和25～27.5％阴离子磷脂；其中，所述透明质酸和所述鱼精蛋白的总用量为0.5～0.85％。
其中，所述蛋白质药物的等电点可为9.8～10.8，例如10.3。
其中，所述蛋白质药物的分子量可为10～14kDa，例如12.4kDa。
其中，所述蛋白质药物可为细胞色素C。
其中，所述蛋白质药物的用量可为32～34％，例如33.07％。
其中，所述透明质酸的用量可为0.3～0.5％，例如0.44％。
其中，所述鱼精蛋白的用量可为0.1～0.3％，例如0.2％。
其中，所述“载脂蛋白和/或其模拟肽”的用量可为3.5～4.5％，例如4.1％。
其中，所述“载脂蛋白和/或其模拟肽”可为ApoE及其模拟肽，ApoA-I、ApoA-II、ApoA-IV及其模拟肽，ApoC-I、ApoC-II、ApoC-III及其模拟肽中的一种或多种，例如ApoE。
其中，所述电中性磷脂可为磷脂酰胆碱。所述磷脂酰胆碱优选为DMPC。
其中，所述电中性磷脂的用量可为35～37％，例如36.09％。
其中，所述阴离子磷脂可为DOPA。
其中，所述阴离子磷脂的用量可为25～27％，例如26.1％。
其中，所述透明质酸和所述鱼精蛋白的总用量可为0.5～0.8％。例如0.64％。
其中，所述纳米复合物的粒径可为25～38nm，例如31.11±3.44nm。
其中，所述纳米复合物的Zeta电位可为-25～-15mV，例如-21.03±2.47mV。
作为一个优选方案，当所述蛋白质药物的等电点为7～9、分子量为140～180kDa时，所述纳米复合物包括5～43％蛋白质药物、0.04～0.9％透明质酸、1～14％鱼精蛋白、2.5～20％“载脂蛋白和/或其模拟肽”、23.5～46％电中性磷脂和16～32％阴离子磷脂；其中，所述透明质酸和所述鱼精蛋白的总用量为1～13.5％。
其中，所述蛋白质药物的等电点可为7.5～8.5，例如8。
其中，所述蛋白质药物的分子量可为150～170kDa，例如160kDa。
其中，所述蛋白质药物可为IgG抗体。
其中，所述蛋白质药物的用量可为6％、20％、33％或者41％。
其中，所述透明质酸的用量可为0.06％、0.09％、0.15％、0.41％或者0.78％。
其中，所述鱼精蛋白的用量可为1.3％、1.7％、2％、4.3％或者13％。
其中，所述“载脂蛋白和/或其模拟肽”的用量可为2.85％、2.92％、3.94％、7.21％、19.29％。
其中，所述“载脂蛋白和/或其模拟肽”可为ApoE及其模拟肽，ApoA-I、ApoA-II、ApoA-IV及其模拟肽，ApoC-I、ApoC-II、ApoC-III及其模拟肽中的一种或多种，例如ApoE或ApoA-I模拟肽。
其中，所述电中性磷脂可为磷脂酰胆碱。所述磷脂酰胆碱优选为DMPC。
其中，所述电中性磷脂的用量可为24.36％、34.22％、41.21％、42.34％、或者45％。
其中，所述阴离子磷脂可为DOPA。
其中，所述阴离子磷脂的用量可为17.64％、24.78％、29.79％、30％或者30.66％。
其中，所述透明质酸和所述鱼精蛋白的总用量可为1.71％、1.79％、2.15％、5.08％或者13.06％。
其中，所述纳米复合物的粒径可为15～95nm，例如26.52±4.31nm、27.38±7.83nm、 37.98±14.29nm或者74.20±14.23nm。
其中，所述纳米复合物的Zeta电位可为-70～-20mV，例如-57.33±2.31、-55.20±10.74、-43.87±9.68或者-31.57±4.67mV。
作为一个优选方案，当所述蛋白质药物的等电点为8.3～10.3、分子量为15～40kDa时，所述纳米复合物包括1～3％蛋白质药物、0.2～0.6％透明质酸、4.5～6.5％鱼精蛋白、2.5～5％“载脂蛋白和/或其模拟肽”、54～57％电中性磷脂和32～35％阴离子磷脂；其中，所述透明质酸和所述鱼精蛋白的总用量为5～7％。
其中，所述蛋白质药物的等电点可为8.8～9.8，例如9.3。
其中，所述蛋白质药物的分子量可为20～33kDa，例如26kDa。
其中，所述蛋白质药物可为神经生长因子β-NGF。
其中，所述蛋白质药物的用量可为1.5～2.2％，例如1.78％。
其中，所述透明质酸的用量可为0.3～0.5％，例如0.43％。
其中，所述鱼精蛋白的用量可为5～6％，例如5.53％。
其中，所述“载脂蛋白和/或其模拟肽”的用量可为3～4.5％，例如3.82％。
其中，所述“载脂蛋白和/或其模拟肽”可为ApoE及其模拟肽，ApoA-I、ApoA-II、ApoA-IV及其模拟肽，ApoC-I、ApoC-II、ApoC-III及其模拟肽中的一种或多种，例如ApoE。
其中，所述电中性磷脂可为磷脂酰胆碱。所述磷脂酰胆碱优选为DMPC。
其中，所述电中性磷脂的用量可为54.5～56％，例如55.32％。
其中，所述阴离子磷脂可为DOPA。
其中，所述阴离子磷脂的用量可为32.5～34％，例如33.12％。
其中，所述透明质酸和所述鱼精蛋白的总用量可为5.5～6.5％，例如5.96％。
作为一个优选方案，当所述蛋白质药物的等电点为6～8.5、分子量为30～50kDa时，所述纳米复合物包括15～20％蛋白质药物、0.05～0.8％透明质酸、0.8～1.6％鱼精蛋白、3～6％“载脂蛋白和/或其模拟肽”、44～47％电中性磷脂和29～33％阴离子磷脂；其中，所述透明质酸和所述鱼精蛋白的总用量为0.3～2％。
其中，所述蛋白质药物的等电点可为6.5～8，例如7.2。
其中，所述蛋白质药物的分子量可为35～45kDa，例如40kDa。
其中，所述蛋白质药物可为活性酶类蛋白药物HRP。
其中，所述蛋白质药物的用量可为16.5～19％，例如17.73％。
其中，所述透明质酸的用量可为0.1～0.3％，例如0.14％。
其中，所述鱼精蛋白的用量可为0.8～1.5％，例如0.83％。
其中，所述“载脂蛋白和/或其模拟肽”的用量可为4～5％，例如4.58％。
其中，所述“载脂蛋白和/或其模拟肽”可为ApoE及其模拟肽，ApoA-I、ApoA-II、ApoA-IV及其模拟肽，ApoC-I、ApoC-II、ApoC-III及其模拟肽中的一种或多种，例如ApoE。
其中，所述电中性磷脂可为磷脂酰胆碱。所述磷脂酰胆碱优选为DMPC。
其中，所述电中性磷脂的用量可为45～46％，例如45.61％。
其中，所述阴离子磷脂可为DOPA。
其中，所述阴离子磷脂的用量可为30.5～32％，例如31.11％。
其中，所述透明质酸和所述鱼精蛋白的总用量可为0.5～1.5％，例如0.97％。
其中，所述纳米复合物的粒径可为20～57nm，例如37.22±7.28nm。
其中，所述纳米复合物的Zeta电位可为-55～-15mV，例如-48.87±1.95mV。
作为一个优选方案，当所述蛋白质药物的等电点为4.4～6.4、分子量为210～255kDa时，所述纳米复合物包括10～33％蛋白质药物、0.2～3％透明质酸、3.5～7％鱼精蛋白、3～6％“载脂蛋白和/或其模拟肽”、32～42％电中性磷脂和22～39％阴离子磷脂；其中，所述透明质酸和所述鱼精蛋白的总用量为4～8％。
其中，所述蛋白质药物的等电点可为4.9～5.9，例如5.4。
其中，所述蛋白质药物的分子量可为220～250kDa。
其中，所述蛋白质药物可为过氧化氢酶(CAT)。
其中，所述蛋白质药物的用量可为11～32％，例如12.06或者30.9％。
其中，所述透明质酸的用量可为0.3～2.5％，例如0.5％或者1.53％。
其中，所述鱼精蛋白的用量可为4～5.5％，例如4.85％或者5.1％。
其中，所述“载脂蛋白和/或其模拟肽”的用量可为4～5％，例如4.43％或者4.5％。
其中，所述“载脂蛋白和/或其模拟肽”可为ApoE及其模拟肽，ApoA-I、ApoA-II、ApoA-IV及其模拟肽，ApoC-I、ApoC-II、ApoC-III及其模拟肽中的一种或多种，例如ApoE。
其中，所述电中性磷脂可为磷脂酰胆碱。所述磷脂酰胆碱优选为DMPC。
其中，所述电中性磷脂的用量可为33～41％，例如34.2％或者39.91％。
其中，所述阴离子磷脂可为DOPA。
其中，所述阴离子磷脂的用量可为24～38％，例如24.8％或者37.22％。
其中，所述透明质酸和所述鱼精蛋白的总用量可为5～7％，例如5.6％或者6.38％。
其中，所述纳米复合物的粒径可为55～70nm，例如63.62±1.97nm。
其中，所述纳米复合物的Zeta电位可为-25～-15mV，例如-19.43±1.96mV。
本发明的第二个目的在于，提供一种所述纳米复合物的制备方法，其通过下述方法 一或方法二进行制备：
方法一：
S1.所述蛋白质药物、所述透明质酸和所述鱼精蛋白混合形成纳米凝胶；
S2.所述脂质成分通过常规方法制备脂质体；
S3.所述纳米凝胶与所述脂质体共孵育形成含有纳米凝胶的脂质体；
S4.所述含有纳米凝胶的脂质体和“所述载脂蛋白和/或其模拟肽”的混合物通过自组装形成所述纳米复合物；
方法二：
S1.所述蛋白质药物、所述透明质酸和所述鱼精蛋白混合形成纳米凝胶；
S2.将所述脂质成分通过常规方法制备脂质体；
S3.所述纳米凝胶与所述脂质体通过微流控芯片制备含有纳米凝胶的脂质体，超滤去除溶剂后，制得包载蛋白药物的脂质体；
S4.所述包载蛋白药物的脂质体和“所述载脂蛋白和/或其模拟肽”的混合物通过自组装形成所述纳米复合物。
方法一或方法二，S1中，所述混合的操作和方法可为本领域常规。
方法一或方法二，S2中，所述常规方法可为本领域常规脂质体的制备方法，例如薄膜水化法、注入法、复溶法、熔融法或者冷冻干燥法。
作为一个优选方案，方法一，S2中，所述脂质体可通过下述步骤制得：所述脂质成分溶解在有机溶剂中，然后在旋转蒸发器上蒸发除去溶剂，使脂质成分在器壁上形成薄膜，再加入适量缓冲溶液，通过振摇使之充分水合分散，得到脂质体。可通过超声仪探头超声进一步减少脂质体粒径。
作为一个优选方案，方法二，S2中，所述脂质体可通过下述步骤制得：将所述脂质成分溶于乙醇，形成脂质体。
方法一，S3中，所述孵育的操作和方法可为本领域常规。
方法一或方法二，S4中，所述自组装的操作和方法可为本领域常规，例如可将混合物在置于震荡摇床于120rpm，37℃孵育36h。
方法二，S3，所述纳米凝胶与所述脂质体通过微流控芯片制备含有纳米凝胶的脂质体的方法可为本领域常规方法。
本发明的第三个目的在于，提供一种所述纳米复合物在所述蛋白质药物递送中的应用。
作为一个优选方案，所述纳米复合物可用于蛋白质药物的细胞内递送。
作为一个优选方案，所述纳米复合物可用于蛋白质药物的体内递送。
作为一个优选方案，所述纳米复合物可用于蛋白质药物的脑内递送。
本发明中，若无特殊说明，“％”均是指的质量百分数。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：
本发明纳米复合物包括特定用量的蛋白质药物、透明质酸、鱼精蛋白和重组脂蛋白，其通过合适比例的透明质酸、鱼精蛋白构成适合蛋白质药物的微环境，通过重组脂蛋白实现蛋白质药物高效的细胞内、体内乃至脑内递送。
本发明纳米复合物为不同理化性质(例如分子量10-255KDa和PI 4-11)的蛋白质药物提供通用型载体，实现有效的细胞内递送和体内递送，所采用技术具备普适性，可有效解决现有蛋白质药物(包括分子量、PI超出实施例的蛋白质药物)体内外输送不足的技术问题。
附图说明
图1为Hela细胞对不同处方牛血清白蛋白摄取情况的荧光分布图。
图2为Hela细胞对不同处方藻红蛋白摄取情况的荧光分布图。
图3为U87细胞对不同处方细胞色素C的摄取情况的荧光分布图。
图4为HT22细胞对不同处方Alexa Fluor488-IgG的摄取情况的荧光分布图。
图5为C6细胞对不同处方载Alexa Fluor488-IgG纳米复合物的摄取情况的荧光分布图。
图6为载神经生长因子(NGF)的纳米复合物促PC12细胞分化。图6A为不同制剂促PC12细胞分化的神经突起长度统计；图6B为不同制剂促PC12细胞分化的神经突起分支点统计；图6C为不同制剂促PC12细胞分化的形态图。
图7为BV2细胞对不同处方过氧化氢酶的摄取情况的荧光分布图。
图8为CAT-HA-PRTM-rHDL有效提高细胞内过氧化氢酶水平(图8A)，并有效降低H 2O 2引起的细胞内ROS水平增加(图8B)。
图9为CAT-HA-PRTM-rHDL有效抑制由H 2O 2诱导的细胞膜损伤(图9A)，并有效提高细胞活力(图9B)。
图10为C57小鼠的皮质损伤模型中，尾静脉给予荧光染料DiR标记的载透明质酸、 鱼精蛋白、CAT的重组脂蛋白纳米复合物(CAT-HA-PRTM-rHDL)和载透明质酸、鱼精蛋白、CAT的脂质体(CAT-HA-PRTM-LIPO)，给药4h后取小鼠脏器进行活体成像，评价制剂的体内分布及入脑情况。
图11为构建CX3CR1-GFP小鼠的皮质损伤模型，通过尾静脉给予荧光染料DiI标记的不同制剂，给药4h后取小鼠脑进行冰冻切片，评价载CAT的纳米复合物在活体内被脑内小胶质细胞摄取情况。
图12为构建C57小鼠皮质损伤模型，Sham组为假损伤对照组，建模后连续给药1周，Morris水迷宫实验考察载带CAT的不同制剂对CCI模型小鼠潜伏期、穿越平台的次数以及在目标象限的时间的影响。
图13为应用SOD小鼠模型，连续给药并同时评价小鼠运动能力，转棒实验、抓杆实验和后肢握紧实验考察载带CAT的不同制剂对SOD转基因小鼠运动能力的影响。
图14为Hela细胞对载带或未载蛋白的空白载体摄取情况的荧光分布图(图14A)和载带或未载蛋白的空白载体在小鼠的皮质损伤模型中脑内分布情况(图14B)。
具体实施方式
下述实施例中使用的仪器均为：
旋转蒸发仪(RE-52CS-1，上海亚荣生化仪器厂，中国)
超声仪(JY92-II，宁波新芝生物科技股份有限公司，中国)
透射电镜(H-7650，Hitachi，日本)
冷冻电镜(FEI Tecnai F20，Holland)
激光粒度仪(Zetasizer Nano ZS90 ZEN3590，Malvern，英国)
酶标仪(Thermo，美国)
激光共聚焦显微镜(TCS SP8，Leica，德国)。
实施例1 载牛血清白蛋白的纳米复合物的制备、表征及其细胞内蛋白递送
(1)制备：
①将牛血清白蛋白、透明质酸和鱼精蛋白按照不同质量比共同孵育形成载蛋白复合物(FITC-BSA-HA-PRTM)；
②采用薄膜水化法制备脂质体：称取脂质(中性磷脂DMPC和/或卵磷脂lecithin、阳离子磷脂DOTAP、和阴离子磷脂DOPA和/或阴离子鞘脂单唾液酸四己糖神经节苷脂GM1和/或DMPA、DPPA、DSPA)，置于500mL圆底烧瓶中，加入2mL乙醚，挥干除去磷脂中的水分，再加入氯仿溶液2mL，置旋转蒸发仪上抽真空1h。加入4mL 0.01M  PBS溶液(pH 7.4)，于40℃水浴间歇振摇10min至薄膜水化脱落得到脂质体。超声仪探头超声进一步减小脂质体粒径，得到脂质体。
③将脂质体与FITC-BSA-HA-PRTM按不同比例共孵育形成包载蛋白质药物的脂质体(FITC-BSA-HA-PRTM-LIPO)(表1-2)；将载脂蛋白(ApoE)加入到上述脂质体溶液中，轻轻混匀，置于震荡摇床于120rpm，37℃孵育36h，得到载牛血清白蛋白的纳米复合物(FITC-BSA-HA-PRTM-rHDL)，其中阴离子脂质DOPA替换为DMPA或者DPPA或者DSPA得到载牛血清白蛋白的纳米复合物分别为FITC-BSA-HA-PRTM-rHDL-1、FITC-BSA-HA-PRTM-rHDL-2、FITC-BSA-HA-PRTM-rHDL-3。
(2)表征
将载牛血清白蛋白的纳米复合物通过磷钨酸负染，采用透射电镜观察形态。进一步制样后采用冷冻电镜观察结构。激光粒度仪测定其粒径和表面电位。酶标仪检测将载牛血清白蛋白的纳米复合物对蛋白药物的包封率和载药量。
(3)细胞内蛋白递送效果评价
通过激光共聚焦显微镜观察载牛血清白蛋白的纳米复合物细胞内蛋白递送情况。人源宫颈癌细胞Hela细胞以50000个/孔密度接种于共聚焦皿，培养24h。吸弃原有培液，加入500μL载绿色荧光蛋白标记各蛋白质药物。
实验组：牛血清白蛋白(FITC-BSA)的重组脂蛋白纳米复合物(FITC-BSA-HA-PRTM-rHDL)。
对照组分别为：①游离的FITC-BSA蛋白；②透明质酸、鱼精蛋白、FITC-BSA复合物(FITC-BSA-HA-PRTM)，即按照步骤(1)的第①步的制备方法制得的载蛋白复合物；③不含透明质酸、鱼精蛋白的载BSA重组脂蛋白纳米复合物(FITC-BSA-rHDL)，其制备方法除了不添加透明质酸、鱼精蛋白以外，其它制备方法均与步骤(1)相同；④载透明质酸、鱼精蛋白、FITC-BSA的脂质体(FITC-BSA-HA-PRTM-LIPO)，其制备方法除了不添加载脂蛋白以外，脂质体的制备方法均与步骤(1)第①②步相同；⑤FITC-BSA+商业化蛋白转染试剂(FITC-BSA-Pulsin)；⑥含不同阴离子的牛血清白蛋白(FITC-BSA)的重组脂蛋白纳米复合物，其制备方法同FITC-BSA-HA-PRTM-rHDL，除了将DOPA分别替换成DMPA或者DPPA或者DSPA得到载牛血清白蛋白的纳米复合物分别为FITC-BSA-HA-PRTM-rHDL-1、FITC-BSA-HA-PRTM-rHDL-2、FITC-BSA-HA-PRTM-rHDL-3。实验组和对照组(给药浓度为20μg/mL，按FITC-BSA蛋白质量计算)分别在37℃孵育4h。然后3.7％甲醛于37℃固定10min，Hoechest染核10min，PBS洗3次后，共聚焦拍摄定性观察，采用Image J软件对细胞摄取FITC-BSA进行半定量分析。
结果显示，合适比例的FITC-BSA-HA-PRTM-rHDL粒径均在100nm以下，有利于制剂透过体内生物膜屏障；而蛋白和HA、PRTM的复合物FITC-BSA-HA-PRTM粒径较大(198.0±3.10nm)，随时间延长(4℃静置过夜)进一步聚集，沉淀现象较严重，当检测其粒径时，检测仪器提示不符合检测要求，无法有效检测。相比FITC-BSA、FITC-BSA-HA-PRTM、FITC-BSA-rHDL、FITC-BSA-HA-PRTM-LIPO，优化的FITC-BSA-HA-PRTM-rHDL处方更有效地将BSA递送进入细胞(细胞摄取平均光密度值更高)，且较均匀分布于胞浆(如表1、图1)。相比之下，虽然市售蛋白转染制剂组显示更强的细胞相关荧光强度，但多数荧光信号分布在细胞外(图1)。同时，不同的阴离子脂质DMPA或者DPPA或者DSPA形成的蛋白纳米复合物均可高效递送蛋白至细胞胞浆。出乎意料的是，在FITC-BSA-HA-PRTM-rHDL处方中，含阳离子脂质DOTAP的处方(配方1)蛋白的细胞摄取量低于含阴离子脂质处方(表2)。
表1 不同处方牛血清白蛋白纳米复合物表征和Hela细胞摄取蛋白的定量数据
Figure PCTCN2022071526-appb-000001
Figure PCTCN2022071526-appb-000002
备注：①表1中处方组成FITC-BSA-HA-PRTM-rHDL为表2相中配方6的处方组成。②“未检测”是指未做相关检测。③Zeta电位表示纳米粒的电势，其由组成纳米载体的材料决定。④***与FITC-BSA-HA-PRTM-rHDL组存在显著性差异； ###与FITC-BSA组存在显著性差异。
图1为Hela细胞对不同处方牛血清白蛋白摄取情况。游离绿色荧光蛋白标记牛血清白蛋白(FITC-BSA)；载透明质酸、鱼精蛋白、FITC-BSA的重组脂蛋白纳米复合物(FITC-BSA-HA-PRTM-rHDL)；透明质酸、鱼精蛋白、FITC-BSA复合物(FITC-BSA-HA-PRTM)；不含透明质酸、鱼精蛋白的载BSA重组脂蛋白纳米复合物(FITC-BSA-rHDL)；载透明质酸、鱼精蛋白、FITC-BSA的脂质体(FITC-BSA-HA-PRTM-LIPO)和FITC-BSA+商业化蛋白转染试剂(FITC-BSA-Pulsin)。
表2 不同FITC-BSA-HA-PRTM-rHDL处方牛血清白蛋白纳米复合物表征和C6细胞摄取蛋白的定量数据
Figure PCTCN2022071526-appb-000003
Figure PCTCN2022071526-appb-000004
备注：①表2中配方1、2、7为对照配方，配方3-6为本发明实施例配方。②制剂稳定性评价方法为：4℃下静置12h。③组分EPC为阳离子脂质。配方7中，制剂稳定性中“易沉降”是指的4℃保存12h肉眼可见沉降，粒径增大为1000nm以上。④配方3-6稳定性较好，在温度4℃下静置1个月后，粒径和电势稳定，与放置前相比均无变化。⑤统计分析以处方1为对照，***P<0.001与配方1存在显著性差异。⑥表2中，卵磷脂lecithin是指的蛋黄卵磷脂，将其等量替换为氢化大豆卵磷脂，效果相当。
由表2可知，配方3-6中，配方6细胞摄取平均光密度值最大，表示其细胞摄取效率最高，效果最优。
实施例2 包载藻红蛋白的纳米复合物的制备、表征及其细胞内蛋白递送
(1)制备：
①将藻红蛋白(PE)与透明质酸和鱼精蛋白按照不同质量比共同孵育形成载蛋白复合物(PE-HA-PRTM)；
②取适量磷脂(两性磷脂DMPC和阴离子磷脂DOPA)组合溶于乙醇相，得脂质体；
③取适量PE-HA-PRTM与上述脂质体通过微流控芯片制备载蛋白脂质体(PE-HA-PRTM-LIPO)，超滤去除乙醇溶液；去除乙醇后的脂质体加载脂蛋白和/或其模拟肽自组装得到由透明质酸、鱼精蛋白、重组脂蛋白构成的载PE的纳米复合物(PE-HA-PRTM-rHDL)。
(2)表征
将载PE的纳米复合物通过磷钨酸负染，透射电镜观察形态。进一步制样后冷冻电镜观察其结构。激光粒度仪测定其粒径和表面电位。酶标仪检测将载PE的纳米复合物对蛋白药物的包封率和载药量。
(3)细胞内蛋白递送效果评价
通过激光共聚焦显微镜观察载PE的纳米复合物细胞内蛋白递送情况。人源宫颈癌细胞Hela细胞或脑胶质瘤细胞C6以50000/孔密度接种于共聚焦皿，培养24h。吸弃原有培液，加入500μL载PE的重组脂蛋白纳米复合物(PE-HA-PRTM-rHDL)。对照组分别游离的PE；透明质酸、鱼精蛋白和PE复合物(PE-HA-PRTM)；不含透明质酸、鱼精蛋白的载PE重组脂蛋白纳米复合物(PE-rHDL)；载透明质酸、鱼精蛋白、PE复合物的脂质体(PE-HA-PRTM-LIPO)和PE+商业化蛋白转染试剂(PE-Pulsin)(给药浓度为10μg/mL，按PE质量计算)，37℃孵育4h。然后3.7％甲醛于37℃固定10min，Hoechest染核10min，PBS洗3次后，共聚焦拍摄定性观察，采用Image J软件对细胞摄取FITC-PE进行半定量分析。
结果显示，合适比例的PE-HA-PRTM-rHDL粒径在100nm以下(表3-4)，而透明质酸、鱼精蛋白、PE组成的载蛋白复合物(PE-HA-PRTM)粒径大(788.1±60.98nm)，随时间延长(4℃静置过夜)进一步聚集，沉淀现象较严重，当检测其粒径时，检测仪器提示不符合检测要求，无法有效检测。相比PE、PE-HA-PRTM、PE-rHDL、PE-HA-PRTM-LIPO，优化的PE-HA-PRTM-rHDL处方更有效地将PE递送进入细胞，且较均匀分布于胞浆(表3、图2)。同样地，虽然市售蛋白转染制剂组显示更强的细胞相关荧光强度，但多数荧光信号分布在细胞外(图2)。同样地，在PE-HA-PRTM-rHDL处方中，含阳离子脂质的处方蛋白的细胞摄取量低于含阴离子脂质处方。
表3 不同处方藻红蛋白纳米复合物表征和Hela细胞摄取蛋白的定量数据
Figure PCTCN2022071526-appb-000005
Figure PCTCN2022071526-appb-000006
备注：“未检测”是指未做相关检测。统计分析以PE-HA-PRTM-rHDL作为对照，***P<0.001与PE-HA-PRTM-rHDL存在显著性差异。
图2为Hela细胞对不同处方藻红蛋白摄取情况。藻红蛋白(PE)；载透明质酸、鱼精蛋白、PE的重组脂蛋白纳米复合物(PE-HA-PRTM-rHDL)；透明质酸、鱼精蛋白、PE复合物(PE-HA-PRTM)；不含透明质酸、鱼精蛋白的载PE重组脂蛋白纳米复合物(PE-rHDL)；载透明质酸、鱼精蛋白、PE的脂质体(PE-HA-PRTM-LIPO)和PE+商业化蛋白转染试剂(PE-Pulsin)。
表4 不同PE-HA-PRTM-rHDL处方藻红蛋白纳米复合物表征和C6细胞摄取蛋白的定量数据
Figure PCTCN2022071526-appb-000007
备注：①表4中，配方1-2为对照配方，配方3-7为本发明实施例配方。②制剂稳定性评价方法为：4℃下静置12h。③配方2中，“易沉降”是指4℃保存12h肉眼可见沉降，粒径增大为1000nm以上。④配方3-7稳定性较好，在温度4℃下静置1个月后，粒径和电势稳定，与放置前相比均无变化。⑤统计分析以配方1为对照，***P<0.001与配方1存在显著性差异。
由表4可知，配方3-7中，配方7细胞摄取平均光密度值最大，表示其细胞摄取效率最高，效果最优。
实施例3 包载细胞色素C的纳米复合物的制备、表征及其细胞内蛋白递送
(1)制备：
①将荧光标记的细胞色素C与透明质酸和鱼精蛋白按照不同质量比共同孵育形成载蛋白复合物(FITC-CC-HA-PRTM)；
②采用薄膜水化法制备脂质体：称取脂质(中性磷脂DMPC和阴离子磷脂DOPA组合)置于500mL圆底烧瓶中，加入2mL乙醚，挥干除去磷脂中的水分，再加入氯仿溶液，置旋转蒸发仪上抽真空1h。加入4mL 0.01M PBS溶液(pH 7.4)，于40℃水浴间歇振摇10min至薄膜水化脱落得到脂质体。探头超声进一步减小脂质体粒径，得到脂质体。
③将上述制备的脂质体与按不同比例共孵育形成包载蛋白质药物脂质体(FITC-CC-HA-PRTM-LIPO)；将ApoE加入到纳米复合物溶液中，轻轻混匀，置于震荡摇床于120rpm，37℃孵育36h，得到载细胞色素C的纳米复合物(FITC-CC-HA-PRTM-rHDL)。
(2)表征
将载细胞色素C的纳米复合物通过磷钨酸负染，透射电镜观察形态。进一步制样后冷冻电镜观察其结构。激光粒度仪测定其粒径和表面电位。酶标仪检测将载细胞色素C的纳米复合物对蛋白药物的包封率和载药量。
(3)细胞内蛋白递送效果评价
通过激光共聚焦显微镜观察载细胞色素C的纳米复合物细胞内蛋白递送情况。脑胶质瘤细胞系U87以50000/孔密度接种于共聚焦皿，培养24h。吸弃原有培液，加入500μL载绿色荧光蛋白标记细胞色素C(FITC-CC)的重组脂蛋白纳米复合物(FITC-CC-HA-PRTM-rHDL)，对照组分别游离的FITC-CC蛋白；透明质酸、鱼精蛋白、FITC-CC复合物(FITC-CC-HA-PRTM)；不含透明质酸、鱼精蛋白的载CC重组脂蛋白纳米复合物(FITC-CC-rHDL)；载透明质酸、鱼精蛋白、FITC-CC的脂质体(FITC-CC-HA-PRTM-LIPO) 和FITC-CC+商业化蛋白转染试剂(FITC-CC-Pulsin)(给药浓度为20μg/mL，按FITC-CC蛋白质量计算)，37℃孵育6h。然后3.7％甲醛于37℃固定10min，Hoechest染核10min，PBS洗3次后，共聚焦拍摄定性观察，采用Image J软件对细胞摄取FITC-CC进行半定量分析。
结果显示，合适比例的FITC-CC-HA-PRTM-rHDL粒径在100nm以下。相比FITC-CC、FITC-CC-HA-PRTM、FITC-CC-rHDL、FITC-CC-HA-PRTM-LIPO和市售蛋白转染制剂组，优化的FITC-CC-HA-PRTM-rHDL处方更有效地将CC递送进入细胞，且较均匀分布于胞浆(表5、图3)。
表5 不同处方载细胞色素C纳米复合物表征和U87细胞摄取蛋白的定量数据
Figure PCTCN2022071526-appb-000008
备注：①“未检测”是指未做相关检测。②处方组成中，FITC-CC-HA-PRTM-rHDL稳定性较好，在温度4℃下静置1个月后，粒径和电势稳定，与放置前相比均无变化。③统计分析，以FITC-CC-HA-PRTM-rHDL作为对照。***P<0.001与FITC-CC-HA-PRTM-rHDL组存在显著性差异。
图3为U87细胞对不同处方细胞色素C的摄取情况的荧光分布图。绿色荧光蛋白标 记细胞色素C(FITC-CC)；载透明质酸、鱼精蛋白、FITC-CC复合物的重组脂蛋白纳米复合物(FITC-CC-HA-PRTM-rHDL)；透明质酸、鱼精蛋白、FITC-CC复合物(FITC-CC-HA-PRTM)；不含透明质酸、鱼精蛋白的载FITC-CC重组脂蛋白纳米复合物(FITC-CC-rHDL)；载透明质酸、鱼精蛋白、FITC-CC复合物的脂质体(FITC-CC-HA-PRTM-LIPO)和FITC-CC+商业化蛋白转染试剂(FITC-CC-Pulsin)。
实施例4 包载IgG抗体的纳米复合物的制备、表征及其细胞内蛋白递送
(1)制备：
①将荧光标记抗体Alexa Fluor488-IgG与透明质酸和鱼精蛋白按照不同质量比共同孵育形成载蛋白复合物(Alexa Fluor488-IgG-HA-PRTM)。
②采用薄膜水化法制备脂质体：称取脂质(两性磷脂DMPC、阳离子磷脂DOTAP和阴离子磷脂DOPA组合)置于500mL圆底烧瓶中，加入2mL乙醚，挥干除去磷脂中的水分，再加入氯仿溶液，置旋转蒸发仪上抽真空1h。加入4mL 0.01M PBS溶液(pH7.4)，于40℃水浴间歇振摇10min至薄膜水化脱落得到脂质体。探头超声进一步减小脂质体粒径，得到脂质体。
③将上述制备的脂质体与Alexa Fluor488-IgG-HA-PRTM按不同比例共孵育形成包载蛋白质药物的脂质体(Alexa Fluor488-IgG-HA-PRTM-LIPO)；将ApoE或ApoA Ⅰ模拟肽(Ac-FAEKFKEAVKDYFAKFWD)加入到上述载蛋白的脂质体溶液中，轻轻混匀，置于震荡摇床于120rpm，37℃孵育36h，得到载Alexa Fluor488-IgG抗体的纳米复合物(Alexa Fluor488-IgG-HA-PRTM-rHDL)。
(2)表征
将载Alexa Fluor488-IgG抗体的纳米复合物通过磷钨酸负染，透射电镜观察形态。进一步制样后冷冻电镜观察其结构。激光粒度仪测定其粒径和表面电位。酶标仪检测载Alexa Fluor488-IgG抗体的纳米复合物对蛋白药物的包封率和载药量。
(3)细胞内蛋白递送效果评价
通过激光共聚焦显微镜观察载Alexa Fluor488-IgG抗体的纳米复合物细胞内蛋白递送情况。人源宫颈癌细胞Hela细胞以50000/孔密度接种于共聚焦皿，培养24h。吸弃原有培液，加入500μL载Alexa Fluor488-IgG抗体(Alexa Fluor488-IgG)的重组脂蛋白纳米复合物(Alexa Fluor488-IgG-HA-PRTM-rHDL)，对照组分别游离的Alexa Fluor488-IgG蛋白；透明质酸、鱼精蛋白、Alexa Fluor488-IgG复合物(Alexa Fluor488-IgG-HA-PRTM)；不含透明质酸、鱼精蛋白的载Alexa Fluor488-IgG重组脂蛋白纳米复合物(Alexa Fluor488- IgG-rHDL)；载透明质酸、鱼精蛋白、Alexa Fluor488-IgG的脂质体(Alexa Fluor488-IgG-HA-PRTM-LIPO)和Alexa Fluor488-IgG+商业化蛋白转染试剂(Alexa Fluor488-IgG-Pulsin)(给药浓度为10μg/mL，按Alexa Fluor488-IgG蛋白质量计算)，37℃孵育6h。然后3.7％甲醛于37℃固定10min，Hoechest染核10min，PBS洗3次后，共聚焦拍摄定性观察，采用Image J软件对细胞摄取Alexa Fluor488-IgG进行半定量分析。
结果显示，合适比例的Alexa Fluor488-IgG粒径均在100nm以下；而透明质酸、鱼精蛋白、抗体组成的复合物(Alexa Fluor488-IgG-HA-PRTM)粒径为63.48±5.20nm，随时间延长(4℃静置过夜)发生聚集，沉淀现象较严重，检测粒径的仪器提示不符合检测要求，无法进行检测。相比Alexa Fluor488-IgG、Alexa Fluor488-IgG-HA-PRTM、Alexa Fluor488-IgG-rHDL和Alexa Fluor488-IgG-HA-PRTM-LIPO，优化的Alexa Fluor488-IgG-HA-PRTM-rHDL处方更有效地将Alexa Fluor488-IgG递送进入细胞，且较均匀分布于胞浆(表6、图4)。相比之下，虽然市售蛋白转染制剂组显示更强的细胞相关荧光强度，但多数荧光信号分布在细胞外。
表6 不同处方载Alexa Fluor488-IgG纳米复合物表征和Hela细胞摄取蛋白的定量数据
Figure PCTCN2022071526-appb-000009
Figure PCTCN2022071526-appb-000010
备注：①“未检测”是指未做相关检测。②处方组成中，Alexa Fluor488-IgG-HA-PRTM-rHDL两个处方稳定性较好，在温度4℃下静置1个月后，粒径和电势稳定，与放置前相比均无变化。③统计分析时，以Alexa Fluor488-IgG-HA-PRTM-rHDL-1为对照，***P<0.001与Alexa Fluor488-IgG-HA-PRTM-rHDL-1组存在显著性差异。
图4为HT22细胞对不同处方Alexa Fluor488-IgG的摄取情况的荧光分布图。Alexa Fluor488-IgG；载透明质酸、鱼精蛋白、Alexa Fluor488-IgG的重组脂蛋白纳米复合物(Alexa Fluor488-IgG-HA-PRTM-rHDL)；透明质酸、鱼精蛋白、Alexa Fluor488-IgG复合物(Alexa Fluor488-IgG-HA-PRTM)；不含透明质酸、鱼精蛋白的载Alexa Fluor488-IgG重组脂蛋白纳米复合物(Alexa Fluor488-IgG-rHDL)；载透明质酸、鱼精蛋白、Alexa Fluor488-IgG的脂质体(Alexa Fluor488-IgG-HA-PRTM-LIPO)和Alexa Fluor488-IgG+商业化蛋白转染试剂(Alexa Fluor488-IgG-Pulsin)
表7 不同Alexa Fluor488-IgG-HA-PRTM-rHDL处方的纳米复合物表征和C6细胞摄取蛋白的定量数据
Figure PCTCN2022071526-appb-000011
Figure PCTCN2022071526-appb-000012
表中，配方5为对照组，配方1-4均为本发明实施例。***P<0.001，****P<0.0001与配方5存在显著性差异。
图5为C6细胞对不同处方载Alexa Fluor488-IgG纳米复合物的摄取情况的荧光分布图。
实施例5 载带β-NGF的纳米复合物促PC12细胞分化能力评价
选用β-NGF作为神经营养类蛋白药物的研究模型，与实施例1的制备方法相同，分别制备实验组载β-NGF的纳米复合物和对照组，实验组和对照组处方如表8所示。
选取小鼠原代神经元或者PC12细胞接种于96孔板，β-NGF给药浓度为100ng/mL，不同载带蛋白制剂与细胞共同孵育8小时，应用incucyte实时动态细胞成像技术检测β-NGF诱导PC12细胞分化为神经元的过程，并半定量分析由PC12细胞诱导分化的神经元分支点和突触长度的动态变化。
表8 不同处方载NGF纳米复合物表征及其促神经分化活性检测
Figure PCTCN2022071526-appb-000013
Figure PCTCN2022071526-appb-000014
备注：统计分析以NGF-HA-PRTM-rHDL为对照，*P<0.05、**P<0.01或者***P<0.001与NGF-HA-PRTM-rHDL组存在显著性差异。
实验结果如图6所示，载NGF的纳米复合物快速高效促进PC12细胞分化为神经元，神经元分支点和突触长度显著增加，形态类似于神经元。
图6为载神经生长因子(NGF)的纳米复合物促PC12细胞分化。图6A为不同制剂促PC12细胞分化的神经突起长度统计；图6B为不同制剂促PC12细胞分化的神经突起分支点统计；图6C为不同制剂促PC12细胞分化的形态图。载透明质酸、鱼精蛋白、NGF的重组脂蛋白纳米复合物(NGF-HA-PRTM-rHDL)与PC12细胞共同孵育8小时；对照为游离NGF；不含透明质酸、鱼精蛋白的载NGF重组脂蛋白纳米复合物(NGF-rHDL)；空白脂质体(liposome)和培养液空白对照(Control)。
实施例6 载带HRP的纳米复合物胞内酶活性评价
选用HRP作为活性酶类蛋白药物的研究模型，与实施例1的制备方法相同，分别制备实验组载HRP的纳米复合物和对照组，实验组和对照组处方如表9所示。
选取Hela细胞接种于96孔板，HRP给药浓度为10ug/mL，不同载带蛋白制剂与细胞共同孵育6小时，然后PBS漂洗细胞3次，每孔加入200ul TMB显色液，室温避光孵育3-30min直至显色至预期深浅，在650nm测定吸光度值，即指示胞内HRP酶活性水平。
实验结果如表9显示，给予游离的HRP组细胞内辣根过氧化物酶水平最低，载透明质酸、鱼精蛋白、HRP的脂质体可以部分递送HRP酶至细胞内从而提高细胞内酶活性，而载透明质酸、鱼精蛋白、HRP的重组脂蛋白纳米复合物(HRP-HA-PRTM-rHDL)显著提高细胞内辣根过氧化物酶活性。
表9 不同处方载HRP纳米复合物表征及其胞内酶活性检测
Figure PCTCN2022071526-appb-000015
Figure PCTCN2022071526-appb-000016
备注：①“未检测”是指未做相关检测。②统计分析以HRP-HA-PRTM-rHDL为对照，**P<0.01，***P<0.001与HRP-HA-PRTM-rHDL组存在显著性差异。
实施例7 载过氧化氢酶(CAT)的纳米复合物的制备、表征及其细胞内蛋白递送
(1)制备
①将过氧化氢酶(CAT)与透明质酸和鱼精蛋白按照不同质量比共同孵育形成载蛋白复合物(FITC-CAT-HA-PRTM)；
②采用薄膜水化法制备脂质体：称取脂质(中性磷脂DMPC和阴离子磷脂DOPA组合)置于500mL圆底烧瓶中，加入2mL乙醚，挥干除去磷脂中的水分，再加入氯仿溶液2mL，置旋转蒸发仪上抽真空1h。加入4mL 0.01M PBS溶液(pH 7.4)，于40℃水浴间歇振摇10min至薄膜水化脱落得到脂质体。探头超声进一步减小脂质体粒径，得到脂质体。
③将上述制备的脂质体与FITC-CAT-HA-PRTM按不同比例共孵育形成载透明质酸、鱼精蛋白、FITC-CAT的脂质体(FITC-CAT-HA-PRTM-LIPO)；将ApoE加入到上述脂质体溶液中，轻轻混匀，置于震荡摇床于120rpm，37℃孵育36h，得到载过氧化氢酶(CAT)的重组脂蛋白纳米复合物(FITC-CAT-HA-PRTM-rHDL)。
(2)表征
将载过氧化氢酶的纳米复合物通过磷钨酸负染，透射电镜观察形态。进一步制样后冷冻电镜观察其结构。激光粒度仪测定其粒径和表面电位。酶标仪检测将载过氧化氢酶的纳米复合物对蛋白药物的包封率和载药量。
(3)细胞内蛋白递送效果评价
通过激光共聚焦显微镜观察载过氧化氢酶的纳米复合物细胞内蛋白递送情况。小胶质细胞BV2以50000/孔密度接种于共聚焦皿，培养24h。吸弃原有培液，加入500μL载绿色荧光蛋白标记过氧化氢酶(FITC-CAT)的重组脂蛋白纳米复合物(FITC-CAT-HA- PRTM-rHDL)，对照组分别游离的FITC-CAT蛋白；透明质酸、鱼精蛋白、FITC-CAT复合物(FITC-CAT-HA-PRTM)；不含透明质酸、鱼精蛋白的载CAT重组脂蛋白纳米复合物(FITC-CAT-rHDL)；载透明质酸、鱼精蛋白、FITC-CAT的脂质体(FITC-CAT-HA-PRTM-LIPO)和FITC-CAT+商业化蛋白转染试剂(FITC-CAT-Pulsin)(给药浓度为20μg/mL，按FITC-CAT蛋白质量计算)，37℃孵育6h。然后3.7％甲醛于37℃固定10min，Hoechest染核10min，PBS洗3次后，共聚焦拍摄定性观察，采用Image J软件对细胞摄取FITC-CAT进行半定量分析。
结果显示，合适比例的FITC-CAT-HA-PRTM-rHDL粒径在100nm以下，而FITC-CAT-HA-PRTM粒径较大(530.90±311.00nm)，且随时间延长(4℃静置过夜)进一步聚集，沉淀现象较严重，当检测其粒径时，检测仪器提示不符合检测要求，无法有效检测。相比FITC-CAT、FITC-CAT-HA-PRTM、FITC-CAT-rHDL、FITC-CAT-HA-PRTM-LIPO和市售蛋白转染制剂组，优化的FITC-CAT-HA-PRTM-rHDL处方更有效地将CAT递送进入细胞，且较均匀分布于胞浆(表10、图7)。
表10 不同处方载过氧化氢酶的纳米复合物表征和小胶质细胞BV2摄取蛋白的定量数据
Figure PCTCN2022071526-appb-000017
Figure PCTCN2022071526-appb-000018
备注：①“未检测”是指未做相关检测。②统计分析以FITC-CAT-HA-PRTM-rHDL为参照，***P<0.001与FITC-CAT-HA-PRTM-rHDL存在显著性差异。
图7为BV2细胞对不同处方过氧化氢酶的摄取情况的荧光分布图。绿色荧光蛋白标记过氧化氢酶(FITC-CAT)；载透明质酸、鱼精蛋白、FITC-CAT的重组脂蛋白纳米复合物(FITC-CAT-HA-PRTM-rHDL)；透明质酸、鱼精蛋白、FITC-CAT复合物(FITC-CAT-HA-PRTM)；不含透明质酸、鱼精蛋白的载FITC-CAT重组脂蛋白纳米复合物(FITC-CAT-rHDL)；载透明质酸、鱼精蛋白、FITC-CAT的脂质体(FITC-CAT-HA-PRTM-LIPO)和FITC-CAT+商业化蛋白转染试剂(FITC-CAT-Pulsin)。
实施例8 载过氧化氢酶(CAT)的纳米复合物对HT22细胞的转染能力、抗氧化能力及对细胞活力的评价
选用氧化氢酶(CAT)作为蛋白药物的研究模型，与实施例7的制备方法相同，分别制备载氧化氢酶(CAT)的纳米复合物，处方如表11所示。
采用过氧化氢酶检测试剂盒检测载CAT纳米复合物对HT22细胞的转染能力，反应原理为在过氧化氢相对比较充足的情况下，过氧化氢酶可以催化过氧化氢产生水和氧气。残余的过氧化氢在过氧化物酶的催化下可以氧化生色底物，产生红色的产物，最大吸收波长为520nm。用过氧化氢标准品，制作标准曲线，这样就可以计算出样品中的过氧化氢酶在单位时间单位体积内催化了多少量的过氧化氢转变为水和氧气，从而可以计算出样品中过氧化氢酶的酶活力。具体实验方案为将HT22细胞种于12孔板，在二氧化碳培养箱培养24h后给予载带CAT蛋白的不同制剂，包括含透明质酸、鱼精蛋白、CAT的重组脂蛋白纳米复合物(CAT-HA-PRTM-rHDL)及对照组分别为未处理组(Control)、游离的CAT蛋白组和CAT+商业化蛋白转染试剂组(CAT-Pulsin)(给药浓度为20μg/mL，按CAT蛋白质量计算)，37℃孵育6h。裂解细胞，根据操作说明检测细胞内过氧化氢酶水平。
结果如图8A所示，未处理的Control组和给予游离的CAT组细胞内过氧化氢酶水 平最低，商业化蛋白转染试剂Pulsin可以部分递送CAT蛋白至细胞内从而提高细胞内酶活性，而含透明质酸、鱼精蛋白、CAT的重组脂蛋白纳米复合物(CAT-HA-PRTM-rHDL)显著提高细胞内过氧化氢酶活性。
表11 不同处方载过氧化氢酶的纳米复合物提高小胶质细胞BV2内过氧化氢酶活性
Figure PCTCN2022071526-appb-000019
备注：以配方1为对照。*P<0.05或者***P<0.001与配方1存在显著性差异。ns表示配方2与配方1的酶活性水平与无显著性差异。
由于CAT蛋白具有抗氧化功能，采用双氧水诱导细胞内氧化应激水平提高以评价载CAT纳米复合物对细胞内氧化应激水平的调控作用。具体实验方法如下，将HT22细胞种于96孔板，待汇合度达到70％时给予不同的载带CAT蛋白药物制剂，给药4h后，给药4h后弃除制剂溶液，加入荧光探针DCFH-DA与细胞共孵30min，然后弃除探针溶液，加入400μM的双氧水与细胞共孵15min诱导其氧化损伤，通过酶标仪做定量分析。结果如图10所示，Control组为完全未处理组细胞内活性氧水平，单独给予双氧化诱导后细胞内ROS水平激增，而给予CAT-HA-PRTM-rHDL显著降低了由双氧水诱导的细胞ROS水平增加。
图8.CAT-HA-PRTM-rHDL有效提高细胞内过氧化氢酶水平(图8A)，并有效降低H 2O 2引起的细胞内ROS水平增加(图8B)。
乳酸脱氢酶细胞毒性检测试剂盒主要评价细胞膜完整性，也是作为细胞状态评价的指标之一，细胞给药步骤同上，给药4h后弃除制剂溶液，加入双氧水与细胞共孵诱导其氧化损伤，在预定的检测时间点前1h，从细胞培养箱里取出细胞培养板，在“样品最大酶活性对照孔”中加入试剂盒提供的LDH释放试剂，加入量为原有培养液体积的10％。加入LDH释放试剂后，反复吹打数次混匀，然后继续在细胞培养箱中孵育，到达预定时间后，将细胞培养板用多孔板离心机400g离心5min。分别取各孔的上清液120μl，加入到一新的96孔板相应孔中，随即进行样品测定。CCK8的操作步骤基本同上，首先加入不同蛋白制剂与细胞共孵4h后弃除制剂溶液，加入双氧水与细胞共孵诱导其氧化损伤，在预定的检测时间点加入CCK8试剂检测细胞活力。实验结果如图9所示，细胞ROS水 平增加显著降低由双氧水诱导的细胞膜损伤，提高细胞活力水平。
图9.CAT-HA-PRTM-rHDL有效抑制由H 2O 2诱导的细胞膜损伤(图9A)，并有效提高细胞活力(图9B)。
实施例9 载过氧化氢酶(CAT)的纳米复合物在小鼠的体内分布和脑内分布情况评价
制备荧光探针DiR和DiI标记的载透明质酸、鱼精蛋白、CAT的重组脂蛋白纳米复合物(CAT-HA-PRTM-rHDL)(即实施例7的实验组FITC-CAT-HA-PRTM-rHDL)和载透明质酸、鱼精蛋白、CAT的脂质体(CAT-HA-PRTM-LIPO)。同时建立小鼠控制性皮层损伤(Controlled Cortical Injury,CCI)模型。小鼠腹腔注射5％水合氯醛麻醉后，固定于脑立体定向仪，无菌条件下切开头皮，暴露右顶骨，于右侧冠状缝和人字缝之间、中线旁开圆形颅窗，暴露硬脑膜，设置不同打击参数模拟不同程度皮层损伤模型。打击参数：速度1.5m/s，深度1mm，打击头直径2mm，接触时间100ms模拟中度伤情，用于常规脑损伤实验。损伤后闭合颅骨，缝合皮肤。假损伤Sham组小鼠仅切开头皮，暴露右侧顶骨，不予打击损伤。对于制剂的体内分布实验，应用DiR标记的制剂尾静脉给药(按磷脂DMPC给药浓度为20mg/kg计算)，于给药后4h取出心、肝、脾、肺、肾和脑组织，生理盐水冲洗后置于小动物活体成像仪采集图像，观察纳米复合物在体内的分布及其向脑部转运的动态变化。实验结果如图10所示，给予生理盐水组脑内完全无荧光制剂分布，而CAT-HA-PRTM-rHDL脑内荧光强度显著且高度聚集于右脑CCI损伤位置，说明本发明构建的纳米复合物可高效递送目的蛋白入脑并靶向分布于损伤部位。
图10.C57小鼠的皮质损伤模型中，尾静脉给予荧光染料DiR标记的载透明质酸、鱼精蛋白、CAT的重组脂蛋白纳米复合物(CAT-HA-PRTM-rHDL)和载透明质酸、鱼精蛋白、CAT的脂质体(CAT-HA-PRTM-LIPO)，给药4h后取小鼠脏器进行活体成像，评价制剂的体内分布及入脑情况。
实施例10 载过氧化氢酶(CAT)的纳米复合物(即实施例7的实验组FITC-CAT-HA-PRTM-rHDL)在小鼠的脑内分布情况评价
进一步本发明选取小胶质细胞转入绿色荧光蛋白的CX3CR1-GFP转基因小鼠构建CCI模型，同样于CCI后尾静脉给予DiI标记的荧光制剂，通过脑切片实验评价DiI-CAT-HA-PRTM-rHDL制剂在CCI小鼠脑内的分布情况及其被小胶质细胞摄取情况，尾静脉给药3h后，麻醉固定小鼠进行心脏灌流，取荷瘤小鼠完整的大脑，置4％多聚甲醛中后固 定24h，PBS漂洗后依次置于15％和30％蔗糖溶液中脱水至下沉，然后用O.C.T.包埋并–20℃冷冻，作连续冰冻冠状切片，片厚为14μm，PBS漂洗后用100ng/mL DAPI染色10min，PBS漂洗，拭去水渍，磷酸甘油封片，激光共聚焦显微镜观察CAT-HA-PRTM-rHDL脑内分布。实验结果如图11所示，CAT-HA-PRTM-rHDL可以被脑内小胶质细胞高效摄取。
图11.构建CX3CR1-GFP小鼠的皮质损伤模型，通过尾静脉给予荧光染料DiI标记的不同制剂，给药4h后取小鼠脑进行冰冻切片，评价载CAT的纳米复合物在活体内被脑内小胶质细胞摄取情况。
实施例11 载过氧化氢酶(CAT)的纳米复合物(即实施例7的实验组FITC-CAT-HA-PRTM-rHDL)对CCI小鼠空间学习记忆能力的影响
构建小鼠中度CCI脑损模型，尾静脉给予载带CAT的不同制剂(按照CAT计算给药浓度为13300Units/kg)，连续给药7天后采用Morris水迷宫对小鼠进行行为学训练及测试。水迷宫由圆形水池、平台和记录系统三部分组成；水池直径150cm，高50cm，水池分为4个象限(Ⅰ、Ⅱ、Ⅲ和Ⅳ象限)，水池注水30cm深，加入白色食用色素使水变成不透明的白色，使实验小鼠无法直接看见平台以及水池底部；水温保持在25℃左右。水池四周设置空间参照物(门、摄像头及墙上标志等)，且位置保持不变，以供小鼠定位以及记忆平台的位置。圆柱形平台直径9cm，高在29cm，用不反光黑布包裹，放置于Ⅳ象限，平面没于水面下1cm。一摄像头置于水池中央上方，自动采集动物游泳图像，所收集信号直接输入计算机，采用Morris水迷宫视频分析系统2.0监测并记录小鼠的游泳轨迹。定位航行实验(Hidden platform test)在小鼠连续给药7后开始，历时5天；每次训练的入水点为按随机原则排列的4个象限，每次使不同的小鼠在相同的位置，面向池壁放入水中，每相邻两天的入水顺序不同，计算机监测并记录小鼠从入水开始寻找至找到并爬上黑色平台的路线以及所需时间(潜伏期)。每只小鼠每天接受4次训练，每次训练设定的潜伏期为60s，如若小鼠60s内未找到平台，需将其引领到平台，并停留10s,这时潜伏期记为60s，每只小鼠每两次训练间隔为30s；空间探索实验(Probe trial)在5天定位航行试验后，于第6天撤去平台，分别从Ⅱ、Ⅲ象限入水点将小鼠面向池壁放入水中，记录小鼠在60s内目标象限(平台所在象限)的时间百分比以及小鼠搜索平台的轨迹。实验结果如图12所示，随着训练时间增加，小鼠的潜伏期逐渐变短，CAT-HA-PRTM-rHDL显著缩短小鼠寻找平台的潜伏期；同时，空间探索实验显示，给予CAT-HA-PRTM-rHDL的小鼠穿越平台次数和在目标平台停留时间显著增加，提示CAT-HA-PRTM-rHDL 高效递送CAT蛋白至脑损部位缓解由脑损伤引起的氧化应激水平增加，改善小鼠空间学习记忆能力。
图12.构建C57小鼠皮质损伤模型，Sham组为假损伤对照组，建模后连续给药1周，Morris水迷宫实验考察载带CAT的不同制剂对CCI模型小鼠潜伏期、穿越平台的次数以及在目标象限的时间的影响。
实施例12 载过氧化氢酶(CAT)的纳米复合物(即实施例7的实验组FITC-CAT-HA-PRTM-rHDL)对SOD小鼠运动能力和生存期的影响
同实施例10给予转基因小鼠SOD1-G93A(肌萎缩性侧索硬化症模型)CAT-HA-PRTM-rHDL，其中给药组Saline和游离的CAT蛋白作为对照组，SOD小鼠3月龄开始给药，连续每天给药，给药的同时进行小鼠运动能力评价实验，包括转棒实验、抓杆实验和后肢握紧实验。转棒实验具体步骤为：第一周训练小鼠三次，转棒转速为14rpm，以此转速小鼠在转棒上停留180秒定义为无症状，当小鼠在转棒上停留时间小于180秒定义为临床发病时间，小鼠训练一周后即可进行测试实验；抓杆实验为记录小鼠依靠上肢抓杆时长，总检测时间为60秒，记录60秒内小鼠掉落潜伏期；后肢握紧实验为握取小鼠尾巴将小鼠倒挂，录制15秒的视频并记录小鼠在15秒内后肢握紧情况，评分由0-3分评价小鼠后肢握紧程度(与僵硬严重度成正比)。实验结果如图13所示，转棒实验在测试最初阶段各组小鼠在转棒上停留时间相近，随着时间延长(4℃静置过夜)，给予CAT-HA-PRTM-rHDL的小鼠在转棒上停留时间明显延长，而抓杆实验CAT-HA-PRTM-rHDL组小鼠的抓杆时间明显较对照组延长，后肢握紧程度减弱说明制剂组缓解了SOD小鼠瘫痪的病理进程。
图13.应用SOD小鼠模型，连续给药并同时评价小鼠运动能力，转棒实验、抓杆实验和后肢握紧实验考察载带CAT的不同制剂对SOD转基因小鼠运动能力的影响。
实施例13 不含蛋白的空白纳米复合物的制备、表征及其细胞摄取效率和脑内分布
(1)制备：
①将透明质酸和鱼精蛋白按照不同质量比共同孵育形成载复合物(HA-PRTM)；
②采用薄膜水化法制备脂质体：称取脂质(中性磷脂DMPC、和阴离子磷脂DOPA)以及红色荧光染料DiI置于500mL圆底烧瓶中，加入2mL乙醚，挥干除去磷脂中的水分，再加入氯仿溶液2mL，置旋转蒸发仪上抽真空1h。加入4mL 0.01M PBS溶液(pH7.4)，于40℃水浴间歇振摇10min至薄膜水化脱落得到脂质体。超声仪探头超声进一步 减小脂质体粒径，得到载带红色荧光探针的脂质体(DiI-LIPO)。
③将脂质体与HA-PRTM按不同比例共孵育形成不含蛋白质药物的空白脂质体(DiI-HA-PRTM-LIPO)；将载脂蛋白(ApoE)加入到上述脂质体溶液中，轻轻混匀，置于震荡摇床于120rpm，37℃孵育36h，得到不含蛋白质药物的纳米复合物(DiI-HA-PRTM-rHDL)。
(2)表征
将不含蛋白质药物的纳米复合物通过磷钨酸负染，采用透射电镜观察形态。进一步制样后采用冷冻电镜观察结构。激光粒度仪测定其粒径和表面电位。
(3)细胞摄取效率评价
通过激光共聚焦显微镜观察不含蛋白质药物的纳米复合物的细胞摄取情况。人源宫颈癌细胞Hela细胞以50000个/孔密度接种于共聚焦皿，培养24h。吸弃原有培液，加入500μL载红色荧光探针标记的不含蛋白质药物的纳米复合物。
实验组：载红色荧光探针标记的不含蛋白质药物的纳米复合物(DiI-HA-PRTM-rHDL)。对照组：载红色荧光探针标记的含BSA蛋白质药物的纳米复合物(DiI-BSA-HA-PRTM-rHDL)，制备方法同实施例1。实验组和对照组(给药浓度为10μg/mL，按磷脂DMPC质量计算)分别在37℃孵育4h。然后3.7％甲醛于37℃固定10min，Hoechest染核10min，PBS洗3次后，共聚焦拍摄定性观察，采用Image J软件对细胞摄取纳米复合物进行半定量分析。
(3)纳米复合物脑内分布评价
应用C57小鼠构建CCI模型，同样于CCI后尾静脉给予DiI标记的荧光制剂，通过脑切片实验评价DiI-BSA-HA-PRTM-rHDL和DiI-HA-PRTM-rHDL制剂在CCI小鼠脑内的分布情况，尾静脉给药3h后，麻醉固定小鼠进行心脏灌流，取荷瘤小鼠完整的大脑，置4％多聚甲醛中后固定24h，PBS漂洗后依次置于15％和30％蔗糖溶液中脱水至下沉，然后用O.C.T.包埋并–20℃冷冻，作连续冰冻冠状切片，片厚为14μm，PBS漂洗后用100ng/mL DAPI染色10min，PBS漂洗，拭去水渍，磷酸甘油封片，扫描显微镜拍摄观察两种制剂脑内分布。
图14.构建C57小鼠的皮质损伤模型，通过尾静脉给予荧光染料DiI标记的不同制剂，给药4h后取小鼠脑进行冰冻切片，评价载带或者不载带蛋白的纳米复合物在小鼠脑内的分布。
结果如图14所示，含BSA蛋白质药物的纳米复合物(DiI-BSA-HA-PRTM-rHDL)和不含蛋白质药物的空白载体(DiI-HA-PRTM-rHDL)粒径均在100nm以下，有利于制 剂透过体内生物膜屏障，均可以高效被细胞摄取(图14A)；同时，在小鼠皮质损伤模型中，不含蛋白和含蛋白的纳米复合物均可高效靶向分布于脑损伤部位(图14B)。这些发现提示，载蛋白与否均不影响细胞摄取和脑内靶向分布，载体本身和载蛋白处方具有相似的细胞摄取和体内分布行为。
表12 不同处方不含蛋白的纳米复合物表征
Figure PCTCN2022071526-appb-000020
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (10)

  1. 一种纳米复合物，其特征在于，所述纳米复合物包括0～60％蛋白质药物、0.03～15％透明质酸和0.1～20％鱼精蛋白、35～95％脂质成分和2.5～40％“载脂蛋白和/或其模拟肽”；所述脂质成分包括电中性脂质和阴离子脂质；所述透明质酸和所述鱼精蛋白的总用量为0.03～15％；百分比为各组分分别相对于所述纳米复合物的质量百分比。
  2. 如权利要求1所述的纳米复合物，其特征在于，所述纳米复合物中不包括阳离子脂质DOPAT；
    和/或，所述蛋白质药物的分子量为10～255kDa，例如10～16kDa、15～40kDa、30～50kDa、60～80kDa 140～180kDa、200～255kDa或者210～255kDa，优选为10～14kDa、20～33kDa、35～45kDa、60～80kDa、150～170kDa、220～250kDa、220～250kDa，再例如12.4kDa、26kDa、40kDa、69.3kDa、160kDa或者240kDa；
    和/或，所述蛋白质药物的等电点为4～11，例如4～5.3、3.7～5.7、4.4～6.4、6～8.5、7～9、8.3～10.3或者9.3～11，优选4～4.8、4.2～5.5、4.9～5.9、6.5～8、7.5～8.5、8.8～9.8或者9.8～10.8，再例如4.3、4.7、5.4、7.2、8、9.3或者10.3；
    和/或，所述电中性脂质包括两性脂质、非离子型脂质、胆固醇及其衍生物中的一种或多种，优选包括磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰甘油和鞘磷脂中的一种或多种；
    所述磷脂酰胆碱优选为二肉豆蔻酰磷脂酰胆碱DMPC、二棕榈酰磷脂酰胆碱DPPC、二硬脂酰基磷脂酰胆碱DSPC、二油酰基磷脂酰胆碱DOPC、二月桂酰基卵磷脂DLPC、二芥酰磷脂酰胆碱DEPC、1-棕榈酰基-2-油酰基卵磷脂POPC、卵磷脂、豆磷脂、氢化大豆卵磷脂HSPC及其衍生物中的一种或多种，更优选为二肉豆蔻酰磷脂酰胆碱DMPC、蛋黄卵磷脂和氢化大豆卵磷脂HSPC中的一种或多种；
    所述磷脂酰乙醇胺优选为二肉豆蔻酰磷脂酰乙醇胺DMPE、二硬脂酰基磷脂酰乙醇胺DSPE、二棕榈酰磷脂酰乙醇胺DPPE、二油酰基磷脂酰乙醇胺DOPE、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000、二硬脂酰基磷脂酰乙醇胺-聚乙二醇5000、二棕榈酰磷脂酰乙醇胺-聚乙二醇2000、二棕榈酰磷脂酰乙醇胺-聚乙二醇5000及其衍生物中的一种或多种；
    所述磷脂酰甘油优选为二肉豆蔻酰磷脂酰甘油DMPG、二硬脂酰基磷脂酰甘油DSPG、二棕榈酰磷脂酰甘油DPPG、二油酰基磷脂酰甘油DOPG、1-棕榈酰基-2油酰基磷脂酰甘油POPG-Na、蛋黄磷脂酰甘油EPG及其衍生物中的一种或多种；
    优选，当所述电中性脂质为二肉豆蔻酰磷脂酰胆碱DMPC和蛋黄卵磷脂的混合物时，二肉豆蔻酰磷脂酰胆碱DMPC和蛋黄卵磷脂的质量比可为20：(8～12)，例如20:10.94；
    和/或，所述阴离子脂质为阴离子磷脂；所述阴离子磷脂优选包括磷脂酸、磷脂酰肌醇、磷脂酰丝氨酸、心磷脂、溶血磷脂和神经节苷脂中的一种或多种，更优选为磷脂酸和/或神经节苷脂；
    其中，所述磷脂酸优选为二肉豆蔻酰磷脂酸DMPA、二硬脂酰磷脂酸DSPA、二棕榈酰磷脂酸DPPA、二油酰基磷脂酸DOPA及其衍生物中的一种或多种，更优选为二油酰基磷脂酸DOPA；
    所述磷脂酰丝氨酸优选为二油酰基磷脂酰丝氨酸DOPS和/或二棕榈酰磷脂酰丝氨酸DPPS；
    所述的溶血磷脂优选为硬脂酰溶血卵磷脂S-lysoPC、肉豆蔻酰溶血卵磷脂M-LysoPC、棕榈酰溶血卵磷脂P-LysoPC及其衍生物中的一种或多种；
    所述的神经节苷脂优选单唾液酸四己糖神经节苷脂GM1；
    优选，当阴离子磷脂为磷脂酸和神经节苷脂的混合物时，神经节苷脂和磷脂酸质量比可为30.07：(3～7)，例如30.07:5；
    和/或，所述“载脂蛋白和/或其模拟肽”为ApoE及其模拟肽，ApoA-I、ApoA-II、ApoA-IV及其模拟肽，ApoC-I、ApoC-II、ApoC-III及其模拟肽，ApoB及其模拟肽，ApoJ及其模拟肽中的一种或多种，例如ApoE或者ApoA-I模拟肽；
    和/或，所述蛋白质药物的用量为1～43％，例如1.5％、1.78％、2.2％、3％、4％、4.8％、5％、6％、8％、8.5％、9％、10％、11％、12.06％、15％、16％、16.5％、17％、17.73％、19％、20％、22％、28.08％、28.68％、29.31％、30％、31.5％、32％、30.9％、33％、33.07％、34％、34.5％或者41％；
    和/或，所述透明质酸的用量为0.03～5％，例如0.04％、0.05％、0.06％、0.08％、0.09％、0.1％、0.13％、0.14％、0.15％、0.2％、0.24％、0.25％、0.3％、0.33％、0.35％、0.41％、0.43％、0.44％、0.45％、0.47％、0.5％、0.6％、0.78％、0.8％、0.9％、1.53％、2.1％、0.2％、2.1％、2.5％或者3％；
    和/或，所述鱼精蛋白的用量为0.2～16％，例如0.1％、0.2％、0.3％、0.35％、0.8％、0.83％、1％、1.3％、1.5％、1.6％、1.7％、2％、2.34％、2.39％、2.44％、3％、3.26％、3.3％、3.5％、3.7％、4％、4.3％、4.5％、4.85％、5％、5.1％、5.5％、5.53％、6％、6.5％、7％、13％或者14％；
    和/或，所述“载脂蛋白和/或其模拟肽”的用量为2.85％～25％，例如2.92％、3％、3.5％、3.82％、3.94％、4％、4.1％、4.12％、4.43％、4.5％、4.65％、4.68％、4.78％、4.83％、4.89％、5％、5.4％、5.8％、6％、6.35％、6.52％、7.21％、10.35％、16.5％、16.37％、19.29％或者 20％；
    和/或，所述脂质成分的用量为35～90％。
    和/或，所述阴离子脂质的用量为14～40％、例如16、17.64％、22％、24％、24.04％、24.78％、24.8％、25％、25.66％、26％、26.1％、27％、27.22％、27.5％、29.79％、29％、30％、30.5％、30.66％、31％、31.08％、31.11％、32％、31.5％、32％、32.5％、33％、33.06％、33.12％、33.6％、34％、34.44％、35％、35.07％、36％、37.22％、37.72％、37.8％、38％或者39％；
    或者，所述电中性脂质的用量为22～60％，例如23.5％、24.36％、30％、30.94％、32％、33％、34.2％、34.22％、35％，36.09％、37％、37.44％、37.5％、38.25％、39.08％、39.91％、41％、41.21％、42％、42.34％、42.92％、43.5％、44％、45％、45.61％、46％、46.4％、47％、47.56％、48.43％、50％、52.17％、52.2％、53％、54％、54.5％、55.32％、56％或者57％；
    或者，所述透明质酸和所述鱼精蛋白的总用量为0.2～14％，例如0.3％、0.5％、0.64％、0.8％、0.85％、0.97％、1％、1.08％、1.15％、1.2％、1.5％、1.63％、1.71％、1.79％、2％、2.15％、2.35％、2.58％、2.63％、2.68％、3.2％、3.5％、4％、4.17％、5％、5.08％、5.5％、5.6％、5.96％、6.38％、6.5％、7％、8％或者8.6％、13.06％或者13.5％；
    和/或，所述纳米复合物的粒径为10～1000nm，优选为10～100nm；例如12～95nm，再例如20.30±5.89nm、23.79±7.91nm、25nm、26.52±4.31nm、27.31±10.84nm、27.38±7.83nm、27.55±6.99nm、37.98±14.29nm、28.96±8.74nm、31.11±3.44nm、36.30±6.41nm、37.22±7.28nm、37.55±13.73nm、37.63±4.20nm、37.68±2.20nm、38nm、39.12±4.84nm、40.55±7.66nm、55nm、55.75±7.69nm、57nm、60nm、63.62±1.97nm、70nm、74.20±14.23nm或者75.29±14.53nm；
    和/或，所述纳米复合物的Zeta电位为-70～-15mV，例如-65、-64.87±3.30、-63.7±2.66、-58.87±4.90、-57.33±2.31、-56.33±3.26、-55.20±10.74、-52.07±2.15、-50.10±3.18、-48.87±1.95、-45.20±2.15、-44.87±0.45mV、-43.93±14.03、-43.87±9.68、-43.20±2.75mV、-40.23±6.92、-38.27±13.10、-36.83±2.71、-31.57±4.67、-30、-25，-20、-21.03±2.47或者-19.43±1.96mV。
  3. 如权利要求1或2所述的纳米复合物，其特征在于，当所述蛋白质药物的等电点4～5.7，分子量为60～80kDa时，所述纳米复合物包括0～30％蛋白质药物、0.15～2.1％透明质酸、1～6.5％鱼精蛋白、4.65～7％“载脂蛋白和/或其模拟肽”、30～53.5％电中性磷脂和23.5～38.5％阴离子磷脂；所述透明质酸和所述鱼精蛋白的总用量为1～9％；
    所述蛋白质药物的等电点优选为4.2～5.5，例如4.7；
    所述蛋白质药物的分子量优选为60～80kDa，例如69.3kDa；
    所述蛋白质药物优选为牛血清白蛋白；
    所述蛋白质药物的用量优选为0％、9％、16％、22％、28.08％、28.68％、29.31％或者30％；
    所述透明质酸的用量优选为0.15％、0.24％、0.33％、0.35％、0.47％或者2.1％；
    所述鱼精蛋白的用量优选为1％、2％、2.34％、2.39％、2.44％、3.26％、3.7％或者6.5％；
    所述“载脂蛋白和/或其模拟肽”的用量优选为4.65％、4.68％、4.78％、4.83％、4.89％、5.4％、6.35％或者6.52％；
    所述“载脂蛋白和/或其模拟肽”优选为ApoE；
    所述电中性磷脂优选为DMPC和/或蛋黄卵磷脂，或者，DMPC和/或氢化大豆卵磷脂；
    当电中性磷脂为DMPC和蛋黄卵磷脂的混合物时，DMPC和蛋黄卵磷脂的质量比优选为20：(8～12)，例如20:10.94；
    所述电中性磷脂的用量优选为30.94％、37％、37.44％、38.25％、39.08％、43.5％或者48.43％或者52.17％；
    所述阴离子磷脂优选为神经节苷脂和/或磷脂酸；所述磷脂酸优选为二油酰基磷脂酸DOPA；所述的神经节苷脂优选单唾液酸四己糖神经节苷脂GM1；
    当阴离子磷脂为单唾液酸四己糖神经节苷脂GM1和DOPA的混合物时，单唾液酸四己糖神经节苷脂GM1和DOPA质量比优选为30.07：(3～7)，例如30.07:5；
    所述阴离子磷脂的用量优选为24.04％、25.66％、26％、27.22％、31.5％、33.06％、35.07％或者37.72％；
    所述透明质酸和所述鱼精蛋白的总用优选为1.15％、2.35％、2.58％、2.63％、2.68％、4.17％或者8.6％；
    所述纳米复合物的粒径优选为20～95nm，例如27.55±6.99nm、36.30±6.41nm、37.63±4.20nm、37.68±2.20nm、39.12±4.84nm、40.55±7.66nm、55.75±7.69nm、或者75.29±14.53nm；
    所述纳米复合物的Zeta电位优选为-70～-20mV，例如-63.7±2.66、-64.87±3.30、-52.07±2.15、-45.20±2.15、-44.87±0.45mV、-43.20±2.75mV、-40.23±6.92或者-38.27±13.10mV。
  4. 如权利要求1～3中至少一项所述的纳米复合物，其特征在于，当所述蛋白质药物的等电点为4～5.3、分子量为200～255kDa时，所述纳米复合物包括4～17％蛋白质药物、0.05～0.25％透明质酸、1～3.3％鱼精蛋白、4～16.5％“载脂蛋白和/或其模拟肽”、42～53％电 中性磷脂和31～38％阴离子磷脂；所述透明质酸和所述鱼精蛋白的总用量为1～3.5％；
    所述蛋白质药物的等电点优选为4～4.8，例如4.3；
    所述蛋白质药物的分子量优选为220～250kDa，例如240kDa；
    所述蛋白质药物优选为藻红蛋白；
    所述蛋白质药物的用量优选为4.8％、8％、8.5％、11％或者16％；
    所述透明质酸的用量优选为0.08％、0.13％、0.15％或者0.2％；
    所述鱼精蛋白的用量优选为1％、1.5％或者3％；
    所述“载脂蛋白和/或其模拟肽”的用量优选为4.12％、5.8％、10.35％或者16.37％；
    所述“载脂蛋白和/或其模拟肽”优选为ApoE；
    所述电中性磷脂优选为磷脂酰胆碱；所述磷脂酰胆碱优选为DMPC；
    所述电中性磷脂的用量优选为42.92％、43.5％、46.4％、47.56％或者52.2％；
    所述阴离子磷脂优选为DOPA；
    所述阴离子磷脂的用量优选为31.08％、31.5％、33.6％、34.44％或者37.8％；
    所述透明质酸和所述鱼精蛋白的总用量优选为1.08％、1.15％、1.2％、1.63％或者3.2％；
    所述纳米复合物的粒径优选为12～60nm，例如20.30±5.89nm、23.79±7.91nm、27.31±10.84nm、28.96±8.74nm或者37.55±13.73nm；
    所述纳米复合物的Zeta电位优选为-65～-30mV，例如-58.87±4.90、-56.33±3.26、-50.10±3.18、-43.93±14.03或者-36.83±2.71mV。
  5. 如权利要求1～4中至少一项所述的纳米复合物，其特征在于，当所述蛋白质药物为的等电点9.3～11，分子量为8～16kDa时，所述纳米复合物包括31.5～34.5％蛋白质药物、0.25～0.45％透明质酸、0.1～0.35％鱼精蛋白、3～5％“载脂蛋白和/或其模拟肽”、35～37.5％电中性磷脂和25～27.5％阴离子磷脂；所述透明质酸和所述鱼精蛋白的总用量为0.5～0.85％；
    所述蛋白质药物的等电点优选为9.8～10.8，例如10.3；
    所述蛋白质药物的分子量优选为10～14kDa，例如12.4kDa；
    所述蛋白质药物优选为细胞色素C；
    所述蛋白质药物的用量优选为32～34％，例如33.07％；
    所述透明质酸的用量优选为0.3～0.5％，例如0.44％；
    所述鱼精蛋白的用量优选为0.1～0.3％，例如0.2％；
    所述“载脂蛋白和/或其模拟肽”的用量优选为3.5～4.5％，例如4.1％；
    所述“载脂蛋白和/或其模拟肽”优选为ApoE；
    所述电中性磷脂优选为磷脂酰胆碱；所述磷脂酰胆碱优选为DMPC；
    所述电中性磷脂的用量优选为35～37％，例如36.09％；
    所述阴离子磷脂优选为DOPA；
    所述阴离子磷脂的用量优选为25～27％，例如26.1％；
    所述透明质酸和所述鱼精蛋白的总用量优选为0.5～0.8％，例如0.64％；
    所述纳米复合物的粒径优选为25～38nm，例如31.11±3.44nm；
    所述纳米复合物的Zeta电位优选为-25～-15，例如-21.03±2.47。
  6. 如权利要求1～5中至少一项所述的纳米复合物，其特征在于，当所述蛋白质药物的等电点为7～9、分子量为140～180kDa时，所述纳米复合物包括5～43％蛋白质药物、0.04～0.9％透明质酸、1～14％鱼精蛋白、2.5～20％“载脂蛋白和/或其模拟肽”、23.5～46％电中性磷脂和16～32％阴离子磷脂；所述透明质酸和所述鱼精蛋白的总用量为1～13.5％；
    所述蛋白质药物的等电点优选为7.5～8.5，例如8；
    所述蛋白质药物的分子量优选为150～170kDa，例如160kDa；
    所述蛋白质药物优选为IgG抗体；
    所述蛋白质药物的用量优选为6％、20％、33％或者41％；
    所述透明质酸的用量优选为0.06％、0.09％、0.15％、0.41％或者0.78％；
    所述鱼精蛋白的用量优选为1.3％、1.7％、2％、4.3％或者13％；
    所述“载脂蛋白和/或其模拟肽”的用量优选为2.85％、2.92％、3.94％、7.21％、19.29％；
    所述“载脂蛋白和/或其模拟肽”优选为ApoE或ApoA-I模拟肽；
    所述电中性磷脂优选为磷脂酰胆碱，所述磷脂酰胆碱优选为DMPC；
    所述电中性磷脂的用量优选为24.36％、34.22％、41.21％、42.34％、或者45％；
    所述阴离子磷脂优选为DOPA；
    所述阴离子磷脂的用量优选为17.64％、24.78％、29.79％、30％或者30.66％；
    所述透明质酸和所述鱼精蛋白的总用量优选为1.71％、1.79％、2.15％、5.08％或者13.06％；
    所述纳米复合物的粒径优选为15～95nm，例如26.52±4.31nm、27.38±7.83nm、37.98±14.29nm或者74.20±14.23nm；
    所述纳米复合物的Zeta电位优选为-70～-20mV，例如-57.33±2.31、-55.20±10.74、-43.87±9.68或者-31.57±4.67mV。
  7. 如权利要求1～6中至少一项所述的纳米复合物，其特征在于，当所述蛋白质药物的等电点为8.3～10.3、分子量为15～40kDa时，所述纳米复合物包括1～3％蛋白质药物、0.2～0.6％透明质酸、4.5～6.5％鱼精蛋白、2.5～5％“载脂蛋白和/或其模拟肽”、54～57％电中 性磷脂和32～35％阴离子磷脂；所述透明质酸和所述鱼精蛋白的总用量为5～7％；
    所述蛋白质药物的等电点优选为8.8～9.8，例如9.3；
    所述蛋白质药物的分子量优选为20～33kDa，例如26kDa；
    所述蛋白质药物优选为神经生长因子β-NGF；
    所述蛋白质药物的用量优选为1.5～2.2％，例如1.78％；
    所述透明质酸的用量优选为0.3～0.5％，例如0.43％；
    所述鱼精蛋白的用量优选为5～6％，例如5.53％；
    所述“载脂蛋白和/或其模拟肽”的用量优选为3～4.5％，例如3.82％；
    所述“载脂蛋白和/或其模拟肽”优选为ApoE；
    所述电中性磷脂优选为磷脂酰胆碱；所述磷脂酰胆碱优选为DMPC；
    所述电中性磷脂的用量优选为54.5～56％，例如55.32％；
    所述阴离子磷脂优选为DOPA；
    所述阴离子磷脂的用量优选为32.5～34％，例如33.12％；
    所述透明质酸和所述鱼精蛋白的总用量优选为5.5～6.5％，例如5.96％。
  8. 如权利要求1～7中至少一项所述的纳米复合物，其特征在于，当所述蛋白质药物的等电点为6～8.5、分子量为30～50kDa时，所述纳米复合物包括15～20％蛋白质药物、0.05～0.8％透明质酸、0.8～1.6％鱼精蛋白、3～6％“载脂蛋白和/或其模拟肽”、44～47％电中性磷脂和29～33％阴离子磷脂；所述透明质酸和所述鱼精蛋白的总用量为0.3～2％；
    所述蛋白质药物的等电点优选为6.5～8，例如7.2；
    所述蛋白质药物的分子量优选为35～45kDa，例如40kDa；
    所述蛋白质药物优选为活性酶类蛋白药物HRP；
    所述蛋白质药物的用量优选为16.5～19％，例如17.73％；
    所述透明质酸的用量优选为0.1～0.3％，例如0.14％；
    所述鱼精蛋白的用量优选为0.8～1.5％，例如0.83％；
    所述“载脂蛋白和/或其模拟肽”的用量优选为4～5％，例如4.58％；
    所述“载脂蛋白和/或其模拟肽”优选为ApoE；
    所述电中性磷脂优选为磷脂酰胆碱，所述磷脂酰胆碱优选为DMPC；
    所述电中性磷脂的用量优选为45～46％，例如45.61％；
    所述阴离子磷脂优选为DOPA；
    所述阴离子磷脂的用量优选为30.5～32％，例如31.11％；
    所述透明质酸和所述鱼精蛋白的总用量优选为0.5～1.5％，例如0.97％；
    所述纳米复合物的粒径优选为20～57nm，例如37.22±7.28nm；
    所述纳米复合物的Zeta电位优选为-55～-15，例如-48.87±1.95；
    当所述蛋白质药物的等电点为4.4～6.4、分子量为210～255kDa时，所述纳米复合物包括10～33％蛋白质药物、0.2～3％透明质酸、3.5～7％鱼精蛋白、3～6％“载脂蛋白和/或其模拟肽”、32～42％电中性磷脂和22～39％阴离子磷脂；所述透明质酸和所述鱼精蛋白的总用量为4～8％；
    所述蛋白质药物的等电点优选为4.9～5.9，例如5.4；
    所述蛋白质药物的分子量优选为220～250kDa；
    所述蛋白质药物优选为过氧化氢酶CAT；
    所述蛋白质药物的用量优选为11～32％，例如12.06或者30.9％；
    所述透明质酸的用量优选为0.3～2.5％，例如0.5％或者1.53％；
    所述鱼精蛋白的用量优选为4～5.5％，例如4.85％或者5.1％；
    所述“载脂蛋白和/或其模拟肽”的用量优选为4～5％，例如4.43％或者4.5％；
    所述“载脂蛋白和/或其模拟肽”优选为ApoE；
    所述电中性磷脂优选为磷脂酰胆碱；所述磷脂酰胆碱优选为DMPC；
    所述电中性磷脂的用量优选为33～41％，例如34.2％或者39.91％；
    所述阴离子磷脂优选为DOPA；
    所述阴离子磷脂的用量优选为24～38％，例如24.8％或者37.22％；
    所述透明质酸和所述鱼精蛋白的总用量优选为5～7％，例如5.6％或者6.38％；
    所述纳米复合物的粒径优选为55～70nm，例如63.62±1.97nm；
    所述纳米复合物的Zeta电位优选为-25～-15mV，例如-19.43±1.96mV。
  9. 一种如权利要求1～8中至少一项所述的纳米复合物的制备方法，其通过下述方法一或方法二进行制备：
    方法一：
    S1.所述蛋白质药物、所述透明质酸和所述鱼精蛋白混合形成纳米凝胶；
    S2.所述脂质成分通过常规方法制备脂质体；
    S3.所述纳米凝胶与所述脂质体共孵育形成含有纳米凝胶的脂质体；
    S4.所述含有纳米凝胶的脂质体和“所述载脂蛋白和/或其模拟肽”的混合物通过自组装形成所述纳米复合物；
    方法二：
    S1.所述蛋白质药物、所述透明质酸和所述鱼精蛋白混合形成纳米凝胶；
    S2.将所述脂质成分通过常规方法制备脂质体；
    S3.所述纳米凝胶与所述脂质体通过微流控芯片制备含有纳米凝胶的脂质体，超滤去除溶剂后，制得包载蛋白药物的脂质体；
    S4.所述包载蛋白药物的脂质体和“所述载脂蛋白和/或其模拟肽”的混合物通过自组装形成所述纳米复合物。
  10. 一种如权利要求1～8中至少一项所述的纳米复合物在所述蛋白质药物递送中的应用；优选，所述纳米复合物用于所述蛋白质药物的细胞内递送、体内递送或者脑内递送。
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