A fusion protein, high-density lipoprotein containing the fusion protein and bionic nanomedicine, and preparation and application thereof

Abstract

The present invention discloses a fusion protein, a high-density lipoprotein containing the fusion protein, and a bionic nano-drug, as well as preparation and application thereof. The fusion protein comprises an alpha-helical amphipathic polypeptide and a neuron targeting peptide. The high-density lipoprotein containing the fusion protein is used as a bionic nano-carrier to achieve brain-targeted delivery of the drug through intravenous administration, which saves manpower, material resources and financial resources compared with in situ administration to the brain, and significantly improves patient compliance.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a fusion protein, a high-density lipoprotein containing the fusion protein, and a bionic nano-medicine, as well as preparation and application thereof. Background Art

[0002] Among common central nervous system diseases, traumatic brain injury (TBI) has the highest incidence. The main cause of neurological dysfunction in TBI patients is the loss of high-level neural activity and learning and memory abilities caused by massive neuronal damage and loss. Under pathological conditions, the impaired neuroprotective function of endogenous neurotrophic factors and their exhaustion in brain injury lesions are the key to the lack of intrinsic motivation for neural regeneration after brain injury. Therefore, how to efficiently deliver neurotrophic factors to promote neural regeneration and repair is the key to improving the prognosis of neurological function in patients with central nervous system injuries.

[0003] At present, there are no clinical neurotrophic factor-related drugs for the treatment of central nervous system injuries, no effective drugs for central nervous system nerve regeneration and repair treatment, and no corresponding effective carriers for the intracerebral delivery of neurotrophic factors in the market. The patents currently applied for and disclosed are mainly for the carriers of neurotrophic factors and their applications in vitro or in peripheral nerve injury models, such as CN103656623A, which prepares nano-microspheres loaded with neurotrophic factors and can effectively promote cell axon growth in the form of sustained release. CN111297513A discloses an artificial nerve conduit loaded with nutrient factors, which is characterized by being composed of an external acylated aminopolysaccharide tube and a biological fiber loaded with a sustained release layer of neurotrophic factors filled in the tube. CN116459329A prepares a nanocomposite material for promoting neurovascular reconstruction and its preparation method and application, but due to the lack of targeting, it is necessary to adopt an in situ administration method. Summary of the invention

[0004] In order to solve the problem of lack of targeting in existing central nervous system nerve regeneration and repair drugs, the present invention provides a fusion protein, high-density lipoprotein containing the fusion protein, and bionic nanomedicine, and preparation and application thereof. The present invention achieves efficient blood-brain barrier crossing and efficient targeting of brain damaged tissues and damaged neurons by preparing a bionic nanocarrier carrying neurotrophic factors, and achieves nerve regeneration and repair by accurately releasing neurotrophic factors at the site of brain damage, thereby improving the prognosis of central nervous system damage.

[0005] The present invention simulates the natural microenvironment of neurotrophic factors based on the bionic principle, uses cell matrix components hyaluronic acid and protamine, and self-assembles with neurotrophic factors to form a bionic core. The cell matrix-like bionic core imitates the natural neurotrophic factor storage environment to effectively protect the biological activity of neurotrophic factors and realize the universal carrying of neurotrophic factors. Then, a reconstituted high-density lipoprotein (rHDL) shell is constructed, and the bionic core is self-assembled through electrostatic, hydrogen bonding and other effects to form a core-shell structure, which protects the stability of neurotrophic factors and realizes their long circulation in the body. At the same time, combined with the nerve targeting peptide RVG, the α-helical peptide fusion technology is used to modify the nerve targeting peptide RVG on the surface of rHDL to construct a bionic carrier carrying neurotrophic factors, realize the efficient targeted release of neurotrophic factors in the damaged lesions in the brain, and promote nerve regeneration and repair. The design of the bionic nanocarrier breaks through the bottleneck of neurotrophic factor delivery in the brain, and provides a new idea for the treatment of nerve regeneration and repair of TBI and other brain diseases.

[0006] The first aspect of the present invention provides a fusion protein comprising an α-helical amphipathic polypeptide and a neuron targeting peptide.

[0007] In some preferred embodiments of the present invention, the α-helical amphipathic polypeptide is selected from one or more of ApoA-II, ApoC-III, ApoE and ApoA-1; the neuron targeting peptide is RVG and / or a mimetic peptide of RVG.

[0008] In the present invention, the RVG mimetic peptide refers to a polypeptide fragment derived from the rabies virus glycoprotein (RVG), which is highly neurotropic and can specifically recognize and bind to nicotinic acetylcholine receptors in the central nervous system, penetrate the blood-brain barrier through a receptor-mediated transcytosis mechanism, and deliver drugs or other therapeutic molecules directly into the brain parenchyma.

[0009] In some more preferred embodiments of the present invention, the α-helical amphipathic polypeptide is ApoA-1; the neuron targeting peptide is RVG; wherein the amino acid sequence of ApoA-1 is as shown in SEQ ID NO: 3 or has 75%, 80%, 85%, 90%, 95% or 99% identity with the amino acid sequence as shown in SEQ ID NO: 3; the amino acid sequence of the RVG targeting peptide is as shown in SEQ ID NO: 4 or has 75%, 80%, 85%, 90%, 95% or 99% identity with the amino acid sequence as shown in SEQ ID NO: 4.

[0010] In some embodiments of the present invention, the α-helical amphipathic polypeptide and the neuron targeting peptide are connected via a linker sequence or a linker fragment.

[0011] In some preferred embodiments of the present invention, the linking sequence is selected from any one or more of GGGGS, GSL, GSGS, GSG and GGG; the linking fragment includes (PEG)n, wherein n is an integer of 6-12, for example, 6, 8, 9 or 12.

[0012] In some more preferred embodiments of the present invention, the linker sequence is GGG; the linker fragment is (PEG)n; wherein n is an integer of 6-12, for example, 6, 8, 9 or 12.

[0013] In a specific embodiment of the present invention, the linker fragment is PEG12.

[0014] In some embodiments of the present invention, the amino acid sequence of the fusion protein is shown as SEQ ID NO: 1 or SEQ ID NO: 2.

[0015] The second aspect of the present invention provides a recombinant high-density lipoprotein, wherein the recombinant high-density lipoprotein comprises liposomes, apolipoproteins and the fusion protein according to the first aspect of the present invention;

[0016] In some preferred embodiments of the present invention, the lipids of the liposomes are selected from one or more of DMPC (dimyristoylphosphatidylcholine), DOPE (dioleoylphosphatidylethanolamine), DOTAP ((2,3-dioleyloxypropyl)trimethylammonium chloride), DOPA (dioleoylphosphatidic acid), DMPA (dimyristoylphosphatidic acid sodium salt), DPPA (dipalmitoylphosphatidic acid), DSPA (distearoylphosphatidic acid sodium salt) and DOPS (dioleoylphosphatidylserine); the apolipoproteins are selected from one or more of ApoE3, ApoA-1 and ApoJ.

[0017] In a specific embodiment of the present invention, the lipid is DMPC and / or DOPA, such as DMPC and DOPA, or DOTAP and DOPA; and the apolipoprotein is ApoE3.

[0018] In some further more preferred embodiments of the present invention, the molar ratio of DMPC to DOPA is 0.5-5:1; the molar ratio of DOTAP to DOPA is 0.5-5:1.

[0019] In a specific embodiment of the present invention, the molar ratio of DMPC to DOPA is 1.5:1; the molar ratio of DOTAP to DOPA is 1.5:1.

[0020] In the present invention, the apolipoprotein and the fusion protein as described in the first aspect of the present invention are embedded and attached to the surface of the liposome via an α-helical structure.

[0021] The third aspect of the present invention provides a method for preparing recombinant high-density lipoprotein, the method comprising: (1) mixing the liposome and the apolipoprotein and then co-incubating to obtain the lipoprotein;

[0022] (2) Co-incubating the lipoprotein with the fusion protein described in the first aspect of the present invention to obtain the recombinant high-density lipoprotein.

[0023] In some embodiments of the present invention, the liposome is prepared by thin film hydration of the lipid; the lipid of the liposome is selected from one or more of DMPC, DOPE, DOTAP, DOPA, DMPA, DPPA, DSPA and DOPS; the apolipoprotein is selected from one or more of ApoE3, ApoA-1 and ApoJ; the co-incubation satisfies one or more of the following conditions: 100-200 rpm, for example, 120 rpm; 30-37°C, for example, 37 o C; 18-30 h, for example 24 h.

[0024] In some preferred embodiments of the present invention, the lipid is DMPC and / or DOPA, such as DMPC and DOPA, or DOTAP and DOPA; and the apolipoprotein is ApoE3.

[0025] In a specific embodiment of the present invention, the molar ratio of the liposome, the apolipoprotein and the fusion protein as described in the first aspect of the present invention is 1000-2000:1-100:1, for example, 1500:2:1; the molar ratio of DMPC and DOPA is 0.5-5:1, for example, 1.5:1; the molar ratio of DOTAP and DOPA is 0.5-5:1, for example, 1.5:1.

[0026] The fourth aspect of the present invention provides a recombinant high-density lipoprotein, which is prepared by the method described in the third aspect of the present invention.

[0027] The fifth aspect of the present invention provides a bionic nanomedicine, which is a core-shell structure, and comprises a drug nanogel and a recombinant high-density lipoprotein from the inside to the outside; wherein the recombinant high-density lipoprotein is selected from any one of the following groups:

[0028] (1) The recombinant high-density lipoprotein according to the second or fourth aspect of the present invention;

[0029] (2) Contains lipids and apolipoproteins;

[0030] (3) contains lipids and neurotargeting peptides;

[0031] Wherein, the lipid is selected from one or more of DMPC, DOPE, DOTAP, DOPA, DMPA, DPPA, DSPA and DOPS; the apolipoprotein is selected from one or more of ApoE3, ApoA-1 and ApoJ; and the neurotargeting peptide is RVG and / or a mimetic peptide of RVG.

[0032] In a specific embodiment of the present invention, the lipid is DMPC and / or DOPA, such as DMPC and DOPA, or DOTAP and DOPA; the apolipoprotein is ApoE3; and the neurotargeting peptide is RVG.

[0033] In the present invention, the molar ratio of DMPC to DOPA is 0.5-5:1, for example, 1.5:1; the molar ratio of DOTAP to DOPA is 0.5-5:1, for example, 1.5:1; the recombinant high-density lipoprotein is wrapped as a shell in the outer layer of the drug nanogel.

[0034] In some embodiments of the present invention, the drug nanogel comprises hyaluronic acid, protamine and a drug.

[0035] In some embodiments of the present invention, the drug is a biomacromolecule drug.

[0036] In some preferred embodiments of the present invention, the biomacromolecule drug is a protein drug and / or a nucleic acid drug.

[0037] In the present invention, the protein drugs are protein products derived from animals or plants and developed through applied biotechnology with certain biological activities for the prevention, treatment and diagnosis of human, animal and plant diseases, including one or more selected from interferon, cytokine, protein hormone, peptide, antibody and vaccine.

[0038] In the present invention, the nucleic acid drug refers to nucleic acid (including nucleotides and deoxynucleotides) extracted from the cells of certain animals and microorganisms, or a substance with a nucleic acid structure, including a nucleotide and a deoxynucleotide structure, prepared by artificial synthesis, and having a certain pharmacological effect, which is called a nucleic acid drug or a nucleic acid biochemical drug. The nucleic acid drug includes one or more selected from nucleotide drugs, nucleic acid drugs and drugs containing different base compounds, such as Aptamer, Antigene, Ribozyme, Antisensenucleic acid or RNA interfering agent.

[0039] In some more preferred embodiments of the present invention, the protein drug is a neurotrophic factor; and the nucleic acid drug is RNA.

[0040] In a specific embodiment of the present invention, the neurotrophic factor is NGF and / or BDNF, for example, NGF; the gene drug includes one or more selected from siRNA, shRNA, microRNA, mRNA, long non-coding RNA and DNA; for example, siRNA.

[0041] The sixth aspect of the present invention provides a method for preparing a bionic nanomedicine, the method comprising: mixing and assembling liposomes loaded with drug nanogel and apolipoprotein and then co-incubating with the fusion protein as described in the first aspect of the present invention.

[0042] In some preferred embodiments of the present invention, the incubation satisfies one or more of the following conditions: 100-200 rpm, such as 120 rpm; 30-37° C., such as 37 o C; 18-30 h, for example 24 h.

[0043] In some preferred embodiments of the present invention, the method further comprises: incubating the drug nanogel and the liposome at 20-25° C. for 15-40 min to obtain the liposome loaded with the drug nanogel; for example, 25° C. for 30 min.

[0044] In some more preferred embodiments of the present invention, the method further comprises: obtaining the drug nanogel by incubating the drug, hyaluronic acid and protamine at 20-25° C. for 5-15 min; for example, 25° C. for 10 min.

[0045] In some further more preferred embodiments of the present invention, the method further comprises: preparing liposomes by a thin film hydration method of the lipids.

[0046] In the present invention, the steps of the thin film hydration method are: adding anhydrous ether to the lipid, evaporating under vacuum, adding chloroform solution, placing a rotary evaporator to evacuate for 1 hour, and after hydration, intermittently shaking in a 40°C water bath for 5 minutes until the thin film is hydrated and falls off to obtain the liposome. Probe ultrasound further reduces the liposome particle size to obtain the liposome.

[0047] In some embodiments of the present invention, the drug is a biomacromolecule drug; the lipid is selected from one or more of DMPC, DOPE, DOTAP, DOPA, DMPA, DPPA, DSPA and DOPS; the apolipoprotein is selected from one or more of ApoE3, ApoA-1 and ApoJ;

[0048] In some preferred embodiments of the present invention, the biomacromolecule drug is a protein drug and / or a nucleic acid drug; the lipid is DMPC and / or DOPA, such as DMPC and DOPA, or DOTAP and DOPA; and the apolipoprotein is ApoE3.

[0049] In some more preferred embodiments of the present invention, the protein drug includes one or more selected from interferon, cytokine, protein hormone, polypeptide, antibody and vaccine, such as neurotrophic factor; the nucleic acid drug includes one or more selected from nucleotide drugs, nucleic acid drugs and drugs containing different base compounds, such as RNA, Aptamer, antigene, ribozyme, antisense nucleic acid or RNA interfering agent.

[0050] In some further more preferred embodiments of the present invention, the neurotrophic factor is NGF and / or BDNF, for example, NGF; the nucleic acid drug includes one or more selected from siRNA, shRNA, microRNA, mRNA, long non-coding RNA and DNA; for example, siRNA; the molar ratio of the liposome, apolipoprotein and fusion protein is 1000-2000:1-100:1, for example, 1500:2:1; the molar ratio of DMPC and DOPA is 0.5-5:1, for example, 1.5:1; the molar ratio of DOTAP and DOPA is 0.5-5:1, for example, 1.5:1; the molar ratio of the drug, hyaluronic acid and protamine is 400-800:2-8:50-100, for example, 631:2:77.

[0051] The seventh aspect of the present invention provides a bionic nanomedicine, which is prepared by the method described in the sixth aspect of the present invention.

[0052] The eighth aspect of the present invention provides a pharmaceutical composition, which comprises one or more of the fusion protein as described in the first aspect of the present invention, the recombinant high-density lipoprotein as described in the second aspect or the fourth aspect of the present invention, and the bionic nanomedicine as described in the fifth aspect or the seventh aspect of the present invention.

[0053] The ninth aspect of the present invention provides the use of one or more of the fusion protein as described in the first aspect of the present invention, the recombinant high-density lipoprotein as described in the second aspect of the present invention, the recombinant high-density lipoprotein as described in the fourth aspect of the present invention, the bionic nanomedicine as described in the fifth aspect of the present invention, and the bionic nanomedicine as described in the seventh aspect in the preparation of neural regeneration and repair drugs or drugs for treating brain diseases.

[0054] In some embodiments of the present invention, the brain disease is traumatic brain injury. Based on the common sense in the art, the above-mentioned preferred conditions can be combined arbitrarily to obtain the preferred embodiments of the present invention.

[0055] The reagents and raw materials used in the present invention are commercially available.

[0056] The positive and progressive effects of the present invention are:

[0057] The present invention prepares a fusion protein and uses recombinant high-density lipoprotein containing the fusion protein as a bionic nanocarrier. The greatest advantage of the fusion protein is that it can carry a variety of different types of drugs, has universal applicability, and can achieve brain-targeted drug delivery through intravenous administration. Compared with in situ brain administration, it saves manpower, material resources and financial resources, and significantly improves patient compliance.

[0058] The design of the bionic nanocarrier loaded with neurotrophic factors prepared by the present invention breaks through the bottleneck of neurotrophic factor delivery in the brain, provides a synergistic treatment strategy for activating endogenous nerve regeneration ability through targeted delivery of neurotrophic factors, and will provide new technology for nerve regeneration treatment of diseases related to brain nerve damage.

[0059] The bionic nanocarriers carrying neurotrophic factors prepared by the present invention are all made of bionic materials, the raw materials are easily available, and the natural components in the human body are highly restored, and have good biological safety. The preparation process of the bionic nanocarrier is simple, does not require complicated operation procedures and high-end instruments and equipment, has low manufacturing costs, and is easy to mass produce. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 The morphology of biomimetic nanocarriers loaded with NGF is shown by particle size analyzer and transmission electron microscopy. (A) Light scattering particle size distribution; (B) Zeta potential distribution; (C) Transmission electron microscopy image; Scale bar: 50 nm.

[0061] Figure 2 The biomimetic nanocarriers loaded with NGF induced PC12 cell differentiation. (A) Differentiation images of PC12 cells after treatment with different preparations; (B) Image J analysis of the differentiation ratio of PC12 cells after treatment with different preparations. The positive control was free NGF; the simple differentiation medium was the blank control (Control). MAP2 antibody was used to label PC12 cell dendrites, blue fluorescence: cell nucleus; green fluorescence: MAP2. Scale bar: 100 μm. *p<0.05, indicating a significant difference from the free NGF group.

[0062] Figure 3The targeting effect of bionic nanocarriers loaded with NGF on brain lesions in mice with cortical injury model is shown. (A) Brain distribution of bionic nanocarriers loaded with NGF, bionic nanocarriers labeled with DIR; (B) Semi-quantitative analysis of brain fluorescence by Image J; (C) Confocal laser scanning microscopy analysis of the targeting effect of bionic nanocarriers loaded with NGF on neurons in frozen sections of CCI model mice, using Alexa Fluor 647 to label NGF protein. Blue fluorescence: cell nucleus; green fluorescence: neurons; red fluorescence: NGF. White arrows indicate NGF protein taken up by neuronal cells. Scale bar: 100 μm. (D) Elisa quantitative analysis of NGF concentration in brain injury lesions of CCI model mice after tail vein administration of different preparations. *p<0.05, ****p<0.0001, indicating significant differences compared with the group injected with free NGF.

[0063] Figure 4 The cortical injury model of C57 mice was established, and the sham group was used as the sham injury control group. After modeling, continuous medication for 2 weeks improved the motor function of mice. (A) Flowchart of the construction of cortical injury model and behavioral evaluation of C57 mice. (B) mNSS scores were performed on CCI model mice from 1 day to 14 days after injury. (C) Rotarod fatigue test was performed on CCI model mice from 1 day to 7 days after injury. *p<0.05, **p<0.01, ****p<0.0001, indicating significant differences compared with the CCI+PBS group.

[0064] Figure 5 A C57 mouse cortical injury model was established, and the sham group was a sham injury control group. After modeling, continuous medication for 4 weeks improved the memory function of mice. (A) Schematic diagram of the mouse novel object recognition experiment to investigate the short-term memory function of mice. (B) Cognitive index of novel object recognition in mice. (C) Schematic diagram of the Morris water maze experiment in mice to investigate the effects of different preparations loaded with NGF on the long-term learning and memory function of CCI model mice. (D) Representative trajectory diagram of the learning and testing periods of the mouse water maze experiment. (E) Statistical diagram of the latency of CCI model mice to find the platform 23-26 days after injury. (F) Statistical diagram of the average time spent exploring the quadrant where the platform is located in the water maze experiment of CCI model mice. (G) Statistical diagram of the average swimming speed recorded in the water maze experiment of CCI model mice. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, indicating significant differences compared with the CCI+PBS group. #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001, indicating significant differences compared with the CCI+rHDL-core-RVG group.

[0065] Figure 6The in vivo safety evaluation of biomimetic nanocarriers loaded with NGF is shown. (A) A C57 mouse cortical injury model was established, and the Sham group was a sham injury control group. After modeling, the drug was continuously administered for 4 weeks. HE staining images of the organs of mice in each group. Scale bar: 100 μm. (B) The serum ALT, AST, BUN, CREA, CK, and LDH levels of mice in each group after 4 weeks of administration.

[0066] Figure 7 The storage stability of the NGF-loaded biomimetic nanoformulation was demonstrated.

[0067] Figure 8 The effect of the linker sequence on the targeting of NGF-loaded biomimetic nanoformulations is shown.

[0068] Fig. 9 The results show that the bionic nanocarrier has high loading efficiency for siRNA.

[0069] Fig.10 The results show that the biomimetic nanocarrier has high efficiency in loading miRNA. DETAILED DESCRIPTION

[0070] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0071] Example 1 Preparation and characterization of bionic nanocarriers carrying neurotrophic factors

[0072] (1) Preparation

[0073] The thin film hydration method was used to prepare empty liposomes: 10 μmol of lipids (DMPC and DOPA in a molar ratio of 1.5:1, Avanti Polar Lipids, Alabaster, AL, USA) were weighed and placed in a 500 mL round-bottom flask, anhydrous ether was added, and the water in the phospholipids was removed by vacuum evaporation. Then, chloroform solution was added, and the evaporator was placed in a rotary evaporator and vacuumed for 1 h. Then, 4 mL of triple distilled water was added for hydration, and the liposomes were obtained by intermittent shaking in a 40°C water bath for 5 min until the thin film was hydrated and fell off. The liposome particle size was further reduced by probe ultrasound to obtain empty liposomes (lipo).

[0074] The neurotrophic factor core was prepared by self-assembly. The neurotrophic factor NGF (0.0631 μmoL) (Sino Biological, Beijing, China) was incubated with hyaluronic acid (0.0002 μmoL) (Sigma-Aldrich (StLouis, MO, USA) and protamine (0.0077 μmoL) (Sigma-Aldrich, St Louis, MO, USA) at room temperature (20-25°C) for 10 min to form the neurotrophic factor core (NGF core).

[0075] Empty liposomes (10 μmoL) were incubated with the core loaded with neurotrophic factor at room temperature (20-25°C) for 30 minutes to form nanoliposomes loaded with neurotrophic factor core (NGF core-lipo).

[0076] Apolipoprotein ApoE3 (0.0145 μmoL) (PEPROTECH, Rocky Hill, NJ, USA) was added to the above nanoliposome solution and placed on a shaker at 120 rpm, 37 o C for 24 h to form lipoprotein containing neurotrophic factor core (NGF core-rHDL).

[0077] The APOE3 sequence is as follows (SEQ ID NO: 5):

[0078] MKVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVCGRLVQYRGEVQAMLGQSTEELRVRLASHLRKLRKRLL RDADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVRAATVGSLAGQPLQERAQAWGERLRARMEEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWAGLVEKVQAAVGTSAAPVPSDNH

[0079] The fusion protein of ApoA-1 and neuron targeting peptide RVG was synthesized by solid phase peptide synthesis, and its sequence was Ac-FAEKFKEAVKDYFAKFWD-GGG-GNSARKGRSNTFIDCPTGPRPNEPMWITY (SEQ ID NO: 1) or Ac-FAEKFKEAVKDYFAKFWD-PEG12-GNSARKGRSNTFIDCPTGPRPNEPMWITY (SEQ ID NO: 2), wherein Ac is acetyl.

[0080] ApoA-1: FAEKFKEAVKDYFAKFWD (SEQ ID NO: 3)

[0081] RVG targeting peptide: GNSARKGRSNTFIDCPTGPRPNEPMWITY (SEQ ID NO: 4)

[0082] The above lipoproteins were incubated with the neurotropic peptide RVG (0.0065 μmoL) at 37°C and 120 rpm for 24 h to form a bionic nanocarrier containing a neurotrophic factor core (NGF core-rHDL-RVG).

[0083] (2) Characterization

[0084] The biomimetic nanocarriers carrying the NGF core were negatively stained with phosphotungstic acid and the morphology was observed by transmission electron microscopy. The dynamic light scattering particle size and surface potential were measured by laser particle size analyzer. Figure 1 Under the electron microscope, the bionic nanocarrier with NGF core was spherical with uniform particle size, about 40 nm-50 nm. Its surface potential was about -30 mV as measured by laser particle size analyzer.

[0085] Example 2 Bionic nanocarriers loaded with neurotrophic factors induce PC12 cell differentiation

[0086] PC12 cells were cultured with proliferation medium (RPMI1640 medium + 10% horse serum + 5% fetal bovine serum + 1% double antibody). The cells were plated at a density of 50% on a PLL-coated 96-well plate. After 24 hours of proliferation culture, they were replaced with differentiation medium (RPMI1640 medium + 1% horse serum (without fetal bovine serum)) and cultured for 12 hours for starvation treatment, followed by drug administration. The NGF administration concentration was 100 ng / mL, and different NGF preparations were co-incubated with cells for 5 days, followed by discarding the medium, washing with PBS 3 times, fixing with 4% paraformaldehyde for 15 min, and then permeabilizing and blocking with 0.3% TritonX-100. Primary antibody staining: Incubate with diluted MAP2 antibody (1:1000) overnight at 4°C. Pour out the solution and wash the cells 3 times with PBS for 5 min each time. Secondary antibody staining: Incubate with Alexa fluor 488-conjugated secondary antibody (1:1000) at room temperature in the dark for 1 h, then wash the cells 3 times with PBS in the dark for 5 minutes each time. Use fluorescence microscopy to observe the morphology of PC12 cells differentiated into neuron-like cells induced by different NGF preparations, and use Image J to semi-quantitatively analyze the proportion of neurons induced by PC12 cells. The experimental results are shown in Figure 2 As shown, the bionic nanocarrier loaded with NGF core efficiently promoted the differentiation of PC12 cells into neuron-like cells, with increased branch points and synapse length, and a morphology similar to that of neurons. The differentiation-promoting ratio of the bionic nanocarrier loaded with NGF core was comparable to that of free NGF.

[0087] Example 3 Study on the targeting of bionic nanocarriers loaded with neurotrophic factors to brain injury lesions and neurons in CCI model mice

[0088] First, NGF labeled with Alexa Fluor647 fluorescent probe was prepared, and biomimetic nanocarriers and control preparations loaded with NGF were prepared in the same manner as in Example 1. The preparation solution was concentrated using an ultrafiltration centrifuge tube with a molecular weight cutoff of 30 kD, and NGF was administered via the tail vein at a dose of 2.5 mg / kg. A controlled cortical injury (CCI) model in mice was constructed. After weighing C57BL / 6 male mice, anesthesia was induced using 3% isoflurane, and then the mouse head was fixed on a stereotaxic head frame, and anesthesia was maintained using 1.5% isoflurane. The surgical area was prepared, the hair in the surgical area was shaved, and iodine was used for disinfection. The scalp was incised under sterile conditions to expose the right parietal bone, the periosteum was scraped off with a sterile cotton swab soaked in hydrogen peroxide, and the right parietal bone was drilled using a grinding drill. The center of the drilling was located 2 mm behind the anterior fontanelle, 2 mm away from the midline, and the drilling diameter was 4 mm. A circular bone window was opened to expose the dura mater. The striking parameters were set as follows: speed 4.5 m / s, depth 1.5 mm, striking head diameter 2.5 mm, contact time 100 ms, to simulate moderate injury. After injury, the skull was closed, the skin was sutured, and the incision was disinfected. In the sham injury group, only the scalp was incised to expose the right parietal bone, and no striking injury was given. The preparation solution labeled with Alexa Fluor647 probe was administered via the tail vein. Four hours after administration, the mice were anesthetized by isoflurane inhalation and killed. The brain tissue was removed, rinsed with saline, and placed in a small animal in vivo imaging device to collect images. The experimental results are shown in the figure. Figure 3 As shown in AB, free NGF is less distributed in brain injury lesions, the enrichment of NGF core-loaded liposomes (NGF core-lipo) and NGF core-loaded recombinant high-density lipoproteins (NGF core-rHDL) in brain injury lesions gradually increases, while the accumulation of RVG-modified NGF core-loaded lipoprotein administration group (NGF core-rHDL-RVG) in brain injury lesions is significantly increased. The above results indicate that compared with free NGF and carriers without RVG targeting peptide modification, NGF core-loaded lipoproteins modified with RVG targeting peptide can efficiently penetrate the blood-brain barrier and be distributed to brain injury sites.

[0089] In order to evaluate the distribution of RVG targeting peptide-modified NGF-core-loaded lipoproteins in brain injury lesions of CCI model mice and their targeting to neurons at the brain slice level, transgenic mice Thy1-GFP expressing green fluorescent protein in neurons were used to prepare NGF labeled with Alexa Fluor 647 fluorescent probe, and bionic nanocarriers loaded with NGF and control preparations were prepared in the same way as in Example 1. The preparation solution of Alexa Fluor647 probe fluorescent labeling was administered through the tail vein. Four hours after administration, the mice were anesthetized by isoflurane inhalation. The mice were fixed, the chest cavity was cut open and the heart was fully exposed. The scalp needle was inserted into the left ventricle, and then the right auricle was cut open. The mice were immediately perfused with normal saline until the outflowing perfusate was bloodless. Then, the heart was perfused and fixed with 4% paraformaldehyde solution until the liver, limbs, and tail became hard. The brain tissue was placed in formalin solution and fixed for 48 hours, and then placed in 15% and 30% sucrose solution gradient dehydration to the bottom. The brain tissue was embedded in OCT and frozen at -20℃. Continuous brain coronal sections were made with a freezing microtome with a thickness of 20 μm. The brain sections were rinsed with PBS, the cell nuclei were stained with DAPI for 10 minutes, and then rinsed again with PBS. The water stains were wiped off, and the anti-fluorescence quencher was added to seal the sections. The brain sections were observed under a laser confocal microscope. The experimental results are shown in the figure. Figure 3 As shown in Figure C, free NGF is less distributed in brain injury lesions and its distribution in the brain is scattered and non-targeted, while the accumulation of NGF core-rHDL-RVG in brain injury lesions is significantly increased and is mainly distributed around neurons. The above results indicate that compared with free NGF, lipoproteins containing NGF core modified with RVG targeting polypeptide efficiently penetrate the blood-brain barrier and are targeted to brain injury sites and enriched around neurons.

[0090] Elisa was used to detect the concentration of NGF in the brain of CCI model mice 4 hours after different NGF preparations were administered through the tail vein. The bionic nanocarriers loaded with NGF and the control preparations were prepared in the same way as in Example 1. The preparation solution was concentrated using an ultrafiltration centrifuge tube with a molecular weight cutoff of 30 kD, and NGF was administered through the tail vein at a dose of 2.5 mg / kg. The mouse CCI model was constructed as described above, and different groups of bionic nanocarriers of NGF were administered through the tail vein after injury recovery. The mice were anesthetized by isoflurane inhalation and killed 4 hours after administration. The brain tissue was removed after perfusion with 0.9% saline, and the cortical tissue and hippocampal tissue on the injured side were separated on ice. The tissue was dissolved with protein lysis buffer, and 100 μL of 3% SDS lysis buffer was added for every 10 mg of tissue. Then, an equal volume of Triton X-100 lysis buffer (containing inhibitors) was added. The tissue was homogenized with a homogenizer, lysed on ice for 10 min, and then centrifuged at 4°C, 15,000 rpm for 20 min. The supernatant was collected and the NGF content was determined by ELISA. The experimental results are shown in Figure 3As shown in D, consistent with the results of in vivo imaging, the concentration of free NGF was lowest in the brain injury lesions, while the concentration of NGF core-rHDL-RVG group in the brain injury lesions was significantly increased. The above results also prove that lipoproteins carrying NGF core modified with RVG targeting polypeptide can efficiently pass through the blood-brain barrier and be targeted to the brain injury site.

[0091] Example 4 Bionic nanocarriers loaded with neurotrophic factors improve motor ability of CCI model mice

[0092] The bionic nanocarriers carrying the NGF core and the control preparations were prepared in the same manner as in Example 1, and the mNSS neurological function score and the Rotarod test were used to evaluate the improvement of the motor ability of the bionic nanocarriers carrying the NGF core in the CCI model mice. Before starting behavior, the mice need to be placed in the room where the behavioral test is performed for 2-3 hours to adapt to the behavioral environment. It is necessary to ensure that the environment is relatively quiet (with white noise of lower decibels) and dim (the light source needs to be away from the behavioral device). The behavioral instrument needs to be smeared with 75% alcohol before and during the experiment to eliminate the odor, feces, etc. brought by the previous experimental subject. CCI model mice were randomly divided into groups, 8 in each group, and the tail vein was administered as follows: 1. Sham + saline in the sham injury group; 2. CCI + saline in the control group; 3. CCI + NGF; 4. CCI + core-rHDL-RVG; 5. CCI + NGF core-rHDL-RVG. Modified neurological severity score (mNSS) tests were performed 1, 3, 7, and 14 days after administration. The mNSS score includes the evaluation of movement, sensation, balance, and reflexes (normal neurological function is 0 points; the most severe neurological impairment is 10 points). If a specific task cannot be completed or a reflex of the test is lost, 1 point is given. Therefore, the higher the score, the more severe the neurological impairment. 1-4 points, mild impairment; 5-6 points, moderate impairment; 7-8 points, severe impairment; 9 points, moribund state; 10 points, mouse death. mNSS scoring criteria: 1. The mouse has hemiplegia or monoplegia; 2. The mouse passes a 3 cm wide balance beam; 3. The mouse passes a 2 cm wide balance beam; 4. The mouse passes a 1 cm wide balance beam; 5. The mouse maintains balance on a 0.7 cm wide balance beam; 6. The mouse maintains balance on a 0.5 cm wide round stick; 7. The mouse can find the exit in a circle with a diameter of 30 cm within 2 minutes; 8. The mouse walks in a straight line; 9. The mouse has exploratory behavior; 10. The mouse has a startle reflex. The experimental results are as follows Figure 4As shown in B, the motor function score of the Sham group was the best, and the change was not large, proving that surgical operations such as opening the bone window had no effect on the motor ability of mice. The mNSS score of mice in the control group CCI+PBS was the worst, and the recovery speed was the slowest. The mNSS score and recovery speed of the free NGF group were similar to those of the control group; the motor function recovery speed of mice in the CCI+empty vector core-rHDL-RVG group was accelerated, but there was no significant difference at each time point compared with the control group; the mNSS score of mice in the CCI+NGF core-rHDL-RVG group further improved, and the motor ability of mice recovered faster. Compared with the control group, there was a significant difference in motor function from the 7th day. It proves that NGF core-rHDL-RVG can improve the motor ability of CCI model mice.

[0093] Rotarod fatigue test was performed on days 1, 2, 3, 5, and 7 after administration. 3 days of training were required before the test. Mice were required to complete 5 rotarod tests on the test day, and the data that did not meet the requirements (such as running in the opposite direction, accidental falling, etc.) were removed and the average value was taken. Mice were allowed to rest for 20 min between each experiment. Training: On the first day of rotarod training, the rotarod speed was set to 20 r / min, the rotation time was 5 min, and the operation mode was to uniformly accelerate from 5 r / min to 20 r / s within 150 s and then maintain a uniform speed; on the second day of rotarod training, the rotarod speed was set to 30 r / min, and the rotation time was 5 min. The operation mode was to uniformly accelerate from 5 r / min to 30 r / s within 150 s and then maintain a uniform speed; on the third day of rotarod training, the rotarod speed was set to 40 r / min, and the rotation time was 5 min. The operation mode is to uniformly accelerate from 5 r / min to 40 r / s within 150 s and then maintain a constant speed; after 3 days of training, mice that cannot learn to rotate the rod and mice whose continuous exercise time does not meet the standard are eliminated. Test: After training, the maximum rotation speed is set to 40 r / min, and the operation mode is the same as before. The continuous exercise time of the mouse on the rotating rod before falling is recorded. If the mouse does not fall for 5 minutes, it is recorded as 5 minutes, and the experiment is ended. The experimental results are as follows Figure 4As shown in C, the mice in the sham group basically maintained without falling for 5 minutes, proving that surgical operations such as opening bone windows have no effect on motor function. The mice in the control group CCI+PBS had the shortest time to fall from the rotating rod, and gradually recovered over time after injury, with the slowest recovery rate. The falling time of mice in the CCI+free NGF group was similar to that of the control group; the motor function recovery rate of mice in the CCI+empty vector core-rHDL-RVG group was accelerated, and the falling time was significantly different from that of the control group from the third day after injury, proving that core-rHDL-RVG also has the ability to partially restore the motor function of mice; the falling time of mice in the CCI+NGF core-rHDL-RVG group was better, and the mice's motor ability recovered faster. Compared with the control group, there was a significant difference in the falling time from the third day. It proves that NGF core-rHDL-RVG can improve the motor ability of CCI model mice.

[0094] Example 5 Bionic nanocarriers loaded with neurotrophic factors improve spatial learning and memory abilities of CCI model mice

[0095] The novel object recognition test (NORt) and Morris water maze test (Morris water maze) were used to evaluate the improvement of the spatial learning and memory ability of CCI model mice by biomimetic nanocarriers carrying NGF core. The same grouping was performed as in Example 4. The novel object recognition test was performed 21 days after administration. NORt is a delicate and sensitive behavioral method that uses the natural instinct of rodents to approach and explore novel objects to detect animal recognition memory. The number, time and distance of the exploration of new and old objects by mice were used to detect the cognitive status of mice. If the cognitive ability of mice is poor, there is no difference in the exploration of new and old objects; if the cognitive ability of mice is normal, the exploration of new objects is longer than that of old objects. The recognition index (RI) calculation formula is: RI = new object contact time / (new object contact time + old object contact time) * 100%. A set of equipment for new object recognition: including a square activity field, camera equipment, objects: wooden blocks of different colors and shapes that are odorless, not smooth, and cannot be moved by mice at will. For 3 days before the start of training and testing, the mice were stroked every day to avoid stress. At the beginning of training, objects A and B were placed on the left and right ends of one side wall respectively. The mouse was placed in the arena with its back facing the two objects, and the distance between the tip of the mouse's nose and the two objects should be the same. 10 minutes after being placed in the arena, the contact between the mouse and the two objects was recorded. During the testing phase, object B in the arena was replaced with object C of a different color and shape, and the mouse was still placed with its back facing the two objects, with the tip of its nose at the same distance from the two objects. 10 minutes after being placed in the arena, the contact between the mouse and the two objects was recorded. The experimental results are as follows. Figure 5As shown in B, the cognitive index of mice in the CCI+NGF core-rHDL-RVG group was significantly improved, which was similar to that of the Sham group.

[0096] Morris water maze test was performed 28 days after drug administration. The composition of the water maze: the pool is 150 cm in diameter, 50 cm in height, 30 cm in depth, and the water temperature is 25±1℃. The circumference of the pool is divided into four water entry points, and their connecting lines divide the circular pool into four quadrants. A 9 cm white platform is placed in the center of the fourth quadrant. The platform is about 1 cm below the water surface. The bottom of the pool, the platform and the four walls are dyed opaque white with food dye to make the platform invisible. Spatial reference objects (doors, cameras, wall signs, etc.) are set around the pool and their positions remain unchanged for mice to locate and remember the position of the platform. The camera is placed above the center of the pool to automatically collect images of animal swimming. The collected signals are directly input into the computer, and the Morris water maze video analysis system is used to monitor and record the swimming trajectory of mice. Positioning navigation test: used to evaluate the spatial learning ability of mice, which lasts for 5 days. The platform was fixed in the center of quadrant IV, and mice were placed in the water facing the pool wall from the water entry points of the four quadrants according to the principle of randomness. The computer monitored and recorded the route, time required (latency) and swimming speed of the mice from entering the water to finding and climbing onto the platform. If the mouse fails to find the platform within 60 s, it must be led to the platform and stay for 10 s. At this time, the latency is recorded as 60 s. Each mouse was trained 4 times a day. In the spatial exploration experiment, after 5 days of positioning and navigation tests, the platform was removed on the 6th day, and the mice were placed in the water from the water entry point of quadrant II facing the pool wall. The exploration time of the mice in the target quadrant (the quadrant where the platform is located) within 60 s and the trajectory of the mice searching for the platform were recorded. The experimental results are as follows: Figure 5 As shown in the CG, there was no difference in the swimming speed of each group of mice during the training period 28 days after modeling, indicating that the test results of each group of mice were not affected by motor function. As the training time increased, the latency of mice in the CCI+Saline group, CCI+NGF group and CCI+core-rHDL-RVG group did not show a significant decrease. The latency of mice in the CCI+NGF core-rHDL-RVG group gradually decreased with the extension of training time, which was significantly lower than that of mice in the CCI model group; at the same time, the spatial exploration experiment showed that the target platform residence time of mice given core-rHDL-RVG and NGF core-rHDL-RVG increased significantly, and the platform residence time of the NGF core-rHDL-RVG group was also significantly higher than that of the empty vector core-rHDL-RVG group, indicating that NGF core-rHDL-RVG efficiently delivers NGF to the brain damage site to promote nerve damage repair and improve the spatial learning and memory ability of mice.

[0097] Example 6 Biosafety Evaluation of Bionic Nanocarriers Carrying Neurotrophic Factors

[0098] Similar to Example 5, the mice were killed after the behavioral experiment. The hearts were perfused with 0.9% saline and 4% paraformaldehyde for fixation. The hearts, livers, spleens, lungs and kidneys were removed and HE staining was performed. The experimental results are shown in Figure 6 As shown in A, no obvious organ damage was observed in the mice in each drug-treated group compared with the sham group, indicating that the biomimetic nanocarrier has good safety. The serum of the mice was taken before the mice were killed, and the biochemical indicators were tested on the machine, including the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), blood creatinine (CREA), creatine kinase (CK), and lactate dehydrogenase (LDH). ALT and AST levels indicate liver function levels, BUN and CREA indicate kidney function levels, and CK and LDH indicate heart damage. The experimental results are shown in Figure 6 As shown in B, there was no significant difference in the biochemical indicators of each drug-treated group compared with the sham group, which also suggests that the bionic nanocarrier has good safety.

[0099] Example 7 Effects of other liposome components on neurotrophic factor loading

[0100] The same method as in Example 1 was used to prepare a bionic nanoparticle preparation loaded with NGF. The combination of shell phospholipids (DMPC + DOPA with a molar ratio of 1.5:1) was replaced with a combination of (DOTAP + DOPA with a molar ratio of 1.5:1). The feed ratio and the preparation process were kept the same as in Example 1. After the preparation of the bionic nanoparticle preparation loaded with NGF was completed, the preparation characterization results were as follows: Figure 7 As shown, the bionic nanoformulation loaded with NGF prepared by the combination of (DOTAP+DOPA) underwent obvious precipitation after being stored at 4 degrees for 24 hours, while the bionic nanoformulation loaded with NGF prepared by the combination of (DMPC+DOPA) remained clear after being stored at 4 degrees for one month, and its pharmaceutical properties remained stable.

[0101] Example 8 Effect of the linker sequence on the targeting of NGF-loaded biomimetic nanoformulations

[0102] As in Example 1, a bionic nanoformulation loaded with NGF was prepared, and the effects of two linker sequences, GGG and PEG12, on NGF targeting primary neurons were compared.

[0103] Primary neuron culture: After anesthesia with anhydrous ether, SD rats at 13-15 days of pregnancy were sterilized with alcohol. The abdomen was cut open with sterile surgical scissors, and one to two strings of uterus were taken out and placed in a 10 cm culture dish, and then moved into the clean bench for operation. The uterus was cut open, the fetus was taken out, the part below the head was cut off, the mouse brain was split into two halves along the midline of the brain, the cerebral cortex was taken out, and placed in DMEM (operation on ice). The vascular membrane and other accompanying brain tissues were carefully peeled off, and the cortex was cut into pieces with scissors. The cut tissue was aspirated into a plate containing 5 mL of digestion solution that had been filtered and sterilized, and digested at 37°C for 10-15 min, with slight shaking every 3 min to ensure uniform digestion. The digestion solution was transferred to a 50 mL centrifuge tube containing 10 mL of stop solution to stop digestion, 10-20 μL of DNase was added, and the cells were dispersed by pipetting 10 times. Let it stand for 2 min, and the precipitated tissue blocks were aspirated and discarded. The supernatant was centrifuged at 1500-1600 rpm for 5 min. Pour off the supernatant, keep the precipitate, add 5 mL of adhesion buffer, and mix the precipitate by blowing. Count under a microscope, dilute to an appropriate concentration with adhesion buffer, seed on a culture plate or dish (coated with poly-lysine in advance), and place in a 5% CO2 cell culture incubator at 37°C. 96-well plate seeding density: about 15,000 cells / well. After 8-10 hours of culture, replace the culture medium with neuronal culture medium. Replace 1 / 3 of the neuronal culture medium every 5-7 days. Use for experiments after 7 days of culture.

[0104] The efficiency of targeted delivery of NGF to primary neurons by bionic nanoparticles loaded with NGF was observed by laser confocal microscopy. Primary neurons were seeded in a confocal dish at a density of 10,000 per well and cultured for 24 h. The original culture medium was discarded, and 500 μL of bionic nanoparticles loaded with NGF labeled with red fluorescent DiI was added. o C for 3 h. Then, 3.7% formaldehyde was added at 37 o C fixation for 10 min, Hoechest nucleus staining for 10 min, PBS washing 3 times, and confocal imaging observation. The experimental results are shown in Figure 8 It shows that when the linker sequence of the RVG peptide is PEG12, its preparation can be efficiently taken up by primary neurons and evenly distributed in the branches of neurons; when the linker sequence of the RVG peptide is GGG, its preparation can also be taken up by primary neurons, but the overall fluorescence is low and some cells do not take up the preparation.

[0105] Example 9 Investigation of the loading efficiency of biomimetic nanocarriers for siRNA and miRNA

[0106] Similar to Example 1, bionic nanoparticle preparations loaded with siRNA and miRNA (Ribobio, Guangzhou, China) were prepared to investigate the universality of bionic nanocarrier drug delivery.

[0107] The efficiency of biomimetic nanoparticles loaded with siRNA and miRNA in delivering gene drugs to neural cells was observed by laser confocal microscopy. BV2 cells were seeded in a confocal dish at a density of 50,000 / well and cultured for 24 h. The original culture medium was discarded, and 500 μL of biomimetic nanoparticles loaded with FAM-siRNA or CY3-miRNA were added. o C for 3 h. Then, 3.7% formaldehyde was added at 37 o C fixation for 10 min, Hoechest nucleus staining for 10 min, PBS washing 3 times, and confocal imaging observation. The experimental results are shown in Fig. 9 and Fig.10 It was shown that, compared with free gene drugs and liposomes without targeting peptide modification, the bionic nanocarrier efficiently delivered siRNA and miRNA to BV2 cells, confirming the universal drug loading performance of this bionic nanocarrier.

Claims

1. A fusion protein, characterized in that The fusion protein comprises an α-helical amphipathic polypeptide and a neuron targeting peptide; the α-helical amphipathic polypeptide is ApoA-1; the neuron targeting peptide is RVG; wherein the amino acid sequence of the ApoA-1 is shown in SEQ ID NO: 3; the amino acid sequence of the RVG targeting peptide is shown in SEQ ID NO: 4; the α-helical amphipathic polypeptide and the neuron targeting peptide are connected by a connecting fragment; the connecting fragment is PEG12.

2. The fusion protein according to claim 1, characterized in that The amino acid sequence of the fusion protein is shown in SEQ ID NO:

2.

3. A recombinant high-density lipoprotein, characterized in that: The recombinant high-density lipoprotein comprises liposomes, apolipoproteins and the fusion protein according to claim 1 or 2; The lipids of the liposome are DMPC and DOPA; the apolipoprotein is selected from one or more of ApoE3, ApoA-1 and ApoJ.

4. The recombinant high-density lipoprotein according to claim 3, characterized in that The molar ratio of DMPC to DOPA is (0.5-5):

1.

5. A method for preparing recombinant high-density lipoprotein, characterized in that: The method comprises: (1) mixing liposomes and apolipoproteins and then co-incubating them to obtain lipoproteins; the lipids of the liposomes are DMPC and DOPA; the apolipoproteins are selected from one or more of ApoE3, ApoA-1 and ApoJ; (2) Co-incubating the lipoprotein with the fusion protein according to claim 1 or 2 to obtain the recombinant high-density lipoprotein.

6. The method according to claim 5, characterized in that The method further comprises: the liposome is prepared by thin film hydration method of the lipid; the co-incubation satisfies one or more of the following conditions: 100-200 rpm; 30-37° C.; 18-30 h.

7. The method according to claim 5, characterized in that The molar ratio of the liposome, the apolipoprotein and the fusion protein is (1000-2000):(1-100):1; the molar ratio of DMPC and DOPA is (0.5-5):

1.

8. A bionic nanomedicine, characterized in that: The bionic nanomedicine is a core-shell structure, and comprises drug nanogel and recombinant high-density lipoprotein from the inside to the outside; wherein the recombinant high-density lipoprotein comprises the recombinant high-density lipoprotein as claimed in claim 3 or 4.

9. The bionic nano drug according to claim 8, characterized in that: The recombinant high-density lipoprotein acts as a shell and is wrapped in the outer layer of the drug nanogel.

10. The bionic nano drug according to claim 8, characterized in that: The drug nanogel contains hyaluronic acid, protamine and drugs; the drugs are biomacromolecule drugs.

11. The bionic nano drug according to claim 10, characterized in that: The biomacromolecule drug is a protein drug and / or a nucleic acid drug.

12. The bionic nano drug according to claim 11, characterized in that: The protein drug comprises one or more selected from cytokines, protein hormones, polypeptides, antibodies and vaccines.

13. A method for preparing bionic nanomedicine, characterized in that: The method comprises: mixing and co-incubating liposomes loaded with drug nanogels and apolipoproteins, and then co-incubating with the fusion protein according to claim 1 or 2; the lipids of the liposomes are DMPC and DOPA; and the apolipoproteins are selected from one or more of ApoE3, ApoA-1 and ApoJ.

14. The method according to claim 13, wherein: The co-incubation meets one or more of the following conditions: 100-200 rpm; 30-37° C.; 18-30 h.

15. The method according to claim 13, wherein: The method further comprises: incubating the drug nanogel and the liposome at 20-25° C. for 15-40 min to obtain the liposome loaded with the drug nanogel.

16. The method according to claim 13, wherein: The method further comprises: obtaining the drug nanogel by incubating the drug, hyaluronic acid and protamine at 20-25° C. for 5-15 min; the drug is a biomacromolecule drug.

17. The method according to claim 13, characterized in that The method further comprises: preparing liposomes by thin film hydration method.

18. The method according to claim 16, characterized in that The biomacromolecule drug is a protein drug and / or a nucleic acid drug.

19. The method according to claim 18, characterized in that The protein drug comprises one or more selected from cytokines, protein hormones, polypeptides, antibodies and vaccines.

20. The method of claim 13, wherein: The molar ratio of the liposome, apolipoprotein and fusion protein is (1000-2000):(1-100):1; the molar ratio of DMPC and DOPA is (0.5-5):1; the molar ratio of the drug, hyaluronic acid and protamine is (400-800):(2-8):(50-100).

21. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises one or more of the fusion protein according to claim 1 or 2, the recombinant high-density lipoprotein according to claim 3 or 4, and the bionic nanomedicine according to any one of claims 8 to 12.

22. Use of one or more of the fusion protein according to claim 1 or 2, the recombinant high-density lipoprotein according to claim 3 or 4, and the bionic nanomedicine according to any one of claims 8 to 12 in the preparation of a drug for treating brain diseases; the brain disease is traumatic brain injury.

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