Nucleic acid brain targeting delivery carrier as well as preparation method and application thereof

Abstract

The invention provides a nucleic acid brain targeting delivery carrier as well as a preparation method and application thereof. The nucleic acid brain targeting delivery carrier is composed of a nucleic acid delivery polymer and brain targeting amphiphilic polypeptide. The brain-targeted amphiphilic polypeptide interacts with the nucleic acid delivery polymer to form a vesicle structure, and nucleic acid is carried in the vesicle. The brain-targeted amphiphilic polypeptide is formed by fusing brain-targeted peptide and hydrophobic polypeptide, and the brain-targeted peptide is distributed on the surface of the nucleic acid brain-targeted delivery carrier so as to realize nucleic acid brain-targeted delivery. The nucleic acid brain targeted delivery carrier provided by the invention is used for efficiently delivering a nucleic acid drug to a brain region in a targeted manner and realizing nucleic acid expression, so that the technical problem that the nucleic acid drug is difficult to penetrate through a blood brain barrier to realize expression in the brain is solved.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid drug delivery, and particularly relates to a nucleic acid brain-targeted delivery vector, a preparation method thereof, and an application thereof. Background Art

[0002] Brain diseases such as brain tumors, central nervous system infections, chronic pain, drug addiction, epilepsy, cyclic migraine, neurodegenerative diseases, schizophrenia, etc. have a great impact on human health. However, most active drugs cannot penetrate the blood-brain barrier (BBB), making it difficult to prevent, diagnose, and treat many brain diseases.

[0003] Methods for overcoming the BBB to increase drug delivery to the brain can be classified into two major categories: invasive and non-invasive, according to the administration route. Invasive administration methods may cause brain infections, damage to the BBB, and surgical injuries, and the administration methods are complex. Non-invasive administration methods mainly include structural modification of drugs, chemical delivery systems, carrier-mediated transport, endocytic transport, and nasal drug delivery systems, etc. Chinese Patent CN101897669A discloses a brain-targeted drug delivery system, which includes a mediating molecule, a carrier, and a drug. The mediating molecule is a fatty acid, and a polycationic macromolecule is used as the carrier. The two are covalently bonded into nanoparticles or micelles, and the drug is loaded by encapsulation or adsorption.

[0004] On the other hand, nucleic acid therapy refers to introducing exogenous normal nucleic acids into target cells to correct or compensate for diseases caused by defective and abnormal nucleic acids. The core of realizing nucleic acid therapy lies in adopting specific delivery systems and technologies to deliver nucleic acids into cells to achieve therapeutic effects. Cell transfection technology is an important technology in molecular biology and cell biology research. Specifically, it refers to a professional technology that enables exogenous molecules (such as nucleic acids) to be introduced into specific eukaryotic receptor cells, enabling eukaryotic cells to obtain new genetic markers. Cell transfection technology has a wide range of applications in life science research. It can not only be used to analyze the functions of genes and gene products in cells, but also has good application prospects in drug research and development and production, such as the production of cell lines, drug screening, nucleic acid therapy, vaccine production, etc. The methods of cell transfection are diverse and can be divided into: biological methods, chemical methods, and physical methods according to the preparation methods. In the application of biological methods, cell transfection mediated by viruses as vectors has the characteristics of high transfection efficiency and convenient application, but its application is also limited by factors such as loading capacity and safety. For the viral vector AAV used in gene therapy, most of it relies on natural serotypes of AAV, and these serotypes have highly overlapping tropisms and can widely infect cells, but lack targeting. In the application of chemical methods, cell transfection mediated by liposomes has the characteristics of easy preparation, biodegradability, low immunogenicity, and large capacity for target genes. At the same time, it also has disadvantages such as low tolerance and low transfection efficiency. In particular, lipid-based nanocarriers generally also have the defect of difficult extrahepatic targeting and it is difficult to achieve effective breakthroughs technically. Chinese invention patent CN111632039A discloses a multifunctional nanodelivery system composed of an amphiphilic polymer and a plasmid gene delivery polymer, which has a hydrophilic outer shell and a hydrophobic inner core. Plasmid gene binding sites are also distributed on the hydrophilic outer shell, and the plasmid binding sites are provided by the plasmid gene delivery polymer for binding plasmid genes, hydrophobic drugs, and contrast agents. The hydrophobic drugs and contrast agents are encapsulated in the hydrophobic inner core of the nanodelivery system to achieve the simultaneous delivery of plasmid genes, hydrophobic drugs, and contrast agents. This method can be used for the functional regulation and tracing of therapeutic cells such as stem cells.

[0005] Meanwhile, in the prior art, nerve growth factors, immunomodulatory factors, etc. have protective or reparative effects on brain injuries. For example, nerve growth factors have the function of neuroprotection after brain injury, and immunomodulatory factors have the function of repairing brain disease injuries. However, the lack of an effective method for systemic delivery of nerve growth factor nucleic acids to ischemic regions in the prior art has hindered their clinical application.

[0006] Based on the problems existing in the prior art and with further technical improvements on the basis of the prior art, the present invention combines a brain-targeting peptide and a nucleic acid delivery polymer to achieve accurate and efficient brain-targeted delivery and expression, thereby overcoming the technical problem of difficult targeting of delivery vectors in the prior art. Summary of the Invention

[0007] In view of this, the object of the present invention is to provide a nucleic acid brain-targeted delivery vector, its preparation method and application, which have the characteristics of good brain targeting and biocompatibility.

[0008] In order to achieve the above object, the present invention provides the following technical solutions.

[0009] As the first aspect of the invention, the present invention provides a nucleic acid brain-targeted delivery vector, which is composed of a nucleic acid delivery polymer and a brain-targeted amphiphilic polypeptide; the brain-targeted amphiphilic polypeptide interacts with the nucleic acid delivery polymer to form a vesicle structure, and the nucleic acid is carried inside the vesicle.

[0010] Preferably, the brain-targeted amphiphilic polypeptide is formed by fusing a brain-targeted peptide and a hydrophobic polypeptide, and the brain-targeted peptide is distributed on the surface of the nucleic acid brain-targeted delivery vector to achieve brain-targeted delivery of the nucleic acid.

[0011] Preferably, the brain-targeted peptide includes but is not limited to at least one of brain-targeting functional peptides such as Angiopep-2, transferrin, lactoferrin, LDLR ligand, apolipoprotein E short peptide, HIR ligand, nAchR targeting peptide, THR, RVG29, Peptide-22, ApoE(159~167), etc.

[0012] Preferably, the hydrophobic polypeptide includes but is not limited to polypeptides containing structures such as α-helix, β-sheet, β-hairpin, etc., and it fuses with the above-mentioned brain-targeted peptide to form an amphiphilic polypeptide.

[0013] As a preferred embodiment, the brain-targeted amphiphilic polypeptide includes but is not limited to any one of Angiopep-2 amphiphilic polypeptide, nAchR amphiphilic polypeptide, RVG amphiphilic polypeptide, etc.

[0014] Preferably, the nucleic acid delivery polymer is slightly soluble in water, has lipophilicity, and has nucleic acid binding sites.

[0015] Preferably, the nucleic acid delivery polymer is poly-β-amino ester; the molecular weight is 100~200000 Da.

[0016] Preferably, the nucleic acid includes any one or a combination of two or more of small molecule messenger nucleic acid, mRNA, interfering gene plasmid, therapeutic protein gene plasmid and reporter gene plasmid.

[0017] As another aspect of the invention, the present invention also provides a method for preparing the aforementioned nucleic acid brain-targeted delivery vector, which is characterized in that the nucleic acid brain-targeted delivery vector is prepared by mixing a nucleic acid delivery polymer and a brain-targeting amphiphilic polypeptide; specifically comprising the following steps: S1. Dissolve the nucleic acid in an aqueous sodium acetate solution to obtain solution A; S2. Dissolve the nucleic acid delivery polymer and the brain-targeting amphiphilic polypeptide in an organic solvent to obtain solution B; S3. Under an ultrasonic environment, add solution A to solution B to obtain solution C; S4. Under an ultrasonic environment, add solution C to water to obtain solution D, and purify solution D to obtain the brain-targeted nucleic acid delivery vector.

[0018] Preferably, in S1, the concentration of the aqueous sodium acetate solution is 30 mM and it does not contain RNase.

[0019] Preferably, the concentration of the nucleic acid in solution A is 400 ng / μL.

[0020] Preferably, in S2, the organic solvent includes any one or a combination of several of ethanol, dichloromethane, chloroform, tetrahydrofuran, dimethyl sulfoxide, N,N-dimethylformamide, etc., but is not limited thereto.

[0021] Preferably, the mass ratio of the nucleic acid delivery polymer to the brain-targeting amphiphilic polypeptide is 0.01:1 to 1:200.

[0022] Preferably, the concentration of the nucleic acid delivery polymer is 100 mg / mL.

[0023] Preferably, the concentration of the brain-targeting amphiphilic polypeptide is 10 mg / mL.

[0024] Preferably, the mass ratio of the nucleic acid to the delivery vector is 0.01:1 to 1:100.

[0025] Preferably, in S3, the volume ratio of solution A to solution B is 0.01:1 to 1:100.

[0026] The time taken for slowly adding solution A to solution B is 0.1 to 20 min.

[0027] Preferably, the mixing method of solution A and solution B includes any one of ultrasonic dispersion method, microfluidic method, ultrasonic emulsification method or mechanical stirring method.

[0028] Preferably, in S4, the time taken for slowly adding solution C to water is 0.1 to 20 min.

[0029] The volume ratio of the solution C to the solution D is 0.01:1 to 1:100.

[0030] Preferably, after the solution C and the solution D are mixed to be homogeneous, the purification treatment is carried out.

[0031] Preferably, the mixing method of the solution C and the solution D includes any one of ultrasonic dispersion method, microfluidic method, ultrasonic emulsification method or mechanical stirring method.

[0032] Preferably, the treatment time of ultrasonic is 1 to 60 min.

[0033] Preferably, the purification treatment is ultrafiltration purification treatment.

[0034] As a preferred embodiment, the brain-targeted amphiphilic polypeptide is obtained by fusing the brain-targeting peptide and the hydrophobic polypeptide through a linking peptide.

[0035] As a preferred embodiment, the sequence of the linking peptide is GGGGS.

[0036] Preferably, the preparation method of the brain-targeted amphiphilic polypeptide includes any one of solid-phase peptide synthesis (SPPS), liquid-phase peptide synthesis (LPPS) or enzymatic synthesis. As one of the purposes of the invention, the present invention also provides the use of the aforementioned nucleic acid brain-targeted delivery vector in delivering a drug to a brain region.

[0037] Preferably, the use includes that the drug containing the nucleic acid brain-targeted delivery vector can be targeted and delivered to the brain region by intravenous injection.

[0038] The beneficial technical effects obtained by the present invention: (1) By adopting the technical scheme of the present invention, a nucleic acid brain-targeted delivery vector is provided, which can efficiently target and deliver nucleic acid drugs to the difficult-to-transfect brain region, and overcome the technical problem of difficult penetration of the blood-brain barrier during the delivery of nucleic acid drugs.

[0039] (2) The components of the nucleic acid brain-targeted delivery vector of the present invention are all biodegradable and do not affect cell functions. The results of cell-level proliferation toxicity detection show that the cell survival rate exceeds 90%, and it has excellent biocompatibility. Moreover, poly-β-amino ester has acid-responsive characteristics, which is beneficial to the lysosomal escape of the nucleic acid brain-targeted delivery vector and nucleic acid after being phagocytosed by cells, and promotes the release of nucleic acid into the cytoplasm, thereby realizing efficient cell transfection.

[0040] (3) The raw materials of the nucleic acid brain-targeted delivery vector provided by the technical solution of the present invention are simple and easy to obtain, very safe, the operation process is convenient and controllable, suitable for industrial production, and the drug containing the targeted delivery vector can be targeted to the brain region by intravenous injection, and the operation is convenient and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 It is a schematic diagram of the simulated structure of the transfection reagent prepared by the present invention.

[0042] Figure 2 It is a transmission electron micrograph of the brain-targeted nucleic acid delivery vector prepared in Example 1 of the present invention.

[0043] Figure 3 It is the result of cell viability detection after the brain-targeted nucleic acid delivery vector in Example 1 of the present invention was incubated with brain microvascular endothelial cells for 12 hours.

[0044] Figure 4 It is a result graph of nucleic acid expression detected by bioluminescence after the brain-targeted nucleic acid delivery vector in Example 2 of the present invention encapsulated the red firefly luciferase plasmid nucleic acid and was injected into the blood of mice through the tail vein for 24 h. DETAILED DESCRIPTION OF THE INVENTION

[0045] In view of the defects of the prior art, the inventors of the present case have proposed the technical solution of the present invention through long-term research and a large number of practices. It is mainly composed of a nucleic acid delivery polymer and a brain-targeted amphiphilic polypeptide. The brain-targeted amphiphilic polypeptide is mainly formed by fusing a brain-targeted peptide and a hydrophobic polypeptide. It interacts with the nucleic acid delivery polymer to form a vesicle structure. The nucleic acid is carried in the vesicle, and the brain-targeted peptide is distributed on the surface of the delivery vector to achieve brain-targeted delivery of nucleic acid. The delivery vector of the present invention has excellent brain-targeting function, can effectively deliver therapeutic nucleic acid to the brain, and has good biocompatibility.

[0046] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative work belong to the scope of protection of the present invention.

[0047] One aspect of the embodiments of the present invention provides a nucleic acid brain-targeted delivery vector for targeting and delivering nucleic acid drugs into the brain, which includes: mainly composed of a nucleic acid delivery polymer and a brain-targeted amphiphilic polypeptide; the brain-targeted amphiphilic polypeptide is mainly formed by fusing a brain-targeted peptide and a hydrophobic polypeptide. Refer to Figure 1, which is a schematic structural simulation diagram of the nucleic acid brain-targeted delivery vector provided by the present invention. As can be seen from the figure, the nucleic acid brain-targeted delivery vector mainly consists of a nucleic acid delivery polymer, a brain-targeting amphiphilic polypeptide, and a nucleic acid. The nucleic acid delivery polymer and the brain-targeting amphiphilic polypeptide interact to form a vesicle structure, and the nucleic acid is carried inside the vesicle.

[0048] In some more specific embodiments, the nucleic acid delivery polymer is a poly-β-amino ester with a molecular weight of 100 - 200,000 Da.

[0049] In some more specific embodiments, the brain-targeting peptide includes at least one of short peptides with brain-targeting functions such as Angiopep-2, transferrin, lactoferrin, LDLR ligand, apolipoprotein E short peptide, HIR ligand, nAchR-targeting peptide, THR, RVG29, Peptide-22, ApoE(159 - 167), etc.

[0050] In some more specific embodiments, the hydrophobic polypeptide includes, but is not limited to, polypeptides containing structures such as α-helix, β-sheet, β-hairpin, etc., which are fused with the above-mentioned brain-targeting peptide to form an amphiphilic polypeptide.

[0051] In some more specific embodiments, the brain-targeting amphiphilic polypeptide is obtained by fusing the brain-targeting peptide and the hydrophobic polypeptide through a linker peptide.

[0052] In some more specific embodiments, the sequence of the linker peptide is GGGGS.

[0053] In some more specific embodiments, the brain-targeting amphiphilic polypeptide includes, but is not limited to, any one of Angiopep-2 amphiphilic polypeptide, nAchR amphiphilic polypeptide, RVG amphiphilic polypeptide, etc.

[0054] In some more specific embodiments, the nucleic acid includes: small molecule messenger nucleic acid, mRNA, interfering gene plasmid, therapeutic protein nucleic acid plasmid, and reporter gene plasmid, dsRNA, antisense nucleic acid, antisense oligonucleotide, microRNA, antisense microRNA, microRNA inhibitor, microRNA activator, and immunostimulatory nucleic acid.

[0055] Furthermore, the delivery vector is prepared by ultrasonic emulsification method, mechanical stirring method, ultrasonic dispersion method or microfluidic method from the nucleic acid delivery polymer and the brain-targeting amphiphilic polypeptide.

[0056] The delivery vector of the present invention is a vesicle structure formed by the interaction between the brain-targeting amphiphilic polypeptide and the nucleic acid delivery polymer. The nucleic acid is carried inside the vesicle, and the brain-targeting peptide is distributed on the surface of the delivery vector. The preparation method includes the following steps: Step 1: Dissolve the nucleic acid in an aqueous sodium acetate solution free of RNase to obtain Solution A; Step 2: Dissolve the poly-β-amino ester and the brain-targeting amphiphilic polypeptide in an organic solvent to obtain Solution B; Step 3: Under an ultrasonic environment, slowly add Solution A to Solution B to obtain Solution C; Step 4: Under an ultrasonic environment, slowly add Solution C to water to obtain Solution D, and purify Solution D to obtain the brain-targeting nucleic acid delivery carrier.

[0057] In some more specific embodiments, the organic solvent is a volatile organic solvent, including any one or a combination of two or more of ethanol, tetrahydrofuran, dimethyl sulfoxide, dichloromethane, chloroform, N,N-dimethylformamide, and is not limited thereto.

[0058] In some more specific embodiments, the preparation method includes: adding Solution B to Solution A for mixing and homogenizing, and ultrasonic treatment.

[0059] In some more specific embodiments, the preparation method includes: adding water to Solution C under stirring conditions.

[0060] In some more specific embodiments, the mass ratio of the nucleic acid to the delivery carrier is 0.001:1 to 1:100.

[0061] In some more specific embodiments, the volume ratio of Solution A to Solution B is 0.01:1 to 1:100.

[0062] In some more specific embodiments, the mixing method of Solution A and Solution B includes any one of ultrasonic dispersion method, microfluidic method, ultrasonic emulsification method or mechanical stirring method.

[0063] In some more specific embodiments, the volume ratio of Solution C to Solution D is 0.01:1 to 1:100.

[0064] In some more specific embodiments, after Solution C and Solution D are mixed to be homogeneous, the purification treatment is carried out.

[0065] In some more specific embodiments, the mixing method of Solution C and Solution D includes any one of ultrasonic dispersion method, microfluidic method, ultrasonic emulsification method or mechanical stirring method.

[0066] Furthermore, the time of the ultrasonic treatment is 1 - 60 min; preferably 2 - 10 min; more preferably 3 min.

[0067] Further, the purification treatment is ultrafiltration purification treatment.

[0068] In some embodiments, the mass ratio of the nucleic acid to the delivery vector may be 1:(1 - 200).

[0069] The technical solutions of the present invention will be further described in detail below in conjunction with several preferred embodiments and the accompanying drawings. These embodiments are implemented on the premise of the technical solutions of the present invention, and detailed implementation manners and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0070] In the following examples, the experimental materials used can be purchased from conventional biochemical reagent companies without special instructions.

[0071] Example 1 Based on the neuroprotective functions of nerve growth factors such as NGF (GenBank: AAH32517.2), NT3 (GeneID: 4908), NTFs (GeneID: 441549), BDNF (GenBank: CAA62632.1), Nrf2 (GenBank: AAB32188.1), and GDNF (Gene ID: 2668) after brain injury, through nucleic acid delivery technology, a brain-targeted NGF-mRNA delivery vector was prepared and used to target-transfect NGF-mRNA into the brains of mice, enabling the expression of neuroprotective factors in the brain to achieve neuroprotective functions, overcoming the technical obstacle in the prior art of lacking the technology to systemically deliver nerve growth factor nucleic acids to the ischemic area. Through the above strategy, it is expected to realize the clinical application of nerve growth factor nucleic acids in the treatment of brain diseases.

[0072] Specifically, the preparation method of the brain-targeted NGF-mRNA delivery vector includes the following steps: (1) Dissolve 450 μg of NGF-mRNA (purchased from Ascentage) in 10 mL of RNase-free sodium acetate aqueous solution to obtain solution A; (2) Dissolve 30 mg of poly-β-amino ester and 200 mg of Angiopep-2 amphiphilic polypeptide (amino acid sequence: TFFYGGSRGKRNNFKTEEY) in tetrahydrofuran to obtain solution B; (3) Under an ultrasonic environment, slowly add solution A to solution B to obtain solution C; (4) Under an ultrasonic environment, slowly add solution C to water to obtain solution D, and purify solution D to obtain the brain-targeted NGF delivery vector. Through intravenous injection, the brain-targeted NGF delivery vector is injected into the blood and further enters the brain to exert neuroprotective functions.

[0073] See Figure 2 , which is the transmission electron micrograph of the brain-targeted NGF-mRNA delivery vector prepared in this example; as can be seen from the figure, the diameter of the vesicles is about 100 nm.

[0074] See Figure 3 , which is the cell viability test result after incubating the brain-targeted nucleic acid delivery vector prepared in this example with brain microvascular endothelial cells for 12 hours (the blank control is PBS buffer). As can be seen from the figure, the delivery process has little effect on cell viability, indicating that the brain-targeted nucleic acid delivery vector has good biocompatibility.

[0075] Example 2 In this example, a brain-targeted red firefly luciferase plasmid (RfLuc plasmid, GenBank: AAA89082) delivery vector was prepared by the following method and used to evaluate the brain-targeted delivery efficiency of the delivery vector, including the following steps: (1) Dissolve 300 μg of RfLuc plasmid in 10 mL of RNase-free sodium acetate aqueous solution to obtain solution A; (2) Dissolve 30 mg of poly-β-amino ester and 200 mg of nAchR amphiphilic polypeptide (amino acid sequence: AGSRILMWPGR) in tetrahydrofuran to obtain solution B; (3) Under ultrasonic environment, slowly add solution A to solution B to obtain solution C; (4) Under ultrasonic environment, slowly add solution C to water to obtain solution D, and purify solution D to obtain the brain-targeted RfLuc delivery vector. Through tail vein injection, the brain-targeted RfLuc delivery vector was injected into the blood of mice and further entered the mouse brain to express RfLuc, which was detected by a bioluminescence instrument.

[0076] See Figure 4 , which is the result diagram of nucleic acid expression detected by bioluminescence after the brain-targeted nucleic acid delivery vector of this example encapsulated the red firefly luciferase plasmid and was injected into the blood of mice through the tail vein for 24 h. As can be seen from the figure, the red firefly luciferase plasmid is expressed in the mouse brain, indicating that the brain-targeted nucleic acid delivery vector successfully delivered the red firefly luciferase plasmid to the mouse brain.

[0077] Example 3 The immune response plays a key role in the pathological process of brain disease injury repair. It is an effective strategy to enhance its immune regulatory function by using nucleic acid delivery technology to express immune regulatory factors (such as IL-6 (GeneID: 3569), IL-17 (GenBank: AAC50341.1) and IL-2 (GenBank: AAB46883.1), etc.) in the brain.

[0078] In this embodiment, a brain-targeted IL-6-mRNA delivery vector is prepared by the following method, which is expected to be used for immunomodulatory treatment of brain injury repair, including the following steps: (1) Dissolve 600 μg of IL-6-mRNA (purchased from Ascent) in 10 mL of RNase-free sodium acetate aqueous solution to obtain solution A; (2) Dissolve 100 mg of poly-β-amino ester and 200 mg of RVG amphiphilic polypeptide (amino acid sequence: YTIWMPENPRPGTPCDIFTNSRGKRASNGLLLLLL) in tetrahydrofuran to obtain solution B; (3) Under ultrasonic environment, slowly add solution A to solution B to obtain solution C; (4) Under ultrasonic environment, slowly add solution C to water to obtain solution D, and purify solution D to obtain the brain-targeted IL-6-mRNA delivery vector. By intravenous injection, the brain-targeted IL-6-mRNA delivery vector is injected into the blood, which can target and deliver nucleic acids into the brain region for enhancing the brain immunomodulatory function.

[0079] In addition, the inventors of this case also referred to the foregoing embodiments, and conducted experiments with other raw materials, process operations, and process conditions described in this specification, and all obtained relatively ideal results.

[0080] All aspects, embodiments, features, and examples of the present invention should be considered illustrative in all respects and are not intended to limit the present invention. The scope of the present invention is only defined by the claims. Without departing from the spirit and scope of the claimed invention, those skilled in the art will understand other embodiments, modifications, and uses.

[0081] The use of the title of the present invention does not mean limiting the present invention; the content of the invention and the specific implementation part can be applied to any aspect, embodiment, or feature of the present invention.

[0082] Throughout the present invention, where a composition is described as having, comprising, or including a specific component, or where a process is described as having, comprising, or including a specific process step, it is contemplated that the compositions taught by the present invention also consist essentially of or consist of the recited components, and the processes taught by the present invention also consist essentially of or consist of the recited process steps or groups of process steps.

[0083] Unless otherwise specifically stated, the use of the terms "include (include, includes, including)", "have (have, has, or having)" should generally be understood as open-ended and non-restrictive.

[0084] It should be understood that the order of each step or the order of performing a specific action is not of great importance as long as the teachings of the present invention remain operable. In addition, two or more steps or actions can be carried out simultaneously.

[0085] The above are only the preferred embodiments of the present invention, which do not thereby limit the protection scope of the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any changes, modifications, substitutions, integrations, and parameter changes made to these embodiments within the spirit and principle of the present invention by means of conventional substitutions or capable of achieving the same functions without departing from the principle and spirit of the present invention fall within the protection scope of the present invention.

Claims

1. A nucleic acid brain-targeted delivery vector, which consists of a nucleic acid delivery polymer and a brain-targeting amphiphilic polypeptide; the brain-targeting amphiphilic polypeptide interacts with the nucleic acid delivery polymer to form a vesicle structure, and the nucleic acid is carried inside the vesicle.

2. The nucleic acid brain-targeted delivery vector according to claim 1, wherein The brain-targeting amphiphilic polypeptide is formed by fusing a brain-targeting peptide and a hydrophobic polypeptide, and the brain-targeting peptide is distributed on the surface of the nucleic acid brain-targeted delivery vector to achieve the brain-targeted delivery of nucleic acid and nucleic acid expression in the brain region.

3. The nucleic acid brain-targeted delivery vector according to claim 2, wherein The brain-targeting peptide includes at least one of brain-targeting functional peptides such as Angiopep-2, transferrin, lactoferrin, LDLR ligand, apolipoprotein E short peptide, HIR ligand, nAchR targeting peptide, THR, RVG29, Peptide-22, ApoE(159~167), etc.; And / or, the hydrophobic polypeptide includes polypeptides containing α-helix structure, β-sheet structure, and β-hairpin structure.

4. The nucleic acid brain-targeted delivery vector according to claim 2, wherein the brain-targeting amphiphilic polypeptide includes any one of Angiopep-2 amphiphilic polypeptide, nAchR amphiphilic polypeptide, and RVG amphiphilic polypeptide.

5. The nucleic acid brain-targeted delivery vector according to any one of claims 1-4, characterized in that, The nucleic acid delivery polymer is slightly soluble in water, has lipophilicity, and has nucleic acid binding sites; The nucleic acid delivery polymer is poly-β-amino ester; the molecular weight is 100~200000 Da.

6. The nucleic acid brain-targeted delivery vector according to any one of claims 1-4, characterized in that, The nucleic acid includes any one or a combination of two or more of small molecule messenger nucleic acid, mRNA, interfering gene plasmid, therapeutic protein gene plasmid, and reporter gene plasmid.

7. A method for preparing a nucleic acid brain-targeted delivery vector according to any one of claims 1-6, characterized in that, The nucleic acid brain-targeted delivery vector is prepared by mixing a nucleic acid delivery polymer and a brain-targeting amphiphilic polypeptide; specifically includes the following steps: S1. Dissolve the nucleic acid in an aqueous sodium acetate solution to obtain solution A; S2. Dissolve the nucleic acid delivery polymer and the brain-targeting amphiphilic polypeptide in an organic solvent to obtain solution B; S3. Under an ultrasonic environment, add solution A to solution B to obtain solution C; S4. Under an ultrasonic environment, add solution C to water to obtain solution D, and purify solution D to obtain the brain-targeted nucleic acid delivery vector.

8. The preparation method according to claim 7, wherein in S1, the concentration of the aqueous sodium acetate solution is 30 mM and it does not contain RNase; the concentration of the nucleic acid in solution A is 400 ng / μL; in S2, the organic solvent includes any one of ethanol, dichloromethane, chloroform, tetrahydrofuran, dimethyl sulfoxide, and N,N-dimethylformamide; and / or, the mass ratio of the nucleic acid delivery polymer to the brain-targeting amphiphilic polypeptide is 0.01:1~1:200; the concentration of the nucleic acid delivery polymer is 100 mg / mL; the concentration of the brain-targeting amphiphilic polypeptide is 10 mg / mL; and / or, the mass ratio of the nucleic acid to the delivery vector is 0.01:1~1:100; in S3, the volume ratio of solution A to solution B is 0.01:1~1:100; the time taken for slowly adding solution A to solution B is 0.1~20 min; The mixing method of the solution A and the solution B includes any one of ultrasonic dispersion method, microfluidic method, ultrasonic emulsification method or mechanical stirring method; In S4, the time taken for slowly adding the solution C into water is 0.1 - 20 min; The volume ratio of the solution C to the solution D is 0.01:1 - 1:100; After the solution C and the solution D are mixed to be homogeneous, the purification treatment is carried out; The mixing method of the solution C and the solution D includes any one of ultrasonic dispersion method, microfluidic method, ultrasonic emulsification method or mechanical stirring method; The treatment time of ultrasound is 1 - 60 min; The purification treatment is ultrafiltration purification or dialysis purification treatment.

9. The preparation method according to claim 7 or 8, characterized in that, The preparation method of the brain-targeting amphiphilic polypeptide includes any one of solid-phase synthesis method (SPPS), liquid-phase synthesis method (LPPS) or enzymatic synthesis method.

10. Use of a nucleic acid brain-targeting delivery vector as described in any one of claims 1 - 6 in the preparation of a drug for targetedly delivering nucleic acid into the brain region.

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