CN107937443B - Self-assembly nano preparation suitable for nucleic acid transfection and preparation and application thereof - Google Patents

Self-assembly nano preparation suitable for nucleic acid transfection and preparation and application thereof Download PDF

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CN107937443B
CN107937443B CN201711110814.0A CN201711110814A CN107937443B CN 107937443 B CN107937443 B CN 107937443B CN 201711110814 A CN201711110814 A CN 201711110814A CN 107937443 B CN107937443 B CN 107937443B
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polyethyleneimine
self
adamantane
beta
nucleic acid
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CN107937443A (en
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于倩茹
彭金良
徐宇虹
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Shanghai Jiaotong University
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
    • C08B37/0012—Cyclodextrin [CD], e.g. cycle with 6 units (alpha), with 7 units (beta) and with 8 units (gamma), large-ring cyclodextrin or cycloamylose with 9 units or more; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/333—Polymers modified by chemical after-treatment with organic compounds containing nitrogen
    • C08G65/33303—Polymers modified by chemical after-treatment with organic compounds containing nitrogen containing amino group
    • C08G65/3331—Polymers modified by chemical after-treatment with organic compounds containing nitrogen containing amino group cyclic
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    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/34—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from hydroxy compounds or their metallic derivatives
    • C08G65/46—Post-polymerisation treatment, e.g. recovery, purification, drying
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
    • C08G73/02—Polyamines
    • C08G73/028—Polyamidoamines
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G81/00—Macromolecular compounds obtained by interreacting polymers in the absence of monomers, e.g. block polymers
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G2650/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G2650/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule characterized by the type of post-polymerisation functionalisation
    • C08G2650/04—End-capping

Abstract

The invention belongs to the technical field of nucleic acid drug carriers, and particularly relates to a self-assembled nano preparation suitable for nucleic acid transfection, and preparation and application thereof. The self-assembled nano preparation comprises the following components: beta-cyclodextrin modified polyethyleneimine, adamantane terminated polyethylene glycol and adamantane modified polyamidoamine. The invention discovers for the first time that when polyethyleneimine in beta-cyclodextrin modified polyethyleneimine adopts polyethyleneimine with low molecular weight, the formed self-assembled nano preparation has a very good transfection effect on nucleic acid molecules.

Description

Self-assembly nano preparation suitable for nucleic acid transfection and preparation and application thereof
Technical Field
The invention belongs to the technical field of nucleic acid drug carriers, and particularly relates to a self-assembled nano preparation suitable for nucleic acid transfection, and preparation and application thereof.
Background
Malignant tumor is a leading cause of death, and is threatening the health of human body, and has become a common disease and a frequently encountered disease. The mortality rate of malignant tumor exceeds cardiovascular disease, and is also called as three major challenges of modern medicine together with viral disease and senile disease. For malignant tumors, the method still cannot overcome the defect, and has no complete treatment means, and only can be used for conventional methods such as surgical excision, chemotherapy, radiotherapy, immunotherapy and the like. However, with the continuous and deep understanding of human tumor diseases, breakthroughs have been made in the field of immunotherapy in recent years.
Tumor immunotherapy has attracted considerable attention recently and is a focus and hot spot in the field of tumor therapy. The immunotherapy applies immunological principle and method to improve the immunogenicity of tumor cells and the sensitivity to killing of effector cells, stimulate and enhance the anti-tumor immune response of organisms, and applies immune cells and effector molecules to be infused into a host body to cooperate with the immune system of the organisms to kill tumors and inhibit the growth of the tumors. The tumor immunotherapy is expected to become an innovation in the field of tumor therapy after surgery, chemotherapy, radiotherapy and targeted therapy due to the excellent curative effect and the tumor immunotherapy. In recent years, in the field of immunotherapy, the elimination of cancer cells by genetically modifying lymphocytes including T cells as "live drugs" is a new direction of cancer therapy. For example, the specific process of T cell therapy is mainly divided into nine steps: (1) leukocytes are isolated from the blood of the patient. (2) Lymphocytes are enriched by countercurrent centrifugal elutriation or other methods. (3) The enriched lymphocytes are stimulated with a specific antigen in an in vitro culture environment. (4) At the same time, a gene fragment encoding a CAR or TCR is introduced via the vector. (5) These lymphocytes are split and proliferated in a bioreactor. (6) When lymphocytes reach sufficient numbers, T cells that do not express the predetermined CAR or TCR are washed away. (7) The concentrated cell sample is subjected to a quality check. (8) Then frozen to form the final product for delivery to clinics. (9) The physician at the clinic will thaw the product and return it to the patient. One of the most critical steps in the overall therapeutic process is the introduction of the gene fragment by the vector.
At present, the common lymphocyte transfection methods include virus mediated methods, common electroporation techniques, non-viral vector transfection methods and the like. The virus-mediated method is time-consuming and labor-consuming, has the defects of biological potential safety hazard, high cost and the like, and the defects of unpredictable immune response and the like caused by virus infection cannot be ignored. The general electroporation technology generally has the problem of low transfection efficiency on primary cells related to immunity, and downstream experiments cannot be carried out. Therefore, non-viral vectors with high safety, low side effects and low production cost are increasingly favored, but because T cells are suspension cells and because of some biological properties of T cells, no effective non-viral vectors are currently available for efficient delivery of gene fragments into T cells.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention aims to provide a self-assembled nano preparation suitable for nucleic acid transfection and preparation and application thereof.
In order to achieve the above objects and other related objects, the present invention adopts the following technical solutions:
in a first aspect of the invention, there is provided a self-assembled nano-formulation comprising the components: beta-cyclodextrin modified polyethyleneimine, adamantane terminated polyethylene glycol and adamantane modified polyamidoamine.
Preferably, the beta-cyclodextrin modified polyethyleneimine may be a polyethyleneimine grafted with beta-cyclodextrin.
Preferably, the polyethyleneimine prior to grafting is a low molecular weight polyethyleneimine.
Further preferably, the molecular weight of polyethyleneimine before grafting may be in the range of 400 to 2000. More preferably, the molecular weight of polyethyleneimine before grafting may be in the range of 600 to 1800. Still more preferably, the polyethyleneimine before grafting may have a molecular weight in the range of 800.
Preferably, in the beta-cyclodextrin grafted polyethyleneimine, one or more beta-cyclodextrin molecules are grafted on each polyethyleneimine long chain. Furthermore, 4-7 beta-cyclodextrin molecules are grafted on each polyethyleneimine long chain.
Preferably, in the adamantane-terminated polyethylene glycol, adamantane may be attached to the end of the polyethylene glycol through an amide bond.
Preferably, the molecular weight of the polyethylene glycol before the end capping may be 2000 to 5000.
Preferably, the adamantane-modified polyamidoamine may be an adamantane-linked polyamidoamine.
Preferably, the adamantane can be attached to the amine group of the polyamidoamine through an isocyanate group.
Preferably, one or more adamantanes may be attached to each polyamidoamine.
Preferably, the polyamidoamine can be a 1-generation polyamidoamine.
Preferably, in the self-assembled nano preparation, the molar ratio of the beta-cyclodextrin modified polyethyleneimine, the adamantane modified polyamide-amine and the adamantane terminated polyethylene glycol is (12-72): (10-20): (12-48).
In a second aspect of the present invention, there is provided a method for preparing the self-assembled nano-formulation, comprising: the components are taken according to the proportion, mixed and self-assembled to obtain the self-assembled nano preparation.
Preferably, the adamantane-terminated polyethylene glycol is obtainable by a process comprising the steps of: adamantane was attached to the end of polyethylene glycol by an amide reaction.
Preferably, the amino group on adamantane can be amide-reacted with the carboxyl group on polyethylene glycol to obtain adamantane-terminated polyethylene glycol.
Preferably, the adamantane-modified polyamidoamine is obtainable by a process comprising the steps of: 1-adamantane isocyanate is reacted with a polyamidoamine, to which adamantane can be attached via an isocyanate group.
Preferably, the beta-cyclodextrin modified polyethyleneimine is obtainable by a process comprising the steps of: reacting the beta-cyclodextrin with an activating agent to obtain activated beta-cyclodextrin, and grafting the activated beta-cyclodextrin onto PEI to obtain beta-cyclodextrin modified polyethyleneimine.
Preferably, the activator may be N, N' -carbonyldiimidazole or p-toluenesulfonyl chloride.
Preferably, the beta-cyclodextrin modified low molecular weight polyethyleneimine, when prepared, can be activated with N, N' -carbonyldiimidazole.
A third aspect of the present invention provides a self-assembled nano-formulation obtained by the aforementioned production method.
In a fourth aspect, the invention provides the use of the aforementioned self-assembled nano-formulation in the preparation of a nucleic acid transfection or delivery vector.
The nucleic acid includes, but is not limited to, various nucleic acid drugs such as antisense nucleic acid, ribozyme, aptamer, interfering RNA, sgRNA and the like, expression vectors such as plasmid DNA or mRNA thereof, and plasmid DNA or mRNA expressing various bioactive molecules.
The transfection or delivery may be to cells in vitro or to cells in vivo.
The transfected subject is a lymphocyte, preferably a T lymphocyte.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, through screening and optimizing the components of the self-assembly nanometer preparation based on the cationic component, the Polyethyleneimine (PEI) modified by cyclodextrin with different molecular weights and the polyamidoamine dendritic Polymer (PAMAM) modified by adamantane with different generations are mixed according to a certain proportion and are automatically assembled into the nanometer particle transport nucleic acid segment, so that the nucleic acid drug can be effectively transported, the toxic and side effects of the carrier can be reduced, and the high-level transfection effect on lymphocyte can be realized.
Drawings
FIG. 1A: beta-Cyclodextrin modified polyethyleneimine (CD-PEI) from example 1800) The synthesis reaction formula (2).
FIG. 1B: beta-Cyclodextrin modified polyethyleneimine (CD-PEI) from example 110000) The synthesis reaction formula (2).
FIG. 2A: beta-cyclodextrin modified Low molecular weight polyethyleneimine (CD-PEI) from example 1800) Hydrogen spectrum of Nuclear Magnetic Resonance (NMR).
FIG. 2B: NMR spectrum of 6-O- (p-toluenesulfonate) - β -cyclodextrin (6-OTs- β -CD) in example 1.
FIG. 2C: beta-Cyclodextrin modified polyethyleneimine (CD-PEI) from example 110000) Hydrogen spectrum of Nuclear Magnetic Resonance (NMR).
FIG. 3: the reaction scheme for the synthesis of adamantane-terminated polyethylene glycol (Ad-PEG) in example 2.
FIG. 4: nuclear magnetic resonance hydrogen spectra of adamantane-polyethylene glycol in example 2.
FIG. 5: the reaction scheme for the synthesis of adamantane-modified polyamidoamine (Ad-PAMAM) in example 3.
FIG. 6: particle size distribution of self-assembled nucleic acid nanopreparations in example 4.
FIG. 7: biological transmission electron microscopy characterization of each assembled nucleic acid nanoformulation in example 4.
FIG. 8A: CD-PEI800 containing self-assembled nucleic acid nanoformulation DNA @ SNP800 in water stability change characterization.
FIG. 8B: and (3) characterizing the stability change of the self-assembly nucleic acid nano preparation DNA @ SNP10000 containing CD-PEI10000 in water.
FIG. 9: the gel electrophoresis result of the self-assembled nucleic acid nano preparation is shown, wherein A represents marker, B represents naked PGL3 plasmid DNA, C represents PGL3DNA @ liposome, D represents PGL3DNA @ SNP800, and E represents PGL3DNA @ SNP 10000.
FIG. 10A: results of transfection of luciferase expression plasmid (PGL3) mediated by self-assembled nucleic acid nanoformulation in example 7.
FIG. 10B: luciferase expression plasmid (PGL3) transfection results mediated by different low molecular weight polyethyleneimine self-assembled nucleic acid nanoformulations in example 7.
FIG. 10C: luciferase plasmid DNA (PGL3) cell transfection results mediated by self-assembled nucleic acid nano-preparations with different nitrogen-phosphorus ratios.
FIG. 10D: the composition of the adamantane-modified polyamidoamine in example 7 was changed, and the resulting self-assembled nucleic acid nanoformulation mediated transfection of luciferase plasmid DNA (PGL3) cells was performed.
FIG. 10E: the composition of the adamantane-terminated polyethylene glycol was changed in example 7, and the resulting self-assembled nucleic acid nanoformulation mediated transfection of luciferase plasmid DNA (PGL3) cells was performed.
FIG. 11: example 7 transfection results of self-assembled nucleic acid Nanodiagnets-mediated Green fluorescent protein expression plasmid (pGFP), wherein a-c represent CD-PEI-based10000The self-assembled nucleic acid nano-preparation group has GFP positive cell number and fluorescence intensity, and d to f represent the fluorescence intensity based on CD-PEI800The number of GFP positive cells and the fluorescence intensity of the self-assembled nucleic acid nano-preparation group of (a).
Detailed Description
Self-assembling nano preparation
The self-assembled nano preparation comprises the following components: beta-cyclodextrin modified polyethyleneimine, adamantane terminated polyethylene glycol and adamantane modified polyamidoamine.
The beta-cyclodextrin modified polyethyleneimine may be a polyethyleneimine grafted with beta-cyclodextrin.
Grafting of beta-cyclodextrin onto polyethyleneimine to obtain polyethyleneimine grafted with beta-cyclodextrin is well known to those skilled in the art.
The polyethyleneimine before grafting is low molecular weight polyethyleneimine. Further, the molecular weight of the low molecular weight polyethyleneimine may be in the range of 400 to 2000. Further, the molecular weight of the low molecular weight polyethyleneimine may be in a range of 600 to 1800. In some embodiments, the molecular weight of the low molecular weight polyethyleneimine may be in the range of 600 to 800, or 800 to 1800.
In one embodiment, the molecular weight of polyethyleneimine before grafting is 600, 800, 1800, but the molecular weight is not limited to 600, 800, 1800.
In the polyethyleneimine grafted with the beta-cyclodextrin, each polyethyleneimine long chain is grafted with one or more than one beta-cyclodextrin molecules. Furthermore, each polyethyleneimine long chain can be grafted with 4-7 beta-cyclodextrin molecules. In one embodiment, a long polyethyleneimine chain to which 5 β -cyclodextrin molecules are grafted is exemplified, but the grafting ratio is not limited thereto.
The structural schematic diagram of the beta-cyclodextrin modified polyethyleneimine can be seen in fig. 1A.
Capping of polyethylene glycol with adamantane is well known to those skilled in the art. For example, in the adamantane-terminated polyethylene glycol, adamantane may be attached to the end of the polyethylene glycol through an amide bond.
The molecular weight of the polyethylene glycol before blocking can be 2000-5000. In one embodiment, the polyethylene glycol is exemplified to have a molecular weight of 5000, but is not limited thereto.
A schematic of the structure of the adamantane-terminated polyethylene glycol can be seen in fig. 3.
The adamantane-modified polyamidoamine can be a polyamidoamine with adamantane attached.
The attachment of adamantanes to polyamidoamines is well known to those skilled in the art. For example, adamantane can be attached to the amine group of the polyamide-amine through an isocyanate group.
One or more adamantanes may be attached to each polyamidoamine. For example, 1 to 8 adamantanes may be attached to each polyamidoamine. Further, 4 to 8 adamantanes may be attached to each polyamidoamine. In one embodiment of the present invention, polyamidoamine having 8 adamantane molecules bonded thereto is exemplified, but the present invention is not limited thereto.
The polyamidoamine can be a 1-generation polyamidoamine. The polyamidoamine can have a molecular weight of 1457.90. The examples of the invention illustrate that the polyamidoamine can be the first generation of polyamidoamine dendrimers with a 1, 4-diaminobutane core and amine ends. The polyamidoamines are commercially available from Dendritic Nanotechnologies, Inc (Mount plexant, MI).
In the self-assembly nano preparation, the molar ratio range of the beta-cyclodextrin modified polyethyleneimine, the adamantane modified polyamide-amine and the adamantane terminated polyethylene glycol is (12-72): (10-20): (12-48).
In the self-assembly nano preparation, the molar ratio range of the beta-cyclodextrin modified polyethyleneimine, the adamantane modified polyamide-amine and the adamantane terminated polyethylene glycol is (24-60): (10-20): (12-48).
In some embodiments, the molar ratio between the beta-cyclodextrin modified polyethyleneimine, the adamantane modified polyamidoamine, and the adamantane terminated polyethylene glycol may be in the range of (36-60): (10-20): (12-48).
In some embodiments, the molar ratio between the beta-cyclodextrin modified polyethyleneimine, the adamantane modified polyamidoamine, and the adamantane terminated polyethylene glycol can range from (36-48): (10-20): (12-48).
In some embodiments, the molar ratio between the beta-cyclodextrin modified polyethyleneimine, the adamantane modified polyamidoamine, and the adamantane terminated polyethylene glycol may be in the range of (48 to 60): (10-20): (12-48).
In some embodiments, the molar ratio between the beta-cyclodextrin modified polyethyleneimine, the adamantane modified polyamidoamine, and the adamantane terminated polyethylene glycol may be in the range of (36-60): 10: 12.
in summary, the self-assembled nano-preparation is a self-assembled nano-preparation system, and the self-assembled nano-preparation system comprises three parts of molecular modules a, B, C, a having specific structures and molecular sizes, wherein a is adamantane-terminated polyethylene glycol for self-assembly polymerization termination of the nano-preparation and improving biocompatibility and circulation time of the self-assembled nucleic acid preparation, B is adamantane-modified polyamide-amine for binding nucleic acid molecules and controlling particle sizes of the self-assembled nucleic acid preparation, and C is beta-cyclodextrin-modified low-molecular-weight polyethyleneimine for binding nucleic acid molecules and promoting endosome release of the nucleic acid molecules.
Preparation method of self-assembled nano preparation
The preparation method of the self-assembled nano-scale comprises the following steps: the components are taken according to the proportion, mixed and self-assembled to obtain the self-assembled nano preparation.
Wherein the adamantane-terminated polyethylene glycol is obtainable by a process comprising the steps of: adamantane was attached to the end of polyethylene glycol by an amide reaction.
That is, an amino group on adamantane and a carboxyl group on polyethylene glycol may be subjected to an amide reaction to obtain adamantane-terminated polyethylene glycol.
The adamantane-modified polyamidoamine can be obtained by a process comprising the steps of: 1-adamantane isocyanate is reacted with a polyamidoamine, to which adamantane can be attached via an isocyanate group.
The beta-cyclodextrin modified polyethyleneimine may be obtained by a method comprising the steps of: reacting the beta-cyclodextrin with an activating agent to obtain activated beta-cyclodextrin, and grafting the activated beta-cyclodextrin onto PEI to obtain beta-cyclodextrin modified polyethyleneimine.
The activator may be N, N' -carbonyldiimidazole or p-toluenesulfonyl chloride.
When preparing the beta-cyclodextrin modified low molecular weight polyethyleneimine, N' -carbonyldiimidazole can be used for activating the beta-cyclodextrin.
Use of a self-assembling nano-formulation in the preparation of a nucleic acid transfection or delivery vector.
The beta cyclodextrin modified low molecular weight polyethyleneimine, the adamantane terminated polyethylene glycol and the adamantane modified polyamide-amine are self-assembled into the nano preparation through the molecular recognition of adamantane and cyclodextrin. The self-assembled nucleic acid nano preparation can be used for loading and conveying nucleic acid molecules. By mixing the self-assembly preparation and the nucleic acid molecules, the nucleic acid molecules are wrapped inside the self-assembly nano preparation through the charge effect, and the self-assembly nano preparation prepared by optimizing the molecular sizes and the proportions of different components has a good cell nucleic acid transportation effect. The self-assembly nano preparation is used as a carrier of nucleic acid molecules, maintains the low toxicity of low molecular weight polyethyleneimine, solves the problems of low efficiency when the traditional low molecular weight polyethyleneimine is used for loading nucleic acid molecules and is used for cell transfection and the high toxicity when the high molecular weight polyethyleneimine is used as a nucleic acid carrier material, and realizes the high-efficiency loading of nucleic acid molecules and the safety and high efficiency in cell transfection. Meanwhile, the preparation method of the self-assembly nucleic acid nano preparation has simple process, low cost, convenience and feasibility.
The nucleic acid molecule may be a gene drug. The nitrogen-phosphorus proportion range between the beta-cyclodextrin-polyethyleneimine in the self-assembly nucleic acid nano preparation and the nucleic acid molecule is (4-24): 1. further, the nitrogen-phosphorus proportion range between the beta-cyclodextrin-polyethyleneimine in the self-assembled nucleic acid nano preparation and the nucleic acid molecule can be (8-20): 1. still further, the nitrogen-phosphorus proportion range between the beta-cyclodextrin-polyethyleneimine in the self-assembly nucleic acid nano preparation and the nucleic acid molecule can be (12-20): 1. in some embodiments, the ratio of nitrogen to phosphorus between β -cyclodextrin-polyethyleneimine and the nucleic acid molecule in the self-assembled nucleic acid nanoformulation can range from (12-16): 1. in some embodiments, the nitrogen-to-phosphorus ratio between the β -cyclodextrin-polyethyleneimine and the nucleic acid molecule in the self-assembled nucleic acid nanoformulation may be in the range of (16-20): 1.
the nucleic acid molecule is selected from, but not limited to, various nucleic acid molecules such as antisense nucleic acid, ribozyme, aptamer, interfering RNA, sgRNA, and the like, expression vectors such as plasmid DNA or mRNA thereof, and vectors such as plasmid DNA or mRNA thereof expressing various bioactive molecules, and the like, and transfection thereof.
The transfection or delivery may be to cells in vitro or to cells in vivo. The transfected subject is a lymphocyte, preferably a T lymphocyte.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. Test methods in which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under conditions recommended by the respective manufacturers.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: a LABORATORY MANUAL, Second edition, Cold Spring Harbor LABORATORY Press, 1989and Third edition, 2001; ausubel et al, Current PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; (iii) METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, chromatography Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.
EXAMPLE 1 Synthesis of beta-cyclodextrin modified Low molecular weight polyethyleneimine CD-PEI
Mono, beta-cyclodextrin modified low molecular weight polyethylenimine CD-PEI (M)W800) preparation of
The synthetic reaction scheme is detailed in FIG. 1A.
Specifically, 0.42g (0.37mmol) of beta-cyclodextrin and 0.80g (5.2mmol) of N, N' -Carbonyldiimidazole (CDI) (English name, full name, CDI) are dissolved in 6mL of dry N, N-dimethylformamide solution, and the mixture is stirred for reaction for 1 hour at room temperature under the protection of nitrogen and in the dark. After the reaction was completed, precipitation was carried out with cold ether, and centrifugation was carried out at 5000rpm for 5min to obtain the product CD-CDI, which was dissolved in 5ml of a dry dimethyl sulfoxide solution.
1.5g (2.5mmol) of a low molecular weight polyethyleneimine (M) are takenW800) was dissolved in 3ml of a dry dimethyl sulfoxide solution, CD-CDI dissolved in dimethyl sulfoxide was slowly added dropwise over 90 minutes, and 0.3ml of a triethylamine solution was added to react for 5 hours. Dialyzing the reaction solution with a dialysis membrane with molecular weight cutoff of 3,500 with deionized water, and freeze-drying the dialyzed product to obtain cyclodextrin-polyethyleneimine 800 (CD-PEI)800) Dissolving appropriate amount of product in D2O for nmr hydrogen spectroscopy, as shown in fig. 2A, the results indicate successful attachment of the cyclodextrin to the polyethyleneimine.
Di, beta-cyclodextrin-high molecular weight polyethylenimine CD-PEI (M)W＝10000)
The synthetic reaction scheme is detailed in FIG. 1B.
Specifically, 34.4g (0.03mol) of beta-cyclodextrin is dissolved in 400mL of 1% sodium hydroxide solution in mass fraction in a 500mL round-bottom flask, stirred until colorless, and then the round-bottom flask is placed in an ice-water bath for precooling. 22mL of acetonitrile solution containing 5.8g (0.03mol) of p-toluenesulfonyl chloride (p-Ttscl) was added dropwise over 160.0min to the round bottom flask reactor and stirring was continued overnight at room temperature. Filtering the reaction solution, collecting the filtrate, acidifying the filtrate by using 1mol/L hydrochloric acid until the pH value is 2-3, standing the filtrate overnight at the temperature of 4 ℃, filtering the filtrate to obtain a white solid, and recrystallizing and purifying the white solid twice by using distilled water to obtain a pure 6-OTs-beta-CD product.
The appropriate amount of 6-OTs-beta-CD product was dissolved in DMSO-d6, and the product 6-OTs-beta-CD was characterized and confirmed by NMR spectroscopy, as shown in FIG. 2B.
100mg of high molecular weight PEI (30% wt, M)W10kD,0.01mmol) was dissolved in 100mL DMSO solution, transferred to a 500mL round bottom flask, 1.29g (1mmol) of 6-OTs- β -CD was added, and the reaction was stirred at 70 ℃ for 3 days. Dialyzing the reaction solution with a dialysis membrane with molecular weight cutoff of 3,500 with deionized water for 6 days, timely replacing the dialysate, and freeze-drying the final dialyzed product to obtain the product CD-PEI10000。
Taking a proper amount of CD-PEI10000The product is dissolved in D2And (3) detecting the nuclear magnetic resonance hydrogen spectrum in the O, as shown in figure 2C, and indicating that the cyclodextrin is successfully connected to the polyethyleneimine.
Example 2 Synthesis of adamantane-terminated polyethylene glycol Ad-PEG
The synthesis reaction formula is shown in detail in figure 3.
Specifically, Adamantane (Adamantane, Ad). 9.39mg (0.05mmol) of adamantane hydrochloride with the molecular weight of 187.74g/mol is weighed and dissolved in 3ml dichloromethane solution, 5.06mg (0.05mmol) of triethylamine is added as a reaction catalyst, 50mg (MW 5000,0.01mmol) of polyethylene glycol succinimide is added, a reactor is stirred and reacted for 2h under the condition of room temperature, and nitrogen gas is filled for protection during the reaction. After the reaction, the reaction mixture was placed in a 20ml eggplant-shaped flask, and the methylene chloride solvent was distilled off under reduced pressure to dry the solvent to a dry state to give a white solid. Adding appropriate amount of deionized water into eggplant-shaped bottle to dissolve white solid at 1 × 104Centrifuging at rpm for 15min, collecting supernatant, adding into dialysis membrane with molecular weight cutoff of 3500, and removingAnd (3) dialyzing with sub-water overnight, freeze-drying the dialyzed solution to obtain white powder, namely the product adamantane-terminated polyethylene glycol, and detecting by using a nuclear magnetic resonance hydrogen spectrum, wherein a detection result shows that adamantane molecules are successfully connected to the polyethylene glycol as shown in figure 4.
EXAMPLE 3 Synthesis of adamantane-modified polyamidoamine Ad-PAMAM
The synthetic reaction scheme is detailed in FIG. 5.
Specifically, adamantane, abbreviated Ad. The polyamidoamine PAMAM used in this example was a first generation polyamide dendrimer with a 1, 4-diaminobutane core and amine ends (PAMAM, G1). The polyamidoamine PAMAM is commercially available from Dendritic Nanotechnologies, Inc (Mount plexant, MI).
100mg (20% wt, 0.07mmol) of the methanolic polyamidoamine solution was added to a round-bottomed flask, the methanolic solution was removed by distillation under reduced pressure, the viscous solid was redissolved in 10ml of dry tetrahydrofuran solution, 244.6mg (1.4mmol) of 1-isocyano adamantane was dissolved in 10ml of dry tetrahydrofuran solution, the dissolved 1-isocyano adamantane solution was added to the polyamidoamine solution, the reaction was stirred at room temperature for 2h and the solution was removed by distillation under reduced pressure. And adding 100ml of ether solution into the reaction container to generate a white solid, washing the precipitate obtained after suction filtration for 3 times by using 100ml of ether, and drying to obtain the product, namely the adamantane modified polyamide-amine Ad-PAMAM. According to data analysis, 8 adamantane molecules were attached to each polyamidoamine.
EXAMPLE 4 preparation of self-assembled nucleic acid Nanoparticulates (DNA @ SNP)
600ul of deionized water was added to a 1.5ml EP tube, and the components of the self-assembled nucleic acid nanopreparation were added sequentially, Ad-PEG1.69nmol, Ad-PAMAM1.39nmol, vortexed for 15s, allowed to stand for 2 minutes, plasmid DNA 6ug, vortexed for 15s, and finally CD-PEI 1.5ul (10mg/ml) of different molecular weights was immediately vortexed for 30 seconds, and allowed to stand for 20 minutes to obtain the assembled nucleic acid nanopreparation (DNA @ SNPs).
At this time, the nitrogen-phosphorus ratio range between the beta-cyclodextrin-polyethyleneimine in the self-assembled nano preparation and the encapsulated nucleic acid molecule is 12: 1, the molar ratio of the beta-cyclodextrin modified polyethyleneimine, the adamantane modified polyamidoamine, and the adamantane terminated polyethylene glycol in the self-assembled nano preparation is 36: 10: 12.
the characterization was performed by using a laser scattering particle size analyzer (see FIG. 6) and a 120kV biological transmission electron microscope (Tecnai G2spirit Biotwin) (see FIG. 7, a and b indicate self-assembled nucleic acid nanopreparations containing CD-PEI800, and c and d indicate self-assembled nucleic acid nanopreparations containing CD-PEI 10000). Experimental results show that the particle size of the self-assembly nucleic acid nano preparation is about 100nm, the distribution is uniform, and the appearance is regular.
Example 5 stability Studies of self-assembled nucleic acid Nanodiulation
Self-assembled nucleic acid nanopreparations were prepared according to the method of example 4 above, and sampled at time points of 0 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, etc. and the particle size and the number of particles of the self-assembled nucleic acid nanopreparations were measured using Nanosight. As shown in fig. 8A and fig. 8B, the experiment results show that the number of particles and the particle size of the self-assembled nucleic acid nano-formulation do not change significantly at different time points, indicating that the self-assembled nucleic acid nano-formulation prepared by us is stable.
Example 6 study of nucleic acid molecule Loading by self-assembling nucleic acid Nanodiormulations
The invention provides application research of a self-assembled nucleic acid nano preparation as a carrier to nucleic acid drug transportation, wherein the application research comprises the capability of the carrier to package nucleic acid drugs and the capability of whether cells can be transfected or not.
Whether gene therapy is effective includes a number of influencing factors. Firstly, the first influencing factor is whether a nucleic acid drug is effectively wrapped by a drug carrier; transfection cannot be achieved if the nucleic acid cannot be encapsulated. When a non-viral vector is used, transfection activity is only possible when negatively charged nucleic acid drug is electrostatically bound to the carrier material to form complex particles. Therefore, the ability to efficiently package nucleic acid drugs is one of the important properties of a transport vehicle. The carrier material for nucleic acid medicine transfection and nucleic acid medicine are assembled together by means of mutual attraction of positive and negative charges, and the charge ratio plays an important role due to the action of the charges, namely the nitrogen/phosphorus (N/P) ratio, wherein N represents amino in the carrier and can generate a unit of positive charge; p is a phosphate group in a nucleic acid drug molecule and can generate a unit negative charge. The N/P ratio reflects the charge ratio of the carrier to the nucleic acid drug, and it must be experimentally found that when the carrier/nucleic acid drug ratio is large or small, the nucleic acid drug can be sufficiently packaged. In the present invention, we use a simpler and more intuitive mass ratio to represent the carrier/nucleic acid drug ratio, which is more accurate in practical applications, and the carrier/nucleic acid drug ratio refers to the mass ratio of the two.
In a preferred embodiment, the invention determines the DNA entrapment capacity of various carrier materials prepared by the invention through an agarose gel retardation experiment, and the experiment result shows that: each support material is capable of binding DNA completely.
On the other hand, whether the nucleic acid drug can exert the curative effect is the most important reason that the transportation carrier can transfect the nucleic acid drug into cells and exert the effect, and the transfection capability of the self-assembled nano preparation to the nucleic acid drug is determined by a cell transfection experiment. The plasmid DNA is used as a nucleic acid drug model, and experimental results show that the vector material can successfully transfer the plasmid DNA into cells, but the transfection efficiency of different vector materials to the plasmid DNA is different due to different molecular weights, composition ratios and charges. When the carrier material is the same and the ratio of the carrier material to DNA is different, the difference in transfection effect tends to be different depending on the kind of the material.
Weighing 0.6g of agarose into a conical flask, adding 60mL of 1 XTAE buffer solution into the conical flask, heating the conical flask by a microwave oven until the agarose is dissolved, cooling the temperature to about 60 ℃, adding 6 microliter of Gelred staining solution, shaking up gently to avoid bubbles, pouring the agarose solution into an agarose gel plate after shaking up, standing for 30 minutes until the agarose gel is solidified, taking out gently, and putting the agarose solution into the 1 XTAE buffer solution for later use. The prepared self-assembly nucleic acid nano preparation is loaded with 300ng of plasmid DNA added into each well, 1 XTAE is used as electrophoresis buffer solution, electrophoresis is carried out for 60 minutes under the voltage of 90V, and the gel imaging system is used for photographing and observation. As shown in fig. 9, the gel electrophoresis results of the self-assembled nucleic acid nanopreparations showed that no significant free DNA escaped from the self-assembled nucleic acid nanopreparations compared to the naked DNA loading group, and the fluorescence intensity of DNA was also darker since DNA was encapsulated inside SNPs, indicating that DNA was successfully loaded in the self-assembled nucleic acid nanopreparations.
Example 7 cell transfection experiment
Human acute T cell leukemia cell line Jurkat was cultured in RPMI1640 containing 10% fetal bovine serum at 37 deg.C and 5% CO2Culturing in an incubator, and selecting Jurkat cells in logarithmic growth phase during experiment.
One, self-assembly nucleic acid nano preparation mediated luciferase plasmid DNA (PGL3) cell transfection
Jurkat cell preparation: the cells were counted and 1ml (1X 10)6Pieces/ml) of cell suspension was added to 24-well plates, three parallel wells per set.
Group setting: positive control (Lipofectamine TM2000 group); CD-PEI800DNA Complex group (CD-PEI)800) (ii) a CD-PEI based800Self-assembling nucleic acid nano-preparation Set (SNP)800Group) based on CD-PEI10000Self-assembling nucleic acid nano-preparation Set (SNP)10000Groups). And replacing the marks in the drawings according to the above abbreviation.
Preparation of experimental samples: CD-PEI based800Self-assembling nucleic acid nano-preparation Set (SNP)800Group) based on CD-PEI10000Self-assembling nucleic acid nano-preparation Set (SNP)10000Group) was prepared as described above. Lipofectamine (TM) 2000 group and CD-PEI800the/DNA complex was prepared according to the product description and literature methods.
Transfection experiments: jurkat cell suspension (1ml) was added to a 24-well plate, centrifuged at 1200rpm at 4 ℃ for 5 minutes, the medium was aspirated, 200ul of the experimental sample loaded with 2ug of plasmid DNA was added to each well, and after 4 hours of incubation in an incubator, 800ul of 10% FBS RPMI1640 medium was added. After the cell culture plate was placed in an incubator and cultured for 24 hours, expression of the Luciferase gene was detected with reference to the instructions of the Luciferase Assay System. As shown in FIG. 10, the results of the experiment revealed that the Lipofectamine (TM) 2000 control group had higher fluorescenceLuciferase expression, and CD-PEI800DNA Complex group (CD-PEI)800) And based on CD-PEI10000Self-assembling nucleic acid nano-preparation Set (SNP)10000Group) had very low luciferase expression levels, and was based on CD-PEI800The self-assembled nucleic acid nanoformulation group of (2) showed very high luciferase expression, and was significantly higher than the Lipofectamine TM2000 control group. The result shows that the nucleic acid delivery effect of the self-assembled nucleic acid nano preparation based on cyclodextrin-low molecular weight polyethyleneimine is obviously better than that of the current commonly used transfection reagent Lipofectamine TM2000, CD-PEI800Or based on macromolecular polyethyleneimine nanoparticles, etc.
Second, cell transfection of self-assembled nucleic acid nano-preparation mediated green fluorescent protein plasmid DNA (pGFP)
Jurkat cell preparation: the cells were counted and 1ml (1X 10)6Pieces/ml) of cell suspension was added to 24-well plates, three parallel wells per set.
Group setting: positive control (Lipofectamine TM2000 group); CD-PEI based800Self-assembling nucleic acid nano-preparation Set (SNP)800Group), Experimental group 2 (CD-PEI)10000Groups).
Preparation of experimental samples: CD-PEI based800Self-assembling nucleic acid nano-preparation Set (SNP)800Group) was prepared as described above. The experiment was performed using the general transfection reagent Lipofectamine (TM) 2000 as a positive control, and the protocol of the experiment was according to the Lipofectamine (TM) 2000 protocol.
Transfection experiments: jurkat cell suspension (1ml) was added to a 24-well plate, centrifuged at 1200rpm at 4 ℃ for 5 minutes, the medium was aspirated, 200ul of DNA loaded with 2ug of plasmid was added to each well, and after 4 hours of culture in an incubator, 800ul of RPMI1640 medium with 10% FBS was added. The cell culture plate was placed in an incubator and cultured for another 48 hours, and then the expression of GFP in the cells was observed by a fluorescence microscope. Experimental results show that the CD-PEI is based on800The GFP positive cell number and fluorescence intensity of the self-assembly nucleic acid nano preparation group are obviously higher than those of a Lipofectamine TM2000 control group, and the self-assembly nucleic acid nano preparation has higher cell transfection efficiency than that of the current commercial reagent。
In addition, in beta-cyclodextrin-polyethyleneimine, the molecular weight of polyethyleneimine before grafting is changed, and other conditions are not changed, different self-assembled nano preparations are prepared according to the preparation of self-assembled nucleic acid nano preparation (DNA @ SNP) in example 4, and luciferase plasmid DNA (PGL3) cell transfection mediated by the self-assembled nucleic acid nano preparation in example 7 is referred to, and as a result, as shown in FIG. 10B, it is known that when the molecular weight of polyethyleneimine before grafting is 600-1800, good transfection of nucleic acid molecules can be achieved.
In addition, different self-assembled nano-formulations were prepared by varying the nitrogen-phosphorus ratio between β -cyclodextrin-polyethyleneimine and the entrapped nucleic acid molecule in the self-assembled nano-formulations, as described in example 4 for the preparation of self-assembled nucleic acid nano-formulations (DNA @ SNP), when the nitrogen-phosphorus ratio between β -cyclodextrin-polyethyleneimine and the entrapped nucleic acid molecule in the self-assembled nano-formulations ranged from 4: 1, the molar ratio of the beta-cyclodextrin modified polyethyleneimine, the adamantane modified polyamidoamine and the adamantane terminated polyethylene glycol in the self-assembled nano preparation is 12: 10: 12; when the nitrogen-phosphorus proportion range between the beta-cyclodextrin-polyethyleneimine in the self-assembly nano preparation and the encapsulated nucleic acid molecules is 8: 1, the molar ratio of the beta-cyclodextrin modified polyethyleneimine, the adamantane modified polyamidoamine, and the adamantane terminated polyethylene glycol in the self-assembled nano preparation is 24: 10: 12; when the proportion of nitrogen and phosphorus between the beta-cyclodextrin-polyethyleneimine and the encapsulated nucleic acid molecules in the self-assembled nano preparation is within 12: 1, the molar ratio of the beta-cyclodextrin modified polyethyleneimine, the adamantane modified polyamidoamine and the adamantane terminated polyethylene glycol in the self-assembled nano preparation is 36: 10: 12; when the nitrogen-phosphorus proportion range between the beta-cyclodextrin-polyethyleneimine in the self-assembly nano preparation and the encapsulated nucleic acid molecule is 16: 1, the molar ratio of the beta-cyclodextrin modified polyethyleneimine, the adamantane modified polyamidoamine, and the adamantane terminated polyethylene glycol in the self-assembled nano preparation is 48: 10: 12; when the nitrogen-phosphorus proportion range between the beta-cyclodextrin-polyethyleneimine in the self-assembly nano preparation and the encapsulated nucleic acid molecules is 20: 1, the molar ratio of the beta-cyclodextrin modified polyethyleneimine, the adamantane modified polyamidoamine and the adamantane terminated polyethylene glycol in the self-assembled nano preparation is 60: 10: 12; when the nitrogen-phosphorus ratio range between the beta-cyclodextrin-polyethyleneimine in the self-assembled nano preparation and the encapsulated nucleic acid molecule is 24: 1, the molar ratio of the beta-cyclodextrin modified polyethyleneimine, the adamantane modified polyamidoamine, and the adamantane terminated polyethylene glycol in the self-assembled nano preparation is 72: 10: 12.
referring to the transfection results of luciferase plasmid DNA (PGL3) cells mediated by the self-assembled nucleic acid nano-preparation in example 7, as shown in fig. 10C, it can be seen that when β -cyclodextrin-polyethyleneimine in the self-assembled nano-preparation is in a nitrogen-phosphorus ratio range with the entrapped nucleic acid molecules (12-20): 1, the molar ratio of the beta-cyclodextrin modified polyethyleneimine, the adamantane modified polyamide-amine and the adamantane terminated polyethylene glycol in the self-assembled nano preparation is (36-60): 10: 12, can realize good transfection to nucleic acid molecules.
Then, the compositional changes of the adamantane-modified polyamidoamine were examined, and the resulting self-assembled nucleic acid nanopreparations mediated transfection of luciferase plasmid DNA (PGL3) cells resulted in the molar ratio of 36: (10-20): 12, the transfection of nucleic acid molecules can be realized well.
Next, the compositional changes of the adamantane-terminated polyethylene glycol were examined, and the resulting self-assembled nucleic acid nanopreparations mediated transfection of luciferase plasmid DNA (PGL3) cells, and the results are shown in fig. 10E when the molar ratio between β -cyclodextrin modified polyethyleneimine, adamantane-modified polyamidoamine, and adamantane-terminated polyethylene glycol in the self-assembled nanopreparations was 36: 10: (12-48), the transfection on the nucleic acid molecules can be realized well.
While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that the foregoing and other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.

Claims (6)

1. A self-assembly nano preparation is composed of beta-cyclodextrin modified polyethyleneimine, adamantane-terminated polyethylene glycol and adamantane-modified polyamide-amine, wherein the molar ratio range of the beta-cyclodextrin modified polyethyleneimine, the adamantane-modified polyamide-amine and the adamantane-terminated polyethylene glycol is (12-72): (10-20): (12-48) of a first step,
the beta-cyclodextrin modified polyethyleneimine is characterized in that the beta-cyclodextrin-grafted polyethyleneimine is selected from polyethyleneimine, wherein one or more beta-cyclodextrin molecules are grafted on each polyethyleneimine long chain in the beta-cyclodextrin-grafted polyethyleneimine, the polyethyleneimine before grafting is low-molecular-weight polyethyleneimine, and the molecular weight range of the polyethyleneimine before grafting is 600-1800; wherein: the beta-cyclodextrin modified polyethyleneimine is obtained by a method comprising the following steps: reacting beta cyclodextrin with an activating agent to obtain activated beta-cyclodextrin, and grafting the activated beta-cyclodextrin onto polyethyleneimine to obtain beta-cyclodextrin modified polyethyleneimine, wherein the activating agent is N, N' -carbonyldiimidazole or p-toluenesulfonyl chloride;
the adamantane-modified polyamide-amine is polyamide-amine connected with adamantane, and each polyamide-amine is connected with one or more than one adamantane; adamantane is linked to the amine group of the polyamidoamine through an isocyanate group; the polyamidoamine is a 1-generation polyamidoamine.
The molecular weight of the polyethylene glycol before blocking is 2000-5000.
2. The self-assembling nano-formulation of claim 1, wherein: in the adamantane-terminated polyethylene glycol, adamantane is linked to the end of the polyethylene glycol through an amide bond.
3. A method for preparing a self-assembled nano-formulation as claimed in any one of claims 1 to 2, comprising: the components are taken according to the proportion, mixed and self-assembled to obtain the self-assembled nano preparation.
4. Use of the self-assembled nano-preparation according to any one of claims 1 to 2 or the self-assembled nano-preparation prepared by the method according to claim 3 for preparing a nucleic acid transfection or delivery vector.
5. The use according to claim 4, wherein the transfected subject is a lymphocyte.
6. Use according to claim 5, characterized in that the lymphocytes are T lymphocytes.
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