CN116459352B - DNA condensation system, non-unwinding cyclic compound, preparation method and application in preparation of gene therapy medicine - Google Patents
DNA condensation system, non-unwinding cyclic compound, preparation method and application in preparation of gene therapy medicine Download PDFInfo
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- CN116459352B CN116459352B CN202211725400.XA CN202211725400A CN116459352B CN 116459352 B CN116459352 B CN 116459352B CN 202211725400 A CN202211725400 A CN 202211725400A CN 116459352 B CN116459352 B CN 116459352B
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
- A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
- A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
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- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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Abstract
The invention relates to a DNA condensation system, a non-unwinding cyclic compound, a preparation method and application thereof in preparing gene therapy drugs, wherein the DNA condensation system can condense DNA molecules into nano particles through the recombination of polyethylene glycol-lateral amino polyamino acid block copolymer and DNA molecules ions to form the non-unwinding cyclic compound of DNA and polymer, thereby being beneficial to maximally preserving the biological activity of the DNA, improving the endocytosis efficiency of cells and increasing the circulation time of the DNA in organisms through carrying a polyethylene glycol protective shell. The DNA condensation system and the corresponding non-unwinding cyclic compound can be widely applied to gene therapy.
Description
Technical Field
The invention relates to the technical field of gene delivery, in particular to a DNA condensation system, a non-unwinding annular compound, a preparation method and application thereof in preparing a gene therapy drug.
Background
Methods for treating certain human genetic diseases by correcting or compensating for human genes by introducing exogenous genes are called gene therapy strategies. Gene therapy has considerable advantages in the theoretical level, and can permanently cure certain diseases by clinically implementing simple gene therapy. Although the process from theory to clinical application is tortuous and lengthy, with the development of gene vectors, the rise of chimeric antigen receptor T (CAR-T) cell immunotherapy and the breakthrough of genome editing technology, gene therapy has gradually become an important method for disease treatment in recent years, providing a brand-new therapeutic option for a plurality of medical fields. Since the direct injection of naked DNA into a living body has many limitations, such as easy degradation of nucleic acid in the living body, low uptake of nucleic acid molecules by cells, etc., and limits the practical application of therapeutic genes, the use of gene vectors to transport therapeutic genes to lesion sites, thereby improving the utilization rate of therapeutic genes in the living body, is a key to the future of gene therapy.
Recombinant viral vectors with replication defects are the first molecular tool to efficiently transfer genes of interest to human cells. Although viruses have complex structures and life cycles, many of which are pathogens, several viruses are efficient gene delivery tools. Non-viral vectors are generally not nearly as efficient as viral vectors because copies of the gene must be presented to the target cell to express one or several copies in the nucleus. For viral vectors, the usual approach is to remove unwanted or pathogenic genes while preserving the efficiency of gene delivery, expression and persistence at the appropriate location. At the same time, viral vectors are also required to overcome many common problems, such as removal of toxic genes, lack of replication-competent parental viruses and the effect of the genes of interest on viral vector components. The virus gene vector can realize the high-efficiency expression of DNA, but the safety of the virus vector is reduced due to the higher immunogenicity brought by the unique structure of the virus and the integrated mutation effect of the gene. At the same time, production of viral vectors is difficult, which also makes it difficult for viral vectors to support the development of future gene therapies. Thus, currently, viral vectors that can be used are very limited.
Therefore, the development and development of non-viral vectors have become important points of research in gene therapy, wherein cationic polymer vectors have become research hot spots in non-viral vectors due to the advantages of controllable chemical structure, easy chemical modification, wide raw material sources, mass production and the like.
Disclosure of Invention
Aiming at the problems that the uptake of nucleic acid molecules by cells is relatively low, the circulation time of naked DNA in vivo is short, the double-stranded structure of the DNA is easy to be damaged in the condensation embedding process and the like in gene therapy, the invention aims at providing a DNA condensation system, a non-unwinding annular compound, a preparation method and application in preparing gene therapy medicaments.
In a first aspect of the invention, there is provided a DNA condensation system comprising the following components: block copolymers, DNA, salt ions and water;
Wherein the concentration of the salt ions is 150-1200 mmol/L;
the block copolymer is provided with a polyethylene glycol block and a lateral amino type polyamino acid block, wherein the lateral amino type polyamino acid block is at least one of a polylysine block and a polyornithine block, and 0-45% of lateral groups-NH 2 of the lateral amino type polyamino acid block are replaced by-SH; wherein the weight average molecular weight of the polyethylene glycol block is 5 kDa-20 kDa, and the weight average molecular weight of the side amino type polyamino acid block is 3.5 kDa-15 kDa.
In some embodiments, the block copolymer has a structure represented by formula (1):
In the formula (1), q is an integer selected from 200 to 250; y is an integer selected from 35 to 50;
R 1 is C 1-3 alkyl;
L 1 is C 1-6 alkylene;
Z 1 is-NH- -O-NH-or-O-;
Any one L 2 is independently butylene or propylene;
Either R 2 is independently-NH 2 or R 3; wherein, R 3 has the structure-NH-Z 3-L3 -SH, wherein L 3 is C 2-6 alkylene, Z 3 is-C (=nh) -, -C (=o) -or-C (=o) -NH-, wherein ". X" represents the L 2 direction; in the formula (1), the number of R 3 is n and the ratio of n to y is 0-45%.
In some embodiments, the DNA condensation system satisfies any one or more of the following characteristics:
The salt ions are sodium ions;
The concentration of the salt ions is 500-700 mmol/L; preferably 550-650 mmol/L
The pH of the DNA condensation system is 7.2-7.6.
In some embodiments, the DNA condensation system satisfies any one or more of the following characteristics:
The molar ratio of the positive charges of the amino groups in the block copolymer to the negative charges of the phosphate groups in the DNA is recorded as N/P, and the N/P is 1-16, preferably 1-2;
the concentration of the DNA is 20-150 ng/. Mu.L; preferably 25 to 50 ng/. Mu.L.
In some embodiments, the DNA condensation system satisfies any one or more of the following characteristics:
The length of the DNA is 3 k-20 k bp;
The lateral amino type polyamino acid block is a polylysine block;
the weight average molecular weight of the polyethylene glycol block is 6 kDa-12 kDa;
The weight average molecular weight of the side amino type polyamino acid block is 4 kDa-13 kDa;
0 to 42% of the pendant groups-NH 2 of the pendant amino-type polyamino acid block are replaced by-SH.
In some embodiments, the ratio of n to y is 0 to 42%;
optionally, the ratio of n to y is 0 or 8% to 42%.
In some embodiments, the DNA condensation system satisfies any one or a combination of any more of the following characteristics:
L 2 are the same;
R 2 are the same;
q is an integer selected from 225 to 230;
y is an integer selected from 36 to 44;
R 1 is methyl;
L 1 is 1, 2-ethylene, 1, 3-propylene or 1, 4-butylene;
z 1 is-NH-;
L 2 is 1, 4-butylene;
Z 3 is-C (=nh) -;
L 3 is 1, 2-ethylene, 1, 3-propylene or 1, 4-butylene.
In some embodiments, the DNA condensation system satisfies either or both of the following characteristics:
L 1 is 1, 2-ethylene;
l 3 is 1, 3-propylene.
In a second aspect of the present invention, there is provided a non-unwinding cyclic complex formed by complexing a block copolymer with DNA; wherein the block copolymer is as defined in the first aspect of the invention.
In some embodiments, the non-helic cyclic complex is prepared from the DNA condensation system of the first aspect of the invention.
In some embodiments, either or both of the following features are satisfied:
The hydrodynamic diameter of the non-unwinding annular compound is 180-190 nm;
At least a portion of the DNA has a complete double-stranded structure;
the non-unwinding cyclic complex contains cross-linked disulfide bonds.
In a third aspect of the present invention, there is provided a method of preparing a non-unwinding cyclic complex comprising the steps of: vortex mixing DNA and block copolymer in the presence of 150-1200 mmol/L salt ion, and letting stand the prepared mixed system; wherein the block copolymer is the condensation system of the first aspect of the present invention.
In some embodiments, the methods of preparation meet one or more of the following characteristics:
the vortex mixing time is 1-5 seconds;
the standing temperature is 4-25 ℃;
standing for 0.5-24 h;
After the standing is finished, the method further comprises the following steps: dialyzing with water;
the mixed system formed by the DNA and the block copolymer is the DNA condensation system of the first aspect of the invention.
In some embodiments, the pendant amino-type polyamino acid block contains-SH in its side chain structure; the block copolymer is pretreated with a disulfide bond reducing agent prior to mixing the DNA and the block copolymer.
In some embodiments, the disulfide bond reducing agent is selected from one or more of dithiothreitol, glutathione, and TCEP.
In a fourth aspect of the present invention, there is provided the use of a DNA condensation system according to the first aspect of the present invention, or a non-helicitic cyclic complex according to the second aspect of the present invention, or a non-helicitic cyclic complex prepared by a preparation method according to the third aspect of the present invention, in the preparation of a gene therapy drug.
In some embodiments, the medicament is for treating at least one of the following diseases: ischemic diseases, tumors and ocular neovascular diseases.
In a fifth aspect of the invention there is provided the use of a block copolymer as a non-viral gene vector or in gene delivery, said use not being for diagnostic or therapeutic purposes; the block copolymer is as defined in the first aspect of the invention;
the application is based on any one of the following systems or substances:
the DNA condensation system according to the first aspect of the present invention;
the non-unwinding cyclic complex according to the second aspect of the invention; and
The non-unwinding cyclic compound prepared by the preparation method of the third aspect of the invention.
The DNA condensation system provided by the invention can condense DNA molecules into nano particles through the ion composite action of polyethylene glycol-lateral amino polyamino acid block copolymer (PEG-PNAA) and DNA molecules to form a cyclic non-unwinding compound of DNA and polymer, thereby being beneficial to keeping the complete double-chain structure of DNA, keeping the bioactivity of DNA to the maximum, improving the endocytosis efficiency of cells and prolonging the circulation time of DNA in organisms through carrying a polyethylene glycol protective shell. The DNA condensation system and the annular compound can be widely applied to gene therapy.
The pendant amino groups of PEG-PNAA may or may not be replaced by-SH, herein designated PEG-PNAA (SH), herein designated PEG-PNAA (NH 2). By substituting a part of side group-NH 2 of the side amino type polyamino acid block with-SH, the obtained sulfhydryl modified polyethylene glycol-side amino type polyamino acid block copolymer can be named as PEG-PNAA (SH), and the block copolymer can crosslink the-SH in the formed nano-composite to form disulfide bonds, so that the extracellular stability of the nano-composite can be improved, the disulfide bonds are reduced in response to intracellular glutathione when the nano-composite enters cells, the dissociation of the nano-composite in the cells is promoted, the release of DNA in the cells is promoted, the stable existence in blood circulation is realized, and the DNA can be released rapidly when the nano-composite reaches the target cells.
When the pendant amino-based polyamino acid block is a polylysine block, PEG-PNAA is designated as PEG-PLys, PEG-PNAA (SH) is designated as PEG-PLys (SH), and PEG-PNAA (NH 2) is designated as PEG-PLys (NH 2). Further, the use of PEG-PNAA (NH 2) and/or PEG-PNAA (SH)) and pDNA (plasmid DNA) to condense into a cyclic complex in a directed manner can significantly increase the transfection efficiency of non-viral vector carrying DNA.
In addition, the circular DNA condensate provided by the invention is a circular self-winding condensate, wherein the double-chain structure of the DNA is not damaged, and the circular DNA condensate is beneficial to the transcription of the DNA in the later period.
Drawings
In order to more clearly illustrate the technical solution in the embodiments of the present application and to more fully understand the present application and its advantageous effects, the following brief description will be given with reference to the accompanying drawings, which are required to be used in the description of the embodiments. It is evident that the figures in the following description are only some embodiments of the application, from which other figures can be obtained without inventive effort for a person skilled in the art. It should be further noted that the drawings are drawn in a simplified form and serve only to facilitate a convenient and clear illustration of the application. The various dimensions of each of the components shown in the figures are arbitrarily, may be exact or may not be drawn to scale. For example, the dimensions of the elements are exaggerated in some places in the drawings for clarity of illustration. Unless otherwise indicated, the various elements in the drawings are not drawn to scale. The present application is not limited to each size of each component.
Wherein like reference numerals refer to like parts throughout the following description.
FIG. 1 is a schematic diagram of the regulation of the properties of a DNA condensate and the synthesis of a circular DNA condensate and a schematic diagram of the treatment of ischemic diseases of the lower limbs; in the system, PEG-PNAA is taken as PEG-PLys (SH), and salt ions are taken as sodium ions as examples; wherein PLASMID DNA is plasmid DNA; condensing the DNA in a folding manner to form an unwinding state, and not forming the DNA in a winding manner to form an unwinding state; PEG-PLys (SH) is only an example, where PEG has a degree of polymerization of 228, unsubstituted lysine residue has a degree of polymerization of m, SH substituted lysine residue has a degree of polymerization of n, see formula (1B);
FIG. 2 shows 1 H NMR spectra of PEG-PLys (NH 2) and PEG-PLys (SH) according to an embodiment of the invention; a) 1 HNMR nuclear magnetic resonance spectrum for PEG-PLys (NH 2), b) 1 H NMR nuclear magnetic resonance spectrum for PEG-PLys (SH);
FIG. 3 shows the results of the analysis of the capacity of the cationic carrier to bind pDNA and the stability of the cationic carrier to bind pDNA in example 2 of the present invention: a) Gel blocking electrophoresis patterns of PEG-PLys (SH) for different N/P; b) Gel blocking electrophoretogram for PEG-PLys (SH)/pDNA incubated at different heparin sodium concentrations;
FIG. 4 shows the results of a test for studying DNA complexes using a transmission electron microscope, DLS measurement and reaction with S1 nuclease in example 2 of the present invention; a) Corresponding rod-like complexes; b) Corresponding cyclic complexes; scale bar: 500nm;
Fig. 5 shows the evaluation results of the structural stability of cyclic pDNA condensate under heparin treatment at different concentrations in example 3 of the present invention: a) PEG-PLys (NH 2) and pDNA condensate; b) Disulfide-crosslinked pDNA condensate of PEG-PLys (SH) in the absence of GSH; c) pDNA condensate of PEG-PLys (SH) under GSH (10 mM) incubation;
FIG. 6 shows the nuclease resistance test of the DNA complex of example 3 according to the present invention, wherein the ordinate is REMAINING INTACT DNA, i.e. the percentage of intact DNA remaining;
FIG. 7 is a cytotoxicity study of different N/P complexes in example 3 of the present invention, with the ordinate showing cell viability (Cellular viability);
FIG. 8 shows the cell uptake efficiency of rod and ring condensates with and without disulfide crosslinks [ ss (+) ] of example 3 of the present invention; a) Quantifying cell uptake efficiency by flow cytometry, wherein pDNA is a Cy 5-labeled pDNA condensate (×p <0.001, t-test); b) Observing the distribution of pDNA in cells 24 hours after incubation of the DNA condensate using laser confocal; upper, pDNA; lower, annular pDNA condensate; pDNA is marked by red fluorescence, cell nucleus is marked by blue fluorescent substance, and the proportion is 20 μm; displayed as a gray scale map;
FIG. 9 shows the expression efficiency of Luc in HUVECs for rod-and cyclic-pLuc-condensate in example 3 of the present invention (< 0.001 in p, t-test); the ordinate axis is gene expression activity in RLU (relative light unit);
FIG. 10 is a graph showing the result of the proliferation of hind limb blood vessels in the mice in example 4 of the present invention;
FIG. 11 shows immunofluorescent staining of mouse gastrocnemius muscle in example 4 of the present invention; the nuclei were labeled with blue fluorescent material, the VEGF was labeled with green fluorescent material, and the CD31 was labeled with red fluorescent material, shown as a gray scale;
FIG. 12 shows immunofluorescent staining of calf muscle of hind limb of mice in example 4 of the present invention: a) VEGF expression is quantified, the ordinate is VEGF expression (unit RFU, relative fluorescence unit), and the corresponding groups from left to right are saline, pVEGF and toroidal condensates; b) Quantitatively estimating the blood vessel density based on CD31 positive, wherein the groups corresponding to the blood vessel density from left to right are saline, pVEGF and toroidal condensates;
FIG. 13 is a graph showing the results of treatment 28 days after ligation in the investigation of the condition of necrosis of hind limb in a relief mouse according to example 4 of the present invention;
FIG. 14 is a transmission electron micrograph of the coagulated form of DNA at different salt (NaCl) concentrations in example 5 of the present invention;
FIG. 15 shows the results of cell-free transcription level test of DNA condensate at different salt (NaCl) concentrations in example 5 of the present invention;
FIG. 16 shows the results of cell transfection activity test of DNA condensate at different salt (NaCl) concentrations in example 5 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the drawings, embodiments and examples. It should be understood that these embodiments and examples are provided solely for the purpose of illustrating the invention and are not intended to limit the scope of the invention in order that the present disclosure may be more thorough and complete. It will also be appreciated that the present invention may be embodied in many different forms and is not limited to the embodiments and examples described herein, but may be modified or altered by persons skilled in the art without departing from the spirit of the invention, and equivalents thereof are also intended to fall within the scope of the invention. For example, features illustrated or described as part of one embodiment can be combined with another embodiment in a suitable manner to yield a new embodiment. Furthermore, in the following description, numerous specific details are set forth in order to provide a more thorough understanding of the invention, it being understood that the invention may be practiced without one or more of these details.
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. The terminology used herein in the description of the invention is for the purpose of describing the embodiments and examples only and is not intended to be limiting of the invention.
Terminology
Unless otherwise indicated or contradicted, terms or phrases used herein have the following meanings:
The term "and/or," "and/or," as used herein, includes any one of two or more of the listed items in relation to each other, as well as any and all combinations of the listed items in relation to each other, including any two of the listed items in relation to each other, any more of the listed items in relation to each other, or all combinations of the listed items in relation to each other. It should be noted that, when at least three items are connected by a combination of at least two conjunctions selected from the group consisting of "and/or", "and/or", it should be understood that, in the present application, the technical solutions include technical solutions that all use "logical and" connection, and also include technical solutions that all use "logical or" connection. For example, "a and/or B" includes a combination of A, B and a and B in three parallel schemes. For another example, the technical schemes of "a, and/or B, and/or C, and/or D" include any one of A, B, C, D (i.e., the technical schemes of all "logical or" connections), also include any and all combinations of A, B, C, D, i.e., the combinations of any two or three of A, B, C, D, and also include four combinations of A, B, C, D (i.e., the technical schemes of all "logical and" connections).
In the present application, when at least three features are connected by a combination of at least two conjunctions selected from the group consisting of "and/or", "and/or", the expression "having one or more features" corresponds to, for example, "TA, and/or, TB, and/or, TC, and/or, TD" corresponds to "having one or more of the following features: TA, TB, TC, and TD).
The term "plural", and the like in the present invention refers to, unless otherwise specified, a number of 2 or more. For example, "one or more" means one kind or two or more kinds.
In the present invention, unless otherwise indicated, "one or more" means any one of the listed items or any combination of the listed items. Similarly, "one or more" and the like are otherwise indicated for the case of "one or more", and the same is understood unless otherwise indicated.
As used herein, "a combination thereof," "any combination thereof," and the like include all suitable combinations of any two or more of the listed items.
The "suitable" in the "suitable combination manner", "suitable manner", "any suitable manner" and the like herein refers to the fact that the technical scheme of the present invention can be implemented, the technical problem of the present invention is solved, and the technical effect expected by the present invention is achieved.
Herein, "preferred", "better", "preferred" are merely to describe better embodiments or examples, and it should be understood that they do not limit the scope of the invention. If there are multiple "preferences" in a solution, if there is no particular description and there is no conflict or constraint, then each "preference" is independent of the others.
In the present invention, "further," "still further," "special," "for example," "such as," "example," "illustrated," etc. are for descriptive purposes to indicate that there is a relationship between different solutions in the preceding and following contexts, but should not be construed as limiting the preceding solution or the scope of the invention. In the present invention, a (e.g., B), where B is one non-limiting example of a, is understood not to be limited to B, unless otherwise stated.
In the present application, "optional" means optional or not, that is, means any one selected from two parallel schemes of "with" or "without". If multiple "alternatives" occur in a technical solution, if no particular description exists and there is no contradiction or mutual constraint, then each "alternative" is independent. In the present application, "optionally containing", and the like are described, meaning "containing or not containing". "optional component X" means that component X is present or absent, or that component X is present or absent.
In the present invention, the terms "first", "second", "third", "fourth", etc. are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or quantity, nor as implying an importance or quantity of a technical feature being indicated. Moreover, the terms "first," "second," "third," "fourth," and the like are used for non-exhaustive list description purposes only, and are not to be construed as limiting the number of closed forms.
In the invention, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.
In the present invention, a numerical range (i.e., a numerical range) is referred to, and, unless otherwise indicated, a distribution of optional values within the numerical range is considered to be continuous and includes two numerical endpoints (i.e., a minimum value and a maximum value) of the numerical range, and each numerical value between the two numerical endpoints. When a numerical range merely points to integers within the numerical range, unless expressly stated otherwise, both endpoints of the numerical range are inclusive of the integer between the two endpoints, and each integer between the two endpoints is equivalent to the integer directly recited. When multiple numerical ranges are provided to describe a feature or characteristic, the numerical ranges may be combined. In other words, unless otherwise indicated, the numerical ranges disclosed herein are to be understood as including any and all subranges subsumed therein. The "numerical value" in the numerical interval may be any quantitative value, such as a number, a percentage, a proportion, or the like. "numerical intervals" allows for the broad inclusion of numerical interval types such as percentage intervals, proportion intervals, ratio intervals, and the like.
The temperature parameter in the present invention is not particularly limited, and may be a constant temperature treatment or may vary within a predetermined temperature range. It should be appreciated that the constant temperature process described allows the temperature to fluctuate within the accuracy of the instrument control. Allows for fluctuations within a range such as + -5 ℃, + -4 ℃, + -3 ℃, + -2 ℃, + -1 ℃.
In the present invention, the term "room temperature" or "normal temperature" generally means 4℃to 35℃such as 20.+ -. 5 ℃. In some embodiments of the invention, "room temperature" or "normal temperature" refers to 10 ℃ to 30 ℃. In some embodiments of the invention, "room temperature" or "normal temperature" refers to 20 ℃ to 30 ℃.
In the present invention, referring to a unit of a data range, if a unit is only carried behind a right end point, the units indicating the left and right end points are the same. For example, 3 to 5h means that the units of the left end point "3" and the right end point "5" are both h (hours).
In the present application, where numerical values or numerical ranges are referred to, unless otherwise indicated, it is understood that the numerical ranges cover reasonable divisors of the two endpoints, and that the fluctuation range due to the divisors can be included in the indicated numerical range. That is, in the present application, "N1" and "about N1" have the same meaning, and are used interchangeably, and "N1 to N2" and "about N1 to about N2" have the same meaning, and are used interchangeably, wherein N1 and N2 are two unequal values, unless otherwise indicated; about values within the divisor range are also intended to be included within the range that is dictated by the numerical range due to one or more factors of reasonable deviation, instrument control accuracy, and the like permitted in the art. For example, reference to "a temperature of 20 ℃ to 30 ℃ may be understood as" about 20 ℃ to about 30 ℃; further, in the case of the endpoints of "20 ℃ and their approximate numbers of.+ -. 1 ℃, the approximate numbers of 19 ℃ and 19.5 ℃ in the approximate number range indicated by" about 20 ℃ should be included in the range indicated by 20 ℃ to 30 ℃.
Herein, unless otherwise specified, "about" means that the fluctuation range is within a range of a certain amplitude above and below the present number, and the fluctuation range may be different depending on the type and the numerical value of the present number. For example, it is allowed to be within a range of + -10%, + -5%, + -2%, + -1%, etc. Such as about 200 microns, may represent a material selected from 200 + -20 microns.
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Unless otherwise indicated to the contrary by the intent and/or technical aspects of the present application, all references to which this application pertains are incorporated by reference in their entirety for all purposes. When reference is made to a cited document in the present application, the definitions of the relevant technical features, terms, nouns, phrases, etc. in the cited document are also incorporated. In the case of the cited documents, examples and preferred modes of the cited relevant technical features are also incorporated into the present application by reference, but are not limited to being able to implement the present application. It should be understood that when a reference is made to the description of the application in conflict with the description, the application is modified in light of or adaptive to the description of the application.
In the present invention, the method flow involves a plurality of steps, and the steps are not strictly limited in order to be performed in other orders than that shown unless explicitly stated otherwise herein. Moreover, any step may include a plurality of sub-steps or a plurality of stages, which are not necessarily performed at the same time, but may be performed at different times, the order of which is not necessarily sequential, and may be performed in rotation or alternately or simultaneously with other steps or a part of the sub-steps or stages of other steps.
The mass or weight of the related components mentioned in the description of the embodiments of the present invention may refer not only to the specific content of each component, but also to the proportional relationship of the mass or weight of each component, so long as the content of the related component in the description of the embodiments of the present invention is scaled up or down within the scope of the disclosure of the embodiments of the present invention. Specifically, the mass or weight described in the specification of the embodiment of the present invention may be a unit well known in the chemical industry such as mu g, mg, g, kg.
The non-viral vector loads and condenses DNA mainly by ionic recombination between the positive charge of the non-viral vector and the negative charge of the nucleic acid molecule. Researchers develop different strategies aiming at the problems of particle size, biocompatibility, stability and the like of a non-viral vector and DNA complex, and all achieve a certain effect. However, the low transfection efficiency of genes by non-viral vectors is a major obstacle to the development of non-viral vectors compared to viral vectors. The inventor of the present application has found that the DNA is compressed in the virus in a ring configuration, but the structure of a complex formed by a non-viral vector and the DNA is disordered, and the double-stranded DNA is easy to unwind in the embedding condensation process, so that the biological activity of the DNA is affected. Based on this, the inventors of the present application propose the following idea: the condensing shape of the cationic vector and the DNA complex is controlled to simulate the condensing form of viruses on DNA, so that the gene transfection efficiency of the non-viral vector can be improved, and the support is provided for the development of the non-viral vector in gene therapy.
There are one or more of the following problems in current gene therapy:
(1) The problem of relatively low uptake of nucleic acid molecules by cells;
(2) The bare DNA has short circulation time in the body and is easy to degrade;
(3) The DNA nanocomposite formed by the block copolymer and DNA needs to satisfy the following conditions: stably exists in the blood circulation and can rapidly release DNA when reaching the target cells;
(4) The transfection efficiency by using non-viral vector gene is not high.
Aiming at the problems of poor transfection efficiency, high toxicity and the like of the traditional polymer gene vector, the application provides a high-efficiency strategy for improving the transfection efficiency of the vector and reducing the cytotoxicity. Depending on the nature of the biological microenvironment or intracellular environment, researchers have developed a variety of gene delivery vectors, but the transfection efficiency of non-viral vectors is still not high compared to viral gene vectors. The condensed structure of DNA is regularly packed into chromosome in organism, and is enclosed in inner shell in virus body in circular mode. The inventors of the present application speculate that the structure of the cationic polymer vector for condensation of DNA is generally similar to a linear or spherical structure, the internal double-stranded DNA structure is destroyed, and the double-stranded DNA is unwound into single-stranded DNA, which may be an important cause of low transfection efficiency of the non-viral vector. Thus, in contrast to DNA packaging in viral vectors, it was found that DNA is packaged in a spiral wound fashion within the viral envelope. Based on this, it is envisaged that this regular winding pattern is critical to the biological function of the DNA.
In the present invention, the effect of the cyclic DNA condensate on the DNA transfection efficiency was examined, and the effect of the cyclic DNA condensate on angiogenesis was studied.
In the present invention, entrapment of DNA is achieved by complexing with a cationic compound to neutralize its negative charge, and the DNA then undergoes a volumetric transformation from an expanded coiled to compact form, a process known as DNA condensation. The invention also researches the process of adding a neutral hydrophilic polyethylene glycol (PEG) block on the DNA condensation cation chain segment, condensing the block cation monomer and DNA, and controlling the condensation process by adjusting the ionic strength in the ion composite interaction process so as to realize ordered condensation of the DNA condensate. The DNA directional virus-like is condensed into a ring-shaped condensate, so that the efficient expression of genes is realized. Referring to FIG. 1, the following process is shown in FIG. 1 taking PEG-PNAA as an example of PEG-PLys (SH): the cation PLys and the anion plasmid DNA cause DNA condensation through ion recombination, so that a nano core-shell structure taking PLys and DNA as inner cores and PEG as outer shells is formed; wherein, based on the regulation of the ionic strength in the ion composite solution, the DNA condensation path can be the folding of DNA molecules or the winding of DNA molecules, wherein, partial DNA in the 'rod-shaped' condensation body formed in a folding way generates double-chain unwinding, and the DNA in the 'ring-shaped' condensation body formed in a winding way does not generate double-chain unwinding; in addition, the PLys (SH) introduced with the mercapto can realize the crosslinking of the condensate based on the manner of covalent bonding of the mercapto-mercapto to form disulfide bonds, so that the stability of the condensate structure in a physiological environment is improved; further, by injecting a plasmid DNA circular condensate capable of expressing Vascular Endothelial Growth Factor (VEGF) into the ischemic site of the lower limb, the revascularization is stimulated, and the blood circulation of the ischemic site of the lower limb can be effectively improved.
Based on this, in a first aspect of the present invention, there is provided a DNA condensation system comprising the following components: block copolymers, DNA, salt ions and water;
Wherein the concentration of the salt ions is 150-1200 mmol/L;
The block copolymer has a polyethylene glycol block and a side amino type polyamino acid block, wherein the side amino type polyamino acid block is at least one of a polylysine block and a polyornithine block, and a side group-NH 2 of the side amino type polyamino acid block is substituted or unsubstituted by-SH.
The block copolymer has a polyethylene glycol block and a pendant amino type polyamino acid block, denoted PEG-PNAA. When the pendant amino-type polyamino acid block is a polylysine block, the block copolymer is designated PEG-PLys.
In some embodiments, a DNA condensation system is provided that includes the following components: block copolymers, DNA, salt ions and water;
Wherein the concentration of the salt ions is 150-1200 mmol/L;
The block copolymer is provided with a polyethylene glycol block and a lateral amino type polyamino acid block, wherein the lateral amino type polyamino acid block is at least one of a polylysine block and a polyornithine block, and 0-45% of lateral groups-NH 2 of the lateral amino type polyamino acid block are replaced by-SH; further, the weight average molecular weight of the polyethylene glycol block may be 5kDa to 20kDa, and the weight average molecular weight of the pendant amino polyamino acid block may be 3.5kDa to 15kDa.
The side chain of the side amino polyamino acid block of the block copolymer contains a large amount of amino groups (-NH 2), and is a cationic polymer, a cationic carrier and further a cationic non-viral carrier. The DNA condensation system provided by the invention can condense DNA molecules into nano particles through the ion composite action of polyethylene glycol-lateral amino polyamino acid block copolymer (PEG-PNAA) and DNA molecules to form a cyclic compound of DNA and polymer, thereby improving the endocytosis efficiency of cells and increasing the circulation time of the DNA in organisms through carrying a polyethylene glycol protective shell. The DNA condensation system and the annular compound can be widely applied to gene therapy. PEG in the chimeric copolymer is uncharged and cannot act with DNA and PLys, so that embedding condensation of a single pDNA molecule is facilitated by virtue of the space shielding effect of PEG, and if the block polymer omits a polyethylene glycol block, a plurality of pDNA molecules exist in a single nanoparticle, so that the aim of uniform condensation is not facilitated. In addition, highly biocompatible PEG can enhance the safety of the condensate, and if the block polymer omits the polyethylene glycol block, the exposed cationic polylysine block (PLys) may have a tendency to hemolyze.
In some embodiments, the pendant amino-type polyamino acid block is a polylysine block.
When the side amino type polyamino acid block is a polylysine block, the DNA condensation system provided by the invention can condense DNA molecules into nano particles through the ion composite action of polyethylene glycol-polylysine block copolymer (PEG-PLys) and DNA molecules to form a cyclic compound of DNA and polymer, so that the endocytosis efficiency of cells is improved, and the circulation time of the DNA in organisms is prolonged through carrying a polyethylene glycol protective shell. The DNA condensation system and the annular compound can be widely applied to gene therapy.
The inventor of the present application also confirms that the block copolymer provided by the present application has good complex condition as a cationic carrier and DNA and can be in a single dispersion system through Dynamic Light Scattering (DLS) test.
Further, gel blocking experiments and heparin competition experiments prove that the cationic vector provided by the invention has good binding capacity and protective capacity on plasmid DNA.
In addition, when the cationic polymer provided by the invention is used for compositing with DNA, the cell uptake efficiency can be obviously improved, and the cationic polymer also has good biocompatibility.
The inventors of the present application, through extensive experimental exploration, have unexpectedly found that the salt ion concentration is critical to whether the DNA condensation system is capable of being oriented into a loop. Experiments prove that the different salt ion concentrations can lead the compound of the DNA and the polymer to present different shapes, and the rod-shaped, annular and fibrous DNA condensate can be prepared respectively by adjusting the salt ion concentration during the compounding, and the shapes can be observed by a transmission electron microscope (SEM). Wherein the rod-shaped condensate is formed by regularly folding double-stranded DNA with rigidity, and the end double strand of the DNA fold is dissociated into single strands; the circular DNA condensation body formed by the DNA condensation system of the application is based on the winding of DNA, and a complete double-chain structure can be reserved in the forming process.
In some embodiments, the salt ions include one or both of sodium ions and potassium ions.
In some embodiments, the salt ion is sodium ion, potassium ion, or a combination thereof.
In some embodiments, the salt ion is sodium ion, and rod-shaped, ring-shaped and fiber-shaped DNA condensate can be prepared respectively by adjusting the concentration of sodium ion during compounding.
In some embodiments, the concentration of the salt ion is 150 to 1200mmol/L (i.e., 150 to 1200 mM), and further may be 500 to 700
The mM may be 550 to 650mM. The concentration of the salt ion may be selected from any one of the following concentrations or from a range of any two of the following concentrations: 150mM, 200mM, 250mM, 300mM, 350mM, 400mM, 450mM, 500mM, 550mM, and the like,
600MM, 650mM, 700mM, 750mM, 800mM, 850mM, 900mM, 950mM, 1000mM, 1100mM, 1200mM, etc., may also be selected from any suitable range of: 450-800 mM, etc.
In some embodiments, the pH of the DNA condensation system is between 7.2 and 7.6. Non-limiting examples are 7.2, 7.4, 7.5, 7.6, etc. The pH of the DNA condensation system can be adjusted by NaOH and HCl. If the pH is too high, basic amino acids cannot be ionized, the structure of DNA may be irreversibly damaged, if the pH is too low, DNA cannot be ionized, and the structure of DNA may be damaged.
The pendant amino groups of PEG-PNAA may or may not be replaced by-SH, herein designated PEG-PNAA (SH), herein designated PEG-PNAA (NH 2). When the pendant amino-based polyamino acid block is a polylysine block, PEG-PNAA is designated as PEG-PLys, PEG-PNAA (SH) is designated as PEG-PLys (SH), and PEG-PNAA (NH 2) is designated as PEG-PLys (NH 2).
In the present invention, the PEG-PNAA may be PEG-PNAA (NH 2) or PEG-PNAA (SH), unless otherwise specified. In the present invention, PEG-PLys may be PEG-PLys (NH 2) or PEG-PLys (SH), unless otherwise specified.
In some embodiments, a portion of the pendant groups-NH 2 of the pendant amino-type polyamino acid block are not substituted with-SH, at which point the block copolymer is designated PEG-PNAA (NH 2). In some embodiments, the pendant amino-type polyamino acid block is a polylysine block, a portion of the pendant groups-NH 2 of the polylysine block are not substituted with-SH, and the block copolymer is designated PEG-PLys (NH 2).
In some embodiments, a portion of the pendant groups-NH 2 of the pendant amino-type polyamino acid block are substituted with-SH, when the block copolymer is denoted PEG-PNAA (SH). In some embodiments, the pendant amino-type polyamino acid block is a polylysine block, a portion of the pendant groups-NH 2 of the polylysine block are replaced with-SH, and the block copolymer is referred to as PEG-PLys (SH).
By substituting a part of side group-NH 2 of the side amino type polyamino acid block with-SH, the obtained sulfhydryl modified polyethylene glycol-side amino type polyamino acid block copolymer can be named as PEG-PNAA (SH), and the block copolymer can crosslink the-SH in the formed nano-composite to form disulfide bonds, so that the extracellular stability of the nano-composite can be improved, the disulfide bonds are reduced in response to intracellular glutathione when the nano-composite enters cells, the dissociation of the nano-composite in the cells is promoted, the release of DNA in the cells is promoted, the stable existence in blood circulation is realized, and the DNA can be released rapidly when the nano-composite reaches the target cells.
When the side amino type polyamino acid block is a polylysine block, by replacing a part of side groups-NH 2 of the polylysine block with-SH, the obtained sulfhydryl modified polyethylene glycol-polylysine block copolymer can be named as PEG-PLys (SH), and the block copolymer can crosslink the-SH in the formed nano-composite to form disulfide bonds, so that the extracellular stability of the nano-composite can be improved, the disulfide bonds are reduced in response to intracellular glutathione when the nano-composite enters cells, the dissociation of the nano-composite in the cells is promoted, the release of DNA in the cells is promoted, the stable existence in blood circulation is realized, and the DNA can be released rapidly when the nano-composite reaches target cells.
Further, the use of PEG-PNAA (NH 2) and/or PEG-PNAA (SH)) and pDNA (plasmid DNA) to condense into a cyclic complex in a directed manner can significantly increase the transfection efficiency of non-viral vector carrying DNA.
When the side amino type polyamino acid block is a polylysine block, PEG-PLys (PEG-PLys (NH 2) and/or PEG-PLys (SH)) and pDNA (plasmid DNA) are directionally condensed into a circular compound, so that the transfection efficiency of the non-viral vector carrying DNA can be remarkably improved.
When a part of the side group-NH 2 of the side amino-group polyamino acid block is substituted with-SH, the substitution rate (denoted as R S or SH substitution rate) of the side group-NH 2 of the side amino-group polyamino acid block by-SH is not liable to be excessively large, otherwise, the disulfide bond crosslinking degree may be excessively high, and it is difficult to release pDNA in cells.
In some embodiments, the SH substitution rate is equal to or less than 45% (i.e., R S is 0 to 45%), and further, the SH substitution rate may be selected from any one of the following percentages or from a range ：0、1％、2％、3％、4％、5％、6％、7％、8％、9％、10％、11％、12％、14％、15％、16％、8％、20％、22％、24％、25％、26％、28％、30％、32％、33％、34％、35％、36％、38％、40％、42％、44％、45％ constituted by any two of the following percentages, and may be selected from any one of the following ranges ：5％～45％、8％～45％、10％～45％、15％～45％、20％～45％、30％～45％、35％～45％、0～42％、5％～42％、8％～42％、10％～42％、15％～42％、20％～42％、30％～42％、35％～42％、35％～42％、35％～40％, and the like. In some embodiments, R S is 0 or 8% to 42%. In some embodiments, R S is 0 or 32% to 42%.
The inventor of the present application verifies that disulfide bonds of a block copolymer PEG-PNAA (SH), which may be PEG-PLys (SH), are dissociated in a reducing environment existing in cells through in vitro level experiments on DNA condensate, so that DNA can be released; the DNA condensate crosslinked using disulfide bonds exhibits excellent nuclease degradation resistance and has good stability. In addition, when the cationic polymer PEG-PNAA (SH) and DNA are used for compounding, the cell uptake efficiency is obviously improved, and good biocompatibility is shown. In addition, the transfection effect analysis of the DNA condensate in different forms also shows that the cyclic condensate crosslinked by disulfide bonds has better transfection effect than that of the condensate which is not substituted by-SH, and the transfection effect of PEG-PNAA (SH) with proper SH substitution rate is better than that of PEG-PNAA (NH 2) (which can be PEG-PLys (NH 2)).
In some embodiments, the polyethylene glycol block and the pendant amino-type polyamino acid block each have a suitable molecular weight. The weight average molecular weight of the polyethylene glycol block can be 5kDa to 20kDa, and the weight average molecular weight of the side amino type polyamino acid block can be 3.5kDa to 15kDa. The molecular weight of the polyethylene glycol block and the lateral amino type polyamino acid block has a certain influence on the regulation of the shape of the DNA condensate. The inventors of the present application have also found that the shape of the condensate has a certain influence on the gene transfection efficiency, and therefore, the efficient expression of the non-viral vector DNA in an organism can be achieved by condensing the DNA into a circular DNA condensate by directional condensation as a way of condensing the DNA in a pseudo-viral body.
In some embodiments, the weight average molecular weight of the polyethylene glycol block may be from 5kDa to 20kDa, and may also be selected from any one of the following molecular weights or from the interval consisting of any two of the following molecular weights: 5kDa, 6kDa, 7kDa, 8kDa, 9kDa, 10kDa, 12kDa, 14kDa, 15kDa, 16kDa, 18kDa, 20kDa, etc., and may be selected from any of the following ranges: 5kDa to 15kDa, 5kDa to 12kDa, 6kDa to 20kDa, 6kDa to 15kDa, 6kDa to 12kDa, 8kDa to 20kDa, 8kDa to 15kDa, 8kDa to 12kDa, etc.
In some embodiments, the weight average molecular weight of the pendant amino-type polyamino acid block (which may be a polylysine block) may be 3.5kDa to 15kDa, and may also be selected from any one of the following molecular weights or from the interval consisting of any two of the following molecular weights: 3.5kDa, 4kDa, 4.5kDa, 5kDa, 5.5kDa, 6kDa, 6.5kDa, 7kDa, 8kDa, 9kDa, 10kDa, 12kDa, 14kDa, 15kDa, etc., and may be selected from any of the following ranges ：3.5kDa～12kDa、3.5kDa～10kDa、3.5kDa～8kDa、4kDa～15kDa、4kDa～12kDa、4kDa～10kDa、4kDa～8kDa、4kDa～6.5kDa、8kDa～12kDa、8kDa～10kDa, etc.
In some embodiments, the polyethylene glycol block has a weight average molecular weight of 6kDa to 12kDa; the weight average molecular weight of the side amino type polyamino acid block (which can be a polylysine block) is 4kDa to 13kDa.
The polyethylene glycol block and the pendant amino polyamino acid block (which may be a polylysine block) may be covalently linked by any suitable divalent linking group. The polyethylene glycol block may be attached to the N-or C-terminus of the pendant amino-type polyamino acid block. The N-terminal refers to the end containing-NH 2 and the C-terminal refers to the end containing-COOH.
The main chain terminal-NH 2 and the side chain terminal-NH 2 of the side amino type polyamino acid block (which can be polylysine) have different activities and can be protected by different amino protecting groups, so that the polyethylene glycol chain segment can be connected to the N end or the C end of the side amino type polyamino acid block (which can be polylysine block) in a fixed point mode.
The terminal hydroxyl group of polyethylene glycol may be modified to-NH 2, which may be covalently linked to the C-terminus of a pendant amino polyamino acid block (which may be a polylysine block), to form an amide bond-NH-C (=o) -, where-NH-is proximal to one side of the polyethylene glycol block.
The terminal hydroxyl group of polyethylene glycol may also be modified to a carboxyl group-COOH or an activated carboxyl group (e.g., an active ester group, further such as a succinimide active ester group), etc., and then covalently linked to the N-terminus of a pendant amino-type polyamino acid block (which may be a polylysine block), an amide bond-C (=O) -NH-or-O-C (=O) -NH-or the like may be formed, wherein, -NH-is near one side of the pendant amino polyamino acid block.
Non-limiting examples of succinimide reactive ester groups include succinimidocarbonyl (-CO-NHS,) Succinimidyl carbonate group (-OC (=o) -NHS,) Etc.
In some embodiments, the polyethylene glycol block and the pendant amino polyamino acid block (which may be a polylysine block) are linked by-NH-C (=o) -, -C (=o) -NH-, or-O-C (=o) -NH-, wherein x is near one side of the polyethylene glycol block.
In some embodiments, the block copolymer has a structure represented by formula (1):
In the formula (1), q and y are each independently a positive integer; further alternatively, q is an integer selected from 200 to 250, and y is an integer selected from 35 to 50;
r 1 is alkyl;
l 1 is alkylene;
Z 1 is-NH- -O-NH-or-O-;
any one L 2 is independently alkylene;
Either R 2 is independently-NH 2 or R 3; wherein, R 3 has the structure-NH-Z 3-L3 -SH, wherein L 3 is alkylene, Z 3 is-C (=nh) -, -C (=o) -or-C (=o) -NH-, wherein ". X" represents the direction of L 2; in the formula (1), the number of R 3 is n and the ratio of n to y is denoted as n/y, wherein n/y can be more than or equal to 0.
L 2 in the formula (1) may be the same or different.
R 2 in the formula (1) may be the same or different.
In some embodiments, the block copolymer has either or both of the following features:
L 2 are the same;
R 2 are identical.
In some embodiments, L 2 are all the same.
In some embodiments, R 2 are all the same.
Wherein n/y is equal in value to R S described above, i.e., corresponds to the substitution rate of-NH 2 in the side chain of the-SH contralateral amino polyamino acid block (which may be a polylysine block). The value of n/y may be selected from any of the foregoing R S. For example, in some embodiments, the ratio of n to y is 0 to 42%, and further, the ratio of n to y may be 0 or 32% to 42%.
If the lysine residues in the polylysine block are epsilon, e.g., -C (=O) -C (R 2)-CH2CH2CH2CH2 NH-, this may lead to reduced biocompatibility.
Herein, the term "alkyl" refers to a monovalent residue of a saturated hydrocarbon containing a primary (positive) carbon atom, or a secondary carbon atom, or a tertiary carbon atom, or a quaternary carbon atom, or a combination thereof, losing one hydrogen atom. Phrases containing this term, for example, "C 1～9 alkyl" refers to an alkyl group containing from 1 to 9 carbon atoms, which at each occurrence may be, independently of one another, C 1 alkyl, C 2 alkyl, C 3 alkyl, C 4 alkyl, C 5 alkyl, C 6 alkyl, C 7 alkyl, C 8 alkyl or C 9 alkyl. suitable examples include, but are not limited to: methyl (Me, -CH 3), ethyl (Et, -CH 2CH3), 1-propyl (n-Pr, n-propyl, -CH 2CH2CH3), 2-propyl (i-Pr, i-propyl), -CH (CH 3)2), 1-butyl (n-Bu, n-butyl, -CH 2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, i-butyl, -CH 2CH(CH3)2), 2-butyl (s-Bu, s-butyl, -CH (CH 3)CH2CH3), a catalyst for the preparation of a pharmaceutical composition, 2-methyl-2-propyl (t-Bu, t-butyl, -C (CH 3)3), 1-pentyl (n-pentyl, -CH 2CH2CH2CH2CH3), 2-pentyl (-CH (CH 3) CH2CH2CH 3), 3-pentyl (-CH (CH 2CH3)2), a process for preparing the same, 2-methyl-2-butyl (-C (CH 3)2CH2CH3), 3-methyl-2-butyl (-CH (CH 3)CH(CH3)2), 3-methyl-1-butyl (-CH 2CH2CH(CH3)2), 2-methyl-1-butyl (-CH 2CH(CH3)CH2CH3), 1-hexyl (-CH 2CH2CH2CH2CH2CH3), 2-hexyl (-CH (CH 3)CH2CH2CH2CH3), 3-hexyl (-CH (CH 2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C (CH 3)2CH2CH2CH3)), a catalyst, 3-methyl-2-pentyl (-CH (CH 3)CH(CH3)CH2CH3), 4-methyl-2-pentyl (-CH (CH 3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C (CH 3)(CH2CH3)2), 2-methyl-3-pentyl (-CH (CH 2CH3)CH(CH3)2)), a catalyst for the preparation of a pharmaceutical composition, 2, 3-dimethyl-2-butyl (-C (CH 3)2CH(CH3)2), 3-dimethyl-2-butyl (-CH (CH 3)C(CH3)3 and octyl (- (CH 2)7CH3)).
The term "alkylene" as used herein refers to a hydrocarbon group derived by removing one more hydrogen atom on an alkyl basis to form a center having two monovalent radicals, which may be a saturated branched alkyl group or a saturated straight chain alkyl group. For example, "C 1～C9 alkylene" means that the alkyl moiety contains from 1 to 9 carbon atoms and, at each occurrence, can be, independently of one another, C 1 alkylene, C 2 alkylene, C 3 alkylene, C 4 alkylene, C 5 alkylene, C 6 alkylene, C 7 alkylene, C 8 alkylene or C 9 alkylene. Suitable examples include, but are not limited to: methylene (-CH 2 -), 1-ethyl (-CH (CH 3) -), 1, 2-ethyl (-CH 2CH2 -), 1-propyl (-CH (CH 2CH3) -), and, 1, 2-propyl (-CH 2CH(CH3) -), 1, 3-propyl (-CH 2CH2CH2 -) and 1, 4-butyl (-CH 2CH2CH2CH2 -).
In some embodiments, R 1 is alkyl, further can be C 1-3 alkyl, further can be methyl.
In some embodiments, L 1 is alkylene, further may be C 1-6 alkylene, further may be C 1-5 alkylene, further may be C 1-4 alkylene, further may be C 1-3 alkylene, further may be any of C 2-6 alkylene, C 2-5 alkylene, C 2-4 alkylene, and the like. In some embodiments, L 1 is 1, 2-ethylene, 1, 3-propylene, or 1, 4-butylene. In some embodiments, L 1 is 1, 2-ethylene.
In some embodiments of the present invention, in some embodiments, Z 1 is-NH- -O-NH-or-O-. In some embodiments, Z 1 is-NH-or-O-NH-. In some embodiments, Z 1 is-NH-.
In some embodiments, any one L 2 is independently alkylene, further any one L 2 may be independently butylene or propylene. In some embodiments, L 2 is 1, 4-butylene, in which case the repeat units of the pendant amino-type polyamino acid block are lysine residues or lysine residues substituted with R 3. Further, L 2 in the formula (1) may be the same or different. In some embodiments, L 2 in formula (1) are all the same.
In some embodiments, any one R 2 is independently-NH 2 or R 3; wherein, R 3 has the structure-NH-Z 3-L3 -SH, wherein L 3 is alkylene, Z 3 is-C (=nh) -, -C (=o) -or-C (=o) -NH-, wherein "×" denotes the direction of L 2. In some embodiments, Z 3 is-C (=nh) -or-C (=o) -. In some embodiments, Z 3 is-C (=nh) -. Further, R 2 in formula (1) may be the same or different. In some embodiments, R 2 in formula (1) are all the same.
In some embodiments, L 3 is alkylene, further may be C 2-6 alkylene, and may also be any of C 2-5 alkylene, C 2-4 alkylene, and the like. In some embodiments, L 3 is 1, 2-ethylene, 1, 3-propylene, or 1, 4-butylene. L 3 in the formula (1) may be the same or different. In some embodiments, L 3 in formula (1) are all the same. In some embodiments, L 3 is 1, 2-ethylene. In some embodiments, L 3 is 1, 3-propylene.
In some embodiments, in formula (1),
R 1 is C 1-3 alkyl;
L 1 is C 1-6 alkylene;
Z 1 is-NH- -O-NH-or-O-;
Any one L 2 is independently butylene or propylene;
Either R 2 is independently-NH 2 or R 3; wherein, R 3 has the structure-NH-Z 3-L3 -SH, wherein L 3 is C 2-6 alkylene, Z 3 is-C (=nh) -, -C (=o) -or-C (=o) -NH-, wherein ". X" represents the L 2 direction; in the formula (1), the number of R 3 is n, the ratio of n to y is more than or equal to 0, and further can be 0-45%; the SH substitution rate may also be selected from any suitable value or range as previously described.
In some embodiments, in formula (1), q is a positive integer, further may be an integer selected from 200 to 250, further may be an integer selected from 225 to 230. Wherein q may be any one of the following integers or a range selected from any two of the following integers: 200. 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, etc.
In some embodiments, in formula (1), y is a positive integer, and further may be an integer selected from 35 to 50. Wherein y may be any one of the following integers or a range selected from any two of the following integers: 35. 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, etc., may also be selected from any suitable range: 36 to 44, 42 to 44, 40 to 44, etc.
In some embodiments, the DNA condensation system satisfies any one or a combination of any more of the following characteristics:
q is an integer selected from 225 to 230;
y is an integer selected from 36 to 44;
R 1 is methyl;
L 1 is 1, 2-ethylene, 1, 3-propylene or 1, 4-butylene;
z 1 is-NH-;
L 2 is 1, 4-butylene;
Z 3 is-C (=nh) -;
L 3 is 1, 2-ethylene, 1, 3-propylene or 1, 4-butylene.
In some embodiments, the DNA condensation system satisfies either or both of the following characteristics:
L 1 is 1, 2-ethylene;
l 3 is 1, 3-propylene.
In some embodiments, the molar ratio of the positive amino charges in the block copolymer to the negative phosphate charges in the DNA is N/P, wherein N/P is an integer selected from 1 to 16, can be selected from 1 to 2, can be selected from any one of the following ratios or a range formed by any two ratios: 1.0, 1.2, 1.4, 1.5, 1.6, 1.7, 1.8, 2.0, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, etc., may also be selected from any suitable ranges 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, etc. If N/P is too large, an excessive amount of basic amino acids causes cytotoxicity, and if N/P is too small, the negative charge of DNA is not completely neutralized, and the stability of the DNA condensate is also poor.
In some embodiments, the concentration of the DNA is 20 to 150 ng/. Mu.L. The concentration may be selected from any one of the following concentrations, or from a range ：25ng/μL、26ng/μL、28ng/μL、30ng/μL、32ng/μL、35ng/μL、36ng/μL、40ng/μL、45ng/μL、50ng/μL、60ng/μL、70ng/μL、0ng/μL、90ng/μL、100ng/μL、110ng/μL、120ng/μL、130ng/μL、140ng/μL、150ng/μL formed by any two of the following concentrations, and the like, and may be selected from any suitable range as follows: 25-50 ng/. Mu.L, 25-40 ng/. Mu.L, etc.
In some embodiments, the DNA is 3k to 20 kbp (i.e., 3000 to 20000 bp) in length. 1bp represents 1 base pair, and 1 kbp represents 1000 base pairs. In the present invention, any suitable length of DNA may be selected. The length of the DNA may be selected from any one of the following lengths or from a region ：3k bp、4k bp、5k bp、6k bp、7k bp、8k bp、9k bp、10k bp、11k bp、12k bp、13k bp、14k bp、15k bp、16k bp、17k bp、18k bp、19k bp、20k bp composed of any two of the following lengths
In some embodiments, the DNA is plasmid DNA (pDNA, plasmid DNA).
The DNA condensation system provided by the invention can form a ring-shaped DNA condensation body, which is a ring-shaped self-winding condensation body, wherein the double-chain structure of DNA is not damaged, and the DNA condensation system is beneficial to the later-stage DNA transcription. In other shapes of the condensate, the double-stranded structure of the DNA is destroyed to a different extent, and the DNA is unwound into a single strand, and the biological activity may be inferior. For example, the DNA in the formed rod-shaped DNA aggregate is subject to unwinding, whereas the double-stranded structure of the DNA in the formed circular DNA aggregate is not subject to unwinding.
In a second aspect of the present invention, there is provided a cyclic complex formed by compounding a block copolymer and DNA; wherein the block copolymer is as defined in the first aspect of the invention. The cyclic complex is a non-unwinding cyclic complex.
In the present invention, unless otherwise indicated, "no helic" in "no helic cyclic complex" means: in the condensation process of the DNA, the original double-stranded DNA can not be unwound to Cheng Shanlian DNA, and the original double-stranded DNA structure is still maintained.
In some embodiments, the cyclic complex is prepared from the DNA condensation system of the first aspect of the invention.
In some embodiments, at least a portion of the DNA has a complete double-stranded structure. The circular complex formed by the DNA condensation system of the invention is based on the entanglement of the DNA itself, and the complete double-stranded structure is maintained in the forming process.
In some embodiments, the DNA in the circular complex has complete double strand, i.e., no helicity.
In some embodiments, the DNA is plasmid DNA.
In some embodiments, the hydrodynamic diameter of the cyclic complex is 180 to 190nm.
In some embodiments, the cyclic complex contains cross-linked disulfide bonds and may be prepared using PEG-PNAA (SH), such as PEG-PLys (SH).
In a third aspect of the present invention, there is provided a method of preparing a cyclic complex comprising the steps of: mixing DNA and block copolymer in the presence of salt ion in proper concentration and letting stand the mixture; wherein the block copolymer is as defined in the first aspect of the invention.
The species of salt ion may be as defined in the first aspect of the invention.
In some embodiments, a method of preparing a cyclic complex is provided, comprising the steps of: mixing DNA and block copolymer in the presence of 150-1200 mmol/L salt ion, and letting stand the prepared mixed system; wherein the block copolymer is as defined in the first aspect of the invention. In some embodiments, the DNA and the block copolymer are vortexed.
In some embodiments, the vortex mixing time is from 1 to 5 seconds.
In some embodiments, the resting temperature is from 4 to 25 ℃. The rest temperature may be any one of the following temperatures or a range of any two temperatures selected from the group consisting of: 4 ℃,5 ℃,6 ℃,7 ℃,8 ℃,9 ℃,10 ℃,12 ℃,14 ℃,15 ℃, 16 ℃, 18 ℃, 20 ℃, 22 ℃, 24 ℃, 25 ℃, and the like.
In some embodiments, the resting time is from 0.5 to 24 hours. The rest time may be any one of the following or a section selected from any two of the following: 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 7h, 18h, 19h, 20h, 22h, 24h, etc., may also be selected from any suitable range of: 1-24 h, etc.
In some embodiments, after the end of the standing, the method further comprises the steps of: with water dialysis, ultrapure water dialysis may be used.
In some embodiments, the mixed system of the DNA and the block copolymer is the DNA condensation system of the first aspect of the invention.
In some embodiments, the methods of preparation meet one or more of the following characteristics:
the vortex mixing time is 1-5 seconds;
the standing temperature is 4-25 ℃;
standing for 0.5-24 h;
After the standing is finished, the method further comprises the following steps: the water dialysis can be adopted, and the ultrapure water dialysis can be adopted;
the mixed system formed by the DNA and the block copolymer is the DNA condensation system of the first aspect of the invention.
In some embodiments, the pendant amino-type polyamino acid block (which may be polylysine) contains-SH in its side chain structure; further, the block copolymer may be pretreated with a disulfide bond reducing agent prior to mixing the DNA and the block copolymer to form naked-SH.
In some embodiments, the disulfide bond reducing agent is selected from one or more of dithiothreitol, glutathione, and TCEP. Wherein TCEP is a conventional disulfide bond reducing agent: tris (2-carboxyethyl) phosphine.
In some embodiments, when pretreatment of the block copolymer with a disulfide reducing agent is employed, HEPES-NaOH may be added to control the system pH, and further, the system pH may be controlled to any suitable pH or pH range as described above.
In another aspect of the present invention, there is also provided a method for preparing the block copolymer of the first aspect of the present invention.
In some embodiments, PEG-PNAA block copolymers (which may be PEG-PLys block copolymers) are prepared by ring-opening polymerization of carboxyanhydrides using amino-functionalized polyethylene glycols (PEG), and further, PEG-PNAA (SH) (which may be PEG-PLys (SH)) may also be prepared by modification of pendant amino-type polyamino acid blocks (which may be polylysine blocks) with-SH.
In some embodiments, taking the side amino polyamino acid block as an example of a polylysine block, PEG-Plys, including PEG-PLys (NH 2) and PEG-PLys (SH), are prepared by the following synthetic route. In this non-limiting example, for formula (1), the polyethylene glycol polymerization degree (i.e., q) is 228, the number of lysine residues in peg-PLys (NH 2) is n+m=y, where n is a positive integer; in PEG-PLys (SH), the SH substitution rate of polylysine side chains rs=n/y.
In a fourth aspect of the invention, there is provided the use of a DNA condensation system according to the first aspect of the invention, or a cyclic complex according to the second aspect of the invention, or a cyclic complex prepared by a preparation method according to the third aspect of the invention, in the preparation of a gene therapy drug.
Experiments prove that the imitated virus condensate provided by the application has application potential in promoting blood vessel regeneration, and can provide a new idea for non-viral vectors in gene delivery.
In some embodiments, the medicament is for treating at least one of the following diseases: ischemic diseases, tumors and ocular neovascular diseases.
The inventors of the present application have also conducted studies on the ability of DNA condensation systems to promote angiogenesis. By establishing a model of the ischemia of the hind limb of the mouse, carrying out in-situ treatment on the mouse by injecting the annular DNA condensate expressing the vascular endothelial growth factor plasmid (pVEGF) at multiple points, the experimental result shows that the capability of promoting the regeneration of blood vessels of the annular DNA condensate of pVEGF is obvious, the ischemia of the lower limb can be improved, and a certain application potential is shown in the aspect of relieving the treatment of ischemic diseases.
In a fifth aspect of the invention there is provided the use of a block copolymer as a non-viral gene vector or in gene delivery, either for diagnostic or therapeutic purposes or not; the block copolymer is as defined in the first aspect of the invention.
In some embodiments, the application is based on any one of the following systems or substances:
the DNA condensation system according to the first aspect of the present invention;
the cyclic complex of the second aspect of the invention; and
The cyclic compound prepared by the preparation method of the third aspect of the invention.
Examples of such non-diagnostic and therapeutic purposes are nucleic acid transfection.
Some specific examples are provided below.
Embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples, in which specific conditions are not noted, are preferably referred to the guidelines given in the present invention, and may be according to the experimental manual or conventional conditions in the art, the conditions suggested by the manufacturer, or the experimental methods known in the art.
In the specific examples described below, the measurement parameters relating to the raw material components, unless otherwise specified, may have fine deviations within the accuracy of weighing. Temperature and time parameters are involved, allowing acceptable deviations from instrument testing accuracy or operational accuracy.
In the following examples, reference is made to Table 1 for sources of raw materials, which are commercially available unless otherwise indicated. In table 1, CP represents chemical purity.
TABLE 1 sources of raw materials
TABLE 2 Instrument
The length of DNA used in each of the following examples was in the range of 3k to 20 kbp unless otherwise stated.
EXAMPLE 1 Synthesis of cationic Polymer (Block copolymer PEG-PNAA according to the present invention)
Based on ring-opening polymerization of carboxyl ring anhydride, amino functionalized PEG molecule is used as initiator to initiate polymerization of monomer [ Lys (TFA) -NCA ] to prepare PEG-PLys segmented copolymer, wherein the polymerization degree of PLys can be finely controlled by the addition amount of monomer Lys (TFA) -NCA. In addition, -SH is introduced into the cation PLys fragment to achieve crosslinking of the DNA complex to achieve stabilization of the condensed core.
In this example, the method for synthesizing polyethylene glycol polylysine block copolymer (PEG-PLys) comprises the following steps: the block cationic polymers PEG-PLys, including PEG-PLys (NH 2) and PEG-PLys (SH), were prepared by ring-opening polymerization of Lys (TFA) -NCA using omega-NH 2 end groups of alpha-methoxy-omega-aminopolyethylene glycol (weight average molecular weight M w: 10000 Da) as the starting groups, followed by removal of the TFA protecting groups with NaOH.
In this example, the synthetic routes for the cationic polymers PEG-PLys (NH 2) and PEG-PLys (SH) are shown below and are designated synthetic route I.
Specifically, a certain amount of mPEG-NH 2 (100 mg, 10. Mu. Mol) powder was weighed, a small amount of methylene chloride was added to facilitate dissolution, and then 5mL of benzene was added to dissolve. Freezing with liquid nitrogen, and vacuum drying. The dried mPEG-NH 2 was transferred to a glove box in an anhydrous and anaerobic environment, dissolved by adding a small amount of DMF, 60. Mu. Mol of Lys (TFA) -NCA was weighed and dissolved by adding DMF. The Lys (TFA) -NCA solution and the mPEG-NH 2 solution were mixed well. The reaction was carried out at 30℃for 72h. After the reaction was completed, the reaction mixture was precipitated three times with glacial ethyl ether, and the precipitate was placed in a vacuum drying oven and thoroughly dried to remove ethyl ether. The solution was dissolved in methanol using 1N NaOH. Stirred at 30 ℃ overnight to remove the protection of the amine group by the trifluoroacetyl group. After the reaction was completed, the solution was dialyzed in 0.01N hydrochloric acid solution using a dialysis bag having a molecular weight cut-off of 3500Da for 48 hours and then in ultrapure water for 48 hours. Lyophilizing to obtain PEG-PLys powder.
In this case, the synthesis of PEG-PLys (SH) was carried out according to scheme I above. The block copolymer of PEG-PLys is synthesized by ring-opening polymerization with alpha-methoxy omega-aminopolyethylene glycol (mPEG-NH 2, weight average molecular weight M w of 10 kDa) as initiator and Lys (TFA) -NCA monomer as monomer. The TFA residue is hydrolyzed by an alkaline method to obtain a PEG-PLys (NH 2) segmented copolymer, and the results of corresponding functional group nuclear magnetic resonance hydrogen spectrum 1 H NMR and peak position are shown as a) in figure 2, wherein the chemical shift of a characteristic peak of polyethylene glycol-CH 2CH2 O-is about 3.7ppm, and the chemical shift of a characteristic peak of polylysine- (CH 2)3-CH2NH2) is 1.3-1.9ppm. The polymerization degree of lysine was calculated to be about 43 and the yield was 82% from the characteristic peak of lysine. That is, y=43 in the formula (1).
Primary amines in the lysine residue of PEG-PLys (NH 2) readily react with cyclic imine esters via ring opening, forming sulfhydryl groups via amidino linkages, thus giving PEG-PLys (SH). PEG-PLys (NH 2) was used to react with 2-iminothiolane to introduce thiol groups to give PEG-PLys (SH). By nuclear magnetic resonance hydrogen spectroscopy 1 H NMR, it was observed that the reaction of primary amine groups of polylysine was very evident: the epsilon-methylene protons of lysine residues migrate to the low field (e to f) and furthermore a proton signal (g-i) characteristic of the gamma-mercaptopropyl CH 2 unit occurs. The SH substitution per PEG-PLys (SH) (R S =n/y) was determined from the peak intensity ratio of β -, γ -and δ -methylene protons of Lys ((CH 2)3, γ=1.3 to 1.9 ppm) to protons of trimethylene units of mercaptopropyl (HS- (CH 2)3, δ=2.1 ppm to 2.8 ppm) according to 1 HNMR spectrum shown in fig. 2 b).
Example 2 shape control and characterization of DNA complexes
2.1. Preparation of vector/Gene Complex and control of Properties
Entrapment of DNA by cationic polymer carriers is accomplished by complexing with a cationic polymer, which then neutralizes the negative charge in the DNA, and after complexing with the DNA, the DNA undergoes a volumetric transition from an expanded coiled shape to a compact form, a process known as DNA condensation. The complexing process can be controlled by adjusting the complexing ionic strength between the polycation block and the DNA, thereby realizing the directional condensation of the DNA condensate.
Method for analyzing the ability of cationic vector to bind pDNA: the prepared polycation block copolymer (PEG-PLys (SH)) was first dissolved in 10mM HEPES-NaOH (100mmol DTT,pH7.4) solution without NaCl concentration, wherein the concentration of NaCl was 0mmol, 600mmol. Incubate at 25℃for 1h. Thus, disulfide bonds in the carrier polymer are sufficiently reduced to mercapto groups so that the carrier can be sufficiently bonded to the pDNA. After the incubation is completed, the cationic carrier solution and the pDNA solution are mixed at different N/P ratios (molar ratio of amino positive charge in the polycationic carrier to phosphate group negative charge in the pDNA). The specific operation is as follows: the pDNA solution was adjusted to a 50 ng/. Mu.L solution at a concentration using 10mM HEPES-NaOH (NaCl concentration 0mmol, 600 mmol), and then the polymer solution was added to the pDNA solution according to a volume ratio of polymer solution to pDNA solution of 1:2 for different N/P, and the two were rapidly and uniformly mixed (the concentration of pDNA in the final complex solution was 33.3 ng/. Mu.L). Then, the solution was dialyzed with ultrapure water for 12 hours, and the solution was dialyzed 2 times to remove ions. Subsequently, the complex was crosslinked by dialysis against 0.1% (v/v) dimethyl sulfoxide (DMSO) solution, with water change over 12 hours and 3 times. After three times of dialysis with pure water, excess DMSO was removed. Finally, the vector/gene complexes with different shapes are obtained.
In order to verify the binding capacity and the protective capacity of the synthesized polycation gene vector to the gene, the protective capacity of the polycation gene vector to the DNA can be observed by observing the blocking condition of the DNA by using an agarose gel electrophoresis experiment. Heparin sodium is used as a polyanion and has stronger cation binding capacity, so that DNA is competed from a condensed body formed by a polycation carrier and the DNA. Thus, co-incubation of heparin sodium with the circular DNA condensate can verify the stability of the DNA condensate. The vector PEG-PLys (SH) was complexed with pDNA under different N/P (0, 1,2,3,4, 5) conditions and the binding capacity of the vector to DNA was verified by gel blocking experiments.
The results can be seen in fig. 3 a). When N/P is 1, the DNA is totally blocked in the well, indicating that the cationic polymer PEG-PLys (SH) can fully bind to negative charge pDNA under the N/P, and has good binding capacity with pDNA. When N/P is less than 1, negative charges in DNA are not completely neutralized and can still migrate in an electric field; when N/P is 1 or more, negative charges in DNA are completely neutralized and do not migrate in an electric field.
2.2. Stability investigation of vector/Gene complexes
According to the method of the above 2.1. Part preparation of the complex, a PEG-PLys (SH)/pDNA complex having N/P of 1, 2, 3,4, 5, respectively, was prepared. 30. Mu.L each (containing 1. Mu.g of pDNA) was taken and analyzed by agarose gel electrophoresis.
The electrophoresis conditions were: 1.0% agarose gel, 1% of 0.1. Mu.g/ML GELSTAIN was added as a stain to color the DNA. Electrophoresis was performed in 1×TAE electrophoresis buffer. Adding Loading Buffer of one sixth of the materials before electrophoresis, mixing, applying voltage of 100V, electrophoresis time of 30min, and observing and photographing in gel imaging system after electrophoresis
The stability investigation method of the vector/gene complex is as follows: preparation of PEG-Phys (SH)/pDNA complexes with an N/P ratio of 2, 30. Mu.L each (containing 1. Mu.g of pDNA) was added to heparin sodium solutions of different concentrations (final concentrations of 0, 0.1, 0.5, 1.0, 2.0, 5.0mg/mL, respectively) and incubated in an incubator at 37℃for 2h, and agarose gel electrophoresis analysis was performed under the same electrophoresis conditions as above.
To verify the stability of the circular DNA condensate in physiological environment, the stability of the carrier PEG-PLys (SH) binding to DNA was further studied by heparin competition experiments, and the results can be seen in fig. 3 b. Even under the maximum concentration of heparin sodium (5 mg/mL), the release of DNA can be ignored, which proves that the condensate formed by PEG-PLys (SH) and pDNA has extremely high stability, can resist the exchange of complex polyanion in organisms, can prolong the blood circulation time in the bodies, can enhance the long-acting circulation capacity and tissue accumulation capacity of nano particles in the bodies, and can lay a good foundation for the long-acting expression of DNA in specific parts.
2.3. Water and particle size testing and Transmission Electron Microscopy (TEM)
Water and particle size testing: after preparing a DNA aggregate according to the above method, water and particle size (size) of the complex were measured using Zetasizer Nano ZS nm particle sizer.
TEM test: the morphology of the nanocomposite was observed by transmission electron microscopy at an accelerating voltage of 125 KV. And (3) cleaning the TEM carbon film copper net for 10s by using an ion sputtering instrument, uniformly mixing Uranium Acetate (UA) (2%w/v) staining solution and the compound solution according to the volume ratio of 1:1, immersing the copper net in the mixed solution for 30s, taking out the copper net, sucking the excessive compound solution by using filter paper, and naturally airing and testing.
The test results can be seen in fig. 4 a) and b).
PDNA@PEG-PLys (SH) complexed in sodium chloride (0 mM) exhibited a uniform rod-like morphology, termed rod-like condensate, as in FIG. 4 a); the pDNA@PEG-PLys (SH) complexed in sodium chloride (600 mM) takes on a cyclic morphology, called cyclic condensate, as shown in FIG. 4 b). The rod-shaped condensate is formed by regularly folding double-stranded DNA having rigidity, and is mainly characterized in that the end double strand of the folded DNA is dissociated into single strands, and the circular DNA condensate is formed by winding the DNA itself, so that the whole double-stranded structure is maintained during the formation.
The result of cleavage of single-stranded DNA by S1 nuclease shows that the circular condensate shows a complete DNA structure after incubation with S1 nuclease, and that the DNA is not unwound. In addition, it was also observed that the rod-shaped DNA condensate showed a fragmented structure of DNA after incubation with S1 nuclease. The DNA fragments are generated because double-stranded DNA is unwound into single-stranded DNA at the end after being regularly folded in the process of forming a rod-shaped condensate. Therefore, it is considered that the double-stranded structure of the DNA is not destroyed after the circular self-entangled condensate is formed by the pDNA, which is advantageous for the transcription of the DNA in the later stage. In addition, dynamic light scattering intensity (DLS) measurements were consistent with transmission electron microscopy measurements, with hydrodynamic diameters of rod-like and ring-like aggregates of approximately 177nm and 186nm, respectively.
In vitro performance detection of DNA condensation bodies
3.1. Response of circular DNA condensate complex to reducing environment
First, circular DNA condensates of pDNA@PEG-PLys (NH 2) and pDNA@PEG-PLys (SH) with N/P of 2 were prepared according to the method of 2.1 in example 2, incubated with heparin sodium at different concentrations, and then subjected to agarose gel electrophoresis.
The reduction environment in the cell is simulated by 10mmol of Glutathione (GSH), disulfide bonds in the annular DNA condensate of pDNA@PEG-PLys (SH) are cross-linked, the annular DNA condensate is reduced into mercaptan, heparin sodium with different concentrations is added for incubation, and agarose gel electrophoresis is carried out.
In addition to the need for stable presence outside the cell, the DNA condensate should also be susceptible to dissociation within the cell to release DNA for subsequent transcription and translation processes. Since a large amount of reduced glutathione exists in the cell (the concentration is 10-100 mM), and the concentration of extracellular glutathione is only 10-100 mu M, the formed DNA condensate can be kept stable in the extracellular environment by using a disulfide bond crosslinking strategy, and the disulfide bond is reduced to mercaptan in the specific reduction environment in the cell, so that the condensate loses covalent crosslinking effect, and the DNA condensate is easy to exchange with polyanions existing in the cell, thereby releasing DNA.
The results are shown in FIG. 5.
In FIG. 5 a), in the absence of cross-linking of disulfide bonds, DNA condensate has been released at heparin sodium concentrations of 0.5 mg/ml; in FIG. 5 b), the amount of DNA released is still small even if heparin sodium concentration is as high as 5.0mg/ml in the presence of disulfide bonds; in FIG. 5 c), DNA release was initiated at 0.5mg/mL heparin sodium after GSH (10 mM) treatment. Therefore, the disulfide-bond crosslinked pDNA condensate provided by the invention can have good stability outside cells, has reduction response capability in cells, and is easy to dissociate and release pDNA, so that transfection of the DNA condensate in the cells can be promoted.
DNA Complex resistance experiments against nucleases
The DNA-containing sample was mixed with DNase I reaction solution (DNase I:0.01U, magnesium chloride: 25mM, tris-HCl buffer: 10mM pH 7.4,pDNA:33.3ng/. Mu.L) containing the desired components, incubated at 37℃and after a predetermined reaction time, the enzyme reaction was stopped by adding saturated EDTA solution, and then the plasmid DNA payload was released from the self-assembly by adding dithiothreitol (DTT: 100 mM) and saturated anionic dextran sulfate. After overnight incubation, the reaction was collected for qRT-PCR detection to quantify pDNA.
The inventors of the present application considered that nuclease degradation in vivo was the most important cause of failure in gene delivery, and in order to enhance the stability of DNA condensate, to prevent premature degradation of DNA condensed in the inner core by nucleases, the present application used a disulfide-bond crosslinking strategy. The cyclic DNA condensate formed by PEG-PLys (NH 2) or PEG-PLys (SH) and DNA is respectively placed in a simulated physiological microenvironment (heparin sodium: 1.0mg/ml, DNase I: 0.01U) for incubation, and the DNA treated by different incubation times is quantified by qRT-CPR.
The results are shown in FIG. 6 with the ordinate REMAINING INTACT DNA, i.e. the percentage of intact DNA remaining.
After 30min DNase I treatment, the naked DNA was rapidly degraded, while the pDNA condensate formed for PEG-PLys (NH 2) still had a DNA content of 24% after 30min DNase I treatment;
In addition, the disulfide-crosslinked pDNA condensate can remarkably prolong the tolerance time of DNA to DNase I, and DNA can still be detected after the DNA condensate is incubated with DNase I for 120min, such as a pDNA@PEG-PLys (SH) experimental group.
These results indicate that the condensation of pDNA with PEG-PLys (SH) can significantly enhance the protection against DNA.
3.3. Cytotoxicity test of vector and Complex
Cytotoxicity of the PEG-PLys (SH)/pDNA complex was detected by MTT method. Cells cultured in culture flasks (used when the cells grew to 90%) were used. Cells were digested and the pellet of cells was collected by centrifugation. Adding culture medium containing serum and double antibody, and suspending again to obtain cell suspension. Cell counts were then performed using a cell counter to adjust the cell concentration to 1X 10 5 cells/ml and 100. Mu.1 cell suspension was added to each well in a 96-well plate. Placing into incubator, 37 deg.C, 5% CO 2, culturing for 24 hr, and adding medicine. 5 duplicate wells were set for each drug concentration. After 24h incubation with drug, 100. Mu.L of MTT solution (0.5 mg/mL after dilution with DMEM medium) was added to each well. Incubation was continued for 4h at 37℃and the supernatant was carefully removed from the wells. 100 mu L of DMSO is added into each hole, and the mixture is sufficiently vibrated to sufficiently dissolve the purple formazan. The absorbance at 570nm was measured using a microplate reader and the reference wavelength was 630nm.
The formula for cell viability is:
Cell viability (%) = (OD experimental hole -OD Blank hole for experiment )/(OD Control wells -OD Control blank wells ) ×100%
Toxicity of cationic polymer gene delivery systems to cells is an important parameter in evaluating the safety of gene delivery systems. Since positively charged cations can interact with negatively charged cell surface proteins and proteoglycans, cell membrane destabilization and cell necrosis are induced. Thus, this section uses thiazole blue colorimetry (MTT) to detect the activity of cells. The principle is as follows: mitochondria of living cells can produce succinate dehydrogenase, can reduce MTT to purple crystalline formazan, and deposit in cells. Therefore, the relative survival rate of the cells can be judged according to the content of the generated formazan, so that the toxicity of the drug to the cells can be judged. Generally, as N/P increases, the cations of the gene delivery system increase, which tends to disrupt the cell membrane and cause cell death.
The results can be seen in fig. 7.
The cell viability was examined using HUVECs cells, and the results showed that with increasing N/P, the toxicity of the DNA condensate to the cells did not change significantly, and even with a maximum N/P of 6, the relative activity of the cells was still above 80%, and experimental results showed that the pDNA condensate prepared using PEG-PLys (SH) was able to shield excessive cations of polylysine due to the presence of the PEG outer layer, which served as a protective shell, and the condensate remained with good biosafety while maintaining a higher N/P (reflecting stable DNA binding ability).
3.4. Cell uptake assay
Before performing an uptake experiment of cells, the labeled DNA was first specified according to the product in the Cy5 nucleic acid labeling kit, and then the labeled DNA was mixed with unlabeled DNA (w/w=1:10) and a DNA complex with N/P of 2 was prepared. HUVECs cells were inoculated at a concentration of 1X 10 5/mL into a confocal laser culture dish, incubated at 37℃under 5% CO 2 for 24 hours, the medium was discarded, and fresh medium (2 mL) containing 120. Mu.L of vector-DNA complex (4. Mu.g DNA) was added. Culturing in incubator at 37deg.C under 5% CO 2 for 4 hr, and observing the intake under confocal laser microscope.
The groups are respectively as follows:
pDNA: only DNA was added, no block polymer was added;
ss (-). Using PEG-PLys (NH 2), cross-linking without disulfide bonds;
ss (+): using PEG-PLys (SH), cross-linked with disulfide bonds;
rod like condensates: a rod-shaped condensate;
toroidalcondensates: the DNA ring condensate formed by self-winding.
HUVECs cells were used to assess the cellular uptake efficiency of circular DNA condensate.
The results can be seen in fig. 8.
After the PEG-PLys (SH) condenses the pDNA, the uptake efficiency of the cells to the pDNA can be obviously enhanced.
In addition, disulfide-crosslinked pDNA condensate significantly enhanced the uptake capacity of cells, about 8-fold enhancement in uptake efficiency, compared to disulfide-free crosslinked pDNA condensate (either rod-like or cyclic pDNA condensate). It is presumed that the disulfide crosslinking increases the structural stability of the pDNA condensate, thereby increasing the cell uptake capacity of the pDNA condensate.
Furthermore, it was found that the uptake efficiency of the rod-shaped DNA aggregate and the circular DNA aggregate was the same. Meanwhile, the distribution of circular DNA condensate in cells was also observed using a laser confocal microscope (b) in fig. 8), while barely distributed pDNA in cells.
3.5. In vitro transfection ability analysis of differently shaped DNA complexes
Circular and rod-shaped DNA condensates of pDNA@PEG-PLys (NH 2) and pDNA@PEG-PLys (SH) were prepared using pCAG-luc luciferase plasmid. HUVECs cells were seeded at a concentration of 1X 10 5 cells/mL in 96-well plates, 100. Mu.L of complete medium was added to each well, and incubated for 24 hours. The medium in the wells was discarded, washed two to three times with sterile PBS, then DMEM medium without serum and diabody was added, incubated at 37 ℃ for 4 hours at 5% CO 2, then DNA complexes were prepared according to the procedure of 3.1. In example 3, DNA condensate containing 1 μg of pCAG-luc plasmid was added to the corresponding wells, 5 multiplex wells were set per group, and then incubated at 37 ℃ for 5-8 hours at 5% CO 2. The DMEM medium without serum and antibiotics was changed to complete medium and the culture was continued for 48 hours at 37 ℃ with 5% co 2. 100. Mu.L (150. Mu.g/mL) of luciferase substrate was added to each well, and after culturing in a cell culture incubator for 30min, chemiluminescent quantification of luciferase expression was performed using a microplate reader.
Human venous endothelial cells (HUVECs) cells were used to assess the cell transfection efficiency of circular DNA condensates.
The gene expression efficiency of the pDNA condensate was quantified using pDNA encoding a luciferase reporter. The experimental results confirm that the gene expression efficiency for naked pDNA is negligible, while the transfection efficiency of DNA condensate without disulfide cross-linking is also low. However, the transfection efficiency was significantly improved for pDNA condensate with disulfide cross-linking. In addition, it was also observed that the gene expression level of the circular pDNA condensate was significantly higher than that of the rod-shaped condensate, about 4.8-fold.
See fig. 9.
Since the cell uptake efficiency of the cyclic condensate and the rod-like condensate are not very different, the excellent gene expression efficiency of the cyclic condensate may be attributed to the fact that the cyclic condensate can promote transcription activity. Since the transcription process is performed along the DNA, it can be reasonably assumed that the process is continued along the circular structure of the DNA. It is also possible that the transcription process is performed along the direction of the shaft in the rod-like structure, but the end integrity of the DNA double-stranded structure of the rod-like condensate is damaged, so that the transcription process is disturbed when it proceeds to the end of the rod. Therefore, the DNA circular condensate (toroidal condensates) formed by self-winding has higher integrity of DNA based on the influence of transcription process, thereby having higher gene expression efficiency than the rod-shaped condensate (rod-like condensates).
EXAMPLE 4 analysis of the promotion of lower limb revascularization by circular DNA condensate
VEGF gene cyclic virosomal pro-angiogenic experiment
The established hind limb ischemia model mice are randomly divided into 3 groups, namely, group A, group B, group C and 4 mice in each group, wherein the group A carries out injection treatment of pVEGF@PEG-PLys (SH) circular imitated virus complex (100 μl, DNA concentration is 100 ng/. Mu.l, N/P value is 2), the group B carries out pVEGF injection (100 μl, DNA concentration is 100 ng/. Mu.l), and the group C carries out normal saline injection (100 μl), and the injection modes are local muscle multipoint injection of the affected limb. Then, on the 14 th day, 21 th day and 28 th day of treatment, subcutaneous tissues of hind limbs of the mice are observed, and the conditions of angiogenesis are observed, and meanwhile, calf muscles of hind limbs of the mice on the 28 th day are taken for immunofluorescence staining, so that secretion of VEGF and CD 31 are observed.
Condition of murine hindlimb pro-angiogenesis:
Analysis of the circular DNA condensate for promoting revascularization of lower limbs of mice, a multipoint injection method was adopted for the gastrocnemius tissue part of the lower calf part at the femoral artery ligation part of the mice. The hind limb ischemia mice model is divided into 3 groups, wherein, group 1, the control group, 100 μl physiological saline is injected, and the marker is saline; group 2, negative control, 100. Mu.l empty plasmid (100 ng/. Mu.l) was injected, labeled pVEGF; group 3, VEGF treated (positive control), injected with 100. Mu.L of circular DNA condensate (plasmid concentration 100 ng/. Mu.L), labeled toroidal condensates; on the 14 th day, 21 st day and 28 th day of treatment, the hindlimb subcutaneous tissues of the mice are separated, and the hindlimb vascular proliferation condition of the mice is observed.
The results can be seen in fig. 10.
From the experimental results, it can be seen that the proliferation of blood vessels of the circular DNA condensate injected at the 14 th day of treatment is obvious, and the blood vessels of the mice injected with the physiological saline group are not proliferated. At day 21, the normal saline group, the blood vessels did not proliferate, and the blood vessels were gradually absorbed due to the blood vessel ligation, and at day 28, no identifiable minute blood vessels were substantially seen, whereas the pVEGF group and the circular DNA condensate both generated distinct minute blood vessels.
4.2. Immunofluorescence staining result of mice hindlimb gastrocnemius
For immunofluorescent staining, paraffin sections of muscle tissue of the ischemic limb of the mice were stained with antibodies specifically targeting VEGF-Sub>A and CD31 (in immunohistochemistry, CD31 is used primarily to demonstrate the presence of endothelial cell tissue, assess angiogenesis) in order to recognize expressed VEGF and new blood vessels.
The testing method comprises the following steps: after dewaxing of the prepared paraffin sections, incubation was performed with VEGF-Sub>A and CD31 primary antibodies, followed by Sub>A secondary antibody reaction labeled with Cy3 (primary antibody to VEGF-Sub>A) and FITC (primary antibody to CD 31). Sections were observed under a fluorescence microscope and images were acquired. (DAPI ultraviolet excitation wavelength 330-380nm, emission wavelength 420nm, blue light emission, FITC excitation wavelength 465-495nm, emission wavelength 515-555nm, green light emission, cy3 excitation wavelength 510-560nm, emission wavelength 590nm, red light emission).
To further verify the pro-angiogenic status and VEGF expression, immunofluorescent staining was used to observe the amount of vascular VEGF expression in tissue cells, as well as the pro-angiogenic status. On day 28 of treatment, gastrocnemius muscle extracts of mice were fixed with paraformaldehyde buffer and then subjected to paraffin fluorescence immunohistochemical staining.
The results can be seen in fig. 11.
Expression of VEGF and CD31 was hardly observed for sections of the saline-injected group. The expression level of the simultaneously injected pVEGF groups was confirmed to be very low. In contrast, VEGF and CD31 immunofluorescent staining of the injected circular DNA condensate group can be clearly observed. At the same time, the produced VEGF and CD31 were quantitatively analyzed.
The results can be seen in fig. 12. The results of FIG. 12 are consistent with the immunofluorescence staining results, and the VEGF expression level is improved by about 3 times after the annular pVEGF condensate is injected, and the vascular density is improved by about 3 times, so that the annular DNA condensate prepared by the invention has stronger capability of inducing vascular regeneration.
4.3. Relieving the necrotic condition of the hind limb of the mouse
See fig. 13 for ischemic necrosis of the hind limb of the mice observed after the twenty-eighth day of treatment.
As a result, it was found that for pVEGF groups, the hind limbs of the mice had significant necrosis. In the ring-shaped pVEGF condensed body group, the hind limb had no necrotic condition, which was the same as the limb condition without ischemia. Compared with the angiogenesis status of mice, the necrosis status of pVEGF groups is mainly due to the fact that the blood vessels of the hind limbs are less generated, so that the oxygen and nutrient supply of the ischemic hind limbs is insufficient. In contrast, the group treated by injecting the ring-shaped pVEGF condensate had more angiogenesis in the hindlimb and had more perfect collateral circulation, so that there was almost no necrosis of the limb due to ischemia.
Example 5 investigation of the Effect of salt ion concentration
5.1. Different sodium ion concentrations
The experimental method comprises the following steps: the method of example 2 examined the shape control and properties of the DNA complex at different sodium ion concentrations.
The results can be seen in fig. 14 and table 3.
The results show that adjusting the salt concentration can regulate the DNA aggregation form, and the cyclic DNA aggregation appears at the concentration of 500-1200 mM. Further, in this example, the proportion of the cyclic coacervate formed was highest at a salt concentration of 600 mM.
Table 3 shows the number ratio of DNA condensation bodies of different condensation forms at different sodium ion concentrations.
Table 3.
| NaCl(mM) | Annular (%) | Stick shape (%) | Other (%) |
| 0 | 1.1 | 95.3 | 3.6 |
| 150 | 33.4 | 62.3 | 4.3 |
| 300 | 53.4 | 41.9 | 4.7 |
| 450 | 63.8 | 35.4 | 0.8 |
| 600 | 88.4 | 9.5 | 2.1 |
| 700 | 80.1 | 16.7 | 3.2 |
| 800 | 72.4 | 24.2 | 3.4 |
| 900 | 60.3 | 37.9 | 1.8 |
| 1000 | 41.2 | 56.9 | 1.9 |
| 1200 | 32.3 | 64.2 | 3.5 |
| 1500 | 28.3 | 68.3 | 3.4 |
5.2. Cell-free transcriptional level testing
The expression efficiency of cell-free genes was assessed using a TNT rapid transcription/translation coupled system (Promega co., USA) using a luciferase T7 control DNA encoding a luciferase gene. The resulting mixed solution was mixed with a solution from a cell-free system (TnT T7 Quick Master Mix) by mixing naked pDNA and polymer micelles to give a mixed solution (each sample contains 1.33 μg of pDNA), and the mixture was incubated at 37 ℃ for 90 minutes according to the specification. After addition of Luciferase substrate (Luciferase ASSAY REAGENT, promega co., USA), luciferase expression was assessed using a Mithras LB 940 photometer (Berthold Technologies, germany).
The results are shown in FIG. 15.
The regulation of salt concentration can regulate the DNA aggregation form, and when the salt concentration is 600mM, the transcription level of the formed pDNA polymer micelle is highest.
5.3. Cell transfection Activity assay
Polymer micelles prepared at different salt concentrations of pDNA (plasmid DNA encoded by EGFP (enhanced green fluorescent protein)) were injected into the cytoplasm of HUVECs cells using a micromanipulator (Micromanipulator NI 2) and a microinjector (Microinjector FemtoJet (Eppendorf, germany)). The conditions set at the time of injection were P i＝100hPa,Pc = 30hPa, time 0.1s. The total volume injected into the cytoplasm was estimated by co-injecting Texas Red-labeled dextran (Texas Red-labeled dextran) mw=70,000 (a charge neutral fluorescent dye) with a polymer micelle solution through a glass microtube. After injection, cells were incubated at 37℃for 24 hours under 5% CO 2, and fluorescence intensity of EGFP was measured by fluorescence microscopy. The expression level of the green fluorescent protein gene was calculated from the following formula:
Wherein, I green is the fluorescence intensity of EGFP, I back,green is the fluorescence intensity of background under the detection wavelength, I red is the fluorescence intensity of Texas Red-labeled dextran (Texas Red-labeled dextran), I back,red is the exposure time of EGFP and Texas Red respectively for the fluorescence intensity t GFP and t Texas-red of background under the detection wavelength. The values calculated from the above formula give the gene expression activity of the pDNA polymer micelle under different conditions.
The results can be seen in fig. 16.
The adjustment of the salt concentration can regulate the DNA condensation state, and the gene expression activity level of the formed pDNA polymer micelle is strongest when the salt concentration is 600 mM.
The technical features of the above embodiments and examples may be combined in any suitable manner, and for brevity of description, all of the possible combinations of the technical features of the above embodiments and examples are not described, however, as long as there is no contradiction between the combinations of the technical features, they should be considered to be within the scope described in the present specification.
The above examples merely illustrate several embodiments of the present invention, which facilitate specific and detailed understanding of the technical solutions of the present invention, but should not be construed as limiting the scope of protection of the present invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Further, it is understood that various changes and modifications of the present invention may be made by those skilled in the art after reading the above teachings, and equivalents thereof are intended to fall within the scope of the present invention. It should also be understood that, based on the technical solutions provided by the present invention, those skilled in the art obtain technical solutions through logical analysis, reasoning or limited experiments, all of which are within the scope of protection of the appended claims. The scope of the patent is therefore intended to be covered by the appended claims, and the description and drawings may be interpreted as illustrative of the contents of the claims.
Claims (21)
1. A DNA condensation system, characterized by comprising the following components: block copolymers, DNA, sodium chloride and water; the DNA is plasmid DNA, and at least one part of the DNA has a complete double-stranded structure; sodium chloride provides salt ions, wherein the salt ions are sodium ions;
Wherein the concentration of the salt ions is 150-1200 mmol/L;
The block copolymer is provided with a polyethylene glycol block and a lateral amino type polyamino acid block, wherein the lateral amino type polyamino acid block is at least one of a polylysine block and a polyornithine block, and 8% -45% of lateral groups-NH 2 of the lateral amino type polyamino acid block are replaced by-SH; wherein the weight average molecular weight of the polyethylene glycol block is 5 kDa-20 kDa, and the weight average molecular weight of the side amino type polyamino acid block is 3.5 kDa-15 kDa;
the DNA condensation system can form a cyclic compound formed by compounding the block polymer and the DNA;
The molar ratio of the positive charges of the amino groups in the block copolymer to the negative charges of the phosphate groups in the DNA is recorded as N/P, and the N/P is 1-16;
The pH of the DNA condensation system is 7.2-7.6.
2. The DNA condensation system of claim 1, wherein the block copolymer has a structure represented by formula (1):
In the formula (1), q is an integer selected from 200 to 250; y is an integer selected from 35 to 50;
R 1 is C 1-3 alkyl;
L 1 is C 1-6 alkylene;
Z 1 is-NH- -O-NH-or-O-;
Any one L 2 is independently butylene or propylene;
Either R 2 is independently-NH 2 or R 3; wherein, R 3 has the structure-NH-Z 3-L3 -SH, wherein L 3 is C 2-6 alkylene, Z 3 is-C (=nh) -, -C (=o) -or-C (=o) -NH-, wherein ". X" represents the L 2 direction; in the formula (1), the number of R 3 is n and the ratio of n to y is 8-45%.
3. The DNA condensation system according to claim 1, wherein the concentration of the salt ion is 500 to 700mmol/L.
4. The DNA condensation system according to claim 3, wherein the concentration of the salt ion is 550 to 650mmol/L.
5. The DNA condensation system according to claim 1, wherein the concentration of the DNA is 20 to 150 ng/. Mu.L.
6. The DNA condensation system according to claim 5, wherein any one or more of the following features are satisfied:
the N/P is 1-2;
The concentration of the DNA is 25-50 ng/. Mu.L.
7. The DNA condensation system according to claim 1, wherein any one or more of the following features are satisfied:
The length of the DNA is 3 k-20 k bp;
The lateral amino type polyamino acid block is a polylysine block;
the weight average molecular weight of the polyethylene glycol block is 6 kDa-12 kDa;
The weight average molecular weight of the side amino type polyamino acid block is 4 kDa-13 kDa;
30-45% of the side groups-NH 2 of the side amino polyamino acid block are replaced by-SH.
8. The DNA condensation system of claim 2, wherein the ratio of n to y is between 35% and 40%.
9. The DNA condensation system of claim 2, wherein the ratio of n to y is 8% to 42%.
10. The DNA condensation system according to any one of claims 2, 8 to 9, characterized in that any one or a combination of any plurality of the following features is satisfied:
L 2 are the same;
R 2 are the same;
q is an integer selected from 225 to 230;
y is an integer selected from 36 to 44;
R 1 is methyl;
L 1 is 1, 2-ethylene, 1, 3-propylene or 1, 4-butylene;
z 1 is-NH-;
L 2 is 1, 4-butylene;
Z 3 is-C (=nh) -;
L 3 is 1, 2-ethylene, 1, 3-propylene or 1, 4-butylene.
11. The DNA condensation system of claim 10, wherein either or both of the following characteristics are satisfied:
L 1 is 1, 2-ethylene;
l 3 is 1, 3-propylene.
12. A non-unwinding cyclic compound is characterized by being formed by compounding a block copolymer and DNA; wherein the DNA is plasmid DNA; the block copolymer being as defined in any one of claims 1 to 11; the molar ratio of the positive charges of the amino groups in the block copolymer to the negative charges of the phosphate groups in the DNA is recorded as N/P, and the N/P is 1-16; the non-unwinding cyclic compound contains cross-linked disulfide bonds;
the non-unwinding cyclic compound is prepared from the DNA condensation system according to any one of claims 1 to 11.
13. The non-helic cyclic complex according to claim 12, wherein the non-helic cyclic complex has a hydrodynamic diameter of 180 to 190nm.
14. The helicless circular complex according to claim 12, wherein the DNA in the helicless circular complex has a complete double strand.
15. A method for preparing a non-unwinding cyclic compound, comprising the steps of: vortex mixing DNA and block copolymer in the presence of 150-1200 mmol/L salt ion, and letting stand the prepared mixed system; wherein the block copolymer is as defined in any one of claims 1 to 11, the salt ion is a sodium ion, the sodium ion being derived from sodium chloride; the DNA is plasmid DNA;
The molar ratio of the positive charges of the amino groups in the block copolymer to the negative charges of the phosphate groups in the DNA is recorded as N/P, and the N/P is 1-16;
The pH of the DNA condensation system is 7.2-7.6.
16. The production method according to claim 15, wherein any one or any one of the following characteristics is satisfied:
the vortex mixing time is 1-5 seconds;
the standing temperature is 4-25 ℃;
standing for 0.5-24 h;
After the standing is finished, the method further comprises the following steps: dialyzing with water;
The mixed system of the DNA and the block copolymer is the DNA condensation system according to any one of claims 1 to 11.
17. The method of claim 15, wherein the block copolymer is pretreated with a disulfide bond reducing agent prior to mixing the DNA and the block copolymer.
18. The method of claim 17, wherein the disulfide bond reducing agent is selected from one or more of dithiothreitol, glutathione, and TCEP.
19. Use of the DNA condensation system according to any one of claims 1 to 11, or the non-helicitic cyclic complex according to any one of claims 12 to 14, or the non-helicitic cyclic complex prepared by the method of preparation according to any one of claims 15 to 18, in the preparation of a gene therapy drug; the gene in the gene therapy medicine is VEGF gene, and the gene therapy medicine is used for promoting blood vessel regeneration.
20. The use of claim 19, wherein said pro-angiogenic comprises a pro-angiogenic of the lower extremities.
21. Use of a DNA condensation system according to any one of claims 1 to 11, or a non-helic cyclic complex according to any one of claims 12 to 14, or a non-helic cyclic complex prepared by a method according to any one of claims 15 to 18, for gene delivery, characterized in that the use is not diagnostic or therapeutic.
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