CN111718494A

CN111718494A - Reduction responsive hyperbranched poly-beta-amino ester with high-efficiency gene delivery capacity and preparation method and application thereof

Info

Publication number: CN111718494A
Application number: CN202010526961.1A
Authority: CN
Inventors: 殷黎晨; 王笑; 梁秋君
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2020-06-09
Filing date: 2020-06-09
Publication date: 2020-09-29

Abstract

The invention provides reduction-responsive hyperbranched poly-beta-amino ester with high-efficiency gene delivery capacity and a preparation method and application thereof, wherein the polymer is polymerized by a Michael addition method of A2+ B3+ C2 to form a hyperbranched structure. Compared with a linear structure, the branched structure can enhance the interaction between the polymer and the nucleic acid molecule, obviously improve the gene condensation capability of the polymer, and simultaneously increase the cellular uptake by enhancing the interaction with a cell membrane. The main chain of the polymer has a reduction-response group (disulfide bond), and under the action of GSH in damaged vascular endothelial cells, poly beta-amino ester can be rapidly degraded in cytoplasm to release the entrapped gene drug (ICAM-1 siRNA), so that high-efficiency transfection of genes is realized, and the material toxicity is reduced. These properties of the poly-beta-amino esters make them promising for the development of biomedical materials, especially in the field of gene delivery.

Description

Reduction responsive hyperbranched poly-beta-amino ester with high-efficiency gene delivery capacity and preparation method and application thereof

Technical Field

The invention relates to the field of gene loading and delivery, in particular to reduction-responsive hyperbranched poly-beta-amino ester with high gene delivery capacity, a preparation method and application thereof, and application thereof in siRNA transfection.

Background

Genetic vectors are important tools for loading nucleic acid molecules, delivering them into target cells, and successfully expressing them. Genetic vector materials can be divided into two broad categories, viral vectors and non-viral vectors. The viral vector has the advantage of high transfection efficiency, but the defects of high immunogenicity, high carcinogenic risk, low gene loading and the like of the viral vector seriously restrict the application and development of the viral vector in the field of medicine. Based on this, non-viral gene vectors are gradually gaining attention and development. The common non-viral gene vectors mainly include liposome, cationic polymer, polysaccharide, etc. The existing gene vectors are limited by the structure, and often need higher mass ratio to effectively condense nucleic acid molecules, which also brings higher cytotoxicity. Based on the above, the development of the stimulus-responsive degradable cationic polymer can solve the above-mentioned contradiction while realizing the stable entrapment of the nucleic acid drug.

Disclosure of Invention

The invention aims to provide hyperbranched poly-beta-amino ester with reduction responsiveness, which can be used as a carrier of nucleic acid drugs and has good biocompatibility, reduction sensitivity and high gene transfection capacity; and provides a preparation method of the hyperbranched reduction-responsive poly-beta-amino ester combined nucleic acid molecule and application of the hyperbranched reduction-responsive poly-beta-amino ester combined nucleic acid molecule in a nucleic acid drug delivery system.

The invention provides hyperbranched poly-beta-amino ester with reduction responsiveness, which is polymerized by a Michael addition method and then is terminated by using micromolecule amine as a terminating agent; the cationic poly-beta-amino ester has a hyperbranched structure, and a main chain has a reduction-sensitive group.

The invention adopts the following technical scheme:

a reduction-responsive hyperbranched poly-beta-amino ester has a structure shown in a formula (I);

in the structure shown in the formula (I), 8-15, y is 7-14, and z is 6-10; preferably, x is 9-11, y is 8-10, and z is 7-9.

The invention discloses a preparation method of the reduction responsive hyperbranched poly-beta-amino ester, which takes 2, 2-dithiodiethanol diacrylate, amino alcohol, trimethylolpropane triacrylate and an amino compound as raw materials to prepare the reduction responsive hyperbranched poly-beta-amino ester through reaction.

The invention discloses a preparation method of a nano-drug, which comprises the following steps: 2, 2-dithiodiethanol diacrylate, amino alcohol, trimethylolpropane triacrylate and an amino compound are taken as raw materials to react to prepare reduction responsive hyperbranched poly beta-amino ester; and (3) obtaining the nano-drug from the reduction-responsive hyperbranched poly-beta-amino ester compound drug.

The amino alcohol has the following chemical formula:

in the present invention, R₁The small molecule group with hydroxyl can be as follows:

the amine-based compound has the following chemical structural formula:

in the present invention, R₂Is a small molecular group with an amine group, R₂The structure can be as follows:

the invention provides a preparation method of a polymer poly beta-amino ester with a structure shown as a formula (II), which takes poly beta-amino ester with a structure shown as a formula (II) as an example, and comprises the following steps: 2, 2-dithiodiethanol diacrylate, 4-amino-1-butanol, trimethylolpropane triacrylate and 1- (3-aminopropyl) -4-methylpiperazine are used as raw materials to prepare hyperbranched poly beta-amino ester with reduction (GSH) responsiveness through reaction.

The invention discloses a preparation method of a nano-drug, which comprises the following steps: 2, 2-dithiodiethanol diacrylate, 4-amino-1-butanol, trimethylolpropane triacrylate and 1- (3-aminopropyl) -4-methylpiperazine are used as raw materials to prepare reduction responsive hyperbranched poly beta-amino ester through reaction; the reducing responsive hyperbranched poly beta-amino ester compound nucleic acid molecule obtains a nano-drug; the specific method is that the reduction responsive hyperbranched poly-beta-amino ester is dissolved in acetic acid buffer solution, then nucleic acid solution is added, and then the nano-drug is obtained after incubation at 37 ℃.

In the invention, 2, 2-dithiodiethanol diacrylate, amino alcohol and trimethylolpropane triacrylate are heated to react, then an amino compound is added, and then the reaction is carried out at room temperature to prepare reduction responsive hyperbranched poly beta-amino ester; the molar ratio of the 2, 2-dithiodiethanol diacrylate to the trimethylolpropane triacrylate, the amino alcohol and the amine compound is 0.83: 0.25: 1; the heating reaction is carried out for 14-18 h at 50 ℃; the reaction time at room temperature is 10-15 hours. For example, 2-dithiodiethanol diacrylate, 4-amino-1-butanol and trimethylolpropane triacrylate react at 50 ℃, then 1- (3-aminopropyl) -4-methylpiperazine is added, and the reaction is carried out at room temperature to prepare reduction responsive hyperbranched poly beta-amino ester; preferably, the molar ratio of the 2, 2-dithiodiethanol diacrylate to the trimethylolpropane triacrylate, the 4-amino-1-butanol and the 1- (3-aminopropyl) -4-methylpiperazine is 0.83: 0.25: 1; the reaction time is 16 h at 50 ℃; the reaction time at room temperature was 12 h.

In the invention, bis (2-hydroxyethyl) disulfide and acryloyl chloride are used as raw materials, triethylamine is used as a catalyst, and 2, 2-dithiodiethanol diacrylate is prepared.

Specifically, the preparation method of the polymer poly beta-amino ester with the structure of the formula (I) comprises the following steps:

(1) the method comprises the following steps of (1) reacting bis (2-hydroxyethyl) disulfide and acryloyl chloride serving as raw materials with triethylamine serving as a catalyst to prepare 2, 2-dithiodiethanol diacrylate;

(2) 2, 2-dithiodiethanol diacrylate, trimethylolpropane triacrylate, amino alcohol and amino compound are used as raw materials to react to prepare the reduction responsive hyperbranched poly-beta-amino ester.

The specific reaction described above can be represented as follows:

in the above technical scheme:

in the step (1), the solvent for reaction is anhydrous Tetrahydrofuran (THF) solution, and the reaction is carried out for 24 hours at room temperature; the obtained product is treated with Na₂CO₃Washing the solution and distilled water respectively, drying with anhydrous magnesium sulfate, and removing the solvent by rotary evaporation; the crude product was purified by silica gel column chromatography (developing solvent: n-hexane/ethyl acetate = 16/1); the chemical structural formula of the 2, 2-dithiodiethanol diacrylate is as follows:

the reduction responsive hyperbranched poly-beta-amino ester provided by the invention can be self-assembled with nucleic acid molecules to form a nano-drug, so that the invention discloses a nano-drug which is obtained by the reduction responsive hyperbranched poly-beta-amino ester compound drug.

In the present invention, the drug is a nucleic acid molecule.

In the invention, the nucleic acid medicament is siRNA, and can specifically degrade target genes and inhibit the expression of the target genes.

In the invention, the mass ratio of the reduction-responsive hyperbranched poly beta-amino ester to the nucleic acid molecule is (10-100): 1, the preferable mass ratio is (20-50): 1, and the more preferable mass ratio is (30-40): 1.

in the invention, the particle size of the nano-drug is 120-600 nm, the preferable particle size is 120-200 nm, and the more preferable particle size is 120-150 nm.

The invention discloses an application of the reduction responsive hyperbranched poly-beta-amino ester in preparing a drug carrier or a nano-drug; or the application of the nano-drug in the preparation of gene drugs. The drug carrier is preferably a gene drug carrier, and the nano-drug is a gene drug.

The main advantages of the invention are:

(1) ① poly β -amino ester with a hyperbranched structure has positive charges with higher density, can obviously enhance the electrostatic interaction between the polymer and nucleic acid molecules, and effectively condenses gene drugs under the lower mass ratio of the polymer to the nucleic acid molecules;

the branched polymer having a three-dimensional structure may be modified at its terminal with a group having a specific structure and function to impart various specific functions to the polymer.

(2) The reduction responsive hyperbranched poly-beta-amino ester can break the main chain of the polymer under the GSH condition in damaged cells, and realize the rapid degradation of the polymer, thereby remarkably improving the transfection efficiency and simultaneously reducing the cytotoxicity of the material.

(3) The reduction-responsive hyperbranched poly beta-amino ester has high positive charge density, promotes the interaction between the material and the cell membrane, and further greatly improves the endocytosis efficiency of the material by the cell.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:

FIG. 1 shows the preparation of (2, 2-dithiodiethanol diacrylate, SSDA) in example I¹H NMR spectrum;

FIG. 2 shows the results of example two (BPAE-SS)¹H NMR spectrum;

FIG. 3 is a photograph of comparative example I (BPAE-CC)¹H NMR spectrum;

FIG. 4 is the GPC results of example two, comparative example one, and comparative example two;

FIG. 5 is a gel electrophoresis image of example two and comparative example two;

FIG. 6 is a graph of particle size and potential after siRNA entrapment in example 6;

FIG. 7 is a gel electrophoresis chart of polymer encapsulating siRNA at different mass ratios after GSH treatment;

FIG. 8 is a graph of particle size after encapsulation of siRNA before and after GSH treatment for example two and comparative example one;

FIG. 9 is a graph showing the release of siRNA treated with heparin sodium after complexing with siRNA in example two and comparative example one;

FIG. 10 is a graph showing the cellular uptake efficiency of the complexes after siRNA was encapsulated by example two, comparative example one, comparative example two and PEI;

FIG. 11 is a graph of endosome escape of complexes in RCMECs versus intracellular release of siRNA;

FIG. 12 is the cytotoxicity of example two, comparative example one, comparative example two and PEI at different polymer/siRNA mass ratios;

FIG. 13 is a graph showing the gene expression level of ICAM-1 in cells after complex treatment;

FIG. 14 is an ICAM-1 gene expression level at the damaged myocardial tissue of rats after the administration of the complex;

FIG. 15 shows the expression levels of ICAM-1 and the inflammatory factors tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) protein at the injured myocardial tissue of rats after the administration of the complex;

FIG. 16 is a TTC staining pattern of rat hearts after compound administration;

FIG. 17 is an ultrasonic image of rat heart after administration of the complex.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.

The poly beta-amino ester is a high-efficiency and low-toxicity cationic gene delivery carrier, the main chain of the poly beta-amino ester contains a hydrolyzable disulfide bond, tertiary amine with positive charges can form a nano-complex with nucleic acid molecules with negative charges through electrostatic adsorption, and meanwhile, excessive positive charges on the surface of a polymer are mainly utilized to be combined with cell membranes to promote endocytosis of cells, so that the poly beta-amino ester can be used for intracellular delivery of the nucleic acid molecules; researches show that compared with a linear structure, the polymer structure can obviously enhance the interaction between the polymer and nucleic acid molecules, improve the gene condensation capability and promote the cellular uptake by enhancing the interaction with cell membranes. Therefore, the poly beta-amino ester with the hyperbranched structure can be more effectively combined with nucleic acid molecules to realize high-efficiency transfection of genes, and meanwhile, the polymer with stimulus responsiveness can realize quick and controllable release of the nucleic acid molecules.

The specific procedures and tests involved in the examples of the present invention are routine in the art, and all materials are commercially available, wherein ICAM-1 siRNA is obtained from the Gima gene and SD male rats are obtained from Shanghai Spiker animal laboratories, Inc.

Example one

The method for preparing the reduction-responsive hyperbranched poly-beta-amino ester comprises the following steps:

bis (2-hydroxyethyl) disulfide (7.7 g, 50 mmol) and triethylamine (18.75 mL, 150 mmol) were dissolved in 200mL anhydrous Tetrahydrofuran (THF). Acryloyl chloride (12.2 mL, 150 mmol) in dry THF (50 mL) was added dropwise over 2h under nitrogen, and the reaction was carried out at room temperature for 24 h. Na (Na)₂CO₃The solution (0.2M, 5 × 100 mL) and distilled water (3 × 100 mL) were washed separately, dried over anhydrous magnesium sulfate, rotary evaporated to remove the solvent and the crude product was purified by silica gel column chromatography (developing solvent: n-hexane/ethyl acetate = 16/1, v/v) to give 2, 2-dithiodiethanol diacrylate (SSDA) as a pale yellow oil (8.1 g, 31 mmol, yield 62%). deuterochloroform nuclear magnetic resonance, which is shown in fig. 1 as its nuclear magnetic spectrum.

Example two

2, 2-Dithiodiethanol diacrylate (217 mg, 0.83 mmol), trimethylolpropane triacrylate (TMPTA, 74 mg, 0.25 mmol) and 4-amino-1-butanol (89 mg, 1 mmol) were reacted at 50 ℃ for 16 hours without solvent. A solution of 1- (3-aminopropyl) -4-methylpiperazine (MPZ, 157 mg, 1 mmol) in methylene chloride (1 mL) was added and the mixture was reacted at room temperature for 12 hours. It was then precipitated three times with ether and the solvent was removed in vacuo to give a yellow viscous oil, BPAE-SS, yield 90%. And (3) performing nuclear magnetic resonance by using deuterated chloroform, wherein the nuclear magnetic spectrum of the figure 2 is shown. The BPAE-SS chemical structural formula (x is 9-11, y is 8-10, and z is 7-9) is as follows:

the reaction at 50 ℃ for 16 hours was changed to 60 ℃ for 8 hours, and the remainder was unchanged to obtain BPAE-SS in a yield of 75%.

Comparative example 1

1, 6-hexanediol diacrylate (188 mg, 0.83 mmol), trimethylolpropane triacrylate (74 mg, 0.25 mmol) and 4-amino-1-butanol (89 mg, 1 mmol) were added to 50^oC, after reacting for 16 h, adding a dichloromethane solution (1 mL) of 1- (3-aminopropyl) -4-methylpiperazine (157 mg, 1 mmol), and reacting at room temperature overnight to obtain insensitive hyperbranched poly β -amino ester (BPAE-CC) as a positive control, wherein the nuclear magnetism is performed by deuterochloroform, and the nuclear magnetism spectrogram is shown in the attached figure 3:

comparative example No. two

2, 2-Dithiodiethanol diacrylate (314 mg, 1.2 mmol) and 4-amino-1-butanol (89 mg, 1 mmol) were present at 50^oAfter 24 h of reaction with C, a solution of 1- (3-aminopropyl) -4-methylpiperazine (157 mg, 1 mmol) in methylene chloride (1 mL) was added and the reaction was allowed to proceed overnight at room temperature to give a Linear Polymer (LPAE) as a positive control. The chemical structural formula is as follows:

FIG. 4 is the GPC results of example two, comparative example one, and comparative example two; data analysis showed no significant difference in molecular weight for example two, comparative example one and comparative example two.

Example preparation, characterization and application of triple siRNA-entrapped nano-drug

Preparing an acetic acid buffer solution (pH = 5.2) of poly beta-amino ester (BPAE-SS) of example II with a concentration of 1 mg/mL and an aqueous DEPC solution of ICAM-1 siRNA (siICAM-1) with a concentration of 0.1 mg/mL respectively; the poly β -amino ester and siRNA were then mixed at different weight ratios (10/1, 20/10, 30/1, 40/1, 50/1, 80/1, and 100/1), the mixture was vortexed for 10 seconds, and then incubated at 37 ℃ for 30 min to form a poly β -amino ester/siRNA complex (BPAE-SS/siRNA). And then adding 2% agarose gel electrophoresis into the sample hole, running for 20 min at 90V, imaging by a gel imaging system, and determining the loading efficiency of the siRNA.

The particle size and potential of the complex at different weight ratios of poly β -amino ester/siRNA was determined by Dynamic Light Scattering (DLS).

The poly-beta-amino ester was pre-treated with glutathione (GSH, 5mM, 1 h, RT), then the poly-beta-amino ester and siRNA were mixed in different weight ratios (10/1, 20/10, 30/1, 40/1, 50/1, 80/1, and 100/1), the mixture was vortexed for 10 seconds, and then incubated at 37 ℃ for 30 min to form a poly-beta-amino ester/siRNA complex. The encapsulation efficiency of siRNA was determined by agarose gel electrophoresis and the particle size variation of the complexes was studied by DLS.

To the prepared complexes (BPAE/siRNA = 30) was added heparin sodium at different concentrations, 37^oC, incubation for 1 h, and an agarose gel electrophoresis experiment to study siRNA release of the complex before and after GSH treatment.

Rat Cardiac Microvascular Endothelial Cells (RCMECs) were cultured at 1.5 × 10⁵The cells were plated at a density of one well in 12-well plates and cultured for 24 hours. The medium was changed to serum-free DMEM (1 mL/well), and the complex (polymer/FAM-siRNA = 30, 1 μ g siRNA/well) was added to blank cells without any treatment. 37^oC incubation for 4 hours, abandoning the culture medium, rinsing with PBS (20U/mL) containing heparin sodium three times, digesting and collecting cells, and detecting the cell uptake of FAM-siRNA (a commercial product, purchased from Germa gene) by a flow cytometer.

Confocal laser scanning microscope observation of the endosome escape of the complex and intracellular release of siRNA RCMECs cells were treated with 4 × 10⁴cells/dish density was seeded on glass-bottom cell culture dishes (. phi. = 20 mm) and cultured for 24 hours. The medium was replaced with serum-free DMEM, and BPAE-SS/FAM-siRNA complex (1. mu.g FAM-siRNA/dish), 37 was added^oC was incubated for 4 hours, rinsed three times with sodium heparin in PBS (20U/mL), stained with Hoechst 33258 (5. mu.g/mL, 30 min) and Lysotracker Red (200 nM, 1 h), respectively, and the cells were visualized by confocal fluorescence microscopy.

RCMECs cells were cultured at 1.5 × 10⁴The density of individual cells/well was seeded in 96-well plates,after 24 hours of culture, the medium was changed to serum-free DMEM and complexes (0.1. mu.g siRNA/well), 37, were added^oC incubation for 4 h. The medium was changed to DMEM containing 10% FBS, and the culture was continued for 20 hours, and the cell viability was measured by MTT assay.

RCMECs cells were cultured at 2 × 10⁵The density of individual cells/well was plated in 6-well plates and cultured for 24 hours. Medium was replaced with serum-free DMEM, and complexes (2. mu.g siRNA/well) were added 37^oC incubation for 4 h. The medium was changed to DMEM containing 10% FBS, the culture was continued for 20 hours, LPS (300 ng/mL) was added for stimulation for 6 hours, Trizol reagent was added to extract total RNA, and the expression level of the gene of interest (ICAM-1) was analyzed by a real-time PCR system.

The polymer/siRNA complex was injected tail vein at a dose of 400 μ g/kg (BPAE-SS/siRNA = 30, w/w) according to a conventional method by ligating the left coronary artery of rat heart to induce an ischemia reperfusion injury model. After 24 h of administration, the rat was sacrificed and the heart was removed, washed with PBS, total RNA of ischemic cardiac tissue was extracted with Trizol reagent, and the expression level of the target gene (ICAM-1) was analyzed by a real-time PCR system and Western Blot experiment; 7 days after administration, the rats were sacrificed and the hearts were removed, washed with PBS, cut into pieces of about 2 mm thickness, placed in 1% TTC phosphate staining solution, 37^oC water bath for 20 min, 4% formaldehyde solution fixed overnight. Scanning the sheet tissue by a scanner, analyzing a scanning image by ImageJ, and calculating the myocardial infarction area; after 3 days of administration, the rats were anesthetized and fixed with pentobarbital sodium (5%, 1.5 mL/kg) by intraperitoneal injection, and the breasts were dehaired, cardiac ultrasonic diagnosis was performed, and left ventricular contractile function of the heart was evaluated by detecting the cardiac Ejection Fraction (EF) and left ventricular minor axis shortening rate (FS) of the rats.

Replacing the reduction-responsive hyperbranched poly-beta-amino ester (BPAE-SS) of the second example with the insensitive hyperbranched poly-beta-amino ester (BPAE-CC) of the first comparative example and the linear poly-beta-amino ester (LPAE) of the second comparative example, and comparing the two with the existing polymer to perform a parallel comparison experiment; the results are as follows:

FIG. 5 is a gel electrophoresis chart of example two and comparative example two at different polymer/siRNA mass ratios, and data analysis shows that example two can completely encapsulate siRNA when the mass ratio is greater than or equal to 10, which indicates that the hyperbranched poly-beta-amino ester in example two has significantly improved siRNA condensation capability. In contrast, the polymer BPAE-NB disclosed in example two of CN110746599A needs to be at a mass ratio of 15 or more to completely encapsulate the same siRNA.

FIG. 6 is a graph of particle size and potential after siRNA entrapment in an example, and data analysis shows that a poly-beta-amino ester and a nucleic acid drug of the present invention have a positive charge when the mass ratio is 5, a surface potential of about 7-25 mV, and a particle size of about 120-150 nm.

FIG. 7 is a gel electrophoresis chart of siRNA entrapment at different mass ratios after GSH pretreatment in example two and comparative example, and data analysis shows that after GSH treatment, in example two, siRNA can not be completely condensed at a mass ratio of 50, while in comparative example one, the ability of condensing siRNA before and after treatment is not changed, thus demonstrating the reduction sensitivity of example two/siRNA complex.

FIG. 8 is a graph of particle size of example two and comparative example one after wrapping siRNA before and after GSH treatment, and data analysis shows that after GSH treatment, the particle size of example two (BPAE-SS complex) is significantly increased and siRNA condensation ability is significantly reduced, while the particle size of comparative example one (BPAE-CC complex) is unchanged, indicating that BPAE-SS has reduction sensitivity.

FIG. 9 is a graph showing the release of siRNA treated with sodium heparin after complexing with siRNA in example two and comparative example one. from FIG. 9, it is found that more siRNA molecules are competitively released when the sodium heparin concentration is increased. After GSH treatment of the polymer, the concentration of heparin sodium required by siRNA release in the compound of the second example is greatly reduced, while the siRNA release behavior of the compound of the first comparative example is not changed, which shows that the second example has good reduction response type, thereby promoting the release of gene drugs.

FIG. 10 shows the cellular uptake efficiency of the complexes in RCMECs cells after siRNA encapsulation by example two, comparative example one, comparative example two and PEI, and the data analyzed shows that example two has higher endocytosis rate than comparative example one, comparative example two and PEI, wherein the blank is a non-staining cell used for debugging the flow instrument. The same cell uptake efficiency experiment was performed, comparing to 78.6% of the endocytosis rate of the polymer BPAE-NB disclosed in CN110746599A example two after wrapping siRNA.

FIG. 11 is a graph of endosome escape of complexes in RCMECs versus intracellular release of siRNA. Analysis of the data revealed that a greater amount of green fluorescence in the cells after treatment with the compound of example two compared to the compound of comparative example two, indicated that it was more taken up by the cells, and that there was a significant separation of green fluorescence from red fluorescence, indicating that the compound could effectively escape the endosome/lysosome.

FIG. 12 is a graph of the cytotoxicity of example two, comparative example one, comparative example two and PEI at different polymer/siRNA mass ratios. As can be seen from fig. 12, the cell viability after the example two/siRNA complex treatment is about 100%, the cell viability after the comparative example two/siRNA complex treatment is greater than or equal to 90%, and no significant cytotoxicity is shown, while the cell viability after the comparative example one/siRNA complex treatment is significantly reduced and significant cytotoxicity is shown. This indicates that the example dimer polymer can be degraded into low molecular weight fragments under the stimulation of intracellular GSH, effectively reducing the cytotoxicity of the transfected material, and greatly improving the biocompatibility.

FIG. 13 is a graph showing the gene expression level of ICAM-1 in RCMECs cells after complex treatment. Data analysis shows that the second/siICAM-1 complex in the example can inhibit 75% of ICAM-1 mRNA expression in RCMECs cells, and the silencing effect of the second/siICAM-1 complex is obviously superior to that of a commercial reagent PEI. Whereas the gene silencing efficiency of comparative example I/SIICAM-1 was about 50%; the gene silencing efficiency of comparative example II/SIICAM-1 was only 30%; the control refers to cells treated with PBS, the PCR experiment uses the cells treated with PBS as a control group, and the gene expression level is recorded as 100%.

FIG. 14 is an ICAM-1 gene expression level at the damaged myocardial tissue of rats after the administration of the complex. Data analysis can show that after the second example/siICAM-1 compound is administered, the expression level of ICAM-1 mRNA in damaged cells is reduced to 21 percent, which is obviously lower than that of the first comparative example/siICAM-1 and the second comparative example/siICAM-1, and the excellent siRNA delivery and silencing efficiency of the second example nano-drug is demonstrated; wherein the control is rat myocardial tissue injected with PBS.

FIG. 15 shows the expression levels of ICAM-1 and the inflammatory factors tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) protein in rat injured myocardial tissue after the administration of the complex. As can be seen from data analysis, the band gray scale of histone administered in example II/SIICAM-1 is significantly reduced, which indicates that the ICAM-1 protein expression level is significantly reduced, the protein expression level is lower than that of comparative example I/SIICAM-1 and comparative example II/SIICAM-1, and the result is consistent with the mRNA expression level; wherein the control is rats in PBS administration group (essentially, rats treated by injecting PBS after the inflammation model is established).

FIG. 16 is a graph of TTC staining of rat hearts after compound administration. After TTC staining, normal tissue appeared red and infarcted tissue appeared white. Data analysis can show that compared with the first comparative example and the second comparative example, the white part of the heart of the rat in the second example/SIICAM-1 administration group is obviously reduced, and the myocardial infarction area is only 6 percent, which proves that the compound can effectively reduce the myocardial infarction area and relieve heart injury; the control is the same as that in FIG. 15.

FIG. 17 is an ultrasonic image of rat hearts after compound administration, wherein Ejection Fraction (EF) and left ventricular short axis shortening (FS) are important parameters for assessing left ventricular function. Data analysis shows that the EF and FS of rats in the example two/siICAM-1 administration group are 84% and 47%, which are significantly higher than those in the comparative example one and the comparative example two, and have no significant difference compared with the normal group. The results indicate that the second/siICAM-1 complex of the example can effectively deliver ICAM-1 siRNA, and inhibit inflammatory reaction and remarkably improve the contraction function of the left ventricle by silencing the expression of ICAM-1.

The polymer has a hyperbranched structure and a group responsive to reduction conditions, can be used as a delivery carrier of nucleic acid, has reduction responsiveness, high efficient gene transfection efficiency and good biocompatibility, and has good application prospect in nucleic acid drugs, particularly siRNA drug delivery systems.

Claims

1. A reduction-responsive hyperbranched poly- β -amino ester characterized in that: the poly beta-amino ester has the following chemical structural formula:

wherein x is 8-15, y is 7-14, and z is 6-10; r is the following group:

。

2. the reduction-responsive hyperbranched poly β -amino ester according to claim 1, wherein R is₁Is a small molecular group with hydroxyl, R₂Is a small molecular group with an amine group.

3. The reduction-responsive hyperbranched poly- β -amino ester according to claim 1, characterized in that the preparation method of the reduction-responsive hyperbranched poly- β -amino ester comprises the following steps: 2, 2-dithiodiethanol diacrylate, amino alcohol, trimethylolpropane triacrylate and an amino compound are taken as raw materials to react to prepare the reduction responsive hyperbranched poly beta-amino ester.

4. The reduction-responsive hyperbranched poly (beta-amino ester) according to claim 3, wherein the reduction-responsive hyperbranched poly (beta-amino ester) is prepared by reacting 2, 2-dithiodiethanol diacrylate, aminoalcohol, trimethylolpropane triacrylate under heating, adding an amine compound, and reacting at room temperature.

5. The reduction-responsive hyperbranched poly- β -amino ester according to claim 4, wherein the molar ratio of 2, 2-dithiodiethanol diacrylate to trimethylolpropane triacrylate, amino alcohol, and amine compound is 0.83: 0.25: 1; the heating reaction is carried out for 14-18 h at 50 ℃; the reaction time at room temperature is 10-15 hours.

6. The reduction-responsive hyperbranched poly-beta-amino ester according to claim 2, wherein 2, 2-dithiodiethanol diacrylate is prepared using bis (2-hydroxyethyl) disulfide and acryloyl chloride as raw materials and triethylamine as a catalyst.

7. The method for preparing the reduction-responsive hyperbranched poly-beta-amino ester according to claim 1, which comprises the following steps: 2, 2-dithiodiethanol diacrylate, amino alcohol, trimethylolpropane triacrylate and an amino compound are taken as raw materials to react to prepare the reduction responsive hyperbranched poly beta-amino ester.

8. The preparation method of the nano-drug is characterized by comprising the following steps: 2, 2-dithiodiethanol diacrylate, amino alcohol, trimethylolpropane triacrylate and an amino compound are taken as raw materials to react to prepare reduction responsive hyperbranched poly beta-amino ester; and (3) obtaining the nano-drug from the reduction-responsive hyperbranched poly-beta-amino ester compound drug.

9. The method for preparing the nano-drug according to claim 8, wherein the nano-drug has a particle size of 120 to 150 nm; the Zeta potential of the nano-drug is 7-25 mV; the drug is a nucleic acid; the mass ratio of the reduction-responsive hyperbranched poly beta-amino ester to the nucleic acid is (10-100) to 1.

10. Use of the reduction-responsive hyperbranched poly- β -amino ester of claim 1 for the preparation of a pharmaceutical carrier or a nano-drug.