Dopamine functional poly (beta-amino ester) and preparation method and application thereof
阅读说明：本技术 一种多巴胺功能化聚（β-氨基酯）及其制备方法和应用 (Dopamine functional poly (beta-amino ester) and preparation method and application thereof ) 是由 申有青 刘衍朋 周珠贤 于 2020-12-29 设计创作，主要内容包括：本发明涉及生物技术领域,公开了一种多巴胺功能化聚(β-氨基酯)及其制备方法和应用,该聚(β-氨基酯)结构式如下,其制备方法是将1,4-丁二醇双丙烯酸酯和含氨基单体进行聚合反应；再采用小分子胺对聚合物进行封端；最后对多巴胺酚羟基脱保护,得到多巴胺功能化聚(β-氨基酯；将多巴胺引入到PBAE中,并采用三价铁离子与其络合,形成包封型纳米粒子结构,提高复合物的稳定性,获得转染效率高且细胞毒性小的基因转染载体,使得其在非病毒基因载体方面具有潜在的临床应用价值。(The invention relates to the technical field of biology, and discloses a dopamine functionalized poly (beta-amino ester), a preparation method and application thereof, wherein the poly (beta-amino ester) has the following structural formula, and the preparation method comprises the steps of carrying out polymerization reaction on 1, 4-butanediol diacrylate and amino-containing monomers; then, the polymer is terminated by adopting micromolecular amine; and introducing dopamine into PBAE, and complexing with ferric ions to form an encapsulated nanoparticle structure, thereby improving the stability of the compound, obtaining a gene transfection vector with high transfection efficiency and low cytotoxicity, and having potential clinical application value in the aspect of non-viral gene vectors.)
1. A dopamine-functionalized poly (β -amino ester), characterized by the structural formula:
wherein R is1One selected from 5-amino-1-pentanol, N-dimethylethylenediamine and N, N-diethylethylenediamine; r2One selected from dopamine, N-aminopropylmorpholine and 1- (3-aminopropyl) -4-methylpiperazine; m/n is 1: 1-9.
2. The dopamine-functionalized poly (β -amino ester) according to claim 1, wherein the molecular weight of the dopamine-functionalized poly (β -amino ester) is 2000-20000 Da.
3. The process for the preparation of dopamine-functionalized poly (β -aminoesters) according to claim 1 or 2, characterized in that it comprises the following steps:
(1) protecting phenolic hydroxyl of dopamine;
(2) carrying out polymerization reaction on 1, 4-butanediol diacrylate and amino-containing monomer;
(3) then, adopting small molecular amine to carry out end capping on the polymer prepared in the step (2);
(4) deprotecting phenolic hydroxyl of dopamine in the polymer subjected to end capping in the step (3) to obtain the dopamine functional poly (beta-amino ester);
the amino-containing monomer is a composition of dopamine and at least one of 5-amino-1-pentanol, N-dimethylethylenediamine and N, N-diethylethylenediamine; the molar ratio of dopamine in the amino-containing monomer is 10-50%.
4. The method for preparing dopamine-functionalized poly (β -aminoester) according to claim 3, wherein in step (1), the phenolic hydroxyl group protecting agent of dopamine is selected from trimethylchlorosilane or dimethyl-t-butylchlorosilane; the reaction solvent comprises at least one of dichloromethane, tetrahydrofuran, acetonitrile and N, N-dimethylformamide; the reaction temperature is 0-30 ℃, and the reaction time is 10-15 h.
5. The preparation method of the dopamine-functionalized poly (beta-amino ester) according to claim 3, wherein in the step (2), the molar ratio of the 1, 4-butanediol diacrylate to the amino-containing monomer is 1.1-1.2: 1; the temperature of the polymerization reaction is 70-90 ℃, and the time is 12-48 h.
6. The method for preparing a dopamine-functionalized poly (β -aminoester) according to claim 3, wherein in step (3), the small molecule amine comprises at least one of dopamine, N-aminopropylmorpholine, 1- (3-aminopropyl) -4-methylpiperazine; the molar ratio of the micromolecule amine to the polymer is 1.8-2.2: 1; the solvent for the end-capping reaction is at least one of dimethyl sulfoxide, tetrahydrofuran and N, N-dimethylformamide, and the reaction temperature is 5-30 ℃; the reaction time is 2-6 h;
in the step (4), the adopted deprotection agent comprises at least one of boron tribromide, tetrabutylammonium fluoride and potassium carbonate; the molar ratio of the deprotection agent to the polymer is 4-6: 1; the reaction solvent comprises any one of dimethyl sulfoxide, tetrahydrofuran and N, N-dimethylformamide, the reaction time is 2-6 h, and the reaction temperature is 5-30 ℃.
7. A complex comprising the dopamine functionalized poly (β -amino ester) of claim 1 or 2 and siRNA.
8. A method for preparing a compound according to claim 7, comprising the steps of: adding dopamine functional poly (beta-amino ester) and siRNA into 25mM acetic acid-sodium acetate buffer solution with the pH value of 5.0, swirling for 5-30 s, standing for 5-30 min, and adding iron ions to obtain the compound.
9. The method for preparing the complex according to claim 7, wherein the concentration of the siRNA is 0.05-0.2 μmol/L, and the N/P ratio of the dopamine functionalized poly (β -amino ester) to the siRNA is 1-40: 1.
10. Use of a complex according to claim 7 for the preparation of a gene therapy drug or a gene silencing kit.
Technical Field
The invention relates to the technical field of biology, in particular to dopamine functionalized poly (beta-amino ester) and a preparation method and application thereof.
Background
Interfering rna (rnai) is a naturally occurring cellular mechanism that ultimately leads to the knock-down of specific genes. The targeted gene knockdown delivered by siRNA has potential clinical application value in the aspect of treating diseases caused by abnormal gene expression. However, safe and efficient intracellular siRNA delivery remains challenging. Current research has used carrier materials (liposomes, inorganic materials and polymers) that can deliver DNA for delivery of siRNA. Two major delivery obstacles characteristic of siRNA are: nanoparticle stability and cytoplasmic targeting. Since siRNA is about 200 times smaller than DNA plasmid and relatively rigid, its electrostatic interaction with cationic polymer is low, preventing both from self-assembling to form stable and small-sized nanoparticles. On the other hand, siRNA only efficiently enters the cell, degrading mRNA in the cytoplasmic matrix.
Poly (beta-amino ester, PBAE) is a cationic polymer which is easy to synthesize and has strong structural adaptability, and the PBAE structure contains ester bonds and can be hydrolyzed in vivo to reduce cytotoxicity. CN111718494A discloses a reduction responsive hyperbranched poly-beta-amino ester with high-efficiency gene delivery capability, a preparation method and application thereof, wherein the polymer is polymerized by a Michael addition method of 'A2 + B3+ C2' to form the polymer with 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-1siRNA), so that high-efficiency transfection of genes is realized, and the material toxicity is reduced.
CN106554499A discloses a disulfide bond-containing poly (beta-amino ester) polymer gene vector. The poly (beta-amino ester) polymer is a cationic polymer prepared by addition polymerization reaction, can be combined with plasmid DNA with negative electricity in vitro through electrostatic interaction, and further transports the DNA into cells to realize gene delivery. The poly (beta-amino ester) polymer introduces disulfide bonds, so that the poly (beta-amino ester) polymer has a redox response mechanism in cells, and is favorable for degradation of the polymer and sufficient release of DNA in the cells. Cytotoxicity experiments and cell transfection experiments show that the disulfide bond-containing poly (beta-amino ester) polymer gene vector has lower cytotoxicity and higher transfection efficiency, and is obviously higher than conventional commercial gene transfection reagents.
In the prior art, the main concern about improving the transfection efficiency is mainly focused on improving the molecular weight, positive charge and branching degree of a polymer, but the excessive positive charge can cause that the formed nanoparticles are too compact after being wrapped by siRNA, the nanoparticles cannot be fully released in cells after endocytosis, and in addition, the large molecular weight can also cause the increase of cytotoxicity. Therefore, the problem of improving siRNA delivery by instability and efficient intracellular release of gene vectors and siRNA nanoparticles still needs to be continuously explored.
Disclosure of Invention
The invention provides a novel poly (beta-amino ester) for synthesizing a high-efficiency, low-toxicity and stable gene transfection vector, dopamine is introduced into a cationic polymer and is complexed with metal ions to form a stable gene transfection vector in serum, so that the stability of the vector in the in-vivo transportation process is improved, and the gene transfection efficiency is further improved.
In order to achieve the purpose, the invention adopts the technical scheme that:
a dopamine functionalized poly (β -amino ester) having the structural formula:
wherein R is1One selected from 5-amino-1-pentanol, N-dimethylethylenediamine and N, N-diethylethylenediamine; r2One selected from dopamine, N-aminopropylmorpholine and 1- (3-aminopropyl) -4-methylpiperazine; m/n is 1: 1-9.
The molecular weight of the dopamine functionalized poly (beta-amino ester) is 2000-20000 Da. Too small molecular weight cannot form a complex with siRNA, too large molecular weight increases toxicity, and the complex is tightly packed and cannot release siRNA.
The invention also provides a preparation method of the dopamine functional poly (beta-amino ester), which comprises the following steps:
(1) protecting phenolic hydroxyl of dopamine;
(2) carrying out polymerization reaction on 1, 4-butanediol diacrylate and amino-containing monomer;
(3) then, adopting small molecular amine to carry out end capping on the polymer prepared in the step (2);
(4) deprotecting phenolic hydroxyl of dopamine in the polymer subjected to end capping in the step (3) to obtain the dopamine functional poly (beta-amino ester);
the amino-containing monomer is a composition of dopamine and at least one of 5-amino-1-pentanol, N-dimethylethylenediamine and N, N-diethylethylenediamine; the molar ratio of dopamine in the amino-containing monomer is 10-50%. The invention increases dopamine structure based on the fact that the tertiary amine structure in poly (beta-amino ester) can form a compound with the charge of siRNA with negative electricity, and the formed compound further increases the size stability of the compound through the complexation of ferric ions and phenolic hydroxyl groups, so that the toxicity of the polymer is reduced and the gene transfection efficiency is increased. When the content of dopamine is too high, the complexing effect of the compound and iron ions is high, so that the size of the compound is large, and the product effect is not ideal. When the dopamine content is too low, the dopamine cannot be effectively complexed with iron ions, and the dimensional stability of the compound is poor.
In the step (1), the specific steps for protecting the phenolic hydroxyl group of dopamine are as follows: dissolving dopamine hydrochloride and an acid-binding agent in a solvent, slowly dropwise adding a phenolic hydroxyl protective agent of dopamine, and after the reaction is finished, carrying out rotary evaporation, washing and drying on a product to obtain a phenolic hydroxyl protected dopamine monomer.
In the step (1), the phenolic hydroxyl protective agent of dopamine is selected from trimethylchlorosilane or dimethyl tert-butylchlorosilane; the reaction solvent comprises at least one of dichloromethane, tetrahydrofuran, acetonitrile and N, N-dimethylformamide; the reaction temperature is 0-30 ℃, and the reaction time is 10-15 h. The preferred reaction time is 12 h. This step is aimed at protecting the two phenolic hydroxyl groups of dopamine first.
The acid-binding agent comprises any one of imidazole, triethylamine and pyridine, and preferably, the acid-binding agent is imidazole.
In the step (2), the 1, 4-butanediol diacrylate and the amino-containing monomer are reacted under the solvent-free condition with the molar ratio of 1.1-1.2: 1; the temperature of the polymerization reaction is 70-90 ℃, and the time is 12-48 h; preferably, the reaction temperature is 90 ℃ and the reaction time is 24 h. The monomers polymerized to form dopamine-grafted poly (. beta. -amino ester).
In the step (3), the small molecular amine comprises at least one of dopamine, N-aminopropylmorpholine and 1- (3-aminopropyl) -4-methylpiperazine; the solvent for the end-capping reaction is at least one of dimethyl sulfoxide, tetrahydrofuran and N, N-dimethylformamide, and the reaction temperature is 5-30 ℃; the reaction time is 2-6 h. The molar ratio of the micromolecule amine to the polymer is 1.8-2.2: 1. The polymer after the end capping has higher stability in the compound formed at the later stage.
In the step (4), the adopted deprotection agent comprises at least one of boron tribromide, tetrabutylammonium fluoride and potassium carbonate; the molar ratio of the deprotection agent to the polymer is 4-6: 1; the reaction solvent comprises at least one of dimethyl sulfoxide, tetrahydrofuran and N, N-dimethylformamide, the reaction time is 2-6 h, and the reaction temperature is 5-30 ℃.
The invention also provides a compound comprising the dopamine functionalized poly (beta-amino ester) and siRNA.
The invention also provides a preparation method of the compound, which comprises the following steps: adding dopamine functional poly (beta-amino ester) and siRNA into 25mM acetic acid-sodium acetate buffer solution with the pH value of 5.0, swirling for 5-30 s, standing for 5-30 min, and adding iron ions to obtain the compound.
The concentration of the siRNA is 0.05-0.2 mu mol/L, and the N/P ratio of the dopamine functional poly (beta-amino ester) to the siRNA is 1-40: 1. Preferably, the concentration of the siRNA is 0.1. mu. mol/L.
The invention also provides the application of the compound in the preparation of gene therapy medicines or gene silencing kits.
Compared with the prior art, the invention has the following beneficial effects:
dopamine is introduced into PBAE, and ferric ions are adopted to complex with the PBAE to form an encapsulated nanoparticle structure, so that the stability of the compound is improved; and reducing iron ions under the action of glutathione in cytoplasm and the like after the compound enters cells to release siRNA, so as to obtain a gene transfection vector with high transfection efficiency and low cytotoxicity, and the gene transfection vector has potential clinical application value in the aspect of non-viral gene vectors.
Drawings
FIG. 1 shows the nuclear magnetic spectra of PBAE 1-3 prepared in examples 1-3.
FIG. 2 is a nuclear magnetic spectrum of PBAE1, PBAE4, and PBAE5 prepared in examples 1,4, and 5.
FIG. 3 shows the results of comparing the cytotoxicity of PBAE 1-5 prepared in examples 1-5 with that of the commercial transfection reagent Lipofectamine 2000.
FIG. 4 shows the particle size of the complexes formed by PBAE 1-5 and siRNA prepared in examples 1-5.
FIG. 5 is a graph showing the potentials of complexes formed between PBAE 1-5 prepared in examples 1-5 and siRNA.
Fig. 6 shows the particle size stability of complexes formed by PBAE1, PBAE4, and PBAE5 prepared in examples 1,4, and 5 with siRNA.
FIG. 7 shows the results of HeLa-GFP cell transfection of complexes formed between PBAE 1-5 prepared in examples 1-5 and a commercial transfection reagent Lipofectamine2000 and siRNA.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Those skilled in the art should understand that they can make modifications and equivalents without departing from the spirit and scope of the present invention, and all such modifications and equivalents are intended to be included within the scope of the present invention.
The chemicals in the examples were all from commercially available chemical reagents, siRNA was from the company biosciences, custom made, and siGFP sequence: 5'-CAA GCU GAC CCU GAA GUU CTT-3', respectively; 5'-GAA CUU CAG GGU CAG CUU GTT-3' are provided.
Example 1
(1) Dopamine phenolic hydroxyl protection: dopamine hydrochloride (2mmol) and imidazole (2.4mmol) were dissolved in dichloromethane solution (10mL), dimethyl tert-butylchlorosilane (4mmol) was added dropwise in an ice bath, and the reaction was stirred at 25 ℃ for 12 h. After the reaction, carrying out rotary evaporation on the mixed solution to obtain a crude product, and washing, separating, rotary evaporation and drying the solution to obtain a phenolic hydroxyl protected dopamine monomer;
(2) mixing the dopamine monomer (0.5mmol) prepared in the step (1) with 5-amino-1-pentanol (2mmol), and stirring and reacting the mixture with 1, 4-butanediol diacrylate (2.75mmol) in dimethyl sulfoxide at 90 ℃ for 24 hours to obtain a polymer;
(3) dissolving the polymer (1mmol) prepared in the step (2) in tetrahydrofuran solution (4mL), adding 2mmol of N-aminopropylmorpholine into a reaction system, and stirring at room temperature for 4 hours to obtain a capped polymer;
(4) adding the terminated 1mmol of polymer into a tetrahydrofuran (5mL) solution of 5mmol of tetrabutylammonium fluoride, and stirring for 4h to finally obtain dopamine functionalized poly (beta-amino ester) PBAE 1.
Examples 2 to 3
According to the preparation process of example 1, the amounts of dopamine monomer and 5-amino-1-pentanol in step (2) are replaced by 1mmol and 1.5mmol, 1.25mmol and 1.25mmol, respectively, and the rest steps are unchanged, so that dopamine functionalized poly (beta-amino ester) PBAE2 and PBAE3 are obtained.
Examples 4 to 5
According to the preparation process of example 1, the 5-amino-1-pentanol in step (1) was replaced with N, N-dimethylethylenediamine and N, N-diethylethylenediamine, respectively, and the remaining steps were unchanged, to give dopamine-functionalized poly (. beta. -amino ester) PBAE4 and PBAE5, respectively.
Comparative example 1
Commercial transfection reagent Lipofectamine2000 was used as a comparative example.
Nuclear magnetic characterization
Performing nuclear magnetic hydrogen spectrum characterization on the PBAE 1-5 polymers prepared in the embodiment, wherein the deuterated reagent is CDCl3The results are shown in FIG. 1 and FIG. 2, and the chemical shifts of the main structure peaks of PBAE 1-5 polymer are as follows: 1.2-1.45(m, -CH)2),1.6(s,-COOCH2CH2),2.25-2.5(m,-NCH2),2.6-2.7(m,-COOCH2NCH2),3.4(s,-N(CH2)2),4.0(s,-COOCH2). Peaks appearing at chemical shifts 6.0-7.0 of PBAE 1-3 are hydrogen on a dopamine benzene ring, and the peak intensity gradually increases with the increase of the proportion of dopamine in a polymer; PBAE 4-NCH occurs in the presence of N, N-dimethylethylenediamine at 1.33Peak(s). PBAE 5-NCH appears in the presence of N, N-diethylethylenediamine at 1.02CH3Peak(s).
Cytotoxicity test
The cytotoxicity of PBAE 1-5 prepared in examples 1-5 was measured by tetrazolium salt (MTT) colorimetric method, and the commercial product Lipofectamine2000 was used as a positive control. Hela cells were cultured in DMEM medium containing 10% FBS (containing 100U/mL penicillin and 100. mu.g/mL streptomycin), and placed at 37 ℃ in 5% CO2In an incubator. Cells in logarithmic growth phase were taken at 5X 10 per well3The individual cells were seeded in 96-well plates in a volume of 180. mu.L per well. Plates were transferred to incubators and incubated overnight. Different doses of PBAE or control groups of 20. mu.L were added to each well and incubated for 48 h. mu.L (5mg/mL) of MTT solution was added to each well and incubated for 4 hours. The culture supernatant was carefully pipetted into the wells and 100. mu.L of dimethyl sulfoxide was added to each well and incubated for 30 minutes at room temperature. After shaking, the absorbance values at 562nm and 620nm of each well were measured by a microplate reader, and the cytotoxicity of each group was calculated.
The test results are shown in fig. 3, compared with comparative example 1, the PBAE polymers have the cell activity of more than 80% under the condition of the highest use concentration (10 mug/mL), which indicates that the PBAE series polymers prepared have lower cytotoxicity.
Example 6
Respectively dissolving dopamine functionalized poly (beta-amino ester) PBAE 1-5 prepared in examples 1-5 and a commercial transfection reagent Lipofectamine2000 in dimethyl sulfoxide to prepare a mother solution of 100mg/mL, dissolving siRNA in an acetic acid-sodium acetate buffer solution (pH 5.0,10mM), preparing polymer solutions according to different N/P ratios, mixing the polymer solutions with the siRNA solutions in an equal volume, adding iron ions (the molar ratio of the iron ions to the dopamine is 1:3), immediately vortex and oscillate for 10s, standing at room temperature for 10min, comprehensively screening and analyzing the early-stage particle size and PDI thereof, and performing particle size and potential test by a particle size analyzer Zetasizer according to the ratio of N/P5.
The particle size test results are shown in FIG. 4, the particle sizes of nanoparticles formed by PBAE 1-5 are all in the range of 100-250 nm, and the particle size of PBAE1 is the smallest in PBAE 1-3. The content of dopamine in the PBAE structure is increased, the stronger the complexing with iron ions is, the stronger the aggregation capability of the nano particles is, and the particle size is increased; in PBAE 3-5, the particle size of PBAE5 is the smallest, which indicates that the hydrophobic structure is beneficial for the nanoparticles to form a more stable structure. The potential test result of the nanoparticles is shown in FIG. 5, which indicates that the Zeta potentials of PBAE 1-5 are all between 5-20 mV, and the gene delivery and transfection are facilitated.
Particle size stability test
The complexes PBAE1, PBAE4, PBAE5 prepared in examples 1,4 and 5 and Lipo2000 of comparative example 1 were placed in HEPES buffer solution at pH 7.4, respectively, and the particle size of the complexes was tested at intervals to observe their stability. The results are shown in fig. 6, where the particle size of nanoparticles formed from PBAE1 and PBAE5 changed less over time than commercial reagents, indicating better dimensional stability.
Application example
Complex in vitro cell transfection-green fluorescent protein gene transfection:
HeLa cells were cultured in 24-well plates at a cell density of 8X 104And each hole is 1mL of culture medium, and the cells are placed in an incubator and cultured for 24 h. The medium in each well was replaced with serum-free medium and a solution of prepared N/P ═ 5 nanocomplexes (siRNA: siGFP) was added and incubated for 4 h. The medium was then discarded and replaced with serum-containing medium and the culture continued for 48 h. The culture medium is discarded, the cells are digested by pancreatin and collected into a centrifuge tube, washed twice by PBS solution, and finally suspended in 0.5mL of PBS solution and detected by a flow cytometer. The excitation wavelength of the green fluorescent protein is 488 nm. In the experiment, the positive control was Lipofectamine 2000/siGFP complex, and the negative control was cells without any treatment.
The test results are shown in FIG. 7, and the Control group is a blank Control group without gene transfection, and the relative GPF fluorescence values of FIG. 7 are obtained by comparing other experimental groups with the blank Control group. Compared with a commercial reagent, PBAE 1-5 has the advantages that the transfection capability of PBAE1 and PBAE5 is slightly superior to that of Lipofectamine2000, the stability of the nanoparticles is superior to that of the commercial transfection reagent, and the potential application prospect is achieved.