WO2021063376A1 - 具有高效基因递送能力的UV光响应性超支化聚β-氨基酯及其制备方法与应用 - Google Patents

具有高效基因递送能力的UV光响应性超支化聚β-氨基酯及其制备方法与应用 Download PDF

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WO2021063376A1
WO2021063376A1 PCT/CN2020/119120 CN2020119120W WO2021063376A1 WO 2021063376 A1 WO2021063376 A1 WO 2021063376A1 CN 2020119120 W CN2020119120 W CN 2020119120W WO 2021063376 A1 WO2021063376 A1 WO 2021063376A1
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light
nitro
poly
amino ester
amino
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PCT/CN2020/119120
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French (fr)
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殷黎晨
曹德盛
段善洲
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苏州大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
    • C08G73/02—Polyamines
    • C08G73/0206—Polyalkylene(poly)amines
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
    • C08G73/02—Polyamines
    • C08G73/0206—Polyalkylene(poly)amines
    • C08G73/0213—Preparatory process
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G83/00—Macromolecular compounds not provided for in groups C08G2/00 - C08G81/00
    • C08G83/002—Dendritic macromolecules
    • C08G83/005—Hyperbranched macromolecules
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Definitions

  • the present invention relates to the field of gene loading and delivery, in particular to the UV light-responsive hyperbranched poly- ⁇ -aminoester with high-efficiency gene delivery ability, its preparation method and application, and its application to the transfection of DNA and siRNA.
  • Gene carrier materials are important tools for loading genes, delivering them into target cells and successfully expressing them.
  • Gene carrier materials can be divided into two types: viral vectors and non-viral vectors.
  • Viral vectors have the advantage of high transfection efficiency, but their own shortcomings such as high immunogenicity, high carcinogenic risk and low gene loading have severely restricted their application and development. Based on this, non-viral vectors have gradually gained attention and development.
  • Commonly used non-viral vectors include liposomes, nanoparticles, cationic polymers and polysaccharides.
  • Existing gene vectors are limited by their structure and require a higher mass ratio to effectively condense nucleic acids, which may bring about higher cytotoxicity.
  • the purpose of the present invention is to provide a hyperbranched poly ⁇ -amino ester with UV light responsiveness; the polymer can be used as a carrier of nucleic acid and has good biocompatibility, UV light sensitivity and high gene transfection ability, It also provides a preparation method of the above-mentioned hyperbranched UV light-responsive poly- ⁇ -amino ester-binding nucleic acid and its application in a nucleic acid drug delivery system.
  • the present invention provides a hyperbranched poly- ⁇ -amino ester with UV light responsiveness. After being polymerized by the Michael addition method, a small molecule amine is used as a capping agent to block the end; the cationic poly- ⁇ -amino ester of the present invention has hyperbranched Structure, with UV sensitive groups on the main chain.
  • the present invention adopts the following technical scheme: a UV light-responsive hyperbranched poly- ⁇ -amino ester, which has a structure represented by formula (I).
  • x is 7-15, y is 7-13, and z is 6-10; preferably, x is 9-11, y is 8-10, and z is 7-9.
  • the present invention provides a method for preparing the above-mentioned polymer poly ⁇ -amino ester with the structure of formula (I), which comprises the following steps: using 4-amino-1-butanol, 2-nitro-1,3-diacrylate m-phenylene Dimethyl, trimethylolpropane triacrylate and 1-(3-aminopropyl)-4-methylpiperazine are used as raw materials to prepare UV light-responsive hyperbranched poly-aminoester.
  • the present invention discloses a preparation method of nano-medicine, which comprises the following steps: using 4-amino-1-butanol, 2-nitro-1,3-diacrylate m-phthalic acid methyl ester, trimethylolpropane triacrylic acid Ester and 1-(3-aminopropyl)-4-methylpiperazine as raw materials to prepare UV light-responsive hyperbranched poly ⁇ -aminoester by reaction; the UV light-responsive hyperbranched poly ⁇ -aminoester compound drug Obtain the nano-medicine; specifically, dissolve the UV light-responsive hyperbranched poly- ⁇ -amino ester in an acetic acid buffer solution, add the nucleic acid solution, and then incubate at 37° C. to obtain the nano-medicine.
  • 4-amino-1-butanol, 2-nitro-1,3-diacrylate m-phthalate, and trimethylolpropane triacrylate are reacted at 50-60°C before adding 1-( 3-Aminopropyl)-4-methylpiperazine, and then react at room temperature to prepare UV light-responsive hyperbranched poly ⁇ -amino ester; preferably, 2-nitro-1,3-diacrylate isophthalic acid methyl ester
  • the molar ratio with trimethylolpropane triacrylate, 4-amino-1-butanol and 1-(3-aminopropyl)-4-methylpiperazine is 0.6: 0.25:1:1; 50 ⁇ 60
  • the reaction time at °C is 5-8 h; the reaction time at room temperature is 12 hours.
  • 2,6-dimethylnitrobenzene and oxidant are used as raw materials to prepare 2-nitro-1, 3-phthalic acid; 2-nitro-1, 3-phthalic acid and borane
  • the oxidant is potassium permanganate; under the amine catalyst, 2-nitro-1,3-benzenedimethanol and acryloyl chloride are used as raw materials to prepare 2-nitro-1,3-diacrylic acid in the reaction solvent. M-phthalate.
  • the preparation method of the polymer poly- ⁇ -amino ester with the structure of formula (I) of the present invention is as follows: (1) Using 2,6-dimethylnitrobenzene and potassium permanganate as raw materials, the preparation of 2- Nitro-1, 3-phthalic acid.
  • Step (1) the reaction solvent is water, the reaction reflux was 95 o C 24 h, the resulting product was adjusted to pH 1 with hydrochloric acid, the reaction is carried out in the presence of sodium hydroxide; the 2 -The chemical structure of nitro-1, 3-phthalic acid is: .
  • step (2) anhydrous tetrahydrofuran is used as the reaction solvent, and the reaction is performed at room temperature for 48 hours.
  • the chemical structural formula of the 2-nitro-1,3-benzenedimethanol is: .
  • step (3) the reaction solvent is dichloromethane, and the catalyst is triethylamine; the structural formula of the 2-nitro-1,3-diacrylate meta-phthalic acid is: .
  • the UV light-responsive hyperbranched poly- ⁇ -amino ester provided by the present invention can self-assemble with nucleic acid to form a nano-medicine. Therefore, the present invention discloses a nano-medicine, which is obtained from the above-mentioned UV-light-responsive hyperbranched poly- ⁇ -amino ester composite medicine .
  • the drug is a nucleic acid; the nucleic acid is selected from DNA and RNA.
  • the DNA is plasmid DNA, which can express protein or be transcribed into small interfering RNA.
  • the mass ratio of UV light-responsive hyperbranched poly- ⁇ -aminoester to nucleic acid is (0.5-50):1, the preferred mass ratio is (10-50):1, and the more preferred mass ratio is (15 ⁇ 30): 1.
  • the particle size of the nanomedicine is 120 ⁇ 800 nm, and the preferred particle size is 120 ⁇ 200 nm, the more preferred particle size is 120 to 170 nm.
  • the present invention discloses the application of the above-mentioned UV light-responsive hyperbranched poly-aminoester in the preparation of drug carriers or the application in the preparation of nano-medicine; or the application of the above-mentioned nano-medicine in the preparation of gene medicine.
  • the main advantages of the present invention are: (1) The present invention introduces trimethylolpropane triacrylate into the poly ⁇ -amino ester structure, so that the polymer has a hyperbranched structure, and the hyperbranched structure has the following advantages: 1 Hyperbranched structure The poly ⁇ -amino ester with higher density of tertiary amines can significantly enhance the electrostatic interaction between the polymer and nucleic acid molecules, and effectively condense nucleic acid molecules at a lower mass ratio; The branched polymer with a three-dimensional structure can be modified with terminal groups with specific structures and functions to give the polymer various specific functions.
  • the UV light-responsive hyperbranched poly-aminoester of the present invention can break the polymer main chain under the trigger of external UV light to realize the degradation of the polymer, thereby reducing the toxicity of the material and significantly improving the transfection efficiency.
  • the UV light-responsive hyperbranched poly- ⁇ -aminoester of the present invention has a high positive charge density, which promotes the interaction between the material and the cell membrane, thereby promoting the endocytosis of the material.
  • Fig. 1 is the 1H NMR spectrum of Example 1 2-nitro-1,3-diacrylate m-phthalic acid.
  • Figure 2 shows the 1H NMR spectrum of Example 2.
  • Figure 3 shows the 1H NMR spectrum of Comparative Example 1.
  • Figure 4 shows the 1H NMR spectrum of Comparative Example 2.
  • Figure 5 shows the GPC results of Example 2, Comparative Example 1, and Comparative Example 2.
  • Fig. 6 shows the GPC spectra of Example 2 and Comparative Example 1 before and after UV light irradiation.
  • Fig. 7 is a graph of the particle size and potential after DNA encapsulation in Example 2.
  • Fig. 8 is a gel electrophoresis diagram of Example 2 and Comparative Example 2 of DNA encapsulated under different mass ratios.
  • Fig. 9 is a gel electrophoresis diagram of Example 2 and Comparative Example 1 before and after the encapsulated DNA is illuminated under different mass ratios.
  • Fig. 10 is a diagram of DNA wrapping after ethidium bromide treatment after wrapping DNA in Example 2, Comparative Example 1 and Comparative Example 2.
  • Figure 11 is a graph of DNA release after treatment with sodium heparin after DNA in Example 2.
  • Fig. 12 shows the gene transfection on HeLa cells, in Example 2, Comparative Example 1 after wrapping the DNA with the complex under UV irradiation at different times.
  • Fig. 13 shows the cell uptake efficiency of the complexes on HeLa cells in Example 2, Comparative Example 2, and PEI-encapsulated DNA.
  • Fig. 14 is a fluorescence image of cell escape before and after UV irradiation in Example 2 on HeLa cells.
  • Figure 15 shows the cytotoxicity of Example 2, Comparative Example 1 and Comparative Example 2 before and after UV irradiation on HeLa cells at different concentrations.
  • Figure 16 shows the cytotoxicity of the complexes with DNA wrapped in different proportions on HeLa cells in Example 2, Comparative Example 1, under different UV light time.
  • Fig. 17 is a graph of particle size and potential after encapsulating siRNA in Example 2.
  • Figure 18 is a gel electrophoresis diagram of Example 2 and Comparative Example 2 encapsulating siRNA under different mass ratios.
  • Figure 19 is a gel electrophoresis diagram of Example 2 and Comparative Example 1 before and after encapsulating siRNA under different mass ratios.
  • Figure 20 shows the cell uptake efficiency of the complex after the siRNA is wrapped in PEI in Example 2, Ratio 2 and PEI on HeLa cells.
  • Figure 21 shows the HeLa cell, example two, comparative example one, example two and PEI wrapped survivin The relative survivin mRNA expression level of cells before and after light.
  • Figure 22 shows the cell viability of HeLa cells after transfection of Example 2/siSur complex and Example 2/siNC complex with different concentrations on HeLa cells after illumination.
  • the poly- ⁇ -aminoester of the present invention is a kind of effective cationic gene delivery vector, and its main chain contains hydrolyzable ester bonds, and the positively charged tertiary amine can electrostatically interact with the negatively charged nucleic acid to form a nanocomposite.
  • the cationic polymer can effectively condense nucleic acid molecules through its own positive charge and the negative charge of the nucleic acid molecule through electrostatic interaction.
  • the polymer structure of the present invention can significantly enhance the interaction between the polymer and nucleic acid molecules, improve gene condensation ability, and at the same time can increase cell uptake by enhancing the interaction with the cell membrane. Therefore, the poly- ⁇ -aminoester with a hyperbranched structure can more effectively bind nucleic acid molecules to achieve efficient gene transfection, and at the same time, the polymer can achieve a more controlled release of nucleic acid molecules.
  • all the raw materials are commercially available products, among which the DNA is plasmid DNA, the plasmid containing luciferase expression extracted from E. coli, the conventional product, Survivin siRNA was purchased from Gemma Gene.
  • the method for preparing UV light-responsive hyperbranched poly- ⁇ -aminoester is as follows: add 2,6-dimethylnitrobenzene (15g, 0.15mol) to 95 o C NaOH solution, stir well, and then add high manganese Potassium acid (66 g, 0.418 mol) was refluxed for another 24 h; then the mixture was cooled to room temperature, filtered, and the filtrate was adjusted to pH 1 with hydrochloric acid to obtain 2-nitro-1,3-phthalic acid as a white solid product.
  • Treat the polymer with UV light (365 nm, 20 mW/cm2, 5 min) and use the GPC method to determine the molecular weight of the polymer before and after treatment.
  • 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) are in The reaction was carried out at 60 o C for 8 h without solvent.
  • Add (3-aminopropyl)-4-methylpiperazine (157 mg, 1 mmol) in dichloromethane (1 mL), and react at room temperature overnight.
  • the glacial ether is settled three times, and the solvent is removed under vacuum conditions to obtain a yellow viscous oily substance, which is an insensitive hyperbranched poly- ⁇ -amino ester (BPAE-CC).
  • BPAE-CC hyperbranched poly- ⁇ -amino ester
  • deuterated chloroform was used for NMR.
  • Figure 3 shows the NMR spectrum. Its chemical structure is as follows: .
  • Figure 5 shows the GPC results of Example 2, Comparative Example 1 and Comparative Example 2; data analysis shows that the molecular weights of Example 2, Comparative Example 1 and Comparative Example 2 have no significant difference.
  • BPAE-NB Poly ⁇ -amino ester
  • DLS Dynamic light scattering
  • YOYO-1 (20 ⁇ mol) to label DNA (each 50 bp DNA is labeled with a dye molecule), and then prepare poly- ⁇ -aminoester/YOYO-1-DNA complexes according to different weight ratios.
  • HeLa cells were seeded into a 96-well plate at 1 ⁇ 10 4 cells per well, and then cultured in DMEM medium containing 10% FBS for 24 h, replaced with serum-free medium, and then 0.1 ⁇ g YOYO-1-DNA/well Add the complex to the concentration.
  • YOYO-1-DNA After incubating for 4 h at 37°C, the cells were washed four times with PBS containing heparin sodium (20 U/mL) to remove uninfected cells. The complex was then lysed with RIPA lysis buffer for 20 min at room temperature.
  • HeLa cells were seeded into 96-well plates at 2.5 ⁇ 10 4 cells per well and cultured for 24 hours. Then change to serum-free medium and add different concentrations of poly- ⁇ -amino ester. After incubating for 4 h at 37 °C, discard the complex and replace it with 10% FBS in DMEM medium and culture for 20 h. The cell survival rate was determined by MTT method.
  • HeLa cells were seeded into 96-well plates at 2.5 ⁇ 10 4 cells per well and cultured for 24 h. Then change to a serum-free medium, and add poly- ⁇ -aminoester/DNA complexes in different weight ratios at a concentration of 0.3 ⁇ g DNA/well. After incubating for 4 h at 37 °C, discard the complex and replace it with 10% FBS in DMEM medium and culture for 20 h. The cell survival rate was determined by MTT method.
  • Example 2 After UV irradiation in Example 2, the chromatographic peak of Example 2 almost completely disappeared, while the chromatographic peak of Comparative Example 1 did not change significantly. This shows that the molecular weight of Example 2 is significantly reduced after UV light treatment, while the molecular weight of Comparative Example 1 is basically unchanged, which proves the UV light sensitivity of BPAE-NB.
  • Fig. 7 is a graph of particle size and potential after DNA is wrapped in Example 2. According to data analysis, the poly- ⁇ -aminoester and nucleic acid drug of the present invention change from negative charge to positive charge when the mass ratio is 2, and the surface potential is about 15 ⁇ 35 mV, particle size is about 120 ⁇ 170 nm.
  • Fig. 8 is a gel electrophoresis diagram of Example 2 and Comparative Example 2 under different mass ratios. Data analysis shows that when the mass ratio is greater than or equal to 0.5, Example 2 can completely encapsulate DNA. It shows that the poly- ⁇ -amino ester in Example 2 has stronger binding ability with DNA.
  • Figure 9 shows the gel electrophoresis diagrams of Example 2 and Comparative Example 1 before and after illumination under different mass ratios. According to data analysis, after illumination, Example 2 can condense DNA only when the mass ratio is 5, while Comparative Example 1 is in DNA can be condensed at the same ratio before and after light. Prove the UV light sensitivity of Example 1/DNA complex.
  • Figure 10 is the DNA package diagram of Example 2, Comparative Example 1 and Comparative Example 2 treated with ethidium bromide after wrapping DNA. From Figure 10, it is found that Example 2 and Comparative Example 1 are as high as the DNA mass ratio of 2 Nearly 90% of the loading rate, and the comparative example 2 can only achieve a loading rate of more than 90% when the ratio of DNA mass is 15%.
  • Fig. 11 is a graph of DNA release after treatment with heparin sodium in Example 2. It is found from Fig. 11 that when the concentration of positively charged heparin sodium is increased, more DNA will be competitively released. After UV light irradiation, when the concentration of heparin sodium is 0.02 mg/mL, it can compete for most of the DNA, which is beneficial to the light-controlled release of the drug of the present invention after reaching the diseased site.
  • FIG. 12 shows the HeLa cell, Example 2, Comparative Example 1 The gene transfection of the complex after UV irradiation at different times after wrapping DNA. From Figure 12, it can be found that Example 2 and Comparative Example 1 are in the same state after UV irradiation. At the same mass ratio of nucleic acid drugs, Example 2 has a higher transfection efficiency, especially when UV light is irradiated for 5 minutes, the transfection efficiency is nearly 10 times higher than that of Comparative Example 1, showing that UV light is responsive to polymer. Promoting effect of ⁇ -amino ester transfection.
  • Figure 13 shows the cell uptake efficiency of the complexes in Example 2, Example 2 and PEI wrapped DNA on HeLa cells. From Figure 13, it is found that Example 2 has a better cell uptake efficiency than Comparative Example 2 and PEI.
  • Figure 14 is a fluorescence image of cell escape before and after UV illumination in Example 2 on HeLa cells. It can be seen from Figure 14 that after illumination, the overlap of red and green fluorescence is reduced, and the co-localization rate is significantly reduced, which proves that more complexes escape Extrusion of endosomes is beneficial to promote gene transfection.
  • Figure 15 shows the cytotoxicity of HeLa cells with different concentrations of Example 2, Comparative Example 1 and Comparative Example 2 before and after UV irradiation. It can be seen from Figure 15 that as the concentration increases, compared with Comparative Example 1/DNA complex The example 2/DNA complex has a higher cell survival rate after UV irradiation than when there is no light, which proves that the material in Example 2 degrades after UV irradiation, and the detailed toxicity is reduced.
  • Figure 16 shows the cytotoxicity of the complexes with DNA encapsulated in different proportions on HeLa cells in Example 2, Comparative Example 1. From Figure 16, it can be found that, compared to the complexes of Comparative Example 1, with With the prolongation of the illumination time, the survival rate of the cells incubated with the complex of Example 2 increased significantly, which proved that the toxicity of the complex of Example 2/DNA after light was reduced and the biocompatibility was improved.
  • siRNA siRNA
  • siRNA and poly ⁇ -amino ester in different weight ratios (1/2, 1/5, 1/10, 1/15, 1/20 and 1/30), vortex the mixture for 10 s, and then incubate at 37°C Incubate for 30 min to form a poly- ⁇ -aminoester/siRNA complex.
  • DLS Dynamic light scattering
  • the medium contained 10% FBS in DMEM medium, light (365 nm, 20 mW/cm 2 , 5 min), the cells were cultured for 20 h, and the expression level of Survivin mRNA was detected by real-time quantitative fluorescent PCR.
  • Example 2 of the particle size and potential after encapsulating siRNA; data analysis shows that the polymer of the present invention When the mass ratio of ⁇ -amino ester and nucleic acid drug is 5, it changes from negative to positive, and the surface potential is about 12 ⁇ 35 mV, the particle size is about 130 ⁇ 180 nm.
  • Example 18 is a gel electrophoresis diagram of Example 2 and Comparative Example 2 of encapsulating siRNA under different mass ratios; according to analysis, when the mass ratio is greater than or equal to 15, Example 2 can completely encapsulate siRNA. It shows that the poly- ⁇ -amino ester in Example 2 has stronger binding ability with siRNA.
  • Figure 19 shows the gel electrophoresis diagrams of Example 2 and Comparative Example 1 before and after encapsulating siRNA under different mass ratios. According to analysis, after light, Example 2 cannot effectively condense siRNA when the weight ratio is 30. In Comparative Example 1, siRNA can be condensed at the same weight ratio before and after light. The UV light sensitivity of the second embodiment/siRNA complex is proved.
  • Figure 20 shows the cell uptake efficiency of the complexes after siRNA encapsulated in Example 2, Comparative Example 2 and PEI on HeLa cells; according to analysis, Example 2 has a better cell uptake efficiency than Comparative Example 2 and PEI.
  • Figure 21 shows the relative Survivin mRNA expression levels of the HeLa cells in Example 2, Comparative Example 1, Comparative Example 2, and PEI wrapped siSur before and after light. In the figure, HeLa cells without any treatment are used as a control. According to the analysis, compared with Comparative Example 1, Comparative Example 2 and PEI, Example 2 can be The expression level of survivn mRNA was down-regulated by more than 70%, while in Example 2, the level of survivn mRNA could only be down-regulated by about 50% without light.
  • FIG. 22 shows the cell viability of HeLa cells in HeLa cells with different concentrations of Example 2/siSur complex and Example 2/siNC complex after being transfected with HeLa cells after light; according to analysis, after light, use Example 2
  • the cell viability of Hela cells transfected with the /siSur complex decreased significantly with the increase of the concentration of the complex, while in Example 2 the /siNC complex did not show detailed cytotoxicity when the encapsulated siRNA concentration was 3 ⁇ g/mL. Therefore, it was confirmed that the successful down-regulation of Survivin mediated by BPAE-NB can significantly kill HeLa cells.
  • the UV light-responsive hyperbranched poly- ⁇ -aminoester provided by the present invention has a hyperbranched structure and a UV light-responsive group that can be used as a nucleic acid carrier and has good biocompatibility and UV light sensitivity As well as high gene transfection ability, it has good application prospects in nucleic acid drug delivery systems.

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Abstract

具有高效基因递送能力的UV光响应性超支化聚β-氨基酯及其制备方法与应用，所述聚β-氨基酯以4-氨基-1-丁醇、2-硝基-1，3-二丙烯酸间苯二甲酯、三羟甲基丙烷三丙烯酸酯和1-(3-氨基丙基)-4-甲基哌嗪为原料，通过"A2+B3+C2"迈克尔加成方法聚合而成，使其具有超支化结构。相比于线性结构，支化结构可增强聚合物与核酸分子的相互作用，显著提高基因缩合能力，同时可通过增强与细胞膜的相互作用增加细胞摄取。所述聚β-氨基酯的主链上具有UV响应性基团，在UV光照射下所述聚β-氨基酯在细胞内吞后可迅速被降解，并释放出包载基因，能够实现基因的高效转染，并降低材料毒性。这些性能使其在生物医用材料尤其是基因递送领域中具有巨大的开发前景。

Description

具有高效基因递送能力的UV光响应性超支化聚β-氨基酯及其制备方法与应用 技术领域
本发明涉及基因负载和递送领域，具体涉及具有高效基因递送能力的UV光响应性超支化聚β-氨基酯及其制备方法与应用，并应用于DNA和siRNA的转染。
 背景技术
基因载体材料是用于负载基因，将其递送入目标靶细胞并成功表达的重要工具。基因载体材料可以分为病毒载体和非病毒载体两类。病毒载体具有高转染效率的有点，但其本身的免疫原性高，致癌风险高以及基因装载量低等缺点严重制约了其应用和发展。基于此，非病毒载体逐渐得到重视和发展。常用的非病毒载体有脂质体、纳米颗粒、阳离子聚合物和多糖等。现有基因载体受到其结构的限制需要较高的质量比才能有效缩合核酸，由此可能带来较高的细胞毒性。
 技术问题
本发明的目的是提供一种具有UV光响应性超支化聚β-氨基酯；该聚合物可以用作核酸的载体且具有良好的生物相容性、UV光敏感性以及高基因转染能力，并提供上述超支化UV光响应的聚β-氨基酯结合核酸的制备方法和在核酸药物递送系统中的应用。
本发明提供一种具有UV光响应性超支化的聚β-氨基酯，通过迈克尔加成方法聚合后，使用小分子胺作为封端剂封端；本发明的阳离子聚β-氨基酯具有超支化结构，主链上具有UV光敏感的基团。
 技术解决方案
本发明采用如下技术方案：一种UV光响应性超支化聚β-氨基酯，具有式（I）所示的结构。
Figure 376733dest_path_image001
所述式I所示的结构中，x为7～15，y为7～13，z为6～10；优选的，x为9～11，y为8～10，z为7～9。
本发明提供了上述具有式（Ⅰ）结构的聚合物聚β-氨基酯的制备方法，包括以下步骤：以4-氨基-1-丁醇、2-硝基-1,3-二丙烯酸间苯二甲酯、三羟甲基丙烷三丙烯酸酯和1-（3-氨基丙基）-4-甲基哌嗪为原料，反应制备UV光响应性超支化聚β-氨基酯。
本发明公开了一种纳米药物的制备方法，包括以下步骤：以4-氨基-1-丁醇、2-硝基-1,3-二丙烯酸间苯二甲酯、三羟甲基丙烷三丙烯酸酯和1-（3-氨基丙基）-4-甲基哌嗪为原料，反应制备UV光响应性超支化聚β-氨基酯；所述UV光响应性超支化聚β-氨基酯复合药物得到纳米药物；具体的，将UV光响应性超支化聚β-氨基酯溶解于醋酸缓冲溶液中，再加入核酸溶液，然后于37 ℃孵育，得到纳米药物。
 本发明中，4-氨基-1-丁醇、2-硝基-1,3-二丙烯酸间苯二甲酯、三羟甲基丙烷三丙烯酸酯于50～60℃反应后再加入1-（3-氨基丙基）-4-甲基哌嗪，然后室温反应，制备UV光响应性超支化聚β-氨基酯；优选的，2-硝基-1,3-二丙烯酸间苯二甲酯与三羟甲基丙烷三丙烯酸酯以及4-氨基-1-丁醇、1-（3-氨基丙基）-4-甲基哌嗪的摩尔比为0.6﹕0.25﹕1﹕1；50～60℃反应的时间为5~8 h；室温反应的时间为12小时。
本发明中，以2,6-二甲基硝基苯和氧化剂为原料，制备2-硝基-1, 3-苯二甲酸；以2-硝基-1, 3-苯二甲酸和硼烷为原料，制备2-硝基-1, 3-苯二甲醇；以2-硝基-1,3-苯二甲醇和丙烯酰氯为原料，制备2-硝基-1,3-二丙烯酸间苯二甲酯。优选的，氧化剂为高锰酸钾；在胺催化剂下，以2-硝基-1,3-苯二甲醇和丙烯酰氯为原料，在反应溶剂中制备2-硝基-1,3-二丙烯酸间苯二甲酯。
具体的，本发明具有式（Ⅰ）结构的聚合物聚β-氨基酯的制备方法如下：（1）以2,6-二甲基硝基苯和高锰酸钾为原料，反应制备2-硝基-1, 3-苯二甲酸。
（2）以2-硝基-1, 3-苯二甲酸和硼烷为原料，反应制备2-硝基-1, 3-苯二甲醇。
（3）以2-硝基-1,3-苯二甲醇和丙烯酰氯为原料，反应制备2-硝基-1,3-二丙烯酸间苯二甲酯。
（4）以4-氨基-1-丁醇、2-硝基-1,3-二丙烯酸间苯二甲酯、三羟甲基丙烷三丙烯酸酯和1-（3-氨基丙基）-4-甲基哌嗪为原料反应制备UV光响应性超支化聚β-氨基酯。
上述技术方案中：步骤（1）中，所述反应的溶剂为水，反应为95 oC回流反应24 h, 所得产物用盐酸调节pH至1，反应在氢氧化钠存在下进行；所述2-硝基-1, 3-苯二甲酸的化学结构式为：
Figure 320419dest_path_image002
。
步骤（2）中,以无水四氢呋喃为反应溶剂，反应为室温条件下反应48h, 所述2-硝基-1, 3-苯二甲醇的化学结构式为:
Figure 348418dest_path_image003
。
步骤（3）中, 反应溶剂为二氯甲烷，催化剂为三乙胺；所述2-硝基-1,3-二丙烯酸间苯二甲酯的结构式为：
Figure 326738dest_path_image004
。
上述具体反应可表示如下：
Figure 250831dest_path_image005
  
Figure 947392dest_path_image006
。
本发明提供的UV光响应性超支化聚β-氨基酯可以与核酸自组装形成纳米药物，因此本发明公开了一种纳米药物，由上述UV光响应性超支化聚β-氨基酯复合药物得到。
本发明中，药物为核酸；所述核酸选自DNA、RNA。
本发明中，所述DNA为质粒DNA，可以表达蛋白质或转录成小分子干扰RNA。
本发明中，UV光响应性超支化聚β-氨基酯与核酸的质量比为（0.5~50）：1，优选的质量比为（10~50）：1，更优选的质量比为（15~30）：1。
本发明中，所述纳米药物的粒径为120~800 nm，优选的粒径为120~200 nm，更优选的粒径为120~170 nm。
本发明公开了上述UV光响应性超支化聚β-氨基酯在制备药物载体中的应用或者在制备纳米药物中的应用；或者上述纳米药物在制备基因药物中的应用。
 有益效果
本发明的主要优势在于：（1）本发明在聚β-氨基酯结构中引入三羟甲基丙烷三丙烯酸酯，使得聚合物具有超支化的结构，超支化结构具有如下优点：①超支化结构的聚β-氨基酯具有更高密度的叔胺可明显增强聚合物与核酸分子的静电相互作用，在较低的质量比下有效缩合核酸分子；
Figure 247923dest_path_image007
三维结构的支化聚合物可修饰上具有特定结构和功能的末端基团，赋予聚合物各种特定的功能。
（2）本发明UV光响应性超支化聚β-氨基酯在外界UV光的触发下可以使得聚合物主链断裂，实现聚合物的降解，从而降低了材料毒性，显著提高转染效率。
（3）本发明UV光响应性超支化聚β-氨基酯正电荷密度高，促进了材料与细胞膜的相互作用，进而促进材料内吞。
 附图说明
附图用来提供对本发明的进一步理解，并且构成说明书的一部分，与下面的具体实施方式一起用于解释本发明，但并不构成对本发明的限制。在附图中：图1为实施例一2-硝基-1,3-二丙烯酸间苯二甲酯的1H NMR谱。
图2为实施例二的1H NMR谱。
图3为对比例一的1H NMR谱。
图4为对比例二的1H NMR谱。
图5为实施例二、对比例一和对比例二的GPC结果。
图6为实施例二和对比例一的在UV光照射前后的GPC谱图。
图7为实施例二包裹DNA后的粒径和电势图。
图8为实施例二和对比例二在不同质量比下包裹DNA的凝胶电泳图。
图9为实施例二和对比例一在不同质量比下包裹DNA光照前后的凝胶电泳图。
图10为实施例二、对比例一和对比例二包裹DNA后的溴化乙锭处理的DNA包裹图。
图11为实施例二DNA后的肝素钠处理的DNA释放图。
图12为HeLa细胞上，实施例二，对比例一包裹DNA后复合物在不同时间UV光照后的基因转染。
图13为HeLa细胞上，实施例二、对比例二和PEI包裹DNA后复合物的细胞摄取效率。
图14为HeLa细胞上，实施例二UV光照前后的细胞逃逸的荧光图。
图15为HeLa细胞上，不同浓度的实施例二，对比例一和对比例二在UV光照前后的细胞毒性。
图16为HeLa细胞上，实施例二，对比例一以不同比例包裹DNA后的复合物在不同UV光照时间下的细胞毒性。
图17实施例二包裹siRNA后的粒径和电势图。
图18为实施例二和对比例二在不同质量比下包裹siRNA的凝胶电泳图。
图19为实施例二和对比例一在不同质量比下包裹siRNA光照前后的凝胶电泳图。
图20为HeLa细胞上，实施例二、比例二和PEI包裹siRNA后复合物的细胞摄取效率。
图21为HeLa细胞上，实施例二，对比例一、比例二和PEI包裹survivin siRNA在光照前后，细胞的相对survivin mRNA表达水平。
图22为HeLa细胞上，在光照后不同浓度的实施例二/siSur复合物以及实施例二/siNC复合物转染Hela细胞后的细胞活力。
 本发明的实施方式
本发明的聚β-氨基酯是一类有效的阳离子基因递送载体，其主链上含有可水解的酯键，带有正点荷的叔胺可以与带负电的核酸静电相互作用形成纳米复合物，其中阳离子聚合物通过自身的正电荷与核酸分子的负电荷通过静电相互作用能够有效地缩合核酸分子，同时主要利用聚合物表面过量的正电荷与细胞膜结合，促进内吞，因此可以用于核酸的递送；研究发现，相比于其他结构，本发明聚合物结构可显著增强聚合物与核酸分子的相互作用，提高基因缩合能力，同时可通过增强与细胞膜的相互作用增加细胞摄取。因此具有超支化结构的聚β-氨基酯可以更加有效的结合核酸分子，实现基因的高效转染，同时聚合物可以实现核酸分子更加可控的释放。
本发明实施例中，所有原料都是市购产品，其中DNA为质粒DNA，从大肠杆菌中提取的含luciferase表达的质粒，常规产品，Survivin siRNA购自吉玛基因。
实施例一。
制备UV光响应性超支化聚β-氨基酯的方法，具体如下：将2,6-二甲基硝基苯（15g, 0.15mol）加入到95 oC NaOH溶液中搅拌均匀，然后加入高锰酸钾（66 g, 0.418 mol），再回流24 h；随后将混合物冷却至室温、过滤，将滤液用盐酸调节pH至1，得到白色固体产物2-硝基-1, 3-苯二甲酸。
冰浴条件下，在氮气的保护下用注射器将1.0 mol硼烷 (在四氢呋喃络合物中, 400mL) 加入2-硝基-1, 3-苯二甲酸 (16.0 g, 76 mmol) 溶于无水四氢呋喃的溶液中，得到混合物；将混合物加热至室温，搅拌48h；然后滴加甲醇 (40mL)，再过滤并在真空条件下干燥，然后将干燥物溶解在乙酸乙酯中，并用涤饱和NaCl溶液 (4 × 100 mL) 洗涤，有机相用无水硫酸镁干燥12 h，然后在真空下除去溶剂，再将得到的黄色固体经硅胶层析进一步纯化（展开剂：正己烷/乙酸乙酯 = 1/1），得到产物将2-硝基-1, 3-苯二甲醇。
在氮气保护的条件下，将三乙胺 (100 mmol) 滴加到2-硝基-1,3-苯二甲醇(7.3 g, 40 mmol) 的无水二氯甲烷 (50 mL) 溶液中，然后用注射器加入丙烯酰氯，得到混合物，混合物在室温下搅拌18 h后过滤，滤液在真空条件下干燥，用乙酸乙酯溶解干燥物，并用饱和氯化钠溶液 (3 × 100 mL) 洗涤，将有机层用无水硫酸镁干燥12 h，然后在真空下除去溶剂，得到的黄色固体经硅胶层析 （展开剂：正己烷/乙酸乙酯 = 1/1），进一步纯化得到2-硝基-1,3-二丙烯酸间苯二甲酯，氘代二甲基亚砜打核磁，附图1为其核磁谱图。
实施例二。
将2-硝基-1,3-二丙烯酸间苯二甲酯（174.6 mg, 0.6 mmol）、三羟甲基丙烷三丙烯酸酯（74 mg，0.25 mmol）和4-氨基-1-丁醇（89 mg，1 mmol）混合，在无溶剂的条件下60℃反应8 h；然后加入（3-氨基丙基）-4-甲基哌嗪（157 mg，1 mmol）的二氯甲烷溶液（1 mL），室温反应12h；然后用冰乙醚沉降三次，真空条件下除去溶剂，得到黄色粘稠油状物，即UV光响应性超支化聚β-氨基酯（BPAE-NB），总收率72%。氘代氯仿打核磁，附图2为其核磁谱图，x为9～11，y为8～10，z为7～9。BPAE-NB化学结构式如下：
Figure 549592dest_path_image008
。
将聚合物用UV光处理（365 nm, 20 mW/cm2，5 min）并用GPC法测定聚合物处理前后的分子量。
对比例一。
1，6-己二醇二丙烯酸酯（188 mg，0.83 mmol）、三羟甲基丙烷三丙烯酸酯（74 mg，0.25 mmol）和4-氨基-1-丁醇（89 mg，1 mmol）在无溶剂的条件下60 oC 反应8 h。加入（3-氨基丙基）-4-甲基哌嗪（157 mg，1 mmol）的二氯甲烷溶液（1 mL），室温反应过夜。冰乙醚沉降三次，真空条件下除去溶剂，得到黄色粘稠油状物，即得到不敏感超支化聚β-氨基酯（BPAE-CC）。作为阳性对照，氘代氯仿打核磁，附图3为其核磁谱图。其化学结构式如下：
Figure 706904dest_path_image009
。
对比例二。
2-硝基-1,3-二丙烯酸间苯二甲酯（349 mg，1.2 mmol）和4-氨基-1-丁醇（89 mg，1 mmol）在无溶剂的条件下50 oC 反应12 h。加入（3-氨基丙基）-4-甲基哌嗪（157 mg，1 mmol）的二氯甲烷溶液（1 mL），室温反应过夜。冰乙醚沉降三次，真空条件下除去溶剂，得到黄色粘稠油状物，即得到UV光响应性线性聚β-氨基酯 （LPAE-NB）。作为阳性对照，氘代氯仿打核磁，附图4为其核磁谱图。其化学结构式如下：
Figure 828443dest_path_image010
。
图5为实施例二、对比例一和对比例二的GPC结果；数据分析表明实施例二、对比例一和对比例二的分子量无显著差异。
对比例三 。
将2-硝基-1，3-二丙烯酸间苯二甲酯（174.6 mg, 0.6 mmol，实施例一制备）、三羟甲基丙烷三丙烯酸酯（74 mg，0.25 mmol）和4-氨基-1-丁醇（89 mg，1 mmol），在无溶剂的条件下70℃反应8 h。加入（3-氨基丙基）-4-甲基哌嗪（157 mg，1 mmol）的二氯甲烷溶液（1 mL），室温反应12 h。冰乙醚沉降三次，真空条件下除去溶剂，得到黄色凝胶状物，该凝胶不溶于醋酸缓冲液等溶液，无法进行后期的表征实验以及核酸包覆。
实施例三载DNA纳米药物的制备以及表征、性能。
配制实施例二的聚β-氨基酯（BPAE-NB）浓度为1 mg/mL的醋酸缓冲溶液（pH = 5.2）和DNA（质粒DNA，从大肠杆菌中提取的含luciferase表达的质粒，常规产品）浓度为0.1 mg/mL 的DEPC水溶液；然后DNA和聚β-氨基酯按不同重量比（1/0.5，1/1 ，1/5，1/10和 1/15）混合，将混合物涡旋10秒，然后在37℃下孵育30 min，以形成聚β-氨基酯/DNA复合物。上样1%琼脂糖凝胶电泳上样孔内，100 V 下运行 40 min，溴化乙锭染色显示，凝胶成像系统成像，测定DNA的包裹效率。
将DNA和聚β-氨基酯按不同重量比（1/0.2，1/0.5，1/1 ，1/2和 1/5）混合，将混合物涡旋10秒，然后在37℃下孵育30 min，以形成聚β-氨基酯/DNA复合物。将聚β-氨基酯/DNA复合物分为两组，一组进行光照（365 nm, 20 mW/cm 2 5 min），一组不进行光照，上样1%琼脂糖凝胶电泳上样孔内，100 V 下运行 40 min，溴化乙锭染色显示，凝胶成像系统成像，测定DNA的包裹效率。
利用动态光散射（DLS）评估聚β-氨基酯/DNA在不同重量比混合下，复合物的粒径和电位。
将EB溶液与DNA以重量比为10:1混合。并在室温下孵育1小时。然后按照聚β-氨基酯/DNA不同重量比将实施例加入到EB/DNA混合物中，进一步将混合物放在室温下孵育30 min，通过酶标仪测定其荧光强度（λex = 510 nm，λem = 590 nm）定量测定DNA包裹效率。
将肝素钠加入到复合物溶液中形成一系列最终浓度，并将此溶液放在37℃下孵育1 h，最后按照上述EB排阻实验过程来测定DNA释放效率。
HeLa细胞以每孔1×10 4个接种到96孔板内，然后在含有10％FBS的DMEM培养基中培养24 h。然后培养基替换成无血清的DMEM，并按照0.1 µg DNA/每孔的浓度加入聚β-氨基酯/DNA复合物（w/w=15/1）。37 ℃孵育4 h后，移除培养基，替换成新鲜培养基，将细胞分为两组，一组进行光照（365 nm, 20 mW/cm 2，5 min），一组不进行光照，之后进一步孵育20 h。使用荧光素酶试剂盒测定荧光素酶表达，并使用BCA试剂盒测定细胞蛋白浓度，评价其基因转染效率。
用YOYO-1（20 μmol）来标记DNA （每50 bp DNA被一个染料分子标记），然后按照不同重量比来制备聚β-氨基酯/YOYO-1-DNA复合物。HeLa细胞以每孔1×10 4个接种到96孔板内，然后在含有10 % FBS的DMEM培养基中培养24 h，换成无血清培养基，然后按照0.1 μg YOYO-1-DNA/孔的浓度加入复合物。以PEI/YOYO-1-DNA复合物（w/w=1:1）作为对照，37℃下孵育4 h后，用含有肝素钠（20 U/mL）的PBS洗涤细胞四次以除去未进入的复合物，随后在室温下用RIPA裂解缓冲液裂解 20 min。可以通过分光荧光测定法（λex = 485 nm，λem = 530 nm）定量YOYO-1-DNA荧光强度，并使用BCA试剂盒测定蛋白质的浓度，评价细胞摄取水平。
共聚焦激光扫描显微镜观察复合物的内涵体逃逸与DNA 胞内释放Hela细胞以4 × 10 4 细胞/皿的密度接种于玻璃底细胞培养皿（Ф = 20mm），培养24 h。将培养基换成无血清DMEM，加入RBITC-聚合物/YOYO-1-DNA复合物（w/w = 15，1 μg YOYO-1-DNA /皿），37 oC 孵育4 h，含肝素钠的PBS（20 U/mL）润洗三次，Hochest 33258 染色（5 μg/mL，30 min），荧光共聚焦显微镜观察细胞，ImageJ 计算YOYO-1-DNA 和RBITC-聚合物的共定位率。其荧光强度通过激光共聚焦显微镜进行观察。
HeLa细胞以每孔2.5×10 4个接种到96孔板内，培养24小时。然后换成无血清培养基，加入不同浓度的聚β-氨基酯。37 ℃下孵育4 h后，弃复合物，换成10％FBS的DMEM培养基中培养20 h后。通过MTT法测定细胞存活率。
HeLa细胞以每孔2.5×10 4个接种到96孔板内，培养24 h。然后换成无血清培养基，按照0.3 µg DNA/每孔的浓度加入不同重量比的聚β-氨基酯/DNA复合物。37 ℃下孵育4 h后，弃复合物，换成10％FBS的DMEM培养基中培养20 h后。通过MTT法测定细胞存活率。
将实施例二的UV光响应性超支化聚β-氨基酯 （BPAE-NB）更换为对比例一的UV光不敏感超支化聚β-氨基酯（BPAE-CC）以及对比例二的UV光响应性线性聚β-氨基酯 （LPAE-NB），并与现有聚合物进行比较，结果如下：图6为实施例二和对比例一的在UV光照射（365 nm, 20 mW/cm 2 5 min）前后的GPC谱图，利用图进行数据分析，实施例二在UV光照后，实施例二的色谱峰几乎完全消失，而对比例一的色谱峰未出现明显变化。这说明UV光照处理后实施例二的分子量显著减小，而对比例一的分子量基本不变，证明了BPAE-NB 的UV光敏感性。
图7为实施例二包裹DNA后的粒径和电势图，数据分析得出，本发明的聚β-氨基酯和核酸药物质量比为2时就从负电荷变为正电荷，表面电势约为15~35 mV，粒径约为120~170 nm。
图8为实施例二和对比例二在不同质量比下的凝胶电泳图，数据分析得出，在质量比大于等于0.5时，实施例二可以完全包载住DNA。说明实施例二中的聚β-氨基酯具有和DNA更强的结合能力。
图9为实施例二和对比例一在不同质量比下光照前后的凝胶电泳图，数据分析得出，在光照后，实施例二在质量比为5时才能缩合DNA，而对比例一在光照前后可在相同比例下缩合DNA。证明实施例一/DNA复合物的UV光敏感性。
图10为实施例二、对比例一和对比例二包裹DNA后的溴化乙锭处理的DNA包裹图，从图10中发现实施例二和对比例一在和DNA质量比为2时就高达将近90%的包载率，而对比例二在和DNA质量比为15时才能达到90%以上的包载率。
图11为实施例二DNA后的肝素钠处理的DNA释放图，从图11中发现，当增加正电荷的肝素钠浓度，会有更多的DNA被竞争释放下来。在UV光照射后，肝素钠浓度为0.02mg/mL时就能竞争下大部分的DNA，利于本发明药物到达病症部位后的光控释放。
图12为HeLa细胞上，实施例二，对比例一包裹DNA后复合物在不同时间UV光照后的基因转染，从图12中可以发现在UV光照后实施例二在和对比例一在和核酸药物相同质量比的时候，实施例二拥有更高的转染效率，尤其是在UV光照射5分钟，转染效率要比对比例一高出将近10倍，显示出UV光响应性对聚β-氨基酯转染效果的促进作用。
图13为HeLa细胞上，实施例二、比例二和PEI包裹DNA后复合物的细胞摄取效率，从图13中发现，实施例二拥有比对比例二和PEI更好的细胞摄取效率。
图14为HeLa细胞上，实施例二UV光照前后的细胞逃逸的荧光图，从图14中可以发现光照后，红色和绿色荧光重叠减少，共定位率明显降低，证明有更多的复合物逃逸出内涵体，有利于促进基因转染。
图15为HeLa细胞上，不同浓度的实施例二，对比例一和对比例二在UV光照前后的细胞毒性，从图15中可以发现随着浓度的上升，相比对比例一/DNA复合物，实施例二/DNA复合物在UV光照后相比不光照时拥有更多的细胞存活率，证明实施例二在UV光照后材料降解，明细毒性降低。
图16为HeLa细胞上，实施例二，对比例一以不同比例包裹DNA后的复合物在不同UV光照时间下的细胞毒性，从图16中可以发现，相比对比例一复合物，随着光照时间的延长，实施例二复合物孵育的细胞存活率显著上升，证明实施例二/DNA复合物在光照后毒性降低，生物相容性提高。
实施例四 载siRNA纳米药物的制备以及表征、性能。
将实施例二的聚β-氨基酯溶解于醋酸钠缓冲溶液（pH = 5.2）配制浓度为1 mg/mL的溶液，配置Survivin siRNA（siSur，购自吉玛基因（上海，中国）） 浓度为0.1 mg/mL 的DEPC水溶液；然后将siRNA和聚β-氨基酯按不同重量比（1/10，1/20，1/50，1/70，1/100，1/120和 1/150）混合，将混合物涡旋10 s，然后在37℃下孵育30 min，以形成聚β-氨基酯/siRNA复合物。上样1%琼脂糖凝胶电泳上样孔内，100 V 下运行 40 min，溴化乙锭染色显示，凝胶成像系统成像，测定DNA的包裹效率。
将siRNA和聚β-氨基酯按不同重量比（1/2，1/5，1/10，1/15，1/20和 1/30）混合，将混合物涡旋10 s，然后在37℃下孵育30 min以形成聚β-氨基酯/siRNA复合物。将β-氨基酯/siRNA复合物分为两组，一组进行光照（365 nm, 20 mW/cm 2, 5 min），一组不进行光照，上样2%琼脂糖凝胶电泳上样孔内，90 V 下运行 20 min，溴化乙锭染色显示，凝胶成像系统成像，测定siRNA的包裹效率。
利用动态光散射（DLS）评估聚β-氨基酯/siRNA在不同重量比混合下，复合物的粒径和电位。
按照不同重量比来制备β-氨基酯/ FAM-siRNA复合物,Hela细胞以每孔1×10 4个接种到96孔板内，然后在含有10％ FBS的DMEM培养基中培养24 h。然后培养基替换成无血清的DMEM，将复合物4按照每孔 0.1 μg FAM-siRNA 加入到孔中，以PEI/FAM-siRNA复合物（w/w=5:1）作为对照，孵育 4 h。用含肝素钠的缓冲液润洗 3 次，加入 RIPA 裂解液（100 µL）裂解，酶标仪测定 FAM-siRNA 的含量（λ ex = 480 nm，λ em = 530 nm），BCA 试剂盒测定细胞内蛋白含量，以研究β-氨基酯/siRNA的细胞摄取效率。
Hela细胞以每孔5×10 5个接种到6孔板内, 然后在含有10％ FBS的DMEM培养基中培养24 h。然后培养基替换成无血清的DMEM，将β-氨基酯/ siSur复合物（w/w=30:1）按照2 μg /mL siRNA 的浓度加入到孔中，以PEI/siSur复合物（w/w=5:1）、β-氨基酯/ siNC复合物（w/w=30:1）作为对照，孵育 4 h。随后培养基含有10％ FBS的DMEM培养基，光照（365 nm, 20 mW/cm 2，5 min），细胞继续培养20 h，实时定量荧光PCR检测Survivin mRNA表达水平。
HeLa细胞以每孔2.5×10 4个接种到96孔板内，培养24 h。然后换成无血清培养基，按照每孔不同的siRNA浓度加入实施例二/siSur复合物（w/w = 30/1）。以实施例二/siNC复合物（w/w = 30/1）作为对照，37 ℃下孵育4小时，光照（365 nm, 20 mW/cm 2，5 min）后弃复合物，换成10％FBS的DMEM培养基中培养48 h后。通过MTT法测定细胞存活率。
将实施例二的UV光响应性超支化聚β-氨基酯 （BPAE-NB）更换为对比例一的UV光不敏感超支化聚β-氨基酯（BPAE-CC）以及对比例二的UV光响应性线性聚β-氨基酯 （LPAE-NB），并与现有聚合物进行比较，结果如下：图17实施例二包裹siRNA后的粒径和电势图；数据分析得出，本发明的聚β-氨基酯和核酸药物质量比为5时就从负电荷变为正电荷，表面电势约为12~35 mV，粒径约为130~180 nm。图18为实施例二和对比例二在不同质量比下包裹siRNA的凝胶电泳图；据分析得出，在质量比大于等于15时，实施例二可以完全包载住siRNA。说明实施例二中的聚β-氨基酯具有和siRNA更强的结合能力。图19为实施例二和对比例一在不同质量比下包裹siRNA光照前后的凝胶电泳图；据分析得出，在光照后，实施例二在重量比为30时都不能有效缩合siRNA，而对比例一在光照前后可在相同重量比例下缩合siRNA。证明实施例二/siRNA复合物的UV光敏感性。图20为HeLa细胞上，实施例二、对比例二和PEI包裹siRNA后复合物的细胞摄取效率；据分析得出，实施例二拥有比对比例二和PEI更好的细胞摄取效率。 图21为HeLa细胞上，实施例二，对比例一、比例二和PEI包裹siSur在光照前后，细胞的相对Survivin mRNA表达水平。图中以不做任何处理的HeLa细胞作为对照。据分析得出，与对比例一、对比例二和PEI相比，实施例二在光照后能将 survivn mRNA 表达水平下调70%以上，而实施例二在不光照下只能下调50%左右的survivn mRNA 水平，这证实了UV光照射大大增强了BPAE-NB介导的基因沉默，这是光触发细胞内siRNA释放的结果，证明了实施例二的聚β-氨基酯在基因递送方面的优势。图22为HeLa细胞上，在光照后不同浓度的实施例二/siSur复合物以及实施例二/siNC复合物转染Hela细胞后的细胞活力；据分析得出，在光照后，用实施例二/siSur复合物转染的Hela细胞的细胞活力随着复合物浓度的增加显著降低，而实施例二/siNC复合物在包裹的siRNA浓度为3μg/mL下都没有显示出明细的细胞毒性。因此，证实了BPAE-NB介导的Survivin的成功下调可显著杀伤HeLa细胞。
本发明提供的UV光响应性超支化聚β-氨基酯，该聚合物具有超支化的结构以及UV光响应的基团可以用作核酸的载体且具有良好的生物相容性、UV光敏感性以及高基因转染能力，在核酸药物递送系统中具有良好的应用前景。

Claims (10)

  1. 一种 UV光响应性超支化聚β-氨基酯，其特征在于：所述聚β-氨基酯具有式（I）所示的结构：
    Figure 485922dest_path_image001
    所述式I所示的结构中，x为7～15，y为7～13，z为6～10。
  2. 权利要求1所述UV光响应性超支化聚β-氨基酯的制备方法，其特征在于，包括以下步骤：以4-氨基-1-丁醇、2-硝基-1,3-二丙烯酸间苯二甲酯、三羟甲基丙烷三丙烯酸酯和1-（3-氨基丙基）-4-甲基哌嗪为原料，反应制备UV光响应性超支化聚β-氨基酯。
  3. 根据权利要求2所述UV光响应性超支化聚β-氨基酯的制备方法，其特征在于，4-氨基-1-丁醇、2-硝基-1,3-二丙烯酸间苯二甲酯、三羟甲基丙烷三丙烯酸酯于50～60℃反应后再加入1-（3-氨基丙基）-4-甲基哌嗪，然后室温反应，制备UV光响应性超支化聚β-氨基酯。
  4. 根据权利要求2所述UV光响应性超支化聚β-氨基酯的制备方法，其特征在于，2-硝基-1,3-二丙烯酸间苯二甲酯与三羟甲基丙烷三丙烯酸酯以及4-氨基-1-丁醇、1-（3-氨基丙基）-4-甲基哌嗪的摩尔比为0.6﹕0.25﹕1﹕1；50～60℃反应的时间为5～8 h；室温反应的时间为10～15 h；以2,6-二甲基硝基苯和氧化剂为原料，制备2-硝基-1, 3-苯二甲酸；以2-硝基-1, 3-苯二甲酸和硼烷为原料，制备2-硝基-1, 3-苯二甲醇；以2-硝基-1,3-苯二甲醇和丙烯酰氯为原料，制备2-硝基-1,3-二丙烯酸间苯二甲酯。
  5. 根据权利要求4所述UV光响应性超支化聚β-氨基酯的制备方法，其特征在于，氧化剂为高锰酸钾；在胺催化剂下，以2-硝基-1,3-苯二甲醇和丙烯酰氯为原料，在反应溶剂中制备2-硝基-1,3-二丙烯酸间苯二甲酯。
  6. 一种纳米药物的制备方法，其特征在于，权利要求1所述UV光响应性超支化聚β-氨基酯复合药物得到纳米药物。
  7. 一种纳米药物, 其特征在于，所述纳米药物由权利要求1所述UV光响应性超支化聚β-氨基酯复合药物得到。
  8. 根据权利要求7所述纳米药物，其特征在于，所述药物为核酸；所述聚UV光响应性超支化β-氨基酯与核酸的质量比为（0.5～50）∶1。
  9. 根据权利要求7所述纳米药物，其特征在于，所述纳米药物的粒径为120～170 nm；所述纳米药物的Zeta电势为15～35 mV。
  10. 权利要求1所述UV光响应性超支化聚β-氨基酯在制备药物载体中的应用或者在制备纳米药物中的应用；或者权利要求7所述纳米药物在制备基因药物中的应用。
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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004083450A2 (en) * 2003-03-21 2004-09-30 The General Hospital Corporation D/B/A Massachusetts General Hospital Hyperbranched dendron and methods of synthesis and use thereof
CN102627767A (zh) * 2012-03-29 2012-08-08 华南理工大学 基于聚-β氨基酯的pH响应无规共聚物及其制法和应用
CN103243122A (zh) * 2013-04-03 2013-08-14 上海交通大学 含可降解亚胺键的核酸物质的载体、其制备方法及应用
US20170216455A1 (en) * 2014-08-06 2017-08-03 National University Of Ireland, Galway HYPERBRANCHED POLY (ß-AMINO ESTER) FOR GENE THERAPY
CN108524946A (zh) * 2018-04-04 2018-09-14 苏州大学 三元复合物纳米药物及其制备方法以及在制备光可控释放纳米递送体系中的应用
CN109988780A (zh) * 2019-03-19 2019-07-09 苏州大学 基于甲基丙烯酸缩水甘油酯的高性能基因载体及其应用
CN110746599A (zh) * 2019-09-30 2020-02-04 苏州大学 具有高效基因递送能力的UV光响应性超支化聚β-氨基酯及其制备方法与应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006118547A1 (en) * 2005-04-29 2006-11-09 Agency For Science, Technology And Research Hyperbranched polymers and their applications
WO2007143659A2 (en) * 2006-06-05 2007-12-13 Massachusetts Institute Of Technology Crosslinked, degradable polymers and uses thereof
WO2016081790A1 (en) * 2014-11-20 2016-05-26 Momentive Performance Materials Inc. Moisture curable compositions
CN109797167B (zh) * 2019-01-31 2020-06-12 广州鼓润医疗科技有限公司 一种靶向敲除人乳头瘤病毒urr基因的质粒、系统、制剂及制备方法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004083450A2 (en) * 2003-03-21 2004-09-30 The General Hospital Corporation D/B/A Massachusetts General Hospital Hyperbranched dendron and methods of synthesis and use thereof
CN102627767A (zh) * 2012-03-29 2012-08-08 华南理工大学 基于聚-β氨基酯的pH响应无规共聚物及其制法和应用
CN103243122A (zh) * 2013-04-03 2013-08-14 上海交通大学 含可降解亚胺键的核酸物质的载体、其制备方法及应用
US20170216455A1 (en) * 2014-08-06 2017-08-03 National University Of Ireland, Galway HYPERBRANCHED POLY (ß-AMINO ESTER) FOR GENE THERAPY
CN108524946A (zh) * 2018-04-04 2018-09-14 苏州大学 三元复合物纳米药物及其制备方法以及在制备光可控释放纳米递送体系中的应用
CN109988780A (zh) * 2019-03-19 2019-07-09 苏州大学 基于甲基丙烯酸缩水甘油酯的高性能基因载体及其应用
CN110746599A (zh) * 2019-09-30 2020-02-04 苏州大学 具有高效基因递送能力的UV光响应性超支化聚β-氨基酯及其制备方法与应用

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