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CN101696272A - Degradable material having multiple sensitive properties, manufacturing method thereof and use thereof - Google Patents

Degradable material having multiple sensitive properties, manufacturing method thereof and use thereof Download PDF

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CN101696272A
CN101696272A CN200910217778A CN200910217778A CN101696272A CN 101696272 A CN101696272 A CN 101696272A CN 200910217778 A CN200910217778 A CN 200910217778A CN 200910217778 A CN200910217778 A CN 200910217778A CN 101696272 A CN101696272 A CN 101696272A
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degradable material
oligoethylenimine
cross
linked
sensitive properties
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陈学思
李非凡
田华雨
王献红
景遐斌
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Changchun Institute of Applied Chemistry of CAS
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Changchun Institute of Applied Chemistry of CAS
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Abstract

本发明涉及一种可降解的具有多重敏感性能的材料、制法和应用。将寡聚乙烯亚胺与N,N′-双丙烯酰胱胺溶解于有机溶剂中,在加热条件下交联形成网状聚合物,再与N-异丙基丙烯酰胺共同溶于去离子水中,加热反应制得目标产物。通过本发明制备的交联网状聚合物同时具有对环境pH、温度和氧化还原敏感的多重性能,水溶液的可见光透过率随温度的升高,由100%逐渐降低为零,并且其最低临界共溶温度随着溶液pH值的升高而降低。在还原环境中,二硫键断裂,迅速降解为小分子化合物,随着温度的升高,可见光下溶液的透过率保持不变。该材料还能够携带正电荷,是一种新型的多重敏感的聚阳离子基因载体,细胞毒性小,具有广泛的应用前景。The invention relates to a degradable material with multiple sensitive properties, a preparation method and an application. Dissolve oligoethylenimine and N,N'-bisacrylcystamine in an organic solvent, cross-link under heating conditions to form a network polymer, and then dissolve in deionized water together with N-isopropylacrylamide , heating the reaction to obtain the target product. The cross-linked polymer prepared by the present invention has multiple properties sensitive to environmental pH, temperature and redox. The visible light transmittance of the aqueous solution gradually decreases from 100% to zero with the increase of temperature, and its minimum critical The melting temperature decreases as the pH of the solution increases. In a reducing environment, the disulfide bonds are broken and rapidly degraded into small molecular compounds. As the temperature increases, the transmittance of the solution under visible light remains unchanged. The material can also carry positive charges, is a new type of multi-sensitive polycationic gene carrier, has low cytotoxicity, and has wide application prospects.

Description

一种可降解的具有多重敏感性能的材料、制法和应用A degradable material, preparation method and application with multiple sensitive properties

技术领域 technical field

本发明涉及一种可降解的具有多重敏感性能的材料、制法和应用,一种基于寡聚乙烯亚胺、N-异丙基丙烯酰胺和N,N′-双丙烯酰胱胺的网状交联,可降解且具有pH、温度和氧化还原多重敏感聚合物及制法和其在基因传递中的应用。The invention relates to a degradable material with multiple sensitive properties, a preparation method and an application, a network based on oligoethylenimine, N-isopropylacrylamide and N, N'-bisacryloylcystamine Cross-linked, degradable and multi-sensitive polymer with pH, temperature and redox, its preparation method and its application in gene delivery.

背景技术 Background technique

近年来,智能材料的研究引起了科研人员的极大兴趣(1-3)。具有多重响应性的智能材料一直是该领域的一个热点,目前研究较多的是具有双重响应性的智能高分子材料,如pH-温度敏感高分子材料、温度-氧化还原敏感聚合物、电场-磁场敏感聚合物等,而两重响应性以上的多重敏感材料的研究较少,合成一般比较复杂,并且大多都是不可降解的聚合物(4-6),因此可降解的具有多重敏感性能的材料合成引起了该领域的众多关注。In recent years, the study of smart materials has aroused great interest of researchers (1-3). Smart materials with multiple responsiveness have always been a hot spot in this field. At present, more researches are on smart polymer materials with dual responsiveness, such as pH-temperature-sensitive polymer materials, temperature-redox-sensitive polymers, electric field- Magnetic field-sensitive polymers, etc., while the research on multiple sensitive materials with more than double responsiveness is less, the synthesis is generally more complicated, and most of them are non-degradable polymers (4-6), so degradable materials with multiple sensitive properties Materials synthesis has attracted numerous attentions in this field.

聚(N-异丙基丙烯酰胺)是一种简单而有效的温度敏感材料,然而,由于聚(N-异丙基丙烯酰胺)主链为聚烷烃(7-8),不可降解,极大地限制了其在生物医学领域的应用。Poly(N-isopropylacrylamide) is a simple and effective temperature-sensitive material, however, since the main chain of poly(N-isopropylacrylamide) is polyalkane (7-8), it is not degradable, greatly This limits its application in the biomedical field.

聚乙烯亚胺具有正电荷携带能力,在污水处理(9)和基因转染(10)领域获得了广泛应用。但聚乙烯亚胺是不可降解聚合物,因此限定了其在生物医学领域的应用,而寡聚乙烯亚胺由于分子量小,生物可以排泄出去,并且同样具有pH缓冲能力和正电荷携带能力,因此在生物医学领域获得了广泛关注(11)。而可降解且同时具有三重及以上敏感性能的基因载体材料未见报道。Polyethyleneimine has a positive charge-carrying ability and has been widely used in the fields of sewage treatment (9) and gene transfection (10). However, polyethyleneimine is a non-degradable polymer, so its application in the biomedical field is limited, and oligoethyleneimine can be excreted by organisms due to its small molecular weight, and it also has pH buffering capacity and positive charge carrying capacity, so in The field of biomedicine has gained a lot of attention (11). However, gene carrier materials that are degradable and have triple and above sensitive properties have not been reported.

发明内容 Contents of the invention

为了解决已有技术存在的问题,本发明通过将寡聚乙烯亚胺与N,N′-双丙烯酰胱胺交联形成网状聚合物,然后N-异丙基丙烯酰胺单体与聚合物表面氨基发生Michael加成反应连接到聚合物表面,制得的材料同时具有对环境pH、温度和氧化还原敏感的多重性能,在还原环境中可以降解,并且能够携带正电荷,是一种新型的多重敏感的聚阳离子基因载体,具有广泛的应用前景。In order to solve the problems in the prior art, the present invention cross-links oligoethylenimine and N, N'-bisacrylcystamine to form a network polymer, and then N-isopropylacrylamide monomer and polymer The surface amino group undergoes Michael addition reaction to connect to the surface of the polymer. The prepared material has multiple properties sensitive to environmental pH, temperature and redox. It can be degraded in a reducing environment and can carry positive charges. It is a new type of The multi-sensitive polycationic gene carrier has broad application prospects.

本发明涉及一种可降解的具有多重敏感性能的材料、制法和应用。The invention relates to a degradable material with multiple sensitive properties, a preparation method and an application.

一种可降解的具有多重敏感性能的材料,其结构式如下:A degradable material with multiple sensitive properties, its structural formula is as follows:

Figure G2009102177787D0000021
Figure G2009102177787D0000021

优选分子量为423~1800的寡聚乙烯亚胺。Oligoethylenimines with a molecular weight of 423-1800 are preferred.

一种可降解的具有多重敏感性能的材料的制备方法的步骤和条件如下:The steps and conditions of a preparation method of a degradable material with multiple sensitive properties are as follows:

(1)按照寡聚乙烯亚胺在有机溶剂中物质的量浓度为0.1~0.5mol/L,将寡聚乙烯亚胺和有机溶剂投料到干燥反应器内,搅拌溶解,然后按照寡聚乙烯亚胺与N,N′-双丙烯酰胱胺物质的量比为2∶3~3∶2,称取N,N′-双丙烯酰胱胺加入到反应器内,混合均匀,升温至25~70℃反应2~4天,旋蒸,浓缩反应液,于0℃的正己烷中沉降,真空干燥,得到的粗产物溶于去离子水中,于截留分子量为3,500的透析袋中对去离子水透析4天,G4砂芯漏斗过滤,冻干,得到中间产物交联寡聚乙烯亚胺(简称CEI);所述的有机溶剂为氯仿与甲醇的体积配比为1∶2~4∶1的混合溶剂;(1) According to the concentration of oligoethylene imine in the organic solvent is 0.1 ~ 0.5mol/L, feed the oligoethylene imine and organic solvent into the drying reactor, stir and dissolve, and then The ratio of amine to N, N'-bisacryloylcystamine is 2:3~3:2, weigh N,N'-bisacryloylcystamine and add it into the reactor, mix well, and heat up to 25~ Reaction at 70°C for 2 to 4 days, rotary evaporation, concentration of the reaction solution, sedimentation in n-hexane at 0°C, vacuum drying, the obtained crude product was dissolved in deionized water, and deionized water Dialyzed for 4 days, filtered through a G4 sand core funnel, and freeze-dried to obtain the intermediate product cross-linked oligoethyleneimine (CEI for short); the organic solvent is chloroform and methanol with a volume ratio of 1:2 to 4:1. Mixed solvent;

(2)按照交联寡聚乙烯亚胺在去离子水中物质的量为0.01~0.1mol/L,将交联寡聚乙烯亚胺和去离子水投料到干燥反应器内,搅拌溶解,然后按照交联寡聚乙烯亚胺与N-异丙基丙烯酰胺物质的量比为1∶50~1∶300,把N-异丙基丙烯酰胺加入到反应器内,混合均匀,于20~60℃反应3~4天,将反应液置于截留分子量为3,500的透析袋中对去离子水透析4天,G4砂芯漏斗过滤,冻干,得到一种N-异丙基丙烯酰胺修饰的交联寡聚乙烯亚胺(简称OEN)。其为可降解的具有多重敏感性能的材料。(2) According to the amount of cross-linked oligoethylenimine in deionized water is 0.01 ~ 0.1mol/L, feed cross-linked oligoethyleneimine and deionized water into the drying reactor, stir to dissolve, and then The ratio of cross-linked oligoethylenimine to N-isopropylacrylamide is 1:50~1:300. Add N-isopropylacrylamide into the reactor, mix well, and put it at 20~60℃ After reacting for 3 to 4 days, the reaction solution was placed in a dialysis bag with a molecular weight cut-off of 3,500 and dialyzed against deionized water for 4 days, filtered through a G4 sand core funnel, and freeze-dried to obtain a cross-linked N-isopropylacrylamide-modified Oligoethylene imine (referred to as OEN). It is a degradable material with multiple sensitive properties.

一种可降解的具有多重敏感性能的材料的多重敏感性能表征如下:The multiple sensitive properties of a degradable material with multiple sensitive properties are characterized as follows:

(1)温度敏感性和pH敏感性表征(1) Characterization of temperature sensitivity and pH sensitivity

配制N-异丙基丙烯酰胺修饰的交联寡聚乙烯亚胺在pH=8~13的缓冲水溶液,该溶液浓度为0.5~20mg/ml;用紫外可见分析光谱测试其在0.5℃/min匀速升温条件下,可见光波长下的透过率随温度的变化。Prepare N-isopropylacrylamide-modified cross-linked oligoethyleneimine buffered aqueous solution at pH = 8-13, the solution concentration is 0.5-20mg/ml; use UV-visible analysis spectrum to test its constant speed at 0.5°C/min The transmittance at visible wavelengths as a function of temperature at elevated temperatures.

(2)氧化还原敏感性表征(2) Redox Sensitivity Characterization

配制N-异丙基丙烯酰胺修饰的交联寡聚乙烯亚胺在pH=8~13的缓冲水溶液,该溶液浓度为0.5~20mg/ml;按照N-异丙基丙烯酰胺修饰的交联寡聚乙烯亚胺与二硫苏糖醇物质的量比为5∶1~20∶1加入二硫苏糖醇,室温下搅拌30min,用紫外可见分析光谱测试其在0.5℃/min匀速升温条件下,可见光波长下的透过率随温度的变化。Prepare N-isopropylacrylamide-modified cross-linked oligoethylenimine buffered aqueous solution at pH = 8-13, the solution concentration is 0.5-20 mg/ml; according to N-isopropylacrylamide modified cross-linked oligo The ratio of polyethyleneimine to dithiothreitol is 5:1-20:1. Add dithiothreitol, stir at room temperature for 30 minutes, and use UV-visible analysis spectrum to test it under the condition of 0.5°C/min uniform temperature rise. , the transmittance at visible wavelengths as a function of temperature.

一种可降解的具有多重敏感性能的材料在体外或体内转染中作为基因传递载体。A degradable material with multiple sensitive properties is used as a gene delivery vehicle in in vitro or in vivo transfection.

一种可降解的具有多重敏感性能的材料在体外转染中作为基因传递载体的用法,其步骤和条件如下:The use of a degradable material with multiple sensitive properties as a gene transfer carrier in in vitro transfection, the steps and conditions are as follows:

(1)细胞的培养(1) Cell culture

将细胞置于含体积分数为10%胎牛血清的培养液中,在37℃含体积分数为5%二氧化碳的孵箱中连续培养;The cells were placed in a culture solution containing 10% fetal bovine serum by volume, and continuously cultured in an incubator containing 5% carbon dioxide by volume at 37°C;

(2)体外转染(2) In vitro transfection

转染前24小时内,取对数生长期细胞,胰酶消化后用达尔伯克改良伊格尔(DMEM)培养基稀释,按每孔1×104细胞的密度接种于96孔培养板,置于37℃含体积分数为5%二氧化碳的孵箱中继续培养至汇合度达到80~90%,转染时,吸弃前一天加注的细胞培养板中的培养液,用磷酸盐缓冲液(PBS)洗涤两次后,基因组转染的复合物颗粒以及无血清或者含有体积分数为10%胎牛血清的DMEM培养基至终体积200μl,继续培养24小时;Within 24 hours before transfection, cells in the logarithmic growth phase were collected, digested with trypsin, diluted with Dulbecco’s modified Eagle (DMEM) medium, and seeded in a 96-well culture plate at a density of 1× 104 cells per well. Place in an incubator at 37°C containing 5% carbon dioxide by volume and continue culturing until the confluence reaches 80-90%. (PBS) After washing twice, the complex particles of genome transfection and the DMEM medium without serum or containing 10% fetal bovine serum to a final volume of 200 μl were cultured for 24 hours;

(3)体外转染的观测(3) Observation of in vitro transfection

取出培养板,将其置于荧光激发显微镜下观察转染效果,并拍照记录;Take out the culture plate, place it under a fluorescence excitation microscope to observe the transfection effect, and take pictures for record;

(4)体外转染效率的测定(4) Determination of in vitro transfection efficiency

取出培养板,吸去培养液,用PBS洗涤2次,加入细胞裂解液裂解,然后加入荧光素酶底物,用照度计测定转染效率;Take out the culture plate, suck off the culture medium, wash twice with PBS, add cell lysate to lyse, then add luciferase substrate, and measure the transfection efficiency with an illuminance meter;

(5)细胞毒性测试(5) Cytotoxicity test

采用噻唑蓝(MTT)比色法比较评价基因载体材料的细胞毒性。The cytotoxicity of gene carrier materials was compared and evaluated by thiazolium blue (MTT) colorimetry.

实验前24小时内,取对数生长期细胞,胰酶消化后用DMEM培养基稀释,按每孔1×104细胞的密度接种于96孔培养板,置于37℃含体积分数为5%二氧化碳的孵箱中继续培养至汇合度到达80~90%,将不同浓度的材料与细胞共同培养24小时后,每孔分别加入20μl含质量分数为0.5%MTT的PBS溶液,混合物在37℃继续作用4小时,加入200μl二甲基亚砜溶解MTT甲臢结晶10分钟,然后用酶标仪测试每孔的吸收,测试波长选用492nm,细胞存活率按下公式计算:Within 24 hours before the experiment, cells in the logarithmic growth phase were taken, digested with trypsin, diluted with DMEM medium, seeded in a 96-well culture plate at a density of 1× 104 cells per well, and placed at 37°C with a volume fraction of 5%. Continue culturing in a carbon dioxide incubator until the confluence reaches 80-90%. After co-cultivating cells with different concentrations of materials for 24 hours, add 20 μl of PBS solution with a mass fraction of 0.5% MTT to each well, and the mixture continues at 37°C. After acting for 4 hours, add 200 μl dimethyl sulfoxide to dissolve MTT formazan crystals for 10 minutes, then use a microplate reader to test the absorption of each well, the test wavelength is 492nm, and the cell survival rate is calculated according to the formula:

细胞存活率(%)=(Asample/Acontrol)×100Cell viability (%)=(A sample /A control )×100

Asample是转染后的细胞样品孔的吸收,Acontrol是不与复合物溶液作用的细胞样品孔的吸收,每组实验重复三次。A sample is the absorption of the cell sample well after transfection, and A control is the absorption of the cell sample well that does not interact with the complex solution. Each group of experiments was repeated three times.

一种可降解的具有多重敏感性能的材料在体内转染中作为基因传递载体的用法,其步骤和条件如下:The use of a degradable material with multiple sensitive properties as a gene transfer carrier in in vivo transfection, the steps and conditions are as follows:

取5周的巴比赛裸鼠,在腹侧皮下接种人宫颈癌上皮细胞的细胞株,待肿瘤直径长至0.4cm后,随机分为两组,每组6只,分别在瘤内注射含5μg荧光素酶质粒的基因组转染的复合物颗粒溶液100μl。第二天重复注射一次,注射后48小时,用精诺真活体成像仪观测体内转染效果。在进行活体成像观察之前,将小鼠麻醉。麻醉5分钟后,向小鼠体内注入200μl荧光素酶底物,10分钟后,用活体成像系统观察体内转染效果。Take 5-week-old Basset nude mice, subcutaneously inoculate the cell line of human cervical cancer epithelial cells on the ventral side, and divide them into two groups at random after the tumor diameter grows to 0.4 cm. Genomic transfection complex particle solution of luciferase plasmid was 100 μl. The injection was repeated the next day, and 48 hours after the injection, the in vivo transfection effect was observed with a Jinguogen live imager. Mice were anesthetized prior to intravital imaging observations. After 5 minutes of anesthesia, 200 μl of luciferase substrate was injected into the mice, and 10 minutes later, the in vivo transfection effect was observed with an in vivo imaging system.

有益效果:本发明制备的材料同时具有温度敏感、pH敏感、氧化还原敏感三种响应性能,在pH=8~13的缓冲水溶液配置浓度为0.5~20mg/ml的溶液,该溶液的可见光透过率随温度的升高,由100%逐渐降低为零,并且其最低临界共溶温度随着溶液pH值的升高而降低,随着溶液浓度的升高也降低。当加入二硫苏糖醇后,二硫键断裂,迅速降解为小分子化合物,随着温度的升高,可见光下溶液的透过率保持不变。并且具有携带正电荷的能力,能够作为可降解且同时具有三重及以上敏感性能的基因载体材料实现基因转染,细胞毒性在浓度10μg/ml以上的同等浓度下远小于PEI25K。Beneficial effects: the material prepared by the present invention has three response properties: temperature sensitive, pH sensitive, and redox sensitive. A solution with a concentration of 0.5 to 20 mg/ml is prepared in a buffered aqueous solution with a pH of 8 to 13, and the visible light of the solution is transmitted through The rate decreases gradually from 100% to zero with the increase of temperature, and its minimum critical solution temperature decreases with the increase of solution pH value, and decreases with the increase of solution concentration. When dithiothreitol is added, the disulfide bond breaks and rapidly degrades into small molecular compounds. As the temperature increases, the transmittance of the solution under visible light remains unchanged. And it has the ability to carry positive charges, and can be used as a gene carrier material that is degradable and has triple or above sensitive properties to realize gene transfection. The cytotoxicity is much lower than that of PEI25K at the same concentration of 10 μg/ml or more.

附图说明 Description of drawings

图1是浓度为5mg/ml的pH=9.2的缓冲液的溶液透过率随温度的变化曲线图。从左至右依次为OEN-2-1/1-300,OEN-2-1/1-200,OEN-2-1/1-100。Fig. 1 is a graph showing the variation of solution permeability with temperature of a buffer solution with a concentration of 5 mg/ml at pH = 9.2. From left to right are OEN-2-1/1-300, OEN-2-1/1-200, OEN-2-1/1-100.

图2是浓度为5mg/ml的OEN-2-1/1-300在不同pH的缓冲溶液中的溶液透过率随温度的变化曲线图。从左至右依次为pH=10.0,pH=8.8,pH=8.1。Fig. 2 is a graph showing the variation of solution permeability with temperature of OEN-2-1/1-300 with a concentration of 5 mg/ml in buffer solutions of different pH. From left to right, pH=10.0, pH=8.8, pH=8.1.

图3是pH=9.2的OEN-2-1/1-300的不同浓度溶液的透过率随温度的变化曲线图。从左至右依次为pH=10mg/ml,pH=5mg/ml,pH=1mg/ml。Fig. 3 is a graph showing the variation of transmittance with temperature of different concentration solutions of OEN-2-1/1-300 with pH=9.2. From left to right, pH=10mg/ml, pH=5mg/ml, pH=1mg/ml.

图4是浓度为5mg/ml的OEN-2-1/1-300溶液在pH=9.0加入二硫苏糖醇反应前后的溶液透过率随温度的变化曲线图。曲线为加入二硫苏糖醇之前的溶液透过率随温度的变化趋势;水平直线为加入二硫苏糖醇之后的溶液透过率随温度的变化趋势。Fig. 4 is a graph showing the variation of solution permeability with temperature before and after adding dithiothreitol to the OEN-2-1/1-300 solution with a concentration of 5 mg/ml at pH = 9.0. The curve is the variation trend of solution permeability with temperature before adding dithiothreitol; the horizontal straight line is the variation trend of solution permeability with temperature after adding dithiothreitol.

图5是OEN-3-3/2-50/GFP重量比为15/1时,使用HeLa细胞体外转染24h的效果图。Fig. 5 is an effect diagram of HeLa cells transfected in vitro for 24 hours when the weight ratio of OEN-3-3/2-50/GFP is 15/1.

具体实施方式 Detailed ways

实施例1:用分子量为423的线形寡聚乙烯亚胺制备目标产物Example 1: Preparation of the target product with a linear oligoethylenimine with a molecular weight of 423

按照寡聚乙烯亚胺在有机溶剂中物质的量浓度为0.2mol/L,称取4.23g分子量为423的线形寡聚乙烯亚胺溶于50ml氯仿与甲醇的等体积体积混合溶剂,然后分别按照表1的寡聚乙烯亚胺与N,N′-双丙烯酰胱胺物质的量比,称取N,N′-双丙烯酰胱胺加入到反应器内,混合均匀,升温至45℃反应4天,旋蒸,浓缩反应液,于1000ml 0℃的正己烷中沉降,真空干燥,将得到的粗产物溶于去离子水中,于截留分子量为3,500的透析袋中对去离子水透析4天,G4砂芯漏斗过滤,冻干,得到中间产物交联寡聚乙烯亚胺。称取0.5g交联寡聚乙烯亚胺按0.02mol/L溶于去离子水中,然后按照分别按照表1的交联寡聚乙烯亚胺与N-异丙基丙烯酰胺物质的量比,把N-异丙基丙烯酰胺加入到反应器内,混合均匀,于45℃下反应3天,将反应液置于截留分子量为3,500的透析袋中对去离子水透析4天,G4砂芯漏斗过滤,冻干,分别得到目标产物N-异丙基丙烯酰胺修饰的交联寡聚乙烯亚胺。According to the concentration of oligoethyleneimine in the organic solvent, the concentration of the substance is 0.2mol/L, and 4.23g of linear oligoethyleneimine with a molecular weight of 423 is dissolved in an equal volume mixed solvent of 50ml of chloroform and methanol, and then respectively according to The ratio of oligoethylenimine to N, N'-bisacryloylcystamine in Table 1, weigh N, N'-bisacryloylcystamine and add it into the reactor, mix well, and heat up to 45°C for reaction For 4 days, spin evaporate, concentrate the reaction solution, settle in 1000ml of n-hexane at 0°C, dry in vacuum, dissolve the obtained crude product in deionized water, and dialyze against deionized water in a dialysis bag with a molecular weight cut-off of 3,500 for 4 days , filtered through a G4 sand core funnel, and freeze-dried to obtain the intermediate product cross-linked oligoethylenimine. Weigh 0.5g of cross-linked oligoethyleneimine and dissolve it in deionized water at 0.02mol/L, then according to the ratio of cross-linked oligoethyleneimine and N-isopropylacrylamide in Table 1 respectively, put Add N-isopropylacrylamide into the reactor, mix well, react at 45°C for 3 days, place the reaction solution in a dialysis bag with a molecular weight cut-off of 3,500 and dialyze against deionized water for 4 days, filter with a G4 sand core funnel , freeze-dried to obtain the target product N-isopropylacrylamide modified cross-linked oligopolyethyleneimine respectively.

表1:用分子量为423的线形寡聚乙烯亚胺制备目标产物Table 1: Preparation of target products with linear oligoethylenimine with a molecular weight of 423

  目标产物编号target product number   寡聚乙烯亚胺与N,N′-双丙烯酰胱胺物质的量比The ratio of oligoethylenimine to N,N'-bisacryloylcystamine   交联寡聚乙烯亚胺与N-异丙基丙烯酰胺物质的量比The molar ratio of cross-linked oligoethylenimine to N-isopropylacrylamide   分子量molecular weight   PDIPDI   OEN-1-2/3-50OEN-1-2/3-50   2/32/3   1/501/50   42304230   1.451.45   OEN-1-1/1-50OEN-1-1/1-50   1/11/1   1/501/50   82008200   1.501.50   OEN-1-3/2-50OEN-1-3/2-50   3/23/2   1/501/50   1042010420   1.621.62

  目标产物编号target product number   寡聚乙烯亚胺与N,N′-双丙烯酰胱胺物质的量比The ratio of oligoethylenimine to N,N'-bisacryloylcystamine   交联寡聚乙烯亚胺与N-异丙基丙烯酰胺物质的量比The molar ratio of cross-linked oligoethylenimine to N-isopropylacrylamide   分子量molecular weight   PDIPDI   OEN-1-2/3-100OEN-1-2/3-100   2/32/3   1/1001/100   66706670   1.551.55   OEN-1-1/1-100OEN-1-1/1-100   1/11/1   1/1001/100   95009500   1.651.65   OEN-1-3/2-100OEN-1-3/2-100   3/23/2   1/1001/100   1292012920   1.741.74   OEN-1-2/3-300OEN-1-2/3-300   2/32/3   1/3001/300   92009200   1.561.56   OEN-1-1/1-300OEN-1-1/1-300   1/11/1   1/3001/300   1030010300   1.631.63   OEN-1-3/2-300OEN-1-3/2-300   3/23/2   1/3001/300   1340013400   1.681.68

实施例2:用分子量为600的超支化寡聚乙烯亚胺制备目标产物Embodiment 2: prepare target product with molecular weight being 600 hyperbranched oligoethylenimine

按照寡聚乙烯亚胺在有机溶剂中物质的量浓度为0.2mol/L,称取6g分子量为600的超支化寡聚乙烯亚胺溶于50ml氯仿与甲醇的等体积体积混合溶剂,然后分别按照表2的寡聚乙烯亚胺与N,N′-双丙烯酰胱胺物质的量比,称取N,N′-双丙烯酰胱胺加入到反应器内,混合均匀,升温至45℃反应4天,旋蒸,浓缩反应液,于1000ml0℃的正己烷中沉降,真空干燥,将得到的粗产物溶于去离子水中,于截留分子量为3,500的透析袋中对去离子水透析4天,G4砂芯漏斗过滤,冻干,得到中间产物交联寡聚乙烯亚胺。称取0.5g交联寡聚乙烯亚胺按0.02mol/L溶于去离子水中,然后分别按照表2的交联寡聚乙烯亚胺与N-异丙基丙烯酰胺物质的量比,把N-异丙基丙烯酰胺加入到反应器内,混合均匀,于45℃下反应3天,将反应液置于截留分子量为3,500的透析袋中对去离子水透析4天,G4砂芯漏斗过滤,冻干,分别得到目标产物N-异丙基丙烯酰胺修饰的交联寡聚乙烯亚胺。According to the amount concentration of oligopolyethyleneimine in the organic solvent, the concentration of the substance is 0.2mol/L, and 6g of hyperbranched oligopolyethyleneimine with a molecular weight of 600 is dissolved in an equal-volume mixed solvent of 50ml chloroform and methanol, and then respectively according to The ratio of oligoethylenimine to N,N'-bisacrylcystamine in Table 2, weigh N,N'-bisacrylcystamine and add it to the reactor, mix well, and heat up to 45°C for reaction For 4 days, spin evaporate, concentrate the reaction solution, settle in 1000ml of n-hexane at 0°C, and vacuum-dry. G4 sand core funnel was filtered and freeze-dried to obtain the intermediate product cross-linked oligoethylenimine. Weigh 0.5g of cross-linked oligoethylene imine and dissolve it in deionized water at 0.02mol/L, and then mix N - Add isopropylacrylamide into the reactor, mix well, react at 45°C for 3 days, place the reaction solution in a dialysis bag with a molecular weight cut-off of 3,500 and dialyze against deionized water for 4 days, filter with a G4 sand core funnel, Freeze-dry to obtain the target product N-isopropylacrylamide modified cross-linked oligopolyethyleneimine respectively.

表2:用分子量为600的超支化寡聚乙烯亚胺制备目标产物Table 2: Preparation of target products with hyperbranched oligoethylenimine with a molecular weight of 600

  目标产物编号target product number   寡聚乙烯亚胺与N,N′-双丙烯酰胱胺物质的量比The ratio of oligoethylenimine to N,N'-bisacryloylcystamine   交联寡聚乙烯亚胺与N-异丙基丙烯酰胺物质的量比The molar ratio of cross-linked oligoethylenimine to N-isopropylacrylamide   分子量molecular weight   PDIPDI   OEN-2-2/3-50OEN-2-2/3-50   2/32/3   1/501/50   63406340   1.481.48 OEN-2-1/1-50OEN-2-1/1-50 1/11/1 1/501/50 95309530 1.551.55   OEN-2-3/2-50OEN-2-3/2-50   3/23/2   1/501/50   1240012400   1.601.60

  目标产物编号target product number   寡聚乙烯亚胺与N,N′-双丙烯酰胱胺物质的量比The ratio of oligoethylenimine to N,N'-bisacryloylcystamine   交联寡聚乙烯亚胺与N-异丙基丙烯酰胺物质的量比The molar ratio of cross-linked oligoethylenimine to N-isopropylacrylamide   分子量molecular weight   PDIPDI OEN-2-2/3-100OEN-2-2/3-100 2/32/3 1/1001/100 87208720 1.461.46   OEN-2-1/1-100OEN-2-1/1-100   1/11/1   1/1001/100   1050010500   1.701.70 OEN-2-3/2-100OEN-2-3/2-100 3/23/2 1/1001/100 1632016320 1.651.65 OEN-2-2/3-300OEN-2-2/3-300 2/32/3 1/3001/300 1063010630 1.591.59 OEN-2-1/1-300OEN-2-1/1-300 1/11/1 1/3001/300 1330013300 1.621.62 OEN-2-3/2-300OEN-2-3/2-300 3/23/2 1/3001/300 1842018420 1.711.71

实施例3:用分子量为1800的超支化寡聚乙烯亚胺制备目标产物。Example 3: The target product was prepared with a hyperbranched oligoethylenimine with a molecular weight of 1800.

按照寡聚乙烯亚胺在有机溶剂中物质的量浓度为0.1mol/L,称取9g分子量为1800的超支化寡聚乙烯亚胺溶于50ml氯仿与甲醇的等体积体积混合溶剂,然后分别按照表3的寡聚乙烯亚胺与N,N′-双丙烯酰胱胺物质的量比,称取N,N′-双丙烯酰胱胺加入到反应器内,混合均匀,升温至45℃反应4天,旋蒸,浓缩反应液,于1000ml0℃的正己烷中沉降,真空干燥,将得到的粗产物溶于去离子水中,于截留分子量为3,500的透析袋中对去离子水透析4天,G4砂芯漏斗过滤,冻干,得到中间产物交联寡聚乙烯亚胺。称取0.5g交联寡聚乙烯亚胺按0.02mol/L溶于去离子水中,然后分别按照表3的交联寡聚乙烯亚胺与N-异丙基丙烯酰胺物质的量比,把N-异丙基丙烯酰胺加入到反应器内,混合均匀,于45℃下反应3天,将反应液置于截留分子量为3,500的透析袋中对去离子水透析4天,G4砂芯漏斗过滤,冻干,得到目标产物N-异丙基丙烯酰胺修饰的交联寡聚乙烯亚胺,投料比例见表3。According to the amount concentration of oligopolyethyleneimine in the organic solvent, the concentration of the substance is 0.1mol/L, and 9g of hyperbranched oligopolyethyleneimine with a molecular weight of 1800 is dissolved in an equal-volume mixed solvent of 50ml chloroform and methanol, and then respectively according to The ratio of oligoethylenimine to N,N'-bisacryloylcystamine in Table 3, weigh N,N'-bisacryloylcystamine and add it to the reactor, mix well, and heat up to 45°C for reaction For 4 days, spin evaporate, concentrate the reaction solution, settle in 1000ml of n-hexane at 0°C, and vacuum-dry. G4 sand core funnel was filtered and freeze-dried to obtain the intermediate product cross-linked oligoethylenimine. Weigh 0.5g of cross-linked oligoethylene imine and dissolve it in deionized water at 0.02mol/L, and then mix N - Add isopropylacrylamide into the reactor, mix well, react at 45°C for 3 days, place the reaction solution in a dialysis bag with a molecular weight cut-off of 3,500 and dialyze against deionized water for 4 days, filter with a G4 sand core funnel, Freeze-dry to obtain the target product N-isopropylacrylamide modified cross-linked oligoethylenimine, and the feeding ratio is shown in Table 3.

表3:用分子量为1800的超支化寡聚乙烯亚胺制备目标产物Table 3: Preparation of target products with hyperbranched oligoethylenimine with a molecular weight of 1800

  目标产物编号target product number   寡聚乙烯亚胺与N,N′-双丙烯酰胱胺物质的量比The ratio of oligoethylenimine to N,N'-bisacryloylcystamine   交联寡聚乙烯亚胺与N-异丙基丙烯酰胺物质的量比The molar ratio of cross-linked oligoethylenimine to N-isopropylacrylamide   分子量molecular weight   PDIPDI   OEN-3-2/3-50OEN-3-2/3-50   2/32/3   1/501/50   75207520   1.521.52 OEN-3-1/1-50OEN-3-1/1-50 1/11/1 1/501/50 1065010650 1.721.72 OEN-3-3/2-50OEN-3-3/2-50 3/23/2 1/501/50 1350013500 1.591.59   OEN-3-2/3-100OEN-3-2/3-100   2/32/3   1/1001/100   1025010250   1.631.63   OEN-3-1/1-100OEN-3-1/1-100   1/11/1   1/1001/100   1430014300   1.761.76   OEN-3-3/2-100OEN-3-3/2-100   3/23/2   1/1001/100   1520015200   1.651.65   OEN-3-2/3-300OEN-3-2/3-300   2/32/3   1/3001/300   1268012680   1.521.52   OEN-3-1/1-300OEN-3-1/1-300   1/11/1   1/3001/300   1725017250   1.601.60   OEN-3-3/2-300OEN-3-3/2-300   3/23/2   1/3001/300   1958019580   1.791.79

实施例4:目标产物温度敏感性表征Embodiment 4: Characterization of target product temperature sensitivity

分别称取25mg实施例1-3得到的各种目标产物,分别溶于5ml的pH=9.2的缓冲液中,分别得到5mg/ml的溶液,用紫外可见分析光谱分别测试其在0.5℃/min匀速升温条件下,550nm波长处的透过率随温度的变化,见图1。Weigh 25 mg of various target products obtained in Examples 1-3, respectively, dissolve them in 5 ml of pH=9.2 buffer solution, respectively obtain 5 mg/ml solutions, and test their temperature at 0.5 °C/min by UV-visible analysis spectrum respectively. Under the condition of uniform temperature rise, the transmittance at the wavelength of 550nm varies with temperature, as shown in Figure 1.

实施例5:目标产物OEN-2-1/1-300的pH敏感性表征Example 5: Characterization of the pH sensitivity of the target product OEN-2-1/1-300

称取25mg OEN-2-1/1-300,分别溶于5ml的pH=8.2、8.8、9.2、10.0的缓冲液中,得到5mg/ml的溶液,用紫外可见分析光谱测试其在0.5℃/min匀速升温条件下,550nm波长处的透过率随温度的变化,见图2。Weigh 25mg of OEN-2-1/1-300, dissolve them in 5ml of buffer solution with pH=8.2, 8.8, 9.2, and 10.0 respectively to obtain a 5mg/ml solution, and test its temperature at 0.5℃/ml by UV-visible analysis spectrum. Under the condition of uniform temperature rise in min, the transmittance at 550nm wavelength varies with temperature, see Figure 2.

实施例6:目标产物OEN-2-1/1-300的氧化还原敏感性表征Example 6: Redox Sensitivity Characterization of the Target Product OEN-2-1/1-300

称取25mg OEN-2-1/1-300,溶于5ml的pH=9.0的缓冲液中,得到5mg/ml的溶液。另称取25mg的OEN-2-1/1-300,溶于5ml的pH=9.0的缓冲液中,得到5mg/ml的溶液,然后加入3mg二硫苏糖醇,室温下搅拌30min。用紫外可见分析光谱测试这两种溶液在0.5℃/min匀速升温条件下,550nm波长处的透过率随温度的变化,见图4。Weigh 25mg of OEN-2-1/1-300 and dissolve in 5ml of pH=9.0 buffer to obtain a 5mg/ml solution. Another 25 mg of OEN-2-1/1-300 was weighed and dissolved in 5 ml of pH=9.0 buffer to obtain a 5 mg/ml solution, then 3 mg of dithiothreitol was added and stirred at room temperature for 30 min. The transmittance at a wavelength of 550nm varies with the temperature of the two solutions under the condition of a constant temperature increase of 0.5°C/min using ultraviolet-visible analysis spectroscopy, as shown in Figure 4.

实施例7:目标产物OEN-2-1/1-300的温度敏感性随浓度变化的表征Example 7: Characterization of the temperature sensitivity of the target product OEN-2-1/1-300 as a function of concentration

称取5mg,25mg,50mg,100mgOEN-2-1/1-300,分别溶于5ml的pH=9.2的缓冲液中,分别得到1mg/ml,5mg/ml,10mg/ml,20mg/ml的溶液,用紫外可见分析光谱测试其在0.5℃/min匀速升温条件下,550nm波长处的透过率随温度的变化,见图3。Weigh 5mg, 25mg, 50mg, 100mg of OEN-2-1/1-300 and dissolve in 5ml of pH=9.2 buffer to obtain 1mg/ml, 5mg/ml, 10mg/ml, 20mg/ml solutions respectively , using ultraviolet-visible analysis spectrum to test its transmittance at 550nm wavelength with temperature under the condition of 0.5°C/min constant temperature rise, as shown in Figure 3.

实施例8:目标产物介导荧光素酶质粒对人子宫颈癌细胞(HeLa细胞)的体外转染Example 8: In vitro transfection of target product-mediated luciferase plasmids to human cervical cancer cells (HeLa cells)

1、HeLa细胞的培养1. Culture of HeLa cells

取HeLa细胞置于含体积分数为10%胎牛血清的培养液中,在37℃含体积分数为5%二氧化碳的孵箱中连续培养。HeLa cells were placed in culture medium containing 10% fetal bovine serum by volume, and continuously cultured in an incubator containing 5% carbon dioxide by volume at 37°C.

2、体外转染2. In vitro transfection

转染前24小时内,取对数生长期HeLa细胞,胰酶消化后用DMEM稀释,按每孔1×104细胞的密度接种于96孔培养板,置于5%CO2、37℃孵箱中继续培养至汇合度到达80~90%。转染时,吸弃前一天加注的细胞培养板中的培养液,更换200μl/孔含有10%胎牛血清的新鲜DMEM培养液,再选用绿色荧光蛋白质粒(GFP)分别加入实施例1-3得到的各种目标产物与GFP及PEI25k/GFP的复合物颗粒进行转染对比,继续培养24小时。Within 24 hours before transfection, HeLa cells in the logarithmic growth phase were collected, digested with trypsin, diluted with DMEM, seeded in a 96-well culture plate at a density of 1×10 4 cells per well, and incubated in 5% CO 2 at 37°C Continue culturing in the box until the confluence reaches 80-90%. During transfection, the culture solution in the cell culture plate added the day before was sucked and discarded, and 200 μl/well was replaced with fresh DMEM culture solution containing 10% fetal bovine serum. 3 The obtained various target products were transfected with GFP and PEI25k/GFP complex particles for comparison, and cultured for 24 hours.

3、体外转染的观测3. Observation of in vitro transfection

取出培养板,将其置于荧光激发显微镜下观察转染效果,并拍照记录,见图5,说明目标产物能够作为基因载体在体外细胞内实现转染。Take out the culture plate, place it under a fluorescence excitation microscope to observe the transfection effect, and take pictures and record it, as shown in Figure 5, which shows that the target product can be used as a gene carrier to achieve transfection in cells in vitro.

实施例9:目标产物的细胞毒性测试Embodiment 9: Cytotoxicity test of target product

1、HeLa细胞的培养1. Culture of HeLa cells

取HeLa细胞置于含体积分数为10%胎牛血清的培养液中,在37℃含体积分数为5%二氧化碳的孵箱中连续培养。HeLa cells were placed in culture medium containing 10% fetal bovine serum by volume, and continuously cultured in an incubator containing 5% carbon dioxide by volume at 37°C.

2、毒性测试2. Toxicity test

采用MTT的方法比较评价实施例1-3得到的各种目标产物和PEI25k的材料细胞毒性。实验前24小时内,取对数生长期的HeLa细胞,胰酶消化后用DMEM稀释,按每孔1×104细胞的密度接种于96孔培养板,置于37℃含体积分数为5%二氧化碳的孵箱中继续培养至汇合度达到80~90%。将不同浓度的材料与细胞共同培养24小时后,每孔分别加入20μl含质量分数为0.5%MTT的PBS溶液。混合物在37℃继续作用4小时,加入200μl二甲基亚砜溶解MTT甲臢结晶10分钟。然后用酶标仪测试每孔的吸收,测试波长选用492nm。细胞存活率按下公式计算:The MTT method was used to compare and evaluate the cytotoxicity of various target products obtained in Examples 1-3 and PEI25k. Within 24 hours before the experiment, take the HeLa cells in the logarithmic growth phase, trypsinize them, dilute them with DMEM, inoculate them in a 96-well culture plate at a density of 1× 104 cells per well, and store them at 37°C with a volume fraction of 5%. Continue culturing in a carbon dioxide incubator until the confluence reaches 80-90%. After co-cultivating the materials with different concentrations for 24 hours, 20 μl of PBS solution containing 0.5% MTT was added to each well. The mixture was continued at 37°C for 4 hours, and 200 µl of dimethyl sulfoxide was added to dissolve the MTT formazan crystals for 10 minutes. Then use a microplate reader to test the absorption of each well, and the test wavelength is 492nm. Cell viability was calculated according to the formula:

细胞存活率(%)=(Asample/Acontrol)×100Cell viability (%)=(A sample /A control )×100

Asample是转染后的细胞样品孔的吸收,Acontrol是不与复合物溶液作用的细胞样品孔的吸收,每组实验重复三次,所得目标产物的细胞毒性在浓度10μg/ml以上的同等浓度下远小于PEI25K。A sample is the absorption of the cell sample well after transfection, and A control is the absorption of the cell sample well that does not interact with the complex solution. Each group of experiments is repeated three times, and the cytotoxicity of the obtained target product is at the same concentration above 10 μg/ml The lower is much smaller than PEI25K.

实施例10:OEN-3-3/2-50和OEN-3-1/1-100介导体内转染实验Example 10: OEN-3-3/2-50 and OEN-3-1/1-100 mediate in vivo transfection experiments

取5周的巴比赛裸鼠,在腹侧皮下接种人宫颈癌上皮细胞的细胞株,待肿瘤直径长至0.4cm后,随机分为两组,每组6只,分别在瘤内注射含5μg荧光酶质粒(pGL3)的OEN-3-3/2-50/pGL3、OEN-3-1/1-100/pGL3与PEI25K/pGL3复合物颗粒溶液100μl。第二天重复注射一次,注射后48小时,用精诺真活体成像仪观测体内转染效果。在进行活体成像观察之前,将小鼠麻醉。麻醉5分钟后,向小鼠体内注入200μl荧光素酶底物。10分钟后,用活体成像系统观察体内转染效果。Take 5-week-old Basset nude mice, subcutaneously inoculate the cell line of human cervical cancer epithelial cells on the ventral side, and divide them into two groups at random after the tumor diameter grows to 0.4 cm. 100 μl of OEN-3-3/2-50/pGL3, OEN-3-1/1-100/pGL3 and PEI25K/pGL3 complex particle solution of luciferase plasmid (pGL3). The injection was repeated the next day, and 48 hours after the injection, the in vivo transfection effect was observed with a Jinguogen live imager. Mice were anesthetized prior to intravital imaging observations. After 5 min of anesthesia, inject 200 μl of luciferase substrate into the mouse. After 10 minutes, observe the transfection effect in vivo with an intravital imaging system.

本发明涉及的参考文献:References involved in the present invention:

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Claims (5)

1.一种可降解的具有多重敏感性能的材料,其特征在于,其结构式如下:1. A degradable material with multiple sensitive properties is characterized in that its structural formula is as follows:
Figure F2009102177787C0000011
Figure F2009102177787C0000011
2.如权利要求1所述的一种可降解的具有多重敏感性能的材料,其特征在于,寡聚乙烯亚胺的分子量为423~1800。2. A degradable material with multiple sensitive properties as claimed in claim 1, characterized in that the molecular weight of the oligoethylenimine is 423-1800. 3.权利要求1所述的一种可降解的具有多重敏感性能的材料的制备方法,其特征是步骤和条件如下:3. the preparation method of a kind of degradable material with multiple sensitive properties as claimed in claim 1 is characterized in that steps and conditions are as follows: (1)按照寡聚乙烯亚胺在有机溶剂中物质的量浓度为0.1~0.5mol/L,将寡聚乙烯亚胺和有机溶剂投料到干燥反应器内,搅拌溶解,然后按照寡聚乙烯亚胺与N,N′-双丙烯酰胱胺物质的量比为2∶3~3∶2,称取N,N′-双丙烯酰胱胺加入到反应器内,混合均匀,升温至25~70℃反应2~4天,旋蒸,浓缩反应液,于0℃的正己烷中沉降,真空干燥,得到的粗产物溶于去离子水中,于截留分子量为3,500的透析袋中对去离子水透析4天,G4砂芯漏斗过滤,冻干,得到中间产物交联寡聚乙烯亚胺;所述的有机溶剂为氯仿与甲醇的体积配比为1∶2~4∶1的混合溶剂;(1) According to the concentration of oligoethylene imine in the organic solvent is 0.1 ~ 0.5mol/L, feed the oligoethylene imine and organic solvent into the drying reactor, stir and dissolve, and then The ratio of amine to N, N'-bisacryloylcystamine is 2:3~3:2, weigh N,N'-bisacryloylcystamine and add it into the reactor, mix well, and heat up to 25~ Reaction at 70°C for 2 to 4 days, rotary evaporation, concentration of the reaction solution, sedimentation in n-hexane at 0°C, vacuum drying, the obtained crude product was dissolved in deionized water, and deionized water Dialyzed for 4 days, filtered through a G4 sand core funnel, and freeze-dried to obtain the intermediate product cross-linked oligoethylenimine; the organic solvent is a mixed solvent with a volume ratio of chloroform and methanol of 1:2 to 4:1; (2)按照交联寡聚乙烯亚胺在去离子水中物质的量为0.01~0.1mol/L,将交联寡聚乙烯亚胺和去离子水投料到干燥反应器内,搅拌溶解,然后按照交联寡聚乙烯亚胺与N-异丙基丙烯酰胺物质的量比为1∶50~1∶300,把N-异丙基丙烯酰胺加入到反应器内,混合均匀,于20~60℃反应3~4天,将反应液置于截留分子量为3,500的透析袋中对去离子水透析4天,G4砂芯漏斗过滤,冻干,得到N-异丙基丙烯酰胺修饰的交联寡聚乙烯亚胺。(2) According to the amount of cross-linked oligoethylenimine in deionized water is 0.01 ~ 0.1mol/L, feed cross-linked oligoethyleneimine and deionized water into the drying reactor, stir to dissolve, and then The ratio of cross-linked oligoethylenimine to N-isopropylacrylamide is 1:50~1:300. Add N-isopropylacrylamide into the reactor, mix well, and put it at 20~60℃ After reacting for 3 to 4 days, the reaction solution was placed in a dialysis bag with a molecular weight cut-off of 3,500 and dialyzed against deionized water for 4 days, filtered through a G4 sand core funnel, and freeze-dried to obtain N-isopropylacrylamide-modified cross-linked oligomer Ethylene imine. 4.权利要求1所述的一种可降解的具有多重敏感性能的材料在基因传递中的应用,其特征在于,可降解的具有多重敏感性能的材料在体外转染中作为基因传递载体。4. The application of a degradable material with multiple sensitive properties in gene transfer according to claim 1, characterized in that the degradable material with multiple sensitive properties is used as a gene transfer carrier in in vitro transfection. 5.权利要求1所述的一种可降解的具有多重敏感性能的材料在基因传递中的应用,其特征在于,可降解的具有多重敏感性能的材料在体内转染中作为基因传递载体。5. The application of a degradable material with multiple sensitive properties in gene transfer according to claim 1, characterized in that the degradable material with multiple sensitive properties is used as a gene transfer carrier in vivo transfection.
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