寡聚荷正电氨基酸修饰的基因递送纳米脂质载体的构建和初步评价
发布时间：2019-05-24 19:40
【摘要】：目的： 脂质体已经成为生物医药技术领域里，最主要的药物、基因、影像剂的重要载体，是一种非病毒载体类载体，在基因传递中具有显著优势。但仍然存在生物大分子负载率低、摄取效率低、胞内释放性能欠佳、内涵体逃逸障碍等问题。因此，本文重在设计一种多能高效的生物大分子输送载体。 本文合成了能高效提高转染效率的DC-Chol，以及赋予脂质体pH敏感性能的CHEMS，对其化学结构进行表征。将DSPE-PEG(2000)与CPP共轭连接，得到导向性脂材DSPE-PEG-CPP，以提高脂质体对细胞的摄取能力。构建了一种能使siRNA冲破内涵体逃逸障碍，避免被溶酶体灭活的CPP修饰载压缩物脂质体CL2，和一种CPP修饰的非载压缩脂质体CL1，并对其进行表征和初步的评价。 方法：1胆固醇氯甲酸酯和过量N，N-二甲基乙二胺分别溶于无醇氯仿中，0-4℃冰水浴条件下反应。TLC监测反应进程，待胆固醇氯甲酸酯反应完全后终止反应（约2h），旋蒸挥干溶剂，干燥后用热绝对乙醇重结晶两次纯化并真空干燥。2胆固醇与过量的丁二酸酐溶于20mL甲苯， DMAP作为催化剂，加热回流反应3h。旋转蒸发挥干溶剂，干燥后重结晶两次，得到灰色产品。3Mal-PEG-DSPE用氯仿-甲醇（3:1）溶解，旋转成膜，HBS(pH6.5)溶液3.0mL水化，和CPP肽溶液（5mg溶于HBS(pH6.5)2.0mL）混合，搅拌反应48h，加入半胱氨酸适量继续搅拌4h，透析2d，冷冻干燥。4空白脂质体单因素考察，通过固定膜材总量；DOPE:CHEMS比例的筛选；添加Chol对制剂的影响；水化液选择；超声时间的选择，最终确定空白脂质体制备方法。5构建非压缩载药脂质体（NL1/CL1）和载压缩物脂质体（NL2和CL2），并进粒径、电位和电镜表征。 制备方法如下： 分别以DSPE-PEG、DSPE-PEG（CPP）成膜，DEPC水溶液水化，超声得胶束M1、M2。 非压缩载药脂质体（NL1/CL1）：以SPC/Chol/DC-Chol为脂材成膜，含siRNA的DEPC溶液水化脱膜，水浴超声10-15min，得到普通载药脂质体。通过后插入法分别与胶束M1和M2孵育4h，，得NL1和CL1。 载压缩物脂质体（NL2和CL2）：以DOPE/CHEMS/Chol为脂材成膜，DEPC水化液脱膜，超声得空白脂质体，与siRNA压缩物混合孵育后,冰浴条件调至pH7.4-7.8，再通过后插入法制备NL2和CL2。6以流式细胞术分析包载siRNA的NL1＆CL1和包载压缩siRNA的NL2＆CL2处理的MCF-7细胞，以阳性细胞百分数和胞内平均荧光强度（MFI）为考察指标，评价细胞摄取效率。 结果：1合成DC-Chol：白色固体粉末1.621g,产率达到60.3%;熔点:107-108℃;TLC(氯仿-甲醇=65：35（v/v）,10%H2SO4显色)， Rf=0.56，显示为纯品;质谱图中m/z501.6，与DC-Chol的理论计算值501.80（M+H+）相符；1HNMR：δ,2.301（s，6H）；δ，2.495（t，2H）； δ，3.305，3.296（q，2H）；δ，5.365（s，1H)。证明产物结构正确。2合成CHEMS：将无水乙醇重结晶干燥后，得到浅灰色固体粉末0.49g，产率78%；熔点：173-176℃；TLC测定（氯仿：甲醇（10:1，v/v），新生成斑点Rf=0.10，显示为纯品；质谱图中有离子峰m/z485.5，与CHEMS的理论计算值为485.5（M-H+）相符。证明产物结构正确。3合成DSPE-PEG-CPP：得到白色絮状物；通过Ellman试剂测得CPP反应率为84.4%；MS+检测，分子离子峰m/z4469.17，与设计肽平理论计算值相对应。证明产物为目标化合物为DSPE-PEG-CPP。4通过单因素考察，确定脂质体制备工艺： 精密量取DOPE、CHEMS、Chol按5:2:2（mol/mol/mol）成膜，加入37℃DEPC水溶液2.0mL水化，超声10-15min，得到带有淡蓝色乳光的半透明体系，测粒径。5构建了非压缩载药脂质体（NL1/CL1）和载压缩物脂质体（NL2和CL2），并进粒径、电位和电镜表征。 NL1、CL1的粒径分别为226.8nm、262.4nm；后者PdI较小，粒径分布窄;Zeta分别为37.0mV、55.7mV，体系稳定；电镜下观察，粒径约200nm左右。 NL2、CL2粒径分别为289.8nm、293.2nm；两组制剂PdI都很小，粒径分布窄；Zeta分别为-29.6mV、-29.2mV，脂质体表面带负电，体系趋于稳定;电镜下观察，粒径约200nm左右。6采用流式细胞分析术分析经药物制剂处理的细胞悬液 非压缩制剂组N-L和C-L进入细胞的荧光强度X-mean分别为9.1,10.4，是阴性对照组和游离siRNA组的20倍左右，由此说明CPP修饰非压缩载药脂质体（C-L制剂）、普通修饰非压缩载药脂质体（N-L）均提高了细胞摄取率，CPP修饰非压缩载药脂质体具有更高的细胞摄取率。 载压缩物制剂组N-L’、C-L’阳性细胞百分比为6.4%、7.9%，其阳性细胞百分数均有显著变化，提高细胞摄取效率约10倍，表明载压缩物制剂均提高了细胞摄取效率，CPP修饰载压缩物脂质体具有更高的细胞摄取率。 结论： 实验结果表明，DC-Chol以及CHEMS合成路线可靠，产率分别为60.3%、78%，产品纯度较高，重现性高。 由Ellman试剂判断反应进程，质谱测定得出的分子离子峰推测产物结构，成功地将CPP共轭连接到DSPE-PEG（2000）中的PEG远端，得到增强细胞穿透能力的导向性脂材DSPE-PEG（2000）-CPP。 成功地制备了四组递送siRNA的递送纳米脂质载体CPP修饰非压缩载药脂质体（NL1）、普通修饰非压缩载药脂质体（NL2）、普通修饰在压缩物脂质体（CL1）、CPP修饰载压缩物脂质体（CL2）。 四组制剂均提高了细胞摄取效率。普通脂质体与CPP修饰的脂质体的细胞摄取相对较高，但差异并不显著。导致这个结果的可能的原因是，如DSPE-PEG-CPP合成产物中有较多剩余脂材原料，反应条件有待进一步优化，产物纯度有待提高。
[Abstract]:Purpose: The liposome has become one of the most important carriers of medicine, gene and image agent in the technical field of biological medicine, However, there are still some questions about the low loading rate of the biological macromolecule, the low uptake efficiency, the poor in-cell release performance, the obstacle of the escape of the connotation, etc. Therefore, this paper focuses on the design of a multi-functional and efficient biological macromolecule transport carrier In this paper, we synthesize the DC-Chol which can improve the efficiency of the transfection, and the CHEMS which gives the pH-sensitive properties of the liposomes to the chemical structure. Line characterization. The DSPE-PEG (2000) was co-linked to the CPP to obtain a guided lipid, DSPE-PEG-CPP, to increase the uptake of the liposome to the cells. The invention provides a CPP modified carrier liposome CL2 which can cause the siRNA to break through the connotation body to escape, avoid the CPP modified carrier liposome CL2 which is inactivated by the lysosome, and a CPP modified non-loaded compressed liposome CL1, The method comprises the following steps:1-cholesterol chloroformate and an excess of N, N-dimethyl ethylenediamine are respectively dissolved in the alcohol-free chloroform, and the ice water bath at the temperature of 0-4 DEG C under conditions, the reaction is monitored by TLC, the reaction process is monitored by TLC, the reaction is terminated after the reaction of the chloroformate is complete (about 2 hours), the solvent is evaporated and dried, the solvent is recrystallized twice with hot absolute ethanol and dried in vacuum, and the cholesterol and the excess of the butylenic acid are dissolved in 20 mL of toluene and the DMAP As a catalyst, heat back The reaction was carried out for 3 h. The dry solvent was evaporated and recrystallized twice to obtain the gray product. The 3Mal-PEG-DSPE was dissolved with chloroform-methanol (3:1), the film was spin-formed, the HBS (pH 6.5) solution was hydrated, and the CPP peptide solution (5 mg dissolved in HBS (pH 6.5) 2.0 mL) was mixed, stirred for 48 h, and a proper amount of cysteine was added for stirring for 4 h. , dialyzing for 2d, freeze-drying.4 blank liposome single-factor investigation, through fixed membrane material total amount; DOPE: CHEMS ratio selection; addition of Chol on preparation; hydration liquid selection; ultrasound The preparation method of the blank liposome is finally determined by the time selection, and the non-compressed drug-loaded liposome (NL1/ CL1) and the carrier-loaded liposome (NL2 and CL2) are constructed, and the particle size and the potential are kept. And electron microscopy. The preparation method comprises the following steps: respectively forming DSPE-PEG and DSPE-PEG (CPP), and hydrating the DEPC aqueous solution, Acoustic micelle M1, M2. Non-compressed drug-loaded liposomes (NL1/ CL1): film-forming with SPC/ Chol/ DC-Chol, water-bath ultrasonic 10-15 mi, water bath ultrasound 10-15 mi, n, the general drug-carrying liposome is obtained, and the drug-carrying liposome is respectively incubated with the micelles M1 and M2 through the post-insertion method, and 4 hours to obtain NL1 and CL1, carrying the compressed object liposome (NL2 and CL2), taking DOPE/ CHEMS/ Chol as a lipid material to form a film, decoating the DEPC hydration liquid, performing ultrasonic to obtain a blank liposome, and mixing the liposome with the siRNA compact, and then carrying out ice bath The conditions were adjusted to pH 7.4-7.8, and then NL2 and CCL2 were prepared by the post-insertion method. The NL1 & CL1 and NL2 & CL2-treated MCF-7 cells of the encapsulated siRNA were analyzed by flow cytometry, and the percentage of positive cells and the intracellular mean fluorescence intensity (MFI) were The results were as follows:1. Synthesis of DC-Chol: white solid powder 1.621 g, yield of 60.3%; melting point:107-108 鈩
本文编号：2485128
[Abstract]:Purpose: The liposome has become one of the most important carriers of medicine, gene and image agent in the technical field of biological medicine, However, there are still some questions about the low loading rate of the biological macromolecule, the low uptake efficiency, the poor in-cell release performance, the obstacle of the escape of the connotation, etc. Therefore, this paper focuses on the design of a multi-functional and efficient biological macromolecule transport carrier In this paper, we synthesize the DC-Chol which can improve the efficiency of the transfection, and the CHEMS which gives the pH-sensitive properties of the liposomes to the chemical structure. Line characterization. The DSPE-PEG (2000) was co-linked to the CPP to obtain a guided lipid, DSPE-PEG-CPP, to increase the uptake of the liposome to the cells. The invention provides a CPP modified carrier liposome CL2 which can cause the siRNA to break through the connotation body to escape, avoid the CPP modified carrier liposome CL2 which is inactivated by the lysosome, and a CPP modified non-loaded compressed liposome CL1, The method comprises the following steps:1-cholesterol chloroformate and an excess of N, N-dimethyl ethylenediamine are respectively dissolved in the alcohol-free chloroform, and the ice water bath at the temperature of 0-4 DEG C under conditions, the reaction is monitored by TLC, the reaction process is monitored by TLC, the reaction is terminated after the reaction of the chloroformate is complete (about 2 hours), the solvent is evaporated and dried, the solvent is recrystallized twice with hot absolute ethanol and dried in vacuum, and the cholesterol and the excess of the butylenic acid are dissolved in 20 mL of toluene and the DMAP As a catalyst, heat back The reaction was carried out for 3 h. The dry solvent was evaporated and recrystallized twice to obtain the gray product. The 3Mal-PEG-DSPE was dissolved with chloroform-methanol (3:1), the film was spin-formed, the HBS (pH 6.5) solution was hydrated, and the CPP peptide solution (5 mg dissolved in HBS (pH 6.5) 2.0 mL) was mixed, stirred for 48 h, and a proper amount of cysteine was added for stirring for 4 h. , dialyzing for 2d, freeze-drying.4 blank liposome single-factor investigation, through fixed membrane material total amount; DOPE: CHEMS ratio selection; addition of Chol on preparation; hydration liquid selection; ultrasound The preparation method of the blank liposome is finally determined by the time selection, and the non-compressed drug-loaded liposome (NL1/ CL1) and the carrier-loaded liposome (NL2 and CL2) are constructed, and the particle size and the potential are kept. And electron microscopy. The preparation method comprises the following steps: respectively forming DSPE-PEG and DSPE-PEG (CPP), and hydrating the DEPC aqueous solution, Acoustic micelle M1, M2. Non-compressed drug-loaded liposomes (NL1/ CL1): film-forming with SPC/ Chol/ DC-Chol, water-bath ultrasonic 10-15 mi, water bath ultrasound 10-15 mi, n, the general drug-carrying liposome is obtained, and the drug-carrying liposome is respectively incubated with the micelles M1 and M2 through the post-insertion method, and 4 hours to obtain NL1 and CL1, carrying the compressed object liposome (NL2 and CL2), taking DOPE/ CHEMS/ Chol as a lipid material to form a film, decoating the DEPC hydration liquid, performing ultrasonic to obtain a blank liposome, and mixing the liposome with the siRNA compact, and then carrying out ice bath The conditions were adjusted to pH 7.4-7.8, and then NL2 and CCL2 were prepared by the post-insertion method. The NL1 & CL1 and NL2 & CL2-treated MCF-7 cells of the encapsulated siRNA were analyzed by flow cytometry, and the percentage of positive cells and the intracellular mean fluorescence intensity (MFI) were The results were as follows:1. Synthesis of DC-Chol: white solid powder 1.621 g, yield of 60.3%; melting point:107-108 鈩
本文编号：2485128
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