甘露糖受体介导的树突状细胞靶向载体的构建与评价
发布时间：2018-11-17 20:33
【摘要】： 背景与目的:机体抗肿瘤的免疫机制十分复杂,它涉及多种免疫成分,包括体液免疫和细胞免疫。它们互相协同杀伤肿瘤细胞。一般认为,以T细胞、NK细胞、巨噬细胞和树突状细胞为主的细胞免疫发挥着重要的作用。如果能研究一种可靶向树突状细胞或者巨噬细胞的给药系统,则有望提高抗原靶向树突状细胞或者巨噬细胞的能力,增加抗原提呈,促进免疫应答,增强抗肿瘤作用。本文根据树突状细胞和巨噬细胞表面有甘露糖受体的表达,制备了含有模型蛋白(卵清蛋白,OVA)的甘露糖修饰的壳聚糖包衣的乳酸-羟基乙酸共聚物(PLGA)纳米粒,研究其靶向树突状细胞和巨噬细胞的能力。 方法:合成了甘露糖修饰的壳聚糖之后,采用复乳法制备了含有OVA的PLGA纳米粒,壳聚糖(CS)以及甘露糖修饰的壳聚糖(MAN-CS)包衣的PLGA纳米粒。研究了不同因素,对纳米粒粒径、载药量和体外释放的影响。接着考察了纳米粒的浓度和孵育时间对于巨噬细胞的毒性的影响,以及通过荧光酶标仪、荧光显微镜、流式细胞仪和共聚焦显微镜考察不同孵育温度、时间、浓度,甘露糖对于巨噬细胞摄取的影响,和纳米粒在细胞内的定位。最后分离小鼠骨髓的单个核细胞,体外诱导其分化形成树突状细胞;通过流式细胞仪分析树突状细胞对纳米粒的摄取,以及激光共聚焦显微镜(CLSM)考察纳米粒在细胞内的定位。 结果:通过红外光谱、核磁图以及元素分析确认了已合成出甘露糖修饰的壳聚糖。PLGA纳米粒制备的最优处方为内水相pH值4.0,PLGA浓度为30mg·mL-1,外水相PVA浓度为5%,油水相比为1:3,超声次数为120次。使用最优处方制得的纳米粒粒径在255nm左右,OVA的包封率约为70%,载药量约为8%。CS包衣纳米粒的粒径和zeta电位,随着CS浓度的增加而增加。在内水相中加入PEG1000、外水相添加蔗糖、CS和MAN-CS包衣均能较少PLGA纳米粒的突释。纳米粒对巨噬细胞基本不产生毒性。4℃时,巨噬细胞的吞噬受到抑制;8h时,巨噬细胞对于纳米粒的摄取已接近饱和;在实验的浓度范围内,巨噬细胞对于游离的FITC-OVA和纳米粒的摄取是逐渐增加的。甘露糖能抑制了MAN-CS包衣的PLGA纳米粒的摄取。荧光酶标仪、流式细胞仪结果表明,巨噬细胞对MAN-CS包衣的纳米粒摄取最多;CLSM表明纳米粒的主要定位在细胞质内。小鼠骨髓的单个核细胞在体外用GM-CSF和IL-4诱导可分化为未成熟树突状细胞,其表面高表达CD11c;流式细胞仪的结果表明,树突状细胞对含有FITC-OVA纳米粒的摄取比游离的FITC-OVA多,且对MAN-CS包衣的纳米粒摄取最多。 结论:本文通过单因素考察确定了PLGA纳米粒制备的最优处方,使用此处方制备纳米粒重现性好;以PLGA纳米粒制备的最优处方为基础,采用复乳法成功地制备出含有OVA的CS以及MAN-CS包衣的PLGA纳米粒。MTT实验表明纳米粒的毒性较低。体外细胞实验表明MAN-CS包衣的PLGA纳米粒能有效地被树突状细胞和巨噬细胞摄取。
[Abstract]:BACKGROUND & OBJECTIVE: The anti-tumor immune mechanism of the body is very complex, and it involves many kinds of immune components, including humoral immunity and cell immunity. They co-act with each other to kill tumor cells. It is generally believed that cellular immunity, which is dominated by T cells, NK cells, macrophages and dendritic cells, plays an important role. if a drug delivery system is capable of targeting dendritic cells or macrophages, it is expected to improve that ability of the antigen to target the dendritic cell or the macrophage, increase the antigen presentation, promote the immune response, and enhance the anti-tumor effect. according to the expression of the mannose receptor on the surface of the dendritic cells and the macrophages, the lactic acid-glycolic acid copolymer (PLGA) nanoparticles containing the mannose-modified chitosan-coated chitosan coated with the model protein (ovalbumin, OVA) are prepared, The ability to target dendritic cells and macrophages was investigated. Methods: After the chitosan-modified chitosan was synthesized, PLGA nanoparticles, chitosan (CS) and mannoose-modified chitosan (MAN-CS)-coated PLG were prepared by complex emulsion method. A nanoparticles were studied. The particle size, drug loading and in vitro release of the nanoparticles were studied. The effects of the concentration of the nanoparticles and the incubation time on the toxicity of the macrophages were then examined, and the different incubation temperatures, time, concentration, mannose for macrophages were examined by means of a fluorescence enzyme marker, a fluorescence microscope, a flow cytometer, and a confocal microscope. the effect of taking, and the nano-particles in the cell, In vitro, the individual nuclear cells of the bone marrow of the mouse were isolated and their differentiation was induced to form a dendritic cell. The uptake of the nanoparticles by the dendritic cells was analyzed by flow cytometry, and the nano-particles were examined by a laser confocal microscope (CLSM). Internal positioning. Result: The synthesized output is confirmed by the infrared spectrum, the nuclear magnetic diagram, and the element analysis the optimal formulation prepared by the mannoose modified chitosan and the PLGA nano-particle is the internal water phase pH value of 4.0, the PLGA concentration is 30mg/ mL-1, the concentration of the external water phase PVA is 5%, the oil-water ratio is 1: 3, The ultrasonic frequency was 120 times. The particle size of the nanoparticles was about 255nm, the encapsulation efficiency of OVA was about 70%, and the drug loading was about 8%. The particle size and zeta potential of the CS-coated nanoparticles were as follows: The increase of S concentration was increased. The addition of PEG1000 and the addition of sucrose, CS and MAN-CS in the internal water phase could be less P The release of the LGA nanoparticles. The nanoparticles were substantially free of toxicity to the macrophages. At 4 鈩，
本文编号：2338966
[Abstract]:BACKGROUND & OBJECTIVE: The anti-tumor immune mechanism of the body is very complex, and it involves many kinds of immune components, including humoral immunity and cell immunity. They co-act with each other to kill tumor cells. It is generally believed that cellular immunity, which is dominated by T cells, NK cells, macrophages and dendritic cells, plays an important role. if a drug delivery system is capable of targeting dendritic cells or macrophages, it is expected to improve that ability of the antigen to target the dendritic cell or the macrophage, increase the antigen presentation, promote the immune response, and enhance the anti-tumor effect. according to the expression of the mannose receptor on the surface of the dendritic cells and the macrophages, the lactic acid-glycolic acid copolymer (PLGA) nanoparticles containing the mannose-modified chitosan-coated chitosan coated with the model protein (ovalbumin, OVA) are prepared, The ability to target dendritic cells and macrophages was investigated. Methods: After the chitosan-modified chitosan was synthesized, PLGA nanoparticles, chitosan (CS) and mannoose-modified chitosan (MAN-CS)-coated PLG were prepared by complex emulsion method. A nanoparticles were studied. The particle size, drug loading and in vitro release of the nanoparticles were studied. The effects of the concentration of the nanoparticles and the incubation time on the toxicity of the macrophages were then examined, and the different incubation temperatures, time, concentration, mannose for macrophages were examined by means of a fluorescence enzyme marker, a fluorescence microscope, a flow cytometer, and a confocal microscope. the effect of taking, and the nano-particles in the cell, In vitro, the individual nuclear cells of the bone marrow of the mouse were isolated and their differentiation was induced to form a dendritic cell. The uptake of the nanoparticles by the dendritic cells was analyzed by flow cytometry, and the nano-particles were examined by a laser confocal microscope (CLSM). Internal positioning. Result: The synthesized output is confirmed by the infrared spectrum, the nuclear magnetic diagram, and the element analysis the optimal formulation prepared by the mannoose modified chitosan and the PLGA nano-particle is the internal water phase pH value of 4.0, the PLGA concentration is 30mg/ mL-1, the concentration of the external water phase PVA is 5%, the oil-water ratio is 1: 3, The ultrasonic frequency was 120 times. The particle size of the nanoparticles was about 255nm, the encapsulation efficiency of OVA was about 70%, and the drug loading was about 8%. The particle size and zeta potential of the CS-coated nanoparticles were as follows: The increase of S concentration was increased. The addition of PEG1000 and the addition of sucrose, CS and MAN-CS in the internal water phase could be less P The release of the LGA nanoparticles. The nanoparticles were substantially free of toxicity to the macrophages. At 4 鈩，
本文编号：2338966
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