微流控技术制备siRNA纳米载体的研究

发布时间：2018-08-20 08:55

【摘要】：纳米载体作为基因药物的有效递送系统自问世以来吸引大批科研人员的关注,然而在过去的50年里一直发展缓慢,主要归结于纳米制剂制备技术的落后。传统的制备技术重现性差,操作繁琐复杂,缺乏精确可控等缺点导致制备的纳米载体分散性差,批次差异大,严重的限制了纳米医学的发展。现如今,可控性好、重现性高、稳定性好的微流控技术已开始用于纳米载体的生产制备过程中,提高制备效率、缩短制备时间、简化操作工序并制备出分散均一狭小的纳米颗粒。本论文将阳离子脂质与聚乙烯亚胺联合应用构建多聚物-脂质复合纳米载体(Polymer-lipid hybrid nanoparticles,P/LNPs),并设计两种微流控芯片(MF1,MF2)制备P/LNPs(P/LNPs-MF1,P/LNPs-MF2)用于递送VEGF siRNA,并考察P/LNPs-MF的载体稳定性、安全性以及递送siRNA能力,而乙醇注入方法(BM)制备的P/LNPs(P/LNPs-BM)则作为阳性对照。论文具体内容概括如下:1.P/LNPs-MF处方组成和制备工艺优化本文首先对微流控芯片技术制备的P/LNPs-MF进行处方组成和制备工艺的优化,以期制备出大小均匀分散的P/LNPs-MF。实验结果显示,DODMA,EggPC,Cholesterol,PEI和DSPE-mPEG2000以40/19/35/5/1的处方比例,在0.8mg/ml的汇合流速下与siRNA混合,并经过超声后处理,能制备出大小合适、分布均匀、稳定性好的P/LNPs-MF纳米粒,其中以P/LNPs-MF2效果最佳,粒径为119.8nm,PDI为0.096,电位为11.3mV,血清孵育12h后仍稳定,4℃放置30天也不发生明显变化,而传统制法的P/LNPs-BM纳米粒则粒径较大且呈多分散性分布。2.P/LNPs-MF的体外抗肿瘤评价其次,本文对微流控芯片技术制备的载siRNA的P/LNPs-MF进行体外细胞实验的考察,以评价P/LNPs-MF载体安全性以及递送siRNA的转染入胞效率、基因沉默效率和肿瘤细胞抑制效率。MTT实验结果显示,各空白P/LNPs细胞存活率均在80%以上,相反地,载siRNA的P/LNPs-MF则发挥显著的抑制作用,相比于P/LNPs-BM仅20%左右的抑制效率,P/LNPs-MF2作用48h能抑制多达50%的癌细胞活性,是前者效率的两倍;流式细胞仪与激光共聚焦显微镜观察结果显示P/LNPs-MF2在A549、HepG-2和MCF-7各细胞系中细胞摄取效果最高,而P/LNPs-BM与P/LNPs-MF1的效果相似且均低于P/LNPs-MF2;qRT-RCR测定VEGF mRNA结果表示,载siRNA的P/LNPs-MF2能抑制62.2%VEGF mRNA的表达,其抑制效果分别比P/LNPs-MF1与P/LNPs-BM多12.6%和19.9%;Western-blot测定VEGF蛋白结果与qRT-RCR一致,均证明了P/LNPs-MF2的基因沉默效率比P/LNPs-MF1与P/LNPs-BM更显著。3.P/LNPs-MF的体内抗肿瘤评价最后,本文对微流控芯片技术制备的载siRNA的P/LNPs-MF进行体内动物实验的考察,以评价P/LNPs-MF在体内药物代谢作用、肿瘤生长抑制作用、基因沉默作用以及安全毒副作用。药代学参数结果显示,P/LNPs-MF2避免了网状内皮系统的消除作用并延长了siRNA在体内的循环时间,这将有利于增加siRNA在肿瘤部位的积累;体内肿瘤抑制作用结果表明,P/LNPs-MF2能抑制肿瘤体积至300mm3左右,持续给药后肿瘤生长停滞并出现负增长,最终肿瘤大小缩小至0.23g,而P/LNPs-BM抑制效果不明显,肿瘤体积达到800mm3左右;P/LNPs-MF2作用后肿瘤组织切片观察发现肿瘤细胞出现大面积变形和死亡,而P/LNPs-BM只杀灭了小部分的肿瘤细胞;体内基因沉默研究结果进一步显示载siRNA的P/LNPs-MF2显著的抑制VEGF mRNA和蛋白表达水平,诱导强烈的基因沉默作用。此外,裸鼠的生理体征、各器官组织切片及血清参数均表明P/LNPs在体内安全可靠,是安全高效的siRNA递送载体。综上所述,通过微流控技术构建的P/LNPs-MF纳米载体,在载体理化性质、siRNA递送效率等方面,均优于传统乙醇注入技术制备的P/LNPs-BM。可见,微流控芯片作为一种新的纳米制剂制备技术,促进了纳米给药系统的发展进步,为纳米制剂下一步的放大生产以及临床研究提供了技术支持。
[Abstract]:Nanocarriers as an effective delivery system for gene drugs have attracted the attention of a large number of researchers since their appearance. However, the development of nanocarriers has been slow in the past 50 years, mainly due to the backwardness of the preparation technology of nanoparticles. Nowadays, microfluidic technology with good controllability, reproducibility and stability has been used in the production and preparation of nanocarriers, which can improve the preparation efficiency, shorten the preparation time, simplify the operation procedure and prepare uniform and narrow nanoparticles. Polymer-lipid hybrid nanoparticles (P/LNPs) were constructed by the combination of cationic lipids and polyethylenimide. Two microfluidic chips (MF1, MF2) were designed to prepare P/LNPs (P/LNPs-MF1, P/LNPs-MF2) for the delivery of VEGF siRNA. The stability, safety and performance of P/LNPs-MF were investigated. P/LNPs-BM prepared by ethanol injection method (BM) was used as the positive control. The main contents of this paper are summarized as follows: 1. The formulation and preparation process of P/LNPs-MF prepared by microfluidic chip technology were optimized to prepare uniformly dispersed P/LNPs-MF. The experimental results showed that DODMA, EggPC, Cholesterol, PEI and DSPE-mPEG2000 were mixed with siRNA at the confluence flow rate of 0.8mg/ml at the prescription ratio of 40/19/35/5/1. After ultrasonic treatment, P/LNPs-MF nanoparticles with proper size, uniform distribution and good stability were prepared. Among them, P/LNPs-MF2 had the best effect, with a particle size of 119.8nm, PDI of 0.096, and potential of 0.8mg/ml. At 11.3 mV, the serum remained stable after incubation for 12 hours, and there was no significant change after incubation at 4 C for 30 days. However, the P/LNPs-BM nanoparticles prepared by traditional methods had larger particle size and more dispersive distribution. MTT assay showed that the survival rates of all blank P/LNPs cells were above 80%. On the contrary, the siRNA-loaded P/LNPs-MF showed a significant inhibitory effect, which was only 20% higher than that of P/LNPs-BM. The results of flow cytometry and confocal laser microscopy showed that P/LNPs-MF2 had the highest uptake effect in A549, HepG-2 and MCF-7 cell lines, while P/LNPs-BM and P/LNPs-MF1 had the similar effect and were lower than P/LNPs-MF2 in the determination of VEGF mRNA by qRT-RCR. P/LNPs-MF2 of iRNA could inhibit the expression of 62.2% VEGF mRNA, and the inhibitory effect was 12.6% and 19.9% more than that of P/LNPs-MF1 and P/LNPs-BM, respectively. Western-blot assay of VEGF protein was consistent with qRT-RCR, which proved that the gene silencing efficiency of P/LNPs-MF2 was more significant than that of P/LNPs-MF1 and P/LNPs-BM. 3.P/LNPs-MF in vivo anti-tumor evaluation of microfluidics-MF. The siRNA-loaded P/LNPs-MF prepared by controlled-chip technique was investigated in vivo in order to evaluate the drug metabolism, tumor growth inhibition, gene silencing and safety toxicity of P/LNPs-MF. Pharmacokinetic parameters showed that P/LNPs-MF2 avoided the elimination of reticuloendothelial system and prolonged siRNA in vivo. The results of in vivo tumor inhibition showed that P/LNPs-MF2 could inhibit the tumor volume to about 300 mm3. After continuous administration of P/LNPs-MF2, the tumor growth was stagnated and negative growth was observed, and the tumor size was reduced to 0.23 g. However, the inhibition effect of P/LNPs-BM was not obvious, and the tumor volume reached 800 mm3 left. Right; P/LNPs-MF2 induced large area of deformation and death of tumor cells, while P/LNPs-BM killed only a small part of tumor cells. In vivo gene silencing further showed that siRNA-loaded P/LNPs-MF2 significantly inhibited the expression of VEGF mRNA and protein, and induced strong gene silencing. The physiological signs, tissue sections and serum parameters of nude mice showed that P/LNPs were safe and reliable in vivo and were safe and efficient siRNA delivery vectors. It can be seen that microfluidic chip, as a new preparation technology of nanoparticles, promotes the development and progress of nanodrug delivery system, and provides technical support for the next step of nanoparticles scale-up production and clinical research.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R943

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