多功能性玉米醇溶蛋白基递送系统的构建及生物学评价

发布时间：2018-01-14 10:17

本文关键词：多功能性玉米醇溶蛋白基递送系统的构建及生物学评价　出处：《华中农业大学》2017年博士论文　论文类型：学位论文

【摘要】：天然高分子凭借其生物相容性好、可降解、易修饰等多种性质,已成为制备纳米材料的主要基材。通过自组装制备的天然高分子纳米载体更是由于其安全无毒、制备简单、表面易修饰、载药量高、控释性能好等优势,在药物及营养素递送方面受到越来越多的关注。目前,提高纳米粒子的稳定性和功能性,并实现高效靶向递送,已成为递送领域研究的研究热点。本文以玉米醇溶蛋白(zein)为主要基材,通过自组装的方式与两种不同形式的多糖(阴离子多糖—羧甲基纤维素钠和阳离子多糖—季铵盐壳聚糖)自组装形成纳米粒子。以疏水性药物紫杉醇(PTX)和疏水性营养素姜黄素(Cur)为模型负载物,研究纳米运载体系对疏水性物质的包封、保护以及体外缓释的性质,通过细胞实验验证载药纳米粒子的生物学特性。为进一步提高抗癌药物的疗效、降低毒副性,赋予运载体系定向性释放的性质,在已制备的纳米粒子表面形成一层单宁酸-金属离子包被膜,通过膜的pH-响应性崩解,赋予纳米粒子pH-响应性释药的性质。本文主要研究结果如下:1.选择PTX作为疏水模型药物,通过自组装方法制备负载PTX的玉米醇溶蛋白/羧甲基纤维素钠纳米粒子(PTX-zein/CMC纳米粒子)。结果显示当蛋白质与多糖质量比为1:3,投药量为80μg/mL时,所得纳米粒子粒径为159.4 nm,载药率高达93.5%。在此条件下可得到分散均匀的球形纳米粒子,且其具有良好的pH稳定性和储藏稳定性。体外缓释实验表明,相比于游离药物的快速释放,载药纳米粒子呈现明显的缓释特性。细胞吞噬实验表明,药物敏感的HepG2细胞和药物抗性的MCF-7细胞对香豆素6标记的纳米粒子的细胞吞噬均呈时间依赖性。同时,对于药物抗性的MCF-7细胞,在高给药浓度下,载药纳米粒子比游离药物显示更强的细胞毒性。细胞凋亡实验显示,对于抗性细胞,载药纳米粒子组引起的细胞凋亡比例大于游离药物的。免疫荧光实验表明载药纳米粒子和游离的PTX均是通过促进微管的增生导致细胞凋亡。2.研究阳离子多糖(季铵盐壳聚糖,HTCC)与zein的组装行为,利用自组装制备纳米粒子,用于疏水性营养素Cur的负载。通过对壳聚糖季铵化改性,大大提高了壳聚糖在中性条件以及碱性条件下的溶解性。改变蛋白质/多糖质量比可以得到不同粒径、不同多分散系数(PDI)、不同负载率的纳米粒子。当固定多糖为HTCC1(Mw 8.708×103),蛋白质/多糖质量比为1:1时,所得纳米粒子的负载率最高。与单纯的zein纳米粒子相比,形成的zein/HTCC纳米粒子分布更加均匀,形状更加规整。将Cur负载于纳米粒子可显著提高其光、热稳定性,且在相同的处理条件下相比于游离Cur具有更强的抗氧化活性。3.为了进一步提高构建的递送体系的高效性,在之前的研究基础上,引入了具有pH响应性的响应因子。基于单宁酸(TA)分子与蛋白质之间具有较强相互作用的邻苯三酚结构及其优异的金属螯合能力,以金属离子为交联剂在zein纳米粒子表面形成一层金属-有机涂层,成功构建了一种新型的具有pH响应性的纳米载体(zein-TA/metal纳米粒子)。通过透射电子显微镜(TEM)对其微观形貌进行观察,发现这种杂化纳米粒子具有更佳的分散性且粒径更加均一,克服了大部分纳米粒子在培养基中不稳定的问题。傅里叶红外光谱(FT-IR)分析表明,TA与金属离子之间存在较强的配位作用,同时TA与蛋白质之间也存在着氢键相互作用与静电相互作用。使用X射线光电子能谱(XPS)对纳米粒子表面元素进行分析,金属离子特征峰的出现以及C/O比值的显著变化均有效证明TA与金属离子的引入。随后以阿霉素(DOX)为模型药物,对其包封率、缓释性能进行研究。结果显示,相比于zein纳米粒子,包被纳米粒子对药物的包封率显著提高且在中性条件下(pH=7.4)具有良好的缓释性能,阻止了突释现象的发生。同时,由于TA-CuII复合膜具有pH响应性,赋予包被纳米粒子良好的pH响应性释药的性质。细胞吞噬实验表明,与游离的DOX相比,通过内吞作用进入细胞的纳米粒子速度较缓慢;对于包被的纳米粒子,由于对药物的pH响应性释放,孵育较长时间才能观察到细胞内DOX的红色荧光。位于包被纳米粒子表面的TA可以原位还原金纳米粒子,该体系为癌症的光热疗法提供了新思路。4.为了能更加精细的调控纳米载体的pH响应性,利用TA对基体的粘附性以及金属螯合能力、氨基与金属之间的配位作用,在zein纳米粒子中引入含有氨基的HTCC,在制备的zein/HTCC纳米粒子表面包被一层金属-有机涂层。同时利用TA-金属离子以及氨基-金属离子之间配位键的pH响应性,赋予纳米粒子pH响应释药的特性。与zein/HTCC纳米粒子相比,形成的包被纳米粒子(zein-HTCC/TA-metal纳米粒子)分散性更好,粒径分布更加均匀,形状更加规整。FT-IR研究表明,TA与金属离子之间存在较强的配位作用;同时TA,HTCC与蛋白质之间也存在着静电相互作用。XPS研究进一步验证了包被纳米粒子表面金属离子和TA的存在。以DOX为药物模型研究发现,包被的纳米粒子对药物的包封率相比于zein/HTCC纳米粒子也有显著的提高。体外缓释实验表明以不同金属离子作为交联中心及金属离子添加量的变化均会导致载药纳米粒子释放行为的变化。与zein-TA/metal纳米粒子相比,zein-HTCC/TA-metal纳米粒子呈现出更优异的刺激响应性。细胞吞噬实验显示未经包被的纳米粒子存在明显的突释现象,在培养基中即可快速释放DOX。对于包被纳米粒子,由于对药物的pH响应性释放,孵育较长时间才能观察到较强的荧光。同时引入稀土元素,使其同时具有pH响应性和生物成像的功能。5.考察金属离子种类及化学计量比对TA-金属离子包被纳米粒子(zein-TA/metal)性质的影响。金属离子种类可显著影响壳层厚度致使纳米粒子的粒径不同;金属离子浓度的增加会引起包被纳米粒子粒径的增加。在较低金属离子浓度下,包被的纳米粒子在培养基中的稳定性大大提到。但是对于TA-CuII包被的纳米粒子,随着金属离子浓度的增大,形成的纳米粒子在培养基中稳定性减小。随后的FT-IR分析得出金属离子的含量显著影响TA与金属离子的配位作用,从而影响包被纳米粒子吸收峰的移动;XPS进一步验证包被纳米粒子中金属离子以及TA的存在,且金属离子的浓度影响包被纳米粒子表面元素含量。细胞吞噬实验表明细胞对zein纳米粒子和zein-TA/metal纳米粒子的吞噬均呈现时间及浓度依赖性。细胞对包被纳米粒子表现出更快的吞噬速率以及更高的吞噬量。金属离子种类以及添加量影响细胞对纳米粒子的吞噬效率。通过激光共聚焦显微镜(CLSM)观察短时间细胞吞噬,细胞对包被纳米粒子粘附性更强,纳米粒子进入细胞速率更快。网格蛋白介导的吞噬作用是包被纳米粒子进入细胞的主要途径。同时包被纳米粒子进入细胞,细胞膜穴样凹陷介导的吞噬作用也可能起到一定作用。对于zein纳米粒子,胞饮作用对于纳米粒子进入细胞至关重要。进入到细胞内的纳米粒子可以和细胞质内溶酶体共定位,观察发现纳米粒子没有进入核区。
[Abstract]:Natural polymer with its good biocompatibility, biodegradable, easy modification and other properties, has become the main material in the preparation of nanomaterials. The natural polymer nanoparticles prepared by self assembly method is due to its non-toxic, simple preparation, easy modification, high load capacity, good controlled-release performance and other advantages. In drug and nutrient delivery has attracted more and more attention. At present, to improve the stability and function of nanoparticles, and to achieve efficient targeted delivery, has become a hot research topic in the field of research. Based on the delivery of zein (zein) as the main material, polysaccharide and two kinds of different forms by self-assembly the way (anionic polysaccharide sodium carboxymethyl cellulose and cationic polysaccharide chitosan quaternary ammonium salt) self-assembled into nanoparticles. The hydrophobic drug paclitaxel (PTX) and hydrophobic nutrients of curcumin (Cur) as the model load. The nano delivery system for hydrophobic material encapsulation, protection and properties of in vitro release, the biological characteristics of cells through the experiment of drug loaded nanoparticles. In order to further improve the efficacy of anticancer drugs, reduce toxicity, given the nature of the system carrying directional release, forming a layer of tannic acid metal ion coating film in nanoparticles the surface has been prepared by the pH- response, membrane disintegration, give pH- response properties of nanoparticles drug release. The main results of this paper are as follows: 1. PTX was selected as the hydrophobic drug model by self-assembly preparation method of PTX loaded zein / sodium carboxymethyl cellulose nanoparticles (PTX-zein/CMC NPs). The results showed when the polysaccharide and protein mass ratio was 1:3, the dosage is 80 g/mL, the particle size is 159.4 nm, the drug loading rate of 93.5%. under this condition can be evenly dispersed The spherical nanoparticles, and it has pH stability and good storage stability. The in vitro release experiment showed that, compared to the rapid release of free drug, drug loaded nanoparticles exhibit remarkable sustained-release property. Phagocytosis experiments show that nanoparticles drug sensitive HepG2 cells and drug resistance of MCF-7 cells to coumarin 6 phagocytic cells were labeled time dependent. At the same time, the drug resistance of MCF-7 cells in high concentration, drug loaded nanoparticles showed stronger cytotoxicity than the free drug. Experiments showed that apoptosis, resistance to cell apoptosis, the proportion of drug loaded nanoparticles was caused by higher than the free drug. Immunofluorescence experiments showed that the drug loaded nanoparticles and free PTX is the cause of apoptosis of.2. cationic polysaccharide by promoting the proliferation of microtubules (quaternary ammonium salt of chitosan, HTCC) and zein group with behavior, Prepared by the self-assembly of nanoparticles with hydrophobic nutrients for a load of Cur. Based on chitosan quaternary ammonium modified, greatly improving the solubility of chitosan in neutral condition and alkaline condition. Change the protein / polysaccharide ratio can be obtained with different particle size, different polydispersity index (PDI), nanoparticles different loading rate. When the fixed polysaccharide is HTCC1 (Mw 8.708 * 103), the protein / polysaccharide ratio was 1:1, the nanoparticles load rate was the highest. Compared with pure zein nanoparticles, zein/HTCC nanoparticles formed distribution more uniform, more regular shape. The Cur loaded in the nanoparticles can significantly improve the light the thermal stability and antioxidant activity of.3., and at the same conditions compared to the free Cur is more efficient in order to further improve the delivery system, based on previous study, introduced with pH The response factor of tannic acid (TA). Based on the adjacent benzene three phenol structure has a strong interaction between molecules and proteins and excellent metal chelating capacity to metal ions as crosslinking agent to form a layer of metal organic coating on the surface of zein nanoparticles, we successfully constructed a new nano carrier with pH response of (zein-TA/metal nanoparticles). By means of transmission electron microscopy (TEM) on the morphology observation, found that the hybrid nanoparticles have better dispersion and more uniform particle size, overcome most of nanoparticles in the culture medium of the unstable problem. Fourier transform infrared spectroscopy (FT-IR) analysis shows that there is a strong coordination between TA with metal ions, and between TA and protein also exist with the electrostatic interactions of hydrogen bonding interactions. Using X ray photoelectron spectroscopy (XPS) on the surface of nanoparticles The element analysis, the characteristics of the metal ion peaks appear and significant changes in the ratio of C/O were proved effective introduction of TA and metal ions. Then to adriamycin (DOX) as a model drug, the encapsulation efficiency of slow release performance. The results showed that compared to zein nanoparticles coated nanoparticles on drug encapsulation efficiency significantly higher in neutral condition (pH=7.4) has a good sustained release performance and preventing the occurrence of burst release phenomenon. At the same time, because of the TA-CuII composite film with pH response, give the package is good response properties of pH nanoparticles release. Phagocytosis experiments show that, compared with the free DOX by endocytosis. The slow speed of cells into nanoparticles; for coated nanoparticles, due to drug release of pH response, a long time to incubate observed red fluorescence of intracellular DOX in coated nanoparticles. TA in situ reduction of gold nanoparticles, the system provides a new way to control.4. nanoparticles more precise pH response for photothermal therapy of cancer, the use of TA on the adhesion of the metal and metal chelating ability, amino group and the coordination effect, introducing HTCC containing amino group of zein nanoparticles. In the preparation of zein/HTCC nanoparticles coated by a layer of metal organic coating. At the same time using TA- between metal ions and amino metal ion coordination of the pH response, given the response characteristics of drug release of pH nanoparticles. Compared with the zein/ HTCC nanoparticles, forming coated nanoparticles (zein-HTCC/TA-metal NPs) better dispersion, particle size distribution is more uniform, more regular shape of.FT-IR show that there is a strong coordination between TA and metal ions; while TA, HTCC and protein between there are static .XPS electric interaction studies further validated coated nanoparticles of metal ions and the presence of TA. The study found that drug model with DOX as coated nanoparticles on drug encapsulation efficiency compared to zein/HTCC nanoparticles also have improved significantly. The in vitro release experiment showed that different metal ions as the change amount of crosslinking and metal center ion will lead to changes in the release behavior of drug loaded nanoparticles. Compared with zein-TA/metal nanoparticles, zein-HTCC/TA-metal nanoparticles exhibited excellent response. Phagocytosis experiments showed that coated nanoparticles without obvious burst release phenomenon, in the culture medium can quickly release for DOX. coated nanoparticles, due to drug pH the response of release, incubation time to strong fluorescence was observed. While the introduction of rare earth elements, which also has pH response And biological imaging function.5. the effects of different metal ions and stoichiometric TA- nanoparticles coated by metal ions (zein-TA/metal). The effects of properties of various metal ions can significantly affect the shell thickness of the particle size is different; the metal ion concentration will cause the coating increased the diameter of nanoparticles in low concentration of metal ions. Next, coated nanoparticles in culture medium greatly stability mentioned. But for TA-CuII coated nanoparticles, with the increase of the concentration of metal ions, the formation of nanoparticles in the culture medium reduced stability. Then FT-IR analysis content that metal ions significantly TA and metal ion coordination effect, thus affecting the package mobile is the absorption peak of XPS nanoparticles coated nanoparticles; further validation of metal ions and the existence of TA, and the effects of metal ions concentration. Coated nanoparticles surface elements content. Experiments show that phagocytosis of phagocytic cells of zein nanoparticles and zein-TA/metal nanoparticles showed a time and concentration dependent. Cells on the coated nanoparticles showed a faster rate and higher amount of phagocytosis phagocytosis. Phagocytic cells on the efficiency of nanoparticles effect of metal ion species and concentration by laser scanning. Confocal microscopy (CLSM) observation of short time phagocytic cells, cells on the coated nanoparticles adhesion stronger, nanoparticles into cells faster. Clathrin mediated phagocytosis is the main way to coated nanoparticles enter cells. At the same time coated nanoparticles into cells, phagocytosis of caveolae mediated may also play a role for the zein nanoparticles, pinocytosis for nanoparticles into cells into cells is essential. The nanoparticles can be Co located with the lysosomes in the cytoplasm. It is found that the nanoparticles do not enter the nuclear area.

【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TQ460.1

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