本文關鍵詞：多功能性玉米醇溶蛋白基遞送系統(tǒng)的構建及生物學評價　出處：《華中農業(yè)大學》2017年博士論文　論文類型：學位論文

  更多相關文章： 玉米醇溶蛋白 多糖 單寧酸 自組裝 pH-響應性 細胞成像 吞噬機理

【摘要】：天然高分子憑借其生物相容性好、可降解、易修飾等多種性質,已成為制備納米材料的主要基材。通過自組裝制備的天然高分子納米載體更是由于其安全無毒、制備簡單、表面易修飾、載藥量高、控釋性能好等優(yōu)勢,在藥物及營養(yǎng)素遞送方面受到越來越多的關注。目前,提高納米粒子的穩(wěn)定性和功能性,并實現(xiàn)高效靶向遞送,已成為遞送領域研究的研究熱點。本文以玉米醇溶蛋白(zein)為主要基材,通過自組裝的方式與兩種不同形式的多糖(陰離子多糖—羧甲基纖維素鈉和陽離子多糖—季銨鹽殼聚糖)自組裝形成納米粒子。以疏水性藥物紫杉醇(PTX)和疏水性營養(yǎng)素姜黃素(Cur)為模型負載物,研究納米運載體系對疏水性物質的包封、保護以及體外緩釋的性質,通過細胞實驗驗證載藥納米粒子的生物學特性。為進一步提高抗癌藥物的療效、降低毒副性,賦予運載體系定向性釋放的性質,在已制備的納米粒子表面形成一層單寧酸-金屬離子包被膜,通過膜的pH-響應性崩解,賦予納米粒子pH-響應性釋藥的性質。本文主要研究結果如下:1.選擇PTX作為疏水模型藥物,通過自組裝方法制備負載PTX的玉米醇溶蛋白/羧甲基纖維素鈉納米粒子(PTX-zein/CMC納米粒子)。結果顯示當蛋白質與多糖質量比為1:3,投藥量為80μg/mL時,所得納米粒子粒徑為159.4 nm,載藥率高達93.5%。在此條件下可得到分散均勻的球形納米粒子,且其具有良好的pH穩(wěn)定性和儲藏穩(wěn)定性。體外緩釋實驗表明,相比于游離藥物的快速釋放,載藥納米粒子呈現(xiàn)明顯的緩釋特性。細胞吞噬實驗表明,藥物敏感的HepG2細胞和藥物抗性的MCF-7細胞對香豆素6標記的納米粒子的細胞吞噬均呈時間依賴性。同時,對于藥物抗性的MCF-7細胞,在高給藥濃度下,載藥納米粒子比游離藥物顯示更強的細胞毒性。細胞凋亡實驗顯示,對于抗性細胞,載藥納米粒子組引起的細胞凋亡比例大于游離藥物的。免疫熒光實驗表明載藥納米粒子和游離的PTX均是通過促進微管的增生導致細胞凋亡。2.研究陽離子多糖(季銨鹽殼聚糖,HTCC)與zein的組裝行為,利用自組裝制備納米粒子,用于疏水性營養(yǎng)素Cur的負載。通過對殼聚糖季銨化改性,大大提高了殼聚糖在中性條件以及堿性條件下的溶解性。改變蛋白質/多糖質量比可以得到不同粒徑、不同多分散系數(PDI)、不同負載率的納米粒子。當固定多糖為HTCC1(Mw 8.708×103),蛋白質/多糖質量比為1:1時,所得納米粒子的負載率最高。與單純的zein納米粒子相比,形成的zein/HTCC納米粒子分布更加均勻,形狀更加規(guī)整。將Cur負載于納米粒子可顯著提高其光、熱穩(wěn)定性,且在相同的處理條件下相比于游離Cur具有更強的抗氧化活性。3.為了進一步提高構建的遞送體系的高效性,在之前的研究基礎上,引入了具有pH響應性的響應因子�；趩螌幩�(TA)分子與蛋白質之間具有較強相互作用的鄰苯三酚結構及其優(yōu)異的金屬螯合能力,以金屬離子為交聯(lián)劑在zein納米粒子表面形成一層金屬-有機涂層,成功構建了一種新型的具有pH響應性的納米載體(zein-TA/metal納米粒子)。通過透射電子顯微鏡(TEM)對其微觀形貌進行觀察,發(fā)現(xiàn)這種雜化納米粒子具有更佳的分散性且粒徑更加均一,克服了大部分納米粒子在培養(yǎng)基中不穩(wěn)定的問題。傅里葉紅外光譜(FT-IR)分析表明,TA與金屬離子之間存在較強的配位作用,同時TA與蛋白質之間也存在著氫鍵相互作用與靜電相互作用。使用X射線光電子能譜(XPS)對納米粒子表面元素進行分析,金屬離子特征峰的出現(xiàn)以及C/O比值的顯著變化均有效證明TA與金屬離子的引入。隨后以阿霉素(DOX)為模型藥物,對其包封率、緩釋性能進行研究。結果顯示,相比于zein納米粒子,包被納米粒子對藥物的包封率顯著提高且在中性條件下(pH=7.4)具有良好的緩釋性能,阻止了突釋現(xiàn)象的發(fā)生。同時,由于TA-CuII復合膜具有pH響應性,賦予包被納米粒子良好的pH響應性釋藥的性質。細胞吞噬實驗表明,與游離的DOX相比,通過內吞作用進入細胞的納米粒子速度較緩慢;對于包被的納米粒子,由于對藥物的pH響應性釋放,孵育較長時間才能觀察到細胞內DOX的紅色熒光。位于包被納米粒子表面的TA可以原位還原金納米粒子,該體系為癌癥的光熱療法提供了新思路。4.為了能更加精細的調控納米載體的pH響應性,利用TA對基體的粘附性以及金屬螯合能力、氨基與金屬之間的配位作用,在zein納米粒子中引入含有氨基的HTCC,在制備的zein/HTCC納米粒子表面包被一層金屬-有機涂層。同時利用TA-金屬離子以及氨基-金屬離子之間配位鍵的pH響應性,賦予納米粒子pH響應釋藥的特性。與zein/HTCC納米粒子相比,形成的包被納米粒子(zein-HTCC/TA-metal納米粒子)分散性更好,粒徑分布更加均勻,形狀更加規(guī)整。FT-IR研究表明,TA與金屬離子之間存在較強的配位作用;同時TA,HTCC與蛋白質之間也存在著靜電相互作用。XPS研究進一步驗證了包被納米粒子表面金屬離子和TA的存在。以DOX為藥物模型研究發(fā)現(xiàn),包被的納米粒子對藥物的包封率相比于zein/HTCC納米粒子也有顯著的提高。體外緩釋實驗表明以不同金屬離子作為交聯(lián)中心及金屬離子添加量的變化均會導致載藥納米粒子釋放行為的變化。與zein-TA/metal納米粒子相比,zein-HTCC/TA-metal納米粒子呈現(xiàn)出更優(yōu)異的刺激響應性。細胞吞噬實驗顯示未經包被的納米粒子存在明顯的突釋現(xiàn)象,在培養(yǎng)基中即可快速釋放DOX。對于包被納米粒子,由于對藥物的pH響應性釋放,孵育較長時間才能觀察到較強的熒光。同時引入稀土元素,使其同時具有pH響應性和生物成像的功能。5.考察金屬離子種類及化學計量比對TA-金屬離子包被納米粒子(zein-TA/metal)性質的影響。金屬離子種類可顯著影響殼層厚度致使納米粒子的粒徑不同;金屬離子濃度的增加會引起包被納米粒子粒徑的增加。在較低金屬離子濃度下,包被的納米粒子在培養(yǎng)基中的穩(wěn)定性大大提到。但是對于TA-CuII包被的納米粒子,隨著金屬離子濃度的增大,形成的納米粒子在培養(yǎng)基中穩(wěn)定性減小。隨后的FT-IR分析得出金屬離子的含量顯著影響TA與金屬離子的配位作用,從而影響包被納米粒子吸收峰的移動;XPS進一步驗證包被納米粒子中金屬離子以及TA的存在,且金屬離子的濃度影響包被納米粒子表面元素含量。細胞吞噬實驗表明細胞對zein納米粒子和zein-TA/metal納米粒子的吞噬均呈現(xiàn)時間及濃度依賴性。細胞對包被納米粒子表現(xiàn)出更快的吞噬速率以及更高的吞噬量。金屬離子種類以及添加量影響細胞對納米粒子的吞噬效率。通過激光共聚焦顯微鏡(CLSM)觀察短時間細胞吞噬,細胞對包被納米粒子粘附性更強,納米粒子進入細胞速率更快。網格蛋白介導的吞噬作用是包被納米粒子進入細胞的主要途徑。同時包被納米粒子進入細胞,細胞膜穴樣凹陷介導的吞噬作用也可能起到一定作用。對于zein納米粒子,胞飲作用對于納米粒子進入細胞至關重要。進入到細胞內的納米粒子可以和細胞質內溶酶體共定位,觀察發(fā)現(xiàn)納米粒子沒有進入核區(qū)。
[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.

【學位授予單位】：華中農業(yè)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：TQ460.1

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