磁性介孔氧化硅納米顆粒載帶短鏈核酸和蛋白藥物及其生物醫(yī)學(xué)應(yīng)用
發(fā)布時間：2018-09-01 06:46
【摘要】：介孔氧化硅納米顆粒由于具有極大的比表面積、孔容，可調(diào)的介孔孔徑和易被修飾的表面特性，在藥物轉(zhuǎn)運系統(tǒng)研究中越來越受到人們的關(guān)注，其中針對小分子抗炎藥物或化療藥物的載帶得到了快速發(fā)展，但是針對生物分子藥物的裝載與傳輸研究則顯得較為滯后。 本工作選取了三類具有代表性的短鏈核酸和蛋白生物分子，利用三種不同孔徑大�。�2.7nm，4.3nm，6.1nm）的磁性介孔二氧化硅納米顆粒（M-MSNs），分別實現(xiàn)了對短鏈siRNA （~20bp），單鏈DNA（CpG寡核苷酸,~20nt）和蛋白藥物（尿激酶，簡稱UK，~50kD）的載帶并擴展了其復(fù)合體在腫瘤基因沉默治療，腫瘤免疫治療和靶向溶栓領(lǐng)域的應(yīng)用。相對于傳統(tǒng)脂質(zhì)體試劑載帶核酸（具有脂質(zhì)體毒性大，顆粒尺寸分布范圍廣等缺陷）和尿激酶靜脈注射治療血管栓塞（具有藥物劑量大，溶血等副作用缺陷）的方案，M-MSNs納米藥物復(fù)合體更有望被用于體內(nèi)的治療研究。 第二章中，我們主要整理了整個論文所涉及的三種孔徑的M-MSNs的材料特性。首先，我們制備了三種孔徑的磁性介孔二氧化硅納米顆粒，并研究了其形貌特征，孔徑分布，磁化曲線等特性。還研究了2.7nm孔徑的M-MSNs經(jīng)siRNA載帶并經(jīng)聚乙烯亞胺（PEI）包被形成的復(fù)合體的形貌特征和粒徑分布。另外，經(jīng)氨基修飾或進一步聚乙二醇（PEG）修飾孔徑為4.3nm的M-MSNs的形貌，Zeta電位和粒徑分布也被進行了表征。 第三章中，我們在具有較強疏水性的溶液環(huán)境中實現(xiàn)了M-MSNs的介孔孔道對siRNA的裝載，并在裝載siRNA后的M-MSNs表面包覆陽離子型高分子聚乙烯亞胺（PEI）,于是構(gòu)建了基于M-MSNs的siRNA轉(zhuǎn)運載體（M-MSN_siRNA@PEI）。后續(xù)實驗證明，M-MSN_siRNA@PEI的干擾效率具有很強的溶酶體逃逸動力學(xué)依賴性，于是我們發(fā)掘了該系統(tǒng)在RNAi作用發(fā)揮中所遇到的瓶頸，即大量siRNA會被束縛于細胞中的內(nèi)涵-溶酶體內(nèi)，從而無法參與細胞質(zhì)中誘導(dǎo)基因沉默的過程。最后，我們對M-MSN_siRNA@PEI轉(zhuǎn)運載體進行了表面功能化處理——連接KALA多肽，這種修飾提高了轉(zhuǎn)運載體的溶酶體逃逸能力，并因此顯著增強了其誘導(dǎo)細胞內(nèi)源基因的RNA干擾效率。 第四章中，我們合成了氨基(APTES)修飾的帶正電荷的M-MSNs，簡稱M-MSN-A，為了使其應(yīng)對更復(fù)雜的體內(nèi)環(huán)境，我們將其進一步修飾聚乙二醇高分子（PEGylation）所得到的顆粒簡稱為M-MSN-P。然后，我們研究了M-MSN-A和M-MSN-P兩顆粒對CpG吸附和釋放的規(guī)律，以及兩者攜帶CpG后對巨噬細胞RAW264.7內(nèi)吞的影響，并對內(nèi)吞現(xiàn)象從顆粒降解和顆粒內(nèi)吞方面做出解釋。我們還進一步檢驗了該載體系統(tǒng)與腫瘤化療藥物共同殺傷腫瘤細胞的效果。最后，我們研究了該載體系統(tǒng)在小鼠體內(nèi)刺激免疫反應(yīng)的能力，與體外實驗結(jié)果進行分析比對，并為后續(xù)腫瘤模型小鼠的治療研究提供數(shù)據(jù)支持。 第五章中，我們根據(jù)高分子藥物擴散定律（菲克定律），建立了平板溶栓數(shù)學(xué)理論模型，得出可被用來極其方便地檢測酶活性的參數(shù)。我們還利用不同活性藥物（尿激酶，UK）檢驗此模型的實用性。接下來，我們研究了溶栓藥物與M-MSNs之間的吸附和脫附規(guī)律，通過參閱文獻，我們發(fā)現(xiàn)我們材料的吸附和脫附曲線跟文獻中理論公式具有很高的貼合度。為了檢驗該載體系統(tǒng)的在體內(nèi)的靶向溶栓效果，我們建立了流體栓塞模型來進行模擬，發(fā)現(xiàn)了M-MSNs/UK復(fù)合物相對于游離UK藥物，顯著提高了溶栓效率（3.5倍），證明了M-MSNs磁靶向溶栓應(yīng)用的可行性。同時，，我們還利用小介孔（3.7nm）磁性介孔氧化硅顆粒與我們所采用的6.1nm的M-MSNs詳細比對了溶栓吸附、脫附行為和溶栓效率，發(fā)現(xiàn)我們6.1nm孔徑的M-MSNs具有更強的緩釋效能和更長的緩釋時間，并因此而認為藥物分子與介孔材料介孔孔洞尺寸上相互匹配是介孔有效載帶藥物的前提。
[Abstract]:Mesoporous silica nanoparticles have attracted more and more attention in drug delivery systems due to their large specific surface area, pore volume, adjustable mesoporous diameter and surface properties. Among them, the carrier bands for small molecules of anti-inflammatory drugs or chemotherapeutic drugs have been developed rapidly, but for the loading of biomolecular drugs. And transmission research is lagging behind.
In this work, three representative short-chain nucleic acids and protein biomolecules were selected, and magnetic mesoporous silica nanoparticles (M-MSNs) with different pore sizes (2.7 nm, 4.3 nm, 6.1 nm) were used to carry and amplify short-chain siRNA (~20bp), single-stranded DNA (CpG oligonucleotide, ~20nt) and protein drugs (UK, ~50kD) respectively. Comparing with traditional liposome reagent-loaded nucleic acid (with liposome toxicity, wide particle size distribution and other defects) and urokinase intravenous injection (with drug dosage, hemolysis and other side effects defects) in the treatment of vascular embolism, its application in tumor gene silencing therapy, tumor immunotherapy and targeted thrombolysis has been expanded. The M-MSNs nanocomposite is more likely to be used for therapeutic research in vivo.
In the second chapter, we mainly summarized the material properties of the three pore sizes of M-MSNs. Firstly, we prepared magnetic mesoporous silica nanoparticles with three pore sizes, and studied their morphology, pore size distribution, magnetization curves and other characteristics. In addition, the morphology, Zeta potential and particle size distribution of M-MSNs with 4.3 nm pore size modified by amino group or polyethylene glycol (PEG) were also characterized.
In the third chapter, we realized the loading of siRNA by the mesoporous channels of M-MSNs in a highly hydrophobic solution environment, and coated the surface of M-MSNs with cationic polyethylenimide (PEI). So we constructed a siRNA transporter based on M-MSNs (M-MSN_siRNA@PEI). The subsequent experiments proved that M-MSN_siRNA@PEI was dry. Disturbance efficiency is strongly dependent on lysosome escape kinetics, so we have uncovered the bottleneck of the system in the role of RNAi, that is, a large number of siRNA will be bound to the cell's content-lysosome, thus unable to participate in the process of inducing gene silencing in the cytoplasm. Finally, we carried out the M-MSN_siRNA@PEI transporter. Surface functionalization, linking KALA peptides, enhances the lysosome escape ability of the transporter and thus significantly enhances its RNA interference efficiency in inducing endogenous genes in cells.
In the fourth chapter, we synthesized APTES-modified positively charged M-MSNs, or M-MSN-A for short. In order to cope with more complex in vivo environment, the particles obtained by further modification of polyethylene glycol polymer (PEGylation) were referred to as M-MSN-P. Then, we studied the adsorption and release rules of M-MSN-A and M-MSN-P particles for CPG. We also examined the effect of CpG carrier system and tumor chemotherapeutics on the cytotoxicity of RAW264.7 macrophages. Finally, we studied the effect of CpG carrier system on the immunoreactivity in mice. The corresponding ability was analyzed and compared with the experimental results in vitro, and provided data support for the follow-up study of tumor model mice.
In the fifth chapter, according to the law of polymer drug diffusion (Fick's law), we established a mathematical model of plate thrombolysis, and obtained the parameters which can be used to detect enzyme activity conveniently. We also tested the practicability of this model with different active drugs (urokinase, UK). Next, we studied the absorption between thrombolytic drugs and M-MSNs. By referring to the literature, we found that the adsorption and desorption curves of our materials were highly compatible with the theoretical formulas in the literature. In order to test the thrombolytic effect of this carrier system in vivo, we established a fluid embolization model to simulate the adsorption and desorption of M-MSNs/UK complex, which was significantly different from free UK drug. The feasibility of magnetic targeted thrombolysis of M-MSNs was proved by improving the thrombolytic efficiency (3.5 times). At the same time, we compared the thrombolytic adsorption, desorption behavior and thrombolytic efficiency of M-MSNs with small mesoporous (3.7 nm) magnetic mesoporous silica particles with that of M-MSNs with 6.1 nm, and found that our 6.1 nm pore size M-MSNs had better thrombolytic efficiency. Therefore, it is considered that the matching of drug molecules with mesoporous materials is the prerequisite for effective drug delivery.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R318.08;TB383.1
本文編號：2216432
[Abstract]:Mesoporous silica nanoparticles have attracted more and more attention in drug delivery systems due to their large specific surface area, pore volume, adjustable mesoporous diameter and surface properties. Among them, the carrier bands for small molecules of anti-inflammatory drugs or chemotherapeutic drugs have been developed rapidly, but for the loading of biomolecular drugs. And transmission research is lagging behind.
In this work, three representative short-chain nucleic acids and protein biomolecules were selected, and magnetic mesoporous silica nanoparticles (M-MSNs) with different pore sizes (2.7 nm, 4.3 nm, 6.1 nm) were used to carry and amplify short-chain siRNA (~20bp), single-stranded DNA (CpG oligonucleotide, ~20nt) and protein drugs (UK, ~50kD) respectively. Comparing with traditional liposome reagent-loaded nucleic acid (with liposome toxicity, wide particle size distribution and other defects) and urokinase intravenous injection (with drug dosage, hemolysis and other side effects defects) in the treatment of vascular embolism, its application in tumor gene silencing therapy, tumor immunotherapy and targeted thrombolysis has been expanded. The M-MSNs nanocomposite is more likely to be used for therapeutic research in vivo.
In the second chapter, we mainly summarized the material properties of the three pore sizes of M-MSNs. Firstly, we prepared magnetic mesoporous silica nanoparticles with three pore sizes, and studied their morphology, pore size distribution, magnetization curves and other characteristics. In addition, the morphology, Zeta potential and particle size distribution of M-MSNs with 4.3 nm pore size modified by amino group or polyethylene glycol (PEG) were also characterized.
In the third chapter, we realized the loading of siRNA by the mesoporous channels of M-MSNs in a highly hydrophobic solution environment, and coated the surface of M-MSNs with cationic polyethylenimide (PEI). So we constructed a siRNA transporter based on M-MSNs (M-MSN_siRNA@PEI). The subsequent experiments proved that M-MSN_siRNA@PEI was dry. Disturbance efficiency is strongly dependent on lysosome escape kinetics, so we have uncovered the bottleneck of the system in the role of RNAi, that is, a large number of siRNA will be bound to the cell's content-lysosome, thus unable to participate in the process of inducing gene silencing in the cytoplasm. Finally, we carried out the M-MSN_siRNA@PEI transporter. Surface functionalization, linking KALA peptides, enhances the lysosome escape ability of the transporter and thus significantly enhances its RNA interference efficiency in inducing endogenous genes in cells.
In the fourth chapter, we synthesized APTES-modified positively charged M-MSNs, or M-MSN-A for short. In order to cope with more complex in vivo environment, the particles obtained by further modification of polyethylene glycol polymer (PEGylation) were referred to as M-MSN-P. Then, we studied the adsorption and release rules of M-MSN-A and M-MSN-P particles for CPG. We also examined the effect of CpG carrier system and tumor chemotherapeutics on the cytotoxicity of RAW264.7 macrophages. Finally, we studied the effect of CpG carrier system on the immunoreactivity in mice. The corresponding ability was analyzed and compared with the experimental results in vitro, and provided data support for the follow-up study of tumor model mice.
In the fifth chapter, according to the law of polymer drug diffusion (Fick's law), we established a mathematical model of plate thrombolysis, and obtained the parameters which can be used to detect enzyme activity conveniently. We also tested the practicability of this model with different active drugs (urokinase, UK). Next, we studied the absorption between thrombolytic drugs and M-MSNs. By referring to the literature, we found that the adsorption and desorption curves of our materials were highly compatible with the theoretical formulas in the literature. In order to test the thrombolytic effect of this carrier system in vivo, we established a fluid embolization model to simulate the adsorption and desorption of M-MSNs/UK complex, which was significantly different from free UK drug. The feasibility of magnetic targeted thrombolysis of M-MSNs was proved by improving the thrombolytic efficiency (3.5 times). At the same time, we compared the thrombolytic adsorption, desorption behavior and thrombolytic efficiency of M-MSNs with small mesoporous (3.7 nm) magnetic mesoporous silica particles with that of M-MSNs with 6.1 nm, and found that our 6.1 nm pore size M-MSNs had better thrombolytic efficiency. Therefore, it is considered that the matching of drug molecules with mesoporous materials is the prerequisite for effective drug delivery.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R318.08;TB383.1
【引證文獻】
相關(guān)博士學(xué)位論文 前1條
1 徐陽;磁性功能材料的制備及其在復(fù)雜樣品預(yù)處理中的應(yīng)用研究[D];吉林大學(xué);2014年
本文編號：2216432
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