磁性介孔氧化硅纳米颗粒载带短链核酸和蛋白药物及其生物医学应用
发布时间：2018-09-01 06:46
【摘要】：介孔氧化硅纳米颗粒由于具有极大的比表面积、孔容，可调的介孔孔径和易被修饰的表面特性，在药物转运系统研究中越来越受到人们的关注，其中针对小分子抗炎药物或化疗药物的载带得到了快速发展，但是针对生物分子药物的装载与传输研究则显得较为滞后。 本工作选取了三类具有代表性的短链核酸和蛋白生物分子，利用三种不同孔径大小（2.7nm，4.3nm，6.1nm）的磁性介孔二氧化硅纳米颗粒（M-MSNs），分别实现了对短链siRNA （~20bp），单链DNA（CpG寡核苷酸,~20nt）和蛋白药物（尿激酶，简称UK，~50kD）的载带并扩展了其复合体在肿瘤基因沉默治疗，肿瘤免疫治疗和靶向溶栓领域的应用。相对于传统脂质体试剂载带核酸（具有脂质体毒性大，颗粒尺寸分布范围广等缺陷）和尿激酶静脉注射治疗血管栓塞（具有药物剂量大，溶血等副作用缺陷）的方案，M-MSNs纳米药物复合体更有望被用于体内的治疗研究。 第二章中，我们主要整理了整个论文所涉及的三种孔径的M-MSNs的材料特性。首先，我们制备了三种孔径的磁性介孔二氧化硅纳米颗粒，并研究了其形貌特征，孔径分布，磁化曲线等特性。还研究了2.7nm孔径的M-MSNs经siRNA载带并经聚乙烯亚胺（PEI）包被形成的复合体的形貌特征和粒径分布。另外，经氨基修饰或进一步聚乙二醇（PEG）修饰孔径为4.3nm的M-MSNs的形貌，Zeta电位和粒径分布也被进行了表征。 第三章中，我们在具有较强疏水性的溶液环境中实现了M-MSNs的介孔孔道对siRNA的装载，并在装载siRNA后的M-MSNs表面包覆阳离子型高分子聚乙烯亚胺（PEI）,于是构建了基于M-MSNs的siRNA转运载体（M-MSN_siRNA@PEI）。后续实验证明，M-MSN_siRNA@PEI的干扰效率具有很强的溶酶体逃逸动力学依赖性，于是我们发掘了该系统在RNAi作用发挥中所遇到的瓶颈，即大量siRNA会被束缚于细胞中的内涵-溶酶体内，从而无法参与细胞质中诱导基因沉默的过程。最后，我们对M-MSN_siRNA@PEI转运载体进行了表面功能化处理——连接KALA多肽，这种修饰提高了转运载体的溶酶体逃逸能力，并因此显著增强了其诱导细胞内源基因的RNA干扰效率。 第四章中，我们合成了氨基(APTES)修饰的带正电荷的M-MSNs，简称M-MSN-A，为了使其应对更复杂的体内环境，我们将其进一步修饰聚乙二醇高分子（PEGylation）所得到的颗粒简称为M-MSN-P。然后，我们研究了M-MSN-A和M-MSN-P两颗粒对CpG吸附和释放的规律，以及两者携带CpG后对巨噬细胞RAW264.7内吞的影响，并对内吞现象从颗粒降解和颗粒内吞方面做出解释。我们还进一步检验了该载体系统与肿瘤化疗药物共同杀伤肿瘤细胞的效果。最后，我们研究了该载体系统在小鼠体内刺激免疫反应的能力，与体外实验结果进行分析比对，并为后续肿瘤模型小鼠的治疗研究提供数据支持。 第五章中，我们根据高分子药物扩散定律（菲克定律），建立了平板溶栓数学理论模型，得出可被用来极其方便地检测酶活性的参数。我们还利用不同活性药物（尿激酶，UK）检验此模型的实用性。接下来，我们研究了溶栓药物与M-MSNs之间的吸附和脱附规律，通过参阅文献，我们发现我们材料的吸附和脱附曲线跟文献中理论公式具有很高的贴合度。为了检验该载体系统的在体内的靶向溶栓效果，我们建立了流体栓塞模型来进行模拟，发现了M-MSNs/UK复合物相对于游离UK药物，显著提高了溶栓效率（3.5倍），证明了M-MSNs磁靶向溶栓应用的可行性。同时，，我们还利用小介孔（3.7nm）磁性介孔氧化硅颗粒与我们所采用的6.1nm的M-MSNs详细比对了溶栓吸附、脱附行为和溶栓效率，发现我们6.1nm孔径的M-MSNs具有更强的缓释效能和更长的缓释时间，并因此而认为药物分子与介孔材料介孔孔洞尺寸上相互匹配是介孔有效载带药物的前提。
[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.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R318.08;TB383.1
本文编号：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.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R318.08;TB383.1
【引证文献】
相关博士学位论文 前1条
1 徐阳;磁性功能材料的制备及其在复杂样品预处理中的应用研究[D];吉林大学;2014年
本文编号：2216432
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