可在体实现胞内直接投递的新型细胞穿膜肽的构建、筛选和穿膜机制研究
发布时间：2018-10-15 09:33
【摘要】：细胞穿膜肽是一种能够自主并携带多种货物分子进入细胞的短肽，具有低生物毒性、高运载效率、高生物相容性、能够突破血脑屏障等多种优势，是近20年来迅速发展的一种新型药物载体。 然而，细胞穿膜肽的研究和应用中也面临着一些问题。第一，细胞穿膜肽的穿膜机制目前尚没有得到完全的认识。第二，在体应用细胞穿膜肽时往往选择更加稳定的D型穿膜肽，但是D型穿膜肽的毒性更大，同时手性变化对于穿膜肽性质的影响也存在争论。第三，也是令研究者最关注的一点，在应用细胞穿膜肽转运货物分子进入细胞时，特别是在在体水平给药时，货物分子会被困在囊泡(胞吞泡)中无法行使功能甚至被降解失活，从而降低了转运药物分子的实际效力。目前，尚无在体水平实现细胞穿膜肽于胞质中直接投递的报道。在在体水平实现无囊泡束缚的、直接的胞内投递已成为当前细胞穿膜肽发展应用的一个重要热点。 针对上述问题，本文构建了一类混合不同比例D/L型精氨酸的新型寡聚精氨酸类穿膜肽，系统分析了该系列穿膜肽在不同孵育环境下的穿膜性质，总结了D型氨基酸对于富含精氨酸类穿膜肽的穿膜性质的影响规律。基于这种规律，本文从这一系列穿膜肽中筛选得到了一种同时具有低生物毒性和高穿膜效率的寡聚精氨酸穿膜肽；基于该穿膜肽，本文首次实现了在体给药后的细胞穿膜肽无囊泡束缚的胞内直接投递。进一步的，依赖于这一系列具有不同穿膜性质的穿膜肽，本文对这类新型寡聚精氨酸细胞穿膜肽的穿膜机理进行了系统研究和阐述。本文具体研究内容如下： (1)为了系统研究氨基酸手性变化对于细胞穿膜肽的影响，本文构建了一类基于混合D/L型精氨酸的新型寡聚精氨酸穿膜肽系列，并对其进行表征。 (2)分析了不同条件下，该系列穿膜肽的亚细胞分布、穿膜效率、生物毒性（包括细胞毒性和系统毒性）和生物稳定性（包括酶稳定性和血清稳定性）。总结了D型精氨酸对于这类穿膜肽的穿膜性质变化的影响规律。依据于这种规律性，在所设计的细胞穿膜肽系列中筛选获得了一种同时具有高穿膜效率、低生物毒性，以及能够分别在细胞水平和活体水平实现胞质内直接投递的细胞穿膜肽(rR)3R2。 (3)通过对细胞穿膜肽穿膜机制的系统研究，证明富含精氨酸类的细胞穿膜肽具有2种独立的穿膜机制：胞吞作用穿膜机制及特殊的直接穿膜机制。其中，直接穿膜机制是一种依赖于细胞能量和细胞膜电位的从细胞膜边缘开始的快速穿膜过程。基于该机制，，穿膜肽能够从细胞膜边缘开始，直接进行胞质内投递，并在细胞内弥散均匀分布。
[Abstract]:Cellular transmembrane peptide is a kind of short peptide that can enter cells independently and carry many kinds of cargo molecules. It has many advantages, such as low biotoxicity, high transport efficiency, high biocompatibility, and the ability to break through the blood-brain barrier and so on. It is a new drug carrier developed rapidly in the past 20 years. However, there are some problems in the research and application of cell-penetrating peptides. First, the transmembrane mechanism of cellular peptides has not been fully understood. Secondly, the more stable D-type transmembrane peptides are often used in vivo, but the toxicity of D-type transmembrane peptides is more serious, and the influence of chiral changes on the properties of transmembrane peptides is also controversial. Third, which is also of great concern to researchers, is the use of cell-penetrating peptides to transport cargo molecules into cells, especially at the level of drug delivery in vivo. Cargo molecules are trapped in vesicles (vesicles) and can not function or even deactivate, thus reducing the actual effectiveness of drug delivery molecules. At present, there are no reports of cell transmembrane peptide delivered directly in the cytoplasm at the level of body. Direct intracellular delivery without vesicle binding has become an important focus in the development and application of cellular membrane peptides. In order to solve the above problems, a new type of poly-arginine transmembrane peptides mixed with different ratios of D / L type arginine was constructed, and the transmembrane properties of the series of transmembrane peptides under different incubation conditions were systematically analyzed. The effects of D-amino acids on the transmembrane properties of arginine-rich transmembrane peptides were summarized. Based on this rule, we have obtained an oligomeric arginine transmembrane peptide with low biotoxicity and high membrane penetration efficiency from the series of transmembrane peptides. In this paper, we first realized the direct delivery of cellular membrane penetrating peptide without vesicle binding after in vivo administration. Furthermore, depending on this series of transmembrane peptides with different transmembrane properties, the mechanism of the transmembrane peptides of this new type of oligoarginine cells has been systematically studied and explained in this paper. The main contents of this paper are as follows: (1) in order to systematically study the effect of amino acid chiral changes on cellular transmembrane peptides, a new type of oligomeric arginine transmembrane peptides based on mixed D / L arginine was constructed. (2) the subcellular distribution, transmembrane efficiency, biotoxicity (including cytotoxicity and systemic toxicity) and biological stability (including enzyme stability and serum stability) of this series of transmembrane peptides were analyzed under different conditions. The effect of D type arginine on the transmembrane properties of this kind of transmembrane peptide was summarized. Based on this regularity, a novel cell membrane penetrating peptide series with high membrane penetration efficiency and low biotoxicity was obtained. And the cellular transmembrane peptide (rR) 3R2, which can be delivered directly into the cytoplasm at the cell level and in vivo, respectively. (3) the mechanism of cell transmembrane peptide transmembrane is studied systematically. The results showed that the membrane penetrating peptides rich in arginine had two independent transmembrane mechanisms: cytosolic transmembrane mechanism and special direct transmembrane mechanism. The mechanism of direct transmembrane penetration is a rapid transmembrane process which depends on cell energy and cell membrane potential from the edge of cell membrane. Based on this mechanism, transmembrane peptides can be delivered directly into the cytoplasm from the edge of the cell membrane and distribute evenly in the cells.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R318.04
[Abstract]:Cellular transmembrane peptide is a kind of short peptide that can enter cells independently and carry many kinds of cargo molecules. It has many advantages, such as low biotoxicity, high transport efficiency, high biocompatibility, and the ability to break through the blood-brain barrier and so on. It is a new drug carrier developed rapidly in the past 20 years. However, there are some problems in the research and application of cell-penetrating peptides. First, the transmembrane mechanism of cellular peptides has not been fully understood. Secondly, the more stable D-type transmembrane peptides are often used in vivo, but the toxicity of D-type transmembrane peptides is more serious, and the influence of chiral changes on the properties of transmembrane peptides is also controversial. Third, which is also of great concern to researchers, is the use of cell-penetrating peptides to transport cargo molecules into cells, especially at the level of drug delivery in vivo. Cargo molecules are trapped in vesicles (vesicles) and can not function or even deactivate, thus reducing the actual effectiveness of drug delivery molecules. At present, there are no reports of cell transmembrane peptide delivered directly in the cytoplasm at the level of body. Direct intracellular delivery without vesicle binding has become an important focus in the development and application of cellular membrane peptides. In order to solve the above problems, a new type of poly-arginine transmembrane peptides mixed with different ratios of D / L type arginine was constructed, and the transmembrane properties of the series of transmembrane peptides under different incubation conditions were systematically analyzed. The effects of D-amino acids on the transmembrane properties of arginine-rich transmembrane peptides were summarized. Based on this rule, we have obtained an oligomeric arginine transmembrane peptide with low biotoxicity and high membrane penetration efficiency from the series of transmembrane peptides. In this paper, we first realized the direct delivery of cellular membrane penetrating peptide without vesicle binding after in vivo administration. Furthermore, depending on this series of transmembrane peptides with different transmembrane properties, the mechanism of the transmembrane peptides of this new type of oligoarginine cells has been systematically studied and explained in this paper. The main contents of this paper are as follows: (1) in order to systematically study the effect of amino acid chiral changes on cellular transmembrane peptides, a new type of oligomeric arginine transmembrane peptides based on mixed D / L arginine was constructed. (2) the subcellular distribution, transmembrane efficiency, biotoxicity (including cytotoxicity and systemic toxicity) and biological stability (including enzyme stability and serum stability) of this series of transmembrane peptides were analyzed under different conditions. The effect of D type arginine on the transmembrane properties of this kind of transmembrane peptide was summarized. Based on this regularity, a novel cell membrane penetrating peptide series with high membrane penetration efficiency and low biotoxicity was obtained. And the cellular transmembrane peptide (rR) 3R2, which can be delivered directly into the cytoplasm at the cell level and in vivo, respectively. (3) the mechanism of cell transmembrane peptide transmembrane is studied systematically. The results showed that the membrane penetrating peptides rich in arginine had two independent transmembrane mechanisms: cytosolic transmembrane mechanism and special direct transmembrane mechanism. The mechanism of direct transmembrane penetration is a rapid transmembrane process which depends on cell energy and cell membrane potential from the edge of cell membrane. Based on this mechanism, transmembrane peptides can be delivered directly into the cytoplasm from the edge of the cell membrane and distribute evenly in the cells.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R318.04
【共引文献】
相关期刊论文 前10条
1 李剑峰;蒋晨;;脑胶质瘤靶向药物递送系统[J];国际药学研究杂志;2013年05期
2 谢向阳;林雯;李e
本文编号：2272115
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