细胞穿透肽应用的研究进展

细胞穿透肽是一类具有特殊细胞膜穿透作用的短肽,研究发现其为药物大分子物质的细胞内转运提供了有力工具,现已得到了广泛关注和大量研究.一方面,近年来发现,细胞穿透肽的应用面临着药物释放率、代谢降解、细胞系的分化状态和Rho-GTPases活性的依赖性等问题,从而阻碍了其研究开发的进程.另一方面,细胞穿透肽在肿瘤治疗、抗菌和抗炎靶向药物等方面的研究却取得了显著的进展,甚至进入了临床前和临床研究阶段.本文围绕细胞穿透肽在应用开发中所面临的挑战和机遇两方面,综述了近期的研究进展,并对细胞穿透肽在新领域中的应用进行了展望.     

细胞穿透肽设计及肿瘤靶向治疗

细胞穿透肽是近年来发现的具有穿透生物膜功能,并能介导大分子物质跨膜转导的一类小分子短肽。该肽段以其转导效率高,速度快,生物活性好,对细胞损害小等特点,成为药物导向治疗方法研究领域的热点。肿瘤靶向治疗的一个局限性是不能使药物有效地进入肿瘤细胞内,极大地降低了肿瘤靶向药物治疗的疗效。因此,如何使抗癌药物特定输送至肿瘤细胞群是当前亟需研究设计的课题,本文就特异性靶向穿膜肽在肿瘤靶向治疗方面的设计、应用作一综述。     

编者按: 转运——小干扰RNA应用领域的瓶颈问题

自20世纪末RNA干扰现象(RNA interference)及其作用机制被发现以来,外源性的小干扰RNA(siRNA)已广泛地用于从基础研究到临床实践的多个领域。但,如何有效地、特异地将siRNA输送进入靶细胞,始终是使用者关注的重点,并已逐步成为siRNA应用于临床治疗的瓶颈问题之一。目前开展研究的siRNA转运方法主要包括3类：a. 通过与配基偶联实现siRNA的转运;b. 将siRNA包载于纳米颗粒等中经内吞进入细胞; c. 载体与细胞膜融合释放所载siRNA进入细胞。 本刊在这期中特意选择了3篇文章组成了一个小专题来介绍与探讨siRNA的输送问题。梁伟等应邀撰写了题为《siRNA脂质纳米输送载体的研究进展》的综述,介绍了siRNA输送载体的基本要求,特别是脂质纳米载体(lipid-based siRNA delivering systems)的设计和构筑原则,以及这类载体的研发现状和应用前景;张洪杰等结合自身的研发工作撰写了《细胞穿透肽及其结构改造在siRNA传递中的应用》一文,从发现、毒副作用、传递机制、结构修饰与传递功能改进等几个方面,系统评述了细胞穿透肽(cell penetrating peptides)在siRNA传递方面研究与应用的进展;张兴梅等在《适配子介导的siRNA转运》一文中重点介绍了基于适配子(aptamer)的siRNA转运系统的转运机制、近期研究进展和应用前景。3篇文章各有侧重,反映了当前siRNA转运研究中几个比较活跃的领域的研究进展,希望能对广大读者有所帮助。     

细胞穿膜肽研究应用的新进展

细胞穿膜肽(Cell-penetrating peptides,CPPs)是一类能够穿过细胞膜或组织屏障的短肽。CPPs可通过内吞和直接穿透等机制运载蛋白质、RNA、DNA等生物大分子进入细胞内发挥其效应功能。相比于其他非天然的化学分子,CPPs具有生物相容性佳、对细胞造成的毒性小、完成入胞转运后可降解、并能与生物活性蛋白直接融合重组表达等优点,因此成为以胞内分子为靶标的药物递送技术发展的重要工具,并在生物医学研究领域具有良好的应用前景。文中针对CPPs的分类特点、入胞转运机制及其治疗应用的新近研究进展进行综述和讨论。     

穿膜肽的研究现状及应用

以细胞内物质为靶标的药物(大分子、蛋白质、多肽及核酸)只有穿透细胞膜才能进一步发挥其药效。细胞穿透多肽(穿膜肽)是由少于30个氨基酸残基组成的小肽,它们能够通过与细胞膜相互作用而穿透细胞膜这一天然屏障。穿膜肽大致分为宿主防御肽、基于信号序列的穿膜肽和富含精氨酸的穿膜肽;穿膜肽进入细胞的机制尚未完全阐明,存在倒置微团模型、地毯式模型及打孔模型等假说。穿膜肽能够携带各种物质进入细胞的特性受到人们的关注。我们就穿膜肽的种类、穿膜机制,及其在生物影像学和生物递送系统中的应用做一综述。     

细胞穿透肽在白菜小孢子中的内化及其效率的探究

该研究以大白菜‘94-323’为材料,通过分析细胞穿透肽(cell-penetrating peptide,CPP)在其小孢子细胞中的内化及其效率,证明了细胞穿透肽Tat2可以克服花粉壁的障碍,独自或者转导GUS酶和DNA(环型质粒DNA、线型质粒DNA)等大分子进入大白菜小孢子中;同时研究了Tat2介导的DNA在白菜小孢子中内化效率的影响因素。结果表明:内化效率与Tat2/DNA复合物和小孢子共育时间正相关,随复合物浓度的升高而增加,最适共育温度为33°C,与转导DNA片段的长度关系不明显。该研究首次证明,Tat2可以作为纳米载体传递大分子进入双子叶植物小孢子细胞,GUS酶的内化频率可达到18%,可为细胞穿透肽介导的芸薹属小孢子转基因技术的建立提供直接的实验依据。     

以两亲性细胞膜穿透肽为基础的siRNA递送策略

小干扰RNA(si RNA)的发展给针对病理障碍特异性基因的靶向治疗带来了巨大的希望。然而,细胞膜对带负电荷分子的低渗透性,细胞对si RNA的摄取能力差,成为了si RNA临床应用的主要障碍。虽然学者们提出了一系列si RNA递送的方法,但是截止到目前,仍没有递送si RNA的通用方法。细胞膜穿透肽(cell-penetrating peptides,CPPs)的发现为si RNA非侵袭性的进入细胞提供了一种非常有前景的运载工具,已被成功地应用于治疗性si RNA分子的体内和体外实验的递送。最近,一种新的以两亲性CPPs为基础的si RNA递送系统-CADY(a secondary amphipathic peptide,Ac-GLWRALWRLLRSLWRLLWRA-cysteamide)受到了高度关注,它能与si RNA形成稳定的非共价复合物,并在原代和悬浮细胞系中高效地递送si RNA,具有极高的应用前景。     

植物非细胞自主性小RNA分子研究进展

非细胞自主性是RNA干扰的主要特点之一,表现为沉默效应可以在细胞、组织和生物个体间传递和扩散,可移动的小RNA分子在这种非细胞自主性的沉默扩散中发挥了核心作用。近年来的研究表明小RNA分子可以与转录因子、多肽和植物激素一样传递胞间信息,并以其特有的方式调控发育模式、响应环境胁迫、增强病毒抗性和维持转座子的沉默。综述了近年来在植物非细胞自主性RNAi研究中取得的主要进展,主要介绍了通过韧皮部和胞间连丝途径传递沉默信号的各种小RNA分子及其生物学作用、非细胞自主性小RNA的分子特征和运输效率的调控,并对存在的问题及其研究前景进行了展望。     

GLP-1与2型糖尿病的研究进展

胰高血糖素样肽-1(glucagon-like peptide-1,GLP-1)具有促进胰岛素分泌、保护胰岛β细胞、降低食欲等多种重要功能,在控制体内血糖平衡的过程中发挥重要作用。2型糖尿病的发生伴随着胰高血糖素样肽-1生物应答功能受损。胰高血糖素样肽-1类似物、胰高血糖素样肽-1受体激动剂和胰高血糖素样肽-1降解酶抑制剂等糖尿病治疗药物已在临床上表现出很好的应用前景。     

细胞穿透肽:一种新型非病毒载体

细胞膜的选择通透性对维持细胞内环境的稳定起着非常重要的作用,但细胞膜的这种特性限制了一些生物大分子和药物进入细胞内,不利于对一些细胞内疾病的诊断和药物靶向治疗的应用。如何将一些具有诊断和治疗潜力的生物大分子、药物通过细胞膜进入细胞内一直是医学界研究的热点和难点。细胞穿透肽是一类能够携带多肽、蛋白质、核酸、纳米颗粒、病毒颗粒及药物等穿过细胞膜进入细胞,导致完整载物内化的短肽,为生物大分子和药物进入细胞内部提供了有力的运载工具,其作为载体具有的高转导效率和低毒性特点,已经得到了广泛关注和大量研究。目前,细胞穿透肽作为生物分子和药物细胞内化的运载体已经在荧光成像,肿瘤治疗,抗炎治疗及药物靶向治疗中发挥了潜在的诊断和治疗作用,显示出其诱人的应用前景。     

siRNA delivery using amphipathic cell-penetrating peptides into human hepatoma cells

Cell-penetrating peptides (CPPs) are an attractive tool for delivering membrane-impermeable compounds, including anionic biomacromolecules such as DNA and RNA, into living cells. Amphipathic helical peptides composed of hydrophobic amino acids and cationic amino acids are typical CPPs. In the current study, we designed amphipathic helical 12-mer peptides containing α,α-disubstituted α-amino acids (dAAs), which are known to stabilize peptide secondary structures. The dominant secondary structures of peptides in aqueous solution differed according to the introduced dAAs. Peptides containing hydrophobic dAAs and adopting a helical structure exhibited a good cell-penetrating ability. As an application of amphipathic helical peptides, small interfering RNA (siRNA) delivery into living human hepatoma cells was investigated. One of the peptides containing dAAs dipropylglycine formed stable complexes with siRNA at appropriate zeta-potential and size for intracellular siRNA delivery. This peptide showed effective RNA interference efficiency at short peptide length and low concentrations of peptide and siRNA. These findings will be helpful for the design of amphipathic helical CPPs as intracellular siRNA delivery.     

A gene delivery system for insect cells mediated by arginine-rich cell-penetrating peptides

Most bioactive macromolecules, such as protein, DNA and RNA, basically cannot permeate into cells freely from outside the plasma membrane. Cell-penetrating peptides (CPPs) are a group of short peptides that possess the ability to traverse the cell membrane and have been considered as candidates for mediating gene and drug delivery into living cells. In this study, we demonstrate that three arginine-rich CPPs (SR9, HR9 and PR9) are able to form stable complexes with plasmid DNA and deliver DNA into insect Sf9 cells in a noncovalent manner. The transferred plasmid DNA containing enhanced green fluorescent protein (EGFP) and red fluorescent protein (RFP) coding regions could be expressed in cells functionally assayed at both the protein and RNA levels. Furthermore, treatment of cells with CPPs and CPP/DNA complexes resulted in a viability of 84-93% indicating these CPPs are not cytotoxic. These results suggest that arginine-rich CPPs appear to be a promising tool for insect transgenesis.     

PEP and CADY-mediated delivery of fluorescent peptides and proteins into living cells

Cell-penetrating peptides (CPPs) constitute a family of peptides with the characteristic ability to cross biological membranes and deliver cargo into the intracellular milieu. Several CPPs have been proposed for delivery of polypeptides and proteins into cells through either of two strategies: covalent or complexed in a non-covalent fashion. Members of the PEP family are primary amphipathic peptides which have been shown to deliver peptides and proteins into a wide variety of cells through formation of non-covalent complexes. CADY is a secondary amphipathic peptide which has been demonstrated to deliver short nucleic acids, in particular siRNA with high efficiency. Here we review the characteristics of the PEP and CADY carriers and describe a novel derivative of CADY termed CADY2, which also presents sequence similarities to Pep1. We have compared Pep1, CADY and CADY2 in their efficiency to interact with and internalize short fluorogenic peptides and proteins into cultured cells, and provide evidence that CADY2 can interact with proteins and peptides and deliver them efficiently into living cells, similar to Pep1, but in contrast to CADY which is unable to deliver any peptide, even short negatively charged peptides. This is the first study to investigate the influence of the cargo on the interactions between PEP and CADY carriers, thereby providing novel insights into the physicochemical parameters underlying interactions and cellular uptake of peptides and proteins by these non-covalent CPPs.     

Identification of a novel cell-penetrating peptide from human phosphatidate phosphatase LPIN3

Biomolecules such as proteins, DNA, and RNA are macromolecules and can not cross the cell membrane. However, cell-penetrating peptide (CPP) has been shown to deliver therapeutic biomolecules successfully into cells. The various and widely used CPPs including TAT, VP22, and Antp are mostly non-human originated CPPs, and are limited by their potential toxicity and immunogenicity. We report here on a newly identified novel cell-penetrating sequence (LPIN; RRKRRRRRK) from the nuclear localization sequence (NLS) of human nuclear phosphatase, LPIN3. LPIN-EGFP recombinant protein was concentration- and time-dependently delivered into cells and localized to the nucleus as well as the cytoplasm. It penetrated the cell membrane by lipid raft-mediated endocytosis by binding to heparan sulfate proteoglycan. LPIN-EGFP was successfully delivered into primary mouse splenocytes in vitro and it could be delivered into various tissues including liver, kidney, and intestine in mice after intra-peritoneal injection. This research suggests that LPIN-CPP could be used in a drug delivery system to deliver therapeutic biomolecules including peptides, proteins, DNA, and RNA and without the limitations of non-human originated CPPs such as TAT-CPP.     

siRNA纳米递送系统研究进展

小干扰RNA (small interfering RNA,siRNA)是RNA干扰的引发物,激发与之互补的目标mRNA沉默,对基因调控及疾病治疗有重要意义。siRNA作为药物需要克服血管屏障、实现细胞内吞及溶酶体逃逸,同时还需要避免核酸酶作用下发生降解。因此,设计合适的纳米载体以帮助siRNA成功递送进细胞并发挥作用是目前siRNA药物发展的重要目标。纳米载体的材料种类、尺寸、结构、表面修饰等精确设计是实现siRNA药物成功递送的重要因素。随着研究的深入和应用的发展,siRNA药物纳米载体的精确控制制备、精准靶向递送及多功能化取得了较好的成果。本文围绕siRNA药物纳米载体,对siRNA药物应用及其递送困难、siRNA药物纳米载体主要设计策略、目前siRNA药物上市情况进行介绍,同时对其未来发展方向进行展望。     

Influence of the Dabcyl group on the cellular uptake of cationic peptides: short oligoarginines as efficient cell-penetrating peptides

Cell-penetrating peptides (CPPs) are promising delivery vehicles. These short peptides can transport wide range of cargos into cells, although their usage has often limitations. One of them is the endosomatic internalisation and thus the vesicular entrapment. Modifications which increases the direct delivery into the cytosol is highly researched area. Among the oligoarginines the longer ones (n > 6) show efficient internalisation and they are well-known members of CPPs. Herein, we describe the modification of tetra- and hexaarginine with (4–((4–(dimethylamino)phenyl)azo)benzoyl) (Dabcyl) group. This chromophore, which is often used in FRET system increased the internalisation of both peptides, and its effect was more outstanding in case of hexaarginine. The modified hexaarginine may enter into cells more effectively than octaarginine, and showed diffuse distribution besides vesicular transport already at low concentration. The attachment of Dabcyl group not only increases the cellular uptake of the cell-penetrating peptides but it may affect the mechanism of their internalisation. Their conjugates with antitumor drugs were studied on different cells and showed antitumor activity.

     

Cell-penetrating peptides: from molecular mechanisms to therapeutics

The recent discovery of new potent therapeutic molecules which do not reach the clinic due to poor delivery and low bioavailability have made the delivery of molecules a keystone in therapeutic development. Several technologies have been designed to improve cellular uptake of therapeutic molecules, including CPPs (cell-penetrating peptides), which represent a new and innovative concept to bypass the problem of bioavailability of drugs. CPPs constitute very promising tools and have been successfully applied for in vivo. Two CPP strategies have been described to date; the first one requires chemical linkage between the drug and the carrier for cellular drug internalization, and the second is based on the formation of stable complexes with drugs, depending on their chemical nature. The Pep and MPG families are short amphipathic peptides, which form stable nanoparticles with proteins and nucleic acids respectively. MPG- and Pep-based nanoparticles enter cells independently of the endosomal pathway and efficiently deliver cargoes, in a fully biologically active form, into a large variety of cell lines, as well as in animal models. This review focuses on the structure-function relationship of non-covalent MPG and Pep-1 strategies, and their requirement for cellular uptake of biomolecules and applications in cultured cells and animal models.     

Glycosylated cell-penetrating peptides and their conjugates to a proapoptotic peptide: preparation by click chemistry and cell viability studies

Cell-penetrating peptides (CPPs), which are usually short basic peptides, are able to cross cell membranes and convey bioactive cargoes inside cells. CPPs have been widely used to deliver inside cells peptides, proteins, and oligonucleotides; however, their entry mechanisms still remain controversial. A major problem concerning CPPs remains their lack of selectivity to target a specific type of cell and/or an intracellular component. We have previously shown that myristoylation of one of these CPPs affected the intracellular distribution of the cargo. We report here on the synthesis of glycosylated analogs of the cell-penetrating peptide (R6/W3): Ac-RRWWRRWRR-NH₂. One, two, or three galactose(s), with or without a spacer, were introduced into the sequence of this nonapeptide via a triazole link, the Huisgen reaction being achieved on a solid support. Four of these glycosylated CPPs were coupled via a disulfide bridge to the proapoptotic KLAK peptide, (KLAKLAKKLAKLAK), which alone does not enter into cells. The effect on cell viability and the uptake efficiency of different glycosylated conjugates were studied on CHO cells and were compared to those of the nonglycosylated conjugates: (R6/W3)S-S-KLAK and penetratinS-S-KLAK. We show that glycosylation significantly increases the cell viability of CHO cells compared to the nonglycosylated conjugates and concomitantly decreases the internalization of the KLAK cargo. These results suggest that glycosylation of CPP may be a key point in targeting specific cells.