综述: 细胞穿透肽及其结构改造在siRNA传递中的应用

小RNA药物应用于临床的主要技术瓶颈在于如何高效、低毒地将小RNA分子传递到它发挥功能的场所．基于细胞穿透肽在小RNA透皮给药的临床应用中所取得的进展,本文系统评述了近年来细胞穿透肽在小RNA的体内、体外传递方面的研究动态,分析了细胞穿透肽的结构改造对肽/小RNA复合物转染进入细胞发挥功能的影响,展望了细胞穿透肽作为小RNA的体内药物传递载体的发展方向．     

Transduction of peptides and proteins into live cells by cell penetrating peptides

Internalization of peptides and proteins into live cells is an essential prerequisite for studies on intracellular signal pathways, for treatment of certain microbial diseases and for signal transduction therapy, especially for cancer treatment. Cell penetrating peptides (CPPs) facilitate the transport of cargo-proteins through the cell membrane into live cells. CPPs which allow formation of non-covalent complexes with the cargo are used primarily in this study due to the relatively easy handling procedure. Efficiency of the protein uptake is estimated qualitatively by fluorescence microscopy and quantitatively by SDS-PAGE. Using the CPP cocktail JBS-Proteoducin, the intracellular concentrations of a secondary antibody and bovine serum albumin can reach the micromolar range. Internalization of antibodies allows mediation of intracellular pathways including knock down of signal transduction. The high specificity and affinity of antibodies makes them potentially more powerful than siRNA. Thus, CPPs represent a significant new possibility to study signal transduction processes in competition or in comparison to the commonly used other techniques. To estimate the highest attainable intracellular concentrations of cargo proteins, the CPPs are tested for cytotoxicity. Cell viability and membrane integrity relative to concentration of CPPs are investigated. Viability as estimated by the reductive activity of mitochondria (MTT-test) is more sensitive to higher concentrations of CPPs versus membrane integrity, as measured by the release of dead cell protease. Distinct differences in uptake efficiency and cytotoxic effects are found using six different CPPs and six different adhesion and suspension cell lines.     

Peptide internalization enabled by folding: triple helical cell‐penetrating peptides

Cell‐penetrating peptides (CPPs) are known as efficient transporters of molecular cargo across cellular membranes. Their properties make them ideal candidates for in vivo applications. However, challenges in the development of effective CPPs still exist: CPPs are often fast degraded by proteases and large concentration of CPPs required for cargo transporting can cause cytotoxicity. It was previously shown that restricting peptide flexibility can improve peptide stability against enzymatic degradation and limiting length of CPP peptide can lower cytotoxic effects. Here, we present peptides (30‐mers) that efficiently penetrate cellular membranes by combining very short CPP sequences and collagen‐like folding domains. The CPP domains are hexa‐arginine (R₆) or arginine/glycine (RRGRRG). Folding is achieved through multiple proline–hydroxyproline–glycine (POG [proline‐hydroxyproline‐glycine])_n repeats that form a collagen‐like triple helical conformation. The folded peptides with CPP domains are efficiently internalized, show stability against enzymatic degradation in human serum and have minimal toxicity. Peptides lacking correct folding (random coil) or CPP domains are unable to cross cellular membranes. These features make triple helical cell‐penetrating peptides promising candidates for efficient transporters of molecular cargo across cellular membranes. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

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。     

Cellular uptake and biophysical properties of galactose and/or tryptophan containing cell-penetrating peptides

Glycosylated cell penetrating peptides (CPPs) have been conjugated to a peptide cargo and the efficiency of cargo delivery into wild type Chinese hamster ovary (CHO) and proteoglycan deficient CHO cells has been quantified by MALDI-TOF mass spectrometry and compared to tryptophan- or alanine containing CPPs. In parallel, the behavior of these CPPs in contact with model membranes has been characterized by different biophysical techniques: Differential Scanning and Isothermal Titration Calorimetries, Imaging Ellipsometry and Attenuated Total Reflectance IR spectroscopy. With these CPPs we have demonstrated that tryptophan residues play a key role in the insertion of a CPP and its conjugate into the membrane: galactosyl residues hampered the internalization when introduced in the middle of the amphipathic secondary structure of a CPP but not when added to the N-terminus, as long as the tryptophan residues were still present in the sequence. The insertion of these CPPs into membrane models was enthalpy driven and was related to the number of tryptophans in the sequence of these secondary amphipathic CPPs. Additionally, we have observed a certain propensity of the investigated CPP analogs to aggregate in contact with the lipid surface.     

PNA-based antisense oligonucleotides for micrornas inhibition in the absence of a transfection reagent

MicroRNAs (miRNAs) are noncoding RNAs approximately 22 nucleotides in length that play a major role in the regulation of important biological processes, including cellular development, differentiation, and apoptosis. Antisense oligonucleotides against miRNAs are useful tools for studying the biological mechanisms and therapeutic targets of miRNAs. Various antisense oligonucleotides chemistries, including peptide nucleic acids (PNAs), have been developed to enhance nuclease-resistance and affinity and specificity for miRNA targets. PNAs have a greater specificity and affinity for DNA and RNA than do natural nucleic acids, and they are resistant to nucleases-an essential property of an miRNA inhibitor that will be exposed to cellular nucleases. However, the main limiting factor in the use of PNAs is their reduced penetration into cells. Recently, several cell-penetrating peptides (CPPs) have been investigated as a means to overcome the limited penetration of PNAs. Here, we evaluated the ability of 11 CPPs to transport PNAs inside cells in the absence of transfection reagents and then investigated the ability of these CPPs to inhibit miRNAs. Of the 11 CPPs tested, Tat-modified-conjugated PNA showed the most effective penetration into cells in the absence of transfection reagents and most effectively inhibited miRNAs. Our data demonstrate that Tat-modified-conjugated CPP is the most suitable for supporting PNA-mediated miRNA inhibition.     

Identification and characterization of a novel cell-penetrating peptide

Cell-penetrating peptides (CPPs) are short amino acid sequences that promote their own translocation across cell plasma membrane. When linked with cargo such as polypeptides, nucleic acid, or liposomes, CPPs can facilitate the transport of these entities across the cell membrane. Therefore, CPPs are receiving increased interest in drug delivery and gene therapy. The majority of CPPs identified so far are polycationic peptides which interact with heparin sulfate chains of plasma membrane for internalization. Here, we report the identification and characterization of a conformationally constrained 13 amino acid peptide (CVQWSLLRGYQPC, designated as S41) which is clearly distinct from classical polycationic peptides. Immunofluorescence assay was employed to test the cellular uptake of S41 in mouse neuroblastoma cell line Neuro2A (N2A) and rat cerebellar granule neurons (CGNs). Internalization of S41 was further examined in N2A cells by means of mutational analysis, flow cytometry and confocal microscopy. Our results demonstrate that S41 can enter cells through lipid rafts dependent endocytosis.     

A plasmid display platform for the selection of peptides exhibiting a functional cell-penetrating phenotype

Cell-penetrating peptides (CPPs) represent a promising nonviral platform for the delivery of therapeutic cargos to cells and tissues. However, these peptides are often nonspecific, and their mechanism of action is still a subject of debate, which hinders the design of new CPPs. The alternative to rational protein design is the combinatorial approach to protein engineering, whereby large libraries of peptides are created and a screening or selection procedure is used to identify members with the desired phenotype(s). Here we describe a novel procedure for selecting peptides with a CPP phenotype using a plasmid display (PD) platform to link the peptides to their encoding DNA sequences. The PD system is based on genetic fusions to a DNA binding domain. The plasmid was designed to concomitantly express a fluorescent reporter protein to serve as a mock therapeutic cargo indicating its functional delivery into a cell. We have demonstrated this selection strategy using a control CPP (the TAT peptide) in the PC12 neuronal-like cell line. In the absence of transfection reagents, TAT was unable to deliver the protein/DNA complexes. The inclusion of the HA2 peptide from the hemagglutinin protein and the addition of polyethylenimine (PEI) were similarly ineffective. The addition of Lipofectamine, however, enabled the TAT-mediated delivery of the protein/DNA complexes, which was significantly better than control experiments without a CPP. This new PD selection platform will be a valuable new approach for use in identifying unique CPPs from randomized libraries with novel abilities and specificities.     

The agony of choice: how to find a suitable CPP for cargo delivery

Successful and effective cellular delivery remains a main obstacles in the medical field. The use of cell‐penetrating peptides (CPPs) has become one of the most important tools for the internalisation of a wide range of molecules including pharmaceuticals. It is still difficult to choose one CPP for one biological application because there is no ubiquitous CPP meeting the diverse requirements. In our case, we are looking for a suitable CPP to deliver the pro‐apoptotic KLA peptide (KLAKLAKKLAKLAK) by a simple co‐incubation strategy. For that reason, we selected three different cell lines (fibroblastic, cancerous and macrophagic cells) and studied the uptake and subcellular localisation of six different CPPs alone as well as mixed with the KLA peptide. Furthermore, we used the CPPs with a carboxyamidated or a carboxylated C‐terminus and analysed the impact of the C‐termini on internalisation and cargo delivery. We could clearly showed that the cellular CPP uptake is not only dependent on the used CPP and cell line but also highly affected by its chemical nature of the C‐terminus (uptake: carboxyamidated CPPs > carboxylated CPPs) and can influence its cellular localisation. We successfully delivered the KLA peptide in the three cell lines and learned that here as well, the C‐terminus is crucial for an effective peptide delivery. Finally, we induced apoptosis in mouse leukaemic monocyte macrophage (RAW 264.7) and in human breast adenocarcinoma (MCF‐7) cells using the mixture of amidated MPG peptide : KLA and in african green monkey kidney fibroblast (Cos‐7) cells using carboxylated integrin peptide : KLA. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Effects of cargo molecules on the cellular uptake of arginine-rich cell-penetrating peptides

The identification of cell-penetrating peptides (CPPs) as vectors for the intracellular delivery of conjugated molecules such as peptides, proteins, and oligonucleotides has emerged as a significant tool to modulate biological activities inside cells. The mechanism of CPP uptake by the cells is still unclear, and appears to be both endocytotic and non-endocytotic, depending on the CPP and cell type. Moreover, it is also unknown whether cargo sequences have an effect on the uptake and cellular distribution properties of CPP sequences. Here, we combine results from quantitative fluorescence microscopy and binding to lipid membrane models to determine the effect of cargo peptide molecules on the cellular uptake and distribution of the arginine-rich CPPs, R₇, and R₇W, in live cells. Image analysis algorithms that quantify fluorescence were used to measure the relative amount of peptide taken up by the cell, as well as the extent to which the uptake was endocytotic in nature. The results presented here indicate that fusion of arginine-rich CPPs to peptide sequences reduces the efficiency of uptake, and dramatically changes the cellular distribution of the CPP from a diffuse pattern to one in which the peptides are mostly retained in endosomal compartments.     

Cellular delivery of small interfering RNA by a non-covalently attached cell-penetrating peptide: quantitative analysis of uptake and biological effect

Cell-penetrating peptides (CPPs) have evolved as promising new tools to deliver nucleic acids into cells. So far, the majority of these delivery systems require a covalent linkage between carrier and cargo. To exploit the higher flexibility of a non-covalent strategy, we focused on the characterisation of a novel carrier peptide termed MPGα, which spontaneously forms complexes with nucleic acids. Using a luciferase-targeted small interfering RNA (siRNA) as cargo, we optimised the conditions for MPGα-mediated transfection of mammalian cells. In this system, reporter gene activity could be inhibited up to 90% with an IC₅₀ value in the sub-nanomolar range. As a key issue, we addressed the cellular uptake mechanism of MPGα/siRNA complexes applying various approaches. First, transfection of HeLa cells with MPGα/siRNA complexes in the presence of several inhibitors of endocytosis showed a significant reduction of the RNA interference (RNAi) effect. Second, confocal laser microscopy revealed a punctual intracellular pattern rather than a diffuse distribution of fluorescently labelled RNA-cargo. These data provide strong evidence of an endocytotic pathway contributing significantly to the uptake of MPGα/siRNA complexes. Finally, we quantified the intracellular number of siRNA molecules after MPGα-mediated transfection. The amount of siRNA required to induce half maximal RNAi was 10000 molecules per cell. Together, the combination of methods provided allows for a detailed side by side quantitative analysis of cargo internalisation and related biological effects. Thus, the overall efficiency of a given delivery technique as well as the mechanism of uptake can be assessed.     

Prediction of Cell-Penetrating Peptides

Cell-penetrating peptides, CPPs, are used as delivery vectors for pharmacologically interesting substances, such as antisense oligonucleotides, proteins and peptides. We present a general principle for designing cell-penetrating peptides derived from naturally occurring proteins as well as from randomly generated polyamino acid sequences. Thereby, we introduce a novel pharmacological principle for identification of cell-penetrating peptides for which the applications can be numerous, including cellular transduction vectors and mimics of intracellular protein–protein interactions. The methods of identifying a CPP comprises assessing the averaged bulk property values of the defined sequence, and ensuring that they fall within the bulk property value interval obtained from the training set. Despite this simplistic approach, the search criteria proved useful for finding CPP properties in either proteins or random sequences. We have experimentally verified cell-penetrating properties of 10–20-mer peptides derived from naturally occurring proteins as well as from random poly-amino acids. We note that since CPPs can be found in part of the protein sequences that may govern protein interactions, it is possible to produce cell-penetrating protein agonists or antagonists.     

新型细胞穿膜肽的鉴定方法与其在抗肿瘤治疗中的应用

如何将生物活性分子高效投递到靶标的细胞和组织仍然是生物治疗领域研究人员面临的难题之一。直到细胞穿膜肽(cell penetrating peptides, CPPs)的出现,其可介导多种外源性功能分子(核酸、多肽、蛋白质和化学药物)进入细胞,而且不影响外源活性分子的功能发挥。另外,CPPs在传递外源活性成分进入肿瘤组织和细胞方面表现出更具应用前景的优势。因此,通过对CPPs的分类、鉴定方法、穿膜机制、其在抗肿瘤治疗中的最新应用以及尚需要解决的问题进行综述,以期为新型CPPs的鉴定和其抗肿瘤治疗策略提供参考。     

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.     

Effects of cargo molecules on the cellular uptake of arginine-rich cell-penetrating peptides

The identification of cell-penetrating peptides (CPPs) as vectors for the intracellular delivery of conjugated molecules such as peptides, proteins, and oligonucleotides has emerged as a significant tool to modulate biological activities inside cells. The mechanism of CPP uptake by the cells is still unclear, and appears to be both endocytotic and non-endocytotic, depending on the CPP and cell type. Moreover, it is also unknown whether cargo sequences have an effect on the uptake and cellular distribution properties of CPP sequences. Here, we combine results from quantitative fluorescence microscopy and binding to lipid membrane models to determine the effect of cargo peptide molecules on the cellular uptake and distribution of the arginine-rich CPPs, R7, and R7W, in live cells. Image analysis algorithms that quantify fluorescence were used to measure the relative amount of peptide taken up by the cell, as well as the extent to which the uptake was endocytotic in nature. The results presented here indicate that fusion of arginine-rich CPPs to peptide sequences reduces the efficiency of uptake, and dramatically changes the cellular distribution of the CPP from a diffuse pattern to one in which the peptides are mostly retained in endosomal compartments.     

Identification of a Short Cell-Penetrating Peptide from Bovine Lactoferricin for Intracellular Delivery of DNA in Human A549 Cells

Cell-penetrating peptides (CPPs) have been shown to deliver cargos, including protein, DNA, RNA, and nanomaterials, in fully active forms into live cells. Most of the CPP sequences in use today are based on non-native proteins that may be immunogenic. Here we demonstrate that the L5a CPP (RRWQW) from bovine lactoferricin (LFcin), stably and noncovalently complexed with plasmid DNA and prepared at an optimal nitrogen/phosphate ratio of 12, is able to efficiently enter into human lung cancer A549 cells. The L5a CPP delivered a plasmid containing the enhanced green fluorescent protein (EGFP) coding sequence that was subsequently expressed in cells, as revealed by real-time PCR and fluorescent microscopy at the mRNA and protein levels, respectively. Treatment with calcium chloride increased the level of gene expression, without affecting CPP-mediated transfection efficiency. Zeta-potential analysis revealed that positively electrostatic interactions of CPP/DNA complexes correlated with CPP-mediated transport. The L5a and L5a/DNA complexes were not cytotoxic. This biomimetic LFcin L5a represents one of the shortest effective CPPs and could be a promising lead peptide with less immunogenic for DNA delivery in gene therapy.     

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.     

Exogenous nucleic acids aggregate in non-P-body cytoplasmic granules when transfected into cultured cells

To modulate gene expression in research studies or in potential clinical therapies, transfection of exogenous nucleic acids including plasmid DNA and small interference RNA (siRNA) are generally performed. However, the cellular processing and the fate of these nucleic acids remain elusive. By investigating the cellular behavior of transfected nucleic acids using confocal imaging, here we show that when siRNA was co-transfected into cultured cells with other nucleic acids, including single-stranded RNA oligonucleotides, single and double-stranded DNA oligonucleotides, as well as long double-stranded plasmid DNA, they all aggregate in the same cytoplasmic granules. Interestingly, the amount of siRNA aggregating in granules was found not to correlate with the gene silencing activity, suggesting that assembly of cytoplasmic granules triggered by siRNA transfection may be separable from the siRNA silencing event. Our results argue against the claim that the siRNA-aggregating granules are the functional site of RNA interference (RNAi). Taken together, our studies suggest that, independent of their types or forms, extraneously transfected nucleic acids are processed through a common cytoplasmic pathway and trigger the formation of a new type of cytoplasmic granules “transfection granules”.