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.     

Cell-penetrating peptides: [corrected] from inception to application

Despite continuing advances in the development of macromolecules, including peptides, proteins, and oligonucleotides, for therapeutic purposes, the successful application of these hydrophilic molecules has so far been hampered by their inability to efficiently traverse the cellular plasma membrane. The discovery of a class of peptides (cell-penetrating peptides, CPPs) with the ability to mediate the non-invasive and efficient import of a whole host of cargoes, both in vitro and in vivo, has provided a new means by which the problem associated with cellular delivery can be circumvented. A complete understanding of the translocation mechanism(s) of CPPs has so far proven elusive. Initial studies indicated an ATP-independent, non-endocytotic mechanism, dependent on direct peptide-membrane interactions, making it an enticing challenge from a biophysical point of view. However, recent evidence cast doubt on many of the earlier results, and led to a re-evaluation of the translocation mechanism of CPPs. In this review a brief history of the field will be given, followed by an introduction to some of the better known and more widely used CPPs, including some of their current applications, and finally a discussion of the translocation mechanism(s) and the controversies surrounding it.     

Cell-penetrating peptides: Application in vaccine delivery

Recent years have seen a surge in interest in cell-penetrating peptides (CPP) as an efficient means for delivering therapeutic targets into cellular compartments. The cell membrane is impermeable to hydrophilic substances yet linking to CPP can facilitate delivery into cells. Thus the unique translocatory property of CPP ensures they remain an attractive carrier, with the capacity to deliver cargoes in an efficient manner having applications in drug delivery, gene transfer and DNA vaccination. Fundamental for an effective vaccine is the delivery of antigen epitopes to antigen-presenting cells, ensuing processing and presentation and induction of an immune response. Vaccination with proteins or synthetic peptides incorporating CTL epitopes have proven limited due to the failure for exogenous antigens to be presented efficiently to T cells. Linking of antigens to CPP overcomes such obstacles by facilitating cellular uptake, processing and presentation of exogenous antigen for the induction of potent immune responses. This review will encompass the various strategies for the delivery of whole proteins, T cell epitopes and preclinical studies utilizing CPP for cancer vaccines.     

Cell-penetrating peptides: a comparative membrane toxicity study

Cell-penetrating peptides (CPPs) constitute a new class of delivery vectors with high pharmaceutical potential. However, the abilities of these peptides to translocate through cell membranes can be accompanied by toxic effects resulting from membrane perturbation at higher peptide concentrations. Therefore, we investigated membrane toxicity of five peptides with well-documented cell-penetrating properties, pAntp(43-58), pTAT(48-60), pVEC(615-632), model amphipathic peptide (MAP), and transportan 10, on two human cancer cell lines, K562 (erythroleukemia) and MDA-MB-231 (breast cancer), as well as on immortalized aortic endothelial cells. We studied the effects of these five peptides on the leakage of lactate dehydrogenase and on the fluorescence of plasma membrane potentiometric dye bis-oxonol. In all cell lines, pAntp(43-58), pTAT(48-60), and pVEC(615-632) induced either no leakage or low leakage of lactate dehydrogenase, accompanied by modest changes in bis-oxonol fluorescence. MAP and transportan 10 caused significant leakage; in K562 and MDA-MB-231 cells, 40% of total lactate dehydrogenase leaked out during 10 min exposure to 10 microM of transportan 10 and MAP, accompanied by a significant increase in bis-oxonol fluorescence. However, none of the CPPs tested had a hemolytic effect on bovine erythrocytes comparable to mastoparan 7. The toxicity profiles presented in the current study are of importance when selecting CPPs for different applications.     

Cell-penetrating peptides as antifungals towards Malassezia sympodialis

Aim: To determine whether different antimicrobial peptides (AMPs) and cell‐penetrating peptides (CPPs) are able to inhibit the growth of the commensal yeast Malassezia sympodialis, which can act as a trigger factor in different skin disorders, such as atopic eczema (AE), seborrhoeic eczema (SE) and dandruff. Methods and results: The antifungal activity of 21 different AMPs and CPPs was investigated by microdilution assay and plate counting to determine the number of colony forming units. Five CPPs and one AMP showed fungicidal activity at submicromolar concentrations. Importantly, no membrane damage on human keratinocytes was detected after peptide treatment. Conclusions: Several CPPs, while being nontoxic to mammalian cells, possess growth inhibitory activity on the very stringent yeast M. sympodialis. Significance and impact of study: Our findings that five CPPs and one AMP that are harmless towards mammalian cells act as antifungal agents against M. sympodialis opens up the possibility to use these in the treatment for AE, SE and dandruff. To our knowledge, this is the first time peptides have been identified as antifungal agents against M. sympodialis. Further studies to elucidate the mechanism are warranted.     

Cell-penetrating peptides derived from viral capsid proteins

Cell-penetrating peptides (CPP) can translocate across the cell membrane and have been extensively studied for the delivery of proteins, nucleic acids, and therapeutics in mammalian cells. However, characterizations of CPP in plants have only recently been initiated. We showed that the intact virion and a recombinant capsid protein (CaP) from a plant-infecting nonenveloped icosahedral RNA virus, Brome mosaic virus (BMV), can penetrate the membranes of plant protoplasts but are trapped by the extracellular matrix. Furthermore, a 22-residue peptide derived from the N-terminal region of the CaP (CPNT) can enter barley protoplasts and cells of intact barley and Arabidopsis roots. An inhibitor of the macropinocytosis reduced CPNT entry, while treatment with NiCl(2) changed the cellular localization of CPNT. CPNT increased uptake of the green flourescent protein (GFP) into the cell when covalently fused to GFP or when present in trans of GFP. The BMV CPNT overlaps with the sequence known to bind BMV RNA, and it can deliver BMV RNAs into cells, resulting in viral replication, as well as deliver double-stranded RNAs that can induce gene silencing.     

Cell-penetrating and cell-targeting peptides in drug delivery

During the last decade, the potential of peptides for drug delivery into cells has been highlighted by the discovery of several cell-penetrating peptides (CPPs). CPPs are very efficient in delivering various molecules into cells. However, except in some specific cases, their lack of cell specificity remains the major drawback for their clinical development. At the same time, various peptides with specific binding activity for a given cell line (cell-targeting peptides) have also been reported in the literature. One of the goals of the next years will be to optimize the tissue and cell delivery of therapeutic molecules by means of peptides which combine both targeting and internalization advantages. In this review, we describe the main strategies that are currently in use or likely to be employed in the near future to associate both targeting and delivery properties.     

Cell-penetrating peptides. A reevaluation of the mechanism of cellular uptake

Cellular uptake of a family of cationic cell-penetrating peptides (examples include Tat peptides and penetratin) have been ascribed in the literature to a mechanism that does not involve endocytosis. In this work we reevaluate the mechanisms of cellular uptake of Tat 48-60 and (Arg)(9). We demonstrate here that cell fixation, even in mild conditions, leads to the artifactual uptake of these peptides. Moreover, we show that flow cytometry analysis cannot be used validly to evaluate cellular uptake unless a step of trypsin digestion of the cell membrane-adsorbed peptide is included in the protocol. Fluorescence microscopy on live unfixed cells shows characteristic endosomal distribution of peptides. Flow cytometry analysis indicates that the kinetics of uptake are similar to the kinetics of endocytosis. Peptide uptake is inhibited by incubation at low temperature and cellular ATP pool depletion. Similar data were obtained for Tat-conjugated peptide nucleic acids. These data are consistent with the involvement of endocytosis in the cellular internalization of cell-penetrating peptides and their conjugates to peptide nucleic acids.     

The other side of the coin: Hypersociability

Affiliative social motivation and behavior, that is, sociability that includes attachment, prosocial behavior (sharing, caring and helping) and empathy (the ability to understand and share the feelings of others), has high variability in the human population, with a portion of people outside of the normal range. While psychiatric disorders and autism spectrum disorders are typically associated with a deficit in social behavior, the opposite trait of hypersociability and indiscriminate friendliness are exhibited by individual with specific neurodevelopmental disorders and following early adverse care. Here we discuss both genetic and environmental factors that cause or increase the risk for developing pathological hypersociability from human to rodent models.     

Tissue-based class control: the other side of tolerance

In this Essay, we offer a new perspective on how immune responses are regulated. We do not cover how they are turned on and off, but focus instead on the second major aspect of an immune response: the control of effector class. Although it is generally thought that the class of an immune response is tailored to fit the invading pathogen, we suggest here that it is primarily tailored to fit the tissue in which the response occurs. To this end, we cover such topics as the nature of T helper (T(H)) cell subsets (current and yet to be discovered), the nature of privileged sites, the difference between oral tolerance and oral vaccination, why the route of immunization matters, whether the T(H)1-type response is really the immune system's primary defense, and whether there might be a different role for some regulatory T cells.     

Cell-penetrating peptides split into two groups based on modulation of intracellular calcium concentration

Cell-penetrating peptides (CPPs) promote the uptake of different cargo molecules, e.g. therapeutic compounds, making the harnessing of CPPs a promising strategy for drug design and delivery. However, the internalization mechanisms of CPPs are still under discussion, and it is not clear how cells compensate the disturbances induced by peptides in the plasma membrane. In this study, we demonstrate that the uptake of various CPPs enhances the intracellular Ca(2+) levels in Jurkat and HeLa cells. The elevated Ca(2+) concentration in turn triggers plasma membrane blebbing, lysosomal exocytosis, and membrane repair response. Our results indicate that CPPs split into two major classes: (i) amphipathic CPPs that modulate the plasma membrane integrity inducing influx of Ca(2+) and activating downstream responses starting from low concentrations; (ii) non-amphipathic CPPs that do not evoke changes at relevant concentrations. Triggering of the membrane repair response may help cells to replace distorted plasma membrane regions and cells can recover from the influx of Ca(2+) if its level is not drastically elevated.     

提高细胞膜穿透肽应用效应的几种策略

细胞膜穿透肽（cell-penetrating peptide,CPP）的发现,为向细胞内输送各种大分子生物活性物质提供了一个有力的运送载体,相比于以前的各种运送载体,它具有操作简便、相对安全和毒副作用小等优点,但同时也存在着诸如从内含体释放效率低、细胞内定位不够精确以及缺乏组织细胞靶向性等缺陷,这也是目前国内外对于CPP的研究热点。