Differentiation Restricted Endocytosis of Cell Penetrating Peptides in MDCK Cells Corresponds with Activities of Rho-GTPases

Purpose Cellular entry of biomacromolecules is restricted by the barrier function of cell membranes. Tethering such molecules to cell penetrating peptides (CPPs) that can translocate cell membranes has opened new horizons in biomedical research. Here, we investigate the cellular internalization of hCT(9-32)-br, a human calcitonin derived branched CPP, and SAP, a γ-zein related sequence. Methods Internalization of fluorescence labelled CPPs was performed with both proliferating and confluent MDCK cells by means of confocal laser scanning microscopy (CLSM) and fluorescence activated cell sorting (FACS) using appropriate controls. Internalization was further elaborated in an inflammatory, IFN-γ/TNF-αa induced confluent MDCK model mimicking inflammatory epithelial pathologies. Activities of active form Rho-GTPases (Rho-A and Rac-1) in proliferating and confluent MDCK cells were monitored by pull-down assay and Western blot analysis. Results We observed marked endocytic uptake of the peptides into proliferating MDCK by a process suggesting both lipid rafts and clathrin-coated pits. In confluent MDCK, however, we noted a massive but compound-unspecific slow-down of endocytosis. This corresponded with a down-regulation of endocytosis by Rho-GTPases, previously identified to be intimately involved in endocytic traffic. In fact, we found endocytic internalization to relate with active Rho-A; vice versa, MDCK cell density, degree of cellular differentiation and endocytic slow-down were found to relate with active Rac-1. To our knowledge, this is the first study to cast light on the previously observed differentiation restricted internalization of CPPs into epithelial cell models. In the inflammatory IFN-γ/TNF-αa induced confluent MDCK model mimicking inflammatory epithelial pathologies, CPP internalization was enhanced in a cytokine concentration-dependent way resulting in maximum enhancement rates of up to 90%. We suggest a cytokine induced redistribution of lipid rafts in confluent MDCK to cause this enhancement. Conclusion Our findings emphasize the significance of differentiated cell models in the study of CPP internalization and point towards inflammatory epithelial pathologies as potential niche for the application of CPPs for cellular delivery. This work was supported by the Commission of the European Union (EU project on Quality of Life and Management of Living Resources, Project No. QLK2-CT-2001-01451.     

Cell penetrating peptides: the potent multi-cargo intracellular carriers

ABSTRACT

Introduction: Cell penetrating peptides (CPPs) known as protein translocation domains (PTD), membrane translocating sequences (MTS), or Trojan peptides (TP) are able to cross biological membranes without clear toxicity using different mechanisms, and facilitate the intracellular delivery of a variety of bioactive cargos. CPPs could overcome some limitations of drug delivery and combat resistant strains against a broad range of diseases. Despite delivery of different therapeutic molecules by CPPs, they lack cell specificity and have a short duration of action. These limitations led to design of combined cargo delivery systems and subsequently improvement of their clinical applications.

Areas covered: This review covers all our studies and other researchers in different aspects of CPPs such as classification, uptake mechanisms, and biomedical applications.

Expert opinion: Due to low cytotoxicity of CPPs as compared to other carriers and final degradation to amino acids, they are suitable for preclinical and clinical studies. Generally, the efficiency of CPPs was suitable to penetrate the cell membrane and deliver different cargos to specific intracellular sites. However, no CPP-based therapeutic approach has approved by FDA, yet; because there are some disadvantages for CPPs including short half-life in blood, and nonspecific CPP-mediated delivery to normal tissue. Thus, some methods were used to develop the functions of CPPs in vitro and in vivo including the augmentation of cell specificity by activatable CPPs, specific transport into cell organelles by insertion of corresponding localization sequences, incorporation of CPPs into multifunctional dendrimeric or liposomal nanocarriers to improve selectivity and efficiency especially in tumor cells.     

Evaluating the translocation properties of a new nuclear targeted penetrating peptide using two fluorescent markers

Human-derived cell penetrating peptides (CPPs) have attracted much more attentions than other CPPs which are limited by their potential toxicity and immunogenicity. Previously, we identified a novel human-originated CPP (named heparin-binding domain (HBD) in this article), which derived from the C-terminus of human extracellular superoxide dismutase, and demonstrated HBD is an efficient vector for delivering exogenous drug molecules such as apoptin into HeLa cells. In this study, we found this novel CPP showed differentiated efficiency in several tested cell lines. Heparin competitive inhibition experiment and heparanase pre-incubation experiment showed cell surface polysaccharides play an important role for the transmembrane transport. The results of endocytosis inhibitors suggested that HBD penetrates the cell membrane via a direct translocation, which is different from that of TAT, a classical clathrin-mediated endocytosis. HBD could deliver up to 90 kD protein cargoes into cells. Different conjugated modes with cargo molecules greatly affect their translocation efficiency. HBD also showed significant nuclear transport capacity when it was incubated with HeLa cells. Furthermore, the core region for HBD possessing membrane-penetrating ability was identified by deletion analyses. These results would be helpful for developing HBD as a new nuclear delivery tool for therapeutic biomolecules.     

On the biomedical promise of cell penetrating peptides: limits versus prospects

The cell membrane poses a substantial hurdle to the use of pharmacologically active biomacromolecules that are not per se actively translocated into cells. An appealing approach to deliver such molecules involves tethering or complexing them with so-called cell penetrating peptides (CPPs) that are able to cross the plasma membrane of mammalian cells. The CPP approach is currently a major avenue in engineering delivery systems that are hoped to mediate the non-invasive import of problematic cargos into cells. The large number of different cargo molecules that have been efficiently delivered by CPPs ranges from small molecules to proteins and even liposomes and particles. With respect to the involved mechanism(s) there is increasing evidence for endocytosis as a major route of entry. Moreover, in terms of intracellular trafficking, current data argues for the transport to acidic early endosomal compartments with cytosolic release mediated via retrograde delivery through the Golgi apparatus and the endoplasmic reticulum. The focus of this review is to revisit the performance of cell penetrating peptides for drug delivery. To this aim we cover both accomplishments and failures and report on new prospects of the CPP approach. Besides a selection of successful case histories of CPPs we also review the limitations of CPP mediated translocation. In particular, we comment on the impact of (i) metabolic degradation, (ii) the cell line and cellular differentiation state dependent uptake of CPPs, as well as (iii) the regulation of their endocytic traffic by Rho-family GTPases. Further on, we aim at the identification of promising niches for CPP application in drug delivery. In this context, as inspired by current literature, we focus on three principal areas: (i) the delivery of antineoplastic agents, (ii) the delivery of CPPs as antimicrobials, and (iii) the potential of CPPs to target inflammatory tissues.     

Fatty acyl moieties: improving Pro‐rich peptide uptake inside HeLa cells

Abstract: In the field of drug delivery there has been a continuous study of powerful delivery systems to aid non permeable drugs in reaching their intracellular target. Among the systems explored are cell penetrating peptides (CPPs), which first garnered interest a decade ago when the interesting translocation properties of the pioneer CPPs Tat and Antp were described. A new family of CPPs has recently been described as non cytotoxic Pro‐rich vectors with favorable profiles for internalization in HeLa cells. Fatty acyl moieties that can tune a peptide's interaction with the lipophilic environment of a cell membrane have been incorporated into the Pro‐rich sequence. Improvements in cellular uptake of peptides modified with fatty acyl groups, as studied by confocal microscopy and flow cytometry, as well as the results obtained by the interaction of these peptides with a model dioleoylphosphatidylcholine (DOPC) membrane and transmission electron microscopy (TEM), illustrate the importance of the fatty acyl moieties for efficient internalization.     

Cell penetrating peptides: a comparative transport analysis for 474 sequence motifs

Delivering reagents into cells is a key demand in molecular medicine. The vehicle of choice is often cell penetrating peptides (CPPs), which can ferry conjugated cargo across membranes. Although numerous peptides have been shown to promote such uptake events, there has been no comprehensive comparison of individual performance under standardized conditions. We have devised a method to rapidly analyze the ability of a multitude of CPP conjugates to carry a model cargo into HeLa cells. Sequence information for 474 CPPs was collected from literature sources, and the respective peptides were synthesized and modified with carboxyfluorescein (FAM) as model cargo. All candidates were evaluated in an identical uptake test, and transport was quantified using cellular fluorescence intensities. Substantial differences in the ability to carry the fluorophore into the cells were observed, with transport performance differing by a factor of 70 between the best CPP investigated and cargo alone. Strong correlations were observed between uptake efficiency and both sequence length and the presence of positive net charge. A compilation of the 20 top performers with regard to cargo delivery performance and cell compatibility is provided.     

Oligoarginine-PEG-lipid particles for gene delivery

Cell-penetrating peptides (CPPs) are small peptides that can facilitate the uptake of macromolecular drugs, such as proteins or nucleic acids, into mammalian cells. Cytosolic delivery of CPPs could be beneficial to bypass conventional endocytosis in order to avoid degradation in the lysosomes. Oligoarginine conjugates have characteristics similar to CPPs in terms of cell translocation and are used in the intracellular delivery of plasmid DNA. In these cases, oligoarginine length and/or charge are important factors in the cellular uptake of oligoarginine alone. The arginine moiety of oligoarginine-modified particles may also be a decisive factor for vectors to deliver plasmid DNA. Oligoarginine-PEG-lipids can form self-assembled particles and modify the surface of lipid- and polymer-based particles. This review focuses on the influence of: i) oligoarginine-modified particles such as micelles, liposomes and polymer-based particles; ii) the morphology of oligoarginine-PEG-lipid complexed with plasmid DNA by decreasing the charge ratio; and iii) the oligoarginine length in the complex on its cellular uptake, transfection efficiency and uptake mechanism. The oligoarginine length of oligoarginine-modified particle complexed with plasmid DNA governs the cellular uptake pathway that determines the destiny of intracellular trafficking and finally transfection efficiency. The new aspects of surface-functionalized particle vectors with oligoarginine are discussed.     

Cell penetrating peptides for tumor targeting

Anticancer drug delivery has been hindered due to cell membrane permeability and the lack of a selective marker for tumor cells. Cell permeability is related to the bioavailability of drugs and has therefore been considered to be an essential step for achieving therapeutic efficacy. While different types of transporters currently exist, cell penetrating peptides (CPPs) have become one of the most popular and effective tools for intracellular drug delivery. Most of the original CPPs are short peptides with basic residues. The mechanism of CPP cell entry remains to be established; however, the CPPs can deliver any type of molecular cargo including solid nanoparticles. Herein, this paper will discuss the classification of CPPs, the mechanism of cell entry, the application of CPPs in tumor therapy, and recent advances in targeted cell penetration that involve CPPs.     

Reviewing biophysical and cell biological methodologies in cell-penetrating peptide (CPP) research

The discovery of cell-penetrating peptides (CPPs), which have the ability to translocate across the plasma membranes of mammalian cells, has led to widespread optimism for delivery of problematic therapeutic cargoes to cells. These cargoes include peptide, protein, and nucleic acid biopharmaceuticals and even nano-sized vectors such as liposomes and nanoparticles. Research on CPPs includes biophysical studies of membrane models to investigate fundamental principles of CPP-lipid membrane interactions as well as cell studies focusing on the efficiency of uptake, mechanisms of translocation, and toxicity. However, both lines of research have suffered from misinterpretation as well as premature extrapolations. In this review, we provide a critical evaluation of the potential and limitations of selected biophysical methodologies such as fluorescence spectroscopy, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy, atomic-force microscopy (AFM), and non-spectroscopic methods. We include a discussion of the most important bilayer membrane models in CPP research. We then evaluate important cell biological methodologies, in particular confocal laser scanning microscopy (CLSM) and fluorescence-associated cell sorting (FACS) in combination with various techniques to distinguish between translocated and non-translocated CPPs. Moreover, we discuss the diverse methodologies for tracing the pathways of CPP translocation and their routes of intracellular trafficking.     

Cell permeable cocaine esterases constructed by chemical conjugation and genetic recombination

Cocaine esterase (CocE) is the most efficient cocaine-metabolizing enzyme tested in vivo to date, displaying a rapid clearance of cocaine and a robust protection against cocaine's toxicity. Two potential obstacles to the clinical application of CocE, however, lie in its proteolytic degradation and induced immune response. To minimize these potential obstacles, we attempted nondisruptive cell encapsulation by creating a cell permeable form of CocE, which was achieved by covalently linking a thermally stable CocE mutant (dmCocE) with cell penetrating peptides (CPPs). Two types of CPPs, Tat and the low molecular weight protamine (LMWP), were used in this study. Two types of disulfide-bridged chemical conjugates, Tat-S-S-dmCocE and LMWP-S-S-dmCocE, were synthesized and then purified by heparin affinity chromatography. In addition, four recombinant CPP-dmCocE fusion proteins, Tat-N-dmCocE, LMWP-N-dmCocE, dmCocE-C-Tat, and dmCocE-C-LMWP, were constructed, expressed in Escherichia coli, and purified as soluble proteins. Among these six CPP-dmCocE variants, LMWP-S-S-dmCocE showed the highest cocaine-hydrolyzing activity, and dmCocE-C-Tat had the highest production yield. To evaluate their cellular uptake behavior, a covalently linked fluorophore (FITC) was utilized to visualize the cellular uptake of all six CPP-dmCocE variants in living HeLa cells. All the six variants exhibited cellular uptake, but their intracellular distribution phenotypes differed. While the chemical conjugates showed primarily cytoplasmic distribution, which was likely due to the reduction of the disulfide linkage between CPP and dmCocE, all the other four recombinant fusion proteins displayed both nuclear and cytoplasmic localization, with dmCocE-C-CPP exhibiting higher cytoplasmic distribution during cellular uptake. Based on a balanced consideration of essentials for clinical application, including parameters such as high cocaine-hydrolyzing efficiency, large production yield, major cytoplasmic distribution, etc., the dmCocE-C-Tat fusion protein seems to be the best candidate from this investigation. Further in vivo studies of the cell-encapsulated dmCocE-C-Tat in hydrolyzing cocaine and alleviating immunogenicity and proteolytic degradation in established, clinically relevant mouse models are currently underway in our laboratories. Findings from this research are not only useful for developing other new CPP-CocE constructs but also valuable for establishing a nondisruptive cell-encapsulation technology for other protein therapeutics that are known to be immunogenic for direct clinical application.     

Cell-penetrating peptides: mechanism and kinetics of cargo delivery

Cell-penetrating peptides (CPPs) are short peptides of less than 30 amino acids that are able to penetrate cell membranes and translocate different cargoes into cells. The only common feature of these peptides appears to be that they are amphipathic and net positively charged. The mechanism of cell translocation is not known but it is apparently receptor and energy independent although, in certain cases, translocation can be partially mediated by endocytosis. Cargoes that are successfully internalized by CPPs range from small molecules to proteins and supramolecular particles. Most CPPs are inert or have very limited side effects. Their penetration into cells is rapid and initially first-order, with half-times from 5 to 20 min. The size of smaller cargoes does not affect the rate of internalization, but with larger cargoes, the rate is substantially decreased. CPPs are novel vehicles for the translocation of cargo into cells, whose properties make them potential drug delivery agents, of interest for future use.     

Cellular Uptake But Low Permeation of Human Calcitonin-Derived Cell Penetrating Peptides and Tat(47-57) Through Well-Differentiated Epithelial Models

PURPOSE: To investigate whether cell penetrating peptides (CPP) derived from human calcitonin (hCT) possess, in addition to cellular uptake, the capacity to deliver their cargo through epithelial barriers. METHODS: Cellular uptake of hCT(9-32) and permeation of six hCT-derived peptides, namely, hCT(9-32), hCT(12-32), hCT(15-32), hCT(18-32), hCT(21-32), and a random sequence of hCT(9-32) were evaluated in fully organized confluent Madin-Darby canine kidney (MDCK), Calu-3, and TR146 cell culture models. For comparison, Tat(47-57) and penetratin(43-58) were investigated. The peptides were N-terminally labeled with carboxyfluorescein (CF). Uptake in the well-differentiated epithelial models was observed by confocal laser scanning microscopy (CLSM), whereas permeation through the models was analyzed by reversed-phase (RP)-HPLC. RESULTS: In MDCK epithelium hCT(9-32), Tat(47-57) and penetratin(43-58) demonstrated punctuated cytoplasmic distribution. In Calu-3, Tat(47-57) and penetratin(43-58) were simultaneously localized in a punctuated cytoplasmic and paracellular distribution, whereas hCT(9-32) showed strict paracellular distribution. By contrast, in TR146 cells, Tat(47-57) was located strictly paracellularily, whereas penetratin(43-58) showed a punctuated cytoplasmic pattern and hCT(9-32) both. The transepithelial permeability of all tested peptides and their cargo was lower than that of paracellular markers. CONCLUSIONS: The CPP uptake pattern depends on both the type of peptide and the cell culture model. In general, the investigated CPP have no apparent potential for systemic drug delivery across epithelia. Nevertheless, distinct patterns of cellular distribution may offer a potential for localized epithelial delivery.     

Cell‐Penetrating Peptides as Carriers for Oral Delivery of Biopharmaceuticals

Oral delivery of biopharmaceuticals, for example peptides and proteins, constitutes a great challenge in drug delivery due to their low chemical stability and poor permeation across the intestinal mucosa, to a large extent limiting the mode of administration to injections, which is not favouring patient compliance. Nevertheless, cell‐penetrating peptides (CPPs) have shown promising potential as carriers to overcome the epithelium, and this minireview highlights recent knowledge gained within the field of CPP‐mediated transepithelial delivery of therapeutic peptides and proteins from the intestine. Two approaches may be pursued: co‐administration of the carrier and therapeutic peptide in the form of complexes obtained by simple bulk mixing, or administration of covalent conjugates demanding more advanced production methodologies. These formulation approaches have their pros and cons, and which is to be preferred depends on the physicochemical properties of both the specific CPP and the specific cargo. In addition to the physical epithelial barrier, a metabolic barrier must be overcome in order to obtain CPP‐mediated delivery of a cargo drug from the intestine, and a number of strategies have been employed to delay enzymatic degradation of the CPP. The mechanisms by which CPPs translocate across membranes are not fully understood, but possibly involve endocytosis as well as direct translocation, and the CPP‐mediated transepithelial delivery of cargo drugs thus likely involves similar mechanisms for the initial membrane interaction and translocation. However, the mechanisms responsible for transcytosis of the cargo drug, if taken up by an endocytic mechanism, or direct translocation across the epithelium are so far not known.     

Cell penetrating peptides for in vivo molecular imaging applications

Cell penetrating peptides (CPPs) are a relatively new class of peptides that have the promising capability to cross cell membranes. While details remain to be resolved, various non-receptor-mediated endocytic pathways likely contribute most to the cell penetrating properties of these peptides. CPPs have been used to deliver many different cargos - ranging from radionuclides and other peptides to antibodies and nanoparticles - into cells. Besides many different drug delivery applications, CPPs have also seen a limited use in molecular imaging. Molecular imaging of intracellular and intranuclear targets, by techniques such as SPECT, PET, optical imaging, and MRI, relies heavily on the delivery of contrast agents to the cytoplasm and/or nuclei of the target tissue. Therefore, the number of applications in molecular imaging of intracellular targets has remained relatively low, because of the effective barrier presented by the cell membrane. One of the key strategies to overcome this challenge is the introduction of membrane-transducing peptides in the design of new contrast agents. This review presents an overview of the literature on CPPs, focusing on their use for molecular imaging. Applications using proteins and peptides, DNA/RNA, and CPP-loaded cells as the imaging agents will be looked at. Moreover, the difficulties and pitfalls regarding the use of CPPs in molecular imaging will be discussed.     

Cell Penetrating Peptides: Intracellular Pathways and Pharmaceutical Perspectives

Cell penetrating peptides, generally categorized as amphipathic or cationic depending on their sequence, are increasingly drawing attention as a non-invasive delivery technology for macromolecules. Delivery of a diverse set of cargo in terms of size and nature ranging from small molecules to particulate cargo has been attempted using different types of cell penetrating peptides (CPPs) in vitro and in vivo. However, the internalization mechanism of CPPs is an unresolved issue to date, with dramatic changes in view regarding the involvement of endocytosis as a pathway of internalization. A key reason for the lack of consensus on the mechanism can be attributed to the methodology in deciphering the internalization mechanism. In this review, we highlight some of the methodology concerns, focus more on the internalization pathway and also provide a novel perspective about the intracellular processing of CPPs, which is a crucial aspect to consider when selecting a cell penetrating peptide as a drug delivery system. In addition, recent applications of cell penetrating peptides for the delivery of small molecules, peptides, proteins, oligonucleotides, nanoparticles and liposomes have been reviewed.     

Quantitative assessment of the cell penetrating properties of RI-Tat-9: evidence for a cell type-specific barrier at the plasma membrane of epithelial cells

Penetration of epithelial cells represents the rate-determining step for the absorption of many drugs and pharmaceutical macromolecules such as proteins and nucleic acid therapeutics. While the potential of using cell-penetrating peptides (CPPs) to facilitate absorption has been increasingly recognized, the mechanism of cell penetration and the uptake into certain cells have recently been called into question due to methodological artifacts. Therefore, the objective of this study was to quantitatively assess the ability of RI-Tat-9, a proteolytically stable CPP, to penetrate epithelial cell monolayers. The permeability of RI-Tat-9 with two epithelial cell lines, Madin-Darby canine kidney (MDCK) and Caco-2 cells, was comparable to the leakiness of the respective intact monolayers. Microscopic imaging showed that fluorescence-tagged RI-Tat-9 did not enter these cells, further supporting a paracellular transport mechanism. Although insufficient data were generated in these studies to generalize the observed phenomenon, the entry of RI-Tat-9 into nonepithelial T lymphocytic MT2 cells, possibly by endocytosis, suggested that a cell type-specific barrier might exist that controlled uptake of RI-Tat-9 by cells. Compared to that in MT2 and HeLa cells, the active uptake of the peptide into MDCK monolayers was much slower and showed no dependence of cell energy. Furthermore, the equilibrium binding of RI-Tat-9 to MDCK cells at 0 degrees C was indicative of an interaction with a nonspecific receptor. A correlation between binding density and concentration difference across a leaky separation barrier suggested that repulsion of free peptide molecules by bound peptide molecules at the MDCK monolayer surface may be significant at micromolar concentrations. The results of this study quantitatively show that Tat CPP uptake into two commonly used epithelial cell types is minimal and possibly cell type-specific. Implications for Tat CPP-assisted drug delivery are discussed.     

穿膜肽的研究进展

穿膜肽是一些具有细胞膜穿透能力的小分子多肽,可有效携带比其分子质量大100倍的外源性疏水大分子进入细胞,并对宿主细胞没有显著毒副作用.穿膜肽存在多种穿膜机制,诸如直接渗透到细胞膜,通过易位形成的一个暂时性的结构和内吞作用介导入膜等.具体的穿膜机制还不清楚,但普遍认为穿膜肽与细胞表面负电荷物质的直接接触是必不可少的.由于穿膜肽的穿膜性质,其在分子生物学、药学、细胞生物学、疫苗学甚至影像学上有广泛的应用前景.本文对近年来穿膜肽的结构、穿膜机制和应用方面的研究进展进行了综述.     

细胞穿膜肽在癌症治疗中的应用

细胞膜有选择渗透性,能阻止大多数外来因子进入细胞。然而许多治疗因子必须穿过细胞膜才能进入细胞内起作用。细胞穿膜肽是一组简短的阳离子序列,它具有很强的穿膜能力。自从1988年发现细胞穿膜肽后,细胞穿膜肽被用于携带各种物质,如小分子、核酸、抗体、纳米粒等进入细胞膜。本文重点介绍了近几年来关于细胞穿膜肽穿膜机制的研究进展,以及细胞穿膜肽作为运载体携带抗癌药物靶向进入肿瘤细胞,从而实现对肿瘤细胞的靶向性,同时可克服肿瘤的多药抗药性等研究进展。     

Transfection efficiency and toxicity of polyethylenimine in differentiated Calu-3 and nondifferentiated COS-1 cell cultures

In the present study, we evaluated polyethylenimine (PEI) of different molecular weights (MWs) as a DNA complexing agent for its efficiency in transfecting nondifferentiated COS-1 (green monkey fibroblasts) and well-differentiated human submucosal airway epithelial cells (Calu-3). Studying the effect of particle size, zeta potential, presence of serum proteins or chloroquine, it appeared that transfection efficiency depends on the experimental conditions and not on the MW of the PEI used. Comparing transfection efficiencies in both cell lines, we found that PEI was 3 orders of magnitude more effective in COS-1 than in Calu-3 cells, because Calu-3 cells are differentiated and secrete mucins, which impose an additional barrier to gene delivery. Transfection efficiency was strongly correlated to PEI cytotoxicity. Also, some evidence for PEI-induced apoptosis in both cell lines was found. In conclusion, our results indicate that PEI is a useful vector for nonviral transfection in undifferentiated cell lines. However, results from studies in differentiated bronchial epithelial cells suggest that PEI has yet to be optimized for successful gene therapy of cystic fibrosis (CF).     

Recent advances in the use of cell-penetrating peptides for medical and biological applications

The selective permeability of the plasma membrane prohibits most exogenous agents from gaining cellular access. Since many therapeutics and reporter molecules must be internalized for activity, crossing the plasma membrane is essential. A very effective class of transporters harnessed for this purpose are cell penetrating peptides (CPPs), a group of short cationic sequences with a remarkable capacity for membrane translocation. Since their discovery in 1988, CPPs have been employed for the delivery of a wide variety of cargo including small molecules, nucleic acids, antibodies and nanoparticles. This review describes recent advances in the use of CPPs for biological and therapeutic applications. In particular, an emphasis is placed on novel systems and insights acquired since 2006. Basic research on CPPs has recently yielded techniques that provide further information on the controversial mechanism of CPP uptake and has also resulted in the development of new model membrane systems to evaluate these mechanisms. In addition, recent use of CPPs for the development of new cellular imaging tools, biosensors, or biomolecular delivery systems have been highlighted. Lastly, novel peptide delivery vectors, designed to tackle some of the drawbacks of CPPs and enhance their versatility, will be described. This review will illustrate the diverse applications for which CPPs have been harnessed and also demonstrate the remarkable advancements these peptides have facilitated in cell biology.