Cell-penetrating Peptide pVEC Research Articles

Curcumin Displays Enhanced Solubility and Antibacterial Activities When Complexed with the Cell Penetrating Peptide pVEC

Curcumin Displays Enhanced Solubility and Antibacterial Activities When Complexed with the Cell Penetrating Peptide pVEC

Design and Synthesis of a Peptide-Based Glioma-Targeted Drug Delivery Vector gHope2.

In the current chapter, we are detailing the synthesis path of a tumor-targeted, CPP-functionalized chemotherapeutic drug, as well as in vitro validation of the targeting and cell penetrating functionalities of the construct. The design of targeted drug delivery vehicle is based on a new glioma-specific homing peptide that has been conjugated to doxorubicin. Further functionalization with an 18-amino acid cell penetrating peptide pVEC was achieved, a CPP that was chosen because of its high cell penetrating efficacy and low toxicity. The three elements were combined into one drug delivery construct gHope2, and its tumor-homing and cell penetrating activity was demonstrated in human glioma cell line U87.

Development of a pVEC peptide-based ribonucleoprotein (RNP) delivery system for genome editing using CRISPR/Cas9 in Chlamydomonas reinhardtii

Recent technical advances related to the CRISPR/Cas9-based genome editing system have enabled sophisticated genome editing in microalgae. Although the demand for research on genome editing in microalgae has increased over time, methodological research has not been established to date for the delivery of a ribonucleoprotein (Cas9/sgRNA complex) using a cell penetrating peptide into microalgal cell lines. Here, we present a ribonucleoprotein delivery system for Chlamydomonas reinhardtii mediated by the cell penetrating peptide pVEC (LLIILRRRIRKQAHAHSK) which is in a non-covalent form. Using this technically simple method, the ribonucleoprotein was successfully delivered into C. reinhardtii. Gene Maa7 and FKB12 were disrupted, and their distinguishing patterns of Indel mutations were analyzed with the observation of several insertions of sequences not originating from the genome DNA, such as chloroplast DNA, into the expected loci. In addition, the cytotoxicity of Cas9 and the ribonucleoprotein was investigated according to the concentration and time in the algal cells. It was observed that Cas9 alone without the sgRNA induces a more severe cytotoxicity compared to the ribonucleoprotein. Our study will not only contribute to algal cell biology and its genetic engineering for further applications involving various organisms but will also provide a deeper understating of the basic science of the CRISPR/Cas9 system.

Membrane Active Peptides and Their Biophysical Characterization.

In the last 20 years, an increasing number of studies have been reported on membrane active peptides. These peptides exert their biological activity by interacting with the cell membrane, either to disrupt it and lead to cell lysis or to translocate through it to deliver cargos into the cell and reach their target. Membrane active peptides are attractive alternatives to currently used pharmaceuticals and the number of antimicrobial peptides (AMPs) and peptides designed for drug and gene delivery in the drug pipeline is increasing. Here, we focus on two most prominent classes of membrane active peptides; AMPs and cell-penetrating peptides (CPPs). Antimicrobial peptides are a group of membrane active peptides that disrupt the membrane integrity or inhibit the cellular functions of bacteria, virus, and fungi. Cell penetrating peptides are another group of membrane active peptides that mainly function as cargo-carriers even though they may also show antimicrobial activity. Biophysical techniques shed light on peptide–membrane interactions at higher resolution due to the advances in optics, image processing, and computational resources. Structural investigation of membrane active peptides in the presence of the membrane provides important clues on the effect of the membrane environment on peptide conformations. Live imaging techniques allow examination of peptide action at a single cell or single molecule level. In addition to these experimental biophysical techniques, molecular dynamics simulations provide clues on the peptide–lipid interactions and dynamics of the cell entry process at atomic detail. In this review, we summarize the recent advances in experimental and computational investigation of membrane active peptides with particular emphasis on two amphipathic membrane active peptides, the AMP melittin and the CPP pVEC.

Insights into membrane translocation of the cell-penetrating peptide pVEC from molecular dynamics calculations

Discovery of cargo carrying cell-penetrating peptides has opened a new gate in the development of peptide-based drugs that can effectively target intracellular enzymes. Success in application and development of cell-penetrating peptides in drug design depends on understanding their translocation mechanisms. In this study, our aim was to examine the bacterial translocation mechanism of the cell-penetrating pVEC peptide (LLIILRRRIRKQAHAHSK) using steered molecular dynamics (SMD) simulations. The significance of specific residues or regions for translocation was studied by performing SMD simulations on the alanine mutants and other variants of pVEC. Residue-based analysis showed that positively charged residues contribute to adsorption to the lipid bilayer and to electrostatic interactions with the lipid bilayer as peptides are translocated. Translocation takes place in three main stages; the insertion of the N-terminus into the bilayer, the inclusion of the whole peptide inside the membrane and the exit of the N-terminus from the bilayer. These three stages mirror the three regions on pVEC; namely, the hydrophobic N-terminus, the cationic midsection, and the hydrophilic C-terminus. The N-terminal truncated pVEC, I3A, L5A, R7A mutants and scramble-pVEC make weaker interactions with the lipids during translocation highlighting the contribution of the N-terminal residues and the sequence of the structural regions to the translocation mechanism. This study provides atomistic detail about the mechanism of pVEC peptide translocation and can guide future peptide-based drug design efforts.

Analysis of Membrane Translocation Simulations using Dimensionality Reduction

In understanding biological processes and biosystems, molecular motion at the atomic level plays a critical role. Computational studies on molecular motion are commonly based on simulations of molecular models, which give a huge number of conformations in the form of Cartesian coordinates for each of the atoms as output (Plaku, Stamati, Clementi, & Kavraki, 2007; Stamati, Clementi, & Kavraki, 2010). In order to quantitatively characterize a computer simulation and extract the important information of the motion of the system, a low-dimensional embedding such that the properties of the underlying manifold is preserved should be defined. Mathematically, finding a set of coordinates in which very few of them show significant variation and the others may said to be inconsiderable is called dimensionality reduction problem. The commonly used dimensionality reduction techniques, principal component analysis (PCA) and multidimensional scaling (MDS) are simple and easy to apply, affordable in the means of computing time and resource and efficiently determine the true structure of data in the high-dimensional input space (Tenenbaum, de Silva, & Langford, 2000). In an effort to characterize the translocation of the 18-residue long cell-penetrating peptide pVEC (LLIILRRRIRKQAHAHSK) through a lipid bilayer, we performed steered molecular dynamic (SMD) simulations, in which force is applied on the peptide to move it from one side of the membrane to the other. We examined the simulation trajectories using linear and nonlinear dimensionality reduction techniques to obtain a description of the underlying energy landscape for the nonlinear process of membrane uptake.

Bacterial Uptake of the Cell-Penetrating Peptide pVEC

The discovery of cell-penetrating peptides (CPPs), which can translocate through the cell membrane without the need for a receptor, has led to an increased interest in the design and development of peptide based drug delivery methods. CPPs are peptides of fewer than 30 residues derived from natural or unnatural proteins or chimeric sequences. The 18 amino acid long pVEC peptide is derived from murine vascular endothelial cadherin (VE-cadherin) protein, which mediates physical contact between adjacent cells by homophilic dimerization. The pVEC peptide has been shown to cross the cell membrane. The pVEC sequence, LLIILRRRIRKQAHAHSK, has a hydrophobic N-terminus and a positively charged midsection. The residues contributing to the uptake of pVEC across the membrane were previously examined by alanine mutagenesis studies (Elmquist, A., M. Hansen, et al., 2006, Biochimica et biophysica acta, 1758(6): 721-729) and the N-terminal residues were shown to be crucial in cellular uptake. Here, we use simulations to study the bacterial uptake of pVEC at atomic detail. Steered molecular dynamics simulations on the original pVEC and its various mutants are performed in an effort to determine the important peptide - membrane interactions on a residue basis and to elucidate the mechanism whereby pVEC passes through the membrane.

Structural rearrangements and chemical modifications in known cell penetrating peptide strongly enhance DNA delivery efficiency

Amphipathic peptides with unusual cellular translocation properties have been used as carriers of different biomolecules. However, the parameters which control the delivery efficiency of a particular cargo by a peptide and the selectivity of cargo delivery are not very well understood. In this work, we have used the known cell penetrating peptide pVEC (derived from VE-cadherin) and systematically changed its amphipathicity (from primary to secondary) as well as the total charge and studied whether these changes influence the plasmid DNA condensation ability, cellular uptake of the peptide–DNA complexes and in turn the efficiency of DNA delivery of the peptide. Our results show that although the efficiency of DNA delivery of pVEC is poor, modification of the same peptide to create a combination of nine arginines along with secondary amphipathicity improves its plasmid DNA delivery efficiency, particularly in presence of an endosomotropic agent like chloroquine. In addition, presence of histidines along with 9 arginines and secondary amphipathicity shows efficient DNA delivery with low toxicity even in absence of chloroquine in multiple cell lines. We attribute these enhancements in transfection efficiency to the differences in the mechanism of complex formation by the different variants of the parent peptide which in turn are related to the chemical nature of the peptide itself. These results exhibit the importance of understanding the physicochemical parameters of the carrier and complex in modulating gene delivery efficiency. Such studies can be helpful in improving peptide design for delivery of different cargo molecules.

Design of a Tumor-Homing Cell-Penetrating Peptide

Chemotherapy is often limited by toxicity to normal cells. Therefore, an ideal anticancer drug should discriminate between normal tissue and tumors. This would require a target receptor molecule mostly present in tumors. The cyclic peptide cCPGPEGAGC (PEGA) is a homing peptide that has previously been shown to accumulate in breast tumor tissue in mice. PEGA peptide does not cross the plasma membrane per se; however, when attached to the cell-penetrating peptide pVEC, the conjugate is taken up by different breast cancer cells in vitro. Additionally, the homing capacity of the PEGA- pVEC is conserved in vivo, where the conjugate mainly accumulates in blood vessels in breast tumor tissue and, consequently is taken up. Furthermore, we show that the efficacy of the anticancer drug, chlorambucil, is increased more than 4 times when the drug is conjugated to the PEGA- pVEC chimeric peptide. These data demonstrate that combining a homing sequence with a cell-penetrating sequence yields a peptide that combines the desirable properties of the parent peptides. Such peptides may be useful in diagnostics and delivery of therapeutic agents to an intracellular location in a specific tumor target tissue.

Structure–activity relationship study of the cell-penetrating peptide pVEC

The peptide pVEC is a recently described cell-penetrating peptide, derived from the murine vascular endothelial-cadherin protein. In order to define which part of this 18-amino acid long peptide is important for the cellular translocation, we performed a structure–activity relationship study of pVEC. Together with the l-alanine substituted peptides, the retro- pVEC, D- pVEC and the scramble pVEC are studied for comparison. The peptide analogues are labeled with carboxyfluorescein at the N-terminus for monitoring the cellular uptake into human Bowes melanoma cells with different efficacy. We show that all the Fl- pVEC analogues internalize in live Bowes melanoma cells. l-Alanine substitution of the five respective N-terminal hydrophobic amino acids significantly decreases the translocation property, while replacing of Arg 6, Arg 8 or Ser 17 by alanine enhances the uptake. The uptake of pVEC is significantly reduced by treatment with an endocytosis inhibitor wortmannin. Treatment with heparinase III, nystatin and EIPA had no effect on the peptide uptake. The data presented here show that the N-terminal hydrophobic part of pVEC is crucial for efficient cellular translocation.

The Cell Penetrating Peptides pVEC and W2-pVEC Induce Transformation of Gel Phase Domains in Phospholipid Bilayers without Affecting Their Integrity

The cell penetrating peptide (CPP) pVEC has been shown to translocate efficiently the plasma membrane of different mammalian cell lines by a receptor-independent mechanism without exhibiting cellular toxicity. This ability renders CPPs of broad interest in cell biology, biotechnology, and drug delivery. To gain insight into the interaction of CPPs with biomembranes, we studied the interaction of pVEC and W2-pVEC, an Ile --> Trp modification of the former, with phase-separated supported phospholipid bilayers (SPB) by atomic force microscopy (AFM). W2-pVEC induced a transformation of dipalmitoyl phosphatidylcholine (DPPC) domains from a gel phase state via an intermediate state with branched structures into essentially flat bilayers. With pVEC the transformation followed a similar pathway but was slower. Employing fluorescence polarization, we revealed the capability of the investigated peptides to increase the fluidity of DPPC domains as the underlying mechanism of transformation. Due to their tighter packing, sphingomyelin (SM) domains were not transformed. By combination, AFM observations, dynamic light scattering studies, and liposome leakage experiments indicated that bilayer integrity was not compromised by the peptides. Transformation of gel phase domains in SPB by CPPs represents a novel aspect in the discussion on uptake mechanisms of CPPs.