Extracellular Microvesicles Modified with Arginine-Rich Peptides for Active Macropinocytosis Induction and Delivery of Therapeutic Molecules.

Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), transfer bioactive molecules from donor to recipient cells in various pathophysiological settings, thereby mediating intercellular communication. Despite their significant roles in extracellular signaling, the cellular uptake mechanisms of different EV subpopulations remain unknown. In particular, plasma membrane-derived MVs are larger vesicles (100 nm to 1 µm in diameter) and may serve as efficient molecular delivery systems due to their large capacity; however, because of size limitations, receptor-mediated endocytosis is considered an inefficient means for cellular MV uptake. This study demonstrated that macropinocytosis (lamellipodia formation and plasma membrane ruffling, causing the engulfment of large fluid volumes outside cells) can enhance cellular MV uptake. We developed experimental techniques to induce macropinocytosis-mediated MV uptake by modifying MV membranes with arginine-rich cell-penetrating peptides for the intracellular delivery of therapeutic molecules.

Prediction of blood-brain barrier penetrating peptides based on data augmentation with Augur.

BACKGROUND: The blood-brain barrier serves as a critical interface between the bloodstream and brain tissue, mainly composed of pericytes, neurons, endothelial cells, and tightly connected basal membranes. It plays a pivotal role in safeguarding brain from harmful substances, thus protecting the integrity of the nervous system and preserving overall brain homeostasis. However, this remarkable selective transmission also poses a formidable challenge in the realm of central nervous system diseases treatment, hindering the delivery of large-molecule drugs into the brain. In response to this challenge, many researchers have devoted themselves to developing drug delivery systems capable of breaching the blood-brain barrier. Among these, blood-brain barrier penetrating peptides have emerged as promising candidates. These peptides had the advantages of high biosafety, ease of synthesis, and exceptional penetration efficiency, making them an effective drug delivery solution. While previous studies have developed a few prediction models for blood-brain barrier penetrating peptides, their performance has often been hampered by issue of limited positive data. RESULTS: In this study, we present Augur, a novel prediction model using borderline-SMOTE-based data augmentation and machine learning. we extract highly interpretable physicochemical properties of blood-brain barrier penetrating peptides while solving the issues of small sample size and imbalance of positive and negative samples. Experimental results demonstrate the superior prediction performance of Augur with an AUC value of 0.932 on the training set and 0.931 on the independent test set. CONCLUSIONS: This newly developed Augur model demonstrates superior performance in predicting blood-brain barrier penetrating peptides, offering valuable insights for drug development targeting neurological disorders. This breakthrough may enhance the efficiency of peptide-based drug discovery and pave the way for innovative treatment strategies for central nervous system diseases.

Lipid-Based Nanoparticle Functionalization with Coiled-Coil Peptides for In Vitro and In Vivo Drug Delivery.

ConspectusFor the delivery of drugs, different nanosized drug carriers (e.g., liposomes, lipid nanoparticles, and micelles) have been developed in order to treat diseases that afflict society. Frequently, these vehicles are formed by the self-assembly of small molecules to encapsulate the therapeutic cargo of interest. Over decades, nanoparticles have been optimized to make them more efficient and specific to fulfill tailor-made tasks, such as specific cell targeting or enhanced cellular uptake. In recent years, lipid-based nanoparticles in particular have taken center stage; however, off-targeting side effects and poor endosomal escape remain major challenges since therapies require high efficacy and acceptable toxicity.To overcome these issues, many different approaches have been explored to make drug delivery more specific, resulting in reduced side effects, to achieve an optimal therapeutic effect and a lower required dose. The fate of nanoparticles is largely dependent on size, shape, and surface charge. A common approach to designing drug carriers with targeting capability is surface modification. Different approaches to functionalize nanoparticles have been investigated since the attachment of targeting moieties plays a significant role in whether they can later interact with surface-exposed receptors of cells. To this end, various strategies have been used involving different classes of biomolecules, such as small molecules, nucleic acids, antibodies, aptamers, and peptides.Peptides in particular are often used since there are many receptors overexpressed in different specific cell types. Furthermore, peptides can be produced and modified at a low cost, enabling high therapeutic screening. Cell-penetrating peptides (CPPs) and cell-targeting peptides (CTPs) are frequently used for this purpose. Less studied in this context are fusogenic coiled-coil peptides. Lipid-based nanoparticles functionalized with these peptides are able to avoid the endolysosomal pathway; instead such particles can be taken up by membrane fusion, resulting in increased delivery of payload. Furthermore, they can be used for targeting cells/organs but are not directed at surface-exposed receptors. Instead, they recognize complementary peptide sequences, facilitating their uptake into cells.In this Account, we will discuss peptides as moieties for enhanced cytosolic delivery, targeted uptake, and how they can be attached to lipid-based nanoparticles to alter their properties. We will discuss the properties imparted to the particles by peptides, surface modification approaches, and recent examples showing the power of peptides for in vitro and in vivo drug delivery. The main focus will be on the functionalization of lipid-based nanoparticles by fusogenic coiled-coil peptides, highlighting the relevance of this concept for the development of future therapeutics.

Negative lipid membranes enhance the adsorption of TAT-decorated elastin-like polypeptide micelles.

A cell-penetrating peptide (CPP) is a short amino-acid sequence capable of efficiently translocating across the cellular membrane of mammalian cells. However, the potential of CPPs as a delivery vector is hampered by the strong reduction of its translocation efficiency when it bears an attached molecular cargo. To overcome this problem, we used previously developed diblock copolymers of elastin-like polypeptides (ELPBCs), which we end functionalized with TAT (transactivator of transcription), an archetypal CPP built from a positively charged amino acid sequence of the HIV-1 virus. These ELPBCs self-assemble into micelles at a specific temperature and present the TAT peptide on their corona. These micelles can recover the lost membrane affinity of TAT and can trigger interactions with the membrane despite the presence of a molecular cargo. Herein, we study the influence of membrane surface charge on the adsorption of TAT-functionalized ELP micelles onto giant unilamellar vesicles (GUVs). We show that the TAT-ELPBC micelles show an increased binding constant toward negatively charged membranes compared to neutral membranes, but no translocation is observed. The affinity of the TAT-ELPBC micelles for the GUVs displays a stepwise dependence on the lipid charge of the GUV, which, to our knowledge, has not been reported previously for interactions between peptides and lipid membranes. By unveiling the key steps controlling the interaction of an archetypal CPP with lipid membranes, through regulation of the charge of the lipid bilayer, our results pave the way for a better design of delivery vectors based on CPPs.

Cell-Penetrating and Enzyme-Responsive Peptides for Targeted Cancer Therapy: Role of Arginine Residue Length on Cell Penetration and In Vivo Systemic Toxicity.

For the improved delivery of cancer therapeutics and imaging agents, the conjugation of cell-penetrating peptides (CPPs) increases the cellular uptake and water solubility of agents. Among the various CPPs, arginine-rich peptides have been the most widely used. Combining CPPs with enzyme-responsive peptides presents an innovative strategy to target specific intracellular enzymes in cancer cells and when combined with the appropriate click chemistry can enhance theranostic drug delivery through the formation of intracellular self-assembled nanostructures. However, one drawback of CPPs is their high positive charge which can cause nonspecific binding, leading to off-target accumulation and potential toxicity. Hence, balancing cell-specific penetration, toxicity, and biocompatibility is essential for future clinical efficacy. We synthesized six cancer-specific, legumain-responsive RnAANCK peptides containing one to six arginine residues, with legumain being an asparaginyl endopeptidase that is overexpressed in aggressive prostate tumors. When conjugated to Alexa Fluor 488, R1-R6AANCK peptides exhibited a concentration- and time-dependent cell penetration in prostate cancer cells, which was higher for peptides with higher R values, reaching a plateau after approximately 120 min. Highly aggressive DU145 prostate tumor cells, but not less aggressive LNCaP cells, self-assembled nanoparticles in the cytosol after the cleavage of the legumain-specific peptide. The in vivo biocompatibility was assessed in mice after the intravenous injection of R1-R6AANCK peptides, with concentrations ranging from 0.0125 to 0.4 mmol/kg. The higher arginine content in R4-6 peptides showed blood and urine indicators for the impairment of bone marrow, liver, and kidney function in a dose-dependent manner, with instant hemolysis and morbidity in extreme cases. These findings underscore the importance of designing peptides with the optimal arginine residue length for a proper balance of cell-specific penetration, toxicity, and in vivo biocompatibility.

Interactions and Transport of a Bioconjugated Peptide Targeting the Mitomembrane.

The Szeto-Schiller (SS) peptides are a subclass of cell-penetrating peptides that can specifically target mitochondria and mediate conditions caused by mitochondrial dysfunction. In this work, we constructed an iron-chelating SS peptide and studied its interaction with a mitochondrial-mimicking membrane using atomistic molecular dynamics (MD) simulations. We report that the peptide/membrane interaction is thermodynamically favorable, and the localization of the peptide to the membrane is driven by electrostatic interactions between the cationic residues and the anionic phospholipid headgroups. The insertion of the peptide into the membrane is driven by hydrophobic interactions between the aromatic side chains in the peptide and the lipid acyl tails. We also probed the translocation of the peptide across the membrane by applying nonequilibrium steered MD simulations and resolved the translocation pathway, free energy profile, and metastable states. We explored four distinct orientations of the peptide along the translocation pathway and found that one orientation was energetically more favorable than the other orientations. We tested a significantly slower pulling velocity on the most thermodynamically favorable system and compared metastable states during peptide translocation. We found that the peptide can optimize hydrophobic interactions with the membrane by having aromatic side chains interacting with the lipid acyl tails instead of forming π-π interactions with each other. The mechanistic insights emerging from our work will potentially facilitate improved peptide design with enhanced activity.

PractiCPP: a deep learning approach tailored for extremely imbalanced datasets in cell-penetrating peptide prediction.

MOTIVATION: Effective drug delivery systems are paramount in enhancing pharmaceutical outcomes, particularly through the use of cell-penetrating peptides (CPPs). These peptides are gaining prominence due to their ability to penetrate eukaryotic cells efficiently without inflicting significant damage to the cellular membrane, thereby ensuring optimal drug delivery. However, the identification and characterization of CPPs remain a challenge due to the laborious and time-consuming nature of conventional methods, despite advances in proteomics. Current computational models, however, are predominantly tailored for balanced datasets, an approach that falls short in real-world applications characterized by a scarcity of known positive CPP instances. RESULTS: To navigate this shortfall, we introduce PractiCPP, a novel deep-learning framework tailored for CPP prediction in highly imbalanced data scenarios. Uniquely designed with the integration of hard negative sampling and a sophisticated feature extraction and prediction module, PractiCPP facilitates an intricate understanding and learning from imbalanced data. Our extensive computational validations highlight PractiCPP's exceptional ability to outperform existing state-of-the-art methods, demonstrating remarkable accuracy, even in datasets with an extreme positive-to-negative ratio of 1:1000. Furthermore, through methodical embedding visualizations, we have established that models trained on balanced datasets are not conducive to practical, large-scale CPP identification, as they do not accurately reflect real-world complexities. In summary, PractiCPP potentially offers new perspectives in CPP prediction methodologies. Its design and validation, informed by real-world dataset constraints, suggest its utility as a valuable tool in supporting the acceleration of drug delivery advancements. AVAILABILITY AND IMPLEMENTATION: The source code of PractiCPP is available on Figshare at https://doi.org/10.6084/m9.figshare.25053878.v1.

Nature-inspired peptide of MtDef4 C-terminus tail enables protein delivery in mammalian cells.

Cell-penetrating peptides show promise as versatile tools for intracellular delivery of therapeutic agents. Various peptides have originated from natural proteins with antimicrobial activity. We investigated the mammalian cell-penetrating properties of a 16-residue peptide with the sequence GRCRGFRRRCFCTTHC from the C-terminus tail of the Medicago truncatula defensin MtDef4. We evaluated the peptide's ability to penetrate multiple cell types. Our results demonstrate that the peptide efficiently penetrates mammalian cells within minutes and at a micromolar concentration. Moreover, upon N-terminal fusion to the fluorescent protein GFP, the peptide efficiently delivers GFP into the cells. Despite its remarkable cellular permeability, the peptide has only a minor effect on cellular viability, making it a promising candidate for developing a cell-penetrating peptide with potential therapeutic applications.

Cell-penetrating peptides for transmucosal delivery of proteins.

Enabling non-invasive delivery of proteins across the mucosal barriers promises improved patient compliance and therapeutic efficacies. Cell-penetrating peptides (CPPs) are emerging as a promising and versatile tool to enhance protein and peptide permeation across various mucosal barriers. This review examines the structural and physicochemical attributes of the nasal, buccal, sublingual, and oral mucosa that hamper macromolecular delivery. Recent development of CPPs for overcoming those mucosal barriers for protein delivery is summarized and analyzed. Perspectives regarding current challenges and future research directions towards improving non-invasive transmucosal delivery of macromolecules for ultimate clinical translation are discussed.

Combined biolistic and cell penetrating peptide delivery for the development of scalable intradermal DNA vaccines.

Physical-based gene delivery via biolistic methods (such as the Helios gene gun) involve precipitation of nucleic acids onto microparticles and direct transfection through cell membranes of exposed tissue (e.g. skin) by high velocity acceleration. The glycosaminoglycan (GAG)-binding enhanced transduction (GET) system exploits novel fusion peptides consisting of cell-binding, nucleic acid condensing, and cell-penetrating domains, which enable enhanced transfection across multiple cell types. In this study, we combined chemical (GET) and physical (gene gun) DNA delivery systems, and hypothesized the combination would generate enhanced distribution and effective uptake in cells not initially transfected by biolistic penetration. Physicochemical characterization, optimization of bullet contents and transfection experiments in vitro in cell monolayers and engineered tissue demonstrated these formulations transfected efficiently, including DC2.4 dendritic cells. We incorporated these formulations into a biolistic format for gene gun by forming fireable dry bullets obtained via lyophilization (freeze drying). This system is simple and with enhanced scalability compared to conventional methods to generate bullets. Flushed GET bullet contents retained their ability to mediate transfection (17-fold greater and 13-fold greater reporter gene expression than standard spermidine bullets in the absence and presence of serum, respectively). Fired GET bullets in vitro (in cells and collagen gels) and in vivo (mice) showed increased reporter gene transfection compared to untreated controls, whilst maintaining cell viability in vitro and having no obvious toxicity in vivo. Lastly, a SARS-CoV-2 plasmid DNA vaccine with spike (S) protein-receptor binding domain (S-RBD) was delivered by gene gun using GET bullets. Specific T cell and antibody responses comparable to the conventional system were generated. The non-physical and physical combination of GET­gold-DNA carriers using gene gun shows potential as an alternative DNA delivery method that is scalable for mass deployable vaccination and intradermal gene delivery.

Membrane Binding Strength vs Pore Formation Cost─What Drives the Membrane Permeation of Nanoparticles Coated with Cell-Penetrating Peptides?

Cell-penetrating peptides (CPPs) enable the transport of nanoparticles through cell membranes. Using molecular simulations, we conduct an in-depth investigation into the thermodynamic forces governing the passive translocation of CPP-coated nanoparticles across lipid bilayers, contrasting their behavior with that of bare particles to dissect the contribution of the peptides. Our analysis unveils a distinctive two-stage translocation mechanism, where the adsorption energy of the particles overcomes the cost of forming a hydrophilic transmembrane pore. Proper evaluation of the translocation mechanisms is only possible when using two reaction coordinates, in particular, one that explicitly includes the density of the lipids on the binding site of the particle. An analysis of adsorption and activation free energies in terms of a simple kinetic model provides a clearer understanding of the CPP effect. Experimental validation using nonendocytic cells confirms the superior membrane permeation of CPP-coated particles. Our findings have implications for the rational design of more efficient cell-permeating particles.

Controlled exchange of protein and nucleic acid signals from and between synthetic minimal cells.

Synthetic minimal cells are a class of bioreactors that have some, but not all, functions of live cells. Here, we report a critical step toward the development of a bottom-up minimal cell: cellular export of functional protein and RNA products. We used cell-penetrating peptide tags to translocate payloads across a synthetic cell vesicle membrane. We demonstrated efficient transport of active enzymes and transport of nucleic acid payloads by RNA-binding proteins. We investigated influence of a concentration gradient alongside other factors on the efficiency of the translocation, and we show a method to increase product accumulation in one location. We demonstrate the use of this technology to engineer molecular communication between different populations of synthetic cells, to exchange protein and nucleic acid signals. The synthetic minimal cell production and export of proteins or nucleic acids allows experimental designs that approach the complexity and relevancy of natural biological systems. A record of this paper's transparent peer review process is included in the supplemental information.

Integrin Facilitates the Internalization of TAT Peptide Conjugated to RGD Motif in Model Lipid Membranes.

In recent years, targeted drug delivery has attracted a great interest for enhanced therapeutic efficiency, with diminished side effects, especially in cancer therapy. Cell penetrating peptides (CPPs) like HIV1-TAT peptides, appear to be the perfect vectors for translocating drugs or other cargoes across the plasma membrane, but their application is limited mostly due to insufficient specificity for intended targets. Although these molecules were successfully used, the mechanism by which the peptides enter the cell interior still needs to be clarified. The tripeptide motif RGD (arginine-glycine-aspartate), found in extracellular matrix proteins has high affinity for integrin receptors overexpressed in cancer and it is involved in different phases of disease progression, including proliferation, invasion and migration. Discovery of new peptides with high binding affinity for disease receptors and permeability of plasma membranes is desirable for both, development of targeted drug delivery systems and early detection and diagnosis. To complement the TAT peptide with specific targeting ability, we conjugated it with an integrin-binding RGD motif. Although the idea of RGD-CPPs conjugates is not entirely new,[1] here we describe the permeability abilities and specificity of integrin receptors of RGD-TAT peptides in model membranes. Our findings reveal that this novel RGD sequence based on TAT peptide maintains its ability to permeate lipid membranes and exhibits specificity for integrin receptors embedded in giant unilamellar vesicles. This promising outcome suggests that the RGD-TAT peptide has significant potential for applications in the field of targeted drug delivery systems.

Engraulisin: A novel marine derived cell penetrating peptide with activity against drug resistant bacteria.

Cell penetrating peptides (CPP) with their intrinsic ability to penetrate plasma membranes facilitate intracellular uptake of various macromolecules. Although a substantial number of CPPs have been reported over the last three decades, the number is still inadequate when compared to the theoretically feasible peptides with similar physicochemical composition. Marine organisms, due to their hostile environment, are an immense source of several high-valued therapeutically relevant peptides. Various marine derived antibacterial, antimycotic and anticancer peptides have demonstrated improved activity in comparison to peptides of terrestrial origin. While a significant number of marine bioactive peptides exist, cell penetrating peptides from marine organisms remain unravelled. In this study, we report Engraulisin from Engraulis japonicus, a computationally derived novel cell penetrating peptide of marine origin. Engraulisin manifest successful uptake in mammalian cells at 5 µM concentration with negligible cytotoxicity observed through MTT assay. Analysis of its cellular uptake mechanism revealed significant inhibition at 4 °C suggesting endocytosis as the major route of cellular entry. Interestingly, the novel peptide also demonstrated selective antimicrobial activity against Methicillin-resistant Staphylococcus aureus (MRSA). Additionally, molecular dynamics simulation with POPC and POPG bilayer system unveiled significance of positively charged residues in forming a stable membrane interaction. Engraulisin represents a novel marine-derived cell penetrating peptide which can be explored for cellular delivery of pharmaceutically relevant molecules.

Targeting NANOG and FAK via Cx26-derived Cell-penetrating Peptides in Triple-negative Breast Cancer.

Triple-negative breast cancer (TNBC) represents the most lethal and treatment-resistant breast cancer subtype with limited treatment options. We previously identified a protein complex unique to TNBC composed of the gap junction protein connexin 26 (Cx26), the pluripotency transcription factor NANOG, and focal adhesion kinase (FAK). We sought to determine whether a peptide mimetic of the interaction region of Cx26 attenuated tumor growth in preclinical models. We designed peptides based on Cx26 juxtamembrane domains and performed binding experiments with NANOG and FAK using surface plasmon resonance. Binding studies revealed that the Cx26 C-terminal tail and intracellular loop bound to NANOG and FAK with submicromolar-to-micromolar affinity and that a 5-amino acid sequence in the C-terminal tail of Cx26 (RYCSG) was sufficient for binding. Peptides with high affinity were engineered with a cell-penetrating antennapedia sequence and assessed in functional assays including cell proliferation, tumorsphere formation, and in vivo tumor growth, and downstream signaling changes were measured. The cell-penetrating Cx26 peptide (aCx26-pep) disrupted self-renewal while reducing nuclear FAK and NANOG and inhibiting NANOG target gene expression in TNBC cells but not luminal mammary epithelial cells. In vivo, aCx26-pep reduced tumor growth and proliferation and induced cell death. Here, we provide proof-of-concept that a Cx26 peptide-based strategy inhibits growth and alters NANOG activity specifically in TNBC, indicating the therapeutic potential of this targeting approach.

Transdermal Properties of Cell-Penetrating Peptides: Applications and Skin Penetration Mechanisms.

Cell-penetrating peptides (CPPs) consist of 5-30 amino acids with intracellular transduction abilities and diverse physicochemical properties, origins, and sequences. Although recent developments in bioinformatics have facilitated the prediction of CPP candidates with the potential for transduction into cells, the mechanisms by which CPPs penetrate cells and various tissues have not yet been elucidated at the molecular interaction level. Recently, the skin-penetrating ability of CPPs has gained wide attention and emerged as a simple and effective strategy for the delivery of macromolecules into the skin. Studies on the skin structure have suggested that the penetration potential of CPPs is based on the molecular interactions and characteristics of the lipid lamellar structure between corneocytes in the stratum corneum. This review provides a brief overview of the general properties, transduction mechanisms, applications, and safety issues of CPPs, focusing on CPPs with transdermal properties, that are currently being used to develop therapeutics and cosmetics.

Cell-Penetrating Peptide-Bismuth Bicycles.

Cell-penetrating peptides (CPPs) play a significant role in the delivery of cargos into human cells. We report the first CPPs based on peptide-bismuth bicycles, which can be readily obtained from commercially available peptide precursors, making them accessible for a wide range of applications. These CPPs enter human cells as demonstrated by live-cell confocal microscopy using fluorescently labelled peptides. We report efficient sequences that demonstrate increased cellular uptake compared to conventional CPPs like the TAT peptide (derived from the transactivating transcriptional activator of human immunodeficiency virus 1) or octaarginine (R8 ), despite requiring only three positive charges. Bicyclization triggered by the presence of bismuth(III) increases cellular uptake by more than one order of magnitude. Through the analysis of cell lysates using inductive coupled plasma mass spectrometry (ICP-MS), we have introduced an alternative approach to examine the cellular uptake of CPPs. This has allowed us to confirm the presence of bismuth in cells after exposure to our CPPs. Mechanistic studies indicated an energy-dependent endocytic cellular uptake sensitive to inhibition by rottlerin, most likely involving macropinocytosis.

Dual-drug controllable co-assembly nanosystem for targeted and synergistic treatment of hepatocellular carcinoma.

The effectiveness of chemotherapeutic agents for hepatocellular carcinoma (HCC) is unsatisfactory because of tumor heterogeneity, multidrug resistance, and poor target accumulation. Therefore, multimodality-treatment with accurate drug delivery has become increasingly popular. Herein, a cell penetrating peptide-aptamer dual modified-nanocomposite (USILA NPs) was successfully constructed by coating a cell penetrating peptide and aptamer onto the surface of sorafenib (Sora), ursolic acid (UA) and indocyanine green (ICG) condensed nanodrug (USI NPs) via one-pot assembly for targeted and synergistic HCC treatment. USILA NPs showed higher cellular uptake and cytotoxicity in HepG2 and H22 cells, with a high expression of epithelial cell adhesion molecule (EpCAM). Furthermore, these NPs caused more significant mitochondrial membrane potential reduction and cell apoptosis. These NPs could selectively accumulate at the tumor site of H22 tumor-bearing mice and were detected with the help of ICG fluorescence; moreover, they retarded tumor growth better than monotherapy. Thus, USILA NPs can realize the targeted delivery of dual drugs and the integration of diagnosis and treatment. Moreover, the effects were more significant after co-administration of iRGD peptide, a tumor-penetrating peptide with better penetration promoting ability or programmed cell death ligand 1 (PD-L1) antibody for the reversal of the immunosuppressive state in the tumor microenvironment. The tumor inhibition rates of USILA NPs + iRGD peptide or USILA NPs + PD-L1 antibody with good therapeutic safety were 72.38 % and 67.91 % compared with control, respectively. Overall, this composite nanosystem could act as a promising targeted tool and provide an effective intervention strategy for enhanced HCC synergistic treatment.

A molecular dynamics study of cell-penetrating peptide transportan-10 (TP10): Binding, folding and insertion to transmembrane state in zwitterionic membrane.

Transportan 10 (TP10) is a 21-residue, cationic, α-helical cell-penetrating peptide that can be used as a delivery vector for various bioactive molecules. Based on recent confocal microscopy studies, it is believed that TP10 can translocate across neutral lipid membrane passively, possibly as a monomer, without the formation of permanent pore. Here, we performed extensive molecular dynamics (MD) simulations of TP10W (Y3W variant of TP10) to find the microscopic details of binding, folding and insertion of TP10W to transmembrane state in POPC bilayer. Binding study with CHARMM36 force field showed that TP10W initially binds to the membrane surface in unstructured configuration, but it spontaneously folds into α-helical conformation under the lipid head groups. Further insertion of TP10W, changing from a surface bound state to a vertically oriented transmembrane state, was investigated via umbrella simulations. The resulting free energy profile shows a relatively small barrier between two states, suggesting a possible translocation pathway as a monomer. In fact, unbiased simulation of transmembrane TP10W revealed how a charged Lys side chain can move from one leaflet to the other without a significant free energy cost. Finally, we compared the results of TP10W simulations with those of point mutated variants (TP10W-K12A18 and TP10W-K19L) to understand the effect of charge distribution on the peptide. It was observed that such a conservative mutation can cause noticeable changes in the conformations of both surface bound and transmembrane states. The results of present study will be discussed in relation to the experimentally observed activities of TP10W against neutral membrane.