Peptide Carrier Research Articles

The design and delivery of a PKA inhibitory polypeptide to treat SCA1.

Spinocerebellar ataxia-1 (SCA1) is a neurodegenerative disease that primarily targets Purkinje cells (PCs) of the cerebellum. The exact mechanism of PC degeneration is unknown, however, it is widely believed that mutant ataxin-1 becomes toxic because of the phosphorylation of its serine 776 (S776) residue by cAMP-dependent protein kinase A (PKA). Therefore, to directly modulate mutant ATXN1 S776 phosphorylation and aggregation, we designed a therapeutic polypeptide to inhibit PKA. This polypeptide comprised of a thermally responsive elastin-like peptide (ELP) carrier, which increases peptide half-life, a PKA inhibitory peptide (PKI), and a cell-penetrating peptide (Synb1). We observed that our therapeutic polypeptide, Synb1-ELP-PKI, inhibited PKA activity at concentrations similar to the PKI peptide. Additionally, Synb1-ELP-PKI significantly suppressed mutant ATXN1 S776 phosphorylation and intranuclear inclusion formation in cell culture. Further, Synb1-ELP-PKI treatment improved SCA1 PC morphology in cerebellar slice cultures. Furthermore, the Synb1-ELP peptide carrier crossed the blood-brain barrier and localized to the cerebellum via the i.p. or intranasal route. Here, we show the intranasal delivery of ELP-based peptides to the brain as a novel delivery strategy. We also demonstrate that our therapeutic polypeptide has a great potential to target the neurotoxic S776 phosphorylation pathway in the SCA1 disease.

Local gene silencing in plants via synthetic dsRNA and carrier peptide

Quick and facile transient RNA interference (RNAi) is one of the most valuable plant biotechnologies for analysing plant gene functions. To establish a novel double-strand RNA (dsRNA) delivery system for plants, we developed an ionic complex of synthetic dsRNA with a carrier peptide in which a cell-penetrating peptide is fused with a polycation sequence as a gene carrier. The dsRNA-peptide complex is 100-300nm in diameter and positively charged. Infiltration of the complex into intact leaf cells of Arabidopsis thaliana successfully induced rapid and efficient down-regulation of exogenous and endogenous genes such as yellow fluorescent protein and chalcone synthase. The present method realizes quick and local gene silencing in specific tissues and/or organs in plants.

Peptide carrier-mediated non-covalent delivery of unmodified cisplatin, methotrexate and other agents via intravenous route to the brain.

BackgroundRapid pre-clinical evaluation of chemotherapeutic agents against brain cancers and other neurological disorders remains largely unattained due to the presence of the blood-brain barrier (BBB), which limits transport of most therapeutic compounds to the brain. A synthetic peptide carrier, K16ApoE, was previously developed that enabled transport of target proteins to the brain by mimicking a ligand-receptor system. The peptide carrier was found to generate transient BBB permeability, which was utilized for non-covalent delivery of cisplatin, methotrexate and other compounds to the brain.ApproachBrain delivery of the chemotherapeutics and other agents was achieved either by injecting the carrier peptide and the drugs separately or as a mixture, to the femoral vein. A modification of the method comprised injection of K16ApoE pre-mixed with cetuximab, followed by injection of a ‘small-molecule’ drug.Principal findingsSeven-of-seven different small molecules were successfully delivered to the brain via K16ApoE. Depending on the method, brain uptake with K16ApoE was 0.72–1.1% for cisplatin and 0.58–0.92% for methotrexate (34-50-fold and 54–92 fold greater for cisplatin and methotrexate, respectively, with K16ApoE than without). Visually intense brain-uptake of Evans Blue, Light Green SF and Crocein scarlet was also achieved. Direct intracranial injection of EB show locally restricted distribution of the dye in the brain, whereas K16ApoE-mediated intravenous injection of EB resulted in the distribution of the dye throughout the brain. Experiments with insulin suggest that ligand-receptor signaling intrinsic to the BBB provides a natural means for passive transport of some molecules across the BBB.SignificanceThe results suggest that the carrier peptide can non-covalently transport various chemotherapeutic agents to the brain. Thus, the method offers an avenue for pre-clinical evaluation of various small and large therapeutic molecules against brain tumors and other neurological disorders.

Sonogashira, CuAAC, and Oxime Ligations for the Synthesis of MnI Tricarbonyl PhotoCORM Peptide Conjugates

AbstractIn this work, facial tricarbonylmanganese(I) complexes [Mn(bpeaCH2C6H4R)(CO)3]PF6 incorporating a functionalized 2,2‐bis(pyrazolyl)ethylamine (bpea) ligand with R = I, C≡CH, and CHO have been explored for their utility in bioorthogonal coupling to carrier peptides bearing N‐terminal alkyne, azide, and aminoxy residues. As a model system, the transforming growth factor β‐recognizing (TGF‐β) peptide sequence R′‐Leu‐Pro‐Leu‐Gly‐Asn‐Ser‐His‐OH was used in which R′ is the reactive group complementary to the metal complex functionality. The use of catalyst‐free oxime ligation gave the most stable conjugate with no degradation observed by HPLC over 96 h even after repeated freeze–thaw cycles. Both the parent complex as well as the functionalized peptide were investigated for photoactivated CO delivery to heme proteins by using the myoglobin assay and found to have essentially identical release properties. This work has established a new strategy for the conjugation of photoactivatable CO‐releasing molecules (PhotoCORMs) to biological carrier systems.

Contrasting effects of cysteine modification on the transfection efficiency of amphipathic peptides

Delivery of DNA to cells remains a key challenge towards development of gene therapy. A better understanding of the properties involved in stability and transfection efficiency of the vector could critically contribute to the improvement of delivery vehicles. In the present work we have chosen two peptides differing only in amphipathicity and explored how presence of cysteine affects DNA uptake and transfection efficiency. We report an unusual observation that addition of cysteine selectively increases transfection efficiency of secondary amphipathic peptide (Mgpe-9) and causes a drop in the primary amphipathic peptide (Mgpe-10). Our results point the effect of cysteine is dictated by the importance of physicochemical properties of the carrier peptide. We also report a DNA delivery agent Mgpe-9 exhibiting high transfection efficiency in multiple cell lines (including hard-to-transfect cell lines) with minimal cytotoxicity which can be further explored for in vivo applications.

The tetrapeptide core of the carrier peptide Xentry is cell-penetrating: novel activatable forms of Xentry.

Here we describe a structure-function analysis of the cell-penetrating peptide Xentry derived from the X-protein of the hepatitis B virus. Remarkably, the tetrapeptide core LCLR retains the cell-penetrating ability of the parental peptide LCLRPVG, as either an L- or D-enantiomer. Substitution of the cysteine with leucine revealed that the cysteine is essential for activity. In contrast, the C-terminal arginine could be substituted in the L-isomer with lysine, histidine, glutamic acid, glutamine, and asparagine, though the resulting peptides displayed distinct cell-type-specific uptake. Substitution of the leucines in the D-isomer with other hydrophobic residues revealed that leucines are optimal for activity. Surprisingly, linear di- and tetra-peptide forms of Xentry are not cell-permeable. Protease-activatable forms of Xentry were created by fusing Xentry to itself via a protease-cleavable peptide, or by attaching a heparin mimic peptide to the N-terminus. These novel activatable forms of Xentry were only taken up by MCF-7 cells after cleavage by matrix metalloproteinase 9, and could be used to deliver drugs specifically to tumours.

Shuttle-cargo fusion molecules of transport peptides and the hD2/3 receptor antagonist fallypride: a feasible approach to preserve ligand-receptor binding?

To determine if the conjugation of a small receptor ligand to a peptidic carrier to potentially facilitate transport across the blood-brain barrier (BBB) by "molecular Trojan horse" transcytosis is feasible, we synthesized several transport peptide-fallypride fusion molecules as model systems and determined their binding affinities to the hD2 receptor. Although they were affected by conjugation, the binding affinities were found to be still in the nanomolar range (between 1.5 and 64.2 nM). In addition, homology modeling of the receptor and docking studies for the most potent compounds were performed, elucidating the binding modes of the fusion molecules and the structure elements contributing to the observed high receptor binding. Furthermore, no interaction between the hybrid compounds and P-gp, the main excretory transporter of the BBB, was found. From these results, it can be inferred that the approach to deliver small neuroreceptor ligands across the BBB by transport peptide carriers is feasible.

Intracellular Delivery of Anionic meso-Tetraarylporphyrin–Per-O-methylated β-Cyclodextrin Supramolecular Complexes by an Oligoarginine Carrier Peptide

Abstract A 1:2 inclusion complex composed of 5-[4-(tetraglutaminylglutaramido)phenyl]-10,15,20-tris(4-sulfonatophenyl)porphyrin and per-O-methylated β-cyclodextrin interacted electrostatically with a dodecaarginine in aqueous solution. Such a ternary complex dispersed in a serum-containing culture medium was efficiently taken up by HeLa cells. This cellular uptake was achieved owing to the interaction between the tetraglutamate chain of the porphyrin and dodecaarginine carrier peptide.

Analysis of the uptake of native versus modified albumin by renal tubular epithelial cells (LB59)

Albumin is most abundant protein in blood and plays important homeostatic roles in the regulation of oncotic pressure and as a carrier of many small proteins, peptides and hormones. Albumin in the circulation can be covalently modified at surface lysine residues and thus the circulation contains a mixture of native albumin (i.e. not modified) and modified albumin. A significant amount of albumin is lost from the blood each day as a result of the filtration occurring in the capillaries of the renal glomeruli. This filtered albumin in the early filtrate is thought to be mostly taken up by and degraded in the early renal proximal tubular epithelial cells, while a small proportion is excreted in the final urine. This degradation of albumin by proximal tubules operates via the megalin scavenger receptor and an endocytic pathway.A major gap in our knowledge is whether the megalin‐based albumin uptake is a non‐selective process, or whether megalin has a significantly different affinity for modified versus native albumin.Using a well‐established albumin uptake assay in Opossum Kidney (OK) cells, modified albumin probes (Alexa488‐BSA and FITC‐BSA) were shown taken up into OK cells via the endocytic pathway. This uptake was efficiently competed by a molar excess of modified albumin, whereas an excess of rHA had little effect upon the uptake, suggesting a difference in binding affinity. The addition of 14C‐rHA to OK cells resulted in a very low percentage (3%) of the probe being taken up by cells compared to a 30% uptake using Alexa488‐BSA. In addition, the uptake of 14C‐rHA was not saturated in a dose‐response assay suggesting non‐specific binding. This was confirmed when an inhibitor of the endocytic pathway failed to inhibit the uptake of 14C‐rHA by OK cells.The results indicate that the megalin pathway has a much greater affinity for modified compared to native albumin. This may be an important mechanism for the removal of modified albumin from the body as it can exert toxic effects.

A Hitchhiker's Guide to the Blood–brain Barrier: In Trans Delivery of a Therapeutic Enzyme

Enzyme replacement therapy (ERT) for the systemic disease associated with lysosomal storage disorders (LSDs) has been an unequivocal success.1 Unfortunately, similar success has not been realized for the central nervous system (CNS) pathology associated with LSDs—in large part because the intravenously administered lysosomal enzymes are unable to cross the blood–brain barrier (BBB). In this issue of Molecular Therapy, Meng et al. report their achievement of supraphysiological levels of the lysosomal enzyme tripeptidyl peptidase I (TPP1) in the brains of young adult mice with the neurodegenerative LSD known as late infantile neuronal ceroid lipofuscinosis (LINCL; late infantile Batten disease).2 Remarkably, therapeutic levels of activity in the brain were achieved following an intravenous injection in young adult animals with an intact BBB. This was accomplished via coinjection of TPP1 and a carrier peptide capable of crossing the BBB.3 The enzyme and carrier peptide were not covalently linked, suggesting that this approach could be useful for a wide range of CNS disorders. Although both the safety and applicability of this approach to other systems must be determined, it could represent an important step forward for the delivery of large molecules across the BBB.

ZEBRA cell‐penetrating peptide as an efficient delivery system in Candida albicans

There is increasing interest in drug delivery systems, such as nanoparticles, liposomes, and cell-penetrating peptides, for the development of new antimicrobial treatments. In this study, we investigated the transduction capacity of a carrier peptide derived from the Epstein-Barr virus ZEBRA protein in the pathogenic fungus Candida albicans. ZEBRA-minimal domain (MD) was able to cross the cell wall and cell membrane, delivering eGFP to the cytoplasm. Uptake into up to 70% of the cells was observed within two hours, without toxicity. This new delivery system could be used in C. albicans as a carrier for different biological molecules including peptides, proteins, and nucleic acids. Thereby, in antifungal therapy, MD may carry promising bioactive fungal inhibitors that otherwise penetrate poorly into the cells. Furthermore, MD will be of interest for deciphering molecular pathways involving cell-cycle control in yeast or signaling pathways. Short interfering peptides could be internalized using MD, providing new tools for the inhibition of metabolic or signaling cascades essential for the growth and virulence of C. albicans, such as yeast-to-hyphae transition, cell wall remodeling, stress signaling and antifungal resistance. These findings create new possibilities for the internalization of cargo molecules, with applications for both treatment and functional analyses.

Computational Characterization of the Disordered Ensembles of Vasopressin and Oxytocin

Vasopressin and oxytocin are intrinsically disordered cyclic nanopeptides belonging to a family of neurohypophysial hormones. Vasopressin is an antidiuretic, regulating the retention of water and salts in mammals by indirectly promoting the insertion of aquaporin-2 channels into the epithelial cells of kidney nephron collecting ducts. Oxytocin, in contrast, is a neurotransmitter responsible for inducing labor and the subsequent lactation, as well as modulating some social behaviors. Although unique in their functions, these peptides only differ by two residues and both feature a tocin ring formed by the disulfide bridge between 1st and 6th cysteine residues. This structural similarity was experimentally linked to inhibition of activity at vasopressin receptors by the present oxytocin. Conversely, previous studies have also shown that single-residue mutations in both peptides have a significant impact on their receptor specificities.In this study we perform molecular dynamics (MD) simulations of wild type and mutant oxytocin and vasopressin in order to characterize their structural ensembles and relate them to the observed variation in activity. The generated ensembles of Y21H and P26L mutants of vasopressin and Q23T and Q23T,P26G mutants of oxytocin reveal significant population shifts compared to the wild type peptides. Here we present the classification of these populations based on the distribution of radius of gyration and deformation of the tocin ring, and attempt to relate the structural changes to the experimentally determined peptide activity and carrier protein binding affinity.

Design and implementation of a high yield production system for recombinant expression of peptides

BackgroundMaking peptide pharmaceuticals involves challenging processes where many barriers, which include production and manufacture, need to be overcome. A non common but interesting research area is related to peptides with intracellular targets, which opens up new possibilities, allowing the modulation of processes occurring within the cell or interference with signaling pathways. However, if the bioactive sequence requires fusion to a carrier peptide to allow access into the cell, the resulting peptide could be such a length that traditional production could be difficult. The goal of the present study was the development of a flexible recombinant expression and purification system for peptides, as a contribution to the discovery and development of these potentially new drugs.ResultsIn this work, a high throughput recombinant expression and purification system for production of cell penetrating peptides in Escherichia coli has been designed and implemented. The system designed produces target peptides in an insoluble form by fusion to a hexahistidine tagged ketosteroid isomerase which is then separated by a highly efficient thrombin cleavage reaction procedure. The expression system was tested on the anticancer peptides p53pAnt and PNC27. These peptides comprise the C-terminal region and the N-terminal region of the protein p53, respectively, fused by its carboxyl terminal extreme to the cell penetrating peptide Penetratin. High yields of purified recombinant fused peptides were obtained in both cases; nevertheless, thrombin cleavage reaction was successful only for p53pAnt peptide release. The features of the system, together with the procedure developed, allow achievement of high production yields of over 30 mg of highly pure p53pAnt peptide per g of dry cell mass. It is proposed that the system could be used for production of other peptides at a similar yield.ConclusionsThis study provides a system suitable for recombinant production of peptides for scientific research, including biological assays.

Susceptibility of Intracellular Coxiella burnetii to Antimicrobial Peptides in Mouse Fibroblast Cells

Q fever is a zoonotic disease caused by Coxiella burnetii, an obligate intracellular bacterium that resides inside a phagolysosome-like niche. Chronic Q fever is typified by endocarditis, and is treated with multiple antibiotics for at least 18 months. The discovery of clinical C. burnetii isolates resistant to the first-line antibiotic doxycycline, and the problematic nature of chronic Q fever treatment have demonstrated the need for improved treatment regimes. To search for alternative antimicrobial agents, we assessed the effect of 26 antimicrobial peptides (AMPs) on the intracellular growth of C. burnetii in L929 cells at a concentration of 25 µM or their maximal non-cytotoxic concentration. Among the peptides tested, A3-APO, Cath-BF, δ-Hemolysin, Octa-1, P5 and Pleurocidin were able to significantly reduce both the total bacterial cell number and the host cell bacterial burden (average bacterial number per host cell). Combining selected AMPs with Chariot, a non-covalent carrier peptide, did not increase treatment potency when non-cytotoxic concentrations were used, with the exception of P5, which remained active at a concentration of 1.6 µM (1.8 µg/mL). Combining AMPs with each other did not further improve AMP potency, with some treatment combinations increasing the growth rate of C. burnetii by >3-fold. This is the first description of AMP cellular penetration to exhibit inhibitory affect on intracellular C. burnetii growth. These results are the first step in the development of a non-traditional antibiotic treatment for Q fever.

The feasibility of rabies virus-vectored immunocontraception in a mouse model

Immunocontraceptive vaccines may be an alternative to surgical sterilization. Dual rabies vaccination and dog population management is a helpful tool for rabies prevention. A synthetic gonadotropin-releasing hormone (GnRH) peptide coupled to a carrier protein or T cell epitope is efficacious in inducing immunocontraception in a variety of mammals. However, virus-vectored GnRH recombinant vaccines have advantages over the conjugation method. In a previous in vitro study, we were able to insert a GnRH-coding sequence into the rabies virus (RABV) glycoprotein (G) gene, and the recombinant viruses grew to high titers in cells. Here, we further focused on the RABV G in accepting various copy numbers of GnRH. We demonstrated although RABV G protein with up to 4 copies of GnRH was well expressed, the recombinant virus was recovered only when 2 copies of GnRH (20 amino acids) were incorporated into the G, indicating a possible insertion limit in making a full infectious clone. The investigation provides insight into the utility of RABV G as a carrier for small peptides and its suitability for vaccine studies. Following our previous study, we selected ERAg3p/2GnRH and tested the construct in mice. The vaccine induced ⩾80% infertility after three doses without any adjuvant, in live (8 of 10 mice infertility) or inactivated (13 of 14 mice infertility) formulations; while the pregnancy rate was 100% (10 of 10 mice) in the controls. This initial success of immunocontraception in mice is promising, and we are now optimizing the vaccine formulation by using adjuvants and exploring novel delivery methods to minimize the dosage.

Calcium Orthophosphate-Based Bioceramics.

Various types of grafts have been traditionally used to restore damaged bones. In the late 1960s, a strong interest was raised in studying ceramics as potential bone grafts due to their biomechanical properties. A bit later, such synthetic biomaterials were called bioceramics. In principle, bioceramics can be prepared from diverse materials but this review is limited to calcium orthophosphate-based formulations only, which possess the specific advantages due to the chemical similarity to mammalian bones and teeth. During the past 40 years, there have been a number of important achievements in this field. Namely, after the initial development of bioceramics that was just tolerated in the physiological environment, an emphasis was shifted towards the formulations able to form direct chemical bonds with the adjacent bones. Afterwards, by the structural and compositional controls, it became possible to choose whether the calcium orthophosphate-based implants remain biologically stable once incorporated into the skeletal structure or whether they were resorbed over time. At the turn of the millennium, a new concept of regenerative bioceramics was developed and such formulations became an integrated part of the tissue engineering approach. Now calcium orthophosphate scaffolds are designed to induce bone formation and vascularization. These scaffolds are often porous and harbor different biomolecules and/or cells. Therefore, current biomedical applications of calcium orthophosphate bioceramics include bone augmentations, artificial bone grafts, maxillofacial reconstruction, spinal fusion, periodontal disease repairs and bone fillers after tumor surgery. Perspective future applications comprise drug delivery and tissue engineering purposes because calcium orthophosphates appear to be promising carriers of growth factors, bioactive peptides and various types of cells.

Blood–Brain Barrier Transport Pathways for Cytoprotective Thiols

The purpose of this study is to further define transport pathways for biological thiols by blood-brain barrier (BBB) endothelial cells, as a means of identifying endogenous cytoprotective mechanisms and potential therapeutic protocols for oxidative injury. Similar low-affininty, high-capacity passive carriers for glutathione (GSH) were observed at both the luminal (blood-facing) and abluminal (brain-facing) plasma membranes of BBB endothelial cells. These carriers are voltage dependent, favoring outward movement of intact peptide across both membrane domains, including efflux at the luminal plasmalemma where γ-glutamyl transpeptidase is located. Although present at both cell surfaces, the carriers are distributed unequally, with more appearing in the abluminal membrane. By contrast, high-affinity, low-capacity sodium-dependent GSH cotransport (Na-GSH) is observed only at the abluminal membrane, indicative of an inwardly directed active peptide carrier at the brain-facing plasma membrane. Treatment of cultured BBB endothelial cells with the GSH precursor γ-glutamyl-cysteine reduces cell damage under conditions simulating ischemia and reperfusion. These findings are consistent with the presence of (1) a typical γ-glutamyl cycle at the luminal membrane of BBB endothelial cells, (2) a significant efflux pathway at the abluminal membrane allowing passive movement of BBB GSH into brain extracellular fluid, (3) a Na-dependent, brain-to-blood pathway for transcellular transport of GSH, and (4) a mechanism for cytoprotection by γ-glutamyl cysteine, under conditions of ischemia and reperfusion.

Magnetic Nanoparticles as Mediators of Ligand-Free Activation of EGFR Signaling

BackgroundMagnetic nanoparticles (NPs) are of particular interest in biomedical research, and have been exploited for molecular separation, gene/drug delivery, magnetic resonance imaging, and hyperthermic cancer therapy. In the case of cultured cells, magnetic manipulation of NPs provides the means for studying processes induced by mechanotransduction or by local clustering of targeted macromolecules, e.g. cell surface receptors. The latter are normally activated by binding of their natural ligands mediating key signaling pathways such as those associated with the epidermal growth factor (EGFR). However, it has been reported that EGFR may be dimerized and activated even in the absence of ligands. The present study assessed whether receptor clustering induced by physical means alone suffices for activating EGFR in quiescent cells.Methodology/Principal FindingsThe EGFR on A431 cells was specifically targeted by superparamagnetic iron oxide NPs (SPIONs) carrying either a ligand-blocking monoclonal anti-EGFR antibody or a streptavidin molecule for targeting a chimeric EGFR incorporating a biotinylated amino-terminal acyl carrier peptide moiety. Application of a magnetic field led to SPION magnetization and clustering, resulting in activation of the EGFR, a process manifested by auto and transphosphorylation and downstream signaling. The magnetically-induced early signaling events were similar to those inherent to the ligand dependent EGFR pathways. Magnetization studies indicated that the NPs exerted magnetic dipolar forces in the sub-piconewton range with clustering dependent on Brownian motion of the receptor-SPION complex and magnetic field strength.Conclusions/SignificanceWe demonstrate that EGFR on the cell surface that have their ligand binding-pocket blocked by an antibody are still capable of transphosphorylation and initiation of signaling cascades if they are clustered by SPIONs either attached locally or targeted to another site of the receptor ectodomain. The results suggest that activation of growth factor receptors may be triggered by ligand-independent molecular crowding resulting from overexpression and/or sequestration in membrane microdomains.

Nanostructured materials based on mesoporous silica and mesoporous silica/apatite as osteogenic growth peptide carriers

The aim of this work was the preparation of inorganic mesoporous materials from silica, calcium phosphate and a nonionic surfactant and to evaluate the incorporation and release of different concentrations of osteogenic growth peptide (OGP) for application in bone regeneration. The adsorption and release of the labeled peptide with 5,6-carboxyfluorescein (OGP-CF) from the mesoporous matrix was monitored by fluorescence spectroscopy. The specific surface area was 880 and 484m2g−1 for pure silica (SiO) and silica/apatite (SiCaP), respectively; the area influenced the percentage of incorporation of the peptide. The release of OGP-CF from the materials in simulated body fluid (SBF) was dependent on the composition of the particles, the amount of incorporated peptide and the degradation of the material. The release of 50% of the peptide content occurred at around 4 and 30h for SiCaP and SiO, respectively. In conclusion, the materials based on SiO and SiCaP showed in vitro bioactivity and degradation; thus, these materials should be considered as alternative biomaterials for bone regeneration.

A novel peptide carrier for efficient targeting of antigens and nucleic acids to dendritic cells

Dendritic cells (DCs) initiate host immune responses by presenting captured antigens to naive T cells. Hence, DC-binding peptides may be used for antigen targeting to boost naive and memory immune responses. By biopanning peptide phage libraries on human monocyte-derived DCs, we identified novel DC-binding peptides. One of the selected phages, displaying the NW peptide (NWYLPWLGTNDW), bound DCs with high affinity, and its binding was inhibited by the corresponding synthetic peptide. Antigenic peptides or proteins conjugated to the NW peptide bound to DCs and were internalized without negative effects on DC phenotype and function. Ex vivo targeted delivery of CMV-pp65 peptides to DCs via the NW peptide increased T-cell responses in HLA-A2(+)/CMV(+) donors compared to untargeted peptides (P<0.001). Stimulation of CD45RO-depleted peripheral blood mononuclear cells from CMV(-) donors with the NW-pp65 fusion peptides expanded pp65-specific precursor T cells. Moreover, the NW peptide mediated small interfering RNA delivery to DCs, and a significant gene silencing was obtained. Collectively, the data reveal that proteins and nucleic acids can be directed to DCs through the NW peptide, enabling effective uptake and functional effects such as T-cell activation in the context of MHC class I and II molecules.