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Enhanced Live-Cell Delivery of Synthetic Proteins Assisted by Cell-Penetrating Peptides Fused to DABCYL.

ABSTRACT: Live-cell delivery of a fully synthetic protein having selectivity towards a particular target is a promising approach with potential applications for basic research and therapeutics. Cell-penetrating peptides (CPPs) allow the cellular delivery of proteins but mostly result in endosomal entrapment, leading to lack of bioavailability. Herein, we report the design and synthesis of a CPP fused to 4-((4-(dimethylamino)phenyl)azo)benzoic acid (DABCYL) to enhance cellular uptake of fluorescently labelled synthetic protein analogues in low micromolar concentration. The attachment of cyclic deca-arginine (cR10) modified with a single lysine linked to DABCYL to synthetic ubiquitin (Ub) and small ubiquitin-like modifier-2 (SUMO-2) scaffolds resulted in a threefold higher uptake efficacy in live cells compared to the unmodified cR10. We could also achieve cR10DABCYL-assisted delivery of Ub and a Ub variant (Ubv) based activity-based probes for functional studies of deubiquitinases in live cells.

SUBMITTER: Mandal S

PROVIDER: S-EPMC8048964 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Corrigendum: Structure–Uptake Relationship Study of DABCYL Derivatives Linked to Cyclic Cell‐Penetrating Peptides for Live‐Cell Delivery of Synthetic Proteins

Project description: Not available

| S-EPMC10545550 | biostudies-literature

Influence of the Dabcyl group on the cellular uptake of cationic peptides: short oligoarginines as efficient cell-penetrating peptides.

Project description:Cell-penetrating peptides (CPPs) are promising delivery vehicles. These short peptides can transport wide range of cargos into cells, although their usage has often limitations. One of them is the endosomatic internalisation and thus the vesicular entrapment. Modifications which increases the direct delivery into the cytosol is highly researched area. Among the oligoarginines the longer ones (n > 6) show efficient internalisation and they are well-known members of CPPs. Herein, we describe the modification of tetra- and hexaarginine with (4-((4-(dimethylamino)phenyl)azo)benzoyl) (Dabcyl) group. This chromophore, which is often used in FRET system increased the internalisation of both peptides, and its effect was more outstanding in case of hexaarginine. The modified hexaarginine may enter into cells more effectively than octaarginine, and showed diffuse distribution besides vesicular transport already at low concentration. The attachment of Dabcyl group not only increases the cellular uptake of the cell-penetrating peptides but it may affect the mechanism of their internalisation. Their conjugates with antitumor drugs were studied on different cells and showed antitumor activity.

| S-EPMC8241751 | biostudies-literature

Magnetic Nanoparticle Assisted Self-assembly of Cell Penetrating Peptides-Oligonucleotides Complexes for Gene Delivery.

Project description:Magnetic nanoparticles (MNPs, Fe3O4) incorporated into the complexes of cell penetrating peptides (CPPs)-oligonucleotides (ONs) promoted the cell transfection for plasmid transfection, splice correction, and gene silencing efficiencies. Six types of cell penetrating peptides (CPPs; PeptFect220 (denoted PF220), PF221, PF222, PF223, PF224 and PF14) and three types of gene therapeutic agents (plasmid (pGL3), splicing correcting oligonucleotides (SCO), and small interfering RNA (siRNA) were investigated. Magnetic nanoparticles incorporated into the complexes of CPPs-pGL3, CPPs-SCO, and CPPs-siRNA showed high cell biocompatibility and efficiently transfected the investigated cells with pGL3, SCO, and siRNA, respectively. Gene transfer vectors formed among PF14, SCO, and MNPs (PF14-SCO-MNPs) showed a superior transfection efficiency (up to 4-fold) compared to the noncovalent PF14-SCO complex, which was previously reported with a higher efficiency compared to commercial vector called Lipofectamine™2000. The high transfection efficiency of the new complexes (CPPs-SCO-MNPs) may be attributed to the morphology, low cytotoxicity, and the synergistic effect of MNPs and CPPs. PF14-pDNA-MNPs is an efficient complex for in vivo gene delivery upon systemic administration. The conjugation of CPPs-ONs with inorganic magnetic nanoparticles (Fe3O4) may open new venues for selective and efficient gene therapy.

| S-EPMC5567346 | biostudies-literature

Synthetic molecular evolution of hybrid cell penetrating peptides.

Project description:Peptides and analogs such as peptide nucleic acids (PNA) are promising tools and therapeutics, but the cell membrane remains a barrier to intracellular targets. Conjugation to classical cell penetrating peptides (CPPs) such as pTat48-60 (tat) and pAntp43-68 (penetratin) facilitates delivery; however, efficiencies are low. Lack of explicit design principles hinders rational improvement. Here, we use synthetic molecular evolution (SME) to identify gain-of-function CPPs with dramatically improved ability to deliver cargoes to cells at low concentration. A CPP library containing 8192 tat/penetratin hybrid peptides coupled to an 18-residue PNA is screened using the HeLa pTRE-LucIVS2 splice correction reporter system. The daughter CPPs identified are one to two orders of magnitude more efficient than the parent sequences at delivery of PNA, and also deliver a dye cargo and an anionic peptide cargo. The significant increase in performance following a single iteration of SME demonstrates the power of this approach to peptide sequence optimization.

| S-EPMC6028423 | biostudies-literature

Delivery of therapeutic oligonucleotides with cell penetrating peptides.

Project description:Oligonucleotide-based drugs have received considerable attention for their capacity to modulate gene expression very specifically and as a consequence they have found applications in the treatment of many human acquired or genetic diseases. Clinical translation has been often hampered by poor biodistribution, however. Cell-penetrating peptides (CPPs) appear as a possibility to increase the cellular delivery of non-permeant biomolecules such as nucleic acids. This review focuses on CPP-delivery of several classes of oligonucleotides (ONs), namely antisense oligonucleotides, splice switching oligonucleotides (SSOs) and siRNAs. Two main strategies have been used to transport ONs with CPPs: covalent conjugation (which is more appropriate for charge-neutral ON analogues) and non-covalent complexation (which has been used for siRNA delivery essentially). Chemical synthesis, mechanisms of cellular internalization and various applications will be reviewed. A comprehensive coverage of the enormous amount of published data was not possible. Instead, emphasis has been put on strategies that have proven to be effective in animal models of important human diseases and on examples taken from the authors' own expertise.

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Cell-Penetrating Dabcyl-Containing Tetraarginines with Backbone Aromatics as Uptake Enhancers.

Project description:Cell-penetrating peptides represent an emerging class of carriers capable of effective cellular delivery. This work demonstrates the preparation and investigation of efficient CPPs. We have already shown that the presence of 4-((4-(dimethylamino)phenyl)azo)benzoic acid (Dabcyl) and Trp greatly increase the uptake of oligoarginines. This work is a further step in that direction. We have explored the possibility of employing unnatural, aromatic amino acids, to mimic Trp properties and effects. The added residues allow the introduction of aromaticity, not as a side-chain group, but rather as a part of the sequence. The constructs presented exceptional internalization on various cell lines, with an evident structure-activity relationship. The CPPs were investigated for their entry mechanisms, and our peptides exploit favorable pathways, yet one of the peptides relies highly on direct penetration. Confocal microscopy studies have shown selectivity towards the cell lines, by showing diffuse uptake in FADU cells, while vesicular uptake takes place in SCC-25 cell line. These highly active CPPs have proved their applicability in cargo delivery by successfully delivering antitumor drugs into MCF-7 and MDA-MB-231 cells. The modifications in the sequences allow the preparation of short yet highly effective constructs able to rival the penetration of well-known CPPs such as octaarginine (Arg8).

| S-EPMC9864790 | biostudies-literature

Ocular Delivery of Therapeutic Agents by Cell-Penetrating Peptides.

Project description:Cell-penetrating peptides (CPPs) are short peptides with the ability to translocate through the cell membrane to facilitate their cellular uptake. CPPs can be used as drug-delivery systems for molecules that are difficult to uptake. Ocular drug delivery is challenging due to the structural and physiological complexity of the eye. CPPs may be tailored to overcome this challenge, facilitating cellular uptake and delivery to the targeted area. Retinal diseases occur at the posterior pole of the eye; thus, intravitreal injections are needed to deliver drugs at an effective concentration in situ. However, frequent injections have risks of causing vision-threatening complications. Recent investigations have focused on developing long-acting drugs and drug delivery systems to reduce the frequency of injections. In fact, conjugation with CPP could deliver FDA-approved drugs to the back of the eye, as seen by topical application in animal models. This review summarizes recent advances in CPPs, protein/peptide-based drugs for eye diseases, and the use of CPPs for drug delivery based on systematic searches in PubMed and clinical trials. We highlight targeted therapies and explore the potential of CPPs and peptide-based drugs for eye diseases.

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Cell-Penetrating Peptides-Based Liposomal Delivery System Enhanced Immunogenicity of Peptide-Based Vaccine against Group A Streptococcus.

Project description:Peptide-based vaccine development represents a highly promising strategy for preventing Group A Streptococcus (GAS) infection. However, these vaccines need to be administered with the help of a delivery system and/or immune adjuvant. Cell-penetrating peptides (CPPs) have been used as a powerful tool for delivering various therapeutic agents, including peptides, as they can overcome the permeability barrier of cell membranes. Here, we used CPPs to deliver our lead lipopeptide-based vaccine (LCP-1). CPPs were anchored through a spacer to LCP-1-bearing multilamellar and unilamellar liposomes and administered to Swiss outbred mice. Tat47-57 conjugated to two palmitic acids via a (Gly)6 spacer (to form a liposome-anchoring moiety) was the most efficient system for triggering immune responses when combined with multilamellar liposomes bearing LCP-1. The immunostimulatory potential of a variety of other CPPs was examined following intranasal administration in mice. Among them, LCP-1/liposomes/Tat47-57 and LCP-1/liposomes/KALA induced the highest antibody titers. The antibodies produced showed high opsonic activity against clinically isolated GAS strains D3840 and GC2 203. The use of the CPP-liposome delivery system is a promising strategy for liposome-based GAS vaccine development.

| S-EPMC8151947 | biostudies-literature

Dynamic measurements of membrane insertion potential of synthetic cell penetrating peptides.

Project description:Cell penetrating peptides (CPPs) have been established as excellent candidates for mediating drug delivery into cells. When designing synthetic CPPs for drug delivery applications, it is important to understand their ability to penetrate the cell membrane. In this paper, anionic or zwitterionic phospholipid monolayers at the air-water interface are used as model cell membranes to monitor the membrane insertion potential of synthetic CPPs. The insertion potential of CPPs having different cationic and hydrophobic amino acids were recorded using a Langmuir monolayer approach that records peptide adsorption to model membranes. Fluorescence microscopy was used to visualize alterations in phospholipid packing due to peptide insertion. All CPPs had the highest penetration potential in the presence of anionic phospholipids. In addition, two of three amphiphilic CPPs inserted into zwitterionic phospholipids, but none of the hydrophilic CPPs did. All the CPPs studied induced disruptions in phospholipid packing and domain morphology, which were most pronounced for amphiphilic CPPs. Overall, small changes to amino acids and peptide sequences resulted in dramatically different insertion potentials and membrane reorganization. Designers of synthetic CPPs for efficient intracellular drug delivery should consider small nuances in CPP electrostatic and hydrophobic properties.

| S-EPMC3918496 | biostudies-literature

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Project description:Small interfering RNA (siRNA) is a promising molecule for gene therapy, but its therapeutic administration remains problematic. Among the recently proposed vectors, cell-penetrating peptides show great promise in in vivo trials for siRNA delivery. Human protein DMBT1 (deleted in malignant brain tumor 1) is a pattern recognition molecule that interacts with polyanions and recognizes and aggregates bacteria. Taking advantage of these properties, we investigated whether specific synthetic DMBT1-derived peptides could be used to formulate nanoparticles for siRNA administration. Using an electrophoretic mobility shift assay and UV spectra, we identified two DMBT1 peptides that could encapsulate the siRNA with a self- and co-assembly mechanism. The complexes were stable for at least 2 hr in the presence of either fetal bovine serum (FBS) or RNase A, with peptide-dependent time span protection. ?-potential, circular dichroism, dynamic light scattering, and transmission electron microscopy revealed negatively charged nanoparticles with an average diameter of 10-800 nm, depending on the reaction conditions, and a spherical or rice-shaped morphology, depending on the peptide and ?-helix conformation. We successfully transfected human MCF7 cells with fluorescein isothiocyanate (FITC)-DMBT1-peptide-Cy3-siRNA complexes. Finally, DMBT1 peptides encapsulating an siRNA targeting a fluorescent reporter gene showed efficient gene silencing in MCF7-recombinant cells. These results lay the foundation for a new research line to exploit DMBT1-peptide nanocomplexes for therapeutic siRNA delivery.

| S-EPMC5514624 | biostudies-literature

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