ABSTRACT: Cytosolic delivery of peptides remains a challenging task owing to their susceptibility to enzymatic degradation and the existence of multiple intracellular barriers. Here, we report a new strategy to address these issues by decoration of a fluorous tag on the terminal of cargo peptides. The fluorous-tagged peptides were assembled into nanostructures, efficiently internalized by cells via several endocytic pathways and released into the cytosol after endosomal escape. They were relatively stable against enzymatic degradation and showed much higher efficiency than nonfluorinated analogs and cell penetrant peptide-conjugated ones. The proposed strategy also efficiently delivered a proapoptotic peptide into specific sites in the cells and restored the function of cargo peptide after cytosolic delivery. The fluorous-tagged proapoptotic peptide efficiently inhibited tumor growth in vivo. This study provides an efficient fluorination strategy to promote the cytosolic delivery of peptides.
Project description:Cationic peptides termed protein transduction domains (PTDs) have been shown to cross biological membranes efficiently. However, proteins transduced by PTDs become entrapped within the endosomal vesicles and are not delivered into organelles. We have developed a novel protein delivery system to enhance the proton sponge effect, which results in rupture of the endosomes, by using a mixture of Wr-T transporter peptide and a commercially available cationic lipid reagent. This peptide and cationic lipid reagent mixture efficiently delivers a variety of cargo proteins into living cells by releasing them from the endosomes.
Project description:In this work, we developed a simple method to load drugs into commercially available contact lenses utilizing fluorous chemistry. We demonstrated this method using model compounds including fluorous-tagged fluorescein and antibiotic ciprofloxacin. We showed that fluorous interactions facilitated the loading of model molecules into fluorocarbon-containing contact lenses, and that the release profiles exhibited sustained release. Contact lenses loaded with fluorous-tagged ciprofloxacin exhibited antimicrobial activity against Pseudomonas aeruginosa in vitro, while no cytotoxicity towards human corneal epithelial cells was observed. To mimic the tear turnover, we designed a porcine eye infection model under flow conditions. Significantly, the modified lenses also exhibited antimicrobial efficacy against Pseudomonas aeruginosa in the ex vivo infection model. Overall, utilizing fluorous chemistry, we can construct a drug delivery system that exhibits high drug loading capacity, sustained drug release, and robust biological activity.
Project description:The promise of oligonucleotide therapeutic agents to perturb expression of disease-related genes remains unrealized, in part due to challenges with functional cellular delivery of these agents. Herein, we describe disulfide-constrained cyclic amphipathic peptides that complex with short-interfering RNA (siRNA) and affect functional cytosolic delivery and knockdown of target gene products in cell culture and in vivo to mouse lung. Reduction of the constraining disulfide bond and subsequent proteolytic clearance of the peptide are key design features that allow unmasking of the siRNA cargo and presentation to the RNA interference machinery.
Project description:Delivering apoptosis inducing peptides to cells is an emerging area in cancer and molecular therapeutics. Here, we have identified an alternative mechanism of action for the proapoptotic chimeric peptide D-NuBCP-9-r8. Integral to D-NuBCP-9-r8 is the Nur-77-derived D-isoform sequence fsrslhsll that targets Bcl-2, and the cell-penetrating peptide (CPP) octaarginine (r8) that is required for intracellular delivery. We find that the N-terminal phenylalanine of fsrslhsll acts in synergy with the cell-penetrating moiety to enhance peptide uptake at low nontoxic levels and cause rapid membrane blebbing and cell necrosis at higher (IC(50)) concentrations. These effects were not observed when a single phenylalanine-alanine mutation was introduced at the N-terminus of D-NuBCP-9-r8. Using primary samples from chronic lymphocytic leukemia (CLL) patients and cancer cell lines, we show that NuBCP-9-r8 induced toxicity, via membrane disruption, is independent of Bcl-2 expression. Overall, this study demonstrates a new mechanism of action for this peptide and cautions its use as a highly specific entity for targeting Bcl-2. For delivery of therapeutic peptides the work emphasizes that key amino acids in cargo, located several residues away from the cell-penetrating sequence, can significantly influence their cellular uptake and mode of action.
Project description:Internalization into cancer cells is a significant challenge in the delivery of many anticancer therapeutics. Drug carriers can address this challenge by facilitating cellular uptake of cytotoxic cargo in the tumor, while preventing cellular uptake in healthy tissues. Here we describe an extrinsically controlled drug carrier, a nanopeptifier, that amplifies cellular uptake by modulating the activity of cell-penetrating peptides with thermally toggled self-assembly of a genetically encoded polypeptide nanoparticle. When appended with a proapoptotic peptide, the nanopeptifier creates a cytotoxic switch, inducing apoptosis only in its self-assembled state. The nanopeptifier provides a new approach to tune the cellular uptake and activity of anticancer therapeutics by an extrinsic thermal trigger.
Project description:Cyclic heptapeptide cyclo(F?RRRRQ) (cF?R4, where ? is l-2-naphthylalanine) was recently found to be efficiently internalized by mammalian cells. In this study, its mechanism of internalization was investigated by perturbing various endocytic events through the introduction of pharmacologic agents and genetic mutations. The results show that cF?R4 binds directly to membrane phospholipids, is internalized into human cancer cells through endocytosis, and escapes from early endosomes into the cytoplasm. Its cargo capacity was examined with a wide variety of molecules, including small-molecule dyes, linear and cyclic peptides of various charged states, and proteins. Depending on the nature of the cargos, they may be delivered by endocyclic (insertion of cargo into the cF?R4 ring), exocyclic (attachment of cargo to the Gln side chain), or bicyclic approaches (fusion of cF?R4 and cyclic cargo rings). The overall delivery efficiency (i.e., delivery of cargo into the cytoplasm and nucleus) of cF?R4 was 4-12-fold higher than those of nonaarginine, HIV Tat-derived peptide, or penetratin. The higher delivery efficiency, coupled with superior serum stability, minimal toxicity, and synthetic accessibility, renders cF?R4 a useful transporter for intracellular cargo delivery and a suitable system for investigating the mechanism of endosomal escape.
Project description:A small tri-beta-peptide library was prepared starting from three enantio- and diastereopure azido acids. Fluorous tagging followed by two cycles of azide reduction, fluorous solid phase extraction (f-SPE), peptide coupling with the original azido acids and f-SPE provided 27 protected azido peptides. Reduction and HPLC purification provided 25 of the 27 targeted tri-beta-peptides in acceptable yields and excellent purities.
Project description:Biologics such as peptides and antibodies are a well-established class of therapeutics. However, their intracellular delivery remains problematic. In particular, methods to efficiently inhibit intra-nuclear targets are lacking. We previously described that Pseudomonas Exotoxin A reaches the nucleoplasm via the endosomes-to-nucleus trafficking pathway. Here, we show that a non-toxic truncated form of PE can be coupled to peptides and efficiently reach the nucleoplasm. It can be used as a Peptide Nuclear Delivery Device (PNDD) to deliver polypeptidic cargos as large as Glutathione- S-transferase (GST) to the nucleus. PNDD1 is a fusion of PNDD to the c-myc inhibitor peptide H1. PNDD1 is able to inhibit c-Myc dependent transcription at nanomolar concentration. In contrast, H1 fused to various cell-penetrating peptides are active only in the micromolar range. PNDD1 attenuates cell proliferation and induces cell death in various tumor cell lines. In particular, several patient-derived Diffuse Large B-Cell Lymphomas cell lines die after exposure to PNDD1, while normal B-cells survive. Altogether, our data indicate that PNDD is a powerful tool to bring active cargo to the nucleus and PNDD1 could be the basis of a new therapy against lymphoma.
Project description:Membrane active peptides are of large interest for development of drug delivery vehicles and therapeutics for treatment of multiple drug resistant infections. Lack of specificity can be detrimental and finding routes to tune specificity and activity of membrane active peptides is vital for improving their therapeutic efficacy and minimize harmful side effects. We describe a de novo designed membrane active peptide that partition into lipid membranes only when specifically and covalently anchored to the membrane, resulting in pore-formation. Dimerization with a complementary peptide efficiently inhibits formation of pores. The effect can be regulated by proteolytic digestion of the inhibitory peptide by the matrix metalloproteinase MMP-7, an enzyme upregulated in many malignant tumors. This system thus provides a precise and specific route for tuning the permeability of lipid membranes and a novel strategy for development of recognition based membrane active peptides and indirect enzymatically controlled release of liposomal cargo.
Project description:We reported recently that certain beta-peptides self-assemble spontaneously into cooperatively folded bundles whose kinetic and thermodynamic metrics mirror those of natural helix bundle proteins. The structures of four such beta-peptide bundles are known in atomic detail. These structures reveal a solvent-sequestered, hydrophobic core stabilized by a unique arrangement of leucine side chains and backbone methylene groups. Here we report that this hydrophobic core can be re-engineered to contain a fluorous subdomain while maintaining the characteristic beta-peptide bundle fold. Like alpha-helical bundles possessing fluorous cores, fluorous beta-peptide bundles are stabilized relative to hydrocarbon analogues and undergo cold denaturation. Beta-peptide bundles with fluorous cores represent the essential first step in the synthesis of orthogonal protein assemblies that can sequester selectively in an interstitial membrane environment.