Generation of endosomolytic reagents by branching of cell-penetrating peptides: tools for the delivery of bioactive compounds to live cells in cis or trans.
ABSTRACT: We describe the synthesis and cellular delivery properties of multivalent and branched delivery systems consisting of cell-penetrating peptides assembled onto a peptide scaffold using native chemical ligation. A trimeric delivery system presenting three copies of the prototypical cell-penetrating peptide TAT shows an endosomolytic activity much higher than its monomeric and dimeric counterparts. This novel reagent promotes the endosomal release of macromolecules internalized into cells by endocytosis, and as a result, it can be used to achieve cytosolic delivery of bioactive but cell-impermeable macromolecules in either cis (covalent conjugation) or trans (simple coincubation).
Project description:Combination therapies consisting of multiple short therapeutic RNAs, such as small interfering RNA (siRNA) and microRNA (miRNA), have enormous potential in cancer treatment as they can precisely silence a specific set of oncogenes and target multiple disease-related pathways. However, clinical use of siRNA/miRNA combinations is limited by the availability of safe and efficient systemic delivery systems with sufficient tumor penetrating and endosomal escaping capabilities. This study reports on the development of multifunctional tumor-penetrating mesoporous silica nanoparticles (iMSNs) for simultaneous delivery of siRNA (siPlk1) and miRNA (miR-200c), using encapsulation of a photosensitizer indocyanine green (ICG) to facilitate endosomal escape and surface conjugation of the iRGD peptide to enable deep tumor penetration. Increased cell uptake of the nanoparticles was observed in both 3D tumor spheroids in vitro and in orthotopic MDA-MB-231 breast tumors in vivo. Using a galectin-8 recruitment assay, we showed that reactive oxygen species generated by ICG upon light irradiation functioned as an endosomolytic stimulus that caused release of the siRNA/miRNA combination from endosomes. Co-delivery of the therapeutic RNAs displayed combined cell killing activity in cancer cells. Systemic intravenous treatment of metastatic breast cancer with the iMSNs loaded with siPlk1 and miR-200c resulted in a significant suppression of the primary tumor growth and in marked reduction of metastasis upon short light irradiation of the primary tumor. This work demonstrates that siRNA-miRNA combination assisted by the photodynamic effect and tumor penetrating delivery system may provide a promising approach for metastatic cancer treatment.
Project description:Fluorescently labeled cell-penetrating peptides can translocate into cells by endocytosis and upon light irradiation, lyse the endocytic vesicles. This photo-inducible endosomolytic activity of Fl-CPPs can be used to efficiently deliver macromolecules such as proteins and nucleic acids and other small organic molecules into the cytosol of live cells. The requirement of a light trigger to induce photolysis provides a more spatial and temporal control to the intracellular delivery process.In this report, we examine the molecular level mechanisms by which cell-penetrating peptides such as TAT when labeled with small organic fluorophore molecules acquire a photo-induced lytic activity using a simplified model of lipid vesicles.The peptide TAT labeled with 5(6)-carboxytetramethylrhodamine binds to negatively charged phospholipids, thereby bringing the fluorophore in close proximity to the membrane of liposomes. Upon light irradiation, the excited fluorophore produces reactive oxygen species at the lipid bilayer and oxidation of the membrane is achieved. In addition, the fluorescent peptide causes aggregation of photo-oxidized lipids, an activity that requires the presence of arginine residues in the peptide sequence.These results suggest that the cell-penetrating peptide plays a dual role. On one hand, TAT targets a conjugated fluorophore to membranes. On the other hand, TAT participates directly in the destabilization of photosensitized membranes. Peptide and fluorophore therefore appear to act in synergy to destroy membranes efficiently.Understanding the mechanism behind Fl-CPP mediated membrane photodamage will help to design optimally photo-endosomolytic compounds.
Project description:HA2-TAT is a peptide-based delivery agent that combines the pH-sensitive HA2 fusion peptide from influenza and the cell-penetrating peptide TAT from HIV. This chimeric peptide is engineered to induce the cellular uptake of macromolecules into endosomes via the TAT moiety and to respond to the acidifying lumen of endosomes to cause membrane leakage and release of macromolecules into cells via the HA2 moiety. The question of how HA2 and TAT affect the properties of one another remains, however, unanswered, and the behavior of the peptide inside endosomes is mostly uncharacterized. To address these issues, the binding and membrane leakage activity of a glutamic acid-enriched analogue E5-TAT was assessed with red blood cells and giant unilamellar vesicles as membrane models for endosomes. Hemolysis and microscopy assays reveal that E5-TAT binds to membranes in a pH-dependent manner and causes membrane leakage by inducing the formation of pores through which macromolecules can escape. The TAT moiety contributes to this activity by causing a shift in the pH response of E5 and by binding to negatively charged phospholipids. On the other hand, TAT binding to glycosaminoglycans reduces the lytic activity of E5-TAT. Addition of TAT to the C-terminus of E5 can therefore either increase or inhibit the activity of E5 depending on the cellular components present at the membrane. Taken together, these results suggest a model for the endosomolytic activity of the peptide and provide the basis for the molecular design of future delivery agents.
Project description:A platform technology has been developed and tested for delivery of intracellular-acting peptides through electrostatically complexed nanoparticles, or nano-polyplexes, formulated from an anionic endosomolytic polymer and cationic therapeutic peptides. This delivery platform has been initially tested and optimized for delivery of two unique vasoactive peptides, a phosphomimetic of heat shock protein 20 and an inhibitor of MAPKAP kinase II, to prevent pathological vasoconstriction (i.e., vasospasm) in human vascular tissue. These peptides inhibit vasoconstriction and promote vasorelaxation by modulating actin dynamics in vascular smooth muscle cells. Formulating these peptides into nano-polyplexes significantly enhances peptide uptake and retention, facilitates cytosolic delivery through a pH-dependent endosomal escape mechanism, and enhances peptide bioactivity in vitro as measured by inhibition of F-actin stress fiber formation. In comparison to treatment with the free peptides, which were endowed with cell-penetrating sequences, the nano-polyplexes significantly increased vasorelaxation, inhibited vasoconstriction, and decreased F-actin formation in the human saphenous vein ex vivo. These results suggest that these formulations have significant potential for treatment of conditions such as cerebral vasospasm following subarachnoid hemorrhage. Furthermore, because many therapeutic peptides include cationic cell-penetrating segments, this simple and modular platform technology may have broad applicability as a cost-effective approach for enhancing the efficacy of cytosolically active peptides.
Project description:Cell-penetrating peptides (CPPs) are well established as delivery agents for otherwise cell-impermeable cargos. CPPs can also theoretically be used to modulate intracellular processes. However, their susceptibility to proteolytic degradation often limits their utility in these applications. Previous studies have explored the consequences for cellular uptake of converting the residues in CPPs from l- to d-stereochemistry, but conflicting results have been reported and specific steps en route to intracellular activity have not been explored. Here we use dimeric fluorescence TAT as a model CPP to explore the broader consequences of l- to d-stereochemical conversion. We show that inversion of chirality provides protease resistance without altering the overall mode of cellular entry, a process involving endocytic uptake followed by endosomal escape and cytosolic access. However, whereas inversion of chirality reduces endocytic uptake, the d-peptide, once in the endosome, is significantly more prone to escape than its l-counterpart. Moreover, the d-peptide is retained in the cytosol of cells for several days, whereas the l-peptide is degraded within hours. Notably, while the l-peptide is relatively innocuous to cells, the d-peptide exerts a prolonged anti-proliferative activity. Together, our results establish connections between chirality, protease resistance, cellular penetration, and intracellular activity that may be useful for the development of future delivery agents with improved properties.
Project description:Ineffective cellular delivery is a common problem in numerous biological applications. Developing delivery reagents that work robustly in a variety of experimental settings remains a challenge. Herein, we report how peptides derived from the prototypical cell penetrating peptide TAT can be used in combination with a small molecule, UNC7938, to deliver macromolecules into the cytosol of cells by a simple co-incubation protocol. We establish successful delivery of peptides, DNA plasmids, and a single-chain variable fragment antibody. We also demonstrate that delivery works in hard-to-transfect mammalian cells and under conditions typically inhibitory to cell-penetrating peptides. Mechanistically, UNC7938 destabilizes the membrane of endosomes. This, in turn, enhances the endosome-leakage activity of cell-penetrating peptides and facilitates the endosomal escape of macromolecules initially internalized by mammalian cells via endocytosis. This combined selective membrane-destabilization represents a new chemical space for delivery tools and provides a novel solution to the problem of endosomal entrapment that often limits the effectiveness of reagent-based delivery approaches.
Project description:Cell-penetrating peptides (CPPs) can transport macromolecular cargos into live cells. However, the cellular delivery efficiency of these reagents is often suboptimal because CPP-cargo conjugates typically remain trapped inside endosomes. Interestingly, irradiation of fluorescently labeled CPPs with light increases the release of the peptide and its cargos into the cytosol. However, the mechanism of this phenomenon is not clear. Here we investigate the molecular basis of the photo-induced endosomolytic activity of the prototypical CPPs TAT labeled to the fluorophore 5(6)-carboxytetramethylrhodamine (TMR).We report that TMR-TAT acts as a photosensitizer that can destroy membranes. TMR-TAT escapes from endosomes after exposure to moderate light doses. However, this is also accompanied by loss of plasma membrane integrity, membrane blebbing, and cell-death. In addition, the peptide causes the destruction of cells when applied extracellularly and also triggers the photohemolysis of red blood cells. These photolytic and photocytotoxic effects were inhibited by hydrophobic singlet oxygen quenchers but not by hydrophilic quenchers.Together, these results suggest that TAT can convert an innocuous fluorophore such as TMR into a potent photolytic agent. This effect involves the targeting of the fluorophore to cellular membranes and the production of singlet oxygen within the hydrophobic environment of the membranes. Our findings may be relevant for the design of reagents with photo-induced endosomolytic activity. The photocytotoxicity exhibited by TMR-TAT also suggests that CPP-chromophore conjugates could aid the development of novel Photodynamic Therapy agents.
Project description:Mitochondrial dysfunction is linked to a variety of human illnesses, but selective delivery of therapeutics into the mitochondrion is challenging. Now a family of amphipathic cell-penetrating motifs (CPMs) is presented, consisting of four guanidinium groups and one or two aromatic hydrophobic groups (naphthalene) assembled through a central scaffold (a benzene ring). The CPMs and CPM-cargo conjugates efficiently enter the interior of cultured mammalian cells and are specifically localized into the mitochondrial matrix, as revealed by high-resolution confocal microscopy. With a membrane-impermeable peptide as cargo, the CPMs exhibited ?170-fold higher delivery efficiency than previous mitochondrial delivery vehicles. Conjugation of a small-molecule inhibitor of heat shock protein 90 to a CPM resulted in accumulation of the inhibitor inside the mitochondrial matrix with greatly enhanced anticancer activity. The CPMs showed minimal effect on the viability or the mitochondrial membrane potential of mammalian cells.
Project description:Iduna is a poly(ADP-ribose) (PAR)-dependent E3 ubiquitin ligase that regulates cellular responses such as proteasomal degradation and DNA repair upon interaction with its substrate. We identified a highly cationic region within the PAR-binding motif of Iduna; the region was similar among various species and showed amino acid sequence similarity with that of known cell-penetrating peptides (CPPs). We hypothesized that this Iduna-derived cationic sequence-rich peptide (Iduna) could penetrate the cell membrane and deliver macromolecules into cells. To test this hypothesis, we generated recombinant Iduna-conjugated enhanced green fluorescent protein (Iduna-EGFP) and its tandem-repeat form (d-Iduna-EGFP). Both Iduna-EGFP and d-Iduna-EGFP efficiently penetrated Jurkat cells, with the fluorescence signals increasing dose- and time-dependently. Tandem-repeats of Iduna and other CPPs enhanced intracellular protein delivery efficiency. The delivery mechanism involves lipid-raft-mediated endocytosis following heparan sulfate interaction; d-Iduna-EGFP was localized in the nucleus as well as the cytoplasm, and its residence time was much longer than that of other controls such as TAT and Hph-1. Moreover, following intravenous administration to C57/BL6 mice, d-Iduna-EGFP was efficiently taken up by various tissues, including the liver, spleen, and intestine suggesting that the cell-penetrating function of the human Iduna-derived peptide can be utilized for experimental and therapeutic delivery of macromolecules.
Project description:Delivery to intracellular target sites is still one of the main obstacles in the development of peptide nucleic acids (PNAs) as antisense-antigene therapeutics. Here, we designed a self-assembled oligonucleotide scaffold that included a central complementary region for self-assembly and lateral regions complementing the PNAs. Assembly of cell-penetrating peptide (CPP)-PNAs on the scaffold significantly promoted endocytosis of PNAs by at least 10-fold in cell cultures, particularly for scaffolds in which the central complementary region was assembled by poly(guanine) and poly(cytosine). The antisense activity of CPP-PNAs increased by assembly on the scaffold and was further enhanced after co-assembly with endosomolytic peptide (EP)-PNA. This synergistic effect was also observed following the assembly of antigene CPP-PNAs\EP-PNAs on the scaffold. However, antigene activity was only observed by targeting episomal viral DNA or transfected plasmids, but not the chromosome in the cell cultures. In conclusion, assembly on oligonucleotide scaffolds significantly enhanced the antisense-antigene activity of PNAs by promoting endocytosis and endosomal escape. This oligonucleotide scaffold provided a simple strategy for assembly of multiple functional peptide-PNA conjugates, expanding the applications of PNAs and demonstrating the potential of PNAs as antiviral therapeutics.