Ability Of Cell-penetrating Peptides Research Articles

β-Lactamase Tools for Establishing Cell Internalization and Cytosolic Delivery of Cell Penetrating Peptides.

The ability of cell penetrating peptides (CPPs) to deliver biologically relevant cargos into cells is becoming more important as targets in the intracellular space continue to be explored. We have developed two assays based on CPP-dependent, intracellular delivery of TEM-1 β-lactamase enzyme, a functional biological molecule comparable in size to many protein therapeutics. The first assay focuses on the delivery of full-length β-lactamase to evaluate the internalization potential of a CPP sequence. The second assay uses a split-protein system where one component of β-lactamase is constitutively expressed in the cytoplasm of a stable cell line and the other component is delivered by a CPP. The delivery of a split β-lactamase component evaluates the cytosolic delivery capacity of a CPP. We demonstrate that these assays are rapid, flexible and have potential for use with any cell type and CPP sequence. Both assays are validated using canonical and novel CPPs, with limits of detection from <500 nM to 1 µM. Together, the β-lactamase assays provide compatible tools for functional characterization of CPP activity and the delivery of biological cargos into cells.

Intranasal administration of elastin-like polypeptide for therapeutic delivery to the central nervous system.

Bypassing the blood–brain barrier is one of the primary considerations when designing compounds intended to function in the central nervous system (CNS). Intranasal (IN) administration of otherwise blood–brain barrier impermeable molecules can result in high CNS concentrations and low systemic accumulation, indicating that IN administration may be a useful method of delivering therapeutics to the CNS. Elastin-like polypeptide (ELP) is a large, non-immunogenic, highly manipulable biopolymer with extensive evidence supporting its use as a carrier with the ability to improve drug pharmacokinetics and drug targeting. The ability of ELP to reach the CNS via IN administration has been shown previously. Previous studies have also identified the ability of cell penetrating peptides (CPPs) to increase the uptake of molecules in some instances, including via the IN route. Here, we compared and contrasted the biodistribution of ELPs with or without addition of the CPPs Tat or SynB1 via both the IN and intravenous routes. Administration of ELP via the IN route led to significant accumulation in the brain, especially in the olfactory bulbs. When injected intravenously, <3% of the ELP signal was present outside the vascular compartment. This contrasted with IN administration, which resulted in 79% of the fluorescence signal localized outside the vascular space. The fusion of Tat or SynB1 significantly altered the biodistribution of ELP, decreasing the total CNS accumulation following IN administration. The addition of CPPs to ELP increased their retention in the nasal epithelium. These results suggest ELP may represent an effective CNS delivery vector without further modification and that the addition of a CPP significantly influences biodistribution.

Translocation mechanism(s) of cell-penetrating peptides: biophysical studies using artificial membrane bilayers.

The ability of cell-penetrating peptides (CPPs) to cross cell membranes has found numerous applications in the delivery of bioactive compounds to the cytosol of living cells. Their internalization mechanisms have been questioned many times, and after 20 years of intense debate, it is now widely accepted that both energy-dependent and energy-independent mechanisms account for their penetration properties. However, the energy-independent mechanisms, named "direct translocation", occurring without the requirement of the cell internalization machinery, remain to be fully rationalized at the molecular level. Using artificial membrane bilayers, recent progress has been made toward the comprehension of the direct translocation event. This review summarizes our current understanding of the translocation process, starting from the adsorption of the CPP on the membrane to the membrane crossing itself. We describe the different key steps occurring before direct translocation, because each of them can promote and/or hamper translocation of the CPP through the membrane. We then dissect the modification to the membranes induced by the presence of the CPPs. Finally, we focus on the latest studies describing the direct translocation mechanisms. These results provide an important framework within which to design new CPPs and to rationalize an eventual selectivity of CPPs in their penetration ability.

Can RNAi-mediated hsp90α knockdown in combination with 17-AAG be a therapy for glioma?

Heat shock protein 90 promotes tumor progression and survival and has emerged as a vital therapeutic target. Previously we reported that the combinatorial treatment of 17AAG/sihsp90α significantly downregulated Hsp90α mRNA and protein levels in Glioblastoma Multiforme (GBM). Here we investigated the ability of cell penetrating peptide (Tat48–60 CPP)-mediated siRNA-induced hsp90α knockdown as a single agent and in combination with 17-allylamino-17-demethoxygeldanamycin (17-AAG) to induce tumor growth inhibition in GBM and whether it possessed therapeutic implications. GBM and non-tumorigenic cells exposed to siRNA and/or 17-AAG were subsequently assessed by qRT-PCR, immunofluorescence, FACS analysis, quantitative Akt, LDH leakage and cell viability assays. PAGE was performed for serum stability assessment. A combination of siRNA/17-AAG treatment significantly induced Hsp90α gene and protein knockdown by 95% and 98%, respectively, concomitant to 84% Akt kinase activity attenuation, induced cell cycle arrest and tumor-specific cytotoxicity by 88%. Efficient complex formation between CPP and siRNA exhibited improved serum stability of the siRNA with minimal intrinsic toxicity in vitro. The preliminary in vivo results showed that combination therapy induced hsp90α knockdown and attenuated Akt kinase activity in intracranial glioblastoma mouse models. The results imply that RNAi-mediated hsp90α knockdown increases 17-AAG treatment efficacy in GBM. In addition, the cytotoxic response observed was the consequence of downregulation of hsp90α gene expression, reduced Akt kinase activity and S-G2/M cell cycle arrest. These results are novel and highlight the ability of Tat to efficiently deliver siRNA in GBM and suggest that the dual inhibition of Hsp90 has therapeutic potentials.

Direct cytosolic delivery of cargoes in vivo by a chimera consisting of D- and L-arginine residues

The ability of cell-penetrating peptides (CPPs) to deliver a range of membrane-impermeable molecules into living cells makes them attractive potential vehicles for therapeutics. However, in vivo, the efficiency of CPP delivery to the cytosol remains unsatisfactory owing to endosomal entrapment and/or systemic toxicity, which severely restrict their bioavailability and efficacy in in vivo applications. In this study, we developed a series of novel chimeras consisting of various numbers of d- and l-arginine residues and investigated their cellular uptake behaviors and systemic toxicities. We demonstrated that the intracellular distribution, uptake efficiency, and systemic toxicity of these oligoarginines were all significantly affected by the number of d-arginine residues in the peptide sequence. We also found that a hybrid peptide, (rR)3R2, possessed low systemic toxicity, high uptake efficiency, and, remarkably, achieved efficient cytosolic delivery not only in cultured cells but also in living tissue cells in mice after intravenous injection, implying that this heterogeneous motif might have promising applications in the delivery of cargoes of small sizes directed to cytosolic targets in vivo. Our studies into the uptake mechanism of (rR)3R2 indicate that its cellular uptake was not affected by pharmacological or physical inhibitors of endocytosis but by the elimination of the membrane potential, suggesting that (rR)3R2 does not enter the cells via endocytosis but rather through direct membrane translocation driven by the membrane potential. The results here might provide useful guidelines for the design and application of CPPs in drug delivery.

Protein Transduction Domain Mimics: The Role of Aromatic Functionality

Cell-penetrating peptides (CPPs), or protein transduction domains (PTDs), are a special class of membrane-active proteins that can cross the cell membrane with unusual efficiency. They have attracted considerable attention because of their ability to readily cross biological membranes, in spite of their highly charged nature. While the exact mechanism of this transport remains under intense investigation, energy-independent pathways are known. Perhaps the clearest example is the ability of CPPs, and their synthetic mimics, to cross model phospholipid bilayer vesicle membranes. One suggested mechanism implies that, in fact, CPPs like polyarginine (pR) need assistance to cross the membrane. It suggests that hydrophobic counterions complex around the guanidinium-rich backbone, thus “coating” the highly cationic structure with lipophilic moieties. This process has been termed “activation”, in which the lipophilic anion acts as an activator. In a series of detailed studies it was shown that aromatic activators outperform aliphatic ones. For example, sodium 4-(pyren-1-yl)butane-1-sulfonate gave an EC50 (effective concentration to obtain 50% activity) of 6.7 mm whereas the value for sodium dodecane-1sulfonate was 16 mm. Among other activators studied, the larger aromatic counterion, coronene, was not better than pyrene; however, a fullerene analogue was surprisingly effective. While this work beautifully demonstrated the role of various counterions for pR activation, it was not clear if this better activation was due to general hydrophobicity or to the aromatic nature of these activators. There is good reason to think that aromatic functional groups may play a special role, beyond their general hydrophobicity. It is well recognized that membrane proteins are enriched in aromatic amino acids at the membrane surface. Their central hydrophobic core, composed mostly of aliphatic residues, is flanked on both sides by “aromatic belts”. Although this belt is predominantly composed of tryptophan and tyrosine, as opposed to phenylalanine, it was shown that aromatic residues, including N-methylindole, have favorable free energies of insertion into the bilayer interface. This rules out a dominant effect of hydrogen bonding. It was suggested that the flat-rigid shape, p-electronic structure, and associated quadrupolar moments provide unique and highly favorable interactions with the bilayer interface. Specific interactions that have been proposed include p-cation, electrostatic, dipole–dipole, and entropic factors related to bilayer perturbation. Even HIV-TAT, the original protein that initiated the field of small PTDs, requires tryptophan (Trp11) for translocation. [10] Moreover, an oligoarginine consisting of seven arginine residues with a C-terminal tryptophan (R7W) and a TAT48–60 peptide with residue 59 substituted with a tryptophan (TAT48–60P59W) exhibit cellular internalization through energy-independent pathways. Another classical CPP, penetratin (Pen), contains two tryptophan residues. Substitution of tryptophan by phenylalanine (Pen2W2F) did not significantly impact cell uptake. Among the aromatic amino acids, phenylalanine has the unique ability to partition at the interface and in the membrane core. In fact, aromatic residues, especially phenylalanine, are most effective at anchoring proteins in the membrane due to their “special ability” to form and stabilize essential interactions with the polar elements of the bilayer. As a result, aromatic functionality could be a critical element facilitating the interactions between CPPs and the bilayer during transduction. In the past few years, we and others have reported polymers designed to mimic the transduction activity of PTDs. More recently, we demonstrated that these protein transduction domain mimics (PTDMs) have “self-activation” properties when hydrophobic alkyl side chains were built into the copolymers. Here, a new series of PTDMs was designed to determine if an aromatic functionality provides better transduction efficiency than aliphatic ones, at the same relative hydrophobicity. Given the importance of aromatic amino acids in membrane proteins and their interactions with the bilayer, it was proposed that aromatic side chains would make better activators, given equal relative hydrophobicity. Although aromatic groups have been studied in peptidebased CPPs, demonstration of the importance of aromatic functionality in these synthetic analogues is critical to establishing them as appropriate mimics, or PTDMs. By using reversed-phase HPLC to determine side-chain hydrophobicity and EC50 values in a classic transduction experiment, it is demonstrated here that it was possible to differentiate between side-chain hydrophobicity and aromaticity. As shown in Table 1, a series of new PTDM polymers was prepared by ring-opening metathesis polymerization (see the Supporting Information for detailed synthesis and characterization of monomers and polymers). Reversed-phase HPLC, commonly used to evaluate relative hydrophobicity, was [*] Dr. A. Som, A. Reuter, Prof. G. N. Tew Polymer Science & Engineering Department University of Massachusetts 120 Governors Drive, Amherst, MA 01003 (USA) E-mail: tew@mail.pse.umass.edu Homepage: http://www.pse.umass.edu/gtew/index.html

Translocation of cell penetrating peptide engrafted nanoparticles across skin layers

The objective of the current study was to evaluate the ability of cell penetrating peptides (CPP) to translocate the lipid payload into the skin layers. Fluorescent dye (DID-oil) encapsulated nano lipid crystal nanoparticles (FNLCN) were prepared using Compritol, Miglyol and DOGS-NTA-Ni lipids by hot melt homogenization technique. The FNLCN surface was coated with TAT peptide (FNLCNT) or control YKA peptide (FNLCNY) and in vitro rat skin permeation studies were performed using Franz diffusion cells. Observation of lateral skin sections obtained using cryotome with a confocal microscope demonstrated that skin permeation of FNLCNT was time dependent and after 24 h, fluorescence was observed upto a depth of 120 μm which was localized in the hair follicles and epidermis. In case of FNLCN and FNLCNY formulations fluorescence was mainly observed in the hair follicles. This observation was further supported by confocal Raman spectroscopy where higher fluorescence signal intensity was observed at 80 and 120 μm depth with FNLCNT treated skin and intensity of fluorescence peaks was in the ratio of 2:1:1 and 5:3:1 for FNLCNT, FNLCN, and FNLCNY treated skin sections, respectively. Furthermore, replacement of DID-oil with celecoxib (Cxb), a model lipophilic drug showed similar results and after 24 h, the CXBNT formulation increased the Cxb concentration in SC by 3 and 6 fold and in epidermis by 2 and 3 fold as compared to CXBN and CXBNY formulations respectively. Our results strongly suggest that CPP can translocate nanoparticles with their payloads into deeper skin layers.

Counterion-activated polyions as soft sensing systems in lipid bilayer membranes: from cell-penetrating peptides to DNA

Activated by amphiphilic anions, cationic oligo- and polymers can phase transfer into and across bulk and lipid bilayer membranes. In biology, this system accounts for the voltage gating of ion channels and the ability of cell-penetrating peptides (CPPs) to cross membrane barriers. In soft nanotechnology, the same system has been used for multianalyte sensing in complex matrices (cholesterol sensing in blood and eggs, for example). Would inversion of all involved charges provide access to similarly versatile functional supramolecular systems? Activation of polyanions by amphiphilic cations is well known as the basis of non-viral gene transfection. Could DNA/RNA nanotechnology thus be applied to produce smart, stimuli-responsive cation transporters that can function as multianalyte sensing in fluorogenic vesicles? Preliminary results suggest that the answer to these questions will be yes.

Amplifier‐Mediated Activation of Cell‐Penetrating Peptides with Steroids: Multifunctional Anion Transporters for Fluorogenic Cholesterol Sensing in Eggs and Blood

Off-the-shelf, prêt-à-porter multianalyte sensing systems are based on the ability of cell-penetrating peptides (CPPs; see picture, blue) to mediate the export of hydrophilic anions (yellow→green) from lipid bilayer vesicles (gray). Covalent capture of hydrophobic analytes (black) with hydrophilic anions (red) produces amphiphilic anions that can activate CPPs.