基于细胞穿膜肽的药物递送研究进展

从细胞穿膜肽(CPP)的分类、内化机制、与货物的连接和应用四个方面讲述目前人们在对细胞穿膜肽的研究上已经取得的成果。细胞穿膜肽是一种能穿过细胞膜的短肽,可分为阳离子型肽、两亲性肽和疏水性肽。细胞穿膜肽的内化机制主要有内吞作用、直接渗透、依赖于糖蛋白的内化机制和依赖于浓度的内化机制等。近年来,人们合成了多种有实际应用价值的CPP-货物复合物,在细胞穿膜肽的应用上,取得了很多进展和突破。科学家们主要研究将细胞穿膜肽应用于药物递送和细胞成像。     

Intrinsically cell-permeable miniature proteins based on a minimal cationic PPII motif

Cell-penetrating peptides (CPPs) provide promising tools for the cellular delivery of molecular cargos ranging in size from small molecules and peptides to proteins and quantum dots. CPPs are typically cationic and/or amphipathic sequences that are unstructured or alpha-helical. We expand the repertoire of cell-penetrating motifs by designing encodable CPPs possessing type-II polyproline (PPII) helical structure. These motifs surpass the uptake efficiency of existing CPPs and are not cytotoxic at concentrations 100 times greater than that necessary for delivery. By replacing the PPII helix of a miniature protein, the motif can endow intrinsic cell permeability without increasing molecular size.     

Energy-independent intracellular gene delivery mediated by polymeric biomimetics of cell-penetrating peptides

Efficient gene transfer into mammalian cells mediated by small molecular amphiphile-polymer conjugates, bile acid-polyethylenimine (BA-PEI), is demonstrated, opening an efficient transport route for genetic materials across the cell membrane. This process occurs without the aid of endocytosis or other energy-consuming processes, thus mimicking macromolecular transduction by cell-penetrating peptides. The exposure of a hydrophilic face of the amphiphilic BA moiety on the surface of BA-PEI/DNA complex that mediates direct contact of the BA molecules to the cell surface seems to play an important role in the endocytosis- and energy-independent internalization process. The new modality of the polymeric biomimetics can be applied to enhanced delivery of macromolecular therapeutics.     

Advance and Designing Strategies in Polymeric Antifungal Agents Inspired by Membrane-Active Peptides

The high-mortality invasive fungal infections seriously threaten the lives of immunocompromised people. Host defense peptides and cell-penetrating peptides are representative membrane-active peptides with different functions. Among them, host defense peptides mimicking is a valid strategy in the design of synthetic antifungal agents. Despite the brilliance in the field of intracellular delivery, the potential of cell-penetrating peptides and their mimics for designing antifungal agents has been overlooked. In this concept article, we describe the structural design of synthetic antifungal polymers as mimics of host defense peptides, and highlight the effectiveness and potential of cell-penetrating peptide-inspired strategy in designing potent and selective antifungal polymeric agents. In addition, an outlook for further expanding the design horizons of antifungal polymers is also presented.     

Recombinant Single-Chain Antibody with the Trojan Peptide Penetratin Positioned in the Linker Region Enables Cargo Transfer Across the Blood–Brain Barrier

Delivery of therapeutic proteins into tissues and across the blood–brain barrier (BBB) is limited by the size and biochemical properties of the proteins. Efficient delivery across BBB is generally restricted to small, highly lipophilic molecules. However, in the last decades, several peptides that can pass cell membranes have been identified. It has been shown that these peptides are also capable of delivering large hydrophilic cargoes into cells and are therefore a powerful biological tool for transporting drugs across cell membranes and even into the brain. We designed and prepared a single-chain antibody fragment (scFvs), specific for the pathological form of the prion protein (PrP^Sc), where a cell-penetrating peptide (CPP) was used as a linker between the two variable domains of the scFv. The intravenously administered recombinant scFv-CPP was successfully targeted to and delivered into mouse brain cells. Our single-chain antibody fragments are of special interest in view of possible therapeutic reagents design not only for prion diseases but also for other neurodegenerative diseases.     

Macrocyclic peptides as regulators of protein-protein interactions

Protein-protein interactions are attractive but challenging targets for drug discovery. Recent technological progress and examples using macrocyclic peptides as protein interaction modulators are reviewed.     

Highly efficient (R-X-R)-type carbamates as molecular transporters for cellular delivery

The (R-X-R) motif-containing arginine-rich peptides are among the most effective cell-penetrating peptides. The replacement of amide linkages in the (R-X-R) motif by carbamate linkages as in (r-ahx-r)(4) or (r-ahx-r-r-apr-r)(2) increases the efficacy of such oligomers several-fold. Internalization of these oligomers in mammalian cell lines occurs by an energy-independent process. These oligomers show efficient delivery of biologically active plasmid DNA into CHO-K1 cells.     

Synthesis of an artificial cell surface receptor that enables oligohistidine affinity tags to function as metal-dependent cell-penetrating peptides

Cell-penetrating peptides and proteins (CPPs) are important tools for the delivery of impermeable molecules into living mammalian cells. To enable these cells to internalize proteins fused to common oligohistidine affinity tags, we synthesized an artificial cell surface receptor comprising an N-alkyl derivative of 3beta-cholesterylamine linked to the metal chelator nitrilotriacetic acid (NTA). This synthetic receptor inserts into cellular plasma membranes, projects NTA headgroups from the cell surface, and rapidly cycles between the plasma membrane and intracellular endosomes. Jurkat lymphocytes treated with the synthetic receptor (10 microM) for 1 h displayed approximately 8,400,000 [corrected]NTA groups on the cell surface. Subsequent addition of the green fluorescent protein AcGFP fused to hexahistidine or decahistidine peptides (3 microM) and Ni(OAc)(2) (100 microM) enhanced the endocytosis of AcGFP by 150-fold (hexahistidine fusion protein) or 600-fold (decahistidine fusion protein) within 4 h at 37 degrees C. No adverse effects on cellular proliferation or morphology were observed under these conditions. By enabling common oligohistidine affinity tags to function as cell-penetrating peptides, this metal-chelating cell surface receptor provides a useful tool for studies of cellular biology [corrected]     

Cell-Penetrating Peptides: Correlation between Peptide-Lipid Interaction and Penetration Efficiency

Cell-penetrating peptides are used in the delivery of peptides and biologics, with some cell-penetrating peptides found to be more efficient than others. The exact mechanism of how they interact with the cell membrane and penetrate it, however, remains unclear. This study attempts to investigate the difference in free energy profiles of three cell-penetrating peptides (TAT, CPP1 and CPP9) with a model lipid bilayer (DOPC) using molecular dynamics pulling simulations with umbrella sampling. Potential mean force (PMF) and free energy barrier between the peptides and DOPC are determined using WHAM analysis and MM-PBSA analysis, respectively. CPP9 is found to have the smallest PMF value, followed by CPP1 and TAT, consistent with the experimental data. YDEGE peptide, however, does not give the highest PMF value, although it is a non-cell-permeable peptide. YDEGE is also found to form water pores, alongside with TAT and CPP9, suggesting that it is difficult to distinguish true water pore formation from artefacts arising from pulling simulations. On the contrary, free energy analysis of the peptide-DOPC complex at the lipid-water interface with MM-PBSA provides results consistent with experimental data with CPP9 having the least interaction with DOPC and lowest free energy barrier, followed by CPP1, TAT and YDEGE. These findings suggest that peptide-lipid interaction at the lipid-water interface has a direct correlation with the penetration efficiency of peptides across the lipid bilayer.     

Fluorophore and dye-assisted dispersion of carbon nanotubes in aqueous solution

DNA short oligo, surfactant, peptides, and polymer-assisted dispersion of single-walled carbon nanotube (SWCNTs) in aqueous solution have been intensively studied. It has been suggested that van der Waals interaction, π-π stacking, and hydrophobic interaction are major factors that account for the SWCNTs dispersion. Fluorophore and dye molecules such as Rhodamine B and fluorescein have both hydrophilic and hydrophobic moieties. These molecules also contain π-conjugated systems that can potentially interact with SWCNTs to induce its dispersion. Through a systematic study, here we show that SWCNTs can be dispersed in aqueous solution in the presence of various fluorophore or dye molecules. However, the ability of a fluorophore or dye molecule to disperse SWCNTs is not correlated with the stability of the fluorophore/dye-SWCNT complex, suggesting that the on-rate of fluorophore/dye binding to SWCNTs may dominate the efficiency of this process. We also examined the uptake of fluorophore molecules by mammalian cells when these molecules formed complexes with SWCNTs. The results can have potential applications in the delivery of poor cell-penetrating fluorophore molecules.     

Myotoxin-3 from the Pacific Rattlesnake Crotalus oreganus oreganus Venom Is a New Microtubule-Targeting Agent

Microtubule targeting agents (MTA) are anti-cancer molecules that bind tubulin and interfere with the microtubule functions, eventually leading to cell death. In the present study, we used an in vitro microtubule polymerization assay to screen several venom families for the presence of anti-microtubule activity. We isolated myotoxin-3, a peptide of the crotamine family, and three isoforms from the venom of the Northern Pacific rattlesnake Crotalus oreganus oreganus, which was able to increase tubulin polymerization. Myotoxin-3 turned out to be a cell-penetrating peptide that slightly diminished the viability of U87 glioblastoma and MCF7 breast carcinoma cells. Myotoxin 3 also induced remodeling of the U87 microtubule network and decreased MCF-7 microtubule dynamic instability. These effects are likely due to direct interaction with tubulin. Indeed, we showed that myotoxin-3 binds to tubulin heterodimer with a Kd of 5.3 µM and stoichiometry of two molecules of peptide per tubulin dimer. Our results demonstrate that exogenous peptides are good candidates for developing new MTA and highlight the richness of venoms as a source of pharmacologically active molecules.     

Ligand exchanged photoluminescent gold quantum dots functionalized with leading peptides for nuclear targeting and intracellular imaging

Ligand exchanged gold quantum dots (GQDs) conjugated with cell-penetrating peptides are a new class of photoluminescent probes for nuclear targeting and intracellular imaging.     

Modular synthesis of photocleavable peptides using click chemistry

A modular synthesis of photocleavable peptides was developed. Peptide based kinase substrates were modified on solid support with a traceless linker derived from 1-(2-nitrophenyl)propargyl alcohol and coupled to azide functionalized cell-penetrating peptides using Cu(I) catalyzed click chemistry.     

Self-assembly of a peptide rod-coil: a polyproline rod and a cell-penetrating peptide Tat coil

Peptide rod-coil molecules, composed of a stiff polyproline rod and a hydrophilic cell-penetrating peptide Tat coil, self-assemble into nanocapsules and mediate efficient intracellular delivery of entrapped hydrophilic molecules.     

Aromatic amino acids providing characteristic motifs in the Raman and SERS spectroscopy of peptides

Raman and surface-enhanced Raman spectroscopies (SERS) are potentially important tools in the characterization of biomolecules such as proteins and DNA. In this work, SERS spectra of three cysteine-containing aromatic peptides: tryptophan-cysteine, tyrosine-cysteine, and phenylalanine-cysteine, bound to Au nanoshell substrates, were obtained, and compared to their respective normal Raman spectra. While the linewidths of the SERS peaks are significantly broadened (up to 70%), no significant spectral shifts (<6 cm (-1)) of the major Stokes modes were observed between the two modalities. We show that the Raman and SERS spectra of penetratin, a cell-penetrating peptide oligomer, can be comprised quite reliably from the spectra of its constituent aromatic amino acids except in the backbone regions where the spectral intensities are critically dependent on the length and conformations of the probed molecules. From this study we conclude that, together with protein backbone groups, aromatic amino acid residues provide the overwhelmingly dominant features in the Raman and SERS spectra of peptides and proteins when present. It follows that the Raman modes of these three small constructed peptides may likely apply to the assignment of Raman and SERS features in the spectra of other peptides and proteins.     

Self-assembling mini cell-penetrating peptides enter by both direct translocation and glycosaminoglycan-dependent endocytosis

A small library of cell-penetrating peptides (CPPs) containing a minimized cationic domain and a lipophilic domain of different size was studied. CPPs that could self-assemble were found to enter cells more efficiently, triggering a glycosaminoglycan-dependent pathway.     

Reactive Oxygen Species (ROS) Activated Liposomal Cell Delivery using a Boronate-Caged Guanidine Lipid

We report boronate-caged guanidine-lipid 1 that activates liposomes for cellular delivery only upon uncaging of this compound by reactive oxygen species (ROS) to produce cationic lipid products. These liposomes are designed to mimic the exceptional cell delivery properties of cell-penetrating peptides (CPPs), while the inclusion of the boronate cage is designed to enhance selectivity such that cell entry will only be activated in the presence of ROS. Boronate uncaging by hydrogen peroxide was verified by mass spectrometry and zeta potential (ZP) measurements. A microplate-based fluorescence assay was developed to study the ROS-mediated vesicle interactions between 1 -liposomes and anionic membranes, which were further elucidated via dynamic light scattering (DLS) analysis. Cellular delivery studies utilizing fluorescence microscopy demonstrated significant enhancements in cellular delivery only when 1 -liposomes were incubated with hydrogen peroxide. Our results showcase that lipid 1 exhibits strong potential as an ROS-responsive liposomal platform for targeted drug delivery applications.     

Design of Oligourea-Based Foldamers with Antibacterial and Antifungal Activities

There is an urgent need to develop new therapeutic strategies to fight the emergence of multidrug resistant bacteria. Many antimicrobial peptides (AMPs) have been identified and characterized, but clinical translation has been limited partly due to their structural instability and degradability in physiological environments. The use of unnatural backbones leading to foldamers can generate peptidomimetics with improved properties and conformational stability. We recently reported the successful design of urea-based eukaryotic cell-penetrating foldamers (CPFs). Since cell-penetrating peptides and AMPs generally share many common features, we prepared new sequences derived from CPFs by varying the distribution of histidine- and arginine-type residues at the surface of the oligourea helix, and evaluated their activity on both Gram-positive and Gram-negative bacteria as well as on fungi. In addition, we prepared and tested new amphiphilic block cofoldamers consisting of an oligourea and a peptide segment whereby polar and charged residues are located in the peptide segment and more hydrophobic residues in the oligourea segment. Several foldamer sequences were found to display potent antibacterial activities even in the presence of 50% serum. Importantly, we show that these urea-based foldamers also possess promising antifungal properties.     

Biomedical applications of functional peptides in nano-systems

During the past decades, functionalized nano-systems were believed in holding the bright future of the nanomaterials in biomedical applications. Due to their excellent biocompatibility, biodegradation capability, and biological activity, functional peptides have been vastly used solitary or employed as functional components in nano-systems for disease treatments. This review focuses on the recent advances on the use of functional peptides as a toolbox to construct various delivery nano-systems for tumor treatments. Arising from the special function contributed to the nano-systems, the functional peptides are mainly divided into three groups, cell-targeting peptides (CTPs), cell-penetrating peptides (CPPs), and environment-sensitive peptides. Within each group, their usage in both organic and inorganic systems is discussed. In particular, strategies used to generate promising therapeutic nano-systems for efficient tumor treatment are also highlighted.     

Uptake of Cell-Penetrating Peptide RL2 by Human Lung Cancer Cells: Monitoring by Electron Paramagnetic Resonance and Confocal Laser Scanning Microscopy

RL2 is a recombinant analogue of a human κ-casein fragment, capable of penetrating cells and inducing apoptosis of cancer cells with no toxicity to normal cells. The exact mechanism of RL2 penetration into cells remains unknown. In this study, we investigated the mechanism of RL2 penetration into human lung cancer A549 cells by a combination of electron paramagnetic resonance (EPR) spectroscopy and confocal laser scanning microscopy. EPR spectra of A549 cells incubated with RL2 (sRL2) spin-labeled by a highly stable 3-carboxy-2,2,5,5-tetraethylpyrrolidine-1-oxyl radical were found to contain three components, with their contributions changing with time. The combined EPR and confocal-microscopy data allowed us to assign these three forms of sRL2 to the spin-labeled protein sticking to the membrane of the cell and endosomes, to the spin-labeled protein in the cell interior, and to spin labeled short peptides formed in the cell because of protein digestion. EPR spectroscopy enabled us to follow the kinetics of transformations between different forms of the spin-labeled protein at a minimal spin concentration (3–16 μM) in the cell. The prospects of applications of spin-labeled cell-penetrating peptides to EPR imaging, DNP, and magnetic resonance imaging are discussed, as is possible research on an intrinsically disordered protein in the cell by pulsed dipolar EPR spectroscopy.