Cell-penetrating peptides as delivery vehicles for biology and medicine

Cell-penetrating peptides (CPPs) have found numerous applications in biology and medicine since the first synthetic cell-permeable sequence was identified two decades ago. Numerous types of drugs have been transported into cells using CPPs, including small-molecule pharmaceuticals, therapeutic proteins, and antisense oligonucleotides. Improved agents for medical imaging have been generated by conjugation with CPPs, with the appended peptides promoting cellular uptake and in some cases, cell-type specificity. Organelle-specific CPPs have also been generated, providing a means to target specific subcellular sites. This review highlights achievements in this area and illustrates the numerous examples where peptide chemistry was exploited as a means to provide new tools for biology and medicine.     

Cellular uptake of an α-AApeptide

Some short and cationic peptides such as the Tat peptide can cross the cell membrane and function as vectors for intracellular delivery. Here we show that an α-AApeptide is able to penetrate the membranes of living cells from an extracellular environment and enter the endosome and cytoplasm of cells. The efficiency of the cellular uptake is comparable to a Tat peptide (48-57) of the same length and is unexpectedly superior to an α-peptide with identical functional groups. The mechanism of uptake is similar to that of the Tat peptide and is through endocytosis by an energy-dependent pathway. Due to the easy synthesis of the α-AApeptides, their resistance to proteolytic hydrolysis, and their low cytotoxicity, α-AApeptides represent a new class of transporters for the delivery of drugs.     

Environment-Responsive Peptide Dimers Bind and Stabilize Double-Stranded RNA

Double-stranded RNAs (dsRNA) possess immense potential for biomedical applications. However, their therapeutic utility is limited by low stability and poor cellular uptake. Different strategies have been explored to enhance the stability of dsRNA, including the incorporation of modified nucleotides, and the use of diverse carrier systems. Nevertheless, these have not resulted in a broadly applicable approach thereby preventing the wide-spread application of dsRNA for therapeutic purposes. Herein, we report the design of dimeric stapled peptides based on the RNA-binding protein TAV2b. These dimers are obtained via disulfide formation and mimic the natural TAV2b assembly. They bind and stabilize dsRNA in the presence of serum, protecting it from degradation. In addition, peptide binding also promotes cellular uptake of dsRNA. Importantly, peptide dimers monomerize under reducing conditions which results in a loss of RNA binding. These findings highlight the potential of peptide-based RNA binders for the stabilization and protection of dsRNA, representing an appealing strategy towards the environment-triggered release of RNA. This can broaden the applicability of dsRNA, such as short interfering RNAs (siRNA), for therapeutic applications.     

Activating peptides for cellular uptake via polymerization into high density brushes

The utility of peptide therapeutics is thwarted by an inability to enter cells, preventing access to crucial intracellular targets. Herein, we describe a simple and potentially widely applicable solution involving the polymerization of a minimally modified amino acid sequence into a high density brush polymer. Specifically, non-cell penetrating peptides can be rendered competent for cell entry by first including a single Arg or Lys in their amino acid sequence, if one is not already present, along with a norbornenyl unit. This modified monomer is then polymerized by ring opening metathesis polymerization (ROMP). To demonstrate the utility of this strategy, a known therapeutic peptide, which does not penetrate cells on its own, was polymerized. The resulting polymer proficiently entered cells while maintaining its intracellular function. We anticipate that this methodology will find broad use in medicine, increasing or enabling the in vivo efficacy of promising peptide therapeutics.     

Cellular import mediated by nuclear localization signal Peptide sequences

The cellular delivery of therapeutic agents and their localization within cells is currently a great challenge in medicinal chemistry. A few cationic peptides have shown a strong propensity to cross the cytoplasmic membrane and enter cells. Nuclear localization signal (NLS) sequences are a class of highly cationic peptides that may be exploited for cellular import of linked cargo. A series of NLS sequence peptides were investigated for entry into different cancer cell lines by flow cytometry and confocal microscopy. All NLS peptides demonstrated rapid accumulation within cells when added to the cellular media. Covalent adducts of proteins and oligonucleotides with NLS peptides were also effectively imported within cells. An understanding of the structural and mechanistic properties of these sequences will provide great potential for the rational design of efficient and selective peptidic delivery systems.     

TAT peptide internalization: seeking the mechanism of entry

During the last decade several peptides have been extensively studied for their ability to translocate across the plasma membrane. These peptides have been called "cell penetrating peptides" (CPP) or "protein transduction domains" (PTD). These peptides also promote the cellular uptake of various cargo molecules. Their mechanism of cellular entry appeared very intriguing since most publications in the field highlighted an energy-independent process. Indeed, cellular uptake of these peptides was still observed by fluorescence microscopy at low temperature or in the presence of several drugs known to inhibit active transport. In addition, internalization was reported to be much faster than known endocytic processes. However the involvement of a specific cellular component responsible for this uptake process appeared unlikely following intensive structure activity relationship studies using a wide panel of Tat analogues. Several reports about a possible artefactual redistribution of CPPs, and their associated cargos, during the cell fixation step commonly used for fluorescence microscopy have recently emerged in the literature. Moreover strong ionic interactions of CPPs with the cell surface also led to an overestimation of the recorded cell-associated fluorescent signal. It now seems well established that arginine-rich peptides are internalized by an energy dependent process involving endocytosis. Whatever the case, however, an increasing number of data indicate that the conjugation of non-permeant molecules to these CPPs allows their cellular uptake and leads to the expected biological responses, thus pointing to the interest of this delivery strategy. However, initial structure activity relationship studies of these CPPs will have to be reconsidered and the relative potency of each peptide (and their analogues) to vectorize the cargos to their most appropriate subcellular compartment will require careful re-evaluation.     

Synthesis and cellular uptake of cell delivering PNA-peptide conjugates

The synthesis and cellular uptake of fluorescently labelled PNA-peptide conjugates is described; Dde/Mmt protected PNA monomers, fully orthogonal to Fmoc chemistry, were used to develop a flexible strategy to give Peptide Nucleic Acids conjugated to tri- and hepta-arginine and the short basic Tat(48-57) peptide as examples of cellular penetrating peptides, thereby allowing efficient cellular delivery of PNA into cells.     

Disulfide Click Reaction for Stapling of S-terminal Peptides

A disulfide click strategy is disclosed for stapling to enhance the metabolic stability and cellular permeability of therapeutic peptides. A 17-membered library of stapling reagents with adjustable lengths and angles was established for rapid double/triple click reactions, bridging S-terminal peptides from 3 to 18 amino acid residues to provide 18- to 48-membered macrocyclic peptides under biocompatible conditions. The constrained peptides exhibited enhanced anti-HCT-116 activity with a locked α-helical conformation (IC₅₀=6.81 μM vs. biological incompetence for acyclic linear peptides), which could be unstapled for rehabilitation of the native peptides under the assistance of tris(2-carboxyethyl)phosphine (TCEP). This protocol assembles linear peptides into cyclic peptides controllably to retain the diverse three-dimensional conformations, enabling their cellular uptake followed by release of the disulfides for peptide delivery.     

Cell-specific delivery of a chemotherapeutic to lung cancer cells

We report that lung cancer-targeting peptides isolated from a peptide library can be used to deliver an active chemotherapeutic in a cell-specific fashion. The peptides were removed from the context of the phage and placed on a pegylated tetrameric scaffold. The tetrameric peptides were shown to block uptake of their cognate phage. The tetrameric peptides were coupled to doxorubicin, and their cytotoxicity against a panel of different cell lines was tested. Our data demonstrate that these targeting peptides can deliver an active anticancer agent in a cell-specific fashion, resulting in an increase of the therapeutic index of the targeted drug compared to systemic delivery. The efficacy of the peptide conjugate correlates to the affinity of the targeting peptide for a particular cell line. As such, we have demonstrated that cell-specific targeted drugs can be synthesized, even when the cell surface target is unknown.     

Evaluation of diverse peptidyl motifs for cellular delivery of semiconductor quantum dots

Cell-penetrating peptides (CPPs) have rapidly become a mainstay technology for facilitating the delivery of a wide variety of nanomaterials to cells and tissues. Currently, the library of CPPs to choose from is still limited, with the HIV TAT-derived motif still being the most used. Among the many materials routinely delivered by CPPs, nanoparticles are of particular interest for a plethora of labeling, imaging, sensing, diagnostic, and therapeutic applications. The development of nanoparticle-based technologies for many of these uses will require access to a much larger number of functional peptide motifs that can both facilitate cellular delivery of different types of nanoparticles to cells and be used interchangeably in the presence of other peptides and proteins on the same surface. Here, we evaluate the utility of four peptidyl motifs for their ability to facilitate delivery of luminescent semiconductor quantum dots (QDs) in a model cell culture system. We find that an LAH4 motif, derived from a membrane-inserting antimicrobial peptide, and a chimeric sequence that combines a sweet arrow peptide with a portion originating from the superoxide dismutase enzyme provide effective cellular delivery of QDs. Interestingly, a derivative of the latter sequence lacking just a methyl group was found to be quite inefficient, suggesting that even small changes can have significant functional outcomes. Delivery was effected using 1 h incubation with cells, and fluorescent counterstaining strongly suggests an endosomal uptake process that requires a critical minimum number or ratio of peptides to be displayed on the QD surface. Concomitant cytoviability testing showed that the QD–peptide conjugates are minimally cytotoxic in the model COS-1 cell line tested. Potential applications of these peptides in the context of cellular delivery of nanoparticles and a variety of other (bio)molecules are discussed.

Stapled Peptides as HIF-1α/p300 Inhibitors: Helicity Enhancement in the Bound State Increases Inhibitory Potency

Protein–protein interactions (PPIs) control virtually all cellular processes and have thus emerged as potential targets for development of molecular therapeutics. Peptide-based inhibitors of PPIs are attractive given that they offer recognition potency and selectivity features that are ideal for function, yet, they do not predominantly populate the bioactive conformation, frequently suffer from poor cellular uptake and are easily degraded, for example, by proteases. The constraint of peptides in a bioactive conformation has emerged as a promising strategy to mitigate against these liabilities. In this work, using peptides derived from hypoxia-inducible factor 1 (HIF-1α) together with dibromomaleimide stapling, we identify constrained peptide inhibitors of the HIF-1α/p300 interaction that are more potent than their unconstrained sequences. Contrary to expectation, the increased potency does not correlate with an increased population of an α-helical conformation in the unbound state as demonstrated by experimental circular dichroism analysis. Rather, the ability of the peptide to adopt a bioactive α-helical conformation in the p300 bound state is better supported in the constrained variant as demonstrated by molecular dynamics simulations and circular dichroism difference spectra.     

Design of Multicomponent Peptide Fibrils with Ordered and Programmable Compositional Patterns

Advanced applications of biomacromolecular assemblies require a stringent degree of control over molecular arrangement, which is a challenge to current synthetic methods. Here we used a neighbor-controlled patterning strategy to build multicomponent peptide fibrils with an unprecedented capacity to manipulate local composition and peptide positions. Eight peptides were designed to have regulable nearest neighbors upon co-assembly, which, by simulation, afforded 412 different patterns within fibrils, with varied compositions and/or peptide positions. The fibrils with six prescribed patterns were experimentally constructed with high accuracy. The controlled patterning also applies to functionalities appended to the peptides, as exemplified by arranging carbohydrate ligands at nanoscale precision for protein recognition. This study offers a route to molecular editing of inner structures of peptide assemblies, prefiguring the uniqueness and richness of patterning-based material design.     

Molecular transporters for peptides: delivery of a cardioprotective epsilonPKC agonist peptide into cells and intact ischemic heart using a transport system, R(7).

BACKGROUND: Recently, we reported a novel oligoguanidine transporter system, polyarginine (R(7)), which, when conjugated to spectroscopic probes (e.g., fluorescein) and drugs (e.g., cyclosporin A), results in highly water-soluble conjugates that rapidly enter cells and tissues. We report herein the preparation of the first R(7) peptide conjugates and a study of their cellular and organ uptake and functional activity. The octapeptide (psi)(epsilon)RACK was selected for this study as it is known to exhibit selective epsilon protein kinase C isozyme agonist activity and to reduce ischemia-induced damage in cardiomyocytes. However, (psi)(epsilon)RACK is not cell-permeable. RESULTS: Here we show that an R(7)-(psi)(epsilon)RACK conjugate readily enters cardiomyocytes, significantly outperforming (psi)(epsilon)RACK conjugates of the transporters derived from HIV Tat and from Antennapedia. Moreover, R(7)-(psi)(epsilon)RACK conjugate reduced ischemic damage when delivered into intact hearts either prior to or after the ischemic insult. CONCLUSIONS: Our data suggest that R(7) converts a peptide lead into a potential therapeutic agent for the ischemic heart.     

Engineered Histidine-Rich Peptides Enhance Endosomal Escape for Antibody-Targeted Intracellular Delivery of Functional Proteins

Currently, the clinical application of protein/peptide therapeutics is mainly limited to the modulation of diseases in extracellular spaces. Intracellular targets are hardly accessed, owing largely to the endosomal entrapment of internalized proteins/peptides. Here, we report a strategy to design and construct peptides that enable endosome-to-cytosol delivery based on an extension of the “histidine switch” principle. By substituting the Arg/Lys residues in cationic cell-penetrating peptides (CPPs) with histidine, we obtained peptides with pH-dependent membrane-perturbation activity. These peptides do not randomly penetrate cells like CPPs, but imitate the endosomal escape of CPPs following cellular uptake. Working with one such 16-residue peptide (hsLMWP) with high endosomal escape capacity, we engineered modular fusion proteins and achieved antibody-targeted delivery of diverse protein cargoes—including the pro-apoptotic protein BID (BH3-interacting domain death agonist) and Cre recombinase—into the cytosol of multiple cancer cell types. After extensive in vitro testing, an in vivo analysis with xenograft mice ultimately demonstrated that a trastuzumab-hsLMWP-BID fusion conferred strong anti-tumor efficacy without apparent side effects. Notably, our fusion protein features a modular design, allowing flexible applications for any antibody/cargo combination of choice. Therefore, the potential applications extend throughout life science and biomedicine, including gene editing, cancer treatment, and immunotherapy.     

Cellular uptake mechanisms and endosomal trafficking of supercharged proteins

Supercharged proteins (SCPs) can deliver functional macromolecules into the cytoplasm of mammalian cells more potently than unstructured cationic peptides. Thus far, neither the structural features of SCPs that determine their delivery effectiveness nor their intracellular fate postendocytosis, has been studied. Using a large set of supercharged GFP (scGFP) variants, we found that the level of cellular uptake is sigmoidally related to net charge and that scGFPs enter cells through multiple pathways, including clathrin-dependent endocytosis and macropinocytosis. SCPs activate Rho and ERK1/2 and also alter the endocytosis of transferrin and EGF. Finally, we discovered that the intracellular trafficking of endosomes containing scGFPs is altered in a manner that correlates with protein delivery potency. Collectively, our findings establish basic structure-activity relationships of SCPs and implicate the modulation of endosomal trafficking as a determinant of macromolecule delivery efficiency.     

净电荷对螺旋型抗癌肽生物活性的影响

以高活性两亲性α-螺旋型阳离子抗癌肽A12L/A20L(多肽P)为模板, 在其亲水面进行氨基酸定点取代, 获得了一系列带有不同净电荷的多肽类似物, 研究了净电荷对螺旋型抗癌肽生物活性的影响. 结果表明, 抗癌肽净电荷的改变对其溶血活性影响较小(最大差异为2倍), 而对抗癌活性和选择性的影响显著(最大差异为10倍). 抗癌肽P的净电荷最适范围为+7到+8, 分子间静电排斥作用的最佳数目为3~5个, 高于或低于此范围, 其抗癌活性和选择性均明显降低. 与人的正常细胞相比, 负电性的癌细胞膜对于抗癌肽的净电荷变化更敏感, 表明两亲性螺旋型抗癌肽针对癌细胞与正常细胞表现出良好的选择特异性.     

Calcium liberates PNAs from endosomes

Many peptides are reported to enhance cellular uptake of peptide nucleic acids and other macromolecules. Cellular uptake, however, is not synonymous with cellular activity. In this issue of Chemistry and Biology, Nielsen and colleagues examine the traffic of PNAs and investigate protocols for improving recognition of target mRNA inside cells.     

Cellular uptake of aminoglycosides, guanidinoglycosides, and poly-arginine

Aminoglycosides (including neomycin B and tobramycin) exhibit poor uptake by eukaryotic cell lines. When the amines of these natural products are converted into guanidine groups, their cellular uptake is dramatically enhanced. We have synthesized BODIPY-containing aminoglycosides and guanidinoglycosides to evaluate their cellular uptake properties. Fluorescence activated cell sorting (FACS) and fluorescence microscopy are used to compare the membrane translocation and the cellular localization of these compounds. Upon guanidinylation, the cellular uptake efficiencies of tobramycin and neomycin B are enhanced by 10-fold and 20-fold, respectively. Guanidino-neomycin B exhibits a highly efficient uptake, superior to a fluorescent poly-arginine peptide. Interestingly, the cellular uptake of this common transduction peptide is inhibited by guanidine-neomycin B, suggesting a similar uptake mechanism for both the arginine-rich peptides and the guanidinoglycosides.     

Bioactive Peptide Brush Polymers via Photoinduced Reversible‐Deactivation Radical Polymerization

Harnessing metal‐free photoinduced reversible‐deactivation radical polymerization (photo‐RDRP) in organic and aqueous phases, we report a synthetic approach to enzyme‐responsive and pro‐apoptotic peptide brush polymers. Thermolysin‐responsive peptide‐based polymeric amphiphiles assembled into spherical micellar nanoparticles that undergo a morphology transition to worm‐like micelles upon enzyme‐triggered cleavage of coronal peptide sidechains. Moreover, pro‐apoptotic polypeptide brushes show enhanced cell uptake over individual peptide chains of the same sequence, resulting in a significant increase in cytotoxicity to cancer cells. Critically, increased grafting density of pro‐apoptotic peptides on brush polymers correlates with increased uptake efficiency and concurrently, cytotoxicity. The mild synthetic conditions afforded by photo‐RDRP, make it possible to access well‐defined peptide‐based polymer bioconjugate structures with tunable bioactivity.     

The relative charge ratio between C and N atoms in amide bond acts as a key factor to determine peptide fragment efficiency in different charge states

The influence of charge state on the peptide dissociation behavior in tandem mass spectrometry (MS/MS) is worthy of discussion. Comparative studies of singly- and doubly-protonated peptide molecules are performed to explore the effect and mechanism of charge state on peptide fragmentation. In view of the charge-directed cleavage of protonated peptides described in the mobile proton model, radiolytic oxidation was applied to change the charge distribution of peptides but retain the sequence. Experimental studies of collision energy-dependent fragmentation efficiencies coupled with quantum chemical calculations indicated that the cleavage of ARRA and its side-chain oxidation products with oxygen atoms added followed a trend that doubly-protonated peptides fragment more easily than singly-protonated forms, while the oxidation product with the guanidine group deleted showed the opposite trend. By analyzing the charge distribution around the amide bonds, we found that the relative charge ratios between C and N atoms (Q_C/Q_N) in the amide bonds provided a reasonable explanation for peptide fragmentation efficiencies. An increase of the Q_C/Q_N value of the amide bond means that a peptide fragments more easily, and vice versa. The results described in this paper provide an experimental and calculation strategy for predicting peptide fragmentation efficiency.