VDAC1-Based Peptides as Potential Modulators of VDAC1 Interactions with Its Partners and as a Therapeutic for Cancer, NASH, and Diabetes.

This review presents current knowledge related to the voltage-dependent anion channel-1 (VDAC1) as a multi-functional mitochondrial protein that acts in regulating both cell life and death. The location of VDAC1 at the outer mitochondrial membrane (OMM) allows control of metabolic cross-talk between the mitochondria and the rest of the cell, and also enables its interaction with proteins that are involved in metabolic, cell death, and survival pathways. VDAC1's interactions with over 150 proteins can mediate and regulate the integration of mitochondrial functions with cellular activities. To target these protein-protein interactions, VDAC1-derived peptides have been developed. This review focuses specifically on cell-penetrating VDAC1-based peptides that were developed and used as a "decoy" to compete with VDAC1 for its VDAC1-interacting proteins. These peptides interfere with VDAC1 interactions, for example, with metabolism-associated proteins such as hexokinase (HK), or with anti-apoptotic proteins such as Bcl-2 and Bcl-xL. These and other VDAC1-interacting proteins are highly expressed in many cancers. The VDAC1-based peptides in cells in culture selectively affect cancerous, but not non-cancerous cells, inducing cell death in a variety of cancers, regardless of the cancer origin or genetics. They inhibit cell energy production, eliminate cancer stem cells, and act very rapidly and at low micro-molar concentrations. The activity of these peptides has been validated in several mouse cancer models of glioblastoma, lung, and breast cancers. Their anti-cancer activity involves a multi-pronged attack targeting the hallmarks of cancer. They were also found to be effective in treating non-alcoholic fatty liver disease and diabetes mellitus. Thus, VDAC1-based peptides, by targeting VDAC1-interacting proteins, offer an affordable and innovative new conceptual therapeutic paradigm that can potentially overcome heterogeneity, chemoresistance, and invasive metastatic formation.

Peptide-Mediated Small Molecule Lysosome-Targeting Chimeras for Targeted Degradation of Membrane and Intracellular Proteins.

Targeted protein degradation through the lysosomal pathway has attracted increasing attention and expanded the scope of degradable proteins. However, the endogenous lysosomal degradation strategies are mainly based on antibodies or nanobodies. Effective small molecule lysosomal degraders are still rather rare. Herein, a new lysosomal degradation approach, termed peptide-mediated small molecule lysosome-targeting chimeras (PSMLTACs), was developed by the incorporation of small molecule ligands with a lysosome-sorting NPGY motif containing the cell-penetrating peptide. PSMLTACs were successfully applied to degrade both membrane and intracellular targets. In particular, the PSMLTAC strategy demonstrated higher degradation efficiency on membrane target PD-L1 and intracellular target PDEδ than corresponding PROTAC degraders. Taken together, this proof-of-concept provides a convenient and effective strategy for targeted protein degradation.

Molecular Modeling of Single- and Double-Hydrocarbon-Stapled Coiled-Coil Inhibitors against Bcr-Abl: Toward a Treatment Strategy for CML.

The chimeric oncoprotein Bcr-Abl is the causative agent of virtually all chronic myeloid leukemias and a subset of acute lymphoblastic leukemias. As a result of the so-called Philadelphia chromosome translocation t(9;22), Bcr-Abl manifests as a constitutively active tyrosine kinase, which promotes leukemogenesis by activation of cell cycle signaling pathways. Constitutive and oncogenic activation is mediated by an N-terminal coiled-coil oligomerization domain in Bcr (Bcr-CC), presenting a therapeutic target for inhibition of Bcr-Abl activity toward the treatment of Bcr-Abl+ leukemias. Previously, we demonstrated that a rationally designed Bcr-CC mutant, CCmut3, exerts a dominant negative effect upon Bcr-Abl activity by preferential oligomerization with Bcr-CC. Moreover, we have shown that conjugation to a leukemia-specific cell-penetrating peptide (CPP-CCmut3) improves intracellular delivery and activity. However, our full-length CPP-CCmut3 construct (81 aa) is encumbered by an intrinsically high degree of conformational variability and susceptibility to proteolytic degradation relative to traditional small-molecule therapeutics. Here, we iterate a new generation of CCmut3 inhibitors against Bcr-CC-mediated Bcr-Abl assembly designed to address these constraints through incorporation of all-hydrocarbon staples spanning i and i + 7 positions in α-helix 2 (CPP-CCmut3-st). We utilize computational modeling and biomolecular simulation to evaluate single- and double-stapled CCmut3 candidates in silico for dynamics and binding energetics. We further model a truncated system characterized by the deletion of α-helix 1 and the flexible loop linker, which are known to impart high conformational variability. To study the impact of the N-terminal cyclic CPP toward model stability and inhibitor activity, we also model the full-length and truncated systems devoid of the CPP, with a cyclized CPP, and with an open-configuration CPP, for a total of six systems that comprise our library. From this library, we present lead-stapled peptide candidates to be synthesized and evaluated experimentally as our next iteration of inhibitors against Bcr-Abl.

An approach for the intracellular delivery of IgG via enzymatic ligation with a cell-permeable attenuated cationic amphiphilic lytic peptide.

Achieving effective intracellular delivery of therapeutic molecules such as antibodies (IgG) is a challenge in biomedical research and pharmaceutical development. Conjugation of IgG with a cell-penetrating peptide is a rational approach. Here, not only the efficacy of the conjugates in internalizing into cells, but also the physicochemical property of the conjugates allowing their solubilized states in solution without forming aggregates are critical. In this study, we have shown that the first requirement can be addressed using a cell-permeable attenuated cationic amphiphilic lytic (CP-ACAL) peptide, L17ER4. The second requirement can be addressed by ligation of IgG to L17ER4 using sortase A, where the use of a linker of appropriate chain length is also important. For evaluation, the intracellular delivery efficacy was studied using conjugate structures with different orientations and conjugation modes of L17ER4 in ligation to a model protein, green fluorescent protein fused to a nuclear localization signal (NLS-EGFP). The effect of tetraarginine positioning in the L17ER4 sequence was also investigated. Following these studies, an optimized peptide sequence containing L17ER4 was ligated to an anti-green fluorescent protein (GFP) IgG bearing a sortase A recognition sequence. Treatment of the cells with the conjugate of anti-GFP IgG and L17ER4 resulted in a high efficiency of cytosolic translocation of the conjugate and the binding to the target protein in the cell without significant aggregate formation. The feasibility of the d-form of L17ER4 as a CP-ACAL was also confirmed.

Carbon dots fluorescence can be used to distinguish isobaric peptide and to monitor protein oligomerization dynamics.

Carbon dots (CD) are widely investigated particles with interesting fluorescent properties which are reported to be used for various purposes, as they are biocompatible, resistant to photobleaching and with tuneable properties depending on the specific CD surface chemistry. In this work, we report on the possibility to use opportunely designed CD to distinguish among isobaric peptides almost undistinguishable by mass spectrometry, as well as to monitor protein aggregation phenomena. Particularly, cell-penetrating peptides containing the carnosine moiety at different positions in the peptide chain produce sequence specific fluorescent signals. Analogously, different insulin oligomerization states can also be distinguished by the newly proposed experimental approach. The latter is here described in details and can be potentially applied to any kind of peptide or protein.

Comparing Cell Penetration of Biotherapeutics across Human Cell Lines.

A major obstacle in biotherapeutics development is maximizing cell penetration. Ideally, assays would allow for optimization of cell penetration in the cell type of interest early in the drug development process. However, few assays exist to compare cell penetration across different cell types independent of drug function. In this work, we applied the chloroalkane penetration assay (CAPA) in seven mammalian cell lines as well as primary cells. Careful controls were used to ensure that data could be compared across cell lines. We compared the nuclear penetration of several peptides and drug-like oligonucleotides and saw significant differences among the cell lines. To help explain these differences, we quantified the relative activities of endocytosis pathways in these cell lines and correlated them with the penetration data. Based on these results, we knocked down clathrin in a cell line with an efficient permeability profile and observed reduced penetration of peptides but not oligonucleotides. Finally, we used small-molecule endosomal escape enhancers and observed enhancement of cell penetration of some oligonucleotides, but only in some of the cell lines tested. CAPA data provide valuable points of comparison among different cell lines, including primary cells, for evaluating the cell penetration of various classes of peptides and oligonucleotides.

Tweaking the NRF2 signaling cascade in human myelogenous leukemia cells by artificial nano-organelles.

NRF2 (nuclear factor erythroid-2-related factor 2) is a key regulator of genes involved in the cell's protective response to oxidative stress. Upon activation by disturbed redox homeostasis, NRF2 promotes the expression of metabolic enzymes to eliminate reactive oxygen species (ROS). Cell internalization of peroxisome-like artificial organelles that harbor redox-regulating enzymes was previously shown to reduce ROS-induced stress and thus cell death. However, if and to which extent ROS degradation by such nanocompartments interferes with redox signaling pathways is largely unknown. Here, we advance the design of H2O2-degrading artificial nano-organelles (AnOs) that exposed surface-attached cell penetrating peptides (CPP) for enhanced uptake and were equipped with a fluorescent moiety for rapid visualization within cells. To investigate how such AnOs integrate in cellular redox signaling, we engineered leukemic K562 cells that report on NRF2 activation by increased mCherry expression. Once internalized, ROS-metabolizing AnOs dampen intracellular NRF2 signaling upon oxidative injury by degrading H2O2. Moreover, intracellular AnOs conferred protection against ROSinduced cell death in conditions when endogenous ROS-protection mechanisms have been compromised by depletion of glutathione or knockdown of NRF2. We demonstrate CPP-facilitated AnO uptake and AnO-mediated protection against ROS insults also in the T lymphocyte population of primary peripheral blood mononuclear cells from healthy donors. Overall, our data suggest that intracellular AnOs alleviated cellular stress by the on-site reduction of ROS.

Delivery of AKT1 phospho-forms to human cells reveals differential substrate selectivity.

Protein kinase B (AKT1) is a serine/threonine kinase that regulates fundamental cellular processes, including cell survival, proliferation, and metabolism. AKT1 activity is controlled by two regulatory phosphorylation sites (Thr308, Ser473) that stimulate a downstream signaling cascade through phosphorylation of many target proteins. At either or both regulatory sites, hyperphosphorylation is associated with poor survival outcomes in many human cancers. Our previous biochemical and chemoproteomic studies showed that the phosphorylated forms of AKT1 have differential selectivity toward peptide substrates. Here, we investigated AKT1-dependent activity in human cells, using a cell-penetrating peptide (transactivator of transcription, TAT) to deliver inactive AKT1 or active phospho-variants to cells. We used enzyme engineering and genetic code expansion relying on a phosphoseryl-transfer RNA (tRNA) synthetase (SepRS) and tRNASep pair to produce TAT-tagged AKT1 with programmed phosphorylation at one or both key regulatory sites. We found that all TAT-tagged AKT1 variants were efficiently delivered into human embryonic kidney (HEK 293T) cells and that only the phosphorylated AKT1 (pAKT1) variants stimulated downstream signaling. All TAT-pAKT1 variants induced glycogen synthase kinase (GSK)-3α phosphorylation, as well as phosphorylation of ribosomal protein S6 at Ser240/244, demonstrating stimulation of downstream AKT1 signaling. Fascinatingly, only the AKT1 variants phosphorylated at S473 (TAT-pAKT1S473 or TAT-pAKT1T308,S473) were able to increase phospho-GSK-3ß levels. Although each TAT-pAKT1 variant significantly stimulated cell proliferation, cells transduced with TAT-pAKT1T308 grew significantly faster than with the other pAKT1 variants. The data demonstrate differential activity of the AKT1 phospho-forms in modulating downstream signaling and proliferation in human cells.

Revealing the dynamic mechanism of cell-penetrating peptides across cell membranes at the single-molecule level.

Cell-penetrating peptides (CPPs) have gained prominence in cellular drug delivery due to their extremely low toxicity and rapid cell internalization properties. Understanding the effect of CPPs' physicochemical properties on trans-membrane behavior will provide a better screening scheme for designing effective CPP-conjugated nano-drugs. Herein, the efficiency of the CPPs interacting with the cell membrane and the subsequent trans-membrane is revealed at the single-molecule level using single-molecule force spectroscopy (SMFS) and force tracing technique based on atomic force spectroscopy (AFM). The dynamic force spectroscopy (DFS) analysis indicates that cationic TAT48-60 and amphipathic MAP are more effective during the interaction with cell membrane due to the strong electrostatic interaction between CPPs and cell membrane. However, for the subsequent trans-membrane process, the hydrophobicity of Pep-7 plays a key role, showing a higher trans-membrane speed at the single-molecule level. Meanwhile, Pep-7 shows lower trans-membrane speed and probability on normal cells (Vero), which makes it more suitable as a peptide-based nano-drug to treat tumors avoiding harming normal cells. The dynamic parameters obtained in this study offer guidance for screening and modifying effective CPPs, targeting specific cell lines or tissues during the nano-drug design.

Investigating the impact of thiol reactivity and disulfide formation on cellular uptake of cell-permeable peptides.

Cell-penetrating peptides (CPPs) have been explored as versatile tools to transport various molecules into cells. The uptake mechanism of CPPs is still not clearly understood and most probably depends on several factors like the nature of the CPP itself, the attached cargo, the investigated cell system, and other experimental conditions, such as temperature and concentration. One of the first steps of internalization involves the interaction of CPPs with negatively charged molecules present at the outer layer of the cell membrane. Recently, thiol-mediated uptake has been found to support the effective translocation of sulfhydryl-bearing substances that would actually not be cell-permeable. Within this work, we aimed to understand the relevance of thiol reactivity for the uptake mechanism of cysteine-containing CPPs that we have developed previously in our group. Therefore, we compared the two peptides, sC18-Cys and CaaX-1, in their single reduced and dimeric disulfide versions. Cytotoxicity, intracellular accumulation, and impact on the internalization process of the disulfides were investigated in HeLa cells. Both disulfide CPPs demonstrated significantly stronger cytotoxic effects and membrane activity compared with their reduced counterparts. Notably, thiol-mediated uptake could be excluded as a main driver for translocation, showing that peptides like CaaX-1 are most likely taken up by other mechanisms.

N-terminal modification of an LAH4-derived peptide increases mRNA delivery in the presence of serum.

The recently developed mRNA-based coronavirus SARS-CoV-2 vaccines highlighted the great therapeutic potential of the mRNA technology. Although the lipid nanoparticles used for the delivery of the mRNA are very efficient, they showed, in some cases, the induction of side effects as well as the production of antibodies directed against particle components. Thus, the development of alternative delivery systems is of great interest in the pursuit of more effective mRNA treatments. In the present work, we evaluated the mRNA transfection capacities of a series of cationic histidine-rich amphipathic peptides derived from LAH4. We found that while the LAH4-A1 peptide was an efficient carrier for mRNA, its activity was highly serum sensitive. Interestingly, modification of this cell penetrating peptide at the N-terminus with two tyrosines or with salicylic acid allowed to confer serum resistance to the carrier.

Cellular Uptake of Cell-Penetrating Peptides Activated by Amphiphilic p-Sulfonatocalix[4]arenes.

We report the synthesis of a series of amphiphilic p-sulfonatocalix[4]arenes with varying alkyl chain lengths (CX4-Cn) and their application as efficient counterion activators for membrane transport of cell-penetrating peptides (CPPs). The enhanced membrane activity is confirmed with the carboxyfluorescein (CF) assay in vesicles and by the direct cytosolic delivery of CPPs into CHO-K1, HCT 116, and KTC-1 cells enabling excellent cellular uptake of the CPPs into two cancer cell lines. Intracellular delivery was confirmed by fluorescence microscopy after CPP entry into live cells mediated by CX4-Cn, which was also quantified after cell lysis by fluorescence spectroscopy. The results present the first systematic exploration of structure-activity relationships for calixarene-based counterion activators and show that CX4-Cn are exceptionally effective in cellular delivery of CPPs. The dodecyl derivative, CX4-C12, serves as best activator. A first mechanistic insight is provided by efficient CPP uptake at 4 °C and in the presence of the endocytosis inhibitor dynasore, which indicates a direct translocation of the CPP-counterion complexes into the cytosol and highlights the potential benefits of CX4-Cn for efficient and direct translocation of CPPs and CPP-conjugated cargo molecules into the cytosol of live cells.

Delivery to, and Reactivation of, the p53 Pathway in Cancer Cells Using a Grafted Cyclotide Conjugated with a Cell-Penetrating Peptide.

Peptides are promising drug modalities that can modulate protein-protein interactions, but their application is hampered by their limited ability to reach intracellular targets. Here, we improved the cytosolic delivery of a peptide blocking p53:MDM2/X interactions using a cyclotide as a stabilizing scaffold. We applied several design strategies to improve intracellular delivery and found that the conjugation of the lead cyclotide to the cyclic cell-penetrating peptide cR10 was the most effective. Conjugation allowed cell internalization at micromolar concentration and led to elevated intracellular p53 levels in A549, MCF7, and MCF10A cells, as well as inducing apoptosis in A549 cells without causing membrane disruption. The lead peptide had >35-fold improvement in inhibitory activity and increased cellular uptake compared to a previously reported cyclotide p53 activator. In summary, we demonstrated the delivery of a large polar cyclic peptide in the cytosol and confirmed its ability to modulate intracellular protein-protein interactions involved in cancer.

Biological importance of arginine: A comprehensive review of the roles in structure, disorder, and functionality of peptides and proteins.

Arginine shows Jekyll and Hyde behavior in several respects. It participates in protein folding via ionic and H-bonds and cation-pi interactions; the charge and hydrophobicity of its side chain make it a disorder-promoting amino acid. Its methylation in histones; RNA binding proteins; chaperones regulates several cellular processes. The arginine-centric modifications are important in oncogenesis and as biomarkers in several cardiovascular diseases. The cross-links involving arginine in collagen and cornea are involved in pathogenesis of tissues but have also been useful in tissue engineering and wound-dressing materials. Arginine is a part of active site of several enzymes such as GTPases, peroxidases, and sulfotransferases. Its metabolic importance is obvious as it is involved in production of urea, NO, ornithine and citrulline. It can form unusual functional structures such as molecular tweezers in vitro and sprockets which engage DNA chains as part of histones in vivo. It has been used in design of cell-penetrating peptides as drugs. Arginine has been used as an excipient in both solid and injectable drug formulations; its role in suppressing opalescence due to liquid-liquid phase separation is particularly very promising. It has been known as a suppressor of protein aggregation during protein refolding. It has proved its usefulness in protein bioseparation processes like ion-exchange, hydrophobic and affinity chromatographies. Arginine is an amino acid, whose importance in biological sciences and biotechnology continues to grow in diverse ways.

Cell-Penetrating Peptides as Vehicles for Delivery of Therapeutic Nucleic Acids. Mechanisms and Application in Medicine.

Currently, nucleic acid therapeutics are actively developed for the treatment and prophylactic of metabolic disorders and oncological, inflammatory, and infectious diseases. A growing number of approved nucleic acid-based drugs evidences a high potential of gene therapy in medicine. Therapeutic nucleic acids act in the cytoplasm, which makes the plasma membrane the main barrier for the penetration of nucleic acid-based drugs into the cell and requires development of special vehicles for their intracellular delivery. The optimal carrier should not only facilitate internalization of nucleic acids, but also exhibit no toxic effects, ensure stabilization of the cargo molecules, and be suitable for a large-scale and low-cost production. Cell-penetrating peptides (CPPs), which match all these requirements, were found to be efficient and low-toxic carriers of nucleic acids. CPPs are typically basic peptides with a positive charge at physiological pH that can form nanostructures with negatively charged nucleic acids. The prospects of CPPs as vehicles for the delivery of therapeutic nucleic acids have been demonstrated in numerous preclinical studies. Some CPP-based drugs had successfully passed clinical trials and were implemented into medical practice. In this review, we described different types of therapeutic nucleic acids and summarized the data on the use of CPPs for their intracellular delivery, as well as discussed, the mechanisms of CPP uptake by the cells, as understanding of these mechanisms can significantly accelerate the development of new gene therapy approaches.

Cardiac-Targeting Peptide: From Discovery to Applications.

Despite significant strides in prevention, diagnosis, and treatment, cardiovascular diseases remain the number one cause of mortality in the United States, with rates climbing at an alarming rate in the developing world. Targeted delivery of therapeutics to the heart has been a lofty goal to achieve with strategies ranging from direct intra-cardiac or intra-pericardial delivery, intra-coronary infusion, to adenoviral, lentiviral, and adeno-associated viral vectors which have preference, if not complete cardio-selectivity, for cardiac tissue. Cell-penetrating peptides (CPP) are 5-30-amino-acid-long peptides that are able to breach cell membrane barriers while carrying cargoes up to several times their size, in an intact functional form. Identified nearly three decades ago, the first of these CPPs came from the HIV coat protein transactivator of transcription. Although a highly efficient CPP, its clinical utility is limited by its robust ability to cross any cell membrane barrier, including crossing the blood-brain barrier and transducing neuronal tissue non-specifically. Several strategies have been utilized to identify cell- or tissue-specific CPPs, one of which is phage display. Using this latter technique, we identified a cardiomyocyte-targeting peptide (CTP) more than a decade ago, a finding that has been corroborated by several independent labs across the world that have utilized CTP for a myriad of different purposes in pre-clinical animal models. The goal of this publication is to provide a comprehensive review of the identification, validation, and application of CTP, and outline its potential in diagnostic and therapeutic applications especially in the field of targeted RNA interference.

Dual-Activity Fluoroquinolone-Transportan 10 Conjugates Offer Alternative Leukemia Therapy during Hematopoietic Cell Transplantation.

Hematopoietic cell transplantation (HCT) is often considered a last resort leukemia treatment, fraught with limited success due to microbial infections, a leading cause of mortality in leukemia patients. To address this critical issue, we explored a novel approach by synthesizing antileukemic agents containing antibacterial substances. This innovative strategy involves conjugating fluoroquinolone antibiotics, such as ciprofloxacin (CIP) or levofloxacin (LVX), with the cell-penetrating peptide transportan 10 (TP10). Here, we demonstrate that the resultant compounds display promising biologic activities in preclinical studies. These novel conjugates not only exhibit potent antimicrobial effects but are also selective against leukemia cells. The cytotoxic mechanism involves rapid disruption of cell membrane asymmetry leading to membrane damage. Importantly, these conjugates penetrated mammalian cells, accumulating within the nuclear membrane without significant effect on cellular architecture or mitochondrial function. Molecular simulations elucidated the aggregation tendencies of TP10 conjugates within lipid bilayers, resulting in membrane disruption and permeabilization. Moreover, mass spectrometry analysis confirmed efficient reduction of disulfide bonds within TP10 conjugates, facilitating release and activation of the fluoroquinolone derivatives. Intriguingly, these compounds inhibited human topoisomerases, setting them apart from traditional fluoroquinolones. Remarkably, TP10 conjugates generated lower intracellular levels of reactive oxygen species compared with CIP and LVX. The combination of antibacterial and antileukemic properties, coupled with selective cytostatic effects and minimal toxicity toward healthy cells, positions TP10 derivatives as promising candidates for innovative therapeutic approaches in the context of antileukemic HCT. This study highlights their potential in search of more effective leukemia treatments. SIGNIFICANCE STATEMENT: Fluoroquinolones are commonly used antibiotics, while transportan 10 (TP10) is a cell-penetrating peptide (CPP) with anticancer properties. In HCT, microbial infections are the primary cause of illness and death. Combining TP10 with fluoroquinolones enhanced their effects on different cell types. The dual pharmacological action of these conjugates offers a promising proof-of-concept solution for leukemic patients undergoing HCT. Strategically designed therapeutics, incorporating CPPs with antibacterial properties, have the potential to reduce microbial infections in the treatment of malignancies.

Delivered complementation in planta (DCIP) enables measurement of peptide-mediated protein delivery efficiency in plants.

Using a fluorescence complementation assay, Delivered Complementation in Planta (DCIP), we demonstrate cell-penetrating peptide-mediated cytosolic delivery of peptides and recombinant proteins in Nicotiana benthamiana. We show that DCIP enables quantitative measurement of protein delivery efficiency and enables functional screening of cell-penetrating peptides for in-planta protein delivery. Finally, we demonstrate that DCIP detects cell-penetrating peptide-mediated delivery of recombinantly expressed proteins such as mCherry and Lifeact into intact leaves. We also demonstrate delivery of a recombinant plant transcription factor, WUSCHEL (AtWUS), into N. benthamiana. RT-qPCR analysis of AtWUS delivery in Arabidopsis seedlings also suggests delivered WUS can recapitulate transcriptional changes induced by overexpression of AtWUS. Taken together, our findings demonstrate that DCIP offers a new and powerful tool for interrogating cytosolic delivery of proteins in plants and highlights future avenues for engineering plant physiology.

Cell penetrating peptides: Highlighting points in cancer therapy.

Cell-penetrating peptides (CPPs), first identified in HIV a few decades ago, deserved great attention in the last two decades; especially to support the penetration of anticancer drug means. In the drug delivery discipline, they have been involved in various approaches from mixing with hydrophobic drugs to the use of genetically conjugated proteins. The early classification as cationic and amphipathic CPPs has been extended to a few more classes such as hydrophobic and cyclic CPPs so far. Developing potential sequences utilized almost all methods of modern science: choosing high-efficiency peptides from natural protein sequences, sequence-based comparison, amino acid substitution, obtaining chemical and/or genetic conjugations, in silico approaches, in vitro analysis, animal experiments, etc. The bottleneck effect in this discipline reveals the complications that modern science faces in drug delivery research. Most CPP-based drug delivery systems (DDSs) efficiently inhibited tumor volume and weight in mice, but only in rare cases reduced their levels and continued further processes. The integration of chemical synthesis into the development of CPPs made a significant contribution and even reached the clinical stage as a diagnostic tool. But constrained efforts still face serious problems in overcoming biobarriers to reach further achievements. In this work, we reviewed the roles of CPPs in anticancer drug delivery, focusing on their amino acid composition and sequences. As the most suitable point, we relied on significant changes in tumor volume in mice resulting from CPPs. We provide a review of individual CPPs and/or their derivatives in a separate subsection.

Screening and characterisation of a novel efficient tumour cell-targeting peptide derived from insulin-like growth factor binding proteins.

Targeted delivery of antitumor drugs is particularly important in tumour treatment. Tumour-targeted peptide is a very effective drug carrier for tumour therapy. Here, we screened and characterised a highly efficient targeted peptide named IHP5, which was derived from insulin-like growth factor binding proteins (IGFBPs). IHP5 exhibited preferential binding to the tested tumour cell lines. The delivery efficiency of IHP5 was higher in various tested tumour cells than in normal cells, especially in the human cervical cancer cell line HeLa, which was 11.7-fold higher than in normal human embryonic kidney cells HEK293. Moreover, the penetration efficiency of IHP5 was 13 times higher than that of the classical cell penetrating peptide TAT in HeLa cells. Detail analysis revealed that IHP5 endocytosis was possibly correlated with acetylated heparan sulphate proteoglycans including phosphatidylinositol proteoglycan 3 (GPC3), phosphatidylinositol proteoglycan 5 (GPC5) and syndecan 2 (SDC2). Subsequently, the introduction of IHP5 enhanced the inhibitory effect of trichosanthin (TCS) on tumour cells, resulting in at least 19-fold increase in tumour cells without enhanced cytotoxicity in normal cells HEK293. These results suggested that IHP5, as a novel tumour cell-targeting penetrating peptide with the ability to target tumour cells, has great potential in drug delivery applications.