The trajectory patterns of single HIV-1 virus-like particle in live CD4 cells: A real time three-dimensional multi-resolution microscopy study using encapsulated nonblinking giant quantum dot.

BACKGROUND: The exploration of virology knowledge was limited by the optical technology for the observation of virus. Previously, a three-dimensional multi-resolution real-time microscope system (3D-MRM) was developed to observe the uptake of HIV-1-tat peptide-modified nanoparticles in cell membrane. In this study, we labeled HIV-1 virus-like particles (VLPs) with passivated giant quantum dots (gQDs) and recorded their interactive trajectories with human Jurkat CD4 cells through 3D-MRM. METHODS: The labeled of gQDs of the HIV-1 VLPs in sucrose-gradient purified viral lysates was first confirmed by Cryo-electronic microscopy and Western blot assay. After the infection with CD4 cells, the gQD-labeled VLPs were visualized and their extracellular and intracellular trajectories were recorded by 3D-MRM. RESULTS: A total of 208 prime trajectories was identified and classified into three distinct patterns: cell-free random diffusion pattern, directional movement pattern and cell-associated movement pattern, with distributions and mean durations were 72.6%/87.6 s, 9.1%/402.7 s and 18.3%/68.7 s, respectively. Further analysis of the spatial-temporal relationship between VLP trajectories and CD4 cells revealed the three stages of interactions: (1) cell-associated (extracellular) diffusion stage, (2) cell membrane surfing stage and (3) intracellular directional movement stage. CONCLUSION: A complete trajectory of HIV-1 VLP interacting with CD4 cells was presented in animation. This encapsulating method could increase the accuracy for the observation of HIV-1-CD4 cell interaction in real time and three dimensions.

Tumor-homing peptide and its utility for advanced cancer medicine.

Cell-penetrating peptides, such as antibodies, have gained great attention as tools for the development of specific delivery systems for payloads, which might be applied as non-invasive carriers in vivo. Among these, tumor-homing peptides recently have been studied for use in tumor medicine. Tumor-homing peptides are oligopeptides, usually consisting of 30 or fewer amino acids that are efficiently and specifically incorporated into tumor cells, suggesting their potential use in establishing novel non-invasive tumor imaging systems for diagnostic and therapeutic applications. Here, we briefly introduce the biological characteristics of our tumor-homing peptides, focusing especially on those developed using a random peptide library constructed using mRNA display technology. The advantage of the tumor-homing peptides is their biological safety, given that these molecules do not show significant cytotoxicity against non-neoplastic cells; lack serious antigenicity, which alternatively might evoke unfavorable immune responses and inflammation in vivo; and are rapidly incorporated into target cells/tissues, with rates exceeding those seen for antibodies. Given their small size, tumor-homing peptides also are easy to modify and redesign. Based on these merits, tumor-homing peptides are expected to find wide application in various aspects of tumor medicine, including imaging diagnostics (eg, with dye-conjugated probes for direct visualization of invasive/metastatic tumor lesions in vivo) and therapeutics (eg, using peptide-drug conjugates [PDCs] for tumor targeting). Although further evidence will be required to demonstrate their practical utility, tumor-homing peptides are expected to show great potential as a next-generation bio-tool contributing to precision medicine for cancer patients.

Modulation of Orai1 by cationic peptides triggers their direct cytosolic uptake.

At concentrations exceeding 10 µM, arginine-rich cell-penetrating peptides (CPPs) trigger a rapid cytoplasmic import that involves activation of acid sphingomyelinase (ASMase). ASMase activation occurs through a variety of stress signals and has also been related to the reorganization of membrane microdomains during entry of pathogens. However, in none of these cases has the initial trigger for ASMase activation been established on a molecular level. We here show that rapid cytosolic CPP import depends upon an increase in intracellular calcium, likely caused by modulation of the Orai1 calcium channel. At low peptide concentration, cytoplasmic import could be induced by thapsigargin, a known activator of Orai1. Compounds known to block Orai1 inhibited rapid uptake. Peptide-mediated modulation of Orai1 involved cell surface sialic acids as inhibition of sialylation as well as chemical blocking of sialic acids reduced rapid cytoplasmic uptake, which could be reconstituted by thapsigargin. These results establish a link between the known propensity of arginine-rich CPPs to interact with the glycocalyx and calcium influx as the initial step triggering direct cytosolic peptide uptake.

Peptide P11 suppresses the growth of human thyroid carcinoma by inhibiting the PI3K/AKT/mTOR signaling pathway.

Thyroid carcinoma is the most common endocrine malignancy, and the incidence of thyroid carcinoma is increasing in recent decades. CYYGQSKYC (P6), a nonapeptide with anti-lymphangiogenic effect by its binding to VEGFR-3 and selectively inhibiting VEGF-C binding to VEGFR-3, could suppress the migration and invasion of cancer cells. LSPPRYP (P9) acts as an effective bFGF/FGFR antagonist and inhibits the growth of the murine melanoma B16-F10 cells. In order to increase the anti-tumor effects of P6 and P9, we connected P6 with P9 via a flexible linker Gly-Gly-Gly (GGG) to reconstruct a novel peptide P11, CYYGQSKYCGGGLSPPRYP. In the present study, the mechanism of action of peptide P11 on the growth of human thyroid carcinoma cells both in vitro and in vivo was determined. Our results showed that peptide P11 inhibited the proliferation, viability, migration, and invasion of human thyroid carcinoma cells. Peptide P11 increased the apoptosis and decreased the protein levels of p-PI3K, p-AKT, and p-mTOR in human thyroid carcinoma cells. In addition, P11 could effectively inhibit the growth of human thyroid carcinoma xenograft tumors in nude mice. In conclusion, peptide P11 could inhibit the growth of human thyroid carcinoma by inhibiting the PI3K/Akt/mTOR signaling pathway. Novel peptides can be designed and applied for the treatment of various types of cancer.

Breach: Host Membrane Penetration and Entry by Nonenveloped Viruses.

Disruption of host membranes by nonenveloped viruses, which allows the nucleocapsid or genome to enter the cytosol, is a mechanistically diverse process. Although the membrane-penetrating agents are usually small, hydrophobic or amphipathic peptides deployed from the capsid interior during entry, their manner of membrane interaction varies substantially. In this review, we discuss recent data about the molecular pathways for externalization of viral peptides amidst conformational alterations in the capsid, as well as mechanisms of membrane penetration, which is influenced by structural features of the peptides themselves as well as physicochemical properties of membranes, and other host factors. The membrane-penetrating components of nonenveloped viruses constitute an interesting class of cell-penetrating peptides, and may have potential therapeutic value for gene transfer.

Breaking in and busting out: cell-penetrating peptides and the endosomal escape problem.

Cell-penetrating peptides (CPPs) have long held great promise for the manipulation of living cells for therapeutic and research purposes. They allow a wide array of biomolecules from large, oligomeric proteins to nucleic acids and small molecules to rapidly and efficiently traverse cytoplasmic membranes. With few exceptions, if a molecule can be associated with a CPP, it can be delivered into a cell. However, a growing realization in the field is that CPP-cargo fusions largely remain trapped in endosomes and are eventually targeted for degradation or recycling rather than released into the cytoplasm or trafficked to a desired subcellular destination. This 'endosomal escape problem' has confounded efforts to develop CPP-based delivery methods for drugs, enzymes, plasmids, etc. This review provides a brief history of CPP research and discusses current issues in the field with a primary focus on the endosomal escape problem, for which several promising potential solutions have been developed. Are we on the verge of developing technologies to deliver therapeutics such as siRNA, CRISPR/Cas complexes and others that are currently failing because of an inability to get into cells, or are we just chasing after another promising but unworkable technology? We make the case for optimism.

Effective Design of Multifunctional Peptides by Combining Compatible Functions.

Multifunctionality is a common trait of many natural proteins and peptides, yet the rules to generate such multifunctionality remain unclear. We propose that the rules defining some protein/peptide functions are compatible. To explore this hypothesis, we trained a computational method to predict cell-penetrating peptides at the sequence level and learned that antimicrobial peptides and DNA-binding proteins are compatible with the rules of our predictor. Based on this finding, we expected that designing peptides for CPP activity may render AMP and DNA-binding activities. To test this prediction, we designed peptides that embedded two independent functional domains (nuclear localization and yeast pheromone activity), linked by optimizing their composition to fit the rules characterizing cell-penetrating peptides. These peptides presented effective cell penetration, DNA-binding, pheromone and antimicrobial activities, thus confirming the effectiveness of our computational approach to design multifunctional peptides with potential therapeutic uses. Our computational implementation is available at http://bis.ifc.unam.mx/en/software/dcf.

p28-Mediated Activation of p53 in G2-M Phase of the Cell Cycle Enhances the Efficacy of DNA Damaging and Antimitotic Chemotherapy.

p28 is an anionic cell-penetrating peptide of 28 amino acids that activates wild-type and mutated p53, leading subsequently to selective inhibition of CDK2 and cyclin A expression and G2-M cell-cycle arrest. In this study, we investigated the cytotoxic effects of p28 treatment alone and in combination with DNA-damaging and antimitotic agents on human cancer cells. p28 enhanced the cytotoxic activity of lower concentrations (IC20-50) of DNA-damaging drugs (doxorubicin, dacarbazine, temozolamide) or antimitotic drugs (paclitaxel and docetaxel) in a variety of cancer cells expressing wild-type or mutated p53. Mechanistic investigations revealed that p28 induced a post-translational increase in the expression of wild-type or mutant p53 and p21, resulting in cell-cycle inhibition at the G2-M phase. The enhanced activity of these anticancer agents in combination with p28 was facilitated through the p53/p21/CDK2 pathway. Taken together, these results highlight a new approach to maximize the efficacy of chemotherapeutic agents while reducing dose-related toxicity. Cancer Res; 76(8); 2354-65. ©2016 AACR.

Extracellular vesicle-mediated delivery of molecular compounds into gametes and embryos: learning from nature.

BACKGROUND: Currently, even the most sophisticated methods of assisted reproductive technology (ART) allow us to achieve live births in only approximately 30% of patients, indicating that our understanding of the fine mechanisms underlying reproduction is far from ideal. One of the main challenges associated with studies of gamete structure and function is that these cells are remarkably resistant towards the uptake of exogenous substances, including 'molecular research tools' such as drugs, biomolecules and intracellular markers. This phenomenon can affect not only the performance of reproductive biology research techniques, but also the outcomes of the in vitro handling of gametes, which forms the cornerstone of ART. Improvement of intra-gamete delivery in a non-aggressive fashion is vital for the investigation of gamete physiology, and the advancement of infertility treatment. In this review, we outline the current state of nanomaterial-mediated delivery into gametes and embryos in vitro, and discuss the potential of a novel exciting drug delivery technology, based upon the use of targeted 'natural' nanoparticles known as extracellular vesicles (EVs), for reproductive science and ART, given the promising emerging data from other fields. METHODS: A comprehensive electronic search of PubMed and Web of Science databases was performed using the following keywords: 'nanoparticles', 'nanomaterials', 'cell-penetrating peptides', 'sperm', 'oocyte', 'egg', 'embryo', 'exosomes', 'microvesicles', 'extracellular vesicles', 'delivery', 'reproduction', to identify the relevant research and review articles, published in English up to January 2015. The reference lists of identified publication were then scanned to extract additional relevant publications. RESULTS: Biocompatible engineered nanomaterials with high loading capacity, stability and selective affinity represent a potential versatile tool for the minimally invasive internalization of molecular cargo into gametes and embryos. However, it is becoming increasingly clear that the translation of these experimental tools into clinical applications is likely to be limited by their non-biodegradable nature. To allow the subsequent use of these methodologies for clinical ART, studies should utilize biodegradable delivery platforms, which mimic natural mechanisms of molecular cargo trafficking as closely as possible. Currently, EVs represent the most physiological intracellular delivery tools for reproductive science and medicine. These natural mediators of cell communication combine the benefits of engineered nanomaterials, such as the potential for in vitro production, targeting and loading, with the essential feature of biodegradability. CONCLUSION: We anticipate that future investigations into the possibility of applying EVs for the intentional intracellular delivery of molecular compounds into gametes and embryos will open new horizons for reproductive science and clinical ART, ultimately leading to improvements in patient care.

Targeting nuclear import shuttles, importins/karyopherins alpha by a peptide mimicking the NFκB1/p50 nuclear localization sequence.

BACKGROUND: We recently reported that a bifunctional nuclear transport modifier (NTM), cSN50.1 peptide, reduced atherosclerosis, plasma cholesterol, triglycerides, and glucose along with liver fat and inflammatory markers, in a murine model of familial hypercholesterolemia. We determined that cSN50.1 improved lipid homeostasis by modulating nuclear transport of sterol regulatory element-binding proteins through interaction with importin ß. Previous studies established that cSN50.1 and related NTMs also modulate nuclear transport of proinflammatory transcription factors mediated by binding of their nuclear localization sequences (NLSs) to importins/karyopherins α. However, selectivity and specificity of NTMs for importins/karyopherins α were undetermined. METHODS AND RESULTS: We analyzed interaction of the NTM hydrophilic module, N50 peptide, derived from the NLS of NFκB1/p50, with endogenous human importins/karyopherins α to determine the mechanism of NTM modulation of importin α-mediated nuclear transport. We show that N50 peptide forms stable complexes with multiple importins/karyopherins α. However, only interaction with importin α5 (Imp α5) displayed specific, high-affinity binding. The 2:1 stoichiometry of the N50-Imp α5 interaction (KD1 = 73 nmol/L, KD2 = 140 nmol/L) indicated occupancy of both major and minor NLS binding pockets. Utilizing in silico 3-dimensional (3-D) docking models and comparative structural analysis, we identified a structural component of the Imp α5 major NLS binding pocket that may stabilize N50 binding. Imp α5 also displayed rapid stimulus-induced turnover, which could influence its availability for nuclear transport during the inflammatory response. CONCLUSIONS: These results provide direct evidence that N50 peptide selectively targets Imp α5, encouraging further refinement of NLS-derived peptides as new tools to modulate inflammatory disorders.

Non-genetic modulation of Notch activity by artificial delivery of Notch intracellular domain into neural stem cells.

Stem cells have become a major focus of scientific interest as a potential source of somatic cell types for biomedical applications. Understanding and controlling the elicitors and mechanisms in differentiation of pluripotent stem cell-derived somatic cell types remains a key challenge. The major types of molecular processes that control cellular differentiation involve evolutionary conserved cell signaling pathways. Notch receptors participate in a wide variety of biological processes, including cell fate decisions of stem cells. This study explores the potential of protein transduction to directly deliver recombinant Notch-1 intracellular domain (NICD) into mammalian cells in order to accomplish transgene-free Notch activation. We engineered a cell-permeant version of NICD and explored its function on mouse and human neural stem cells. We show that NICD transduction modulates known direct and indirect Notch target genes and antagonizes the DAPT-mediated inhibition of Notch signaling on the transcriptional level. Moreover, NICD enhances cell proliferation accompanied by increased cyclin D1 and decreased p27 protein levels. In the absence of growth factors NICD strongly impairs neuronal differentiation while being insufficient to keep cells in a proliferative state. Furthermore, our studies depict NICD protein transduction as a novel tool for a time and dose-dependent non-genetic modulation of Notch signaling to decipher its cellular functions.

Mechanism of ribonuclease A endocytosis: analogies to cell-penetrating peptides.

Pancreatic-type ribonucleases can exert toxic activity by catalyzing the degradation of cellular RNA. Their ability to enter cells is essential for their cytotoxicity. Here, we determine the mechanism by which bovine pancreatic ribonuclease (RNase A) enters human cells. Inhibiting clathrin-dependent endocytosis with dynasore or chlorpromazine decreases RNase A-uptake by ~70%. Limited colocalization between RNase A and transferrin indicates that RNase A is not routed through recycling endosomes. Instead, vesicular staining of RNase A overlaps substantially with that of nona-arginine and the cationic peptide corresponding to residues 47-57 of the HIV-1 TAT protein. At low concentrations (<5 µM), internalization of RNase A and these cell-penetrating peptides (CPPs) is inhibited by chlorpromazine as well as the macropinocytosis inhibitors cytochalasin D and 5-(N-ethyl-N-isopropyl)amiloride to a similar extent, indicative of common endocytic mechanism. At high concentrations, CPPs adopt a nonendocytic mechanism of cellular entry that is not shared by RNase A. Collectively, these data suggest that RNase A is internalized via a multipathway mechanism that involves both clathrin-coated vesicles and macropinosomes. The parallel between the uptake of RNase A and CPPs validates reference to RNase A as a "cell-penetrating protein".

[Development of cell-penetrating peptides as vectors for drug delivery].

Biomacromolecules play an important role in the treatment of many diseases, but as a result of cell membrane serving as the natural barriers, only the small molecular compounds whose molecular weights are smaller than 600 Da can get through cell membrane and enter the cell. In recent years, some short peptides (the length less than 30 amino acids) are found to have the cell-penetrating function, called cell-penetrating peptides (CPPs). They are able to effectively translocate segments of protein, polypeptides, nucleic acid into the cells of many mammal animals with many methods. They have high transduction efficiency and will not lead to cell damage. So, the discovery of CPPs has a very good applicable prospect in such research fields as cell-biology, gene-therapy, drug transduction in vivo, evaluation of clinical medicine and medical immunology. This paper reviews the types and characteristics of CPPs, internalization mechanisms, applications, and their existing problems.