Model membrane systems, membrane-active peptides and application possibilities

The ability of cell-penetrating peptides (CPPs) to translocate a variety of covalently or non-covalently linked cargoes, particularly therapeutics and imaging agents, across the plasma membrane of living cells renders them of broad interest in the medical field as well as in biotechnological development. Thus, the use of CPPs, e.g. in therapeutics, increases the necessity of understanding their mechanism of action, a subject still under debate. For the first step of peptide internalization, the interaction between the positively charged amino acid side chains within the peptide sequence and the anionic constituents of the membrane bilayer is assumed. However, owing to the complexity of biological plasma membranes, studying the mode of action of CPPs on a molecular level is very difficult. With this in mind, model membrane systems, especially large and giant unilamellar vesicles and giant plasma membranes vesicles derived from live cells plasma membranes, were prepared and several techniques were established in order to monitor the initial step of cell entry – binding to and translocation through the cell membrane as well as identification of key components of the plasma membrane required for efficient uptake. The first chapter focuses on the membrane interactions and perturbation mechanism of two structural modified variants of the well-described CPP sC18, a cyclized and a dimeric branched version, with artificial model membranes. Fluorescence analysis demonstrated the importance of negatively charged lipids for an efficient uptake of Arg-rich CPPs, supporting the assumption that electrostatic interactions are required for the initial stage of cell entry. In case of dimeric variants, the strong affinity was further accompanied by intense membrane destabilizing effects, presumably responsible for their lytic behavior as anticancer peptide. Moreover, the cyclization of the peptide backbone turned out to be a promising strategy as well to improve their cell-penetrating capability. Using CD spectroscopy, structural arrangements when in contact with neutral and anionic vesicles further revealed that a certain flexibility and thus the static arrangement of the guanidinium groups within the cycle seem to positively affect peptide-membrane interaction. In summary, these evidences suggest that the internalization of the peptide variants depends highly on the membrane composition of the target cell. In the second chapter, the focus lay on the design and characterization of novel peptide conjugates, composing of a wound healing promoting peptide and sC18 as cell penetrating peptide, with the final goal to further enhance the wound healing activity. Besides, the new conjugate Tylotoin-sC18* exhibited further promising antimicrobial activity and moreover a certain selectivity towards cancer cells, suggesting that Tylotoin-sC18* is a good candidate for further studies and the possible development as a therapeutic agent for infected wounds

Cell membrane softening in human breast and cervical cancer cells

Biomechanical properties are key to many cellular functions such as cell division and cell motility and thus are crucial in the development and understanding of several diseases, for instance cancer. The mechanics of the cellular cytoskeleton have been extensively characterized in cells and artificial systems. The rigidity of the plasma membrane, with the exception of red blood cells, is unknown and membrane rigidity measurements only exist for vesicles composed of a few synthetic lipids. In this study, thermal fluctuations of giant plasma membrane vesicles (GPMVs) directly derived from the plasma membranes of primary breast and cervical cells, as well as breast cell lines, are analyzed. Cell blebs or GPMVs were studied via thermal membrane fluctuations and mass spectrometry. It will be shown that cancer cell membranes are significantly softer than their non-malignant counterparts. This can be attributed to a loss of fluid raft forming lipids in malignant cells. These results indicate that the reduction of membrane rigidity promotes aggressive blebbing motion in invasive cancer cells

Design of a novel cell-permeable chimeric peptide to promote wound healing

Abstract Biological membranes are impermeable to almost all compounds having a molecular weight greater than 500 Da. Recently, cell penetrating peptides (CPPs) as delivery vehicles have attracted great interest in the medical sector for the development of novel therapeutic agents or cosmetic products. Herein, a wound healing promoting sequence, namely Tylotoin, was covalently coupled with a cell penetrating peptide to improve the delivery of Tylotoin across cellular membranes. Indeed, internalization studies indicated that the cellular uptake of these novel peptide conjugates into keratinocytes was significantly improved accompanied by good tolerability. In a scratch wound closure assay used to investigate the wound healing capability, the most promising novel peptide chimera (Tylotoin-sC18*) was found to promote the migration of keratinocytes indicating that the fusion to Tylotoin did not cause any loss in its activity. Even more, proliferative effects on keratinocytes were observed, an important step during the wound healing process. Still more encouraging is the capability of Tylotoin-sC18* to exhibit strong antimicrobial activities since the process of wound healing is often affected by bacterial infections. Owing to their multiple functions, the novel peptide chimera may have potential as future agents for the treatment of infected wounds

Cyclization of a cell-penetrating peptide via click-chemistry increases proteolytic resistance and improves drug delivery

In this work we report synthesis and biological evaluation of a cell-penetrating peptide (CPP), that is partly cyclized via a triazole bridge. Recently, beneficious properties have been reported for cyclized peptides concerning their metabolic stability and intracellular uptake. A CPP based on human calcitonin was used in this study, and side chain cyclization was achieved via copper catalyzed alkyne-azide click reaction. Cell viability studies in several cell-lines revealed no cytotoxic effects. Furthermore, efficient uptake in breast cancer MCF-7 cells could be determined. Moreover, preliminary studies using this novel peptide as drug transporter for daunorubicin were performed. Copyright (c) 2016 European Peptide Society and John Wiley & Sons, Ltd

Design and biological characterization of novel cell-penetrating peptides preferentially targeting cell nuclei and subnuclear regions

Within this study, we report about the design and biological characterization of novel cell-penetrating peptides (CPPs) with selective suborganelle-targeting properties. The nuclear localization sequence N50, as well as the nucleoli-targeting sequence NrTP, respectively, were fused to a shortened version of the cell-penetrating peptide sC18. We examined cellular uptake, subcellular fate and cytotoxicity of these novel peptides, N50-sC18* and NrTP-sC18*, and found that they are nontoxic up to a concentration of 50 or 100 µM depending on the cell lines used. Moreover, detailed cellular uptake studies revealed that both peptides enter cells via energy-independent uptake, although endocytotic processes cannot completely excluded. However, initial drug delivery studies demonstrated the high versatility of these new peptides as efficient transport vectors targeting specifically nuclei and nucleoli. In future, they could be further explored as parts of newly created peptide–drug conjugates

Multivalent magnetic nanoaggregates with unified antibacterial activity and selective uptake of heavy metals and organic pollutants

Covalently functionalized magnetite (Fe3O4) nanoaggregates carrying an imidazolium-derivative (1-hexadecyl-3-vinyl imidazolium bromide, HDVI) and L-cysteine (L-Cys) as surface ligands act as bimodal water-treatment particulate agents (HDVI@L-Cys@PAA@Fe3O4) with high antibacterial efficacy and specific surface adsorption properties. For covalent conjugation of HDVI the polyacrylic acid (PAA)-coated magnetite nanocrystals (PAA@Fe3O4) were functionalized with L-cysteine via carbodiimide coupling (L-Cys@PAA@Fe3O4) having terminal-SH groups that were used for thiol-ene click chemistry. The carefully performed series of water-remediation tests with magnetically separable HDVI@L-Cys@PAA@Fe3O4 nanoaggregates demonstrated their high efficiency in the concomitant removal transition metal ions and organic pollutant without losing the antibacterial effect. Time-dependent adsorption experiments showed high degree (>90%) of trapping and removal activity. Antibacterial action of the HDVI@L-Cys@PAA@Fe3O4 nanoaggregates originates from the amphiphilic structure of HDVI groups capable of penetrating the bacterial cell walls. The presence of surface-bound ligands and conversion efficiency of carbodiimide and click chemistry protocol was verified by FT-IR, elemental and thermogravimetric analysis. The phase, composition morphology and surface charge of nanoaggregates were examined by XRD, SEM/TEM and zeta potential studies, respectively. The experimental findings reported here represent a conceptual advancement in the state-of-the-art magnetic beads developed for water purification or remediation purposes. Our results evidently demonstrate that nanoaggregates are highly effective in unifying bactericidal activity against different microorganism with heavy metal and organic pollutant removal properties. (C) 2020 Elsevier B.V. All rights reserved

Design of CK2 beta-Mimicking Peptides as Tools To Study the CK2 alpha/CK2 beta Interaction in Cancer Cells

The ubiquitously expressed Ser/Thr kinase CK2 is a key regulator in a variety of key processes in normal and malignant cells. Due to its distinctive anti-apoptotic and tumor-driving properties, elevated levels of CK2 have frequently been found in tumors of different origin. In recent years, development of CK2 inhibitors has largely been focused on ATP-competitive compounds; however, targeting the CK2 alpha/CK2 beta interface has emerged as a further concept that might avoid selectivity issues. To address the CK2 subunit interaction site, we have synthesized halogenated CK2 beta-mimicking cyclic peptides modified with the cell-penetrating peptide sC18 to mediate cellular uptake. We investigated the binding of the resulting chimeric peptides to recombinant human CK2 alpha using a recently developed fluorescence anisotropy assay. The iodinated peptide sC18-I-Pc was identified as a potent CK2 alpha ligand (K-i=0.622 mu m). It was internalized in cells to a high extent and exhibited significant cytotoxicity toward cancerous HeLa cells (IC50=37 mu m) in contrast to non-cancerous HEK-293 cells. The attractive features and functionalities of sC18-I-Pc offer the opportunity for further improvement

How to ensure full vaccination? The association of institutional delivery and timely postnatal care with childhood vaccination in a cross-sectional study in rural Bihar, India.

Incomplete and absent doses in routine childhood vaccinations are of major concern. Health systems in low- and middle-income countries (LMIC), in particular, often struggle to enable full vaccination of children, which affects their immunity against communicable diseases. Data on child vaccination cards from a cross-sectional primary survey with 1,967 households were used to assess the vaccination status. The association of timely postnatal care (PNC) and the place of delivery with any-dose (at least one dose of each vaccine) and full vaccination of children between 10-20 months in Bihar, India, was investigated. Bivariate and multivariable logistic regression models were used. The vaccines included targeted tuberculosis, hepatitis B, polio, diphtheria/pertussis/tetanus (DPT) and measles. Moreover predictors for perinatal health care uptake were analysed by multivariable logistic regression. Of the 1,011 children with card verification, 47.9% were fully vaccinated. Timely PNC was positively associated with full vaccination (adjusted odds ratio (aOR) 1.48, 95% confidence interval (CI) 1.06-2.08) and with the administration of at least one dose (any-dose) of polio vaccine (aOR 3.37 95% CI 1.79-6.36), hepatitis B/pentavalent vaccine (aOR 2.11 95% CI 1.24-3.59), and DPT/pentavalent vaccine (aOR 2.29 95% CI 1.35-3.88). Additionally, delivery in a public health care facility was positively associated with at least one dose of hepatitis B/pentavalent vaccine administration (aOR 4.86 95% CI 2.97-7.95). Predictors for timely PNC were institutional delivery (public and private) (aOR 2.7 95% CI 1.96-3.72, aOR 2.38 95% CI 1.56-3.64), at least one ANC visit (aOR 1.59 95% CI 1.18-2.15), wealth quintile (Middle aOR 1.57 95% CI 1.02-2.41, Richer aOR 1.51 95% CI 1.01-2.25, Richest aOR 2.06 95% CI 1.28-3.31) and household size (aOR 0.95 95% CI 0.92-0.99). The findings indicate a correlation between childhood vaccination and timely postnatal care. Further, delivery in a public facility correlates with the administration of at least one dose of hepatitis B vaccine and thus impedes zero-dose vaccination. Increasing uptake of timely PNC, encouraging institutional delivery, and improving vaccination services before discharge of health facilities may lead to improved vaccination rates among children

Elektrokonvulsionstherapie in der Schwangerschaft: Fallbericht und interdisziplinäre Behandlungsvorschläge

Background!#!Psychiatric disorders during pregnancy are common. Electroconvulsive therapy (ECT) can be indicated in severely affective or psychotic disorders with the necessity of a rapid response. Currently available review articles greatly differ in the methodology, leading to divergent conclusions concerning the use of ECT during pregnancy.!##!Objective!#!Description of a new clinical case and interdisciplinary treatment suggestions for the safe application of ECT in pregnancy.!##!Methods!#!Clinical case report and selective review of the literature with special consideration of existing systematic reviews.!##!Results and conclusion!#!This case report shows the potentially high effectiveness and safe administration of ECT in pregnancy for both mother and fetus. The undesired adverse events associated with ECT described in the literature are largely qualitatively congruent with the risks of severe psychotic disorders in pregnancy per se. For a better risk-benefit analysis, larger case control studies would be desirable. Under the premise of a thorough evaluation of the indications, good interdisciplinary coordination and consideration of the specific practical requirements, ECT is a useful therapeutic option in pregnancy

Characterization of a Cell-Penetrating Peptide with Potential Anticancer Activity

Cell-penetrating peptides (CPPs) are still an interesting and viable alternative for drug delivery applications. CPPs contain considerably high amounts of positively charged amino acids, imparting them with cationic character. Tumor cells are characterized by an enhanced anionic nature of their membrane surface, a property that could be used by CPPs to target these cells. We recently identified a branched CPP that displays a high internalization capacity while exhibiting selectivity for certain tumor cell types. In this study we elucidated this observation in greater detail by investigating the underlying mechanism behind the cellular uptake of this peptide. An additional cytotoxicity screen against several cancer cell lines indeed demonstrates high cytotoxic activity against cancer cells over normal fibroblasts. Furthermore, we show that this feature can be used for delivering the anticancer drug actinomycinD with high efficiency in the MCF-7 cancer cell line