69 research outputs found
Quantitative and qualitative effect of gH625 on the nanoliposome-mediated delivery of mitoxantrone anticancer drug to HeLa cells
The present work investigates in vitro the delivery of the anticancer drug mitoxantrone (MTX) to HeLa cancer cells by means of liposomes functionalized with the novel cell penetrating peptide gH625. This hydrophobic peptide enhances the delivery of doxorubicin to the cytoplasm of cancer cells, while the mechanism of this enhancement has not yet been understood. Here, in order to get a better insight into the role of gH625 on the mechanism of liposome-mediated drug delivery, we treated HeLa cells with liposomes functionalized with gH625 and loaded with MTX; liposome were characterized in terms of their physico-chemical properties and drug release kinetics. To quantify the MTX uptake and to study the subcellular drug distribution and interaction, we took advantage of the intrinsic fluorescence of MTX and of the fluorescence-based techniques like fluorescence-activated cell sorting (FACS) and confocal spectral imaging (CSI). gH625 liposomes showed an enhanced staining of the internalized drug is observed mainly in hydrophobic regions of the cytoplasm, where the increased presence of an oxidative metabolite of the drug is observed. MTX delivery with gH625-decorated nanoliposomes enhances the quantity of both the intracellular drug and of its oxidative metabolite and contributes to higher anticancer efficacy of the drug
Tracking the cell cycle origins for escape from topotecan action by breast cancer cells
The anticancer agent topotecan is considered to be S-phase specific. This implies that cancer cells that are not actively replicating DNA could resist the effects of the drug. The cycle specificity of topotecan action was investigated in MCF-7 cells, using time-lapse microscopy to link the initial cell cycle position during acute exposures to topotecan with the antiproliferative consequences for individual cells. The bioactive dose range (0.5–10 μM) for 1-h topotecan exposures was defined by rapid drug delivery and topoisomerase I trapping. Topotecan caused pan-cycle induction and activation of p53. Lineage analysis of the time-lapse sequences identified cells initially in S-phase and G2, and defined the time to mitosis for cells originating from G2, S-phase and G1. Topotecan prevented all mitoses from S-phase cells and G1 cells (half-maximal effects at 0.14 μM and 0.96 μM, respectively). No dose of topotecan completely prevented mitosis among G2 cells, and at saturating doses of topotecan about half the cells of G2 origin continued dividing (the half-maximal effects was at 0.31 μM). Overall, topotecan differentially targeted G1-, S- and G2-phase cells, but many G2 cells were resistant to topotecan, presenting a clear route for cell cycle-mediated drug resistance
Comparability of Raman Spectroscopic Configurations: A Large Scale Cross-Laboratory Study
This is the final version. Available on open access from the American Chemical Society via the DOI in this recordThe variable configuration of Raman spectroscopic platforms is one of the major obstacles in establishing Raman spectroscopy as a valuable physicochemical method within real-world scenarios such as clinical diagnostics. For such real world applications like diagnostic classification, the models should ideally be usable to predict data from different setups. Whether it is done by training a rugged model with data from many setups or by a primary-replica strategy where models are developed on a 'primary' setup and the test data are generated on 'replicate' setups, this is only possible if the Raman spectra from different setups are consistent, reproducible, and comparable. However, Raman spectra can be highly sensitive to the measurement conditions, and they change from setup to setup even if the same samples are measured. Although increasingly recognized as an issue, the dependence of the Raman spectra on the instrumental configuration is far from being fully understood and great effort is needed to address the resulting spectral variations and to correct for them. To make the severity of the situation clear, we present a round robin experiment investigating the comparability of 35 Raman spectroscopic devices with different configurations in 15 institutes within seven European countries from the COST (European Cooperation in Science and Technology) action Raman4clinics. The experiment was developed in a fashion that allows various instrumental configurations ranging from highly confocal setups to fibre-optic based systems with different excitation wavelengths. We illustrate the spectral variations caused by the instrumental configurations from the perspectives of peak shifts, intensity variations, peak widths, and noise levels. We conclude this contribution with recommendations that may help to improve the inter-laboratory studies.COST (European Cooperation in Science and Technology)Portuguese Foundation for Science and TechnologyNational Research Fund of Luxembourg (FNR)China Scholarship Council (CSC)BOKU Core Facilities Multiscale ImagingDeutsche Forschungsgemeinschaft (DFG, German Research Foundation
Synthesis and in vitro evaluation of novel SPION-PEG-gH625 multifunctional nanosystems for cancer theranosis
The research on supermagnetic iron oxide nanoparticles (SPIONs) has been growing exponentially
over the last several years. SPIONs have been established as a promising platform because of their
eclectic biomedical applications such as, cancer theranosis, i.e. therapy, by hyperthermia and/or magnetically
controlled drug delivery, and diagnosis by means of magnetic resonance imaging (MRI)1
.
For these kind of applications, multifunctional SPIONs simultaneously modified with biological ligands,
biocompatible polymers (most often polyethylene glycol PEG), drugs and fluorescence labels
are especially interesting, because they combine the possibility of targeted drug delivery and in vivo
imaging of treated tumors by means of MRI and fluorescence. Once accumulated in tumors, the nanosystems
are expected to recognise cancer cells and to penetrate inside them, in order to enhance the
theranostic potential. However, the intracellular accumulation of the nanosystems depends on their
ability to cross cellular membranes.
In order to explore the possibility to improve the cellular uptake of PEGylated SPIONs, we have
functionnalized the surface of their PEG5000 coating with a membranotropic peptide gH6252
. The nanosystems
contained also fluorescent molecules (cyanine) covalently attached to the SPION core. The
core-shell nanosystems were carefully characterized in terms of their physico-chemical properties
(hydrodynamic size, zeta potential, chemical composition) prior to study the interaction with cancer
cells MDA-MB-231 in vitro. Quantitative determination of the uptaken nanosystems in the cells was
made using both the analysis of the cyanine fluorescence (by flow cytometry) and of the iron content
(by atomic absorption spectrophotometry - SAA). Moreover, we analysed the peptide effect on the
subcellular nanosystems distribution and interaction, using transmission electron microscopy (TEM)
and confocal spectral imaging (CSI) of the cyanine fluorescence. The results showed the capacity of
the peptide gH625 to promote cellular internalization with a possibility of modification of the uptake
mechanism
Enhancing the Potency of Antimicrobial Peptides through Molecular Engineering and Self-Assembly
Healthcare-associated
infections resulting from bacterial attachment
and biofilm formation on medical implants are posing significant challenges
in particular with the emergence of bacterial resistance to antibiotics.
Here, we report the design, synthesis and characterization of self-assembled
nanostructures, which integrate on their surface antibacterial peptides.
The antibacterial WMR peptide, which is a modification of the native
sequence of the myxinidin, a marine peptide isolated from the epidermal
mucus of hagfish, was used considering its enhanced activity against
Gram-negative bacteria. WMR was linked to a peptide segment of aliphatic
residues (AAAAAAA) containing a lipidic tail (C19H38O2) attached to the ε-amino of a terminal
lysine to generate a peptide amphiphile (WMR PA). The self-assembly
of the WMR PA alone, or combined with coassembling shorter PAs, was
studied using spectroscopy and microscopy techniques. The designed
PAs were shown to self-assemble into stable nanofiber structures and
these nanoassemblies significantly inhibit biofilm formation and eradicate
the already formed biofilms of Pseudomonas aeruginosa (Gram-negative bacteria) and Candida albicans (pathogenic fungus) when compared to the native WMR peptide. Our
results provide insights into the design of peptide based supramolecular
assemblies with antibacterial activity, and establish an innovative
strategy to develop self-assembled antimicrobial materials for biomedical
applications
Design and Validation of Nanofibers Made of Self-Assembled Peptides to Become Multifunctional Stimuli-Sensitive Nanovectors of Anticancer Drug Doxorubicin
Self-assembled peptides possess remarkable potential as targeted drug delivery systems and key applications dwell anti-cancer therapy. Peptides can self-assemble into nanostructures of diverse sizes and shapes in response to changing environmental conditions (pH, temperature, ionic strength). Herein, we investigated the development of self-assembled peptide-based nanofibers (NFs) with the inclusion of a cell-penetrating peptide (namely gH625) and a matrix metalloproteinase-9 (MMP-9) responsive sequence, which proved to enhance respectively the penetration and tumor-triggered cleavage to release Doxorubicin in Triple Negative Breast Cancer cells where MMP-9 levels are elevated. The NFs formulation has been optimized via critical micelle concentration measurements, fluorescence, and circular dichroism. The final nanovectors were characterized for morphology (TEM), size (hydrodynamic diameter), and surface charge (zeta potential). The Doxo loading and release kinetics were studied in situ, by optical microspectroscopy (fluorescence and surface-enhanced Raman scattering–SERS). Confocal spectral imaging of the Doxo fluorescence was used to study the TNBC models in vitro, in cells with various MMP-9 levels, the drug delivery to cells as well as the resulting cytotoxicity profiles. The results confirm that these NFs are a promising platform to develop novel nanovectors of Doxo, namely in the framework of TNBC treatment
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