Elastic Liposomes Containing Calcium/Magnesium Ferrite Nanoparticles Coupled with Gold Nanorods for Application in Photothermal Therapy.

This work reports on the design, development, and characterization of novel magneto-plasmonic elastic liposomes (MPELs) of DPPC:SP80 (85:15) containing Mg0.75Ca0.25Fe2O4 nanoparticles coupled with gold nanorods, for topical application of photothermal therapy (PTT). Both magnetic and plasmonic components were characterized regarding their structural, morphological, magnetic and photothermal properties. The magnetic nanoparticles display a cubic shape and a size (major axis) of 37 ± 3 nm, while the longitudinal and transverse sizes of the nanorods are 46 ± 7 nm and 12 ± 1.6 nm, respectively. A new methodology was employed to couple the magnetic and plasmonic nanostructures, using cysteine as bridge. The potential for photothermia was evaluated for the magnetic nanoparticles, gold nanorods and the coupled magnetic/plasmonic nanoparticles, which demonstrated a maximum temperature variation of 28.9 °C, 33.6 °C and 37.2 °C, respectively, during a 30 min NIR-laser irradiation of 1 mg/mL dispersions. Using fluorescence anisotropy studies, a phase transition temperature (Tm) of 35 °C was estimated for MPELs, which ensures an enhanced fluidity crucial for effective crossing of the skin layers. The photothermal potential of this novel nanostructure corresponds to a specific absorption rate (SAR) of 616.9 W/g and a maximum temperature increase of 33.5 °C. These findings point to the development of thermoelastic nanocarriers with suitable features to act as photothermal hyperthermia agents.

Development of pH-Sensitive Magnetoliposomes Containing Shape Anisotropic Nanoparticles for Potential Application in Combined Cancer Therapy.

Late diagnosis and systemic toxicity associated with conventional treatments make oncological therapy significantly difficult. In this context, nanomedicine emerges as a new approach in the prevention, diagnosis and treatment of cancer. In this work, pH-sensitive solid magnetoliposomes (SMLs) were developed for controlled release of the chemotherapeutic drug doxorubicin (DOX). Shape anisotropic magnetic nanoparticles of magnesium ferrite with partial substitution by calcium (Mg0.75Ca0.25Fe2O4) were synthesized, with and without calcination, and their structural, morphological and magnetic properties were investigated. Their superparamagnetic properties were evaluated and heating capabilities proven, either by exposure to an alternating magnetic field (AMF) (magnetic hyperthermia) or by irradiation with near-infrared (NIR) light (photothermia). The Mg0.75Ca0.25Fe2O4 calcined nanoparticles were selected to integrate the SMLs, surrounded by a lipid bilayer of DOPE:Ch:CHEMS (45:45:10). DOX was encapsulated in the nanosystems with an efficiency above 98%. DOX release assays showed a much more efficient release of the drug at pH = 5 compared to the release kinetics at physiological pH. By subjecting tumor cells to DOX-loaded SMLs, cell viability was significantly reduced, confirming that they can release the encapsulated drug. These results point to the development of efficient pH-sensitive nanocarriers, suitable for a synergistic action in cancer therapy with magnetic targeting, stimulus-controlled drug delivery and dual hyperthermia (magnetic and plasmonic) therapy.

Common polymorphic OTC variants can act as genetic modifiers of enzymatic activity.

Understanding the role of common polymorphisms in modulating the clinical phenotype when they co-occur with a disease-causing lesion is of critical importance in medical genetics. We explored the impact of apparently neutral common polymorphisms, using the gene encoding the urea cycle enzyme, ornithine transcarbamylase (OTC), as a model system. Distinct combinations of genetic backgrounds embracing two missense polymorphisms were created in cis with the pathogenic p.Arg40His replacement. In vitro enzymatic assays revealed that the polymorphic variants were able to modulate OTC activity both in the presence or absence of the pathogenic lesion. First, we found that the combination of the minor alleles of polymorphisms p.Lys46Arg and p.Gln270Arg significantly enhanced enzymatic activity in the wild-type protein. Second, enzymatic assays revealed that the minor allele of the p.Gln270Arg polymorphism was capable of ameliorating OTC activity when combined in cis with the pathogenic p.Arg40His replacement. Structural analysis predicted that the minor allele of the p.Gln270Arg polymorphism would serve to stabilize the OTC wild-type protein, thereby corroborating the results of the experimental assays. Our findings demonstrate the potential importance of cis-interactions between common polymorphic variants and pathogenic missense mutations and illustrate how standing genetic variation can modulate protein function.

Antimicrobial activity of Mycobacteriophage D29 Lysin B during Mycobacterium ulcerans infection.

Buruli Ulcer (BU) is a cutaneous disease caused by Mycobacterium ulcerans. The pathogenesis of this disease is closely related to the secretion of the toxin mycolactone that induces extensive destruction of the skin and soft tissues. Currently, there are no effective measures to prevent the disease and, despite availability of antibiotherapy and surgical treatments, these therapeutic options are often associated with severe side effects. Therefore, it is important to develop alternative strategies for the treatment of BU. Endolysins (lysins) are phage encoded enzymes that degrade peptidoglycan of bacterial cell walls. Over the past years, lysins have been emerging as alternative antimicrobial agents against bacterial infections. However, mycobacteria have an unusual outer membrane composed of mycolylarabinogalactan-peptidoglycan. To overcome this complex barrier, some mycobacteriophages encode a lipolytic enzyme, Lysin B (LysB). In this study, we demonstrate for the first time that recombinant LysB displays lytic activity against M. ulcerans isolates. Moreover, using a mouse model of M. ulcerans footpad infection, we show that subcutaneous treatment with LysB prevented further bacterial proliferation, associated with IFN-γ and TNF production in the draining lymph node. These findings highlight the potential use of lysins as a novel therapeutic approach against this neglected tropical disease.

Combination of etoposide and fisetin results in anti-cancer efficiency against osteosarcoma cell models.

Osteosarcoma chemotherapy is often limited by chemoresistance, resulting in poor prognosis. Combined chemotherapy could, therefore, be used to prevent resistance to chemotherapeutics. Here, the effects of fisetin on osteosarcoma cells were investigated, as well as cytostatic potential in combination with the anti-cancer drug etoposide. For this, different osteosarcoma cell lines were treated with fisetin, with etoposide and with respective combinations. Fisetin was associated with decrease in colony formation in Saos-2 and in U2OS cells but not in MG-63 cells. Notwithstanding, upon evaluation of cellular growth by crystal violet assay, MG-63 and Saos-2 cells showed decreased cell proliferation at 40 and 20 µM fisetin, respectively. Depending on the relative concentrations, fisetin:etoposide combinations showed negative-to-positive interactions on the inhibition of cell proliferation. In addition, fisetin treatment up to 50 µM for 48 h resulted in G2-phase cell cycle arrest. Regardless of the combination, fisetin:etoposide increased % cells in G2-phase and decreased % cells in G1-phase. In addition, mixtures with more positive combined effects induced increased % cells in S-phase. Compared to etoposide treatment, these combinations resulted in decreased levels of cyclins B1 and E1, pointing to the role of these regulators in fisetin-induced cell cycle arrest. In conclusion, these results show that the combination of fisetin with etoposide has higher anti-proliferative effects in osteosarcoma associated with cell cycle arrest, allowing the use of lower doses of the chemotherapeutic agent, which has important implications for osteosarcoma treatment.

Hesperetin-etoposide combinations induce cytotoxicity in U2OS cells: Implications on therapeutic developments for osteosarcoma.

Osteosarcoma chemotherapy has improved survival rates, however, chemoresistance and drug toxicity still limit therapy. Drug combinations may overcome these limitations by allowing fewer chemoresistant cells to survive. The aim of this study was to evaluate the cytotoxic potential of hesperetin to osteosarcoma and to analyze the cell cycle effects of combinations of hesperetin with chemotherapeutic agents. For this, the U2OS human osteosarcoma cell line was exposed to hesperetin or hesperetin combined with etoposide or doxorubicin in defined proportions. Hesperetin was less cytotoxic compared to chemotherapeutic agents, as shown by cell growth, viability and clonogenic assays. Notwithstanding, hesperetin combined with etoposide showed additive effects on the inhibition of cell growth. Furthermore, hesperetin induced G2-phase arrest, associated with decreased gene expression of cyclins B1 and E1 and cyclin-dependent kinases 1 and 2. The combination with higher additive effect resulted in higher percentage of cells in G2-phase, showing that G2-phase arrest is associated with cytotoxicity. Moreover, hesperetin induced cytostatic effects. In conclusion, our results suggest that G2-phase arrest is an important step for hesperetin-induced cytotoxicity in U2OS cells. Hesperetin shows potential cytotoxicity when combined with etoposide, which may have implications on therapeutic developments for osteosarcoma.