Marilisa Pedroso Nogueira Gaeti

Toxicity evaluation of the photoprotective compound LQFM048: Eye irritation, skin toxicity and genotoxic endpoints

A new molecule, LQFM048, originally designed through molecular hybridization using green chemistry approach, is in development as a photoprotective agent. Eye irritation, skin toxicity and genotoxicity evaluations are mandatory for predicting health risks. In this context, the purpose of this study was to investigate the eye irritation potential of LQFM048 by combining Short Time Exposure (STE), Bovine Corneal Opacity and Permeability (BCOP) associated with corneal histomorphometry and Hens Egg Test-Chorioallantoic Membrane (HET-CAM). Additionally, skin toxicity was evaluated by interleukin-18 production in the HaCaT keratinocyte, Local Lymph Node Assay (LLNA:BrdU-ELISA) method, 3T3 Neutral red uptake (NRU) assay and in vivo phototoxicity test. Genotoxic potential of LQFM048 was also analyzed by cytokinesis-block micronucleus assay (MNvit test-cytoB) in HepG2 cells. Our results showed that LQFM048 did not induce eye irritation and it was classified as UN GHS No Category for both STE and BCOP assays and non-irritating for HET-CAM test. LQFM048 showed non-potential skin sensitization with stimulation index (SI=0.7) in the LLNA:BrdU-ELISA method. Corroborating in vivo tests, it did not promote significant cytotoxicity in HaCaT cells and it showed similar levels of IL-18 when compared to control. Furthermore, LQFM048 induced non-phototoxic potential with photo-irritation factor (PIF) and mean photo effect (MPE) of 1 and -0.138, respectively, for 3T3 cells. Similarly, it was not phototoxic for in vivo testing with or without exposure to UVA, showing SI values of 1 and 1.2, respectively. The micronucleus test showed that LQFM048 was not genotoxic, under the conditions tested.In conclusion, LQFM048, a heterocyclic compound obtained through an environmentally acceptable simple synthetic route, seems to be safe for human use, especially for the development of a new sunscreen product, since it is neither an eye irritant, nor a contact allergen, nor mutagenic and nor phototoxic.

Microstructured liposome subunit vaccines reduce lung inflammation and bacterial load after Mycobacterium tuberculosis infection

BACKGROUND Tuberculosis is a disease affecting millions of people throughout the world. One of the main problems in controlling the disease is the low efficacy of the Bacillus Calmette-Guérin (BCG) vaccine in protecting young adults. The development of new vaccines that induce a long-lasting immune response or that stimulate the immunity induced by BCG may improve the control of tuberculosis. METHODS The use of microstructured liposomes containing HspX, with or without MPL or CpG DNA adjuvants, as vaccines for tuberculosis was evaluated. The HspX-specific humoral and cellular immune responses to the different vaccine formulations were compared. RESULTS All vaccines containing liposome microparticles and HspX were immunogenic. Vaccines formulated with CpG DNA and HspX induced the strongest humoral and cellular immune responses, mainly by inducing interferon-γ and tumor necrosis factor-α expression by both CD4(+) and CD8(+) T cells. HspX and MPL mainly induced CD8(+) T-cell activation and specific humoral responses. When evaluated the protective efficacy of the formulations against Mycobacterium tuberculosis challenge, the microstructured liposome containing L-HspX and L-HspX-CPG DNA reduced both lung inflammatory lesions and the bacterial load. CONCLUSION We have thus demonstrated, for the first time, the use of microstructured liposomes as an adjuvant and delivery system for a vaccine formulation against tuberculosis.

Characterization, nanoparticle self-organization, and Monte Carlo simulation of magnetoliposomes.

In this work we have developed and implement a new approach for the study of magnetoliposomes using Monte Carlo simulations. Our model is based on interaction among nanoparticles considering magnetic dipolar, van der Waals, ionic-steric, and Zeeman interaction potentials. The ionic interaction between nanoparticles and the lipid bilayer is represented by an ionic repulsion electrical surface potential that depends on the nanoparticle-lipid bilayer distance and the concentration of ions in the solution. A direct comparison among transmission electron microscopy, vibrating sample magnetometer, dynamic light scattering, nanoparticle tracking analysis, and experimentally derived static magnetic birefringence and simulation data allow us to validate our implementation. Our simulations suggest that confinement plays an important role in aggregate formation.

4-Nerolidylcatechol: apoptosis by mitochondrial mechanisms with reduction in cyclin D1 at G0/G1 stage of the chronic myelogenous K562 cell line

Abstract Context: 4-Nerolidylcatechol (4-NRC) has showed antitumor potential through apoptosis. However, its apoptotic mechanisms are still unclear, especially in leukemic cells. Objectives: To evaluate the cytotoxic potential of 4-NRC and its cell death pathways in p53-null K562 leukemic cells. Materials and methods: Cytotoxicity of 4-NRC (4.17–534.5 μM) over 24 h of exposure was evaluated by MTT assay. 4-NRC-induced apoptosis in K562 cells was investigated by phosphatidylserine (PS) externalization, cell cycle, sub-G1, mitochondrial evaluation, cytochrome c, cyclin D1 and intracellular reactive oxygen species (ROS) levels, and caspase activity analysis. Results: IC50 values obtained were 11.40, 27.31, 15.93 and 15.70 μM for lymphocytes, K562, HL-60 and Jurkat cells, respectively. In K562 cells, 4-NRC (27 μM) promoted apoptosis as verified by cellular morphological changes, a significant increase in PS externalization and sub-G1 cells. Moreover, it significantly arrested the cells at the G0/G1 phase due to a reduction in cyclin D1 expression. These effects of 4-NRC also significantly promoted a reduction in mitochondrial activity and membrane depolarization, accumulation of cytosolic cytochrome c and ROS overproduction. Additionally, it triggered an increase in caspases -3/7, -8 and -9 activities. When the cells were pretreated with N-acetyl-l-cysteine ROS scavenger, 4-NRC-induced apoptosis was partially blocked, which suggests that it exerts cytotoxicity though not exclusively through ROS-mediated mechanisms. Discussion and conclusion: 4-NRC has antileukemic properties, inducing apoptosis mediated by mitochondrial-dependent mechanisms with cyclin D1 inhibition. Given that emerging treatment concepts include novel combinations of well-known agents, 4-NRC could offer a promising alternative for chemotherapeutic combinations to maximize tumour suppression.

In Vivo Vaginal Fungal Load Reduction After Treatment with Itraconazole-Loaded Polycaprolactone-Nanoparticles

Itraconazole (ITZ) has a broad spectrum of action and is commonly used for the treatment of fungal infections. Topic administration of ITZ is a promising strategy to improve vulvovaginal candidiasis treatment, which can be further optimized by its encapsulation in nanoparticles to increase drug delivery and reduce ITZ toxicity. In this work, we designed polycaprolactone nanoparticles containing ITZ and evaluated in vivo the efficacy of this yet unexplored approach. Nanocapsules (ITZ-NC) and nanospheres (ITZ-NS) were obtained by nanoprecipitation. ITZ-NC presented encapsulation efficiency of 99%, mean diameter of 190 nm, PDI 0.1 and zeta potential of -15 mV. ITZ-NS showed encapsulation efficiency of 97%, mean diameter of 120 nm, PDI 0.1 and zeta potential of -10 mV. Both particles were efficiently freeze-dried using 10% trehalose + 10% sucrose. Nanoparticles were then incorporated in a viscous formulation for vaginal application in female Balb/C mice infected with Candida albicans. Fungal load was significantly reduced in infected animals after treatment with ITZ-NC but not with ITZ-NS, compared to animals treated with ITZ solution. Histological analysis showed a clear difference between vaginal tissues of ITZ-NC and ITZ-NS and ITZ solution-treated animals, which correlated with IL-1β and TNF-α quantification. Animals treated with ITZ-NC showed reduced cytokine levels and healthy tissue characteristics, while animals treated with ITZ-NS and ITZ solution showed increased IL-1β and TNF-α levels and typical tissue inflammation. Our results demonstrate the potential of ITZ-NC to improve the treatment of vulvovaginal candidiasis after topical application in the vagina, opening new perspectives for the treatment of this disease.