Sens-ocular model: Cell-based assay to evaluate eye stinging potential of chemicals and baby cosmetic formulations.

The TRPV1 receptor, which is known to contribute significantly to pain perception, has recently been identified as a useful tool for predicting eye stinging potential in cosmetics. In this study, HEK-293 cells with high TRPV1 expression were utilized to evaluate calcium influx related to receptor activation triggered by chemicals and cosmetic formulations. The cells were exposed to increasing concentrations of substances to cause or not some aggression to the eye, and TRPV1 activity was assessed by measuring intracellular FURA-2 AM fluorescence signal. To confirm TRPV1 channel activation, capsazepine, a capsaicin antagonist, was employed in addition to using capsaicin as a positive control. The study's results indicate that this novel model can identify compounds known to cause some aggression to the eye, such as stinging, considering a cut-off value of 60% of Ca2+ influx exposed to the lowest evaluated concentration (0.00032%). When applied to the cosmetic baby formulation, although the presented model exhibited higher sensitivity by classifying as stinging formulations that had previously undergone clinical testing and were deemed non-stinging, the assay could serve as a valuable in vitro tool for predicting human eye stinging sensation and can be used as a tier 1 in an integrated testing strategy.

The Application of New Approach Methodologies in Respiratory Disease Research: Their Role in Improving Translational Medicine from Bench to Bedside.

The SARS-CoV-2 outbreak focused global attention on the shortcomings of the drug discovery process. It led to its acceleration in several areas, particularly in the processes associated with the development and approval of COVID-19 vaccines. This situation contrasts with the low approval rates of new drugs for respiratory system diseases (e.g. asthma, chronic obstructive pulmonary disease, cancer, tuberculosis), which are leading causes of morbidity and mortality worldwide. In this context, innovation in respiratory system drug discovery is surely needed, and it is most likely to succeed through the use of preclinical models that are cost-effective, high-throughput and generate predictive human-relevant outcomes. Here, we highlight several non-animal new approach methodologies (NAMs) and their applications in respiratory research. We describe their potential uses for efficacy and toxicity assessments, to optimise the drug development process and reduce the high failure rates in clinical trials.

Paclitaxel-Loaded Lipid-Coated Magnetic Nanoparticles for Dual Chemo-Magnetic Hyperthermia Therapy of Melanoma.

Melanoma is the most aggressive and metastasis-prone form of skin cancer. Conventional therapies include chemotherapeutic agents, either as small molecules or carried by FDA-approved nanostructures. However, systemic toxicity and side effects still remain as major drawbacks. With the advancement of nanomedicine, new delivery strategies emerge at a regular pace, aiming to overcome these challenges. Stimulus-responsive drug delivery systems might considerably reduce systemic toxicity and side-effects by limiting drug release to the affected area. Herein, we report the development of paclitaxel-loaded lipid-coated manganese ferrite magnetic nanoparticles (PTX-LMNP) as magnetosomes synthetic analogs, envisaging the combined chemo-magnetic hyperthermia treatment of melanoma. PTX-LMNP physicochemical properties were verified, including their shape, size, crystallinity, FTIR spectrum, magnetization profile, and temperature profile under magnetic hyperthermia (MHT). Their diffusion in porcine ear skin (a model for human skin) was investigated after intradermal administration via fluorescence microscopy. Cumulative PTX release kinetics under different temperatures, either preceded or not by MHT, were assessed. Intrinsic cytotoxicity against B16F10 cells was determined via neutral red uptake assay after 48 h of incubation (long-term assay), as well as B16F10 cells viability after 1 h of incubation (short-term assay), followed by MHT. PTX-LMNP-mediated MHT triggers PTX release, allowing its thermal-modulated local delivery to diseased sites, within short timeframes. Moreover, half-maximal PTX inhibitory concentration (IC50) could be significantly reduced relatively to free PTX (142,500×) and Taxol® (340×). Therefore, the dual chemo-MHT therapy mediated by intratumorally injected PTX-LMNP stands out as a promising alternative to efficiently deliver PTX to melanoma cells, consequently reducing systemic side effects commonly associated with conventional chemotherapies.

Folate-Targeted PEGylated Magnetoliposomes for Hyperthermia-Mediated Controlled Release of Doxorubicin.

Doxorubicin (DOX) is a chemotherapeutic agent commonly used for the treatment of solid tumors. However, the cardiotoxicity associated with its prolonged use prevents further adherence and therapeutic efficacy. By encapsulating DOX within a PEGylated liposome, Doxil® considerably decreased DOX cardiotoxicity. By using thermally sensitive lysolipids in its bilayer composition, ThermoDox® implemented a heat-induced controlled release of DOX. However, both ThermoDox® and Doxil® rely on their passive retention in tumors, depending on their half-lives in blood. Moreover, ThermoDox® ordinarily depend on invasive radiofrequency-generating metallic probes for local heating. In this study, we prepare, characterize, and evaluate the antitumoral capabilities of DOX-loaded folate-targeted PEGylated magnetoliposomes (DFPML). Unlike ThermoDox®, DOX delivery via DFPML is mediated by the heat released through dynamic hysteresis losses from magnetothermal converting systems composed by MnFe2O4 nanoparticles (NPs) under AC magnetic field excitation-a non-invasive technique designated magnetic hyperthermia (MHT). Moreover, DFPML dismisses the use of thermally sensitive lysolipids, allowing the use of simpler and cheaper alternative lipids. MnFe2O4 NPs and DFPML are fully characterized in terms of their size, morphology, polydispersion, magnetic, and magnetothermal properties. About 50% of the DOX load is released from DFPML after 30 min under MHT conditions. Being folate-targeted, in vitro DFPML antitumoral activity is higher (IC50 ≈ 1 µg/ml) for folate receptor-overexpressing B16F10 murine melanoma cells, compared to MCF7 human breast adenocarcinoma cells (IC50 ≈ 4 µg/ml). Taken together, our results indicate that DFPML are strong candidates for folate-targeted anticancer therapies based on DOX controlled release.

Correlation of hair cortisol and interleukin 6 with structural change in the active progression of keratoconus.

PURPOSE: To evaluate interleukin (IL) and hair cortisol concentrations (HCCs) in progressive keratoconus (KC) and compare them with KC-stable eyes and healthy control, and to determine the correlation of these inflammatory mediators and HCCs and their relationship with structural damage represented by increased corneal curvature. SETTING: University of Sao Paulo, Brazil. DESIGN: Prospective observational comparative study. METHODS: 133 eyes of 74 patients were included. The concentrations of tear cytokines: IL1B, IL6, IL8, IL10, IL12p70, and tumor necrosis factor α were obtained by capillary flow and measured using a flow cytometer. HCCs were determined from the most proximal hair segment as an index of cumulative secretion and measured by liquid chromatography mass spectrometry. RESULTS: 133 eyes of 74 patients. Only IL6 was increased in progressive KC tears compared with stable KC (6.59 ± 3.25 pg/mL vs 4.72 ± 1.91 pg/mL; P < .0001) with a positive correlation between IL6 and maximum keratometry (Kmax) (P < .0001). Progressive KC exhibited significantly higher HCC than stable KC (0.624 ± 0.160 ng/mg vs 0.368 ± 0.0647 ng/mg; P < .0001) and healthy controls (0.624 ± 0.160 ng/mg vs 0.351 ± 0.0896 ng/mg; P < .0001). There was a significant correlation between HCC and Kmax (P < .0001). CONCLUSIONS: KC eyes that are progressing have a higher concentration of IL6 and long-term cortisol than patients with stable forms of KC; second, there is a significant correlation between this increase in IL6 and cortisol with corneal structural damage. Finally, there is a meaningful relationship between this interleukin and the previous few months' cortisol levels.

Application of the adverse outcome pathway framework for investigating skin sensitization potential of nanomaterials using new approach methods.

BACKGROUND: Currently, considerable efforts to standardize methods for accurate assessment of properties and safety aspects of nanomaterials are being made. However, immunomodulation effects upon skin exposure to nanomaterial have not been explored. OBJECTIVES: To investigate the immunotoxicity of single-wall carbon nanotubes, titanium dioxide, and fullerene using the current mechanistic understanding of skin sensitization by applying the concept of adverse outcome pathway (AOP). METHODS: Investigation of the ability of nanomaterials to interact with skin proteins using the micro-direct peptide reactivity assay; the expression of CD86 cell surface marker using the U937 cell activation test (OECD No. 442E/2018); and the effects of nanomaterials on modulating inflammatory response through inflammatory cytokine release by U937 cells. RESULTS: The nanomaterials easily internalized into keratinocytes cells, interacted with skin proteins, and triggered activation of U937 cells by increasing CD86 expression and modulating inflammatory cytokine production. Consequently, these nanomaterials were classified as skin sensitizers in vitro. CONCLUSIONS: Our study suggests the potential immunotoxicity of nanomaterials and highlights the importance of studying the immunotoxicity and skin sensitization potential of nanomaterials to anticipate possible human health risks using standardized mechanistic nonanimal methods with high predictive accuracy. Therefore, it contributes toward the applicability of existing OECD (Organisation for Economic Co-operation and Development) testing guidelines for accurate assessment of nanomaterial skin sensitization potential.

Anti-inflammatory and antinociceptive activity profile of a new lead compound - LQFM219.

LQFM219 is a molecule designed from celecoxibe (COX-2 inhibitor) and darbufelone (inhibitor of COX-2 and 5-LOX) lead compounds through a molecular hybridisation strategy. Therefore, this work aimed to investigate the antinociceptive and anti-inflammatory activities of this new hybrid compound. The acute oral systemic toxicity of LQFM219 was evaluated via the neutral red uptake assay. Acetic acid-induced abdominal writhing and CFA-induced mechanical hyperalgesia were performed to evaluate the antinociceptive activity, and the anti-oedematogenic activity was studied by CFA-induced paw oedema and croton oil-induced ear oedema. Moreover, the acute anti-inflammatory activity was determined by carrageenan-induced pleurisy. In addition, cell migration, myeloperoxidase enzyme activity, and TNF-α and IL-1ß levels were determined in pleural exudate. Moreover, a redox assay was conducted using electroanalytical and DPPH methods. The results demonstrated that LQFM219 was classified as GHS category 4, and it showed better free radical scavenger activity compared to BHT. Besides, LQFM219 decreased the number of writhings induced by acetic acid and the response to the mechanical stimulus in the CFA-induced mechanical hyperalgesia test. Furthermore, LQFM219 reduced oedema formation, cell migration, and IL-1ß and TNF-α levels in the pleural cavity and inhibited myeloperoxidase enzyme activity. Thus, our study provides that the new pyrazole derivative, LQFM219, demonstrated low toxicity, antinociceptive and anti-inflammatory potential in vitro and in vivo.

A new corneal epithelial biomimetic 3D model for in vitro eye toxicity assessment: Development, characterization and applicability.

In vitro eye toxicity assessment using reconstructed corneal epithelial models has emerged highlighting its applicability domain for Classification and Labeling of products and chemicals. However, due to bureaucratic issues, such models are not commercially available in Brazil and Latin America. In this work, we developed, characterized and evaluated the applicability of a new corneal epithelial biomimetic model using a cell lineage for in vitro eye toxicity assessment. The reconstructed tissue was obtained through the cultivation of HaCaT cells in an air-liquid interface, which presented morphology and biomarkers expression such as cytokeratin, CD44, and Ki-67 similar to human tissue. Furthermore, tissue viability was evaluated after exposure of the epithelial model to isolated chemicals from different Globally Harmonized System (GHS) eye irritation categories, and it has been demonstrated to be a suitable endpoint for classification of test materials, allowing discrimination between irritant and non-irritant chemicals. Furthermore, the model showed suitability for testing "real-life mixtures", once it identified irritant products between the analyzed eyebrow henna samples commercially labeled as non-irritants. This reproducible and low-cost epithelial corneal model presents features very important for Brazil and South America for R&D&I with no unnecessary animal experimentation.

Protective effect of sucupira oil nanoemulsion against oxidative stress in UVA-irradiated HaCaT cells.

OBJECTIVES: Bioactive molecules derived from natural products combine the ability to absorb UV light and act as antioxidants. We developed an oil-based sucupira (native species of the Brazilian cerrado) nanoemulsion (NE) using a high-energy emulsification method and assessed its effectiveness in vitro. METHODS: An easily scalable high-pressure homogenization method was used to prepare the formulation. NE droplets mean diameter, pH, stability, conductivity and morphology were analysed. Formulation bioactivity was assessed using HaCaT cells. KEY FINDINGS: The formulation presented suitable pH and size for topic administration and was stable for over 90 days upon storage at 4, 25 and 45°C. The NE showed protective effect against oxidative stress and reduced levels of UVA-induced pro-inflammatory cytokines IL-6 and IL-8. CONCLUSIONS: A novel, stable and easily prepared formulation was obtained for encapsulation of sucupira oil. The protective effect of the formulation by cytokine inhibition in the early stage of the inflammatory process was shown in vitro. Combined with the antioxidant effect by inhibition of reactive oxygen species, the use of sucupira oil NE for prevention and treatment of UVA-induced stress could contribute to decrease the effects of UV radiation on skin ageing.

Translation, adaptation and validation of a Portuguese version of the Moorehead-Ardelt Quality of Life Questionnaire II.

The prevalence of obesity has increased worldwide. An assessment of the impact of obesity on health-related quality of life (HRQoL) requires specific instruments. The Moorehead-Ardelt Quality of Life Questionnaire II (MA-II) is a widely used instrument to assess HRQoL in morbidly obese patients. The objective of this study was to translate and validate a Portuguese version of the MA-II.The study included forward and backward translations of the original MA-II. The reliability of the Portuguese MA-II was estimated using the internal consistency and test-retest methods. For validation purposes, the Spearman's rank correlation coefficient was used to evaluate the correlation between the Portuguese MA-II and the Portuguese versions of two other questionnaires, the 36-item Short Form Health Survey (SF-36) and the Impact of Weight on Quality of Life-Lite (IWQOL-Lite).One hundred and fifty morbidly obese patients were randomly assigned to test the reliability and validity of the Portuguese MA-II. Good internal consistency was demonstrated by a Cronbach's alpha coefficient of 0.80, and a very good agreement in terms of test-retest reliability was recorded, with an overall intraclass correlation coefficient (ICC) of 0.88. The total sums of MA-II scores and each item of MA-II were significantly correlated with all domains of SF-36 and IWQOL-Lite. A statistically significant negative correlation was found between the MA-II total score and BMI. Moreover, age, gender and surgical status were independent predictors of MA-II total score.A reliable and valid Portuguese version of the MA-II was produced, thus enabling the routine use of MA-II in the morbidly obese Portuguese population.