Navigating towards improved cytotoxicity assessment in nanomedicine development: Shifting from colorimetric to fluorescence-based assays

It is well known that the characterization of nanomedicines can pursue different levels of complexity, both in the development stage and in the quality control process [1]. In line with physicochemical aspects, even more obstacles are encountered in biological safety assessment, while anticipation of their immunogenic potential represents an additional challenge. Moreover, interactions between the test reagents and the nanomaterial have been identified as one of the most important issues in toxicity testing that influence market authorization of nanomedicines, which ought to be resolved [1]. The European Nanomedicine Characterization Laboratory – the reference laboratory for nanomedicines, provides protocols for 2 colorimetric cytotoxicity assays employing LLC-PK1 (porcine kidney epithelial cells) and Hep-G2 (human hepatocarcinoma cells) cell lines. However, the latest recommendations in the field underline the demand for enhancing the testing procedures, while proposing incorporation of immune cells as target cell lines for toxicity evaluation, aiming to provide more reliable conclusions on nanomedicine safety in preclinical level. In this study 2 inherently different types of pharmaceutical nanosystemes were selected: nanoemulsion (NE) and solid lipid nanoparticles (LNP) and subjected to a set of orthogonal toxicity evaluation assays. Adjusted WST-1 (assessing mitochondrial activity as an indicator of cellular well-being) and LDH (lactate dehydrogenase release evaluation as an indicator of cell membrane damage) assays have been performed as the colorimetric tests, while propidium-iodide (PI)-based assay was developed as a fluore-scent counterpart (able to directly distinguish live from dead cells), using RAW 246.7 cell line (murine macrophages – immune system cell line). Starting concentration of the tested nanoformulations was 50 % v/v, and they were subsequently diluted with the factor of 2, to create a total of 8 concentrations. Incubation time was 4 h. Presented assays rely on completely different biological bases. Therefore, their careful combination can address some shortcomings in the in vitro evaluations established so far. Although similar toxicity trends were observed regardless the assay used, it was evident that the LDH assay required specific consideration. Since the supernatant is the subject of the analysis (not the cells directly), containing not only the enzyme of interest, but also the nanoformulations, in the wells corresponding to the 3 highest concentration of the NE/LNP pronounced scattering effects were observed. Such an event could be easily overlooked, potentially affecting the conclusions. However, it was overcome by careful design of control and blank wells (each test concentration was coupled with its own blank well containing no cells, but the same concentration of the NE/LNP in the culture medium). In contrast, absorbance measurements in WST-1 assay were performed in the absence of the NE/LNP, avoiding any interactions or scattering effects. Finally, developed PI-based assay proved to be the most relevant method. Based on the penetration of PI into the dead cell only, attaching to their DNA, the concentration of the dead vs. live cells could be directly estimated. What is more, after the incubation time, the measurements can be performed in the nanoformulation-free environment, surpas-sing the potential interactions. Notably, cell viability obtained in the PI-based assay followed the same trend as in the WST-1 assay but with significant difference in the obtained values for the first 3 concentrations (Figure 1).10th IAPC Meeting, Book of Abstract

Toll-like receptor 2 regulates the barrier function of human bronchial epithelial monolayers through atypical protein kinase C zeta, and an increase in expression of claudin-1

We investigated the role of Toll-like receptor (TLR) 2 in maintaining the integrity of the airway epithelial barrier using the human bronchial epithelial cell line Calu-3. Activation of TLR2 by its ligands, Pam3CysSK4 and Peptidoglycan showed a concentration dependent increase in epithelial barrier function, as measured by transepithelial electrical resistance (TEER). This was confirmed by a decrease in paracellular flux of fluorescein sodium. This TLR2 induced increase in TEER was significantly reduced by pretreatment with polyclonal anti-human TLR2-neutralizing antibody. TLR2 stimulation in Calu-3 cell monolayers resulted in an increased expression of the tight junction proteins claudin-1 and ZO-1, and a decreased expression of occludin, at both the mRNA and protein levels. A pseudosubstrate inhibitor to PKCζ significantly prevented the TLR2 mediated increase in barrier function. It also prevented the increase in claudin-1 in a concentration dependent manner up to 1 µM. TLR2 stimulation led to an increase in phosphorylation of atypical PKC ζ, which was prevented by the pseudosubstrate inhibitor in a concentration dependent manner. Taken together, our observations support a model whereby increased tight junction barrier function induced by activation of TLR2 occurs through increased expression of claudin-1, and through modulation of PKC ζ activity

Highly Potent Cationic Chitosan Derivatives Coupled to Antimicrobial Peptide Dendrimers to Combat Pseudomonas Aeruginosa.

The burden of bacterial wound infections has considerably increased due to antibiotic resistance to most of the currently available antimicrobial drugs. Herein, we report for the first time a chemical coupling of two cationic N-aryl (pyridyl and aminocinnamyl) chitosan derivatives to antimicrobial peptide dendrimers (AMPDs) of different generations (first, second and third) via thioether-haloacetyl reaction. The new chitosan-AMPD conjugates showed high selectivity by killing Pseudomonas aeruginosa and very low toxicity towards mammalian cells, as well as extremely low hemolysis to red blood cells. Electron microscopy revealed that the new chitosan derivatives coupled to AMPD destroyed both the inner and outer membranes of Gram-negative P. aeruginosa. Moreover, chitosan-AMPD conjugates showed synergetic effects within extremely low concentrations. The new chitosan-AMPD conjugates can be used as potent antimicrobial therapeutic agents, to eradicate pathogens such as those present in acute and chronic infected wounds. This article is protected by copyright. All rights reserved

Multifunctional PLGA-Based Nanoparticles Encapsulating Simultaneously Hydrophilic Antigen and Hydrophobic Immunomodulator for Mucosal Immunization

We describe here the development of nanoparticles made from poly­(lactic-<i>co</i>-glycolic acid) (PLGA) able to deliver an encapsulated antigen with a Toll-Like Receptor-7 (TLR-7) agonist as immunostimulatory signal and coated with a muco-adhesive chitosan-derivate layer. The potential to stimulate an immune response of these vaccine formulations in the absence or presence of the TLR-7 agonist at the systemic and mucosal level were evaluated in mice following subcutaneous or nasal administrations. Intranasally immunized mice developed a high systemic immune response equivalent to mice injected subcutaneously. However, mucosal immune responses were only induced at local and distal sites in mucosally immunized animals. The adjuvant effect of imiquimod on the polarization of the immune response was only detected at local sites, which tends to increase safety of this vaccine delivery system

Wound Healing Promotion by Hyaluronic Acid: Effect of Molecular Weight on Gene Expression and In Vivo Wound Closure

Hyaluronic acid (HA) has been known to play an important role in wound healing process. However, the effect of molecular weight (MW) of exogenously administered HA on the wound healing process has not been fully understood. In this study, we investigated HA with different MWs on wound healing process using human epidermal keratinocytes and dermal fibroblasts. Cell proliferation and migration ability were assessed by water soluble tetrazolium (WST) assay and wound scratch assay. We examined the effect of HA addition in a full-thickness wound model in mice and the gene expression related to wound healing. Proliferation and migration of HaCaT cells increased with the increase of MW and concentration of HA. Interleukin (IL-1β), IL-8 and vascular endothelial growth factor (VEGF) as well as matrix metalloproteinase (MMP)-9 and MMP-13 were significantly upregulated by high molecular weight (HMW) HA in keratinocytes. Together with VEGF upregulation and the observed promotion of HaCaT migration, HA with the MW of 2290 kDa may hold potential to improve re-epithelialization, a critical obstacle to heal chronic wounds

Recommandations sur la mesure de la pression artérielle. Consensus d’experts de la Société française d’hypertension artérielle, filiale de la Société française de cardiologie

International audienceSince 2016, the French Society of Hypertension has warned about the decline in the management of high blood pressure in France: stagnation, or even decreased number of people who know their blood pressure level, take a treatment and are controlled. These results are lower than those observed in many other countries. Blood pressure is measured with an old method in the doctor's office. Accepted, simple and cost-effective, this method is currently unavoidable for reasons of feasibility and social habit. It has been used in observational and intervention studies that are the basis of the medical reasoning for screening, treatment and drug control of hypertension. In practice, it is too often poorly applied and unpredictable. It is now necessary to measure blood pressure in mmHg using a validated oscillometric automatic device coupled to a specific upper arm cuff adapted to the arm circumferences for the diagnosis and monitoring of high blood pressure in the doctor's office and at home. The auscultatoric measurement is only recommended if any doubt about the reliability of the electronic measurement. Blood pressure measurement is basically performed on both arms to detect asymmetry and then on the arm with the highest blood pressure. It is performed in sitting or lying position after a few minutes of rest without speaking and without having smoked and then in standing position to diagnose orthostatic hypotension, especially in elderly, diabetic and multi-medicated subjects. The blood pressure measurement during the consultation must be repeated and include at least 3 consecutive measurements at one minute intervals. The average of the last 2 measurements determines the blood pressure level. It is recommended to perform BP measurements outside the medical environment for the diagnosis and monitoring of hypertension; Home BP measurement is preferred to ambulatory blood pressure measurement for practical reasons unless otherwise specified. The home blood pressure measurement should include three measurements in the morning at breakfast and three measurements in the evening before bedtime at one minute intervals for at least three days. Prior training must be provided. In treated hypertensive patients, a masked hypertension should be considered as an uncontrolled hypertension and antihypertensive therapy adapted accordingly. The measurement of central BP pressure (aorta) should be limited to clinical research

Increased risk of severe COVID-19 in hospitalized patients with SARS-CoV-2 Alpha variant infection: a multicentre matched cohort study

International audienceBackground: The impact of the variant of concern (VOC) Alpha on the severity of COVID-19 has been debated. We report our analysis in France.Methods: We conducted an exposed/unexposed cohort study with retrospective data collection, comparing patients infected by VOC Alpha to contemporaneous patients infected by historical lineages. Participants were matched on age (± 2.5 years), sex and region of hospitalization. The primary endpoint was the proportion of hospitalized participants with severe COVID-19, defined as a WHO-scale > 5 or by the need of a non-rebreather mask, occurring up to day 29 after admission. We used a logistic regression model stratified on each matched pair and accounting for factors known to be associated with the severity of the disease.Results: We included 650 pairs of patients hospitalized between Jan 1, 2021, and Feb 28, 2021, in 47 hospitals. Median age was 70 years and 61.3% of participants were male. The proportion of participants with comorbidities was high in both groups (85.0% vs 90%, p = 0.004). Infection by VOC Alpha was associated with a higher odds of severe COVID-19 (41.7% vs 38.5%-aOR = 1.33 95% CI [1.03-1.72]).Conclusion: Infection by the VOC Alpha was associated with a higher odds of severe COVID-19