Impact of the chemical composition of poly-substituted hydroxyapatite particles on the in vitro pro-inflammatory response of macrophages

International audienceTo improve the biological properties of calcium phosphate (CaP) bone substitute, new chemical compositions are under development. In vivo such materials are subject to degradation that could lead to particles release and inflammatory reactions detrimental to the bone healing process. This study aimed at investigating the interactions between a murine macrophage cell line (RAW 264.7) and substituted hydroxyapatite particles presenting promising biological properties. Micron size particles of stoichiometric and substituted hydroxyapatites (CO 3 substitution for PO 4 and OH; SiO 4 substitution for PO 4; CO 3 and SiO 4 co-substitution) were obtained by aqueous precipitation followed by spray drying. Cells, incubated with four doses of particles ranging from 15 to 120 µg/mL, revealed no significant LDH release or ROS production, indicating no apparent cytotoxicity and no oxidative stress. TNF-α production was independent of the chemistry of the particles; however the particles elicited a significant dose-dependent pro-inflammatory response. As micron size particles of these hydroxyapatites could be at the origin of inflammation, attention must be paid to the degradation 2 behavior of substituted hydroxyapatite bone substitute in order to limit, in vivo, the generation of particulate debris

Quantification of nanoparticle endocytosis based on double fluorescent pH-sensitive nanoparticles

International audienceAmorphous silica is a particularly interesting material because of its inertness and chemical stability. Silica nanoparticles have been recently developed for biomedical purposes but their innocuousness must be carefully investigated before clinical use. The relationship between nanoparticles physicochemical features, their uptake by cells and their biological activity represents a crucial issue, especially for the development of nanomedicine. This work aimed at adapting a method for the quantification of nanoparticle endocytosis based on pH-sensitive and double fluorescent particles. For that purpose, silica nanoparticles containing two fluorophores: FITC and pHrodoTM were developed, their respective fluorescence emission depends on the external pH. Indeed, FITC emits a green fluorescence at physiological pH and pHrodoTM emits a red fluorescence which intensity increased with acidification. Therefore, nanoparticles remained outside the cells could be clearly distinguished from nanoparticles uptaken by cells as these latter could be spotted inside cellular acidic compartments (such as phagolysosomes, micropinosomes...). Using this model, the endocytosis of 60 nm nanoparticles incubated with the RAW 264.7 macrophages was quantified using time-lapse microscopy and compared to that of 130 nm submicronic particles. The amount of internalized particles was also evaluated by fluorimetry. The biological impact of the particles was also investigated in terms of cytotoxicity, pro-inflammatory response and oxidative stress. Results clearly demonstrated that nanoparticles were more uptaken and more reactive than submicronic particles. Moreover, we validated a method of endocytosis quantification

Quantification of microsized fluorescent particles phagocytosis to a better knowledge of toxicity mechanisms

International audienceBackground: The use of micro- or nanometric particles is in full expansion for the development of new technologies. These particles may exhibit variable toxicity levels depending on their physicochemical characteristics. We focused our attention on macrophages (MA), the main target cells of the respiratory system responsible for the phagocytosis of the particles. The quantification of the amount of phagocytosed particles seems to be a major element for a better knowledge of toxicity mechanisms. The aim of this study was to develop a quantitative evaluation of uptake using both flow cytometry (FCM) and confocal microscopy to distinguish entirely engulfed fluorescent microsized particles from those just adherent to the cell membrane and to compare these data to in vitro toxicity assessments. Methods: Fluorescent particles of variable and well-characterised sizes and surface coatings were incubated with MA (RAW 264.7 cell line). Analyses were performed using confocal microscopy and FCM. The biological toxicity of the particles was evaluated [lactate dehydrogenase (LDH) release, tumor necrosis factor (TNF)-α, and reactive oxygen species (ROS) production]. Results and conclusion: Confocal imaging allowed visualization of entirely engulfed beads. The amount of phagocytic cells was greater for carboxylate 2-µm beads (49±11%) than for amine 1-µm beads (18±5%). Similarly, side scatter geometric means, reflecting cellular complexity, were 446±7 and 139±12, respectively. These results confirm that the phagocytosis level highly depends on the size and surface chemical groups of the particles. Only TNF-α and global ROS production varied significantly after 24-h incubation. There was no effect on LDH and H2O2 production

Quantitative cellular uptake of double fluorescent core-shelled model submicronic particles

International audienceThe relationship between particles' physicochemical parameters, their uptake by cells and their degree of biological toxicity represent a crucial issue, especially for the development of new technologies such as fabrication of micro- and nanoparticles in the promising field of drug delivery systems. This work was aimed at developing a proof-of-concept for a novel model of double fluorescence submicronic particles that could be spotted inside phagolysosomes. Fluorescein isothiocyanate (FITC) particles were synthesized and then conjugated with a fluorescent pHrodo™ probe, red fluorescence of which increases in acidic conditions such as within lysosomes. After validation in acellular conditions by spectral analysis with confocal microscopy and dynamic light scattering, quantification of phagocytosis was conducted on a macrophage cell line in vitro. The biological impact of pHrodo functionalization (cytotoxicity, inflammatory response, and oxidative stress) was also investigated. Results validate the proof-of-concept of double fluorescent particles (FITC + pHrodo), allowing detection of entirely engulfed pHrodo particles (green and red labeling). Moreover incorporation of pHrodo had no major effects on cytotoxicity compared to particles without pHrodo, making them a powerful tool for micro- and nanotechnologies

Size of submicrometric and nanometric particles affect cellular uptake and biological activity of macrophages in vitro

International audienceBackground: Micrometric and nanometric particles are increasingly used in different fields and may exhibit variable toxicity levels depending on their physicochemical characteristics. The aim of this study was to determine the impact of the size parameter on cellular uptake and biological activity, working with well-characterized fluorescent particles. We focused our attention on macrophages, the main target cells of the respiratory system responsible for the phagocytosis of the particles. Methods: FITC fluorescent silica particles of variable submicronic sizes (850, 500, 250 and 150 nm) but with similar surface coating (COOH) were tailored and physico-chemically characterized. These particles were then incubated with the RAW 264.7 macrophage cell line. After microscopic observations (SEM, TEM, confocal), a quantitative evaluation of the uptake was carried out. Fluorescence detected after a quenching with trypan blue allows us to distinguish and quantify entirely engulfed fluorescent particles from those just adhering to the cell membrane. Finally, these data were compared to the in vitro toxicity assessed in terms of cell damage, inflammation and oxidative stress (evaluated by LDH release, TNF-α and ROS production respectively). Results and conclusion: Particles were well characterized (fluorescence, size distribution, zeta potential, agglomeration and surface groups) and easily visualized after cellular uptake using confocal and electron microscopy. The number of internalized particles was precisely evaluated. Size was found to be an important parameter regarding particles uptake and in vitro toxicity but this latter strongly depends on the particles doses employed

Development of innovative pH sensor nanoparticles: a new approach to evaluate phagocytosis

International audienceMacrophages (MA) from the respiratory tract constitute the first defense line against inhaled nanoparticles (NP) thanks to their phagocytic activity. The toxicity of NP mainly depends on their physicochemical characteristics (size, morphology, chemical surface groups...). However the relationship between the number of phagocytosed NP and their cellular activity is still unclear. In this context the accurate quantification of internalized NP by macrophages could allow a better assessment of NP toxicity and uptake mechanisms. The investigation of a relationship between NP internalization, physicochemical parameters and the degree of toxicity represents a crucial issue

Modèle de quantification de la phagocytose à partir de particules fluorescentes

International audienceLes particules industrielles fines et ultrafines peuvent être inhalées et atteindre les alvéoles pulmonaires où elles interagiront préférentiellement avec des macrophages alvéolaires dotés d'une activité de phagocytose qui leur permet d'internaliser les particules étrangères afin de les éliminer

Functional mechanisms underlying pleiotropic risk alleles at the 19p13.1 breast-ovarian cancer susceptibility locus

A locus at 19p13 is associated with breast cancer (BC) and ovarian cancer (OC) risk. Here we analyse 438 SNPs in this region in 46,451 BC and 15,438 OC cases, 15,252 BRCA1 mutation carriers and 73,444 controls and identify 13 candidate causal SNPs associated with serous OC (P=9.2 × 10-20), ER-negative BC (P=1.1 × 10-13), BRCA1-associated BC (P=7.7 × 10-16) and triple negative BC (P-diff=2 × 10-5). Genotype-gene expression associations are identified for candidate target genes ANKLE1 (P=2 × 10-3) and ABHD8 (P<2 × 10-3). Chromosome conformation capture identifies interactions between four candidate SNPs and ABHD8, and luciferase assays indicate six risk alleles increased transactivation of the ADHD8 promoter. Targeted deletion of a region containing risk SNP rs56069439 in a putative enhancer induces ANKLE1 downregulation; and mRNA stability assays indicate functional effects for an ANKLE1 3′-UTR SNP. Altogether, these data suggest that multiple SNPs at 19p13 regulate ABHD8 and perhaps ANKLE1 expression, and indicate common mechanisms underlying breast and ovarian cancer risk

National trends in total cholesterol obscure heterogeneous changes in HDL and non-HDL cholesterol and total-to-HDL cholesterol ratio : a pooled analysis of 458 population-based studies in Asian and Western countries

Background: Although high-density lipoprotein (HDL) and non-HDL cholesterol have opposite associations with coronary heart disease, multi-country reports of lipid trends only use total cholesterol (TC). Our aim was to compare trends in total, HDL and nonHDL cholesterol and the total-to-HDL cholesterol ratio in Asian and Western countries. Methods: We pooled 458 population-based studies with 82.1 million participants in 23 Asian and Western countries. We estimated changes in mean total, HDL and non-HDL cholesterol and mean total-to-HDL cholesterol ratio by country, sex and age group. Results: Since similar to 1980, mean TC increased in Asian countries. In Japan and South Korea, the TC rise was due to rising HDL cholesterol, which increased by up to 0.17 mmol/L per decade in Japanese women; in China, it was due to rising non-HDL cholesterol. TC declined in Western countries, except in Polish men. The decline was largest in Finland and Norway, at similar to 0.4 mmol/L per decade. The decline in TC in most Western countries was the net effect of an increase in HDL cholesterol and a decline in non-HDL cholesterol, with the HDL cholesterol increase largest in New Zealand and Switzerland. Mean total-to-HDL cholesterol ratio declined in Japan, South Korea and most Western countries, by as much as similar to 0.7 per decade in Swiss men (equivalent to similar to 26% decline in coronary heart disease risk per decade). The ratio increased in China. Conclusions: HDL cholesterol has risen and the total-to-HDL cholesterol ratio has declined in many Western countries, Japan and South Korea, with only a weak correlation with changes in TC or non-HDL cholesterol.Peer reviewe