Morphological and Mechanical Characterization of Extracellular Vesicles and Parent Human Synoviocytes under Physiological and Inflammatory Conditions.

The morphology of fibroblast-like synoviocytes (FLS) issued from the synovial fluid (SF) of patients suffering from osteoarthritis (OA), rheumatoid arthritis (RA), or from healthy subjects (H), as well as the ultrastructure and mechanical properties of the FLS-secreted extracellular vesicles (EV), were analyzed by confocal microscopy, transmission electron microscopy, atomic force microscopy, and tribological tests. EV released under healthy conditions were constituted of several lipid bilayers surrounding a viscous inner core. This "gel-in" vesicular structure ensured high mechanical resistance of single vesicles and good tribological properties of the lubricant. RA, and to a lesser extent OA, synovial vesicles had altered morphology, corresponding to a "gel-out" situation with vesicles surrounded by a viscous gel, poor mechanical resistance, and poor lubricating qualities. When subjected to inflammatory conditions, healthy cells developed phenotypes similar to that of RA samples, which reinforces the importance of inflammatory processes in the loss of lubricating properties of SF.

Synovial Extracellular Vesicles: Structure and Role in Synovial Fluid Tribological Performances.

The quality of the lubricant between cartilaginous joint surfaces impacts the joint's mechanistic properties. In this study, we define the biochemical, ultrastructural, and tribological signatures of synovial fluids (SF) from patients with degenerative (osteoarthritis-OA) or inflammatory (rheumatoid arthritis-RA) joint pathologies in comparison with SF from healthy subjects. Phospholipid (PL) concentration in SF increased in pathological contexts, but the proportion PL relative to the overall lipids decreased. Subtle changes in PL chain composition were attributed to the inflammatory state. Transmission electron microscopy showed the occurrence of large multilamellar synovial extracellular vesicles (EV) filled with glycoprotein gel in healthy subjects. Synovial extracellular vesicle structure was altered in SF from OA and RA patients. RA samples systematically showed lower viscosity than healthy samples under a hydrodynamic lubricating regimen whereas OA samples showed higher viscosity. In turn, under a boundary regimen, cartilage surfaces in both pathological situations showed high wear and friction coefficients. Thus, we found a difference in the biochemical, tribological, and ultrastructural properties of synovial fluid in healthy people and patients with osteoarthritis and arthritis of the joints, and that large, multilamellar vesicles are essential for good boundary lubrication by ensuring a ball-bearing effect and limiting the destruction of lipid layers at the cartilage surface.

Comparing visual inspection methods for parenteral products in hospital pharmacy: between reliability, cost, and operator formation considerations.

INTRODUCTION: The COVID-19 pandemic has led to unforeseen and novel manifestations, as illustrated by the management of drug shortages through the development of hospital production of sterile pharmaceutical preparations (P2S). Visual inspection of P2S is a release control whose methods are described in monographs of the European Pharmacopoeia (2.9.20) and the United States Pharmacopeia (1790). However, these non-automated visual methods require training and proficiency testing of personnel. The main objective of this work was to compare the reliability and speed of analysis of two visual methods and an automated method for detecting visible particles by image analysis in P2S. Furthermore, these methods were used to evaluate sources of particulate contamination during pre-production processes (washing, disinfection, depyrogenation) and production (filling, capping). MATERIALS AND METHODS: Three pharmacy technicians examined 41 clear glass vials of type I, 10 and/or 50 mL through manual visual inspection (MVI), semi-automated (SAVI), and automated (AVI) inspection. The vials were distributed as follows: (i) 16 vials of water for injection containing either glass particles (224 µm or 600 µm), stopper fragments, or textile fibres; (ii) five sterile injectable specialties; (iii) 20 vials of water for injection prepared under different pre-production conditions. RESULTS AND DISCUSSION: MVI and SAVI detected 100% of visible particles compared with 28% for AVI, which showed a deficiency in detecting textile fibres. All three methods correctly analysed P2S that did not contain visible particles. The three methods detected particles in vials maintained under International Organization for Standardization (ISO) 9 pre-production conditions. However, detections by (i) MVI and SAVI, and by (ii) AVI of particles contained in vials maintained under ISO 8 pre-production conditions were deemed satisfactory and unsatisfactory, respectively. CONCLUSION: The importance of visual inspection of P2S requires rapid, sensitive, and reliable detection methods. In this context, MVI and SAVI have proven to be more effective than AVI for a more competitive financial, training, and implementation investment.

Effects of pro-inflammatory cytokines and cell interactions on cell area and cytoskeleton of rheumatoid arthritis synoviocytes and immune cells.

Rheumatoid synovitis is infiltrated by immune cells that interact with synoviocytes, leading to the pannus formation. Inflammation or cell interaction effects are mainly evaluated with cytokine production, cell proliferation or migration. Few studies interest on cell morphology. Here, the purpose was to deepen some morphological changes of synoviocytes or immune cells under inflammatory conditions. Inflammatory cytokines, IL-17 and TNF that are largely involved in RA pathogenesis, induced a change in synoviocyte morphology, inducing a retracted cell with higher number of pseudopodia. Several morphological parameters decreased in inflammatory conditions: cell confluence, area and motility speed. The same impact on cell morphology was observed in co-culture of synoviocytes and immune cells in inflammatory/non-inflammatory conditions or with cell activation (miming the in vivo situation), affecting both cell types: synoviocytes were retracted and inversely immune cells proliferated, indicating that cell activation induced a morphological change of cells. In contrast, with RA but not control synoviocytes, cell interactions were not sufficient to affect PBMC and synoviocyte morphology. The morphological effect came only from the inflammatory environment. These findings reveal that the inflammatory environment or cell interactions induced massive changes in control synoviocytes, with cell retraction and increase of pseudopodia number, leading to better interactions with other cells. Except in the case of RA, the inflammatory environment was absolutely required for such changes.

Predictive stability, novel HPLC-MS analysis and semi-automatic compounding process for the emergency implementation of a production line of pancuronium in injectable solution.

During the early months of the COVID-19 pandemic, the international medical product supply chain was tight, causing breaks in the availability of neuromuscular blocking agents essential for the treatment of patients in intensive care units. The present study describes the pharmaceutical development of an injectable 2 mg/mL solution of pancuronium bromide (PC) in a very short lapse of time. The sterile solution was compounded into a good manufacturing practice grade A clean room, filtered (0.2 µm) and filled into 10 mL type I glass, manually sealed with bromobutyl rubber stoppers. A novel HPLC-MS stability indicating method for pancuronium quantification and its degradation product was developed and validated. This fast, sensitive and straightforward method was used to study the stability of the formulation using a semi-predictive method, enabling a very fast attribution of a temporary shelf-life, which was confirmed by a classic prospective stability study. The production line and the analytical tools set-up were performed in six weeks and the semi-predictive stability study was conducted in 90 days, allowing us to predict a shelf life, which was successfully confirmed by prospective study. In conclusion, using innovative methods, we were able to rapidly overcome the shortage of a critical drug.

Biological Applications and Toxicity Minimization of Semiconductor Quantum Dots.

The extraordinary potential of semiconductor quantum dots (QDs) has resulted in their widespread application in various fields, from engineering technology and the development of laboratory techniques to biomedical imaging and therapeutic strategies. However, the toxicity of QDs remains a concern and has limited their applications in human health. Better understanding of the behavior of QDs as it relates to their composition will enable the exploration of their limitations and development of a strategy to control their toxicity for potential therapeutic applications. Here, we describe approaches to minimize their toxicities according to the specific cell type, organ, or animal species, summarizing recent promising research at the cellular, organ, and whole-organism level.

Dual 0.02% chlorhexidine digluconate - 0.1% disodium EDTA loaded thermosensitive ocular gel for Acanthamoeba keratitis treatment.

Poor bioavailability and low residence time limit the efficiency of conventional biguanide-based eye drops against Acanthamoeba keratitis. The aim of this work was to formulate an original anti-amoebic thermoreversible ocular gel combining biguanide and metalloproteases inhibitor - chelating agent. Chlorhexidine digluconate (CHX)-ethylenediaminetetraacetic acid disodium salt (Na2EDTA) were compounded in poloxamer 407 saline solution. 0.02% CHX - 0.1% Na2EDTA loaded thermosensitive ocular gel exhibited appropriate pH (5.73 ±â€¯0.06), iso-osmolality (314 ±â€¯5 mOsm/kg), viscosity (ranged between 15 and 25 mPa.s) and thermal gelation (26.5 °C and 33 °C) properties. Bioadhesion of gel was successfully tested onto isolated bovine eyes as well as the assessment of CHX penetration into the cornea. Intracorneal CHX concentration was found greater than trophozoite minimum amoebicidal concentration and minimal cysticidal concentration after 15-min and 2-h ocular exposure, respectively, while any CHX permeation through the cornea was detected (<51 ng/cm2/h). Improvement of CHX ocular bioavailability was attributed to probable solubilization of tear film lipid layer by poloxamer. In vitro efficiency of CHX-Na2EDTA ocular gel was confirmed from the drastic reduction of trophozoite and cyst survival (to 25% and 2%, respectively), confirming the potential of the multicomponent pharmaceutical material strategy for the treatment of Acanthamoeba keratitis.

Production and stability study of a hospital parenteral nutrition solution for neonates.

Standard parenteral nutrition solutions are mixtures comprising interacting components that may degrade themselves over time. The objective of this study was to investigate the physicochemical and microbiological stability of a hospital preparation for parenteral nutrition in neonatology. The analyses were performed throughout the storage of the preparations at 2-8 °C (up to 4 months). The extent of stability was based on the determination of amino acids dosage, visual and physicochemical properties (glucose and electrolytes concentrations, pH and osmolality measurements, particle counting) and microbiological analysis (sterility test). A thermal degradation of ascorbic acid was conducted to evaluate the antioxidant properties of the parenteral mixture. Physicochemical and microbiological controls were found to comply with the specifications. Amino acids showed a good stability throughout the 4months storage except for cysteine, which was progressively degraded to cystine, conferring a yellow coloration to parenteral solutions. Parenteral nutrition standards solutions remain stable for 4 months at 2-8 °C, ensuring safe administration in preterm infants.

Live-stream characterization of cadmium-induced cell death using visible CdTe-QDs.

Characterization of cell death currently requires the use of indirect markers, which has largely limited the ability to monitor cell death processes inside the cell. Here, we introduce a new method for the characterization of cell death mechanisms using cadmium telluride quantum dots (CdTe-QDs). Using visible CdTe-QDs with mesenchymal cells (e.g. synoviocytes), live-stream imaging allowed for visualization of cadmium-induced cell death, combining characteristics of apoptosis and autophagy. Initially, similar anti-proliferative effect was observed between 10 µg/ml Cd2+ and CdTe-QDs at 24 h (cell index/cell density ratio decreased from 0.6 to -16.6, p < 0.05) using techniques that do not require the capacity of CdTe-QDs. Apoptosis was confirmed by the quantification of morphological parameters (reduced surface area, increased cell thickness) and positive labeling with annexin V. Autophagy was confirmed by monodansylcadaverine staining, identifying similar autophagic vacuoles with both Cd2+ and CdTe-QD. However, QD imaging allowed for visualization of cadmium elements inside cell structures and their kinetic changes leading to cell death. Cell death characteristics were similar in inflammatory and non-inflammatory environment but were induced up to 4 h earlier in the former. Therefore, live-stream imaging of a visible cytotoxic agent has useful applications not currently possible with indirect methods, including chronological monitoring of cell death.

Formulation, stability testing, and analytical characterization of melatonin-based preparation for clinical trial.

A new institutional clinical trial assessed the improvement of sleep disorders in 40 children with autism treated by immediate-release melatonin formulation in different regimens (0.5 mg, 2 mg, and 6 mg daily) for one month. The objectives of present study were to (i) prepare low-dose melatonin hard capsules for pediatric use controlled by two complementary methods and (ii) carry out a stability study in order to determine a use-by-date. Validation of preparation process was claimed as ascertained by mass uniformity of hard capsules. Multicomponent analysis by attenuated total reflectance Fourier transformed infrared (ATR-FTIR) of melatonin/microcrystalline cellulose mixture allowed to identify and quantify relative content of active pharmaceutical ingredients and excipients. Absolute melatonin content analysis by high performance liquid chromatography in 0.5 mg and 6 mg melatonin capsules was 93.6%±4.1% and 98.7%±6.9% of theoretical value, respectively. Forced degradation study showed a good separation of melatonin and its degradation products. The capability of the method was 15, confirming a risk of false negative <0.01%. Stability test and dissolution test were compliant over 18 months of storage with European Pharmacopoeia. Preparation of melatonin hard capsules was completed manually and melatonin in hard capsules was stable for 18 months, in spite of low doses of active ingredient. ATR-FTIR offers a real alternative to HPLC for quality control of high-dose melatonin hard capsules before the release of clinical batches.