Extracellular vesicles from activated platelets possess a phospholipid-rich biomolecular profile and enhance prothrombinase activity.

BACKGROUND: Extracellular vesicles (EVs), in particular those derived from activated platelets, are associated with a risk of future venous thromboembolism. OBJECTIVES: To study the biomolecular profile and function characteristics of EVs from control (unstimulated) and activated platelets. METHODS: Biomolecular profiling of single or very few (1-4) platelet-EVs (control/stimulated) was performed by Raman tweezers microspectroscopy. The effects of such EVs on the coagulation system were comprehensively studied. RESULTS: Raman tweezers microspectroscopy of platelet-EVs followed by biomolecular component analysis revealed for the first time 3 subsets of EVs: (i) protein rich, (ii) protein/lipid rich, and (iii) lipid rich. EVs from control platelets presented a heterogeneous biomolecular profile, with protein-rich EVs being the main subset (58.7% ± 3.5%). Notably, the protein-rich subset may contain a minor contribution from other extracellular particles, including protein aggregates. In contrast, EVs from activated platelets were more homogeneous, dominated by the protein/lipid-rich subset (>85%), and enriched in phospholipids. Functionally, EVs from activated platelets increased thrombin generation by 52.4% and shortened plasma coagulation time by 34.6% ± 10.0% compared with 18.6% ± 13.9% mediated by EVs from control platelets (P = .015). The increased procoagulant activity was predominantly mediated by phosphatidylserine. Detailed investigation showed that EVs from activated platelets increased the activity of the prothrombinase complex (factor Va:FXa:FII) by more than 6-fold. CONCLUSION: Our study reports a novel quantitative biomolecular characterization of platelet-EVs possessing a homogenous and phospholipid-enriched profile in response to platelet activation. Such characteristics are accompanied with an increased phosphatidylserine-dependent procoagulant activity. Further investigation of a possible role of platelet-EVs in the pathogenesis of venous thromboembolism is warranted.

Dynamics of mitochondrial membranes under photo-oxidative stress with high spatiotemporal resolution.

In our study, we harnessed an original Enhanced Speed Structured Illumination Microscopy (Fast-SIM) imaging setup to explore the dynamics of mitochondrial and inner membrane ultrastructure under specific photo-oxidation stress induced by Chlorin-e6 and light irradiation. Notably, our Fast-SIM system allowed us to observe and quantify a distinct remodeling and shortening of the mitochondrial structure after 60-80 s of irradiation. These changes were accompanied by fusion events of adjacent inner membrane cristae and global swelling of the organelle. Preceding these alterations, a larger sequence was characterized by heightened dynamics within the mitochondrial network, featuring events such as mitochondrial fission, rapid formation of tubular prolongations, and fluctuations in cristae structure. Our findings provide compelling evidence that, among enhanced-resolution microscopy techniques, Fast-SIM emerges as the most suitable approach for non-invasive dynamic studies of mitochondrial structure in living cells. For the first time, this approach allows quantitative and qualitative characterization of successive steps in the photo-induced oxidation process with sufficient spatial and temporal resolution.

Menstrual hygiene products: A practice evaluation.

OBJECTIVE: The choice and use of a type of hygienic protection depends on many factors. Due to growing media interest, the field of hygienic protection is evolving, however, to date no study has been carried out on this subject in France. The objective of this study was to evaluate women's practices regarding the use of hygienic protection. MATERIAL AND METHODS: From 2 June 2019 to 4 January 2020, 1,153 patients responding to a self-report questionnaire were included in a prospective, cross-sectional, observational, single-center study. The aim of the study was to describe women's practices with regard to menstrual hygiene products and the factors determining their choices, as well as their knowledge of the potential risks associated with these protections and their sources of information. RESULTS: Disposable sanitary pads were preferred by 930/1148 (81%) of patients, and menstrual tampons were used half as much (525/1150 (45.6%) of women surveyed)). The new menstrual hygiene products (washable sanitary pads, menstrual panties, and menstrual cups) were used by only 51/1150 (4.4%); 20/1149 (1.7%); 108/1150 (9.4%) of the patients; however, among the 92/1136 (8.1%) of the patients who had recently changed the type of protection, these new protections were the most popular because they were considered more ecological and less harmful to health. Menstrual hygiene products were perceived as a health risk for 924/1129 (81.8%) of patients. Menstrual toxic shock syndrome was knowledeg in only 473/1133 (41.7%) of patients. This lack of knowledge could lead to risky behavior. The majority of patients said they were not informed about hygiene protection, with only 151//1108 (13.6%) having discussed the subject with a health professional, yet 973/1067 (91.2%) wanted more information. CONCLUSION: This is the first French study on menstrual hygiene products. It showed that traditional sanitary protection was still the most widely used, but there was a growing awareness among patients about the products they used and their potential health risks as well as the consequences for the environment. Patients wanted to receive information on the subject from health professionals as well as manufacturers in order to be able to choose the product deemed the most suitable and in which they have confidence.

Magnetic resonance imaging evaluation of cervical length by the women's age: a retrospective cohort study.

OBJECTIVE: The anthropometric characteristics of the uterus evolve with pubertal development in girls. It is therefore permissible to ask until these anthropometric characteristics change, in order to know if the cervical length criterion defined for preterm delivery threats is applicable to all ages. The main objective of our study was to analyze the evolution of cervical length with the women's age outside pregnancy to overcome factors related to pregnancy that can affect cervical length. MATERIAL AND METHODS: This retrospective descriptive study over a period of 1 year from March 2017 to March 2018. The cervical length measurements were performed by Magnetic Resonnance Imaging. The cervical length was defined by sagittal T2-weighted magnetic resonance imaging (MRI) as the distance on a straight line between the external cervical os (at the point of divergence of the anterior and posterior lips) and the internal cervical os identified by an intersection between the line of the hypersignal of the glandular epithelium and a line passing through the isthmus. RESULTS: A total of 209 patients were included. The cervical length ranged from 25.2 mm on average in children under 16 years (23.6-27.1 mm) to 39.7 mm between 36 and 40 years (27.9 -58.9 mm). There was a linear association between age and cervical length, irrespective of maternal anthropometric data (Pearson's coefficient ρ = 0.43, 95% CI 0.32-0.54 (p < 0.01). In multivariate analysis, the only factors associated with cervical length were women's age (p < 0.01) and the prior delivery (p < 0.01). CONCLUSION: The cervical length is correlated with women age and the prior delivery.

Mitochondrial cristae modeled as an out-of-equilibrium membrane driven by a proton field.

As the places where most of the fuel of the cell, namely, ATP, is synthesized, mitochondria are crucial organelles in eukaryotic cells. The shape of the invaginations of the mitochondria inner membrane, known as a crista, has been identified as a signature of the energetic state of the organelle. However, the interplay between the rate of ATP synthesis and the crista shape remains unclear. In this work, we investigate the crista membrane deformations using a pH-dependent Helfrich model, maintained out of equilibrium by a diffusive flux of protons. This model gives rise to shape changes of a cylindrical invagination, in particular to the formation of necks between wider zones under variable, and especially oscillating, proton flux.

Lipid Unsaturation Properties Govern the Sensitivity of Membranes to Photoinduced Oxidative Stress.

Unsaturated lipid oxidation is a fundamental process involved in different aspects of cellular bioenergetics; dysregulation of lipid oxidation is often associated with cell aging and death. To study how lipid oxidation affects membrane biophysics, we used a chlorin photosensitizer to oxidize vesicles of various lipid compositions and degrees of unsaturation in a controlled manner. We observed different shape transitions that can be interpreted as an increase in the area of the targeted membrane followed by a decrease. These area modifications induced by the chemical modification of the membrane upon oxidation were followed in situ by Raman tweezers microspectroscopy. We found that the membrane area increase corresponds to the lipids' peroxidation and is initiated by the delocalization of the targeted double bonds in the tails of the lipids. The subsequent decrease of membrane area can be explained by the formation of cleaved secondary products. As a result of these area changes, we observe vesicle permeabilization after a time lag that is characterized in relation with the level of unsaturation. The evolution of photosensitized vesicle radius was measured and yields an estimation of the mechanical changes of the membrane over oxidation time. The membrane is both weakened and permeabilized by the oxidation. Interestingly, the effect of unsaturation level on the dynamics of vesicles undergoing photooxidation is not trivial and thus carefully discussed. Our findings shed light on the fundamental dynamic mechanisms underlying the oxidation of lipid membranes and highlight the role of unsaturations on their physical and chemical properties.

Nonequilibrium fluctuations of lipid membranes by the rotating motor protein F1F0-ATP synthase.

ATP synthase is a rotating membrane protein that synthesizes ATP through proton-pumping activity across the membrane. To unveil the mechanical impact of this molecular active pump on the bending properties of its lipid environment, we have functionally reconstituted the ATP synthase in giant unilamellar vesicles and tracked the membrane fluctuations by means of flickering spectroscopy. We find that ATP synthase rotates at a frequency of about 20 Hz, promoting large nonequilibrium deformations at discrete hot spots in lipid vesicles and thus inducing an overall membrane softening. The enhanced nonequilibrium fluctuations are compatible with an accumulation of active proteins at highly curved membrane sites through a curvature-protein coupling mechanism that supports the emergence of collective effects of rotating ATP synthases in lipid membranes.

Berberine as a photosensitizing agent for antitumoral photodynamic therapy: Insights into its association to low density lipoproteins.

Recent years have seen a growing interest in Berberine, a phytochemical with multispectrum therapeutic activities, as anti-tumoral agent for photodynamic therapy (PDT). In this context, low density lipoproteins (LDL) play a key role in the delivery of the photosensitizer in tumor cells. We correlate the physicochemical parameters of the berberine association to LDL with the influence of LDL-delivery on its accumulation in a glioma cell line and on its photo-induced activity in view of antitumor PDT. Our results evidence an important binding of 400 berberine molecules per LDL. Changes in berberine and apoprotein fluorescence suggest different fixation types, involving various LDL compartments including the vicinity of the apoprotein. The berberine association to LDL does not affect their recognition by the specific B/E receptors, of which over-expression increases the cellular uptake of LDL-preloaded berberine. Fluorescence microscopy evidences the mitochondrial labeling of the glioma model cells, with no significant modification upon LDL-delivery. Moreover, the cellular delivery of berberine by LDL increases its photocytotoxic effects on such cells. So, this research illustrates the potential of berberine as a photosensitizing agent for PDT, in particular due to their behavior towards LDL as plasma vehicles, and gives insights into its mechanisms of cell uptake.

Release kinetics of an amphiphilic photosensitizer by block-polymer nanoparticles.

Block-polymer nanoparticles are now well-known candidates for the delivery of various non-soluble drugs to cells. The release of drugs from these nanoparticles is a major concern related to their efficiency as nanovectors and is still not completely deciphered. Various processes have been identified, depending of both the nature of the block-polymer and those of the drugs used. We focused our interest on an amphiphilic photosensitizer studied for photodynamic treatments of cancer, Pheophorbide-a (Pheo). We studied the transfer of Pheo from poly(ethyleneglycol-b-ϵ-caprolactone) nanoparticles (I) to MCF-7 cancer cells and (II) to models of membranes. Altogether, our results suggest that the delivery of the major part of the Pheo by the nanoparticles occurs via a direct transfer of Pheo from the nanoparticles to the membrane, by collision. A minor process may involve the internalization of a small amount of the nanoplatforms by the cells. So, this research illustrates the great care necessary to address the question of the choice of such nanocarriers, in relation with the properties - in particular the relative hydrophobicity - of the drugs encapsulated, and gives elements to predict the mechanism and the efficiency of the delivery.

Intracellular Monitoring of AS1411 Aptamer by Time-Resolved Microspectrofluorimetry and Fluorescence Imaging.

Time-resolved microspectrofluorimetry and fluorescence microscopy imaging-two complementary fluorescence techniques-provide important information about the intracellular distribution, level of uptake and binding/interactions inside living cell of the labeled molecule of interest. They were employed to monitor the "fate" of AS1411 aptamer labeled by ATTO 425 in human living cells. Confocal microspectrofluorimeter adapted for time-resolved intracellular fluorescence measurements by using a phase-modulation principle with homodyne data acquisition was employed to obtain emission spectra and to determine fluorescence lifetimes in U-87 MG tumor brain cells and Hs68 non-tumor foreskin cells. Acquired spectra from both the intracellular space and the reference solutions were treated to observe the aptamer localization and its interaction with biological structures inside the living cell. The emission spectra and the maximum emission wavelengths coming from the cells are practically identical, however significant lifetime lengthening was observed for tumor cell line in comparison to non-tumor one.

Combining magnetic nanoparticles with cell derived microvesicles for drug loading and targeting.

Inspired by microvesicle-mediated intercellular communication, we propose a hybrid vector for magnetic drug delivery. It consists of macrophage-derived microvesicles engineered to enclose different therapeutic agents together with iron oxide nanoparticles. Here, we investigated in vitro how magnetic nanoparticles may influence the vector effectiveness in terms of drug uptake and targeting. Human macrophages were loaded with iron oxide nanoparticles and different therapeutic agents: a chemotherapeutic agent (doxorubicin), tissue-plasminogen activator (t-PA) and two photosensitizers (disulfonated tetraphenyl chlorin-TPCS2a and 5,10,15,20-tetra(m-hydroxyphenyl)chlorin-mTHPC). The hybrid cell microvesicles were magnetically responsive, readily manipulated by magnetic forces and MRI-detectable. Using photosensitizer-loaded vesicles, we showed that the uptake of microvesicles by cancer cells could be kinetically modulated and spatially controlled under magnetic field and that cancer cell death was enhanced by the magnetic targeting. From the clinical editor: In this article, the authors devised a biogenic method using macrophages to produce microvesicles containing both iron oxide and chemotherapeutic agents. They showed that the microvesicles could be manipulated by magnetic force for targeting and subsequent delivery of the drug payload against cancer cells. This smart method could provide a novel way for future fight against cancer.

Magnetic and photoresponsive theranosomes: translating cell-released vesicles into smart nanovectors for cancer therapy.

Cell-released vesicles are natural carriers that circulate in body fluids and transport biological agents to distal cells. As nature uses vesicles in cell communication to promote tumor progression, we propose to harness their unique properties and exploit these biogenic carriers as Trojan horses to deliver therapeutic payloads to cancer cells. In a theranostic approach, cell-released vesicles were engineered by a top-down procedure from precursor cells, previously loaded with a photosensitizer and magnetic nanoparticles. The double exogenous cargo provided vesicles with magnetic and optical responsiveness allowing therapeutic and imaging functions. This new class of cell-derived smart nanovectors was named "theranosomes". Theranosomes enabled efficient photodynamic tumor therapy in a murine cancer model in vivo. Moreover the distribution of this biogenic vector could be monitored by dual-mode imaging, combining fluorescence and MRI. This study reports the first success in translating a cell communication mediator into a smart theranostic nanovector.

Impact of photosensitizers activation on intracellular trafficking and viscosity.

The intracellular microenvironment is essential for the efficiency of photo-induced therapies, as short-lived reactive oxygen species generated must diffuse through their intracellular surrounding medium to reach their cellular target. Here, by combining measurements of local cytoplasmic dissipation and active trafficking, we found that photosensitizers activation induced small changes in surrounding viscosity but a massive decrease in diffusion. These effects are the signature of a return to thermodynamic equilibrium of the system after photo-activation and correlated with depolymerization of the microtubule network, as shown in a reconstituted system. These mechanical measurements were performed with two intracellular photosensitizing chlorins having similar quantum yield of singlet oxygen production but different intracellular localizations (cytoplasmic for mTHPC, endosomal for TPCS2a). These two agents demonstrated different intracellular impact.

Flavin conjugates for delivery of peptide nucleic acids.

Oligonucleotides and their analogues, such as peptide nucleic acids (PNAs), can be used in chemical strategies to artificially control gene expression. Inefficient cellular uptake and inappropriate cellular localization still remain obstacles in biological applications, however, especially for PNAs. Here we demonstrate that conjugation of PNAs to flavin resulted in efficient internalization into cells through an endocytic pathway. The flavin-PNAs exhibited antisense activity in the sub-micromolar range, in the context of a treatment facilitating endosomal escape. Increased endosomal release of flavin conjugates into the cytoplasm and/or nucleus was shown by chloroquine treatment and also--when the flavin-PNA was conjugated to rhodamine, a mild photosensitizer--upon light irradiation. In conclusion, an isoalloxazine moiety can be used as a carrier and attached to a cargo biomolecule, here a PNA, for internalization and functional cytoplasmic/nuclear delivery. Our findings could be useful for further design of PNAs and other oligonucleotide analogues as potent antisense agents.

Photo-dynamic induction of oxidative stress within cholesterol-containing membranes: shape transitions and permeabilization.

Photochemical internalization is a drug delivery technology employing a photo-destabilization of the endosomes and the photo-controlled release of endocyted macromolecules into the cytosol. This effect is based on the ability of some photosensitizers to interact with endosomal membranes and to photo-induce damages leading to its breakdown. The permeabilization efficiency is not quantitatively related to the importance of the damages, but to their asymmetric repartition within the leaflets. Using unilamellar vesicles and a chlorin, we studied the effect of the membrane's cholesterol content on its photo-permeabilization. First, the affinity of the chlorin for membranes was studied. Then, we asymmetrically oxidized the membranes. For DOPC/CHOL GUVs, we observed different shape transitions, in accordance with an increase followed by a decrease of the membrane effective curvature. These modifications are delayed by the cholesterol. Finally, the photo-permeabilization of GUVs occurs, corresponding to a pore formation due to the membrane tension, resulting from vesicles buddings. Cholesterol-rich GUVs permeabilization occurs after a lag, and is less important. These results are interpreted regarding both (i) the cholesterol-induced tightening of the lipids, its consequences on physical parameters of the membrane and on oxidation rate and (ii) the suggested ability of cholesterol to flip rapidly and then to relax the differential density-based stress accumulated during membrane bending.

Hypericin incorporation and localization in fixed HeLa cells for various conditions of fixation and incubation.

Hypericin is a photosensitizer expressing high affinity for cancerous cells in vivo. Diagnosis of cancer based on hypericin fluorescence imaging has been successfully assessed in several clinical trials. Our final objective will be to evaluate the potential of hypericin fluorescence imaging to improve the efficacy of cervical cancer diagnosis performed on fixed cell smears obtained from liquid-based cytology. For this purpose, the mechanism of hypericin incorporation and localization in fixed HeLa cells using different incubation media and fixation conditions was investigated. Since the duration of fixation may play an important role, the influence of fixation time on hypericin incorporation in fixed HeLa cells was studied. The uptake and distribution of hypericin in fixed HeLa cells were found to be strongly dependent on the hypericin incubation medium: for a polar organic solvent such as the alcohol-based fixative, the localization was essentially perinuclear and nuclear; for cell culture medium supplemented with serum, the localization was cytoplasmic and non-specific; the highest incorporation was observed for the serum-free culture medium but mainly as non-fluorescent aggregates. The hypericin aggregation in the incubation medium, the passive diffusion and the partitioning between the cells and hypericin carriers seemed to be the major factors accounting for these results. The localization was found to be weakly dependent on fixation time, whereas fluctuations of hypericin fluorescence at short fixation time and stabilization after two days of fixation were observed. These results suggest that the fixed cells reached a steady state after two days of fixation.

Interaction dynamics of hypericin with low-density lipoproteins and U87-MG cells.

The natural photosensitizer hypericin exhibits potent properties for tumor diagnosis and photodynamic therapy. Fluorescent properties of hypericin along with various technical approaches have been used for dynamic studies of its interaction with low-density lipoprotein and U87 glioma cells. Evidences for hypericin release from low-density lipoprotein towards cells plasmatic membrane are addressed. Subsequent subcellular bulk flow redistribution leading to non-specific staining of intracellular membranes compartment were observed within cells. It was shown, that monomers of hypericin are the only redistributive forms. Increasing concentration of hypericin leads to the formation of non-fluorescent aggregates within low-density lipoprotein as well as within the U87 cells, and can preclude its photosensitizing activities. However, the aggregation process can only account for a part of the observed emission decrease. As shown by the excited state lifetime measurements, this fluorescence quenching actually results from a combination of aggregation process and energy transfer from monomers to aggregates. In all experiments, hydrophobic character of hypericin appears as the driving force of its redistribution process.

Asymmetric oxidation of giant vesicles triggers curvature-associated shape transition and permeabilization.

Oxidation of unsaturated lipids is a fundamental process involved in cell bioenergetics as well as in cell death. Using giant unilamellar vesicles and a chlorin photosensitizer, we asymmetrically oxidized the outer or inner monolayers of lipid membranes. We observed different shape transitions such as oblate to prolate and budding, which are typical of membrane curvature modifications. The asymmetry of the shape transitions is in accordance with a lowered effective spontaneous curvature of the leaflet being targeted. We interpret this effect as a decrease in the preferred area of the targeted leaflet compared to the other, due to the secondary products of oxidation (cleaved-lipids). Permeabilization of giant vesicles by light-induced oxidation is observed after a lag and is characterized in relation with the photosensitizer concentration. We interpret permeabilization as the opening of a pore above a critical membrane tension, resulting from the budding of vesicles. The evolution of photosensitized giant vesicle lysis tension was measured and yields an estimation of the effective spontaneous curvature at lysis. Additionally photo-oxidation was shown to be fusogenic.

Photosensitizing properties of chlorins in solution and in membrane-mimicking systems.

The photosensitizing properties of three chlorins, meso-tetra(3-hydroxyphenyl)chlorin (m-THPC), chlorin e6 (Ce6) and meso-tetraphenylchlorin substituted by two adjacent sulfonated groups (TPCS(2a)) are compared in solution and when incorporated in dioleoyl-sn-phosphatidylcholine (DOPC) liposomes. In solution, the three chlorins possess a similar efficacy to generate singlet oxygen (quantum yield approximately 0.65). The formation of conjugated dienes was used to determine their ability to induce the peroxidation of methyl linoleate as a target of singlet oxygen. In ethanol solution, the apparent quantum yield for this process is the same for the three chlorins and its value agrees with that expected from the known rates for the decay of singlet oxygen and its reaction with methyl linoleate. When incorporated in liposomes, the order of efficacy is m-THPC > TPCS(2a) > Ce6. This order is tentatively assigned to the relative embedment of the photosensitizer within the lipidic bilayer, TPCS(2a) and Ce6 being anchored by their negative chains nearer to the water-lipid interface. The photoinduced permeation of the lipidic bilayer by these chlorins was investigated by measuring the release of carboxyfluorescein entrapped into DOPC liposomes. The charged chlorins, in particular TPCS(2a), are the most efficient, a result discussed in relation with the technology of photochemical internalization, PCI.

Influence of surface energy distribution on neuritogenesis.

PC12 cells are a useful model to study neuronal differentiation, as they can undergo terminal differentiation, typically when treated with nerve growth factor (NGF). In this study we investigated the influence of surface energy distribution on PC12 cell differentiation, by atomic force microscopy (AFM) and immunofluorescence. Glass surfaces were modified by chemisorption: an aminosilane, n-[3-(trimethoxysilyl)propyl]ethylendiamine (C(8)H(22)N(2)O(3)Si; EDA), was grafted by polycondensation. AFM analysis of substrate topography showed the presence of aggregates suggesting that the adsorption is heterogeneous, and generates local gradients in energy of adhesion. PC12 cells cultured on these modified glass surfaces developed neurites in absence of NGF treatment. In contrast, PC12 cells did not grow neurites when cultured in the absence of NGF on a relatively smooth surface such as poly-L-lysine substrate, where amine distribution is rather homogeneous. These results suggest that surface energy distribution, through cell-substrate interactions, triggers mechanisms that will drive PC12 cells to differentiate and to initiate neuritogenesis. We were able to create a controlled physical nano-structuration with local variations in surface energy that allowed the study of these parameters on neuritogenesis.