A systematic review and meta-analysis of clinical trials assessing safety and efficacy of human extracellular vesicle-based therapy.

Nowadays, it has become clear that extracellular vesicles (EVs) are not a cellular waste disposal vesicle but are an essential part of an intercellular communication system. Besides the use of EVs in biomarker studies and diagnostics, the potential of EV-therapeutics has been seen by many. They provide unique properties for disease therapy, including strong immune-modulatory actions, the possibility of engineering, low immunogenicity, and the capability of crossing biological barriers. Proof-of-concept of EV-therapeutics for various pathologies has been achieved in preclinical studies. However, clinical trials with EVs have only been emerging slowly. Here, we aim to provide a comprehensive overview of the current state-of-the-art concerning clinical studies using EVs in human therapy. By approaching the current knowledge in a systematic manner, we were able to include 21 reports for meta-analysis of safety and evaluation of efficacy outcomes. Overall, we have shown that EV-based therapy is safe with a low incidence of serious adverse events (SAE; 0.7% (95%-CI: 0.1-5.2%), and adverse events (AE; 4.4% (95%-CI: 0.7-22.2%). Subgroup analysis showed no significant difference in SAE when comparing autologous versus allogeneic administration, as well as engineered versus non-engineered EV products. A significantly higher number of AE was seen in autologous versus allogeneic administration. However, the clinical relevance remains questionable. Evaluation of the clinical outcomes of immunostimulatory, immunosuppressive or regenerative EV-therapies indicated improvement in the majority of treated patients. Despite these promising results, data need to be approached with caution due to a high heterogeneity in the EVs manufacturing methods, study design, and reporting of (S)AE. Overall, we conclude that EV-based therapy is safe and presents a promising opportunity in therapy. More efforts are needed in the standardization and harmonization of reporting of EV isolation and characterization data as well as in the reporting of (S)AE to allow inter-study comparison.

A Nanoplasmonic Assay for Point-of-Care Detection of Mannose-Binding Lectin in Human Serum.

Mannose-binding lectin (MBL) activates the complement system lectin pathway and subsequent inflammatory mechanisms. The incidence and outcome of many human diseases, such as brain ischemia and infections, are associated with and influenced by the activity and serum concentrations of MBL in body fluids. To quantify MBL levels, tests based on ELISA are used, requiring several incubation and washing steps and lengthy turnaround times. Here, we aimed to develop a nanoplasmonic assay for direct MBL detection in human serum at the point of care. Our assay is based on gold nanorods (GNRs) functionalized with mannose (Man-GNRs) via an amphiphilic linker. We experimentally determined the effective amount of sugar linked to the nanorods' surface, resulting in an approximate grafting density of 4 molecules per nm2, and an average number of 11 to 13 MBL molecules binding to a single nanoparticle. The optimal Man-GNRs concentration to achieve the highest sensitivity in MBL detection was 15 µg·mL-1. The specificity of the assay for MBL detection both in simple buffer and in complex pooled human sera was confirmed. Our label-free biosensor is able to detect MBL concentrations as low as 160 ng·mL-1 within 15 min directly in human serum via a one-step reaction and by using a microplate reader. Hence, it forms the basis for a fast, noninvasive, point-of-care assay for diagnostic indications and monitoring of disease and therapy.

The use of a complex tetra-culture alveolar model to study the biological effects induced by gold nanoparticles with different physicochemical properties.

A substantial increase in engineered nanoparticles in consumer products has been observed, heightening human and environmental exposure. Inhalation represents the primary route of human exposure, necessitating a focus on lung toxicity studies. However, to avoid ethical concerns the use of in vitro models is an efficient alternative to in vivo models. This study utilized an in vitro human alveolar barrier model at air-liquid-interface with four cell lines, for evaluating the biological effects of different gold nanoparticles. Exposure to PEGylated gold nanospheres, nanorods, and nanostars did not significantly impact viability after 24 h, yet all AuNPs induced cytotoxicity in the form of membrane integrity impairment. Gold quantification revealed cellular uptake and transport. Transcriptomic analysis identified gene expression changes, particularly related to the enhancement of immune cells. Despite limited impact, distinct effects were observed, emphasizing the influence of nanoparticles physicochemical parameters while demonstrating the model's efficacy in investigating particle biological effects.

Manufacture of extracellular vesicles derived from mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) are a promising therapy for various diseases ranging from ischemic stroke to wound healing and cancer. Their therapeutic effects are mainly mediated by secretome-derived paracrine factors, with extracellular vesicles (EVs) proven to play a key role. This has led to promising research on the potential of MSC-EVs as regenerative, off-the-shelf therapeutic agents. However, the translation of MSC-EVs into the clinic is hampered by the poor scalability of their production. Recently, new advanced methods have been developed to upscale MSC cultivation and EV production yields, ranging from new cell culture devices to priming procedures. This review gives an overview of these innovative strategies for manufacturing MSC-EVs.

Cell culture-derived extracellular vesicles: Considerations for reporting cell culturing parameters.

Cell culture-conditioned medium (CCM) is a valuable source of extracellular vesicles (EVs) for basic scientific, therapeutic and diagnostic applications. Cell culturing parameters affect the biochemical composition, release and possibly the function of CCM-derived EVs (CCM-EV). The CCM-EV task force of the Rigor and Standardization Subcommittee of the International Society for Extracellular Vesicles aims to identify relevant cell culturing parameters, describe their effects based on current knowledge, recommend reporting parameters and identify outstanding questions. While some recommendations are valid for all cell types, cell-specific recommendations may need to be established for non-mammalian sources, such as bacteria, yeast and plant cells. Current progress towards these goals is summarized in this perspective paper, along with a checklist to facilitate transparent reporting of cell culturing parameters to improve the reproducibility of CCM-EV research.

Intravesicular Genomic DNA Enriched by Size Exclusion Chromatography Can Enhance Lung Cancer Oncogene Mutation Detection Sensitivity.

Extracellular vesicles (EVs) are cell-derived structures surrounded by a lipid bilayer that carry RNA and DNA as potential templates for molecular diagnostics, e.g., in cancer genotyping. While it has been established that DNA templates appear on the outside of EVs, no consensus exists on which nucleic acid species inside small EVs (<200 nm, sEVs) are sufficiently abundant and accessible for developing genotyping protocols. We investigated this by extracting total intravesicular nucleic acid content from sEVs isolated from the conditioned cell medium of the human NCI-H1975 cell line containing the epidermal growth factor (EGFR) gene mutation T790M as a model system for non-small cell lung cancer. We observed that mainly short genomic DNA (<35−100 bp) present in the sEVs served as a template. Using qEV size exclusion chromatography (SEC), significantly lower yield and higher purity of isolated sEV fractions were obtained as compared to exoEasy membrane affinity purification and ultracentrifugation. Nevertheless, we detected the EGFR T790M mutation in the sEVs' lumen with similar sensitivity using digital PCR. When applying SEC-based sEV separation prior to cell-free DNA extraction on spiked human plasma samples, we found significantly higher mutant allele frequencies as compared to standard cell-free DNA extraction, which in part was due to co-purification of circulating tumor DNA. We conclude that intravesicular genomic DNA can be exploited next to ctDNA to enhance EGFR T790M mutation detection sensitivity by adding a fast and easy-to-use sEV separation method, such as SEC, upstream of standard clinical cell-free DNA workflows.

Polymer-tethered glycosylated gold nanoparticles recruit sialylated glycoproteins into their protein corona, leading to off-target lectin binding.

Upon exposure to biological fluids, the fouling of nanomaterial surfaces results in non-specific capture of proteins, which is particularly important when in contact with blood for in vivo and ex vivo applications. It is crucial to evaluate not just the protein components but also the glycans attached to those proteins. Polymer-tethered glycosylated gold nanoparticles have shown promise for use in biosensing/diagnostics, but the impact of the glycoprotein corona has not been established. Here we investigate how polymer-tethered glycosylated gold nanoparticles interact with serum proteins and demonstrate that the protein corona introduces new glycans and hence off-specific targeting capability. Using a panel of RAFT-derived polymers grafted to the gold surface, we show that the extent of corona formation is not dependent on the type of polymer. In lectin-binding assays, a glycan (galactose) installed on the chain-end of the polymer was available for binding even after protein corona formation. However, using sialic-acid binding lectins, it was found that there was significant off-target binding due to the large density of sialic acids introduced in the corona, confirmed by western blotting. To demonstrate the importance, we show that the nanoparticles can bind Siglec-2, an immune-relevant lectin post-corona formation. Pre-coating with (non-glycosylated) bovine serum albumin led to a significant reduction in the total glycoprotein corona. However, sufficient sialic acids were still present in the residual corona to lead to off-target binding. These results demonstrate the importance of the glycans when considering the protein corona and how 'retention of the desired function' does not rule out 'installation of undesired function' when considering the performance of glyco-nanomaterials.

Pathways Related to NLRP3 Inflammasome Activation Induced by Gold Nanorods.

The widespread and increasing use of engineered nanomaterials (ENM) increases the risk of human exposure, generating concern that ENM may provoke adverse health effects. In this respect, their physicochemical characteristics are critical. The immune system may respond to ENM through inflammatory reactions. The NLRP3 inflammasome responds to a wide range of ENM, and its activation is associated with various inflammatory diseases. Recently, anisotropic ENM have become of increasing interest, but knowledge of their effects on the immune system is still limited. The objective of the study was to compare the effects of gold ENM of different shapes on NLRP3 inflammasome activation and related signalling pathways. Differentiated THP-1 cells (wildtype, ASC- or NLRP3-deficient), were exposed to PEGylated gold nanorods, nanostars, and nanospheres, and, thus, also different surface chemistries, to assess NLRP3 inflammasome activation. Next, the exposed cells were subjected to gene expression analysis. Nanorods, but not nanostars or nanospheres, showed NLRP3 inflammasome activation. ASC- or NLRP3-deficient cells did not show this effect. Gene Set Enrichment Analysis revealed that gold nanorod-induced NLRP3 inflammasome activation was accompanied by downregulated sterol/cholesterol biosynthesis, oxidative phosphorylation, and purinergic receptor signalling. At the level of individual genes, downregulation of Paraoxonase-2, a protein that controls oxidative stress, was most notable. In conclusion, the shape and surface chemistry of gold nanoparticles determine NLRP3 inflammasome activation. Future studies should include particle uptake and intracellular localization.

Macrophages Release Extracellular Vesicles of Different Properties and Composition Following Exposure to Nanoparticles.

Extracellular vesicles are membrane-bound carriers with complex cargoes, which play a major role in intercellular communication, for instance, in the context of the immune response. Macrophages are known to release extracellular vesicles in response to different stimuli, and changes in their size, number, and composition may provide important insights into the responses induced. Macrophages are also known to be highly efficient in clearing nanoparticles, when in contact with them, and in triggering the immune system. However, little is known about how the nature and composition of the vesicles released by these cells may vary upon nanoparticle exposure. In order to study this, in this work, alveolar-like macrophages were exposed to a panel of nanoparticles with varying surface and composition, including amino-modified and carboxylated polystyrene and plain silica. We previously showed that these nanoparticles induced very different responses in these cells. Here, experimental conditions were carefully tuned in order to separate the extracellular vesicles released by the macrophages several hours after exposure to sub-toxic concentrations of the same nanoparticles. After separation, different methods, including high-sensitivity flow cytometry, TEM imaging, Western blotting, and nanoparticle tracking analysis, were combined in order to characterize the extracellular vesicles. Finally, proteomics was used to determine their composition and how it varied upon exposure to the different nanoparticles. Our results show that depending on the nanoparticles' properties. The macrophages produced extracellular vesicles of varying number, size, and protein composition. This indicates that macrophages release specific signals in response to nanoparticles and overall suggests that extracellular vesicles can reflect subtle responses to nanoparticles and nanoparticle impact on intercellular communication.

Characterising extracellular vesicles from individual low volume cerebrospinal fluid samples, isolated by SmartSEC.

Extracellular vesicles (EVs) are suggested to have a role in the progression of neurodegeneration, and are able to transmit pathological proteins from one cell to another. One of the biofluids from which EVs can be isolated is cerebrospinal fluid (CSF). However, so far, few studies have been performed on small volumes of CSF. Since pooling of patient samples possibly leads to the loss of essential individual patient information, and CSF samples are precious, it is important to have efficient techniques for the isolation of EVs from smaller volumes. In this study, the SmartSEC HT isolation kit from System Biosciences has been evaluated for this purpose. The SmartSEC HT isolation kit was used for isolation of EVs from 500 µL starting volumes of CSF, resulting in two possible EV fractions of 500 µL. Both fractions were characterised and compared to one another using a whole range of characterisation techniques. Results indicated the presence of EVs in both fractions, albeit fraction 1 showed more reproducible results over the different characterisation methods. For example, CMG (CellMask Green membrane stain) fluorescence nanotracking analysis (NTA), ExoView, and the particles/µg ratio demonstrated a clear difference between fraction 1 and 2, where fraction 1 came out as the one where most EVs were eluted with the least contamination. In the other methods, this difference was less noticeable. We successfully performed complementary characterisation tests using only 500 µL of CSF starting volume, and, conclude that fraction 1 consisted of sufficiently pure EVs for further biomarker studies. This means that future EV extractions may be based upon smaller CSF quantities, such as from individual patients. In that way, patient samples do not have to be pooled and individual patient information can be included in forthcoming studies, potentially linking EV content, size and distribution to individualised neurological diagnoses.

Synthetic Image Rendering Solves Annotation Problem in Deep Learning Nanoparticle Segmentation.

Nanoparticles occur in various environments as a consequence of man-made processes, which raises concerns about their impact on the environment and human health. To allow for proper risk assessment, a precise and statistically relevant analysis of particle characteristics (such as size, shape, and composition) is required that would greatly benefit from automated image analysis procedures. While deep learning shows impressive results in object detection tasks, its applicability is limited by the amount of representative, experimentally collected and manually annotated training data. Here, an elegant, flexible, and versatile method to bypass this costly and tedious data acquisition process is presented. It shows that using a rendering software allows to generate realistic, synthetic training data to train a state-of-the art deep neural network. Using this approach, a segmentation accuracy can be derived that is comparable to man-made annotations for toxicologically relevant metal-oxide nanoparticle ensembles which were chosen as examples. The presented study paves the way toward the use of deep learning for automated, high-throughput particle detection in a variety of imaging techniques such as in microscopies and spectroscopies, for a wide range of applications, including the detection of micro- and nanoplastic particles in water and tissue samples.

Characterization of Gold Nanorods Conjugated with Synthetic Glycopolymers Using an Analytical Approach Based on spICP-SFMS and EAF4-MALS.

A new comprehensive analytical approach based on single-particle inductively coupled plasma-sector field mass spectrometry (spICP-SFMS) and electrical asymmetric-flow field-flow-fractionation combined with multi-angle light scattering detection (EAF4-MALS) has been examined for the characterization of galactosamine-terminated poly(N-hydroxyethyl acrylamide)-coated gold nanorods (GNRs) in two different degrees of polymerization (DP) by tuning the feed ratio (short: DP 35; long: DP 60). spICP-SFMS provided information on the particle number concentration, size and size distribution of the GNRs, and was found to be useful as an orthogonal method for fast characterization of GNRs. Glycoconjugated GNRs were separated and characterized via EAF4-MALS in terms of their size and charge and compared to the bare GNRs. In contrast to spICP-SFMS, EAF4-MALS was also able of providing an estimate of the thickness of the glycopolymer coating on the GNRs surface.

The polymeric glyco-linker controls the signal outputs for plasmonic gold nanorod biosensors due to biocorona formation.

Gold nanorods (GNRs) are a promising platform for nanoplasmonic biosensing. The localised surface plasmon resonance (LSPR) peak of GNRs is located in the near-infrared optical window and is sensitive to local binding events, enabling label-free detection of biomarkers in complex biological fluids. A key challenge in the development of such sensors is achieving target affinity and selectivity, while both minimizing non-specific binding and maintaining colloidal stability. Herein, we reveal how GNRs decorated with galactosamine-terminated polymer ligands display significantly different binding responses in buffer compared to serum, due to biocorona formation, and how biocorona displacement due to lectin binding plays a key role in their optical responses. GNRs were coated with either poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA) or poly(N-hydroxyethyl acrylamide) (PHEA) prepared via reversible addition-fragmentation chain-transfer (RAFT) polymerisation and end-functionalised with galactosamine (Gal) as the lectin-targeting unit. In buffer Gal-PHEA-coated GNRs aggregated upon soybean agglutinin (SBA) addition, whereas Gal-PHPMA-coated GNRs exhibited a red-shift of the LSPR spectrum without aggregation. In contrast, when incubated in serum Gal-PHPMA-coated nanorods showed no binding response, while Gal-PHEA GNRs exhibited a dose-dependent blue-shift of the LSPR peak, which is the opposite direction (red-shift) to what was observed in buffer. This differential behaviour was attributed to biocorona formation onto both polymer-coated GNRs, shown by differential centrifugal sedimentation and nanoparticle tracking analysis. Upon addition of SBA to the Gal-PHEA coated nanorods, signal was generated due to displacement of weakly-bound biocorona components by lectin binding. However, in the case of Gal-PHPMA which had a thicker corona, attributed to lower polymer grafting densities, addition of SBA did not lead to biocorona displacement and there was no signal output. These results show that plasmonic optical responses in complex biological media can be significantly affected by biocorona formation, and that biocorona formation itself does not prevent sensing so long as its exact nature (e.g. 'hard versus soft') is tuned.

Release and cytotoxicity screening of the printer emissions of a CdTe quantum dots-based fluorescent ink.

The fluorescent properties of cadmium telluride (CdTe) containing quantum dots (QDs) have led to novel products and applications in the ink and pigment industry. The toxic effects of the emissions associated to the use of printing ink containing CdTe QDs might differ from those of conventional formulations which do not integrate nanoparticles, as CdTe QDs might be emitted. Within this work, the airborne emissions of a water-soluble fluorescent ink containing polyethylene glycol (PEG)-coated CdTe QDs of 3-5 nm diameter have been characterized and studied under controlled conditions during household inkjet printing in a scenario simulating the use phase. Subsequently, the cytotoxicological potential of atomized CdTe QDs ink in an acute exposure regimen simulating an accidental, worse-case scenario has been evaluated in vitro at the air-liquid interface (ALI) using the pulmonary cell line BEAS-2B. Endpoints screened included cell viability, oxidative stress and inflammatory effects. We have observed that CdTe QDs ink at 54.7 ng/mL decreased cell viability by 25.6 % when compared with clean air after 1h of exposure; a concentration about 65 times higher was needed to observe a similar effect in submerged conditions. However, we did not observe oxidative stress or inflammatory effects. The present study integrates the development of scenarios simulating the use phase of nano-additivated inks and the direct cell exposure for in vitro effects assessment, thus implementing a life-cycle oriented approach in the assessment of the toxicity of CdTe QDs.

Comparison of extracellular vesicle isolation and storage methods using high-sensitivity flow cytometry.

Extracellular vesicles (EVs) are of interest for a wide variety of biomedical applications. A major limitation for the clinical use of EVs is the lack of standardized methods for the fast and reproducible separation and subsequent detection of EV subpopulations from biofluids, as well as their storage. To advance this application area, fluorescence-based characterization technologies with single-EV resolution, such as high-sensitivity flow cytometry (HS-FCM), are powerful to allow assessment of EV fractionation methods and storage conditions. Furthermore, the use of HS-FCM and fluorescent labeling of EV subsets is expanding due to the potential of high-throughput, multiplex analysis, but requires further method development to enhance the reproducibility of measurements. In this study, we have applied HS-FCM measurements next to standard EV characterization techniques, including nanoparticle tracking analysis, to compare the yield and purity of EV fractions obtained from lipopolysaccharide-stimulated monocytic THP-1 cells by two EV isolation methods, differential centrifugation followed by ultracentrifugation and the exoEasy membrane affinity spin column purification. We observed differences in EV yield and purity. In addition, we have investigated the influence of EV storage at 4°C or -80°C for up to one month on the EV concentration and the stability of EV-associated fluorescent labels. The concentration of the in vitro cell derived EV fractions was shown to remain stable under the tested storage conditions, however, the fluorescence intensity of labeled EV stored at 4°C started to decline within one day.

Improving Quality in Nanoparticle-Induced Cytotoxicity Testing by a Tiered Inter-Laboratory Comparison Study.

The quality and relevance of nanosafety studies constitute major challenges to ensure their key role as a supporting tool in sustainable innovation, and subsequent competitive economic advantage. However, the number of apparently contradictory and inconclusive research results has increased in the past few years, indicating the need to introduce harmonized protocols and good practices in the nanosafety research community. Therefore, we aimed to evaluate if best-practice training and inter-laboratory comparison (ILC) of performance of the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay for the cytotoxicity assessment of nanomaterials among 15 European laboratories can improve quality in nanosafety testing. We used two well-described model nanoparticles, 40-nm carboxylated polystyrene (PS-COOH) and 50-nm amino-modified polystyrene (PS-NH2). We followed a tiered approach using well-developed standard operating procedures (SOPs) and sharing the same cells, serum and nanoparticles. We started with determination of the cell growth rate (tier 1), followed by a method transfer phase, in which all laboratories performed the first ILC on the MTS assay (tier 2). Based on the outcome of tier 2 and a survey of laboratory practices, specific training was organized, and the MTS assay SOP was refined. This led to largely improved intra- and inter-laboratory reproducibility in tier 3. In addition, we confirmed that PS-COOH and PS-NH2 are suitable negative and positive control nanoparticles, respectively, to evaluate impact of nanomaterials on cell viability using the MTS assay. Overall, we have demonstrated that the tiered process followed here, with the use of SOPs and representative control nanomaterials, is necessary and makes it possible to achieve good inter-laboratory reproducibility, and therefore high-quality nanotoxicological data.

Jejunal villus absorption and paracellular tight junction permeability are major routes for early intestinal uptake of food-grade TiO2 particles: an in vivo and ex vivo study in mice.

BACKGROUND: Food-grade TiO2 (E171 in the EU) is widely used as a coloring agent in foodstuffs, including sweets. Chronic dietary exposure raises concerns for human health due to proinflammatory properties and the ability to induce and promote preneoplastic lesions in the rodent gut. Characterization of intestinal TiO2 uptake is essential for assessing the health risk in humans. We studied in vivo the gut absorption kinetics of TiO2 in fasted mice orally given a single dose (40 mg/kg) to assess the ability of intestinal apical surfaces to absorb particles when available without entrapment in the bolus. The epithelial translocation pathways were also identified ex vivo using intestinal loops in anesthetized mice. RESULTS: The absorption of TiO2 particles was analyzed in gut tissues by laser-reflective confocal microscopy and ICP-MS at 4 and 8 h following oral administration. A bimodal pattern was detected in the small intestine: TiO2 absorption peaked at 4 h in jejunal and ileal villi before returning to basal levels at 8 h, while being undetectable at 4 h but significantly present at 8 h in the jejunal Peyer's patches (PP). Lower absorption occurred in the colon, while TiO2 particles were clearly detectable by confocal microscopy in the blood at 4 and 8 h after treatment. Ex vivo, jejunal loops were exposed to the food additive in the presence and absence of pharmacological inhibitors of paracellular tight junction (TJ) permeability or of transcellular (endocytic) passage. Thirty minutes after E171 addition, TiO2 absorption by the jejunal villi was decreased by 66% (p < 0.001 vs. control) in the presence of the paracellular permeability blocker triaminopyrimidine; the other inhibitors had no significant effect. Substantial absorption through a goblet cell (GC)-associated pathway, insensitive to TJ blockade, was also detected. CONCLUSIONS: After a single E171 dose in mice, early intestinal uptake of TiO2 particles mainly occurred through the villi of the small intestine, which, in contrast to the PP, represent the main absorption surface in the small intestine. A GC-associated passage and passive diffusion through paracellular TJ spaces between enterocytes appeared to be major absorption routes for transepithelial uptake of dietary TiO2.

Angiogenic Effects of Human Dental Pulp and Bone Marrow-Derived Mesenchymal Stromal Cells and their Extracellular Vesicles.

Blood vessel formation or angiogenesis is a key process for successful tooth regeneration. Bone marrow-derived mesenchymal stromal cells (BM-MSCs) possess paracrine proangiogenic properties, which are, at least partially, induced by their extracellular vesicles (EVs). However, the isolation of BM-MSCs is associated with several drawbacks, which could be overcome by MSC-like cells of the teeth, called dental pulp stromal cells (DPSCs). This study aims to compare the angiogenic content and functions of DPSC and BM-MSC EVs and conditioned medium (CM). The angiogenic protein profile of DPSC- and BM-MSC-derived EVs, CM and EV-depleted CM was screened by an antibody array and confirmed by ELISA. Functional angiogenic effects were tested in transwell migration and chicken chorioallantoic membrane assays. All secretion fractions contained several pro- and anti-angiogenic proteins and induced in vitro endothelial cell motility. This chemotactic potential was higher for (EV-depleted) CM, compared to EVs with a stronger effect for BM-MSCs. Finally, BM-MSC CM, but not DPSC CM, nor EVs, increased in ovo angiogenesis. In conclusion, we showed that DPSCs are less potent in relation to endothelial cell chemotaxis and in ovo neovascularization, compared to BM-MSCs, which emphasizes the importance of choice of cell type and secretion fraction for stem cell-based regenerative therapies in inducing angiogenesis.

Time-resolved characterization of the mechanisms of toxicity induced by silica and amino-modified polystyrene on alveolar-like macrophages.

Macrophages play a major role in the removal of foreign materials, including nano-sized materials, such as nanomedicines and other nanoparticles, which they accumulate very efficiently. Because of this, it is recognized that for a safe development of nanotechnologies and nanomedicine, it is essential to investigate potential effects induced by nano-sized materials on macrophages. To this aim, in this work, a recently established model of primary murine alveolar-like macrophages was used to investigate macrophage responses to two well-known nanoparticle models: 50 nm amino-modified polystyrene, known to induce cell death via lysosomal damage and apoptosis in different cell types, and 50 nm silica nanoparticles, which are generally considered non-toxic. Then, a time-resolved study was performed to characterize in detail the response of the macrophages following exposure to the two nanoparticles. As expected, exposure to the amino-modified polystyrene led to cell death, but surprisingly no lysosomal swelling or apoptosis were detected. On the contrary, a peculiar mitochondrial membrane hyperpolarization was observed, accompanied by endoplasmic reticulum stress (ER stress), increased cellular reactive oxygen species (ROS) and changes of metabolic activity, ultimately leading to cell death. Strong toxic responses were observed also after exposure to silica, which included mitochondrial ROS production, mitochondrial depolarization and cell death by apoptosis. Overall, these results showed that exposure to the two nanoparticles led to a very different series of intracellular events, suggesting that the macrophages responded differently to the two nanoparticle models. Similar time-resolved studies are required to characterize the response of macrophages to nanoparticles, as a key parameter in nanosafety assessment.