Bridging communities in the field of nanomedicine.

An early dialogue between nanomedicine developers and regulatory authorities are of utmost importance to anticipate quality and safety requirements for these innovative health products. In order to stimulate interactions between the various communities involved in a translation of nanomedicines to clinical applications, the European Commission's Joint Research Centre hosted a workshop titled "Bridging communities in the field of Nanomedicine" in Ispra/Italy on the 27th -28th September 2017. Experts from regulatory bodies, research institutions and industry came together to discuss the next generation of nanomedicines and their needs to obtain regulatory approval. The workshop participants came up with recommendations highlighting methodological gaps that should be addressed in ongoing projects addressing the regulatory science of nanomedicines. In addition, individual opinions of experts relevant to progress of the regulatory science in the field of nanomedicine were summarised in the format of a survey.

The agglomeration state of nanoparticles can influence the mechanism of their cellular internalisation.

BACKGROUND: Significant progress of nanotechnology, including in particular biomedical and pharmaceutical applications, has resulted in a high number of studies describing the biological effects of nanomaterials. Moreover, a determination of so-called "critical quality attributes", that is specific physicochemical properties of nanomaterials triggering the observed biological response, has been recognised as crucial for the evaluation and design of novel safe and efficacious therapeutics. In the context of in vitro studies, a thorough physicochemical characterisation of nanoparticles (NPs), also in the biological medium, is necessary to allow a correlation with a cellular response. Following this concept, we examined whether the main and frequently reported characteristics of NPs such as size and the agglomeration state can influence the level and the mechanism of NP cellular internalization. RESULTS: We employed fluorescently-labelled 30 and 80 nm silicon dioxide NPs, both in agglomerated and non-agglomerated form. Using flow cytometry, transmission electron microscopy, the inhibitors of endocytosis and gene silencing we determined the most probable routes of cellular uptake for each form of tested silica NPs. We observed differences in cellular uptake depending on the size and the agglomeration state of NPs. Caveolae-mediated endocytosis was implicated particularly in the internalisation of well dispersed silica NPs but with an increase of the agglomeration state of NPs a combination of endocytic pathways with a predominant role of macropinocytosis was noted. CONCLUSIONS: We demonstrated that the agglomeration state of NPs is an important factor influencing the level of cell uptake and the mechanism of endocytosis of silica NPs.

Adaptation of targeted nanocarriers to changing requirements in antimalarial drug delivery.

The adaptation of existing antimalarial nanocarriers to new Plasmodium stages, drugs, targeting molecules, or encapsulating structures is a strategy that can provide new nanotechnology-based, cost-efficient therapies against malaria. We have explored the modification of different liposome prototypes that had been developed in our group for the targeted delivery of antimalarial drugs to Plasmodium-infected red blood cells (pRBCs). These new models include: (i) immunoliposome-mediated release of new lipid-based antimalarials; (ii) liposomes targeted to pRBCs with covalently linked heparin to reduce anticoagulation risks; (iii) adaptation of heparin to pRBC targeting of chitosan nanoparticles; (iv) use of heparin for the targeting of Plasmodium stages in the mosquito vector; and (v) use of the non-anticoagulant glycosaminoglycan chondroitin 4-sulfate as a heparin surrogate for pRBC targeting. The results presented indicate that the tuning of existing nanovessels to new malaria-related targets is a valid low-cost alternative to the de novo development of targeted nanosystems.

Quantification of the cellular dose and characterization of nanoparticle transport during in vitro testing.

BACKGROUND: The constant increase of the use of nanomaterials in consumer products is making increasingly urgent that standardized and reliable in vitro test methods for toxicity screening be made available to the scientific community. For this purpose, the determination of the cellular dose, i.e. the amount of nanomaterials effectively in contact with the cells is fundamental for a trustworthy determination of nanomaterial dose responses. This has often been overlooked in the literature making it difficult to undertake a comparison of datasets from different studies. Characterization of the mechanisms involved in nanomaterial transport and the determination of the cellular dose is essential for the development of predictive numerical models and reliable in vitro screening methods. RESULTS: This work aims to relate key physico-chemical properties of gold nanoparticles (NPs) to the kinetics of their deposition on the cellular monolayer. Firstly, an extensive characterization of NPs in complete culture cell medium was performed to determine the diameter and the apparent mass density of the formed NP-serum protein complexes. Subsequently, the kinetics of deposition were studied by UV-vis absorbance measurements in the presence or absence of cells. The fraction of NPs deposited on the cellular layer was found to be highly dependent on NP size and apparent density because these two parameters influence the NP transport. The NP deposition occurred in two phases: phase 1, which consists of cellular uptake driven by the NP-cell affinity, and phase 2 consisting mainly of NP deposition onto the cellular membrane. CONCLUSION: The fraction of deposited NPs is very different from the initial concentration applied in the in vitro assay, and is highly dependent of the size and density of the NPs, on the associated transport rate and on the exposure duration. This study shows that an accurate characterization is needed and suitable experimental conditions such as initial concentration of NPs and liquid height in the wells has to be considered since they strongly influence the cellular dose and the nature of interactions of NPs with the cells.

Simultaneous determination of size and quantification of silica nanoparticles by asymmetric flow field-flow fractionation coupled to ICPMS using silica nanoparticles standards.

This work proposes the use of multimodal mixtures of monodispersed silica nanoparticles (SiO2-NPs) standards for the simultaneous determination of size and concentration of SiO2-NPs in aqueous suspensions by asymmetric flow field-flow fractionation (AF4) coupled to inductively coupled plasma mass spectrometry (ICPMS). For such a purpose, suspensions of SiO2-NPs standards of 20, 40, 60, 80, 100, and 150 nm were characterized by transmission electronic microscopy (TEM), centrifugal liquid sedimentation (CLS), dynamic light scattering (DLS) and by measuring the Z-potential of the particles as well as the exact concentration of NPs by offline ICPMS. An online AF4-ICPMS method which allowed the separation of all the different sized SiO2-NPs contained in the mixture of standards was developed and the analytical figures of merit were systematically evaluated. The method showed excellent linearity in the studied concentration range (0.1-25 mg L(-1)), limits of detection between 0.16 and 0.3 mg L(-1) for smaller and greater particles, respectively, besides a satisfactory accuracy. AF4 calibration with particles with identical nature to those to be analyzed, also permitted accurate size determination in a pragmatic way. Similarly, by using prechannel calibration with NPs for mass determination it was possible to overcome common quantification problems associated with losses of material during the separation and size-dependent effects. The proposed methodology was successfully applied to the characterization in terms of size and concentration of aqueous test samples containing SiO2-NPs with monomodal size distributions.

Application of heparin as a dual agent with antimalarial and liposome targeting activities toward Plasmodium-infected red blood cells.

Heparin had been demonstrated to have antimalarial activity and specific binding affinity for Plasmodium-infected red blood cells (pRBCs) vs. non-infected erythrocytes. Here we have explored if both properties could be joined into a drug delivery strategy where heparin would have a dual role as antimalarial and as a targeting element of drug-loaded nanoparticles. Confocal fluorescence and transmission electron microscopy data show that after 30 min of being added to living pRBCs fluorescein-labeled heparin colocalizes with the intracellular parasites. Heparin electrostatically adsorbed onto positively charged liposomes containing the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane and loaded with the antimalarial drug primaquine was capable of increasing three-fold the activity of encapsulated drug in Plasmodium falciparum cultures. At concentrations below those inducing anticoagulation of mouse blood in vivo, parasiticidal activity was found to be the additive result of the separate activities of free heparin as antimalarial and of liposome-bound heparin as targeting element for encapsulated primaquine. FROM THE CLINICAL EDITOR: Malaria remains an enormous global public health concern. In this study, a novel functionalized heparin formulation used as drug delivery agent for primaquine was demonstrated to result in threefold increased drug activity in cell cultures, and in a murine model it was able to provide these benefits in concentrations below what would be required for anticoagulation. Further studies are needed determine if this approach is applicable in the human disease as well.

The SARS-CoV-2 Nucleoprotein Induces Innate Memory in Human Monocytes.

The interaction of SARS-CoV-2 with the human immune system is at the basis of the positive or negative outcome of the infection. Monocytes and macrophages, which are major innate immune/inflammatory effector cells, are not directly infected by SARS-CoV-2, however they can react to the virus and mount a strong reaction. Whether this first interaction and reaction may bias innate reactivity to re-challenge, a phenomenon known as innate memory, is currently unexplored and may be part of the long-term sequelae of COVID-19. Here, we have tested the capacity of SARS-CoV-2 and some of its proteins to induce innate memory in human monocytes in vitro. Our preliminary results show that the Spike protein subunits S1 and S2 and the entire heat-inactivated virus have no substantial effect. Conversely, monocytes pre-exposed to the nucleocapsid N protein react to subsequent viral or bacterial challenges with an increased production of anti-inflammatory IL-1Ra, a response profile suggesting a milder response to new infections.

Biophysical investigations of GBV-C E1 peptides as potential inhibitors of HIV-1 fusion peptide.

Five peptide sequences corresponding to the E1 protein of GBV-C [NCCAPEDIGFCLEGGCLV (P7), APEDIGFCLEGGCLVALG (P8), FCLEGGCLVALGCTICTD (P10), QAGLAVRPGKSAAQLVGE (P18), and AQLVGELGSLYGPLSVSA (P22)] were synthesized because they were capable of interfering with the HIV-1 fusion peptide (HIV-1 FP)-vesicle interaction. In this work the interaction of these peptides with the HIV-1 FP, as well as with membrane models, was analyzed to corroborate their inhibition ability and to understand if the interaction with the fusion peptide takes place in solution or at the membrane level. Several studies were carried out on aggregation and membrane fusion, surface Plasmon resonance, and conformational analysis by circular dichroism. Moreover, in vitro toxicity assays, including cytotoxicity studies in 3T3 fibroblasts and hemolysis assays in human red blood cells, were performed to evaluate if these peptides could be potentially used in anti-HIV-1 therapy. Results show that P10 is not capable of inhibiting membrane fusion caused by HIV-1 and it aggregates liposomes and fuses membranes, thus we decided to discard it for futures studies. P18 and P22 do not inhibit membrane fusion, but they inhibit the ability of HIV-1 FP to form pores in bilayers, thus we have not discarded them yet. P7 and P8 were selected as the best candidates for future studies because they are capable of inhibiting membrane fusion and the interaction of HIV-1 FP with bilayers. Therefore, these peptides could be potentially used in future anti-HIV-1 research.

Artificial intelligence for imaging-based COVID-19 detection: Systematic review comparing added value of AI versus human readers.

PURPOSE: A growing number of studies have examined whether Artificial Intelligence (AI) systems can support imaging-based diagnosis of COVID-19-caused pneumonia, including both gains in diagnostic performance and speed. However, what is currently missing is a combined appreciation of studies comparing human readers and AI. METHODS: We followed PRISMA-DTA guidelines for our systematic review, searching EMBASE, PUBMED and Scopus databases. To gain insights into the potential value of AI methods, we focused on studies comparing the performance of human readers versus AI models or versus AI-supported human readings. RESULTS: Our search identified 1270 studies, of which 12 fulfilled specific selection criteria. Concerning diagnostic performance, in testing datasets reported sensitivity was 42-100% (human readers, n = 9 studies), 60-95% (AI systems, n = 10) and 81-98% (AI-supported readers, n = 3), whilst reported specificity was 26-100% (human readers, n = 8), 61-96% (AI systems, n = 10) and 78-99% (AI-supported readings, n = 2). One study highlighted the potential of AI-supported readings for the assessment of lung lesion burden changes, whilst two studies indicated potential time savings for detection with AI. CONCLUSIONS: Our review indicates that AI systems or AI-supported human readings show less performance variability (interquartile range) in general, and may support the differentiation of COVID-19 pneumonia from other forms of pneumonia when used in high-prevalence and symptomatic populations. However, inconsistencies related to study design, reporting of data, areas of risk of bias, as well as limitations of statistical analyses complicate clear conclusions. We therefore support efforts for developing critical elements of study design when assessing the value of AI for diagnostic imaging.

Assessing the safety and efficacy of TAVR compared to SAVR in low-to-intermediate surgical risk patients with aortic valve stenosis: An overview of reviews.

BACKGROUND: Transcatheter aortic valve replacement (TAVR) was initially introduced to treat patients with aortic valve stenosis (AS) at high-risk for surgical aortic valve replacement (SAVR). Today, there is ample evidence supporting TAVR in high-risk groups. However, in recent years TAVR has been extended to low-to intermediate risk groups and relevant clinical evidence is still emerging, leaving some uncertainties. METHODS: To obtain information on TAVR versus SAVR in low-to intermediate risk groups, we conducted an overview of systematic reviews following PRISMA guidelines and based on a systematic search of EMBASE, MEDLINE, Cochrane and CRD databases. We focused on systematic reviews assessing mortality and VARC 2 as clinical outcomes. RESULTS: The majority of the 11 systematic reviews included in our study reported no differences in mortality between TAVR and SAVR at short and long-term follow-up times. Two reviews that included the most recent RCTs on low-risk patients reported a decreased mortality risk with TAVR at one-year follow-up. Regarding the secondary endpoints of stroke and MI, the majority of studies presented similar results for TAVR and SAVR. Acute Kidney Injury, Bleeding Complications, Atrial Fibrillation were less frequent with TAVR, with lower risk of Permanent Pacemaker Implantation and Aortic Regurgitation with SAVR. CONCLUSIONS: Our overview indicated that TAVR is a promising intervention for low-to-intermediate surgical risk patients; however additional evidence from longer term follow-up is needed to confirm these findings. This overview highlights inconsistencies about reporting and presentation of data, most notably limited clarity on effects of risk of bias on trial results.

Ongoing inflammation enhances the toxicity of engineered nanomaterials: Application of an in vitro co-culture model of the healthy and inflamed intestine.

Chronic inflammatory conditions can negatively impact intestinal barrier function and affect the epithelium's interaction with nano-sized materials. We demonstrate the application of a Caco-2/THP-1 co-culture mimicking the intestine in healthy (i.e. stable) or inflamed state in nanotoxicological research. The co-cultures were exposed to non-toxic concentrations of silver nanoparticles (AgNPs) or silver nitrate (AgNO3) for 24 h. The barrier integrity and cytokine release as well as necrotic and apoptotic cell death were investigated. AgNPs and AgNO3 most strongly affected the inflamed co-culture. Higher concentrations of AgNPs induced a significant increase in barrier integrity in the inflamed but not the stable co-culture. Necrotic and apoptotic cell death was detected in both conditions but were significantly more pronounced in the inflamed condition. The exposure to AgNO3 affected barrier integrity in all experimental set-ups, but caused nuclear condensation only in the Caco-2 monoculture and the inflamed co-culture. AgNPs reduced the release of monocyte chemoattractant protein-1 in the stable model. Clear differences were observed in the effects of AgNPs and AgNO3 in relation to the model's health status. The results suggest an increased vulnerability of the inflamed epithelial barrier towards AgNPs underlining the importance to consider the intestinal health status in the safety assessment of nanomaterials.

Overview of the blood compatibility of nanomedicines: A trend analysis of in vitro and in vivo studies.

As nanomedicines have the potential to address many currently unmet medical needs, the early identification of regulatory requirements that could hamper a smooth translation of nanomedicines from the laboratory environment to clinical applications is of utmost importance. The blood system is especially relevant as many nanomedicinal products that are currently under development are designed for intravenous administration and cells of the blood system will be among the first biological systems exposed to the injected nanomedicine. This review collects and summarizes the current knowledge related to the blood compatibility of nanomedicines and nanomaterials with a potential use in biomedical applications. Different types of nanomedicines were analyzed for their toxicity to the blood system, and the role of their physicochemical properties was further elucidated. Trends were identified related to: (a) the nature of the most frequently occurring blood incompatibilities such as thrombogenicity and complement activation, (b) the contribution of physicochemical properties to these blood incompatibilities, and (c) the similarities between data retrieved from in vivo and in vitro studies. Finally, we provide an overview of available standards that allow evaluating the compatibility of a material with the blood system. This article is categorized under: Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials Therapeutic Approaches and Drug Discovery > Emerging Technologies Toxicology and Regulatory Issues in Nanomedicine > Regulatory and Policy Issues in Nanomedicine.

Vial sonication and ultrasonic immersion probe sonication to generate stable dispersions of multiwall carbon nanotubes for physico-chemical characterization and biological testing.

Nanotechnology is considered to be a key enabling technology and in recent years there has been much growth in the use of nanostructured materials in industrial applications and in consumer products. It is, therefore, important that prior to being commercialized in consumer products, engineered nanomaterials are subjected to a thorough physico-chemical characterization as part of broader risk assessment to evaluate their possible effects on human health and the environment. The proper dispersion of nanomaterials sourced as powders becomes a first crucial step in the characterization process. This paper focuses on the dispersion of multiwall carbon nanotubes - often hydrophobic and tangled - since it may be challenging to re-disperse them effectively in aqueous media prior to characterization. A comparison has been made of non-contact vial sonication and immersion probe sonication using tannic acid as a dispersant. Transmission electron microscopy and UV-Vis spectroscopy were the techniques used to evaluate the dispersions. We used High Content Imaging and Colony Forming Efficiency to perform in vitro cytotoxicity studies on Human Alveolar Epithelial cells. It was found that both sonication treatments produce equivalent stable dispersions. No cytotoxic effects from MWCNTs were observed although some toxicity was observed and attributed to excess of the tannic acid dispersant.

Are existing standard methods suitable for the evaluation of nanomedicines: some case studies.

The use of nanotechnology in medical products has been demonstrated at laboratory scale, and many resulting nanomedicines are in the translational phase toward clinical applications, with global market trends indicating strong growth of the sector in the coming years. The translation of nanomedicines toward the clinic and subsequent commercialization may require the development of new or adaptation of existing standards to ensure the quality, safety and efficacy of such products. This work addresses some identified needs, and illustrates the shortcomings of currently used standardized methods when applied to medical-nanoparticles to assess particle size, drug loading, drug release and in vitro safety. Alternative physicochemical, and in vitro toxicology methods, with the potential to qualify as future standards supporting the evaluation of nanomedicine are provided.

Proteomics study of silver nanoparticles on Caco-2 cells.

Silver nanoparticles (AgNPs) have been incorporated into several consumer products. While these advances in technology are promising and exciting, the effects of these nanoparticles have not equally been studied. Due to the size, AgNPs can penetrate the body through oral exposure and reach the gastrointestinal tract. The present study was designed as a comparative proteomic analysis of Caco-2 cells, used as an in vitro model of the small intestine, exposed to 30 nm citrate stabilized-silver nanoparticles (AgNPs) for 24 or 72 h. Using two complementary proteomic approaches, 2D gel-based and label-free mass spectrometry, we present insight into the effects of AgNPs at proteins level. Exposure of 1 or 10 µg/mL AgNPs to Caco-2 cells resulted in 56 and 88 altered proteins at 24 h and 72 h respectively, by 2D gel-based technique. Ten of these proteins were found to be common between the two time-points. Using label-free mass spectrometry technique, 291 and 179 altered proteins were found at 24 h and 72 h, of which 24 were in common. Analysis of the proteomes showed several major biological processes altered, from which, cell cycle, cell morphology, cellular function and maintenance were the most affected.

Polyamidoamine Nanoparticles for the Oral Administration of Antimalarial Drugs.

Current strategies for the mass administration of antimalarial drugs demand oral formulations to target the asexual Plasmodium stages in the peripheral bloodstream, whereas recommendations for future interventions stress the importance of also targeting the transmission stages of the parasite as it passes between humans and mosquitoes. Orally administered polyamidoamine (PAA) nanoparticles conjugated to chloroquine reached the blood circulation and cured Plasmodium yoelii-infected mice, slightly improving the activity of the free drug and inducing in the animals immunity against malaria. Liquid chromatography with tandem mass spectrometry analysis of affinity chromatography-purified PAA ligands suggested a high adhesiveness of PAAs to Plasmodium falciparum proteins, which might be the mechanism responsible for the preferential binding of PAAs to Plasmodium-infected erythrocytes vs. non-infected red blood cells. The weak antimalarial activity of some PAAs was found to operate through inhibition of parasite invasion, whereas the observed polymer intake by macrophages indicated a potential of PAAs for the treatment of certain coinfections such as Plasmodium and Leishmania. When fluorescein-labeled PAAs were fed to females of the malaria mosquito vectors Anopheles atroparvus and Anopheles gambiae, persistent fluorescence was observed in the midgut and in other insect's tissues. These results present PAAs as a versatile platform for the encapsulation of orally administered antimalarial drugs and for direct administration of antimalarials to mosquitoes, targeting mosquito stages of Plasmodium.

Development of an in vitro co-culture model to mimic the human intestine in healthy and diseased state.

The intestine forms the largest interface between the environment and the human organism. Its integrity and functioning are crucial for the uptake of nutrients while preventing access of harmful antigens. Inflammatory conditions can significantly change the normal functioning of the intestine. In vitro models that adequately reproduce both healthy and inflamed intestinal tissue could provide a useful tool for studying the mechanisms of intestinal inflammation and investigating new therapeutic drugs. We established a co-culture of Caco-2 and PMA-differentiated THP-1 cells that mimics the intestine in healthy and controlled inflamed states. In homoeostatic conditions without stimulation, Caco-2 and THP-1 cells were co-cultured for 48h without affecting the barrier integrity and with no increase in the release of cytokines, nitric oxide or lactate dehydrogenase. To simulate the inflamed intestine, the Caco-2 barrier was primed with IFN-γ and THP-1 cells were pre-stimulated with LPS and IFN-γ. In these conditions a significant but temporary reduction in barrier integrity was measured, and large concentrations of pro-inflammatory cytokines and cytotoxicity markers detected. With its ability to feature numerous hallmarks of intestinal inflammation the presented co-culture model of epithelial cells and macrophages offers a unique possibility to study exposure effects in relation to the health status of the intestine.

Role of the crystalline form of titanium dioxide nanoparticles: Rutile, and not anatase, induces toxic effects in Balb/3T3 mouse fibroblasts.

The wide use of titanium dioxide nanoparticles (TiO2 NPs) in industrial applications requires the investigation of their effects on human health. In this context, we investigated the effects of nanosized and bulk titania in two different crystalline forms (anatase and rutile) in vitro. By colony forming efficiency assay, a dose-dependent reduction of the clonogenic activity of Balb/3T3 mouse fibroblasts was detected in the presence of rutile, but not in the case of anatase NPs. Similarly, the cell transformation assay and the micronucleus test showed that rutile TiO2 NPs were able to induce type-III foci formation in Balb/3T3 cells and appeared to be slightly genotoxic, whereas anatase TiO2 NPs did not induce any significant neoplastic or genotoxic effect. Additionally, we investigated the interaction of TiO2 NPs with Balb/3T3 cells and quantified the in vitro uptake of titania using mass spectrometry. Results showed that the internalization was independent of the crystalline form of TiO2 NPs but size-dependent, as nano-titania were taken up more than their respective bulk materials. In conclusion, we demonstrated that the cytotoxic, neoplastic and genotoxic effects triggered in Balb/3T3 cells by TiO2 NPs depend on the crystalline form of the nanomaterial, whereas the internalization is regulated by the particle size.

A combined proteomics and metabolomics approach to assess the effects of gold nanoparticles in vitro.

Omics technologies, such as proteomics or metabolomics, have to date been applied in the field of nanomaterial safety assessment to a limited extent. To address this dearth, we developed an integrated approach combining the two techniques to study the effects of two sizes, 5 and 30 nm, of gold nanoparticles (AuNPs) in Caco-2 cells. We observed differences in cells exposed for 72 h to each size of AuNPs: 61 responsive (up/down-regulated) proteins were identified and 35 metabolites in the cell extract were tentatively annotated. Several altered biological pathways were highlighted by integrating the obtained multi-omics data with bioinformatic tools. This provided a unique set of molecular information on the effects of nanomaterials at cellular level. This information was supported by complementary data obtained by immunochemistry, microscopic analysis, and multiplexed assays. A part from increasing our knowledge on how the cellular processes and pathways are affected by nanomaterials (NMs), these findings could be used to identify specific biomarkers of toxicity or to support the safe-by-design concept in the development of new nanomedicines.