Interlaboratory evaluation of a digital holographic microscopy–based assay for label-free in vitro cytotoxicity testing of polymeric nanocarriers

State-of-the-art in vitro test systems for nanomaterial toxicity assessment are based on dyes and several staining steps which can be affected by nanomaterial interference. Digital holographic microscopy (DHM), an interferometry-based variant of quantitative phase imaging (QPI), facilitates reliable proliferation quantification of native cell populations and the extraction of morphological features in a fast and label- and interference-free manner by biophysical parameters. DHM therefore has been identified as versatile tool for cytotoxicity testing in biomedical nanotechnology. In a comparative study performed at two collaborating laboratories, we investigated the interlaboratory variability and performance of DHM in nanomaterial toxicity testing, utilizing complementary standard operating procedures (SOPs). Two identical custom-built off-axis DHM systems, developed for usage in biomedical laboratories, equipped with stage-top incubation chambers were applied at different locations in Europe. Temporal dry mass development, 12-h dry mass increments and morphology changes of A549 human lung epithelial cell populations upon incubation with two variants of poly(alkyl cyanoacrylate) (PACA) nanoparticles were observed in comparison to digitonin and cell culture medium controls. Digitonin as cytotoxicity control, as well as empty and cabazitaxel-loaded PACA nanocarriers, similarly impacted 12-h dry mass development and increments as well as morphology of A549 cells at both participating laboratories. The obtained DHM data reflected the cytotoxic potential of the tested nanomaterials and are in agreement with corresponding literature on biophysical and chemical assays. Our results confirm DHM as label-free cytotoxicity assay for polymeric nanocarriers as well as the repeatability and reproducibility of the technology. In summary, the evaluated DHM assay could be efficiently implemented at different locations and facilitates interlaboratory in vitro toxicity testing of nanoparticles with prospects for application in regulatory science.publishedVersio

Pre-validation of a reporter gene assay for oxidative stress for the rapid screening of nanobiomaterials

Engineered nanomaterials have been found to induce oxidative stress. Cellular oxidative stress, in turn, can result in the induction of antioxidant and detoxification enzymes which are controlled by the nuclear erythroid 2-related factor 2 (NRF2) transcription factor. Here, we present the results of a pre-validation study which was conducted within the frame of BIORIMA (“biomaterial risk management”) an EU-funded research and innovation project. For this we used an NRF2 specific chemically activated luciferase expression reporter gene assay derived from the human U2OS osteosarcoma cell line to screen for the induction of the NRF2 mediated gene expression following exposure to biomedically relevant nanobiomaterials. Specifically, we investigated Fe3O4-PEG-PLGA nanomaterials while Ag and TiO2 “benchmark” nanomaterials from the Joint Research Center were used as reference materials. The viability of the cells was determined by using the Alamar blue assay. We performed an interlaboratory study involving seven different laboratories to assess the applicability of the NRF2 reporter gene assay for the screening of nanobiomaterials. The latter work was preceded by online tutorials to ensure that the procedures were harmonized across the different participating laboratories. Fe3O4-PEG-PLGA nanomaterials were found to induce very limited NRF2 mediated gene expression, whereas exposure to Ag nanomaterials induced NRF2 mediated gene expression. TiO2 nanomaterials did not induce NRF2 mediated gene expression. The variability in the results obtained by the participating laboratories was small with mean intra-laboratory standard deviation of 0.16 and mean inter laboratory standard deviation of 0.28 across all NRF2 reporter gene assay results. We conclude that the NRF2 reporter gene assay is a suitable assay for the screening of nanobiomaterial-induced oxidative stress responses

Label-Free Digital Holographic Microscopy for In Vitro Cytotoxic Effect Quantification of Organic Nanoparticles

Cytotoxicity quantification of nanoparticles is commonly performed by biochemical assays to evaluate their biocompatibility and safety. We explored quantitative phase imaging (QPI) with digital holographic microscopy (DHM) as a time-resolved in vitro assay to quantify effects caused by three different types of organic nanoparticles in development for medical use. Label-free proliferation quantification of native cell populations facilitates cytotoxicity testing in biomedical nanotechnology. Therefore, DHM quantitative phase images from measurements on nanomaterial and control agent incubated cells were acquired over 24 h, from which the temporal course of the cellular dry mass was calculated within the observed field of view. The impact of LipImage™ 815 lipidots® nanoparticles, as well as empty and cabazitaxel-loaded poly(alkyl cyanoacrylate) nanoparticles on the dry mass development of four different cell lines (RAW 264.7, NIH-3T3, NRK-52E, and RLE-6TN), was observed vs. digitonin as cytotoxicity control and cells in culture medium. The acquired QPI data were compared to a colorimetric cell viability assay (WST-8) to explore the use of the DHM assay with standard biochemical analysis methods downstream. Our results show that QPI with DHM is highly suitable to identify harmful or low-toxic nanomaterials. The presented DHM assay can be implemented with commercial microscopes. The capability for imaging of native cells and the compatibility with common 96-well plates allows high-throughput systems and future embedding into existing experimental routines for in vitro cytotoxicity assessment

Standardization of an in vitro assay matrix to assess cytotoxicity of organic nanocarriers: a pilot interlaboratory comparison.

Nanotechnologies such as nanoparticles are established components of new medical devices and pharmaceuticals. The use and distribution of these materials increases the requirement for standardized evaluation of possible adverse effects, starting with a general cytotoxicity screening. The Horizon 2020 project "Regulatory Science Framework for Nano(bio)material-based Medical Products and Devices (REFINE)" identified in vitro cytotoxicity quantification as a central task and first step for risk assessment and development for medical nanocarriers. We have performed an interlaboratory comparison on a cell-assay matrix including a kinetic lactate dehydrogenase (LDH) release cell death and WST-8 cell viability assay adapted for testing organic nanocarriers in four well-characterized cell lines of different organ origins. Identical experiments were performed by three laboratories, namely the Biomedical Technology Center (BMTZ) of the University of Münster, SINTEF Materials and Chemistry (SINTEF), and the National Institute for Public Health and the Environment (RIVM) of the Netherlands according to new standard operating procedures (SOPs). The experiments confirmed that LipImage™ 815 lipidots® are non-cytotoxic up to a concentration of 128 µg/mL and poly(alkyl cyanoacrylate) (PACA) nanoparticles for drug delivery of cytostatic agents caused dose-dependent cytotoxic effects on the cell lines starting from 8 µg/mL. PACA nanoparticles loaded with the active pharmaceutical ingredient (API) cabazitaxel showed a less pronounced dose-dependent effect with the lowest concentration of 2 µg/mL causing cytotoxic effects. The mean within laboratory standard deviation was 4.9% for the WST-8 cell viability assay and 4.0% for the LDH release cell death assay, while the between laboratory standard deviation was 7.3% and 7.8% for the two assays, respectively. Here, we demonstrated the suitability and reproducibility of a cytotoxicity matrix consisting of two endpoints performed with four cell lines across three partner laboratories. The experimental procedures described here can facilitate a robust cytotoxicity screening for the development of organic nanomaterials used in medicine

Standardization of an in vitro assay matrix to assess cytotoxicity of organic nanocarriers: a pilot interlaboratory comparison

Nanotechnologies such as nanoparticles are established components of new medical devices and pharmaceuticals. The use and distribution of these materials increases the requirement for standardized evaluation of possible adverse effects, starting with a general cytotoxicity screening. The Horizon 2020 project “Regulatory Science Framework for Nano(bio)material-based Medical Products and Devices (REFINE)” identified in vitro cytotoxicity quantification as a central task and first step for risk assessment and development for medical nanocarriers. We have performed an interlaboratory comparison on a cell-assay matrix including a kinetic lactate dehydrogenase (LDH) release cell death and WST-8 cell viability assay adapted for testing organic nanocarriers in four well-characterized cell lines of different organ origins. Identical experiments were performed by three laboratories, namely the Biomedical Technology Center (BMTZ) of the University of Münster, SINTEF Materials and Chemistry (SINTEF), and the National Institute for Public Health and the Environment (RIVM) of the Netherlands according to new standard operating procedures (SOPs). The experiments confirmed that LipImage™ 815 lipidots® are non-cytotoxic up to a concentration of 128 µg/mL and poly(alkyl cyanoacrylate) (PACA) nanoparticles for drug delivery of cytostatic agents caused dose-dependent cytotoxic effects on the cell lines starting from 8 µg/mL. PACA nanoparticles loaded with the active pharmaceutical ingredient (API) cabazitaxel showed a less pronounced dose-dependent effect with the lowest concentration of 2 µg/mL causing cytotoxic effects. The mean within laboratory standard deviation was 4.9% for the WST-8 cell viability assay and 4.0% for the LDH release cell death assay, while the between laboratory standard deviation was 7.3% and 7.8% for the two assays, respectively. Here, we demonstrated the suitability and reproducibility of a cytotoxicity matrix consisting of two endpoints performed with four cell lines across three partner laboratories. The experimental procedures described here can facilitate a robust cytotoxicity screening for the development of organic nanomaterials used in medicine

The surgical safety checklist and patient outcomes after surgery: a prospective observational cohort study, systematic review and meta-analysis

© 2017 British Journal of Anaesthesia Background: The surgical safety checklist is widely used to improve the quality of perioperative care. However, clinicians continue to debate the clinical effectiveness of this tool. Methods: Prospective analysis of data from the International Surgical Outcomes Study (ISOS), an international observational study of elective in-patient surgery, accompanied by a systematic review and meta-analysis of published literature. The exposure was surgical safety checklist use. The primary outcome was in-hospital mortality and the secondary outcome was postoperative complications. In the ISOS cohort, a multivariable multi-level generalized linear model was used to test associations. To further contextualise these findings, we included the results from the ISOS cohort in a meta-analysis. Results are reported as odds ratios (OR) with 95% confidence intervals. Results: We included 44 814 patients from 497 hospitals in 27 countries in the ISOS analysis. There were 40 245 (89.8%) patients exposed to the checklist, whilst 7508 (16.8%) sustained ≥1 postoperative complications and 207 (0.5%) died before hospital discharge. Checklist exposure was associated with reduced mortality [odds ratio (OR) 0.49 (0.32–0.77); P\u3c0.01], but no difference in complication rates [OR 1.02 (0.88–1.19); P=0.75]. In a systematic review, we screened 3732 records and identified 11 eligible studies of 453 292 patients including the ISOS cohort. Checklist exposure was associated with both reduced postoperative mortality [OR 0.75 (0.62–0.92); P\u3c0.01; I2=87%] and reduced complication rates [OR 0.73 (0.61–0.88); P\u3c0.01; I2=89%). Conclusions: Patients exposed to a surgical safety checklist experience better postoperative outcomes, but this could simply reflect wider quality of care in hospitals where checklist use is routine

Prospective observational cohort study on grading the severity of postoperative complications in global surgery research

Background The Clavien–Dindo classification is perhaps the most widely used approach for reporting postoperative complications in clinical trials. This system classifies complication severity by the treatment provided. However, it is unclear whether the Clavien–Dindo system can be used internationally in studies across differing healthcare systems in high- (HICs) and low- and middle-income countries (LMICs). Methods This was a secondary analysis of the International Surgical Outcomes Study (ISOS), a prospective observational cohort study of elective surgery in adults. Data collection occurred over a 7-day period. Severity of complications was graded using Clavien–Dindo and the simpler ISOS grading (mild, moderate or severe, based on guided investigator judgement). Severity grading was compared using the intraclass correlation coefficient (ICC). Data are presented as frequencies and ICC values (with 95 per cent c.i.). The analysis was stratified by income status of the country, comparing HICs with LMICs. Results A total of 44 814 patients were recruited from 474 hospitals in 27 countries (19 HICs and 8 LMICs). Some 7508 patients (16·8 per cent) experienced at least one postoperative complication, equivalent to 11 664 complications in total. Using the ISOS classification, 5504 of 11 664 complications (47·2 per cent) were graded as mild, 4244 (36·4 per cent) as moderate and 1916 (16·4 per cent) as severe. Using Clavien–Dindo, 6781 of 11 664 complications (58·1 per cent) were graded as I or II, 1740 (14·9 per cent) as III, 2408 (20·6 per cent) as IV and 735 (6·3 per cent) as V. Agreement between classification systems was poor overall (ICC 0·41, 95 per cent c.i. 0·20 to 0·55), and in LMICs (ICC 0·23, 0·05 to 0·38) and HICs (ICC 0·46, 0·25 to 0·59). Conclusion Caution is recommended when using a treatment approach to grade complications in global surgery studies, as this may introduce bias unintentionally

Critical care admission following elective surgery was not associated with survival benefit: prospective analysis of data from 27 countries

This was an investigator initiated study funded by Nestle Health Sciences through an unrestricted research grant, and by a National Institute for Health Research (UK) Professorship held by RP. The study was sponsored by Queen Mary University of London