Nile Red Research Articles

Nile Red Fluorescence: Where's the Twist?

Nile Red is a fluorescent dye used extensively in bioimaging due to its strong solvatochromism. The photophysics underpinning Nile Red's fluorescence has been disputed for decades, with some studies claiming that the dye fluoresces from two excited states and/or that the main emissive state is twisted and intramolecular charge-transfer (ICT) in character as opposed to planar ICT (PICT). To resolve these long-standing questions, a combined experimental and theoretical study was used to unravel the mechanism of Nile Red's fluorescence. Time-resolved fluorescence measurements indicated that Nile Red emission occurs from a single excited state. Theoretical calculations revealed no evidence for a low-lying TICT state, with the S1 minimum corresponding to a PICT state. Ultrafast pump-probe spectroscopic data contained no signatures associated with an additional excited state involved in the fluorescence decay of Nile Red. Collectively, these data in polar and nonpolar solvents refute dual fluorescence in Nile Red and definitively demonstrate that emission occurs from a PICT state.

Dual-Responsive Nanoparticles for Smart Drug Delivery: A NIR Light-Sensitive and Redox-Reactive PEG-PCL-Based System.

Stimuli-responsive polymeric nanoparticles (NPs) can serve as smart drug delivery systems (DDSs) by triggering drug release upon external or internal stimuli. A dual-responsive DDS made of a triblock poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) (PCL-SS-PEG-SS-PCL) copolymer, bearing disulfide bonds between PCL and PEG, was synthesized. The copolymer was functionalized with coumarin and sensitive to near-infrared (NIR) light irradiation, while the S-S bonds could be cleaved by GSH (10 mM). Characterization was achieved by nuclear magnetic resonance, size exclusion chromatography, and Fourier transform infrared analyses. Nile Red (NR)-loaded NPs were prepared through self-assembly of the copolymer in water and analyzed by dynamic light scattering and field-emission scanning electron microscopy. The NR release upon ultraviolet (UV)/NIR light irradiation as well as by GSH concentrations was monitored by using fluorescence spectroscopy, while simultaneous exposure to UV/NIR light and intracellular GSH concentration led to faster NR release. AlamarBlue assay showed satisfactory cell viability of the NR-loaded NPs, while their cellular uptake in human dermal fibroblast cells was investigated by fluorescence microscopy and fluorescence emission measurements.

A Novel Poly(ε-Caprolactone)-Based Photo-Crosslinkable Liquid Copolymer as a Versatile Drug Delivery Platform

Background/Objectives: Hydrophobic semi-solid or liquid biodegradable polymers have shown unique advantages as injectable matrices for sustained release of a wide range of drugs. Here we report the design, synthesis, and characterization of a new low-melt liquid copolymer based on poly(ε-caprolactone) (PCL) and establish its utility as a versatile delivery platform. Methods: The copolymer, mPA20, consisting of short PCL blocks connected via acid-labile acetal linkages, was synthesized using a one-pot reaction and its properties were comprehensively characterized. Results: mPA20 is an amorphous, injectable liquid at physiological temperature and can undergo pH-sensitive hydrolytic degradation. mPA20 bearing methacrylate end groups can be photo-crosslinked into solid matrices with tunable mechanical properties. A hydrophobic fluorophore, Nile Red (NR), was solubilized in mPA20 without any solvent. Sustained release of NR into aqueous medium was achieved using mPA20, either as an injectable liquid depot or a photo-crosslinked solid matrix. Further, mPA20 self-emulsified in water to form nanodroplets, which were subsequently photo-crosslinked into nanogels. Both the nanodroplets and nanogels mediated efficient intracellular delivery of NR with no cytotoxicity. Conclusions: mPA20, a new photo-crosslinkable, hydrophobic liquid copolymer with pH-sensitive degradability, is highly adaptable as either an injectable or implantable depot or nanoscale carrier for the controlled release and intracellular delivery of poorly soluble drugs.

From microplastics to pixels: testing the robustness of two machine learning approaches for automated, Nile red-based marine microplastic identification.

Despite the urgent need for accurate and robust observations of microplastics in the marine environment to assess current and future environmental risks, existing procedures remain labour-intensive, especially for smaller-sized microplastics. In addition to this, microplastic analysis faces challenges due to environmental weathering, impacting the reliability of research relying on pristine plastics. This study addresses these knowledge gaps by testing the robustness of two automated analysis techniques which combine machine learning algorithms with fluorescent colouration of Nile red (NR)-stained particles. Heterogeneously shaped uncoloured microplastics of various polymers-polyethylene (PE), polyethylene terephthalate (PET), polypropylene (PP), polystyrene (PS), and polyvinyl chloride (PVC)-ranging from 100 to 1000µm in size and weathered under semi-controlled surface and deep-sea conditions, were stained with NR and imaged using fluorescence stereomicroscopy. This study assessed and compared the accuracy of decision tree (DT) and random forest (RF) models in detecting and identifying these weathered plastics. Additionally, their analysis time and model complexity were evaluated, as well as the lower size limit (2-4µm) and the interoperability of the approach. Decision tree and RF models were comparably accurate in detecting and identifying pristine plastic polymers (both > 90%). For the detection of weathered microplastics, both yielded sufficiently high accuracies (> 77%), although only RF models were reliable for polymer identification (> 70%), except for PET particles. The RF models showed an accuracy > 90% for particle predictions based on 12-30 pixels, which translated to microplastics sized < 10µm. Although the RF classifier did not produce consistent results across different labs, the inherent flexibility of the method allows for its swift adaptation and optimisation, ensuring the possibility to fine-tune the method to specific research goals through customised datasets, thereby strengthening its robustness. The developed method is particularly relevant due to its ability to accurately analyse microplasticsweathered under various marine conditions, as well as ecotoxicologically relevant microplastic sizes, making it highly applicable to real-world environmental samples.

Fluorescent Antimicrobial Peptides Based on Nile Red: Effect of Conjugation Site and Chemistry on Wash-Free Staining of Bacteria.

Fluorescent probes for bacterial detection can be obtained by conjugating antimicrobial peptides with fluorescent dyes. However, little is known about the effect of the conjugation site and linker chemistry on staining efficiency. We synthesized three conjugates of the antimicrobial peptide ubiquicidin with the environmentally sensitive fluorophore Nile Red that differed by the attachment site and the chemical composition of the linker. We showed that incorporating fluorophore as a minimalistic non-natural amino acid resulted in a superior probe compared with the typically used bioconjugation approaches. The new peptide-based probe named UNR-1 displayed red fluorescence and enabled robust wash-free staining of Gram-positive and Gram-negative bacteria. The probe exhibited selectivity over mammalian cells and enabled rapid fluorescence detection of bacteria by fluorescence microscopy and flow cytometry in an add-and-read format. Our results may foster the development of next-generation fluorescent AMPs for clinical laboratory diagnostics and medical imaging.

Pervasive Microplastic Ingestion by Commercial Fish Species from a Natural Lagoon Environment

Microplastics have emerged as a significant global environmental concern in the recent decade. The aim of this study was to elucidate microplastic contamination of commercial fish species in a natural lagoon environment. Microplastic contamination was examined in the gastrointestinal tracts and gills of 157 commercial fish from 18 species with varying feeding habits in a vital and sensitive lagoon ecosystem, which connects to the Indian ocean. Microplastics were extracted using digestion, followed by stereomicroscopic inspection using Nile Red stain, and identified via μ-FTIR analysis. Over half of studied fishes ingested microplastics (54.14%). Filaments (50%) and blue items (43%) were the most commonly ingested. Of all the fish species, Eubleekeria splendens had the highest average concentration of microplastics in GIT (1.41 ± 2.52 items/g w.w. tissues), although no statistically significant difference in amount of ingested microplastics (items/g w.w. tissues) was observed among species. The highest concentrations of inhaled microplastics were recorded in Sillago vincenti (1.38 ± 1.30 items/g w.w. tissues). The majority of the extracted microplastics (33%) belonged in the size class 500–1500 μm with rayon, polyethylene terephthalate, and polypropylene as the primary polymers. This study found no correlation between microplastic ingestion and fish species and feeding habits, but a positive correlation with fish size was observed. These findings reveal widespread microplastic contamination in edible fish, posing potential risks to commercially important species due to increasing pollution in lagoon ecosystems.

Nosema ceranae infection reduces the fat body lipid reserves in the honeybee Apis mellifera

Nosema ceranae is an intestinal parasite frequently found in Apis mellifera colonies. This parasite belongs to Microsporidia, a group of obligate intracellular parasites known to be strongly dependent on their host for energy and resources. Previous studies have shown that N. ceranae could alter several metabolic pathways, including those involved in the nutrient storage. To explore the impact of N. ceranae on the fat body reserves, newly emerged summer bees were experimentally infected, and we measured (1) the lipid percentage of the abdominal fat body at 2-, 7- and 14-days post-inoculation (p.i.) using diethyl ether lipid extraction, (2) the triglyceride and protein concentrations by spectrophotometric assay methods, and (3) the amount of intracellular lipid droplets in trophocytes at 14- and 21-days p.i. using Nile Red staining. Comparing the three methods used to evaluate lipid stores, our data revealed that Nile Red staining seemed to be the simplest, fastest and reliable method. Our results first revealed that the percentage of fat body lipids significantly decreased in infected bees at D14 p.i. The protein stores did not seem to be affected by the infection, while triglyceride concentration was reduced by 30% and lipid droplet amount by 50% at D14 p.i. Finally, a similar decrease in lipid droplet reserves in response to N. ceranae infection was observed in bees collected in fall.

Plasmonic Coacervate as a Droplet-Based SERS Platform for Rapid Enrichment and Microanalysis of Hydrophobic Payloads.

A novel and simple coacervate microdroplet-based detection platform for the quantification of trace hydrophobic analytes is presented. Herein, taking advantage of the effective encapsulation and enrichment performance of the condensed coacervates, plasmonic metallic silver nanoparticles (AgNPs) and target hydrophobic analytes are simultaneously concentrated into a single microdroplet. The coencapsulation of AgNPs within coacervates promotes the formation of aggregates with a lot of "hot spots" for surface-enhanced Raman scattering (SERS) enhancement, facilitating the sensitive analysis of hydrophobic analytes by SERS technology. Such plasmonic coacervates are easily prepared and exhibit good reproducibility and signal uniformity. Optimized SERS performance by modulating the volume of encapsulated AgNPs enables quantitative determination of hydrophobic analytes of Nile Red, chlorpyrifos, benzo[e]pyrene, 20 and 50 nm polystyrene nanoplastics with low detection limits of 10-12 M, 10-9 M, 10-10 M, 0.05 ppb, and 0.5 ppb, and an approximately linear correlation between SERS signals and the analytical concentrations. This study opens a new convenient SERS platform for the ultrasensitive detection of hydrophobic hazardous substances, potentially becoming a rapid analysis method for extensive applications ranging from food safety to environment monitoring.

Optimised methods to image hepatic lipid droplets in zebrafish larvae.

The optical transparency of zebrafish larvae enables visualization of subcellular structures in intact organs, and these vertebrates are widely used to study lipid biology and liver disease. Lipid droplet (LD) presence is a prevalent feature of healthy cells but under conditions such as nutrient excess, toxicant exposure or metabolic imbalance, LD accumulation in hepatocytes can be a harbinger of more severe forms of liver disease. We undertook a comprehensive analysis of approaches useful to investigate LD distribution and dynamics in physiological and pathological conditions in the liver of zebrafish larvae. This comparative analysis of the lipid dyes oil red O (ORO), Nile Red (NR), LipidTox and LipidSpot as well as transgenic LD reporters that rely on EGFP fusions of the LD decorating protein perilipin 2 (PLIN2) demonstrate the strengths and limitations of each approach. These protocols are amenable to detection methods ranging from low resolution stereomicroscopy to confocal imaging, which enables measurements of hepatic LD size, number and dynamics at cellular resolution in live and fixed animals. This resource will benefit investigators studying LD biology in zebrafish disease models.

Female-age-dependent changes in the lipid fingerprint of the mammalian oocytes.

Can oocyte functionality be assessed by observing changes in their intracytoplasmic lipid droplets (LDs) profiles? Lipid profile changes can reliably be detected in human oocytes; lipid changes are linked with maternal age and impaired developmental competence in a mouse model. In all cellular components, lipid damage is the earliest manifestation of oxidative stress (OS), which leads to a cascade of negative consequences for organelles and DNA. Lipid damage is marked by the accumulation of LDs. We hypothesized that impaired oocyte functionality resulting from aging and associated OS could be assessed by changes in LDs profile, hereafter called lipid fingerprint (LF). To investigate if it is possible to detect differences in oocyte LF, we subjected human GV-stage oocytes to spectroscopic examinations. For this, a total of 48 oocytes derived from 26 young healthy women (under 33 years of age) with no history of infertility, enrolled in an oocyte donation program, were analyzed. Furthermore, 30 GV human oocytes from 12 women were analyzed by transmission electron microscopy (TEM). To evaluate the effect of oocytes' lipid profile changes on embryo development, a total of 52 C57BL/6 wild-type mice and 125 Gnpat+/- mice were also used. Human oocytes were assessed by label-free cell imaging via coherent anti-Stokes Raman spectroscopy (CARS). Further confirmation of LF changes was conducted using spontaneous Raman followed by Fourier transform infrared (FTIR) spectroscopies and TEM. Additionally, to evaluate whether LF changes are associated with developmental competence, mouse oocytes and blastocysts were evaluated using TEM and the lipid dyes BODIPY and Nile Red. Mouse embryonic exosomes were evaluated using flow cytometry, FTIR and FT-Raman spectroscopies. Here we demonstrated progressive changes in the LF of oocytes associated with the woman's age consisting of increased LDs size, area, and number. LF variations in oocytes were detectable also within individual donors. This finding makes LF assessment a promising tool to grade oocytes of the same patient, based on their quality. We next demonstrated age-associated changes in oocytes reflected by lipid peroxidation and composition changes; the accumulation of carotenoids; and alterations of structural properties of lipid bilayers. Finally, using a mouse model, we showed that LF changes in oocytes are negatively associated with the secretion of embryonic exosomes prior to implantation. Deficient exosome secretion disrupts communication between the embryo and the uterus and thus may explain recurrent implantation failures in advanced-age patients. Due to differences in lipid content between different species' oocytes, the developmental impact of lipid oxidation and consequent LF changes may differ across mammalian oocytes. Our findings open the possibility to develop an innovative tool for oocyte assessment and highlight likely functional connections between oocyte LDs and embryonic exosome secretion. By recognizing the role of oocyte LF in shaping the embryo's ability to implant, our original work points to future directions of research relevant to developmental biology and reproductive medicine. This research was funded by National Science Centre of Poland, Grants: 2021/41/B/NZ3/03507 and 2019/35/B/NZ4/03547 (to G.E.P.); 2022/44/C/NZ4/00076 (to M.F.H.) and 2019/35/N/NZ3/03213 (to Ł.G.). M.F.H. is a National Agency for Academic Exchange (NAWA) fellow (GA ULM/2019/1/00097/U/00001). K.F. is a Diamond Grant fellow (Ministry of Education and Science GA 0175/DIA/2019/28). The open-access publication of this article was funded by the Priority Research Area BioS under the program "Excellence Initiative - Research University" at the Jagiellonian University in Krakow. The authors declare no competing interest. N/A.

A novel fluorescent probe for viscosity and polarity detection in real tobacco root cells and biological imaging.

The disruption of lipid droplet function is associated with the pathogenesis of various diseases. Clarifying the response behavior of lipid droplets to the microenvironment at the cellular level is of great significance. Plant lipids not only exist in phospholipids in cell membranes, but also in aromatic essential oils. Monitoring the level of lipid droplets in plant cells using fluorescent probes provides a simple method for screening lipid-rich varieties. We synthesized a polarity-viscosity responsive coumarin fluorescent probe, Cou-CN, which achieved sensitive detection of polarity and viscosity in dilute solution environments by constructing this simple probe with ICT and TICT properties and verifying it using Gaussian computational simulation. Cou-CN exhibited good lipid droplet illumination effects in HepG2 cells with a correlation coefficient of 0.92 compared to the commercial lipid droplet dye BODIPY. Additionally, co-staining the probe with the lipophilic commercial dye Nile Red in tobacco root stem seedling cells resulted in a high correlation coefficient of 0.9.

The evaluation of Starmerella magnoliae X3 as a biodiesel feedstock based on triacylglycerol (TAG) production, lipid productivity, and fatty acid profile under nitrogen limitation and acidic pH conditions.

The effects of four initial culture pH values (3, 4, 5, and 6) and nitrogen limitation on growth, TAG accumulation, lipid production, fatty acid profile, and estimated biodiesel quality of Starmerella magnoliae X3 were investigated. TAG and lipid levels were measured by Nile Red fluorescence and sulfo-phospho-vanilin (SPV) techniques, respectively. The results showed that a combination of nitrogen limitation and acidic pH significantly (p < 0.05) increased TAG accumulation, total lipid contents, and lipid productivity in Starmerella magnoliae X3 compared to the control group. Under nitrogen limitation, the highest TAG accumulation was achieved at initial pHs of 3 and 5 after 72h of cultivation, and the highest lipid productivity (0.306g L-1 d-1) was observed after 48h at pH 3; the major fatty acids at the four pH values were oleic acid (63.6%-64%), palmitoleic acid (11.3%-12.5%), stearic acid (9.7%-11.4%), and palmitic acid (9.4%-10%). In addition, both stresses were associated with lower iodine value and higher cetane number of the biodiesel compared to the control. These findings suggest that cultivation in a low-nitrogen medium at an initial pH of 3 or 5 holds promise in increasing TAG production in Starmerella magnoliae X3.

Cascade Fluorescence Modulation in Photochromic Microcapsules.

Certain derivatives of terarylene are able to undergo a highly efficient oxidative cycloreversion cascade effect, a ring opening reaction with quantum yields above unity, resulting in a colored-to-colorless transition in solution. In the presence of chloroform, high-energy UV and X-rays can trigger this phenomenon, potentially acting as a visual detection system for ionizing radiation. However, chloroform is sensitive to different irradiation wavelengths without distinction, making it difficult to adapt to a reusable device. Chlorobenzene was chosen as an alternative halogenated solvent, as it offers wavelength selectivity between photocyclization and cascade effect cycloreversion. Nile Red was also incorporated into the system with the aim of improving the sensitivity of the visual detection via fluorescence photoswitching. Finally, microencapsulation of both terarylene and Nile Red was targeted to obtain both the cascade effect and photoswitching in a single system. In microcapsules made from a Pickering emulsion, this terarylene-Nile Red system showed high fatigue resistance to repeated photocyclization and cycloreversion irradiation, giving access to repeated ON/OFF fluorescence photoswitching. The cascade effect was also successfully demonstrated along with fluorescence recovery, showing the versatility of the two phenomena in different media.

Impact of seasonal changes and environmental conditions on suspended and inhalable microplastics in urban air

Microplastics (MPs) are ubiquitous environmental pollutants extensively detected in atmospheric environments. Airborne MPs have raised concerns due to their transport and potential health risks of inhalation exposure. However, the factors influencing airborne MPs, particularly their concentrations and shapes suspended in urban air, remain unclear. We investigated MPs in total suspension particles with one-year measurements in Taipei City and identified their features using Nile Red staining combined with fluorescence microscopy and micro-Fourier transform infrared (μFTIR) spectroscopy. This study quantified the mean number concentration of total MPs as approximately 6.0 #/m³. We observed that MP abundance varied seasonally, with higher levels in the warm season than in the cold. A similar trend was noted for polymer types. Fragment-like MPs were the predominant shape, mainly found in polystyrene (PS), polyethylene (PE), and polypropylene (PP), while fibrous MPs, detected mostly as polyethylene terephthalate (PET) and polyamide (PA), were primarily observed at sizes greater than 300 μm. Both fiber and fragment-like MPs were positively associated with particle mass concentration, temperature, ultraviolet (UV) index, and wind speed, but negatively correlated with relative humidity and rainfall. Fibrous MPs were more affected by environmental factors than fragment-like MPs. Meteorological changes significantly influenced suspended MPs more than human activity within the city.

Optimization and Synthesis of Nano-Niosomes for Encapsulation of Triacontanol by Box-Behnken Design.

Triacontanol is a long-chain primary alcohol derived from policosanol, known for its diverse biological activities, including functioning as a plant growth regulator and exhibiting anti-inflammatory and antitumoral effects. However, its application is limited due to its high hydrophobicity, resulting in poor absorption and reduced therapeutic effectiveness. A potential solution to this problem is the use of niosomes. Niosomes are carriers composed of non-ionic surfactants, cholesterol, charge-inducing agents, and a hydration medium. They are effective in encapsulating drugs, improving their solubility and bioavailability. The objective of this study was to optimize and synthesize nano-niosomes for the encapsulation of triacontanol. Niosomes were synthesized using a thin-film hydration method combined with ultrasonication, following a Box-Behnken design. Niosomes were characterized using various techniques including dynamic light scattering, Fourier-transform infrared spectroscopy (FTIR), confocal microscopy, high-resolution scanning electron microscopy, and transmission electron microscopy (TEM). Formulation 14 of niosomes achieved the desired size, polydispersity index (0.198 ± 0.008), and zeta potential (-31.28 ± 1.21). FTIR analysis revealed a characteristic signal in the 3400-300 cm-1 range, indicating intermolecular interactions due to a bifurcated hydrogen bond between cholesterol and S60. Confocal microscopy confirmed the presence of triacontanol through Nile Red fluorescence. TEM revealed the spherical structure of niosomes.

Silibinin inhibits PM2.5-induced liver triglyceride accumulation through enhancing the function of mitochondrial Complexes I and II.

The standardized extract of milk thistle seeds, known as silibinin, has been utilized in herbal medicine for over two centuries, with the aim of safeguarding the liver against the deleterious effects of various toxic substances. However, the role of silibinin in Particulate Matter (PM2.5)-induced intrahepatic triglyceride accumulation remains unclear. This study seeks to investigate the impact of silibinin on PM2.5-induced intrahepatic triglyceride accumulation and elucidate potential underlying mechanisms. A model of intrahepatic triglyceride accumulation was established in male C57BL/6J mice through intratracheal instillation of PM2.5, followed by assessment of liver weight, body weight, liver index, and measurements of intrahepatic triglycerides and cholesterol after treatment with silibinin capsules. Hep G2 cells were exposed to PM2.5 suspension to create an intracellular triglyceride accumulation model, and after treatment with silibinin, cell viability, intracellular triglycerides and cholesterol, fluorescence staining for Nile Red (lipid droplets), and DCFH-DA (Reactive Oxygen Species, ROS), as well as proteomics, real-time PCR, and mitochondrial function assays, were performed to investigate the mechanisms involved in reducing triglycerides. PM2.5 exposure leads to triglyceride accumulation, increased ROS production, elevated expression of inflammatory factors, decreased expression of antioxidant factors, and increased expression of downstream genes of aryl hydrocarbon receptor. Silibinin can partially or fully reverse these factors, thereby protecting cells and animal livers from PM2.5-induced damage. In vitro studies show that silibinin exerts its protective effects by preserving oxidative phosphorylation of mitochondrial complexes I and II, particularly significantly enhancing the function of mitochondrial complex II. Succinate dehydrogenase (mitochondrial complex II) is a direct target of silibinin, but silibinin A and B exhibit different affinities for different subunits of complex II. Silibinin improved the accumulation of intrahepatic triglycerides induced by PM2.5, and this was, at least in part, explained by an enhancement of oxidative phosphorylation in mitochondrial Complexes I and II.

Cell Membrane-Camouflaged Biomimetic Magnetic Fluorescent Nanoparticles with Enhanced Stability for High-Performance Lead Drug Discovery.

Biomimetic nanoengineering empowers nanoparticles with enhanced biointerfacial capabilities by directly utilizing cell membranes (CMs) of natural origin. This top-down technique provides a powerful tool for the screening of potentially active compounds in complex matrices. Herein, cartilaginous end plate (CEP) cell membrane biomimetic Nile red (NR)-loaded zeolitic imidazolate frameworks-8 (ZIF-8) modified magnetic graphene oxide (CEP/MGO-ZIF-8-NR) nanocomposites with enhanced stability were accurately prepared by chemical bonding and used as a drug discovery platform for the specific identification and effective extraction of drug leads with anti-intervertebral disc degeneration (IDD) in Yaobitong capsules (YBTCs). The constructed CEP/MGO-ZIF-8-NR exhibited excellent magnetic properties, fluorescence properties, and stability. In addition, drug binding experiments showed that the CEP/MGO-ZIF-8-NR nanocomposites possessed higher adsorption capacity, faster adsorption rate, and superior selectivity compared with uncoated MGO-ZIF-8-NR. Ultimately, four potential bioactive molecules, including ginsenoside Ro, ginsenoside Rg1, astringin, and chikusetsusaponin V methyl ester, were successfully screened and identified in vitro from YBTC. The results of the CCK-8 assay and BrdU ELISA kit showed that the screened compounds promoted CEP cell proliferation in a concentration-dependent manner. Cellular distribution experiments revealed that CEP/MGO-ZIF-8-NR could rapidly escape from lysosomes and into the cytoplasm. And the pharmacological activity of these compounds was further confirmed by real-time cytomorphological imaging of CEP cells by confocal laser scanning microscopy (CLSM). Overall, this surface engineering strategy endows bioaffinity sample pretreatment materials with tremendous versatility, improves drug screening efficiency, and broadens the horizons and methodologies for drug lead discovery.

Dysmetabolism of Peripheral Blood Monocytes in Type 1 Diabetes Mellitus.

The level of ROS (fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate) and lipid content (fluorescent lipophilic dye Nile Red) in the peripheral blood monocyte fraction from patients with type 1 diabetes mellitus and healthy volunteers were assessed by flow cytofluorimetry. The number of CD36+ monocytes was assessed using specific antibodies. In patients with type 1 diabetes mellitus, the levels of ROS and intracellular lipids in monocytes and the number of cells expressing CD36 fatty acid translocase were elevated. These results indicate metabolic changes in the peripheral blood cells of patients with carbohydrate metabolism disorders and can be considered as possible prognostic markers for the development of type 1 diabetes mellitus complications.

FLIM nanoscopy resolves the structure and preferential adsorption in the co-nonsolvency of PNIPAM microgels in methanol-water

Polymer microgels are swollen macromolecular networks with a typical size of hundred of nanometers to several microns that show an extraordinary open and responsive architecture to different external stimuli, being therefore important candidates for nanobiotechnology and nanomedical applications such as biocatalysis, sensing and drug delivery. It is therefore crucial to understand the delicate balance of physical-chemical interactions between the polymer backbone and solvent molecules that to a high extent determine their responsivity.In particular, the co-nonsolvency effect of poly(N-isopropylacrylamide) in aqueous alcohols is highly discussed, and there is a disagreement between molecular dynamics (MD) simulations (from literature) of the preferential adsorption of alcohol on the polymer chains and the values obtained by several empirical methods that mostly probe the bulk solvent properties.It is our contention that the most efficacious method for addressing this problem requires a nanoscopic method that can be combined with spectroscopy and record fluorescence spectra and super-resolved fluorescence lifetime images of microgels labeled covalently with the solvatochromic dye Nile Red. By employing this approach, we could simultaneously resolve the structure of sub-micron size objects in the swollen and in the collapsed state and estimate the solvent composition inside of them in ▪–▪ mixtures for two very different polymer architectures. We found an outstanding agreement between the MD simulations and our results that estimate a co-solvent molar fraction excess of approximately 3 with a very flat profile in the lateral direction of the microgel.

Optimising microplastics analysis for quantifying and identifying microplastic fibres in laundry wastewater

Current methods for measuring microplastic fibres (MPF) are cumbersome, time consuming and unscalable for routine high throughput analysis. This study reports a method for rapidly extracting, quantifying and analysing MPFs in laundry wastewater with several key improvements which vastly enhance overall efficiency and scalability of analysis. FT-IR surveying is employed as a preliminary step in analysis to quickly determine what polymers are present in a sample prior to fluorescence treatment. Using random quadrating, whole 25 mm filter membranes were surveyed in <30 min with high recovery rates. In industrial laundry wastewater samples, polyester was the most common MPF, however acrylic, nylon, cotton and rayon were all ubiquitous. The study also demonstrates that an excitation wavelength of 365 nm was optimal for fluorescing PET fibres like polyester which were stained with Nile Red, but not 495 nm, which is commonly used in microplastic analysis. Finally, a custom ImageJ macro was written to automatically enumerate and describe MPFs on filter membranes using just a single stitched fluorescence image. In just a few seconds, concentrations of up to 40,000 fibres/L were analysed in industrial laundry wastewater samples with a lower particle size limit of 20 μm. This study highlights the need for more optimised and scalable analysis workflows which maintain high levels of reliability and accuracy.