Search Results (108)

Sorting and isolating specific cells from heterogeneous populations are crucial for many biomedical applications, including drug discovery and medical diagnostics. Conventional methods such as Fluorescent Activated Cell Sorting (FACS) and Magnetic Activated Cell Sorting (MACS) face limitations in throughput, cost, and the ability to separate subtly different cells. Cell partitioning in Aqueous Two-Phase Systems (ATPSs) offers a biocompatible and cost-effective alternative, particularly when combined with continuous-flow microfluidics. However, it remains challenging to rationally design microfluidic ATPS devices and operation to separate cells with similar origin but different phenotypes. In this paper, using a model ATPS, polyethylene glycol (PEG)—Dextran (Dex) system, and model cells, human chondrocytes (hChs), and carboxylated polystyrene (PS) microparticles, we systematically characterized the material properties affecting cell partitioning in ATPSs, such as surface energies of the solutions and cells and solution viscosities. We developed an energy balance approach between interfacial energy and viscous dissipation to estimate the interface translocation dynamic of cells partitioning into the preferred phase. Combining the experimental measurement and the energy balance model, our calculation reveals that the time required for complete cell partitioning at the ATPS interface can be exploited in microfluidic ATPS devices to separate hChs with different phenotypes (healthy and diseased). We expect our dynamic energy approach to provide a basis and a design strategy for optimizing microfluidic ATPS devices to achieve the efficient separation of phenotypically similar cell populations and further expand the potential of microfluidic cell separation. Full article

The production of water-in-water emulsion droplets, the coalescence of which is prevented by adding oil-in-water micrometric droplets, is reported. Hexadecane (O) and cetyl trimethyl ammonium bromide (CTAB) were added to a W/W emulsion made of dextran (Dex)-enriched droplets in a Polyethyleglycol (PEG)-enriched continuous phase, and the mixture was further sonicated. Using Nile red to label the oil droplets enabled the observation of their presence at the surface of Dex droplets (5 µm), allowing for stabilizing them, preventing coalescence of the W/W emulsion, and mimicking W/O/W double emulsions. The addition of sulfate derivative of Dextran (DexSulf) allowed for stable droplets of a slightly larger diameter. By contrast, the addition of carboxymethyl Dextran (CMDex) destabilized the initial aqueous double-like emulsion, yielding sequestration of the oil droplets within the Dex-rich phase. Interestingly, addition of DexSulf to that unstable emulsion re-yielded stable droplets. Similar findings (destabilization) were obtained when adding sodium dodecyl sulfate (SDS) to the initial double-like emulsion, which reformed stable droplets when adding positively charged Dextran (DEAEDex) derivatives. The use of fluorescently (FITC) labeled derivatives of Dextran (Dex, CMDex, DEAEDex, and DexSulf) allowed us to follow their position within, out of, or at the interface of droplets in the above-mentioned mixtures. These findings are expected to be of interest in the field of materials chemistry. Full article

The functional properties of proteins are closely related to their structure and conformation. The effects of glycosylation and pH on the structural and conformational changes in whey protein isolate (WPI) were investigated using multispectral technology. More and higher-molecular-weight molecules of WPI–dextran conjugates (WDCs) with increased degrees of glycosylation (DGs) in SDS-PAGE occurred at the expense of band intensities of α-lactalbumin, β-lactoglobulin, and bovine serum albumin. The higher wavenumber shift in FTIR peaks of WPI after glycosylation in the Amide I, II, and III regions and the decrease in its intensity occurred. The maximum absorption wavelength (λ_max) of UV-Vis spectra of WPI before and after glycosylation in the range of 260–290 nm showed no significant difference in a pH range of 2.0–10.0. Moreover, the UV-Vis absorption intensities of WDCs at λ_max around 278 nm were highly and positively correlated with their DGs. The λ_max and intensities of total intrinsic fluorescence spectra of Tyr and Trp residues in WDCs with an increase in DGs had an obvious redshift and decrease, respectively. Although the intensities of synchronous fluorescence spectra of individual Tyr or Trp residues in WDCs with an increase in DGs also gradually decreased, the λ_max of the former and latter had a blueshift and redshift, respectively. UV-Vis absorption and fluorescence spectroscopies indicated that the changes in the λ_max and intensity of WPI were closely related to the protonation states of carbonyl groups and free amino groups and the degree of glycosylation. This work may be beneficial for understanding the structural and conformational changes in proteins by measuring the microenvironment around Tyr and/or Trp residues in proteins using UV-Vis absorption and synchronous fluorescence spectroscopies, providing a promising technique for quantitatively monitoring the degree of glycosylation (DG) in a rapid and practical way without any chemical reagents using UV-Vis absorption spectroscopy. Full article

In this study, we report the synthesis and characterization of Fe₃O₄ nanoparticles coated with dextran. The structural and optical properties of the Dx:Fe₃O₄ synthesized composites were investigated by Fourier Transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD) and UV–Vis absorption spectroscopy. For the first time in this paper, the photophysics of Dx:Fe₃O₄ composites in water is studied using fluorescence and phosphorescence molecular spectrometry. An analysis of the absorption spectra of the Dx:Fe₃O₄ composite reveals the broad absorption bands with maxima at wavelengths of 227 nm, 264 nm, and 340 nm. Dx:Fe₃O₄ composite nanoparticles in water exhibit strong fluorescence with a quantum yield of 0.24% in contrast to 0.07% for dextran. Phosphorescence spectra confirm the formation of new emission bands within the Dx:Fe₃O₄ solution evidenced by the maxima shift for both dextran and Dx:Fe₃O₄ composites. Full article

Lipids are the primary components of cell membranes, and their properties can be temporarily modified by microplasma-generated species to enhance drug uptake. The ability of microplasmas to influence membrane dynamics has made them effective tools for facilitating drug uptake into cells. Despite this, the effect of microplasma irradiation on cell membranes is yet to be investigated. We investigated the effects of microplasma irradiation on fluorescein isothiocyanate-dextran 150 (FD-150) uptake in Human Promyelocytic Leukemia (HL-60) cells, with the focus on transmembrane potential and lipid order changes. Plasma was applied to HL-60 cells for five, seven, and ten minutes. Fluorescence intensity measurements showed that an uptake of FD-150 increased with treatment time, before declining at ten minutes of treatment. Following treatment, transmembrane potential analysis indicated transient hyperpolarization followed by gradual depolarization until 60 min, corresponding to increased FD-150 absorption. Analysis of the lipid order showed a more disordered membrane state, with the most pronounced changes observed at ten minutes. The increase in lipid disorder increases membrane permeability while excessive disruption of the lipid order impairs cell viability. These findings demonstrate the potential of plasma-generated reactive species in modulating membrane characteristics for intracellular drug delivery. Full article

The colloidal long-storage stability of nanosized drugs is a crucial factor for pharmacology, as they require much time for robust estimation. The application of bioavailable magnetic nanosuspensions in theranostics is limited by incomplete information about their colloidal stability in the internal media of human organisms. A method for the accelerated temperature stress “aging” of magnetic nanosized suspensions is proposed for the rapid assessment and prediction of the colloidal stability over time of nanosized iron oxide suspensions stabilized by albumin HSA. Colloidal stability is assessed using dynamic light scattering (DLS), fluorescence spectroscopy, electrophoresis, and ion monitoring methods during short- and long-term storage. Rapid assessment is achieved by short high-temperature (70 °C) processing of carboxymethyl-dextran-coated nanosol in the presence of albumin. The role of albumin in the sustained stability of superparamagnetic iron oxide particles (SPIONs) was studied under conditions mimicking blood plasma (pH = 7.4) and endolysosomal cell compartments (pH = 5.5). According to the fluorescence quenching and DLS data, colloidal stability is ensured by the formation of an HSA corona on carboxymethyl-dextran-coated SPIONs and their process of clustering. In the presence of albumin, the colloidal stability of nanoparticles is shown to increase from 80 to 121 days at a storage temperature of 8 °C The prognostic shelf life of magnetic nanosol is estimated by calculating the Van’t Hoff’s relation for the rate of chemical reactions. The validity of using the Van’t Hoff’s rule is confirmed by the agreement of the calculated activation energy at 8 °C and 70 °C. The developed method of the accelerated aging of nanoparticles can not only be employed for the estimation of the shelf life of magnetic nanoparticles coated with HSA in vitro but also for assessing the stability of SPIONs applied in vivo. Full article

The glycosylation process plays a crucial role in the structural integrity and biological activity of glycoproteins, where glycans are attached to a protein backbone. There are many kinds of glycans, the most common being N-glycans, which can be arranged into three classes, that is, complex, hybrid, and high mannoses, forming a structurally very diverse set of polar compounds that are difficult to detect and separate. Most commonly, N-glycans are labeled before separation by charged or fluorescence tags for better MS or fluorescence detection, respectively. This study examines the influence of ionic strength and organic modifier selection on the separation of fluorescently labeled dextran ladders in Hydrophilic Interaction Liquid Chromatography (HILIC). Using a Glycan BEH Amide column and varying the ammonium formate buffer concentration along with acetonitrile and methanol ratios, we investigated analyte retention, separation efficiency, and post-column conductivity changes. Our findings reveal that changes in the ionic strength of the mobile phase do not contribute to changes in selectivity, neither when acetonitrile nor methanol were used as organic modifiers to the mobile phase. However, the addition of methanol significantly changes the separation mechanism where two different prevailing separations mechanisms can be identified. It was assumed that the addition of methanol influences the folding pattern of dextrans around the permanent positive charge on the added tag, which influences the changes of separation selectivity. This work presents a systematic approach to altering mobile phase composition (buffer concentration, organic modifier type) to control retention and selectivity in complex glycan analysis. The discovery that methanol significantly alters separation behavior provides a potential new method for refining HILIC separations of polar compounds. Full article

The antifreeze and antioxidant capacities of tilapia (Oreochromis mossambicus) gelatin hydrolysates were investigated, after glycosylation with saccharides of varying molecular weights, to enhance their functional properties to widen its commercial application in frozen aquatic products. Glycosylation was conducted by mixing gelatin hydrolysates with ribose, glucose, maltose, and dextran (20 kDa) at a 1:1 mass ratio; the glycosylation products had a pH of 10 and were incubated at 80 °C for 1 h. The results showed that the glycosylation degree ranked as: ribose > glucose > maltose > dextran. The mass spectrometry analysis showed that 17, 32, and 5 glycosylation sites were identified for ribose, glucose, and maltose, respectively, suggesting a molecular weight-dependent effect. Spectroscopic analyses, including ultraviolet and infrared spectroscopy, revealed that the gelatin hydrolysate structure was expanded, with chromophores in hydrophilic environments; a blue shift in the amide A and II bands confirmed that the amino group was involved. Fluorescence spectroscopy showed conformational changes with a red shift at 303.4 nm and a reduction in intensity. Antifreeze activity, such as catalase freezing protection and shrimp surimi protein stability, and antioxidant activity, including radical scavenging and metal ion chelation, were significantly improved. Ribose exhibited the strongest effects, followed by maltose and glucose. These results demonstrate the potential of glycosylation to improve gelatin hydrolysates for functional applications. Full article

Objectives: This work aimed at investigating the effect of different microneedle-based strategies on the permeation of high-molecular-weight model molecules (fluorescently labeled dextrans (FDs), 70 and 150 kDa) across the buccal mucosa. Methods: Two different approaches were evaluated: (1) stainless steel microneedles (MNs) of 500 µm height used for tissue pre-treatment; and (2) soluble microneedles of different lengths (150, 500, and 800 µm), made of polyvinylpyrrolidone and FDs, prepared using the solvent casting technique. Porcine esophageal epithelium was used as a model for the buccal mucosa. Results: The application of soluble MNs promoted high-molecular-weight dextran transport across pig esophageal epithelium. The transport was proportional to MN length, with a minimum of 500 µm, regardless of the molecular weight of the FDs. The use of solid MNs of the same length to pre-treat the tissue, followed by the application of a solution of the permeant, did not produce the same effect in terms of onset of permeation, which was found to be much slower. Conclusions: The results obtained show that by applying soluble MNs of appropriate length (500 and 800 µm), the transport of high-molecular-weight dextrans (70 and 150 kDa) across and into the mucosal tissue occurs very rapidly. The multiphoton microscopy analysis confirmed the presence of holes in the tissue and the presence of fluorescein-labeled dextrans. Full article

Glioblastoma (GBM) is a lethal primary brain cancer with a 5.6% five-year survival rate. Tumor treating fields (TTFields) are alternating low-intensity electric fields that have demonstrated a GBM patient survival benefit. We previously reported that 0.5–24 h of TTFields exposure resulted in an increased uptake of FITC-dextran fluorescent probes (4–20 kDa) in human GBM cells. However, this approach, in which a fluorescence plate-based detector is used to evaluate cells attached to glass coverslips, cannot distinguish FITC-dextran uptake in live vs. dead cells. The goal of the study was to report the optimization and validation of two independent methods to quantify human GBM cell membrane permeabilization induced by TTFields exposure. First, we optimized flow cytometry by measuring mean fluorescence intensity at 72 h for 4 kDa (TTFields 6726 ± 958.0 vs. no-TTFields 5093 ± 239.7, p = 0.016) and 20 kDa (7087 ± 1137 vs. 5055 ± 897.8, p = 0.031) probes. Second, we measured the ratio of lactate dehydrogenase (LDH) to cell viability (measured using the CellTiter-Glo [CTG] viability assay); the LDH/CTG ratio was higher under TTFields (1.47 ± 0.15) than no-TTFields (1.08 ± 0.08) conditions, p < 0.0001. The findings using these two independent methods reproducibly demonstrated their utility for time-dependent evaluations. We also showed that these methods can be used to relate the cell membrane-permeabilizing effects of the non-ionizing radiation of TTFields to that of an established cell membrane permeabilizer, the non-ionic detergent Triton-X-100. Evaluating carboplatin ± TTFields, the LDH/CTG ratio was significantly higher in the TTFields vs. no-TTFields condition at each carboplatin concentration (0–30 µM), p = 0.014. We successfully optimized and validated two cost-effective methods to reproducibly quantify TTFields-induced human GBM cancer cell membrane permeabilization. Full article

The accurate assessment of drug concentrations in biodistribution studies is crucial for evaluating the efficacy and toxicity of compounds in drug development. As the concentration of biologics in plasma can be higher than in tissue due to their potentially low volume of distribution, transcardiac perfusion is commonly employed to reduce the influence of excess drugs in residual blood. However, there is a lack of consistency in the literature on the conditions and methods of perfusion. To enhance blood removal during transcardiac perfusion, sodium nitrite (NaNO₂), a vasodilator, has been widely used with concentrations up to 5% in publications. However, we found that such high NaNO₂ could disrupt the BBB during perfusion, which should be avoided in experiments. In this study, we examined the impact of various vasodilators on blood–brain barrier integrity and vascular permeability using the ratio of FITC-Dextran to Texas Red-Dextran (FITC/Texas Red). Additionally, we optimized perfusion conditions—including euthanasia method and perfusion flow rate—based on hemoglobin levels and the FITC/Texas Red ratio in tissues. Despite the superiority of NaNO₂ in terms of solubility and cost over other vasodilators, we found that 2% NaNO₂ disrupted blood–brain barrier integrity, significantly altering the FITC/Texas Red ratio. In contrast, 100 mM NaNO₂ did not significantly affect this ratio. Moreover, under Ketamine/Xylazine (Ket/Xyl) anesthesia, which reduced blood clot formation compared to CO₂ euthanasia, 100 mM NaNO₂ achieved the lowest hemoglobin levels in the brain. Compared to other vasodilators and the PBS control group, 100 mM NaNO₂ decreased the tissue/plasma ratio (K_p,t) but not brain/plasma ratio (K_p,b) of hIgG1 and human transferrin. We have developed a method to efficiently evaluate blood–brain barrier integrity during transcardiac perfusion. The combination of Ket/Xyl anesthesia and 100 mM NaNO₂ effectively removes residual blood from tissues without significantly affecting blood vessel permeability. Full article

This study explores the potential of cold atmospheric plasma (CAP) to facilitate the delivery of large-molecule drugs to the brain. The blood–brain barrier (BBB) restricts the passage of most drugs, hindering treatment for neurological disorders. CAP generates reactive oxygen and nitrogen species (RONS) that may disrupt the BBB’s tight junctions, potentially increasing drug permeability. An in vitro BBB model and an immortalized cell line (bEND.3) were used in this experiment. Fluorescein isothiocyanate dextran (FD-4), a model drug, was added to the cells to determine drug permeability. Custom microplasma was used to produce reactive oxygen species (ROS). Trans-endothelial electrical resistance (TEER) measurements assessed the integrity of the BBB after the CAP treatment. A decrease in TEER was observed in the CAP-treated group compared to the controls, suggesting increased permeability. Additionally, fluorescence intensity measurements from the basal side of the trans-well plate indicated higher drug passage in the CAP-treated group. Moreover, the higher presence of ROS in the plasma-treated cells confirmed the potential of CAP in drug delivery. These findings suggest that CAP may be a promising approach for enhancing brain drug delivery. Full article

To tease apart differences between conventional cage (CC) and cage-free (CF) housing systems, this study focuses on the effects of excreta exposure and age by comparing microbial communities, intestinal permeability, and corticosterone in hens in enriched colonies (EC) and CF housing systems during early- and late-lay. Hens were randomly selected from two rooms of CF (n = 20) and EC (n = 20) at 35 and 76 weeks of age. One hour following an oral gavage of fluorescein isothiocyanate dextran (FITC-D), hens were euthanized, and ileal contents and blood were collected. Serum FITC-D using a fluorescent spectrophotometer and corticosterone using a commercial competitive ELISA kit were analyzed. Following DNA isolation from the ileum contents, the V4 region of the 16S rRNA gene was sequenced. Sequence data were filtered in Mothur v1.43.0, followed by de novo operational taxonomic unit (OTU) clustering and classifying with the SILVA SSU v138 reference database. Serum FITC-D was altered by housing type, age of hens, and the interaction between housing type and age of hens (p < 0.001), with 76-week-old hens housed in EC having the highest FITC-D. Corticosterone increased with age (p = 0.023). Microbial community diversity measurements favored hens housed in the CF housing system as ileal contents tended to have increased species evenness (p = 0.008) and greater alpha diversity (p = 0.006). The majority of the over-representation of OTUs were associated with peak lay. Full article

Zeolitic imidazolate framework-8 (ZIF-8) nanoparticles (NPs) are gaining traction in tumor theranostics for their effectiveness in encapsulating both imaging agents and therapeutic drugs. While typically, similar hydrophilic molecules are encapsulated in either pure aqueous or organic environments, few studies have explored co-encapsulation of chemotherapeutic drugs and imaging agents with varying hydrophilicity and, consequently, constructed multifunctional ZIF-8 composite NPs for acid-responsive, near-infrared fluorescence imaging/chemotherapy combined tumor theranostics. Here, we present a one-pot method for the synthesis of uniform Cy5.5&DOX@ZIF-8 nanoparticles in mixed solvents, efficiently achieving simultaneous encapsulation of hydrophilic doxorubicin (DOX) and hydrophobic Cyanine-5.5 (Cy5.5). Surface decoration with dextran (Dex) enhanced colloidal stability and biocompatibility. The method significantly facilitated co-loading of Cy5.5 dyes and DOX drugs, endowing the composite NPs with notable fluorescent imaging capabilities and pH-responsive chemotherapy capacities. In vivo near-infrared fluorescence (NIRF) imaging in A549 tumor-bearing mice demonstrated significant accumulation of Cy5.5 at tumor sites due to enhanced permeability and retention (EPR) effects, with fluorescence intensities approximately 48-fold higher than free Cy5.5. Enhanced therapeutic efficiency was observed in composite NPs compared to free DOX, validating tumor-targeted capability. These findings suggest ZIF-8-based nanomedicines as promising platforms for multifunctional tumor theranostics. Full article

Blood–brain barrier (BBB) dysfunction is a key feature in neuroimmunological and neurodegenerative diseases. In this study, we developed a microfluidic human BBB-on-a-chip to model barrier dysfunction and immune cell migration using immortalized TY10 brain endothelial cells, pericytes, and astrocytes. It was found that immortalized TY10 brain endothelial cells developed a microvascular structure under flow. Pericytes were localized on the basal side surrounding the TY10 microvascular structure, showing an in vivo-like structure. Barrier integrity increased under co-culture with pericytes. In addition, both ethylenediaminetetraacetic acid (EDTA) and anti-Claudin-5 (CLDN5) neutralizing antibody caused a decrease in the transendothelial electrical resistance (TEER). EDTA caused the leakage of 20 kDa dextran, suggesting different effects on the BBB based on the mechanism of action, whereas anti-CLDN5 antibody did not cause leakage. In the tri-culture model, human T cells migrated through endothelial vessels towards basal C-X-C motif chemokine ligand 12 (CXCL12). The live-imaging analysis confirmed the extravasation of fluorescence-labelled T cells in a CXCL12-concentration- and time-dependent manner. Our BBB model had an in vivo-like structure and successfully represented barrier dysfunction and transendothelial T cell migration. In addition, our study suggests that the inhibition of CLDN5 attenuates the BBB in humans. This platform has various potential uses in relation to the BBB in both drug discovery research and in elucidating the mechanisms of central nervous system diseases. Full article