High biocompatible FITC-conjugated silica nanoparticles for cell labeling in both in vitro and in vivo models.

Fluorescence nanosilica-based cell tracker has been explored and applied in cell biological research. However, the aggregation of these nanoparticles at physiological pH is still the main limitation. In this research, we introduced a novel fluorescence nano-based cell tracker suitable for application in live cells. The silica-coated fluorescein isothiocyanate isomer (FITC-SiO2) nanoparticles (NPs) were modified with carboxymethylsilanetriol disodium salt (FITC-SiO2-COOH), integrating the dianion form of FITC molecules. This nanosystem exhibited superior dispersion in aqueous solutions and effectively mitigated dye leakage. These labeled NPs displayed notable biocompatibility and minimal cytotoxicity in both in vitro and in vivo conditions. Significantly, the NPs did not have negative implications on cell migration or angiogenesis. They successfully penetrated primary fibroblasts, human umbilical vein endothelial cells and HeLa cells in both 2D and 3D cultures, with the fluorescence signal enduring for over 72 h. Furthermore, the NP signals were consistently observed in the developing gastrointestinal tract of live medaka fish larvae for extended periods during phases of subdued digestive activity, without manifesting any apparent acute toxicity. These results underscore the promising utility of FITC-SiO2-COOH NPs as advanced live cell trackers in biological research.

ß-CATENIN stabilizes HIF2 through lncRNA and inhibits intravenous immunoglobulin immunotherapy.

Introduction: Tumor-initiating cells (TICs) are rare, stem-like, and highly malignant. Although intravenous hepatitis B and C immunoglobulins have been used for HBV and HCV neutralization in patients, their tumor-inhibitory effects have not yet been examined. Hepatitis B immunoglobulin (HBIG) therapy is employed to reduce hepatocellular carcinoma (HCC) recurrence in patients after living donor liver transplantations (LDLT). Hypothesis: We hypothesized that patient-derived intravenous immunoglobulin (IVIG) binding to HCC associated TICs will reduce self-renewal and cell viability driven by ß-CATENIN-downstream pathways. ß-CATENIN activity protected TICs from IVIG effects. Methods: The effects of HBIG and HCIG binding to TICs were evaluated for cell viability and self-renewal. Results: Inhibition of ß-CATENIN pathway(s) augmented TIC susceptibility to HBIG- and HCIG-immunotherapy. HBV X protein (HBx) upregulates both ß-CATENIN and NANOG expression. The co-expression of constitutively active ß-CATENIN with NANOG promotes self-renewal ability and tumor-initiating ability of hepatoblasts. HBIG bound to HBV+ cells led to growth inhibition in a TIC subset that expressed hepatitis B surface antigen. The HBx protein transformed cells through ß-CATENIN-inducible lncRNAs EGLN3-AS1 and lnc-ß-CatM. Co-expression of constitutively active ß-CATENIN with NANOG promoted self-renewal ability of TICs through EGLN3 induction. ß-CATENIN-induced lncRNAs stabilized HIF2 to maintain self-renewal of TICs. Targeting of EGLN3-AS1 resulted in destabilization of EZH2-dependent ß-CATENIN activity and synergized cell-killing of TICs by HBIG or HCIG immunotherapy. Discussion: Taken together, WNT and stemness pathways induced HIF2 of TICs via cooperating lncRNAs resulting in resistance to cancer immunotherapy. Therefore, therapeutic use of IVIG may suppress tumor recurrence through inhibition of TICs.

Inhibition of human cervical cancer development through p53-dependent pathways induced by the specified triple helical ß-glucan.

This study demonstrates that the purified ß-glucan (LNT) with a triple helix and relatively narrow molecular weight distribution, extracted and purified from artificially cultured Lentinus edodes, showed a significant cervical cancer inhibition with little cytotoxicity against normal cells in vitro and in vivo. From the in vitro data, the potential mechanism of anti-cervical cancer was preliminarily revealed as follows: LNT was firstly recognized by the human cervical cancer cell line of Hela and induced cell proliferation inhibition through p21 and apoptosis via a mitochondrion-dependent pathway by targeting the tumor suppressor of p53, indicated by an increase in reactive oxygen species (ROS) generation and a loss of mitochondrial membrane potential (Δψm), in a significant dosage-dependent manner. Meanwhile, LNT repressed tumor growth with an inhibition ratio of 61.2 % and induced tumor cell apoptosis through endogenous MDM2/p53/Bax/mitochondrion signal pathway by up-regulating the expression of p53, Bax, Cyt. c, caspase 9, and caspase 3, as well as down-regulating Bcl-2, MDM2, and PARP1 levels in Hela cells-transplanted BALB/c nude mice. This study provides a scientific basis for the clinical treatment of cervical cancer with LNT as a potential drug candidate characterized by the triple helix and specified molecular weight with a relatively narrow distribution.

Dye-Doped Polymeric Microplastics: Light Tools for Bioimaging in Test Organisms.

Ecosystems around the world are experiencing a major environmental impact from microplastic particles (MPs 0.1 µm-1 mm). Water, sediments, and aquatic biota show the widespread presence of this pollutant. However, MPs are rarely used in laboratory studies as they are scarcely available for purchase or expensive, especially if one wishes to trace the particle with a dye or fluorescent. Furthermore, existing preparation techniques have limited application in biological studies. In this work, we propose a new, easy, and cheap way to prepare fluorescent MPs. The protocol is based on the osmosis method in order to obtain spherical polymeric particles of P(S-co-MMA), with 0.7-9 micron diameter, made fluorescent because dye-doped with rhodamine B isothiocyanate (RITC) or fluorescein isothiocyanate (FITC). The dye loading was studied and optimized, and the MPs-dye conjugates were characterized by UV-vis FTIR and XPS spectrometry and scanning electron microscopy (SEM). Furthermore, preliminary tests on aquatic organisms demonstrated the possible use of these fluorescent MPs in bioimaging studies, showing their absorption/adsorption by duckweeds (Lemna minuta) and insect larvae (Cataclysta lemnata).

Characterization and optimization of fluorescein isothiocyanate labeling of humanized h2E2 anti-cocaine mAb.

Fluorescein isothiocyanate (FITC) is widely used to fluorescently label reactive lysine residues on proteins, including antibodies. The rate and extent of labeling varies with reaction conditions, concentration of label, and the concentration and nature of the protein. Fluorescently labeled proteins are very useful, and one use for FITC labeled mAbs is development of assays to measure anti-mAb antibodies produced in vivo during treatment with antibody therapeutics. Our laboratory has developed a humanized anti-cocaine mAb (h2E2) intended for the treatment of cocaine use disorders. Thus, a well characterized FITC labeled h2E2 mAb is needed to quantitate possible anti-mAb antibodies. The time course of labeling and the relative incorporation of FITC into the heavy and light chains, as well as into the Fab and Fc portions of the mAb, was assessed. A novel use of differential scanning fluorimetry in the absence of any extrinsic fluorophore was developed and demonstrated to be capable of measuring antigen (cocaine) binding. In addition, the effect of increasing degrees of labeling by FITC on the thermodynamic parameters driving the binding of cocaine to the mAb was assessed via isothermal titration calorimetry (ITC). This binding technique, unlike others developed recently to measure cocaine binding, is not dependent on, or subject to interference by, the absorbance or fluorescence of the incorporated FITC label. The methods and results reported herein guide the optimization of FITC labeling needed for anti-mAb assays and other assays important for the development of therapeutic mAbs, which are some of the most specific and clinically useful drugs available.

Characterisation of Primary Human Hippocampal Astrocyte Cell Culture Following Exposure to Hypoxia.

Background: The present study aimed to understand the characterisation of human hippocampal astrocyte following hypoxia exposure. Based on the preliminary screening, 15 min was chosen as the time point and the cells were exposed to different oxygen percentages. Methods: The Trypan blue viability assay used to examine cell death. Immunofluorescence assay, glial fibrillary acidic protein (GFAP) was used to portray the morphology of astrocytes. The hypoxia-inducible factor 1 (HIF-1) staining was performed to confirm hypoxia induced cell death and there was a dramatic expression of HIF-1α displayed in exposed astrocyte cells compared to the control. In molecular level, genes were chosen, such as glyceraldehyde 3-phosphate dehydrogenase (GAPDH), GFAP, HIF-1α and B-cell lymphoma 2 (Bcl-2) and ran the reverse transcription-polymerase chain reaction (RT-PCR). Results: Microscope revealed a filamentous and clear nucleus appearance in a control whereas the rupture nuclei with no rigid structure of the cell were found in the 3% oxygen. The control and hypoxia cells were also stained with the annexin V-fluorescein isothiocyanate (annexin V-FITC). Fluorescence microscope reveals astrocyte cells after hypoxia showed higher expression of nuclei but not in control. Merging PI and FITC showed the differences of nuclei expression between the control and hypoxia. In the molecular analysis, there were significant changes of GFAP, HIF-1α and Bcl-2 in hypoxia exposed cells when compared to the control group. Conclusion: Cells that were exposed to hypoxia (3% oxygen for 15 min) clearly showed damage. General view of human hippocampal astrocyte genomic response to hypoxia was obtained.

Inhibition of SARS-CoV-2 nucleocapsid protein-RNA interaction by guanosine oligomeric RNA.

The interaction of the ß-coronavirus severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) nucleocapsid (N) protein with genomic RNA is initiated by specific RNA regions and subsequently induces the formation of a continuous polymer with characteristic structural units for viral formation. We hypothesized that oligomeric RNAs, whose sequences are absent in the 29.9-kb genome sequence of SARS-CoV-2, might affect RNA-N protein interactions. We identified two such hexameric RNAs, In-1 (CCGGCG) and G6 (GGGGGG), and investigated their effects on the small filamentous/droplet-like structures (< a few µm) of N protein-genomic RNA formed by liquid-liquid phase separation. The small N protein structures were sequence-specifically enhanced by In-1, whereas G6 caused them to coalesce into large droplets. Moreover, we found that a guanosine 12-mer (G12, GGGGGGGGGGGG) expelled preexisting genomic RNA from the small N protein structures. The presence of G12 with the genomic RNA suppressed the formation of the small N protein structures, and alternatively apparently altered phase separation to induce the formation of large droplets with unclear phase boundaries. We showed that the N-terminal RNA-binding domain is required for the stability of the small N protein structures. Our results suggest that G12 may be a strong inhibitor of the RNA-N protein interaction.

Spatiotemporal changes of tissue glycans depending on localization in cardiac aging.

Heart failure is caused by various factors, making the underlying pathogenic mechanisms difficult to identify. Since cardiovascular disease tends to worsen over time, early diagnosis is key for treatment. In addition, understanding the qualitative changes in the heart associated with aging, where information on the direct influences of aging on cardiovascular disease is limited, would also be useful for treatment and diagnosis. To fill these research gaps, the focus of our study was to detect the structural and functional molecular changes associated with the heart over time, with a focus on glycans, which reflect the type and state of cells. METHODS: We investigated glycan localization in the cardiac tissue of normal mice and their alterations during aging, using evanescent-field fluorescence-assisted lectin microarray, a technique based on lectin-glycan interaction, and lectin staining. RESULTS: The glycan profiles in the left ventricle showed differences between the luminal side (medial) and wall side (lateral) regions. The medial region was characterized by the presence of sialic acid residues. Moreover, age-related changes in glycan profiles were observed at a younger age in the medial region. The difference in the age-related decrease in the level of α-galactose stained with Griffonia simplicifolia lectin-IB4 in different regions of the left ventricle suggests spatiotemporal changes in the number of microvessels. CONCLUSIONS: The glycan profile, which retains diverse glycan structures, is supported by many cell populations, and maintains cardiac function. With further research, glycan localization and changes have the potential to be developed as a marker of the signs of heart failure.

In vitro and in vivo evaluation of cerium oxide nanoparticles in respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is the most common cause of viral bronchiolitis among children worldwide, yet there is no vaccine for RSV disease. This study investigates the potential of cube and sphere-shaped cerium oxide nanoparticles (CNP) to modulate reactive oxygen (ROS) and nitrogen (RNS) species and immune cell phenotypes in the presence of RSV infection in vitro and in vivo. Cube and sphere-shaped CNP were synthesized by hydrothermal and ultrasonication methods, respectively. Physico-chemical characterization confirmed the shape of sphere and cube CNP and effect of various parameters on their particle size distribution and zeta potential. In vitro results revealed that sphere and cube CNP differentially modulated ROS and RNS levels in J774 macrophages. Specifically, cube CNP significantly reduced RSV-induced ROS levels without affecting RNS levels while sphere CNP increased RSV-induced RNS levels with minimal effect on ROS levels. Cube CNP drove an M1 phenotype in RSV-infected macrophages in vitro by increasing macrophage surface expression of CD80 and CD86 with a concomitant increase in TNFα and IL-12p70, while simultaneously decreasing M2 CD206 expression. Intranasal administration of sphere and cube-CNP were well-tolerated with no observed toxicity in BALB/c mice. Notably, cube CNP preferentially accumulated in murine alveolar macrophages and induced their activation, avoiding enhanced uptake and activation of other inflammatory cells such as neutrophils, which are associated with RSV-mediated inflammation. In conclusion, we report that sphere and cube CNP modulate macrophage polarization and innate cellular responses during RSV infection.

Barrier-penetrating liposome targeted delivery of basic fibroblast growth factor for spinal cord injury repair.

Nanoparticle technologies offer a non-invasive means to deliver basic fibroblast growth factor (bFGF) for the treatment of spinal cord injury (SCI). However, the inability of bFGF to accumulate at the injury site and inefficient penetration across the blood-spinal cord barrier (BSCB) remain challenges. The present study describes a dual-targeting liposome (bFGF@Lip-Cp&Rp) with injury lesion targeting and BSCB-penetrating capability to deliver bFGF for SCI treatment. The CAQK peptide (Cp) with injury lesion targeting ability and R2KC peptide (Rp) with BSCB-penetrating capability were grafted onto the liposomes for a flexible and non-invasive drug delivery systems preparation. Results exhibit that the dual-targeted liposomes could significantly cross the BSCB and accumulate at the injury site. During the early stage of SCI, bFGF@Lip-Cp&Rp promotes repair of BSCB and facilitates M2-polarization of macrophages. Regular delivery of bFGF@Lip-Cp&Rp increase HUVECs tube formation and angiogenesis, ameliorate the microenvironment of lesion site, suppress the neuronal apoptosis and axonal atrophy in SCI rats. Importantly, continuous treatment of bFGF@Lip-Cp&Rp supports the restoration of limb motor function in SCI rats. In summary, this research implies that the injury site-targeting and BSCB-penetrating liposomes could be a promising therapeutic approach for the treatment of SCI.

Comparison of Acoustofluidic and Static Systems for Ultrasound-Mediated Molecular Delivery to T Lymphocytes.

Continuous-flow acoustofluidic technologies can potentially improve processing of T lymphocytes for cell therapies by addressing the limitations with viral and non-viral delivery methods. The objective of this study was to assess the intracellular delivery efficiency with acoustofluidic treatment compared with that of static ultrasound treatment. Optimization of parameters in acoustofluidic and static configurations was performed by assessing intracellular delivery of a fluorescent compound (calcein) in viable human Jurkat T lymphocytes. Ultrasound pressure and the concentration of cationic phospholipid-coated microbubbles influenced calcein delivery in both systems. In the static system, a treatment time of 45 s increased molecular delivery compared with 0-30 s (p < 0.01). Refined parameters were used to assess molecular delivery of small and large compounds (0.6-kDa calcein and 150-kDa fluorescein isothiocyanate-dextran, respectively) after ultrasound treatment with the acoustofluidic or static systems. Molecular delivery was similar with refined parameters for acoustofluidic treatment and static treatment (p > 0.05), even though acoustofluidic treatment had lower microbubble concentration (24 µg/mL vs. 94 µg/mL) and shorter treatment time (∼2-3 s vs. 45 s). This study indicates that the acoustofluidic system can significantly enhance intracellular molecular delivery, which could potentially enable acoustofluidic cell transfection during continuous flow processing for manufacture of cell therapies or other applications.

Ratiometric fluorescence immunoassay of SARS-CoV-2 nucleocapsid protein via Si-FITC nanoprobe-based inner filter effect.

The global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has necessitated rapid, easy-to-use, and accurate diagnostic methods to monitor the virus infection. Herein, a ratiometric fluorescence enzyme-linked immunosorbent assay (ELISA) was developed using Si-fluorescein isothiocyanate nanoparticles (FITC NPs) for detecting SARS-CoV-2 nucleocapsid (N) protein. Si-FITC NPs were prepared by a one-pot hydrothermal method using 3-aminopropyl triethoxysilane (APTES)-FITC as the Si source. This method did not need post-modification and avoided the reduction in quantum yield and stability. The p-nitrophenyl (pNP) produced by the alkaline phosphatase (ALP)-mediated hydrolysis of p-nitrophenyl phosphate (pNPP) could quench Si fluorescence in Si-FITC NPs via the inner filter effect. In ELISA, an immunocomplex was formed by the recognition of capture antibody/N protein/reporter antibody. ALP-linked secondary antibody bound to the reporter antibody and induced pNPP hydrolysis to specifically quench Si fluorescence in Si-FITC NPs. The change in fluorescence intensity ratio could be used for detecting N protein, with a wide linearity range (0.01-10.0 and 50-300 ng/mL) and low detection limit (0.002 ng/mL). The concentration of spiked SARS-CoV-2 N protein could be determined accurately in human serum. Moreover, this proposed method can accurately distinguish coronavirus disease 2019 (COVID-19) and non-COVID-19 patient samples. Therefore, this simple, sensitive, and accurate method can be applied for the early diagnosis of SARS-CoV-2 virus infection. Electronic Supplementary Material: Supplementary material (characterization of Si-FITC NPs (FTIR spectrum, XRD spectra, and synchronous fluorescence spectra); condition optimization of ALP response (fluorescence intensity ratio change); mechanism investigation of ALP response (fluorescence lifetime decay curves and UV-vis absorption spectra); detection of N protein using commercial ELISA Kit; analytical performance of assays for ALP detection or SARS-CoV-2 N protein detection; and determination results of SARS-CoV-2 N protein in human serum) is available in the online version of this article at 10.1007/s12274-022-4740-5.

Establishment and evaluation of in vitro galectin-3 inhibitor screening model / 药学学报

Galectin-3 (Gal-3) belongs to the galectin family and is specific in binding β-galactoside. Through its C-terminal domain, Gal-3 binds to the galactoside group of the glycosylated insulin receptor (IR) and inhibits IR signaling pathway, which leads to the insulin resistance. Thus, Gal-3 is a potential therapeutic target for the treatment of insulin resistance and type 2 diabetes. Here we report a simple Gal-3 screening model based on the property that Gal-3 binds to the galactoside. We expressed and purified human Gal-3 in Escherichia coli (E.coli), and labeled it with fluorescein isothiocyanate (FITC) in vitro. After incubating FITC labeled Gal-3 (Gal-3-FITC) with PANC-1 cells, which express glycosylated membrane protein, PANC-1 cells started to show green fluorescent signal due to the Gal-3-FITC binding to the glycosylated membrane protein. Gal-3 inhibitor disrupts the binding of Gal-3-FITC and PANC1 cells, subsequently leads to the decrease of the fluorescent signal in PANC-1 cells. We can evaluate the inhibitory efficiency of Gal-3 inhibitors through measurement of the fluorescent signal. Further studies show this model is simple, stable, and repeatable with a Z' factor between 0.7 and 0.85. In sum, we have successfully established an in vitro high-throughput screening model for Gal-3 inhibitors.

Evaluation of the physico-chemical properties of potato starch-based foods and their interactions with milk protein and soybean oil.

Physico-chemical properties of potato starch-based foods (PSBF) interacted with milk protein (MP), and soybean oil (SBO) were investigated. Microstructures, rheological properties, and chemical bonding among those ingredients were determined. An emulsion-filled gel, in which oil droplets stabilized by MP and/or amylose-lipid complex (ALC) dispersed in a starch gel structure of PSBF was revealed by confocal laser scanning microscopy. Starch-starch, protein-oil, and protein-protein played the dominant interactions while ALC and starch-protein interaction were subordinates. Rheological data showed that MP induced a thinning effect on starch gel, while SBO seemed to reinforce the solid-like properties of the gel. The chemical interactions analyzed by FTIR, Raman, and X-ray diffraction suggested that these foods were lack in non-covalent crosslinks and were dominated by diverse physical interactions. However, the different preparation of such foods could induce chemical binding in a different way and MP and SBO could affect the properties of PSBF in this study.

Formulation of Chitosan Microparticles for Enhanced Intranasal Macromolecular Compound Delivery: Factors That Influence Particle Size during Ionic Gelation.

Therapeutic macromolecules (e.g., protein and peptide drugs) present bioavailability challenges via extravascular administration. The nasal route presents an alternative non-invasive route for these drugs, although low bioavailability remains challenging. Co-administration of permeation enhancers is a promising formulation approach to improve the delivery of poorly bioavailable drugs. The aim of this study was to prepare and characterize chitosan microparticulate formulations containing a macromolecular model compound (fluorescein isothiocyanate dextran 4400, FD-4) and a bioenhancer (piperine). Ionic gelation was used to produce chitosan microparticle delivery systems with two distinct microparticle sizes, differing one order of magnitude in size (±20 µm and ±200 µm). These two microparticle delivery systems were formulated into thermosensitive gels and their drug delivery performance was evaluated across ovine nasal epithelial tissues. Dissolution studies revealed a biphasic release pattern. Rheometry results demonstrated a sol-to-gel transition of the thermosensitive gel formulation at a temperature of 34 °C. The microparticles incorporating piperine showed a 1.2-fold increase in FD-4 delivery across the excised ovine nasal epithelial tissues as compared to microparticles without piperine. This study therefore contributed to advancements in ionic gelation methods for the formulation of particulate systems to enhance macromolecular nasal drug delivery.

Osteogenic differentiation of pre-conditioned bone marrow mesenchymal stem cells with Nisin on modified poly-L-lactic-acid nanofibers.

Introduction: Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) are undifferentiated cells with self-renewing ability and multi-lineage differentiation beneficial for regenerative medicine. Nano scaffolds are novel materials employed in bone repair and regeneration. Nisin is a prebiotic that can increase stem cells' lifespan and proliferation. This study attempted to provide a proper strategy for bone marrow mesenchymal stem cells differentiation into the Osteocytes on a Poly-L-lactic-acid (PLLA) scaffold after pretreating with Nisin. Methods: MSC osteogenic differentiation was evaluated by measuring Calcium, Alkaline phosphatase, and quantitative tests such as Real-Time PCR, Acridine Orange, Alizarin Red, Von Kossa, and others. Results: The result of the MTT test showed that the optimal dose of Nisin prebiotic for the MSCs' preconditioning was 200 IU/mL on the 1st, 3rd, and 5th days of culture. Real-time PCR data indicated that the expression rate of ALP, Osteonectin, Osteocalcin, and Collagen I have increased in the presence of Nisin, while the RUNX-2 gene expression has decreased. Furthermore, the results of Alizarin Red and Von Kossa tests, as well as Scanning electron microscopy (SEM), revealed that the cell proliferation in the preconditioned samples with Nisin increased significantly. Conclusions: The study concluded that the cell proliferation and differentiation increased in samples pretreated with Nisin on the PLLA Nano scaffolds.

Preparation of Fluorescently Labeled Chitosan-Quercetin Drug-Loaded Nanoparticles with Excellent Antibacterial Properties.

In recent years, quercetin plays an increasingly important role in the medical field. However, the absorption and effect of quercetin as a drug in vivo are limited due to its extremely poor solubility in water. In addition, chitosan nanoparticles can deliver poorly soluble drugs as drug delivery carriers. Herein, chitosan nanoparticles were prepared by oxidative degradation and ionic cross-linking technology to study the drug loading properties of quercetin. On the other hand, the application of chitosan for fluorescent materials can improve the biocompatibility of fluorescent materials and increase the adsorption of fluorescent materials. Fluorescently labeled chitosan nanoparticles, especially chitosan microsphere fluorescent probes prepared using the abundant amino groups on chitosan chains to react with fluorescein isothiocyanate (FTIC), have been widely used as fluorescent probes in biomarkers and medical diagnostics. Therefore, chitosan-quercetin (CS-QT) drug-loaded nanoparticles are labeled with FITC, and the drug-loaded rate, encapsulation efficiency, and antioxidant properties were investigated. The drug-loaded rate of the sample reaches 8.39%, the encapsulation rate reaches 83.65%, and exhibits good antioxidant capacity. The fluorescence aperture of the obtained sample was consistent with the inhibition zone, which could realize the visualization of the antibacterial performance of the sample. The fluorescent-labeled nano-system exhibit superior antibacterial properties, which provide a strategy for observing the release and function of drugs.

Microbiome profiling of nasal extracellular vesicles in patients with allergic rhinitis.

Background: Nasal microbiota is crucial for the pathogenesis of allergic rhinitis (AR), which has been reported to be different from that of healthy individuals. However, no study has investigated the microbiota in nasal extracellular vesicles (EVs). We aimed to compare the microbiome composition and diversity in EVs between AR patients and healthy controls (HCs) and reveal the potential metabolic mechanisms in AR. Methods: Eosinophil counts and serum immunoglobulin E (IgE) levels were measured in patients with AR (n = 20) and HCs (n = 19). Nasal EVs were identified using transmission electron microscopy and flow cytometry. 16S rRNA sequencing was used to profile the microbial communities. Alpha and beta diversities were analyzed to determine microbial diversity. Taxonomic abundance was analyzed based on the linear discriminant analysis effect size (LEfSe). Microbial metabolic pathways were characterized using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUst2) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Results: Eosinophils, total serum IgE, and IgE specific to Dermatophagoides were increased in patients with AR. Alpha diversity in nasal EVs from patients with AR was lower than that in HCs. Beta diversity showed microbiome differences between the AR and HCs groups. The microbial abundance was distinct between AR and HCs at different taxonomic levels. Significantly higher levels of the genera Acetobacter, Mycoplasma, Escherichia, and Halomonas were observed in AR patients than in HCs. Conversely, Zoogloea, Streptococcus, Burkholderia, and Pseudomonas were more abundant in the HCs group than in the AR group. Moreover, 35 microbial metabolic pathways recognized in AR patients and HCs, and 25 pathways were more abundant in the AR group. Conclusion: Patients with AR had distinct microbiota characteristics in nasal EVs compared to that in HCs. The metabolic mechanisms of the microbiota that regulate AR development were also different. These findings show that nasal fluid may reflect the specific pattern of microbiome EVs in patients with AR.

An in vitro human mammary epithelial cell permeability assay to assess drug secretion into breast milk.

Determining the amount of a drug transferred into breast milk is critical for benefit-risk analysis of breastfeeding when a lactating mother takes medications. In this study, we developed a human mammary epithelial cell (MEC)-based permeability assay to assess drug permeability across the mammary epithelium. Human MEC cell MCF10F formed tight junctions when cultured on Transwells with culture medium containing insulin, hydrocortisone and epidermal growth factor (EGF). Formation of integral cell barrier and morphology of the cells were confirmed by assessing trans-epithelial electrical resistance (TEER), flux of fluorescent tracers and imaging with transmission electron microscopy (TEM). MCF10F cells showed consistent P-glycoprotein (P-gp) transporter expression when culturing on Transwell inserts versus on petri dish. A few P-gp transporter drug substrates were used to estimate the permeability from this assay. Human plasma and breast milk were used as incubation medium in basolateral and apical chambers respectively to mimic physiological conditions. The predicted milk to plasma (M/P) ratios were reasonably good. The current effort to develop the MEC-based permeability assay to facilitate M/P ratio prediction showed promising results. This assay may have a potential to be developed as a useful in vitro technique for determining the transfer of small-molecule therapeutic drugs into breast milk.

Effect of type of fatty acid attached to chitosan on walnut oil-in-water Pickering emulsion properties.

The effect of fatty acid type bonded to chitosan on the emulsifying properties of chitosan-based particles was investigated. Capric acid, myristic acid, and stearic acid were attached to chitosan chains. Longer fatty acids in the structure of chitosan lead to the better and more uniform formation of chitosan nanogels. The contact angle of chitosan, chitosan-capric acid, chitosan-myristic acid and chitosan-stearic acid were found to be 52.5°, 60.0°, 65.1° and 72.5°, respectively. Different chitosan nanogels were used to stabilize walnut oil emulsions, and the emulsion stabilized with chitosan-stearic acid nanogels had the lowest creaming index (15.2%). Stabilized emulsions with chitosans attached to longer chain acids were more adapted to the mechanism of Pickering emulsions, in addition to having higher viscosity as well as more gel-like behavior. In general, this study showed that emulsifying properties of chitosan could be improved by increasing the number of fatty acid carbons bonded to chitosan.