Visual Detection of LPS at the Femtomolar Level Based on Click Chemistry-Induced Gold Nanoparticles Electrokinetic Accumulation.

Lipopolysaccharide (LPS) presents a significant threat to human health. Herein, a novel method for detecting LPS was developed by coupling hybridization chain reaction (HCR), gold nanoparticles (AuNPs) agglutination (AA) triggered by a Cu(I)-catalyzed azide-alkyne cycloaddition click chemistry (CuAAC), and electrokinetic accumulation (EA) in a microfluidic chip, termed the HCR-AA-EA method. Thereinto, the LPS-binding aptamer (LBA) was coupled with the AuNP-coated Fe3O4 nanoparticle, which was connected with the polymer of H1 capped on CuO (H1-CuO) and H2-CuO. Upon LPS recognition by LBA, the polymers of H1- and H2-CuO were released into the solution, creating a "one LPS-multiple CuO" effect. Under ascorbic acid reduction, CuAAC was initiated between the alkyne and azide groups on the AuNPs' surface; then, the product was observed visually in the microchannel by EA. Finally, LPS was quantified by the integrated density of AuNP aggregates. The limit of detections were 29.9 and 127.2 fM for water samples and serum samples, respectively. The levels of LPS in the injections and serum samples by our method had a good correlation with those from the limulus amebocyte lysate test (r = 0.99), indicating high accuracy. Remarkably, to popularize our method, a low-cost, wall-power-free portable device was developed, enabling point-of-care testing.

PCR-Free, Label-Free, and Centrifugation-Free Diagnosis of Multiplex Antibiotic Resistance Genes by Combining mDNA-Au@Fe3O4 from Heating Dry and DNA Concatamers with G-Triplex.

Accurate identification of antibiotic resistance genes (ARGs) is crucial for improving treatment and controlling the spread of antibiotic-resistant bacteria (ARB). Herein, a novel PCR-free, centrifugation-free, and label-free magnetic fluorescent biosensor (MFB) was developed by combining polyA-medium DNA-polyT (mDNA, which contained a partial sequence of a target DNA), gold nanoparticle (AuNP)-anchored magnetic nanoparticle (Au@Fe3O4), complementary strand DNA (CS) of the target DNA, DNA concatamer with G-triplex (G3), and thioflavin T (ThT). Thereinto, Au@Fe3O4 nanoparticles were first capped by mDNA strands within 20 min using a simple hot drying method, and then CS was added and hybridized with mDNA on Au@Fe3O4. Second, a DNA concatamer was used to bind with CS on Au@Fe3O4. When an ARG was present in the sample, the CS would recognize it and release the DNA concatamer into solution by a toehold-mediated strand displacement reaction. Finally, under magnetic separation, the free DNA concatamers with G3 were taken out easily and bound with ThT, resulting in strong fluorescence signals. The fluorescence intensity of ThT was positively correlated with the concentration of the ARG. The whole analysis was accomplished within 1.5 h using 96-well plates. Remarkably, our MFB was universal; eight ARGs were detected by replacing the corresponding mDNA and CS in this study. To verify the practicability of our method, 12 clinically isolated strains were analyzed. The results of the MFB method were in good agreement with those of the quantitative real-time PCR method with an area under the curve of 0.92 (95% confidence interval: 0.8479 to 0.9932), sensitivity of 92.00%, and specificity of 91.55%. Above all, the MFB assay established here is simple, low-cost, and universal and has great potential for applications in the identification of ARGs.

High-performance cation electrokinetic concentrator based on a γ-CD/QCS/PVA composite and microchip for evaluating the activity of P-glycoprotein without any interference from serum albumin.

The development of cation electrokinetic concentrators (CECs) has been hindered by the lack of commercial anion-exchange membranes (AEMs). This paper introduces a γ-cyclodextrin-modified quaternized chitosan/polyvinyl alcohol (γ-CD/QCS/PVA) composite as an AEM, which is combined with a microchip to fabricate a CEC. Remarkably, the CEC only concentrates cationic species, thereby overcoming the interference of the highly abundant, negatively charged serum albumin in the blood sample. P-Glycoprotein (P-gp) is recognized as an efflux transporter protein that influences the pharmacokinetics (PK) of various compounds. The CEC was used to evaluate the activity of P-gp by detecting the positively charged rhodamine 123 (Rho123 is a classical substrate of P-gp) with no interference from serum albumin in the serum sample. Using the CEC, the enrichment factor (EF) of Rho123 exceeded 105-fold under DC voltage application. The minimal sample consumption of the CEC (<10 µL) enables reduction of animal sacrifice in animal experiments. Here, the CEC has been applied to evaluate the transport activity of P-gp in in vitro and in vivo experiments by detecting Rho123 in the presence of P-gp inhibitors or agonists. The results are in good agreement with those reported in previous reports. Therefore, the CEC presents a promising application potential, owing to its simple fabrication process, high sensitivity, minimal sample consumption, lack of interference from serum albumin and low cost.

Iron oxide nanoparticles loaded with paclitaxel inhibits glioblastoma by enhancing autophagy-dependent ferroptosis pathway.

OBJECTIVE: To explore inhibitory effect of iron oxide nanoparticles loaded with paclitaxel (IONP@PTX) on glioblastoma (GBM) and its potential mechanism. METHODS: IONP@PTX was synthesized and the characteristics were assessed by chemico-physical analysis and observed directly under transmission electron microscope. U251 cells and HMC3 cells were separately incubated with IONP@PTX and PTX, and then cell viability was detected by Cell Counting Kit-8. The capacity of cell invasive and migration was verified by Scratch wound healing and Transwell migration and invasion assays. Expressions levels of autophagy and ferroptosis biomarkers were demonstrated by Western blotting assay. Intracellular reactive oxygen species (ROS) and lipid peroxidation were detected with DCFH-DA and C11-BODIPY staining, respectively. Intracellular concentration of iron ions was quantified spectrophotometrically. Moreover, inhibitory effect of IONP@PTX on GBM was evaluated by monitoring tumor growth and the toxicity of IONP@PTX was evaluated by measuring the body weight and index of liver and spleen in the mice bearing GBM xenograft. RESULTS: The successfully synthesized IONP@PTX possesses a hydrate diameter about 36 nm and a core diameter around 10 nm. IONP@PTX exerted an inhibitory effect on U251 cells, but had little effect on HMC3 cells compared with PTX alone. In addition, IONP@PTX inhibited the capacity of cell migration and invasion, increased the levels of iron ions, ROS and lipid peroxidation, enhanced the expression of autophagy-related protein Beclin1 and LC3II, and suppressed the expression of p62 and ferroptosis-related protein GPX4 in vitro compared with control group. Moreover, administration of IONP@PTX suppressed tumor volume of GBM xenografts and decreased the expression level of GPX4 protein in tumor tissues in comparison with control group (All P < 0.05). Intriguingly, the effect of IONP@PTX on GBM could be weakened by additional 3-MA or enhanced by additional rapamycin in vitro and in vivo (P < 0.05). More importantly, IONP@PTX had no obvious toxic effect on mice bearing GBM xenograft. CONCLUSION: IONP@PTX inhibits GBM growth by enhancing autophagy-dependent ferroptosis pathway, thus it might become a potential ferroptosis-inducing agent for ferroptosis-based tumor therapy.

A systematic review and meta-analysis of fluorescent-guided resection and therapy-based photodynamics on the survival of patients with glioma.

Glioma is the most common primary central nervous system tumor; many methods are currently being used to research and treat glioma. In recent years, fluorescent-guided resection (FGR) and photodynamic therapy (PDT) have become hot spots in the treatment of glioma. Based on the existing literatures regarding the FGR enhancing resection rate and regarding efficacy of PDT for the treatment of glioma, this paper made a systematic review of FGR for gross total resection of patients and the PDT for the survival of patients with glioma. Meta-analysis of eligible studies was performed to derive precise estimation of PDT on the prognosis of patients with glioma by searching all related literatures in PubMed, EMBASE, Cochrane, and Web of Science databases, and further to evaluate (GTR) under FGR and the efficacy of PDT therapy, including 1-year and 2-year survival rates, overall survival (OS), and progression-free survival (PFS). According to the inclusion and exclusion criteria, a total of 1294 patients with glioma were included in the final analysis of 31 articles, among which a 73.00% (95% CI, 68.00 ~ 79.00%, P < 0.01) rate of GTR in 27 groups included in 23 articles was reported for those receiving FGR. The OS was 17.78 months (95% CI, 8.89 ~ 26.67, P < 0.01) in 5 articles on PDT-treated patients with glioma, and the mean difference of OS was 6.18 (95% CI, 3.3 ~ 9.06, P < 0.01) between PDT treatment and conventional glioma surgery, showing a statistically significant difference (P < 0.01). The PFS was 10.82 months (95% CI, 7.04 ~ 14.61, P < 0.01) in 5 articles on PDT-treated patients with glioma. A 1-year survival rate of 59.00% (95% CI, 38.00 ~ 77.00%, P < 0.01) in 10 groups included in 8 articles and 2-year survival rate of 25.00% (95% CI, 15.00 ~ 36.00%, P < 0.01) in 7 groups included in 6 articles were reported for those with PDT. FGR and PDT are feasible for treatment of patients with glioma, because FGR can effectively increase the resection rate, at the same time, PDT can prolong the survival time. However, due to the limitation of small sample size in the existing studies, larger samples and randomized controlled clinical trials are needed to analyze the resection under FGR and efficacy of PDT in patients with glioma.

Exosomes synergized with PIONs@E6 enhance their immunity against hepatocellular carcinoma via promoting M1 macrophages polarization.

BACKGROUND: Hepatocellular carcinoma (HCC) is easy to relapse after resection for its lack of anti-tumor immunity due to pro-tumorigenesis by promoting M2 type macrophage polarization. Recent studies have shown that exosomes are closely related to the occurrence and development of HCC. Antigenic exosomes from HCC are able to polarize into alternatively activated macrophages M2, but do not stimulate M1 macrophages polarization. Iron oxide nanoparticles (IONs) have been demonstrated to be able to promote M1 macrophages polarization. This research was to explore exosomes as vehicles to synergize with pegylated IONs loaded with chlorin e6 (PIONs@E6) to enhance their immunity against HCC via promoting M1 macrophages polarization. MATERIALS AND METHODS: PIONs@E6 was synthesized and then characterized by chemico-physical analysis, transmission electron microscope (TEM), respectively. After characterization of PIONs-contained exosomes by TEM, and then the exosomal surface specific molecules CD9 and CD63 were determined by Western Blotting assay. Markers of M1 macrophage polarization in vitro and in vivo were analyzed by enzyme linked immunosorbent assay (ELISA) and flow cytometry, respectively. Intracellular reactive oxygen species (ROS) in macrophages were analyzed using a Spectra Max fluorescence microplate reader. Inhibitory effect of PIONs-contained exosomes on HCC was evaluated by monitoring tumor growth in an in vivo xenograft mice model. RESULTS: PIONs@E6 showed good water solubility with a core diameter around 10 nm and a hydrate diameter around 37 nm. The expression of exosome specific markers CD9 and CD63 was kept at a high level. PIONs-contained exosomes can dose-dependently promote M1 macrophages polarization in vitro and in vivo. Of note, PIONs-contained exosomes could initiate a significantly higher level of ROS in macrophages and remarkably inhibit the tumor growth in mice bearing HCC xenograft. CONCLUSION: Exosomes as vehicles could be synergized with PIONs@E6 to enhance their immunity against HCC via promoting M1 macrophages polarization.

Ultrasmall iron oxide nanoparticles induced ferroptosis via Beclin1/ATG5-dependent autophagy pathway.

Iron-based nanoparticles, which could elicit ferroptosis, is becoming a promising new way to inhibit tumor cell growth. Notably, ultrasmall iron oxide nanoparticles (USIONPs) have been found to upregulate the autophagy process in glioblastoma (GBM) cells. Whether USIONPs could also elicit ferroptosis and the relationship between the USIONPs-induced autophagy and ferroptosis need to be explored. In the current study, our synthesized USIONPs with good water solubility could significantly upregulate the ferroptosis markers in GBM cells, and downregulate the expression of anti-ferroptosis genes. Interestingly,ferrostatin-1 could reverse USIONPs- induced ferroptosis, but inhibitors of apoptosis, pyroptosis, or necrosis could not. Meanwhile, autophagy inhibitor 3-methyladenine could also reverse the USIONPs-induced ferroptosis. In addition, shRNA silencing of upstream genes Beclin1/ATG5 of autophagy process could significantly reverse USIONPs-induced ferroptosis, whereas overexpression of Beclin1/ATG5 of autophagy process could remarkably promote USIONPs-induced ferroptosis. Furthermore, lysosome inhibitors could significantly reverse the USIONPs-induced ferroptosis. Collectively, these facts suggest that USIONPs-induced ferroptosis is regulated via Beclin1/ATG5-dependent autophagy pathway.