Nicorandil Regulates Ferroptosis and Mitigates Septic Cardiomyopathy via TLR4/SLC7A11 Signaling Pathway.

This study mainly explored the role of nicorandil in regulating ferroptosis and alleviating septic cardiomyopathy through toll-like receptor (TLR) 4/solute carrier family 7 member 11 (SLC7A11) signaling pathway. Twenty-four male SD rats were randomly divided into control, Nic (nicorandil), LPS (lipopolysaccharide), and LPS + Nic groups and given echocardiography. A detection kit was applied to measure the levels of lactic dehydrogenase (LDH), cardiac troponin I (cTnI), and creatine kinase-MB (CK-MB); HE staining and the levels of glutathione (GSH), malondialdehyde (MDA), total iron, and Fe2+ of myocardial tissues were detected. Moreover, the expression of TLR4 and SLC7A11 were measured by qRT-PCR and the proteins regulating ferroptosis (TLR4, SLC7A11, GPX4, ACSL4, DMT1, Fpn, and TfR1) were checked by western blot. Myocardial cells (H9C2) were induced with lipopolysaccharide (LPS) and transfected with si-TLR4 or SLC7A11-OE. Then, the viability, ferroptosis, and TLR4/SLC7A11 signaling pathway of cells were examined. Nicorandil could significantly increase left ventricular (LV) ejection fraction (LVEF) while reduce LV end-diastolic volume (LVEDV) and LV end-systolic volume (LVESV). Also, it greatly reduced the levels of LDH, cTnI, and CK-MB; alleviated the pathological changes of myocardial injury; notably decreased MDA, total iron, and Fe2+ levels in myocardial tissues; and significantly increased GSH level. Besides, nicorandil obviously raised protein levels of GPX4, Fpn, and SLC7A11, and decreased protein levels of ACSL4, DMT1, TfR1, and TLR4. After knockdown of TLR4 or overexpression of SLC7A11, the inhibition effect of nicorandil on ferroptosis was strengthened in LPS-induced H9C2 cells. Therefore, nicorandil may regulate ferroptosis through TLR4/SLC7A11 signaling, thereby alleviating septic cardiomyopathy.

Gelatinase-responsive biodegradable targeted microneedle patch for abscess wound treatment of S. aureus infection.

Abscess wound caused by bacterial infection is usually difficult to heal, thus greatly affect people's quality of life. In this study, a biodegradable drug-loaded microneedle patch (MN) is designed for targeted eradication of S. aureus infection and repair of abscess wound. Firstly, the bacterial responsive composite nanoparticle (Ce6@GNP-Van) with a size of about 182.6 nm is constructed by loading the photosensitizer Ce6 into gelatin nanoparticle (GNP) and coupling vancomycin (Van), which can specifically target S. aureus and effectively shield the phototoxicity of photosensitizer during delivery. When Ce6@GNP-Van is targeted and enriched in the infected regions, the gelatinase secreted by the bacteria can degrade GNP in situ and release Ce6, which can kill the bacteria by generating ROS under laser irradiation. In vivo experiments show that the microneedle is basically degraded in 10 min after inserting into skin, and the abscess wound is completely healed within 13 d after applying Ce6@GNP-Van-loaded MN patch to the abscess wound of the bacterial infected mice with laser irradiation, which can simultaneously achieve the eradication of biofilm and subsequent wound healing cascade activation, showing excellent synergistic antibacterial effect. In conclusion, this work establishes a synergistic treatment strategy to facilitate the repair of chronic abscess wound.

A novel vascular-targeting peptide for gastric cancer delivers low-dose TNFα to normalize the blood vessels and improve the anti-cancer efficiency of 5-fluorouracil.

Various vascular-targeted agents fused with tumor necrosis factor α (TNFα) have been shown to improve drug absorption into tumor tissues and enhance tumor vascular function. TCP-1 is a peptide selected through in vivo phage library biopanning against a mouse orthotopic colorectal cancer model and is a promising agent for drug delivery. This study further investigated the targeting ability of TCP-1 phage and peptide to blood vessels in an orthotopic gastric cancer model in mice and assessed the synergistic anti-cancer effect of 5-fluorouracil (5-FU) with subnanogram TNFα targeted delivered by TCP-1 peptide. In vivo phage targeting assay and in vivo colocalization analysis were carried out to test the targeting ability of TCP-1 phage/peptide. A targeted therapy for improvement of the therapeutic efficacy of 5-FU and vascular function was performed through administration of TCP-1/TNFα fusion protein in this model. TCP-1 phage exhibited strong homing ability to the orthotopic gastric cancer after phage injection. Immunohistochemical staining suggested that and TCP-1 phage/TCP-1 peptide could colocalize with tumor vascular endothelial cells. TCP-1/TNFα combined with 5-FU was found to synergistically inhibit tumor growth, induce apoptosis and reduce cell proliferation without evident toxicity. Simultaneously, subnanogram TCP-1/TNFα treatment normalized tumor blood vessels. Targeted delivery of low-dose TNFα by TCP-1 peptide can potentially modulate the vascular function of gastric cancer and increase the drug delivery of chemotherapeutic drugs.

Fabrication of ZnO Nanowires Arrays by Anodization and High-Vacuum Die Casting Technique, and Their Piezoelectric Properties.

In this investigation, anodic aluminum oxide (AAO) with arrayed and regularly arranged nanopores is used as a template in the high-vacuum die casting of molten zinc metal (Zn) into the nanopores. The proposed technique yields arrayed Zn nanowires with an aspect ratio of over 600. After annealing, arrayed zinc oxide (ZnO) nanowires are obtained. Varying the anodizing time yields AAO templates with thicknesses of approximately 50 µm, 60 µm, and 70 µm that can be used in the fabrication of nanowires of three lengths with high aspect ratios. Experimental results reveal that a longer nanowire generates a greater measured piezoelectric current. The ZnO nanowires that are fabricated using an alumina template are anodized for 7 h and produce higher piezoelectric current of up to 69 pA.

The current role and therapeutic targets of vitamin D in gastrointestinal inflammation and cancer.

Vitamin D, beyond its classical roles in the regulation of calcium and phosphorus homeostasis and bone metabolism, has been implicated in multiple pathological processes, including progression from inflammation to cancer development and also involvement in autoimmune diseases as well as cardiovascular disorders. In this review, we shall discuss the different roles of vitamin D and its therapeutic targets in different gastrointestinal diseases, focusing on colorectal cancer (CRC) and inflammatory bowel disease (IBD). To this end, vitamin D deficiency has been identified as a risk factor of CRC. On the other hand the active metabolite of vitamin D, 1, 25- dihydroxyvitamin D3 (1, 25(OH)2D3) has multiple anti-cancerous benefits including inhibition of proliferation, induction of apoptosis, promotion of differentiation and suppression of angiogenesis in tumors. In IBD, vitamin D is involved in the pathogenic process through the normalization of immune responses in the colon. With these experimental findings, well-designed and large-scale clinical trials are warranted to further define the therapeutic action of vitamin D in the prevention and/or treatment of IBD and further on CRC in humans.

Simultaneous detection of dual single-base mutations by capillary electrophoresis using quantum dot-molecular beacon probe.

Here a novel capillary electrophoresis (CE) for simultaneous detection of dual single-base mutations using quantum dot-molecular beacon (QD-MB) probe is described. Two QD-MB probes were designed using 585 and 650-nm emitting CdTe QDs which were covalently conjugated to MBs with different DNA oligonucleotide sequences by amide linkage and streptavidin-biotin binding, respectively. The hybridizations of QD-MB probes with different DNA targets were then monitored by CE, and results indicated that the two QD-MB probes specifically hybridized with their complementary DNA sequences, respectively. Target DNA identification was observed to have a high sensitivity of 16.2 pg in CE. Furthermore, the simultaneous detection of dual single-base mutations in a given DNA oligonucleotide was successfully achieved in CE using above two QD-MB probes. This novel CE-assisted QD-MB biosensor offers a promising approach for simultaneous detection of multiple single-base mutations, and exhibits potential capability in the single nucleotide polymorphism (SNP) analysis and high-sensitivity DNA detection.

Special method to prepare quantum dot probes with reduced cytotoxicity and increased optical property.

Quantum dots (QDs) are widely used in the life sciences because of their novel physicochemical properties. However, the cytotoxity of these nonoparticles have attracted great attention recently because this has not been well resolved. Four probes were synthesized by chemical coupling and protein denaturation with CdSeZnS, CdTe QDs, and transferrin. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and capillary electrophoresis were used to verify the conjugation of these luminescent probes. The cytotoxicity of these four luminescent probes and the original QDs were evaluated in HeLa cells. The results showed that over 92% of HeLa cells were still alive after being exposed to 3.2-microM CdSeZnS QDs capped with denatured transferrin for 72 h. Furthermore, while the probe preparation was very simple, the photoluminescence quantum yield of this probe was 7% higher than the original CdSeZnS QDs. This provides a new way for exploiting QD probes with low cytotoxicity, which will expand applications of nanocomposite assembly in biolabeling and imaging.

High-sensitivity quantum dot-based fluorescence resonance energy transfer bioanalysis by capillary electrophoresis.

Here a new method for high-sensitivity quantum dot (QD)-based fluorescence resonance energy transfer (FRET) bioanalysis was developed. In this method, capillary electrophoresis (CE) with fluorescence detection was applied. The FRET system consisted of water-soluble 532-nm emitting CdTe QDs donor and 632-nm emitting CdSe/ZnS QDs acceptor which were covalently conjugated with mouse IgG and goat anti-mouse IgG, respectively. The bio-affinity between antigen and antibody brought two kinds of QDs close enough to make the FRET happen between them. In the CE experiments, highly efficient separation of donor-acceptor immunocomplexes was obtained, and the process of FRET was monitored. Results showed that FRET efficiency obtained by CE (38.56-69.58%) improved substantially in comparison with that obtained by ensemble measurement (12.77-52.37%). The high efficient separation of donor-acceptor immunocomplexes and the possible conformation change of antigen and antibody, contributes to the lower analysis uncertainty (variance) and higher FRET efficiency obtained in CE and consequentially, this makes the analysis of FRET more sensitive. This novel CE-based technique can be easily extended to other FRET system based on QDs and may have potential application in the study of biomolecule conformation change.

A highly efficient capillary electrophoresis-based method for size determination of water-soluble CdSe/ZnS core-shell quantum dots.

This paper describes a highly efficient method for size determination of water-soluble CdSe/ZnS core-shell quantum dots (QDs) by capillary electrophoresis (CE) using polymer additive as sieving medium. The influence of some factors, such as kinds and concentrations of the sieving medium, pH, concentrations of the background electrolyte (BGE) and applied voltage, on the separation of QDs was investigated. Under the optimal separation conditions, four different sized QDs were successfully separated, and the relative standard deviation (RSD) of the migration times for these QDs was below 1.013%. In addition, an equation was fit by taking into account the correlation existing between the electrophoretic mobilities and the sizes of a set of QDs. The feasibility of this equation to measure the sizes of other QDs was confirmed by comparison with the sizes obtained by transmission electron microscopy (TEM) experiment. This work offers a novel method for size determination of QDs, and provides an important reference on the study of QDs based on CE.

Enhanced optical property of Au coated polystyrene beads for multi-color quantum dots encoding.

A novel quantum dots (QDs) fluorescent encoding method was demonstrated in this paper by using Au coated polystyrene (Au @ PS) beads. In the experiments, Au nanoparticles were deposited onto the polystyrene bead to form a stable Au coating through Layer-by-Layer assembly, and the surface morphology of the Au @ PS beads was studied by Scanning Electronic Microscope (SEM). Furthermore, the QDs encoding abilities, including the loading of QDs, the anti-photo bleaching ability and the multi-color encoding feasibility were studied using Au @ PS beads. The QDs leakage from doped QDs encoded Au @ PS was also studied. This study shows that Au particles improve the QDs encoding performance and the QD-encoded Au @ PS beads are the ideal material for the optical encoding.

Study on molecular interactions between proteins on live cell membranes using quantum dot-based fluorescence resonance energy transfer.

Mouse anti-human CD71 monoclonal antibody (anti-CD71) was conjugated with red quantum dots (QDs; 5.3 nm, emission wavelength lambda(em) = 614 nm) and used to label HeLa cells successfully. Then green QD-labeled goat anti-mouse immunoglobulin G (IgG; the size of the green QDs was 2.2 nm; lambda(em) = 544 nm) was added to bind the red-QD-conjugated anti-CD71 on the cell surface by immunoreactions. Such interaction between anti-CD71 and IgG lasted 4 min and was observed from the fluorescence spectra: the fluorescence intensity of the "red" peak at 614 nm increased by 32%; meanwhile that of the "green" one at 544 nm decreased by 55%. The ratio of the fluorescence intensities (I(544 nm)/I(614 nm)) decreased from 0.5 to 0.2. The fluorescence spectra as well as cell imaging showed that fluorescence resonance energy transfer took place between these two kinds of QDs on the HeLa cells through interactions between the primary antibody and the secondary antibody.

Modification of CdTe quantum dots as temperature-insensitive bioprobes.

CdTe quantum dots (QDs) were synthesized in aqueous solution with 3-mercaptopropionic acid (MPA) as the stabilizer. The photoluminescence (PL) of CdTe QDs (3.5nm) is found to be temperature-dependent: as the temperature arising from 278K to 323K, the PL intensity declines to 50.2% of its original and PL emission peak shows obvious red-shift ( approximately 7nm). After modification of the QDs surface with denatured ovalbumin, the PL is more temperature-insensitive than before. The PL intensity retains more than 70% of its original and the emission peak shows less red-shift ( approximately 2nm). Moreover, it is found that the PL intensity and wavelength of denatured ovalbumin coated CdTe QDs are reversible during heating (323K)-cooling (278K) cycles. All the studies provide an important theoretical basis for searching temperature-insensitive bioprobes.

Influence of quantum dot's quantum yield to chemiluminescent resonance energy transfer.

The resonance energy transfer between chemiluminescence donor (luminol-H2O2 system) and quantum dots (QDs, emission at 593 nm) acceptors (CRET) was investigated. The resonance energy transfer efficiencies were compared while the oil soluble QDs, water soluble QDs (modified with thioglycolate) and QD-HRP conjugates were used as acceptor. The fluorescence of QD can be observed in the three cases, indicating that the CRET occurs while QD acceptor in different status was used. The highest CRET efficiency (10.7%) was obtained in the case of oil soluble QDs, and the lowest CRET efficiency (2.7%) was observed in the QD-HRP conjugates case. This result is coincident with the quantum yields of the acceptors (18.3% and 0.4%). The same result was observed in another similar set of experiment, in which the amphiphilic polymer modified QDs (emission at 675 nm) were used. It suggests that the quantum yield of the QD in different status is the crucial factor to the CRET efficiency. Furthermore, the multiplexed CRET between luminol donor and three different sizes QD acceptors was observed simultaneously. This work will offer useful support for improving the CRET studies based on quantum dots.

Solubilization and bioconjugation of QDs and their application in cell imaging.

Quantum dot (QD) solubilization, conjugation with biomolecules, column purification, and labeling of human HepG2 cells with transferrin-QD (Tf-QD) conjugates are reported in detail in this article. Water-soluble QDs (WQDs) were obtained using sodium thiolycolate to replace the surface ligand tri-n-octylphosphine oxide (TOPO) on the surface of oil-soluble QDs, and Tf-QD conjugates were produced by coupling Tf to WQD. The resulting Tf-QDs were characterized by UV and luminescence spectrophotometry and purified by Sephadex column. The results indicate that Tf has been conjugated to QD successfully. Based on transferrin/transferrin-receptor-mediated delivery system, the Tf-QD conjugates were used to label human HepG2 cells. After a short incubation, the QDs were mainly localized to the membrane of cells. After 12-h incubation, QDs appear mainly in the cytoplasm portion. However, QDs were not found in the nucleus of the cells. Furthermore, the fluorescence intensity of QDs in the cells reduces gradually over time, and fluorescence cannot be observed after 10 days. However, the growth of the labeled cells was not markedly affected by the toxicity of QDs, and they are alive for 10 days. These results can be used for further application of QDs in bioscience.

Optical encoding of microbeads based on silica particle encapsulated quantum dots and its applications.

A novel method concerning the coding technology of polystyrene beads with Si encapsulated quantum dot (QD) particles (Si@QDs particles) is studied in this paper. In the reverse microemulsion system containing tetraethoxysilane (TEOS), water-soluble QDs (emission peak at 600 nm) were enveloped within the silica shell, forming Si@QDs particles. The Si@QDs particles were characterized by TEM, showing good uniform size, with an average diameter of about 167.0 nm. In comparison with the pure water-soluble QDs, the encapsulation of water-soluble QDs in the silica shell led to an enhancement in anti-photobleaching by providing inert barriers for the QDs. Images presented by SEM and confocal laser scanning microscopy demonstrated that the Si@QDs particles were equably coated on the surface of carboxyl functionalized polystyrene (PS) beads. Then, with the assistance of ethyl-3-(dimethyl aminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS), human IgG could be successfully crosslinked to Si@QDs particle coated PS-COOH beads. Furthermore, the Si@QDs coated PS-COOH beads with human IgG were examined in immunoassay experiments, and the results indicated that these beads could be applied in the specific recognition of goat-anti-human IgG in solution. This investigation is expected to provide a new route to bead coding in the field of suspension microarrays, based on the use of QDs.

Multi-color encoding of polystyrene microbeads with CdSe/ZnS quantum dots and its application in immunoassay.

Different diameter CdSe/ZnS semiconductor nanocrystals (average diameter: from approximately 3.5 to approximately 20 nm), quantum dots (QDs) were synthesized by changing the nucleation time, using organometallic reagents. These quantum dots possess narrow and symmetrical fluorescent emissions. The emission wavelengths of these composite dots span most of the visible spectrum from 500 through 700 nm. Furthermore, it is found that the quantum dots with an emission at approximately 590 nm, tend to have a good quantum yield (such as Phi(590)=43.5%). While the emission wavelength of prepared CdSe/ZnS QDs shifts toward blue or red from 590 nm, the quantum yield tends to decrease. After that, optical encoding of microbeads with these quantum dots was carried out, and the spectra of encoded beads were identified. The result indicates that, to identify the encoded beads with different emission wavelengths and emission intensities, it is needed to acquire and differentiate the spectra of beads. After immobilized with human IgG, the encoded beads were used to detect the corresponding antibody in solution. The result indicates that the encoded beads can detect the antibody signal effectively. And the effective detection range of the antibody is about 2-15 microM.

Bioconjugate recognition molecules to quantum dots as tumor probes.

Transferrin and mouse anti-human CD71 monoclonal antibody were respectively conjugated covalently to the core/shell CdSe/ZnS quantum dots with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydrocylsulfo-succinimide (Sulfo-NHS). The conjugation worked well and the bioactivities of these macromolecules still remained, which was verified by column filtration, sodium dodecyl sulfate polyacrylamide gel electrophoresis, absorption spectra, fluorescence spectra, and circular dichroism spectrometry. Thus, these two kinds of quantum dot conjugates were used to recognize the tumor cells involved. In case of pseudo positivity, FITC-labeling secondary antibody IgG was used, and the results showed that as-prepared fluorescent quantum dot bioprobes were highly specific to tumor cells.

Purification of denatured bovine serum albumin coated CdTe quantum dots for sensitive detection of silver(I) ions.

CdTe quantum dots (QDs) were synthesized in aqueous solution with 3-mercaptopropionic acid as the stabilizer. Chemically reduced bovine serum albumin (BSA) was used to modify the surface of the QDs. Experimental results showed that the denatured BSA (dBSA) could be effectively conjugated to the surface of CdTe QDs. Column chromatography was used to purify the conjugates and determine the optimal ratio of dBSA to QDs. Further experimental results showed that the conjugation of QDs by dBSA efficiently improved the photoluminescence quantum yield, the chemical stability of QDs and their stability against photobleaching. A facile and sensitive method for determination of silver(I) ions was proposed based on the fluorescence quenching of the dBSA-QDs. Under the optimal conditions, the relative fluorescence intensity decreased linearly with the concentration of the silver(I) ions in the range 0.08-10.66 microM. The detection limit was 0.01 microM. This study provides a new method for the detection of metal cations.

A feasible and quantitative encoding method for microbeads with multicolor quantum dots.

Multicolor encoded beads were achieved by incorporating two color core-shell quantum dots (QDs) (CdSe/ZnS) to commercial polystyrene (PS) beads. By controlling the concentration ratios of the two quantum dots (QDs) in doping solutions, a series of codes with different intensity ratios were obtained. Based on the multiple encoded carboxylic modified polystyrene beads, fluorescent dyes labeled antibodies were distinguished successfully on the beads' surface. It suggests that the encoded beads from this method have the practicability in biological applications and chemical analysis.

Characterization of the coupling of quantum dots and immunoglobulin antibodies.

Water-soluble quantum dots (QDs) were used to label goat anti-human immunoglobulin antibodies (Abs), and the labeling process was characterized by column purification. The QDs obtained in organic solvent were modified with mercaptoacetic acid (MAA) and became water-soluble. These water-soluble QDs were linked to the antibodies using the coupling reagents ethyl-3-(dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The linking process was shown to be effective by ultra-filter centrifugation and column purification. After comparing the quantities of Abs and water-soluble QDs involved in the linking reaction via column purification, it was found that a molar Abs:QD ratio of >1.2 resulted in most of the water-soluble QDs becoming covalently linked to the Abs. The circular dichroism (CD) spectra of Abs and QD-Ab conjugates were very similar to each other, indicating that the secondary structure of Abs remained largely intact after the conjugation. Finally, antigen (Ag)-antibody (Ab) recognition reactions perfomed on the surface of a glass slide showed that the conjugate retained the activity of Abs. This work lends support to the idea of linking biomolecules to QDs, and thus should aid the application of QDs to the life sciences.