RESUMO
The sensitivity and accuracy of fluorescence probes for biological samples are affected by not only interfering molecule compounds but also the nonspecific adsorption of proteins and other macromolecules. Herein, fluorescence probe based on zwitterionic sulfobetaine methacrylate polymer (PSBMA) as an antibiofouling layer and amino boric acid carbon dots encapsulated in the metal-organic framework UiO-66-NH2 (UiO-66-NH2/BN-CDs) as a target recognition site was designed for the detection of baicalin (BAI). Owing to the introduction of BN-CDs into UiO-66-NH2 with high specific surface area, the prepared UiO-66-NH2/BN-CDs@PSBMA probe exhibited a high adsorption capacity of 78.9 mg g-1, while presented fluorescence enhancing and superior fluorescence selectivity to BAI at excitation and emission wavelengths of 400 and 425 nm, respectively. Connecting PSBMA with good hydrophilicity to UiO-66-NH2, resulted in an anti-protein capacity of over 96.3 %, effectively inhibiting protein interference with the fluorescence signal. By virtue of its good antibiofouling and recognizing capacities, the fluorescence probe exhibited a satisfactory detection range of 10-80 nmol L-1, with a fairly low detection limit of 0.0064 µmol L-1. Using the method to detect BAI in Goji berry, Sophora and Yinhuang oral solution, demonstrating its potential for the accurate and quantitative detection of BAI in complex biological samples.
Assuntos
Ácidos Borônicos , Carbono , Flavonoides , Corantes Fluorescentes , Estruturas Metalorgânicas , Pontos Quânticos , Corantes Fluorescentes/química , Estruturas Metalorgânicas/química , Ácidos Borônicos/química , Carbono/química , Flavonoides/química , Flavonoides/análise , Pontos Quânticos/química , Incrustação Biológica/prevenção & controle , Polímeros/química , Espectrometria de Fluorescência/métodos , Limite de Detecção , Metacrilatos/química , Adsorção , Betaína/química , Betaína/análogos & derivados , Ácidos FtálicosRESUMO
Polar stationary phases were prepared by grafting hydrophilic acrylamide (Am) polymer brushes with post modification of carbon dots (CDs) and silicon dots (SiDs) onto SiO2 particles. The prepared stationary phases, SiO2-PAm-CDs, SiO2-PAm-CDs/SiDs, and SiO2-PAm-SiDs, were packed as chromatographic columns, respectively. Using nucleic bases, organic acids, and ß-agonists as target substances to investigate the influence of chromatographic conditions on retention and separation, the packed columns showed the partitioning and adsorption of mixed retention behavior in hydrophilic interaction liquid chromatography mode and successfully separated the polar compounds. Most importantly, under per aqueous liquid chromatography mode (using 100% water as mobile phase), those columns still had good separation ability toward nucleic bases, ß-agonist, and organic acids. Because AM is a temperature-sensitive monomer, the resulting van't Hoff curves exhibited a nonlinear relationship, having temperature-responsive chromatographic characteristic under pure water separation. Hence, building on temperature-sensitive characteristics and pure water of separation conditions, the separation selectivity toward hydrophilic compounds greatly improved. Compared with the commercial hydrophilic columns, the efficiency of our developed column had the superior ability in separation and detection of betaine in Goji berry with the enhanced resolution achieved in the proposed green separation method (just using pure water as mobile phase).
RESUMO
A method based on novel restricted access materials (RAMs) for the determination of neonicotinoid pesticides in Goji samples using offline and online solid phase extraction (SPE) coupled with high-performance liquid chromatography (LC). RAMs were synthesized using poly(chloromethylstyrene-co-divinylbenzene) (PVBC/DVB) microspheres as substrate, styrene (St) and n-vinylpyrrolidone (NVP) were first copolymerized on the interior to construct adsorption sites, and sulfobetaine methacrylate (SBMA) was then polymerized on the exterior to form exclusion sites via two-step surface initiated-atom transfer polymerization. The prepared PVBC/DVB@poly(St-co-NVP)@poly(SBMA) RAMs could efficiently extract neonicotinoid pesticides and automatically exclude proteins. Under the optimized conditions, the developed methods of offline (magnetic SPE and SPE column) and online extraction coupled with LC both using PVBC/DVB@poly(St-co-NVP)@poly(SBMA) RAMs as the extractant, exhibit a wide linearity, low limits of detection and limit of quantification and good inter-day and intra-day precision with satisfactory recoveries. Among these methods, online extraction coupled with LC based on novel RAMs exhibits clear advantages for the determination of neonicotinoid pesticides in Goji samples has clear advantages, such as simple operation by direct injection, short extraction times, and high accuracy with less human error.
Assuntos
Praguicidas , Polímeros , Humanos , Polímeros/química , Praguicidas/análise , Adsorção , Extração em Fase Sólida/métodos , Estireno , Cromatografia Líquida de Alta Pressão/métodosRESUMO
In this study, a developed chromatographic stationary phase combines the high selectivity of mixed-mode retention with a temperature-responsive property to boost separation efficiency. Copolymer brushes were grafted onto silica gels through surface initiated-atom transfer radical polymerization by polymerizing two types of monomer, temperature-responsive vinylcaprolactam (VCl) and quinine (Qun) containing benzopyridine, a tertiary ammonium positive center, and hydroxyl groups. The obtained silica@poly(Qun-co-VCl) stationary phases were packed as a chromatographic column, and the retention behavior of hydrophobic polycyclic aromatics, highly polar nucleosides, charged organic acids and ß-agonists was studied for this column under different separation modes. The ability to separate different types of analyte shows that the silica@poly(Qun-co-VCl) column provides multiple hydrophobic, hydrophilic and electrostatic interactions toward analytes, achieving the separation of various compounds in one column. In addition, temperature-dependent resolution of polycyclic aromatics, nucleosides, organic acids and ß-agonists was investigated using modulation of the column temperature, and the column exhibited adjustable separation selectivity by simply changing the column temperature. These results demonstrate that the grafting of copolymer brushes on a silica surface, consisting of temperature-responsive poly-VCl and multifunctional groups of poly-Qun, is useful as a mixed-mode chromatographic stationary phase for thermally-modulated multiple interactions. Additionally, this column was also used for the quantitative detection of uridine and inosine from cordyceps.
RESUMO
High-selectivity and high-exclusion restricted access materials (RAMs) benefit the analysis of biological samples. Herein, triblock copolymer-functionalized poly(4-vinylbenzyl chloride-co-divinylbenzene) (PVBC/DVB) microspheres were prepared via the sequential surface-initiated atom radical polymerization of hydrophobic styrene (St), ionic vinylimidazole (VIm), and zwitterionic sulfobetaine methacrylate (SBMA), affording RAMs with multiple interaction-adsorption sites and zwitterionic polymer exclusion sites on the internal and external surfaces of PVBC/DVB. The preferential extraction of fluoroquinolones (FQs) is realized based on the hydrophobic/π-π/ion exchange interactions due to the grafted poly-St-VIm, and the zwitterionic poly-SBMA block in the triblock copolymers can efficiently exclude various proteins. A sensitive detection method for FQs in chicken was established by solid phase extraction with RAMs as adsorbent combined with UPLC-MS/MS, achieving wide linearity (2.0-200.0 ng mL-1), low limit of detection (0.5 µg kg-1) and limit of quantification (1.5 µg kg-1), and good inter- and intraday precision with satisfactory recoveries (104.1%-117.7% and 115.3%-121.2% with RSDs < 12%).
Assuntos
Fluoroquinolonas , Polímeros , Fluoroquinolonas/análise , Cromatografia Líquida , Polímeros/química , Espectrometria de Massas em Tandem , Extração em Fase Sólida/métodosRESUMO
Despite its clinical success, chimeric antigen receptor T (CAR T)-cell immunotherapy remains limited in solid tumors, owing to the harsh physical barriers and immunosuppressive microenvironment. Here a CAR-T-cell-based live microrobot (M-CAR T) is created by decorating CAR T with immunomagnetic beads using click conjugation. M-CAR Ts are capable of magnetic-acoustic actuation for precision targeting and in situ activation of antitumor immune responses. Sequential actuation endows M-CAR Ts with magnetically actuated anti-flow and obstacle avoidance as well as tissue penetration driven by acoustic propulsion, enabling efficient migration and accumulation in artificial tumor models. In vivo, sequentially actuated M-CAR Ts achieves long-distance targeting and accumulate at the peritumoural area under programmable magnetic guidance, and subsequently acoustic tweezers actuate M-CAR Ts to migrate into deep tumor tissues, resulting in a 6.6-fold increase in accumulated exogenous CD8+ CAR T cells compared with that without actuation. Anti-CD3/CD28 immunomagnetic beads stimulate infiltrated CAR T proliferation and activation in situ, significantly enhancing their antitumor efficacy. Thus, this sequential-actuation-guided cell microrobot combines the merits of autonomous targeting and penetration of intelligent robots with in situ T-cell immunoactivation, and holds considerable promise for precision navigation and cancer immunotherapies.
Assuntos
Neoplasias , Receptores de Antígenos Quiméricos , Humanos , Neoplasias/terapia , Fenômenos Magnéticos , Imunoterapia Adotiva , Linfócitos T , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Sonodynamic therapy (SDT), a combination of low-intensity ultrasound with a sonosensitizer, has been explored as a promising alternative for cancer therapy. However, condensed extracellular matrix (ECM) resulting in poor perfusion and extreme hypoxia in solid tumor potentially compromises effective SDT. Herein, we develop a novel cleavable collagenase-assistant and O2-supplied nanosonosensitizer (FePO2@HC), which is embedded through fusing collagenase (CLG) and human serum albumin (HSA), followed by encapsulating Ferric protoporphyrin (FeP) and dioxygen. As a smart carrier, HSA is stimuli-responsive and collapsed by reduced glutathione (GSH) overexpressed in tumor, resulting to the release of the components in FePO2@HC. The released CLG acting as an artificial scissor, degrades the collagen fibers in tumor, thus, breaking tumor tissue and enhancing FePO2 accumulation in tumor inner with higher than that without CLG. Simultaneously, oxygen molecules are released from FePO2 in hypoxic environment and alleviate the tumor hypoxia. As a sonosensitizer, FeP is subsequently irradiated by ultrosound wave (US) and activates surrounding dioxygen to generate amount of singlet oxygen (1O2). Contributed from the ECM-degradation, such SDT-based nanosystem with increased sonosensitizer permeability and oxygen content highly improved the tumor inhibition efficacy without toxic effects. This study presents a new paradigm for ECM depletion-based strategy of deep-seated penetration, and will expand the nanomedicine application of metalloporphyrin sonosensitizers in SDT.
Assuntos
Metaloporfirinas , Nanopartículas , Neoplasias , Terapia por Ultrassom , Humanos , Neoplasias/terapia , Neoplasias/patologia , Oxigênio/metabolismo , Colagenases , Linhagem Celular Tumoral , Espécies Reativas de OxigênioRESUMO
Although chimeric antigen receptor T (CAR T) cell immunotherapy has demonstrated remarkable success in clinical, therapeutic effects are still limited in solid tumor due to lack of activated T cell infiltration in immunosuppression of tumor microenvironment. Herein, we develop IL-12 nanostimulant-engineered CAR T cell (INS-CAR T) biohybrids for boosting antitumor immunity of CAR T cells via immunofeedback. As stimulating nanochaperone, IL-12-loaded human serum albumin (HSA) nanoparticles are effectively conjugated onto CAR T cells via bioorthogonal chemistry without influencing their antitumor capabilities. IL-12 is responsively released from INS-CAR T biohybrids in presence of the increased thiol groups on cell-surface triggered by tumor antigens. In return, released IL-12 obviously promotes the secretion of CCL5, CCL2 and CXCL10, which further selectively recruits and expands CD8+ CAR T cells in tumors. Ultimately, the immune-enhancing effects of IL-12 nanochaperone significantly boost CAR T cell antitumor capabilities, dramatically eliminated solid tumor and minimized unwanted side effects. Hence, immunofeedback INS-CAR T biohybrids, which include INS that serves as an intelligent 'nanochaperone', could provide a powerful tool for efficient and safe antitumor immunotherapy.
Assuntos
Neoplasias , Receptores de Antígenos Quiméricos , Humanos , Imunoterapia , Imunoterapia Adotiva , Interleucina-12 , Neoplasias/terapia , Linfócitos T , Microambiente Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Chimeric antigen receptor T cell (CAR-T) therapy has shown remarkable clinical success in eradicating hematologic malignancies. However, hostile microenvironment in solid tumors severely prevents CAR-T cells migrating, infiltrating, and killing. Herein, a nanoengineered CAR-T strategy is reported for enhancing solid tumor therapy through bioorthogonal conjugation with a nano-photosensitizer (indocyanine green nanoparticles, INPs) as a microenvironment modulator. INPs engineered CAR-T biohybrids (CT-INPs) not only retain the original activities and functions of CAR-T cells, but it is further armed with fluorescent tracing and microenvironment remodeling abilities. Irradiated with laser, CT-INPs demonstrate that mild photothermal intervention destroys the extracellular matrix, expanded blood vessels, loosened compact tissue, and stimulated chemokine secretion without damping CAR-T cell activities. Those regulations induce an immune-favorable tumor microenvironment for recruitment and infiltration of CT-INPs. CT-INPs triggered photothermal effects collapse the physical and immunological barriers of solid tumor, and robustly boosted CAR-T immunotherapy. Therefore, CAR-T biohybrids provide reliable treatment strategy for solid tumor immunotherapy via microenvironment reconstruction.
Assuntos
Neoplasias , Receptores de Antígenos Quiméricos , Humanos , Imunoterapia Adotiva , Neoplasias/terapia , Linfócitos T , Microambiente TumoralRESUMO
The adoptive transfer of chimeric antigen receptor-T (CAR-T) cells has shown remarkable clinical responses in hematologic malignancies. However, unsatisfactory curative results and side effects for tumor treatment are still unsolved problems. Herein we develop a click CAR-T cell engineering strategy via cell glycometabolic labeling for robustly boosting their antitumor effects and safety in vivo. Briefly, paired chemical groups (N3/BCN) are separately incorporated into CAR-T cell and tumor via nondestructive intrinsic glycometabolism of exogenous Ac4GalNAz and Ac4ManNBCN, serving as an artificial ligand-receptor. Functional groups anchored on cell surface strengthen the interaction of CAR-T cell and tumor via bioorthogonal click chemistry, further enhancing specific recognition, migration and selective antitumor effects of CAR-T cells. In vivo, click CAR-T cell completely removes lymphoma cells and minimizes off-target toxicity via selective and efficient bioorthogonal targeting in blood cancer. Surprisingly, compared to unlabeled cells, artificial bioorthogonal targeting significantly promotes the accumulation, deep penetration and homing of CAR-T cells into tumor tissues, ultimately improving its curative effect for solid tumor. Click CAR-T cell engineering robustly boosts selective recognition and antitumor capabilities of CAR T cells in vitro and in vivo, thereby holding a great potential for effective clinical cell immunotherapy with avoiding adverse events in patients.
RESUMO
Erv1 (EC number 1.8.3.2) is an essential mitochondrial enzyme catalyzing protein import and oxidative folding in the mitochondrial intermembrane space. Erv1 has both oxidase and cytochrome c reductase activities. While both Erv1 and cytochrome c were reported to be membrane associated in mitochondria, it is unknown how the mitochondrial membrane environment may affect the function of Erv1. Here, in this study, we used liposomes to mimic the mitochondrial membrane and investigated the effect of liposomes and cardiolipin on the folding and function of yeast Erv1. Enzyme kinetics of both the oxidase and cytochrome c reductase activity of Erv1 were studied using oxygen consumption analysis and spectroscopic methods. Our results showed that the presence of liposomes has mild impacts on Erv1 oxidase activity, but significantly inhibited the catalytic efficiency of Erv1 cytochrome c reductase activity in a cardiolipin-dependent manner. Taken together, the results of this study provide important insights into the function of Erv1 in the mitochondria, suggesting that molecular oxygen is a better substrate than cytochrome c for Erv1 in the yeast mitochondria.
Assuntos
Cardiolipinas/química , Lipossomos/química , Proteínas Mitocondriais/química , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/química , Dobramento de Proteína , Proteínas de Saccharomyces cerevisiae/química , Cardiolipinas/farmacologia , Cinética , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The mitochondrial import and assembly (MIA) pathway plays a vitally important role in import and oxidative folding of mitochondrial proteins. Erv1, a member of the FAD-dependent Erv1/ALR disulphide bond generating enzyme family, is a key player of the MIA pathway. Although considerable progress has been made, the molecular mechanism of electron transfer within Erv1 is still not fully understood. The reduction potentials of the three redox centres were previously determined to be -320 mV for the shuttle disulphide, -150 mV for the active-site disulphide and -215 mV for FAD cofactor. However, it is unknown why FAD of Erv1 has such a low potential compared with other sulfhydryl oxidases, and why the shuttle disulphide has a potential as low as many of the stable structural disulphides of the substrates of MIA pathway. In this study, the three reduction potentials of Erv1 were reassessed using the wild-type and inactive mutants of Erv1 under anaerobic conditions. Our results show that the standard potentials for the shuttle and active-site disulphides are approximately -250 mV and -215 ~ -260 mV, respectively, and the potential for FAD cofactor is -148 mV. Our results support a model that both disulphide bonds are redox-active, and electron flow in Erv1 is thermodynamically favourable. Furthermore, the redox behaviour of Erv1 was confirmed, for the first time using Mia40, the physiological electron donor of Erv1. Together with previous studies on proteins of MIA pathway, we conclude that electron flow in the MIA pathway is a thermodynamically favourable, smoothly downhill process for all steps. DATABASE: Erv1: EC 1.8.3.2.
Assuntos
Mitocôndrias/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas Mitocondriais/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Oxirredutases/genética , Proteínas de Saccharomyces cerevisiae/genética , Mitocôndrias/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/metabolismo , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Dobramento de Proteína , Transporte Proteico/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Yeast (Saccharomyces cerevisiae) essential for respiration and viability 1 (Erv1; EC number 1.8.3.2), a member of the flavin adenine dinucleotide-dependent Erv1/ALR disulphide bond generating enzyme family, works together with Mia40 to catalyse protein import and oxidative folding in the mitochondrial intermembrane space. Erv1/ALR functions either as an oxidase or cytochrome c reductase by passing electrons from a thiol substrate to molecular oxygen (O2 ) or cytochrome c, respectively. However, the substrate specificity for oxygen and cytochrome c is not fully understood. In this study, the oxidase and cytochrome c reductase kinetics of yeast Erv1 were investigated in detail, under aerobic and anaerobic conditions, using stopped-flow absorption spectroscopy and oxygen consumption analysis. Using DTT as an electron donor, our results show that cytochrome c is ~ 7- to 15-fold more efficient than O2 as electron acceptors for yeast Erv1, and that O2 is a competitive inhibitor of Erv1 cytochrome c reductase activity. In addition, Mia40, the physiological thiol substrate of Erv1, was used as an electron donor for Erv1 in a detailed enzyme kinetic study. Different enzyme kinetic kcat and Km values were obtained with Mia40 compared to DTT, suggesting that Mia40 modulates Erv1 enzyme kinetics. Taken together, this study shows that Erv1 is a moderately active enzyme with the ability to use both O2 and cytochrome c as the electron acceptors, indicating that Erv1 contributes to mitochondrial hydrogen peroxide production. Our results also suggest that Mia40-Erv1 system may involve in regulation of the redox state of glutathione in the mitochondrial intermembrane space. ERV1: EC number 1.8.3.2.
Assuntos
Mitocôndrias/química , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Dobramento de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Cinética , Proteínas Mitocondriais/química , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/química , Transporte Proteico , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
Surface-enhanced Raman spectroscopy (SERS) has exhibited great potential in protein identification and quantification. However, the poor spectral reproducibility, originating from random protein immobilization on SERS substrates, still makes it challenging for SERS to probe protein functions without any extrinsic Raman labels. Here, in our study, spacer molecules between proteins and SERS substrates are optimized for both biocompatible protein immobilization and Raman scattering enhancement. We have accordingly prepared iminodiacetic acid (IDA)-functionalized silver substrates, which are used for capturing His-tagged proteins via nickel-imidazole coordination. The controlled immobilization enables excellent SERS spectral reproducibility as evidenced by 6 polypeptides. Furthermore, the interactions between two model proteins, Erv1C (C-terminal domain of flavine adenine dinucleotide-dependent mitochondrial cytochrome c reductase Erv1) and AFP (alpha-fetoprotein), and their ligands Cyt c (cytochrome c) and ATRA (all-trans-retinoic acid) are examined, respectively. The results indicate that the IDA-functionalized silver substrates enable controlled protein immobilization and allow label-free protein function investigation by SERS. As a proof-of-concept study, the proposed functionalized SERS-active substrates combined with immobilized metal-affinity chromatography will be useful for mechanism studies on protein-ligand interactions, which is crucially important for understanding the structural basis of protein functional versatility and will contribute to the fields of drug design and biotechnology.
Assuntos
Proteínas/metabolismo , Análise Espectral Raman/métodos , Animais , Citocromos c/metabolismo , Humanos , Iminoácidos/química , Proteínas Imobilizadas/metabolismo , Ligantes , Modelos Moleculares , Peptídeos/metabolismo , Prata/química , Propriedades de Superfície , alfa-Fetoproteínas/metabolismoRESUMO
BACKGROUND: The Chinese Hui ethnic group has diverse origins, including Arab, Persian, Central Asian, and Mongol. The standardized mortality rate of gastric cancer in the Hui population is higher than the overall Chinese population. In this study, we investigated whether COX-2-765G>C polymorphism, an extensively studied polymorphism, contributes to gastric cancer and its precursor lesions (GPL) in the Chinese Hui ethnic group. MATERIALS AND METHODS: COX-2-765G>C polymorphism was determined by pyrosequencing in 100 gastric cancer cases, 102 gastric cancerand its precursor lesions cases and 105 controls. Data were statistically analyzed using Chi-square tests and logistic regression models. RESULTS: Among the Chinese Hui ethnic group COX-2 -765 C allele carriers were at increased risk for gastric cancer (OR=1.977, 95%CI=1.104-3.541). We also found an interaction between COX-2 -765 C carriers and Helicobacter pylori infection and eating pickled vegetables. CONCLUSIONS: Our findings suggest a multi-step process of gene-environment interaction contributes to gastric carcinogenesis.