Am T‐Stück angesetzt: Poly‐T‐Einzelstrang‐DNA (blau, siehe Schema) dient als Templat für die Bildung fluoreszierender Kupfernanopartikel (CuNPs, rote Kugeln). Größe und Fluoreszenz der CuNPs sind über die Länge der Poly‐T‐Sequenz einstellbar. Andere Einzelstrang‐DNAs (grün) sind keine geeigneten Template für CuNPs und können daher zum Aufbau von Nanostrukturen mit wechselnden metallierten und nichtmetallierten Bereichen genutzt werden.
Measuring pH in living cells is of great importance for better understanding cellular functions as well as providing pivotal assistance for early diagnosis of diseases. In this work, we report the first use of a novel kind of label-free carbon dots for intracellular ratiometric fluorescence pH sensing. By simple one-pot hydrothermal treatment of citric acid and basic fuchsin, the carbon dots showing dual emission bands at 475 and 545 nm under single-wavelength excitation were synthesized. It is demonstrated that the fluorescence intensities of the as-synthesized carbon dots at the two emissions are pH-sensitive simultaneously. The intensity ratio (I475 nm/I545 nm) is linear against pH values from 5.2 to 8.8 in buffer solution, affording the capability as ratiometric probes for intracellular pH sensing. It also displays that the carbon dots show excellent reversibility and photostability in pH measurements. With this nanoprobe, quantitative fluorescence imaging using the ratio of two emissions (I475 nm/I545 nm) for the detection of intracellular pH were successfully applied in HeLa cells. In contrast to most of the reported nanomaterials-based ratiometric pH sensors which rely on the attachment of additional dyes, these carbon-dots-based ratiometric probes are low in toxicity, easy to synthesize, and free from labels.
Measuring the levels of Fe in human body has attracted considerable attention for health monitoring as it plays an essential role in many physiological processes. In this work, we reported a selective fluorescent nanoprobe for Fe detection in biological samples based on ultrabright N/P codoped carbon dots. By employing adenosine 5'-triphosphate (ATP) as the carbon, nitrogen, and phosphorus source, the N/P codoped carbon dots could be simply prepared through hydrothermal treatment. The obtained carbon dots exhibited high quantum yields up to 43.2%, as well as excellent photostability, low toxicity, and water solubility. Because of the Fe-O-P bonds formed between Fe and the N/P codoped carbon dots, this nanoprobe showed high selectivity toward Fe against various potential interfering substances in the presence of EDTA. The fluorescence quenching of as-fabricated carbon dots was observed with the increasing Fe concentration, and the calibration curve displayed a wide linear region over the range of 1-150 μM with a detection limit of 0.33 μM. The satisfactory accuracy was further confirmed with the river samples and ferrous sulfate tablets, respectively. With the above outstanding properties, these N/P codoped carbon dots were successfully applied for direct detection of Fe in biological samples including human blood serum and living cells. As compared to the most reported carbon dots-based Fe sensors, this nanoprobe showed high fluorescence, good accuracy, and excellent selectivity, which presents the potential practical application for diagnosis of Fe related disease.
This paper proposed a natural gelatin capped mesoporous silica nanoparticles (MSN@Gelatin) based pH-responsive delivery system for intracellular anticancer drug controlled release. In this system, the gelatin, a proteinaceous biopolymer derived from the processing of animal collagen, was grafted onto the MSN to form a capping layer via temperature-induced gelation and subsequent glutaraldehyde mediated cross-linking, resulting in gelatin coated MSN. At neutral pH, the gelatin capping layer could effectively prohibit the release of loaded drug molecules. However, the slightly acidic environment would lead to enhanced electrostatic repulsion between the gelatin and MSN, giving rise to uncapping and the subsequent controlled release of the entrapped drug. As a proof-of-concept, doxorubicin (DOX) was selected as the model anticancer drug. The loading and pH-responsive release experiments demonstrated that the system had excellent loading efficiency (47.3 mmol g(-1) SiO2), and almost no DOX was leaked at neutral. After being in the slightly acidic condition, the DOX release from the DOX-loaded MSN@Gelatin (DOX/MSN@Gelatin) occurred immediately. The cellular uptake and release studies using Hep-G2 hepatoma cells indicated that the DOX/MSN@Gelatin could be endocytosed and accumulated within lysosomes. Triggered by acidic endosomal pH, the intracellular release of the loaded DOX was obviously eventuated. Further cell viability results demonstrated that DOX/MSN@Gelatin exhibited dose-dependent toxicity and high killing efficacy (IC50 = 17.27 ± 0.63 μg mL(-1)), whereas the MSN@Gelatin showed negligible cytotoxicity (IC50 > 100 μg mL(-1)). This biocompatible and effective delivery system will provide great potential for developing delivery of cancer therapeutic agents.
Adenosine-5'-triphosphate (ATP) is a multifunctional nucleotide, which plays a vital role in many biological processes, including muscle contraction, cells functioning, synthesis and degradation of important cellular compounds, and membrane transport. Thus, the development of ATP-responsive controlled release system for bioorganism application is very significative. Here, an original and facile ATP-responsive controlled release system consisting of mesoporous silica nanoparticles (MSN) functionalized with an aptamer as cap has been designed. In this system, the ATP aptamer was first hybridized with arm single-stranded DNA1 (arm ssDNA1) and arm single-stranded DNA2 (arm ssDNA2) to form the sandwich-type DNA structure and then grafted onto the MSN surface through click chemistry approach, resulting in blockage of pores and inhibition of guest molecules release. In the presence of ATP, the ATP aptamer combined with ATP and got away from the pore, leaving the arm ssDNA1 and ssDNA2 on the surface of MSN. The guest molecules can be released because single-stranded DNA is flexible. The release of the guest molecules from this system then can be triggered by the addition of ATP. As a proof-of-principle, Ru(bipy)(3)(2+) was selected as the guest molecules, and the ATP-responsive loading and release of Ru(bipy)(3)(2+) have been investigated. The results demonstrate that the system had excellent loading efficiency (215.0 μmol g(-1) SiO(2)) and the dye release percentage can reach 83.2% after treatment with 20 mM ATP for 7 h. Moreover, the ATP-responsive behavior shows high selectivity with ATP analogues. However, the leakage of Ru(bipy)(3)(2+) molecule is neglectable if ATP was not added, indicating an excellent capping efficiency. Interestingly, this system can respond not only to the commercial ATP but also to the ATP extracted from living cells. By the way, this system is also relatively stable in mouse serum solution at 37 °C. This proof of concept might promote the application of ATP-responsive devices and can also provide an idea to design various target-responsive systems using other aptamers as cap.
We present here a label-free and turn-on aptamer strategy for cancer cell detection based on the recognition-induced conformation alteration of aptamer and hybridization-induced fluorescence enhancement effect of DNA-silver nanoclusters (DNA-Ag NCs) in proximity of guanine-rich DNA sequences. In this strategy, two tailored DNA probes were involved. One is designed as a hairpin-shaped structure consisting of a target specific aptamer sequence at the 3'-end, a guanine-rich DNA sequence, and an arm segment at the 5'-end (denote as recognition probe). The other, serving as a signal probe, contains a sequence for Ag NCs templated synthesis and a link sequence complementary to the arm segment of the recognition probe. Recognizing and binding of the aptamer to cancer cells enforces the recognition probe to undergo a conformational alteration and then initiates hybridization between the arm segment of the recognition probe and the link sequence of the signal probe. The Ag NCs are then close to the guanine-rich DNA, leading to an enhanced fluorescence readout. As proof-of-concept, the CCRF-CEM cancer cell detection were performed by using the specific aptamer, sgc8c. It was demonstrated that this strategy could specially image the CCRF-CEM cells. Determination by flow cytometry allowed for detection of as low as 150 CCRF-CEM cells in 200 μL binding buffer. The general applicability of the strategy is also achieved in the successful detection of Ramos cells. These results implied that this strategy holds considerable potential for simple, sensitive, universal, and specific cancer cell detection with no required washing and separation steps.
Purpose : Exosomes (EXs) have been increasingly recognized as natural nanoscale vehicles for microRNA (miRNA)-based cell-cell communication and an ideal source of miRNA biomarkers in bodily fluids. Current methods allow bulk analysis of the miRNA contents of EXs, but these approaches are not suitable for the in situ stoichiometry of exosomal miRNAs and fail to reveal phenotypic heterogeneity at the single-vesicle level. This study aimed to develop a single vesicle-based, mild, precise, but versatile method for the in situ quantitative and stoichiometric analysis of exosomal miRNAs. Methods : A total internal reflection fluorescence (TIRF)-based single-vesicle imaging assay was developed for direct visualization and quantification of the single-vesicles of EXs and their miRNA contents in serum microsamples. The assay uses co-delivery of inactive split DNAzymes and fluorescence-quenched substrates into nanosized EXs treated with streptolysin O to produce a target miRNA-activated catalytic cleavage reaction that amplifies the readout of fluorescence signal. We perform the in situ quantitative and stoichiometric analysis of serum exosomal hsa-miRNA-21 (miR-21), a common cancer biomarker, by using the developed TIRF imaging assay. Results : The TIRF imaging assay for serum exosomal miR-21 can distinguish cancer patients from healthy subjects with better performance than conventional real-time polymerase chain reaction (PCR) assay. The exosomal miR-21 level in serum is also informative for monitoring tumor progression and responses to treatment. Moreover, the TIRF assays can readily determine the precise stoichiometry of target exosomal miRNA contents in situ by delivering molecular beacon (MB) probes into EXs. Conclusions : The created TIRF imaging platform shows high applicability to serve as a universal and useful tool for the single-vesicle in situ quantitative and stoichiometric analysis of other disease-associated exosomal miRNAs markers and provide valuable insight into the physiological relevance of EX-mediated miRNA communication.
Photodynamic therapy (PDT) has been applied in cancer treatment by converting O 2 into reactive singlet oxygen ( 1 O 2 ) to kill cancer cells. However, the effectiveness of PDT is limited by the fact that tumor hypoxia causes an inadequate O 2 supply, and the overexpressed glutathione (GSH) in cancer cells consumes reactive oxygen species. Herein, a multifunctional hybrid system is developed for selective and highly efficient PDT as well as gene-silencing therapy using a novel GSH-activatable and O 2 /Mn 2+ -evolving nanocomposite (GAOME NC). This system consists of honeycomb MnO 2 (hMnO 2 ) nanocarrier loaded with catalase, Ce6, and DNAzyme with folate label, which can specifically deliver payloads into cancer cells. Once endocytosed, hMnO 2 carriers are reduced by the overexpressed GSH to Mn 2+ ions, resulting in the reduction of GSH level and disintegration of GAOME NC. The released catalases then trigger the breakdown of endogenous H 2 O 2 to generate O 2 , which is converted by the excited Ce6 into 1 O 2 . The self-sufficiency of O 2 and consumption of GSH effectively enhance the PDT efficacy. Moreover, DNAzyme is freed for gene silencing in the presence of self-generated Mn 2+ ions as cofactors. The rational synergy of enhanced PDT and gene-silencing therapy remarkably improve the in vitro and in vivo therapeutic efficacy of cancers.
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