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L-glutamate is an important component of protein. It can prevent gastrointestinal damage caused by NSAIDs. We constructed two-phase enteric-coated granules of aspirin and L-glutamate compound by extrusion spheronization method and fluidized bed coating. The subliminal effective dose of L-glutamate is 100 mg/kg tested by model of gastric ulcer of rats induced by aspirin and drug administration. HPLC-UV and UV-Vis methods were adopted to determine content and cumulative release of aspirin and L-glutamate as quality analysis method indexes. The prescription and process optimization were carried out with yield, sphericity and dissolution. The two-phase compound granules have good sphericity of 0.93 ± 0.05 (aspirin pellets) and 0.94 ± 0.02 (L-glutamate pellets), content of salicylic acid (0.24 ± 0.03)%, dissolution of aspirin (2.36 ± 0.11)%. Quality evaluation and preliminary stability meet the commercial requirements. The stored environment of compound preparation should be sealed in a cool and dark place., (© 2021. The Author(s).)

Photothermal therapy (PTT) is a promising tumor treatment modality, but its efficacy is strictly hindered by abnormally upregulated heat shock proteins (HSPs) in tumor cells under heat stress. Herein, we developed a flower-like MnO2-coated polydopamine (PDA@MnO2) core-shell nanoplatform with the surface adsorption of HSP70-silencing DNAzyme (DZ) for enhanced PPT. The PDA core acted as a robust photothermal agent, and also as a reductant to allow the surface growth of MnO2via an in situ reduction of KMnO4. The MnO2 shell enabled a rapid and efficient adsorption of DZ, and more importantly, acted as a metal reservoir to release Mn2+ in response to intracellular stimuli for the in situ activation of DZ, which addressed the key limitation of DZ for biological applications, i.e., metal-dependent activity. As a result, HSP70 was remarkably suppressed for improved PTT efficacy upon laser irradiation, which was explicitly demonstrated both in vitro and in vivo. Upon intravenous injection, the nanosystem could effectively accumulate in the tumor, and impose potent PTT for complete tumor elimination via inducing tumor cell apoptosis, but without any noticeable toxicity. This work provides a promising nanosystem for enhanced PTT via silencing resistance-related genes, and offers ideas for the design of self-activated gene therapy platforms using DZ.

The combination of chemotherapy and photodynamic therapy provides a promising approach for enhanced tumor eradication by overcoming the limitations of each individual therapeutic modality. However, tumor is pathologically featured with extreme hypoxia together with the adaptable overexpression of anti-oxidants, such as glutathione (GSH), which greatly restricts the therapeutic efficiency. Here, a combinatorial strategy was designed to simultaneously relieve tumor hypoxia by self-oxygenation and reduce intracellular GSH level to sensitize chemo-photodynamic therapy. In our system, a novel multi-functional nanosystem based on MnO 2 -doped graphene oxide (GO) was developed to co-load cisplatin (CisPt) and a photosensitizer (Ce6). With MnO 2 doping, the nanosystem was equipped with intelligent functionalities: (1) catalyzes the decomposition of H 2 O 2 into oxygen to relieve the tumor hypoxia; (2) depletes GSH level in tumor cells, and (3) concomitantly generates Mn 2+ to proceed Fenton-like reaction, all of which contribute to the enhanced anti-tumor efficacy. Meanwhile, the surface hyaluronic acid (HA) modification could facilitate the targeted delivery of the nanosystem into tumor cells, thereby resulting in amplified cellular toxicity, as well as tumor growth inhibition in nude mice model. This work sheds a new light on the development of intelligent nanosystems for synergistic combination therapy via regulating tumor microenvironment., (© 2021 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V.)

Multi-modal nanomedicines that synergistically combine chemo-, gene-, and photothermal therapy have shown great potential for cancer treatment. In this study, a core-shell nanosystem-based on a zinc oxide (ZnO) nanocore and a polydopamine (PDA) shell was constructed to integrate chemo- (doxorubicin, DOX), gene- (DNAzyme, DZ), and photothermal (PDA layer) therapy in one system. Instead of small interfering RNAs, we employed DZ for tumor-related gene (survivin) regulation owing to its higher stability, biocompatibility, and predictable activity. DOX and amino-modified DZ were loaded onto the PDA shell via physisorption and covalent conjugation, respectively. Specifically, the ZnO nanocore was designed as a metal cofactor reservoir to release Zn 2+ in response to intracellular stimuli, which triggered the activation of DZ for gene silencing after endocytosis into cells. Both in vitro and in vivo experiments demonstrated the enhanced anti-tumor efficacy of these multifunctional nanocomposites and highlighted the advantages of these nano-drug delivery systems to alleviate the side effects of DOX. This study provides a strategy for synergistic cancer therapy via chemo/gene/photothermal combination and offers a strategy to harness DZ as a gene-silencing tool for disease treatment in combination with other therapeutic modalities. STATEMENT OF SIGNIFICANCE: In this work, we constructed a core-shell nanosystem containing a zinc oxide (ZnO) nanocore and a polydopamine (PDA) outer layer, which integrated chemo- (doxorubicin, DOX), gene- (DNAzyme, DZ), and photothermal (PDA layer) therapies for multimodal cancer therapy. Specifically, the ZnO core was incorporated to solve the key issue of DZ for gene silencing applications, which acted as the metal cofactor reservoir to release Zn 2+ inside cells for effective DZ activation. In addition, the PDA shell could detoxify the ZnO by scavenging the reactive oxygen species produced by ZnO, thus increasing the biocompatibility of the nanocarrier. This work solves the key issue of DZ for RNAi-based applications, offers a platform to combine DZ with other therapeutic modalities, and also provides a smart strategy to achieve triggered activation of biocatalytic reactions for therapeutic applications., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020. Published by Elsevier Ltd.)

Functional DNA includes aptamers and DNAzymes, and metal ions are often important for achieving the chemical functions of such DNA. Biosensors based on functional DNA have mainly been tested in aqueous buffers. By introducing organic solvents with much lower dielectric constants, the interaction between metal ions and DNA can be significantly enhanced, and this might affect the performance of DNA-based biosensors. In this work, the effect of ethanol on the activity of the EtNa DNAzyme was studied for Ca 2+ detection. With 30 % ethanol, the sensor has a detection limit of 1.4 μm Ca 2+ , which is a 16-fold improvement relative to that in water. This EtNa DNAzyme is unique because other tested DNAzymes are all inhibited by 50 % ethanol. Finally, by using the EtNa DNAzyme as a scaffold, the adenosine monophosphate (AMP) aptamer was inserted to construct an aptazyme, which allowed the measurement of AMP in ethanol. In summary, this study has reported the most sensitive DNA-based sensor for Ca 2+ , and its sensitivity and selectivity can approach those of proteins or small-molecule ligands. This work also provides a way to measure aptamer binding in organic solvents., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Our view of the interaction between Na + and nucleic acids was changed by a few recently discovered Na + -specific RNA-cleaving DNAzymes. In addition to nonspecific electrostatic interactions, highly specific recognition is also possible. Herein, two such DNAzymes, named EtNa and Ce13d, are compared to elucidate their mechanisms of Na + binding. Mutation studies indicate that they have different sequence requirements. Phosphorothioate (PS) substitution at the scissile phosphate drops the activity of EtNa 140-fold, and it cannot be rescued by thiophilic Cd 2+ or Mn 2+ , whereas the activity of PS-modified Ce13d can be rescued. Na + -dependent activity assays indicate that two Na + ions bind cooperatively in EtNa, and each Na + likely interacts with a nonbridging oxygen atom in the scissile phosphate, whereas Ce13d binds only one Na + ion in a well-defined Na + aptamer, and this Na + ion does not directly interact with the scissile phosphate. Both DNAzymes display a normal pH-rate profile, with a single deprotonation reaction required for catalysis. For EtNa, Na + fails to protect the conserved nucleotides from dimethyl sulfate attack, and no specific Na + binding is detected by 2-aminopurine fluorescence, both of which are different from those observed for Ce13d. This work suggests that EtNa binds Na + mainly through its scissile phosphate without significant involvement of the nucleotides in the enzyme strand, whereas Ce13d has a well-defined aptamer for Na + binding. Therefore, DNA has at least two distinct ways to achieve highly selective Na + binding., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Recently, a few Na + -specific RNA-cleaving DNAzymes have been reported, and a Na + aptamer was identified from the NaA43 and Ce13d DNAzymes. These DNAzymes and the embedded aptamer have been used for Na + detection. In this work, we studied the Na + -dependent folding of the Ce13d DNAzyme using fluorescence resonance energy transfer (FRET). When a FRET donor and an acceptor were respectively labeled at the ends of the DNAzyme, Na + failed to induce an obvious end-to-end distance change, suggesting a rigid global structure. To relax this rigidity, the Ce13d DNAzyme was systematically split at various sites on both the enzyme and the substrate strands. The Na + binding activity of the split structures was characterized by 2-aminopurine fluorescence, enzymatic activity, Tb 3+ -sensitized luminescence, and DMS footprinting. Among the various constructs, the only one that retained Na + binding was the split at the cleavage site, and this construct was further labeled with two dyes near the split site. This FRET result showed Na + -dependent folding with a K d of 26 mM Na + . This study provides important structural information related to Na + binding to this new aptamer, which might also be useful for future work in biosensor design.

DNA-sensitized Tb 3+ luminescence spectroscopy is a powerful method for probing nucleic acids and developing biosensors. Its performance in organic solvents has yet to be explored. In this study, Tb 3+ luminescence with nucleosides, nucleotides and DNA oligonucleotides in various organic solvents is studied. Tb 3+ emission with single nucleotides is quenched up to 88% in dimethyl formamide (DMF), while its emission with nucleosides is enhanced. For the four 15-mer DNA homopolymers, the strongest absolute emission enhancement was achieved with C 15 . Similar emission properties are observed in other solvents including DMF, DMSO, acetonitrile methanol, ethanol, isopropanol and ethylene glycol. A few DNAzymes are tested as random DNA sequences all showing 1.4-6.9-fold emission enhancement in ethanol. A previously reported optimized sequence in water (G 3 T) 5 is further enhanced by the solvents. Using this sequence, a detection limit of 5.5 nm Hg 2+ is achieved in 25% ethanol solution. A similar Hg 2+ sensitivity is also observed in a lake water mixed with ethanol. Luminescence lifetime is longer in DMF than in water. This study indicates that DNA-sensitized Tb 3+ luminescence can be measured in water miscible solvents and most likely, with even stronger emission than that in water., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Most metal sensors are designed for a strong binding affinity toward target metal ions, and the underlying principle relies on binding thermodynamics. The kinetic aspect of binding, however, was rarely explored for sensing. In this work, the binding kinetics of 19 common or toxic metal ions are compared based on a fluorescence quenching assay using DNA oligonucleotides as ligands. Among these metals, Cr 3+ shows uniquely slow fluorescence quenching kinetics, and the quenched fluorescence cannot be recovered by EDTA or sulfide. Most other metals quenched fluorescence instantaneously and can be fully recovered by these metal chelators. Various factors such as DNA sequence and length, chelating agent, pH, and fluorophore type were studied to understand the binding mechanism, leading to a unique two-stage binding model for Cr 3+ . This system has a wide dynamic range of up to 50 μM Cr 3+ and a low limit of detection of 80 nM. It is also useful for measuring Cr 3+ in lake water. This work proposes a new metal sensor design by monitoring binding kinetics with Cr 3+ being a primary example.

Heavy metal detection is a key topic in analytical chemistry. DNA-based metal recognition has advanced significantly producing many specific metal ligands, such as thymine for Hg 2+ and cytosine for Ag + . For practical applications, however, robust sensors that can work in a diverse range of salt concentrations need to be developed, while most current sensing strategies cannot meet this requirement. In this work, 2-aminopurine (2AP) is used as a fluorescence label embedded in the middle of four 10-mer DNA homopolymers. 2AP can be quenched up to 98% in these DNA without an external quencher. The interaction between 2AP and all common metal ions is studied systematically for both free 2AP base and 2AP embedded DNA homopolymers. With such low background, Hg 2+ induces up to 14-fold signal enhancement for the poly-T DNA, and Ag + enhances up to 10-fold for the poly-C DNA. A detection limit of 3nM is achieved for both metals. With these four probes, silver and mercury can be readily discriminated from the rest. A comparison with other signaling methods was made using fluorescence resonance energy transfer, graphene oxide, and SYBR Green I staining, respectively, confirming the robustness of the 2AP label. Detection of Hg 2+ in Lake Huron water was also achieved with a similar sensitivity. This work has provided a comprehensive fundamental understanding of using 2AP as a label for metal detection, and has achieved the highest fluorescence enhancement for non-protein targets., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A previous study of two RNA-cleaving DNAzymes, NaA43 and Ce13d, revealed the possibility of a common Na(+) aptamer motif. Because Na(+) binding to DNA is a fundamental biochemical problem, the interaction between Ce13d and Na(+) was studied in detail by using sensitized Tb(3+) luminescence spectroscopy. Na(+) displaces Tb(3+) from the DNAzyme, and thus quenches the emission from Tb(3+) . The overall requirement for Na(+) binding includes the hairpin and the highly conserved 16-nucleotide loop in the enzyme strand, along with a few unpaired nucleotides in the substrate. Mutation studies indicate good correlation between Na(+) binding and cleavage activity, thus suggesting a critical role of Na(+) binding for the enzyme activity. Ce13d displayed a Kd of ∼20 mm with Na(+) (other monovalent cations: 40-60 mm). The Kd values for other metal ions are mainly due to non-specific competition. With a single nucleotide mutation, the specific Na(+) binding was lost. Another mutant improved Kd to 8 mm with Na(+) . This study has demonstrated a Na(+) aptamer with important biological implications and analytical applications. It has also defined the structural requirements for Na(+) binding and produced an improved mutant., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Chromium is a very important analyte for environmental monitoring, and developing biosensors for chromium is a long-standing analytical challenge. In this work, in vitro selection of RNA-cleaving DNAzymes was carried out in the presence of Cr(3+) . The most active DNAzyme turned out to be the previously reported lanthanide-dependent Ce13d DNAzyme. Although the Ce13d activity was about 150-fold lower with Cr(3+) than that with lanthanides, the activity of lanthanides and other competing metals was masked by using a phosphate buffer; this left Cr(3+) as the only metal that could activate Ce13d. With 100 μm Cr(3+) , the cleavage rate is 1.6 h(-1) at pH 6. By using a molecular beacon design, Cr(3+) was measured with a detection limit of 70 nm, which was significantly lower than the United States Environmental Protection Agency (EPA) limit (11 μm). Cr(4+) was measured after reduction by NaBH4 to Cr(3+) , and it could be sensed with a similar detection limit of 140 nm Cr(4+) ; this value was lower than the EPA limit of 300 nm. This sensor was tested for chromium speciation analysis in a real sample, and the results supported its application for environmental monitoring. At the same time, it has enhanced our understanding of the interactions between chromium and DNA., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Most previously reported RNA-cleaving DNAzymes require only a single divalent metal ion for catalysis. We recently reported a general trivalent lanthanide-dependent DNAzyme named Ce13d. This work shows that Ce13d requires both Na(+) and a trivalent lanthanide (e.g. Ce(3+)), simultaneously. This discovery is facilitated by the sequence similarity between Ce13d and a recently reported Na(+)-specific DNAzyme, NaA43. The Ce13d cleavage rate linearly depends on the concentration of both metal ions. Sensitized Tb(3+) luminescence and DMS footprinting experiments indicate that the guanines in the enzyme loop are important for Na(+)-binding. The Na(+) dissociation constants of Ce13d measured from the cleavage activity assay, Tb(3+) luminescence and DMS footprinting are 24.6, 16.3 and 47 mM, respectively. Mutation studies indicate that the role of Ce(3+) might be replaced by G23 in NaA43. Ce(3+) functions by stabilizing the transition state phosphorane, thus promoting cleavage. G23 competes favorably with low concentration Ce(3+) (below 1 μM). The G23-to-hypoxanthine mutation suggests the N1 position of the guanine as a hydrogen bond donor. Together, Ce13d has two distinct metal binding sites, each fulfilling a different role. DNAzymes can be quite sophisticated in utilizing metal ions for catalysis and molecular recognition, similar to protein metalloenzymes., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Enzymes working in organic solvents are important for analytical chemistry, catalysis, and mechanistic studies. Although a few protein enzymes are highly active in organic solvents, little is known regarding nucleic acid-based enzymes. Herein, we report the first RNA-cleaving DNAzyme, named EtNa, that works optimally in concentrated organic solvents containing only monovalent Na(+). The EtNa DNAzyme has a rate of 2.0 h(-1) in 54% ethanol (with 120 mM NaCl and no divalent metal ions), and a Kd of 21 mm Na(+). It retains activity even in 72% ethanol as well as in DMSO. With 4 mm Na(+), the rate in 54% ethanol is >1000-fold higher than that in water. We also demonstrated the use of EtNa to measuring the ethanol content in alcoholic drinks. In total, this DNAzyme has three unique features: divalent metal independent activity, Na(+) selectivity among monovalent metals, and acceleration by organic solvents., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)