Spectroscopic, calorimetric and cytotoxicity studies on the combined binding of daunorubicin and acridine orange to a DNA tetrahedron.

Chemotherapy-phototherapy (CTPT) combination drugs co-loaded by targeted DNA nanostructures can achieve controlled drug delivery, reduce toxic side effects and overcome multidrug resistance. Herein, we constructed and characterized a DNA tetrahedral nanostructure (MUC1-TD) linked with the targeting aptamer MUC1. The interaction of daunorubicin (DAU)/acridine orange (AO) alone and in combination with MUC1-TD and the influence of the interaction on the cytotoxicity of the drugs were evaluated. Potassium ferrocyanide quenching analysis and DNA melting temperature assays were used to demonstrate the intercalative binding of DAU/AO to MUC1-TD. The interactions of DAU and/or AO with MUC1-TD were analyzed by fluorescence spectroscopy and differential scanning calorimetry. The number of binding sites, binding constant, entropy and enthalpy changes of the binding process were obtained. The binding strength and binding sites of DAU were higher than those of AO. The presence of AO in the ternary system weakened the binding of DAU to MUC1-TD. In vitro cytotoxicity studies demonstrated that the loading of MUC1-TD augmented the inhibitory effects of DAU and AO and the synergistic cytotoxic effects of DAU + AO on MCF-7 cells and MCF-7/ADR cells. Cell uptake studies showed that the loading of MUC1-TD was beneficial in promoting the apoptosis of MCF-7/ADR cells due to its enhanced targeting to the nucleus. This study has important guiding significance for the combined application of DAU and AO co-loaded by DNA nanostructures to overcome multidrug resistance.

A pH-sensitive DNA tetrahedron for targeted release of anthracyclines: Binding properties investigation and cytotoxicity evaluation.

The anticancer efficacy of chemotherapeutic agents can be enhanced by the loading of DNA nanostructures, which is closely related to their interactions. This study achieved pH-responsive and targeted anthracycline delivery using i-motif and MUC1 aptamer co-modified DNA tetrahedron (MUC1-TD). The thermodynamic parameters for the binding of doxorubicin (DOX) and epirubicin (EPI) to MUC1-TD at pHs 7.4 and 5.0 were obtained. The smaller binding constant and the number of binding sites at pH 5.0 than at pH 7.4 indicated that acidic conditions favored the release of DOX and EPI loaded by MUC1-TD. The binding affinity of DOX was stronger than that of EPI at the same pH value due to their different chemical stereostructures. The intercalative binding mechanism was verified. In vitro release experiments revealed that acid pH and deoxyribonuclease I accelerated the release of DOX and EPI. The faster release rate of EPI than DOX was related to their binding affinity. In vitro cytotoxicity and cell uptake experiments revealed that the cytotoxicity of DOX and EPI loaded by MUC1-TD to MCF-7 cells was significantly higher than that to L02 cells. This work will provide theoretical guidance for the application of pH-responsive MUC1-TD nanocarriers in the field of pharmaceutics.

Multispectroscopic and synergistic antioxidant study on the combined binding of caffeic acid and (-)-epicatechin gallate to lysozyme.

The binding of caffeic acid (CA) and/or (-)-epicatechin gallate (ECG) to lysozyme was investigated by multispectroscopic methods and molecular docking. The effects of the single and combined binding on the structure, activity and stability of lysozyme and the synergistic antioxidant activity of CA and ECG were also studied. Fluorescence quenching spectra, time-resolved fluorescence spectra, and UV-vis absorption difference spectra all ascertained the static quenching mechanism of lysozyme by CA/ECG. Thermodynamic parameters indicated that CA and ECG competitively bound to lysozyme, and CA had a stronger binding affinity, which was consistent with the results of molecular docking. Hydrogen bonding, van der Waals' force and electrostatic interaction were the main driving forces for the binding process. Synchronous fluorescence spectra displayed that the interaction of CA/ECG exposed the tryptophan residues of lysozyme to a more hydrophilic environment. Circular dichroism spectroscopy, Fourier transform infrared spectroscopy and dynamic light scattering indicated that the binding of CA and/or ECG to lysozyme resulted in the change of the secondary structure and increased the particle size of lysozyme. The binding of CA and/or ECG to lysozyme inhibited the enzyme activity and enhanced the thermal stability of lysozyme. The combined application of CA and ECG showed antioxidant synergy which was influenced by the encapsulation of lysozyme and cellular uptake. In summary, this work provides theoretical guidance for lysozyme as a carrier for the combined application of CA and ECG.

Closing-upon-repair DNA tetrahedron nanoswitch for FRET imaging the repair activity of 8-oxoguanine DNA glycosylase in living cells.

In situ imaging the repair activity of 8-oxoguanine (8-OG) DNA glycosylase in living cells is important as it is associated with genetic mutation. However, the existing imaging methods confront the interference of intracellular nuclease and resulting in false positive signal. Here, a closing-upon-repair DNA tetrahedron nanoswitch (CRTN) was designed for FRET imaging the repair activity of 8-OG DNA glycosylase in living cells with high specificity and accuracy. CRTN comprised a DNA tetrahedron, a recognition strand modified with 8-OG bases, and a reporting strand designed as hairpin structure and labeled with Cy3/Cy5 dual fluorophores. Initially, the DNA tetrahedron was linked with the reporting strand hybridized to the recognition strand, separating the Cy3 donor and Cy5 acceptor into FRET-invalid distance. Upon repair the 8-OG bases by 8-OG DNA glycosylase, CRTN could undergo a structure change from the open to closed state. Specifically, the reporting strand was dissociated from the recognition strand under the action of 8-OG DNA glycosylase and folded into hairpin structure, bringing the Cy3 donor and Cy5 acceptor into FRET-valid proximity with the generation of FRET signal, which could prevent false positive signal arising from nuclease degradation. CRTN exhibited the feasibility for detecting 8-OG DNA glycosylase activity in vitro with good sensitivity and selectivity. More importantly, CRTN could enter cells without any transfection for FRET imaging the repair activity of intracellular 8-OG DNA glycosylase with high specificity and accuracy. This approach provided a promising tool for deeper understanding 8-OG DNA glycosylase function and further studying genetic mutation-related diseases.