Self-assembly of Hyaluronic Acid-Cu-Quercetin flavonoid nanoparticles: synergistic chemotherapy to target tumors

Abstract

Background: In this study, a natural compound quercetin (Qu) was investigated for its various antitumor effects. However, due to its poor water solubility and low bioavailability, its clinical application is limited. To overcome this constraint, a modification was to Qu, which resulted in the creation of novel flavonoid self-assembling nanoparticles (HCQ NPs).

Methods: HCQ NPs were synthesized by a self-assembly method and characterized using transmission electron microscopy, the Malvern Zetasizer instrument, X-ray photoelectron spectroscopy (XPS), the ultraviolet-visible spectrophotometric method (UV-vis), Fourier transform infrared (FITR) and inductively coupled plasma mass spectrometry. Extracellular, methylene blue spectrophotometric analysis was used to determine the ability of HCQ NPs to react with different concentrations of H₂O₂ to form hydroxyl radicals (^•OH). Intracellular, DCFH-DA staining was used to detect the ability of HCQ NPs to react with H₂O₂ to generate reactive oxygen species. Flow cytometry was used to detect the uptake of HCQ NPs by MDA-MB-231 cells at different time points. The biocompatibility of HCQ NPs was evaluated using the Cell Counting Kit-8 (CCK-8) assay. Calcein AM/PI double staining and the CCK-8 assay were used to evaluate the synergistic antitumor effect of HCQ NPs and H₂O₂.

Results: HCQ NPs showed uniformly sized analogous spherical shapes with a hydrodynamic diameter of 55.36 ± 0.27 nm. XPS revealed that Cu was mainly present as Cu²⁺ in the HCQ NPs. UV-vis absorption spectrum of the characteristic peak of HCQ NPs was located at 296 nm. Similarly, FTIR spectroscopy revealed a complex formation of Qu and Cu²⁺ that substantially changed the wavenumber of the 4-position C = O characteristic absorption peak. Based on the proportion of Qu and Cu²⁺ (1:2), the total drug loading of Qu and Cu²⁺ in the HCQ NPs for therapeutic purposes was calculated to be 9%. Methylene blue spectrophotometric analysis of ^•OH indicated that Cu can lead to the generation of ^•OH by triggering Fenton-like reactions. HCQ NPs rapidly accumulated in MDA-MB-231 cells with the extension of time, and the maximum accumulation concentration was reached at about 0.5 h. Calcein AM/PI double staining and CCK-8 revealed synergistic antitumor effects of HCQ NPs including the chemotherapeutic effect of Qu and chemodynamic therapy by Cu²⁺ in a simulated tumor microenvironment. HCQ NPs demonstrated very low toxicity in LO2 cells in the biocompatibility experiment.

Conclusion: This study show cases a new method of creating self-assembled flavonoid HCQ NPs that show great for fighting cancer.

Keywords: Fenton-like reaction; Flavonoid self-assembled nanoparticles; Quercetin; Quercetin-copper complex; Synergistic antitumor.