Colorimetric determination of tumor cells via peroxidase-like activity of a cell internalizable nanozyme: Hyaluronic acid attached-silica microspheres containing accessible magnetite nanoparticles

Abstract

A simple colorimetric protocol for determination of tumor cell concentration was developed based on a cell internalizable nanozyme functionalized with hyaluronic acid (HA) as a ligand sensitive to CD44 receptors of tumor cells. Monodisperse-porous silica microspheres containing immobilized Fe3O4 nanoparticles (Fe3O4@SiO2 microspheres) were synthesized 6.25 μm in size, by “staged sol-gel templating protocol”. HA attached form of Fe3O4@SiO2 microspheres (HA@Fe3O4@SiO2 microspheres) was obtained via carbodiimide activation. HA@Fe3O4@SiO2 microspheres were effectively uptaken by human cervical cancer (HeLa) cells and primary brain tumor cells (T98 G glioblastoma) via pinocytosis in Dulbecco's modified eagle's medium, depending on the interaction of HA with CD44 receptors. The number of HA@Fe3O4@SiO2 microspheres uptaken by HeLa and T98 G cells increased with the increasing cell concentration. HA@Fe3O4@SiO2 microspheres also acted as a nanozyme and exhibited peroxidase-like activity due to Fe3O4 nanoparticles tightly immobilized within their porous interiors in the accessible form. The decrease of peroxidase-like activity of nanozyme due to the specifically uptaken microspheres was used as a new tool for determination of tumor cell concentration. In the developed colorimetric protocol, the constant number of HA@Fe3O4@SiO2 microspheres were interacted with HeLa and T98 G cell suspensions prepared with different cell concentrations for completion of pinocytosis. The synthetic substrate (3,3’,5,5’-tetramethylbenzidine, TMB) was converted to a colored product (oxidized TMB) by peroxidase-like activity of both cell internalized and non-internalized microspheres. The apparent peroxidase-like activity was determined by measuring visible region absorbance of reaction medium after removal of HA@Fe3O4@SiO2 microspheres. The observed peroxidase-like activity (i.e. the visible region absorbance) exhibited almost a linear decrease with the increasing tumor cell concentration. The increase in the number of HA@Fe3O4@SiO2 microspheres uptaken by tumor cells with the increasing cell concentration was the reason of the decrease observed in the apparent peroxidase-like activity of nanozyme. The proof of concept, “the decrease of peroxidase-like activity of cell internalizable nanozyme depending upon the increasing number of uptaken microspheres” was termed as a general tool utilized for determination of various kinds of tumor cells, for the first time. Proposed method is a promising start for development of facile liquid biopsy protocols instead of currently used complex systems.