CD44 targeted nanozymes for detecting circulating tumor cells in liquid biopsy of breast cancer

Breast cancer is the second leading cause of cancer related deaths wordwide. Metastasis and tumor relapse are the major challenges in treatment of breast cancer. Early diagnosis is a very critical step in the management of breast cancer. Liquid bioposy is an emerging technique for rapid detection of cancer biomarkers (eg., circulating tumor cells). Compelling body of evidences suggest circulating tumor cells (CTCs) which overexpress CD44 are responsible for metastasis and tumor relapse of breast cancer.  In the present study, a simple colorimetric protocol for determination of CTC concentration was developed based on a cell internalizable nanozyme functionalized with chondroitin (CS) as a ligand for CD44 receptors of CTCs. Mesoporous silica microspheres embedded CuSO₄nanoparticles (CuSO₄@SiO2 microspheres) were synthesized by sol-gel technique. They were found to be 9.15μm in size as evident from DLS. Then the CS was conjugated on the surface of CuSO₄@SiO2 microspheres using carbodiimide linkage to obtain CD44 targeted nanoparticles (CD44-CuSO₄@SiO2 microspheres). CD44-CuSO₄@SiO2 microspheres were effectively uptaken by MDA-MB231 and MCF-7 breast cancer cells by CD44 receptor mediated endocytosis in Dulbecco's modified eagle's medium and exhibited peroxidase-like activity. CD44-CuSO₄@SiO2 microspheres, therefore, regarded as  a nanozyme for determination of CTC concentration.

In the developed colorimetric protocol, the constant number of CD44-CuSO₄@SiO2 microspheres were interacted with MDA-MB231 and MCF-7 cell suspensions at  different cell concentrations. The chemical substrate (3, 3’,5,5’-tetramethylbenzidine, TMB) was converted to a coloured product upon oxidation (TMBox) by peroxidase-like activity of CD44-CuSO₄@SiO2 microspheres. The apparent peroxidase-like activity was determined by measuring absorbance of reaction medium after removal of CS at 652nm.

Peroxidase-like activity (absorbance @ 652nm) is decreased with the increasing CTC concentration. The increase in the number of CD44-CuSO₄@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. In conclusion“ peroxidase-like activity of cell internalizable nanozyme decreased with increase in concentration of  CTCs”.  Proposed method is a promising start for development of facile liquid biopsy protocols instead of currently used complex systems.

Key words: Nanozyme; liquid biopsy; peroxidase like activity; breast cancer; CD44; circulating tumor cells