Tumor-acidity activated surface charge conversion of two-photon fluorescent nanoprobe for enhanced cellular uptake and targeted imaging of intracellular hydrogen peroxide

Abstract

Elevated levels of intracellular hydrogen peroxide (H₂O₂) are closely related to the development of cancers. Specific imaging of H₂O₂ in tumor sites would be significant not only for cancer diagnosis but also for gaining a deep understanding of the role of H₂O₂ in cancer. However, traditional fluorescent probes based only on responses to overexpression levels of H₂O₂ in cancer cells are insufficient to distinguish cancer cells from other unhealthy or healthy cells in complex biological systems. Herein, we developed a smart, two-photon fluorescent GC–NABP nanoprobe with pH-dependent surface charge conversion for tumor-targeted imaging of H₂O₂. The nanoprobe was constructed by the self-assembly of amphiphilic GC–NABP, which was synthesized by grafting the hydrophobic, H₂O₂-responsive and two-photon fluorophore, NABP, onto hydrophilic biopolymer glycol chitosan (GC). Taking advantage of pH-titratable amino groups on GC, the nanoprobe had the capability of surface charge conversion from negative at physiologic pH to positive in the acidic tumor microenvironment. The positive charge of the nanoprobe promoted electrostatic interactions with cell membranes, leading to enhanced cellular uptake in acidic environment. Upon cellular uptake, the high level of H₂O₂ in tumor cells triggered boronate deprotections of the nanoprobe, generating a “turn-on” fluorescence emission for H₂O₂ imaging. The nanoprobe exhibited good sensitivity and selectivity to H₂O₂ with a detection limit down to 110 nM in vitro. The results from flow cytometry and two-photon fluorescence imaging of H₂O₂ in living cells and tissues evidenced the enhanced cellular uptake and targeted imaging of intracellular H₂O₂ in acidic environment. Compared to control nanoparticles that lack pH sensitivity, our nanoprobe showed enhanced accumulation in tumor sites and was applied to targeted imaging of H₂O₂ in a tumor-bearing mouse model. This work demonstrates that the nanoprobe GC–NABP holds great promise for tumor-specific imaging of cellular H₂O₂, providing a potential tool to explore the role of H₂O₂ in tumor sites.