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

Elevated levels of intracellular hydrogen peroxide (HO) are closely related to the development of cancers. Specific imaging of HO in tumor sites would be significant not only for cancer diagnosis but also for gaining a deep understanding of the role of HO in cancer. However, traditional fluorescent probes based only on responses to overexpression levels of HO 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 HO. The nanoprobe was constructed by the self-assembly of amphiphilic GC-NABP, which was synthesized by grafting the hydrophobic, HO-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 HO in tumor cells triggered boronate deprotections of the nanoprobe, generating a "turn-on" fluorescence emission for HO imaging. The nanoprobe exhibited good sensitivity and selectivity to HO with a detection limit down to 110 nM . The results from flow cytometry and two-photon fluorescence imaging of HO in living cells and tissues evidenced the enhanced cellular uptake and targeted imaging of intracellular HO 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 HO in a tumor-bearing mouse model. This work demonstrates that the nanoprobe GC-NABP holds great promise for tumor-specific imaging of cellular HO, providing a potential tool to explore the role of HO in tumor sites.