A pH-responsive hybrid fluorescent nanoprober for real time cell labeling and endocytosis tracking

Hydrophilic, fluorescent hybrid nanoprobes (NDI@HNPs) encapsulated with the hydrophobic pH-responsive fluorophore (N,N′-di-n-dodecyl-2,6-di(4-methyl-piperazin-1-yl)naphthalene-1,4,5,8-tetracarboxylic acid diimide, NDI) for recognizing and mapping the route of cell phagocytosis have been fabricated based on the self-assembly of amphiphilic diblock copolymer PS-b-PAA and the subsequent shell cross-linking with 3-mercaptopropyltrimethoxy silane (MPTMS). The as-synthesized NDI@HNPs has a typical spherical morphology of 46 nm in diameter with excellent monodispersity in aqueous solution. The NDI@HNPs probe exhibits extremely low cytotoxicity, fast real time pH response and enhanced fluorescence intensity under acidic environment with respect to the corresponding free dye in highly polar aqueous system because of the encapsulation of NDI molecules inside nanoparticle cores with weak polarity environment. The fluorescence intensity of NDI@HNPs is enhanced by 55-fold upon changing from neutral (pH = 7.4) or basic (pH = 8.4) to acid (pH = 3.4) in aqueous system, in contrast to the serious fluorescence quenching of free NDI in the same medium, which can exactly meet the physiological pH range in cells. The favorably long emission wavelength is beneficial to the low scattering and minimal interfering requirements to fluorescent bioimaging. Moreover, functionalization with rapid cell-penetrating peptides (HIV-1 TAT) allows them to overcome the physiological and biological barriers during the phagocytosis process. Its characteristic fluorescent response to pH benefits the intracellular labeling and organelle targeting, realizing the real time tracking of the probe entry into cancer cells, the accumulation into the endolysosome and the further escape.