Nanoparticle (NP)-based targeted drug delivery is intended to transport therapeutically active molecules to specific cells and particular intracellular compartments. However, there is limited knowledge regarding the complete route of NPs in this targeting scenario. In this study, simultaneously performing motion and dynamic pH sensing using single-particle tracking (SPT) leads to an alternative method of gaining insights into the mesoporous silica nanoparticle's (MSN) journey in targeting lysosome. Two different pH-sensitive dyes and a reference dye are incorporated into mesoporous silica nanoparticles (MSNs) via co-condensation to broaden the measurable pH range (pH 4−7.5) of the nanoprobe. The phosphonate, amine, and lysosomal sorting peptides (YQRLGC) are conjugated onto the MSN's surface to study intracellular nano-biointeractions of two oppositely charged and lysosome-targetable MSNs. The brightness and stability of these MSNs allow their movement and dynamic pH evolution during their journey to be simultaneously monitored in real time. Importantly, a multidimensional analysis of MSN's movement and local pH has revealed new model intracellular dynamic states and distributions of MSNs, previously inaccessible when using single parameters alone. A key result is that YQRLGC-conjugated MSNs took an alternative route to target lysosomes apart from the traditional one, which sped up to 4 h and enhanced their targeting efficiency (up to 32%). The findings enrich our understanding of the intracellular journey of MSNs. This study offers complementary information on correlating the surface design with the full pathway of nanoparticles to achieve targeted delivery of therapeutic payload.
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