Dissection the endocytic routes of viral capsid proteins-coated upconversion nanoparticles by single-particle tracking

Real-time exploring the cellular endocytic pathway of viral capsid proteins (VCPs) functionalized nanocargos at the single-particle level can provide deep insight into the kinetic information involved in virus infection. In this work, porcine circovirus type 2 (PCV2) VCPs with different functions are modified onto the surface of upconversion nanoparticles (VCPs-UCNPs) to investigate the cellular internalization process in real-time. Clathrin-mediated endocytosis is found to be the essential uptake mechanism for these VCPs-UCNPs. Besides, it is verified that P₁-UCNPs (PCV2 VCPs with nuclear localization signal, namely P₁) can be easily assembled close to the perinuclear area, which is different from that of P₂-UCNPs (PCV2 VCPs without nuclear localization signal, namely P₂). Interestingly, multistep entry processes are observed. Particularly, confined diffusion is observed during the transmembrane process. The intracellular transport of VCPs-UCNPs is dependent on microtubules toward the cell interior. During this process, P₁-UCNPs display increased velocities with active transport, while diffusion much faster around the perinuclear area. But for P₂-UCNPs, there are only two phases involved in their endocytosis process. This study presents distinct dynamic mechanisms for the nanocargos with different functions, which would make a useful contribution to the development of robust drug delivery systems.