Single-chain nanoparticles (SCNPs) are a type of polymeric nanoparticles formed by collapsing/folding individual polymer chains via intra-molecular interactions. Their emergence holds promise to construct sub-20 nm polymeric nanoparticles with defined structure. Challenges remain for controllable construction of SCNPs. First, soft nanostructures like SCNPs are prone to deformation and cannot be well represented by geometrical terms such as size and shape; instead, a topological description is more proper. Second, SCNP formation by stochastic pairing of the functionalities from the same linear precursor leads to stochastic outcomes. Versatile synthetic approaches as well as novel analytical tools are needed to gain control on the SCNP topologies. Third, it remains an open question how SCNPs' topology affects their properties, such as their interaction with cells. This thesis investigates SCNP folding and their interaction with cells from the topological point of view. We first show that the SCNP topology can be tuned. The effect from two key factors (the initial chain conformation and the length of the cross-linkers) on the formation of SCNPs were experimentally investigated. A suite of analytical tools was developed and applied to characterize the SCNP topology, which was then correlated to SCNPs' cytotoxicity profile. We then show that cellular uptake discriminates SCNP topological isomers. SCNPs cross-linked with disulfide bonds were transformed into topological isomers by reshuffling the disulfide pairs. The nuances of different topologies were captured by the unparallel sensitivity of analytical ultracentrifugation. The glucocorticoid induced GFP translocation assays showed that the SCNPs' topology was essential for their ability to access the cytosol. The overall work presented in the thesis provides new perspectives on SCNPs' formation, characterization, and interaction with cells from the topological point of view.