Nanocarrier-Based Programmed Nucleus/Mitochondria Delivery via Guanidinium Density-Dependent Switching of Cellular Uptake Pathway

Abstract Efficient cytosolic access and organelle-specific targeting of polymeric nanocarriers remain unresolved challenges in nanomedicine, largely due to limited control over nano-bio interfacial interactions and intracellular trafficking pathways. Conventional cationic vectors bearing amine or ammonium functionalities often rely on endocytic uptake. In contrast, guanidinium groups, structurally analogous to the side chain of arginine, enable unique interactions with anionic phospholipids and sulfated proteoglycans on cell surfaces that promote direct membrane translocation. Here, we establish guanidinium surface density as a chemically tunable switch that governs both the cellular entry mechanism and the intracellular fate of polymeric nanocarriers. Amphiphilic poly(succinimide)-derived micellar nanocarriers with systematically varied guanidinium densities, PG0.1 (low), PG0.5 (moderate), and PG1.0 (high) were engineered, forming uniform ∼20–30 nm assemblies in aqueous medium. Across this series, all nanocarriers exhibited rapid cellular entry dominated by direct membrane translocation, bypassing classical endocytic routes. Strikingly, guanidinium density dictated organelle-specific trafficking: PG1.0 showed pronounced nuclear accumulation. PG0.5 displayed time-dependent cytosol-to-nucleus redistribution, while PG0.1 preferentially associated with mitochondria. Mechanistic investigations reveal that membrane translocation is accompanied by nanocarrier deformation and partial disassembly, enabling redistribution of polymer segments within intracellular environments. This redistribution is further directed by interaction with intracellular compartments, where PG0.1 localization is governed by mitochondrial membrane potential, while PG1.0 engages importin α/β-mediated nuclear transport pathways. PG0.1 shows preferential mitochondrial association, while PG1.0 exhibits a stronger tendency for nuclear accumulation under reductionist buffer conditions. These findings provide a mechanistically grounded design framework for engineering nanocarriers with programmable intracellular destinations, advancing the rational development of next-generation nanomedicines.