Polymer-Functionalized Liposomes as Universal Nanocarriers for Drug Delivery: Single Particle Insights on Size-Dependent Performance and Intracellular Behavior.

Nanomedicine requires smart delivery systems that are precise, robust, and universal. While liposomes are established vehicles in drug delivery, their full potential is challenged by limited stability, leakage, insufficient response, and limited insight into particle size-dependent performance. Here, we provide polymer-modified liposomes (PMLs), engineered for high structural integrity, broad cargo compatibility, and stimulus-responsive cargo release. We thoroughly characterize PMLs at the single particle level, shedding light on key structure-function relationships within polydisperse formulations, revealing that small vesicles (<100 nm) displayed significantly higher cargo packing densities, while release was independent of vesicle size. PMLs display high versatility, effectively encapsulating cargo types ranging from positively or negatively charged small molecules to oligonucleotides and proteins. Studies on PMLs' interaction with the cell membrane show that PMLs maintain a high internalization rate in HeLa and hCMEC/D3 brain cells and achieve an ∼50% reduction in cell viability within 24 h when loaded with the anticancer drug 5-fluorouracil. Finally, PMLs successfully deliver siRNA targeting eGFP in HEK293-d2eGFP cells, achieving a 10-12% knockdown of eGFP expression, as resolved by machine learning-driven single-cell analysis. This work establishes a framework for PMLs' high-resolution functional profiling and opens the way for the next generation rational design of tunable PMLs for drug delivery.