ROPISA Strategy for In‐Situ Loading in Polypeptide Nanoparticles

We report a ring-opening polymerization induced self-assembly (ROPISA) synthetic strategy for in-situ encapsulation of fluorescent dye molecules in poly(ʟ-serine) based polypeptide nano-assemblies and demonstrate their cellular bioimaging application. A bulky ʟ-serine N-carboxyanhydride monomer is tailor-made and polymerized using PEG-amine as hydrophilic macroinitiator in water at pH 8.5 to obtain polypeptide block copolymer as stable dispersions in the form of opalescent solutions. Both water soluble fluorescent dyes like Rhodamine B, HPTS and water insoluble fluorescent dye like Nile red are readily encapsulated in-situ in the ROPISA process which afforded stable fluorescent polypeptide nanoformulation for direct application in biological system. The polypeptide nanoparticle dispersion is found to be stable, and they are found to have spherical nanoparticle morphology of 25 nm in size. Both the nascent and fluorescent dye encapsulated polypeptide nanoparticles were found to be nontoxic to mammalian cells up to 100 µg/mL and non-hemolytic to Red Blood Cells. These polypeptide nanoparticles were readily endocytosed across the cell membrane and internalized in the cytosol, and the proof-of-concept was established by confocal microscopy. This newly developed in-situ ROPISA process for fluorescent dye loading opens up new platform for polypeptide nano-formulations for application in both material and biomedical fields.