Protein Corona Impacts Surface Properties of Layer-By-Layer Nanoparticles

When introduced into biological mediums, a layer of proteins known as the protein corona (PC) adsorbs to the surface of nanosystems. In recent years, the effects of PC in the therapeutic outcome of drugs and drug carriers such as nanoparticles (NPs) has become more evident, impacting their stability, cellular uptake, circulation time, and immunotoxicity. NPs constructed via the layer-by-layer (LbL) method show promise as a modular theragnostic platform that uses a variety of charged polymers, giving rise to tunable surface properties that can influence targeting capacity and biodistribution. We hypothesized that PC is likely affected by this variable surface chemistry and may contribute to the performance of LbL NP in vivo. However, PC formation on LbL NPs remains to be characterized.

Here, we report a reproducible method to isolate and characterize the PC on LbL NPs, then an evaluation on how the presence of PC impacts in vitro cellular uptake. First, we prepared a library of LbL NPs with different outer polymers (polypeptides, polysaccharides, and synthetic hydrocarbon backbones) based on their therapeutic potential in different disease models, together with PEGylated liposomes as the gold standard. These NPs were incubated in a biologically relevant medium, then subjected to an optimized ultrafiltration method to remove unbound protein for characterization of the isolated PC. Particle size and surface charge before and after PC isolation, quantified by dynamic light scattering, showed moderate size increases, while also maintaining surface charges. Differences in the amounts of proteins associated with LbL NP were observed, with polypeptides showing lower protein concentration than PEGylated liposomes. Further studies revealed differences in cellular uptake in both an ovarian cancer cell line and in macrophage. In particular, for the best performing layer, a pre-formed PC increased the uptake by the cancer cell line, while decreased the uptake by macrophages. These findings indicate that further characterization can and should be carried out for the PC on LbL NPs to determine how protein corona influences the fate of these NPs, providing a key step toward accelerating their clinical translation.