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Biomaterials

Breaking Barriers: Biomaterials Revolutionizing Drug Delivery Strategies

Protein Corona Impacts Targeting and Affinity of Layer-by-Layer Nanoparticles

Saturday, October 14, 2023
3:30 PM – 3:45 PM PDT
Location: 331 (Center, Level 3)
Introduction: After nanoparticles are immersed in biological fluids, such as blood, a layer of proteins, lipids, and other biomolecules adsorbs the surface of the nanocarrier forming a protein corona. The protein corona is known to have an effect on targeting, transport, and the elicited immune response. This phenomenon is influenced by nanoparticle properties such as charge and surface chemistry, as well as biological environment properties including pH and composition. Layer-by-layer nanoparticles (LbL NPs) are effective carriers for targeted therapeutic delivery due to their highly tunable surface properties. Previously our lab reported LbL NPs with different outer layer chemistries had varying trafficking in vitro and differences in biodistribution. We hypothesize this is partly due to differences in proteins adsorbed on LbL NPs surfaces as outer chemistries change. This study aims to characterize protein coronas on LbL NPs and understand the effects on biological outcomes of NP targeting and affinity in vitro.

Materials & Methods: A library of LbL NPs with varying outer layer surface chemistries was generated (Fig A). Spent media was collected from RAW 264.7 macrophages containing cell-secreted biomolecules and 10% FBS, to use as a physiologically relevant biological fluid. LbL NPs were incubated in spent media for 30 minutes at 37°C while shaking to form a protein corona. Unbound proteins were separated from adsorbed proteins using centrifugal ultrafiltration. To characterize the protein corona, NP size and surface charge were measured using dynamic light scattering, and protein concentrations and composition were analyzed using nanodrop and SDS-PAGE, respectively. To assess how protein corona affects targeting and affinity, macrophages were dosed with LbL NPs with a pre-formed protein corona and uptake was analyzed using flow cytometry and confocal microscopy.

Results & Discussion: We assessed changes in properties of various LbL NPs after incubation in spent media. Following incubation, NP sizes dramatically increased for some surface chemistries, while for others these was little to no change in size. (Fig B). NPs with anionic outer layers were less negative while NPs with cationic outer layers charge converted to negative. Collectively, this suggests NPs interacting with proteins, and demonstrates how a protein corona can alter intrinsic LbL NP properties, implicating the importance of outer layer chemistry selection. Although concentration of proteins in the protein corona (Fig C) varied between different surface chemistries, similar banding patterns on SDS-PAGE suggest similar proteins are interacting with LbL NPs. Pre-formed protein coronas on LbL NPs reduced uptake in macrophages. Confocal microscopy (Fig D) further revealed LbL NPs with anionic acrylic and polypeptide polymer outer layers were internalized, however those with the carboxylated polysaccharide outer layer had lowest internalization with some NPs localized to the cell membrane.

Conclusions: We identified that protein corona on LbL NP alters surfaces properties and affects biological outcomes in vitro with differences between varying outer layer chemistries. These LbL NPs are commonly used in our lab for delivering therapeutics to treat various diseases including ovarian cancers and glioblastoma. Characterizing the protein corona on these LbL NPs is essential to tuning targeting specificity which can improve biodistribution and circulation times. Based on SDS-PAGE, we hypothesize globulins and apolipoproteins are part of the protein corona composition; future analysis with mass spectroscopy will be used to confirm protein identities. Overall, understanding how NPs interact with biological environments can help us improve clinical translation of nanomedicines.