Biophysical Considerations in the Rational Design and Cellular Targeting of Flexible Polymeric Nanoparticles

It is becoming evident that engineered physicochemical characteristics of nanoparticles (NPs) are essential to improve their biological function for their cellular delivery and uptake. How NP mechanical properties impact multivalent ligand-receptor mediated binding to cell surfaces, the avidity of NP adhesion to cells, and cooperative effects due to crowding remain largely unknown or unquantified, and how tuning NPs' stiffness impacts their propensity for internalization is not clear. Here we focus on exploring the binding mechanisms of three distinct NPs that differ in type and rigidity (core-corona flexible NP, rigid NP, and rigid-tethered NP) but are otherwise similar in size and ligand surface density; moreover, for the case of flexible NP, we tune NP stiffness by varying the internal crosslinking density. We employed our recent spatial biophysical modeling of NP binding to membranes together with thermodynamic analysis powered by free energy calculations and show that efficient cellular targeting and uptake of NP functionalized with targeting ligand molecules can be shaped by factors including NP flexibility and crowding, receptor-ligand binding avidity, state of the membrane cytoskeleton, and curvature inducing proteins. Owing to this multitude of factors, we demonstrate that the binding avidity of a flexible NP depends on engineered changes in NP flexibility in a non-intuitive fashion because of significant enthalpy entropy compensations arising from multiple competing terms associated with NP, receptor density, and membrane. Analyses of the individual enthalpic and entropic contributions associated with NP, membrane, and receptor-ligand binding and receptor diffusion collectively illuminate this complex dependence of avidity on crosslinking. We find that the NP binding avidity can be engineered in a crowded environment by tuning their flexibility. We also find that when the cell membrane is bound to the cytoskeletal proteins via the pinning sites, the pinning sites' presence does not limit the binding avidity of both flexible and rigid-tethered NPs. Furthermore, we show that the membrane tension and pinning density, and the stiffness of flexible NPs can be tuned to alter the adhesion energy landscape and eventually improve the adhesion efficiency of flexible NPs. We also probed the effects of curvature-inducing proteins and receptor-ligand binding interactions on NP uptake. We found that complete uptake of both rigid-tethered NPs, and flexible NPs can be achieved by tuning NP stiffness and membrane tension even under moderate levels of ligand-densities in use for physiologically relevant applications. These findings provide strong evidence that NP flexibility is an important design parameter for rationally engineering NP targeting and uptake in a crowded cellular adhesion microenvironment.