Introduction of optimized folate group modification on lipid-coated magnetic nanobeads enables targeting of folate...

The functionalization with targeting ligands is a strategy that is often employed to guide nanocarriers to specific target cells. While there is a large variety of different targeting molecules, folate stands out as especially promising. This is because of its high solubility in water, easy experimental handling, and the fact that folate receptor expressing cells present interesting targets for immunomodulation. While folate receptor alpha can be used to target epithelial tumors, folate receptor beta allows targeting of macrophages that are associated to tumors or inflammation sites. However, while several folate-functionalized nanocarriers (NCs) have been reported, studies have almost exclusively focused on targeting outcome, neglecting the effect of folate functionalization on NC colloidal stability. Here, we optimized a two-step functionalization strategy to simultaneously balance both effective targeting and high colloidal stability. Specifically, we optimized the number of functional folate groups depending on overall NC concentration. Here, we observed that high folate group densities required medium NC concentrations, while there was no influence on stability over time. We first validated our strategy using model liposomes and subsequently with a more complex platform consisting of lipid-coated nanobeads (silica-embedded SPIONs), enabling selective targeting and immunomodulation of profibrotic macrophages in relevant in vitro and in vivo models of lung fibrosis. Folate-functionalized nanocarriers selectively targeted FRβ-expressing M2 macrophages in vitro and accumulated in macrophages in a mouse model of lung injury progressing to pulmonary fibrosis whereas unmodified nanocarriers appeared predominantly as isolated particles with no clear cellular association.