Formation of the biomolecular corona ultimately determines the successful application of nanoparticles in vivo. Adsorption of biomolecules such as proteins is an inevitable process that takes place instantaneously upon contact with physiological fluid (e.g. blood). Therefore, strategies are needed to control this process in order to improve the properties of the nanoparticles and to allow targeted drug delivery. Here, we show that the design of the protein corona by a pre-formed protein corona with tailored properties enables targeted cellular interactions. Nanoparticles were pre-coated with immunoglobulin depleted plasma to create and design a protein corona that reduces cellular uptake by immune cells. It was proven that a pre-formed protein corona remains stable even after nanoparticles were re-introduced to plasma. This opens up the great potential to exploit protein corona formation, which will significantly influence the development of novel nanomaterials.

Attaching targeting ligands on the surface of self-assembled drug delivery systems is the key request for a controlled transport of the drug to a desired location. Most commonly, the amphiphilic molecules (block-copolymers, lipids etc.) are therefore pre-functionalized before the self-assembly takes place. However, this strategy cannot be applied, if it interferes with the self-assembly process, if the introduced functional groups react with loaded cargo or if natural carriers like extracellular vesicles should be functionalized. Here, we present the site-specific coupling of antibodies to the surface of amino group-terminated liposomes via bio-orthogonal copper-free click chemistry after liposome formation. The present primary amino groups were functionalized with a linker carrying a strained alkyne group for a bio-orthogonal strain-promoted alkyne–azide cycloaddition (SPAAC) reaction where Cu(I) as a catalyst can be avoided. Antibodies were site-specifically functionalized with azide moieties along the Fc region to avoid interference with the antigen binding sites. The liposome surface functionalization reaction was optimized by precisely analyzing the number of available functional groups (both amine and alkyne), which often represents a challenge for self-assembled systems. By finally confirming the successful antibody coupling, we provide a facile and robust functionalization strategy, which can be applied to a wide range of self-assembled systems and desired targeting antibodies maintaining physiological conditions throughout the procedure.

Adsorption of blood proteins to the surface of nanocarriers is known to be the critical factor influencing cellular interactions and eventually determining the successful application of nanocarriers as drug carriers in vivo. There is an increasing number of reports summarizing large data sets of all identified corona proteins. However, to date our knowledge about the multiple mechanisms mediating interactions between proteins and nanocarriers is still limited. In this study, we investigate the influence of protein structure on the adsorption process and focus on the effect of heat inactivation of serum and plasma, which is a common cell culture procedure used to inactivate the complement system. As in general routine lab procedure, heat inactivation was performed at 56 °C for 30 min in order to denature heat labile proteins. When nanocarriers were exposed to native versus heat inactivated serum, we saw that the cellular uptake by macrophages was significantly affected. These results were then correlated with an altered corona composition that depended on the treatment of the protein source. In summary, we were able to prove that the protein structure is one of the key parameters determining protein corona formation.

Understanding the behavior of nanoparticles upon contact with a physiological environment is of urgent need in order to improve their properties for a successful therapeutic application. Most commonly, the interaction of nanoparticles with plasma proteins are studied under in vitro conditions. However, this has been shown to not reflect the complex situation after in vivo administration. Therefore, here we focused on the investigation of magnetic nanoparticles with blood proteins under in vivo conditions. Importantly, we observed a radically different proteome in vivo in comparison to the in vitro situation underlining the significance of in vivo protein corona studies. Next to this, we found that the in vivo corona profile does not significantly change over time. To mimic the in vivo situation, we established an approach, which we termed “ex vivo” as it uses whole blood freshly prepared from an animal. Overall, we present a comprehensive analysis focusing on the interaction between nanoparticles and blood proteins under in vivo conditions and how to mimic this situation with our ex vivo approach. This knowledge is needed to characterize the true biological identity of nanoparticles.

Safe and functional delivery of delicate mRNA molecules to target tissues is a crucial step in the development of effective vaccines and therapeutic interventions. Lipid nanoparticles (LNP) are the most clinically advanced delivery vehicles for mRNA drugs and crucially rely on ionizable cationic lipids. However, the structure‐function relationships between ionizable lipids and efficient in‐vivo mRNA delivery remain poorly understood. Here, we focus on the rational design and sequential structural optimization of our ionizable lipids that performed well in vitro, but not in vivo. Through two distinct iterative optimization cycles—targeting the lipid tail and the headgroup—we studied how fusogenicity and pKa of ionizable lipids contribute to LNP delivery performance, in vivo. By engineering lipids with both unsaturated tails and more hydrophobic amino headgroups, we achieved both significant improvement of protein expression in vitro, reduced hemolysis risk, and more than 200‐fold improvement of in vivo mRNA delivery. When compared head‐to‐head to a market‐approved LNP benchmark, the newly developed ionizable lipids/LNP resulted in equally highly efficient in vivo mRNA delivery, with strong liver and spleen tropism upon intravenous injection, while matching the safety of the approved platform. Our findings are pivotal for the development of next‐generation mRNA‐LNP therapies and vaccines.