with cells, [10] can shield recognition units, [15] but its role for biodistribution and circulation is still far from well understood. [8,16] Except for liposomes, other types of nano-sized drug delivery systems, such as polymeric micelles or more complex polymer constructs like cylindrical polymer brushes [17] have been so far hardly investigated with respect to protein corona formation (for comparison between the structures of these nanoparticles and the colloidal nanoparticles discussed above, see Figures S1 and S2 in the Supporting Information). [8,[18][19][20] However, the former are interesting, since polymeric micelles are in advanced stages of clinical testing (e.g., CPC634 (phase II) [21] and NC-6004 Nanoplatin (phase III) [22] ).To evaluate the formation of a protein corona, separation of the nanoparticle-protein-complex from unbound proteins used for incubation or upon in vivo exposure becomes a necessity. The isolation of incubated nanoparticles (colloids and inorganic nanoparticles) from unbound proteins was mainly performed by centrifugation, which is a separation method based on differences in density. [23] Thus, this method allows only the purification of nanoparticles with a higher density but can hardly be used for low density particles, such as polymeric micelles and polymer brushes. [24] Only recently, size exclusion techniques such as size exclusion chromatography and asymmetrical flow field-flow fractionation (AF4) have been employed for this purpose. [19,25,26] AF4 is a separation technique, which can be applied for the separation of particle and protein mixtures in the size range between 1 nm and 1 µm. [27,28] It consists of a separation channel with a flow gradient in which the particles are separated by an additional vertical force field depending on their diffusion coefficient. [29,30] During an AF4 measurement, the injected particles are pushed by the vertical cross flow toward the membrane at the bottom of the channel. Due to Brownian motion, the particles are diffusing back into the middle of the channel. Since smaller particles are faster than larger ones (because of their higher diffusion coefficient), they are concentrating faster in the middle, thus eluting first through the channel outlet. In contrast to conventional size exclusion chromatography, in AF4 the contact to the interface and the shear forces are substantially reduced, which leads to very mild separation conditions, minimizing perturbations of a potential protein corona. [26,31] Based on this method, Landfester and coworkers recently fully characterized the protein corona of Lutensol AT50-coated polystyrene nanoparticles and PEG functionalized liposomes, identifying all adsorbed proteins. [19,26] Here, we present a purification procedure based on AF4 for the separation of smaller polymeric architectures (R h : 20-30 nm) that are hardly separable by centrifugation. We isolated polymeric nanoparticles from unbound blood plasma components and characterized them by dynamic light scattering, gel electrophoresis and mass s...

A recent paper demonstrated that the formation of a protein corona is not a general property of all types of nanosized objects. In fact, it varies between a massive aggregation of plasma proteins onto the nanoparticle down to traces (e.g., a few proteins per 10 nanoparticles), which can only be determined by mass spectrometry in comparison to appropriate negative controls and background subtraction. Here, differences between various types of nanosized objects are discussed in order to determine general structure–property‐relations from a physico‐chemical viewpoint. It is highlighted that “not all nanoparticles are alike” and shown that their internal morphology, especially the difference between a strongly hydrated/swollen shell versus a sharp “hard” surface and its accessibility, is most relevant for biomedical applications.

Despite considerable progress in the design of multifunctionalized nanoparticles (NPs) that selectively target specific cell types, their systemic application often results in unwanted liver accumulation. The exact mechanisms for this general observation are still unclear. Here we asked whether the number of cell-targeting antibodies per NP determines the extent of NP liver accumulation and also addressed the mechanisms by which antibody-coated NPs are retained in the liver. We used polysarcosine-based peptobrushes (PBs), which in an unmodified form remain in the circulation for >24 h due to the absence of a protein corona formation and low unspecific cell binding, and conjugated them with specific average numbers (2, 6, and 12) of antibodies specific for the dendritic cell (DC) surface receptor, DEC205. We assessed the time-dependent biodistribution of PB−antibody conjugates by in vivo imaging and flow cytometry. We observed that PB− antibody conjugates were trapped in the liver and that the extent of liver accumulation strongly increased with the number of attached antibodies. PB−antibody conjugates were selectively captured in the liver via Fc receptors (FcR) on liver sinusoidal endothelial cells, since systemic administration of FcR-blocking agents or the use of F(ab′) 2 fragments prevented liver accumulation. Cumulatively, our study demonstrates that liver endothelial cells play a yet scarcely acknowledged role in liver entrapment of antibody-coated NPs and that low antibody numbers on NPs and the use of F(ab′) 2 antibody fragments are both sufficient for cell type-specific targeting of secondary lymphoid organs and necessary to minimize unwanted liver accumulation.

Most nanomaterials acquire a protein corona upon contact with biological fluids. The magnitude of this effect is strongly dependent both on surface and structure of the nanoparticle. To define the contribution of the internal nanoparticle structure, protein corona formation of block copolymer micelles with poly(N‐2‐hydroxypropylmethacrylamide) (pHPMA) as hydrophilic shell, which are crosslinked—or not—in the hydrophobic core is comparatively analyzed. Both types of micelles are incubated with human blood plasma and separated by asymmetrical flow field‐flow fractionation (AF4). Their size is determined by dynamic light scattering and proteins within the micellar fraction are characterized by gel electrophoresis and quantified by liquid chromatography‐high‐resolution mass spectrometry‐based label‐free quantitative proteomics. The analyses reveal only very low amounts of plasma proteins associated with the micelles. Notably, no significant enrichment of plasma proteins is detectable for core‐crosslinked micelles, while noncrosslinked micelles show a significant enrichment of plasma proteins, indicative of protein corona formation. The results indicate that preventing the reorganization of micelles (equilibrium with unimers) by core‐crosslinking is crucial to reduce the interaction with plasma proteins.

Fast and bioorthogonally reacting nanoparticles are attractive tools for biomedical applications such as tumor pretargeting. In this study, we designed an amphiphilic block copolymer system based on HPMA using different strategies to introduce the highly reactive click units 1,2,4,5-tetrazines (Tz) either at the chain end (Tz-CTA) or statistical into the hydrophobic block. This reactive group undergoes a rapid, bioorthogonal inverse electron-demand Diels–Alder reaction (iEDDA) with trans-cyclooctenes (TCO). Subsequently, this polymer platform was used for the preparation of different Tz-covered nanoparticles, such as micelles and colloids. Thereby it was found that the reactivity of the polymeric micelles is comparable to that of the low molar mass tetrazines. The core-cross-linked micelles can be successfully conjugated at rather low concentrations to large biomacromolecules like antibodies, not only in physiological buffer, but also in human blood plasma, which was confirmed by fluorescence correlation spectroscopy (FCS).