For nanocarriers with low protein affinity, we show that the interaction of nanocarriers with cells is mainly affected by the density, the molecular weight, and the conformation of polyethylene glycol (PEG) chains bound to the nanocarrier surface. We achieve a reduction of nonspecific uptake of ovalbumin nanocarriers by dendritic cells using densely packed PEG chains with a “brush” conformation instead of the collapsed “mushroom” conformation. We also control to a minor extent the dysopsonin adsorption by tailoring the conformation of attached PEG on the nanocarriers. The brush conformation of PEG leads to a stealth behavior of the nanocarriers with inhibited uptake by phagocytic cells, which is a prerequisite for successful in vivo translation of nanomedicine to achieve long blood circulation and targeted delivery. We can clearly correlate the brush conformation of PEG with inhibited phagocytic uptake of the nanocarriers. This study shows that, in addition to the surface’s chemistry, the conformation of polymers controls cellular interactions of the nanocarriers.

Albumin-based protein nanocarriers obtained by TAD click chemistry have been widely exploited as drug delivery systems, since they show excellent degradability, low toxicity, but at the same time provide high loading capacity and relevant uptake into cells.

When nanoparticles (NPs) are introduced to a biological fluid, different proteins (and other biomolecules) rapidly get adsorbed onto their surface, forming a protein corona capable of giving to the NPs a new "identity" and determine their biological fate. Protein-nanoparticle conjugation can be used in order to promote specific interactions between living systems and nanocarriers. Non-covalent conjugates are less stable and more susceptible to desorption in biological media, which makes the development of engineered nanoparticle surfaces by covalent attachment an interesting topic. In this work, the surface of poly(globalide-co-ε-caprolactone) (PGlCL) nanoparticles containing double bonds in the main polymer chain is covalently functionalized with bovine serum albumin (BSA) by thiol-ene chemistry, producing conjugates which are resistant to dissociation. The successful formation of the covalent conjugates is confirmed by flow cytometry (FC) and fluorescence correlation spectroscopy (FCS). Transmission electron microscopy (TEM) allows the visualization of the conjugate formation, and the presence of a protein layer surrounding the NPs can be observed. After conjugation with BSA, NPs present reduced cell uptake by HeLa and macrophage RAW264.7 cells, in comparison to uncoated NP. These results demonstrate that it is possible to produce stable conjugates by covalently binding BSA to PGlCL NP through thiol-ene reaction.

be observed by electron microscopy (EM). Correlative light and electron microscopy (CLEM) combine the strengths of both techniques and contribute to a precise localization of the nanocarriers in the cell. Using fluorescent nanodiamonds, for example, can serve as a fluorescent marker [11,12] These markers have the advantage of being biocompatible and can be easily modified to achieve a certain surface functionalization. [13] CLEM preparation ideally features a fluorophore with a stable inherent fluorescence suitable for confocal laser scanning microscopy (cLSM) which, in combination, has a contrasting component suitable for transmission electron microscopy (TEM). Another approach is using inorganic, fluorescent nanoparticles. Quantum dots are the commonly used fluorescent markers for intracellular tracking, but they are not as bright as NPLs for both one photon and two photon excitation. [19] Moreover, due to their small size and spherical shape it can be challenging to identify quantum dots-but also the larger, rectangular NPLs-in a cellular environment by TEM. [14,15] This might involve additional elemental analysis (e.g., energy dispersive x-ray spectroscopy [EDS] or electron energy loss spectroscopy [EELS]) to confirm the identity of the quantum dots to ultimately locate the nanocarrier. [16][17][18] Here, we develop a model system consisting of an organic nanocapsule (NC) labeled with a fluorescent labeling system. As fluorescent marker we utilize large (20-50 nm), rectangular CdSe-CdZnS nanoplatelets (NPLs). We encapsulated these NPLs into biocompatible NCs by a polyaddition reaction at the droplet interface in an inverse miniemulsion. We used NCs made of bovine serum albumin (BSA), crosslinked at the interface with toluene diisocyanate (TDI), forming a dense polymeric shell. [6,7] The NPLs were added to the aqueous dispersed phase during the miniemulsion process, leading to the encapsulation of the NPLs into the NCs. The key to success of the NPL markers hinged on the protective coating on the NPLs. [19] This coating makes NPLs both easy to disperse in aqueous medium and protects the NPLs' surface from major damage during the encapsulation process, thus leading to high fluorescence after encapsulation. The potential toxicity of CdSe nanoparticles is not an issue at this stage. The fluorescent marker can either be easily replaced or even omitted completely.To follow the intracellular pathway of the protein NCs, RAW264.7 macrophages were incubated with these NCs followed by different techniques like flow cytometry, cLSM, and This work analyzes the intracellular fate of protein-based nanocarriers along their endolysosomal pathway by means of correlative light and electron microscopy methods. To unambiguously identify the nanocarriers and their degradation remnants in the cellular environment, they are labeled with fluorescent, inorganic nanoplatelets. This allows tracking the nanocarriers on their intracellular pathway by means of electron microscopy imaging. From the present data, it is possible to identify d...