Susanne Schöttler

Protein adsorption is required for stealth effect of poly(ethylene glycol)- and poly(phosphoester)-coated nanocarriers

The current gold standard to reduce non-specific cellular uptake of drug delivery vehicles is by covalent attachment of poly(ethylene glycol) (PEG). It is thought that PEG can reduce protein adsorption and thereby confer a stealth effect. Here, we show that polystyrene nanocarriers that have been modified with PEG or poly(ethyl ethylene phosphate) (PEEP) and exposed to plasma proteins exhibit a low cellular uptake, whereas those not exposed to plasma proteins show high non-specific uptake. Mass spectrometric analysis revealed that exposed nanocarriers formed a protein corona that contains an abundance of clusterin proteins (also known as apolipoprotein J). When the polymer-modified nanocarriers were incubated with clusterin, non-specific cellular uptake could be reduced. Our results show that in addition to reducing protein adsorption, PEG, and now PEEPs, can affect the composition of the protein corona that forms around nanocarriers, and the presence of distinct proteins is necessary to prevent non-specific cellular uptake.

Protein Corona of Nanoparticles: Distinct Proteins Regulate the Cellular Uptake

Understanding nanoparticle-protein interactions is a crucial issue in the development of targeted nanomaterial delivery. Besides unraveling the composition of the nanoparticles protein coronas, distinct proteins thereof could control nanoparticle uptake into specific cell types. Here we differentially analyzed the protein corona composition on four polymeric differently functionalized nanoparticles by label-free quantitative mass spectrometry. Next, we correlated the relative abundance of identified proteins in the corona with enhanced or decreased cellular uptake of nanoparticles into human cancer and bone marrow stem cells to identify key candidates. Finally, we verified these candidate proteins by artificially decorating nanoparticles with individual proteins showing that nanoparticles precoated with the apolipoproteins ApoA4 or ApoC3 significantly decreased the cellular uptake, whereas precoating with ApoH increased the cellular uptake.

Super liquid-repellent gas membranes for carbon dioxide capture and heart–lung machines

In a gas membrane, gas is transferred between a liquid and a gas through a microporous membrane. The main challenge is to achieve a high gas transfer while preventing wetting and clogging. With respect to the oxygenation of blood, haemocompatibility is also required. Here we coat macroporous meshes with a superamphiphobic—or liquid repellent—layer to meet this challenge. The superamphiphobic layer consists of a fractal-like network of fluorinated silicon oxide nanospheres; gas trapped between the nanospheres keeps the liquid from contacting the wall of the membrane. We demonstrate the capabilities of the membrane by capturing carbon dioxide gas into a basic aqueous solution and in addition use it to oxygenate blood. Usually, blood tends to clog membranes because of the abundance of blood cells, platelets, proteins and lipids. We show that human blood stored in a superamphiphobic well for 24 h can be poured off without leaving cells or adsorbed protein behind.

Controlling the Stealth Effect of Nanocarriers through Understanding the Protein Corona.

The past decade has seen a significant increase in interest in the use of polymeric nanocarriers in medical applications. In particular, when used as drug vectors in targeted delivery, nanocarriers could overcome many obstacles for drug therapy. Nevertheless, their application is still impeded by the complex composition of the blood proteins covering the particle surface, termed the protein corona. The protein corona complicates any prediction of cell interactions, biodistribution, and toxicity. In particular, the unspecific uptake of nanocarriers is a major obstacle in clinical studies. This Minireview provides an overview of what we currently know about the characteristics of the protein corona of nanocarriers, with a focus on surface functionalization that reduces unspecific uptake (the stealth effect). The ongoing improvement of nanocarriers to allow them to meet all the requirements necessary for successful application, including targeted delivery and stealth, are further discussed.

Aggregation Behavior of Polystyrene-Nanoparticles in Human Blood Serum and its Impact on the in vivo Distribution in Mice

The interactions between nanoparticles (NPs) and proteins in complex biological application media such as blood serum are capable of inducing aggregate formation which can lead to subsequent changes in biological activity. Here, we correlate surface charge, aggregation-tendency, and surface serum protein adsorption with cellular uptake and biodistribution in mice. Polystyrene-based NPs (80 - 170 nm) with different surface functionalizations were synthesized and incubated with human serum. Interaction of NPs with serum proteins and aggregate formation were analyzed by mass spectrometryanalysis and dynamic light-scattering. Influence of surface functionalization on specific cellular uptake and organdistribution was characterized. Localization and organ targeting of intravenously applied NPs preferentially depended on their aggregationbehavior in the presence of serum. Whereas strongly aggregating particles mainly located to liver, non-aggregating particles distributed to all organs. Determination of aggregate formation of NPs in the presence of serum and further analysis of the protein corona allows for pre-selection of NPs for in vivo application.

Protein corona composition of poly(ethylene glycol)- and poly(phosphoester)-coated nanoparticles correlates strongly with the amino acid composition of the protein surface

Extensive molecular dynamics simulations reveal that the interactions between proteins and poly(ethylene glycol) (PEG) can be described in terms of the surface composition of the proteins. PEG molecules accumulate around non-polar residues while avoiding the polar ones. A solvent-accessible-surface-area model of protein adsorption accurately fits a large set of data on the composition of the protein corona of poly(ethylene glycol)- and poly(phosphoester)-coated nanoparticles recently obtained by label-free proteomic mass spectrometry.

Polyurethane Dispersions with Peptide Corona: Facile Synthesis of Stimuli-Responsive Dispersions and Films

Peptide-polymer hybrid particles of submicron size yielding stimuli-responsive macroscopic films are presented. A thermoplastic polyurethane (PU) carrying polysiloxane and polyester soft segments serves as core material to obtain flexible, yet semicrystalline films with temperature-sensitivity. The synthesis is based on the high-sheer emulsification of isocyanate-terminated PU prepolymers, which in our model system purposefully lack any ability of colloidal self-stabilization. While emulsification in water leads to immediate coagulation, stable dispersions of polyurethane nanoparticles were formed in aqueous solutions of a hydrolyzed protein from wool. A comparison of dispersion and film properties to nonreactive, otherwise identical dispersions suggests covalent attachment of the peptide to the PU backbone. We show that the colloidal stability of the hybrid particles is completely governed by the peptide corona, and hence pH-triggered coagulation can be employed to induce particle deposition and film formation. Differential scanning calorimetry confirms partial crystallinity in the film and reveals strongly modified crystallization behavior due to the peptide.

Protein machineries defining pathways of nanocarrier exocytosis and transcytosis

The transport of nanocarriers through barriers like the gut in a living organism involves the transcytosis of these nanocarriers through the cell layer dividing two compartments. Understanding how this process works is not only essential to further developing strategies for a more effective nanocarrier transport system but also for providing fundamental insights into the barrier function as a means of protection against micro- and nanoplastics in the food chain. We therefore set out to investigate the different uptake mechanisms, intracellular trafficking and the routes for exocytosis for small polystyrene nanoparticles (PS-NPs ca. 100 nm) as mimicking nanocarriers in a Caco-2 cell model for gut-blood transition. We used label-free, quantitative mass spectrometry (MS) for determining the proteome that adhered to transversed nanoparticles. From this rich proteomics dataset, as well as previous studies, we generated stable-transfected Caco-2 cell lines carrying the green fluorescent protein (GFP) coupled to proteins of interest for uptake, early, late and exocytotic endosomes. We detected the spatial and temporal overlap of such marked endosomes with the nanocarrier signal in confocal laser scanning and super-resolution microscopy. There was a clear distinction in the time course of nanoparticle trafficking between groups of proteins for endocytosis, intracellular storage and putatively transcytosis and we identified several key transcytotic markers like Rab3 and Copine1. Moreover, we postulate the necessity of a certain protein composition on endosomes for successful transcytosis of nanocarriers. Finally, we define the two-sided impasse of the lysosome as a dead end for nano-plastic and the limit of nanocarriers in the 100 nm range. STATEMENT OF SIGNIFICANCE Here we focus on mechanisms of transcytosis and how we can follow these with methods not used before. First, we use mass spectrometry of transcytosed nanoparticles to pick proteins of the transcytosis machinery describing key proteins involved. We can detect the complex mixtures of proteins. As this is a dynamic process involving whole families of proteins interacting with each other and as this is an orchestrated process we coined the term protein machineries for this active interplay. By genetically modifying the proteins attaching GFP we are able to follow the transcytosis pathway. We evaluate the process in a quantitative manner over time. This reveals that the most obvious obstacle to transcytosis is a routing of the nanocarriers to the lysosomes.