Protein adsorption is required for stealth effect of poly(ethylene glycol)- and poly(phosphoester)- coated nanocarriers Susanne Schöttler 1,2 , Greta Becker 1 , Svenja Winzen 1 , Tobias Steinbach 1 , Kristin Mohr 1 , Katharina Landfester 1 , Volker Mailänder 1,2 * † and Frederik R. Wurm 1 * † 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. A dsorption of proteins from physiological fluids to nano- carriers leads to the formation of a protein shell 1,2 . This rapidly forming protein corona has previously been shown to be responsible for the biological fate of nanocarriers 3–5 . Poly- (ethylene glycol) (PEG) is widely used to suppress any non-specific protein adsorption, and PEGylated drugs and (nano)carriers show longer blood half-lives and less non-specific cellular uptake com- pared to unmodified drugs 6,7 . This is a prerequisite for specific targeting 8,9 . For surfaces, PEGylation is also the typical approach to reduce protein adsorption 10,11 . This feature, usually referred to as the ‘stealth’ effect, is generally explained by the high level of hydration of the hydrophilic polyether backbone, which also pre- vents protein adsorption on typically hydrophobic polymer surfaces by means of steric repulsion 12,13 . When the stealth properties of PEG are discussed it is often neglected that even if the overall protein adsorption is reduced, it cannot be fully suppressed; a certain amount of serum proteins, for example, is always detected on the drug carriers, thus altering the surface properties 14,15 . PEG is a non-biodegradable polyether and its accumulation in the body must be prevented. This is especially important when drug–PEG conjugates are administered for chronic diseases over a period of several years. In such cases, an accumulation of PEG in the body is likely, and can cause unwanted side effects 13 . Indeed, the development of PEG antibodies 16,17 and severe hypersensitivity reactions have been reported 18 . Antibody formation can lead to an accelerated blood clear- ance following repeated systemic administration 19 . Furthermore, PEG has been shown to trigger complement activation, which can lead to anaphylactic reactions in sensitive individuals 20 . These factors have given rise to a search for alternatives to PEG, and other biocompa- tible polymers have been recognized to improve the in vivo proper- ties of pharmaceuticals. For example, zwitterionic molecules such as polybetaines or polysaccharides can also generate hydrophilic shells when coupled to nanoparticles 21 . Degradable polymers that have been proposed as PEG alternatives include hydroxyethyl starch (HES), polysialic acid and dextrin 13 . However, another very interest- ing polymer class—the poly(phosphoester)s (PPEs)—has never been investigated with respect to stealth behaviour. In recent years, we have been studying PPEs with respect to novel synthetic protocols and biomedical applications 22,23 . The chemical structure of PPEs is highly modular, they are degradable, and their degra- dation products and time can be adjusted by means of precise chemistry 24,25 . Several studies have dealt with the preparation of PPE-based drug carriers, but none has investigated the protein interactions of the hydrophilic PPEs with blood proteins 26,27 . Three distinct findings are reported herein. First, the polymer- modified nanocarriers (with PEG and PPE) exhibit decreased protein adsorption after incubation in human plasma compared to unmodified particles. Second, and more importantly, mass spec- trometric analysis of the protein corona generated on the particles’ surface after plasma incubation revealed a similar pattern of proteins on PEGylated and ‘PPEylated’ surfaces. Clusterin—also termed apo- lipoprotein J (ApoJ)—was identified as a major component on both surfaces. Third, interestingly, a high non-specific cellular uptake for both polymer-modified nanocarriers was found without previous plasma incubation, indicating the requirement for distinct proteins to prevent non-specific cellular uptake. This non-specific cellular uptake could eventually be reduced by non-covalent ‘pre-loading’ with clusterin onto the nanocarriers, demonstrating a strong effect of reduced cell internalization. Unravelling the effects of protein type and stealth polymer structure will produce new efficient drug delivery devices. PEEP as novel stealth polymers The nanocarriers (Fig. 1) used in this study were monodisperse polystyrene nanoparticles with diameters of ∼100 nm, which were prepared by free radical terpolymerization of three monomers 1 Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany. 2 Johannes Gutenberg University Mainz, University Medical Center, Department of Dermatology, Langenbeckstr. 1, 55131 Mainz, Germany. † These authors jointly supervised this work. *e-mail: mailaender@mpip-mainz.mpg.de; wurm@mpip-mainz.mpg.de ARTICLES PUBLISHED ONLINE: 15 FEBRUARY 2016 | DOI: 10.1038/NNANO.2015.330 NATURE NANOTECHNOLOGY | ADVANCE ONLINE PUBLICATION | www.nature.com/naturenanotechnology 1 © 2016 Macmillan Publishers Limited. All rights reserved