German Edition: DOI: 10.1002/ange.201502398 Nanocarriers International Edition: DOI: 10.1002/anie.201502398 Carbohydrate-Based Nanocarriers Exhibiting Specific Cell Targeting with Minimum Influence from the Protein Corona** Biao Kang, Patricia Okwieka, Susanne Schçttler, Svenja Winzen, Jens Langhanki, Kristin Mohr, Till Opatz, Volker Mailänder, Katharina Landfester,* and FrederikR. Wurm* Abstract: Whenever nanoparticles encounter biological fluids like blood, proteins adsorb on their surface and form a so- called protein corona. Although its importance is widely accepted, information on the influence of surface functional- ization of nanocarriers on the protein corona is still sparse, especially concerning how the functionalization of PEGylated nanocarriers with targeting agents will affect protein corona formation and how the protein corona may in turn influence the targeting effect. Herein, hydroxyethyl starch nanocarriers (HES-NCs) were prepared, PEGylated, and modified on the outer PEG layer with mannose to target dendritic cells (DCs). Their interaction with human plasma was then studied. Low overall protein adsorption with a distinct protein pattern and high specific affinity for DC binding were observed, thus indicating an efficient combination of “stealth” and targeting behavior. Nanomedicine is a key technology for the 21 st century. Besides the initial development of various nanocarrier systems and the development of specific targeting, the development of protein-repellent surfaces is of high impor- tance. When synthetic nanocarriers enter biological fluids, it is known that, due to high surface energy and hydrophobic interactions, they strongly adsorb plasma proteins. [1] Many researchers have proposed that the in vivo fate of any nanocarrier is determined by this protein “corona”, formed post injection, instead of the intrinsic properties of the (mostly polymeric) nanocarrier. [2] PEGylation (PEG = poly- ethylene glycol) is the state-of-the-art approach to reducing nonspecific interactions with plasma proteins; this effect is often termed the “stealth effect”. [3] However, the stealth effect alone is not enough : specific targeting agents have to be attached additionally in order to reach the binding target. [1a] But how does this additional moiety interact with plasma proteins? A new corona could be generated, thereby altering the in vivo performance by covering and deactivating the targeting group. The formation of a protein corona around single-compo- nent nanoparticles (NPs), such as polystyrene, [2c,g, 4] zinc oxide, [5] silica, [2g, 5, 6] gold, [2o, 7] silver, [2o] and titanium dioxi- de [2n, 5] nanoparticles, has been extensively studied, while the surface modification of nanoparticles with PEG [8] and zwit- terionic agents [9] has been proven to effectively reduce the protein absorption. However, the combination of “stealth” behavior with “on top” attachment of specific targeting groups has not been studied. The protein interactions of a PEGylated nanocarrier before and after the attachment of an additional targeting group are of central importance for the generation of efficient specific cellular uptake after blood contact. For the first time, we compare the blood plasma interaction of PEGylated nanocarriers with that of nano- carriers that carry mannose groups attached to the PEG chains. Isothermal titration calorimetry (ITC), dynamic light scattering (DLS), and cellular uptake studies before and after incubation with human plasma were performed and the influence on the targeting of dendritic cells was evaluated. In addition, proteomic mass spectrometry revealed a distinct pattern of plasma proteins still present on all of the nano- carriers that does not hamper the specific lectin binding of mannose. We have been studying hydroxyethyl starch nanocapsules (HES-NCs) as biodegradable nanocarriers intensively. [10] They are prepared through an inverse miniemulsion method, can be loaded with hydrophilic cargo, and their diameters can be adjusted precisely. More recently, well- controlled PEGylation of HES-NCs was accomplished through a number of different methods. [11] Herein, we extend these methods to generate nanocarriers that can be further functionalized at the outer layer with specific target- ing groups. PEG diisocyanate (OCN-PEG 110 -NCO, M n = 5000 g mol À1 ) reacts with the surface hydroxy groups of the polysaccharide and subsequently with the targeting groups. After one of the isocyanate groups has reacted with the surface, the reactivity of the other one is drastically decreased owing to steric hindrance and loss of mobility. If an excess of PEG diisocyanate is used, there is a further reduction in the formation of cyclic species and the second isocyanate group [*] B. Kang, P. Okwieka, S. Schçttler, S. Winzen, Dr. K. Mohr, Dr. V. Mailänder, Prof.Dr. K. Landfester, Dr. F.R. Wurm Max Planck Institute for Polymer Research Ackermannweg 10, 55128 Mainz (Germany) E-mail: landfester@mpip-mainz.mpg.de wurm@mpip-mainz.mpg.de P. Okwieka, Dr. V. Mailänder Department of Hematology, Medical Oncology, and Pneumology University Medical Center Mainz Langenbeckstr. 1, 55131 Mainz (Germany) Dipl.-Chem. J. Langhanki, Prof. Dr. T. Opatz Institute of Organic Chemistry, University of Mainz Duesbergweg 10–14, 55128 Mainz (Germany) [**] Financial support by the BMBF (Cluster CI3) and the DFG (Colaborative Research Projct SFB1066) is highly appreciated. We would like to thank Christine Rosenauer for the DLS measurement, and Katja Klein for the synthesis of polystyrene nanoparticles used in this study. F.R.W. thanks the Max Planck Graduate Center for support. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201502398. A ngewandte Chemi e 1 Angew. Chem. Int. Ed. 2015, 54,1–6  2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim These are not the final page numbers! Ü Ü