The authors thank Dr. uptake into DCs is achieved after incubation Deoxygalactonojirimycin HCl in human blood plasma, proving an efficient combination of Deoxygalactonojirimycin HCl stealth and targeting after simple adsorption of the PPEs. This strategy can transform any nanocarrier into DC\targeting by noncovalent adsorption of PPEs and will aid in developing novel immunotherapies. 1.5 and molar masses between 5200 and 9000 g mol?1 (Table S1, Supporting Information). Phos\S(2) and Phos\S(3) were reacted with mercaptopropyl\\d\mannopyranoside (2), resulting in the mannosylated amphiphiles Phos\S(2)\Man ( 0.001***). These experiments clearly prove that the prepared PPE increased a receptor\mediated uptake of nanocarriers into DCs. As a complementary approach, the mannose receptor was blocked with soluble antibodies (Figure S29, Supporting Information). If cells were treated with antibodies prior to uptake analysis, cellular interactions of NCs coated with mannose PPE amphiphiles were also reduced, proving the receptor\mediated uptake of mannose\PPEylated NCs. The previous cell experiments confirmed the targeting under artificial conditions in cell culture medium, but in the absence blood plasma proteins. The loss of specific targeting after blood incubation had been reported as plasma proteins tend to bury the targeting groups.[qv: 6a,13] Also in our case, after incubation into human blood plasma, the cellular uptake of the nanocarriers was influenced by the protein corona: Flow cytometry experiments indicated that PS nanocarriers, which were coated with both mannosylated amphiphiles, exhibited a lower cell internalization after the protein corona formation compared to the protein\free scenario (Figure 4 b). This effect was more pronounced for Phos\S(3)\Man compared to Phos\S(2)\Man, probably due to the overall stronger cell uptake Deoxygalactonojirimycin HCl in the former case ( 0.001***). However, a significantly higher amount of the mannosylated NCs was still selectively taken up into the DCs as compared to Phos\S(1)\coated NCs. The low protein adsorption onto PPEylated surfaces still guaranteed access to the mannose units and resulted in a cellular uptake into the DCs. As PPEs were recently identified by our group as potential stealth polymers that reduce unspecific cell uptake by selected adsorption of lipoproteins,22 we conducted a detailed proteomic analysis of the mannose\modified PPEylated NCs. All identified proteins were classified into eight different classes depending on their biological function (Figure ?(Figure4c,d).4c,d). In general, the amount of adsorbed protein and the overall corona composition were very similar for the mannosylated and non\mannosylated NCs (Figure S30, Supporting Information). The corona of Phos\S(3)\coated PS NCs was enriched with lipoproteins (19%) compared to NCs coated with Phos\S(1) (9%) or Phos\S(2) (13%) (Figure ?(Figure3d,3d, and Figures S31CS33, Supporting Information). In previous studies, we found that lipoproteins, in particular clusterin, interacted with PEGylated and PPEylated NCs and reduced the cellular interactions with macrophages.[qv: 22a] Their presence in the corona explains the reduced uptake Rabbit polyclonal to ZW10.ZW10 is the human homolog of the Drosophila melanogaster Zw10 protein and is involved inproper chromosome segregation and kinetochore function during cell division. An essentialcomponent of the mitotic checkpoint, ZW10 binds to centromeres during prophase and anaphaseand to kinetochrore microtubules during metaphase, thereby preventing the cell from prematurelyexiting mitosis. ZW10 localization varies throughout the cell cycle, beginning in the cytoplasmduring interphase, then moving to the kinetochore and spindle midzone during metaphase and lateanaphase, respectively. A widely expressed protein, ZW10 is also involved in membrane traffickingbetween the golgi and the endoplasmic reticulum (ER) via interaction with the SNARE complex.Both overexpression and silencing of ZW10 disrupts the ER-golgi transport system, as well as themorphology of the ER-golgi intermediate compartment. This suggests that ZW10 plays a criticalrole in proper inter-compartmental protein transport Deoxygalactonojirimycin HCl and indicates that corona proteins can shield the targeting ligand, but do not suppress the interaction with the mannose receptor. Interestingly, the internalization of the PMMA NCs seemed to be relatively unaltered in the presence of blood proteins (Figure ?(Figure4a).4a). We even detected an enhanced cellular uptake ( 0.001***) for PMMA NCs coated with Phos\S(2)\Man after corona formation. This effect can be explained by the distinct corona composition. We detected a higher amount of immunoglobulins (15%) on the surface of Phos\S(2)\Man\coated NCs compared to Phos\S(1)\Man (6%) or Phos\S(3)\Man ones (9%). Immunoglobulins are referred to Deoxygalactonojirimycin HCl the class of opsonins, which are known to enhance interaction with immune cells.23 Therefore, the interaction of Phos\S(2)\Man\coated PMMA NCs and moDCs could be favored. Open in a separate window Figure 4 a,b) Cell interactions of NCs after blood incubation: PPE\coated PS or PMMA NCs were exposed to human blood plasma and the cellular uptake (150 g mL?1, 2 h) toward dendritic cells was quantified via flow cytometry. Values are expressed as mean SD from triplicates. c,d) Protein corona analysis from mass spectrometry based proteomics. All identified proteins were classified into eight different protein classes depending on their biological function. A full list of all detected proteins is.