Noncovalent Targeting of Nanocarriers to Immune Cells with Polyphosphoester‐Based Surfactants in Human Blood Plasma

Abstract Dendritic cells (DCs) are part of the immune system and can internalize pathogens by carbohydrate receptors. The uptake induces maturation and migration of the DCs resulting in an adaptive immune response by presenting antigens to T‐cells. Thus, targeted delivery to DCs is a powerful tool for immunotherapy. However, in blood, specific targeting is challenging as blood proteins adsorb to the nanocarriers and mask the targeting molecules. Additionally, covalent coupling of targeting groups to nanocarriers requires new chemistry for each nanocarrier, while a general strategy is missing. A general protocol by noncovalent adsorption of mannosylated polyphosphoesters (PPEs) on the nanocarriers' surface resulting in specific uptake into DCs combined with low protein adsorption of PPEs is presented. PPEs with hydrophobic anchors and multiple mannose units are reported and adsorbed to different model nanocarriers. Their protein corona remain similar to pure stealth nanocarriers and prove only low uptake into nontargeted cells (monocytes). Due to the “stealth” properties of PPEs, a high specific uptake into DCs is achieved after incubation in human blood plasma, proving an efficient combination of “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.


Material and methods
General Information. All chemicals and solvents were purchased from Sigma-Aldrich, Acros Organics or Fluka and used as received unless otherwise stated. Deuterated solvents were purchased from Sigma-Aldrich and used as received. The eluents for column chromatography (cyclohexane and ethyl acetate) were distilled prior to use. Deutero chloroform was stored over alumina (Brockmann activity I). The HSQC sepctra are phase-sensitive (opposite signs for CH/CH 3 and CH 2 ).The 1 H and 13 C chemical shifts (δ) were referenced to the residual solvent signal as internal standard (DMSO-d 6 : δ = 2.50 ppm and 39.52 ppm, CD 3 OD: δ = 3.31 ppm and 49.00 ppm for 1 H and 13 C NMR, respectively). [1] Coupling constants (J) are reported in Hz (splitting abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad; and combinations thereof). 1 H, 13 C and 31 P NMR spectra of mannose derivative (3) and all polymers were recorded using a Bruker Avance III 250, a Bruker Avance 300 or a Bruker Avance III 500 and processed with MestReNova. All 31 P NMR spectra were recorded decoupled from protons. All spectra were referenced internally to residual proton signals of the deuterated solvent. 2D spectra were processed with the topspin 3.5 software.
For the diffusion measurements (DOSY) [2] a 5 mm triple resonance BBFO 1 H/X probe equipped with a z-gradient on the 500 MHz Bruker AVANCE III system was used. For the diffusion measurements a 2D sequence (DOSY, dstebpgp3s) with a stimulated echo was used. [3] The temperature was kept at 298.3 K and regulated by a standard 1 H methanol NMR sample using the topspin 3.5s software (Bruker). The control of the temperature was realized with a VTU (variable temperature unit) and an accuracy of +/-0,1K. The diffusion time was 30-70 ms and the gradient length to 1.4 ms. The relaxation delay between scans was 1.5-2 s. The gradient strength was calibrated by analysis of a sample of 2H 2 O/1H 2 O at a defined temperature and comparison with the theoretical diffusion coefficient of Cell culture. Monocytes and monocyte derived dendritic cells were isolated from human buffy coats according to the vote of the local ethics committee and the Declaration of Helsinki as previously reported [4] . Briefly, peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll separation and plated into 6-well plates in DC-medium (RPMI + 2% human serum, 100 IU/mL penicillin, 100 μg/mL streptomycin). Cells were incubated for 1 h at 37 °C and non-adherent cells were further discarded by washing the plates with PBS (3-4 times).
Adherent cells were either directly frozen and further used for monocyte experiments or differentiated into monocyte derived dendritic cells (moDCs). For generate moDCs, cells were incubated with 800 IU/mL granulocyte-macrophage colony-stimulating factor (GMCSF) and 500 IU/mL interleukin IL4 (both PromoCell). Afterwards 48 h the medium was refreshed with 1600 IU/mL GMCSF and 500 IU/mL IL4 and incubation for 72 h was followed. MoDCs were detached from the wells with 0.5 mM PBS-EDTA solution.
Surface marker. 100 000 cells were resuspended in 100 µL PBS and the following antibodies were used to label the surface marker for the mannose receptor (APC anti-human CD206 MMR Antibody (Biolegend) and APC anti-human CD209 MMR Antibody (Biolegend)).  Statistical analysis: An unpaired student´s t-test assuming equal variances was performed.
The p values were defined as followed: *p < 0.05, **p < 0.01, ***p < 0.001. Not significant differences are labelled as n.s. In solution digestion. To remove SDS, protein solutions were applied to Pierce detergent removal columns (Thermo Fisher). Digestion was performed to previously estabilished 6 protocols. [5], [6] A protein:trypsin ratio of 50:1 was chosen and the reaction was carried out over 14 h at 37°C.

Liquid chromatography coupled to mass spectrometry (LC-MS analysis).
Peptide samples were diluted with with 0.1% formic acid. Hi3 Ecoli was added at a concentration of 50 fmol/µL for absolute protein quantification [7] . Protein identification. To identify proteins, a reviewed human data base (Uniprot) was downloaded and analysis was carried out with Progenesis QI (2.0). Parameters for protein identification were set as described before [8] . The human data base was modified with the sequence information of Hi3 Ecoli standard for absolute protein quantification. The amount of each protein in fmol was obtained via the TOP3/Hi3 approach [9] .
The mixture was filtered over a bed of Celite, which was washed thoroughly with toluene.