Introducing carbohydrate patterning in mannose-decorated supramolecular assemblies and hydrogels

Benzene-1,3,5-tricarboxamide (BTA) glyco-monomers containing one, two or three mannose units are synthesized and formulated into differently patterned supramolecular glycopolymers through homo-assembly or co-assembly with non-functionalized BTAs. Unfortunately, no cellular activity could be detected. Excitingly, these glyco-BTA monomers could be formulated into hydrogels, paving the way for (immune) cell culture.


Static Light Scattering (SLS): Measurements were performed on an ALV Compact Goniometer (CGS-3)
Multi-Detector (MD-4) equipped with an ALV-7004 Digital Multiple Tau Real Time Correlator and an Nd-YAG laser operating at a wavelength of 532 nm. The scattering intensity was detected over the angular range of 30−150° with steps of 5° or 10 °, with 10 runs of 10 s per angle. Samples were prepared at a 250 µM or 0.8 mM concentration of nBTA in MQ water, and measured in light scattering tubes with an outer diameter of 1 cm. As a reference, samples of only the corresponding solvent and only toluene were measured. Water was filtered with a 0.2 μm syringe filter (Supor membrane, PALL Corporation), and toluene was filtered with a 0.2 μm syringe filter (PTFE membrane, Whatman). The measurements were analyzed with AfterALV (1.0d, Dullware) to remove measurements showing obvious scattering from dust.
The Rayleigh ratio as a function of the angle was computed using the equation below with toluene as a Ultraviolet-visible (UV-vis) absorbance spectra: UV-vis spectra were recorded on and a Jasco V-650 UVvis spectrometer or a Jasco V-750 UV-vis spectrometer with a Jasco ETCT-762 temperature controller.
Transmission electron cryo-microscopy: Vitrified films were prepared in a 'Vitrobot' instrument (FEI Vitrobot TM Mark IV, FEI Company) at 22 °C and at a relative humidity of 100%. In the preparation chamber of the 'Vitrobot' samples were applied on grids, which were surface plasma treated just prior to use (Cressington 208 carbon coater operating at 5 mA for 40 s). Excess sample was removed by blotting using filter paper with a blotting force of -1, and the thin film thus formed was plunged (acceleration about 3 g) into liquid ethane just above its freezing point. Liquid samples were vitrified on Quantifoil grids (R 2/2, Quantifoil Micro Tools GmbH) by applying 3 µL of the sample and blotting away the sample for 3 s. Samples with a high viscosity were vitrified within 15 min after preparing the samples. Vitrified films were prepared by applying 2 µL of the sample on Lacey grids (LC200-Cu, Electron Microscopy Sciences) and blotting away the sample for 5 s. Due to the high viscosity of the sample not all material could be blotted away, resulting in a thick layer of vitrified water and only 10-20% of the grids could be imaged for those samples. Vitrified films were transferred into the vacuum of a CryoTITAN equipped with a field emission gun that was operated at 300 kV, a post-column Gatan energy filter with a slit width of 20 eV, and a 2048 x 2048 Gatan CCD camera. Vitrified films were observed in the CryoTITAN microscope at temperatures below -170 °C.
Micrographs were taken at low dose conditions, starting at a magnification of 6500 with a defocus setting of -40 µm, and at a magnification of 24000 with a defocus setting of -10 µm or -5 µm. The contrast was increased with 20% for all images. Total internal reflection fluorescence (TIRF) microscopy: TIRF images were acquired with a Nikon N-STORM microscopy system. Sample was excited using 561 nm laser. Fluorescence was collected using a Nikon×100, 1.4NA oil immersion objective and passed through a quad-band pass dichroic filter (97335 Nikon). Images were recorded with an EMCCD camera (ixon3, Andor, pixel size 0.17 μm). The samples were imaged in a μ-Slide 8 Well plate with No. 1.5 coverslip bottom suitable for microscopy.
Cell viability -resazurin assay: Resazurin assays were performed to assess whether the molecules showed toxic effects to the cells. To this end, RAW264.7 macrophages were seeded at a density of 5 × 10 4 cells/well in a 96-well plate, respectively, and were maintained under standard culturing conditions until cells were grown to 60-70% confluence. Next, the cells were grown in the presence of the UV-sterilized mannose BTAs in 100 µL fresh culture medium for an additional 24 h. To assess cell viability, 44 µM resazurin in fresh culture medium was added to the cells. The cells were incubated under standard culturing conditions for 3 h. Viable cells convert non-fluorescent resazurin into fluorescent resorufin (λex = 530 nm, λem = 590 nm), which was measured on a Synergy HT plate reader in 90 µL in triplicate per well. Each condition was tested 3 times. Background subtraction (i.e. wells containing only medium, without cells) was carried out for all measurements. Cell viability in percentage is presented relative to the viability of RAW264.7 macrophages that were cultured in untreated medium, which was set at 100% cell viability. Cells exposed to 0.5 v/v% Triton X-100 in phosphate buffered saline (PBS) were used as negative control.
Quantification of CD206 expression on cell membrane -flow cytometry: Cells were trypsinized, centrifuged at 300 rpm for 5 min and resuspended in PBS with 2 v/v% bovine serum albumin (BSA) to reduce non-specific binding. For the RAW264.7 macrophages (3.33 × 10 5 cells per sample), the cells were first blocked with 5 v/v% BSA in PBS to reduce non-specific binding. The sample of interest was incubated with an anti-mouse allophycocyanin (APC) primary anti-CD206 antibody (2 µg/mL) for 30 min at room temperature in PBS containing 2 v/v% BSA, while the isotype control sample was incubated with a rat, APC isotype control antibody (not specific for CD206) at similar concentrations to correct for non-specific binding of the antibody. The samples were centrifuged at 300 rpm for 5 min after each antibody incubation step and washed with PBS. Unstained cells were used to correct for background and autofluorescence of the cells. All antibodies were purchased from Biolegend. The obtained data was analyzed using Flow Jo software (Tree Star, Ashland, OR, USA).
Quantification of IL-10 cytokine release -ELISA: ELISA assays. To this end, RAW264.7 macrophages were seeded at a density of 5 × 10 4 cells/well in a 96-well plate. Simultaneously with cell seeding, UV-sterilized mannose BTAs, poly-mannose or LPS were added in a total of 100 µL culture medium, and the cells were maintained under standard culturing conditions for 24 h. After 24 h, 90 µL of the cell medium was collected and quantified for IL-10 using ELISA (Biolegend, IL-10 mouse Max Deluxe) according to the manufacturer's protocol. If necessary, the samples containing cell medium were stored at -80 °C until the ELISA experiment was performed. A standard curve was measured ranging from 0 -2000 pg/mL IL-10 and the data was fitted using a 2 nd degree polynomial fit. Non-stimulated, untreated cells were used as negative control and cells stimulated with LPS as positive control. 1 Internalization studies -fluorescent confocal microscopy: RAW 264.7 macrophages were seeded in Ibidi µ-angiogenesis slides prior to the experiment and allowed to adhere. Stock solutions of the mannose BTA assemblies containing BTA-Cy5 2 (5%) for visualization were prepared at 250 µM stock concentration and allowed to incubate overnight in the dark. The next day, the assemblies were added to the cells (5 µL with 45 µL medium) to obtain a final concentration of 25 µM BTAs. The cells with the BTA assemblies were incubated for 2 h at 37 °C with 5 % CO2. Next, the samples were washed with PBS, followed by fixation for 10 min with 3.7 v/v% formaldehyde and washing with PBS twice afterwards. Then, the cells were stained with a lysosomal (green) and cell (blue) mask for 1 h from Invitrogen according to the manufacturer's procedure. Finally, the samples were washed with PBS. Immediately thereafter, the samples were imaged Rheology: Rheological measurements of mannose BTA samples were carried out on a discovery hybrid rheometer (DHR-3, TA Instruments). Hydrogels were prepared according to the sample preparation method and scooped when possible or else pipetted (M3 BTA) onto the rheometer. Hydrogels were analyzed using a cone plate geometry (diameter=25 mm) at a gap height of 49 μm. All measurements were performed in duplo (except for the M1 BTA concentration series) and measured at room temperature. A solvent trap was used to minimize sample drying during the measurements. Strain sweep experiments were measured at strains ranging from 1 to 100%, at a constant frequency of 1 rad/s. Frequency sweeps were performed at frequencies ranging from 100 to 0.1 rad/s, at a constant strain of 1%. The self-healing experiment was performed 1 h after gel preparation. The gel was subjected to the following cycle: 1% strain at 1 rad/s for 50 s, 1000% strain at 1 rad/s for 50 s to achieve gel rupture; 1% strain at 1 rad/s until the gel was self-healed to its initial stiffness.      71.58, 71.55, 71.53, 71.39, 71.15, 68.63, 67.77, 62.96, 62.22, 41.23, 30.72, 30.69, 30.68, 30.58, 30.              The loss factor (tan(δ)), i.e. the ratio of viscous to elastic response, measured at different concentrations. B) Self-healing rheological experiment of M1 BTA hydrogel. The gel is first subjected to 1% strain, after which the strain is increased to 1000% to completely break the gel. After removing the extreme strain, the gel self-heals to its original stiffness. 1 rad/s is applied during the whole cycle. The BTA assemblies were exposed to the cells in complete culture medium for 24 h. The cell viability is normalized on cells that were maintained in untreated medium (i.e. medium control). Exposure of the cells to 0.5 v/v% Triton X-100 in PBS was used as a negative control.      S23. Representative graphs of ELISA assay to quantify the release of IL-10 in RAW 264.7 macrophages after treatment with different concentrations of homo-assemblies (top) and their co-assemblies (bottom) (50,000 RAW 264.7 macrophages/well in 96-well plate). The BTA assemblies were exposed to the cells in complete culture medium for 24 h. Untreated cells and wells containing only medium were both used as negative controls. Fig. S24. ELISA assay to quantify the release of IL-10 in RAW 264.7 macrophages after treatment with mannan (poly-mannose) and different LPS concentrations as controls (50,000 RAW 264.7 macrophages/well in 96-well plate). The BTA assemblies were exposed to the cells in complete culture medium for 24 h. Untreated cells and wells containing only medium were both used as negative controls.