Principles of glycocalyx engineering with hydrophobic-anchored synthetic mucins

The cellular glycocalyx is involved in diverse biological phenomena in health and disease. Yet, molecular level studies have been challenged by a lack of tools to precisely manipulate this heterogeneous structure. Engineering of the cell surface using insertion of hydrophobic-terminal materials has emerged as a simple and efficient method with great promise for glycocalyx studies. However, there is a dearth of information about how the structure of the material affects membrane insertion efficiency and resulting density, the residence time of the material, or what types of cells can be utilized. Here, we examine a panel of synthetic mucin structures terminated in highly efficient cholesterylamide membrane anchors for their ability to engineer the glycocalyx of five different cell lines. We examined surface density, residence time and half-life, cytotoxicity, and the ability be passed to daughter cells. We report that this method is robust for a variety of polymeric structures, long-lasting, and well-tolerated by a variety of cell lines.


Fluorophore labeling
AZDye 594 NHS ester was dissolved at 10 mg/mL in dimethylsulfoxide. Polymer was dissolved at the maximum concentration for solubility in MilliQ water. An aliquot from the polymer stock solution was transferred to a 4 mL vial equipped with a stir bar. A 1 M stock solution of NaHCO3 was used to adjust the polymer solution in the 4 mL vial to a 0.2 M NaHCO3 concentration. A volume of the AZDye 594 NHS ester solution was added to the 4 mL vial corresponding to 5 equivalents of fluorophore per polymer chain. The solution was shielded from light and allowed to react overnight. The following day, unreacted fluorophore and salts were removed by spin filtration using Amicon Ultra-15 MWCO 3kDa filters and spun down 5 times, diluting in MilliQ water each time.

Fluorophore labeling efficiency determination
To determine the fluorophore labeling efficiency, labeled polymers were dissolved at 100 µM in MilliQ water and analyzed on a Nanodrop 2000 Spectrophotometer using the proteins and labels procedure. The spectrophotometer determines the concentration of the selected fluorophore. For example, if the spectrophotometer determines the [AZDye 594] as 55.0 µM, the labeling efficiency would be reported as 55%.

General glycocalyx engineering procedure for imaging
All imaging studies were conducted with adherent cell types. First, AZDye 594-labeled polymers were dissolved in complete adherent cell media at 10 µM and sterile filtered through a 0.2 µm membrane. Cells were trypsinized and neutralized with complete media according to ATCC guidelines. 100,000 cells/sample were pelleted by centrifugation at 100 xg for 5 minutes. Cells were suspended at 10 6 cells/mL in polymer-free media as a mock-engineered control or media containing polymer. Cells were incubated, covered, at room temperature for one hour. Post-incubation, cells were pelleted, washed with PBS, resuspended in 500 µL complete media, and plated on a 24-well plate. All cells were left to grow at 37 °C. At timepoints 24, 48, 72, and 96 hours following polymer treatment, cells were imaged with a brightfield/fluorescent microscope. Separate wells of treated and control cells were also nuclear-stained with Hoescht 33342 24 hours post-incubation and imaged. Studies were run in duplicate, with n = 4 for image analysis. Fluorescence and cell margins (from brightfield images) were measured with ImageJ via thresholding and pixel quantification. AZDye TM 594 fluorescence was normalized to # of cells (via cell margin quantification) and plotted against time. An exponential decay fit was applied in Excel and used to estimate the half-life. Natural log plots were also created in Excel to evaluate the exponential fit.

General glycocalyx engineering procedure for flow cytometry
Flow cytometry was used to conduct: 1) concentration studies in HEK 293 cells to determine ability to control cell surface density, 2) persistence studies with suspension cell types (i.e. Raji and Jurkat), 3) density studies analyzed post-polymer incubation with both suspension and adherent cells, and 4) wash studies in Raji cells with 50% glycosylated 26mer and 92mer (see Supplementary Figure 17). AZDye 594-labeled polymers were dissolved in complete media, at 10 µM for most studies, and sterile filtered through a 0.2 µm membrane. To correlate polymer concentration to density on membrane, concentrations of 1, 2, 5, 10, 25, and 50 µM were prepared. At this time, cells were trypsinized if necessary. Cells were pelleted and suspended in media +/-polymer at 10 7 cells/mL. Cells were incubated, covered, at room temperature for one hour. Post-incubation, cells were pelleted, washed with PBS (and washed further for wash studies), and either resuspended in PBS for immediate flow analysis or in complete media for expansion and later flow preparation. To examine persistence, every 24 hours, expanded cells were counted and 0.5-110 6 cells/sample were pelleted, washed once with PBS, and resuspended in PBS for flow analysis. DAPI (0.1 µg/mL from ThermoFisher # 62247) was added as a live/dead discriminator. Flow cytometry data was acquired with at least 10,000 events for all samples. The gating tree was as follows: 1) FSC/SSC to exclude debris, 2) singlet gate (FSC-height vs. FSC-area), 3) live gate (DAPI negative), and 4) SSC/PE-Texas Red (AZDye TM 594 positive), when applicable. See Supplementary Figure 1 for gating example. Data were analyzed by taking median fluorescence intensity (MFI) values, adjusting for fluorophore conjugation efficiency by dividing raw MFI by fractional conjugation efficiency, and averaged. Most data were collected in duplicate, and data variance and significance was analyzed via standard error and p-values calculated by ANOVA and Tukey testing. Exponential decay fits were applied to persistence data and used to estimate the half-life. Natural log plots were also created in Excel to evaluate the exponential fit. To compare density of incorporation across all cell types, RFU data was normalized to the volume of the cell. The surface areas of cells in suspension were quantified in image J. Assuming a spherical shape, volumes (V) were calculated from surface area (SA) via V = (4/3)*π*[sqrt(SA/π)] 3 and normalized against resulting values for HEK 293 cells. Data normalized against surface area is also shown in the SM (Fig 12).

CCK-8 cytotoxicity assay
Polymer-coated HEK 293 cells were analyzed via CCK-8 assay 4 days after polymer incubation. Positive/live control cells were prepared at time of incubation. One hour prior to assay, a negative/dead cell control was prepared by treating cells with 1% Triton X-100 in complete media. Following this, 50 µL/well CCK-8 reagent was added to cells and also to media as a blank (volume of reagent scaled for 24-well plates). Cells were incubated with reagent for 3 hrs at 37°C and absorbance was read at 450 nm on a SpectraMax M2 Microplate Reader. Data were collected in triplicate, normalized against the blank, averaged, and plotted with associated standard deviation. An ANOVA and Tukey-test were conducted.

Transferrin colocalization study
One million cells, either Raji or HEK 293, were coated with 10 µM AZDye TM 594-50% glycosylated 92mer in serum-free media according to above protoco. CF488A-transferrin was added to a final concentration of 30 μg/mL for the last 30 minutes of a 1-hour incubation with polymer. Media was exchanged for complete media, and cells were incubated at 37°C for 15 minutes to allow for transferrin trafficking. Cells were washed with PBS, allowed to sediment onto coverslips at 1106 cells/mL PBS, fixed with 4% paraformaldehyde, and fluorescently imaged. Transferrin colocalization data was used to estimate the fraction of polymer inserted in cell membrane, as compared to internalized polymer, for both HEK 293 and Raji cells. Fluorescence owing to CF488A-transferrin (F488) excitation and, separately, AZDye TM 594polymer excitation (F594) was thresholded and pixel-quantified in ImageJ. The percentage of polymer on the cell membrane was calculated by [(F594-F488)/F594]*100% in Excel with an n = 10 per cell line, and averages and standard deviations were also quantified.

Polymer distribution among daughter cells
For this study, suspended Raji cells were analyzed via flow cytometry and adherent HEK 293 via imaging. Raji cells were incubated at 10 7 cells/mL with 10 µM AZDye TM 594-50% glycosylated 92mer and 5 µM CellTracker TM Green CMFDA (Thermo C2925) in serum-free media for one-hour at room temperature. Unstained and single-color controls were also prepared at this time. Post-incubation, cells were washed with PBS, resuspended in complete media, and plated. At 1 hr, 24 hr, 48 hr, and 72 hr post-incubation, 110 6 cells/sample were pelleted, washed once with PBS, and resuspended in PBS for flow analysis. Flow cytometry data was acquired with at least 10,000 events for all samples. The distribution of polymer to daughter cells was evaluated via population comparison in FlowJo. Similarly, 150,000 adherent HEK 293 cells were incubated with 300 µL 10 µM AZDye TM 594-50% glycosylated 92mer and 25 µM CellTracker TM Blue CMHC (Thermo C2111) in serum-free media for one-hour at room temperature. Polymer-free and unlabeled controls were also prepared at this time. Postincubation, cells were washed with PBS, plated in complete media, and fluorescently imaged 24 and 48 hours after treatment.

Lectin binding
Raji cells were first incubated at 10 7 cells/mL with 10 µM AZDye TM 594-labeled polymer panel for one hour at room temperature. Post-incubation, cells were washed once with PBS. Polymertreated cells were then incubated with 5 µg/mL helix pomatia lectin-FITC (EY Labs F-3601-1) in 1% BSA in PBS (Mg ++ and Ca ++ ) on ice for one hour. Cells were then washed twice and resuspended in 1% BSA in PBS. DAPI (0.1 µg/mL from ThermoFisher # 62247) was added as a live/dead discriminator. Flow cytometry data was acquired with at least 10,000 events for all samples. Polymer-treated cells were prepared in duplicate and unstained, single-color, and polymer-free lectin controls were also prepared. FITC-anti-human CD37 antibody (Biolegends 356304) was used at 10 µg/mL to prepare a FITC single-color control. Data were analyzed by taking median fluorescence intensity (MFI) values associated with lectin binding and polymer coating, normalized against background lectin labeling and efficiency of fluorophore labeling where applicable, normalized against one another to account for differentiating amounts of polymer on cell surface, averaged, and plotted with standard error.  Figure 3D in manuscript, but this plot also includes incorporation density directly post-incubation with polymer (time = 0 hr). B) Natural log plot of the data, demonstrating that polymer residence exponentially decays from ~ days 1-5 while exhibiting faster decay after initial incubation and slower decay after day 5. Data were collected via flow cytometry for 10 days post-incubation with polymer and averaged median fluorescence intensities are plotted with their associated standard error with n = 2. Supplementary Figure 18. Data showing effect of additional PBS washes on polymer density on cell surface. A) compares density of incorporation vs. additional washing of our lowest-density polymer, AZDye TM 594-50% glycosylated 26 mer with AZDye TM 594-50% glycosylated 92mer, which has comparable density to all other polymers. B) outlines the amount retained on the cell surfaces post-washing. It is evident that 50% glycosylated 26mer is particularly susceptible to washing (almost 64% loss vs. 21% loss of 50% glycosylated 92mer. C-F) show comparison of cell populations labeled either with C-D) 50% glycosylated 92mer or E-F) 50% glycosylated 26mer after C) & E) initial wash or D) & F) first additional wash. Cells coated with 50% glycosylated 92mer have little spread increase after wash, while 50% glycosylated 26mer has inherently more spread and also is enhanced with washing.

Supplementary
Supplementary Figure 19. Helix pomatia agglutinin binding efficiency to polymers in panel, collected via flow cytometry. Plot shows median fluorescence intensity (MFI) values associated with lectin binding that were normalized against background lectin labeling and against efficiency of polymer coating and averaged. Associated standard error is also plotted. Data corresponds to that in Figure 5A in manuscript, but this plot also encompasses data for the 50% glycosylated 26mer, which is less densely populated on cell surface. Figure 20. Dynamic light scattering curves for 50% glycosylated 26mer and 92mer, shown with A) linear X-axis or B) logarithmic X-axis. Polymers were suspended in 1X DPBS at 1µM and analyzed on a a Malvern Zetasizer Nano ZS (backscatter at angle of 173°).  Table 2. Half-life and associated standard error for all polymers that were run in our initial panel as well as our final, published panel.