Glycodendrimers: tools to explore multivalent galectin-1 interactions

Four generations of lactose-functionalized polyamidoamine (PAMAM) were employed to further the understanding of multivalent galectin-1 mediated interactions. Dynamic light scattering and fluorescence microscopy were used to study the multivalent interaction of galectin-1 with the glycodendrimers in solution, and glycodendrimers were observed to organize galectin-1 into nanoparticles. In the presence of a large excess of galectin-1, glycodendrimers nucleated galectin-1 into nanoparticles that were remarkably homologous in size (400–500 nm). To understand augmentation of oncologic cellular aggregation by galectin-1, glycodendrimers were used in cell-based assays with human prostate carcinoma cells (DU145). The results revealed that glycodendrimers provided competitive binding sites for galectin-1, which diverted galectin-1 from its typical function in cellular aggregation of DU145 cells.


Table of Contents
Page General experimental protocol S2 Fluorescence microscopy protocol S2-S6 Fluorescence microscopy statistical analysis S3-S4 Fluorescence microscopy calibration curve and S4-S5 micrographs for standard fluorescent microsphere Dynamic light scattering protocol S7-S10 Cell-based assay protocol S10-S11 Representative cell images S12-S15 Saturation curve for 1 S15 Control experiments for cell based assays S16 Solution preparation for cell based assays S17 References for the supporting information S17 S2

General methods
Galectin-1 was provided by Dr. Linda Baum and Mabel Pang of UCLA in stock aliquots of 6.4 and 7.2 mg/mL and stored at −78 °C. General reagents were purchased from Life Technologies, Inc. PAMAM dendrimers were purchased from Dendritech.

Nanoparticle formation Fluorescence microscopy
Fluorescent chromophores and fluorescent microsphere standards (FluoSpheres ® ) were purchased from Molecular Probes™. Fluorescent images were captured on an Olympus BX-61 motorized microscope with MicroSuite™ software with a 100× oil immersion objective. Exposure time was optimized at 2 ms. Gimp 2.8 image manipulation software was used to combine images for subsequent particle perimeter (pixel) analysis. Pixcavator 6.0 Image Analysis software (Intelligent Perception) was used to measure particle perimeter in terms of pixel count. Particles below a threshold of 80 "round" as calculated by Pixcavator were excluded from analysis.
To a final concentration of galectin-1 of 40 µM, varying amounts of glycodendrimer (0.18, 4.5, and 13 µM; final concentration) were added to achieve the following ratios of galectin-1 to glycodendrimer: (a) 220:1; (b) 9:1; and (c) 3:1. PBS was added to bring the total volume of the solution to 100 µL. Assays were incubated for 1 hour at room temperature (19 °C). Each sample was scanned in triplicate, at intervals of approximately five minutes and vortexed between scans. The average measured S3 diameter and polydispersity of the fluorescently labeled galectin-1/glycodendrimer aggregates is provided in Table S1. Fluorescence microscopy data was analyzed for statistically significant differences in aggregate size at the varying ratios ( Figure S1). For each compound, statistically significant changes in aggregate size were determined by comparing the size of aggregates formed at 220:1 to 9:1 and those formed at 9:1 to 3:1 (Table S3). In addition, for each compound, aggregates formed in the presence of a slight excess of galectin-1 (3:1) were analyzed for statistical significance with respect to aggregates formed in the presence of a large excess (220:1). The size of galectin-1 aggregates formed with compounds 2, 3, and 4, respectively, were compared at 220:1. Figure S1: Average diameter (nm) of multivalent galectin-1 nanoparticles formed with multivalent glycodendrimers. Nanoparticle diameter (nm) was measured in the presence of galectin-1 at 220 molar excess, a 9 molar excess, and a 3 molar excess with compounds 2 (purple), 3 (red), and 4 (blue), respectively. Statistical analyses were performed using a paired two-tailed student's T-Test. Symbols (* and ♦) are used to indicate a statistically significant change in the size aggregates as the ratio of galectin-1 changes relative to a compound. The symbols *, **, and *** indicate a statistical significance of p < 0.05, p < 0.01, and p < 0.001, respectively, by comparing aggregates formed at 9:1 with respect to aggregates formed at 220:1, and by comparing aggregates formed at 3:1 with respect to those formed at 9:1. The symbols ♦, ♦♦, and ♦♦♦ indicate a statistical significance of p < 0.05, p < 0.01, and p < 0.001, respectively, by comparing aggregates formed at 3:1 with respect to those formed at 220:1. NS represents nonsignificant difference in aggregate size measured at 220:1 for all generations determined by ANOVA.

Pixel (count) to size (nm) calibration curve
Fluorescent microsphere standards (FluoSpheres ® , Molecular Probes™) were used to establish a calibration curve to convert the measured particle perimeter (pixels) to diameter (nm) at the optimized exposure time ( Figure S2). Representative images of the fluorescent microsphere standards are shown Figure S3a-c. Table S2 shows the effective diameter of the fluorescent microspheres as measured with DLS. S5 Figure S2: Calibration curve correlating measured pixels to reported particle diameter (nm).   Prior to mixing the reagents, all solutions were filtered using a 0.22 µm millipore filter

Lactose inhibition
A lactose inhibition assay was performed in which monomeric lactoside was co-  Table S4.  (Particles of a diameter 6.8 ± 0.38 nm as measured by NNLS analysis agree with the reported value for polyhydroxylated G(6)-PAMAM dendrimer) 1 .
Monomeric lactoside was dissolved in filtered PBS buffer to afford a 100 µM solution.

Cell-based assays
The human prostate carcinoma cell line DU145 was purchased from ATCC ® (HTB-81™). Cellular aggregation assays were observed on a Jenco™ USA microscope with 10× objective and a Canon Powershot A630 camera at 4× magnification. All reagents for cell-based media were purchased from Gibco ® by Life Technologies™, except NaHCO 3 , which was purchased from Fisher Scientific.
Adhered cells were removed with 0.5 mM EDTA and collected in a 15 mL Falcon tube.
After sufficient centrifugation to form a cell pellet, the EDTA solution was removed and the pellet was re-suspended in 1 mL serum free media (SFM). Cells were counted using a hemocytometer, following a 10× dilution in SFM of a 10 µL aliquot of the cell suspension and a subsequent 2× dilution in Trypan Blue (TPB) of 10 µL aliquot of the previously diluted sample. Centrifugation and dilution of the cells in SFM were performed to achieve a concentration of 16 × 10 6 cells/mL. 15 µL of the cell suspensions were added to each Eppendorf for a final concentration approximately 240,000 cells/70 µL. For all assays, other than the untreated control, a final galectin-1 concentration of 3.7 µM was used. The volume of glycodendrimer (compounds 1-4) added was doubled at each stage in the assay, and SFM was added to bring the final volume to 70 µL. Table S5 below contains the volume of the reagents added for the untreated control (Tube 1), galectin-1 with increasing glycodendrimer concentrations (Tubes 2-5), and galectin-1 control (Tube 6). S11 Table S5: Cell-based assay. Tube 1: untreated DU145 control; Tubes 2-5: DU145 cells with galectin-1 and increasing glycodendrimer; and Tube 6: galectin-1 treated control.  Figure S8, and the graph shown in Figure   S9 reveals a dose responsive inhibition mechanism with 1.

Controls
A series of controls were performed to investigate the binding enhancement and the specificity of inhibition using lactose-functionalized dendrimers.

Lactose inhibition
To assess the multivalent enhancement in inhibiting cancer cell adhesion achieved with the dendrimer scaffold, a control assay was performed using monomeric lactoside.
Figure S10: Inhibition of DU145 cellular adhesion by monomeric lactose. Inhibition of cancer cell adhesion by monomeric lactose was observed at 6 mM lactose.

Solution preparation
Assays were run in a final volume of 70 µL. Galectin-1 was prepared at 0.5 mg/mL in PBS buffer (pH 7.4, 15 mM NaCl) from a 7.2 mg/mL stock solution. Glycodendrimer For the DU145 complete media with 10% fetal bovine serum (FBS), to SFM, 5 mL vitamin, 5 mL streptavidin/penicillin mixture, 10 mL essential amino acid, 5 mL nonessential amino acid, and 50 mL FBS were added and filled to 500 mL with SFM and sterilized using a sterile filter unit.