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S1 Supporting Information for Macrophage Uptake of Janus Particles Depends Upon Janus Balance Yuan Gao and Yan Yu Department Of

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S1 Supporting Information for Macrophage Uptake of Janus Particles Depends Upon Janus Balance Yuan Gao and Yan Yu Department Of Supporting Information for Macrophage Uptake of Janus Particles Depends upon Janus Balance Yuan Gao and Yan Yu* Department of Chemistry, Indiana University, Bloomington, Indiana 47405, United States *Corresponding author: [email protected] Contents Experimental Section Supplementary Figures S1-S3 S1 Experimental Section 1. Cells and reagents Monodisperse silica particles were purchased from Spherotech (Lake Forest, IL). Sylgard 184 silicone elastomer kit was purchased from Dow Corning (Midland, MI). Pluronic F- 127, (3-Aminopropyl) triethoxysilane (APTES), biotin N-hydroxysuccinimide ester (biotin-NHS), bovine serum albumin (BSA) and immunoglobulin G (IgG) from rabbit serum were purchased from Sigma-Aldrich (St. Louis, MO). Biotinylated bovine serum albumin (BSA-biotin) was purchased from Thermo Scientific (Waltham, MA). Streptavidin (SA), streptavidin Alexa Fluor-568 conjugate, Fluo-4 acetoxymethyl ester (Fluo-4) and Alexa Fluor-568 succinimidyl ester were purchased from Life Technologies (Grand Island, NY). VivoTrack 680 NIR fluorescence imaging agent was purchased from Perkin Elmer (Waltham, MA). BSA-biotin Alexa Fluor-568 conjugate was synthesized from BSA-biotin and Alexa Fluor-568 succinimidyl ester. IgG was biotinylated with biotin-NHS. Phospholipids 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-lissamine rhodamine B sulfonyl (RhB- PE) were purchased from Avanti Polar Lipid (Alabaster, AL). RAW264.7 macrophage cells were purchased from ATCC (Manassas, VA). RAW264.7 macrophage cells were cultured in DMEM complete growth media supplemented with 10% fetal bovine serum (FBS), 0.11 mg/ml (1 mM) sodium pyruvate, 100 units/ml penicillin and 100 μg/ml streptomycin. Ringer’s imaging buffer (pH = 7.2, 155 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 2 mM NaH2PO4, 10 mM HEPES, 10 mM glucose) was used for all live-cell imaging. 2. Fabrication of Janus particles by micro-contact printing (µCP) Silica particles (1.6 µm and 3 µm in diameter) were pretreated with piranha solution (3:1 H2SO4: 30% H2O2) and rinsed with deionized water. Particle monolayers were made on pre-cleaned glass microscope slides with methods as described elsewhere1 and particle monolayers were treated with piranha solution before printing. The µCP procedure was modified from previously reported methods.2,3 To prepare the polydimethylsiloxane (PDMS) stamps, monomers and curing agent from the Sylgard 184 silicone elastomer kit S2 were mixed at a ratio of 2:1 (w:w) in a petri dish, degassed and baked for 8 hours at 70 °C in a vacuum oven. To render the PDMS stamp hydrophilic, which is critical for efficient transfer of proteins, the stamping surface of the PDMS stamp was pretreated with a modified piranha solution (1:1 H2SO4:30% H2O2) for 3 min, rinsed thoroughly with deionized water, and incubated with 30 µg/ml BSA-biotin or BSA-biotin Alexa Fluor-568 conjugate in 1× phosphate buffered saline (PBS) solution for 20 min. After sequential rinsing with PBS solution and deionized water, the stamp was dried under a strong stream of filtered air and pressed against the particle monolayer immediately. A constant mechanical pressure of 1.5×104 Pa was applied for 3 min before the stamp was peeled off. Particles adhered to the PDMS stamp were harvested by brief sonication in 1× PBS solution containing 10 µM BSA. To attach IgG onto particle surfaces, harvested particles were sequentially incubated with 10 µM BSA for 1 hour, 100 nM streptavidin for 1.5 hours and 10 nM biotinylated IgG for 1.5 hours. 3. Fabrication of particles uniformly coated with IgG Pretreated silica particles were incubated in an anhydrous tetrahydrofuran solution containing 2% (v/v) APTES for 30 min, thoroughly washed with ethanol, and dried in oven at 120 °C for 2 hours. Biotinylation was performed by incubation of particles with 2 mM biotin-NHS in sodium bicarbonate buffer (pH = 8.2) for 1 hour. To attach IgG onto particle surface, particles were incubated with 10 µM BSA for 1 hour, 100 nM streptavidin or streptavidin Alexa Fluor-568 conjugate for 1.5 hours, and 2 nM IgG-biotin for 1.5 hours sequentially. The same procedure but without the IgG conjugation was used to fabricate non-coated control particles. To vary the total number of IgG on the particle surface, IgG-biotin was mixed with BSA-biotin at various ratios, but the total concentration of both biotinylated proteins was kept the same at 2 nM. S3 4. Quantification of ligand patch size (Janus balance) Janus particles that were coated with BSA-biotin Alexa Fluor-568 conjugate were imaged with the epi-fluorescence imaging mode on a Nikon A1R-A1 microscope system that is equipped with a Nikon 100× oil-immersed objective and a Hamamatsu C11440 camera (Light Microscope Imaging Center, Indiana University). To ensure the imaging focal plane is at the center of the IgG patch on each particle, we took a stack of epi- fluorescence images along the z axis with a 0.2-μm step size (controlled by a motorized piezo stage) and selected the most in-focus image for quantification of the patch size. Because epi-fluorescence microscopy generates a 2-dimensional projection of a 3- dimensional specimen within a given depth of field, the protein patches may appear non- spherical depending on the orientation of the particles (Figure 1b). In spite of the different shapes, the longest dimension of each projected image equals the diameter of the protein patch and does not change with particle orientation. Due to the diffraction limit, the epi-fluorescence images appear larger than the actual size of the patches. A minimal intensity threshold, defined as 1/3 of the maximum intensity of the patch, was used to determine the edge of each protein patch in the epi-fluorescence images. The longer axis of each 2-D projection was determined by using ImageJ and its length was used as the diameter (a) of each protein patch. The arc angle of the patch, θ, was calculated based on the following equation: θ = 2arcsin(a/2r) (1) in which a is the diameter of the patch and r is the radius of the particle. 5. Quantification of particle internalization efficiency RAW264.7 macrophage cells were seeded on glass coverslips in imaging chambers and allowed to spread and grow for at least 36 hours before imaging. Macrophages were serum starved for 3 hours prior to all cell experiments. A typical confluency of cells during experiment was 30% and the particle to cell number ratio was kept as 8:1 in all experiments. To label the macrophage cell membrane with VivoTrack 680, cells were incubated with 2 µg/ml VivoTrack 680 for 25 min at room temperature in Ringer’s buffer. S4 After washing with Ringer’ buffer for three times, macrophages were incubated with particles for 15 min at 37 °C in Ringer’s buffer to allow particle internalization before samples were fixed with 2% (w/v) paraformaldehyde (PFA) on ice. We chose 15 min as the incubation time because we observed that if particles were attached to cell membrane but not internalized within the first 15 min, they were rarely internalized afterwards. To distinguish internalized particles from the ones that are outside of cells, both particles and macrophages were imaged with 3-D confocal laser scanning fluorescence microscopy. Entire cells were scanned along the z-axis with a 0.5-µm step size. Internalization efficiency (% internalization) was defined as the ratio of the number of internalized particles to the number of all particles that were either internalized or attached to the cell membrane. For Janus particles, samples were imaged with both 3-D confocal scanning fluorescence microscopy and epi-fluorescence imaging in order to identify particle internalization and to quantify ligand patch size simultaneously. 6. Quantification of IgG surface density on particles The IgG surface density was determined by a quantitative fluorescence microscopy method reported previously.4 The first step of this method is to obtain a calibration curve of fluorescence intensity versus surface density by using a standard sample in which the surface density of fluorescently labeled molecules on particles is known. Silica particles (diameter = 1.6 μm) that were coated with a lipid bilayer containing a known fraction of rhodamine B-labeled lipids were used as the standard. 100-nm lipid vesicles (DOPC with a small fraction of RhB-PE) were prepared with the extrusion method (instruction available at www.avantilipids.com). Lipid vesicles were mixed with pre-cleaned silica particles for 2 hours at room temperature to prepare the lipid bilayer-coated particles. We varied the molar ratio of RhB-PE to DOPC to achieve different surface densities of RhB- PE (50, 100, 200, 300 RhB-PE/µm2), given that the average surface area of a DOPC lipid is 0.72 nm2.5 The standard particles were imaged with epi-fluorescence microscopy. The average fluorescence intensity per pixel (IRhB) of lipid bilayer-coated particles was analyzed with ImageJ, plotted against the surface density of RhB-PE (σRhB-PE). A linear regression was fitted with Origin software and the slope of the fitted linear line (s) was S5 obtained (Figure S2 SI). The second step is to measure the fluorescence intensity of the labeled IgG antibodies on particles. Janus particles and control particles that were uniformly coated with IgG Alexa Fluor-568 conjugate were imaged with epi-fluorescence microscopy under the same imaging acquisition settings as with the standard. The average fluorescence intensity per pixel (IAlexa568) was obtained in ImageJ. The third step is to measure the scaling factor F that allows direct comparison between the fluorescence intensity of Alexa Fluor-568 to the standard calibration curve of RhB-PE by taking in account the spectral difference between the two fluorophores.
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