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Anisotropic Ligand Nanogeometry Modulates the Adhesion and Polarization State of Macrophages

Material implants trigger host reactions generated by cells, such as macrophages, which display dynamic adhesion and polarization including M1 inflammatory state and M2 anti-inflammatory state. Creating materials that enable diverse nanoscale display of integrin-binding groups, such as RGD ligand, can unravel nanoscale recruitment and ligation of integrin, which modulate cellular adhesion and activation. Here, we synthesized gold nanorods (GNRs) with various nanoscale anisotropies (i.e., aspect ratios, ARs), but in similar surface areas, and controlled their substrate conjugation to display an anisotropic ligand nanogeometry without modulating ligand density. Using nanoscale immunolabeling, we demonstrated that highly anisotropic ligand-coated GNRs ("AR4" and "AR7") facilitated the recruitment of integrin β1 on macrophages to their nanoscale surfaces. Consequently, highly anisotropic GNRs (e.g., "AR4" and "AR7") elevated the adhesion and M2 state of macrophages, with the inhibition of their M1 state in the culture and mice, entailing rho-associated protein kinase. This nanoscale anisotropic nanogeometry provides a novel and critical parameter to be considered in the generation of biomaterials to potentially modulate host reactions to the implants for immunomodulatory tissue regeneration.


Synthesis of gold nanorods (GNRs) with various anisotropies without altering their nanoscale surface areas.
GNRs with various anisotropies (aspect ratios: ARs), but in similar surface areas were synthesized by precisely tuning Au 3+ , Ag + , and binary surfactant mixtures. Maintaining similar surface areas of the GNRs in various anisotropy were critical to control similar surface density of ligand to be subsequently conjugated to the GNRs on the substrate. Hexadecyltrimethylammonium bromide (CTAB)-capped GNRs in various aspect ratio (AR = 2, 4, or 7) were synthesized with the precise control in their dimensions (length and diameter) to obtain the GNRs in various anisotropy, but in similar surface area. We first synthesized gold seed solution. 2.5 mL of 0.5 mM HAuCl4 (Sigma) solution was added into 2.5 mL of 0.2 M CTAB (TCI) solution in 20 mL glass vial. To this mixture solution, 1 mL of fresh 0.006 M NaBH4 (Sigma) was injected. The mixture was subjected to vigorous stirring for 2 min and allowed to age at 29 o C for 30 min to obtain gold seed solution.
To synthesize the GNRs with an aspect ratio of 2 (AR2), a single surfactant of CTAB was used. 0.08 g sodium salicylic acid and 0.9 g CTAB were dissolved in 25 mL of deionized (DI) water. 0.6 mL of 4 mM AgNO3 was added into this mixture, which was then left undisturbed for 15 min. To synthesize the AR4 and AR7 GNRs, a binary surfactant mixture (CTAB and sodium oleate) was used for both GNRs, but with different amounts of AgNO3 to prepare different aspect ratio of AR4 vs. AR7 GNRs. 0.1234 g sodium oleate and 0.9 g CTAB were dissolved in 25 mL of DI water. To this mixture solution, 1.6 or 2.4 mL of 4 mM AgNO3 was added to prepare the AR4 or AR7 GNRs, respectively, and then left undisturbed for 15 min. 25 mL of 1 mM HAuCl4 solution was added to this solution and stirred for 90 min. 210 µL of HCl was added to this mixture and stirred for 15 min. 125 µL of 0.064 M ascorbic acid solution was quickly injected into this reaction mixture and stirred for 30 s. To this solution, 80 µL of the gold seed solution was added. This 3 solution was then stirred for another 30 s and then left the mixture undisturbed at 29 o C for 12 h to obtain the AR4 or AR7 GNRs.
Citrate-capped gold nanospheres (GNSs) were also synthesized as a control to the GNRs. In particular, GNSs were prepared to exhibit similar surface area to that of the GNRs and they were referred to the GNRs in aspect ratio of 1 (AR1). We used the cycle by cycle method to prepare the GNSs in approximate diameter of 45 nm. Briefly, 28.9 mL of DI water was added to a three-necked flask, which was allowed to boil. 1.1 mL trisodium citrate solution was then injected into this boiling water. To this solution, 0.2 mL of 0.025 M HAuCl4 (Sigma) solution was injected and this mixture solution was allowed to react for 15 min under stirring. The reaction mixture was allowed to cool down to 90 o C. To this mixture solution, 0.2 mL of 0.025 M HAuCl4 was injected and this mixture was allowed to react for 30 min. We repeated this step two times. 11 mL of resultant mixture was extracted to which 10.6 mL of DI water was added. When the temperature of this mixture solution reached 85 o C, we added 0.4 mL of 60 mM trisodium citrate into this mixture and subsequently injected 0.2 mL of 0.025 M HAuCl4 three times, which were then allowed to react for 30 min. We repeated this step six times to obtain the GNSs in approximate diameter of 42 nm.

Transmission electron microscopy (TEM) of the GNRs with various anisotropies.
TEM imaging (Hitachi) was performed to characterize the GNRs with various aspect ratios (AR = 1, 2, 4, or 7) at an accelerating voltage of 120 kV. The approximate size (length and diameter) and surface area of the GNRs in various aspect ratio (assuming spherical or cylindrical shape) were determined with the Image J software based on 100 different nanoparticles and presented as the mean ± standard error. These quantifications confirmed systematic preparation of the GNRs with various anisotropies without altering their nanoscale surface areas.

UV-Vis spectrophotometer measurement of the GNRs.
The extinction spectra of the GNRs with various anisotropies were measured to examine their major absorption peaks that typically exhibit red shifts with increasing aspect ratio of the GNRs.
UV-Vis spectrophotometer (Cary 5000, Agilent) was used to characterize the extinction spectra of the GNRs (AR1, AR2, AR4, or AR7) in 1-cm-path length quartz cuvette within the wavelength range from 400 to 1350 nm.

Ligand exchange processes of GNRs with various anisotropies to allow their uniform substrate
coupling.
Prior to substrate coupling of the GNRs with various anisotropies, we performed ligand exchange processes of the GNRs to allow their uniform coupling to the substrate. The CTAB-capped GNRs (AR2, AR4, or AR7) underwent ligand exchange processes that serially replace CTAB with polystyrene sulfonate (PSS) and then further with citrate. 160 mL of CTAB-capped GNRs were concentrated to approximately 10 mL in a stirred ultrafiltration cell using a cellulose membrane filter and then diluted with DI water to 100 mL (approximately 168 µg/mL). The citrate-capped GNR suspensions were centrifuged, dispersed in 0.15% Na-PSS to 100 mL (approximately 135 µg/mL), and allowed to sit for 1 h. The PSS-capped GNR suspensions were centrifuged, dispersed in 30 mL of 5 mM sodium citrate, and allowed to sit for 12 h to obtain citrate-capped GNRs (approximately 125 µg/mL), which were subsequently used for uniform substrate coupling.

Zeta potential measurement of GNRs with various anisotropies to characterize their ligand exchange processes.
Zeta potential measurement was performed to determine the changes in surface charges of the GNRs in various anisotropy through their serial ligand exchange processes from CTAB to PSS and then to citrate. Photon correlation spectroscopy (Brookhaven Instruments Corporation) was used for measurements.

Substrate coupling of GNRs displaying anisotropic ligand nanogeometry without changing either macroscale or nanoscale ligand density.
GNRs with various anisotropies after undergoing ligand exchange processes to obtain citratecapped GNRs were systematically coupled to a substrate (culture-grade glass coverslips in 22 mm x 22 mm) to display anisotropic ligand nanogeometry without modulating macroscale and nanoscale ligand density. The glass substrate was treated with a mixture of HCl and methanol (1:1) for 30 min to wash any organic residues on the substrate surface and was then washed with DI water. The hydroxyl group on the glass substrate was activated by immersing the substrate in sulfuric acid for 1 h and then washing it with DI water. The activated substrate was thiolated by the treatment with 0.5 mM mercaptopropylsilatrane in methanol for 30 min in the dark and 5 subsequently washed with methanol. The thiol-presenting substrate was then immersed in 1 nM citrate-capped GNR solution and incubated for various time ranging from 30 min to 3 h depending on various anisotropy (i.e. aspect ratio) of the GNRs to obtain similar density of the substratecoupled GNRs in various nanoscale anisotropy, which was then washed with DI water.
The GNR-coupled substrate was blocked by PEGylation on the remaining area of the substrate that was not covered with the GNRs to minimize the non-specific macrophage adhesion. The GNR-coupled substrate was incubated in 100 µM maleimide-poly(ethylene glycol)-CH3 in DI water for 2 h in the dark, which was subsequently washed with DI water. The blocked GNRcoupled substrate was incubated in thiol-bearing RGD peptide (GCGYGRGDSPG, Mn = 1025.06 Da, GenScript) and 5 mM tris(2-carboxyethyl)phosphine hydrochloride in phosphate-buffered saline (PBS) for 16 h to obtain the RGD-bearing GNR-coupled substrate in various nanoscale RGD ligand anisotropies. As a control, we used a scrambled RGD peptides (RGE peptides, GCGYGRGESPG, GenScript).
Scanning electron microscopy (SEM) and energy dispersive spectra (EDS) to characterize the substrate decorated with anisotropic ligand-bearing GNRs.
SEM imaging (SU8000 Series, Hitachi High-Tech Ltd.) was carried out to characterize the substrate coupled with anisotropic RGD ligand-bearing GNRs with various aspect ratios (AR = 1, 2, 4, or 7). The substrate-coupled GNR density was calculated from 10 random areas by using Image J software and presented as the mean ± standard error. These quantifications confirmed systematic substrate coupling of the GNRs in various anisotropy in a monolayer without altering their macroscopic and nanoscale density. EDS analysis was carried out by following a previous report 1 to confirm substrate coupling of the GNRs by detecting gold (Au) element.

Quantification of RGD ligand conjugated to the substrate-coupled GNRs with various anisotropies.
The density of the substrate-conjugated RGD ligand was determined by following Ellman's assay (Sigma). Briefly, the GNR-coupled substrate was incubated in thiol-bearing RGD peptide solution.
After this incubation, the remaining solution containing unreacted thiol-bearing RGD peptide (20 µl) was collected and mixed with an assay buffer (100 µl) containing 1 mM EDTA (Sigma-Aldrich) as an antioxidant in sodium phosphate dibasic (0.1 M, pH = 8, Sigma-Aldrich). This mixture 6 solution was added into a detection buffer (50 µl) containing 0.5 mg/mL Ellman reagent (Sigma-Aldrich) dissolved in an assay buffer for the reaction for 10 min. RGD peptide solution incubated without substrate coupling of the GNRs was included as positive control, whereas DI water was used as a negative control. The reacted mixture solution was subjected to the reading of the

Synthesis of gold nanospheres (GNSs) used for nanoscale immunolabeling.
GNSs used for nanoscale immunolabeling were synthesized in a size (approximately 13 nm) significantly smaller than those of the GNRs with various anisotropy to clearly differentiate the GNSs (used for immunolabeling) from the GNRs. Briefly, 1 mM HAuCl4·3H2O in 50 mL of DI 7 water was boiled with vigorous stirring. To this boiling solution, 1% (w/v) sodium citrate solution in 5 mL of DI water was rapidly added to this boiling solution. The solution was boiled for 15 min and then was cooled to 25 o C to obtain the resultant GNS solution (10 nM) before the GNSs were coated with secondary antibody.

Dynamic light scattering (DLS) of the GNSs.
DLS analysis was carried out with photon correlation spectroscopy (Brookhaven Instruments Corporation) to confirm a narrow distribution in the diameter of the GNSs to clearly differentiate the GNSs (used for immunolabeling) from the GNRs with various anisotropies. The DLS measurement was performed for the GNS suspension in DI water.

GNS.
The HAADF-STEM imaging at atomic resolution (JEOL JEM-ARM200F) was carried out to clearly identify the GNS in high magnification as previously described, 2 which was used for immnuolabeling. The GNS was identified by quantifying the lattice spacing between periodic lattice fringes with Image J software. and washed with DI water. To enhance the contrast of the labeled cells, they were stained with 1% osmium tetroxide in PIPES buffer for 1 h and washed with PIPES buffer for 2 min six times. To prepare samples with nanoscale immunolabeling before SEM imaging, we performed critical point dehydration to dry all the substrate samples including macrophages, which were recruited to the GNRs and subsequently labelled with antibody-coated gold nanospheres. Afterwards, we mounted the samples onto copper studs and sputtered them with gold/palladium approximately for 30 s with rotation to obtain 2-3 nm-thick conductive layer. SEM imaging was subsequently performed to characterize the recruitment of integrin of macrophages to the surface of the anisotropic RGD ligand-bearing GNRs on the substrate. In the SEM images, red pseudocolor was assigned to the cells, whereas yellow pseudocolor was assigned to the GNSs used for nanoscale immunolabeling.

Immunofluorescent staining of adherent macrophages.
The adhesion and polarization of macrophages modulated by anisotropic ligand nanogeometry were analyzed by immunofluorescent staining by following previously reported procedures. 3 In brief, the macrophages were fixed with 4% (w/v) paraformaldehyde at 25 °C for 10 min and The background of images was identically subtracted from the acquired images between the compared groups by using ImageJ software (rolling ball radius of 750 pixels).

Quantitative cell analysis based on immunofluorescent staining.
The adhesion and polarization of macrophages modulated by nanoscale ligand anisotropy were quantified for immunofluorescently stained images by using ImageJ software. To determine the adherent macrophage density, the number of macrophages was counted from four different DAPIstained images. To determine the area and aspect ratio (major axis/minor axis) of the adherent macrophages, F-actin images of approximately 30 cells from four different images were used. The density, area, and aspect ratio of the adherent host macrophages were calculated from F-actin images of host cells expressing either M1-specific indicator (iNOS) or M2-specific indicator (Arg-1). To calculate the density of the adherent host neutrophils, host cells expressing neutrophilspecific indicator (NIMP-R14) from four different images were counted.

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR).
The polarization state of macrophages modulated by anisotropic ligand nanogeometry was quantitatively analyzed by the qRT-PCR by following a previous report. 4 After macrophages were cultured in either M1 or M2 stimulators, they were collected to analyze quantitative expression levels of M1-specific indicators (iNOS and CD80) or M2-specific indicators (Arg-1 and Ym2).
RNA from macrophages were extracted with TRIzol by following the manufacturer's protocol.
For each sample, 800 ng of RNA was subjected to reverse transcription to cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific). The Applied Biosystems 7300 Real Time system was used to run real-time PCR reactions with TaqMan assays. The expression of each target gene was presented after it was normalized to that of corresponding housekeeping gene (GAPDH). The expression level of macrophages was subsequently normalized to that of the control group as described in figure captions and presented as relative fold expression.
The adhesion and polarization of host macrophages modulated by anisotropic ligand nanogeometry.
The adhesion and polarization of host macrophages modulated by the substrate displaying nanoscale ligand anisotropy were examined. 20 three-month-old BALB/c mice were used for surgery after the approval of the Institutional Animal Care and Use Committee at the Chinese University of Hong Kong. Each mouse was anesthetized by the intraperitoneal supply of ketamine (100 mg/kg) and xylazine (10 mg/kg). A 1.5 cm-long incision was then made in the back of each mouse. Each substrate displaying nanoscale ligand anisotropy was implanted into the subcutaneous pocket and the skin was closed. For 10 mice, we subsequently supplied each 100 ng of IL-4 and IL-13 (M2 stimulators) onto the implanted substrates (groups with M2 stimulator supply), whereas the other 10 mice had no supply of these stimulators. The mice were then housed in the cages and sacrificed at 24 h after surgery. The implanted substrates were retrieved for immunofluorescent staining analysis for the adhesion and polarization of host cells as well as qRT-PCR analysis for their state.

Statistical analyses.
All the experiments shown in this study were repeated at least twice independently. All the quantitative statistical analyses were carried out with Graphpad Prism 5.00 software. In the statistical analyses, p-values less than 0.05 were considered statistically significant differences between the compared groups, to which different alphabetical letters (a, b, c, or d) were assigned.
Same alphabetical letters represent statistically non-significant differences between the compared groups.