Anisotropic Ligand Nanogeometry Modulates the Adhesion and

Letter Cite This: Nano Lett. XXXX, XXX, XXX−XXX

pubs.acs.org/NanoLett

Anisotropic Ligand Nanogeometry Modulates the Adhesion and Polarization State of Macrophages Heemin Kang,†,¶ Siu Hong Dexter Wong,†,¶ Qi Pan,∥,⊥ Gang Li,∥,⊥,# and Liming Bian*,†,∇,○,◆ †

Department of Biomedical Engineering, The Chinese University of Hong Kong, Hong Kong, China Department of Orthopaedics & Traumatology, Faculty of Medicine, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong, China ⊥ Stem Cells and Regenerative Medicine Laboratory, Lui Che Woo Institute of Innovative Medicine, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong, China # The CUHK-ACC Space Medicine Centre on Health Maintenance of Musculoskeletal System, The Chinese University of Hong Kong, Hong Kong, China ∇ Translational Research Centre of Regenerative Medicine and 3D Printing Technologies of Guangzhou Medical University, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China ○ China Orthopedic Regenerative Medicine Group (CORMed), Hangzhou, Zhejiang, China ◆ Shenzhen Research Institute, The Chinese University of Hong Kong, Hong Kong, China

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S Supporting Information *

ABSTRACT: 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. KEYWORDS: anisotropic nanogeometry, nanoscale immunolabeling, integrin recruitment, macrophage adhesion, macrophage polarization Implanted materials provoke host reactions,5 which can be modulated by the materials displaying bioactive molecules that modulate the adhesion as well as functional activation of immune cells,10 such as macrophage states. The adhesion of macrophages is modulated by the interaction between integrin and bioactive ligand groups, including integrin-binding RGD in the nanofibrous adhesive proteins, such as fibronectin.11 This anisotropy of cell-adhesive microenvironment was shown to modulate the development of polarized adhesive structures in

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acrophages are types of immune cells that govern host immune systems, homeostasis, disease development, and immunomodulatory regeneration of damaged or diseased tissue.1−3 Macrophages can be functionalized into M1 inflammatory state or M2 anti-inflammatory state.4 Material implants induce host reactions that are modulated by the adhesion and functional state of immune cells, including adhesion and polarization state of host macrophages, which are important to be controlled.5−7 Hence, strategic material development to manipulate nanoscale display of bioactive moieties can modulate diverse interactions between materials and host cells, and this is a powerful method to manipulate host reactions to implanted materials.8,9 © XXXX American Chemical Society

Received: December 26, 2018 Revised: January 27, 2019

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DOI: 10.1021/acs.nanolett.8b05150 Nano Lett. XXXX, XXX, XXX−XXX

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(A) Gold nanorods (GNRs) with various anisotropies were synthesized and conjugated to a substrate to display anisotropic ligand nanogeometry, but in similar ligand density. (B) Anisotropic ligand nanogeometry modulated nanoscale integrin recruitment, adhesion, and state of macrophages, entailing ROCK molecule.

cells that influence cellular functions.12 Spatial micropatterning of bioactive groups, such as RGD or fibronectin, has been shown to spatially confine adherent cell shape within diversely shaped micropatterns that modulate cell phenotypes, including macrophage polarization states.13−15 Decoration of material surface with nanomaterials16 displaying various bioactive molecules,17−19 such as DNA aptamers,20 adhesive proteins,21 surface receptors,22 antibodies,23,24 or antigens,25 was shown to modulate the adhesion or function of cells involved in the immune system. Material development to display bioactive ligand in a novel nanogeometry may modulate integrin recruitment of host macrophages and thus their adhesion as well as functional state. Material development to enable diverse nanoscale display of bioactive groups, including RGD ligand, spatially or dynam-

ically, can unravel how integrin is recruited to nanoscale ligand surface that mediates integrin-ligand binding to modulate cell adhesion and subsequent function. Integrin−ligand binding occurs in nanoscale; therefore, controlling nanoscale interactions between cell surface receptors and ligand is critical to understanding cell−material interactions.26 In addition, controlling ligand display is also important to the accurate understanding of nanoscale cell−material interactions.27 Various spatial distribution of RGD-displaying nanoparticles28 on material surface has been shown to regulate cell adhesion by modulating RGD density and nanospacing,29 nanospacing with dynamic changes,30 nanospacing with micropattern size,31 or local vs global density.32 Nanoscale RGD clustering was also reported to modulate cell adhesion.33 We demonstrated that the manipulation of in situ assembly,34 physical uncaging,35 B


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Figure 1. Substrate coating of gold nanorods (GNRs) to display anisotropic ligand nanogeometry without modulating ligand density. (A) Transmission electron micrographs of the GNRs with various aspect ratios (“AR1”, “AR2”, “AR4”, or “AR7”). The aspect ratio, dimensions (length/diameter), and surface area of individual GNRs were accordingly calculated. Scale bars: 100 nm. (B) Scanning electron microscope images and representative energy dispersive spectrum of substrate-conjugated GNRs with various anisotropy. The densities of the substrate-conjugated GNRs or RGD coated on the substrate-conjugated GNRs were accordingly calculated. Scale bars: 500 nm. Data indicate the means ± standard error of the mean.

tether compliance,36 oscillation,37 or tether strength38 of RGDdisplaying nanoparticles modulated cell adhesion. A recent study showed that macroscale RGD uncaging modulated the adhesion of host macrophages, which did not explore the modulation of their polarization state.39 Controlling the nanoscale anisotropy in the ligand geometry, without modulating RGD density, can enable the investigation of the effect of ligand nanogeometry on the adhesion and state of host macrophages. In this study, we prepared the GNRs with various anisotropies but with similar surface areas by tuning the amounts of Au3+, Ag+, and binary surfactants used in the synthesis of the GNRs (Scheme 1A). We coated the substrate

with these GNRs displaying various anisotropy by tuning ligand exchange processes of the GNRs and substrate coating reaction conditions. These substrate-conjugated GNRs were further coated with RGD to display ligands in an anisotropic nanogeometry without modulating ligand density. Using nanoscale immune-characterization, highly anisotropic liganddisplaying GNRs were shown to directly recruit the integrin β1 of macrophages to their nanoscale surface that elevated the adhesion and M2 state of macrophages, in the culture and mice (Scheme 1B). The findings from this study can aid the material development to achieve desired host reactions for enhancing implant integration or immunomodulatory tissue-regenerative processes. C


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Citrate-capped GNRs in various anisotropy were conjugated to thiol-displaying substrate through the reaction of gold and thiol. The GNRconjugated substrate was then PEGylated. RGD peptide was conjugated to surface of the GNRs on the substrate through the reaction of gold and thiol to display anisotropic RGD ligand nanogeometry without modulating ligand density.

ments of the extinction spectra of the GNRs with various anisotropy revealed obvious red shifts of the main peaks with increasing nanoanisotropy of the GNRs (Supplementary Figure S1). These findings confirmed the control over the surface area of the GNRs displaying various anisotropies and therefore the quantity of the conjugated ligand density. To aid uniform substrate coating of the GNRs in various anisotropy, CTAB-capped GNRs underwent ligand exchange processes to serially replace CTAB with polystyrenesulfonate (PSS) and then with citrate. TEM images and zeta potential measurements of the GNRs through the serial ligand exchange processes revealed similar morphology and distinct changes in surface charges of the GNRs, thereby confirming the effective ligand exchange processes (Supplementary Figure S2). The citrate-capped GNRs with different anisotropies obtained from the ligand exchange treatments were then conjugated to the substrate to display anisotropic ligand nanogeometry with no modulation of ligand density (Scheme 2). We grafted the citrate-capped GNRs in various anisotropy to thiol-displaying substrate through the reaction of gold and thiol by controlling and varying the reaction time depending on various anisotropies of the GNRs to obtain similar density of the substrate-grafted GNRs in various nanoscale anisotropy. The non-GNR-coated area in the substrate was PEGylated to block the non-specific adhesion of macrophages. Surface of the

We prepared the substrate displaying anisotropic ligand nanogeometry first by synthesizing GNRs with various anisotropies, without modulating their nanoscale surface areas. Hexadecyltrimethylammonium bromide (CTAB)-capped GNRs were synthesized with the control in their dimensions (length and diameter) to obtain the GNRs with various anisotropies (i.e., aspect ratio; AR = 1, 2, 4, or 7) but similar surface areas. To synthesize the “AR2” GNRs, a unary surfactant of CTAB was used, whereas binary surfactant mixture (CTAB and sodium oleate) was used to synthesize the “AR4” and “AR7” GNRs with different amounts of AgNO3. We also prepared citrate-capped gold nanospheres (GNSs) to exhibit similar surface area to that of the GNRs, which are hereafter referred to the “AR1” GNRs. Transmission electron microscope (TEM) images revealed uniform dimensions of the “AR1” GNRs with a diameter of 42.3 ± 2.8 nm and the other GNRs with their length and diameter of 57.7 ± 4.2 and 29.5 ± 1.5 nm (“AR2”), 83.9 ± 5.9 and 21.1 ± 1.3 nm (“AR4”), or 109.1 ± 3.3 and 15.3 ± 0.9 nm (“AR7”) (Figure 1A). The surface areas of the GNRs with various anisotropy ranged from 5250 to 5550 μm2 in all groups with no significant differences (Figure 1A). We used this range of surface area since the diameters of the GNRs with high anisotropy roughly matched the size of integrin receptors (∼10 nm) to achieve better isotropic receptor recruitment to the surface of GNRs via the GNR-mediated anisotropic ligand display. UV−vis measureD


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Figure 2. Integrin of macrophages was recruited to highly anisotropic ligand-displaying GNRs to elevate their adhesion. (A) Vinculin, actin, and nuclei stained in adhered cells at 12 h after culture. AR indicates various aspect ratio (“AR1”, “AR2”, “AR4”, or “AR7”) of the substrate-conjugated GNRs. Scale bars: 50 μm. (B) Schematic representation and (C) scanning electron micrographs (SEM) of immunocharacterization of integrin of macrophages recruited to nanoscale surface of anisotropic ligand-displaying GNRs on the substrate. Secondary antibody-coated GNSs labeled integrin β1 of adherent macrophages after incubating macrophages in primary antibody against integrin β1. In the SEM images, red color was assigned to the cells, whereas yellow color was assigned to the GNSs used for immunocharacterization to clearly visualize the recruited integrin to the nanoscale surface of highly anisotropic ligand-displaying GNRs. Scale bar: 100 nm.

densities conjugated to the GNRs on the substrate, and they approximately ranged from 1.2 to 1.4 RGD molecules/nm2 across all groups without significant differences (Figure 1B). These confirm the control over the substrate conjugation of the GNRs and subsequent RGD ligands to display anisotropic ligand nanogeometry without modulating ligand density. Recent studies demonstrated the modulation of cellular adhesion by varying density and interparticle spacing of RGD-coated nanoparticles.29−31 Comparatively, we prepared low density of the substrate-conjugated GNRs in our present study because this low density helped to better investigate the

GNRs on the substrate was then coated with RGD peptides through the reaction of gold and thiol. Scanning electron microscopy (SEM) images revealed homogeneously populated GNRs on the surface of substrate displaying various anisotropic nanogeometry (Figure 1B). Representative energy dispersive spectrum of the GNR-coated substrate showed the detection of the Au element arising from the substrateconjugated GNRs (Figure 1B). The density of the substrateconjugated GNRs in various anisotropy was determined to range approximately from 19 to 21 GNRs/μm2 in all groups with no significant differences. We further calculated the RGD E


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Figure 3. M1 state of macrophages was restrained by high ligand anisotropy when cultured with M1 stimulators. (A) The expression of M1-specific indicators (iNOS/CD80) or M2-specific indicators (Arg-1/Ym2) by qRT-PCR and (B) iNOS, Arg-1, and nuclei stained in cells after 36 h of culture with M1 stimulators. AR represents various aspect ratios (“AR1”, “AR2”, “AR4”, or “AR7”) of the substrate-conjugated GNRs. Scale bar: 50 μm. Data indicate the means ± standard deviations.

Figure S5). We evaluated adhesive structures in macrophages and observed that prevalent development of actin filament and vinculin in the adherent macrophages with higher cell area and aspect ratio in the “AR4” and “AR7”, which were considerably less pronounced in the “AR1” and “AR2” (Figure 2A and Supplementary Figure S4). Considerable difference was not observed in this quantification between the “AR1” and “AR2”. These findings clearly reveal that highly anisotropic nanogeometry display of ligand can elevate the adhesion of macrophages. We next investigated the mechanism underlying the influence of nanoanisotropic ligand display on the adhesion of macrophages. Nanoscale examination of cell−material interfaces is critical in unraveling the interactions between subcellular components and nanomaterials.40,41 To this end,

effect of anisotropic ligand nanogeometry on the modulation of macrophage adhesion and polarization. We next investigated whether manipulating the anisotropic ligand nanogeometry can modulate the adhesive structure development on macrophages. At 12 h after culture, cells were found to adhere significantly more in the “AR4” and “AR7” than the “AR1” and “AR2” (Figure 2A and Supplementary Figure S3). For example, 58% and 68% more adhered cells were observed in the “AR4” and “AR7”, respectively, than in the “AR2” (Supplementary Figure S3). Without conjugated RGD peptide on the GNRs, the substrate did not effectively support the adhesion of macrophages (Supplementary Figure S4). Coating the AR7 GNRs with RGE peptide (a scrambled RGD) significantly decreased macrophage adhesion, compared to the AR7 GNRs with functional RGD (Supplementary F


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Figure 4. M2 state of macrophages was elevated by high ligand anisotropy when cultured with M2 stimulators. (A) The expression of M2-specific indicators (Arg-1/Ym2) or M1-specific indicators (iNOS/CD80) by qRT-PCR and (B) Arg-1, iNOS, and nuclei stained in cells when cultured with M2 stimulators for 36 h. AR indicates various aspect ratios (“AR1”, “AR2”, “AR4”, or “AR7”) of the substrate-conjugated GNRs. Scale bar: 50 μm. Data indicate the means ± standard deviations.

we performed immunolabeling to characterize integrin recruitment to the surface of anisotropic ligand-displaying GNRs on the substrate. For nanoscale immunocharacterization, we prepared GNSs in a size around 13 ± 2 nm (Supplementary Figure S6), which was substantially smaller than that of the GNRs with various anisotropy used for substrate conjugation. We then coated these GNSs with secondary antibody to specifically label integrin in adherent macrophages (Figure 2B). The adherent macrophages on the substrate displaying anisotropic ligand-displaying GNRs were first incubated with primary antibody of integrin β1. The secondary antibodycoated GNSs subsequently labeled the integrin β1 in adherent macrophages. Representative SEM images revealed that the integrin β1 (labeled by the GNSs in yellow color) in adherent macrophages (colored in red) was effectively recruited to the

surface of the ligand-displaying GNRs with high nanoanisotropy (“AR4” and “AR7”) (Figure 2B). Contrastively, negligible GNSs labeling integrin β1 were found on the surface of the GNRs in the “AR1” and “AR2”. These indicate that highly anisotropic ligand-displaying GNRs on the substrate guided the recruitment of integrin β1 in cell adhesion structures, thereby elevating macrophage adhesion. We observed significant differences in integrin recruitment and adhesion of macrophages modulated by the nanoscale anisotropy of the ligand-displaying GNRs between high anisotropy (“AR4” and “AR7” groups) and low anisotropy (“AR1” and “AR2” groups). Therefore, we found the critical nanoscale anisotropy at the aspect ratio between 2 and 4 of the GNRs to modulate cell adhesion. It was previously found that there is a critical interparticle spacing (around 70 nm) of the G


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Figure 5. High ligand anisotropy entails ROCK in elevating the adhesion and M2 state of macrophages. (A) ROCK2 and nuclei stained in cells when cultured without stimulators, or with M1 or M2 stimulators for 36 h. (B) Actin and nuclei in cells without stimulators, iNOS, Arg-1, and nuclei in cells with M1 stimulators, or Arg-1, iNOS, and nuclei in cells with M2 stimulators. AR represents various aspect ratio (“AR1” or “AR7”) of the substrate-grafted GNRs. The “AR7” was also cultured with a ROCK inhibitor, Y27632 (“AR7 + Y27632”). Scale bars: 50 μm. The areas and aspect ratios of the cells were accordingly calculated. The expression of M2-specific indicators (Arg-1/Ym2) analyzed by qRT-PCR after the culture with M2 stimulators. Data indicate the means ± standard deviations.

adhesive structures (Figure 2 and Supplementary Figure S3), and these adhesive features were reported to elevate their M2 state.13 The states of adherent macrophages on the substrate displaying nanoscale ligand anisotropy was analyzed at 36 h after culture with M1 or M2 stimulators. Macrophages were cultured with M1 stimulators, and then the expression of M1specific indicators (iNOS/CD80) was investigated by qRTPCR. Both iNOS and CD80 expression were significantly hindered in the “AR4” and “AR7” in comparison to their

ligand-displaying gold nanoparticles to modulate cell adhesion.29 In our present study, we suggest the nanoscale ligand anisotropy displayed by the anisotropic nanomaterials as a novel and critical parameter that effectively modulates cell adhesion. The adhesive structures of macrophages can modulate polarization states of macrophages with M1 or M2 inducers.13 In this study, we showed that the integrin recruitment was elevated by high ligand anisotropy to aid the formation of H


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Figure 6. Adhesion of host macrophages was elevated by high ligand anisotropy along with the suppression of their M1 state. (A) Schematics of the substrate subcutaneously implanted, which displays nanoscale ligand anisotropy. (B) Actin, iNOS, and nuclei stained in host cells at 24 h of surgery. AR indicates various aspect ratio (“AR1” or “AR7”) of the substrate-conjugated GNRs. Scale bar: 20 μm. (C) The densities, areas, and aspect ratios of host macrophages were accordingly calculated. The expression of M1-specific indicators (iNOS/CD80) or M2-specific indicators (Arg-1/Ym2) of the host cells. Data indicate the means ± standard error of the mean.

expression in the “AR1” (Figure 3A). The expression of these M1-specific indicators in the “AR1” and “AR2” were comparable. Considerable difference in the expression of M2-specific indicators (Arg-1/Ym2) was not observed in all groups with various ligand anisotropy (Figure 3A). The inhibition of M1 state by highly anisotropic ligand nanogeometry was further tested by staining cells for iNOS and Arg-1. Strongly positive iNOS signal was observed in the “AR1” and “AR2”, which was considerably higher than that in the “AR4” and “AR7” (Figure 3B). These confirm that the M1

state of adhered macrophages was restrained by high ligand anisotropy. We next tested the culture of macrophages with M2 stimulators. Significantly increased expression of Arg-1 in the “AR4” by 185% and “AR7” by 204% as well as Ym2 in the “AR4” by 114% and the “AR7” by 230% were observed in comparison to their expression in the “AR1”, which was comparable to that in the “AR2” (Figure 4A). The expression of M1-specific indicators (iNOS/CD80) was comparable in all groups with various ligand anisotropy (Figure 4A). Strongly positive Arg-1 signal was observed in the “AR4” and “AR7”, I


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Figure 7. Adhesion and M2 state of macrophages were both elevated by high ligand anisotropy with M2 stimulators. (A) Schematics of the substrate subcutaneously implanted, which displays anisotropic ligand nanogeometry to modulate host macrophages with supplied M2 stimulators, such as interleukin-4 (IL-4) and interleukin-13 (IL-13). (B) Actin, Arg-1, and nuclei stained in host cells after 24 h of surgery. AR represents various aspect ratio (“AR1” or “AR7”) of the substrate-grafted GNRs. Scale bar: 20 μm. The densities, areas, and aspect ratios of host macrophages were accordingly calculated. The expression level of M2-specfic indicators (Arg-1/Ym2) or M1-specfiic indicators (iNOS/CD80) of host cells. Data indicate the means ± standard error of the mean.

whereas minimal Arg-1 signal was shown in the “AR1” and “AR2” (Figure 4B). These confirm that the facilitated integrin recruitment and development of adhesive structures in macrophages by high ligand anisotropy further elevated their M2 anti-inflammatory state, with the suppression of their M1 inflammatory state. We further delved into the role of adhesive structure development in macrophages in modulating their states. We investigated how ROCK2 is involved in highly anisotropic

ligand nanogeometry-mediated adhesion and M2 state of macrophages. We cultured macrophages without stimulators, or with M1 or M2 stimulators, and then stained the cells for ROCK2 after the culture on the substrate displaying representative ligand nanoanisotropy (i.e., AR1 vs AR7). Considerably higher expression of ROCK2 was observed in the “AR7” than in the “AR1” (Figure 5A). Y27632 was added to inhibit ROCK, and consequently, significantly reduced area and aspect ratio of adhered macrophages were observed in the J


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Nano Letters “AR7 + Y27632”, in comparison to those in the “AR7” (Figure 5B). Further, qRT-PCR and staining outcomes showed that with M2 stimulators, the elevated M2 state of adhered macrophages in the “AR7” was significantly reduced in the “AR7 + Y27632” without considerable changes in the M1 state (Figure 5B and Supplementary Figure S7). Conversely, with M1 stimulators, the expression of M1 state was elevated in the “AR7 + Y27632” in comparison to the “AR7” with no considerable differences in the M2 state (Figure 5B and Supplementary Figures S8−9). These prove that ROCK functioned as an essential converter in modulating adhesion and M2 state of macrophages with an anisotropic display of cell adhesive ligands. We next examined whether anisotropic ligand nanogeometry can control the development of adhesive structures and polarization states of host macrophages. Host reactions to implanted materials are modulated by functional immune cells, such as polarized macrophages.5,8,42 Thus, controlling the display of bioactive ligand can achieve desirable reactions to the implanted materials. The substrate displaying nanoscale ligand anisotropy was implanted into mice to evaluate their recruitment and modulation of host cells (Figure 6A). After 24 h of surgery, the adhesion and states of recruited host macrophages were evaluated by staining host cells for actin with iNOS or Arg-1. The density, area, and aspect ratio of adherent host macrophages all increased in the “AR7” in comparison to the “AR1” (Figure 6B−C and Supplementary Figure S10). In the “AR1”, higher iNOS and lower Arg-1 signals in the cells were observed than in the “AR7” (Figure 6B−C and Supplementary Figure S10). Substantially increased iNOS and CD80 expression by qRT-PCR43 and lower neutrophil adhesion were observed in the “AR1” in comparison to the “AR7”, with no considerable difference in the Arg-1 and Ym2 expression (Figure 6C and Supplementary Figure S11A− B). These confirm that the M1 inflammatory state of macrophages was restrained by high ligand anisotropy. Since functionally modulated macrophages into M2 state can aid immunomodulatory repair of tissue,8 anisotropic ligand nanogeometry with M2 stimulators was used to examine the modulation of M2 macrophages in the mice (Figure 7A). After 24 h of surgery, the density, area, and aspect ratio of host macrophages were considerably elevated in the “AR7” in comparison to the “AR1” (Figure 7B−C). In the “AR7″, higher Arg-1 and lower iNOS fluorescence intensities in the cells were observed than in the “AR1” (Figure 7B and Supplementary Figure S12). Further, considerable increases in the Arg-1 expression by 154% and Ym2 expression by 142% as well as neutrophil adhesion were observed in “AR7” in comparison to the “AR1” with no substantial difference in the iNOS and CD80 expression (Figure 7C and Supplementary Figure S13A−B). These confirm that the adhesion and M2 antiinflammatory state of macrophages was elevated by high ligand anisotropy, which can potentially provide guidance to the development of implant biomaterials to aid immunomodulatory tissue regeneration. In summary, we demonstrated the manipulation of anisotropic nanogeometries of the GNRs while controlling similar surface areas. We further showed the manipulation of substrate conjugation of these GNRs to display anisotropic ligand nanogeometry without modulating ligand density. Using nanoscale immunolabeling with GNSs and antibodies, we demonstrated that highly anisotropic ligand-displaying GNRs directly recruited the integrin β1 of macrophages and increased

integrin ligation on their surface, and this elevated the adhesion and M2 state of macrophages with the inhibition of their M1 state, entailing ROCK pathways. This strategy for manipulating nanoscale ligand anisotropic display can enable the manipulation of a myriad of host cells.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.8b05150.

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Characterizations of GNRs with various anisotropies used for uniform substrate conjugation and GNSs used for nanoscale immunocharacterization (by UV−vis spectroscopy, TEM, zeta potential, HAADF-STEM, and DLS); the adhesion, polarization, and ROCK signaling of macrophages modulated by the substrate displaying anisotropic ligand nanogeometry; adhesion of host macrophages and neutrophils, and macrophage state modulated by the substrate displaying nanoscale ligand anisotropy (by staining and quantification) (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Heemin Kang: 0000-0003-2694-9882 Siu Hong Dexter Wong: 0000-0001-7920-4599 Liming Bian: 0000-0003-4739-0918 Author Contributions ¶

H.K. and S.H.D.W. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Project 31570979 is supported by the National Natural Science Foundation of China. This work is supported by a General Research Fund grant from the Research Grants Council of Hong Kong (project no. 14220716); the Health and Medical Research Fund, the Food and Health Bureau, the Government of the Hong Kong Special Administrative Region (reference no.: 04152836); the Chow Yuk Ho Technology Centre for Innovative Medicine, and The Chinese University of Hong Kong. The work was partially supported by Hong Kong Research Grants Council Theme-based Research Scheme (ref. T13-402/17-N). The work was partially supported by grants from Research Grants Council of the Hong Kong Special Administrative Region, China (Project No. 14120118, 14160917, 9054014 N_CityU102/15, T13-402/17-N); National Natural Science Foundation of China (81772404, 81430049, and 81772322); and Hong Kong Innovation Technology Commission Funds (ITS/UIM-305). This study was also supported in part by SMART program, Lui Che Woo Institute of Innovative Medicine.

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REFERENCES

(1) Murray, P. J.; Wynn, T. A. Protective and Pathogenic Functions of Macrophage Subsets. Nat. Rev. Immunol. 2011, 11, 723−737.

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Anisotropic Ligand Nanogeometry Modulates the Adhesion and

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