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Magnetic Manipulation of Reversible Nanocaging Controls In Vivo Adhesion and Polarization of Macrophages Heemin Kang, Hee Joon Jung, Sung Kyu Kim, Dexter Siu Hong Wong, Sien Lin, Gang Li, Vinayak P. Dravid, and Liming Bian ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b02226 • Publication Date (Web): 16 May 2018 Downloaded from http://pubs.acs.org on May 16, 2018

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Magnetic Manipulation of Reversible Nanocaging Controls In Vivo Adhesion and Polarization of Macrophages Heemin Kang,† Hee Joon Jung,‡,§,∥ Sung Kyu Kim,‡,§,∥ Dexter Siu Hong Wong,† Sien Lin,#,¶,□ Gang Li,#,¶,◊ Vinayak P. Dravid,‡,§,∥ and Liming Bian○,†,□,⌠,±,* †

Department of Biomedical Engineering, The Chinese University of Hong Kong, Hong Kong,

China. ‡

Department of Materials Science and Engineering, Northwestern University, Evanston, IL, USA.

§

International Institute for Nanotechnology, Evanston, IL, USA.

∥NUANCE #

Center, Northwestern University, Evanston, IL, USA.

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 Shenzhen Research Institute, Shenzhen, China. □

Department of Pharmacology, Guangdong Key Laboratory for Research and Development of

Natural Drugs, Guangdong Medical University, Zhanjiang, Guangdong, China. o

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. ⌠

Shenzhen Research Institute, The Chinese University of Hong Kong, China.

±

China Orthopedic Regenerative Medicine Group (CORMed), Hangzhou, Zhejiang, China.

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ABSTRACT Macrophages are key immune cells that perform various physiological functions, such as the maintenance of homeostasis, host defense, disease progression, and tissue regeneration. Macrophages adopt distinctly polarized phenotypes, such as pro-inflammatory M1 phenotype or anti-inflammatory (pro-healing) M2 phenotype, to execute disparate functions. The remotely controlled reversible uncaging of bioactive ligands, such as Arg-Gly-Asp (RGD) peptide, is an appealing approach for temporally regulating the adhesion and resultant polarization of macrophages on the implants in vivo. Here, we utilize physical and reversible uncaging of RGD by a magnetic field that allows facile tissue penetration. We first conjugated a RGD-bearing gold nanoparticle (GNP) to the substrate, and then a magnetic nanocage (MNC) to the GNP via a flexible linker to form the heterodimeric nanostructure. We magnetically manipulated nanoscale displacement of MNC and thus its proximity to the GNP, to reversibly uncage and cage RGD. The uncaging of RGD temporally promoted the adhesion and subsequent M2 polarization of macrophages, while inhibiting their M1 polarization, both in vitro and in vivo. The RGD uncaging-mediated adhesion and M2 polarization of macrophages involved rho-associated protein kinase (ROCK) signaling. This study demonstrates physical and reversible uncaging of RGD to regulate the adhesion and polarization of host macrophages in vivo. This approach of magnetically regulating the heterodimer conformation for physical and reversible uncaging of RGD, offers the promising potential to manipulate inflammatory or tissue-regenerative immune responses to the implants in vivo. KEYWORDS: magnetic nanocaging, reversible caging, heterodimer, remote manipulation, macrophage adhesion, macrophage polarization


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Macrophages play crucial roles as immune cells in regulating immune systems, homeostasis, tissue regeneration, and disease progression, such as cancer, atherosclerosis, and obesity.1-4 Macrophages can be activated and polarized to adopt different phenotypes to perform their proinflammatory functions (classically activated M1 phenotypes) or anti-inflammatory and prohealing functions (alternatively activated M2 phenotypes).5 Macrophage polarization is traditionally considered to be regulated by soluble signals, such as chemokines,6 but emerging studies have shown the roles of physical and mechanical cues of extracellular matrix (ECM)7 in regulating macrophage polarization. Therefore, various biomaterials have been designed to modulate immune functions in vivo,8-15 including macrophage M2 polarization to mediate tissue regeneration.13,16-20 Host macrophages dynamically interact with the ECM that regulates their adhesion and polarization, and such interactions are mediated by the dynamic binding of integrin and ligand motifs in the ECM proteins, such as RGD tripeptide sequences (Arg-Gly-Asp) in vivo.21,22 The decoration of RGD peptides on biomaterials23,24 can control desirable host immune responses of biomaterial implants. The remote and reversible manipulation of RGD presentation offers versatile potential for the temporal regulation of immune responses in vivo. Cell-material interactions have been shown to be modulated by the application of various external stimuli, such as enzymes,25,26 light,22 and magnetic field.27,28 Photochemical reactions have predominantly been used to cleave,29,30 oscillate,31 or uncage RGD22 to regulate cell-material interactions. Notably, Lee et al. recently demonstrated the uncaging of RGD via ultraviolet lightmediated photochemical reactions to non-reversibly modulate the adhesion of macrophages in vivo, but not their polarization.22 Furthermore, light is significantly absorbed by living tissues, so its penetration depth is limited and is potentially cytotoxic to living cells in vivo. Alternatively, a magnetic field has been used for remotely and physically manipulating the movement of magnetic nanoparticles,32-36 and safe and prolonged applications in vivo,37,38 including its clinical use,39 owing to its readily penetrative nature through living tissues with significantly lower absorption than light. Our own studies have shown that the magnetic field tuned the tether compliance40 or oscillation28 of RGD-bearing magnetic nanoparticles on biomaterials to modulate cellular adhesion. However, there have been no precedent studies of remotely,


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physically, and reversibly manipulating the uncaging of RGD to modulate the adhesion and polarization of host macrophages in vivo. The adhesion of macrophages modulates their polarization phenotypes. While pro-healing M2 macrophages exhibit pronounced adhesion with pervasive actin assembly, pro-inflammatory M1 macrophages show localized actin clusters.41-43 Recent study by McWhorter et al. showed that modulating adherent shape of macrophages regulated their polarization with elongated shape and prevalent actin assembly enhanced M2 polarization and suppressed M1 polarization.43 Furthermore, rho-associated protein kinase (ROCK) signaling that regulates cytoskeletal arrangement and contractility, was recently shown to mediate M2 polarization of macrophages and inhibiting ROCK pathway switched M2 polarization of macrophages to M1 phenotype.44 Various nanoarrangement of RGD-coated static nanoparticles, such as gold nanoparticles (GNPs), has been designed on biomaterial surfaces to modulate cellular adhesion by varying their nanospacing and density,45 order and disorder,46 and coupling strength.47 Nanoparticles on biomaterial surfaces have also been decorated with various bioactive motifs, such as adhesive proteins48 or peptides,49 surface receptors,50 or antibodies,51 to modulate the adhesion or activation of immune cells. Furthermore, the formation of heterodimer nanoparticles, including magnetic nanoparticles, on biomaterial surfaces could open up opportunities to magnetically control the interactions between biomaterials and macrophages, which has not been demonstrated before. In this study, we flexibly grafted dynamic magnetic nanoparticles as the magnetic nanocage (MNC) to the underlying RGD-coated GNP, to form (RGD-GNP)-MNC heterodimers on the substrate surface (Scheme 1). In the absence of magnetic field, the MNC is placed adjacent to the RGD-bearing GNP, thus physically caging RGD. Under the magnetic field, the MNC is dislodged away from the RGD-bearing GNP, thereby uncaging RGD to be physically accessible to the integrin of macrophages that mediates the formation of adhesive structures in macrophages. This physical and reversible RGD uncaging temporally enhanced the adhesion and M2 polarization of macrophages, but suppressed their M1 polarization, both in vitro and in vivo, through ROCK signaling. This remote, non-contact, tissue-penetrative, and cytocompatible


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control of physical and reversible RGD caging holds promising potential to temporally manipulate inflammation or tissue regeneration in vivo.

RESULTS AND DISCUSSION To magnetically manipulate nanoscale movement of the MNC relative to the RGD-coated GNP in the heterodimeric nanostructure, we prepared the (RGD-GNP)-MNC heterodimer on the substrate by serially coupling the GNP and MNC to the substrate. We first prepared and characterized the GNP.52,53 Transmission electron micrographs (TEM) of the GNPs showed that the spherical GNPs and they were 12 ± 2 nm in diameter (Supplementary Figure S1A). Furthermore, dynamic light scattering (DLS) analysis indicated that the hydrodynamic size of the GNPs was 13 ± 3 nm, consistent with the TEM characterization (Supplementary Figure S1B). We next prepared the magnetic nanoparticles to be used as the MNC.54 TEM of the citratecapped MNC showed a spherical morphology, with a 34 ± 7 nm diameter (Supplementary Figure S2A). DLS analysis also showed the MNC to be 36 ± 8 nm (Supplementary Figure S2B). These analyses confirmed that the MNCs are considerably larger than the GNPs so that one GNP can typically accommodate only one MNC, thereby yielding the heterodimer structure, and this crucial design feature allowed the successful nanocaging of the GNP by the MNC in the absence of an external magnetic field. In addition, x-ray diffraction spectra showed that the diffraction peaks of the MNC corresponded to those of magnetite (Fe3O4) (Supplementary Figure S3), as reported previously.54 We characterized the magnetic property of the MNC. Vibrating sample magnetometer hysteresis measurement revealed that the MNC displayed superparamagnetic characteristics with saturation magnetization (Ms) of 52 emu/g (Supplementary Figure S4). This superparamagnetic property of the MNC is essential to the temporal control of the reversible uncaging and caging of the RGD-bearing GNP with and without the application of the magnetic field, respectively. We next functionalized MNC with a long flexible polymer linker (thiol-poly[ethylene glycol] [PEG], molecular weight: 5 kDa) to graft it to the GNP on the substrate through the Au-S bond, allowing us to magnetically manipulate the reversible movement of the MNC relative to the GNP. 5 ACS Paragon Plus Environment

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Fourier transform infrared spectroscopy measurements revealed that the absorption peaks of the citrate-capped MNC, corresponded to the Fe-O bond detected at 583 cm-1, the C-H bond at 2918 cm-1, and the FeO-H bond at 3921 cm-1 (Supplementary Figure S5). The thiol-PEGylated MNC showed the absorption peaks corresponding to the citrate-capped MNC, as well as those indicative of the C-O-C bond at 1098 cm-1 and amide bond at 1592 cm-1, confirming the successful coating of the flexible linker to the MNC. Zeta potential measurements further confirmed the successful thiol-PEGylation of the MNC. The citrate-capped MNC conferred a negative surface charge with the zeta potential of -22.6 ± 4.9 mV (Supplementary Figure S6). The zeta potential of the thiol-PEGylated MNC shifted to -14.9 ± 4.1 mV, suggesting that the negative surface charge on the citrate-capped MNC was shielded when it was tethered to the thiol-PEG. Next, we prepared the (RGD-GNP)-MNC heterodimeric nanostructure on the substrate by sequentially grafting the RGD-bearing GNP and thiol-PEGylated MNC, as described in the Scheme 2. TEM image shows the homogeneous distribution of the tethered GNPs in a monolayer on the substrate (Figure 1A). The density of the GNPs that bear the RGD peptide, was measured to be 54 ± 12 particles/µm2. The density and nanospacing of RGD on the substrate play a crucial role in regulating cellular adhesion.45,55 Compared with these previous reports, we used a relatively lower density of RGD in this study because this allowed the effective and optimal regulation of the adhesion and polarization of macrophages by physically and reversibly uncaging RGD. Energy dispersive spectra (EDS) analysis further confirmed the successful grafting of the GNPs by the detection of Au element (Supplementary Figure S7). The (RGDGNP)-MNC heterodimers on the substrate were prepared by coupling the RGD peptide bearing a cysteine residue, and thiol-PEGylated MNC to the GNP conjugated on the substrate (Scheme 2). TEM image revealed a monolayer of uniformly distributed heterodimeric nanoparticle on the substrate, with the GNP shown in dark shade and MNC shown in bright shade (Figure 1A). EDS analysis corroborated the successful coupling of the GNP by detecting Au element as well as the thiolated MNC by detecting Fe and S element (Supplementary Figure S8). Scanning transmission electron microscopy (STEM) and EDS mapping further confirmed the direct coupling between the GNP and thiolated MNC in the heterodimeric nanostructure


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(Supplementary Figure S9). We quantified that 83% of the GNP was approximately coupled with the MNC, allowing the effective nanocaging of the GNP by the MNC. We next used in situ magnetic atomic force microscopy (AFM) to demonstrate the physical and reversible nanocaging of RGD via magnetic manipulation of the movement of MNC relative to the GNP. With the permanent magnet used in this study, we measured the magnetic field strength as the distance between the end of the magnet and the measurement point was increased. We found an inverse relationship, in which the magnetic field strength decreased with increasing distance from the magnet, and was 121 ± 6 mT at a distance of 1 cm (Supplementary Figure S10). We placed the magnet at approximately 1 cm from the heterodimeric substrate during in vitro cell cultures, and the bottom of the mouse cage during the in vivo experiment in this study. A previous study showed that PEG molecule in high molecular weight exhibits a highly flexible nature such that it can be stretched into a longer chain similar to a spring or coiled back into a shorter chain reversibly, based on an entropic elasticity.56 We hypothesized that such flexible nature of the PEG could allow reversible tuning of the proximity of the MNC to the RGDbearing GNP by the magnetic field, thereby reversible RGD uncaging. We placed the magnet on one side of the (RGD-GNP)-MNC heterodimer-grafted substrate to attract the MNC away from the GNP that separates the heterodimer, thereby uncaging RGD (Scheme 1). Conversely, we removed the magnet to restore the proximate position of the MNC relative to the GNP, thereby re-caging RGD. We characterized the heterodimeric nanostructure in the presence of an external magnetic field (i.e. uncaged state of RGD). AFM image showed the separated larger MNC and smaller GNP in the heterodimeric nanostructure, suggesting the uncaging of RGD (Supplementary Figure S11). The height profile along a linear scan of the partitioned heterodimer further confirmed the size of the GNP to be around 12 nm based on the lower peak and the larger size of the MNC at around 35 nm based on the higher peak, consistent with our findings in both the TEM and STEM images. We used a tapping mode for AFM imaging to minimize the disturbance to the heterodimeric nanostructure (i.e. conformation) caused by the scanning cantilever probe, and found that it was negligible when the heterodimer was serially scanned (Supplementary Figure S12). We then examined whether the partitioned conformation of the heterodimer could be reversibly controlled. We removed the magnet and found that the MNC physically shielded the underlying GNP, suggesting the re-caging of RGD (Figure 1B). 7 ACS Paragon Plus Environment

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We found that this uncaging and caging can be repeatedly controlled by the presence and absence of the external magnetic field, respectively, indicative of the effective reversible RGD uncaging. These findings further suggest that tuning reversible RGD uncaging by the magnetic field can regulate the physical accessibility of RGD to the integrin on macrophages, modulating their adhesion. We next evaluated whether the reversible RGD uncaging on the heterodimer-grafted substrate could regulate the binding of integrin to RGD to modulate the adhesion of macrophages. We adjusted the magnetic field externally by placing or removing the magnet on one side of the substrate (hereafter referred to as “RGD uncaging” and “RGD caging”, respectively) and monitored the macrophage adhesion. We blocked the substrate with PEG molecules to minimize non-specific macrophage adhesion (Scheme 2). After 12 h of culture in basal growth medium, “RGD uncaging” group yielded a significantly higher density of adherent macrophages than the “RGD caging” group (Figure 2A). Quantification of the adhered cell density revealed 81% more adherent cells in the “RGD uncaging” group than the “RGD caging” group (Figure 2B). This indicates the efficacy of the RGD uncaging in enhancing the exposure of RGD and thus its physical accessibility of RGD for integrin binding, thereby facilitating the macrophage adhesion. To further evaluate this RGD uncaging-mediated macrophage adhesion through the displacement of the MNC, we performed a series of control experiments. We prepared the GNP-MNC heterodimer-grafted substrate, but with no RGD conjugated to the GNP, and monitored the macrophage adhesion. We observed considerably low level of adhesion irrespective of the uncaging or caging of the GNP, suggesting that RGD tethering is required to modulate macrophage adhesion by the MNC (Supplementary Figure S13A-B). This also indicates that the passivation of the substrate by the PEG molecules effectively minimized non-specific macrophage adhesion. We also prepared the (RGD-GNP)-grafted substrate without the MNC. We detected substantial adhesion of macrophages irrespective of the application of the magnetic field, suggesting that the MNC-mediated uncaging and caging of RGD are essential for the magnetic manipulation of macrophage adhesion (Supplementary Figure S14A-B). Since the morphology and the assembly of the actin filaments of the adhered macrophages modulate their subsequent polarization,43 we further analyzed the adhesive structures of the macrophages. After 12 h in culture, the adhered macrophages showed pervasive actin assembly and vinculin 8 ACS Paragon Plus Environment

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expression in the cytoplasm in the “RGD uncaging” group, suggesting that the RGD uncaging facilitated the robust adhesion of macrophages (Figure 2A). Furthermore, the “RGD uncaging” group displayed significantly greater cell area by 100% and a more elongated morphology with a higher aspect ratio by 155% than the “RGD caging” group (Figure 2B). These different adhesive structures of the macrophages under RGD uncaging vs. caging were not observed in the absence of RGD conjugation to the GNP (Supplementary Figure S13A-B) or MNC conjugation to the RGD-GNP (Supplementary Figure S14A-B). Macrophages display dynamic adhesion and polarization on implants that yield distinct timedependent immune functions, including pro-inflammatory or pro-regenerative functions.8 Therefore, the remote control of the reversible adhesion and polarization of macrophages can offer substantial opportunities to manipulate time-regulated immune responses to implants. Therefore, we next investigated whether physical and reversible RGD uncaging can mediate the reversible adhesion of macrophages during 24 h in culture. We magnetically controlled the “RGD caging” or “RGD uncaging” continuously for the entire 24 h period. We applied “RGD caging” for the initial 12 h and then switched to “RGD uncaging” for the subsequent 12 h (“RGD caging-uncaging”). We also applied “RGD uncaging” for the initial 12 h and then switched to “RGD caging” for the following 12 h (“RGD uncaging-caging”). After 24 h of culture, continuous “RGD uncaging” (without switching) significantly enhanced the adhesion of macrophages compared with that in the “RGD caging” group, consistent with the distinct trend observed after 12 h. (Figure 2A-B). When “RGD uncaging” was switched to “RGD caging”, the adherent macrophages displayed significantly reduced cell area (by 39%) and aspect ratio (by 46%), compared with the cells in the continuous “RGD uncaging” group (Figure 2A-B). This suggests that the reversible RGD caging slightly disrupted the macrophage adhesion that was established under the initial “RGD uncaging”. Furthermore, upon temporal switching from “RGD caging” to “RGD uncaging” (“RGD caging-uncaging”), the adherent macrophages displayed increased cell area (by 82%) and aspect ratio (by 117%), together with the morphological change from round to elongated cells, compared with the cells in the continuous “RGD caging” group (Figure 2A-B). This finding indicates that the reversible RGD uncaging successfully restored the adhesive structures of the macrophages after the initial temporal suppression of their adhesion under the “RGD caging”. This physical and reversible nature of the 9 ACS Paragon Plus Environment

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RGD uncaging can offer a powerful tool to reversibly regulate the adhesion of macrophages to modulate their polarization in a non-contact manner, without altering the chemical structure that occurs during the light-based photochemical uncaging of RGD. Macrophage polarization is traditionally considered to be regulated by chemokines.6 Recent studies have also suggested emerging roles for the adhesive structures of macrophages, such as their cytoplasmic actin cytoskeletons and cell shape, in regulating their polarization.7,43 In particular, a number of studies have shown that a pronounced adhesive structure with prevalent actin assembly is present in M2 macrophages.41-43 An elongated morphology and the assembly of cytoplasmic actin cytoskeletons in macrophages promote their pro-regenerative M2 polarization, while inhibiting their pro-inflammatory M1 polarization.43 Therefore, we hypothesized that the significantly enhanced adhesion of macrophages induced by the reversible RGD uncaging could further promote their M2 polarization. The macrophages were allowed to adhere to the (RGDGNP)-MNC heterodimer-grafted substrate for the initial 12 h under basal growth medium and were then cultured for another 24 h under M1- or M2-polarizing medium that contain corresponding M1- or M2-polarizing chemokines, respectively. We applied the “RGD uncaging” or “RGD caging” for the entire 36 h period, or temporally switched from the RGD caging in the initial 12 h to the RGD uncaging in the following 24 h (“RGD caging-uncaging”). After the culture in M1-polarizing medium, we carried out gene expression analyses for M1 polarization markers (iNOS and CD80) after 36 h. The “RGD uncaging” group exhibited significantly downregulated expression of both iNOS by 72% and CD80 by 67%, compared with that in the “RGD caging” group (Figure 3A). In addition, the “RGD caging-uncaging” group exhibited the expression of M1 markers comparable to that in the “RGD uncaging” group. In contrast, the expression of M2 markers (Arg-1 and Ym2) remained similar in all the groups (Figure 3A). We further confirmed the reversible RGD uncaging-mediated suppression of M1 polarization with immunofluorescent co-staining against M1 (iNOS) and M2 marker (Arg-1). The staining intensities against iNOS were significantly higher in the majority of cells in the “RGD caging” group than the “RGD uncaging” and “RGD caging-uncaging” groups, whereas staining against Arg-1 remained predominantly negative (Figure 3B). These findings suggest that the reversible RGD uncaging effectively inhibited the polarization of macrophages toward the 10 ACS Paragon Plus Environment

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M1 phenotype. Next, we cultured macrophages in M2-polarizing medium. Both the “RGD uncaging” and “RGD caging-uncaging” groups displayed significantly upregulated expression of both Arg-1 (by 255% and 211%, respectively), and Ym2 (by 186% and 214%, respectively), compared with that in the “RGD caging” group (Figure 4A). No significant differences in the expression of M1 markers (iNOS and CD80) were detected in any of the groups. Immunofluorescent staining against Arg-1 was positive in the majority of cells in the “RGD uncaging” and “RGD caging-uncaging” groups, in contrast to the minimal staining in the majority of cells in the “RGD caging” group (Figure 4B). The staining against iNOS was predominantly negative in all groups. Taken together, these findings suggest that the reversible RGD uncaging, which facilitated the substantial development of adhesive structures on the macrophages, efficiently stimulated their polarization toward the pro-regenerative M2 phenotype, while inhibiting their pro-inflammatory M1 polarization. ROCK plays key roles in regulating adhesive structures and actin cytoskeletal organization and contractility.57 A recent study by Zandi et al. revealed that ROCK signaling mediates the M2 polarization of macrophages.44 That study also showed that ROCK functions as a molecular switch, and the inhibition of the ROCK signaling switched the polarization of macrophages from M2 to M1. Therefore, we postulate that the reversible RGD uncaging-mediated development of adhesive structures on macrophages could promote their M2 polarization via ROCK signaling. We first investigated the adhesion of macrophages under the “RGD caging” and “RGD uncaging” for 24 h in basal growth medium. We also investigated the adhesion of macrophages under the RGD uncaging in the presence of Y27632, a ROCK inhibitor (“RGD uncaging + Y27632”). In the “RGD uncaging” group, the adherent macrophage area increased significantly (by 84%) and aspect ratio increased significantly (by 160%), compared with those in the “RGD caging” group (Figure 5A-B). Notably, the supplementation with the ROCK inhibitor (“RGD uncaging + Y27632”) significantly reduced the macrophage adhesion area (by 36%) and aspect ratio (by 35%), compared with those in the “RGD uncaging” group (Figure 5A-B). This suggests that the inhibition of ROCK signaling hindered the RGD uncaging-mediated adhesion of macrophages. We next examined whether the suppression of macrophage adhesion by the ROCK inhibition under the RGD uncaging, further modulates the polarization fates of the macrophages. This was examined after the cells were cultured in basal growth medium for 12 h, followed by the culture 11 ACS Paragon Plus Environment

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in either M1- or M2-polarizing medium for 24 h. After 36 h in culture with either M1- or M2polarizing medium, higher expression of ROCK2, known to mediate the M2 macrophage polarization, was observed in the “RGD uncaging” group than in the “RGD caging” group (Supplementary Figure S15). Furthermore, after 36 h in culture with M2-polarizing medium, the “RGD uncaging” group displayed highly positive Arg-1 staining in the majority of cells, in contrast to the minimal staining in the “RGD caging” group (Figure 5A). The RGD uncagingmediated M2 polarization was abrogated by ROCK inhibition, yielding only half the number of Arg-1-positive cells compared with those in the “RGD uncaging” group. Concomitantly, the gene expression of the Arg-1 and Ym2 in the “RGD uncaging + Y27632” group was significantly downregulated (by 39% and 51%, respectively), compared with those in the “RGD uncaging” group (Figure 5B). The expression levels of the iNOS and CD80 remained similar in all the groups (Supplementary Figure S16). After 36 h of culture with M1-polarizing medium, the “RGD uncaging” group exhibited minimally positive iNOS staining, in contrast to highly positive staining in nearly all the cells in the “RGD caging” group (Figure 5A). The “RGD uncaging + Y27632” group exhibited positive iNOS staining in the majority of cells. Furthermore, the gene expression of the iNOS and CD80 in the “RGD uncaging + Y27632” group was significantly upregulated (by 165% and 131%, respectively), compared with that in the “RGD uncaging” group (Figure 5B), whereas no significant differences were observed in the expression of the Arg-1 and Ym2 in all the groups (Supplementary Figure S17). These findings collectively suggest the roles of ROCK in regulating the RGD uncaging-mediated adhesion of macrophages and their subsequent M2 polarization, consistent with the previous report.44 Host macrophages respond immunologically to implants in vivo through their dynamic adhesion and polarization states. In particular, macrophages are known to shape the regenerative niche when they adopt the M2 polarization phenotype.3,8,13 The implants are initially subject to the innate immune responses in which macrophages play a key role. Temporally controlling the adhesion and M2 polarization states of macrophages in the early post-implantation period is a vital requirement for the success of tissue repair because unbalanced macrophage polarization can lead to inflammation and fibrous tissue formation. The remote manipulation of the RGD uncaging has been predominantly demonstrated via photochemical reactions. Compared with the light-based approach, the utility of a magnetic field in controlling physical and reversible RGD 12 ACS Paragon Plus Environment

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uncaging to mediate the adhesion and M2 polarization of macrophages, has the advantage of reaching deep interior anatomical sites in a non-invasive and cytocompatible manner. Therefore, we next examined whether the physical and reversible RGD uncaging can modulate the adhesion and polarization of recruited host macrophages in vivo, during 24 h of implantation. The (RGDGNP)-MNC heterodimer-grafted substrate was subcutaneously implanted into Balb/c mice and temporally applied the reversible RGD uncaging by placing or removing a magnet, approximately 1 cm distant from the cage containing mice (Figure 6A). We identified the adherent host macrophages as those cells expressing either an M1 polarization marker (iNOS) or an M2 polarization marker (Arg-1). We analyzed the adhesion of these macrophages by examining their cytoplasmic actin. We induced the “RGD uncaging” or “RGD caging” for the entire 24 h post-implantation period, or temporally switched from the RGD caging during the initial 12 h to the RGD uncaging during the subsequent 24 h (“RGD caging-uncaging”). At 24 h after implantation, the adhesion of the host macrophages was significantly promoted in the “RGD uncaging” group, with the macrophage density increased by 93%, area by 54%, and aspect ratio by 121%, relative to those in the “RGD caging” group (Figure 6B and Supplementary Figure S18). The reversible RGD uncaging in the “RGD caging-uncaging” group facilitated the macrophage adhesion consistently with that in the “RGD uncaging” group. This suggests that the reversible RGD uncaging allows temporal regulation of macrophage adhesion in vivo. In addition, the “RGD caging” group showed highly positive iNOS staining in nearly all the cells, but minimally positive Arg-1 staining (Figure 6B and Supplementary Figure S18). In stark contrast, the “RGD uncaging” and “RGD caging-uncaging” groups displayed minimally positive iNOS staining in the majority of cells, but strongly positive Arg-1 staining in a small population of cells. We further examined the polarization of the host cells with a quantitative gene expression analysis, according to a previous report.58 The “RGD uncaging” and “RGD caging-uncaging” groups displayed significantly downregulated iNOS expression (by 62% and 55%, respectively), and CD80 (by 61% and 65%, respectively), compared with those in the “RGD caging” group (Figure 6C). The expression of Arg-1 and Ym2 remained non-significantly different in all the groups (Supplementary Figure S19). These findings collectively indicate that the reversible RGD uncaging efficiently promoted the adhesion of the host macrophages and inhibited their M1 polarization. We also identified the adherent host neutrophils as the cells 13 ACS Paragon Plus Environment

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expressing NIMP-R14 (a neutrophil marker). Analogous to the macrophages, the “RGD uncaging” and “RGD caging-uncaging” groups significantly enhanced the adhesion of the host neutrophils in higher density (by 94% and 90%, respectively), than observed in the “RGD caging” group (Supplementary Figure S20A-B). Macrophages and neutrophils are the predominant innate immune cells recruited to implants during the early post-implantation period.22,59 The reversible RGD uncaging promoted the adhesion of host macrophages and successfully suppressed their M1 polarization. The temporal control of the adhesion and M2 polarization of macrophages is crucial in shaping a regenerative niche for tissue repair. However, the reversible RGD uncaging alone did not significantly induce M2 polarization in the recruited host macrophages. This could be due to the dominant acute inflammation within 24 h after implantation of biomaterials, and the lack of anti-inflammatory M2-polarizing signals, consistent with a previous report.22 We further investigated whether the effect of the RGD uncaging on inducing M2 macrophage polarization, can be amplified by the injection of M2-polarizing cytokines (IL-4 and IL-13), while inhibiting their M1 polarization (Figure 7A). A recent study showed that biomaterials worked synergistically with M2-polarizing IL-4 cytokine to induce M2 polarization of host macrophages at 24 h after treatment.20 At 24 h after implantation, the adhesion of the host macrophages was significantly higher in the “RGD uncaging” group (by 94%) and “RGD caging-uncaging” group (by 99%) than in the “RGD caging” group (Figure 7BC). The area and aspect ratio of the adherent host macrophages were also consistently higher in the “RGD uncaging” group than in the “RGD caging” group (Figure 7C). Strikingly, in the presence of M2-polarizing cytokines, the reversible RGD uncaging (in both the “RGD uncaging” and “RGD caging-uncaging” groups) displayed highly positive Arg-1 staining in the majority of cells, but minimal iNOS staining (Figure 7B and Supplementary Figure S21). In contrast, the “RGD caging” group showed minimal Arg-1 staining, but moderately positive iNOS staining. The “RGD uncaging” and “RGD caging-uncaging” groups displayed significantly upregulated expression of Arg-1 (by 154% and 192%, respectively), and CD80 (by 185% and 167%, respectively), compared with that in the “RGD caging” group (Figure 7C). The expression of “iNOS” and “CD80” did not differ significantly amongst the groups (Supplementary Figure S22). The “RGD uncaging” and “RGD caging-uncaging” groups also significantly promoted the adhesion of the NIMP-R14-positive host neutrophils at higher densities (by 76% and 87%, 14 ACS Paragon Plus Environment

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respectively), than in the “RGD caging” group (Supplementary Figure S23A-B). Neutrophils are known to play roles not only in the inflammation, but also in early phagocytosis of cell and tissue debris that is critical in promoting M2 macrophage-mediated tissue repair.60 Understanding how these neutrophils could influence M2 macrophage polarization and M2 macrophage-mediated tissue repair under the RGD uncaging, warrants further investigation. These findings collectively suggest that the reversible RGD uncaging acts synergistically with M2-polarizing cytokines, to temporally promote not only the adhesion of the host macrophages, but also their M2 polarization, while partly suppressing their M1 polarization during 24 h after implantation. Recent studies suggested that controlling early immune responses to biomaterials is critical in regulating subsequent foreign body reactions, such as fibrous capsule formation, or tissue repair in the long-term period. For example, a previous study showed that inhibiting the interactions between biomaterial implant and inflammatory cells, such as macrophages, during 24 h after implantation, effectively suppressed fibrous capsule formation at 28 days after implantation.22 Furthermore, a recent study revealed that suppressing pro-inflammatory M1 macrophages and promoting pro-healing M2 macrophages during 1 to 4 days after treatment with biomaterials, significantly improved cardiac repair at 14 days after implantation.20 These results also suggest that the remote, non-invasive, tissue-penetrative, and cytocompatible control of physical and reversible RGD caging afforded by our magnetic strategy can potentially mediate tissue repair in deep interior anatomical sites over prolonged periods.

CONCLUSION In summary, we have demonstrated the formation of the (RGD-GNP)-MNC heterodimeric nanostructure on a substrate. The MNC was coupled to the underlying RGD-bearing GNP via a long flexible linker. Thus, the MNC was positioned close to the RGD-GNP to physically nanocage RGD. However, under an external magnetic field, the MNC was dislodged away from the RGD-GNP, thereby uncaging RGD. This physical and reversible RGD uncaging effectively promoted the adhesion and M2 polarization of macrophages, while inhibiting their M1 polarization, both in vitro and in vivo. This RGD uncaging-mediated adhesion and M2 polarization of macrophages involved ROCK signaling. We demonstrated that physical and reversible RGD uncaging temporally regulated the adhesion and polarization of the recruited host macrophages in vivo. The tissue-penetrative and cytocompatible nature of the magnetic field 15 ACS Paragon Plus Environment

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used to remotely manipulate reversible RGD uncaging offers a highly appealing strategy of manipulating host responses of the implants to potentially facilitate tissue repair, while suppressing inflammation. Furthermore, the RGD ligand that coats the GNP, which was effectively and reversibly uncaged by the MNC, can be readily replaced with a myriad of bioactive peptides or proteins, such as avidin or streptavidin,61 which can regulate various cellular responses, allowing the widespread applications of this heterodimeric system.

METHODS Synthesis of gold nanoparticles (GNPs). GNPs were synthesized52 to prepare GNP-grafted substrate to which magnetic nanocage (MNC) is subsequently grafted to form the (RGD-GNP)MNC heterodimer. In brief, 0.88 mM HAuCl4·3H2O in 20 mL of deionized (DI) water was heated with vigorous stirring until the solution boils. To this boiling solution, 1% (w/v) sodium citrate solution in 2.4 mL of DI water was added. The boiling of the solution was maintained for 20 min. The boiling solution gradually exhibited reddish color. Subsequently, the boiling solution was cooled down to room temperature. This GNP stock solution was diluted with 1% (w/v) sodium citrate solution to obtain 10 nM GNP solution prior to the grafting of the GNP to the substrate. Synthesis of magnetic nanocages (MNCs). MNCs were synthesized as citrate-capped iron oxide nanoparticles.54 In brief, 1 mmol of C6H5Na3O7·2H2O, 4 mmol of NaOH, and 0.2 mol of NaNO3 were added into 19 mL of DI water. The mixture solution was heated with vigorous stirring until solution boils. The boiling of the solution gradually yielded a pellucid solution. To form iron oxide nanoparticles, 0.6 mmol of FeSO4·4H2O in 1 mL of DI water was added into the mixture solution. The boiling of the solution was maintained for 1 h. Subsequently, the boiling solution was cooled down to room temperature. The mixture solution was washed with DI water three times through the collection of the MNCs by a permanent magnet, which was readily dispersed in DI water. Functionalization of MNC with a long and flexible linker molecule. MNCs were functionalized by a flexible polymer linker (thiol-PEG) to be grafted to the GNP-substrate and magnetically manipulate reversible movements of the MNC relatively to the GNP. 1 mL of 16 ACS Paragon Plus Environment

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citrate-capped MNCs were diluted with 9 mL of DI water, in which 1 mg of amine-poly(ethylene glycol)-thiol (thiol-PEG; Mn = 5 kDa; JenKem) was dissolved. The citrate group of the MNCs reacted with amino group of the thiol-PEG through EDC and NHS activation. To the mixture solution, 50 mg of EDC was added and vigorously stirred for 10 min. Subsequently, 10 mg of NHS was added into the mixture solution and vigorously stirred for 16 h under dark condition. The mixture solution was washed with DI water three times through the collection of the thiolPEGylated MNCs by a permanent magnet, which was readily dispersed in DI water. Transmission electron microscopy (TEM). TEM imaging (CM-200, Philips, Oregon, USA) was carried out to characterize the size and morphology of the GNP and MNC as well as (RGDGNP)-MNC heterodimer grafted to the substrate at an accelerating voltage of 100 kV. The GNP and MNC in the heterodimer was identified by different sizes and shades with smaller GNP in darker shade and larger MNC in brighter shade. Dynamic light scattering (DLS) and zeta potential. DLS analysis (Brookhaven Instruments Corporation) was performed to characterize the size (hydrodynamic diameter) distribution of the GNP and MNC in suspension in DI water. Zeta potential measurement was carried out to examine changes in the surface charges of the MNC before and after thiol-PEGylation. Powder X-ray diffraction (XRD). The MNCs were analyzed by powder XRD to examine the crystalline phase of the iron oxide nanoparticles. The lyophilized MNCs were densely packed on the sample holder and subject to scanning by using Smartlab diffractometer (Rigaku, Cu target, 40 kV, 80 mA). The samples were scanned at diffraction angle from 20 to 70 degree at 10 degree/min. The diffraction peaks of the magnetite was used to identify the peaks of the samples. Vibrating sample magnetometer (VSM) and magnetic field strength measurement. The MNCs were analyzed by VSM to examine their magnetic properties. The magnetic field strength was applied from – 6 kOe to 6 kOe in the hysteresis loop at 300 K (PPMS Model 6000, Quantum Design) to characterize the superparamagnetic properties of the MNCs. The measured magnetic moment in the hysteresis loop was normalized to the dry weight of the MNCs. The magnetic


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field strength of the permanent magnet was measured as a function of distance from the magnet (410 Gaussmeter, Lake Shore). Fourier transform infrared spectroscopy (FTIR). FTIR analysis (IRTracer-100, Shimadzu) was conducted to characterize changes in the chemical bonds of the MNCs before and after thiolPEGylation. The lyophilized MNCs were densely packed into the KBr pellet prior to the measurement. GNP-grafted substrate. Prior to magnetic nanocaging of the adhesive RGD ligand, GNPgrafted substrate was prepared where GNP is later subject to the coating with RGD. Glass coverslips in culture-grade (22 mm X 22 mm) were used as a substrate for in vitro culture. The glass substrate was soaked in a mixture of HCl and methanol (1:1) for 30 min to wash off any organic impurities from the substrate surface and washed with DI water three times. Prior to thiolization, hydroxyl group of the glass substrate was activated by soaking the substrate in sulfuric acid for 1 h and washing with DI water. The activated substrate was incubated in 0.5 mM mercaptopropylsilatrane (MPS) in methanol for 1 h under dark condition and washed with methanol. The thiolized substrate was subsequently incubated in 0.1 nM GNP solution for 15 min to obtain GNP-grafted substrate and washed with DI water. Prior to the coating of the GNP with RGD peptide, the GNP-grafted substrate was blocked with PEGylation to minimize nonspecific macrophage adhesion. The substrate was incubated in 100 µM Maleimide-poly(ethylene glycol) (Mn = 750 Da)-CH3 in DI water for 2 h under dark condition and washed with DI water. Heterodimeric substrate. The GNP-grafted substrate was further coated with the thiolated RGD peptide and flexible linker-conjugated MNC (thiol-PEGylated MNC) to obtain the (RGD-GNP)MNC heterodimer-grafted substrate. The GNP-decorated substrate was incubated in 1 mL of the thiol-PEGylated MNC solution and 0.14 mM thiolated RGD peptide (GCGYGRGDSPG, Mn = 1.025 kDa, GenScript) in presence of 10 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) to prevent disulfide formation for 2 h under dark condition and subsequently washed with DI water.


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Energy dispersive spectra (EDS) and EDS mapping by scanning transmission electron microscopy (STEM). STEM and EDS mapping as well as TEM and EDS spectra were used to characterize the GNP- and heterodimer (GNP and thiolated MNC)-grafted substrate. The substrates were thoroughly dried in air and coated with platinum for 30 s using a sputter coater. EDS spectral analysis was performed to characterize the grafting of the GNP (Au element) and thiolated MNC (Fe and S element). STEM and EDS mapping were used to probe direct grafting between the GNP and thiolated MNC through Au-S bond on the substrate. In situ magnetic atomic force microscopy (AFM). The physical and reversible nanocaging of the RGD ligand was demonstrated by in situ magnetic AFM imaging. The imaging (Bruker) was carried out in a tapping mode at 25 oC, to minimize the movement of the heterodimer nanoparticles by the scanning cantilever. AFM cantilever (Bruker) with a spring constant of 0.4 N/m was used. In situ AFM imaging was performed on the identical scan area with and without the application of external magnetic field by a permanent magnet. In the absence of the magnet, the MNC is situated adjacent to the RGD-bearing GNP, thereby physically caging RGD. With the magnet placed below and on one side of the substrate, the MNC is drawn away from the RGD-bearing GNP, thereby physically uncaging RGD. GNP in smaller size was used as a nonmagnetic reference nanoparticle, to characterize the magnetically controlled nanoscale displacement of the MNC in larger size. With significant size of the GNP and MNC, the heterodimer nanostructure was confirmed by the height profile along a linear scan. In vitro macrophage adhesion and polarization under reversible RGD uncaging. The temporally regulated adhesion and polarization of macrophages on the (RGD-GNP)-MNC heterodimer-grafted substrate was evaluated with physical and reversible RGD uncaging for in vitro culture. Macrophages (passage 5, RAW 264.7 from ATCC) were seeded onto the PEGblocked heterodimer-grafted substrate at the density of 50,000 cells/cm2 and cultured in basal growth medium, consisting of high glucose DMEM, 10% (v/v) heat inactivated fetal bovine serum, and 50 U/mL penicillin/streptomycin at 37 oC and 5% CO2. The time-regulated adhesion of the macrophages on the heterodimer-grafted substrate was examined for 12 h and 24 h under the switching of the placement and removal of the magnet on one side of the substrate in approximately 1 cm distance, to allow uncaging or caging of the RGD, respectively. A series of 19 ACS Paragon Plus Environment

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control experiments were also conducted. The heterodimer-grafted substrate, but without RGD peptide coating to the GNPs on the substrate, was used to evaluate non-specific macrophage adhesion under the uncaging and caging GNP without RGD. The RGD-bearing GNP-grafted substrate, but without magnetic nanocage, was used to examine macrophage adhesion in the absence of the nanocaging, with and without external magnetic field. The time-regulated polarization of the adherent macrophages was evaluated after 36 h of culture, for initial 12 h of culture in basal growth medium for macrophage adhesion and subsequent 24 h of culture in the co-supplementation of 10 ng/mL lipopolysaccharide (LPS) and 10 ng/mL recombinant interferon-gamma (IFN-γ) for M1-polarizing medium or in the co-supplementation of 20 ng/mL interleukin-4 (IL-4) and 20 ng/mL interleukin-13 (IL-13) for M2-polarizing medium.43 During the initial 12 h of culture and following 24 h of culture, reversible RGD uncaging and caging were temporally regulated to examine the adhesion and resultant polarization of macrophages. The role of adhesive structures of macrophages in regulating their polarization under the RGD uncaging, was investigated in the presence and absence of 50 µM Y27632 (Abcam), rho-associated kinase (ROCK) inhibitor.

Immunofluorescent staining analyses of macrophages and corresponding quantifications. The temporally regulated adhesion and polarization of macrophages on the (RGD-GNP)-MNC heterodimer-grafted

substrate

under

reversible

RGD

uncaging,

were

analyzed

by

immunofluorescent staining. Briefly, the macrophages were fixed in 4% (w/v) paraformaldehyde at 25 °C for 15 min and washed with PBS three times. The fixed macrophages were permeabilized with 0.25% (v/v) Triton-X (Sigma Aldrich) in PBS at 25 °C for 10 min. 1% (w/v) bovine serum albumin (BSA) in PBS was then applied to block the treated cells at 37 °C for 1 h. The blocked cells were incubated in the blocking buffer containing primary antibodies against vinculin (1:400, Sigma Aldrich), iNOS (M1 polarization marker, 1:100, Santa Cruz Biotechnology), or Arginase-1 (M2 polarization marker, 1:100, Abcam) at 4 °C for 16 h and washed with PBS containing 0.5% Tween 20 three times. The in vivo adhered host neutrophils were also incubated against NIMP-R14 (Neutrophil marker, 1:100, Santa Cruz Biotechnology). The cells were subsequently incubated in the blocking buffer containing secondary antibodies (1:250, Thermo Scientific) and phalloidin (1:100, Molecular Probes) at 25 °C for 1 h and washed 20 ACS Paragon Plus Environment

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with PBS containing 0.5% Tween 20. The nuclei of macrophages were stained by incubating the cells in DAPI (1:1000, Molecular Probes) in PBS at 25 °C for 10 min and washing with PBS three times. The stained cells were mounted onto glass slides and imaged using confocal microscope (Nikon), while the images were acquired under the same exposure conditions for the compared groups. The background of images was identically subtracted for the images in the compared groups by using ImageJ software (rolling ball radius of 750 pixels). The adhesion and polarization of the macrophages on the heterodimer-grafted substrate under reversible RGD uncaging, were quantified by using immunofluorescently stained images with Image J software. To calculate the adhered macrophage density, the number of cellular nuclei from the 10 DAPI-stained images was counted. To quantify the area and aspect ratio (major axis/minor axis) of the adhered macrophages, 30 phalloidin-stained F-actin images from each cell were used. The density, area, and aspect ratio of the in vivo adhered host macrophages, were determined for the cells exhibiting the expression of either M1 polarization marker (iNOS) or M2 polarization marker (Arg-1) by F-actin images. To determine the density of the in vivo adhered host neutrophils, the cells showing the expression of neutrophil marker (NIMP-R14) were counted. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The polarization of macrophages on the heterodimer-grafted substrate under reversible RGD uncaging, was quantitatively analyzed by the qRT-PCR. Following the culture of macrophages under either M1- or M2-polarizing medium, the cells were collected to analyze quantitative gene expression levels for M1 polarization markers (iNOS and CD80) or M2 polarization markers (Arg-1 and Ym2). TRIzol method was used to extract RNA from the macrophage cultures by following the manufacturer’s protocol. For each sample, 800 ng of RNA was subject 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 normalized to that of corresponding housekeeping gene (GAPDH). The expression level of the macrophages was then normalized to that of the control group as detailed in the figure captions and presented as relative fold expression. 21 ACS Paragon Plus Environment

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In vivo macrophage adhesion and polarization under reversible RGD uncaging. The in vivo adhesion and polarization of host macrophages on the heterodimer-grafted substrate were examined under reversible RGD uncaging. 20 three-month-old BALB/c mice were used with the approval of the Institutional Animal Care and Use Committee at the Chinese University of Hong Kong. The mice were anaesthetized through the intraperitoneal administration of ketamine (100 mg/kg) and xylazine (10 mg/kg). 2 cm-long incision was subsequently made in the back of the mice. The heterodimer-grafted silicon substrate was implanted into the subcutaneous pocket and skin was closed. 10 mice received the injection of each 100 ng of IL-4 and IL-13 (M2-polarizing cytokines) onto the implanted substrate, whereas the other 10 mice received no injection. Following the surgery, the mice were housed in the confined cages and subject to the placement or removal of the magnet approximately 1 cm below the bottom of the confined cages containing the mice, which allowed uncaging or caging of RGD, respectively. Following 24 h of implantation, the implanted substrate was retrieved for immunofluorescent staining analyses of the adhesion and polarization of host cells as well as qRT-PCR analyses of their polarization. Statistical analyses. All the experiments shown in this study were repeated at least two times independently. All the quantitative statistical analyses were performed by using Graphpad Prism 5.00 software. Statistically significant differences were assigned with different alphabetic letters for p-values less than 0.05. Two groups at the same time point were compared by two-tailed Student’s t-test. Multiple groups at the same time point were compared by one-way analysis of variance (ANOVA) with Tukey-Kramer post-hoc test.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Characterizations of GNP and MNC prior to their heterodimerization on the substrate, direct coupling between the GNP and thiolated MNC in the heterodimeric nanostructure on the substrate (by Scanning Transmission Electron Microscopy, Energy dispersive Spectra mapping, and in situ magnetic Atomic Force Microscopy), magnetic field strength of the magnet, 22 ACS Paragon Plus Environment

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macrophage adhesion on the heterodimer-grafted substrate in the absence of RGD conjugation to the GNP or MNC, quantification of M1 or M2 polarization with a ROCK inhibitor, quantification and immunofluorescent staining of M1 or M2 polarization of in vivo adherent host macrophages or neutrophils with or without in vivo injection of IL-4 and IL-13.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected].

ORCID Heemin Kang: 0000-0003-2694-9882 Vinayak P. Dravid: 0000-0002-6007-3063 Liming Bian: 0000-0003-4739-0918 The authors declare no conflict of interest.

ACKNOWLEDGEMENTS 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. 14202215, 14220716); the Health and Medical Research Fund, the Food and Health Bureau, the Government of the Hong Kong Special Administrative Region (reference no.: 04152836, 03140056); the project BME-p3-15 of the Shun Hing Institute of Advanced Engineering, The Chinese University of Hong Kong; the Chow Yuk Ho Technology Centre for Innovative Medicine, The Chinese University of Hong Kong. The work was partially supported by grants from Research Grants Council of the Hong Kong Special Administrative Region, China (Project No. 14119115, 14160917, 9054014 N_CityU102/15, T13-402/17-N); National Natural Science Foundation of China (81772404, 81430049 and 81772322); 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. This work made use of the EPIC facility of Northwestern University’s NUANCE Center, which has received support from the Soft and Hybrid Nanotechnology Experimental (SHyNE) Resource (NSF ECCS23 ACS Paragon Plus Environment

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1542205); the MRSEC program (NSF DMR-1720139) at the Materials Research Center; the International Institute for Nanotechnology (IIN); the Keck Foundation; and the State of Illinois, through the IIN.

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Figure Legends Scheme 1. Summary of the experimental procedure used in this study. Magnetic nanocage (MNC) was conjugated to an underlying RGD-coated gold nanoparticle (GNP) via a long flexible linker, to form the (RGD-GNP)-MNC heterodimer nanostructure. The displacement and thus proximity of the magnetic nanocage (MNC) relative to the RGD-GNP, were magnetically controlled during reversible RGD uncaging. The reversible RGD uncaging was used to temporally manipulate the adhesion and polarization of macrophages, both in vitro and in vivo.

Scheme 2. Summary of the experimental procedure used to form heterodimeric nanostructure via a flexible linker. Schematic representation of the serial coupling of the gold nanoparticle (GNP) and magnetic nanocage (MNC) to prepare the (RGD-GNP)-MNC 30 ACS Paragon Plus Environment

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heterodimer. The GNP was grafted to the thiolated glass substrate through the Au-S bond. Both thiol-PEGylated MNC and thiolated RGD peptide were then grafted to the GNP via the Au-S bond. The thiol-PEG was used as a flexible linker to allow the movement of the MNC relative to the RGD-bearing GNP in response to an external magnetic field.

Figure 1. In situ magnetic manipulation of reversible nanocaging of the RGD-bearing GNP. (A) Transmission electron microscopic images of a monolayer of the GNP- or (RGD-GNP)MNC heterodimer-grafted substrate. Scale bars represent 100 nm. In high-magnification images, scale bars indicate 10 nm. (B) In situ magnetic atomic force microscopic (AFM) images of the (RGD-GNP)-MNC heterodimer-grafted substrate used to demonstrate the magnetic manipulation of the reversible nanocaging of the RGD-bearing GNP, between the RGD caging and uncaging. The AFM images were obtained by scanning an identical area with or without the application of the external magnetic field. Blue dotted lines were drawn across the centers of the RGD-GNPs as a non-magnetic reference to estimate nanoscale movement of the MNC and its proximity to the RGD-GNPs. Scale bar represents 20 nm.

Figure 2. Magnetically controlled RGD uncaging promotes time-regulated reversible adhesion of macrophages. (A) Immnuofluorescent staining micrographs against vinculin (green), actin (red), and nuclei (blue) in macrophages as a function of culture time (12 h or 24 h). Macrophages adhered to the (RGD-GNP)-MNC heterodimer-grafted substrate under RGD caging or uncaging for 24 h (“RGD caging” and “RGD uncaging” groups, respectively), RGD caging for the initial 12 h and uncaging for the following 12 h (“RGD caging-uncaging” group), or RGD uncaging for the initial 12 h and caging for the following 12 h (“RGD uncaging-caging” group). Scale bars indicate 50 µm. (B) Corresponding quantification of the density, area, and aspect ratio of the adherent macrophages. Data are displayed as mean ± standard errors (n=30). Different alphabetical letters (a, b, c, and d) indicate statistically significant differences (p < 0.05), as determined by two-tailed Student’s t-test or one-way ANOVA with Tukey-Kramer post-hoc test. Same alphabetical letters indicate statistically non-significant differences between the compared groups.


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Figure 3. Time-regulated RGD uncaging in M1-polarizing medium suppresses M1 polarization of macrophages. (A) Quantitative gene expression of M1 polarization markers (iNOS and CD80) and M2 polarization markers (Arg-1 and Ym2), and (B) immunofluorescent staining micrographs of macrophages against iNOS (green), Arg-1 (red), and nuclei (blue) after 36 h in culture. The macrophages were allowed to adhere to the (RGD-GNP)-MNC heterodimergrafted substrate under RGD caging or uncaging for the initial 12 h in basal growth medium and for the following 24 h in M1-polarizing medium (“RGD caging” and “RGD uncaging” group, respectively), or RGD caging for the initial 12 h and uncaging for the following 24 h (“RGD caging-uncaging” group). Data are shown as means ± standard errors (n=3). Gene expression data are presented as relative fold expression of target genes (iNOS, CD80, Arg-1, and Ym2) after normalization to the “RGD uncaging” group. Scale bars indicate 50 µm. Multiple groups were compared by one-way ANOVA with Tukey-Kramer post-hoc test. Different alphabetical letters (a and b) were assigned to statistically significant differences with p-values less than 0.05. N.S. represents statistically non-significant differences between the compared groups.

Figure 4. Temporally regulated RGD uncaging in M2-polarizing medium stimulates M2 polarization phenotype of macrophages. (A) Quantitative gene expression analysis of M2 polarization markers (Arg-1 and Ym2) and M1 polarization markers (iNOS and CD80), and (B) immunofluorescent staining images of macrophages against Arg-1 (green), iNOS (red), and nuclei (blue) of the macrophages after 36 h in culture. Macrophages were allowed to adhere to the (RGD-GNP)-MNC heterodimer-decorated substrate under RGD caging or uncaging for the initial 12 h under basal growth medium and the subsequent 24 h in M2-polarizing medium (“RGD caging” and “RGD uncaging” group, respectively), or RGD caging for the initial 12 h and uncaging for the subsequent 24 h (“RGD caging-uncaging” group). Data are displayed as means ± standard errors (n=3). Gene expressions are presented as fold expression of genes of interest (Arg-1, Ym2, iNOS, and CD80) after the normalization to the “RGD caging” group. Scale bars represent 50 µm. One-way ANOVA with Tukey-Kramer post-hoc test was used to statistically compare various groups. Different alphabetical letters (a and b) were assigned to statistically significant differences (p < 0.05). N.S. indicates statistically non-significant differences between the compared groups.


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Figure 5. The RGD uncaging-mediated adhesion and M2 polarization of macrophages involve ROCK signaling. (A) Immnuofluorescent staining images against actin (red) and nuclei (blue) under basal medium cultures after 24 h, iNOS (green), Arg-1 (red), and nuclei (blue) under M1-polarizing medium cultures after 36 h, or Arg-1 (green), iNOS (red), and nuclei (blue) under M2-polarzing medium cultures after 36 h. For the polarization cultures, the cells were initially cultured in basal medium for 12 h and then polarized under either M1- or M2-polarizing medium for another 24 h. The macrophages were cultured on the (RGD-GNP)-MNC heterodimer-grafted substrate under RGD caging or uncaging (“RGD caging” and “RGD uncaging” group, respectively), or RGD uncaging in the presence of Y27632, the ROCK inhibitor ("RGD uncaging + Y27632” group). Scale bars indicate 50 µm. (B) Corresponding quantifications of the area and aspect ratio of the adhered macrophages. Data are displayed as mean ± standard errors (n=30). Quantitative gene expressions of M1 polarization markers (iNOS and CD80) or M2 polarization markers (Arg-1 and Ym2). Data are displayed as means ± standard errors (n=3). Gene expression data are presented as relative fold expression of target genes after the normalization to the “RGD uncaging” group for the iNOS and CD80, or the “RGD caging” group for the Arg-1 and Ym2. Various groups were compared by one-way ANOVA with Tukey-Kramer post-hoc test. Different alphabetical letters (a, b, and c) were assigned to statistically significant differences with p values less than 0.05. Same alphabetical letters represent statistically non-significant differences between the compared groups.

Figure 6. Magnetically controlled reversible RGD uncaging promotes the adhesion of host macrophages and inhibits their M1 polarization in vivo. (A) Schematic presentation of the subcutaneously implanted (RGD-GNP)-MNC heterodimer-grafted substrate under reversible RGD uncaging to regulate the adhesion and polarization of host macrophages. (B) Immunofluorescent staining against actin (green), iNOS (red), and nuclei (blue) of the adherent cells following 24 h of implantation under RGD caging or uncaging (“RGD caging” and “RGD uncaging” groups, respectively), or temporal switching of the RGD caging for the initial 12 h and uncaging for the subsequent 12 h (“RGD caging-uncaging” group). Scale bars indicate 20 µm. (C) Corresponding quantification of the density, area, and aspect ratio of the in vivo adhered macrophages after 24 h. Data are displayed as mean ± standard errors (n=30). Quantitative gene expressions of M1 polarization markers (iNOS and CD80) for the in vivo adhered cells after 24 h. 33 ACS Paragon Plus Environment

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Data are shown as means ± standard errors (n=3). Gene expression data are shown as relative fold expression of target genes after the normalization to the “RGD uncaging” group. Various groups were compared by one-way ANOVA with Tukey-Kramer post-hoc test. Different alphabetical letters (a and b) were assigned to statistically significant differences with p values less than 0.05. Same alphabetical letters indicate statistically non-significant differences between the compared groups.

Figure 7. Magnetic manipulation of reversible RGD uncaging promotes the adhesion of host macrophages and their M2 polarization in vivo synergistically with M2-polarizing cytokines. (A) Schematic representation of subcutaneously implanted heterodimer-decorated substrate under reversible RGD uncaging to regulate the adhesion and polarization of host macrophages synergistically with IL-4 and IL-13 injected onto the substrate. (B) Immunofluorescent staining micrographs against actin (green), Arg-1 (red), and nuclei (blue) following 24 h of implantation under RGD caging or uncaging (“RGD caging” and “RGD uncaging” group, respectively), or temporal change of the RGD caging for the initial 12 h and uncaging for the following 12 h (“RGD caging-uncaging” group). Scale bars represent 20 µm. (C) Corresponding quantification of cell density, area, and aspect ratio of the in vivo adhered macrophages after 24 h. Data are displayed as mean ± standard errors (n=30). Quantitative gene expressions of M2 polarization markers (Arg-1 and Ym2) for the in vivo adhered cells after 24 h. Data are shown as means ± standard errors (n=3). Gene expressions are presented as relative fold expression of genes of interest (Arg-1 and Ym2) after the normalization to the “RGD caging” group. One-way ANOVA and the Tukey-Kramer post-hoc test were used to compare various groups. Different alphabetical letters (a and b) were assigned to indicate statistically significant p-values (p < 0.05). Same alphabetical letters indicate statistically non-significant differences between the compared groups.


Page 35 of 44 Scheme 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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B

2000

Adherent cell aspect ratio (Major axis/minor axis)

Adherent cell area ( m2)

Basal medium

b

1500 a

a

1000 500 0

8

b

6 4

c a

2 0



iNOS Fold Expression

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

5

5 4

5

a a

3 2

b

b

4

4

2

5

5

a c

3 b

Ym2

Arg-1

CD80

3 2

c a

3 2

1

1

1

1

0

0

0

0


b

4

a a

Figure 6

ACS Nano

Page 42 of 44

A

24 h

Adhered macrophage area ( m 2)

b

1000

b

b

800 a

600 400 200 0

CD80

b

4

a

3 2

Actin/iNOS/Nuclei

Nuclei

iNOS

b

b

1

ACS Paragon Plus Environment 0

Macrophage elongation factor

Actin

Fold Expression

Fold Expression

Adhered macrophage density (Cells/cm2)

1 2 3 4 5 6 7 8 9 10 11 12B 13 14 15 Reversible RGD caging 16 12 h 24 h 17 0 h 18 19 20 21 Caging 22 23 24 25 26 27 28 Uncaging 29 30 31 32 33 34 Caging Uncaging 35 36 37 38 39 C 40 41 10000 b 42 43 8000 44 6000 a 45 46 4000 47 2000 48 49 0 50 51 iNOS 52 4 53 a 54 3 55 56 2 57 b 58 1 59 60 0

6 4 2 0

b

a

b

Figure Page 43 of 44

7

ACS Nano

A

24 h (IL-4 and IL-13 injection)

Adhered macrophage area ( m 2)

b

b

1000 800 600

a

400 200 0

Ym2 b

4

b

b

3 2

Actin/Arg-1/Nuclei

Nuclei

Arg-1

a

1

ACS Paragon Plus Environment 0

b

Macrophage elongation factor

Actin

Fold Expression

Fold Expression

Adhered macrophage density (Cells/cm 2)

1 2 3 4 5 6 7 8 9 10 11 12B 13 14 15 Reversible RGD caging 16 12 h 24 h 17 0 h 18 19 20 21 Caging 22 23 24 25 26 27 28 Uncaging 29 30 31 32 33 34 Caging Uncaging 35 36 37 38 39 C 40 41 b 10000 42 43 8000 44 6000 a 45 46 4000 47 2000 48 49 0 50 51 Arg-1 52 4 53 b 54 3 55 56 2 57 a 58 1 59 60 0

8 b

6 4 2 0

a

b

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

333x178mm (150 x 150 DPI)


Page 44 of 44

1 Magnetic Manipulation of Reversible ... - ACS Publications

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