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Remote Manipulation of Ligand Nano-Oscillations Regulates Adhesion and Polarization of Macrophages In Vivo Heemin Kang, Sungkyu Kim, Dexter Siu Hong Wong, Hee Joon Jung, Sien Lin, Kaijie Zou, Rui Li, Gang Li, Vinayak P. Dravid, and Liming Bian Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.7b03405 • Publication Date (Web): 06 Sep 2017 Downloaded from http://pubs.acs.org on September 7, 2017

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Remote Manipulation of Ligand Nano-Oscillations Regulates Adhesion and Polarization of Macrophages In Vivo Heemin Kang,†,⊥ Sungkyu Kim,§,∥,#,⊥ Dexter Siu Hong Wong,† Hee Joon Jung,§,∥,# Sien Lin,¶ Kaijie Zou,¶ Rui Li,† 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 and Traumatology, Faculty of Medicine, The Chinese University

of Hong Kong, Hong Kong, China. ∆

Shun Hing Institute of Advanced Engineering, The Chinese University of Hong Kong, Hong

Kong, China. ⌠

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

◊

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

□

Centre for Novel Biomaterials, The Chinese University of Hong Kong, Hong Kong, China.

Corresponding Authors *E-mail: [email protected]. *E-mail: [email protected].

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ABSTRACT Macrophages play crucial roles in various immune-related responses, such as host defense, wound healing, disease progression, and tissue regeneration. Macrophages perform distinct and dynamic functions in vivo, depending on their polarization states, such as the pro-inflammatory M1 phenotype and pro-healing M2 phenotype. Remote manipulation of the adhesion of host macrophages to the implants and their subsequent polarization in vivo can be an attractive strategy to control macrophage polarization-specific functions, but has rarely been achieved. In this study, we grafted RGD ligand-bearing superparamagnetic iron oxide nanoparticles (SPIONs) to a planar matrix via a long flexible linker. We characterized the nanoscale motion of the RGDbearing SPIONs grafted to the matrix, in real-time by in situ magnetic Scanning Transmission Electron Microscopy (STEM) and in situ Atomic Force Microscopy. The magnetic field was applied at various oscillation frequencies to manipulate the frequency-dependent ligand nanooscillation speeds of the RGD-bearing SPIONs. We demonstrate that a low oscillation frequency of the magnetic field stimulated the adhesion and M2 polarization of macrophages, whereas a high oscillation frequency suppressed the adhesion of macrophages, but promoted their M1 polarization, both in vitro and in vivo. Macrophage adhesion was also temporally regulated by switching between the low and high frequencies of the oscillating magnetic field. To the best of our knowledge, this is the first demonstration of the remote manipulation of the adhesion and polarization phenotype of macrophages, both in vitro and in vivo. Our system offers the promising potential to manipulate host immune responses to implanted biomaterials, including inflammation or tissue reparative processes, by regulating macrophage adhesion and polarization. KEYWORDS: ligand nano-oscillations, SPION, remote manipulation, macrophage adhesion, macrophage polarization


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Macrophages play pivotal roles in maintaining homeostasis, immune functions, and the progression of pathologies, such as cancer and atherosclerosis, and tissue regeneration.1-3 Macrophages possess an ability to polarize into different phenotypes to perform their distinct functions, including classically activated pro-inflammatory M1 phenotype and alternative prohealing M2 phenotype. Regulating macrophage polarizations to modulate their immune functions and elucidating the roles of polarization-regulating proteins have recently received a surge of interests.1,2,4-8 Furthermore, designing biomaterials that can modulate the adhesion and polarization of host macrophages is important to eliciting the desirable host responses and maintaining in vivo function of the implanted biomaterials, but this still remains a challenge.9-11 Emerging studies have showed the potential of engineering biomaterials to modulate the host responses by immune cells, such as macrophage M2 polarization, to regulate tissue regenerative processes.11-14 The in vivo macrophage behaviors are known to be regulated by the interaction between the cell surface receptors, such as integrin, and the ligand motifs, such as RGD sequences (Arg-Gly-Asp), present in the adhesive extracellular matrix proteins, including fibronectin and collagen.15 Thus, developing biomaterials with the adhesive ligand is an attractive approach to control the adhesion and functional activation of macrophages. Furthermore, the adherent cell shape of macrophages differs, depending on their polarization states: M1 macrophages exhibit clustered actin structures, whereas M2 macrophages display prevalent and pronounced actin organization.10,16-18 Recently, the micropatterning of the adhesive protein, such as fibronectin, which binds to integrin β1 of macrophages,19 has been shown to regulate the adherent cell morphology of macrophages and their polarization in vitro.16 Specifically, the elongated cell shape exhibiting pronounced actin cytoskeleton, promoted M2 polarization, while inhibiting M1 polarization. Furthermore, nanomaterials have been engineered, including surface coating with ligands,20 to modulate cellular behaviors, both in vitro and in vivo.21,22 The nanopatterning of the adhesive molecules,23 receptors,24 antibodies,25 or antigens26 has been successfully fabricated to control the adhesion and/or activation of immune cells, such as monocytes,23 neutrophils,24 or T cells.25,26 These findings suggest that the nano-arrangement of the adhesive ligand may modulate the adhesion and polarization of macrophages.


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The in vivo host responses to implanted biomaterials entail dynamic interactions between the biomaterials and immune cells, such as macrophages, through integrin and adhesive ligands.15 The remote manipulation of the ligand presentation of the biomaterials can be an appealing approach to dynamically controlling the adhesion and phenotype of host macrophages. The external control of cell-biomaterial interactions through ligand presentation has been largely demonstrated by the use of various light sources of different wavelengths, such as ultraviolet (UV),27,28 visible,29 and near-infrared light,30,31 and diverse light-responsive molecules, including photolabile molecules27,28,30 and photoisomerizable molecules.29,31 A recent study showed that the transdermal application of UV light for 10 min, with the estimated attenuation of 90% in the light intensity, modulated the in vivo adhesion of macrophages, but not their polarization.27 However, there have been no precedent reports of remotely manipulating the presentation of the ligand to modulate the adhesion as well as polarization of host macrophages in vivo. The magnetic field allows facile tissue penetration and thus has been harnessed for the remote manipulation of the motion of magnetic nanoparticles,32,33 for in vivo applications34 and clinics.35 We recently demonstrated that the static magnetic field can modulate the adhesion and differentiation of stem cells in vitro by altering the ligand tether mobility.36 In addition to the static application of the magnetic field, dynamic magnetic field is readily applicable with the spatiotemporal changes in the magnetic field, thereby allowing the temporal regulation on the dynamic motion of magnetic nanoparticles.32 For instance, the oscillatory motion of magnetic nanoparticles was dynamically manipulated by the external application of an oscillating magnetic field.37 These reports suggest that the application of an oscillating magnetic field may be utilized to remotely manipulate the motion of the magnetic nanoparticles decorated with the ligands in vivo to regulate the adhesion and polarization of host macrophages. Here, we present a strategy in which an oscillating magnetic field is used to remotely manipulate the nanoscale motion of superparamagnetic nanoparticles (SPIONs). The SPIONs conjugated with the RGD ligand were grafted to the planar matrix via a long (5 kDa) flexible polymeric linker (Scheme 1). Our findings showed that altering the ligand nano-oscillations by adjusting the oscillation frequencies of magnetic field had significant impact on the adhesion and polarization phenotypes of the plated macrophages in vitro and recruited host macrophages in vivo. Dynamic application of an oscillating magnetic field with time-controlled switching of the 4 ACS Paragon Plus Environment

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oscillation frequencies allowed the temporal regulation on the macrophage adhesion. As a noninvasive and non-contact approach without using sophisticated instruments, this remote manipulation of ligand nano-oscillations in vivo to regulate the adhesion and polarization of host macrophages on the implanted matrix via dynamic magnetic field is a promising technique for modulating immune responses to the implants and regenerative processes. We first fabricated the RGD ligand-tethered SPIONs and examined the ability to remotely manipulate the ligand oscillations at various speeds. We characterized the iron oxide phase of the SPIONs. Powder X-ray diffraction pattern showed the defined diffraction peaks, which coincided with those of the crystalline phase of the magnetite in the composition of Fe3O4 (Figure 1A), consistent with the previous report.38 We prepared SPION core-amino silica shell structure to further conjugate long flexible PEG linker to the amino group. We then examined the magnetic properties of the SPION alone and SPION@silica and found that both nanoparticles exhibit superparamagnetic properties with the overlapped hysteresis loops by vibrating sample magnetometer measurements (Figure 1B). The superparamagnetic properties of the SPION@silica allow the nanoparticles to respond reversibly with changes in the oscillations of the magnetic field, and are therefore desirable for achieving temporal control over their oscillations by adjusting the oscillation mode of magnetic field. We further measured the magnetic field strength as a function of distance from the permanent magnet, used for both in vitro and in vivo experiments in this study. The magnetic field strength decreased with increasing distance from the magnet (Supplementary Figure S1). In particular, the magnetic field strength, at approximately 1 cm distance from the magnet, was 124 ± 8 mT. The magnet was placed at approximately 1 cm distance from the in vitro cultures and in vivo mouse cage bottom, and subject to the oscillations in various frequencies to modulate the motion of the SPION@silica. The SPION and SPION@silica both exhibited the spherical shape with the diameter of 13 ± 1 nm and 42 ± 3 nm, respectively, as revealed by Transmission Electron Microscopy (TEM) images (Figure 1C). As shown by Dynamic Light Scattering analysis, the hydrodynamic size of the SPION and SPION@silica was 14 ± 3 nm and 45 ± 7 nm, respectively (Figure 1D), consistent with the size determined from the TEM images. We further coupled the flexible PEG linker to the SPION@silica and then RGD peptide to the PEG linker. The surface charges of the SPION@silica and PEGylated SPION, as measured by zeta potential, were 23.1 ± 3.7 mV and 5 ACS Paragon Plus Environment

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17.1 ± 5.6 mV, respectively, suggesting that the PEG linker shielded the positive charge of the amino group of the silica surface (Supplementary Figure S2). The coupling of the RGD peptide to the PEGylated SPION further changed the surface charge to -8.3 ± 4.1 mV. The chemical bond analysis by Fourier transform infrared spectroscopy further confirmed the coupling of PEG linker and RGD ligand. The absorption peak at 578 cm-1 for the SPION corresponds to the Fe-O stretching bond (Supplementary Figure S3), in good agreement with the previous report.39 The absorption peaks at 802 cm-1 and 1054 cm-1 for the RGD-bearing SPIONs, correspond to the SiO bonds, and another peaks at 1523 cm-1 and 1634 cm-1 correspond to amide bonds, indicating the presence of covalent coupling in the RGD-bearing SPIONs. We next characterized the tunability of the oscillation speeds of the RGD ligand-tethered SPIONs under the application of the oscillating magnetic field. The fluorescent RGD-bearing SPIONs were subject to the oscillatory motion by externally applying the magnetic field via oscillating the permanent magnet at various frequencies, and the real-time motion was tracked by the confocal microscope imaging, analogous to the previous report of microscopic tracking of the motion of nanoparticle.40 The oscillatory motion speeds of the RGD-bearing SPIONs increased with the increasing oscillation frequencies of the magnetic field, as shown in the movies and the quantification of the motion speeds (Supplementary Movies S1-4 and Figure 1E). Next, we tethered the RGD-bearing SPIONs to the planar matrix and characterized their in situ magnetic nanoscale motion as tethered to the matrix. We used a long flexible PEG linker with the molecular weight of 5 kDa to tether the SPIONs to the matrix, for facile motion of the SPIONs. The PEGylated SPIONs were grafted to the thiolated matrix (Supplementary Figure S4). Concomitantly, the surface hydrophilicity, measured as the water contact angle, decreased from 64 ± 3 o to 52 ± 5 o, which could be attributed to the hydrophilicity of the PEG linker molecule (Supplementary Figure S5A-B). The coupling of the RGD peptide to the PEGylated SPIONs further reduced the contact angle to 41 ± 2 o. We next visualized how the RGD-bearing SPIONs are grafted to the matrix. The RGD-bearing SPIONs were found to be randomly scattered on the matrix, as revealed by Scanning electron microscopy and two-dimensional (2D) Atomic force microscopy (AFM) images, with the quantified the nanoparticle density of 13 ± 2 and 15 ± 3 per µm2, respectively (Figure 2A-B). Furthermore, three-dimensional (3D) AFM images revealed the diameter of matrix-tethered RGD-bearing SPIONs to be approximately 45 nm (Figure 2C). The 6 ACS Paragon Plus Environment

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nano-arrangement of the adhesive ligands, such as their density and nanospacing, has been used to modulate the cellular adhesion,23,41-47 but not yet for the macrophages. Consistent with those reports on other cell types, we also found that the ligand density also played a role in regulating macrophage adhesion (data not shown), and this warrants further detailed investigations. Compared to these reports, we used a relatively lower density of the RGD-bearing SPIONs (approximately 13-15 particles/µm2) in this work to better elucidate the effect of ligand nanooscillations on macrophage adhesion and polarization. We further characterized the nanoscale motion of the RGD-bearing SPIONs on the matrix by applying dynamic magnetic field and utilizing in situ magnetic AFM imaging. Since non-magnetic reference is required to analyze the motion of the RGD-SPIONs, we co-conjugated the RGD-SPIONs in spherical shape together with the gold nanorods (approximately 76 nm in the long axis and 21 nm in the short axis) on the same matrix (Figure 2D). Under the static magnetic field, the SPIONs scanned on the same area exhibited the displacement less than 2 nm, relative to the nanorods (Supplementary Figure S6), by using the tapping mode so as not to disturb the position of the SPIONs by the scanning tip. In contrast, with the change in the magnetic field, the SPIONs exhibited the displacement of approximately 12.3 ± 3.1 nm (calculated from eight measurements). The size of integrin receptor that binds to the RGD ligand is reported to be around 10 nm,48,49 similar to the nanodisplacement of the SPIONs controlled by the dynamic magnetic field. In this study, we tethered the RGD ligands to the SPIONs via long flexible PEG linker (5 kDa). A recent work by Attwood et al. estimated the length of this PEG linker (5 kDa) to be approximately 38.2 nm.50 In this study, we were only able to determine the nanoscale displacement of the SPIONs on the matrix, but not that of the RGD ligands, by dynamic magnetic field. Since the RGD ligands were tethered to the SPIONs via PEG linker, the actual displacement of the RGD ligands could be in a larger length scale than that of the SPIONs under the motion. This added length scale to the SPIONs by the PEG linker could serve as an important design parameter in manipulating the macrophage adhesion by the ligand nano-oscillations, to be described later in the text. We next examined the real-time nanoscale motion of the RGD-SPIONs on the matrix by in situ magnetic High-Angle Annular Dark-Field Scanning Transmission Electron Microscopy (HAADF-STEM) imaging. We developed the method to tether the curved magnet to the tungsten tip of the electrical biasing holder, of which we controlled the continuous motion inside the STEM chamber for real-time imaging (Supplementary Figure S7). Adjacent to the RGD-SPIONs on the matrix, the motion of 7 ACS Paragon Plus Environment

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the magnet was continuously manipulated to exert dynamic magnetic field, under vacuum condition. The real-time motion of the RGD-bearing SPIONs on the matrix during the continuous movement of the magnet was shown in the movie (Supplementary Movie S5). These findings could suggest that tuning dynamic nanoscale motion of the RGD-bearing SPIONs may modulate the interaction between the RGD ligand and integrin of macrophages, and thus their adhesion. Next, we dynamically applied magnetic field at various oscillation frequencies, which could modulate the nano-oscillation speeds of the RGD-bearing SPIONs on the matrix, to evaluate the ability to regulate the adhesion of macrophages. We externally applied the magnetic field by moving the magnet approximately 1 cm distant from the matrix, at three different oscillation frequencies (0.1, 0.5, and 2 Hz) for the three groups, “Low frequency”, “Medium frequency”, and “High frequency”, respectively. We included the controls with static magnetic field, “Zero frequency” group as well as “No magnetic field” group. To minimize the non-specific adhesion of macrophages, we blocked the planar matrix with bovine serum albumin. After 24 h of culture in basal medium, the oscillation frequency-dependent ligand motion led to substantially different density of adherent cells (Figure 3A). The “Zero frequency” group exhibited similar extent of adhered cells compared with the “No magnetic field” group, suggesting non-oscillating magnetic field does not alter the adhesion of macrophages. Strikingly, compared to the “Zero frequency” group, “Low frequency” group significantly promoted the density of adherent cells, “Medium frequency” group exhibited similar level of cellular adhesion, and “High frequency” group significantly inhibited cellular adhesion. We further performed quantitative cell analysis based on the fluorescently stained images. Compared to “Zero frequency” group, “Low frequency” group exhibited 41% more cell numbers, while “High frequency” group showed 30% less cell numbers (Figure 3B). We next investigated whether these frequency-dependent disparate levels of cellular adhesion were driven by the RGD ligand-specific binding of the macrophages. We fabricated the matrix coated with the SPIONs, but without the RGD conjugation. We found that the adhesion densities of macrophages were considerably low, compared to the groups with the RGD conjugation as described above, and not significantly different amongst all the frequency groups (Supplementary Figure S8A-B). These findings suggest that blocking the matrix with BSA suppresses, but does not entirely prevent the non-specific (RGD-independent) adhesion of 8 ACS Paragon Plus Environment

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macrophages. Albeit with some degree of the non-specific adhesion of macrophages, the adhesion of macrophages was effectively manipulated by the frequency-dependent nanooscillations of the RGD ligands, in the presence of BSA blocking agent. We further examined the effect of the oscillating magnetic field on the adhesion of macrophages in the absence of magnetically responsive SPIONs on the matrix. We replaced the SPION@silica nanoparticles used in this study, by non-magnetically responsive pure silica nanoparticles coated with the RGD ligands on the matrix. We found that the size (41 ± 4 nm) of pure silica nanoparticles and their density as tethered to the matrix (Supplementary Figure S9A-B) are comparable to those of the SPION@silica nanoparticles. We applied the oscillating magnetic field and observed no significant differences in the number of the adhered macrophages amongst all the groups, i.e. “Zero frequency”, “Low frequency”, “Medium frequency”, “High frequency, and “No magnetic field” groups (Supplementary Figure S9C-D). We also evaluated the adhesion of macrophages to the matrix devoid of any conjugated nanoparticles under oscillating magnetic field. We observed the low degree of macrophage adhesion with no significant differences among all the groups (Supplementary Figure S10A-B). These findings suggest that the oscillating magnetic field does not significantly influence the adhesion of macrophages, in the absence of magnetically responsive RGD-bearing SPIONs. In addition to the adherent cell density, we analyzed the adhesive structure of macrophages by the staining of actin filaments and vinculin as well as quantification of the adherent cell area and shape (by elongation factor). “Low frequency” group exhibited 56% higher cell spread area and more elongated cell shape (139% higher elongation factor) than “Zero frequency” group (Figure 3A-B). Concomitantly, “Low frequency” group exhibited prevalent and pronounced cytoplasmic actin cytoskeletons and vinculin, suggesting substantial development of cell adhesion structures. In stark contrast, “High frequency” group exhibited 23% lower cell spread area and rounder cell morphology (52% lower elongation factor) than “Zero frequency” group, while “Zero frequency”, “Medium frequency”, and “High frequency” groups all exhibited the formation of actin clusters. We next investigated the ability of integrin β1 to bind to the oscillating RGD ligand, which is known to regulate the adhesion of macrophages.15,19 We performed the immunofluorescent staining against integrin β1 after supplying integrin β1 to the matrix with the oscillating RGD ligand. We found significantly higher and lower fluorescent intensity for the 9 ACS Paragon Plus Environment

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“Low frequency” and “High frequency” group, respectively, compared with the “Zero frequency” and “Medium frequency” group (Supplementary Figure S11A-B). These findings suggest that “Low frequency” and “High frequency” group promoted and inhibited the binding of integrin β1 to the oscillating RGD ligand, respectively. This is consistent with the previous observation of promoted and suppressed cellular adhesion by low and high frequency, respectively (Figure 3AB), suggesting that the oscillating magnetic field modulated integrin binding to the RGD ligand to regulate the macrophage adhesion. Having examined the distinct levels of adhesion of macrophages depending on the oscillation frequencies of the magnetic field, we evaluated whether the temporal change of the oscillation frequencies can further regulate the adhesion of macrophages. Time-regulated cellular adhesion has been demonstrated by the light-mediated photocleavage27,28,30 or photoisomerization.31,51 While these approaches entail chemical alterations of the ligand-presenting structure to modulate cell adhesion, the magnetic field can offer an alternative physical method to temporally tune the adhesion. We alternated between “Low frequency” and “High frequency” group and monitored the change in the adhesion of macrophages. We applied “Low frequency” for 12 h and then “High frequency” for another 12 h: “Low-High” group. We also applied “High frequency” for 12 h and then “Low frequency” for another 12 h: “High-Low” group. As described before, “Low frequency” and “High frequency” stimulated and suppressed the adhesion of macrophages for the initial 12 h of culture, respectively. Under the changed oscillation frequencies for another 12 h of culture, interestingly, the cellular adhesion also changed. Compared to initial 12 h of culture under “Low frequency”, another 12 h of culture under “High frequency” (i.e. “Low-High” group) led to some degree of loss in the adhesion with the decreased cell spread area by 11% and the reduced degree of elongation by 43% (Figure 4A-B). For the “High-Low” group, compared to initial 12 h of culture under “High frequency”, another 12 h of culture under “Low frequency” resulted in enhanced adhesion with the increased cell number by 107%, 58% higher cell spread area, and elevated degree of elongation by 135%. The findings suggest the potential utility of the magnetic field for temporal regulation of macrophage adhesion and potentially macrophage phenotypes.


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The adhesive structure of macrophages, such as actin assembly and cell morphology, has been reported to synergistically regulate the polarization phenotype of macrophages in the presence of either M1-inducing or M2-inducing cytokines.16 Since we observed the frequency-dependent distinct levels of macrophage adhesion, in the high and low frequency group analogous to the adhesion structure of M1 and M2 macrophages, respectively, we further investigated whether such regulation of cell adhesion can influence the polarization of macrophages. We cultured the macrophages in basal growth medium for 12 h to allow cells to adhere and then in the presence of either M1-inducing factors (lipopolysaccharide and interferon-γ) or M2-inducing cytokines (interleukin-4 and interleukin-13; IL-4 and IL-13) for another 24 h.16 The planar matrix bearing the RGD-bearing SPIONs was subject to various oscillation frequencies of the magnetic field throughout the whole 36 h of culture time. After the culture under M1-inducing medium, we first performed quantitative gene expression analyses for M1 polarization markers (iNOS and CD80). Compared to the “Zero frequency” group, the macrophages cultured under “Low frequency” of oscillating magnetic field exhibited significantly downregulated expression of iNOS by 52% and CD80 by 54% (Supplementary Figure S12A). In contrast, compared with the “Zero frequency” group, the “High frequency” group exhibited significantly upregulated expression of iNOS by 124% and CD80 by 281%. Under the same culture conditions with M1-inducing medium, no significant differences in the gene expression of M2 markers (Arginase-1 and Ym2) were found among all the groups (Supplementary Figure S12A). To further confirm oscillation frequencydependent polarization, we performed co-staining of M1 marker (iNOS) and M2 marker (Arginase-1). Only less than half of the adherent macrophages in the “Low frequency” group exhibited positive iNOS staining (Figure 5A). In stark contrast, almost all the cells in the “High frequency” group exhibited positive staining against iNOS, while the majority of the cells in the “No magnetic field”, “Zero frequency”, and “Medium frequency” groups stained positive against iNOS. All the groups showed minimal Arginase-1 staining. Next, we investigated how the oscillation frequency-dependent disparate adhesive structure influences the macrophage polarization to M2 phenotype in the presence of M2-inducing cytokines. Quantitative gene expression analyses revealed that M2 markers (Arginase-1 and Ym2) were significantly upregulated in the “Low frequency” group, compared with all other groups (Supplementary Figure S12B). No significant differences in the expression levels of M1 11 ACS Paragon Plus Environment

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markers (iNOS and CD80) were found amongst all the other groups (Supplementary Figure S12B). Immunofluorescent staining against Arginase-1 revealed the highly positive staining for the “Low frequency” group, while all the other groups exhibited considerably less intense staining (Figure 5B). Concomitantly, the staining against iNOS remained minimal in all the groups. These findings collectively suggest that “High frequency” group stimulated macrophage polarization toward pro-inflammatory M1 phenotype, while “Low frequency” group suppressed M1 polarization and promoted the polarization toward pro-healing M2 phenotype. The exact mechanisms underlying the distinct oscillation frequency-dependent regulation on macrophage adhesion and their polarization remain elusive. However, we attribute these results to the roles of cell shape and actin assembly in the macrophage phenotype, consistent with the previous reports showing that the elongated morphology of macrophages with the substantial actin assembly, inhibited the M1 polarization, but promoted M2 polarization.16 A previous study has demonstrated the manipulation of the motion of the magnetic nanoparticles in vivo,34 and we next evaluated the effect of the oscillation frequency-dependent motion of RGD-bearing SPIONs on the adhesion and polarization of the recruited host macrophages in vivo. We subcutaneously implanted the planar matrix coated with the RGD-bearing SPIONs into BALB/c mice and housed the mice in the confined cages mounted on top of the permanent magnet oscillating at various frequencies, approximately in 1 cm distance (Figure 6A). After 24 h of implantation, we analyzed the adhesion and polarization of the host macrophages. We used BALB/c mice that previously showed the recruitment and adhesion of host macrophages to the subcutaneously implanted matrix27,52 to evaluate the remote manipulation of the adhesion and polarization of host macrophages by using our system. We co-stained actin with M1 marker (iNOS) or M2 marker (Arginase-1) to analyze the cytoskeletal structure and phenotype of macrophages, where the cells with the expression of either iNOS or Arginase-1 were used to identify and quantify the macrophages. The matrix decorated with a monolayer of uniformly distributed RGD-bearing SPIONs (Supplementary Figure S13) exhibited the oscillation frequency-dependent adherent host cell number and morphology (Figure 6B and Supplementary Figure S14). The “No magnetic field” and “Zero frequency” group mostly round adherent macrophages that stained positive for iNOS, but minimal to negative for Arginase-1 (Supplementary Figure S15). Compared to the “Zero frequency” group, “High frequency” group 12 ACS Paragon Plus Environment

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exhibited almost all round macrophages (46% lower elongation factor) in 35% lower adherent cell number and they expressed iNOS, not Arginase-1. In stark contrast, “Low frequency” group stimulated the adhesion of host macrophages and led to 36% higher cell number, 67% higher cell area, and 108% higher elongation factor, compared with the “Zero frequency” group. Interestingly, the adherent host macrophages for the “Low frequency” group exhibited minimal positivity for iNOS, but some expression of Arginase-1. We further performed quantitative gene expression analyses for the polarization markers of the in vivo adhered cells, as previously reported.53 The gene expression for iNOS and CD80 was found to be significantly upregulated for the “High frequency” group, compared with all the other groups (Supplementary Figure S14). In contrast, the gene expression for Arginase-1 and Ym2 remained not significantly different amongst all the groups (Supplementary Figure S16). In addition to the host macrophages, neutrophils are also primary immune cells that are recruited within 24 h of implantation of biomaterials.19,27 Therefore, we stained against NIMP-14, the neutrophil marker, and found that the host neutrophils also adhered to the matrices in all the groups, with higher adherent cell number in the “Low frequency” group than all other groups (Supplementary Figure S17A-B). These are consistent with a previous report showing predominant M1-polarized macrophages along with neutrophils adhered to the RGD-bearing implanted biomaterials after 24 h of implantation.27 Collectively, these findings suggest that “Low frequency” and “High frequency” group stimulated and suppressed the adhesion of host macrophages, respectively, similar to the trend observed in the in vitro culture. The roundest shape of host macrophages in the “High frequency” promoted their M1 polarization. The “Low frequency” group appeared to induce the expression of M2 macrophages, but not considerably, possibly due to the acute inflammation dominant within the 24 h of implantation in the absence of any external intervention. Therefore, we performed another in vivo experiment using the same procedures as before, but further injected M2-inducing cytokines, IL-4 and IL-13, onto the implanted matrix immediately after implantation, to evaluate whether the oscillation frequencies can modulate the M2 polarization of the host macrophages (Figure 6A). The in vivo administration of M2-polarizing cytokines, such as IL-453 and IL-1354, was previously shown to induce the polarization of host macrophages into M2 phenotype. The trend of the oscillatory frequency-dependent adhesion of host macrophages 13 ACS Paragon Plus Environment

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with the injection of M2-inducing cytokines (Figure 6C and Supplementary Figure S18) was similar to the results obtained without injecting the exogenous cytokines (Figure 6B and Supplementary Figure S14). The “No magnetic field” and “Zero frequency” group exhibited a majority of round adherent macrophages in the mixture of Arginase-1-positive cells (Figure 6C) and iNOS-positive cells (Supplementary Figure S19). Compared to the “Zero frequency” group, “High frequency” group exhibited nearly all round macrophages (44% lower elongation factor) and 36% lower adherent cell number. These cells remained nearly negative for Arginase-1, but highly positive for iNOS. Compared with the “Zero frequency” group, “Low frequency” group enhanced macrophage adhesion with 38% higher adherent cell number, 88% higher cell area, and 46% lower elongation factor, and the majority of cells stained highly positive against Arginase-1, but minimally positive for iNOS. The quantitative gene expression analyses corroborated the pattern observed with the immunofluorescent staining. The gene expression for Arginase-1 and Ym2 was significantly upregulated for the “Low frequency” group, compared with all other groups (Supplementary Figure S18), while the expression for iNOS and CD80 was generally lower in the “Low frequency” group, but higher in the “High frequency” group (Supplementary Figure S20). The adhesion of NIMP-14-positive host neutrophils was also found in all the groups with higher adherent cell number in the “Low frequency” group than all other groups (Supplementary Figure S21A-B). Taken together, these findings suggest that “Low frequency” group with M2-inducing cytokines synergistically stimulated M2 polarization phenotype of host macrophages in vivo, while “High frequency” group promoted M1 polarization of host macrophages albeit with the addition of exogenous M2-inducing cytokines. The injection of M2-polarizing cytokines positively induced M2 polarization of the host macrophages, particularly for the “Low frequency” group. These results demonstrate the capability to remotely regulate host macrophage polarization in vivo with the oscillating magnetic field. Furthermore, the structure of RGD ligand-coated silica shell of SPIONs used in this study can be further modified as controlled delivery vehicles for immunoregulatingcytokines, such as porous silica shell nanoparticle,53 and this will further enhance the regulation of macrophage polarization to influence fibrous encapsulation of biomaterials or pro-healing immune response-mediated tissue reparative responses in vivo.


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To recapitulate the study, we showed a strategy for remotely manipulating the nano-oscillations of the adhesive ligands with magnetically responsive SPIONs, simply by adjusting the oscillating frequency of magnetic field. High frequency oscillations suppressed the adhesion of macrophages and promoted pro-inflammatory M1 polarization phenotype in the in vitro and in vivo microenvironments. In stark contrast, low frequency oscillations promoted the adhesion and pro-healing M2 polarization of the macrophages, in vitro and in vivo. Reversible macrophage adhesion was also demonstrated by switching the frequencies during the cell adhesion. To the best of our knowledge, this study is the first to show the remote regulation of adhesion and polarization phenotypes of macrophages by using dynamic magnetic field, both in vitro and in vivo. The in vivo regulation of macrophage adhesion and polarization can modulate the immune responses to implanted biomaterials, thus offering promising potential in enhancing regenerative therapies. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Characterizations of the surface charges and chemical bonds of the SPIONs and RGD-bearing SPIONs, the changes in the surface chemistry and surface hydrophilicity for the matrix-grafting of the RGD-bearing SPIONs, characterization control of nanoscale displacement of the matrixgrafted RGD-bearing SPIONs, macrophage adhesion on the oscillating non-RGD-bearing SPIONs, integrin β1 binding analysis to the oscillating RGD-bearing SPIONs, distribution of RGD-bearing SPIONs on the matrix used for in vivo implantation, host macrophage polarization and neutrophils analyses by the quantitative gene expression and immunofluorescent staining with and without the injection of M2-polarizing cytokines, movies for the frequency-dependent oscillations of the RGD-bearing SPIONs, and the movie for the real-time nanoscale motion of the RGD-bearing SPIONs on the matrix by in situ magnetic High-Angle Annular Dark-Field Scanning Transmission Electron Microscopy (HAADF-STEM) imaging with the set-up descriptions. AUTHOR INFORMATION 15 ACS Paragon Plus Environment

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Corresponding Authors *E-mail: [email protected]. *E-mail: [email protected]. ORCID Heemin Kang: 0000-0003-2694-9882 Vinayak P. Dravid: 0000-0002-6007-3063 Liming Bian: 0000-0003-4739-0918 Author Contributions ⊥Contributed equally to this work. The authors declare no conflict of interest. ACKNOWLEDGEMENTS Project 31570979 is supported by the National Natural Science Foundation of China. The work described in this paper is supported by a General Research Fund grant from the Research Grants Council of Hong Kong [Project No. 14202215, 14220716]. This work is supported by the Health and Medical Research Fund, the Food and Health Bureau, the Government of the Hong Kong Special Administrative Region [Reference No.: 02133356, 03140056]. This research is supported by the Chow Yuk Ho Technology Centre for Innovative Medicine (The Chinese University of Hong Kong). This work was performed at the EPIC facility of Northwestern University’s NUANCE Center with the support from the Soft and Hybrid Nanotechnology Experimental (SHyNE) Resource (NSF ECCS-1542205); the MRSEC program (NSF DMR1121262) at the Materials Research Center; the International Institute for Nanotechnology (IIN); the Keck Foundation; and the State of Illinois, through the IIN. We gratefully thank Mr. Gang Yang for the assistance in the experiments.


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Figure Legends Scheme 1. The outline of the experimental protocol used in this report. The RGD-bearing superparamagnetic nanoparticles (SPIONs) were coupled to the matrix via flexible polymeric linker. By oscillating the magnet (thus, magnetic field) in various frequencies, the nanoscale motion of the RGD-bearing SPIONs was remotely manipulated to regulate the adhesion and polarization of the plated macrophages in vitro and the recruited host macrophages in vivo. Figure 1. Remotely manipulable ligand oscillation speeds of the RGD-bearing SPIONs. (A) Xray diffraction pattern of the SPIONs. The diffraction peaks of the magnetite phase of the iron oxide was used to assign the planar indices. (B) Magnetic property, (C) transmission electron microscopic images, and (D) dynamic light scattering of the SPIONs and the SPION core and the SPION core-silica shells. The magnetic moment was shown after the normalization to the dry weight. Scale bar represents 20 nm. (E) Quantification of the oscillatory motion speeds of the RGD-bearing SPIONs at various oscillation frequencies (0, 2.5, 5, or 12.5 mHz) of the magnetic field, calculated from the real-time confocal microscopy imaging. Data are shown as mean ± standard errors (n=30). Figure 2. In situ imaging of the nanoscale displacement of the matrix-tethered RGD-bearing SPIONs. (A) Scanning electron microscopy image and (B) two-dimensional (2D) and (C) threedimensional (3D) atomic force microscopy (AFM) image of the matrix-tethered RGD-bearing 21 ACS Paragon Plus Environment

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SPIONs in the homogeneously distributed monolayer. Scale bars indicate 1 µm. (D) Transmission electron microscopy image of the non-magnetic gold nanorods. Scale bar indicates 50 nm. (E) In situ AFM images of scanning the identical area under the external magnetic field applied from the magnet in the opposite positions outside the scanning area of image (i) vs. image (ii). The RGD-bearing SPIONs were differentially identified by the spherical shape from the non-magnetic reference, gold nanorods in the aspect ratio of 4. Blue dotted lines indicate the reference line drawn along the longer axis of the gold nanorods, to characterize the relative displacement of the SPIONs. Black dotted lines and red arrows indicate the centers of the SPIONs and the direction of movement of the SPIONs in the left image, respectively, to show the relative displacement of the SPIONs in the right image. Scale bars represent 50 nm. Figure 3. Tuning ligand oscillations by the oscillation frequencies modulate the adhesion of macrophages. (A) Immnuofluorescent staining micrographs of vinculin (green), actin (red), and nuclei (blue) after 24 h of macrophage culture on the matrix bearing RGD ligand-grafted SPION under the application of various oscillation frequencies of the magnetic field (“Zero frequency”, “Low frequency”, “Medium frequency”, and “High frequency”, corresponding to 0, 0.1, 0.5, and 2 Hz, respectively). The control includes the “No magnetic field” group. Scale bars indicate 20 µm. (B) Corresponding quantifications of adherent cell density, area, and elongation factor. Data are displayed as mean ± standard errors (n=30). Different letters represent statistically significant differences (p < 0.05) as determined by one-way ANOVA with Tukey-Kramer post-hoc test. Figure 4. Time-regulated ligand oscillations by the oscillation frequencies dynamically tune the adhesion of macrophages. (A) Immunofluorescent staining images of vinculin (green), actin (red), and nuclei (blue) after 12 h and 24 h of macrophage culture on the matrix coated with RGD ligand-coupled SPION under temporal switching of the applied oscillation frequencies of the magnetic field. The oscillation frequencies were maintained at low frequency (0.1 Hz) or high frequency (2 Hz) for 12 h of culture, and they were switched from low to high frequency (“Low-High frequency”) or high to low frequency (“High-Low frequency”) and the culture continued for another 12 h, until the total of 24 h of culture. Control groups under either “Low frequency” or “High frequency” that were not switched during 24 h of culture were also included. Scale bars represent 50 µm. (B) Corresponding quantification of adherent cell area and 22 ACS Paragon Plus Environment

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elongation factor. Data are shown as mean ± standard errors (n=30). Different letters indicate statistically significant differences (p < 0.05) of the same group between different culture time, as determined by two-tailed Student’s t-test. Figure 5. Low-frequency ligand oscillation inhibits M1 polarization of macrophages, but promotes their M2 polarization. Immunofluorescent staining against (A) iNOS (green), arginase1 (red), and nuclei (blue) of the macrophages cultured in M1-polarizing medium or (B) arginase1 (green), iNOS (red), and nuclei (blue) of the macrophages cultured in M2-polarizing medium. The cultures were subject to the application of various oscillation frequencies of the magnetic field (“Zero frequency”, “Low frequency”, “Medium frequency”, and “High frequency”, corresponding to 0, 0.1, 0.5, and 2 Hz, respectively) with the control of the “No magnetic field” group. The cells were cultured for 12 h in basal growth medium and subsequently for another 24 h under M1-polarizing medium, in the presence of M1-polarizating co-stimulators, 10 ng/mL lipopolysaccharide (LPS) and 10 ng/mL recombinant interferon-gamma (IFN-γ), or under M2polarizing medium, in the presence of M2-polarizating co-stimulators, 20 ng/mL interleukin-4 (IL-4) and 20 ng/mL interleukin-13 (IL-13). Scale bars represent 50 µm. Figure 6. High frequency ligand oscillation suppresses the adhesion of macrophages, but promotes their M1 polarization, whereas low frequency ligand oscillation promotes the adhesion and M2 polarization of macrophages in vivo. (A) Schematic presentation of the subcutaneously implanted matrix coated with the RGD-bearing SPIONs under the oscillations of the magnet in various frequencies, to examine the adhesion and polarization of the host macrophages, with and without the injection of M2-polarizing cytokines onto the matrix. Immunofluorescent staining against (B) actin (green), iNOS (red), and nuclei (blue) of the cells adhered to the implanted matrix bearing RGD ligand without the injection of M2-polarizing cytokines as well as (C) actin (green), iNOS (red), and nuclei (blue) of the matrix-adhered cells with the injection of M2polarizing cytokines. The cells were analyzed after 24 h of implantation under the application of various oscillation frequencies of the magnetic field (“Zero frequency”, “Low frequency”, and “High frequency”, corresponding to 0, 0.1, and 2 Hz, respectively) with the control of the “No magnetic field” group. Scale bars represent 20 µm. For the injection of M2-polarizing cytokines,


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each 100 ng of interleukin-4 and interleukin-13 (M2 polarization co-stimulators) were injected onto the implanted matrix immediately after the implantation.


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Scheme 1

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Nano Letters

Figure 3 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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A Actin/Nuclei

Vinculin

Vinculin

Low frequency Medium frequency High frequency

Oscillating magnetic field

Basal medium

Zero frequency

No magnetic field

Actin/Nuclei

Vinculin/Actin/ Nuclei

15000 a 10000 5000 0

b a

a c

2000

b 1500

a

a

1000 500 0 ACS Paragon Plus Environment

a c

Elongation factor

20000

Adhered cell area (m 2)

Adhered cell density (Cells/cm2)

B

b

8 6 4

a

a

a c

2 0

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Nano Letters

Figure 4 A 0h

12 h

24 h

Vinculin

Actin/Nuclei

Vinculin

Low

Frequency of oscillating magnetic field

Basal medium

24 h

12 h Actin/Nuclei

High

Low

High

High

Low

12 h

20000

Adhered cell area (m 2)

Adhered cell density (cells/cm2)

B 24 h

15000

b

10000

a

5000 0 Low

Elongation factor

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

High

Low High High Low

24 h

12 h

10

a

8

b

6 b

4

a

2 0 Low

High

Low High High Low ACS Paragon Plus Environment

12 h

2000

24 h a

1500

b

b a

1000 500 0 Low

High

Low High High Low

Figure 5 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

Nano Letters

A

B


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Nano Letters

Figure 6

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

A

OR

Without injection of M2-polarizing cytokines

B

Injection of M2-polarizing cytokines

C


Nano Letters

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

For TOC only 312x123mm (150 x 150 DPI)


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Remote Manipulation of Ligand Nano-Oscillations ... - ACS Publications

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