Remote Control of Multimodal Nanoscale Ligand Oscillations

Subscriber access provided by UNIVERSITY OF ADELAIDE LIBRARIES

Article

Remote Control of Multi-Modal Nanoscale Ligand Oscillations Regulates In Vivo Stem Cell Adhesion and Differentiation Heemin Kang, Dexter Siu Hong Wong, Xiaohui Yan, Hee Joon Jung, Sungkyu Kim, Sien Lin, Kongchang Wei, Gang Li, Vinayak P. Dravid, and Liming Bian ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b02857 • Publication Date (Web): 25 Aug 2017 Downloaded from http://pubs.acs.org on August 27, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Nano is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 41

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

ACS Nano

Remote Control of Multi-Modal Nanoscale Ligand Oscillations Regulates In Vivo Stem Cell Adhesion and Differentiation Heemin Kang,†,⊥ Dexter Siu Hong Wong,†,⊥ Xiaohui Yan,‡ Hee Joon Jung,§,∥,# Sungkyu Kim,§,∥,# Sien Lin,¶ Kongchang Wei,† Gang Li,¶ Vinayak P. Dravid,§,∥,#,* and Liming Bian†,∆,⌠,◊,□,* †

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

China. ‡

Department of Physics, 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.

1 ACS Paragon Plus Environment

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

Page 2 of 41

ABSTRACT Cellular adhesion is regulated by the dynamic ligation process of surface receptors, such as integrin, to adhesive motifs, such as Arg-Gly-Asp (RGD). Remote control of adhesive ligand presentation using external stimuli is an appealing strategy for the temporal regulation of cellimplant interactions in vivo and was recently demonstrated using photochemical reaction. However, the limited tissue penetration of light potentially hampers the widespread applications of this method in vivo. Here, we present a strategy for modulating the nanoscale oscillations of an integrin ligand simply and solely by adjusting the frequency of an oscillating magnetic field to regulate the adhesion and differentiation of stem cells. A superparamagnetic iron oxide nanoparticle (SPION) was conjugated with the RGD ligand and anchored to a glass substrate by a long flexible polyethylene glycol (PEG) linker to allow the ligand oscillatory motion of the ligand to be magnetically tuned. In situ magnetic scanning transmission electron microscopy (STEM) and atomic force microscopy (AFM) imaging confirmed the nanoscale motion of the substrate-tethered RGD-grafted SPION. Our findings show that ligand oscillations under a low oscillation frequency (0.1 Hz) of the magnetic field promoted integrin-ligand binding and the formation and maturation of focal adhesions and therefore the substrate adhesion of stem cells, while ligands oscillating under high frequency (2 Hz) inhibited integrin ligation and stem cell adhesion, both in vitro and in vivo. Temporal switching of the multi-modal ligand oscillations between low and high frequency modes reversibly regulated stem cell adhesion. The ligand oscillations further induced the stem cell differentiation and mechanosensing in the same frequency-dependent manner. Our study demonstrates a non-invasive, penetrative, and tunable approach to regulate cellular responses to biomaterials in vivo. Our work not only provides additional insight into the design considerations of biomaterials to control cellular adhesion in vivo, but also offers a platform to elucidate the fundamental understanding of the dynamic integrin-ligand binding that regulates the adhesion, differentiation, and mechanotransduction of stem cells. KEYWORDS: integrin ligand oscillations, SPION, multi-modal control, mesenchymal stem cells, in vivo cell adhesion, stem cell differentiation


Page 3 of 41

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

ACS Nano

Many cell behaviors and functions, such as adhesion, are dynamically regulated by the interactions between cells and the extracellular matrix (ECM) in vivo.1,2 Such cell-ECM interactions are mediated by the dynamic binding of cell surface receptors, such as integrin, to their recognition ligands, such as the RGD tripeptide sequences (Arg-Gly-Asp), present in celladhesive ECM components including fibronectin, vitronectin, and collagen.3,4 Integrins are heterodimeric proteins with two membrane-spanning subunits, which regulate intracellular mechanotransduction through dynamic links to adhesive ligands.5-7 The ligation of integrins to their adhesive ligands triggers the formation of cell focal adhesions, which link to the actin cytoskeleton and mediate mechanotransduction signaling. The decoration of integrin ligands such as RGD peptide on biomaterials, particularly synthetic materials lacking such integrin recognition motifs, is essential for conferring the required bioactivity and eliciting the desirable host responses of biomaterial implants. Integrins undergo free diffusion and immobilization cycles in regulating focal adhesions through their dynamic nanoscale organizations.8

The focal adhesion-mediated cell traction force is

highly dynamic and undergoes temporal changes that are attributed to the dynamic integrinligand coupling.7,9 It was recently shown that the magnitude of cell adhesion forces on fibronectin or an anti-integrin β1-coated surface displayed oscillatory cycles at a specific frequency.10 Such prior reports imply that dynamic materials with nanoscale oscillations of integrin ligands could be harnessed to regulate cellular adhesion. Dynamic presentation of cell-adhesive ligands offers great potential utility in modulating multifaceted cell-biomaterial interactions.11-14 A recent study showed that the mobile RGD ligand tethered to biomaterials can be harnessed to modulate integrin-ligand binding, and therefore cell adhesion in a dynamic fashion.15 Furthermore, the remote control of the presentation of integrin ligand can offer versatile opportunities for the temporal regulation of the cell-adhesive microenvironment.11,16 Such potential has been predominantly realized with in vitro culture platforms by the application of various external stimuli,11 such as electrical potential,17,18 heat,19 enzyme,20 and light.21-27 Among these different stimuli, light, with its potential in vivo applicability, has been most extensively exploited to modulate cellular adhesion and detachment via photochemical reactions, such as the photocleavage of caging molecules21-23 3 ACS Paragon Plus Environment

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

Page 4 of 41

or linker moieties,24,25 or photo-induced isomerization.26,27 Lee et al. recently reported the utilization of light for the temporal regulation of cellular adhesion in vivo.23 However, light that can trigger photochemical reactions is readily absorbed by living tissues, thus resulting in limited penetration depth and potential harmful effects on living cells in vivo. Alternatively, a magnetic field has been utilized to remotely control the motion of magnetic nanoparticles and can penetrate living tissues with significantly reduced absorption, thus allowing widespread and prolonged (longer than hourly scale) applications in vivo.16,28-30 A high-intensity magnetic field (3 Tesla) has been applied for the magnetic resonance imaging of patients, and no harmful effects have been reported.31 Our own study also showed that a static magnetic field can be used to tune the tether compliance of integrin ligands on a substrate to modulate cell adhesion for in vitro culture.32 In addition, oscillating magnetic fields have been applied to manipulate the oscillatory responses of magnetic nanoparticles.30 Thus, harnessing the advantages of oscillating magnetic fields and nanotechnology to modulate the dynamic presentation of integrin ligands can offer a versatile strategy to regulate cell-material interactions in vivo. In this study, we developed a system in which the nanoscale motion of an integrin ligand can be remotely controlled simply by tuning the frequency of an oscillating magnetic field as graphically illustrated in Scheme 1. Specifically, a RGD ligand-tethered superparamagnetic iron oxide nanoparticle (SPION) was coupled to a substrate by a long flexible polyethylene glycol (PEG) linker (Mn = 5000 Da) to allow its controllable motion. We demonstrate that changing the frequency of the applied oscillating magnetic field led to significant changes in the motion speed of the ligand-grafted SPION, and this suggests that a magnetic field can be used to regulate the nanoscale motion of substrate-tethered ligand-grafted SPION in a frequency-dependent manner. Our results show that stem cell adhesion was regulated solely by altering the frequency of the oscillating magnetic field, both in vitro and in vivo. We also show the temporal regulation of stem cell adhesion and differentiation by switching the oscillation frequency of the magnetic field during the cellular adhesion processes. RESULTS AND DISCUSSION Tunable oscillatory motion speeds of adhesive ligand-bearing SPION. Bioadhesive ligand oscillations were mediated by the oscillating movement of SPION conjugated with the RGD 4 ACS Paragon Plus Environment

Page 5 of 41

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

ACS Nano

ligand. X-ray diffraction spectra revealed that the diffraction peaks of the SPION corresponded to those of magnetite (Fe3O4) (Figure 1A), as reported previously.33 Transmission electron micrographs (TEM) of the SPIONs showed that the size of the SPIONs was 15 ± 3 nm (Supplementary Figure S1A), which increased to 40 ± 4 nm upon amino-functionalized silica coating (Figure 1B). Further, dynamic light scattering (DLS) measurement revealed that the hydrodynamic sizes of the bare and silica-coated SPION were 14 ± 2 nm and 46 ±, 5 nm respectively, which is consistent with the TEM observations (Figure 1B and Supplementary Figure S1B). We then characterized the magnetic properties of the bare and silica-coated SPION. Vibrating sample magnetometer hysteresis measurement showed that both the aminofunctionalized silica-coated SPIONs (Figure 1D) and bare SPIONs (Supplementary Figure S1C) displayed superparamagnetic characteristics, consistent with a previous finding33 with saturation magnetization (Ms) of 21 and 51 emu/g, respectively. This finding suggests that the magnetic responses of the SPIONs can be solely controlled by an external magnetic field, with complete reversibility. We next characterized the conjugation of the long flexible linker molecule and the subsequent grafting of the RGD ligands onto the silica-coated SPIONs. Zeta potential measurements revealed sequential changes in the surface charges in each step of the molecular tethering process. The amino-silica coating conferred a positive surface charge of 20.6 ± 6.1 mV, which differed from the bare SPION of -1.3 ± 1.1 mV (Supplementary Figure S2A). We further grafted the flexible linker of maleimide-polyethylene glycol-N-hydroxysuccinimide ester, with a large molecular weight (Mn = 5000 Da), to the amino-functionalized surface of the SPIONs, resulting in a negative surface charge of -17.5 ± 4.7 mV. We subsequently tethered the RGD peptide onto the PEGylated surface of the SPIONs, yielding a surface charge of -7.7 ± 4.8 mV. In addition to the analysis of the changes in the surface charges, Fourier transform infrared spectroscopy measurements revealed that Fe-O bonds of the bare SPIONs detected at 582 cm-1 (Supplementary Figure S2B), consistent with a previous report.34 The RGD ligand-tethered SPIONs exhibited the absorption peaks at 795 cm-1 and 1065 cm-1, indicative of Si-O bonds, and peaks at 1539 cm-1 and 1648 cm-1, suggestive of amide II and amide I bond, respectively, and these collectively confirmed the successful tethering of the RGD peptide to the SPIONs.


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

Page 6 of 41

Following the characterization of the bioadhesive ligand-bearing SPION, we characterized the controllable motion of the SPION under an oscillating magnetic field. We fluorescently labeled the SPION with rhodamine isothiocyanate. We externally applied an oscillating magnetic field by oscillating a permanent magnet at various frequencies ranging from 0 to 12.5 mHz and monitored the motion trajectory of the SPION. The red-fluorescent SPION displayed an oscillating motion under the oscillating magnetic field and the motion speeds of SPION increased as the oscillation frequency (i.e. oscillation speed) of the magnetic field increased (Figure 1E-F). The real-time motion of the SPION under an oscillating magnetic field is shown in the movies, on which bright field images were overlain as references (Supplementary Movies S1-S4). Such trackable motion of the nanoparticle was previously demonstrated using an optical microscope.35 Such controlled motion speeds of the SPION, achieved by changing the frequency of the applied oscillating magnetic field, could be extrapolated to tuning the nanoscale motion speeds of the SPION tethered to a substrate via long flexible linker, in a frequency-dependent manner.

In situ magnetic imaging of nanoscale motion and integrin binding of adhesive ligandbearing SPION tethered onto a substrate. Having verified the tunable motion speeds of the RGD ligand-SPION by modulating the oscillation frequencies of the applied magnetic field, we further conjugated such the ligand-SPION to a substrate and examined whether the controllable motions of the substrate-tethered SPION under various magnetic field oscillations are achievable. To allow the nanoscale motion of the substrate-tethered SPION under a magnetic field, we utilized a long flexible linker, PEG (Mn = 5000 Da). Tethering of the RGD ligand to a substrate via a PEG molecule of such a high molecular weight is known to inhibit cell adhesion.36 We pretreated the glass substrate to present thiol groups for the subsequent serial conjugations of the PEGylated SPIONs and RGD ligand (Supplementary Figure S3). We measured the water contact angles to analyze the changes in the surface chemistry of the substrates (Supplementary Figure S4A-B). The contact angle measurements revealed that the thiolization of the glass substrate with mercaptopropylsilatrane (MPS) molecules reduced the surface hydrophilicity with increasing the water contact angle from 34.7 ± 2.4

o

to 61.7 ± 2.3 o. Following the grafting of PEGylated

SPION onto the substrate, surface hydrophilicity increased with a decreased contact angle to 42.0 ± 2.8 o owing to the hydrophilic nature of PEG molecule. We further tethered RGD peptide 6 ACS Paragon Plus Environment

Page 7 of 41

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

ACS Nano

to the PEGlyated SPION and observed the contact angle of 39.6 ± 1.4 o. We further assessed the distribution and topography of tethered RGD ligand-SPION on the substrate. Scanning electron microscopy (SEM) images show a homogeneous distribution of tethered ligand-SPION in a monolayer onto the substrate (Figure 2A). The density of the ligand-tethered SPION was measured as 12 ± 2 particles/µm2. Atomic force microscopy (AFM) analysis showed the substrate-tethered SPION with lateral and vertical diameters of around 40 nm (Figure 2B), consistent with prior TEM analysis. Thus, we suggest that such tethered SPIONs present RGD ligand clusters37 that have been shown to regulate cellular adhesion and differentiation depending on various parameters, such as the coupling strength,38 spacing,39 and order/disorder.40 We next characterized the in situ nanoscale motion of the ligand-SPION under an oscillating magnetic field by in situ AFM imaging. We co-conjugated gold nanorods onto thiolated substrate through Au-S bond, and the gold nanorods possess an aspect ratio of 4, in stark contrast to spherical shape of the SPION, and serve as a non-magnetic reference. TEM imaging revealed the gold nanorod exhibited the dimension of approximately 78 nm × 18 nm (Supplementary Figure S5A). Furthermore, we used tapping mode of AFM imaging to minimize the non-magnetic motion of the nanoparticles that could be caused by the scanning cantilever probe, and the nonmagnetic movement was negligible within 1 nm-scale (Supplementary Figure S5B). We subsequently characterized the magnetic motion of the ligand-SPION against the reference nanorod under magnetic field applied in different locations and found that the center of the ligand-SPION relative to the gold nanorod under oscillating magnetic field was shifted (Figure 2C) by approximately 9.4 ± 2.1 nm through quantification of 10 movements. Interestingly, such nanoscale ligand displacement is comparable to the dimension of integrin, which is approximately 10 nm.39,41,42 Furthermore, we characterized the nanoscale motion of the ligandSPION by in situ magnetic scanning transmission electron microscopy (STEM) imaging. We constructed the set-up by attaching the magnet to the in situ electrical biasing holder to manipulate magnetic field inside the STEM chamber during in situ STEM imaging (Supplementary Figure S6A-B). We characterized the magnetic motion of the ligand-SPION by changes in their inter-particle spacing under magnetic field applied in three different locations as revealed by relative positional changes of upper ligand-SPIONs to lower ligand-SPION through the movement of both upper and lower ligand-SPIONs (Figure 2D and Supplementary Figure 7 ACS Paragon Plus Environment

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

Page 8 of 41

S7). The inter-particle spacing between the centers of ligand-SPIONs was found to change approximately by 11.2 ± 3.3 nm through quantification of 10 movements. We demonstrated that the motion speed of the non-substrate-tethered ligand-SPION was closely dependent on the oscillation frequency of the magnetic field (Figure 1E-F and Supplementary Movies S1-4). Therefore, we postulate that the frequency of magnetic field oscillation will influence the oscillation speed of the tethered RGD-bearing SPION, and this may affect the integrin-RGD binding and therefore cell adhesion and spreading on the substrate. To test our hypothesis, we examined whether the oscillation speed of the tethered ligand-SPION under various oscillation frequencies of the applied magnetic field would modulate integrin-ligand binding, which is known to mediate focal adhesion and mechanosensing. We applied magnetic field of various oscillation frequencies (0, 0.1, 0.5, or 2 Hz; hereafter referred to as “Stationary”, “Slow”, “Moderate”, and “Fast”, respectively) to the substrate conjugated with RGD-bearing SPIONs and monitored the binding of integrin β1 to the RGD ligand. Immunofluorescent staining of integrin β1 revealed that “Slow” ligand oscillation significantly promoted integrin binding with ligand, evident as more speckles of fluorescent staining, whereas “Fast” ligand oscillation significantly inhibited integrin ligation, compared with those in the “Stationary” and “Moderate” groups (Figure 2E-F). Altering integrin ligand oscillation speeds regulates adhesion of stem cells. We next evaluated whether the application of magnetic field at various frequencies can regulate the adhesion of human mesenchymal stem cells (hMSCs) on the substrate coupled with RGDbearing SPIONs. We coated the RGD-bearing substrate with bovine serum albumin (BSA) to minimize non-specific cellular adhesion. We also included controls of RGD-bearing substrate in the absence of applied magnetic field and substrate devoid of coupled RGD-SPION (hereafter named as “No magnet” and “No RGD”, respectively). After 12 h of culture, altering RGD ligand oscillation speed by adjusting the oscillation frequencies of magnetic field yielded strikingly disparate levels of cellular adhesion, analogous to the integrin-ligand binding results described above. Compared to the “Stationary” control under non-oscillating static magnetic field, “Slow” ligand oscillation significantly promoted 8 ACS Paragon Plus Environment

Page 9 of 41

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

ACS Nano

cellular adhesion, whereas “Fast” ligand oscillation inhibited cell adhesion (Figure 3A). Quantification of density of cellular adhesion corroborated the observations described above showing 50% more and 49% less adherent cells in “Slow” and “Fast” groups, respectively, compared to “Stationary” group (Figure 3B). We also applied the magnetic field of more extreme oscillation frequencies (0.02 Hz and 5 Hz) and observed that the density of cellular adhesion was comparable to the “Slow” group (0.1 Hz) and the “Fast” group (2 Hz), respectively (Supplementary Figure S8A-D). Furthermore, we observed no significant difference in the number of adherent cells amongst “No magnet”, “Stationary”, and “Moderate” groups, but all these three groups displayed more adherent cells than the “No RGD” control, suggesting the efficacy of RGD ligand to mediate cell adhesion in the presence of BSA blocking agent. As a control, we used PEG as a blocking agent and found that “Slow” and “Fast” ligand oscillation also significantly promoted and inhibited cellular adhesion, respectively, compared with the “Stationary” control, similarly to the results obtained on the BSA-blocked substrates (Supplementary Figure S8E-F). To further evaluate such ligand oscillation-mediated stimulation and suppression of stem cell adhesion, we performed a series of control experiments by using SPION-tethered substrate, but without the conjugation of RGD ligand (Supplementary Figure S9A) or with the conjugation of scrambled RGD sequences, such as RAD and RGE. We found no significant differences in cellular adhesion among all groups regardless of the oscillating magnetic field frequency, in the absence of RGD conjugation (Supplementary Figure S9B-C) or the conjugation of RAD or RGE (Supplementary Figure S10A-D). This finding suggests that RGD ligand tethering to SPION is required to modulate stem cell adhesion through the oscillating motion of SPION. To examine whether the oscillating magnetic field affects cellular adhesion independent of the magnetic field-mediated ligand motion, we prepared RGD-bearing pure silica nanoparticles that do not respond to the magnetic field. We found that pure silica nanoparticles exhibited the size (approximately 40 nm) comparable to that of the silica-coated SPION used in this study (Supplementary Figure S11A), and the density of the conjugated pure silica nanoparticles was comparable to that of the silica-coated SPION (Supplementary Figure S11B). In the presence of substrate-tethered RGD-bearing silica nanoparticles, we observed no significant difference in the number of adherent cells among “No magnet”, “Stationary”, “Slow”, and “Fast” groups, suggesting that the oscillating magnetic field itself does not affect the cellular adhesion (Supplementary Figure S11C-D). 9 ACS Paragon Plus Environment

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

Page 10 of 41

Because integrin-ligand binding mediates focal adhesion (FA) formation by recruiting FA adaptor molecules, such as vinculin, we examined cell spreading area and focal adhesion development after 12 h and 48 h of culture. The disparate adherent cell density correlated to different extent of cell spreading. “Slow” and “Fast” ligand oscillations resulted in significantly larger (approximately by 168%) or smaller (approximately by 30%) cell spread area, respectively, as compared to the “Stationary” and “Moderate” oscillation groups (Figure 3A-B). The number of focal adhesions per cell quantified by vinculin staining43 correlated positively with the cell spread area (Supplementary Figure S12A). Quantification of the aspect ratio (length/width) of adherent cells revealed the inverse relationship between cell aspect ratio and cell spread area/number of focal adhesions (Supplementary Figure S12B). “Slow” and “Fast” ligand oscillations resulted in significantly smaller and larger aspect ratio, respectively. The focal adhesion of cells is dynamically regulated by integrin ligation events. The “Slow” (0.1 Hz) nanoscale oscillation motion of the tethered RGD ligand yielded the optimal integrin binding and cell adhesion to the substrate. A recent report showed that the magnitude of cell adhesion forces to fibronectin or anti-integrin β1-coated surface exhibit oscillatory cycles at 0.01-0.02 Hz.10 In correlation with dynamic nature of integrin ligation processes, we suggest that “Slow” ligand oscillation increases the likelihood that integrin will contact the ligand and thus stimulate cell adhesion, analogous to a previous report.15 In stark contrast, “Fast” ligand oscillation (2 Hz) was found to significantly inhibit integrin ligation and cellular adhesion, thus exhibiting minimal vinculin expression and highly elongated cell shape. Such observation could be attributed to poor integrin binding with highly oscillatory ligand. Collectively, ligand oscillations at various frequencies (“Slow”, “Moderate”, and “Fast”) mediated by remotely and continuously applied magnetic field offer a powerful tool with which to regulate cell adhesion and spreading in a physical and non-contact manner. Temporal switching of ligand oscillations modulates focal adhesion of stem cells. We next evaluated whether the temporal switching of the oscillation frequency of the magnetic field can regulate stem cell adhesion. We observed the distinct cell adhesion under “Slow” and “Fast” RGD ligand oscillations induced by the oscillating magnetic field and superparamagnetic 10 ACS Paragon Plus Environment

Page 11 of 41

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

ACS Nano

characteristics of the SPIONs. Therefore, we switched the oscillation frequencies of the magnetic field from “Slow” to “Fast” ligand oscillations (“Slow-Fast”) or from “Fast” to “Slow” ligand oscillations (“Fast-Slow”) after 12 h of culture and further cultured for another 36 h, until 48 h of total culture time. We included non-switched continuous “Slow” or “Fast” ligand oscillations for 48 h of culture as control groups (Figure 4A). After 12 h of culture, non-switched continuous “Slow” and “Fast” ligand oscillations resulted in significantly enhanced or inhibited adhesion of stem cells, respectively, and such distinct trend persisted until 48 h of culture (Figure 4A-B). Upon temporal switching from “Slow” ligand oscillation to “Fast” ligand oscillation after 12 h of culture (“Slow-Fast” group), the spreading area and the number of focal adhesions of stem cells significantly decreased (by 46% and 68%, respectively) at 48 h of culture, compared to 12 h of culture, and cells switched their morphology from spread to elongated shape (with 208% increase in aspect ratio). The temporally switched “Fast-Slow” group exhibited a reverse trend as compared to “Slow-Fast” group, with increase in the spreading area and the number of focal adhesions as well as switch from elongated to spread morphology. Such findings indicate that the temporal control over oscillation speeds of cell adhesive ligands enabled reversible cell adhesion and spreading. Integrin-ligand binding is highly dynamic during cellular adhesion7-9 and reversible cellular adhesion was previously demonstrated via photochemical reactions.26,27 Compared to the light-based approach, the utility of magnetic field to regulate cell adhesion and spreading has the advantage of longer working distance without being obstructed by physical barriers such as biological tissues, thereby allowing potentially widespread in vivo applications. Furthermore, the magnetic control over ligand oscillation speeds also affords fine tuning on the extent of cell adhesion and spreading by adjusting the magnetic field oscillation frequency. This allows investigation on the behaviors of cells with different levels of adhesion free from other confounding factors, such as ligand density and substrate stiffness. In addition, switching of the magnetic field oscillation frequency at selected time points provides the opportunity to study the effect of temporal changes in cell spreading on cell behaviors.


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

Page 12 of 41

Tuning ligand oscillations modulate stem cell differentiation. Having examined the effect of RGD ligand oscillation speeds on cellular adhesion, we evaluated whether such dynamic cellligand interactions could influence the differentiation of hMSCs. The directed differentiation of stem cells is essential to the success of regenerative therapies and is dictated by the responses of cells to microenvironmental cues, such as the physicochemical properties of the matrix12,13,44-47 and soluble factors.48-51 The adhesion and morphology of stem cells are known to play an important role in regulating mechanotransduction and differentiation of stem cells.52-54. hMSCs were cultured in basal growth medium for 12 h to allow cellular adhesion and subsequently cultured in osteogenic induction medium for 7 d to examine their osteogenesis. We included the groups with temporal switching of ligand oscillations, “Slow-Fast” and “Fast-Slow” groups, at 12 h of culture to analyze differentiations at 7 d, and the continuous “Fast” ligand oscillation group was excluded owing to significantly inhibited cellular adhesion. Immunofluorescent staining for RUNX2, a master regulator in osteogenic differentiation, revealed that “Slow” RGD ligand oscillation led to the highest level of RUNX2 expression in hMSCs (Figure 5A). Quantification of nuclear to cytoplasmic fluorescence ratio corroborated such pattern of expression level with “Slow” group 222% higher than “Stationary” group (Figure 5B). In addition, switching ligand oscillation from “Slow” to “Fast” mode after 12 h (“Slow-Fast” group) resulted in lower RUNX2 expression at day 7 (approximately by 54%) compared to the continuous “Slow” oscillation group. Interestingly, switching from “Fast” to “Slow” ligand oscillation (“Fast-Slow” group) led to higher RUNX2 expression comparable to that of the “Slow” group. These findings indicate that “Slow” ligand oscillation helped to recover the adhesion, spreading, and differentiation potential of stem cells following the initial temporal suppression of cellular adhesion under “Fast” ligand oscillation. We further performed alkaline phosphatase (ALP) staining to characterize the osteogenesis of stem cells under altered ligand oscillations. The degree of ALP staining was found to correlate to the level of RUNX2 nuclear localization, further confirming the effect of tuning ligand oscillations on stem cell differentiation. We then investigated the role of mechanotransduction in regulating such disparate differentiation potential because focal adhesion is known to mediate mechanosensing of stem cells. 12 ACS Paragon Plus Environment

Page 13 of 41

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

ACS Nano

Immunostaining of Yes-associated protein (YAP), a key regulator of mechanotransduction, at day 2 revealed more pronounced nuclear localization in the “Slow” and “Fast-Slow” group compared to other control groups (Figure 5A-B), confirming the enhanced mechanotransduction signaling in hMSCs under “Slow” ligand oscillations. Immunofluorescent staining of transcriptional co-activator with PDZ-binding motif (TAZ) showed higher nuclear localization in the “Slow” group than the “No Magnet”, “Stationary”, and “Moderate” groups, further confirming

that

the

“Slow”

ligand

oscillation

promoted

the

mechanotransduction

(Supplementary Figure S13A-B). Therefore, we collectively suggest that the remote control of ligand oscillation speeds via magnetic field regulates the adhesion, spreading, mechanosensing, and consequently differentiation of stem cells.

Remotely tuning RGD ligand oscillation speeds modulates in vivo substrate adhesion of stem cells. We further evaluated whether the remote control of RGD ligand oscillation speeds could regulate stem cell adhesion in vivo. We implanted substrate bearing the RGD-SPION into subcutaneous pocket of nude mice and injected hMSCs onto the substrate. We externally applied magnetic field of varying oscillation frequencies to the implantation area, while keeping the mice under anesthesia for 4 h before extracting the implanted substrate to examine cell adhesion. Significantly more adhered cells were found in “Slow” ligand oscillation, “Stationary”, and “No magnet” groups (approximately 1400 - 1550 cells/cm2), compared to the minimal cellular adhesion observed in “Fast” oscillation and “No RGD” groups (approximately 550 – 650 cells/cm2) (Figure 6A-B). We next identified cell species by immunofluorescent staining of human-specific nuclear antigen and found that all in vivo adhered cells were human donor cells (Figure 6A). This could be attributable to the insufficient time (4 h) for recruitment and subsequent adhesion of host cells. The adhesion of recruited immune cells including neutrophils and macrophages onto RGD-bearing biomaterials was observed following 24 h of implantation.23 Furthermore, “Slow” ligand oscillation significantly increased the number of focal adhesion site as evidenced by vinculin staining (approximately by 236% – 433% higher) and spread size of adherent cells (approximately by 291% – 445% higher), compared to all other groups (Figure 6A-B). In contrast, the “Fast” group exhibited 65% less focal adhesions and 33% smaller cell size than those of the “Stationary” group. These findings demonstrate that remote control of ligand oscillations via magnetic field to regulate stem cell adhesion remains effective under the 13 ACS Paragon Plus Environment

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

Page 14 of 41

dynamic and complex in vivo environment. To the best of our knowledge, such multi-modal (“Slow” and “Fast” ligand oscillations) remote regulation of cellular adhesion in vivo by adjusting the oscillation frequency of external magnetic field has not been demonstrated before. The non-invasive, penetrative, and tunable nature of external magnetic field potentially allows temporal manipulation of cell adhesion to implants in deep interior anatomic sites in vivo. Having verified the effective ligand oscillation-mediated control over stem cell adhesion in vivo, we evaluated whether pronounced spreading and focal adhesion of the in vivo adhered stem cells under “Slow” ligand oscillation stimulate their mechanosensing in vivo. The nuclear to cytoplasmic ratio of YAP in the “Slow” group was found to be significantly higher than that in the other groups showing minimal nuclear staining, thereby indicating the possible involvement of focal adhesion-associated mechanotransduction signaling in vivo (Supplementary Figure S14A-B). CONCLUSION In summary, this study demonstrates an approach to controlling the nanoscale motion of the integrin-binding ligands by adjusting the oscillation frequency of an applied magnetic field and shows that the motion speeds of the ligands significantly influence the adhesion and differentiation of stem cells. Specifically, RGD ligand-coupled SPION was bound to a substrate with long and flexible polymer linker. The controllable motion speeds of the RGD ligandcoupled SPION in a oscillation frequency-dependent manner (demonstrated by fluorescent imaging) led to their nanoscale motion upon substrate tethering (demonstrated by in situ magnetic STEM and AFM imaging). Remotely inducing slow or fast ligand oscillations by tuning the oscillation frequency of the magnetic field significantly promoted or inhibited integrin ligation and the adhesion of stem cells, respectively, both in vitro and in vivo, thereby significantly regulating the osteogenic differentiation of the stem cells. Temporal switching of the multi-modal ligand oscillation speed reversibly regulated cellular adhesion. The speeds of the ligand oscillations significantly affected the differentiation and mechanosensing of the stem cells. Such controlled nanoscale ligand oscillations can be harnessed to shed light on the dynamic processes of integrin ligation to extracellular matrix. Furthermore, such a non-invasive, tissue penetrative, cytocompatible, and tunable approach by the application of a magnetic field, holds 14 ACS Paragon Plus Environment

Page 15 of 41

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

ACS Nano

great potential utility to manipulate and study of the cellular adhesion and responses to implant biomaterials in the host microenvironment in vivo. METHODS Preparation of superparamagnetic iron oxide nanoparticle (SPION). To remotely control integrin ligand-tethered nanoparticle, SPION was prepared by following a previously reported procedure.55 Briefly, 36.5 g (120 mmol) of sodium oleate and 10.8 g (40 mmol) of iron (III) chloride hexahydrate were dissolved in a mixture of 80 mL of ethanol, 60 mL of deionized (DI) water, and 140 mL of heptane at 70 oC for 4 h under inert atmosphere. The iron-oleate containing heptane layer was separated and washed with 40 mL of DI water followed by the evaporation of heptane. 36 g (40 mmol) of the dried iron-oleate was mixed with 5.7 g (20 mmol) of oleic acid and 200 g of 1-octadecene at 100 oC for 5 min. The mixture was further heated to 320 oC and kept for 30 min. The mixture was then cooled to 25 oC and 500 mL of ethanol was added. The obtained SPION was washed with ethanol and dispersed in heptane. Functionalization of SPIONs. To couple long flexible polymer chain to SPION, SPION was functionalized through aminosilica coating (SPION@SiO2). 30 mg of SPION was dispersed in 100 mL of cyclohexane, 20 mL of Triton-X, 20 mL of 1-hexanol, 2 mL of NH4OH, and 4 mL of DI water and stirred for 30 min. To the mixture, 100 µL of tetraethyl orthosilicate (TEOS) was added and stirred for 10 min. (3Aminopropyl)triethoxysilane (APTES) was further added to the mixture and stirred for 6 h. As a control, this protocol was also used to prepare pure silica nanoparticles in the absence of SPION. 100 mL of acetone was added to the mixture that was washed with acetone and DMF. The resultant amino-silica coated SPION was collected by a permanent magnet. Conjugation of flexible polymer linker to functionalized SPION. To tether functionalized SPION (SPION@SiO2) onto a substrate and subsequently maneuver the motion, long flexible polymer linker was conjugated. 20 mg of amino-silica coated SPION was suspended in 1 mL of DMF to which 10 mg of Maleimide-Polyethylene glycol (Mn = 5000 Da)NHS ester (JenKem Technology) and 0.2% N,N-Diisopropylethylamine (DIPEA) was added.


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

Page 16 of 41

The suspension was stirred for 16 h under dark condition, collected by a permanent magnet, and then washed with DMF and DMSO. The resultant PEGylated SPION was later used for conjugation onto a substrate. For characterization of integrin ligand-tethered PEGylated SPION, the suspension in DMSO was mixed with 0.14 mM thiolated RGD peptides (GCGYGRGDSPG; Mn = 1025.06 Da, GenScript), 0.2% DIPEA, and 10 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) for 16 h under gentle stirring and dark condition and washed with DMSO and DI water. As a control, we conjugated scrambled RGD peptides (GCGYGRADSPG or GCGYGRGESPG, GenScript). X-ray diffraction (XRD). To characterize crystalline phase of the synthesized magnetic nanoparticles, XRD spectra was measured. The magnetic nanoparticles were subject to flash-freezing and lyophilization. The dried particles were mounted and scanned by using Smartlab (Rigaku) diffractometer with Cu target (40 kV, 80 mA) at a diffraction angle ranging from 20 to 70 degree at 10 degree/min. Transmission electron microscopy (TEM). To characterize the size and morphology of the SPION before and after amino-silica coating, TEM imaging was performed by using CM-200 (Philips, Oregon, USA) at an accelerating voltage of 100 kV. The size of the SPION was quantified by Image J from 20 different particles in 3 different images and presented as mean ± standard errors. Dynamic light scattering (DLS) and zeta potential. To determine the size distribution profile and changes in surface charges of the SPION before and after amino-silica coating, DLS and zeta potential measurements were performed by using photon correlation spectroscopy (Brookhaven Instruments Corporation). The SPION was suspended in DI water and subject to the measurements. Vibrating sample magnetometer (VSM) measurement. To evaluate the magnetic property of the SPION, VSM measurement (PPMS Model 6000 Quantum Design) was conducted. The measurement was conducted at 300 K under applied 16 ACS Paragon Plus Environment

Page 17 of 41

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

ACS Nano

magnetic field from – 6000 Oe to 6000 Oe. The magnetic moment (emu/g) of hysteresis loop was presented after normalization of dry weight of SPION. Fourier Transform Infrared Spectroscopy (FTIR). To evaluate changes in chemical bond characteristics of the SPION before and after RGD ligand tethering, FTIR was conducted by using IRTracer-100 (Shimadzu). The SPION was subject to flash-freezing and lyophilization prior to the measurement. Synthesis of fluorescent SPION. To fluorescently track motion of SPION, we conjugated fluorescent molecule to SPION. 10 mg of Rhodamine isothiocyanate (RITC) was added into suspension of 20 mg of amino-silica coated SPION in 1 mL of DMF. The mixture was stirred for 2 d under dark condition and washed with DMF. Maleimide-Polyethylene glycol-NHS ester coupled with RGD peptide was added to the RITC-conjugated amino-silica coated SPION with 0.2% DIPEA and kept for 16 h. The resulting fluorescent RGD ligand-bearing SPION was washed with DMF and DI water for fluorescent imaging. Confocal microscopy imaging of oscillating fluorescent SPION. To examine tunable oscillatory motion of the SPION dependent upon the frequencies of oscillating magnetic field, confocal microscopy imaging was performed. The RGD-bearing fluorescent SPION was suspended in DI water. The fluorescent image with bright field image as a reference was taken on the same area every 10 s while the external magnetic field was applied by oscillating a permanent magnet (40 mT) at frequencies of 0, 2.5, 5, and 12.5 mHz. 20 timelapse snapshots of oscillating SPION were used to quantify frequency-dependent average motion speed of the SPION and generate movies demonstrating the oscillatory motion of the SPION at various frequencies. Coupling of flexible chain-tethered SPION to a substrate and further grafting of integrin ligand. To conjugate flexible chain-tethered (PEGylated) SPION to a substrate, culture-grade glass coverslips (22 mm X 22 mm) were used. The glass substrates were immersed in the mixture of HCl and MeOH (1:1) for 30 min to remove any organic impurities from the surface of substrates 17 ACS Paragon Plus Environment

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

Page 18 of 41

and subsequently washed with DI water. For thiolization, the substrates were immersed in 1 mM mercaptopropylsilatrane (MPS) in methanol for 30 min under dark condition and then washed with methanol. The thiolized substrate was soaked in 1 mL of PEGylated SPION in DMSO containing 0.2% DIPEA under dark condition for 16 h and washed with DMSO. To further conjugate RGD ligand to the PEGylated SPION-coated substrate, the substrate was immersed in 0.14 mM RGD peptides in 1 mL of DMSO containing 0.2% DIPEA and 10 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) for 16 h under gentle rotation and dark condition and washed with DMSO and DI water. Contact angle measurement. To evaluate changes in surface chemistry of the substrate upon surface modifications, water contact angle measurements were carried out. The substrates were dried at 37 oC for 3 h. Approximately 2 µL of water droplet was applied onto the substrates and the surface of substrates were imaged at fixed position by using contact angle analyzer (JC2000C1, Powerrach Ltd). Contact angle was quantified from 4 different images and presented as mean ± standard errors. Scanning electron microscopy (SEM). To visualize conjugation of RGD ligand-SPION (RGD-PEG-SPION@SiO2) onto the glass substrate, SEM imaging (SU8000 Series, Hitachi High-Tech Ltd., UK) was carried out. The substrate was rinsed with DI water and dried at 37 oC for 3 h. The dried substrate was goldcoated using a sputter coater and imaged at an accelerating voltage of 15 kV. The density of the substrate-coupled RGD ligand-SPION was quantified by Image J from 10 different images and presented as mean ± standard errors. In situ Atomic force microscopy (AFM). To visualize 3D distribution of tethered integrin ligand-SPION (RGD-PEG-SPION@SiO2) onto the glass substrate as well as characterize movement of tethered ligand-SPION, AFM imaging (Bruker) was performed in tapping mode at 25 oC. The spring constant of the AFM cantilever (Bruker) was 0.4 N/m. 3D height image was obtained in a scan size of 5 X 5 µm. 18 ACS Paragon Plus Environment

Page 19 of 41

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

ACS Nano

To visualize and quantify the movement of tethered ligand-SPION in situ, gold nanorod (aspect ratio of 4) was co-conjugated to the substrate that served as non-magnetic reference by following previously reported protocol.56 Briefly, 0.7 g of hexadecyltrimethylammonium bromide (CTAB) and 0.1234 g of sodium oleate (NaOL, TCI America) were dissolved in 25 mL of DI water at 30 o

C and 1.8 mL of AgNO3 was added to the mixture. The mixture was stirred for 15 min and 25

mL of 1 mM Au3+ from gold (III) chloride trihydrate (HAuCl4·3H2O, Sigma) was injected into the solution. The mixture was stirred for 90 min and 210 µL of 37% HCl was added. After 15 min of stirring, 125 µL of 64 mM ascorbic acid and seed solution (the mixture of 2.5 mL of 200 mM CTAB, 2.5 mL of 0.5 mM Au3+, and 0.3 mL of 10 mM chilled sodium borohydride that was aged for 2 h) were added to the mixture and kept for 12 h. Thiolated glass substrates (SPION conjugated) were immersed in 0.5 nM gold nanorods solution for 2 h and washed with DI water thoroughly and dried under nitrogen. In situ AFM imaging was performed on the same scan area with external magnetic field applied by a permanent magnet (40 mT) placed at two opposite outer sides of the scan area to visualize and quantify the movement of tethered ligand-SPION from the reference gold nanorod. Approximately 10 movements were quantified between the centers of ligand-SPION and presented as mean ± standard errors. In situ magnetic high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM). To characterize nanoscale motion of the RGD ligand-SPION conjugated to the substrate, in situ HAADF-STEM imaging was performed by using JEOL 2100F with 1nm probe size, condenser aperture of 20 µm, and collection angle of 80~150 mrad for Z contrast. We constructed a set-up by tethering a flexible curved rubber magnet (sized to 1mm thick and 3mm long) securely to a mobile tungsten STM tip of in situ electrical biasing holder (Nanofactory ST-1000) that can move controllably inside STEM chamber during in situ TEM imaging. A 50 nm-thick silicon oxide TEM grid (Dune Sciences, cat. no. NG02-011A), used as a substrate for conjugation of the RGD ligand-SPION, was fixed at the other electrode side. The crooked tip part of the rubber magnet in contact with the TEM grid was relocated at three different positions to manipulate 19 ACS Paragon Plus Environment

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

Page 20 of 41

local magnetic field near individual SPIONs, monitored by HAADF-STEM to characterize interparticle movement. Note that each STEM image has at least 5 - 10 min interval after relocation of magnetic tip to correct ronchigram (beam astigmatism) because local magnetic field variation distorts electron beam condition significantly. Approximately 10 movements were quantified between the centers of ligand-SPIONs and presented as mean ± standard errors. Integrin β1 binding to oscillating RGD ligands. To evaluate integrin β1 binding onto oscillating RGD ligands in various frequencies, RGD ligand-SPION tethered substrate was incubated in 50 µg/mL of integrin β1 in PBS at 37 oC for 30 min under the applied magnetic field by a permanent magnet (40 mT) in oscillation frequencies of 0, 0.1, 0.5, and 2 Hz by electrical set-up. The incubated substrate was fixed and subject to immunofluorescent staining of integrin β1 to examine the bound integrin β1 onto oscillating RGD ligand. In vitro culture. To examine cellular adhesion on oscillating RGD ligands in varying frequencies, RGD ligandSPION coupled (culture-grade) substrate was sterilized and blocked with 1% bovine serum albumin (BSA, Sigma-Aldrich) at 37 oC for 1 h or 0.1% Maleimide-Polyethylene glycol (Mn = 5000 Da)-OH (JenKem Technology) for 1 h as a control. The blocked substrate was immediately subject to seeding of human mesenchymal stem cells (hMSCs; passage 4, Lonza) at plating density of 5000 cells/cm2 and cultured in basal growth medium (High glucose DMEM, 10% [v/v] fetal bovine serum, 4 mM L-glutamine, and 50 U/mL penicillin/streptomycin; Thermo Scientific) at 37 oC and 5% CO2. The adhesion of stem cells onto oscillating RGD ligands for 12 h and 48 h under the applied magnetic field by oscillating a permanent magnet (40 mT) at frequencies of 0, 0.1, 0.5, and 2 Hz by electrical set-up. For temporal switch of oscillation frequency was performed after 12 h of cell plating between 0.1 Hz and 2 Hz. The differentiation of stem cells following their focal adhesion was examined by changing basal growth medium to osteogenicinducing medium [basal growth medium containing 10 mM β-glycerophosphate (Sigma-Aldrich), 50 µM ascorbic acid-2-phosphate (Sigma-Aldrich), and 100 nM dexamethasone (Sigma-Aldrich) after 12 h of cell adhesion. The osteogenic-inducing medium was replenished every two days and differentiation was analyzed after 7 days of culture. 20 ACS Paragon Plus Environment

Page 21 of 41

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

ACS Nano

Immunofluorescent staining. The cell cultures were fixed by using 4% (w/v) paraformaldehyde at 25 °C for 15 min and washed with PBS. The fixed cells were permeabilized with 0.25% (v/v) Triton-X (Sigma-Aldrich) in PBS at 25 °C for 10 min. The treated cells were blocked with 1% (w/v) BSA at 37 °C for 1 h. The blocked cells were incubated with primary antibody against integrin β1 (1:100, mouse, Santa Cruz Biotechnology), vinculin (1:400, mouse, Sigma-Aldrich), RUNX2 (1:100, mouse, Santa Cruz Biotechnology), YAP (1:100, mouse, Santa Cruz Biotechnology), or TAZ (1:100, mouse, Santa Cruz Biotechnology) in blocking solution at 4 °C for 16 h and washed with PBS containing 0.5% Tween 20 three times. The cells were incubated with secondary antibody (1:250; Thermo Scientific) and phalloidin (1:100, Molecular Probes) in the blocking solution at 25 °C for 1 h and washed with PBS containing 0.5% Tween 20. The nuclei were stained by using DAPI (1:1000, Molecular Probes) in PBS at 25 °C for 10 min and washed with PBS. The stained cells were mounted onto glass slides and visualized by using confocal microscope (Nikon). The images were acquired using the same exposure conditions for all groups. The background of images was identically subtracted for all images by using ImageJ software with rolling ball radius of 750 pixels. Fluorescent staining analyses. To quantify integrin binding as well as cell adhesion and differentiation, immunofluorescently stained images were utilized for the analyses by Image J. To quantify the integrin-ligand binding, the fluorescent intensities of integrin β1 staining from 6 different images were calculated by using histogram function in Image J. To calculate adhered cell density, the number of cell nuclei from 10 different images was counted. To quantify cell area, F-actin staining from each cell of approximately 30 cells from 5 different images was used. To determine the number of focal adhesion, vinculin staining greater than 1 µm2 from each cell of approximately 30 cells from 5 different images was counted by following previously established procedure.43 To calculate cell shape factor, F-actin staining from each cell of approximately 30 cells from 5 different images was used. To determine nuclear localization of osteogenic marker (RUNX2) and mechanotransduction regulator (YAP), ratiometric analyses of nuclear fluorescence intensity to


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

Page 22 of 41

cytoplasmic fluorescence intensity (excluding nuclei area) were performed from each cell of approximately 30 cells from 5 different images. The data are presented as mean ± standard errors. Alkaline phosphatase (ALP) staining. The hMSCs were analyzed for RGD-ligand oscillation frequency-dependent osteogenic differentiation by alkaline phosphatase (ALP) staining after 7 days of culture under osteogenic induction medium. The hMSCs were fixed in 4% (w/v) paraformaldehyde for 2 min at room temperature that preserves ALP activity and washed with PBS. The fixed cells were stained with mixture of 4% (v/v) naphthol AS-MX phosphate (Sigma-Aldrich) and 0.06% (w/v) Fast Blue RR Salt solution (Sigma-Aldrich) in DI water for 45 min at 37 oC under dark condition and washed with PBS. The stained cells were visualized using a microscope under A filter in color mode. Subcutaneous implantation. To examine stem cell adhesion onto oscillating RGD ligands at various frequencies in vivo, 10 3month-old nude mice were used with the approval of the Institutional Animal Care and Use Committee at the Chinese University of Hong Kong. The nude mice were subject to inhalation anesthesia with 3% isoflurane and 2 cm-long incision was made in the back of the mice. The ligand-SPION grafted substrate following BSA coating was subcutaneously implanted and skin was carefully closed. hMSCs were subcutaneously injected onto the substrate approximately at 20 k/cm2 under oscillating magnetic field in various frequencies. Following 4 h of stem cell injection, the implanted substrate was collected for immunostaining analyses of cell adhesion and mechanosensing. The in vivo adhered cells were analyzed by immunofluorescent staining (including human nuclei antigen, 1:100, Millipore for identifying human donor cells) through the same procedures described in analyzing in vitro cultured cells. Statistical analyses. All experiments were repeated at least two times. Using Graphpad Prism 5, statistical significances were assigned for p-values less than 0.05. One-way analysis of variance (ANOVA) with Tukey-Kramer post-hoc test was used to compare multiple groups at the same time point. Two-tailed Student’s t-test was used to compare two groups at the same time point.


Page 23 of 41

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

ACS Nano

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Characterizations of the bare SPIONs and RGD ligand tethering to the SPIONs, substrate tethering of the RGD ligand-bearing SPIONs, characterization set-up of nanoscale motion of the substrate-tethered RGD-bearing SPION by in situ atomic force microscopy or scanning transmission electron microscopy, cellular adhesion control experiments with different oscillation frequencies of the magnetic field, poly(ethylene glycol) blocking method, the absence of conjugated RGD ligand, conjugated RAD or RGE, or pure silica nanoparticle, focal adhesion analysis, immunofluorescent staining for TAZ and YAP and movies for the oscillatory motion of the RGD-bearing SPIONs. The authors declare no conflict of interest. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]. *E-mail: [email protected]. ORCID Liming Bian: 0000-0003-4739-0918 Author Contributions ⊥Contributed

equally to this work.

ACKNOWLEDGEMENTS This work was supported by a General Research Fund grant from the Research Grants Council of Hong Kong (Project No. 14202215), the National Natural Science Foundation of China (Project No. 31570979), the Health and Medical Research Fund, the Food and Health Bureau, the Government of the Hong Kong Special Administrative Region (Reference No. 03140056). This 23 ACS Paragon Plus Environment

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

Page 24 of 41

research was also supported by project BME-p3-15 of the Shun Hing Institute of Advanced Engineering and 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. REFERENCES 1.

Wolfenson, H.; Lavelin, I.; Geiger, B., Dynamic Regulation of the Structure and

Functions of Integrin Adhesions. Dev. Cell 2013, 24, 447-458. 2.

Rozario, T.; DeSimone, D. W., The Extracellular Matrix in Development and

Morphogenesis: A Dynamic View. Dev. Biol. 2010, 341, 126-140. 3.

Evans, E. A.; Calderwood, D. A., Forces and Bond Dynamics in Cell Adhesion. Science

2007, 316, 1148-1153. 4.

Ruoslahti, E.; Pierschbacher, M. D., New Perspectives in Cell Adhesion: RGD and

Integrins. Science 1987, 238, 491-497. 5.

Jalali, S.; del Pozo, M. A.; Chen, K.-D.; Miao, H.; Li, Y.-S.; Schwartz, M. A.; Shyy, J.

Y.-J.; Chien, S., Integrin-Mediated Mechanotransduction Requires Its Dynamic Interaction with Specific Extracellular Matrix (ECM) Ligands. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 1042-1046. 6.

Kim, S.-H.; Turnbull, J.; Guimond, S., Extracellular Matrix and Cell Signalling: The

Dynamic Cooperation of Integrin, Proteoglycan and Growth Factor Receptor. J. Endocrinol. 2011, 209, 139-151. 7.

Zhang, Y.; Ge, C.; Zhu, C.; Salaita, K., DNA-Based Digital Tension Probes Reveal

Integrin Forces During Early Cell Adhesion. Nat. Commun. 2014, 5. 8.

Rossier, O.; Octeau, V.; Sibarita, J.-B.; Leduc, C.; Tessier, B.; Nair, D.; Gatterdam, V.;

Destaing, O.; Albiges-Rizo, C.; Tampé, R., Integrins β1 and β3 Exhibit Distinct Dynamic Nanoscale Organizations Inside Focal Adhesions. Nat. Cell Biol. 2012, 14, 1057-1067. 9.

Balaban, N. Q.; Schwarz, U. S.; Riveline, D.; Goichberg, P.; Tzur, G.; Sabanay, I.;

Mahalu, D.; Safran, S.; Bershadsky, A.; Addadi, L., Force and Focal Adhesion Assembly: A


Page 25 of 41

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

ACS Nano

Close Relationship Studied Using Elastic Micropatterned Substrates. Nat. Cell Biol. 2001, 3, 466-472. 10.

Zhu, Y.; Qiu, H.; Trzeciakowski, J. P.; Sun, Z.; Li, Z.; Hong, Z.; Hill, M. A.; Hunter, W.

C.; Vatner, D. E.; Vatner, S. F., Temporal Analysis of Vascular Smooth Muscle Cell Elasticity and Adhesion Reveals Oscillation Waveforms That Differ with Aging. Aging Cell 2012, 11, 741750. 11.

Robertus, J.; Browne, W. R.; Feringa, B. L., Dynamic Control Over Cell Adhesive

Properties Using Molecular-Based Surface Engineering Strategies. Chem. Soc. Rev. 2010, 39, 354-378. 12.

Mager, M. D.; LaPointe, V.; Stevens, M. M., Exploring and Exploiting Chemistry at the

Cell Surface. Nat. Chem. 2011, 3, 582-589. 13.

Stevens, M. M.; George, J. H., Exploring and Engineering the Cell Surface Interface.

Science 2005, 310, 1135-1138. 14.

Pan, G.; Guo, Q.; Ma, Y.; Yang, H.; Li, B., Thermo‐Responsive Hydrogel Layers

Imprinted with RGDS Peptide: A System for Harvesting Cell Sheets. Angew. Chem. Int. Ed. 2013, 52, 6907-6911. 15.

Seo, J.-H.; Kakinoki, S.; Inoue, Y.; Yamaoka, T.; Ishihara, K.; Yui, N., Inducing Rapid

Cellular Response on RGD-Binding Threaded Macromolecular Surfaces. J. Am. Chem. Soc. 2013, 135, 5513-5516. 16.

Dobson, J., Remote Control of Cellular Behaviour with Magnetic Nanoparticles. Nat.

Nanotechnol. 2008, 3, 139-143. 17.

Ng, C. C. A.; Magenau, A.; Ngalim, S. H.; Ciampi, S.; Chockalingham, M.; Harper, J. B.;

Gaus, K.; Gooding, J. J., Using An Electrical Potential to Reversibly Switch Surfaces Between Two States for Dynamically Controlling Cell Adhesion. Angew. Chem. Int. Ed. 2012, 51, 77067710. 18.

Yeo, W.-S.; Yousaf, M. N.; Mrksich, M., Dynamic Interfaces Between Cells and

Surfaces: Electroactive Substrates That Sequentially Release and Attach Cells. J. Am. Chem. Soc. 2003, 125, 14994-14995. 19.

Li, L.; Wu, J.; Gao, C., Gradient Immobilization of A Cell Adhesion RGD Peptide on

Thermal Responsive Surface for Regulating Cell Adhesion and Detachment. Colloid Surface B 2011, 85, 12-18. 25 ACS Paragon Plus Environment

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

20.

Page 26 of 41

Roberts, J. N.; Sahoo, J. K.; McNamara, L. E.; Burgess, K. V.; Yang, J.; Alakpa, E. V.;

Anderson, H. J.; Hay, J.; Turner, L.-A.; Yarwood, S. J., Dynamic Surfaces for the Study of Mesenchymal Stem Cell Growth Through Adhesion Regulation. ACS Nano 2016, 10, 6667-6679. 21.

Ohmuro‐Matsuyama, Y.; Tatsu, Y., Photocontrolled Cell Adhesion on a Surface

Functionalized with a Caged Arginine‐Glycine‐Aspartate Peptide. Angew. Chem. Int. Ed. 2008, 47, 7527-7529. 22.

Petersen, S.; Alonso, J. M.; Specht, A.; Duodu, P.; Goeldner, M.; del Campo, A.,

Phototriggering of Cell Adhesion by Caged Cyclic RGD Peptides. Angew. Chem. Int. Ed. 2008, 47, 3192-3195. 23.

Lee, T. T.; García, J. R.; Paez, J. I.; Singh, A.; Phelps, E. A.; Weis, S.; Shafiq, Z.;

Shekaran, A.; Del Campo, A.; García, A. J., Light-Triggered In Vivo Activation of Adhesive Peptides Regulates Cell Adhesion, Inflammation and Vascularization of Biomaterials. Nat. Mater. 2015, 14, 352-360. 24.

Li, W.; Wang, J.; Ren, J.; Qu, X., Near-Infrared Upconversion Controls Photocaged Cell

Adhesion. J. Am. Chem. Soc. 2014, 136, 2248-2251. 25.

Wirkner, M.; Alonso, J. M.; Maus, V.; Salierno, M.; Lee, T. T.; García, A. J.; Del Campo,

A., Triggered Cell Release from Materials Using Bioadhesive Photocleavable Linkers. Adv. Mater. 2011, 23, 3907-3910. 26.

Li, W.; Chen, Z.; Zhou, L.; Li, Z.; Ren, J.; Qu, X., Noninvasive and Reversible Cell

Adhesion

and

Detachment

Via

Single-Wavelength

Near-Infrared

Laser

Mediated

Photoisomerization. J. Am. Chem. Soc. 2015, 137, 8199-8205. 27.

Liu, D.; Xie, Y.; Shao, H.; Jiang, X., Using Azobenzene‐Embedded Self‐Assembled

Monolayers To Photochemically Control Cell Adhesion Reversibly. Angew. Chem. Int. Ed. 2009, 48, 4406-4408. 28.

Colombo, M.; Carregal-Romero, S.; Casula, M. F.; Gutiérrez, L.; Morales, M. P.; Böhm,

I. B.; Heverhagen, J. T.; Prosperi, D.; Parak, W. J., Biological Applications of Magnetic Nanoparticles. Chem. Soc. Rev. 2012, 41, 4306-4334. 29.

Carregal-Romero, S.; Guardia, P.; Yu, X.; Hartmann, R.; Pellegrino, T.; Parak, W. J.,

Magnetically Triggered Release of Molecular Cargo from Iron Oxide Nanoparticle Loaded Microcapsules. Nanoscale 2015, 7, 570-576.


Page 27 of 41

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

ACS Nano

30.

Corten, C. C.; Urban, M. W., Repairing Polymers Using Oscillating Magnetic Field. Adv.

Mater. 2009, 21, 5011-5015. 31.

Rössler, K.; Donat, M.; Lanzenberger, R.; Novak, K.; Geissler, A.; Gartus, A.; Tahamtan,

A.; Milakara, D.; Czech, T.; Barth, M., Evaluation of Preoperative High Magnetic Field Motor Functional MRI (3 Tesla) in Glioma Patients by Navigated Electrocortical Stimulation and Postoperative Outcome. J. Neurol. Neurosur. Ps. 2005, 76, 1152-1157. 32.

Wong, D. S.; Li, J.; Yan, X.; Wang, B.; Li, R.; Zhang, L.; Bian, L., Magnetically Tuning

Tether Mobility of Integrin Ligand Regulates Adhesion, Spreading, and Differentiation of Stem Cells. Nano Lett. 2017, 17, 1685–1695. 33.

Yan, X.; Zhou, Q.; Yu, J.; Xu, T.; Deng, Y.; Tang, T.; Feng, Q.; Bian, L.; Zhang, Y.;

Ferreira, A., Magnetite Nanostructured Porous Hollow Helical Microswimmers for Targeted Delivery. Adv. Funct. Mater. 2015, 25, 5333-5342. 34.

Larsen, E. K.; Nielsen, T.; Wittenborn, T.; Birkedal, H.; Vorup-Jensen, T.; Jakobsen, M.

H.; Østergaard, L.; Horsman, M. R.; Besenbacher, F.; Howard, K. A., Size-Dependent Accumulation of PEGylated Silane-Coated Magnetic Iron Oxide Nanoparticles in Murine Tumors. ACS Nano 2009, 3, 1947-1951. 35.

Ma, X.; Hahn, K.; Sanchez, S., Catalytic Mesoporous Janus Nanomotors for Active

Cargo Delivery. J. Am. Chem. Soc. 2015, 137, 4976-4979. 36.

Attwood, S. J.; Cortes, E.; Haining, A. W. M.; Robinson, B.; Li, D.; Gautrot, J.; del Río

Hernández, A., Adhesive Ligand Tether Length Affects the Size and Length of Focal Adhesions and Influences Cell Spreading and Attachment. Sci. Rep. 2016, 6. 37.

Maheshwari, G.; Brown, G.; Lauffenburger, D. A.; Wells, A.; Griffith, L. G., Cell

Adhesion and Motility Depend on Nanoscale RGD Clustering. J. Cell Sci. 2000, 113, 1677-1686. 38.

Choi, C. K. K.; Xu, Y. J.; Wang, B.; Zhu, M.; Zhang, L.; Bian, L., Substrate Coupling

Strength of Integrin-Binding Ligands Modulates Adhesion, Spreading, and Differentiation of Human Mesenchymal Stem Cells. Nano Lett. 2015, 15, 6592-6600. 39.

Ye, K.; Wang, X.; Cao, L.; Li, S.; Li, Z.; Yu, L.; Ding, J., Matrix Stiffness and Nanoscale

Spatial Organization of Cell-Adhesive Ligands Direct Stem Cell Fate. Nano Lett. 2015, 15, 4720-4729.


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

40.

Page 28 of 41

Huang, J.; Grater, S. V.; Corbellini, F.; Rinck, S.; Bock, E.; Kemkemer, R.; Kessler, H.;

Ding, J.; Spatz, J. P., Impact of Order and Disorder in RGD Nanopatterns on Cell Adhesion. Nano Lett. 2009, 9, 1111-1116. 41.

Xiong, J.-P.; Stehle, T.; Diefenbach, B.; Zhang, R.; Dunker, R.; Scott, D. L.; Joachimiak,

A.; Goodman, S. L.; Arnaout, M. A., Crystal Structure of the Extracellular Segment of Integrin αVβ3. Science 2001, 294, 339-345. 42.

Xiong, J.-P.; Stehle, T.; Zhang, R.; Joachimiak, A.; Frech, M.; Goodman, S. L.; Arnaout,

M. A., Crystal Structure of the Extracellular Segment of Integrin αVβ3 in Complex with An ArgGly-Asp Ligand. Science 2002, 296, 151-155. 43.

Deeg, J. A.; Louban, I.; Aydin, D.; Selhuber-Unkel, C.; Kessler, H.; Spatz, J. P., Impact

of Local Versus Global Ligand Density on Cellular Adhesion. Nano Lett. 2011, 11, 1469-1476. 44.

Bian, L.; Guvendiren, M.; Mauck, R. L.; Burdick, J. A., Hydrogels That Mimic

Developmentally Relevant Matrix and N-cadherin Interactions Enhance MSC Chondrogenesis. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 10117-10122. 45.

Cha, C.; Liechty, W. B.; Khademhosseini, A.; Peppas, N. A., Designing Biomaterials To

Direct Stem Cell Fate. ACS Nano 2012, 6, 9353-9358. 46.

Gaharwar, A. K.; Mihaila, S. M.; Swami, A.; Patel, A.; Sant, S.; Reis, R. L.; Marques, A.

P.; Gomes, M. E.; Khademhosseini, A., Bioactive Silicate Nanoplatelets for Osteogenic Differentiation of Human Mesenchymal Stem Cells. Adv. Mater. 2013. 47.

Xavier, J. R.; Thakur, T.; Desai, P.; Jaiswal, M. K.; Sears, N.; Cosgriff-Hernandez, E.;

Kaunas, R.; Gaharwar, A. K., Bioactive nanoengineered hydrogels for bone tissue engineering: a growth-factor-free approach. ACS Nano 2015, 9, 3109-3118. 48.

Kang, H.; Shih, Y.-R. V.; Nakasaki, M.; Kabra, H.; Varghese, S., Small Molecule–

Driven Direct Conversion of Human Pluripotent Stem Cells into Functional Osteoblasts. Sci. Adv. 2016, 2, e1600691. 49.

Xu, J.; Li, J.; Lin, S.; Wu, T.; Huang, H.; Zhang, K.; Sun, Y.; Yeung, K. W.; Li, G.; Bian,

L., Nanocarrier‐Mediated Codelivery of Small Molecular Drugs and siRNA To Enhance Chondrogenic Differentiation and Suppress Hypertrophy of Human Mesenchymal Stem Cells. Adv. Funct. Mater. 2016, 26, 2463-2472.


Page 29 of 41

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

ACS Nano

50.

Min, J.; Choi, K. Y.; Dreaden, E. C.; Padera, R. F.; Braatz, R. D.; Spector, M.; Hammond,

P. T., Designer Dual Therapy Nanolayered Implant Coatings Eradicate Biofilms and Accelerate Bone Tissue Repair. ACS Nano 2016, 10, 4441-4450. 51.

Choi, C. K. K.; Li, J.; Wei, K.; Xu, Y. J.; Ho, L. W. C.; Zhu, M.; To, K. K.; Choi, C. H. J.;

Bian, L., A Gold@Polydopamine Core–Shell Nanoprobe for Long-Term Intracellular Detection of MicroRNAs in Differentiating Stem Cells. J. Am. Chem. Soc. 2015, 137, 7337-7346. 52.

McBeath, R.; Pirone, D. M.; Nelson, C. M.; Bhadriraju, K.; Chen, C. S., Cell Shape,

Cytoskeletal Tension, and RhoA Regulate Stem Cell Lineage Commitment. Dev. Cell 2004, 6, 483-495. 53.

Mahmoudi, M.; Bonakdar, S.; Shokrgozar, M. A.; Aghaverdi, H.; Hartmann, R.; Pick, A.;

Witte, G.; Parak, W. J., Cell-Imprinted Substrates Direct the Fate of Stem Cells. ACS Nano 2013, 7, 8379-8384. 54.

Connelly, J. T.; Gautrot, J. E.; Trappmann, B.; Tan, D. W.-M.; Donati, G.; Huck, W. T.;

Watt, F. M., Actin and Serum Response Factor Transduce Physical Cues from the Microenvironment To Regulate Epidermal Stem Cell Fate Decisions. Nat. Cell Biol. 2010, 12, 711-718. 55.

Bloemen, M.; Brullot, W.; Luong, T. T.; Geukens, N.; Gils, A.; Verbiest, T., Improved

Functionalization of Oleic Acid-Coated Iron Oxide Nanoparticles for Biomedical Applications. J. Nanopart. Res. 2012, 14, 1100. 56.

Yang, H.; Chen, Z.; Zhang, L.; Yung, W. Y.; Leung, K. C. F.; Chan, H. Y. E.; Choi, C. H.

J., Mechanism for the Cellular Uptake of Targeted Gold Nanorods of Defined Aspect Ratios. Small 2016, 12, 5178-5189.


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

Page 30 of 41

Figure Legends Scheme 1. Summary of experimental procedures used in this study. Remote control of multimodal ligand oscillations was used to regulate the adhesion and differentiation of stem cells by altering the oscillation frequency of the magnetic field. Superparamagnetic iron oxide nanoparticle (SPION), functionalized with amino-silica coating, was conjugated to a glass substrate with long flexible polyethylene glycol linker (Mn = 5000 Da) to which the RGD ligand was grafted. Such platform was used to control stem cell adhesion both in vitro and in vivo, and subsequent differentiation. Figure 1. Tunable oscillation speeds of RGD ligand-tethered SPION. (A) X-ray diffraction spectra of superparamagnetic iron oxide nanoparticle (SPION). Crystalline plane indices were assigned from the diffraction peaks of magnetite (Fe3O4). (B) Transmission electron micrograph, (C) dynamic light scattering analysis, and (D) vibrating sample magnetometer measurement of RGD ligand-tethered SPION. Scale bar indicates 50 nm. The magnetization was normalized to the dry weight of the SPION. (E) Time-lapse snapshots of the tunable oscillatory motions of 30 ACS Paragon Plus Environment

Page 31 of 41

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

ACS Nano

RGD-bearing SPION under an oscillating magnetic field at various frequencies (2.5, 5, or 12.5 mHz). Blue dotted circles highlight red-fluorescent SPION. Blue arrow indicates the direction of the moving SPION. Scale bar represents 20 µm. (F) Quantified average motion speeds of the SPION under oscillatory magnetic fields of various frequencies. Data are displayed as mean ± standard errors (n=20).

Figure 2. In situ magnetic imaging of the nanoscale motion and integrin binding of the RGD ligand tethered onto the substrate. (A) Scanning electron micrograph and (B) 3D atomic force micrograpy (AFM) image for a monolayer of RGD ligand-SPION coated substrate. The scale bars represent 1 µm. (C) In situ AFM images scanned on the identical area with external magnetic field applied at two opposite outer sides of the scan area. Spherical particle represents SPION, while utilizing Au nanorod (aspect ratio of 4) as a non-magnetic reference. Black dotted lines were drawn along the long axis of nanorod to characterize the movement of the SPION relative to the nanorod. Red dotted lines were drawn across the centers of the SPIONs to show the movement of the SPIONs. White arrow indicates the direction of moving SPION from the left to right image. The scale bars indicate 50 nm. (D) In situ magnetic high-angle annular darkfield scanning transmission electron microscopy (HAADF-STEM) images scanned on the identical area with localized magnetic field manipulated by a magnetic tip relocated at three different positions with approximately10 min time interval near SPIONs inside the STEM chamber. Gray dotted lines were drawn across the center of lower SPIONs to show the relative positional changes of upper SPIONs through the movement of both upper and lower SPIONs. Red dotted lines were drawn across the centers of the upper SPIONs to show relative movement of the SPIONs. Red arrows indicate the direction of positional changes of upper SPIONs relative to lower SPIONs from the left to middle to right image. The scale bars indicate 50 nm. (E) Immunofluorescent staining images of integrin β1 binding to the substrate-tethered “Stationary”, “Slow”, “Moderate”, and “Fast” ligand-SPION oscillations under various oscillation frequencies (0, 0.1, 0.5, and 2 Hz) of applied magnetic field, respectively, and (F) corresponding quantification of fluorescence intensities. Red arrows indicate ligand-bound integrin clusters. Scale bars represent 50 µm. Data are shown as mean ± standard errors (n=6). Different letters indicate statistical significances (p < 0.05) as determined by one-way ANOVA with TukeyKramer post-hoc test. 31 ACS Paragon Plus Environment

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

Page 32 of 41

Figure 3. Ligand oscillations regulate stem cell adhesion with oscillation frequencydependency. (A) Time-dependent immnuofluorescent staining micrographs of vinculin (green), actin (red), and nuclei (blue) after 12 h and 48 h of hMSC culture under “Stationary”, “Slow”, “Moderate”, and “Fast” ligand-SPION oscillations by applying various oscillation frequencies (0, 0.1, 0.5, and 2 Hz) of magnetic field, respectively, with “No RGD” and “No magnet” controls. Scale bars represent 100 µm. (B) Corresponding quantifications of adhered cell density and area. Data are shown as mean ± standard errors (n=30). Different letters represent statistical significances (p < 0.05) at the same culture time as determined by one-way ANOVA with Tukey-Kramer post-hoc test.

Figure 4. Time-regulated switching of the ligand oscillations allows reversible focal adhesion of stem cells. (A) Time-dependent immnuofluorescent staining micrographs of vinculin (green), actin (red), and nuclei (blue) after 12 h and 48 h of hMSC culture under ligandSPION oscillations with reversible switching for oscillation frequencies between “Slow” oscillation (0.1 Hz) and “Fast” oscillation (2 Hz) of the magnetic field after 12 h of culture for “Slow-Fast” and “Fast-Slow” groups. Non-switched “Slow” and “Fast” oscillation groups are included as a control. Scale bars indicate 50 µm. (B) Corresponding quantification of adhered cell area, focal adhesion number, and cell shape factor. Data are shown as mean ± standard errors (n=30). Different letters indicate statistical significances (p < 0.05) at the same culture time as determined by one-way ANOVA with Tukey-Kramer post-hoc test.

Figure 5. Modulating ligand oscillations regulate stem cell differentiation through involvement of mechanosensing. (A) Immunofluorescent staining against RUNX2 (green), actin (red), and nuclei (blue) and ALP staining as well as YAP (green), actin (red), and nuclei (blue)

after 7 d and 2 d of hMSC culture, respectively, under “Stationary”, “Slow”, and

“Moderate” oscillations by applying various oscillation frequencies (0, 0.1, and 0.5 Hz) of magnetic field, respectively, with “No magnet” control. The groups include time-regulated switch groups “Slow-Fast” and “Fast-Slow” at 12 h of culture. Scale bars indicate 100 µm. (B) Corresponding ratiometric analyses of nuclear to cytoplasmic fluorescence intensities for RUNX2 and YAP. Data are presented as mean ± standard errors (n=30). Different letters indicate 32 ACS Paragon Plus Environment

Page 33 of 41

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

ACS Nano

statistical significances (p < 0.05) as determined by one-way ANOVA with Tukey-Kramer posthoc test. Figure 6. Remote control of ligand oscillations regulate stem cell adhesion in vivo. (A) Immnuofluorescent staining micrographs of human-specific nuclear antigen (HuNu; green) or vinculin (green), actin (red), and nuclei (blue) after 4 h of in vivo hMSC injection onto the implanted substrate under “Stationary”, “Slow”, and “Fast” ligand oscillations by applying various oscillation frequencies (0, 0.1, and 2 Hz) of magnetic field, respectively, with “No RGD” and “No magnet” controls. Scale bars indicate 100 µm. (B) Corresponding quantification of adhered cell density, area, focal adhesion, and aspect ratio. Data are presented as mean ± standard errors (n=30). Different letters indicate statistical significances (p < 0.05) as determined by one-way ANOVA with Tukey-Kramer post-hoc test.


Scheme 1

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

ACS Paragon Plus Environment

Page 34 of 41

Page 35 of 41

ACS Nano

Figure 1 B

A Intensity (a.u.)

(311)

(220)

20

30

(400) (222)

40

(511) (440) (422)

50

60

70

2 (Degree)

D Magnetic moment (emu/g)

C

Intensity (%)

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

50 40 30 20 10 0

0

20

40

60

80

100

15 0 -15 -30 -6000 -3000

0

3000 6000

Magnetic field strength (Oe)

Size (d, nm) E

F 30 s

60 s RGD-SPION speed ( m/s)

0s Oscillating magnetic field

30

2.5 mHz

5 mHz

2.0 1.5 1.0 0.5 0.0 0

2.5

5

12.5

Magnet oscillation frequency (mHz)

12.5 mHz


ACS Nano

Page 36 of 41

Figure 2 B

A

40 nm 0

1 µm

C

D

SPION

SPION SPIONs

Au nanorod

SPIONs

SPIONs

Au nanorod

E

F Integrin

400 300 200 100 0

Moderate

b

500

Fast


a

a c

Oscillating magnetic field

Slow Fluorescence intensity (a.u.)

Stationary


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

Stationary Slow Moderate Fast

Page 37 of 41

ACS Nano

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

A

12 h

48 h

Vinculin/Actin/ Nuclei

Vinculin

Actin/Nuclei

Slow Moderate

Adhered cell density (Cells/cm2)

B 5000

c

4000 b

3000 2000

b

b

a a

1000 0

Adhered cell area (m 2)

Fast


Stationary

No magnet

No RGD

Actin/Nuclei

15000 10000 5000 a

b b

b a

d

e e

0 12 h

12 h

f

c


48 h

e d


ACS Nano

Figure 4 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 0 h 12 h

Page 38 of 41

48 h

12 h

48 h


Vinculin


Vinculin

slow

fast

fast

slow

15000

c

c

a

a 10000

e 5000

d

b

b

0 12 h

48 h

150 c 100 a

c

a

50

e b

b

d

Aspect ratio (Major/minor axis)

B

fast

2 Focal adhesion number Adhered cell area (m )


slow

15 b

b

10

d d

5 a

a

c

c

0

0 12 h

48 h


12 h

48 h

Page 39 of 41

ACS Nano

Figure 5 A

0h

RUNX2

12 h

7d

0h

Actin/Nuclei

YAP

ALP

No magnet

Stationary

slow


Moderate

slow

fast

fast

slow

b

15

b

10 a

5 a 0

a

a

Nuclear/cytoplasmic YAP ratio

B Nuclear/cytoplasmic RUNX2 ratio

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

b

4 3

a

b a

a

2 1 0


a

12 h

48 h

Actin/Nuclei

ACS Nano

Figure 6

A

Vinculin

Actin/Nuclei

HuNu


Fast

Slow

Stationary

No magnet

No RGD

Actin/Nuclei


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

Page 40 of 41

b

b

b

1500 1000

a a

500 0 60

b

40 20

a

a

a c

0

Adhered cell area (m 2)

2000

Aspect ratio (Major/minor axis)

Focal adhesion number

Adhered cell density (Cells/cm2)

B 10000

In vivo adhesion

b

8000 6000 4000 a

a

a c

2000 0

15 c

10 a a

5 0


a b

Page 41 of 41

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

ACS Nano

358x184mm (150 x 150 DPI)


Remote Control of Multimodal Nanoscale Ligand Oscillations

Recommend Documents