Supporting Information

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1 Supporting Information Magnetic Manipulation of Reversible Nanocaging Controls In Vivo Adhesion and Polarization of Macrophages Heemin Kang, Hee Joon Jung,,, Sung Kyu Kim,,, Dexter Siu Hong Wong, Sien Lin, #,, Gang Li, #,, Vinayak P. Dravid,,, and Liming Bian,,,,±,* Department of Biomedical Engineering, The Chinese University of Hong Kong, Hong Kong, China. Department of Materials Science and Engineering, Northwestern University, Evanston, IL, USA. International Institute for Nanotechnology, Evanston, IL, USA. NUANCE Center, Northwestern University, Evanston, IL, USA. # Department of Orthopaedics & Traumatology, Faculty of Medicine, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong, China. Stem Cells and Regenerative Medicine Laboratory, Lui Che Woo Institute of Innovative Medicine, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong, China. The CUHK-ACC Space Medicine Centre on Health Maintenance of Musculoskeletal System, The Chinese University of Hong Kong Shenzhen Research Institute, Shenzhen, China. Department of Pharmacology, Guangdong Key Laboratory for Research and Development of Natural Drugs, Guangdong Medical University, Zhanjiang, Guangdong, China. o Translational Research Centre of Regenerative Medicine and 3D Printing Technologies of Guangzhou Medical University, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China. Shenzhen Research Institute, The Chinese University of Hong Kong, China. ± China Orthopedic Regenerative Medicine Group (CORMed), Hangzhou, Zhejiang, China. 1

2 Supplementary Figure Legends Supplementary Figure S1. Characterization of the gold nanoparticles (GNPs). (A) Transmission electron micrograph and (B) dynamic light scattering analysis of the size distribution of the GNPs. Scale bar indicates 20 nm. 2

3 Supplementary Figure S2. Characterization of the magnetic nanocages (MNCs). (A) Transmission electron micrograph and (B) dynamic light scattering analysis of the size distribution of the MNCs. Scale bar represents 20 nm. 3

4 Supplementary Figure S3. X-ray diffraction spectra of the magnetic nanocage (MNC). The diffraction peaks were assigned according to the crystalline plane indices of those from magnetite phase (Fe3O4) of the iron oxide nanoparticles. Supplementary Figure S4. Superparamagnetic property of the magnetic nanocage (MNC) by vibrating sample magnetometer measurement. The magnetic moment was presented after normalization to the dry weight of the MNC. 4

5 Supplementary Figure S5. Functionalization of the magnetic nanocage (MNC) with a flexible polymer. Fourier transform infrared spectroscopy of the MNC before and after thiol-pegylation. Chemical bonds were assigned to the peaks in the absorption spectra. Supplementary Figure S6. Coating of the magnetic nanocage (MNC) with a flexible molecule. Zeta potential measurement of the MNC before and after thiol-pegylation. Data are shown as mean ± standard errors (n=3). 5

6 Supplementary Figure S7. Energy dispersive spectroscopy of the GNP-grafted substrate. Au element confirmed the successful grafting of the GNP to the substrate. Supplementary Figure S8. Energy dispersive spectra of the (RGD-GNP)-MNC heterodimergrafted substrate. Au element indicates the grafting of the GNP to the substrate, whereas elemental Fe and S confirms the simultaneous grafting of the thiolated MNC to the substrate. 6

7 Supplementary Figure S9. Direct coupling between the GNP and thiolated MNC on the substrate. Scanning transmission electron microscope image and corresponding elemental dispersive spectroscopy mapping of the heterodimer (Au element for GNP and, Fe and S elements for thiolated MNC). Supplementary Figure S10. Magnetic field strength as a function of distance from the permanent magnet. Data are shown as mean ± standard errors (n=3). 7

8 Supplementary Figure S11. Characterization of the nanostructure of the heterodimer grafted onto the substrate by atomic force microscopy (AFM) imaging. AFM image of the (RGD-GNP)-MNC heterodimer-grafted substrate and the corresponding height profile of a linear scan across the heterodimer, along the direction indicated by the white dotted line. Scale bar represents 20 nm. GNP and MNC were differentiated by their significantly different nanosizes (smaller GNP and larger MNC). Supplementary Figure S12. Control experiments for atomic force microscopy (AFM) imaging characterization of heterodimer-grafted substrate. AFM images of the (RGD-GNP)- MNC heterodimer-grafted substrate scanned on an identical area, under the continuous application of a magnetic field. Black dotted line was drawn across the centers of the RGD-GNPs as a nonmagnetic reference. Red dotted line was drawn across the centers of the magnetic nanocage (MNC) to confirm the negligible displacement of the MNC under the continuous application of the magnetic field. Scale bar represents 20 nm. 8

9 Supplementary Figure S13. Minimal non-specific macrophage adhesion on the GNP-MNC heterodimer-grafted substrate, without RGD conjugation to the GNP. (A) Immnuofluorescent staining images against vinculin (green), actin (red), and nuclei (blue) in macrophages after 12 h in culture. Macrophages were cultured on the GNP-MNC heterodimer-decorated substrate under caging or uncaging of the GNP without RGD ( Caging without RGD and Uncaging without RGD group, respectively). Scale bars represent 50 µm. (B) Corresponding quantifications of the density, area, and aspect ratio of the macrophages. Data are shown as mean ± standard errors (n=30). N.S. represents statistically non-significant differences found between the compared groups. 9

10 Supplementary Figure S14. No magnetic control of macrophage adhesion on the (RGD- GNP)-grafted substrate, in the absence of the magnetic nanocage (MNC). (A) Immnuofluorescent staining micrographs against vinculin (green), actin (red), and nuclei (blue) in macrophages after 12 h of culture. Macrophages were adhered on the (RGD-GNP)-coupled substrate with and without the application of an external magnetic field ( + Magnetic field and - Magnetic field groups, respectively). (B) Corresponding quantifications of the adherent macrophage density, area, and aspect ratio. Data are presented as mean ± standard errors (n=30). N.S. indicates a statistically non-significant difference between the compared groups. 10

11 Supplementary Figure S15. The RGD uncaging promotes the expression of ROCK2 in adherent macrophages. Immnuofluorescent staining images against ROCK2 (green) and nuclei (blue) under basal medium cultures after 24 h, M1-polarizing medium cultures after 36 h, or M2- polarzing medium cultures after 36 h. For the polarization cultures, the cells were initially cultured in basal medium for 12 h and then polarized under either M1- or M2-polarizing medium for another 24 h. The macrophages were cultured on the (RGD-GNP)-MNC heterodimer-grafted substrate under RGD caging or uncaging ( RGD caging and RGD uncaging group, respectively). Scale bars represent 50 µm. 11

12 Supplementary Figure S16. Quantitative gene expressions of M1 polarization markers (inos and CD80) in adherent macrophages in M2 polarization culture in the presence and absence of Y27632, a ROCK inhibitor. Macrophages were adhered onto the heterodimer-grafted substrate under RGD caging or uncaging ( RGD caging and RGD uncaging groups, respectively), or RGD uncaging in the presence of Y27632 ("RGD uncaging + Y27632 group). Macrophages were initially cultured in basal medium for 12 h and then polarized in M2-polarizing medium for another 24 h. Data are displayed as means ± standard errors (n=3). Gene expression data are presented as relative fold expression of the gene of interest (inos and CD80) after normalization to the RGD caging group. Various groups were compared with one-way ANOVA with Tukey-Kramer post-hoc test. N.S. indicates statistically non-significant differences. 12

13 Supplementary Figure S17. Quantitative gene expression of M2 polarization markers (Arg- 1 and Ym2) in adherent macrophages in M1 polarization cultures in the presence and absence of Y27632, a ROCK inhibitor. Macrophages adhered to the heterodimer-grafted substrate under RGD caging or uncaging ( RGD caging and RGD uncaging groups, respectively), or RGD uncaging in the presence of Y27632 ("RGD uncaging + Y27632 group). Macrophages were initially cultured in basal medium for 12 h and then polarized in M1-polarizing medium for another 24 h. Data are shown as means ± standard errors (n=3). Gene expression data are shown as relative fold expression of genes of interest (Arg-1 and Ym2) after the normalization to the RGD uncaging group. Various groups were compared by one-way ANOVA with Tukey- Kramer post-hoc test. N.S. indicates statistically non-significant differences. 13

14 Supplementary Figure S18. M2 polarization of the in vivo adherent host macrophages under reversible RGD uncaging. Immunofluorescent staining micrographs of actin (green), Arg-1 (red), and nuclei (blue) for the adherent cells at 24 h after the subcutaneous implantation of the (RGD- GNP)-MNC heterodimer-grafted substrate under RGD caging or uncaging ( RGD caging and RGD uncaging group, respectively), or RGD caging for the initial 12 h and uncaging for the subsequent 12 h ( RGD caging-uncaging group). Scale bars indicate 20 µm. 14

15 Supplementary Figure S19. Quantification of M2 polarization of in vivo adherent cells under reversible RGD uncaging. Quantitative gene expression of M2 polarization markers (Arg-1 and Ym2) in adherent cells at 24 h after the subcutaneous implantation of the heterodimer-grafted substrate under RGD caging or uncaging ( RGD caging or RGD uncaging group, respectively), or RGD caging for the initial 12 h and uncaging for the following 12 h ( RGD caging-uncaging group). Data are shown as means ± standard errors (n=3). Gene expression data are shown as the relative fold expression of the gene of interest (Arg-1 and Ym2) after the normalization to the RGD uncaging group. Various groups were compared by one-way ANOVA with Tukey-Kramer post-hoc test. N.S. indicates statistically non-significant differences. 15

16 Supplementary Figure S20. Characterization of in vivo adherent host neutrophils under reversible RGD uncaging. (A) Immunofluorescent staining micrographs of NIMP-R14 (green) and nuclei (blue) for the adhered cells at 24 h after the subcutaneous implantation of the heterodimer-decorate substrate under RGD caging or uncaging ( RGD caging or RGD uncaging group, respectively), or RGD caging for the initial 12 h and uncaging for the subsequent 12 h ( RGD caging-uncaging group). Scale bars represent 20 µm. (B) Corresponding quantification of the density of in vivo adherent host neutrophils. Data are shown as mean ± standard errors (n=30). Various groups were compared by one-way ANOVA with Tukey-Kramer post-hoc test. Different alphabetical letters (a and b) were assigned to statistically significant differences with p- values less than Same alphabetical letters indicate statistically non-significant differences. 16

17 Supplementary Figure S21. Remote control of the RGD uncaging with M2-polarizing cytokines suppresses M1 polarization of in vivo adherent host macrophages. Immunofluorescent staining micrographs of actin (green), inos (red), and nuclei (blue) of cells adherent to the subcutaneously implanted heterodimer-coupled substrate under reversible RGD uncaging with the injection of IL-4 and IL-13 onto the substrate after 24 h. Experimental conditions include RGD caging or uncaging ( RGD caging and RGD uncaging group, respectively), or RGD caging for the initial 12 h and uncaging for the following 12 h ( RGD caging-uncaging group). Scale bars represent 20 µm. 17

18 Supplementary Figure S22. Quantitative analysis of M1 polarization of adherent cells in vivo under reversible RGD uncaging with M2-polarizing cytokines. Quantitative gene expression of M1 polarization markers (inos and CD80) in cells adhering to the subcutaneously implanted heterodimer-coupled substrate following 24 h under RGD caging or uncaging ( RGD caging and RGD uncaging group, respectively), or RGD caging for the initial 12 h and uncaging for the subsequent 12 h ( RGD caging-uncaging group). Data are shown as means ± standard errors (n=3). Gene expressions are presented as relative fold expression of target genes after the normalization to the RGD caging group. Various groups were compared by one-way ANOVA with Tukey-Kramer post-hoc test. N.S. indicates statistically non-significant differences. 18

19 Supplementary Figure S23. Identification of in vivo adherent host neutrophils in the presence of M2-polarizing cytokines under reversible RGD uncaging. (A) Immunofluorescent staining images of NIMP-R14 (green) and nuclei (blue) of cells adherent to the subcutaneously implanted heterodimer-coupled substrate after 24 h. Experimental conditions include RGD caging or uncaging ( RGD caging and RGD uncaging group, respectively), or RGD caging for the initial 12 h and uncaging for the following 12 h ( RGD caging-uncaging group). Scale bars indicate 20 µm. (B) Corresponding quantification of the density of the in vivo adherent neutrophils. Data are presented as mean ± standard errors (n=30). One-way ANOVA with Tukey-Kramer post-hoc test was used to compare various groups. Different alphabetical letters (a and b) were assigned to statistically significant differences with p-values less than Same alphabetical letters indicate statistically non-significant differences. 19