Supplementary Information

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1 Supplementary Information Nanofibrous Spongy Microspheres to Distinctly Release mirna and Growth Factors to Enrich Regulatory T Cells and Rescue Periodontal Bone Loss Zhongning Liu, Xin Chen, Zhanpeng Zhang, Xiaojin Zhang, Laura Saunders, Yongsheng Zhou, and Peter X. Ma Supplementary Figures Supplementary Figure S1. MSNs and their mesoporous structure characterization. Transmission electron micrographs (TEM) of unmodified MSNs (a), PLGA and amino 1

2 group functionalized MSNs (b). The diameter distribution of MSNs measured using dynamic light scattering (DLS) (c) and pore volume distributions of the MSNs before and after functionalization (d). The diameter of the MSNs is around 300 nm, with an average pore size of around 15 nm. There are scattered areas with PLGA modification that improved the MSN attachment to the NF-SMS but did not appear to substantially affect the overall particle size or porosity of these MSNs. Supplementary Figure S2. FT-IR spectra and H NMR spectra of the amino functionalized multi-armed cationic polymer HP (H40-PEI-PEG). a b Supplementary Figure S3. The size (a) and zeta potential (b) of HP/miR-10a polyplexes at various N/P ratios. Above N/P ratio of 1.0, the mirna was fully encapsulated by the multi-armed cationic polymer. Each data point represents the mean ± standard deviation (n = 3). 2

3 Supplementary Figure S4. Agarose gel electrophoresis of different HP/miRNA at various N/P ratios using 1% agarose in Tris-acetate running buffer. Supplementary Figure S5. Transfection of HP/Cy3 pre-stained mirna polyplexes into total T cells at 24 h. The red fluorescence shows that the cationic HP can capture and effectively transfect mirna into T cells. 3

4 Supplementary Figure S6. After transfection of HP/miR-10a polyplexes into total T cells for 1 day and 3 days, the mir-10a gene expression levels were significantly higher than those in the corresponding mirna negative control groups. Supplementary Figure S7. FT-IR spectra of amino group modified MSNs (black curve), PLGA and amino group functionalized MSNs (red curve). Supplementary Figure S8. The H&E staining show the injection sites, where the microspheres remained under the gingival tissue. 4

5 Supplementary Figure S9. The role of IRS-1 in regulating Foxp3 expression was examined using Western blotting. a) The intracellular IRS-1 was substantially downregulated and the Foxp3 level was upregulated by si-irs-1 compared with its negative control si-nc. b) The IRS-1 level was increased and the Foxp3 level was decreased by IRS-1 plasmid (pcdna3.1-irs-1) compared with the control plasmid (pcdna3.1) group. Supplementary Figure S10. The biocompatibility of NF-SMS for T cells was tested by using Cell Counting Kit-8. The results show that the microspheres had no detectable level of inhibitory effect on T cells. 5

6 Supplementary Figure S11. The gene expression profile of gingival tissues was quantified using real-time PCR. The results show that these additional inflammatory cytokines IL6, IL8, IFN-γ and MCP-1 were lower in the combined mir-10a/il-2/tgf-β release group than in all other groups. 6

7 Supplementary Figure S12. The bone resorption of various control groups in a mouse periodontal disease model (after ligation): untreated - no treatment after ligation; MS only NF-SMS vehicle injection without loading of any biologics; Mir10a bolus HP/miR-10a polyplex injection; IL-2/TGF-β - bolus IL-2/TGF-β combination injection without any vehicle; Mir10a/IL-2/TGF-β bolus HP/miR-10a polyplex and IL-2/TGFβ combination injection. MicroCT results show bone loss and the bone volume between the first and second molars of these control groups in the periodontitis model. Supplementary Methods Transmission electron microscopy (TEM) images were recorded on a Philips CM200 transmission electron microscope operated at 200 kv. For the TEM observation, samples were obtained by dropping 5 μl of solution onto carbon-coated copper grids. All the TEM images were visualized without staining. The surface areas were calculated using the Brunauer Emmett Teller (BET) method, and the pore size distributions were calculated using the Barrett Joyner Halenda (BJH) method. The ultraviolet-visible (UV-Vis) spectra were measured with dilute aqueous solution in a 2-mm thick quartz cell using a Hitachi U-2910 spectrophotometer. The dynamic light scattering (DLS) and zeta potentials were measured using a Delsa Nano C particle analyzer (Beckman Coulter, USA) running Delsa Nano software and using 4 mw He- Ne laser operating at a wavelength of 633 nm and avalanche photodiode (APD) detector. The infrared (IR) spectra were obtained using AVATAR 320 FT-IR using KBr pellets. 7

8 1H NMR experiments were performed using a Bruker DRX400 spectrometer operating at 400 MHz. The mirna loading content in the NF-SMS was about nmol/mg. First, 1.0 mg of PLGA MS and MSNs co-loaded PLLA NF-SMS containing polymer/mirna polyplex was dissolved in 1.0 ml of dichloromethane. Then, 0.5 ml of RNase-free water was added to extract mirna. The extraction process was repeated five times. The mirna concentration was measured using a ThermoElectron 3001 Varioskan Flash Spectral Scanning Microplate Reader. The loading contents of IL-2 and TGF-β in the NF-SMS were determined to be about 0.45 µg/mg and 0.87 µg/mg, which were measured with a human IL-2 enzyme-linked immunosorbent assay (ELISA) kit (PeproTech, USA) and a human TGF-β ELISA kit (Boster, China), respectively, after the dissolution and extraction process similar to that for mirna. In this procedure, 2 ml aqueous sodium hydroxide solution (1 mol/l) was used to dissolve MSNs and extract protein. To test the transfection efficiency of HP/miRNA polyplexes into T cells, Cy3 Dye- Labeled Pre-miR Negative Control (Ambion, USA) and the HP solution (1.0 mg/ml) were mixed and incubated at room temperature for 30 min for HP/miRNA polyplex formation. Then, the polyplexes were added into the culture medium of total T cells. After 24h of incubation, the cells were fixed, mounted with Vectashield mounting medium containing DAPI (Vector Laboratories, USA), and observed under a laser scanning confocal microscope (Nikon TS-100, Tokyo, Japan). The fluorescence results show that the cationic HP can capture and transfer mirna into T cells (Figure S5). To further verify the transfection efficiency, total T cells were cultured with HP/miR-10a complexes for 1 day and 3 days. After incubation, total RNA of the cells was extracted, the mir-10a gene expression was examined using real-time PCR. The gene expression results show that the delivery system could transfect the mir-10a into T cells efficiently (Figure S6). 8

9 Periodontal disease model was established to examine the effect of multifunctionalized spongy microspheres on rescuing bone loss due to periodontitis. We established a ligature-induced mouse periodontal disease model by ligaturing between first and second molars. We injected 3 µl microspheres into the gingival margin between the first and second maxillary molars of 8-week-old C57BL/6 mice. To visualize microspheres in periodontal tissues, the formalin-fixed maxillae were decalcified, dehydrated, embedded in paraffin, sectioned at a thickness of 5 μm, and stained with hemotoxylin and eosin (H&E). The H&E staining show that the injected microspheres could be kept locally under the gingival tissues in the periodontal disease model (Figure S8). 9