Additional file 1 of Metformin and histone deacetylase inhibitor based anti-inflammatory nanoplatform for epithelial-mesenchymal transition suppression and metastatic tumor treatment

Additional file 1: Scheme S1 Synthetic route of pro-DHA. Fig. S1 Characterization of the HDAC inhibitor pro-DHA. The 1H-NMR spectrums of A propofol, B DHA and C pro-DHA in CDCl3. D The ESI-MS result of pro-DHA. Fig. S2 TEM images of A HAOPTs and B OPTs with wider view. Fig. S3 In vivo fluorescence imaging at 2, 6, 12 and 24 h after intravenous injection with OPTs-DiR, DexOPTs-DiR and HAOPTs-DiR. The white dotted ring represents orthotopic breast tumor tissues. Fig. S4 Cell cytotoxicity curves of A free OA-Met, B free pro-DHA, pro-DHA in HAOPs, C free triptolide, triptolide in HAOTs, triptolide in DexOPTs and triptolide in HAOPTs on 4T1 cells. Data were represented as mean ± SD (n = 3). Fig. S5 Quantitative analysis of cancer stem cell-like 4T1 cells with CD44+/CD24-/low phenotype in the flow cytometry assay. Data were represented as mean ± SD (n = 3), *P < 0.05, **P < 0.01, ****P < 0.0001. Fig. S6 Quantitative analysis of M2 macrophage subpopulation in the flow cytometry assay. Data were represented as mean ± SD (n = 3), *P < 0.05, ***P < 0.001, ****P < 0.0001. Fig. S7 Images of breast tumor tissues stained by immunohistochemical staining with A Collagen I and B MMP-9. The brown regions represent positive areas of Collagen I or MMP-9, Scale bar, 100 µm. Fig. S8 The image of ex vivo primary breast tumors. Fig. S9 Representative images of primary tumor sections analyzed by H&E staining after all treatments. The green dotted line separates necrotic area from normal sites. Scale bar, 200 µm. Fig. S10 HAOPTs promoted cell apoptosis in primary breast tumor tissues. A Representative images of apoptotic tumor tissues (green) after treatments of free OA-Met, free pro-DHA, free triptolide, HAOPs, HAOTs, DexOPTs and HAOPTs. Scale bar, 100 µm. B Quantitative analysis of TUNEL positive area. Data were represented as mean ± SD (n = 5), **P < 0.01, ***P < 0.001, ****P < 0.0001. Fig. S11 Mice body weight curves during the treatment period of free OA-Met, free pro-DHA, free triptolide, HAOPs, HAOTs, DexOPTs and HAOPTs. Data were represented as mean ± SD (n = 5). Fig. S12 H&E staining images of major organ sections after treatments of free OA-Met, free pro-DHA, free triptolide, HAOTs, DexOPTs and HAOPTs. The black arrows and plus signs in liver section indicated typical liver injuries and representative inflammatory cell infiltration, respectively. Scale bar, 200 µm. Fig. S13 Blood chemistry analysis of A AST, B ALT and C ALP in mice serum. Data were represented as mean ± SD (n = 5), ****P < 0.0001, compared with the saline group. Table S1 Characterization of OPTs, DexOPTs and HAOPTs. Table S2 Loading capacity and encapsulation efficiency of pro-DHA in OPTs, DexOPTs and HAOPTs. Table S3 Loading capacity and encapsulation efficiency of triptolide in OPTs, DexOPTs and HAOPTs. Table S4 Optimization of the preparation condition of HAOPTs. Table S5 Pharmacokinetic parameters of various DiR labeled formulations in SD rats