Additional file 1 of Facile fabrication of multi-pocket nanoparticles with stepwise size transition for promoting deep penetration and tumor targeting

Additional file 1: Fig. S1. 1H NMR spectrum (400 MHz) of mPEG-LA conjugates in CDCl3. Fig. S2. SEM image (A), size (B) and zeta potential (C) of the self-assembly of mPEG-LA conjugates (NPs, 100 g mL−1). Fig. S3. Size distribution of MPNs at different concentrations. Fig. S4. Fluorescence spectra of a mixed solution of NPs (100 μg mL−1)and MPNs (100 μg mL−1) prepared with 0.1 wt% DiO/DiI tracing the development of FRET between two dyes. Fig. S5. Critical aggregation concentration of MPNs at (A) pH 7.4 or (B) 6.5. Fig. S6. Fluorescence spectra of a mixed solution of MPNs (100 μg mL−1, pH 6.5) prepared with 0.1 wt% DiO/DiI tracing the development of FRET between two dyes over time. Fig. S7. Flow cytometric histogram profiles of 4T1 cells treated with MPNs, DOX, D-NPs and D-MPNs for 3 h (DOX concentration: 10 μg mL−1). Fig. S8. CLSM images of 4T1 cells incubated with DOX, D-NPs, D-MPNs and DOX·HCl for 1 h (DOX dosage: 2 μg mL−1). Hoechst 33342 was used to stain the cell nuclei. Scale bar: 10 μm. Fig. S9. Quantitative assessment of penetration of DOX formulations into 4T1 cells. The cells were incubated with DOX, D-NPs and D-MPNs at a DOX concentration of 5 μg mL−1 for 2 h. Fig. S10. IOD (Integrated optical density) of immunohistochemical images for saline, MPNs, DOX·HCl, D-NPs and D-MPNs groups. The apoptotic rates of tumor sections based on TUNEL images (A), tumor microvessel density based on CD31 images (B) and Ki67 positive cells (C) of tumor sections were calculated with Image-Pro Plus 6.0 software. Data were presented as mean ± SD (n = 3), *p < 0.01, **p < 0.005. Fig. S11. Body weight changes of 4T1-bearing BALB/c mice after administration with saline, MPNs, DOX·HCl, D-NPs and D-MPNs (n = 8, dosage: 5 mg DOX kg−1 mouse body weight, *p < 0.01). The arrows indicated the time points for intravenous injection. Fig. S12. Routine blood analysis results of the mice collected on the 12th day after intravenous injection of saline, MPNs, DOX·HCl, D-NPs or D-MPNs. The results show mean and standard deviation of white blood cells (WBCs), red blood cell (RBC), hemoglobin (HGB) and platelets (PLT). Fig. S13. Histological examination of major organs separated from 4T1-bearing BALB/c mice after administration with saline, MPNs, DOX·HCl, D-NPs and D-MPNs for 18 days. Scale bar: 100 μm. Fig. S14. TUNEL staining of heart (×200) of 4T1-bearing BALB/c mice after administration with saline, MPNs, DOX·HCl, D-NPs and D-MPNs for 18 days. The apoptotic cells (red arrow) and normal cells were stained brown and blue, respectively. Table S1. Pharmacokinetic parameters of DOX·HCl, D-NPs and D-MPNs after intravenous administration at an equivalent dose of 10 mg DOX/kg mouse body weight (n = 3 per group).