1：Experimental Design and Method Selection:

The study involved synthesizing hydrophobic SPIO NPs via thermal decomposition, coating them with DSPE-PEG 2000 and DOX using thin-film hydration, and loading ICG into the lipid shell. Methods included characterization of NPs, in vitro biocompatibility and cytotoxicity assays (MTT), and in vivo imaging (fluorescence and MR) and efficacy evaluations in animal models.

2：Sample Selection and Data Sources:

Human umbilical vein endothelial cells (HUVECs) and U251 glioma cells were used for in vitro studies. Orthotopic glioma models were established in BALB/c nude mice and Wistar rats using C6 glioma cells.

3：List of Experimental Equipment and Materials:

Equipment included a rotary evaporator, fluorescence spectrophotometer (Varian Cary Eclipse), TEM (Hitachi H-7650), DLS analyzer (ZetaPALS), ICP-OES (PerkinElmer Optima 5,300 DV), CLSM (Olympus FV300), microplate reader (BioTek ELx800), IVIS spectrum imaging system (PerkinElmer), and 3.0 T MR system (Philips Achieva). Materials included iron tri(acetyl acetonate), oleylamine, oleic acid, DSPE-PEG 2000, DOX.HCl, MTT, DMEM, FBS, trypsin-EDTA, streptomycin, penicillin, chloroform, PBS, dialysis bags, and various chemicals from suppliers like J&K Chemical, Advanced Vehicle Technology, Meilune Biological Technology, Sigma-Aldrich, and Thermo Fisher Scientific.

4：0 T MR system (Philips Achieva). Materials included iron tri(acetyl acetonate), oleylamine, oleic acid, DSPE-PEG 2000, DOX.HCl, MTT, DMEM, FBS, trypsin-EDTA, streptomycin, penicillin, chloroform, PBS, dialysis bags, and various chemicals from suppliers like J&K Chemical, Advanced Vehicle Technology, Meilune Biological Technology, Sigma-Aldrich, and Thermo Fisher Scientific. Experimental Procedures and Operational Workflow:

4. Experimental Procedures and Operational Workflow: NPs were synthesized, characterized for size, zeta potential, and drug loading. In vitro studies involved MTT assays for biocompatibility and cytotoxicity, and cellular uptake via CLSM. In vivo studies included fluorescence and MR imaging to assess BBB penetration and tumor accumulation, and antitumor efficacy was evaluated by monitoring tumor size, survival, and histology.

5：Data Analysis Methods:

Data were analyzed using one-way ANOVA and Student's t-tests, with statistical significance set at P<0.05. Results are presented as mean ± SD.