1：Experimental Design and Method Selection:

The study involved designing and synthesizing paclitaxel dimer (PTX-s-s-PTX) and a two-photon photosensitizer (2PE-PS), which were coassembled into nanoparticles (Co-NPs) using a nanoprecipitation method. The rationale was to create stable nanoparticles that enable synergistic chemo-photodynamic therapy. Theoretical models included self-assembly principles for nanoparticle formation and photodynamic therapy mechanisms for reactive oxygen species (ROS) generation.

2：Sample Selection and Data Sources:

Samples included synthesized PTX-s-s-PTX and 2PE-PS, with nanoparticles formed in aqueous solutions. Biological samples included HeLa and HepG2 cancer cell lines for in vitro studies and male nude mice with HeLa tumor xenografts for in vivo studies. Data were acquired through spectroscopic, microscopic, and cytotoxicity assays.

3：List of Experimental Equipment and Materials:

Equipment: Bruker NMR-400 DRX spectrometer for 1H NMR, Bruker autoflex III smartbeam MALDI-TOF/TOF mass spectrometer for MS, Shimadzu UV-2450 PC UV/Vis spectrophotometer for UV-vis, PerkinElmer LS-55 Spectrofluorophotometer for fluorescence, Malvern Zeta-sizer Nano for DLS and zeta potential, JEOL JEM-1011 TEM for morphology, Zeiss LSM 700 CLSM for imaging, fluorescence microscope (Nikon Eclipse TE2000-U), micro plate reader for MTT assay, flow cytometer. Materials: PTX-s-s-PTX, 2PE-PS, DCC, DMAP, paclitaxel, 3,3-dithiodipropionic acid, sodium hydroxide, cyclopentanone, MTT, Lyso-Tracker Red, Live-Dead Cell Staining Kit, DPBF, DCFH-DA, AO, calcein-AM, PI, DAPI, tetrahydrofuran, DMF, DMSO, fetal bovine serum, cell culture media.

4：Experimental Procedures and Operational Workflow:

Synthesis of PTX-s-s-PTX and 2PE-PS followed literature methods. Nanoparticles were prepared by dropwise adding THF solutions into water, evaporating THF, and dialyzing. Characterization included TEM, DLS, UV-vis, fluorescence, and zeta potential. Singlet oxygen detection used DPBF degradation under 808 nm laser. Intracellular studies involved cell incubation with Co-NPs, irradiation, staining with probes (e.g., DCFH-DA for ROS, Lyso-Tracker Red for lysosomes, AO for permeability), and imaging with CLSM or fluorescence microscope. Cytotoxicity was assessed via MTT assay and live/dead staining after irradiation. In vivo studies involved intratumoral injection of Co-NPs in mice, irradiation, and monitoring tumor volume and weight.

5：Data Analysis Methods:

Data analysis included statistical methods (e.g., significance tests with p-values), quantification of fluorescence intensity, IC50 calculation from cytotoxicity data, and image analysis for colocalization and cell viability. Software tools were not specified, but instruments like CLSM and flow cytometer provided quantitative outputs.