1：Experimental Design and Method Selection:

The study involved the synthesis of P-4 through a multi-step chemical reaction process, followed by self-assembly into nanoparticles (NPs) for enhanced solubility and targeting. Methods included UV-vis-NIR spectroscopy, fluorescence spectroscopy, NMR, MALDI-TOF MS, DLS, TEM, and various biological assays to characterize properties and therapeutic effects.

2：Sample Selection and Data Sources:

HeLa cells were used for in vitro studies, and nude mice with HeLa tumor xenografts were used for in vivo experiments. Samples included P-4 and its intermediates, P-4 NPs in PBS at different pH levels, and biological reagents from commercial sources.

3：List of Experimental Equipment and Materials:

Equipment included UV-vis-NIR spectrometer (Shimadzu), Fluorescence F-7000 spectrometer (HITACHI), NMR spectrometer (Bruker DRX), MALDI-TOF MS (Bruker autoflex speed), DLS (Malvern Zeta Sizer), TEM (JEM-2010FEF), confocal fluorescence microscope (Olympus IX 70), inverted fluorescence microscope (Nikon), Flow Cytometry System, IR thermal camera (FLIR System), 660-nm diode-laser, and in vivo bioluminescence imaging system. Materials included fetal bovine serum (FBS), MTT, various trackers (Lyso-Tracker, Mito-Tracker, ER-Tracker), DAPI, DPBF, SOSG, and chemical agents from Sigma-Aldrich and GIBCO.

4：Experimental Procedures and Operational Workflow:

Synthesis of P-4 involved reactions with specific reagents under controlled conditions. P-4 NPs were prepared by self-assembly in PBS. Characterizations included absorbance, fluorescence, size, and stability measurements. In vitro assays involved cell culture, cytotoxicity (MTT), apoptosis (flow cytometry), ROS detection, subcellular localization, and cellular uptake. In vivo studies included fluorescence imaging, pharmacokinetics, antitumor assays, and histology.

5：Data Analysis Methods:

Data were analyzed using standard equations for quantum yields and photothermal conversion efficiency. Statistical analysis included calculation of IC50, Pearson’s correlation coefficient, and pharmacokinetic parameters using DAS 2.0 software.