Chemiluminescence-initiated and in situ-enhanced photoisomerization for tissue-depth-independent photo-controlled drug release

Tissue-penetration-depth-independent self-luminescence is highly expected to perform photoisomerization-related bioapplications in vivo to overcome the limitation of shallow tissue-penetration from external photoexcitation.

= singlet, d = doublet, t = triplet, q = quartet, m = multiplet. Mass spectra were obtained on a matrix-assisted laser desorption/ionization time of flight mass spectrometry MS (MALDI-TOF, Bruker AutoFlex III system). The ultraviolet-visible (UV-vis) absorption, fluorescence and chemiluminiscence spectra were measured by a Shimadzu UV-vis spectrophotometer (UV-3600) and fluorescence spectrophotometer with a photomultiplier tube detector, respectively. The molecular weights (Mn) and polydispersities (PDI) of the polymers were determined by gel permeation chromatography (GPC) analysis on a Shim-pack GPC-80X column with water as the S3 eluent at a flow rate of 1.0 mL min -1 . Dynamic light scattering (DLS) was performed on a particle size analyzer (NanoBrook 90Plus, Brookhaven Instruments Corporation).
Transmission electron microscopy (TEM) images were performed on a HT7700 transmission electron microscope operating at 100 kV. The methyl thiazolyl tetrazolium (MTT) assay was performed by a PowerWave XS/XS2 microplate spectrophotometer (BioTek, Winooski, VT). Images were acquired by IVIS living imaging system.

Statistical analysis
Statistical analyses of the data were performed with the SPSS 12.0 software.
Measurement data are expressed as the mean ± SD. For cell viability assays, a Student's t-test was applied to identify significant differences. Multiple group comparison was conducted by one-way analysis of variance (ANOVA). A value of p < 0.05 was considered statistically significant.

Methods
Synthetic route to the compound PEAZO and PCD. 1,2

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The resulting solution was cooled to room temperature and extracted with ethyl acetate.
After the organic layer was dried with anhydrous magnesium sulfate, the solvent was removed by evaporation. The product was purified by column chromatogrqaphy (silica gel, ethyl acetate: hexane = 1:1 as eluent) to yield 2.0 g (58%) of orange powder. 1

Synthesis of 7 (EAZO).
Monomer 7 was synthesized according to previous report. 6 (9.0 g, 20 mmol), tosyl chloride (11.40 g, 60 mmol) and pyridine (20 mL) were dissolved in dichloromethane (250 mL). The resulting solution was stirred at 20 °C for 16 h. The reaction mixture was diluted with water (200 mL). The organic layer was washed with equivalent volumes of water several times. The organic layer was washed with brine twice and dried over magnesium sulfate, the solvent was evaporated under reduced pressure, and the crude product was used the next step. The crude product was used the next step.
Afterward, the reaction mixture was allowed to cool to room temperature and was diluted with ethyl acetate (250 mL

Synthesis of PEAZO.
Polymer PEAZO was synthesized according to previous report. 2

Synthesis of PCD.
Polymer PCD was synthesized according to previous report. 2

The emission-reabsorption studies
The emission-reabsorption process using an experimental setup as previously reported. 4 CPT, CPPO, PCD and H2O2 (2 mM) in beaker to produce blue CL, and the generated 4T1 Tumor Animal models.
In vivo blood elimination kinetics. Methods were demonstrated as previous reports.

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The jugular vein of Female BALB/c nude mice bearing 4T1 tumors (~90 mm 3