A Self‐Degradable Conjugated Polymer for Photodynamic Therapy with Reliable Postoperative Safety

Abstract As a noninvasive therapeutic technique, photodynamic therapy (PDT) has attracted numerous research interests for cancer therapy. Nevertheless, the residual photosensitizers (PSs) still produce reactive oxygen species (ROS) and damage normal cells under sunlight after PDT, which limits their practical application in clinic. Herein, the authors propose a self‐degradable type‐I PS based on conjugated polymer, which is composed of aggregation‐induced emission (AIE) and imidazole units. Due to the effective conjugated skeleton and unique AIE properties, thus‐obtained polymers can effectively generate superoxide radical (O2 −•) through the type‐I process under light irradiation, which is ideal for hypoxic tumors treatment. Intriguingly, under light irradiation, O2 −• produced by the conjugated polymers can further lead to the self‐degradation of the polymer to form nontoxic micro‐molecules. It not only helps to resolve the potential phototoxicity problems of residual PSs, but also can accelerate the metabolism of the conjugated polymers to avoid the potential biotoxicity of drug accumulation. This work develops a self‐degradable type‐I PS, which can turn off the generation of ROS in time after PDT, providing a novel strategy to balance the PDT effect and postoperative safety.

Triphenylamine (500 mg, 2 mmol) was dissolved in dry DMF (10 mL). The phosphoryl chloride (20 mL) was added dropwise to the mixture at 0 °C, and the mixture was stirred for 1 h at room temperature. Then, the reaction mixture was heated to 80 °C for 8 h under the N2 atmosphere.
Finally, the reaction solution was poured into ice-cold water (20 mL) to obtain the yellow precipitate. The residue was purified by column chromatography using ethyl acetate-petroleum ether as the eluent to give TPA-CHO as a yellow solid. 1

Synthesis of TPA-yen
The mixture of TPA-CHO (300 mg, 1 mmol) and 4-Ethynylphenylacetonitrile (141 mg, 1 mmol) were dissolved in anhydrous methanol (30 mL) and removed into reaction bulb. Then, the piperidine (5 drops) was added and the mixture was heated to reflux for 8 h. The methanol solution was removed by rotary evaporation and the crude product was separated by column chromatography on silica gel using ethyl acetate-petroleum ether. 1

Synthesis of CP1
The preparation of CP1 is through Sonogashira polymerization. TPA-yen (54 mg, 0.1mmol), 2,5-Diiodo-1-methylimidazole (33 mg, 0.1mmol), Pd(PPh3)2Cl2 (0.5 mg) and CuI (2mg) were dissolved well in ethyl acetate (5 mL). After degassing for 30 min by bubbling with N2, triethylamine (0.5 mL) was injected to initiate the Sonogashira reaction and the mixture was kept at 75 °C for 6h. Then, the reaction mixture would precipitate a red solid, which could be separated via centrifugation. The final product was purified by washing with ethyl acetate three times.

Synthesis of CP2
The preparation of CP2 was similar to CP1.

Synthesis of CP-NPs
Briefly, CP1 (3mg) and F127 (60mg) were dissolved well in DMSO (3mL). Then, the mixture solution was dropwise added into 10 mL PBS under sonication. After sonication for 1 h, the solution was dialyzed against PBS for 3 days to remove DMSO (MWCO: 3000 Da). Finally, the concentration of CP1 encapsulated in the nanoparticles was calculated by UV/VIS/NIR spectrophotometer at 450 nm. After being purified by a 0.2 μm filter, the resultant sample CP-NPs was stored at 4 °C for further usage.

ROS generation measurement in aqueous media
2′, 7′-dichlorodi-hydrofluorescein diacetate (DCFH-DA) (1 mM in Ethanol) was treated with NaOH (1 mM in H2O) for 30 min to obtain the DCFH. The final concentration of DCFH was 50 μM. Then, the 1 μg/mL CP1 solution (stock solution: 1mg/mL in DMSO) require a selfdegradable preprocessing under the white light irradiation (100 mW/cm 2 ) for 0, 3 and 10 min and let stand in dark for 1 h. Afterward, CP1 with different irradiation time were added into the solution of DCFH for ROS generation measurement. The fluorescence signals were recorded under 20 mW/cm 2 white light irradiation every 30 s. The maximal emission wavelength of the DCFH solution was at 526 nm with an excitation wavelength of 488 nm.

•OH generation measurement in aqueous media
The •OH generation measurements were conducted using hydroxyphenyl fluorescein (HPF) as the Indicator. The 1 μg/mL CP1 solution (stock solution: 1 mg/mL in DMSO) require a selfdegradable preprocessing under the white light irradiation (100 mW/cm 2 ) for 0, 3 and 10 min and let stand in dark for 1 h. Afterward, CP1 with different pre-irradiation time were added into the PBS buffer solution containing 10 μM HPF (stock solution: 5 mM in DMF). The fluorescence signals were recorded under 20 mW/cm 2 white light irradiation every 30 s. The maximal emission wavelength of HPF solution was at 514 nm with an excitation wavelength of 480 nm.

O2 generation measurement in aqueous media
The 1 O2 generation measurements were conducted using and 9,10-anthracenediylbis(methylene) dimalonic acid (ABDA) as the indicator. The 1 μg/mL CP1 solution (stock solution: 1mg/mL in DMSO) require a self-degradable preprocessing under the white light irradiation (100 mW/cm 2 ) for 0, 3 and 10 min and let stand in dark for 1 h. Afterward, CP1 with different pre-irradiation time were added into PBS buffer solution containing 20 μM ABDA (stock solution: 10 mM in DMSO). The absorption spectra were recorded under 20 mW/cm 2 white light irradiation every 30 s. The absorbance decline relative to the initial value at 380 nm was recorded to indicate the decomposition rates of ABDA ( 1 O2 generation rate).

Cytotoxicity evaluation of CP-NPs
The cell viability values of CP-NPs were determined via a counting kit-8 (CCK-8) assay using HeLa and 4T1 cells. Cells were planted in 96-well plates with a density of 10000 cells per well and cultured one day. Prepared CP-NPs solution requires a self-degradable preprocessing under the white light irradiation (100 mW/cm 2 ) for 0, 3, and 10 min and let stand in dark for 3 h. Then, the preprocessed CP-NPs with a series of concentrations from 5 to 80 μg/mL were added and incubated at 37 °C for 24 h in dark. After that, 10 μL of CCK-8 dyes and 100 μL of Dulbecco's Modified Eagle's Medium (DMEM) cell culture media were added to each well and incubated for 2 h at 37 °C. The absorbance of sample and control wells at 450 nm was recorded via a microplate reader. Cell viability was calculated based on the absorbance data. Three replicate wells were used for each control and test concentrations per microplate, and the experiment was repeated three times.

Confocal microscopic imaging
Hela cells were seeded in a confocal imaging dish maintained at 37 °C under a humidified condition of 5% CO2. Until the cell density reached 60-70% confluence, HeLa cells were first incubated with a culture medium containing 20 µg/mL CP-NPs for 0 and 2 h. Then, the old culture medium was removed and a fresh culture medium containing 1 µg/mL Hoechst33258 was added. After 30 min of culture, cell images were conducted with CLSM. For CP-NPs, the excitation wavelength was 450 nm and the emission filter was 580-620 nm. For Hoechst33258, the excitation wavelength was 405nm and the emission filter was 430-480 nm.

General ROS detection in vitro
HeLa cells were incubated in a confocal imaging dish until the cell density reached 60-70% confluence. Prepared CP-NPs solution requires a self-degradable preprocessing under the white light irradiation (100 mW/cm 2 ) for 0, 3, and 10 min and let stand in dark for 1 h. Then, HeLa cells were cultured with a medium containing 20 µg/mL preprocessed CP-NPs solution for 4 h and treated with a medium containing 10 μM DCFH-DA solution for 30 min at 37 °C, followed by white light irradiation at 20 mW/cm 2 for 3 min. Afterward, the signal intensity was recorded by CLSM at excitation of 488 nm laser. Light alone and CP-NPs alone were stained like the above procedures. The emission filter was 495-550 nm.

O2 −• detection in vitro
HeLa cells were incubated in a confocal imaging dish until the cell density reached 60-70% confluence. Prepared CP-NPs solution requires a self-degradable preprocessing under the white light irradiation (100 mW/cm 2 ) for 0, 3, and 10 min and let stand in dark for 1 h. Then, HeLa cells were cultured with a medium containing 20 µg/mL preprocessed CP-NPs solution for 4 h and treated with medium containing 10 μM DHE solution for 30 min at 37 °C, followed by white light irradiation at 20 mW/cm 2 for 3 min. Afterward, the signal intensity was recorded by CLSM at excitation of 488 nm laser. Light alone and CP-NPs alone were stained like the above procedures. The emission filter was 580-650 nm.

PDT evaluation in vitro
Hela cells and 4T1 cells were respectively planted in 96-well plates with a density of 10000 cells per well and cultured one day. Prepared CP-NPs solution requires a self-degradable preprocessing under the white light irradiation (100 mW/cm 2 ) for 0, 3, and 10 min and let stand in dark for 1 h Then, the preprocessed CP-NPs (20 μg/mL) were added and incubated at 37 °C for 4 h in dark. After exposure to white light irradiation (20 mW/cm 2 ) for 3 min, the cells were further seeded at 37 °C to 4 h. CCK-8 assay was consistent with the description in Cytotoxicity evaluation of CP-NPs.

Live/dead cell co-staining assay
HeLa cells and 4T1 cells were respectively incubated in a confocal imaging dish until the cell density reached 60-70% confluence. Prepared CP-NPs solution requires a self-degradable preprocessing under the white light irradiation (100 mW/cm 2 ) for 0, 3, and 10 min and let stand in dark for 1 h. Then, the preprocessed CP-NPs (20 μg/mL) were added and incubated at 37 °C for 4 h in dark. After exposure to white light irradiation (20 mW/cm 2 ) for 3 min, the cells were further seeded at 37 °C to 4 h. Afterward, the cells were stained with medium containing 2 μM Calcein-AM (AM) and 4 μM propidium iodide (PI) for 15 min. The cell imaging was recorded via a fluorescence microscope. PBS alone, light alone, and CP-NPs alone were stained like the above procedures.

In vivo imaging
All in vivo fluorescent images were recorded on the IVIS Lumina II In Vivo Imaging System (PerkinElmer). The nude mice bearing subcutaneous 4T1 tumor were given CP-NPs (200 μg/mL, 25μL/50mm 3 tumor) via intratumor injection. After injection of CP-NPs for 30 min, the tumor area of mice in the light group is irradiated by white light (200 mW/cm 2 ) for 20 min and the mice in no light group are untreated. Then fluorescent images were captured by the Lumina II at various time points. At 12 h of treatment with CP-NPs, the major organs and tumor tissues were collected for the imaging study.

PDT in vivo
All nude mice bearing subcutaneous 4T1 tumor mice were randomly divided into four groups (n = 5): "PBS", "PBS + Light", "CP-NPs", and "CP-NPs + Light". Thereinto, the intratumoral injected dose of PBS and CP-NPs (200 μg/mL) is 25 μL/50 mm 3 tumor. After intratumoral injection, mice in "PBS + Light" and "CP-NPs + Light" groups were irradiated with a white light of 200 mW/cm 2 for 20 min, while other groups are not administered. During the treatment period, the weight and tumor size of mice in each group were measured once every 3 days for 18 days. Then the volume of tumors was calculated using the formula: V = (a × b 2 )/2 where a is the length diameter and b is the width of the tumor.
On day 19, mice were sacrificed, and the tumor and major organs (heart, liver, spleen, lungs, and kidneys) were harvested. The collected tissues were immersed in 4% buffered paraformaldehyde, which was embedded into paraffin for further hematoxylin and eosin (H&E) staining. The histopathological changes were evaluated using an optical microscope.                     Figure 5A and S20, respectively.