Suppressing ACQ of molecular photosensitizers by distorting the conjugated-plane for enhanced tumor photodynamic therapy

Non-AIE-type molecular photosensitizers (PSs) suffer from the aggregation-caused-quenching (ACQ) effect in an aqueous medium due to the strong hydrophobic and π–π interactions of their conjugated planes, which significantly hinders the enhancement of tumor photodynamic therapy (PDT). So far, some ionic PSs have been reported with good water-solubility, though the ACQ effect can still be induced in a biological environment rich in ions, leading to unsatisfactory in vivo delivery and fluorescence imaging performance. Hence, designing molecular PSs with outstanding anti-ACQ properties in water is highly desirable, but it remains a tough challenge for non-AIE-type fluorophores. Herein, we demonstrated a strategy for the design of porphyrin-type molecular PSs with remarkable solubility and anti-ACQ properties in an aqueous medium, which was assisted by quantum chemical simulations. It was found that cationic branched side chains can induce serious plane distortion in diphenyl porphyrin (DPP), which was not observed for tetraphenyl porphyrin (TPP) with the same side chains. Moreover, the hydrophilicity of the chain spacer is also crucial to the plane distortion for attaining the desired anti-ACQ properties. Compared to ACQ porphyrin, anti-ACQ porphyrin displayed type-I ROS generation in hypoxia and much higher tumor accumulation efficacy by blood circulation, leading to highly efficient in vivo PDT for hypoxic tumors. This study demonstrates the power of sidechain chemistry in tuning the configuration and aggregation behaviors of porphyrins in water, offering a new path to boost the performance of PSs to fulfill the increasing clinical demands on cancer theranostics.


Intracellular ROS detection
2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-DA) was used as indicators for overall ROS generation, which can be converted to DCF and emit green fluorescence at 525 nm when oxidized by ROS.HeLa cells were seeded in cell culture dish and incubated overnight in normoxia or hypoxia condition, followed by adding 3C solution (1 μg/mL) and incubating another 12 h under the same conditions.After rinse with PBS, the cells were incubated with 10 μM DCFH-DA for 20 min.The treated cells were exposed to irradiation for 10 min with a 635 nm laser (500 mW/cm 2 ).After irradiation, the fluorescence imaging was taken on CLSM.

Intracellular 1 O 2 detection
The intracellular 1 O 2 detection followed the same procedure as detailed in 1.7 for ROS by using singlet oxygen sensor green (SOSG) as the indicator instead of using DCFH-DA.The working concentration of SOSG is chosen at 5 μM.

Intracellular OH• detection
Hydroxyphenyl fluorescein (HPF) was used to detect the production of OH•.The testing process of OH• is the same as that detailed in 1.7 for detecting ROS.The working concentration of SOSG is chosen at 50 μM.
Then, the cell images were collected on a CLSM.

Calcein-AM/PI staining of HeLa cells
The HeLa cells were seeded on 20 mm confocal dishes and incubated overnight.Then the cells were incubated with different concentrations of 3C solutions (0,5 μg/mL) for 12 h.After that, the confocal dishes were irradiated with a LED light for 20 min or kept in dark.Then, the cells were stained with a PBS solution containing 2 μM Calcein-AM and 8 μM PI.Confocal images were taken to observe the survival and death of cells.

Cyclic voltammetry measurement
The cyclic voltammetry measurement of 3C (20 μM) was conducted in a three-electrode system.H 2 O was selected as a supporting electrolyte.Glassy carbon, Ag/AgCl, Pt wire were employed as work electrode, reference electrode and counter electrode respectively, with a scan rate of 100 mV•s -1 .

Computational detail
Quantum chemical calculations were used to determine the geometry and electronic structure of porphyrin molecules.The calculations were performed with ORCA 5.0.3 1 All calculated structures were verified as true minima by the absence of negative eigenvalues in the harmonic vibrational frequency analysis.Tighter than default convergence criteria (tightopt), grid values (grid5, finalgrid6) were chosen for both the optimization of the structural parameters and the scf (tightscf).The geometry optimizations were performed at the B3LYP-D3/def2-TSVP level of theory 2,3 .The RIJCOSX approximation and the related auxiliary basis set def2/J were used to speed up the calculations 4,5 .Molecular orbitals and optimized geometries were visualized with IBOView 6 .The water solvent effects are implicitly considered by the universal solvation model 7 .

Animal model
All experiments related to animals were implemented in accordance to the protocols approved by the local Ethical Committee in compliance with the China Animal Management Regulations.The female BALB/c mice were purchased from Jinan Pengyue Laboratory Animal Breeding Co. Ltd.Cell suspension was prepared by subculture and amplification of 4T1 cell line, and the suspension was inoculated subcutaneously with 5.0 × 10 7 cells mL −1 in BALB/c mice.

In vivo fluorescence imaging
4T1 Tumor-bearing BALB/c mice were intravenously injected with 3C or TPP 1 for in vivo imaging.The distribution of drugs in mice was recorded with an IVIS spectrum imaging system.Then, at 24 h postinjection, the mice were sacrificed, and their harvested organs (liver, lung, spleen, kidney, and heart) and tumors were imaged under the imaging system.

In vivo photodynamic therapy (PDT)
The mice were divided into three groups randomly for treatment.1) saline solution injection only (control group); 2) 3C aqueous solution injection only; 3) 3C aqueous solution plus laser irradiation (experimental group).
3C saline solutions (1 μmol/kg) were injected via tail vein of the mice at 0, 2, 4 day.After a 24-hour cycle, control group and experimental group were irradiated with a 635 nm laser for 10 min (500 mW/cm 2 ).The tumor size (tumor size = width × width × length / 2.) and body weights of the mice were measured and recorded for 2 weeks.
Then, tumors and main organs (liver, lung, spleen, kidney, and heart) were dissected from the mice for histological analysis by hematoxylin-eosin (H&E) staining.The synthesis methods of the alkylation agent and compound b in this paper refer to literatures. 8,9 eral procedure 1 for synthesis of compound b

General Synthesis Methods
The hydroxybenzaldehyde and alkylation agent was dissolved in acetone or DMF, respectively.Then, the two solutions were mixed and stirred at room temperature under a nitrogen atmosphere for 20 min, followed by adding potassium carbonate.The resulting mixture was refluxed and monitored by TLC until the reaction was finished.Then, the mixture was diluted with dichloromethane (DCM).The organic phase was dried over anhydrous Na 2 SO 4 and evaporated under reduced pressure.The crude product was then purified by column chromatography with silica gel.General procedure 2 for synthesis of compound c.
The compound c were synthesized according to the well-developed method.Dipyrromethane (0.16 mmol) and the corresponding substituted aldehydes (b 1 -b 8 , 0.16 mmol) were dissolved in DCM (20 mL) and stirred in dark at room temperature under a nitrogen atmosphere for 20 min.Trifluoroacetic acid (TFA, 0.256 mmol) was added subsequently.The mixture was stirred overnight and then DDQ (0.256 mmol) was added.After stirring for another hour, triethylamine (40 μL) was added to quench TFA.The mixture was evaporated under reduced pressure, and the residue was purified by column chromatography with silica gel using dichloromethane/ methanol (40:1) as the eluent.The solid obtained from above was dissolved in dioxane (4 mL) followed by the addition of concentrated HCl solution (2 mL) at 0 ℃.Then, the resulting mixture was shielded from light and stirred overnight at room temperature.The reaction mixture was added dropwise into cold acetone (20 mL) for precipitation.The obtained green precipitates were then centrifugated and redissolved into pH=3-4 deionized water.The final product was obtained as a green solid after freeze-drying.Finally, the green solid was dissolved in pH=2-3 deionized water and adjusted to a concentration of 2 mmol/L as stock solution.

References
Scheme S1.General synthetic route of targeted porphyrins.

3 .
The photophysical and photochemical properties of the porphyrins.

Figure S3 .
Figure S3.UV−vis absorption spectra of different porphyrins in different solvents.(red line: H 2 O; blue line: MeOH)

Figure S12 .
Figure S12.Hematoxylin and eosin (H&E)-stained histological section of heart, spleen, kidney, liver, lung, and tumor tissues obtained from mice of the control and PDT groups.Scale bar: 50 μm.