Enhanced photodynamic therapy for skin cancer: using

pegylated carbon nitride nanosheets combined with

TMPyP4.

Qian Huang1,2,3,4 MD, Liying Hao1 MD, Ronghui Zhou1 PhD, Yanjing Li1 MD, Qirong Li1 MD,

Bofeng Zhu2,3,5, Xiaoxiao Cai1,* PhD

1State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases,

West China Hospital of Stomatology, Sichuan University, Chengdu, 610041, China

2Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, College of

Stomatology, Xi’an Jiaotong University, Xi’an, 710004, China

3Clinical Research Center of Shaanxi Province for Dental and Maxillofacial Diseases, College of

Stomatology, Xi’an Jiaotong University, Xi’an, 710004, China

4Department of Implant Dentistry, Stomatologic Hospital, Xi'an Jiaotong University, Xi'an,

710004, China.

5Department of Forensic Genetics, School of Forensic Medicine, Southern Medical University,

Guangzhou 510515, P. R. China

*Correspondence: X.X. Cai. Tel: +86 28 85503579; Fax: +86 28 85503579

E-mail: xcai@scu.edu.cn

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Supplementary Materials

Materials

Carboxyl-capped four-arm-poly ethylene glycol (4arm-PEG-COOH, M=10000 Dalton) and

N-hydroxysuccinimide-capped four-arm-poly ethylene glycol (4arm-PEG-NHS, M=10000

Dalton ） was obtained from Shanghai Yare Biotech, Inc. PBS (pH=7.40), Dulbecco’s

Modified Eagle’s Medium-high glucose （ H-DMEM ） and Penicillin/Streptomycin

Solution(100X) was purchased from HyClone. Fetal bovine serum (FBS), 0.25% Trypsin-

EDTA was bought from Gibico. Cell Counting Kit-8 (CCK-8) was bought from Dojindo

Laboratories. Ultrafiltration centrifugal tube was bought from Millipore, America. Phalloidin-

FITC was bought from MedChem Express. DCFH-DA Reactive Oxygen Species Detection

Kit was bought from Beyotime Biotechnology. Antifade Mounting Medium was bought from

Solarbio. BALB/c Nude Mice was obtained from Dashuo Biotechnology, China. 5, 10, 15,

20-tetra-(N-methy1-4-pyridyl) porphyrin (TMPyP4) was obtained from APExBIO

Technology. Monoclonal Antibody to Caspase-3, Bax and GAPDH was bought from Abcam

Inc, Alexa Fluor® 594-AffiniPure Goat Anti-Rabbit IgG(H+L) was bought from Jackson

ImmunoResearch Laboratories, Inc, and HRP-conjugated Goat Anti-Rabbit IgG was bought

from Signalway Antibody.

Synthesis of AGCN-PEG

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Firstly, 1mg AGCN-NS was dissolved in 2 mL PBS solution (pH=7), then add 100mg N-

hydroxysuccinimide-capped four-arm-poly ethylene glycol (4arm-PEG-NHS), adjust the final

pH to 7, stir overnight at room temperature. The obtained poly ethylene glycol modified

amino-rich g-C3N4 nanosheets (AGCN-PEG) was purified using ultrafiltration centrifuge tube

(100KD, 3000r/s, 3min), which was applied to remove excess four-arm polyethylene glycol.

Characterization of the Materials

The morphology and size of the material were characterized by atomic force microscopy

(AFM, SPM-9600, Shimadzu Corporation, Japan). Samples were diluted and dried on mica

sheets at room temperature, then the mica sheets bearing the samples were measured under

the AFM.

Fourier transform infrared spectroscopy (FTIR) was carried out on fourier transform infrared

spectrometer (Thermo Nicolet IS10). The main procedures are as follows: taking appropriate

samples and drying KBr, pouring them into agate grinding bowl, fully grinding and evenly

mixing the powder, finally pouring the powder into the mould and pressing them into

transparent sheets. After the instrument is blank calibrated, the pressed sheets are tested. The

scanning range is 500 cm−1 to 4000 cm−1 .

Electrical characteristics of AGCN-NS and CGCN-NS were determined using nanoparticle

size potential analyzer (Malvern Zetasizer Nano ZS90, Malvern Instruments Co., Ltd.), the

concentration of the samples tested were 0.5 mg/mL.

Cell culture

Epidermoid carcinoma cell line (A431) was obtained from cell bank of Shanghai Institutes for

Biological Sciences, Chinese Academy of Sciences and human dermal fibroblasts (HDFs)

was obtained form ScienceCell Research Laboratories. These two cells were cultured in high

glucose DMEM supplemented with 10% FBS and 1% penicillin-streptomycin, at 37 °C with

5% CO2/95% air. Experiment were performed at 75% confluence of the cell layer.

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In vitro confocal fluorescence microscopy

A431 cells were inoculated in a confocal dish with glass bottom, the cell density was 2.5 x

104/mL and the medium volume was 2 mL. After 24 hours, the old medium was replaced with

new culture medium containing 100 μg/ml AGCN-NS and CGCN-NS. After 24 hours of

incubation, the cells were washed with PBS three times and fixed with 4% paraformaldehyde

for 30 minutes, then after washing with PBS three times, the rhodamine labelled phalloidin

(Phalloidin-FITC, MCE company) was added and kept on the 37 shaking bed for 30℃

minutes, at last, the samples were washed with PBS three times after dyeing, adding antifade

mounting medium (Solarbio). Fluorescence images were then captured using a laser-scanning

confocal microscope (TCS SP8; Leica, Wetzlar, Germany).

Photodynamic killing effects of AGCN-NS and reAGCN-PEG on A431 cells

A431 cells were inoculated into 24-well plate at a cell density of 5 ×103 cells/cm2 and medium

volume of 1 mL. After 2 days of incubation, one group was incubated with 50 μg AGCN-NS

and the other group was treated with 50 μg of AGCN-PEG. After incubating for 24 hours, the

medium was replaced and irradiated with blue light with a wavelength of 405 nm and average

intensity of 20 mW/cm2 for 5 minutes. After incubating for 24 hours, the medium was

discarded again. The adherent cells were gently washed with PBS for three times, then serum-

free medium containing 10% CCK-8 reagent was added to each sample. After incubating at

37 for 2 hours, 100 μL of the supernatant was absorbed from each pore of the 24-well℃

plate and added into the corresponding pore of a new 96-well plates. Varioskan Flash 3001

was used to measure the optical density of samples in the new plate at 450 nm, four samples

were set up in each group to get the average value.

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Measurement of ROS in A413 cells after PDT by AGCN-NS and AGCN-PEG.

A431 cells were inoculated into 24-well plate at a cell density of 5 ×103 cells/cm2 and medium

volume of 1 mL. After 2 days of incubation, one group were incubated with 50 μg AGCN-NS

and the other group were treated with 50 μg of AGCN-PEG. After incubating for 24 hours, the

medium was replaced and irradiated with blue light with a wavelength of 405 nm and average

intensity of 20 mW/cm2 for 5 minutes. Then the culture medium was discarded again. The

adherent cells were gently washed with PBS for 3 times and then added DCFH-DA (DCFH-

DA Reactive Oxygen Detection Kit: Beyotime Biotechnology) diluted by serum-free medium,

the samples were incubated at 37 for 20 minutes. Fluorescence microscope was applied to℃

get fluorescence image of cells at an excitation wavelength of 488 nm and emission

wavelength of 525 nm.

Test of AGCN-PEG’s affinity to A431 Cells

A431 cells (5 ×103 cells/cm2) and HDFs cells (5 ×103 cells/cm2) were seeded into the 24-well

plate (the volume of culture medium was 1 mL). After 48 hours of incubation, 50 μg AGCN-

PEG were added. After incubation for 4 hours, the culture medium was discarded and the

adherent cells were gently washed with PBS for 3 times, fixed with 4% polyformaldehyde for

30 minutes, then washed with PBS for 3 times. Fluorescence microscopy was used to observe

and take pictures.

Measurement of ROS in A413 cells after PDT by AGCN-PEG, TMPyP4 and

AGCN-PEG-TMPyP4 in normal and hypoxic conditions.

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A431 cells were inoculated into 24-well plate at a cell density of 5 ×10 3 cells/cm2 with a

medium volume of 1 mL. the cells were cultured in normal conditions (37 °C, 5% CO2, 20%

O2) and hypoxic conditions (37 °C, 5% CO2, 1% O2). After 2 days of incubation, the samples

in each culture conditions were divided into 3 groups: group 1 was incubated with 50 μg

AGCN-PEG, group 2 was treated with 10 μg of TMPyP4 and group 3 was incubated with 50

μg AGCN-PEG and 10 μg of TMPyP4. then the samples were irradiated with blue light with

a wavelength of 405 nm and average intensity of 20 mW/cm2 for 5 minutes. Then the culture

medium was discarded again. The adherent cells were gently washed with PBS for 3 times

and then added DCFH-DA (DCFH-DA Reactive Oxygen Detection Kit: Beyotime

Biotechnology) diluted by serum-free medium, the samples were incubated at 37 for 20℃

minutes. Fluorescence microscope was applied to get fluorescence image of cells at a

excitation wavelength of 488 nm and emission wavelength of 525 nm.

Photodynamic treatment in vitro

A431 cells were inoculated into 6-well plate (for western blot analysis) or confocal dish (for

immunofluorescence staining.) at a cell density of 2.5 ×104 cells/cm2 with a medium volume

of 2 mL. After 24 hours of incubation in hypoxic incubator (C02: 5%, 02: 1%, T=37 ), the℃

samples were divided into 4 groups: untreated group: discarded culture medium and add 2 mL

fresh culture medium; AGCN-PEG group: discarded the medium, add 2 mL fresh medium

containing 50 μg/mL AGCN-PEG; TMPyP4 group: discarded the medium and add 2 mL fresh

medium containing 10 ug/mL TMPyP4; AGCN-PEG and TMPyP4 combined treatment

group: discarded the medium, add 2 mL fresh medium containing 50 μg/mL AGCN-PEG and

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10 μg/mL TMPyP4.

Protein extraction and western blotting in hypoxic condition.

Four groups were irradiated with blue light (wavelength 405 mm) with an average intensity of

20mW/cm2 for 2 minutes, then cultured under the original conditions for 24 hours, and

washed with PBS gently for 3 times. Total proteins were extracted using Tissue or Cell Total

Protein Extraction Kit (KeyGEN) according to the manufacturer’s instructions. Then samples

were then analyzed using SDS-PAGE electrophoresis and then probed with with primary

antibodies (anti-BAX ， 1:500, rabbit mAb, ab53154, anti-caspase-3 (1:1000, rabbit mAb,

ab184787), anti-GAPDH (1:3000, rabbit mAb, ab181602) all from abcam UK). Then HRP-

conjugated Goat Anti-Rabbit IgG (Signalway Antibody, America) was applied to probe the

primary antibody, after the stripes were exposed and developed, ImageJ was used to make

semi-quantitative analysis of gray value.

Immunofluorescence staining.

Four groups were irradiated with blue light (wavelength 405 mm) with an average intensity of

20 mW/cm2 for 2 minutes, then cultured under the original conditions for 24 hours. Cells in

confocal dishes were washed thrice with PBS and fixed in paraformaldehyde (4%) for 10 min

at 4 . After washed thrice with PBS, cells were permeabilized in 0.5% Triton X/PBS for 20℃

min at room temperature and washed, then blocked in 5% goat serum for 1 h and incubated

with primary antibodies ( anti-BAX, 1: 250, rabbit mAb, ab53154, anti-caspase-3, 1: 200

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rabbit mAb ab13847, all from abcam UK) overnight at 4 . Cells were then washed with℃

PBS for three times after rewarming for 0.5 h and incubated with the secondary antibody

(Alexa Fluor® 594-AffiniPure Goat Anti-Rabbit IgG(H+L), Jackson ImmunoResearch

Laboratories) for 1 h at room temperature. the samples were washed with PBS three times,

then adding rhodamine labelled phalloidin (Phalloidin-FITC, MCE company). After washing,

adding antifade mounting medium (Solarbio). Fluorescence images were then captured using

a laser-scanning confocal microscope (TCS SP8; Leica, Wetzlar, Germany).

Photodynamic treatment in vivo

Balb/c nude mice (4 weeks, about 20g, male, SPF grade) were purchased from Dashuo

Biotechnology Co Ltd (Chengdu, China). The Animal Laboratory was provided by

Experimental Animal Center, West China Hospital, Sichuan University (Chengdu,China). The

protocols used were approved by the Ethics Committee of West China Hospital of

Stomatology (number: WCHSIRB-D-2017-159). A431 cells were cultured in H-DMEM+10%

FBS. When the cells were 60%-80% confluence, cells were digested, centrifuged and

resuspended in serum-free medium. Then the nude mouse were injected cell suspension (2.0 ×

107/ml, 0.1ml for each mice) under the skin in the left axillary. After a few weeks, nude mice

with single growth tumors of the same size were selected to conduct the experiment. The

experimental mice were divided into 4 groups: Control group: injected 50 μL PBS solution;

AGCN-PEG group: inject 50 μL PBS solution containing 100 μg AGCN-PEG; TMPyP4

group: inject 50 μL PBS solution containing 50 μg TMPyP4; Combination therapy group:

inject 50 μL PBS solution containing 100 μg AGCN-PEG and 50 μg TMPyP4.

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Intratumoral injection was conducted every three days. after 14 hours of injection, the

tumors were irradiated with blue laser (wavelength: 405 nm, intensity: 200 mw ) for 15

minutes. The nude mice were treated with photodynamic therapy under anesthesia.

The size of tumors was observed every three days, and the long diameter (a) and short

diameter (b) of tumors were measured and recorded. The formula for calculating volume (Vt)

was Vt= ab2/2, and the relative volume of tumors (RTV) was calculated using RTV=Vt/V0

(V0 was the initial volume of tumors at the beginning of the experimental). After treatment,

nude mouse were euthanized by cervical dislocation. The transplanted tumor tissues and main

organs (heart, brain, liver, kidney and lung), HE staining were carried out

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Supplementary Figure Legends

Supplementary Figure 1: Histologic appearance of main organs at the 13th day of PDT treatment in different groups: PBS, AGCN-PEG, TMPyP4, AGCN-PEG+TMPyP4. Micrographs of H&E staining of main organs (heart, liver, spleen, lung, kidney) of the nude mice in the experiment after 13 days of treatment with various materials, all the pictures are displayed in the same scale.

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