A Membrane‐Targeting Photosensitizer with Aggregation‐Induced Emission Characteristics for Highly Efficient Photodynamic Combat of Human Coronaviruses

Abstract COVID‐19 pandemic, caused by severe acute respiratory syndrome coronavirus 2, has resulted in global social and economic disruption, putting the world economy to the largest global recession since the Great Depression. To control the spread of COVID‐19, cutting off the transmission route is a critical step. In this work, the efficient inactivation of human coronavirus with photodynamic therapy (PDT) by employing photosensitizers with aggregation‐induced emission characteristics (DTTPB) is reported. DTTPB is designed to bear a hydrophilic head and two hydrophobic tails, mimicking the structure of phospholipids on biological membranes. DTTPB demonstrates a broad absorption band covering the whole visible light range and high molar absorptivity, as well as excellent reactive oxygen species sensitizing ability, making it an excellent candidate for PDT. Besides, DTTPB can target membrane structure, and bind to the envelope of human coronaviruses. Upon light irradiation, DTTPB demonstrates highly effective antiviral behavior: human coronavirus treated with DTTPB and white‐light irradiation can be efficiently inactivated with complete loss of infectivity, as revealed by the significant decrease of virus RNA and proteins in host cells. Thus, DTTPB sensitized PDT can efficiently prevent the infection and the spread of human coronavirus, which provides a new avenue for photodynamic combating of COVID‐19.


Materials and general instruments
All chemical reagents were obtained from J&K Scientific, and used without further purification. CCK-8 kit was purchased from MedChemExpress. Minimum essential medium (MEM) was purchased from Gibco. Phosphate buffered saline (PBS), penicillin and streptomycin were purchased from Invitrogen. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), Trypsin-EDTA and CellMask™ Green were purchased from Thermo Fisher Scientific. The antibody against Coronavirus Antibody OC-43 strain was purchased from Millipore (541-8F).
The FMDV 3D protein antibody was homemade. HRP Goat Anti-Mouse IgG (H+L) (ABclonal) was used as the secondary antibody. Antibody against GAPDH was obtained from Proteintech (Wuhan, China). Milli-Q water was supplied by Milli-Q Plus System (Millipore Corporation, United States). All the solvents for optical spectroscopic studies were HPLC or spectroscopic grade.
Thin-layer chromatography analyses were performed on silica gel GF 254. Column chromatography purification was carried out on silica gel (200-300 mesh). NMR spectra were recorded using a Bruker AMX-400. Chemical shifts were given in ppm relative to the internal reference TMS, CD3OD or DMSO-d6 as the internal standard.
The following abbreviations were used in 1 H NMR: s = singlet; d = doublet; t = triplet; q = quartet; m = multiplet. High resolution mass spectra were recorded on a Bruker Daltonics Bio TOF mass spectrometer. Fluorescence spectra were obtained using a Horiba Duetta spectrofluorometer with a 10 mm quartz cuvette. UV-Vis absorption spectra were recorded on a Hitachi PharmaSpec UV-1900 UV-Visible spectrophotometer. Fluorescence images were collected on Olympus IX71 inverted fluorescence microscope or Zeiss LSM 880 confocal laser scanning microscope.

Cell and virus culture
HeLa cells, NIH-3T3 cells and HEK-293T cells were cultured in DMEM containing 10% fetal bovine serum (FBS) (Thermo Fisher Scientific) and 1% antibiotic (penicillin and streptomycin) in a 5% CO2 humidified incubator at 37 o C.

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While HepG2 cells were grown in RPMI 1640 medium (Thermo Fisher Scientific) with 10% fetal bovine serum and 1% antibiotics (penicillin and streptomycin) at 37 °C in a 5% CO2/95% air incubator. Once the cells reached 80%-90% confluence, they were dissociated into single cells with 0.05% Trypsin-EDTA at 37 o C for 5 min and passaged at a ratio of 1:6-1:19 in a new cell culture dish.
Human embryonic lung fibroblast cells line (MRC-5) and baby hamster kidney cell line (BHK-21) were provided by the China Center for Type Culture Collection. MRC-5 and BHK-21 cells were cultured in MEM medium supplemented with 10% fetal bovine serum, 100 units mL -1 penicillin and 100 μg mL -1 streptomycin in a 5% CO2 humidity incubator at 37℃. One day before the infection experiment, the cells were seeded into a 24-well cell culture plate to approximately 70-80% confluence and then infected with virus. The live viruses were incubated with 0.01, 0.05, 0.3, 0.8, 1.0, 5.0, 7.5, 10 and 15 μM of DTTPB. In a typical experiment, 2 μL of a 10 mM stock solution of DTTPB in DMSO were diluted to 1 mL with cell culture medium, followed by further dilution to desired concentration. For the ROS sensitizing, after mixing with the DTTPB, each well of virus was exposed to white light (9 mW/cm 2 ) for 20 min, another two plates with cells were kept in dark as control.

Fluorescence imaging
Cells were stained with CellMask™ Green (Thermo Fisher Scientific, 10 min) and 5 M DTTPB (1h) at 37°C. After the incubation, the cells were washed with DPBS for three times. Confocal imaging was performed using the Zeiss LSM 880 confocal laser scanning microscope equipped with a Plan-Apochromat 63×/1.4 NA oil objective lens, a photo-multiplier tube and a Gallium arsenide phosphide detector driven by the ZEN software (Carl Zeiss). The 561 nm laser and 620-720 nm emission filter were used for DTTPB. The 488 nm laser and 510-550 nm emission filters were used for CellMask™ Green. Digital images were captured and processed by ZEN software (ZEN 2.5 lite) in grayscale and pseudo color.

Viral infectivity assay
To characterize the effects of DTTPB on virus, cells were infected with FMDV,

RNA extraction and real-time quantitative polymerase chain reaction (qPCR)
After viral infection, cells were collected and RNAs were extracted with TRIzol reagent (Thermo Fisher Scientific) according to the manufacturer protocol. The integrity and purity of RNAs were tested using 1.5% agarose gels, NANO DROP2000 (Thermo), and Agilent 2100. cDNA synthesis was performed according to standard protocols. Briefly, 1 μg RNA was reverse-transcribed using an Oligo(dT)15 Primer Primers of qPCR were designed with Primer Premier 6.0 software (Primer, Canada).
The qPCR primers used are listed in Table S1. Each qPCR reaction mixture Each sample was subjected to three independent replicates. All assays were performed with three independent biological replicates. Data analyses were performed using Rotor-Gene 4.6.

Immunofluorescence
Cells grown in 24-well plates were washed twice with PBS and fixed with 4%

Western blot analysis
Cells underwent different treatments, were washed twice with PBS and proteins were extracted with 2 × SDS buffer and then boiled for 5 min. After centrifuging for 10 min at 10,000 × g, the supernatant was subjected to a 10% SDS-PAGE gel for electrophoresis followed by electrotransfered onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad) for Western blotting. Prior to the detection of target proteins with specific protein antibody, the transferred membranes were blocked with 5% skim milk (BD Biosciences) for 1 h at room temperature to reduce non-specific binding.
Excess milk was washed away three times, and immunoblotting was performed with FMDV 3D or GAPDH protein antibody (1:2000) followed by HRP-conjugated AffiniPure secondary antibody (1:10,000) to develop the blotting results. After washing three times with TBST (containing 0.15 % Tween-20), membrane-bound antibodies were detected using Immobilon Western Chemiluminescent HRP Substrate S9 (Millipore). Results were presented using the GelDoc XR System (Bio-Rad), and all experiments were repeated independently three times.

TCID 50 assays
MRC-5 cells were incubated in 96-well plates at a concentration of 5000 cells/well, and the cells could be used for TCID50 when the confluence reached over 70%. And, the virus stock solution was diluted with a 10-fold gradient. The dilution factor for FMDV, HCoV-OC43, and HCoV-229E, the dilution factor was from 10 1 to 10 4 , from 10 1 to 10 4 , and from 10 1 to 10 3 , respectively. Afterwards, the supernatants in the 96-

PDI of viruses on filter paper
Sterilized filter papers were cut into small pieces with sizes of 1 cm × 1 cm, and then randomly divided into control groups and experimental groups with three independent replicates. 10 μL of ethanol containing different concentrations of DTTPB was added S10 to filter paper with completely immerse. After air-drying for more than 12 h, the filter papers were placed in 24-well plate and incubated with FMDV, HCoV-OC43 or HCoV-229E for 10 min, and then treated with or without white-light irradiation (9 mW/cm 2 ) for 20 min. The BHK-21 or MRC-5 cells were infected with FMDV, HCoV-OC43 or HCoV-229E separately for 24 h. The replications were then determined by qPCR. In brief, an equal number of cells were plated in 24-well plates.
When cells reached 70-80% confluence, they were infected with FMDV, HCoV-OC43 or HCoV-229E. After 24 hours, cells were collected and RNA was extracted with TRIzol reagent (Takara) according to the manufacturer protocol, followed by qPCR analysis of RNA copies. S11

NMR and Mass Spectra
Figure S1 1 H NMR of PY-BC in CD3OD.