Chemiluminescent organic nanophotosensitizer for a penetration depth independent photodynamic therapy

Photodynamic therapy initiated by external photoexcitation is a clinically-approved therapeutic paradigm, but its practical application has been severely hindered by the shallow penetration of light. Here, we describe a penetration-independent PDT modality using a chemiluminescent organic nanophotosensitizer, which is activated by hydrogen peroxide instead of external photoexcitation.


Materials
All chemicals and reagents were purchased from Sigma-Aldrich or Fisher Scientific unless otherwise specified and were used without further purification. DSPE-mPEG2000 was obtained from Avanti (Alabaster, AL, USA). Live & Dead Viability Kit was purchased from KeyGEN BioTECH.

Instrument
Steady-state UV absorption spectrum data was obtained on a SHIMADZU UV-3600 PLUS UV-Vis-NIR spectrophotometer; Transmission electron microscopy (TEM) images were performed on a HT7700 transmission electron microscope operating at 100 kV. Dynamic light scattering (DLS) was performed on a particle size analyzer Electronic Supplementary Material (ESI) for RSC Advances. This journal is © The Royal Society of Chemistry 2020 (NanoBrook 90Plus, Brookhaven Instruments Corporation). Cell death images were observed with an inverted laser scanning confocal microscope (Zeiss LSM 710 Meta NLO).

Preparation of C NPs
C NPs were prepared by self-assembly of PLGA, Luminol and DSPE-mPEG2000 through a one-step nanoprecipitation method. Briefly, PLGA r was first dissolved in an acetonitrile solution (2 mg/ml). Then, the pH of 1 mg/mL Luminol aqueous solution was adjusted to 7 using NaOH solution, and 2 mg/mL HRP aqueous solution was added. Add the prepared PLGA acetonitrile solution to the Luminol/HRP aqueous solution to obtain a Luminol/ HRP/ PLGA solution. The solution of Luminol/HRP/PLGA was then added dropwise to the 2 mg/mL DSPE-mPEG2000 aqueous solution under gentle stirring. The mixed solution was sonicated for 3 minutes and then gently stirred at room temperature for 4 h. C NPs were obtained after high-speed centrifugal purification to remove residual organic solvents and free molecules.

Measurement of singlet oxygen generation
Luminol (2 mg/mL) and HRP (0.4 mg/mL) were added to an aqueous solution of a singlet oxygen indicator ADMA at 1 mg/mL, and the initial ultraviolet absorption was measured. Subsequently, H 2 O 2 was added to the mixed solution, and the ultraviolet absorption spectrum was measured every 2 minutes. The operation method of the C NPs experimental group is the same, that is, the added Luminol and HRP are replaced with C NPs.
In this way, we can compare the efficiency of the Luminol-H 2 O 2 -HRP chemiluminescence system and C NPs to generate singlet oxygen by the decline rate of the UV absorption peak at ADMA 261 nm.

Live & dead cell assay
Hela cells were seeded in a confocal dish and incubated in DMEM medium containing 10% fetal bovine serum and 1% streptomycin/ penicillin at 37 °C, 5% CO 2 for 24 h. Then, the medium was aspirated,

Antibacterial effect
The Escherichia coli were used in analysis of antibacterial effect based on the morphological characteristics of the colonies. Escherichia coli were obtained from ATCC. E. coli were cultured in LB broth at 37° C until OD 600 =0.6, and further add 0.1 mL of Luminol (2 mg / mL), 0.1 mL of HRP (0.4 mg / mL) or 50 μL of H 2 O 2 (3%) to the 2ml bacterial solution, respectively, furthermore continue to shake in the incubator for 4 h, and then diluted 10 -0 , 10 -1 , 10 -2 , 10 -3 , with sterile physiological saline. 6 microliters of each dilution were inoculated to solid Medium LB. The bacteria were grown at 37C for 12 h and their growth was monitored every 3h. At the same time, quantitative analysis of the bacteria after treatment by counting method. Take the average bacterial growth number (N Control ) of the control group as 100%, that is, the bacteriostatic rate (A) is 0%, and the average colony number (N 1 ) in the fourth column of the experimental group is obtained by counting. By formula: The antibacterial effect of the experimental group was calculated. The antibacterial effect repeated four times.