Singlet oxygen sensor green (sosg)

The production of singlet oxygen generated from the HIPPNPs upon light illumination was measured by using singlet oxygen sensor green (SOSG; Life Technologies, Carlsbad, CA) as the fluorescent probe. According to the manufacturer manual, 200-μL PBS containing HIPPNPs with 0 (no HIPPNP), 1.25, 2.5, 5, 10, and 25 μM ICG equivalent were separately mixed with 200-μL SOSG (10 μM), followed by incubation at room temperature in dark for 10 min. 200 μL of each sample was then transferred into one well of a 96-wells culture plate and irradiated by using a 808-nm continuous wave (CW) laser with intensity of 6 W/cm². The level of SOSG-induced fluorescence in each group was detected by using a spectrofluorometer performed with 488 and 525 nm of excitation and emission wavelength, respectively, every 60 sec for 5 min. The productions of singlet oxygen generated from the freely dissolved ICG in PBS with 2.5 and 25 μM were treated as the controls.

HPF (H36004) and SOSG (S36002) were purchased from Life Technologies and were used as directed by the manufacturer’s protocol. Working solutions were prepared at 10 µM in DPBS or 5 µM TRIS buffer (pH 7.5) for HPF and SOSG, respectively, using 10 µM concentrations of the appropriate photosensitizer. All sample irradiation was carried out using 660 nm-centered LED-generated light (M660L3, Thorlabs) at an irradiance of 100 mW/cm². Irradiation of 80 µL sample volumes was carried out in clear plastic 96-well plates, with the emission of the ROS probes measured with a plate reader (SpectraMax m5, Molecular Devices). Experiments were performed in triplicate and experimental values (N = 3) were averaged and background-subtraction was performed.

UV absorption spectra of ICG were measured with UV spectrophotometer (Shimadzu, UV-2450, Kyoto, Japan). The UV absorption of ICG (at 780 nm) and the fluorescence of SOSG (λ_ex/λ_em = 504/525 nm) were measured by a microplate reader. Laser irradiation was performed by two laser devices (660 nm, 1 w/cm² and 532 nm, 0.1 w/cm²). Ce6, ICG and Eosin Y were purchased from Sigma-Aldrich Co. LLC (Shanghai, China). SOSG was purchased from Life Technologies (Shanghai, China).

The production of singlet oxygen generated from the HIDPPNPs under 808-nm laser irradiation with an intensity of 6 W/cm² was measured using singlet oxygen sensor green (SOSG; Life Technologies, Carlsbad, CA) as the fluorescent probe according to the manufacturer’s instructions. The level of SOSG-induced fluorescence in each group was detected using a spectrofluorometer with 488 and 525 nm of excitation and emission wavelengths, respectively, every 60 sec for 5 min and was quantitatively represented by RFUs.

The fluorescence intensity of SOSG (Thermo Fisher Scientific, Waltham, MA, USA) oxidized by ¹O₂ was determined from the fluorescence of the HPLC peaks. The photocatalyst (2 µM) and SOSG (10 µM) were added to 50% CH₃CN solution in 10 mM MES buffer (pH 7.4) in a 1.5 mL tube. The solution was irradiated with blue light (RELYON, Twin LED light, 455 nm) for 30 s on ice. After irradiation, the solution was diluted 2.6-fold with 0.1% aqueous formic acid and analyzed using HPLC. Analytical HPLC was carried out on a JASCO PU-4580 HPLC Pump, JASCO LG-4580 Quaternary Gradient Unit (Tokyo, Japan), and JASCO DG-4580 Degassing Unit with a JASCO MD-2018 Plus Photodiode Array Detector, JASCO CO-4060 Column Oven, JASCO As-455 HPLC Autosampler, and JASCO LC-NetII/ADC Interface Box using a C18 reverse phase column (Inertsil ODS-4, 150 × 4.6 mm, 5 μm (GL Science, Inc., Tokyo, Japan)). The HPLC conditions were as follows: mobile phase A, 0.1% formic acid in H₂O, mobile phase B, 0.1% formic acid in CH₃CN. 0−5 min, 5% B; 5−27 min, 5−100% B; 27−32 min, 100% B. The fluorescence of the separated peaks was detected using HPLC (Ex 504 nm/Em 525 nm).

PTX (API, purity > 99%) and IR780 iodide were purchased from Meilun Biotechnology Co., Ltd. (Dalian, China) and Merck KGaA (Darmstadt, Germany), respectively. PTX Injection (solution) was purchased from Jiangsu Osaikang Pharmaceutical Co., Ltd. (Nanjing, China). HA (240 kDa) was obtained from Freda Pharmaceutical Group Co., Ltd. (Ji’nan, China). Dioleoyl phosphoethanolamine (DOPE) and phosphatidylcholine (Lipoid S100) were purchased from Lipoid GmbH (Ludwigshafen, Germany). HA-DOPE was previously synthesized in our laboratory; the structure verification results are shown in Additional file 1: Figure S7 [79 (link)]. Glycerol dioleate was procured from Macklin Inc. (Shanghai, China). Western Blot Kit was purchased from Elabscience Biotechnology Co., Ltd. (Wuhan, China). Cell Counting Kit-8 (CCK-8) and DCFH-DA were obtained from Beyotime Biotechnology (Shanghai, China). SOSG and fetal bovine serum were purchased from ThermoFisher Scientific (New York, NY, USA). Other chemicals (for cell culture and labeling) were purchased from Jiangsu KeyGEN BioTECH Co., Ltd. (Nanjing, China) and Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Antibodies are listed in Additional file 1: Table S3.