4. Experimental Section
4.1 Materials
Folic Acid (FA), PEG-bis(amine) (Mn: 3.4 kDa), β-benzyl-L-aspartate (BLA), Triethylamine (TEA), MTT, PBS, and Sodium Bicarbonate were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Triphosgene was purchased from Aldrich Chemical Co. (Milwaukee, WI, USA). N-hydroxysuccinimide (NHS) and N, N’-dicyclohexylcarbodiimide were purchased from Fluka (Buchs, Switzerland). 3TT-IC-4Cl was provided by SunaTech Inc. (Suzhou, China). Indocyanine Green (ICG) was purchased from Adamas (Shanghai, China). CHCl3 was purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). DMSO was purchased from Fuchen Chemical Reagent Co.,Ltd (Tianjin, China). Chloroform-d was purchased from Tenglong Weibo Technology Co., Ltd (Qingdao, China). DMSO-d6 was purchased from Ningbo Cuiying Chemical Technology Co., Ltd (Ningbo, China). Dulbecco’s modified Eagle’s medium (DMEM), Fetal Bovine Serum (FBS), Penicillin, and Streptomycin were purchased from GibcoBRL (Invitrogen Corp., CA, USA). All other chemicals were of analytical grade and used as received without further purification.
4.2 Characterization
The chemical structure was determined by 400 MHz 1H NMR (AVANCE III HD 400MHz, Bruker, Fällanden, Switzerland) using CHCl3-d and DMSO-d6 as the solvent. The photophysical properties of samples in aqueous solution were confirmed by UV-visible spectrophotometry (UV-2550, Shimadzu, Tokyo, Japan) and fluorescence spectrophotometer (F-4600, Hitachi, Tokyo, Japan). The morphologies, sizes, and size distributions of nanoparticles were determined by transmission electron microscopes (TEM) (TECNAI G2 Spirit TWIN, FEI, Hillsboro, FL, USA) and dynamic light scattering (DLS) (Zetasizer Nano ZS90, Malvern Instruments Co, Malvern, UK) at 25ºC using a He-Ne laser (633 nm) as a light source. The temperature was monitored by IR thermal camera (TiS65, Fluke, Everett, WA, USA). The NIR laser (880 nm) used in this study was purchased from Beijing Laserwave Optoelectronics Technology Co., Ltd. (LWIRL880-20W-F, Laserwave, Beijing, China).
4.3 Preparation of TNPs
In order to prepare TNPs, first, the amphiphilic block copolymer FA-PEG-PBLA10 used for 3TT-IC-4Cl encapsulation was synthesized by ring-opening polymerization as our previous reported41. The chemical structure of FA-PEG-PBLA10 was confirmed by 1H NMR (400 MHz, DMSO). Then, the TNPs were prepared by nanoprecipitation method. Briefly, 5 mg 3TT-IC-4Cl was dissolved in 1 mL THF, then, the 3TT-IC-4Cl solution was added into to 50 mL FA-PEG-PBLA10 solution (0.5 mg/mL in DMSO) dropwise, and then the mixture was transfer to dialysis tubs (Cut-off 3.5 K Mw) to remove THF and DMSO, followed by freeze drying, the TNPs were obtained.
3.4 Photothermal Effect
To confirm the PTT application potential, the photohermal property of TNPs was investigated, a series concentrations of TNPs (0, 30, 90, 180, and 250 μg/mL) in water were irradiated by 880 nm laser (0.7 W/cm2) for 720 s, the temperature of TNPs solution was recorded by IR thermal camera every 30 s. In addition, the constant concentration (180 μg/mL) of TNPs were irradiated by 880 nm laser for 720 s with various power densities (0.3, 0.5, 0.8, and 1.5 W/cm2) was investigated by same method.
3.5 Stability of TNPs
In order to investigated the stability of TNPs, TNPs (180 μg/mL, 30 μg/mL free 3TT-IC-4Cl equiv.), and free ICG (30 μg/mL) were irradiated with 880 nm laser (0.7 W/cm2) for 5 min, then the laser was turned off and the sample was cooled to the room temperature naturally, the temperature of samples were recorded using the IR thermal camera every 30 s. Subsequently, the procedures were repeated four times.
3.6 In vitro Phototoxicity and Biocompatibility of TNPs
HeLa cells (1×104 cells/well) were seeded onto 96-well plates in 200 μl DMEM and allowed to attach for 24 h. After cell attachment, the medium was replaced with 100 μl of fresh medium containing FA-PEG-PBLA10 and TNPs with a series of concentration (0, 30, 60, 90, 120, 180, and 250 μg/mL), and then incubated for 4 h. The cells were washed with PBS and replace with fresh DMEM. The samples were irradiated with a laser (880 nm, 0.7 mW/cm2) for 5 min. Then, irradiated cells were incubated at 37ºC for 24 h and cell viability was evaluated by MTT assay. Data presented are averaged results of quadruplicate experiments. For biocompatibility, HeLa cells (1×104 cells/well) were seeded onto 96-well plates in 200 μl DMEM and allowed to attach for 24 h. After cell attachment, the medium was replaced with 100 μl of fresh medium containing FA-PEG-PBLA10 and TNPs with a series of concentration (0, 30, 60, 90, 120, 180, and 250 μg/mL), and then incubated for 24 h. The cell viability was evaluated by MTT assay. Data presented are averaged results of quadruplicate experiments.