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.