W. Bian et al.
Bioorganic Chemistry 113 (2021) 104954
of mitochondria and induce cell necrosis with hyperpyrexia. In a series of in
vitro and in vivo tests, Mito-BWQ showed high PCE, light-induced cyto-
toxicity, good biocompatibility, and admirable fluorescence imaging
capability and tumor growth suppression, which will be a good choice for
PTT. And to the best of our knowledge, this work is the first to report OSPAs
based on the thiazole orange maternal unit with mitochondria as the pri-
mary site of action for enhanced PTT of tumors.
t = ꢀ
τ
s In (θ)
(A4)
θ is a dimensionless parameter, known as the driving force temper-
ature, as calculated using equation (A5).
T ꢀ TSurr
θ =
(A5)
TMax ꢀ TSurr
Tmax and TSurr are the maximum steady state temperature and the
2. Materials and methods
environmental temperature, respectively.
2.1. Materials
2.5. Cell culture and confocal microscopy studies
In this study, all chemicals and reagents were purchased from com-
mercial in AR grade and were used without further purifications.
phosphate-buffered saline (PBS, pH = 7.4), Fetal bovine serum (FBS),
DMEM mediu, trypsinCell Counting Kit (CCK-8), 4,6-diamidino-2 phe-
nylindole (DAPI) were purchased from Gibco, USA. Calcein-AM and
propidium iodide (PI) were purchased from YEASEN (Shanghai, China).
All other reagents were purchased from Aladdin.
MCF-7 cells and U87 cells were incubated in DMEM medium sup-
plemented with 1% penicillin–streptomycin (v/v) and 10% (v/v) FBS at
37℃ under a humidified atmosphere with 5% CO2. The cells were
treated and incubated with Mito-BWQ at 37℃ under 5% CO2 during the
time mentioned in the text. For the confocal microscopic samples, the
cells were passed and plated on glass bottomed dishes. The cells were
washed three times with phosphate buffered saline and then imaged
after further incubation in colorless serum-free media for 30 min.
Fluorescence microscopy images of labeled cells were obtained with
spectral confocal microscopes (*/LSM 800 With Airscan). Tracking dyes
for co-localization experiments were bought from Invitrogen. Other in-
formation is available in the figure captions.
2.2. Synthesis of OSPA (photo-thermal thiazoleorange)
The putative mitochondria-targeted thiazoleorange (Mito-BWQ) and
a nontargeted thiazoleorange (BWQ) control were synthesized as out-
lined in Fig. 2. Briefly, the synthesis entailed alkylation reaction of 1, 4-
dibromobutane with triphenyl phosphine gives 1 and the reaction with
2-methylbenzothiazole gives 2. 3 was obtained by alkylation reaction of
4-chloro-dimethylquinoline with p-bromomethylbenzoic acid, and then
2 was added to methanol, and triethylamine (TEA) was added as catalyst
2.6. In vitro PTT.
The cell viability assays was performed by a standard CCK-8 assay.
MCF-7 cells and U87 cells (104 cells/well) were seeded on 96-well plate
and incubated for 24 h at 37 ◦C. The cells were treated with different
for reaction, so that the π-conjugated system was expanded, and the
target compound was obtained and gave 1H NMR, 13C NMR spectral and
mass spectrum analytical data consistent with their proposed structures
(Figure S2-S10).
concentrations of Mito-BWQ (0, 3, 6, 12, 25, 50 μg/mL) and incubated
for 4 h at 37 ◦C. After incubation, the cells were washed three times and
irradiated different times (0, 1, 2, 3, 4, and 5 min) with a 600 nm laser at
1.5 W/cm2 . On the one hand, MCF-7 cells (2.0 × 105 per dish) were
seeded on 35 mm confocal dishes and allowed to stabilize for 24 h. The
cells were then treated with different concentration Mito-BWQ (0, 3, 6,
12, 25, and 50 ug/ml) in 1 mL culture medium. After 4 h incubation, the
cells were irradiated with a 600 nm laser (1.5 W/cm2) for 5 min to
induce photothermal cytotoxicity. The mode of cell death was examined
by using Calcein AM/PI staining kit, and the cells were imaged by
OLYMPUS 20,112,167 inverted fluorescence microscope.
2.3. Calculation of the fluorescence quantum yield
The fluorescence quantum yields of Mito-BWQ and BWQ were
calculated the standard of fluorescein (Φ = 0.95) in the condition of 1%
NaOH ethanol, and ΦX were measured according to the following
equation[7]:
(
)(
)
GradX
GradST
ηx2
ΦX = ΦST
(A1)
η2ST
Where, the subscript ST is the standard and the subscript × is the test
sample. Φ represents the fluorescence quantum yield value. Grad is the
slope of the curve with the integrated fluorescence intensity as the
2.7. Animals and tumor Models.
Female Balb/c nude mice (18–20 g) were housed under aseptic
conditions in small animal isolators with free access to food and water.
All care and procedures of animals were approved by the University
Ethics Committee for the use of experimental animals. To establish
tumor xenograft models, MCF-7 cell suspensions (1 × 106 cells) were
subcutaneously injected
ordinate, the ultraviolet absorbance as the abscissa, and
refractive index of the solvent used.
η is the
2.4. Calculation of the photothermal conversion efficiency
The photothermal conversion efficiencies (
cording to a previously described method[36]:
η
) were measured ac-
into the flank of nude mice. A caliper was used to measure tumor
sizes, and tumor volume (mm3) was calculated as (tumor major axis) ×
(tumor minor axis)2 /2.
hs(TMax ꢀ TSurr) ꢀ QDis
η
=
(A2)
I(1 ꢀ 10ꢀ A730
)
2.8. In vivo FL
h is the heat transfer coefficient, s is the surface area of the container,
All the animal experiments were approved by the Guanzhong Med-
ical Laboratory Animal Center. Balb/c nude mice (6–8 weeks) with
subcutaneous-tumors in the lower right side of the back area were used
as the animal models. The imaging experiments were carried out when
the subcutaneous-tumors grew to 60 mm3 in diameter. The Mito-BWQ
(1 mg/kg) was intratumorally injected into mice with normal saline as
an aqueous solution (n = 5 in each group), and then FL images at
different time points were acquired on an in vivo FL imaging system (FX
PRO, Bruker).
and the value of hs is determined from the equation (A3). QDis represents
heat dissipated from the laser mediated by the solvent and container. I is
the laser power and A is the absorbance at 600 nm.
mC
hs =
(A3)
τ
s
m is the mass of the solution containing the photoactive material, C is
the specific heat capacity of the solution, and τS is the associated time
constant, which can be determined from equation (A4).
3