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Journal of Materials Chemistry B
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nanoparticles coated with oleic acid/ligand and drug-loading respiratory detector to avoid unexpected death.VTiehweArdticeletaOinlleinde
DOI: 10.1039/D0TB00017E
nanoparticles. Dynamic light scattering (DLS) was used to therapeutic strategy is illustrated in Scheme 3.
measure the size distribution and Zeta-potential of the empty
and drug-loading nanoparticles. A UV-vis spectrometer was
employed to determine the drug loading rate.
2.8 Antitumor efficacy in vitro
Mia Paca-2 cells were seeded in a 24-well plate with a 5 × 104
cells/well density and cultured overnight in an adherent state.
GEM or pGEM (500 μL, 40 μg/mL) was added to the wells and
cultured for 4 h. PBS was used to rinse the cells three times and
DMEM (500 μL, containing 10% FBS) was added to each well.
For the irradiated group, a 730-nm laser (180 mW/cm2) was
used to treat the cells for 10 min. All groups were cultured at 37
°C for 24 h and rinsed three times with PBS. Following the steps
specified by the Annexin V-FITC kit, binding buffer (250 μL),
Annexin V-FITC (2.5 μL), and propidium iodide (2.5 μL) was Scheme 3. The treatment was performed 10 days after tumour implantation, with a PTT
24 h after the drug-managed intravenous injection. If necessary, these treatments were
repeated every 4 days. Luciferin bioluminescence imaging was performed every week to
monitor tumour development.
added subsequently. After 15 min, the medium was rinsed and
the cells were observed via confocal microscopy, where the
λex/em wavelengths for Annexin V-FITC and propidium iodide
are 488/500–550 and 543/600–680 nm, respectively.
2.9 Animal preparation
Herein, the survival duration was not monitored because the
PADC tumours of saline group grew so rapidly that after 3 weeks
post implantation, they reached diameters of ˃2 cm.
Considering the guideline request of Affidavit Animal Ethical
The protocol used to establish an orthotopic nude mouse model
of pancreatic cancer was reported previously18. All animals
received human care in accordance with the National Institute
Welfare, all mice were executed after 2 weeks and the anti-
of Health (NIH) Policy on the Care and Use of Laboratory
tumour effect was evaluated by comparing the excised tumour
Animals and the protocol was approved by the Animal Use and
tissues. The tumour volume was calculated using the formula of
Care Committee of Ruijin Hospital, School of Medicine,
long diameter multiplied by the square of the short diameter
Shanghai Jiaotong University.
divided by 2.
2.10 In vivo distribution by IVIS
2.12 Apoptosis tumour detection
The in vivo distribution experiments were performed using mice
with in situ pancreatic cancer 14 days post implantation. NGP
(200 μL NGP, 5.0 mg/mL) was administrated via intravenous
injection. After 6, 24, and 48 h, the IVIS system for small animals
Fluorescein labelled dUTP can link to the 3’-OH moiety of
cleaved DNA in apoptosis tumour tissue under the action of the
TdT enzyme and can be observed using fluorescence
microscopy. Almost no 3’-OH DNA is observed in normal DNA
was applied to track the in vivo signal from NaLuF4:Nd@NaLuF4
and no fluorescein labelled dUTP can be formed. This strategy
nanoparticles, with an excitation wavelength of 808 nm and
can be applied for detecting apoptosis in tumour cells.
power of 200 mW/cm2 with a 920 nm filer lens. D-luciferin in
The sliced samples were carefully marked prior to evaluation.
PBS 7.4 (containing 4% paraformaldehyde) was used as a tissue
PBS (200 μL, 15 mg/mL) was injected into the mice to measure
the in situ tumour volume and location in full-band acquisition
fixation solution, PBS 7.4 (containing 1% Triton X-100) was used
mode after 10–15 min.
as a cellular membrane permeation fluid, and 3% H2O2 in PBS
was used as a sealing fluid. The TdT enzyme solution was freshly
2.11 Anti-tumour efficacy in vivo
prepared by adding FITC-12-dUTP (1.0 µL) and TdT enzyme (4.0
µL) into an equilibration buffer (45 μL) in the dark.
Tumour-bearing mice (24×) were divided into 6 groups:
NGP+PTT (drug-delivery chemotherapy combined with
photothermal therapy); NGP (drug-delivery chemotherapy);
NP+PTT (photothermal therapy); NP (empty nanocarriers); GEM
(commercial GEM); and NS (saline as a negative group).
Tumour-bearing mice were treated with GEM (20 mg/kg) or
pGEM (26 mg/kg) via intravenous injection 10/14/18/22 days
post tumour implantation, and the NGP+PTT/NP+PTT groups
received photothermal treatment (λex=730 nm, 10 min, 200
mW/cm2) 24 h after injection. All mice were observed under the
IVIS Spectrum 10/17/24 days post implantation and the tumour
signal was quantified using Living Image software 4.2. During
the photothermal treatment and IVIS observation period, the
mice were anaesthetized with isoflurane and monitored using a
The TUNEL kit protocol was followed. Briefly, the air-dried sliced
samples were immersed in tissue fixation solution and matured
for 25 min at 25 °C, rinsed with PBS three times (5 min each).
The samples were then immersed in the cellular membrane
permeation fluid for 5 min and rinsed with PBS three times (5
min each). The samples were subsequently immersed in the
sealing fluid for 10 min at 25 °C and rinsed with PBS three times
(5 min each). The liquids of the samples were removed by
bibulous paper. Each sample was mixed with TdT enzyme
solution (50 µL) and covered with glass for 60 min at 37 °C. The
samples were subsequently rinsed with PBS three times (5 min
each). The samples were then stained with DAPI following a
similar procedure.
2.13 Statistical analysis
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Ding, Xiaoyi
