2. Results and Discussion
2.1 Synthesis and Characterization of TNPs
A novel PTAs with 880nm triggered A-D-A structure non-fullerene
molecules, 3TT-IC-4Cl, which includes three fused
thieno[3,2-b]thiophene as the central core and difluoro substituted
indanone as the end groups40 was selected for PTT. In
order to effectively utilize 3TT-IC-4Cl for tumor therapy. An
amphiphilic block copolymer (FA-PEG-PBLA10) was
synthesized as our previous reported method41 and used
for 3TT-IC-4Cl encapsulation, 3TT-IC-4Cl was encapsulated in
FA-PEG-PBLA10 by nano-precipitation and dialysis process
to form stable nanoparticles (TNPs), as shown in Figure 1, PBLA segment
of copolymer as a reservoir for 3TT-IC-4Cl storage in the inner core,
PEG segment as the out shell to improve solubility, stability and
biocompatibility of TNPs, active targeting ligand FA was introduced to
the surface of nanoparticles to enhance selectivity of nanoparticles,
the chemical structure was confirmed by 1H NMR, as
shown in Figure 2A, the characteristic peaks a and b are belong to
FA-PEG-PBLA10, and the characteristic peaks c, d, e, f,
g, and h attribute to 3TT-IC-4Cl, respectively. It indicated that the
3TT-IC-4Cl was encapsulated in FA-PEG-PBLA10successfully, the encapsulation rate (93.5%) was calculated by the
relative intensity ratio of the methylene proton of PEG at 3.5 ppm and
the proton of the alkane chain of in 3TT-IC-4Cl at about 1 ppm.
For nanomedicine used in cancer therapy, the size, morphology, and
stability are the key properties that influence in vivo performance.
These factors affect the biodistribution and circulation time of the
drug carriers. Stable and smaller particles have reduced uptake by the
RES and provide efficient passive tumor-targeting ability via an
enhanced permeation and retention (EPR) effect42. The
morphology of TNPs was evaluated by TEM, as shown in Figure 3. The TNPs
were submicron in size and uniform nearly spherical with no aggregation
between nanoparticles observed due to the polymer modification, the
average diameter was 150 nm. DLS measurements showed average
hydrodynamic diameter of TNPs is about 200 nm (Figure 3, inset), a
suitable size for passive targeting ability through EPR effect. The size
distribution of TNPs maintained a narrow and monodisperse unimodal
pattern.
2.2 Optical properties of TNPs
The optical properties of TNPs was investigated by UV-vis absorption
spectra and fluorescence spectra (Figure 4A and Figure 4B), for free
3TT-IC-4Cl in CHCl3 solution, it shows strong absorption
at 772 nm and a maximal fluorescence at about 840 nm. However, after
formation nanoparticles, the TNPs aqueous solution exhibit strong
absorption at 874 nm, the significant red shift would due to the π-π
stacking of 3TT-IC-4Cl during the nanoparticles formation, this result
would conducive to trigger TNPs by 880 nm NIR light source for
phototherapy of tumor in deep tissue. In the other hand, compare to free
3TT-IC-4Cl in CHCl3 solution, the TNPs aqueous solution
nearly no fluorescence signal is observed due to the 3TT-IC-4Cl
aggregation during the nanoparticles formation, which would
significantly increases nonradiative heat generation, and enhance PTT
efficiency18, 43.
2.3 Photothermal Properties of TNPs in Vitro
To investigate the photothermal conversion property of the TNPs, the
temperature of TNPs aqueous solution with a series of concentrations
(from 0 to 250 μg/mL) under the 880 nm laser irradiation (0.7
W/cm2) for 15 min was monitored (Figure 5A), the
related infrared (IR) thermal images of TNPs aqueous solution were
showed in Figure 5C. As shown in the Figures, the temperature increased
significantly as TNPs concentration increased. It is noted that the TNPs
at 90 μg/mL exhibit effective hyperthermia (>50 ℃), which
is sufficient to induce apoptosis or necrosis of cancer
cells44. The relationship between temperature of TNPs
aqueous solution (180 μg/mL) and different laser power (from 0.3 to 1.5
W/cm2) was future measured, as shown in Figure 5B, the
temperature of the TNPs aqueous solution depends on the laser power. The
related infrared (IR) thermal images of TNPs aqueous solution were
showed in Figure 5D. In the other hand, we also investigated the
photothermal conversion efficiency of TNPs through a cycle of heat-up
and cooling using previous reported method (Figure
S1)45. The photothermal conversion efficiency of the
TNPs was 31.5%, which is more higher than other PTAs such as cyanine
dyes (e.g.,≈26.6%) and gold nanorods (e.g.,≈21.0%)22,
46, 47. The strong absorption and high photothermal conversion
efficiency of TNPs in the NIR region provided the potential of
photothermal treatment of cancer.
2.4 Photothermal stability of TNPs
The photothermal stability is an important parameter of photothermal
drugs for PTT applications, it would be crucial for clinical application
and therapeutic efficiency. The photothermal stability of TNPs was
evaluated by monitoring its ability to maintain the temperature
elevation. As shown in Figure 6A, the TNPs was irradiated at 0.7
W/cm2 for 5 min, then the laser was turned off,
following the samples were cooling down to the room temperature, the
temperature was recorded by IR thermal camera throughout the process,
this irradiation/cooling procedures were repeated five times, as Figure
6A shows, TNPs displayed negligible change in their temperature
elevation after five irradiation/cooling cycles. However, the
temperature elevation of free ICG decreased significantly after one
irradiation/cooling cycle. In the other hand, we also observed the
changes in the color of samples, as shown in Figure 6B, after 5 min
irradiation, the color of free ICG solution changed observably, but the
TNPs exhibit no change after 30 min irradiation. These results indicated
the TNPs exhibit excellent photothermal stability.
2.5 In Vitro Cell Test
In order to investigate the feasibility of TNPs as nano photothermal
agents for PTT, the in vitro cytotoxicity of TNPs was
investigated by MTT assay and the average cell viability was monitored.
For biocompatibility test, the dark toxicity of TNPs was investigated.
As shown in Figure 7A, both FA-PEG-PBLA10 and TNPs
exhibited no significant dark toxicity. As the concentration increased,
the average cell viability was greater than 90% even when cells were
treated with 250 μg/mL of TNPs. For the phototoxicity test, we
investigated the concentration dependent (0, 30, 60, 90, 120, 180, and
250 μg/mL) cytotoxicity of TNPs with 880 nm laser irradiation. As shown
in Figure 7B, after irradiation at 0.7 W/cm2 for 5
min, the cell viability gradually decreased as the TNPs concentration
increased. Taken together, these results indicate that the TNPs could
considerably enhance the efficiency of PTT for tumor in deep tissue,
even at low concentrations.