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.