880 nm NIR-Triggered Organic Small Molecular-Based Nanoparticles for Photothermal Therapy of Tumor

Photothermal therapy (PTT) has received constant attention as an efficient cancer therapy method due to locally selective treatment, which is not affected by the tumor microenvironment. In this study, a novel 880 nm near-infrared (NIR) laser-triggered photothermal agent (PTA), 3TT-IC-4Cl, was used for PTT of a tumor in deep tissue. Folic acid (FA) conjugated amphiphilic block copolymer (folic acid-polyethylene glycol-poly (β-benzyl-L-aspartate)10, FA-PEG-PBLA10) was employed to encapsulate 3TT-IC-4Cl by nano-precipitation to form stable nanoparticles (TNPs), and TNPs exhibit excellent photothermal stability and photothermal conversion efficiency. Furthermore, the in vitro results showed TNPs display excellent biocompatibility and significant phototoxicity. These results suggest that 880 nm triggered TNPs have great potential as effective PTAs for photothermal therapy of tumors in deep tissue.


Introduction
Phototherapy has attracted extensive attention in recent years as a powerful cancer treatment method due to characteristics such as convenience, noninvasiveness, locally selective treatment, negligible drug resistance and minimized adverse side effects [1]. Photodynamic therapy (PDT) and photothermal therapy (PTT) are two typical phototherapy approaches, PTT is based on the photothermal agents (PTA), which are preferentially taken up and retained by diseased tissue; then after excitation by appropriate wavelength laser, the PTA convert light to heat to induce cancer cell apoptosis or necrosis. Compared to PDT, PTT is not affected by the tumor microenvironment, such as the local oxygen level, so PTT has received increasing attention and developed rapidly in recent years.

Characterization
The chemical structure was determined by 400 MHz 1 H NMR (AVANCE III HD 400 MHz, Bruker, Fällanden, Switzerland) using CHCl 3 -d and DMSO-d 6 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 25 • C 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).

Preparation of TNPs
In order to prepare TNPs, first, the amphiphilic block copolymer FA-PEG-PBLA 10 used for 3TT-IC-4Cl encapsulation was synthesized by ring-opening polymerization as our previous reported [48]. The chemical structure of FA-PEG-PBLA 10 was confirmed by 1 H NMR (400 MHz, DMSO). Then, the TNPs were prepared by the 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-PBLA 10 solution (0.5 mg/mL in DMSO) dropwise, and then the mixture was transfered to dialysis tubs (Cut-off 3.5 K Mw) to remove THF and DMSO, followed by freeze drying, after which the TNPs were obtained.

Photothermal Effect
To confirm the PTT application potential, the photothermal property of TNPs was investigated, and a series of concentrations of TNPs (0, 30, 90, 180 and 250 µg/mL) in water were irradiated by 880 nm laser (0.7 W/cm 2 , where, the power densities (W/cm 2 ) = laser beam power/laser beam area) for 720 s, the temperature of TNPs solution was recorded by an IR thermal camera every 30 s. In addition, the constant concentration (180 µg/mL) of TNPs were irradiated by an 880 nm laser for 720 s with various power densities (0.3, 0.5, 0.8 and 1.5 W/cm 2 ) was investigated by the same method.

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 an 880 nm laser (0.7 W/cm 2 ) for 5 min; then the laser was turned off and the sample was cooled to the room temperature naturally, and the temperature of samples was recorded using the IR thermal camera every 30 s. Subsequently, the procedures were repeated four times.

In Vitro Phototoxicity and Biocompatibility of TNPs
HeLa cells (provided by Dingguo Biology Technology Co., Ltd., 1 × 10 4 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-PBLA 10 (the polymer dispersed in aqueous medium) 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/cm 2 ) for 5 min. Then, irradiated cells were incubated at 37 • C for 24 h and cell viability was evaluated by MTT assay. Data presented are averaged results of quadruplicate experiments. For biocompatibility, HeLa cells (1 × 10 4 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-PBLA 10 and TNPs with a series of concentration (0, 30, 60, 90, 120, 180 and 250 µg/mL), and then they were incubated for 24 h. The cell viability was evaluated by an MTT assay. Data presented are averaged results of quadruplicate experiments.

Synthesis and Characterization of TNPs
A novel PTA with an 880 nm-triggered A-D-A structure non-fullerene molecule, 3TT-IC-4Cl, which included three fused thieno [3,2-b]thiophene as the central core and difluoro substituted indanone as the end group [47] was selected for PTT. In order to effectively utilize 3TT-IC-4Cl for tumor therapy. An amphiphilic block copolymer (FA-PEG-PBLA 10 ) was synthesized as in our previous reported method [48] and used for 3TT-IC-4Cl encapsulation, 3TT-IC-4Cl was encapsulated in FA-PEG-PBLA 10 by nano-precipitation and a dialysis process to form stable nanoparticles (TNPs), as shown in Figure 1, the PBLA segment of the copolymer was used as a reservoir for 3TT-IC-4Cl storage in the inner core, the PEG segment was used as the outer shell to improve solubility, stability and biocompatibility of TNPs, the active targeting ligand FA was introduced to the surface of nanoparticles to enhance selectivity of nanoparticles, the chemical structure was confirmed by 1 H NMR, as shown in Figure 2A, and the characteristic peaks a and b are belong to FA-PEG-PBLA 10 , 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-PBLA 10 successfully, 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, size, morphology and stability are the key properties that influence in vivo performance. These factors affect the bio-distribution and circulation time of the drug carriers. Stable and suitable-sized particles have reduced uptake by the reticuloendothelial systems (RES) and provide efficient passive tumor targeting ability via an enhanced permeation and retention (EPR) effect [49]. The incomplete tumor vasculature results in leaky vessels with gap sizes of 100 nm to 2 µm depending on the tumor type, and some studies have shown that particles with diameters of <200 nm are more effective [49,50]. The morphology of TNPs was evaluated by TEM, as shown in Figure 3. The TNPs were submicron in size and uniform and nearly spherical with no aggregation between nanoparticles observed due to the polymer modification, the average diameter was 150 nm. DLS measurements showed average hydrodynamic diameters of TNPs were about 200 nm ( Figure 3, inset), a suitable size for passive targeting ability through the EPR effect. The size distribution of TNPs maintained a narrow and monodisperse unimodal pattern. Zeta potential of TNPs was measured as shown in Figure S1. It was shown that TNPs have negative surface charges, and zeta potential is about −13.2 mV. The zeta potential of TNPs showed that it would more stable against aggregation. Furthermore, the size of TNPs in DMEM remains almost same within 60 days ( Figure S2).

Synthesis and Characterization of TNPs
A novel PTA with an 880 nm-triggered A-D-A structure non-fullerene molecule, 3TT-IC-4Cl, which included three fused thieno[3,2-b]thiophene as the central core and difluoro substituted indanone as the end group [47] 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 in our previous reported method [48] and used for 3TT-IC-4Cl encapsulation, 3TT-IC-4Cl was encapsulated in FA-PEG-PBLA10 by nano-precipitation and a dialysis process to form stable nanoparticles (TNPs), as shown in Figure 1, the PBLA segment of the copolymer was used as a reservoir for 3TT-IC-4Cl storage in the inner core, the PEG segment was used as the outer shell to improve solubility, stability and biocompatibility of TNPs, the active targeting ligand FA was introduced to the surface of nanoparticles to enhance selectivity of nanoparticles, the chemical structure was confirmed by 1 H NMR, as shown in Figure 2A, and 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-PBLA10 successfully, 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, size, morphology and stability are the key properties that influence in vivo performance. These factors affect the bio-distribution and circulation time of the drug carriers. Stable and suitable-sized particles have reduced uptake by the reticuloendothelial systems (RES) and provide efficient passive tumor targeting ability via an enhanced permeation and retention (EPR) effect [49]. The incomplete tumor vasculature results in leaky vessels with gap sizes of 100 nm to 2 μm depending on the tumor type, and some studies have shown that particles with diameters of <200 nm are more effective [49,50]. The morphology of TNPs was evaluated by TEM, as shown in Figure 3. The TNPs were submicron in size and uniform and nearly spherical with no aggregation between nanoparticles observed due to the polymer modification, the average diameter was 150 nm. DLS measurements showed average hydrodynamic diameters of TNPs were about 200 nm ( Figure 3, inset), a suitable size for passive targeting ability through the EPR effect. The size distribution of TNPs maintained a narrow and monodisperse unimodal pattern. Zeta potential of TNPs was measured as shown in Figure S1. It was shown that TNPs have negative surface charges, and zeta potential is about −13.2 mV. The zeta potential of TNPs showed that it would more stable against aggregation. Furthermore, the size of TNPs in DMEM remains almost same within 60 days ( Figure S2).

Optical Properties of TNPs
The optical properties of TNPs were investigated by UV-vis absorption spectra and fluorescence spectra ( Figure 4A,B), for free 3TT-IC-4Cl in CHCl3 solution, and it shows strong absorption at 772 nm and a maximal fluorescence at about 840 nm. However, after the formation of nanoparticles, the TNPs aqueous solution exhibits strong absorption at 874 nm, the significant red shift was due to the π-π stacking of 3TT-IC-4Cl during the nanoparticles formation and this result would be conducive to trigger TNPs by an 880 nm NIR light source for the phototherapy of the tumor in deep tissue. On the other hand, compared to free 3TT-IC-4Cl in CHCl3 solution, in the TNPs aqueous solution, nearly no fluorescence signal was observed due to the 3TT-IC-4Cl aggregation during the nanoparticle formation, which would significantly increase non-radiative heat generation and enhance PTT efficiency [20,51].

Optical Properties of TNPs
The optical properties of TNPs were investigated by UV-vis absorption spectra and fluorescence spectra ( Figure 4A,B), for free 3TT-IC-4Cl in CHCl 3 solution, and it shows strong absorption at 772 nm and a maximal fluorescence at about 840 nm. However, after the formation of nanoparticles, the TNPs aqueous solution exhibits strong absorption at 874 nm, the significant red shift was due to the π-π stacking of 3TT-IC-4Cl during the nanoparticles formation and this result would be conducive to trigger TNPs by an 880 nm NIR light source for the phototherapy of the tumor in deep tissue. On the other hand, compared to free 3TT-IC-4Cl in CHCl 3 solution, in the TNPs aqueous solution, nearly no fluorescence signal was observed due to the 3TT-IC-4Cl aggregation during the nanoparticle formation, which would significantly increase non-radiative heat generation and enhance PTT efficiency [20,51]. fluorescence spectra ( Figure 4A,B), for free 3TT-IC-4Cl in CHCl3 solution, and it shows strong absorption at 772 nm and a maximal fluorescence at about 840 nm. However, after the formation of nanoparticles, the TNPs aqueous solution exhibits strong absorption at 874 nm, the significant red shift was due to the π-π stacking of 3TT-IC-4Cl during the nanoparticles formation and this result would be conducive to trigger TNPs by an 880 nm NIR light source for the phototherapy of the tumor in deep tissue. On the other hand, compared to free 3TT-IC-4Cl in CHCl3 solution, in the TNPs aqueous solution, nearly no fluorescence signal was observed due to the 3TT-IC-4Cl aggregation during the nanoparticle formation, which would significantly increase non-radiative heat generation and enhance PTT efficiency [20,51].

Photothermal Properties of TNPs In Vitro
To investigate the photothermal conversion property of the TNPs, the temperature of TNP aqueous solution with a series of concentrations (from 0 to 250 μg/mL) under the 880 nm laser irradiation (0.7 W/cm 2 ) for 15 min was monitored ( Figure 5A), and the related infrared (IR) thermal images of TNPs aqueous solution were showed in Figure 5C. As

Photothermal Properties of TNPs In Vitro
To investigate the photothermal conversion property of the TNPs, the temperature of TNP aqueous solution with a series of concentrations (from 0 to 250 µg/mL) under the 880 nm laser irradiation (0.7 W/cm 2 ) for 15 min was monitored ( Figure 5A), and 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 TNP concentration increased. It is noted that the TNPs at 90 µg/mL exhibit effective hyperthermia (>50 • C), which is sufficient to induce apoptosis or necrosis of cancer cells [52]. The relationship between temperature of TNPs aqueous solution (180 µg/mL) and different laser power (from 0.3 to 1.5 W/cm 2 ) was future measured, as shown in Figure 5B, and the temperature of the TNPs aqueous solution depends on the laser power. The related infrared (IR) thermal images of TNP aqueous solution were showed in Figure 5D. On the other hand, we also investigated the photothermal conversion efficiency of TNPs through a cycle of heat-up and cooling using the previously reported method ( Figure S3) [53]. The photothermal conversion efficiency of the TNPs was 31.5%, which is higher than other PTAs such as cyanine dyes (e.g., ≈26.6%) and gold nanorods (e.g., ≈21.0%) [24,54,55]. The strong absorption and high photothermal conversion efficiency of TNPs in the NIR region provided the potential of photothermal treatment of cancer. mal images of TNP aqueous solution were showed in Figure 5D. On the other hand, we also investigated the photothermal conversion efficiency of TNPs through a cycle of heatup and cooling using the previously reported method ( Figure S3) [53]. The photothermal conversion efficiency of the TNPs was 31.5%, which is higher than other PTAs such as cyanine dyes (e.g., ≈26.6%) and gold nanorods (e.g., ≈21.0%) [24,54,55]. The strong absorption and high photothermal conversion efficiency of TNPs in the NIR region provided the potential of photothermal treatment of cancer.

Photothermal Stability of TNPs
The photothermal stability is an important parameter of photothermal drugs for PTT applications, and it would be crucial for clinical applications 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 were irradiated at 0.7 W/cm 2 for 5 min, then the laser was turned off, the following samples were cooled down to room temperature, the temperature was recorded by IR thermal camera throughout the process,

Photothermal Stability of TNPs
The photothermal stability is an important parameter of photothermal drugs for PTT applications, and it would be crucial for clinical applications 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 were irradiated at 0.7 W/cm 2 for 5 min, then the laser was turned off, the following samples were cooled down to 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, and 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. On the other hand, we also observed the changes in the color of the samples, as shown in Figure 6B, and 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. this irradiation/cooling procedures were repeated five times, as Figure 6A shows, and 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. On the other hand, we also observed the changes in the color of the samples, as shown in Figure 6B, and 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.

In Vitro Cell Test
In order to investigate the feasibility of TNPs as nano photothermal agents for PTT, in vitro cytotoxicity of TNPs was investigated by MTT assay and the average cell viability was monitored. For a 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/cm 2 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.

Conclusions
In summary, an 880 nm NIR laser that triggered TNPs as PTA for photothermal therapy of a tumor in deep tissue was developed. In this work, a novel PTA, 3TT-IC-4Cl, was

In Vitro Cell Test
In order to investigate the feasibility of TNPs as nano photothermal agents for PTT, in vitro cytotoxicity of TNPs was investigated by MTT assay and the average cell viability was monitored. For a biocompatibility test, the dark toxicity of TNPs was investigated. As shown in Figure 7A, both FA-PEG-PBLA 10 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/cm 2 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. this irradiation/cooling procedures were repeated five times, as Figure 6A shows, and 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. On the other hand, we also observed the changes in the color of the samples, as shown in Figure 6B, and 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.

In Vitro Cell Test
In order to investigate the feasibility of TNPs as nano photothermal agents for PTT, in vitro cytotoxicity of TNPs was investigated by MTT assay and the average cell viability was monitored. For a 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/cm 2 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.

Conclusions
In summary, an 880 nm NIR laser that triggered TNPs as PTA for photothermal therapy of a tumor in deep tissue was developed. In this work, a novel PTA, 3TT-IC-4Cl, was

Conclusions
In summary, an 880 nm NIR laser that triggered TNPs as PTA for photothermal therapy of a tumor in deep tissue was developed. In this work, a novel PTA, 3TT-IC-4Cl, was selected and used for PTT; it included three fused thieno [3,2-b]thiophene as the central core and difluoro-substituted indanone as the end group. After encapsulation by the FA-PEG-PBLA 10 block copolymer and forming nanoparticles, the TNP aqueous solution exhibited strong absorption at 880 nm due to the π-π stacking. DLS and TEM measurements showed that the TNPs have a spherical shape and narrow size distribution with a mean diameter of 150 nm. TNPs exhibit excellent photothermal stability and high photothermal conversion efficiency after 880 nm laser irradiation. In the in vitro test, TNPs display excellent biocompatibility and significant phototoxicity. Therefore, the 880 nmtriggered TNPs have great potential as an effective PTA for the photothermal therapy of tumor in deep tissue.