Nanococktail Based on AIEgens and Semiconducting Polymers: A Single Laser Excited Image-Guided Dual Photothermal Therapy

Semiconducting polymers (SPs)-based dual photothermal therapy (PTT) obtained better therapeutic effect than single PTT due to its higher photothermal conversion efficiency. However, most dual PTT need to use two lasers for heat generation, which brings about inconvenience and limitation to the experimental operations. Herein, we report the development of “nanococktail” nanomaterials (DTPR) with 808 nm-activated image-guided dual photothermal properties for optimized cancer therapy. Methods: In this work, we co-encapsulated AIEgens (TPA-BDTO, T) and SPs (PDPPP, P) by using maleimide terminated amphiphilic polymer (DSPE-PEG2000-Mal, D), then further conjugated the targeting ligands (RGD, R) through “click” reaction. Finally, such dual PTT nanococktail (termed as DTPR) was constructed. Results: Once DTPR upon irradiation with 808 nm laser, near-infrared fluorescence from T could be partially converted into thermal energy through fluorescence resonance energy transfer (FRET) between T and P, coupling with the original heat energy generated by the photothermal agent P itself, thus resulting in image-guided dual PTT. The photothermal conversion efficiency of DTPR reached 60.3% (dual PTT), much higher as compared to its inherent photothermal effect of only 31.5% (single PTT), which was further proved by the more severe photothermal ablation in vitro and in vivo upon 808 nm laser irradiation. Conclusion: Such smart “nanococktail” nanomaterials could be recognized as a promising photothermal nanotheranostics for image-guided cancer treatment.

Polymer molecular weight was measured with PL-GPC 220 high temperature chromatograph equipped with an IR5 detector at 140 °C; polystyrene was utilized as the calibration standard and 1,2,4-trichlorobenzene as eluent (1 mL/min). UV-Vis absorption spectra were taken on an Agilent Cary 60 UV/Visible Spectrometer. All the fluorescence measurements were performed on a FS5 spectrofluorometer (Edinburgh Instruments), except the two-photon Fluorescence spectra were recorded on a Horiba Fluoromax-4 fluorescence spectrophotometer. The dynamic light scattering (DLS) size distribution and zeta potential of particles were recorded by a Malvern Instruments Zetasizer Nano ZS90. Field emission highresolution scanning electron microscope (FESEM) were recorded by Hitachi SU-8010.
Transmission electron microscope (TEM) images were obtained with a FEI Tecnai G2 12 TEM instrument with an accelerating voltage of 100 kV. MTT assay was obtained on an Infinite M200 PRO Microplate Reader (Tecan Austria). Confocal laser scanning microscopy images were recorded using LSM 880 confocal microscopy (Carl Zeiss), equipped with a FemtoSecond Laser (Coherent Inc.). A 20 × oil or 63 × oil objective was used in the imaging process.

Detection of ROS in Solution
The ROS generation was studied by using ABDA as an indicator as the absorbance of ABDA decreases upon reaction with ROS. 10 L of ABDA solution (4.5 mg/mL in DMSO) was added to different nanoparticles, then 1 mL mixed solution in Petri dish was irradiated with 808 nm laser (1.1 W/cm 2 ). The decomposition of ABDA was monitored by the absorbance decrease. The absorbance of ABDA at 400 nm was recorded for different durations of 808 nm laser irradiation to obtain the decay rate of the photosensitizing process.

Photothermal Performance of Nanoparticles
Photothermal performance of nanoparticles was measured and analyzed by irradiating a 1.5 mL EP tube containing 100 µL different nanoparticles dispersion with different concentrations. NIR laser was produced using an 808 nm power multimode pump laser (Changchun New Industries Optoelectronics Tech Cc., Ltd, china). The temperature and thermal images of the irradiated aqueous dispersion and cells were recorded on an infrared thermal imaging instrument (FLIR TM E85 camera, Japan).

Cellular Imaging
For confocal laser scanning microscopy (CLSM) imaging, cells were seeded into cell culture dishes at a density of 2×10 4 in growth medium (DMEM supplemented with 10% FBS).
After an overnight incubation, the cells were washed with phosphate buffer saline (PBS, pH 7.2-7.4) for three times. A solution of the indicated nanoparticles in medium was then added, and the cells were incubated in a 5% CO 2 atmosphere at 37 °C for further usage. For coculture cells CLSM, the same number (2×10 4 of each dish) of GFP-SKOV-3 cells and MCF-7 cells were co-culture in the medium of DMEM medium at 37 °C in a humidified atmosphere containing 5% CO 2 .

Endocytosis Mechanism
To study the endocytosis mechanism of DTPR nanoparticles, SKOV-3 cells were incubated with DTPR at 4 °C to study the effect of low temperature on the cellular endocytosis of the nanoagents. Besides, we investigated the possible endocytosis pathways of DTPR nanoparticles using the following inhibitors: filipin (1 g/ml), chlorpromazine (10 g/ml), EIPA (10 g/ml). After separately pre-incubating of these inhibitors for 30 min in serum-free medium, cells were then treated with DTPR nanoparticles (10 g/mL) for 4 h, and then analyzed by CLSM imaging.

Cytotoxicity Studies
The metabolic activities of SKOV-3 cells were evaluated using MTT assays. SKOV-3 cells were seeded in 96-well plates at an intensity of 5 × 10 3 cells/mL, respectively. After 80% confluence, the old medium was replaced by different nanoparticles in DMEM suspension at various concentrations. After 24 h incubation, the suspensions were replaced by fresh DMEM containing 10% FBS and 1% penicillin-streptomycin. The selected wells were exposed to 808 nm laser irradiation (1.1 W/cm 2 , 3 min). The cells were further cultured for 24 h, and then washed with 1× PBS buffer and 20 L of freshly prepared MTT (5 mg/mL) solution was added into each well. The MTT medium solution was carefully removed after 3 h incubation in the incubator. DMSO (150 L) was then added into each well and the plate was gently shaken for 10 min at room temperature to dissolve all the precipitates formed. The absorbance 6 of MTT at 570 nm was monitored by the microplate reader. Cell viability was expressed by the ratio of the absorbance of the cells incubated with nanoparticles to that of the cells incubated with culture medium only.

PTT-mediated Apoptosis Assay
SKOV-3 cells were incubated with nanoparticles (40 g/mL) for 4 h. After washing three times using PBS, the cells were treated with 808 nm laser irradiation (1.1 W/cm 2 , 5 min), the area of illumination was controlled by masking. After removal of the medium, the adherent cells were rinsed with PBS for three times. Then SKOV-3 cells were stained with CMFDA for 15 min and washed three times with fresh culture medium to remove dead cells. Fluorescent images were visualized under fluorescent microscope.

Hemolysis Test
The release of hemoglobin from BALB/c nude mice blood cells was used to evaluate the hemolytic activities of DR, DTR, DPR, and DTPR nanoparticles by spectrophotometry. The blood samples were centrifugated and resuspended in normal saline to get the red blood cells (RBCs 2%). 100 L RBCs suspension mixed with 100 L ultrapure water and 100 L normal saline solution were regarded as positive control (producing 100% hemolysis) and negative control (producing no hemolysis), respectively. Whereafter, 100 L of different nanoparticles solutions were added into the mixture of 100 L RBCs suspension, respectively. After kept at 37 °C for 3 h, all the samples were centrifuged. The absorbance of supernatants was measured with UV spectrophotometer and the normal saline was used as blank. The hemolysis ratio of RBSs was calculated using the following formula: hemolysis (%) = (A sample -A negative ) / (A positive -A negative ) × 100%, where A sample , A negative , and A positive refered to the absorption of nanoparticles sample solution, negative control, and positive control at 570 nm, respectively. the tumors and main organs (heart, liver, spleen, lung and kidney) were monitored by the IVIS Spectrum (PerkinElmer) (λ ex =535 nm, λ em =680 nm). To examine the targeting of RGD peptides, SKOV-3 tumor-bearing mice were tail vein injected with 200 L, 100 g/mL DTP and DTPR, respectively. Post-injection 12 h, tumors were also monitored by the IVIS Spectrum (λ ex =535 nm, λ em =680 nm).

Photothermal Imaging
SKOV-3 tumor-bearing mice were tail vein injected with 200 L PBS, DR, DTR, DPR and DTPR (100 µg/mL), respectively. Post-injection 12 h, tumors of the mice were irradiated for 6 min with 808 nm laser (0.8 W/cm 2 ). The temperature changes were recorded with a Thermal imaging equipment.

Therapeutic Effect In vivo
BALB/c nude mice bearing SKOV-3 tumors were invoked as model. When tumors grew up to about 15-20 mm 3 in volume, the mice were randomly assigned in nine groups: (a) PBS only group, (b) PBS+808 nm laser group, (c) DR only group, (d) DR+808 nm laser group, (e) DTR +808 nm laser group, (f) DPR only group, (g) DPR +808 nm laser group, (h) DTPR only group, (i) DTPR +808 nm laser group. Then 200 L nanoparticles were tail vein injected once 8 per three days. For PTT groups, tumors were exposed to 808 nm laser at 0.8 W/cm 2 for 6 min.
After treatments, body weight and tumor sizes of each mouse were monitored by a digital caliper at a unique time point every day. To further explore the effect of therapy, 11 days after various treatments, the mice were sacrificed, and the tumors and other major organs (heart, liver, spleen, lung, and kidney) were collected for further analysis. The tissue sections were stained with hematoxylin-eosin (H&E) following the standard protocol and then pathologically examined under a microscope.                          Photos of tumors after various treatments were taken at day 11 (n = 5). (C) Body weight changes of SKOV-3 tumor-bearing mice that received various treatments as indicated (n = 5).
(D) Histopathological examinations via H&E staining of tumor, heart, kidneys, liver, lung, spleen of various treatments groups as indicated. Scale bars: 50 m.