Dopamine hydrochloride and PTX were purchased from Sigma Aldrich Co., Ltd. Coumarin 6, IR780, and Pluronic F127 were purchased from Aladdin Co., Ltd. CCK-8 kit was purchased from Topscience Inc. DAPI kit was purchased from Biotech Ltd. The apoptosis detection kit was purchased from Beyotime Inc. Biotechnology. IL-6 detection kit and TNF-α test kit were purchased from Wuhan Servicebio Ltd. The other chemical reagents were purchased from Sinopharm Lnc. The Huh-7, BEL-7402, HepG-2, and HL-7702 Cell lines were all derived from ATCC. The BALB/C mice and BALB/c-nu/nu mice were purchased from Beijing HFK Biotechnology Ltd. The CB-17 SCID mouse was purchased from Charles River (Beijing) Experimental Animal Technology Ltd. Unless otherwise stated, all the chemicals and reagents were of analytical grade and used as received.

In a round bottom flask, 4 ml of Tris HCl (0.5 mg/ml, water) was placed. A total of 1 mg of PTX and 4 mg of Pluronic F127 was dissolved in 200 μl of DMSO. The solution was added dropwise to the Tris HCl solution under stirring (800 rpm). And then, 100 μL of IR780 (20 mg/ml, DMSO) was added to the solution. The solution was stirred continuously for 20 min before being treated by a 150 W ultrasound for 5 min. After that, 1 ml of dopamine hydrochloride solution (10 mg/ml, water) was slowly added to the solution above. After stirring for 5 min, the solution was sealed and rotated in the dark for more than 72 h. Then, the solvent and soluble impurities were removed by dialysis against the Tris HCl solution, and the precipitations were removed by low-speed centrifugation to obtain the final DIST NPs solution.

The particle size and zeta-potential distributions of DIST NPs were detected by the dynamic light scattering method. The morphology of DIST NPs was observed by a transmission electron microscope (TEM, Tecnai G2 F20 U-TWIN, FEI, USA). The DIST NPs were decomposed in an acidic environment to release PTX and IR780. Chloroform was used to extract the two substances. The concentrations of PTX and IR780 were detected by HPLC and fluorescence spectrophotometry, respectively. Then, the encapsulation rate and drug loading capacity of DIST NPs were calculated. The particle size changes of DIST NPs in PBS, complete medium, and fetal bovine serum were continuously measured to determine their colloidal stability in different physiological environments. The release degree of DIST NPs under different pH and temperature conditions were detected by the dialysis method. In evaluation of the photothermal conversion, different parameters, such as concentrations and irradiation time, were set, and temperature was measured by a thermal imager (E4, Teledyne FLIR, USA). The wavelength of the laser is 808 nm. In vivo evaluation of thermal conversion was performed in the BALB/c mouse model. The mice were depilated by a cream for a more effective observation. The DIST NPs was subcutaneously injected in the crotch of mouse. Subsequently, the 808-nm laser was used to irradiate the injection area. The infrared thermal imager was employed to measure the temperature.

After referring to a variety of preparation protocols, DIST NPs were labeled with coumarin 6 to have a green fluorescence (24, 36). BEL-7402 cells were then planted in 3.5-cm glass-bottom dishes (1 × 10⁵ cells/dish) and cultured at 37°C and 5% CO₂ for 48 h. Then, the cells were incubated with fluorescence-labeled DIST NPs and observed at different time points by a laser scanning confocal microscope (TCS SPT, Leica, Germany). In order to determine the internalization effect of DIST NPs, the cells were treated with sodium azide, an endocytosis inhibitor, and incubated with DIST NPs for confocal observation and comparison.

The cell inhibition of DIST NPs in vitro was tested by CCK-8 assay. The hepatic carcinoma cell lines (Huh-7, BEL-7402, HepG2) and a normal hepatic cell line (HL-7702) in their logarithmic growth phase were inoculated into 96-well plates (8 × 10³ cells/well). Then, the DIST NPs, PTX, IR780, and PDA were added, respectively. The cells were cultured at 37°C and 5% CO₂. After the cells in the control wells reached 90% confluence, the obsolete medium in the well was removed, and the colorless cultured medium containing 10% CCK-8 was added. The cells were incubated for 2 h before being put it into a plate reader (ELx800, BioTek, USA) to measure the absorbance at 450 nm each well for the cell viability calculation.

The combined treatment of PTT-chemotherapy was evaluated by CCK-8 assay, colony formation test, and flow cytometry method. The cell lines were Huh-7, BEL-7402, and HepG2. In the CCK-8 assay, the cells were inoculated into 96-well plates (4 × 10⁴/well) at 37°C and 5% CO₂ for 24 h. After the DIST NPs, PTX, IR780, and PDA were added, the cells were irradiated with an 808-nm laser, and then incubated for 6 h. The culture medium was replaced by the colorless culture medium containing CCK-8. The cells were incubated for 2 h before being put into a plate reader to measure the absorbance at 450 nm for the cell viability calculation. In the colony formation test, 500 μl of cell suspension (2,000 cells/ml) was added into a 1.5-ml centrifuge tube. The sample was added, and laser irradiation was performed, and then the cell suspension was transferred to a 6-cm plate and cultured for five days. After that, the colony was stained for observation. After the cells were treated with PTT, chemotherapy, and combination therapy, the cells were stained with a cell apoptosis kit and tested by a flow cytometer (Accuri C6 Plus, BD, USA).

DIST NPs are supposed to be injected intravenously, and their circulation time should be long in the body. Therefore, it is necessary to evaluate whether the DIST NPs and their components can cause hemolysis. A 2% erythrocyte suspension was prepared and divided into groups. Then, the DIST NPs and their main components were added to the test groups, respectively. The water, saline, and 0.1% Triton X-100 were added to the control groups, respectively. The red blood cells were incubated at 37°C for 2 h. The supernatant was replaced by the same amount of water after centrifugation before another 4 h of incubation. The absorbance wavelength at 540 nm in each group was measured by a microplate reader, and the hemolysis rate was calculated.

In the acute toxicity test, 40 BALB/c mice, half male and half female, weighing about 20 g, were selected and randomly divided into four groups. DIST NPs, PTX, IR780, and PDA were intravenously injected into the mice, respectively. All the samples injected were adjusted to the equivalent PTX concentration of 5 mg/kg or IR780 concentration of 10 mg/kg. The mice were prohibited from food and water 6 h before injection and were resumed for 2 h after the injection. The mice were observed continuously for 14 d. The symptoms were recorded, and the survival rate was calculated. Finally, the surviving mice were euthanized. The main organs were taken for pathological analysis.

The levels of interleukin-6 (IL-6) and tumor necrosis factor (TNF-α) in the blood of mice were tested by the ELISA kit to evaluate whether the DIST NPs can cause a systemic inflammatory reaction in vivo. The grouping is the same as the acute toxicity test except that there were four mice in each group. The samples were injected through the tail vein, and the blood samples were collected 24 h after injection.

All the animal experiments in this study were approved by the Laboratory Animal Administration Committee of Xi’an Medical University. The protocols for the animal experiments followed the Guidelines for the Use and Care of Experimental Animals at Xi’an Medical University. The Animal Ethics Approved Document Number is XY-AUC-2020-341.

Four-week-old female BALB/c-nu/nu mice were reared in an SPF room for five days for acclimation. Then, 150 μl of the BEL-7402 cell suspension (1 × 10⁶/ml) was injected into the crotch of each mouse. The tumor-bearing mice were used for subsequent experiments after their tumors grew to a suitable volume.

The orthotopic hepatic carcinoma model was established by CB-17 SCID mice. The 6-week-old female mice were reared in the SPF room for five days. After their abdominal hairs were removed, the mice were given general anesthesia through the inhalation of isoflurane and were fixed on the operating platform. A vertical incision with a length of 1 cm was cut below 1 cm of the xiphoid process. The peritoneum was cut, and the wound was expanded with a retractor. A small wound with 3–5 mm length and 1–2 mm depth was gently drawn on the surface of the liver. The fresh tumor tissue obtained from the subcutaneous tumor was planted in the wound. The wound on the liver was bonded with surgical glue. After the major wound was disinfected and sutured, lidocaine was injected subcutaneously by the side of the wound for analgesia. The mice were placed in a clean cage for observation and were given high nutritional feed. Their wounds were disinfected every day. After their wounds recovered, the mice were continually checked by abdominal touch. Once the clear indurations were found, the mice were used for subsequent experiments.

In the NIFI investigation, two orthotopic hepatic carcinoma model mice were injected with the DIST NPs and the same concentration of IR780 solution, respectively. The distribution of fluorescent signals in mice was continuously observed by the IVIS imaging system (PE, USA). The excitation and emission wavelengths were 780 nm and 845 nm, respectively. After the observation, the mice were euthanized, and the main organs and tumor tissues were taken out to analyze the distribution of the DIST NPs in the tissues.

In the PAI experiments, two subcutaneous tumor-bearing mice were injected with the DIST NPs and the same concentration of IR780 solution, respectively. The coupling agent was evenly coated on the mice. Wrapped with film and maintained with anesthesia, the mice were fixed in the supporting device and placed in the water tank for PAI. The excitation wavelength was 780 nm, and the emission range was 800–900 nm.

A total of 25 single side tumor-bearing mice were divided into five groups. The treatment methods were normal saline, IR780 + laser, PTX, IR780 + PTX + laser, and DIST NPs + laser. The dosage was calculated according to the concentrations of PTX and IR780, which were 1 mg/kg and 2 mg/kg, respectively. The dosage was determined in the pre-experiment, which could effectively demonstrate the difference of the treatment effect between the treatments. The ratio of IR780 and PTX causes the preparation craft of DIST NPs, that is 2:1. The administration was through intravenous injection. The laser irradiation time after injection was determined according to the experimental results of in vivo imaging. The irradiation wavelength, power, and time were 808 nm, 1 W/cm², and 2 min, respectively. The whole treatment process was conducted twice a week. The size of tumors and the weight of mice were measured continually, and the tumor area was photographed. After 21 d, the mice were euthanized, and the tumor tissues were taken, weighed, and photographed.

The two-way ANOVA and t-test were used for statistical analysis. The software was GraphPad prism 5.0. The data of independently repeated experiments were presented as the mean values ± standard deviation (SD). A P-value less than 0.05 indicated a statistical difference.

As shown in Figures 1A, B , the size and surface potential distributions of the DIST NPs are 103.4 ± 13.3 nm and -16.7 ± 3.8 mV, respectively. The TEM results in Figure 1C show that the DIST NPs have a spherical shape, mono-dispersity, and uniform particle size. Further observation by high-resolution TEM shows that the DIST NPs are dense solid spheres ( Figure 1D ). The result preliminarily determined that the DIST NPs were successfully constructed. The overall particle size meets the requirements of effectively entering the tumor tissue through the enhanced permeability and retention (EPR) effect. Their surfaces are relatively smooth and negatively charged, indicating that they can effectively escape from the clearance by the reticuloendothelial system in vivo and are expected to maintain a long circulation time. The encapsulation rates of DIST NPs for PTX and IR780 are 93.4 ± 3.1% and 92.3 ± 4.8%, respectively. The drug loading capacities of the DIST NPs for PTX and IR780 were 12.7 ± 4.1% and 24.9 ± 5.8%, respectively. The stability evaluation results of the DIST NPs are shown in Figures 1E–G . The size of the DIST NPs did not change significantly under various simulated physiological environments, indicating a good colloidal stability of the DIST NPs.

According to the in vivo delivery requirements, the DIST NPs should have long circulation and pH/temperature responding release abilities. Figure 2A shows the IR780 release curve of the DIST NPs in PBS at room temperature. Free IR780 exhibited an apparent burst release in which most of the IR780 was released within 2 h. However, only approximately 12% of IR780 in the DIST NPs was released in 72 h, indicating a good encapsulation stability of the DIST NPs. Figure 2B is the release curve of the DIST NPs under different pH environments. The release degree gradually increased with the decrease of the pH value. This scenario demonstrates that the DIST NPs can effectively release their loaded drugs under the acidic environment in tumor tissues and cells. Figure 2C shows the release curve of the DIST NPs at different temperatures. The temperature elevation promoted the release, indicating that the increase of temperature is also conducive to the drug release of the DIST NPs during PTT. Figure 2D shows the photothermal conversion results of the DIST NPs. With the same concentration, the temperature of the DIST NPs solution gradually increases with the extension of the irradiation time. The maximum temperature was 65°C. As shown in Figure 2E , there is a concentration-effect relationship between the concentration and temperature of the DIST NPs under the same irradiation power and time length. The 243 μg/ml DIST NPs had a maximum temperature of 64°C. The photothermal conversion effects in vivo are shown in Figures 2F, G . It can be seen that the DIST NPs can still absorb the laser energy and generate heat under the skin of mice. Their temperature reached more than 55°C in 3 min under irradiation. The temperature rise exhibited a positive relationship both with the concentration and irradiation time. These results indicate that the DIST NPs can be used for photothermal treatment in vivo.

According to our previous study, DIST NPs were labeled with a green fluorescence for confocal microscopic observation (36). The results show that the DIST NPs can effectively enter the BEL-7402 cells. As shown in Figure 3A , with the extension of the incubation time, the intensity of intracellular fluorescent signal gradually increased, gathering in the cytoplasm. In the quantitative analysis of fluorescent signals ( Figure 3B ), the fluorescent intensity of the cytoplasm is gradually enhanced compared with that of DAPI in the nucleus. In order to prove that the DIST NPs entered the cells through endocytosis, sodium azide was used to pretreat the cells. As shown in Figure 3C , the fluorescent signal in the sodium azide-treated cells was significantly weaker than in the untreated cells. There is a significant difference between the two groups. The results show that the DIST NPs entered the cells through endocytosis and gradually released the drugs in the cytoplasm.

The CCK-8 assay results are shown in Figure 4A . PDA and IR780 showed no significant toxicity in all four cell lines. The inhibitory effects of PTX and DIST NPs in all four cell lines rose with the increase of their concentrations and with no significant difference between them. The results showed that the DIST NPs effectively released the loaded PTX after entering the cells, performing a cell inhibitory effect. The PTT was introduced in the later CCK-8 assay ( Figure 4B ). With the 808-nm laser irradiation, the DIST NPs, IR780 and PDA groups showed apparent cell inhibitory effects. It is worth noting that the cell survival rate of the DIST NPs treatment group was lower than those of the IR780 + laser irradiation group and PTX treatment group. These results fully prove that the DIST NPs can effectively play the synergistic effect of PTT and chemotherapy in cells. The colony formation assay results ( Figure 4C ) further prove that the DIST NPs had excellent synergistic therapeutic effects. There was no significant difference in the numbers of clones between the DIST NPs group and the PPT + IR780 + laser irradiation group. The results of flow cytometry analysis ( Figure 4D ) show that the DIST NPs can effectively induce cell apoptosis, of which, the therapeutic effect is better than that of chemotherapy or PTT alone.

Before testing the in vivo imaging and antitumor effect, the in vivo safety of the DIST NPs needs to be evaluated. It is notable to point at whether the DIST NPs and their main components would cause hemolysis after intravenous injection. The results are shown in Figure 5A . The DIST NPs and their main components, PDA, IR780 PTX, and Pluronic F127, did not cause a significant erythrocyte rupture, while the hemolysis rate of the positive control group was 90%. The results show that the DIST NPs have an injection safety. The acute toxicity of the DIST NPs was evaluated by BALB/c mice. As shown in Figure 5B , within 14 d, no mouse died in the DIST NPs-treated group, and no adverse symptoms such as convulsion, depression, and appetite loss were found in mice except a few mice in the PTX-treated group. The survival statuses of mice in the PDA- and IR780-treated groups were also acceptable. Only three mice in the PTX-treated group died, and some mice had symptoms such as anorexia, weight loss, and mental malaise. In the pathological analysis of the mice organs ( Figure 5C ), the DIST NPs group mice only had a little lymphocyte infiltration in the liver, and no significant pathological changes were found in the other main organs. The blood concentration of two inflammatory factors, IL-6 and TNF-α, were measured ( Figures 5D, E ). Only two inflammatory factors of the PTX- and Pluronic F127-treated mice increased to a certain level, and the other groups, including a control group treated with normal saline, maintained a low inflammatory factors concentration. These results prove that the DIST NPs have a good biocompatibility in vivo and can be safely applied.

Firstly, the NIFI of the DIST NPs was evaluated on the orthotopic hepatic carcinoma model mice. As Figure 6A shows, in the DIST NPs-injected mice, the fluorescent signals began to accumulate in the abdomen 1 h after injection. The fluorescent intensity increased continuously over time, reached its highest at 24 h, and maintained through to 48 h, then decreased slowly. The fluorescent signal in the tumor was maintained until 72 h, showing an excellent in vivo NIFI effect. Moreover, the result also demonstrated that the DIST NPs could significantly prolong the internal circulation time of IR780. This property is crucial for the application of in vivo monitoring. After euthanasia, the residual signals in the main organs and tumors were detected ( Figure 6B ). There are only weak signals in the lungs and tumors of the mouse injected with IR780. In contrast, the DIST NPs-treated mice have apparent signals in the tumors and livers. However, the signals of metastatic lesions and spleen are relatively low. The results show that the DIST NPs can prolong the circulation time of drugs and can be effectively enriched in liver tumors, showing a good NIFI ability.

PAI results are shown in Figure 7A . After injection, no obvious PA signal was found in the tumor area of the two mice. Later, the PA signal of the IR780-treated mice was consistently weak. In contrast, the PA signal of the DIST NPs-treated mice gradually gathered in the tumor over time. At 12 h, an obvious PA signal was detected in the tumor. At 24 h, the tumor PA signal was further enhanced. The PA signal reached its highest point at 36 h and began to drop gradually after that. At 48 h, there was still a weak signal in the tumor area. In this case, the PA signal did not only originate from IR780, but also came from PDA. The PDA was gradually degraded in the tumor tissue; therefore, the weakening of the signal was consequent on the decomposition of PDA. The quantitative results of the PA signal intensity are shown in Figure 7B . The PA signal changing trends of the two groups were significantly different. The signal of the DIST NPs group is much higher than that of the free IR780 group, even the PDA has been degraded. The results show that the DIST NPs can effectively realize tumor PAI.

The antitumor effect of DIST NPs in vivo was evaluated by the subcutaneous tumor model of hepatic carcinoma. Figure 8A shows the tumor growth curve. The graph indicates that the free PTX-treated mice did not show a significant antitumor effect at the dose of 1 mg/kg. IR780 also failed to inhibit tumor growth even with the 808-nm laser irradiation assistance, leaving only a few burning marks on some tumors. A similar situation prevails in the chemotherapy + PTT combined treatment group, there was no apparent inhibitory effect either. No significant difference in the tumor growth rate was found between the above three groups and the normal saline group. In contrast, the DIST NPs group showed an excellent antitumor effect. The tumors of four of the five mice were completely ablated. The average tumor volume was close to the lowest value after 10 d. Figure 8B shows the photographs of all the mice during the treatment. The tumors of mice in the DIST NPs group showed burning signs very early. After the scab, the treated area had recovered. However, the tumors of the other four groups kept on growing. Figure 8C shows the photographs of all the tumors taken out of the mice after euthanasia. Only very tiny tumors were found in the DIST NPs group. There is no significant tumor volume difference between the other four groups. The tumor weight results ( Figure 8D ) also show the same scenario. In addition, the average body weight of mice in the DIST NPs group was decreased in the first week, and then it continuously increased until the end of the experiment. However, the bodyweight of mice in the other groups decreased significantly ( Figure 8E ). These results further prove the therapeutic effect and in vivo safety of the DIST NPs.