Biodegradable black phosphorus-based nanospheres for in vivo photothermal cancer therapy

Photothermal therapy (PTT) offers many advantages such as high efficiency and minimal invasiveness, but clinical adoption of PTT nanoagents have been stifled by unresolved concerns such as the biodegradability as well as long-term toxicity. Herein, poly (lactic-co-glycolic acid) (PLGA) loaded with black phosphorus quantum dots (BPQDs) is processed by an emulsion method to produce biodegradable BPQDs/PLGA nanospheres. The hydrophobic PLGA not only isolates the interior BPQDs from oxygen and water to enhance the photothermal stability, but also control the degradation rate of the BPQDs. The in vitro and in vivo experiments demonstrate that the BPQDs/PLGA nanospheres have inappreciable toxicity and good biocompatibility, and possess excellent PTT efficiency and tumour targeting ability as evidenced by highly efficient tumour ablation under near infrared (NIR) laser illumination. These BP-based nanospheres combine biodegradability and biocompatibility with high PTT efficiency, thus promising high clinical potential.

reported previously 1,2 and BSA is used to conjugate with the AuNRs using a method described previously 2,3 . (c) Relative viability of the MCF7 cells after incubation with BPQDs/PLGA NSs and AuNRs with different concentrations (same absorption at 808 nm) for 4 h and irradiated with the 808 nm laser (1 W/cm 2 ) for 10 min. (d) Corresponding fluorescence images of the cells stained with calcein AM (live cells, green fluorescence) and PI (dead cells, red fluorescence).

Added in the revised manuscript (Line 2, Page 10):
"In the next step, the PTT efficiency of the BPQDs/PLGA NSs was compared with that of gold nanorods (AuNRs), one of the common photothermal agents. On account of the large NIR extinction coefficient and high photothermal conversion efficiency of the BPQDs 39 , the BPQDs/PLGA NSs are more efficient in increasing the solution temperature than the AuNRs ( Supplementary Fig. 6). In the cell photothermal experiments, both of the NSs (containing only 10 ppm of BPQDs) and AuNRs (72.4 ppm) can kill the cancer cells almost completely, but it is clear that less BPQDs are needed. These results confirm the suitability of BPQDs/PLGA NSs as an efficient PTT agent."

Added in the revised manuscript (Line 13, Page 14):
"The AuNRs were employed as a positive control in the photothermal experiments ( Supplementary Fig.   9). Under the same irradiation condition, the tumor temperature of the mice injected with 100 μL of the AuNRs (3.62 mg/mL) increases to 54.4 °C, which is lower than that induced by the NSs. These results indicate the high efficiency of the BPQDs/PLGA NSs as a PTT agent for in vivo tumor ablation." Added in the "Methods" section (Line 7, Page 21): "The AuNRs were employed as a positive control in the in vitro photothermal experiments. The MCF7 cells was incubated with AuNRs (concentrations of 0, 7. 2, 18.1, 36.2, and 72.4 ppm) for 4 h at 37 °C and then irradiated with the 808 nm laser (1 W/cm 2 ) for 10 min. The corresponding fluorescence images of the cells and cell viability were assessed by the above method."  "In vivo biodistribution. Since significant amounts of P exist in the animal body, it is very difficult to direct obtain biodistribution information of the BP-based materials ( Supplementary Fig. 7). Therefore, in order to study the in vivo behavior of the BPQDs/PLGA NSs, Cy5.5, a commonly used NIR fluorescent dye 61 was utilized to label the BPQDs/PLGA NSs by entrapping it into the NSs using the oil-in-water emulsion solvent evaporation method mentioned above. The synthesized Cy5.5-labeled BPQDs/PLGA NSs with similar size of the BPQDs/PLGA NSs exhibit bright and stable fluorescence at about 695 nm ( Supplementary Fig. 8) enabling non-invasive monitoring and quantitative examination of the NSs biodistribution in the mice. Hence, the Balb/c nude mice bearing MCF7 breast tumors are intravenously injected with the Cy5.5-labeled BPQDs/PLGA NSs (100 μL of 1 mg BP/mL for each mouse) for the biodistribution examinations.
The pharmacokinetics profile of the Cy5.5-labeled BPQDs/PLGA NSs was examined by fluorometry to determine the concentrations in blood at different time intervals post-injection (Fig. 6a). Blood circulation of the NSs obeys the typical two compartment model. After the first phase (distribution phase, with a rapid decline) with a half-life of only 1.50 ± 0.21 h, the NSs in circulating blood exhibit a long second phase (elimination phase, the predominant process for NSs clearance) with a half-life of 22.66 ± 3.65 h. The volume of distribution (V) is measured to be 2.31 ± 0.72 mL and the area under curve (AUC) is 0.65 ± 0.11 mg·h/mL. The long blood circulation of the NSs delays the macrophage clearance in reticuloendothelial systems (RES) 62 , favoring enhanced tumor targeting by the EPR effect.
The biodistribution of the Cy5.5-labeled BPQDs/PLGA NSs in the mice is directly observed by fluorescence imaging. As shown in Fig A quantitative biodistribution analysis of the Cy5.5-labeled BPQDs/PLGA NSs in mice is conducted (Fig. 6e). The tumor and major organs were collected from the mice, weighed, and solubilized by a lysis buffer at different time intervals post-injection. The homogenized tissue lysates were diluted and measured by fluorometry to quantitatively determine the NSs concentrations. At 24 h post-injection, large NSs concentrations can be found from not only the tumor, but also organs including the liver, spleen, and kidney as consistent with the above ex vivo fluorescence examination. Uptake of the NSs by the liver and spleen may be due to RES absorption 62 , while the kidney uptake can be associated with possible renal excretion 63 . Even so, considerable uptake of the NSs by the tumor can be achieved on account of the EPR effect 21 ." "Pharmacokinetic and biodistribution analysis. In the biodistribution and pharmacokinetic analysis, fluorescent labeled BPQDs/PLGA NSs were prepared by adding 0.1 mg/mL of Cy5.5 to the BPQDs/PLGA solution in DCM followed by oil-in-water emulsion solvent evaporation described above.
The excess dye molecules were removed by centrifugation and washed away with water more than 5 times until no noticeable color change was observed from the supernatant fluids followed by resuspension in PBS.
The female Balb/c nude mice (6 weeks old) were purchased from Slac Laboratory Animal Co.Ltd (Hunan, China). In the pharmacokinetic analysis, blood circulation was assessed by drawing 10 μL of blood from the tail veins of the Balb/c nude mice at certain time intervals post-injection of the Cy5.5-labeled BPQDs/PLGA NSs. Each blood sample was dissolved in 1 mL of the lysis buffer (the same as the above used) and the concentration of the NSs in the blood was determined from the fluorescence spectrum acquired on a Fluoromax 4 fluorometer (Horiba Jobin Yvon, France). A series of dilutions of the was performed to obtain a standard calibration curve. The blank blood sample without injection was measured to determine the blood auto-fluorescence level, which was subtracted from the fluorescence intensities of injected samples during concentration calculation. The pharmacokinetic parameters such as half-life (t 1/2 ), V, and AUC were determined using a Microsoft add-in tool, PKSolver 67 .
In the in vivo fluorescence imaging experiments, the Balb/c nude mice bearing MCF7 breast tumors were intravenously injected with the Cy5.5-labeled BPQDs/PLGA NSs (100 μL of 1 mg BP/mL for each mouse) and examined by a fluorescence (Xenogen IVIS-Spectrum) imaging system as a function of time for up to 48 h. NIR light with a peak wavelength of 675 nm was used as the excitation source and in vivo spectral imaging with the Cy5.5 bandpass emission filter (680 nm to 720 nm) was carried out for an exposure time of 200 ms for each image frame. All the images were captured using identical system settings and auto-fluorescence was removed using the spectral unmixing software.
In the ex vivo fluorescence imaging experiments, the NSs-treated mice were sacrificed by cervical dislocation and the corresponding major organs and tissues including the liver, spleen, kidney, heart, stomach, lung, intestine, and tumor were collected and imaged immediately afterwards. The tumors were fixed in 10% neutral buffered formalin and embedded in paraffin. Sections of whole tumor were stained using DAPI (shown in blue) to label all nuclei of tumor cells. The fluorescence images of the tumor sections were acquired on the Leica DM4000B fluorescence microscope (Leica, Nussloch, Germany).
In the quantitative biodistribution analysis, the NSs-treated mice were sacrificed and the organs/tissues were weighed and solubilized by a lysis buffer (1% SDS, 1% Triton X-100, 40 mM Tris Acetate) using a PowerGen homogenizer (Fisher Scientific). The clear homogeneous tissue lysates were diluted 100 times to avoid light scattering and self-quenching during fluorescence measurement. The fluorescence intensities of both the standard samples and real tissues were adjusted to be in the linear range by appropriate dilution and subjected to fluorometry to quantitatively determine the NSs concentrations. The organs and tissues from a control mouse without injection of the NSs were collected and used as controls to subtract the autofluorescence background in various tissues. The samples were measured in triplicate to ensure reproducibility and accuracy. The biodistribution of the NSs in the various organs of the mice was calculated and presented as the percentage of injected dose per gram of tissue (%ID/g)."

Added in the revised manuscript (Line 3, Page 16):
"The anticancer efficiency is further analyzed by a TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling) assay, which is generally utilized to detect the intratumoral levels of apoptosis. As shown in Supplementary Fig. 12, no or few TUNEL-positive cells (shown in green) are observed from the PBS, PLGA, and BPQDs groups, while significant colocalization of nuclei (DAPI staining, shown in blue) and TUNEL-positive apoptotic cells (shown in green) can be observed from the BPQDs/PLGA NSs group. Moreover, the apoptosis examination at macro-organizational level (about 10 mm 2 ) shows that the TUNEL-positive apoptotic cells are distributed throughout the tumor section ( Supplementary Fig. 13). These results indicate that the NSs-mediated PTT can induce cancer cell death by activating apoptosis in the tumor. " Added in the "Methods" section (Line 1, Page 25): "Apoptosis detection. The tumors were collected from the Balb/c nude mice treated with PBS, PLGA NSs, BPQDs and BPQDs/PLGA NSs 24 h after the treatment. The individual tumors were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 5 micrometers, and stained using the TUNEL technique using the In Situ Cell Death Detection Kit (Roche Applied Science, Germany). The experimental procedures were in accordance with the manufacturer's instructions. DAPI was used to stain the sections in the absence of light to label the apoptotic cells and cellular DNA. The fluorescence images were taken on the Leica DM4000B fluorescence microscope (Leica, Nussloch, Germany)." Comment 5: The in vivo study was done against MCF7 tumors for 14 days and the mice showed tumor free survival for 40 days. However, the pictures in figure 5c do not support these observations as the mice treated with the particles still show large tumors. Moreover, MCF7 tumor model may not be appropriate for studying photothermal therapy, as it is prepared in mice with a deficient immune system.
Thus, the activity of the particles should be studied in a second model with competent immune system and relevant to photothermal therapy to validate the applicability of the particles. Figure 5c show the black scar induced by the PTT at the tumor site, which may be mistaken for a "large tumor". To avoid such misunderstanding, we have prolonged the examination time until the scar at the tumor site is completely cured. Moreover, BALB/c mice with a competent immune system have been added as another animal model (for another major cancer: B16 melanoma tumors) to examine the applicability of the nanospheres-mediated photothermal therapy.

Reply 5: The original pictures in
Corresponding in vitro experiments of B16 melanoma cells are also added. "To further demonstrate the applicability of the NSs-mediated PTT, BALB/c mice, with a competent immune system, were employed as another animal model in the photothermal treatments. As shown in Supplementary Fig. 11, the BPQDs/PLGA NSs also exhibit excellent photothermal efficacy to kill the melanoma tumor in the BALB/c mice without causing obvious toxic side effects. The results demonstrate that the NSs-mediated PTT is suitable for such two kinds of animals with different immune systems." "The depth of photothermal damage is investigated in the tumor bearing nude mice with the tumor volume as large as 1000 mm 3 . After the photothermal treatment, intratumoral apoptosis of the tumor sections at different depths was detected by a TUNEL assay (Supplementary Fig. 14). Most cancer cells undergo apoptosis in the tumor sections at depths of no more than 6 mm. Although evident depth-dependent decay of the PTT efficiency is observed when the depth is over 6 mm, significant apoptosis of cells can still be found from the section at the depth of 10 mm. The considerable photothermal damage to deep tissues stems from the excellent PTT efficiency of the NSs and high tissue penetration ability of NIR light. It should be noted that although the penetration depth of NIR light is limited to be no more than 10 mm 21 , clinical photothermal treatment of deep tumors is still achievable with the aid of specialized medical devices such as endoscopic ones in combination with optical fibers as well as implanted NIR devices 65,66 ."

Added in the "Methods" section (Line 7, Page 25):
"To further investigate the depth of the photothermal damage, the tumor bearing nude mice with a tumor volume as large as 1000 mm 3 was illuminated with the 808 nm NIR laser (1 W/cm 2 ) for 10 min.

Comment 7:
The toxicity from the particles was completely ignored. The authors should demonstrate the safety of these particles without irradiation, with irradiation, and by exposing the mice to daylight (as a main issue for photosensitizer molecules is the side effects arising after sun exposure).

Reply 7:
We have examined the in vivo toxicity of the nanospheres by performing the hematological, blood biochemical, and histological analyses. Acoording to your suggestion, the mice in these examinations have been randomly divided into 4 groups and subjected to variable conditions, including: (1) Control group without any treatment, (2) NSs directly intravenously injected into the mice, (3) NSs intravenously injected into the mice after 808 nm laser irradation for 10 min, and (4) NSs intravenously injected into the mice which are then exposed to artificial daylight for 24 h. The corresponding results and discussion have beed added to the revised manuscript. "In vivo toxicity. The in vivo toxicology of the BPQDs/PLGA NSs is investigated systematically. Sixty healthy female Balb/c mice (6 weeks old) were randomly divided into 4 groups and subjected to variable conditions, including: (1) Control group without any treatment, (2) NSs directly intravenously injected into the mice, (3) NSs intravenously injected into the mice after 808 nm laser irradiation for 10 min, and (4) NSs intravenously injected into the mice which are then exposed to artificial daylight for 24 h. The injection dose of the NSs is about 10 mg BP/kg and hematological, blood biochemical, and histological analyses were performed at time points of 1, 7, and 28 days post-injection.
The standard hematology markers including the white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets (PLT), and hematocrit (HCT) were measured ( Fig. 5a). Compared to the control group, all the parameters in the three NSs-treated groups at all time points appear to be normal and the differences between are not statistically significant (P value > 0.05).
These results indicate that the BPQDs/PLGA NSs do not cause obvious infection and inflammation in the treated mice 59 .
Blood biochemical analysises were carried out and various parameters including alanine transaminase (ALT), aspartate transaminase (AST), total protein (TP), globulin (GLB), total bilirubin (TBIL), blood urea nitrogen (BUN), creatinine (CREA), and albumin (ALB) were examined (Fig. 5b). Compared to the control group, no meaningful difference can be observed from the three NSs-treated groups at all time points. Hence, the NSs treatment does not affect the blood chemistry of mice. Furthermore, since ALT, AST, and CREA are closely related to the functions of the liver and kidney of mice 59 , the results demonstrate that the NSs induce no obvious hepatic and kidney toxicity in mice.
Finally, the corresponding histological changes of organs were checked by immunohistochemistry using major organs including the liver, spleen, kidney, heart, and lung collected and sliced for hematoxylin and eosin (H&E) staining (Fig. 5c). No noticeable signal of organ damage can be observed during the whole treatment period from all the groups suggesting no apparent histological abnormalities or lesions in the NSs-treated groups for the test dose.
According to above analyses, inappreciable toxicity is observed from the BPQDs/PLGA NSs regardless of NIR laser irradiation. Even if the NSs-treated mice are under artificial daylight illumination for 24 h, no significant toxic side effects can be found, indicating that the NSs induce no evident phototoxicity which has generally been observed from many photosensitizer molecules 60  to variable conditions. This include: (1) Control group without any treatment, (2) NSs directly intravenously injected into the mice, (3) NSs intravenously injected into the mice after 808 nm laser irradiation for 10 min, and (4) NSs intravenously injected into the mice which are then exposed to artificial daylight for 24 h. The mice were sacrificed at various time points after injection (1, 7, and 28 days, five mice per group at each time point). About 0.8 mL of blood were collected from each mouse to conduct complete blood panel analysis and serum biochemistry assay at the Shanghai Research Center for Biomodel Organism. The major organs (liver, spleen, kidney, heart, and lung) were harvested, fixed in 10% neutral buffered formalin, processed routinely into paraffin, sectioned at 8 μm, stained with H&E, and examined by digital microscopy."

Comment 8:
The authors mentioned that the higher efficacy of their particles over the BPQDs is because the 100 nm size of the nanoparticles is more appropriate for efficient tumor targeting compared to the ultrasmall size of BPQDs (3 nm). However, this observation is biased from the experimental setting. Accordingly, the dose and time for irradiation (24 h after injection) of the antitumor experiment were more suitable for the nanoparticles than for the BPQDs. If the experiment is performed with higher BPQDs doses and earlier irradiations, the results could provide better antitumor effect for BPQDs than for PLGA/BPQDs, making the development of PLGA/BPQDs unnecessary. The authors should demonstrate that at the optimal conditions for each formulation, i.e. dose and irradiation timing for BPQDs and PLGA/BPQDs, the PLGA/BPQDs still perform better than BPQDs.
Reply 8: Per your suggestion, we have examined the time-dependent temperature increase in the tumor-bearing nude mice after separate injection of the BPQDs and BPQDs/PLGA NSs with different concentrations (0.5, 1.0, 2.0 and 3.0 mg BP/mL) and irradiated with the NIR laser at different times (1,4,8,12,24 and 48 h) post-injection. The corresponding results have beed added to the revised manuscript. Supplementary Information (Supplementary Figure 10 Time-dependent temperature increase in the tumor-bearing nude mice after separate intravenous injection of the BPQDs and BPQDs/PLGA NSs with different concentrations (0.5, 1, 2 and 3 mg BP/mL) and irradiation with the 808 nm laser (1 W/cm 2 ) at different time points (1, 4, 8, 12, 24 and 48 h) after injection. The color bars refer to the relative temperature.

Added in the revised manuscript (Line 17, Page 14 to Line 6, Page 15):
"To further evaluate the influence of PLGA encapsulation on the in vivo PTT efficiency of the BPQDs, the BPQDs and BPQDs/PLGA NSs with different concentrations (0.5, 1.0, 2.0 and 3.0 mg BP/mL) were injected intravenously into the tumor-bearing nude mice, which were irradiated with the NIR laser at different times (1,4,8,12,24 and 48 h) post-injection. As shown in Supplementary Fig. 10, the BPQDs/PLGA NSs produce larger tumor temperature increase than the bare BPQDs under all conditions. The better in vivo PTT efficiency of the BPQDs/PLGA NSs than bare BPQDs pertaining to tumor ablation can be attributed to two factors. Firstly, the BPQDs/PLGA NSs have better stability than the bare BPQDs and so can maintain the photothermal performance during circulation in the body.
Secondly, compared to the ultrasmall BPQDs with a size of about 3 nm 16 , the size of the BPQDs/PLGA NSs of about 100 nm is more appropriate for efficient tumor targeting and retension during the long blood circulation in the body."

Added in the "Methods" section (Line 12, Page 24):
"To further compare the photothermal effects between the BPQDs and BPQDs/PLGA NSs in details, photothermal experiments with different injection concentrations (0.5, 1, 2 and 3 mg BP/mL) and irradiation time (1,4,8,12,24 and 48 h post-injection) were performed. The temperature of the tumors and infrared thermographic maps were obtained by the infrared thermal imaging camera. " Comment 9: English should be carefully revised. There are too many misspellings throughout the manuscript.

Reply 9:
We have revised the manuscript carefully to correct misspelling.

Replies to the 3 rd reviewer's comments (NCOMMS-16-03858)
Comment 1: The data cannot support the conclusions very well, especially the excellent tumor targeting ability and biocompatibility of BPQDs/PLGA nanosphere in vivo. In addition, the in vivo studies on the unique biodegradability and excellent biocompatibility of BPQDs/PLGA nanosphere are important for its further application. Therefore, the distribution and metabolism of BPQDs/PLGA nanosphere in mice should be investigated.  "In vivo biodistribution. Since significant amounts of P exist in the animal body, it is very difficult to direct obtain biodistribution information of the BP-based materials ( Supplementary Fig. 7). Therefore, in order to study the in vivo behavior of the BPQDs/PLGA NSs, Cy5.5, a commonly used NIR fluorescent dye 61 was utilized to label the BPQDs/PLGA NSs by entrapping it into the NSs using the oil-in-water emulsion solvent evaporation method mentioned above. The synthesized Cy5.5-labeled about 695 nm ( Supplementary Fig. 8) enabling non-invasive monitoring and quantitative examination of the NSs biodistribution in the mice. Hence, the Balb/c nude mice bearing MCF7 breast tumors are intravenously injected with the Cy5.5-labeled BPQDs/PLGA NSs (100 μL of 1 mg BP/mL for each mouse) for the biodistribution examinations.
The pharmacokinetics profile of the Cy5.5-labeled BPQDs/PLGA NSs was examined by fluorometry to determine the concentrations in blood at different time intervals post-injection (Fig. 6a) A quantitative biodistribution analysis of the Cy5.5-labeled BPQDs/PLGA NSs in mice is conducted (Fig. 6e). The tumor and major organs were collected from the mice, weighed, and solubilized by a lysis buffer at different time intervals post-injection. The homogenized tissue lysates were diluted and measured by fluorometry to quantitatively determine the NSs concentrations. At 24 h post-injection, large NSs concentrations can be found from not only the tumor, but also organs including the liver, spleen, and kidney as consistent with the above ex vivo fluorescence examination. Uptake of the NSs by the liver and spleen may be due to RES absorption 62 , while the kidney uptake can be associated with possible renal excretion 63 . Even so, considerable uptake of the NSs by the tumor can be achieved on account of the EPR effect 21 .
Since the NSs in the physiological medium can maintain their integrity (Fig. 3c,d) without causing evident fluorescence decrease of the entrapped Cy5.5 ( Supplementary Fig. 8) for 7 days, the fluorescence examinations were further used to estimate the time-dependent residual amounts of the NSs in mice during the 7 days post-injection. The residual ratios were calculated by normalizing the total residual amounts in these organs and tissues to initial total amounts. It can be calculated that the residual ratio of the NSs decreases from 90.1%ID/g at day 1 (24 h) to only 29.9%ID/g at day 7, suggesting the possibility of the NSs to be partially metabolized. It is known that such nanoparticles is generally difficult to be completely metabolized and excreted from the body directly. However, the aforementioned biodegradability of the NSs enables harmless clearance from the body in a reasonable period of time (for example, several months)." "Pharmacokinetic and biodistribution analysis. In the biodistribution and pharmacokinetic analysis, fluorescent labeled BPQDs/PLGA NSs were prepared by adding 0.1 mg/mL of Cy5.5 to the BPQDs/PLGA solution in DCM followed by oil-in-water emulsion solvent evaporation described above.
The excess dye molecules were removed by centrifugation and washed away with water more than 5 times until no noticeable color change was observed from the supernatant fluids followed by resuspension in PBS.
The female Balb/c nude mice (6 weeks old) were purchased from Slac Laboratory Animal Co.Ltd (Hunan, China). In the pharmacokinetic analysis, blood circulation was assessed by drawing 10 μL of blood from the tail veins of the Balb/c nude mice at certain time intervals post-injection of the Cy5.5-labeled BPQDs/PLGA NSs. Each blood sample was dissolved in 1 mL of the lysis buffer (the same as the above used) and the concentration of the NSs in the blood was determined from the fluorescence spectrum acquired on a Fluoromax 4 fluorometer (Horiba Jobin Yvon, France). A series of dilutions of the was performed to obtain a standard calibration curve. The blank blood sample without injection was measured to determine the blood auto-fluorescence level, which was subtracted from the fluorescence intensities of injected samples during concentration calculation. The pharmacokinetic parameters such as half-life (t 1/2 ), V, and AUC were determined using a Microsoft add-in tool, PKSolver 67 .
In the in vivo fluorescence imaging experiments, the Balb/c nude mice bearing MCF7 breast tumors were intravenously injected with the Cy5.5-labeled BPQDs/PLGA NSs (100 μL of 1 mg BP/mL for each mouse) and examined by a fluorescence (Xenogen IVIS-Spectrum) imaging system as a function of time for up to 48 h. NIR light with a peak wavelength of 675 nm was used as the excitation source and in vivo spectral imaging with the Cy5.5 bandpass emission filter (680 nm to 720 nm) was carried out for an exposure time of 200 ms for each image frame. All the images were captured using identical system settings and auto-fluorescence was removed using the spectral unmixing software.
In the ex vivo fluorescence imaging experiments, the NSs-treated mice were sacrificed by cervical dislocation and the corresponding major organs and tissues including the liver, spleen, kidney, heart, stomach, lung, intestine, and tumor were collected and imaged immediately afterwards. The tumors were fixed in 10% neutral buffered formalin and embedded in paraffin. Sections of whole tumor were stained using DAPI (shown in blue) to label all nuclei of tumor cells. The fluorescence images of the tumor sections were acquired on the Leica DM4000B fluorescence microscope (Leica, Nussloch, Germany).
In the quantitative biodistribution analysis, the NSs-treated mice were sacrificed and the organs/tissues were weighed and solubilized by a lysis buffer (1% SDS, 1% Triton X-100, 40 mM Tris Acetate) using a PowerGen homogenizer (Fisher Scientific). The clear homogeneous tissue lysates were diluted 100 times to avoid light scattering and self-quenching during fluorescence measurement. The fluorescence intensities of both the standard samples and real tissues were adjusted to be in the linear range by appropriate dilution and subjected to fluorometry to quantitatively determine the NSs concentrations. The organs and tissues from a control mouse without injection of the NSs were collected and used as controls to subtract the autofluorescence background in various tissues. The samples were measured in triplicate to ensure reproducibility and accuracy. The biodistribution of the NSs in the various organs of the mice was calculated and presented as the percentage of injected dose per gram of tissue (%ID/g)." can kill the cancer cells almost completely, but it is clear that less BPQDs are needed. These results confirm the suitability of BPQDs/PLGA NSs as an efficient PTT agent."  9). Under the same irradiation condition, the tumor temperature of the mice injected with 100 μL of the AuNRs (3.62 mg/mL) increases to 54.4 °C, which is lower than that induced by the NSs. These results indicate the high efficiency of the BPQDs/PLGA NSs as a PTT agent for in vivo tumor ablation." Added in the "Methods" section (Line 7, Page 21): "The AuNRs were employed as a positive control in the in vitro photothermal experiments. The MCF7 cells was incubated with AuNRs (concentrations of 0, 7. 2, 18.1, 36.2, and 72.4 ppm) for 4 h at 37 °C and then irradiated with the 808 nm laser (1 W/cm 2 ) for 10 min. The corresponding fluorescence images of the cells and cell viability were assessed by the above method." Added in the "Methods" section (Line 8, Page 24): "The AuNRs (100 μL, 3.62 mg/mL) were employed as a positive control in the photothermal