Nanoparticles from Ancient Ink Endowing a Green and Effective Strategy for Cancer Photothermal Therapy in the Second Near-Infrared Window.

Photothermal therapy (PTT) in the second near-infrared window (NIR-II, 1000-1350 nm) has presented great superiority in cancer treatment recently. However, it is generally limited to a few photothermal agents and most of them often suffer from intricate design and complicated synthesis. Herein, by subtly extracting nanoparticles from ancient ink (AINPs), a versatile AINP dispersion with definite ingredients, good biosafety, and excellent photothermal effect in the NIR-II window was obtained. In vivo trials demonstrated that the obtained AINP dispersion provides a promising alternative for tumor sentinel lymph node (SLN) mapping. Besides, under the guidance of photoacoustic imaging, the metastatic SLNs could be accurately eliminated by NIR-II laser irradiation. The preliminary biosafety of AINP dispersion has also been systematically confirmed. Therefore, we believe this work would provide a green and effective strategy for PTT of tumor in the NIR-II window.

Introduction

Photothermal therapy (PTT), which utilizes focused hyperthermia by photothermal agents (PTAs) under near-infrared (NIR) laser irradiation, is a clinically promising strategy in tumor treatments.[1−3] Different from traditional cancer treatments, PTT exhibits numerous advantages including simple procedures, noninvasive, and high spatiotemporal selectivity.[4,5] In the past decade, a large number of different nanomaterials have been investigated as PTAs for PTT of cancer.[6−13] However, most of the previous work primarily focused on PTT in the first NIR window (NIR-I, 700–950 nm), which often suffered from the limited penetration depth.

In comparison with the NIR-I window, the second NIR window (NIR-II, 1000–1350 nm) is more feasible for clinical PTT due to the deeper tissue penetration and larger maximum permissible exposure (MPE) dose to human skin (0.33 W/cm2 for 808 nm laser while 1 W/cm2 for 1064 nm laser).[14−16] Therefore, massive efforts have been devoted to exploring novel PTAs for NIR-II PTT recently.[17−24] For example, by controlling the interfaces of noble metal and adjusting the coupling effect of localized surface plasmon resonance, Jiang et al. successfully constructed a dual plasmonic Au–Cu9S5 hybrid nanostructure, which exhibited strong absorbance during the NIR-II region.[17] Shi et al. also developed a liquid exfoliation method and prepared a two-dimensional niobium carbide for PTT of cancer in both the NIR-I and the NIR-II window.[23] Very recently, some organic molecules or semiconducting polymers composed of electron-delocalized π-conjugated structures have also demonstrated great potential in the NIR-II PTT of cancer.[25−29] However, for future clinical translation, these artificial nanomaterials often suffer from elaborate design and complicated synthesis, which limit their industrial development. From this point, some natural nanomaterials such as humic acid,[30] human hair,[31] endogenous biliverdin,[32] and cuttlefish ink[33] with easy synthetic procedures, good biocompatibility, and excellent therapeutic efficiency may become new candidate agents for future clinical applications, although their PTT still presented a poor efficiency in the NIR-II window.

Ancient ink, as a glorious calligraphy material derived from the incomplete combustion of natural plant, has been used for centuries and played an important role in the creation of human civilization.[34] The benefits of the ancient ink such as the nanosized structure, carbon element composition, and its formation mechanism of resonance-stabilized hydrocarbonradical chain reactions were gradually revealed by the efforts of contemporary scientists.[35,36] It subsequently opens a promising prospect for diverse applications including flexible batteries,[37] solar energy conversion,[38] and three-dimensional electrodes.[39] Besides, in many world-renowned medical books, such as Compendium of Materia Medica,[34] ancient ink was ever recorded as a nontoxic medicinal herb for hemostasis, which convincingly demonstrates that it possesses excellent biocompatibility. However, further applications of ancient ink for modern biomedicine, especially for PTT of cancer in the NIR-II window, have not yet been explored to the best of our knowledge.[35]

Herein, inspired by our tentative discovery that ancient ink manifested a distinct wide absorption band even in the NIR-II region like most other PTAs, a new conceptual biomedical application of ancient ink for NIR-II PTT of cancer was thus proposed. As shown in Scheme , the ancient ink nanoparticles (AINPs) were first extracted using the traditional ink production process and then modified with poly(vinylpyrrolidone) (PVP), a Food and Drug Administration (FDA) approved pharmaceutical adjuvant,[40] to obtain the final AINP dispersion through simple hydrothermal treatment. Interestingly, the AINP dispersion that we obtained presents certain ingredients (only ink nanoparticles and PVP), good biocompatibility, and excellent photothermal performance in the NIR-II window. More importantly, it proposes a very promising strategy for sentinel lymph node (SLN) mapping and NIR-II PTT of tumors in future clinical practice and promotes further exploration of biomedical applications of ancient ink.

Scheme 1

Illustration of Nanoparticles from Ancient Ink (AINPs) for Cancer Sentinel Lymph Node (SLN) Mapping and Photothermal Therapy (PTT) in the Second Near-Infrared (NIR-II) Window

As revealed by photoacoustic (PA) imaging, AINP dispersion after being injected to the primary tumor on the right hind foot pad could effectually transfer into the SLNs via the lymphatic vessels. NIR-II PTT was then implemented by exposing SLNs to a 1064 nm laser.

Results and Discussion

Preparation and Characterization of AINP Dispersion

The raw AINPs prepared by incomplete combustion of pinewood or tung oil in kiln are generally of soot form with a deep black color (Scheme ).[34] To explore the elemental constituents of AINPs, X-ray photoelectron spectroscopy (XPS) was first conducted. As confirmed in Figure a, the AINPs mainly consisted of carbon and oxygen elements, and the carbon element dominated the constituents with up to 90.5%. From the X-ray diffraction (XRD) pattern (Figure b), it revealed a similar broad peak at ≈25°, which was consistent with the carbon crystal plane (002), which was a typical characteristic of graphitic carbon materials.[8,37] Then, a Raman spectroscope, a general instrument to study the carbonaceous materials, was subsequently used to investigate its structural feature. As shown in Figure c, the D band (∼1300 cm–1) and G band (∼1600 cm–1) peaks were clearly observed, which confirmed the constituents of both amorphous carbon and graphene sheet-like structure in AINPs.[41] From the Fourier transform infrared (FT-IR) spectrum (Figure S1), it also indicates the presence of −OH (3350–3570 cm–1), C–H (2835 and 2916 cm–1), C=O (1632 cm–1), and C–O–C (1091 cm–1) functional groups on the surface of AINPs. To explore the nanoparticle morphology and features of AINPs, scanning electron microscopy (SEM) was then carried out. SEM images (Figure d) revealed a morphology in good accord with our desired nanosized structure, and the corresponding element-mapping images showed a uniform distribution of elements C (Figure e) and O (Figure f) in AINPs. However, the severe aggregation, as shown in SEM images (Figure d), and the poor water dispersibility of AINPs (Figure g) limited their further applications, especially in biological medicine.[42]

Figure 1

Characterization of AINP dispersion. (a) XPS, (b) XRD, and (c) Raman spectrum of AINPs. (d) SEM and its corresponding (e) carbon and (f) oxygen element distribution of AINPs, scale bar: 200 nm. (g) Photographs of raw AINPs and AINP dispersion (by PVP modification) in different physiological environments after 3 days. (h) Transmission electron microscope (TEM) photograph of AINP dispersion. (i) Size distribution of AINP dispersion by dynamic light scattering (DLS).

To solve this problem, PVP, a widely used stabilizer approved by the FDA was then used.[40] AINP dispersion was obtained by simple sonication and subsequent hydrothermal treatment of AINPs and PVP. Compared with the original AINPs, the obtained AINP dispersion by PVP modification was very stable in various physiological environments. No aggregation was observed even after storage for 3 days in water, phosphate buffer saline (PBS), and Roswell Park Memorial Institute (RPMI) 1640 (Figure g). From the transmission electron microscope (TEM) image, we could observe that the AINP dispersion primarily consists of homogeneous ink nanoparticles (Figure h). It further demonstrated that the AINPs after PVP modification presented good dispersibility in water with a hydrodynamic diameter of about 150 nm according to dynamic light scattering (DLS) (Figure i). Next, the NIR absorption ability of AINPs was investigated using a UV–vis–NIR spectrometer. From Figure a, the AINPs presented a distinct wide absorption band in the NIR region similar to other carbon nanomaterials[7−9] and still maintained intense absorption even in the NIR-II window (1000–1350 nm). Considering the higher MPE power density and the deeper tissue penetration of the NIR-II window than the traditional NIR-I window,[14−16] a 1064 nm laser was then selected to investigate its photothermal property. As shown in Figure b, the temperature of AINP dispersion increased obviously with the increasing concentration of AINPs when irradiated under a 1064 nm laser using the MPE power density (1 W/cm2). For example, the temperature could reach promptly from 25 to 70.5 °C (100 μg/mL) after continuous irradiation for 5 min, which was much higher than that of the 808 nm laser if irradiated under its corresponding MPE power density (0.33 W/cm2) (Figure c). The temperature change of AINP dispersion under different laser irradiations at power densities of 0.33 and 1 W/cm2 was also conducted, which indicated that AINPs possessed a parallel photothermal effect under 808 or 1064 nm laser irradiation using the same power density (Figure S2). To evaluate the photothermal stability of AINPs, the temperature profiles of AINP dispersion were recorded during circular heating and cooling processes. No distinct decrease was observed during the whole successive cycle experiment (Figure S3). In the meantime, insignificant variation was also manifested in the UV–vis–NIR spectra after repeated irradiation (Figure S4). Using the reported method,[43] the photothermal conversion efficiency of AINPs in 1064 nm was further calculated to be 48% (Figure S5), which was superior to many PTAs that are currently used in the NIR-II window (Table S1). Besides the desired physicochemical properties and excellent photothermal performance, long-term stability is also a highly important property when it comes to real clinical application. Interestingly, we found that the UV–vis–NIR spectral property of AINPs was quite stable during the experiment (Figure S6). It barely exhibited a change in the photothermal effect of AINPs even when stored for 90 days (Figure d), demonstrating that AINPs maintained a stable photothermal performance. The ζ-potential (Figure e) and the hydrodynamic diameter (Figure f) of AINP dispersion were also stable during the storage time, which were crucial prerequisites for clinical storage. To sum up, the prominent photothermal performance in the NIR-II window as well as the stable physicochemical properties of AINP dispersion together make it a promising PTA candidate for future cancer treatment.

Figure 2

Photothermal performance and physicochemical stability of AINP dispersion. (a) UV–vis–NIR absorption spectra of AINP dispersion (50 μg/mL). (b) Temperature variation of AINP dispersion with different concentrations exposed to a 1064 nm laser (1 W/cm2, 5 min). (c) Temperature variation of AINP dispersion (100 μg/mL) under irradiation by a 1064 nm laser (1 W/cm2, 5 min) and an 808 nm laser (0.33 W/cm2, 5 min). (d) Photothermal effect, (e) ζ-potential, and (f) size distribution change of AINP dispersion at 0th and 90th day.

Cytotoxicity and In Vitro Photothermal Therapy

According to the traditional literature on medicine, ancient ink was not only used in calligraphy but was also a good medicinal herb. That ancient ink was pungent and warm, nontoxic, and mainly used in the treatment of hemostasis was recorded in Compendium of Materia Medica, a world influential traditional medical book.[34] Hence, we speculated that the AINP dispersion (only consists of ink nanoparticles and FDA approved PVP) that we obtained would possess low pan class="Disease">toxicity and good biocompatibility. To confirm our assumption, normal cells (HEK-293T cells) and cancerous cells (CT-26 cells) were selected to verify the safety of AINP dispersion by cell counting kit-8 (CCK-8) assay. As shown in Figure a, even when the AINP concentration was up to 400 μg/mL, both HEK-293T cells and CT-26 cells still maintained over 90% cell viabilities after 24 h incubation. These consequences confirmed the negligible cytotoxicity and good biocompatibility of the obtained AINP dispersion. Next, we evaluated the PTT therapeutic efficacy of AINP dispersion in vitro on CT-26 cells. As shown in Figure b, the cells treated with various concentrations of AINPs demonstrated a distinct concentration-dependent cell viability decrease after 1064 nm laser irradiation (1 W/cm2, 5 min). For instance, the cell viability decreased over 87.4% in AINPs + 1064 nm laser group when the concentration of AINPs increased to 100 μg/mL, while it decreased only 15.4% in the same concentration under irradiation for 5 min by 808 nm laser with its corresponding MPE power density (0.33 W/cm2). This considerable difference on cell viability decrease could be primarily ascribed to the higher MPE power density of the NIR-II window than the traditional NIR-I window. For a more visual representation of PTT therapeutic efficacy, confocal laser scanning microscopy (CLSM) imaging with calcein acetoxymethyl ester (calcein-AM, labeling living cells with green fluorescence) and propidium iodide (PI, labeling dead cells with red fluorescence) staining were also carried out. From the CLSM images (Figure c), the results showed that CT-26 cells in AINPs + 1064 nm laser group presented strong red fluorescence, which indicated that the AINPs could efficiently kill tumor cells under 1064 nm laser exposure. However, cells in the control group, 1064 nm laser group, AINP group, and AINPs + 808 nm laser group presented intense green fluorescence, which demonstrated that a large proportion of cells were still alive. Meanwhile, flow cytometry was also conducted at each treatment group for quantitative analysis of cell apoptosis. As shown in Figure d, the apoptosis rate of cells in AINPs + 1064 nm laser group could reach up to 99.4%, indicating that almost all of the cell death was induced via AINP-mediated photothermal ablation. In conclusion, all of these in vitro cell experiments consistently indicated that the AINP dispersion that we obtained possessed ignorable cytotoxicity and was able to effectively kill cancer cells by its prominent photothermal ability, especially in the NIR-II window.

Figure 3

In vitro photothermal efficacy of AINP dispersion. (a) Cell viability of CT-26 cancer cells and HEK-293T normal cells after being cultured with different concentrations of AINP dispersion for 24 h. (b) Respective cell viability of CT-26 cancer cells after being treated with AINP dispersion at different concentrations followed by laser irradiation (1 W/cm2 for 1064 nm and 0.33 W/cm2 for 808 nm) for 5 min. (c) CLSM images stained with calcein-AM (green fluorescence, live cells) and PI (red fluorescence, dead cells). (d) Flow cytometry analyses of CT-26 cancer cells after different treatments. Scale bar: 100 μm.

Photoacoustic (PA) Imaging of Tumor Sentinel Lymph Nodes

As we all know, metastasis has become the biggest challenge in cancer diagnosis and treatment and is also the leading cause of cancer mortality in clinics.[44] During the early stage of metastasis, the sentinel lymph nodes (SLNs) near the primary tumor are generally the major targets of tumor cells through the lymphatic vessel.[45] Hence, it is very clinically significant to recognize the locations of SLNs and then remove them selectively to prevent further metastasis. However, the accurate and rapid location of SLNs with low tissue damage is challenging in current clinical practice and limited to few strategies such as blue dye staining, radio-colloid tracers, or just their simple combination.[46]

PA imaging has aroused great interest in the last few years, as promising imaging technology.[1,47] It is based on the thermoelastic expansion generated by the PA imaging contrast agent and subsequent acoustic wave detection.[47] Compared with conventional computed tomography (CT) and magnetic resonance imaging (MRI), PA imaging integrates deep penetration, simple operation, and high sensitivity, which presents great superiority in SLN mapping.[47,48] Interestingly, we found that most PTAs were also latent PA imaging contrast agents on account of the strong absorption in the NIR window.[8,9] As shown in Figure a, the in vitro PA imaging capacity of AINP dispersion was evaluated, and the PA signal enhanced distinctly with the increasing concentration of AINPs (Figure b). In addition, it was reported that nanoscale agents after being injected into the primary tumor could effectually migrate into the SLNs along the lymphatic vessels.[49] Therefore, AINP dispersion was supposed to be an excellent nanoprobe to locate the SLNs through PA imaging. In the following trials, Balb/c mice were inoculated with CT-26 cancer cells on the hind foot pad and allowed to develop both the primary tumors and metastatic tumors in SLNs. Subsequently, the primary tumors of these mice were injected with AINP dispersion. At the same time, the PA signals in the SLN region were recorded at various points of time (0, 10, 30, 60, 120, and 180 min) under a PA imaging scanner. As shown in Figure c, the PA signals of the SLN region gradually showed up at 10 min post the injection of AINP dispersion and exhibited increased PA signals over time, indicating the successful translocation of the injected AINPs from the primary tumor to the SLNs. The PA signal intensity in the SLN region reached its highest level at around 120 min after the primary tumors were injected with AINP dispersion. Meanwhile, the quantification value of the PA signal at the SLN region was also obtained. For instance, the PA intensity of the SLN region before and after being injected into the primary tumors for 120 min was calculated as 0.25 ± 0.03 and 2.57 ± 0.23, respectively (Figure d). After exposed by slight anatomy, the SLNs were also distinguished clearly from the surrounding tissues due to the transference and retention of dark black AINPs (Figure S7). In conclusion, the high spatial resolution of PA imaging and the subsequent black staining of SLNs offered by the AINP dispersion were confirmed to be a valid and accurate strategy for tumor SLNs mapping in future clinical practice.

Figure 4

In vitro and in vivo PA imaging. (a) PA signal intensity of AINP dispersion in vitro at various excitation wavelengths. (b) Linear relation between the PA signal in vitro versus different concentrations of AINP dispersion. (c) PA imaging and (d) intensity in sentinel lymph nodes (SLNs) at different time points. The whole image shows the right hind foot pad of nude mice and the PA positive region consistent with the SLN region. (e) Thermal images and local temperature of SLNs after being exposed to laser irradiation (1 W/cm2 for 1064 nm and 0.33 W/cm2 for 808 nm) for 10 min.

PA Imaging-Guided NIR-II PTT of Tumor SLNs

Next, PA imaging was applied to guide the PTT of SLN metastasis in colorectal tumor. The primary tumor of mice growing on the hind foot pad was first injected with a certain amount of AINP dispersion (5 mg/mL, 50 μL). At 120 min post injection, the SLNs in the popliteal site were exposed continuously to a 1064 nm laser for 10 min under its MPE power density (1 W/cm2). At the same time, an infrared imaging device was also used to record the temperature change in real time. As observed in Figure e, the regional temperature of SLNs in AINPs + 1064 nm laser group could increase quickly from 30.6 to 51.0 °C in 5 min and continued to maintain this hyperthermia for the remaining time, which was sufficient to ablate the cancer tissues.[50] Nevertheless, the temperature in the PBS group after the same laser power irradiation presented negligible temperature variation and the temperature only reached to 35.0 °C, which would hardly cause any damage to the tumor cells.[51] To further confirm the superiority of PTT in the NIR-II window, the SLNs after intratumor injection of AINP dispersion were also exposed to an 808 nm laser with its corresponding MPE power density (0.33 W/cm2). As shown in Figure e, the temperature of the SLN region only increases a little, which was much less than that of the AINPs + 1064 nm laser group (1 W/cm2). The poor performance of PTT effect in the NIR-I window could be ascribed to the lower MPE and insufficient tissue penetration, as the SLNs were generally located several millimeters beneath the skin.[51]

Afterward, we assessed the therapeutic efficacy of PTT in vivo. Mice with manifestation of SLN metastasis were randomly divided into five treatment groups: (1) control group, (2) PBS + 1064 nm laser group (1 W/cm2, 10 min), (3) AINP group, (4) AINPs + 1064 nm laser group (1 W/cm2, 10 min), and (5) AINPs + 808 nm laser group (0.33 W/cm2, 10 min). PTT was conducted at 120 min after the primary tumors were injected with AINP dispersion (5 mg/mL, 50 μL). At the 14th day, these mice were sacrificed by anesthetization, and their SLNs were then dissected. The weights of SLNs for Groups I, II, III, IV, and V were 24.66 ± 7.29, 24.74 ± 6.57, 21.58 ± 2.48, 8.18 ± 1.51, and 19.9 ± 7.41 mg, respectively (Figure a). As expected, the noteworthy therapeutic effect was obtained in Group IV (AINPs + 1064 nm laser group) and was better than all of the other groups including the AINPs + 808 nm laser group. To further evaluate the therapeutic efficacy of PTT, hematoxylin and eosin (H&E) staining was then conducted. As observed in Figure e, a mass of necrosis and pyknosis (typical thermal damage features of cells) emerged in Group IV. On the contrary, the cells with intact morphology were presented in the remaining four groups (Figure b–d,f). These results consistently demonstrated that our obtained AINP dispersion exhibited a great therapeutic effect for cancer PTT in the NIR-II window.

Figure 5

In vivo photothermal therapy. (a) Photograph and weight of sentinel lymph nodes (SLNs) from different treatments. H&E staining of SLNs from (b) control group, (c) 1064 nm laser group, (d) AINP group, (e) AINPs + 1064 nm laser group, and (f) AINPs + 808 nm laser group (1 W/cm2 for 1064 nm and 0.33 W/cm2 for 808 nm, 10 min); all of the scale bars are 100 μm. *p < 0.05, **p < 0.01, and ***p < 0.001.

Systematic In Vivo Biocompatibility Assay of AINP Dispersion

At last, to promote further clinical translation, the toxicological mechanism of AINP dispersion was also carefully conducted. Twenty healthy mice (∼4 weeks) were randomly divided into four groups, including the control and three AINP-treated groups at different doses (10, 50, and 100 mg/kg). After intravenous injection of AINP dispersion, the body weights of mice in each group were measured every 3 days. No apparent abnormal behavior and body weight loss of mice were monitored in each group (Figure S8). Afterward, these mice were sacrificed after one-mouth feeding. Their key organs were collected for H&E staining, and the blood indexes were measured for biochemical analysis. As shown in Figure , no obvious cell/tissue damage was observed in these major organs even after intravenous injection of AINP dispersion with elevated dosages up to 100 mg/kg, which was much higher than our therapeutic dose (12.5 mg/kg) and that of other nanoparticles’ reported dose.[11,20] The major blood indexes of the mice including alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CREA), albumin (ALB), globulin (GLOB), white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), red cell distribution width (RDW-SD), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), blood platelet (PLT), and platelet distribution width (PDW) in AINP-treated groups also presented an ignorable change to those in the control group (Figure ). Therefore, these in vivo results strongly manifest the low toxicity and excellent biosafety of AINP dispersion, which would pave the way for their future potential clinical application.

Figure 6

H&E staining of the main organs of mice 30 days after the intravenous injection of AINP dispersion at elevated doses. All of the scale bars are 100 μm.

Figure 7

Relevant blood indexes of mice from each group at different AINP doses of 0, 10, 50, and 100 mg/kg after intravenous administration and further feeding for 30 days.

Conclusions

We have subtly taken advantage of the versatile ancient ink to develop a green and effective strategy for PTT of tumor in the NIR-II window. By simply leveraging PVP as the stabilizer, a stable AINP dispersion with good water dispersity was prepared through hydrothermal treatment. The AINP dispersion demonstrated excellent photothermal performance and high photothermal conversion efficiency in the NIR-II window. Besides, the desired black staining and the favorable PA imaging capacity make it an ideal candidate for tumor SLN mapping. As revealed by PA imaging, the AINP dispersion after being injected to the primary tumors could effectually transfer into SLNs, which further promises the great potential for PA imaging-guided NIR-II PTT of tumor SLN metastasis. More importantly, both the preliminary in vitro and in vivo trials together demonstrated that our obtained AINP dispersion possessed benign biosafety.

However, to finally realize clinical and translational applications of AINPs, more pharmacology and toxicology of AINP dispersion are obligatory. In addition, to achieve a preferable therapeutic effect, the combination of AINPs together with the low-boiling-point phase change agent by leveraging injectable in situ forming thermal responsive hydrogel might also be carefully investigated in future work to realize PTT and thermal mechanical destruction synergistic therapy.

Experimental Section

Materials and Animals

AINPs were provided by Anhui Jixi Medicine Ink Factory, China. Poly(vinylpyrrolidone) (PVP, MW ≈ 58 000) was obtained from Sigma-Aldrich. Dulbecco’s modified Eagle’s medium (DMEM) and RPMI-1640 medium were obtained from GE Healthcare Life Science. PI, calcein-AM, and CCK-8 were purchased from Beyotime Institute of Biotechnology. Balb/c mice were provided by the Shanghai BK Lab. The animal protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Fudan University. All experiments were performed in accordance with relevant guidelines and regulations.

Preparation of AINP Dispersion

The AINP dispersion was prepared by a modified hydrothermal reaction. Briefly, raw AINPs (100 mg) were added into a PVP–water solution (10 mL, 10 mg/mL) under sonication. After the AINPs were completely dispersed, the resulting solution was transferred into a Teflon-lined stainless-steel autoclave (30 mL) and heated at 160 °C. The autoclave was cooled to room temperature 6 h later, and the AINP dispersion was then obtained.

Cell Cytotoxicity and In Vitro PTT Assay

Approximately 1.0 × 104 CT-26 cells and HEK-293T cells were plated in 96-well plates and cultured at the standard cell culture environment for 24 h. The cells were mixed with different samples (0, 12.5, 25, 50, 100, 200, and 400 μg/mL, counted by AINPs) for 24 h to evaluate the cytotoxicity of AINP dispersion. For the in vitro PTT assay, CT-26 cells were incubated in 96-well plates (2.0 × 105 cells per well) and cultured for 12 h. Then, the AINP dispersion (12.5, 25, 50, and 100 μg/mL, counted by AINPs) was added to each plate and incubated for another 4 h, followed by 5 min irradiation (808 nm: 0.33 W/cm2; 1064 nm: 1 W/cm2). Finally, the CLSM imaging and flow cytometry analysis was also conducted, and the detailed processes were provided in our previous work.[11]

In Vitro and In Vivo PA Imaging

CT-26 SLN metastases were first induced by subcutaneous injection of 3 × 106 CT-26 cells suspended in 50 μL of PBS into the right hind foot pad of nude mice.[52,53] At the 30th day after inoculation, the mice with spherical hard lumps in their popliteal fossa were selected as the CT-26 SLN metastases for future experiments. The PA imaging of AINP dispersion was conducted using the PA imaging system, and the detailed processes were provided in our previous work.[11]

PA Imaging-Guided PTT of SLNs

Mice (N = 35) exhibiting SLN metastases were divided randomly into the following five groups: control group, AINP group, 1064 nm laser (10 min, 1 W/cm2) group, AINPs + 1064 nm laser (10 min, 1 W/cm2) group, and AINPs + 808 nm laser (10 min, 0.33 W/cm2) group. The NIR thermal imaging device was used to record the temperature change. After 14 days, all of the mice were euthanized and the SLNs of mice were dissected and fixed in 4% formalin solution for further evaluation.

Systematic In Vivo Biocompatibility Assay of AINP Dispersion

Twenty healthy mice (∼4 weeks) were randomly assigned to four groups, including control and three AINP-treated groups at different doses (10, 50, and 100 mg/kg, counted by AINPs). After the intravenous injection of AINP dispersion, the body weight of the mice was measured every 2 days for 1 month. After that, the major organs from each group were collected for H&E staining, and their blood samples were collected to conduct the complete blood panel test.

Statistical Analysis

GraphPad Prism 5 software was used for statistical analysis. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was used to determine differences between groups. *p < 0.05, **p < 0.01, and ***p < 0.001 were considered statistically significant and n.s. represented no significance.