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Self-Assembled CuCo2S4 Nanoparticles for Efficient Chemo-Photothermal Therapy of Arterial Inflammation

Cardiovascular disease caused by atherosclerosis (AS) seriously affects human health. Photothermal therapy (PTT) brings hope to the diagnosis and treatment of AS, with the development of nanotechnology. To improve treatment efficiency, self-assembled CuCo2S4 nanocrystals (NCs) were developed as a drug-delivery nanocarrier, triggered by near-infrared (NIR) light for efficient chemophotothermal therapy of arterial inflammation. The as-prepared self-assembled CuCo2S4 NCs exhibited excellent biocompatibility and a very high chloroquine (CL)-loading content. In addition, the self-assembled CuCo2S4 NCs/CL nanocomposites showed good photothermal performance, due to strong absorption in the NIR region, and the release of CL from the NCs/CL nanocomposites was driven by NIR light. When illuminated by NIR light, both PTT from the NCs and chemotherapy from the CL were simultaneously triggered, resulting in killing macrophages with a synergistic effect. Moreover, chemo-photothermal therapy with CuCo2S4 NCs/CL nanocomposites showed an effective therapeutic effect for arterial inflammation, in vivo. Our work demonstrated that chemo-photothermal therapy could be a promising strategy for the treatment of arterial inflammation against atherosclerosis.


Introduction
Cardiovascular disease caused by atherosclerosis (AS) seriously affects human health. Disability and fatality rates are continually increasing, but the current treatment methods exhibit varying numbers of deficiencies, such as severe surgical trauma and poor chemotherapy effect [1]. Atherosclerosis is a chronic inflammatory disease, leading to stenosis and occlusion of the lumen. Its pathological manifestations include macrophage enrichment, foam-cell formation, and endothelial-cell dysfunction. It is an important mechanism for macrophages to regulate the inflammatory response of the blood-vessel wall and the formation of AS plaques by phagocytosing lipids and releasing a large number of inflammatory factors to interact with the blood-vessel wall. It has been demonstrated that macrophages play an important role in the formation, development and instability of AS plaques, and promoting their apoptosis can inhibit plaque progression [2]. In addition, the main clinical treatment of AS is balloon dilation and stent implantation, alleviating the symptoms of ischemia, but at the same time activating macrophages to further aggravate the inflammation of the vessel wall and promote the pathological remodeling of the arterial wall [3][4][5]. Therefore, inhibiting the activation, infiltration and proliferation indicated that the self-assembled CuCo 2 S 4 NCs were characterized by a hollow diskshape with a size of 750nm ( Figure 1A). Moreover, the size and unique morphology were further confirmed by SEM ( Figure S1A). Strong light-absorption within the NIR region is an essential feature of the biomaterials used for NIR-induced PTT. As shown in Figure 1B, the UV-vis-NIR absorbance spectrum of the self-assembled CuCo 2 S 4 NCs exhibited a strong light-absorption within the range of 780 nm to 1100 nm. Moreover, the self-assembled CuCo 2 S 4 NCs were detected by powder X-ray diffraction (XRD) to depict the crystallographic structure, and the pattern was well indexed to the phase of cubic spinel CuCo 2 S 4 ( Figure S1B). Before the drug-loading procedure, the surface area and pore volume of the self-assembled CuCo 2 S 4 NCs were measured using the BJH and BET methods, to assess their drug-loading property. The results revealed that the surface area and pore size was 315.08 m 2 g −1 and 13.62 nm, respectively ( Figure 1C,D), suggesting that the self-assembled CuCo 2 S 4 NCs could provide sufficient space for further drug delivery. The encapsulation efficiency and loading content of the CL into the self-assembled CuCo 2 S 4 NCs were determined to be as high as 81.5% and 23.7% (by weight), respectively.

Results and Discussion
The self-assembled CuCo2S4 NCs was prepared using a facile one-pot hydrothermal method for PTT and drug delivery. The results of transmission electron microscopy (TEM) indicated that the self-assembled CuCo2S4 NCs were characterized by a hollow disk-shape with a size of 750nm ( Figure 1A). Moreover, the size and unique morphology were further confirmed by SEM ( Figure S1A). Strong light-absorption within the NIR region is an essential feature of the biomaterials used for NIR-induced PTT. As shown in Figure 1B, the UV-vis-NIR absorbance spectrum of the self-assembled CuCo2S4 NCs exhibited a strong light-absorption within the range of 780 nm to 1100 nm. Moreover, the self-assembled CuCo2S4 NCs were detected by powder X-ray diffraction (XRD) to depict the crystallographic structure, and the pattern was well indexed to the phase of cubic spinel CuCo2S4 ( Figure S1B). Before the drug-loading procedure, the surface area and pore volume of the self-assembled CuCo2S4 NCs were measured using the BJH and BET methods, to assess their drug-loading property. The results revealed that the surface area and pore size was 315.08 m 2 g −1 and 13.62 nm, respectively ( Figure 1C,D), suggesting that the self-assembled CuCo2S4 NCs could provide sufficient space for further drug delivery. The encapsulation efficiency and loading content of the CL into the self-assembled CuCo2S4 NCs were determined to be as high as 81.5% and 23.7% (by weight), respectively. To construct the relationship between laser irradiation and temperature of the self-assembled CuCo2S4 NCs/CL nanocomposites, the aqueous dispersions of the materials at the concentrations of 0, 25, 50, and 100 ppm were continually irradiated by an 808 nm wavelength laser. The results showed a positive correlation between the duration of To construct the relationship between laser irradiation and temperature of the selfassembled CuCo 2 S 4 NCs/CL nanocomposites, the aqueous dispersions of the materials at the concentrations of 0, 25, 50, and 100 ppm were continually irradiated by an 808 nm wavelength laser. The results showed a positive correlation between the duration of laser irradiation and the temperature of the NCs/CL nanocomposites. Notably, after 5-min laser irradiation, the temperature of the aqueous dispersions with a concentration of 100 ppm reached 60 • C ( Figure 2A). Furthermore, under the condition of 5-min laser irradiation, the concentration and temperature of self-assembled CuCo 2 S 4 NCs exhibited an approximate direct proportional-correlation ( Figure S2). The release of CL from the self-assembled CuCo 2 S 4 NCs/CL nanocomposites at pH 7.4 with or without NIR irradiation was assessed, to determine the drug-release property of the CuCo 2 S 4 NCs/CL nanocomposites. Compared with no NIR irradiation, CL was released faster from the materials under NIR irradiation ( Figure 2B). Ten hours after NIR irradiation, >60% CL was released, whereas approximately only 15% CL was released without NIR irradiation. Therefore, the CuCo 2 S 4 NCs/CL nanocomposites showed NIR-triggered drug-release properties, and were suitable for subsequent in vitro and in vivo experiments. laser irradiation and the temperature of the NCs/CL nanocomposites. Notably, after 5-min laser irradiation, the temperature of the aqueous dispersions with a concentration of 100 ppm reached 60℃ ( Figure 2A). Furthermore, under the condition of 5-min laser irradiation, the concentration and temperature of self-assembled CuCo2S4 NCs exhibited an approximate direct proportional-correlation ( Figure S2). The release of CL from the self-assembled CuCo2S4 NCs/CL nanocomposites at pH 7.4 with or without NIR irradiation was assessed, to determine the drug-release property of the CuCo2S4 NCs/CL nanocomposites. Compared with no NIR irradiation, CL was released faster from the materials under NIR irradiation ( Figure 2B). Ten hours after NIR irradiation, >60% CL was released, whereas approximately only 15% CL was released without NIR irradiation. Therefore, the CuCo2S4 NCs/CL nanocomposites showed NIR-triggered drug-release properties, and were suitable for subsequent in vitro and in vivo experiments. Chronic inflammatory-reaction mediated by macrophages is the central part of the pathological and physiological mechanism of AS [16][17][18][19]. On the one hand, macrophages are able to take up lipids and turn them into foam cells, which constitute the principal component of AS plaque [20]. On the other hand, inflammatory macrophages secrete inflammatory cytokines including IL-1, TNF-α, and IL-6, and exacerbate the inflammatory reaction of the artery wall [21]. Raw264.7, a mouse macrophage cell-line, characterized by significant phagocytosis and pseudopodial movement, was widely used in the research of the phenotype and the function of the macrophage [22]. The results of cellular immunofluorescence revealed that Raw264.7 was highly positive for the macrophage marker CD68 ( Figure S3). The uptake of biomaterials by macrophages is the premise of our treatment strategy for AS. To confirm that CuCo2S4 NCs/CL could be specifically phagocyted by macrophages, Raw264.7 cells were incubated with CuCo2S4 NCs/CL, and then TEM was used to observe the phagocytosis process. Compared with the blank control ( Figure 3A,B), self-assembled CuCo2S4 NCs/CL were obviously phagocyted by Raw264.7, with no damage to the normal cell structure ( Figure 3C,D). To calculate the optimal concentration of CuCo2S4 NCs/CL for the treatment of AS in vivo, CCK-8 and Calcein AM/PI staining assays were then conducted. In order to avoid cell damage while achieving the best therapeutic effect of the materials, we determined their maximum safe-dose on Raw264.7 cells. According to the results of the CCK-8 assay, when Raw264.7 cells were incubated with CuCo2S4 NCs/CL at the concentration >120 ppm, cell viability was significantly decreased ( Figure 4A). However, the cell viability at the concentrations of CuCo2S4 NCs/CL ≤ 80 ppm showed no obvious difference. Therefore, 80 ppm was the optimal concentration of CuCo2S4 NCs/CL, and was used for the following experiments. Chronic inflammatory-reaction mediated by macrophages is the central part of the pathological and physiological mechanism of AS [16][17][18][19]. On the one hand, macrophages are able to take up lipids and turn them into foam cells, which constitute the principal component of AS plaque [20]. On the other hand, inflammatory macrophages secrete inflammatory cytokines including IL-1, TNF-α, and IL-6, and exacerbate the inflammatory reaction of the artery wall [21]. Raw264.7, a mouse macrophage cell-line, characterized by significant phagocytosis and pseudopodial movement, was widely used in the research of the phenotype and the function of the macrophage [22]. The results of cellular immunofluorescence revealed that Raw264.7 was highly positive for the macrophage marker CD68 ( Figure S3). The uptake of biomaterials by macrophages is the premise of our treatment strategy for AS. To confirm that CuCo 2 S 4 NCs/CL could be specifically phagocyted by macrophages, Raw264.7 cells were incubated with CuCo 2 S 4 NCs/CL, and then TEM was used to observe the phagocytosis process. Compared with the blank control ( Figure 3A,B), self-assembled CuCo 2 S 4 NCs/CL were obviously phagocyted by Raw264.7, with no damage to the normal cell structure ( Figure 3C,D). To calculate the optimal concentration of CuCo 2 S 4 NCs/CL for the treatment of AS in vivo, CCK-8 and Calcein AM/PI staining assays were then conducted. In order to avoid cell damage while achieving the best therapeutic effect of the materials, we determined their maximum safe-dose on Raw264.7 cells. According to the results of the CCK-8 assay, when Raw264.7 cells were incubated with CuCo 2 S 4 NCs/CL at the concentration >120 ppm, cell viability was significantly decreased ( Figure 4A). However, the cell viability at the concentrations of CuCo 2 S 4 NCs/CL ≤ 80 ppm showed no obvious difference. Therefore, 80 ppm was the optimal concentration of CuCo 2 S 4 NCs/CL, and was used for the following experiments. To explore the effect of CuCo 2 S 4 NCs/CL on the cell viability of macrophages, Raw264.7 cells incubated with 80 ppm of CL, CuCo 2 S 4 NCs, CuCo 2 S 4 NCs/CL were stained with Calcein AM/PI. As shown in Figure 4B, after 808 nm NIR laser irradiation, almost all cells were live, with green fluorescence in the control group, but in the CL and CuCo 2 S 4 NCs group approximately 40% of cells were dead, with red fluorescence. Notably, after NIR irradiation, >90% of Raw264.7 were dead in the CuCo 2 S 4 NCs/CL group, indicating that PTT could eliminate inflammatory macrophages. The results of the CCK-8 assay further confirmed the excellent PTT efficacy of CuCo 2 S 4 NCs/CL, which was consistent with previous conclusions ( Figure 4C). Accumulation of inflammatory macrophages in the artery wall causes an abnormal immune response and a disturbance of lipid metabolism, leading to the pathogenesis of AS [23]. The elimination of inflammatory macrophages or inhibition of immune response mediated by macrophages is a promising therapeutic target for AS [24][25][26][27]. The current results demonstrated that self-assembled CuCo 2 S 4 NCs/CL had the dual effects of drug delivery and PTT, which could be utilized to reduce arterial inflammation and relieve AS.
To explore the effect of CuCo2S4 NCs/CL on the cell viability of macropha cells incubated with 80 ppm of CL, CuCo2S4 NCs, CuCo2S4 NCs/CL wer Calcein AM/PI. As shown in Figure 4B, after 808 nm NIR laser irradiation, were live, with green fluorescence in the control group, but in the CL and group approximately 40% of cells were dead, with red fluorescence. Nota irradiation, >90% of Raw264.7 were dead in the CuCo2S4 NCs/CL group, PTT could eliminate inflammatory macrophages. The results of the CCKconfirmed the excellent PTT efficacy of CuCo2S4 NCs/CL, which was c previous conclusions ( Figure 4C). Accumulation of inflammatory macro artery wall causes an abnormal immune response and a disturbance of lip leading to the pathogenesis of AS [23]. The elimination of inflammatory m inhibition of immune response mediated by macrophages is a promisi target for AS [24][25][26][27]. The current results demonstrated that self-assem NCs/CL had the dual effects of drug delivery and PTT, which could be uti arterial inflammation and relieve AS.  We used the combination of carotid artery wire-injury and high-fat feeding methods to fully mimic arterial inflammation and AS in ApoE −/− mice, as previously described [28]. Two weeks after the surgery, 100 µL of CL, CuCo 2 S 4 NCs, or CuCo 2 S 4 NCs/CL (80 ppm) was locally injected into the inflammation site surrounding the carotid artery, and PBS was used as the control ( Figure S4). Twelve hours after injection, an 808 nm NIR laser was applied to the body-surface projection of the carotid arteries for 5 min. Two weeks later, the mice were sacrificed and their blood, carotid arteries and vital organs were harvested. To confirm the efficacy of PTT in vivo, the carotid arteries were made into paraffin slices, and HE and immunofluorescence staining were then performed on these slices. The vascular smooth-muscle-cell (SMCs) marker, α-SMA, was used to indicate SMCs in the media of the carotid artery, and the macrophage marker CD68 was used to label inflammatory macrophages infiltrating the artery walls. As shown in Figure 5A,C, significant macrophage infiltration in the control group was observed, and the number of macrophages in the artery wall was decreased in the CL and CuCo 2 S 4 NCs group after PTT. Moreover, compared with the previous three groups, the infiltrated macrophages were significantly reduced in the CuCo 2 S 4 NCs/CL group. The results demonstrated that self-assembled CuCo 2 S 4 NCs/CL could act as a highly efficient drug carrier and photothermal agent for the alleviation of arterial inflammation.  We used the combination of carotid artery wire-injury and high-fat feeding metho to fully mimic arterial inflammation and AS in ApoE −/− mice, as previously described [2 Two weeks after the surgery, 100 μL of CL, CuCo2S4 NCs, or CuCo2S4 NCs/CL (80 pp was locally injected into the inflammation site surrounding the carotid artery, and P was used as the control ( Figure S4). Twelve hours after injection, an 808 nm NIR las was applied to the body-surface projection of the carotid arteries for 5 min. Two wee later, the mice were sacrificed and their blood, carotid arteries and vital organs we harvested. To confirm the efficacy of PTT in vivo, the carotid arteries were made in paraffin slices, and HE and immunofluorescence staining were then performed on the slices. The vascular smooth-muscle-cell (SMCs) marker, α-SMA , was used to indica SMCs in the media of the carotid artery, and the macrophage marker CD68 was used label inflammatory macrophages infiltrating the artery walls. As shown in Figure 5A significant macrophage infiltration in the control group was observed, and the number macrophages in the artery wall was decreased in the CL and CuCo2S4 NCs group af PTT. Moreover, compared with the previous three groups, the infiltrated macrophag were significantly reduced in the CuCo2S4 NCs/CL group. The results demonstrated th Atherosclerotic plaques and pathological hyperplasia of the intima and media could lead to thrombosis or stenosis [29]. To evaluate the lumen stenosis and the intima and media thickness of carotid arteries, HE staining and morphological analysis was conducted. The results revealed that, compared with the control, the degree of lumen stenosis and the intima and media thickness were partially decreased in the carotid arteries treated with CL and CuCo 2 S 4 NCs ( Figure 5A,B). However, CuCo 2 S 4 NCs/CL combined with 808 nm NIR laser irradiation significantly improved lumen area and inhibited intima and media hyperplasia, which could increase blood supply to vital organs, and thus reduce the possibility of a fatal ischemic event resulting from AS. Moreover, significant complications of AS such as bleeding or thrombosis were not found during the whole experimental period. The above results demonstrated that self-assembled CuCo 2 S 4 NCs/CL with irradiation using the NIR laser showed great potential for use in chemo-photothermal therapy for arterial inflammation of AS. Atherosclerotic plaques and pathological hyperplasia of the intima and media could lead to thrombosis or stenosis [29]. To evaluate the lumen stenosis and the intima and media thickness of carotid arteries, HE staining and morphological analysis was conducted. The results revealed that, compared with the control, the degree of lumen stenosis and the intima and media thickness were partially decreased in the carotid arteries treated with CL and CuCo2S4 NCs ( Figure 5A,B). However, CuCo2S4 NCs/CL combined with 808 nm NIR laser irradiation significantly improved lumen area and inhibited intima and media hyperplasia, which could increase blood supply to vital organs, and thus reduce the possibility of a fatal ischemic event resulting from AS. Moreover, significant complications of AS such as bleeding or thrombosis were not found during the whole experimental period. The above results demonstrated that self-assembled CuCo2S4 NCs/CL with irradiation using the NIR laser showed great potential for use in chemo-photothermal therapy for arterial inflammation of AS.
Good biocompatibility is required for the in vivo application of nanotherapy. To detect any tissue toxicity of self-assembled CuCo2S4 NCs/CL, HE staining was conducted Good biocompatibility is required for the in vivo application of nanotherapy. To detect any tissue toxicity of self-assembled CuCo 2 S 4 NCs/CL, HE staining was conducted on the vital organs, including heart, kidney, liver, lung, and spleen. No significant morphological or pathological features were found among the CuCo 2 S 4 NCs/CL, the CuCo 2 S 4 NCs, the CL and the control groups ( Figure 6A). Moreover, no cell necrosis, tissue fibrosis or inflammation infiltration was observed in these tissues. The renal and liver function of the experimental mice were assessed by the detection of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CR), and blood urea nitrogen (BUN). No significant difference was observed between these four groups in terms of these blood parameters ( Figure 6B). These results demonstrated that the CuCo 2 S 4 NCs/CL had no organ toxicity and no effect on the normal physiological function of the body. In summary, self-assembled CuCo 2 S 4 NCs/CL was proven to be an ideal therapeutic agent for the treatment of arterial inflammation and AS, with great efficacy and safety. summary, self-assembled CuCo2S4 NCs/CL was proven to be an ideal therapeutic agent for the treatment of arterial inflammation and AS, with great efficacy and safety.

Synthesis of Self-Assembled CuCo2S4 NCs/CL Nanocomposites
A total of 1.5 mmol of thiourea, 0.5 mmol of CoCl2•6H2O, 0.25 mmol of CuCl2•2H2O, and 1 g of poly (vinyl pyrrolidone) were fully dissolved in pure water (40 mL). The resulting solution was kept at 160 °C for 20 h in a hydrothermal reactor. The NCs were obtained by centrifuge and washing with water. CL loading was achieved by mixing CuCo2S4 NCs and CL under stirring, overnight. The final products were obtained by centrifuge and washing with water.

Characterization
The shape and size of self-assembled CuCo2S4 NCs can be detected by a transmission electron microscope (TEM). The absorption spectrum of the self-assembled CuCo2S4

Synthesis of Self-Assembled CuCo 2 S 4 NCs/CL Nanocomposites
A total of 1.5 mmol of thiourea, 0.5 mmol of CoCl 2 ·6H 2 O, 0.25 mmol of CuCl 2 ·2H 2 O, and 1 g of poly (vinyl pyrrolidone) were fully dissolved in pure water (40 mL). The resulting solution was kept at 160 • C for 20 h in a hydrothermal reactor. The NCs were obtained by centrifuge and washing with water. CL loading was achieved by mixing CuCo 2 S 4 NCs and CL under stirring, overnight. The final products were obtained by centrifuge and washing with water.

Characterization
The shape and size of self-assembled CuCo 2 S 4 NCs can be detected by a transmission electron microscope (TEM). The absorption spectrum of the self-assembled CuCo 2 S 4 NCs was measured by the UV-vis spectrophotometer. The phase of self-assembled CuCo 2 S 4 NCs was detected by an X-ray diffractometer (XRD). The release of CL from the self-assembled CuCo 2 S 4 NCs with or without 808 nm NIR laser irradiation was studied using a UV-vis spectrophotometer.

CL Release In Vitro
The CL-loaded self-assembled CuCo 2 S 4 NCs (NCs/CL) were collected using centrifugation, and washed three times with PBS solution to remove the unbound CL at different time-points. All the supernatant solution was collected together and measured, using a UV-Vis spectrophotometer, to calculate the amount of CL payload in the selfassembled CuCo 2 S 4 NCs. The encapsulation efficiency of the self-assembled CuCo 2 S 4 NCs = (weight of CL loaded into the self-assembled CuCo 2 S 4 NCs)/(initial weight of CL). The loading content of the self-assembled CuCo 2 S 4 NCs = (weight of CL loaded into the self-assembled CuCo 2 S 4 NCs)/(weight of the self-assembled CuCo 2 S 4 NCs + CL loaded into the self-assembled CuCo 2 S 4 NCs).The release of CL from the self-assembled CuCo 2 S 4 nanomaterials with or without 808 nm NIR laser irradiation was studied. The NCs/CL was dispersed in PBS solution at pH 7.4. The laser-triggered drug-release experiments were performed by irradiating the liquid dispersion for 5 min under stirring. The liquid dispersion was then centrifuged and the supernatant was collected. The amount of released CL in the supernatant was determined by using a UV-Vis-NIR spectrophotometer. At certain time intervals, the above operation was repeated.

Cell Culture and TEM Detection
Raw264.7 cells were cultured in DMEM with 4.5 g/L of glucose supplemented with 10% FBS and 1% penicillin and streptomycin at 37 • C and 5% CO 2 . Raw264.7 cells were identified by immunofluorescent staining with the macrophage-specific antibody CD68. After being cocultured with or without CuCo 2 S 4 NCs/CL (80 µg mL −1 ) for 12 h, the Raw264.7 cells were collected and then observed under TEM [30].

Cell Viability
The cytotoxicity of CuCo 2 S 4 NCs/CL was firstly assessed in vitro. Raw264.7 macrophages were seeded at a density of 1 × 10 5 on a 96-well plate, and cultured for 12 h. The cells were then incubated with various concentrations of CuCo 2 S 4 NCs/CL (0, 20, 40, 80, 120, 200, 400, 800 µg mL −1 ) for 24 h, and a CCK-8 cell proliferation assay was performed to evaluate the cell viability, as described in a previous study [12]. The maximum safe concentration was considered as the optimal concentration, and used for the following studies. Next, the Raw264.7 cells were co-cultured with PBS and 80 µg mL −1 of CL, CuCo 2 S 4 NCs, and CuCo 2 S 4 NCs/CL for 12 h, followed with 808 nm NIR laser irradiation with a power density of 0.56 W cm −2 for 5 min. After washing with PBS, the cells were incubated with CCK-8 agent for 1 h, and the absorbance at 450 nm wavelength was detected. In another set of experiments, Raw264.7 cells were stained with calcein AM/PI and observed under an immunofluorescence microscope (LSM 510 META, Carl-Zeiss-Strasse, Germany).

Animal Model and In Vivo Nanotherapy
All animal experiments were approved by the Animal Ethics Committee of the First Affiliated Hospital of Bengbu Medical College (No. ChiCTR2000040024). Apolipoprotein E knockout (ApoE −/− ) mice (eight-week-old, male) were fed with a high-fat diet (0.15% cholesterol, 21% fat). The mice were acclimatized to the new environment for 1 week before surgery. The carotid artery wire-injury method was used to induce atherogenesis [28]. Two weeks after the surgery, PBS, CL, CuCo 2 S 4 NCs, or CuCo 2 S 4 NCs/CL dissolved in PBS (100 µL, 80 µg mL −1 ) were subcutaneously injected around the carotid artery of the mice (n = 8 for each group). Twelve hours after the injection, an 808 nm NIR laser with a power density of 0.56 W cm −2 was applied at the injection site for 5 min, as previously described [11].

Histological and Blood Analysis
Two weeks after PTT, the mice were euthanized with a pentobarbital overdose and their blood, carotid arteries and vital organs were harvested. Frozen sections of the carotid arteries and paraffin-embedded sections of both carotid arteries and vital organs were prepared. For immunofluorescence analysis, macrophages, smooth muscle cells (SMC) and nuclei were stained with CD68, α-SMA, and DAPI, respectively. The paraffin-embedded sections of the carotid arteries were stained with hematoxylin/eosin (HE). The thickness of the carotid intima and media were analyzed using ImageJ Pro Plus software (Media Cybernetics, Rockville, MD, USA). Furthermore, the sections of heart, kidney, liver, lung, and spleen from the mice were stained with HE to assess the tissue toxicity of CuCo 2 S 4 NCs/CL. In addition, the blood samples were obtained for liver-and kidney-function detection.

Statistics
All quantitative data from the experiments were reported in the form of mean ± standard deviation (SD). The Student's t-test or a one-way analysis of variance (ANOVA) was applied to determine the statistical significance (p < 0.05) of the data. All experiments were repeated three times in every group.

Conclusions
In conclusion, we developed self-assembled CuCo 2 S 4 nanocrystals (NCs) as a drugdelivery nanocarrier triggered by near-infrared (NIR) light for the efficient chemophotothermal therapy of arterial inflammation. The as-prepared self-assembled CuCo 2 S 4 NCs exhibited a high chloroquine (CL)-loading efficiency, due to the mesoporous structure. In addition, the self-assembled CuCo 2 S 4 NCs showed good photothermal performance, resulting from the strong NIR absorption, and the release of CL from the NCs/CL nanocomposites was driven by NIR light. When illuminated by NIR light, both PTT from the NCs and chemotherapy from the CL were simultaneously triggered, resulting in killing macrophages with a synergistic effect. Moreover, chemo-photothermal therapy with CuCo 2 S 4 NCs/CL nanocomposites showed an effective therapeutic effect for arterial inflammation, in vivo. Our work demonstrated that CuCo 2 S 4 NCs/CL nanocomposites under the action of the NIR laser could be used in the efficient treatment of arterial inflammation, against AS.