A cos-peg-pix-sa nanocomposite and a preparation method and application thereof

Abstract

The application discloses a CoS-PEG-PPIX-SA nanocomposite as well as a preparation method and application thereof. The CoS-PEG-PPIX-SA nanocomposite comprises a CoS-PEG nanocomposite, a photosensitizer and a targeting agent. The CoS-PEG nanocomposite comprises CoS nanosheets and polyethylene glycol (PEG) wrapped on surfaces of the CoS nanosheets. The photosensitizer and the targeting agent are modified on surfaces of the CoS-PEG. The photosensitizer is protoporphyrin (PPIX), and the targeting agent is sialic acid (SA). CoS has CAT activity, which enhances O2 supply in a tumor microenvironment, and improves the curative effect of PDT. Meanwhile, CoS releases Co in situ in the TME due to its excellent chemical dynamic performance, triggers a Fenton-like reaction, and promotes H2O2 in the tumor microenvironment to generate highly toxic ·OH. 2+ In addition, the strong absorption of CoS to near-infrared light and the photo-thermal conversion capability of CoS can effectively convert photon energy into local thermal energy, so that the temperature of a lesion part reaches a treatment threshold. The photo-thermal effect can also significantly accelerate the kinetics of the Fenton-like reaction, thereby enhancing the CDT efficiency, and realizing the synergistic anti-tumor effect of CDT / PTT / PDT.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine materials technology, and more specifically, to a CoS-PEG-PPIX-SA nanocomposite, its preparation method, and its application in anti-breast cancer drugs. Background Technology

[0002] Currently, surgery, radiotherapy, and chemotherapy are the three main treatments for breast cancer. However, these traditional methods have significant limitations, such as significant side effects, low targeting accuracy, and high invasiveness, which seriously affect treatment outcomes and patients' quality of life. Therefore, there is an urgent need to develop new strategies for treating breast cancer to maximize treatment effectiveness. In recent years, non-invasive treatment methods based on nanotechnology, such as chemodynamic therapy (CDT), photothermal therapy (PTT), and photodynamic therapy (PDT), have provided new ideas for breast cancer treatment due to their advantages such as strong targeting, low side effects, and good penetration.

[0003] Chemodynamic therapy (CDT) utilizes Fenton-like reactions involving metal ions to generate highly toxic hydroxyl radicals (·OH), thereby inducing tumor cell apoptosis. Photothermal therapy (PTT) uses photothermal conversion materials to convert light energy into heat energy, raising the temperature at the tumor site and thus killing tumor cells. Photodynamic therapy (PDT) uses light of a specific wavelength to excite a photosensitizer, causing it to react with oxygen (O2) to generate singlet oxygen (·OH). 1 O2, thereby killing tumor cells. PDT has attracted much attention due to its advantages such as being non-invasive, having low systemic toxicity, and good tissue selectivity. However, since the efficacy of PDT is highly dependent on O2 concentration, and the tumor microenvironment (TME) is usually severely hypoxic, this hypoxic state not only limits the generation of singlet oxygen (O2) by PDT, but also... 1 The ability of oxygen (O2) may also promote tumor invasion and metastasis through pathways such as activating hypoxia-inducible factor (HIF-1α).

[0004] Therefore, designing nanomaterials that combine oxygen production and multimodal synergistic therapy capabilities holds promise as an ideal carrier for improving the treatment outcomes of breast cancer. Summary of the Invention

[0005] The purpose of this invention is to provide a CoS-PEG-PPIX-SA nanocomposite, its preparation method and application, to overcome the shortcomings of existing technologies such as poor efficacy of single therapy and limited efficacy of PDT due to tumor hypoxia.

[0006] To achieve the above objectives, according to one aspect of the present invention, a CoS-PEG-PPIX-SA nanocomposite is provided, comprising a CoS-PEG nanocomposite, a photosensitizer, and a targeting agent; The CoS-PEG nanocomposite comprises CoS nanosheets and polyethylene glycol (PEG) coated on the surface of the CoS nanosheets. The photosensitizer and targeting agent are modified on the CoS-PEG surface; the photosensitizer is protoporphyrin (PPIX), and the targeting agent is sialic acid (SA).

[0007] According to another aspect of the present invention, a method for preparing the CoS-PEG-PPIX-SA nanocomposite described above is provided, comprising: Step 1: CoS nanosheets are dispersed in an aqueous solution of NH2-PEG-COOH to obtain CoS-PEG. Step 2: Protoporphyrin (PPIX), (3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) are dispersed in dimethyl sulfoxide (DMSO) to obtain an activated PPIX solution. CoS-PEG is then mixed with the activated protoporphyrin (PPIX) solution and reacted to obtain CoS-PEG-PPIX. Step 3: CoS-PEG-PPIX is dispersed in deionized water, and (3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are added to obtain an activated CoS-PEG-PPIX solution. Sialic acid (SA) is dissolved in deionized water and mixed with the activated CoS-PEG-PPIX solution to obtain the target product CoS-PEG-PPIX-SA nanocomposite.

[0008] As a preferred embodiment, in step one, the method for preparing CoS nanosheets is as follows: Co(NO3)2·6H2O, thioacetamide (TAA) and polyvinylpyrrolidone (PVP) are dissolved in deionized water, and then NaOH aqueous solution is added under stirring conditions to obtain a mixture solution. The mixture solution is then subjected to a hydrothermal reaction to obtain CoS nanosheets.

[0009] In a preferred embodiment, the temperature of the hydrothermal reaction is 100°C.

[0010] In a preferred embodiment, the mass ratio of Co(NO3)2·6H2O, thioacetamide (TAA), and polyvinylpyrrolidone (PVP) is 291:150:200.

[0011] In a preferred embodiment, the concentration of the NH2-PEG-COOH aqueous solution in step one is 2.0 mg / mL.

[0012] In a preferred embodiment, in steps two and three, the mass ratio of (3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) to N-hydroxysuccinimide (NHS) is 1:1.

[0013] In a preferred embodiment, the reaction temperature in steps one, two, and three is room temperature.

[0014] According to another aspect of the present invention, the application of the CoS-PEG-PPIX-SA nanocomposite in the preparation of a synergistic treatment for breast cancer is provided, wherein the synergistic treatment is a combination of chemodynamic therapy (CDT), photothermal therapy (PTT), and photodynamic therapy (PDT).

[0015] According to another aspect of the invention, a medicament for the synergistic treatment of breast cancer is provided, comprising the CoS-PEG-PPIX-SA nanocomposite and a pharmaceutically acceptable carrier.

[0016] According to the CoS-PEG-PPIX-SA nanocomposite provided by the present invention, protoporphyrin acts as a photosensitizer, reacting with O2 under light irradiation to generate... 1 O2 is used to achieve the therapeutic effect of PDT. CoS has CAT activity, which enhances the O2 supply in the tumor microenvironment, further improving the efficacy of PDT. At the same time, CoS also releases Co in situ in the TME due to its excellent chemodynamic properties. 2+ Triggering a Fenton-like reaction, CoS promotes the formation of highly toxic ·OH from H2O2 in the tumor microenvironment. Furthermore, CoS's strong absorption of near-infrared light and its photothermal conversion capabilities effectively convert photon energy into localized heat, bringing the lesion site to the therapeutic threshold temperature. This photothermal effect also significantly accelerates the Fenton-like reaction kinetics, thereby enhancing CDT efficiency and achieving a synergistic anti-tumor effect of CDT / PTT / PDT. This multi-effect synergistic strategy fully leverages the complementary advantages of different treatment modalities, opening up new directions for developing highly effective and low-toxicity novel tumor treatment regimens. Attached Figure Description

[0017] Figure 1 To study the preparation of CPPS and its application mechanism in synergistic therapy for breast cancer; Figure 2 TEM plots for CoS(a) (with inset of HRTEM plot of CoS), CP(b) (with inset of HRTEM plot of CoS), CPP(c) (with inset of HRTEM plot of CoS), and CPPS(d) (with inset of HRTEM plot of CoS). Figure 3XRD patterns (a) and XPS patterns of CoS, CP, CPP, and CPPS; fine spectra of CPPS: (c) Co2p, (d) S2p, (e) C1s, and (f) O1s. Figure 4 (a) UV-Vis-NIR spectrum, (b) fluorescence spectrum, (c) infrared spectrum, and (d) zeta potential spectrum of CoS, CP, CPP, and CPPS. Figure 5 In the table, (a) shows the in vitro generation of hydroxyl radicals in the TA+H2O group, TA+H2O2 group, CPPS+H2O2 group, TA+CPPS group, and TA+CPPS+H2O2 group; (b) shows the generation of hydroxyl radicals in CPPS under different pH conditions. Figure 6 In the figure, (a) shows the temperature change curves after irradiation with H2O, CoS, CP, CPP, and CPPS; (b) shows the temperature change curves under irradiation with different concentrations of 808 nm (1.8 W) laser; (c) shows the temperature change curves of CPPS under irradiation with different power densities of 808 nm laser; (d) shows the thermal cycling curve of CPPS under irradiation with 808 nm laser; and (e) shows the infrared thermal imaging of H2O, CoS, CP, CPP, and CPPS under irradiation with 808 nm laser (1.8 W) over time. Figure 7 In the figure, (a) is a single heating and cooling curve of CPPS under irradiation with 808 nm laser (1.8 W); (b) is a linear relationship between cooling time and -ln(θ). Figure 8 The curves showing the change of absorbance of ABDA at 380 nm with illumination time under different conditions are shown. Figure 9 The O2 release curve of CPPS; Figure 10 Survival rates of HUVEC and MCF-7 cells after incubation with different concentrations of CPPS for 24 h; Figure 11 The hemolysis rate of different concentrations of CPPS (10, 25, 50, 100, 150, 200 μg / mL); Figure 12 Fluorescence images of MCF-7 cells after CPPS treatment for 0, 2, 4, and 6 h (scale bar = 50 μm). Figure 13 Fluorescence images of HUVEC and MCF-7 cells after incubation with CPPS (scale bar = 50 μm). Figure 14In the image, (a) shows the ROS production of MCF-7 cells after incubation in different treatment groups (scale bar = 50 μm); (b) shows the quantitative analysis of cell fluorescence intensity in each group (***). p <0.001); Figure 15 The survival rates of MCF-7 cells incubated with CPPS+H2O2, CPPS+808 nm NIR, CPPS+660 nm NIR, and CPPS+H2O2+808 nm NIR+660 nm NIR groups (***) p <0.001); Figure 16 In the figure, (a) shows the killing effect of PBS group, NIR+H2O2 group, CPPS group, CPPS+H2O2 group, CPPS+808 nm NIR group, CPPS+660 nm NIR group, and CPPS+H2O2+808 nm NIR+660 nm NIR group on MCF-7 cells (scale bar = 200 μm); (b) shows the statistical analysis of cell fluorescence quantification in each group. Figure 17 Apoptosis (a) and apoptosis rate (b) of MCF-7 cells after incubation in the PBS group, NIR+H2O2 group, CPPS group, CPPS+H2O2 group, CPPS+808 nm NIR group, CPPS+660 nm NIR group, and CPPS+H2O2+808 nm NIR+660 nm NIR group. p <0.001); Figure 18 Fluorescence images of AO-stained MCF-7 cells in different treatment groups (scale bar = 50 μm). Figure 19 The damage to mitochondrial membrane potential in MCF-7 cells by the PBS group, NIR+H2O2 group, CPPS group, CPPS+H2O2 group, CPPS+808 nm NIR group, CPPS+660 nm NIR group, and CPPS+H2O2+808 nm NIR+660 nm NIR group (scale bar = 20 μm). Figure 20 Western blot analysis of the effect of CPPS on the expression and quantification of the apoptosis-related protein Caspace-3 in MCF-7 cells (***) p <0.001); Figure 21 In the image, (a) shows the distribution of CPPS in mice at different time points after injection; (b) shows fluorescence images of major organs and tumors in mice 9 hours after CPPS injection. Figure 22Infrared thermal imaging of mice at different time points after intravenous injection of Saline, CoS, CP, CPP, and CPPS via the tail vein; Figure 23 In the image, (a) is a basic flowchart of in vivo anti-tumor therapy; (b) mouse body weight change curves, (c) relative tumor volume detection, and (d) tumor weight changes in different treatment groups during treatment; (e) mouse images and (f) images of ex vivo tumors after 14 days of treatment in different treatment groups; and (g) HE staining of mouse tumor tissue (*** p <0.001); Figure 24 Blood and biochemical indicators of mice in each group; Figure 25 HE staining was performed on normal tissues (heart, liver, spleen, lung, and kidney) of mice in each group. Detailed Implementation

[0018] The basic concept of this invention is to encapsulate polyethylene glycol (PEG) on the surface of CoS nanosheets with strong near-infrared absorption capabilities to further enhance the water solubility and stability of CoS. A photosensitizer, protoporphyrin (PPIX), and a targeting agent, sialic acid (SA), are then modified onto the CoS-PEG surface via an amide reaction to form a CoS-PEG-PPIX-SA nanocomposite (CPPS). Through the retention (EPR) effect and the targeting effect of SA, CPPS can rapidly enter the tumor site. The photothermal effect generated under 808 nm laser irradiation can effectively kill cancer cells. Simultaneously, the photothermal effect promotes the peroxidase and catalase catalytic activity of CPPS, generating more ·OH and O2 to enhance the ability of CPPS to generate singlet oxygen under 660 nm laser irradiation, achieving a synergistic effect of CDT / PTT / PDT therapy, thereby significantly inhibiting tumor growth without significant toxicity to normal tissues. The constructed CPPS nanocomposite material can achieve synergistic CDT / PTT / PDT therapy, providing a new strategy and direction for multimodal precision treatment of breast cancer.

[0019] 1. Preparation of CoS-PEG-PPIX-SA (CPPS) Preparation of CoS: Co(NO3)2·6H2O (291 mg), TAA (150 mg), and PVP (200 mg) were dissolved in deionized water (50.0 mL). NaOH aqueous solution (0.5 mol / L, 12.0 mL) was added under magnetic stirring to form a mixture solution. The mixture solution was heated and maintained at 100 °C for 60 min. During the reaction, the solution color changed from blue-green to black, indicating the formation of CoS nanosheets. After cooling to room temperature, the precipitate was collected by centrifugation (10000 rpm, 10 min). The precipitate was washed with deionized water and anhydrous ethanol.

[0020] Preparation of CoS-PEG (CP): The prepared CoS nanosheets were redispersed in a beaker containing an aqueous solution of NH2-PEG-COOH (2.0 mg / mL) and stirred overnight. Excess NH2-PEG-COOH molecules were removed by centrifugation (12000 rpm, 10 min), and the product was repeatedly washed with deionized water and freeze-dried to obtain CP.

[0021] Preparation of CoS-PEG-PPIX (CPP): 5 mg PPIX, 15 mg EDC, and 15 mg NHS were dispersed in a beaker containing DMSO and reacted for 30 min to activate PPIX. The CoS-PEG solution was then mixed with the above reaction system and stirred overnight. After centrifugation (15000 rpm, 10 min), the precipitate was collected, washed several times with DMSO, anhydrous ethanol, and deionized water, and lyophilized to obtain CPP, which was stored at 4°C protected from light.

[0022] Preparation of CoS-PEG-PPIX-SA (CPPS): CPP was dispersed in a beaker containing 10 mL of deionized water at room temperature. 24 mg of EDC and 24 mg of NHS were added and stirred for 30 min to obtain activated CPP. 20 mg of SA was dissolved in 10 mL of deionized water and mixed with the activated CPP solution. After stirring thoroughly for 12 h, the solution was dialyzed for 6 h (MWCO, 1000 Da) to obtain CPPS. The CPPS was then lyophilized and stored at 4 °C for later use.

[0023] 2. Characterization of CPPS The morphologies of CoS, CP, CPP, and CPPS were characterized using transmission electron microscopy. Figure 2 As can be seen in (a), the prepared CoS has a plate-like structure with a lattice spacing of 0.238 nm, corresponding to the (400) crystal plane. Figure 2 As shown in (bd), CP, CPP, and CPPS all exhibit regular sheet-like structures, indicating that the modification of PEG and the loading of PPIX and SA did not change the surface morphology of CoS.

[0024] Figure 3(a) shows the XRD patterns of CoS, CP, CPP, and CPPS. As shown in the figure, CoS exhibits strong diffraction peaks at 31.5°, 38.2°, and 55.1°, corresponding to the (311), (400), and (440) crystal planes of CoS, respectively. These characteristic peaks are consistent with the crystal structure of CoS. After coating the CoS surface with PEG, typical PEG diffraction peaks appear at 19.3° and 23.5°, corresponding to the (120) and (112) crystal planes of PEG. In CPP, a wide range of diffraction peaks appear in the 20~30° range, indicating that PPIX usually exists in an amorphous or low-crystallinity form. In CPPS, the crystal form did not change significantly after surface modification with SA, indicating that the modification with SA did not have a significant impact on the crystal structure of the material. The chemical composition and surface element valence states of CoS, CP, CPP, and CPPS were analyzed by XPS. Figure 3 (b) shows the XPS spectra of CoS, CP, CPP, and CPPS, revealing that the main elements contained in CoS, CP, CPP, and CPPS are Co, S, C, and O. Figure 3 As shown in the Co2p spectrum in (c), the peaks at 780.4 eV and 796.7 eV correspond to Co 2+ Co2p 3 / 2 With Co2p 1 / 2 Orbit. Furthermore, the 784.8 and 802.2 eV peaks correspond to two widely reported satellite peaks in the Co2p spectrum. In the S2p spectrum, the peaks at 162.0 eV and 161.0 eV correspond to S2... 2- and S 2- The spin-orbit coupling, the peak at 163.5 eV corresponds to CS bonds, and the peak at 168.5 eV corresponds to SO bonds. The formation of SO bonds may be due to the surface oxidation of nanoparticles. Figure 3 (d)). In the C1s spectrum ( Figure 3 In (e), the peak at 284.6 eV corresponds to the C=C / CC bond, while the peaks at 288.6 eV and 285.7 eV correspond to the C=O and CS bonds. Figure 3 (f) is the O1s spectrum of CPPS, with peaks at 531.1 eV and 533.4 eV at which they are attributed to C=O and CO bonds, respectively.

[0025] Figure 4(a) shows the UV-Vis and NIR absorption spectra of CoS, CP, CPP, and CPPS. CoS exhibits significant and widespread absorption characteristics in the near-infrared region. The UV-Vis and NIR absorption spectra of CP show that PEG encapsulation did not affect the characteristic UV absorption peaks of CoS. After loading PPIX onto the CP surface, a porphyrin Shore absorption band appeared at 405 nm, along with a weak Q band in the 500 nm–630 nm range; these characteristic peaks are consistent with the absorption characteristics of PPIX. The UV-Vis and NIR absorption spectrum of CPPS at 405 nm did not change significantly, indicating that SA modification did not alter the characteristic UV absorption peaks of CPPS. The optical properties of CPPS after surface modification with the photosensitizer PPIX were analyzed by fluorescence spectroscopy. Figure 4 As shown in (b), at the maximum excitation wavelength of 405 nm, the maximum emission peak of CPPS is located at 630 nm, indicating that CPPS retains the fluorescence properties of PPIX. The surface functional groups of CoS, CP, CPP, and CPPS were characterized by FT-IR. Figure 4 (c)). CoS at 528 cm -1 and 627 cm -1 Stretching vibration peaks of Co-S and cobalt atoms on the CoS surface were observed at 1100 cm⁻¹. -1 2900 cm -1 3400 cm -1 The broad peak at 996 cm⁻¹ corresponds to the stretching vibrations of COC, CH, and NH, confirming the presence of PEG in the composite material. CPP at 996 cm⁻¹... -1 and 3000cm -1 The absorption peak at 1725 cm⁻¹ is attributed to the pyrrole ring in PPIX and the NH and CH stretching vibrations of the pyrrole ring. -1 The absorption peak at 1650 cm⁻¹ is due to the C=O stretching vibration. CPP and CPPS also show absorption peaks at 1650 cm⁻¹. -1 The absorption peak at [location] is attributed to the C=O stretching vibration of the amide I band, confirming that PPIX and SA are loaded onto the CP surface via amide bonds formed through an amide reaction. Figure 4 (d) The Zeta potential diagrams for CoS, CP, CPP, and CPPS can be seen. The potential of CoS is -11.1 mV. After PEG coating, the potential of CP changes from -11.1 mV to 1.36 mV. After loading PPIX, the potential of CPP decreases to -13.1 mV. This may be due to the loading of negatively charged PPIX onto CPP. After surface modification with SA, the potential of CPPS is -16.93 mV.

[0026] 3. In vitro performance study of CPPS 3.1 Determination of in vitro chemokinetic properties of CPPS The ability of CPPS to generate hydroxyl radicals was determined using terephthalic acid (TA) as a fluorescent probe. The experiment consisted of five groups: ①TA + H₂O, ②TA + H₂O₂, ③CPPS + H₂O₂, ④TA + CPPS, and ⑤TA + CPPS + H₂O₂. The concentrations of TA and H₂O₂ were 5 mM and 10 mM, respectively, and the concentration of the nanomaterial was 100 μg / mL. The fluorescence intensity at 430 nm was measured after 30 min.

[0027] To further investigate the effect of pH on the generation of hydroxyl radicals, mixed solutions of TA (5 mM), H2O2 (10 mM), and CPPS (100 μg / mL) were prepared using PBS at different pH values ​​(5.5, 6.5, and 7.4) and shaken on a shaker at 37°C. The supernatant was aspirated every 2 min, and the fluorescence intensity at 430 nm was measured.

[0028] Terephthalic acid (TA) was used as a fluorescent probe to evaluate the chemokinetic properties of CPPS. Figure 5 As shown in (a), no obvious fluorescence signals were observed in the TA+H2O group, TA+H2O2 group, CPPS+H2O2 group, and TA+CPPS group, while the TA+CPPS+H2O2 group produced a significant fluorescence signal at 435 nm. This indicates that the TA+CPPS+H2O2 group generates hydroxyl radicals, which... Figure 5 (b) It is evident that the solution with pH 5.5 generated the most hydroxyl radicals at the same time interval. This result indicates that an acidic environment can significantly promote the catalytic generation of hydroxyl radicals from H₂O₂ by CPPS, thereby enhancing its chemodynamic therapeutic effect. In conclusion, CPPS exhibits excellent chemodynamic properties in vitro and can effectively catalyze the generation of hydroxyl radicals from H₂O₂.

[0029] 3.2 Evaluation of CPPS in vitro photothermal performance To explore the photothermal properties of CPPS, equal volumes of secondary water and CPPS solutions of different concentrations (50, 100, 150, and 200 μg / mL) were continuously irradiated for 10 min under an 808 nm laser (1.8 W). A 200 μg / mL CPPS solution was continuously irradiated for 10 min with 808 nm lasers of different power densities (0.5, 1, 1.5, and 1.8 W), and the temperature was recorded. Subsequently, the temperature changes of H2O, CoS, CP, CPP, and CPPS (200 μg / mL) under 808 nm laser irradiation (1.8 W) over 10 min were investigated, with solution temperatures recorded every 30 s. Furthermore, the photothermal stability of CPPS was evaluated and the photothermal conversion efficiency was calculated using five laser on / off cycles (10 min irradiation with the light source on, 10 min cooling with the light source off). η During the experiment, an infrared thermal imager (PS400) was used to monitor the temperature changes of H2O, CoS (200 μg / mL), CP (200 μg / mL), CPP (200 μg / mL), and CPPS (200 μg / mL) solutions, and the data were recorded every 2 minutes.

[0030] Formula (1-1) Formula (1-2) Formula (1-3) Formula (1-4) Formula (1-5) in ，h The heat transfer coefficient is... S The surface area of ​​the cuvette. T max The highest temperature of the solution. T surr For ambient temperature, Q dis The heat absorbed by the water and the quartz cuvette itself under laser irradiation. I It's the laser power. A The absorbance of the material at the laser wavelength is denoted as . τ s Let be the system heat transfer time constant. m For the mass of the solution, C water The specific heat capacity of water, t For time.

[0031] To investigate the in vitro photothermal effects of different materials, an 808 nm laser was selected as the light source, and thermocouple probes were used to detect the temperature change curves of different materials after laser irradiation. Figure 6 (a) Experimental results show that the temperature of H2O did not increase significantly after 10 min of laser irradiation, while the temperatures of CoS, CP, CPP, and CPPS increased to 61.8℃, 57.6℃, 56.0℃, and 55.0℃, respectively, after 10 min of laser irradiation. This result indicates that CPPS has good photothermal properties, and its photothermal heating capacity was not affected by the loading of PEG, PPIX, and SA. Figure 6 (b) and Figure 6 (c) shows that the temperature rise of CPPS increases significantly with increasing concentration and laser power, indicating that the material exhibits concentration- and laser power-dependent temperature rise. To verify the photothermal stability of CPPS, five heating and cooling cycles were performed on it. Figure 6(d) The results showed that the temperature of the CPPS solution remained stable throughout multiple cycles, demonstrating its good photothermal stability. Figure 6 (e) shows the infrared thermal images of H2O, CoS, CP, CPP, and CPPS under laser irradiation. This result confirms that CPPS has good photothermal imaging capabilities.

[0032] To further evaluate the photothermal conversion performance of CPPS, the linear relationship between the heating and cooling curves of CPPS and the cooling time and -ln(θ) was analyzed. Figure 7 By fitting the cooling curve, the τ of CPPS was obtained. s The value is 198. Calculations show that the photothermal conversion efficiency of CPPS is 53.6%, demonstrating its excellent photothermal effect and its potential as a photothermal material.

[0033] 3.3 Determination of in vitro photodynamic properties of CPPS The ABDA probe was used to detect singlet oxygen generated by CPPS during photodynamic therapy. 1 O2). When ABDA and 1 When O2 reacts, its absorbance at 380 nm decreases. Monitoring changes in absorbance can indirectly reflect… 1 O2 generation was investigated. The experiment consisted of five groups: ① ABDA+NIR group, ② ABDA+CoS+NIR group, ③ ABDA+CP+NIR group, ④ ABDA+CPP+NIR group, and ⑤ ABDA+CPPS+NIR group. The ABDA concentration was 0.05 mg / mL. In groups ②, ③, ④, and ⑤, the nanomaterial concentration was 100 μg / mL. The nanomaterials were irradiated with a 660 nm laser in the dark. The absorbance at 380 nm was measured and recorded at different time points (0, 5, 10, 15, 20, 25, and 30 min) after the start of irradiation, and the data were plotted.

[0034] like Figure 8 As shown, after 10 min of 660 nm laser irradiation, the UV absorbance values ​​of the ABDA+NIR group, ABDA+CoS+NIR group, and ABDA+CP+NIR group did not show a significant decreasing trend. However, the UV absorbance values ​​of the ABDA+CPP+NIR group and ABDA+CPPS+NIR group at 380 nm decreased significantly, indicating that CPPS has good ROS generation ability and can effectively respond to 660 nm laser irradiation.

[0035] 3.4 CPPS in vitro oxygen production To investigate the O2 release capacity of the material, 100 μg / mL of CoS and CPPS were added to a 1 mM H2O2 solution, and the changes in O2 concentration in the solution were monitored in real time using a dissolved oxygen meter (JPBJ-608).

[0036] To verify the characteristics of CPPS in catalyzing the decomposition of H2O2 to produce oxygen and its synergistic effect on PDT, the oxygen generation of different materials was quantitatively analyzed using a dissolved oxygen analyzer. For example... Figure 9 As shown, the oxygen concentration in the H2O2 group remained almost constant. However, the oxygen concentration increased rapidly after adding CoS and CPPS to the H2O2 solution, respectively. These results demonstrate that CoS and CPPS have good oxygen-producing capabilities, both decomposing H2O2 into O2 through the catalase activity of CoS.

[0037] 4. Cellular experiments with CPPS 4.1 Cytotoxicity assay The cytotoxicity of different concentrations of CPPS solution (0, 10, 20, 40, 60, 80, 100 μg / mL) was evaluated using the MTT assay. MCF-7 cells and HUVEC cells were seeded in 96-well plates and cultured for 24 h. The supernatant was discarded, and the cells were washed 2-3 times with PBS. Different concentrations of CPPS solution (0, 10, 20, 40, 60, 80, 100 μg / mL) were added to the 96-well plates, and the cells were cultured for another 24 h. The absorbance (OD) value of each well was measured using the MTT assay.

[0038] The therapeutic effect was evaluated by measuring the cell viability of each group. The experiment was divided into four groups: ① CPPS+H2O2 (CDT) group, ② CPPS+808 nm NIR (PTT) group, ③ CPPS+660 nm NIR (PDT) group, and ④ CPPS+H2O2+808 nm NIR+660 nm NIR (CDT+PDT+PTT) group. MCF-7 cells in each group were incubated with different concentration gradients of CPPS (0, 10, 20, 40, 60, 80, 100 μg / mL) for 24 h. Groups ② and ③ were irradiated with 808 nm and 660 nm lasers for 20 min, respectively, while group ④ was irradiated with 808 nm and 660 nm lasers simultaneously for 20 min. The same procedure was followed to evaluate the cell viability of each group.

[0039] To assess the cytotoxicity of CPPS, HUVEC and MCF-7 cells were used as models, and cell viability was detected using the MTT assay. Figure 10 As shown, when the CPPS concentration reached 100 μg / mL, the survival rate of both HUVEC cells and MCF-7 cells remained above 80%. This indicates that CPPS has good biocompatibility.

[0040] 4.2 Hemolysis test After centrifuging whole blood, the red blood cell pellet was collected, washed with physiological saline until the supernatant was colorless, and resuspended in physiological saline. Eight centrifuge tubes were used, with 500 μL of red blood cell suspension added to each tube. A positive control group (deionized water) and a negative control group (physiological saline) were set up as comparisons of the CPPS solution. Different concentrations of CPPS solution (10, 25, 50, 100, 150, 200 μg / mL) were added to the remaining tubes. The tubes were incubated at 37℃ for 1 h. The tubes were centrifuged (1300 rpm, 10 min) and photographed. The absorbance of the supernatant at 570 nm was measured using a microplate reader, and the hemolysis rate was calculated according to formula (1-6).

[0041] Formula (1-6) The blood compatibility of CPPS was investigated using a hemolysis test. Different concentrations of CPPS suspensions were co-incubated with red blood cells. Figure 11 As shown, no hemolysis was observed in the red blood cells of the negative control group, while significant hemolysis was observed in the red blood cells of the positive control group. No significant hemolysis was observed in any of the CPPS experimental groups at different concentrations. The calculated hemolysis rate for each experimental group was less than 5%, verifying that CPPS has good blood compatibility.

[0042] 4.3 Cell uptake experiment Using MCF-7 cells as a tumor cell model, the uptake effect of CPPS by the cells was evaluated. MCF-7 cells were uniformly seeded in confocal dishes and cultured for 24 h. Subsequently, they were incubated with CPPS (100 μg / mL) for different times (0, 2, 4, 6 h). After incubation, the supernatant was discarded, and the cells were washed 2-3 times with PBS. 1 mL of PBS was added, and images of cell uptake were acquired using a laser confocal microscope (CLSM).

[0043] like Figure 12 As shown, after 2 hours of incubation with CPPS, weak red fluorescence was observed in MCF-7 cells. The fluorescence gradually increased with prolonged incubation, reaching a stable state after 6 hours. The red fluorescence mainly originated from PPIX in CPPS, demonstrating that CPPS can be effectively taken up by MCF-7 cells.

[0044] The uptake of CPPS by HUVEC and MCF-7 cells was observed by laser confocal microscopy. Figure 13 The results showed that CPPS could be effectively taken up by MCF-7 cells, while normal cells took up less CPPS.

[0045] 4.4 Intracellular reactive oxygen species detection To further evaluate the chemodynamic and photodynamic properties of CPPS on tumor cells, the ROS production of CPPS in cells was detected using the DCFH-DA probe. Cells were divided into six groups: ①PBS, ②NIR+H2O2, ③CPPS, ④CPPS+H2O2, ⑤CPPS+660 nm NIR, and ⑥CPPS+H2O2+660 nm NIR. In groups ⑤ and ⑥, the H2O2 concentration was 50 mM, and in groups ③-⑥, the CPPS concentration was 100 μg / mL. Cells in each group were incubated with DCFH-DA (10 μM) for 30 min. Intracellular fluorescence was observed using CLSM at an excitation wavelength of 488 nm, and fluorescence intensity was analyzed using ImageJ software.

[0046] Depend on Figure 14 As can be seen, no significant fluorescence signal was observed after incubation of MCF-7 cells in the PBS group, NIR+H2O2 group, and CPPS group. Weak green fluorescence appeared in the CPPS+H2O2 group and the CPPS+660 nm NIR group, indicating that CDT and PDT induced a small amount of ROS generation. Notably, the green fluorescence was significantly enhanced in the CPPS+H2O2+660 nm NIR group, indicating that the synergistic effect of CDT and PDT significantly increased the level of intracellular ROS. This is because CPPS reacts with H2O2 to generate O2, increasing the level of ROS generated by PDT, thus more effectively exerting its anti-tumor effect. This experiment verifies the high efficiency of CPPS in synergistic chemodynamic and photodynamic therapy.

[0047] 4.5 In vitro antitumor effect of CPPS To evaluate the killing effect of different treatment groups on MCF-7 cells, the MTT assay was used to detect cell viability in each group. MCF-7 cells were incubated for 24 h with PBS, NIR+H2O2, CPPS (100 μg / mL), and CPPS (100 μg / mL)+H2O2 (50 mM)+808 nm NIR+660 nm NIR, respectively. Before the end of incubation, the CPPS+H2O2+808 nm NIR+660 nm NIR group was irradiated with 808 nm and 660 nm lasers for 20 min, respectively. After incubation, cells were washed 2-3 times with PBS, stained with acridine orange for 10 min, and images were acquired under CLSM.

[0048] like Figure 15As shown, the cell viability rates of the CPPS+H2O2 group, the CPPS+808 nm NIR group, and the CPPS+660 nm NIR group at a concentration of 100 μg / mL were 65.66%, 59.72%, and 58.98%, respectively, indicating that single treatment methods have a certain inhibitory effect on cell proliferation. However, the cell viability rate of the CPPS+H2O2+808 nm NIR+660 nm NIR group at a concentration of 100 μg / mL was significantly reduced to 24.82%. This result indicates that the synergistic effect of chemodynamic therapy, photothermal therapy, and photodynamic therapy can more effectively inhibit the proliferation of MCF-7 cells, demonstrating a significant therapeutic advantage.

[0049] In vitro cell apoptosis was assessed using live and dead cell staining assays. MCF-7 cells were randomly divided into seven groups and seeded in laser confocal dishes for comparative treatment: ① PBS group, ② NIR+H2O2 group, ③ CPPS group, ④ CPPS+H2O2 group, ⑤ CPPS+808 nm NIR group, ⑥ CPPS+660 nm NIR group, and ⑦ CPPS+H2O2+808 nm NIR+660 nm NIR group. The concentration of CPPS was 100 μg / mL for all groups, and the concentration of H2O2 was 50 mM. Groups ⑤, ⑥, and ⑦ were continuously irradiated with 808 nm and 660 nm lasers, respectively, for 20 min. After treatment, cells were washed with PBS, and live / dead cells were identified using Calcein-AM / PI simultaneous staining. Fluorescence images were obtained using CLSM.

[0050] Depend on Figure 16 As shown in (a), cells in the PBS group and the NIR+H2O2 group exhibited green fluorescence, indicating high cell viability and almost no cell death, while very little cell death was observed in the CPPS group. Cells in the CPPS+H2O2 group, CPPS+808nm NIR group, CPPS+660 nm NIR group, and CPPS+H2O2+808 nm NIR+660 nm NIR group showed different degrees of red and green fluorescence, while the red fluorescence intensity of the CPPS+H2O2+808 nm NIR+660 nm NIR group was significantly enhanced, indicating that CPPS has a good anti-tumor effect on cells. Figure 16 (b) shows the fluorescence intensity quantification of each group in the live-dead cell experiment. The fluorescence intensity quantification values ​​of the CPPS group, CPPS+H2O2 group, CPPS+808 nm NIR group, CPPS+660 nm NIR group, and CPPS+H2O2+808 nm NIR+660 nm NIR group are 4.59%, 30.65%, 38.89%, 42.83%, and 83.82%, respectively.

[0051] To evaluate the effects of different treatment groups on cell apoptosis, the Annexin V-FITC-PI apoptosis assay kit was used. MCF-7 cells were divided into seven groups for treatment: ① PBS group, ② NIR+H2O2 group, ③ CPPS group, ④ CPPS+H2O2 group, ⑤ CPPS+808 nm NIR group, ⑥ CPPS+660 nm NIR group, and ⑦ CPPS+H2O2+808 nm NIR+660 nm NIR group. The concentration of CPPS was 100 μg / mL for all groups, and the concentration of H2O2 was 50 mM. Groups ⑤, ⑥, and ⑦ were continuously irradiated with 808 nm and 660 nm lasers, respectively, for 20 min. After treatment, cells were digested with trypsin and collected. After washing with PBS, 500 μL of Annexin V-FITC-PI staining solution was added, and the cells were incubated in the dark for 15 min. Flow cytometry was used to detect cell apoptosis.

[0052] The results are as follows Figure 17 As shown, the apoptosis rates in the PBS group, NIR+H2O2 group, and CPPS group were all below 10%, indicating that apoptosis was not significantly induced in these groups. The apoptosis rates in the CPPS+H2O2 group, CPPS+808 nm NIR group, CPPS+660 nm NIR group, and CPPS+H2O2+808 nm NIR+660 nm NIR group were 35%, 35.64%, 46.9%, and 81.04%, respectively. This may be because CPPS can enter MCF-7 cells, and the photothermal effect generated under 808 nm laser irradiation can effectively kill cells; at the same time, photothermal activity can promote the catalytic activity of CPPS enzymes, catalyzing the production of more ·OH and O2 from H2O2, while O2 enhances the ability of CPPS to produce singlet oxygen under 660 nm laser. Under the combined effect of these mechanisms, the synergistic therapeutic effect of CDT, PTT, and PDT is significantly better than that of single therapy, further validating the application potential of CPPS in breast cancer treatment.

[0053] 4.6 Lysosomal membrane permeation experiment AO staining can detect the integrity of lysosomal membranes in MCF-7 cells. AO indicators typically show green fluorescence in the cytoplasm and nucleus, and red fluorescence in lysosomes. The disappearance of red fluorescence in the cells indicates that the lysosomal membrane has been damaged. Figure 18As shown, green and red fluorescence were simultaneously detected in cells from the PBS, NIR+H2O2, and CPPS groups, indicating that the integrity of the lysosomal membrane was not significantly damaged. However, almost no red fluorescence was observed in the CPPS+H2O2+NIR group, indicating severe damage to the lysosomal membrane. This may be because CPPS can rapidly enter the lysosomes of MCF-7 cells, and the photothermal effect generated under 808 nm laser irradiation can effectively destroy the lysosomal membrane. Furthermore, CPPS exhibits strong catalytic activity in the lysosomal environment (pH=4.5), catalyzing the production of ·OH and O2 from the high concentration of H2O2 in the tumor. Simultaneously, under 660 nm laser irradiation and self-oxygenated conditions, CPPS can enhance the production of singlet oxygen. These mechanisms work together to ultimately disrupt the lysosomal membrane structure, leading to lysosomal damage and subsequently inducing apoptosis.

[0054] 4.7 Detection of mitochondrial membrane potential damage To assess mitochondrial membrane potential damage, the mitochondrial membrane potential of cells in different treatment groups was measured using a mitochondrial membrane potential assay kit (JC-1). MCF-7 cells were cultured in confocal dishes for 24 h and incubated with CPPS (100 μg / mL) for 24 h. After discarding the culture medium, JC-1 staining solution was added, and the cells were incubated in the dark for 25 min. After staining, the cells were washed with PBS, and images were acquired under CLSM.

[0055] Mitochondria are organelles enclosed by a double membrane, primarily located in energy-demanding regions of the cell. They are the cell's energy supply centers and the main site of cellular aerobic respiration. Previous studies have shown that mitochondrial damage is a key and representative marker of apoptosis, and changes in mitochondrial membrane potential (MMP) can be used to assess mitochondrial damage. JC-1 is a fluorescent probe capable of entering the mitochondrial interior and accumulating in a potential-dependent manner, which can be used to detect changes in MMP. To further explore the mechanism of action of CPPS on apoptosis, we used the fluorescent probe JC-1 to assess the degree of MMP damage caused by CPPS. Figure 19As shown, the PBS group, NIR+H2O2 group, and CPPS group exhibited strong red fluorescence, indicating that these groups did not damage the mitochondrial membrane potential of the cells. However, the CPPS+H2O2 group, CPPS+808 nm NIR group, CPPS+660 nm NIR group, and CPPS+H2O2+808 nm NIR+660 nm NIR group showed significantly enhanced green fluorescence, indicating that these groups led to a decrease in mitochondrial membrane potential and impaired mitochondrial function, ultimately resulting in apoptosis. This confirms that the induction of apoptosis during treatment is achieved through the mitochondrial pathway.

[0056] 4.8 Western Blot Experiment To investigate the molecular mechanism of CPPS-induced apoptosis, the expression level of the apoptosis-related protein Caspase-3 in MCF-7 cells was detected by Western blotting. When the cell density reached 70%, cells were treated with PBS and CPPS + H2O2 + 808 nm NIR + 660 nm NIR, respectively. After treatment, cells were lysed with lysis buffer and centrifuged at 12000 rpm for 10 min at 4°C, and the supernatant was collected. Protein concentration was determined using a BCA protein assay kit. Equal amounts of total protein were separated by 12% SDS-PAGE electrophoresis and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk for 2 h, incubated overnight at 4°C with the corresponding primary antibody, washed with TBST, and incubated with horseradish peroxidase-labeled secondary antibody at room temperature for 1 h. Chemiluminescence signals were detected using a Tanon 5200 Multi fully automated chemiluminescence / fluorescence image analysis system.

[0057] Caspase-3 is a key executor in the early stages of apoptosis; its activation marks the entry of the apoptotic process into an irreversible phase. Figure 20 As shown, compared with the control group, the expression of Caspase-3 in MCF-7 cells treated with CPPS was significantly increased. This may be because CPPS, through the combined effects of chemodynamic therapy, photothermal therapy, and photodynamic therapy, damages mitochondria, releasing cytochrome C into the cytoplasm, further promoting the activation of Caspase-3, thereby leading to apoptosis of tumor cells.

[0058] 5. Evaluation of the in vivo antitumor effect of CPPS Establishment of a mouse tumor model: A mouse tumor model was established by injecting a suspension of 4T1 cells into the right axilla of six-week-old female Balb / c mice. The tumor model was established when the average tumor volume reached 100 mm². 3 Then, subsequent animal experiments began.

[0059] 5.1 Mouse fluorescence imaging The uptake and distribution of CPPS in animals were investigated using a small animal in vivo imaging system. To evaluate the accumulation and tissue distribution of CPPS at tumor sites, two tumor-bearing mice were injected with CPPS solution (1 mg / mL, 100 μL) via the tail vein. Images were acquired at different time points (0, 3, 6, 9, 12, 24, and 48 h) after CPPS injection. Nine h after injection, the other mouse was sacrificed, and its tumor and major organs were dissected and analyzed using in vitro fluorescence imaging.

[0060] like Figure 21 As shown in (a), after CPPS injection, mice initially showed a significant fluorescent signal in the liver. Over time, CPPS gradually accumulated in the tumor, with the fluorescent signal increasing progressively in the tumor region. Nine hours after injection, the fluorescent signal in the tumor reached its peak, indicating that CPPS accumulation in the tumor area was most significant at this time. In the in vivo antitumor activity study, near-infrared laser irradiation was performed at 9 hours after CPPS injection. Further observation revealed that the fluorescent signal in the tumor gradually weakened at 12 hours. At 24 hours, the fluorescent signal in the tumor became weak, with only the liver showing a significant fluorescent signal; at 48 hours, the fluorescent signal in the liver essentially disappeared. CPPS exhibited efficient tumor accumulation, confirming its good tumor targeting and metabolic clearance capabilities. Furthermore, to further verify the accumulation of CPPS in the tumor, mice were dissected at the 9-hour mark when the fluorescent signal was strongest, and major organs and tumor tissues were collected for in vitro fluorescence imaging analysis (e.g., CPPS in tumors). Figure 21 (b) The results showed that the results of ex vivo imaging were highly consistent with those of in vivo imaging, further confirming that CPPS mainly accumulated in the tumor region, while its distribution in other major organs was relatively small.

[0061] 5.2 Mouse photothermal imaging Infrared thermal imaging was used to evaluate the photothermal effects of each material group in vivo. In the in vivo thermal imaging experiment, five tumor-bearing mice were selected and injected via tail vein with saline, CoS, CP, CPP, and CPPS (1 mg / mL, 100 μL), respectively. Nine hours after injection, the tumor sites were irradiated with an 808 nm laser (1.8 W, 10 min), and the temperature changes in the tumor area during irradiation were recorded in real time using infrared thermal imaging.

[0062] like Figure 22 As shown, the temperature at the tumor site in the Saline group did not change significantly. Mice in the CoS, CP, CPP, and CPPS groups showed significant temperature increases under laser irradiation, but the magnitude of the temperature increase varied among the groups. Among them, the temperature increase at the tumor site was most significant in mice injected with CPPS. This may be due to the targeting effect of SA, which causes CPPS to accumulate in large quantities at the tumor site, thereby achieving efficient photothermal conversion, resulting in a more significant temperature increase in this group compared to other groups. Therefore, CPPS has good photothermal imaging capabilities and can achieve effective accumulation and photothermal conversion at the mouse tumor site.

[0063] 5.3 Evaluation of antitumor effects in mice To evaluate the antitumor effect of CPPS, a 4T1 tumor-bearing mouse model was established ( Figure 23 (a) When the tumor volume reaches 100 mm 3 In vivo antitumor treatment was initiated. Mice were randomly divided into 7 groups (n=5): ①PBS group, ②NIR group, ③CoS group, ④PPIX+660 nm NIR group, ⑤CPPS+808 nm NIR group, ⑥CPPS+660 nm NIR group, and ⑦CPPS+808 nm NIR+660 nm NIR group. The nanomaterials were injected into the mice via the tail vein. The concentration of the nanomaterials in all experimental groups was 1 mg / mL, and the injection volume was 100 μL, administered every other day for a total of seven administrations. Nine hours after each injection, groups ④-⑦ underwent laser irradiation of the tumor site for 30 minutes. During the treatment period, tumor volume and mouse weight were recorded every two days.

[0064] When the tumor volume in mice reaches 100 mm 3 Different groups of materials were injected via the tail vein to treat tumors, and tumor size and mouse weight were monitored every two days. Figure 23 (b) It can be seen that the weight of the mice did not fluctuate significantly during the treatment period. Figure 23 (c) and Figure 23 (d) Shows the changes in relative tumor volume and tumor weight in mice during treatment. Compared to other treatment groups, the relative tumor volume and weight of CPPS+808 nm NIR+660 nm NIR not only did not increase, but were also significantly ablated, indicating that CPPS exhibits excellent anti-tumor ability at the in vivo level. Images of mice 14 days after treatment ( Figure 23 (e) and images of ex vivo tumors ( Figure 23 (f) This further confirms the significant effect of CPPS nanocomposites in inhibiting breast cancer tumor growth. For example... Figure 23As shown in (g), partial ablation of tumor tissues was observed in the CoS group, PPIX+660 nm NIR group, CPPS+808 nm NIR group, and CPPS+660 nm NIR group. The tumor tissues in the CPPS+808 nm NIR+660 nm NIR group showed significant large-area ablation. These results further confirm that CPPS can effectively inhibit tumor cell growth, induce tumor cell apoptosis, and demonstrate excellent anti-tumor effects.

[0065] 5.4 In vivo biosafety evaluation Fourteen days after treatment, blood samples were collected from mice for routine blood tests and liver and kidney function tests. Mice were euthanized, and major organs (heart, liver, spleen, lungs, and kidneys) and tumor tissue were removed. Tissue samples were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. After hematoxylin-eosin (HE) staining, the samples were observed under a microscope and images were acquired.

[0066] The biocompatibility of CPPS nanocomposites was assessed using routine blood tests and biochemical indicators. Figure 24 The results of blood routine tests (including WBC, RBC, PLT, HCT, MCH, HGB, MCV, MCHC) and liver and kidney markers (including AST, ALT, TP, ALB, CREA, UREA) in mice of each group are shown. No significant differences were found between the treatment group and the control group in any of the indicators, indicating that CPPS did not induce significant inflammatory responses or liver and kidney dysfunction in mice, demonstrating good biocompatibility.

[0067] like Figure 25 As shown, HE staining results of the heart, liver, spleen, lungs, and kidneys of mice showed no obvious physiological morphological abnormalities in the major organs of mice, indicating that the materials in each group have good biocompatibility with normal mouse tissues.

Claims

1. A CoS-PEG-PPIX-SA nanocomposite, characterized in that: Including CoS-PEG nanocomposites, photosensitizers, and targeting agents; The CoS-PEG nanocomposite comprises CoS nanosheets and polyethylene glycol (PEG) coated on the surface of the CoS nanosheets. The photosensitizer and targeting agent are modified on the CoS-PEG surface; the photosensitizer is protoporphyrin (PPIX), and the targeting agent is sialic acid (SA).

2. The method for preparing the CoS-PEG-PPIX-SA nanocomposite according to claim 1, characterized in that, include: Step 1: CoS nanosheets are dispersed in an aqueous solution of NH2-PEG-COOH to obtain CoS-PEG. Step 2: Protoporphyrin (PPIX), (3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) are dispersed in dimethyl sulfoxide (DMSO) to obtain an activated PPIX solution. CoS-PEG is then mixed with the activated protoporphyrin (PPIX) solution and reacted to obtain CoS-PEG-PPIX. Step 3: CoS-PEG-PPIX is dispersed in deionized water, and (3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are added to obtain an activated CoS-PEG-PPIX solution. Sialic acid (SA) is dissolved in deionized water and mixed with the activated CoS-PEG-PPIX solution to obtain the target product CoS-PEG-PPIX-SA nanocomposite.

3. The method according to claim 2, characterized in that: In step one, the preparation method of CoS nanosheets is as follows: Co(NO3)2·6H2O, thioacetamide (TAA) and polyvinylpyrrolidone (PVP) are dissolved in deionized water, and then NaOH aqueous solution is added under stirring to obtain a mixture solution. The mixture solution is then subjected to a hydrothermal reaction to obtain CoS nanosheets.

4. The method according to claim 3, characterized in that: The hydrothermal reaction was carried out at a temperature of 100°C.

5. The method according to claim 3 or 4, characterized in that: The mass ratio of Co(NO3)2·6H2O, thioacetamide (TAA), and polyvinylpyrrolidone (PVP) is 291:150:

200.

6. The method according to claim 5, characterized in that: In step one, the concentration of the NH2-PEG-COOH aqueous solution is 2.0 mg / mL.

7. The method according to claim 2 or 6, characterized in that: In steps two and three, the mass ratio of (3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) to N-hydroxysuccinimide (NHS) is 1:

1.

8. The method according to claim 7, characterized in that: In steps one, two, and three, the reaction temperature is room temperature.

9. The use of the CoS-PEG-PPIX-SA nanocomposite according to claim 1 in the preparation of a drug for the synergistic treatment of breast cancer, wherein the synergistic treatment is a combination of chemodynamic therapy (CDT), photothermal therapy (PTT), and photodynamic therapy (PDT).

10. A drug for the synergistic treatment of breast cancer, characterized in that: It includes the CoS-PEG-PPIX-SA nanocomposite as described in claim 1 and a pharmaceutically acceptable carrier.

Images