CCS nano particle combining CuS, Cu < 2 + > and salvianolic acid B as well as preparation method and application of CCS nano particle

Abstract

The invention discloses CCS nanoparticles combining CuS, Cu < 2 + > and salvianolic acid B as well as a preparation method and application of the CCS nanoparticles, and belongs to the technical field of nano-drugs. The CCS NPs prepared by the invention has oxidation-reduction activity of Cu < 2 + > and Cu < + > at the same time, and can efficiently catalyze and generate a variety of active oxygen in illumination or tumor microenvironment. The multi-way ROS generation not only destroys lipid, nucleic acid and protein of cancer cells, but also can overcome the limitation of hypoxia of a tumor microenvironment, so that the treatment effect is enhanced. Through the synergistic effect of the SAB, the CCS NPs not only can remove harmful cell factors in a tumor microenvironment and improve the inflammatory state through ROS, but also can induce tumor cells to be 'copper death' through the oxidation-reduction reaction of Cu < + >. The comprehensive regulation of the tumor microenvironment provides a new treatment strategy for tumors which are difficult to treat.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanomedicine, and specifically relates to a method for combining CuS, Cu 2+ CCS nanoparticles of tannic acid B and salvianolic acid B, and preparation method and application thereof. Background Art

[0002] At present, many anti-tumor treatment methods, including photothermal therapy (PTT), photodynamic therapy (PDT), chemodynamic therapy (CDT) and drug therapy, often have the problem of insufficient efficacy of a single treatment method. For example, PDT depends on the oxygen supply in the tumor tissue, while the tumor microenvironment is usually in a hypoxic state; the ROS production efficiency of CDT is limited by the distribution of catalysts and reaction conditions. Therefore, developing new treatment methods to regulate tumor metabolic pathways and promote programmed cell death of tumor cells will effectively reduce the toxic side effects of traditional treatments, alleviate and reverse tumor resistance, and prolong patient survival.

[0003] Copper ion (Cu 2+ ) in tumor treatment mainly depends on its catalytic generation of reactive oxygen species (ROS). ROS has significant biological effects and can damage cell lipids, nucleic acids and proteins, leading to cell dysfunction and even inducing cell death. Studies have shown that excessive Cu 2+ It can significantly increase the level of ROS in the body, thereby destroying the function of cancer cells and inducing apoptosis and autophagy in a variety of cancer cells including breast cancer and lung cancer. 2+ , Cu + Due to its unique redox activity, it is more conducive to the precise elimination of cancer cells through the "copper death" mechanism, providing a new direction and idea for anti-tumor treatment. In the field of photothermal therapy, copper sulfide (CuS) nanomaterials have attracted widespread attention due to their unique optical properties and excellent biocompatibility. CuS nanoparticles have high photothermal conversion efficiency under near-infrared light (NIR) excitation, and can accurately heat and destroy tumor tissues, thereby achieving non-invasive and targeted tumor hyperthermia. In addition, CuS has good metabolic properties and low toxicity, making it a photothermal therapeutic agent with clinical potential. The single photothermal therapy function of CuS also has certain limitations: its therapeutic effect depends on the illumination conditions, the treatment depth is limited, and it is difficult to completely eliminate tumor tissue. More importantly, CuS itself has a weak ROS production ability and poor therapeutic effect on the hypoxic tumor microenvironment, which greatly limits its application in a single treatment mode. Therefore, in order to improve the treatment efficiency, CuS needs to work synergistically with other treatment methods.

[0004] Salvianolic acid B (SAB) is a natural polyphenol active substance with significant anti-inflammatory, antioxidant and anti-tumor activities. Studies have shown that salvianolic acid B can not only effectively inhibit tumor growth by regulating multiple signaling pathways, but also improve the inflammatory state in the tumor microenvironment and reduce the invasiveness of tumors. There are some challenges when salvianolic acid B is used alone as a drug, such as poor stability in the body and low bioavailability, which makes it difficult to achieve effective tumor-targeted delivery, limiting its clinical application potential.

[0005] The above existing technologies have significant limitations in the following aspects: 1. Limitation of ROS generation. 2+ Cu + Therapeutic methods that catalyze the generation of ROS have the problem of low therapeutic efficiency. Existing technologies generally lack the ability to generate ROS in multiple ways, especially under conditions of hypoxia in the tumor microenvironment, and their therapeutic effects are significantly limited. 2. Limitations of photothermal therapy. Copper sulfide (CuS) nanomaterials have attracted attention due to their high photothermal conversion efficiency, but their photothermal treatment depth depends on near-infrared light irradiation conditions, making it difficult to completely eliminate tumors, and the therapeutic effect is easily affected by the location of the tumor and the depth of light penetration. 3. The complexity of the tumor microenvironment. The hypoxia and inflammatory state of the tumor microenvironment significantly affect the therapeutic effect. Existing treatment methods generally fail to effectively combine the anti-inflammatory and ROS generation mechanisms of action, resulting in reduced treatment efficiency. 4. Biosafety and stability issues. The potential toxicity risks and insufficient chemical stability of conventional CuS nanomaterials after metabolism in the body limit their widespread clinical application. To overcome these limitations, multifunctional synergistic treatment strategies have gradually become a research hotspot. Summary of the invention

[0006] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0007] In order to overcome the above limitations, the present invention has developed a novel nanostructured carbon nanotube with copper sulfide (CuS) as the core, salvianolic acid B (SAB) and copper ions (Cu 2+ ) as the shell layer of multifunctional synergistic nanoparticles (CCS NPs).

[0008] The present invention combines copper sulfide nanoparticles (CuS NPs) and a shell layer formed by combining natural polyphenolic compound salvianolic acid B (SAB) and copper ions. By integrating the light-heat conversion properties of CuS NPs with SAB and copper ions (Cu 2+) to construct a synergistic treatment system that combines precision treatment and microenvironment regulation. 2+ Combined with other materials, the synergistic effect can significantly improve the tumor treatment effect. 2 + -SAB, CCS NPs) not only have Cu 2+ and Cu + The ability to catalyze the production of multiple ROS also enables the combined therapeutic effect of chemodynamic therapy (CDT) and photodynamic therapy (PDT) due to its unique nanostructure. This synergistic ROS production property has shown significant advantages in inhibiting tumor growth and provides a new strategy for the development of highly effective anti-tumor nanomedicines.

[0009] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0010] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for combining CuS, Cu 2+ and CCS nanoparticles of salvianolic acid B.

[0011] In order to solve the above technical problems, the present invention provides the following technical solutions, including: the CCS nanoparticles are copper sulfide@copper ion-salvianolic acid B nanoparticles, which are composed of copper sulfide, salvianolic acid B and Cu 2+ The solution is reacted to obtain;

[0012] Wherein, the copper sulfide, salvianolic acid B and Cu 2+ The mass ratio is 1:0.5~2:0.4~1.6.

[0013] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for combining CuS, Cu 2+ and a method for preparing CCS nanoparticles of tannic acid B.

[0014] In order to solve the above technical problems, the present invention provides the following technical solutions, including:

[0015] Cupric chloride and polyvinyl pyrrolidone are mixed in deionized water, and sodium hydroxide solution and hydrazine hydrate solution are added under stirring to form a cuprous oxide suspension; sodium sulfide aqueous solution is then added, and the reaction is continued at 50-80°C with stirring for 1-3 hours; after the reaction is completed, the precipitate is cooled, centrifuged and washed to obtain copper sulfide nanoparticles, which are recorded as CuS NPs;

[0016] After copper sulfide is prepared into an aqueous solution, it is added to the salvianolic acid B aqueous solution under stirring, and then a cupric chloride solution is added and stirring is continued. The precipitate is separated by centrifugation, washed to remove impurities, and freeze-dried to obtain copper sulfide@copper ion-salvianolic acid B nanoparticles, recorded as CCS NPs.

[0017] As a preferred embodiment of the method for preparing CCS nanoparticles of the present invention, the CCS NPs are of a core-shell structure, wherein the core is copper sulfide and the shell is Cu 2+ and salvianolic acid B.

[0018] As a preferred embodiment of the method for preparing CCS nanoparticles of the present invention, the particle size of the CCS NPs is 400-2000 nm.

[0019] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a CCS nano drug for use in the preparation of anti-tumor drugs, drug delivery, and tumor cell killing.

[0020] The purpose of the present invention is to overcome the deficiencies in the prior art and provide an anti-tumor drug comprising the CCSNPs and other pharmaceutically acceptable adjuvants.

[0021] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for killing tumor cells.

[0022] As a preferred embodiment of the tumor cell killing method of the present invention, the CCS NPs are combined with near-infrared light irradiation to provide local high temperature to accurately kill tumor cells.

[0023] As a preferred embodiment of the tumor cell killing method of the present invention, the near-infrared light irradiation has a wavelength of 808 nm and an illumination intensity of 0.2 to 1.0 W / cm 2 The illumination time is 2 to 10 minutes, and the killing effect is consolidated by multiple cycles of the killing method.

[0024] As a preferred embodiment of the tumor cell killing method of the present invention, the concentration of the CCS NPs is 12.5-200 μg / mL.

[0025] As a preferred embodiment of the tumor cell killing method of the present invention, the CCS NPs react with CuS core through the photothermal effect. 2+ , and the SAB shell regulate the tumor microenvironment.

[0026] Beneficial effects of the present invention:

[0027] (1) Synergistically enhanced ROS generation ability. +and Cu 2+ The coexistence of 2 and 3 makes the nanoparticles have dual-path ROS generation ability, which can generate ·OH through Fenton reaction and O through redox cycle. 2 · - Combined with near-infrared light irradiation, it further promotes ROS generation, enhances the oxidative stress level of tumor cells, and significantly improves the treatment efficiency.

[0028] (2) Multifunctional synergistic therapy. The present invention combines the multimodal synergistic effects of photothermal therapy and chemodynamic therapy. CCS NPs achieve efficient photothermal conversion under near-infrared light excitation. 2+ and Cu + The redox reaction can continuously generate ROS, achieving a deep synergistic effect of photothermal therapy (PTT) and chemodynamic therapy (CDT). Experiments have shown that the synergistic effect significantly reduces the survival rate of cancer cells, effectively overcoming the limitations of a single treatment method.

[0029] (3) Enhanced targeting and reduced side effects: Compared with traditional photothermal therapy or single chemotherapy drugs, CCS nanoparticles can be used to 2+ The targeting effect of CuS and SAB enhances the aggregation at the tumor site and reduces the side effects on normal tissues. The photothermal effect of CuS can be precisely concentrated at the tumor site, improving the local effect of treatment and reducing systemic side effects.

[0030] (4) Regulate the tumor microenvironment and improve the therapeutic effect. The shell of the nanoparticles introduces salvianolic acid B (SAB), which has anti-inflammatory and antioxidant properties and can effectively regulate the tumor microenvironment. SAB inhibits pro-inflammatory signaling pathways, reduces the level of inflammatory factors, and promotes oxidative stress in tumor tissues, thereby improving the targetedness and efficiency of treatment.

[0031] (5) Good biocompatibility and chemical stability. CCS NPs use an optimized CuS core in their design, which significantly improves their chemical stability and metabolic properties, reducing the risk of toxicity. The introduction of salvianolic acid B further improves the biocompatibility of the nanoparticles, giving them good safety in the body and providing a guarantee for clinical application.

[0032] (6) Optimize the synthesis process to achieve large-scale preparation. 2+ The combined process of the two methods and the one-step assembly strategy not only improve the production efficiency but also ensure the structural uniformity and functional stability of the particles. The simplified process makes it more suitable for large-scale production and meets the potential needs of clinical transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work. Among them:

[0034] Figure 1 The low-magnification TEM image (a), high-magnification TEM image (b), high-resolution TEM image (c) and element distribution map (d) of CCS NPs prepared in Example 1 of the present invention.

[0035] Figure 2 This is the UV-visible absorption spectrum of CCS NPs prepared in Example 1 of the present invention.

[0036] Figure 3 This is a particle size distribution diagram of CCS NPs and CuS NPs prepared in Example 1 of the present invention.

[0037] Figure 4 This is a Zeta potential analysis diagram of CCS NPs and CuS NPs prepared in Example 1 of the present invention.

[0038] Figure 5 X-ray diffraction patterns of CCS NPs and CuS NPs prepared in Example 1 of the present invention.

[0039] Figure 6 This is a comparison chart of the UV-visible absorption spectra of CCS NPs and CuS NPs prepared in Example 1 of the present invention within the range of 300 to 900 nm.

[0040] Figure 7 Comparison of Fourier transform infrared spectra of SAB, CuS NPs and CCS NPs.

[0041] Figure 8 Different concentrations of CCS NPs (12.5-200 μg / mL) at 1.0 W / cm 2 Temperature rise curve under near-infrared light (808nm).

[0042] Fig. 9 At a fixed concentration (200 μg / mL), different light intensities (0.2-1.0 W / cm 2 ) on the photothermal effect of CCS NPs.

[0043] Fig.10 For CCS NPs at 1.0 W / cm 2 Photothermal stability test under illumination.

[0044] Fig.11 These are infrared thermal imaging photos of different concentrations.

[0045] Fig.12 These are infrared thermal imaging photos under different light intensity conditions.

[0046] Fig.13 For CCS NPs at 1.0 W / cm 2 Calculation results of photothermal conversion efficiency under illumination.

[0047] Fig.14 Schematic diagram of the CDT (chemodynamic therapy) process.

[0048] Fig.15 UV-Vis absorption spectra under different experimental conditions.

[0049] Fig.16 Schematic diagram of the PDT (photodynamic therapy) process.

[0050] Fig.17 The fluorescence spectrum changes with time.

[0051] Fig.18 The effects of different concentrations (25-200 μg / mL) of CuS NPs and CCS NPs on cell viability.

[0052] Fig.19 The figures are the hemolysis rate data at different concentrations (25-400 μg / mL) and the corresponding experimental photos showing the results of the hemolysis test.

[0053] Fig. 20 The figure shows the cell killing effect of CuS and CCS nanoparticles with different concentrations under the condition of near-infrared (NIR) light.

[0054] Fig.21 (a) The 2p spectrum of Cu and (b) S in CuS NPs, (c) The 2p spectrum of Cu and (d) S in CCS NPs. DETAILED DESCRIPTION

[0055] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0056] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0057] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0058] Copper sulfide@copper ion-salvianolic acid B (CuS@Cu 2+ -SAB, abbreviated as CCS), nanoparticles (NPs), and salvianolic acid B (SAB).

[0059] The raw materials used in the present invention are all commercially available unless otherwise specified.

[0060] The performance of the material prepared in the embodiment of the present invention was tested as follows:

[0061] 1. Extracellular hydroxyl radicals (·OH) and singlet oxygen ( 1 O 2 ) generated detection:

[0062] Tetramethylbenzidine (TMB) was used as a probe to detect the generation of ·OH in the solution. In this experiment, the pH-dependent generation of ·OH was detected. Nanoparticles (NPs) were added to TMB solutions with different pH values ​​(7.4, 6.5). In the specific experimental steps, 500 μL of H was added to the experimental group containing 500 μL of solution. 2 O 2 and 50 μL of TMB. Subsequently, the mixture was incubated for 10 minutes in dark conditions. After that, the absorbance was measured using a wavelength of 652 nm. The experimental materials were divided into three groups, namely: 1) CuS NPs with a concentration of 200 μg / mL, 2) CCS NPs with a concentration of 200 μg / mL, and 3) CCS NPs with a concentration of 200 μg / mL under 808 nm laser irradiation. 2 O 2 The treated TMB solution was used as the control group. To ensure the accuracy and reliability of the experiment, each group of samples was tested in parallel 3 times.

[0063] 1 O 2 The generation of CCS nanoparticles was detected by recording the absorbance change of diphenylisobenzofuran (DPBF) at 415 nm. 1 mL of 200 μg / mL CCS nanoparticles was mixed with 15 μL of 1.25 mg / mL DPBF and the mixture was heated at 808 nm with a power of 0.6 W / cm 2 The power density was irradiated for different time periods. To ensure accuracy and reliability, each group of samples was tested three times in parallel.

[0064] 2. Photothermal performance testing:

[0065] To investigate the photothermal properties of CCS nanoparticles, a FLIR E5 infrared imaging camera (FLIR Systems AB, Sweden) was used to record the photothermal activity of CCS nanoparticles at different power densities (0.2, 0.4, 0.6, and 0.8 W / cm 2 The temperature changes of CCS nanoparticle solutions with different concentrations were recorded every 20 seconds under 808nm laser irradiation. The final photothermal treatment condition was 0.6W / cm 2 The power density was irradiated for 10 minutes. The same photothermal treatment conditions were used in subsequent in vitro and in vivo experiments.

[0066] For the photothermal stability, the CCS nanoparticle solution was subjected to a power density of 0.6 W / cm 2 The samples were irradiated with 808 nm laser for 10 minutes and then cooled to room temperature. This process was repeated for four cycles and the temperature was recorded every 20 seconds. The photothermal conversion efficiency (η) of CCS nanoparticles was calculated according to the following formula:

[0067]

[0068] Where h: thermal conductivity of the sample (W / (m 2 K)); S: surface area of ​​the sample (m 2 );

[0069] T max : The highest temperature of the sample under illumination (K); T surr : Temperature of the environment where the sample is located (K); Q S : Heat loss of the sample in other forms (W); I: Light intensity of the laser irradiating the sample (W / m 2 );A 808 : absorbance of the sample at 808 nm wavelength; 10 -A808 is the transmittance, which represents the proportion of light remaining through the sample at a wavelength of 808 nm, 10 -A808 The smaller the value, the more light the sample absorbs. -A808 It is the absorbance of the sample to 808nm light.

[0070] Example 1

[0071] This embodiment provides a copper sulfide@copper ion-salvianolic acid B (CuS@Cu 2+ The preparation method of CCS nanoparticles (NPs) adopts the method of coordinated coordination and physical adsorption, specifically:

[0072] The cupric chloride solution (67 g / L, 100 μL) was mixed with polyvinyl pyrrolidone PVP (0.24 g) in deionized water (25 mL), and magnetic stirring was performed at room temperature. Sodium hydroxide solution (pH = 9, 25 mL) and 80 wt.% hydrazine hydrate solution (6.4 μL) were added in sequence to form a cuprous oxide suspension. Then, a sodium sulfide aqueous solution (320 mg / mL, 200 μL) was added to the cuprous oxide suspension and stirred at 60 ° C for 2 h. After the reaction was completed, the mixture was cooled to room temperature, and the precipitate was separated by centrifugation (11000 r / min, 10 min), and then washed twice with deionized water to obtain pure copper sulfide nanoparticles (CuS NPs).

[0073] CuS NPs were prepared into a copper sulfide aqueous solution (1 mg / mL, 60 mL) and added to a salvianolic acid B aqueous solution (7.5 mg / mL, 8 mL) under gentle stirring. Then, copper chloride solution (25 mg / mL, 2 mL) was added to the mixture and stirred for 10 min (i.e., copper sulfide, salvianolic acid B and CuS NPs were mixed). 2+ The mass ratio of the precipitate to the precipitate was 1:1:0.83). Afterwards, the precipitate was separated by centrifugation and washed three times with deionized water to remove impurities. Finally, the washed precipitate was freeze-dried to obtain CCS NPs.

[0074] Figure 1 (a) is the low-magnification transmission electron microscopy (TEM) characterization of CCS NPs, which shows that these nanoparticles have a relatively regular core-shell structure and uniform particle size distribution; Figure 1 (b) High-magnification transmission electron microscopy characterization of CCS NPs, with a shell thickness of 33.8 nm; Figure 1 (c) High-resolution transmission electron microscopy (HRTEM) characterization of CCS NPs shows that the interplanar spacing is 0.325 nm, which has clear crystal structure characteristics. Figure 1 (d) is the element distribution map, showing the distribution of copper (Cu), sulfur (S), oxygen (O) and nitrogen (N).

[0075] Figure 2 is the ultraviolet-visible absorption spectrum (UV-Vis) of CCS. As the concentration increases, the absorption peak gradually increases, indicating good concentration dependence.

[0076] Figure 3 The particle size distribution diagram (DLS) shows that the particle size distribution of CuS is concentrated at about 200nm, while the particle size distribution of CCS increases significantly to about 1000nm. Figure 4For Zeta potential analysis, CuS is about -30 mV, and the surface potential of CCS decreases slightly, but still remains in the negative range, indicating that it has a certain colloidal stability; Figure 5 XRD patterns of CuS and CCS.

[0077] Figure 6 The UV-visible absorption spectra of CuS and CCS in the range of 300-900nm are compared. The absorption peak intensity of CCS is higher than that of CuS, especially in the range of 700-900nm, indicating that the introduction of the shell layer significantly enhances the light absorption capacity of the material. Figure 7 Comparison of Fourier transform infrared (FTIR) spectra of SAB, CuS and CCS. The absorption peak characteristics of CCS overlap with the characteristic peaks of SAB and CuS, indicating the successful coating of SAB by shell and the interaction between shell and core.

[0078] CCS NPs have excellent photothermal properties, including good concentration and power dependence, cycling stability, and efficient photothermal conversion characteristics, which make them have great potential in applications such as photothermal therapy. Figure 8 to Figure 13 Demonstrated photothermal properties of CCS NPs:

[0079] Figure 8 The results show that CCS NPs with a concentration of 12.5 to 200 μg / mL can effectively inhibit the ionization of CCS at 1.0 W / cm 2 Temperature rise curve under near-infrared light (808nm). The results show that the photothermal effect of CCS NPs is positively correlated with concentration. As the concentration increases, the temperature rises more significantly. The 200μg / mL sample reaches about 60°C within 600 seconds, while the temperature change of the PBS control group is minimal, verifying the photothermal conversion ability of CCSNPs.

[0080] Fig. 9 We further investigated the effect of different light intensities (0.2-1.0 W / cm 2 ) on the photothermal effect of CCS NPs. With the increase of light intensity, the temperature rise of the sample is significantly accelerated, 1.0W / cm 2 The maximum temperature under different light intensities exceeded 60 °C, indicating that light intensity is an important factor affecting the photothermal properties of CCS NPs.

[0081] Fig.10 CCS NPs were shown to be able to withstand 1.0 W / cm 2 Photothermal stability test under illumination. After four on / off cycles, the temperature change remained consistent without significant attenuation, indicating that CCS NPs have excellent photothermal cycling stability.

[0082] Fig.11 and Fig.12 The infrared thermal imaging photos under different concentrations and light intensities show the trend of the photothermal effect changing with time and conditions. These thermal imaging results are consistent with the temperature curve, further verifying the photothermal performance of CCS NPs.

[0083] Fig.13 For CCS NPs at 1.0 W / cm 2 Calculation results of photothermal conversion efficiency under illumination. Through linear fitting of the heating and cooling process, the photothermal conversion efficiency of CCS is 73.14%, and the fitting degree R 2 =0.998, indicating that CCS NPs have high photothermal conversion efficiency.

[0084] CCS NPs have the ability to simultaneously generate multiple ROS, including ·OH and 1 O 2 This synergistic ROS production property gives it a significant advantage in tumor treatment and can achieve the combined therapeutic effect of CDT and PDT. Figure 14 to Figure 17 The ROS-generating properties of CCSNPs were demonstrated.

[0085] Fig.14 This is a schematic diagram of the CDT (chemodynamic therapy) process. In this process, TMB (tetramethylbenzidine) is used as an indicator and can be detected by Cu 2+ and H 2 O 2 The hydroxyl radical (·OH) produced by the reaction is oxidized to the blue oxidized TMB (oxTMB). This reaction mechanism demonstrates the catalytic effect of CCS NPs in CDT.

[0086] Fig.15 The UV-Vis absorption spectra under different experimental conditions. The curve shows a characteristic absorption peak around 650nm, which is the typical absorption of oxTMB and confirms the generation of ·OH. The difference in absorption intensity under different conditions reflects the change in the efficiency of ROS generation.

[0087] Fig.16 Schematic diagram of the PDT (photodynamic therapy) process. In this process, DPBF (1,3-diphenylisobenzofuran) is used as singlet oxygen ( 1 O 2 ) probe molecules can be 1 O 2 Oxidized to DBB (diphenylbenzofuran). This reaction is used to detect the PDT process 1 O 2 The production of.

[0088] Fig.17The fluorescence spectrum of DPBF changes with time. As the reaction time increases (0-10 min), the fluorescence intensity of DPBF decreases gradually, indicating that 1 O 2 The fluorescence intensity decreases and the CCSNPs are continuously produced and react with DPBF. 1 O 2 ability.

[0089] CCS NPs have good biocompatibility and hemocompatibility, and show significant antitumor effects under NIR light irradiation, demonstrating their potential application as photothermal therapeutic agents. Figures 18 to 20 The antitumor effect of CCS NPs in vitro was demonstrated.

[0090] Fig.18 The effect of different concentrations (25-200 μg / mL) of CuS NPs and CCS NPs on cell viability. The data show that even at a higher concentration (200 μg / mL), both nanoparticles exhibited low cytotoxicity and the cell survival rate was maintained above 75%. This result confirms the good biocompatibility of the material.

[0091] Fig.19 The results of the hemolysis test are shown, including the hemolysis rate data at different concentrations (25-400 μg / mL) and the corresponding experimental photos. The blood precipitation at the bottom of the test tube indicates that the membrane structure of the red blood cells remains intact and no hemolysis occurs. In contrast, the ultrapure water group caused the red blood cells to rupture due to the hypotonic environment, showing hemolysis. The hemolysis rate at all tested concentrations was less than 2%, far below the safety standard (5%), indicating that CCS NPs have excellent blood compatibility and are suitable for biomedical applications.

[0092] Fig. 20 The cell-killing effects of different concentrations of CuS NPs and CCS NPs were compared with and without near-infrared (NIR) light. The results showed that cell survival was high when the nanoparticles were used alone. After the addition of NIR light, cell survival was significantly reduced, especially at high concentrations (200 μg / mL). The photothermal therapy effect of the CCS group was better than that of the CuS group, reducing cell survival to below about 20% at a concentration of 200 μg / mL.

[0093] XPS analysis of CCS NPs. Fig.21 (a) and (b) are the 2p spectra of Cu and S in CuS NPs, respectively; (c) and (d) are the 2p spectra of Cu and S in CCS NPs, respectively. Figure (c) shows that compared with CuS, the Cu 2p spectra of CCS samples have a higher concentration of Cu 2+ and Cu +The characteristic peaks are still visible, but Cu + This indicates that in CCS NPs, Cu + The proportion of SAB may increase, thus contributing more significantly to its catalytic activity and the "copper death" mechanism. (d) This indicates that SAB coating may change the chemical state of sulfur on the surface of nanoparticles, which helps them to coordinate ROS production and improve the tumor microenvironment.

[0094] Example 2

[0095] The difference between this embodiment and embodiment 1 is that the copper sulfide, salvianolic acid B and Cu 2+ The mass ratio of is 1:0.5:0.4, and the rest of the preparation process is the same as that in Example 1 to obtain CCS NPs.

[0096] Through testing, the particle size of CCS NPs is about 1000nm.

[0097] Example 3

[0098] The difference between this embodiment and embodiment 1 is that the copper sulfide, salvianolic acid B and Cu 2+ The mass ratio of is 1:2:1.6, and the rest of the preparation process is the same as that in Example 1 to obtain CCS NPs.

[0099] Through testing, the particle size of CCS NPs is about 1000nm.

[0100] Comparative Example 1

[0101] The difference between this comparative example and Example 1 is that the mass of PVP is adjusted to 0.03 g, and the rest of the preparation process is the same as that of Example 1 to prepare CCS NPs.

[0102] The particle size distribution of CCS NPs is not uniform. When the amount of PVP is insufficient, the protection of the particle surface is insufficient, which makes the nanoparticles easily aggregate and form particles of different sizes. This aggregation phenomenon may lead to uneven particle size, and then generate larger particles or aggregates.

[0103] Comparative Example 2

[0104] The difference between this comparative example and Example 1 is that the volume of the sodium hydroxide solution is adjusted to 5 mL (pH=9), and the rest of the preparation process is the same as that of Example 1, and a cuprous oxide suspension cannot be formed.

[0105] When NaOH dissolves in a solution, it makes the solution alkaline and provides OH - These OH - With Cu 2+ Reaction to form copper hydroxide Cu(OH)2 Precipitation, followed by Cu(OH) 2 In the presence of hydrazine hydrate, it is further reduced to cuprous oxide (Cu 2 O), Cu(OH) 2 It forms a precipitate in water. To obtain Cu 2 O suspension, Cu 2+ Under higher pH conditions, Cu 2 O nanoparticles and keep them uniformly dispersed in the solution. If the amount of NaOH solution added is insufficient, the pH value of the solution is not enough to promote the Cu(OH) 2 The formation and further reduction to Cu 2 O, a uniform suspension will not be formed.

[0106] Comparative Example 3

[0107] The experimental groups of this comparative example are as follows:

[0108] CuS NPs group: Tumor cells were treated with different concentrations (25, 50, 100, 200 μg / mL) of CuS NPs.

[0109] CCS NPs group: Tumor cells were treated with different concentrations (25, 50, 100, 200 μg / mL) of CCS NPs.

[0110] NIR irradiation group: The tumor cells in the above two groups were treated with near-infrared light (808nm, 1.0W / cm 2 , irradiation for 10 min).

[0111] Blank control group: only culture medium treatment, without nanoparticles and NIR irradiation.

[0112] The operation is as follows:

[0113] Tumor cells (such as HepG2) were cultured in DMEM medium containing 10% fetal bovine serum until the logarithmic growth phase. The cells were seeded in 96-well plates (1×10 4 / well), and different concentrations of CuS NPs or CCS NPs were added and incubated for 6 hours. The drug-containing medium in the well plate was replaced with fresh medium. Subsequently, the NIR group was irradiated with 808 nm (1.0 W / cm 2, 10min), and the non-NIR group did not receive any treatment. The cells were incubated for 18 hours. The MTT method was used to detect cell viability: MTT reagent was added, and after incubation for another 4 hours, DMSO was used to dissolve the formazan crystals and the absorbance at 490nm was measured. The cell viability of each group was calculated (%) = (absorbance of the experimental group / absorbance of the blank control group) × 100%. The data were expressed as mean ± standard deviation, and the differences between the groups were compared using t-test (p < 0.05 was significant).

[0114] 1) Cell survival rate of nanoparticles alone

[0115] CuS NPs group: When the concentration of CuS NPs was 25 μg / mL, the survival rate of tumor cells was 98.5%±3.5%, and when the concentration increased to 200 μg / mL, the survival rate of tumor cells was still 50.8%±5.6%, indicating that CuS NPs had limited cytotoxicity to tumor cells under this condition.

[0116] CCS NPs group: When the concentration of CCS NPs was 25 μg / mL, the survival rate of tumor cells was 91.3%±2.8%; when the concentration was 200 μg / mL, the survival rate of tumor cells decreased to 41.5%±4.3%. Compared with CuS NPs, CCS NPs had a lower survival rate of tumor cells at the same concentration, indicating that Cu 2+ / SAB shell enhances the cytotoxicity of the material.

[0117] 2) Synergistic effect under NIR irradiation

[0118] When the concentration of CuS NPs was 25 μg / mL, the survival rate of tumor cells was 85.2% ± 3.2%; when the concentration increased to 200 μg / mL, the survival rate of tumor cells decreased to 24.2% ± 4.9%. In comparison, when the concentration of CCS NPs was 25 μg / mL, the survival rate of tumor cells was 79.6% ± 3.2%; when the concentration was 200 μg / mL, the survival rate of tumor cells decreased significantly to 8.7% ± 2.2%. This shows that CCS NPs exhibit more significant cytotoxicity than CuS NPs under NIR. This shows that the photothermal effect (CuS heat generation) and chemical toxicity (Cu 2+ / SAB release) synergistically, significantly enhancing the inhibitory effect on tumor cell growth.

[0119] 3) Statistical analysis

[0120] The CCS NPs+NIR group showed significant difference compared with other groups (p<0.01), verifying the synergistic anti-tumor mechanism.

[0121] In summary, the present invention has developed a copper sulfide (CuS) core, copper ions (Cu 2+) and salvianolic acid B (SAB) as the shell of multifunctional synergistic nanoparticles (CCS NPs). Copper ions and salvianolic acid B are co-coated on the surface of copper sulfide nanoparticles through interaction. The network structure (Cu 2+ -SAB). Therefore, copper ions and salvianolic acid B are co-coated on the surface of copper sulfide, synergistically enhancing the therapeutic effect of nanoparticles. 2+ The distribution of SAB in the shell may be uniform, forming a stable structure, which not only helps the generation of ROS, but also improves the tumor microenvironment and enhances the therapeutic effect.

[0122] CuS is used as the core material, and its excellent photothermal effect under near-infrared light irradiation can effectively photothermally kill tumor cells. This solution precisely controls the photothermal effect to provide local high temperature to accurately destroy tumor cells and avoid damage to normal tissues.

[0123] Cu 2+ The SAB shell has a synergistic effect. 2+ Combined with SAB. Cu 2+ It can improve the biocompatibility of nanoparticles and may play an important regulatory role in the tumor microenvironment through its chemical properties; SAB, as a natural polyphenol, has anti-inflammatory and anti-tumor properties, can effectively regulate the tumor microenvironment, reduce oxidative stress, and enhance immune response.

[0124] Design of drug delivery system: The present invention uses CuS as the core, Cu 2+ The core-shell structure design with SAB as the shell enables the drug delivery system to not only deliver targeted drugs to tumor tissues, but also effectively regulate the tumor microenvironment. This design improves the targeting of treatment, reduces side effects, and enhances the regulation of the tumor microenvironment.

[0125] The present invention combines copper sulfide nanoparticles (CuS NPs) with copper ions (Cu 2+ ) and the natural polyphenol compound salvianolic acid B (SAB) for composite design. By integrating the photothermal conversion properties of CuS NPs with the multiple biological functions of SAB, the aim is to construct a synergistic treatment system that combines precision treatment and microenvironment regulation. 2+ Combined with salvianolic acid B, the synergistic effect can significantly improve the tumor treatment effect. The prepared CCS NPs not only have Cu 2+ and Cu + The ability to catalyze the production of multiple ROS also enables the combined therapeutic effect of chemodynamic therapy and photodynamic therapy due to its unique nanostructure. This synergistic ROS production property has shown significant advantages in inhibiting tumor growth and provides a new strategy for the development of highly effective anti-tumor nanomedicines.

[0126] The CCS NPs prepared by the present invention can be combined with other means to achieve multifunctional synergistic treatment. For example, by combining photothermal therapy with anti-tumor and anti-inflammatory functions, the present invention can simultaneously achieve precise photothermal killing of tumor cells and regulation of the microenvironment. CCS NPs significantly reduce the activity of tumor cells under NIR light, and its effect is better than that of simple CuS or Cu 2+ Treatment group. This shows that photothermal therapy and chemodynamic therapy have significant synergistic effects, which can jointly inhibit the growth of cancer cells through different mechanisms and achieve higher treatment efficiency.

[0127] Compared with traditional photothermal therapy or single chemotherapy drugs, CCS nanoparticles can 2+ The targeting effect of CuS and SAB enhances the aggregation at the tumor site and reduces the side effects on normal tissues. The photothermal effect of CuS can be precisely concentrated at the tumor site, improving the local effect of treatment and reducing systemic side effects.

[0128] Existing photothermal therapy usually ignores the role of tumor microenvironment, while the CCS nanoparticles in the present invention have Cu 2+ and Cu + The redox activity of α-hydroxyl radicals (·OH) and superoxide anions (O 2 · - ). This multi-pathway ROS production not only destroys the lipids, nucleic acids and proteins of cancer cells, but also overcomes the limitation of hypoxia in the tumor microenvironment, thereby enhancing the therapeutic effect. Through the synergistic effect of SAB, CCS NPs can not only remove harmful cytokines in the tumor microenvironment through ROS and improve the inflammatory state, but also promote the formation of new tumor microenvironmental inflammatory cells through Cu. + The redox reaction of the tumor microenvironment induces "copper death" of tumor cells. This comprehensive regulation of the tumor microenvironment provides a new treatment strategy for difficult-to-treat tumors (such as hypoxic tumors).

[0129] CCS NPs showed extremely low toxicity to normal cells without NIR light, and the hemolysis rate of red blood cells was far below the cytotoxicity threshold (<5%). This shows that CCS NPs have good biocompatibility and low toxicity, and have significant advantages in biosafety, laying the foundation for its further in vivo application.

[0130] The present invention has an innovative core-shell structure design. Through the core-shell structure design of CCS nanoparticles, the present invention effectively combines photothermal therapy and anti-tumor therapy, and improves drug loading performance and therapeutic effect through the synergistic effect of shell materials. This design is the first in the prior art and provides new ideas and application prospects.

[0131] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. Combination of CuS, Cu 2+ and CCS nanoparticles of salvianolic acid B, characterized in that: The CCS nanoparticles are copper sulfide@copper ion-salvianolic acid B nanoparticles, which are composed of copper sulfide, salvianolic acid B and Cu 2+ The solution is reacted to obtain; Wherein, the copper sulfide, salvianolic acid B and Cu 2+ The mass ratio is 1:0.5~2:0.4~1.

6.

2. A method of combining CuS, Cu 2+ The method for preparing CCS nanoparticles containing salvianolic acid B is characterized by: include, Cupric chloride and polyvinyl pyrrolidone are mixed in deionized water, and sodium hydroxide solution and hydrazine hydrate solution are added under stirring to form a cuprous oxide suspension; sodium sulfide aqueous solution is then added, and the reaction is continued at 50-80°C with stirring for 1-3 hours; after the reaction is completed, the precipitate is cooled, centrifuged and washed to obtain copper sulfide nanoparticles, which are recorded as CuS NPs; After copper sulfide is prepared into an aqueous solution, it is added to the salvianolic acid B aqueous solution under stirring, and then a cupric chloride solution is added and stirring is continued. The precipitate is separated by centrifugation, washed to remove impurities, and freeze-dried to obtain copper sulfide@copper ion-salvianolic acid B nanoparticles, recorded as CCS NPs.

3. The combination of CuS, Cu as claimed in claim 2 2+ The method for preparing CCS nanoparticles containing salvianolic acid B is characterized by: The CCS NPs are core-shell structures, wherein the core is copper sulfide and the shell is Cu 2+ and salvianolic acid B.

4. The combination of CuS, Cu as claimed in claim 2 2+ The method for preparing CCS nanoparticles containing salvianolic acid B is characterized by: The particle size of the CCS NPs is 400-2000 nm.

5. Use of the CCS nano drug as claimed in claim 1 in the preparation of anti-tumor drugs, drug delivery, and tumor cell killing.

6. An anti-tumor drug, characterized in that: Comprising the CCS NPs according to claim 1 and other pharmaceutically acceptable adjuvants.

7. A method for killing tumor cells, characterized in that: The CCS NPs described in claim 1 provide local high temperature and precise killing of tumor cells in combination with near-infrared light irradiation.

8. The method for killing tumor cells according to claim 7, characterized in that: The near-infrared light irradiation has a wavelength of 808 nm and a light intensity of 0.2 to 1.0 W / cm 2 The illumination time is 2 to 10 minutes, and the killing effect is consolidated by multiple cycles of the killing method.

9. The method for killing tumor cells according to claim 7, characterized in that: The concentration of the CCS NPs is 12.5-200 μg / mL.

10. The method for killing tumor cells according to claim 7, characterized in that: The CCS NPs react with CuS core via the photothermal effect. 2+ , and the SAB shell regulate the tumor microenvironment.

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