Preparation method and application of copper selenide / polydopamine / new indocyanine green composite nanoparticles

Abstract

The invention relates to a preparation method of copper selenide / polydopamine / new indocyanine green composite nanoparticles, which comprises the following steps: 1) mixing Se powder, NaBH4 and deionized water to react for 3-5 hours in a room-temperature inert gas atmosphere to obtain a solution A; cuCl2.H2O and PVP are uniformly dispersed in deionized water, a NaOH solution is added for a reaction for 3-8 min, an ascorbic acid solution is added for a reaction for 3-8 min, then a solution A is added for a reaction for 15-30 min, centrifugation and washing are performed, and hollow Cu2-xSe is obtained; and 2) dispersing the hollow Cu2-xSe in a Tris buffer solution, then adding dopamine hydrochloride and IR820, shaking in a dark place at room temperature for 1.5-3 hours, centrifuging and washing to obtain the hollow Cu2-xSe composite nanoparticle. The hollow Cu2-xSe composite nanoparticle can play a good effect in the aspect of tumor treatment.

Description

Technical Field

[0001] This invention belongs to the field of nanoparticle preparation and photothermal and photodynamic therapy for tumor synergistic treatment. Specifically, it relates to a method for preparing and applying copper selenide / polydopamine / neoindocyanine green composite nanoparticles. These composite nanoparticles are composed of copper selenide / polydopamine / neoindocyanine green and can be induced by near-infrared laser. Through pH response, they synergistically kill tumor cells in combination with photothermal and photodynamic therapy, achieving a good tumor-killing effect. Background technology. Cancer therapy is a crucial area of ​​nanomedicine application, focusing on developing nanoscale materials to improve the effectiveness and safety of cancer treatments. Nanoparticles can serve as a therapeutic platform, leveraging their small size, targeting capabilities, and drug-carrying capacity for targeted drug delivery, precise tumor diagnosis, and combined therapies using multiple methods, such as photothermal, photodynamic, chemotherapy, and radiotherapy. Specific nanoparticles, such as copper selenide, can absorb near-infrared light and convert it into heat energy, achieving localized thermal ablation of tumors. Nanoparticles loaded with photosensitizers generate reactive oxygen species under light irradiation, which can destroy tumor structures and precisely locate lesions through photodynamic therapy. These emerging treatment methods can effectively address the shortcomings of traditional cancer treatments, adding new vitality to current research in the field of cancer therapy.

[0003] Nanomaterials hold immense potential in the biomedical field. Their unique physicochemical properties, such as high specific surface area, small size effect, and tunable surface charge, offer novel avenues for clinical tumor treatment. Nanomaterials can serve as carriers of photothermal agents and photosensitizers, enabling targeted delivery to tumor tissues, increasing drug accumulation at the tumor site, and enhancing therapeutic efficacy. Furthermore, nanomaterials can be designed and modified to integrate multiple functions, such as possessing both photothermal conversion and photosensitizing properties, further improving the efficiency of combined therapy. Photothermal therapy combined with photodynamic therapy using nanomaterials has become a current research hotspot in tumor treatment. Photothermal therapy (PTT) and photodynamic therapy (PDT) are two emerging tumor treatment methods. PTT utilizes photothermal conversion materials to convert light energy into heat energy, causing irreversible damage to tumor cells through localized high temperatures, thereby achieving the goal of tumor treatment. The advantage of PTT is that it can use variable-level external laser irradiation to precisely target tumors while avoiding damage to surrounding healthy tissues. PDT, on the other hand, uses the combined action of photosensitizers, specific wavelength light, and oxygen to generate cytotoxic reactive oxygen species (ROS) to destroy tumor cells. During the process of excited-state electrons returning to the ground state through phosphorescence, most photosensitizers transfer energy to triplet oxygen. 3 O2) to produce singlet oxygen ( 1O2 is essential for photodynamic therapy (PDT), so most PTTs rely on oxygen. A major problem with PTT compared to laser irradiation is its low light penetration depth, which can lead to insufficient ablation of malignant tumors outside the irradiated area. Hypoxia is a major limiting factor for PTT efficacy, and the tumor microenvironment and bacterial biofilms are typical hypoxic environments, significantly restricting the anti-tumor effects of PTT. However, combining photothermal and PTT can compensate for their respective shortcomings. The heat generated by photothermal therapy can accelerate blood flow to improve the hypoxic environment of the tumor and alter cell membrane permeability, offering advantages such as increased photosensitizer infiltration rate and improved target selectivity in PTT. When photothermal therapy is used alone for anti-tumor purposes, it easily induces heat resistance in tumor cells, but PTT can enhance the sensitivity of target cells to thermal stimulation and further kill heat-resistant tumor cells. Furthermore, both photothermal and PTT are photoexcited, making the combined phototherapy approach easier to implement.

[0004] Commonly used photothermal conversion materials include noble metal nanomaterials (such as gold nanoparticles and silver nanoparticles), carbon-based nanomaterials (such as graphene and carbon nanotubes), and semiconductor nanomaterials (such as copper sulfide and bismuth selenide). These materials possess unique optical properties, exhibiting strong absorption in the near-infrared region (700-1100 nm), and near-infrared light has good tissue penetration capabilities, allowing it to penetrate deep into tumor tissue. Photosensitizers commonly used in photodynamic therapy include porphyrins, phthalocyanines, chlorophylls, and indocyanine green. Due to the differences in vascular structure and metabolic characteristics between tumor tissue and normal tissue, these photosensitizers are more likely to accumulate at the tumor site through enhanced penetration and retention (EPR) effects. Research on nanomaterials for integrated photothermal and photodynamic therapy platforms is emerging in large numbers. Researchers have prepared gold nanoparticles loaded with photosensitizers, achieving simultaneous photothermal and photodynamic therapy through near-infrared light irradiation, significantly enhancing the killing effect on tumor cells. For example, an H-TiO2@PDA@ICG@NPe6@Lipo nanocomposite, prepared by polydopamine (PDA) modification, combined with indocyanine green (ICG) and NPe6 photosensitizer, and then encapsulated in thermosensitive liposomes, combined photothermal and photodynamic therapy. Results showed that, without any cytotoxicity, the component exhibited substantial improvements in spectral response range, photothermal conversion efficiency, and light absorption performance through modification and photosensitizer application. Other studies have found that copper phosphide nanoparticles can be used for combined photothermal and photodynamic tumor therapy, achieving significant tumor-killing effects.

[0005] Based on the above research, this invention will prepare a copper selenide / polydopamine / neoindocyanine green composite nanoparticle that combines photothermal and photodynamic therapy, which can exert good effects in the treatment of tumors. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a copper selenide / polydopamine / neoindocyanine green composite nanoparticle that combines photothermal and photodynamic therapy. This composite nanoparticle can exert good effects in the treatment of tumors.

[0007] The present invention also provides a method for preparing the above-mentioned copper selenide / polydopamine / neoindocyanine green composite nanoparticles and their applications.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing copper selenide / polydopamine / neoindocyanine green composite nanoparticles, comprising the following steps: 1) Under an inert gas atmosphere (such as nitrogen or argon) at room temperature, Se powder, NaBH4, and deionized water are mixed and reacted for 3-5 hours to obtain solution A; CuCl2·H2O and polyvinylpyrrolidone (PVP) are uniformly dispersed in deionized water, NaOH solution is added and reacted for 3-8 minutes, ascorbic acid solution is added and reacted for 3-8 minutes, then solution A is added and reacted for 15-30 minutes. After centrifugation and washing, hollow Cu is obtained. 2-x Se; 2) Hollow Cu 2-x Se was dispersed in Tris buffer, then dopamine hydrochloride and new indocyanine green (IR820) were added, and the mixture was shaken in the dark at room temperature for 1.5-3 h. After centrifugation and washing, the product was obtained.

[0009] Specifically, in step 1), the molar ratio of Se powder to NaBH4 can be 1:0.9-1.

[0010] Specifically, in step 1), the molar ratio of Se powder, CuCl2·H2O and PVP can be 1:0.5-0.6:5-6.

[0011] Specifically, in step 1), the molar ratio of Se powder to NaOH can be 1:2-2.4.

[0012] Furthermore, in step 1), the molar ratio of Se powder to ascorbic acid can be 1:0.8-1.2.

[0013] Specifically, in step 2), hollow Cu 2-x The mass ratio of Se to dopamine hydrochloride can be 1:8-12.

[0014] Furthermore, in step 2), the hollow Cu 2-x The mass ratio of Se to IR820 can be 1:12-15.

[0015] This invention provides copper selenide / polydopamine / neoindocyanine green composite nanoparticles prepared by the above-described preparation method.

[0016] This invention also provides the application of the above-mentioned copper selenide / polydopamine / neoindocyanine green composite nanoparticles in the preparation of antitumor drugs.

[0017] Specifically, as a preferred option, the hollow Cu 2-x Se nanospheres were prepared by the following steps: First, nitrogen gas was purged for 5 minutes to remove oxygen from the flask. Then, 50 mL of deionized water, 19 mg of NaBH4, and 39.5 mg of Se powder were added sequentially, and the mixture was purged with nitrogen for 3-5 hours to obtain solution A. 0.048 g of NaOH and 0.1056 g of AA (ascorbic acid) were weighed and dissolved in 6 mL of DI water respectively and set aside for later use. 0.051 g of CuCl2·H2O was dissolved in 150 mL of deionized water and stirred evenly on a magnetic stirrer. 0.3 g of PVP was added, and stirring continued until completely dissolved. Then, NaOH solution was added dropwise, at which point the solution turned blue. The reaction continued for 5 minutes, followed by the addition of AA solution dropwise. Stirring continued for 5 minutes (at which point the solution changed from blue to green to yellow). Solution A was then added, and the reaction was carried out for 20 minutes. The product was collected by centrifugation and washed three times each with alcohol and deionized water. The hollow Cu nanospheres were then... 2-x Se was dispersed in 3 mL of water and prepared to a concentration of 1 mg / mL. -1 The dispersion was prepared and stored for later use.

[0018] Next, Cu 2-x Synthesis of Se / PDA / IR820 composite nanoparticles: Take 1 mg / mL hollow Cu 2-x 1 mL of Se dispersion was centrifuged, the supernatant was removed, and then Cu was added. 2-x Se precipitate was dispersed in 0.5 mL Tris 8.5 buffer, followed by the addition of 10 mg dopamine hydrochloride and 15 mg IR820. The mixture was incubated in the dark on a shaker for 2 h, centrifuged, and washed three times to obtain Cu. 2-x Se / PDA / IR820 composite nanoparticles were dispersed in water and stored for later use. Polydopamine (PDA) formed a hollow mesoporous Cu nanoparticle through layer-by-layer self-assembly or interfacial polymerization. 2-x The complete encapsulation layer of Se; the photosensitizer, neo-indocyanine green (IR820), can not only be embedded in Cu through physical adsorption. 2-x Within the hollow mesoporous structure of Se, high drug loading is achieved by utilizing the high specific surface area of ​​the mesoporous structure. Simultaneously, the encapsulation of PDA prevents premature leakage, and the drug can be anchored to the PDA surface through π-π stacking. Stable binding is achieved by utilizing the abundant phenolic hydroxyl groups and o-phenylenediamine structures in the PDA molecule. The entire therapeutic system achieves highly efficient loading of IR820 and combines photothermal and photodynamic therapy, overcoming the limitations of single-treatment methods.

[0019] This invention relates to copper selenide / polydopamine / neoindocyanine green (Cu 2-x In Se / PDA / IR820) composite nanoparticles, polydopamine PDA is incorporated into hollow mesoporous Cu. 2-x A complete encapsulation layer is formed on the surface of Se; the photosensitizer IR820 can not only be physically adsorbed and embedded in Cu 2-x Within the hollow mesoporous structure of Se, a high drug loading capacity is achieved by utilizing the high specific surface area of ​​the mesoporous structure. At the same time, the encapsulation of PDA prevents premature leakage, and it can also be anchored to the surface of PDA through π-π stacking. Stable binding is achieved by utilizing the abundant phenolic hydroxyl groups and o-phenylenediamine structure in the PDA molecule.

[0020] The composite nanoparticle platform constructed in this invention: Cu 2-x Se / PDA / IR820, made of Cu with a hollow mesoporous structure 2-x The system comprises Se, a PDA encapsulation layer, and a highly efficient photosensitizer IR820, forming a combined photothermal and photodynamic therapy system. Material Cu 2-X Se is a hollow sphere that can kill cancer cells through photothermal action under 808nm near-infrared light irradiation. Neo-indocyanine green (IR820) is a derivative of the photosensitizer indocyanine green (ICG) that can generate ROS under near-infrared light irradiation, inducing tumor cell death through photodynamic therapy. Polydopamine has strong adhesive properties, which can not only enhance Cu... 2-x The photothermal effect of Se can also better satisfy Cu 2-x Se combines with IR820. It also exhibits pH responsiveness, allowing it to be degraded by acid in the acidic tumor microenvironment, releasing a large amount of the photosensitizer IR820 to achieve a therapeutic effect.

[0021] Numerous studies have shown that copper selenide can generate heat under near-infrared light irradiation, achieving photothermal therapy. It exhibits excellent light absorption and emission capabilities in terms of photothermal performance. However, solid copper selenide suffers from drawbacks such as small specific surface area, low contact efficiency with tumor cells and reactants, and limited catalytic activity and photothermal conversion efficiency. Furthermore, it has been found to possess certain tumor-therapeutic properties. Therefore, to overcome these limitations, this application considered whether hollow copper selenide could achieve the desired effect. Exploration revealed that hollow copper selenide has a larger specific surface area and increased active sites. Simultaneously, the hollow cavity enhances light absorption and heat accumulation, enabling highly efficient loading of photosensitizers or drugs, thereby strengthening damage to tumor cells. These advantages precisely compensate for the limitations of solid copper selenide. In addition, the photosensitizer neo-indocyanine green IR820 has absorption and emission wavelengths in the near-infrared region. Light in this band penetrates deeply into biological tissues, making it suitable for tumor photodynamic therapy. Meanwhile, polydopamine exhibits excellent adhesion and can be pH-responsive, meaning that an increase in environmental acidity can trigger acid degradation of the PDA coating, releasing a large amount of IR820 encapsulated within the nanospheres. The incompletely degraded PDA coating can also remain anchored to some remaining hollow Cu. 2-x The Se surface continues to exert its anti-tumor effect under laser irradiation. Therefore, the Cu of this invention... 2-x Se / PDA / IR820 features a thinner shell and a larger specific surface area, enhancing photothermal conversion efficiency and overcoming the drawbacks of solid copper selenide, such as small specific surface area, low contact efficiency, and limited drug loading. This invention increases the specific surface area and drug loading of the material, improving photothermal conversion efficiency and the efficient loading of the photosensitizer IR820. This invention exhibits pH responsiveness, enabling precise and controllable drug release. This invention combines photothermal ablation and ROS killing, achieving a combined photothermal and photodynamic therapy approach, possessing significant practicality and broad application value. The preparation method of this invention is simple, yields high output, and is suitable for large-scale production.

[0022] Compared with the prior art, the present invention has the following advantages and beneficial effects: This invention combines the photosensitizer IR820 and hollow mesoporous Cu with photothermal effect. 2-x Se, combined with PDA, exhibits high photothermal conversion efficiency, and its significantly increased specific surface area enables more efficient absorption of near-infrared light. Cu 2-x The Se cavity can also achieve high drug loading. The highly adhesive PDA will also adhere the photosensitizer IR820 to its surface, allowing IR820 to achieve a greater loading capacity. Furthermore, this invention is pH responsive; in the acidic tumor microenvironment, the PDA continuously degrades, and the photosensitizer loaded within the cavity achieves controlled release. 2-xThe Se cavity decomposes in an acidic environment to produce divalent copper ions, which can consume excess glutathione (GSH) in tumor cells, thereby inhibiting tumor cell growth and proliferation and further enhancing the therapeutic effect on tumors. Simultaneously, this invention is a multifunctional nanoplatform integrating photothermal ablation and ROS killing, achieving a combination of photothermal and photodynamic therapy methods.

[0023] Cu prepared by this invention 2-x Se / PDA / IR820 composite nanoparticles possess excellent photothermal properties; this application uses Cu... 2-x Se / PDA / IR820 composite nanoparticles were exposed to an 808nm laser using a 1W cm⁻¹ laser. -2 Irradiation with a power of [power value missing] resulted in a temperature increase of 62.8°C within 5 minutes at a concentration of 100 μg / mL. Furthermore, at the same concentration, the temperature gradually increased with prolonged irradiation time until it stabilized. Through five cycles, the Cu [structure / effect / process] of this application [was observed / achieved / tested]. 2-x The Se / PDA / IR820 composite nanoparticles maintained a high temperature response, indicating their excellent thermal stability. Finally, the Cu... 2-x The photothermal conversion efficiency of the Se / PDA / IR820 composite nanoparticles under optimal conditions is 44.56%, directly reflecting the great potential of this material in photothermal applications. In vitro cell experiments show that these composite nanoparticles possess excellent tumor-killing activity; under laser irradiation, as Cu... 2- x Increasing the concentration of Se / PDA / IR820 significantly decreased cell viability, with an inhibition rate of 98.80% at a drug concentration of 100 μg / mL, significantly higher than the control group without laser irradiation. Through ROS flow cytometry, we found that Cu... 2-x The Se / PDA / IR820 combined light irradiation group induced apoptosis of 76.32% through ROS production, indicating that the addition of IR820 significantly increased ROS production and thus induced cell death. In vivo experimental results further demonstrate the effectiveness of the proposed composite nanoparticle Cu... 2-x The anti-tumor effect of Se / PDA / IR820 under laser irradiation was demonstrated by the fact that the size and volume of tumors in mice in the combined nanoparticle and light-irradiation group were significantly lower than those in other groups. H&E staining of pathological slides of the mouse tumors revealed that the necrotic area in the tumor tissue of the combined nanoparticle and light-irradiation group was also significantly larger than that in other groups, indicating a better tumor-suppressive effect. Therefore, this composite nanoparticle can significantly kill tumor cells under laser irradiation, achieving a combination of photothermal and photodynamic therapy. Attached Figure Description

[0024] Figure 1 The present invention relates to copper selenide / polydopamine / neoindocyanine green composite nanoparticles (Cu 2-xA schematic diagram of the synthesis process of Se / PDA / IR820; Figure 2 To prepare hollow Cu 2-x Transmission electron microscopy (TEM) image of Se nanoparticles. The image clearly shows a hollow structure, confirming the presence of hollow Cu. 2-x Successful synthesis of Se nanoparticles; Figure 3 To prepare the composite nanoparticles Cu 2-x The transmission electron microscope image of Se / PDA / IR820 clearly shows the PDA encapsulation, proving the successful synthesis of the composite nanoparticles; Figure 4 Cu composite nanoparticles of different concentrations 2-x Photothermal temperature rise curve of Se / PDA / IR820 under NIR (808 nm, 1W cm-2) irradiation; Figure 5 To test the effects of near-infrared irradiation on PBS and Cu composite nanoparticles at a concentration of 50 μg / mL. 2-x Thermal images of Se / PDA / IR820 over time; Figure 6 Cu composite nanoparticles 2-x Photothermal stability of Se / PDA / IR820 after 5 photothermal cycles; Figure 7 To combine Cu nanoparticles of different concentrations 2-x A comparison of the viability of esophageal squamous cell carcinoma cells (KYSE150) and human umbilical vein endothelial cells (HUVEC) after co-incubation with Se / PDA / IR820 for 6 hours. Figure 8 Control group (left) and Cu composite nanoparticles under laser irradiation 2-x A fluorescence image of ROS generated by the Se / PDA / IR820 (right). Detailed Implementation

[0025] The technical solution of the present invention will be further described in detail below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto.

[0026] In the following examples, all raw materials used are common commercially available products that can be purchased directly, or can be prepared using conventional techniques in the art.

[0027] Example 1 A method for preparing copper selenide / polydopamine / neoindocyanine green composite nanoparticles, comprising the following steps: 1) Hollow Cu 2-x Preparation of Se; First, purge the oxygen from the bottle with nitrogen for 5 minutes. Then, add 50 mL of deionized water, 19 mg (0.5 mmol) of NaBH4, and 39.5 mg (0.5 mmol) of Se powder sequentially. Purge with nitrogen for 3 hours to obtain solution A. Weigh 0.048 g (1.2 mmol) of NaOH and 0.1056 g (0.6 mmol) of ascorbic acid AA and dissolve them in 6 mL of dihydrate (DI) for later use. Dissolve 0.051 g (0.3 mmol) of CuCl2·H2O in 150 mL of deionized water and stir evenly on a magnetic stirrer. Add 0.3 g (3 mmol) of PVP and continue stirring until completely dissolved. Then, add NaOH solution dropwise. The solution turns blue. Continue the reaction for 5 minutes. Add AA solution dropwise and stir for another 5 minutes (the solution changes from blue to green to yellow). Add solution A and react for 20 minutes. Centrifuge to collect the product. Wash three times each with alcohol and deionized water to obtain the hollow Cu product. 2-x Se (transmission electron microscope image as shown) Figure 2 (As shown).

[0028] 2) Put 1mg of empty Cu 2-x Se was dispersed in 0.5 mL Tris 8.5 buffer, then 10 mg of dopamine hydrochloride and 15 mg of neoindocyanine green (IR820) were added. The mixture was incubated at room temperature in the dark for 2 h with a shaker, followed by centrifugation and washing to obtain Cu. 2-x Se / PDA / IR820 composite nanoparticles (transmission electron microscopy image shown) Figure 3 (As shown).

[0029] This application further verified through experiments the copper selenide / polydopamine / neoindocyanine green (Cu) prepared according to the present invention. 2-x The efficacy of Se / PDA / IR820 composite material in tumor treatment applications.

[0030] Experimental Example 1 This application evaluated the prepared Cu 2-x The heating effect of Se / PDA / IR820 composite nanoparticles at concentrations of 10, 20, 50, and 100 μg / mL was measured. A power density of 1.0 W / cm³ was used. -2 The laser was irradiated with an 808 nm near-infrared laser, and the temperature was recorded every 30 seconds using an infrared thermal imager for a total of 10 minutes. Temperature rise curves for different concentrations were plotted, and the results are shown below. Figure 4 and 5 .

[0031] like Figure 4 As shown, Cu 2-xThe heating effect of Se / PDA / IR820 composite nanoparticles showed a concentration-dependent effect, and 100 μg mL of Cu 2-x The temperature of the Se / PDA / IR820 dispersion can rise to 64°C within 10 minutes.

[0032] Thermal imaging can visually display PBS and Cu at a concentration of 50 μg / mL. 2-x The temperature rise of both Se / PDA / IR820 composite nanoparticles. For example... Figure 5 As shown, Cu 2-x The temperature of the Se / PDA / IR820 composite nanoparticles gradually increased over time under laser irradiation, reaching 51.2℃ from room temperature within 5 minutes, while the temperature of PBS only increased slightly from room temperature to 32℃ during the same period. These results demonstrate that Cu 2-x Se / PDA / IR820 composite nanoparticles exhibit good heating effect.

[0033] Experiment Example 2 This application verifies the material's stability under laser irradiation using photothermal curves over five cycles. For example... Figure 6 As shown, after multiple light-cooling cycles, the composite nanoparticles can still maintain a high temperature response. The highest temperatures achievable in the five heating-cooling cycles are not significantly different, indicating that the synthesized Cu... 2-x Se / PDA / IR820 nanoparticles exhibit excellent thermal stability.

[0034] Experimental Example 3 Esophageal squamous cell carcinoma cells (KYSE150) are a cell line derived from human esophageal squamous cell carcinoma, and human umbilical vein endothelial cells (HUVECs) are a cell line derived from the endothelium of human umbilical veins. Both KYSE150 and HUVECs were purchased from the Cell Bank of the Chinese Academy of Sciences (ATCC). KYSE150 was cultured in 1640 medium containing 10% fetal bovine serum (FBS). HUVECs were cultured in DMEM medium containing 10% FBS. For KYSE150 cells grown to the logarithmic growth phase, first aspirate the culture medium, wash twice with 2 mL PBS, then add 1 mL of trypsin containing 0.25% EDTA, shake well until the bottom of the dish is covered, and incubate in a 37°C CO2 incubator for 5 minutes. After complete digestion, add 2 mL of 1640 medium containing 10% FBS to the dish to stop digestion, repeatedly pipette the bottom of the dish, collect the liquid in the dish into a 5 mL EP tube, and centrifuge at 1200 rpm for 5 min. Then remove the supernatant (the digestion of HUVEC cells is the same as KYSE150, except that the culture medium used is DMEM medium containing 10% FBS). Then resuspend in complete culture medium and count, according to 5 × 10⁶. 3Cells were evenly seeded into 96-well plates at a density of [number] cells / well. After the cells had fully adhered the following day, the original culture medium was discarded, and 100 μL of Cu (containing serum-free medium at concentrations of 0, 5, 10, 15, 20, 25, 30, and 40 μg / mL) was added to each well. 2-x Se / PDA / IR820 solution. After incubation for 24 hours, the original culture medium was discarded and the cells were washed with PBS. 100 μL of CCK-8 working solution diluted 10-fold was added, and incubation continued for 2 hours in a CO2 incubator. The OD value at 450 nm was then measured using a microplate reader, and the cell viability of HUVECs and KYSE150 cells was calculated. Cell viability (%) = (OD experimental group - OD background) / (OD control group - OD background) × 100%. Results are shown below. Figure 7 .

[0035] Cu prepared by this invention 2-x The Se / PDA / IR820 composite nanoparticles exhibit good biocompatibility with esophageal squamous cell carcinoma (KYSE150) and human umbilical vein endothelial cells (HUVECs). HUVECs are a typical representative of vascular endothelial cells, and the vascular endothelium is one of the first structures to come into contact with blood and tissues after biomaterials are implanted in the body. HUVECs have a well-defined endothelial cell function, and the effect of the material on endothelial cells can be directly reflected by detecting the cell viability of HUVECs, thereby assessing the biosafety of the material. By co-culturing the composite nanoparticles with esophageal squamous cell carcinoma (KYSE150) and HUVECs for 24 hours without light exposure, and detecting the viability of the two cell types, we found that the cell viability of both cell types did not decrease significantly with increasing composite nanoparticle concentration. When the material concentration was 40 μg / mL, the cell viability was still around 75% after 24 hours, and the difference in viability between the two cell types was not significant (e.g., ...). Figure 7 (As shown). The composite nanoparticles Cu of this invention 2-x Se / PDA / IR820 had no significant effect on HUVECs, indicating that it can easily pass through blood vessels and reach tumor tissue via blood circulation. Furthermore, it had no killing effect on tumor cells without light exposure, and the composite nanoparticles could not exert anti-tumor effects without near-infrared light irradiation. Therefore, the above research demonstrates that the Cu composite nanoparticles of this invention... 2-x Se / PDA / IR820 has good biocompatibility.

[0036] Experiment Example 4: Detection of ROS Level: This experiment was divided into a control group and a control group. 2-xTwo groups were used: one using Se / PDA / IR820+L laser. KYSE150 cells were cultured at a density of 1.5 × 10⁻⁶. 5 Cells were seeded at a density of 10 cells / well in 12-well plates and incubated overnight in a CO2 incubator before being treated with Cu... 2-x Se / PDA / IR820+L group with 1.0 W cm -2 Irradiation with an 808 nm laser was performed, followed by a 5-minute irradiation at 42 °C, and then incubation for 10 hours. The sample was then stained with 300 μL of DCFH-DA, incubated at 37 °C for 20 minutes, washed three times with PBS, and finally observed under an EVOS 7000 fluorescence microscope. Experimental results are shown below. Figure 8 .

[0037] like Figure 8 As shown, when using 1.0 W cm -2 After irradiation with an 808 nm laser for 5 minutes, compared with the control group, Cu 2-x The Se / PDA / IR820+L group produced a large amount of reactive oxygen species (ROS). This indicates that the Cu prepared in this invention... 2-x Se / PDA / IR820 can exert a photodynamic antitumor effect by generating a large amount of ROS under laser irradiation.

Claims

1. A method for preparing copper selenide / polydopamine / neoindocyanine green composite nanoparticles, characterized in that, Includes the following steps: 1) Under an inert gas atmosphere at room temperature, Se powder, NaBH4, and deionized water are mixed and reacted for 3-5 hours to obtain solution A; CuCl2·H2O and PVP are uniformly dispersed in deionized water, NaOH solution is added and reacted for 3-8 minutes, ascorbic acid solution is added and reacted for 3-8 minutes, then solution A is added and reacted for 15-30 minutes. After centrifugation and washing, hollow Cu is obtained. 2-x Se; 2) Hollow Cu 2-x Se was dispersed in Tris buffer, then dopamine hydrochloride and IR820 were added, and the mixture was shaken in the dark at room temperature for 1.5-3 h. After centrifugation and washing, the product was obtained.

2. The preparation method of copper selenide / polydopamine / neoindocyanine green composite nanoparticles as described in claim 1, characterized in that, In step 1), the molar ratio of Se powder to NaBH4 is 1:0.9-1.

3. The preparation method of copper selenide / polydopamine / neoindocyanine green composite nanoparticles as described in claim 1, characterized in that, In step 1), the molar ratio of Se powder, CuCl2·H2O and PVP is 1:0.5-0.6:5-6.

4. The preparation method of copper selenide / polydopamine / neoindocyanine green composite nanoparticles as described in claim 1, characterized in that, In step 1), the molar ratio of Se powder to NaOH is 1:2-2.

4.

5. The preparation method of copper selenide / polydopamine / neoindocyanine green composite nanoparticles as described in claim 1, characterized in that, In step 1), the molar ratio of Se powder to ascorbic acid is 1:0.8-1.

2.

6. The method for preparing copper selenide / polydopamine / neoindocyanine green composite nanoparticles as described in claim 1, characterized in that, In step 2), hollow Cu 2-x The mass ratio of Se to dopamine hydrochloride is 1:8-12.

7. The preparation method of copper selenide / polydopamine / neoindocyanine green composite nanoparticles as described in claim 1, characterized in that, In step 2), hollow Cu 2-x The mass ratio of Se to IR820 is 1:12-15.

8. Copper selenide / polydopamine / neoindocyanine green composite nanoparticles prepared by any of the preparation methods described in claims 1 to 8.

9. The use of the copper selenide / polydopamine / neoindocyanine green composite nanoparticles according to claim 8 in the preparation of antitumor drugs.

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