Nano-particle, preparation method thereof and application of nano-particle in tumor photothermal-immune combined therapy

Abstract

The invention discloses a nanoparticle and a preparation method and application thereof in tumor photothermal-immune combined therapy, the target nanoparticle ICG-Cu-NLG8189-coated NK is prepared by using Cu < 2 + > as a medium to coordinate and bind ICG and NLG8189 through a metal coordination driven self-assembly method and modifying an NK cell membrane on the outer layer, and the nanoparticle is used for tumor therapy. The method has the advantages that the implementation of local photothermal therapy is accurately guided, tumor cells are induced to generate ICD, the immune response of tumor parts is activated, the local immunosuppression state in the tumor microenvironment is relieved, the activation of the tumor immune microenvironment is realized, and the systemic anti-tumor effect is realized in cooperation with ICD caused by photothermal.

Description

Technical Field

[0001] The present invention relates to the field of nanoparticle synthesis and application, specifically to a nanoparticle and a preparation method thereof and an application in combined photothermal-immunotherapy of tumors, in particular to a method for preparing nanoparticles by metal coordination driven self-assembly and an application thereof, in particular to a nanoparticle for precise combined photothermal-immunotherapy of tumors under the guidance of NIR-Ⅱ fluorescence imaging and a preparation method thereof and an application thereof. Background Art

[0002] Nowadays, the morbidity and mortality of malignant tumors remain high, seriously threatening human life. Traditional tumor treatments include surgery, radiotherapy and chemotherapy, which have many limitations such as large trauma and serious adverse reactions.

[0003] As a non-invasive treatment method, photothermal therapy can convert light energy into heat energy through photosensitizers under the irradiation of an external light source, thereby killing tumor cells that are more sensitive to temperature and achieving the purpose of treatment. Compared with traditional treatment methods, photothermal therapy has many advantages such as strong targeting, few adverse reactions, short treatment time, and repeatable treatment. In addition, the immunogenic cell death (ICD) of tumor cells caused by photothermal therapy can activate local immune responses, and synergize with immunotherapy to produce a "1+1>2" effect. Among many photosensitizers, indocyanine green (ICG) has been approved by the FDA for clinical use. As a photosensitizer, it has a high photothermal conversion efficiency and is a star "diagnostic and therapeutic small molecule" with both imaging diagnosis and photothermal therapy effects. As a light-emitting element, it has a high quantum yield and has good luminescence and imaging effects.

[0004] Good photothermal therapy is inseparable from the precise delineation of the treatment target area. Only by accurately applying the light source to lock the irradiation range can tumor cells be killed to the greatest extent and the surrounding normal tissues be protected, thereby reducing adverse reactions. As a relatively new imaging method, optical imaging technology has the advantages of no ionizing radiation, intuitive and easy operation compared to traditional imaging methods such as X-ray, ultrasound, CT and MRI. Its high sensitivity and imaging resolution have unique advantages in real-time in vivo observation of tumors. Compared with visible light, near-infrared fluorescence (NIR) has higher sensitivity and deeper penetration depth. In particular, the near-infrared region II (NIR-Ⅱ) has the uniqueness of a "tissue transparent window" compared to the near-infrared region I (NIR-Ⅰ), and has lower scattering and absorption in tissues, which can further improve the spatial resolution and tissue penetration depth of imaging, "light up" the tumor site, and provide a reliable basis for the delineation of the photothermal therapy target area. However, as a local treatment method, the scope of photothermal therapy is limited to the tumor site. Although it can induce ICD, the immune activation effect is very limited. In addition, the suppressive immune microenvironment of the "cold tumor" makes it difficult to achieve systemic tumor treatment.

[0005] As an emerging systemic therapy, immunotherapy brings hope to cancer patients. As researchers continue to deepen their research on the tumor immune microenvironment, they found that the overexpression of indoleamine-(2,3)-dioxygenase (IDO) in the tumor microenvironment is the key reason for the formation of the tumor suppressive immune microenvironment. IDO can catalyze the conversion of tryptophan to kynurenine, causing a lack of tryptophan in the microenvironment, thereby inhibiting the activity of effector T cells and NK cells, while enhancing the role of Treg cells, providing tumors with an opportunity for immune escape. NLG8189 (Indoximod, 1-MT) is a methylated tryptophan. As an IDO pathway inhibitor, it can effectively reverse the immunosuppressive effect mediated by IDO, reverse the suppressive immune microenvironment, and turn "cold tumors" into "hot tumors". It can work in synergy with ICD caused by photothermal therapy to induce systemic anti-tumor immunity in vivo. Summary of the invention

[0006] Based on the defects of the above-mentioned prior art, the present invention proposes an innovative treatment strategy: to achieve precise photothermal-immunotherapy of tumors under the guidance of NIR-II imaging. Specifically, by constructing ICG-Cu-NLG8189@NK nanoparticles, precise photothermal therapy guided by NIR-II fluorescence imaging technology is achieved, and the local "pro-activation" effect of ICD induced by photothermal therapy is combined with the systemic "anti-inhibition" effect of NLG8189 to activate the tumor immune microenvironment, thereby achieving systemic anti-tumor effects and greatly improving the effect of tumor treatment.

[0007] The specific technical solutions are as follows:

[0008] A nanoparticle, wherein the nanoparticle is Cu 2+ Spherical self-assembled nanoparticles ICG-Cu-NLG8189@NK are used as a medium to coordinate and bind ICG and NLG8189 and the outer layer modifies the NK cell membrane.

[0009] An application of nanoparticles, the application of the spherical self-assembled nanoparticles ICG-Cu-NLG8189@NK in nano-diagnostic and therapeutic agents for combined photothermal-immunotherapy of tumors.

[0010] Furthermore, the diagnostic and therapeutic agent is used in precise photothermal-immunotherapy guided by NIR-II fluorescence imaging of tumors.

[0011] Furthermore, the diagnostic and therapeutic agent is used in inhibiting tumor growth.

[0012] Furthermore, the spherical self-assembled nanoparticles ICG-Cu-NLG8189@NK are used in activating tumor immune microenvironment.

[0013] Furthermore, the spherical self-assembled nanoparticles ICG-Cu-NLG8189@NK are used in constructing a comprehensive precision diagnosis and treatment system.

[0014] Furthermore, the system includes utilizing the near-infrared fluorescence imaging function of the nanoparticles to accurately delineate the photothermal target area.

[0015] Still further, the system includes triggering an immune response at the tumor site via local photothermal therapy.

[0016] Furthermore, the system includes using the action of NLG8189 to reverse the immunosuppressive microenvironment at the tumor site and further activate the systemic immune response.

[0017] A method for preparing nanoparticles, characterized in that it comprises the following steps:

[0018] S1, according to 2mg ICG: 0.2mg CuCl 2· Disperse ICG and CuCl2.5H2O in 5 mL of anhydrous ethanol and stir on a magnetic stirrer for 8-14 h.

[0019] S2, centrifuging the reaction solution at a speed of 15000 rpm and a temperature of 0°C for 20 min, removing the supernatant to obtain a precipitate;

[0020] S3, dissolving the precipitate in anhydrous ethanol and fully dispersing it by ultrasonication;

[0021] S4, weighing NLG8189 in a ratio of 1 mg NLG8189: 0.5 mL anhydrous ethanol and dissolving it in anhydrous ethanol, fully dispersing it by ultrasonication, mixing the two, and stirring them on a magnetic stirrer for 8-14 h to obtain a reaction solution A;

[0022] S5, centrifuging the reaction solution at a speed of 15000 rpm and a temperature of 0°C for 20 min, removing the supernatant to obtain a precipitate, adding ultrapure water to the precipitate, fully dispersing the precipitate by ultrasonication, and placing the precipitate on a magnetic stirrer and stirring at a low speed for 4 h to obtain a reaction solution B;

[0023] S6, take the NK cell membrane, add 2 mL of sterile PBS, and use an ultrasonic disruptor to perform intermittent ultrasonication for 1 min in an ice bath to obtain reaction solution C;

[0024] S7, mixing the reaction solution B and the reaction solution C of S5 and S6, and performing ultrasonic dispersion for 10 minutes to obtain a reaction solution D;

[0025] S8, placing the reaction solution D of S7 in a liposome extruder, and sequentially extruding through 0.8 μm, 0.45 μm, and 0.22 μm filter membranes to obtain the target nanoparticles ICG-Cu-NLG8189@NK.

[0026] Working principle of the invention: This application prepares a Cu 2+ Spherical self-assembled nanoparticles with an outer layer that modify the NK cell membrane and coordinate ICG and NLG8189 are used as a medium. The near-infrared fluorescence imaging function of the nanoparticles is used to accurately delineate the photothermal target area, and local photothermal therapy is used to trigger the immune response in the tumor site. With the help of NLG8189, the immunosuppressive microenvironment of the tumor site is reversed, and the systemic immune response is further activated. The comprehensive and precise diagnosis and treatment from target area positioning to local photothermal therapy to systemic immune activation provides a new and effective strategy for tumor treatment.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) Preparation of a diagnostic and therapeutic nanoparticle with efficient tumor enrichment effect

[0029] ICG-Cu-NLG8189@NK;

[0030] (2) It can accurately guide the implementation of local photothermal therapy, induce ICD in tumor cells, and activate the immune response at the tumor site;

[0031] (3) The immune microenvironment regulation effect of NLG8189 can help relieve the local immunosuppression in the tumor microenvironment, activate the tumor immune microenvironment, and cooperate with the ICD caused by photothermal therapy to achieve a systemic anti-tumor effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Electron microscopy of ICG-Cu-NLG8189, ICG-Cu-NLG8189@NK and mapping element analysis of ICG-Cu-NLG8189@NK.

[0033] Figure 2 are the UV absorption spectra and infrared spectra of different samples; where:

[0034] (A) is the UV absorption spectra of ICG, NLG8189, ICG-NLG8189, ICG-Cu-NLG8189, and ICG-Cu-NLG8189@NK;

[0035] (B) is NLG8189, CuCl 2· 5H2O, NLG8189 and CuCl 2· 5H2O powder mixture and NLG8189-Cu 2+ Infrared absorption spectrum of the complex.

[0036] Figure 3 is the in vitro photothermal heating curve of ICG-Cu-NLG8189@NK, where:

[0037] (A) is the photothermal heating curve of ICG-Cu-NLG8189@NK at different concentrations;

[0038] (B) is the photothermal heating curve of ICG-Cu-NLG8189@NK at different powers;

[0039] (C) is the photothermal stability curve.

[0040] Figure 4 This is a diagram showing the effect of cell uptake and killing of 4T1 cells; among which:

[0041] (A) Fluorescence microscopy images of cells after ICG-Cu-NLG8189@NK and 4T1 cells were co-incubated for 0, 1, 2, 4, and 6 h.

[0042] (B) Flow cytometry was used to detect apoptosis of 4T1 cells after different treatments. L stands for Laser, i.e. laser irradiation.

[0043] Figure 5 This is a laser confocal image of immunofluorescence detection of immunogenic death markers; among them:

[0044] (A) is the immunofluorescence image of Calreticulin (CRT);

[0045] (B) is the immunofluorescence image of High Mobility Group Protein 1 (HMGB1).

[0046] Figure 6 In vivo fluorescence imaging and thermal imaging;

[0047] (A) In vivo fluorescence imaging images of mice with subcutaneous tumor transplantation at different time points after tail vein injection of ICG, ICG-NLG8189@NK, ICG-Cu-NLG8189, and ICG-Cu-NLG8189@NK;

[0048] (B) In vivo thermal imaging images of mice with subcutaneous tumor transplantation under 808 nm laser irradiation after tail vein injection of PBS, ICG, ICG-NLG8189@NK, and ICG-Cu-NLG8189@NK.

[0049] Figure 7 This is a graph showing the results of a unilateral tumor inhibition experiment; among which:

[0050] (A) Tumor growth curves of mice in different groups;

[0051] (B) White light photographs of representative mice in each group on day 16.

[0052] Figure 8 The results of biosafety testing; among them:

[0053] (A) In vitro hemolysis assay of nanoparticles at different concentrations;

[0054] (B) The results of liver and kidney function tests of mouse serum collected after photothermal-immunotherapy based on ICG-Cu-NLG8189@NK nanoparticles. DETAILED DESCRIPTION

[0055] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings. It should be understood that these descriptions are only exemplary and are not intended to limit the scope of the present invention. In addition, in the following invention, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention.

[0056] Example 1: Preparation of Nanoparticles

[0057] The nanoparticles of the present application are a kind of Cu 2+ The spherical self-assembled nanoparticles with ICG and NLG8189 as the medium and the outer layer modified NK cell membrane mainly drive the self-assembly through metal coordination. The specific steps are as follows:

[0058] (1) Weigh 2 mg ICG and 0.2 mg CuCl 2· 5H2O was dispersed in 5mL of anhydrous ethanol and stirred on a magnetic stirrer for 8-14h.

[0059] (2) The reaction solution was centrifuged at 15,000 rpm and 0°C for 20 min.

[0060] (3) After centrifugation, the supernatant was carefully removed and the precipitate was dissolved in 0.5 mL of anhydrous ethanol and ultrasonicated for about 30 seconds to fully disperse it; 1 mg of NLG8189 was weighed and dissolved in 0.5 mL of anhydrous ethanol and ultrasonicated for about 30 seconds to fully disperse it. The two were mixed and stirred on a magnetic stirrer for 8-14 hours.

[0061] (4) The reaction solution was centrifuged at 15,000 rpm, 0°C, and for 20 min.

[0062] (5) After centrifugation, carefully remove the supernatant to obtain the precipitate, add 1 mL of ultrapure water, and disperse thoroughly by ultrasonic dispersion for about 1 min. Place on a magnetic stirrer and stir at low speed for 4 h.

[0063] (6) Weigh 0.5 mg of NK cell membranes in an EP tube, add 2 mL of sterile PBS, and use an ultrasonic disruptor to sonicate for 1 min in an ice bath.

[0064] (7) The reaction solutions in (5) and (6) were mixed and dispersed by ultrasonic for 10 min.

[0065] (8) The reaction solution was placed in a liposome extruder and extruded through 0.8 μm, 0.45 μm, and 0.22 μm filter membranes in sequence to obtain the target nanoparticles ICG-Cu-NLG8189@NK.

[0066] Example 2: Characterization of synthesized ICG-Cu-NLG8189@NK nanoparticles

[0067] (1) Morphology and elemental composition of ICG-Cu-NLG8189@NK

[0068] The synthesized nanoparticles were diluted in a solution at an appropriate concentration and dropped onto a copper grid to dry. Then, transmission electron microscopy images were collected on a JEM-2100F transmission electron microscope, and images of mapping element distribution were collected on a Talos F200 field emission transmission electron microscope.

[0069] like Figure 1The electron microscopy results on the left show that the core nanoparticles of ICG-Cu-NLG8189 are uniformly spherical, well dispersed, and have a particle size of about 190nm. Electron microscopy of ICG-Cu-NLG8189@NK shows that a thin film with a thickness of about 12nm has been successfully modified on its surface, indicating the successful modification of the NK cell membrane. Due to the modification of the outer cell membrane, the particle size of ICG-Cu-NLG8189@NK is slightly increased compared to ICG-Cu-NLG8189, about 200nm. Further characterization is performed, and the results are as follows Figure 1 The mapping element analysis on the right shows that C, N, O, Cu, and S elements are evenly distributed in the nanoparticles, which indicates that in Cu 2+ Under the mediation of , ICG and NLG8189 successfully self-assembled to form spherical solid nanoparticles. In addition, the representative element of the cell membrane, P, was evenly distributed on the surface of the spherical particles, which further demonstrated the successful modification of the NK cell membrane on the surface of the nanoparticles.

[0070] (2) Ultraviolet absorption spectrum and infrared spectrum detection

[0071] After different samples were dissolved in ultrapure water, their UV absorption spectra were measured on a Cary 5000 UV-visible near-infrared spectrophotometer. Different sample powders were measured on a Nicoletis 50 Fourier transform infrared spectrometer for infrared spectra.

[0072] like Figure 2 As shown in A, the characteristic ultraviolet absorption peak of ICG is about 780nm. In comparison, the characteristic ultraviolet absorption peaks of ICG-NLG8189 and ICG-Cu-NLG8189 are obviously red-shifted, which indicates that Cu 2+ And NLG8189 can interact with ICG. In addition, compared with ICG-Cu-NLG8189, the UV absorption spectrum of ICG-Cu-NLG8189@NK did not change significantly, which indicates that the outer layer modification of NK cell membrane does not affect the interaction and coordination self-assembly between ICG and NLG8189. 2+ Whether there is interaction between them, NLG8189, CuCl 2· 5H2O, NLG8189 and CuCl 2· 5H2O powder mixture and NLG8189-Cu 2+ Infrared spectrum of the complex. Figure 2 As shown in B, NLG8189 and CuCl 2· The infrared spectrum of the mixture of 5H2O powder is pure NLG8189 and CuCl 2·The weighted average of pure 5H2O shows no characteristic peaks and no shift. This indicates that after physical mixing, NLG8189 and CuCl 2· 5H2O does not have any interaction force. In contrast, NLG8189-Cu 2+ The infrared absorption spectrum of the complex showed characteristic peaks at 1690nm and 1610nm, which further confirmed the affinity of NLG8189 with Cu 2+ The existence of interaction forces between them.

[0073] (3) Study on the photothermal performance of ICG-Cu-NLG8189@NK

[0074] The in vitro photothermal properties of ICG-Cu-NLG8189@NK nanoparticles were detected by thermal imaging.

[0075] The results are as follows Figure 3 As shown in A, at a fixed laser irradiation power, as the concentration of ICG-Cu-NLG8189@NK nanoparticles increases, the photothermal heating rate and platform temperature gradually increase. At a fixed ICG-Cu-NLG8189@NK nanoparticle concentration, as the 808nm laser irradiation power increases, the photothermal heating rate and final temperature of these nanoparticles continue to rise (3B). In addition, the excellent photothermal stability of ICG-Cu-NLG8189@NK nanoparticles was further verified by the temperature rise and fall experiment of continuously turning on the laser (3C).

[0076] Example 3: Cell uptake and cell killing experiments

[0077] In the cell uptake detection experiment, 4T1 cells were evenly plated in a 12-well plate containing a cell slide at an appropriate density and incubated for 12 hours to allow the cells to adhere to the wall. The culture medium was then discarded and replaced with fresh culture medium containing ICG-Cu-NLG8189@NK. After incubation for 0, 1, 2, 4, and 6 hours, the culture medium was discarded, and the cells were washed 3 times with PBS, fixed with paraformaldehyde, stained with wheat germ agglutinin (WGA)-Rho for cell membrane, stained with DAPI for cell nucleus, and then sealed with anti-fluorescence quencher, and observed and photographed under a Leica upright fluorescence microscope. In the flow cytometry apoptosis detection experiment, the AnnexinV FITC / PIKit kit was used to detect cell apoptosis according to the instructions.

[0078] like Figure 4 A laser confocal microscope image of cell uptake shows that the uptake of nanoparticles in cells gradually increases over time. Flow cytometry results show that compared with other groups, the ICG-Cu-NLG8189@NK nanoparticle group can show a significant cell killing effect under laser irradiation (4B).

[0079] Example 4: Photothermal therapy induced ICD detection

[0080] Using immunofluorescence technology, the changes of cell immunogenic death markers CRT and HMGB1 were detected. Specifically, 4T1 cells were evenly plated in a 12-well plate containing a cell slide at an appropriate density and incubated for 12 hours to allow the cells to adhere to the wall. After that, the culture medium was discarded and replaced with fresh culture medium containing different samples for co-incubation. Then, the group that needed to be illuminated was irradiated with 808nm laser. After 4 hours, the cells were washed with PBS, fixed with 4% paraformaldehyde, and permeabilized with 0.1% TritonX-100 solution (permeabilization is not required for observing CRT, but permeabilization is required for observing HMGB1). Then, they were placed in 3% BSA blocking buffer for 1 hour to block nonspecific binding. Then, they were incubated with primary antibodies against CRT or HMGB1 (CRT: ab92516, 1:500; HMGB1: ab18256, 1:1000) at 4°C overnight. Subsequently, they were incubated with secondary antibodies conjugated to AlexaFluor488 for 1 hour at room temperature. After washing with PBS, the cell nuclei were stained with DAPI, and finally the slides were sealed with anti-fluorescence quencher and observed and photographed under a Nikon laser confocal microscope.

[0081] like Figure 5 As shown in the figure, the fluorescence signal of ICG-Cu-NLG8189@NK nanoparticles is enhanced under light conditions, indicating that it can significantly induce CRT eversion (5A); at the same time, the fluorescence signal of HMGB1 is weakened, indicating that it can promote the migration of HMGB1 in the nucleus (5B). These results show that the nanoparticles can effectively induce the immunogenic death of tumor cells under light conditions and activate the immune response.

[0082] Example 5: Study on tumor targeting and photothermal heating ability of ICG-Cu-NLG8189@NK nanoparticles in mice

[0083] 100 μL (7×10 6 mL -1 )4T1 cells were injected into the right hind limb of mice to construct a subcutaneous transplanted tumor mouse model. After 7 days, when the tumor grew to a certain size, different samples were injected into the mouse through the tail vein for in vivo fluorescence imaging using a 900 / 1700 NIR-II fluorescence imaging system, or in vivo thermal imaging using a thermal imager under 808nm laser irradiation.

[0084] The results are as follows Figure 6As shown in the figure, after being injected into mice through the tail vein, the nanoparticles can achieve long-term and efficient enrichment at the tumor site, thereby achieving accurate tumor imaging and efficient drug delivery, which is also the key to subsequent accurate diagnosis and treatment (6A). The in vivo thermal imaging results verified its powerful in vivo photothermal heating performance, which is due to the efficient enrichment of nanoparticles at the tumor site and the excellent in vivo heating performance of nanoparticles (6B).

[0085] Example 6: Tumor inhibition experiment

[0086] 100 μL (7×10 6 mL -1 )4T1 cells were injected into the right hind limb of mice to construct a subcutaneous transplanted tumor mouse model. When the tumor grew to about 80-100 mm 3 Different samples were injected into mice via the tail vein and the tumor sites of mice that needed to be irradiated were irradiated with laser. The tumor size (volume = length × width) of the mice was measured the next day. 2 × 0.5) and body weight once for 16 days, and white light photos of the mice were taken on the 16th day.

[0087] The results are as follows Figure 7 As shown in the figure, from the tumor growth curve, it can be seen that under laser irradiation, ICG-Cu-NLG8189@NK nanoparticles showed a more significant tumor growth inhibition effect compared with pure ICG and nanoparticles without copper ions (7A). In addition, the excellent tumor growth inhibition effect of the ICG-Cu-NLG8189@NK+L group can be intuitively seen from the white light image of mice on the 16th day (7B). This strongly proves the advantages of photothermal-immunotherapy based on ICG-Cu-NLG8189@NK in tumor treatment.

[0088] Example 7: Biosafety Assessment of Nanoparticles

[0089] In the hemolysis experiment, blood from healthy mice was washed with PBS and centrifuged to separate red blood cells, and the red blood cells were redissolved in PBS to form a red blood cell suspension. PBS solutions of different concentrations of ICG-Cu-NLG8189@NK were mixed with the red blood cell suspension, with PBS as the negative control group and pure water as the positive control group. After a period of time, the supernatant was observed by centrifugation. In the mouse liver and kidney function evaluation experiment, whole blood was collected from the mice in each group after treatment through the eye sockets, and after standing and coagulation, the upper serum was centrifuged to detect various indicators of liver and kidney function.

[0090] The results are as follows Figure 8As shown, the results of the in vitro hemolysis experiment showed that compared with the PBS group, the red blood cells in the ICG-Cu-NLG8189@NK group did not show obvious hemolytic effect (8A). In addition, the serum samples of mice in different groups after treatment were tested. The test results showed that the liver and kidney functions of mice in the ICG-Cu-NLG8189@NK group and the various treatment component treatment groups were within the normal range (8B). This to a certain extent shows that the nanoparticles have good biosafety, providing important safety guarantees for their future clinical applications.

[0091] It should be understood that the above specific embodiments of the present invention are only used for illustrative inventions or to explain the principles of the present invention, and do not constitute limitations on the present invention. Therefore, any modifications, equivalent substitutions, improvements, etc. made without departing from the spirit and scope of the present invention should be included in the protection scope of the present invention. In addition, the appended claims of the present invention are intended to cover all changes and modifications that fall within the scope and boundaries of the appended claims, or the equivalent forms of such scope and boundaries.

Claims

1. A nanoparticle, characterized in that: The nanoparticles are Cu 2+ Spherical self-assembled nanoparticles ICG-Cu-NLG8189@NK are used as a medium to coordinate and bind ICG and NLG8189 and the outer layer modifies the NK cell membrane.

2. A use of a nanoparticle according to claim 1, characterized in that: The spherical self-assembled nanoparticles ICG-Cu-NLG8189@NK are used in nano-diagnostic and therapeutic agents for combined photothermal-immunotherapy of tumors.

3. The use according to claim 2, characterized in that: The diagnostic and therapeutic agent is used in precise photothermal-immunotherapy guided by NIR-II fluorescence imaging of tumors.

4. The use according to claim 2, characterized in that: The use of the diagnostic and therapeutic agent in inhibiting tumor growth.

5. A use of a nanoparticle according to claim 1, characterized in that: The application of the spherical self-assembled nanoparticles ICG-Cu-NLG8189@NK in activating tumor immune microenvironment.

6. A use of a nanoparticle according to claim 1, characterized in that: The application of the spherical self-assembled nanoparticles ICG-Cu-NLG8189@NK in constructing a comprehensive precision diagnosis and treatment system.

7. The use according to claim 6, characterized in that: The system includes utilizing the near-infrared fluorescence imaging function of nanoparticles to accurately delineate the photothermal target area.

8. The use according to claim 6, characterized in that: The system involves triggering an immune response at the tumor site through local photothermal therapy.

9. The use according to claim 6, characterized in that: The system includes using the action of NLG8189 to reverse the immunosuppressive microenvironment at the tumor site and further activate the systemic immune response.

10. A method for preparing nanoparticles according to claim 1, characterized in that: The following steps are involved: S1, according to 2mg ICG: 0.2mg CuCl 2· Disperse ICG and CuCl2.5H2O in 5 mL of anhydrous ethanol and stir on a magnetic stirrer for 8-14 h. S2, centrifuging the reaction solution at a speed of 15000 rpm and a temperature of 0°C for 20 min, removing the supernatant to obtain a precipitate; S3, dissolving the precipitate in anhydrous ethanol and fully dispersing it by ultrasonication; S4, weighing NLG8189 in a ratio of 1 mg NLG8189: 0.5 mL anhydrous ethanol and dissolving it in anhydrous ethanol, fully dispersing it by ultrasonication, mixing the two, and stirring them on a magnetic stirrer for 8-14 h to obtain a reaction solution A; S5, centrifuging the reaction solution at a speed of 15000 rpm and a temperature of 0°C for 20 min, removing the supernatant to obtain a precipitate, adding ultrapure water to the precipitate, fully dispersing the precipitate by ultrasonication, and placing the precipitate on a magnetic stirrer and stirring at a low speed for 4 h to obtain a reaction solution B; S6, take the NK cell membrane, add 2 mL of sterile PBS, and use an ultrasonic disruptor to perform intermittent ultrasonication for 1 min in an ice bath to obtain reaction solution C; S7, mixing the reaction solution B and the reaction solution C of S5 and S6, and performing ultrasonic dispersion for 10 minutes to obtain a reaction solution D; S8, placing the reaction solution D of S7 in a liposome extruder, and sequentially extruding through 0.8 μm, 0.45 μm, and 0.22 μm filter membranes to obtain the target nanoparticles ICG-Cu-NLG8189@NK.

Images