An iridium-loaded long afterglow nanocomposite, its preparation method and application

Abstract

The present invention discloses an iridium-loaded long afterglow nanocomposite, its preparation method and application. The nanocomposite is a core-shell structure formed by loading metallic iridium on the surface of a long afterglow nanomaterial. The core-shell structure consists of an inner core and a shell surrounding the core. The core is the long afterglow nanomaterial, and the shell is exosomes derived from and extracted from milk. The metallic iridium is loaded on the surface of the long afterglow nanomaterial in the form of single atoms or atomic clusters. The nanocomposite of the present invention can achieve tumor afterglow, computed tomography and photothermal multimodal imaging, and at the same time can effectively inhibit tumor growth by synergistically combining photothermal and catalytic treatment methods to achieve efficient tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to an iridium-loaded long afterglow nanocomposite and its preparation method and application. Background Art

[0002] As one of the major global public health problems, malignant tumors seriously endanger human health. The diagnosis and treatment of cancer urgently need to be further improved and solved. Multifunctional nanoprobes have received great attention in cancer diagnosis and treatment. Multifunctional nanoprobes can be used for imaging or detection in multiple modes, such as fluorescence imaging, X-ray computed tomography (CT), magnetic resonance imaging (MRI), etc. In addition, multifunctional nanoprobes can have multiple treatment capabilities, such as photothermal therapy, photodynamic therapy, chemotherapy, etc., enabling multifunctional nanoprobes to achieve highly sensitive diagnosis and synergistic treatment of tumors. Developing high-performance integrated diagnosis and treatment nanoprobes has become a very important frontier research direction in the fields of biomedicine, chemistry, materials science, etc.

[0003] In recent years, long afterglow nanomaterials, as a new type of optical material, have attracted extensive research interest. It is a material that can store excitation energy (such as X-rays, ultraviolet rays, and infrared rays) and continue to emit light after the excitation light stops. Near-infrared emitting long afterglow nanomaterials (PLNPs) have the outstanding advantage of being free from in-situ excitation, which can effectively avoid the autofluorescence or background interference of biological tissues itself and achieve high signal-to-noise ratio imaging. In practical imaging applications, when the afterglow is weak, white light or red light-emitting diodes (LEDs) can be used for repeated excitation, so that the imaging time is no longer limited to the afterglow time of the material, further expanding the application range of long afterglow nanomaterials. Summary of the Invention

[0004] The purpose of the present invention is to provide an iridium-loaded long afterglow nanocomposite and its preparation method and application, which realizes the loading of single iridium atoms or atom clusters on the surface of long afterglow nanomaterials and can be used for tumor afterglow / computed tomography / photothermal multimodal imaging and synergistic photothermal / catalytic therapy.

[0005] The present invention adopts the following technical scheme: An iridium-loaded long afterglow nanocomposite is a core-shell structure formed by loading metal iridium on the surface of a long afterglow nanomaterial. The core-shell structure is composed of an inner core and a shell surrounding the core. The core is a long afterglow nanomaterial, and the shell is exosomes derived from milk and extracted from milk. The metal iridium is loaded on the surface of the long afterglow nanomaterial in the form of single atoms or atom clusters.

[0006] Furthermore, the mass fraction of iridium in the iridium-loaded long afterglow nanocomposite is 3% of the iridium-loaded long afterglow nanocomposite.

[0007] Furthermore, the hydrated particle size of the core-shell structure is 57 ± 20 nm.

[0008] Furthermore, the long afterglow nanomaterial is Zn 1.2 Ga 1.6 Ge 0.2 O4:Cr 3+ 0.0075 。

[0009] A preparation method of an iridium-loaded long afterglow nanocomposite consists of the following steps:

[0010] Step 1: Dissolve urea in an IrCl3·xH2O aqueous solution and stir.

[0011] Step 2: Add the long afterglow nanomaterial and perform ultrasonic treatment.

[0012] Step 3: Vigorously stir under high temperature conditions and collect the solid precipitate.

[0013] Step 4: Vacuum dry the solid precipitate and then calcine it to obtain the iridium-loaded long afterglow nanomaterial.

[0014] Step 5: Incubate the iridium-loaded long afterglow nanomaterial and exosomes for 40 min, and then synthesize the exosome-coated iridium-loaded long afterglow nanocomposite by ultrasonic fragmentation.

[0015] Furthermore, the mass ratio of the added urea, IrCl3·xH2O, and long afterglow nanomaterial is 75:4:25.

[0016] Furthermore, in Step 5, the ratio of the number of exosomes to the added amount of the iridium-loaded long afterglow nanomaterial is 5×10 8 pieces / mg.

[0017] An application of a long afterglow nanocomposite in tumor multimodal imaging; the multimodal imaging includes afterglow imaging, nuclear magnetic imaging, and photoacoustic imaging.

[0018] An application of a long afterglow nanocomposite in the preparation of an anti-tumor nanodrug for synergistic photothermal therapy and catalytic therapy.

[0019] The beneficial effects of the present invention are as follows:

[0020] 1. In the nanocomposite of the present invention, iridium metal exists in the form of single atoms or atomic clusters on the surface of the long afterglow nanomaterial, effectively improving the peroxidase-like activity of the iridium-loaded long afterglow nanomaterial; it can catalyze the release of hydroxyl radicals to kill tumor cells, and synergistically improve the treatment effect with photothermal therapy, realizing tumor catalytic therapy based on nanozymes.

[0021] 2. The shell of the present invention is exosomes derived from milk. The exosome membrane can increase the biocompatibility of the complex, prolong the blood circulation time of the nano-drug, enable the nano-complex to be more and more safely enriched at the tumor site, and increase the targeting of imaging and treatment and the utilization efficiency of the nano-complex;

[0022] 3. The nano-complex of the present invention can achieve tumor afterglow, computed tomography and photoacoustic multimodal imaging, and at the same time can effectively inhibit tumor growth by synergistic photothermal and catalytic treatment methods, realizing efficient tumor treatment. Brief Description of the Drawings

[0023] Figure 1 It is a morphological characterization diagram of the long afterglow nano-material (PLNP) under a transmission electron microscope;

[0024] Figure 2 It is a spherical aberration corrected high angle annular dark field image-scanning transmission electron image (AC-HAADF-STEM), high angle annular dark field image-scanning transmission electron image (HAADF-STEM) and Zn, Ga, Ge, Cr and Ir element distribution diagrams of the iridium-loaded long afterglow nano-material (Ir / PLNP);

[0025] Figure 3 It is a morphological characterization diagram of exosomes under a transmission electron microscope;

[0026] Figure 4 It is a transmission electron microscope image of the iridium-loaded long afterglow nano-complex (Ir / PLNP@EVs);

[0027] Figure 5 It is an excitation-emission spectrum diagram of the long afterglow nano-material (PLNP), where λ em = 696 nm;

[0028] Figure 6 It is an emission spectrum diagram of the iridium-loaded long afterglow nano-material (Ir / PLNP);

[0029] Figure 7 It is an afterglow spectrum diagram of the iridium-loaded long afterglow nano-material (Ir / PLNP);

[0030] Figure 8 It is an afterglow imaging diagram of the iridium-loaded long afterglow nano-material (Ir / PLNP) after being excited by an ultraviolet lamp for 5 minutes and by an LED light for 1 minute respectively;

[0031] Figure 9 It is a cell labeling diagram of the iridium-loaded long afterglow nano-complex (Ir / PLNP@EVs);

[0032] Figure 10Tumor-bearing mice were respectively injected with iridium-loaded long afterglow nanomaterials (Ir / PLNP) and iridium-loaded long afterglow nanocomposites (Ir / PLNP@EVs) via the tail vein, and in vivo afterglow imaging maps were collected at 5 min, 1 h, 2 h, 4 h, and 6 h;

[0033] Figure 11 Tumor-bearing mice were respectively injected with iridium-loaded long afterglow nanomaterials (Ir / PLNP) and iridium-loaded long afterglow nanocomposites (Ir / PLNP@EVs) via the tail vein, and afterglow imaging maps of their major organs and tumor tissues were collected after 6 h;

[0034] Figure 12 Computer tomography (CT) maps of iridium-loaded long afterglow nanocomposites (Ir / PLNP@EVs) at different concentrations and a linear graph of the corresponding CT values;

[0035] Figure 13 CT maps of tumor-bearing mice injected with iridium-loaded long afterglow nanocomposites (Ir / PLNP@EVs) in situ before and after injection;

[0036] Figure 14 Photothermal imaging maps of tumor-bearing mice injected with iridium-loaded long afterglow nanocomposites (Ir / PLNP@EVs) in situ before and after injection;

[0037] Figure 15 Absorption spectra of long afterglow nanomaterials (PLNP) and iridium-loaded long afterglow nanocomposites (Ir / PLNP@EVs);

[0038] Figure 16 Photothermal curves of aqueous dispersions (1 mL) of long afterglow nanomaterials (PLNP) and iridium-loaded long afterglow nanomaterials (Ir / PLNP) and water under 808 nm laser irradiation (10 min, 1.0 W / cm 2 )

[0039] Figure 17 Characterization diagrams of the peroxidase-like activity of iridium-loaded long afterglow nanomaterials (Ir / PLNP) at different temperatures;

[0040] Figure 18 Characterization diagrams of the peroxidase-like activity of iridium-loaded long afterglow nanomaterials (Ir / PLNP) with or without 808 nm laser irradiation under different pH conditions;

[0041] Figure 19 Results diagrams of the catalysis of 3,3',5,5'-tetramethylbenzidine by iridium-loaded long afterglow nanomaterials (Ir / PLNP) with or without 808 nm laser irradiation under different pH conditions;

[0042] Figure 20Use the CCK-8 kit to evaluate the cytotoxicity of iridium-loaded long afterglow nanocomposites (Ir / PLNP@EVs).

[0043] Figure 21 Use the CCK-8 kit to evaluate the killing effect diagram of iridium-loaded long afterglow nanocomposites (Ir / PLNP@EVs) on mouse breast cancer cells (4T1 cells) after different treatments, ***p < 0.001;

[0044] Figure 22 Use the CCK-8 kit to evaluate the killing effect diagram of iridium-loaded long afterglow nanocomposites (Ir / PLNP@EVs) on human breast cancer cells (MDA-MB-231 cells) after different treatments, *p < 0.5, ***p < 0.001;

[0045] Figure 23 Laser confocal microscopy imaging diagram of mouse breast cancer cells (4T1 cells) after different treatments stained with calcein-AM and propidium iodide (PI), the scale bar is 10 μm;

[0046] Figure 24 Hemolysis analysis of iridium-loaded long afterglow nanocomposites (Ir / PLNP@EVs);

[0047] Figure 25 Hematoxylin-eosin staining images of major organs of mice after 30 days of drug administration and normal mice, the scale bar is 100 μm;

[0048] Figure 26 Weight changes of mice within 14 days after different treatments;

[0049] Figure 27 Changes in tumor volume during treatment under different treatment methods, *p < 0.5, **p < 0.01, ***p < 0.001;

[0050] Figure 28 Final mouse tumor diagrams after different treatment methods. Specific implementation manners

[0051] The present invention will be described in detail below in conjunction with the accompanying drawings and specific implementation manners.

[0052] It should be noted that the structures, ratios, sizes, etc. shown in the drawings of this specification are only used to cooperate with the content disclosed in the specification for those skilled in this technology to understand and read, and are not used to limit the implementable conditions of the present invention. Therefore, they do not have technical essence. Any modification of the structure, change of the proportional relationship or adjustment of the size, without affecting the effects that the present invention can produce and the purposes that can be achieved, should still fall within the scope covered by the technical content disclosed by the present invention.

[0053] Near-infrared long afterglow nanomaterials, denoted as PLNP, have the prominent advantage of being free from in-situ excitation, which can effectively avoid the autofluorescence of biological tissues themselves or background interference and achieve high signal-to-noise ratio imaging. In actual imaging applications, when the afterglow is weak, white light or red LED lights can be used for repeated excitation, so that the imaging time is no longer limited to the afterglow time of the material, further expanding the application scope of long afterglow nanomaterials. Controlling the composition, particle size of the material and surface modification endows long afterglow nanomaterials with multiple functions and can be used in many fields such as medical imaging, disease diagnosis and treatment.

[0054] The present invention discloses an iridium-loaded long afterglow nanocomposite, which is a core-shell structure formed by loading iridium metal on the surface of a long afterglow nanomaterial. The core-shell structure consists of an inner core and a shell surrounding the core. The core is a long afterglow nanomaterial, and the shell is exosomes derived from milk and extracted from milk. The iridium metal is loaded on the surface of the long afterglow nanomaterial in the form of single atoms or atomic clusters.

[0055] The mass fraction of iridium in the iridium-loaded long afterglow nanocomposite is 3%. The hydrated particle size of the core-shell structure is 57 ± 20 nm. The long afterglow nanomaterial is Zn 1.2 Ga 1.6 Ge 0.2 O4:Cr 3+ 0.0075 。

[0056] The present invention also discloses a preparation method of an iridium-loaded long afterglow nanocomposite, which consists of the following steps:

[0057] Step 1: Dissolve urea in an IrCl3·xH2O aqueous solution and stir.

[0058] Step 2: Add the long afterglow nanomaterial and perform ultrasonic treatment.

[0059] Step 3: Vigorously stir under high temperature conditions and collect the solid precipitate.

[0060] Step 4: Vacuum dry the solid precipitate and then calcine it to obtain the iridium-loaded long afterglow nanomaterial.

[0061] Step 5: Incubate the iridium-loaded long afterglow nanomaterial and exosomes for 40 min, and then synthesize the exosome-coated iridium-loaded long afterglow nanocomposite by ultrasonic fragmentation method.

[0062] Among them, the mass ratio of urea, IrCl3·xH2O, and the added long afterglow nanomaterial is 75:4:25.

[0063] In Step 5, the ratio of the number of exosomes to the added amount of the iridium-loaded long afterglow nanomaterial is 5×108 per mg

[0064] The present invention also discloses an application of the long afterglow nanocomposite in tumor multimodal imaging; the multimodal imaging includes afterglow imaging, nuclear magnetic imaging, and photoacoustic imaging.

[0065] The present invention also discloses an application of the long afterglow nanocomposite in preparing an anti-tumor nanomedicine for synergistic photothermal therapy and catalytic therapy.

[0066] Example 1

[0067] The symbolic meanings in this example are as follows:

[0068] The long afterglow nanomaterial is denoted by PLNP, the iridium-loaded long afterglow nanomaterial is denoted by Ir / PLNP, the exosome is denoted by EVs, and the iridium-loaded long afterglow nanocomposite is denoted by Ir / PLNP@EVs.

[0069] The long afterglow nanomaterial selected in this example is Zn 1.2 Ga 1.6 Ge 0.2 O4:Cr 3+ 0.0075 , or it can be replaced with other long afterglow nanomaterials, but it should ensure the same near-infrared luminescence and long afterglow time.

[0070] The raw material for loading iridium in this example is IrCl3·xH2O, which can be replaced with other metal raw materials, but it should ensure the same growth rate, enzyme-like activity, photothermal properties, and computed tomography imaging ability.

[0071] In this example, the exosome with the shell derived from milk can also be replaced with other liposomes or exosomes from other sources, but it should ensure that it can extend the circulation time of the nanomedicine in the blood and enable it to accumulate more in the tumor site.

[0072] 1. Preparation of near-infrared long afterglow nanomaterial (PLNP)

[0073] Gallium oxide is dissolved in 3% ammonium hydroxide and made up to a 0.5 mol / L solution. At the same time, prepare zinc nitrate solution (1 mol / L), gallium nitrate solution (1 mol / L), and chromium nitrate solution (0.1 mol / L). Take 4.8 mmol Zn 2+ , 6.4 mmol Ga 3+ , 1.6 mmol Ge 4+ , 0.03 mmol Cr 3+Place it in a round-bottom flask. Under vigorous stirring, adjust the pH of the solution to 8.0 with ammonium hydroxide (28%, wt) to form a white suspension. After vigorously stirring for 3 h, transfer it to a polytetrafluoroethylene autoclave. After hydrothermal treatment at 220 °C for 10 h, wait for the reactants to cool to room temperature, and collect the white precipitate by centrifugation. The white precipitate is the long-afterglow nanomaterial. The obtained Figure 1 transmission electron microscopy images show that the morphology of PLNP is relatively regular and has an obvious lattice structure.

[0074] Disperse the long-afterglow nanomaterial (PLNP) in ultrapure water and detect the fluorescence excitation and emission spectra. According to the Figure 5 excitation and emission spectra of PLNP shown as follows, it can be found that PLNP has three relatively large fluorescence excitation peaks at 254 nm, 410 nm and 570 nm, and has the maximum emission peak at 694 nm.

[0075] 2. Preparation of iridium-loaded long-afterglow nanomaterial (Ir / PLNP)

[0076] Weigh 0.15 g of urea, add it to the IrCl3·xH2O solution (0.1 mol / L, 0.25 mL), add water to 25 mL and stir for 30 min; then add 50 mg of long-afterglow nanomaterial (PLNP), ultrasonically treat for 2 h, and then vigorously stir and react at 98 °C for 1 h, and collect the solid precipitate. After vacuum drying, calcine at 750 °C for 2 h to obtain the iridium-loaded long-afterglow nanocomposite material. As Figure 2 shown, in the low-magnification mode, Ir / PLNP shows the typical morphology of PLNP in the HAADF-STEM image, and the distributions of Zn, Ga, Ge and Ir elements are clear and uniform, and no nanoparticles other than PLNP are found; as the magnification increases, there are highly dispersed isolated recognizable bright spots on the long-afterglow surface of Ir / PLNP in the AC-HAADF-STEM image, which is attributed to the loading of iridium metal.

[0077] Disperse the iridium-loaded long-afterglow nanomaterial (Ir / PLNP) in ultrapure water and detect the fluorescence emission spectrum and afterglow spectrum. According to the Figure 6 fluorescence emission image of Ir / PLNP shown as follows, it can be found that the position of the maximum excitation peak of Ir / PLNP is the same as that of the single PLNP, and it has an afterglow emission in the near-infrared region of about 694 nm. According to the Figure 7 afterglow image of Ir / PLNP shown as follows, Ir / PLNP shows an afterglow emission of 600 s after being irradiated with a 254 nm ultraviolet lamp for 5 min.

[0078] The afterglow signal of the iridium-loaded long-afterglow nanomaterial (Ir / PLNP) powder is collected by a small animal imager, as shown in the Figure 8As shown, Ir / PLNP not only exhibits long-lasting afterglow luminescence after irradiation with a 254 nm ultraviolet lamp, but also can be activated by a 650 nm LED lamp to restore its afterglow luminescence, indicating that Ir / PLNP has good afterglow luminescence performance, and the imaging time is no longer limited to the afterglow time, having high application value in in vitro and in vivo imaging.

[0079] 2.1 Peroxidase-like activity of Ir / PLNP

[0080] To investigate the peroxidase-like activity of iridium-loaded long afterglow nanomaterials (Ir / PLNP), the optimal reaction temperature of its peroxidase-like enzyme was first screened. As Figure 17 shown, the optimal reaction temperature of Ir / PLNP is 45 °C, which is equivalent to the temperature after irradiating Ir / PLNP with an 808 nm laser for 10 min. Therefore, the peroxidase-like activity of Ir / PLNP under different pH conditions under 808 nm laser irradiation was further studied. As Figures 18 - 19 shown, consistent with the expected results, 808 nm laser irradiation increased the peroxidase-like activity of the material in the pH range of 2.6 to 5.0. These results demonstrate that 808 nm laser irradiation can enhance the peroxidase-like activity of Ir / PLNP, and Ir / PLNP is expected to achieve tumor catalytic therapy based on nanozymes.

[0081] 3. Preparation of iridium-loaded long afterglow nanocomposites (Ir / PLNP@EVs)

[0082] Weigh 2 mg of iridium-loaded long afterglow nanomaterials (Ir / PLNP) and add it to 1 mL of Dulbecco's phosphate buffered saline (DPBS) containing milk-derived exosomes (10 9 particles / mL) to obtain a mixture with a ratio of the number of exosomes to the added amount of iridium-loaded long afterglow nanomaterials of 5×10 8 particles / mg. The morphology of the exosomes is shown in Figure 3 as cup-shaped or saucer-shaped structures. After co-incubating the iridium-loaded long afterglow nanomaterials and exosomes on ice for 40 min, they were treated on an ultrasonic disruptor for 8 min to obtain the iridium-loaded long afterglow nanocomposites (Ir / PLNP@EVs) shown in the appendix Figure 4 .

[0083] 3.1 Cell labeling with Ir / PLNP@EVs

[0084] After incubating mouse breast cancer cells (4T1 cells) with iridium-loaded long afterglow nanocomposites (Ir / PLNP@EVs) for 1.5 h, Hoechst produced by Thermo Fisher Scientific and lysosomal green fluorescent probe (Lyso Tracker Green DND-26) were added for co-incubation for 30 min. Then, the culture medium was removed and the cells were washed twice with Dulbecco's phosphate buffered saline (DPBS). The labeling of cells with the three fluorescent dyes was observed under a laser confocal microscope. As shown in the attached Figure 9 The results of Ir / PLNP@EVs labeling live cells in Figure Figure 9 show that: under 405 nm excitation light, cells emit blue fluorescence of Hoechst; under 488 nm excitation light, cells emit green fluorescence of the lysosomal green fluorescent probe; under 552 nm excitation light, cells emit red fluorescence of Ir / PLNP@EVs. After superimposing the three different fluorescences, it was found that the blue fluorescence was located in the nucleus, and the red fluorescence and green fluorescence overlapped to appear yellow, with a high co-localization efficiency, proving that Ir / PLNP@EVs can enter the intracellular lysosomes.

[0085] 3.2 Ability of Ir / PLNP@EVs in in vivo afterglow imaging

[0086] To further verify the ability of iridium-loaded long afterglow nanocomposites (Ir / PLNP@EVs) in in vivo afterglow imaging, tumor-bearing mice were intravenously injected with iridium-loaded long afterglow nanomaterials (Ir / PLNP) and Ir / PLNP@EVs via the tail vein. After 5 min, 1 h, 2 h, 4 h, and 6 h after injection, the afterglow signals of the mice were collected. After sacrificing the mice, their main organs (heart, liver, spleen, lung, kidney, and tumor) were collected for imaging. The mice and organs were pre-excited with a 650 nm LED lamp, and afterglow imaging was performed under an IVIS imaging system.

[0087] As shown in the attached Figure 10 The afterglow imaging of mice in Figure Figure 10 shows that compared with Ir / PLNP, Ir / PLNP@EVs can rapidly accumulate at the tumor site within 1 h after injection, and with the passage of time, the afterglow signal at the tumor site of the mice gradually increases. As shown in Figure 11 The afterglow imaging of the mice's organs in Figure Figure 11 shows that there are strong afterglow signals in the liver and spleen (reticuloendothelial system) and lungs of both groups of mice, and the afterglow signal of the mice injected with Ir / PLNP@EVs mainly accumulates in the liver. In addition, the afterglow signal at the tumor site of the mice injected with Ir / PLNP@EVs is stronger, proving that the material encapsulated by exosomes derived from milk can reach the tumor site more.

[0088] 3.3 Computed tomography (CT) imaging ability of Ir / PLNP@EVs

[0089] To verify the computed tomography (CT) imaging ability of iridium-loaded long afterglow nanocomposites (Ir / PLNP@EVs), dispersions of Ir / PLNP@EVs with different concentrations were selected for CT imaging. As Figure 12 shown, with the increase in iridium concentration, the CT imaging effect of Ir / PLNP@EVs became more obvious. To further verify the in vivo CT imaging ability of Ir / PLNP@EVs, tumor-bearing mice injected in situ with Ir / PLNP@EVs were subjected to CT imaging before and after injection. As Figure 13 shown, the tumor is indicated by the circle. After Ir / PLNP@EVs entered the tumor site, the CT imaging signal of the mice increased significantly, making the tumor site clearer.

[0090] 3.4 In vivo photothermal imaging ability of Ir / PLNP@EVs

[0091] To verify the in vivo photothermal imaging ability of iridium-loaded long afterglow nanocomposites (Ir / PLNP@EVs), Ir / PLNP@EVs were injected into the tumor sites of tumor-bearing mice, and normal saline was injected into the control group. Images were collected on a thermal imager under the irradiation of an 808 nm laser, as Figure 14 shown, and the photothermal imaging effect of the group injected with Ir / PLNP@EVs was better.

[0092] From the above verification, it can be seen that iridium-loaded long afterglow nanocomposites (Ir / PLNP@EVs) can be used for afterglow, CT, and photothermal multimodal imaging, making up for the limitations of single imaging methods and achieving highly sensitive imaging of tumors.

[0093] 3.5 Absorption spectra of Ir / PLNP@EVs and PLNP

[0094] The absorption spectra of iridium-loaded long afterglow nanocomposites (Ir / PLNP@EVs) and long afterglow nanomaterials (PLNP) were collected. The results are as Figure 15 shown. Compared with PLNP, Ir / PLNP@EVs had a higher absorption value at 808 nm, indicating that Ir / PLNP@EVs had the potential for photothermal conversion under the irradiation of an 808 nm laser.

[0095] To further verify the photothermal conversion ability of Ir / PLNP@EVs, 1 mL of an Ir / PLNP aqueous dispersion (1 mg / mL) was irradiated with an 808 nm laser for 10 min, and the temperature was recorded every minute during the irradiation. PLNP and water were set as the control groups. As Figure 16 shown, after 10 min of irradiation, the temperature of Ir / PLNP rose to 47.8 °C, while there was no obvious temperature increase in PLNP and water, proving that Ir / PLNP had a high photothermal conversion ability, which was mainly attributed to iridium metal and was expected to achieve tumor photothermal therapy.

[0096] 3.6 Cytotoxicity of Ir / PLNP@EVs

[0097] Good biocompatibility is crucial for treatment at the tumor cell level. Therefore, the cytotoxicity of iridium-loaded long afterglow nanocomposites (Ir / PLNP@EVs) was verified. The cytotoxicity of Ir / PLNP@EVs against four types of cells was evaluated using the Cell Counting Kit-8 (CCK-8). Mouse breast cancer cells (4T1 cells), human hepatocellular carcinoma cells (HepG2), human breast cancer cells (MDA-MB-231 cells), and mouse fibroblasts (L929 cells) were passaged into 96-well plates and cultured overnight. Then, different concentrations of Ir / PLNP@EVs were added in the form of medium replacement and cultured for 24 h or 48 h. Finally, CCK-8 was added, and the absorbance of the cells at 450 nm was measured after 2 h of incubation. As Figure 20 shown, even at a high concentration of 200 μg / mL, the survival rate of the four types of cells was still greater than 80%, demonstrating that Ir / PLNP@EVs has low cytotoxicity.

[0098] 3.7 Tumor cell killing effect of Ir / PLNP@EVs

[0099] To evaluate the tumor cell killing effect of iridium-loaded long afterglow nanocomposites (Ir / PLNP@EVs), mouse breast cancer cells (4T1 cells) and human breast cancer cells (MDA-MB-231 cells) were passaged into 96-well plates and cultured overnight.

[0100] Then, the two types of cells were each divided into four groups: incubated with different concentrations of Ir / PLNP@EVs for 24 h; co-incubated with different concentrations of Ir / PLNP@EVs and 100 μM hydrogen peroxide for 24 h; co-incubated with different concentrations of Ir / PLNP@EVs and 100 μM hydrogen peroxide for 24 h, and irradiated with an 808 nm laser for 10 min at 18 h of co-incubation.

[0101] Finally, the cell survival rate was detected using the CCK-8 kit. As Figure 21 shown, the cytotoxicity of Ir / PLNP@EVs alone against cells is low. The killing effect of Ir / PLNP@EVs on 4T1 cells is weak in the presence of hydrogen peroxide. The killing effect on cells is stronger when laser is applied on the basis of incubation with Ir / PLNP@EVs. The killing effect is the strongest when Ir / PLNP@EVs is applied in the presence of hydrogen peroxide and laser irradiation, and the cell survival rate drops to 19%.

[0102] As Figure 22As shown, different treatments had the same killing effect on MDA-MB-231. The application of Ir / PLNP@EVs in the presence of hydrogen peroxide and external laser irradiation had the strongest killing effect on cells, and the cell survival rate dropped to 24%.

[0103] 3.8. Cell-killing effect of Ir / PLNP@EVs

[0104] In order to more intuitively evaluate the cell-killing effect of iridium-loaded long-lasting nanocomposites (Ir / PLNP@EVs), the cells after different treatments were co-stained with fluorescein diacetate dye (FDA) and propidium iodide (PI). Figure 23 As shown in the figure, compared with the control group, the killing ability of 4T1 cells by 808nm laser alone, hydrogen peroxide alone, Ir / PLNP@EVs alone, or hydrogen peroxide and 808nm laser was very weak, and the cells showed strong green fluorescence, indicating that the cells were alive. Consistent with the results of CCK-8 method, the killing effect of Ir / PLNP@EVs and laser irradiation in the presence of hydrogen peroxide was the strongest, showing strong red fluorescence. The above results prove that the photothermal and catalytic synergistic therapy based on Ir / PLNP@EVs has good therapeutic effect.

[0105] 3.9 Hemolysis experiment and tissue section analysis of Ir / PLNP@EVs

[0106] Good biocompatibility is crucial for tumor treatment, so hemolysis experiments and tissue section analysis were performed before in vivo treatment. Figure 24 As shown in the figure, when the concentration of Ir / PLNP@EVs was as high as 200μg / mL, the hemolysis rate was still less than 1%. The main organs (heart, liver, spleen, lung, and kidney) of mice injected with Ir / PLNP@EVs by tail vein were collected on the 30th day to make tissue sections, which were stained with hematoxylin-eosin. Figure 25 As shown in the figure, compared with the control group, the mice injected with Ir / PLNP@EVs via tail vein showed no obvious inflammation or tissue damage in various organs. This proves that Ir / PLNP@EVs has good biocompatibility, so Ir / PLNP@EVs has high safety in vivo application.

[0107] 3.10 Synergistic tumor therapeutic effect of Ir / PLNP@EVs

[0108] In order to evaluate the synergistic tumor therapeutic effect of iridium-loaded long-lasting nanocomposites (Ir / PLNP@EVs), 4T1 tumor-bearing mice were randomly divided into 4 groups (3 mice in each group) and given different treatments. Figure 26 As shown in the attached figure, no significant changes in the body weight of mice in the four groups were found during the treatment.Figures 27 - 28 As shown, in the treatment group irradiated only with 808 nm laser, the tumors of the mice increased rapidly, and the tumor size was similar to that of the control group after 14 days. Therefore, light irradiation had no obvious inhibitory effect on tumor growth.

[0109] Injecting only Ir / PLNP@EVs had a certain inhibitory effect on tumors. In the experimental group irradiated with 808 nm laser after injecting Ir / PLNP@EVs, the tumors became significantly smaller, and the final tumors were extremely small or disappeared, proving that under the irradiation of 808 nm laser, Ir / PLNP@EVs had good photothermal and nanozyme-based catalytic synergistic therapeutic effects on tumors.

[0110] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An iridium-loaded long afterglow nanocomposite, characterized in that, It is a core-shell structure formed by loading iridium metal on the surface of a long-afterglow nanomaterial. The core-shell structure consists of an inner core and a shell surrounding the core. The core is a long-afterglow nanomaterial, and the shell is exosomes derived from and extracted from milk. The iridium metal is loaded on the surface of the long-afterglow nanomaterial in the form of single atoms or atom clusters; the long-afterglow nanomaterial is Zn 1.2 Ga 1.6 Ge 0.2 O4:Cr 3 + 0.0075 .

2. The iridium-loaded long afterglow nanocomposite according to claim 1, characterized in that, The mass fraction of iridium in the iridium-loaded long afterglow nanocomposite is 3% of the iridium-loaded long afterglow nanocomposite.

3. The iridium-loaded long afterglow nanocomposite according to claim 1, wherein The hydrated particle size of the core-shell structure is 57 ± 20 nm.

4. A method for preparing an iridium-loaded long afterglow nanocomposite as described in claim 1, characterized in that, It consists of the following steps: Step 1: Dissolve urea in an IrCl3·xH2O aqueous solution and stir. Step 2: Add the long afterglow nanomaterial and perform ultrasonic treatment. Step 3: Vigorously stir under high-temperature conditions and then collect the solid precipitate. Step 4: Vacuum-dry the solid precipitate and then calcine it to obtain the iridium-loaded long afterglow nanomaterial. Step 5: Incubate the iridium-loaded long afterglow nanomaterial and exosomes for 40 min, and then synthesize the exosome-coated iridium-loaded long afterglow nanocomposite by ultrasonic fragmentation.

5. The preparation method of an iridium-loaded long afterglow nanocomposite according to claim 4, characterized in that, The mass ratio of the added urea, IrCl3·xH2O, and long afterglow nanomaterial is 75:4:

25.

6. The preparation method of an iridium-loaded long afterglow nanocomposite according to claim 4, characterized in that, In step 5, the ratio of the number of the exosomes to the addition amount of the iridium-loaded long afterglow nanomaterials is 5×10 8 pieces / mg.

7. Use of a long afterglow nanocomposite according to any one of claims 1-3 or a long afterglow nanocomposite prepared by the preparation method of claims 4, 5 or 6 in the preparation of an anti-tumor nanomedicine for synergistic photothermal therapy and catalytic therapy.

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