A tannic acid iron modified dual photosensitizer loaded upconversion nanoparticle, preparation method and application

Abstract

The present invention provides a tannic acid iron modified dual photosensitizer loaded upconversion nanoparticle, a preparation method and an application. The nanoparticle consists of a four-layer core-shell structure upconversion nanoparticle, loaded with dual photosensitizers Ce6 and MC540, and adsorbed on the surface with a tannic acid iron complex. The diameter of the nanoparticle is 100nm. The preparation method includes the preparation of multi-layer core-shell structure upconversion nanoparticles, the loading of dual photosensitizers, and the combination of tannic acid iron complexes. The preparation method of the present invention is simple, can effectively load dual photosensitizers, and has efficient photodynamic therapy performance under 808nm near-infrared light irradiation. The tannic acid iron complex responds to the tumor microenvironment to produce chemodynamic therapy and produce oxygen, thereby sensitizing photodynamic therapy and realizing chemodynamic photodynamic synergistic tumor therapy. Further induce tumor immunogenic cell death, combined with immune checkpoint inhibitors to sensitize anti-tumor immunotherapy. At the same time, the nanoparticle has the performance of computed tomography and magnetic resonance imaging of the tumor site.

Description

Technical Field

[0001] The present invention belongs to the field of biological nanomaterials, and relates to a tannic acid iron modified dual photosensitizer loaded upconversion nanoparticle and its preparation method and application. Specifically, the present invention relates to the preparation and application performance evaluation of a nanotherapy platform that has the synergistic treatment function of chemodynamic therapy and photodynamic therapy, produces tumor immunogenic cell death at the same time, and can realize sensitized anti-tumor immunity. Background Art

[0002] Malignant tumors are major diseases that seriously threaten human health. Currently, the means of treating malignant tumors include surgical resection, chemotherapy, radiotherapy, immunotherapy, etc. However, the above methods all have certain limitations, such as advanced tumors cannot be surgically removed, chemotherapy has systemic toxicity and is prone to drug resistance, the hypoxic tumor microenvironment restricts the efficacy of radiotherapy, and immunotherapy has poor responsiveness and is expensive. Therefore, it is urgent to explore new tumor treatment strategies. The occurrence and development of tumors are closely related to the unlimited proliferation of tumor cells and their escape from the immune surveillance system. The mobility and loss of contact inhibition of tumor cells enable them to have unlimited proliferation ability. At the same time, tumor cells evade the monitoring and clearance of the body's immune system through various mechanisms. Therefore, the ideal tumor treatment strategy is to kill tumor cells and activate the body's anti-tumor immune response at the same time to achieve comprehensive tumor treatment.

[0003] With the development of nanotechnology, it has been widely used in biomedical fields such as tumor diagnosis and treatment, opening up a variety of tumor treatment strategies, including photothermal therapy, photodynamic therapy, chemodynamic therapy, etc. Photodynamic therapy (PDT) refers to the use of lasers of specific wavelengths to irradiate photosensitizers, which transfer energy to oxygen molecules to produce reactive oxygen species (ROS) to kill tumors. The photosensitizers used in traditional PDT require ultraviolet-visible light excitation, and the tissue penetration depth of ultraviolet-visible light is insufficient, which greatly limits its application in the treatment of solid or deep tumors. The near-infrared region is called the "optical window" of biological tissues, and near-infrared light (NIR) can effectively penetrate tissues. Through upconversion nanoparticles (UCNPs), NIR is converted into visible light or ultraviolet light, thereby exciting photosensitizers and achieving PDT. Through reasonable UCNP structural design and optimization of energy transfer efficiency, the efficiency of upconversion PDT can be improved. Chemodynamic therapy (CDT) is an emerging tumor treatment method that uses Fenton's reagent to react with H2O2 in the tumor site to produce hydroxyl radicals, which kill the tumor.

[0004] Both PDT and CDT treatment systems have the potential to induce immunogenic cell death (ICD) in tumor cells. Studies have shown that PDT can effectively induce ICD in tumor cells, but the efficiency of ICD induction is restricted in the hypoxic tumor microenvironment. In addition, CDT can be combined with other methods such as chemotherapy to induce ICD in tumor cells. Therefore, the CDT / PDT synergistic tumor treatment system can not only improve the efficiency of PDT-induced ICD by improving the hypoxic environment, but also CDT / PDT has the potential to synergistically induce ICD.

[0005] Currently, the methods used for tumor imaging in clinical practice include fluorescence, computed tomography (CT), magnetic resonance imaging (MRI), etc. An integrated diagnosis and treatment system with the above tumor imaging capabilities has significant advantages in the precise treatment of tumors.

[0006] In summary, how to prepare nanoparticles that combine the above effects is of great significance. Summary of the invention

[0007] The purpose of the present invention is to provide a tannic acid iron modified dual photosensitizer loaded upconversion nanoparticles (UCS-PS-FeTA) and its preparation method and application in view of the deficiencies of the prior art. In the present invention, four-layer core-shell structure upconversion nanoparticles (UCNPs) are first prepared, and after modification, dual photosensitizers dihydrochlorin E6 (chlorin-e6, Ce6) and merocyanine 540 (merocyanine 540, MC540) are loaded, and tannic acid iron complexes are further adsorbed on the surface to form new composite nanoparticles (UCS-PS-FeTA), wherein the absorption peak of the dual photosensitizer completely covers the UCNP emission peak, which can improve the PDT efficacy under near-infrared light induction, and the FeTA tumor microenvironment responsively induces CDT and oxygen production, realizing CDT / PDT synergistic treatment of tumors. Tumor cell ICD is further induced, and immune checkpoint inhibitors are combined to enhance the anti-tumor immune response. The system has both CT and MRI multimodal tumor imaging performance, realizing integrated diagnosis and treatment.

[0008] The objective of the present invention is achieved through the following technical solutions:

[0009] A tannic acid iron modified dual photosensitizer loaded upconversion nanoparticle comprises an upconversion nanoparticle and two photosensitizers and a tannic acid iron complex loaded on the surface of the upconversion nanoparticle, wherein the two photosensitizers are MC540 and Ce6 respectively, and the upconversion nanoparticle is a four-layer core-shell structure of NaGdF4:Yb,Tm(49,1%)@NaGdF4:Nd,Yb(10,10%)@NaGdF4:Yb,Er(30,5%)@NaGdF4; wherein the two numerical values ​​in brackets represent the doping ratios of the two substances before the brackets respectively, for example, Yb,Tm(49,1%) means that the molar doping ratio of the two rare earth elements Yb and Tm is 49:1, and so on.

[0010] A method for preparing the above-mentioned ferric tannate modified dual photosensitizer-loaded upconversion nanoparticles comprises the following steps:

[0011] (1) 15 mg of ligand-free upconversion nanoparticles, 20 mL of ethanol, 0.8 mL of ammonia water, and 20 μL of tetraethyl orthosilicate were dispersed in a 40 mg / mL aqueous solution of polyvinyl pyrrolidone in sequence, and then 50 μL of 3-aminopropyltriethoxysilane was added and reacted for 4 hours to obtain silica-coated upconversion nanoparticles UCNP@SiO2;

[0012] (2) 0.2 mL 15 mg / mL UCNP@SiO2, 0.5 mL 1 mg / mL Ce6 acetone solution, and 0.5 mL 1 mg / mL MC540 aqueous solution were dispersed in deionized water, mixed evenly, and centrifuged. The product was dispersed in 1 mL deionized water, and then 70 μL 24 mM tannic acid aqueous solution and 70 μL 24 mM FeCl3 aqueous solution were added to form a mixed solution. The above mixed solution was vortexed for 30 seconds and ultrasonically treated for 2 minutes, and then centrifuged and washed to obtain tannic acid iron modified dual photosensitizer loaded upconversion nanoparticles.

[0013] Furthermore, in step (1), the upconversion nanoparticles are prepared by the following method:

[0014] (1) 5 mM Ln2O3 and 50% trifluoroacetic acid aqueous solution were mixed and added into a flask, wherein the molar ratio of Ln2O3 to trifluoroacetic acid was 1:1.5, and heated to 110°C, and stirred continuously and condensed under reflux until the solution became clear, and then the excess trifluoroacetic acid was evaporated to obtain Ln(CF3COO)3 dissolved in 50 mL of deionized water.

[0015] (2) 2 mL of 0.2 M Gd(CH3COO)3 aqueous solution, 1.96 mL of 0.2 M Yb(CH3COO)3 aqueous solution, 0.04 mL of 0.2 M Tm(CH3COO)3 aqueous solution, 6 mL of oleic acid, and 14 mL of octadecene were mixed in a flask and stirred continuously. After heating to remove water, the temperature was raised to 150°C and maintained for 1 hour. After cooling to room temperature, 8 mL of 0.4 M NH4F methanol solution and 4 mL of 0.5 M NaOH methanol solution were added and the temperature was maintained at 50°C for 30 minutes. After heating to remove methanol and evacuating the mixture, the temperature was raised to 300°C in a nitrogen atmosphere and the reaction was carried out for 1.5 hours. After cooling to room temperature, washing with ethanol and centrifuging, NaGdF4:Yb,Tm (49,1%) nanoparticles were obtained and dispersed in 8 mL of cyclohexane.

[0016] (3) 0.8 mmol of Gd(CF3COO)3, 0.8 mmol of Nd(CF3COO)3, 0.8 mmol of Yb(CF3COO)3, 0.8 mmol of Na(CF3COO), 2 mL of NaGdF4:Yb,Tm(49,1%) cyclohexane solution, 6.34 mL of oleic acid, and 6.4 mL of octadecene were added to a flask, stirred and heated continuously, cyclohexane was removed under vacuum, and the temperature was raised to 310°C under nitrogen atmosphere for 45 minutes. Cooled to room temperature, ethanol was added for washing and centrifugation to obtain NaGdF4:Yb,Tm(49,1%)@NaGdF4:Nd,Yb(10,10%) and dispersed in 8 mL of cyclohexane.

[0017] (4) 1.6 mmol of Gd(CF3COO)3, 1.6 mmol of Nd(CF3COO)3, 1.6 mmol of Yb(CF3COO)3, 1.6 mmol of Na(CF3COO), 2 mL of NaGdF4:Yb,Tm(49,1%)@NaGdF4:Nd,Yb(10,10%) cyclohexane solution, 12.68 mL of oleic acid, and 12.8 mL of octadecene were added to a flask, stirred and heated continuously, cyclohexane was removed under vacuum, and the temperature was raised to 310°C under nitrogen atmosphere for 45 minutes. Cooled to room temperature, ethanol was added for washing and centrifugation to obtain NaGdF4:Yb,Tm(49,1%)@NaGdF4:Nd,Yb(10,10%)@NaGdF4:Yb,Er(30,5%) and dispersed in 8 mL of cyclohexane.

[0018] (5) 0.8 mmol of Gd(CF3COO)3, 0.8 mmol of Na(CF3COO), 4 mL of NaGdF4:Yb,Tm(49,1%)@NaGdF4:Nd,Yb(10,10%)@NaGdF4:Yb,Er(30,5%) cyclohexane solution, 6.34 mL of oleic acid, and 6.4 mL of 1-octadecene were added to a flask, stirred continuously and heated to 110°C, cyclohexane was removed under vacuum, and the temperature was raised to 310°C under nitrogen atmosphere for 45 minutes. Cooled to room temperature, ethanol was added for washing and centrifugation to obtain NaGdF4:Yb,Tm(49,1%)@NaGdF4:Nd,Yb(10,10%)@NaGdF4:Yb,Er(30,5%)@NaGdF4, i.e., the final upconversion nanoparticles.

[0019] Furthermore, the ligand-free upconversion nanoparticles are prepared by the following method:

[0020] 1 mL of 36 mg / mL cyclohexane solution of upconversion nanoparticles was added to 5 mL of acetone and centrifuged to obtain the precipitate, which was redispersed in 6 mL of acetone, and 0.6 mL of 12 M concentrated hydrochloric acid was added. The solution was ultrasonicated for 30 minutes and then centrifuged. After washing with acetone three times, ligand-free upconversion nanoparticles were formed.

[0021] An application of the above-mentioned iron tannate modified dual photosensitizer loaded upconversion nanoparticles includes: preparing a chemodynamic photodynamic synergistic therapy preparation for tumors and / or preparing a preparation for computed tomography (MRI) and magnetic resonance imaging (CT) at the tumor site.

[0022] Among them, in the UCNP loaded with dual photosensitizers of the present invention, the Yb group and the Gd group in the UCNP have CT and MRI imaging capabilities. Therefore, it has the potential for multimodal tumor imaging. 3+ The complex with TA is pH responsive. TA is tightly complexed under neutral conditions and decomposes under acidic conditions, activating TA to Fe 3+ The reduction of Fe 2+ , Fe 2+ and Fe 3+ All are common Fenton reagents, Fe 2+ The CDT efficiency is significantly better than that of Fe 3+ , while in an environment rich in H2O2, Fe 3+ It can further react with H2O2 to produce oxygen, enhancing the PDT efficiency. 2+ / Fe 3+The conversion not only reacts with H2O2 to produce CDT, but also produces oxygen in the presence of H2O2 to promote PDT, thereby achieving CDT / PDT synergistic treatment of tumors.

[0023] The tannic acid iron-modified dual photosensitizer-loaded upconversion nanoparticles of the present invention can also be used in combination with immune checkpoint inhibitors to enhance anti-tumor immunity.

[0024] The beneficial effect of the present invention is that under the irradiation of 808nm near-infrared light, the UCS-PS-FeTA composite nanoparticles excite the dual photosensitizers Ce6 and MC540 through UCNP upconversion, producing efficient PDT. FeTA responds to the acidic microenvironment of the tumor and decomposes to produce Fe 2+ , producing highly efficient chemodynamic therapy, while producing oxygen, promoting the efficiency of PDT, and achieving CDT / PDT synergistic treatment of tumors. It further induces ICD, increases tumor immunogenicity, and combines with immune checkpoint inhibitors to enhance the effect of tumor immunotherapy. In addition, it has the tumor imaging performance of both CT and MRI, which is of great significance in tumor diagnosis and treatment.

[0025] In the present invention, a chemodynamic therapy and photodynamic therapy synergistic treatment platform is realized through composite nanoparticles, anti-tumor immune response is activated, and diagnosis and treatment integration under multimodal imaging is realized. The preparation method of the present invention has simple process, low price, good biosafety, and strong clinical application potential and value. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The present invention is further described below in conjunction with the accompanying drawings and embodiments;

[0027] Figure 1 is the scanning electron microscope (SEM) image of UCNP (scale bar: 50 nm);

[0028] Figure 2 SEM image of UCS (scale bar: 50 nm);

[0029] Figure 3 is the hydrodynamic radius of UCS and UCS-PS-FeTA composite nanoparticles;

[0030] Figure 4 FTIR spectra of Ce6, MC540, UCS, and UCS-Ce6 / MC540 composite nanoparticles;

[0031] Figure 5 is the Zeta potential of UCNP and its composite nanoparticles during modification;

[0032] Figure 6 Element distribution map of UCS-PS-FeTA composite nanoparticles (scale bar: 50 nm);

[0033] Figure 7 This is the dissolved oxygen curve of UCS-PS-FeTA composite nanoparticles in the solution under acidic environment and the presence of H2O2;

[0034] Figure 8 ESR detection of UCS-PS-FeTA composite nanoparticles under different conditions · OH and 1 O2 generation;

[0035] Fig. 9 The cell activity of normal mouse liver cells AML12 after incubation with different concentrations of UCS-PS-FeTA for 24 hours;

[0036] Fig.10 The cell activity of mouse liver cancer cell Hepa1-6 under UCS-PS-FeTA, UCS-PS+NIR, and UCS-PS-FeTA+NIR conditions;

[0037] Fig.11 To detect the apoptosis of Hepa1-6 cells under UCS-PS-FeTA, UCS-PS+NIR, and UCS-PS-FeTA+NIR conditions using flow cytometry;

[0038] Fig.12 To detect the statistical analysis of apoptosis of Hepa1-6 cells under UCS-PS-FeTA, UCS-PS+NIR, and UCS-PS-FeTA+NIR conditions using flow cytometry;

[0039] Fig.13 To detect the intracellular ROS production of Hepa1-6 cells under UCS-PS-FeTA, UCS-PS+NIR, and UCS-PS-FeTA+NIR conditions using flow cytometry;

[0040] Fig.14 To detect the statistical analysis of intracellular ROS production in Hepa1-6 cells under UCS-PS-FeTA, UCS-PS+NIR, and UCS-PS-FeTA+NIR conditions using flow cytometry;

[0041] Fig.15 Confocal microscopy was used to observe the intracellular CRT efflux of Hepa1-6 cells under UCS-PS-FeTA, UCS-PS+NIR, and UCS-PS-FeTA+NIR conditions; (G1: control, G2: UCS-PS-FeTA, G3: UCS-PS+NIR, G4: UCS-PS-FeTA+NIR) (Scale bar: 50 μm)

[0042] Fig.16 Confocal microscopy was used to observe the efflux of HMGB1 from the nucleus of Hepa1-6 cells under UCS-PS-FeTA, UCS-PS+NIR, and UCS-PS-FeTA+NIR conditions; (G1: control, G2: UCS-PS-FeTA, G3: UCS-PS+NIR, G4: UCS-PS-FeTA+NIR) (Scale bar: 50 μm)

[0043] Fig.17 The weight changes of mice in different groups;

[0044] Fig.18 The changes of tumor volume during treatment in different groups of mice;

[0045] Fig.19 The tumor masses of mice in different groups after treatment;

[0046] Fig. 20 Optical images of tumors in different groups of mice after treatment;

[0047] Fig.21 CRT immunofluorescence staining of tumor tissues in different groups (scale bar: 100 μm);

[0048] Fig. 22 These are optical photos of proximal tumors in different groups of mice after treatment when UCS-PS-FeTA composite nanoparticles were combined with PD-L1 antibodies;

[0049] Fig.23 The proximal tumor mass of mice in different groups after treatment when UCS-PS-FeTA composite nanoparticles were combined with PD-L1 antibody;

[0050] Fig.24 The changes in proximal tumor volume of mice in different groups after treatment when UCS-PS-FeTA composite nanoparticles were combined with PD-L1 antibodies;

[0051] Fig.25 Optical photos of distal tumors in different groups of mice after treatment when UCS-PS-FeTA composite nanoparticles were combined with PD-L1 antibodies;

[0052] Fig.26 The distal tumor mass of mice in different groups after treatment when UCS-PS-FeTA composite nanoparticles were combined with PD-L1 antibody;

[0053] Fig. 27 The changes in distal tumor volume of mice in different groups after treatment when UCS-PS-FeTA composite nanoparticles were combined with PD-L1 antibodies;

[0054] Fig.28 The in vitro MRI T1-weighted signals of UCS-PS-FeTA composite nanoparticles with different concentrations; the concentrations of gadolinium ions in the UCS-PS-FeTA composite nanoparticles from left to right are 0.03125, 0.0625, 0.125, 0.25, and 0.5 mM, respectively;

[0055] Fig.29 MRI T1-weighted signals of tumor sites in Hepa1-6 tumor-bearing mice before, and 1, 2, and 3 hours after intravenous injection of UCS-PS-FeTA composite nanoparticles;

[0056] Fig.30 In vitro CT imaging signals of UCS-PS-FeTA composite nanoparticles with different concentrations; the concentrations of UCS-PS-FeTA composite nanoparticles from left to right are 0.625, 1.25, 2.5, 5, and 10 mg / mL, respectively;

[0057] Fig.31 The CT imaging signals of the tumor site in Hepa1-6 tumor-bearing mice before and 1, 2, and 3 hours after intravenous injection of UCS-PS-FeTA composite nanoparticles. DETAILED DESCRIPTION

[0058] The present invention is only necessarily described below in conjunction with the accompanying drawings and the following embodiments. It should be understood that the accompanying drawings and the following embodiments are only used to illustrate the present invention, rather than to limit the present invention.

[0059] The present invention provides a composite nanoparticle, which first synthesizes a four-layer core-shell structure UCNP nanoparticle, covers the surface with a SiO2 layer, and performs amino modification at the same time. Further, the dual photosensitizers MC540 and Ce6 are loaded by electrostatic adsorption, and the tannic acid iron complex is adsorbed on the surface to form the final composite nanoparticle.

[0060] The up-conversion nanoparticles of the present invention are NaGdF4:Yb,Tm(49,1%)@NaGdF4:Nd,Yb(10,10%)@NaGdF4:Yb,Er(30,5%)@NaGdF4 with a four-layer core-shell structure. 3+ As a sensitizing ion, it effectively absorbs 808nm excitation light; Yb 3+ As a bridge for energy migration, it transfers energy to the shells on both sides through the interface, effectively stimulating Tm 3+ and Er 3+ Gd 3+ As a matrix ion, it can assist long-distance energy transfer and effectively transfer energy to the outermost layer. The loaded dual photosensitizers Ce6 and MC540 can achieve Tm 3+ and Er 3+The maximum coverage of luminescence produces efficient PDT. Ferric tannate decomposes in the acidic tumor microenvironment, activating TA to Fe 3+ The reduction of Fe 2+ , chemodynamic therapy with H2O2 production in the tumor microenvironment, while Fe 3+ It can also further react with H2O2 to produce oxygen, promote the efficiency of PDT, and achieve CDT / PDT synergistic treatment of tumors.

[0061] The four-layer core-shell structure UCNP nanoparticle size of the present invention is about 60nm, and has a nano effect. 3+ -Yb 3+ -Tm 3+ and Nd 3+ -Yb 3+ -Er 3+ The upconversion system emits multiple visible / ultraviolet emission peaks.

[0062] To further load the photosensitizers necessary for photodynamic therapy and the Fenton reagent required for chemodynamic therapy, UCNPs need to be modified and modified before functional factor loading. Specifically, 1 mL of UCNP cyclohexane solution was added to 5 mL of acetone, centrifuged, and the precipitate was dispersed in 6 mL of acetone to form a suspension (6 mg / mL), and 0.6 mL of concentrated hydrochloric acid (12M) was added. After ultrasonication for 30 minutes, it was centrifuged. Wash with acetone and centrifuge for 3 times, dispersed in deionized water to form ligand-free UCNP. 15 mg of ligand-free UCNP was added to 5 mL of polyvinyl pyrrolidone aqueous solution (40 mg / mL) and ultrasonicated for 30 minutes. Add 20 mL of ethanol and stir for 30 minutes. Add 0.8 mL of ammonia water and stir for 30 minutes. Add 20 μL of tetraethyl orthosilicate and stir for 12 hours. Add 50 μL of 3-aminopropyltriethoxysilane and stir for 4 hours. After centrifugation, the product was washed three times with deionized water and ethanol, and dispersed in 3 mL of deionized water to form UCNP@SiO2 (UCS) coated with silica and modified with amino groups on the surface.

[0063] 0.2mL 15mg / mL UCS aqueous solution, 0.5mL Ce6 acetone solution (1mg / mL), 0.5mL MC540 aqueous solution (1mg / mL) were mixed, ultrasonicated for 30 minutes, centrifuged, and the product was dispersed in 1mL deionized water to obtain UCS-Ce6 / MC540 (UCS-PS) composite nanoparticles loaded with dual photosensitizers MC540 and Ce6. 70μL tannic acid aqueous solution (24mM) and 70μL FeCl3 aqueous solution (24mM) were added, vortexed quickly for 30 seconds, and ultrasonicated for 2 minutes. Centrifuged, the product was washed 3 times with deionized water and dispersed in deionized water. UCS-PS-FeTA composite nanoparticles loaded with dual photosensitizers MC540 and Ce6 and adsorbed on the surface of tannic acid iron complex were formed.

[0064] The present invention is described in detail below by way of examples. It should be understood that the following examples are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention all belong to the scope of protection of the present invention. The specific process parameters of the following examples are also only an example within a suitable range, that is, those skilled in the art can make a selection within a suitable range through the description herein, and are not limited to the specific values ​​​​exemplified below.

[0065] Example 1: Preparation of multilayer core-shell structured UCNP nanoparticles

[0066] Ln2O3 (5mM, Ln = Gd, Yb, Nd, Er) and trifluoroacetic acid (35mmol, 50wt% aqueous solution) were added to a flask, wherein the molar ratio of Ln2O3 to trifluoroacetic acid was 1:1.5, heated to 110°C, stirred continuously and condensed under reflux until the solution became clear, and the excess trifluoroacetic acid was evaporated to obtain Ln(CF3COO)3. Ln(CF3COO)3 was dissolved in 50mL of deionized water for later use.

[0067] Add 2mL 0.2M Gd(CH3COO)3 aqueous solution, 1.96mL 0.2M Yb(CH3COO)3 aqueous solution, 0.04mL 0.2M Tm(CH3COO)3 aqueous solution to the flask, then add 6mL oleic acid and 14mL octadecene, continue stirring, and heat to 130℃ to remove water. After the water is removed, heat to 150℃ and keep for 1 hour. Cool to room temperature, add a mixed methanol solution of NH4F (8mL, 0.4M) and NaOH (4mL, 0.5M), and keep warm at 50℃ for 30 minutes. Heat to 100℃ to remove methanol and evacuate, then heat to 300℃ in a nitrogen atmosphere for 1.5 hours. Cool to room temperature, add ethanol to wash and centrifuge 3 times, and store in 8mL cyclohexane.

[0068] Add 0.8 mmol of Ln(CF3COO)3 (Ln=Gd, Nd, Yb), 0.8 mmol of Na(CF3COO), 2 mL of NaGdF4:Yb, Tm (49, 1%) cyclohexane solution, 6.34 mL of oleic acid, and 6.4 mL of octadecene to a flask, stir continuously and heat to 110°C, remove cyclohexane under vacuum, heat to 310°C under nitrogen atmosphere and react for 45 minutes. Cool to room temperature, add ethanol to wash and centrifuge 3 times, and store in 8 mL of cyclohexane.

[0069] 1.6 mmol of Ln(CF3COO)3 (Ln=Gd, Nd, Yb), 1.6 mmol of Na(CF3COO), 2 mL of NaGdF4:Yb,Tm(49,1%)@NaGdF4:Nd,Yb(10,10%) cyclohexane solution, 12.68 mL of oleic acid, and 12.8 mL of 1-octadecene were added to the flask, stirred continuously and heated to 110°C, cyclohexane was removed under vacuum atmosphere, and the temperature was raised to 310°C under nitrogen atmosphere for 45 minutes. Cooled to room temperature, washed with ethanol, centrifuged 3 times, and stored in 8 mL of cyclohexane.

[0070] 0.8 mmol of Gd(CF3COO)3, 0.8 mmol of Na(CF3COO), 4 mL of NaGdF4:Yb,Tm(49,1%)@NaGdF4:Nd,Yb(10,10%)@NaGdF4:Yb,Er(30,5%) cyclohexane solution, 6.34 mL of oleic acid, and 6.4 mL of 1-octadecene were added to the flask, stirred continuously and heated to 110°C, cyclohexane was removed under vacuum atmosphere, and the temperature was raised to 310°C under nitrogen atmosphere for 45 minutes. Cooled to room temperature, washed with ethanol, centrifuged 3 times, and stored in 4 mL of cyclohexane.

[0071] like Figure 1 As shown, the synthesized UCNPs have a multilayer core-shell structure with good dispersion and an average diameter of about 60 nm.

[0072] Example 2: Preparation of UCS-PS-FeTA composite nanoparticles

[0073] 1mL of UCNP cyclohexane solution was added to 5mL of acetone and centrifuged. The precipitate was dispersed in 6mL of acetone to form a suspension. 0.6mL of concentrated hydrochloric acid (12M) was added and the suspension was centrifuged after ultrasonication for 30 minutes. Wash with acetone and centrifuge for 3 times, disperse in deionized water to form ligand-free UCNP. 15mg of ligand-free UCNP was added to 5mL of polyvinylpyrrolidone aqueous solution (40mg / mL) and ultrasonicated for 30 minutes. 20mL of ethanol was added and stirred for 30 minutes. 0.8mL of ammonia water was added and stirred for 30 minutes. 20μL of tetraethyl orthosilicate was added and stirred for 12 hours. 50μL of 3-aminopropyltriethoxysilane was added and stirred for 4 hours. Centrifuge, wash the product with deionized water and ethanol for 3 times, disperse in 3mL of deionized water to form silica-coated and surface-amino-modified nanoparticles UCNP@SiO2 (UCS).

[0074] 0.2mL 15mg / mL UCS aqueous solution, 0.5mL Ce6 acetone solution (1mg / mL), 0.5mL 1mg / mL MC540 aqueous solution were mixed, ultrasonicated for 30 minutes, centrifuged, and the product was dispersed in 1mL deionized water. 70μL tannic acid aqueous solution (24mM) and 70μL FeCl3 aqueous solution (24mM) were added, vortexed quickly for 30 seconds, and ultrasonicated for 2 minutes. Centrifuged, the product was washed 3 times with deionized water and dispersed in deionized water. UCS-PS-FeTA composite nanoparticles loaded with dual photosensitizers MC540 and Ce6 and adsorbed on the surface of tannic acid iron complex were formed.

[0075] like Figure 2 , the nanoparticles UCS still maintain good dispersibility, with a diameter of about 80nm. Figure 3 As shown in Figure 2, after loading the dual photosensitizers and adsorbing the FeTA complex on the surface, the hydrodynamic radius of the UCS-PS-FeTA composite nanoparticles increased compared to that of the UCS nanoparticles. Figure 4 The FTIR results show that the characteristic peak of the carboxyl group of Ce6 (1710 cm -1 ) and the CN vibration peak of MC540 (1710cm -1 ) appeared on the spectrum of UCS-Ce6 / MC540, proving the successful loading of the two photosensitizers. Zeta potential analysis revealed that the UCNP surface was positively charged (+43.0mV). The surface of the silica layer was rich in carboxyl groups, which made the USC surface negatively charged (-39.1mV), and after amino modification, the surface was positively charged (+13.3mV). After loading the dual photosensitizers and FeTA, the UCS-PS-FeTA surface finally had a negative charge (-36.3mV)( Figure 5). The element distribution analysis of UCS-PS-FeTA composite nanoparticles detected Na, Gd, F, Yb, Nd in UCNP, Si, O in silica-coated nanoparticles, Fe ( Figure 6 ).

[0076] Example 3: Performance testing of UCS-PS-FeTA composite nanoparticles

[0077] The dissolved oxygen content in the UCS-PS-FeTA solution was measured using a dissolved oxygen meter in acidic (pH=5.8) and H2O2 (1 mM) conditions to characterize its oxygen production performance.

[0078] Electron spin spectroscopy (ESR) was used to detect ROS species, where DMPO and TEMP were used to detect hydroxyl radicals ( 。 OH) and singlet oxygen ( 1 O2). Three test groups were set up in PBS with a pH value of 5.8, namely 1. DMPO group, 2. UCS-PS-FeTA+H2O2 group, and 3. UCS-PS-FeTA+H2O2+near-infrared light group.

[0079] like Figure 7 As shown, there is no obvious oxygen production in the H2O2 solution. When H2O2 is added to the UCS-PS-FeTA solution, dissolved oxygen is immediately detected. At the same time, oxygen is continuously generated during the 300-second observation period. ESR is used to detect the types of ROS produced by UCS-PS-FeTA under the action of 808nm near-infrared light. UCS-PS-FeTA+H2O2 shows a 1:2:2:1 peak belonging to OH, and UCS-PS-FeTA+H2O2 shows a 1:2:2:1 peak belonging to OH under near-infrared light irradiation. 1 The peaks of 1:1:1 of O2 and 1:2:2:1 of OH indicate that the composite nanoparticles have CDT / PDT synergistic therapeutic potential under near-infrared light irradiation ( Figure 8 ).

[0080] Example 4: Biocompatibility of UCS-PS-FeTA composite nanoparticles

[0081] The biocompatibility of UCS-PS-FeTA composite nanoparticles was investigated at the cellular level.

[0082] UCS-PS-FeTA composite nanoparticles were co-incubated with normal mouse liver cells AML12 for 24 hours. Even at a concentration of 500 μg / mL, cell survival was not significantly affected, indicating that UCS-PS-FeTA composite nanoparticles have good biocompatibility ( Fig. 9 ).

[0083] Example 5: In vitro tumor therapeutic effect of UCS-PS-FeTA composite nanoparticles

[0084] The in vitro tumor therapeutic effect of UCS-PS-FeTA composite nanoparticles was investigated at the cellular level using mouse liver cancer cells Hepa1-6. UCS-PS-FeTA composite nanoparticles were co-incubated with Hepa1-6 for 24 hours, and about 50% of the cells survived under the single CDT action. Since UCS-PS composite nanoparticles are not modified with ferric tannate, they only have PDT effect under the action of 808nm NIR, and about 50% of the cells survive. Under the irradiation of 808nm NIR, only about 20% of the cells survived under the action of UCS-PS-FeTA composite nanoparticles, reflecting the synergistic effect of CDT / PDT ( Fig.10 ). Flow cytometry was used to detect cell apoptosis after different treatments. It was found that UCS-PS-FeTA and UCS-PS under 808nm NIR caused about 25% and 30% of cell apoptosis, respectively, indicating the effects of single CDT and single PDT. UCS-PS-FeTA under 808nm NIR can cause about 50% of cell apoptosis, showing a synergistic therapeutic effect of CDT / PDT ( Fig.11 , Fig.12 ). Flow cytometry was used to detect the production of intracellular ROS after different treatments. It was found that the ROS produced by single CDT and PDT was twice that of the control group, while the ROS produced by the synergistic effect of CDT / PDT was four times that of the control group ( Fig.13 , Fig.14 ). Using fluorescence confocal experiments, it was found that there was almost no Calreticulin (CRT) on the surface of tumor cell membranes in the control group. After treatment with UCS-PS-FeTA and UCS-PS under 808nm NIR, the distribution of CRT on the surface of tumor cell membranes increased. The CRT fluorescence intensity was the strongest after treatment with UCS-PS-FeTA under 808nm NIR ( Fig.15 In addition, HMGB1 in the control group was located in the cell nucleus. After treatment with UCS-PS-FeTA and UCS-PS under 808nm NIR, the HMGB1 positive ratio in the cell nucleus decreased. After treatment with UCS-PS-FeTA under 808nm NIR, the HMGB1 positive ratio in the cell nucleus decreased further ( Fig.16 ). When tumor cells produce immunogenic cell death, CRT is transported from the endoplasmic reticulum to the cell membrane, and HMGB1 is expelled from the cell nucleus. This indicates that UCS-PS-FeTA composite nanoparticles induce significant immunogenic cell death in tumor cells under the action of near-infrared light.

[0085] Example 6: In vivo tumor therapeutic effect of UCS-PS-FeTA composite nanoparticles

[0086] The UCS-PS-FeTA composite nanoparticles were used to investigate the in vivo tumor treatment of Hepa1-6 liver cancer cell subcutaneous tumor-bearing mice. The liver cancer tumor-bearing mice were randomly divided into four groups for in vivo treatment: 1. Intravenous injection of PBS (control group); 2. Intravenous injection of UCS-PS-FeTA (UCS-PS-FeTA group); 3. Intravenous injection of UCS-PS and NIR irradiation (UCS-PS+NIR group); 4. Intravenous injection of UCS-PS-FeTA and NIR irradiation (UCS-PS-FeTA+NIR group). The treatment system was intravenously injected on day 0. The amount of UCS-PS in the treatment system remained the same. The intravenous injection dose was 35 mg / kg. 808nm NIR irradiation was performed 2 hours after injection. The conditions involving 808nm NIR irradiation were 1.0w cm -2 Irradiation for 5 minutes. The treatment cycle was 2 weeks, and the weight and tumor volume of mice were measured every 2 days. After 2 weeks of treatment, there was no significant difference in the weight of mice among the groups, indicating that UCS-PS-FeTA has good in vivo biocompatibility ( Fig.17 ). Both the UCS-PS-FeTA group and the UCS-PS+NIR group had a certain degree of tumor growth inhibition effect. The tumor growth of mice in the UCS-PS-FeTA+NIR group was significantly inhibited ( Fig.18 ). According to the optical photos of the tumor and the tumor mass results, it was also shown that the UCS-PS-FeTA+NIR group had the strongest anti-tumor effect ( Fig.19 , Fig. 20 ). Groups 2 and 3 represent single chemodynamic therapy and photodynamic therapy, respectively, and group 4 represents the synergistic effect of chemodynamic therapy and photodynamic therapy, indicating that the synergistic effect of chemodynamic therapy and photodynamic therapy of UCS-PS-FeTA under NIR irradiation is excellent in treating tumors. Immunofluorescence staining was further performed on the tumor tissue to observe the ICD changes of tumor cells in vivo. Compared with the UCS-PS-FeTA group and the UCS-PS+NIR group, the fluorescence intensity of CRT on the tumor cell membrane surface of the UCS-PS-FeTA+NIR group was the strongest, and the effect of inducing ICD was the most obvious ( Fig.21 ).

[0087] A bilateral Hepa1-6 liver cancer cell subcutaneous tumor-bearing model was established, and UCS-PS-FeTA was combined with PD-L1 antibody to evaluate the systemic antitumor immune response. Bilateral subcutaneous tumor-bearing mice were randomly divided into 4 groups: 1. intravenous injection of PBS, 2. PD-L1 antibody, 3. UCS-PS-FeTA near infrared, 4. UCS-PS-FeTA combined with near infrared and PD-L1 antibody. Groups 2 and 4 were intravenously injected with PD-L1 antibody (2 mg / kg) on ​​days 1, 4, and 7, respectively. After intravenous injection of UCS-PS-FeTA in groups 3 and 4, only the left tumor site (primary tumor) was irradiated with near infrared, and no treatment was performed on the right tumor site (distal tumor). The results showed that UCS-PS-FeTA combined with near infrared had an inhibitory effect on the growth of primary tumors and had a better inhibitory effect on the growth of distant tumors. The combined treatment of PD-L1 antibody and UCS-PS-FeTA with near infrared had a more obvious inhibitory effect on the growth of primary and distant tumors ( Fig. 22 , Fig.23 ). Photos of tumor tissue and tumor weight showed similar trends, indicating that the anti-tumor effects of PD-L1 antibody and UCS-PS-FeTA combined with near-infrared were enhanced ( Fig.24 , Fig.25 , Fig.26 , Fig. 27 ).

[0088] This indicates that UCS-PS-FeTA composite nanoparticles have excellent tumor-killing effects and are of great significance in tumor treatment.

[0089] Example 7: In vivo tumor imaging effect of UCS-PS-FeTA composite nanoparticles

[0090] The CT and MRI tumor imaging effects of UCS-PS-FeTA composite nanoparticles were investigated. In vitro, UCS-PS-FeTA has the potential to be used as an MRI contrast agent. With the increase of concentration, the T1 signal is significantly enhanced ( Fig.28 ). In vivo MRI imaging capability was tested in Hepa1-6 tumor-bearing mice. One hour after injection of UCS-PS-FeTA 35mg / kg, the T1 signal at the tumor site was significantly higher than before injection. The signal only slightly decreased 3 hours after injection ( Fig.29 ). In addition, UCS-PS-FeTA can be used as a CT contrast agent. As the concentration increases, the CT value is significantly enhanced ( Fig.30 ). Compared with before intravenous injection, the CT signal of the tumor site was significantly enhanced 1 hour after injection of UCS-PS-FeTA 35mg / kg, and the signal decreased slightly 3 hours after injection ( Fig.31 ). In vivo MRI and CT imaging showed the diagnostic effect of UCS-PS-FeTA composite nanoparticles.

Claims

1. A dual photosensitizer-loaded upconversion nanoparticle modified with iron tannate, characterized in that: The invention comprises upconversion nanoparticles and two photosensitizers and tannic acid iron complex loaded on the surface of the upconversion nanoparticles, wherein the two photosensitizers are MC540 and Ce6 respectively, and the upconversion nanoparticles are NaGdF4:Yb,Tm@NaGdF4:Nd,Yb@NaGdF4:Yb,Er@NaGdF4 with a four-layer core-shell structure, and the surface is coated with SiO2, and amino modification is performed at the same time; wherein in the NaGdF4:Yb,Tm layer, the molar doping ratio of the two rare earth elements Yb and Tm is 49:1; in the NaGdF4:Nd,Yb layer, the molar doping ratio of the two rare earth elements Nd and Yb is 10:10; and in the NaGdF4:Yb,Er layer, the molar doping ratio of the two rare earth elements Yb and Er is 30:

5.

2. A method for preparing the ferric tannate modified dual photosensitizer-loaded upconversion nanoparticles according to claim 1, characterized in that: The following steps are involved: (1) 15 mg of ligand-free upconversion nanoparticles, 20 mL of ethanol, 0.8 mL of ammonia water, and 20 μL of tetraethyl orthosilicate were dispersed in 5 mL of a 40 mg / mL aqueous solution of polyvinyl pyrrolidone in sequence, and then 50 μL of 3-aminopropyltriethoxysilane was added and reacted for 4 hours to obtain silica-coated upconversion nanoparticles UCNP@SiO2; (2) Disperse 0.2 mL of 15 mg / mL UCNP@SiO2 aqueous solution, 0.5 mL of 1 mg / mL Ce6 acetone solution, and 0.5 mL of 1 mg / mL MC540 aqueous solution in deionized water, mix well, and centrifuge. Disperse the product in 1 mL of deionized water, then add 70 μL of 24 mM tannic acid aqueous solution and 70 μL of 24 mM FeCl3 aqueous solution to form a mixed solution. Vortex the mixed solution for 30 seconds and ultrasonically treat for 2 minutes, then centrifuge and wash to obtain tannic acid iron-modified dual photosensitizer-loaded upconversion nanoparticles.

3. The preparation method according to claim 2, characterized in that: In the step (1), the upconversion nanoparticles are prepared by the following method: (1) mixing 5 mM Ln2O3 and 50% trifluoroacetic acid aqueous solution, wherein the molar ratio of Ln2O3 to trifluoroacetic acid is 1:1.5, heating to 110°C, continuously stirring and condensing under reflux until the solution becomes clear, and evaporating excess trifluoroacetic acid to obtain Ln(CF3COO)3, wherein Ln is Gd, Yb, Nd or Er; (2) 2 mL of 0.2 M Gd(CH3COO)3 aqueous solution, 1.96 mL of 0.2 M Yb(CH3COO)3 aqueous solution, 0.04 mL of 0.2 M Tm(CH3COO)3 aqueous solution, 6 mL of oleic acid, and 14 mL of octadecene were mixed and stirred continuously, heated to remove water, and then heated to 150°C for 1 hour; cooled to room temperature, 8 mL of 0.4 M NH4F methanol solution and 4 mL of 0.5 M NaOH methanol solution were added, and the mixture was kept at 50°C for 30 minutes; heated to remove methanol and evacuated, and then heated to 300°C in a nitrogen atmosphere for reaction for 1.5 hours; cooled to room temperature, washed with ethanol, and centrifuged to obtain NaGdF4:Yb,Tm nanoparticles, which were dispersed in 8 mL of cyclohexane; the molar doping ratio of the two rare earth elements Yb and Tm in the NaGdF4:Yb,Tm nanoparticles was 49:1; (3) 0.8 mmol of Gd(CF3COO)3, 0.8 mmol of Nd(CF3COO)3, 0.8 mmol of Yb(CF3COO)3, 0.8 mmol of Na(CF3COO) and 2 mL of NaGdF4:Yb,Tm nanoparticle cyclohexane solution, 6.34 mL of oleic acid and 6.4 mL of octadecene were mixed and stirred continuously and heated, and the cyclohexane was removed under vacuum atmosphere, and the temperature was raised to 310°C under nitrogen atmosphere for reaction for 45 minutes; cooled to room temperature, washed with ethanol and centrifuged to obtain NaGdF4:Yb,Tm@NaGdF4:Nd,Yb and dispersed in 8 mL of cyclohexane; the molar doping ratio of the two rare earth elements Nd and Yb in NaGdF4:Nd,Yb was 10:10; (4) 1.6 mmol of Gd(CF3COO)3, 1.6 mmol of Nd(CF3COO)3, 1.6 mmol of Yb(CF3COO)3, 1.6 mmol of Na(CF3COO), 2 mL of NaGdF4:Yb,Tm@NaGdF4:Nd,Yb cyclohexane solution, 12.68 mL of oleic acid, and 12.8 mL of octadecene were added to a flask, stirred and heated continuously, cyclohexane was removed under vacuum, and the temperature was raised to 310°C under nitrogen atmosphere for reaction for 45 minutes; cooled to room temperature, washed with ethanol, and centrifuged to obtain NaGdF4:Yb,Tm@NaGdF4:Nd,Yb@NaGdF4:Yb,Er and dispersed in 8 mL of cyclohexane; in NaGdF4:Yb,Er, the molar doping ratio of the two rare earth elements Yb and Er was 30:5; (5) 0.8 mmol of Gd(CF3COO)3, 0.8 mmol of Na(CF3COO), 4 mL of NaGdF4:Yb,Tm@NaGdF4:Nd,Yb@NaGdF4:Yb,Er cyclohexane solution, 6.34 mL of oleic acid, and 6.4 mL of 1-octadecene were mixed, stirred continuously, and heated to 110°C. The cyclohexane was removed under vacuum atmosphere, and the mixture was heated to 310°C under nitrogen atmosphere for 45 minutes. The mixture was cooled to room temperature, washed with ethanol, and centrifuged to obtain NaGdF4:Yb,Tm@NaGdF4:Nd,Yb@NaGdF4:Yb,Er@NaGdF4, i.e., the final upconversion nanoparticles.

4. The preparation method according to claim 3, characterized in that: The ligand-free upconversion nanoparticles were prepared by the following method: 1 mL of 36 mg / mL cyclohexane solution of upconversion nanoparticles was added to 5 mL of acetone and centrifuged to obtain the precipitate, which was redispersed in 6 mL of acetone, and 0.6 mL of 12 M concentrated hydrochloric acid was added. The solution was ultrasonicated for 30 minutes and then centrifuged. The ligand-free upconversion nanoparticles were formed after washing with acetone three times.

5. An application of the ferric tannate modified dual photosensitizer-loaded upconversion nanoparticles according to claim 1, comprising: Preparation of chemodynamic and photodynamic synergistic therapy preparations for tumors and / or preparations for computer tomography and magnetic resonance imaging at tumor sites.

Images