Preparation method and application of hypoxic prodrug-loaded up-conversion nanoparticles

Abstract

The invention relates to a preparation method and application of hypoxic prodrug loaded up-conversion nanoparticles. The nanoparticles are formed by compounding nanocrystals based on up-conversion nanoparticles with an organic photosensitizer and loading glycyrrhizic acid and hypoxic prodrugs. The preparation method comprises the following steps: preparing the up-conversion nanoparticles, compounding the organic photosensitizer, and loading glycyrrhizic acid and hypoxic prodrug. The preparation method is simple, the photosensitizer and the hypoxic prodrug can be effectively loaded, and the photodynamic therapy material has efficient photodynamic therapy performance under the irradiation of 808nm near-infrared light. Glycyrrhizic acid enables nanoparticles to functionalize and target mitochondria, a hypoxic environment caused by photodynamic therapy activates a hypoxic prodrug, and DNA damage of tumor cells is caused. The tumor immunogen cell death is further caused, the tumor immunotherapy curative effect of the immune checkpoint inhibitor is enhanced, and a new strategy is provided for tumor immunotherapy.

Description

Technical Field

[0001] This invention belongs to the field of bionanomaterials and relates to upconversion nanoparticles loaded with hypoxia prodrugs, their preparation methods, and applications. Specifically, the invention relates to the preparation and application performance evaluation of a nanotherapeutic platform that synergizes photodynamic therapy with chemotherapy, simultaneously targeting tumor cell mitochondria to induce DNA damage and immunogenic cell death, thereby sensitizing immune checkpoint inhibitors. Background Art

[0002] Photodynamic therapy (PDT) uses non-toxic photosensitizers to generate toxic reactive oxygen species (ROS) under light, directly eliminating tumor cells in a spatially controlled manner. Oxidative damage induced by PDT triggers immunogenic cell death (ICD), manifested by the release of damage-associated molecular patterns (DAMPs) that promote the maturation of dendritic cells, macrophage polarization, and antigen presentation to cytotoxic T cells.

[0003] Most organic photosensitizers used for PDT are activated by UV-visible light, which can only treat superficial diseases due to the strong absorption and scattering of biological tissues. In contrast, near-infrared (NIR) light provides deeper tissue penetration due to the weakening of light absorption and scattering, making it more suitable for PDT of deep tumors. Although NIR-responsive photosensitizers have been developed, challenges such as photostability, biocompatibility and complex molecular synthesis remain to be solved. Another strategy involves combining photostable lanthanide-based nanomaterials with organic photosensitizers, using lanthanides such as Nd 3+ and Yb 3 + The NIR responsiveness of the NIR-induced PDT can be enhanced, and it can be combined with other treatment methods such as chemotherapy and immunotherapy to significantly expand its application in tumor diagnosis and treatment.

[0004] As central organelles for cellular metabolism and oxygen consumption, mitochondria play a key role in dynamic therapy research. Inhibiting mitochondrial physiological functions is an effective way to conserve intracellular oxygen during PDT. The ROS generated by PDT damages mitochondria, further amplifying oxidative stress and leading to cell apoptosis. Furthermore, mitochondria are closely linked to immune responses and play a crucial role in cell proliferation, apoptosis, and calcium homeostasis.

[0005] In summary, how to prepare a mitochondria-targeted nanoparticle with synergistic PDT effect is of great significance. Summary of the Invention

[0006] The purpose of the present invention is to provide an upconversion nanoparticle ((NZ @TG ) and its preparation method and application. In the present invention, upconversion nanoparticles NaGdF4:Nd are first prepared, and the organic photosensitizer ZnPcC4 is compounded with the upconversion nanoparticles through carboxyl coordination. The amphiphilic DSPE-PEG5K is used to transfer the hydrophobic upconversion nanoparticles and the organic photosensitizer complex into an aqueous solution to enhance its biocompatibility. At the same time, the mitochondrial targeting ligand glycyrrhizic acid and the hypoxia prodrug TH302 are loaded to form a new type of composite nanoparticles ((NZ @TG ). Under the action of glycyrrhizic acid, NZ @TG Targeting mitochondria in tumor cells; under 808nm NIR irradiation, upconversion nanoparticles interact with organic photosensitizers to produce efficient ROS, achieving excellent PDT effect. ROS damages mitochondrial oxidative stress, thereby alleviating the local hypoxic environment of mitochondria and promoting the efficacy of PDT. After the efficient PDT action, intracellular hypoxia is aggravated, thereby activating the hypoxia prodrug TH302 and inducing DNA damage. Mitochondrial oxidative stress and DNA damage further induce tumor cell ICD, activate systemic anti-tumor immune response, and enhance the efficacy of immune checkpoint inhibitors. Therefore, NZ targeting mitochondria @TG It has a cascade reaction effect of PDT and hypoxia prodrug, and can also promote anti-tumor immune response.

[0007] The object of the present invention is achieved through the following technical solutions:

[0008] A type of upconversion nanoparticle loaded with a hypoxia prodrug is composed of a composite of upconversion nanoparticles, an organic photosensitizer, an amphiphilic surfactant, glycyrrhizic acid and the hypoxia prodrug. Among them, the organic photosensitizer mainly absorbs near-infrared light, the upconversion nanoparticles assist the organic photosensitizer in absorbing near-infrared light, and the amphiphilic surfactant assists the hydrophobic upconversion nanoparticles and the organic photosensitizer in forming a hydrophilic complex to enhance its biocompatibility; glycyrrhizic acid is responsible for targeting mitochondria, and the hypoxia prodrug induces DNA damage under hypoxic conditions.

[0009] Furthermore, the mass ratio of upconversion nanoparticles and organic photosensitizer was 100:1, and the effective loading rates of glycyrrhizic acid and hypoxia prodrug were over 17.7% and 42.2%, respectively.

[0010] Furthermore, the organic photosensitizer is ZnPcC4, the upconversion nanoparticles are NaGdF4:Nd, the hypoxic prodrug is TH302, and the amphiphilic surfactant is DSPE-PEG5K.

[0011] A method for preparing upconversion nanoparticles loaded with hypoxia prodrugs comprises the following steps:

[0012] (1) A tetrahydrofuran solution of upconversion nanoparticles and a methanol solution of an organic photosensitizer are mixed, and the mixture is mixed overnight under N2 protection. The complex of the nanoparticles and the organic photosensitizer is collected and redispersed in chloroform to obtain a chloroform solution of the complex; wherein the volume ratio of the tetrahydrofuran solution of the upconversion nanoparticles to the methanol solution of the organic photosensitizer is 5:1; the volume of chloroform is twice the volume of methanol; the concentration of the organic photosensitizer in the methanol solution of the organic photosensitizer is 0.5 mg / mL, and the mass ratio of the upconversion nanoparticles to the organic photosensitizer is 100:1;

[0013] (2) The chloroform solution of the complex was added dropwise to the chloroform solution containing the amphiphilic surfactant, and glycyrrhizic acid and TH302 were added at the same time. After the addition of glycyrrhizic acid and TH302, the concentration was at least 0.048 mg / L; then, the mixture was stirred for 1 hour under N2 protection and heated at 50°C until the solvent was completely evaporated; the precipitate was redispersed in phosphate buffer solution and purified by ultrafiltration to obtain upconversion nanoparticles loaded with hypoxic prodrug; wherein the volume ratio of the chloroform solution of the complex to the chloroform solution containing the amphiphilic surfactant was 1:9; and the concentration of the amphiphilic surfactant in the chloroform solution containing the amphiphilic surfactant was 8 mg / mL.

[0014] Furthermore, in step (1), the upconversion nanoparticles are prepared by the following method:

[0015] The upconversion nanoparticles are NaGdF4:Nd, which are prepared by the following method:

[0016] 5mmoL Ln(CH3CO2)3 was mixed with 20mL oleic acid and 20mL 1-octadecene, heated to 150℃ under N2 atmosphere and kept warm for 2 hours to form a lanthanide oleate complex; then cooled to 110℃ and vacuumed for 30 minutes; then, 10mmoL NaHF2 powder was added under N2 atmosphere and vacuumed for 15 minutes; under N2 protection, the mixture was heated to 310℃ and kept warm for 40 minutes to obtain upconversion nanoparticles; wherein Ln=Gd, Nd, Gd:Nd=49:1.

[0017] An application of the above-mentioned upconversion nanoparticles loaded with hypoxia prodrugs includes: preparing a photodynamic chemotherapy synergistic treatment preparation for tumors and / or its role in sensitizing immune checkpoint inhibitors.

[0018] Among them, the present invention is a kind of up-conversion nanoparticles loaded with hypoxia prodrug, which shows excellent singlet oxygen generation efficiency under 808nm NIR irradiation and has excellent PDT effect. @TG With its mitochondrial targeting properties, PDT around mitochondria impairs mitochondrial function, alleviating the local hypoxic microenvironment, thereby triggering a positive feedback loop of PDT, promoting the accumulation of ROS and increasing oxidative stress. The hypoxia induced by PDT facilitates the activation of the hypoxic prodrug TH302, leading to DNA damage in tumor cells. This allows for synergistic photodynamic therapy and chemotherapy in the treatment of tumors.

[0019] Furthermore, the upconversion nanoparticles loaded with hypoxia prodrugs of the present invention can also be combined with immune checkpoint inhibitors to enhance the immunotherapy efficacy.

[0020] The beneficial effects of the present invention are: @TG Under 808nm NIR irradiation, the nanoparticles produce highly effective PDT through the interaction between upconversion nanoparticles and organic photosensitizers. Glycyrrhizic acid targets tumor cell mitochondria, generating PDT around them, inhibiting mitochondrial function and alleviating the hypoxic microenvironment, creating a positive feedback loop that boosts PDT efficiency. The intracellular hypoxic environment generated by efficient PDT activates the hypoxic prodrug TH302, causing DNA damage in tumor cells, thereby forming a synergistic photodynamic chemotherapy treatment. DNA damage and oxidative stress induce ICD in tumor cells, increasing tumor immunogenicity and sensitizing immune checkpoint inhibitors to enhance the efficacy of tumor immunotherapy, which is of great significance in tumor treatment.

[0021] In this invention, composite nanoparticles are used to achieve a synergistic chemotherapy and photodynamic therapy platform, activating systemic anti-tumor immune responses and sensitizing immune checkpoint inhibitors. The invention has a simple and feasible preparation process, low cost, excellent biosafety, and excellent clinical translation potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The present invention will be further described below with reference to the accompanying drawings and examples;

[0023] Figure 1 For NZ @TG FTIR spectrum of

[0024] Figure 2 For NZ @TG Hydrodynamic diameter diagram and transmission electron microscopy image (scale bar: 20 nm);

[0025] Figure 3The absorption spectra of DPBF degradation caused by singlet oxygen generated by NZ under 808nm NIR irradiation and the DPBF oxidation results of ZnPcC4, NaGdF4:Nd and NZ as the irradiation time changes;

[0026] Figure 4 The electron spin resonance spectrum of TEMP as a spin trap shows the singlet oxygen generated by NZ under 808nm NIR irradiation;

[0027] Figure 5 NZ shown by fluorescence confocal microscopy @TG Mitochondrial targeting results (scale bar: 20 μm);

[0028] Figure 6 Hep1-6 mouse liver cancer cells were cultured in NZ and NZ @T , NZ @TG Statistical graph of cell activity under different conditions;

[0029] Figure 7 In order to detect the expression of Hep1-6 cells in NZ and NZ by flow cytometry @T , NZ @TG Results of cell apoptosis under different conditions;

[0030] Figure 8 In order to detect the expression of Hep1-6 cells in NZ and NZ by flow cytometry @T , NZ @TG Statistical analysis results of cell apoptosis under different conditions;

[0031] Figure 9 Hep1-6 cells in NZ, NZ @T , NZ @TG Statistical results of mitochondrial ROS levels under different conditions;

[0032] Figure 10 Hep1-6 cells in NZ, NZ @T , NZ @TG GPX3 level statistical result graph under certain conditions;

[0033] Figure 11 Hep1-6 cells in NZ, NZ @T , NZ @TG Statistical results of Nrf-2 levels under different conditions;

[0034] Figure 12 Hep1-6 cells in NZ, NZ @T , NZ @TG Statistical results of SOD levels under different conditions;

[0035] Figure 13 In order to detect the expression of Hep1-6 cells in NZ and NZ by flow cytometry @T , NZ @TG Results of cell cycle arrest under different conditions;

[0036] Figure 14 In order to detect the expression of Hep1-6 cells in NZ and NZ by flow cytometry @T , NZ @TG Statistical analysis results of cell cycle arrest under different conditions;

[0037] Figure 15 To examine the Hep1-6 cells in NZ and NZ using transmission electron microscopy @T , NZ @TG Results of mitochondrial damage under different conditions;

[0038] Figure 16 Hep1-6 cells in NZ, NZ @T , NZ @TG Results of intracellular ATP under different conditions;

[0039] Figure 17 To observe Hep1-6 cells in NZ and NZ by confocal microscopy @T , NZ @TG Results of intracellular CRT efflux and nuclear HMGB1 efflux under different conditions (scale bar: 50 μm);

[0040] Figure 18 ELISA was used to detect the expression of Hep1-6 cells in NZ and NZ @T , NZ @TG Figure 3 shows the quantitative analysis results of HSP70 and HSP90 in the cell supernatant under different conditions;

[0041] Figure 19 Fluorescence imaging of tumors during treatment in different groups of mice;

[0042] Figure 20 The graph shows the results of fluorescence quantitative analysis of mice in different groups during treatment;

[0043] Figure 21 The results of immunofluorescence staining of HMGB1 and HSP90 in tumor tissues of different groups (scale bar: 50 μm);

[0044] Figure 22 The figure shows the quantitative analysis results of HMGB1 immunofluorescence staining of tumor tissues in different groups;

[0045] Figure 23 The figure shows the quantitative analysis results of HSP90 immunofluorescence staining of tumor tissues in different groups;

[0046] Figure 24 Figure 2 shows the results of CD8 / CD4 immunofluorescence staining of tumor tissues in different groups (scale bar: 100 μm);

[0047] Figure 25 For NZ @TG Fluorescence imaging of mouse tumors in combination with PD-L1 antibodies;

[0048] Figure 26 For NZ @TG Fluorescence quantitative analysis results in combination with PD-L1 antibody;

[0049] Figure 27 For NZ @TG Survival curve of mice treated with PD-L1 antibody;

[0050] Figure 28 For NZ @TG HE staining results of tumors combined with PD-L1 antibody (scale bar: 100 μm);

[0051] Figure 29 For NZ @TG Figure 3. CD8 / CD4 immunofluorescence staining results of tumors in combination with PD-L1 antibody (Scale bar: 100 μm). DETAILED DESCRIPTION

[0052] The present invention will be described below with reference to 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.

[0053] The present invention provides composite nanoparticles comprising upconversion nanoparticles loaded with a hypoxia prodrug. The upconversion nanoparticles, NaGdF4:Nd, are synthesized and then composited with an organic photosensitizer, ZnPcC4. The nanoparticles are then modified with the amphiphilic DSPE-PEG5K and loaded with the mitochondrial-targeting ligand glycyrrhizic acid and the hypoxia prodrug TH302 to form novel composite nanoparticles.

[0054] The interfacial energy transfer between upconversion nanoparticles NaGdF4:Nd and ZnPcC4 is the basis for the generation of singlet oxygen. The energy of the lowest excited triplet state of ZnPcC4 (ΔE S0-T1 , ~1.13eV) and Nd 3+ of 4 F 3 / 2 The excited state (~1.43eV) is very close, which makes the energy from Nd 3+ The triplet energy transfer from Nd to ZnPcC4 is possible. 3+Energy harvested from 808nm photons is transferred to the triplet state of ZnPcC4, generating singlet oxygen. Glycyrrhizic acid enables the composite nanoparticles to target tumor cell mitochondria. 808nm NIR light induces PDT, impairing mitochondrial function and alleviating the hypoxic microenvironment, thereby promoting PDT. The intracellular hypoxia induced by efficient PDT activates the hypoxic prodrug TH302, inducing DNA damage in tumor cells, thereby achieving synergistic photodynamic therapy with chemotherapy.

[0055] The present invention first prepares NaGdF4:Nd nanoparticles. An organic photosensitizer and other functional factors are further loaded. Specifically, a 50 mg solution of NaGdF4:Nd nanoparticles is precipitated with ethanol, resuspended in 5 mL of tetrahydrofuran, and mixed with 1 mL of a 0.5 mg / mL methanol solution of ZnPcC4. The mixture is stirred overnight under nitrogen protection. The resulting complex of the nanoparticles and the organic photosensitizer, NaGdF4:Nd-ZnPcC4, is collected by centrifugation at 20,000 rpm / min for 15 minutes and redispersed in 2 mL of chloroform.

[0056] 400 μL of a nanoparticle-organic photosensitizer complex, NaGdF4:Nd-ZnPcC4, in chloroform was added dropwise to 3.6 mL of chloroform containing 40 mg of DSPE-PEG5K. Simultaneously, 100 μL of a 2 mg / L solution of glycyrrhizic acid in methanol and 40 μL of a 5 mg / L solution of TH302 in DMSO were added. The mixture was stirred under nitrogen for 1 hour and heated at 50°C until the solvent evaporated completely. The precipitate was dispersed in 2 mL of phosphate buffer and purified by ultrafiltration to collect the complex, NaGdF4:Nd-ZnPcC4@TH302 / GA / DSPE-PEG.

[0057] 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 fall within the scope of protection of the present invention. The specific process parameters, etc. of the following examples are only examples within a suitable range, and those skilled in the art can select within a suitable range through the description herein, and are not limited to the specific numerical values ​​exemplified below. In the following examples, unless otherwise specified, indicators (such as protein expression levels, ATP content, etc.) are all carried out using relevant detection reagents in accordance with the corresponding operating instructions.

[0058] Example 1: NZ @TG Preparation and performance testing

[0059] 5 mmol Ln(CH3CO2)3 (Ln=Gd, Nd, Gd:Nd=49:1) was mixed with 20 mL oleic acid and 20 mL 1-octadecene and heated to 150°C under a nitrogen atmosphere for 2 hours to remove moisture, forming a lanthanide oleate complex. The mixture was then cooled to 110°C under vacuum for 30 minutes. Under nitrogen, 10 mmol NaHF2 powder was added to the reaction mixture, and the mixture was vacuumed for 15 minutes. Under nitrogen, the mixture was heated to 310°C and held for 40 minutes. The resulting NaGdF4:Nd nanoparticles were collected by ethanol precipitation and centrifugation at 4000 rpm for 8 minutes. The reaction mixture was redispersed in cyclohexane, washed three times with ethanol, and redispersed in 10 mL cyclohexane for storage.

[0060] A cyclohexane solution of 50 mg of NaGdF4:Nd nanoparticles was precipitated with ethanol, resuspended in 5 mL of tetrahydrofuran, and mixed with 1 mL of a 0.5 mg / mL methanol solution of ZnPcC4. The mixture was stirred overnight under nitrogen. The resulting complex of nanoparticles and organic photosensitizer, NaGdF4:Nd-ZnPcC4 (NZ), was collected by centrifugation at 20,000 rpm / min for 15 minutes and redispersed in 2 mL of chloroform.

[0061] 400 μL of chloroform solution of the nanoparticles and organic photosensitizer complex NaGdF4:Nd-ZnPcC4 was added dropwise to 3.6 mL of chloroform solution containing 40 mg of DSPE-PEG5K. At the same time, 100 μL of 2 mg / L glycyrrhizic acid methanol solution and 40 μL of 5 mg / L TH302 DMSO solution were added. The mixture was stirred for 1 h under N2 protection and heated at 50 ° C until the solvent was completely evaporated. The precipitate was dispersed in 2 mL of phosphate buffer solution and purified by ultrafiltration to collect the complex NaGdF4:Nd-ZnPcC4@TH302 / GA / DSPE-PEG (NZ @TG ).

[0062] like Figure 1 The FTIR spectrum test results show that the characteristic peaks of TH302, GA, ZnPcC4, NaGdF4:Nd, and DSPE-PEG5K appear in NZ @TG The successful loading of functional factors is proved on the spectrum. Figure 2 As shown, NZ @TG It has good dispersibility in water and has a diameter of about 21 nm.

[0063] The production of ROS was detected by taking advantage of the fact that DPBF can be oxidized by ROS. Electron spin spectroscopy (ESR) was used to detect the type of ROS, and TEMP was used to detect singlet oxygen ( 1Three detection groups were set up, all under near-infrared light conditions, namely 1. ZnPcC4 group, 2. NaGdF4:Nd group, and 3. NZ group.

[0064] like Figure 3 As shown in Figure 2, the decrease in the absorption of DPBF at 415 nm confirms that ROS generated by NZ under near-infrared light irradiation oxidizes DPBF. At the same time, a peak of 1:1:1 belonging to singlet oxygen is shown ( Figure 4 ).

[0065] Example 2: NZ @TG In vitro tumor therapeutic effects

[0066] Using mouse liver cancer cells Hep1-6 to explore NZ at the cellular level @TG In vitro tumor therapeutic effect. 5-Carboxyfluorescein was labeled with NZ @TG and New Zealand @T (Only add the same amount of TH302 during the complexation) to detect the intracellular distribution of nanoparticles. @TG and New Zealand @T After incubation with Hep1-6 cells at a ratio of 1 mg nanoparticles per 100,000 cells for 12 h, confocal imaging showed that NZ @TG There was obvious colocalization between the green fluorescence of 5-carboxyfluorescein and the red fluorescence of the mitochondrial tracer mitotracker, indicating that NZ @TG Under the action of glycyrrhizic acid, it can effectively target mitochondria in Hep1-6 cells ( Figure 5 ). Further, the CCK-8 assay (according to the reagent instructions) was used to evaluate the NZ under 808nm NIR irradiation. @TG Only about 40% of Hep1-6 cells survived; while NZ and NZ @T After treatment, approximately 60% and 50% of Hep1-6 cells survived, respectively. @TG The combination of mitochondrial targeting ligand and hypoxia activated prodrug TH302 enables it to interact with NZ and NZ @T group has superior therapeutic performance ( Figure 6 ). Flow cytometry was used to detect NZ and NZ @T and New Zealand @TG The results of cell apoptosis after different treatments (incubation with Hep1-6 cells at a ratio of 1 mg nanoparticles per 100,000 cells for 12 h) are shown in Figure 2. Figure 7-8 , NZ under 808nm NIR irradiation @TG caused about 25% of cells to apoptosis, compared with NZ and NZ @T The apoptotic rate of treated cells increased significantly ( Figure 7, 8). Flow cytometry was further used to detect the production of ROS in cells after different treatments. It was found that NZ and NZ @T The ROS produced by NZ was twice that of the blank control group NC, while @TG The ROS produced by the treatment was 2.5 times that of the blank control group ( Figure 9 The accumulation of ROS in tumor cells causes a large amount of oxidative stress damage. The expression levels of oxidative stress-related proteins in tumor cells were detected using corresponding protein detection reagents, such as Figure 10-12 GPX3, Nrf-2, and SOD were all upregulated. Figure 9 The results were consistent. Detection of tumor cell cycle, such as Figure 13-14 , in NZ containing the drug TH302 @T , NZ @TG The most serious cell tissues in the G2 / M stage were observed in both groups. DNA damage caused by the hypoxia prodrug TH302 inhibited the rapid proliferation of tumor cells, playing a key role in alleviating tumor progression and creating a therapeutic window. According to transmission electron microscopy images, NZ @TG Treatment resulted in severe mitochondrial damage, including mitochondrial swelling, disrupted cristae, decreased cristae density, and damaged mitochondrial-endoplasmic reticulum interface, accompanied by vesicle formation ( Figure 15 The structural damage leads to impaired mitochondrial function and a significant decrease in ATP production in tumor cells ( Figure 16 ). Local oxidative damage within tumor cells also triggers a positive feedback loop, promoting the generation of ROS and enhancing the oxidative stress response.

[0067] PDT-induced oxidative damage can promote ICD, trigger the release of DAMPs and activate anti-tumor immune responses. To verify this effect, key DAMPs, including calreticulin (CRT), high-mobility group box 1 (HMGB1) and heat shock proteins (HSP70 and HSP90), were evaluated in Hep1-6 cells after various treatments. @TG The group showed the strongest fluorescence signals of CRT and HMGB1 ( Figure 17 ). ELISA results showed that compared with other groups, NZ @TG The levels of HSP70 and HSP90 in the cell supernatant of the group were significantly increased ( Figure 18 These results indicate that NIR-responsive PDT targeting mitochondria can induce strong ICD.

[0068] Example 3: NZ @TG In vivo tumor therapeutic effects

[0069] H22 hepatoma cells were used to establish liver tumors in situ in mice. @TGThe in vivo tumor treatment study was conducted as follows: H22 cells were injected into the liver of mice orthotopically and then treated one week later. The liver cancer-bearing mice were randomly divided into four groups for in vivo treatment: 1. Intratumoral injection of PBS (control group); 2. Intratumoral injection of NZ (NZ group); 3. Intratumoral injection of NZ @T (NZ @T group); 4. Intratumoral injection of NZ @TG (NZ @TG The NZ dosage of the treatment system remained the same at 25 mg / ml. 808 nm NIR irradiation was performed 4 hours after intratumoral injection. The condition involving 808 nm NIR irradiation was 2.0 w cm -2 Irradiation for 8 minutes. The treatment cycle is 2 weeks, and regular fluorescence imaging of mouse tumors is performed to monitor tumor growth. TG Mice bearing orthotopic tumors treated with WT mice still showed excellent tumor suppression on day 7 after treatment, while the control group exhibited rapid tumor growth and metastasis throughout the abdominal cavity ( Figure 19 、 20 ). Groups 2 and 3 represent PDT and PDT combined with hypoxic prodrug treatment, respectively. Group 4 represents PDT targeting tumor cell mitochondria combined with hypoxic prodrug treatment. @TG The synergistic effects of mitochondrial targeting, PDT and hypooxidative prodrugs have shown excellent tumor treatment effects.

[0070] Immunofluorescence staining was further performed on the tumor tissue to observe the changes in ICD of tumor cells in vivo. @TG The HMGB1 and HSP90 fluorescence intensities of the group were the strongest, the proportion of positive tumor cells was the highest, and the effect of inducing ICD was the most obvious ( Figure 21-23 ). Fluorescence staining of tumor tissue revealed that NZ @TG CD8 / CD4 + The highest proportion of T cells indicated successful activation of systemic anti-tumor immune response ( Figure 24 ).

[0071] Example 4: NZ @TG In vivo tumor therapeutic effects in combination with immune checkpoint inhibitors

[0072] Combining PDT therapy targeting mitochondrial enhancement with immune checkpoint inhibitors may further enhance the anti-tumor immune effect. H22 liver cancer cells were used to establish liver cancer in situ in mice. Treatment was performed one week after in situ injection of H22 cells into the liver of mice. Liver cancer-bearing mice were randomly divided into four groups for in vivo treatment: 1. Intratumoral injection of PBS (control group); 2. Intravenous injection of PD-L1 antibody group (anti-PD-L1 group); 3. Intratumoral injection of NZ @TG (NZ@TG group); 4. Intratumoral injection of NZ @TG + Intravenous PD-L1 antibody group (NZ @TG +anti-PD-L1 group), the dose of intravenous PD-L1 was 2ml / kg, and the dose of NZ was injected into the tumor @TG的 The dose of NZ was consistent with that in Example 3, which was 25 mg / mL. Groups 3 and 4 were irradiated with 808 nm NIR. The treatment system was injected intratumorally on day 0, and 808 nm NIR irradiation was performed 4 hours after the intratumoral injection. The conditions for 808 nm NIR irradiation were 2.0 w cm -2 Irradiation for 8 minutes. Group 2 and Group 4 received intravenous injection of anti-PD-L1 antibody on day 1, day 4, and day 7. The treatment cycle was 2 weeks, and tumor fluorescence imaging of mice was performed regularly to monitor tumor growth. According to the results of bioluminescence imaging, NZ @TG The combined PD-L1 antibody treatment group effectively inhibited tumor progression ( Figure 25 、 26 According to the mouse survival curve analysis, the combined treatment also significantly improved the survival rate of tumor-bearing mice ( Figure 27 Histological analysis of mouse tumors showed extensive apoptosis in the combined treatment group ( Figure 28 ), CD4 and CD8 immunofluorescence staining showed that T cell infiltration was enhanced after combined treatment ( Figure 29 ), confirmed that NZ @TG Induced immune activation sensitizes the efficacy of immune checkpoint inhibition.

Claims

1. An upconversion nanoparticle loaded with a hypoxia prodrug, characterized in that: It is composed of upconversion nanoparticles, organic photosensitizers, amphiphilic surfactants, glycyrrhizic acid and hypoxic prodrugs. Among them, the organic photosensitizer mainly absorbs near-infrared light, and the upconversion nanoparticles assist the organic photosensitizer in absorbing near-infrared light. The amphiphilic surfactant assists the hydrophobic upconversion nanoparticles and the organic photosensitizer to form a hydrophilic complex, thereby enhancing its biocompatibility; glycyrrhizic acid is responsible for targeting mitochondria, and the hypoxic prodrug induces DNA damage under hypoxic conditions.

2. The upconversion nanoparticles loaded with hypoxia prodrug according to claim 1, characterized in that: The mass ratio of upconversion nanoparticles and organic photosensitizers was 100:1, and the effective loading rates of glycyrrhizic acid and hypoxia prodrug were over 17.7% and 42.2%, respectively.

3. The upconversion nanoparticles loaded with hypoxia prodrug according to claim 1, characterized in that: The organic photosensitizer is ZnPcC4, the upconversion nanoparticles are NaGdF4:Nd, the hypoxic prodrug is TH302, and the amphiphilic surfactant is DSPE-PEG5K.

4. A method for preparing upconversion nanoparticles loaded with hypoxia prodrug according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) A tetrahydrofuran solution of upconversion nanoparticles and a methanol solution of an organic photosensitizer are mixed, and the mixture is mixed overnight under N2 protection. The complex of the nanoparticles and the organic photosensitizer is collected and redispersed in chloroform to obtain a chloroform solution of the complex; wherein the volume ratio of the tetrahydrofuran solution of the upconversion nanoparticles to the methanol solution of the organic photosensitizer is 5:1; the volume of chloroform is twice the volume of methanol; the concentration of the organic photosensitizer in the methanol solution of the organic photosensitizer is 0.5 mg / mL, and the mass ratio of the upconversion nanoparticles to the organic photosensitizer is 100:1; (2) The chloroform solution of the complex was added dropwise to the chloroform solution containing the amphiphilic surfactant, and glycyrrhizic acid and TH302 were added at the same time. After the addition of glycyrrhizic acid and TH302, the concentration was at least 0.048 mg / L; then, the mixture was stirred for 1 hour under N2 protection and heated at 50°C until the solvent was completely evaporated; the precipitate was redispersed in phosphate buffer solution and purified by ultrafiltration to obtain upconversion nanoparticles loaded with hypoxic prodrug; wherein the volume ratio of the chloroform solution of the complex to the chloroform solution containing the amphiphilic surfactant was 1:9; and the concentration of the amphiphilic surfactant in the chloroform solution containing the amphiphilic surfactant was 8 mg / mL.

5. The preparation method according to claim 4, characterized in that In step (1), the upconversion nanoparticles are NaGdF4:Nd, which are prepared by the following method: 5mmoL Ln(CH3CO2)3 was mixed with 20mL oleic acid and 20mL 1-octadecene, heated to 150℃ under N2 atmosphere and kept warm for 2 hours to form a lanthanide oleate complex; then cooled to 110℃ and vacuumed for 30 minutes; then, 10mmoL NaHF2 powder was added under N2 atmosphere and vacuumed for 15 minutes; under N2 protection, the mixture was heated to 310℃ and kept warm for 40 minutes to obtain upconversion nanoparticles; wherein Ln=Gd, Nd, Gd:Nd=49:

1.

6. Use of the upconversion nanoparticles loaded with hypoxia prodrug according to any one of claims 1 to 3, characterized in that: include: Preparation of photodynamic therapy and chemotherapy synergistic treatment preparations for tumors and / or preparations for sensitizing tumor immunotherapy.

7. The use according to claim 6, characterized in that The photodynamic therapy and chemotherapy synergistic treatment preparations for tumors and / or the preparations for sensitizing tumor immunotherapy also include immune checkpoint inhibitors.

Images