Nanoreactor for enhancing immunotherapy as well as preparation method and application of nanoreactor

Abstract

The invention discloses a nanoreactor for enhancing immunotherapy as well as a preparation method and application of the nanoreactor. The nanoreactor LNP / GOx / TF / Mn has a core-shell structure, a liposome wrapping a PD-1 / PD-L1 small-molecule inhibitor is taken as a core, the surface of the liposome is modified with a metal-polyphenol network, glucose oxidase is embedded into the metal-polyphenol network, and a MnO2 shell layer is plated on the metal-polyphenol network. The nanoreactor can prolong the in-vivo circulation time of the liposome, the liposome is stabilized by a multi-layer structure, the outer-layer structure design is linked with one another, a cascade reaction is generated, and the purpose of chemodynamic therapy (CDT) / starvation therapy (ST) / immunotherapy (IT) synergistic treatment of tumors is achieved.

Description

Technical Field

[0001] This invention relates to the field of biomedical nanomaterials technology, and in particular to a nanoreactor for enhancing immunotherapy, its preparation method, and its application. Background Technology

[0002] Thyroid cancer, the most common malignant tumor of the endocrine system, originates from thyroid follicular epithelium or parafollicular cells and is mainly divided into four types: papillary carcinoma, follicular carcinoma, medullary carcinoma, and undifferentiated carcinoma. Among them, papillary carcinoma has the highest incidence and a relatively optimistic prognosis, while undifferentiated carcinoma is extremely malignant and progresses rapidly. Although surgical resection, radioactive iodine therapy, and endocrine therapy constitute the traditional treatment system, the limitations of these therapies are becoming increasingly apparent when facing advanced or refractory thyroid cancer, and patients urgently need more effective treatment options.

[0003] Liposomes, as closed vesicles composed of a phospholipid bilayer, possess a highly promising drug delivery system due to their structural similarity to biological membranes. They can efficiently encapsulate hydrophilic, hydrophobic, and amphiphilic drugs while exhibiting excellent biocompatibility, targeting, and sustained-release properties. Through surface modification and the attachment of targeting ligands, liposomes can actively recognize antigens or receptors on the surface of thyroid cancer cells; simultaneously, leveraging the high permeability and retention effect (EPR) unique to tumor tissue, they achieve passive targeted delivery. This dual-targeting mechanism allows drugs to precisely accumulate at the tumor site, enhancing therapeutic efficacy while significantly reducing toxic side effects on normal tissues. Based on these unique advantages, liposomes demonstrate broad application prospects in the treatment of thyroid cancer. Despite the numerous advantages liposomes have demonstrated in the treatment of thyroid cancer, their application still faces a series of challenges. The stability of liposomes is a primary concern. The phospholipid bilayer structure is susceptible to environmental factors; changes in temperature and pH can alter liposome membrane fluidity, leading to drug leakage, aggregation, or even rupture. This not only reduces drug delivery efficiency but may also release drugs into non-tumor sites, increasing toxic side effects. Simultaneously, liposomes are easily recognized and cleared by the reticuloendothelial system (RES) in vivo, shortening their circulation time and affecting the effective delivery of drugs to tumor tissues.

[0004] Traditional liposomes, used only as a single drug delivery device in thyroid cancer treatment, are ill-suited to address the complexities of the disease and suffer from short circulation and poor targeting. To overcome these limitations, transforming liposomes into multifunctional nanoreactors has become a cutting-edge research direction. Through structural and functional optimization, the goal is to extend circulation time and enable combined treatment with multiple approaches. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and provide a nanoreactor that enhances immunotherapy, which has multiple inhibitory effects on thyroid cancer cells, including the efficacy of chemodynamic therapy (CDT) / starvation therapy (ST) / immunotherapy (IT), high drug absorption rate, and few side effects.

[0006] A second objective of this invention is to provide a method for preparing the nanoreactor.

[0007] A third objective of this invention is to provide the application of the nanoreactor in the preparation of medicaments for treating tumors.

[0008] A fourth objective of this invention is to provide a tumor treatment drug.

[0009] The above-mentioned objective of this invention is achieved through the following technical solution: A nanoreactor LNP / GOx / TF / Mn has a core-shell structure, with a liposome encapsulating a PD-1 / PD-L1 small molecule inhibitor as its core. The surface of the liposome is modified with a metal-polyphenol network, in which glucose oxidase GOx is embedded and coated with a MnO2 shell.

[0010] Considering the strong adhesive properties of metal-polyphenols, which can construct networks with excellent bioadhesion and self-assemble onto the surface of nanocarriers to form stable shells, this invention modifies liposomes with metal-polyphenols. On the one hand, this enhances their stability, reduces the risk of clearance by the reticuloendothelial system, and significantly prolongs in vivo circulation time. On the other hand, the metal-polyphenol shell can serve as a multifunctional platform, integrating chemotherapy drugs, gene therapy carriers, and photothermal conversion materials, thus endowing liposomes with multiple therapeutic effects, including chemotherapy, gene therapy, and photothermal therapy. The liposome-based nanoreactors modified with metal-polyphenols combine stable in vivo circulation performance with multi-effect therapeutic advantages, achieving precise sustained drug release and integrating multiple treatment methods, providing a highly efficient and flexible new solution for the comprehensive treatment of thyroid cancer. The nanoreactor of this invention can prolong the circulation time of liposomes in vivo. The liposomes are stabilized by a multilayer structure, with the outer layer structure designed in an interlocking manner to generate a cascade reaction. It can synergistically treat tumors through chemodynamic therapy (CDT), starvation therapy (ST), and immunotherapy (IT). The principle is as follows: the MnO2 shell of the LNP / GOx / TF / Mn nanoreactor reacts with excess GSH in the tumor microenvironment to generate Mn. 2+ The MnO2 shell breaks down, releasing GOx. GOx catalyzes the reaction of glucose, oxygen, and water to produce H2O2. 2+The liposomes react with H2O2 in a Fenton-like reaction, producing ROS, which induces tumor cell death. This process is known as starvation therapy and chemodynamic therapy. After the liposome's stable layer is disrupted, PD-1 / PD-L1 small molecule inhibitors are gradually released. These inhibitors block the binding between PD-1 and PD-L1 in the tumor, achieving immune checkpoint blockade (ICB) and thus enabling T cell-mediated tumor cell death, thereby enhancing immunotherapy.

[0011] Furthermore, the MnO2 shell of the nanoreactor also has a coating agent layer, namely, the nanoreactor LNP / GOx / TF / Mn / coating agent.

[0012] Preferably, the coating agent includes, but is not limited to, bovine serum albumin (BSA), silk fibroin (SF), hyaluronic acid (HA), etc.

[0013] Furthermore, the coating layer is also modified with tumor-targeting molecules. Depending on the coating agent used, specific tumor-targeting molecules can be modified on the surface of the nanoreactor. These tumor-targeting molecules include, but are not limited to, RGD short peptides, T7 short peptides, and folic acid.

[0014] Furthermore, the particle size of the nanoreactor is 100~300nm.

[0015] Furthermore, the PD-1 / PD-L1 small molecule inhibitors described in this invention include, but are not limited to, BMS-1, BMS-202, and brominated benzyl ether derivatives.

[0016] The present invention also provides a method for preparing the above-mentioned nanoreactor LNP / GOx / TF / Mn, comprising the following steps: S1. Preparation of organic phase: PD-1 / PD-L1 inhibitory molecules, metal salts, phospholipids and cholesterol are added to an organic solvent and fully dissolved to obtain a homogeneous organic phase; S2. Preparation of aqueous phase: Polyphenols and glucose oxidase are dissolved in ultrapure water and stirred evenly to form an aqueous phase; S3. Mixing and self-assembly: The prepared organic phase is slowly added dropwise to the aqueous phase while being mixed. After the addition is complete, the organic solvent is removed. S4. Separation and purification: Centrifuge the reaction solution after the above treatment, collect the precipitate, wash it, and you will get LNP / GOx / TF; S5. Polyallylamine hydrochloride treatment: Add the polyallylamine hydrochloride solution to the prepared LNP / GOx / TF solution and mix at room temperature; S5. KMnO4 redox treatment: KMnO4 solution was added to the solution after polyallylamine hydrochloride treatment in step S5, and the mixture was stirred until the solution color turned dark brown. The solution was centrifuged, washed, and the centrifuged precipitate was collected to obtain the nanoreactor LNP / GOx / TF / Mn.

[0017] Furthermore, the method also includes dissolving the precipitate obtained in step S5 in water, adding a solution containing a coating agent, mixing at room temperature to allow the coating agent to fully coat the particles, and obtaining LNP / GOx / TF / Mn / coating agent.

[0018] Furthermore, it also includes activating the surface groups of LNP / GOx / TF / Mn / coating agents and then coupling them with tumor-targeting molecules.

[0019] Furthermore, the metal salt mentioned in step S1 includes, but is not limited to, iron salts, such as FeCl3.

[0020] Furthermore, the phospholipids mentioned in step S1 include, but are not limited to, one or more of DPPC, DSPE, and DPPG.

[0021] Furthermore, the organic solvent mentioned in step S1 includes, but is not limited to, tetrahydrofuran.

[0022] Furthermore, the polyphenols mentioned in step S2 include, but are not limited to, tannic acid (TA).

[0023] As a preferred embodiment, the present invention also provides a method for preparing the LNP / GOx / TF / Mn nanoreactor, comprising the following steps: Step 1, Preparation of LNP / GOx / TF Preparation of the organic phase: PD-1 / PD-L1 inhibitory molecules, FeCl3, DPPC, DSPE, DPPG and cholesterol were added to tetrahydrofuran and dissolved completely to obtain a homogeneous organic phase; Preparation of the aqueous phase: Tannic acid (TA) and GOx are dissolved in ultrapure water and stirred until homogeneous to form an aqueous phase; Mixing and self-assembly: The prepared organic phase was slowly added dropwise to the aqueous phase while stirring; after the addition was complete, stirring was continued to allow the tetrahydrofuran to fully volatilize. Separation and purification: The reaction solution after the above treatment is placed in a centrifuge, centrifuged, and the precipitate obtained after centrifugation is collected. The precipitate is washed with ultrapure water to obtain LNP / GOx / TF. Step 2, prepare LNP / GOx / TF / Mn Polyallylamine hydrochloride (PAH) treatment: Take PAH solution and add it to the LNP / GOx / TF solution prepared above. Stir at room temperature to allow PAH and LNP / GOx / TF to mix and adsorb fully. KMnO4 redox treatment: Add KMnO4 solution to the PAH-treated solution, continue stirring at room temperature for a certain time until the solution turns dark brown, centrifuge and wash with pure water, and collect the centrifuged precipitate; Coating agent treatment: The above precipitate is dissolved in water, and a solution containing a coating agent is added. Stirring is continued at room temperature for a certain period of time to allow the coating agent to fully coat the particles. Separation and washing: Centrifuge the solution after the above treatment and collect the precipitate; wash the precipitate multiple times with ultrapure water to obtain LNP / GOx / TF / Mn.

[0024] Preferably, in step 1, the mass ratio of PD-1 / PD-L1 inhibitory small molecules, FeCl3, DPPC, DSPE, DPPG and cholesterol is 0.5~5:0.5~5:1~10:1~10:1~10:0.1~1; the mass ratio of TA and GOx is 2~20:0.1~1.

[0025] Preferably, in step 2, the mass fraction of PAH solution is 5%, the mass fraction of KMnO4 solution is 2%, the mass fraction of the coating agent is 1% when bovine serum albumin (BSA), 2% when silk fibroin (SF), and 2% when hyaluronic acid (HA), and more preferably, the molecular weight (MW) of the coating agent is 6000DA when HA is used.

[0026] Preferably, in step 2, the mass ratio of LNP / GOx / TF, PAH, KMnO4 and coating agent is 0.5~5:5~50:2~20:50~500.

[0027] As a preferred embodiment, the present invention also provides a method for modifying the surface of an LNP / GOx / TF / Mn nanoreactor with a thyroid cancer-targeting molecule (RGD short peptide). Specifically, the carboxyl groups on the surface of the LNP / GOx / TF / Mn coating agent are activated into an active ester intermediate using EDC and NHS, and then coupled with the amino group of the RGD short peptide to obtain an LNP / GOx / TF / Mn nanoreactor modified with the RGD short peptide targeting thyroid cancer. Modification of other targeting molecules can be achieved using the same principle and method.

[0028] Preferably, the mass ratio of the LNP / GOx / TF / Mn nanoreactor, EDC, NHS and RGD short peptide is 1~10:0.2~2:0.2~2:0.5~5.

[0029] The present invention also provides the application of any of the above-described nanoreactors LNP / GOx / TF / Mn in the preparation of antitumor drugs.

[0030] Furthermore, the tumor is thyroid cancer.

[0031] The present invention also provides a tumor treatment drug containing any of the above-described nanoreactors LNP / GOx / TF / Mn.

[0032] Compared with the prior art, the present invention has the following beneficial effects: The nanoreactor prepared in this invention can carry PD-1 / PD-L1 small molecule inhibitors, improving the water solubility of these drugs, reducing toxicity and side effects, and enhancing the bioavailability of chemotherapy drugs. It can effectively block the binding between PD-1 and PD-L1 in tumors, achieving immune checkpoint blockade (ICB) effects. The nanoreactor prepared in this invention can responsively dissociate and release drugs in the tumor microenvironment. The slightly acidic environment of the tumor can cause protonation of some groups on the coating agent molecular chain, leading to changes in intramolecular and intermolecular hydrogen bonding, thereby causing the coating layer to dissociate. The exposed MnO2 layer reacts in the tumor's excessive GSH environment to generate Mn. 2+ This leads to the disintegration of the MnO2 layer, and the liposomes gradually release their encapsulations after exposure, thereby achieving precise drug delivery. The nanoreactor prepared by this invention can prolong the circulation time of liposomes in vivo. The liposomes are stabilized by a multi-layered structure, and the outer layer structure is designed with interlocking links to generate a cascade reaction, achieving the goal of synergistic treatment of tumors by chemodynamic therapy (CDT), starvation therapy (ST), and immunotherapy (IT). Attached Figure Description

[0033] Figure 1 This is a transmission electron microscope image of the nanoreactor.

[0034] Figure 2 The effect of nanoreactors on the activity of thyroid cancer cells (K1).

[0035] Figure 3 The effect of nanoreactors on reactive oxygen species in thyroid cancer cells (K1).

[0036] Figure 4 This is a statistical analysis of the distribution of nanoreactors in organs within a nude mouse model of thyroid cancer.

[0037] Figure 5 The effect of nanoreactors on the tumor size of a nude mouse model of thyroid cancer.

[0038] Figure 6 The effect of nanoreactors on the survival rate of nude mice with thyroid cancer.

[0039] Figure 7 Statistical analysis of the antitumor immune effect of nanoreactors on nude mice with thyroid cancer: mature dendritic cells. Detailed Implementation

[0040] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0041] Unless otherwise specified, all reagents, materials, and equipment used in the embodiments of the invention are commercially available; unless otherwise specified, all test methods are conventional test methods in the art. Example

[0042] This embodiment provides a nanoreactor for enhancing immunotherapy, and its preparation method includes the following steps: An organic phase was prepared by dissolving 5 mg BMS-1 ​​(PD-1 / PD-L1 inhibitory small molecule), 5 mg FeCl3, 10 mg DPPC, 10 mg DSPE, 10 mg DPPG, and 1 mg cholesterol in 10 mL of tetrahydrofuran. Simultaneously, an aqueous phase was prepared by dissolving 20 mg tannic acid (TA) and 1 mg GOx in 50 mL of ultrapure water. The organic phase was slowly added dropwise to the aqueous phase at 30 °C and stirred for 3 h to volatilize the tetrahydrofuran. The reaction solution was then centrifuged at 13000 rpm for 15 min, the precipitate was collected, washed three times with ultrapure water, and dispersed in 50 mL of ultrapure water to obtain BMS-1@LNP / GOx / TF (BLGTF). Further, 2.5 mL of 20 mg / mL PAH solution was added to the BLGTF solution, and the mixture was stirred at room temperature for 30 min. Then, 2 mL of 10 mg / mL KMnO4 solution was added, and stirring continued for 1 h until the solution turned dark brown. Finally, 25 mL of 20 mg / mL SF coating agent solution was added, and the mixture was stirred for 1 h. The mixture was then centrifuged again at 13000 rpm for 15 min, and the precipitate was collected. After washing three times with ultrapure water, the precipitate was dispersed in 50 mL of ultrapure water to obtain BMS-1@LNP / GOx / TF / Mn / SF (BLGTFMS). 10 mg of BLGTFMS was dissolved in pure water. The surface SF was activated with 2 mg EDC and 2 mg NHS to form an active ester intermediate, which was then coupled with 0.5 mg of RGD short peptide amino groups to obtain the RGD short peptide-modified BMS-1@LNP / GOx / TF / Mn / SF-RGD nanoreactor (BLGTFMSR) targeting thyroid cancer. Example

[0043] This embodiment provides a method for preparing a nanoreactor that enhances immunotherapy, which is basically the same as that in Example 1, except for the following raw material feeding amounts: 0.5mg BMS-1, 0.5mg FeCl3, 1mg DPPC, 1mg DSPE, 1mg DPPG, 0.1mg cholesterol, 2mg TA, 0.1mg GOx, 5mg PAH, 2mg KMnO4, 50mg SF, 0.2mg EDC, 0.2mg NHS, 0.1mg RGD short peptide.

[0044] Example 3: Method for detecting BLGTFMSR particle size and drug loading rate The specific test method for particle size analysis is as follows: The particle size distribution of the sample was measured using dynamic light scattering (DLS). In the experiment, a Malvern Zetasizer Nano-ZS 90 particle size analyzer was used to determine the particle size distribution of the BLGTFMSR aqueous dispersion (1 mg / mL), and the test temperature was 25℃. The results are shown in Table 1.

[0045] The specific test methods for drug loading and encapsulation efficiency are as follows: (1) Preparation of standard solution: Accurately weigh BMS-1 ​​standard, dissolve it in DMSO, and prepare a standard solution of 5~100 μg / ml; (2) The standard solution and BLGTFMSR dispersion (1 mg / mL) were detected by HPLC. The peak area of ​​BMS-1 ​​in the standard solution was used as the ordinate and the concentration (μg / mL) was used as the abscissa to perform linear regression and obtain the linear regression equation. The corresponding peak area of ​​the sample solution was substituted into the linear regression equation to calculate the BMS-1 ​​content of BLGTFMSR, and thus the drug content in the carrier was calculated.

[0046] The drug loading and encapsulation efficiency were calculated using the following formulas. Three parallel tests were conducted, and the average value was taken. The results of the drug loading and encapsulation efficiency are shown in Table 1 below.

[0047]

[0048] The results are shown in Table 1. The particle size of BLGTFMSR prepared in each example was between 200 and 250 nm, and the PDI was around 0.2, indicating good dispersibility. The drug loading rate was similar in all examples, indicating that different feed weights did not affect the drug loading at this feed ratio.

[0049] The BLGTFMSR nanoreactor prepared in Example 1 was used as the sample. The BLGTFMSR was dissolved in sterile PBS, and then the BLGTFMSR solution was diluted with complete culture medium to BMS-1 ​​final concentrations of 0.1, 1, 10, and 100 μg / mL. K1 cells were cultured at 8 × 10⁸ cells per well. 3The culture medium was seeded at a density of [number] cells / wells into 96-well plates. After incubation for 24 h, the original culture medium was discarded. 100 μL of the prepared BLGTFMSR solution was added to each well, with six parallel wells for each concentration. A blank control group was also included. After another 24 h of incubation, 1 mL of 10% CCK-8 solution was added to each well. The plates were incubated at 37°C with 5% CO2 for a period of time, and the OD value was measured at 450 nm using a multi-mode microplate reader.

[0050]

[0051] The results are as follows Figure 2 As shown, the higher the BMS-1 ​​concentration, the lower the K1 cell survival rate. Compared with the BMS-1 ​​group and the BMS-1@LNP group, BLGTFMSR showed significantly enhanced cytotoxicity. This is because BLGTFMSR triggers a Fenton-like reaction in the tumor microenvironment through starvation therapy and chemodynamic therapy. The strong oxidizing •OH induces tumor cell apoptosis by damaging DNA and inactivating proteins, and synergistically exerts in vitro antitumor activity with the ICB effect of BMS-1.

[0052] Using the BLGTFMSR prepared in Example 1 as a sample, K1 cells were cultured at 4 × 10⁻⁶. 5 Cells were seeded at a density of cells / well in 6-well plates and cultured under appropriate conditions (37 °C, 5% CO2) for 24 h. The original culture medium was removed, and 1 mL of medium containing BLGTFMSR was added, followed by incubation for 2 h, 4 h, and 8 h, respectively. The culture medium was then discarded, and the cells were cultured for 30 min with fresh DCFH-DA (10 μM) medium instead of the original medium. Cells were then digested and collected, and the fluorescence intensity of ROS was measured by flow cytometry; higher fluorescence intensity indicated higher ROS content.

[0053] The results are as follows Figure 3 As shown, the fluorescence intensity of intracellular ROS in the PBS, BMS-1, and BMS-1@LNP groups remained relatively constant over time, almost zero. Conversely, the fluorescence intensity of intracellular ROS in the BLGTFMSR group increased over time, indicating that BLGTFMSR can generate a large amount of ROS intracellularly. This is due to the uptake of BLGTFMSR by the cells, leading to increased intracellular GOX and Mn. 2+ As accumulation increases, H2O2 and Mn produced by GOx catalysis react with each other. 2+ A Fenton-like reaction occurs, resulting in a large amount of ROS.

[0054] The BLGTFMSR prepared in Example 1 was used as the sample.

[0055] Four-week-old BALB / c nude mice were selected, carefully fed, and observed for one week. After the nude mice stabilized, K1 cell suspension was added at 1×10⁻⁶.7 Cells were inoculated at a density of 1 cell / mouse on the right posterior dorsal side of nude mice until the tumor diameter was approximately 100 mm. 3 Drug administration began at the specified time. Tumor-bearing nude mice were intravenously injected with Cy5-LGTFMSR (Cy5 replacing BMS-1) and Cy5@LNP. The nude mice were anesthetized at 6 h, 12 h, 24 h, and 48 h after administration, and the distribution of nanoparticles in the nude mice was detected using a small animal in vivo imaging system.

[0056] Figure 4 The statistical graphs of fluorescence intensity at the heart, liver, spleen, lung, kidney, and tumor sites in nude mice show that the relative fluorescence intensity at the tumor sites in nude mice injected with Cy5-LGTFMSR was significantly higher than that at other sites, indicating that the nanoparticles of this invention have significant targeting ability to tumor sites. Furthermore, the Cy5@LNP group showed almost zero fluorescence after 48 hours, while the Cy5-LGTFMSR group exhibited a longer fluorescence retention time, with a small amount of fluorescence signal still detectable after 48 hours, indicating that the nanoreactor can effectively protect and prolong the retention time of the encapsulated drug in vivo.

[0057] The BLGTFMSR prepared in Example 1 was used as the sample.

[0058] Nude mice with tumor volumes meeting the requirements were randomly divided into four groups of five mice each: PBS, BMS-1, BMS-1@LNP, and BLGTFMSR. Each mouse was injected via tail vein with 100 μL of one of the following samples: PBS, BMS-1 ​​(10 mg / kg), BMS-1@LNP (10 mg / kg), or BLGTFMSR (10 mg / kg), every other day. The mice's health was monitored daily, and the length and diameter of the tumors were measured every other day to monitor tumor volume (tumor volume = length × width). 2 (× 0.5), and the 28-day survival rate of the nude mice was calculated.

[0059] The results are as follows Figure 5 , Figure 6 As shown, compared to the PBS group, the BMS-1 ​​group, BMS-1@LNP group, and BLGTFMSR group exhibited anti-tumor effects. Compared to the BMS-1 ​​group and BMS-1@LNP group, the BLGTFMSR group showed a stronger inhibitory effect on tumor growth. This is because the nanoreactor has a targeted effect; starvation and chemodynamic effects produce a large amount of ROS to kill tumor cells, while simultaneously inducing tumor cell immunogenic cell death (ICD), promoting CRT exposure, HMGB1 and ATP release. ICD and BMS-1-mediated PD-L1 immune checkpoint blockade exert an effective synergistic effect. Furthermore, BLGTFMSR significantly improved the survival rate of tumor-bearing nude mice.

[0060] Mature dendritic cells (DCs) are considered a marker of immune activation. CD11c was detected by flow cytometry in extracted cells from treated mouse tumor tissue. + CD86 + The proportion of dendritic cell subsets is used to assess the maturation status of dendritic cells (DCs). For example... Figure 7 As shown, the content of mature DCs in the tumor tissue of mice in the PBS group was low, while the degree of DC maturation in the BMS-1 ​​group, BMS-1@LNP group and BLGTFMSR group was significantly increased, especially the BLGTFMSR group had the highest number of mature DCs. This indicates that the BLGTFMSR group can effectively promote DC maturation and activate a strong anti-tumor immune response.

[0061] In summary, BLGTFMSR is a targeted nanoreactor for the synergistic treatment of thyroid cancer using chemodynamic therapy (CDT), starvation therapy (ST), and immunotherapy (IT).

[0062] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0063] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A nanoreactor LNP / GOx / TF / Mn, characterized in that, The nanoreactor LNP / GOx / TF / Mn has a core-shell structure, with liposomes encapsulating PD-1 / PD-L1 small molecule inhibitors as the core. The surface of the liposomes is modified with a metal-polyphenol network, and glucose oxidase is embedded in the metal-polyphenol network and coated with a MnO2 shell.

2. The nanoreactor LNP / GOx / TF / Mn according to claim 1, characterized in that, The nanoreactor also has a coating agent layer on its MnO2 shell.

3. The nanoreactor LNP / GOx / TF / Mn according to claim 1, characterized in that, The coating layer is also modified with tumor-targeting molecules.

4. The nanoreactor LNP / GOx / TF / Mn according to claim 1, characterized in that, The nanoreactor has a particle size of 100~300nm.

5. The method for preparing the LNP / GOx / TF / Mn nanoreactor according to claim 1, characterized in that, Includes the following steps: S1. Preparation of organic phase: PD-1 / PD-L1 inhibitory molecules, metal salts, phospholipids and cholesterol are added to an organic solvent and fully dissolved to obtain a homogeneous organic phase; S2. Preparation of aqueous phase: Polyphenols and glucose oxidase are dissolved in ultrapure water and stirred evenly to form an aqueous phase; S3. Mixing and self-assembly: The prepared organic phase is slowly added dropwise to the aqueous phase while being mixed. After the addition is complete, the organic solvent is removed. S4. Separation and purification: Centrifuge the reaction solution after the above treatment, collect the precipitate, wash it, and you will get LNP / GOx / TF; S5. Polyallylamine hydrochloride treatment: Add the polyallylamine hydrochloride solution to the prepared LNP / GOx / TF solution and mix at room temperature; S5. KMnO4 redox treatment: KMnO4 solution was added to the solution after polyallylamine hydrochloride treatment in step S5, and the mixture was stirred until the solution color turned dark brown. The solution was centrifuged, washed, and the centrifuged precipitate was collected to obtain the nanoreactor LNP / GOx / TF / Mn.

6. The preparation method according to claim 5, characterized in that, It also includes dissolving the precipitate obtained in step S5 in water, adding a solution containing a coating agent, mixing at room temperature to allow the coating agent to fully coat the particles, and obtaining LNP / GOx / TF / Mn / coating agent.

7. The preparation method according to claim 6, characterized in that, It also includes activating the surface groups of LNP / GOx / TF / Mn / coating agents and then coupling them with tumor-targeting molecules.

8. The use of the nanoreactor LNP / GOx / TF / Mn according to any one of claims 1 to 4 in the preparation of antitumor drugs.

9. The application according to claim 8, characterized in that, The tumor is thyroid cancer.

10. A tumor treatment drug, characterized in that, It contains the LNP / GOx / TF / Mn nanoreactor described in any one of claims 1 to 4.

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