Antigen-capturing and mannose receptor-targeting nanomedicine, preparation method, and application thereof

Abstract

This invention belongs to the field of biomedicine and specifically relates to an antigen-capturing and mannose receptor-targeting nanodrug, its preparation method, and its application. This nanodrug can capture tumor-associated antigens released after thermal ablation of cancer cells and, in combination with an FTO inhibitor, deliver them to tumor-infiltrating dendritic cells. After thermal ablation, the synergistic effect of the nanodrug and immune checkpoint blockade therapy inhibits distal tumor growth and improves long-term survival.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and more specifically relates to an antigen-capturing and mannose receptor-targeting nanomedicine and its preparation method and application. Background Art

[0002] Currently, thermal ablation therapy is widely used in the treatment of hepatocellular carcinoma (HCC) due to its minimally invasive nature. As an inducing factor of immunogenic cell death (ICD), thermal ablation therapy can cause the release of tumor-associated antigens (TAAs), thereby activating anti-tumor immune responses. However, in actual clinical treatment, the prognosis of most HCC patients after thermal ablation therapy is not very good, and most of them will die due to tumor metastasis and recurrence. Immune checkpoints, as protective molecules in the human immune system, can interrupt the immune response and keep the activation of the immune system within the normal range, and have a certain therapeutic effect on tumors (Wang Fei, Zhou Zubang. Research progress on ultrasound-guided thermal ablation combined with immune checkpoint inhibitors for the treatment of hepatocellular carcinoma [J]. Chinese Journal of Interventional Imaging and Therapy, 2019, 16(11):4.).

[0003] However, in actual clinical treatment, the simultaneous introduction of immune checkpoint blockade (ICB) therapy after thermal ablation can eliminate the T cell-mediated killing response to tumor cells. Because the immune response induced by thermal ablation is too weak, the combined use of thermal ablation and ICB therapy may result in ineffective tumor suppression, allowing distant tumors to continue to grow and reproduce. Therefore, research on how to effectively enhance the anti-tumor efficacy of the combined therapy is of great clinical significance. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of existing thermal ablation therapy combined with ICB therapy, which may result in ineffective tumor suppression and allow distal tumors to still grow and reproduce, and to provide an antigen-capturing and mannose receptor-targeted nanomedicine that can be combined with thermal ablation therapy.

[0005] The purpose of the present invention is to provide a method for preparing the antigen-capturing and mannose receptor-targeting nanomedicine.

[0006] Another object of the present invention is to provide applications of the antigen-capturing and mannose receptor-targeting nanomedicine.

[0007] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0008] The invention discloses an antigen-capturing and mannose receptor-targeting nanomedicine. The nanomedicine encapsulates an FTO inhibitor in mesoporous polydopamine (MPDA) nanoparticles, uses maleimide (mal) as an antigen-capturing agent, uses mannose as a ligand targeting dendritic cells, and is anchored on the MPDA surface via a polyethylene glycol (PEG) linker.

[0009] Tumor antigen-capturing nanoparticles can enhance the antigen uptake efficiency and anti-tumor response of dendritic cells (DCs). However, tumor-infiltrated dendritic cells (TIDCs) often exhibit an immunosuppressive phenotype, resulting in a failure of anti-tumor activity even when tumor antigens are engulfed. Therefore, it is crucial to study how to effectively activate TIDCs after tumor thermal ablation to enhance the anti-tumor immune response of ICB.

[0010] To address the above problems, the present invention provides an antigen-capturing and mannose receptor-targeted nanodrug, which, when injected into the tumor, can capture TAAs released by tumor thermal ablation through mal group reaction and be taken up and internalized by TIDCs through mannose (Man)-mediated targeting. TAAs and FTO inhibitors are delivered to TIDCs, promoting DCs maturation by increasing RNA m6A methylation levels, activating anti-tumor immunity after HCC thermal ablation, and improving ICB-mediated anti-tumor immune responses.

[0011] Furthermore, the FTO inhibitor is FB23-2 or entacapone.

[0012] Furthermore, the polyethylene glycol is amino polyethylene glycol; the amino polyethylene glycol can be connected to mannose or maleimide, and the amino group carried by the amino polyethylene glycol can be covalently connected to the catechol group of the MPDA@FTO inhibitor.

[0013] In addition, the present invention also claims a method for preparing the antigen-capturing and mannose receptor-targeting nanomedicine, which specifically comprises the following steps:

[0014] S1. Fully dissolve the template and dopamine hydrochloride, add an emulsifier and mix evenly (ultrasonicate in a water bath to form a uniform white emulsion), and add ammonia water dropwise while stirring to completely react to obtain mesoporous polydopamine MPDA;

[0015] S2. Prepare an aqueous solution of the MPDA obtained in step S1, add the FTO inhibitor to fully load it, and wash with water to remove the unloaded FTO inhibitor to obtain the MPDA@FTO inhibitor;

[0016] S3. The MPDA@FTO inhibitor obtained in step S2 is prepared into an aqueous solution, the pH is adjusted to 9-11, polyethylene glycol maleimide and polyethylene glycol mannose are added, the coupling reaction is completed, and the unloaded polyethylene glycol molecules are removed by washing.

[0017] Furthermore, in step S1, the template is PEO-PPO-PEO triblock copolymer F127.

[0018] Furthermore, in step S1, the emulsifier is 1,3,5-trimethylbenzene (TMB).

[0019] Furthermore, in step S1, the reaction temperature is 45-55° C. Preferably, the reaction time is 1-3 hours; more preferably, the reaction time is 2 hours.

[0020] Furthermore, in step S2, the loading temperature is room temperature and the loading time is 40 to 55 hours; more preferably, the loading time is 48 hours.

[0021] Furthermore, in step S2, the mass ratio of the MPDA to the FTO inhibitor is (0.1-3):1; preferably, the mass ratio of the MPDA to the FTO inhibitor is (1-3):1; more preferably, the mass ratio of the MPDA to the FTO inhibitor is 1:1.

[0022] In addition, the present invention also claims the use of the antigen-capturing and mannose receptor-targeting nanomedicine in the preparation of anticancer drugs.

[0023] Furthermore, the cancer includes hepatocellular carcinoma, cholangiocarcinoma, and colorectal cancer.

[0024] Furthermore, the anticancer drug is used in combination with thermal ablation therapy.

[0025] The present invention has the following beneficial effects:

[0026] The present invention provides an antigen-capturing and mannose receptor-targeting nanodrug, which can capture tumor-associated antigens released after thermal ablation of cancer cells and deliver them in combination with FTO inhibitors to tumor-infiltrating dendritic cells. After thermal ablation treatment, the synergistic effect of the nanodrug and immune checkpoint blockade therapy can inhibit the growth of distal tumors and improve long-term survival rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Transmission electron microscopy (TEM) image of the nanomedicine M / m-MP@F.

[0028] Figure 2The release amount of nanodrug M / m-MP@F in acidic environment: (A) UV absorption curve of nanodrug; (B) release efficiency curve of FB23-2.

[0029] Figure 3 Statistical graph of the detection results of HMGB1 and HSP60 contents.

[0030] Figure 4 Statistical graph of the relative mRNA levels of m6A in different treatment groups assessed using an m6A RNA methylation quantification kit (n=3; means±SD).

[0031] Figure 5 The figure is a statistical graph of the expression percentage of CD80+CD86+ in different treatment groups detected by flow cytometry (gate CD11c+).

[0032] Figure 6 (A) CLSM lymph node sections after intratumoral injection of M / m-MP@NR and m-MP@NR into the right tumor show the colocalization of CD11c and nanomedicine on DCs, and the fluorescence intensity distribution across the cells along the selected green and red dashed lines; Figure 6 (B) is a statistical graph of fluorescence intensity values ​​with green dashed line; Figure 6 (C) is a statistical graph of the fluorescence intensity values ​​indicated by the red dotted line.

[0033] Figure 7 (A) Schematic diagram of the progress of anti-distant tumor research after thermal ablation of the primary tumor; Figure 7 (B) Distal tumor volumes of mice receiving different treatments (PBS, M / M-mp, m-MP@F, M-MP@F, and M / m-MP@F); Figure 7 (C) is a statistical graph of the cumulative survival rate of mice. DETAILED DESCRIPTION

[0034] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0035] The abbreviations used are: M for mannose; Ag for antigen; m for maleimide group; MP for mesoporous polydopamine (MPDA); @ for encapsulation; and F for FB23-2.

[0036] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0037] Example 1: An antigen-capturing and mannose receptor-targeted nanomedicine M / m-MP@F

[0038] The preparation of the antigen-capturing and mannose receptor-targeting nanomedicine M / m-MP@F specifically includes the following steps:

[0039] S1. Preparation of MPDA: 0.1 g of block copolymer F127 and 0.15 g of dopamine hydrochloride were fully dissolved in a mixture of deionized water and anhydrous ethanol (1:1, 10 mL). 160 μL of TMB was added, and the mixture was sonicated in a water bath for 6 min to form a uniform white emulsion. 375 μL of ammonia was added dropwise while stirring, and the mixture was reacted at 50°C for 2 h. The mixture was centrifuged at 9000 rpm for 10 min, and the precipitate was washed three times with ethanol and then water to obtain MPDA.

[0040] S2. Preparation of MPDA@FB23-2 (MP@F): The MPDA obtained in step S1 was prepared into an aqueous solution. 0.2 mL of FB23-2 dissolved in DMSO (mass ratio, MPDA:FB23-2=1:1) was added to 1.8 mL of MPDA aqueous solution (100 μg / mL). The mixture was stirred at room temperature for 48 h (DMSO:H2O=1:9, 2 mL). The mixture was washed by centrifugation with deionized water to remove unloaded FB23-2 to obtain MPDA@FB23-2 (MP@F).

[0041] S3. Preparation of Man / mal-MPDA@FB23-2 (M / m-MP@F) (using aminopolyethylene glycol mannose and aminopolyethylene glycol maleimide (HN2-PEG2000-Man, NH2-PEG2000-mal) as modifiers, covalently linked to the catechol groups on the MP@F surface through their amino groups): 1 mL of the MP@F (1 mg / mL) solution obtained in step S2 was placed in a 5 mL centrifuge tube, and 1 mM NaOH solution was added dropwise to adjust the pH to 10; 5 mg of NH2-PEG2000-Man and 5 mg of NH2-PEG2000-mal were dissolved in 1 mL of deionized ice water and sonicated for 2 min, then added to the above-mentioned MP@F solution after pH adjustment, diluted to 5 mL with pure water, and stirred overnight. The resulting product was centrifuged and washed three times at 9000 rpm to remove excess unloaded PEG molecules to obtain Man / mal-MPDA@FB23-2 (M / m-MP@F) nanomedicine.

[0042] Characterization of nanomedicine M / m-MP@F:

[0043] The particle size and morphology of MP@F, nanomedicine M / m-MP@F and the interaction of nanomedicine M / m-MP@F with antigen protein (chicken ovalbumin, OVA) were observed by transmission electron microscopy (TEM). Figure 1 As shown in the figure, it can be seen that the antigen protein OVA successfully binds to the surface of M / -m-MP@F.

[0044] Dynamic light scattering (DLS) detection showed that the particle size of the nanodrug M / m-MP@F increased after binding with OVA, and the surface negative charge also increased.

[0045] The nanomedicine M / m-MP@F was analyzed by UV spectroscopy. The results are shown in Figure 2 A, As can be seen from the figure, FB23-2 and M / m-MP@F reached the maximum absorption peak near 300nm, indicating that FB23-2 was encapsulated on M / m-MP@F.

[0046] The UV absorption value of FB23-2 in each component was determined by UV spectrophotometer. Figure 2 B. As can be seen from the figure, the nanocarrier has a strong acid-responsive drug release ability. Under pH 5.0 conditions (in lysosomes), it can fully release the loaded FTO inhibitor FB23-2, achieving the effect of targeted tumor treatment.

[0047] The protein content of high-mobility group protein B1 (HMGB1) and heat shock protein 60 (HSP60) in tumor cell lysates after ablation was analyzed by ELISA: Hepa1-6 cells were heated using microwave antenna technology (MWA) of a thermal ablation instrument, with real-time temperature monitoring. The temperature was maintained at 60°C for 2 minutes. M / m-MP@F and M-MP@F were added and incubated for 10 minutes. After removing the cells, the nanomedicines were collected. HMGB1 and HSP60 captured on the nanomedicines were detected using ELISA kits. The results are shown in Figure 2. Figure 3 As shown in the figure, it can be seen that after ablation treatment, the cells release a large amount of HSP60 and HMGB1, and the nanodrug M / m-MP@F can capture high levels of HSP60 and HMGB1 due to the presence of maleimide groups.

[0048] Example 2 Nanodrug M / m-MP@F promotes DC maturation by increasing m6A modification levels

[0049] 1. Experimental methods:

[0050] Bone marrow-derived dendritic cells (BMDCs) were cultured at a rate of 1×10 5Cells were seeded at a density of 100 cells / mL on a 6-well plate and cultured for 24 h. The cells were divided into four groups and treated with PBS+M / Ag-m-MP (named M / Ag-m-MP after M / m-MP@F captured the antigen), METTL3 siRNA (small interfering RNA of METTL3) (METTL3 KD)+M / Ag-m-MP, FTO siRNA (small interfering RNA of FTO) (FTO KD)+M / Ag-m-MP, and M / Ag-m-MP@F (small interfering RNA concentration of 120 nM; FTO concentration of 5 μM). After incubation for 24 h, total RNA was isolated from BMDCs cells, and the mA (N6-methyladenine) level in BMDCs was determined using the EpiQuik mA RNA methylation quantification kit according to the manufacturer's instructions.

[0051] The FTO enzyme prevents m6A modification on RNA by demethylating m6A. FTO inhibitors can be used to inhibit FTO activity, enhancing m6A modification of RNA in DCs and inducing DC maturation. Tumor-associated antigens (TAAs) released by tumor ablation are also adsorbed onto the functionalized MP@F (M / Ag-m-MP@F), providing effective antigen delivery. The FTO inhibitor-loaded, antigen-captured nanocarriers, M / Ag-m-MP@F, were incubated with BMDCs, and total RNA was extracted to analyze m6A mRNA expression levels.

[0052] Experimental results: Figure 4 As shown, cells treated with METTL3 siRNA showed downregulated m6A modification levels, while cells treated with FTO siRNA or M / Ag-m-MP@F showed enhanced m6A modification levels.

[0053] 2. Experimental methods:

[0054] BMDCs were cultured at a rate of 1×10 5 The cells were seeded at a density of 100 cells / mL on a 6-well plate and cultured for 12 h. The cells were then divided into 6 groups and treated with PBS, M / m-MP (without FTO inhibitor drug encapsulation), M / Ag-m-MP, M / m-MP@F, M / Ag-m-MP@F and Ag-m-MP@F (Ag is antigen, m represents maleimide group, MP represents mesoporous polydopamine (MPDA), @ represents encapsulation, and F represents FB23-2), respectively. After incubation for 24 h, BMDCs were washed with PBS and incubated with fluorescently labeled flow cytometry antibodies CD11c, CD80 and CD86 at room temperature for 30 min. After washing with PBS, the expression of surface molecules was detected by flow cytometry.

[0055] Experimental results: Figure 5As shown in the results, the maturation level of BMDCs induced by M / Ag-m-MP (34.0%) was higher than that of the M / m-MP-treated group (13.5%) and the M / m-MP@F-treated group (17.3%). In addition, the maturation level of BMDCs treated with M / Ag-m-MP@F was the highest (68.6%), while that of BMDCs treated with Ag-m-MP@F was lower (39.3%), which may be due to insufficient endocytosis caused by non-targeted nanoparticles.

[0056] The above results indicate that the nanomedicine M / m-MP@F delivery vector prepared in this application captures TAAs and is internalized into DCs through mannose targeting, while the FTO inhibitor on the nanocarrier can promote antigen presentation and stimulate DCs maturation by upregulating the m6A modification level.

[0057] Example 3 Study on the migration of nanomedicine to lymph nodes in vivo

[0058] Experimental method: Nile red (NR)-labeled nanomedicines (M / m-MP@NR and m-MP@NR) were intratumorally injected into the subcutaneous tumor of the right leg of C57BL / 6j mice (Nile red concentration was 300 nM), and then the lymph node sections at different time points after injection were observed using laser scanning confocal microscopy (CLSM).

[0059] The results are as follows Figure 6 As shown in the figure, 6h and 12h after the injection of m-MP@NR (non-targeted drug loaded with Nile red), the green fluorescence-labeled DCs in the inguinal lymph showed less red fluorescence (red arrow), indicating that few m-MP@NR carriers were transferred to the lymph nodes through the lymphatic system; on the contrary, in the animals injected with the M / m-MP@NR (targeted drug loaded with Nile red) group (yellow arrow), more red fluorescence was observed overlapping with green fluorescence (yellow arrow), indicating that the mannose-targeted nanomedicine entered the lymph nodes through DCs.

[0060] Example 4 Establishment of Subcutaneous Distal Tumor Model and Treatment Analysis

[0061] Experimental methods: Six days after the establishment of the bilateral subcutaneous tumor model, Heap1-6 tumor-bearing mice were intratumorally injected with nanomedicines (PBS, M / m-MP, m-MP@F, M-MP@F, and M / m-MP@F) into the right tumor for thermal ablation treatment. After treatment in different groups, all mice were given 5 mg / kg of aPD-L1 antibody every 3 days, and the distal tumor size was monitored every 3 days during treatment.

[0062] Experimental results: In the subcutaneous distal tumor model ( Figure 7(A) After thermal ablation of the primary tumor, the recurrence rates of the m-MP@F and M-MP@F groups were lower than those of the PBS or M / m-MP groups, and no tumor recurrence was observed in the M / m-MP@F group. Compared with PBS, the M / m-MP group only slightly inhibited the growth of subcutaneous distal tumors (secondary tumors). In addition, the m-MP@F, M-MP@F, and M / m-MP@F groups exhibited more pronounced inhibitory effects on the growth of subcutaneous distal tumors, with M / m-MP@F showing the most significant inhibitory effect. ( Figure 7 (B)), moreover, the M / m-MP@F group also showed better effects in prolonging animal survival ( Figure 7 (C)).

[0063] The above embodiments are preferred implementations of the present invention, but the implementations of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. An antigen-capturing and mannose receptor-targeting nanomedicine, characterized in that: The nanomedicine encapsulates the FTO inhibitor in mesoporous polydopamine (MPDA) nanoparticles, uses maleimide as an antigen capture agent, uses mannose as a ligand targeting dendritic cells, and is anchored on the MPDA surface via a polyethylene glycol (PEG) linker; the FTO inhibitor is FB23-2 or entacapone; Specifically, the nanomedicine is prepared by a method comprising the following steps: S1. Fully dissolve the template and dopamine hydrochloride, add an emulsifier and mix evenly, and dropwise add ammonia water while stirring to complete the reaction to obtain mesoporous polydopamine MPDA; S2. Prepare an aqueous solution of the MPDA obtained in step S1, add a FTO inhibitor to fully load it, and wash it to obtain an MPDA@FTO inhibitor; the mass ratio of the MPDA to the FTO inhibitor is (0.1-3):1; S3. Prepare the MPDA@FTO inhibitor obtained in step S2 into an aqueous solution, adjust the pH to 9-11, add polyethylene glycol maleimide and polyethylene glycol mannose, complete the coupling reaction, and wash to obtain the product.

2. The nanomedicine according to claim 1, characterized in that In step S1, the template is PEO-PPO-PEO triblock copolymer F127.

3. The nanomedicine according to claim 1, characterized in that In step S1, the emulsifier is 1,3,5-trimethylbenzene.

4. The nanomedicine according to claim 1, characterized in that In step S1, the reaction temperature is 45-55°C.

5. Use of the antigen-capturing and mannose receptor-targeting nanomedicine according to any one of claims 1 to 4 in the preparation of an anti-hepatocellular carcinoma drug.

6. The application according to claim 5, characterized in that The anti-hepatocellular carcinoma drug is used in combination with thermal ablation therapy.

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