Responsive antigen-capturing nano-platform, and preparation method and application thereof

Abstract

The present application relates to the technical field of tumor immunotherapy. The present application provides a responsive antigen capture nano platform, a preparation method and application thereof. The product of the present application is applied by systemic administration such as intravenous injection; can specifically respond to peroxynitrite in the tumor microenvironment, realize the precise activation of the tumor site; can covalently capture tumor-related antigens through high efficiency, deliver the antigens to antigen presenting cells, so as to enhance the anti-tumor immune response; combined with photodynamic therapy can significantly inhibit tumor growth.

Description

Technical Field

[0001] This invention relates to the field of tumor immunotherapy technology, and in particular to a responsive antigen capture nanoplatform, its preparation method, and its application. Background Technology

[0002] Cancer immunotherapy is an important approach to cancer treatment, and antigen-capturing nanomaterials have shown great potential in this field. However, existing antigen-capturing strategies suffer from limitations such as reliance on local tumor administration and limited antigen-binding affinity, which significantly restrict their clinical application.

[0003] Traditional antigen-capturing nanomaterials typically bind tumor-associated antigens (TAAs) through non-covalent interactions (such as hydrophobic interactions and electrostatic interactions), which suffers from drawbacks such as poor binding stability and susceptibility to interference from other proteins in the tumor microenvironment. Furthermore, these materials usually require local injection into the tumor, resulting in poor therapeutic efficacy for heterogeneously distributed or difficult-to-locate tumors, and are prone to off-target effects.

[0004] Although some studies have attempted to use maleimide-modified platforms to covalently bind TAAs, these platforms lack tumor microenvironment-specific activation mechanisms, still require local administration, and may non-specifically bind to albumin in the blood, affecting treatment efficacy and safety.

[0005] Therefore, developing an activated covalent antigen capture nanoplatform that can be administered systemically, has high spatiotemporal selectivity, and strong antigen binding ability is key to solving the above problems. Summary of the Invention

[0006] The purpose of this invention is to provide a responsive antigen capture nanoplatform, its preparation method, and its application, so as to overcome the defects of existing antigen capture nanomaterials, such as reliance on local drug delivery, low antigen binding affinity, and significant off-target effects.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a method for preparing a responsive antigen-capturing nanoplatform, comprising the following steps:

[0009] (1) DSPE-PEG 2000 -FPB and DSPE-PEG 2000 The mixtures were then dissolved together in tetrahydrofuran to obtain a mixed solution.

[0010] (2) The mixture obtained above was added dropwise to water, and ultrasonic treatment was performed during the dropwise process. Then, nitrogen was used to purge the solvent and the mixture was filtered to obtain a responsive antigen capture nanoplatform.

[0011] Step (1) of the DSPE-PEG 2000 -FPB and the amphiphilic polymer DSPE-PEG 2000 The mass ratio of the mixture is (0.5~1.5):(0.5~1.5);

[0012] The volume ratio of the mixture to water in step (2) is 1:(8-10).

[0013] Preferably, the DSPE-PEG described in step (1) 2000 -FPB, DSPE-PEG 2000 The ratio of the nitrate to tetrahydrofuran is (2-3) mg: (2-3) mg: 1 ml.

[0014] Preferably, the rate at which the mixture is added in step (2) is 0.5 to 2 drops / s; the power of the ultrasonic treatment is 100 to 200W; and after the addition is completed, the ultrasonic treatment continues for 2 to 4 minutes.

[0015] Preferably, the filtration includes sequential membrane filtration and ultrafiltration, wherein the pore size of the membrane filtration is 0.22 to 0.45 μm, and the molecular weight cutoff of the ultrafiltration is 100 kDa.

[0016] This invention also provides DSPE-PEG 2000 As a preferred method for synthesizing FPB, the DSPE-PEG... 2000 - The synthetic route of FPB is as follows Figure 1 As shown, the synthesis method is as follows:

[0017] The DSPE-PEG 2000 The preparation method of -FPB includes the following steps:

[0018] S1) Diethylaminosulfur trifluoride was mixed with a dichloromethane solution of methyl 4-bromo-3-(hydroxymethyl)benzoate and reacted at 0°C for 20–40 min. The mixture was stirred at room temperature for 10–14 h. After washing, concentration and purification by silica gel column chromatography, methyl 4-bromo-3-(fluoroethyl)benzoate was obtained.

[0019] S2) Methyl 4-bromo-3-(fluoroethyl)benzoate, bis(pinacolyl)diboron, [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride and potassium acetate were dissolved in N,N-dimethylformamide and reacted at 98-102℃ for 7-9 h under argon protection. After washing, concentration and purification by silica gel column chromatography, FPB-COOCH3 was obtained.

[0020] S3) Dissolve FPB-COOCH3 in a tetrahydrofuran / water mixture, mix with an aqueous lithium hydroxide solution, react for 10-16 h, and then neutralize, extract, dry, and perform silica gel column chromatography to obtain FPB-COOH;

[0021] S4) FPB-COOH, dicyclohexylcarbodiimide, and N-hydroxysuccinimide were dissolved in dichloromethane and reacted under a nitrogen atmosphere for 50–70 min. Then, DSPE-PEG was added. 2000 The dichloromethane solution containing -NH2 was reacted for 40–60 hours, filtered, dialyzed, and lyophilized to obtain DSPE-PEG. 2000 -FPB.

[0022] Preferably, the concentration of the lithium hydroxide aqueous solution is 0.8–1.2 M.

[0023] This invention provides a responsive antigen-capturing nanoplatform prepared by the aforementioned method.

[0024] This invention provides the application of the aforementioned responsive antigen-capturing nanoplatform in the preparation of antitumor drugs.

[0025] Preferably, the tumor is a solid tumor.

[0026] The present invention provides a responsive antigen capture nano-drug delivery system, wherein the responsive antigen capture nanoplatform is loaded with a drug and / or a photosensitizer.

[0027] The responsive antigen-capturing nanoplatform of the present invention can load anti-tumor drugs, including but not limited to chemotherapeutic drugs and photosensitizers.

[0028] The beneficial effects of this invention are as follows:

[0029] 1. It can be administered systemically without local tumor injection, and is suitable for tumors that are difficult to locate or have a heterogeneous distribution. It is administered via intravenous injection, and the tumor site is then treated with light after administration.

[0030] 2. It specifically responds to ONOO- in the tumor microenvironment, precisely activates and covalently binds to TAAs at the tumor site, and reduces off-target effects;

[0031] 3. By combining photodynamic therapy with released TAAs, and simultaneously capturing the released TAAs in situ on the tumor, the inhibitory effect of photodynamic therapy on bladder tumors can be enhanced. Attached Figure Description

[0032] Figure 1 DSPE-PEG in Example 1 2000 - Synthetic route of FPB;

[0033] Figure 2 The 1H NMR spectrum of methyl 4-bromo-3-(fluoroethyl)benzoate in Example 1;

[0034] Figure 3The 1H NMR spectrum of compound FPB-COOCH3 in Example 1;

[0035] Figure 4 The 1H NMR spectrum of compound FPB-COOH in Example 1;

[0036] Figure 5 DSPE-PEG in Example 1 2000 -FPB's proton NMR spectrum;

[0037] Figure 6 The 1H NMR spectrum of TD in Example 2;

[0038] Figure 7 The 1H NMR spectrum of TD-TK in Example 2;

[0039] Figure 8 The 1H NMR spectrum of TDR848 in Example 2;

[0040] Figure 9 Representative dynamic light scattering results for a responsive antigen-capturing nanoplatform;

[0041] Figure 10 Quantitative analysis of particle size and captured proteins of different nanoparticles before and after the addition of TAAs;

[0042] Figure 11 The protein capture rate of different nanoparticles in TAA solutions with different protein concentrations;

[0043] Figure 12 To analyze the expression of CD80, CD86 and MHC-II in BMDCs in different groups and to quantify the positive expression levels;

[0044] Figure 13 The curves show the changes in tumor volume over time in different groups of tumor-bearing mice. Detailed Implementation

[0045] This invention provides a method for preparing a responsive antigen capture nanoplatform, comprising the following steps: (1) preparing DSPE-PEG 2000 -FPB and DSPE-PEG 2000 (1) Mix and dissolve together in tetrahydrofuran to obtain a mixture; (2) Add the above-obtained mixture dropwise to water, accompanied by ultrasonic treatment during the dropwise addition, and then perform nitrogen purging to remove the solvent and filtration to obtain a responsive antigen capture nanoplatform; the DSPE-PEG obtained in step (1) 2000 -FPB and the amphiphilic polymer DSPE-PEG 2000The mass ratio of the mixture is (0.5-1.5):(0.5-1.5); the volume ratio of the mixture to water in step (2) is 1:(8-10).

[0046] In this invention, DSPE-PEG is used. 2000 -FPB and DSPE-PEG 2000 The mixtures are combined and dissolved together in tetrahydrofuran to obtain a mixed solution. In this invention, the DSPE-PEG... 2000 -FPB is a self-synthesized amphiphilic polymer, and the DSPE-PEG is... 2000 This is a commercially available amphiphilic polymer. In this invention, the DSPE-PEG is... 2000 -FPB and the amphiphilic polymer DSPE-PEG 2000 The preferred mass ratio of the mixture is (0.5–1.5):(0.5–1.5), more preferably (0.8–1.2):(0.8–1.2), and even more preferably 1:1. In this invention, the DSPE-PEG... 2000 -FPB, DSPE-PEG 2000 The preferred ratio of the product to tetrahydrofuran is (2-3) mg:(2-3) mg:1 ml, more preferably (2.3-2.7) mg:(2.3-2.7) mg:1 ml, and even more preferably 2.5 mg:1 ml.

[0047] In this invention, the above-obtained mixture is added dropwise to water, accompanied by ultrasonic treatment during the dropwise addition. Then, nitrogen purging is performed to remove the solvent, followed by filtration to obtain a responsive antigen-capturing nanoplatform. In this invention, the volume ratio of the mixture to water is preferably 1:(8-10), more preferably 1:(8.5-9.5), and even more preferably 1:9. In this invention, the water is preferably ultrapure water; the dropwise addition rate of the mixture is preferably 0.5-2 drops / s, more preferably 1 drop / s; the dropwise addition process is accompanied by ultrasonic treatment, the ultrasonic power being 100-200W, preferably 120-180W, more preferably 150W. After the dropwise addition is completed, ultrasonic treatment continues for 2-4 minutes, preferably 2.5-3.5 minutes, more preferably 3 minutes. The ultrasonic treatment is preferably performed using an ultrasonic cell disruptor, with the ultrasonic probe preferably immersed in ultrapure water at a distance of 0.8-1.2 cm from the bottom of the bottle. After the ultrasonic treatment, the present invention performs nitrogen purging to remove the solvent (residual tetrahydrofuran); the nitrogen purging time is preferably 3-5 h, more preferably 3.5-4.5 h, and even more preferably 4 h.

[0048] After the nitrogen purging is completed, the present invention performs filtration; the filtration includes sequential membrane filtration and ultrafiltration, wherein the pore size of the membrane filtration is preferably 0.22-0.45 μm, more preferably 0.45 μm; the molecular weight cutoff of the ultrafiltration is preferably 100 kDa, and the ultrafiltration is preferably performed using an ultrafiltration tube, specifically by transferring the liquid to the ultrafiltration tube for centrifugation, wherein the centrifugal force is preferably 3000-5000 g, more preferably 3500-4500 g, and even more preferably 4000 g, and the centrifugation time is preferably 5-8 min; if the volume after centrifugation is less than the original volume of the mixture, ultrapure water is used to make up to the original volume of the mixture, thereby obtaining a responsive antigen capture nanoplatform.

[0049] This invention also provides DSPE-PEG 2000 As a preferred method for synthesizing FPB, the DSPE-PEG... 2000 - The synthetic route of FPB is as follows Figure 1 As shown, the synthesis method is as follows:

[0050] The DSPE-PEG 2000 The preparation method of -FPB includes the following steps:

[0051] S1) Diethylaminosulfur trifluoride is mixed with a dichloromethane solution of methyl 4-bromo-3-(hydroxymethyl)benzoate and reacted at 0°C for 20–40 min, preferably under argon protection; then stirred at room temperature for 10–14 h, preferably 11–13 h, more preferably 12 h; then washed, concentrated, and purified by silica gel column chromatography to obtain methyl 4-bromo-3-(fluoroethyl)benzoate.

[0052] S2) Methyl 4-bromo-3-(fluoroethyl)benzoate, bis(pinacolyl)diboron, [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride and potassium acetate were dissolved in N,N-dimethylformamide and reacted at 98-102°C for 7-9 h under argon protection, preferably at 100°C for 8 h; then washed, concentrated and purified by silica gel column chromatography to obtain FPB-COOCH3;

[0053] S3) FPB-COOCH3 is dissolved in a tetrahydrofuran / water mixture and mixed with an aqueous lithium hydroxide solution. The mixture is reacted for 10–16 h. The concentration of the aqueous lithium hydroxide solution is preferably 0.8–1.2 M, more preferably 0.9–1.1 M, and even more preferably 1.0 M. Then, after neutralization, extraction, drying, and silica gel column chromatography, FPB-COOH is obtained.

[0054] S4) FPB-COOH, dicyclohexylcarbodiimide, and N-hydroxysuccinimide are dissolved in dichloromethane and reacted under a nitrogen atmosphere for 50–70 min, preferably 55–65 min, more preferably 60 min, and then DSPE-PEG is added. 2000 The dichloromethane solution containing -NH2 is reacted for 40–60 h, preferably 45–50 h, and more preferably 48 h; then filtered, dialyzed, and lyophilized to obtain DSPE-PEG. 2000 -FPB.

[0055] The present invention also provides a responsive antigen-capturing nanoplatform prepared by the aforementioned preparation method.

[0056] This invention also provides the application of the aforementioned responsive antigen-capturing nanoplatform in the preparation of antitumor drugs. In this invention, the type of tumor is preferably a solid tumor.

[0057] This invention also provides a responsive antigen capture nano-drug delivery system, wherein a drug and / or photosensitizer are loaded onto the responsive antigen capture nanoplatform. In this invention, the drug includes a chemotherapeutic drug; in this invention, the mass ratio of the responsive antigen capture nanoplatform, the drug, and the photosensitizer is preferably (0.5–1.5):(0.5–1.5):(0.5–1.5), more preferably (0.8–1.2):(0.8–1.2):(0.8–1.2), and even more preferably 1:1:1. In this invention, the preparation method of the responsive antigen capture nano-drug delivery system is described above for the responsive antigen capture nanoplatform, the difference being that in step 1), the drug and / or photosensitizer, DSPE-PEG, are loaded... 2000 -FPB, DSPE-PEG 2000 The mixture is then dissolved in tetrahydrofuran to obtain a solution. During nanoparticle formation, the hydrophobic distearate phosphatidylethanolamine (DSPE) domains intertwine with the drug or photosensitizer to form a core, while the hydrophilic polyethylene glycol (PEG) chains form a protective shell and extend into the aqueous phase, thereby stabilizing the nanoparticles and preventing further aggregation.

[0058] The specific embodiments of the present invention will be further described below with reference to the examples. The following examples are only used to illustrate the technical embodiments of the present invention more clearly, and should not be used to limit the scope of protection of the present invention.

[0059] Example 1: DSPE-PEG 2000 -FPB Synthesis

[0060] Synthetic routes such as Figure 1 As shown, the details are as follows:

[0061] 1) Under argon protection, diethylaminosulfur trifluoride (800 μL, 6.15 mmol) was added to dichloromethane containing methyl 4-bromo-3-(hydroxymethyl)benzoate (500 mg, 2.05 mmol) as the starting material. The reaction was carried out at 0 °C for 0.5 h. The reaction mixture was stirred at room temperature for 12 h, then washed with deionized water and saturated brine, and the organic phase was collected, dried, and concentrated. The crude product was purified by silica gel column chromatography to give methyl 4-bromo-3-(fluoroethyl)benzoate (346 mg, yield 68.54%). The 1H NMR spectrum is shown below. Figure 2 As shown.

[0062] 2) Methyl 4-bromo-3-(fluoroethyl)benzoate (330 mg, 1.34 mmol), bis(pinacolyl)diboron (511 mg, 2.01 mmol), [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (95 mg, 0.13 mmol), and potassium acetate (394 mg, 4.02 mmol) were dissolved in N,N-dimethylformamide (10 mL) and reacted at 100 °C for 8 h under argon protection. After the reaction was completed, the mixture was cooled to room temperature, diluted with diethyl ether, washed with deionized water and saturated sodium chloride, and separated. The organic phase was collected, dried, and concentrated. The crude product was purified by silica gel column chromatography to give compound FPB-COOCH3 (209 mg, yield 53.02%). The 1H NMR spectrum is shown below. Figure 3 As shown.

[0063] 3) A 1M aqueous solution of lithium hydroxide (1.5 mL, 1.50 mmol) was added to a solution of compound FPB-COOCH3 (151 mg, 0.51 mmol) dissolved in tetrahydrofuran / water (0.6 mL / 0.2 mL). The reaction mixture was stirred overnight at room temperature, then diluted with water, neutralized with 1M hydrochloric acid in an ice bath, extracted with ethyl acetate (10 mL), and the organic phase was collected and dried over anhydrous sodium sulfate. The crude product was subjected to silica gel column chromatography to give compound FPB-COOH (34.22 mg, yield 24%), and its 1H NMR spectrum is shown below. Figure 4 As shown.

[0064] 4) FPB-COOH (31 mg, 0.11 mmol), dicyclohexylcarbodiimide (45 mg, 0.22 mmol), and N-hydroxysuccinimide (25 mg, 0.22 mmol) were dissolved in dichloromethane (10 mL) and placed in a flask. The mixture was stirred for 1 h under a nitrogen atmosphere to prepare activated FPB-COOH. Next, 150 mg of DSPE–PEG2000–NH2 (44 mg, 0.44 mmol) was dissolved in dichloromethane (10 mL) and added to the flask. Finally, the reaction mixture was stirred for another 48 h under a nitrogen atmosphere. After the reaction was complete, the white precipitate was removed by filtration. The obtained DSPE-PEG... 2000 - The FPB solution was added to deionized water and dialyzed for 48 hours to completely remove excess FPB-COOH and other byproducts. The liquid in the dialysis bag was then freeze-dried to obtain pure DSPE-PEG. 2000 -FPB.

[0065] Results: For DSPE-PEG 2000 -FPB was used to characterize its structure using 1H NMR spectroscopy, which confirmed the correctness of the structure. Figure 5 (As shown).

[0066] Example 2

[0067] Preparation and size and morphology characterization of responsive antigen-capturing nanoplatforms

[0068] The synthesis steps of the aggregation-induced emission photosensitizer TDR848 are as follows:

[0069] The raw material TPE-DPA-CHO was synthesized with reference to the literature: Gao Z, Jia S, Ou H, et al. An ActivableNear-Infrared Afterglow Theranostic Prodrug with Self-SustainableMagnification Effect of Immunogenic Cell Death[J]. Angewandte ChemieInternational Edition, 2022, 61(40):e202209793.

[0070] 1) TPE-DPA-CHO (1.10 mmol, 580 mg) and rotannin-3-acetic acid (1.32 mmol, 252 mg) were dissolved in glacial acetic acid, and ammonium acetate (3.30 mmol, 254 mg) was added. The mixture was heated under reflux with stirring for 24 h. After cooling to room temperature, the product precipitated. The solid was filtered and washed with ethyl acetate / petroleum ether (v / v = 1:3), anhydrous ethanol, and deionized water, respectively. Finally, it was dried in a vacuum drying oven to obtain pure TD (621 mg, 80.64%). The 1H NMR spectrum is shown below. Figure 6 As shown.

[0071] 2) Compound TD (420 mg, 0.60 mmol), 2,2'-[propane-2,2-diylbis(thionidyl)]diethanol (353 mg, 1.80 mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (230 mg, 1.20 mmol), and 4-dimethylaminopyridine (7 mg, 0.06 mmol) were dissolved in anhydrous dichloromethane. The reaction mixture was stirred at room temperature under a nitrogen atmosphere for 24 h. After the reaction was complete, the reaction mixture was diluted with dichloromethane and washed with saturated sodium chloride solution. The aqueous phase was then repeatedly extracted with dichloromethane, and the organic phase was separated and collected using a separatory funnel. The organic phase was dried over anhydrous sodium sulfate, concentrated on a rotary evaporator, and then purified by silica gel column chromatography to give pure TD-TK (346 mg, 65.7%). The 1H NMR spectrum is shown below. Figure 7 As shown.

[0072] 3) TD-TK (263 mg, 0.30 mmol), diisopropylethylamine (55 mg, 1.20 mmol), and 4-dimethylaminopyridine (3 mg, 0.03 mmol) were dissolved in ultra-dry dichloromethane. Under nitrogen atmosphere and stirring at 0 °C, a solution of 4-nitrobenzenechloroformate (120 mg, 0.60 mmol) in ultra-dry dichloromethane was slowly added dropwise, and stirring continued for 4 h at room temperature. After the reaction was complete, the mixture was concentrated under vacuum and then redissolved with N,N-dimethylformamide. R848 (377 mg, 1.20 mmol) and ultra-dry triethylamine (121 mg, 1.20 mmol) were then added to the reaction mixture, and stirring continued for 24 h. The reaction mixture was concentrated under vacuum. The organic phase was extracted and separated with dichloromethane and saturated brine, dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain the crude product. Finally, the crude product was purified by silica gel column chromatography to obtain pure TDR848 (238 mg, 65.2%). The 1H NMR spectrum is shown below. Figure 8 As shown.

[0073] Preparation of nanoparticles: First, various compounds were weighed into centrifuge tubes according to different formulations:

[0074] The formulation of PEG nanoparticles is: 5mg DSPE-PEG 2000 ;

[0075] The formulation of Mal nanoparticles is: 2.5 mg DSPE-PEG 2000 and 2.5mg DSPE-PEG 2000 -Mal;

[0076] The formulation of FPB nanoparticles (i.e., responsive antigen capture nanoplatforms) is: 2.5 mg DSPE-PEG. 2000 and 2.5mg DSPE-PEG 2000 -FPB;

[0077] The formulation of photosensitizer@FPB nanoparticles (i.e., responsive antigen capture nanoparticle drug delivery system) is: 2.5 mg DSPE-PEG. 2000 2.5mg DSPE-PEG 2000 -FPB and 1 mg of the aggregation-induced emission photosensitizer TDR848 prepared above;

[0078] Next, add 1 mL of tetrahydrofuran to the centrifuge tube and immediately tighten the cap (for the mixture).

[0079] Next, centrifuge tubes were placed in a floating foam tube rack and then into an ultrasonic cleaning tank filled with water. The ultrasonic cleaning tank was run for 10 minutes to allow all components to dissolve completely. Then, a 250 mL beaker was filled with crushed ice, and a 20 mL glass bottle was inserted into the ice, ensuring it was firmly fixed in the center, with the insertion depth close to the bottle opening. 9 mL of deionized water was added to the glass bottle, and the beaker was then placed in an ultrasonic cell disruptor. The position of the ultrasonic probe was adjusted so that it was submerged in the deionized water and 1 cm from the bottom of the bottle. During ultrasonic treatment, the completely dissolved mixture was added dropwise to the glass bottle at a rate of one drop per second using a disposable syringe. The addition rate of the tetrahydrofuran solution does not need to be strictly controlled, but a steady dropwise addition is recommended. After all the solution has been added, ultrasonic treatment was continued for 3 minutes until a clear, transparent liquid was observed. The mixture was then purged with nitrogen for 4 hours to remove the tetrahydrofuran, ensuring the liquid surface fluctuated but did not splash. Finally, the solution was filtered using a 0.45 μm syringe filter. Transfer the filtered solution to an ultrafiltration tube (molecular weight cutoff 100 kDa). Centrifuge at approximately 4,000 g for 8 min, and finally concentrate to 1.0 mL. If the volume is less than 1.0 mL, add deionized water to a 1.0 mL volumetric flask to bring the volume to 1.0 mL.

[0080] Hydrated particle size and distribution: Add 0.9 mL of deionized water to the polystyrene particle size analyzer. Add 0.1 mL of the nanoparticle solution prepared according to the above method to the 0.9 mL water and mix well. Insert the analyzer into the sample cell of the nanoparticle size analyzer, and determine the size and distribution information of the nanoparticles based on the principle of dynamic light scattering.

[0081] Result: From Figure 9 As can be seen, the responsive antigen capture nanoplatform exhibits a narrow size distribution, and its hydrodynamic diameter was measured to be about 100 nm by dynamic light scattering experiments. This size endows the responsive antigen capture nanoplatform with the ability to enter tumor tissue through the gaps in tumor blood vessels and remain there for a long time.

[0082] Example 3

[0083] Research on antigen capture by responsive antigen-capturing nanoplatform

[0084] First, to obtain TAAs, MB49 bladder cancer cells (purchased from ATCC) were resuspended in PBS at 37°C at a cell density of 1×10⁻⁶ cells / mL. 7 / mL. Cells were then rapidly frozen in liquid nitrogen and thawed at 37°C (5 min each time, for a total of 5 thaws). Next, the cell lysate was centrifuged at 200g for 5 min to remove insoluble cell debris. The collected TAA supernatant was used to determine protein concentration using the BCA method, and the protein solution was diluted to 1 mg / mL, 0.5 mg / mL, and 0.2 mg / mL for subsequent experiments.

[0085] Next, in order to prepare FPB ONOO- Add 1 mL of ONOO- (1 mM) to 1 mL of FPB (5 mg / mL) and react in a constant temperature shaker (37℃, 100 rpm) for 0.5 h. Then centrifuge the reaction solution for 5 min using a 2 mL ultrafiltration centrifuge tube (molecular weight cutoff 10 kDa). Add 1 mL of deionized water to the ultrafiltration tube and centrifuge again for 5 min (repeat 3 times) to remove excess ONOO- and avoid interference with subsequent experimental results. The ultrafiltration purified FPB... ONOO- Adjust the solution to 1 mL, maintaining a concentration of 5 mg / mL. Prepare before use and use immediately after preparation.

[0086] Once preparation was complete, the particle size change of the nanoparticles before and after antigen capture was determined using a nanoparticle size analyzer based on the principle of dynamic light scattering. Additionally, the amount of protein bound to the nanoparticles was determined using the BCA method. The specific procedure was as follows: nanoparticles (PEG, Mal, FPB, and FPB) at a concentration of 2 mg / mL were... ONOO-The nanoparticles were mixed with 1 mg / mL TAA supernatant at a volume ratio of 1:1 and incubated together at 37°C for 2 h. Then, they were added to an ultrafiltration tube (molecular weight cutoff 300 kDa) for ultrafiltration. After centrifugation, the supernatant contained the captured protein nanoparticles, while the filtrate contained the uncaptured protein. The amount of protein captured by the nanoprobe was calculated by subtracting the amount of uncaptured protein from the amount of protein in the supernatant before capture. All measurements are the average of three independent measurements.

[0087] Results: Different nanoparticles bind to TAAs and FPB through different mechanisms. ONOO- Mal binds to proteins through activated covalent interactions, while PEG, as a negative control, should have minimal interaction with proteins. Figure 10 As shown, after co-incubation with TAAs, Mal and FPB ONOO- The particle size increased significantly, confirming the successful capture of TAAs. Furthermore, the FPB per milligram... ONOO- Mal, FPB, and PEG bound 409.7 μg, 355.9 μg, 63.6 μg, and 35.3 μg of protein, respectively. The ability of FPB to capture TAAs was significantly enhanced by 6.44-fold after reacting with ONOO-. These results indicate that FPB possesses the ability to capture TAAs in response to the overexpression of the tumor inflammatory microenvironment biomarker ONOO-.

[0088] Comparative Example 1

[0089] Comparison of the responsive antigen-capturing nanoplatform prepared in this invention with the antigen-capturing nanoplatform Mal

[0090] To further compare Mal and FPB ONOO- The sensitivity of capturing TAAs was further improved by using Mal and FPB at a concentration of 2 mg / mL. ONOO- The protein was mixed with 1 mg / mL, 0.5 mg / mL, and 0.2 mg / mL TAAs at a volume ratio of 1:1 and incubated at 37°C for 2 h. The amount of protein captured was determined according to the above method, and the protein capture rate was calculated by dividing the amount of captured protein by the amount of added protein.

[0091] Result: As Figure 11 As shown, especially at lower protein concentrations (100 μg / mL), FPB ONOO- The protein capture rate of the FPB group was significantly higher than that of the Mal group, demonstrating that FPB ONOO- It exhibits higher sensitivity in capturing TAAs. This is related to the fact that the methylenequinone functional group obtained after the reaction of FPB with ONOO- has higher nucleophilic activity than maleimide and can covalently bind to a wider variety of amino acids.

[0092] Example 4

[0093] Evaluation of BMDCs' Maturation and Cross-Presentation Capabilities

[0094] Bone marrow-derived dendritic cells (BMDCs) were extracted from the medullary canals of the mouse femur and tibia according to standard procedures. 5 × 10⁶ cells were extracted per well. 4 Cells were seeded in 12-well plates and cultured in 1640 medium for 24 h. At this point, photosensitizer@FPB and MB49 bladder cancer cells (purchased from ATCC) were co-cultured for 4 h, followed by exposure to white light (0.25 W / cm²). 2 Cells were irradiated for 10 min to generate tumor fragments. A solution containing TAAs was obtained by centrifugation and collection of the supernatant, and then mixed with an equal volume of 1 mg / mL nanoparticles (PEG, FPB, Mal, and FPB) at room temperature. ONOO- After a period of pre-incubation, the cells were co-cultured with BMDCs for 24 hours, and then collected by centrifugation. The collected BMDCs were stained with anti-CD11c-FITC, anti-CD86-APC, anti-CD80-PE, and anti-MHC-II-APC for 30 minutes on ice. Afterwards, the cells were collected by centrifugation, and 0.5 mL of PBS was added for flow cytometry detection of CD80, CD86, and MHC-II expression on the surface of BMDCs.

[0095] Result: As Figure 12 As shown, after cell supernatant and FPB ONOO- CD80 in the mixture treatment group + and CD86 + The proportion of cells in the total number of DCs was significantly higher than that of the groups treated with cell supernatant and the mixture of cell supernatant with PEG, FPB and Mal, respectively, indicating that FPB-based treatment... ONOO- The antigen mixture significantly enhanced the maturation of BMDCs. More importantly, based on FPB... ONOO- The antigen mixture can also effectively promote cross-presentation of antigens and upregulate the expression level of MCH-II on the surface of BMDCs.

[0096] Example 5

[0097] Evaluation of anti-tumor effects

[0098] 8×10 5 MB49 bladder cancer cells were subcutaneously injected into the right abdomen of C57BL / 6J mice. On day 7, when the tumor volume reached approximately 50 mm... 3Mice bearing tumors were randomly divided into four groups (n=5 per group): 1) saline group, 2) photosensitizer@FPB group, 3) photosensitizer@PEG + light group, and 4) photosensitizer@FPB + light group. On days 7, 9, and 11, mice in each group were intravenously injected with saline (group 1), photosensitizer@FPB (groups 2 and 4, dose calculated as photosensitizer: 10 mg / kg), and photosensitizer@PEG (group 3, dose calculated as photosensitizer: 10 mg / kg), respectively. Mice in groups 3 and 4 were exposed to white light (0.5 W / cm²) 6 hours after injection. 2 (10 min). Tumor volume was then continuously monitored, and according to the animal experimental standards of this study, when the tumor volume reached 1500 mm², [the tumor was considered to have reached this value]. 3 At that time, the mice will be euthanized.

[0099] Result: As Figure 13 As shown, compared with the "photosensitizer@PEG+light" treatment group, the "photosensitizer@FPB+light" treatment significantly inhibited tumor growth, indicating that the therapy based on the antigen-capturing nanoplatform FPB has the effect of enhancing the anti-tumor efficacy of photodynamic therapy.

[0100] In summary, this invention provides a responsive antigen-capturing nanoplatform, its preparation method, and its applications. This nanoplatform exhibits high spatial specificity and antigen-capturing sensitivity. High spatial specificity specifically manifests in its ability to activate antigen capture only upon stimulation by the overexpressed biomarker ONOO- in the tumor microenvironment, avoiding premature binding to proteins other than TAAs before reaching the tumor site. High antigen-capturing sensitivity is another significant advantage, thanks to its protein binding via methylenequinone (QM). QM has higher nucleophilic reactivity than maleimide (Mal) and can covalently bind to a wider range of amino acids. Its high spatial specificity and sensitivity make it suitable for intravenous administration. Animal experiments show that intravenous injection of the photosensitizer @FPB can enrich it at the tumor site and significantly enhance the efficacy of antitumor immunotherapy. The preparation method of the responsive antigen-capturing nanoplatform described in this invention is simple and economical, effectively overcoming the limitations of traditional intratumoral injection, and providing a promising and feasible technical platform for the development of in situ tumor vaccines.

[0101] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a responsive antigen-capturing nanoplatform, characterized in that, Includes the following steps: (1) DSPE-PEG 2000 -FPB and DSPE-PEG 2000 The mixtures were then dissolved together in tetrahydrofuran to obtain a mixed solution. (2) The mixture obtained above is added dropwise to water, accompanied by ultrasonic treatment during the dropwise process. Then, nitrogen is used to purge the solvent and the mixture is filtered to obtain a responsive antigen capture nanoplatform. Step (1) of the DSPE-PEG 2000 -FPB and the amphiphilic polymer DSPE-PEG 2000 The mass ratio of the mixture is (0.5~1.5):(0.5~1.5). The volume ratio of the mixture to water in step (2) is 1:(8~10); The DSPE-PEG 2000 The preparation method of -FPB includes the following steps: S1) Diethylaminosulfur trifluoride was mixed with a dichloromethane solution of methyl 4-bromo-3-(hydroxymethyl)benzoate and reacted at 0°C for 20-40 min, followed by stirring at room temperature for 10-14 h. After washing, concentration, and purification by silica gel column chromatography, methyl 4-bromo-3-(fluoroethyl)benzoate was obtained. S2) Methyl 4-bromo-3-(fluoroethyl)benzoate, bis(pinacolyl)diboron, [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride and potassium acetate were dissolved in N,N-dimethylformamide and reacted at 98-102℃ for 7-9 h under argon protection. After washing, concentration and purification by silica gel column chromatography, FPB-COOCH3 was obtained. S3) Dissolve FPB-COOCH3 in a tetrahydrofuran / water mixture, mix with an aqueous lithium hydroxide solution, react for 10-16 h, and then neutralize, extract, dry, and perform silica gel column chromatography to obtain FPB-COOH; S4) Dissolve FPB-COOH, dicyclohexylcarbodiimide, and N-hydroxysuccinimide in dichloromethane and react under a nitrogen atmosphere for 50-70 min, then add DSPE-PEG. 2000 The dichloromethane solution containing -NH2 was reacted for another 40–60 hours. After filtration, the solution was dialyzed and lyophilized to obtain DSPE-PEG. 2000 -FPB.

2. The preparation method according to claim 1, characterized in that, The DSPE-PEG described in step (1) 2000 -FPB, DSPE-PEG 2000 The ratio of the nitrate to tetrahydrofuran is (2~3) mg: (2~3) mg: 1 ml.

3. The preparation method according to claim 2, characterized in that, In step (2), the rate of adding the mixed liquid is 0.5 to 2 drops / s; the power of the ultrasonic treatment is 100 to 200 W, and after the addition is completed, the ultrasonic treatment continues for 2 to 4 minutes.

4. The preparation method according to claim 3, characterized in that, The filtration in step (2) includes sequential membrane filtration and ultrafiltration. The pore size of the membrane filtration is 0.22~0.45μm, and the molecular weight cutoff of the ultrafiltration is 100kDa.

5. The preparation method according to claim 1, characterized in that, The concentration of the lithium hydroxide aqueous solution is 0.8~1.2M.

6. The responsive antigen-capturing nanoplatform prepared by the preparation method according to any one of claims 1 to 5.

7. The application of the responsive antigen-capturing nanoplatform according to claim 6 in the preparation of antitumor drugs.

8. The application according to claim 7, characterized in that, The tumor is a solid tumor.

9. A responsive antigen-capturing nanomedicine delivery system, characterized in that, The drug and / or photosensitizer are loaded onto the responsive antigen capture nanoplatform as described in claim 6.

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