Responsive antigen capture nano platform as well as preparation method and application thereof

Abstract

The invention relates to the technical field of tumor immunotherapy. The invention provides a response type antigen capture nano platform as well as a preparation method and application thereof. The product disclosed by the invention is applied through intravenous injection and other systemic administration modes; peroxynitrite in a tumor microenvironment can be specifically responded, and accurate activation of a tumor part is realized; tumor-associated antigens can be efficiently and covalently captured, and the antigens are delivered into antigen presenting cells, so that the anti-tumor immune response is enhanced; the tumor growth can be obviously inhibited by combining photodynamic therapy.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of tumor immunotherapy, and particularly relates to a responsive antigen-capturing nano-platform and a preparation method and application thereof. BACKGROUND

[0002] Cancer immunotherapy is an important means of current tumor treatment, and antigen-capturing nano-materials show great potential in cancer immunotherapy. However, existing antigen-capturing strategies have problems such as dependence on local tumor administration and limited antigen binding affinity, which greatly limit their clinical application.

[0003] Traditional antigen-capturing nano-materials are mostly combined with tumor-associated antigens (TAAs) through non-covalent interactions (such as hydrophobic interactions and electrostatic interactions), which have defects such as poor binding stability and easy interference by other proteins in the tumor microenvironment. At the same time, these materials usually need to be injected locally in the tumor, and the treatment effect is poor for tumors with heterogeneous distribution or difficult to locate, and off-target effects are easy to occur.

[0004] Although some studies have tried to use maleimide-modified platforms to covalently bind TAAs, such platforms lack a tumor microenvironment-specific activation mechanism, still need local administration, and may non-specifically bind to albumin in the blood, affecting treatment effect and safety.

[0005] Therefore, developing an activated covalent antigen-capturing nano-platform that can be administered systemically, has high spatiotemporal selectivity and strong antigen binding capacity, is the key to solving the above problems. SUMMARY

[0006] The present application aims to provide a responsive antigen-capturing nano-platform and a preparation method and application thereof, to overcome the defects of existing antigen-capturing nano-materials such as dependence on local administration, low antigen binding affinity, and obvious off-target effects.

[0007] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0008] The present application provides a preparation method of a responsive antigen-capturing nano-platform, comprising the following steps:

[0009] (1) mixing DSPE-PEG 2000 -FPB and DSPE-PEG 2000 , and dissolving them in tetrahydrofuran to obtain a mixed solution;

[0010] (2) adding the mixed solution obtained above dropwise into water, accompanied by ultrasonic treatment during the adding process, and then sequentially performing nitrogen blowing to remove the solvent and filtering to obtain the responsive antigen-capturing nano-platform;

[0011] DSPE-PEG described in step (1) 2000 -FPB and 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 mixed solution to water in step (2) is 1:(8-10).

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

[0014] Preferably, the speed of adding the mixed solution in step (2) 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 is continued for 2 to 4 minutes.

[0015] Preferably, the filtration comprises membrane filtration and ultrafiltration performed sequentially, the pore size of the membrane filtration is 0.22 to 0.45 μm, and the molecular weight cut-off of the ultrafiltration is 100 kDa.

[0016] The present invention also provides DSPE-PEG 2000 -FPB synthesis method, as preferably, the DSPE-PEG 2000 -FPB synthesis route such as Figure 1 As shown, the synthesis method is as follows:

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

[0018] S1) mixing diethylaminosulfur trifluoride with a dichloromethane solution of methyl 4-bromo-3-(hydroxymethyl)benzoate, reacting at 0°C for 20-40 minutes, stirring at room temperature for 10-14 hours, washing, concentrating, and purifying by silica gel column chromatography to obtain methyl 4-bromo-3-(fluoroethyl)benzoate;

[0019] S2) dissolving methyl 4-bromo-3-(fluoroethyl)benzoate, bis(pinacolato)diboron, [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride, and potassium acetate in N,N-dimethylformamide, reacting at 98-102° C. under argon protection for 7-9 hours, washing, concentrating, and purifying by silica gel column chromatography to obtain FPB-COOCH3;

[0020] S3) dissolving FPB-COOCH3 in a tetrahydrofuran / water mixture, mixing with a lithium hydroxide aqueous solution, reacting for 10 to 16 hours, neutralizing, extracting, drying, and silica gel column chromatography to obtain FPB-COOH;

[0021] S4) FPB-COOH, dicyclohexyl carbodiimide and N-hydroxysuccinimide were dissolved in dichloromethane, reacted for 50-70 min under nitrogen atmosphere, then DSPE-PEG 2000 -NH2 solution in dichloromethane was added and the reaction was continued for 40-60 h, after filtration, dialysis and lyophilization, DSPE-PEG 2000 -FPB was obtained.

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

[0023] The application provides a responsive antigen-capturing nano platform prepared by the preparation method.

[0024] The application provides application of the responsive antigen-capturing nano platform in preparation of an anti-tumor drug.

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

[0026] The application provides a responsive antigen-capturing nano drug delivery system, which is loaded with a drug and / or a photosensitizer by the responsive antigen-capturing nano platform.

[0027] The responsive antigen-capturing nano platform can load an anti-tumor drug, including but not limited to a chemotherapy drug and a photosensitizer.

[0028] The application has the following beneficial effects:

[0029] 1. The drug can be administered systemically without the need for local injection of the tumor, and is suitable for tumors that are difficult to locate or have heterogeneous distribution, and the drug is administered by intravenous injection, and after administration, the tumor site is treated with light;

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

[0031] 3. The drug can release TAAs in combination with photodynamic therapy, and at the same time, the released TAAs are captured in situ in the tumor, thereby enhancing the inhibitory effect of photodynamic therapy on bladder tumors. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 The synthesis route of DSPE-PEG 2000 -FPB in Example 1 is shown in the following scheme;

[0033] Figure 2 The nuclear magnetic resonance hydrogen spectrum of 4-bromo-3-(fluoroethyl) benzoic acid methyl ester in Example 1 is shown in the following scheme;

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

[0035] Figure 4 is the hydrogen nuclear magnetic resonance spectrum of the compound FPB-COOH in Example 1;

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

[0037] Figure 6 is the H NMR spectrum of TD in Example 2;

[0038] Figure 7 is the hydrogen nuclear magnetic resonance spectrum of TD-TK in Example 2;

[0039] Figure 8 is the hydrogen nuclear magnetic resonance spectrum of TDR848 in Example 2;

[0040] Figure 9 Representative dynamic light scattering results of the responsive antigen capture nanoplatform;

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

[0042] Figure 11 is the protein capture rate of different nanoparticles in TAAs solutions with different protein concentrations;

[0043] Figure 12 The expression of CD80, CD86 and MHC-II by BMDCs in different groups and the quantitative analysis of the positive expression levels;

[0044] Figure 13 Figure 2 is the curve of tumor volume change over time in tumor-bearing mice in different groups. DETAILED DESCRIPTION

[0045] The present invention provides a method for preparing a responsive antigen capture nano-platform, comprising the following steps: (1) DSPE-PEG 2000 -FPB and DSPE-PEG 2000 Mix and dissolve them in tetrahydrofuran to obtain a mixed solution; (2) add the mixed solution obtained above to water, and ultrasonicate it during the addition process, then blow nitrogen to remove the solvent, and filter to obtain a responsive antigen capture nanoplatform; the DSPE-PEG in step (1) 2000 -FPB and amphiphilic polymer DSPE-PEG 2000The mass ratio of the mixture is (0.5-1.5): (0.5-1.5); the volume ratio of the mixed liquid to water in step (2) is 1: (8-10).

[0046] In the present invention, DSPE-PEG 2000 -FPB and DSPE-PEG 2000 Mix and dissolve in tetrahydrofuran to obtain a mixed solution. 2000 -FPB is an amphiphilic polymer synthesized by ourselves, and the DSPE-PEG 2000 It is a commercial amphiphilic polymer. In the present invention, the DSPE-PEG 2000 -FPB and amphiphilic polymer DSPE-PEG 2000 The mixing mass ratio is preferably (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 the present invention, the DSPE-PEG 2000 -FPB, DSPE-PEG 2000 The ratio of tetrahydrofuran to iodine is preferably (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 the present invention, the mixed solution obtained above is added dropwise to water, and ultrasonic treatment is performed during the addition process, and then nitrogen is purged to remove the solvent and filtered to obtain a responsive antigen capture nano-platform. In the present invention, the volume ratio of the mixed solution to water is preferably 1: (8-10), more preferably 1: (8.5-9.5), and even more preferably 1: 9. In the present invention, the water is preferably ultrapure water; the speed of adding the mixed solution dropwise is preferably 0.5-2 drops / s, more preferably 1 drop / s; the addition process is accompanied by ultrasonic treatment, and the power of the ultrasonic treatment is 100-200W, preferably 120-180W, more preferably 150W. After the addition is completed, the ultrasonic treatment is continued for 2-4min, preferably 2.5-3.5min, and more preferably 3min. The ultrasonic treatment is preferably performed using an ultrasonic cell disruptor, and the ultrasonic probe is preferably immersed in ultrapure water and 0.8-1.2 cm away from the bottom of the bottle. In the present invention, after the ultrasonic treatment, nitrogen purging is performed to remove the solvent (residual tetrahydrofuran); the nitrogen purging time is preferably 3 to 5 hours, more preferably 3.5 to 4.5 hours, and even more preferably 4 hours.

[0048] After the nitrogen purge is completed, the present invention performs filtration; the filtration includes membrane filtration and ultrafiltration performed in sequence, the pore size of the membrane filtration is preferably 0.22-0.45 μm, and 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, the liquid is transferred to the ultrafiltration tube for centrifugation, and the centrifugal force of the centrifugation is preferably 3000-5000 g, more preferably 3500-4500 g, and even more preferably 4000 g. The centrifugation time is preferably 5-8 min; if the volume after the centrifugation is less than the original volume of the mixed liquid, ultrapure water is added to make up to the original volume of the mixed liquid, and a responsive antigen capture nanoplatform is obtained.

[0049] The present invention also provides DSPE-PEG 2000 -FPB synthesis method, as preferably, the DSPE-PEG 2000 -FPB synthesis route such as Figure 1 As shown, the synthesis method is as follows:

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

[0051] S1) mixing diethylaminosulfur trifluoride with a dichloromethane solution of methyl 4-bromo-3-(hydroxymethyl)benzoate, reacting at 0°C for 20 to 40 minutes, preferably under argon protection; then stirring at room temperature for 10 to 14 hours, preferably 11 to 13 hours, more preferably 12 hours; then washing, concentrating, and purifying by silica gel column chromatography to obtain methyl 4-bromo-3-(fluoroethyl)benzoate;

[0052] S2) dissolving methyl 4-bromo-3-(fluoroethyl)benzoate, bis(pinacolato)diboron, [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride and potassium acetate in N,N-dimethylformamide, reacting at 98-102° C. for 7-9 h, preferably at 100° C. for 8 h under argon protection; then washing, concentrating and purifying by silica gel column chromatography to obtain FPB-COOCH3;

[0053] S3) dissolving FPB-COOCH3 in a tetrahydrofuran / water mixture, mixing with a lithium hydroxide aqueous solution, and reacting for 10 to 16 hours, wherein the concentration of the lithium hydroxide aqueous solution is preferably 0.8 to 1.2 M, more preferably 0.9 to 1.1 M, and even more preferably 1.0 M; then neutralizing, extracting, drying, and silica gel column chromatography to obtain FPB-COOH;

[0054] S4) FPB-COOH, dicyclohexylcarbodiimide and N-hydroxysuccinimide are dissolved in dichloromethane and reacted under nitrogen atmosphere for 50 to 70 minutes, preferably 55 to 65 minutes, more preferably 60 minutes, and then DSPE-PEG is added. 2000 -NH2 dichloromethane solution, continue the reaction for 40 to 60 hours, preferably 45 to 50 hours, more preferably 48 hours; then filter, dialyze, and freeze-dry to obtain DSPE-PEG 2000 -FPB.

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

[0056] The present invention also provides the use of the responsive antigen capture nano-platform in the preparation of anti-tumor drugs. In the present invention, the type of the tumor is preferably a solid tumor.

[0057] The present invention also provides a responsive antigen capture nano drug delivery system, in which the responsive antigen capture nano platform is loaded with drugs and / or photosensitizers. In the present invention, the drugs include chemotherapy drugs; in the present invention, the mass ratio of the responsive antigen capture nano platform, drugs and photosensitizers is preferably (0.5-1.5): (0.5-1.5): (0.5-1.5), further preferably (0.8-1.2): (0.8-1.2): (0.8-1.2), and further preferably 1:1:1. In the present invention, the preparation method of the responsive antigen capture nano drug delivery system refers to the description of the above-mentioned responsive antigen capture nano platform, the difference being that in step 1), the drugs and / or photosensitizers, DSPE-PEG 2000 -FPB, DSPE-PEG 2000 The two components are mixed and dissolved in tetrahydrofuran to obtain a mixture. During the nanoparticle formation process, the hydrophobic distearoylphosphatidylethanolamine (DSPE) domain entangles with the drug or photosensitizer to form a core, while the hydrophilic polyethylene glycol (PEG) chain forms a protective shell that extends into the aqueous phase, thereby stabilizing the nanoparticles and preventing further aggregation.

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

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

[0060] Synthesis route 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 the starting material 4-bromo-3-(hydroxymethyl)benzoic acid methyl ester (500 mg, 2.05 mmol) in dichloromethane, and 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 liquid was separated. The organic phase was collected and dried and concentrated. The crude product was purified by silica gel column chromatography to obtain 4-bromo-3-(fluoroethyl)benzoic acid methyl ester (346 mg, yield 68.54%). The H NMR spectrum was as follows: Figure 2 shown.

[0062] 2) Methyl 4-bromo-3-(fluoroethyl)benzoate (330 mg, 1.34 mmol), bis(pinacolato)diboron (511 mg, 2.01 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (95 mg, 0.13 mmol) and potassium acetate (394 mg, 4.02 mmol) were dissolved in N,N-dimethylformamide (10 mL) and heated to 100 ° C under argon protection for 8 hours. After the reaction was completed, it was cooled to room temperature, diluted with ether, washed with deionized water and saturated sodium chloride, and the organic phase was collected and dried and concentrated. The crude product was purified by silica gel column chromatography to obtain compound FPB-COOCH3 (209 mg, yield 53.02%). The nuclear magnetic resonance hydrogen spectrum was as follows Figure 3 shown.

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

[0064] 4) FPB-COOH (31 mg, 0.11 mmol), dicyclohexyl carbodiimide (45 mg, 0.22 mmol) and N-hydroxysuccinimide (25 mg, 0.22 mmol) were dissolved in dichloromethane (10 mL) in a flask and stirred for 1 h under 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 continued to stir for 48 h under nitrogen atmosphere and the reaction was completed after which the white precipitate was removed by filtration. The prepared DSPE-PEG 2000 -FPB solution was added to deionized water and dialyzed in deionized water for 48 h to completely remove excess FPB-COOH and other byproducts. The liquid in the dialysis bag was lyophilized to obtain pure DSPE-PEG 2000 -FPB.

[0065] Results: The structure of DSPE-PEG 2000 -FPB was verified to be correct by nuclear magnetic resonance hydrogen spectrum characterization Figure 5

[0066] Example 2

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

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

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

[0070] ​1) Dissolve TPE-DPA-CHO (1.10 mmol, 580 mg) and rhodanine 3-acetic acid (1.32 mmol, 252 mg) in glacial acetic acid and add ammonium acetate (3.30 mmol, 254 mg). Heat under reflux for 24 h while stirring. After cooling to room temperature, the product will precipitate. After filtering to obtain a solid, the solid is washed with ethyl acetate / petroleum ether (v / v = 1:3), anhydrous ethanol and deionized water respectively. Finally, dry in a vacuum drying oven to obtain pure TD (621 mg, 80.64%). The H NMR spectrum is as follows Figure 6 shown.

[0071] 2) Compound TD (420 mg, 0.60 mmol), 2,2'-[propane-2,2-diylbis(sulfanediyl)]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. Under a nitrogen atmosphere, the reaction was stirred at room temperature for 24 hours. After the reaction was completed, the reaction mixture was diluted with dichloromethane and washed with a saturated sodium chloride solution. Then, the aqueous phase was 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 obtain pure TD-TK (346 mg, 65.7%). H NMR spectrum is as follows Figure 7 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. A solution of 4-nitrobenzene chloroformate (120 mg, 0.60 mmol) in ultra-dry dichloromethane was slowly added dropwise under nitrogen at 0°C with stirring, and stirring was continued at room temperature for 4 h. After the reaction was complete, the mixture was concentrated under vacuum and then redissolved in N,N-dimethylformamide. R848 (377 mg, 1.20 mmol) and ultra-dry triethylamine (121 mg, 1.20 mmol) were then added to the reaction solution, and stirring was continued for 24 h. The reaction mixture was concentrated under vacuum. The mixture was extracted with dichloromethane and saturated brine, and the organic phase was separated, 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%). Figure 8 shown.

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

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

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

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

[0077] The formula of photosensitizer@FPB nanoparticles (i.e., responsive antigen capture nanodelivery system) is: 2.5mg 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 cap the tube tightly (mixed solution).

[0079] Next, place the centrifuge tube in a floating foam tube rack and place it in an ultrasonic cleaning tank filled with water. Run the ultrasonic cleaning tank for 10 minutes to allow all components to fully dissolve. Next, fill a 250 mL beaker with crushed ice and insert a 20 mL glass bottle into the crushed ice, ensuring it is securely centered and inserted close to the bottle's opening. Add 9 mL of deionized water to the glass bottle and place the beaker in an ultrasonic cell disruptor. Adjust the ultrasonic probe position so that it is immersed in the deionized water and 1 cm from the bottom of the bottle. While ultrasonication is running, use a disposable laboratory syringe to add the completely dissolved mixture dropwise to the glass bottle at a rate of one drop per second. 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, continue ultrasonication for 3 minutes until a clear, transparent liquid is observed. Subsequently, purge the mixture with nitrogen for 4 hours to remove the tetrahydrofuran, ensuring that the liquid surface fluctuates but does not splash. The solution is then 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 minutes to concentrate to a final volume of 1.0 mL. If the volume is less than 1.0 mL, use a 1.0 mL volumetric flask to add deionized water to 1.0 mL.

[0080] Hydrated Particle Size and Distribution: Add 0.9 mL of deionized water to a polystyrene particle size analyzer. Add 0.1 mL of the nanoparticle solution prepared according to the above method to the 0.9 mL of water and mix thoroughly. Insert the analyzer into the sample well of a nanoparticle size analyzer, and measure the size and distribution of the nanoparticles using dynamic light scattering.

[0081] Results: From Figure 9 It can be seen that the responsive antigen capture nanoplatform exhibits a narrow size distribution, and the dynamic light scattering experiment measures its hydrodynamic diameter to be around 100 nm. This size gives the responsive antigen capture nanoplatform the ability to enter the tumor tissue through the gaps in tumor blood vessels and remain there for a long time.

[0082] Example 3

[0083] Study on antigen capture using responsive antigen capture 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 7 / mL. Subsequently, the cells were quickly 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 protein concentration of the collected TAAs supernatant was determined 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, to prepare FPB ONOO- , add 1mL of ONOO- (1mM) to 1mL of FPB (5mg / mL), place it in a constant temperature shaker (37℃, 100rpm) to react for 0.5h, and then centrifuge the reaction solution for 5min using a 2mL ultrafiltration centrifuge tube (molecular weight cutoff 10kDa). Then add 1mL of deionized water to the ultrafiltration tube and centrifuge again for 5min (repeat 3 times) to remove excess ONOO- and avoid its interference with subsequent experimental results. ONOO- The solution is diluted to 1 mL, maintaining a concentration of 5 mg / mL. Prepare immediately before use and use immediately.

[0086] After preparation, the change in the particle size of the nanoparticles before and after antigen capture was measured by the principle of dynamic light scattering in a nanoparticle size analyzer. In addition, the amount of protein bound to the nanoparticles was determined using the BCA method. The specific operation was as follows: the nanoparticles (PEG, Mal, FPB and FPB) with a concentration of 2 mg / mL were added. ONOO-) were mixed with 1 mg / mL TAA supernatant in a 1:1 volume ratio and incubated at 37°C for 2 hours. The mixture was then added to an ultrafiltration tube (molecular weight cutoff, 300 kDa) for ultrafiltration. After centrifugation, the upper layer of liquid was the nanoparticles containing captured proteins, and the lower filtrate was the uncaptured proteins. 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 TAAs and FPB via different mechanisms ONOO- Mal binds to proteins through activatable covalent interactions, Mal binds to proteins through non-activatable covalent interactions, and PEG serves as a negative control, which 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. ONOO- , Mal, FPB, and PEG were bound to 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 increased by 6.44-fold after reacting with ONOO-. These results indicate that FPB has the ability to capture TAAs in response to ONOO-, a biomarker overexpressed in the tumor inflammatory microenvironment.

[0088] Comparative Example 1

[0089] Comparison between the responsive antigen capture nanoplatform prepared by the present invention and the antigen capture nanoplatform Mal

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

[0091] Results: As Figure 11 As shown, especially at lower protein concentration (100 μg / mL), FPB ONOO- The protein capture rate of the group was significantly higher than that of the Mal group, proving that FPB ONOO- It has higher sensitivity in capturing TAAs. This is related to the fact that the functional group methylene quinone obtained after the reaction of FPB with ONOO- has higher nucleophilic reactivity than maleimide and can covalently bind to more types of amino acids.

[0092] Example 4

[0093] Evaluation of BMDCs maturation and cross-presentation ability

[0094] Bone marrow-derived dendritic cells (BMDCs) were isolated from the medullary cavity of mouse femur and tibia according to standard procedures. 5×10 4 The cells were seeded in a 12-well plate and cultured in 1640 medium for 24 h. At this time, photosensitizer@FPB and MB49 bladder cancer cells (purchased from ATCC) were co-cultured for 4 h, and then a white light flashlight (0.25 W / cm 2 The cells were irradiated for 10 min to generate tumor fragments. The supernatant was collected by centrifugation to obtain a solution containing TAAs, which was then mixed with an equal volume of 1 mg / mL nanoparticles (PEG, FPB, Mal, and FPB) at room temperature. ONOO- After preincubation with PBS, cells were co-cultured with BMDCs for 24 hours and then harvested by centrifugation. The harvested BMDCs were stained with anti-CD11c-FITC, anti-CD86-APC, anti-CD80-PE, and anti-MHC-II-APC on ice for 30 minutes. The cells were then harvested by centrifugation and added with 0.5 mL of PBS for flow cytometry analysis of CD80, CD86, and MHC-II expression on the BMDC surface.

[0095] Results: As Figure 12 As shown, the cell supernatant and FPB ONOO- CD80 in the mixture-treated group + and CD86 + The proportion of cells in the total number of DCs cells was significantly higher than that in the groups treated with cell supernatant and the mixture of cell supernatant and PEG, FPB and Mal, respectively, indicating that the FPB-based ONOO- The antigen mixture can significantly enhance the maturation of BMDCs. More importantly, FPB-based ONOO- The antigen mixture can also effectively promote the 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. After 7 days, when the tumor volume reached about 50 mm 3The tumor-bearing mice were randomly divided into 4 groups (5 mice in each group), which were named as: 1) "normal saline" group, 2) "photosensitizer@FPB" group, 3) "photosensitizer@PEG+light" group and 4) "photosensitizer@FPB+light" group. The mice in each group were intravenously injected with normal saline (Group 1), photosensitizer@FPB (Groups 2 and 4, the dose calculated as photosensitizer was 10 mg / kg), photosensitizer@PEG (Group 3, the dose calculated as photosensitizer was 10 mg / kg) on ​​the 7th, 9th and 11th days, respectively. The mice in Groups 3 and 4 were irradiated with white light (0.5 W / cm 2 The volume of the tumor was then continuously monitored, and according to the animal experiment standards of this study, when the tumor volume reached 1500 mm 3 Mice will be euthanized when .

[0099] Results: 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 antigen capture nanoplatform FPB-based therapy has enhanced the anti-tumor efficacy of photodynamic therapy.

[0100] In summary, the present invention provides a responsive antigen capture nanoplatform, its preparation method and application. The nanoplatform has high spatial specificity and antigen capture sensitivity. The high spatial specificity is specifically manifested in that its antigen capture ability can only be activated under the stimulation of the biomarker ONOO- overexpressed in the tumor microenvironment, avoiding premature binding to other proteins other than TAAs before reaching the tumor site. High antigen capture sensitivity is another major advantage, which is due to the fact that it is bound to the protein through quinone methylene QM. QM has higher nucleophilic reactivity than maleimide Mal and can covalently bind to more types of amino acids. Due to its high spatial specificity and sensitivity, it has the potential for intravenous administration. Animal experiments show that after intravenous injection, the photosensitizer @FPB can be enriched in the tumor site and significantly enhances the effect of anti-tumor immunotherapy. The preparation method of the responsive antigen capture nanoplatform described in the present invention is simple and economical, and effectively overcomes the limitations of traditional intratumoral injection, providing a promising and feasible technical platform for the development of in situ tumor vaccines.

[0101] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing a responsive antigen capture nanoplatform, characterized in that: The steps include: (1) DSPE-PEG 2000 -FPB and DSPE-PEG 2000 mix, and dissolve together in tetrahydrofuran to obtain a mixed solution; (2) adding the mixed solution obtained above dropwise to water, accompanied by ultrasonic treatment during the addition process, and then purging the solvent with nitrogen and filtering to obtain a responsive antigen capture nanoplatform; DSPE-PEG described in step (1) 2000 -FPB and 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 mixed solution to water in step (2) is 1:(8-10).

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

3. The preparation method according to claim 2, characterized in that The speed of adding the mixed solution in step (2) is 0.5 to 2 drops / s; the power of the ultrasonic treatment is 100 to 200 W. After the addition is completed, the ultrasonic treatment is continued for 2 to 4 minutes.

4. The preparation method according to claim 3, characterized in that The filtration includes membrane filtration and ultrafiltration performed sequentially, the pore size of the membrane filtration is 0.22-0.45 μm, and the molecular weight cut-off of the ultrafiltration is 100 kDa.

5. The preparation method according to claim 1, characterized in that The DSPE-PEG 2000 The preparation method of FPB comprises the following steps: S1) mixing diethylaminosulfur trifluoride with a dichloromethane solution of methyl 4-bromo-3-(hydroxymethyl)benzoate, reacting at 0°C for 20-40 minutes, and stirring at room temperature for 10-14 hours; washing, concentrating, and purifying by silica gel column chromatography to obtain methyl 4-bromo-3-(fluoroethyl)benzoate; S2) dissolving methyl 4-bromo-3-(fluoroethyl)benzoate, bis(pinacolato)diboron, [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride, and potassium acetate in N,N-dimethylformamide, reacting at 98-102° C. under argon protection for 7-9 hours, washing, concentrating, and purifying by silica gel column chromatography to obtain FPB-COOCH3; S3) dissolving FPB-COOCH3 in a tetrahydrofuran / water mixture, mixing with a lithium hydroxide aqueous solution, reacting for 10 to 16 hours, neutralizing, extracting, drying, and silica gel column chromatography to obtain FPB-COOH; S4) FPB-COOH, dicyclohexylcarbodiimide and N-hydroxysuccinimide were dissolved in dichloromethane and reacted under nitrogen atmosphere for 50-70 min, and then DSPE-PEG was added. 2000 -NH2 dichloromethane solution, continue the reaction for 40 to 60 hours, filter, dialyze and freeze-dry to obtain DSPE-PEG 2000 -FPB.

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

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

8. Use of the responsive antigen capture nanoplatform according to claim 7 in the preparation of anti-tumor drugs.

9. The use according to claim 8, characterized in that The type of tumor is a solid tumor.

10. A responsive antigen capture nano drug delivery system, characterized in that: The responsive antigen-capturing nanoplatform according to claim 7 is loaded with drugs and / or photosensitizers.

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