A nano-necroptosis drug targeting CD47 protein and preparation and application thereof

Abstract

The application discloses a nano-focal anaphylaxis drug targeting CD47 protein and preparation and application thereof, and belongs to the technical field of drugs. The nano-focal anaphylaxis drug is a micellar nanoparticle formed by self-assembly of an amphiphilic carrier material and a focal anaphylaxis prodrug, the amphiphilic carrier material is composed of an amphiphilic polymer with a hydrophilic end modified with a CD47 targeting peptide and an amphiphilic polymer without modification, and the focal anaphylaxis prodrug is a prodrug formed by reduction response chemical bond connection of lonidamide or 3-bromopyruvic acid. The nano-focal anaphylaxis drug provided by the application can realize precise tumor treatment, the CD47 targeting peptide realizes efficient targeted delivery and accumulation of the drug in the tumor, the release of the focal anaphylaxis prodrug is triggered by higher reduction medium level in tumor tissues, and tumor cells are induced to undergo focal anaphylaxis. In normal tissues, the nano drug cannot induce normal cell focal anaphylaxis, and therefore the tumor selectivity of the nano drug is significantly enhanced.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to nano-pyroptosis drugs with CD47 protein targeting and redox response, their preparation methods, and anti-tumor applications. Background Technology

[0002] Cancer, especially malignant cancer, has become the second leading cause of disease seriously threatening human health. Immunotherapy, an emerging approach to cancer treatment, offers new directions for cancer treatment and even cure. Pyroptosis, as an important branch of immunotherapy, is receiving increasing attention from researchers. Unlike apoptosis, pyroptosis is an immunogenic cell death mechanism that primarily relies on Gasdermin (GSDM) family proteins to perform its function. These proteins are cleaved by certain activated cysteine-containing aspartic proteases (such as caspase 1 / 3). The GSDM-N-terminal fragment produced after cleavage by caspases drives the formation of pores in the cell membrane, causing the cell to absorb water and burst, thus triggering tumor cell pyroptosis. Simultaneously, pyroptosis releases damage-associated molecular pattern (DAMP) molecules such as high-mobility group box 1 (HMGB1) and interleukin-1β (IL-1β) as tumor-associated antigens, thereby restoring tumor immunogenicity and activating the body's own anti-tumor immune response, ultimately effectively inhibiting tumor growth, recurrence, and metastasis.

[0003] Currently reported pyroptosis inducers include only a few chemotherapeutic drugs (such as paclitaxel and doxorubicin), epigenetic drugs (CD73 inhibitor MethADP), and photosensitizers (such as cinnamoyl-18). However, these pyroptosis inducers generally lack selectivity. While killing tumor cells through pyroptosis, they also induce pyroptosis in normal cells, leading to pyroptosis-related damage and toxic side effects in normal tissues. Furthermore, due to their high osmotic pressure in vivo, these small-molecule pyroptosis agents are easily metabolized by the kidneys and rapidly eliminated from the body, resulting in a very short retention time. Simultaneously, lacking tumor-specific targeting, they can easily infiltrate both normal and tumor tissues indiscriminately. When pyroptosis drugs are taken up more by normal cells, further increases in drug concentration are needed to achieve better efficacy, which in turn greatly increases systemic toxicity.

[0004] Therefore, constructing nanomedicines that specifically target tumor tissues and selectively induce pyroptosis in tumor cells is an effective way to solve the problems of poor tumor selectivity and low tumor targeting of pyroptosis drugs.

[0005] CD47 is a transmembrane glycoprotein, also known as integrin-associated protein (IAP), overexpressed on the surface of most malignant tumor cells, with a molecular weight of 52 kDa. Highly expressed CD47 protein in tumor cells can prevent tumor cells from being phagocytosed by macrophages by binding to signal regulatory protein α (SIRPα) on the surface of macrophages and releasing a "don't eat me" signal. Therefore, current anti-tumor research based on CD47 protein mainly focuses on anti-CD47 antibodies that block the CD47 / SIRPα interaction, CD47 fusion proteins, and small molecule inhibitors of CD47. However, research on active targeted nanodelivery systems based on CD47 has not been reported.

[0006] Therefore, developing a novel nanomaterial that can both target tumor CD47 signaling and induce efficient tumor pyroptosis, thereby satisfying the aforementioned goals of tumor-specific targeting and selective therapy, is a problem that needs to be solved by those skilled in the art. Summary of the Invention

[0007] The purpose of this invention is to provide a novel nanomedicine that targets tumor-induced pyroptosis. This nanomedicine can utilize the highly expressed CD47 protein and high content of reducing mediators on the tumor surface to specifically target tumor tissue and release the drug in response to the tumor microenvironment, thereby inducing tumor-specific pyroptosis.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] This invention provides a nano-pyroptosis drug targeting CD47 protein. The drug is a micelle nanoparticle formed by the self-assembly of an amphiphilic carrier material and a pyroptosis prodrug. The amphiphilic carrier material consists of an amphiphilic polymer with a CD47-targeting peptide modified at its hydrophilic end and an unmodified amphiphilic polymer. The pyroptosis prodrug is a prodrug formed by the linkage of chlordamine or 3-bromopyruvate through reduction-responsive chemical bonds.

[0010] This invention utilizes an amphiphilic polymer carrier to encapsulate chlordamine prodrug or 3-bromopyruvate prodrug. During the self-assembly of the amphiphilic polymer in an aqueous environment to form micelles, its hydrophobic ends interact with the hydrophobic prodrug through hydrophobic interactions, as well as intermolecular π-π stacking and hydrogen bonding, thereby encapsulating the chlordamine prodrug or 3-bromopyruvate prodrug inside the polymer micelles, thus obtaining the aforementioned nanomedicine.

[0011] Preferably, the hydrophobic segment of the amphiphilic polymer is distearylphosphatidylethanolamine, and the hydrophilic segment is polyethylene glycol. More preferably, the amphiphilic polymer is distearylphosphatidylethanolamine-methoxy polyethylene glycol 2000.

[0012] Based on the high expression of CD47 protein on the surface of tumor cells, this invention modifies the surface of a drug delivery system with CD47 targeting peptides to endow it with the ability to target tumors. After the nanomedicine enters the tumor tissue, the high level of CD47 protein on the cell surface enhances the ability of nanoparticles to selectively target tumors, thereby greatly improving the accumulation and uptake of nanoparticles in tumors.

[0013] The CD47 targeting peptide is a polypeptide molecule that specifically binds to the CD47 protein. Preferably, the amino acid sequence of the CD47 targeting peptide is shown in SEQ ID NO.1.

[0014] In constructing an amphiphilic polymer with a CD47-targeting peptide, this invention uses a double bond addition reaction between a thiol group and maleic anhydride to connect the hydrophilic segment of the polymer to the CD47-targeting peptide. This polymer component plays a decisive role in the selective targeting of tumors by nanoparticles.

[0015] Preferably, the amphiphilic polymer with the hydrophilic end modified with the CD47 targeting peptide has the structural formula shown in Formula (I).

[0016]

[0017] This invention uses a mixture of amphiphilic polymers modified with CD47 targeting peptides at their hydrophilic ends and unmodified amphiphilic polymers as drug carriers. Preferably, the mass ratio of the amphiphilic polymer modified with CD47 targeting peptides to the unmodified amphiphilic polymers in the amphiphilic carrier material is 9-15:16-25.

[0018] In constructing the nano-drug delivery system, this invention uses a structurally modified chemotherapeutic drug precursor as the encapsulated drug. This type of prodrug itself has low activity, but after being metabolized in vivo, it becomes an active original drug, which can reduce the systemic toxic side effects of the drug.

[0019] In this invention, chlordamine or 3-bromopyruvate is used as a drug to induce pyroptosis in tumor cells. In the prior art, chlordamine and 3-bromopyruvate are used as glycolysis inhibitors for antitumor effects. This invention has found that chlordamine and 3-bromopyruvate can achieve antitumor effects by inducing pyroptosis in tumor cells.

[0020] This invention employs reduction-responsive chemical bonds to structurally modify chlordamine or 3-bromopyruvic acid, forming a prodrug by linking two molecules of chlordamine or 3-bromopyruvic acid via reduction-responsive chemical bonds. Studies have shown that the reduction-responsive chemical bonds of the prodrug break under high-concentration reducing media, leading to the disintegration of micelle nanoparticles. During this process, the drug is rapidly and efficiently released and activated, thereby inducing pyroptosis in tumor cells.

[0021] The chemical bonds used in the reduction response can be, but are not limited to, disulfide bonds (-S S-), diselenide bonds (-Se Se-), monosulfide bonds (-S-), monoselenide bonds (-Se-), etc.

[0022] Preferably, the structural formula of the chlordamine prodrug is shown in formula (II), and the structural formula of the 3-bromopyruvate prodrug is shown in formula (III).

[0023]

[0024] As a preferred option, the mass ratio of the amphiphilic carrier material to the pyroptosis prodrug is 25–40:1–6.

[0025] The present invention also provides a method for preparing the aforementioned nano-pyroptosis drug targeting CD47 protein. The method includes using polymer micelle preparation methods such as solvent replacement, dialysis, ultrasound, or liquid membrane to self-assemble an amphiphilic carrier material and a pyroptosis prodrug in water to form micelle-type nanoparticles, thereby obtaining the nano-pyroptosis drug.

[0026] The liquid film method includes: first, dissolving the amphiphilic carrier material and the pyroptosis precursor in a good solvent, then removing the solvent by rotary evaporation to form a liquid film, then adding water to the liquid film under ultrasonic conditions, and subsequently the product self-assembling to form the nanomedicine.

[0027] Preferably, the mass ratio of unmodified amphiphilic polymer, amphiphilic polymer modified with CD47 targeting peptide, and pyroptosis prodrug is 16:9:5. The drug encapsulation efficiency of the amphiphilic polymer increases with increasing polymer dosage, but excessive polymer dosage leads to waste. Within this mass ratio range, a high drug encapsulation rate and polymer utilization rate can be guaranteed.

[0028] The present invention also provides the application of the aforementioned pyroptosis nanoparticle drug targeting CD47 protein in the preparation of antitumor drugs, wherein the drug induces pyroptosis of tumor cells to achieve the therapeutic purpose of antitumor treatment.

[0029] Compared to normal tissues, tumor cells highly express CD47 protein and reducing mediators. The nano-pyroptosis drug provided by this invention, on the one hand, increases drug targeting and accumulation in tumor tissues through CD47-targeting peptides; on the other hand, the disulfide bonds in the nano-drug, under the action of reducing mediators highly expressed in tumor cells, promote the release and activation of pyroptosis prodrugs in tumor tissues.

[0030] Furthermore, the tumors include, but are not limited to, colon cancer and breast cancer.

[0031] The beneficial effects of this invention are as follows:

[0032] The nano-pyroptosis drug provided by this invention enables precise tumor treatment. The highly expressed CD47 protein in tumor cells interacts with the CD47-targeting peptide in the nanomedicine, achieving efficient targeted delivery and accumulation of the drug within the tumor. Upon reaching the tumor tissue, a high level of reducing mediators triggers the release and activation of the pyroptosis prodrug, inducing pyroptosis in tumor cells. In contrast, the nanomedicine does not induce pyroptosis in normal tissues, thus significantly enhancing the tumor selectivity of the nanomedicine and achieving precise tumor treatment. Attached Figure Description

[0033] Figure 1 The images show the 1H NMR spectrum of DSPE-PEG2000-4N1K in Example 1, where A is the 1H NMR spectrum of 4N1K, B is the 1H NMR spectrum of DSPE-PEG2000-MAL, and C is the 1H NMR spectrum of the synthesized product.

[0034] Figure 2 The image shows the 1H NMR spectrum of the chloridamine prodrug in Example 1.

[0035] Figure 3 This is a particle size distribution diagram obtained by dynamic light scattering of the nano-pyroptosis drug CLNDN in water in Example 1.

[0036] Figure 4 This is a transmission electron microscope (TEM) image of the nano-pyroptosis drug CLNDN in Example 1.

[0037] Figure 5 This is a drug release curve of the nano-pyroptosis drug CLNDN in Example 1.

[0038] Figure 6 The uptake of the nano-pyroptosis drug CLNDN by CT26 tumor cells at different time points was shown, with the freeCe6 group serving as a positive control. Ce6 The LNDN group consisted of Ce6-labeled chlordamine prodrug nanoparticles without CD47 targeting activity, with the drug added to them. Ce6 The CLNDN group consists of Ce6-labeled chlordamine prodrug nanoparticles with CD47-targeting activity. Ce6 The CLNDN+anti-CD4 group consists of a drug-addition group containing chlordamine prodrug nanoparticles with CD47 targeting activity, which are pre-incubated with anti-CD47 antibody and then labeled with Ce6.

[0039] Figure 7 This describes the accumulation of the nano-pyroptosis drug CLNDN in tumor tissue.

[0040] Figure 8To observe the pyroptosis induced by the nano-pyroptosis drug CLNDN using confocal microscopy, PBS was used as the control group, Free LND was chlordamine monotherapy, LNDN was chlordamine prodrug nanoparticles without CD47 targeting, and CLNDN group was chlordamine prodrug nanoparticles with CD47 targeting.

[0041] Figure 9 To detect the expression of pyroptosis-related proteins after CLNDN treatment using Western blotting. PBS served as the control group, LNDN consisted of chlordamine prodrug nanoparticles without CD47 targeting, and the CLNDN group consisted of chlordamine prodrug nanoparticles with CD47 targeting.

[0042] Figure 10 This is a tumor growth curve in an experiment where the nano-pyroptosis drug CLNDN inhibited tumors in Balb / c mice bearing CT26 colon cancer cells.

[0043] Figure 11 This is a graph showing the change in body weight of Balb / c mice during the experiment of inhibiting tumors of CT26 colon cancer cells in Balb / c mice using the nano-pyroptosis drug CLNDN in Example 1.

[0044] Figure 12 This is a particle size distribution diagram obtained by dynamic light scattering of the nano-pyroptosis drug CLNDN in water in Example 2.

[0045] Figure 13 Example 3 illustrates the detection of pyroptosis-related protein expression after CBPN treatment using Western blotting. PBS served as the control group, BPN consisted of 3-bromopyruvate prodrug nanoparticles without CD47 targeting activity, and the CBPN group consisted of 3-bromopyruvate prodrug nanoparticles with CD47 targeting activity. Detailed Implementation

[0046] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0047] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0048] DSPE-PEG2000, Molecular Formula: C 45 H 87 NNaO 37 P, molecular weight: 2765.12, CAS number: 147867-65-0, structural formula as follows:

[0049]

[0050] The amino acid sequence of the CD47 targeting peptide (4N1K polypeptide) is shown in SEQ ID NO.1, specifically: Lys-Arg-Phe-Tyr-Val-Val-Met-Try-Lys-Lys.

[0051] DSPE-PEG2000-MAL and thiol-capped 4N1K peptide were purchased from Shanghai Aivito Co., Ltd.; chlordamine and 3-bromopyruvic acid were purchased from Shanghai Anaiji Chemical Reagent Co., Ltd.; CD47 antibody was purchased from BioXcell; and CT26 colon cancer cells were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0052] Example 1

[0053] 1. Preparation of an amphiphilic polymer (DSPE-PEG2000-4N1K) containing CD47 targeting peptide

[0054] DSPE-PEG2000-MAL (288 mg, 0.1 mmol) was dissolved in 1 mL of DMF solution, then added to 2 mL of PBS solution (1×, pH 7.4) containing 147 mg, 0.12 mmol of thiol-terminated 4N1K peptide. The mixture was vortexed until homogeneous and reacted at 4 °C for 24 hours. After the reaction, the sample was dialyzed three times with 1 L of methanol, with a molecular weight cutoff of 1 kDa in the dialysis bag. The reaction procedure is as follows:

[0055]

[0056] Polymers are characterized by nuclear magnetic resonance, such as Figure 1 As shown, the proton NMR spectrum of the obtained polymer differs from that of either of the two raw materials. Specifically, the H peaks between 8.4 and 6.0 are the H peaks of the aromatic rings on the polypeptide and the -NH- peak on DSPE-PEG2000, while the peaks between 5.0 and 0 are the H peaks of the fatty chains on the polypeptide and DSPE-PEG2000. This indicates that an amphiphilic polymer containing a CD47-targeting peptide was successfully prepared.

[0057] 2. Preparation of nano-pyroptosis drug CLNDN

[0058] (1) Preparation of chlordamine prodrug containing disulfide bonds:

[0059] Clonidamine (64.88 mg, 0.2 mmol), 4-dimethylaminopyridine (DMAP, 0.23 mg, 0.01 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 57.5 mg, 0.3 mmol) were dissolved in 5 mL of anhydrous N,N-dimethylformamide (DMF) solution. The mixture was stirred at room temperature for 1 h. Then, 1 mL of 2,2'-dithiodiethanol in DMF solution was added to the above system, and the reaction was continued for 12 h. After the reaction was complete, the mixture was extracted with dichloromethane / water solution, and the organic solvent was evaporated and purified by column chromatography to obtain the clodinidamine prodrug in a yield of 67%. The reaction process is as follows:

[0060]

[0061] The above products were characterized by nuclear magnetic resonance, such as Figure 2 As shown, specifically, 1 ¹H NMR (400MHz, CDCl₃): δ 8.25–8.23 (d, J = 8 Hz, 2H), 7.42–7.31 (m, 8H), 7.08–7.06 (d, J = 8 Hz, 2H), 6.69–6.67 (d, J = 8 Hz, 2H), 5.76 (s, 4H), 4.77–4.74 (t, J = 12 Hz, 4H), 3.22–3.19 (d, J = 12 Hz, 4H). This demonstrates the successful preparation of the chlordamine prodrug.

[0062] (2) Nanoparticle pyroptosis drug CLNDN was prepared via thin-film-hydration method:

[0063] DSPE-PEG2000-4N1K (9 mg), DSPE-PEG2000 (16 mg), and chlordamine prodrug (5 mg) were dissolved in 3 mL of a mixture of dichloromethane and methanol (volume ratio 2:1) and placed in a 25 mL flask. The solvent was evaporated to form a thin film at the bottom of the flask, and then 10 mL of water was added. The mixture was sonicated in a water bath for 5-10 minutes and concentrated by ultrafiltration to finally obtain a nano-pyroptosis drug micelle (CLNDN) solution.

[0064] The particle size of the above products was determined by dynamic light scattering, such as... Figure 3 As shown, the nanoparticles prepared by the thin-film-hydration method have a particle size of approximately 100 nm. The polydispersity index (PDI) is 0.15. Furthermore, transmission electron microscopy revealed that the nanoparticles are spherical particles with a particle size of approximately 100 nm. Figure 4 ).

[0065] When used in vivo, an appropriate particle size can promote a longer circulation time of nanoparticles in the blood and facilitate their accumulation within tumors. The nanoparticles prepared in this embodiment have a particle size of approximately 100 nm, which can prevent premature renal clearance and also prevent the clearance of large particles by the liver's cellular clearance system, making them suitable for in vivo application.

[0066] 3. In vitro release of CLNDN nanomedicines

[0067] Reduction-responsive release capability is a very important part of evaluating the in vivo application of CLNDN. Good reduction-responsive release capability can ensure the full activation of pyroptosis drugs and the subsequent generation of pyroptosis toxicity.

[0068] CLNDN nanoparticles (2 mL, 1 mg / mL chlordamine) were sealed in a dialysis bag with a molecular weight cutoff of 3500 Da and incubated at different pH values ​​in 50 mL of PBS containing 1% Tween 80, both with and without 5 mM dithiothreitol (DTT). 500 μL of dialysate was collected from outside the dialysis bag at regular intervals, and the concentration of chlordamine was determined by HPLC.

[0069] like Figure 5 As shown, in 5 mM DTT (pH 6.5), after 48 hours, approximately 83.9% of the chlordamine prodrug encapsulated in the nanoparticles was reduced to chlordamine and released. In contrast, in an environment without DTT (pH 7.4), only 14.2% was released.

[0070] 4. Targeted delivery of CLNDN in vitro and in vivo

[0071] (1) In vitro targeting effect of CLNDN: The targeting effect of CLNDN containing CD47 targeting group, LNDN without CD47 targeting group (prepared by DSPE-PEG2000 and chlordamine prodrug via thin film-hydration method) and CLNDN+CD47 antibody was evaluated by detecting the endocytosis content of tumor cells.

[0072] CT26 colon cancer cells were seeded at a density of 50,000 cells per well in 12-well plates and incubated overnight. Cells were then exposed to various porphyrin (Ce6)-labeled drugs for different durations, and the endocytosis of CT26 colon cancer cells in response to different drug treatments was detected by flow cytometry.

[0073] like Figure 6As shown, tumor cells take up significantly more CLNDN than LNDN, and the increased uptake by tumor cells caused by CD47 targeting in CLNDN can be blocked by CD47 antibody, indicating that CLNDN has a CD47-dependent drug delivery effect on CT26 colon cancer cells.

[0074] (2) Targeted delivery of CLNDN in vivo: The content of CLNDN in small animal tumor tissue was detected to evaluate the targeted delivery of CLNDN containing CD47 targeting group and CLNDN without CD47 targeting group.

[0075] 500,000 CT26 colon cancer cells were subcutaneously injected into Balb / C mice until the tumors reached 60 mm. 3 Next, Ce6-labeled CLNDN and LNDN nanoparticles were injected via the tail vein (Ce6 concentration of 0.5 mg / mL). The accumulation of nanoparticles in tumor tissue was then assessed by detecting the fluorescence content in the tumors of each group of mice using a small animal in vivo fluorescence spectrometer.

[0076] like Figure 7 As shown, CLNDN exhibits significantly stronger fluorescence in tumor tissue than LNDN, indicating that CLNDN also has a CD47-dependent drug delivery effect in CT26 colon cancer tumor tissue.

[0077] 5. CLNDN induces pyroptosis in tumor cells.

[0078] (1) Observation of cell morphology: Cells were seeded at a density of 50,000 cells per well in confocal culture dishes and incubated overnight. Cells were exposed to various drug treatment groups and incubated for 2 hours. Then, the morphological changes of CT26 colon cancer cells were observed and photographed using a fluorescence confocal microscope.

[0079] like Figure 8 As shown, tumor cells incubated with both CLNDN and LNDN exhibited the "bubbling" phenomenon, with the CLNDN group showing a more pronounced effect than the LNDN group. This preliminarily suggests that CLNDN can induce pyroptosis in CT26 colon cancer cells.

[0080] (2) The N-terminal cleavage effect of CLNDN on GSDME protein:

[0081] Cells were seeded at a density of 50,000 cells per well in 12-well plates and incubated overnight. Different groups of drugs were added to the wells. After 2 hours of incubation, the cells were lysed with lysis buffer, and then Western blotting was used to detect the N-terminal cleavage of GSDME protein and the activation of other pyroptosis-related proteins (caspase 3).

[0082] like Figure 9As shown, GSDME protein underwent significant N-terminal cleavage in tumor cells treated with CLNDN and LNDN, and Caspase3 was significantly activated. The phenomenon was more pronounced in the CLNDN group than in the LNDN group, further demonstrating that CLNDN can induce pyroptosis in CT26 colon cancer cells.

[0083] 6. Tumor-suppressing effect of CLNDN in the CT26 tumor model of Balb / C mice

[0084] Balb / C mice were subcutaneously injected with 50 × 10 4 One CT26 cell. The tumor volume reached 60 mm. 3 At approximately 10:00 AM, mice were randomly assigned to three treatment groups (n=5): PBS, LNDN, and CLNDN. The equivalent dose of chlordamine was 10 mg / kg. The drug was administered via tail vein injection every two days for a total of four doses. Tumor volume (mm²) was calculated using the formula. 3 Tumor volume = (shortest diameter) 2 ×(longest diameter)×0.5.

[0085] The tumor suppression was evaluated in a Balb / C mouse CT26 subcutaneous tumor model. Figure 10 As shown, CLNDN exhibited a more significant tumor-suppressive effect compared to the PBS and LNDN groups. Meanwhile, the body weight of mice in the CLNDN group did not change significantly. Figure 11 ).

[0086] Example 2

[0087] Preparation of nano-pyroptosis drug CLNDN by co-precipitation method:

[0088] The DSPE-PEG2000-4N1K (9 mg) and DSPE-PEG2000 (16 mg) prepared in Example 1 were dissolved in 200 μL of methanol solution as solution A. The chlordamine prodrug (5 mg) prepared in Example 1 was dissolved in 50 μL of DMSO solution as solution B. Then, solutions A and B were simultaneously added dropwise to 10 mL of vigorously stirred aqueous solution. After the addition was completed, stirring was continued for half an hour. Then, ultrafiltration and centrifugation were performed to obtain nano-pyroptosis drug micelle solution.

[0089] The particle size of the above products was determined by dynamic light scattering, such as... Figure 12 As shown, the nanoparticles prepared by the co-precipitation method have a particle size of approximately 102 nm. The polydispersity index (PDI) is 0.16. This particle size is suitable for in vivo applications.

[0090] Example 3

[0091] I. Preparation of Nano-Pyroplasmic Drug CBPN

[0092] (1) Preparation of 3-bromopyruvic acid prodrug containing disulfide bonds:

[0093] 3-Bromopyruvic acid (33.39 mg, 0.2 mmol), 4-dimethylaminopyridine (DMAP, 0.23 mg, 0.01 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 57.5 mg, 0.3 mmol) were weighed and dissolved in 5 mL of anhydrous N,N-dimethylformamide (DMF) solution. After stirring at room temperature for 1 h, 1 mL of 2,2'-dithiodiethanol in DMF solution was added to the above system, and the reaction was continued for 12 h. After the reaction was complete, the solution was extracted with dichloromethane / water solution, the organic solvent was evaporated, and the solution was purified by column chromatography to obtain chlordamine prodrug in 60% yield. The reaction process is as follows:

[0094]

[0095] (2) Nanoparticle pyroptosis drug CBPN was prepared via thin-film-hydration method:

[0096] DSPE-PEG2000-4N1K (9 mg), DSPE-PEG2000 (16 mg), and 3-bromopyruvic acid prodrug (5 mg) were dissolved in 3 mL of a mixture of dichloromethane and methanol (volume ratio 2:1) and placed in a 25 mL flask. The solvent was evaporated to form a thin film at the bottom of the flask, and then 10 mL of water was added. The mixture was sonicated in a water bath for 5-10 minutes and concentrated by ultrafiltration to finally obtain nano-pyroptosis drug micelle solution (CBPN).

[0097] II. CBPN induces pyroptosis in tumor cells

[0098] CBPN's effect on the N-terminal cleavage of GSDME protein: CT26 colon cancer cells were seeded at a density of 50,000 cells per well in 12-well plates and incubated overnight. Different groups of the drug were added to the wells. After 2 hours of incubation, the cells were lysed with lysis buffer, and then Western blotting was used to detect the N-terminal cleavage of GSDME protein and the activation of other pyroptosis-related proteins (caspase 3).

[0099] The results are as follows Figure 13 As shown, GSDME protein underwent significant N-terminal cleavage in tumor cells treated with CBPN, and Caspase3 was significantly activated, indicating that CBPN can induce pyroptosis in CT26 colon cancer cells.

Claims

1. A nanopyroptotic drug targeting CD47 protein, characterized in that, The drug is a micellar nanoparticle formed by self-assembly of an amphiphilic carrier material and a pyroptosis prodrug, the amphiphilic carrier material is composed of an amphiphilic polymer modified with a CD47-targeting peptide at a hydrophilic end and an amphiphilic polymer without modification, and the pyroptosis prodrug is a prodrug formed by a reduction-responsive chemical bond connecting of lonidamide or 3-bromopyruvic acid; The structural formula of the amphiphilic polymer modified with a CD47-targeting peptide at a hydrophilic end is shown as formula (I), 2. The nanopyroptotic drug targeting CD47 protein of claim 1, wherein, The mass ratio of the amphiphilic polymer modified with a CD47-targeting peptide at a hydrophilic end to the amphiphilic polymer without modification in the amphiphilic carrier material is 9-15:16-25.

3. The nanopyroptotic drug targeting CD47 protein of claim 1, wherein, The structural formula of the lonidamide prodrug is shown as formula (II), and the structural formula of the 3-bromopyruvic acid prodrug is shown as formula (III), 4. The nanopyroptotic drug targeting CD47 protein of claim 1 or 3, wherein, The mass ratio of the amphiphilic carrier material to the pyroptosis prodrug is 25-40:1-6.

5. The method of claim 1-4, wherein the method of preparing a nanocytorrhexis drug targeting CD47 protein is characterized in that, The micellar nanoparticle formed by self-assembly of the amphiphilic carrier material and the pyroptosis prodrug in water is prepared by using a solvent replacement method, a dialysis method, an ultrasonic method or a liquid membrane method.

6. The use of the nanopyroptotic drug targeting CD47 protein according to any one of claims 1-4 in the preparation of an antitumor drug, characterized in that, The drug induces pyroptosis of tumor cells, and the tumor is colon cancer or breast cancer.

Images