A polymer with pyroptosis execution protein function and preparation method and application thereof

Abstract

The application relates to the technical field of medicines, and discloses a polymer with pyroptosis execution protein function and a preparation method and application thereof. The polymer has the structure shown in the description, wherein R1 is as shown in the description, R2=-CO(CH2) n CH3.In the application, EPL is taken as a skeleton, a structural unit capable of specifically combining with an anionic lipid is grafted, and a fatty chain is obtained, the polymer can quickly break the inner side of a cell membrane, induce cell blebbing, release inflammatory factors, play a similar GSDM function, make cells die in a similar pyroptosis mode, exhibit excellent anti-tumor effects, and can combine with an anionic lipid cardiolipin on the outer side of a bacterial membrane, so that the bacterial membrane is broken, and the bacteria can be killed in a short time, and excellent antibacterial treatment effects are achieved. The polymer has strong scientific research value and good clinical transformation prospects.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a polymer with pyroptosis-executing protein function, its preparation method, and its application. Background Technology

[0002] Pyroptosis is an immunogenic cell death process that can transform cold tumors into hot tumors, and therefore holds great promise in tumor immunotherapy. However, its occurrence depends on the participation of gasdermins. When activated, gasdermins (GSDMs) family proteins are cleaved into the active ingredient N-Gasdermins (N-GSDMs), which can bind to anionic lipid phospholipids (mainly phosphatidylserine (PS)) on the inner side of the cell membrane through electrostatic interactions. This leads to oligomerization on the cell membrane, creating pores and causing changes in osmotic pressure between the inside and outside of the cell. This induces cell vacuole and releases inflammatory factors, resulting in the cell exhibiting a pyroptotic phenotype (The gasdermins, a protein family executing cell death and inflammation. Nat Rev Immunol 2020, 20(3), 143.). Therefore, the expression of pyroptosis executive proteins is a prerequisite for pyroptosis. Existing technologies also include compounds that promote the expression of pyroptosis executive proteins. For example, CN 116514738 A discloses a pyroptosis prodrug that can target lysosomes. Under light, the reactive oxygen species generated can activate Caspase-1 to cleave GSDMD protein, create pores in the cell membrane, and induce pyroptosis.

[0003] Unfortunately, tumor cells can evade this death mechanism by selectively silencing the expression of GSDMs-related proteins, thus placing the tumor in an immunosuppressive microenvironment, which greatly limits the application of pyroptosis strategies in tumor immunotherapy (Gasdermin E suppresses tumor growth by activating anti-tumourimmunity. Nature 2020, 579(7799), 415.).

[0004] Activated pyroptosis-executing proteins (N-GSDMs) can bind not only to the intracellular anionic lipid PS, but also directly to the extracellular cardiolipin, directly creating pores in the bacterial membrane to eliminate bacteria and prevent infection. However, abnormal binding of GSDM proteins in vivo can also lead to various problems such as inflammatory storms and thrombosis (Channelling inflammation: gasdermins in physiology and disease. Nat Rev Drug Discov 2021, 20(5), 384.).

[0005] Therefore, it is desirable to design a polymer that possesses a similar ability to bind to anionic lipids as activated N-GSDMs, binding to PS on the inner side of the cell membrane even in tumor cells that do not express GSDMs. This would cause the tumor cell membrane to rupture and vesicle, releasing its contents and inducing pyroptosis-like immunogenic cell death to activate the immune system. Simultaneously, this polymer could also bind to cardiolipin on bacterial membranes, causing membrane rupture and killing bacteria. Obtaining a pyroptosis-like polymer with both anti-tumor and antibacterial effects would be of great significance for research in tumor immunotherapy. Summary of the Invention

[0006] This invention addresses the problem of pyroptosis being dependent on the expression of GSDM family proteins by providing a GSDM analog that can be rapidly internalized by cells and bind to PS on the inner side of the cell membrane, inducing cell rupture, vesicle formation, and the release of inflammatory factors. This results in a pyroptosis-like phenotype in cells that do not express GSDM family proteins, and can serve as a substitute for GSDM proteins for the study of cell death behavior.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A polymer with pyroptosis-executing protein function has the following structure:

[0009]

[0010] in, R2=-CO(CH2) n CH3; X=2~15, y=10~30, Z=0~10, n=1~18.

[0011] Inspired by the membrane-breaking mechanism of Gasdermin protein, this invention designs a polymer with highly efficient binding ability to acidic phospholipids (phosphatidylserine, PS) on the inner side of the cell membrane. Using commercially available ε-polylysine (EPL) as the backbone, structural units that specifically bind to anionic lipids are grafted into its structure. Utilizing the multivalent covalent effect of the polymer and the DPA-Zn group (R1) in the structure, this polymer can be rapidly integrated into the cell and bind to PS on the inner side of the cell membrane, disrupting the cell membrane potential, causing membrane rupture on the inner side of the cell membrane, inducing cell vesicle extrusion, and releasing inflammatory factors. Thus, in cells that do not express GSDMs family proteins, it functions similarly to pyroptosis-executing proteins GSDMs, causing cell death through a pyroptosis-like process. Therefore, this polymer can serve as a substitute for GSDMs proteins for the study of cell death behavior.

[0012] On the other hand, because this polymer can induce immunogenic cell death, it can effectively activate tumor immunity, exhibiting excellent anti-tumor effects and showing great promise for application in tumor immunotherapy. Furthermore, this polymer can also bind to the anionic lipid cardiolipin on the outer surface of bacterial membranes, thereby rupturing the bacterial membrane and killing bacteria in a short time, demonstrating excellent antibacterial therapeutic effects.

[0013] When Z = 0, the polymer structure is as follows, named PDPA-Zn:

[0014]

[0015] When Z is not 0, the polymer structure is as follows, named DPDPA-Zn:

[0016]

[0017] This invention introduces aliphatic chains into the aforementioned polymer backbone, meaning Z is not 0, which increases the polymer's ability to interact with cell membranes and can significantly shorten the material's membrane rupture time. Therefore, Z not being 0 is more preferable.

[0018] Preferably, X = 2-10, Y = 10-30, Z = 2-10, and n = 8-16.

[0019] When X < 2, the number of DPA grafted units in the polymer is small, and the binding ability with PS is weak. Due to spatial limitations, the number of X modifications can only be up to about 10. Therefore, the preferred range of X is 2-10.

[0020] There is also a balance to be struck regarding the number of Z modifications. Too few modifications result in weak interactions with the cell membrane, while too many modifications make the polymer structure hydrophobic and insoluble in water. Therefore, as a preferred option, the range of Z in this patent is limited to 2-10.

[0021] The remaining amount of free -NH2, i.e., the number of Y atoms, is determined by the number of X and Z atoms. Preferably, in this patent, the range is defined as Y = 10-30.

[0022] More preferably, n = 8-12, and even more preferably n = 10.

[0023] The present invention also provides a method for preparing the polymer having the pyroptosis-executing protein function, comprising the steps of:

[0024] Step 1: Mix α,α′-dichloro-p-xylene (Dx) and dimethylpyridinium (DPA) and react them under the action of a catalyst to obtain DX-DPA monomer;

[0025] Step 2: Dissolve ε-polylysine (EPL) in a solvent, add the DX-DPA monomer, and polymerize under the action of a catalyst to obtain polymer PDPA;

[0026] When Z=0, in step 3, the PDPA and zinc salt are dissolved in a solvent, stirred and mixed, the solvent is removed, water is added for dialyzing, and the polymer PDPA-Zn with pyroptosis-executing protein function is obtained by freeze drying.

[0027] The reaction formula is as follows:

[0028]

[0029] When Z is not 0, step 3 is replaced by the following steps:

[0030] Step 3-1: Dissolve the PDPA in an inorganic alkaline solution, and add ClCO(CH2) dropwise under ice bath conditions. n CH3, after the reaction is complete, dialyze and freeze-dry to obtain the polymer DPDPA containing aliphatic chains; n=1-18;

[0031] Step 3-2: Dissolve DPDPA and zinc salt in a solvent, stir and mix, remove the solvent, dialyze with water, and freeze dry to obtain the polymer DPDPA-Zn with pyroptosis-executing protein function.

[0032] The reaction formula is as follows:

[0033]

[0034] In step 1, the molar ratio of α,α′-dichloro-p-xylene and dimethylpyridinium is 2-2.5:1; to ensure that only one benzyl chloride in α,α′-dichloro-p-xylene reacts, the molar equivalent of α,α′-dichloro-p-xylene and dimethylpyridinium is kept greater than 2 during the reaction.

[0035] The catalyst mentioned in step 1 includes any one or more of potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, and potassium hydroxide.

[0036] The solvent used in step 1 includes any one or more of dichloromethane, trichloromethane, tetrahydrofuran, N,N-dimethylpyridinium chloride, and dimethyl sulfoxide.

[0037] The reaction time for step 1 is 24-72 hours, and the reaction temperature is room temperature.

[0038] The catalyst in step 2 includes any one or more of potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, and potassium hydroxide.

[0039] In steps 2, 3, and 3-1, the solvents independently include one or more of methanol, ethanol, and water;

[0040] The reaction conditions for step 2 are 40-60℃ for 24-72 hours;

[0041] In step 2, the molar ratio of amino groups to DX-DPA monomers in ε-polylysine is 1:1 to 1:10.

[0042] In step 3, the amount of zinc salt used is 1-5 equivalents of the DPA monomer grafted onto PDPA; the reaction conditions for step 3 are 2-4 hours at room temperature.

[0043] In step 3-1, the free amino groups in PDPA react with ClCO(CH2). n The molar ratio of CH3 is 1:1-1:10, and the reaction conditions after the addition are 2-4 hours at room temperature.

[0044] In step 3-1, the inorganic base includes one or more of sodium hydroxide, sodium bicarbonate, sodium carbonate, and potassium carbonate;

[0045] In step 3-2, the amount of zinc salt used is 1-5 equivalents of the DPA monomer grafted onto DPAPA; the reaction conditions for step 3-2 are 2-4 hours at room temperature.

[0046] The zinc salt includes one or more of zinc nitrate, zinc sulfate, and zinc chloride.

[0047] During dialysis, dialysis bags are used to remove excess Zn with a molecular weight cutoff range of 1K-5K. 2+ .

[0048] The present invention also provides the use of the polymer having pyroptosis executive protein function in the preparation of a pyroptosis induction kit, wherein the generation of pyroptosis is independent of the pyroptosis executive protein.

[0049] The present invention also provides a pyroptosis induction kit, comprising the polymer having the function of the pyroptosis executive protein.

[0050] The present invention also provides the use of the polymer having pyroptosis-executing protein function in the preparation of antitumor drugs.

[0051] The present invention also provides the use of the polymer having pyroptosis-executing protein function in the preparation of antibacterial drugs.

[0052] The present invention also provides an antibacterial or antitumor drug, the active ingredient of which includes the polymer having the function of pyroptosis-executing protein.

[0053] Compared with the prior art, the present invention has the following beneficial effects:

[0054] (1) This invention relates to the synthesis of a polymer that can bind to phosphatidylserine on the inner side of the cell membrane. The polymer can be rapidly integrated into the cell, disrupting the cell membrane potential, thereby causing the cell membrane to rupture on the inner side, inducing cell vesicle extrusion, and releasing inflammatory factors, thereby exerting a function similar to pyroptosis-executing proteins GSDMs, causing the cell to die in a pyroptosis-like manner, and exhibiting excellent anti-tumor effects.

[0055] (2) The polymer synthesized in this invention has the ability to efficiently break cell membranes, induce cell vesicles and release inflammatory factors, thereby making the cells exhibit a pyroptosis-like phenotype. Moreover, this phenomenon does not depend on the participation of intracellular pyroptosis executive proteins GSDMs, which can overcome the inherent defect that tumor cells or bacteria do not express pyroptosis executive proteins and therefore cannot undergo pyroptosis. It can be used for the study of the mechanism of pyroptosis death pathway, such as preparing a kit for pyroptosis detection.

[0056] (3) The polymer synthesized in this invention can bind to the anionic lipid cardiolipin on the outer side of the bacterial membrane, thereby causing the bacterial membrane to rupture and killing the bacteria in a short time, thus having excellent antibacterial therapeutic effect.

[0057] (4) Pyroptosis-executing proteins require a complex intracellular cleavage activation process. Therefore, even in cell lines expressing pyroptosis-executing proteins, pyroptosis takes several hours to occur and the incidence rate is low. The polymer designed and synthesized in this invention can rapidly enter tumor cells without relying on the activation of upstream signaling pathways. Therefore, it can induce cell or bacterial membrane rupture within minutes, achieving the effect of killing tumor cells or bacteria.

[0058] Therefore, the cell membrane rupture polymer designed and synthesized in this patent has not only strong scientific research value, such as for studying cell membrane rupture and bubbling behavior, but also has good clinical translation prospects and significant application value. Attached Figure Description

[0059] Figure 1 The NMR spectra of DX-DPA, raw material EPL, and polymers PDPA-Zn and DPDPA-Zn synthesized in Example 1 are shown.

[0060] Figure 2 In Example 1(D), PDPA-Zn releases Tb from liposomes by binding to PS in liposomes. 3+ The release curve.

[0061] Figure 3 To observe the film-breaking ability of DPDPA-Zn using transmission electron microscopy in Example 2.

[0062] Figure 4 This is a scanning electron microscope image of the permeabilization of gastric cancer cells using Example 3(D) PDPA-Zn.

[0063] Figure 5 This is an optical microscope image of cells induced to bleb out using Example 4(D) PDPA-Zn.

[0064] Figure 6 The curve of intracellular inflammatory factor release induced by PDPA-Zn in Example 5(D) is shown.

[0065] Figure 7 The tumor inhibition curve of PDPA-Zn in B16F10 subcutaneous tumor is shown in Example 6(D).

[0066] Figure 8 The tumor inhibition curve of PDPA-Zn applied in Example 6(D) is shown in the subcutaneous tumor of 4T1 breast cancer.

[0067] Figure 9 This is a scanning electron microscope image of the cell membrane disruption of bacteria using Example 7(D) PDPA-Zn. Detailed Implementation

[0068] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Modifications or equivalent substitutions made by those skilled in the art based on their understanding of the technical solutions of this invention, without departing from the spirit and scope of the invention, should be covered within the protection scope of this invention.

[0069] Unless otherwise specified, all raw materials used in the following specific embodiments were purchased from the market. The chemical raw materials used in the examples were derived from Adamas-beta reagents, other organic reagents were purchased from Sinopharm Chemical Co., Ltd. or Maclean's, the cell lines used were all from the ATCC cell bank, and other raw materials not specified were purchased from the market.

[0070] Example 1: Synthesis of polymers PDPA-Zn and DPDPA-Zn

[0071] 1) Synthesis of DX-DPA

[0072]

[0073] Dx (8.8 g, 50.4 mmol) and DPA (5.0 g, 25.2 mmol) were dissolved sequentially in 20 mL of dichloromethane. Anhydrous K₂CO₃ (5.52 g, 40 mmol) was added to the mixture, and the mixture was stirred at room temperature for 48 h under a nitrogen atmosphere. The reaction of DPA was monitored by TLC to determine if it had reacted completely. Subsequently, the reaction mixture was concentrated by rotary evaporation, and the resulting residue was separated by silica gel column chromatography:methanol (20:1, v / v) to obtain a pale yellow oily Dx-DPA (5.1 g, 60%). 1 H-NMR (400MHz, CDCl3): δppm: 1 H NMR(400MHz, CDCl3)δ8.63–8.35(m,wH),7.62(tdd,4H),7.41(d,2H),7.32–7.20(m, 2H),7.16–6.94(m,2H),4.50(d,J=19.8Hz,1H),3.82(s,2H),3.71(d,J=13.4Hz,1H).

[0074] 2) Synthesis of PDPA

[0075] The resulting Dx-DPA (2.0 g, 6 mmol) and EPL (2.5 g, 0.625 mmol) had the following NMR spectra: Figure 1 The product was dissolved in 50 mL of anhydrous methanol. Anhydrous K₂CO₃ (2.5 g, 18 mmol) was added to the mixture, and the mixture was stirred at 50 °C for 48 h. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The product was then reprecipitated in cold ethanol (100 mL × 5) to give a yellow solid PDPA (60%), as shown in the following reaction formula:

[0076]

[0077] The NMR spectrum of the product PDPA is as follows: Figure 1 As shown. The grafting rate of DPA is based on... 1 H-NMR calculations showed a grafting rate of 4.6, i.e., x = 4.6.

[0078] c) Synthesis of DPDPA

[0079] The obtained PDPA (250 mg) was dissolved in 10 mL of NaOH solution (1 N), and lauroyl chloride (20 μL) was added dropwise to the solution cooled to 0 °C. After stirring overnight at room temperature, the mixture was dialyzed against deionized water (1 L × 4) (MWCO, 3.5 kDa) and lyophilized to obtain a pale yellow powder DPDPA (200 mg). The reaction formula is as follows:

[0080]

[0081] polymer DPDPA 1 H-NMR NMR spectrum as follows Figure 1 As shown, the dodecyl content is calculated to be 5.9, i.e., z = 5.9.

[0082] d) Synthesis of PDPA-Zn and DPDPA-Zn

[0083] The synthesized PDPA and DPDPA were dissolved in methanol and then mixed with Zn in a molar ratio of 1:1. 2+ DPA (grafted units of DPA) in a 2:1 ratio was mixed with Zn(NO3)2·6H2O. The mixture was stirred at room temperature for 3 hours. The solvent was removed by rotary evaporation to obtain the crude product. The crude product was dialyzed against deionized water (1 L × 3) (MWCO, 3.5 kDa) to remove excess Zn. 2+ Freeze-drying yielded purified PDPA-Zn and DPDPA-Zn yellow powders.

[0084] The polymers PDPA-Zn and DPDPA-Zn prepared in Example 1 were used in application tests to verify their effects, with blank samples or EPL as controls, as follows:

[0085] Application Example 1Tb 3+ Release experiment

[0086] Membrane perforation capacity was assessed using a standard liposome leakage assay (Nature 2016, 535(7610), 153). Tb-embedded lipids were prepared by hydrating specific lipids (2.5 μmol myristoyl phosphatidylcholine (DMPC) or myristoyl phosphatidylcholine / phospholipid (DMPC / DSPS) = 4:1) in a preparation buffer containing 20 mM sodium citrate and 15 mM TbCl3 (10 mM 4-hydroxyethylpiperazine ethanesulfonic acid, pH = 7.4, 150 mM NaCl). 3+ Liposomes were obtained by passing the suspension through a 200 nm polycarbonate membrane 21 times. Free TbCl3 was removed by dialysis and dextran G-25 desalting column. Subsequently, Tb-containing liposomes were... 3+Liposomes (lipid concentration: 50 μM) were suspended in 5 mL of HEPES buffer containing 50 μM pyridine-2,6-dicarboxylic acid (PDA) and treated with 10 μg / mL polymer. The fluorescence spectrum excited at 276 nm was recorded using a SepectraMax iD5 microplate reader.

[0087] The degree of liposome leakage is calculated using the formula: t(%) = 100 × (F t -F t0 ) / (F t100 -F t0 ), where F t0 Tb when adding polymer 3+ The initial fluorescence signal of liposomes in PDA-containing buffer, Ft represents the fluorescence signal recorded at each time point, F t100 To add 0.1% Triton X-100 (a commonly used nonionic surfactant and emulsifier) ​​as Tb 3+ Positive control with complete release.

[0088] The results are as follows Figure 2 As shown, DPDPA-Zn exhibited the strongest membrane-breaking effect, causing approximately 80% of liposome membranes to rupture within 2 hours, while EPL and PDPA-Zn showed relatively weaker membrane-breaking abilities. Furthermore, the membrane-breaking ability of the polymers depended on the PS content in the liposomes; none of the three polymers could lyse DMPC liposomes without PS.

[0089] Application Example 2: Electron microscopy observation of the ability of DPDPA-Zn to permeate PS-containing liposomes

[0090] Prepared DMPC or DMPC / DSPS = 4:1 liposomes (liposome concentration: 50 μM) were pretreated with polymer (10 μg / mL) for 15 min. Subsequently, the liposome-polymer suspension negatively stained with diluted uranyl acetate (20 μL) was loaded onto a 150-mesh carbon copper grid, air-dried for 1 min, and then blotted dry with clean filter paper to form a thin layer on the grid surface. Images were recorded using a transmission electron microscope (Thermo Scientific Talos L120C) at 120 kV.

[0091] The results are as follows Figure 3 As shown, DPDPA-Zn exhibits a significant membrane-breaking effect for DMPC / DSPS liposomes; however, DPDPA-Zn has no membrane-breaking effect for DMPC liposomes, further demonstrating that the membrane-breaking ability of the polymer depends on the binding of PS.

[0092] Example 3: SEM observation of the membrane-breaking ability of (D)PDPA-Zn;

[0093] AGS or HeLa cells were seeded in 12-well plates at a density of 3 × 10⁶ cells / well. 4 Cells / well, incubated for 24 h. The culture medium was replaced with 1 mL of fresh, FBS-free medium and treated with the polymer prepared in this example (50 μg / mL) for 1 h or 4 h. Untreated cells served as controls. Cells were collected after treatment and washed three times with PBS. The resulting cells were fixed overnight at 4°C with glutaraldehyde solution (2.5% in PBS). After removing the fixative, the cells were washed three times with PBS and dehydrated with a series of ethanol solutions of varying concentrations (50, 60, 70, 80, 90, 95, 100%). The sample was resuspended in 50 μl of 100% ethanol, and the ethanol-containing sample was carefully added dropwise onto a gold-plated slide and air-dried for 12 h, followed by SEM observation.

[0094] The results are as follows Figure 4 As shown, the cells in the blank control group and the EPL treatment group have uniformly distributed microvilli on their surface and are spherical, while the cells in the PDPA-Zn and DPDPA-Zn treatment groups have sparse microvilli on their surface and become flattened due to the loss of cell contents.

[0095] Application Example 4: Observation of (D) PDPA-Zn-induced cell bubbling ability using optical microscopy

[0096] To observe morphological changes in cells, AGS and HeLa cells were seeded in 12-well plates. After 24 hours, the cells were treated with the appropriate polymer (50 μg / mL) for 1 hour or 4 hours, and then static bright-field images of the cells were captured using an Olympus IX71.

[0097] The results are as follows Figure 5 As shown, DPDPA-Zn induces pyroptosis-like blistering in approximately 80% of cells within 15 minutes of addition, while PDPA-Zn induces blistering at a much slower rate, with only about 50% of cells blistering after 4 hours. EPL, on the other hand, does not induce pyroptosis-like blistering.

[0098] Application Example 5: Release of intracellular inflammatory factors:

[0099] AGS or HeLa cells were seeded in 12-well plates at a density of 3 × 10⁶ cells / well. 4 Cells / well, incubated for 24 h. The culture medium was replaced with 1 mL of fresh, FBS-free medium and treated with a polymer (50 μg / mL) for 1 h or 4 h. LDH release was determined using an LDH cytotoxicity assay kit (Beyotime, C0016). Interleukin-1β (IL-1β) and high-mobility group box 1 (HMGB-1) release were detected using an IL-1β and HMGB-1 enzyme-linked immunosorbent assay (ELISA) kit.

[0100] The results are as follows Figure 6 As shown, treatment with PDPA-Zn and DPDPA-Zn significantly increased the levels of HMGB-1 and IL-1β in the matrix of both the AGS and HeLa groups, with DPDPA-Zn exhibiting a more pronounced inducing effect. The levels of HMGB-1 and IL-1β in the matrix of the EPL-treated group showed no significant difference compared to the blank control group. These results indicate that DPDPA-Zn and PDPA-Zn, while inducing pyroptosis and vesicle formation, also led to the release of inflammatory factors.

[0101] Application Example 6(D) Verification of the tumor-suppressing effect of PDPA-Zn on subcutaneous tumors of 4T1 breast cancer

[0102] Female C57BL / 6 or BALB / c homozygous athymic nude mice, 6 to 8 weeks old, were subcutaneously inoculated with B16-F10 or 4T1 cells (5 × 10⁻⁶ cells) on the right ventral side. 5 (Cells / mouse). When the tumor size reaches ~100mm 3 Mice were randomly divided into 5 groups (n=6 / group). Intratumoral injections of PBS, EPL, PDPA-Zn, or DPDPA-Zn were administered at a dose of 25 mg / kg. Mice in the PD-1 antibody treatment group received an intraperitoneal injection of 0.2 mL of PD-1 antibody. Injections were administered every two days for a total of four times. Tumor length and width, as well as mouse weight, were measured after each injection. Tumor volume (V) was calculated using the formula: V(mm²) = ... 3 )=L(mm)×W 2 (mm 2 )×0.5.

[0103] The results are as follows Figure 7 , Figure 8 As shown, EPL exhibited only weak inhibitory activity against both B16-F10 and 4T1 tumor cells, while both DPDPA-Zn and PDPA-Zn showed significant inhibitory effects. After treatment ended, tumor volume in the PDPA-Zn-treated group began to increase again, while the tumor volume in the DPDPA-Zn-treated group did not rebound, indicating that DPDPA-Zn had a stronger tumor-suppressing effect. Furthermore, no significant difference was observed in mouse body weight, suggesting that both PDPA-Zn and DPDPA-Zn treatments have a certain degree of safety.

[0104] Application Example 7

[0105] (D) Antibacterial experiment of PDPA-Zn against Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA)

[0106] Escherichia coli and drug-resistant Staphylococcus aureus MRSA (10) 6The bacteria were treated with the appropriate polymers (concentrations: 0, 1, 5, 10 μg / mL) for 0.5 h. After that, the treated bacteria were evenly spread on plates and incubated for 24 h. The colony formation was then photographed and counted.

[0107] The results are as follows Figure 9 As shown, electron microscopy results indicate that both PDPA-Zn and DPDPA-Zn exhibit membrane-disrupting effects against E. coli and MRSA. Electron microscopy revealed significant morphological damage to the bacterial surfaces in the PDPA-Zn and DPDPA-Zn-treated groups, showing marked differences compared to the control group. Colony statistics showed that both PDPA-Zn and DPDPA-Zn possess potent antibacterial abilities, with significantly better antibacterial effects than EPL.

Claims

1. A polymer having pyroptosis-executing protein function, characterized in that, It has the following structure: Where, R1= R2 = -CO(CH2) n CH3; X=2~15, y = 10~30, Z = 0~10, n = 1~18.

2. The polymer with pyroptosis-executing protein function according to claim 1, characterized in that, Z is not 0.

3. The polymer with pyroptosis-executing protein function according to claim 1, characterized in that, X =2~10, Y=10~30, Z=2~10, n=8~16.

4. The method for preparing the polymer with pyroptosis-executing protein function according to any one of claims 1 to 3, characterized in that, Including the following steps: Step 1: Mix α,α′-dichloro-p-xylene and dimethylpyridinium in a solvent and react them under the action of a catalyst to obtain DX-DPA monomer; Step 2: Dissolve ε-polylysine in a solvent, add the DX-DPA monomer, and polymerize under the action of a catalyst to obtain polymer PDPA; When Z=0, in step 3, the PDPA and zinc salt are dissolved in a solvent, stirred and mixed to react, the solvent is removed, water is added for dialyzing, and freeze-drying is performed to obtain the polymer PDPA-Zn with pyroptosis-executing protein function. When Z is not 0, step 3 is replaced by the following steps: Step 3-1: Dissolve the PDPA in an inorganic alkaline solution, and add ClCO(CH2) dropwise under ice bath conditions. n CH3, after the reaction is complete, dialyze and freeze-dry to obtain the polymer DPDPA containing aliphatic chains; n=1~18; Step 3-2: Dissolve DPDPA and zinc salt in a solvent, stir and mix to react, remove the solvent, dialyze with water, and freeze dry to obtain the polymer DPDPA-Zn with pyroptosis-executing protein function; The structures of DX-DPA, PDPA, PDPA-Zn, DPDPA, and DPDPA-Zn are shown below: 。 5. The method for preparing the polymer with pyroptosis-executing protein function according to claim 4, characterized in that, In step 1, the molar ratio of α,α′-dichloro-p-xylene and dimethylpyridinium is 2-2.5:1; And / or, the catalyst in step 1 includes any one or more of potassium carbonate, sodium bicarbonate, triethylamine, sodium hydroxide, and 4-dimethylaminopyridine; And / or, the solvent used in step 1 includes any one or more of dichloromethane, trichloromethane, tetrahydrofuran, N,N-dimethylformamide, and dimethyl sulfoxide; And / or, the reaction time in step 1 is 24-72 h, and the reaction temperature is room temperature; And / or, the catalyst in step 2 includes any one or more of potassium carbonate, sodium carbonate, sodium hydroxide, sodium bicarbonate, and potassium bicarbonate; And / or, in steps 2, 3 and 3-1, the solvent independently includes one or more of methanol, ethanol and water; And / or, the reaction conditions for step 2 are 40-60 °C for 24-72 h; And / or, in step 2, the molar ratio of amino groups to DX-DPA monomers in ε-polylysine is 1:1 to 1:10; And / or, in step 3, the amount of zinc salt used is 1-5 equivalents of the DPA units grafted onto the PDPA; the reaction conditions for step 3 are 2-4 h at room temperature. And / or, in step 3-1, the amino group in PDPA reacts with ClCO(CH2). n The molar ratio of CH3 was 1:1 to 1:10, and the reaction conditions after the addition were 2-4 h at room temperature. And / or, in step 3-1, the inorganic base includes one or more of sodium hydroxide, sodium bicarbonate, sodium carbonate, and potassium carbonate; And / or, in step 3-2, the amount of zinc salt used is 1-5 equivalents of the DPA unit grafted onto DPAPA; the reaction conditions for step 3-2 are 2-4 h at room temperature; And / or, the zinc salt includes one or more of zinc nitrate, zinc sulfate, and zinc chloride.

6. The use of the polymer with pyroptosis executive protein function according to any one of claims 1-3 in the preparation of a pyroptosis induction kit, wherein the generation of pyroptosis is independent of the pyroptosis executive protein.

7. A pyroptosis induction kit, characterized in that, The polymer having pyroptosis-executing protein function as described in any one of claims 1-3.

8. The use of the polymer with pyroptosis-executing protein function according to any one of claims 1-3 in the preparation of antitumor drugs.

9. The use of the polymer with pyroptosis-executing protein function according to any one of claims 1-3 in the preparation of antibacterial drugs.

10. A drug for antibacterial or antitumor purposes, characterized in that, Its active ingredients include the polymer with pyroptosis-executing protein function as described in any one of claims 1-3.

Images