Stimuli-responsive nanocapsules for delivering protacs, methods of making and use

Abstract

This invention belongs to the field of pharmaceutical formulation technology, and relates to a stimulus-responsive nanocapsule for delivering PROTAC, its preparation method, and its application. First, this invention incorporates a tryptophan analog conjugate into the nanocapsule drug delivery system, enabling it to act as a ligand specifically targeting glioma lesions. Second, disulfide bonds are introduced into the nanocapsule to enhance its intratumoral high-glutathione response function. Furthermore, under the influence of high glutathione, the nanocapsule releases the PROTAC drug, exerting a highly efficient effect in degrading proteins and inhibiting tumor cells. Simultaneously, the large-scale consumption of glutathione enhances oxidative stress and promotes reactive oxygen species damage to DNA. The nanocapsule prepared by this invention possesses both GSH-stimuli responsiveness and the specific recognition and phagocytic capacity of L-amino acid transporter 1, overcoming the shortcomings of existing anti-glioma therapies and achieving targeted accumulation of glioma lesions, on-demand drug release, and enhanced clinical efficacy of existing glioma radiotherapy and chemotherapy.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical formulation technology, and relates to a stimulus-responsive nanocapsule for delivering PROTAC, its preparation method, and its application. Background Technology

[0002] Gliomas, the most common primary malignant brain tumors, have a five-year survival rate of less than 5%. Single-therapy approaches are insufficient to inhibit their development and progression; clinically, a combination of radiotherapy and chemotherapy is generally used to prolong patient survival. Radiotherapy uses high-energy rays, including X-rays, to damage DNA, while chemotherapy uses DNA alkylating agents such as temozolomide. Therefore, DNA damage is the primary means of clinical treatment for gliomas; however, current clinical efficacy is not ideal, largely because gliomas possess the ability to repair damaged DNA. Thus, the key to glioma treatment lies in inhibiting DNA damage repair.

[0003] Studies have shown that BRD4, a key protein in DNA damage repair and an epigenetic regulator, is highly expressed in gliomas. Therefore, targeting BRD4 holds promise as a potential sensitization mechanism for glioma treatment. However, BRD4 lacks small molecule inhibitors and is traditionally considered an undrugifiable protein. Unlike the site-occupying inhibition mechanism of small molecule inhibitors, PROTACs, through ubiquitination labeling of target proteins, can efficiently degrade "undrugifiable" target proteins.

[0004] However, PROTACs are characterized by their large molecular weight, poor solubility, and poor permeability, posing a challenge in reaching the brain and necessitating the design of intelligent drug delivery systems to address this issue. Therefore, how to address the treatment challenges of gliomas and achieve efficient DNA damage by integrating with the tumor microenvironment is a key scientific question in clinical treatment; utilizing intelligent drug delivery systems to deliver PROTACs and enhance DNA damage is one of the key strategies for sensitizing radiotherapy and chemotherapy in clinical practice. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a stimulus-responsive nanocapsule for delivering PROTAC and its preparation method. Taking into full account the relevant pathological factors of glioma (including high concentrations of glutathione), this invention first selects a PROTAC drug with highly efficient degradation of the target protein BRD4; then, it synthesizes a PEG-based polyamino acid polymer, coupling PEG with a tryptophan analog via a click reaction and polymerizing it with amino acids to obtain a highly biocompatible polymer; using the amphiphilic polymer, the hydrophobic PROTAC drug is encapsulated into the nanoparticle core, and then a cross-linking reaction is used to form nanocapsules, enhancing the overall stability of the formulation and endowing it with lesion-responsive properties.

[0006] This invention first incorporates a tryptophan analogue conjugate into a nanocapsule drug delivery system, enabling it to act as a ligand specifically targeting glioma lesions. Second, due to the glutathione-responsive function of disulfide bonds, introducing disulfide groups within the nanocapsule allows it to exert a high glutathione response within the tumor. Furthermore, under high glutathione conditions, the nanocapsule releases PROTAC drugs, exhibiting highly efficient protein degradation and tumor cell inhibition, while simultaneously enhancing oxidative stress and promoting reactive oxygen species-induced DNA damage due to the large-scale consumption of glutathione. Therefore, the nanocapsules prepared in this invention possess both GSH-stimulated responsiveness and L-amino acid transporter 1-specific recognition and phagocytic capacity, overcoming the shortcomings of existing anti-glioma therapies and achieving targeted accumulation of glioma lesions, on-demand drug release, and enhanced clinical efficacy of existing glioma radiotherapy and chemotherapy.

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

[0008] A stimulus-responsive nanocapsule for delivering PROTAC is a nanocapsule containing PROTAC drug formed by cross-linking a polymer backbone material under the action of a cross-linking agent containing disulfide bonds.

[0009] The polymer backbone material includes polyethylene glycol-polylysine-polyphenylalanine polymer and tryptophan analog-modified polyethylene glycol-polylysine-polyphenylalanine polymer.

[0010] The disulfide-containing crosslinking agent is a DTSSP crosslinking agent;

[0011] The PROTAC drug is SIS or ARV-825.

[0012] Preferably, the chemical formula of the polyethylene glycol-polylysine-polyphenylalanine polymer is as shown in Formula I:

[0013]

[0014] Formula I

[0015] The chemical formula of the tryptophan analog-modified polyethylene glycol-polylysine-polyphenylalanine polymer is shown in Formula II:

[0016]

[0017] Formula II

[0018] More preferably, the nanocapsules have a diameter of 100-110 nm, a zeta potential of 0-2 mV, a drug loading of 7.0% for SIS, and an encapsulation efficiency of 67.89%.

[0019] The method for preparing the above-mentioned stimulus-responsive nanocapsule for delivering PROTAC includes the following steps:

[0020] (1) N 6 -benzyloxycarbonyl-L-lysine and triphosgene were mixed and protected with argon; then anhydrous tetrahydrofuran was added and reacted at 50 °C for 3 h; cooled to room temperature, the insoluble matter was removed by filtration, the filtrate was slowly added dropwise into pre-cooled anhydrous n-hexane, filtered, the product was collected, and dried under vacuum to obtain Lys(Cbz)-NCA;

[0021] (2) Weigh L-phenylalanine and triphosgene and mix them under argon protection; then add anhydrous tetrahydrofuran and react at 50 °C for 3 h; cool to room temperature, filter to remove insoluble matter, slowly drop the filtrate into pre-cooled anhydrous n-hexane, stir, filter, collect the product, and dry under vacuum to obtain Phe-NCA.

[0022] (3) CH3O-PEG112-NH2 and Lys(Cbz)-NCA were protected with argon gas; then anhydrous DMF was added, and the mixture was reacted at 50 °C for 48 h to obtain reaction solution one; Phe-NCA was dissolved in anhydrous DMF and added to reaction solution one, and the mixture was reacted at 50 °C for 48 h to obtain reaction solution two; a DMF solution of acetic anhydride and triethylamine was added to reaction solution two, and the mixture was reacted at room temperature for 3 h; after the reaction was terminated, the resulting solution was dialyzed in pure water for 24 h; after freeze-drying, the product mPEG was obtained. 112 -pLys(Cbz)-pPhe-CH3;

[0023] (4) mPEG 112 -pLys(Cbz)-pPhe-CH3 was dissolved in trifluoroacetic acid under stirring, followed by the addition of HBr solution, and the reaction was carried out at room temperature for 1.5 h. The reaction system was then dialyzed, freeze-dried, and the product mPEG was collected. 112 -pLys-pPhe-CH3;

[0024] (5) Disperse 1-methyl-L-tryptophan in a mixed solution of pure water and tetrahydrofuran, add NaOH, stir to dissolve, add (Boc)2O dropwise, react at 0 ℃ for 10 min, react at 25 ℃ for 24 h; evaporate the solvent to obtain the reaction mixture; acidify the reaction mixture with hydrochloric acid until the solid content no longer increases; extract the reaction mixture with ethyl acetate; dry the organic layer with anhydrous Na2SO4; evaporate the organic solvent; and vacuum dry to obtain the product Boc-N-1-methyl-L-tryptophan (MLT-Boc);

[0025] (6) MLT-Boc, 1-hydroxybenzotriazole (HOBT), and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU) were activated in anhydrous DMF and an ice bath for 40 min under argon protection; then propargylamine and DMF solution of N,N-diisopropylethylamine (DIPEA) were added, and the temperature was gradually restored to room temperature for 24 h; the product 1-MLT(Boc)-propargyl was separated by column chromatography with a mobile phase of dichloromethane / ethyl acetate at a volume ratio of 90 / 10.

[0026] (7) Dissolve N3-PEG112-NH2 and 1-MLT(Boc)-propargyl in anhydrous DMF, then add a DMF solution of CuI and DIPEA, under high-purity argon protection, and finally add sodium ascorbate (VcNa); react overnight at room temperature in the dark. After the reaction is complete, put the reaction solution into a dialysis bag and dialyze it in a PBS solution containing 10 mM EDTA-2Na at pH 7.4 for 24 h until the red color of the system disappears and turns yellow. Then transfer the dialysis bag to pure water and dialyze for 24 h. Freeze-dry to obtain the pale yellow product MLT-Boc-PEG. 112 -NH2;

[0027] (8) MLT-Boc-PEG 112 -NH2 and Lys(Cbz)-NCA were dissolved in anhydrous DMF under high-purity argon protection and reacted at 50 °C for 48 h; then, Phe-NCA was added to a DMF solution and reacted at 50 °C for 48 h; cold ether precipitation yielded the product MLT-Boc-PEG. 112 -pLys(Cbz)-pPhe-NH2;

[0028] (9) MLT-Boc-PEG 112 -pLys(Cbz)-pPhe-NH2 was dissolved in trifluoroacetic acid, HBr was added, and the reaction was carried out with stirring at room temperature for 1.5 h. The reaction system was dialyzed and freeze-dried to obtain the pale yellow product MLT-PEG. 112 -pLys-pPhe-NH2;

[0029] (10) Preparation of nanocapsules by nanoprecipitation method: mPEG with a concentration of 20 mg / mL was prepared respectively. 112 -pLys-pPhe-CH3, MLT-PEG 112 - DMSO solution of pLys-pPhe-NH2 material and SIS drug; take mPEG 112 DMSO solution of -pLys-pPhe-CH3, MLT-PEG 112A DMSO solution of -pLys-pPhe-NH2 and a DMSO solution of SIS were mixed; then added dropwise to pure water; the mixture was stirred for 4 hours; the DMSO was removed by dialyzing for 12 hours, with the water changed every 4 hours, to obtain MPLP@SIS nanoparticles; a DTSSP solution was added to the MPLP@SIS nanoparticle formulation, and the reaction was carried out at room temperature for 6 hours, followed by dialyzing with pure water for 12 hours to remove excess DTSSP, to obtain MPL. (SS) P@SIS nanocapsules.

[0030] Preferably, the N mentioned in step (1) 6 The molar ratio of -benzyloxycarbonyl-L-lysine to triphosgene is 2.5 : 1.

[0031] Preferably, the molar ratio of L-phenylalanine to triphosgene in step (2) is 2:1.

[0032] Preferably, the molar ratio of Lys(Cbz)-NCA, Phe-NCA and CH3O-PEG-NH2 in step (3) is 12:20:1; and the molar ratio of acetic anhydride, triethylamine and CH3O-PEG-NH2 is 5:3:1.

[0033] Preferably, in step (4), 200 mg of the mPEG is... 112 The solid -pLys(Cbz)-pPhe-CH3 was dissolved in 10 mL of trifluoroacetic acid, and then 200 µL of HBr was added to react, wherein HBr was a 33 wt.% acetic acid solution.

[0034] Preferably, in step (5), the volume ratio of pure water to tetrahydrofuran is 1:1, and the reactant amounts are: 400 mg of 1-methyl-L-tryptophan, 350 µL of (Boc)2O, 1 mol / L of NaOH, and 1 mol / L of hydrochloric acid.

[0035] Preferably, HOBT and HBTU in step (6) act as amide condensing agents to promote the amide reaction; DIPEA is an alkaline organic catalyst for the condensation reaction; the molar ratio of MLT-Boc to propargylamine is 1:1.2; and the molar ratio of MLT-Boc to HOBT, HBTU, and DIPEA is 1:1.1:1.1:2.4.

[0036] Preferably, in step (7), the molar ratio of N3-PEG112-NH2 and 1-MLT(Boc)-propyne is 1:2; the molar ratio of N3-PEG112-NH2 to CuI, DIPEA, and VcNa is 1:1:1:15; CuI acts as a catalyst; the excess VcNa prevents CuI oxidation; excess copper ions are removed through complexation during EDTA-2Na dialysis; the resulting product is MLT-Boc-PEG. 112 -NH2 is pale yellow.

[0037] Preferably, the Lys(Cbz)-NCA, Phe-NCA and MLT-Boc-PEG described in step (8) 112 The molar ratio of -NH2 is 12 : 20 : 1.

[0038] Preferably, in step (9), 200 mg of MLT-PEG is used. 112 The solid -pLys(Cbz)-pPhe-NH2 was dissolved in 10 mL of trifluoroacetic acid, and then 200 µL of HBr (a 33 wt.% acetic acid solution) was added to react.

[0039] Preferably, the mPEG described in step (10) 112 -pLys-pPhe-CH3, MLT-PEG 112 The mass ratio of -pLys-pPhe-NH2 to SIS was 9:1:1; the DTSSP solution solvent was PBS at pH 12.1, and the concentration of the DTSSP solution was 20 mg / mL. 1 mL of nanoparticle formulation was added to 100 µL of DTSSP solution, reacted for 6 h, and dialyzed for 12 h to remove excess DTSSP, yielding MPL. (SS) P@SIS nanocapsules.

[0040] The above-mentioned stimulus-responsive nanocapsule for delivering PROTAC is used in the treatment of glioma.

[0041] The dialysis method is as follows: the product is placed in a dialysis bag with a molecular weight cutoff of 3500, immersed in water, and dialyzed to remove impurities.

[0042] The room temperature mentioned in this invention refers to 25°C.

[0043] Compared with the prior art, the beneficial effects of the present invention include:

[0044] (1) The nanocapsule carrier described in this invention does not produce significant toxicity to the body and has good biocompatibility;

[0045] (2) The nanocapsules prepared by the present invention have small particle size and uniform morphology. The capsule skeleton can react with GSH to have lesion response and release drugs.

[0046] (3) The nanocapsules prepared in this invention can be used as nanomedicines for the treatment of gliomas and are suitable for intravascular injection. The nanocapsules can target L-amino acid transporter 1 and cross the blood-brain barrier with the help of tryptophan analogs and accumulate in glioma lesions. Under the stimulation of high concentration of glutathione in glioma lesions, the nanocapsules selectively release PROTAC drugs and play a role in degrading target proteins. At the same time, the nanocapsule carrier enhances the level of reactive oxygen species and can play a role in enhancing DNA damage. Attached Figure Description

[0047] Figure 1 The non-targeted polymer mPEG in Example 1 112 Synthetic route of -pLys-pPhe-CH3;

[0048] Figure 2 The targeted polymer MLT-PEG in Example 1 112 Synthetic route of -pLys-pPhe-NH2;

[0049] Figure 3 For mPEG 112 -pLys-pPhe-CH3 1H NMR spectrum;

[0050] Figure 4 MLT-PEG 112 -pLys-pPhe-NH2 1H NMR spectrum;

[0051] Figure 5 This is a schematic diagram illustrating the preparation of the targeted nanocapsules in Example 1;

[0052] Figure 6 The dynamic light scattering particle size distribution of the targeted nanocapsules in Example 1;

[0053] Figure 7 The potential distribution of the targeted nanocapsules in Example 1;

[0054] Figure 8 The image shows the transmission electron microscope (TEM) morphology of the targeted nanocapsules from Example 1.

[0055] Figure 9 The drug release curves of the non-targeted nanoparticles and nanocapsules in the PBS system in Example 1 are shown.

[0056] Figure 10 The drug release curves of the targeted nanocapsules in Example 1 under stimulation with different concentrations of DTT are shown.

[0057] Figure 11 The degradation effect of the targeted nanocapsules of Example 1 on the target protein BRD4 in glioma cells G422 at different concentrations is shown.

[0058] Figure 12 The inhibitory effect of the targeted nanocapsule carrier of Example 1 on G422 glioma cells at different concentrations is shown.

[0059] Figure 13 The effect of different drug administration groups on the survival rate of GBM model mice in Example 1;

[0060] Figure 14 The effect of different drug administration groups on the body weight of GBM model mice in Example 1;

[0061] Where G1: saline group; G2: MPL (SS) P@SIS nanocapsule group; G3: X-ray + TMZ group; G4: MPL (SS) P@SIS nanocapsules + TMZ group; G5: MPL (SS) P@SIS Nanocapsules + X-ray assembly; G6: PL (SS) P@SIS non-targeted nanocapsules + X-ray + TMZ combination; G7: MPL (SS) P@SIS nanocapsules + X-rays + TMZ group. Detailed Implementation

[0062] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, the embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0063] Example 1

[0064] A method for preparing a stimulus-responsive nanocapsule for delivering PROTAC includes the following steps:

[0065] (1) Polymer mPEG 112 Synthesis of -pLys-pPhe-CH3

[0066] according to Figure 1 The synthesis route is as follows, and the synthesis is carried out sequentially:

[0067] Synthesis of a. Lys(Cbz)-NCA

[0068] 3 g N 6-Benzyloxycarbonyl-L-lysine (10.71 mmol, 1 equiv.) and 1.27 g triphosgene (4.28 mmol, 0.4 equiv.) were added to a 100 mL two-necked flask, and the gas in the flask was displaced using an argon-filled balloon. Then, 50 mL of anhydrous tetrahydrofuran was added to the flask with stirring using a syringe. The system was initially a white, turbid liquid, which was then transferred to a 50 °C oil bath and reacted for 3 h. After the reaction was complete, the system became a colorless, transparent solution. It was allowed to cool to room temperature, and the residue was removed by filtration. The reaction solution was then slowly added dropwise to a beaker containing 500 mL of pre-cooled (-20 °C) anhydrous n-hexane using a plastic dropper. During the addition, the system was stirred to allow for precipitation. The precipitate was then obtained by filtration, washed with a small amount of n-hexane, compacted, and dried overnight. After complete drying, the solid was weighed, the yield was calculated, and it was characterized by 1H NMR.

[0069] b. Synthesis of Phe-NCA

[0070] 3 g of L-phenylalanine (18.16 mmol, 1 equiv.) and 2.7 g of triphosgene (9.10 mmol, 0.5 equiv.) were weighed into a 100 mL two-necked flask, and the gas in the flask was displaced using an argon-filled balloon. Then, 60 mL of anhydrous tetrahydrofuran was added to the flask while stirring using a syringe. The system was initially a white, turbid liquid. The mixture was then transferred to a 50 °C oil bath and reacted for 3 h. After the reaction was complete, the system became a colorless, transparent solution. The solution was allowed to stand at room temperature and filtered to remove any remaining insoluble matter. The reaction mixture was then slowly added dropwise to a beaker containing 600 mL of pre-cooled (-20 °C) anhydrous n-hexane using a plastic dropper. During the addition, the system was stirred to allow for the precipitation of a flocculent precipitate. The precipitate was then filtered to obtain a white solid, washed with n-hexane, compacted, and dried. After complete drying, the solid was weighed, the yield was calculated, and the solid was characterized by 1H NMR spectroscopy.

[0071] c. mPEG 112 Synthesis of -pLys(Cbz)-pPhe-CH3

[0072] Weigh 200 mg CH3O-PEG112-NH2 (0.04 mmol, 1 equiv.) and 148 mg of compound Lys(Cbz)-NCA (0.48 mmol, 12 equiv.) into a 100 mL two-necked flask, and replace the gas in the flask with argon gas. Next, 20 mL of anhydrous DMF was added to the two-necked flask while stirring using a syringe. The system then became a colorless and transparent solution, which was transferred to a 50 °C oil bath and reacted for 48 h. 153 mg of compound Phe-NCA (0.8 mmol, 20 equiv.) was dissolved in anhydrous DMF and injected into a 100 mL two-necked flask. The system again became a colorless and transparent solution, and the reaction was carried out at 50 °C for 48 h. Then, 20 μL of acetic anhydride (0.20 mmol, 21 mg, 5 equiv.) and 17.5 μL of triethylamine (0.12 mmol, 13 mg, 3 equiv.) were added, and the reaction was carried out at room temperature for 3 h. After the reaction was terminated, the solution was transferred to a dialysis bag and dialyzed in a large volume of pure water for 24 h (with water changed three times at equal time intervals). The suspension was then transferred to a plastic tube, freeze-dried, and a white powdery solid was obtained. The weight was used to calculate the yield, and the solid was characterized by 1H NMR spectroscopy.

[0073] d. mPEG 112 Synthesis of -pLys-pPhe-CH3

[0074] Weigh out 200 mg mPEG 112 -pLys(Cbz)-pPhe-CH3 was added to a single-necked flask, and 10 mL of trifluoroacetic acid was added with stirring to dissolve the polymer. The system was initially a pale yellow solution. Then, 200 μL of HBr (33 wt.%, acetic acid solution) was added. A large amount of carbon dioxide gas was generated, and the yellow color deepened. The flask was sealed with a glass stopper and stirred at room temperature for 1.5 h. The reaction system was then dialyzed, and after freeze-drying, a white powdery solid was obtained. The yield was calculated by weighing and identified by 1H NMR spectroscopy. The results are as follows: Figure 3 As shown in the image.

[0075] (2) Targeting polymer MLT-PEG 112 Synthesis of -pLys-pPhe-NH2

[0076] according to Figure 2 The synthesis route is as follows, and the synthesis is carried out sequentially:

[0077] a. Synthesis of Boc-N-1-methyl-L-tryptophan

[0078] 400 mg of 1-methyl-L-tryptophan was suspended in a 1:1 mixture of pure water and tetrahydrofuran. 6 mL of 1 mol / L NaOH was added, and the mixture was stirred to dissolve. 350 µL of (Boc)₂O was added dropwise. The reaction mixture was reacted at 0 °C for 10 min and then at 25 °C for 24 h. The solvent was evaporated, and the residue was cooled in an ice bath. The reaction mixture was acidified with 1 mol / L hydrochloric acid until the solid content no longer increased. The reaction mixture was extracted with ethyl acetate. The organic layer was dried over anhydrous Na₂SO₄. The solvent was evaporated, and the mixture was dried under vacuum to obtain Boc-N-1-methyl-L-tryptophan.

[0079] b. Synthesis of 1-MLT(Boc)-propargyl

[0080] MLT-Boc (100 mg), HOBT (42.6 mg), and HBTU (119.2 mg) were placed in a two-necked flask, and 10 mL of anhydrous DMF was added at once. The mixture was activated in an ice bath for 40 min. Prolylamine (19 mg) and DIPEA (89 mg) were dissolved in anhydrous DMF and added to the system. The ice bath was removed, and the mixture was gradually brought to room temperature. The reaction was allowed to proceed for 24 h. The product 1-MLT(Boc)-propyl was isolated under the condition that the mobile phase was dichloromethane / ethyl acetate = 90 / 10.

[0081] c. MLT-Boc-PEG 112 Synthesis of -NH2

[0082] Weigh 102 mg N3-PEG112-NH2 and 14.5 mg 1-MLT(Boc)-propargyl into a 50 mL two-necked flask, add 2 mL of anhydrous DMF solution, and dissolve CuI (3.8 mg) and DIPEA (2.59 mg) in the anhydrous DMF solution. Add DIPEA and CuI, protect with high-purity argon gas, and finally add 59.4 mg VcNa. React overnight (12 h) at room temperature in the dark. After the reaction is complete, transfer the reaction solution into a dialysis bag and dialyze it in 5 L of PBS (pH 7.4) containing 10 mM EDTA-2Na for 24 h until the red color disappears and turns into a very weak yellow. Then transfer the dialysis bag to pure water and dialyze for 24 h. Freeze-dry to obtain the pale yellow product MLT-Boc-PEG. 112 -NH2.

[0083] d. MLT-Boc-PEG 112 Synthesis of -pLys(Cbz)-pPhe-NH2

[0084] Weigh out 200 mg of MLT-Boc-PEG 112-NH2 (0.04 mmol, 1 equiv.) and 148 mg Lys(Cbz)-NCA (0.48 mmol, 12 equiv.) were added to a two-necked flask, and the mixture was sealed with high-purity argon to isolate it from air. 20 mL of anhydrous DMF was added, and the mixture was reacted in an oil bath at 50 °C for 48 h. 153 mg Phe-NCA (0.80 mmol, 20 equiv.) was dissolved in 20 mL of anhydrous DMF and added to the mixture. The mixture was reacted at 50 °C for 48 h. The product MLT-Boc-PEG was obtained by cold ether precipitation. 112 -pLys(Cbz)-pPhe-NH2.

[0085] e. MLT-PEG 112 Synthesis of -pLys-pPhe-NH2

[0086] Weigh out 200 mg of MLT-Boc-PEG 112 In a single-necked flask, 10 mL of trifluoroacetic acid was added with stirring to dissolve the polymer, resulting in a pale yellow solution. 200 μL of HBr (33 wt.% acetic acid solution) was then added, producing a large amount of carbon dioxide gas and deepening the yellow color. The flask was sealed with a glass stopper and stirred at room temperature for 1.5 h. The reaction system was dialyzed and freeze-dried to obtain the product MLT-PEG. 112 -pLys-pPhe-NH2 was analyzed by 1H NMR spectroscopy, and the results are as follows: Figure 4 As shown in the image.

[0087] (3) Preparation of nanocapsules by nanoprecipitation method

[0088] according to Figure 5 Preparation of nanocapsules

[0089] a. Preparation of MPLP@SIS nanoparticles

[0090] mPEG 112 -pLys-pPhe-CH3, MLT-PEG 112 - pLys-pPhe-NH2 material and SIS drug were dissolved in DMSO to prepare a concentration of 20 mg / mL; 180 µL of mPEG solution was taken. 112 DMSO solution of -pLys-pPhe-CH3, 20 µL of solution containing MLT-PEG 112A DMSO solution containing -pLys-pPhe-NH2 and 20 µL of DMSO solution containing SIS were mixed; the mixed DMSO solution was added dropwise to 1.8 mL of pure water; the mixture was stirred for 4 hours; and the DMSO was removed by dialyzing with pure water for 12 hours, with the water being changed every 4 hours. This yielded MPLP@SIS nanoparticles.

[0091] b. MPL (SS) Preparation of P@SIS nanocapsules

[0092] DTSSP was dissolved in PBS at pH 12.1 to a concentration of 20 mg / mL. 1 mL of the nanoparticle formulation was added to 100 µL of DTSSP solution, reacted for 6 h, and then dialyzed for 12 h to remove excess DTSSP, yielding MPL. (SS) P@SIS nanocapsules.

[0093] The following tests were performed on the above nanocapsules:

[0094] Figure 6 Dynamic light scattering particle size of PROTAC nanocapsules; visible: MPL (SS) The P@SIS nanocapsules have a particle size of approximately 100 nm, which is significantly smaller than that of nanoparticles.

[0095] Figure 7 The zeta potential distribution of PROTAC nanocapsules; it can be seen that the zeta potential of MPLP@SIS nanoparticles is approximately 25 mV. (SS) The zeta potential of P@SIS nanocapsules is approximately 2 mV, which is significantly lower than that of nanoparticles.

[0096] Figure 8 The image shows the morphology of the PROTAC nanocapsules under a transmission electron microscope. As can be seen, the nanoparticles are nearly spherical, with uniform morphology and a particle size of about 100 nm.

[0097] Preparation of non-targeted nanocapsules for studying drug release properties

[0098] a. Preparation of PLP@SIS nanoparticles

[0099] The mPEG112-pLys-pPhe-CH3 material and the SIS drug were dissolved in DMSO to prepare a concentration of 20 mg / mL. 200 µL of the DMSO solution containing mPEG112-pLys-pPhe-CH3 and 20 µL of the DMSO solution containing SIS were mixed. The mixed DMSO solution was added dropwise to 1.8 mL of pure water. The mixture was stirred for 4 hours. The DMSO was removed by dialyzing with pure water for 12 hours, with the water changed every 4 hours. This yielded PLP@SIS nanoparticles.

[0100] b. Preparation of PL(SS)P@SIS nanocapsules

[0101] DTSSP was dissolved in PBS at pH 12.1 to a concentration of 20 mg / mL. 1 mL of the nanoparticle formulation was added to 100 µL of DTSSP solution, reacted for 6 h, and dialyzed for 12 h to remove excess DTSSP, thus obtaining PL(SS)P@SIS nanocapsules.

[0102] Figure 9 Drug release curves of nanoparticles and nanocapsules in PBS system; the release rate of nanocapsules in PBS system was 30% after 12 hours. Compared with nanoparticles, cross-linking gives nanocapsules stronger stability.

[0103] Figure 10 The figures show the drug release curves of PROTAC nanocapsules stimulated by different concentrations of DTT. 10 mM DTT simulates the high GSH state in the tumor region. It can be seen that as time goes by, the nanocapsules release more and more PROTAC drug SIS under DTT stimulation, and high concentrations of DTT can significantly promote the release of SIS drug from the nanocapsules.

[0104] Figure 11 The degradation effect of nanocapsules on the target protein BRD4 in glioma cells G422 at different concentrations; MPL is visible. (SS) P@SIS nanocapsules can effectively degrade the target protein BRD4, exhibiting the strongest degradation effect at a concentration of 100 nM.

[0105] Figure 12 The inhibitory effect of nanocapsule carriers on glioma cells G422 at different concentrations was investigated. It can be seen that even at a concentration of 250 µg / mL, the nanocapsule carriers did not have significant toxicity to the cells, indicating that the nanocapsule carriers themselves do not produce significant toxicity to the body and have good biocompatibility.

[0106] Figure 13 Effects of different drug administration groups on the survival rate of GBM-modeled mice; It can be seen that: MPL nanocapsules (SS) P@SIS combined with X-ray radiotherapy and TMZ chemotherapy achieved the highest survival rate, indicating that nanocapsules are beneficial for the therapeutic effect of radiotherapy and chemotherapy on gliomas.

[0107] Figure 14 Effects of different drug administration groups on body weight in GBM-modeled mice; It can be seen that: MPL nanocapsules (SS) P@SIS combined with X-ray radiotherapy and TMZ chemotherapy resulted in minimal weight loss, indicating that nanocapsules are beneficial for the therapeutic effect of radiotherapy and chemotherapy on gliomas.

[0108] Example 2

[0109] A method for preparing a stimulus-responsive nanocapsule for delivering the PROTAC drug ARV-825 includes the following steps:

[0110] (1) Preparation of nanoparticles loaded with ARV-825

[0111] mPEG 112 -pLys-pPhe-CH3, MLT-PEG 112 - pLys-pPhe-NH2 material and ARV-825 drug were dissolved in DMSO to prepare a concentration of 20 mg / mL; 180 µL of mPEG solution was taken. 112 DMSO solution of -pLys-pPhe-CH3, 20 µL of solution containing MLT-PEG 112 A DMSO solution containing -pLys-pPhe-NH2 and 20 µL of DMSO solution containing ARV-825 were mixed; the mixed DMSO solution was added dropwise to 1.8 mL of pure water; the mixture was stirred for 4 hours; and the DMSO was removed by dialyzing with pure water for 12 hours, with the water being changed every 4 hours. This yielded MPLP@ARV nanoparticles with targeting function.

[0112] (2) Preparation of nanocapsules loaded with ARV-825

[0113] DTSSP was dissolved in PBS at pH 12.1 to a concentration of 20 mg / mL. 1 mL of the nanoparticle formulation was added to 100 µL of DTSSP solution, reacted for 6 h, and then dialyzed for 12 h to remove excess DTSSP. This yielded MPL with brain-targeting function. (SS) P@ARV nanocapsules.

[0114] (3) Characterizing MPL (SS) P@ARV Nanocapsules

[0115] MPL (SS) The P@ARV nanocapsules have a particle size of approximately 110 nm, a zeta potential of approximately 0-5 mV, a drug loading of approximately 5.0% for ARV-825, and an encapsulation efficiency of approximately 52.3%.

Claims

1. A stimulus-responsive nanocapsule for delivering PROTAC, characterized in that, It is a nanocapsule encapsulating PROTAC drug formed by cross-linking a polymer backbone material under the action of a cross-linking agent containing disulfide bonds; The polymer backbone material includes polyethylene glycol-polylysine-polyphenylalanine polymer and tryptophan analog-modified polyethylene glycol-polylysine-polyphenylalanine polymer. The disulfide-containing crosslinking agent is a DTSSP crosslinking agent; The PROTAC drug is ARV-825; The chemical formula of the polyethylene glycol-polylysine-polyphenylalanine polymer is shown in Formula I: Formula I The chemical formula of the tryptophan analog-modified polyethylene glycol-polylysine-polyphenylalanine polymer is shown in Formula II: Formula II.

2. The stimulus-responsive nanocapsule for delivering PROTAC according to claim 1, characterized in that, The nanocapsules have a diameter of 100-110 nm and a zeta potential of 0-2 mV.

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