Ultrasonic response type microsphere preparation entrapped with protein degradation agent as well as preparation method and application of ultrasonic response type microsphere preparation

Abstract

The invention provides an ultrasonic response type microsphere preparation entrapped with a protein degradation agent as well as a preparation method and application of the ultrasonic response type microsphere preparation. The microsphere is of a microsphere structure entrapped with a protein degradation agent and formed by a conjugate of a biodegradable polymer and a sound-sensitive agent and the protein degradation agent. According to the invention, the targeted protein degradation agent is entrapped in a biodegradable novel polymer matrix, so that the slow release effect of the medicine is realized, and the long-acting effect is achieved at a specific part in the body. The microsphere combines the respective advantages of an ultrasonic technology and a targeted protein degradation technology, and the two technologies work together, so that the controllability of drug release is remarkably improved, a synergistic effect is achieved, and the treatment effect is improved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a novel drug delivery system. More specifically, this invention relates to an injectable microsphere formulation with ultrasound-responsive properties, which encapsulates protein-degrading agents such as protein-degrading targeting chimeras, enabling spatiotemporally specific, on-demand, and controllable drug release triggered by exogenous ultrasound signals. This invention also relates to a method for preparing the microsphere formulation and its use in the preparation of cancer treatment drugs. Background Technology

[0002] Polylactic acid-glycolic acid copolymer (PLGA) and polylactic acid (PLA) microspheres, as biodegradable polymeric carriers, have been widely used in long-acting sustained-release drug systems. Their advantages include good biocompatibility, controllable degradation cycles, and a history of FDA-approved clinical applications. Traditional PLGA / PLA microspheres, by adjusting the polymer molecular weight, ratio, and microsphere structure, can achieve slow drug release over days to months. However, the release behavior of these microspheres is mostly driven by passive diffusion and matrix erosion, lacking spatiotemporal controllability. For protein degraders with narrow therapeutic windows and requiring on-demand administration, the "sustained release" mode of traditional PLGA / PLA microspheres may lead to initial burst release, insufficient release later, or failure to respond to the dynamic needs of the disease, thereby limiting efficacy and increasing systemic toxicity.

[0003] To address the uncontrollable release issues of traditional microspheres, stimulus-responsive carriers have become a research hotspot. Among them, ultrasound-responsive microspheres, by incorporating acoustically sensitive materials or designing cavity structures, can undergo cavitation, phase transitions, or structural rupture under external ultrasound, achieving rapid drug release to the target area. In recent years, solid PLGA / PLA-based ultrasound microspheres, by doping with acoustically sensitive agents, have achieved higher drug loading release under low-frequency ultrasound triggering. However, research has mostly focused on chemotherapeutic drugs or gene fragments, with release mechanisms dependent on the mechanical or thermal effects of ultrasound. Systematic optimization of the physicochemical properties of protein degraders (such as high hydrophobicity, large molecular size, and activity retention requirements) has not yet been conducted.

[0004] Protein degraders are bifunctional molecules that degrade target proteins via the ubiquitin-proteasome pathway. However, their clinical application is limited by low oral bioavailability, off-target toxicity from systemic exposure, and lack of tissue selectivity. Although PLGA / PLA microspheres can improve their pharmacokinetics, they cannot achieve precise activation at the lesion site. While ultrasound-responsive technology can provide spatiotemporal controlled release, few studies have combined it with protein degraders. Existing ultrasound microsphere systems mostly focus on hydrophilic drugs or small molecules, making it difficult to efficiently encapsulate hydrophobic protein degraders while maintaining their conformational stability. Furthermore, how to coordinate material design with ultrasound parameters to achieve "on-demand release" of protein degraders and ultrasound-assisted therapy (such as the sonodynamic effect enhancing cellular proteasome activity) remains an unexplored technological gap.

[0005] In summary, no existing technology possesses an intelligent delivery system that integrates the biocompatibility of PLGA / PLA microspheres, the spatiotemporal controlled release capability responsive to ultrasound, and the efficient encapsulation and activity protection of protein degrading agents. Developing such a platform is of great significance for promoting the clinical translation of protein degrading agents and achieving precision medicine. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method for preparing an ultrasound-responsive microsphere formulation encapsulating a protein-degrading agent for controlled release. This method encapsulates the targeted protein-degrading agent within a novel biodegradable polymer matrix, achieving sustained drug release and allowing it to exert a long-lasting effect at specific sites in vivo. By combining the advantages of ultrasound technology and targeted protein degradation technology, these microspheres work synergistically to significantly improve the controllability of drug release and enhance therapeutic efficacy.

[0007] Therefore, one aspect of the present invention provides an ultrasound-responsive microsphere formulation of a protein-degrading agent for controlled release, wherein the microsphere is a protein-degrading agent-encapsulated microsphere structure formed by a conjugate of a biodegradable polymer and a sonic sensitizer and a protein-degrading agent. The ultrasound-responsive microsphere formulation of the present invention, encapsulating a protein-degrading agent, enables spatiotemporal specificity and on-demand controlled release of the drug upon triggering by an exogenous ultrasound signal.

[0008] In some embodiments, the microsphere structure may be a solid microsphere or a porous microsphere. A solid microsphere refers to a micron-sized spherical particle with a dense, continuous internal structure and essentially free of interconnected pores. A porous microsphere refers to a micron-sized spherical particle with a large number of pore structures (open or closed pores) on its interior or surface, and the pores can be interconnected to form a three-dimensional porous network.

[0009] In some embodiments, the conjugate of the biodegradable polymer and the sound-sensitizer refers to a composite material system in which the sound-sensitizer molecule is stably linked to the main chain or side chain of the biodegradable polymer through covalent chemical bonds, thereby forming a composite material system in which the sound-sensitizer function is gradually released or activated in vivo as the polymer degrades.

[0010] In some embodiments, the sonosensitive agent refers to an agent that can be activated and undergo physical or chemical energy conversion under ultrasonic stimulation, thereby generating active species (such as singlet oxygen¹O₂, hydroxyl radical·OH, superoxide anion·O₂). - These are substances or materials that trigger specific biological effects (such as cell damage, signal regulation, or drug release). Examples include porphyrins, bodily oils, and cyanides.

[0011] In some embodiments, the sound-sensing agent is selected from the following compounds:

[0012] .

[0013] In some embodiments, the biodegradable polymer may refer to a polyester synthetic polymer. In a preferred embodiment, the biodegradable polymer may be selected from polylactic acid-glycolic acid copolymer (PLGA) or polylactic acid (PLA).

[0014] In some embodiments, the molar ratio of the sound-sensitizing agent to the biodegradable polymer is 1:1 to 1:5.

[0015] In some embodiments, the mass ratio of the conjugate to the protein degrading agent is 10-50, for example, 20, 30, 40, etc.

[0016] In some embodiments, the coupling of the biodegradable polymer and the sound-sensitizing agent is a coupling of polylactic acid-glycolic acid copolymer (PLGA) and sound-sensitizing agent (R) as shown in Formula 1a (PLGA-R), or a coupling of polylactic acid (PLA) and sound-sensitizing agent (R') as shown in Formula 1b (PLA-R'):

[0017] Formula 1a

[0018] Formula 1b in, In Formula 1a, the degree of polymerization of the polylactic-co-glycolic acid copolymer (PLGA) is x:y = 90-10:10-90; for example, in some embodiments, x is 50, 75, 100, etc.; y is 50, 30, 25, etc. In one embodiment, x is 100; y is 30. In Formula 1b, polylactic acid (PLA) is levorotatory, dextrorotatory, or racemic, with an intrinsic viscosity (IV) of 0.1~1.0 dL / g, preferably 0.2~0.6 dL / g, and a degree of polymerization z selected from an integer of 70~2100, preferably 550, 600, or 650. R and R' are ultrasonic-responsive groups derived from a sonicator, and specifically, each is independently selected from the structures shown below: , , .

[0019] In some embodiments, preferably, PPa is used as a sonicating agent to provide an ultrasonically responsive group.

[0020] In some embodiments, the protein degrading agent includes a protein-targeting chimera, a molecular gel, and a prodrug thereof.

[0021] In some embodiments, the protein-targeting chimera includes: BRD4 degraders, such as ARV-771 and ARV-825; RAPR degraders, such as MS8815; RIPK1 degraders, such as RI-ICR-5; EZH2 degraders, such as 180055, etc.

[0022] In some embodiments, the protein-targeting chimera is selected from the following compounds: , , , .

[0023] In some embodiments, the molecular adhesive includes a BRD4 degrader, such as TMX-1, ZZ7-16-073, JP-2-197, or JQ1.

[0024] In some embodiments, the molecular adhesive is selected from the following compounds: , , , .

[0025] In some embodiments, the molecular adhesive is preferably JP-2-197.

[0026] In some embodiments, preferably, an ARV-825 modified prodrug is used as a protein degrading agent, wherein the ARV-825 modified prodrug is selected from the following compounds: , , , .

[0027] Another aspect of the present invention provides a pharmaceutical composition comprising: The above-mentioned ultrasonically responsive microsphere formulations encapsulating protein degrading agents; and Optionally, a pharmaceutically acceptable carrier.

[0028] Another aspect of the present invention provides a method for preparing the above-mentioned ultrasonically responsive microsphere formulation loaded with protein degrading agent, comprising the following steps: S1, by covalently linking the sound-sensitizer molecule to the main chain or side chain of the biodegradable polymer, a conjugate of the biodegradable polymer and the sound-sensitizer is obtained. S2, using the conjugate of the biodegradable polymer and the sonic sensitizer obtained in S1 and the protein degrader, ultrasonic responsive microspheres loaded with the protein degrader are prepared by an emulsification solvent evaporation method.

[0029] In some embodiments, S1 includes: dissolving a sound-sensitizing agent and an equimolar amount of a carboxyl activator in an organic solvent, sealing the solution, reacting it at an arbitrary temperature between 0-40°C for 0.5-6 hours, then adding an equimolar amount of a biodegradable polymer, reacting it at an arbitrary temperature between 0-40°C for 0.5-48 hours, dialyzing it with an organic solvent (dialysis bag with a molecular weight of 1000-1500), dialyzing it for 5-20 hours, replacing the dialysate with purified water, dialyzing it for 24-36 hours, pre-freezing the reaction product (e.g., -80°C) for 12-24 hours, and then freeze-drying it for 24-48 hours to obtain a conjugate of the biodegradable polymer and the sound-sensitizing agent.

[0030] In some embodiments, in S1, the carboxyl activator is one or more selected from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, dicyclohexylcarbodiimide, 1-hydroxybenzotriazole, N-hydroxysuccinimide, N,N-diisopropylethylamine, and 4-dimethylaminopyridine (DMAP).

[0031] In some embodiments, in S1, the organic solvent may be the same or different and each independently selected from one or more of methanol, acetonitrile, dichloromethane, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide and dimethyl sulfoxide.

[0032] In some embodiments, preferably, in S1, the organic solvent is selected from dichloromethane and ethyl acetate.

[0033] In some embodiments, preferably, in S1, PLGA is used as a biodegradable polymer, wherein the molecular weight of the PLGA is 19-118 kDa.

[0034] In some implementations, S2 is selected from either method one or method two: Method 1, the preparation method of porous microspheres includes: 1) Dissolve the protein degrading agent in an organic solvent to form a drug solution; dissolve the coupling compound of the biodegradable polymer and the sonic sensitizer in an organic solvent to form a polymer solution; mix the drug solution and the polymer solution to form an oil phase (O); dissolve the pore-forming agent in water to obtain an inner aqueous phase solution (W1); dissolve the surfactant in water to form an outer aqueous phase solution (W2). 2) Add the aqueous phase solution (W1) to the oil phase (O) and sonicate using a cell disruptor to obtain a white W1 / O colostrum; 3) Slowly pour the W1 / O colostrum into the external aqueous phase solution (W2) of the surfactant, and homogenize or shear disperse it evenly to form W1 / O / W2 embryo microspheres; 4) Disperse the solution containing W1 / O / W2 embryo microspheres in water and stir to solidify; 5) Collect the solidified microspheres by centrifugation or filtration, and wash them with deionized water to remove residual surfactants and organic solvents; 6) Pre-freeze at -20~-80℃ for 2~24 days and then freeze-dry to obtain ultrasonically responsive porous microspheres loaded with protein degrading agents; Method 2, the preparation method of solid microspheres includes: 1) Dissolve the protein degrading agent in an organic solvent to form a drug solution; dissolve the conjugate of the biodegradable polymer and the sound sensitizer in an organic solvent to form a polymer solution; mix the drug solution and the polymer solution to form an oil phase (O); dissolve the surfactant in water to form an external aqueous phase solution (W2). 2) The oil phase is dispersed in an external aqueous solution (W), and homogenized or sheared to form O / W embryo microspheres; 3) Disperse the solution containing O / W embryo microspheres in water and stir to solidify; 4) Collect the solidified microspheres by centrifugation or filtration, and wash them with deionized water to remove residual surfactants and organic solvents; 5) Pre-freeze at -20~-80℃ for 2~24 days and then freeze-dry to obtain ultrasonically responsive solid microspheres loaded with protein degradation agents.

[0035] In the preparation method of the present invention, the structures of the sound-sensitive agent, the biodegradable polymer, and the protein degrading agent are as described above, and will not be repeated here.

[0036] In some embodiments, preferably, the acoustic sensitizer is PPa, and the biodegradable polymer is PLGA or PLA; more preferably, the molar ratio of the acoustic sensitizer to the biodegradable polymer is 1:1 to 1:5.

[0037] In some embodiments, in S2, in both method one and method two, the organic solvent may be the same or different and each independently may be one or more selected from methanol, acetonitrile, dichloromethane, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide and dimethyl sulfoxide.

[0038] In some embodiments, preferably, in S2, the concentration of the conjugate of the biodegradable polymer and the sound-sensitive agent is 1% to 10% (g / mL), more preferably 5% to 10% (g / mL).

[0039] In some embodiments, preferably, in step 2) of method one, the volume ratio of the internal aqueous phase solution to the oil phase is 1:20 to 1:100, the power of the cell disruptor is 100w to 120w, and the total ultrasound time is 1-10s.

[0040] In some embodiments, preferably, in step 3) of method one, the volume ratio of the external aqueous phase solution to the oil phase is 10:1 to 40:1.

[0041] In some embodiments, preferably, the homogenization or shearing speed in step 3) of method one and step 2) of method two is 9000 rpm to 10000 rpm, the homogenization time is 30 s to 2 min, and the conditions are ice bath.

[0042] In some implementations, preferably, the curing time in step 4) of method one and step 3) of method two is 5 to 15 hours.

[0043] In some embodiments, preferably, the preparation method of a microsphere formulation for controlled release of protein degradation via ultrasound according to the present invention includes the following steps: 2 mg of protein degradation agent JP-2-197 molecular gel and 50 mg of PLGA-PPa are weighed and dissolved separately in 500 μL of dichloromethane and then mixed. 50 μL of 30 mg / mL ammonium bicarbonate solution is added, and the mixture is ultrasonically sonicated in an ice bath at 120 W for 2 seconds followed by a 2-second pause, repeated three times to form a white O / W1 primary emulsion. The emulsion is then homogenized at 9000 rpm and added to 12.5 mL of 1% PVA solution W2 in an ice bath, dispersed for 1 min, to form W1 / O / W2 microspheres. These microspheres are poured into 400 mL of distilled water, stirred at 100 rpm for 5 h to solidify, centrifuged to collect the microspheres, and washed three times with deionized water to remove residual surfactants and organic solvents. After pre-freezing at -80°C for approximately 12 h, the microspheres are freeze-dried in a freeze dryer to obtain microsphere powder.

[0044] In some embodiments, preferably, in S2, the microsphere particle size is 1~200μm; more preferably, in S2, the microsphere particle size is 20~80μm.

[0045] Another aspect of the present invention provides the use of the above-described ultrasonically responsive microsphere formulation containing the protein degrading agent or the above-described pharmaceutical composition in the preparation of a medicament.

[0046] In some embodiments, the drug may be a drug for treating cancer, preferably, the cancer includes metastatic or drug-resistant solid malignant tumors, such as breast cancer, lung cancer, liver cancer, stomach cancer, head and neck cancer, cervical cancer, pancreatic cancer, ovarian cancer, colon cancer, and prostate cancer. Attached Figure Description

[0047] Figure 1 The 1H NMR spectrum of PLGA-PPa prepared in Example 1.

[0048] Figure 2 The images shown are scanning electron microscope (SEM) images of the microsphere morphology. Specifically, a is an SEM image of the microsphere morphology in Example 2; b is an SEM image of the microsphere morphology in Example 3; c is an SEM image of the microsphere morphology in Example 4; and d is an SEM image of the microsphere morphology in Example 5.

[0049] Figure 3The images show the Western blot (WB) plots of the JP-2-197 molecular adhesive-loaded ultrasonic-responsive microspheres concentration gradient at the cellular level, illustrating the degradation effect on target proteins. Figure a shows the degradation effect of the JP-2-197 molecular adhesive-loaded ultrasonic-responsive microspheres on breast cancer cells (4T1 cell line), and Figure b shows its quantitative analysis. Figure c shows the degradation effect of the JP-2-197 molecular adhesive-loaded ultrasonic-responsive microspheres on colon cancer cells (MC38 cell line), and Figure d shows its quantitative analysis. Figure e shows the degradation effect of the JP-2-197 molecular adhesive-loaded ultrasonic-responsive microspheres on melanoma cells (B16 cell line), and Figure f shows its quantitative analysis.

[0050] Figure 4 This is a diagram of drug release in response to ultrasound in Example 7. US represents ultrasound treatment.

[0051] Figure 5 The images show the microsphere morphology in Example 8. Figure a shows the microsphere morphology under an optical microscope, and Figure b shows the microsphere morphology under a fluorescence inverted microscope.

[0052] Figure 6 This is a diagram showing the sustained-release effect of microspheres in Example 8.

[0053] Figure 7 Electron microscopy image of the morphology of PLA-PPa ultrasonically responsive porous microspheres coated with JP-2-197 molecular adhesive prepared in Example 9.

[0054] Figure 8 This is a schematic diagram of the microspheres of the present invention and their preparation process. The microspheres are composed of a protein-encapsulated protein degrading agent formed by a coupling of a biodegradable polymer and a sound-sensitizing agent, and are formed into solid or porous microspheres by an emulsification solvent evaporation method. The solid microspheres have a dense, continuous internal structure and are essentially free of interconnected pores. The porous microspheres exhibit a structure with a large number of pores (open or closed pores) inside or on the surface, and the pores can be interconnected to form a three-dimensional porous network.

[0055] Figure 9a shows the mass spectrum of ARV-825 and... Figure 9b The 1H NMR spectrum of JP-2-197. Detailed Implementation

[0056] The following detailed description of specific embodiments further illustrates the above-mentioned content of the present invention, but it should not be construed as limiting the scope of protection of the present invention in any way. All technical solutions implemented based on the above-mentioned content of the present invention fall within the scope of the present invention.

[0057] In the following embodiments, unless otherwise stated, the test methods are generally carried out under conventional conditions or conditions recommended by the manufacturer; the raw materials and reagents are all available commercially available.

[0058] The main equipment and instruments used in this invention and related information: (1) High-speed shearing machine, instrument model T10; (2) Ultrasonic cell disruptor, instrument model Scientz-IID; (3) Freeze dryer, instrument model SCIENTZ-10N; (4) Magnetic stirrer (digital display heating type), instrument model MS-H-PRO+; (5) Ultrapure water system, model Merck Milli-Q; (6) Centrifuges, instrument models 5804R and 5425R; (7) Small animal live optical imaging system, instrument model Revvity.

[0059] The 4T1, MC38, B16, and MDA-MB-231 cells used in the examples were all purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0060] The biological reagents used in the examples, including DMEM basal medium, trypsin, trypsin-EDTA solution, penicillin-streptomycin solution, bovine serum albumin (BSA), tris-hydroxymethylaminomethane (Tris base), sodium dodecyl sulfate (SDS), TBST buffer (10 ×), PBS buffer (1 ×), rainbow prestained protein marker (10-250 kDa), PAGE gel rapid preparation kit, and BCA protein assay kit, were all purchased from Dalian Meilun Biotechnology Co., Ltd.

[0061] ARV-825 and JP-2-197 were synthesized in this laboratory (see [link]). Figure 9a and Figure 9b : Figure 9a This is the mass spectrum of ARV-825. Figure 9b (This is the 1H NMR spectrum of JP-2-197). 1,1'-Octadecyl-3,3,3',3'-Tetramethylindole tricarbonyl iodide (DIR) and pyromethesphalophylloic acid-ALPHA (PPa) were purchased from Dalian Meilun Biotechnology Co., Ltd. Polyvinyl alcohol (PVA) was purchased from Sinopharm Chemical Reagent Group. Polylactic acid (PLA) and polylactic-glycolic acid (PLGA) was purchased from Jinan Daigang Bioengineering Co., Ltd.

[0062] Female BALB / c mice (6-8 weeks old) were purchased from the Shanghai Laboratory Animal Center (IACUC approval number 2025-09-YHJ-19) and housed at the SPF Animal Center of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

[0063] Example 1: Preparation of PLGA-PPa

[0064] Dissolve 2 mg PPa, 0.76 mg 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), and 0.5 mg DMAP in 2 mL of dichloromethane (DCM) and add to a round-bottom flask (to activate the carboxyl group) for 2 hours. Then add 100 mg PLGA (dissolved in 1 mL DCM) and react overnight at room temperature. Dialyze using DMF (dialysis bag 1000-1500), change the water, pre-freeze at -80°C for 3 hours, and lyophilize overnight. The 1H NMR spectrum of PLGA-PPa is shown in [reference needed]. Figure 1 .

[0065] Example 2: Preparation of ARV-825 PLGA-PPa Ultrasonic Responsive Solid Microspheres (Target Protein Chip) 2 mg of ARV-825 and 50 mg of PLGA-PPa prepared in Example 1 were weighed and dissolved in 500 μL of dichloromethane respectively. The solutions were then mixed and added to 12.5 mL of 1% PVA aqueous solution in an ice bath under a homogenizer at 9000 rpm. The mixture was dispersed for 1 min to form O / W microspheres. These microspheres were then poured into 400 mL of distilled water and stirred at 100 rpm for 5 h to solidify. The microspheres were then collected by centrifugation and washed three times with deionized water to remove residual PVA surfactant and organic solvent. After pre-freezing at -80℃ for approximately 12 h, the microspheres were freeze-dried to obtain microsphere powder. Electron micrographs of the microsphere morphology are shown below. Figure 2 As shown in Figure a.

[0066] Example 3: Preparation of ARV-825 PLGA-PPa ultrasonically responsive porous microspheres (targeting protein chimera) Weigh 2 mg of ARV-825 and 50 mg of PLGA-PPa prepared in Example 1. Dissolve each in 500 μL of dichloromethane and mix. Add 50 μL of 30 mg / mL ammonium bicarbonate solution (W1) to the mixture. Sonicate the mixture three times in an ice bath at 120 W for 2 seconds followed by a 2-second pause, forming a white W1 / O primary emulsion. Homogenize the emulsion at 9000 rpm and add it to 12.5 mL of 1% PVA aqueous solution in an ice bath (W2). Disperse for 1 min to form W1 / O / W2 microspheres. Pour the microspheres into 400 mL of distilled water and stir at 100 rpm for 5 h to solidify. Collect the microspheres by centrifugation and wash three times with deionized water to remove residual PVA surfactant and organic solvent. Pre-freeze at -80°C for about 12 h and then freeze-dry to obtain microsphere powder. Electron micrographs of the microsphere morphology are shown below. Figure 2 As shown in b.

[0067] Example 4: Preparation of ultrasonically responsive solid microspheres of molecular adhesive JP-2-197 PLGA-PPa 2 mg of JP-2-197 and 50 mg of PLGA-PPa prepared in Example 1 were weighed and dissolved in 500 μL of dichloromethane respectively. The mixture was then added to 12.5 mL of 1% PVA aqueous solution in an ice bath at 9000 rpm and dispersed for 1 min to form O / W microspheres. These microspheres were then poured into 400 mL of distilled water and stirred at 100 rpm for 5 h to solidify. The microspheres were then collected by centrifugation and washed three times with deionized water to remove residual PVA surfactant and organic solvent. After pre-freezing at -80℃ for 24 h, the microspheres were freeze-dried to obtain microsphere powder. Electron micrographs of the microsphere morphology are shown below. Figure 2 As shown in c.

[0068] Example 5: Preparation of ultrasonically responsive porous microspheres of molecular adhesive JP-2-197 PLGA-PPa 2 mg of JP-2-197 and 50 mg of PLGA-PPa were weighed and dissolved separately in 500 μL of dichloromethane, then mixed. 50 μL of 30 mg / mL ammonium bicarbonate solution (W1) was added, and the mixture was sonicated in an ice bath at 120 W for 2 seconds followed by a 2-second pause, repeated three times to form a white W1 / O primary emulsion. This emulsion was then homogenized at 9000 rpm and added to 12.5 mL of 1% PVA solution in an ice bath (W2), and dispersed for 1 min to form W1 / O / W2 microspheres. These microspheres were poured into 400 mL of distilled water and stirred at 100 rpm for 5 h to solidify. The microspheres were then collected by centrifugation and washed three times with deionized water to remove residual PVA surfactant and organic solvent. After pre-freezing at -80℃ for approximately 30 min, the microspheres were freeze-dried to obtain microsphere powder. Electron micrographs of the microsphere morphology are shown below. Figure 2 As shown in d.

[0069] Example 6: Degradation effect of different concentrations of JP-2-197 molecular glue-loaded ultrasonically responsive microspheres on target proteins at the cellular level (WB diagram) 1. Culture 4T1, MC38 and B16 cells in DMEM + 10% FBS (gibco) until the viability is above 90%.

[0070] 2. Press 4×10 5 1 cell / well seeded into a 6-well plate.

[0071] 3. After 24 h of incubation, microspheres of different concentrations were administered at concentrations of 0, 0.5, 15, and 10 μg / mL, with an ultrasonic power of 0.2 W / cm². 2Ultrasonic frequency: 1.0 MHz, Duty Cycle: 50%, Total ultrasound duration: 3 min (30 s intervals, 30 s intervals after 30 s intervals, 6 intervals in total).

[0072] 4. After culturing for 24 hours, total protein was extracted from 4T1, MC38, and B16 cells, and their degradation ability against target proteins was characterized by Western blotting (WB). The results are shown in [Figure number missing]. Figure 3 .like Figure 3 As shown, the degradation effect of microspheres loaded with JP-2-197 on BRD4 at the 4T1, MC38, and B16 cell levels under ultrasound treatment increased with increasing concentration.

[0073] Example 7: Evaluation of Ultrasonic Response Drug Release The microspheres prepared in Examples 2-5 were placed in release medium (PBS, pH 7.4) and in vitro drug release experiments were conducted at 37 °C under constant-rate oscillation conditions. Samples were taken at preset time points, and an equal volume of fresh medium was added to maintain a constant volume. During the experiment, the samples were subjected to a short-duration ultrasonic pulse treatment (1.0 MHz, 1.0–1.5 W / cm²) at 4 h and 24 h of incubation, respectively. 2 The ultrasound treatment was conducted at a duty cycle of 50% for 1 minute. Samples were taken immediately before and after the ultrasound treatment to record instantaneous release changes. No ultrasound was applied during the remaining time, allowing the microspheres to remain in a naturally released state. The collected supernatant was used to determine the drug content using appropriate quantitative methods, and the cumulative release rate was calculated. Finally, a step-like release curve was obtained under two ultrasound pulse stimuli to evaluate the temporal controllability of ultrasound-induced drug release. The ultrasound-response drug release curve is shown below. Figure 4 .like Figure 4 As shown, compared with the microspheres without ultrasound, the drugs in all four examples were released in a significantly responsive manner under the action of ultrasound; and the porous microspheres released more drugs and at a faster rate than the solid microspheres.

[0074] Example 8: Evaluation of the sustained-release effect of microspheres Weigh 2 mg of DIR and 50 mg of PLGA-PPa prepared in Example 1. Dissolve each in 500 μL of dichloromethane and mix thoroughly. Add the mixture to 12.5 mL of 1% PVA aqueous solution in an ice bath using a homogenizer at 9000 rpm and disperse for 1 min to form O / W microspheres. Pour the mixture into 400 mL of distilled water and stir at 100 rpm for 5 h to solidify. Collect the microspheres by centrifugation and wash three times with deionized water to remove residual PVA surfactant and organic solvent. Pre-freeze at -80℃ for about 12 h and then freeze-dry in a freeze dryer to obtain microsphere powder. The morphology of the microspheres is as follows. Figure 5 Figure a shows the morphology of the microspheres under an optical microscope, and Figure b shows the morphology of the microspheres under a fluorescence inverted microscope.

[0075] DIR and PLGA-PPa microspheres containing an equal amount of DIR were administered intranasally to BALB / c mice at a dose of 2.5 mg / kg. Fluorescence intensity was observed in vivo at 0 min, 20 min, 2 h, 4 h, 17 h, 24 h, 46 h, and 9 days. The sustained-release effect of the microspheres is shown in the figure. Figure 6 .like Figure 6 As shown, the DIR dye exhibited fluorescence fading after 4 hours, while PLGA-PPa microspheres loaded with the same amount of DIR still showed significant fluorescence on day 9. This demonstrates its sustained-release effect.

[0076] Example 9: Preparation of ultrasonically responsive porous microspheres of molecular adhesive JP-2-197 PLA-PPa Weigh 2 mg of JP-2-197 and 50 mg of PLA-PPa (PLA-PPa was prepared using the same method as in Example 1, except that PLA with a molecular weight of 11 kDa was used instead of PLAG, wherein the molar ratio of PLA to PPa was 1:1). Dissolve both in 500 μL of dichloromethane and mix them. Add 50 μL of 30 mg / mL ammonium bicarbonate solution (W1) to the mixture. Sonicate the mixture in an ice bath at 120 W for 2 seconds followed by 2 seconds, repeating this process three times to form a white W1 / O primary emulsion. Add the emulsion to 12.5 mL of 1% PVA aqueous solution in an ice bath (W2) using a homogenizer at 9000 rpm and disperse for 1 min to form W1 / O / W2 microspheres. The microspheres were poured into 400 mL of distilled water and stirred at 100 rpm for 5 hours to solidify. The microspheres were then collected by centrifugation and washed three times with deionized water to remove residual surfactants and organic solvents. After pre-freezing at -80°C for approximately 30 minutes, the microspheres were freeze-dried to obtain microsphere powder. Electron micrographs of the microsphere morphology are shown below. Figure 7 As shown.

Claims

1. An ultrasound-responsive microsphere formulation loaded with a protein degrading agent, wherein the microsphere is a microsphere structure loaded with a protein degrading agent formed by a conjugate of a biodegradable polymer and a sound-sensitive agent and a protein degrading agent.

2. The ultrasonically responsive microsphere formulation of the protein-degrading agent according to claim 1, wherein: The microsphere structure is a solid microsphere or a porous microsphere; and / or The aforementioned conjugate of the biodegradable polymer and the sound-sensing agent refers to a composite material system in which the sound-sensing agent molecule is stably linked to the main chain or side chain of the biodegradable polymer via covalent chemical bonds, forming a composite material system that can gradually release or activate the function of the sound-sensing agent in vivo as the polymer degrades; and / or The sonosensitive agent refers to an agent that, under ultrasonic stimulation, can be activated and undergo physical or chemical energy conversion to produce active species (such as singlet oxygen¹O₂, hydroxyl radical·OH, superoxide anion·O₂). - Substances or materials that trigger specific biological effects (such as cell damage, signal regulation, drug release), such as porphyrins, bodily oils, cyanides; and / or The biodegradable polymer refers to polyester-based synthetic polymers; and / or The molar ratio of the sound-sensitizing agent to the biodegradable polymer is 1:1 to 1:5; and / or The mass ratio of the coupling agent to the protein degrading agent is 10-50, for example, 20, 30, 40, etc.

3. The ultrasound-responsive microsphere formulation according to claim 1 or 2, wherein: The sound-sensing agent is selected from the following compounds: ; and / or The biodegradable polymer is selected from polylactic acid-glycolic acid copolymer (PLGA) or polylactic acid (PLA). Preferably, the coupling of the biodegradable polymer and the sound-sensitizing agent is a coupling of polylactic acid-glycolic acid copolymer (PLGA) and sound-sensitizing agent (R) as shown in Formula 1a (PLGA-R), or a coupling of polylactic acid (PLA) and sound-sensitizing agent (R') as shown in Formula 1b (PLA-R'): (PLGA) Formula 1a (PLA) Formula 1b in, In Formula 1a, the degree of polymerization of polylactic-co-glycolic acid copolymer (PLGA) is x:y = 90-10:10-90; for example, x is 50, 75, 100, etc.; y is 50, 30, 25, etc.; specifically, x is 100; y is 30. In Formula 1b, polylactic acid (PLA) is levorotatory, dextrorotatory, or racemic, with an intrinsic viscosity (IV) of 0.1~1.0 dL / g, preferably 0.2~0.6 dL / g, and a degree of polymerization z selected from an integer of 70~2100, preferably 550, 600, or 650. R and R' are ultrasonic-responsive groups derived from a sonicator, and specifically, each is independently selected from the structures shown below: ， ， 。 4. The ultrasound-responsive microsphere formulation according to any one of claims 1-3, wherein, The protein degrading agents include protein-targeting chimeras, molecular gels, and their prodrugs. Preferably, the protein-targeting chimera comprises: a BRD4 degrader, such as ARV-771 or ARV-825; a RAPR degrader, such as MS8815; a RIPK1 degrader, such as RI-ICR-5; and an EZH2 degrader, such as 180055. More preferably, the protein-targeting chimera is selected from the following compounds: ， ， ， ， Preferably, the molecular adhesive includes a BRD4 degrading agent, such as TMX-1, ZZ7-16-073, JP-2-197, or JQ1. More preferably, the molecular adhesive is selected from the following compounds: ， ， ， ， Preferably, an ARV-825 modified prodrug is used as a protein degrading agent, wherein the ARV-825 modified prodrug is selected from the following compounds: ， ， ， 。 5. A pharmaceutical composition comprising: The ultrasound-responsive microsphere formulation according to any one of claims 1-4, and Optionally, a pharmaceutically acceptable carrier.

6. A method for preparing an ultrasound-responsive microsphere formulation according to any one of claims 1-4, comprising the following steps: S1, by covalently linking the sound-sensitizer molecule to the main chain or side chain of the biodegradable polymer, a conjugate of the biodegradable polymer and the sound-sensitizer is obtained. S2, using the conjugate of the biodegradable polymer and the sonic sensitizer obtained in S1 and the protein degrader, ultrasonic responsive microspheres loaded with the protein degrader are prepared by an emulsification solvent evaporation method.

7. The method according to claim 6, wherein, S1 comprises: dissolving a sound-sensitizing agent and an equimolar amount of a carboxyl activator in an organic solvent, sealing the solution, and reacting it at any temperature between 0-40°C for 0.5-6 hours. Then, an equimolar amount of a biodegradable polymer is added, and the reaction is continued at any temperature between 0-40°C for 0.5-48 hours. The mixture is then dialyzed with an organic solvent (dialysis bag with a molecular weight of 1000-1500). After dialysis for 5-20 hours, the dialysate is replaced with purified water. After dialysis for 24-36 hours, the reaction product is pre-frozen (e.g., -80°C) for 12-24 hours, followed by lyophilization to obtain the conjugate of the biodegradable polymer and the sound-sensitizing agent. Preferably, in S1, the carboxyl activator is one or more selected from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, dicyclohexylcarbodiimide, 1-hydroxybenzotriazole, N-hydroxysuccinimide, N,N-diisopropylethylamine, and 4-dimethylaminopyridine (DMAP). Preferably, in S1, the organic solvents are the same or different and each independently is one or more selected from methanol, acetonitrile, dichloromethane, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, and dimethyl sulfoxide, etc. More preferably, the organic solvent is selected from dichloromethane and ethyl acetate. Preferably, in S1, PLGA is used as a biodegradable polymer, wherein the molecular weight of the PLGA is 15-120 kDa.

8. The method according to claim 6 or 7, wherein, S2 is selected from either Method 1 or Method 2: Method 1, the preparation method of porous microspheres includes: 1) Dissolve the protein degrading agent in an organic solvent to form a drug solution; dissolve the coupling compound of the biodegradable polymer and the sonic sensitizer in an organic solvent to form a polymer solution; mix the drug solution and the polymer solution to form an oil phase (O); dissolve the pore-forming agent in water to obtain an inner aqueous phase solution (W1); dissolve the surfactant in water to form an outer aqueous phase solution (W2). 2) Add the aqueous phase solution (W1) to the oil phase (O) and sonicate using a cell disruptor to obtain a white W1 / O colostrum; 3) Slowly pour the W1 / O colostrum into the external aqueous phase solution (W2) of the surfactant, and homogenize or shear disperse it evenly to form W1 / O / W2 embryo microspheres; 4) Disperse the solution containing W1 / O / W2 embryo microspheres in water and stir to solidify; 5) Collect the solidified microspheres by centrifugation or filtration, and wash them with deionized water to remove residual surfactants and organic solvents; 6) Pre-freeze at -20~-80℃ for 2~24h and then freeze-dry to obtain ultrasonically responsive porous microspheres loaded with protein degrading agents; Method 2, the preparation method of solid microspheres includes: 1) Dissolve the protein degrading agent in an organic solvent to form a drug solution; dissolve the conjugate of the biodegradable polymer and the sound sensitizer in an organic solvent to form a polymer solution; mix the drug solution and the polymer solution to form an oil phase (O); dissolve the surfactant in water to form an external aqueous phase solution (W). 2) The oil phase is dispersed in an external aqueous solution (W), and homogenized or sheared to form O / W embryo microspheres; 3) Disperse the solution containing O / W embryo microspheres in water and stir to solidify; 4) Collect the solidified microspheres by centrifugation or filtration, and wash them with deionized water to remove residual surfactants and organic solvents; 5) Pre-freeze at -20~-80℃ for 2~24h and then freeze-dry to obtain ultrasonically responsive solid microspheres loaded with protein degradation agents; Preferably, the sound-sensitizing agent is PPa, and the biodegradable polymer is PLGA or PLA. More preferably, the molar ratio of the sound-sensitizing agent to the biodegradable polymer is 1:1 to 1:

5. Preferably, in methods one and two, the organic solvents are the same or different and each independently is one or more selected from methanol, acetonitrile, dichloromethane, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, and dimethyl sulfoxide. Preferably, in S2, the concentration of the coupling compound between the biodegradable polymer and the sound-sensitive agent is 1% to 10% (g / mL), more preferably 5% to 10% (g / mL). Preferably, the mass ratio of the coupling agent to the protein degrading agent is 10-50, for example, 20, 30, 40, etc. Preferably, in step 2) of method one, the volume ratio of the internal aqueous phase solution to the oil phase is 1:20 to 1:100, the power of the cell disruptor is 100W to 120W, and the total ultrasonication time is 1-10s. Preferably, in step 3) of method one, the volume ratio of the external aqueous phase solution to the oil phase is 10:1 to 40:

1. Preferably, the homogenization or shearing speed in step 3) of method one and step 2) of method two is 9000 rpm to 10000 rpm, the homogenization time is 30 s to 2 min, and the conditions are an ice bath. Preferably, the curing time in step 4) of method one and step 3) of method two is 5~15h.

9. The method according to any one of claims 6-8, comprising the following steps: weighing 2 mg of protein degrading agent JP-2-197 molecular gel and 50 mg of PLGA-PPa, dissolving each in 500 μL of dichloromethane and mixing them, then adding 50 μL of 30 mg / ml ammonium bicarbonate solution. The microspheres were ultrasonically disrupted in an ice bath at 120 W for 2 seconds followed by a 2-second pause, for a total of three times, to form a white O / W1 pre-emulsion. The emulsion was then homogenized at 9000 rpm and added to 12.5 ml of 1% PVA solution W2 in an ice bath. The mixture was dispersed for 1 min to form W1 / O / W2 microspheres. These microspheres were then poured into 400 ml of distilled water and stirred at 100 rpm for 5 h to solidify. The microspheres were then collected by centrifugation and washed three times with deionized water to remove residual surfactants and organic solvents. After pre-freezing at -80°C for 12 h, the microspheres were freeze-dried in a freeze dryer to obtain microsphere powder.

10. The use of the ultrasound-responsive microsphere formulation according to any one of claims 1-4, the pharmaceutical composition according to claim 5, or the ultrasound-responsive microsphere formulation prepared by the method according to any one of claims 6-9 in the preparation of a drug. Preferably, the drug is a drug used to treat cancer. More preferably, the cancer includes metastatic or drug-resistant solid malignancies, such as breast cancer, lung cancer, liver cancer, stomach cancer, head and neck cancer, cervical cancer, pancreatic cancer, ovarian cancer, colon cancer, and prostate cancer.

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