Protein degradation chimera with mitochondrial targeted degradation function as well as preparation method and application of protein degradation chimera

Abstract

The invention relates to the technical field of biomedical material preparation, in particular to a protein degradation chimera with a mitochondrial targeted degradation function as well as a preparation method and application of the protein degradation chimera. The chemical formula of the protein degradation chimera disclosed by the invention is C45H63BFN5O10S. According to the ROS response activated mitochondrial protein degradation chimera designed by the invention, space-time selective activation of a tumor part is realized through combined administration of the lipid nanoparticles and the microneedle patch, mitochondrial related proteins can be efficiently degraded, ICD can be induced, and an anti-tumor immune curative effect can be remarkably enhanced. The system provides a new material and a new strategy for tumor immunotherapy, and has good clinical transformation potential.

Description

Technical Field

[0001] This invention relates to the field of biomedical material preparation technology, and in particular to a protein degradation chimera with mitochondrial-targeted degradation function, its preparation method and application. Background Technology

[0002] Mitochondria are important double-membrane organelles in eukaryotic cells, serving as cellular energy factories and playing crucial roles in vital biological processes such as programmed cell death regulation, signal transduction, cell communication, and immune stress. Recent studies have shown that mitochondria are key hubs for enhancing the immune response to "cold tumors." Mitochondrial dysfunction in tumor cells can drive immunogenic cell death (ICD) by releasing damage-associated molecular patterns (DAMPs), inhibiting tumor growth and metastasis, and reshaping the tumor immune microenvironment. Therefore, mitochondria are important targets for enhancing tumor immunogenicity and synergizing with current immunotherapies. To date, more than 1,000 proteins have been identified in mitochondria, playing key roles in cellular energy metabolism, apoptosis regulation, and redox homeostasis. Therefore, influencing mitochondrial homeostasis in tumor cells by degrading mitochondrial proteins is one of the important pathways to inhibit tumor growth and metastasis and regulate the tumor immune microenvironment. Based on this, the development of novel and effective drug molecules that target and degrade mitochondrial proteins and the study of their mechanisms of action are of great importance and urgency in the field of tumor immunotherapy.

[0003] Proteolysis-targeting chimeras (PROTACs) are an emerging class of targeted protein degradation drugs that have been extensively studied and applied in recent years to treat various diseases, including cancer. PROTACs utilize the intracellular natural ubiquitin-proteasome system (UPS) to tag the protein to be degraded with ubiquitin, enabling it to be recognized and hydrolyzed by the proteasome. Compared to traditional strategies using small molecule inhibitors, PROTACs can more thoroughly block the biological function of the target protein, circumvent drug resistance caused by binding site mutations, and achieve the degradation of "undruggable" proteins that are difficult to target with traditional small molecules.

[0004] However, PROTACs still face numerous challenges in clinical applications. First, their poor artificial controllability and spatiotemporal selectivity lead to uncontrolled degradation, potentially causing significant toxicity to normal cells and reducing therapeutic specificity. Furthermore, the large molecular weight (>700 Da) of PROTACs results in strong hydrophobicity, poor permeability, and low bioavailability. Therefore, developing on / off strategies or employing delivery systems to achieve tumor-targeted selective delivery and activation will help improve the therapeutic efficacy, specificity, and safety of PROTACs, laying the foundation for their widespread clinical application. In addition, because mitochondria lack proteasomes, and PROTACs targeting mitochondrial proteins require personalized high-throughput screening of different proteins to obtain effective point-of-care (POI) ligands, there is currently very little research using PROTAC technology to degrade specific proteins within mitochondria, a membrane-bound organelle. Therefore, given the crucial role of mitochondrial protein dysfunction in tumor growth and immune regulation, constructing a controllable degradation-targeting chimeric mitochondrial organelle-related protein hydrolysis solution is a key problem that needs to be addressed in this field. Summary of the Invention

[0005] The purpose of this invention is to provide a ROS-responsive mitochondrial protein degradation chimera that uses trifluoromethyl 2-thiophenecarboxylate (TTFA) as a reactive oxygen species (ROS) inducer to efficiently degrade mitochondrial-related proteins and induce ICD. Combined with a nanodelivery and microneedle drug delivery system, it can achieve tumor site-specific activation and enhance anti-tumor immunotherapy.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a protein degradation chimera with mitochondrial-targeted degradation function, as shown in Formula I:

[0008]

[0009] The present invention also provides the application of the protein degradation chimera in the preparation of tumor immunotherapy drugs.

[0010] This invention also provides a method for preparing a protein degradation chimera with mitochondrial-targeted degradation function, comprising the following steps:

[0011] (1) 2,2-Dimethyl-4-oxo-3,8,11,14-tetraoxo-5-azahexadecanoic acid, E3 ubiquitin ligase ligand, N,N-diisopropylethylamine, hexafluorophosphate-O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea and N,N-dimethylformamide were mixed and reacted for 50-70 min to obtain intermediate C2;

[0012] (2) Mix intermediate C2 with trifluoroacetic acid and react to obtain intermediate C3;

[0013] (3) Intermediate C3, phenylboronic acid ester, hexafluorophosphate-O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea, N,N-diisopropylethylamine and mixed solvent were mixed and reacted for 2.5-3.5 h. The mixture was then extracted, washed with water, dried, concentrated and purified to obtain the protein degradation chimera.

[0014] Preferably, the E3 ubiquitin ligase ligand in step (1) is (2S,4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide;

[0015] The molar ratio of 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxo-5-azahexadecanoic acid, E3 ubiquitin ligase ligand, N,N-diisopropylethylamine, and hexafluorophosphate-O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea is 1-2:1-2:1-2:1-2;

[0016] The molar volume ratio of 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxo-5-azahexadecanoic acid to N,N-dimethylformamide is 1–2 mmol: 5 mL.

[0017] The reaction temperature in step (1) is 20–30°C;

[0018] In step (2), the molar volume ratio of intermediate C2 to trifluoroacetic acid is 1 mmol: 2.5–3.5 mL.

[0019] The reaction temperature in step (2) is 20–30°C;

[0020] The molar ratio of intermediate C3, phenylboronic acid ester, hexafluorophosphate-O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea and N,N-diisopropylethylamine in step (3) is 1:1:1 to 1.5:2.0 to 2.6.

[0021] The mixed solvent in step (3) is a solution obtained by mixing dichloromethane and N,N-dimethylformamide in a volume ratio of 4 to 6:1;

[0022] In step (3), the molar volume ratio of intermediate C3 to mixed solvent is 1 mmol: 9-11 mL.

[0023] Preferably, the phenylboronic ester in step (3) is 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)-3-(difluoromethyl)benzoic acid; the preparation method is as follows:

[0024] 1) Methyl 4-bromo-3-methylbenzoate, N-bromosuccinimide, azobisisobutyronitrile and carbon tetrachloride were mixed and reacted for 8-12 h to obtain a reaction solution. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain a concentrate. The concentrate was purified to obtain compound 1.

[0025] 2) Mix compound 1, silver sulfate and dioxane aqueous solution, react for 50-70 min to obtain reaction solution, filter the reaction solution and take the filtrate; extract the filtrate with ethyl acetate, take the organic phase, wash the organic phase, dry, filter, concentrate the filtrate under reduced pressure and purify to obtain compound 2;

[0026] 3) Compound 2, diethylaminosulfur trifluoride and dichloromethane were mixed and reacted for 8-12 h to obtain a reaction solution; the organic phase in the reaction solution was washed, dried, filtered, and the filtrate was concentrated under reduced pressure and purified to obtain compound 3;

[0027] 4) Compound 3, bis(pinacol)diboron, potassium acetate, [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride and N,N-dimethylformamide were mixed and reacted for 2.5-3.5 h to obtain the reactant; the reactant was mixed with the catalyst and reacted for 3-5 h to obtain the reaction solution; the reaction solution was diluted with diethyl ether to obtain the diluted solution; the organic phase in the diluted solution was washed, dried, filtered, and the filtrate was concentrated under reduced pressure and purified to obtain compound 4;

[0028] 5) Mix compound 4, tetrahydrofuran aqueous solution and lithium hydroxide aqueous solution, react for 8-12 h to obtain a reaction solution, dilute the reaction solution to obtain a diluted solution; neutralize the diluted solution to obtain the extract; extract the extract with ethyl acetate, take the organic phase, dry and concentrate to obtain phenylboronic acid ester.

[0029] Preferably, the mass ratio of methyl 4-bromo-3-methylbenzoate, N-bromosuccinimide, and azobisisobutyronitrile in step 1) is 2–2.5: 1.5–2.3: 0.15–0.18.

[0030] The mass-to-volume ratio of methyl 4-bromo-3-methylbenzoate to carbon tetrachloride is 2–2.5 g: 25 mL;

[0031] In step 2), the mass ratio of compound 1 to silver sulfate is 300–312:495–502.

[0032] The mass-to-volume ratio of compound 1 to dioxane aqueous solution is 300–312 mg: 20 mL;

[0033] The volume ratio of dioxane to water in an aqueous solution of dioxane is 1-2:1-2;

[0034] In step 3), the mass-to-volume ratio of compound 2 and diethylaminosulfur trifluoride is 230–240 mg: 370–390 μL.

[0035] The mass-to-volume ratio of compound 2 to dichloromethane is 230–240 mg: 14–16 mL;

[0036] In step 4), the mass ratio of compound 3, bis(pinacol)diboron, potassium acetate, and [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride is 180–190: 225–230: 215–225: 25–35.

[0037] The mass-to-volume ratio of compound 3 and N,N-dimethylformamide is 180–190 mg: 2–3 mL;

[0038] In step 5), the mass-to-volume ratio of compound 4 and the tetrahydrofuran aqueous solution is 14.5–15.5 mg: 0.8 mL.

[0039] The mass-to-volume ratio of compound 4 to the lithium hydroxide aqueous solution was 14.5–15.5 mg: 140–160 μL.

[0040] The present invention also provides a lipid nanoparticle of a protein degradation chimera with mitochondrial targeted degradation function, wherein the lipid nanoparticle of the protein degradation chimera contains the protein degradation chimera, an amphiphilic copolymer, 1,2-dioleoyl-3-trimethylammonium propane and 1,2-distearate-sn-propanetriyl-3-phosphocholine;

[0041] The mass ratio of the protein degradation chimera, the amphiphilic copolymer, 1,2-dioleoyl-3-trimethylammonium propane, and 1,2-distearate-sn-propanetriyl-3-phosphocholine is 0.8–1.2:8–12:1–3:1–3;

[0042] The amphiphilic copolymer is distearate phosphatidylethanolamine-polyethylene glycol 2000.

[0043] The present invention also provides the application of the lipid nanoparticles in the preparation of tumor immunotherapy drugs.

[0044] The present invention also provides a microneedle patch for activating the protein degradation chimera, which is a microneedle patch loaded with 2-thiophenecarboxylic acid trifluoromethyl ester based on a hyaluronic acid and polyvinyl alcohol composite system.

[0045] The present invention also provides a tumor immunotherapy drug, comprising the protein degradation chimera or the lipid nanoparticles and the microneedle patch;

[0046] The tumors include colorectal cancer and / or breast cancer.

[0047] The method of the present invention has the following advantages:

[0048] This invention is the first to construct a spatiotemporally selective mitochondrial protein degradation chimera through a ROS-responsive activation strategy. By combining nanoparticle delivery with microneedle patches, it achieves tumor site-specific activation, which can efficiently degrade mitochondrial-related proteins and induce ICD, thus exerting significant anti-tumor immunotherapeutic effects.

[0049] The protein degradation chimera constructed in this invention exhibits high spatiotemporal selectivity, activating only at the tumor site via ROS, reducing damage to normal cells and improving therapeutic specificity. It eliminates the need for ligand selection, achieving multi-target degradation through covalent binding of ROS-responsive groups to neighboring proteins, overcoming the limitation of "undruggable" proteins. Furthermore, it synergistically enhances ICD; mitochondrial protein degradation induces mitochondrial damage, promoting DAMPs release and enhancing antigen presentation and T-cell infiltration. Finally, the combination of nanoparticles and microneedles improves bioavailability and delivery efficiency, ensuring highly efficient activation at the tumor site.

[0050] The ROS-responsive mitochondrial protein degradation chimera designed in this invention exhibits excellent mitochondrial targeting and protein degradation capabilities. This system overcomes the problems of poor spatiotemporal selectivity and low bioavailability of traditional PROTACs, promoting the infiltration and killing function of immune cells at tumor sites. This provides new research ideas and effective technical support for novel tumor immunotherapy drugs, mitochondrial protein degradation, and treatment options for colorectal cancer and breast cancer. Attached Figure Description

[0051] Figure 1 The preparation route for phenylboronic acid esters;

[0052] Figure 2 The preparation route for protein degradation chimeras;

[0053] Figure 3 The 1H spectrum of the protein degradation chimera;

[0054] Figure 4 HRMS spectrum of protein degradation chimera;

[0055] Figure 5 Bright-field microscopic images of cancer cells after ROS activation by TTFA-generated protein degradation chimeras and CCK8 detection results;

[0056] Figure 6 To detect, using laser confocal microscopy, the calreticulin eversion induced in cancer cells by ROS activation generated by TTFA in protein degradation chimeras.

[0057] Figure 7 To analyze the levels of HSP70 and HMGB1 induced by the protein degradation chimera after ROS activation by TTFA in cancer cells;

[0058] Figure 8 The level of ATP release induced by protein degradation chimeras in cancer cells after ROS activation by TTFA-generated ROS.

[0059] Figure 9 To detect changes in mitochondrial membrane potential in cancer cells induced by ROS activation generated by TTFA using laser confocal microscopy.

[0060] Figure 10 To detect the specific binding of biotin-modified protein degradation chimeras to mitochondrial proteins after ROS activation by TTFA using laser confocal microscopy and conlocalization.

[0061] Figure 11 Particle size distribution and transmission electron microscopy images of lipid nanoparticles carrying protein degradation chimeras;

[0062] Figure 12 Scanning electron microscope image of a TTFA-loaded microneedle patch;

[0063] Figure 13 The fluorescence enrichment of lipid nanoparticles carrying protein degradation chimeras in the main tissues and tumors of tumor-bearing mice;

[0064] Figure 14 The results of tumor inhibition by lipid nanoparticles combined with TTFA microparticles in the treatment of breast cancer and colorectal cancer were statistically analyzed, along with changes in body weight in mice.

[0065] Figure 15 TUNEL staining results of tumor tissue from breast cancer treated with lipid nanoparticles combined with TTFA microtargets. Detailed Implementation

[0066] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0067] Example 1

[0068] Preparation of protein degradation chimeras with mitochondrial-targeted degradation function

[0069] 1. Preparation of phenylboronic acid ester (4-(4,4,5,5-tetramethyl-1,3,2-dioxaboran-2-yl)-3-(difluoromethyl)benzoic acid)

[0070] 1) Preparation of compound 1

[0071] Methyl 4-bromo-3-methylbenzoate (2.3 g, 10 mmol), N-bromosuccinimide (1.9 g, 11 mmol), and azobisisobutyronitrile (164 mg, 1 mmol) were added to carbon tetrachloride (25 mL). The mixture was heated under argon protection and refluxed for 10 h. After the reaction was complete, the reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The concentrate was purified by silica gel column chromatography (elution: n-hexane / ethyl acetate = 15:1) to give compound 1 as a pale solid, in 71% yield.

[0072] 2) Preparation of compound 2

[0073] Compound 1 (308 mg, 1.0 mmol) obtained in step 1) was dissolved in an aqueous solution of dioxane (dioxane to water 1:1 v / v, 20 mL), and silver sulfate (Ag₂SO₄, 499 mg, 1.6 mmol) was added. The mixture was heated under reflux for 60 min in the dark. After cooling, the reaction solution was filtered, and the filtrate was extracted with ethyl acetate (20 mL). The combined organic phases were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The concentrate was purified by silica gel column chromatography to give compound 2 as a white solid in 96% yield.

[0074] 3) Preparation of compound 3

[0075] Compound 2 (235.4 mg, 0.96 mmol) obtained in step 2) was dissolved in anhydrous dichloromethane (15 mL), and diethylaminosulfur trifluoride (DAST, 380 μL, 2.88 mmol) was slowly added dropwise under ice bath conditions at 0 °C. The reaction system was brought to room temperature and stirred overnight. After the reaction was completed, the organic phase was washed successively with deionized water (20 mL) and saturated brine (20 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The concentrate was purified by silica gel column chromatography (eluent: n-hexane / ethyl acetate = 20:1) to give the fluorinated product compound 3 in 78% yield.

[0076] 4) Preparation of compound 4

[0077] Compound 3 (185.1 mg, 0.75 mmol) obtained in step 3), bis(pinacol)diboron (B2Pin2, 228.6 mg, 0.90 mmol), potassium acetate (220.8 mg, 2.25 mmol), and [1,1'-bis(diphenylphosphine)ferrocene]palladium(II) dichloride (Pd(dppf)Cl2, 30.6 mg, 0.0375 mmol) were added to anhydrous N,N-dimethylformamide (DMF, 2.5 mL). The mixture was heated to 100 °C and reacted for 3 h under an inert gas atmosphere. Subsequently, the same amount of [1,1'-bis(diphenylphosphine)ferrocene]palladium(II) dichloride (Pd(dppf)Cl2) catalyst (30.6 mg) was added to the reaction system, and the reaction was continued at 100 °C for 4 h. After the reaction was complete, the mixture was cooled to room temperature, diluted with diethyl ether (30 mL), and washed successively with deionized water (20 mL) and saturated brine (20 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrate was purified by silica gel column chromatography (eluent: n-hexane / ethyl acetate = 10:1) to give compound 4 as a light-colored oil, in 26% yield.

[0078] 5) Preparation of compound 5

[0079] Compound 4 (15.1 mg, 0.05 mmol) obtained in step 4) was dissolved in 0.8 mL of tetrahydrofuran aqueous solution (tetrahydrofuran to water ratio 6:2) under ice bath conditions at 0 °C, and 150 μL (0.15 mmol) of 1 M lithium hydroxide aqueous solution was slowly added. The reaction system was brought to room temperature and stirred for 10 h. After the reaction was completed, water (1 mL) was added for dilution, and the mixture was neutralized with 1 M hydrochloric acid solution under ice bath conditions. The product was extracted with ethyl acetate, and the organic phase was dried and concentrated to give the target intermediate compound 5, which is a phenylboronic ester containing a free carboxyl group (4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)-3-(difluoromethyl)benzoic acid), with a yield of 91%. This crude product was used directly for subsequent coupling reactions without purification.

[0080] The specific preparation process is as follows: Figure 1 As shown.

[0081] 2. Preparation of protein degradation chimeras

[0082] 2,2-Dimethyl-4-oxo-3,8,11,14-tetraoxo-5-azahexadecanoic acid (1.63 mmol), E3 ubiquitin ligand (2S,4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl)phenyl)ethyl)pyrrolidine-2-carboxamide (1.63 mmol), N,N-diisopropylethylamine (DIPEA) (1.63 mmol), and HATU coupling agent (hexafluorophosphate-O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea) (1.63 mmol) were mixed in N,N-dimethylformamide (DMF) (5 mL) and reacted at 25 °C for 60 min. After the reaction was complete, the reaction solution was extracted with an organic solvent (ethyl acetate). The resulting organic phase was washed successively with water and saturated brine, then dried with anhydrous sodium sulfate and other drying agents. Finally, the solution was filtered and the solvent was removed by rotary evaporation under reduced pressure to obtain the crude product. Then, it was purified by rapid column chromatography (gradient elution: 0% to 10% methanol in ethyl acetate) to give intermediate C2, which is a light brown solid with a yield of 50%.

[0083] Intermediate C2 was mixed with trifluoroacetic acid (TFA) at a ratio of 1 mmol:3 mL and deprotected at 25 °C to give intermediate C3 containing free amino and free carboxyl groups. Subsequently, compound 5 (phenylboronic acid ester) (0.2 mmol) and intermediate C3 (0.2 mmol) were coupled for 3 h in a mixed solvent of anhydrous dichloromethane and DMF (5:1 volume ratio) in the presence of hexafluorophosphate-O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea HATU (0.24 mmol) and N,N-diisopropylethylamine DIPEA (0.4 mmol). After the reaction, the mixture was extracted with ethyl acetate, washed with water, and the organic phase was dried over anhydrous sodium sulfate. The dried organic phase was evaporated and concentrated, and purified by silica gel column chromatography (eluent: dichloromethane / methanol). The purified product was collected and dried under vacuum to obtain the pure protein degradation chimeric product. A pale yellow solid, with a yield of 41%.

[0084] Specific preparation methods are as follows: Figure 2 As shown.

[0085] The structure and molecular weight of the protein degradation chimera were characterized by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry, and the results are as follows: Figures 3-4 As shown.

[0086] Figures 3-4As shown, the product structure was confirmed by 1H NMR and HRMS: 1H NMR (400MHz, DMSO-d6) δ 8.98 (s, 1H), 8.67 (t, J = 5.5Hz, 1H), 8.43 (d, J = 7.9Hz, 1H), 7.94 (s, 1H), 7.88-7.81 (m, 1H), 7.78 (d, J = 7.3Hz, 1H), 7.48-0.41 (m, 2H), 7.40-7.30 (m, 3H), 5.72 (s, 1H), 5.60 (s, 1H), 4.89 (t, J = 7.2Hz, 1H), 4.54 (d, J = 9.6Hz, 1H),4.44(t,J=8.1Hz,1H),4.28(dq,J=5.6,3.1,2.5Hz,1H),3.95(s,2H),3.62-3.53(m,4H),3.46-3.38(m,4H),2.45(s, 3H),2.10-2.00(m,1H),1.82-1.71(m,1H),1.36(d,J=7.0Hz,3H),1.32-1.29(m,12H),0.93(s,9H).HRMS(ESI)m / z[M+Na] + 918.4270 (calcd for C) 45 H 63 BFN5NaO 10 S), the Na ions in this detection result are automatically added during the mass spectrometry detection process for neutralization reaction, and the final chemical formula of the protein degradation chimera is C. 45 H 63 BFN5O 10 S was measured to be 918.4275. The above data matches the expected structure.

[0087] Protein degradation chimeras are as shown in Formula I:

[0088]

[0089] Preparation of active solution:

[0090] 4.47 mg of the pure protein degradation chimera powder was dissolved in 100 μL of dimethyl sulfoxide to a final concentration of 50 mM. In subsequent experiments, the prepared mitochondrial protein degradation chimera solution was diluted appropriately as needed.

[0091] Experimental Example 1

[0092] Evaluation of the responsive activation of protein degradation chimeras and their killing effect on tumor cells

[0093] EMT6 breast cancer cells were used at a rate of 5 × 10 4Cells were seeded at a density of / wells in 12-well plates and cultured overnight. Four treatment groups were then established: a. blank control group; b. Example group (protein degradation chimera) (20 μM); c. 2-thiophenecarboxylic acid trifluoromethyl ester (TTFA) group (1 mM); d. Protein degradation chimera pre-incubation for 6 h followed by TTFA group (Example + TTFA). Cell morphology was observed using an optical microscope, and cell viability was detected using the CCK8 assay. The results showed... Figure 5 As shown, treatment with the protein degradation chimera alone or TTFA alone had no significant effect on cell viability. However, when the protein degradation chimera and TTFA were treated together, the survival rate dropped to 35%. Bright-field cell images further corroborated the cytotoxicity of the combined treatment. This indicates that the protein degradation chimera can efficiently induce tumor cell death after TTFA activation.

[0094] Experiment Example 2

[0095] Assessment of the ability of protein degradation chimeras to induce immunogenic cell death (ICD) in tumor cells

[0096] EMT6 tumor cells were injected at a rate of 5 × 10 4 After seeding cells at the specified density in 12-well plates and culturing overnight, the protein degradation chimera and TTFA were treated with cells as described in Example 1. After incubation, the cell supernatant was collected, and the ATP release levels of different groups were quantitatively detected using a kit; or, the cells were centrifuged at 12000 rpm for 30 min at 4°C, the supernatant was collected, loading buffer was added, and the mixture was boiled for 5 min to prepare supernatant protein samples. Western blot was used to examine the levels of HMGB1 and HSP70 proteins released by the cells. Tumor cells were seeded at 5 × 10⁻⁶ cells / well. 4 Cells were seeded at a density of / wells in confocal microscopy dishes and cultured overnight. Different experimental groups (blank control group, Example (protein degradation chimera), TTFA, Example + TTFA (protein degradation chimera + TTFA)) were set up for drug treatment. After incubation, cells were fixed with 4% paraformaldehyde at room temperature for 20 min, incubated overnight at 4°C with anti-Calreticulin primary antibody, and then stained with Alexa Fluor 633goat anti-rabbit secondary antibody at room temperature for 2 h, followed by DAPI staining for 10 min. The occurrence of Ecto-CRT in cells of different treatment groups was observed and analyzed using confocal microscopy. Results are as follows: Figure 6 As shown, laser scanning confocal microscopy (CLSM) results indicate that the combination of protein degradation chimera and TTFA leads to increased localization of the ICD marker calreticulin on the EMT6 tumor cell membrane (ecto-CRT). Figure 7 , 8The results showed that a large number of other DAMPs, including HMGB1, HSP70 and ATP, were also found in the cell supernatant, demonstrating that the protein degradation chimera can be effectively activated by mitochondrial-generated ROS in TTFA-treated cells, driving the ICD effect in tumor cells.

[0097] Experimental Example 3

[0098] Verification of the effects of protein degradation chimeras on mitochondrial damage and their co-localization with mitochondria.

[0099] EMT6 cells were seeded in confocal microscope dishes. The confocal microscope dishes were treated with a combination of biotin and TTFA as described in Experiment 1. An untreated group, a group treated with only the biotin, and a group treated with only TTFA served as controls. To detect changes in mitochondrial membrane potential, JC-1 staining working solution was added to the dishes after incubation, and the dishes were incubated at 37°C for 20 min. Finally, Hoechst 33342 staining was performed for 10 min to label the cell nuclei. The effect of the activated biotin on mitochondrial membrane potential was observed and analyzed under a confocal microscope. To verify at the cellular level whether the protein-binding function of the biotin was activated in the mitochondrial region, thereby binding to mitochondrial proteins, a biotin-modified biotin was synthesized. The biotin and TTFA were used to treat the confocal microscope dishes, with the biotin treated alone serving as a control. After incubation, cells were fixed with 4% paraformaldehyde for 20 min at room temperature, and then permeabilized with 0.5% Triton-X100 to remove unbound biotin chimeras. Cells were then blocked with 5% BSA, incubated overnight at 4°C with the mitochondrial marker anti-TOMM20 primary antibody, and then incubated at room temperature with Alexa Fluor 633goat anti-rabbit secondary antibody and streptavidin AlexaFluro488 for 1 h. Finally, DAPI staining was performed for 10 min, and the co-localization of biotin chimeras with mitochondria was observed and analyzed under a confocal microscope. Figure 9 As shown, the CLSM results indicate that after the protein degradation chimera was effectively activated by mitochondrial-generated ROS in TTFA-treated EMT6 cells, the number of JC-1 fluorescent probe monomers increased (enhanced green fluorescence), indicating a decrease in mitochondrial membrane potential and suggesting that it caused more severe mitochondrial damage. Furthermore, from... Figure 10It can be seen that, compared with the unactivated biotin alone, the biotin in TTFA-treated cells has a high degree of overlap with the mitochondrial localization, indicating that the biotin with the responsive group phenylboronic acid ester as the target protein binding ligand can be effectively activated in the mitochondria, the main ROS-producing region, and achieve selective binding with mitochondrial-related proteins.

[0100] Experiment Example 4

[0101] Preparation and characterization of lipid nanoparticles encapsulating protein degradation chimeras and TTFA microneedles (TTFA@MN) containing activated protein degradation chimera lipid nanoparticles.

[0102] 1 mg of the protein-degrading chimera prepared in Example 1, 10 mg of the amphiphilic copolymer distearylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), 2 mg of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), and 2 mg of 1,2-distearyl-sn-propanetriyl-3-phosphocholine (DSPC) were dissolved in 1 mL of tetrahydrofuran (THF) solution. The mixture was then added dropwise to 10 mL of PBS (pH 7.4, 0.01 M) under sonication using a micro-tip sonicator to obtain lipid nanoparticles containing the protein-degrading chimera of Example 1. The lipid nanoparticles were transferred to a dialysis bag and dialyzed overnight to remove organic solvents, excess lipids, and proteins from the solution. The solution was then repeatedly ultrafiltered and centrifuged at 5000 rpm (with a molecular weight cutoff of 100 kDa) to concentrate it to the target concentration. After filtration and purification through a 0.45 μm filter membrane, purified lipid nanoparticles were finally obtained.

[0103] The prepared lipid nanoparticles were characterized by monitoring the particle size of the prepared lipid nanoparticles using a dynamic light scattering instrument; the lipid nanoparticles were dropped onto a carbon support film copper grid, and the morphology of the nanoparticles was observed by transmission electron microscopy.

[0104] Before preparing the microneedle patch, a 4% hyaluronic acid (HA) solution and a 20% polyvinyl alcohol (PVA) solution were prepared and stored at room temperature. 300 μl of the 4% HA solution was placed in a 1.5 mL centrifuge tube, and TTFA was added according to a predetermined ratio. The mixture was repeatedly blown and aspirated to mix thoroughly, then transferred to a microneedle molding mold. Due to the high viscosity of HA, the mold was centrifuged at 3500 rpm for 25 min to ensure the drug fully filled the mold and eliminate air bubbles. The mold was then leveled using 4% HA and dried in a 30°C oven for 2 h. This leveling-drying cycle was repeated three times to form a dense substrate structure. A 20% PVA solution was then dropped onto the mold surface to cover the microneedle array, and dried under the same conditions to form a mechanically reinforcing layer. After drying, the microneedle array was flexibly separated to avoid structural damage. The morphology of the microneedles was characterized using scanning electron microscopy. Figure 11 As shown, the lipid nanoparticles encapsulating the protein degradation chimera of Example 1 had a particle size of approximately 119 nm, as determined by dynamic light scattering testing and transmission electron microscopy (TEM). The microneedle patch loaded with TTFA was characterized using scanning electron microscopy. Figure 12 The results demonstrate that the microneedle patch was successfully prepared.

[0105] Experimental Example 5

[0106] Validation of tumor-targeted drug delivery

[0107] To verify the tumor-targeting properties of the lipid nanoparticles prepared in Example 4, the fluorescent dye Dil was added during the preparation of the lipid nanoparticles. Balb / c mice were used to establish a subcutaneous EMT6 tumor-bearing model. The Dil-containing lipid nanoparticles were injected into the tumor-bearing mice via the tail vein. Subsequently, at six time points (0h, 2h, 6h, 12h, 24h, and 48h), the mice were euthanized by dislocation, and tissues such as the heart, liver, spleen, lung, kidney, and tumor were collected. Xenogen... Fluorescence imaging was performed using the LuMina II small animal in vivo imaging system to determine the tumor enrichment level of protein-degraded chimeric lipid nanoparticles. Results are as follows: Figure 13 As shown, the protein-degrading chimeric lipid nanoparticles with nanoscale dimensions effectively accumulated in mouse tumors through enhanced permeability and retention effect (EPR), reaching maximum fluorescence intensity 24 hours post-injection. Therefore, TTFA-loaded microneedle patches were inserted into mouse tumor tissue 24 hours after tail vein injection of the protein-degrading chimeric lipid nanoparticles.

[0108] Heart means heart; Liver means liver; Spleen means spleen; Lung means lung; Kidney means kidney; Tumor means tumor.

[0109] Experimental Example 6

[0110] In vivo antitumor effects of combined therapy

[0111] Balb / c mice were used to construct breast cancer and colorectal cancer tumor models, respectively, using EMT6 and CT26 cells. EMT6 and CT26 cells were cultured in vitro. When the cells reached 80% confluence, they were digested, centrifuged, and resuspended in PBS. The cell concentration was then adjusted to 4 × 10⁻⁶ cells / mL. 7 Prepare a concentration of [value] / mL and store on ice. Anesthetize mice using a 3% isoflurane inhalation anesthesia system. Inoculate 50 μL of sterile CT26 or EMT6 cell suspension into the second pair of mammary fat pads on the right side of the mice. Wait for the tumor to grow to 80 mm. 3 Subsequent experiments were conducted around the time of the event. Tumor-bearing mice were randomly divided into four groups (a. control; b. lipid nanoparticles prepared in Example 4; c. TTFA@MN (microneedle patch prepared in Example 4); d. lipid nanoparticles prepared in Example 4 combined with TTFA@MN (microneedle patch prepared in Example 4)). Specifically, lipid nanoparticles were injected into tumor-bearing mice via the tail vein. Then, based on the optimal tumor enrichment time selected in the above protocol, TTFA-loaded microneedle patches were directly attached to the tumor site to responsively activate the drug-loaded nanoparticles enriched at the tumor site. Tumor volume and body weight were then monitored and recorded, and tumor tissue sections were prepared for analysis of apoptosis and proliferation. Results are as follows: Figure 14 As shown, the combined treatment of lipid nanoparticles and TTFA@MN has excellent anti-tumor therapeutic effects in both breast cancer and colorectal cancer, and there is no significant difference in the weight data of mice between the treatment group and the control group, proving that this strategy has no physiological toxicity to mice. Figure 15 Immunostaining results showed that the combined treatment with lipid nanoparticles and TTFA microneedle patches significantly increased tumor apoptosis and necrosis.

[0112] As demonstrated by the above embodiments, the ROS-responsive mitochondrial protein degradation chimera designed in this invention achieves spatiotemporal selective activation at the tumor site through combined administration of lipid nanoparticles and microneedle patches. This results in efficient degradation of mitochondrial-related proteins, induction of ICD, and significant enhancement of anti-tumor immunotherapy efficacy. This system provides new materials and strategies for tumor immunotherapy and possesses excellent clinical translational potential.

[0113] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A protein degradation chimera with mitochondrial-targeted degradation function, characterized in that, The protein degradation chimera is shown in formula (I):

2. The use of the protein degradation chimera according to claim 1 in the preparation of tumor immunotherapy drugs.

3. A method for preparing a protein degradation chimera with mitochondrial-targeted degradation function, characterized in that, Includes the following steps: (1) 2,2-Dimethyl-4-oxo-3,8,11,14-tetraoxo-5-azahexadecanoic acid, E3 ubiquitin ligase ligand, N,N-diisopropylethylamine, hexafluorophosphate-O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea and N,N-dimethylformamide were mixed and reacted for 50-70 min to obtain intermediate C2; (2) Mix intermediate C2 with trifluoroacetic acid and react to obtain intermediate C3; (3) Intermediate C3, phenylboronic acid ester, hexafluorophosphate-O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea, N,N-diisopropylethylamine and mixed solvent were mixed and reacted for 2.5-3.5 h. The mixture was then extracted, washed with water, dried, concentrated and purified to obtain the protein degradation chimera.

4. The preparation method according to claim 3, characterized in that, The E3 ubiquitin ligase ligand in step (1) is (2S,4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide; The molar ratio of 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxo-5-azahexadecanoic acid, E3 ubiquitin ligase ligand, N,N-diisopropylethylamine, and hexafluorophosphate-O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea is 1-2:1-2:1-2:1-2; The molar volume ratio of 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxo-5-azahexadecanoic acid to N,N-dimethylformamide is 1–2 mmol: 5 mL. The reaction temperature in step (1) is 20–30°C; In step (2), the molar volume ratio of intermediate C2 to trifluoroacetic acid is 1 mmol: 2.5–3.5 mL. The reaction temperature in step (2) is 20–30°C; The molar ratio of intermediate C3, phenylboronic acid ester, hexafluorophosphate-O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea and N,N-diisopropylethylamine in step (3) is 1:1:1 to 1.5:2.0 to 2.

6. The mixed solvent in step (3) is a solution obtained by mixing dichloromethane and N,N-dimethylformamide in a volume ratio of 4 to 6:1; In step (3), the molar volume ratio of intermediate C3 to mixed solvent is 1 mmol: 9-11 mL.

5. The preparation method according to claim 3, characterized in that, The phenylboronic acid ester mentioned in step (3) is 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)-3-(difluoromethyl)benzoic acid; The preparation method is as follows: 1) Methyl 4-bromo-3-methylbenzoate, N-bromosuccinimide, azobisisobutyronitrile and carbon tetrachloride were mixed and reacted for 8-12 h to obtain a reaction solution. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain a concentrate. The concentrate was purified to obtain compound 1. 2) Mix compound 1, silver sulfate and dioxane aqueous solution, react for 50-70 min to obtain reaction solution, filter the reaction solution and take the filtrate; extract the filtrate with ethyl acetate, take the organic phase, wash the organic phase, dry, filter, concentrate the filtrate under reduced pressure and purify to obtain compound 2; 3) Compound 2, diethylaminosulfur trifluoride and dichloromethane were mixed and reacted for 8-12 h to obtain a reaction solution; the organic phase in the reaction solution was washed, dried, filtered, and the filtrate was concentrated under reduced pressure and purified to obtain compound 3; 4) Compound 3, bis(pinacol)diboron, potassium acetate, [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride and N,N-dimethylformamide were mixed and reacted for 2.5-3.5 h to obtain the reactant; the reactant was mixed with the catalyst and reacted for 3-5 h to obtain the reaction solution; the reaction solution was diluted with diethyl ether to obtain the diluted solution; the organic phase in the diluted solution was washed, dried, filtered, and the filtrate was concentrated under reduced pressure and purified to obtain compound 4; 5) Mix compound 4, tetrahydrofuran aqueous solution and lithium hydroxide aqueous solution, react for 8-12 h to obtain a reaction solution, dilute the reaction solution to obtain a diluted solution; neutralize the diluted solution to obtain the extract; extract the extract with ethyl acetate, take the organic phase, dry and concentrate to obtain phenylboronic acid ester.

6. The preparation method according to claim 5, characterized in that, The mass ratio of methyl 4-bromo-3-methylbenzoate, N-bromosuccinimide, and azobisisobutyronitrile in step 1) is 2–2.5: 1.5–2.3: 0.15–0.

18. The mass-to-volume ratio of methyl 4-bromo-3-methylbenzoate to carbon tetrachloride is 2–2.5 g: 25 mL; In step 2), the mass ratio of compound 1 to silver sulfate is 300–312:495–502. The mass-to-volume ratio of compound 1 to dioxane aqueous solution is 300–312 mg: 20 mL; The volume ratio of dioxane to water in an aqueous solution of dioxane is 1-2:1-2; In step 3), the mass-to-volume ratio of compound 2 and diethylaminosulfur trifluoride is 230–240 mg: 370–390 μL. The mass-to-volume ratio of compound 2 to dichloromethane is 230–240 mg: 14–16 mL; In step 4), the mass ratio of compound 3, bis(pinacol)diboron, potassium acetate, and [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride is 180–190: 225–230: 215–225: 25–35. The mass-to-volume ratio of compound 3 and N,N-dimethylformamide is 180–190 mg: 2–3 mL; In step 5), the mass-to-volume ratio of compound 4 and the tetrahydrofuran aqueous solution is 14.5–15.5 mg: 0.8 mL. The mass-to-volume ratio of compound 4 to the lithium hydroxide aqueous solution was 14.5–15.5 mg: 140–160 μL.

7. A lipid nanoparticle of a protein degradation chimera with mitochondrial-targeted degradation function, characterized in that, The lipid nanoparticles of the protein degradation chimera include the protein degradation chimera of claim 1, an amphiphilic copolymer, 1,2-dioleoyl-3-trimethylammonium propane, and 1,2-distearate-sn-propanetriyl-3-phosphocholine; The mass ratio of the protein degradation chimera, the amphiphilic copolymer, 1,2-dioleoyl-3-trimethylammonium propane, and 1,2-distearate-sn-propanetriyl-3-phosphocholine is 0.8–1.2:8–12:1–3:1–3; The amphiphilic copolymer is distearate phosphatidylethanolamine-polyethylene glycol 2000.

8. The use of the lipid nanoparticles according to claim 7 in the preparation of tumor immunotherapy drugs.

9. A microneedle patch for activating the protein degradation chimera of claim 1, characterized in that, It is a microneedle patch loaded with 2-thiophenecarboxylic acid trifluoromethyl ester, prepared based on a composite system of hyaluronic acid and polyvinyl alcohol.

10. A tumor immunotherapy drug, characterized in that, Includes the protein degradation chimera of claim 1, the lipid nanoparticles of claim 7, and the microneedle patch of claim 9; The tumors include colorectal cancer and / or breast cancer.

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