Preparation method and application of liposome vesicle-encapsulated targeted protein degradation nanoparticles

Abstract

The present invention discloses a method for preparing liposome-encapsulated targeted protein degradation nanoparticles and their application. The present invention blends a polymetformin-based protein degradation targeted chimera with hyaluronic acid to form polyprotein degradation targeted chimera nanoparticles through electrostatic adsorption. The liposome vesicles are then blended with the polyprotein degradation targeted chimera nanoparticles and co-extruded to obtain liposome-encapsulated targeted protein degradation nanoparticles. The liposome-encapsulated targeted protein degradation nanoparticles designed by the present invention can overcome the hook effect of the protein degradation targeted chimera in tumor cells and achieve long-term circulation of the protein degradation targeted chimera in the body, thereby maximizing the in vivo utilization of the protein degradation targeted chimera and enhancing the anti-tumor effect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a preparation method and application of liposome vesicle-encapsulated targeted protein degradation nanoparticles. Background Art

[0002] Cancer is the leading cause of death worldwide and the most common disease, impacting human health and socioeconomic well-being. Therefore, cancer control measures are increasingly necessary.

[0003] Current cancer treatments include surgery, chemotherapy, radiotherapy, targeted therapy, bone marrow transplantation, and immunotherapy. Targeted therapy involves specifically binding to specific oncogenic sites at the cellular molecular level, disrupting their biological functions and inducing specific cell death. A novel targeted therapy strategy, protein degradation targeting chimeras (PROTACs), has attracted significant interest due to their potential for therapeutic protein regulation. PROTACs are heterobifunctional small molecules composed of two ligands: one ligand targets the target protein, while the other recruits an E3 ubiquitin ligase. When PROTACs simultaneously bind to the target protein and the E3 ligase, they induce ubiquitination of the target protein, leading to its subsequent degradation by the ubiquitin-proteasome system (UPS). Compared to small molecule inhibitors, PROTACs offer advantages such as high selectivity, the ability to overcome drug resistance, and the potential for targeted undruggability. However, their large molecular weight, poor water solubility, and low cellular permeability limit their in vivo application. While significant progress has been made in the development of protein degradation-targeted chimeras, challenges remain, such as poor efficacy as a single therapy and poor clinical translation. Compared to current conventional tumor radiotherapy and chemotherapy, the applicability of protein degradation-targeted chimeras is far from sufficient, necessitating the development of more protein degradation platforms.

[0004] Nanoparticle delivery systems, due to their small size and ability to effectively penetrate lesions, have found widespread application in cancer treatment. These systems can enhance drug efficacy and reduce toxicity by enhancing drug stability, reducing adverse reactions, and modulating pharmacokinetic profiles. Therefore, nanoparticle-based drug delivery approaches can overcome many of the obstacles faced by traditional therapies and are emerging as novel diagnostic and therapeutic strategies. Summary of the Invention

[0005] To address the issues of high off-target potential, limited hook effects, and poor pharmacokinetics associated with protein degradation-targeted chimeras, the present invention provides a method for preparing liposome-encapsulated targeted protein degradation nanoparticles and their application. By overcoming the off-target potential and hook effects of protein degradation-targeted chimeras in tumor cells and achieving prolonged circulation within the body, the present invention significantly increases the in vivo utilization of protein degradation-targeted chimeras and exerts a robust anti-tumor effect.

[0006] The method for preparing liposome vesicle-encapsulated targeted protein degradation nanoparticles of the present invention comprises the following steps:

[0007] Step 1: A carboxylated targeting small molecule and polymetformin are mixed and dissolved in an organic solvent, and reacted at a specific temperature and with a catalyst to obtain a targeting small molecule-polymetformin conjugate 1. A carboxylated recruitment small molecule and polymetformin are mixed and dissolved in an organic solvent, and reacted at a specific temperature and with a catalyst to obtain a recruitment small molecule-polymetformin conjugate 2. Conjugates 1 and 2 are blended to obtain a positively charged mixed solution of a polyprotein degradation-targeted chimera. The targeting small molecule, recruitment small molecule, and polymetformin are generally linked via chemical bonds.

[0008] Step 2: Blending the positively charged mixed solution of the polyprotein degradation targeting chimera with the hyaluronic acid solution to obtain negatively charged polyprotein degradation targeting chimera nanoparticles, wherein the negatively charged polyprotein degradation targeting chimera nanoparticles are formed by electrostatic adsorption of the linkers 1 and 2 and the hyaluronic acid.

[0009] Step 3: The negatively charged polyprotein degradation targeted chimeric nanoparticles are mixed with liposome vesicles, and the mixture is repeatedly extruded through a micro-extruder equipped with a nanoscale polycarbonate membrane to obtain liposome vesicle-encapsulated targeted protein degradation nanoparticles.

[0010] Furthermore, in step 1, the carboxylated targeting small molecule and polymetformin are mixed and dissolved in DMSO at a mass ratio of 4~0.5:2~0.5, and reacted at 0~60°C in the presence of a catalyst for 12~48 hours to obtain a targeting small molecule and polymetformin linker 1; the carboxylated recruitment small molecule and polymetformin are mixed and dissolved in DMSO at a mass ratio of 3~0.5:2~0.5, and reacted at 0~60°C in the presence of a catalyst for 12~48 hours to obtain a recruitment small molecule and polymetformin linker 2; at 10°C~40°C, linkers 1 and 2 are respectively dissolved in deionized water, blended, and ultrasonicated for 1~20 minutes to obtain a positively charged mixed solution of polyprotein degradation targeting chimera.

[0011] The catalyst is a condensation agent, which is one or more selected from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, and N,N'-dicyclohexylcarbodiimide.

[0012] The targeted small molecule is selected from one or more of MK1775, 4-hydroxytamoxifen, palbociclib, JQ1, dasatinib, gefitinib, enzalutamide, NLG919, indomethacin, olaparib, and RSL3.

[0013] The recruiting small molecule is selected from one or more of pomalidomide, lenalidomide, thalidomide, VH032, and VH298.

[0014] The drug loading rate of the targeted small molecule and polymetformin linker 1 is 5% to 95%.

[0015] The drug loading rate of the recruited small molecule and polymetformin linker 2 is 5% to 95%.

[0016] The drug loading rate calculation formula is: .

[0017] Furthermore, in step 2, the positively charged mixed solution of the polyprotein degradation targeting chimera is rapidly added to the hyaluronic acid solution at 10°C to 40°C, and ultrasonicated for 1 to 20 minutes to uniformly mix to obtain negatively charged polyprotein degradation targeting chimera nanoparticles. The mass ratio of the polyprotein degradation targeting chimera to hyaluronic acid is generally between 0.5:0.1 and 10.

[0018] Furthermore, step 3 includes the following steps:

[0019] 3a, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), (2,3-dioleoyl-propyl)-trimethylammonium-chloride (DOTAP), N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-mPEG 2000 ) are dissolved in chloroform in a molar ratio of 1-12:0.5-6:0.5-10. After the reaction system is uniform, the organic solvent is dried using a rotary evaporator to obtain a dry lipid film, which is then ultrasonically hydrated in a deionized water solution to obtain a lipid suspension, which is repeatedly extruded through a micro-extruder equipped with a nanoscale polycarbonate membrane to obtain liposome vesicles.

[0020] 3b. Negatively charged polyprotein degradation targeted chimeric nanoparticles dissolved in deionized water and liposome vesicles are mixed, ultrasonicated for 1 to 20 minutes to mix evenly, and repeatedly extruded through a micro-extruder equipped with a nanoscale polycarbonate membrane to obtain liposome vesicle-encapsulated targeted protein degradation nanoparticles.

[0021] The mass ratio of the polyprotein degradation targeted chimeric nanoparticles to the liposome vesicles is generally between 0.2~1:0.5~5.

[0022] The polycarbonate membrane is a polycarbonate membrane used in conjunction with a liposome extruder produced by Avanti Polar Lipids, and the size range used is 100 nm to 1000 nm.

[0023] The invention relates to the use of targeted protein degradation nanoparticles encapsulated by liposome vesicles in the preparation of anti-tumor drugs.

[0024] The beneficial effects of the present invention are embodied in:

[0025] The present invention designs a liposome vesicle-encapsulated targeted protein degradation nanoparticle, wherein the polyprotein degradation targeting chimera based on polymethformin provides effective multi-ligand targeting and multi-site recruitment functions, can target multiple target proteins in tumor cells, and recruit multiple E3 ubiquitin ligases to perform protein ubiquitination, and finally be recognized and degraded by the ubiquitin-proteasome system. By being encapsulated in liposome vesicles, the polymeric protein degradation targeting chimera can avoid unstable diffusion in the blood circulation, enabling it to better remain in the tumor site and be internalized by tumor cells. Compared with small molecule-based protein degradation targeting chimeras, the polyprotein degradation targeting chimera can have the advantages of efficient protein degradation effect, long blood circulation time, and targeted tumor accumulation. The polyprotein degradation targeting chimera provides a universal platform for the precise construction of protein degradation targeting chimeras and provides a promising model for the future clinical translation application of protein degradation targeting chimeras. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The preparation route of the targeted protein degradation nanoparticles encapsulated by liposome vesicles in the examples of the present invention is schematically shown.

[0027] Figure 2 The synthetic route of the PolyPROTAC component in the examples of the present invention is schematically shown.

[0028] Figure 3 The hydrogen nuclear magnetic resonance spectrum of the intermediate 1 prepared in the embodiment of the present invention is schematically shown.

[0029] Figure 4 The ultraviolet absorption spectrum of the intermediate 1 prepared in the embodiment of the present invention is schematically shown.

[0030] Figure 5 The hydrogen nuclear magnetic resonance spectrum of the intermediate 2 prepared in the embodiment of the present invention is schematically shown.

[0031] Figure 6 The hydrogen nuclear magnetic resonance spectrum of the intermediate 3 prepared in the embodiment of the present invention is schematically shown.

[0032] Figure 7 The hydrogen nuclear magnetic resonance spectrum of intermediate 4 prepared in an embodiment of the present invention is schematically shown.

[0033] Figure 8 The ultraviolet absorption spectrum of intermediate 4 prepared in an example of the present invention is schematically shown.

[0034] Figure 9 The hydrogen nuclear magnetic resonance spectrum of the intermediate 5 prepared in the embodiment of the present invention is schematically shown.

[0035] Figure 10 The hydrogen nuclear magnetic resonance spectrum of intermediate 6 prepared in an example of the present invention is schematically shown.

[0036] Figure 11 The hydrogen nuclear magnetic resonance spectrum of the intermediate 7 prepared in the embodiment of the present invention is schematically shown.

[0037] Figure 12 The hydrogen nuclear magnetic resonance spectrum of the intermediate 8 prepared in an embodiment of the present invention is schematically shown.

[0038] Figure 13 The hydrogen nuclear magnetic resonance spectrum of intermediate 9 prepared in an example of the present invention is schematically shown.

[0039] Figure 14 The ultraviolet absorption spectrum of intermediate 9 prepared in an embodiment of the present invention is schematically shown.

[0040] Figure 15 The figure schematically shows the dynamic light scattering particle size, PDI, Zeta potential and transmission electron microscopy characterization of the negatively charged PolyPROTAC-NP nanoparticles prepared in an embodiment of the present invention.

[0041] Figure 16 The dynamic light scattering particle size, PDI, Zeta potential and transmission electron microscopy characterization of Lipo-PolyPROTAC nanoparticles prepared in an embodiment of the present invention are schematically shown.

[0042] Figure 17 The figure schematically shows the cytotoxicity experimental results of Lipo-PolyPROTAC nanoparticles prepared in an embodiment of the present invention.

[0043] Figure 18The figure schematically shows the Western Blot experimental results of protein degradation by Lipo-PolyPROTAC nanoparticles prepared in an embodiment of the present invention.

[0044] Figure 19 The figure schematically shows a dosing plan diagram of the Lipo-PolyPROTAC nanoparticles prepared in an embodiment of the present invention.

[0045] Figure 20 The schematic diagram shows the efficacy experiment of Lipo-PolyPROTAC nanoparticles prepared in the embodiment of the present invention, including the tumor growth curve.

[0046] Figure 21 The biosafety experiment of Lipo-PolyPROTAC nanoparticles prepared in an embodiment of the present invention is schematically shown, including the weight gain curve of mice. DETAILED DESCRIPTION

[0047] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments, but they are not intended to limit the scope of the present invention.

[0048] Example 1: Synthesis of PolyMet (Intermediate 1)

[0049] like Figure 2 As shown in the synthetic route, a 50 mL flask was charged with 0.1 g of linear polyethyleneimine (PEI), 1 g of dicyandiamide, and 10 mL of 2M HCl solution. A reflux system was then added and the mixture was reacted in a 100°C oil bath for 24 h. After the reaction, the pH of the reaction solution was adjusted to alkaline (pH = 12) with 1 M NaOH solution. The solution was then ultrafiltered multiple times through a 3000 MW ultrafiltration tube until the volume was reduced to 1-2 mL. The solution was then transferred to a 10 mL centrifuge tube and lyophilized to obtain intermediate 1 as a white solid.

[0050] By nuclear magnetic resonance spectroscopy ( 1 The chemical structure of the intermediate 1 synthesized in Example 1 was characterized by H-NMR) and UV absorption spectroscopy.

[0051] Figure 2 The synthetic route of the PolyPROTAC component in the examples of the present invention is schematically shown.

[0052] Figure 3 The H NMR spectrum of the intermediate 1 prepared in the embodiment of the present invention is schematically shown. Figure 3 It can be seen that in the D2O reagent, there is a specific peak at δ 2.5-4.0, which is similar to metformin (Met).

[0053] Figure 4The UV absorption spectrum of the intermediate 1 prepared in the embodiment of the present invention is schematically shown. The product solvent is a deionized water solution with a pH of 14. Figure 4 It can be seen that PolyMet has an absorption peak similar to that of metformin (Met).

[0054] Example 2: Synthesis of Intermediate 2

[0055] like Figure 2 As shown in the synthetic route, a 10 mL flask was charged with 2-(2,6-dioxopiperidin-3-yl)-4-fluoro-2,3-dihydro-1H-isoindole-1,3-dione (0.2 mmol, 55.24 mg), glycine tert-butyl ester (0.24 mmol, 31.48 mg), and N,N-diisopropylethylamine (DIPEA) (0.4 mmol, 51.70 mg). The mixture was dissolved in dimethyl sulfoxide (DMSO) (2 mL), sealed, and the temperature was set at 90°C for 24 h. After completion of the reaction, saturated ammonium chloride solution was added to quench the reaction. The product was extracted with ethyl acetate and dried on a rotary evaporator. The product was then dissolved in 2-3 mL of ethyl acetate and separated on a silica gel plate using a 3:4 ratio of cyclohexane:ethyl acetate. The separated silica gel powder was scraped off and rinsed with ethyl acetate. The ethyl acetate solution containing the pure product was dried on a rotary evaporator to obtain intermediate 2 as a yellow oil in a yield of 44.3%.

[0056] By nuclear magnetic resonance spectroscopy ( 1 The chemical structure of the intermediate 2 synthesized in Example 2 was characterized by H-NMR) and mass spectrometry.

[0057] Figure 5 The H NMR spectrum of the intermediate 2 prepared in the embodiment of the present invention is schematically shown. 1 The H NMR and mass spectrometry data are as follows:

[0058] 1H NMR (400 MHz, DMSO) δ 11.13 (s, 1H), 7.59 (dd,J= 8.3, 7.4 Hz,1H), 7.09 (d,J= 7.0 Hz, 1H), 6.98 (d,J= 8.6 Hz, 1H), 6.86 (t,J= 6.0 Hz, 1H),5.08 (dd,J= 12.9, 5.4 Hz, 1H), 4.10 (d,J= 6.0 Hz, 2H), 2.90 (ddd,J= 17.4,14.1, 5.4 Hz, 1H), 2.68 – 2.52 (m, 2H), 2.11 – 2.00 (m, 1H), 1.44 (s, 10H).m / z 388.1507 (M+H) + .

[0059] Example 3: Synthesis of Carboxylated Pomalidomide (Intermediate 3)

[0060] like Figure 2 As shown in the synthetic route, the obtained intermediate 2 (0.2 mmol, 77.48 mg) was dissolved in 2 mL of dichloromethane (DCM) and transferred to a 10 mL flask. 1 mL of trifluoroacetic acid was added, the flask was sealed, and the reaction was allowed to proceed at room temperature for 2 h. After the reaction, the mixture was dried on a rotary evaporator and washed several times with DCM to precipitate a yellow solid. Finally, the mixture was dried in a vacuum oven to obtain intermediate 3, a yellow solid with a yield of 85.4%.

[0061] By nuclear magnetic resonance spectroscopy ( 1 The chemical structure of intermediate 3 synthesized in Example 3 was characterized by H-NMR) and mass spectrometry.

[0062] Figure 6 The H NMR spectrum of the intermediate 3 prepared in the embodiment of the present invention is schematically shown. 1 The H NMR and mass spectrometry data are as follows:

[0063] 1H NMR (400 MHz, DMSO) δ 11.13 (s, 1H), 7.59 (dd,J= 8.2, 7.5 Hz, 1H), 7.08 (d,J= 7.0 Hz, 1H), 6.99 (d,J= 8.6 Hz, 1H), 6.86 (t,J= 5.7 Hz, 1H), 5.08(dd,J= 12.9, 5.3 Hz, 1H), 4.11 (d,J= 5.8 Hz, 2H), 2.95 – 2.82 (m, 1H), 2.58(dd,J= 19.1, 10.4 Hz, 2H), 2.11 – 1.98 (m, 1H).m / z 330.0726 (MH) - .

[0064] Example 4: Synthesis of PolyPomalidomide (Intermediate 4)

[0065] like Figure 2 As shown in the synthetic route, intermediate 3 (0.5 mmol, 150.64 mg), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (0.5 mmol, 97.85 mg) and N-hydroxysuccinimide (NHS) (0.5 mmol, 57.54 mg) were added to a 50 mL flask, dissolved in 10 mL DMSO, and reacted at room temperature for 1 h. Then, intermediate 1 (100 mg) was added and reacted at room temperature for 48 h. After the reaction, the reaction solution was placed in a 3.5K dialysis bag and dialyzed for about a week. Finally, it was placed in a freeze dryer and dried for 48 h to obtain yellow solid intermediate 4 (i.e., linker 2).

[0066] By nuclear magnetic resonance spectroscopy ( 1 The chemical structure of intermediate 4 synthesized in Example 4 was characterized by H-NMR) and UV absorption spectroscopy.

[0067] Figure 7 The H NMR spectrum of the intermediate 4 prepared in the embodiment of the present invention is schematically shown. Figure 7 It can be seen that in the DMSO-d6 reagent, there are specific peaks at δ 6.5-8.0 and 0.5-4.0, which are similar to those of pomalidomide.

[0068] Figure 8 The UV absorption spectrum of the intermediate 4 prepared in the embodiment of the present invention is schematically shown. The product solvent is a deionized water solution with a pH of 7. Figure 8It can be seen that PolyPomalidomide has an absorption peak similar to that of Pomalidomide. The drug loading rate of the conjugate of Pomalidomide and PolyMetformin is 22.5%.

[0069] Example 5: Synthesis of Intermediate 5

[0070] like Figure 2 As shown in the synthetic route, 1,2-dihydro-1-[6-(1-hydroxy-1-methylethyl)-2-pyridine]-6-(methylthio)-2-(2-propen-1-yl)-3H-pyrazolo[3,4-D]pyrimidin-3-one (0.1 mmol, 35.74 mg) and 3-chloroperoxybenzoic acid (mCPBA) (0.11 mmol, 22.33 mg) were first added to a 10 mL flask, and then dissolved in 2 mL of toluene. After reacting at room temperature for 1 h, DIPEA (0.2 mmol, 25.85 mg) and tert-butyl 4-(4-aminophenyl)piperazine-1-carboxylate (0.12 mmol, 33.28 mg) were added and reacted at room temperature for 18 h. After the reaction was completed, saturated ammonium chloride solution was added to quench the reaction, and the product was extracted with ethyl acetate and dried on a rotary evaporator. 2-3 mL of ethyl acetate was added for dissolution, and separation was performed on a silica gel plate. The chromatographic solution was cyclohexane:ethyl acetate in a ratio of 2:3. The silica gel powder was scraped off and rinsed with ethyl acetate. The ethyl acetate solution containing the pure product was dried on a rotary evaporator to obtain intermediate 5 as a brown oil with a yield of 66.4%.

[0071] By nuclear magnetic resonance spectroscopy ( 1 The chemical structure of intermediate 5 synthesized in Example 5 was characterized by H-NMR) and mass spectrometry.

[0072] Figure 9 The H NMR spectrum of the intermediate 5 prepared in the embodiment of the present invention is schematically shown. 1 The H NMR and mass spectrometry data are as follows:

[0073] 1H NMR (400 MHz, DMSO) δ 10.22 (s, 1H), 8.85 (s, 1H), 8.07 (s, 1H), 7.76 (d,J= 7.8 Hz, 1H), 7.62 (d,J= 7.6 Hz, 3H), 6.96 (d,J= 8.8 Hz, 2H), 5.68(ddt,J= 16.3, 10.3, 6.0 Hz, 1H), 5.37 (s, 1H), 5.00 (dd,J= 10.2, 1.1 Hz, 1H), 4.83 (dd,J= 17.1, 1.2 Hz, 1H), 4.70 (d,J= 5.1 Hz, 2H), 3.48 (s, 4H), 3.06 (s,4H), 1.48 (s, 6H), 1.43 (s, 9H).m / z 609.2881 (M+Na) + .

[0074] Example 6: Synthesis of Intermediate 6

[0075] like Figure 2 As shown in the synthetic route, the obtained product intermediate 5 (0.1 mmol, 58.67 mg) was dissolved in 3 mL of DCM and transferred into a 10 mL flask. 1 mL of trifluoroacetic acid was added and the mixture was reacted at room temperature for 3 h. After the reaction, the mixture was dried on a rotary evaporator, washed repeatedly with DCM, and dried on a rotary evaporator. Finally, the mixture was dried in a vacuum drying oven to obtain the intermediate 6 as a brown oil with a yield of 80.5%.

[0076] By nuclear magnetic resonance spectroscopy ( 1 The chemical structure of intermediate 6 synthesized in Example 6 was characterized by H-NMR) and mass spectrometry.

[0077] Figure 10 The H NMR spectrum of the intermediate 6 prepared in the embodiment of the present invention is schematically shown. 1 The H NMR and mass spectrometry data are as follows:

[0078] 1H NMR (400 MHz, DMSO) δ 10.24 (s, 1H), 9.04 (s, 1H), 8.86 (s, 1H), 8.05 (d,J= 7.2 Hz, 1H), 7.76 (d,J= 7.9 Hz, 1H), 7.62 (d,J= 7.6 Hz, 3H), 7.00(d,J= 8.8 Hz, 2H), 5.80 – 5.55 (m, 1H), 5.40 (s, 1H), 5.00 (d,J= 10.1 Hz,1H), 4.83 (d,J= 17.1 Hz, 1H), 4.70 (d,J= 4.8 Hz, 2H), 3.32 (d,J= 4.7 Hz, 4H),3.28 (s, 4H), 1.47 (s, 6H).m / z 487.2549 (M+H) + .

[0079] Example 7: Synthesis of Intermediate 7

[0080] like Figure 2 As shown in the synthetic route, intermediate 6 (0.05 mmol, 24.33 mg), tert-butyl 3-bromopropionate (0.1 mmol, 20.90 mg), and K2CO3 (0.15 mmol, 20.73 mg) were added to a 10 mL flask. The mixture was dissolved in 3 mL of N,N-dimethylformamide (DMF) and allowed to react at 80°C for 12 h. After completion of the reaction, the mixture was quenched with saturated ammonium chloride solution, extracted with ethyl acetate, and dried on a rotary evaporator. Dissolved in 2-3 mL of ethyl acetate, the mixture was separated on a silica gel plate using a 10:1 ratio of ethyl acetate to methanol. The silica gel powder was scraped off and rinsed with ethyl acetate. The ethyl acetate solution containing the pure product was dried on a rotary evaporator to afford intermediate 7 as a brown oil in a yield of 46.3%.

[0081] By nuclear magnetic resonance spectroscopy ( 1 The chemical structure of intermediate 7 synthesized in Example 7 was characterized by HPLC (H-NMR) and mass spectrometry.

[0082] Figure 11 The H NMR spectrum of the intermediate 7 prepared in the embodiment of the present invention is shown schematically. 1 The H NMR and mass spectrometry data are as follows:

[0083] 1H NMR (400 MHz, DMSO) δ 10.19 (s, 1H), 8.83 (s, 1H), 8.06 (s, 1H), 7.75 (d,J= 7.8 Hz, 1H), 7.61 (d,J= 7.7 Hz, 3H), 6.93 (d,J= 8.8 Hz, 2H), 5.66(ddt,J= 16.3, 10.4, 6.0 Hz, 1H), 5.37 (s, 1H), 5.00 (d,J= 10.2 Hz, 1H), 4.82(d,J= 17.1 Hz, 1H), 4.69 (d,J= 5.1 Hz, 2H), 3.09 (s, 4H), 2.55 (s, 6H), 2.42(s, 2H), 1.46 (s, 6H), 1.41 (s, 9H).m / z 615.3378 (M+H) + .

[0084] Example 8: Synthesis of Carboxylated MK1775 (Intermediate 8)

[0085] like Figure 2 As shown in the synthetic route, the intermediate 7 (0.05 mmol, 30.74 mg) was dissolved in 3 mL of DCM and transferred to a 10 mL flask. 1 mL of trifluoroacetic acid was added and the mixture was reacted at room temperature for 3 h. After the reaction, the mixture was dried on a rotary evaporator, washed repeatedly with DCM, and dried on a rotary evaporator. Finally, the mixture was dried in a vacuum drying oven to obtain the intermediate 8 as a brown oil with a yield of 83.6%.

[0086] By nuclear magnetic resonance spectroscopy ( 1 The chemical structure of intermediate 8 synthesized in Example 8 was characterized by H-NMR) and mass spectrometry.

[0087] Figure 12 The H NMR spectrum of the intermediate 8 prepared in the embodiment of the present invention is shown schematically. 1 The H NMR and mass spectrometry data are as follows:

[0088] 1H NMR (400 MHz, DMSO) δ 10.18 (s, 1H), 8.83 (s, 1H), 8.06 (s, 1H), 7.75 (d,J= 7.7 Hz, 1H), 7.61 (d,J= 7.7 Hz, 3H), 6.93 (d,J= 8.9 Hz, 2H), 5.66(ddt,J= 16.3, 10.3, 6.0 Hz, 1H), 5.36 (s, 1H), 4.99 (dd,J= 10.2, 1.0 Hz, 1H), 4.82 (dd,J= 17.1, 1.1 Hz, 1H), 4.68 (d,J= 5.2 Hz, 2H), 3.12 (s, 4H), 2.69(dd,J= 14.8, 7.8 Hz, 2H), 2.62 (s, 4H), 2.48 – 2.44 (m, 2H), 1.46 (s, 6H).m / z557.2626 (MH) - .

[0089] Example 9: Synthesis of PolyMK1775 (Intermediate 9)

[0090] like Figure 2 As shown in the synthetic route, a 50 mL flask was added with intermediate 8 (0.1 mmol, 55.864 mg), EDCI (0.5 mmol, 97.85 mg) and NHS (0.5 mmol, 57.54 mg), respectively, and 10 mL DMSO was added to dissolve the mixture. The mixture was reacted at room temperature for 1 h, and then intermediate 1 (50 mg) was added. The mixture was reacted at 60°C for 48 h. After the reaction, the reaction solution was placed in a 3.5K dialysis bag and dialyzed for about a week. Finally, the reaction solution was placed in a freeze dryer and dried for 48 h to obtain a brown solid intermediate 9 (i.e., linker 1).

[0091] By nuclear magnetic resonance spectroscopy ( 1 The chemical structure of intermediate 9 synthesized in Example 9 was characterized by H-NMR) and UV absorption spectroscopy.

[0092] Figure 13 The H NMR spectrum of the intermediate 9 prepared in the embodiment of the present invention is schematically shown. Figure 13 It can be seen that in DMSO-d6 reagent, there are specific peaks at δ 4.0-10.0, 0.5-3.5, similar to MK1775

[0093] Figure 14 The UV absorption spectrum of the intermediate 9 prepared in the embodiment of the present invention is schematically shown. The product solvent is a deionized water solution with a pH of 7. Figure 14It can be seen that PolyMK1775 has an absorption peak similar to that of MK1775. The drug loading efficiency of the conjugate of MK1775 and polymetformin is 9.5%.

[0094] Example 10: Preparation of Lipo-PolyPROTAC Nanoparticles

[0095] like Figure 1 As shown in the preparation route, at room temperature, PolyMK1775 and PolyPomalidomide were blended in a mass ratio of 4:1 and dissolved in deionized water to obtain a 0.5 mg / ml concentration of a positively charged PolyPROTAC mixed solution.

[0096] The positively charged PolyPROTAC mixed solution and the hyaluronic acid (HA) solution were evenly blended at a mass ratio of 0.5:0.9, and ultrasonicated for 5 minutes to obtain negatively charged PolyPROTAC-NP nanoparticles.

[0097] 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), (2,3-dioleoyl-propyl)-trimethylammonium-chloride (DOTAP), N-(carbonyl-methoxy polyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-mPEG 2000 ) were dissolved in chloroform in a molar ratio of 6:0.5:0.5. After the reaction system was uniform, the organic solvent was dried using a rotary evaporator to obtain a dry lipid film, which was then ultrasonically hydrated in a deionized water solution to obtain a lipid suspension, which was repeatedly extruded through a micro-extruder equipped with a 1000 nm polycarbonate membrane to obtain liposome vesicles.

[0098] The negatively charged PolyPROTAC-NP nanoparticles and liposome vesicles were mixed at a mass ratio of 0.35:1.1, sonicated for 10 minutes, and repeatedly extruded through a micro-extruder equipped with a 200 nm nanoscale polycarbonate membrane to obtain a liposome vesicle-encapsulated targeted protein degradation nanoparticle Lipo-PolyPROTAC.

[0099] The negatively charged PolyPROTAC-NP nanoparticles prepared in Example 10 were characterized by nanoparticle size and Zeta potential analyzer (DLS) and transmission electron microscopy. The results are as follows: Figure 15 shown.

[0100] The Lipo-PolyPROTAC nanoparticles prepared in Example 10 were characterized by nanoparticle size and Zeta potential analyzer (DLS) and transmission electron microscopy. The results are as follows: Figure 16 shown.

[0101] Figure 1 The preparation route of the targeted protein degradation nanoparticles encapsulated by liposome vesicles in the examples of the present invention is schematically shown.

[0102] Figure 15 The following diagram schematically shows the dynamic light scattering particle size, PDI, Zeta potential and transmission electron microscopy characterization of the negatively charged PolyPROTAC-NP nanoparticles prepared in the embodiment of the present invention. Figure 15 It can be seen that the dynamic light scattering particle size of PolyPROTAC-NP nanoparticles is 157.2nm, PDI is 0.195, and Zeta potential is -19.0mV.

[0103] Figure 16 The following schematically shows the dynamic light scattering particle size, PDI, Zeta potential and transmission electron microscopy characterization of Lipo-PolyPROTAC nanoparticles prepared in an embodiment of the present invention. Figure 16 It can be seen that the dynamic light scattering particle size of Lipo-PolyPROTAC nanoparticles is 88.22nm, PDI is 0.199, and Zeta potential is -9.74mV.

[0104] Example 11: Toxicity Experiment of Lipo-PolyPROTAC Nanoparticles

[0105] 4T1 cells were first seeded in a 96-well plate (3000 cells per well). After 12 hours, the cells adhered to the wall and were incubated with Lipo-PolyMet, Lipo-PolyMK1775, Lipo-PolyPomalidomide, and Lipo-PolyPROTAC for 24 hours. The concentration gradient was set to 0, 50, 100, 200, 400, 800, 1000, 2000, 4000, and 5000 mM. The drugs were then aspirated and MTT working solution was added (100 ul of 0.5 mg / mL MTT working solution per well). After incubation for 4 hours, the MTT working solution was aspirated and DMSO was added (150 ul of DMSO per well). Finally, the UV absorbance of each well at 490 nm was measured.

[0106] Figure 17 The cytotoxicity test results of Lipo-PolyPROTAC nanoparticles prepared in the embodiment of the present invention are schematically shown. Figure 17 The data show that compared with the control groups such as Lipo-PolyMet, Lipo-PolyMK1775, and Lipo-PolyPomalidomide, Lipo-PolyPROTAC has a stronger effect in killing tumor cells.

[0107] Example 12: Protein degradation effect of Lipo-PolyPROTAC nanoparticles

[0108] First, 4T1 cells were seeded in 10 cm culture dishes (5*10 5 cells), and after 12 hours of cell attachment, the cells were treated with Lipo-PolyPROTAC nanoparticles for 24 hours at a concentration gradient of 0, 1, 2, 3, 4, and 5 μM. The cells were then lysed with RIPA buffer, and the protein was extracted. Finally, the degradation effect of Wee1 protein was determined by Western Blot.

[0109] Figure 18 The Western Blot experimental results of protein degradation by Lipo-PolyPROTAC nanoparticles prepared in an embodiment of the present invention are schematically shown. Figure 18 It can be seen that Lipo-PolyPROTAC nanoparticles exhibit significant protein degradation effect and have good dose dependence.

[0110] Example 13: Animal Efficacy Experiment of Lipo-PolyPROTAC Nanoparticles

[0111] Mouse 4T1 cells (25*10 4 ) were subcutaneously inoculated into the lower flank of 6-week-old female BALB / c mice. When the tumor reached 50-100 mm3, PBS, Lipo-PolyMK1175 + Lipo-PolyPomalidomide, and Lipo-PolyPROTAC nanoparticles were subcutaneously injected every three days for a total of three doses. The dosing schedule is detailed in

[15] . Figure 19 .

[0112] Figure 19 The figure schematically shows a dosing plan diagram of the Lipo-PolyPROTAC nanoparticles prepared in an embodiment of the present invention.

[0113] The dose of Lipo-PolyMK1775 + Lipo-Poly Pomalidomide and Lipo-Poly PROTAC groups was 5 mg / kg. The inhibitory effect of Lipo-Poly PROTAC nanoparticles on tumor growth was verified by comparing changes in tumor volume.

[0114] Figure 20 The schematic diagram shows the efficacy experiment of Lipo-PolyPROTAC nanoparticles prepared in the embodiment of the present invention, including the tumor growth curve. Figure 20It can be seen that according to the two-way ANOVA statistical analysis, the differences in tumor size between the groups were: there was no significant difference between the PBS group and the Lipo-PolyMK1775 + Lipo-PolyPomalidomide group, *p = 0.0390 between the PBS group and the Lipo-PolyPROTAC group, and **p = 0.0024 between the Lipo-PolyMK1775 + Lipo-PolyPomalidomide group and the Lipo-PolyPROTAC group.

[0115] Figure 21 The biosafety experiment of Lipo-PolyPROTAC nanoparticles prepared in the embodiment of the present invention is schematically shown, including the weight growth curve of mice. Figure 21 It can be seen that there is no obvious biological toxicity reflected in the changes in mouse body weight.

[0116] Therefore, it can be concluded that Lipo-PolyPROTAC nanoparticles exhibited strong antitumor effects and good biosafety compared with the control group.

[0117] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing liposome-encapsulated targeted protein degradation nanoparticles, characterized in that The steps include: Step 1: The carboxylated targeting small molecule and polymetformin are mixed and dissolved in DMSO at a mass ratio of 4-0.5:2-0.5, and reacted at 0-60°C in the presence of a catalyst for 12-48 hours to obtain a targeting small molecule and polymetformin conjugate 1; the carboxylated recruitment small molecule and polymetformin are mixed and dissolved in DMSO at a mass ratio of 3-0.5:2-0.5, and reacted at 0-60°C in the presence of a catalyst for 12-48 hours to obtain a recruitment small molecule and polymetformin conjugate 2; the conjugates 1 and 2 are separately dissolved in deionized water at 10-40°C, blended, and then sonicated to obtain a positively charged mixed solution of a polyprotein degradation targeting chimera; Step 2: blending the positively charged mixed solution of the polyprotein degradation targeting chimera with a hyaluronic acid solution to obtain negatively charged polyprotein degradation targeting chimera nanoparticles; Step 3: mixing the negatively charged polyprotein degradation targeted chimeric nanoparticles with liposome vesicles, and repeatedly extruding through a micro-extruder equipped with a nanoscale polycarbonate membrane to obtain liposome vesicle-encapsulated targeted protein degradation nanoparticles; In step 1, the catalyst is a condensation agent selected from one or more of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, and N,N'-dicyclohexylcarbodiimide; In step 1, the targeting small molecule is MK1775, and the recruiting small molecule is pomalidomide.

2. The preparation method according to claim 1, wherein: In step 2: at 10°C~40°C, the positively charged mixed solution of the polyprotein degradation targeting chimera is quickly added to the hyaluronic acid solution, and ultrasonically mixed to obtain negatively charged polyprotein degradation targeting chimera nanoparticles; the blending mass ratio of the polyprotein degradation targeting chimera to hyaluronic acid is 0.5:0.1~10.

3. The preparation method according to claim 1, wherein: Step 3 includes the following steps: 3a. 1,2-dioleoyl-sn-glycero-3-phosphocholine, (2,3-dioleoyl-propyl)-trimethylammonium chloride, and N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine are dissolved in chloroform, mixed uniformly, and the organic solvent is removed by rotary evaporation to obtain a dry lipid film. The lipid film is then ultrasonically hydrated in deionized water to obtain a lipid suspension, which is repeatedly extruded through a micro-extruder equipped with a nanoscale polycarbonate membrane to obtain liposome vesicles. 3b. Negatively charged polyprotein degradation targeted chimeric nanoparticles dissolved in deionized water and liposome vesicles are mixed, ultrasonically mixed, and repeatedly extruded through a micro-extruder equipped with a nanoscale polycarbonate membrane to obtain liposome vesicle-encapsulated targeted protein degradation nanoparticles.

4. The preparation method according to claim 3, wherein: 1,2-Dioleoyl-sn-glycero-3-phosphocholine, (2,3-dioleoyl-propyl)-trimethylammonium chloride, and N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine were dissolved in chloroform at a molar ratio of 1-12:0.5-6:0.5-10.

5. The preparation method according to claim 3, wherein: The mass ratio of the polyprotein degradation targeted chimeric nanoparticles to liposome vesicles was 0.2~1:0.5~5.

6. Use of targeted protein degradation nanoparticles encapsulated by liposome vesicles prepared according to the preparation method of any one of claims 1 to 5 in the preparation of anti-breast cancer drugs.

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