Preparation method and application of targeted protein degradation nanoparticles wrapped by liposome vesicles

Abstract

The invention discloses a preparation method and application of targeted protein degradation nanoparticles wrapped by liposome vesicles. The preparation method comprises the following steps: blending a protein degradation targeting chimera based on polydimethyldiguanide with hyaluronic acid, and forming polyprotein degradation targeting chimera nanoparticles through an electrostatic adsorption effect; and blending the liposome vesicles with the polyprotein degradation targeting chimera nanoparticles, and carrying out co-extrusion to obtain the targeting protein degradation nanoparticles wrapped by the liposome vesicles. The targeting protein degradation nano-particles wrapped by the liposome vesicles can overcome the hook effect of the protein degradation targeting chimera in tumor cells and achieve long circulation of the protein degradation targeting chimera in a living body, the in-vivo utilization rate of the protein degradation targeting chimera can be increased to the maximum extent, and the anti-tumor effect is enhanced.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically 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, which has always affected human health and social and economic stability. According to statistics from the World Health Organization (WHO), there will be nearly 20 million new cancer cases and nearly 10 million cancer deaths in 2022. And demographic-based forecasts show that by 2050, the number of new cancer cases per year will reach 35 million, an increase of 77% over the 2022 level. Therefore, cancer control measures are becoming increasingly necessary.

[0003] Current treatments for tumors include surgery, chemotherapy, targeted therapy, bone marrow transplantation, and immunotherapy. Among them, targeted therapy is a method of specifically binding to a clear oncogenic site at the cellular molecular level, destroying its original biological effect, and inducing specific cell death. A new targeted therapy strategy, protein degradation targeted chimera (PROTAC), has attracted great interest due to its potential in therapeutic regulation of proteins. Protein degradation targeted chimera is a heterobifunctional small molecule composed of two ligands: one ligand targets the target protein, and the other ligand recruits the E3 ubiquitin ligase. When the protein degradation targeted chimera binds to the target protein and the E3 ligase at the same time, it can induce ubiquitination of the target protein, which is then degraded by the ubiquitin-proteasome system (UPS). Compared with small molecule inhibitors, protein degradation targeted chimera has the advantages of high selectivity, overcoming drug resistance, and non-drugability of the target. However, due to its large molecular weight, it has problems such as poor water solubility and low cell permeability, which limits the application of protein degradation targeted chimera in vivo. Although the development of protein degradation targeted chimeras has made great progress, there are still problems such as poor efficacy of protein degradation targeted chimeras as a single therapy and poor clinical translation. Compared with the current traditional tumor radiotherapy and chemotherapy, the applicability of protein degradation targeted chimeras is far from enough, and more protein degradation platforms need to be developed.

[0004] Nano-delivery systems are widely used in the field of cancer treatment due to the advantages of small nanoparticle size and the ability to effectively enter the lesion area. Nano-delivery systems can improve the efficacy and reduce biological toxicity by enhancing drug stability, reducing adverse reactions and regulating pharmacokinetic characteristics. Therefore, drug delivery based on nano-delivery can overcome many obstacles faced by traditional therapies and is becoming a new approach for diagnostic and therapeutic strategies. Summary of the invention

[0005] In order to solve the problems of high off-target property, limited hook effect and poor pharmacokinetics of protein degradation targeted chimeras, the present invention provides a preparation method and application of liposome vesicle-encapsulated targeted protein degradation nanoparticles. The present invention overcomes the off-target property and hook effect of protein degradation targeted chimeras in tumor cells and realizes the long circulation of protein degradation targeted chimeras in the body, thereby greatly increasing the in vivo utilization rate of protein degradation targeted chimeras and exerting a good anti-tumor effect.

[0006] The method for preparing the targeted protein degradation nanoparticles encapsulated by liposome vesicles of the present invention comprises the following steps: Step 1: Mix and dissolve the carboxylated targeting small molecule and polymetformin in an organic solvent, react under a certain temperature and catalyst conditions to obtain a targeting small molecule and polymetformin conjugate 1; mix and dissolve the carboxylated recruitment small molecule and polymetformin in an organic solvent, react under a certain temperature and catalyst conditions to obtain a recruitment small molecule and polymetformin conjugate 2; blend the conjugates 1 and 2 to obtain a positively charged mixed solution of a polyprotein degradation targeting chimera. The targeting small molecule, the recruitment small molecule and polymetformin are generally connected by chemical bonds.

[0007] 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.

[0008] Step 3: The negatively charged polyprotein degradation targeted chimeric nanoparticles are mixed with liposome vesicles, and are repeatedly extruded through a micro extruder equipped with a nanoscale polycarbonate membrane to obtain liposome vesicle-encapsulated targeted protein degradation nanoparticles.

[0009] Further, 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 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; at 10°C~40°C, conjugates 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.

[0010] 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.

[0011] The targeting small molecule is selected from one or more of MK1775, 4-hydroxytamoxifen, palbociclib, JQ1, dasatinib, gefitinib, enzalutamide, NLG919, indomethacin, olaparib, and RSL3.

[0012] The recruiting small molecule is selected from one or more of pomalidomide, lenalidomide, thalidomide, VH032, and VH298.

[0013] The drug loading rate of the targeted small molecule and polymetformin linker 1 is 5% to 95%.

[0014] The drug loading rate of the recruited small molecule and polymetformin linker 2 is 5% to 95%.

[0015] The drug loading rate calculation formula is: .

[0016] Further, in step 2: at 10°C to 40°C, the positively charged mixed solution of the polyprotein degradation targeting chimera is quickly added to the hyaluronic acid solution, ultrasonicated for 1 to 20 minutes, and mixed evenly 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.

[0017] Further, step 3 includes the following steps: 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 ) is 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. The lipid film 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.

[0018] 3b. The negatively charged polyprotein degradation targeted chimera nanoparticles dissolved in deionized water and liposome vesicles are mixed, ultrasonicated for 1 to 20 minutes, mixed evenly, and repeatedly extruded through a micro extruder equipped with a nanoscale polycarbonate membrane to obtain liposome vesicle-encapsulated targeted protein degradation nanoparticles.

[0019] The mass ratio of the polyprotein degradation targeted chimeric nanoparticles to the liposome vesicles is generally between 0.2~1:0.5~5.

[0020] The polycarbonate membrane is a polycarbonate membrane matched with a liposome extruder produced by Avanti Polar Lipids, and the size specification range used is 100nm-1000nm.

[0021] The invention discloses an application of the targeted protein degradation nanoparticles encapsulated by liposome vesicles in the preparation of anti-tumor drugs.

[0022] The beneficial effects of the present invention are embodied in: The present invention designs a targeted protein degradation nanoparticle encapsulated by liposome vesicles, wherein the polyprotein degradation targeted chimera based on polymethformin provides the functions of effective multi-ligand targeting and multi-site recruitment, can target multiple target proteins in tumor cells, and recruit multiple E3 ubiquitin ligases for protein ubiquitination, and finally be recognized and degraded by the ubiquitin-proteasome system. Through the encapsulation of liposome vesicles, the polymerized protein degradation targeted chimera can avoid unstable diffusion in the blood circulation, which promotes its better retention in the tumor site and endocytosis by tumor cells. Compared with the protein degradation targeted chimera based on small molecules, the polyprotein degradation targeted chimera can have the advantages of efficient protein degradation effect, long blood circulation time and targeted tumor accumulation. The polyprotein degradation targeted chimera provides a universal platform for the precise construction of protein degradation targeted chimera, and provides a promising model for the future clinical transformation application of protein degradation targeted chimera. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The preparation route of the targeted protein degradation nanoparticles encapsulated by liposome vesicles in the embodiments of the present invention is schematically shown.

[0024] Figure 2 The synthetic route of the PolyPROTAC component in the examples of the present invention is schematically shown.

[0025] Figure 3 The hydrogen nuclear magnetic resonance spectrum of the intermediate 1 prepared in the embodiment of the present invention is schematically shown.

[0026] Figure 4 The ultraviolet absorption spectrum of the intermediate 1 prepared in the embodiment of the present invention is schematically shown.

[0027] Figure 5 The hydrogen nuclear magnetic resonance spectrum of the intermediate 2 prepared in the embodiment of the present invention is schematically shown.

[0028] Figure 6 The hydrogen nuclear magnetic resonance spectrum of the intermediate 3 prepared in the embodiment of the present invention is schematically shown.

[0029] Figure 7 The hydrogen nuclear magnetic resonance spectrum of the intermediate 4 prepared in the embodiment of the present invention is schematically shown.

[0030] Figure 8 The ultraviolet absorption spectrum of intermediate 4 prepared in an embodiment of the present invention is schematically shown.

[0031] Fig. 9 The hydrogen nuclear magnetic resonance spectrum of the intermediate 5 prepared in the embodiment of the present invention is schematically shown.

[0032] Fig.10 The hydrogen nuclear magnetic resonance spectrum of the intermediate 6 prepared in the embodiment of the present invention is schematically shown.

[0033] Fig.11 The hydrogen nuclear magnetic resonance spectrum of the intermediate 7 prepared in the embodiment of the present invention is schematically shown.

[0034] Fig.12 The hydrogen nuclear magnetic resonance spectrum of the intermediate 8 prepared in the embodiment of the present invention is schematically shown.

[0035] Fig.13 The hydrogen nuclear magnetic resonance spectrum of the intermediate 9 prepared in the embodiment of the present invention is schematically shown.

[0036] Fig.14 The ultraviolet absorption spectrum of intermediate 9 prepared in an embodiment of the present invention is schematically shown.

[0037] Fig.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.

[0038] Fig.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.

[0039] Fig.17 The figure schematically shows the cytotoxicity experimental results of Lipo-PolyPROTAC nanoparticles prepared in an embodiment of the present invention.

[0040] Fig.18The figure schematically shows the results of a Western Blot experiment of protein degradation by Lipo-PolyPROTAC nanoparticles prepared in an embodiment of the present invention.

[0041] Fig.19 The schematic diagram of the dosing plan of Lipo-PolyPROTAC nanoparticles prepared in an embodiment of the present invention is schematically shown.

[0042] Fig. 20 The efficacy experiment of Lipo-PolyPROTAC nanoparticles prepared in an embodiment of the present invention is schematically shown, including a tumor growth curve.

[0043] Fig.21 The biosafety experiment of Lipo-PolyPROTAC nanoparticles prepared in an embodiment of the present invention is schematically shown, including a mouse weight growth curve. DETAILED DESCRIPTION

[0044] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but is not intended to limit the scope of the present invention. Example 1: Synthesis of PolyMet (Intermediate 1)

[0045] like Figure 2 As shown in the synthetic route, a 50 mL flask was added with 0.1 g of linear polyethyleneimine (PEI), 1 g of dicyandiamide and 10 mL of 2M HCI solution, and a reflux device was added to react 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 a 1M NaOH solution, and ultrafiltered multiple times with a 3000 MW ultrafiltration tube to ultrafilter the solution to 1-2 mL, and then transferred to a 10 mL centrifuge tube for freeze-drying to finally obtain a white solid intermediate 1.

[0046] By nuclear magnetic resonance spectroscopy ( 1 H-NMR) and UV absorption spectra were used to characterize the chemical structure of the intermediate 1 synthesized in Example 1.

[0047] Figure 2 The synthetic route of the PolyPROTAC component in the examples of the present invention is schematically shown.

[0048] Figure 3 The hydrogen nuclear magnetic resonance 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 of δ 2.5-4.0, which is similar to metformin (Met).

[0049] 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). Example 2: Synthesis of Intermediate 2

[0050] like Figure 2 As shown in the synthetic route, a 10 mL flask was taken, 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) were added respectively, and dissolved with dimethyl sulfoxide (DMSO) (2 mL), sealed, and the temperature was set at 90 ° C. The reaction was allowed to proceed for 24 hours. After the reaction was completed, a saturated ammonium chloride solution was added to quench the reaction, and the mixture was extracted with ethyl acetate. After extraction, the mixture was dried on a rotary evaporator, and then 2-3 mL of ethyl acetate was added to dissolve the mixture, and the mixture was separated on a silica gel plate. The chromatographic solution was cyclohexane: ethyl acetate = 3:4. The silica gel powder of the separated product was scraped off and rinsed with ethyl acetate. The ethyl acetate solution containing the pure product was dried on a rotary evaporator to obtain a yellow oily intermediate 2 with a yield of 44.3%.

[0051] By nuclear magnetic resonance spectroscopy ( 1 H-NMR) and mass spectrometry were used to characterize the chemical structure of intermediate 2 synthesized in Example 2.

[0052] Figure 5 The hydrogen nuclear magnetic resonance spectrum of the intermediate 2 prepared in the embodiment of the present invention is schematically shown. 1 H NMR nuclear magnetic resonance data and mass spectrum data are as follows: 1 H 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) + . Example 3: Synthesis of Carboxylated Pomalidomide (Intermediate 3)

[0053] like Figure 2 As shown in the synthetic route, the obtained intermediate 2 (0.2mmol, 77.48mg) was dissolved in 2mL of dichloromethane (DCM), transferred into a 10mL flask, and then 1mL of trifluoroacetic acid was added, sealed, and reacted at room temperature for 2h. After the reaction, it was dried by a rotary evaporator, and then DCM was added for multiple washings to precipitate a yellow solid, and finally placed in a vacuum oven to dry to obtain a yellow solid intermediate 3, with a yield of 85.4%.

[0054] By nuclear magnetic resonance spectroscopy ( 1 H-NMR) and mass spectrometry were used to characterize the chemical structure of intermediate 3 synthesized in Example 3.

[0055] Figure 6 The hydrogen nuclear magnetic resonance spectrum of the intermediate 3 prepared in the embodiment of the present invention is schematically shown. 1 H NMR nuclear magnetic resonance data and mass spectrum data are as follows: 1 H 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) - . Example 4: Synthesis of PolyPomalidomide (Intermediate 4)

[0056] like Figure 2 As shown in the synthetic route, take a 50mL flask, add intermediate 3 (0.5mmol, 150.64mg), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (0.5mmol, 97.85mg) and N-hydroxysuccinimide (NHS) (0.5mmol, 57.54mg), respectively, dissolve in 10mL DMSO, react at room temperature for 1h, then add intermediate 1 (100mg), react at room temperature for 48h, after the reaction is completed, put the reaction solution into a 3.5K dialysis bag for dialyzing for about a week, and finally put it into a freeze dryer for drying for 48h to obtain a yellow solid intermediate 4 (i.e., linker 2).

[0057] By nuclear magnetic resonance spectroscopy ( 1 H-NMR) and UV absorption spectra were used to characterize the chemical structure of intermediate 4 synthesized in Example 4.

[0058] Figure 7 The hydrogen nuclear magnetic resonance 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, 0.5-4.0, which are similar to Pomalidomide.

[0059] 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 8 It 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%. Example 5: Synthesis of Intermediate 5

[0060] like Figure 2As shown in the synthetic route, take a 10 mL flask, first add 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-chloroperbenzoic acid (mCPBA) (0.11 mmol, 22.33 mg), then dissolve in 2 mL of toluene, react at room temperature for 1 h, then add DIPEA (0.2 mmol, 25.85 mg) and tert-butyl 4-(4-aminophenyl)piperazine-1-carboxylate (0.12 mmol, 33.28 mg), and react 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 to dissolve the product and separated on a silica gel plate. The chromatographic liquid was cyclohexane:ethyl acetate = 2:3. After scraping off the product silica gel powder, it was rinsed with ethyl acetate. The ethyl acetate solution containing the pure product was dried on a rotary evaporator to obtain a brown oily intermediate 5 with a yield of 66.4%.

[0061] By nuclear magnetic resonance spectroscopy ( 1 The chemical structure of intermediate 5 synthesized in Example 5 was characterized by H-NMR) and mass spectrometry.

[0062] Fig. 9 The hydrogen nuclear magnetic resonance spectrum of the intermediate 5 prepared in the embodiment of the present invention is schematically shown. 1 H NMR nuclear magnetic resonance data and mass spectrum data are as follows: 1 H 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, 1.1Hz, 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) + . Example 6: Synthesis of Intermediate 6

[0063] like Figure 2 As shown in the synthetic route, the 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 reacted at room temperature for 3 h. After the reaction, it was dried using a rotary evaporator, washed repeatedly with DCM and then dried using a rotary evaporator. Finally, it was dried in a vacuum drying oven to obtain a brown oily intermediate 6 with a yield of 80.5%.

[0064] By nuclear magnetic resonance spectroscopy ( 1 H-NMR) and mass spectrometry were used to characterize the chemical structure of intermediate 6 synthesized in Example 6.

[0065] Fig.10 The hydrogen nuclear magnetic resonance spectrum of the intermediate 6 prepared in the embodiment of the present invention is schematically shown. 1 H NMR nuclear magnetic resonance data and mass spectrum data are as follows: 1 H 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) + . Example 7: Synthesis of Intermediate 7

[0066] like Figure 2 As shown in the synthetic route, take a 10mL flask, add intermediate 6 (0.05mmol, 24.33mg), tert-butyl 3-bromopropionate (0.1mmol, 20.90mg) and K2CO3 (0.15mmol, 20.73mg), dissolve with 3mL N,N-dimethylformamide (DMF), and react at 80℃ for 12h. After the reaction, quench with saturated ammonium chloride solution, extract with ethyl acetate, dry on a rotary evaporator, add 2-3mL ethyl acetate to dissolve, separate on a silica gel plate, the chromatographic liquid is ethyl acetate: methanol = 10:1, scrape off the product silica gel powder, rinse with ethyl acetate, and dry the ethyl acetate solution containing the pure product on a rotary evaporator to obtain brown oily intermediate 7 with a yield of 46.3%.

[0067] By nuclear magnetic resonance spectroscopy ( 1 The chemical structure of intermediate 7 synthesized in Example 7 was characterized by H-NMR) and mass spectrometry.

[0068] Fig.11 The hydrogen nuclear magnetic resonance spectrum of the intermediate 7 prepared in the embodiment of the present invention is schematically shown. 1 H NMR nuclear magnetic resonance data and mass spectrum data are as follows: 1 H 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) + . Example 8: Synthesis of carboxylated MK1775 (Intermediate 8)

[0069] 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 into a 10 mL flask. 1 mL of trifluoroacetic acid was added and reacted at room temperature for 3 h. After the reaction, it was dried on a rotary evaporator, washed repeatedly with DCM and dried on a rotary evaporator. Finally, it was dried in a vacuum drying oven to obtain a brown oily intermediate 8 with a yield of 83.6%.

[0070] By nuclear magnetic resonance spectroscopy ( 1 The chemical structure of intermediate 8 synthesized in Example 8 was characterized by H-NMR) and mass spectrometry.

[0071] Fig.12 The hydrogen nuclear magnetic resonance spectrum of the intermediate 8 prepared in the embodiment of the present invention is schematically shown. 1 H NMR nuclear magnetic resonance data and mass spectrum data are as follows: 1 H 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, 1.0Hz, 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 / z 557.2626 (MH) - . Example 9: Synthesis of PolyMK1775 (Intermediate 9)

[0072] like Figure 2As shown in the synthesis route, take a 50mL flask, add intermediate 8 (0.1mmol, 55.864mg), EDCI (0.5mmol, 97.85mg) and NHS (0.5mmol, 57.54mg) respectively, add 10mL DMSO to dissolve, react at room temperature for 1h, then add intermediate 1 (50mg), react at 60℃ for 48h. After the reaction, put the reaction solution into a 3.5K dialysis bag for dialyzation for about one week, and finally put it into a freeze dryer for drying for 48h to obtain a brown solid intermediate 9 (i.e., linker 1).

[0073] By nuclear magnetic resonance spectroscopy ( 1 H-NMR) and UV absorption spectra were used to characterize the chemical structure of intermediate 9 synthesized in Example 9.

[0074] Fig.13 The hydrogen nuclear magnetic resonance spectrum of the intermediate 9 prepared in the embodiment of the present invention is schematically shown. Fig.13 It can be seen that in the DMSO-d6 reagent, there are specific peaks of δ 4.0-10.0, 0.5-3.5, similar to MK1775 Fig.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. Fig.14 It can be seen that PolyMK1775 has an absorption peak similar to that of MK1775. The drug loading rate of the conjugate of MK1775 and polymetformin is 9.5%. Example 10: Preparation of Lipo-PolyPROTAC Nanoparticles

[0075] 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 PolyPROTAC positively charged mixed solution.

[0076] The PolyPROTAC positively charged mixed solution and the hyaluronic acid (HA) solution were evenly mixed at a mass ratio of 0.5:0.9, and ultrasonicated for 5 minutes to obtain negatively charged PolyPROTAC-NP nanoparticles.

[0077] 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) 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. The lipid film 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.

[0078] The negatively charged PolyPROTAC-NP nanoparticles were mixed with liposome vesicles at a mass ratio of 0.35:1.1, ultrasonicated for 10 minutes, and repeatedly extruded through a micro-extruder equipped with a 200nm nanoscale polycarbonate membrane to obtain a liposome vesicle-encapsulated targeted protein degradation nanoparticle Lipo-PolyPROTAC.

[0079] 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: Fig.15 shown.

[0080] 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: Fig.16 shown.

[0081] Figure 1 The preparation route of the targeted protein degradation nanoparticles encapsulated by liposome vesicles in the embodiments of the present invention is schematically shown.

[0082] Fig.15 The following 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. Fig.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.

[0083] Fig.16 The dynamic light scattering particle size, PDI, Zeta potential and transmission electron microscopy characterization of Lipo-PolyPROTAC nanoparticles prepared in the embodiment of the present invention are schematically shown. Fig.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. Example 11: Toxicity experiment of Lipo-PolyPROTAC nanoparticles

[0084] First, 4T1 cells were 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, respectively. The concentration gradient was set to 0, 50, 100, 200, 400, 800, 1000, 2000, 4000, and 5000 mM. Then, the drugs were 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.

[0085] Fig.17 The cytotoxicity test results of Lipo-PolyPROTAC nanoparticles prepared in the embodiment of the present invention are schematically shown. Fig.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. Example 12: Protein degradation effect of Lipo-PolyPROTAC nanoparticles

[0086] First, 4T1 cells were seeded in 10 cm culture dishes (5*10 5 cells), and the cells were allowed to adhere to the wall after 12 hours. The cells were treated with Lipo-PolyPROTAC nanoparticles for 24 hours, and the concentration gradient was set to 0, 1, 2, 3, 4, and 5 uM. The cells were then lysed with RIPA lysis buffer, and the protein was extracted. Finally, the degradation effect of Wee1 protein was determined by Western Blot.

[0087] Fig.18 The Western Blot experimental results of protein degradation by Lipo-PolyPROTAC nanoparticles prepared in the embodiment of the present invention are schematically shown. Fig.18 It can be seen that Lipo-PolyPROTAC nanoparticles exhibit significant protein degradation effect and have good dose dependence. Example 13: Animal efficacy experiment of Lipo-PolyPROTAC nanoparticles

[0088] 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 3 doses. The dosing schedule is detailed in Fig.19 .

[0089] Fig.19 The schematic diagram of the dosing plan of Lipo-PolyPROTAC nanoparticles prepared in an embodiment of the present invention is schematically shown.

[0090] The dose of Lipo-PolyMK1775 + Lipo-PolyPomalidomide group and Lipo-PolyPROTAC group was 5 mg / kg. The effect of Lipo-PolyPROTAC nanoparticles in inhibiting tumor growth was verified by comparing the changes in tumor volume.

[0091] Fig. 20 The efficacy experiment of Lipo-PolyPROTAC nanoparticles prepared in the embodiment of the present invention is schematically shown, including the tumor growth curve. Fig. 20 It 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.

[0092] Fig.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. Fig.21 It can be seen that no obvious biological toxicity is reflected in the changes in mouse body weight.

[0093] Therefore, it can be concluded that Lipo-PolyPROTAC nanoparticles exhibited strong antitumor effects and good biosafety compared with the control group.

[0094] 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 description is only a specific embodiment of the present invention and is 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 vesicle-encapsulated targeted protein degradation nanoparticles, characterized in that The steps include: Step 1: Mix and dissolve the carboxylated targeting small molecule and polymetformin in an organic solvent, and react under a certain temperature and catalyst conditions to obtain a linker 1 of the targeting small molecule and polymetformin; Mix and dissolve the carboxylated recruitment small molecule and polymetformin in an organic solvent, and react under a certain temperature and catalyst conditions to obtain a linker 2 of the recruitment small molecule and polymetformin; Blend the linkers 1 and 2 to obtain a positively charged mixed solution of a polyprotein degradation targeting chimera; Step 2: mixing the positively charged mixed solution of the polyprotein degradation targeted chimera with the hyaluronic acid solution to obtain negatively charged polyprotein degradation targeted chimera nanoparticles; Step 3: The negatively charged polyprotein degradation targeted chimeric nanoparticles are mixed with liposome vesicles, and are repeatedly extruded through a micro extruder equipped with a nanoscale polycarbonate membrane to obtain liposome vesicle-encapsulated targeted protein degradation nanoparticles.

2. The preparation method according to claim 1, characterized in that: 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 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; at 10°C~40°C, conjugates 1 and 2 are respectively dissolved in deionized water, blended and sonicated to obtain a positively charged mixed solution of polyprotein degradation targeting chimera.

3. The preparation method according to claim 2, characterized in that: 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.

4. The preparation method according to claim 2, characterized in that: The targeting small molecule is selected from one or more of MK1775, 4-hydroxytamoxifen, palbociclib, JQ1, dasatinib, gefitinib, enzalutamide, NLG919, indomethacin, olaparib, and RSL3; the drug loading rate of the targeting small molecule and the polymetformin connector 1 is 5% to 95%.

5. The preparation method according to claim 2, characterized in that: The recruited small molecule is selected from one or more of pomalidomide, lenalidomide, thalidomide, VH032, and VH298; the drug loading rate of the recruited small molecule and the polymetformin connector 2 is 5% to 95%.

6. The preparation method according to claim 1, characterized in that: 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.

7. The preparation method according to claim 1, characterized in that: Step 3 includes the following steps: 3a. Dissolve 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 in chloroform, mix well, and then remove the organic solvent by rotary evaporation to obtain a dry lipid film, then ultrasonically hydrate the lipid film in a deionized water solution to obtain a lipid suspension, and repeatedly extrude the lipid suspension through a micro extruder equipped with a nanoscale polycarbonate membrane to obtain liposome vesicles; 3b. The negatively charged polyprotein degradation targeted chimera nanoparticles dissolved in deionized water and liposome vesicles are mixed, ultrasonically mixed evenly, and repeatedly extruded through a micro extruder equipped with a nanoscale polycarbonate membrane to obtain liposome vesicle-encapsulated targeted protein degradation nanoparticles.

8. The preparation method according to claim 7, characterized in that: 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.

9. The preparation method according to claim 7, characterized in that: The mass ratio of the polyprotein degradation targeted chimeric nanoparticles and liposome vesicles is 0.2~1:0.5~5.

10. Use of targeted protein degradation nanoparticles encapsulated by liposome vesicles prepared according to any one of the preparation methods of claims 1 to 9 in the preparation of anti-tumor drugs.

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