CN118203646A

CN118203646A - Targeted protein degradation chimera molecule self-assembled nano material, preparation method and application

Info

Publication number: CN118203646A
Application number: CN202410614511.6A
Authority: CN
Inventors: 刘沙; 王晓雯; 张晓瑜; 楚永超
Original assignee: Yantai Center For Disease Control And Prevention; Yantai University
Current assignee: Yantai Center For Disease Control And Prevention; Yantai University
Priority date: 2024-05-17
Filing date: 2024-05-17
Publication date: 2024-06-18

Abstract

The invention belongs to the technical field of pharmaceutical preparations, and particularly discloses a targeting protein degradation chimeric molecule self-assembled nanomaterial and a preparation method and application thereof. The self-assembled nano material of the target protein degradation chimeric molecule comprises MAL-PEG-DSPE and a target protein degradation chimeric molecule (PROTAC) for degrading bFGF; the targeting protein degradation chimeric molecule self-assembly nano material is formed by self-assembling the PROTAC and the MAL-PEG-DSPE. The structural formula of PROTAC is as follows: . The invention also provides a preparation method of the targeting protein degradation chimeric molecule self-assembled nanomaterial, and the surface is further modified with the sulfhydryl cetuximab to form a drug delivery system of targeting tumor cells. The targeting protein degradation chimera molecule self-assembled nano material has a certain killing effect on non-small cell lung cancer cell A549 cells.

Description

Targeted protein degradation chimera molecule self-assembled nano material, preparation method and application

Technical Field

The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a targeting protein degradation chimeric molecule self-assembled nanomaterial and a preparation method of the targeting protein degradation chimeric molecule self-assembled nanomaterial. The invention also relates to application of the targeting protein degradation chimeric molecule self-assembled nanomaterial.

Background

Basic fibroblast growth factor (bFGF) is a potent pro-angiogenic factor. By activating Fibroblast Growth Factor Receptors (FGFR), including FGFR1, FGFR2, FGFR3, FGFR4, exerting their pro-angiogenic effects, it has been demonstrated that they play a dual role in angiogenesis, on the one hand, can be involved in various pathophysiological processes such as angiogenesis and tissue repair, regulate immune cell activity, and affect immune system function. On the other hand, the polypeptide can provide nutrition support and a metastasis way for tumor cells, and is highly expressed in various malignant tumors. Research shows that bFGF has close relation with the occurrence and development of non-small cell lung cancer. For the occurrence of such conditions, methods of developing inhibitors may be employed for treatment. Four types of FGFR inhibitors have been marketed worldwide, namely erdasatinib, pemitinib, infliximab and fubat. However, due to off-target FGFR inhibitors and FGFR gene mutation, side effects such as hyperphosphatemia are caused, so that clinical treatment is limited. Therefore, it is important to degrade the bFGF target protein.

In the prior art, a heptapeptide (the sequence is PLLQATL) can block bFGF and has a certain treatment effect on breast cancer and colorectal cancer. However, polypeptide drugs generally have the problems of unstable physicochemical properties, short half-life, difficulty in permeation through cell membranes and the like, and limit the clinical application of the polypeptide drugs.

Targeting protein degradation chimera (PROTAC) is a technology that utilizes the ubiquitin-proteinase system (UPS) to degrade target proteins. PROTAC molecules consist of three parts, namely a target protein ligand, a connector Linker and an E3 ubiquitin ligase ligand. Target protein ligands include small molecules and polypeptides for targeting and capturing target proteins, E3 ubiquitin ligase ligands are responsible for specific recruitment of E3 ubiquitin ligase, linker is used to link these two ligands to form a stable ternary complex, degrading proteins by using ubiquitin-proteinase system. The PROTAC technology is based on the principle that an E3 ubiquitin ligase ligand binds to the E3 ligase, a target protein ligand binds to the target protein, the E3 ligase is recruited to the vicinity of the target protein through a linker, and the target protein is labeled with a ubiquitination tag, and then degraded through a ubiquitin-proteinase system. PROTAC has the characteristics of small dosage, stable structure, high targeting degradation protein precision and the like, but PROTAC generally has molecular weight of more than 1000 daltons, so that the water solubility, cell permeability, bioavailability and other properties of the targeting degradation protein are influenced.

Therefore, in the prior art, the targeting protein degradation chimeric molecule PROTAC of bFGF is lacking, and further application thereof in the direction of treating tumor is limited due to the large molecular weight of PROTAC, low solubility in water, poor targeting in vivo and inherent toxicity of nano-carrier.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a targeting protein degradation chimera molecule self-assembled nano material, a preparation method and application thereof, designs PROTAC molecules for directly degrading bFGF protein, self-assembles PROTAC and maleimide-polyethylene glycol-distearoyl phosphatidylethanolamine by utilizing the advantage of PROTAC self-structure to form a nano material with uniform particle size, simple components and high drug load, and the nano material can be further connected with sulfhydryl cetuximab with a tumor targeting function to form a nano system for precisely targeting tumor positions, and is applied to the field of medicine.

The technical scheme of the invention is as follows:

A self-assembled nanomaterial of a target protein degradation chimeric molecule, the self-assembled nanomaterial of the target protein degradation chimeric molecule comprises maleimide-polyethylene glycol-distearoyl phosphatidylethanolamine and a target protein degradation chimeric molecule for degrading basic fibroblast growth factor; the target protein degradation chimeric molecule self-assembled nanomaterial is prepared by self-assembling the target protein degradation chimeric molecule and the maleimide-polyethylene glycol-distearoyl phosphatidylethanolamine;

The structural formula of the targeting protein degradation chimeric molecule is as follows:

。

preferably, the targeting protein degradation chimeric molecule self-assembled nanomaterial further comprises a thiolated cetuximab; the sulfhydryl cetuximab is covalently connected with the maleimide-polyethylene glycol-distearoyl phosphatidylethanolamine.

The preparation method of the targeting protein degradation chimera molecule self-assembled nano material comprises the following steps:

The first step: dissolving thalidomide in N, N-dimethylformamide, adding N, N-diisopropylethylamine and 6-aminocaproic acid-2-methylpropan-2-yl ester into the solution, reacting for 10-12 hours at 110-130 ℃, adding water, extracting with ethyl acetate, washing, drying, filtering, concentrating to obtain a residue, and purifying to obtain an intermediate;

And a second step of: dissolving the intermediate prepared in the first step in dichloromethane, adding trifluoroacetic acid into the solution, reacting for 10-12 hours at room temperature, ending the reaction, and concentrating to obtain a crude product intermediate;

and a third step of: dissolving the crude product intermediate prepared in the second step in N, N-dimethylformamide, sequentially adding heptapeptide, 1-hydroxybenzotriazole, N-diisopropylethylamine and 1- (3-dimethylaminopropyl) -3-ethylcarbodimethylamine into the solution, stirring for 10-12 hours, filtering, washing and drying to obtain a target protein degradation chimeric molecule;

Fourth step: dissolving the targeting protein degradation chimeric molecule prepared in the third step and maleimide-polyethylene glycol-distearoyl phosphatidylethanolamine in absolute ethyl alcohol, removing an organic solvent by rotary evaporation, adding phosphate buffer solution for hydration, and passing through a microporous filter membrane to obtain the targeting protein degradation chimeric molecule self-assembled nanomaterial.

Further preferably, the mass ratio of thalidomide, N-diisopropylethylamine and 6-aminocaproic acid-2-methylpropan-2-yl ester in the first step is 1 (0.9-1.1): 0.6-0.8); the mass ratio of the crude product intermediate to the heptapeptide in the third step is (1:2) - (1:5); in the fourth step, the mass ratio of the targeting protein degradation chimeric molecule to the maleimido-polyethylene glycol-distearoyl phosphatidylethanolamine is in the range of 1: (4.5 to 5.5).

Further preferably, the mass ratio of the targeting protein degrading chimeric molecule to the maleimido-polyethylene glycol-distearoyl phosphatidylethanolamine in the fourth step is in the range of 1:5.

The preparation method of the self-assembled nanomaterial of the target protein degradation chimeric molecule with covalently linked sulfhydryl cetuximab comprises the following steps:

Fourth step: dissolving the targeting protein degradation chimeric molecule prepared in the third step and maleimide-polyethylene glycol-distearoyl phosphatidylethanolamine in absolute ethyl alcohol, removing an organic solvent by rotary evaporation, adding phosphate buffer solution for hydration, then co-incubating with the sulfhydryl cetuximab for 12-14 hours, and passing through a microporous filter membrane to obtain the target protein degradation chimeric molecule self-assembled nanomaterial with the sulfhydryl cetuximab in covalent connection.

Further preferably, the mass ratio of the targeting protein degrading chimeric molecule to the maleimido-polyethylene glycol-distearoyl phosphatidylethanolamine in the fourth step is in the range of 1:5, a step of; after hydration, the cells were incubated with mercaptocetuximab for a further 12h.

The application of the targeting protein degradation chimeric molecule self-assembled nanomaterial in preparing medicaments for inhibiting non-small cell lung cancer.

The application of the self-assembled nano material of the target protein degradation chimeric molecule prepared by the preparation method of the self-assembled nano material of the target protein degradation chimeric molecule in preparing medicaments for inhibiting non-small cell lung cancer.

Compared with the prior art, the invention has the beneficial effects that:

1. The self-assembled nano material of the target protein degradation chimera molecule designed and synthesized by the invention can be used for directly degrading basic fibroblast growth factor (bFGF), ensures the stability of the medicine to a certain extent and avoids the drug resistance caused by FGFR mutation.

2. The self-assembled nano material of the target protein degradation chimeric molecule designed and synthesized by the invention improves the cell permeability and in-vivo anti-tumor effect of the target protein degradation chimeric molecule, has high drug-loading rate, has the particle size of less than 200nm, and can be targeted to tumor tissues by injection.

Drawings

FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of PROTAC compounds prepared according to the present invention.

FIG. 2 is a diagram showing the degradation of bFGF target protein in A549 cells by PROTAC compounds prepared according to the present invention.

FIG. 3 is a graph showing the particle size distribution of PROTAC self-assembled nanomaterial obtained in Experimental example 2 in the third embodiment of the present invention.

FIG. 4 is a graph showing the particle size distribution of PROTAC self-assembled nanomaterial obtained in Experimental example 4 in the third embodiment of the present invention.

Fig. 5 is a transmission electron microscope image of PROTAC self-assembled nanomaterial obtained in experimental example 2 in the third embodiment of the present invention.

Fig. 6 is a transmission electron microscope image of PROTAC self-assembled nanomaterial obtained in experimental example 4 in the third embodiment of the present invention.

FIG. 7 is a graph showing the weight of a fourth tumor according to the embodiment of the present invention.

FIG. 8 is a line graph of a four tumor growth line according to an embodiment of the present invention.

Detailed Description

The invention is further illustrated below in conjunction with examples and experimental data.

The invention provides a self-assembled nanomaterial of a target protein degradation chimeric molecule (PROTAC), which comprises maleimide-polyethylene glycol-distearoyl phosphatidylethanolamine and a target protein degradation chimeric molecule (PROTAC) for degrading basic fibroblast growth factor (bFGF); the self-assembled nano material of the target protein degradation chimeric molecule (PROTAC) is formed by self-assembling the target protein degradation chimeric molecule (PROTAC) with the maleimide group-polyethylene glycol-distearoyl phosphatidylethanolamine (MAL-PEG-DSPE);

。

EXAMPLE one, PROTAC preparation of Compounds

The first step: thalidomide (2.0 g) as compound 1 was dissolved in N, N-dimethylformamide of 25 mL, N-diisopropylethylamine (1.87 g) and tert-butyl 6-aminocaproate (1.49 g) as compound 2 were added to the solution, and 12 h was reacted at 120 ℃,50 mL water was added to the reaction system, and extraction was performed with ethyl acetate (50 mL ×3). The combined organic layers were washed with 50 mL% saturated brine, dried over anhydrous sodium sulfate, and concentrated by filtration to give a residue. The crude product was purified by column chromatography (silica, petroleum ether: ethyl acetate=5:1 to 1:2). Intermediate (1.093 g, 34% yield) was obtained as compound 3 as a white oil.

Wherein, the structural formula of the compound 1 is as follows:

。

wherein, the structural formula of the compound 2 is as follows:

。

Wherein the intermediate of the compound 3 is named as 6- { [2- (2, 6-dioxy hexahydropyridin-3-yl) -1, 3-dioxy-2, 3-dihydro-1H-isoindol-4-yl ] amino } hexanoic acid-2-methylpropan-2-yl ester, and the structural formula is as follows:

。

and a second step of: the intermediate (1.093 g) prepared in the first step was dissolved in 20 dichloromethane mL, 4 mL trifluoroacetic acid was added to the solution, the reaction was completed at room temperature for 12 h, and the reaction system was concentrated under reduced pressure to obtain the crude product intermediate (1 g) as compound 4 as a white solid.

Wherein the crude intermediate of Compound 4 is named 6- { [2- (2, 6-dioxo-hexahydropyridin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-4-yl ] amino } hexanoic acid of the formula:

。

And a third step of: the crude intermediate (1.0 g) prepared in the second step was dissolved in 20. 20 mL of N, N-dimethylformamide, heptapeptide (sequence PLLQATL) (2.0 g) as compound 5, 1-hydroxybenzotriazole (384 mg), N-diisopropylethylamine (734 mg) and 1- (3-dimethylaminopropyl) -3-ethylcarbodimethylamine (546 mg) were sequentially added to the solution, stirred at room temperature for 10 hours, and the reaction mixture was filtered, washed three times with 20 mL of N, N-dimethylformamide and three times with 20 mL of methanol. The final product PROTAC compound (350. 350 mg, 98.6% purity) was obtained after drying in vacuo.

Wherein, the structural formula of the heptapeptide is as follows:

。

The thalidomide and the tert-butyl 6-aminohexanoate-aminocaproate related in this example are commercial compounds available in the market, and were purchased from Shanghai Yuan Ye Biotechnology Co., ltd, shanghai Jizhui Biotechnology Co., ltd, and heptapeptide (sequence PLLQATL) was synthesized by Sanguisia medical technology (Shanghai) Limited.

The reaction equations of the first to third steps are as follows:

；

DMF, TFA, DCM, HOBt, EDCI, DIEA, RT in the above equation represents N, N-dimethylformamide, trifluoroacetic acid, dichloromethane, 1-hydroxybenzotriazole, 1- (3-dimethylaminopropyl) -3-ethylcarbodimethylamine, N-diisopropylethylamine, and normal temperature, respectively.

FIG. 1 shows the successful synthesis of PROTAC compounds .PROTAC：1H NMR: (400 MHz, DMSO-d6), δ ppm 12.46 (s, 0.46H), 11.08 (s, 1H), 8.24-8.00 (m, 1H), 7.96-7.92 (m, 2H), 7.89-7.84 (m, 1H), 7.81-7.74 (m, 1H), 7.72-7.65 (m, 1H), 7.60-7.56 (m, 1H), 7.18 (s, 1H), 7.10-7.06 (m, 1H), 7.02 (d, J = 6.8 Hz, 1H), 6.74 (s, 1H), 6.53 (s, 1H), 5.07-5.02 (m, 1H), 4.40-4.31 (m, 2H), 4.28-4.14 (m, 6H), 3.95-3.92 (m, 2H), 3.58-3.53 (m, 1H), 3.48-3.41 (m, 1H), 3.34-3.29 (m, 2H), 2.93-2.84 (m, 1H), 2.61-2.54 (m, 1H), 2.30 (t, J = 7.2 Hz, 1H), 2.12-1.98 (m, 4H), 1.91-1.82 (m, 3H), 1.77-1.73 (m, 1H), 1.68-1.35 (m, 15H), 1.31-1.19 (m, 5H), 1.05 (d, J = 6.4 Hz, 3H), 0.89-0.80 (m, 18H).

Experimental example of degradation Effect of PROTAC Compounds on bFGF protein

A549 cells are inoculated on a six-hole plate in advance, and when the cells grow to about 80%, the cells are washed 3 times by using precooled Phosphate Buffer (PBS), 100 mu L of cell lysate (RIPA) (1 mu L of protease inhibitor is contained) is added into each hole, the cells are lysed for 3-5 min on ice, scraped by a cell scraper, and transferred into a 1.5 mL centrifuge tube. Continuing to lyse 30min, then centrifuging 10 and min at 12000 and rpm at 4 ℃, and collecting the supernatant to obtain the total protein solution. Protein concentration was determined using BCA method. Preparing 15% of separating gel, adding tetramethyl ethylenediamine (TEMED), mixing, pouring gel, slowly pouring pure water into the upper layer of separating gel, separating gel solid about 30min, and pouring the upper water. Preparing 5% concentrated gel, adding TEMED, mixing, pouring the gel, inserting a comb into the concentrated gel, pulling out the comb after solidification, adding 5 mu L of PROTAC samples prepared in the first example with the concentration of 20 mu g/mL and untreated control samples into the holes, and adding 3 mu L of markers (the markers represent protein molecular weight standards) into gel holes beside the samples. The separation gel voltage was set at 90V and the concentration gel voltage was set at 150V, and the electrophoresis was stopped until the electrophoresis reached about 1 and cm at the bottom of bromophenol blue.

A polyvinylidene fluoride (PVDF) film (0.45 μm) of moderate size was prepared, and before use, 2 min was activated with methanol, and the separation gel was placed in a transfer solution, and transfer conditions were set to 300 mA constant flow, 30 min. The transferred PVDF membrane was placed in a TBST incubation tank for a quick wash, added with 5% milk, placed on a shaker and blocked at room temperature for 30 min. The anti-bFGF is diluted according to the proportion of 1:1000, PVDF membrane is put into primary antibody working solution, and a shaking table is incubated at 4 ℃ for overnight. The secondary antibody is prepared by diluting goat anti-rabbit coupled with horseradish peroxide according to the proportion of 1:5000, and placing PVDF film into secondary antibody working solution, and incubating at room temperature for 30 min. And taking out the PVDF film, sucking the liquid on the dry film, adding the film into the mixed chemiluminescent liquid, and placing the film into a chemiluminescent instrument for development after the reaction is 1 min. The bar graph is shown in fig. 2. As can be seen from fig. 2, the PROTAC group prepared in example one has reduced bFGF expression compared with the control group, which initially indicates that the PROTAC compound synthesized in the present invention is capable of degrading bFGF protein to some extent.

Degradation rate = [1- (PROTAC/β -actin PROTAC)/(control/β -actin control) ] ×100%.

Wherein PROTAC groups: group PROTAC gray values expressed by bFGF;

Beta-actin PROTAC group: group PROTAC gray values expressed by bFGF;

control group: gray values expressed by bFGF of control group;

beta-actin control group: gray values expressed by bFGF of control group;

The degradation rate of the PROTAC groups prepared in the first example on bFGF protein is calculated to be 74.9%.

Preparation and verification examples of self-assembled nanomaterial of examples III and PROTAC

Experimental example 1: dissolving 1mg PROTAC mg of maleimide-polyethylene glycol-distearoyl phosphatidylethanolamine (MAL-PEG-DSPE) in absolute ethanol, removing organic solvent by rotary evaporation at 45 ℃, adding 3mL of phosphate buffer solution for hydration, and filtering with a microporous filter membrane.

Experimental example 2: dissolving 1mg PROTAC mg of maleimide-polyethylene glycol-distearoyl phosphatidylethanolamine (MAL-PEG-DSPE) in absolute ethanol, rotationally evaporating at 45 ℃ to remove organic solvent, adding 3mL of phosphate buffer solution for hydration, and filtering with microporous membrane.

Experimental example 3: dissolving 1mg PROTAC mg of maleimide-polyethylene glycol-distearoyl phosphatidylethanolamine (MAL-PEG-DSPE) in absolute ethanol, removing organic solvent by rotary evaporation at 45 ℃, adding 3mL of phosphate buffer solution for hydration, and filtering with microporous filter membrane.

Experimental example 4: dissolving 1mg PROTAC mg of maleimide-polyethylene glycol-distearoyl phosphatidylethanolamine (MAL-PEG-DSPE) in absolute ethyl alcohol, rotationally evaporating at 45 ℃ to remove an organic solvent, adding 3mL of phosphate buffer solution for hydration, incubating with the thiolated cetuximab for 12 hours, and enabling the thiolated cetuximab to be covalently connected with the maleimide-polyethylene glycol-distearoyl phosphatidylethanolamine (MAL-PEG-DSPE), and filtering by a microporous filter membrane.

Comparative experiment example 1: dissolving 1mg PROTAC and 2mg of polyethylene glycol-polylactic-co-glycolic acid (PEG-PLGA) in dichloromethane, removing the organic solvent by rotary evaporation at 45 ℃, adding 3mL of phosphate buffer solution for hydration, and filtering by a microporous filter membrane.

Comparative experiment example 2: dissolving 1mg PROTAC and 10mg of polyethylene glycol-polylactic-co-glycolic acid (PEG-PLGA) in dichloromethane, removing the organic solvent by rotary evaporation at 45 ℃, adding 3mL of phosphate buffer solution for hydration, and filtering by a microporous filter membrane.

Comparative experiment example 3: dissolving 1mg PROTAC mg polyethylene glycol-polylactic-co-glycolic acid (PEG-PLGA) in methanol, rotary evaporating at 45deg.C to remove organic solvent, adding 3mL phosphate buffer solution for hydration, and filtering with microporous membrane.

Comparative experiment example 4: dissolving 1mg PROTAC mg of maleimide-polyethylene glycol-distearoyl phosphatidylethanolamine (MAL-PEG-DSPE) in a mixed solution of acetone and ethyl acetate (acetone: ethyl acetate=1:2), removing the organic solvent by rotary evaporation at 45 ℃, adding 3mL of phosphate buffer solution for hydration, and filtering by a microporous filter membrane to obtain the modified starch.

Comparative experiment example 5: dissolving 1mg of thalidomide and 5mg of maleimide-polyethylene glycol-polylactic-co-glycolic acid (MAL-PEG-PLGA) in absolute ethyl alcohol, rotationally evaporating at 45 ℃ to remove an organic solvent, adding 3mL of phosphate buffer solution for hydration, and filtering by a microporous filter membrane to obtain the modified polylactic acid.

The solutions obtained in examples 1 to 4 and comparative examples 1 to 5 were monitored for encapsulation efficiency and particle size using an ultraviolet spectrophotometer and a particle sizer, as shown in Table 1.

Table 1 encapsulation efficiency and particle size of each experimental example

As shown in Table 1, the result is that PROTAC is easy to be encapsulated due to the acting force between the supramolecules of PROTAC and MAL-PEG-DSPE in the PROTAC self-assembled nanomaterial prepared by the invention, the encapsulation rate is high, the encapsulation rate can reach about 87% at most, and the particle size is about 200 nm.

As can be seen from the comparison of the test examples 1 to 3, the highest encapsulation rate of the test example 2 is the best test example. The morphology of the solution obtained in experimental example 2 was observed by transmission electron microscopy, see fig. 3 and 5.

Experimental example 4 self-assembled nanomaterial was covalently linked PROTAC with thiolated cetuximab, and the resulting solution was observed for morphology by transmission electron microscopy, see fig. 4 and 6.

Example IV, PROTAC in vivo anti-tumor Activity measurement of self-assembled nanomaterials

Taking A549 cells in logarithmic phase, centrifuging at 1000 r/min for 5 min, discarding supernatant, washing with PBS for three times, and re-suspending with appropriate amount of PBS at 1×10 ⁷/mL. 100 mu L of the cell suspension is inoculated to the upper part of the left armpit of a male nude mouse, the nude mouse is continuously fed in a feeding room with proper temperature and humidity, and the administration is started when the tumor volume is more than 70 mm ³.

Nude mice were randomly divided into 4 groups of 3 mice each. The intraperitoneal injection administration was performed at a dose of 4 mg/kg, and physiological saline, PROTAC compound solution prepared in example one, PROTAC self-assembled nanomaterial solution prepared in experimental example 2 in example three, and PROTAC self-assembled nanomaterial solution covalently linked to cetuximab thiol prepared in experimental example 4 in example three, respectively, were injected, and physiological saline was used as a control group. Dosing (i.v.) was performed every other day for 6 times. The animals were kept in a suitable temperature and humidity feeding house, and the weight and the length and width of the tumor were measured before each administration. The nude mice were sacrificed 15 days from the start of dosing, tumors were dissected and removed, and weighed and recorded after saline washout. All animal experiments and research procedures were performed according to the corresponding national standards.

Tumor growth inhibition = (1-experimental group tumor weight/physiological saline group tumor weight) ×100%.

And (3) calculating to obtain: the tumor inhibition rate of PROTAC compound prepared in example one is 39%, the tumor inhibition rate of PROTAC self-assembled nanomaterial prepared in experiment 2 in example three is 58%, the tumor inhibition rate of PROTAC self-assembled nanomaterial covalently linked with cetuximab prepared in experiment 4 in example three is 67%, compared with normal saline group, it is shown that PROTAC and self-assembled nanomaterial thereof have a certain in vivo anti-tumor capability, and the tumor inhibition rate is compared with: thiolated cetuximab covalently linked PROTAC self-assembled nanomaterial > PROTAC self-assembled nanomaterial > PROTAC compound.

Tumor volume (mm ³) =length×width ²/2.

The in vivo anti-tumor test results are shown in fig. 7 and 8 (p is equal to or less than 0.05, p is equal to or less than 0.01, p is equal to or less than 0.001, and p is equal to or less than 0.0001). Both fig. 7 and 8 intuitively show that the PROTAC and its self-assembled nanomaterial of the present invention can inhibit tumor growth to some extent compared to the physiological saline group.

Claims

1. A targeting protein degradation chimera molecule self-assembly nano material is characterized in that:

The self-assembled nanomaterial of the target protein degradation chimeric molecule comprises maleimide-polyethylene glycol-distearoyl phosphatidylethanolamine and the target protein degradation chimeric molecule for degrading basic fibroblast growth factor; the target protein degradation chimeric molecule self-assembled nanomaterial is prepared by self-assembling the target protein degradation chimeric molecule and the maleimide-polyethylene glycol-distearoyl phosphatidylethanolamine;

。

2. The targeted protein degradation chimera molecule self-assembled nanomaterial of claim 1, wherein: the targeting protein degradation chimeric molecule self-assembled nanomaterial also comprises sulfhydryl cetuximab; the sulfhydryl cetuximab is covalently connected with the maleimide-polyethylene glycol-distearoyl phosphatidylethanolamine.

3. The method for preparing the targeting protein degradation chimeric molecule self-assembled nanomaterial of claim 1, characterized by comprising the following steps:

4. A method of preparation as claimed in claim 3, wherein: in the first step, the mass ratio of thalidomide, N-diisopropylethylamine and 6-aminocaproic acid-2-methylpropan-2-yl ester is 1 (0.9-1.1) (0.6-0.8); the mass ratio of the crude product intermediate to the heptapeptide in the third step is (1:2) - (1:5); in the fourth step, the mass ratio of the targeting protein degradation chimeric molecule to the maleimido-polyethylene glycol-distearoyl phosphatidylethanolamine is in the range of 1: (4.5 to 5.5).

5. The method of manufacturing according to claim 4, wherein: in the fourth step, the mass ratio of the targeting protein degradation chimeric molecule to the maleimido-polyethylene glycol-distearoyl phosphatidylethanolamine is in the range of 1:5.

6. The method for preparing the targeting protein degradation chimeric molecule self-assembled nanomaterial of claim 2, comprising the steps of:

7. The method of manufacturing according to claim 6, wherein: in the first step, the mass ratio of thalidomide, N-diisopropylethylamine and 6-aminocaproic acid-2-methylpropan-2-yl ester is 1 (0.9-1.1) (0.6-0.8); the mass ratio of the crude product intermediate to the heptapeptide in the third step is (1:2) - (1:5); in the fourth step, the mass ratio of the targeting protein degradation chimeric molecule to the maleimido-polyethylene glycol-distearoyl phosphatidylethanolamine is in the range of 1: (4.5 to 5.5).

8. The method of manufacturing according to claim 7, wherein: in the fourth step, the mass ratio of the targeting protein degradation chimeric molecule to the maleimido-polyethylene glycol-distearoyl phosphatidylethanolamine is in the range of 1:5, a step of; after hydration, the cells were incubated with mercaptocetuximab for a further 12h.

9. Use of the targeted protein degradation chimera molecule self-assembled nanomaterial of claim 1 or 2 in the preparation of a medicament for inhibiting non-small cell lung cancer.

10. Use of the self-assembled nanomaterial of the target protein degradation chimera molecule prepared by the method for preparing the self-assembled nanomaterial of the target protein degradation chimera molecule according to any one of claims 3 to 8 in preparation of drugs for inhibiting non-small cell lung cancer.