Membrane-coated PROTAC magnetic nanoparticles capable of enhancing technical efficiency of targeted protein degradation chimera as well as preparation method and application of membrane-coated PROTAC magnetic nanoparticles
By coating tumor cell membranes on PROTAC magnetic nanoparticles and loading BRD4 inhibitors, the off-target effect and monitoring problems of PROTAC technology when targeting tumors are solved, and the protein degradation and real-time monitoring of breast cancer tumors is achieved efficiently, enhancing the Anti-tumor effect.
Patent Information
- Application Number
- CN202411301560.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-18
- Publication Date
- 2024-12-17
AI Technical Summary
PROTAC technology has off-target effects when targeting tumor tissues, making it difficult to pass through the cell membrane, unclear distribution in the body, and the treatment effect cannot be monitored in real time, resulting in bottlenecks in its development.
Using magnetic mesoporous material as a carrier, the BRD4 protein degradation inhibitor ARV-825 was loaded, and the tumor cell membrane was coated on the surface. The membrane-coated PROTAC magnetic nanoparticles were prepared by co-precipitation method and cell membrane coating technology to achieve efficient targeting. Breast cancer tumors, combined with simulated fasting to regulate tumor cell energy metabolism, improve the efficiency of target protein degradation.
It has achieved efficient protein degradation of breast cancer tumors, enhanced anti-tumor effects, and monitored tumor distribution in real time through nuclear magnetic resonance MRI, improving the visualization and effect of treatment.
Smart Images
Figure CN119139261A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of magnetic nanoparticles, and in particular to a membrane-coated PROTAC magnetic nanoparticle capable of enhancing the efficiency of targeted protein degradation chimera technology, and a preparation method and application thereof. Background Art
[0002] The protein degradation targeting chimera (PROTAC) technology utilizes the ubiquitination-proteasome system naturally present in cells to achieve specific and efficient degradation of disease-related target proteins through a bifunctional hybrid compound that is different from antibodies and traditional small molecule inhibitors, thereby achieving the treatment of diseases. Compared with monoclonal antibodies or small molecule targeted drugs, the biggest advantage of PROTAC technology is that it changes the target from "non-drugable" to "drugable". Traditional monoclonal antibodies or small molecule targeted drugs need to have a strong binding to the target protein, usually to the active site, but 80% of the proteins in human cells lack such sites, and PROTAC only needs to weakly bind to the target protein to specifically "mark" it. Therefore, ~80% of the undruggable proteins in the proteome can be solved by PROTAC technology without developing drug resistance, and have good clinical application prospects.
[0003] Although PROTAC technology has great potential for clinical application, its development has encountered bottlenecks. For example, it is necessary to optimize the structure of PROTAC compounds multiple times to determine the optimal linker length and composition, so that the E3 ligase and the target protein are in a suitable spatial conformation to form an effective ternary complex; secondly, traditional small molecule PROTACs cannot target tumor tissues, so off-target effects may occur. Although peptide PROTACs have high targeting specificity and good biocompatibility, they are difficult to pass through the cell membrane and enter the cell. In addition, the in vivo distribution of PROTACs is still unclear, and the therapeutic effect cannot be monitored and evaluated in real time. Therefore, it is of great significance to develop PROTAC magnetic nanoparticles with efficient protein targeting. Summary of the invention
[0004] The present invention aims to provide a membrane-coated PROTAC magnetic nanoparticle that can enhance the efficiency of targeted protein degradation chimera technology and a preparation method and application thereof, so as to achieve the purpose of efficiently degrading target proteins.
[0005] To achieve the above objectives, the present invention adopts the following technical scheme: a membrane-coated PROTAC magnetic nanoparticle that can enhance the efficiency of targeted protein degradation chimera technology, with a magnetic mesoporous material as a carrier, a BRD4 protein degradation inhibitor loaded on the carrier, and a tumor cell membrane coated on the surface of the carrier.
[0006] Preferably, as an improvement, the BRD4 protein degradation inhibitor is ARV-825.
[0007] Preferably, as an improvement, the magnetic mesoporous material is magnetic mesoporous silicon.
[0008] Preferably, as an improvement, the tumor cell membrane is an MDA-MB-231 breast cancer cell membrane.
[0009] Preferably, as an improvement, the membrane-coated PROTAC magnetic nanoparticles are spherical structures with a particle size of 104±0.2 nm.
[0010] Preferably, as an improvement, a method for preparing membrane-coated PROTAC magnetic nanoparticles that can enhance the efficiency of targeted protein degradation chimera technology is prepared by co-precipitation method combined with cell membrane coating technology.
[0011] Preferably, as an improvement, a method for preparing membrane-coated PROTAC magnetic nanoparticles that can enhance the efficiency of targeted protein degradation chimera technology comprises the following steps:
[0012] Step 1, coprecipitation: dissolving ferroferric oxide in anhydrous ethanol, adding alkaline solution thereto, and then dropping deionized water to carry out coprecipitation reaction to obtain system I;
[0013] Step 2: Surface modification: adding ethyl silicate aqueous solution to system I for surface modification;
[0014] Step 3, separation and washing: the product obtained in step 2 is repeatedly washed with anhydrous ethanol and double distilled water, centrifuged, and dried for later use;
[0015] Step 4: Cell membrane preparation: resuspend the tumor cells, freeze and thaw repeatedly, remove the supernatant by centrifugation, extract the tumor cell membrane, wash it with sterile PBS and set aside;
[0016] Step 5, membrane coating: resuspend the product obtained in step 3 and the product obtained in step 4 in deionized water, stir and mix, reverse-extrude the above solution with a liposome extruder, and then centrifuge and wash with anhydrous ethanol and double distilled water to obtain membrane-coated PROTAC magnetic nanoparticles.
[0017] Preferably, as an improvement, in step one, the alkaline solution is ammonium hydroxide; the coprecipitation is carried out under sonication conditions, the reaction temperature is 37° C., the reaction time is 10 min, and the pH is 10.0; in step two, the surface modification treatment time is 24 h.
[0018] Preferably, as an improvement, in step 4, the freeze-thaw conditions are carried out in liquid nitrogen and a water bath at 37° C., and the number of repeated freeze-thaw cycles is 5 times.
[0019] Preferably, as an improvement, a membrane-coated PROTAC magnetic nanoparticle that can enhance the efficiency of targeted protein degradation chimera technology is used in the preparation of anti-tumor drugs.
[0020] The principle and advantages of this solution are: in actual application, this technical solution optimizes the carrier material, the type of loaded drug and the type of coated cell membrane as a whole, uses magnetic mesoporous silicon nanoparticles as a carrier, selects the PROTAC small molecule drug ARV-825, which has entered the clinical phase 2 experiment, and loads it into the magnetic mesoporous silicon. Then, a layer of MDA-MB-231 breast cancer cell membrane is coated on its surface, so that it can efficiently target breast cancer tumors and enter the tumor to achieve the degradation of bromodomain protein 4 (BRD4) protein, thereby exerting an anti-tumor effect. At the same time, combined with a simulated fasting diet, it regulates the energy metabolism of tumor cells, promotes the increase of ubiquitination levels in tumor cells, further improves the degradation efficiency of the target protein BRD4, and enhances the anti-tumor effect. In addition, under nuclear magnetic resonance MRI imaging, real-time monitoring and visualization of tumors can be achieved. In the technical research and development stage, in the coprecipitation reaction stage, by using ethyl silicate to modify the surface of nanoparticles, a layer of silicon dioxide (SiO2) coating can be formed on the surface of magnetic nanoparticles. This coating can protect the magnetic core and prevent it from agglomerating or oxidizing during subsequent processing and application. In addition, the silicon dioxide (SiO2) coating can increase the hydrophilicity of nanoparticles and improve their dispersibility in aqueous media, which is conducive to subsequent processing and application. Not only that, the silicon dioxide coating can also provide good biocompatibility and reduce the toxicity of nanoparticles to organisms. Moreover, through research, it is found that the silicon dioxide (SiO2) coating has a key influence on the magnetic size of magnetic nanoparticles. The addition amount and mass ratio of ferroferric oxide to ethyl silicate are one of the difficulties of this technical solution. By adjusting the addition amount and reaction conditions of ethyl silicate, the size and morphology of magnetic nanoparticles can be controlled, thereby obtaining a nanoparticle material that meets the use requirements of this technical solution. This technical solution tried different mass ratios of Si:Fe (15:1; 20:1; 10:1; 20:3), and finally found that the best nanoparticle morphology and particle size were the most uniform when the ratio was 20:1; in addition, this solution also studied the different mass ratios of the step three product SiO2-Fe3O4 and the step four product cell membrane ((6:1; 12:1; 18:1; 24:1), and finally found that the tumor cell membrane was coated the most when the ratio was 12:1, thereby achieving better targeting efficiency. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 This is a transmission electron microscopy image of membrane-coated PROTAC magnetic nanoparticles in an embodiment of the present invention.
[0022] Figure 2This is the particle size distribution diagram of membrane-coated PROTAC magnetic nanoparticles in the embodiment of the present invention
[0023] Figure 3 These are confocal images of MDA-MB-231 breast cancer cells after being treated with different groups according to the embodiments of the present invention.
[0024] Figure 4 This is a Western protein immunoblot analysis of total ubiquitinated proteins in 143B and MDA-MB-231 tumor cells after co-culture in FMD medium for different time periods according to an embodiment of the present invention.
[0025] Figure 5 The Western blot analysis of BRD 4, BRD 3, BRD 2 and c-MYC in MDA-MB 231 tumor cells after treatment in the examples of the present invention.
[0026] Figure 6 It is the cell survival rate of MDA-MB-231 cells after being treated with each group of nanoparticles in the embodiment of the present invention.
[0027] Figure 7 The T2-weighted MRI images of tumor-bearing mice at different times after injection of nanoparticles in the present invention. Left: 143B tumor; right: MDA-MB-231 tumor.
[0028] Figure 8 This is the average tumor growth curve of MDA-MB-231 tumors after treatment in different groups in the examples of the present invention. DETAILED DESCRIPTION
[0029] The following is further described in detail through specific implementations, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following implementations are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods; the materials, reagents, etc. used can all be obtained from commercial channels.
[0030] A membrane-coated PROTAC magnetic nanoparticle that can enhance the efficiency of targeted protein degradation chimera technology, using magnetic mesoporous silica nanoparticles as carriers, carrying a BRD4 inhibitor (ARV-825) on the carrier, and the surface of the carrier is coated with a tumor cell membrane (MDA-MB-231 breast cancer cell membrane); the particle size of the membrane-coated PROTAC magnetic nanoparticle is 104±0.2nm.
[0031] A method for preparing membrane-coated PROTAC magnetic nanoparticles that can enhance the efficiency of targeted protein degradation chimera technology is prepared by co-precipitation method and cell membrane coating technology, and specifically comprises the following steps:
[0032] Step 1, coprecipitation: weigh 100 mg of ferroferric oxide, dissolve it in 17.5 mL of anhydrous ethanol, add 5 mL of ammonium hydroxide, then slowly drop 22.5 mL of deionized water, place it in an ultrasonicator for 10 minutes, the reaction temperature is 37 ° C, and the pH is 10.0;
[0033] Step 2: Surface modification: slowly drip ethyl silicate aqueous solution into the solution obtained in step 1, and slowly stir for 24 hours;
[0034] Step 3, separation and washing: the product obtained in step 2 is repeatedly washed with anhydrous ethanol and double distilled water, centrifuged, and dried for later use;
[0035] Step 4: Cell membrane preparation: resuspend the MDA-MB-231 tumor cell membrane in 10% sterile PBS solution, and freeze-thaw repeatedly for 5 times in liquid nitrogen and water bath at 37°C, then centrifuge to remove the supernatant, and then wash 3 times with sterile PBS for later use;
[0036] Step 5, membrane coating: resuspend the product obtained in step 3 and the product obtained in step 4 in deionized water, stir and mix, reverse press the above solution 8 times with a liposome extruder, and then centrifuge and wash with anhydrous ethanol and double distilled water to obtain membrane-coated PROTAC magnetic nanoparticles.
[0037] Experimental Example 1 Transmission Electron Microscope Scanning
[0038] The membrane-coated PROTAC magnetic nanoparticles prepared in this example were subjected to transmission electron microscopy scanning and DLS particle size detection. The results are as follows: Figure 1 , Figure 2 As shown: The particle size of the membrane-coated PROTAC magnetic nanoparticles is 104±0.2nm, and the structure is uniformly spherical ( Figure 1 The scale bar is 100 nm).
[0039] Experimental Example 2
[0040] By setting up control groups (simple culture medium group, Cy5.5-labeled magnetic mesoporous silica), the efficient targeting of membrane-coated PROTAC magnetic nanoparticles combined with simulated fasting diet (FMD) was verified at the cellular level.
[0041] The experimental method is as follows:
[0042] (1) Experimental groups:
[0043] Ctrl control group;
[0044] Si-Fe@BRD4 control group;
[0045] Si-Fe@BRD4+FMD control group;
[0046] NP@M231 (Si-Fe@BRD4@M 231 ) Experimental group;
[0047] NP@M 231 (Si-Fe@BRD4@M 231 )+FMD experimental group;
[0048] (2) MDA-MB-231 breast cancer cells were digested with trypsin to form a single cell suspension, and 1×10 5 Cells were cultured overnight in a 37°C incubator to allow them to fully adhere to the wall.
[0049] (3) Fasting diet (FMD) simulation, that is, cells were cultured in vitro with 1640 medium containing 1% fetal bovine serum, 1% penicillin-streptomycin and 0.5 g / L glucose in an incubator maintained at a constant temperature of 37°C and an atmosphere of 5% CO2.
[0050] For the FMD treatment group, breast cancer MDA-MB-231 cells were treated with FMD medium for 24 h, and then the Cy5.5-fluorescently labeled nanoparticles in the above groups were added and cultured for a further 2 h.
[0051] The experimental results are as follows Figure 3 As shown, the results showed that membrane-coated PROTAC magnetic nanoparticles can effectively target breast cancer MDA-MB-231 cells, and their targeting ability is further enhanced when combined with simulated fasting FMD.
[0052] Experimental Example 3
[0053] Under simulated fasting diet conditions, protein immunoblotting was used to detect the ubiquitination level in MDA-MB-231 breast cancer cells and 143B human osteosarcoma cells. The specific experimental method was as follows: MDA-MB-231 breast cancer cells and 143B human osteosarcoma cells were incubated under FMD conditions for 0, 6, 12, 24, 36, 48, and 60 hours, respectively, and then the cell proteins were extracted. Protein immunoblotting was used to detect the expression of ubiquitinated proteins in MDA-MB-231 breast cancer cells and 143B human osteosarcoma cells after incubation for different time periods under FMD conditions.
[0054] The experimental results are as follows Figure 4 As shown, the results showed that: with the increase of incubation time, the ubiquitination level in MDA-MB-231 breast cancer cells and 143B human osteosarcoma cells increased, reaching a peak at 48 hours.
[0055] Experimental Example 4
[0056] PROTAC magnetic nanoparticles coated with different concentrations were incubated in normal culture medium or simulated fasting FMD culture medium, and the degradation effect on the target protein BRD4 was detected by western blotting.
[0057] Experimental design: Different concentrations (0.1, 0.3, 0.6 μM) of membrane-coated PROTAC magnetic nanoparticles were incubated with breast cancer MDA-MB-231 cells in normal culture medium or simulated fasting FMD culture medium for 24 hours. Then the cell proteins were extracted and the expression levels of BRD4, BRD3, BRD2 and c-MYC proteins in MDA-MB-231 breast cancer cells were detected by western blotting.
[0058] The experimental results are as follows Figure 5 As shown, the results showed that membrane-coated PROTAC magnetic nanoparticles combined with simulated fasting FMD can achieve efficient degradation of the target protein BRD4, and can also reduce the expression levels of its upstream and downstream proteins such as BRD3, BRD2 and c-MYC.
[0059] Experimental Example 5
[0060] Breast cancer MDA-MB-231 cells were incubated with different concentrations (0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10 μM) of membrane-coated PROTAC magnetic nanoparticles in normal culture medium or simulated fasting FMD culture medium for 24 hours, and then the cell proliferation was detected by CCK-8 method.
[0061] Note: The fasting simulated diet (FMD) medium for cell experiments was prepared as follows: 1% FSB, 0.5 g / L glucose and 1% penicillin-streptococcus were added to RPMI 1640 medium to prepare the fasting simulated diet (FMD) medium.
[0062] The experimental results are as follows Figure 6 As shown: It was found that the membrane-coated PROTAC magnetic nanoparticles combined with the simulated fasting diet (FMD) group had very high tumor cell toxicity.
[0063] Experimental Example 6
[0064] Tumor-bearing nude mice were injected with membrane-coated PROTAC magnetic nanoparticles through the tail vein under normal diet and fasting simulated FMD diet conditions, and nuclear magnetic resonance imaging was performed before injection, 2h, 6h, 14h and 26h after injection. Figure 7As shown, the results showed that the membrane-coated PROTAC magnetic nanoparticles coated with the MDA-MB-231 tumor cell membrane can efficiently target the MDA-MB-231 tumor tissue, significantly reduce its magnetic resonance signal, and visualize the distribution of membrane-coated PROTAC magnetic nanoparticles in the tumor tissue in real time.
[0065] Experimental Example 7 In vivo animal experiment
[0066] Experimental design: A subcutaneous transplant tumor model of MDA-MB-231 breast cancer was established, and then the tumor-bearing mice were randomly divided into eight groups, with five mice in each group. The experimental grouping of the eight groups is as follows:
[0067] Normal saline group, normal saline + FMD diet group, BRD4-P simple inhibitor group, BRD4-P simple inhibitor + FMD diet group, nBRD@M 143 Membrane-coated PROTAC magnetic nanoparticles, nBRD@M 143 Membrane-coated PROTAC magnetic nanoparticles + FMD diet group, nBRD@M 231 Membrane-coated PROTAC magnetic nanoparticles and nBRD@M 143 Membrane-coated PROTAC magnetic nanoparticles + FMD diet group.
[0068] The FMD setting scheme for in vivo animal experiments is as follows: mice are housed in clean cages to avoid eating residual food. Then they are fed 50% low-calorie feed on the first day, followed by 90% low-calorie feed on the second and third days, and 100% calorie feed on the fourth day. The above 4 days are one FMD cycle. During the FMD treatment, mice consumed all the caloric diet provided and showed no signs of anorexia. The tumor volume method is long diameter × short diameter × short diameter / 2. The tumor size of each group was measured every two days.
[0069] The experimental results are as follows Figure 8 As shown, the results showed that compared with other control groups, the tumor volume of the membrane-coated PROTAC magnetic nanoparticles combined with the simulated fasting diet group was the smallest and could effectively inhibit tumor growth.
[0070] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.
Claims
1. A membrane-coated PROTAC magnetic nanoparticle that can enhance the efficiency of targeted protein degradation chimera technology, characterized in that: A magnetic mesoporous material is used as a carrier, a BRD4 protein degradation inhibitor is loaded on the carrier, and a tumor cell membrane is coated on the surface of the carrier.
2. A membrane-coated PROTAC magnetic nanoparticle that can enhance the efficiency of targeted protein degradation chimera technology according to claim 1, characterized in that: The BRD4 protein degradation inhibitor is ARV-825.
3. A membrane-coated PROTAC magnetic nanoparticle that can enhance the efficiency of targeted protein degradation chimera technology according to claim 2, characterized in that: The magnetic mesoporous material is magnetic mesoporous silicon.
4. A membrane-coated PROTAC magnetic nanoparticle capable of enhancing the efficiency of targeted protein degradation chimera technology according to claim 3, characterized in that: The tumor cell membrane is an MDA-MB-231 breast cancer cell membrane.
5. A membrane-coated PROTAC magnetic nanoparticle capable of enhancing the efficiency of targeted protein degradation chimera technology according to claim 4, characterized in that: The membrane-coated PROTAC magnetic nanoparticles are spherical structures with a particle size of 104±0.2nm.
6. A method for preparing a membrane-coated PROTAC magnetic nanoparticle capable of enhancing the efficiency of targeted protein degradation chimera technology according to any one of claims 1 to 5, characterized in that: It was prepared by co-precipitation method combined with cell membrane coating technology.
7. A method for preparing a membrane-coated PROTAC magnetic nanoparticle capable of enhancing the efficiency of targeted protein degradation chimera technology according to claim 6, characterized in that: The steps include: Step 1, coprecipitation: dissolving ferroferric oxide in anhydrous ethanol, adding alkaline solution thereto, and then dropping deionized water to carry out coprecipitation reaction to obtain system I; Step 2: Surface modification: adding ethyl silicate aqueous solution to system I for surface modification; Step 3, separation and washing: the product obtained in step 2 is repeatedly washed with anhydrous ethanol and double distilled water, centrifuged, and dried for later use; Step 4: Cell membrane preparation: resuspend the tumor cell membrane, freeze and thaw repeatedly, remove the supernatant by centrifugation, extract the tumor cell membrane, wash it with sterile PBS and set aside; Step 5, membrane coating: resuspend the product obtained in step 3 and the product obtained in step 4 in deionized water, stir and mix, reverse-extrude the above solution with a liposome extruder, and then centrifuge and wash with anhydrous ethanol and double distilled water to obtain membrane-coated PROTAC magnetic nanoparticles.
8. A method for preparing a membrane-coated PROTAC magnetic nanoparticle that can enhance the efficiency of targeted protein degradation chimera technology according to claim 7, characterized in that: In step 1, the alkaline solution is ammonium hydroxide; the coprecipitation is carried out under sonication conditions, the reaction temperature is 37° C., the reaction time is 10 min, and the pH is 10.0; in step 2, the surface modification treatment time is 24 h.
9. A method for preparing a membrane-coated PROTAC magnetic nanoparticle capable of enhancing the efficiency of targeted protein degradation chimera technology according to claim 8, characterized in that: In step 4, the freeze-thaw conditions are liquid nitrogen and a water bath at 37°C, and the number of repeated freeze-thaw cycles is 5 times.
10. Use of a membrane-coated PROTAC magnetic nanoparticle capable of enhancing the efficiency of targeted protein degradation chimera technology according to any one of claims 1 to 5 in the preparation of anti-tumor drugs.