A highly targeted composite nanoparticle preparation for treating tumors and a preparation method thereof

Abstract

The present invention belongs to the technical field of anti-tumor nano-preparations. The present invention provides a method for preparing a highly targeted composite nano-preparation for treating tumors, the preparation method comprising the following steps: (1) fusing thylakoid membranes and cancer cell membranes into a hybrid membrane at a weight ratio of 1:3 to 5, and then incubating the hybrid membrane in a monomethoxy polyethylene glycol solution to obtain a hybrid membrane complex; (2) coating nanoparticles carrying doxorubicin with the hybrid membrane complex. The nano-preparation obtained by the present invention has excellent targeting for tumors, and can achieve excellent anti-tumor effects while carrying a relatively small amount of doxorubicin; at the same time, the preparation method of the nano-preparation of the present invention is simple and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-tumor nano-preparations, and in particular relates to a highly targeted composite nano-preparation for treating tumors and a preparation method thereof. Background Art

[0002] In the field of tumor treatment, the use of membrane materials to prepare anti-tumor preparations has attracted increasing attention from researchers. Some members of the inventor team have published a targeted drug delivery bionic nanomotor and its preparation method and application in the Chinese patent CN114366811B. The nanomotor in this patent is obtained by coating drug-loaded nanoparticles with a hybrid cell membrane composed of cancer cell membranes, white blood cell membranes and red blood cell membranes, and then fixing them with urease. The inventor found that after Erastin is loaded with a nanomotor, the anti-cancer effect can be greatly improved under the action of ultrasound. Although this patent has good effects, it still needs to be fixed with urease, and the operation is relatively cumbersome. In addition, the active pharmaceutical ingredients used are not widely used in clinical practice, and there are certain deficiencies in the industrial application value.

[0003] After that, based on the research of this patent, the inventor team further studied the effects of different membrane materials in preparing anti-tumor preparations, and focused on developing preparations that can carry commonly used anti-tumor drugs and have excellent anti-tumor effects.

[0004] At present, when developing pharmaceutical preparations based on membrane materials, the field mainly focuses on animal-derived membrane materials, and there are not many studies on plant-derived membrane materials. In December 2022, Dr. Lin Xianfeng and Professor Fan Shunwu’s team from the Department of Orthopedics of the Run Run Shaw Hospital Affiliated to Zhejiang University School of Medicine, and Professor Tang Ruikang’s team from the Department of Chemistry of Zhejiang University, for the first time, achieved cross-species delivery of plant thylakoids into aging and diseased cells in animals, providing a corresponding research basis for the application of plant-derived membrane materials in the field of biomedicine. To this end, the inventors considered hybridizing and fusing thylakoid membranes and animal-derived membrane materials, and developing corresponding biopharmaceutical preparation materials. As far as the inventors know, research in this area is still in its infancy.

[0005] Doxorubicin is an anti-tumor antibiotic that inhibits the synthesis of nucleic acid, the genetic material of cancer cells, and has a wide anti-tumor spectrum, killing a variety of tumor cells. However, the current preparations carrying doxorubicin often require a higher dose when treating tumors, making the side effects that may occur during the use of the drug a problem that people have to face. Although people have made a lot of achievements in developing pharmaceutical preparations that reduce the amount of doxorubicin used, such as Chinese patent CN108853056B, it is still unknown whether a pharmaceutical preparation with such an effect can be prepared by using plant-derived membrane materials. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention aims to provide an anti-tumor nanoformulation and preparation method which has a simple preparation scheme, good tumor targeting, uses plant-derived membrane materials as raw materials, and can reduce the dosage of doxorubicin. To this end, the present invention adopts the following technical solutions:

[0007] A method for preparing a highly targeted composite nanoformulation for treating tumors, the method comprising the following steps:

[0008] (1) fusing the thylakoid membrane and the cancer cell membrane into a hybrid membrane at a weight ratio of 1:3 to 5, and then incubating the hybrid membrane in a monomethoxy polyethylene glycol solution to obtain a hybrid membrane complex;

[0009] (2) Nanoparticles carrying doxorubicin are coated with hybrid membrane complexes.

[0010] Furthermore, in step (1), the thylakoid membrane is the thylakoid membrane of spinach.

[0011] Furthermore, the preparation method of the thylakoid membrane is: wash the spinach, then homogenize it, filter it and take the filtrate, and then centrifuge to separate the chloroplasts; resuspend the obtained chloroplasts in a hypotonic lysis buffer and then centrifuge, remove the matrix extract in the supernatant, ultrasonically treat it in water and then centrifuge to take the precipitate to obtain the thylakoid membrane.

[0012] Furthermore, the weight ratio of thylakoid membrane to cancer cell membrane was 1:4.

[0013] Furthermore, when preparing the hybrid membrane, the thylakoid membrane and the cancer cell membrane are extruded and fused through a polycarbonate porous membrane.

[0014] Furthermore, in preparing the hybrid membrane complex, the hybrid membrane was placed in a monomethoxy polyethylene glycol solution at a solid-liquid weight ratio of 1:20 and incubated at 37° C. and 200 rpm for 60 minutes.

[0015] Furthermore, the nanoparticles carrying doxorubicin are liposomes carrying doxorubicin.

[0016] Furthermore, the preparation method of the liposome carrying doxorubicin is as follows: soybean lecithin and cholesterol are dissolved in dichloromethane at a weight ratio of 5:1, and then the dichloromethane is removed by reduced pressure rotary evaporation at 37°C to form an oil film; then 300mmol / L ammonium sulfate solution is added, and then ultrasonic treatment is performed at a power of 300W for 30 minutes to obtain an emulsion, and after impurities are removed by 0.45μm and 0.22μm filter membranes in turn, blank liposomes are obtained and dialyzed; doxorubicin solution is added to the dialyzed liposomes, and incubated at 60°C for 30 minutes.

[0017] Furthermore, when the nanoparticles carrying doxorubicin are coated with the hybrid membrane complex, the nanoparticles carrying doxorubicin are mixed with an excess of the hybrid membrane complex, and then ultrasonically treated at 300 W for 3 minutes under ice bath conditions, and then passed through a polycarbonate porous membrane with a pore size of 400 nm and 200 nm, and then centrifuged to obtain the nanoparticles.

[0018] The present invention also provides a highly targeted composite nano preparation for treating tumors prepared by the above preparation method.

[0019] Beneficial effects of the present invention:

[0020] The nano preparation obtained by the present invention has excellent targeting to tumors and can achieve excellent anti-tumor effects while carrying a relatively small amount of doxorubicin. Meanwhile, the preparation method of the nano preparation of the present invention is simple and has good application prospects. DETAILED DESCRIPTION

[0021] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by technical personnel in this field based on the above invention content still fall within the scope of protection of the present invention.

[0022] Example 1

[0023] 1.1 Raw materials:

[0024] Spinach: commercially available; monomethoxypolyethylene glycol: purchased from Xi'an Qiyue Biotechnology Co., Ltd.; doxorubicin: doxorubicin hydrochloride, purchased from Sigma-Aldrich; dichloromethane and ammonium sulfate: laboratory-owned.

[0025] Preparation method of cancer cell membrane: prepare Hepes B (2.38g / L Hepes, 0.476g / LMgCl2, 0.292g / LEDTA, 0.154g / LDTT, 0.746g / L KCl, pH 7.6) and Hepes C (11.914g / L Hepes, 5.844g / L NaCl, 13.492g / L KCl, pH 7.6) buffer; culture HepG2 cells in a culture dish, collect a certain number of cancer cells by centrifugation after digestion, suspend them in Hepes B buffer and 1% protease inhibitor mixture, cool them in an ice bath for 5 minutes, homogenize the cells several times, and remove the cell nucleus. Repeat the above steps and finally collect 8-10mL of supernatant; prepare sucrose solutions with concentrations of 30%, 40%, and 55% (w / v) with Hepes B buffer, and add them to centrifuge tubes in descending order of concentration, and slowly add the collected supernatant to the sucrose density gradient column; centrifuge at high speed at 4°C, collect the required sample band, blow it repeatedly with Hepes C buffer, and centrifuge; finally, resuspend the obtained cancer cell membrane in Hepes C buffer and store it for later use.

[0026] The preparation method of red blood cell membrane refers to the preparation method of cancer cell membrane, the difference lies in the different cell sources.

[0027] Preparation of thylakoid membrane: Wash the spinach, then place it in a juicer, add homogenization buffer (20mM Tricine, 0.4M sucrose, 2mM ascorbic acid, 2mM MgCl2, 40mM NaCl, 0.2% BSA, pH 7.8) for homogenization, then filter and take the filtrate, and then centrifuge to separate the chloroplasts; the obtained chloroplasts are resuspended in hypotonic lysis buffer (10mM HEPES, pH 8.0) and then centrifuged. After removing the matrix extract in the supernatant, ultrasonically treat it in water and centrifuge to take the precipitate to obtain the thylakoid membrane.

[0028] 2. Preparation of highly targeted composite nanoparticle preparations for treating tumors:

[0029] (1) The thylakoid membrane and cancer cell membrane were ultrasonically treated at a weight ratio of 1:4 at 300 W for 3 minutes; then, they were extruded and fused through 400 nm and 200 nm polycarbonate porous membranes, and the hybrid membranes were collected by centrifugation;

[0030] (2) preparing a 50 mmol / L monomethoxy polyethylene glycol aqueous solution, placing the obtained hybrid membrane in an excess of monomethoxy polyethylene glycol aqueous solution at a solid-liquid weight ratio of 1:20, incubating at 37° C. and 200 rpm for 60 minutes, and then collecting the hybrid membrane complex by centrifugation;

[0031] (3) Dissolve soybean lecithin and cholesterol in dichloromethane at a weight ratio of 5:1, and then remove dichloromethane by vacuum rotary evaporation at 37°C to form an oil film; then add 300mmol / L ammonium sulfate solution, and then perform ultrasonic treatment at 300W power for 30 minutes to obtain an emulsion, which is filtered through 0.45μm and 0.22μm filter membranes to remove impurities, obtain blank liposomes, and perform dialysis treatment; add doxorubicin solution to the dialyzed liposomes, and incubate at 60°C for 30 minutes to obtain liposomes carrying doxorubicin, and control the drug loading of doxorubicin to be 0.5% w / w;

[0032] (4) The nanoparticles carrying doxorubicin were mixed with an excess of the hybrid membrane complex, and then subjected to ultrasonic treatment at 300 W for 3 minutes in an ice bath, and then passed through a polycarbonate porous membrane with a pore size of 400 nm and 200 nm, and then centrifuged to obtain the nanoparticles. When used, the nanoparticles were resuspended in PBS at a weight fraction of 10%.

[0033] Example 2

[0034] The raw materials used are consistent with those in Example 1.

[0035] Preparation of highly targeted composite nanoformulations for tumor treatment:

[0036] (1) The thylakoid membrane and cancer cell membrane were ultrasonically treated at a weight ratio of 1:3 at 300 W for 3 minutes; then, they were extruded and fused through 400 nm and 200 nm polycarbonate porous membranes, and the hybrid membranes were collected by centrifugation;

[0037] (2) preparing a 50 mmol / L monomethoxy polyethylene glycol aqueous solution, placing the obtained hybrid membrane in an excess of monomethoxy polyethylene glycol aqueous solution at a solid-liquid weight ratio of 1:20, incubating at 37° C. and 200 rpm for 60 minutes, and then collecting the hybrid membrane complex by centrifugation;

[0038] (3) Dissolve soybean lecithin and cholesterol in dichloromethane at a weight ratio of 5:1, and then remove dichloromethane by vacuum rotary evaporation at 37°C to form an oil film; then add 300mmol / L ammonium sulfate solution, and then perform ultrasonic treatment at 300W power for 30 minutes to obtain an emulsion, which is filtered through 0.45μm and 0.22μm filter membranes to remove impurities, obtain blank liposomes, and perform dialysis treatment; add doxorubicin solution to the dialyzed liposomes, and incubate at 60°C for 30 minutes to obtain liposomes carrying doxorubicin, and control the drug loading of doxorubicin to be 0.5% w / w;

[0039] (4) The nanoparticles carrying doxorubicin were mixed with an excess of the hybrid membrane complex, and then subjected to ultrasonic treatment at 300 W power for 3 minutes in an ice bath, and then passed through a polycarbonate porous membrane with a pore size of 400 nm and 200 nm, and then centrifuged to obtain the nanoparticles. When used, the nanoparticles were resuspended in PBS at a weight fraction of 10%.

[0040] Example 3

[0041] The raw materials used are consistent with those in Example 1.

[0042] Preparation of highly targeted composite nanoformulations for tumor treatment:

[0043] (1) The thylakoid membrane and cancer cell membrane were ultrasonically treated at a weight ratio of 1:5 for 3 minutes at 300 W power; then, they were extruded and fused through 400 nm and 200 nm polycarbonate porous membranes, and the hybrid membranes were collected by centrifugation;

[0044] (2) preparing a 50 mmol / L monomethoxy polyethylene glycol aqueous solution, placing the obtained hybrid membrane in an excess of monomethoxy polyethylene glycol aqueous solution at a solid-liquid weight ratio of 1:20, incubating at 37° C. and 200 rpm for 60 minutes, and then collecting the hybrid membrane complex by centrifugation;

[0045] (3) Dissolve soybean lecithin and cholesterol in dichloromethane at a weight ratio of 5:1, and then remove dichloromethane by vacuum rotary evaporation at 37°C to form an oil film; then add 300mmol / L ammonium sulfate solution, and then perform ultrasonic treatment at 300W power for 30 minutes to obtain an emulsion, which is filtered through 0.45μm and 0.22μm filter membranes to remove impurities, obtain blank liposomes, and perform dialysis treatment; add doxorubicin solution to the dialyzed liposomes, and incubate at 60°C for 30 minutes to obtain liposomes carrying doxorubicin, and control the drug loading of doxorubicin to be 0.5% w / w;

[0046] (4) The nanoparticles carrying doxorubicin were mixed with an excess of the hybrid membrane complex, and then subjected to ultrasonic treatment at 300 W power for 3 minutes in an ice bath, and then passed through a polycarbonate porous membrane with a pore size of 400 nm and 200 nm, and then centrifuged to obtain the nanoparticles. When used, the nanoparticles were resuspended in PBS at a weight fraction of 10%.

[0047] Comparative Example 1

[0048] Compared with Example 1, the hybrid membrane was not incubated with the monomethoxy polyethylene glycol solution in this comparative example, and the other raw materials used were the same as those in Example 1.

[0049] When preparing the composite nanoformulation, the scheme adopted is as follows:

[0050] (1) The thylakoid membrane and cancer cell membrane were ultrasonically treated at a weight ratio of 1:4 for 3 minutes at 300 W power; then, they were extruded and fused through 400 nm and 200 nm polycarbonate porous membranes, and the hybrid membranes were collected by centrifugation;

[0051] (2) Dissolve soybean lecithin and cholesterol in dichloromethane at a weight ratio of 5:1, and then remove dichloromethane by vacuum rotary evaporation at 37°C to form an oil film; then add 300 mmol / L ammonium sulfate solution, and then perform ultrasonic treatment at 300 W power for 30 minutes to obtain an emulsion, which is filtered through 0.45 μm and 0.22 μm filter membranes to remove impurities, obtain blank liposomes, and perform dialysis treatment; add doxorubicin solution to the dialyzed liposomes, and incubate at 60°C for 30 minutes to obtain liposomes carrying doxorubicin, and control the drug loading of doxorubicin to be 0.5% w / w;

[0052] (3) The nanoparticles carrying doxorubicin were mixed with an excess of the hybrid membrane, and then subjected to ultrasonic treatment at 300 W for 3 minutes in an ice bath, and then passed through a polycarbonate porous membrane with a pore size of 400 nm and 200 nm, and then centrifuged to obtain the nanoparticles. When used, the nanoparticles were resuspended in PBS at a weight fraction of 10%.

[0053] Comparative Example 2

[0054] Compared with Example 1, in this comparative example, leukocyte membrane is further added when preparing the hybrid membrane. The method of obtaining the leukocyte membrane refers to the preparation method of the cancer cell membrane in Example 1. The raw materials used in this comparative example are consistent with those in Example 1.

[0055] When preparing the composite nanoformulation, the scheme adopted is as follows:

[0056] (1) Thylakoid membranes, cancer cell membranes and leukocyte membranes were ultrasonically treated at a weight ratio of 1:4:2 at 300 W for 3 minutes; then, they were extruded and fused through 400 nm and 200 nm polycarbonate porous membranes, and the hybrid membranes were collected by centrifugation;

[0057] (2) preparing a 50 mmol / L monomethoxy polyethylene glycol aqueous solution, placing the obtained hybrid membrane in an excess of monomethoxy polyethylene glycol aqueous solution at a solid-liquid weight ratio of 1:20, incubating at 37° C. and 200 rpm for 60 minutes, and then collecting the hybrid membrane complex by centrifugation;

[0058] (3) Dissolve soybean lecithin and cholesterol in dichloromethane at a weight ratio of 5:1, and then remove dichloromethane by vacuum rotary evaporation at 37°C to form an oil film; then add 300mmol / L ammonium sulfate solution, and then perform ultrasonic treatment at 300W power for 30 minutes to obtain an emulsion, which is filtered through 0.45μm and 0.22μm filter membranes to remove impurities, obtain blank liposomes, and perform dialysis treatment; add doxorubicin solution to the dialyzed liposomes, and incubate at 60°C for 30 minutes to obtain liposomes carrying doxorubicin, and control the drug loading of doxorubicin to be 0.5% w / w;

[0059] (4) The nanoparticles carrying doxorubicin were mixed with an excess of the hybrid membrane complex, and then subjected to ultrasonic treatment at 300 W for 3 minutes in an ice bath, and then passed through a polycarbonate porous membrane with a pore size of 400 nm and 200 nm, and then centrifuged to obtain the nanoparticles. When used, the nanoparticles were resuspended in PBS at a weight fraction of 10%.

[0060] Comparative Example 3

[0061] Compared with Example 1, the monomethoxy polyethylene glycol aqueous solution was replaced by polyethylene glycol aqueous solution in this comparative example, and the other raw materials were the same as those in Example 1. The polyethylene glycol used in this comparative example was purchased from Xi'an Jinxiang Pharmaceutical Excipients Co., Ltd., model 1500.

[0062] When preparing the composite nanoformulation, the scheme adopted is as follows:

[0063] (1) The thylakoid membrane and cancer cell membrane were ultrasonically treated at a weight ratio of 1:4 for 3 minutes at 300 W power; then, they were extruded and fused through 400 nm and 200 nm polycarbonate porous membranes, and the hybrid membranes were collected by centrifugation;

[0064] (2) preparing a 50 mmol / L polyethylene glycol aqueous solution, placing the obtained hybrid membrane in an excess of polyethylene glycol aqueous solution at a solid-liquid weight ratio of 1:20, incubating at 37° C. and 200 rpm for 60 minutes, and then collecting the hybrid membrane complex by centrifugation;

[0065] (3) Dissolve soybean lecithin and cholesterol in dichloromethane at a weight ratio of 5:1, and then remove dichloromethane by vacuum rotary evaporation at 37°C to form an oil film; then add 300mmol / L ammonium sulfate solution, and then perform ultrasonic treatment at 300W power for 30 minutes to obtain an emulsion, which is filtered through 0.45μm and 0.22μm filter membranes to remove impurities, obtain blank liposomes, and perform dialysis treatment; add doxorubicin solution to the dialyzed liposomes, and incubate at 60°C for 30 minutes to obtain liposomes carrying doxorubicin, and control the drug loading of doxorubicin to be 0.5% w / w;

[0066] (4) The nanoparticles carrying doxorubicin were mixed with an excess of the hybrid membrane complex, and then subjected to ultrasonic treatment at 300 W for 3 minutes in an ice bath, and then passed through a polycarbonate porous membrane with a pore size of 400 nm and 200 nm, and then centrifuged to obtain the nanoparticles. When used, the nanoparticles were resuspended in PBS at a weight fraction of 10%.

[0067] Experimental example

[0068] After HepG2 cells (human liver cancer cells) were revived and digested, 100 μL containing 5×10 6 The suspension of 10 cells was subcutaneously injected into the right groin of nude mice (4 weeks old, 15 ± 2 g) to establish a transplanted tumor model. 3 When the mice were about 10 minutes old, they were randomly divided into 8 groups, namely, saline group, doxorubicin group, Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2 and Comparative Example 3, and each group was injected with saline, doxorubicin or composite nano-preparation, respectively. 100 μL of each group of preparations, saline or doxorubicin (4 mg / kg) was injected intravenously into nude mice. The time of the first injection was set as day 0, and the size of the nude mouse tumor was measured every 3 days. The calculation formula of tumor volume is: V = (L × W 2 ) / 2, where L and W represent the longest diameter and shortest diameter of the tumor measured by vernier calipers, respectively. The experimental results are shown in Table 1.

[0069] Table 1

[0070]

[0071] As shown in Comparative Example 2 in Table 1, unlike the patent CN114366811B previously applied by the inventor, when the present invention uses leukocyte membrane and cancer cell membrane as the raw materials of the hybrid membrane, the anti-tumor effect of the obtained preparation is greatly reduced. The reason for this needs further study, and the reason may be that the related proteins of the leukocyte membrane and one or some substances of the thylakoid membrane have antagonistic effects in providing tumor targeting receptors or signals.

[0072] It is also worth noting that whether the hybrid membrane is incubated and modified with monomethoxy polyethylene glycol is also critical, which can be seen from the results of Comparative Examples 1 and 3.

Claims

1. A method for preparing a highly targeted composite nanoformulation for treating tumors, characterized in that: The preparation method comprises the following steps: (1) fusing the thylakoid membrane and the cancer cell membrane into a hybrid membrane at a weight ratio of 1:3 to 5, and then incubating the hybrid membrane in a monomethoxy polyethylene glycol solution to obtain a hybrid membrane complex; (2) Nanoparticles carrying doxorubicin are coated with hybrid membrane complexes.

2. The method for preparing a highly targeted composite nanoformulation for treating tumors according to claim 1, characterized in that: In step (1), the thylakoid membrane is the thylakoid membrane of spinach.

3. The method for preparing a highly targeted composite nano-preparation for treating tumors according to claim 1 or 2, characterized in that: The preparation method of the thylakoid membrane is as follows: the spinach is washed, then homogenized, filtered, and the filtrate is collected, and then centrifuged to separate the chloroplasts; the obtained chloroplasts are resuspended in a hypotonic lysis buffer and then centrifuged, the matrix extract in the supernatant is removed, and then the precipitate is collected after ultrasonic treatment in water, thereby obtaining the thylakoid membrane.

4. The method for preparing a highly targeted composite nanoformulation for treating tumors according to claim 3, characterized in that: The weight ratio of thylakoid membrane to cancer cell membrane is 1:

4.

5. The method for preparing a highly targeted composite nano-preparation for treating tumors according to claim 4, characterized in that: When preparing the hybrid membrane, the thylakoid membrane and the cancer cell membrane are extruded and fused through a polycarbonate porous membrane.

6. The method for preparing a highly targeted composite nano-preparation for treating tumors according to claim 1 or 5, characterized in that: In preparing the hybrid membrane complex, the hybrid membrane was placed in a monomethoxy polyethylene glycol solution at a solid-liquid weight ratio of 1:20 and incubated at 37° C. and 200 rpm for 60 minutes.

7. The method for preparing a highly targeted composite nano-preparation for treating tumors according to claim 1 or 5, characterized in that: The nanoparticles carrying doxorubicin are liposomes carrying doxorubicin.

8. The method for preparing a highly targeted composite nano-preparation for treating tumors according to claim 7, characterized in that: The preparation method of the liposome carrying doxorubicin is as follows: soybean lecithin and cholesterol are dissolved in dichloromethane at a weight ratio of 5:1, and then the dichloromethane is removed by reduced pressure rotary evaporation at 37°C to form an oil film; then 300mmol / L ammonium sulfate solution is added, and then ultrasonic treatment is performed at a power of 300W for 30 minutes to obtain an emulsion, and after impurities are removed by 0.45μm and 0.22μm filter membranes in turn, blank liposomes are obtained and dialyzed; doxorubicin solution is added to the dialyzed liposomes, and the liposomes are incubated at 60°C for 30 minutes to obtain the emulsion.

9. The method for preparing a highly targeted composite nano-preparation for treating tumors according to claim 8, characterized in that: When the nanoparticles carrying doxorubicin are coated with the hybrid membrane complex, the nanoparticles carrying doxorubicin are mixed with an excess of the hybrid membrane complex, and then ultrasonically treated at 300 W for 3 minutes in an ice bath, and then passed through a polycarbonate porous membrane with a pore size of 400 nm and 200 nm, and then centrifuged to obtain the nanoparticles.

10. A highly targeted composite nanoformulation for treating tumors, characterized in that: The composite nano preparation is prepared by the preparation method according to any one of claims 1 to 9.

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