Stem cell membrane nanocarrier for tumor-targeted magnetic diagnosis and chemotherapy, and its preparation method and application

Abstract

The present invention discloses a stem cell membrane nanocarrier for tumor-targeted magnetic diagnosis and chemotherapy and a preparation method thereof. The stem cell membrane nanocarrier is a nanocarrier formed by stem cell membrane-coated magnetic nanoparticle clusters and chemotherapy drugs. The magnetic nanoparticle clusters are obtained using an alternating magnetic field and temperature control. The magnetic nanoparticle clusters and chemotherapy drugs are jointly loaded into the stem cell membrane with the help of a static magnetic field or an alternating magnetic field. The prepared stem cell membrane nanocarrier has outstanding advantages such as large loading capacity, synergy of magnetic therapy and chemotherapy, and integrated diagnosis and treatment, and has great clinical potential for targeted magnetic diagnosis and chemotherapy.

Description

Technical Field

[0001] The present invention relates to the field of nanocarriers for tumor diagnosis and treatment, and in particular to stem cell membrane nanocarriers for tumor-targeted magnetic diagnosis and treatment and chemotherapy, as well as to a preparation method and application of the carriers. Background Art

[0002] In the field of targeted tumor therapy, whether it's passive targeting through osmotic retention or active targeting through designing specific ligands for receptors overexpressed on the tumor surface, the true targeting rate of nanodrugs after entry into the body is less than 0.5%. Compared to other drug delivery systems that directly aggregate to the tumor site through the bloodstream, cell-loaded nanodrug systems can achieve precise delivery to the tumor site by stimulating it with high concentrations of chemical factors and other components in the tumor area. It has been confirmed that various stem cells are highly susceptible to induced migration to the tumor site by chemical factors such as CXC motif-containing chemical factors and soluble tumor-derived factors. Immunohistochemical studies have shown that MSCs can uniformly infiltrate tumor tissue and secrete vesicles to deliver nanodrugs, ensuring that the loaded nanodrugs are fully and evenly dispersed throughout the tumor cell population. However, due to the uncertainty of the final differentiation and metabolic fate of transplanted stem cells in vivo, stem cell-targeted tumor therapy has several significant drawbacks. For example, studies have shown that stem cells can promote antigen presentation, inducing T cell activation and immune rejection, and there is also a risk of malignant tumor formation. These safety concerns have severely hampered the clinical application of stem cell therapy.

[0003] As the mechanisms of stem cell therapy deepen, stem cell membranes and extracellular vesicles secreted by stem cells are increasingly being recognized as promising alternatives to stem cells themselves as drug delivery vehicles for clinical translation. Because these membrane structures lack a nucleus and do not transcribe reactive proteins, they prevent excessive immune responses and the development of malignancies. However, the vesicle membranes still contain functional molecules such as proteins and polysaccharides, preserving the ability of stem cells to home to tumors. Therefore, stem cell-derived membranes have the potential to be safer and more effective for pharmaceutical applications. Fabricating these membrane structures at the nanoscale can impart unique nanoscale effects, further expanding the applications of stem cell membranes as novel targeted anti-tumor nanocarriers. However, current understanding of stem cell membrane nanocarriers, including loading methods, cargo types, and loading capacity, remains limited, resulting in a lack of a truly effective nanoplatform for integrated cancer diagnosis and treatment.

[0004] Iron-based nanomedicines are among the few inorganic metal nanomaterials currently in clinical use. They have been successfully used in medical practices such as magnetic resonance imaging enhancement and magnetothermal therapy for tumors. However, the imaging resolution and magnetothermal properties of currently marketed iron-based nanomedicines are still far from being suitable for integrated clinical anti-tumor diagnosis and treatment. This is primarily due to the limited crystal and magnetic properties of the materials, as well as the challenges of low targeted delivery efficiency. Summary of the Invention

[0005] In view of this, one of the objects of the present invention is to provide a stem cell membrane nanocarrier for tumor-targeted magnetic diagnosis and chemotherapy. The carrier is based on stem cell membranes and high-performance magnetic nanoparticle clusters, and is combined with magnetic-mediated membrane labeling technology to prepare a stem cell membrane nanocarrier that can be used for tumor-targeted magnetic therapy. Compared with ordinary stem cell membrane carriers, it has outstanding advantages such as large load capacity, synergy of magnetic therapy and chemotherapy, and integrated diagnosis and treatment. The second object of the present invention is to provide a preparation method of the stem cell membrane nanocarrier for tumor-targeted magnetic diagnosis and chemotherapy. The third object of the present invention is to provide the application of the stem cell membrane nanocarrier in the preparation of tumor-targeted magnetic therapy.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] 1. Stem cell membrane nanocarriers for tumor-targeted magnetic diagnosis and chemotherapy, wherein the stem cell membrane nanocarriers are nanocarriers formed by stem cell membrane-coated magnetic nanoparticle clusters and chemotherapy drugs.

[0008] Preferably, the stem cell membrane is made from any one or more of bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, and neural stem cells.

[0009] Preferably, the magnetic nanoparticle clusters are formed by inducing the binding of magnetic nanoparticles under an alternating magnetic field and regulating the temperature of the reaction solution.

[0010] Preferably, the magnetic nanoparticles are selected from one or more of ferroferric oxide, gamma ferric oxide, manganese ferrite, zinc ferrite, cobalt ferrite and cobalt tetroxide and are prepared by high-temperature pyrolysis.

[0011] Preferably, the chemotherapy drug is selected from any one or more of doxorubicin, hydroxycamptothecin, paclitaxel, cisplatin and 5-fluorouracil.

[0012] Preferably, the method of coating the stem cell membrane with magnetic nanoparticle clusters and chemotherapy drugs is to load the magnetic nanoparticle clusters and chemotherapy drugs into the stem cell membrane by means of a static magnetic field or an alternating magnetic field, and obtain a stem cell membrane nanocarrier after purification.

[0013] 2. The method for preparing the stem cell membrane nanocarrier for tumor-targeted magnetic diagnosis and chemotherapy comprises the following steps:

[0014] 1) Preparation of stem cell membrane: The stem cells are subjected to hypotonic treatment, homogenization, sonication and filtration to obtain stem cell membrane;

[0015] 2) Preparation of magnetic nanoparticle clusters: preparing magnetic nanoparticles by high-temperature pyrolysis of a magnetic compound, and then using an alternating magnetic field to induce and control the temperature of the reaction solution to form magnetic nanoparticle clusters;

[0016] 3) Preparation of stem cell membrane nanocarriers: Mixing magnetic nanoparticle clusters and chemotherapy drugs with stem cell membranes, loading the magnetic nanoparticle clusters and chemotherapy drugs into the stem cell membranes under the induction of a static magnetic field or an alternating magnetic field, and obtaining stem cell membrane nanocarriers after purification.

[0017] Preferably, in step 1) of the present invention, the size of the stem cell membrane is 100-300 nm.

[0018] Preferably, in step 2) of the present invention, the central intensity of the alternating magnetic field is 10-90 mT.

[0019] Preferably, in step 2) of the present invention, the temperature of the reaction solution is 10-50°C.

[0020] Preferably, in step 3) of the present invention, the magnetic field strength is 100-300 mT.

[0021] 3. Application of the stem cell membrane nanocarrier in the preparation of tumor-targeted magnetic therapy.

[0022] The beneficial effects of the present invention are as follows: the present invention discloses a stem cell membrane nanocarrier for tumor-targeted magnetic diagnosis and chemotherapy, the magnetic nanoparticle clusters obtained by the carrier using an alternating magnetic field and temperature control have good magnetic response performance (saturation magnetization intensity reaches 170.6emu / g), which is significantly higher than ordinary magnetic nanoparticles (saturation magnetization intensity reaches 51.7emu / g). A stem cell membrane nanocarrier with a high iron content (iron concentration of 15mg / mL) was prepared using a magnetic field induced labeling method, while the iron content of the stem cell membrane nanocarrier prepared without the aid of a magnetic field was only 6.8mg / mL. Compared with the stem cell membrane carrier loaded with ordinary magnetic nanoparticles, the stem cell membrane carrier loaded with magnetic nanoparticle clusters has significantly enhanced T2 magnetic resonance imaging performance (38mM -1 s -1 with 74mM -1 s -1 ) and magnetocaloric properties (specific absorption coefficients of 86 W / g and 151 W / g), and has great clinical potential for targeted magnetic diagnosis and chemotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to make the purpose, technical solutions and beneficial effects of the present invention more clear, the present invention provides the following drawings for illustration:

[0024] Figure 1 Transmission electron microscopy images of magnetic nanoparticle clusters;

[0025] Figure 2 A comparison of the iron content of different stem cell membrane nanocarriers;

[0026] Figure 3 This is a comparison diagram of the hysteresis loops of stem cell membrane nanocarriers;

[0027] Figure 4 Comparison of T2 magnetic resonance relaxation curves of stem cell membrane nanocarriers;

[0028] Figure 5 This is a comparison diagram of the magnetothermal heating curves of stem cell membrane nanocarriers;

[0029] Figure 6 Comparative photos of hematoxylin-eosin-stained tumor tissue pathological sections after in vivo treatment with stem cell membrane nanocarriers;

[0030] Figure 7 This is a comparison of tumor volumes after in vivo treatment with stem cell membrane nanocarriers;

[0031] Figure 8 Comparison photos of tumor-bearing mice after in vivo treatment with stem cell membrane nanocarriers;

[0032] Figure 9 This is a schematic diagram of the preparation of stem cell membrane nanocarriers and the process of magnetic diagnosis and chemotherapy of tumors. DETAILED DESCRIPTION

[0033] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0034] Example 1. Extraction and preparation of stem cell membranes

[0035] Umbilical cord blood mesenchymal stem cells were dispersed in a physiological buffer solution, ethylenediaminetetraacetic acid and protease inhibitors were added at 4°C, and the mixture was shaken at 130 rpm for 90 minutes. The mixture was then homogenized 30 times at 3000 rpm. The treated turbid liquid was centrifuged at high speed at 4°C for 40 minutes (30,000 rpm) to collect the bottom precipitate. The precipitate was transferred to a physiological buffer solution and ultrasonically disrupted for 5 hours (200 watts, ultrasonication for 3 seconds, and pause for 6 seconds) to prepare a nanosized stem cell membrane.

[0036] Example 2: Preparation of magnetic nanoparticle clusters

[0037] Ferric acetylacetonate (2 mmol) and manganese acetylacetonate (1 mmol) were dissolved in benzyl ether (15 mL), and oleic acid (10 mmol) and oleylamine (20 mmol) were added. After thorough mixing, the mixture was heated to 220°C at a rate of 3.5°C per minute under nitrogen bubbling. After maintaining this temperature for 30 minutes, the temperature was further increased to 290°C at a rate of 3.5°C per minute and maintained at this temperature for 1 hour. The mixture was allowed to cool naturally to room temperature, and anhydrous ethanol (15 mL) was added. The mixture was then magnetically separated and washed to obtain manganese ferrite nanoparticles, which were then dispersed in 20 mL of n-hexane. Distearoylphosphatidylethanolamine-polyethylene glycol 2000 (80 mg) was dissolved in dichloromethane (3 mL), and the prepared manganese ferrite nanoparticle dispersion (Fe content 8 mg / mL, 2 mL) was added. The mixture was mixed by ultrasonication for 1 hour, and the solvent was removed by vacuum rotary evaporation at 50°C. Purified water (1 mL) was added, and the mixture was placed in an alternating magnetic field coil (center strength 50 mT), and then placed in a 50°C water bath. Under the action of the alternating magnetic field and the water bath, the temperature was lowered at a rate of 2°C per minute until 10°C, resulting in magnetic manganese ferrite nanoparticle clusters. The structure and morphology are shown in the attached figure. Figure 1 ,The results showed that nanoparticle clusters were formed.

[0038] Example 3 Stem cell membrane co-loaded with magnetic nanoparticle clusters and chemotherapy drugs

[0039] Hydroxycamptothecin (2 mg / mL, 2 mL) methanol solution and magnetic manganese ferrite nanoparticle clusters (Fe content 5 mg / mL, 4 mL) were slowly added dropwise to the stem cell membrane buffer prepared in Example 1. Place vertically in the center of a static magnetic field (field strength 200 mT) and maintain ultrasound at 4 ° C for 2 hours. The dispersion was centrifuged at high speed (10,000 rpm, 4 ° C) for 20 minutes to remove the unloaded drug and magnetic particles in the supernatant. The bottom precipitate was redispersed to obtain a stem cell membrane nanocarrier.

[0040] The method is the same as above, but the difference is that ordinary magnetic nanoparticles are loaded, that is, the step of using an alternating magnetic field to induce binding and regulate the temperature of the reaction solution to form magnetic nanoparticle clusters is not performed.

[0041] The iron content of the prepared stem cell membrane nanocarriers was tested as follows: Figure 2 As shown, the results showed that the iron concentration of the prepared stem cell membrane nanocarriers was 15 mg / mL, while the iron content of the stem cell membrane nanocarriers prepared without the aid of a magnetic field was only 6.8 mg / mL.

[0042] Magnetic response properties of stem cell membrane nanocarriers Figure 3The results show that the magnetic nanoparticle clusters obtained by alternating magnetic field and temperature control have good magnetic response performance, with a saturation magnetization intensity of 170.6emu / g, which is significantly higher than that of ordinary magnetic nanoparticles (saturation magnetization intensity of 51.7emu / g); T2 magnetic resonance imaging results are shown as follows Figure 4 The results showed that the stem cell membrane carrier loaded with magnetic nanoparticle clusters had significantly enhanced T2 magnetic resonance imaging performance. The T2 magnetic resonance imaging performance of the stem cell membrane nanocarrier was 74mM -1 s -1 , which is 38mM better than the stem cell membrane nanocarrier loaded with ordinary magnetic nanoparticles -1 s -1 The results of the magnetothermal heating curve are as follows: Figure 5 As shown, the results show that the stem cell membrane carrier loaded with magnetic nanoparticle clusters has higher magnetocaloric performance, with a specific absorption coefficient of 151W / g, while the stem cell membrane nanocarrier loaded with ordinary magnetic nanoparticles is only 86W / g.

[0043] The prepared drug-loaded stem cell membrane nanocarriers are used for targeted chemotherapy. The principle is as follows Figure 9 As shown. This method uses temperature control and magnetic field induction of alternating magnetic fields to obtain magnetic manganese ferrite nanoparticle clusters with significantly enhanced magnetism. At the same time, the magnetic nanoparticle clusters and chemotherapy drugs are loaded into the stem cell membrane with the help of magnetic field induction, thereby preparing a stem cell membrane nanocarrier with stable structure and uniform dispersion. The prepared stem cell membrane nanocarrier loaded with drugs and magnetic manganese ferrite nanoparticle clusters was subjected to targeted magnetic therapy and chemotherapy in tumor-bearing mice. Compared with the magnetic manganese ferrite nanoparticle clusters alone, it showed an excellent inhibitory effect on the tumor at the end of the treatment, which was reflected in the obvious destruction of the tumor tissue ( Figure 6 ) and a significant reduction in tumor volume ( Figure 7 and Figure 8 ).

[0044] The above embodiments are merely preferred embodiments for the purpose of fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

Claims

1. Stem cell membrane nanocarrier for tumor-targeted magnetic diagnosis and chemotherapy, characterized in that: Prepared by the following method: (1) Preparation of stem cell membrane: Umbilical cord blood mesenchymal stem cells were dispersed in physiological buffer, ethylenediaminetetraacetic acid and protease inhibitors were added at 4 °C, and the mixture was placed in a shaker at 130 rpm for 90 minutes; then the mixture was homogenized 30 times at 3000 rpm; the treated turbid liquid was centrifuged at 4 °C and 30,000 rpm for 40 minutes, and the bottom precipitate was collected; the precipitate was transferred to physiological buffer and ultrasonically disrupted at 200 watts for 5 hours, with each ultrasonication for 3 seconds and a pause of 6 seconds during the ultrasonication, to prepare a nano-sized stem cell membrane. (2) Preparation of magnetic nanoparticle clusters: 2 mmol of ferric acetylacetonate and 1 mmol of manganese acetylacetonate were dissolved in 15 mL of benzyl ether, and 10 mmol of oleic acid and 20 mmol of oleylamine were added; after thorough mixing, the temperature was raised to 220 °C at a rate of 3.5 °C per minute under nitrogen flow, and the temperature was maintained for 30 minutes, and then the temperature was raised to 290 °C at a rate of 3.5 °C per minute and maintained at this temperature for 1 hour; the mixture was cooled to room temperature naturally, 15 mL of anhydrous ethanol was added, and magnetic separation and washing were performed to obtain manganese ferrite nanoparticles, which were dispersed in 20 mL of n-hexane; 80 mg of distearoylphosphatidylethanolamine-polyethylene glycol 2000 was dissolved in 3 mL of dichloromethane, and 2 mL of the prepared manganese ferrite nanoparticle dispersion with an Fe content of 8 mg / mL was added, mixed and ultrasonicated for 1 hour, and the solvent was removed by vacuum rotary evaporation at 50 °C; 1 mL of purified water was added, and the mixture was transferred into a central intensity 50 mT alternating magnetic field coil, and placed outside in a 50 ℃ water bath; under the action of the alternating magnetic field and the water bath cycle, the temperature was lowered at a rate of 2 ℃ per minute until 10 ℃, and magnetic manganese ferrite nanoparticle clusters were obtained, forming magnetic nanoparticle clusters; (3) Preparation of stem cell membrane nanocarriers: 2 mL of 2 mg / mL hydroxycamptothecin methanol solution and 4 mL of magnetic manganese ferrite nanoparticle clusters with an Fe content of 5 mg / mL were slowly added dropwise to the stem cell membrane buffer prepared in step (1), and placed vertically at the center of a static magnetic field with a field strength of 200 mT. Ultrasonication was maintained at 4°C for 2 hours. The dispersion was centrifuged at 4°C and 10,000 rpm for 20 minutes to remove the unloaded drugs and magnetic particles in the supernatant. The bottom precipitate was redispersed to obtain the stem cell membrane nanocarriers.

2. The method for preparing the stem cell membrane nanocarrier for tumor-targeted magnetic diagnosis and chemotherapy according to claim 1, characterized in that: The steps include: (1) Preparation of stem cell membrane: Umbilical cord blood mesenchymal stem cells were dispersed in physiological buffer, ethylenediaminetetraacetic acid and protease inhibitors were added at 4 °C, and the mixture was placed in a shaker at 130 rpm for 90 minutes; then the mixture was homogenized 30 times at 3000 rpm; the treated turbid liquid was centrifuged at 4 °C and 30,000 rpm for 40 minutes, and the bottom precipitate was collected; the precipitate was transferred to physiological buffer and ultrasonically disrupted at 200 watts for 5 hours, with each ultrasonication for 3 seconds and a pause of 6 seconds during the ultrasonication, to prepare a nano-sized stem cell membrane. (2) Preparation of magnetic nanoparticle clusters: 2 mmol of ferric acetylacetonate and 1 mmol of manganese acetylacetonate were dissolved in 15 mL of benzyl ether, and 10 mmol of oleic acid and 20 mmol of oleylamine were added; after thorough mixing, the temperature was raised to 220 °C at a rate of 3.5 °C per minute under nitrogen flow, and the temperature was maintained for 30 minutes, and then the temperature was raised to 290 °C at a rate of 3.5 °C per minute and maintained at this temperature for 1 hour; the mixture was cooled to room temperature naturally, 15 mL of anhydrous ethanol was added, and magnetic separation and washing were performed to obtain manganese ferrite nanoparticles, which were dispersed in 20 mL of n-hexane; 80 mg of distearoylphosphatidylethanolamine-polyethylene glycol 2000 was dissolved in 3 mL of dichloromethane, and 2 mL of the prepared manganese ferrite nanoparticle dispersion with an Fe content of 8 mg / mL was added, mixed and ultrasonicated for 1 hour, and the solvent was removed by vacuum rotary evaporation at 50 °C; 1 mL of purified water was added, and the mixture was transferred into a central intensity 50 mT alternating magnetic field coil, and placed outside in a 50 ℃ water bath; under the action of the alternating magnetic field and the water bath cycle, the temperature was lowered at a rate of 2 ℃ per minute until 10 ℃, and magnetic manganese ferrite nanoparticle clusters were obtained, forming magnetic nanoparticle clusters; (3) Preparation of stem cell membrane nanocarriers: 2 mL of 2 mg / mL hydroxycamptothecin methanol solution and 4 mL of magnetic manganese ferrite nanoparticle clusters with an Fe content of 5 mg / mL were slowly added dropwise to the stem cell membrane buffer prepared in step (1), and placed vertically at the center of a static magnetic field with a field strength of 200 mT. Ultrasonication was maintained at 4°C for 2 hours. The dispersion was centrifuged at 4°C and 10,000 rpm for 20 minutes to remove the unloaded drugs and magnetic particles in the supernatant. The bottom precipitate was redispersed to obtain the stem cell membrane nanocarriers.

3. Use of the stem cell membrane nanocarrier according to claim 1 in the preparation of tumor-targeted magnetic therapeutic drugs.

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