Preparation method and application of cell membrane modified transition metal nitride sonodynamic nano-carrier
By preparing cell membrane-modified transition metal nitride nanocarriers, the problems of poor water solubility and insufficient targeting in tumor treatment are solved, and accurate delivery of tumor sites and efficient oxidation damage are achieved, improving the therapeutic effect and safety.
Patent Information
- Application Number
- CN202510410100.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-02
- Publication Date
- 2025-08-12
AI Technical Summary
Existing sound-sensitizers have problems with poor water solubility and limited clinical application of skin phototoxicity in tumor treatment, and traditional treatment methods are difficult to achieve accurate and non-invasive targeted tumor delivery and efficient oxidative damage.
The transition metal nitride nanocarrier modified with cell membrane is designed through the core-shell structure. The outer shell layer is composed of lipids doped with tumor cell membranes, and the inner core layer is composed of transition metal nitride sound-sensitizer and oxygen carrier to achieve ultrasonic responsive release and enhance tumor targeting and therapeutic effects.
Accurate targeted delivery of tumor sites and efficient oxidative damage, reducing toxic and side effects of normal tissues, improving the safety and efficiency of tumor treatment, and reducing drug resistance risks.
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Figure CN120459293A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomaterials, and in particular relates to a preparation method and application of a cell membrane-modified transition metal nitride sonodynamic nanocarrier. Background Art
[0002] Cancer is a major disease that seriously endangers human health. Its core pathological mechanism lies in abnormal cell growth regulation and abnormal metastasis, which leads to damage to the body's normal tissue structure and function. Breakthroughs in modern tumor diagnosis and treatment technologies have significantly improved cancer survival indicators in recent years. It is worth noting that although the prognosis of cancer patients in my country has improved compared with the past, it is still insufficient compared with the international advanced level. This situation highlights the urgency of the research and development of innovative treatment methods. Therefore, based on the biological characteristics of malignant tumors and clinical treatment needs, the establishment of a new precise and minimally invasive treatment system can not only improve the efficiency of tumor cell clearance, but also minimize the systemic adverse reactions caused by traditional therapies, thereby bringing better treatment effects and quality of life to patients.
[0003] The research and development of new tumor treatment strategies is a hot topic at the forefront of current research. Constructing a treatment platform with tumor-targeted recognition capabilities can not only improve treatment efficacy and reduce toxic side effects, but also combine non-invasive technologies to enhance the efficiency of precision treatment. Among the many treatment methods, ultrasound technology has become an ideal choice for tumor sonodynamic therapy due to its unique physical acoustic wave effects, as well as its advantages of convenient operation, real-time monitoring, safety and no radiation. Sonodynamic therapy is a new treatment method that uses the interaction between ultrasound and specific sonosensitizers to treat diseases. It mainly activates sonosensitizers through ultrasound to produce reactive oxygen species, thereby destroying diseased tissue. In addition, sonodynamic therapy has strong tissue penetration and can achieve millimeter-level precision control at the target site, thereby killing breast cancer while minimizing damage to normal tissue. Therefore, sonodynamic therapy has great application potential for the treatment of deep tumors.
[0004] Currently, research on sonosensitizers primarily focuses on organic sonosensitizers and inorganic nanosonosensitizers. While organic sonosensitizers offer high reactive oxygen species (ROS) production rates, their poor water solubility and skin phototoxicity limit their clinical applications. In contrast, inorganic sonosensitizers are highly favored due to their excellent stability and controllable size. Among them, transition metal nitrides, with their superior electrical conductivity, high melting point, and exceptional thermal and chemical stability, hold broad application prospects in energy-related fields such as various batteries, supercapacitors, and electrocatalysis. Due to their efficient sonocatalytic properties, excellent chemical stability, and abundant surface active sites, transition metal nitrides maintain structural integrity under ultrasonic cavitation while catalyzing the generation of large amounts of oxygen free radicals, resulting in significant oxidative damage to tumor cells. Based on these properties, transition metal nitrides, as novel inorganic sonosensitizers, not only enable efficient and stable ROS generation but also enable precise in vivo delivery through nanobiotechnology, thereby establishing an integrated diagnosis and treatment platform for tumor therapy. This strategy not only provides a solid theoretical basis for the non-invasive treatment of tumors such as breast cancer, but also shows broad clinical application prospects and opens up new research directions in the field of tumor treatment. Summary of the Invention
[0005] In view of the above technical background, an anti-tumor treatment platform integrating diagnosis and treatment is constructed based on ultrasound technology. The present invention provides a preparation method and application of a cell membrane-modified transition metal nitride sonodynamic nanocarrier.
[0006] The object of the present invention is achieved as follows: a cell membrane-modified transition metal nitride nanocarrier, characterized in that the carrier has a core-shell structure as an ultrasound-activated inorganic sonosensitizer, wherein the outer shell is composed of tumor cell membrane doped with lipids and the inner core layer is composed of a transition metal nitride sonosensitizer and an oxygen carrier.
[0007] A method for preparing a cell membrane-modified transition metal nitride sonodynamic nanocarrier, characterized in that the method comprises the following steps:
[0008] Step 1): Preparation of nanoparticles loaded with transition metal nitride sonosensitizer and oxygen carrier
[0009] Transition metal nitride is prepared, and then loaded with an oxygen carrier to obtain nanoparticles loaded with the transition metal nitride sonosensitizer and the oxygen carrier.
[0010] Step 2): Isolation and extraction of breast cancer cell membranes
[0011] Breast cancer cell lines were cultured and expanded in vitro, and then breast cancer cell membranes were extracted.
[0012] Step 3): Preparation of cell membrane modified transition metal nitride sonodynamic nanocarriers
[0013] Nanoparticles loaded with sonosensitizers and oxygen carriers are co-blended with lipid-doped tumor cell membranes and extruded, and then filled with oxygen to prepare cell membrane-modified transition metal nitride sonodynamic nanocarriers.
[0014] Preferably, the transition metal nitride is prepared by a high temperature nitridation method, a sol-gel method, or a hydrothermal / solvothermal method.
[0015] Preferably, the transition metal nitride includes but is not limited to: copper nitride, iron nitride, and cobalt nitride.
[0016] Preferably, the cell membrane is derived from one of the mouse breast cancer cell lines 4T1, EMT6, JC, or the human breast cancer cell lines MCF-7, MDA-MB-231, T-47D, BT-474.
[0017] Preferably, the transition metal nitride has a particle size distribution of 10-1000 nm.
[0018] Preferably, the preparation method of the liposome includes but is not limited to: a thin film dispersion method, a co-extrusion method and a microfluidic mixing method.
[0019] Preferably, the lipid components of the liposome include but are not limited to three or more of: soybean lecithin, hydrogenated soybean lecithin, cholesterol, cholesterol ester, and distearoylphosphatidylethanolamine-polyethylene glycol 2000.
[0020] Preferably, the oxygen carrier entrapped in the liposome includes but is not limited to: one of perfluorobutane, perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluorododecane, and perfluoromethylcyclohexane.
[0021] Another object of the present invention is to provide the use of the above-mentioned cell membrane-modified transition metal nitride sonodynamic nanocarrier in tumor treatment.
[0022] Preferably, the cell membrane modified transition metal nitride sonodynamic nanocarrier is characterized in that the sonodynamic nanocarrier can be applied to treatments including but not limited to breast tumors.
[0023] Preferably, the cell membrane modified transition metal nitride sonodynamic nanocarrier is characterized in that the ultrasonic frequency range used is 0.1-30 MHz and the power range is 0.1-5 W / cm 2 , ultrasonic time is 0.1-60min.
[0024] Working principle of the present invention
[0025] The cell membrane-modified transition metal nitride sonodynamic nanocarrier described in the present invention has the following working principles: (1) The modification of the breast cancer cell membrane gives the nanocarrier excellent in vivo stability, prolongs the blood circulation time, and enhances the enrichment efficiency in tumor tissue through homologous targeting, reduces the toxic side effects of normal tissue, and realizes precision treatment; (2) The transition metal nitride not only has excellent catalytic activity, but also can efficiently trigger the sonodynamic effect, synergistically kill tumor cells, and enhance the therapeutic effect; (3) Relying on the loaded sonosensitizer and oxygen carrier, the nanocarrier has a release behavior in response to ultrasonic signals, thereby regulating the tumor microenvironment, improving the anti-tumor efficiency, and reducing the risk of drug resistance, thereby realizing safe and efficient precision treatment.
[0026] Beneficial effects of the present invention:
[0027] 1. The present invention provides a cell membrane-modified transition metal nitride sonodynamic nanocarrier with clear structural components, simple preparation process, and good biocompatibility.
[0028] 2. The cell membrane-modified transition metal nitride sonodynamic nanocarrier provided by the present invention has good tumor targeting ability due to homologous tumor cell membrane modification, and can be effectively enriched at the tumor site to achieve the purpose of precise diagnosis and treatment of tumors.
[0029] 3. The cell membrane-modified transition metal nitride sonodynamic nanocarrier provided by the present invention can effectively release transition metal nitride sonosensitizer and oxygen under external ultrasonic stimulation, greatly improving the sonodynamic treatment effect of tumors, which is an innovation in the field of biomedical engineering. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] Figure 1 The particle size and Zeta potential of the cell membrane-modified transition metal nitride sonodynamic nanocarrier prepared in Example 1 were tested;
[0031] Figure 2 The storage stability test of the cell membrane modified transition metal nitride sonodynamic nanocarrier prepared in Example 1;
[0032] Figure 3 This is the ultrasound response oxygen release curve of the cell membrane modified transition metal nitride sonodynamic nanocarrier prepared in Example 1;
[0033] Figure 4 The in vitro sonodynamic effect test of the cell membrane modified transition metal nitride sonodynamic nanocarrier prepared in Example 1;
[0034] Figure 5 This is the in vitro breast cancer cell killing effect test of the cell membrane modified transition metal nitride sonodynamic nanocarrier prepared in Example 1. DETAILED DESCRIPTION
[0035] The present invention is further described below by way of examples, but the following examples do not limit the scope of rights of this patent.
[0036] Example 1
[0037] The preparation method of the cell membrane modified transition metal nitride sonodynamic nanocarrier is as follows:
[0038] (1) Extraction of tumor cell membranes: 4T1 cells of mouse breast cancer cell line cultured in vitro were collected and pre-cooled Tris-MgCl2(TM) buffer (containing 10 mM Tris and 1 mM MgCl2, pH 7.4) was added to make the final concentration of cells 5×10 7 / mL. The cells were then repeatedly squeezed using a liposome extruder (without a polycarbonate membrane) for 20 cycles. Pre-cooled sucrose solution was added to the collected cell lysate to a final sucrose concentration of 0.25 M. A series of gradient centrifugation operations were then performed at 4°C, including: (1) centrifugation at 3000 rpm for 30 min to obtain a precipitate; (2) re-suspending in pre-cooled TM buffer-sucrose solution (sucrose concentration of 0.25 M), centrifuging at 3000 rpm for 30 min to obtain a precipitate, and thus obtaining 4T1 breast cancer cell membranes.
[0039] (2) Preparation of transition metal nitrides: 0.06 g of copper nitrate trihydrate (Cu(NO3)2·3H2O) was dissolved in a mixed solvent of 5 mL of octadecylamine and 5 mL of octadecene. After the reaction system was fully degassed, it was heated to 150°C under continuous magnetic stirring and maintained for 3 h. At this time, the color of the solution gradually changed from blue to green and finally to yellow. The temperature was then gradually increased to 250°C and maintained for 30 min. Finally, transition metal nitride (copper nitride) nanocubes were isolated and purified by centrifugation and purification steps.
[0040] (3) Preparation of cell membrane modified transition metal nitride sonodynamic nanocarriers: 50 mg of soybean lecithin, 12.5 mg of cholesterol, 5 mg of distearoylphosphatidylethanolamine-polyethylene glycol 2000 and 20 μg of transition metal nitride (copper nitride) were dissolved in a chloroform-methanol mixture (1:4, v / v) and evaporated under reduced pressure in a 45°C water bath to form a dry lipid film. Subsequently, 10 mL of normal saline was added and hydrated in a 42°C water bath for 30 min. The suspension was subjected to probe sonication in an ultrasonic cell disruptor in an ice bath for 90 cycles (ultrasonic power: 2000 W, each ultrasonic cycle contained 1 s of sonication and 2 s of interval). The suspension was then filtered using a 0.22 μm mixed cellulose membrane to remove large particles and unencapsulated transition metal nitride (copper nitride). The prepared nanocarriers were mixed with tetradecafluorohexane (150 μL) and extruded in the order of 800, 400, and 200 nm using a liposome extruder. The mixture was then mixed with the extracted 4T1 breast cancer cell membranes and extruded in the order of 800, 400, and 200 nm. The oxygen chamber was filled with oxygen for 1 min (at an oxygen flow rate of 5 L / min) to obtain cell membrane-modified transition metal nitride sonodynamic nanocarriers.
[0041] Example 2
[0042] Particle size and Zeta potential test of cell membrane modified transition metal nitride sonodynamic nanocarriers:
[0043] The cell membrane modified transition metal nitride sonodynamic nanocarrier prepared in Example 1 was taken and its particle size and potential were measured by dynamic light scattering method. The results are as follows: Figure 1 As shown, the particle size of the sonodynamic nanocarrier is 167.2±3.4 nm and the potential is -17.3±1.09 mV.
[0044] Example 3
[0045] Storage stability test of cell membrane modified transition metal nitride sonodynamic nanocarriers:
[0046] The cell membrane modified transition metal nitride sonodynamic nanocarrier prepared in Example 1 was placed in a 4°C environment, and its particle size was measured by dynamic light scattering method on the 0th, 1st, 3rd, 5th, 7th and 9th day, and the particle size change curve was plotted. The results are shown in Figure 2. Figure 2 As shown in the figure, the particle size of the sonodynamic nanocarriers did not change significantly within 9 days, indicating good storage stability.
[0047] Example 4
[0048] Ultrasonic response oxygen release curve of cell membrane modified transition metal nitride sonodynamic nanocarrier:
[0049] The cell membrane modified transition metal nitride sonodynamic nanocarrier prepared in Example 1 was filled with nitrogen to remove oxygen, and an ultrasonic probe was used to stimulate the system with an ultrasonic signal (0.35 W / cm 2 ,1MHz,3min), the change of oxygen content in the solution is measured by dissolved oxygen electrode, and the oxygen content-time curve is shown in Figure 3 The results show that under ultrasound stimulation, the sonodynamic nanocarrier can quickly release a large amount of oxygen. The oxygen content in the system can reach 10 mg / L within 90 seconds of ultrasound, significantly increasing the oxygen content in the solution medium.
[0050] Example 5
[0051] In vitro sonodynamic effect test of cell membrane modified transition metal nitride sonodynamic nanocarriers:
[0052] The cell membrane modified transition metal nitride sonodynamic nanocarriers and free copper nitride solution prepared in Example 1 were dispersed in air-saturated water and nitrogen-saturated water, respectively. Singlet oxygen indicator SOSG was added for co-incubation. The system was stimulated with an ultrasonic signal (0.35 W / cm 2 ,1MHz,3min), and then detect the change of SOSG fluorescence intensity (Ex=504nm,Em=525nm). Figure 4 As shown, it can be seen that in the absence of oxygen, free copper nitride hardly produces any sonodynamic effect, while the cell membrane-modified transition metal nitride sonodynamic nanocarrier can release a large amount of oxygen under the action of ultrasound, providing raw materials for the sonodynamic effect, thereby significantly increasing the fluorescence intensity of SOSG and having a significant sonodynamic effect.
[0053] Example 6
[0054] Tumor cell killing effect of cell membrane modified transition metal nitride sonodynamic nanocarriers:
[0055] 4T1 cells were cultured at a rate of 1 × 10 4 The cells were plated in a 96-well plate and then cultured in an AnaeroPack hypoxic box at 37°C until they were completely attached. After washing with PBS to remove residual serum and cell debris, PBS, free copper nitride, and cell membrane-modified transition metal nitride sonodynamic nanocarriers were added, respectively. After incubation with 4T1 cells for 10 hours, the supernatant was replaced with incomplete culture medium. Ultrasound (0.35W / cm 2,1MHz) to stimulate the cells for 3 minutes, and the group without ultrasound was used as a control. After culturing for 24 hours, 20μL MTT solution (5mg / mL) was added to each well and incubated at 37℃ for another 4 hours. After aspirating the supernatant with a syringe, 150μL dimethyl sulfoxide was added to completely dissolve the blue-purple formazan crystals, and the absorbance at 570nm was measured with a microplate reader to calculate the cell survival rate. The results are shown in Figure 2. Figure 5 As shown in the figure, it can be seen that the sonodynamic killing effect of free copper nitride is severely hindered in hypoxic environment. However, due to the ultrasound-responsive oxygen supply ability of cell membrane-modified transition metal nitride sonodynamic nanocarriers, the killing effect on breast cancer cells in hypoxic environment is greatly improved.
[0056] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.
Claims
1. A cell membrane-modified transition metal nitride sonodynamic nanocarrier, characterized by: The nanocarrier has a core-shell structure, comprising: (1) an outer shell composed of tumor cell membrane doped with lipids; and (2) an inner core layer composed of a transition metal nitride sonosensitizer and an oxygen carrier.
2. The cell membrane modified sonodynamic therapy nanocarrier according to claim 1, characterized in that: The cell membrane is derived from one of the following breast cancer cell lines: mouse breast cancer cell lines 4T1, EMT6, JC, or human breast cancer cell lines MCF-7, MDA-MB-231, T-47D, BT-474.
3. The cell membrane modified sonodynamic therapy nanocarrier according to claim 1, characterized in that: The transition metal nitride includes but is not limited to copper nitride, iron nitride, and cobalt nitride.
4. The transition metal nitride according to claim 3, characterized in that: Its particle size distribution range is 10-1000nm.
5. The transition metal nitride according to claim 3, wherein: The transition metal nitride is modified with liposomes loaded with an oxygen carrier to enhance water dispersibility; the preparation method of the liposomes includes but is not limited to a thin film dispersion method, a co-extrusion method or a microfluidic mixing method.
6. The liposome according to claim 5, wherein: The lipid components of the liposome include but are not limited to three or more of the following ingredients: soybean lecithin, hydrogenated soybean lecithin, cholesterol, cholesterol ester, and distearoylphosphatidylethanolamine-polyethylene glycol 2000.
7. The liposome according to claim 5, wherein: The oxygen carrier entrapped by the liposome includes but is not limited to one of the following: perfluorobutane, perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluorododecane, and perfluoromethylcyclohexane.
8. The use of the cell membrane modified ultrasound-responsive sonodynamic therapy nanocarrier according to claim 1, characterized in that: The ultrasonic frequency range used is 0.1-30 MHz, and the power range is 0.1-5 W / cm 2 , the ultrasonic action time is 0.1-60min.
9. The use of the cell membrane modified transition metal nitride sonodynamic nanocarrier according to claim 1, characterized in that: The ultrasound-responsive nanocarrier can be applied to the treatment of malignant tumors such as breast tumors, but is not limited thereto.