Drug-loaded Fe3O4 nano-particle composite preparation, drug-loaded Fe3O4 nano-particle composite preparation camouflaged by vesicle membrane, and preparation method and application of drug-loaded Fe3O4 nano-particle composite preparation

Abstract

The invention discloses a drug-loaded Fe3O4 nano-particle composite preparation, and belongs to the technical field of antitumor drugs, the drug-loaded Fe3O4 nano-particle composite preparation comprises a core-empty-shell Fe3O4 nano-particle composite material and drugs filled in gaps of the core-empty-shell Fe3O4 nano-particle composite material, and the core-empty-shell Fe3O4 nano-particle composite material comprises a ZnO shell structure, a Fe3O4 nano-particle core structure and gaps located between the ZnO shell structure and the Fe3O4 nano-particle core structure and used for filling the drugs. The invention also discloses a drug-loaded Fe3O4 nano-particle composite preparation camouflaged by the vesicle membrane, and a corresponding preparation method and application of the drug-loaded Fe3O4 nano-particle composite preparation. The drug-loaded Fe3O4 nanoparticle composite preparation provided by the invention has targeting property, and can effectively kill tumor cells without causing damage to normal tissues. The drug-loaded Fe3O4 nano-particle composite preparation is wrapped by the vesicle membrane derived from the tumor receptor, so that the investigation and removal of the immune system in the tumor receptor are avoided, the blood circulation time of the tumor receptor is greatly prolonged, and the improvement of the anti-tumor medical effect is facilitated.

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-tumor drugs, and specifically relates to a drug-loaded Fe3O4 nanoparticle composite preparation, a drug-loaded Fe3O4 nanoparticle composite preparation disguised by a vesicle membrane, and a preparation method and use thereof. Background Art

[0002] Nanoparticles have already achieved fairly mature development in various fields. However, there are still insurmountable limitations in the biological field that make it difficult to apply them in the clinical field. The main manifestations are: nanomaterials are easily screened and eliminated by the human immune system in the human body and cannot play their due role; the high permeability retention (EPR) effect of nanomaterials is not significant and can easily cause damage to normal cells; nanomaterials have potential toxicity in the body and the nature of being difficult to actively overcome physiological barriers makes it difficult for them to find the real target and accumulate at the target location.

[0003] Fe3O4 is an FDA-approved nanodrug. Due to its unique properties and long blood circulation, it is currently being widely studied to overcome the limitations of the above nanomaterials in clinical application. Functional modification of Fe3O4 nanoparticles to prepare anti-tumor drugs has become a research trend. The use of external camouflage of cell membranes can not only evade clearance by the immune system and obtain longer blood circulation, but also increase tumor targeting ability and enhance tumor magnetic resonance imaging (MRI) and photothermal therapy (PPT) technology. Summary of the invention

[0004] In response to one or more of the above-mentioned defects or improvement needs of the prior art, the present invention provides a drug-loaded Fe3O4 nanoparticle composite preparation, a drug-loaded Fe3O4 nanoparticle composite preparation camouflaged by a vesicle membrane, and a preparation method and use, which can improve the targeting effect on tumor cells and effectively kill tumor cells without causing damage to normal tissues.

[0005] To achieve the above-mentioned purpose, according to the first aspect of the present invention, there is provided a drug-loaded Fe3O4 nanoparticle composite preparation, comprising a core-void-shell Fe3O4 nanoparticle composite material and a drug filled in the voids thereof; the core-void-shell Fe3O4 nanoparticle composite material comprises a ZnO shell structure, a Fe3O4 nanoparticle core structure and a void between the two for filling the drug.

[0006] As a further improvement of the present invention, the core-void-shell Fe3O4 nanoparticle composite material is prepared by solvent thermal decomposition method, sol-gel method, chemical bath deposition method and selective etching method in sequence;

[0007] FeCl36H2O is decomposed by a solvent thermal decomposition method to prepare Fe3O4 nanoparticles; a SiO2 shell layer is coated on the surface of the prepared Fe3O4 nanoparticles by hydrolysis of ethyl silicate by a sol-gel method to prepare Fe3O4@SiO2; a layer of ZnO is deposited on the surface of the prepared Fe3O4@SiO2 by a chemical bath deposition method to obtain Fe3O4@SiO2@ZnO; and a SiO2 layer of the Fe3O4@SiO2@ZnO middle layer is etched by a selective etching method using aqueous ammonia to obtain Fe3O4@clear@ZnO, thereby obtaining the core-void-shell Fe3O4 nanoparticle composite material.

[0008] As a further improvement of the present invention, the drug is the tumor antibody daunorubicin, doxorubicin, epirubicin or aclarubicin, and the weight ratio of the drug to the core-void-shell Fe3O4 nanoparticle composite material is 1:5.

[0009] According to a second aspect of the present invention, a method for preparing a drug-loaded Fe3O4 nanoparticle composite preparation is provided, which is used to prepare the drug-loaded Fe3O4 nanoparticle composite preparation, comprising the following steps:

[0010] (1) Preparation of core-shell Fe3O4 nanoparticle composites

[0011] Dissolve 1.2 g to 1.5 g of FeCl36H2O in 35 mL to 45 mL of ethylene glycol to form a suspension, then add 3 g to 4 g of sodium acetate and 0.8 g to 1.2 g of polyethylene glycol to the suspension, continue stirring for 25 min to 35 min, seal the mixture in an autoclave, heat to 180° C. to 220° C. and maintain for 4 h to 6 h, collect the product Fe3O4, wash and dry to obtain Fe3O4 nanoparticles;

[0012] 0.05 g to 0.15 g of the Fe3O4 nanoparticles are soaked in a mixture of 140 to 180 mL of ethanol, 30 to 55 mL of water and 1 to 3 mL of NH3H2O, and after ultrasonic treatment for 20 min to 40 min, 4 mL to 6 mL of an ethanol solution containing 0.8 mL to 1.0 mL of ethyl silicate is slowly added dropwise, stirred vigorously, and reacted at room temperature for 6 h to 10 h, and the synthesized product is harvested by magnetic separation, and washed and dried to obtain a core-shell Fe3O4@SiO2 nanocomposite material;

[0013] 0.05 g to 0.15 g of the core-shell Fe3O4@SiO2 nanocomposite material is dispersed in 80 mL to 120 mL of deionized water, subjected to ultrasonic treatment for 5 min to 15 min, and then vigorously stirred for 15 min to 25 min, followed by adding 0.60 g to 0.76 g of Zn(Ac)2·2H2O, and adjusting the pH of the dispersion to 10 to 13 with an aqueous ammonia solution to obtain Fe3O4@SiO2@ZnO, followed by selective etching in an aqueous ammonia environment, mechanical stirring for 15 min to 25 min, and ultrasonic treatment at 55°C to 65°C for 1 h to 3 h, and finally separating, rinsing and drying the obtained product to obtain a Fe3O4@clear@ZnO nanocomposite material;

[0014] (2) preparing the drug-loaded Fe3O4 nanoparticle composite preparation;

[0015] 2 mL of 1 mg / mL daunorubicin solution, doxorubicin solution, epirubicin solution or aclarubicin solution is mixed with 4.5 mL to 5.5 mL of the Fe3O4@clear@ZnO nanocomposite dispersion, wherein the dispersion contains 0.01 g of the Fe3O4@clear@ZnO nanocomposite, and the solvent is phosphate buffer; ultrasonic oscillation is performed for 2 min to 4 min, the dispersion is transferred to a centrifuge tube, oscillated at 25 to 37° C. for 22 h to 26 h, centrifuged with phosphate buffer to remove unloaded drugs, and the supernatant and washing liquid are collected to obtain the drug-loaded Fe3O4 nanoparticle composite preparation.

[0016] According to the third aspect of the present invention, there is provided a drug-loaded Fe3O4 nanoparticle composite preparation disguised by a vesicle membrane, comprising the drug-loaded Fe3O4 nanoparticle composite preparation and a vesicle membrane wrapped around the outside thereof.

[0017] According to a fourth aspect of the present invention, there is provided a method for preparing the drug-loaded Fe3O4 nanoparticle composite preparation disguised by a vesicle membrane, comprising the following steps:

[0018] (1) Preparation of core-shell Fe3O4 nanoparticle composites

[0019] Dissolve 1.2 g to 1.5 g of FeCl36H2O in 35 mL to 45 mL of ethylene glycol to form a suspension, then add 3 g to 4 g of sodium acetate and 0.8 g to 1.2 g of polyethylene glycol to the suspension, continue stirring for 25 min to 35 min, seal the mixture in an autoclave, heat to 180° C. to 220° C. and maintain for 4 h to 6 h, collect the product Fe3O4, wash and dry to obtain Fe3O4 nanoparticles;

[0020] 0.05 g to 0.15 g of the Fe3O4 nanoparticles are soaked in a mixture of 140 to 180 mL of ethanol, 30 to 55 mL of water and 1 to 3 mL of NH3H2O, and after ultrasonic treatment for 20 min to 40 min, 4 mL to 6 mL of an ethanol solution containing 0.8 mL to 1.0 mL of ethyl silicate is slowly added dropwise, stirred vigorously, and reacted at room temperature for 6 h to 10 h, and the synthesized product is harvested by magnetic separation, and washed and dried to obtain a core-shell Fe3O4@SiO2 nanocomposite material;

[0021] 0.05 g to 0.15 g of the core-shell Fe3O4@SiO2 nanocomposite material is dispersed in 80 mL to 120 mL of deionized water, subjected to ultrasonic treatment for 5 min to 15 min, and then vigorously stirred for 15 min to 25 min, followed by adding 0.60 g to 0.76 g of Zn(Ac)2·2H2O, and adjusting the pH of the dispersion to 10 to 13 with an aqueous ammonia solution to obtain Fe3O4@SiO2@ZnO, followed by selective etching in an aqueous ammonia environment, mechanical stirring for 15 min to 25 min, and ultrasonic treatment at 55°C to 65°C for 1 h to 3 h, and finally separating, rinsing and drying the obtained product to obtain a Fe3O4@clear@ZnO nanocomposite material;

[0022] (2) preparing the drug-loaded Fe3O4 nanoparticle composite preparation;

[0023] 2 mL of 1 mg / mL daunorubicin solution, doxorubicin solution, epirubicin solution or aclarubicin solution is mixed with 4.5 mL to 5.5 mL of the Fe3O4@clear@ZnO nanocomposite dispersion, wherein the dispersion contains 0.01 g of the Fe3O4@clear@ZnO nanocomposite, and the solvent is phosphate buffer; ultrasonic oscillation is performed for 2 min to 4 min, the dispersion is transferred to a centrifuge tube, oscillated at 25 to 37° C. for 22 h to 26 h, centrifuged with phosphate buffer to remove unloaded drugs, and the supernatant and washing liquid are collected to obtain the drug-loaded Fe3O4 nanoparticle composite preparation.

[0024] (3) Preparation of drug-loaded Fe3O4 nanoparticle composite formulations disguised by vesicle membranes;

[0025] The drug-loaded Fe3O4 nanoparticle composite preparation is mixed with a vesicle membrane, and is repeatedly extruded through holes of 100nm to 300nm to wrap the vesicle membrane on the drug-loaded Fe3O4 nanoparticle composite preparation, thereby preparing the drug-loaded Fe3O4 nanoparticle composite preparation disguised by the vesicle membrane.

[0026] As a further improvement of the present invention, the preparation of the vesicle membrane comprises the following steps:

[0027] The vesicles are purified from the blood of tumor recipients, and then subjected to hypotonic treatment, repeated freeze-thaw or ultrasonic treatment to preliminarily obtain the vesicle membrane. The preliminarily obtained vesicle membrane is then placed on a micro-extruder and extruded multiple times using 400 nm and 200 nm micropores in sequence to obtain the vesicle membrane having a microporous structure.

[0028] As a further improvement of the present invention, the hydrodynamic diameter of the vesicle membrane is 300nm to 400nm, and the zeta potential is -30mV to 26mV.

[0029] According to a fifth aspect of the present invention, there is provided an application of a drug-loaded Fe3O4 nanoparticle composite preparation disguised by a vesicle membrane in the preparation of an anti-tumor drug, the drug-loaded Fe3O4 nanoparticle composite preparation disguised by a vesicle membrane as claimed in claim 5, or the drug-loaded Fe3O4 nanoparticle composite preparation disguised by a vesicle membrane is prepared according to the preparation method of the drug-loaded Fe3O4 nanoparticle composite preparation disguised by a vesicle membrane as claimed in any one of claims 6 to 8,

[0030] The tumors include breast tumors, lung cancer tumors, and malignant lymphomas.

[0031] In general, the above technical solution conceived by the present invention has the following beneficial effects compared with the prior art:

[0032] (1) The drug-loaded Fe3O4 nanoparticle composite preparation of the present invention uses Fe3O4 nanoparticles with high magnetic response as the core and ZnO sensitive to pH as the shell. The drug is combined with the nanocomposite material through the gaps in the middle layer of the nanocomposite material with a core-void-shell structure; since the acidity and alkalinity of normal tissue cells are neutral, the ZnO shell can be kept closed to prevent the antibody drug that flows out due to the decomposition of ZnO from damaging normal tissue cells; while the pH of tumor cells is often acidic, ZnO is decomposed when it encounters it, allowing the antibody drug to flow out and accurately act on tumor cells. Moreover, the Fe3O4 nanoparticle composite material can accurately find and reach the location of tumor lesion cells under the guidance of an external magnetic field, which can achieve local drug accumulation and improve the distribution of drugs in the tumor site. Therefore, the drug-loaded Fe3O4 nanoparticle composite preparation of the present invention has targeting and can effectively kill tumor cells without causing damage to normal tissues.

[0033] (2) The drug-loaded Fe3O4 nanoparticle composite preparation disguised by vesicle membrane of the present invention is a composite preparation of drug-loaded Fe3O4 nanoparticles wrapped by vesicle membrane. Since the vesicle membrane is derived from the tumor receptor, it can inherit the immune escape characteristics of the original cell vesicle membrane. Through the vesicle membrane camouflage, it can easily escape the screening and elimination of the immune system in the tumor receptor, thereby greatly increasing its circulation time in the blood, which helps to improve the anti-tumor medical effect.

[0034] (3) The drug-loaded Fe3O4 nanoparticle composite preparation disguised by vesicle membrane of the present invention comprises, from inside to outside, magnetic Fe3O4 nanoparticles, tumor antibody daunorubicin filled in the gaps, ZnO outer shell structure, and vesicle membrane wrapped in the outermost layer. The inherent superparamagnetism of magnetic Fe3O4 nanoparticles can have better tumor magnetic resonance imaging (MRI) and magnetic hyperthermia (MHT) technology, but they are unstable and easy to aggregate. Platelets, as sentinels in the blood, can identify and interact with tumor cells when they metastasize. The present invention can increase its dispersibility and improve its biocompatibility by surface modification of vesicle membranes derived from tumor receptor blood, so that it has contrast signals and can achieve a better effect of automatically tracking tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a schematic diagram of the preparation process of the drug-loaded Fe3O4 nanoparticle composite preparation in Example 1 of the present invention;

[0036] Figure 2 Schematic diagram of the preparation process of the drug-loaded Fe3O4 nanoparticle composite preparation disguised by vesicle membrane in Example 2 of the present invention;

[0037] Figure 3 This is a TEM image of the drug-loaded Fe3O4 nanoparticle composite preparation prepared in Example 1 of the present invention;

[0038] Figure 4 The results of the in vivo experiments in Example 4 of the present invention prove the targeting and camouflage of the drug-loaded Fe3O4 nanoparticle composite preparation disguised by the vesicle membrane;

[0039] Figure 5 The results of the in vitro experiments in Example 4 of the present invention demonstrate the targeting and camouflage of the drug-loaded Fe3O4 nanoparticle composite preparation disguised by the vesicle membrane. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0041] An embodiment of the present invention provides a drug-loaded Fe3O4 nanoparticle composite preparation, including a core-void-shell Fe3O4 nanoparticle composite material and a drug filled in the voids thereof, wherein the core-void-shell Fe3O4 nanoparticle composite material includes a ZnO shell structure, a Fe3O4 nanoparticle core structure, and a void between the two for filling the drug.

[0042] The drug-loaded Fe3O4 nanoparticle composite preparation of the embodiment of the present invention has magnetic Fe3O4 nanoparticles as the core and pH-sensitive ZnO as the shell. The drug is combined with the nanocomposite material through the gap between the ZnO shell structure of the nanocomposite material of the core-void-shell structure and the Fe3O4 nanoparticle core structure.

[0043] The internal trigger factor of the drug-loaded Fe3O4 nanoparticle composite preparation is the pH value. The low toxicity of the nanomaterial ZnO has significant potential in the biomedical field. ZnO will automatically degrade into zinc ions under acidic conditions and exist stably under neutral conditions. Since tumor tissues are often more acidic than normal tissues, this allows the drug antibodies to be accurately released and act on the tumor site, which can improve the targeting of tumor cells and effectively kill tumor cells without causing damage to normal tissues.

[0044] The external trigger factor of the drug-loaded Fe3O4 nanoparticle composite preparation is the magnetic field light irradiation effect. Fe3O4 is a magnetic nanoparticle that has special magnetic properties such as superparamagnetism and high magnetic susceptibility. At the same time, relying on the high chemical stability, low toxicity, good biocompatibility and easy access of magnetic Fe3O4 nanoparticles, the magnetic Fe3O4 nanoparticle composite material carrying drugs can accurately find and reach the location of tumor lesion cells under the guidance of the external magnetic field, which can achieve local drug accumulation and improve the distribution of drugs in the tumor site. In addition, drug-loaded Fe3O4 can generate heat under the influence of hysteresis loss of the alternating magnetic field applied by the outside world, which can reduce drug toxicity and enhance anti-tumor ability. In magnetic resonance imaging technology (MRI), Fe3O4 nanoparticles can also be used as T1 or T2 contrast agents. In addition, Fe3O4 nanoparticles have the advantages of prolonging blood circulation time, fast clearance, and low side effects.

[0045] The core-shell Fe3O4 nanoparticle composite material of the embodiment of the present invention is prepared by a selective etching method, specifically, by a solvent thermal decomposition method, a sol-gel method, a chemical bath deposition method and a selective etching method in sequence.

[0046] Selective etching is a simple, effective and direct method that can directly control the core diameter, void space and shell thickness. Selective etching is used to remove the SiO2 in the middle layer of Fe3O4@SiO2@ZnO to form a core-void-shell structure nanocomposite with significant characteristics such as a movable core, internal void space and shell multifunctionality, which can achieve the delivery of more antibody drugs. The use of porous ZnO as the shell can increase the carrying capacity of drug antibodies and make the drug antibodies more secure through the holes to avoid the loss of antibody drugs due to mechanical force.

[0047] Specifically, the method for preparing the drug-loaded Fe3O4 nanoparticle composite preparation according to the embodiment of the present invention comprises the following steps:

[0048] (1) Preparation of core-shell Fe3O4 nanoparticle composites

[0049] Dissolve 1.2 g to 1.5 g of FeCl36H2O in 35 mL to 45 mL of ethylene glycol to form a suspension, then add 3 g to 4 g of sodium acetate and 0.8 g to 1.2 g of polyethylene glycol to the suspension, continue stirring for 25 min to 35 min, seal the mixture in an autoclave, heat to 180° C. to 220° C. and maintain for 4 h to 6 h, collect the product Fe3O4, wash and dry to obtain Fe3O4 nanoparticles;

[0050] 0.05 g to 0.15 g of the Fe3O4 nanoparticles are soaked in a mixture of 140 to 180 mL of ethanol, 30 to 55 mL of water and 1 to 3 mL of NH3H2O, and after ultrasonic treatment for 20 min to 40 min, 4 mL to 6 mL of an ethanol solution containing 0.8 mL to 1.0 mL of ethyl silicate is slowly added dropwise, stirred vigorously, and reacted at room temperature for 6 h to 10 h, and the synthesized product is harvested by magnetic separation, and washed and dried to obtain a core-shell Fe3O4@SiO2 nanocomposite material;

[0051] 0.05 g to 0.15 g of the core-shell Fe3O4@SiO2 nanocomposite material is dispersed in 80 mL to 120 mL of deionized water, subjected to ultrasonic treatment for 5 min to 15 min, and then vigorously stirred for 15 min to 25 min, followed by adding 0.60 g to 0.76 g of Zn(Ac)2·2H2O, and adjusting the pH of the dispersion to 10 to 13 with an aqueous ammonia solution to obtain Fe3O4@SiO2@ZnO, followed by selective etching in an aqueous ammonia environment, mechanical stirring for 15 min to 25 min, and ultrasonic treatment at 55°C to 65°C for 1 h to 3 h, and finally separating, rinsing and drying the obtained product to obtain a Fe3O4@clear@ZnO nanocomposite material;

[0052] (2) preparing the drug-loaded Fe3O4 nanoparticle composite preparation;

[0053] 2 mL of a drug solution with a concentration of 1 mg / mL is mixed with 4.5 mL to 5.5 mL of the Fe3O4@clear@ZnO nanocomposite dispersion, wherein the dispersion contains 0.01 g of the Fe3O4@clear@ZnO nanocomposite and the solvent is a phosphate buffer solution; ultrasonic oscillation is performed for 2 min to 4 min, the dispersion is transferred to a centrifuge tube, oscillated at 25 to 37° C. for 22 h to 26 h, and the unloaded drug is removed by centrifugation with a phosphate buffer solution, and the supernatant and the washing liquid are collected to obtain the drug-loaded Fe3O4 nanoparticle composite preparation.

[0054] In a preferred embodiment, the drug is a tumor antibody, daunorubicin, doxorubicin, epirubicin or aclarubicin. Considering that too little dosage of the drug antibody has no significant effect and too much dosage causes waste, the weight ratio of the drug to the core-empty-shell Fe3O4 nanoparticle composite material is 1:5.

[0055] Furthermore, an embodiment of the present invention also provides a drug-loaded Fe3O4 nanoparticle composite preparation disguised by a vesicle membrane, comprising the above-mentioned drug-loaded Fe3O4 nanoparticle composite preparation and a vesicle membrane wrapped around the outside thereof.

[0056] Vesicle membranes are collectively referred to as vesicles and their membrane proteins. The vesicle membranes of this embodiment are derived from platelets collected from the blood of tumor-stricken receptors. The above vesicle membranes are coated on the surface of the prepared drug-loaded Fe3O4 nanoparticle composite preparation to enable it to inherit the long blood circulation and tumor targeting properties of the membrane. Platelets, as sentinels in the blood, can respond to damaged sites and invading microorganisms. Platelets can recognize and interact with tumor cells and have the characteristics of avoiding being cleared by the body's immune system and phagocytes. Therefore, this application uses platelet vesicle membranes derived from tumor receptors to camouflage nanoparticle composite materials, which helps to improve the anti-tumor medical effect.

[0057] Specifically, the method for preparing the vesicle membrane is: first purify the vesicles from the blood of tumor receptors, then obtain the preliminary vesicle membrane through hypotonic treatment, repeated freeze-thaw or ultrasonic treatment, and finally obtain the vesicle membrane with a microporous structure by porous extrusion of the preliminary obtained vesicle membrane.

[0058] Among them, hypotonic treatment utilizes the environment in which cells are easily absorbed and burst to achieve the separation of cell membranes; repeated freezing and thawing utilizes the principle of repeatedly freezing and thawing cell membranes to cause physical changes in the cell membranes through repeated changes in temperature and separate them. Through the above repeated treatments, a relatively pure cell membrane product can be obtained; ultrasonic treatment is to gather the collected vesicle cells, separate the vesicle membranes by ultrasonic separation, and finally obtain the vesicle membranes by centrifugation followed by filtration.

[0059] In an embodiment of the present invention, a method for preparing a composite preparation of drug-loaded Fe3O4 nanoparticles disguised by vesicle membranes is also provided, which specifically comprises the following steps:

[0060] The drug-loaded Fe3O4 nanoparticle composite preparation is prepared according to the above method; the drug-loaded Fe3O4 nanoparticle composite preparation and the vesicle membrane are repeatedly extruded through holes of 100nm to 300nm, and the vesicle membrane is wrapped on the drug-loaded Fe3O4 nanoparticle composite preparation to prepare the drug-loaded Fe3O4 nanoparticle composite preparation disguised by the vesicle membrane.

[0061] In order to better understand and illustrate the technical solution and technical effects of the present application, the following specific embodiments are provided:

[0062] Example 1 Preparation of drug-loaded Fe3O4 nanoparticle composite preparation

[0063] Figure 1 Schematic diagram of the preparation process of the drug-loaded Fe3O4 nanoparticle composite preparation in Example 1 of the present invention.

[0064] (1) Preparation and characterization of core-shell Fe3O4 nanoparticle composites

[0065] (a) Preparation of core-shell Fe3O4 nanoparticle composites

[0066] like Figure 1 As shown, in this embodiment, the core-void-shell Fe3O4 nanoparticle composite material is prepared by solvent thermal decomposition method, sol-gel method 1, chemical bath deposition method 2 and selective etching method 3, wherein the solvent thermal reaction is used to prepare magnetic Fe3O4 nanoparticles.

[0067] Specifically, in this embodiment, the preparation method of the core-shell Fe3O4 nanoparticle composite material is as follows:

[0068] 1.35 g of FeCl36H2O was dissolved in 40 mL of ethylene glycol (EG) under magnetic stirring to form a clear yellow solution. Then 3.5 g of sodium acetate (NaAc) and 1 g of polyethylene glycol (PEG) were added to the above suspension and stirred for 30 min. The mixture was sealed in a Teflon-lined stainless steel autoclave (100 mL), heated to 200 °C and maintained for 5 h, the black product Fe3O4 was collected, and finally washed and dried to obtain magnetic Fe3O4 nanoparticles.

[0069] 0.1 g of Fe3O4 was soaked in a mixture of ethanol (160 mL), water (40 mL) and 2 ml of NH3H2O, and then ultrasonically treated for 30 min. Then, 5 mL of ethanol solution containing ethyl silicate (0.9 mL) was slowly added dropwise, stirred vigorously, and reacted at room temperature for 8 h. Finally, the synthesized Fe3O4@SiO2 nanocomposite was harvested by magnetic separation, rinsed with deionized water and ethanol several times, and then vacuum dried at 60 °C for 12 h. This step corresponds to Figure 1 Sol-gel method in 1.

[0070] 0.1 g of core-shell Fe3O4@SiO2 nanocomposite was dispersed in 100 mL of deionized water, ultrasonicated for 10 min, and then vigorously stirred for 20 min. Then, 0.68 g of Zn(Ac)2·2H2O was added to the suspension, and the pH of the dispersion was adjusted to 11.5 with an ammonia solution. The dispersion was mechanically stirred for 20 min, and then ultrasonicated at 60 °C for 2 h. Finally, the obtained Fe3O4@clear@ZnO was separated, rinsed, and dried. This step corresponds to Figure 1 Chemical bath deposition method 2 and selective etching method 3 in.

[0071] (b) TEM morphology characterization of core-void-shell Fe3O4 nanoparticle composites

[0072] The morphology of the prepared core-shell Fe3O4 nanoparticle composite material was characterized by TEM. Figure 3 As shown, the particle size of the prepared drug-loaded Fe3O4 nanoparticle composite preparation is about 380nm, and it can be seen that there is an obvious gap between the core structure of the Fe3O4 nanoparticle and the ZnO shell structure, indicating that SiO2 was successfully etched by aqueous ammonia. The core-void-shell Fe3O4 nanoparticle composite material was successfully prepared by the above method.

[0073] (2) Preparation of drug-loaded Fe3O4 nanoparticle composite preparations

[0074] Take 2 mL of daunorubicin solution (1 mg / mL) and mix it with 5 mL of Fe3O4@clear@ZnO (0.01 g) dispersion (solvent is phosphate buffer), ultrasonically vibrate for 3 min, transfer the dispersion to a centrifuge tube, vibrate at 37 ° C for 24 h, centrifuge with phosphate buffer (PBS) to remove unloaded daunorubicin, and collect the supernatant and washing solution. This step corresponds to Figure 1 The daunorubicin loading in 4.

[0075] Example 2 Preparation of drug-loaded Fe3O4 nanoparticle composite preparation disguised by vesicle membrane

[0076] Figure 2Schematic diagram of the preparation process of the drug-loaded Fe3O4 nanoparticle composite preparation disguised by vesicle membrane in Example 2 of the present invention.

[0077] (1) Preparation of vesicle membrane

[0078] In this embodiment, the preparation of the vesicle membrane includes the following steps:

[0079] Passivate vesicles from the blood of tumor-affected recipients: extract the blood of tumor-affected recipients, freeze it at 4°C, and centrifuge it at 2000r / min for 10 minutes to remove the white matter at the dividing line and the upper serum part. Add pre-cooled phosphate buffer and perform multiple centrifugation washes, and finally remove the supernatant to obtain vesicle cells; then obtain the preliminary vesicle membrane through repeated freezing and thawing; then place the preliminary vesicle membrane on a micro-extruder and use 400nm and 200nm multi-micropores to perform multiple extrusions to remove the liquid substance in the vesicle to prepare a vesicle membrane with a multi-microporous structure. This process corresponds to Figure 2 Step 5 in the embodiment. The hydrodynamic diameter of the vesicle membrane obtained in this embodiment is 300 nm to 400 nm, and the zeta potential is -30 mV to 26 mV.

[0080] (2) Wrapping the vesicle membrane on the outside of the drug-loaded Fe3O4 nanoparticle composite preparation

[0081] The drug-loaded Fe3O4 nanoparticle composite preparation is mixed with the freshly prepared vesicle membrane and repeatedly extruded through 100nm to 300nm holes. The mechanical force applied during the extrusion process encapsulates the vesicle membrane on the drug-loaded Fe3O4 nanoparticle composite preparation to obtain the drug-loaded Fe3O4 nanoparticle composite preparation disguised by the vesicle membrane. This process corresponds to Figure 2 Follow step 6 in the

[0082] Example 3 Safety evaluation of drug-loaded Fe3O4 nanoparticle composite preparation disguised by vesicle membrane

[0083] The drug-loaded Fe3O4 nanoparticle composite preparation disguised by vesicle membrane prepared in Example 2 was injected into 6 C57BL / 6 mice by different methods such as tail vein injection, subcutaneous injection, and intraperitoneal injection. The injection dose is as shown in the following table. After a single injection, the mice were monitored for one week to observe and monitor the death, allergy and infection of the mice. No adverse reactions such as death, allergy, infection, etc. were observed.

[0084] Injection method Injection dosage Number of deaths Number of allergies Number of infections Tail vein injection Equivalent to daunorubicin 2 mg / kg 0 0 0 Subcutaneous injection Equivalent to daunorubicin 4 mg / kg 0 0 0 Intraperitoneal injection Equivalent to daunorubicin 2 mg / kg 0 0 0

[0085] Example 4 Targeting and camouflage effects of drug-loaded Fe3O4 nanoparticle composite preparations camouflaged by vesicle membranes

[0086] (a) In vivo experiments demonstrated the targeting and camouflage properties of the drug-loaded Fe3O4 nanoparticle composite preparation disguised by vesicle membrane:

[0087] Six mice carrying MCF-7 human breast tumor transplants were divided into two groups, with three mice in each group. They were injected with 50 μg of the same amount of drug-loaded Fe3O4 nanoparticle composite preparation without vesicle membrane camouflage (referred to as Fe3O4-clear-ZnO) and drug-loaded Fe3O4 nanoparticle composite preparation disguised by vesicle membrane (referred to as PLT-Fe3O4-clear-ZnO) through the tail vein. The mice were killed 48 hours after the injection, and the main organs (heart, liver, kidney, spleen, pancreas) and tumors of each mouse were removed and quantitatively analyzed using inductively coupled plasma atomic emission spectrometry (ICP-AES). The results are shown in Figure 2. Figure 4 As shown, it was found that the drug accumulation of PLT-Fe3O4-clear-ZnO was the highest in the tumor site, indicating that PLT-Fe3O4-clear-ZnO had better targeting; after ICP-AES quantitative analysis, the accumulation of PLT-Fe3O4-clear-ZnO in major organs was less, indicating that PLT-Fe3O4-clear-ZnO had acquired the immune escape ability provided by the vesicle membrane of platelet-derived cells and had a certain camouflage ability.

[0088] (b) In vitro experiments demonstrated the targeting and camouflage properties of the drug-loaded Fe3O4 nanoparticle composite preparation disguised by the vesicle membrane:

[0089] Equal amounts of PLT-Fe3O4-clear-ZnO, Fe3O4-clear-ZnO and extracted RAW264.7 mouse macrophages and MCF-7 cancer cells were cultured in vitro. After 24 h of culture, the Fe content was measured and recorded using the ICP-AES method. The results are shown in Figure 2. Figure 5 As shown; the results showed that the expression level of PLT-Fe3O4-clear-ZnO was lowest in mouse RAW-264.7 phagocytes and highest in MCF-7 cancer cells, which well proved that PLT-Fe3O4-clear-ZnO has considerable targeting and camouflage properties.

[0090] Example 5 In vitro or in vivo antitumor effects of drug-loaded Fe3O4 nanoparticle composite preparations disguised by vesicle membranes

[0091] (a) In vitro antitumor effect

[0092] Three different tumor cell lines, HUH-7 liver cancer, A549 lung cancer, and Hct-116 human colon cancer, were cultured in vitro, and the drug-loaded daunorubicin-Fe3O4 nanoparticle composite preparation disguised by vesicle membrane was added, and the corresponding daunorubicin was used as a positive drug control to explore its IC50 The values ​​were verified by experiments, Hct-116 human colon cancer cells, the converted daunorubicin IC50: 1.23μM, A549 human lung cancer cells, the converted daunorubicin IC50: 0.87μM, HUH-7 human liver cancer, the converted daunorubicin IC50: 3.42μM. Therefore, the daunorubicin-Fe3O4 nanoparticle composite preparation disguised by the vesicle membrane has a better tumor killing effect.

[0093] (b) Antitumor effect in vivo

[0094] Three different tumor cell lines of liver cancer, lung cancer and intestinal cancer were inoculated subcutaneously into nude mice to construct a tumor model. After the tumor formed a certain volume, the daunorubicin-Fe3O4 nanoparticle composite preparation was injected into the nude mice through the tail vein, and the tumor size was detected every 3 days. At the same time, daunorubicin was used as a positive drug control. Experimental verification found that the tumor volume of the drug-loaded daunorubicin-Fe3O4 nanoparticle composite preparation camouflaged by the vesicle membrane was more significantly reduced, and it had a better tumor-killing effect.

[0095] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A drug-loaded Fe3O4 nanoparticle composite preparation, characterized in that: The invention comprises a core-void-shell Fe3O4 nanoparticle composite material and drugs filled in the voids thereof; the core-void-shell Fe3O4 nanoparticle composite material comprises a ZnO shell structure, a Fe3O4 nanoparticle core structure and a void between the two for filling drugs.

2. The drug-loaded Fe3O4 nanoparticle composite preparation according to claim 1, characterized in that: The core-shell Fe3O4 nanoparticle composite material is prepared by solvent thermal decomposition, sol-gel method, chemical bath deposition method and selective etching method in sequence; FeCl36H2O is decomposed by a solvent thermal decomposition method to prepare Fe3O4 nanoparticles; a SiO2 shell layer is coated on the surface of the prepared Fe3O4 nanoparticles by hydrolysis of ethyl silicate by a sol-gel method to prepare Fe3O4@SiO2; a layer of ZnO is deposited on the surface of the prepared Fe3O4@SiO2 by a chemical bath deposition method to obtain Fe3O4@SiO2@ZnO; and a SiO2 layer of the Fe3O4@SiO2@ZnO middle layer is etched by a selective etching method using aqueous ammonia to obtain Fe3O4@clear@ZnO, thereby obtaining the core-void-shell Fe3O4 nanoparticle composite material.

3. The drug-loaded Fe3O4 nanoparticle composite preparation according to claim 1, characterized in that: The drug is tumor antibody daunorubicin, doxorubicin, epirubicin or aclarubicin, and the weight ratio of the drug to the core-empty-shell Fe3O4 nanoparticle composite material is 1:

5.

4. A method for preparing a drug-loaded Fe3O4 nanoparticle composite preparation, used for preparing the drug-loaded Fe3O4 nanoparticle composite preparation according to any one of claims 1 to 3, characterized in that: The steps include: (1) Preparation of core-shell Fe3O4 nanoparticle composites Dissolve 1.2 g to 1.5 g of FeCl36H2O in 35 mL to 45 mL of ethylene glycol to form a suspension, then add 3 g to 4 g of sodium acetate and 0.8 g to 1.2 g of polyethylene glycol to the suspension, continue stirring for 25 min to 35 min, seal the mixture in an autoclave, heat to 180° C. to 220° C. and maintain for 4 h to 6 h, collect the product Fe3O4, wash and dry to obtain Fe3O4 nanoparticles; 0.05 g to 0.15 g of the Fe3O4 nanoparticles are soaked in a mixture of 140 to 180 mL of ethanol, 30 to 55 mL of water and 1 to 3 mL of NH3H2O, and after ultrasonic treatment for 20 min to 40 min, 4 mL to 6 mL of an ethanol solution containing 0.8 mL to 1.0 mL of ethyl silicate is slowly added dropwise, stirred vigorously, and reacted at room temperature for 6 h to 10 h, and the synthesized product is harvested by magnetic separation, and washed and dried to obtain a core-shell Fe3O4@SiO2 nanocomposite material; 0.05 g to 0.15 g of the core-shell Fe3O4@SiO2 nanocomposite material is dispersed in 80 mL to 120 mL of deionized water, subjected to ultrasonic treatment for 5 min to 15 min, and then vigorously stirred for 15 min to 25 min, followed by adding 0.60 g to 0.76 g of Zn(Ac)2·2H2O, and adjusting the pH of the dispersion to 10 to 13 with an aqueous ammonia solution to obtain Fe3O4@SiO2@ZnO, followed by selective etching in an aqueous ammonia environment, mechanical stirring for 15 min to 25 min, and ultrasonic treatment at 55°C to 65°C for 1 h to 3 h, and finally separating, rinsing and drying the obtained product to obtain a Fe3O4@clear@ZnO nanocomposite material; (2) preparing the drug-loaded Fe3O4 nanoparticle composite preparation; 2 mL of 1 mg / mL daunorubicin solution, doxorubicin solution, epirubicin solution or aclarubicin solution is mixed with 4.5 mL to 5.5 mL of the Fe3O4@clear@ZnO nanocomposite dispersion, wherein the dispersion contains 0.01 g of the Fe3O4@clear@ZnO nanocomposite, and the solvent is phosphate buffer; ultrasonic oscillation is performed for 2 min to 4 min, the dispersion is transferred to a centrifuge tube, oscillated at 25 to 37° C. for 22 h to 26 h, centrifuged with phosphate buffer to remove unloaded drugs, and the supernatant and washing liquid are collected to obtain the drug-loaded Fe3O4 nanoparticle composite preparation.

5. A drug-loaded Fe3O4 nanoparticle composite preparation disguised by vesicle membrane, characterized in that: It comprises the drug-loaded Fe3O4 nanoparticle composite preparation as described in any one of claims 1 to 3 and a vesicle membrane wrapped on the outside thereof.

6. A method for preparing a composite preparation of drug-loaded Fe3O4 nanoparticles disguised by vesicle membranes as claimed in claim 5, characterized in that: The steps include: (1) Preparation of core-shell Fe3O4 nanoparticle composites Dissolve 1.2 g to 1.5 g of FeCl36H2O in 35 mL to 45 mL of ethylene glycol to form a suspension, then add 3 g to 4 g of sodium acetate and 0.8 g to 1.2 g of polyethylene glycol to the suspension, continue stirring for 25 min to 35 min, seal the mixture in an autoclave, heat to 180° C. to 220° C. and maintain for 4 h to 6 h, collect the product Fe3O4, wash and dry to obtain Fe3O4 nanoparticles; 0.05 g to 0.15 g of the Fe3O4 nanoparticles are soaked in a mixture of 140 to 180 mL of ethanol, 30 to 55 mL of water and 1 to 3 mL of NH3H2O, and after ultrasonic treatment for 20 min to 40 min, 4 mL to 6 mL of an ethanol solution containing 0.8 mL to 1.0 mL of ethyl silicate is slowly added dropwise, stirred vigorously, and reacted at room temperature for 6 h to 10 h, and the synthesized product is harvested by magnetic separation, and washed and dried to obtain a core-shell Fe3O4@SiO2 nanocomposite material; 0.05 g to 0.15 g of the core-shell Fe3O4@SiO2 nanocomposite material is dispersed in 80 mL to 120 mL of deionized water, subjected to ultrasonic treatment for 5 min to 15 min, and then vigorously stirred for 15 min to 25 min, followed by adding 0.60 g to 0.76 g of Zn(Ac)2·2H2O, and adjusting the pH of the dispersion to 10 to 13 with an aqueous ammonia solution to obtain Fe3O4@SiO2@ZnO, followed by selective etching in an aqueous ammonia environment, mechanical stirring for 15 min to 25 min, and ultrasonic treatment at 55°C to 65°C for 1 h to 3 h, and finally separating, rinsing and drying the obtained product to obtain a Fe3O4@clear@ZnO nanocomposite material; (2) preparing the drug-loaded Fe3O4 nanoparticle composite preparation; 2 mL of 1 mg / mL daunorubicin solution, doxorubicin solution, epirubicin solution or aclarubicin solution is mixed with 4.5 mL to 5.5 mL of the Fe3O4@clear@ZnO nanocomposite dispersion, wherein the dispersion contains 0.01 g of the Fe3O4@clear@ZnO nanocomposite, and the solvent is phosphate buffer; ultrasonically oscillate for 2 min to 4 min, transfer the dispersion to a centrifuge tube, oscillate at 25 to 37° C. for 22 h to 26 h, centrifuge with phosphate buffer to remove unloaded drugs, collect the supernatant and washing liquid, and obtain the drug-loaded Fe3O4 nanoparticle composite preparation; (3) Preparation of drug-loaded Fe3O4 nanoparticle composite formulations disguised by vesicle membranes; The drug-loaded Fe3O4 nanoparticle composite preparation is mixed with a vesicle membrane, and is repeatedly extruded through holes of 100nm to 300nm to wrap the vesicle membrane on the drug-loaded Fe3O4 nanoparticle composite preparation, thereby preparing the drug-loaded Fe3O4 nanoparticle composite preparation disguised by the vesicle membrane.

7. The method for preparing the drug-loaded Fe3O4 nanoparticle composite preparation disguised by vesicle membrane according to claim 6, characterized in that: The preparation of the vesicle membrane comprises the following steps: The vesicles are purified from the blood of tumor recipients, and then subjected to hypotonic treatment, repeated freeze-thaw or ultrasonic treatment to preliminarily obtain the vesicle membrane. The preliminarily obtained vesicle membrane is then placed on a micro-extruder and extruded multiple times using 400 nm and 200 nm micropores in sequence to obtain the vesicle membrane having a microporous structure.

8. The method for preparing the drug-loaded Fe3O4 nanoparticle composite preparation disguised by vesicle membrane according to claim 6 or 7, characterized in that: The hydrodynamic diameter of the vesicle membrane is 300nm to 400nm, and the zeta potential is -30mV to 26mV.

9. Use of a drug-loaded Fe3O4 nanoparticle composite preparation disguised by a vesicle membrane in the preparation of an anti-tumor drug, using the drug-loaded Fe3O4 nanoparticle composite preparation disguised by a vesicle membrane as claimed in claim 5, or the drug-loaded Fe3O4 nanoparticle composite preparation disguised by a vesicle membrane is prepared according to the preparation method of the drug-loaded Fe3O4 nanoparticle composite preparation disguised by a vesicle membrane as claimed in any one of claims 6 to 8, characterized in that: The tumors include breast tumors, lung cancer tumors, and malignant lymphomas.

Images