Nanoparticles and their preparation method and application

Abstract

The present invention provides a nanoparticle and a preparation method and application thereof, and relates to the technical field of pharmaceutical preparations. The nanoparticle comprises a nano self-assembled particle and a homotypic tumor cell membrane; wherein the nano self-assembled particle is mainly prepared by self-assembly of digoxin and doxorubicin, and the prepared nano self-assembled particle does not need to introduce a carrier material, and can achieve the effect of high drug loading; at the same time, the surface of the nano self-assembled particle of the present application is coated with a homotypic tumor cell membrane, and since the surface of the tumor cell membrane has a variety of antigens, the nanoparticle of the present application has the characteristics of immune escape and homotypic binding with tumor cells, thereby improving its tumor targeting and escaping the host immune system. Therefore, the nanoparticle prepared by the present application has the advantages of high drug loading and strong tumor targeting, and while improving the existing anti-tumor treatment effect, it has stronger targeting and can effectively reduce the toxicity of the drug to normal tissues.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical preparations, and in particular to a nanoparticle and a preparation method and application thereof. Background Art

[0002] Cancer has become one of the most serious public health problems in the world. With the continuous development of molecular diagnosis and treatment, the diagnosis and treatment of cancer have been greatly improved in the past 10 years. The current treatment methods mainly include radiotherapy, chemotherapy, surgery, gene targeted therapy, etc.

[0003] Many genotoxic anticancer drugs, such as doxorubicin, attack DNA and produce multiple DNA damages on tumor cells to exert their anticancer effects. However, tumor cells can clear DNA damage caused by genotoxic anticancer drugs through the DNA damage repair (DDR) pathway. Therefore, inhibiting DNA damage repair may enhance the anticancer effect of genotoxic anticancer drugs. It has been reported that inhibiting DNA damage repair can make tumor cells sensitive to doxorubicin. Digoxin, as the main drug for the treatment of heart failure for more than two centuries, has been shown to inhibit the repair of double-strand breaks in DNA. Since cancer chemotherapy is often hindered by rapid DNA damage repair (DDR) during and after treatment, DDR targeting has become a powerful supplement to conventional chemotherapy. Studies have shown that digoxin can exert its anti-tumor effect by inhibiting the repair of double-strand and single-strand breaks in DNA.

[0004] However, the concentration range of digoxin in clinical treatment is very narrow (0.5-2.0 ng / mL), and exceeding the therapeutic range will cause toxicity. On the other hand, as a genotoxic anticancer drug, doxorubicin lacks tumor targeting and has cardiotoxicity, which limits its clinical application. Although previous studies have shown that the combined use of digoxin and doxorubicin has a synergistic effect in the treatment of cancer both in vivo and in vitro, its actual therapeutic effect is not ideal.

[0005] Therefore, it is necessary and urgent to research and develop a nanoparticle with the advantages of high drug loading and strong tumor targeting, which can effectively improve the narrow therapeutic window of existing digoxin and the lack of tumor targeting of doxorubicin, and improve the treatment effect of cancer. However, no reports have been seen so far.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The object of the present invention is to provide a nanoparticle, which has the advantages of high drug loading and strong tumor targeting. While improving the existing anti-tumor treatment effect, it has stronger targeting and can effectively reduce the toxicity of the drug to normal tissues; and thus the nanoparticle can be widely used in the preparation process of cancer treatment drugs.

[0008] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0009] In a first aspect, the present invention provides a nanoparticle, wherein the nanoparticle comprises a nano self-assembled particle and a homotypic tumor cell membrane;

[0010] The nano self-assembled particles are mainly made of digoxin and doxorubicin;

[0011] The homologous tumor cell membrane is coated on the surface of the nano self-assembled particles.

[0012] In a second aspect, the present invention provides a method for preparing the above-mentioned nanoparticles, the preparation method comprising the following steps:

[0013] (A) Preparation of self-assembled nanoparticles using precipitation method;

[0014] (B) providing homologous tumor cell membranes, mixing the homologous tumor cell membranes with the nano self-assembled particles prepared in step (A), and then extruding them in stages to obtain nanoparticles.

[0015] In a third aspect, the present invention provides a use of the above-mentioned nanoparticles in the preparation of drugs for treating cancer.

[0016] Preferably, the cancer is non-small cell lung cancer.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The nanoparticles provided by the present invention include nano self-assembled particles and homotypic tumor cell membranes; wherein the nano self-assembled particles are mainly prepared by self-assembly of digoxin and doxorubicin, and the prepared nano self-assembled particles do not need to introduce carrier materials (equivalent to a drug loading rate of 100%), and can achieve the effect of high drug loading; at the same time, the surface of the nano self-assembled particles of the present application is coated with homotypic tumor cell membranes, and because the surface of the homotypic tumor cell membrane has a variety of antigens, the nanoparticles of the present application have the characteristics of immune escape and homotypic binding with tumor cells, thereby improving its tumor targeting and escaping the host immune system. Therefore, the nanoparticles prepared by the present application have the advantages of high drug loading and strong tumor targeting, and while improving the existing anti-tumor treatment effect, have stronger targeting, and can effectively reduce the toxicity of drugs to normal tissues.

[0019] The present invention provides a method for preparing nanoparticles, wherein the method comprises: first preparing nano self-assembled particles by precipitation method; then mixing the same type of tumor cell membrane with the nano self-assembled particles, and then extruding them in stages to obtain nanoparticles. The method has the advantages of simple preparation process and easy operation.

[0020] The nanoparticles provided by the present invention can be widely used in the preparation process of cancer treatment drugs, especially in the preparation process of non-small cell lung cancer treatment drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 A Tyndall effect diagram of the nanoparticles and nano self-assembled particles of the present invention provided in Example 4 of the present invention;

[0023] Figure 2 Transmission electron microscope observation image of the nanoparticles and nano self-assembled particles of the present invention provided in Example 4 of the present invention;

[0024] Figure 3 This is a graph showing the inhibitory effect of the nanoparticles and nano self-assembled particles of the present invention on the proliferation of A549 cells provided in Example 5 of the present invention;

[0025] Figure 4 This is a graph showing the effect of the nanoparticles and nano self-assembled particles of the present invention on the apoptosis induction rate of A549 cells provided in Example 5 of the present invention;

[0026] Figure 5 A fluorescence microscope observation diagram of a zebrafish experiment of the nanoparticles and nano self-assembled particles of the present invention provided in Example 6 of the present invention;

[0027] Figure 6 This is a tumor growth observation diagram of the nanoparticles and nano self-assembled particles of the present invention in Example 6 of the present invention in a nude mouse experiment;

[0028] Figure 7 This is a graph showing the effect of the nanoparticles and nano self-assembled particles of the present invention provided in Example 6 of the present invention on the tumor weight of nude mice;

[0029] Figure 8 This is a diagram showing the effect of tumor volume on nude mice experiments using the nanoparticles and nano self-assembled particles of the present invention provided in Example 6 of the present invention. DETAILED DESCRIPTION

[0030] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] According to one aspect of the present invention, a nanoparticle comprises a nano self-assembled particle and a homotypic tumor cell membrane;

[0032] The nano self-assembled particles are mainly made of digoxin and doxorubicin;

[0033] The homologous tumor cell membrane is coated on the surface of the nano self-assembled particles.

[0034] The nanoparticles provided by the present invention include nano self-assembled particles and homotypic tumor cell membranes; wherein the nano self-assembled particles are mainly prepared by self-assembly of digoxin and doxorubicin, and the prepared nano self-assembled particles do not need to introduce carrier materials (equivalent to a drug loading rate of 100%), and can achieve the effect of high drug loading; at the same time, the surface of the nano self-assembled particles of the present application is coated with homotypic tumor cell membranes, and because the surface of the homotypic tumor cell membrane has a variety of antigens, the nanoparticles of the present application have the characteristics of immune escape and homotypic binding with tumor cells, thereby improving its tumor targeting and escaping the host immune system. Therefore, the nanoparticles prepared by the present application have the advantages of high drug loading and strong tumor targeting, and while improving the existing anti-tumor treatment effect, have stronger targeting, and can effectively reduce the toxicity of drugs to normal tissues.

[0035] In a preferred embodiment of the present invention, the hydrated particle size of the nanoparticles is 150-200 nm; the hydrated particle size of the nano self-assembled particles is 100-150 nm.

[0036] As a preferred embodiment, nanotechnology, as a rapidly developing emerging research field, provides an opportunity for a breakthrough in specific anti-tumor immunotherapy. The nano drug delivery system itself has the characteristics of targeting, slow and controlled release, transdermal, physical response, and long circulation, which can overcome the disadvantages of poor stability, short pharmacological action time, low bioavailability, and large toxic and side effects of traditional drugs. Since nanoparticles have a physical size of suitable size, they will produce an enhanced permeation and retention effect (abbreviated as EPR effect) in tumor blood vessels. The preparation of the drug of the present application as nano-scale nanoparticles has the effect of reducing the nanoparticles reaching normal tissues and increasing the accumulation of nanoparticles in the pathological site of tumor cells, thereby effectively improving the targeting function of the nanoparticles of the present application. At the same time, nanoparticles accumulate in the tumor site in vivo through active or passive targeting, which can reduce the drug dosage and reduce the toxic and side effects of drugs on normal tissue cells. Therefore, the nanoparticles with a hydrated particle size of 150 to 200 nm and the nano self-assembled particles with a hydrated particle size of 100 to 150 nm prepared in the present application can effectively reduce the toxicity of drugs to normal tissues while having stronger tumor targeting.

[0037] Note: The EPR effect, or enhanced permeability and retention effect of solid tumors, refers to the property that molecules or particles of certain sizes tend to accumulate in tumor tissues relative to normal tissues. The microvascular endothelial gaps in normal tissues are dense and structurally intact, and macromolecules and lipid particles are not easy to penetrate the vascular wall. However, solid tumor tissues are rich in blood vessels, with wide gaps in the vascular wall, poor structural integrity, and lack of lymphatic return, resulting in selective high permeability and retention of macromolecules and lipid particles. This phenomenon is called the enhanced permeability and retention effect of solid tumor tissues, or EPR effect for short.

[0038] In a preferred embodiment of the present invention, the homotypic tumor cell membrane comprises a non-small cell lung cancer cell membrane, preferably an A549 tumor cell membrane.

[0039] Preferably, the doxorubicin is doxorubicin hydrochloride.

[0040] According to one aspect of the present invention, a method for preparing the above-mentioned nanoparticles comprises the following steps:

[0041] (A) Preparation of self-assembled nanoparticles using precipitation method;

[0042] (B) providing homologous tumor cell membranes, mixing the homologous tumor cell membranes with the nano self-assembled particles prepared in step (A), and then extruding them in stages to obtain nanoparticles.

[0043] The present invention provides a method for preparing nanoparticles, wherein the method comprises: first preparing nano self-assembled particles by precipitation method; then mixing the same type of tumor cell membrane with the nano self-assembled particles prepared in step (A), and then extruding the nanoparticles in stages to obtain the nanoparticles. The above method has the advantages of simple preparation process and easy operation.

[0044] In a preferred embodiment of the present invention, the step (A) comprises the following steps:

[0045] (a), dissolving digoxin in an organic solvent to obtain a digoxin solution;

[0046] (b), dissolving doxorubicin in deionized water to obtain a doxorubicin solution;

[0047] (c) Under stirring conditions, the digoxin solution obtained in step (a) is first injected into deionized water to obtain solution A; then the doxorubicin solution obtained in step (b) is added to solution A and mixed to obtain nano self-assembled particles.

[0048] In a preferred embodiment of the present invention, the organic solution in step (a) comprises any one of anhydrous ethanol, methanol, isopropanol or n-butanol, preferably anhydrous ethanol;

[0049] Preferably, when the organic solution in step (a) is anhydrous ethanol, the molar concentration of the digoxin solution is 0.1 to 2 mmol / L, preferably 1 mmol / L;

[0050] Preferably, the molar concentration of the doxorubicin solution in step (b) is 0.1 to 10 mmol / L, preferably 1 mmol / L;

[0051] In a preferred embodiment of the present invention, the molar concentration of digoxin in solution A in step (c) is 0.1 mmol / L;

[0052] Preferably, in step (c), the volume ratio of solution A to doxorubicin solution is 18 to 20:1, preferably 19:1;

[0053] Preferably, the reaction conditions for stirring in step (c) at least satisfy at least one of the following:

[0054] The stirring speed is 1000-1200 r / min, the stirring temperature is 48-52°C, and the stirring time is 0.8-1.5 h;

[0055] More preferably, the reaction conditions of stirring in step (c) at least satisfy at least one of the following:

[0056] The stirring speed is 1000 r / min, the stirring temperature is 50°C, and the stirring time is 1 h;

[0057] In a preferred embodiment of the present invention, the step (B) comprises the following steps:

[0058] (d), providing a solution containing homotypic tumor cell membranes, and then mixing the homotypic tumor cell membranes with the nano self-assembled particles prepared in step (c) to obtain a solution B;

[0059] (e) using a liposome extruder, extruding solution B through a filter membrane with a pore size of 200 to 400 nm for 3 to 6 cycles to obtain nanoparticles;

[0060] Preferably, the content of homotype tumor cell membrane in the homotype tumor cell membrane solution in step (d) is 0.1 mg / mL;

[0061] Preferably, the volume ratio of the solution containing homotype tumor cell membranes in step (d) to the nano self-assembled particles prepared in step (c) is 1-10:100-500.

[0062] In a preferred embodiment of the present invention, the preparation method comprises the following steps:

[0063] (a) dissolving digoxin in anhydrous ethanol to obtain a digoxin solution with a molar concentration of 0.1 to 2 mmol / L;

[0064] (b) dissolving doxorubicin in deionized water to obtain a doxorubicin solution having a molar concentration of 0.1 to 10 mmol / L;

[0065] (c), under stirring conditions of a stirring speed of 1000-1200 r / min and a temperature of 48-52° C., first injecting the digoxin solution obtained in step (a) into deionized water to obtain a solution A having a digoxin molar concentration of 0.1 mmol / L; then adding the doxorubicin solution obtained in step (b) into the solution A and stirring for 0.8-1.5 h to obtain nano self-assembled particles; the volume ratio of the solution A to the doxorubicin solution is 18-20:1;

[0066] (d), providing a solution containing homotypic tumor cell membranes at a concentration of 0.1 mg / mL, and then mixing the homotypic tumor cell membranes with the nano self-assembled particles prepared in step (c) at a volume ratio of 1 to 10: 100 to 500 to obtain a solution B;

[0067] (e) using a liposome extruder, extruding solution B through a filter membrane with a pore size of 400 nm for 3 to 6 cycles, and then extruding through a filter membrane with a pore size of 200 nm for 3 to 6 cycles to obtain nanoparticles;

[0068] Preferably, the preparation method comprises the following steps:

[0069] (a) dissolving digoxin in anhydrous ethanol to obtain a digoxin solution with a molar concentration of 1 mmol / L;

[0070] (b) dissolving doxorubicin in deionized water to obtain a doxorubicin solution with a molar concentration of 1 mmol / L;

[0071] (c) under stirring conditions of a stirring speed of 1000 r / min and a temperature of 50° C., first injecting the digoxin solution obtained in step (a) into deionized water to obtain a solution A with a digoxin molar concentration of 0.1 mmol / L; then adding the doxorubicin solution obtained in step (b) into the solution A, stirring for 1 hour, to obtain nano self-assembled particles; the volume ratio of the solution A to the doxorubicin solution is 19:1;

[0072] (d), providing a solution containing homotypic tumor cell membranes at a concentration of 0.1 mg / mL, and then mixing the homotypic tumor cell membranes with the nano self-assembled particles prepared in step (c) at a volume ratio of 1 to 10: 100 to 500 to obtain a solution B;

[0073] (e) Using a liposome extruder, solution B was extruded through a filter membrane with a pore size of 400 nm for 3 cycles, and then through a filter membrane with a pore size of 200 nm for 3 cycles to obtain nanoparticles.

[0074] According to one aspect of the present invention, the above nanoparticles are used in the preparation of drugs for treating cancer.

[0075] Preferably, the cancer is non-small cell lung cancer.

[0076] The nanoparticles provided by the present invention can be widely used in the preparation process of cancer treatment drugs, especially in the preparation process of non-small cell lung cancer treatment drugs.

[0077] The technical solution of the present invention will be further described below in conjunction with embodiments.

[0078] Example 1 Extraction of A549 cell membrane

[0079] A method for extracting A549 cell membrane comprises the following steps:

[0080] (1) Preparation of reagents: Dissolve the membrane protein extraction reagent at room temperature, mix well, take an appropriate amount of the membrane protein extraction reagent and place it on ice. Before use, add PMSF (phenylmethylsulfonyl fluoride) to the membrane protein extraction reagent so that the final concentration of PMSF in the membrane protein extraction reagent is 1 mmol / L.

[0081] (2) Cell preparation: Select A549 cells in the logarithmic growth phase and in good condition, wash once with PBS, scrape the cells, collect the cells by centrifugation, discard the supernatant, and keep the cell pellet for later use. Wash the cells twice with ice PBS and count them. Take 1 mL of membrane protein extraction reagent with PMSF added, add 2×10 7 Mix gently and place on ice for 10 to 15 minutes.

[0082] (3) Cell disruption: Use the freeze-thaw method to disrupt cells. Repeat the freeze-thaw method for the sample in step 2 for three times in a 37°C water bath and in liquid nitrogen. Take a small amount of sample and observe the cell state under a microscope. If the degree of cell disruption is not enough, increase the number of freeze-thaw cycles until the degree of cell disruption is greater than 70%.

[0083] (4) Remove cell nuclei and unbroken cells: Centrifuge at 700 g for 10 min at 4°C. Carefully collect the supernatant and place it in a new centrifuge tube. Do not touch the precipitate.

[0084] (5) Precipitate cell membrane fragments: Centrifuge at 14000 g for 30 min at 4°C to precipitate the cell membrane fragments, add 20 μL DW and mix well for later use, and extract the A549 cell membrane.

[0085] The membrane protein extraction reagent is: Biyuntian, P0033.

[0086] Example 2 Preparation of Nano Self-Assembled Particles

[0087] A method for preparing nano self-assembled particles comprises the following steps:

[0088] (a), dissolving digoxin in anhydrous ethanol to prepare a solution with a final digoxin concentration of 1 mmol / L, and then filtering through a 0.22 μM microporous filter membrane to obtain a 1 mmol / L digoxin anhydrous ethanol solution;

[0089] (b) Add 17 mL of ultrapure water and a stirring bar into an eggplant-shaped bottle and preheat it in a 50°C water bath; under magnetic stirring at 1000 r / min, slowly and uniformly add 2 mL of a fully dissolved 1 mmol / L digoxin anhydrous ethanol solution using a syringe pump, open the lid and continue stirring for 5 min to obtain solution A;

[0090] (c) Dissolve doxorubicin hydrochloride in deionized water to prepare a 1 mmol / L doxorubicin hydrochloride (ADR-HCl) aqueous solution, then add 1 mL of the 1 mmol / L ADR-HCl aqueous solution to solution A, close the lid and continue stirring for 1 hour to obtain nano self-assembled particles.

[0091] Example 3 Preparation of Nanoparticles

[0092] A method for preparing nanoparticles comprises the following steps:

[0093] (d) Collect 4 mL of the nano self-assembled particles obtained in Example 2 using a 5 mL centrifuge tube, then add 12 μL of the A549 cell membrane solution obtained in Example 1, and blow evenly with a pipette to obtain Solution B.

[0094] (e) After cleaning the liposome extruder with ultrapure water, the solution B was extruded through a filter membrane with a pore size of 400 nm for at least 3 cycles using the liposome extruder, and then extruded through a filter membrane with a pore size of 200 nm for at least 3 cycles to obtain nanoparticles.

[0095] Example 4 Characterization of Nanoparticles

[0096] This example performs basic characterization on the nano self-assembled particles prepared in Example 2 and the nanoparticles prepared in Example 3. The specific method is as follows:

[0097] (1) Tyndall effect: The prepared nano-self-assembled particles and nanoparticles are placed in an absorption cell and illuminated by a laser pen to observe the Tyndall effect;

[0098] The Tyndall effect is an important part of colloid chemistry. Some important concepts in nanomaterials are based on the classical theory and mature ideas of colloid chemistry. Colloid, as a dispersed system, consists of two parts: dispersant and dispersoid. When the particle size of the dispersoid is in the range of 1 to 100 nm, the dispersed system is called a colloid. When nanoparticles are dispersed in a liquid phase system, a lyosol is formed. When the particle size is smaller than the visible wavelength (400 to 700 nm), the particles are highly dispersed in the lyosol and have a significant scattering effect, namely the Tyndall effect.

[0099] like Figure 1 As shown, the nano self-assembled particles (DANPs) prepared in Example 2 and the nanoparticles (DAMNPs) prepared in Example 3 both showed the Tyndall phenomenon, and the solution of digoxin plus doxorubicin hydrochloride (D+A) showed no light scattering phenomenon.

[0100] (2) Transmission electron microscopy (TEM) observation: The self-assembled nanoparticles prepared in Example 2 and the nanoparticles prepared in Example 3 were observed using a transmission electron microscope (TEM). Transmission electron microscopy (TEM) is a common and intuitive method for observing the size and morphology of nanoparticles.

[0101] like Figure 2As shown in the transmission electron microscopy photos, it can be seen that the dispersibility of DANPs and DAMNPs is relatively good. The nano self-assembled particles (DANPs) prepared in Example 2 are spherical solid particles with uniform particle size distribution, and the particle size is about 100nm to 150nm. The nanoparticles (DAMNPs) prepared in Example 3 S ) has a larger particle size, and it is obvious that the A549 tumor cell membrane with stronger light transmittance wraps the DANPs with weaker light transmittance inside the particles.

[0102] Example 5 In vitro anti-tumor experiment of nanoparticles

[0103] (I) In order to evaluate the nanoparticles (DAMNP S ) has an inhibitory effect on the proliferation of A549 cells. We used the MTT method to detect cell survival rate. The MTT method mainly uses the succinate dehydrogenase in the mitochondria of living cells to reduce MTT to formazan, which is blue-purple crystals, insoluble in water and deposited in cells. Dead cells will not have this reaction. DMSO can dissolve formazan, and the optical density value can be measured by a microplate reader to indirectly detect cell proliferation and survival. This detection method is low-cost, easy to operate, and highly sensitive.

[0104] Therefore, we used MTT method to detect the effect of digoxin plus doxorubicin hydrochloride (D+A), DANPs, and DAMNPs on the proliferation of A549 cells. The three groups of D+A, DANPs, and DAMNPs were given drugs according to the drug concentration gradient, and the ratio of digoxin (DIG) to doxorubicin (ADR) was 2:1.

[0105] The results are as follows Figure 3 As shown in the figure, 72h after administration, the cytotoxic effect of DAMNPs on A549 cells was dose-dependent. When the drug concentration was low, there was no significant difference in the proliferation inhibition of A549 cells among the three groups, but with the increase of the drug concentration, the proliferation inhibition of DAMNPs was significantly enhanced when the DIG concentration was 0.5μM and the ADR concentration was 0.25μM compared with the other two groups. The results show that the inhibitory effect of DAMNPs on A549 cells is dose-dependent.

[0106] (II) Cell apoptosis refers to the autonomous and orderly cell death through gene regulation in order to ensure that the cells can carry out their own life activities normally. The occurrence of apoptosis is an active death process chosen to better adapt to the living environment. Many studies have shown that inducing tumor cell apoptosis can effectively treat cancer, and a variety of drugs can inhibit tumor cell proliferation by inducing tumor cell apoptosis.

[0107] In order to explore whether the inhibitory effect of D+A, DANPs, and DAMNPs on A549 cell proliferation is caused by cell apoptosis, this example uses APC single staining method to detect the effects of D+A, DANPs, and DAMNPs on A549 cell apoptosis.

[0108] The results are as follows Figure 4 As shown, the apoptosis induction rates of A549 cells in the D+A group, DANPs group, and DAMNPs group were 9.4%, 14.7%, and 17.2%, respectively, which were significantly higher than 5.5% in the control group, among which the apoptosis rate induced by DAMNPs was the highest. The experimental results were tested by t-test using GraphPad Prism 7 software, and the apoptotic cell numbers in the D+A group, DANPs group, and DAMNPs group were significantly different from those in the control group (control).

[0109] Example 6 In vivo anti-tumor experiment of nanoparticles

[0110] (I) Zebrafish experiment:

[0111] (1) Zebrafish breeding: The species used in the experiment was wild-type AB zebrafish, and the circulating water used was at a temperature of (28.5±0.5)℃, a pH value of about 7.2, and a total water hardness of 62 mg / L (measured in terms of CaCO 3 The conductivity was 485μS. The light treatment time was 14h and the night treatment was 10h. Zebrafish were fed with Artemia once in the morning and evening.

[0112] (2) Establishment of zebrafish tumor model

[0113] 1. Obtain zebrafish embryos: The night before the experiment, separate the adult AB zebrafish male and female into a breeding tank at a ratio of 1:1, and place a draw plate in the middle to separate the male and female fish. Draw the plate at 8:00am the next day, and collect the eggs in time 15 minutes after spawning. Rinse the eggs with E3 culture medium, and remove the dead eggs to avoid affecting the healthy eggs. Then place them in a 10cm culture dish, add culture medium containing 0.2mmol / L PTU, and place them in a 32℃ incubator for 48h.

[0114] 2. Preparation of A549 cell suspension: Select A549 cells in good cell condition and in the logarithmic growth phase, wash with PBS, digest with trypsin, and prepare cell suspension with PBS. Then add DIO dye and incubate in 37℃ incubator for 20min, wash both sides with PBS, and prepare 5×10 6 / mL cell suspension.

[0115] 3. Zebrafish embryo microinjection: Zebrafish embryos were anesthetized with 1.2mmol / L tricaine at 48h, and then placed on 2% agarose gel. Use a microinjector to inject about 20nL of cell suspension into the yolk sac of each zebrafish embryo, which contains about 100 cells. After cell injection, the embryos were screened using a fluorescence microscope. Embryos with basically the same fluorescence intensity and cell area size were selected for subsequent experiments. The embryos were transferred to a 24-well plate, 2mL of E3 culture medium containing different drugs was added to each well, and cultured in a 32°C incubator for 48h.

[0116] 4. Grouping and administration method: The embryos were divided into four groups, namely control group, 1.4μM DIG+0.7μM ADR group, DANPs group (1.4μM DIG+0.7μM ADR), and DAMNPs group (1.4μM Digoxin+0.7μM ADR). The administration method was drug immersion method, and the administration lasted for 48 hours. After 48 hours, the fluorescence intensity of A549 cells in zebrafish was observed using a fluorescence microscope.

[0117] The results are as follows Figure 5 As shown, after 48 hours of exposure to fluorescence microscopy, it can be seen that the fluorescence intensity of the tumor in zebrafish in the D+A group, DANPs group and DAMNPs group was significantly reduced compared with the control group, indicating that the three groups can effectively inhibit the proliferation of tumor cells in zebrafish. Compared with the D+A group and DANPs group, the fluorescence intensity of the tumor in DAMNPs was also significantly reduced. Figure 5 The horizontal axis in the middle is the zebrafish photographed under bright field (Bright field) and the DIO group (cell membrane staining) photographed under fluorescence. The first two groups are overlapped, that is, the same fish and the same position are photographed under different light sources (Merge). Figure 5 It can be explained that when DAMNPs act on zebrafish at the same concentration, the effect on tumor cells is stronger than that of the D+A group and DANPs group, and the inhibitory effect is more significant.

[0118] (I) Nude mouse experiment:

[0119] (1) Establishment of subcutaneous transplanted tumor model in mice

[0120] 1. Preparation of A549 cell suspension: Select A549 cells in good cell condition and in the logarithmic growth phase, wash with PBS, digest with trypsin, add culture medium to make cell suspension, count. Then centrifuge at 1000 rpm for 5 min, wash twice with PBS, and make a cell suspension with a density of 1×10 7 / mL of cell suspension.

[0121] 2. Cultivation of transplanted tumors: 200 μL of cell suspension was injected subcutaneously into the right abdomen of nude mice. Approximately 2×10 6 cells.

[0122] 3. For transplanting tumors, sterilize surgical scissors, surgical forceps and inoculation needles in advance. When the tumor grows to the required number and size of tumors for transplantation experiments, remove the tumor tissue from the nude mouse, place it in a culture dish, add physiological saline, and cut the tissue into 20-30 mm 3 The tumors were uniformly sized and then transplanted into healthy BALB / c nude mice of about 4-5 weeks old using an inoculation needle.

[0123] 4. Grouping and administration: When the tumor area of ​​nude mice grows to 80-100mm 3 The mice were randomly divided into four groups: control group, 1mg / kg DIG+0.4mg / kg ADR group, DANPs (1mg / kg DIG+0.4mg / kg ADR) group, and DAMNPs (1mg / kg DIG+0.4mg / kg ADR) group. Each group was injected with the tail vein. The control group was injected with the corresponding volume of saline. The mice were given the drug once every three days. The body weight was weighed and the tumor volume was measured (tumor volume = tumor length × tumor width). 2 / 2). After 15 days, the nude mice were treated, the tumor tissues were taken out, photographed and weighed, and the changes in tumor volume and the average tumor weight of each group at the time of sacrifice were counted. After the end, half of the tumor, heart, liver, spleen, lung and kidney tissues were frozen in a -80℃ refrigerator, and the other half was fixed in 4% paraformaldehyde.

[0124] The results are as follows Figure 6 , Figure 7 and Figure 8 As shown, during the administration process, the tumor volume of the control group increased rapidly with time, and the D+A group and the DANPs group also showed a certain growth trend with time, while the DAMNPs group grew relatively slowly compared with the other three groups. Each group was killed 18 days after administration, and the tumor volume and tumor weight of each group were statistically analyzed. After 18 days of administration, the tumor volume and tumor weight of the D+A group and the DANPs group were reduced compared with the control group, and there was a statistical difference. Compared with the control group, not only was the tumor volume of the DAMNPs group significantly reduced, but the tumor weight was also significantly reduced. Compared with the D+A group and the DANPs group, the tumor volume and tumor weight also showed a significant downward trend and were statistically significant.

[0125] In summary, the nanoparticles prepared in the present application have the advantages of high drug loading and strong tumor targeting. While improving the existing anti-tumor treatment effect, they have stronger targeting and can effectively reduce the toxicity of drugs to normal tissues.

[0126] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nanoparticle, characterized in that: The nanoparticles include nano self-assembled particles and homotypic tumor cell membranes; The nano self-assembled particles are mainly made of digoxin and doxorubicin; The homologous tumor cell membrane is coated on the surface of the nano self-assembled particles; the homologous tumor cell membrane is a non-small cell lung cancer cell membrane; The preparation method of the nanoparticles comprises the following steps: (A) Preparation of self-assembled nanoparticles using precipitation method; (B) providing a homologous tumor cell membrane, mixing the homologous tumor cell membrane with the nano self-assembled particles prepared in step (A), and then extruding them in stages to obtain nanoparticles; Step (A) comprises the following steps: (a), dissolving digoxin in an organic solvent to obtain a digoxin solution; (b), dissolving doxorubicin in deionized water to obtain a doxorubicin solution; (c) under stirring, injecting the digoxin solution obtained in step (a) into deionized water at a uniform rate to obtain solution A; Then, the doxorubicin solution obtained in step (b) is added to solution A and mixed to obtain nano self-assembled particles; In step (c), the digoxin solution is uniformly injected at a rate of 20-30 ml / h; In the step (c), the volume ratio of solution A to doxorubicin solution is 18-20:1; The reaction conditions of stirring in step (c) at least meet the following requirements: The stirring speed is 1000~1200r / min, the stirring temperature is 48~52℃, and the stirring time is 0.8~1.5h.

2. The nanoparticle according to claim 1, characterized in that The hydrated particle size of the nanoparticles is 100-200 nm.

3. The nanoparticle according to claim 1, characterized in that The homotypic tumor cell membrane is A549 tumor cell membrane.

4. A method for preparing nanoparticles according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: (A) Preparation of self-assembled nanoparticles using precipitation method; (B) providing a homologous tumor cell membrane, mixing the homologous tumor cell membrane with the nano self-assembled particles prepared in step (A), and then extruding them in stages to obtain nanoparticles.

5. The method for preparing nanoparticles according to claim 4, characterized in that: The step (A) comprises the following steps: (a), dissolving digoxin in an organic solvent to obtain a digoxin solution; (b), dissolving doxorubicin in deionized water to obtain a doxorubicin solution; (c) Under stirring conditions, the digoxin solution obtained in step (a) is first injected into deionized water at a uniform speed to obtain solution A; then the doxorubicin solution obtained in step (b) is added to solution A and mixed to obtain nano self-assembled particles.

6. The method for preparing nanoparticles according to claim 5, characterized in that: The organic solution in step (a) comprises any one of anhydrous ethanol, methanol, isopropanol or n-butanol.

7. The method for preparing nanoparticles according to claim 5, characterized in that: The molar concentration of digoxin in the solution A of step (c) is 0.1-2 mmol / L.

8. The method for preparing nanoparticles according to claim 4, characterized in that: The step (B) comprises the following steps: (d), providing a solution containing homotypic tumor cell membranes, and then mixing the homotypic tumor cell membranes with the nano self-assembled particles prepared in step (c) to obtain a solution B; (e) Using a liposome extruder, the solution B was graded and extruded through a filter membrane with a pore size of 200 to 400 nm for 3 to 6 cycles to obtain nanoparticles.

9. Use of the nanoparticles according to any one of claims 1 to 3 in the preparation of a drug for treating non-small cell lung cancer.

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