A biomimetic nano delivery system and its preparation method and application

Abstract

The present invention provides a bionic nano delivery system and its preparation method and application, which belongs to the technical field of pharmaceutical preparation. The bionic nano delivery system in the present invention is composed of a black phosphorus nano sheet core and an M1 type macrophage membrane that wraps the black phosphorus nano sheet core, and has a photothermal effect under near-infrared light 780nm~1000nm laser irradiation. The present invention also provides a preparation method and application of the bionic nano delivery system. The bionic nano delivery system of the present invention can actively identify tumor cells and accumulate in cancer cells. After entering the cells, the local tumor tissue is rapidly heated under 808nm laser irradiation, ablating cancer tissue, and accurately and efficiently releasing drugs at the same time, causing cancer cell apoptosis, thereby inhibiting tumor growth. Therefore, the present invention has a very broad application prospect in thermal therapy, chemotherapy, imaging and / or treatment of cancer, hereditary diseases and infectious diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparation, and in particular relates to a bionic nano delivery system and a preparation method and application thereof. Background Art

[0002] Due to the complexity, diversity and heterogeneity of tumors, the most commonly used treatment methods are still surgery, radiotherapy and chemotherapy. However, these traditional methods have certain disadvantages, such as the inability to completely remove malignant tumors by surgery, the serious toxic side effects of radiotherapy and chemotherapy on normal cells, and the multidrug resistance of tumor cells caused by continuous chemotherapy.

[0003] In the past 20 years, nanotechnology and nanomedicine have developed rapidly in cancer diagnosis and treatment, but there is still a gap between the nanoparticle-based therapeutic platform and the goal of clinical cancer treatment. The main challenge is that nanoparticles used in vivo must cross several biological barriers before reaching tumor cells, including systemic blood circulation, tumor accumulation and penetration, cellular endocytosis and intracellular drug release.

[0004] In recent years, nanoparticle-mediated energy conversion therapy has attracted widespread attention, among which photothermal conversion nanoparticle-mediated thermochemotherapy is a new direction for nanomedicine to treat cancer. For example, black phosphorus nanosheets, a two-dimensional (2D) layered nanomaterial with a unique folded honeycomb structure, have a very large specific surface area. Compared with ordinary materials, ultra-thin 2D nanosheets with a thickness of nearly a single atomic layer have many physical and chemical properties that other materials do not have, such as high-efficiency photothermal conversion and drug loading, as well as special biological effects, such as good biocompatibility, cellular endocytosis and biodegradability. The nanoplatform developed on this basis can simultaneously provide heat and chemotherapeutic drugs to tumors for thermochemotherapy. The limitation is that the black phosphorus nanosheets that enter the body have a short circulation time and poor stability. Most of them are metabolized and cleared in organs rich in immune cells such as the liver, kidney, and spleen, and less than 5% of the nanoparticles can enter tumor cells. To overcome the above limitations, we started from the perspective of bionics and tried to construct a multifunctional nanoplatform with long in vivo circulation performance and photothermal therapy mediated by near-infrared lasers, which can be used for chemotherapy, hyperthermia and imaging at the same time.

[0005] Cell membrane engineering provides a top-down modification method for nanoparticles and has become a cutting-edge strategy for improving material interfaces. Cell membrane-coated nanoparticles have been widely studied in recent years due to their biocompatibility, retention of functional properties of the source cell membrane, and adaptability to various therapeutic and imaging applications. These nanoparticles are made of one or more cell membrane coatings, derived from coatings of red blood cells, platelets, white blood cells, cancer cells, and bacteria, and exhibit properties unique to the source cells, such as long circulation, immune escape, and specific targeting. Currently, cell membranes derived from cancer cells, stem cells, red blood cells, and white blood cells have been used to modify nanoparticles and endow them with cell-like functions. Nanosystems modified with leukocyte membranes have the advantage of crossing the vascular barrier, downregulating and reducing immune system recognition, thereby improving the in vivo stability of nanoparticles. For cancer treatment, unlike traditional nanoparticles that rely on the high permeability and retention (EPR) effect and passively aggregate in tumor tissues. The activated M1 macrophage membrane can mediate the host's defense against infection, and at the same time, under the action of monocyte chemoattractant protein 1 (CCL2), it recognizes and captures tumor cells and participates in anti-tumor responses. The black phosphorus nanoparticles wrapped around the M1 macrophage membrane have active targeting capabilities because they can recognize and bind to tumors.

[0006] The existing technology uses bionic red blood cell membranes to wrap perfluorocarbons to alleviate the hypoxic environment at the tumor site. Nano drugs mainly rely on the EPR effect to passively target and accumulate in the tumor, thereby improving the hypoxic environment and synergizing with radiotherapy to treat tumors. The existing technology also uses cancer cell membranes to modify nanoparticles loaded with paclitaxel, but similar chemotherapy drugs often have greater toxicity. The existing technology uses cancer cell membranes to modify magnetic nanoparticles loaded with chemotherapy drugs, but doxorubicin also has greater toxicity, and the loading efficiency of magnetic nanoparticles is difficult to reach a high level.

[0007] Therefore, safer and more effective bionic nanodelivery systems remain a research hotspot in this field. Summary of the invention

[0008] In order to solve the above technical problems, the present invention provides a bionic nano delivery system and its preparation method and application. The cell membrane-modified black scale nanosheets in the present invention have functions similar to those of source cells, such as specific recognition, long-term blood circulation and immune escape. For cancer treatment, nanoparticles camouflaged from activated M1 macrophage membranes show excellent performance in efficient targeted delivery of small molecule compounds, photosensitizers or enzymes to tumors. The present invention constructs a new platform based on drug delivery, which uses black phosphorus nanosheets as the core. Due to the unique folded honeycomb layered structure of black phosphorus nanosheets, its specific surface area is greatly increased, and anti-tumor drugs such as vorinostat can be efficiently and in large quantities loaded, and further camouflaged M1 macrophage membranes. This new type of bionic nanoparticle not only shows highly specific active targeting of tumor cells, but also has immune escape characteristics, which prolongs the in vivo circulation of drugs. By irradiating the tumor site with 808nm near-infrared laser, a high degree of concentration of the tumor site can be achieved to achieve a specific targeting effect. Under the superior photothermal conversion performance of the black-scale nanosheets, the temperature of the local tumor tissue quickly rises to the threshold of 45-50°C and lasts for a period of time, resulting in irreversible damage to the tumor cell membrane and protein denaturation, thereby inhibiting tumor growth and even ablating the tumor.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] The present invention provides a bionic nano-delivery system, which consists of a black phosphorus nano-sheet core and a cell membrane wrapping the black phosphorus nano-sheet core; the black phosphorus nano-sheet has a major diameter range of 80 to 200 nm and a minor diameter range of 50 to 150 nm; the mass ratio of the black phosphorus nano-sheet to the cell membrane is 1:(0.3 to 1); the bionic nano-delivery system has a photothermal effect under near-infrared light 780 nm to 1000 nm laser irradiation.

[0011] Furthermore, the major diameter of the black phosphorus nanosheets ranges from 80 to 150 nm, and the minor diameter ranges from 80 to 100 nm.

[0012] Furthermore, the long diameter range of the black phosphorus nanosheet is 100 nm, and the short diameter range is 100 nm.

[0013] Furthermore, the cell membrane is selected from any one or more of M1 macrophage membrane, Hela cell membrane, NCI-H121010 cell membrane, LLC cell membrane, B16-F10 cell membrane, 4T1 cell membrane, CAL cell membrane and HCT cell membrane.

[0014] Furthermore, the cell membrane is an M1 macrophage membrane.

[0015] Furthermore, the M1 macrophage membrane originates from mice.

[0016] Furthermore, the mass ratio of the black phosphorus nanosheets to the cell membrane is 1:(0.5-1).

[0017] Furthermore, the mass ratio of the black phosphorus nanosheets to the cell membrane is 1:1.

[0018] Furthermore, the bionic nano-delivery system has a photothermal effect under irradiation of near-infrared light 800nm ​​to 850nm laser.

[0019] Furthermore, the bionic nano-delivery system is also loaded with drugs; the drugs are selected from any one or more of small molecule compounds, photosensitizers and enzymes; the small molecule compounds are selected from any one or more of vorinostat, doxorubicin and paclitaxel; the photosensitizers are selected from any one or more of zinc cyanine, hematoporphyrin monomethyl ether and Bodipy dyes; the enzymes are selected from any one or more of deubiquitinating enzymes, cysteine ​​proteases and calpains.

[0020] The present invention also provides a method for preparing the bionic nano delivery system, which comprises the following steps:

[0021] (1) Preparation of black scale nanosheets

[0022] The black phosphorus crystal powder is added to an N-methyl-2-pyrrolidone (NMP) solution supersaturated with sodium hydroxide, the ultrasonic power is set to 250-350W, the ultrasonic is carried out at 4-12°C for 10-11 hours, and then the unpeeled macroscopic black phosphorus crystals are removed by centrifugation at 8500-10500rpm at 3-10°C, the supernatant is collected, and then the solution is centrifuged and washed at 14000-16000rpm at 3-10°C to remove the residual N-methyl-2-pyrrolidone (NMP), and the precipitate obtained is the black scale nanosheet;

[0023] (2) Extraction of cell membrane

[0024] The method is repeated freezing and thawing, the number of repeated freezing and thawing is 3 to 5 times, and the repeated freezing and thawing refers to freezing the cell suspension at -75 to -85°C for 1.5 to 2.5 hours and thawing at room temperature;

[0025] (3) Preparation of biomimetic nanodelivery system by wrapping black scale nanosheets with cell membrane

[0026] The cell membrane prepared in step (2) is added to the black phosphorus nanosheet prepared in step (1) and mixed evenly. After ice bath ultrasonic treatment for 25 to 35 minutes, the mixture is stirred and reacted at 20 to 30° C. for 12 to 48 hours to obtain the bionic nano delivery system.

[0027] Furthermore, the ultrasonic power in step (1) is 300 W, the ultrasonic temperature is 10° C., and the ultrasonic duration is 10 h; the centrifugal speed for removing unpeeled macroscopic black phosphorus crystals is 10,000 rpm, the centrifugal temperature is 4° C., and the centrifugal time is 10 min; the centrifugal speed for removing residual N-methyl-2-pyrrolidone (NMP) is 15,000 rpm, the centrifugal temperature is 4° C., and the centrifugal time is 10 min; deoxygenated ultrapure water is used for washing, and the number of washing times is 3 times.

[0028] Furthermore, the number of repeated freezing and thawing in step (2) is 4 times, and the repeated freezing and thawing refers to freezing the cell suspension at -80°C for 2 hours and thawing at room temperature.

[0029] Furthermore, in step (3), the temperature for continuous stirring reaction is 26-28° C., and the continuous stirring reaction time is 24 h.

[0030] Furthermore, the step (1) also includes a process of loading drugs on the black-scale nanosheets.

[0031] The present invention also provides an application of the bionic nano delivery system or the bionic nano delivery system prepared according to the preparation method in disease hyperthermia, chemotherapy, imaging and / or preparation of therapeutic products and / or drugs.

[0032] Furthermore, the disease treatment drug is specifically a drug that inhibits tumor growth.

[0033] Compared with the prior art, the present invention has the following technical effects:

[0034] (1) The method of the present invention constructs a precise and efficient delivery system that integrates thermal chemotherapy and tumor imaging. It can specifically identify cancer cells and accumulate in cancer cells. After entering the cells, it successfully escapes from the lysosomes and promotes the release of drugs from the carrier under the action of 808nm infrared laser, effectively converting light energy into heat energy, specifically killing cancer cells and ablating cancer tissue, thereby inhibiting tumor growth.

[0035] (2) The bionic nano-delivery system of the present invention has a strong photothermal conversion effect under near-infrared laser irradiation. -2 Under the irradiation conditions of 808nm infrared laser, the temperature of black scale nanosheets with experimental concentrations rapidly increased by 30-45℃ within 6 minutes.

[0036] (3) The present invention is applicable to the thermal treatment of tumors and has broad application prospects in cancer, genetic diseases and infectious diseases. The method of the present invention uses biomimetic nanoparticles to integrate thermal therapy, chemotherapy and tumor imaging, accurately and efficiently deliver drugs, and inhibit the proliferation of cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The preparation and tumor growth inhibition process of the bionic nano delivery system in Example 1 of the present invention;

[0038] Figure 2 The characterization results and in vitro biocompatibility analysis results of the biomimetic nano-delivery system in Example 2 of the present invention;

[0039] Figure 3 The results of the analysis of the loading efficiency of BP on SAHA in Example 3 of the present invention are as follows;

[0040] Figure 4 The cell viability analysis results in Example 4 of the present invention;

[0041] Figure 5 The polarization index analysis results of macrophages in Example 5 of the present invention;

[0042] Figure 6 The results of laser confocal microscopy and flow cytometry of the cell uptake in Example 6 of the present invention;

[0043] Figure 7 The confocal microscopy results of the dose-dependent and time-dependent uptake of LLC cells by MBS system in Example 7 of the present invention;

[0044] Figure 8 This is the in vitro evaluation result of the inhibitory effect of MBS on tumor cells in Example 8 of the present invention;

[0045] Fig. 9 The real-time biodistribution results of MBS in mice in Example 9 of the present invention;

[0046] Fig.10 The results are the evaluation of the MBS tumor inhibition effect and the analysis of changes in serum biochemical indicators in Example 10 of the present invention. DETAILED DESCRIPTION

[0047] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the method, steps or conditions of the present invention are within the scope of the present invention. The reagents and instruments used in the following examples can all be obtained commercially, and the methods used in the examples are consistent with the conventional methods unless otherwise specified.

[0048] Before describing the following embodiments, the terms used herein are defined as follows:

[0049] The term “BP” refers to: black phosphorus nanosheets;

[0050] The term “SAHA” refers to: vorinostat antineoplastic agent;

[0051] The term “M” refers to: M1 macrophage membrane;

[0052] The term “BS” refers to: vorinostat-loaded black phosphorus nanosheets;

[0053] The term “MB” refers to: M1 macrophage membrane-encapsulated black phosphorus nanosheets;

[0054] The term “MBS” is: M1 macrophage membrane-encapsulated vorinostat-loaded black phosphorus nanosheets;

[0055] The term “LBS” refers to: lipo3000-wrapped vorinostat-loaded black phosphorus nanosheets;

[0056] The term "NMP" means: N-methyl-2-pyrrolidone solution;

[0057] The term "Bodipy dyes" refers to: fluoroborate dipyrrole compounds, fluorescent dyes with good optical properties;

[0058] The term “LPS” refers to: lipopolysaccharide;

[0059] The term “FITC” refers to: the fluorescent dye fluorescein isothiocyanate;

[0060] The term “BP / FITC” refers to: black phosphorus nanosheets loaded with Fic;

[0061] The term “Lipo / BP / FITC” refers to: Lipo3000-loaded FITC-loaded black phosphorus nanosheets;

[0062] The term “MB / FITC” refers to: M1 macrophages encapsulated with FITC-loaded black phosphorus nanosheets;

[0063] The term “DiR” refers to: a lipophilic fluorescent dye;

[0064] The term “BP / DiR” refers to: black phosphorus nanosheets loaded with DiR;

[0065] The term “MB / DiR” refers to: DiR-loaded black phosphorus nanosheets wrapped by M1 macrophage membranes;

[0066] The term “CLSM” refers to: confocal laser scanning microscopy;

[0067] The term "FACS" means: flow cytometry.

[0068] The reagents and instruments used in the following examples are as follows:

[0069] Reagents:

[0070] NMP, anhydrous sodium hydroxide, PBS, and DMSO were purchased from Guangzhou Chemical Reagent Factory;

[0071] BALB / c nude mice were purchased from Beijing Sibeifu Biotechnology Co., Ltd.; LLC cells, NIH-3T3 cells, MH-S cells, and MLE-12 cells were purchased from Guangzhou Tuokeda Biotechnology Co., Ltd.; protease inhibitors and plasma protein extraction kits were purchased from Qingke Biotechnology Co., Ltd.

[0072] instrument:

[0073] Transmission electron microscope, purchased from FEI, Japan, model JEM-1400PLUS;

[0074] Laser confocal microscope, purchased from Zeiss, Germany, model LSM880;

[0075] Flow cytometer, purchased from Merck, Germany, model ImageSteamX Mark

[0076] The technical solution of the present invention is further described in detail below in conjunction with embodiments.

[0077] Example 1

[0078] This example is used to illustrate the preparation of the bionic nano delivery system of the present invention.

[0079] (1) Preparation of black-scale nanosheets. Liquid-phase ultrasonic-assisted exfoliation method was used: first, accurately measure 25 mL of NMP solution with a measuring cylinder and pour it into a beaker of appropriate size, then weigh an appropriate amount of anhydrous sodium hydroxide and dissolve it in the NMP solution, making sure that the sodium hydroxide is supersaturated. Accurately weigh 25 mg of black phosphorus crystal powder and add it to the above-mentioned sodium hydroxide-supersaturated NMP solution. The obtained mixed solution was placed in an ultrasonic cleaning machine with an ultrasonic power of 300 W and an ultrasonic process temperature maintained below 10°C for 10 hours. The brown black phosphorus dispersion obtained after 10 hours was centrifuged in an ultracentrifuge with the parameters set to 10,000 rpm and centrifuged at 4°C for 10 minutes to remove the unexfoliated macroscopic black phosphorus crystals, and the supernatant after centrifugation was carefully collected. Subsequently, the collected supernatant containing BPNSs was centrifuged again with the parameters set to 15,000 rpm and centrifuged at 4°C for 10 minutes to remove NMP, and then washed three times with deoxygenated ultrapure water to wash away the residual NMP as much as possible. The obtained BPNSs precipitate was transferred to a brown penicillin bottle and resuspended with an appropriate amount of deoxygenated ultrapure water, protected from light, and stored at -20°C.

[0080] (2) Extraction of M1 macrophage membrane. The M1 macrophage membrane was obtained by repeated freezing and thawing. First, M1 macrophages were collected. Washed three times with PBS solution containing protease inhibitors. Cell membrane and cytoplasmic protein extraction kit A reagent and 1X protease inhibitor were added to the cells, and the cells were lysed at 4°C for 30 minutes, vortexing every 10 minutes. The cell suspension was frozen at -80°C for 2 hours and thawed at room temperature, repeatedly frozen and thawed four times, and ultrasonically treated at 4°C for 5 minutes at a power of 60W. The lysate was centrifuged at 14000rpm and 4°C for 30 minutes, and the resulting precipitate was the cell membrane. The cell membrane was suspended in D-hanks solution and stored at -80°C.

[0081] (3) Prepare black-scale nanosheets loaded with vorinostat: BS (5 ml, 1 mg / ml) and SAHA (1 ml, 2.5 mg / ml) were mixed at a mass ratio of 1:8 and stirred for 24 hours. The precipitate was collected by centrifugation at 15,000 rpm and 4°C for 5 minutes, and resuspended with an appropriate amount of 10% DMSO solution to obtain BS. The BS was stored at 4°C in the dark.

[0082] (5) Preparation of MBS. M1 macrophage membrane (12.1 μl, 8.24 mg / ml), SAHA (400 μl, 2 mg / ml), and BPNSs (50 ul, 2 mg / ml) were mixed at a mass ratio of 1:8:1, and self-assembly was completed by ultrasonication in an ice bath for 30 minutes and stirring at room temperature for 24 hours. Excess membrane material was removed by centrifugation at 15,000 rpm and 4°C for 10 minutes, and the precipitate was resuspended in an appropriate amount of 10% DMSO solution to obtain MBS, which was stored at 4°C in the dark.

[0083] The preparation process of the bionic nano delivery system and the tumor growth inhibition process in this embodiment are as follows: Figure 1 shown.

[0084] Example 2

[0085] In this example, the biomimetic nano-delivery system prepared in Example 1 was characterized and subjected to in vitro biocompatibility analysis. Figure 2 As shown, Figure 2 A shows the transmission electron microscope (TEM) images of BP, BS, and MBS and the particle size (DLS) analysis of BP, BS, and MBS; Figure 2 B is the 7-day stability analysis of MBS in aqueous solution; Figure 2 C is the Zeta potential analysis of BP, BS, and MBS; Figure 2 D is the photothermal stability and photothermal conversion capacity of the BP-based delivery system under repeated irradiation at 808 nm with different laser intensities; Figure 2 E is the photothermal effect caused by different laser powers; Figure 2 F is the photothermal effect caused by different formulations; Figure 2G is the cytotoxicity of MB components at different concentrations.

[0086] The results show that:

[0087] The average particle size of the biomimetic nano-delivery system MBS in the present invention is 200 nm ( Figure 2 A), the surface potential of MBS is -28Mv( Figure 2 C), and MBS can maintain the stability of nanoparticles in aqueous solution within 7 days without any change in particle size and aggregation and sedimentation. Figure 2 B).

[0088] The BP used in the present invention is 0.5W / cm 2 , 1W / cm 2 , 1.5W / cm 2 , 2W / cm 2 After 10 minutes of laser irradiation and four consecutive heating and cooling cycles, the photothermal effect of BP did not change with the number of heating and cooling cycles, indicating that it has a stable and rapid photothermal effect under the stimulation of near-infrared laser ( Figure 2 D) The temperature of BP increased by the photothermal effect is dependent on the laser power and irradiation time. The temperature increases with the increase of laser power and the extension of irradiation time. 2 After 10 minutes of laser irradiation, the temperature rises to about 60°C, indicating that it fully meets the needs of photothermal therapy ( Figure 2 E). The temperature of the sample containing BP rises higher than that of the sample without BP under the same laser power and time, indicating that the BP in the present invention has good photothermal performance ( Figure 2 F); Different concentrations of M and BP have little effect on LLC cell viability. It can be seen that the cell membrane and black phosphorus rice sheet have good biocompatibility with cells ( Figure 2 G).

[0089] Example 3

[0090] This example is used to illustrate the loading efficiency induced by BP. Take 1 ml of vorinostat antitumor drug (SAHA) (2.5 mg) and add it to 5 ml of BP (1 mg / mL) aqueous solution. Then add 4 mL of water and stir for 24 hours in the dark. Then centrifuge the mixture (10 min, 15000 rpm, 4 ° C) to obtain black phosphorus nanosheets BS loaded with SAHA. Then, BS was suspended in water. The loading efficiency was tested, and the results are as follows Figure 3 shown.

[0091] Depend on Figure 3 It can be seen that when the mass ratio of BP to SAHA is 2:1, the loading efficiency of BP can reach 80%, indicating that BP has excellent drug loading efficiency.

[0092] Example 4

[0093] This test example is used to illustrate the analysis of cell viability. LLC cells were cultured at 1×10 5 / well were planted in a 106-well plate and cultured for 12 hours. Cells were treated with different concentrations of SAHA (0.078μg / ml, 0.156μg / ml, 0.310μg / ml, 0.625μg / ml, 1.25μg / ml, 2.5μg / ml, 5μg / ml, 10μg / ml, 20μg / ml, 40μg / ml), and different formulas (NC, NC+L, BP+L, SAHA+L, BS+L, MBS, MBS+L). After 8 hours of drug addition, the cells were irradiated with near-infrared laser for 10 minutes at a distance of 10 cm. After 16 hours of continuous culture, the cells were analyzed by CCK-8 method. The results are shown in Figure 4 As shown, Figure 4 A is the analysis of cell viability after treatment with different concentrations of SAHA for 24 h; Figure 4 B is the analysis of the effects of different formula treatments on cell viability.

[0094] Depend on Figure 4 It can be seen that the killing rate of SAHA on LLC is concentration-dependent. The half lethality of SAHA on LLC cells is 5 μg / ml. This concentration can be used as the concentration for subsequent cell experiments ( Figure 4 A); Compared with other treatments, MBS+L has an inhibition rate of up to 100% on LLC cells, while SAHA-L has an inhibition rate of only 50% on LLC cells, indicating that MBS+L can significantly increase the inhibition rate of SAHA on LLC cells ( Figure 4 B).

[0095] Example 5

[0096] This test example is used to illustrate the polarization index of macrophages. RAW264.7 cells were seeded in a six-well plate at a density of 2×10 5 cells / well. After 6 hours, the cells were incubated with LPS (500 ng / mL) for 3 days to polarize RAW264.7 cells into M1-like macrophages. The polarization index was detected. The results were as follows: Figure 5 As shown, Figure 5 A is the polarization index of cells (iNOS and CD80) detected by western blotting (iNOS and CD80); Figure 5 B is the polarization index (iNOS and TNF-α) detected by real-time quantitative PCR.

[0097] Depend on Figure 5It can be seen that iNOS and CD80 are both characteristic proteins of M1 macrophages. After LPS induction, the results of western blotting show that the expression of iNOS and CD80 proteins is significantly upregulated, indicating that LPS successfully induces RAW264.7 cells into M1 macrophages ( Figure 5 A); The results of real-time quantitative PCR showed that iNOS and TNF-α, the marker genes of M1 macrophages, were significantly upregulated, indicating that LPS successfully induced RAW264.7 cells into M1 macrophages at the gene level ( Figure 5 B).

[0098] Example 6

[0099] This test example is used to illustrate the dose-dependent and time-dependent uptake evaluation of MB / FITC system delivery on LLC cells. LLC cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. The cells were washed with PBS, fixed with 4% paraformaldehyde for 15 minutes, stained with DAPI for 15 minutes, and analyzed by CLSM. The analysis results are shown in Figure 6, where Figure 6 A shows the uptake of nanoparticles by LLC cells incubated with different concentrations (0.62, 1.25, 2.5, 5, and 10 μg / mL) analyzed by laser confocal microscopy. Figure 6 B is the uptake of MB / FITC nanoparticles by LLC cells incubated at 37°C for different times (1, 4, 8, 12 and 24 h) as shown by laser confocal microscopy.

[0100] Depend on Figure 6 It can be seen that the uptake of MB / FITC by LLC cells is time-dependent and concentration-dependent, but when the concentration and time increase to a certain extent, the uptake of MB / FITC by LLC cells no longer increases, which may be related to the efflux mechanism of the cells. Based on the results of this experimental example, we chose to use a concentration of 5 μg / ml and an incubation time of 8 hours as a reference indicator for subsequent cell uptake experiments ( Figure 6 A, 6B).

[0101] Example 7

[0102] This test example is used to illustrate the evaluation of specific cell uptake. Since SAHA itself does not have a fluorescent group, FITC is used as a substitute for SAHA. After 8 hours of cell administration (BP / FITC, Lipo / BP / FITC, MB / FITC), the cells were incubated at 37°C, 5% CO2. The cells were washed with PBS, fixed with 4% paraformaldehyde for 15 minutes, stained with DAPI for 15 minutes, and analyzed by CLSM. In order to quantitatively analyze cell uptake, after the same administration method, the cells were digested with trypsin and collected for flow cytometric analysis. The analysis results are shown in the figure. Figure 7 As shown, Figure 7 A shows the efficient uptake of MB / FITC by cells analyzed by laser confocal microscopy and flow cytometry; Figure 7 B, Laser confocal microscopy and flow cytometry analysis of cellular uptake in different cell lines.

[0103] Depend on Figure 7 It can be seen from the analysis of laser confocal microscopy and flow cytometry that within the same incubation time, LLC cells have the highest uptake of MB / FITC, which is 20% and 10% higher than Lipo / BP / FITC and BP / FITC, respectively. Figure 7 A); After different cells were incubated with the same drug concentration for the same time, LLC cells uptake MB / BP / FITC was about 5-8 times higher than NIH-3T3 cells, MLE-12 cells, and MH-S cells, indicating that MB / FITC has excellent targeting to LLC cells ( Figure 7 B).

[0104] Example 8

[0105] This test example is used to illustrate the in vitro evaluation of the inhibitory effect of MBS on tumor cells. LLC cells were cultured at 1×10 5 / well were planted in confocal culture dishes or 6-well plates and cultured for 12 hours. After different treatments (PBS, PBS-L, BP-L, SAHA-L, BS-L, MBS, MBS-L), near-infrared laser irradiation was used for 10 minutes 8 hours after drug addition, with a laser power of 2W / cm 2 The irradiation distance was 10 cm. After 16 hours of continuous culture, the cells were collected and analyzed by CLSM staining and FACS analysis of apoptosis using the live and dead cell detection kit and Annexin V-FITC / PI cell apoptosis detection kit, respectively. The analysis results are shown in Figure 8 As shown, Figure 8 A is live / dead assay analysis; Figure 8 B: FACS analysis.

[0106] Depend on Figure 8It can be seen that the use of laser alone does not affect cell viability, while BP-L, SAHA-L, BS-L, and MBS will cause apoptosis of some cells. The killing rate of MBS-L on LLC reached 99.3%, indicating that under the targeting effect of the M1 macrophage membrane, more MBS entered the LLC. Under the action of near-infrared laser, BP exerted a photothermal effect and released SAHA at the same time, achieving the effect of synergistic killing of LLC ( Figure 8 A, 8B).

[0107] Example 9

[0108] This experimental example is used to illustrate that MBS can be precisely targeted to tumors in vivo. Tumor-bearing mice were divided into 5 groups (Saline, BP, DiR, BP / DiR, MB / DiR (BP content was 12.5 mg / kg). After intravenous injection of different dosage forms for 12 hours, the mice were imaged in vivo at 633 nm using a small animal in vivo imaging device at 1 hour, 6 hours, 12 hours, 24 hours, and 48 hours. The results are shown in Fig. 9 As shown, Fig. 9 A is the in vivo imaging pictures of mice at different times; Fig. 9 B is the fluorescence intensity of mouse tumor sites at different times; Fig. 9 C is a real-time fluorescent image of various organs (heart, liver, spleen, lung, kidney, and tumor) of the mouse after dissection; Fig. 9 D is a statistical graph of the fluorescence intensity of each organ.

[0109] Depend on Fig. 9 It can be seen that MB / DiR accumulates the most in the tumor site and accumulates for the longest time. A strong fluorescence signal is still observed in the living tumor site 48 hours after the drug enters the body, while no strong signal is observed in the other groups BP, DiR, and BP / DiR. It can also be seen from the dissected tumor that MB / DiR accumulates the most in the tumor site.

[0110] The M1 macrophage membrane can accurately target the tumor site, increase the amount of drugs in the tumor site, prolong the retention time of drugs in the tumor site, and thus enhance the therapeutic effect of the drug.

[0111] Example 10

[0112] This test example is used to illustrate the evaluation of MBS anti-tumor effect and the analysis of changes in serum biochemical indicators. Tumor-bearing mice were injected with different dosage forms through the tail vein (Groups 1 and 2: 200 μl of normal saline; Group 3: 12.5 mg / kg BP nanosheets; Group 4: 5 mg / kg SAHA; Group 5: 12.5 mg / kg BP nanosheets and 5 mg / kg SAHA; Group 6: 12.5 mg / kg BP nanosheets, 5 mg / kg SAHA and 25 mg / kg membrane; Group 7: 12.5 mg / kg BP nanosheets, 5 mg / kg SAHA and 25 mg / kg membrane). After 12 hours, Groups 2, 3, 4, 5 and 7 were treated with 2 W / cm 2 The laser was irradiated for 10 minutes, with the light source 10 cm above the tumor site, and the treatment was performed every 3 days. The tumor inhibition effect was evaluated and the changes in serum biochemical indicators were analyzed. The results are as follows Fig.10 As shown, Fig.10 A shows the changes in tumor volume under different treatments; Fig.10 B is the image of the extracted tumor after different processing; Fig.10 C is TUNNEL analysis of tumor tissue; Fig.10 D is the analysis of the changes in mouse body weight; Fig.10 E is the analysis of serum biochemical indicators.

[0113] Depend on Fig.10 It can be seen that during the 15-day treatment, the tumors in the MBS-L group were completely suppressed and showed no growth trend, while the treatment effects of the other treatment groups (BP-L, BS-L, SAHA-L, MBS) were not ideal, and the tumors showed a growth trend. The tumors in the control group (Saline, Saline-L) also showed a rapid growth trend ( Fig.10 A). It can also be seen from the tumors dissected out at the end that the tumor volume of the MBS-L group mice is the smallest compared with the other treatment groups and the control group, indicating that MBS-L can effectively inhibit the growth of tumor cells in vivo ( Fig.10 B). Analysis of tumor tissue TUNNEL sections showed that apoptotic tumor cells in MBS-L tumor tissue sections reached 100%, while other treatment groups only had 30% or even lower percentages, further demonstrating that MBS-L has a highly effective inhibitory effect on tumor growth ( Fig.10 C). From the recorded weight of mice during the treatment, there was no significant difference in the weight of mice in the MBS-L treatment group compared with the control group, indicating that the safety of MBS-L is guaranteed ( Fig.10 D). There was no significant difference in serum biochemical indexes between MBS-L and control mice, which further demonstrated that MBS-L has good safety ( Fig.10 E).

[0114] In general, the MBS-L drug delivery system can accurately target tumor tissues, increase the accumulation of SAHA in the tumor site, and exert the photothermal effect of BP under the stimulation of 808nm near-infrared laser, destroying the tumor cell membrane and causing irreversible damage to tumor cells. This combination of precise targeting and thermal chemotherapy drug delivery platform can significantly enhance drug efficacy and reduce side effects during chemotherapy.

[0115] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A bionic nano delivery system, characterized in that: The bionic nano delivery system consists of a black phosphorus nanosheet core and a cell membrane wrapping the black phosphorus nanosheet core; the black phosphorus nanosheet has a long diameter range of 80-200 nm and a short diameter range of 50-150 nm; the cell membrane is an M1 macrophage membrane; the mass ratio of the black phosphorus nanosheet to the cell membrane is 1:(0.3-1); the bionic nano delivery system is also loaded with drugs; the bionic nano delivery system has a photothermal effect under near-infrared light 780nm-1000nm laser irradiation; The preparation method of the bionic nano delivery system comprises the following steps: (1) Preparation of black scale nanosheets The black phosphorus crystal powder is added to an N-methyl-2-pyrrolidone (NMP) solution supersaturated with sodium hydroxide, the ultrasonic power is set to 250-350W, the ultrasonic is performed at 4-12°C for 10-11 hours, and then the unpeeled macroscopic black phosphorus crystals are removed by centrifugation at 8500-10500rpm and 3-10°C, the supernatant is collected, and then the solution is centrifuged and washed at 14000-16000rpm and 3-10°C to remove the residual N-methyl-2-pyrrolidone (NMP), and the precipitate obtained is the black scale nanosheet; (2) Extraction of cell membrane The method is repeated freezing and thawing, the number of repeated freezing and thawing is 3 to 5 times, and the repeated freezing and thawing refers to freezing the cell suspension at -75 to -85°C for 1.5 to 2.5 hours and thawing at room temperature; (3) Preparation of biomimetic nanodelivery system using cell membrane-wrapped black scale nanosheets The cell membrane prepared in step (2) is added to the black phosphorus nanosheet prepared in step (1) and mixed evenly, and then subjected to ice bath ultrasonication for 25 to 35 minutes, and then stirred and reacted at 20 to 30° C. for 12 to 48 hours to obtain the bionic nano delivery system; The step (1) also includes a process of loading drugs on the black-scale nanosheets.

2. The biomimetic nano delivery system according to claim 1, characterized in that: The long diameter of the black phosphorus nanosheets is in the range of 80-150 nm, and the short diameter is in the range of 80-100 nm.

3. The biomimetic nano delivery system according to claim 1, characterized in that: The drug is selected from any one or more of small molecule compounds, photosensitizers and enzymes; the small molecule compounds are selected from any one or more of vorinostat, doxorubicin and paclitaxel; the photosensitizer is selected from any one or more of phenocyanine zinc, hematoporphyrin monomethyl ether and Bodipy dyes; the enzyme is selected from any one or more of deubiquitinating enzymes, cysteine ​​proteases and calpains.

4. The method for preparing the biomimetic nano delivery system according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) Preparation of black scale nanosheets The black phosphorus crystal powder is added to an N-methyl-2-pyrrolidone (NMP) solution supersaturated with sodium hydroxide, the ultrasonic power is set to 250-350W, the ultrasonic is performed at 4-12°C for 10-11 hours, and then the unpeeled macroscopic black phosphorus crystals are removed by centrifugation at 8500-10500rpm and 3-10°C, the supernatant is collected, and then the solution is centrifuged and washed at 14000-16000rpm and 3-10°C to remove the residual N-methyl-2-pyrrolidone (NMP), and the precipitate obtained is the black scale nanosheet; (2) Extraction of cell membrane The method is repeated freezing and thawing, the number of repeated freezing and thawing is 3 to 5 times, and the repeated freezing and thawing refers to freezing the cell suspension at -75 to -85°C for 1.5 to 2.5 hours and thawing at room temperature; (3) Preparation of biomimetic nanodelivery system using cell membrane-wrapped black scale nanosheets The cell membrane prepared in step (2) is added to the black phosphorus nanosheet prepared in step (1) and mixed evenly, and then subjected to ice bath ultrasonication for 25 to 35 minutes, and then stirred and reacted at 20 to 30° C. for 12 to 48 hours to obtain the bionic nano delivery system; The step (1) also includes a process of loading drugs on the black-scale nanosheets.

5. The preparation method according to claim 4, characterized in that: The ultrasonic temperature in step (1) is 10°C and the centrifugation temperature is 4°C.

6. The preparation method according to claim 5, characterized in that: The temperature for continuous stirring reaction in step (3) is 26-28° C., and the continuous stirring reaction time is 24 h.

7. Use of the bionic nano delivery system according to any one of claims 1 to 3 or the bionic nano delivery system prepared according to any one of claims 4 to 6 in the preparation of disease imaging and / or treatment products and / or drugs.

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