Multi-stimuli-responsive bionic nano preparation and preparation method thereof

Abstract

The invention provides a multi-stimuli-responsive bionic nano preparation which comprises an inner core and a shell, the inner core is a drug-loaded gold nano cage, drugs can be chemotherapeutic drugs, traditional Chinese medicine active ingredients and the like, the surface of the gold nano cage is connected with beta-cyclodextrin (beta-CD) through glutathione-sensitive gold-sulfur bonds (Au-S) to block the drugs, and the drug-loaded gold nano cage can be used as a chemotherapeutic drug or a traditional Chinese medicine active ingredient. Imine (Ada-PCA) formed by amantadine and protocatechuic aldehyde is inlaid on the surfaces of the nano-particles through host-guest interaction between cyclodextrin and adamantane, and a shell is a hybrid membrane bionic thermosensitive liposome. The invention also provides a preparation method. The nano preparation has multiple stimulation response performance such as temperature, low pH value, glutathione reducing environment and the like; in tumor tissues, near-infrared light excites the gold nanocage to generate heat, so that the bionic thermosensitive liposome is decomposed, pH sensitive bonds on the surface of an inner core are broken in an acid environment, meanwhile, high-concentration glutathione in cells promotes the blocking agent to fall off, accurate release of the medicine in tumor cells is achieved, the treatment effect of the medicine is improved, and toxic and side effects are reduced.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a multi-stimulus responsive bionic nano preparation and a preparation method thereof. Background Art

[0002] Traditional drugs lack targeting, and while killing tumor cells, they will damage normal cells of the body, producing toxic side effects that cannot be ignored. Although liposomes have many advantages as anti-tumor drug carriers and some have been used in clinical practice, there are still many problems. For example, targeted liposomes concentrate at the tumor site but do not improve the anti-tumor effect. There is a mismatch between drug efficacy and targeting. For example, the area under the curve of cisplatin long-circulating liposomes is improved when used as tumor medicine, but the anti-tumor effect is not good. Doxorubicin long-circulating liposomes mainly reduce toxic side effects, and the anti-tumor effect is not significantly different from that of free drugs. In addition, the slow release of long-circulating liposomes is not conducive to the efficacy of the drug.

[0003] In addition, polyethylene glycol (PEG) is widely used to modify nanomaterials such as thermosensitive liposomes. Although PEGylated liposomes are considered to be almost non-immunogenic and have been clinically studied, developed and applied for many years, with its wide application in other fields, it has been found that some healthy people have PEG antibodies, and repeated injections of PEGylated liposomes will cause "accelerated blood clearance" (ABC) phenomenon. This immune response has brought challenges to the development and clinical application of PEGylated preparations. At the same time, some stimuli-responsive nano-delivery systems that target cells have the problem of drug release outside tumor cells. Drugs that diffuse into the blood can cause toxicity to adjacent normal tissues and even cause multidrug resistance. Therefore, the development of intracellular drug precision release systems is crucial in tumor treatment. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a multi-stimulus responsive bionic nanoformulation and a preparation method thereof, so as to solve the problems of the mismatch between efficacy and targeting of the existing anti-tumor drug liposomes, the ABC phenomenon of PEGylated liposomes and toxicity caused by extracellular drug release, and to improve the performance of the nanoformulation by utilizing the stabilizing effect of protocatechuic aldehyde on bionic thermosensitive liposomes.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The invention provides a multi-stimulus responsive bionic nano preparation, which is a bionic nano core-shell system with multi-stimulus responsiveness, comprising an inner core and an outer shell, wherein the inner core is a drug-loaded gold nanocage (AuNCs), the surface of the gold nanocage is connected to beta-cyclodextrin (β-CD) through a glutathione-sensitive gold-sulfur bond (Au-S) to block the drug, the imine (Ada-PCA) formed by adamantane-protocatechuic aldehyde is embedded on the surface of the nanoparticle through the host-guest interaction between cyclodextrin and adamantane, and the outer shell is a hybrid membrane bionic thermosensitive liposome (HMBTL), which comprises a platelet membrane (PM), an erythrocyte membrane (EM) and a thermosensitive liposome (TSL).

[0007] The present invention also provides a method for preparing the above-mentioned multi-stimulus responsive bionic nanoformulation, which comprises the following steps:

[0008] ① Preparation of gold nanocages (AuNCs): silver nitrate, PVP and sodium hydrosulfide solution were added to a flask, stirred to react, and silver nanocubes were obtained; then the silver nanocube dispersion was added to an aqueous solution containing HAuCl4, stirred to react, and gold nanocages were obtained. The LSPR wavelength of the gold nanocages was 750-800nm ​​by controlling the amount of HAuCl4;

[0009] ②Amino modification of gold nanocages: Take the aqueous dispersion of gold nanocages, add mercaptoethylamine, stir, centrifuge and dry;

[0010] ③ Drug loading and encapsulation of gold nanocages: The amino gold nanocages and DOX solution were mixed, stirred, and centrifuged. The precipitate was reacted with carboxymethyl β-cyclodextrin, and the DOX loading and encapsulation efficiency was determined after washing.

[0011] ④Synthesis of the imine structure of adamantaneamine-protocatechuic aldehyde (Ada-PCA): Weigh Ada and PCA, react and treat in DMSO to obtain Ada-PCA;

[0012] ⑤ Construction of drug-loaded gold nanocage core (DOX / AuNCs-β-CD / Ada-PCA) responsive to acidic pH and glutathione (GSH) reducing environment: AuNCs-β-CD loaded with DOX was dispersed in PBS, and then the prepared adamantane-protocatechuic aldehyde imine structure (Ada-PCA) was added, and stirred, centrifuged and washed at room temperature to obtain DOX / AuNCs-β-CD / Ada-PCA;

[0013] ⑥ Preparation of platelet membrane (PM): extract platelet membrane from fresh mouse blood;

[0014] ⑦ Preparation of erythrocyte membrane (EM): extract erythrocyte membrane from fresh mouse blood;

[0015] ⑧ Preparation of platelet-erythrocyte hybrid membrane: the platelet membrane and erythrocyte membrane are mixed in a phosphate buffer solution to obtain a composite membrane solution, and the composite membrane solution is reciprocatedly extruded through a polycarbonate membrane on a liposome extruder to obtain a platelet-erythrocyte hybrid membrane solution;

[0016] ⑨ Preparation of hybrid membrane biomimetic thermosensitive liposomes (HMBTL): HMBTL was prepared by mixing platelet-erythrocyte hybrid membrane, DPPC and MSPC;

[0017] ⑩ Assembly of pH and GSH responsive drug-loaded gold nanocage core (DOX / AuNCs-β-CD / Ada-PCA) and hybrid membrane bionic thermosensitive liposome shell (HMBTL): After breaking the membrane of HMBTL, ultrasonic assembly with gold nanocage was performed.

[0018] Furthermore, the specific steps of step ② aminated modification of gold nanocages are: adding mercaptoethylamine to the gold nanocage aqueous dispersion, stirring at room temperature under nitrogen protection, centrifuging and discarding the supernatant, and vacuum drying for storage.

[0019] Furthermore, the specific steps of drug loading and encapsulation of gold nanocages in step ③ are as follows: doxorubicin hydrochloride (DOX) is loaded into the aminated gold nanocages by diffusion: the aminated gold nanocages are suspended in the DOX solution and stirred, the supernatant and precipitate are collected by centrifugation, and the precipitate is the aminated gold nanocages loaded with DOX, which are then added with excess carboxymethyl β-cyclodextrin activated by EDC / NHS and continuously stirred, the product is collected and washed with ultrapure water, and finally the DOX content in all supernatants is determined by a fluorescence spectrophotometer, and the drug loading and encapsulation rate of DOX in the particles is calculated.

[0020] Furthermore, the specific steps of synthesizing the imine structure of adamantane amine-protocatechuic aldehyde (Ada-PCA) in step ④ are as follows: weigh adamantane methylamine (Ada) and protocatechuic aldehyde (PCA), and dissolve them in DMSO respectively; then mix the two solutions and stir them at 50° C. for 4 hours; after concentrating the solution with a rotary evaporator, wash it with ether and methanol to obtain the final product, which is recorded as Ada-PCA.

[0021] Furthermore, the specific steps of step ⑥ platelet membrane (PM) preparation are as follows: first, fresh blood of mice is collected and carefully placed in an EP tube pre-treated with EDTA, and then placed in a low-temperature environment for centrifugation for 20 minutes to separate platelet-rich plasma; then, the platelet-containing plasma and PBS containing EDTA, prostaglandin E1 and protease inhibitors are fully mixed, and centrifuged again at 3400 rpm for 20 minutes at 4°C to further collect platelet precipitates; thereafter, the obtained platelet EDTA suspension is transferred to a -80°C refrigerator for deep freezing treatment, and then placed at room temperature for natural thawing, and freeze-thaw cycles are repeated for multiple times; after freeze-thawing is completed, centrifuge at 8000 rpm for 5 minutes at a low temperature of 4°C to collect the precipitate, and then the precipitate is carefully washed several times with a PBS solution containing protease inhibitors, and finally a pure platelet membrane is successfully obtained.

[0022] Furthermore, the specific steps of step ⑦ for preparing red blood cell membrane (EM) are as follows: first, fresh blood of mice is collected and placed in a centrifuge tube treated with EDTA (ethylenediaminetetraacetic acid), followed by centrifugation under low temperature conditions, the supernatant is separated and retained, and the precipitate is washed; then, the blood cells are transferred to a new container, a phosphate buffer solution is added, and the container is placed in an ice water bath to fully lyse the red blood cells; thereafter, hemoglobin is removed by high-speed centrifugation, the precipitate is washed several times and centrifuged again, and finally a pure red blood cell membrane is obtained; after the red blood cell membrane is dispersed with a phosphate buffer solution, it is stored in a refrigerator at 4°C for use.

[0023] Furthermore, step ⑨ includes the following specific steps for preparing hybrid membrane biomimetic thermosensitive liposomes (HMBTL): adding platelet-erythrocyte hybrid membrane, DPPC (dipalmitoylphosphatidylcholine) and MSPC (1-myristoyl-2-stearoylphosphatidylcholine) to PBS containing protease inhibitors and a pH value of 7.4, mixing them evenly, and incubating them at 37°C for 20 minutes; then, performing ultrasonic treatment at 37°C and 100 W power for 1 minute, and then continuing to incubate at 37°C for 30 minutes; finally, repeatedly extruding using a polycarbonate membrane with a pore size of 100 nm in a liposome extruder to obtain uniform hybrid membrane biomimetic thermosensitive liposomes (HMBTL).

[0024] Furthermore, the specific steps of step ⑩ assembling the pH and GSH responsive drug-loaded gold nanocage inner core (DOX / AuNCs-β-CD / Ada-PCA) and the hybrid membrane bionic thermosensitive liposome outer shell (HMBTL) are as follows: the hybrid membrane bionic thermosensitive liposome with a suitable phase transition temperature screened in step ⑨ is repeatedly frozen and thawed to break the membrane, and the pH and GSH responsive drug-loaded gold nanocage inner core is added and placed in a small glass bottle. At 18°C ​​and 53kHz, ultrasound is performed at a power of 100 W for 2 minutes to assemble the hybrid membrane bionic nanodrug DOX / AuNCs-β-CD / Ada-PCA / HMBTL with a core-shell structure.

[0025] The beneficial effects of the present invention are:

[0026] 1. The thermosensitive liposomes are modified with a hybrid membrane formed by the red blood cell membrane and the platelet membrane. The long circulation and targeting properties of the nanoformulation are enhanced by taking advantage of the long circulation of the red blood cell membrane and the characteristics of the protein on the platelet membrane, without the need to use PEG and additional target heads to avoid "accelerated blood clearance".

[0027] 2. Protocatechuic aldehyde is cleverly selected to stabilize biomimetic thermosensitive liposomes, reduce the risk of drug release outside tumor cells, and reduce toxicity to normal tissues: the functional groups in the protocatechuic aldehyde molecule interact with the cell membrane and the lipid bilayer of the liposome, regulate the physical properties of the cell membrane and liposome, such as increasing membrane fluidity, while filling the gaps between lipid molecules, enhancing the stability of the lipid bilayer structure, and reducing leakage of contents. During the preparation, storage and in vivo circulation, it helps to maintain the structural integrity and functional stability of the nanoformulation. The properties of protocatechuic aldehyde that inhibit platelet membrane aggregation and promote the orderly arrangement of the lipid bilayer synergize with other components in the nanoformulation to further improve the stability of the entire nanosystem. This is not only conducive to the effective encapsulation and transportation of drugs, but also ensures the performance reliability of nanoformulations in complex physiological environments, providing strong support for the realization of efficient tumor treatment.

[0028] 3. The nanoformulation has multiple stimulus response properties such as temperature, low pH value, and glutathione (GSH) reducing environment. In tumor tissue, near-infrared light stimulates the gold nanocage to generate heat, causing the hybrid membrane bionic thermosensitive liposome to decompose, and the pH-sensitive bonds on the surface of the core to hydrolyze in an acidic environment. At the same time, the high concentration of glutathione in the cell promotes the detachment of the blocking agent, which together achieves the precise release of the drug in the tumor cells, further improving the therapeutic effect of the drug and reducing toxic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Transmission electron microscopy (TEM) images of gold nanocages (AuNCs);

[0030] Figure 2TEM image of the multi-stimulus responsive bionic nanoformulation (DOX / AuNCs-β-CD / Ada-PCA / HMBTL) obtained by assembling the pH and GSH responsive drug-loaded gold nanocage core (DOX / AuNCs-β-CD / Ada-PCA) with the hybrid membrane bionic thermosensitive liposome shell (HMBTL). DETAILED DESCRIPTION

[0031] The technical solution of the present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0032] A multi-stimulus responsive bionic nanoformulation is a bionic nano core-shell system with multi-stimulus responsiveness, comprising an inner core and an outer shell, wherein the inner core is a drug-loaded gold nanocage (AuNCs), the surface of the gold nanocage is connected to β-cyclodextrin (β-CD) through a glutathione-sensitive gold-sulfur bond (Au-S) to block the drug, the imine (Ada-PCA) formed by adamantane-protocatechuic aldehyde is embedded on the surface of the nanoparticle through the host-guest interaction between cyclodextrin and adamantane, and the outer shell is a hybrid membrane bionic thermosensitive liposome (HMBTL), which comprises a platelet membrane (PM), an erythrocyte membrane (EM) and a thermosensitive liposome (TSL).

[0033] A method for preparing a multi-stimulus responsive bionic nanoformulation, the method comprising the following steps:

[0034] ① Preparation of gold nanocages (AuNCs): First, silver nitrate was reduced using polyvinyl pyrrolidone (PVP) and sodium hydrosulfide to form silver nanocubes, which were used as templates. The silver nanocubes were then etched into hollow porous gold nanocages through electrochemical replacement reactions. The amount of HAuCl4 added was adjusted to obtain gold nanocages with a localized surface plasmon resonance (LSPR) wavelength of 750-800nm.

[0035] ②Amino modification of gold nanocages: 15 mL of 3×10 -10 Excess mercaptoethylamine was added to the mol / L gold nanocage aqueous dispersion, stirred at room temperature under nitrogen protection for 4 h, centrifuged and the supernatant was discarded, and vacuum dried for storage.

[0036] ③ Drug loading and encapsulation of gold nanocages: Doxorubicin hydrochloride (DOX) was loaded into the aminated gold nanocages by diffusion: the aminated gold nanocages were suspended in 15 mL of DOX solution (1 mg / mL) and stirred for 24 hours. The supernatant and precipitate were collected by centrifugation. The precipitate was the aminated gold nanocages loaded with DOX, which was then added with excess carboxymethyl β-cyclodextrin activated by EDC / NHS and stirred for 24 hours. The product was collected and washed with ultrapure water for 5 times. Finally, the DOX content in all supernatants was determined by a fluorescence spectrophotometer, and the drug loading and encapsulation efficiency of DOX in the particles was calculated.

[0037] ④ Synthesis of the imine structure of adamantane-protocatechuic aldehyde (Ada-PCA): Weigh 1.1 equivalents of 1-adamantanemethylamine (Ada) and 0.9 equivalents of protocatechuic aldehyde (PCA) and dissolve them in DMSO respectively. Then mix the two solutions and stir them at 50°C for 4 hours. After concentrating the solution with a rotary evaporator, wash it with ether and methanol 5 times to obtain the final product, which is recorded as Ada-PCA.

[0038] ⑤ Construction of drug-loaded gold nanocage core (DOX / AuNCs-β-CD / Ada-PCA) with acidic pH and glutathione (GSH) reducing environment response: AuNCs-β-CD loaded with DOX was dispersed in PBS (10 mL, pH 7.4), and then an excess of previously prepared adamantane-protocatechuic aldehyde imine structure (Ada-PCA, whose phenylimine bond begins to break at acidic pH 6.8) was added and stirred at room temperature for 12 hours. The precipitate was collected by centrifugation and washed 5 times with PBS to obtain a system loaded with doxorubicin.

[0039] ⑥ Preparation of platelet membrane (PM): First, collect fresh blood from mice and carefully place it in a 1.5mL EP tube pre-treated with EDTA, then place it in a low-temperature environment of 4°C and centrifuge it at 1200rpm for 20 minutes to separate platelet-rich plasma. Then, mix the platelet-containing plasma with PBS containing 1mM EDTA, 2mM prostaglandin E1 and 1% protease inhibitors, and centrifuge it again at 3400rpm for 20 minutes at 4°C to further collect platelet precipitates. After that, transfer the obtained platelet EDTA suspension to a -80°C refrigerator for deep freezing, and then place it at room temperature to thaw naturally, and repeat the freeze-thaw cycle several times. After the freeze-thaw cycle is completed, centrifuge it at 8000rpm at a low temperature of 4°C for 5 minutes, collect the precipitate, and then use a PBS solution containing protease inhibitors to carefully wash the precipitate several times, and finally successfully obtain a relatively pure platelet membrane.

[0040] ⑦ Preparation of erythrocyte membrane (EM): First, collect fresh blood from mice and put it into a 1.5mL EP centrifuge tube treated with EDTA (ethylenediaminetetraacetic acid), then centrifuge at 4°C, separate the supernatant and retain it, and wash the precipitate. Subsequently, transfer the blood cells to a new container, add a phosphate buffer solution equivalent to 0.25 times its volume, and place it in an ice water bath to fully lyse the red blood cells. After that, remove hemoglobin by high-speed centrifugation, wash the obtained precipitate several times and centrifuge it again, and finally obtain a relatively pure erythrocyte membrane. After dispersing the erythrocyte membrane with phosphate buffer solution, store it in a refrigerator at 4°C for use.

[0041] ⑧ Preparation of platelet-erythrocyte hybrid membrane: Mix the erythrocyte membrane and platelet membrane in an appropriate amount of phosphate buffer solution. Then, pass the mixed system through 400nm and 200nm polycarbonate membranes in turn, and reciprocate for 40 times in a liposome extruder to obtain a hybrid membrane dispersion of erythrocyte membrane and platelet membrane.

[0042] ⑨ Preparation of hybrid membrane biomimetic thermosensitive liposomes (HMBTL): Add platelet-erythrocyte hybrid membrane, DPPC (dipalmitoylphosphatidylcholine) and MSPC (1-myristoyl-2-stearoylphosphatidylcholine) of different mass ratios to PBS containing protease inhibitors and pH 7.4, mix well, and incubate at 37°C for 20 minutes. Then, perform ultrasonic treatment at 37°C and 100W power for 1 minute, and then continue incubation at 37°C for 30 minutes. Finally, use a polycarbonate membrane with a pore size of 100nm in a liposome extruder to repeatedly extrude to obtain uniform hybrid membrane biomimetic thermosensitive liposomes (HMBTL).

[0043] ⑩ Assembly of pH and GSH responsive drug-loaded gold nanocage inner core (DOX / AuNCs-β-CD / Ada-PCA) and hybrid membrane bionic thermosensitive liposome outer shell (HMBTL): The hybrid membrane bionic thermosensitive liposome with a suitable phase transition temperature screened by the above method was repeatedly frozen and thawed to break the membrane, and the pH and GSH responsive drug-loaded gold nanocage inner core was added and placed in a small glass bottle. At 18°C ​​and 53kHz, ultrasound was performed at a power of 100 W for 2 minutes to assemble the hybrid membrane bionic nanodrug DOX / AuNCs-β-CD / Ada-PCA / HMBTL with a core-shell structure.

[0044] Figure 1 This is a transmission electron microscope (TEM) image of gold nanocages (AuNCs), which have a smooth surface and are not a core-shell structure. Figure 2TEM image of the multi-stimuli responsive bionic nanoformulation (DOX / AuNCs-β-CD / Ada-PCA / HMBTL) obtained by assembling the pH and GSH responsive drug-loaded gold nanocage core (DOX / AuNCs-β-CD / Ada-PCA) with the hybrid membrane bionic thermosensitive liposome shell (HMBTL). Figure 1 In comparison, a light-colored, more uniform hybrid membrane biomimetic thermosensitive liposome shell can be observed on the surface of the gold nanocage core.

[0045] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. All modifications, equivalent substitutions, improvements, etc. within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A multi-stimulus responsive bionic nanoformulation, characterized in that: It is a bionic nano core-shell system with multiple stimulus responsiveness, including an inner core and an outer shell. The inner core is a drug-loaded gold nanocage (AuNCs). The surface of the gold nanocage is connected to β-cyclodextrin (β-CD) through glutathione-sensitive gold-sulfur bonds (Au-S) to block the drug. The imine (Ada-PCA) formed by adamantane-protocatechuic aldehyde is embedded on the surface of the nanoparticle through the host-guest interaction between cyclodextrin-adamantane. The outer shell is a hybrid membrane bionic thermosensitive liposome (HMBTL), which contains platelet membrane (PM), erythrocyte membrane (EM) and thermosensitive liposome (TSL).

2. The method for preparing a multi-stimulus responsive bionic nanoformulation according to claim 1, characterized in that: The method comprises the following steps: ① Preparation of gold nanocages (AuNCs): Add silver nitrate, PVP and sodium hydrosulfide solution into a flask, stir and react to obtain silver nanocubes; then add the silver nanocube dispersion into an aqueous solution containing chloroauric acid (HAuCl4), stir and react to obtain gold nanocages, and control the amount of HAuCl4 to make the LSPR wavelength of the gold nanocages 750-800nm; ②Amino modification of gold nanocages: Take the aqueous dispersion of gold nanocages, add mercaptoethylamine, stir, centrifuge and dry; ③ Drug loading and encapsulation of gold nanocages: The amino gold nanocages and DOX solution were mixed, stirred, and centrifuged. The precipitate was reacted with carboxymethyl β-cyclodextrin, and the DOX loading and encapsulation efficiency was determined after washing. ④Synthesis of the imine structure of adamantaneamine-protocatechuic aldehyde (Ada-PCA): Weigh Ada and PCA, react them in DMSO, and treat to obtain Ada-PCA; ⑤ Construction of drug-loaded gold nanocage core (DOX / AuNCs-β-CD / Ada-PCA) responsive to acidic pH and glutathione (GSH) reducing environment: AuNCs-β-CD loaded with DOX was dispersed in PBS, and then the prepared adamantane-protocatechuic aldehyde imine structure (Ada-PCA) was added, and stirred, centrifuged and washed at room temperature to obtain DOX / AuNCs-β-CD / Ada-PCA; ⑥ Preparation of platelet membrane (PM): extract platelet membrane from fresh mouse blood; ⑦ Preparation of erythrocyte membrane (EM): extract erythrocyte membrane from fresh mouse blood; ⑧ Preparation of platelet-erythrocyte hybrid membrane: platelet membrane and erythrocyte membrane are mixed in phosphate buffer solution to obtain a composite membrane solution, which is reciprocatedly extruded through a polycarbonate membrane on a liposome extruder to obtain a platelet-erythrocyte hybrid membrane solution; ⑨ Preparation of hybrid membrane biomimetic thermosensitive liposomes (HMBTL): HMBTL was prepared by mixing platelet-erythrocyte hybrid membrane, DPPC (dipalmitoylphosphatidylcholine) and MSPC (1-myristoyl-2-stearoylphosphatidylcholine); ⑩ Assembly of pH and GSH responsive drug-loaded gold nanocage core (DOX / AuNCs-β-CD / Ada-PCA) and hybrid membrane bionic thermosensitive liposome shell (HMBTL): After breaking the membrane of HMBTL, ultrasonic assembly with gold nanocage was performed.

3. The method for preparing a multi-stimulus responsive bionic nanoformulation according to claim 1, characterized in that: Step ② The specific steps of amino modification of gold nanocages are: add mercaptoethylamine to the gold nanocage water dispersion, stir at room temperature under nitrogen protection, centrifuge and discard the supernatant, and vacuum dry and store for later use.

4. The method for preparing a multi-stimulus responsive bionic nanoformulation according to claim 1, characterized in that: Step ③ The specific steps of drug loading and encapsulation of gold nanocages are as follows: doxorubicin hydrochloride (DOX) is loaded into the aminated gold nanocages by diffusion; the aminated gold nanocages are suspended in the DOX solution and stirred, and the supernatant and precipitate are collected by centrifugation to obtain the aminated gold nanocages loaded with DOX, which are then added with excess carboxymethyl β-cyclodextrin activated by EDC / NHS and continuously stirred, the products are collected and washed with ultrapure water, and finally the DOX content in all supernatants is determined by a fluorescence spectrophotometer, and the drug loading and encapsulation rate of DOX in the particles is calculated.

5. The method for preparing a multi-stimulus responsive bionic nanoformulation according to claim 1, characterized in that: Step ④ The specific steps for synthesizing the imine structure of adamantane amine-protocatechuic aldehyde (Ada-PCA) are as follows: weigh adamantane methylamine (Ada) and protocatechuic aldehyde (PCA), and dissolve them in DMSO respectively; then mix the two solutions and stir them at 50° C. for 4 hours; concentrate the solution with a rotary evaporator, and wash with ether and methanol to obtain the final product, which is recorded as Ada-PCA.

6. The method for preparing a multi-stimulus responsive bionic nanoformulation according to claim 1, characterized in that: Step ⑥ The specific steps for preparing the platelet membrane (PM) are as follows: first, collect fresh blood from mice and carefully place it in an EP tube that has been pre-treated with EDTA, then place it in a low-temperature environment and centrifuge it for 20 minutes to separate platelet-rich plasma; then, fully mix the platelet-containing plasma and PBS containing EDTA, prostaglandin E1 and protease inhibitors, and centrifuge it again at 3400 rpm for 20 minutes at 4°C to further collect the platelet precipitate; then, transfer the obtained platelet EDTA suspension to a -80°C refrigerator for deep freezing, and then place it at room temperature to thaw naturally, and repeat the freeze-thaw cycle operation for multiple times; after the freeze-thaw is completed, centrifuge it at 8000 rpm for 5 minutes at a low temperature of 4°C, collect the precipitate, and then use a PBS solution containing protease inhibitors to carefully wash the precipitate for multiple times, and finally successfully obtain a pure platelet membrane.

7. The method for preparing a multi-stimulus responsive bionic nanoformulation according to claim 1, characterized in that: Step ⑦ Specific steps for preparing red blood cell membrane (EM): First, collect fresh blood from mice and put it into a centrifuge tube treated with EDTA (ethylenediaminetetraacetic acid), then centrifuge it under low temperature conditions, separate the supernatant and retain it, and wash the precipitate; then, transfer the blood cells to a new container, add phosphate buffer solution, and place it in an ice water bath to fully lyse the red blood cells; then, remove hemoglobin by high-speed centrifugation, wash the obtained precipitate several times and centrifuge it again, and finally obtain pure red blood cell membrane; after dispersing the red blood cell membrane with phosphate buffer solution, store it in a refrigerator at 4°C for use.

8. The method for preparing a multi-stimulus responsive bionic nanoformulation according to claim 1, characterized in that: Step ⑨ The specific steps for preparing hybrid membrane biomimetic thermosensitive liposomes (HMBTL) are as follows: platelet-erythrocyte hybrid membrane, DPPC (dipalmitoylphosphatidylcholine) and MSPC (1-myristoyl-2-stearoylphosphatidylcholine) are added to PBS containing protease inhibitors and a pH value of 7.4, mixed evenly, and incubated at 37° C. for 20 minutes; then, ultrasonic treatment is performed at 37° C. and 100 W power for 1 minute, and then incubated at 37° C. for 30 minutes; finally, repeated extrusion is performed using a polycarbonate membrane with a pore size of 100 nm in a liposome extruder to obtain uniform hybrid membrane biomimetic thermosensitive liposomes (HMBTL).

9. The method for preparing a multi-stimulus responsive bionic nanoformulation according to claim 1, characterized in that: Step ⑩ The specific steps of assembling the pH and GSH responsive drug-loaded gold nanocage inner core (DOX / AuNCs-β-CD / Ada-PCA) and the hybrid membrane bionic thermosensitive liposome outer shell (HMBTL): repeatedly freeze-thaw the hybrid membrane bionic thermosensitive liposome with a suitable phase transition temperature screened in step ⑨ to break the membrane, add the pH and GSH responsive drug-loaded gold nanocage inner core, place it in a small glass bottle, and ultrasonicate it at 18°C ​​and 53kHz for 2 minutes at a power of 100W to assemble the hybrid membrane bionic nanodrug DOX / AuNCs-β-CD / Ada-PCA / HMBTL with a core-shell structure.

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