Multifunctional bionic nano-drug as well as preparation method and application thereof

Abstract

The invention discloses a multifunctional bionic nano-drug and a preparation method and application thereof in the technical field of antitumor drugs. The multifunctional bionic nano-drug comprises a drug-loaded cationic micelle and a tumor cell membrane wrapping the drug-loaded cationic micelle, and the drug-loaded cationic micelle comprises a cationic micelle, a photosensitizer and a fat-soluble HSP90 inhibitor drug. A photosensitizer and a fat-soluble HSP90 inhibitor drug are connected through pi-pi stacking to form a photosensitizer-fat-soluble HSP90 inhibitor drug, the photosensitizer-fat-soluble HSP90 inhibitor drug is loaded in a hydrophobic cavity in the cationic micelle, further, the drug-loaded cationic micelle further comprises HIF-1 alpha siRNA, and the HIF-1 alpha siRNA is adsorbed on the surface of the cationic micelle. The multifunctional bionic nano-drug disclosed by the invention can realize multi-mode combined killing of tumors through low-temperature thermal therapy, ferroptosis and immunotherapy, and can better inhibit the growth of tumor cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-tumor drugs, and in particular relates to a multifunctional bionic nano drug and a preparation method and application thereof. Background Art

[0002] Photodynamic therapy (PDT) and photothermal therapy (PTT) are both phototherapy, which are cell-killing strategies based on photophysical principles. PDT damages cell structures by generating reactive oxygen species (ROS), while PTT kills tumor cells through thermal energy.

[0003] Immunogenic cell death (ICD) occurs by exposing and releasing damage-associated molecular patterns (DAMPs) into the tumor microenvironment (TME), which are recognized by antigen-presenting cells (mainly dendritic cells (DCs)) and trigger immune responses. The exposure and secretion of DAMPs, including adenosine triphosphate (ATP), calreticulin (CRT), and high-mobility group protein B1 (HMGB1), ultimately lead to the maturation of DCs and the activation of CD8 + Recruitment and activation of T cell-mediated immune responses against tumor cells. The ICD effect induced by PDT and PTT can stimulate the immune response to a certain extent.

[0004] Indocyanine green (ICG) is an excellent photosensitizer that produces a large amount of ROS and heat under 808nm laser irradiation, so it can meet the needs of both PDT and PTT treatments. However, ICG alone is not effective in treating tumors. Summary of the invention

[0005] In order to solve the deficiencies in the prior art, the present invention provides a multifunctional bionic nanomedicine and a preparation method and application thereof.

[0006] Biomimetic nanoparticles are hybrid nanostructures in which the outermost layer is decorated with biological ligands or biological cell membranes. This approach has multiple advantages, including escape from biological systems, prolonged residence time in the blood circulation, targeted delivery, and reduced systemic toxicity. Cancer cell membranes are ideal candidates for encapsulating nanoparticles due to the unique feature of cancer cells that enable them to self-target homologous cells.

[0007] Heat shock protein 90 (HSP90) is an important protein that enhances cell heat resistance, and it can also maintain the stability of the organism by forming complexes with other functional molecules. Garcinia acid (GA) is a natural HSP90 inhibitor with anti-tumor, anti-angiogenic and anti-metastatic activities. Ferroptosis usually occurs in the presence of reactive oxygen species, producing toxic lipid peroxides (LPO) that damage structures such as cell membranes and organelle membranes. Glutathione (GSH) is the core substance in the ferroptosis process. It is an important antioxidant that protects cells from oxidative damage and is also a substrate for the synthesis of glutathione peroxidase 4 (GPX4). In the process of ferroptosis, GPX4 is an important enzyme that removes ROS in the cytoplasm.

[0008] HIF1 (hypoxia inducible factor-1), whose Chinese name is hypoxia inducible factor-1, is a heterodimer composed of two basic helix-loop-helix PAS (Per-ARNT-Sim) proteins HIF-1α and HIF-1β.

[0009] The specific technical solution of the present invention is:

[0010] The present invention provides a multifunctional bionic nanomedicine, which comprises a drug-loaded cationic micelle and a tumor cell membrane wrapped outside the drug-loaded cationic micelle, wherein the drug-loaded cationic micelle comprises a cationic micelle, a photosensitizer and a lipophilic HSP90 inhibitor drug, the photosensitizer and the lipophilic HSP90 inhibitor drug are connected by ππ stacking to form a photosensitizer-lipophilic HSP90 inhibitor drug, and the photosensitizer-lipophilic HSP90 inhibitor drug is loaded in a hydrophobic cavity inside the cationic micelle.

[0011] Furthermore, the cationic micelles have a particle size of 90 to 160 nm and a charge of +15 to +30 mV;

[0012] Preferably, the particle size of the cationic micelle is 120 to 160 nm;

[0013] Preferably, the particle size of the cationic micelle is 130 to 150 nm;

[0014] Preferably, the cationic micelles are prepared from lecithin, polyethylene glycol-15 hydroxystearate and DOTAP.

[0015] Furthermore, the photosensitizer is selected from one or more of indocyanine green, Ce6, methylene blue, and acridine orange;

[0016] The fat-soluble HSP90 inhibitor drug is selected from one or more of gambogic acid, gedunin, and 17-DMAG;

[0017] Preferably, the mass ratio of the photosensitizer to the lipophilic HSP90 inhibitor is 0.5-1:1.5-3;

[0018] Preferably, the photosensitizer is selected from indocyanine green;

[0019] Preferably, the lipophilic HSP90 inhibitor drug is selected from gambogic acid.

[0020] Furthermore, the drug-loaded cationic micelles also include HIF-1α siRNA, and the HIF-1α siRNA is adsorbed on the surface of the cationic micelles;

[0021] Preferably, the mass ratio of the cationic micelle to HIF-1α siRNA is 4 to 10:1;

[0022] Preferably, the mass ratio of the cationic micelle to HIF-1α siRNA is 4 to 8:1;

[0023] Preferably, the nucleotide sequence of the HIF-1α siRNA is as shown in SEQ ID NO.1.

[0024] The present invention also provides a method for preparing the multifunctional bionic nanomedicine, comprising the following steps:

[0025] (1) connecting a photosensitizer and a lipophilic HSP90 inhibitor drug via ππ stacking to form a photosensitizer-lipophilic HSP90 inhibitor drug;

[0026] (2) mixing a photosensitizer-lipid-soluble HSP90 inhibitor drug, a lipid material required for preparing cationic micelles, and a first organic solvent, and then preparing drug-loaded cationic micelles by a thin film hydration method;

[0027] (3) dissolving HIF-1α siRNA in a second solvent, and then mixing it with the drug-loaded cationic micelles obtained in step (2), and incubating the mixture by flipping to obtain drug-loaded cationic micelles adsorbing HIF-1α siRNA;

[0028] (4) The cationic micelles loaded with HIF-1α siRNA adsorbed thereon and tumor cell membrane fragments are mixed and self-assembled to obtain the multifunctional bionic nanomedicine according to claim 1.

[0029] Furthermore, the method for forming a photosensitizer-lipid-soluble HSP90 inhibitor drug by ππ stacking connection in step (1) is: dissolving the photosensitizer and the lipid-soluble HSP90 inhibitor drug in DMSO respectively, mixing them and then ultrasonically treating them, and finally freeze-drying them;

[0030] Preferably, the ultrasonic treatment conditions are: ultrasonic power is 150-250 W, preferably 190-220 W, and ultrasonic time is 10-15 min, preferably 11-13 min.

[0031] Furthermore, the lipid materials required for preparing the cationic micelles in step (2) are lecithin, polyethylene glycol-15 hydroxystearate and DOTAP;

[0032] In step (2), the first organic solvent is anhydrous ethanol;

[0033] Preferably, the ratio of the mass of the photosensitizer-lipid-soluble HSP90 inhibitor drug to the volume of the first organic solvent is 5-10:10-20 mg / mL, preferably 5-7.5 mg:10-15 mg / mL;

[0034] Preferably, the mass ratio of the photosensitizer-lipid-soluble HSP90 inhibitor drug, lecithin, polyethylene glycol-15 hydroxystearate and DOTAP is 1-5:10-50:10-50:3-15;

[0035] Preferably, the mass ratio of the photosensitizer-lipid-soluble HSP90 inhibitor drug, lecithin, polyethylene glycol-15 hydroxystearate and DOTAP is 2-4:20-40:20-40:6-12;

[0036] Preferably, the rotary evaporation temperature of the thin film hydration method is 45-55°C, preferably 50°C, and the rotary evaporation speed is 60-80r / min, preferably 65-75r / min; the hydration process conditions of the thin film hydration method are: 10-15mL of ultrapure water, hydration temperature of 45-55°C, preferably 50°C, and hydration time of 30-60min, preferably 40-50min.

[0037] Furthermore, in step (3), the second solvent is a DEPC aqueous solution;

[0038] Preferably, the temperature of the flip incubation in step (3) is 2 to 8°C, preferably 4 to 6°C, the rotation speed is 30 to 40 r / min, and the time is 4 to 6 h.

[0039] Furthermore, the preparation method of tumor cell membrane fragments in step (4) is as follows: the cell fragment solution after ultrasonic disruption is subjected to differential centrifugation to extract membrane fragments, the low-speed centrifugation temperature is 2-8°C, the speed is 2000-4000 r / min, and the time is 10-15 min; the high-speed centrifugation temperature is 2-8°C, the speed is 12000-14000 r / min, and the time is 30-40 min; preferably, the low-speed centrifugation temperature is 4-6°C, the speed is 2500-3000 r / min; the high-speed centrifugation temperature is 4-6°C, the speed is 12500-13000 r / min, and the time is 40-50 min.

[0040] In step (4), the mixed self-assembly is carried out by ultrasonic method, the ultrasonic power is 150-250W, the ultrasonic temperature is 4-6°C, the ultrasonic time is 10-20min, and the ultrasonic time is preferably 14-16min.

[0041] The present invention also provides the use of the multifunctional bionic nano drug in the preparation of anti-tumor drugs.

[0042] Furthermore, the multifunctional bionic nanomedicine is injected into the patient's body and treated under near-infrared light. In the present invention, the wavelength of the near-infrared light is preferably 800-900 nm, more preferably 808 nm.

[0043] The multifunctional bionic nanomedicine provided by the present invention, on the one hand, the photosensitizer can generate heat and ROS under laser irradiation, and simultaneously meet the PDT and PTT treatments; the lipophilic HSP90 inhibitor drugs inhibit the expression of HSP90, thereby increasing the binding of RACK1 to the PAS-A domain of HIF-1α, promoting the degradation of HIF-1α, and the reduction of the intracellular HIF-1α content ultimately affects the expression of the cell surface PDL1 receptor, and enhances the tumor immune checkpoint blocking effect. Further, the HIF-1α siRNA adsorbed on the surface of the cationic micelle inhibits the expression of HIF-1α in tumor cells at the gene level, silences the expression of HIF-1α, and works together with the lipophilic HSP90 inhibitor drugs to reduce the intracellular HIF-1α content, ultimately affecting the expression of the cell surface PDL1 receptor, and enhancing the tumor immune checkpoint blocking effect. On the other hand, the inhibition of HSP90 will also reduce the content of GSH, thereby reducing the content of GPX4, and inducing tumor cell iron death when PDT produces reactive oxygen. Finally, the inhibition of HSP90 will also reduce the heat resistance of tumor cells, achieve low-temperature hyperthermia, reduce damage to normal tissues, and enhance the effect of tumor hyperthermia during the PTT process. The combined action of PTT and PDT will induce a strong ICD effect, ultimately leading to effective immunotherapy.

[0044] The beneficial effects of the present invention are:

[0045] The present invention provides a multifunctional bionic nanomedicine, which includes a drug-loaded cationic micelle and a tumor cell membrane wrapped outside the drug-loaded cationic micelle, wherein the drug-loaded cationic micelle includes a cationic micelle, a photosensitizer and a lipophilic HSP90 inhibitor drug, the photosensitizer and the lipophilic HSP90 inhibitor drug are connected by ππ stacking to form a photosensitizer-lipophilic HSP90 inhibitor drug, the photosensitizer-lipophilic HSP90 inhibitor drug is loaded in the hydrophobic cavity inside the cationic micelle, preferably, the drug-loaded cationic micelle also includes HIF-1α siRNA, and the HIF-1α siRNA is adsorbed on the surface of the cationic micelle. The multifunctional bionic nanomedicine of the present invention gathers around the tumor tissue through active targeting (homologous targeting effect) and passive targeting (EPR effect), and after entering the tumor cell, the photosensitizer generates ROS to oxidize the lipid material of the synthetic micelle under 808nm laser irradiation, releases the anti-tumor drug, and kills the tumor cell. The multifunctional bionic nanomedicine of the present invention can effectively release chemotherapeutic drugs and photosensitizers, accurately act on different sites, improve the tumor microenvironment, cause changes in HSP90 and ferroptosis-related proteins, and jointly reduce the expression of HIF-1α in both protein and gene aspects, realize low-temperature hyperthermia, ferroptosis, and immunotherapy multi-mode combined tumor killing, and better inhibit the growth of tumor cells. In addition, the multifunctional bionic nanomedicine of the present invention has no toxic side effects on normal cells, is easily metabolized in the body, and has good biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The transmission electron microscopy image of the cationic micelle loaded with drug ICG-GA prepared in Example 1;

[0047] Figure 2 This is a transmission electron microscope image of the multifunctional bionic nanoscale drug prepared in Example 1;

[0048] Figure 3 The ROS generation of different materials under 808nm laser irradiation in Example 2;

[0049] Figure 4 The temperature changes of different materials in Example 3 under 808nm laser irradiation;

[0050] Figure 5 This is the survival rate of tumor cells after being treated with different materials in Example 3. DETAILED DESCRIPTION

[0051] In a specific embodiment, the present invention provides a multifunctional bionic nanomedicine, which includes a drug-loaded cationic micelle and a tumor cell membrane wrapped outside the drug-loaded cationic micelle, wherein the drug-loaded cationic micelle includes a cationic micelle, a photosensitizer and a lipophilic HSP90 inhibitor drug, and the photosensitizer and the lipophilic HSP90 inhibitor drug are connected by ππ stacking to form a photosensitizer-lipophilic HSP90 inhibitor drug, and the photosensitizer-lipophilic HSP90 inhibitor drug is loaded in the hydrophobic cavity inside the cationic micelle.

[0052] In a specific embodiment, the cationic micelles have a particle size of 90 to 160 nm and a charge of +15 to +30 mV.

[0053] In one embodiment, the particle size of the cationic micelle is 120 to 160 nm, preferably 130 to 150 nm. In the present invention, the internal hydrophobic cavity of the cationic micelle provides space for loading photosensitizers and other lipophilic drugs.

[0054] In one embodiment, the cationic micelles are prepared from lecithin, polyethylene glycol-15 hydroxystearate and DOTAP.

[0055] In a specific embodiment, the photosensitizer is selected from one or more of indocyanine green, Ce6, methylene blue, and acridine orange, preferably indocyanine green.

[0056] In a specific embodiment, the lipophilic HSP90 inhibitor drug is selected from one or more of gambogic acid, gedunin, and 17-DMAG, preferably gambogic acid.

[0057] In a specific embodiment, the mass ratio of the photosensitizer to the lipophilic HSP90 inhibitor is 0.5-1:1.5-3.

[0058] In a specific embodiment, the drug-loaded cationic micelles also include HIF-1α siRNA, and the negatively charged HIF-1α siRNA is adsorbed on the surface of the cationic micelles by electrostatic adsorption. Preferably, the mass ratio of cationic micelles to HIF-1α siRNA is 4 to 10:1, preferably 4 to 8:1.

[0059] In a specific embodiment, the nucleotide sequence of HIF-1α siRNA is: UCAAGUUGCUGGUCAUCAGTT, as shown in SEQ ID NO.1.

[0060] In the present invention, the cationic micelles have no toxic side effects on normal cells, can accumulate at the tumor site through the dual effects of homologous targeting and passive targeting, and can circulate in the body for a long time due to the camouflage of the cell membrane, and have good biocompatibility.

[0061] In a specific embodiment, the present invention provides a method for preparing the multifunctional bionic nanomedicine, comprising the following steps:

[0062] (1) connecting a photosensitizer and a lipophilic HSP90 inhibitor drug via ππ stacking to form a photosensitizer-lipophilic HSP90 inhibitor drug;

[0063] (2) mixing a photosensitizer-lipid-soluble HSP90 inhibitor drug, a lipid material required for preparing cationic micelles, and a first organic solvent, and then preparing drug-loaded cationic micelles by a thin film hydration method;

[0064] (3) dissolving HIF-1α siRNA in a second solvent, and then mixing it with the drug-loaded cationic micelles obtained in step (2), and incubating the mixture by flipping to obtain drug-loaded cationic micelles adsorbing HIF-1α siRNA;

[0065] (4) The cationic micelles loaded with HIF-1α siRNA adsorbed thereon and tumor cell membrane fragments are mixed and self-assembled to obtain the multifunctional bionic nanomedicine according to claim 1.

[0066] Furthermore, the method for forming a photosensitizer-lipid-soluble HSP90 inhibitor drug by ππ stacking connection in step (1) is: dissolving the photosensitizer and the lipid-soluble HSP90 inhibitor drug in DMSO respectively, mixing them and then ultrasonically treating them, and finally freeze-drying them;

[0067] Preferably, the ultrasonic treatment conditions are: ultrasonic power is 150-250 W, preferably 190-220 W, and ultrasonic time is 10-15 min, preferably 11-13 min.

[0068] Furthermore, the lipid materials required for preparing the cationic micelles in step (2) are lecithin, polyethylene glycol-15 hydroxystearate and DOTAP;

[0069] In step (2), the first organic solvent is anhydrous ethanol;

[0070] Preferably, the ratio of the mass of the photosensitizer-lipid-soluble HSP90 inhibitor drug to the volume of the first organic solvent is 5-10:10-20 mg / mL, preferably 5-7.5 mg:10-15 mg / mL;

[0071] Preferably, the mass ratio of the photosensitizer-lipid-soluble HSP90 inhibitor drug, lecithin, polyethylene glycol-15 hydroxystearate and DOTAP is 1-5:10-50:10-50:3-15;

[0072] Preferably, the mass ratio of the photosensitizer-lipid-soluble HSP90 inhibitor drug, lecithin, polyethylene glycol-15 hydroxystearate and DOTAP is 2-4:20-40:20-40:6-12;

[0073] Preferably, the rotary evaporation temperature of the thin film hydration method is 45-55°C, preferably 50°C, and the rotary evaporation speed is 60-80r / min, preferably 65-75r / min; the hydration process conditions of the thin film hydration method are: 10-15mL of ultrapure water, hydration temperature of 45-55°C, preferably 50°C, and hydration time of 30-60min, preferably 40-50min.

[0074] Furthermore, in step (3), the second solvent is a DEPC aqueous solution;

[0075] Preferably, the temperature of the flip incubation in step (3) is 2 to 8°C, preferably 4 to 6°C, the rotation speed is 30 to 40 r / min, and the time is 4 to 6 h.

[0076] Furthermore, the preparation method of tumor cell membrane fragments in step (4) is as follows: the cell fragment solution after ultrasonic disruption is subjected to differential centrifugation to extract membrane fragments, the low-speed centrifugation temperature is 2-8°C, the speed is 2000-4000 r / min, and the time is 10-15 min; the high-speed centrifugation temperature is 2-8°C, the speed is 12000-14000 r / min, and the time is 30-40 min; preferably, the low-speed centrifugation temperature is 4-6°C, the speed is 2500-3000 r / min; the high-speed centrifugation temperature is 4-6°C, the speed is 12500-13000 r / min, and the time is 40-50 min.

[0077] In step (4), the mixed self-assembly is carried out by ultrasonic method, the ultrasonic power is 150-250W, the ultrasonic temperature is 4-6°C, the ultrasonic time is 10-20min, and the ultrasonic time is preferably 14-16min.

[0078] In the present invention, the multifunctional bionic nanomedicine is injected into the patient's body to treat the tumor under near-infrared light irradiation. In the present invention, the wavelength of the near-infrared light is preferably 800-900 nm, more preferably 808 nm.

[0079] In the present invention, after the multifunctional bionic nanomedicine reaches the tumor site through dual targeting, the multifunctional bionic nanomedicine enters the tumor cell through membrane fusion, and the photosensitizer produces photothermal and photodynamic effects under near-infrared light irradiation, rapidly heating up to 40-60°C, and the ROS produced at the same time will oxidize the lipid material, causing the nano drug delivery system to disintegrate, releasing the HSP90 inhibitor loaded by the drug delivery system, and killing tumor cells. The multifunctional bionic nanomedicine can effectively release HSP90 inhibitor drugs and photosensitizers, accurately act on different sites, improve the photodynamic, photothermal and immunotherapy effects, achieve good multimodal combined treatment, and better inhibit the growth of tumor cells.

[0080] In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be understood as limiting the scope of protection of the present invention. In the embodiments, all raw reagents and materials are commercially available, and the experimental methods without specific conditions are conventional methods and conventional conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0081] Example 1

[0082] (1) Indocyanine green (ICG) and gambogic acid (GA) were connected by ππ stacking to prepare ICG-GA. 1.5 mg ICG and 4 mg GA were weighed, dissolved in DMSO, and prepared into a 100 mM solution, which was mixed. After mixing, the mixture was ultrasonically treated at 190 W for 11 min. The resulting solution was freeze-dried.

[0083] (2) The ππ stacking product ICG-GA obtained after freeze-drying was dissolved in 11 mL of anhydrous ethanol, and 50 mg of lecithin, 50 mg of Solutol HS15, and 15 mg of DOTAP were weighed and added to the anhydrous ethanol for ultrasonic dissolution. The anhydrous ethanol was removed by a rotary evaporator at a rotary evaporation temperature of 50°C and a rotary evaporation speed of 65 r / min. After the ethanol was completely evaporated and a thin film was formed at the bottom of the eggplant-shaped bottle, 10 mL of ultrapure water was added, and the eggplant-shaped bottle was shaken to completely dissolve the formed thin film, and then placed in a 50°C water bath for hydration for 45 minutes to obtain a cationic micelle solution loaded with drug ICG-GA.

[0084] (3) The mass ratio of cationic micelles to HIF-1α siRNA (the nucleotide sequence of HIF-1α siRNA in this embodiment is: UCAAGUUGCUGGUCAUCAGTT; SEQ ID NO. 1) is 4:1. HIF-1α siRNA is dissolved in DEPC aqueous solution and then added to the micelle solution loaded with drug ICG-GA. The mixture is incubated at 4°C and 30 rpm for 4 h to obtain cationic micelles loaded with HIF-1α siRNA.

[0085] (4) The cell fragment solution after ultrasonic disruption of 4T1 mouse breast cancer cells was subjected to differential centrifugation to extract membrane fragments. The low-speed centrifugation time was 10 min at 4°C and 2500 r / min; and the high-speed centrifugation time was 40 min at 4°C and 12500 r / min to obtain cell membrane fragments.

[0086] (5) The cationic micelles loaded with HIF-1α siRNA and cell membrane fragments were mixed and self-assembled, and the cell membrane was coated by ultrasound. The ultrasonic power was 200 W, the temperature was 4°C, and the time was 15 min. Then the obtained solution was dialyzed in a 3500-mesh dialysis bag for 12 h to obtain the multifunctional bionic nanoscale drug CM-ICG / GAM-siRNA (CM-IGM / siRNA).

[0087] The transmission electron microscopy image of the cationic micelle loaded with drug ICG-GA prepared in step (2) is as follows: Figure 1 The transmission electron microscopy image of the multifunctional bionic nanoscale drug prepared in step (5) is shown in FIG. Figure 2 shown.

[0088] Example 2

[0089] (1) ICG-GA was prepared by connecting indocyanine green and gambogic acid by ππ stacking. 1.6 mg ICG and 4.3 mg GA were weighed, dissolved in DMSO, and prepared into a 100 mM solution, which was mixed. After mixing, the mixture was ultrasonically treated at 200 W for 12 min. The resulting solution was freeze-dried.

[0090] (2) The ππ stacking product ICG-GA obtained after freeze-drying was dissolved in 12 mL of anhydrous ethanol, and 55 mg of lecithin, 55 mg of Solutol HS15, and 16.5 mg of DOTAP were weighed and added to the anhydrous ethanol for ultrasonic dissolution. The anhydrous ethanol was removed by a rotary evaporator at a rotary evaporation temperature of 52°C and a rotary evaporation speed of 70 r / min. After the ethanol was completely evaporated and a thin film was formed at the bottom of the eggplant-shaped bottle, 11 mL of ultrapure water was added, and the eggplant-shaped bottle was shaken to completely dissolve the formed thin film, and then placed in a 52°C water bath for hydration for 50 min to obtain a cationic micelle solution loaded with drug ICG-GA.

[0091] (3) The mass ratio of cationic micelles to HIF-1α siRNA (the nucleotide sequence of HIF-1α siRNA in this embodiment is: UCAAGUUGCUGGUCAUCAGTT; SEQ ID NO. 1) is 6:1. HIF-1α siRNA is dissolved in DEPC aqueous solution and then added to the micelle solution loaded with drug ICG-GA. The mixture is incubated at 4°C and 35 rpm for 5 h to obtain cationic micelles loaded with HIF-1α siRNA.

[0092] (4) The cell fragment solution after ultrasonic disruption of 4T1 mouse breast cancer cells was subjected to differential centrifugation to extract membrane fragments. The low-speed centrifugation time was 12 min at 4°C and 2700 r / min; and the high-speed centrifugation time was 45 min at 4°C to obtain cell membrane fragments.

[0093] (5) The cationic micelles loaded with HIF-1α siRNA and cell membrane fragments were mixed and self-assembled, and the cell membrane was coated by ultrasound. The ultrasonic power was 210 W, the temperature was 4° C., and the time was 17 min. The resulting solution was then dialyzed in a 3500-mesh dialysis bag for 14 h to obtain the multifunctional bionic nanoscale drug CM-ICG / GAM-siRNA (CM-IGM / siRNA).

[0094] Since ICG will produce ROS under 808nm laser irradiation, the indicator 1,3-diphenylfuran (1,3-diphenylfuran, DPBF) is used to detect the generation of reactive oxygen species to prove the photodynamic performance. The DPBF experimental detection groups are: DPBF+H2O, DPBF+empty carrier (empty carrier refers to cationic micelles that do not include ICG and GA), DPBF+ICG, DPBF+CM-IGM / siRNA in this example. Each group of samples was added to a centrifuge tube containing 2mL of 100μg / mL DPBF ethanol solution (be careful to avoid light), irradiated with 808nm laser for 5min, and the full spectrum of each group of solutions at wavelengths of 360nm to 450nm was measured with an enzyme reader. The results are as follows Figure 3 shown.

[0095] Example 3

[0096] (1) ICG-GA was prepared by connecting indocyanine green and gambogic acid using ππ stacking. 1.8 mg ICG and 4.6 mg GA were weighed and dissolved in DMSO to prepare a 100 mM solution. After mixing, the mixture was ultrasonically treated at 210 W for 13 min. The resulting solution was freeze-dried.

[0097] (2) The ππ stacking product ICG-GA obtained after freeze-drying was dissolved in 13 mL of anhydrous ethanol, and 60 mg of lecithin, 60 mg of Solutol HS15, and 18 mg of DOTAP were weighed and added to the anhydrous ethanol for ultrasonic dissolution. The anhydrous ethanol was removed by a rotary evaporator at a rotary evaporation temperature of 54°C and a rotary evaporation speed of 75 r / min. After the ethanol was completely evaporated and a thin film was formed at the bottom of the eggplant-shaped bottle, 12 mL of ultrapure water was added, and the eggplant-shaped bottle was shaken to completely dissolve the formed thin film, and then placed in a 54°C water bath for hydration for 55 min to obtain a cationic micelle solution loaded with drug ICG-GA.

[0098] (3) The mass ratio of cationic micelles to HIF-1α siRNA (the nucleotide sequence of HIF-1α siRNA in this embodiment is: UCAAGUUGCUGGUCAUCAGTT; SEQ ID NO. 1) is 8:1. HIF-1α siRNA is dissolved in DEPC aqueous solution and then added to the micelle solution loaded with drug ICG-GA. The mixture is incubated at 4°C and 40 rpm for 6 h to obtain cationic micelles loaded with HIF-1α siRNA.

[0099] (4) The cell fragment solution after ultrasonic disruption of 4T1 mouse breast cancer cells was subjected to differential centrifugation to extract membrane fragments. The low-speed centrifugation time was 14 min at 4°C and 2900 r / min; and the high-speed centrifugation time was 50 min at 4°C and 12900 r / min to obtain cell membrane fragments.

[0100] (5) The cationic micelles loaded with HIF-1α siRNA and cell membrane fragments were mixed and self-assembled, and the cell membrane was coated by ultrasound. The ultrasonic power was 220 W, the temperature was 4° C., and the time was 19 min. The resulting solution was then dialyzed in a 3500-mesh dialysis bag for 16 h to obtain the multifunctional bionic nanoscale drug CM-ICG / GAM-siRNA (CM-IGM / siRNA).

[0101] Since ICG has photothermal properties, four samples of H2O, empty carrier, ICG, and CM-IGM / siRNA in this example were prepared. 1 mL of each sample was placed in a 1.5 mL centrifuge tube and irradiated with an 808 nm laser at a power of 2 W / cm 2 , the irradiation time is 5 minutes. Figure 4 As shown, the nanodrug CM-IGM / siRNA still has excellent photothermal properties after multiple steps of modification.

[0102] The cytotoxicity and proliferation inhibition of 4T1 mouse breast cancer cells by different nano-drug delivery systems were detected by CCK-8 detection kit. The experimental groups were PBS, ICG, IGM (IGM refers to cationic micelles loaded with ICG-GA), IGM / siRNA (IGM / siRNA refers to IGM further adsorbing HIF-1α siRNA), CM-IGM / siRNA in this embodiment, and CM-IGM / siRNA+Laser in this embodiment. The cells were plated on a 96-well plate (50,000 cells / well), and after 24 hours, the cells adhered to the wall, and each nano-drug delivery system was added. The addition amount was 1:10 with a volume ratio of 100 μL of the culture medium, that is, 10 μL of material was added to 100 μL of the culture medium, and 5 parallels were set for each group. After the 96-well plate was placed in a CO2 incubator and continued to be cultured for 4 hours, the group was irradiated with 808nm laser. After 1 hour of illumination, 10 μL of CCK-8 solution was added to each well, and incubated in the cell culture incubator for 1 hour. The absorbance of cells at 450nm was measured by an ELISA instrument to calculate the cell growth and proliferation. Figure 5 shown.

[0103] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A multifunctional bionic nanomedicine, characterized in that: The multifunctional bionic nanomedicine comprises a drug-loaded cationic micelle and a tumor cell membrane wrapped outside the drug-loaded cationic micelle, wherein the drug-loaded cationic micelle comprises a cationic micelle, a photosensitizer and a lipophilic HSP90 inhibitor drug, the photosensitizer and the lipophilic HSP90 inhibitor drug are connected by ππ stacking to form a photosensitizer-lipophilic HSP90 inhibitor drug, and the photosensitizer-lipophilic HSP90 inhibitor drug is loaded in the hydrophobic cavity inside the cationic micelle.

2. The multifunctional bionic nanomedicine according to claim 1, characterized in that: The cationic micelles have a particle size of 90 to 160 nm and a charge of +15 to +30 mV; Preferably, the particle size of the cationic micelle is 120 to 160 nm; Preferably, the cationic micelles are prepared from lecithin, polyethylene glycol-15 hydroxystearate and DOTAP.

3. The multifunctional bionic nanomedicine according to claim 1, characterized in that: The photosensitizer is selected from one or more of indocyanine green, Ce6, methylene blue, and acridine orange; The fat-soluble HSP90 inhibitor drug is selected from one or more of gambogic acid, gedunin, and 17-DMAG; Preferably, the mass ratio of the photosensitizer to the lipophilic HSP90 inhibitor is 0.5-1:1.5-3; Preferably, the photosensitizer is selected from indocyanine green; Preferably, the lipophilic HSP90 inhibitor drug is selected from gambogic acid.

4. The multifunctional bionic nanomedicine according to claim 1, characterized in that: The drug-loaded cationic micelles also include HIF-1α siRNA, and the HIF-1α siRNA is adsorbed on the surface of the cationic micelles; Preferably, the mass ratio of the cationic micelle to HIF-1α siRNA is 4 to 10:1; Preferably, the nucleotide sequence of the HIF-1α siRNA is as shown in SEQ ID NO.

1.

5. A method for preparing the multifunctional bionic nanomedicine according to claim 1, characterized in that: The following steps are involved: (1) connecting a photosensitizer and a lipophilic HSP90 inhibitor drug via ππ stacking to form a photosensitizer-lipophilic HSP90 inhibitor drug; (2) mixing a photosensitizer-lipid-soluble HSP90 inhibitor drug, a lipid material required for preparing cationic micelles, and a first organic solvent, and then preparing drug-loaded cationic micelles by a thin film hydration method; (3) dissolving HIF-1α siRNA in a second solvent, and then mixing it with the drug-loaded cationic micelles obtained in step (2), and incubating the mixture by flipping to obtain drug-loaded cationic micelles adsorbing HIF-1α siRNA; (4) The cationic micelles loaded with HIF-1α siRNA adsorbed thereon and tumor cell membrane fragments are mixed and self-assembled to obtain the multifunctional bionic nanomedicine according to claim 1.

6. The preparation method according to claim 5, characterized in that: The method for forming a photosensitizer-lipid-soluble HSP90 inhibitor drug by ππ stacking connection in step (1) is: dissolving the photosensitizer and the lipid-soluble HSP90 inhibitor drug in DMSO respectively, mixing them and then ultrasonically treating them, and finally freeze-drying them; Preferably, the ultrasonic treatment conditions are: ultrasonic power is 150-250W, and ultrasonic time is 10-15min.

7. The preparation method according to claim 5, characterized in that: The lipid materials required for preparing cationic micelles in step (2) are lecithin, polyethylene glycol-15 hydroxystearate and DOTAP; In step (2), the first organic solvent is anhydrous ethanol; Preferably, the ratio of the mass of the photosensitizer-lipid-soluble HSP90 inhibitor drug to the volume of the first organic solvent is 5-10:10-20 mg / mL, preferably 5-7.5 mg:10-15 mg / mL; Preferably, the mass ratio of the photosensitizer-lipid-soluble HSP90 inhibitor drug, lecithin, polyethylene glycol-15 hydroxystearate and DOTAP is 1-5:10-50:10-50:3-15; Preferably, the mass ratio of the photosensitizer-lipid-soluble HSP90 inhibitor drug, lecithin, polyethylene glycol-15 hydroxystearate and DOTAP is 2-4:20-40:20-40:6-12; Preferably, the rotary evaporation temperature of the thin film hydration method is 45-55°C, and the rotary evaporation speed is 60-80r / min; the hydration process conditions of the thin film hydration method are: 10-15mL of ultrapure water, hydration temperature of 45-55°C, and hydration time of 30-60min.

8. The preparation method according to claim 5, characterized in that: In step (3), the second solvent is a DEPC aqueous solution; Preferably, the temperature of the flip incubation in step (3) is 2 to 8°C, the rotation speed is 30 to 40 r / min, and the time is 4 to 6 hours.

9. The preparation method according to claim 5, characterized in that: The preparation method of tumor cell membrane fragments in step (4) is as follows: extracting membrane fragments from the cell fragment solution after ultrasonic disruption by differential centrifugation, wherein the low-speed centrifugation temperature is 2-8°C, the speed is 2000-4000 r / min, and the time is 10-15 min; the high-speed centrifugation temperature is 2-8°C, the speed is 12000-14000 r / min, and the time is 30-40 min; In step (4), the mixed self-assembly is carried out by ultrasonic method, the ultrasonic power is 150-250W, the ultrasonic temperature is 4-6°C, and the ultrasonic time is 10-20min.

10. Use of the multifunctional bionic nanomedicine according to claim 1 in the preparation of anti-tumor drugs.

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