A hypoxia-responsive bionic nano-delivery system and preparation method thereof

Abstract

The present invention belongs to the field of medical technology, and relates to a hypoxia-responsive bionic nano-delivery system, a preparation method and a use thereof. The delivery system of the present invention uses polyethylene glycol-polylysine hypoxia-responsive copolymer as a nano-core, and wraps a cell membrane in the outer layer of the nano-core by thin film extrusion technology. The hypoxia-responsive bionic delivery system can encapsulate hydrophobic drugs, and its particle size is uniform and stable, the encapsulation rate is good, and it also has good hypoxia response characteristics. Cell experiments show that the hypoxia-responsive bionic delivery system is more easily taken up by 4T1 cells. Animal experimental results show that the delivery system has good targeting in the triple-negative breast cancer lung metastasis model, and can inhibit the growth and lung metastasis of in situ tumors in the triple-negative breast cancer model.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a hypoxia responsive bionic nano delivery system and a preparation method and use thereof. Background Art

[0002] Literature reports that breast cancer is one of the most common malignant tumors in women. Worldwide, breast cancer accounts for 10% of all cancer incidence, 25%-30% of female malignant tumor incidence, and 15% of female tumor-related mortality. Triple negative breast cancer (TNBC) refers to breast cancer that is negative for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (Her-2), accounting for 10%-20% of all breast cancers. Compared with other subtypes of breast cancer, triple negative breast cancer has the characteristics of young onset, high malignancy, easy recurrence and metastasis, and poor survival rate. In addition, due to the lack of ideal targets for endocrine therapy and targeted therapy, triple negative breast cancer has become one of the difficult problems in the current field of breast cancer treatment.

[0003] With the development of diagnostic technology and the improvement of surgical treatment in recent years, carcinoma in situ itself is no longer the main cause of death from breast cancer, and distant metastasis of cancer has become the primary cause of death in TNBC patients. Clinical practice shows that when breast cancer metastasizes, traditional surgical resection therapy can no longer effectively control it, and radiotherapy and chemotherapy have become the few ways to fight tumor metastasis. Radiotherapy can effectively kill tumor cells located at the metastatic site, but it is only effective for local metastatic lesions, and it also causes a certain degree of damage to normal tissues around the metastatic site. Although the radiation range of radiotherapy has been continuously reduced in recent years, the classic large-field irradiation method including regional lymph nodes has been replaced, and the ability to target and kill tumors has been improved, but its adverse reactions are still large, and patients are prone to symptoms such as bone marrow suppression and vomiting. Cytotoxic chemotherapy drugs, as a classic clinical anti-breast cancer metastasis option, can kill tumor cells and inhibit their spread, but unfortunately, due to the lack of the ability of chemotherapy drugs to target tumor tissues and selectively kill tumor cells, their clinical inhibitory metastasis effect is poor and the toxic side effects are serious; in addition, repeated administration of chemotherapy drugs is likely to cause tumor cells to develop multidrug resistance. The above problems greatly limit the clinical application of chemotherapy drugs. According to clinical analysis, the lungs are the organ with the highest probability of breast cancer metastasis, with the probability of lung metastasis being as high as 60%. Therefore, how to effectively inhibit breast cancer lung metastasis is both a key issue in improving the treatment effect of breast cancer and a difficult problem that needs to be overcome urgently.

[0004] In order to solve the problem of breast cancer metastasis, nano drug delivery system came into being. It is mainly composed of artificially synthesized or naturally derived nano carriers and therapeutic active substances (drugs), with a particle size between 1nm-1000nm. Typical nano drug delivery systems include nanoparticles, liposomes, micelles, etc. In the nano drug delivery system, the drug can be encapsulated in the nano carrier or embedded in the surface of the nano carrier. In the circulatory system, the nano drug delivery system is more stable than free drugs, which can reduce the premature release and clearance of drugs. In addition, through the permeation and retention enhancement (EPR) effect, the drug delivery system can passively target and deliver drugs to tumor tissues to improve the anti-tumor effect of drugs. Based on the above advantages, the nano drug delivery system provides a new option for anti-breast cancer metastasis. However, the nano preparations in the prior art still have the defects of low targeting efficiency to metastatic lesions and poor drug concentration in metastatic lesions, which limit the therapeutic effect of breast cancer metastasis.

[0005] Based on the current status of the prior art, the inventors of the present application intend to provide a hypoxia-responsive bionic nano-delivery system and a preparation method and use thereof. Summary of the invention

[0006] The purpose of the present invention is to provide a hypoxia-responsive bionic nano-delivery system based on the current status and defects of the prior art, wherein the delivery system uses a polymer having a hypoxia-responsive function in the metastatic microenvironment as a nano-core and an outer layer coated with a neutrophil membrane having a metastatic targeting function.

[0007] Specifically, the present invention is achieved through the following technologies:

[0008] The invention provides a hypoxia-responsive bionic nano delivery system, which consists of a hypoxia-responsive nano core and a cell membrane shell.

[0009] The hypoxia-responsive nanocore is prepared from a polyethylene glycol-polylysine hypoxia-responsive copolymer, in which lysine is covalently linked to a hypoxia-responsive group, which is a nitroimidazole group. The nanocore can encapsulate hydrophobic drugs.

[0010] The polyethylene glycol-polylysine hypoxia responsive copolymer has the following structural formula:

[0011]

[0012] The hydrophilic segment of the polyethylene glycol-polylysine hypoxia responsive copolymer is polyethylene glycol (PEG), and the lipophilic end is a polylysine covalently linked hypoxia responsive group.

[0013] Preferably, the molecular weight of the hydrophilic end PEG is 5000Da.

[0014] The hydrophobic drug is one or more of paclitaxel, sorafenib, doxorubicin, all-trans retinoic acid, doxorubicin hypoxia-responsive prodrug, all-trans retinoic acid hypoxia-responsive prodrug, coumarin-6, long-chain lipophilic dialkyl carbocyanine dye Dir, long-chain lipophilic dialkyl carbocyanine dye Did, and long-chain lipophilic dialkyl carbocyanine dye DiI.

[0015] In the hypoxia-responsive bionic nano-delivery system, the hydrophobic drug is a combination of all-trans retinoic acid and doxorubicin or a combination of doxorubicin hypoxia-responsive prodrug and all-trans retinoic acid hypoxia-responsive prodrug.

[0016] The all-trans retinoic acid hypoxia-responsive prodrug has the following structural formula:

[0017]

[0018] The cell membrane is a neutrophil membrane.

[0019] The neutrophil membrane is a neutrophil membrane activated by adding TNF-α during in vitro culture.

[0020] The present invention also provides a method for preparing the hypoxia-responsive bionic nano-delivery system, which is a thin film dispersion method, wherein a polyethylene glycol-polylysine hypoxia-responsive copolymer and a hydrophobic drug are dissolved in an organic solvent, the organic solvent is removed by rotary evaporation, aqueous phase ultrasound is added to prepare a nano-core, a cell membrane is added, and a thin film extrusion technology is used to prepare the hypoxia-responsive bionic delivery system.

[0021] The mass ratio of the copolymer to the hydrophobic drug is 1:0.01-0.2.

[0022] The mass ratio of the copolymer to the cell membrane is 1:0.01-2.

[0023] The present invention further provides a use of the hypoxia-responsive bionic nano-delivery system, wherein the hypoxia-responsive delivery system can be used to specifically target metastatic lesions of breast cancer.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] 1. The drug delivery system of the present invention can achieve effective targeting of breast cancer metastases;

[0026] 2. The drug delivery system of the present invention can respond to the hypoxic microenvironment and achieve rapid release of drugs in metastatic lesions;

[0027] 3. Good biological safety after cell membrane wrapping;

[0028] 4. The delivery system of the present invention can achieve the effect of differentiating tumor stem cells and synergistically eliminating tumor cells after simultaneously encapsulating differentiation drugs and chemotherapy drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the synthetic route of polyethylene glycol-polylysine hypoxia-responsive copolymer.

[0030] Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of compound 3.

[0031] Figure 3 This is the hydrogen nuclear magnetic resonance spectrum of compound 4.

[0032] Figure 4 This is the hydrogen nuclear magnetic resonance spectrum of compound 5.

[0033] Figure 5 This is the molecular weight diagram of compound 5 determined by gel permeation chromatography (GPC).

[0034] Figure 6 These are transmission electron microscopy images. Figure A shows the nanocore prepared by polyethylene glycol-polylysine hypoxia-responsive copolymer, Figure B shows the neutrophil membrane vesicles, and Figure C shows the hypoxia-responsive bionic nanodelivery system.

[0035] Figure 7 This is the particle size diagram detected by the particle size analyzer. M-NI is the nanocore prepared by polyethylene glycol-polylysine hypoxia-responsive copolymer, Neutrophil membrane is the neutrophil membrane vesicle, and NM-M-NI is the hypoxia-responsive bionic nanodelivery system.

[0036] Figure 8 is the Zeta potential diagram, M-NI is the nanocore prepared by polyethylene glycol-polylysine hypoxia-responsive copolymer, Neutrophil membrane is the neutrophil membrane vesicle, and NM-M-NI is the hypoxia-responsive bionic nanodelivery system.

[0037] Fig. 9 To simulate the ultraviolet absorption spectrum of the hypoxic environment in vitro, M-NI is a nanocore prepared from a polyethylene glycol-polylysine hypoxia-responsive copolymer, and M-NI+Na2S2O4 is a nanocore prepared from a polyethylene glycol-polylysine hypoxia-responsive copolymer incubated with sodium dithionite.

[0038] Fig.10 To simulate the particle size change diagram of the in vitro hypoxic environment, M-NI is a nanocore prepared by polyethylene glycol-polylysine hypoxia-responsive copolymer, and M-NI+Na2S2O4 is a nanocore prepared by polyethylene glycol-polylysine hypoxia-responsive copolymer and co-incubated with sodium dithionite.

[0039] Fig.11 This is a diagram of 4T1 cell uptake. M-NI is a nanocore prepared from polyethylene glycol-polylysine hypoxia-responsive copolymer, and NM-M-NI is a hypoxia-responsive bionic nanodelivery system.

[0040] Fig.12 This is an in vivo imaging image in the 4T1 lung metastasis model. M-NI is a nanocore prepared from polyethylene glycol-polylysine hypoxia-responsive copolymer, and NM-M-NI is a hypoxia-responsive bionic nanodelivery system.

[0041] Fig.13 Figure 2 is a graph of tumor weight in the 4T1 orthotopic tumor model. Saline is the group injected with normal saline, M-NI is the group injected with the nanocore prepared by polyethylene glycol-polylysine hypoxia-responsive copolymer, and NM-M-NI is the group injected with the hypoxia-responsive bionic nanodelivery system.

[0042] Fig.14 The graph shows the changes in tumor volume in the 4T1 orthotopic tumor model. Saline is the group injected with normal saline, M-NI is the group injected with the nanocore prepared by polyethylene glycol-polylysine hypoxia-responsive copolymer, and NM-M-NI is the group injected with the hypoxia-responsive bionic nanodelivery system.

[0043] Fig.15 The expression of acetaldehyde dehydrogenase (ALDH) in the tumor in the 4T1 orthotopic tumor model. Saline is the group injected with normal saline, M-NI is the group injected with the nanocore prepared by polyethylene glycol-polylysine hypoxia-responsive copolymer, and NM-M-NI is the group injected with the hypoxia-responsive bionic nanodelivery system.

[0044] Fig.16 is the number of lung nodules in the 4T1 lung metastasis model, Saline is the group injected with normal saline, M-NI is the group injected with the nanocore prepared by polyethylene glycol-polylysine hypoxia-responsive copolymer, and NM-M-NI is the group injected with the hypoxia-responsive bionic nanodelivery system. DETAILED DESCRIPTION

[0045] The beneficial effects of the present invention are further described by the following examples. The examples are only for illustrative purposes and do not limit the scope of the present invention. At the same time, obvious changes and modifications made by ordinary technicians in the field according to the present invention are also included in the scope of protection of the present invention.

[0046] Example 1

[0047] Compound 1-2 was synthesized using the method described in the literature (ACS Appl Mater Interfaces, 2015, 7(9): 5444-5453).

[0048] Compound 3: Compound 2 was placed in a round-bottom flask, and 10 ml of trifluoroacetic acid and 0.5 ml of a mixture of hydrobromic acid / acetic acid were added. The mixture was stirred at room temperature for 3 hours. The reaction solution was transferred to a dialysis bag (MW 3500), and dialyzed in 2 L of pure water for 24 hours. The water was changed every 8 hours, and the compound was obtained by freeze-drying.

[0049] 1 H NMR(400MHz,dmso)δ8.06(s,1H),4.23(s,2H),3.47(d,J=27.9Hz,29H),3.24(s,1H),2.75(s,3H),2.5 7–2.42(m,5H),1.89(s,1H),1.62(s,1H),1.52(s,4H),1.31(s,2H),1.23(s,1H),0.04–-0.03(m,1H).

[0050] Example 2

[0051] Compound 4: Weigh 2-hydroxymethyl-1-methyl-5-nitroimidazole, p-nitrophenyl chloroformate, dimethylaminopyridine, and triethylamine, add tetrahydrofuran to dissolve, react at room temperature for 8 hours, and purify by silica gel column chromatography (petroleum ether: ethyl acetate = 4:1) to obtain a pure product.

[0052] 1 H NMR (400MHz, dmso) δ8.34(d,J=8.8Hz,3H),8.13(s,1H),7.60(d,J=8.8Hz,3H),5.47(s,3H),3.96(s,5H),3.36(s,23H),2.50(s,13H).

[0053] Example 3

[0054] Compound 5: Weigh compound 3, compound 4, dimethylaminopyridine, and triethylamine, add N'N-dimethylformamide (DMF) to dissolve, react at room temperature for 8 hours, transfer the reaction solution to a dialysis bag (MW 3500), place it in 1L DMF for 24 hours, and then place it in 2L pure water for 24 hours, and freeze-dry to obtain compound 5.

[0055] 1H NMR(400MHz,dmso)δ7.97(d,J=56.1Hz,47H),7.38(s,1H),7.38(s,24H),5.13(s,46H),4.18(s,9H),3.89(s,68H),3.51(s ,532H),3.35(s,655H),2.94(s,47H),2.51(s,153H),1.63(s,7H),1.54(d,J=49.5Hz,36H),1.36(s,26H),1.24(s,18H).

[0056] Example 4

[0057] Weigh 10 mg of the prepared polyethylene glycol-polylysine hypoxia-responsive copolymer, add 1 ml (DMF) to dissolve, place in a round-bottom flask, remove DMF by oil pump evaporation, add ultrapure water, ultrasonicate for 2 hours, and filter through a 0.45-micron filter membrane to obtain the hypoxia-responsive nanocore.

[0058] Example 5

[0059] To extract neutrophil membrane, take the mouse tibia and femur, separate the tissues, cut the two ends of the tibia and femur, use a 1ml syringe to blow 1640 culture medium into the tibia and femur until the bones turn white, centrifuge at 3000rpm for 3min, discard the culture medium, add 2ml of resuspended cell suspension centrifuge tube, add 2ml 78%, 65%, 52% Percoll separation solution, add cell suspension again, centrifuge at 1500rpm for 30min, and absorb the cell layer. Add RBC lysis buffer to lyse red blood cells, wash 3 times with PBS, and add TNF-α to activate neutrophils. After the cells are lysed by the lysis buffer at 12000rpm for 10min, take the supernatant, ultracentrifuge at 100000xg for 1h, and the lower layer is the neutrophil membrane precipitation.

[0060] Example 6

[0061] Weigh 10 mg of the prepared polyethylene glycol-polylysine hypoxia-responsive copolymer and 1 mg of paclitaxel, add 1 ml of N'N-dimethylformamide (DMF) to dissolve, place in a round-bottom flask, remove DMF by oil pump rotary evaporation, add ultrapure water, ultrasonicate for 2 hours, filter through a 0.45 micron filter membrane, and obtain the hypoxia-responsive nanocore. The paclitaxel encapsulation efficiency is 79.3%. Add 1 mg of the extracted neutrophil membrane, and repeatedly extrude with a film extruder to obtain the hypoxia-responsive bionic delivery system.

[0062] Example 7

[0063] Weigh 10 mg of the prepared polyethylene glycol-polylysine hypoxia-responsive copolymer and 0.1 mg of doxorubicin, add 1 ml of N'N-dimethylformamide (DMF) to dissolve, place in a round-bottom flask, remove DMF by oil pump rotary evaporation, add ultrapure water, ultrasonicate for 2 hours, filter through a 0.45 micron filter membrane, and obtain the hypoxia-responsive nanocore. The doxorubicin encapsulation efficiency is 90.5%. Add 5 mg of the extracted neutrophil membrane, and repeatedly extrude with a film extruder to obtain the hypoxia-responsive bionic delivery system.

[0064] Example 8

[0065] Weigh 10 mg of the prepared polyethylene glycol-polylysine hypoxia-responsive copolymer and 0.5 mg of coumarin-6, add 1 ml of N'N-dimethylformamide (DMF) to dissolve, place in a round-bottom flask, remove DMF by oil pump rotary evaporation, add ultrapure water, ultrasonicate for 2 hours, filter through a 0.45 micron filter membrane, and obtain the hypoxia-responsive nanocore. The coumarin-6 encapsulation efficiency is 62.1%. Add 20 mg of the extracted neutrophil membrane, and repeatedly extrude with a film extruder to obtain the hypoxia-responsive bionic delivery system.

[0066] Example 9

[0067] Weigh 10 mg of the prepared polyethylene glycol-polylysine hypoxia-responsive copolymer and 0.5 mg of sorafenib, add 1 ml of N'N-dimethylformamide (DMF) to dissolve, place in a round-bottom flask, remove DMF by oil pump rotary evaporation, add ultrapure water, ultrasonicate for 2 hours, filter through a 0.45 micron filter membrane, and obtain the hypoxia-responsive nanocore. The sorafenib encapsulation efficiency is 42.8%. Add 0.1 mg of the extracted neutrophil membrane, and repeatedly extrude with a film extruder to obtain the hypoxia-responsive bionic delivery system.

[0068] Example 10

[0069] Weigh 10 mg of the prepared polyethylene glycol-polylysine hypoxia-responsive copolymer and 0.5 mg of all-trans retinoic acid, add 1 ml of N'N-dimethylformamide (DMF) to dissolve, place in a round-bottom flask, remove DMF by oil pump rotary evaporation, add ultrapure water, ultrasonicate for 2 hours, filter through a 0.45 micron filter membrane, and obtain the hypoxia-responsive nanocore, with an encapsulation efficiency of 32.6%. Add 0.1 mg of the extracted neutrophil membrane, and repeatedly extrude with a film extruder to obtain the hypoxia-responsive bionic delivery system.

[0070] Embodiment 11

[0071] Weigh 10 mg of the prepared polyethylene glycol-polylysine hypoxia-responsive copolymer, 0.5 mg of all-trans retinoic acid, and 0.5 mg of doxorubicin, add 1 ml of N'N-dimethylformamide (DMF) to dissolve, place in a round-bottom flask, remove DMF by oil pump rotary evaporation, add ultrapure water, ultrasonicate for 2 hours, filter through a 0.45 micron filter membrane, and obtain hypoxia-responsive nanocores. The encapsulation efficiency of all-trans retinoic acid is 22.6%, and the encapsulation efficiency of doxorubicin is 55.5%. Add 0.1 mg of extracted neutrophil membrane, and repeatedly extrude with a film extruder to obtain a hypoxia-responsive bionic delivery system.

[0072] Example 12

[0073] Weigh 10 mg of the prepared polyethylene glycol-polylysine hypoxia-responsive copolymer, 0.5 mg of all-trans retinoic acid hypoxia-responsive prodrug, and 0.5 mg of doxorubicin hypoxia-responsive prodrug, add 1 ml of N'N-dimethylformamide (DMF) to dissolve, place in a round-bottom flask, remove DMF by oil pump rotary evaporation, add ultrapure water, ultrasonicate for 2 hours, filter through a 0.45 micron filter membrane, and obtain hypoxia-responsive nanocores. The encapsulation efficiency of all-trans retinoic acid hypoxia-responsive prodrug is 59.2%, and the encapsulation efficiency of doxorubicin hypoxia-responsive prodrug is 62.1%. Add 0.1 mg of extracted neutrophil membrane, and repeatedly extrude with a film extruder to obtain a hypoxia-responsive bionic delivery system.

[0074] Example 13

[0075] Weigh 10 mg of the prepared polyethylene glycol-polylysine hypoxia-responsive copolymer and 0.5 mg of the fluorescent probe DiD, add 1 ml of N'N-dimethylformamide (DMF) to dissolve, place in a round-bottom flask, remove DMF by oil pump rotary evaporation, add ultrapure water, ultrasonicate for 2 hours, filter through a 0.45 micron filter membrane, and obtain the hypoxia-responsive nanocore. The encapsulation efficiency of the fluorescent probe DiD is 63.6%, and the encapsulation efficiency of doxorubicin is 55.5%. Add 0.1 mg of the extracted neutrophil membrane, and repeatedly extrude with a film extruder to obtain the hypoxia-responsive bionic delivery system.

[0076] Embodiment 14

[0077] Weigh 10 mg of the prepared polyethylene glycol-polylysine hypoxia-responsive copolymer and 0.5 mg of the fluorescent probe DiR, add 1 ml of N'N-dimethylformamide (DMF) to dissolve, place in a round-bottom flask, remove DMF by oil pump rotary evaporation, add ultrapure water, ultrasonicate for 2 hours, filter through a 0.45 micron filter membrane, and obtain the hypoxia-responsive nanocore. The detection of the fluorescent probe DiR is 62.6%, and the encapsulation rate of doxorubicin is 55.5%. Add 0.1 mg of the extracted neutrophil membrane, and repeatedly extrude with a film extruder to obtain the hypoxia-responsive bionic delivery system.

[0078] Embodiment 15

[0079] Weigh 10 mg of the prepared polyethylene glycol-polylysine hypoxia-responsive copolymer and 0.5 mg of DiR, add 1 ml of N'N-dimethylformamide (DMF) to dissolve, place in a round-bottom flask, remove DMF with an oil pump, add ultrapure water, sonicate for 2 hours, and filter through a 0.45-micron filter membrane.

[0080] Example 16

[0081] Weigh 10 mg of the prepared polyethylene glycol-polylysine hypoxia-responsive copolymer and 0.5 mg of doxorubicin, add 1 ml of N'N-dimethylformamide (DMF) to dissolve, place in a round-bottom flask, remove DMF with an oil pump, add ultrapure water, sonicate for 2 hours, and filter through a 0.45-micron filter membrane.

[0082] Embodiment 17

[0083] Weigh 10 mg of the prepared polyethylene glycol-polylysine hypoxia-responsive copolymer, 0.5 mg of all-trans retinoic acid, and 0.5 mg of doxorubicin, add 1 ml of N'N-dimethylformamide (DMF) to dissolve, place in a round-bottom flask, remove DMF with an oil pump, add ultrapure water, sonicate for 2 hours, and filter through a 0.45-micron filter membrane.

[0084] Verification Example

[0085] Verification Example 1

[0086] The molecular weight of the polyethylene glycol-polylysine hypoxia responsive copolymer (Compound 5) was measured by gel chromatography. The results are as follows: Figure 5 shown.

[0087] Verification Example 2

[0088] The samples were counterstained with phosphotungstic acid and the nanocores prepared in Example 4, the neutrophil membrane vesicles extracted in Example 5, and the hypoxia-responsive biomimetic nanodelivery system prepared in Example 11 were observed using a transmission electron microscope. Figure 6 It can be seen that the prepared hypoxia-responsive bionic nanodelivery system has a double-layer structure.

[0089] Verification Example 3

[0090] The particle size and Zeta potential of the nanocore prepared in Example 4, the neutrophil membrane vesicles extracted in Example 5, and the hypoxia-responsive biomimetic nanoparticles prepared in Example 11 were measured by a nanoparticle size analyzer. Figure 7-8 The particle size of the hypoxia-responsive biomimetic nanodelivery system shown is about 20 nm higher than that of the nanocore, and the Zeta potential is close to that of the neutrophil membrane vesicles.

[0091] Verification Example 4

[0092] Sodium dithionite was used to simulate an in vitro hypoxic environment, and the particle size change was detected by a nanoparticle size analyzer after incubation with Example 11. The results are shown in Figure 10. After the addition of sodium dithionite, the nanocore particle size increased rapidly, increasing by about 500 nm.

[0093] Verification Example 5

[0094] Sodium dithionite was used to simulate the hypoxic environment in vitro, and after incubation with Example 4, the change in absorption wavelength was detected by ultraviolet spectrophotometer. Fig. 9 As shown, after adding sodium dithionite, the absorption wavelength of polyethylene glycol-polylysine oxygen-deficient copolymer was red-shifted.

[0095] Verification Example 6

[0096] 4T1 cells were plated on a 24-well plate, and the preparations obtained in Example 7 and Example 16 were diluted to the same concentration in basal medium, incubated for 0.5 h and 2 h, and the cell uptake was observed under an inverted fluorescence microscope. Fig.11 As shown, the hypoxia-responsive biomimetic nanodelivery system has higher cellular uptake.

[0097] Verification Example 7

[0098] ERFP-4T1 cells were injected into BalB / C mice via the tail vein. A lung metastasis model was formed one week later. Example 14 and Example 15 with the same Dir concentration were injected respectively and the in vivo distribution was observed by in vivo imaging. Fig.12 It was shown that the hypoxia-responsive biomimetic nanodelivery system had more accumulation in the lungs.

[0099] Verification Example 8

[0100] 4T1 cells were injected into the second fat pad on the right side of BalB / C mice to establish an orthotopic breast cancer model in mice. After 14 days, physiological saline, the preparation obtained in Example 11, and the preparation obtained in Example 17 were injected continuously at intervals of 2 days. The tumor volume and body weight of the mice were detected at 35 days. The results are as follows. Figure 13-14 As shown in Figure 2, the hypoxia-responsive bionic nanodelivery system can effectively inhibit tumor growth. The expression of ALDH in in situ tumors was further detected using an ALDH kit. The results are shown in Figure 2. Fig.15 As shown, the hypoxia-responsive bionic nanodelivery system can reduce the expression of ALDH. Since ALDH is a tumor stem cell marker, this suggests that the hypoxia-responsive bionic nanodelivery system can reduce the number of tumor stem cells in tumors.

[0101] Verification Example 9

[0102] 4T1 cells were injected into BalB / C mice via the tail vein. A lung metastasis model was formed one week later. Physiological saline was injected four times with an interval of one day. The number of lung metastatic nodules was detected on the 15th day. The results are as follows: Fig.16 As shown, the hypoxia-responsive biomimetic nanodelivery system can reduce the number of nodules in lung metastases.

Claims

1. A hypoxia-responsive bionic nano-delivery system, characterized in that: The delivery system consists of a hypoxia-responsive nanocore and a cell membrane shell; The hypoxia-responsive nanocore is prepared from a polyethylene glycol-polylysine hypoxia-responsive copolymer, wherein lysine in the copolymer is covalently linked to a hypoxia-responsive group, wherein the hypoxia-responsive group is a nitroimidazole group, and the nanocore can encapsulate a hydrophobic drug; The cell membrane is a neutrophil membrane; The hydrophobic drug is a combination of all-trans retinoic acid and doxorubicin or a combination of doxorubicin hypoxia-responsive prodrug and all-trans retinoic acid hypoxia-responsive prodrug; The polyethylene glycol-polylysine hypoxia responsive copolymer has the following structural formula: ； The hydrophilic end of the polyethylene glycol-polylysine hypoxia responsive copolymer is polyethylene glycol, and the lipophilic end is polylysine covalently linked to the hypoxia responsive group; The molecular weight of the hydrophilic end polyethylene glycol is 5000Da; The hypoxia-responsive bionic nano-delivery system is prepared by the following method: a thin film dispersion method is used to dissolve polyethylene glycol-polylysine hypoxia-responsive copolymer and a hydrophobic drug in an organic solvent, the organic solvent is removed by rotary evaporation, an aqueous phase is added to ultrasonically prepare a nano-core, a cell membrane is added, and a thin film extrusion technology is used to prepare the hypoxia-responsive bionic delivery system.

2. The hypoxia-responsive bionic nano-delivery system according to claim 1, characterized in that: In the preparation method, the mass ratio of the copolymer to the hydrophobic drug is 1:0.01-0.

2.

3. The hypoxia-responsive bionic nano-delivery system according to claim 1, characterized in that: The hypoxia-responsive bionic nano-delivery system is used in the preparation of a drug for specifically targeting metastatic lesions of breast cancer.

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