Homologous targeting neutrophil-liposome system and application thereof in treatment of triple negative breast cancer

Abstract

The invention discloses a homologous targeting neutrophil-liposome system and application thereof in treatment of triple negative breast cancer, the homologous targeting neutrophil-liposome system NE-GI-LNPs is developed and verified for the first time, and the homologous targeting neutrophil-liposome system NE-GI-LNPs can effectively inhibit tumor growth through pyroptosis enhanced anti-tumor immunotherapy. The invention provides a new thought and strategy for research and development of triple negative breast cancer treatment drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular, relates to a homologous targeted neutrophil-liposome system and its application in the treatment of triple-negative breast cancer. Background Art

[0002] Triple Negative Breast Cancer (TNBC) is a special immunohistochemical subtype of breast cancer, defined as a breast cancer with negative expression of estrogen receptor (ER), progesterone receptor (PR) and oncogene human epidermal growth factor receptor-2 (HER2). Epidemiological data show that the incidence of TNBC is young, usually under 40 years old, and mostly young premenopausal women, accounting for about 15%-20% of all breast cancer patients. TNBC patients have a poor prognosis, with a mortality rate of 5% within 40 years after diagnosis, and a high metastasis rate, with about 46% of patients having distant metastasis. Distant metastasis of TNBC usually occurs in the third year after diagnosis, with a median survival time of only 13.3 months after metastasis, and a recurrence rate of up to 25%. The average recurrence time of TNBC patients is 19-40 months, which is much lower than the 35-67 months of non-TNBC patients. In addition, the mortality rate of TNBC within 3 months after recurrence is as high as 75%.

[0003] Due to the special molecular phenotype of TNBC, TNBC is insensitive to endocrine therapy or molecular targeted therapy and often shows resistance to targeted drugs. Therefore, chemotherapy is the main systemic treatment. However, the efficacy of conventional postoperative adjuvant radiotherapy and chemotherapy is limited and often cannot prevent tumor recurrence. Therefore, the development of new treatment options for TNBC is particularly urgent in order to improve patients' prognosis and quality of life. Immunotherapy has shown promising clinical responses and great potential in the treatment of TNBC. Enhancing and activating anti-tumor immunity can help inhibit the progression of TNBC. Summary of the invention

[0004] In view of the above-mentioned technical problems existing in the prior art, the present invention has for the first time developed a new homologous targeted neutrophil-liposome system NE-GI-LNPs and its application in the treatment of triple-negative breast cancer.

[0005] The inventors of the present invention have developed and verified a drug delivery system based on neutrophil light response, tumor targeting and pyroptosis enhancement for the efficient treatment of TNBC. First, liposomes based on TNBC cell membranes are used to load photothermal reagent IR775 and plasmid GSDME (GI-LNPs) that promotes cell pyroptosis. The prepared GI-LNPs are anchored on the surface of neutrophils through biotin-avidin interactions and delivered to tumors through neutrophil infiltration driven by local damage. In addition, under near-infrared (NIR) laser irradiation, IR775 triggers the production of apoptotic protein caspase-3, promotes the cleavage of expressed GSDME, converts the apoptosis of 4T1 cells into pyroptosis, and enhances anti-tumor immunity. In general, the research of the present invention shows that the multifunctional GI-LNPs (NE-GI-LNPs) delivered by neutrophils can effectively inhibit tumor growth through anti-tumor immunotherapy enhanced by pyroptosis.

[0006] The present invention adopts the following technical solutions to achieve the above-mentioned invention objectives:

[0007] The first aspect of the present invention provides a method for preparing a homologous targeted neutrophil-liposome system NE-GI-LNPs.

[0008] Further, the method comprises the following steps:

[0009] DSPE-PEG-Biotin and DSPE-PEI were added to a triple-negative breast cancer cell membrane suspension and co-incubated to prepare a DSPE-PEG-Biotin / DPE-PEI modified triple-negative breast cancer cell membrane;

[0010] Plasmid pGSDME and cyanine dye IR775 are added to the triple-negative breast cancer cell membrane suspension containing the DSPE-PEG-Biotin / DPE-PEI modified, and co-incubated to prepare GI-LNPs;

[0011] DSPE-PEG-STA was added to the neutrophil suspension and co-incubated to prepare neutrophil NE-STA;

[0012] The NE-STA and the GI-LNPs are co-incubated to prepare the neutrophil-liposome system NE-GI-LNPs.

[0013] Further, the molar ratio of DSPE-PEG-Biotin to DSPE-PEI is (5-15): (1-5);

[0014] Optionally, the molar ratio of DSPE-PEG-Biotin to DSPE-PEI is 10:1;

[0015] Optionally, DSPE-PEG-Biotin and DSPE-PEI are added to the triple-negative breast cancer cell membrane suspension and co-incubated for 0.1-5 h;

[0016] Optionally, DSPE-PEG-Biotin and DSPE-PEI are added to the triple-negative breast cancer cell membrane suspension and co-incubated for 1 h;

[0017] Optionally, the triple-negative breast cancer cell membrane is a 4T1 cell membrane.

[0018] The dosages of the plasmids pGSDME and IR775 are 50-150 μg / mL and 10-90 μg / mL, respectively;

[0019] Optionally, the dosages of the plasmids pGSDME and IR775 are 100 μg / mL and 50 μg / mL, respectively;

[0020] Optionally, the dosage of the triple-negative breast cancer cell membrane suspension containing the DSPE-PEG-Biotin / DPE-PEI modified triple-negative breast cancer cell membrane is 1-7 mg / mL;

[0021] Optionally, the dosage of the triple-negative breast cancer cell membrane suspension containing the DSPE-PEG-Biotin / DPE-PEI modified is 4 mg / mL;

[0022] Optionally, the plasmid pGSDME and the cyanine dye IR775 are added to the triple-negative breast cancer cell membrane suspension containing the DSPE-PEG-Biotin / DPE-PEI modified triple-negative breast cancer cell membrane and co-incubated for 0.1-5h;

[0023] Optionally, the plasmid pGSDME and the cyanine dye IR775 are added to the suspension of triple-negative breast cancer cell membranes modified with the DSPE-PEG-Biotin / DPE-PEI and incubated for 1 hour.

[0024] Furthermore, the dosages of DSPE-PEG-STA and neutrophils are 0.05-0.15 mg / mL, (0.1-5)×10 5 / mL;

[0025] Optionally, the dosage of DSPE-PEG-STA and neutrophils is 0.1 mg / mL and 1×10 5 / mL;

[0026] Optionally, DSPE-PEG-STA is added to the neutrophil suspension and co-incubated for 0.1-5 h;

[0027] Optionally, DSPE-PEG-STA is added to the neutrophil suspension and incubated for 1 hour.

[0028] Furthermore, the dosage of NE-STA and GI-LNPs is (0.1-5)×10 6 / mL, (0.5-3.5) mg / mL;

[0029] Optionally, the dosage of NE-STA and GI-LNPs is 1×10 6 / mL, 2mg / mL;

[0030] Optionally, the NE-STA and the GI-LNPs are co-incubated for 1-7 h;

[0031] Optionally, the NE-STA and the GI-LNPs are co-incubated for 4 hours.

[0032] In the present invention, the DSPE-PEG-Biotin refers to phosphatidylethanolamine-polyethylene glycol-biotin (1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-PEG-Biotin), and its corresponding CAS number is 385437-57-0. DSPE-PEG-Biotin consists of three parts: DSPE (distearoyl phosphatidylethanolamine), PEG (polyethylene glycol) and Biotin (biotin). DSPE is a saturated 18C phospholipid, which is commonly used in the synthesis of liposomes; PEG has hydrophilicity and good biocompatibility; biotin has specific affinity and can specifically bind to cell surface receptors. The biotin part in DSPE-PEG-Biotin can specifically bind to avidin or streptavidin, so as to be used to detect target molecules. At the same time, the introduction of PEG chains increases the water solubility and stability of the molecules, and is conducive to long circulation in the body.

[0033] In the present invention, the DSPE-PEI is DSPE-PEI1800, which refers to bis-allyl phosphatidyl myosamine-polyethyleneimine (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethyleneimine)1800]). In a specific embodiment of the present invention, the DSPE-PEI1800 is purchased from Xi'an Qiyue Company, and the product number is R-Z54512-1.8k. DSPE-PEI is a synthetic compound with important applications in the fields of gene delivery and drug delivery. The complex consists of two parts: bis-allyl phosphatidyl (DSPE) and polyethyleneimine (PEI), wherein DSPE (bis-allyl phosphatidyl) is a phosphatidylcholine ester compound with good biocompatibility and biosimilarity. This characteristic makes DSPE widely used in the field of nano drug delivery. As a membrane structure component, it helps to form stable nanoparticles or liposomes, thereby improving the stability and delivery efficiency of drugs. PEI (polyethyleneimine) is a cationic polymer that is widely used to mediate DNA transfection due to its excellent DNA binding ability. Its positive charge can interact electrostatically with the negative charge of DNA to form a stable complex, promoting gene delivery and expression.

[0034] In the present invention, the plasmid pGSDME was purchased from Genomeditech (Shanghai) Company, established using pcDNA3.1 (+), gene ID: NM_018769.4. The plasmid pGSDME is a plasmid expressing the GSDME gene. The full name of the GSDME is Gasdermin E, which is one of the important members of the Gasdermins protein family, has multiple biological functions and potential application values, and its Gene ID in NCBI is 1687. The detailed information of the GSDME gene can be obtained at https: / / www.ncbi.nlm.nih.gov / gene / .

[0035] In the present invention, the cyanine dye IR775 refers to IR775 chloride (2-[2-[2-chloro-3-[2-(1,3-dihydro-1,3,3-trimethyl-2h-indol-2-ylidene)-ethylidene]-1-cyclohexen-1-yl]-ethen yl]-1,3,3-trimethyl-3h-indolium chloride), IR775 is a near-infrared dye with a CAS number of 199444-11-6.

[0036] In the present invention, the DSPE-PEG-STA refers to phosphatidylethanolamine-polyethylene glycol-streptavidin (1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-PEG3400-Streptavidin), which is purchased from Xi'an Qiyue Company, and the product number is R-SS0065-3.4k. The DSPE-PEG-STA consists of three parts: DSPE (distearoyl phosphatidylethanolamine), PEG (polyethylene glycol) and Streptavidin (streptavidin). DSPE is a saturated 18C phospholipid, which is commonly used in the synthesis of liposomes; PEG has hydrophilicity and good biocompatibility; streptavidin has specific affinity and can specifically bind to biotin. The streptavidin part in DSPE-PEG-STA can specifically bind to biotin, so as to be used to detect target molecules. At the same time, the introduction of PEG chains increases the water solubility and stability of the molecules, and is conducive to long circulation in the body.

[0037] In some embodiments, the triple-negative breast cancer cell membrane is a cell membrane derived from a triple-negative breast cancer cell line. Exemplarily, the triple-negative breast cancer cell line includes but is not limited to: triple-negative breast cancer cell line 4T1, triple-negative breast cancer cell line MDA-MB-231, triple-negative breast cancer cell line BT20, triple-negative breast cancer cell line MDA-MB-468, triple-negative breast cancer cell line HCC1937, triple-negative breast cancer cell line Hs578T, triple-negative breast cancer cell line HCC1806, triple-negative breast cancer cell line SUM149PT.

[0038] The second aspect of the present invention provides a homologous targeted neutrophil-liposome system NE-GI-LNPs.

[0039] Furthermore, the neutrophil-liposome system NE-GI-LNPs is prepared by anchoring triple-negative breast cancer cell membrane loaded with IR775 and pGSDME on the surface of neutrophils through biotin-avidin interaction;

[0040] Optionally, the neutrophil-liposome system NE-GI-LNPs are neutrophil-liposome system NE-GI-LNPs prepared by the method described in the first aspect of the present invention.

[0041] The third aspect of the present invention provides a pharmaceutical composition.

[0042] Furthermore, the pharmaceutical composition comprises the neutrophil-liposome system NE-GI-LNPs described in the second aspect of the present invention.

[0043] Furthermore, the pharmaceutical composition may also contain other drugs that can be used for the treatment and / or adjuvant treatment of triple-negative breast cancer.

[0044] Furthermore, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier and / or excipient.

[0045] In some embodiments, the present invention has no particular limitations on the pharmaceutically acceptable carriers and / or excipients, and any pharmaceutically acceptable carriers and / or excipients suitable for combination with the active ingredients provided by the present invention (such as the neutrophil-liposome system NE-GI-LNPs described above) to obtain the pharmaceutical composition or pharmaceutical preparation of the present invention are within the scope of protection of the present invention.

[0046] In some embodiments, specific illustrative examples of the pharmaceutically acceptable carrier and / or excipient include, but are not limited to: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose and methyl cellulose; tragacanth powder; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols, such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers, such as wetting agents, such as sodium lauryl sulfate; colorants; flavorings; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solution; and phosphate buffer, etc.

[0047] A fourth aspect of the present invention provides a pharmaceutical preparation.

[0048] Furthermore, the pharmaceutical preparation comprises the pharmaceutical composition described in the third aspect of the present invention.

[0049] Furthermore, the dosage form of the pharmaceutical preparation includes, but is not limited to, intravenous infusion preparations, in situ injection preparations, oral preparations, tablets, capsule preparations, granule preparations, sustained-release preparations, powders or pills.

[0050] In some embodiments, the present invention has no particular limitation on the dosage form of the pharmaceutical preparation, and the dosage form of the pharmaceutical preparation includes, but is not limited to, intravenous reinfusion preparations, in situ injection preparations, oral preparations, tablets, capsule preparations, granule preparations, sustained-release preparations, powders, and pills. The pharmaceutical preparation can be prepared by a pharmaceutical preparation preparation method known to those skilled in the art.

[0051] In some embodiments, the suitable dosage of the pharmaceutical composition or pharmaceutical preparation described in the present invention can be prescribed in a variety of ways according to factors such as formulation method, administration mode, patient's age, weight, sex, morbidity, diet, administration time, administration route, excretion rate and reaction sensitivity. A skilled physician can usually easily determine the prescription and the dosage that is effective for the desired treatment.

[0052] The fifth aspect of the present invention provides the use of the neutrophil-liposome system NE-GI-LNPs described in the second aspect of the present invention in the preparation of a drug for treating triple-negative breast cancer.

[0053] The sixth aspect of the present invention provides the use of the neutrophil-liposome system NE-GI-LNPs described in the second aspect of the present invention in combination with near-infrared laser in the preparation of a drug for treating triple-negative breast cancer.

[0054] In addition, the present invention also provides a method for treating triple-negative breast cancer, which comprises administering a therapeutically effective amount of the neutrophil-liposome system NE-GI-LNPs of the present invention as described above, the pharmaceutical composition of the present invention as described above, or the pharmaceutical preparation of the present invention as described above to a subject in need, and simultaneously performing near-infrared laser irradiation.

[0055] In some embodiments, the subject of the present invention refers to any animal, and also refers to humans and non-human animals. Non-human animals include all vertebrates, for example, mammals, such as non-human primates (particularly higher primates), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and any livestock or pets; and non-mammals, such as chickens, amphibians, reptiles, etc. In a preferred embodiment, the subject is a human.

[0056] In some embodiments, the treatment described herein generally relates to treating humans or animals (e.g., used by veterinarians), where certain desired therapeutic effects can be achieved, such as inhibiting the development of a disease (including reducing the rate of development, stopping the development), improving the disease, and curing the disease. Treatment as a preventive measure (e.g., prevention) is also included. Use in patients who have not yet developed a disease but are at risk of developing the disease is also included in the term treatment as used herein.

[0057] In some embodiments, the effective amount of the present invention refers to an amount having a therapeutic effect or an amount required to produce a therapeutic effect in a subject. For example, a drug therapeutically or pharmaceutically effective amount refers to the amount of the drug required to produce the desired therapeutic effect, and the therapeutic effect can be reflected by the results of clinical trials, model animal studies and / or in vitro studies. The pharmaceutically effective amount depends on several factors, including but not limited to: characteristic factors of the subject (such as height, weight, sex, age and medication history), the severity of the disease, etc.

[0058] In some embodiments, the administration methods of the neutrophil-liposome system NE-GI-LNPs, pharmaceutical composition or pharmaceutical preparation include, but are not limited to: intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intraperitoneal, intranasal, intrapulmonary, intrarectal, etc. In addition, the active ingredient can be administered by any device capable of being transferred to the target tissue. In a specific embodiment, the pharmaceutical composition or pharmaceutical preparation provided by the present invention can be prepared into various dosage forms according to actual needs, and can be administered by a clinician according to factors such as the type, age, weight and general disease condition of the subject, and the mode of administration to determine the dosage that is beneficial to the patient. The mode of administration can be, for example, injection or any other suitable mode of administration known to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 :Synthesis route of NE-GI-LNPs;

[0060] Figure 2 :Scanning electron microscopy results of neutrophils (NEs) and neutrophils modified with liposomes (NEs-Lip);

[0061] Figure 3 : Gel results (a), potential results (b), UV (c), fluorescence spectra (d) and GSDME cleavage results (ef) determined by Western blotting at different feed ratios of DSPE-PEI / pGSDME;

[0062] Figure 4 : Confocal imaging results of 4T1 after incubation with GI-LNPs;

[0063] Figure 5 :CD80 after different treatments + CD86 + Expression results and MHC II + The expression level of

[0064] Figure 6: The tumor growth curve and weight changes of tumor-bearing mice after different treatments in the triple-negative breast cancer tumor-bearing mouse model, including: (a) schematic diagram of the animal experiment; (bi) detection results of tumor volume in different groups of mice; (j) statistical results of tumor growth in different groups of mice; (l) detection results of weight of different groups of mice. DETAILED DESCRIPTION

[0065] The present invention is further described below in conjunction with specific embodiments, which are only used to explain the present invention and cannot be understood as limiting the present invention. It can be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purpose of the present invention, and the scope of the present invention is defined by the claims and their equivalents.

[0066] The reagents and raw materials used in the present invention are easily obtained by those of ordinary skill in the art, and can be obtained from commercial sources unless otherwise specified. The experimental methods for which specific conditions are not specified in the present invention are usually tested under conventional conditions or under conditions recommended by the manufacturer. In particular, the following examples are only used to illustrate the present invention and should not limit the scope of the present invention in any way. It should be noted that the experimental conditions and results described in the following examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0067] Example 1 Preparation of homologous targeted neutrophil-liposome system NE-GI-LNPs

[0068] The synthetic route of the neutrophil-liposome system NE-GI-LNPs is as follows Figure 1 shown.

[0069] First, the femur and tibia of C57 mice were collected. Then, the ends of the femur and tibia of the mice were removed. Next, the bone marrow mixture was collected by HBSS flushing. The mixed solution was filtered through a 40 μm filter. The collected solution was centrifuged at 1800 rpm for 5 minutes. The supernatant was discarded to obtain a cell pellet. After that, 3 mL of red blood cell lysate was added and then incubated for 5 minutes in the dark. 9 mL of PBS solution was added to interrupt the process. The mixed solution was centrifuged at 1800 rpm for 5 minutes. The obtained cell pellet was resuspended with hanks balanced salt solution (HBSS). Next, 3 mL of 62% separation solution was added to the bottom of the centrifuge tube. At the same time, the cell suspension was kept at the top and the separation solution was kept at the bottom, and then, centrifuged at 2500 rpm at a lifting speed of 0 for 35 minutes. After centrifugation, the top layer of liquid was carefully discarded and the cell pellet was dispersed with 2 mL of HBSS. The dispersed cell suspension was centrifuged at 1800 rpm for 5 minutes to remove the residual separation solution. Neutrophils (NE) were prepared.

[0070] First, 4T1 cell membranes were prepared by repeatedly freezing and thawing 4T1 cells 6 times using liquid nitrogen / 37°C. Then, the cell nucleus and cytoplasm were removed by centrifugation at 14800rpm for 10 minutes to obtain 4T1 cell membranes. The obtained 4T1 cell membranes were washed once with pre-cooled PBS. Then, the 4T1 cell membranes were resuspended in PBS. After the obtained dispersion (4T1 cell membrane, 4 mg / mL) was mixed with DSPE-PEG-Biotin / DSPE-PEI (molar ratio: 10 / 1, total amount 0.1 mg / mL) and incubated for 1 hour (wherein, the DSPE-PEG-Biotin was purchased from Xi'an Qiyue Company, the item number is R-2107-2k; the DSPE-PEI was purchased from Xi'an Qiyue Company, the item number is R-Z54512-1.8k), centrifuged at 1000rpm for 5 minutes to remove the unreacted DSPE-PEG-Biotin and DSPE-PEI. DSPE-PEG-Biotin / DPE-PEI modified 4T1 cell membrane was prepared.

[0071] To prepare GI-LNPs, DSPE-PEG-Biotin / DSPE-PEI (molar ratio: 10 / 1) and 4T1 cell membranes were co-incubated for 1 hour to obtain DSPE-PEG-Biotin / DPE-PEI modified 4T1 cell membranes. Then, plasmid pGSDME (purchased from Genomeditech (Shanghai) Company, established with pcDNA3.1 (+), gene ID: NM_018769.4) and cyanine dye IR775 (CAS No. 199444-11-6) were added to a 4 mg / mL suspension containing DSPE-PEG-Biotin / DSPE-PEI modified 4T1 cell membranes (the specific amounts of plasmid pGSDME and IR775 were 100 μg / mL and 50 μg / mL, respectively) and co-incubated for 1 hour. The suspension was extruded through a 200 nm polycarbonate membrane using a liposome extruder. Finally, the unreacted plasmid pGSDME and IR775 molecules were removed by dialysis to prepare GI-LNPs.

[0072] To prepare NE-STA, the neutrophil suspension prepared above (NEs, 1×10 5 / mL) and 0.1mg / mL DSPE-PEG-STA were co-incubated for 1h. The DSPE-PEG-STA refers to phosphatidylethanolamine-polyethylene glycol-streptavidin (1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-PEG3400-Streptavidin), purchased from Xi'an Qiyue Company, and the product number is R-SS0065-3.4k. Finally, the unreacted DSPE-PEG-STA was removed by centrifugation. NE-STA was prepared.

[0073] To prepare NE-GI-LNPs, 1×10 6 / mL of the NE-STA and 2 mg / mL of the GI-LNPs (1 mL) prepared above were incubated at room temperature for 4 hours. Then, the mixture was centrifuged at 1800 rpm for 5 minutes to remove unbound GI-LNPs. Finally, the neutrophil-liposome system NE-GI-LNPs was prepared.

[0074] NE-G-LNPs and NE-I-LNPs are prepared by methods similar to those described above. The difference is that in the process of preparing NE-G-LNPs, the plasmid pGSDME is added to a suspension containing 4T1 cell membranes modified with DSPE-PEG-Biotin / DSPE-PEI, and then G-LNPs are prepared by an extruder. Other conditions are the same. In the process of preparing NE-I-LNPs, IR775 is added to a suspension containing 4T1 cell membranes modified with DSPE-PEG-Biotin / DSPE-PEI, and then I-LNPs are prepared by an extruder. Other conditions are the same. Example 2 Detection of homologously targeted neutrophil-liposome system NE-GI-LNPs and verification of in vitro and in vivo effects

[0075] 1. Experimental methods

[0076] The scanning electron microscopy results of neutrophil particles (NEs) and neutrophil particles modified with liposomes (NEs-Lip) were observed by scanning electron microscopy.

[0077] By setting different feed ratios of DSPE-PEI / pGSDME (1:2, 1:1, 1.5:1, 2:1, 5:1, 10:1, 20:1), the optimal feed ratio of DSPE-PEI and plasmid pGSDME in the preparation process was explored.

[0078] Zeta potential analyzer was used to detect the membrane potential of different substances (liposome 4T1LNPs prepared based on cell membrane, liposome LNPs modified with DSPE-PEG-Biotin / DSPE-PEI on the surface of 4T1 LNPs, and liposome GI-LNPs further loaded with drug pGSDME / IR775).

[0079] The UV-Vis absorption spectra (UV-Vis) of IR775 before and after loading into liposomes (IR775 and GI-LNPs) were measured using a UV-Vis absorption spectrometer to detect whether loading IR775 into liposomes affects its own photothermal effect.

[0080] The fluorescence spectra (FL) of IR775 before and after loading into liposomes (IR775 and GI-LNPs) were measured using a fluorescence spectrometer to detect whether the emission peak change of the fluorescence spectrum of IR775 loaded into liposomes is conducive to tracer visualization in in vivo experiments.

[0081] The expression level of GSDME protein after transduction into cells was evaluated by changing the molar ratio of DSPE-PEI:pGSDME (2:1, 1.5:1).

[0082] Confocal microscopy was used to detect the confocal imaging results of 4T1 after incubation with GI-LNPs, in which the cell nucleus was stained with Hoechst33342 and the plasmid pGSDME was labeled with the green nucleic acid dye YOYO-1. Before the plasmid pGSDME was introduced into the preparation of GI-LNPs, the nucleic acid dye YOYO-1 was used to label pGSDME, and the cell nucleus was stained with the nuclear dye Hoechst, showing blue.

[0083] First, DC cells (dendritic cells) were prepared by in vitro induction, and the activation effect of cell fragments after different treatments on the induced DC cells was evaluated. Specifically, mice were first euthanized, and their femurs and tibias were obtained. Then, the bone marrow cells were rinsed with RPMI-1640 medium. After centrifugation at 1800rpm for 5 minutes, the cells were collected and cultured in RPMI-1640 medium containing 20% ​​FBS. GM-CSF (10ng / mL) and IL-4 (4ng / mL) were added to induce BMDC (bone marrow-derived dendritic cells) differentiation. BMDC was cultured at 2×10 per well. 5 The number of cells was seeded in a six-well plate and incubated for 24 h. After incubation with the treated 4T1 cells, BMDCs were washed three times with PBS and stained with flow cytometry antibodies (APC-labeled anti-mouse CD80, PE-labeled anti-mouse CD86, and PE-labeled MHC II), followed by flow cytometric analysis.

[0084] For animal experiments, 2 × 10 7 4T1 cells were inoculated into the right mammary pad of each mouse to establish an orthotopic 4T1 xenograft tumor model. The 4T1 tumor-bearing mice were randomly divided into eight groups and intravenously injected with 100 μL PBS (control group), NE-G-LNPs (1×10 6 cells), NE-I-LNPs (1×10 6 cells), NE-GI-LNPs (1×10 6 cells), NIR laser irradiation (808 nm, power 1 W / cm 2 ,10min), NE-G-LNPs+NIR(1×10 6 cells combined with NIR laser irradiation), NE-I-LNPs+NIR (1×10 6 cells combined with NIR laser irradiation), and NE-GI-LNPs+NIR (1×10 6 To evaluate the anticancer ability of various drugs in vivo, the tumor volume and body weight of mice were monitored every other day for 20 days. 2 × length / 2 calculation.

[0085] 2. Experimental results

[0086] Scanning electron microscopy results of neutrophils (NEs) and neutrophils modified with liposomes (NEs-Lip) are shown in Figure 2. Figure 2 As shown, compared with the SEM results of NEs, the SEM results of NEs-Lip showed obvious white granular substances on the surface of NEs, indicating that the liposome Lip was successfully modified on the surface of NEs.

[0087] The gel results, potential results, UV and fluorescence spectra of DSPE-PEI / pGSDME with different feed ratios and the GSDME cleavage results determined by Western blotting are shown in Figure 2. Figure 3 As shown in the figure, the molar ratio of DSPE-PEI to plasmid pGSDME in the preparation process was first explored. It was found that there was an obvious band relative to the free pGSDME. When the molar ratio of DSPE-PEI: pGSDME reached 1.5:1, DSPE-PEI could completely encapsulate the plasmid pGSDME. Therefore, this feed ratio was adopted in subsequent experiments ( Figure 3 a).

[0088] Figure 3b is the measurement of membrane potential of different substances. It was found that the surface membrane potential of liposomes prepared based on cell membrane was about -30mV. After surface modification with DSPE-PEG-Biotin / DSPE-PEI, the membrane potential changed to about +8mV, which indirectly proved that DSPE-PEI was successfully modified on the surface of liposomes. After further loading the drug pGSDME / IR775, the membrane potential changed to about -6mV, indicating that the plasmid pGSDME was successfully loaded on the liposomes.

[0089] By measuring the UV-Vis absorption spectra (IR775 and GI-LNPs) of IR775 before and after loading into liposomes, it was found that the characteristic absorption peak of IR775 at around 775nm had almost no change before and after loading, indicating that loading IR775 into liposomes has almost no effect on its own photothermal effect ( Figure 3 c).

[0090] By measuring the fluorescence spectra (FL) of IR775 before and after loading into liposomes (IR775 and GI-LNPs), it was found that the emission peak of the fluorescence spectrum of IR775 loaded into liposomes was red-shifted from 815nm to about 840nm. The red-shifted fluorescence emission is beneficial for the visualization of tracing in in vivo experiments ( Figure 3 d).

[0091] By changing the molar ratio of DSPE-PEI: pGSDME, the expression level of GSDME protein after transduction into cells was evaluated. The results showed that as the proportion of plasmid pGSDME increased, the expression level of GSDME protein in cells increased significantly ( Figure 3 e), Figure 3 f is correct Figure 3 Statistical value of the grayscale of the protein band in e.

[0092] The confocal imaging results of 4T1 after incubation with GI-LNPs are shown in Figure 2. Figure 4 As shown, the cell nucleus was stained with Hoechst33342, and the plasmid pGSDME was labeled with the green nucleic acid dye YOYO-1. Before the plasmid pGSDME was introduced into the preparation of GI-LNPs, the nucleic acid dye YOYO-1 was used to label pGSDME, and the cell nucleus was stained with the nuclear dye Hoechst, which appeared blue. The confocal results showed that the YOYO-1-labeled pGSDME was mainly located in the cell nucleus, indicating that the plasmid pGSDME was successfully delivered to the cell nucleus through GI-LNPs.

[0093] CD80 after different treatments + CD86 + Expression results and MHC II + The expression level results are as follows Figure 5As shown. First, DC cells were prepared by induction in vitro, and the activation effect of cell fragments after different treatments on the induced DC cells was evaluated. Specifically, the mice were first euthanized, and their femurs and tibias were obtained. Then, the bone marrow cells were washed with RPMI-1640 medium. After centrifugation at 1800rpm for 5 minutes, the cells were collected and cultured in RPMI-1640 medium containing 20% ​​FBS. GM-CSF (10ng / mL) and IL-4 (4ng / mL) were added to induce BMDC differentiation. BMDC were plated at 2×10 5 The number of cells was seeded in a six-well plate and incubated for 24 h. After incubation with the treated 4T1 cells, BMDCs were washed three times with PBS and stained with flow cytometry antibodies (APC-labeled anti-mouse CD80, PE-labeled anti-mouse CD86, and PE-labeled MHC II), followed by flow cytometry analysis. Figure 5 The results showed that NE-GI-LNPs combined with NIR laser irradiation treated cells were incubated with DCs to maximize the induction of DC cell activation (CD11c + CD80 + , CD11c + CD86 + and CD11c + MHC II + The highest ratio), which is beneficial to antigen presentation.

[0094] The growth curve of the tumor and the weight change of the tumor-bearing mice after different treatments in the triple-negative breast cancer tumor-bearing mouse model are shown in Figure 2. Figure 6 For animal experiments, 2 × 10 7 4T1 cells were inoculated into the right mammary pad of each mouse to establish an orthotopic 4T1 xenograft tumor model. The 4T1 tumor-bearing mice were randomly divided into eight groups and intravenously injected with 100 μL PBS (control group), NE-G-LNPs (1×10 6 cells), NE-I-LNPs (1×10 6 cells), NE-GI-LNPs (1×10 6 cells), NIR laser irradiation (808 nm, power 1 W / cm 2 ,10min), NE-G-LNPs+NIR(1×10 6 cells combined with NIR laser irradiation), NE-I-LNPs+NIR (1×10 6 cells combined with NIR laser irradiation), and NE-GI-LNPs+NIR (1×10 6To evaluate the anticancer ability of various drugs in vivo, the tumor volume and body weight of mice were monitored every other day for 20 days. 2 × length / 2 calculation ( Figure 6 a). By testing the tumor volumes of mice in different groups, it was found that NE-GI-LNPs combined with NIR laser irradiation achieved the best tumor killing effect ( Figure 6 bi). The statistical results of tumor growth in different groups also illustrate this point ( Figure 6 j). By testing the body weight of mice in different groups, it was found that there was no significant change ( Figure 6 l), indicating the biosafety of the treatment strategy of NE-GI-LNPs combined with NIR laser irradiation.

Claims

1. A method for preparing homologous targeted neutrophil-liposome system NE-GI-LNPs, characterized in that: The method comprises the following steps: DSPE-PEG-Biotin and DSPE-PEI were added to a triple-negative breast cancer cell membrane suspension and co-incubated to prepare a DSPE-PEG-Biotin / DPE-PEI modified triple-negative breast cancer cell membrane; Plasmid pGSDME and cyanine dye IR775 are added to the triple-negative breast cancer cell membrane suspension containing the DSPE-PEG-Biotin / DPE-PEI modified, and co-incubated to prepare GI-LNPs; DSPE-PEG-STA was added to the neutrophil suspension and co-incubated to prepare neutrophil NE-STA; The NE-STA and the GI-LNPs are co-incubated to prepare the neutrophil-liposome system NE-GI-LNPs.

2. The method according to claim 1, characterized in that The molar ratio of DSPE-PEG-Biotin to DSPE-PEI is (5-15): (1-5); Optionally, the molar ratio of DSPE-PEG-Biotin to DSPE-PEI is 10:1; Optionally, DSPE-PEG-Biotin and DSPE-PEI are added to the triple-negative breast cancer cell membrane suspension and co-incubated for 0.1-5 h; Optionally, DSPE-PEG-Biotin and DSPE-PEI are added to the triple-negative breast cancer cell membrane suspension and co-incubated for 1 h; Optionally, the triple-negative breast cancer cell membrane is a 4T1 cell membrane.

3. The method according to claim 1, characterized in that The dosages of the plasmids pGSDME and IR775 are 50-150 μg / mL and 10-90 μg / mL, respectively; Optionally, the dosages of the plasmids pGSDME and IR775 are 100 μg / mL and 50 μg / mL, respectively; Optionally, the dosage of the triple-negative breast cancer cell membrane suspension containing the DSPE-PEG-Biotin / DPE-PEI modified triple-negative breast cancer cell membrane is 1-7 mg / mL; Optionally, the dosage of the triple-negative breast cancer cell membrane suspension containing the DSPE-PEG-Biotin / DPE-PEI modified is 4 mg / mL; Optionally, the plasmid pGSDME and the cyanine dye IR775 are added to the triple-negative breast cancer cell membrane suspension containing the DSPE-PEG-Biotin / DPE-PEI modified triple-negative breast cancer cell membrane and co-incubated for 0.1-5h; Optionally, the plasmid pGSDME and the cyanine dye IR775 are added to the suspension of triple-negative breast cancer cell membranes modified with the DSPE-PEG-Biotin / DPE-PEI and incubated for 1 hour.

4. The method according to claim 1, characterized in that The dosage of DSPE-PEG-STA and neutrophils is 0.05-0.15 mg / mL, (0.1-5)×10 5 / mL; Optionally, the dosage of DSPE-PEG-STA and neutrophils is 0.1 mg / mL and 1×10 5 / mL; Optionally, DSPE-PEG-STA is added to the neutrophil suspension and co-incubated for 0.1-5 h; Optionally, DSPE-PEG-STA is added to the neutrophil suspension and incubated for 1 hour.

5. The method according to claim 1, characterized in that The dosage of NE-STA and GI-LNPs is (0.1-5)×10 6 / mL, (0.5-3.5) mg / mL; Optionally, the dosage of NE-STA and GI-LNPs is 1×10 6 / mL, 2mg / mL; Optionally, the NE-STA and the GI-LNPs are co-incubated for 1-7 h; Optionally, the NE-STA and the GI-LNPs are co-incubated for 4 hours.

6. A homologous targeted neutrophil-liposome system NE-GI-LNPs, characterized in that: The neutrophil-liposome system NE-GI-LNPs is prepared by anchoring triple-negative breast cancer cell membrane loaded with IR775 and pGSDME on the surface of neutrophils through biotin-avidin interaction; Optionally, the neutrophil-liposome system NE-GI-LNPs are neutrophil-liposome system NE-GI-LNPs prepared by the method according to any one of claims 1-5.

7. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the neutrophil-liposome system NE-GI-LNPs described in claim 6.

8. A pharmaceutical preparation, characterized in that The pharmaceutical preparation comprises the pharmaceutical composition according to claim 7.

9. Use of the neutrophil-liposome system NE-GI-LNPs according to claim 6 in the preparation of a drug for treating triple-negative breast cancer.

10. Use of the neutrophil-liposome system NE-GI-LNPs according to claim 6 in combination with near-infrared laser in the preparation of a drug for treating triple-negative breast cancer.

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