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CN116531522A - A kind of M2 type macrophage targeting nano drug preparation and its application - Google Patents

A kind of M2 type macrophage targeting nano drug preparation and its application Download PDF

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Publication number
CN116531522A
CN116531522A CN202310579553.6A CN202310579553A CN116531522A CN 116531522 A CN116531522 A CN 116531522A CN 202310579553 A CN202310579553 A CN 202310579553A CN 116531522 A CN116531522 A CN 116531522A
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CN
China
Prior art keywords
hemoglobin
drug
nano
corona
doxorubicin
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Pending
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CN202310579553.6A
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Chinese (zh)
Inventor
周东方
白晓春
王玉鹏
梁小玲
王伟
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Southern Medical University
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Southern Medical University
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Priority to CN202310579553.6A priority Critical patent/CN116531522A/en
Publication of CN116531522A publication Critical patent/CN116531522A/en
Pending legal-status Critical Current

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Abstract

本发明涉及一种M2型巨噬细胞靶向的纳米药物制剂及其应用，所述纳米药物制剂包括药物活性成分和纳米载体；其中，所述纳米载体的表面键接有血红蛋白冠。本发明通过血红蛋白修饰含有传统药物活性成分的药物载体，可以起到靶向M2型巨噬细胞，递送氧气或一氧化碳的作用，能够有效的靶向炎症病发部位，提高药物在炎症部位的浓度，达到治疗炎症疾病效果。到达炎症部位的血红蛋白冠纳米药物制剂在炎症微环境的刺激下，快速解体，有利于将所负载的药物快速释放到靶部位，提高治疗效果，降低毒副作用。

The invention relates to an M2 macrophage-targeted nano-medicine preparation and an application thereof. The nano-medicine preparation includes pharmaceutical active ingredients and a nano-carrier; wherein, the surface of the nano-carrier is bonded with a hemoglobin corona. The present invention modifies the drug carrier containing traditional drug active ingredients through hemoglobin, can target M2 type macrophages, deliver oxygen or carbon monoxide, can effectively target the inflammation site, and increase the concentration of the drug in the inflammation site, To achieve the effect of treating inflammatory diseases. The hemoglobin corona nanomedicine preparation that reaches the inflammatory site disintegrates quickly under the stimulation of the inflammatory microenvironment, which is conducive to the rapid release of the loaded drug to the target site, improving the therapeutic effect and reducing toxic and side effects.

Description

一种M2型巨噬细胞靶向的纳米药物制剂及其应用A nanomedicine preparation targeting M2 macrophages and its application

技术领域Technical Field

本发明属于医药化工药物制剂技术领域，具体涉及一种M2型巨噬细胞靶向的纳米药物制剂及其应用。The present invention belongs to the technical field of pharmaceutical chemical drug preparations, and in particular relates to an M2 macrophage-targeted nano drug preparation and an application thereof.

背景技术Background Art

巨噬细胞是固有免疫的重要成员，在机体防御病原微生物感染、肿瘤、过敏性疾病、代谢类疾病、组织损伤修复等发生发展过程中发挥着极其重要的作用。在微环境，尤其是多种细胞因子的影响下，巨噬细胞极化成M1/M2型巨噬细胞，表达相应的特异性基因，行使不同的功能。M1型巨噬细胞主要发挥促炎、杀菌及呈递抗原的功能，M2型巨噬细胞主要起抑炎和组织修复的作用。其中M2型巨噬细胞在多种疾病的发生发展中起重要作用，例如：1)哮喘和过敏性变态反应性哮喘是肺部最常见的慢性炎症性疾病，炎症反应是由Th2淋巴细胞、肥大细胞、嗜酸性粒细胞和巨噬细胞向肺的聚集所致，并与巨噬细胞的M2极化有关。巨噬细胞是过敏性哮喘的重要调节因子，也是肺损伤、纤维化和杯状细胞增生相关炎症反应的启动因子；2)慢性炎症是心血管疾病的主要病因，最终导致动脉粥样硬化。巨噬细胞凋亡是动脉硬化发展各个阶段的一个重要特征，在疾病进展过程中，主要通过M2巨噬细胞，通过内膜吞噬细胞来解决细胞凋亡及其不良影响。此外，M1和M2巨噬细胞的炎症表型并不稳定，这可能反映了单核细胞衍生细胞对微环境变化的可塑性；3)类风湿关节炎是一种慢性自身免疫性疾病,可引起关节炎症和残疾。类风湿关节炎炎症早期阶段，滑膜衬里层巨噬细胞数量增加，滑膜巨噬细胞浸润程度与关节破坏程度相关。M1型巨噬细胞通过抗原递呈激活T细胞，同时还分泌多种炎性细胞因子，募集更多免疫细胞，激活成纤维样滑膜细胞，促进炎性反应。类风湿关节炎炎症消退阶段，M2型巨噬细胞发挥抑制炎性作用。4)结核病是由结核杆菌感染引起的慢性传染病，结核分枝杆菌可能抑制巨噬细胞以避免细胞毒性功能和逃避细胞免疫反应，或使M1极化的巨噬细胞向M2表型转移。尽管在预防、诊断和治疗方面取得了进展，但结核病仍然是世界上最致命的传染病之一；5)肿瘤是目前全球性健康问题，其中浸润到肿瘤内的巨噬细胞在肿瘤的发生发展中发挥着重要的作用，M1型巨噬细胞具有杀伤肿瘤细胞的作用，而M2型巨噬细胞则表现为促进肿瘤生长。巨噬细胞的极化与肿瘤微环境密切相关，在长期肿瘤微环境的作用下，巨噬细胞主要表现为M2型，与肿瘤生长、发展、侵袭和转移密切相关。巨噬细胞向M1型和M2型的极化具有可逆性和可调节性。在肿瘤的各阶段，M1型和M2型巨噬细胞均存在，早期以M1型为主，中晚期以M2型为主。随着肿瘤的进展，M1型逐渐向M2型极化，M2型数量的增多也提示着预后不良。因此，以M2型巨噬细胞为治疗靶点在多种疾病治疗中具有重大价值。Macrophages are important members of innate immunity and play an extremely important role in the development of the body's defense against pathogenic microorganisms, tumors, allergic diseases, metabolic diseases, tissue damage and repair. Under the influence of the microenvironment, especially a variety of cytokines, macrophages polarize into M1/M2 macrophages, express corresponding specific genes, and perform different functions. M1 macrophages mainly play the role of pro-inflammatory, bactericidal and antigen presentation, while M2 macrophages mainly play the role of anti-inflammatory and tissue repair. Among them, M2 macrophages play an important role in the development of many diseases, for example: 1) Asthma and allergic allergic asthma are the most common chronic inflammatory diseases of the lungs. The inflammatory response is caused by the aggregation of Th2 lymphocytes, mast cells, eosinophils and macrophages to the lungs, and is related to the M2 polarization of macrophages. Macrophages are important regulators of allergic asthma and initiators of inflammatory responses related to lung injury, fibrosis and goblet cell hyperplasia; 2) Chronic inflammation is the main cause of cardiovascular disease, ultimately leading to atherosclerosis. Macrophage apoptosis is an important feature of all stages of atherosclerosis development. During the progression of the disease, apoptosis and its adverse effects are mainly resolved by M2 macrophages and intimal phagocytes. In addition, the inflammatory phenotype of M1 and M2 macrophages is not stable, which may reflect the plasticity of monocyte-derived cells to changes in the microenvironment; 3) Rheumatoid arthritis is a chronic autoimmune disease that can cause joint inflammation and disability. In the early stage of rheumatoid arthritis inflammation, the number of macrophages in the synovial lining layer increases, and the degree of synovial macrophage infiltration is related to the degree of joint destruction. M1 macrophages activate T cells through antigen presentation, and also secrete a variety of inflammatory cytokines, recruit more immune cells, activate fibroblast-like synoviocytes, and promote inflammatory responses. In the stage of inflammation resolution in rheumatoid arthritis, M2 macrophages play an anti-inflammatory role. 4) Tuberculosis is a chronic infectious disease caused by infection with Mycobacterium tuberculosis. Mycobacterium tuberculosis may inhibit macrophages to avoid cytotoxic functions and escape cellular immune responses, or cause M1 polarized macrophages to shift to M2 phenotypes. Despite progress in prevention, diagnosis and treatment, tuberculosis remains one of the world's deadliest infectious diseases; 5) Tumors are currently a global health problem, among which macrophages infiltrating into tumors play an important role in the occurrence and development of tumors. M1 macrophages have the function of killing tumor cells, while M2 macrophages promote tumor growth. The polarization of macrophages is closely related to the tumor microenvironment. Under the influence of the long-term tumor microenvironment, macrophages mainly show the M2 type, which is closely related to tumor growth, development, invasion and metastasis. The polarization of macrophages to M1 and M2 types is reversible and adjustable. At all stages of the tumor, both M1 and M2 macrophages exist, with M1 being the main type in the early stage and M2 being the main type in the middle and late stages. As the tumor progresses, M1 gradually polarizes to M2, and the increase in the number of M2 types also indicates a poor prognosis. Therefore, using M2 macrophages as therapeutic targets is of great value in the treatment of various diseases.

目前，靶向M2型巨噬细胞的手段极少，多数以合成靶向多肽修饰的纳米给药系统包括脂质体、微球、纳米粒等，或者以抗体药物偶联物(ADC)为主。但是复杂的合成工艺和高昂成成本，限制其向临床的转化的可行性。因此，目前以M2型巨噬细胞为靶点的治疗因为没有合适的靶向药物而陷入瓶颈。At present, there are very few means to target M2 macrophages. Most of them are nano-drug delivery systems modified with synthetic targeting peptides, including liposomes, microspheres, nanoparticles, etc., or antibody-drug conjugates (ADCs). However, the complex synthesis process and high cost limit the feasibility of its transformation to clinical practice. Therefore, the current treatment targeting M2 macrophages has reached a bottleneck due to the lack of suitable targeted drugs.

CD163是一种仅存在于巨噬细胞及其祖细胞中的表面受体，并且在M2型巨噬细胞中过表达。人体内游离血红蛋白的清除机制是血红蛋白内源性地与血液中结合珠蛋白结合，其复合体可特异性地靶向结合肝脏、脾脏部位的M2型巨噬细胞的CD163受体，从而清除出体内。因此血红蛋白是一种很有潜力的M2型巨噬细胞靶向分子。目前，尚无有关以血红蛋白为靶向修饰的纳米剂型用于炎症疾病治疗的报道。CD163 is a surface receptor that only exists in macrophages and their progenitor cells, and is overexpressed in M2 macrophages. The clearance mechanism of free hemoglobin in the human body is that hemoglobin endogenously binds to haptoglobin in the blood, and the complex can specifically target and bind to the CD163 receptors of M2 macrophages in the liver and spleen, thereby clearing it out of the body. Therefore, hemoglobin is a very potential M2 macrophage targeting molecule. At present, there are no reports on the use of hemoglobin-targeted nanoformulations for the treatment of inflammatory diseases.

发明内容Summary of the invention

本发明通过大量的检索和创造性实验，一种M2型巨噬细胞靶向的纳米药物制剂；该药物制剂具有粒径小、载药量高等特性，可精准靶向发病部位(如结核病、肿瘤)的M2型巨噬细胞，实现药物的靶向递送；然后通过调控M2型巨噬细胞调控发病部位免疫微环境，从而达到治疗炎症相关疾病的效果。The present invention, through a large number of searches and creative experiments, has developed a nano drug preparation targeted at M2 macrophages; the drug preparation has the characteristics of small particle size and high drug loading, and can accurately target M2 macrophages at the site of disease (such as tuberculosis, tumors) to achieve targeted drug delivery; then, by regulating the M2 macrophages, the immune microenvironment at the site of disease is regulated, thereby achieving the effect of treating inflammatory-related diseases.

本发明提供的一种M2型巨噬细胞靶向的纳米药物制剂，包括药物活性成分和纳米载体；其中，所述纳米载体的表面键接有血红蛋白冠。The present invention provides an M2 macrophage-targeted nano drug preparation, comprising a drug active ingredient and a nano carrier; wherein the surface of the nano carrier is bonded with a hemoglobin corona.

其中，所述血红蛋白冠以直接方式键接在纳米载体表面上。Wherein, the hemoglobin corona is directly bonded to the surface of the nanocarrier.

所述血红蛋白冠采用如下方式键接在纳米载体上：血红蛋白通过表面的巯基基团与纳米载体表面的马来酰亚胺进行迈克尔加成反应，形成血红蛋白冠。The hemoglobin corona is bonded to the nanocarrier in the following manner: hemoglobin undergoes Michael addition reaction with maleimide on the surface of the nanocarrier via the thiol groups on the surface to form the hemoglobin corona.

具体的：纳米载体的表面修饰有马来酰亚胺官能基团，血红蛋白可以通过表面的巯基基团与纳米载体表面的马来酰亚胺进行迈克尔加成反应，特异性键合在纳米载体的表面，在纳米载体表面形成一层血红蛋白的外冠，从而能够与血液内内源性结合珠蛋白特异性结合，靶向炎症部位的M2型巨噬细胞。Specifically: The surface of the nanocarrier is modified with maleimide functional groups. Hemoglobin can undergo Michael addition reaction with the maleimide on the surface of the nanocarrier through the surface thiol groups, specifically bond to the surface of the nanocarrier, and form a layer of hemoglobin outer corona on the surface of the nanocarrier, so that it can specifically bind to endogenous haptoglobin in the blood and target M2 macrophages in the inflammatory site.

其中，所述纳米载体的粒径为50～200nm。Wherein, the particle size of the nanocarrier is 50 to 200 nm.

其中，所述纳米载体为键接有血红蛋白冠的脂质体、胶束、纳米囊泡、外泌体、纳米微球、固体脂质纳米粒或纳米凝胶等药物载体输送系统。即为修饰有血红蛋白的脂质体、胶束、纳米囊泡、外泌体、纳米微球、固体脂质纳米粒或纳米凝胶等The nanocarrier is a drug carrier delivery system such as liposomes, micelles, nanovesicles, exosomes, nanospheres, solid lipid nanoparticles or nanogels bonded with a hemoglobin corona.

上述脂质体、胶束、纳米囊泡、外泌体、纳米微球、固体脂质纳米粒或纳米凝胶等药物载体输送系统可按照现有技术中报道过的任何一种方法制备。The above-mentioned drug carrier delivery systems such as liposomes, micelles, nanovesicles, exosomes, nanospheres, solid lipid nanoparticles or nanogels can be prepared according to any method reported in the prior art.

优选的，制备所述脂质体的原料选自胆固醇、二油酰磷脂酰胆碱、磷脂磷脂酰胆碱、磷脂磷脂酰丝氨酸、磷脂酰胆碱、二硬脂酰磷脂酰胆碱、N-羟基琥珀酰亚胺修饰磷脂-聚乙二醇、马来酰亚胺修饰磷脂-聚乙二醇、N-羟基琥珀酰亚胺修饰二硬脂酰基磷脂酰乙醇胺聚乙二醇、马来酰亚胺修饰二硬脂酰基磷脂酰乙醇胺聚乙二醇，或其衍生物中的一种或多种。Preferably, the raw materials for preparing the liposomes are selected from cholesterol, dioleoylphosphatidylcholine, phospholipid phosphatidylcholine, phospholipid phosphatidylserine, phosphatidylcholine, distearoylphosphatidylcholine, N-hydroxysuccinimide-modified phospholipid-polyethylene glycol, maleimide-modified phospholipid-polyethylene glycol, N-hydroxysuccinimide-modified distearoylphosphatidylethanolamine polyethylene glycol, maleimide-modified distearoylphosphatidylethanolamine polyethylene glycol, or one or more of their derivatives.

以脂质体为载体的纳米药物制剂的制备方法可采用如下方式制得：The preparation method of the nano drug preparation using liposome as carrier can be prepared in the following manner:

1)将脂质材料分散在有机溶剂中，为体系A；将药物活性成分分散在有机溶剂中，为体系B；将血红蛋白溶于缓冲盐溶液溶液中(pH为7.0～7.6)，为体系C；1) Dispersing lipid materials in an organic solvent is system A; dispersing active pharmaceutical ingredients in an organic solvent is system B; dissolving hemoglobin in a buffered saline solution (pH 7.0-7.6) is system C;

2)将体系A中加入体系B中，得到体系D；再将体系D通过旋转蒸发除去有机溶剂并在容器壁上形成薄膜；然后用超纯水，使用超声复乳技术，即可得载活性药物的脂质体药物制剂，获得体系E；2) adding system A to system B to obtain system D; then removing the organic solvent from system D by rotary evaporation and forming a thin film on the container wall; then using ultrapure water and ultrasonic emulsion technology to obtain a liposome drug preparation carrying active drugs, obtaining system E;

3)将体系C加入体系E中，在氩气保护状态下，冰浴中搅拌12h，得到体系F；然后将体系F通过葡聚糖凝胶G-250透析除去未键合的游离血红蛋白和未担载的药物，使用0.45μm和0.22μm滤膜过滤，即得到纳米药物制剂(血红蛋白冠脂质体药物制剂)。3) System C was added to system E, and the mixture was stirred in an ice bath for 12 h under argon protection to obtain system F; system F was then dialyzed through dextran gel G-250 to remove unbound free hemoglobin and unloaded drugs, and filtered using 0.45 μm and 0.22 μm filter membranes to obtain a nano drug preparation (hemoglobin corona liposome drug preparation).

优选的，制备胶束(聚合物胶束)的原料选自N-羟基琥珀酰亚胺修饰聚乙二醇-聚乳酸-羟基乙酸或马来酰亚胺修饰聚乙二醇-聚乳酸-羟基乙酸、N-羟基琥珀酰亚胺修饰聚乙二醇-聚己内酯或马来酰亚胺修饰聚乙二醇-聚己内酯、N-羟基琥珀酰亚胺修饰聚乙二醇-聚碳酸酯或马来酰亚胺修饰聚乙二醇-聚碳酸酯中的一种或多种。Preferably, the raw materials for preparing micelles (polymer micelles) are selected from one or more of N-hydroxysuccinimide-modified polyethylene glycol-polylactic acid-glycolic acid or maleimide-modified polyethylene glycol-polylactic acid-glycolic acid, N-hydroxysuccinimide-modified polyethylene glycol-polycaprolactone or maleimide-modified polyethylene glycol-polycaprolactone, N-hydroxysuccinimide-modified polyethylene glycol-polycarbonate or maleimide-modified polyethylene glycol-polycarbonate.

以胶束为载体的纳米药物制剂的制备方法可采用如下方式制得：The preparation method of the nano drug preparation using micelles as carriers can be prepared in the following manner:

1)将聚合物材料分散在有机溶剂中，为体系Ⅰ；将药物活性成分分散在有机溶剂中，为体系Ⅱ；将血红蛋白溶于缓冲盐溶液溶液中(pH为7.0～7.6)，为体系Ⅲ；1) Dispersing the polymer material in an organic solvent is system I; dispersing the active ingredient of the drug in an organic solvent is system II; dissolving hemoglobin in a buffered saline solution (pH 7.0-7.6) is system III;

2)将体系Ⅰ加入体系Ⅱ中，得到体系Ⅳ；将体系Ⅳ逐滴加入到水溶液中，搅拌1小时后加入截留分子量3500的透析袋中，以水为透析介质进行透析，形成包裹疏水药物的聚合物药物制剂，获得体系Ⅴ；2) adding system I into system II to obtain system IV; adding system IV dropwise into the aqueous solution, stirring for 1 hour, adding into a dialysis bag with a molecular weight cutoff of 3500, and dialyzing with water as the dialysis medium to form a polymer drug preparation encapsulating the hydrophobic drug, and obtaining system V;

3)将体系Ⅲ加入至体系Ⅴ中，在氩气保护状态下，冰浴中搅拌12h，得到体系Ⅵ；将体系Ⅵ通过葡聚糖凝胶G-250透析除去未键合的游离血红蛋白和未担载的药物，使用0.45μm和0.22μm滤膜过滤，即得到纳米药物制剂(血红蛋白冠聚合物胶束药物制剂)。3) adding system III to system V, stirring in an ice bath for 12 h under argon protection to obtain system VI; dialyzing system VI through dextran gel G-250 to remove unbound free hemoglobin and unloaded drugs, and filtering using 0.45 μm and 0.22 μm filter membranes to obtain a nano drug preparation (hemoglobin corona polymer micelle drug preparation).

其中，所述药物活性成分包括但不限于为治疗炎症、肿瘤等药物；可以为化学药物，或中药提取物，或蛋白药物，或核酸药物。The active ingredients of the medicine include, but are not limited to, medicines for treating inflammation, tumors, etc.; they can be chemical medicines, or Chinese medicine extracts, or protein medicines, or nucleic acid medicines.

所述药物活性成分为抗肿瘤药物、抗菌药物、关节炎药物、抗病毒药物或维生素。The active ingredients of the medicine are anti-tumor drugs, antibacterial drugs, arthritis drugs, antiviral drugs or vitamins.

具体的，所述药物活性成分选自甲氨蝶呤、来氟米特、羟氯喹、利福平、异烟肼、链霉素、乙胺丁醇、吡嗪酰胺、阿霉素、紫杉醇、吉西他滨、奥沙利铂、多柔比星、柔红霉素、甲氨蝶呤、多西他赛、卡培他滨、胰岛素、PD-1抗体、CTLA-4抗体、小干扰RNA、核酸适配体、维生素C中的一种或多种。Specifically, the active ingredient of the drug is selected from one or more of methotrexate, leflunomide, hydroxychloroquine, rifampicin, isoniazid, streptomycin, ethambutol, pyrazinamide, doxorubicin, paclitaxel, gemcitabine, oxaliplatin, doxorubicin, daunorubicin, methotrexate, docetaxel, capecitabine, insulin, PD-1 antibody, CTLA-4 antibody, small interfering RNA, nucleic acid aptamer, and vitamin C.

其中，所述药物活性与所述纳米载体的重量比为1：4～15；优选的，所述药物活性成分与纳米载体原料的质量比为1:8；优选为1:6～10；更优选为1:8。Wherein, the weight ratio of the drug activity to the nanocarrier is 1:4-15; preferably, the mass ratio of the drug active ingredient to the nanocarrier raw material is 1:8; preferably 1:6-10; more preferably 1:8.

其中，所述血红蛋白为本领域常规的血红蛋白，如牛血红蛋白、小鼠血红蛋白、人血红蛋白；能够靶向炎症、肿瘤部位的M2型巨噬细胞。Wherein, the hemoglobin is conventional hemoglobin in the art, such as bovine hemoglobin, mouse hemoglobin, and human hemoglobin; and it can target M2 macrophages in inflammation and tumor sites.

本发明提供上述任一所述的M2型巨噬细胞靶向的纳米药物制剂在制备治疗炎症疾病上药物或试剂中的应用。The present invention provides use of any of the above-mentioned M2 macrophage-targeted nanomedicine preparations in the preparation of drugs or reagents for treating inflammatory diseases.

该炎症疾病包括病原微生物感染，如细菌、病毒、支原体等引起人体组织渗出、增生、坏死等炎性反应，包括结核病、阴道炎、前列腺炎、肺炎等。非感染性炎症指的是由于物理、化学因素导致的炎症反应，包括风湿性关节炎、狼疮性皮炎等；炎症相关性疾病包括癌症、脑卒中等。The inflammatory diseases include infections caused by pathogenic microorganisms, such as bacteria, viruses, mycoplasma, etc., which cause inflammatory reactions such as exudation, proliferation, and necrosis of human tissues, including tuberculosis, vaginitis, prostatitis, pneumonia, etc. Non-infectious inflammation refers to inflammatory reactions caused by physical and chemical factors, including rheumatoid arthritis, lupus dermatitis, etc.; inflammation-related diseases include cancer, stroke, etc.

即，所述药物或试剂具体为结核病、阴道炎、前列腺炎、肺炎、风湿性关节炎、狼疮性皮炎、癌症、脑卒药物或制剂。That is, the drug or agent is specifically a drug or preparation for tuberculosis, vaginitis, prostatitis, pneumonia, rheumatoid arthritis, lupus dermatitis, cancer, or stroke.

本发明所述的M2型巨噬细胞靶向的纳米药物制剂的制备方法如下：将药物活性成分(治疗性药物)采用物理方式包封在纳米载体中，在将血红蛋白通过化学键合修饰在载药纳米载体表面，制备血红蛋白冠纳米药物制剂。The preparation method of the M2 macrophage-targeted nano drug preparation of the present invention is as follows: the active ingredient of the drug (therapeutic drug) is physically encapsulated in a nano carrier, and hemoglobin is modified on the surface of the drug-loaded nano carrier by chemical bonding to prepare a hemoglobin corona nano drug preparation.

在实际应用中，将所制备的血红蛋白冠纳米药物制剂通过静脉注射到人体内，血液循环过程中，血液中结合珠蛋白与血红蛋白冠纳米药物制剂表面的血红蛋白特异性结合，呈现出新的抗原决定簌，可以特异性识别炎症部位的M2型巨噬细胞表面的CD163抗体，从而主动靶向定位到炎症部位，提高了炎症部位药物浓度，保障了药效的发挥。研究表明，感染性炎症、非感染性炎症以及炎症相关性疾病如肺结核、癌症的发病部位均有大量M1型和M2型巨噬细胞募集，并且疾病的发生与发展与M2型巨噬细胞的炎症调控密切相关，因此利用修饰有血红蛋白冠的纳米药物制剂治疗炎症或炎症相关的疾病如结核病、癌症，可以靶向提高药物在病变部位的蓄积，从而提高药物的疗效，并降低脱靶风险，降低药物的毒副作用。综上所述，担载有治疗性药物的血红蛋白冠的纳米药物制剂，在内源性结合珠蛋白的作用下，可以将治疗性药物靶向递送到炎症相关疾病部位，进行疾病的靶向治疗，抑制疾病的发生和发展。In practical applications, the prepared hemoglobin corona nanopharmaceutical preparation is injected intravenously into the human body. During the blood circulation process, the haptoglobin in the blood specifically binds to the hemoglobin on the surface of the hemoglobin corona nanopharmaceutical preparation, presenting a new antigenic determinant, which can specifically recognize the CD163 antibody on the surface of the M2 macrophages at the site of inflammation, thereby actively targeting the site of inflammation, increasing the drug concentration at the site of inflammation, and ensuring the efficacy of the drug. Studies have shown that a large number of M1 and M2 macrophages are recruited at the site of infectious inflammation, non-infectious inflammation, and inflammation-related diseases such as tuberculosis and cancer, and the occurrence and development of the disease are closely related to the inflammatory regulation of M2 macrophages. Therefore, the use of nanopharmaceutical preparations modified with hemoglobin corona to treat inflammation or inflammation-related diseases such as tuberculosis and cancer can target and increase the accumulation of drugs at the site of lesions, thereby improving the efficacy of the drug, reducing the risk of off-target, and reducing the toxic side effects of the drug. In summary, the nanodrug preparations loaded with therapeutic drugs on the hemoglobin corona can, under the action of endogenous haptoglobin, deliver therapeutic drugs to the sites of inflammation-related diseases in a targeted manner, thereby performing targeted treatment of the disease and inhibiting the occurrence and development of the disease.

本发明所述的纳米载体可按照现有技术中报道过的任何一种方法制备。The nanocarrier of the present invention can be prepared according to any method reported in the prior art.

本发明以血红蛋白冠修饰的纳米载体为递药载体，首先，纳米载体可以保护活性药物逃避网状内皮系统的清除，延长活性药物的循环半衰期，实现药物的可控释放，减少药物泄露所导致的毒副作用。其次，血红蛋白冠修饰的纳米载体可以与血液中结合珠蛋白特异性结合，从而特异性靶向炎症部位的M2型巨噬细胞，提高活性药物在炎症部位的浓度。第三，血红蛋白可以同时传输氧气、一氧化碳、一氧化氮等一种或多种治疗气体，能够在炎症高ROS环境中响应性释放携带的气体，配合活性药物治疗炎症。因此，血红蛋白冠纳米载体可以起到内源特异性靶向递送，降低活性药物对正常细胞、组织毒副作用，与活性药物协同治疗炎症的作用。The present invention uses a hemoglobin corona-modified nanocarrier as a drug delivery carrier. First, the nanocarrier can protect the active drug from escaping the clearance of the reticuloendothelial system, prolong the circulation half-life of the active drug, achieve controlled release of the drug, and reduce the toxic and side effects caused by drug leakage. Secondly, the hemoglobin corona-modified nanocarrier can specifically bind to the haptoglobin in the blood, thereby specifically targeting the M2 macrophages at the site of inflammation and increasing the concentration of the active drug at the site of inflammation. Third, hemoglobin can simultaneously transmit one or more therapeutic gases such as oxygen, carbon monoxide, and nitric oxide, and can responsively release the carried gas in an inflammatory high ROS environment to cooperate with the active drug to treat inflammation. Therefore, the hemoglobin corona nanocarrier can play an endogenous specific targeted delivery role, reduce the toxic and side effects of the active drug on normal cells and tissues, and synergize with the active drug to treat inflammation.

与现有技术相比，本发明具有的优点如下：Compared with the prior art, the present invention has the following advantages:

1.本发明提供的血红蛋白冠修饰的纳米药物制剂，内源性与结合珠蛋白相结合，特异性靶向炎症部位的M2型巨噬细胞，成功解决了活性药物的对炎症部位M2型巨噬细胞的靶向性问题。1. The hemoglobin corona-modified nanopharmaceutical preparation provided by the present invention combines endogenously with haptoglobin and specifically targets M2 macrophages in inflammatory sites, thus successfully solving the problem of targeting of active drugs to M2 macrophages in inflammatory sites.

2.本发明所制备的血红蛋白冠修饰纳米药物制剂，利用纳米载体担载活性药物，保证了较高的载药量，实现对药物的可控释放，减少了活性药物对正常组织的毒副作用。2. The hemoglobin corona modified nano drug preparation prepared by the present invention utilizes nano carriers to carry active drugs, thereby ensuring a higher drug loading amount, achieving controlled release of drugs, and reducing the toxic and side effects of active drugs on normal tissues.

3.本发明所制备的血红蛋白冠修饰的纳米药物制剂，能够同时递送氧气、一氧化碳、一氧化氮等一种或多种治疗气体，因此能够协同活性药物提高炎症治疗效果。3. The hemoglobin corona-modified nanopharmaceutical preparation prepared by the present invention can simultaneously deliver one or more therapeutic gases such as oxygen, carbon monoxide, and nitric oxide, thereby synergizing active drugs to improve the therapeutic effect of inflammation.

4.本发明所使用的纳米载体均是被FDA批准的已经在临床中应用的纳米载体，所使用的血红蛋白是人体内数量最多的功能蛋白，容易从患者体内获得，这使得本发明具备较高的临床开发应用价值。4. The nanocarriers used in the present invention are all FDA-approved nanocarriers that have been used clinically. The hemoglobin used is the most abundant functional protein in the human body and is easy to obtain from the patient's body, which makes the present invention have a high clinical development and application value.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

图1为负载阿霉素的血红蛋白冠脂质体制剂的DLS图；FIG1 is a DLS graph of a hemoglobin corona liposome preparation loaded with doxorubicin;

图2为负载阿霉素的血红蛋白冠脂质体制剂的TEM图片；FIG2 is a TEM image of a hemoglobin corona liposome preparation loaded with doxorubicin;

图3为不同阿霉素/纳米载体投料比条件下的药物担载效率和载药量曲线图；FIG3 is a graph showing drug loading efficiency and drug loading under different doxorubicin/nanocarrier feed ratios;

图4为负载阿霉素的血红蛋白冠脂质体制剂在37℃和4℃条件下PBS(pH7.4)中的稳定性；FIG4 shows the stability of hemoglobin corona liposome preparation loaded with doxorubicin in PBS (pH 7.4) at 37° C. and 4° C.;

图5为负载阿霉素的血红蛋白冠脂质体药物制剂在37℃条件下pH值7.4,，6.5，5.0PBS(10％FBS，0.01M)中的药物释放行为。FIG5 shows the drug release behavior of the hemoglobin corona liposome drug preparation loaded with doxorubicin in PBS (10% FBS, 0.01 M) at pH values of 7.4, 6.5, and 5.0 at 37°C.

图6为小分子阿霉素，脂质体阿霉素(市售)和负载阿霉素的血红蛋白冠脂质体药物制剂的血液药代动力学曲线；FIG6 is a blood pharmacokinetic curve of small molecule doxorubicin, liposomal doxorubicin (commercially available) and hemoglobin corona liposome drug preparation loaded with doxorubicin;

图7为小分子阿霉素，脂质体阿霉素(市售)和负载阿霉素的血红蛋白冠脂质体药物制剂给药24h后主要脏器中药物浓度；FIG7 shows the drug concentrations in major organs of small molecule doxorubicin, liposomal doxorubicin (commercially available) and hemoglobin corona liposome drug preparations loaded with doxorubicin 24 hours after administration;

图8为小分子阿霉素，脂质体阿霉素(市售)和负载阿霉素的血红蛋白冠脂质体药物制剂给药24h后在肺、肝脏和肿瘤中巨噬细胞内的药物荧光强度；FIG8 shows the drug fluorescence intensity in macrophages in the lung, liver and tumor 24 hours after administration of small molecule doxorubicin, liposomal doxorubicin (commercially available) and hemoglobin corona liposome drug preparation loaded with doxorubicin;

图9为原位乳腺癌模型小鼠给药生理盐水，小分子阿霉素，脂质体阿霉素(市售)，携带氧气的负载阿霉素的血红蛋白冠脂质体药物制剂和携带一氧化碳的负载阿霉素的血红蛋白冠脂质体药物制剂肿瘤抑制曲线；FIG9 is a tumor inhibition curve of mice with orthotopic breast cancer model given normal saline, small molecule doxorubicin, liposomal doxorubicin (commercially available), hemoglobin corona liposome drug preparation loaded with doxorubicin carrying oxygen, and hemoglobin corona liposome drug preparation loaded with doxorubicin carrying carbon monoxide;

图10为原位乳腺癌模型小鼠给药小分子阿霉素，脂质体阿霉素(市售)，携带氧气的负载阿霉素的血红蛋白冠脂质体药物制剂和携带一氧化碳的负载阿霉素的血红蛋白冠脂质体药物制剂治疗后小鼠心、肝、脾、肺、肾和肿瘤的H&E切片；FIG10 is H&E sections of the heart, liver, spleen, lung, kidney and tumor of mice with orthotopic breast cancer model treated with small molecule doxorubicin, liposomal doxorubicin (commercially available), hemoglobin corona liposome drug formulation loaded with doxorubicin carrying oxygen and hemoglobin corona liposome drug formulation loaded with doxorubicin carrying carbon monoxide;

图11为原位乳腺癌模型小鼠给药小分子阿霉素，脂质体阿霉素(市售)，携带氧气的负载阿霉素的血红蛋白冠脂质体药物制剂和携带一氧化碳的负载阿霉素的血红蛋白冠脂质体药物制剂治疗后小鼠生存曲线；FIG11 is a survival curve of mice in an orthotopic breast cancer model treated with small molecule doxorubicin, liposomal doxorubicin (commercially available), hemoglobin corona liposome drug formulation loaded with doxorubicin carrying oxygen, and hemoglobin corona liposome drug formulation loaded with doxorubicin carrying carbon monoxide;

图12为负载利福平的血红蛋白冠聚合物胶束制剂的DLS图；FIG12 is a DLS graph of a hemoglobin corona polymer micelle preparation loaded with rifampicin;

图13为负载利福平的血红蛋白冠聚合物胶束制剂的TEM图片；FIG13 is a TEM image of a hemoglobin corona polymer micelle preparation loaded with rifampicin;

图14为不同利福平/纳米载体投料比条件下的药物担载效率和载药量曲线图；FIG14 is a graph showing drug loading efficiency and drug loading under different rifampicin/nanocarrier feed ratios;

图15为负载利福平的血红蛋白冠聚合物胶束制剂在37℃和4℃条件下PBS(pH7.4)中的稳定性；FIG15 shows the stability of rifampicin-loaded hemoglobin corona polymer micelle formulations in PBS (pH 7.4) at 37° C. and 4° C.;

图16为负载利福平的血红蛋白冠聚合物胶束制剂在37℃条件下pH值7.4，6.5，5.0PBS中的药物释放行为；FIG16 shows the drug release behavior of rifampicin-loaded hemoglobin corona polymer micelle preparation in PBS with pH values of 7.4, 6.5, and 5.0 at 37° C.;

图17为小分子利福平，负载利福平的聚合物胶束制剂和负载利福平的血红蛋白冠聚合物胶束制剂的血液药代动力学曲线；FIG17 is a blood pharmacokinetic curve of small molecule rifampicin, a polymer micelle preparation loaded with rifampicin, and a hemoglobin corona polymer micelle preparation loaded with rifampicin;

图18为小分子利福平，负载利福平的聚合物胶束制剂和负载利福平的血红蛋白冠聚合物胶束制剂给药24h后主要脏器中药物浓度；FIG18 shows the drug concentrations in major organs of small molecule rifampicin, rifampicin-loaded polymer micelle preparations, and rifampicin-loaded hemoglobin corona polymer micelle preparations 24 hours after administration;

图19为负载荧光探针的聚合物胶束制剂和负载荧光探针的血红蛋白冠聚合物胶束制剂给药24h后为肺结核结节切片中荧光染料(红色荧光)和CD163(绿色荧光)免疫荧光染色，荧光染料(红色荧光)和CD86(绿色荧光)免疫荧光染色；Figure 19 shows immunofluorescence staining of fluorescent dye (red fluorescence) and CD163 (green fluorescence), and immunofluorescence staining of fluorescent dye (red fluorescence) and CD86 (green fluorescence) in sections of tuberculosis nodules 24 hours after administration of the polymer micelle preparation loaded with fluorescent probes and the hemoglobin corona polymer micelle preparation loaded with fluorescent probes;

图20为原位肺结核模型小鼠给药生理盐水，小分子利福平，负载利福平的聚合物胶束制剂和负载利福平的血红蛋白冠聚合物胶束制剂给药后小鼠肺部荧光海分枝杆菌成像；FIG20 is an imaging of fluorescent Mycobacterium marinum in the lungs of mice with an in situ pulmonary tuberculosis model after administration of normal saline, small molecule rifampicin, polymer micelle preparations loaded with rifampicin, and hemoglobin corona polymer micelle preparations loaded with rifampicin;

图21为原位肺结核模型小鼠给药生理盐水，小分子利福平，负载利福平的聚合物胶束制剂和负载利福平的血红蛋白冠聚合物胶束制剂给药后小鼠肺部细菌平板图。Figure 21 is a bacterial plate image of the lungs of mice with an in situ pulmonary tuberculosis model after administration of normal saline, small molecule rifampicin, rifampicin-loaded polymer micelle preparations, and rifampicin-loaded hemoglobin corona polymer micelle preparations.

具体实施方式DETAILED DESCRIPTION

以下实施例用于说明本发明，但不用来限制本发明的范围。The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

如下血红蛋白采用如下方式进行分离和纯化：The following hemoglobins were isolated and purified as follows:

收集小鼠血液：取50mL烧杯，加入溶有0.6g柠檬酸钠的10mL的生理盐水，再加入10mL的小鼠血液，搅拌。Collect mouse blood: Take a 50 mL beaker, add 10 mL of normal saline containing 0.6 g of sodium citrate, then add 10 mL of mouse blood and stir.

红细胞的洗涤：转移5mL的小鼠血液稀释液到10mL离心管中，5000r/min离心5min，得到上层透明的黄色血浆和下层暗红色的红细胞液体；用胶头滴管吸出上层透明的黄色血浆，把下层暗红色的红细胞液体转入10mL的烧杯，再加入5倍体积的生理盐水，轻轻搅拌10min，转入离心管中，5000r/min离心5min。如此再重复3次，直至上清液不显黄色为止，表明红细胞洗涤干净。Washing of red blood cells: transfer 5mL of mouse blood dilution to a 10mL centrifuge tube, centrifuge at 5000r/min for 5min, and obtain the upper transparent yellow plasma and the lower dark red red blood cell liquid; use a rubber-tipped dropper to suck out the upper transparent yellow plasma, transfer the lower dark red red blood cell liquid to a 10mL beaker, add 5 times the volume of physiological saline, stir gently for 10min, transfer to a centrifuge tube, and centrifuge at 5000r/min for 5min. Repeat this 3 times until the supernatant is no longer yellow, indicating that the red blood cells are washed clean.

血红蛋白的释放：洗涤好的红细胞液约2mL.加入30mL的蒸馏水，置于磁力搅拌器上充分搅拌10min，5000r/min离心8min；上清液为血红蛋白溶液，沉淀物为细胞碎片；用双层纱布过滤。得到含血红蛋白的滤液。Release of hemoglobin: Add 30 mL of distilled water to about 2 mL of washed red blood cell solution, place on a magnetic stirrer and stir thoroughly for 10 minutes, centrifuge at 5000 r/min for 8 minutes; the supernatant is the hemoglobin solution, and the precipitate is the cell debris; filter with double-layer gauze to obtain the filtrate containing hemoglobin.

盐析分离血红蛋白：取2mL滤液，逐渐加入30mL的(NH4)2SO4饱和溶液，边加边摇，溶液出现红色浑浊；转移10mL混浊液到离心管中，10000r/min离心10min.离心管下部沉淀的深红物质就是分离出来的血红蛋白。Salting out to separate hemoglobin: Take 2 mL of filtrate and gradually add 30 mL of saturated (NH4) 2 SO 4 solution, shaking while adding until the solution becomes red and turbid; transfer 10 mL of the turbid solution to a centrifuge tube and centrifuge at 10,000 r/min for 10 min. The dark red substance precipitated at the bottom of the centrifuge tube is the separated hemoglobin.

实施例1Example 1

本实施例提供一种负载阿霉素的血红蛋白冠修饰的脂质体药物制剂(Hb-Lip@DOX)，具体采用如下方式制备：This embodiment provides a hemoglobin corona-modified liposome drug preparation (Hb-Lip@DOX) loaded with doxorubicin, which is prepared in the following manner:

1、荷载阿霉素的马来酰亚胺修饰脂质体(Lip@DOX)的制备1. Preparation of maleimide-modified liposomes loaded with doxorubicin (Lip@DOX)

利用薄膜水化法制备荷载阿霉素的马来酰亚胺修饰脂质体，制备步骤具体为：Maleimide-modified liposomes loaded with doxorubicin were prepared by thin film hydration method, and the preparation steps were as follows:

1)将65mg溴化三甲基-2,3-二油酰氧基丙基铵(DOTAP)、40mg胆固醇(DC-Chol)、35mg马来酰亚胺修饰磷脂-聚乙二醇(Mal-DSPE-PEG2000)溶于30mL二氯甲烷。搅拌进行反应后旋转蒸发除去乙醇并在容器壁上形成薄膜。1) Dissolve 65 mg of trimethyl-2,3-dioleyloxypropylammonium bromide (DOTAP), 40 mg of cholesterol (DC-Chol), and 35 mg of maleimide-modified phospholipid-polyethylene glycol (Mal-DSPE-PEG2000) in 30 mL of dichloromethane. After stirring for reaction, remove ethanol by rotary evaporation and form a film on the container wall.

2)25mg盐酸阿霉素溶于10mL磷酸盐缓冲液。将阿霉素溶液加入薄膜容器中，使薄膜水化脂质体，使用25kHz超声分散，分别过0.45μm和0.22μm微孔滤膜，即可得载阿霉素脂质体药物制剂。2) 25 mg of doxorubicin hydrochloride was dissolved in 10 mL of phosphate buffer. The doxorubicin solution was added to the film container to hydrate the liposomes, and the liposomes were dispersed by 25 kHz ultrasound and filtered through 0.45 μm and 0.22 μm microporous membranes, respectively, to obtain the doxorubicin-loaded liposome drug preparation.

2、负载阿霉素的血红蛋白冠修饰的脂质体药物制剂(Hb-Lip@DOX)的制备2. Preparation of hemoglobin corona-modified liposome drug formulation loaded with doxorubicin (Hb-Lip@DOX)

1)向血红蛋白溶液(60mg/mL)中加入抗坏血酸0.5mg，搅拌10min，密封，使其转化为氧合态血红蛋白；向氧合态血红蛋白溶液(60mg/mL)中通一氧化碳气体20min，密封，使其转化为一氧化碳态血红蛋白；向氧合态血红蛋白溶液(60mg/mL)中通一氧化氮气体20min，密封，使其转化为一氧化氮态血红蛋白。1) Add 0.5 mg of ascorbic acid to the hemoglobin solution (60 mg/mL), stir for 10 min, and seal to convert it into oxygenated hemoglobin; pass carbon monoxide gas to the oxygenated hemoglobin solution (60 mg/mL) for 20 min, and seal to convert it into carbon monoxide hemoglobin; pass nitric oxide gas to the oxygenated hemoglobin solution (60 mg/mL) for 20 min, and seal to convert it into nitric oxide hemoglobin.

2)将1mL氧合态血红蛋白的溶液(60mg/mL)，加入至担载阿霉素的马来酰亚胺修饰脂质体溶液中，氩气或一氧化碳或一氧化氮保护状态下，冰浴中搅拌12h。2) Add 1 mL of oxygenated hemoglobin solution (60 mg/mL) to the maleimide-modified liposome solution loaded with doxorubicin, and stir in an ice bath for 12 h under the protection of argon, carbon monoxide or nitric oxide.

3)将反应后的溶液通过葡聚糖凝胶G-250透析除去游离阿霉素药物和未键合的游离血红蛋白，分别过0.45μm和0.22μm微孔滤膜，即得携带不同气体的负载阿霉素的血红蛋白冠脂质体递药系统，即携带氧气的血红蛋白冠脂质体递药系统(O2Hb-Lip@DOX)、携带一氧化碳的血红蛋白冠脂质体递药系统(COHb-Lip@DOX)、携带一氧化氮的血红蛋白冠脂质体递药系统(NOHb-Lip@DOX)。将制备的最终产物以氩气或一氧化碳或一氧化氮保护，4℃保存。实验评价：3) The solution after the reaction was dialyzed through dextran gel G-250 to remove free doxorubicin and unbound free hemoglobin, and then filtered through 0.45 μm and 0.22 μm microporous membranes, respectively, to obtain hemoglobin corona liposome delivery systems loaded with doxorubicin carrying different gases, namely, hemoglobin corona liposome delivery systems carrying oxygen (O 2 Hb-Lip@DOX), hemoglobin corona liposome delivery systems carrying carbon monoxide (COHb-Lip@DOX), and hemoglobin corona liposome delivery systems carrying nitric oxide (NOHb-Lip@DOX). The final product was protected with argon, carbon monoxide or nitric oxide and stored at 4°C. Experimental evaluation:

1、负载阿霉素的血红蛋白冠修饰的脂质体药物制剂(Hb-Lip@DOX)的粒径形貌检测1. Particle size and morphology detection of hemoglobin corona-modified liposome drug preparation loaded with doxorubicin (Hb-Lip@DOX)

将制备的负载阿霉素的血红蛋白冠脂质体制剂通过动态光散射(DLS)纳米粒度分析仪(NanoBrook 90Plus Zeta)测定粒径分布，结果如图1所示，结果显示，携带不同气体的负载阿霉素的血红蛋白冠脂质体制剂粒径分布在50～250nm。将负载阿霉素的血红蛋白冠脂质体制剂进行透射电镜观察。结果如图2的TEM图片所示，得到的携带不同气体的负载阿霉素的血红蛋白冠脂质体制剂粒径呈球形，粒径在50-250nm之间，分散均匀。The prepared hemoglobin corona liposome preparation loaded with doxorubicin was measured for particle size distribution by a dynamic light scattering (DLS) nanoparticle size analyzer (NanoBrook 90Plus Zeta). The results are shown in FIG1 . The results show that the particle size distribution of the hemoglobin corona liposome preparation loaded with doxorubicin carrying different gases is 50 to 250 nm. The hemoglobin corona liposome preparation loaded with doxorubicin was observed by transmission electron microscopy. As shown in the TEM picture of FIG2 , the obtained hemoglobin corona liposome preparation loaded with doxorubicin carrying different gases has a spherical particle size, a particle size between 50 and 250 nm, and is evenly dispersed.

2、负载阿霉素的血红蛋白冠脂质体制剂(Hb-Lip@DOX)的载药量检测2. Drug loading test of hemoglobin corona liposome preparation loaded with doxorubicin (Hb-Lip@DOX)

将制备的负载阿霉素的血红蛋白冠脂质体制剂使用二氯甲烷进行超声破乳，萃取出包裹的药物。使用高效液相色谱(HPLC)对药物进行定量。阿霉素检测条件：色谱柱：C18柱(150×4.6mm)。流动相：正磷酸(5mol/L)：甲醇：乙腈：异丙醇＝5:3:8:4(v/v)，流速1.0mL/min，荧光检测激发波长450nm，样品进样体积20μL。The prepared hemoglobin corona liposome preparation loaded with doxorubicin was ultrasonically demulsified using dichloromethane to extract the encapsulated drug. The drug was quantified using high performance liquid chromatography (HPLC). Doxorubicin detection conditions: Chromatographic column: C18 column (150×4.6mm). Mobile phase: orthophosphoric acid (5mol/L): methanol: acetonitrile: isopropanol = 5:3:8:4 (v/v), flow rate 1.0mL/min, fluorescence detection excitation wavelength 450nm, sample injection volume 20μL.

实验结果：如图3所示(不同阿霉素/纳米载体原料投料比(投料质量比)条件下的药物担载效率和载药量曲线图)，改变阿霉素/纳米载体原料投料比(阿霉素/血红蛋白+溴化三甲基-2,3-二油酰氧基丙基铵(DOTAP)+胆固醇+马来酰亚胺修饰磷脂-聚乙二醇)1:15，1:10，1:8，1:6，1:4，药物担载效率在26％～100％之间，可以控制载药量在2.5％～6％，当阿霉素/纳米载体原料投料比为1：8时，药物担载效率为48.2％，载药量为5.68％。因此，优选阿霉素/纳米载体原料＝1：8作为最优投药量(即，药物活性成分与血红蛋白修饰脂质体的质量比最优为1:8)。Experimental results: As shown in Figure 3 (curves of drug loading efficiency and drug loading under different doxorubicin/nanocarrier raw material feed ratios (feed mass ratios)), the drug loading efficiency was between 26% and 100% when the doxorubicin/nanocarrier raw material feed ratios (doxorubicin/hemoglobin + trimethyl-2,3-dioleyloxypropylammonium bromide (DOTAP) + cholesterol + maleimide-modified phospholipid-polyethylene glycol) were changed to 1:15, 1:10, 1:8, 1:6, 1:4, and the drug loading efficiency was between 26% and 100%, and the drug loading amount could be controlled between 2.5% and 6%. When the doxorubicin/nanocarrier raw material feed ratio was 1:8, the drug loading efficiency was 48.2% and the drug loading amount was 5.68%. Therefore, doxorubicin/nanocarrier raw material = 1:8 is preferred as the optimal dosage (i.e., the optimal mass ratio of the active drug ingredient to the hemoglobin-modified liposome is 1:8).

其中，载药量＝负载阿霉素血红蛋白冠脂质体制剂内的阿霉素质量/负载阿霉素的血红蛋白冠脂质体制剂总质量Wherein, drug loading = mass of doxorubicin in the hemoglobin corona liposome preparation loaded with doxorubicin / total mass of the hemoglobin corona liposome preparation loaded with doxorubicin

药物担载效率＝负载阿霉素血红蛋白冠脂质体制剂内的阿霉素质量/阿霉素投药质量Drug loading efficiency = mass of doxorubicin in the hemoglobin corona liposome formulation loaded with doxorubicin / mass of doxorubicin administered

3、负载阿霉素的血红蛋白冠修饰的脂质体药物制剂(Hb-Lip@DOX)的稳定性3. Stability of hemoglobin corona-modified liposome drug formulation loaded with doxorubicin (Hb-Lip@DOX)

将制备的负载阿霉素的血红蛋白冠修饰的脂质体药物制剂在37℃条件下pH值7.4的PBS(10％FBS，0.01M)中孵育24h，分别在1h，3h，6h，12h和24h通过动态光散射测定制备的负载阿霉素的血红蛋白冠修饰的脂质体药物制剂的粒径和PDI指数；将制备的负载阿霉素的血红蛋白冠脂质体递药系统在4℃条件下pH值7.4的PBS中孵育7天，分别在1天，2天，3天，4天，5天，6天和7天通过动态光散射测定血红蛋白修饰脂质体药物制剂的粒径和PDI指数。如图4所示，得到的制备的负载阿霉素的血红蛋白冠脂质体制剂在37℃和4℃储存条件下粒径和PDI均保持稳定。The prepared hemoglobin corona modified liposome drug preparation loaded with doxorubicin was incubated in PBS (10% FBS, 0.01M) with a pH value of 7.4 at 37°C for 24h, and the particle size and PDI index of the prepared hemoglobin corona modified liposome drug preparation loaded with doxorubicin were measured by dynamic light scattering at 1h, 3h, 6h, 12h and 24h; the prepared hemoglobin corona liposome drug delivery system loaded with doxorubicin was incubated in PBS with a pH value of 7.4 at 4°C for 7 days, and the particle size and PDI index of the hemoglobin modified liposome drug preparation were measured by dynamic light scattering at 1 day, 2 days, 3 days, 4 days, 5 days, 6 days and 7 days. As shown in Figure 4, the particle size and PDI of the prepared hemoglobin corona liposome drug preparation loaded with doxorubicin remained stable under storage conditions of 37°C and 4°C.

4、负载阿霉素的血红蛋白冠修饰的脂质体药物制剂(Hb-Lip@DOX)的药物释放4. Drug release of doxorubicin-loaded hemoglobin corona-modified liposome drug formulation (Hb-Lip@DOX)

负载阿霉素的血红蛋白冠修饰的脂质体药物制剂在不同pH(7.4、6.5、5.0)的PBS(0.01M、1mL)中溶解，包装在透析管中(MWCO＝3500)。然后将试管浸泡在20mL PBS(pH：7.4、6.5或5.0)中，以80rpm连续摇晃。在不同时间间隔抽出1mL透析液，换上新鲜的PBS溶液。采用高效液相色谱法测定阿霉素的含量。计算阿霉素的相对释放率随时间的变化。实验结果显示负载阿霉素的血红蛋白冠脂质体递药系统在生理环境(pH7.4，0.01M，37℃)中表现出较高的稳定性，48h内只释放了9％的阿霉素；在较低pH 6.5(肿瘤微环境pH),48h内释放了32％的阿霉素；pH 5.0(细胞内/溶酶体pH)条件下，48h内释放了57％以上的阿霉素。图5为血红蛋白冠修饰的脂质体药物制剂在37℃条件下pH值7.4，6.5，5.0PBS(0.01M)中的药物释放行为。The hemoglobin corona-modified liposome drug preparation loaded with doxorubicin was dissolved in PBS (0.01M, 1mL) of different pH (7.4, 6.5, 5.0) and packaged in a dialysis tube (MWCO=3500). The tube was then immersed in 20mL PBS (pH: 7.4, 6.5 or 5.0) and shaken continuously at 80rpm. 1mL of dialysate was drawn out at different time intervals and replaced with fresh PBS solution. The doxorubicin content was determined by high performance liquid chromatography. The relative release rate of doxorubicin was calculated over time. The experimental results showed that the hemoglobin corona liposome drug delivery system loaded with doxorubicin showed high stability in physiological environment (pH7.4, 0.01M, 37℃), and only 9% of doxorubicin was released within 48 hours; at a lower pH of 6.5 (tumor microenvironment pH), 32% of doxorubicin was released within 48 hours; at pH 5.0 (intracellular/lysosomal pH), more than 57% of doxorubicin was released within 48 hours. Figure 5 shows the drug release behavior of the hemoglobin corona modified liposome drug preparation in PBS (0.01M) with pH values of 7.4, 6.5, and 5.0 at 37℃.

5、药代动力学检测5. Pharmacokinetic testing

取balb/c雌性小鼠，体重16-18g，静脉注射小分子药物，脂质体阿霉素(市售)和负载阿霉素的血红蛋白冠脂质体药物制剂(Hb-Lip@DOX)，给药量：阿霉素5mg/kg。再给药1min，5min，10min，20min，30min，1h，2h，4h，8h，12h后，通过眼眶取血得到小鼠血液，放于抗凝管中。Balb/c female mice weighing 16-18g were intravenously injected with small molecule drugs, liposomal doxorubicin (commercially available) and hemoglobin corona liposome drug preparation loaded with doxorubicin (Hb-Lip@DOX), with a dosage of 5 mg/kg doxorubicin. After 1min, 5min, 10min, 20min, 30min, 1h, 2h, 4h, 8h, and 12h of administration, blood was collected from the eye sockets and placed in anticoagulant tubes.

3000r/min离心5min，取最上层血浆。Centrifuge at 3000r/min for 5min and take the top layer of plasma.

使用氯仿对血浆进行萃取，氯仿-甲醇(4:1)萃取液2mL混匀，高速离心(3000rpm，10min)后将下层有机层全部转移至另一试管中，重复萃取三次。将萃取液用氮气吹干，残渣加100μL甲醇溶解，精密吸取20μL通过HPLC进行分析。The plasma was extracted with chloroform, and 2 mL of chloroform-methanol (4:1) extract was mixed and centrifuged at high speed (3000 rpm, 10 min) and the lower organic layer was transferred to another test tube. The extraction was repeated three times. The extract was dried with nitrogen, and the residue was dissolved with 100 μL of methanol, and 20 μL was accurately drawn for analysis by HPLC.

实验结果：通过单室模型计算，小分子阿霉素的血液半衰期为1.3h，脂质体阿霉素(市售)的血液半衰期为8.1h，负载阿霉素的血红蛋白冠脂质体药物制剂(Hb-Lip@DOX)半衰期为6.7h，图6为小分子阿霉素、脂质体阿霉素(市售)和负载阿霉素的血红蛋白冠修饰的脂质体药物制剂(Hb-Lip@DOX)的血液药代动力学曲线。Experimental results: Calculated by the single-compartment model, the blood half-life of small molecule doxorubicin is 1.3h, the blood half-life of liposomal doxorubicin (commercially available) is 8.1h, and the half-life of the hemoglobin corona liposome drug preparation loaded with doxorubicin (Hb-Lip@DOX) is 6.7h. Figure 6 shows the blood pharmacokinetic curves of small molecule doxorubicin, liposomal doxorubicin (commercially available) and hemoglobin corona modified liposome drug preparation loaded with doxorubicin (Hb-Lip@DOX).

6、负载阿霉素的血红蛋白冠修饰的脂质体药物制剂(Hb-Lip@DOX)的原位乳腺癌模型小鼠的相对摄取率和靶向效率。6. Relative uptake rate and targeting efficiency of hemoglobin corona-modified liposome drug formulation loaded with doxorubicin (Hb-Lip@DOX) in orthotopic breast cancer model mice.

一).原位乳腺癌模型小鼠建立：1) Establishment of orthotopic breast cancer model mice:

消化收集4T1小鼠乳腺癌细胞，以106个/mL的密度，接种0.1mL细胞到雌性balb/c小鼠乳房垫下，8天左右形成80mm3大小肿瘤块，12天左右形成200～300mm3大小肿瘤块。4T1 mouse breast cancer cells were digested and collected, and 0.1 mL of cells were inoculated under the mammary pad of female balb/c mice at a density of 10 6 cells/mL. A tumor mass of 80 mm 3 was formed in about 8 days, and a tumor mass of 200-300 mm 3 was formed in about 12 days.

二)肿瘤模型药物分布检测：II) Drug distribution detection in tumor models:

1)荷瘤balb/c小鼠24只。在肿瘤体积长到200～300mm3时，静脉注射小分子药物，脂质体阿霉素(市售)，负载阿霉素的血红蛋白冠脂质体制剂(Hb-Lip@DOX)。给药量：阿霉素5mg/kg，分别在1h，12h，24h和48h取小鼠心、肝、脾、肺、肾及肿瘤。1) 24 tumor-bearing balb/c mice. When the tumor volume grew to 200-300 mm3 , small molecule drugs, liposomal doxorubicin (commercially available), and hemoglobin corona liposome preparation loaded with doxorubicin (Hb-Lip@DOX) were intravenously injected. Dosage: 5 mg/kg doxorubicin. The heart, liver, spleen, lung, kidney and tumor of the mice were taken at 1h, 12h, 24h and 48h.

将各组织高速剪切得到组织匀浆液，再加入氯仿-甲醇(4:1)萃取液2mL混匀，高速离心(3000rpm，10min)后将下层有机层全部转移至另一试管中，重复萃取三次。将萃取液用氮气吹干。残渣加100μL甲醇溶解，精密吸取20μL通过HPLC进行分析。Shear each tissue at high speed to obtain a tissue homogenate, add 2 mL of chloroform-methanol (4:1) extract and mix well. After high-speed centrifugation (3000 rpm, 10 min), transfer the lower organic layer to another test tube and repeat the extraction three times. Blow dry the extract with nitrogen. Dissolve the residue in 100 μL of methanol and accurately draw 20 μL for analysis by HPLC.

实验结果如图7所示：负载阿霉素的血红蛋白冠脂质体制剂给药组比小分子阿霉素给药组和脂质体阿霉素(市售)给药组在肿瘤中蓄积更多，滞留时间更长。小分子阿霉素给药后24h药物在肿瘤的蓄积量为1.97μg DOX/g组织，脂质体阿霉素(市售)给药后24h药物在肿瘤的蓄积量为4.81μg DOX/g组织，负载阿霉素的血红蛋白冠脂质体制剂(Hb-Lip@DOX)给药后24h药物在肿瘤的蓄积量为8.89μg DOX/g组织。The experimental results are shown in Figure 7: The hemoglobin corona liposome preparation loaded with doxorubicin accumulates more in the tumor and has a longer retention time than the small molecule doxorubicin administration group and the liposome doxorubicin (commercially available) administration group. The accumulation of the drug in the tumor 24 hours after the administration of small molecule doxorubicin was 1.97μg DOX/g tissue, the accumulation of the drug in the tumor 24 hours after the administration of liposome doxorubicin (commercially available) was 4.81μg DOX/g tissue, and the accumulation of the drug in the tumor 24 hours after the administration of the hemoglobin corona liposome preparation loaded with doxorubicin (Hb-Lip@DOX) was 8.89μg DOX/g tissue.

三)M2型肿瘤巨噬细胞靶向性检测：III) Targeted detection of M2 tumor macrophages:

比较肝脏、肺组织中和肿瘤中M1型和M2型巨噬细胞在给药后24h对阿霉素的摄取Comparison of doxorubicin uptake by M1 and M2 macrophages in liver, lung tissue and tumors 24 hours after administration

方法：流式细胞仪测定细胞中阿霉素的摄取情况(荧光强度)。Methods: Flow cytometry was used to measure the uptake of doxorubicin in cells (fluorescence intensity).

切除肝脏组织、肾脏组织和肿瘤组织，转移到培养皿中，切成小块(小于1mm3)。将片段悬浮在1mL消化液中(RPMI1640培养基中Ⅰ型胶原酶400μg/mL，IV型胶原酶100μg/mL)，37℃持续搅拌0.5h。然后以1500rpm离心5min收集细胞。Liver tissue, kidney tissue and tumor tissue were removed, transferred to a culture dish, and cut into small pieces (less than 1 mm 3 ). The pieces were suspended in 1 mL of digestion solution (400 μg/mL type I collagenase and 100 μg/mL type IV collagenase in RPMI1640 medium) and stirred continuously at 37°C for 0.5 h. The cells were then collected by centrifugation at 1500 rpm for 5 min.

用200目筛过滤。用红细胞裂解液消化细胞2min，收集细胞到5mL流式管内，350g，5min离心去上清。Filter with a 200-mesh sieve. Digest the cells with red blood cell lysis buffer for 2 minutes, collect the cells into a 5 mL flow tube, and centrifuge at 350g for 5 minutes to remove the supernatant.

加入200μL预冷的PBS重悬细胞，并摇匀，再次350g，5min离心去上清；Add 200 μL of pre-cooled PBS to resuspend the cells, shake well, and centrifuge again at 350 g for 5 min to remove the supernatant;

再加入200μL的PBS重悬细胞，立即上机检测。Add 200 μL of PBS to resuspend the cells and immediately test them.

测定激发波长480nm和发射波长590nm处的平均荧光强度MFI。The mean fluorescence intensity (MFI) was measured at an excitation wavelength of 480 nm and an emission wavelength of 590 nm.

实验结果如图7所示：小分子阿霉素给药后24h药物在肿瘤M1型巨噬细胞中的流式荧光强度MFI：154，M2型巨噬细胞中的流式荧光强度MFI：132；脂质体阿霉素(市售)给药后24h药物在M1型巨噬细胞中的流式荧光强度MFI：440，M2型巨噬细胞中的流式荧光强度MFI：339。负载阿霉素的血红蛋白冠脂质体制剂(Hb-Lip@DOX)给药后24h药物在M1型巨噬细胞中的流式荧光强度MFI：259，M2型巨噬细胞中的流式荧光强度MFI：1033。The experimental results are shown in Figure 7: 24 hours after administration of small molecule doxorubicin, the flow fluorescence intensity of the drug in tumor M1 macrophages was MFI: 154, and the flow fluorescence intensity of the drug in M2 macrophages was MFI: 132; 24 hours after administration of liposomal doxorubicin (commercially available), the flow fluorescence intensity of the drug in M1 macrophages was MFI: 440, and the flow fluorescence intensity of the drug in M2 macrophages was MFI: 339. 24 hours after administration of the hemoglobin corona liposome preparation loaded with doxorubicin (Hb-Lip@DOX), the flow fluorescence intensity of the drug in M1 macrophages was MFI: 259, and the flow fluorescence intensity of the drug in M2 macrophages was MFI: 1033.

小分子阿霉素给药组和脂质体阿霉素(市售)给药组中，药物在肝脏和肺中巨噬细胞内的蓄积较多，在肿瘤中M1型巨噬细胞内和M2型巨噬细胞内的比例接近，没有对M2型肿瘤相关巨噬细胞的特异靶向性。负载阿霉素的血红蛋白冠脂质体制剂(Hb-Lip@DOX)给药组中，药物在肝脏和肿瘤中巨噬细胞内的蓄积增加，特别是在肿瘤中的M2型巨噬细胞内蓄积更多，在M1型巨噬细胞内蓄积较少，证明负载阿霉素的血红蛋白冠脂质体制剂(Hb-Lip@DOX)对M2型肿瘤相关巨噬细胞有特异的靶向性。In the small molecule doxorubicin administration group and the liposomal doxorubicin (commercially available) administration group, the drug accumulated more in macrophages in the liver and lungs, and the ratios in M1 macrophages and M2 macrophages in tumors were similar, with no specific targeting to M2 tumor-associated macrophages. In the doxorubicin-loaded hemoglobin corona liposome preparation (Hb-Lip@DOX) administration group, the drug accumulated more in macrophages in the liver and tumors, especially in M2 macrophages in tumors, and less in M1 macrophages, proving that the doxorubicin-loaded hemoglobin corona liposome preparation (Hb-Lip@DOX) has specific targeting to M2 tumor-associated macrophages.

7、负载阿霉素的血红蛋白冠修饰的脂质体药物制剂(Hb-Lip@DOX)的原位乳腺癌模型小鼠药效学评价7. Pharmacodynamic evaluation of hemoglobin corona-modified liposome drug formulation loaded with doxorubicin (Hb-Lip@DOX) in an orthotopic breast cancer model mouse model

荷瘤小鼠30只，在肿瘤增长到成80mm3大小时，随机分成5组，每组6只，分别为生理盐水组、小分子阿霉素组、脂质体阿霉素(市售)、携带氧气的负载阿霉素的血红蛋白冠脂质体制剂(O2Hb-Lip@DOX)和携带一氧化碳的负载阿霉素的血红蛋白冠脂质体制剂(COHb-Lip@DOX)。给药剂量均为5mg DOX/kg。所有动物分别在0、3、6天给药，共给药3次。每两天对小鼠肿瘤的直径进行测量，肿瘤体积的计算公式为：体积＝长×宽×宽/2。绘制小鼠肿瘤生长曲线，结果见图9。由图9可见，小分子阿霉素组和脂质体阿霉素(市售)仅能一定程度上抑制肿瘤的生长，12天后小鼠肿瘤体积增长至810mm3和630mm3左右，而携带氧气的负载阿霉素的血红蛋白冠脂质体制剂(O2Hb-Lip@DOX)和携带一氧化碳的负载阿霉素的血红蛋白冠脂质体制剂(COHb-Lip@DOX)则能够很好的抑制肿瘤的生长，12天后小鼠肿瘤体积增长至350mm3和240mm3左右。证明本发明所制备的携带不同气体的负载阿霉素的血红蛋白冠脂质体制剂有效的抑制肿瘤生长，并且负载不通气体能够起到不同的抑制效果。Thirty tumor-bearing mice were randomly divided into five groups, each with six mice, when the tumor grew to 80 mm3 , including normal saline group, small molecule doxorubicin group, liposomal doxorubicin (commercially available), hemoglobin corona liposome preparation loaded with doxorubicin carrying oxygen ( O2Hb -Lip@DOX), and hemoglobin corona liposome preparation loaded with doxorubicin carrying carbon monoxide (COHb-Lip@DOX). The dosage was 5 mg DOX/kg. All animals were given the drug on days 0, 3, and 6, for a total of three times. The diameter of the mouse tumor was measured every two days, and the tumor volume was calculated as follows: volume = length × width × width/2. The mouse tumor growth curve was drawn, and the results are shown in Figure 9. As shown in Figure 9, the small molecule doxorubicin group and liposomal doxorubicin (commercially available) can only inhibit tumor growth to a certain extent. After 12 days, the tumor volume of mice increased to about 810 mm 3 and 630 mm 3 , while the hemoglobin corona liposome preparation loaded with doxorubicin carrying oxygen (O 2 Hb-Lip@DOX) and the hemoglobin corona liposome preparation loaded with doxorubicin carrying carbon monoxide (COHb-Lip@DOX) can effectively inhibit tumor growth. After 12 days, the tumor volume of mice increased to about 350 mm 3 and 240 mm 3. It is proved that the hemoglobin corona liposome preparation loaded with doxorubicin carrying different gases prepared by the present invention effectively inhibits tumor growth, and different gas loading can have different inhibitory effects.

小鼠生理生化指标测试：抑瘤实验结束后，将小鼠的血液通过促凝管进行收集。静置30min后，3000rpm离心5min，取上清进行生化指标测试，包括：ALT(谷丙转氨酶)，AST(谷草转氨酶)，CREAT(肌酐)和BUN(尿素氮)。Physiological and biochemical index test of mice: After the tumor inhibition experiment, the blood of mice was collected through a coagulation tube. After standing for 30 minutes, centrifuged at 3000 rpm for 5 minutes, and the supernatant was taken for biochemical index testing, including: ALT (alanine aminotransferase), AST (aspartate aminotransferase), CREAT (creatinine) and BUN (urea nitrogen).

表1抑瘤实验结束后，将小鼠的血液生化指标Table 1 After the tumor inhibition experiment, the blood biochemical indicators of mice

血液生化结果显示，与生理盐水组相比，各给药组的生化指标与肝功能相关的AST，ALT以及与肾功能相关的各种指标包括CREAT和BUN均在正常波动范围内，说明各药物在当前剂量下的长期毒性较小。The blood biochemistry results showed that compared with the normal saline group, the biochemical indices of each medication group, including AST and ALT related to liver function, and various indices related to kidney function including CREAT and BUN, were all within the normal fluctuation range, indicating that the long-term toxicity of each drug at the current dose was relatively small.

病理分析：抑瘤实验结束后，将小鼠各器官(心、肝、脾、肺、肾)和肿瘤，用生理盐水清理干净后，用4％的多聚甲醛溶液固定，进行组织脱水，石蜡包埋。石蜡切片机上切取2μm厚的组织切片后，进行H&E染色。使用显微镜观察切片。结果见图10。由图10可见，各给药组小鼠的心，肝，脾，肺器官均没有受到任何影响，没有观察到病变的发生。而小分子DOX给药组中肾组织均有一定的损伤，其肾小球发生了变小或融合，肾小球的壁有些消失，其他各给药组的肾组织相对正常，并没有发现明显的病变。生理盐水给药组中的肿瘤组织的核几乎完好，没有出现大片的坏死区域，而其他给药组肿瘤组织均出现大量的坏死区域，尤其是携带一氧化碳的负载阿霉素的血红蛋白冠脂质体制剂(COHb-Lip@DOX)组的坏死区域最为明显，进一步说明携带一氧化碳的负载阿霉素的血红蛋白冠脂质体制剂(COHb-Lip@DOX)在对其他器官没有毒副作用的同时，对肿瘤造成最有效的杀伤。Pathological analysis: After the tumor inhibition experiment, the organs (heart, liver, spleen, lung, kidney) and tumors of the mice were cleaned with physiological saline, fixed with 4% paraformaldehyde solution, dehydrated and embedded in paraffin. After cutting 2μm thick tissue sections on a paraffin slicer, H&E staining was performed. The sections were observed under a microscope. The results are shown in Figure 10. As can be seen from Figure 10, the heart, liver, spleen, and lung organs of the mice in each drug-treated group were not affected in any way, and no lesions were observed. The renal tissues in the small molecule DOX-treated group were all damaged to a certain extent, the glomeruli became smaller or fused, and the walls of the glomeruli disappeared to some extent. The renal tissues of the other drug-treated groups were relatively normal, and no obvious lesions were found. The nuclei of the tumor tissue in the saline group were almost intact, without large necrotic areas, while the tumor tissues in the other groups showed a large number of necrotic areas, especially in the COHb-Lip@DOX group, where the necrotic areas were most obvious. This further shows that COHb-Lip@DOX has the most effective killing effect on tumors while having no toxic side effects on other organs.

生存率考察：以肿瘤增长到成80mm3大小时记为第0天，记录各组小鼠的生存时间，绘制生存期曲线，结果见图11。由图11可见，携带不同气体的负载阿霉素的血红蛋白冠脂质体制剂组能够显著延皮下荷瘤模型小鼠的生存时间。证明本发明所制备的携带不同气体的负载阿霉素的血红蛋白冠脂质体制剂有效的抑制肿瘤的发生及发展。Survival rate investigation: The tumor grew to a size of 80 mm3 as day 0, and the survival time of each group of mice was recorded. The survival curve was plotted, and the results are shown in Figure 11. As can be seen from Figure 11, the hemoglobin corona liposome preparation group loaded with doxorubicin and carrying different gases can significantly prolong the survival time of subcutaneous tumor-bearing model mice. It is proved that the hemoglobin corona liposome preparation loaded with doxorubicin and carrying different gases prepared by the present invention effectively inhibits the occurrence and development of tumors.

实施例2本实施例提供一种负载利福平的血红蛋白冠修饰的胶束药物制剂，具体采用如下方式制得：Example 2 This example provides a hemoglobin corona-modified micellar drug preparation loaded with rifampicin, which is prepared in the following manner:

一、负载利福平的血红蛋白冠聚合物胶束制剂(Hb-Micel@Rif)的制备1.荷载利福平的马来酰亚胺修饰聚合物胶束的制备，利用溶剂沉淀法制备荷载利福平的马来酰亚胺修饰聚合物胶束，制备步骤具体为：1. Preparation of rifampicin-loaded hemoglobin corona polymer micelle preparation (Hb-Micel@Rif) 1. Preparation of rifampicin-loaded maleimide-modified polymer micelles. The rifampicin-loaded maleimide-modified polymer micelles were prepared by solvent precipitation method. The specific preparation steps are as follows:

1)将140mg N-羟基琥珀酰亚胺修饰聚乙二醇-聚乳酸-羟基乙酸(Mal-PEG-PLGA)溶于8mL二甲基亚砜(DMSO)中，获得含有马来酰亚胺修饰聚合物胶束的溶液。1) 140 mg of N-hydroxysuccinimide-modified polyethylene glycol-polylactic acid-glycolic acid (Mal-PEG-PLGA) was dissolved in 8 mL of dimethyl sulfoxide (DMSO) to obtain a solution containing maleimide-modified polymer micelles.

2)在上述溶液中，加入1mL含25mg/mL利福平的DMSO溶液，获得聚合物/药物混合溶液。2) Add 1 mL of DMSO solution containing 25 mg/mL rifampicin to the above solution to obtain a polymer/drug mixed solution.

3)将聚合物/药物混合溶液缓慢滴加入30mL去离子水，室温下搅拌1h。3) The polymer/drug mixed solution was slowly added dropwise to 30 mL of deionized water and stirred at room temperature for 1 h.

4)去离子水透析48h，除去未担载的游离利福平药物，分别过0.45μm和0.22μm微孔滤膜，即可得载利福平的聚合物药物制剂。4) The unloaded free rifampicin drug was removed by dialyzing with deionized water for 48 hours, and the rifampicin-loaded polymer drug preparation was obtained by filtering through 0.45 μm and 0.22 μm microporous membranes, respectively.

2.负载利福平的血红蛋白冠聚合物胶束制剂(Hb-Micel@Rif)的制备2. Preparation of rifampicin-loaded hemoglobin corona polymer micelles (Hb-Micel@Rif)

1)向血红蛋白溶液(60mg/mL)中加入抗坏血酸0.5mg，搅拌10min，密封，使其转化为氧合态血红蛋白。1) Add 0.5 mg of ascorbic acid to the hemoglobin solution (60 mg/mL), stir for 10 min, and seal to convert it into oxygenated hemoglobin.

2)将1mL氧合态血红蛋白的溶液(60mg/mL)，加入至担载利福平的马来酰亚胺修饰聚合物胶束溶液中，氩气保护状态下，冰浴中搅拌12h。2) 1 mL of oxygenated hemoglobin solution (60 mg/mL) was added to the maleimide-modified polymer micelle solution loaded with rifampicin, and stirred in an ice bath under argon protection for 12 h.

3)将反应后的溶液通过葡聚糖凝胶G-250透析除去游离利福平药物和未键合的游离血红蛋白，分别过0.45μm和0.22μm微孔滤膜，即得负载利福平的血红蛋白冠聚合物胶束制剂(Hb-Micel@Rif)。将制备的最终产物以氩气保护，4℃保存。3) The solution after the reaction was dialyzed through dextran gel G-250 to remove free rifampicin and unbound free hemoglobin, and then filtered through 0.45 μm and 0.22 μm microporous membranes, respectively, to obtain rifampicin-loaded hemoglobin corona polymer micelle preparation (Hb-Micel@Rif). The final product was protected by argon and stored at 4°C.

二、负载利福平的血红蛋白冠聚合物胶束制剂(Hb-Micel@Rif)的粒径形貌检测2. Particle size and morphology detection of rifampicin-loaded hemoglobin corona polymer micelle preparation (Hb-Micel@Rif)

将制备的负载利福平的血红蛋白冠聚合物胶束制剂通过动态光散射(DLS)纳米粒度分析仪(NanoBrook 90Plus Zeta)测定粒径分布，结果如图12所示，结果显示，负载利福平的血红蛋白冠聚合物胶束制剂粒径分布在50-200nm。将负载阿霉素的血红蛋白冠脂质体递药系统进行透射电镜观察。结果如图13的TEM图片所示，得到的携带不同气体的负载阿霉素的血红蛋白冠脂质体递药系统粒径呈球形，粒径在50-200nm之间，分散均匀。The prepared rifampicin-loaded hemoglobin corona polymer micelle preparation was measured for particle size distribution by a dynamic light scattering (DLS) nanoparticle size analyzer (NanoBrook 90Plus Zeta). The results are shown in FIG12 . The results show that the particle size distribution of the rifampicin-loaded hemoglobin corona polymer micelle preparation is 50-200 nm. The doxorubicin-loaded hemoglobin corona liposome drug delivery system was observed by transmission electron microscopy. As shown in the TEM picture of FIG13 , the obtained hemoglobin corona liposome drug delivery system loaded with doxorubicin and carrying different gases has a spherical particle size, with a particle size between 50-200 nm and uniform dispersion.

三.负载利福平的血红蛋白冠聚合物胶束制剂(Hb-Micel@Rif)的载药量检测3. Drug loading test of rifampicin-loaded hemoglobin corona polymer micelle preparation (Hb-Micel@Rif)

将制备的负载利福平的血红蛋白修饰聚合物胶束制剂使用二氯甲烷进行超声破乳，萃取出包裹的药物。使用高效液相色谱(HPLC)对药物进行定量。The prepared rifampicin-loaded hemoglobin-modified polymer micelle preparation was ultrasonically demulsified using dichloromethane to extract the encapsulated drug. The drug was quantified using high performance liquid chromatography (HPLC).

利福平监测条件：色谱柱：C18柱(150×4.6mm)。流动相：甲醇：0.02mol/L磷酸二氢钾＝75:25(v/v)；流速1.0mL/min；荧光检测激发波长280nm，柱温：室温；样品进样体积20μL。Rifampicin monitoring conditions: Chromatographic column: C18 column (150×4.6 mm). Mobile phase: methanol: 0.02 mol/L potassium dihydrogen phosphate = 75:25 (v/v); flow rate 1.0 mL/min; fluorescence detection excitation wavelength 280 nm, column temperature: room temperature; sample injection volume 20 μL.

实验结果：如图14所示(不同利福平/纳米载体投料比条件下的药物担载效率和载药量曲线图)，改变利福平/纳米载体原料投料比(即，利福平/血红蛋白+聚乙二醇-聚乳酸-羟基乙酸的质量比)1:15，1:10，1:8，1:6，1:4，药物担载效率在35％～100％之间，可以控制载药量在2％～8％，当利福平/纳米载体原料投料比为1:8时，药物担载效率为60.7％，载药量为7.05％。因此，优选利福平/纳米载体原料＝1：8作为最优投药量(图12)(即，药物活性成分与血红蛋白修饰聚合物胶束的质量比最优为1:8)。Experimental results: As shown in Figure 14 (curve diagram of drug loading efficiency and drug loading under different rifampicin/nanocarrier feed ratios), the drug loading efficiency is between 35% and 100% when the rifampicin/nanocarrier feed ratio (i.e., the mass ratio of rifampicin/hemoglobin + polyethylene glycol-polylactic acid-glycolic acid) is changed to 1:15, 1:10, 1:8, 1:6, 1:4, and the drug loading efficiency is between 35% and 100%, and the drug loading can be controlled at 2% to 8%. When the rifampicin/nanocarrier feed ratio is 1:8, the drug loading efficiency is 60.7% and the drug loading is 7.05%. Therefore, it is preferred that rifampicin/nanocarrier raw material = 1:8 is used as the optimal dosage (Figure 12) (i.e., the optimal mass ratio of the active pharmaceutical ingredient to the hemoglobin-modified polymer micelle is 1:8).

其中，载药量＝负载利福平的血红蛋白冠聚合物胶束制剂内的利福平质量/负载利福平的血红蛋白冠聚合物胶束制剂总质量Wherein, drug loading = mass of rifampicin in the hemoglobin corona polymer micelle preparation loaded with rifampicin / total mass of the hemoglobin corona polymer micelle preparation loaded with rifampicin

药物担载效率＝负载利福平的血红蛋白冠聚合物胶束制剂内的利福平质量/利福平投药质量Drug loading efficiency = the mass of rifampicin in the hemoglobin corona polymer micelle preparation loaded with rifampicin / the mass of rifampicin administered

四、负载利福平的血红蛋白冠聚合物胶束制剂(Hb-Micel@Rif)的稳定性4. Stability of rifampicin-loaded hemoglobin corona polymer micelle formulation (Hb-Micel@Rif)

将制备的负载利福平的血红蛋白修饰聚合物胶束制剂在37℃条件下pH值7.4PBS(10％FBS，0.01M)中孵育24h，分别在1h，3h，6h，12h和24h通过动态光散射测定血红蛋白修饰聚合物药物制剂的粒径和PDI指数；将制备的负载利福平的血红蛋白修饰聚合物胶束制剂在4℃条件下pH值7.4PBS(0.01M)中孵育7天，分别在1天，2天，3天，4天，5天，6天和7天通过动态光散射测定负载利福平的血红蛋白修饰聚合物胶束制剂的粒径和PDI指数(聚合物分散性指数，用于描述聚合物分子量分布，PDI越大，分子量分布越宽；PDI越小，分子量分布越均匀)。如图15所示负载利福平的血红蛋白修饰聚合物胶束制剂在37℃条件下，pH值7.4PBS(10％FBS，0.01M)中，和在4℃条件下pH值7.4PBS(10％FBS，0.01M)中的稳定性。结果显示，得到的负载利福平的血红蛋白修饰聚合物胶束制剂在37℃和4℃储存条件下粒径和PDI均保持稳定。The prepared rifampicin-loaded hemoglobin-modified polymer micelle preparation was incubated in PBS (10% FBS, 0.01M) with a pH value of 7.4 at 37°C for 24 h, and the particle size and PDI index of the hemoglobin-modified polymer drug preparation were determined by dynamic light scattering at 1 h, 3 h, 6 h, 12 h and 24 h, respectively; the prepared rifampicin-loaded hemoglobin-modified polymer micelle preparation was incubated in PBS (0.01M) with a pH value of 7.4 at 4°C for 7 days, and the particle size and PDI index (polymer dispersibility index, used to describe the molecular weight distribution of the polymer, the larger the PDI, the wider the molecular weight distribution; the smaller the PDI, the more uniform the molecular weight distribution) of the hemoglobin-modified polymer micelle preparation loaded with rifampicin were determined by dynamic light scattering at 1 day, 2 days, 3 days, 4 days, 5 days, 6 days and 7 days, respectively. As shown in Figure 15, the stability of the hemoglobin modified polymer micelle preparation loaded with rifampicin at 37°C, pH 7.4 PBS (10% FBS, 0.01M), and at 4°C, pH 7.4 PBS (10% FBS, 0.01M). The results show that the particle size and PDI of the obtained hemoglobin modified polymer micelle preparation loaded with rifampicin remain stable under storage conditions of 37°C and 4°C.

五、负载利福平的血红蛋白冠聚合物胶束制剂(Hb-Micel@Rif)的药物释放5. Drug release of rifampicin-loaded hemoglobin corona polymer micelle formulation (Hb-Micel@Rif)

负载利福平的血红蛋白冠聚合物胶束制剂在不同pH(7.4、6.5、5.0)的PBS(0.01M、1mL)中溶解，包装在透析管中(MWCO＝3500)。然后将试管浸泡在20mL PBS(pH：7.4、6.5或5.0)中，以80rpm连续摇晃。在不同时间间隔抽出1mL透析液，换上新鲜的PBS溶液。采用高效液相色谱法测定利福平的含量。计算利福平的相对释放率随时间的变化。实验结果显示负载利福平的血红蛋白冠聚合物胶束制剂在生理环境(pH7.4，0.01M，37℃)中表现出较高的稳定性，48h内只释放了11％的利福平；在较低pH6.5(肺结核结节微环境pH),48h内释放了37％的阿霉素；pH5.0(细胞内/溶酶体pH)条件下，48h内释放了61％以上的阿霉素。图16为负载利福平的血红蛋白冠聚合物胶束制剂在37℃条件下pH值7.4，6.5，5.0PBS(0.01M)中的药物释放行为。The hemoglobin corona polymer micelle preparation loaded with rifampicin was dissolved in PBS (0.01M, 1mL) of different pH (7.4, 6.5, 5.0) and packaged in a dialysis tube (MWCO=3500). The tube was then immersed in 20mL PBS (pH: 7.4, 6.5 or 5.0) and shaken continuously at 80rpm. 1mL of dialysate was drawn out at different time intervals and replaced with fresh PBS solution. The content of rifampicin was determined by high performance liquid chromatography. The relative release rate of rifampicin was calculated over time. The experimental results show that the hemoglobin corona polymer micelle preparation loaded with rifampicin exhibits high stability in physiological environment (pH7.4, 0.01M, 37℃), and only 11% of rifampicin is released within 48 hours; at a lower pH6.5 (microenvironmental pH of tuberculosis nodules), 37% of doxorubicin is released within 48 hours; at pH5.0 (intracellular/lysosomal pH), more than 61% of doxorubicin is released within 48 hours. Figure 16 shows the drug release behavior of the hemoglobin corona polymer micelle preparation loaded with rifampicin in PBS (0.01M) at pH values of 7.4, 6.5, and 5.0 at 37℃.

六、药代动力学检测6. Pharmacokinetic testing

1)取c57雌性小鼠，体重16-18g，静脉注射小分子利福平、负载利福平的聚合物胶束制剂(未修饰血红蛋白冠)和负载利福平的血红蛋白冠聚合物胶束制剂，给药量：利福平5mg/kg。再给药1min，5min，10min，20min，30min，1h，2h，4h，8h，12h后，通过眼眶取血得到小鼠血液，放于抗凝管中。1) C57 female mice weighing 16-18g were intravenously injected with small molecule rifampicin, rifampicin-loaded polymer micelle preparation (unmodified hemoglobin corona) and rifampicin-loaded hemoglobin corona polymer micelle preparation, dosage: rifampicin 5mg/kg. After 1min, 5min, 10min, 20min, 30min, 1h, 2h, 4h, 8h, 12h of administration, blood was collected from the eye sockets and placed in anticoagulant tubes.

2)3000r/min离心5min，取最上层血浆。2) Centrifuge at 3000r/min for 5min and take the top layer of plasma.

3)使用氯仿对血浆进行萃取，氯仿-甲醇(4:1)萃取液2mL混匀，高速离心(3000rpm，10min)后将下层有机层全部转移至另一试管中，重复萃取三次。将萃取液用氮气吹干，残渣加100μL甲醇溶解，精密吸取20μL通过HPLC进行分析。3) Use chloroform to extract the plasma, mix 2 mL of chloroform-methanol (4:1) extract, transfer the lower organic layer to another test tube after high-speed centrifugation (3000 rpm, 10 min), and repeat the extraction three times. Blow dry the extract with nitrogen, add 100 μL of methanol to dissolve the residue, and accurately take 20 μL for analysis by HPLC.

实验结果：通过单室模型计算，小分子利福平的血液半衰期0.91h，负载利福平的聚合物胶束制剂(未修饰血红蛋白冠)半衰期为9.4h，负载利福平的血红蛋白冠聚合物胶束制剂半衰期为7.7h。如图17所示，为小分子利福平，负载利福平的聚合物胶束制剂(未修饰血红蛋白冠)和负载利福平的血红蛋白冠聚合物胶束制剂的血液药代动力学曲线。Experimental results: Calculated by the single-compartment model, the blood half-life of small molecule rifampicin is 0.91h, the half-life of the polymer micelle preparation loaded with rifampicin (unmodified hemoglobin corona) is 9.4h, and the half-life of the hemoglobin corona polymer micelle preparation loaded with rifampicin is 7.7h. As shown in Figure 17, the blood pharmacokinetic curves of small molecule rifampicin, polymer micelle preparation loaded with rifampicin (unmodified hemoglobin corona) and hemoglobin corona polymer micelle preparation loaded with rifampicin are shown.

七、负载利福平的血红蛋白冠聚合物胶束制剂(Hb-Micel@Rif)的原位肺结核模型小鼠的相对摄取率和靶向效率。7. Relative uptake rate and targeting efficiency of rifampicin-loaded hemoglobin corona polymer micelle preparation (Hb-Micel@Rif) in orthotopic tuberculosis model mice.

1、原位肺结核模型小鼠建立：1. Establishment of in situ pulmonary tuberculosis model in mice:

取适量海分枝杆菌菌液，加入到7H9肉汤培养基中，在37℃细菌摇动培养箱中培养15-30天。当达到对数生长期时(OD600＝0.8±0.2)，通过离心收集细菌，在PBS中洗涤两次，并用PBS稀释至最终浓度为2×108CFU/mL。通过静脉注射到雌性C57BL/6小鼠体内(100uL/只)。21天后通过H&E和抗酸染色证实，原位肺结核小鼠模型建立。Take an appropriate amount of Mycobacterium marinum bacterial liquid, add it to 7H9 broth medium, and culture it in a 37°C bacterial shaking incubator for 15-30 days. When the logarithmic growth phase is reached (OD600 = 0.8 ± 0.2), collect the bacteria by centrifugation, wash twice in PBS, and dilute with PBS to a final concentration of 2 × 10 8 CFU / mL. Inject it intravenously into female C57BL / 6 mice (100uL / mouse). After 21 days, H&E and acid-fast staining confirmed that the in situ tuberculosis mouse model was established.

2、肺结核模型药物分布检测2. Drug distribution detection in tuberculosis model

c57小鼠24只，肺部灌注海分枝杆菌后2周静脉注射小分子利福平，负载利福平的聚合物胶束制剂(未修饰血红蛋白冠)，负载利福平的血红蛋白冠聚合物胶束制剂，给药量：利福平5mg/kg，分别在1，12，24和48小时取小鼠心、肝、脾、肺、肾。Twenty-four C57 mice were intravenously injected with small molecule rifampicin, polymer micelle preparation loaded with rifampicin (unmodified hemoglobin corona), and hemoglobin corona polymer micelle preparation loaded with rifampicin 2 weeks after pulmonary perfusion with Mycobacterium marinum. The dosage was 5 mg/kg rifampicin. The heart, liver, spleen, lung, and kidney of the mice were collected at 1, 12, 24, and 48 hours, respectively.

将各组织高速剪切得到组织匀浆液，再加入氯仿-甲醇(4:1)萃取液2mL混匀，高速离心(3000rpm，10min)后将下层有机层全部转移至另一试管中，重复萃取三次。将萃取液用氮气吹干,残渣Each tissue was sheared at high speed to obtain a tissue homogenate, and then 2 mL of chloroform-methanol (4:1) extract was added and mixed. After high-speed centrifugation (3000 rpm, 10 min), the lower organic layer was transferred to another test tube and the extraction was repeated three times. The extract was blown dry with nitrogen, and the residue was

加100μL甲醇溶解，精密吸取20μL通过HPLC进行分析。100 μL of methanol was added to dissolve the solution, and 20 μL was precisely pipetted for analysis by HPLC.

实验结果：负载利福平的血红蛋白冠聚合物胶束制剂给药组比小分子利福平给药组和负载利福平的聚合物胶束制剂(未修饰血红蛋白冠)在M2型巨噬细胞数量较多的肝脏和肺中蓄积更多，滞留时间更长。小分子利福平给药后24h药物在肺的蓄积量为2.02μg利福平/g组织，负载利福平的聚合物胶束制剂(未修饰血红蛋白冠)给药后24h药物在肺的蓄积量为3.22μg利福平/g组织，负载利福平的血红蛋白冠聚合物胶束制剂给药后24h药物在肺的蓄积量为6.79μg利福平/g组织(如图17所示，小分子利福平、负载利福平的聚合物胶束制剂(未修饰血红蛋白冠)和负载利福平的血红蛋白冠聚合物胶束制剂给药24h后主要脏器中药物浓度)。Experimental results: The rifampicin-loaded hemoglobin corona polymer micelle preparation administration group accumulated more in the liver and lungs where the number of M2 macrophages was larger than that of the small molecule rifampicin administration group and the rifampicin-loaded polymer micelle preparation (unmodified hemoglobin corona), and the retention time was longer. The accumulation of the drug in the lungs 24 hours after the administration of small molecule rifampicin was 2.02μg rifampicin/g tissue, the accumulation of the drug in the lungs 24 hours after the administration of the rifampicin-loaded polymer micelle preparation (unmodified hemoglobin corona) was 3.22μg rifampicin/g tissue, and the accumulation of the drug in the lungs 24 hours after the administration of the rifampicin-loaded hemoglobin corona polymer micelle preparation was 6.79μg rifampicin/g tissue (as shown in Figure 17, the drug concentrations in the main organs 24 hours after the administration of small molecule rifampicin, rifampicin-loaded polymer micelle preparation (unmodified hemoglobin corona) and rifampicin-loaded hemoglobin corona polymer micelle preparation).

3、考察结核结节在给药后24h对负载荧光探针的血红蛋白冠聚合物胶束制剂的摄取3. Investigate the uptake of the fluorescent probe-loaded hemoglobin corona polymer micelle preparation by tuberculous nodules 24 hours after administration

c57小鼠肺部灌注海分枝杆菌后2周静脉注射负载荧光探针的聚合物胶束制剂(未修饰血红蛋白冠)，负载荧光探针的血红蛋白冠聚合物胶束制剂，给药24h后，肺组织切片中荧光探针(红色荧光)和CD163(M2型巨噬细胞，绿色荧光)、荧光探针(红色荧光)和CD86(M1型巨噬细胞，绿色荧光)免疫荧光染色。Two weeks after the lungs of c57 mice were perfused with Mycobacterium marinum, they were intravenously injected with polymer micelle preparations loaded with fluorescent probes (unmodified hemoglobin corona). 24 hours after administration, immunofluorescence staining of fluorescent probes (red fluorescence) and CD163 (M2 macrophages, green fluorescence) and fluorescent probes (red fluorescence) and CD86 (M1 macrophages, green fluorescence) were performed in lung tissue sections.

方法：收集肺组织标本，用冰冻仪制作6mm厚的冰冻切片。切片风干至少1h，然后在20℃的丙酮中固定10min，用20％的小鼠血清封闭后，分别与抗CD163的单抗，抗CD86的单抗在4℃孵育过夜，然后与二抗孵育1h。DAPI染色10min后，用PBS洗涤两次，在激光共聚焦显微镜下观察。肺结核结节中制剂荧光探针(红色荧光)和CD163(绿色荧光)，制剂荧光探针(红色荧光)和CD86(绿色荧光)免疫荧光染色。Methods: Lung tissue specimens were collected and frozen sections of 6 mm thick were made using a cryostat. The sections were air-dried for at least 1 hour, then fixed in acetone at 20°C for 10 minutes, blocked with 20% mouse serum, and incubated with anti-CD163 monoclonal antibodies and anti-CD86 monoclonal antibodies at 4°C overnight, and then incubated with secondary antibodies for 1 hour. After DAPI staining for 10 minutes, the sections were washed twice with PBS and observed under a laser confocal microscope. Immunofluorescence staining of preparation fluorescent probe (red fluorescence) and CD163 (green fluorescence), preparation fluorescent probe (red fluorescence) and CD86 (green fluorescence) in pulmonary tuberculosis nodules.

实验结果：负载荧光探针的聚合物胶束制剂(未修饰血红蛋白冠)给药组中，制剂荧光探针红色荧光与CD163绿色荧光，与CD86绿色荧光均高度重合，因此判断负载荧光探针的聚合物胶束制剂(未修饰血红蛋白冠)对M2型巨噬细胞和M1型巨噬细胞没有选择性。负载荧光探针的血红蛋白冠聚合物胶束制剂给药组中，制剂荧光探针红色荧光与CD163绿色荧光高度重合，制剂荧光探针红色荧光与CD86绿色荧光重合性差，因此判断负载利福平的血红蛋白冠聚合物胶束制剂能够靶向结核结节内CD163高表达的M2型巨噬细胞(如图18所示)。八、负载利福平的血红蛋白冠聚合物胶束制剂(Hb-Micel@Rif)的原位肺结核模型小鼠药效学评价Experimental results: In the group of patients treated with the fluorescent probe-loaded polymer micelle preparation (unmodified hemoglobin corona), the red fluorescence of the fluorescent probe of the preparation highly overlapped with the green fluorescence of CD163 and the green fluorescence of CD86. Therefore, it was judged that the polymer micelle preparation (unmodified hemoglobin corona) loaded with fluorescent probe had no selectivity for M2 macrophages and M1 macrophages. In the group of patients treated with the fluorescent probe-loaded hemoglobin corona polymer micelle preparation, the red fluorescence of the fluorescent probe of the preparation highly overlapped with the green fluorescence of CD163, and the red fluorescence of the fluorescent probe of the preparation had poor overlap with the green fluorescence of CD86. Therefore, it was judged that the hemoglobin corona polymer micelle preparation loaded with rifampicin could target M2 macrophages with high expression of CD163 in tuberculosis nodules (as shown in Figure 18). 8. Pharmacodynamic evaluation of the hemoglobin corona polymer micelle preparation loaded with rifampicin (Hb-Micel@Rif) in in situ pulmonary tuberculosis model mice

c57小鼠48只，c57小鼠肺部灌注荧光转染的海分枝杆菌后2周随机分成4组，每组12只，分别为生理盐水组、小分子利福平组、负载利福平的聚合物胶束制剂(未修饰血红蛋白冠)和负载利福平的血红蛋白冠聚合物胶束制剂。给药剂量均为5mg利福平/kg。所有动物分别在0、3、6、9、12、15天给药，共给药6次。每7天每组处死3只小鼠，取肺部进行荧光成像，检测肺结核发展情况，结果见图19。由图19可见，小分子利福平和负载利福平的聚合物胶束制剂(未修饰血红蛋白冠)仅能一定程度上抑制肺结核的发展，3周后小鼠肺部仍然具有强的菌荧光强度，存在大量海分枝杆菌。而负载利福平的血红蛋白冠聚合物胶束制剂则能够很好的抑制结核的发展，3周后小鼠肺部荧光基本消失，海分枝杆菌基本被清除。证明本发明所制备的负载利福平的血红蛋白冠聚合物胶束制剂有效的抑制肺结核发展。48 C57 mice were randomly divided into 4 groups 2 weeks after the lungs of C57 mice were perfused with fluorescently transfected marine mycobacterium, each with 12 mice, namely, normal saline group, small molecule rifampicin group, polymer micelle preparation loaded with rifampicin (unmodified hemoglobin crown) and hemoglobin crown polymer micelle preparation loaded with rifampicin. The dosage was 5 mg rifampicin/kg. All animals were administered on days 0, 3, 6, 9, 12, and 15, for a total of 6 times. Three mice were killed in each group every 7 days, and the lungs were taken for fluorescence imaging to detect the development of pulmonary tuberculosis. The results are shown in Figure 19. As can be seen from Figure 19, small molecule rifampicin and polymer micelle preparation loaded with rifampicin (unmodified hemoglobin crown) can only inhibit the development of pulmonary tuberculosis to a certain extent. After 3 weeks, the mouse lungs still have strong bacterial fluorescence intensity, and there are a large number of marine mycobacteria. The hemoglobin corona polymer micelle preparation loaded with rifampicin can effectively inhibit the development of tuberculosis. After 3 weeks, the fluorescence in the lungs of mice basically disappeared, and Mycobacterium marinum was basically eliminated. This proves that the hemoglobin corona polymer micelle preparation loaded with rifampicin prepared by the present invention effectively inhibits the development of pulmonary tuberculosis.

小鼠肺部CFU涂板：抗结核实验结束后，处死小鼠，取出小鼠肺组织，将器官和组织用组织研磨器研磨，过滤取滤液，连续稀释并在LB固体培养基上涂布。平板置于37℃培养箱中培养。培养1天后，记录菌落数，结果如图9所示。与对照组(生理盐水组)相比，小分子Rif和负载利福平的聚合物胶束制剂(未修饰血红蛋白冠)均能在一定程度上杀伤海分枝结核杆菌，而负载利福平的血红蛋白冠聚合物胶束制剂的则能有效的杀伤海分枝结核杆菌，基本实现了完全抑制。Mouse lung CFU plating: After the anti-tuberculosis experiment, the mice were killed, the mouse lung tissues were taken out, the organs and tissues were ground with a tissue grinder, the filtrate was filtered, and the filtrate was continuously diluted and spread on LB solid culture medium. The plate was placed in a 37°C incubator for culture. After 1 day of culture, the number of colonies was recorded, and the results are shown in Figure 9. Compared with the control group (normal saline group), the small molecule Rif and the polymer micelle preparation loaded with rifampicin (unmodified hemoglobin corona) can kill Mycobacterium tuberculosis to a certain extent, while the hemoglobin corona polymer micelle preparation loaded with rifampicin can effectively kill Mycobacterium tuberculosis, basically achieving complete inhibition.

小鼠生理生化指标测试：抗结核实验结束后，将小鼠的血液通过促凝管进行收集。静置30min后，3000rpm离心5min，取上清进行生化指标测试，包括：AST(谷草转氨酶)，ALT(谷丙转氨酶)，UREA(尿素)，CREA(肌酐)和UA(尿酸)。Physiological and biochemical index test of mice: After the anti-tuberculosis experiment, the blood of mice was collected through a coagulation tube. After standing for 30 minutes, centrifuged at 3000rpm for 5 minutes, and the supernatant was taken for biochemical index testing, including: AST (aspartate aminotransferase), ALT (alanine aminotransferase), UREA (urea), CREA (creatinine) and UA (uric acid).

表2抗结核实验结束后小鼠血液生化指标Table 2 Blood biochemical indicators of mice after the anti-tuberculosis experiment

血液生化结果显示，与生理盐水组相比，各给药组的生化指标与肝功能相关的AST，ALT以及与肾功能相关的各种指标包括CREAT和BUN均在正常波动范围内，说明各药物在当前剂量下的长期毒性较小。The blood biochemistry results showed that compared with the normal saline group, the biochemical indices of each medication group, including AST and ALT related to liver function, and various indices related to kidney function including CREAT and BUN, were all within the normal fluctuation range, indicating that the long-term toxicity of each drug at the current dose was relatively small.

虽然，上文中已经用一般性说明、具体实施方式及试验，对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。Although the present invention has been described in detail above by general description, specific implementation methods and experiments, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, these modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims (10)

1. An M2 type macrophage targeted nano-drug preparation is characterized by comprising a drug active ingredient and a nano-carrier; wherein, the surface of the nano-carrier is bonded with a hemoglobin crown.
2. The nano-drug formulation according to claim 1, wherein the particle size of the hemoglobin modified nano-drug formulation is 50-200 nm.
3. The nano-pharmaceutical formulation according to claim 1 or 2, wherein the hemoglobin crown is bonded to the nano-carrier by: the hemoglobin forms a hemoglobin crown through Michael addition reaction of the thiol group on the surface and maleimide on the surface of the nano-carrier.
4. The nano-drug formulation according to claim 1 or 2, wherein the nano-carrier is a liposome, micelle, nanovesicle, exosome, nanoparticle, solid lipid nanoparticle or nanogel-like drug carrier delivery system bonded with hemoglobin crown.
5. The nano-pharmaceutical formulation according to claim 4, wherein the liposome is prepared from one or more materials selected from cholesterol, dioleoyl phosphatidylcholine, phosphatidylserine, phosphatidylcholine, distearoyl phosphatidylcholine, N-hydroxysuccinimide modified phospholipid-polyethylene glycol, maleimide modified phospholipid-polyethylene glycol, N-hydroxysuccinimide modified distearoyl phosphatidylethanolamine polyethylene glycol, maleimide modified distearoyl phosphatidylethanolamine polyethylene glycol, or derivatives thereof.
6. The nano-drug formulation according to claim 4, wherein the micelle is prepared from one or more materials selected from the group consisting of N-hydroxysuccinimide modified polyethylene glycol-polylactic acid-glycolic acid or maleimide modified polyethylene glycol-polylactic acid-glycolic acid, N-hydroxysuccinimide modified polyethylene glycol-polycaprolactone or maleimide modified polyethylene glycol-polycaprolactone, N-hydroxysuccinimide modified polyethylene glycol-polycarbonate and maleimide modified polyethylene glycol-polycarbonate.
7. The nano-pharmaceutical formulation according to any one of claims 1 to 6, wherein the pharmaceutically active ingredient is selected from one or more of methotrexate, leflunomide, hydroxychloroquine, rifampicin, isoniazid, streptomycin, ethambutol, pyrazinamide, doxorubicin, paclitaxel, gemcitabine, oxaliplatin, doxorubicin, fluorouracil, daunorubicin, methotrexate, docetaxel, capecitabine, insulin, PD-1 antibodies, CTLA-4 antibodies, small interfering RNAs, nucleic acid aptamers, vitamin C.
8. The nano-pharmaceutical formulation according to claim 7, wherein the weight ratio of the pharmaceutical activity to the nano-carrier is 1:4 to 15;
Preferably, the mass ratio of the active pharmaceutical ingredient to the nano-carrier raw material is 1:8; preferably 1:6-10; more preferably 1:8.
9. Use of a nano-pharmaceutical formulation according to any one of claims 1 to 8 for the preparation of a medicament or agent for the treatment of inflammatory diseases.
10. The use according to claim 9, wherein the medicament or agent is in particular tuberculosis, vaginitis, prostatitis, pneumonia, rheumatoid arthritis, lupus dermatitis, cancer, cerebral apoplexy medicament or formulation.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118340742A (en) * 2024-04-24 2024-07-16 首都医科大学附属北京康复医院（北京工人疗养院） A nano drug carrier for targeted regulation of macrophage polarization and its preparation method and application

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118340742A (en) * 2024-04-24 2024-07-16 首都医科大学附属北京康复医院（北京工人疗养院） A nano drug carrier for targeted regulation of macrophage polarization and its preparation method and application
CN118340742B (en) * 2024-04-24 2025-04-04 首都医科大学附属北京康复医院（北京工人疗养院） A nano drug carrier for targeted regulation of macrophage polarization and its preparation method and application

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