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CN114432248B - Targeting composite carrier, drug carrying system, preparation method and application thereof - Google Patents

Targeting composite carrier, drug carrying system, preparation method and application thereof Download PDF

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CN114432248B
CN114432248B CN202210103200.4A CN202210103200A CN114432248B CN 114432248 B CN114432248 B CN 114432248B CN 202210103200 A CN202210103200 A CN 202210103200A CN 114432248 B CN114432248 B CN 114432248B
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cholesterol
peg
dspe
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CN114432248A (en
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武敬亮
王方卿
张波
董金萍
李成垒
李娜
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Weifang University of Science and Technology
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Abstract

The invention discloses a targeting composite carrier, a drug carrying system, a preparation method and application thereof. The composite carrier comprises a first targeting carrier and a second targeting carrier; the particle size of the first targeting carrier is 150 nm-200 nm; the particle size of the second targeting carrier is 25-50 nm. According to the invention, medicines are loaded by different targeting carriers, the tumor microenvironment is regulated and controlled, and the targeting and killing of tumor cells are realized, so that the synergistic anti-tumor effect is achieved, and the problem of tumor drug resistance generated by long-term treatment of a single medicine is solved.

Description

一种靶向复合载体、载药体系、制备方法及其用途A targeted composite carrier, drug-carrying system, preparation method and use thereof

技术领域technical field

本发明涉及一种传送载体以及靶向药物载体,特别涉及一种靶向复合载体、载药体系、制备方法及其用途。The present invention relates to a delivery carrier and a targeted drug carrier, in particular to a targeted composite carrier, a drug-carrying system, a preparation method and an application thereof.

背景技术Background technique

肝细胞癌(HCC)是最常见的恶性肿瘤之一,是癌症相关死亡的主要原因。目前,手术治疗和肝移植是临床治疗肝癌的主要方法,但总体生存率较低,主要原因是恶性增殖和全身转移。随着对癌症研究的深入,肿瘤微环境(tumor microenvironment,TME)为肿瘤细胞的生长、侵袭和转移提供了必要的土壤条件。因此,靶向肿瘤微环境策略是肿瘤治疗的关键。Hepatocellular carcinoma (HCC) is one of the most common malignancies and the leading cause of cancer-related death. At present, surgical treatment and liver transplantation are the main methods for the clinical treatment of liver cancer, but the overall survival rate is low, mainly due to malignant proliferation and systemic metastasis. With the deepening of cancer research, the tumor microenvironment (tumor microenvironment, TME) provides the necessary soil conditions for the growth, invasion and metastasis of tumor cells. Therefore, targeting the tumor microenvironment strategy is the key to tumor therapy.

TME由基质细胞、肿瘤血管、细胞外基质(ECM)和各类因子组成。肿瘤相关成纤维细胞(CAFs)作为TME的重要组成部分,已受到广泛关注。其中,CAFs不仅能分泌胶原沉积ECM,还与肝癌细胞建立“cross-talk”,诱导肝癌细胞耐药和转移。另外,大量ECM不仅能形成物理屏障,还能压迫肿瘤血管,限制药物在肿瘤中的渗透,降低药物生物利用度。因此,抑制CAFs活化和ECM沉积对提高肝癌治疗效果至关重要的。TME is composed of stromal cells, tumor blood vessels, extracellular matrix (ECM) and various factors. Cancer-associated fibroblasts (CAFs), as an important component of the TME, have received extensive attention. Among them, CAFs can not only secrete collagen and deposit ECM, but also establish "cross-talk" with liver cancer cells, inducing drug resistance and metastasis of liver cancer cells. In addition, a large amount of ECM can not only form a physical barrier, but also compress tumor blood vessels, limit the penetration of drugs in tumors, and reduce the bioavailability of drugs. Therefore, inhibiting CAFs activation and ECM deposition is crucial to improve the therapeutic effect of HCC.

辣椒素(CAP)是从红辣椒中提取的一种辛辣的生物碱,具有抗癌、抗增殖和抗氧化等作用。最近研究发现,CAP有效抑制成纤维细胞活化,治疗肝纤维化。此外,替米沙坦(Telmisartan,TEL)是一种血管紧张素Ⅱ受体1(angiotensinⅡreceptor 1,AT1R)拮抗剂,通过转化生长因子-β(transforming growth factor-β,TGF-β)信号通路抑制CAF分泌ECM。Capsaicin (CAP) is a pungent alkaloid extracted from red pepper, which has anticancer, antiproliferative and antioxidant effects. Recent studies have found that CAP can effectively inhibit the activation of fibroblasts and treat liver fibrosis. In addition, Telmisartan (Telmisartan, TEL) is an angiotensin Ⅱ receptor 1 (angiotensin Ⅱ receptor 1, AT1R) antagonist, through the transformation growth factor-β (transforming growth factor-β, TGF-β) signaling pathway inhibition CAFs secrete ECM.

发明内容Contents of the invention

发明目的:为了解决现有技术的问题,本发明提供了一种靶向复合载体、载药体系、制备方法及其用途。Purpose of the invention: In order to solve the problems of the prior art, the present invention provides a targeted composite carrier, drug loading system, preparation method and application thereof.

技术方案:本发明所述的一种复合载体,包括第一靶向载体以及第二靶向载体,所述第一靶向载体包括以下组分:二油酰基磷脂酰乙醇胺、卵磷脂、胆固醇琥珀酸单酯、胆固醇、咪唑基胆固醇以及DSPE-PEG-HA;所述DSPE-PEG-HA通过DSPE-PEG-NH2与透明质酸制备而成;所述咪唑基胆固醇通过氯甲酸胆甾醇和N-(3-氨基丙基)咪唑制备而成;所述第二靶向载体包括以下组分:复合磷脂以及甘氨胆酸,所述复合磷脂包括卵磷脂以及DSPE-PEG-GA;所述第一靶向载体的粒径为150nm~200nm;所述第二靶向载体的粒径为25~50nm。Technical solution: a composite carrier according to the present invention, including a first targeting carrier and a second targeting carrier, the first targeting carrier includes the following components: dioleoylphosphatidylethanolamine, lecithin, cholesterol succinate Acid monoester, cholesterol, imidazolyl cholesterol and DSPE-PEG-HA; the DSPE-PEG-HA is prepared by DSPE-PEG-NH 2 and hyaluronic acid; the imidazolyl cholesterol is prepared by chloroformic acid cholesterol and N - prepared from (3-aminopropyl) imidazole; the second targeting carrier includes the following components: complex phospholipids and glycocholic acid, the complex phospholipids include lecithin and DSPE-PEG-GA; the second targeting carrier The particle diameter of the first targeting carrier is 150nm-200nm; the particle diameter of the second targeting carrier is 25-50nm.

作为本发明一种优选实施方式,所述第一靶向载体包括质量比为10~15:5~10:1~5:1~5:1~5:1~5的二油酰基磷脂酰乙醇胺、卵磷脂、胆固醇琥珀酸单酯、胆固醇、咪唑基胆固醇以及DSPE-PEG-HA。As a preferred embodiment of the present invention, the first targeting carrier includes dioleoylphosphatidylethanolamine with a mass ratio of 10-15:5-10:1-5:1-5:1-5:1-5 , lecithin, cholesterol monosuccinate, cholesterol, imidazolyl cholesterol, and DSPE-PEG-HA.

作为本发明一种具体实施方式,所述第一靶向载体包括质量比为14:6:3:2:2:1的二油酰基磷脂酰乙醇胺、卵磷脂、胆固醇琥珀酸单酯、胆固醇、咪唑基胆固醇以及DSPE-PEG-HA。As a specific embodiment of the present invention, the first targeting carrier includes dioleoylphosphatidylethanolamine, lecithin, cholesterol monosuccinate, cholesterol, Imidazolyl cholesterol and DSPE-PEG-HA.

作为本发明一种优选实施方式,所述第二靶向载体中,卵磷脂与DSPE-PEG-GA的质量比为15~20:1;所述复合磷脂与甘氨胆酸的质量比为1.4~3.5:1。As a preferred embodiment of the present invention, in the second targeting carrier, the mass ratio of lecithin to DSPE-PEG-GA is 15-20:1; the mass ratio of the complex phospholipid to glycocholic acid is 1.4 ~3.5:1.

作为本发明一种具体实施方式,所述第二靶向载体中,卵磷脂与DSPE-PEG-GA的质量比为20:1;所述复合磷脂与甘氨胆酸的质量比为1.7:1。As a specific embodiment of the present invention, in the second targeting carrier, the mass ratio of lecithin to DSPE-PEG-GA is 20:1; the mass ratio of the complex phospholipid to glycocholic acid is 1.7:1 .

作为本发明一种优选实施方式,所述咪唑基胆固醇为氯甲酸胆甾醇和N-(3-氨基丙基)咪唑在三乙胺存在的条件下合成。As a preferred embodiment of the present invention, the imidazolyl cholesterol is synthesized from cholesterol chloroformate and N-(3-aminopropyl)imidazole in the presence of triethylamine.

作为本发明一种优选实施方式,所述DSPE-PEG-NH2的分子量为2000~5000Da,透明质酸的分子量为2000~5000Da。As a preferred embodiment of the present invention, the molecular weight of the DSPE-PEG-NH 2 is 2000-5000 Da, and the molecular weight of the hyaluronic acid is 2000-5000 Da.

作为本发明一种具体实施方式,所述DSPE-PEG-NH2的分子量为2000Da,透明质酸的分子量为5000Da。As a specific embodiment of the present invention, the molecular weight of the DSPE-PEG-NH 2 is 2000Da, and the molecular weight of hyaluronic acid is 5000Da.

本发明所述的药物载体,包括质量比为10~15:5~10:1~5:1~5:1~5:1~5的二油酰基磷脂酰乙醇胺、卵磷脂、胆固醇琥珀酸单酯、胆固醇、咪唑基胆固醇以及DSPE-PEG-HA;所述DSPE-PEG-HA通过DSPE-PEG-NH2与透明质酸制备而成;所述咪唑基胆固醇通过氯甲酸胆甾醇和N-(3-氨基丙基)咪唑制备而成;所述载药体系负载的药物为辣椒素以及替米沙坦。The drug carrier of the present invention comprises dioleoylphosphatidylethanolamine, lecithin, cholesterol succinic acid mono ester, cholesterol, imidazolyl cholesterol and DSPE-PEG-HA; the DSPE-PEG-HA is prepared by DSPE-PEG-NH 2 and hyaluronic acid; the imidazolyl cholesterol is prepared by chloroformic acid cholesterol and N-( 3-aminopropyl) imidazole; the drugs loaded in the drug-carrying system are capsaicin and telmisartan.

本发明所述的药物载体或者为负载阿霉素的药物载体,所述的药物载体包括质量比为1.4~3.5:1的复合磷脂以及甘氨胆酸,所述复合磷脂包括质量比为15~20:1的卵磷脂以及DSPE-PEG-GA;药物载体负载的药物阿霉素与卵磷脂的质量比为1:15~20。The drug carrier of the present invention may be a drug carrier loaded with doxorubicin. The drug carrier includes complex phospholipids and glycocholic acid with a mass ratio of 1.4 to 3.5:1. The complex phospholipids include a mass ratio of 15 to 3.5:1. 20:1 lecithin and DSPE-PEG-GA; the mass ratio of drug doxorubicin loaded on the drug carrier to lecithin is 1:15-20.

本发明所述的复合载体的制备方法,包括以下步骤:The preparation method of the composite carrier of the present invention comprises the following steps:

(S1)第一靶向载体的制备(S1) Preparation of the first targeting carrier

(S11)DSPE-PEG-HA的合成:DSPE-PEG-NH2经过EDC与NHS活化后,与透明质酸在氮气环境下交联反应,得到DSPE-PEG-HA;(S11) Synthesis of DSPE-PEG-HA: After DSPE-PEG-NH 2 is activated by EDC and NHS, it reacts with hyaluronic acid in a nitrogen environment to obtain DSPE-PEG-HA;

(S12)咪唑基胆固醇的合成:在三乙胺的催化作用下,氯甲酸胆甾醇和N-(3-氨基丙基)咪唑合成咪唑基胆固醇;(S12) Synthesis of imidazolyl cholesterol: under the catalysis of triethylamine, chloroformic acid cholesterol and N-(3-aminopropyl) imidazole synthesize imidazolyl cholesterol;

(S13)二油酰基磷脂酰乙醇胺、卵磷脂、胆固醇琥珀酸单酯、胆固醇、咪唑基胆固醇和DSPE-PEG-HA加入氯仿中,用旋转蒸发器去除氯仿,待形成一层均匀的薄膜时,加入PBS溶液水化,随后在冰浴中超声,过滤,得到第一靶向载体;(S13) Add dioleoylphosphatidylethanolamine, lecithin, cholesterol succinate monoester, cholesterol, imidazolyl cholesterol and DSPE-PEG-HA into chloroform, remove chloroform with a rotary evaporator, and when a uniform film is formed, Add PBS solution for hydration, then sonicate in an ice bath, and filter to obtain the first targeting carrier;

(S2)第二靶向载体的制备:将卵磷脂、DSPE-PEG-GA在乙醇中混合,然后慢慢滴注到甘氨胆酸溶液中,乙醇蒸发后,在冰浴中超声,过滤,得到第二靶向载体。(S2) Preparation of the second targeting carrier: mix lecithin and DSPE-PEG-GA in ethanol, then slowly instill into the glycocholic acid solution, after the ethanol evaporates, sonicate in an ice bath, filter, Obtain the second targeting vector.

本发明所述的一种载药体系,包括第一载药体系以及第二载药体系,所述第一载药体系包括第一靶向载体以及经由第一靶向载体负载的辣椒素以及替米沙坦;所述第一靶向载体包括二油酰基磷脂酰乙醇胺、卵磷脂、胆固醇琥珀酸单酯、胆固醇、咪唑基胆固醇以及DSPE-PEG-HA;所述DSPE-PEG-HA通过DSPE-PEG-NH2与透明质酸制备而成;所述咪唑基胆固醇通过氯甲酸胆甾醇和N-(3-氨基丙基)咪唑制备而成;所述第二载药体系包括第二靶向载体以及经由所述第二靶向载体负载的阿霉素;所述第二靶向载体包括复合磷脂以及甘氨胆酸,所述复合磷脂包括卵磷脂以及DSPE-PEG-GA;和/或所述第一载药体系中,辣椒素的载药量为2~3%,替米沙坦的载药量为2~3%;所述第二载药体系中,阿霉素的载药量为2~3%。A drug-carrying system according to the present invention includes a first drug-carrying system and a second drug-carrying system, the first drug-carrying system includes a first targeting carrier and capsaicin loaded via the first targeting carrier and a substitute Misartan; the first targeting carrier includes dioleoyl phosphatidylethanolamine, lecithin, cholesterol succinate monoester, cholesterol, imidazole-based cholesterol and DSPE-PEG-HA; the DSPE-PEG-HA passes through DSPE- Prepared from PEG-NH 2 and hyaluronic acid; the imidazole-based cholesterol is prepared from cholesterol chloroformate and N-(3-aminopropyl) imidazole; the second drug-loading system includes a second targeting carrier and doxorubicin loaded via the second targeting carrier; the second targeting carrier includes complex phospholipids and glycocholic acid, and the complex phospholipids includes lecithin and DSPE-PEG-GA; and/or the In the first drug-loading system, the drug-loaded amount of capsaicin is 2-3%, and the drug-loaded amount of telmisartan is 2-3%; in the second drug-loaded system, the drug-loaded amount of doxorubicin is 2 to 3%.

本发明所述的载药体系的制备方法,包括以下步骤:The preparation method of the drug-carrying system of the present invention comprises the following steps:

(1)第一靶向载体的制备(1) Preparation of the first targeting carrier

(1a)DSPE-PEG-HA的合成:DSPE-PEG-NH2经过EDC与NHS活化后,与透明质酸在氮气环境下交联反应,得到DSPE-PEG-HA;(1a) Synthesis of DSPE-PEG-HA: After DSPE-PEG-NH 2 is activated by EDC and NHS, it reacts with hyaluronic acid in a nitrogen environment to obtain DSPE-PEG-HA;

(1b)咪唑基胆固醇的合成:在三乙胺的催化作用下,氯甲酸胆甾醇和N-(3-氨基丙基)咪唑合成咪唑基胆固醇;(1b) Synthesis of imidazolyl cholesterol: under the catalysis of triethylamine, chloroformic acid cholesterol and N-(3-aminopropyl) imidazole synthesize imidazolyl cholesterol;

(1c)二油酰基磷脂酰乙醇胺、卵磷脂、胆固醇琥珀酸单酯、胆固醇、咪唑基胆固醇、DSPE-PEG-HA、辣椒素以及替米沙坦加入氯仿中,用旋转蒸发器去除氯仿,待形成一层均匀的薄膜时,加入PBS溶液水化,随后在冰浴中超声,过滤,得到第一载药体系;(1c) Dioleoyl phosphatidylethanolamine, lecithin, cholesterol succinate monoester, cholesterol, imidazolyl cholesterol, DSPE-PEG-HA, capsaicin, and telmisartan were added to chloroform, and the chloroform was removed with a rotary evaporator. When a uniform film is formed, add PBS solution for hydration, then sonicate in an ice bath, and filter to obtain the first drug-loading system;

(2)第二靶向载体的制备:将卵磷脂、DSPE-PEG-GA在乙醇中混合,然后慢慢滴注到含有甘氨胆酸以及阿霉素的溶液中,乙醇蒸发后,在冰浴中超声,过滤,得到第二载药体系。(2) Preparation of the second targeting carrier: mix lecithin and DSPE-PEG-GA in ethanol, then slowly drip into the solution containing glycocholic acid and doxorubicin, after the ethanol evaporates, the Ultrasound in the bath, and filter to obtain the second drug-loaded system.

本发明所述的载药体系在制备肝癌药物中的应用。Application of the drug-carrying system of the invention in the preparation of liver cancer drugs.

本发明所述的一种药物组合物,包括辣椒素、替米沙坦以及阿霉素。A pharmaceutical composition of the present invention comprises capsaicin, telmisartan and doxorubicin.

有益效果:(1)本发明提供了一种复合载体(脂质大球+纳米小球)制剂治疗肝癌通过脂质大球(CTHLs)共载TEL和CAP,具有肝癌靶向性和深渗透性,CTHLs经HA修饰,可以被CD44+的CAFs主动摄入;载体中的DOPE促进脂质体和内含体融合;AIM-Chol的质子海绵效应促进TEL和CAP从内含体逃逸,阻断CAFs活化,抑制ECM沉积,提高DGNs的渗透效率;纳米小球(DGNs)负载DOX,具有双pH敏感性和CAFs靶向性,通过CTHLs与DGNs协同作用,抑制肝癌发展(2)本发明的复合载体中,通过甘草次酸(GA)修饰后的DGNs,提高了肝癌细胞摄入效率;(3)本发明通过改变复合磷脂与甘胆酸水合物的比例,制备了粒径30nm的胶束,有利于DOX递送至肝癌组织内部;(4)本发明通过建立新型体内外研究模型,评价复合载体“脂质大球+纳米小球”混合制剂的抗肝癌效果,在细胞水平,模拟肝癌环境,建立由BEL-7402和肝星状细胞(LX-2)组成的共培养模型:BEL-7402+LX-2,通过体外摄取实验评价CTHLs和DGNs的靶向能力,采用3D肿瘤球检测DGNs的渗透能力,并分析混合制剂抗增殖和抗迁移特征。建立“H22+m-HSCs”共植入小鼠模型、原位肝癌模型和肺转移模型,评价混合制剂抗增殖和转移效果;(5)本发明实现了辣椒素、替米沙坦以及阿霉素的联合用药,TEL和CAP联合用药抑制CAFs活化和ECM沉积,有效调控TME,在TME调控的基础上,加入化疗药物阿霉素(DOX),阻断CAFs与肝癌细胞间的“cross-talk”,抑制肝细胞癌的耐药和转移;(6)本发明通过靶向载体的制备,克服了DOX联合TEL和CAP治疗面临的障碍,解决了传统的联合制剂靶向性弱,毒副作用大的缺陷,并且通过靶向载体克服了DOX、TEL和CAP对不同细胞的作用靶点不同,体内药代动力学和组织分布不同,难以发挥协同作用的问题;(7)本发明的载药体系解决了现有技术中ECM形成的物理屏障严重阻碍药物穿透肿瘤深部,导致肝癌复发的问题,通过新的药物给药系统,实现药物的高效给药、协同作用和深入渗透。Beneficial effects: (1) The present invention provides a composite carrier (lipid macrosphere + nanosphere) preparation for the treatment of liver cancer. TEL and CAP are co-loaded by lipid macrospheres (CTHLs), which has liver cancer targeting and deep penetration , CTHLs modified by HA can be actively taken up by CD44 + CAFs; DOPE in the carrier promotes the fusion of liposomes and endosomes; the proton sponge effect of AIM-Chol promotes the escape of TEL and CAP from endosomes and blocks CAFs Activate, inhibit ECM deposition, improve the penetration efficiency of DGNs; nanospheres (DGNs) loaded with DOX, have dual pH sensitivity and CAFs targeting, and inhibit the development of liver cancer through the synergistic effect of CTHLs and DGNs (2) The composite carrier of the present invention Among them, the DGNs modified by glycyrrhetinic acid (GA) improved the uptake efficiency of liver cancer cells; (3) the present invention prepared micelles with a particle size of 30nm by changing the ratio of complex phospholipids to glycocholic acid hydrate. Facilitate the delivery of DOX to the liver cancer tissue; (4) The present invention evaluates the anti-liver cancer effect of the composite carrier "lipid macrosphere + nanosphere" mixed preparation by establishing a new in vivo and in vitro research model, and simulates the liver cancer environment at the cellular level to establish A co-culture model consisting of BEL-7402 and hepatic stellate cells (LX-2): BEL-7402+LX-2, the targeting ability of CTHLs and DGNs was evaluated by in vitro uptake experiments, and the penetration ability of DGNs was detected by 3D tumorspheres , and analyze the anti-proliferation and anti-migration characteristics of the mixed preparation. Establish "H22+m-HSCs" co-implantation mouse model, orthotopic liver cancer model and lung metastasis model, and evaluate the anti-proliferation and metastasis effects of mixed preparations; The combination of TEL and CAP inhibits the activation of CAFs and ECM deposition, and effectively regulates TME. On the basis of TME regulation, the chemotherapeutic drug doxorubicin (DOX) is added to block the "cross-talk" between CAFs and liver cancer cells. ", inhibiting the drug resistance and metastasis of hepatocellular carcinoma; (6) The present invention overcomes the barriers faced by DOX combined with TEL and CAP therapy through the preparation of targeting carriers, and solves the problem of weak targeting and large toxic and side effects of traditional combined preparations. The defects of DOX, TEL and CAP are overcome by the targeting carrier, and the target points of different cells are different, the pharmacokinetics and tissue distribution in the body are different, and the problem that it is difficult to play a synergistic effect; (7) the drug-carrying system of the present invention It solves the problem that the physical barrier formed by the ECM in the prior art seriously hinders the drug from penetrating into the deep part of the tumor, leading to the recurrence of liver cancer. Through the new drug delivery system, the drug can be delivered efficiently, synergistically, and deeply penetrated.

附图说明Description of drawings

图1为本发明载体的制备示意图;Fig. 1 is the preparation schematic diagram of carrier of the present invention;

图2为DSPE-PEG-HA载体材料的合成路线与核磁表征图谱,其中,A图为合成路线,B图中的a为HA特征峰b为PEG特征峰;Figure 2 is the synthesis route and NMR characterization spectrum of DSPE-PEG-HA carrier material, wherein, A is the synthesis route, and B is the characteristic peak of HA and b is the characteristic peak of PEG;

图3为不同磷脂投料比的脂质大球在酸性pH值下粒径变化;Fig. 3 is the particle size change of lipid macrospheres with different phospholipid charging ratios under acidic pH value;

图4为DGNs与CTHLs的表征图,其中,A图为DGNs的粒径分布图,B图为CTHLs的粒径分布图;C图为DGNs在pH7.4条件下的TEM图像;D图为CTHLs在pH7.4条件下的TEM图像;E图为CTHLs在pH5.0条件下的TEM图像;F图为DGNs的7天稳定性;G图为CTHLs的7天稳定性;H图为DGNs的体外释放图;I图为CTHLs在不同pH条件下的体外释放图;Figure 4 is the characterization diagram of DGNs and CTHLs, in which, A is the particle size distribution of DGNs, B is the particle size distribution of CTHLs; C is the TEM image of DGNs at pH7.4; D is CTHLs TEM image at pH 7.4; E is the TEM image of CTHLs at pH 5.0; F is the 7-day stability of DGNs; G is the 7-day stability of CTHLs; H is the DGNs in vitro Release diagram; Figure I is the in vitro release diagram of CTHLs under different pH conditions;

图5为DGNs的体外摄取和DGNs滞留结果,其中,A图为DGNs的体外摄取激光共聚焦图像;B图为流式细胞术对DGNs摄取的定量;C图为流式细胞术检测各种制剂对DGNs滞留的影响;D图为流式细胞术检测各种制剂摄取的定量结果;Figure 5 shows the in vitro uptake of DGNs and the retention of DGNs. Among them, A is the in vitro uptake laser confocal image of DGNs; B is the quantification of DGNs uptake by flow cytometry; C is the detection of various preparations by flow cytometry The influence on the retention of DGNs; Figure D shows the quantitative results of the uptake of various preparations detected by flow cytometry;

图6为LX-2对CTHLs的体外摄取结果,其中,A图为CTHLs的体外摄取激光共聚焦图像;B图为流式细胞术对CTHLs摄取的定量;Figure 6 shows the in vitro uptake of CTHLs by LX-2, where A is the in vitro uptake laser confocal image of CTHLs; B is the quantification of CTHLs uptake by flow cytometry;

图7为DGNs的3D多细胞球体结果,其中,A图为DOX、DNs和DGNs对3D多细胞球体的激光共聚焦图;B图为60μm深度下各组的荧光定量;Figure 7 shows the results of 3D multicellular spheroids of DGNs, in which, A is the laser confocal image of DOX, DNs and DGNs on 3D multicellular spheroids; B is the fluorescence quantification of each group at a depth of 60 μm;

图8为DGNs联合CTHLs的细胞毒性评价,其中,A图为单独BEL-7402模型和LX-2/BEL-7402共培养模型示意图;B图为单独BEL-7402模型的MTT检测;C图为LX-2/BEL-7402共培养模型MTT检测;D图为LX-2/BEL-7402共培养模型的活死染色结果;Figure 8 is the cytotoxicity evaluation of DGNs combined with CTHLs, in which, picture A is a schematic diagram of a single BEL-7402 model and a LX-2/BEL-7402 co-culture model; picture B is the MTT test of a single BEL-7402 model; picture C is LX MTT detection of -2/BEL-7402 co-culture model; D shows the live-death staining results of LX-2/BEL-7402 co-culture model;

图9为DGNs联合CTHLs对肿瘤细胞迁移抑制率的影响,其中,A图为单独BEL-7402模型和LX-2/BEL-7402共培养模型示意图;B图为单独BEL-7402模型的细胞迁移结果;C图为LX-2/BEL-7402共培养模型的细胞迁移结果;D图为单独BEL-7402模型的细胞迁移抑制率;E图为LX-2/BEL-7402共培养模型的细胞迁移抑制率;Figure 9 is the effect of DGNs combined with CTHLs on the inhibition rate of tumor cell migration, in which, Figure A is a schematic diagram of a single BEL-7402 model and a LX-2/BEL-7402 co-culture model; Figure B is the result of cell migration of a single BEL-7402 model ; Picture C is the result of cell migration of LX-2/BEL-7402 co-culture model; Picture D is the inhibition rate of cell migration of single BEL-7402 model; Picture E is the inhibition of cell migration of LX-2/BEL-7402 co-culture model Rate;

图10为DGNs联合CTHLs对细胞迁移机制探究(A)LX-2/BEL-7402共培养模型的transwell结果(B)LX-2/BEL-7402共培养模型的transwell的定量(C)E-Cadherin和Vimentin的Western blot分析;(D)E-Cadherin和Vimentin定量分析;Figure 10 is DGNs combined with CTHLs to explore the mechanism of cell migration (A) Transwell results of LX-2/BEL-7402 co-culture model (B) Quantification of transwell of LX-2/BEL-7402 co-culture model (C) E-Cadherin Western blot analysis of E-Cadherin and Vimentin; (D) quantitative analysis of E-Cadherin and Vimentin;

图11为DGNs和CTHLs在荷瘤小鼠体内的生物分布(A)DGNs和CTHLs在不同时间点的NIR图像(B)不同器官和肿瘤的48h荧光图像(C)在DID波长下不同组织的荧光强度分析(D)在DIR波长下不同组织的荧光强度分析(E)不同深度下游离DID处理组的肿瘤切片(F)不同深度下DID-GNs处理组的肿瘤切片;Figure 11 shows the biodistribution of DGNs and CTHLs in tumor-bearing mice (A) NIR images of DGNs and CTHLs at different time points (B) 48h fluorescence images of different organs and tumors (C) fluorescence of different tissues at DID wavelength Intensity analysis (D) Fluorescence intensity analysis of different tissues under DIR wavelength (E) Tumor slices of free DID treatment group at different depths (F) Tumor slices of DID-GNs treatment group at different depths;

图12为CTHLs对DGNs体内渗透的结果(A)不同处理组的肿瘤切片图像(B)肿瘤切片荧光强度的定量;Figure 12 is the result of in vivo penetration of CTHLs on DGNs (A) images of tumor slices in different treatment groups (B) quantification of fluorescence intensity of tumor slices;

图13为DGNs联合CTHLs对H22皮下移植瘤的抗肿瘤评价(A)H22皮下移植瘤的示意图(B)不同治疗组的瘤体积变化(C)不同治疗组的体重变化(D)不同治疗组的抑瘤率(E)肿瘤照片;Figure 13 is the anti-tumor evaluation of DGNs combined with CTHLs on H22 subcutaneous transplanted tumors (A) Schematic diagram of H22 subcutaneous transplanted tumors (B) Tumor volume changes in different treatment groups (C) Body weight changes in different treatment groups (D) Body weight changes in different treatment groups Tumor inhibition rate (E) Tumor photos;

图14为不同治疗组的肿瘤和主要器官的H&E图像;Figure 14 is the H&E images of tumors and major organs in different treatment groups;

图15为DGNs联合CTHLs对m-HSC/H22皮下移植瘤的抗肿瘤评价(A)m-HSC/H22皮下移植瘤示意图(B)不同治疗组的瘤体积变化(C)不同治疗组的体重变化(D)不同治疗组的抑瘤率(E)肿瘤照片;Figure 15 is the anti-tumor evaluation of DGNs combined with CTHLs on m-HSC/H22 subcutaneous transplanted tumors (A) Schematic diagram of m-HSC/H22 subcutaneous transplanted tumors (B) Tumor volume changes in different treatment groups (C) Body weight changes in different treatment groups (D) tumor inhibition rate (E) tumor photo of different treatment groups;

图16为DGNs联合CTHLs对m-HSC/H22皮下移植瘤治疗效果的评价(A)不同治疗组的肿瘤H&E图片(B)不同治疗组的肿瘤Masson图片(C)不同治疗组的肿瘤的α-SMA免疫组化图片(D)不同治疗组的肿瘤的CD31免疫组化图片(E)肿瘤微血管的数量(F)组织中E-Cadherin和Vimentin的Western blot分析(G)E-Cadherin和Vimentin定量分析;Figure 16 is the evaluation of the therapeutic effect of DGNs combined with CTHLs on m-HSC/H22 subcutaneous transplanted tumors (A) H&E pictures of tumors in different treatment groups (B) Masson pictures of tumors in different treatment groups (C) α- SMA immunohistochemical picture (D) CD31 immunohistochemical picture of tumors in different treatment groups (E) number of tumor microvessels (F) Western blot analysis of E-Cadherin and Vimentin in tissue (G) quantitative analysis of E-Cadherin and Vimentin ;

图17为DGNs联合CTHLs对肝原位移植瘤治疗效果的评价(A)肝原位移植瘤的示意图(B)不同治疗组的肝照片(C)不同治疗组的肿瘤H&E图片(D)不同治疗组的肿瘤Masson图片(E)不同治疗组的肿瘤α-SMA免疫组化图片(F)不同治疗组的肿瘤的CD31免疫组化图片;Figure 17 is the evaluation of the therapeutic effect of DGNs combined with CTHLs on liver orthotopic transplanted tumors (A) schematic diagram of liver orthotopic transplanted tumors (B) liver photos of different treatment groups (C) tumor H&E pictures of different treatment groups (D) different treatments Masson pictures of tumors in one group (E) α-SMA immunohistochemical pictures of tumors in different treatment groups (F) CD31 immunohistochemical pictures of tumors in different treatment groups;

图18为DGNs联合CTHLs对肺转移治疗效果的评价(A)肺转移示意图(B)不同治疗组的体重变化(C)不同治疗组的肺结节数量(D)不同治疗组的肺照片(E)不同治疗组的H&E图片。Figure 18 is the evaluation of the treatment effect of DGNs combined with CTHLs on lung metastases (A) Schematic diagram of lung metastases (B) Body weight changes in different treatment groups (C) Number of lung nodules in different treatment groups (D) Lung photos in different treatment groups (E ) H&E pictures of different treatment groups.

具体实施方式Detailed ways

一、材料来源1. Source of materials

1.1材料1.1 Materials

卵磷脂酰胆碱(SPC)、1,2-二油酰-sn-甘油-3-磷酸乙醇胺(DOPE)和胆固醇均购自A.V.T.制药有限公司(中国,上海)。DOX、CAP、TEL均购自中国大连美伦生物科技有限公司。甘氨胆酸(GAH)和半琥珀酸胆酯(CHEMS)购自Macklin生化有限公司(中国,上海)。DSPE-PEG2000-NH2购自西安瑞禧生物科技有限公司(中国,西安)。抗CD31和抗E-Cadherin等抗体等均购自Abcam公司(美国,MA,USA)。其他化学试剂均为分析级或色谱级。Egg phosphatidylcholine (SPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and cholesterol were purchased from AVT Pharmaceutical Co., Ltd. (Shanghai, China). DOX, CAP, and TEL were purchased from Dalian Meilun Biotechnology Co., Ltd., China. Glycocholic acid (GAH) and cholyl hemisuccinate (CHEMS) were purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). DSPE-PEG 2000 - NH2 was purchased from Xi'an Ruixi Biotechnology Co., Ltd. (Xi'an, China). Anti-CD31 and anti-E-Cadherin antibodies were purchased from Abcam (USA, MA, USA). Other chemical reagents are analytical grade or chromatographic grade.

1.2细胞和动物1.2 Cells and animals

人肝癌细胞系(BEL-7402,HepG2)、人源肝星状细胞(LX-2)、鼠源肝癌细胞(H22)和小鼠源肝细胞(m-HSCs)均来自北京生物技术研究所。BALB/c小鼠(雌性,18~22g)来自中国济南朋悦实验动物中心。所有动物实验均经潍坊医学院动物实验伦理委员会(WFMC,2019-045)批准,实验动物的护理和使用严格遵守中华人民共和国卫生部《动物管理规定》(No.55,2001)。Human hepatoma cell lines (BEL-7402, HepG2), human hepatic stellate cells (LX-2), mouse hepatoma cells (H22) and mouse hepatocytes (m-HSCs) were all from Beijing Institute of Biotechnology. BALB/c mice (female, 18-22 g) were from Pengyue Experimental Animal Center, Jinan, China. All animal experiments were approved by the Animal Experiment Ethics Committee of Weifang Medical College (WFMC, 2019-045), and the care and use of experimental animals strictly complied with the "Animal Management Regulations" (No.55, 2001) of the Ministry of Health of the People's Republic of China.

二、样品制备2. Sample preparation

实施例1:DSPE-PEG-HA的合成Embodiment 1: the synthesis of DSPE-PEG-HA

通过DSPE-PEG2000-NH2与HA(MW 5000)的交联反应,合成了一种新型的功能磷脂:DSPE-PEG-HA。100mg HA溶解于蒸馏水中,加入46.5mg碳化二亚胺(EDC)和34.5mg N-羟基琥珀酰亚胺(NHS)活化。将68mg DSPE-PEG2000-NH2加入到HA溶液中,缓慢搅拌。常温下氮气环境下反应24h。将所得溶液纯化、冻干,用1H NMR测定样品结构,结果如图2所示,通过核磁共振氢谱确定了DSPE-PEG2000-HA聚合物的结构,在2.0ppm处的吸收带属于HA的甲基基团,在3.6ppm处的特征峰为DSPE-PEG2000-NH2的PEG基团。DSPE-PEG2000-HA在3.6ppm和2.0ppm处都有峰表明,HA成功地修饰到DSPE-PEG2000-NH2上。Through the cross-linking reaction of DSPE-PEG 2000 -NH 2 with HA (MW 5000), a new type of functional phospholipid: DSPE-PEG-HA was synthesized. 100mg HA was dissolved in distilled water, and activated by adding 46.5mg carbodiimide (EDC) and 34.5mg N-hydroxysuccinimide (NHS). Add 68 mg of DSPE-PEG 2000 -NH 2 into the HA solution and stir slowly. The reaction was carried out under nitrogen atmosphere at room temperature for 24 h. The resulting solution was purified and freeze-dried, and the structure of the sample was determined by 1 H NMR. The results are shown in Figure 2. The structure of the DSPE-PEG 2000 -HA polymer was determined by H NMR spectroscopy. The absorption band at 2.0ppm belongs to HA The methyl group, the characteristic peak at 3.6ppm is the PEG group of DSPE-PEG 2000 -NH 2 . DSPE-PEG 2000 -HA had peaks at 3.6ppm and 2.0ppm, indicating that HA was successfully modified onto DSPE-PEG 2000 -NH 2 .

实施例2:共载CAP和TEL脂质大球(CTHLs)的制备Example 2: Preparation of co-loaded CAP and TEL lipid macrospheres (CTHLs)

(1)AIM-Chol合成(1) AIM-Chol synthesis

将氯甲酸胆甾醇(0.45g,1mmol)溶解于二氯甲烷(10mL)中。滴加N-(3-氨基丙基)咪唑(0.6mL,4mmol),再加入200μL三乙胺,在冰浴中搅拌10h,反应结束后用真空蒸馏除去溶剂。采用柱层析法对产物进行纯化。在三乙胺的催化作用下,氯甲酸胆甾醇和N-(3-氨基丙基)咪唑生成了AIM-Chol。Cholesterol chloroformate (0.45 g, 1 mmol) was dissolved in dichloromethane (10 mL). Add N-(3-aminopropyl)imidazole (0.6 mL, 4 mmol) dropwise, and then add 200 μL triethylamine, stir in an ice bath for 10 h, and evaporate the solvent in vacuo after the reaction is complete. The product was purified by column chromatography. Under the catalysis of triethylamine, cholesteryl chloroformate and N-(3-aminopropyl)imidazole generate AIM-Chol.

(2)采用薄膜蒸发法pH敏感脂质大球(图1)。首先确定二油酰基磷脂酰乙醇胺(DOPE)、卵磷脂(SPC)的优化比例,按照两者1:3、1:1和7:3比例,加入胆固醇琥珀酸单酯(CHEMS)、胆固醇、咪唑基-胆固醇(AIM-Chol)和DSPE-PEG2000-HA制备脂质体,检测三种脂质体在酸性pH(pH5.5)条件下粒径的变化情况,确定磷脂的投料比。加入辣椒素(CAP)和替米沙坦(TEL)制备共载脂质大球。(2) pH-sensitive lipid macrospheres were prepared by thin-film evaporation (Figure 1). First determine the optimal ratio of dioleoylphosphatidylethanolamine (DOPE) and lecithin (SPC), and add cholesterol succinate monoester (CHEMS), cholesterol, and imidazole according to the ratios of 1:3, 1:1 and 7:3. Liposomes were prepared from base-cholesterol (AIM-Chol) and DSPE-PEG 2000 -HA, and the particle size changes of the three liposomes were detected under acidic pH (pH5.5) conditions to determine the feed ratio of phospholipids. Co-loaded lipid spheroids were prepared by adding capsaicin (CAP) and telmisartan (TEL).

结果分析:通过控制DOPE与SPC的投料比(质量比),DOPE:SPC=7:3,DOPE:SPC=1:1和DOPE:SPC=1:3,制备了三种脂质体。检测不同脂质体在酸性pH值(pH5.5)下粒径的变化情况,确定磷脂的投料比,结果如图3所示,在pH 5.0的条件下,DOPE:SPC为7:3时,脂质体粒径随孵育时间逐渐变大,该投料比具有明显的pH敏感性。因此,我们选择DOPE:SPC=7:3作为酸敏感脂质体的制备处方。Result analysis: By controlling the feed ratio (mass ratio) of DOPE and SPC, DOPE:SPC=7:3, DOPE:SPC=1:1 and DOPE:SPC=1:3, three kinds of liposomes were prepared. Detect the variation of particle size of different liposomes under acidic pH value (pH5.5), determine the feeding ratio of phospholipids, the result is as shown in Figure 3, under the condition of pH 5.0, when DOPE:SPC is 7:3, The particle size of liposomes gradually increased with the incubation time, and the feeding ratio had obvious pH sensitivity. Therefore, we choose DOPE:SPC=7:3 as the preparation prescription of acid sensitive liposome.

(3)CTHLs的最终优化工艺如下:(3) The final optimization process of CTHLs is as follows:

将二油酰基磷脂酰乙醇胺(DOPE)70mg、卵磷脂(SPC)30mg、胆固醇琥珀酸单酯(CHEMS)15mg、胆固醇15mg、咪唑基-胆固醇(AIM-Chol)10mg、DSPE-PEG-HA5mg、辣椒素(CAP)5mg、替米沙坦(TEL)5mg溶解于5mL氯仿中。用旋转蒸发器去除氯仿,旋蒸条件:40℃,20rpm。待形成一层均匀的薄膜时,加入5mL PBS(pH=7.4),水化1小时。最后,在冰浴中超声5min,并依次通过0.45μm和0.22μm聚醚砜膜,获得CTHLs。Dioleoylphosphatidylethanolamine (DOPE) 70mg, lecithin (SPC) 30mg, cholesterol succinate monoester (CHEMS) 15mg, cholesterol 15mg, imidazolyl-cholesterol (AIM-Chol) 10mg, DSPE-PEG-HA 5mg, pepper Dissolve 5 mg of CAP (CAP) and 5 mg of telmisartan (TEL) in 5 mL of chloroform. Chloroform was removed with a rotary evaporator, the rotary evaporation conditions: 40°C, 20rpm. When a uniform film is formed, add 5 mL of PBS (pH=7.4) and hydrate for 1 hour. Finally, CTHLs were obtained by sonicating for 5 min in an ice bath and passing through 0.45 μm and 0.22 μm polyethersulfone membranes in turn.

实施例3:载DOX肝靶向纳米小球(DGNs)的制备Example 3: Preparation of DOX-loaded liver targeting nanospheres (DGNs)

采用乙醇注射法制备负载DOX的纳米小球(图1)。首先将GAH溶解在PBS中,用氢氧化钠溶液调节pH至5.5~6.0。其次,按照GAH与磷脂(SPC)的不同比例(1.2,1.4,1.7,2.0,3.5),将SPC和DSPE-PEG-GA(CN2018113191397实施例1的方法合成)乙醇溶液(DSPE-PEG-GA/SPC的比例为1:20)滴入到GAH溶液中,并加入DOX。乙醇蒸发后,在冰浴中超声,并依次通过0.45μm和0.22μm聚醚砜膜。DOX-loaded nanospheres were prepared by ethanol injection (Figure 1). Firstly, GAH was dissolved in PBS, and the pH was adjusted to 5.5-6.0 with sodium hydroxide solution. Secondly, according to the different ratios (1.2, 1.4, 1.7, 2.0, 3.5) of GAH to phospholipids (SPC), SPC and DSPE-PEG-GA (synthesized by the method of CN2018113191397 Example 1) ethanol solution (DSPE-PEG-GA/ The ratio of SPC is 1:20) was dropped into GAH solution, and DOX was added. After the ethanol evaporated, it was sonicated in an ice bath and passed through 0.45 μm and 0.22 μm polyethersulfone membranes sequentially.

固定SPC和DSPE-PEG-GA的含量(20:1)改变甘氨胆酸的含量,考察复合磷脂/甘氨胆酸质量比对DGNs粒径大小的影响。结果如表1所示。随着甘氨胆酸含量的降低,DGNs的粒径呈增加的趋势。其中复合磷脂/甘氨胆酸质量比为1.7时,所制备的纳米粒约为32nm。该粒径胶束既有利于DGNs在肿瘤部位的渗透有减少了在体外循环时被肾脏代谢。The content of SPC and DSPE-PEG-GA was fixed (20:1) and the content of glycocholic acid was changed to investigate the effect of the mass ratio of compound phospholipid/glycocholic acid on the particle size of DGNs. The results are shown in Table 1. The particle size of DGNs tended to increase with the decrease of glycocholic acid content. Wherein when the mass ratio of complex phospholipid/glycocholic acid is 1.7, the prepared nanoparticles are about 32nm. The size of micelles is not only conducive to the penetration of DGNs in the tumor site, but also reduces the metabolism by the kidneys during extracorporeal circulation.

表1不同比例复合磷脂和甘氨胆酸对DGNs粒径和PDI的影响Table 1 Effects of different ratios of complex phospholipids and glycocholic acid on the particle size and PDI of DGNs

Figure BDA0003493104650000081
Figure BDA0003493104650000081

DGNs的优化工艺为:The optimization process of DGNs is:

将SPC、DOX(SPC:DOX=20:1)和DSPE-PEG-GA在乙醇中混合(GAH/复合磷脂的比例为1:1.7,DSPE-PEG-GA/SPC的比例为1:20),然后慢慢滴注到GAH溶液中。乙醇蒸发后,DGNs在冰浴中超声5min,并依次通过0.45μm和0.22μm聚醚砜膜,获得DGNs。Mix SPC, DOX (SPC:DOX=20:1) and DSPE-PEG-GA in ethanol (the ratio of GAH/complex phospholipid is 1:1.7, the ratio of DSPE-PEG-GA/SPC is 1:20), Then slowly instill into the GAH solution. After ethanol evaporated, DGNs were sonicated in an ice bath for 5 min, and passed through 0.45 μm and 0.22 μm polyethersulfone membranes in turn to obtain DGNs.

三、结果分析3. Results Analysis

3.1分析方法3.1 Analysis method

3.1.1 CTHLs和DGNs的表征3.1.1 Characterization of CTHLs and DGNs

用Malvern Zetasizer Nano ZS90分别测量了CTHLs和DGNs的粒径分布和zeta电位。在4℃条件下保存7天,分别测定CTHLs和DGNs在PBS或RPMI 1640中的粒径变化。用透射电子显微镜(TEM)观察CTHLs和DGNs的形态。紫外分光光度计在480nm处测定DGNs的DOX含量,高效液相色谱(HPLC)在280和295nm处测定CTHLs中的CAP和TEL含量。载药量(LE)和包封率(EE)计算公式如下:The particle size distribution and zeta potential of CTHLs and DGNs were measured with Malvern Zetasizer Nano ZS90, respectively. Stored at 4°C for 7 days, the particle size changes of CTHLs and DGNs in PBS or RPMI 1640 were measured respectively. The morphology of CTHLs and DGNs was observed by transmission electron microscopy (TEM). The DOX content of DGNs was measured by UV spectrophotometer at 480nm, and the CAP and TEL content of CTHLs were measured by high performance liquid chromatography (HPLC) at 280 and 295nm. The formulas for drug loading (LE) and encapsulation efficiency (EE) are as follows:

Figure BDA0003493104650000091
Figure BDA0003493104650000091

Figure BDA0003493104650000092
Figure BDA0003493104650000092

3.1.2 CTHLs和DGNs的体外释药3.1.2 In vitro drug release of CTHLs and DGNs

(1)在不同pH值(pH 7.4,6.5或5.5)的PBS溶液中,评价CTHLs药物释放。将1mLCTHLs加入透析袋(MWCO=3500),释放介质为30mL(PBS,0.5%Tween-80)。然后将释放系统放入37℃恒温震荡箱中。在设定的时间点,取出2mL的释放介质,然后再补充2mL的释放介质,然后分别在280nm和295nm处用高效液相色谱法测定释放CAP和TEL的量。(2)1mL DGNs加入透析袋(MWCO=3500),释放介质(30mL PBS溶液,pH7.4,0.5%Tween-80)中,然后将释放系统放入37℃恒温震荡箱中。在设定的时间点,取出2mL的释放介质,然后再补充2mL的释放介质,用紫外分光光度计在480nm处检测DOX的吸光值。(1) Evaluation of CTHLs drug release in PBS solutions with different pH values (pH 7.4, 6.5 or 5.5). 1 mL of CTHLs was added to the dialysis bag (MWCO=3500), and the release medium was 30 mL (PBS, 0.5% Tween-80). Then the release system was placed in a constant temperature shaking box at 37°C. At the set time point, 2mL of the release medium was taken out, and then 2mL of the release medium was replenished, and then the amount of released CAP and TEL was measured by high performance liquid chromatography at 280nm and 295nm, respectively. (2) Add 1mL DGNs into the dialysis bag (MWCO=3500), release medium (30mL PBS solution, pH7.4, 0.5% Tween-80), and then put the release system into a 37°C constant temperature shaking box. At the set time point, 2 mL of the release medium was taken out, and then 2 mL of the release medium was added, and the absorbance value of DOX was detected at 480 nm with an ultraviolet spectrophotometer.

3.1.3模拟微环境的体外共培养研究模型建立3.1.3 Establishment of in vitro co-culture research model with simulated microenvironment

肝癌微环境中,不仅有肝癌细胞,还有肝星状细胞(HSCs)等基质细胞,而HSCs促进肝癌的增殖和转移。为了模拟真实微环境,我们建立由BEL-7402和LX-2细胞组成的共培养模型。把BEL-7402和LX-2细胞以5:1的比例加入到培养板中,培养24h,就形成共培养细胞模型。为了区分两种细胞,我们用CFSE(一种对活细胞进行荧光标记的新型染料)预处理LX-2细胞成C-LX-2,然后混合培养,制备可分辨的BEL-7402/C-LX-2共培养模型。In the liver cancer microenvironment, there are not only liver cancer cells, but also stromal cells such as hepatic stellate cells (HSCs), and HSCs promote the proliferation and metastasis of liver cancer. In order to simulate the real microenvironment, we established a co-culture model consisting of BEL-7402 and LX-2 cells. Add BEL-7402 and LX-2 cells to the culture plate at a ratio of 5:1 and culture for 24 hours to form a co-culture cell model. To differentiate the two types of cells, we pretreated LX-2 cells into C-LX-2 with CFSE (a novel dye for fluorescent labeling of living cells), and then mixed cultured to prepare distinguishable BEL-7402/C-LX -2 co-culture model.

3.1.4细胞摄取和药物滞留试验3.1.4 Cellular uptake and drug retention test

(1)载DOX纳米小球(DNs)的制备:将SPC和DOX(SPC:DOX=20:1)在乙醇中混合(GAH/SPC的比例为1:1.7),然后慢慢滴注到GAH溶液中。乙醇蒸发后,DNs在冰浴中超声5min,并依次通过0.45μm和0.22μm聚醚砜膜,获得DNs。(1) Preparation of DOX-loaded nanospheres (DNs): SPC and DOX (SPC:DOX=20:1) were mixed in ethanol (the ratio of GAH/SPC was 1:1.7), and then slowly dripped into GAH in solution. After ethanol evaporated, DNs were sonicated in an ice bath for 5 min, and passed through 0.45 μm and 0.22 μm polyethersulfone membranes in turn to obtain DNs.

(2)CAP和TEL这两种药物本身不具备荧光,因此在细胞摄取实验中选用疏水性荧光素C6代替两种药物,分别制备载C6脂质大球(C6-Ls)以及载C6脂质大球(C6-HLs)。载C6脂质大球(C6-Ls)的制备:将二油酰基磷脂酰乙醇胺(DOPE)70mg、卵磷脂(SPC)30mg、胆固醇琥珀酸单酯(CHEMS)15mg、胆固醇15mg、咪唑基-胆固醇(AIM-Chol)10mg、5mg香豆素6(C6)溶解于5mL氯仿中。用旋转蒸发器去除氯仿,旋蒸条件:40℃,20rpm。待形成一层均匀的薄膜时,加入5mL PBS(pH=7.4),水化1小时。最后,在冰浴中超声5min,并依次通过0.45μm和0.22μm聚醚砜膜,获得载C6脂质大球(C6-Ls)。载C6脂质大球(C6-HLs)的制备:将二油酰基磷脂酰乙醇胺(DOPE)70mg、卵磷脂(SPC)30mg、胆固醇琥珀酸单酯(CHEMS)15mg、胆固醇15mg、咪唑基-胆固醇(AIM-Chol)10mg、DSPE-PEG-HA 5mg,5mg香豆素6(C6)溶解于5mL氯仿中。用旋转蒸发器去除氯仿,旋蒸条件:40℃,20rpm。待形成一层均匀的薄膜时,加入5mL PBS(pH=7.4),水化1小时。最后,在冰浴中超声5min,并依次通过0.45μm和0.22μμm聚醚砜膜,获得载C6脂质大球(C6-HLs)。摄入实验:将BEL-7402和LX-2分别接种于细胞培养皿中(5×104细胞/培养皿),检测细胞摄取。BEL-7402细胞培养12h,加入游离DOX(DOX的浓度为10μg/mL)、载DOX纳米小球(DNs)(DOX的浓度为10μg/mL)和GA修饰的纳米小球(DGNs)(DOX的浓度为10μg/mL),共孵育0.5h。LX-2细胞培养12h,加入游离香豆素6(C6,C6的浓度为2μg/mL)、载C6脂质大球(C6-Ls,C6的浓度为2μg/mL)、HA修饰的载C6脂质大球(C6-HLs,C6的浓度为2μg/mL)在不含胎牛血清的DMEM培养基中共培养0.5h。PBS洗三次后,DAPI染色10min。随后,用共聚焦激光扫描显微镜拍照,并通过AccuriC6 Plus流式细胞仪定量。(2) The two drugs, CAP and TEL, do not have fluorescence themselves. Therefore, hydrophobic fluorescein C6 was used instead of the two drugs in the cell uptake experiment to prepare C6-loaded lipid spheres (C6-Ls) and C6-loaded lipids, respectively. Large balls (C6-HLs). Preparation of C6-loaded lipid macrospheres (C6-Ls): Dioleoylphosphatidylethanolamine (DOPE) 70mg, lecithin (SPC) 30mg, cholesterol succinate monoester (CHEMS) 15mg, cholesterol 15mg, imidazolyl-cholesterol (AIM-Chol) 10 mg, 5 mg Coumarin 6 (C6) were dissolved in 5 mL chloroform. Chloroform was removed with a rotary evaporator, the rotary evaporation conditions: 40°C, 20rpm. When a uniform film was formed, 5 mL of PBS (pH=7.4) was added and hydrated for 1 hour. Finally, sonicate for 5 min in an ice bath, and pass through 0.45 μm and 0.22 μm polyethersulfone membranes in turn to obtain C6-loaded lipid macrospheres (C6-Ls). Preparation of C6-loaded lipid macrospheres (C6-HLs): Dioleoylphosphatidylethanolamine (DOPE) 70mg, lecithin (SPC) 30mg, cholesterol succinate monoester (CHEMS) 15mg, cholesterol 15mg, imidazolyl-cholesterol (AIM-Chol) 10 mg, DSPE-PEG-HA 5 mg, 5 mg Coumarin 6 (C6) were dissolved in 5 mL chloroform. Chloroform was removed with a rotary evaporator, the rotary evaporation conditions: 40°C, 20rpm. When a uniform film was formed, 5 mL of PBS (pH=7.4) was added and hydrated for 1 hour. Finally, sonicate for 5 min in an ice bath, and pass through 0.45 μm and 0.22 μm polyethersulfone membranes in turn to obtain C6-loaded lipid macrospheres (C6-HLs). Uptake experiment: BEL-7402 and LX-2 were inoculated in cell culture dishes (5×10 4 cells/culture dish), and the cell uptake was detected. BEL-7402 cells were cultured for 12 h, and free DOX (DOX concentration of 10 μg/mL), DOX-loaded nanospheres (DNs) (DOX concentration of 10 μg/mL) and GA-modified nanospheres (DGNs) (DOX The concentration is 10μg/mL), and incubated for 0.5h. LX-2 cells were cultured for 12 hours, and free coumarin 6 (C6, the concentration of C6 was 2 μg/mL), C6-loaded lipid macrospheres (C6-Ls, the concentration of C6 was 2 μg/mL), and HA-modified C6-loaded Lipid macrospheres (C6-HLs, the concentration of C6 is 2 μg/mL) were co-cultured in DMEM medium without fetal bovine serum for 0.5 h. After washing three times with PBS, stain with DAPI for 10 min. Subsequently, photographs were taken with a confocal laser scanning microscope and quantified by an AccuriC6 Plus flow cytometer.

滞留实验:将BEL-7402/LX-2共培养细胞接种于6孔板(每孔细胞接种量为50万/个),加入DOX(DOX浓度为2μg/mL)、DOX+CAP+TEL(DOX浓度为2μg/mL、CAP浓度为2μg/mL、TEL浓度为2μg/mL)、DGNs(DOX浓度为2μg/mL)、DGNs+CTHLs(DOX浓度为2μg/mL、CAP浓度为2μg/mL、TEL浓度为2μg/mL),孵育12小时。收集细胞并在AccuriC6 Plus流式细胞仪检测细胞内药物量。Retention experiment: BEL-7402/LX-2 co-cultured cells were inoculated in a 6-well plate (500,000 cells per well), and DOX (DOX concentration was 2 μg/mL), DOX+CAP+TEL (DOX Concentration of 2 μg/mL, CAP concentration of 2 μg/mL, TEL concentration of 2 μg/mL), DGNs (DOX concentration of 2 μg/mL), DGNs+CTHLs (DOX concentration of 2 μg/mL, CAP concentration of 2 μg/mL, TEL concentration was 2 μg/mL), and incubated for 12 hours. Cells were collected and intracellular drug levels were detected on an AccuriC6 Plus flow cytometer.

3.1.5体外抗肿瘤实验3.1.5 In vitro anti-tumor experiments

为了更好地评价药物在肿瘤微环境中的作用,我们建立了两种细胞模型:(1)BEL-7402细胞;(2)“BEL-7402+LX-2”共培养细胞。首先将这两种细胞模型分别接种在96孔板中,每个孔加入6×103个细胞,然后在37℃培养24小时。加入DOX、CAP+TEL、DOX+CAP+TEL(药物浓度比DOX:CAP:TEL为1:1:1)、CTHLs、DGNs、DGNs+CTHLs(药物浓度比DOX:CAP:TEL为1:1:1)等制剂组,DOX浓度设定为0.01~10μg/mL,培养48h。然后,在每个孔中加入MTT溶液,再孵育4小时。去除培养基,每孔加入150μLDMSO。应用酶标仪(ELX800,BioTek,USA)检测OD值(490nm),并计算各组的细胞毒性。采用Calcein-AM/PI双染色试剂盒检测BEL-7402细胞和LX-2细胞共培养模型的活死细胞染色特征。In order to better evaluate the effect of drugs in the tumor microenvironment, we established two cell models: (1) BEL-7402 cells; (2) "BEL-7402+LX-2" co-cultured cells. First, the two cell models were seeded in 96-well plates, and 6× 103 cells were added to each well, and then cultured at 37°C for 24 hours. Add DOX, CAP+TEL, DOX+CAP+TEL (drug concentration ratio DOX:CAP:TEL is 1:1:1), CTHLs, DGNs, DGNs+CTHLs (drug concentration ratio DOX:CAP:TEL is 1:1: 1) In the equal preparation group, the DOX concentration was set at 0.01-10 μg/mL, and cultured for 48 hours. Then, MTT solution was added to each well and incubated for another 4 hours. Remove the medium and add 150 μL DMSO to each well. The OD value (490nm) was detected by a microplate reader (ELX800, BioTek, USA), and the cytotoxicity of each group was calculated. The staining characteristics of living and dead cells in the co-culture model of BEL-7402 cells and LX-2 cells were detected by Calcein-AM/PI double staining kit.

3.1.6细胞迁移分析3.1.6 Cell migration analysis

采用划痕试验和transwell实验分析细胞迁移和侵袭特征。(1)细胞划痕试验。将BEL-7402细胞和BEL-7402/C-LX-2混合细胞分别以6×105每孔接种到6孔板中,37℃孵育24小时。用200μL吸管尖划伤细胞,在不同药物(药物在细胞中的终浓度为2μg/mL)形成的无血清培养基中培养24小时。在0小时和24小时用尼康eclipse Ti-S显微镜监测划伤创面并拍照。利用Image J软件测量间隙闭合率。(2)应用Transwell分析细胞侵袭。将BEL-7402/C-LX-2共培养细胞(5×104)接种在上室。培养24h后,将附着于下室的细胞用4%聚甲醛固定,然后用结晶紫染色。随后,使用尼康eclipse Ti-S显微镜捕获细胞。Cell migration and invasion characteristics were analyzed by scratch test and transwell test. (1) Cell scratch test. BEL-7402 cells and BEL-7402/C-LX-2 mixed cells were seeded into 6-well plates at 6×10 5 per well, and incubated at 37°C for 24 hours. The cells were scratched with a 200 μL pipette tip, and cultured in serum-free medium formed by different drugs (the final concentration of the drug in the cells was 2 μg/mL) for 24 hours. Scratch wounds were monitored and photographed with a Nikon eclipse Ti-S microscope at 0 and 24 hours. The gap closure rate was measured by Image J software. (2) Transwell was used to analyze cell invasion. BEL-7402/C-LX-2 co-cultured cells (5×10 4 ) were seeded in the upper chamber. After 24 h of culture, the cells attached to the lower chamber were fixed with 4% paraformaldehyde and then stained with crystal violet. Subsequently, cells were captured using a Nikon eclipse Ti-S microscope.

3.1.7 3D肿瘤球药物渗透分析3.1.7 3D tumorsphere drug penetration analysis

用液体覆盖法构建3D肿瘤球模型。将1.5%低熔点琼脂糖(50μL)加入96孔板中,经紫外线照射灭菌30min后,将2×103个BEL-7402细胞悬液接种于孔板中,孵育10天,使其长成球形。去除介质,分别加入DOX(DOX浓度为10μg/mL)、DNs(DOX浓度为10μg/mL)和DGNs(DOX浓度为10μg/mL)。共孵育8h后,采用共聚焦激光扫描显微镜检测药物在肿瘤球的分布特征。A 3D tumorsphere model was constructed using the liquid overlay method. Add 1.5% low-melting point agarose (50 μL) into a 96-well plate, sterilize it by ultraviolet radiation for 30 min, inoculate 2× 103 BEL-7402 cell suspensions in the well plate, and incubate for 10 days to make it grow into spherical. Remove the medium and add DOX (10 μg/mL DOX concentration), DNs (10 μg/mL DOX concentration) and DGNs (10 μg/mL DOX concentration) respectively. After co-incubating for 8 h, the distribution characteristics of the drug in the tumor spheres were detected by confocal laser scanning microscopy.

3.1.8 Western blot分析抗肝癌机制3.1.8 Western blot analysis of anti-hepatoma mechanism

采用Western blot检测肝癌细胞中E-cadherin,Vimentin等蛋白的表达情况。裂解肿瘤细胞和肝癌组织提取蛋白质,点样在10%或12%SDS-PAGE凝胶上电泳,并转移到PVDF膜上。然后,将PVDF膜用5%的乳剂TBST在室温下密封2小时,然后与一抗在4℃下孵育过夜。随后,加入二抗,室温孵育1h。用超敏ECL化学发光溶液检测蛋白表达情况。Western blot was used to detect the expression of E-cadherin, Vimentin and other proteins in liver cancer cells. The tumor cells and liver cancer tissues were lysed to extract proteins, and the samples were electrophoresed on 10% or 12% SDS-PAGE gel, and transferred to PVDF membrane. Then, PVDF membranes were sealed with 5% emulsion TBST for 2 h at room temperature, and then incubated with primary antibodies overnight at 4 °C. Subsequently, the secondary antibody was added and incubated at room temperature for 1 h. Protein expression was detected with ultra-sensitive ECL chemiluminescent solution.

3.1.9活体成像和组织渗透分析3.1.9 In vivo imaging and tissue penetration analysis

DID-GNs的制备方法:将SPC和DID(DID:SPC=1:30)和DSPE-PEG-GA在乙醇中混合(GAH/SPC的比例为1:1.7,DSPE-PEG-GA/SPC的比例为1:20),然后慢慢滴注到GAH溶液中。乙醇蒸发后,DNs在冰浴中超声5min,并依次通过0.45μm和0.22μm聚醚砜膜,获得DID-GNs。The preparation method of DID-GNs: mix SPC and DID (DID:SPC=1:30) and DSPE-PEG-GA in ethanol (the ratio of GAH/SPC is 1:1.7, the ratio of DSPE-PEG-GA/SPC 1:20), and then slowly drip into the GAH solution. After ethanol evaporated, DNs were sonicated in an ice bath for 5 min, and passed through 0.45 μm and 0.22 μm polyethersulfone membranes sequentially to obtain DID-GNs.

DIR-HLs的制备方法:将二油酰基磷脂酰乙醇胺(DOPE)70mg、卵磷脂(SPC)30mg、胆固醇琥珀酸单酯(CHEMS)15mg、胆固醇15mg、咪唑基-胆固醇(AIM-Chol)10mg、DSPE-PEG-HA5mg、DIR 2mg溶解于5mL氯仿中。用旋转蒸发器去除氯仿,旋蒸条件:40℃,20rpm。待形成一层均匀的薄膜时,加入5mL PBS(pH=7.4),水化1小时。最后,在冰浴中超声5min,并依次通过0.45μm和0.22μm聚醚砜膜,获得DIR-HLs。The preparation method of DIR-HLs: dioleoyl phosphatidylethanolamine (DOPE) 70mg, lecithin (SPC) 30mg, cholesterol succinic acid monoester (CHEMS) 15mg, cholesterol 15mg, imidazolyl-cholesterol (AIM-Chol) 10mg, 5mg of DSPE-PEG-HA and 2mg of DIR were dissolved in 5mL of chloroform. Chloroform was removed with a rotary evaporator, the rotary evaporation conditions: 40°C, 20rpm. When a uniform film is formed, add 5 mL of PBS (pH=7.4) and hydrate for 1 hour. Finally, the DIR-HLs were obtained by sonicating for 5 min in an ice bath and passing through 0.45 μm and 0.22 μm polyethersulfone membranes sequentially.

选用DID和DIR两种荧光物质代替药物,检测CTHLs和DGNs在H22荷瘤小鼠体内的生物分布。将2×106H22细胞悬液皮下注射到BALB/c小鼠中,建立H22荷瘤模型。当肿瘤体积约为200mm3时,将小鼠分为DID、DIR、DID-GNs、DIR-HLs和DID-GNs+DIR-HLs。将不同制剂用无菌PBS(pH=7.4)稀释,使DID和DIR的浓度为60μg/mL,按照小鼠体重每10g尾静脉注射0.1mL。采用近红外荧光成像系统(NIRF)在不同时间点监测DID和DIR在小鼠体内的生物分布。48h后取器官及肿瘤,离体分析荧光强度。Two fluorescent substances, DID and DIR, were used instead of drugs to detect the biodistribution of CTHLs and DGNs in H22 tumor-bearing mice. 2×10 6 H22 cell suspension was subcutaneously injected into BALB/c mice to establish the H22 tumor-bearing model. When the tumor volume was approximately 200 mm, mice were divided into DID, DIR, DID-GNs, DIR-HLs, and DID-GNs+DIR-HLs. Different preparations were diluted with sterile PBS (pH=7.4) to make the concentration of DID and DIR 60 μg/mL, and 0.1 mL was injected into tail vein per 10 g of mouse body weight. The biodistribution of DID and DIR in mice was monitored at different time points using a near-infrared fluorescence imaging system (NIRF). After 48 hours, the organs and tumors were collected, and the fluorescence intensity was analyzed in vitro.

为了评价DGNs的通透性,建立H22荷瘤小鼠模型。当肿瘤体积约200mm3时,将小鼠分为5组:DID、DID-GNs、DID-GNs(TEL预处理)、DID-GNs(TEL+CAP预处理)、DID-GNs(CTHLs预处理)。所有预处理组均经尾静脉连续治疗3天,静脉注射药物剂量:DOX(3mg/kg)、CAP(3mg/kg)、TEL(3mg/kg)。注射DID后12小时处死小鼠,分离肿瘤。然后将肿瘤组织切成10μm厚的切片。肿瘤切片采用DAPI染色,然后使用共聚焦激光扫描显微镜(TCSSP8;徕卡)。To evaluate the permeability of DGNs, an H22 tumor-bearing mouse model was established. When the tumor volume was about 200 mm, mice were divided into 5 groups: DID, DID-GNs, DID-GNs (TEL pretreatment), DID-GNs (TEL+CAP pretreatment), DID-GNs (CTHLs pretreatment) . All pretreatment groups were treated continuously for 3 days through the tail vein, and the drug doses were injected intravenously: DOX (3mg/kg), CAP (3mg/kg), TEL (3mg/kg). Mice were sacrificed 12 hours after DID injection, and tumors were isolated. Tumor tissues were then cut into 10 μm thick sections. Tumor sections were stained with DAPI, followed by a confocal laser scanning microscope (TCSSP8; Leica).

3.1.10建立3种体内抑制瘤模型,全面评价抑瘤效果3.1.10 Establish three in vivo tumor suppression models to comprehensively evaluate the tumor suppression effect

(1)单荷瘤模型。选取雌性BALB/c小鼠,在右侧皮下接种2×106个H22细胞,等肿瘤体积长到约150mm3时,小鼠随机分组(n=5),开展药物治疗。(1) Single tumor bearing model. Female BALB/c mice were selected, and 2×10 6 H22 cells were inoculated subcutaneously on the right side. When the tumor volume grew to about 150 mm 3 , the mice were randomly divided into groups (n=5), and drug treatment was carried out.

(2)共植入模型。选取雌性BALB/c小鼠,按照5:1比例混合H22和m-HSC细胞,在右侧皮下接种2×106个细胞,等肿瘤体积增长到约150mm3时,随机分组(n=5),开展药物治疗。(2) Co-implantation model. Select female BALB/c mice, mix H22 and m-HSC cells at a ratio of 5:1, subcutaneously inoculate 2× 106 cells on the right side, and wait for the tumor volume to grow to about 150mm3 , then randomly group them into groups (n=5) , to carry out drug treatment.

(3)原位移植瘤模型。我们建立了两种皮下肿瘤模型(H22荷瘤模型,m-HSC/H22共植入模型)。用5%水合氯醛麻醉BALB/c小鼠,纵向切开腹腔至暴露出肝脏。用无菌注射器注入肝叶5×105H22细胞(PBS,25μL Matrigel),然后缝合腹部伤口。(3) Orthotopic xenograft model. We established two subcutaneous tumor models (H22 tumor-bearing model, m-HSC/H22 co-implantation model). BALB/c mice were anesthetized with 5% chloral hydrate, and the abdominal cavity was cut longitudinally to expose the liver. Liver lobe 5×10 5 H22 cells (PBS, 25 μL Matrigel) were injected with a sterile syringe, and then the abdominal wound was sutured.

3.1.11皮下移植瘤的抑瘤评价3.1.11 Tumor inhibition evaluation of subcutaneous transplanted tumors

为了评估CTHLs和DGNs的肝癌治疗效果,建立H22皮下荷瘤小鼠模型,肿瘤体积约为150mm3,分为7组(n=5组):(1)生理盐水;(2)CAP+TEL;(3)DOX;(4)DOX+CAP+TEL;(5)CTHLs;(6)DGNs;(7)DGNs+CTHLs。In order to evaluate the therapeutic effects of CTHLs and DGNs on liver cancer, a H22 subcutaneous tumor-bearing mouse model was established. The tumor volume was about 150 mm 3 and divided into 7 groups (n=5 groups): (1) normal saline; (2) CAP+TEL; (3) DOX; (4) DOX+CAP+TEL; (5) CTHLs; (6) DGNs; (7) DGNs+CTHLs.

建立m-HSC/H22共植入模型,当肿瘤达到约150mm3被分成8组(n=5):(1)H22对照组;(2)m-HSC/H22对照组;(3)CAP+TEL;(4)DOX;(5)DOX+CAP+TEL;(6)CTHLs;(7)DGNs;(8)DGNs+CTHLs。The m-HSC/H22 co-implantation model was established, and when the tumor reached about 150 mm 3 was divided into 8 groups (n=5): (1) H22 control group; (2) m-HSC/H22 control group; (3) CAP+ (4) DOX; (5) DOX+CAP+TEL; (6) CTHLs; (7) DGNs; (8) DGNs+CTHLs.

静脉注射药物剂量:DOX(3mg/kg)、CAP(3mg/kg)、TEL(3mg/kg),共7次。药物治疗后每隔一天监测小鼠的肿瘤大小和体重。两周后,处死小鼠,摘取肿瘤并称重。Intravenous drug dosage: DOX (3mg/kg), CAP (3mg/kg), TEL (3mg/kg), 7 times in total. Tumor size and body weight of mice were monitored every other day after drug treatment. Two weeks later, the mice were sacrificed, and the tumors were removed and weighed.

4%多聚甲醛固定肿瘤及重要脏器。然后对肿瘤切片进行H&E染色、免疫组织化学分析、Masson三色染色和western blot分析。4% paraformaldehyde was used to fix tumors and important organs. Then the tumor sections were subjected to H&E staining, immunohistochemical analysis, Masson's trichrome staining and western blot analysis.

3.1.12原位移植瘤的抑瘤评价3.1.12 Tumor inhibition evaluation of orthotopic transplanted tumors

为了进一步评价DGNs和CTHLs的抑瘤效果,我们构建了HCC原位肿瘤模型。2周后,将小鼠分为5组:(1)生理盐水;(2)DOX+CAP+TEL;(3)CTHLs(4)DGNs(5)DGNs+CTHLs。DGNs(DOX浓度为300μg/mL)和CTHLs(CAP浓度为300μg/mL、TEL浓度为300μg/mL)用无菌PBS(pH=7.4)稀释后,按照小鼠体重每10g注射0.1mL。每2天给小鼠注射不同剂型。14天后,对所有动物实施安乐死,分离器官进行分析。To further evaluate the tumor suppressive effects of DGNs and CTHLs, we constructed an orthotopic tumor model of HCC. After 2 weeks, the mice were divided into 5 groups: (1) normal saline; (2) DOX+CAP+TEL; (3) CTHLs (4) DGNs (5) DGNs+CTHLs. DGNs (DOX concentration 300 μg/mL) and CTHLs (CAP concentration 300 μg/mL, TEL concentration 300 μg/mL) were diluted with sterile PBS (pH=7.4), and injected 0.1 mL per 10 g of mouse body weight. Mice were injected with different dosage forms every 2 days. After 14 days, all animals were euthanized and organs were isolated for analysis.

3.1.13肺转移实验3.1.13 Lung metastasis experiment

尾静脉注射2×106H22细胞,构建肝癌肺转移模型。然后,将小鼠随机分为7组(n=3):(1)生理盐水;(2)CAP+TEL;(3)DOX;(4)DOX+CAP+TEL;(5)CTHLs;(6)DGNs;(7)DGNs+CTHLs。DGNs(DOX浓度为300μg/mL)和CTHLs(CAP浓度为300μg/mL、TEL浓度为300μg/mL)用无菌PBS(pH=7.4)稀释后,按照小鼠体重每10g注射0.1mL。每2天注射不同剂型的小鼠。14天后处死小鼠。取下肺并用4%多聚甲醛固定。然后测量转移结节数量,并对肺组织切片进行H&E染色。2×10 6 H22 cells were injected into the tail vein to construct the lung metastasis model of liver cancer. Then, the mice were randomly divided into 7 groups (n=3): (1) normal saline; (2) CAP+TEL; (3) DOX; (4) DOX+CAP+TEL; (5) CTHLs; ) DGNs; (7) DGNs+CTHLs. DGNs (DOX concentration 300 μg/mL) and CTHLs (CAP concentration 300 μg/mL, TEL concentration 300 μg/mL) were diluted with sterile PBS (pH=7.4), and injected 0.1 mL per 10 g of mouse body weight. Mice were injected with different dosage forms every 2 days. Mice were sacrificed 14 days later. The lungs were removed and fixed with 4% paraformaldehyde. Then the number of metastatic nodules was measured, and H&E staining was performed on lung tissue sections.

3.1.14统计分析3.1.14 Statistical analysis

数据以平均数±标准差表示。使用Prism 8.0(GraphPad)进行数据制图和统计分析。两组间差异采用学生t检验或方差分析,以P<0.05为差异有统计学意义。Data are presented as mean ± standard deviation. Data graphing and statistical analysis were performed using Prism 8.0 (GraphPad). Differences between the two groups were analyzed using Student's t-test or analysis of variance, and P<0.05 was considered statistically significant.

3.2结果和讨论3.2 Results and discussion

3.2.1 DGNs和CTHLs的理化性质分析3.2.1 Analysis of physicochemical properties of DGNs and CTHLs

CTHLs和DGNs的表征如表2所示。DGNs的平均粒径约为32nm(图4中A图所示),既避免了粒径过小引起的肾脏代谢,又有利于DGNs向肿瘤深部浸润。DGNs的zeta电位为负。DOX的包封率和载药量分别为88.9%和2.38%。CTHLs的平均粒径为160nm(图4中B图所示),具有负电位的电位特性。CAP的包封率和载药量分别为83.7%和2.79%,TEL的包封率和载药量分别为83.1%和2.77%。TEM图像显示DGNs和CTHLs在pH为7.4时呈规则的球形,而CTHLs在pH为5.0时粒径增大,形状不规则(图4中C图-E图)。其次,研究DGNs和CTHLs在PBS和RPMI1640培养基中的稳定性。如图4中F图和G图所示,结果表明DGNs和CTHLs在7天内保持稳定。The characterization of CTHLs and DGNs is shown in Table 2. The average particle size of DGNs is about 32nm (shown in A in Figure 4), which not only avoids the kidney metabolism caused by too small particle size, but also facilitates the deep infiltration of DGNs into the tumor. The zeta potential of DGNs is negative. The encapsulation efficiency and drug loading of DOX were 88.9% and 2.38%, respectively. The average particle size of CTHLs is 160nm (as shown in Figure 4B), and has a potential characteristic of negative potential. The encapsulation efficiency and drug loading capacity of CAP were 83.7% and 2.79%, respectively, and the encapsulation efficiency and drug loading capacity of TEL were 83.1% and 2.77%. TEM images showed that DGNs and CTHLs were regular spherical at pH 7.4, while CTHLs increased in particle size and irregular in shape at pH 5.0 (Panel C-E in Fig. 4). Second, the stability of DGNs and CTHLs in PBS and RPMI1640 medium was studied. As shown in panels F and G in Fig. 4, the results indicated that DGNs and CTHLs remained stable within 7 days.

DOX从DGNs的释放如图4中H图所示。与游离DOX相比,DGNs在8h内的体外累积释放率较低,说明DGNs具有长循环性。另外,通过测定不同pH条件下CTHLs的释放速率,验证其对pH的敏感性。如图4中I图所示,CAP在pH 5.0下48h内的累积释放率为72.5%,显著高于pH6.5(54.3%)和pH 7.4(42.5%),说明在酸性条件下DOPE具有增强药物释放的作用。TEL的累积释放率在pH 5.0(10.4%)和pH 6.5(42.8%)下显著低于pH 7.4(69.2%),其主要原因是其酸度较弱。因此,pH敏感脂质体将TEL从溶酶体快速释放到细胞质中有利于TEL发挥其作用。The release of DOX from DGNs is shown in panel H in Fig. 4. Compared with free DOX, the in vitro cumulative release rate of DGNs within 8 h was lower, indicating that DGNs have a long circulation. In addition, the sensitivity to pH was verified by measuring the release rate of CTHLs under different pH conditions. As shown in Figure 4, the cumulative release rate of CAP in 48h at pH 5.0 was 72.5%, which was significantly higher than pH 6.5 (54.3%) and pH 7.4 (42.5%), indicating that DOPE has enhanced The effect of drug release. The cumulative release rate of TEL at pH 5.0 (10.4%) and pH 6.5 (42.8%) was significantly lower than that at pH 7.4 (69.2%), mainly due to its weaker acidity. Therefore, the rapid release of TEL from lysosomes into the cytoplasm by pH-sensitive liposomes is beneficial for TEL to exert its effects.

表2 DGNs和CTHLs的表征Table 2 Characterization of DGNs and CTHLs

Figure BDA0003493104650000151
Figure BDA0003493104650000151

3.2.2细胞摄取和药物滞留检测3.2.2 Cellular uptake and drug retention assays

研究显示,纳米载体的粒径、电位、表面修饰等因素对肿瘤细胞的摄取起着重要作用。小于20nm的纳米粒在体内血液循环中被肾脏快速清除,而大于100nm的纳米颗粒则不利于细胞摄取。在本研究中,我们制备了32nm的纳米小球。如图5中A图所示,DNs组比游离DOX组荧光信号更强,说明粒径较小的纳米胶束更容易被细胞吸收。此外,与DNs组相比,DGNs组显示出更强的荧光信号,显示DGNs可能通过GA介导的胞吞作用增加HCC细胞对DOX的摄取。用流式细胞术定量各组的荧光强度,其结果与激光共聚焦结果一致(图5中B图)。我们也检测了LX-2对CTHLs的摄取情况,如图6中A图-B图所示,非靶向脂质体组处理的细胞表现出更强的绿色。这可能是由于CD44受体在LX-2细胞表面过表达,HA修饰的脂质体可以促进受体介导的细胞内吞。Studies have shown that factors such as particle size, potential, and surface modification of nanocarriers play an important role in the uptake of tumor cells. Nanoparticles smaller than 20 nm are rapidly cleared by the kidneys in the blood circulation in vivo, while those larger than 100 nm are not conducive to cellular uptake. In this study, we prepared 32nm nanospheres. As shown in panel A of Figure 5, the fluorescence signal of the DNs group was stronger than that of the free DOX group, indicating that nanomicelles with smaller particle sizes were more easily absorbed by cells. In addition, compared with the DNs group, the DGNs group showed stronger fluorescent signals, indicating that DGNs may increase the uptake of DOX by HCC cells through GA-mediated endocytosis. The fluorescence intensity of each group was quantified by flow cytometry, and the result was consistent with the laser confocal result (Figure 5, B). We also detected the uptake of CTHLs by LX-2, as shown in Figure 6, A-B, the cells treated with non-targeted liposome group showed stronger green color. This may be due to the overexpression of CD44 receptor on the surface of LX-2 cells, and HA-modified liposomes can promote receptor-mediated endocytosis.

研究显示,肝癌微环境中的CAFs能诱导肿瘤细胞的药物外排,降低肿瘤对药物的敏感性。我们建立LX-2/BEL-7402共培养模型,模拟体内环境,检测不同制剂组的药物滞留效果(图5中C图所示)。与单独BEL-7402相比,LX-2/BEL-7402组的荧光信号较弱,说明CAFs可以减少DOX在肿瘤细胞中的积累。有趣的是,在共培养模型中加入CAP和TEL后,各组的荧光信号均显著增强,说明CAP和TEL能有效抑制药物外排。采用流式细胞仪检测细胞内DOX量(图5中D图所示),与CLSM检测结果相一致。Studies have shown that CAFs in the liver cancer microenvironment can induce drug efflux from tumor cells and reduce tumor sensitivity to drugs. We established a LX-2/BEL-7402 co-culture model to simulate the in vivo environment and test the drug retention effect of different preparation groups (shown in C in Figure 5). Compared with BEL-7402 alone, the fluorescence signal of LX-2/BEL-7402 group was weaker, indicating that CAFs can reduce the accumulation of DOX in tumor cells. Interestingly, after adding CAP and TEL to the co-culture model, the fluorescence signals of each group were significantly enhanced, indicating that CAP and TEL can effectively inhibit drug efflux. The amount of DOX in the cells was detected by flow cytometry (shown in D in FIG. 5 ), which was consistent with the results of CLSM detection.

3.2.3检测DGNs在3D肿瘤球的渗透3.2.3 Detection of penetration of DGNs in 3D tumorspheres

为了评价DGNs的穿透能力,建立了3D肿瘤球模型,并采用CLSM的Z-stack扫描监测不同配方的渗透能力。如图7中A图-B图所示,DOX主要分布在3D肿瘤球的外围,而不是内部。相比之下,两个纳米粒组可以渗透到球体内部。而DGNs组在肿瘤深处的荧光强度明显高于DNs组,说明甘草次酸的修饰提高了纳米粒的渗透效果。To evaluate the penetration ability of DGNs, a 3D tumorsphere model was established, and the penetration ability of different formulations was monitored by Z-stack scanning of CLSM. As shown in Figure A-B in Figure 7, DOX is mainly distributed in the periphery of the 3D tumorspheres, rather than inside. In contrast, the two nanoparticle groups can penetrate inside the spheres. However, the fluorescence intensity of the DGNs group in the deep tumor was significantly higher than that of the DNs group, indicating that the modification of glycyrrhetinic acid improved the penetration effect of nanoparticles.

3.2.4 CTHLs增强DGNs的细胞毒性3.2.4 CTHLs enhance the cytotoxicity of DGNs

肿瘤的发生和发展与TME密切相关,TME中的CAFs可以提高肿瘤细胞的耐药性。然而,传统的体外研究模型是肿瘤细胞,无法真实模拟TME。在本研究中,在肝癌细胞(BEL-7402)基础上,我们新建立BEL-7402/LX-2共培养细胞模型,模拟TME,检测不同剂型的体外药物毒性(图8中A图所示)。如图8中B图-C图所示,各组具有明显的浓度依赖性。与单独BEL-7402细胞相比,游离药物组在BEL-7402/LX-2共培养细胞中的IC50显著降低。其中,游离DOX对共培养体系的IC50为7.13μg/mL,比BEL7402细胞高出6.4倍。这是因为激活LX-2降低了BEL-7402对药物的敏感性,导致肿瘤耐药。同时,DGNs+CTHLs组比游离DOX+CAP+TEL组具有更高的毒性。这可能是由于DGNs通过GA受体介导作用增加了药物摄取,这也在细胞摄取研究中得到了证明。活/死染色结果也表明DGNs+CTHLs组比其他制剂组具有更强的促凋亡效果(图8中D图所示)。The occurrence and development of tumors are closely related to TME, and CAFs in TME can increase the drug resistance of tumor cells. However, the traditional in vitro research model is tumor cells, which cannot truly mimic the TME. In this study, on the basis of liver cancer cells (BEL-7402), we newly established a BEL-7402/LX-2 co-culture cell model to simulate the TME to detect the in vitro drug toxicity of different dosage forms (shown in A in Figure 8) . As shown in panels B-C in Figure 8, each group has obvious concentration dependence. Compared with BEL-7402 cells alone, the IC50 of the free drug group in BEL-7402/LX-2 co-cultured cells was significantly lower. Among them, the IC50 of free DOX on the co-culture system was 7.13 μg/mL, which was 6.4 times higher than that of BEL7402 cells. This is because activating LX-2 reduces the sensitivity of BEL-7402 to the drug, leading to tumor resistance. Meanwhile, DGNs+CTHLs group had higher toxicity than free DOX+CAP+TEL group. This may be due to the increased drug uptake by DGNs mediated by GA receptors, which was also demonstrated in cellular uptake studies. The results of live/dead staining also showed that the DGNs+CTHLs group had a stronger pro-apoptotic effect than the other preparation groups (shown in D in Figure 8).

3.2.5 CTHLs通过阻断EMT抑制肝癌细胞迁移3.2.5 CTHLs inhibit the migration of liver cancer cells by blocking EMT

为探讨不同制剂对细胞迁移的影响,我们建立BEL-7402单细胞模型和BEL-7402/LX-2共培养模型(图9中A图所示)。如图9中B图和D图所示,BEL-7402/LX-2共培养细胞的迁移率(49.3%)显著高于单独BEL-7402细胞的迁移率(39.97%),说明LX-2促进了细胞迁移。此外,DGNs+CTHLs组能有效抑制单独BEL-7402和BEL-7402/LX-2的细胞迁移(图9中C图和E图所示),这可能是由于CTHLs联合DGNs治疗能效抑制了LX-2与BEL-7402间的cross-talk,从而有效地抑制了细胞迁移。除细胞划痕研究外,我们采用transwell实验进一步检测细胞侵袭能力。如图10中A图-B图所示,与其他组相比,DGNs+CTHLs组中侵袭的细胞更少,显示脂质大球+纳米小球混合制剂有效抑制了肿瘤的侵袭和迁移。To investigate the effects of different preparations on cell migration, we established a BEL-7402 single-cell model and a BEL-7402/LX-2 co-culture model (shown in Figure 9, panel A). As shown in Figure B and D in Figure 9, the migration rate (49.3%) of BEL-7402/LX-2 co-cultured cells was significantly higher than that of BEL-7402 cells alone (39.97%), indicating that LX-2 promotes cell migration. In addition, the DGNs+CTHLs group can effectively inhibit the cell migration of BEL-7402 and BEL-7402/LX-2 alone (shown in panels C and E in Figure 9), which may be due to the effective inhibition of LX- 2 and BEL-7402 cross-talk, thus effectively inhibiting cell migration. In addition to the cell scratch study, we used the transwell assay to further detect the cell invasion ability. As shown in Figure A-B in Figure 10, compared with other groups, the invasive cells in the DGNs+CTHLs group were less, showing that the mixed preparation of lipid macrospheres + nanospheres effectively inhibited tumor invasion and migration.

研究显示,CAFs通过诱导肿瘤细胞EMT促进肿瘤迁移。我们通过western blot实验检测了EMT相关蛋白的表达。如图10中C图-D图所示,与BEL-7402细胞相比,LX-2/BEL-7402共培养系统中Vimentin表达明显上调,E-Cadherin的表达明显下调,表明CAFs能促进肿瘤细胞的EMT。此外,与对照组相比CAP+TEL组Vimentin表达明显下调,E-Cadherin的表达明显上调,表明CAP联合TEL可以能有效抑制CAFs介导的EMT,抑制肿瘤细胞迁移。Studies have shown that CAFs promote tumor migration by inducing EMT of tumor cells. We detected the expression of EMT-related proteins by western blot experiment. As shown in Figure 10, panels C-D, compared with BEL-7402 cells, the expression of Vimentin in the LX-2/BEL-7402 co-culture system was significantly up-regulated, and the expression of E-Cadherin was significantly down-regulated, indicating that CAFs can promote tumor cell The EMT. In addition, compared with the control group, the expression of Vimentin in the CAP+TEL group was significantly down-regulated, and the expression of E-Cadherin was significantly up-regulated, indicating that CAP combined with TEL can effectively inhibit CAFs-mediated EMT and tumor cell migration.

3.2.6 DGNs和CTHLs在体内组织分布研究3.2.6 Study on tissue distribution of DGNs and CTHLs in vivo

为了评估纳米小球和脂质大球在体内的组织分布,将DID和DIR分别包裹在DGNs和CTHLs中,通过NIRF技术观察DID-GNs和DIR-HLs在体内的生物分布。如图11A所示,在DID激发波长下,观察到DID-GNs聚集在肿瘤部位,这是由于甘草次酸的修饰提高了DID-GNs的肝癌靶向能力。在DIR激发波长下,观察到DIR-HLs也能分布在肿瘤部位,这可能是由于脂质体的EPR作用提高了DIR在肿瘤区域的积累。另外,在肿瘤部位观察到

Figure BDA0003493104650000171
共振能量转移(FRET)效应。在DID激发波长处,与DID-GNs+DIR-HLs组相比,DID-GNs组的DID荧光强度(激发峰为648nm;发射峰为670nm)明显增强。相反,在DIR激发波长处,与DID-GNs+DIR-HLs组相比,DIR-HLs组的DIR荧光强度(FRET受体:激发峰,750nm;发射峰为782nm)明显降低,这说明DID-GNs和DIR-HLs都能在肿瘤部位聚集,有利于发挥剂型的协同抗肿瘤作用。同时,体外荧光定量结果也证实了FRET效应(图11中的B图-D图)。此外,我们对DID-GNs组肿瘤进行冷冻切片,与游离DID组相比,DID-GNs组在肿瘤深部部位的荧光强度更强(图11中E图和F图),表明小粒径的纳米小球更有利于组织渗透。To evaluate the tissue distribution of nanoglobules and lipid macrospheres in vivo, DID and DIR were encapsulated in DGNs and CTHLs, respectively, and the biodistribution of DID-GNs and DIR-HLs in vivo was observed by NIRF technique. As shown in Figure 11A, at the DID excitation wavelength, DID-GNs were observed to accumulate at the tumor site, which was due to the modification of Glycyrrhetinic acid to improve the liver cancer targeting ability of DID-GNs. At the DIR excitation wavelength, it was observed that DIR-HLs could also distribute in the tumor site, which may be due to the EPR effect of liposomes enhancing the accumulation of DIR in the tumor area. In addition, it was observed at the tumor site
Figure BDA0003493104650000171
Resonance Energy Transfer (FRET) Effect. At the DID excitation wavelength, compared with the DID-GNs+DIR-HLs group, the DID fluorescence intensity (excitation peak at 648nm; emission peak at 670nm) in the DID-GNs group was significantly enhanced. On the contrary, at the DIR excitation wavelength, compared with the DID-GNs+DIR-HLs group, the DIR fluorescence intensity (FRET receptor: excitation peak, 750nm; emission peak is 782nm) of the DIR-HLs group was significantly reduced, which indicated that DID- Both GNs and DIR-HLs can accumulate in the tumor site, which is conducive to the synergistic anti-tumor effect of the dosage form. At the same time, the in vitro fluorescence quantitative results also confirmed the FRET effect (Figures B-D in Figure 11). In addition, we performed cryosections of the tumors in the DID-GNs group. Compared with the free DID group, the fluorescence intensity in the deep part of the tumor was stronger in the DID-GNs group (Fig. Small balls are more conducive to tissue penetration.

3.2.7 CTHLs增强DID-GNs在组织的深渗透3.2.7 CTHLs enhance the deep penetration of DID-GNs in tissues

最近的研究表明,肿瘤微环境中的CAFs和ECM抑制了药物在肿瘤组织的渗透,降低了肿瘤治疗效果。因此,通过CTHLs抑制CAFs活化,减少肿瘤微环境中的ECM,可以有效增加药物深渗透。为了证实这一说法,构建H22/m-HSCs共植入荷瘤模型。治疗3天后,观察各治疗组药物渗透情况。如图12中A图-B图所示,与游离DID相比,DID-GNs组的荧光强度更强,这与3D肿瘤球的实验结果一致。此外,加入TEL或TEL+CAP后,肿瘤深处的荧光强度增强,这说明TEL和CAP能够促进微环境疏松,提高药物的通透性。而加入CTHLs后,肿瘤组织内部的荧光更强,说明CTHLs比游离TEL+CAP具有更强的微环境调控效果,有效增强DID-GNs在肝癌组织的深渗透。Recent studies have shown that CAFs and ECM in the tumor microenvironment inhibit the penetration of drugs in tumor tissues and reduce the therapeutic effect of tumors. Therefore, inhibiting the activation of CAFs by CTHLs and reducing the ECM in the tumor microenvironment can effectively increase the deep penetration of drugs. In order to confirm this statement, a H22/m-HSCs co-implantation tumor-bearing model was constructed. After 3 days of treatment, the drug penetration in each treatment group was observed. As shown in panels A-B in Figure 12, compared with free DID, the fluorescence intensity of the DID-GNs group was stronger, which was consistent with the experimental results of 3D tumorspheres. In addition, after adding TEL or TEL+CAP, the fluorescence intensity in the deep tumor was enhanced, which indicated that TEL and CAP can promote the loosening of the microenvironment and improve the permeability of drugs. After adding CTHLs, the fluorescence inside the tumor tissue was stronger, indicating that CTHLs had a stronger microenvironment regulation effect than free TEL+CAP, and effectively enhanced the deep penetration of DID-GNs in liver cancer tissues.

3.2.8 GNs联合CTHLs抑制H22皮下移植瘤发展3.2.8 GNs combined with CTHLs inhibit the development of H22 subcutaneous xenografts

建立H22荷瘤小鼠模型,评价DGNs联合CTHLs的抗肿瘤活性(图13中A图所示)。如图13中B图和D图所示,与生理盐水组相比,药物治疗组显示不同程度的肿瘤生长抑制效果,其中,DGNs+CTHLs组的肿瘤体积远小于DGNs或CTHLs组,表明DGNs联合CTHLs能提高抗肿瘤的效果。组织学分析结果显示,在DGNs+CTHLs组,肿瘤组织有明显的核仁溶解和胞浆空泡化,表明联合治疗组具有较好的抗肿瘤疗效(图13中A图所示)。体重检测结果显示,所有纳米制剂组与对照组相似,而游离DOX组的体重均较低(图13中C图所示)。另外,器官H&E结果显示(图14中A图所示),经纳米制剂处理后,细胞核大小均匀,结构无明显变化,心肌肌纤维排列紧密,形态规则,而DOX治疗组小鼠心肌核固缩或破裂,心肌排列紊乱松散,明显溶出和破裂。以上结果表明,纳米制剂有效降低了DOX的毒副作用。The H22 tumor-bearing mouse model was established to evaluate the anti-tumor activity of DGNs combined with CTHLs (shown in panel A in Figure 13). As shown in Figures B and D in Figure 13, compared with the normal saline group, the drug treatment groups showed different degrees of tumor growth inhibition effects, among which, the tumor volume of the DGNs+CTHLs group was much smaller than that of the DGNs or CTHLs group, indicating that the combination of DGNs and CTHLs CTHLs can enhance the anti-tumor effect. The results of histological analysis showed that in the DGNs+CTHLs group, the tumor tissue had obvious nucleolus lysis and cytoplasmic vacuolation, indicating that the combined treatment group had a better anti-tumor effect (shown in A in Figure 13). The body weight test results showed that all nano preparation groups were similar to the control group, while the body weight of the free DOX group was lower (as shown in Figure 13, panel C). In addition, the organ H&E results showed (shown in Figure A in Figure 14) that after nano-preparation treatment, the size of the nucleus was uniform, the structure did not change significantly, the myocardial muscle fibers were tightly arranged, and the shape was regular, while the myocardium of the mice in the DOX treatment group was pyknotic or Rupture, disordered and loose myocardial arrangement, obvious dissolution and rupture. The above results indicated that the nano-preparation effectively reduced the toxic and side effects of DOX.

3.2.9 CTHLs促进DGNs对m-HSC/H22共植入荷瘤模型的抑制效果3.2.9 CTHLs promote the inhibitory effect of DGNs on the m-HSC/H22 co-implantation tumor-bearing model

肝癌微环境包括肝星状细胞、免疫细胞等多种基质细胞,这些基质细胞在原发肿瘤的发生发展中发挥重要作用。有报道称,肿瘤微环境中的CAFs促进肿瘤细胞增殖和迁移。因此,为了模拟TME,我们将鼠源HSC(m-HSC)与H22细胞混合,共植入小鼠右背侧,建立m-HSC/H22共植入荷瘤模型,验证各制剂的体内抗肿瘤作用(图15中A图所示)。The microenvironment of liver cancer includes a variety of stromal cells such as hepatic stellate cells and immune cells, which play an important role in the occurrence and development of primary tumors. It has been reported that CAFs in the tumor microenvironment promote tumor cell proliferation and migration. Therefore, in order to simulate TME, we mixed murine HSC (m-HSC) and H22 cells, co-implanted in the right dorsal side of mice, established m-HSC/H22 co-implantation tumor-bearing model, and verified the anti-tumor effect of each preparation in vivo (As shown in Figure A in Figure 15).

如图15中B图和D图所示,与传统H22荷瘤小鼠模型相比,m-HSC/H22荷瘤小鼠模型的肿瘤体积明显增大。这是由于TME中的CAFs促进了肿瘤的增殖。此外,与游离药物组相比,DGNs或CTHLs的肿瘤体积均有不同程度的减小,说明脂质大球和纳米小球可显著提高抗肿瘤疗效。H&E染色显示,DGNs或CTHLs的凋亡区域比游离药组明显增多(图16中A图所示),而DGNs+CTHLs联合治疗组对肿瘤生长的抑制作用最强。这可能是由于CTHLs释放CAP和TEL,抑制CAFs的活化,增强了DGNs的肝癌治疗效果。Masson染色和α-SMA免疫组化实验显示(图16中B图-C图),DGNs+CTHLs组比DGNs组的胶原沉积更少,α-SMA阳性区域更小,这表明CTHLs能有效地抑制CAF活化,降低胶原蛋白的生成。另外,CD31蛋白免疫组化实验显示,与游离药相比,DGNs+CTHLs组肿瘤微血管明显减少,表明基于纳米载体的联合治疗策略可抑制肿瘤血管新生(图16中D图-E图)。As shown in panels B and D in Figure 15, compared with the traditional H22 tumor-bearing mouse model, the tumor volume of the m-HSC/H22 tumor-bearing mouse model was significantly increased. This is due to the fact that CAFs in the TME promote tumor proliferation. In addition, compared with the free drug group, the tumor volume of DGNs or CTHLs decreased to varying degrees, indicating that lipid macrospheres and nanospheres can significantly improve the antitumor efficacy. H&E staining showed that the apoptotic area of DGNs or CTHLs was significantly more than that of the free drug group (shown in panel A in Figure 16), and the combined treatment group of DGNs+CTHLs had the strongest inhibitory effect on tumor growth. This may be due to the release of CAP and TEL by CTHLs, which inhibits the activation of CAFs and enhances the therapeutic effect of DGNs on liver cancer. Masson staining and α-SMA immunohistochemical experiments showed (Figure 16 B-C panels), the DGNs+CTHLs group had less collagen deposition than the DGNs group, and the α-SMA positive area was smaller, which indicated that CTHLs can effectively inhibit CAF activation reduces collagen production. In addition, CD31 protein immunohistochemical experiments showed that compared with the free drug, the tumor microvessels in the DGNs+CTHLs group were significantly reduced, indicating that the combined treatment strategy based on nanocarriers can inhibit tumor angiogenesis (Fig. 16, D-E).

为了分析DGNs+CTHLs的抗肿瘤机制,我们进行了Western blot检测。如图16中F图-G图所示,与H22小鼠相比,mHSC/H22小鼠中E-Cadherin的表达下降,Vimentin的表达则相反。这说明,加入m-HSCs细胞可促进HCC细胞EMT。加入药物后,DGNs+CTHLs组的E-Cadherin表达显著升高,Vimentin表达明显降低,提示DGNs联合CTHLs治疗能有效逆转HCC的EMT。In order to analyze the anti-tumor mechanism of DGNs+CTHLs, we performed Western blot detection. As shown in panels F-G in Figure 16, compared with H22 mice, the expression of E-Cadherin in mHSC/H22 mice decreased, and the expression of Vimentin was opposite. This indicated that the addition of m-HSCs cells can promote EMT of HCC cells. After adding drugs, the expression of E-Cadherin in the DGNs+CTHLs group was significantly increased, and the expression of Vimentin was significantly decreased, suggesting that DGNs combined with CTHLs therapy can effectively reverse the EMT of HCC.

3.2.10 DGNs联合CTHLs抑制原位肿瘤3.2.10 DGNs combined with CTHLs inhibit orthotopic tumors

为了深入评价DGNs联合CTHLs的抗HCC效果,我们建立了原位肝癌移植瘤模型(图17中A图所示)。如图17中B图-C图所示,相比于游离DOX+CAP+TEL治疗组,DGNs+CTHLs治疗组的肿瘤面积明显减少,肿瘤凋亡区域更多,表明DGNs联合CTHLs可以有效提高抗肝癌疗效。Masson和免疫组化显示(图16中D图-F图所示),与DGNs治疗组相比,DGNs+CTHLs治疗组的α-SMA阳性区域更小,胶原沉积更少,新生血管减少。这表明CTHLs调控了肿瘤微环境,有效地抑制了微环境中CAFs活化,降低了ECM沉积,减少了新生血管的生成。In order to deeply evaluate the anti-HCC effect of DGNs combined with CTHLs, we established an orthotopic liver cancer xenograft model (shown in panel A in Figure 17). As shown in Figure 17 B-C, compared with the free DOX+CAP+TEL treatment group, the tumor area of the DGNs+CTHLs treatment group was significantly reduced, and the tumor apoptotic area was more, indicating that DGNs combined with CTHLs can effectively improve the anti-inflammatory effect. efficacy of liver cancer. Masson and immunohistochemistry showed (shown in panels D-F in Figure 16) that compared with the DGNs treatment group, the α-SMA-positive area, less collagen deposition, and reduced neovascularization in the DGNs+CTHLs treatment group were shown. This indicates that CTHLs regulate the tumor microenvironment, effectively inhibit the activation of CAFs in the microenvironment, reduce the deposition of ECM, and reduce the formation of new blood vessels.

3.2.11 DGNs联合CTHLs抑制肺转移3.2.11 DGNs combined with CTHLs inhibit lung metastasis

肝癌转移是肝癌预后不良的主要原因。为了检测DGNs联合CTHLs抑制肝癌肺转移的疗效,我们通过静脉注射H22细胞建立了肝癌肺转移模型(图18中A图所示)。图18中B图显示,与对照组相比,纳米制剂组没有明显的体重变化。图18中C图-D图显示肺转移结节情况,DGNs+CTHLs组结节数量比其他组更少,表现出更强的抑制肺转移效果。肺脏H&E染色图像显示(图17中E图所示),各治疗组均有不同程度的肺转移灶,而DGNs+CTHLs组肺转移灶小,提示DGNs联合CTHLs有效抑制肝癌转移。HCC metastasis is the main cause of poor prognosis in HCC. In order to test the efficacy of DGNs combined with CTHLs in inhibiting lung metastasis of liver cancer, we established a lung metastasis model of liver cancer by intravenously injecting H22 cells (shown in panel A in Figure 18). Panel B in Figure 18 shows that, compared with the control group, there was no significant change in body weight in the nano-formulation group. Figures C-D in Figure 18 show the situation of lung metastases, and the number of nodules in the DGNs+CTHLs group is less than that of other groups, showing a stronger effect of inhibiting lung metastasis. The H&E staining images of the lungs showed (as shown in Figure 17, E), that there were different degrees of lung metastases in each treatment group, while the lung metastases were smaller in the DGNs+CTHLs group, suggesting that DGNs combined with CTHLs effectively inhibited liver cancer metastasis.

Claims (11)

1.一种复合载体,其特征在于,包括第一靶向载体以及第二靶向载体,所述第一靶向载体包括以下组分:二油酰基磷脂酰乙醇胺、卵磷脂、胆固醇琥珀酸单酯、胆固醇、咪唑基胆固醇以及DSPE-PEG-HA;所述DSPE-PEG-HA通过DSPE-PEG-NH2与透明质酸制备而成;所述咪唑基胆固醇通过氯甲酸胆甾醇和N-(3-氨基丙基)咪唑制备而成;所述第二靶向载体包括以下组分:复合磷脂以及甘氨胆酸,所述复合磷脂包括卵磷脂以及DSPE-PEG-GA;所述第一靶向载体的粒径为150nm~200nm;所述第二靶向载体的粒径为25~50nm。1. A composite carrier, characterized in that it includes a first targeting carrier and a second targeting carrier, and the first targeting carrier includes the following components: dioleoyl phosphatidylethanolamine, lecithin, cholesterol succinic acid mono ester, cholesterol, imidazolyl cholesterol and DSPE-PEG-HA; the DSPE-PEG-HA is prepared by DSPE-PEG-NH 2 and hyaluronic acid; the imidazolyl cholesterol is prepared by chloroformic acid cholesterol and N-( 3-aminopropyl) imidazole; the second targeting carrier includes the following components: complex phospholipids and glycocholic acid, the complex phospholipids include lecithin and DSPE-PEG-GA; the first targeting The particle diameter of the targeting carrier is 150nm-200nm; the particle diameter of the second targeting carrier is 25-50nm. 2.根据权利要求1所述的复合载体,其特征在于,所述第一靶向载体包括质量比为10~15:5~10:1~5:1~5:1~5:1~5的二油酰基磷脂酰乙醇胺、卵磷脂、胆固醇琥珀酸单酯、胆固醇、咪唑基胆固醇以及DSPE-PEG-HA。2. The composite carrier according to claim 1, wherein the first targeting carrier comprises a mass ratio of 10-15:5-10:1-5:1-5:1-5:1-5 dioleoylphosphatidylethanolamine, lecithin, cholesterol succinate monoester, cholesterol, imidazolyl cholesterol and DSPE-PEG-HA. 3.根据权利要求1所述的复合载体,其特征在于,所述第二靶向载体中,卵磷脂与DSPE-PEG-GA的质量比为15~20:1;所述复合磷脂与甘氨胆酸的质量比为1.4~3.5:1。3. The composite carrier according to claim 1, characterized in that, in the second targeting carrier, the mass ratio of lecithin to DSPE-PEG-GA is 15 to 20:1; the composite phospholipid and glycine The mass ratio of cholic acid is 1.4-3.5:1. 4.根据权利要求1所述的复合载体,其特征在于,所述咪唑基胆固醇为氯甲酸胆甾醇和N-(3-氨基丙基)咪唑在三乙胺存在的条件下合成。4. The composite carrier according to claim 1, wherein the imidazole-based cholesterol is synthesized under the condition that triethylamine exists with cholesterol chloroformate and N-(3-aminopropyl) imidazole. 5.根据权利要求1所述的复合载体,其特征在于,所述DSPE-PEG-NH2的分子量为2000~5000Da,透明质酸的分子量为2000~5000Da。5. The composite carrier according to claim 1, characterized in that, the molecular weight of the DSPE-PEG-NH 2 is 2000-5000 Da, and the molecular weight of the hyaluronic acid is 2000-5000 Da. 6.一种药物载体,其特征在于,所述药物载体包括第一靶向载体以及第二靶向载体,所述第一靶向载体包括质量比为10~15:5~10:1~5:1~5:1~5:1~5的二油酰基磷脂酰乙醇胺、卵磷脂、胆固醇琥珀酸单酯、胆固醇、咪唑基胆固醇以及DSPE-PEG-HA;所述DSPE-PEG-HA通过DSPE-PEG-NH2与透明质酸制备而成;所述咪唑基胆固醇通过氯甲酸胆甾醇和N-(3-氨基丙基)咪唑制备而成;所述第一靶向载体负载的药物为辣椒素以及替米沙坦;所述第二靶向载体包括质量比为1.4~3.5:1的复合磷脂以及甘氨胆酸,所述复合磷脂包括质量比为15~20:1的卵磷脂以及DSPE-PEG-GA,所述第二靶向载体负载的药物为阿霉素。6. A drug carrier, characterized in that the drug carrier includes a first targeting carrier and a second targeting carrier, and the first targeting carrier includes a mass ratio of 10-15:5-10:1-5 :1~5:1~5:1~5 dioleoyl phosphatidylethanolamine, lecithin, cholesterol succinate monoester, cholesterol, imidazolyl cholesterol and DSPE-PEG-HA; the DSPE-PEG-HA passes through DSPE - PEG-NH 2 is prepared with hyaluronic acid; the imidazolyl cholesterol is prepared by chloroformic acid cholesterol and N-(3-aminopropyl) imidazole; the drug loaded on the first targeting carrier is capsicum and telmisartan; the second targeting carrier includes complex phospholipids and glycocholic acid with a mass ratio of 1.4 to 3.5:1, and the complex phospholipids includes lecithin and DSPE with a mass ratio of 15 to 20:1 - PEG-GA, the drug loaded on the second targeting carrier is doxorubicin. 7.根据权利要求1所述的复合载体的制备方法,其特征在于,包括以下步骤:7. The preparation method of composite carrier according to claim 1, is characterized in that, comprises the following steps: (S1)第一靶向载体的制备(S1) Preparation of the first targeting carrier (S11)DSPE-PEG-HA的合成:DSPE-PEG-NH2经过EDC与NHS活化后,与透明质酸在氮气环境下交联反应,得到DSPE-PEG-HA;(S11) Synthesis of DSPE-PEG-HA: After DSPE-PEG-NH 2 is activated by EDC and NHS, it reacts with hyaluronic acid in a nitrogen environment to obtain DSPE-PEG-HA; (S12)咪唑基胆固醇的合成:在三乙胺的催化作用下,氯甲酸胆甾醇和N-(3-氨基丙基)咪唑合成咪唑基胆固醇;(S12) Synthesis of imidazolyl cholesterol: under the catalysis of triethylamine, chloroformic acid cholesterol and N-(3-aminopropyl) imidazole synthesize imidazolyl cholesterol; (S13)二油酰基磷脂酰乙醇胺、卵磷脂、胆固醇琥珀酸单酯、胆固醇、咪唑基胆固醇和DSPE-PEG-HA加入氯仿中,用旋转蒸发器去除氯仿,待形成一层均匀的薄膜时,加入PBS溶液水化,随后在冰浴中超声,过滤,得到第一靶向载体;(S13) Add dioleoylphosphatidylethanolamine, lecithin, cholesterol succinate monoester, cholesterol, imidazolyl cholesterol and DSPE-PEG-HA into chloroform, remove chloroform with a rotary evaporator, and when a uniform film is formed, Add PBS solution for hydration, then sonicate in an ice bath, and filter to obtain the first targeting carrier; (S2)第二靶向载体的制备:将卵磷脂、DSPE-PEG-GA在乙醇中混合,然后慢慢滴注到甘氨胆酸溶液中,乙醇蒸发后,在冰浴中超声,过滤,得到第二靶向载体。(S2) Preparation of the second targeting carrier: mix lecithin and DSPE-PEG-GA in ethanol, then slowly instill into the glycocholic acid solution, after the ethanol evaporates, sonicate in an ice bath, filter, Obtain the second targeting vector. 8.一种载药体系,其特征在于,包括第一载药体系以及第二载药体系,所述第一载药体系包括第一靶向载体以及经由第一靶向载体负载的辣椒素以及替米沙坦;所述第一靶向载体包括二油酰基磷脂酰乙醇胺、卵磷脂、胆固醇琥珀酸单酯、胆固醇、咪唑基胆固醇以及DSPE-PEG-HA;所述DSPE-PEG-HA通过DSPE-PEG-NH2与透明质酸制备而成;所述咪唑基胆固醇通过氯甲酸胆甾醇和N-(3-氨基丙基)咪唑制备而成;所述第二载药体系包括第二靶向载体以及经由所述第二靶向载体负载的阿霉素;所述第二靶向载体包括复合磷脂以及甘氨胆酸,所述复合磷脂包括卵磷脂以及DSPE-PEG-GA。8. A drug-carrying system, characterized in that it includes a first drug-carrying system and a second drug-carrying system, the first drug-carrying system includes a first targeting carrier and capsaicin loaded via the first targeting carrier and Telmisartan; the first targeting carrier includes dioleoyl phosphatidylethanolamine, lecithin, cholesterol succinate monoester, cholesterol, imidazole-based cholesterol and DSPE-PEG-HA; the DSPE-PEG-HA passes through DSPE -prepared from PEG-NH 2 and hyaluronic acid; the imidazolyl cholesterol is prepared from chloroformic acid cholesterol and N-(3-aminopropyl) imidazole; the second drug-carrying system includes a second targeting A carrier and doxorubicin loaded via the second targeting carrier; the second targeting carrier includes complex phospholipids and glycocholic acid, and the complex phospholipids includes lecithin and DSPE-PEG-GA. 9.根据权利要求8所述的载药体系,其特征在于,所述第一载药体系中,辣椒素的载药量为2~3%,替米沙坦的载药量为2~3%;所述第二载药体系中,阿霉素的载药量为2~3%。9. The drug-loading system according to claim 8, characterized in that, in the first drug-loading system, the drug-loading capacity of capsaicin is 2-3%, and the drug-loading capacity of telmisartan is 2-3%. %; in the second drug-loading system, the drug-loading amount of doxorubicin is 2-3%. 10.一种如权利要求8所述的载药体系的制备方法,其特征在于,包括以下步骤:10. A preparation method of the drug-carrying system as claimed in claim 8, characterized in that, comprising the following steps: (1)第一靶向载体的制备(1) Preparation of the first targeting carrier (1a)DSPE-PEG-HA的合成:DSPE-PEG-NH2经过EDC与NHS活化后,与透明质酸在氮气环境下交联反应,得到DSPE-PEG-HA;(1a) Synthesis of DSPE-PEG-HA: After DSPE-PEG-NH 2 is activated by EDC and NHS, it reacts with hyaluronic acid in a nitrogen environment to obtain DSPE-PEG-HA; (1b)咪唑基胆固醇的合成:在三乙胺的催化作用下,氯甲酸胆甾醇和N-(3-氨基丙基)咪唑合成咪唑基胆固醇;(1b) Synthesis of imidazolyl cholesterol: under the catalysis of triethylamine, chloroformic acid cholesterol and N-(3-aminopropyl) imidazole synthesize imidazolyl cholesterol; (1c)二油酰基磷脂酰乙醇胺、卵磷脂、胆固醇琥珀酸单酯、胆固醇、咪唑基胆固醇、DSPE-PEG-HA、辣椒素以及替米沙坦加入氯仿中,用旋转蒸发器去除氯仿,待形成一层均匀的薄膜时,加入PBS溶液水化,随后在冰浴中超声,过滤,得到第一载药体系;(1c) Dioleoylphosphatidylethanolamine, lecithin, cholesterol succinate monoester, cholesterol, imidazolyl cholesterol, DSPE-PEG-HA, capsaicin and telmisartan were added to chloroform, and the chloroform was removed with a rotary evaporator. When a uniform film is formed, add PBS solution for hydration, then sonicate in an ice bath, and filter to obtain the first drug-loading system; (2)第二靶向载体的制备:将卵磷脂、DSPE-PEG-GA在乙醇中混合,然后慢慢滴注到含有甘氨胆酸以及阿霉素的溶液中,乙醇蒸发后,在冰浴中超声,过滤,得到第二载药体系。(2) Preparation of the second targeting carrier: mix lecithin and DSPE-PEG-GA in ethanol, then slowly drip into the solution containing glycocholic acid and adriamycin Ultrasound in the bath, and filter to obtain the second drug-loaded system. 11.如权利要求8~9任一项所述的载药体系在制备肝癌药物中的应用。11. The application of the drug-loading system according to any one of claims 8 to 9 in the preparation of liver cancer drugs.
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