【作者】 杨岚；

【导师】 张志荣；

【作者基本信息】 四川大学 ， 药剂学， 2023， 硕士

【摘要】 乳腺癌作为女性中最常发的恶性肿瘤,致死率高。乳腺癌具有高度异质性,患者之间的临床治疗和预后差异很大。乳腺癌按照受体表达不同有多种分型。三阴性乳腺癌（TNBC）被定义为人表皮生长因子受体-2（HER2）、孕激素（PR）和雌激素（ER）表达阴性的乳腺癌。TNBC主要发生在40岁以下的绝经前年轻女性。与其他乳腺癌亚型相比,TNBC患者的生存时间更短,肿瘤的侵袭性和复发性高。由于其特殊的分子表型,TNBC对内分泌治疗或分子靶向治疗不敏感,所以,放化学治疗是主要的治疗方式。但是术后辅助放化疗的治疗效果较差,并且残留的肿瘤组织最后也会导致复发。因而,需要开发更有效的靶向治疗肿瘤的方案。细胞表面工程是在细胞表面通过连接键（共价结合或受体配体结合等）将外源的生物材料等和细胞连接。目前主要的连接方式有内化、吸附、共价偶联和配体-受体附着。本课题主要利用了生物素和链霉亲和素这对生物系统中已知的最强非共价相互作用(Ka约为10¹⁵ M^-1)。巨噬细胞是免疫系统的重要组成部分,可以通过追踪肿瘤细胞分泌的细胞因子粘附分子以及通过受体配体相互作用而迁移到肿瘤。更重要的是巨噬细胞可以到达肿瘤深处。为此,本课题选用巨噬细胞,将其与纳米材料相互连接。为了将阿霉素脂质体负载到巨噬细胞表面,本课题首先制备了表面修饰生物素的阿霉素脂质体和表面嵌有链霉亲和素的巨噬细胞,通过生物素和链霉亲和素之间的非共价相互作用将脂质体和巨噬细胞相互连接（MA-Lip）,静脉注射给药到荷4T1乳腺癌的BALB/c小鼠。通过观察MA-Lip的分布,发现其主要分布在肿瘤部位。通过监测小鼠肿瘤生长曲线和生存期,发现MA-Lip的药效相比于普通的阿霉素脂质体药效更好。并且MA-Lip的安全性较好。本文通过五部分来介绍MA-Lip的制备表征和治疗4T1肿瘤的实验研究结果。第一部分建立了阿霉素体外样品的HPLC含量检测方法和荧光分光光度计检测方法。通过评价专属性,精密度和回收率,发现这些检测方法均符合方法学的要求,为后续药物定量提供了方法。第二部分制备及表征了表面修饰了生物素的阿霉素脂质体。表面修饰了生物素的阿霉素脂质体采用硫酸铵梯度法制备。制备得到的阿霉素脂质体在透射电镜下呈现双分子层的结构,水合粒径为114.86±2.47 nm,表面带负电,电位为-24.86±2.08 m V,包封率为86.38%,载药量为9.5%。在血浆和PBS中都具有一定的稳定性,与游离阿霉素相比具有一定的缓释效果。第三部分制备及表征了链霉亲和素修饰的巨噬细胞,以及将修饰后的巨噬细胞与生物素修饰的阿霉素脂质体连接。首先构建了表面嵌入DSPE-PEG-STA的巨噬细胞,并对其进行表征,证明DSPE-PEG-STA已成功修饰在巨噬细胞上,并通过测试巨噬细胞表面的抗体以及趋化因子,证明了在细胞表面修饰链霉亲和素不会影响细胞的迁移能力和表面性质。利用生物素和链霉亲和素之间的相互作用,使修饰有生物素的阿霉素脂质体（DOX-Lip）和嵌入链霉亲和素的巨噬细胞相互结合（MA-DOX-Lip）。筛选出RAW264.7:DSPE-PEG-STA:DOX-Lip=5×10⁵:500μg:500μg/m L是最佳比例,并对MA-DOX-Lip通过共聚焦显微镜和扫描电镜进行表征,显示MA-DOX-Lip构建成功。细胞活性实验证明了筛选出的制剂比例中DSPE-PEG-STA和DOX-Lip对巨噬细胞的活性没有影响。摄取实验证明了4T1细胞对MA-DOX-Lip相较于其他脂质体组的摄取更高。在肿瘤球实验中证明了MA-DOX-Lip可以更好地进入肿瘤深处。第四部分对制备的MA-Di D-Lip进行了体内分布研究。在荷瘤小鼠静脉给MA-Di D-Lip后,在12 h的荧光半定量结果显示MA-Di D-Lip在肿瘤的蓄积是普通Di D脂质体的2.94倍。第五部分对MA-DOX-Lip进行了体内药效、免疫学指标和安全性的初步评价。MA-DOX-Lip抑瘤明显强于DOX-Lip和游离的DOX。MA-DOX-Lip显著延长了荷瘤小鼠的生存期到44天。在肿瘤引流淋巴结和肿瘤部位都引起了CD4⁺T细胞、CD8⁺T细胞、DC细胞和NK细胞的增多。制备成MA-DOX-Lip在一定程度上降低了DOX的毒性,安全性较好。综上本文通过利用生物素-链霉亲和素之间的相互作用,在巨噬细胞表面连接阿霉素的脂质体,从而利用了巨噬细胞的肿瘤靶向能力。与传统的脂质体相比,该巨噬细胞-脂质体（MA-DOX-Lip）系统进一步增加了肿瘤部位的阿霉素积累,更深入地渗透到肿瘤组织中,并增强了抗肿瘤免疫反应。结果显示,MA-DOX-Lip系统延长了荷4T1乳腺癌小鼠的存活时间。此外,MA-Lip系统使用具有高度生物相容性和广泛认可的材料,确保了其长期安全性。本研究为三阴性乳腺癌治疗提供了新的方案,提出了一种基于巨噬细胞的药物递送新策略。更多还原

【Abstract】 Triple-negative breast cancer（TNBC）,a subtype of breast cancer that negatively expresses the progesterone receptor（PR）and estrogen receptor（ER）and lacks the human epidermal growth factor-2（HER2）gene,is more aggressive than other breast cancer subtypes.Unlike HER2-targeted therapy for patients with HER2⁺or endocrine therapy for patients with ER⁺and PR⁺,there is no effective targeted therapy for TNBC.Therefore,TNBC is mainly treated by tumor resection and chemotherapy.The recurrence rate of TNBC is also high after these treatments.These all make TNBC difficult to cure.Recently,studies reported a few nanomedicines（e.g.,liposome-based delivery systems）that could improve anti-TNBC efficiency and reduce tumor metastasis by tumor-targeted delivery.Cell surface engineering is to connect exogenous drug materials,etc.,to cells on the cell surface through linkages（covalent binding or receptor ligand binding,etc.）.Currently the main attachment methods are internalization,adsorption,covalent coupling,and ligand-receptor attachment.In this project,biotin and streptavidin,the strongest known non-covalent interaction in biological systems(Ka is about 10¹⁵M^-1),are mainly used.Macrophages are an important part of the immune system and can migrate to tumors by tracking cytokines and adhesion molecules secreted by tumor cells.What’s more,macrophages can reach deep into the tumor.To load doxorubicin liposomes on the surface of macrophages,this subject firstly prepared doxorubicin liposomes with surface-modified biotin and macrophages with streptavidin embedded on the surface.Through non-covalent interactions between biotin and streptavidin（MA-Lip）,liposomes and macrophages are connected to each other（MA-Lip）.Administered intravenously to BALB/c mice bearing 4T1 breast cancer.The distribution of MA-Lip was detected and found to be mainly distributed at the tumor site.The tumor growth curve and survival period of mice were detected,and the drug effect of MA-Lip was found to be better than that of ordinary doxorubicin liposomes.And MA-Lip is more secure.The first part establishes the HPLC content detection method and fluorescence spectrophotometer detection method of doxorubicin in vitro samples.These detection methods all meet the requirements of the methodology.The second part prepared and characterized the surface-modified biotin doxorubicin liposomes.Firstly,doxorubicin liposomes were prepared by ammonium sulfate gradient method.The prepared doxorubicin liposomes were spherical under the electron microscope,with a hydrated particle size of 114.86±2.47 nm,a surface potential of-24.86±2.08 m V,an encapsulation efficiency of 86.38%,and a drug loading capacity of 9.5%.The liposomes can remain stable in both plasma and PBS,and release slowly compared with free doxorubicin.The third part prepared and characterized streptavidin-modified macrophages and linked them with biotin-modified doxorubicin liposomes.Firstly,macrophages embedded with DSPE-PEG-STA were constructed and characterized,which proved that DSPE-PEG-STA was successfully modified on macrophages.By testing the antibodies and chemokines on the surface of macrophages,it is proved that modifying streptavidin on the cell surface will not affect the migration ability and surface properties of cells.Using the interaction between biotin and avidin,biotin-modified doxorubicin liposomes（DOX-Lip）and streptavidin-embedded macrophages（MA-DOX-Lip）were combined with each other.RAW264.7:DSPE-PEG-STA:DOX-Lip=5×10^5:500μg:500μg/m L is the optimal ratio.And MA-DOX-Lip was characterized by confocal microscope and transmission electron microscope,which showed that MA-DOX-Lip was successfully constructed.The cell activity experiment also proved that DSPE-PEG-STA and DOX-Lip had no effect on the activity of macrophages.The uptake experiment also effectively proved that MA-DOX-Lip has a higher uptake ability to 4T1 cells than other liposome groups.In the tumor spheroids experiment,we proved that MA-DOX-Lip can penetrate deep into the tumor.The fourth part studies the in vivo distribution of the prepared MA-DOX-Lip.After intravenous administration of MA-Di D-Lip preparations to tumor-bearing mice,the fluorescence semi-quantitative results at 12 hours showed that the accumulation of MA-Di D-Lip in tumors was 2.94 times that of ordinary Di D liposomes.The fifth part is the preliminary evaluation of MA-DOX-Lip in vivo efficacy,immunological indicators,and safety.MA-DOX-Lip inhibited tumor significantly stronger than DOX-Lip and free DOX.MA-DOX-Lip significantly prolongs the survival of tumor-bearing mice to 44 days.Moreover,CD4⁺T cells,CD8⁺T cells,DC cells,and NK cells were increased in tumor-draining lymph nodes and tumor sites.MA-DOX-Lip reduces the toxicity of DOX.In this study,the tumor-targeting ability of macrophages was utilized by linking liposomes of doxorubicin on the surface of macrophages by utilizing the biotin-streptavidin interaction.Compared with traditional liposomes,this macrophage-liposome（MA-DOX-Lip）system further increased the accumulation of doxorubicin at the tumor site,penetrated deeper into the tumor tissue,and enhanced antitumor immune response.As a result,the MA-DOX-Lip system significantly prolonged the survival rate of 4T1 cell mice.In addition,the MA-Lip system uses highly biocompatible and widely approved materials,ensuring its long-term safety.This study provides a system for triple-negative breast cancer therapy and provides an alternative macrophage-based delivery strategy.更多还原