【作者】 李晓东；

【导师】 刘桂锋；

【作者基本信息】 吉林大学 ， 影像医学与核医学（专业学位）， 2025， 博士

【摘要】 乳腺癌是女性最常见的恶性肿瘤之一。与其他分型乳腺癌患者相比,三阴性乳腺癌（Triple Negative Breast Cancer,TNBC）,具有发病年龄更低,组织学分级更高,侵袭能力更强,耐药性更强,更易复发转移的特点。由于特异性靶点的缺失,使TNBC临床治疗面临巨大挑战。早发现、早治疗对TNBC患者的预后至关重要。因此,为TNBC开发更好的早期诊断和治疗方法,对于改善TNBC患者的生存情况并提高生存率具有重要临床意义。分子影像学（Molecular Imaging）作为一门新兴学科,为肿瘤诊断开辟了新视角,并能够为传统影像技术提供更加精准的诊断策略。构建兼具强肿瘤特异性、高效结合靶点、稳定性好、高生物相容性、体内清除速率快等特点的分子影像探针,利用分子影像技术的肿瘤精准靶向成像策略,对TNBC进行诊疗一体化研究,进而实现TNBC早发现和靶向治疗具有重要意义。趋化因子受体4（Chemokine receptor type 4,CXCR4）和程序性死亡1蛋白配体（Programmed death ligand-1,PD-L1）在包括TNBC在内的多种恶性肿瘤细胞上高表达,是两种重要的肿瘤生物标志物。研究表明,针对CXCR4的拮抗剂和PD-L1的抑制剂在癌症诊疗领域具有巨大潜力。磁共振成像（Magnetic Resonance Imaging,MRI）因其具有出色的软组织对比度,并且可多参数、多序列成像等优点,已被广泛应用于临床上多种疾病的非侵入性诊断。但因其敏感性较低,往往需要借助于对比剂来突出感兴趣区域（Region of interest,ROI）的解剖学和病理学特征,以提高病灶检出能力。因此,构建一种合适的具有主动靶向能力的纳米磁共振（Magnetic Resonance,MR）对比剂,既能够增强肿瘤成像能力,又能够减轻潜在的生物毒性,是临床亟待解决的问题。本实验针对TNBC,以四氟钆酸钠（NaGdF₄）纳米点（Nanodots,NDs）为纳米探针,构建了两种具有主动肿瘤靶向能力的纳米诊疗试剂:CXCR4-NaGdF₄双功能NDs和PDL1-NaGdF₄NDs。细胞及活体实验结果表明,所合成的纳米诊疗试剂不仅具有良好的T₁加权MR肿瘤成像能力,而且能够通过对CXCR4或PD-L1的拮抗作用有效降低肿瘤细胞的活率。另外,CXCR4-NaGdF₄双功能NDs和PDL1-NaGdF₄NDs还具有高效的肾脏清除能力和良好的生物相容性等优点,体外和小动物活体实验结果表明其是一种很有潜力的纳米诊疗试剂。具体研究内容如下:1、采用溶剂热法合成了平均粒径为5±0.5 nm的NaGdF₄NDs。以胰蛋白胨（Tryptone）作为相转移剂,通过Gd-磷酸盐配位反应将疏水的油酸包覆的NaGdF₄NDs（OA-NaGdF₄NDs）转换为亲水的Try-NaGdF₄NDs,进一步利用酰胺化反应将靶向TNBC的CXCR4拮抗多肽或PD-L1抑制多肽修饰到Try-NaGdF₄NDs表面,获得具有分散性好、大小均一、尺寸较小的CXCR4-NaGdF₄双功能NDs和PDL1-NaGdF₄NDs。高分辨透射电子显微镜（High resolution transmission electron microscopy,HRTEM）表征显示所制备的NDs呈球状,并具有良好的晶体结构和单分散性。NDs的大小、晶型和形状在配体交换前后无明显变化。通过减小NDs的粒径能够有效缩短其在体内的滞留时间,从而解决了纳米粒子因尺寸相对较大（粒径>10 nm）,在活体内通过肝脏代谢缓慢（在体内留存时间甚至可达数月之久）而引起的纳米粒子潜在生物毒性的问题。该合成方法简单、不需要太复杂的步骤和昂贵的设备要求,重现性较好,易于未来实现规模化制备。2、通过溶液MRI分析证明CXCR4-NaGdF₄双功能NDs和PDL1-NaGdF₄NDs具有较好的T₁加权成像效果,摩尔纵向弛豫率分别为r₁=21.87 mM^-1S^-1和22.82mM^-1S^-1,高于Try-NaGdF₄NDs(r₁=10.75 mM^-1S^-1)和临床使用的MR对比剂(r₁=3.07 mM^-1S^-1)。3、细胞实验结果表明Try-NaGdF₄NDs没有明显的细胞毒性。CXCR4-NaGdF₄双功能NDs和PDL1-NaGdF₄NDs对MDA-MB-231细胞（TNBC肿瘤细胞）有较强的细胞毒性,共孵育后细胞活率分别降至61%和55%。电感耦合等离子体质谱（Inductively coupled plasma-mass spectrometry,ICP-MS）分析发现与CXCR4-NaGdF₄双功能NDs和PDL1-NaGdF₄NDs共孵育MDA-MB-231细胞中Gd元素的含量明显高于与Try-NaGdF₄NDs共孵育MDA-MB-231细胞中Gd元素含量。这一结果说明CXCR4-NaGdF₄双功能NDs和PDL1-NaGdF₄NDs与MDA-MB-231细胞结合能力较强,能够明显抑制肿瘤细胞增殖。4、通过构建荷MDA-MB-231肿瘤裸鼠模型,考察所制备的Try-NaGdF₄NDs、CXCR4-NaGdF₄双功能NDs和PDL1-NaGdF₄NDs的活体肿瘤靶向性、体内代谢途径、活体肿瘤MRI对比增强效果以及肿瘤抑制情况。经尾静脉分别将上述NDs注射到荷MDA-MB-231肿瘤裸鼠体内,在注射后不同时间点采集T₁加权MR图像。通过分析MR图像发现,注射后1小时,肿瘤部位均开始强化;注射后2小时,强化达到最大值;随着时间的延长,强化程度逐渐减低。说明在2小时肿瘤部位NDs的富集量是最多的。同时,注射NDs后裸鼠肾脏的MR信号显著增强,而肝脏MR信号几乎不变。另外,在同一时间段内,CXCR4-NaGdF₄双功能NDs和PDL1-NaGdF₄NDs对肿瘤的MR增强效果优于Try-NaGdF₄NDs。ICP-MS分析也发现注射后2小时,注射CXCR4-NaGdF₄双功能NDs和PDL1-NaGdF₄NDs裸鼠肿瘤部位的Gd元素含量明显高于注射Try-NaGdF₄NDs裸鼠肿瘤部位的Gd元素含量。上述实验结果说明CXCR4-NaGdF₄双功能NDs和PDL1-NaGdF₄NDs肿瘤富集能力较强,具有显著的肿瘤靶向性,可经肾脏代谢排出体外。肿瘤生长曲线和荷瘤裸鼠存活曲线研究表明CXCR4-NaGdF₄双功能NDs和PDL1-NaGdF₄NDs对MDA-MB-231肿瘤具有显著抑制效果并能够显著延长荷瘤裸鼠的生存时间;PDL1-NaGdF₄NDs抑瘤能力优于CXCR4-NaGdF₄双功能NDs（抑瘤效率:66%肿瘤体积vs 48%肿瘤体积）。血常规分析和主要器官H&E染色实验结果显示,Try-NaGdF₄NDs、CXCR4-NaGdF₄双功能NDs和PDL1-NaGdF₄NDs均无明显活体毒性。上述实验结果表明CXCR4-NaGdF₄双功能NDs和PDL1-NaGdF₄NDs不仅具有高效的肾脏清除能力、出色的肿瘤靶向能力以及优异的生物相容性等优点,而且还具有优异的T₁加权MR成像性能以及良好的肿瘤抑制效果,能够满足临床应用的基本标准,为进一步构建TNBC诊疗纳米药物平台提供了新思路。更多还原

【Abstract】 Breast cancer is one of the most common malignant tumour in female.Compared with other types of breast cancer,triple negative breast cancer（TNBC）has the characteristics of lower age of onset,higher histological grade,more aggressive,stronger drug resistance,and easier recurrence and metastasis.Due to the lack of specific targets,its clinical treatment faces great challenges.It is of great significance to develop better early diagnosis and treatment methods for TNBC to improve the survival rate of patients.As an emerging discipline in recent years,molecular imaging has opened new perspectives for diagnosis of cancer,providing more accurate diagnosis strategy.The precise targeting strategy is the key to achieve early diagnosis of breast cancer.Design of molecular imaging probes needs to consider the properties of strong specificity and efficient binding to targets,good stability,efficient clearance in vivo and non-toxic side effects.Lastly,molecular imaging technology is efficiently used to significantly improve the sensitivity and specificity of the diagnosis of breast cancer.Chemokine receptor type 4（CXCR4）and programmed death ligand-1（PD-L1）are highly expressed on various malignant tumor cells,including TNBC,and are two important tumor biomarkers.Research has shown that CXCR4 antagonists and PD-L1inhibitors have great potential in the field of cancer diagnosis and treatment.Magnetic Resonance Imaging（MRI）has been widely used in non-invasive diagnosis of various diseases in clinical practice due to its excellent soft tissue contrast,multi-parameter and multi-sequence imaging.However,due to its relatively low sensitivity,some contrast agents are often applied to highlight the anatomical and pathological features of the region of interest（ROI）in order to improve the ability to detect lesion.Therefore,the construction of suitable nanoprobes with active targeting ability can not only enhance the tumor imaging ability,but also reduce the potential biological toxicity,which is an urgent problem to be solved in clinical practice.In this study,the sodium tetrafluoro gadolinium（NaGdF₄）nanodots（NDs）were used as nanoprobes to construct two TNBC targeting diagnostic and therapeutic nanoagents:CXCR4-NaGdF₄bifunctional NDs and PDL1-NaGdF₄NDs.The in vitro and in vivo experimental results indicate that the synthesized nanomedicines not only have good T1-weighted Magnetic Resonance（MR）tumor imaging ability,but also can effectively reduce tumor cell viability by antagonizing CXCR4 or PD-L1.In addition,CXCR4-NaGdF₄bifunctional NDs and PDL1-NaGdF₄NDs also have the advantages of efficient renal clearance ability and good biocompatibility,making them a promising diagnostic and therapeutic nanoagents.The detailed research content is as follows:1.NaGdF₄NDs with an average particle size of 5±0.5 nm was synthesized by solvothermal method.Using tryptone as a phase transfer agent,hydrophobic oleic acid coated NaGdF₄NDs（OA-NaGdF₄NDs）were converted into hydrophilic Try-NaGdF₄NDs through Gd phosphate coordination reaction.Furthermore,the CXCR4-NaGdF₄bifunctional NDs and PDL1-NaGdF₄NDs were prepared by modified CXCR4antagonist peptides or PD-L1 inhibitory peptide targeting TNBC onto the surface of Try-NaGdF₄NDs via the amidation reaction.The as-prepared CXCR4 NaGdF₄bifunctional NDs and PDL1 NaGdF₄NDs exhibit good dispersibility and uniform size.High resolution transmission electron microscopy（HRTEM）characterization shows that the as-prepared NDs are spherical with good crystal structure and monodispersity.The size,crystal form,and shape of NDs shows no significant changes before and after ligand exchange.Reducing the particle size of NDs can effectively shorten their retention time in the body.This solves the problem of potential biological toxicity of nanoparticles caused by their relatively large size（particle size>10 nm）and slow liver metabolism in vivo（retention time in vivo can even reach several months）.This synthesis method is simple,does not require too complex steps and expensive equipment requirements,has good reproducibility,and is easy to achieve large-scale preparation in the future.2.The MRI analysis demonstrates that CXCR4-NaGdF₄bifunctional NDs and PDL1-NaGdF₄NDs have good T1-weighted MR imaging effects,with molar longitudinal relaxation rates（r₁）of 21.87 mM^-1S^-1and 22.82 mM^-1S^-1,respectively,higher than those of Try-NaGdF₄NDs(r₁=10.75 mM^-1S^-1)and clinically used MR contrast agents(r₁=3.07 mM^-1S^-1).3.The cell experiment results show that Try-NaGdF₄NDs have negligible cytotoxicity towards MDA-MB-231 cells（TNBC tumor cells）,while CXCR4-NaGdF₄bifunctional NDs and PDL1-NaGdF₄NDs exhibit strong cytotoxicity towards MDA-MB-231 cells.After coculture with CXCR4-NaGdF₄bifunctional NDs and PDL1-NaGdF₄NDs,the viabilities of MDA-MB-231 reduce to61%and 55%,respectively.Inductively coupled plasma mass spectrometry（ICP-MS）analysis reveals that the Gd contents in MDA-MB-231 cells cocultured with CXCR4-NaGdF₄bifunctional NDs and PDL1-NaGdF₄NDs are significantly higher than that in MDA-MB-231 cells cocultured with Try-NaGdF₄NDs.This result indicates that CXCR4-NaGdF₄bifunctional NDs and PDL1-NaGdF₄NDs have strong binding ability to MDA-MB-231 cells and can significantly inhibit tumor cell proliferation.4.The in vivo tumor targeting,in vivo metabolic pathways,in vivo tumor MRI contrast enhancement effect,and tumor inhibition of the prepared Try-NaGdF₄NDs,CXCR4-NaGdF₄bifunctional NDs,and PDL1-NaGdF₄NDs were investigated by constructing an MDA-MB-231 tumor bearing nude mouse model.The above-mentioned NDs were injected into MDA-MB-231 tumor bearing nude mice via the tail vein,and T1-weighted MR images were collected at different time points after injection.It was found that the MR signal in tumor site began to strengthen at one-hour post-injection.The maximum value of reinforcement on MRI is reached at two-hour post-injection,and the degree of reinforcement gradually decreases with the extension of time.The result indicates that the enrichment of NDs at the tumor site is highest at two-hour post-injection.Meanwhile,after injection of NDs,the MR signal of the kidneys in nude mice was significantly enhanced,while the MR signal of the liver remained almost unchanged.In addition,during the same time period,CXCR4-NaGdF₄bifunctional NDs and PDL1-NaGdF₄NDs show stronger MR enhancement effects on tumors than that of Try-NaGdF₄NDs.ICP-MS analysis also found that the Gd content in the tumor site of CXCR4-NaGdF₄bifunctional NDs and PDL1-NaGdF₄NDs injected into nude mice was significantly higher than that of Try-NaGdF₄NDs injected into nude mice,at two-hour post-injection.The above experimental results indicate that CXCR4-NaGdF₄bifunctional NDs and PDL1-NaGdF₄NDs have strong tumor accumulation ability,significant tumor targeting,and can be metabolized and excreted from the body through the kidneys.Studies on tumor growth curves and survival lines of tumor bearing nude mice have shown that CXCR4-NaGdF₄bifunctional NDs and PDL1-NaGdF₄NDs have significant suppression effects on MDA-MB-231 tumors,and can significantly prolong the survival time of tumor bearing nude mice.Specially,the suppression ability of PDL1-NaGdF₄NDs is superior to CXCR4-NaGdF₄bifunctional NDs（anti-tumor efficiency:66%tumor volume vs 48%tumor volume）.The results of blood routine analysis and H&E staining experiments on major organs show that Try-NaGdF₄NDs,CXCR4-NaGdF₄bifunctional NDs,and PDL1-NaGdF₄NDs have no significant in vivo toxicity.In summary,the in vitro and in vivo experimental results indicate that CXCR4-NaGdF₄bifunctional NDs and PDL1-NaGdF₄NDs not only have efficient renal clearance ability,excellent tumor targeting ability,and excellent biocompatibility,but also have excellent T1-weighted MRI ability and good tumor suppression effect.These advantages make that the CXCR4-NaGdF₄bifunctional NDs and PDL1-NaGdF₄NDs meet the basic standards of clinical application,which provides new ideas for further constructing a TNBC diagnosis and treatment nanomedicine platform.更多还原