【作者】 王宁；

【导师】 乔卫红；

【作者基本信息】 大连理工大学 ， 精细化工， 2021， 博士

【摘要】 癌症是严重危害人类健康的疾病之一。目前,手术治疗、放射治疗和化学治疗等单一模式的治疗方式己无法满足人们的需求,双模式协同治疗作用的纳米颗粒,尤其是化疗（CT）与化学动力学治疗（CDT）联合的纳米颗粒,在癌症的治疗中效果极为突出。然而,目前大多数CT/CDT联合的纳米颗粒在治疗癌症的准确性、选择性、高效性和安全性等方面仍需要进一步提升。基于此,本论文旨在集多级靶向、CT与CDT协同、肿瘤微环境时空控制多药释放和核磁共振成像（MRI）功能为一体,设计开发了一系列用于癌症治疗的纳米颗粒,从而增强抗肿瘤效率。（1）设计并合成了五种化合物,包括一种pH响应型前药PEG_2K-NH-N-DOX、一种ROS响应型前药PEG_2K-S-S-CPT-ROS、两种有机CDT试剂TPP-PEG_2K-LND、TPP-PEG_2K-TOS和一种T₁型MRI造影剂Gd-DTPA-N16-16。通过上述分子组装并包封小分子抗肿瘤血管生成药物康普瑞丁（CA4）,成功制备了靶向线粒体、pH/ROS双响应、内源性活性氧（ROS）爆发和时空可控多种药物释放的纳米颗粒TLDCAG,其平均粒径小于200 nm,储存稳定性、血清稳定性和pH/ROS双重响应性良好;体外释药结果表明TLDCAG在微酸性及高ROS浓度下具有时空控制多药释放能力;激光共聚焦和流式细胞仪结果证实了MCF-7细胞摄取TLDCAG呈时间依赖性;证实了TLDCAG对MCF-7细胞线粒体具有靶向能力,以及TLDCAG可以引起MCF-7细胞内源性ROS的爆发并降低其线粒体膜电位;蛋白质印迹法证实了TLDCAG是通过线粒体内源性途径引起MCF-7细胞发生凋亡;TLDCAG的弛豫效率是临床造影剂Gd-DTPA的3.89倍,体内外成像效果显著;细胞毒性、体内抗癌活性实验结果均表明TLDCAG具有较强的抗肿瘤活性,因此,TLDCAG为CT/CDT的协同作用提供了一种有效的策略。（2）设计并合成了一种GSH响应、靶向乳腺癌型前药Bio-PEG_2K-S-S-CPT。通过Bio-PEG_2K-S-S-CPT、PEG_2K-NH-N-DOX、TPP-PEG_2K-TOS、TPP-PEG_2K-LND和Gd-DTPA-N16-16组装并包封CA4,成功制备了乳腺癌肿瘤组织-细胞-线粒体多级靶向、时空控制多种药物释放到相应活性位点、消耗细胞内GSH和内源性ROS爆发的多功能纳米颗粒BDTLAG,其平均粒径为140 nm,储存稳定性、血清稳定性、稀释稳定性和pH/GSH双重响应性良好;体外释药实验表明BDTLAG具有时空可控多药释放能力;激光共聚焦与流式细胞仪数据证实BDTLAG具有多级靶向乳腺癌的能力;激光共聚焦进一步证实BDTLAG明显地引起线粒体超氧化物的产生,确保线粒体中更多ROS物种的形成;蛋白质印迹法证实了BDTLAG是通过线粒体内源性途径引起MCF-7细胞发生凋亡;体内外核磁共振成像结果表明BDTLAG具有较高的弛豫效率和显著的MRI能力;细胞毒性、体内抗癌活性和体内毒性实验证实BDTLAG具有极强的抗肿瘤活性且体内毒性可忽略不计。这种将CT、CDT、消耗细胞内GSH和MR成像功能整合在同一纳米颗粒的策略反映了“一体化”的概念,提高了纳米颗粒治疗癌症时的准确性、选择性、高效性与安全性。（3）设计并合成了一种GSH响应、肝癌靶向型前药GA-PEG_2K-S-S-CPT。通过GA-PEG_2K-S-S-CPT、PEG_2K-NH-N-DOX、TPP-PEG_2K-TOS、TPP-PEG_2K-LND和Gd-DTPA-N16-16的组装,成功制备了具有多级靶向肝癌、时空控制多种药物释放到相应活性位点、诱导内源性ROS爆发和MRI等功能的纳米颗粒CDTLG,其平均粒径为147 nm,储存稳定性、血清稳定性、稀释稳定性和pH/GSH双重响应性良好;激光共聚焦图像、流式细胞仪数据、体内MR成像、体内荧光成像证实CDTLG具有多级靶向肝癌的能力;激光共聚焦图像证实CDTLG可以引起HepG2细胞内源性ROS的爆发并降低其线粒体膜电位;细胞毒性和体内抗肿瘤活性实验证实CDTLG实现了CT/CDT的协同作用并具有极强的抗肿瘤活性;治疗结束后,荷瘤小鼠的体重、血液学和血清生化测试以及荷瘤小鼠各器官H&E染色数据显示CDTLG具有极高的生物安全性。多功能纳米颗粒CDTLG提供了一种增强肝癌治疗效率的策略。更多还原

【Abstract】 Cancer is known as one of the diseases that has seriously endangered the human health.Currently,the available single-mode treatments,such as surgery,radiotherapy and chemotherapy are not able to meet people’s needs.Nanoparticles with dual-mode synergistic therapeutic effects,especially those combined with chemotherapy（CT）and chemodynamic therapy（CDT）,the effect is extremely prominent in the treatment of cancer.However,the accuracy,selectivity,efficiency and safety of most CT/CDT nanoparticles in cancer treatment still needs to be further improved.Based on these mentioned concerns,this thesis aims to design and develop a series of nanoparticles for cancer treatment,which combines the functions of both CT and CDT,multistage-targeting,spatiotemporally controlled release of chemotherapy drug and magnetic resonance imaging（MRI）,so as to enhance the antitumor efficiency.The pH-responsive prodrug(PEG_2K-NH-N-DOX),ROS-responsive prodrug(PEG_2K-S-S-CPT-ROS),organic CDT agents(TPP-PEG_2K-LND,TPP-PEG_2K-TOS),and MRI contrast agents（Gd-DTPA-N16-16）were designed and synthesized.The targeted mitochondria,pH/ROS dual responsive nanoparticle（TLDCAG）with endogenous ROS burst and spatiotemporally controlled multiple drug release ability was prepared by the above-mentioned molecular assembly and encapsulation of the small anti-tumor angiogenic drug combrestatin A4（CA4）.The average particle size of TLDCAG is less than 200 nm,and has outstanding storage stability,serum stability and pH/ROS dual responsiveness.The results of drug release showed that TLDCAG has the ability to control multi-drug release in time and space under slightly acidic and high ROS level.Confocal laser and flow cytometry results demonstrated that the uptake of TLDCAG by MCF-7 cells was time-dependent.Besides,confocal laser results confirmed that TLDCAG has the targeting ability to MCF-7cells mitochondria,and also TLDCAG can cause the outbreak of endogenous ROS in MCF-7cells and reduce its mitochondrial membrane potential.Western blotting confirmed that TLDCAG induced apoptosis of MCF-7 cells through mitochondrial endogenous routes.The relaxation efficiency of TLDCAG is 3.89 times that of the clinical contrast agent Gd-DTPA,and the imaging effect is significant in vivo and in vitro.Both cytotoxicity and in vivo anti-cancer activity test results showed that TLDCAG has excellent anti-tumor activity.Therefore,TLDCAG provides an effective strategy for the synergistic effect of CT/CDT.The GSH-responsive and breast cancer targeting prodrug(Bio-PEG_2K-S-S-CPT)was designed and synthesized.The multifunctional nanoparticle（BDTLAG）with breast cancer tissue-cells-mitochondria targeting,spatiotemporally controlled multiple drugs release to the corresponding active sites,GSH depletion,and endogenous ROS burst induced ability was successfully prepared by the Bio-PEG_2K-S-S-CPT,PEG_2K-NH-N-DOX,TPP-PEG_2K-TOS,TPP-PEG_2K-LND,Gd-DTPA-N16-16 assembly and encapsulation of CA4.The average particle size of BDTLAG was determined as≈140 nm,and has outstanding storage stability,serum stability,dilution stability and pH/GSH dual responsiveness.The results of drug release showed that BDTLAG has the ability to control multi-drug release in time and space.Confocal laser and flow cytometry results demonstrated that BDTLAG has the multistage-targeting ability to breast cancer.Besides,confocal laser results confirmed that BDTLAG could obviously cause mitochondrial superoxide generation to guarantee the formation of much more amount of ROS species in mitochondria.Western blotting confirmed the BDTLAG induced apoptosis of MCF-7 cells through mitochondrial endogenous routes.In vitro and in vivo MRI results showed that BDTLAG has high relaxation efficiency and remarkable MRI ability.Cytotoxicity,anticancer activity and toxicity experiments confirmed that BDTLAG has outstanding antitumor activity and negligible toxicity in vivo.Therefore,such a single nanoparticle that integrates CT,CDT,and MRI functions along with GSH depletion reflects the“all in one”concept and improves the accuracy,selectivity,efficiency and safety of nanoparticles in the treatment of cancer.The GSH-responsive and liver cancer targeting prodrug(GA-PEG_2K-S-S-CPT)was designed and synthesized.The multifunctional nanoparticle（CDTLG）with cancer tissue-cells-mitochondria targeting,spatiotemporally controlled multiple drugs release to the corresponding active sites,MRI and endogenous ROS burst induced ability was prepared by the GA-PEG_2K-S-S-CPT,PEG_2K-NH-N-DOX,TPP-PEG_2K-TOS,TPP-PEG_2K-LND and Gd-DTPA-N16-16 self-assembly.The average particle size of CDTLG was determined as≈147 nm,and has outstanding storage stability,serum stability,dilution stability and pH/GSH dual responsiveness.The results of confocal laser,flow cytometer,in vivo MRI confirmed that CDTLG has the multistage-targeting ability to liver cancer.The confocal laser images confirmed that CDTLG can cause the outbreak of endogenous ROS in HepG2 cells and reduce the mitochondrial membrane potential.Cytotoxicity and anti-tumor activity experiments in vivo confirmed that CDTLG realized the synergistic effect of CT/CDT and has strong antitumor activity.After treatment,the weight,hematology,serum biochemical and H&E staining of various organs of tumor-bearing mice showed that CDTLG has high biological safety.The multifunctional nanoparticle（CDTLG）provides a strategy to improve the treatment efficiency of liver cancer.更多还原