【作者】 杨涛；

【导师】 陈华兵；

【作者基本信息】 苏州大学 ， 药剂学， 2018， 博士

【摘要】 恶性肿瘤严重威胁人类的生命健康。因此,如何实现肿瘤的高效诊断和治疗是当前面临的巨大挑战。近年来,纳米材料领域的飞速发展为肿瘤的诊断与治疗带来新的契机。其中,基于蛋白质的纳米材料因其生物相容性好、制备工艺简单等特点受到广泛关注。影像探针或治疗用药可通过静电吸附或共价偶联等方式整合进蛋白质制得蛋白纳米粒,可有效提高药物负载量、提升药物稳定性、延长药物体内循环时间,显著提升肿瘤成像与治疗效果。然而,使用这些方法制备的蛋白纳米粒尺寸无法得到精确控制,由于尺寸分布较宽且易聚集等特点,巨噬系统对其摄取量高,从而降低肿瘤部位蓄积量,难以满足肿瘤的高效诊疗。因此,如何在温和条件下实现蛋白纳米粒的可控合成是蛋白纳米材料用于肿瘤高效诊疗中亟待解决的关键问题。基于蛋白纳米反应器的仿生合成法是近年来快速发展的一种新型无机纳米粒合成方法,其原理是模拟蛋白质内部空腔中的生物矿化作用,通过蛋白质分子中氨基酸残基与金属离子相互作用(如静电吸附、配位等)形成成核中心,在还原反应、无机沉淀反应等作用下,诱导无机纳米晶在其内腔中成核、生长,形成包载无机纳米晶的蛋白纳米粒。该方法具有粒径可控、反应条件温和、制备工艺简单等特点,可有效提升蛋白纳米粒在肿瘤部位的蓄积量,提高肿瘤成像与治疗效果,实现肿瘤精确诊断与高效治疗,显示出良好的生物应用前景。本论文首先通过人血清白蛋白(human serum albumin,HSA)纳米反应器仿生合成法,制得尺寸可控的载硫化银白蛋白纳米粒(Ag2S@HSA),用于肿瘤部位超灵敏、高分辨的近红外二区荧光、光声双模式成像,同时利用其光热效应,实现高效的肿瘤光热治疗。此外,为解决深部肿瘤解剖学成像对比度不足的问题,我们以肿瘤靶向性转铁蛋白(transferrin,Trf)作为纳米反应器,构建载四氧化三锰转铁蛋白纳米粒(Mn3O4@Trf),用于肿瘤微环境响应性磁共振成像,实现深部肿瘤的高对比度解剖学成像,提高诊断准确性。本论文具体研究内容概述如下:第一章:简述近年来蛋白纳米粒的发展历程,以及蛋白纳米粒在纳米医药领域的研究现状,并在此基础上阐明了本论文的立题依据及研究内容。第二章:以白蛋白作为纳米反应器诱导硫化银在蛋白内腔中可控生长,制备载硫化银白蛋白纳米粒(Ag2S@HSA),并探索其在肿瘤诊疗一体化中的应用。人血清白蛋白作为纳米反应器可在温和条件下诱导Ag2S纳米晶在白蛋白内腔中成核、生长,并通过控制反应时间和原料反应比,实现Ag2S@HSA粒径的精确调控。利用吸收光谱对其生长过程进行监测,发现该过程属于扩散控制的生长动力学行为。在近红外激光照射下,Ag2S@HSA具有尺寸依赖的近红外二区荧光信号及光热效应,且光稳定性良好。Ag2S@HSA可被4T1细胞通过网格蛋白介导的内吞途径摄取,并定位于酸性细胞器溶酶体,光照后对细胞可产生显著杀伤作用。Ag2S@HSA在小鼠体内血液循环时间较长(t1/2β=5.7 h),显示出良好的肿瘤靶向性,并能长期滞留于肿瘤部位。在50μmolkg-1注射剂量下,Ag2S@HSA在4T1皮下瘤动物模型中可实现超灵敏、空间分辨显著的近红外二区荧光和光声双模式成像。在近红外激光激发下,单次光热治疗即可完全消除肿瘤,且对正常组织无明显损伤。此外,Ag2S@HSA具有良好的体内安全性,可在注射后30天从体内清除,避免长期蓄积带来的潜在毒性。第三章:以肿瘤靶向性转铁蛋白作为纳米反应器诱导四氧化三锰在其空腔中成核生长,制得载四氧化三锰转铁蛋白纳米粒(Mn304@Trf),并探索其在肿瘤微环境响应性磁共振成像中的应用。利用转铁蛋白与高锰酸钾发生的氧化还原反应,诱导四氧化三锰纳米晶在蛋白空腔中成核、生长,在温和条件下制得Mn304@Trf,并通过控制反应时间,实现了 Mn304@Trf的可控生长。Mn304@Trf结晶性良好,但仍存在晶格缺陷,该缺陷使其在酸性及还原性环境中大量释放Mn2+,有效提高局部Mn2+浓度,从而大幅提升磁共振造影性能。Mn304@Trf与pH 5.0缓冲溶液共孵育24 h后,其纵向弛豫系数(r1)由0.34mM-1s-1提升至3.2mM-1s-1;还原性环境的协同作用促进了更多的Mn2+释放,从而使其ri进一步增加至7.2mM-1s-1,显示出酸性、还原性双重响应的磁共振信号增强效应。Mn304@Trf具有较长的血液循环时间(t1/2β = 4.5h),通过与转铁蛋白受体(TfR1)的相互作用,能高效蓄积于高表达TfR1的人结肠癌(HT-29)皮下移植瘤,并渗透至肿瘤深部,显示出尺寸依赖的靶向行为。内吞进入溶酶体后,Mn304@Trf在酸性和还原性环境刺激下,将皮下移植瘤和原位肿瘤的磁共振造影信噪比提高至200～250%。此外,在肿瘤瘤内局部注射酸性或还原性溶液后,Mn304@Trf可将肿瘤部位的信噪比进一步提高至400%,产生了高对比度的磁共振成像效果。上述研究表明,Mn304@Trf具有显著的酸性、还原性响应的磁共振成像能力,可有效提高肿瘤解剖学成像的对比度。本论文利用蛋白质作为纳米反应器构建了多种载无机材料蛋白纳米粒,对其在肿瘤多模态成像与光热治疗方面的应用进行了研究,并为该蛋白纳米体系在肿瘤诊疗领域中的应用奠定了一定的科学基础。更多还原

【Abstract】 Malignant tumor is one of the major diseases that seriously threaten human lives and health.Therefore,it is still a great challenge to achieve efficient tumor diagnosis and therapy.In recent years,the rapid development of nanomaterials has brought new opportunities for cancer diagnosis and treatment.Protein-based nanoparticles have been extensively explored as nanomedicine for cancer theranostics.Imaging agents or therapeutic drugs can be incorporated within proteins via electrostatic adsorption,hydrophobic interaction or specific site binding to fabricate nanoparticles with higher drug payload,enhanced drug stability and prolonged blood circulation time,leading to improved cancer diagnostic and therapeutic efficacy.However,these protein-based nanoparticles often exhibited poor tumor target capacity due to lack of size control,thereby leading to insufficient tumor accumulation without satisfied efficacy.Therefore,it’s highly desired to explore and develop facile construction of protein-based nanoparticles under mild conditions with controlled and uniform size for enhanced tumor accumulation,in order to achieve highly efficient tumor diagnosis and therapy.Protein nanoreactor has been exploited as an emerging strategy for inorganic nanomaterials synthesis.In this method,protein molecules are used as hollow nanocages,in which metal ions are interacted with amino acid residues(via electrostatic adsorption,coordination,etc.)to act as nucleation center to facilitate the nanocrystal growth through precipitation or reduction reaction.The biomimetic mineralization process within protein nanoreactor could result in inorganic nanoparticles with protein corona in a facile,mild and controlled manner.The synthesized protein nanoparticles with controlled size exhibit multiple advantages including great biocompatibility,effective cellular uptake as well as tumor accumulation,suggesting potential application in tumor theranostics.Herein,we demonstrate the size-dependent Ag2S@HSA nanoparticles precisely synthesized through carefully controlled growth of Ag2S nanocrystals in hollow human serum albumin(HSA)nanocages,generating sufficient in vivo second near-infrared(NIR-Ⅱ)fluorescence and photoacoustic signals as well as potent hyperthermia at tumors for cancer theranostics.Moreover,tumor targeting ligand transferrin(Trf)is further employed as nanoreactor to synthesize Mn3O4@Trf with tumor microenvironment triggered magnetic resonance(MR)signal amplification for contrast-enhanced tumor imaging with high spatial resolution.These investigations are shown as follows:Chapter 1.This chapter is an overall introduction of protein-based nanoparticles for nanomedicine.Chapter 2.We have successfully synthesized size-dependent Ag2S@HSA with well-defined nanostructure as a theranostic agent for multimodal imaging and simultaneous photothermal therapy.The nucleation and growth of Ag2S nanocrystals in expansive hollow albumin nanocages was triggered by precipitation reaction between Ag+ and S2-in mild conditions.And the size of Ag2S@HSA could be precisely controlled through initial reactant ratio and reaction time.The growth mechanism of Ag2S@HSA was determined to be diffusion-controlled growth progress based on the analysis of reaction kinetics via monitoring the absorbance of Ag2S@HSA after different reaction time.Ag2S@HSA with the hydrodynamic diameter of 36.2 nm produced effective fluorescence in NIR-Ⅱ region,distinct photoacoustic intensity,and potent photothermal conversion in a size-dependent manner under NIR irradiation.Ag2S@HSA were efficiently internalized by 4T1 cells via clathrin-mediated endocytosis and mainly distributed into the lysosomes.Upon NIR irradiation,Ag2S@HSA exhibited intense photo-induced cytotoxicity on 4T1 cells with IC50 of 0.17 mM.Moreover,Ag2S@HSA possessed prolonged blood circulation time with t1/2βof 5.7 h,preferable tumor accumulation and long-term retention in tumor site.Under NIR light exposure,Ag2S@HSA were able to totally ablate the tumors through the photothermal injury without damaging surrounding normal tissues after a single injection at the dose of 50 μmol kg-1.In addition,Ag2S@HSA could be effectively eliminated from the mice after 30 days post-injection,indicating that they can avoid the long-term in vivo retention and thus have no significant safety concern.Chapter 3.Tumor targeting ligand transferrin is further employed to synthesize Mn304@Trf with tumor microenvironment triggered MR signal amplification for tumor detection with high spatial resolution.The nucleation and growth of Mn304 within Trf molecules was triggered through redox reaction between KMnO4 and Trf inside expansive nanocages in mild condition,resulting in Mn3O4@Trf with precisely regulable size through reaction time control.Owing to lattice defect,Mn304@Trf exhibited enhanced Mn2+release behavior in acidic environment,generating higher longitudinal relaxivity(r1)of 3.2 mM"1 s-1 for enhanced MR imaging.The r1 can be further increased to 7.2 mM-1 s-1 with co-incubation of GSH and acidic PBS,exhibiting dually pH/reduction-responsive MR enhancement.Moreover,Mn3O4@Trf showed prolonged blood circulation time with t1/2βof 4.5 h,specific accumulation in TfRl receptor over-expressed tumor(HT-29)in a size-dependent manner as well as deep tumor penetration,leading to enhanced MR imaging capacity with the signal-to-noise(S/N)contrast increased to 200～240%in both subcutaneous and orthotopic tumor model.With additional injection of acidic PBS and GSH directly into tumors,the S/N contrast could be further improved to 400%,thereby generating precise tumor diagnosis with high spatial resolution.Therefore,Mn3O4@Trf with dually pH/reduction-responsive MR signals can be applied for tumor spatial imaging with enhanced MR contrast.In summary,multifunctional nanoparticles synthesized through an optimized biomineralization process in protein nanoreactors are developed for applications in multimodal imaging and photothermal tumor ablation.The biomimetic synthesized nanoparticles via the facile and universal protein nanoreactor method would have promising potentials in the field of cancer theranostics.更多还原