【作者】 王晓雨；

【导师】 曾苏； 唐睿康；

【作者基本信息】 浙江大学 ， 药物化学， 2011， 博士

【摘要】 随着生物学、材料学、医学等学科的发展,病毒不再仅是传统意义上的病原微生物,而逐渐成为在新型材料的构建、载体投递、基因治疗、疫苗开发、生物检测、疾病诊断等方面有着广泛应用的重要生物工具。病毒具有纳米尺寸和结构高度对称等特点、还能大量在生物体内包装生产,能够适用于大规模的功能化修饰。然而目前为止还没有通用性的病毒修饰方法能够完全克服病毒在应用发展中所遇到的困难,例如在对病毒的操作过程中会造成意外感染、而且对病毒的大规模处理耗时很长且十分复杂,此外病毒在生物应用中常受到细胞特异性的限制并且容易在生物体防御机制作用下失活等。因此,我们认为有必要提出一种新的病毒修饰的策略,为其今后生物学和材料学中的应用提供新方法。仿生生物矿化是利用无机材料对有机生命体进行化学生物学修饰的一种理想方法,据此我们提出病毒壳工程概念,通过自组装和生物矿化的方法对病毒进行功能化修饰,构建出具有核壳结构的病毒-材料新型复合体,并结合应用实例对该复合体的理化性质和生物学特性进行了研究,为病毒在投递载体、基因治疗和疫苗开发等方面的应用提供了可行性参考。本文主要内容及学术贡献如下：1以人类黄热病毒疫苗株YF17D为模板,采用层层自主装(LbL)技术获得了操作简便、安全性好的病毒复合物。通过静电作用在病毒表面交替吸附多聚电解质聚烯丙胺盐酸盐(PAH)和聚苯乙烯磺酸钠(PSS)构建核壳结构。通过多聚电解质修饰后,改变了原有病毒的理化性质,能够通过普通离心进行高效富集和分离；多聚电解质的吸附还增加了病毒表面电子密度,这样就可以在非负染色的条件下利用透射显微技术(TEM)直接观察病毒；而且多聚的外壳还增加了病毒表面的物理强度,使病毒颗粒能够在不进行生物固定等条件处理就可以利用扫描电子显微镜(SEM)和原子力显微镜(AFM)进行观察。我们还发现,病毒／聚合物核壳结构具有较好的生物相容性,能够在短时间内被不同种类的细胞内吞从而进入细胞,并且对细胞不产生毒性。同时PAH/PSS外壳能够抑制具有感染性的病毒在细胞内的释放,使得病毒操作的安全性也将得到改善。该方法为以病毒为单元的新型材料的构建提供了新思路。2采用生物矿化的技术在人5型腺病毒(Ad5)颗粒表面引入磷酸钙(CaPi)矿物外壳。该无机外壳能够将病毒表面的蛋白完全掩盖,使得在非生物环境下矿化病毒颗粒能够呈现出与纳米CaPi颗粒相似的理化特性,通过常规方法不能够检测到其中的病毒成分。矿化病毒进入生物体后,由于磷酸钙具有良好的生物相容性,带有磷酸钙外壳的病毒颗粒能够被病毒受体缺陷细胞非特异地内吞,在细胞内矿化外壳则能在内溶酶体酸性的环境下自发降解并释放病毒颗粒使其恢复感染性。通过静脉注射途径,矿化病毒能够进入小鼠血液循环,增强病毒基因在小鼠体内的表达,同时改变并扩大腺病毒的组织嗜性,还能够保护矿化病毒不被体内的抗体中和而失效。这种基于特洛伊木马的病毒生物矿化策略与其他方法比在操作上更为经济、简单,能够克服病毒在应用中的存在的局限性。同时这种方法也改变了人们对病毒及其应用前景的认识。3将Si02纳米颗粒自发组装在Ad5病毒上。病毒表面被SiO2无机相封闭后细胞受体无法识别病毒表面的衣壳蛋白,因此病毒/纳米SiO2复合物不能被细胞内吞。该工作是探索了病毒表面修饰多样化并强调了无机相的重要性。4我们进一步发现病毒矿化技术能够应用于非包膜病毒和包膜病毒,采用生物矿化的方法对有包膜的黄病毒进行修饰,表面经CaPi生物矿化处理的YF17D也发生了相似的理化性质的变化。在细胞水平YF17D外层矿物相可降解并释放病毒颗粒,使其恢复感染性。矿化外壳能够阻止表面抗原与具有表位中和活性的单克隆抗体结合,保护病毒不失活。在动物体内,矿化病毒经由腹腔途径免疫可在小鼠体内产生抗体,其效价高于正常黄热病毒YF17D,证实了矿化处理能够增强病毒的免疫原性。该研究能够为新疫苗剂型的开发提供参考。更多还原

【Abstract】 Viruses have received great attentions in various research fields including biology, material chemistry and medicine sciences. Nowadays, viruses are gradually accepted as potential tools or building blocks for materials fabrications, vector delivery, gene therapy, vaccine development, biological detection device and diagnosis rather than kinds of traditional pathogenic microorganism. Viruses have the characteristics of nano size effects and high symmetric structural properties, etc; they can be scaled up with biological culture, which facilitates the virus engineering and modification in large scale. However, another characteristics of viruses, such as undesirable infections and tedious and time comsuming for scale up. along with inate cellular barriers and nature traps from biological system, result in the inability to engineer viruses as the useful tools and there is no universal strategy to solve these serious problems.A new strategy for virus modification is in keen pursuit for offering new alternatives in both biological and material fields. Biomineralization is now an effective physicochemical tool to engineer living organisms by using non-living materials. Herein, we propose a shellization strategy to fabricate virus core-shell structures using self-assembly and biomineralization technologies, and characterized their physical, chemical and biological properties in combined with practical applications, providing feasible reference for virus applications as delivery vectors, gene therapy and vaccine development for the future.The main contents and academic contributions of this thesis are summarized as follows:1 Using yellow fever vaccine YF17D as template, we develop a facile strategy to fabricate encapsulated virus-polyelectrolyte hybrids using Layer-by-Layer (LbL) method with high efficiency and security. The polyelectrolytes. poly(allylamine) hydrochloride (PAH) and poly(sodium styrene sulfonate (PSS). can adsorb on virus surface altemately through electrostatic interaction to form core-shell structure. The modified viruses have regained different physical and biological properties such as concentration by the normal speed centrifugations and infection recovery, which can be understood by the surface repulsive force variation. They can be observed by transmission electron microscopy (TEM) direactly owing to the increasing of surface electron density by the polyelectrolytes. They can also be examined by scaning electron microscope (SEM) and atomic force microscope (AFM) readily without any biological fixation because of the mechenical strength reinforcement by the chemical compounds. The virus-polyelectrolyte core-shell nanoparticle with good biocompatibility can be rapidly internalized in different kinds of cell lines and have low cytotoxicity. Meanwhile, during the delivery, polyelectrolyte shells inhibit the release of enclosed viruses, which leads to the suppression of infectivity. This polyelectrolyte-based modification provides a new strategy to control the biological security of viruses, which encourages the multiplex applications of viral building blocks.2 Biomineralization treatments are used to confer single virus a mineral envelop. By shielding completely the viral surface protein, the living viruse can be masqueraded as inanimate calcium phosphate (CaPi) nanoparticles and they possess different physical and chemical properties from the native ones, resulting in the undetectable of virus components by using conventional methods. Under intracellular conditions, biocompatibilities of CaPi envelope exploit the similar cellular internalization as the conventional CaPi nanoparticles via a receptor independent endocytosis pathway, and virus release/recovery from mineral phase is induced spontaneously by the demineralization of CaPi inorganic phase under a low-pH condition that exists in endolysosomes after internalization. After intravenous administration, mineralized virus can deliver in circulatory system, transfect encoding genes with enhanced efficiency, expand tropisms of native virus and circumvent the neutralizing antibodies in the pre-immunized system. We suggest that single virus biomineralization is an effective and economic method to provide a "Trojan" strategy for virus to circumvent the limitations of receptors and natural traps, which will change our conventional understandings of viral infection and its multiple applications.3 SiO2 nanoparticle can also be assembled onto the adenovirus surface by non-covalent interactions to form the virus-core/SiO2-shell structures. After such a modification, the virus fails to interact with cellular receptor due to the surface is completely blocked by nano-SiO2, leading to the incapability of cellular internalization. The orderly assembly of nano-SiO2 on virus surface provides the basis for virus surface modification with different materials exploring important roles the inorganic phase playing.4 We confirm that biomineralization treatment is uniform and repeatable. and can be employed for both non-envelope and envelope viruses. Enveloped YF17D is modified by biomineralization technique to facilitate the efficient concentration by regular centrifugation too. During intracellular delivery, mineral phase of mineralized virus is degraded to release infectious virus particle. Mineralized shell can prevent neutralizing of monoclonal antibody to keep virus alive during the delivery. Besides, intraperitoneal injection of the mineralized virus can produce higher titers of antibodies in mice than that of normal YF17D, indicating the immunogenic enhancement of mineral phase in vivo. This study provides a new strategy for developing vaccine delivery system.更多还原