【作者】 杨峰；

【导师】 高申；

【作者基本信息】 第二军医大学 ， 药剂学， 2013， 博士

【摘要】 目前的疫苗接种方式主要为皮下或肌肉注射，存在顺应性差、免疫效率低、易产生耐受等问题，因此顺应性更好、更方便、高效的新型疫苗接种方法和递送系统研究一直是免疫学和药剂学研究的热点。经皮免疫(Transcutaneous immunization，TCI)是近年发展起来的一种新型免疫接种策略，它是将抗原和佐剂局部应用于皮肤而诱导产生全身免疫反应的新方法，可以克服目前注射接种方式存在的缺陷，有着广阔的应用前景。目前限制其应用的主要障碍是皮肤表层角质层的屏障作用，使疫苗的大分子抗原物质难以通过皮肤表层而进入富含抗原提呈细胞（Antigen-presenting cells，APCs）的活性表皮层和真皮层。微针（Microneedles）是指通过微电子机械工艺技术(MEMS)制作的，尺寸在微米级，长度100μm以上呈针状的复杂结构，材料可以为硅、聚合物、金属等。微针作为药物传输系统可有效克服角质层的屏障作用，同时具有精确、无痛、高效、便利的优点，已成为大分子抗原分子的透皮递送的最有效手段。在皮肤活性表皮层和真皮层内分布着大量抗原提呈细胞，主要是树突状细胞（Dendritic cells，DCs）及其未成熟亚型郎格汉氏细胞（Langerhans cells，LCs），它们均可有效的摄取、处理和提呈外来抗原，并在迁移到引流淋巴结后有效激发抗原特异性的细胞和体液免疫应答。因此活性表皮层和真皮层被认为是疫苗接种的最佳部位之一。利用微针给药系统将抗原递送到活性表皮层和真皮层，由其中的抗原提呈细胞捕获处理抗原，发挥抗原提呈作用，可激发有效的全身免疫应答。本研究以鸡卵清白蛋白（Ovalbumin，OVA）为模型抗原，成功构建了可主动靶向DCs的免疫脂质体疫苗递送系统，结合微针透皮给药机制和促进大分子抗原透皮效果研究，开展其作为经皮主动靶向疫苗递送系统体内外特异性靶向DCs能力及机制、免疫脂质体对DCs功能影响及其机制、经皮免疫效果及机制等研究，分析其免疫应答的特点，初步阐明其免疫应答的机制和途径。第一部分中我们采用薄膜分散法成功的制备出了包载OVA抗原的脂质体，再利用巯基化试剂将抗DCs表面特异性CD11c分子的单克隆抗体巯基化，使其与脂质体膜材中的马来酰亚胺基团反应共价连接于脂质体表面，成功的制备纯化了包载OVA抗原主动靶向DCs的免疫脂质体，实验中同时制备了异硫氰酸荧光素标记的OVA蛋白双荧光标记免疫脂质体用于后续靶向性和抗原摄取实验研究。对制备得到免疫脂质体的粒径、形貌、包封率、Zeta电位等进行了表征，测定了单克隆抗体与脂质体的连接率和免疫脂质体表面连接单抗的DCs结合活性。结果表明脂质体表面光滑，粒径分布均匀，平均粒径在145-160nm范围，与未结合单克隆抗体的脂质体相比较，靶向免疫脂质体的平均粒径稍有增大，Zeta电位变化较小，测定得到CD11c单克隆抗体与脂质体连接率为(77.6±3.2)%，流式细胞术检测证明了CD11c单克隆抗体连接到脂质体表面后，仍然保持了与CD11c分子的特异结合活性，激光共聚焦实验也证明了DCs靶向免疫脂质体的成功制备。第二部分探讨了微针作用促进大分子抗原载体透皮作用的效果与机制。研究发现微针处理可大大提高免疫脂质体及其包裹大分子抗原的经皮透过量和皮肤滞留量，表明微针透皮给药方法具有较好的经皮促渗效果。激光共聚焦显微镜对微针处理皮肤后双荧光标记免疫脂质体透皮效果的观察表明，免疫脂质体及其包裹大分子抗原可通过微针阵列在皮肤表面穿刺出的微孔通道进入皮肤内，并可在皮肤近200μm深度观察到显著的荧光，说明免疫脂质体及其包裹的抗原蛋白可有效的被递送进入皮肤表皮层和真皮层上部。而实验中也观察到皮肤毛囊也可作为免疫脂质体及包载抗原进入皮肤的通道。通过台盼蓝染色、HE染色和扫描电镜等方法直观、清晰的表征了使用的金属微针阵列穿刺后皮肤的角质层和真皮层的药物传输通道的形成和微观物理形态改变，印证了微针阵列促进药物透皮的机制。还利用经皮水份流失TEWL测定说明了微针阵列穿刺后表皮透过能力的变化，发现无论是小鼠或人体皮肤，微针处理均可明显增加其TEWL值，表明微针穿刺一定程度破坏了皮肤的表面结构，增加了皮肤的通透性。但同时微针穿刺的皮肤刺激反应评价的结果也说明微针透皮具有较好的使用安全性。第三部分中我们采用流式细胞术和激光共聚焦显微镜观察的方法证明了制备的DCs靶向免疫脂质体体外对DCs具有特异靶向性。同时该主动靶向的免疫脂质体可有效促进DCs表型的成熟，使细胞表面Iab、CD40、CD80、CD86等表面分子的表达提高。同时免疫脂质体还可有效促进DCs对抗原的摄取和吞噬，并刺激DCs分泌功能性细胞因子TNF-α，说明免疫脂质体可有效促进DCs的功能成熟，有利于其免疫提呈功能的发挥。研究结果还表明DCs靶向免疫脂质体促进抗原摄取的机制是免疫脂质体的单克隆抗体与其配体的特异性结合。同时在体实验中观察到DCs可将摄取的免疫脂质体及其包载抗原有效的递送至引流淋巴结和脾脏等次级淋巴器官。第四部分我们以表面特异表达OVA抗原的EG7-OVA肿瘤细胞为模型，通过免疫后小鼠的荷瘤实验，观察了微针作用后DCs靶向免疫脂质体经皮免疫组小鼠的肿瘤生长情况和存活率，结果表明，与阴性对照组相比，DCs靶向免疫脂质体经皮免疫组小鼠的肿瘤生长较缓慢，生存期明显延长。对小鼠体内OVA特异性抗体的产生的测定表明，DCs靶向免疫脂质体经皮免疫有效激发了小鼠体内抗原特异性的体液免疫应答。更有意义的是，研究中发现该免疫脂质体微针经皮免疫在小鼠体内产生了更高的IgG1/IgG2a抗体滴度比，说明该免疫方法激发的免疫应答是Th1偏向的，更有利于细胞免疫的产生。通过进一步检测DCs靶向免疫脂质体经皮免疫在小鼠体内激发细胞免疫的情况，发现该免疫脂质体抗原载体系统微针经皮免疫后，可以强烈活化抗原特异性的T细胞，诱导Th1型细胞因子IFN-γ的产生，产生OVA抗原特异性的CTL，充分激发了细胞免疫应答。与之对应，霍乱毒素CT作为经皮佐剂或直接皮下注射免疫虽然同样可激发Th1型的应答，但其作用机制与DCs靶向免疫脂质体单独经皮免疫可能有一定的不同。本研究在微针给药系统基础上，利用单克隆抗体的主动靶向性，采用化学交联技术将针对DCs表面分子的单克隆抗体连接到脂质体表面形成主动靶向抗原提呈细胞的免疫脂质体，可有效的将免疫脂质体所包裹的大分子抗原特异高效地转运到树突状细胞，增加其对抗原分子的内吞、摄取和提呈，并激发了更有效的免疫应答。该研究结合了微针和免疫脂质体两种技术，发挥微针皮层定位和免疫脂质体特异细胞靶向的特性，实现微针到活性表皮层及单克隆抗体到抗原提呈细胞的二次定位，达到主动性的双重靶向功效，有望成为一种有效的免疫新方法，在抗病毒、抗肿瘤领域有较好的应用前景。更多还原

【Abstract】 Because of poor adaptability, low immune efficiency and easily-generatingdrug-resistance of present approachs for vaccination, it has always been the hot spot tosearch for good adaptative, more convenient novel vaccination and deliver system inimmunology and pharmaceutics. Transcutaneous immunization (TCI) is a novel strategyfor immune vaccination in recent years, which applies antigen and adjuvant locally to skinto reduce systemic immune response. This new method could overcome the drawbacks ofpresent injection vaccination, therefore becomes a promising application. The currentobstacle is the skin barrier function of cuticle, which makes it difficult for macro-molecularantigen to penetrate skin surface and enter the active epidermis layer which is rich inAntigen-presenting cells，APCs. Microneedles are complicated needlelike structures whichare machined by Micro-electro mechanical Systems(MEMS), size in micro-metre andmake from silicon, polymers or metals. As drug delivery system, microneedles couldeffectively overcome the obstacle of the barrier function of cuticle, at the same time,because of its accuracy, painless, high efficiency and convenience, microneedles becomesthe most efficient method in transdermal delivery.There are numerous Antigen-presenting cells locating in the active epidermis layer ofskin. These cells mainly include Dendritic cells(DCs) and immature Langerhans cells(LCs). Both cells can effectively uptake, deal with and present foreign antigens,furthermore, they effectively trigger the antigen-specific cells and humoral immuneresponse after moving into draining lymph node, as a consequence, active epidermis layeris taken as the best position for vaccination. Microneedles can be initially used in activetargeting to active epidermis layer and deliver antigen to active epidermis layer whereAPCs capture antigen and trigger efficient systemic immune response.In this study, immunoliposome delivery system active-targeting to DCs is successfullyconstructed with Ovalbumin (OVA) as the antigen model. Combined with the mechanismof microneedles transdermal drug delivering and the study on enhancing macromoleculetransdermal efficiency, experiments are carried out on its targeting capacity and mechanism, immunoliposome’s effect on DCs’ function and mechanism, the effect and mechanism ofthe immunoliposome to DCs. Based on all above, the characteristics of the immuneresponse is analyzed to search out the mechanism and route to enhance the immuneresponse and practical prospect of this project.Part I:we apply film dispersion method and extrusion method to prepare OVA loadedimmunoliposome, then thiolate the immunoliposome to make it react with the maleimideof the lipospme material to form covalent bond which links CD11c monoclone antibody tothe surface of the liposome, successfully construct and purify OVA loadedimmunoliposome with active targeting to DCs. Furthermore, the immunoliposome ischaracterized with particle size, morphology, encapsulation and zeta potential. The resultsindicate, the liposome has a smooth surface, homogeneous in particle size, with the rangefrom145-160nm. Compared with OVA-liposomes, CD11c-OVA-imliposomes haveincreased particle size, with the zeta potencial (-31.2±1.7)mv. Flow cytometry data indicatethe specific targeting to CD11c is preserved after CD11s monoclonal antibody conjugatingto the surface of the immunoliposomes.Part II: we probe into the effects and mechanism of microneedles’ improvement onmacromolecular antigen’s transdermal behavior. We find out the amount ofimmunoliposomes and the macromolecule and antigen loaded permeating and detainedgreatly increased. That means a good enhancement on trandemal permeation. Marked by2fluorescence and under laser confocal microscopy, observations indicate,immunoliposomes can get into skin through the microtunnels made by microneedle matrix.At a depth of200μm, strong fluorescence is observed, which implies, immunoliposomesand the macromolecule antigen loaded in it are effectively delivered to the skin epidermislayer and the top of the dermis layer. Also in the experiment it is found that pores of skincan be the tunnel to deliver the immunoliposomes.We observe the formation of the drugdelivery channel and the changes of skin microstructure with SEM and dyeing the skinwith trypan blue and HE. TEWL(Transepidermal water loss) data show treating withmicodeedles can increase the TEWL value; compared with the untreated group, treatingwith microneedle matrix greatly increases the transdermal and retention amount of OVA loaded antigen in immunoliposome and shows outstanding transdermal permeability.Meanwhile, results of the evaluation of the irratation response indicatemicroneedletransdermal has good safety in practice.Part III:CD11c-OVA-immunoliposomes’ specific targeting to DCs is proved by flowcytometer and confocal microscopy. We discover the active targeting immunoliposomescan effectively enhance the expression of cell maturity-related molecule such as Iab、CD40、CD80、CD86, therefore promote the mature of DCs to benefit the immunepresenting function.Meanwhile, immunoliposomes can effectively enhance DCs’ uptakeand endocytosis of antigen, also stimulate DCs to secrets functional cell factors TNF-α,which imply immunoliposomes effectively improve the function maturity of the DCs, andbenefit for exerting the immune presenting function. We also find out, the mechanism ofDCs targeting to immunoliposomes and enhancement of antigen uptake is the specificconjugation of monoclonal antibody of immunoliposomes and its ligand. Additionally, wehave observed that DCs can effectively deliver the uptake immunoliposomes and theantigen loaded to draining lymph node, spleen and other secondary lymphoid organ.Part IV: we take advantage of the EG7-OVA cell model whose surface specificallyexpresses OVA，to measure the tumor growth and mice survival rates after applyingmicroneedles loading CD11c-OVA-imliposomes to tumor-bearing mice, herein vivoeffect and immune response character are evaluated. Compared with negative groups,tumors of those mice treated with microneedles loading CD11c-OVA-imliposomes growmuch slower, survival time is prolonged.Determination of specific anti-OVA antibody inmice indicates transdermal vaccination of CD11c-OVA-imliposomes effectively trigger themice’s antigen specific immune response in vivo. More significantly, in the experimentswe discover a higher antibody’s titer ratio of IgG1/IgG2a, which indicates the immuneresponse triggered in this way is Th1polarized, which is beneficial to generating cellularimmune.Further observation on the immunoliposomes targeting to DCs stimulating cellularimmunity of mice in vivo shows, after microneedle transcutaneous immunization, the immunoliposomes antigen system greatly activates the T cell with antigenic specificity,induces Th1cell factor to secrete IFN-γ and generate CTL with OVA antigenic specificityto fully stimulate cellular immune response. Correspondently，although cholera toxin virusas transdermal adjuvant or directly subcutaneous injection can trigger Th1type response,but its mechanism may be differs from immunization of immunoliposomes targeting toDCs transcutaneous immunization alone.Based on microneedle drug delivery system, making use of monoclonal antibody’sactive targeting and adopting chemical crosslinking technique, monoclonal antibodies fromthe surface of DCs are linked to the surface of liposome to form immune liposome whichcan active target to antigen presenting cells. In this way, macromolecules loaded inimmune liposome can be effectively delivered to DCs, then enhance the capacity ofendocytosis, uptake and presenting the antigen molecules to trigger more effective immuneresponse. In this study, microneedles and immunoliposome are combined to exert theaccurate position and immunoliposome’s targeting to specific cells. Two fold localizations(from microneedles to active epidermis layer and from monoclonal antibody to antigenpresenting cells) are achieved, therefore it is a totally new promising and effective immunemethod and route.更多还原