【作者】 骆海明；

【导师】 张智红；

【作者基本信息】 华中科技大学 ， 生物医学工程， 2013， 博士

【摘要】 鼻咽癌是中国南方和东南亚等地域广泛流行的高度恶性肿瘤，具有发病部位隐蔽和易于发生远处转移的特点。远处转移是鼻咽癌治疗失败的主要原因之一。目前对于局部晚期鼻咽癌主要使用以顺铂为基础的联合化疗，但是有30%以上的患者由于发生远处转移而导致治疗失败，其5年总生存率仅为25%-30%。抗EGFR单克隆抗体Cetuximab联合化疗可以一定程度提高复发或转移性鼻咽癌患者的总生存率，但尚存在着人抗鼠抗体（HAMA）反应导致的显著毒副作用问题。与小分子化疗药物相比，基于纳米载体的药物靶向运输具有延长药物的体内循环周期、提高药物的利用率、增加药物的细胞内摄取能力等优点，并在一定程度上降低了药物的毒副作用。因此，研制高效的鼻咽癌靶向纳米药物，对转移性鼻咽癌的治疗具有重要的临床应用价值。本研究取得如下创新结果：（1）发明了一种八价多肽纳米荧光探针，可用于靶向多肽的在体评价，由此鉴定了一条鼻咽癌高特异性的靶向多肽（TTP）。具体方法是，将靶向多肽基因序列偶联到四聚体远红色荧光蛋白（tfRFP）基因序列的碳、氮两端，通过tfRFP成熟后的四聚化效应自组装形成一种八价多肽纳米荧光探针Octa-FNP。在体荧光成像证实，它对鼻咽癌5-8F皮下肿瘤具有高特异性的靶向能力、增强的富集能力和高对比度的肿瘤显像能力，因此利用Octa-FNP可以快速灵敏地评价多肽的靶向能力。本文利用此方法鉴定出一条鼻咽癌高特异性的靶向多肽LTVSPWY（TTP），并通过对125I标记的Octa-FNP进行活体放射成像，同样发现其能够高效蓄积于肿瘤组织内；（2）发明了一种新型的同时具有鼻咽癌协同靶向增强能力和杀伤能力的多肽R4F-TTP。具体方法是将鉴定的TTP多肽与两亲性螺旋多肽R4F亲水端进行偶联。荧光显微成像和流式细胞检测结果均证实，FITC标记的R4F-TTP被5-8F细胞摄取的量是FITC标记的TTP的3.1倍，是FITC标记的R4F的10.4倍。此结果表明R4F-TTP多肽对5-8F细胞确实具有协同增强的靶向能力。碘化丙啶（PI）染色结果证实，R4F-TTP对5-8F细胞还具有选择性杀伤能力。（3）成功研制了一种对鼻咽癌5-8F肿瘤具有高选择性靶向能力和良好治疗效果的超小粒径纳米颗粒。透射电镜和纳米粒度仪结果显示，NP-TTP是一种直径约为11nm的球形纳米颗粒。流式细胞检测结果表明，NP-TTP被5-8F细胞摄取的量是纳米颗粒NP（不含多肽TTP）的20.5倍，是NP-scrTTP（scrTTP为TTP的随机序列多肽）的3.0倍。NP-TTP表现出明显增强的鼻咽癌细胞特异性摄取能力，提示多肽TTP被呈现在纳米颗粒的表面。整体荧光成像结果证实，近红外荧光染料DiR-BOA标记的NP-TTP能够高效蓄积于鼻咽癌5-8F肿瘤内，并有效地扩散到整个肿瘤组织中，DiR-BOA的荧光信号在肿瘤组织与肝、肾等正常脏器之间具有极高的对比度。通过尾静脉连续两次隔天给药，NP-TTP对5-8F肿瘤的生长抑制率为88±7%，对5-8F-mRFP转移性肿瘤也具有明显的抑制效果。NP-TTP对肿瘤生长的这种抑制作用，被证明与其诱导细胞凋亡和自噬体形成密切相关。当NP-TTP同步装载姜黄素后，表现出更强的抑制鼻咽癌5-8F-mRFP肿瘤转移的能力，明显延长了实验小鼠的生存期。综上所述，本文基于四聚体荧光蛋白发明了一种八价多肽纳米荧光探针，鉴定了一条鼻咽癌高特异性的靶向多肽TTP。将TTP与两亲性螺旋多肽R4F偶联所形成的杂交多肽R4F-TTP，不仅具有协同增强鼻咽癌靶向摄取和选择性杀伤的能力，还能控制脂质纳米颗粒形成及其功能。利用R4F-TTP形成的脂质纳米颗粒，具有鼻咽癌高对比度靶向成像的能力和极低毒副作用的靶向治疗效果。本研究为靶向和治疗性多肽的在体评价、肿瘤的靶向成像和诊断以及多策略治疗提供了新方法。更多还原

【Abstract】 Nasopharyngeal carcinoma (NPC) is an epidemic malignant cancer in SouthernChina and Southeast Asia with hidden location and early distant metastasis, which resultsto its treatment failure as one of the main reasons. Cisplatin-based combinationchemotherapy remains the main treatment for locoregional advanced NPC, but over30%of patients fail with local recurrent or distant metastases in the treatment and their5-yearsurvival rate is just25%-30%. Anti-epidermal growth factor receptor (EGFR) monoclonalantibody cetuximab combined with chemotherapy improves the overall survival ofmetastatic NPC patients to some extent but still with side effects induced by humananti-mouse antibody (HAMA) response. Compared with small-molecule chemothera-peutic agents, nanocarrier-based drugs can extend the half-time of drugs in the blood,improve drugs utilization and increase intracellular concentration of drugs but with lowside effects. Thus, it is valuable in clinical applications for developing nanodrugs targetedNPC with high efficiency to treat metastatic NPC. The innovation results in this thesis canbe summarized as follows.1) We develop an octavalent peptide fluorescent nanoprobe (Octa-FNP) which canbe used to evaluate peptides in vivo and utilize it to identify a NPC-targeting peptide TTP.This was accomplished by combining a tetrameric far-red fluorescent protein (tfRFP) as ascaffold with a tumor-targeting peptide to engineer an Octa-FNP. Whole-body imaging invivo demonstrated Octa-FNP could specifically target5-8F subcutaneous tumor withenhanced tumor accumulation and high-contrast tumor imaging which was valuable forassessing the targeting capability of peptides. We used the methods to identify aNPC-targeting peptide LTVSPWY (TTP), which could high efficiently accumulate in thetumor tissue when radioimaging with125I-labeled Octa-FNP.2) We creat a novel NPC-targeting peptide R4F-TTP with synergistic enhancementand selective killing. It was designed by conjugating a tandem NPC-targeted peptide(LTVSPWY) with an amphipathic-helical peptide R4F. Confocal imaging and FACSanalysis demonstrated the uptake of R4F-TTP by5-8F cells after incubated24h is3.1-fold of TTP,10.4-fold of R4F, indicating R4F-TTP exhibit a synergistic effect on enhancing the targeting to5-8F cells. Propidium iodide (PI) staining further demonstratedthe selective killing tumor cells of R4F-TTP.3) We develop a ultra-small nanocarrier for5-8F tumors with high-specific targetingand significant therapeutic effects. Both transmission electron microscopy (TEM) anddynamic light scattering (DLS) revealed NP-TTP with an average size of11nm andspherical structure. FACS analysis demonstrated the uptake of NP-TTP by5-8F cells afterincubated3h is20.5-fold of TTP,3.0-fold of R4F-scrTTP (scrTTP is the scramble peptideof TTP). The enhanced specific uptake of NP-TTP by5-8F cells suggested that targetingpeptide TTP was presented on the surface of the NP-TTP. Whole-body imagingdemonstrated NP-TTP loaded with near infrared fluorescent dyes DiR-BOA could beefficiently accumulated in the5-8F-mRFP tumor and diffused into entire tumor tissue,which resulted in extremely high contrast between the tumor tissue and normal tissues,such as liver and kidney. When administered twice every other day by post-injection,NP-TTP achieved88±7%inhibition rate in subcutaneous growth of5-8F tumors andsignificant higher survival rate when compared with PBS. The inhibition of tumor growthwas demonstrated to be related with the induction of apoptosis and autophagy formationby NP-TTP. When synchronized with curcumin, NP-TTP showed a strong preventiveeffect on5-8F metastasis.In sumarry, we invented an octavalent peptide fluorescent nanoprobe based ontetrameric far-red fluorescent protein and used it to identify a target-specific peptide TTPfor NPC. Conjugating the TTP peptide with an amphipathic-helical peptide R4F createda novel NPC-targeting peptide R4F-TTP, which not only synergistically enhance thetarget-specific cellular upatke by5-8F cells and their selective killing, but only can controlthe formation of lipid nanoparticles and their function. The novel lipid nanoparticle couldachieve high-contrast imaging in vivo and therapeutic effects for5-8F tumors. This studyprovides new methods for the evaluation in vivo of therapeutic and targeted peptides,tumor-targeted imaging for diagnosis and multi-targeted therapies for cancer treatment.更多还原