用于鼻咽癌高效靶向诊疗的多肽—脂质纳米探针的研制
发布时间：2018-11-22 12:49
【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。
[Abstract]:Nasopharyngeal carcinoma (NPC) is a highly malignant tumor, such as China's South and Southeast Asia, which is characterized by the hidden and easy occurrence of distant metastasis. The distant metastasis is one of the main causes of the failure of the treatment of nasopharyngeal carcinoma. Currently, for locally advanced nasopharyngeal carcinoma, combined chemotherapy based on cisplatin is mainly used, but more than 30% of patients have failed due to distant metastasis, and the 5-year overall survival rate is only 25-30%. The combined chemotherapy of anti-EGFR monoclonal antibody, Cetuximab, can improve the overall survival rate of patients with recurrent or metastatic nasopharyngeal carcinoma to a certain extent, but there are significant toxic and side effects caused by the reaction of human anti-mouse antibody (HAMA). Compared with the small-molecule chemotherapy medicine, the drug-targeted transportation based on the nano-carrier has the advantages of prolonging the in-vivo circulation period of the medicine, improving the utilization rate of the medicine, increasing the intracellular uptake capacity of the medicine, and the like, and reducing the toxic and side effect of the medicine to a certain extent. Therefore, it is of great clinical value to develop high-efficiency target nano-drugs for nasopharyngeal carcinoma and to treat metastatic nasopharyngeal carcinoma. The results of this study are as follows: (1) The invention provides an eight-valent polypeptide nano-fluorescent probe, which can be used for the body evaluation of a target polypeptide, thereby identifying a target polypeptide (TT) with high specificity for nasopharyngeal carcinoma, P). The specific method is that the target polypeptide gene sequence is coupled to the carbon and nitrogen ends of the tetramer far red fluorescent protein (tfRFP) gene sequence, and an eight-valence polypeptide nano-fluorescent probe Octa-F is formed by self-assembly by the tetramerization effect after the tfRFP is mature. NP. It is confirmed by the body fluorescence imaging that it has high specific targeting ability, enhanced enrichment ability and high contrast tumor imaging ability for the 5-8F subcutaneous tumor of the nasopharyngeal carcinoma, so the targeting of the polypeptide can be quickly and sensitively evaluated by using the Octa-FNP. Ability. This method is used to identify a highly specific target polypeptide LtWY (TTP) for nasopharyngeal carcinoma, and it is also found that it can accumulate in the tumor group with high efficiency by carrying out in-vivo radiation imaging of the Octa-FNP labeled with 125I. The invention relates to a novel polypeptide R4 with a synergistic targeting and enhancing capability and an anti-killing capability of nasopharyngeal carcinoma, F-TTP. The specific method is to hydrophilic the identified TTP polypeptide and the amphipathic helical polypeptide R4F. The results of the fluorescence microscopy and flow cytometry confirmed that the FITC-labeled R4F-TTP was taken by the 5-8F cell as a 3. 1-fold of the FITC-labeled TTP, which was the FITC-labeled R4F. The results show that the R4F-TTP polypeptide has a synergistic effect on the 5-8F cells. The results showed that R4F-TTP had the choice of 5-8F cells. (3) successfully developed a highly selective targeting ability and good therapeutic effect on the 5-8F tumor of nasopharyngeal carcinoma. The results of transmission electron microscopy and nano-particle size show that the NP-TTP is a kind of nano-particle with a diameter of about 11n. The results of flow cytometry show that the amount of NP-TTP in the 5-8F cells is 20. 5 times that of the NP (not including the polypeptide TTP), which is the random sequence of the NP-scrTTP (sctp). The 3. 0-fold. NP-TTP of the polypeptide showed enhanced specific uptake of the nasopharyngeal carcinoma cells, suggesting that the polypeptide TTP was presented. The results of the overall fluorescence imaging show that the near-infrared fluorescent dye DiR-BOA-labeled NP-TTP can be efficiently accumulated in the 5-8F tumor of the nasopharyngeal carcinoma and effectively diffuse into the whole tumor tissue, and the fluorescence signal of the DiR-BOA is between the tumor tissue and the normal organs such as the liver and the kidney. With very high contrast, the growth inhibition rate of NP-TTP on 5-8F tumor was 88-7%, and the 5-8F-mRFP metastatic tumor was also used for 5-8F-mRFP. The inhibitory effect of NP-TTP on the growth of the tumor is proved to be related to the induction of apoptosis and self-adaptation. It is closely related to the formation of the autophagy. When the NP-TTP is loaded with the curcumin, the ability to inhibit the metastasis of the 5-8F-mRFP tumor of the nasopharyngeal carcinoma is shown to be significantly prolonged. In conclusion, based on the tetramer fluorescent protein, an eight-valent polypeptide nano-fluorescent probe was developed to identify a nasopharyngeal carcinoma (NPC). the hybrid polypeptide R4F-TTP formed by coupling the TTP with the amphipathic helical polypeptide R4F not only has the capability of cooperatively enhancing the target uptake and the selective killing of the nasopharyngeal carcinoma, but also can control the lipid The formation and the function of the quality nano-particles. The lipid nanoparticles formed by the R4F-TTP have the capability of high-contrast target imaging of the nasopharyngeal carcinoma and the extremely low targeted therapeutic effects of toxic and side effects. The present study provides for targeted and therapeutic polypeptides in vivo evaluation, targeted imaging and diagnosis of tumors, and
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R739.63
本文编号：2349422
[Abstract]:Nasopharyngeal carcinoma (NPC) is a highly malignant tumor, such as China's South and Southeast Asia, which is characterized by the hidden and easy occurrence of distant metastasis. The distant metastasis is one of the main causes of the failure of the treatment of nasopharyngeal carcinoma. Currently, for locally advanced nasopharyngeal carcinoma, combined chemotherapy based on cisplatin is mainly used, but more than 30% of patients have failed due to distant metastasis, and the 5-year overall survival rate is only 25-30%. The combined chemotherapy of anti-EGFR monoclonal antibody, Cetuximab, can improve the overall survival rate of patients with recurrent or metastatic nasopharyngeal carcinoma to a certain extent, but there are significant toxic and side effects caused by the reaction of human anti-mouse antibody (HAMA). Compared with the small-molecule chemotherapy medicine, the drug-targeted transportation based on the nano-carrier has the advantages of prolonging the in-vivo circulation period of the medicine, improving the utilization rate of the medicine, increasing the intracellular uptake capacity of the medicine, and the like, and reducing the toxic and side effect of the medicine to a certain extent. Therefore, it is of great clinical value to develop high-efficiency target nano-drugs for nasopharyngeal carcinoma and to treat metastatic nasopharyngeal carcinoma. The results of this study are as follows: (1) The invention provides an eight-valent polypeptide nano-fluorescent probe, which can be used for the body evaluation of a target polypeptide, thereby identifying a target polypeptide (TT) with high specificity for nasopharyngeal carcinoma, P). The specific method is that the target polypeptide gene sequence is coupled to the carbon and nitrogen ends of the tetramer far red fluorescent protein (tfRFP) gene sequence, and an eight-valence polypeptide nano-fluorescent probe Octa-F is formed by self-assembly by the tetramerization effect after the tfRFP is mature. NP. It is confirmed by the body fluorescence imaging that it has high specific targeting ability, enhanced enrichment ability and high contrast tumor imaging ability for the 5-8F subcutaneous tumor of the nasopharyngeal carcinoma, so the targeting of the polypeptide can be quickly and sensitively evaluated by using the Octa-FNP. Ability. This method is used to identify a highly specific target polypeptide LtWY (TTP) for nasopharyngeal carcinoma, and it is also found that it can accumulate in the tumor group with high efficiency by carrying out in-vivo radiation imaging of the Octa-FNP labeled with 125I. The invention relates to a novel polypeptide R4 with a synergistic targeting and enhancing capability and an anti-killing capability of nasopharyngeal carcinoma, F-TTP. The specific method is to hydrophilic the identified TTP polypeptide and the amphipathic helical polypeptide R4F. The results of the fluorescence microscopy and flow cytometry confirmed that the FITC-labeled R4F-TTP was taken by the 5-8F cell as a 3. 1-fold of the FITC-labeled TTP, which was the FITC-labeled R4F. The results show that the R4F-TTP polypeptide has a synergistic effect on the 5-8F cells. The results showed that R4F-TTP had the choice of 5-8F cells. (3) successfully developed a highly selective targeting ability and good therapeutic effect on the 5-8F tumor of nasopharyngeal carcinoma. The results of transmission electron microscopy and nano-particle size show that the NP-TTP is a kind of nano-particle with a diameter of about 11n. The results of flow cytometry show that the amount of NP-TTP in the 5-8F cells is 20. 5 times that of the NP (not including the polypeptide TTP), which is the random sequence of the NP-scrTTP (sctp). The 3. 0-fold. NP-TTP of the polypeptide showed enhanced specific uptake of the nasopharyngeal carcinoma cells, suggesting that the polypeptide TTP was presented. The results of the overall fluorescence imaging show that the near-infrared fluorescent dye DiR-BOA-labeled NP-TTP can be efficiently accumulated in the 5-8F tumor of the nasopharyngeal carcinoma and effectively diffuse into the whole tumor tissue, and the fluorescence signal of the DiR-BOA is between the tumor tissue and the normal organs such as the liver and the kidney. With very high contrast, the growth inhibition rate of NP-TTP on 5-8F tumor was 88-7%, and the 5-8F-mRFP metastatic tumor was also used for 5-8F-mRFP. The inhibitory effect of NP-TTP on the growth of the tumor is proved to be related to the induction of apoptosis and self-adaptation. It is closely related to the formation of the autophagy. When the NP-TTP is loaded with the curcumin, the ability to inhibit the metastasis of the 5-8F-mRFP tumor of the nasopharyngeal carcinoma is shown to be significantly prolonged. In conclusion, based on the tetramer fluorescent protein, an eight-valent polypeptide nano-fluorescent probe was developed to identify a nasopharyngeal carcinoma (NPC). the hybrid polypeptide R4F-TTP formed by coupling the TTP with the amphipathic helical polypeptide R4F not only has the capability of cooperatively enhancing the target uptake and the selective killing of the nasopharyngeal carcinoma, but also can control the lipid The formation and the function of the quality nano-particles. The lipid nanoparticles formed by the R4F-TTP have the capability of high-contrast target imaging of the nasopharyngeal carcinoma and the extremely low targeted therapeutic effects of toxic and side effects. The present study provides for targeted and therapeutic polypeptides in vivo evaluation, targeted imaging and diagnosis of tumors, and
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R739.63
【参考文献】
相关期刊论文 前1条
1 Jill Wykosky;Tim Fenton;Frank Furnari;Webster K. Cavenee;;Therapeutic targeting of epidermal growth factor receptor in human cancer: successes and limitations[J];癌症;2011年01期
本文编号：2349422
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