目的：结直肠癌是一种常见的恶性肿瘤，是威胁人类健康的重大疾病，其发病率和死亡率在所有恶性肿瘤中均位列第三。由于结肠癌的症状比较隐匿，很多患者确诊时已是中晚期。化疗是中晚期结肠癌患者的主要治疗方式，但传统的化疗方案常常伴有明显的毒副作用，严重降低了患者的生存质量。靶向治疗可选择性地将细胞毒性药物递送至肿瘤细胞，而减少对正常组织细胞的损伤，因此在改善化疗疗效、减轻药物的不良反应方面具有重要的应用前景。靶向治疗通常由靶向药物递送系统（Targeted drug delivery system, TDDS）来实现，TDDS由三部分组成：靶向肿瘤的配体、纳米药物载体、细胞毒性药物。

核仁蛋白通常存在于细胞核内，但在很多肿瘤细胞膜表面也有高表达（包括结肠癌细胞），因此被认为是具有重大应用潜能的肿瘤靶向治疗靶标。核酸适配体（Aptamer, Apt）是一类单链寡核苷酸分子，能形成复杂的三维结构，可与相关靶标特异性地结合，并具有低免疫原性、合成修饰简单、成本低廉、分子量小及肿瘤组织穿透力高等优点，因此是重要的新型肿瘤靶向配体。白蛋白是一种内源性蛋白，无免疫原性，可用于构建生物相容性良好的纳米药物载体。多西紫杉醇（Docetaxel, DTX）具有广谱抗癌作用，临床上可用于多种肿瘤的治疗，如宫颈癌、非小细胞肺癌、转移性乳腺癌等。

迄今为止，将靶向核仁蛋白的含多西紫杉醇的白蛋白纳米粒用于治疗结肠癌，尚未见文献报道。AS1411是一种靶向核仁蛋白的核酸适配体。本研究计划构建AS1411介导的包载多西紫杉醇白蛋白纳米颗粒，用于结肠癌治疗，并在细胞系和体内实验中评估其对结肠癌的治疗效果。所选材料均曾被FDA批准用于人体疾病治疗，具有较好的临床应用前景。

方法：将5′端用巯基修饰的核酸适配体通过偶联剂SMCC与白蛋白上的氨基相连。通过自组装法，将多西紫杉醇负载于AS1411核酸适配体功能化的白蛋白中，构成TDDS（Apt-NPs-DTX）。利用透射电镜观察纳米粒的形态。通过动态光散射法检测纳米粒粒径、多分散系数（PDI）和zeta电位。通过高效液相色谱法检测包封率、载药率及药物释放曲线。利用琼脂糖凝胶电泳实验，检测核酸适配体是否成功连接到白蛋白纳米粒上。将白蛋白包载荧光染料，利用荧光显微镜、流式细胞分析技术和激光共聚焦显微镜评估纳米颗粒对CT26结肠癌细胞的靶向性。通过体外杀伤实验检测Apt-NPs-DTX对结肠癌细胞和对照细胞的毒性。通过体内动物实验评估Apt-NPs-DTX的抗肿瘤疗效、对生存期的影响以及毒副作用。

结果：所构建的Apt-NPs-DTX，其平均粒径为62 nm，zeta电位为-31.2 mV。DTX从白蛋白纳米颗粒的释放具有典型的缓释特性。相对于对照细胞，细胞表面高表达核仁蛋白的CT26结肠癌细胞能优先摄取AS1411介导的纳米颗粒。体外细胞杀伤研究表明，Apt-NPs-DTX显著增强了对CT26结肠癌细胞的杀伤力。动物实验表明，与不具靶向性的载药纳米颗粒相比，Apt-NPs-DTX显著提升了抗肿瘤疗效，延长了荷瘤小鼠的存活时间，但没有进一步增加毒副作用。

结论：本研究构建了一种新型的AS1411修饰的包载多西紫杉醇的白蛋白纳米粒子，用于靶向治疗结肠癌。实验结果表明，Apt-NPs-DTX在小鼠体内能显著增强抗肿瘤疗效，在结肠癌的靶向治疗方面具有一定的应用潜能。

Objective:  Colon cancer is a common malignant tumor that poses a great threat to human health. The incidence and mortality of colorectal cancer rank third among all malignant tumors. Because the symptoms of colon cancer are relatively insidious, many patients are already in the middle and advanced stages when they are diagnosed. Chemotherapy is the main treatment for patients with advanced colon cancer. However, traditional chemotherapy regimens are often accompanied by obvious side effects, which seriously reduce the quality of life of patients. Targeted therapy can selectively deliver cytotoxic drugs to tumor cells while reducing damage to normal tissue. Therefore, it has important application prospects in improving the efficacy of chemotherapy and reducing serious adverse effect caused by the drugs. Targeted therapy usually requires a targeted drug delivery system (TDDS), which often includes three components: tumor-targeting ligands, nanocarriers, and cytotoxic drugs.

Nucleolin is usually found in the nucleus, but is also highly expressed on the cell surface of many tumors, including colon cancer. Therefore, nucleolin is a potentially important therapeutic target for targeted tumor therapy. Aptamer is a type of ligands made of single-stranded oligonucleotides. By forming complicated 3D structure, aptamer can bind to molecular targets with high specificity and affinity. Aptamer also has low immunogenicity, low production cost, and small molecular weight, with the advantages of good tumor tissue penetration. As a result, aptamer is regarded as a tumor-targeting ligand with great application potential. Albumin is an endogenous protein with low immunogenicity, and an excellent component for making biocompatible nanoparticles. Docetaxel (DTX) is a broad-spectrum anticancer drug, which is clinically used for treatment of a wide variety of tumors, such as cervical cancer, non-small cell lung cancer, and metastatic breast cancer.

So far, however, aptamer-guided and DTX-loaded albumin nanoparticle has not been evaluated for colon cancer treatment. AS1411 is a nucleolin-binding aptamer that has been approved for clinical trials. In this study, we plan to build a new TDDS with DTX-loaded albumin NP that is AS1411-modified (Apt-NPs-DTX). We plan to study Apt-NPs-DTX’s therapeutic effect on colon cancer with in vitro and in vivo experiments. Of note, AS1411, albumin, and other components of Apt-NPs-DTX have been approved by US FDA for human applications.

Methods: The 5' end of the AS1411 aptamer was modified with a sulfhydryl group,  and was connected to the amino group on the albumin through the coupling agent SMCC. Through the self-assembly method, docetaxel was loaded into the albumin functionalized with AS1411 to form TDDS (Apt-NPs-DTX). The morphology of nanoparticles (NPs) was studied by transmission electron microscope. The particle size, polydispersity coefficient (PDI), and zeta potential of nanoparticles were evaluated by dynamic light scattering method. The encapsulation rate, drug loading rate, and drug release curve were studied by high performance liquid chromatography. Agarose gel electrophoresis was used to evaluate whether the aptamer was connected to the albumin NPs. The albumin NPs was loaded with fluorescent dyes, and flow cytometry and laser confocal microscopy were used to evaluate the targeted delivery of the load to CT26 colon cancer cells. The cellular anticancer efficacy of Apt-NPs-DTX was assayed by in vitro cytotoxicity experiments. In vivo animal experiments were conducted to evaluate the antitumor efficacy of Apt-NPs-DTX, its impact on survival, and side effects.

Results: Apt-NPs-DTX has an average diameter of 62 nm and a zeta potential of -31.2 mV. DTX is released from albumin nanoparticles with a typical sustained-release profile. Compared with control cells, nucleolin-positive CT26 colon cancer cells preferentially ingest aptamer-mediated NPs. In vitro cytotoxicity studies shows that Apt-NPs-DTX significantly enhances the cytotoxicity against CT26 colon cancer cells. Animal experiments shows that, compared with non-targeted nanoparticles, Apt-NPs-DTX significantly improves the antitumor efficacy and prolonged the survival of tumor-bearing mice, without generating extra adverse effect.

Conclusion: In this study, AS1411-modified and docetaxel-loaded NPs are constructed for targeted treatment of colon cancer. The results show that Apt-NPs-DTX can selectively deliver docetaxel to colon cancer cells in vitro, and significantly enhance the antitumor efficacy in vivo, indicating that the TDDS has application potential in  targeted therapy against colon cancer.