桦木酸 (BA) 因其对各种癌症的抗增殖和线粒体途径介导的细胞凋亡诱导作用而得到了深入研究。然而，其溶解度差和脱靶活性限制了其在临床试验中的应用。此外，桦木酸类似物在肿瘤微环境（TME）中的免疫调节作用在很大程度上尚不清楚。在这里，我们设计了一种潜在的结直肠癌 (CRC) 纳米疗法，其中含有一种名为 2c 的铅桦木酸类似物，带有通过连接基连接到 BA 上的 1,2,3-三唑部分，发现比 BA 更有效地抑制 CRC细胞系，并被选在这里进行这项研究。上皮细胞粘附分子 (EpCAM) 在 CRC 细胞膜上高度过表达。折叠成 3D 定义的结构的单链短寡核苷酸序列适体 (Apt) 可用作与靶蛋白特异性结合的靶向配体。 EpCAM 靶向适体设计用于将适体缀合的 2c 负载纳米粒子 (Apt-2cNP) 在 CRC 肿瘤部位进行位点特异性归巢，以增强治疗潜力并减少正常细胞的脱靶毒性。我们研究了适体偶联纳米疗法在CRC-TME中的体外和体内治疗效果和抗肿瘤免疫反应。在对纳米工程适体偶联桦木酸纳米疗法进行表征后，我们评估了治疗效果、肿瘤靶向效率和抗肿瘤免疫反应。我们发现 Apt-2cNP 提高了药物生物利用度，延长了其生物半衰期，提高了抗增殖活性，并最大限度地减少了脱靶细胞毒性。重要的是，在体内 TME 中，Apt-2cNP 显示出有希望的抗肿瘤免疫反应迹象（mDC/pDC 比率增加、M1 巨噬细胞群和 CD8 T 细胞增强）。此外，体内促凋亡基因的上调同时抗凋亡基因的下调以及对癌症组织病理学的显着治愈功效表明，Apt-2cNP 比非适体缀合的纳米粒子和游离药物具有更大的治疗潜力。此外，我们通过 CRC 大鼠模型中的实时成像观察到放射性标记的 Apt-2cNP 更大的肿瘤积累。Apt-2cNP 在 CRC-TME 中增强的治疗功效和强大的抗肿瘤免疫反应是其作为前瞻性药物潜力的有希望的指标。用于治疗 CRC 的治疗剂。然而，还需要进一步研究。© 2024。作者。

Betulinic acid (BA) has been well investigated for its antiproliferative and mitochondrial pathway-mediated apoptosis-inducing effects on various cancers. However, its poor solubility and off-target activity have limited its utility in clinical trials. Additionally, the immune modulatory role of betulinic acid analogue in the tumor microenvironment (TME) is largely unknown. Here, we designed a potential nanotherapy for colorectal cancer (CRC) with a lead betulinic acid analogue, named as 2c, carrying a 1,2,3-triazole-moiety attached to BA through a linker, found more effective than BA for inhibiting CRC cell lines, and was chosen here for this investigation. Epithelial cell adhesion molecule (EpCAM) is highly overexpressed on the CRC cell membrane. A single-stranded short oligonucleotide sequence, aptamer (Apt), that folds into a 3D-defined architecture can be used as a targeting ligand for its specific binding to a target protein. EpCAM targeting aptamer was designed for site-specific homing of aptamer-conjugated-2c-loaded nanoparticles (Apt-2cNP) at the CRC tumor site to enhance therapeutic potential and reduce off-target toxicity in normal cells. We investigated the in vitro and in vivo therapeutic efficacy and anti-tumorigenic immune response of aptamer conjugated nanotherapy in CRC-TME.After the characterization of nanoengineered aptamer conjugated betulinic acid nanotherapy, we evaluated therapeutic efficacy, tumor targeting efficiency, and anti-tumorigenic immune response using cell-based assays and mouse and rat models.We found that Apt-2cNP improved drug bioavailability, enhanced its biological half-life, improved antiproliferative activity, and minimized off-target cytotoxicity. Importantly, in an in vivo TME, Apt-2cNP showed promising signs of anti-tumorigenic immune response (increased mDC/pDC ratio, enhanced M1 macrophage population, and CD8 T-cells). Furthermore, in vivo upregulation of pro-apoptotic while downregulation of anti-apoptotic genes and significant healing efficacy on cancer tissue histopathology suggest that Apt-2cNP had predominantly greater therapeutic potential than the non-aptamer-conjugated nanoparticles and free drug. Moreover, we observed greater tumor accumulation of the radiolabeled Apt-2cNP by live imaging in the CRC rat model.Enhanced therapeutic efficacy and robust anti-tumorigenic immune response of Apt-2cNP in the CRC-TME are promising indicators of its potential as a prospective therapeutic agent for managing CRC. However, further studies are warranted.© 2024. The Author(s).