靶向PSP介导MMP-7和CTSB双酶响应的UCL-MRI双模态纳米探针在结直肠癌发生、转移及药物筛选的研究

Colorectal Cancer (CRC) is one of the most common malignancies of digestive system. Diagnosis of CRC in early stage, detection of micrometastasis and screening of antitumor drugs are of great importance for clinical practice. The extensive studies over the past decades shows that the up regulation of cathepsin B (CTSB) is closely related to the occurrence, infiltration and metastasis of tumor, and the stromal cells secrete relative large amount of activated matrix metalloproteinases -7 (MMP-7) in the process of tumor angiogenesis. In addition, the polypeptide ligand of tumor vascular endothelial growth factor (VEGF), has good tumor homing ability. In this proposal, the -NaGdF4: Yb3+, Ln3+@ NaGdF4 UCNPs with higher upconversion luminescence (UCL) quantum yield in red light region are synthesized by adjusting the elemental types and proportion of doped rare earth ions (Yb3+, Ln3+), and angiogenic factor peptide ligand (PSP) with high affinity to CRC cells are screened through interactions of synthesized peptides with CRC cells. Subsequently, a tumor-targeting and CTSB/MMP-7-responsing nanoprobe (UCNP@SiO2-Peptide-Cy5) are fabricated by simultaneous modification of PSP and Cy5 labeled pH-responsive protease peptide substrate (NC-MMP-CPP-CTSB) on the silica coated UCNP surface. After MMP-7 cleavage, UCNP@SiO2-Peptide-Cy5 has positive surface charge in the weakly acidic tumor microenvironment, which promotes UCNP@SiO2-Peptide-Cy5 to enter the tumor cells and react with cellular CTSP, resulting in significant recovering of the red UCL of UCNP. Orthotopic CRC tumor mouse model is established to test the practicability of the as-constructed UCNP@SiO2-Peptide-Cy5 by in vivo UCL-MRI dual modality imaging. The capacity of UCNP@SiO2-Peptide-Cy5 nanoprobes is further demonstrated by visualization of CRC with small volume and liver CRC metastase. Furthermore, the CTSB inhibitors can be screened by the UCL change of nanoprobe because the recovery ratio of UCNP@SiO2-Peptide-Cy5 is in direct proportion to the activity of CTSB. The project provides a new method and new idea for noninvasive diagnosis of CRC in early stage, detection of CRC micrometastasis and development of tumor-targeting drugs.

早期结直肠癌、肝转移灶的检出以及抗肿瘤药物筛选具有重要临床意义。国内外研究表明组织蛋白酶B（CTSB）上调与肿瘤发生、转移密切相关，血管生成因子多肽配体(PSP)具有良好肿瘤归巢能力，肿瘤分泌大量基质金属蛋白酶-7(MMP-7)并具有高活性。本课题组以CTSB为靶分子，合成在红光区有高UCL量子产率的β-NaGdF4: Yb3+, Ln3+@ NaGdF4 UCNPs；筛选与肿瘤细胞具有高亲和力的PSP，将PSP和Cy5标记具有pH活性的蛋白酶多肽底物同时修饰于SiO2包裹的Yb3+, Ln3+表面，构建靶向PSP介导MMP-7和CTSB双酶响应纳米探针UCNP@SiO2-Peptide-Cy5；进行荷瘤小鼠UCL-MRI双模态成像，实现肿瘤微小灶和肝转移灶可视化；利用纳米探针UCL恢复与CTSB活性成正比关系，筛选CTSB抑制剂，为无创性肿瘤早期诊断和靶向药物设计提供一种新思路。

提高早期结直肠癌的检出并进行药物筛选是临床与科研亟待解决的问题。研究表明组织蛋白酶B（CTSB）上调与肿瘤发生、转移密切相关，血管生成因子多肽配体(PSP)具有良好肿瘤归巢能力，肿瘤分泌大量基质金属蛋白酶-7(MMP-7)并具有高活性。本课题组以 CRC 发生、浸润、转移密切相关的CTSB和MMP-7为靶分子，分别构建了稳定性好且具有靶向性的CTSB响应跨膜纳米探针 USP和MMP-7响应跨膜纳米探针UCNP@SiO2@PSP@MMP-7-Cy5，将两种纳米探针应用于溶液相和结直肠癌HCT116细胞中，实现了对CTSB和MMP-7的高灵敏检测；并通过荷瘤小鼠 UCL-MRI双模态成像，成功实现了早期及原位结直肠癌的精准可视化，为结直肠癌的无创性早期诊断和靶向药物设计提供了一种新思路。

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