高稳定两亲性甘脲纳米组装体构筑及细胞内短链DNA高效递送

doi:10.6023/cjoc202501022

摘要/Abstract

为实现利用小分子组装体将短链DNA高效递送进细胞内的目标, 制备了四个基于甘脲的两亲性分子夹M1~M4. 分子夹的凸面带有两个甲基, 其分子骨架具有高疏水性. 每个分子带有四个阳离子亲水基团, 因此具有两亲性特征, 在水中受疏水作用驱动聚集形成纳米颗粒. 动态光散射实验表明, M1和M2可以被疏水驱动, 在微摩尔浓度就可以聚集形成极其稳定的纳米颗粒. 荧光滴定和电动电位实验显示, 由M1和M2形成的纳米颗粒受离子静电吸引作用驱动, 能够有效吸收和包埋短链DNA. 荧光成像和流式细胞术研究揭示, 该纳米组装体能够将包封的DNA递送到正常细胞和癌症细胞中, 递送率最高达到94%. 体外实验表明, 这两个化合物具有良好的生物相容性和低细胞毒性.

关键词: 自组装, 甘脲, 两亲性, 纳米颗粒, DNA递送

Four glycoluril-based amphiphilic molecular clips (AMCs) M1~M4 have been prepared for intracellular delivery of short DNA. M1~M4 have two methyl groups on its convex surface and four cations on its aromatic side arm, which can be used to construct self-assembled nanoparticles in aqueous solution driven by hydrophobic interaction. Dynamic light scattering experiments show that M1 and M2 can be driven hydrophobically to aggregate into extremely stable nanoparticles in water at the micromolar concentrations. Fluorescence titration and zeta potential experiments support that the nanoparticles formed by M1 and M2 are able to efficiently encapsulate short DNA (sDNA). Fluorescence imaging and flow cytometry studies reveal that their nano sizes enable intracellular delivery of the encapsulated sDNA into both normal and cancer cells, with delivery percentage reaching up to 94%, while in vitro experiments indicate that the two compounds have excellent biocompatibility and low cytotoxicity.

Key words: self-assembly, glycoluril, amphiphilicity, nanoparticle, DNA delivery

引用此文

郭聪颖, 高睿, 李倩, 王辉, 张丹维, 周伟, 黎占亭. 高稳定两亲性甘脲纳米组装体构筑及细胞内短链DNA高效递送[J]. 有机化学, 2025, 45(8): 2945-2952.

Congying Guo, Rui Gao, Qian Li, Hui Wang, Danwei Zhang, Wei Zhou, Zhan-Ting Li. Self-Assembly of Highly Stable Nanoparticles by Amphiphilic Glycolurils for Efficient Intracellular Short DNA Delivery[J]. Chinese Journal of Organic Chemistry, 2025, 45(8): 2945-2952.

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图/表 7

Scheme 1. Compounds M1~M4 and the routes for their preparations

Figure 1. DLS profile of the aqueous solutions of compounds (a) M1 and (b) M2

Figure 2. Tentative mechanism for the formation of nanoparticles by M1~M4 through hydrophobically driven aggregation of the glycoluril unit and their delivery for short DNA

Figure 3. Fluorescence spectra of Cy5-ssDNA-21 (0.25 μmol/L, λex=625 nm) in water with the addition of (a) M1 and (b) M2 (all the experiments were conducted at 25 ℃)

Figure 4. Zeta potentials of the solutions of (a) M1, M1＋Cy5-ssDNA-21 (10 μmol/L), and (b) M2, M2＋Cy5-ssDNA-21 (10 μmol/L) (all the experiments were conducted at 25 ℃)

Figure 5. CLSM images of MCF-7/ADR cells after incubation with Cy5-ssDNA-21, Cy5-ssDNA-21＋M4, Cy5-ssDNA-21＋M3, Cy5-ssDNA-21＋M2, Cy5-ssDNA-21＋M1 and Cy5- ssDNA-21＋Lipo2000 for 2 h ([Cy5-ssDNA-21]=2.5 μg/mL, [M4/M3/M2/M1/Lipo2000]=20.0 μg/mL)

Figure 6. (a) Delivery (internalization) of Cy5-ssDNA-21 (2.5 μg/mL) and Cy5-dsDNA-21 (5 μg/mL) into MCF-7/ADR cell line by M1~M4 (20 μg/mL for ssDNA and 40 μg/mL for dsDNA) and Lipo2000 (20 μg/mL for ssDNA and 40 μg/mL for dsDNA) after incubation in complete 1640 medium for 2 h, and (b) cell viability values (%) of MCF-7/ADR cell line evaluated by CCK-8 proliferation tests versus the incubation concentration of M1~M4 (the cells (≈2×104 per well) were incubated with M1~M4 at 37 ℃ for 24 h. Error bars represent the s.d. of uncertainty for each point)

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