Tammam Salma N, Azzazy Hassan M E, Breitinger Hans G, Lamprecht Alf
Laboratory of Pharmaceutical Technology and Biopharmaceutics, University of Bonn , Bonn 53121, Germany.
Department of Chemistry, The American University in Cairo , Cairo 11835, Egypt.
Mol Pharm. 2015 Dec 7;12(12):4277-89. doi: 10.1021/acs.molpharmaceut.5b00478. Epub 2015 Nov 10.
Many recently discovered therapeutic proteins exert their main function in the nucleus, thus requiring both efficient uptake and correct intracellular targeting. Chitosan nanoparticles (NPs) have attracted interest as protein delivery vehicles due to their biocompatibility and ability to escape the endosomes offering high potential for nuclear delivery. Molecular entry into the nucleus occurs through the nuclear pore complexes, the efficiency of which is dependent on NP size and the presence of nuclear localization sequence (NLS). Chitosan nanoparticles of different sizes (S-NPs ≈ 25 nm; L-NP ≈ 150 nm) were formulated, and they were modified with different densities of the octapeptide NLS CPKKKRKV (S-NPs, 0.25, 0.5, 2.0 NLS/nm(2); L-NPs, 0.6, 0.9, 2 NLS/nm(2)). Unmodified and NLS-tagged NPs were evaluated for their protein loading capacity, extent of cell association, cell uptake, cell surface binding, and finally nuclear delivery efficiency in L929 fibroblasts. To avoid errors generated with cell fractionation and nuclear isolation protocols, nuclear delivery was assessed in intact cells utilizing Förster resonance energy transfer (FRET) fluorometry and microscopy. Although L-NPs showed ≈10-fold increase in protein loading per NP when compared to S-NPs, due to higher cell association and uptake S-NPs showed superior protein delivery. NLS exerts a size and density dependent effect on nanoparticle uptake and surface binding, with a general reduction in NP cell surface binding and an increase in cell uptake with the increase in NLS density (up to 8.4-fold increase in uptake of High-NLS-L-NPs (2 NLS/nm(2)) compared to unmodified L-NPs). However, for nuclear delivery, unmodified S-NPs show higher nuclear localization rates when compared to NLS modified NPs (up to 5-fold by FRET microscopy). For L-NPs an intermediate NLS density (0.9 NLS/nm(2)) seems to provide highest nuclear localization (3.7-fold increase in nuclear delivery compared to High-NLS-L-NPs). Results indicate that a higher NLS density does not result in maximum protein nuclear localization and that a universal optimal density for NPs of different sizes does not exist.
许多最近发现的治疗性蛋白质在细胞核中发挥其主要功能，因此既需要有效的摄取又需要正确的细胞内靶向定位。壳聚糖纳米颗粒（NPs）因其生物相容性以及从内涵体逃逸的能力而作为蛋白质递送载体引起了人们的关注，这为核递送提供了很高的潜力。分子通过核孔复合体进入细胞核，其效率取决于NP的大小和核定位序列（NLS）的存在。制备了不同大小的壳聚糖纳米颗粒（S-NPs≈25nm；L-NP≈150nm），并用不同密度的八肽NLS CPKKKRKV对其进行修饰（S-NPs，0.25、0.5、2.0 NLS/nm²；L-NPs，0.6、0.9、2 NLS/nm²）。对未修饰和带有NLS标签的NPs进行了蛋白质负载能力、细胞结合程度、细胞摄取、细胞表面结合的评估，最后评估了它们在L929成纤维细胞中的核递送效率。为了避免细胞分级分离和核分离方案产生的误差，利用Förster共振能量转移（FRET）荧光测定法和显微镜在完整细胞中评估核递送。尽管与S-NPs相比，L-NPs每个NP的蛋白质负载量增加了约10倍，但由于S-NPs具有更高的细胞结合和摄取能力，因此显示出更好的蛋白质递送效果。NLS对纳米颗粒的摄取和表面结合具有大小和密度依赖性影响，随着NLS密度的增加，NP细胞表面结合普遍减少，细胞摄取增加（与未修饰的L-NPs相比，高NLS-L-NPs（2 NLS/nm²）的摄取增加高达8.4倍）。然而，对于核递送，与NLS修饰的NPs相比，未修饰的S-NPs显示出更高的核定位率（通过FRET显微镜观察高达5倍）。对于L-NPs，中等NLS密度（0.9 NLS/nm²）似乎提供了最高的核定位（与高NLS-L-NPs相比，核递送增加3.7倍）。结果表明，较高的NLS密度并不会导致蛋白质的最大核定位，并且不存在适用于不同大小NPs的通用最佳密度。
