General Hospital of Tianjin Medical University, Tianjin, 300052, China.
Department of Neurology, Tianjin First Central Hospital, Tianjin, 300192, China.
Int J Biol Macromol. 2017 Dec;105(Pt 2):1473-1481. doi: 10.1016/j.ijbiomac.2017.06.061. Epub 2017 Jun 12.
Mechanism study of why astrocytes isolated from experimental autoimmune encephalomyelitis (EAE)-induced B6 mice or after being exposed to inflammatory factors had the highest transfection efficiency to larger-sized, but not compacted, pspCS/pDNA particles.
Phosphorylatable short peptide conjugated chitosan (pspCS) was compounded with plasmid DNA (pDNA) at different N:P ratios to form pspCS/pDNA particles of different size and zeta potentials. These pspCS/pDNA particles were used for the transfection of astrocytes isolated from either EAE induced or healthy B6 mice. Transfection efficiency and cell permeability of the particles were determined by the internalization of radio [H3]-labeled plasmid and the expression of a luciferase reporter gene respectively. Phagocytosis of EAE-astrocytes was determined by the internalization of FITC labeled dextran beads. By comparing the transfection efficiency of differently-sized pspCS/pDNA particles to normal and phagocytic astrocytes, with or without cytochalasin D, a phagocytosis inhibitor, in the presence, the contribution of phagocytosis to cell permeability and transfection efficiency was evaluated.
In vivo EAE-induction or in vitro inflammatory factors treatment transferred normal astrocytes to be phagocytic astrocytes which underwent phagocytosis, had the highest cell permeability and transfection efficiency to larger-sized pspCS/pDNA particles formed at lower N:P ratios. When phagocytosis was inhibited by cytochalasin D, both cell permeability and transfection efficiency of phagocytic astrocytes to larger were significantly decreased. Thereafter, particle size, not zeta potential, was verified as the key factor for determining whether the particles could be phagocytosed. In addition phagocytosis was successfully induced in ARPE-19 cells as well, which also improved the transfection efficiency of larger pspCS/pDNA particles.
A generally accepted concept is that the internalization of cationic polymer/pDNA particles, chitosan-DNA complex for instance, is mainly through the procedure of endocytosis of the transfected cells. More compacted particles with higher zeta potential were used to be considered had higher cell permeability and transfection efficiency. However, here we reported that phagocytosis is another important procedure for determining internalization and transfection efficiency of cationic polymer/pDNA nanoparticles, especially for advanced transfection efficiency of large pspCS/pDNA particles. Thus, for gene delivery applications, the environmental condition of the target cells should be seriously considered for selecting an appropriate gene transfer strategies.
研究实验性自身免疫性脑脊髓炎（EAE）诱导的 B6 小鼠或暴露于炎症因子后的星形胶质细胞转染效率最高的原因，即为什么它们对较大而非紧密的 pspCS/pDNA 颗粒的转染效率最高。
将可磷酸化短肽偶联壳聚糖（pspCS）与质粒 DNA（pDNA）以不同的 N:P 比复合，形成不同大小和 zeta 电位的 pspCS/pDNA 颗粒。将这些 pspCS/pDNA 颗粒用于转染来自 EAE 诱导或健康 B6 小鼠的星形胶质细胞。通过放射性 [H3]-标记的质粒的内化和荧光素酶报告基因的表达分别确定颗粒的转染效率和细胞通透性。通过 FITC 标记的葡聚糖珠的内化来确定 EAE 星形胶质细胞的吞噬作用。通过比较不同大小的 pspCS/pDNA 颗粒对正常和吞噬星形胶质细胞的转染效率，以及在存在和不存在吞噬抑制剂细胞松弛素 D 的情况下，评估吞噬作用对细胞通透性和转染效率的贡献。
体内 EAE 诱导或体外炎症因子处理将正常星形胶质细胞转化为吞噬星形胶质细胞，它们经历吞噬作用，对较低 N:P 比形成的较大 pspCS/pDNA 颗粒具有最高的细胞通透性和转染效率。当吞噬作用被细胞松弛素 D 抑制时，吞噬星形胶质细胞对较大的细胞通透性和转染效率均显著降低。此后，证实颗粒大小而不是 zeta 电位是决定颗粒是否可被吞噬的关键因素。此外，还成功诱导了 ARPE-19 细胞的吞噬作用，这也提高了较大 pspCS/pDNA 颗粒的转染效率。
普遍接受的概念是，阳离子聚合物/pDNA 颗粒（例如壳聚糖-DNA 复合物）的内化主要是通过转染细胞的内吞作用进行的。更紧密的具有更高 zeta 电位的颗粒被认为具有更高的细胞通透性和转染效率。然而，在这里我们报告说，吞噬作用是决定阳离子聚合物/pDNA 纳米颗粒内化和转染效率的另一个重要过程，特别是对于大 pspCS/pDNA 颗粒的高级转染效率。因此，对于基因传递应用，应认真考虑靶细胞的环境条件，以选择合适的基因传递策略。
