Contreras-Ruiz Laura, de la Fuente María, Párraga Jenny E, López-García Antonio, Fernández Itziar, Seijo Begoña, Sánchez Alejandro, Calonge Margarita, Diebold Yolanda
Ocular Surface Group-IOBA, University of Valladolid, Paseo de Belén 17, Valladolid, Spain.
Mol Vis. 2011 Jan 27;17:279-90.
Nanoparticles are a promising alternative for ocular drug delivery, and our group has proposed that they are especially suited for ocular mucosal disorders. The goal of the present study was to determine which internalization pathway is used by cornea-derived and conjunctiva-derived cell lines to take up hyaluronic acid (HA)-chitosan oligomer (CSO)-based nanoparticles (HA-CSO NPs). We also determined if plasmids loaded onto the NPs reached the cell nucleus.
HA-CSO NPs were made of fluoresceinamine labeled HA and CSO by ionotropic gelation and were conjugated with a model plasmid DNA for secreted alkaline phosphatase. Human epithelial cell lines derived from the conjunctiva and the cornea were exposed to HA-CSO NPs for 1 h and the uptake was investigated in living cells by fluorescence microscopy. The influence of temperature and metabolic inhibition, the effect of blocking hyaluronan receptors, and the inhibition of main endocytic pathways were studied by fluorometry. Additionally, the metabolic pathways implicated in the degradation of HA-CSO NPs were evaluated by lysosome identification.
There was intracellular localization of plasmid-loaded HACSO NPs in both corneal and conjunctival cells. The intracellular presence of NPs diminished with time. HA-CSO NP uptake was significantly reduced by inhibition of active transport at 4 °C and by sodium azide. Uptake was also inhibited by blocking hyaluronan receptors with anti-CD44 Hermes-1 antibody, by excess HA, and by filipin, an inhibitor of caveolin-dependent endocytosis. HA-CSO NPs had no effect on cell viability. The transfection efficiency of the model plasmid was significantly higher in NP treated cells than in controls.
HA-CSO NPs were internalized by two different ocular surface cell lines by an active transport mechanism. The uptake was mediated by hyaluronan receptors through a caveolin-dependent endocytic pathway, yielding remarkable transfection efficiency. Most of HA-CSO NPs were metabolized within 48 h. This uptake did not compromise cell viability. These findings further support the potential use of HA-CSO NPs to deliver genetic material to the ocular surface.
纳米颗粒是一种很有前景的眼部给药替代方案，我们团队提出它们特别适用于眼部黏膜疾病。本研究的目的是确定角膜来源和结膜来源的细胞系通过何种内化途径摄取基于透明质酸（HA）-壳聚糖低聚物（CSO）的纳米颗粒（HA-CSO NPs）。我们还确定了负载在纳米颗粒上的质粒是否能到达细胞核。
HA-CSO NPs由荧光胺标记的HA和CSO通过离子凝胶法制成，并与用于分泌碱性磷酸酶的模型质粒DNA偶联。将结膜和角膜来源的人上皮细胞系暴露于HA-CSO NPs 1小时，通过荧光显微镜在活细胞中研究摄取情况。通过荧光测定法研究温度和代谢抑制的影响、透明质酸受体阻断的效果以及主要内吞途径的抑制作用。此外，通过溶酶体鉴定评估参与HA-CSO NPs降解的代谢途径。
负载质粒的HA-CSO NPs在角膜和结膜细胞中均有细胞内定位。纳米颗粒在细胞内的存在随时间减少。通过在4°C抑制主动转运和叠氮化钠可显著降低HA-CSO NPs的摄取。用抗CD44 Hermes-1抗体阻断透明质酸受体、过量的HA以及小窝蛋白依赖性内吞作用的抑制剂制霉菌素也可抑制摄取。HA-CSO NPs对细胞活力无影响。在纳米颗粒处理的细胞中，模型质粒的转染效率显著高于对照组。
HA-CSO NPs通过主动转运机制被两种不同的眼表细胞系内化。摄取由透明质酸受体通过小窝蛋白依赖性内吞途径介导，产生显著的转染效率。大多数HA-CSO NPs在48小时内被代谢。这种摄取不会损害细胞活力。这些发现进一步支持了HA-CSO NPs在向眼表递送遗传物质方面的潜在用途。
