INSERM, U646, F-49933 Angers, France.
Int J Pharm. 2012 Feb 14;423(1):93-101. doi: 10.1016/j.ijpharm.2011.06.001. Epub 2011 Jun 12.
In the context of targeted therapy, we addressed the possibility of developing a drug delivery nanocarrier capable to specifically reach cancer cells that express the most prominent marker associated with cancer stem cell (CSC) phenotype, AC133. For this purpose, 100nm lipid nanocapsules (LNCs) were functionalized with a monoclonal antibody (mAb) directed against AC133 according to two distinct methods: firstly, post-insertion within 100nm LNCs of a lipid poly(ethylene glycol) functionalized with reactive-sulfhydryl maleimide groups (DSPE-PEG(2000)-maleimide) followed by thiolated mAb coupling, and, secondly, creation of a thiolated lipo-immunoglobulin between DSPE-PEG(2000)-maleimide and AC133, then post-inserted within LNCs. Due to the reduced number of purification steps, lower amounts of DSPE-PEG(2000)-maleimide that were necessary as well as lower number of free maleimide functions present onto the surface of immuno-LNC, the second method was found to be more appropriate. Thus, 126nm AC133-LNC with a zeta potential of -22mV while keeping a narrow distribution were developed. Use of the IgG1κ isotype control-immunoglobulins produced similar control IgG1-LNCs. Micro-Bradford colorimetric assay indicated a fixation of about 40 immunoglobulins per LNC. Use of human Caco-2 cells that constitutively express AC133 (Caco-2-AC133(high)) allowed addressing the behavior of the newly functionalized immuno-LNCs. siRNA knockown strategy permitted to obtain Caco-2-AC133(low) for comparison. Immunofluorescence-combined flow cytometry analysis demonstrated that the epitope-recognition function of AC133 antibody was preserved when present on immuno-LNCs. Although grafting of immunoglobulins onto the surface of LNCs repressed their internalization within Caco-2 cells as evaluated by flow cytometry, AC133-specific cellular binding was obtained with AC133-LNC as assessed by computer-assisted fluorescence microscopy. In conclusion, interest of AC133-LNCs as niche carriers is discussed toward the development of CSC targeted chemo- or radio-nanomedicines.
在靶向治疗的背景下，我们研究了开发一种药物递送纳米载体的可能性，该载体能够特异性地到达表达与癌症干细胞（CSC）表型相关的最显著标志物的癌细胞。为此，我们使用两种不同的方法对 100nm 脂质纳米胶囊（LNC）进行了功能化：首先，将带有反应性巯基马来酰亚胺基团的聚乙二醇化脂质（DSPE-PEG（2000）-马来酰亚胺）插入 100nm LNC 中，然后进行硫醇化 mAb 偶联；其次，在 DSPE-PEG（2000）-马来酰亚胺和 AC133 之间创建硫醇化脂免疫球蛋白，然后将其插入 LNC 中。由于纯化步骤减少，所需的 DSPE-PEG（2000）-马来酰亚胺的量也减少，并且免疫 LNC 表面的游离马来酰亚胺基团数量减少，因此发现第二种方法更为合适。因此，开发了具有-22mV 表面等电点和窄分布的 126nm AC133-LNC。使用 IgG1κ 同型对照免疫球蛋白产生了类似的对照 IgG1-LNC。微量 Bradford 比色法分析表明，每个 LNC 固定约 40 个免疫球蛋白。使用人 Caco-2 细胞（其组成性表达 AC133（Caco-2-AC133（高）），可以研究新功能化的免疫 LNC 的行为。siRNA 敲低策略允许获得用于比较的 Caco-2-AC133（低）。免疫荧光-流式细胞术分析表明，当抗体的表位识别功能存在于免疫 LNC 上时，该功能得以保留。尽管通过流式细胞术评估，免疫球蛋白接枝到 LNC 表面会抑制其在 Caco-2 细胞中的内化，但通过计算机辅助荧光显微镜评估，仍可获得针对 AC133-LNC 的 AC133 特异性细胞结合。总之，讨论了 AC133-LNC 作为小生境载体的应用潜力，以开发针对 CSC 的化学或放射纳米药物。
