Resnier Pauline, Lepeltier Elise, Emina Anthea Lucrezia, Galopin Natacha, Bejaud Jérôme, David Stephanie, Ballet Caroline, Benvegnu Thierry, Pecorari Frédéric, Chourpa Igor, Benoit Jean-Pierre, Passirani Catherine
MINT, UNIV Angers, INSERM 1066, CNRS 6021, Université Bretagne Loire Angers France
CRCINA, INSERM, Université d'Angers, Université de Nantes Nantes France.
RSC Adv. 2019 Aug 30;9(47):27264-27278. doi: 10.1039/c9ra03668g. eCollection 2019 Aug 29.
Malignant melanoma is an aggressive tumor, associated with the presence of local and/or distant metastases. The development of gene therapy by the use of small interfering RNA (siRNA) represents a promising new treatment. However, the protection of this biomolecule is necessary in order for it to be intravenously administrated, for example its incorporation into nanomedicines. In parallel to the passive targeting usually obtained by pegylation, various studies have aimed at developing "smart" nanomedicines to efficiently deliver the drug to tumor sites. In this work, siRNA loaded lipid nanocapsules (LNCs) were modified with DSPE-polyethylene glycol (DSPE-PEG), tetraether-PEG (TE-PEG) and/or with an Affitin model, to assay multiple targeting strategies. The uptake of fluorescently labelled LNCs, nanocarrier integrity and siRNA release into human SK-Mel28 melanoma cells were studied by flow cytometry, conventional confocal microscopy and by confocal spectral imaging in a Förster Resonance Energy Transfer (FRET) mode. Surface modified siRNA LNCs were followed after human plasma incubation and after intravenous injection, in order to compare the stealth properties. Finally, the biodistribution of the different siRNA LNCs in healthy and melanoma tumor bearing mice models was assessed by biofluorescence imaging (BFI), to evaluate the potential tumor targeting ability. The post-insertion of DSPE-PEG induced a strong decrease of the internalization into melanoma cells compared to TE-PEG modification. Both PEG polymer decorations induced a great plasma protection of siRNA but only DSPE-PEG led to stealth properties, even at low concentration (5 mM). The Affitin grafting by thiolation of DSPE-PEG was validated on siRNA LNCs. DSPE-PEG-Affitin LNCs were not detected in this melanoma tumor model but did not show unspecific accumulation in organs. DSPE-PEG and TE-PEG LNCs induced a significant intratumoral accumulation of modified LNCs.
恶性黑色素瘤是一种侵袭性肿瘤，与局部和/或远处转移的存在有关。利用小干扰RNA（siRNA）开展基因治疗是一种很有前景的新疗法。然而，为了能够静脉给药，例如将其整合到纳米药物中，保护这种生物分子是必要的。与通常通过聚乙二醇化实现的被动靶向同时，各种研究旨在开发“智能”纳米药物，以有效地将药物递送至肿瘤部位。在这项工作中，用二硬脂酰磷脂酰乙醇胺-聚乙二醇（DSPE-PEG）、四醚-聚乙二醇（TE-PEG）和/或亲和体模型对负载siRNA的脂质纳米囊（LNCs）进行修饰，以测试多种靶向策略。通过流式细胞术、传统共聚焦显微镜以及在Förster共振能量转移（FRET）模式下的共聚焦光谱成像，研究了荧光标记的LNCs的摄取、纳米载体完整性以及siRNA释放到人SK-Mel28黑色素瘤细胞中的情况。在人血浆孵育后和静脉注射后追踪表面修饰的siRNA LNCs，以比较其隐身性能。最后，通过生物荧光成像（BFI）评估不同siRNA LNCs在健康小鼠模型和荷黑色素瘤肿瘤小鼠模型中的生物分布，以评估潜在的肿瘤靶向能力。与TE-PEG修饰相比，DSPE-PEG的插入导致黑色素瘤细胞内化显著减少。两种PEG聚合物修饰都能对siRNA起到很好的血浆保护作用，但只有DSPE-PEG能实现隐身性能，即使在低浓度（5 mM）下也是如此。通过DSPE-PEG的硫醇化进行亲和体接枝在siRNA LNCs上得到验证。在该黑色素瘤肿瘤模型中未检测到DSPE-PEG-亲和体LNCs，但它们在器官中未显示非特异性积累。DSPE-PEG和TE-PEG LNCs诱导修饰后的LNCs在肿瘤内显著积累。
