Laboratory of Organic and Macromolecular Chemistry (IOMC), Friedrich Schiller University Jena, Humboldtstrasse 10, 07743, Jena, Germany.
Klinik für Innere Medizin II, Abteilung Hämatologie und Internistische Onkologie, Universitätsklinikum Jena, Am Klinikum 1, 07747, Jena, Germany.
J Nanobiotechnology. 2021 Mar 6;19(1):70. doi: 10.1186/s12951-021-00801-y.
Although there has been substantial progress in the research field of gene delivery, there are some challenges remaining, e.g. there are still cell types such as primary cells and suspension cells (immune cells) known to be difficult to transfect. Cationic polymers have gained increasing attention due to their ability to bind, condense and mask genetic material, being amenable to scale up and highly variable in their composition. In addition, they can be combined with further monomers exhibiting desired biological and chemical properties, such as antioxidative, pH- and redox-responsive or biocompatible features. By introduction of hydrophobic monomers, in particular as block copolymers, cationic micelles can be formed possessing an improved chance of transfection in otherwise challenging cells. In this study, the antioxidant biomolecule lipoic acid, which can also be used as crosslinker, was incorporated into the hydrophobic block of a diblock copolymer, poly{[2-(dimethylamino)ethyl methacrylate]-b-[n-(butyl methacrylate)-co-(lipoic acid methacrylate)]} (P(DMAEMA-b-[nBMA-co-LAMA])), synthesized by RAFT polymerization and assembled into micelles (LAMA-mic). These micelles were investigated regarding their pDNA binding, cytotoxicity mechanisms and transfection efficiency in K-562 and HEK293T cells, the former representing a difficult to transfect, suspension leukemia cell line. The LAMA-mic exhibited low cytotoxicity at applied concentrations but demonstrated superior transfection efficiency in HEK293T and especially K-562 cells. In-depth studies on the transfection mechanism revealed that transfection efficiency in K-562 cells does not depend on the specific oncogenic fusion gene BCR-ABL alone. It is independent of the cellular uptake of polymer-pDNA complexes but correlates with the endosomal escape of the LAMA-mic. A comparison of the transfection efficiency of the LAMA-mic with structurally comparable micelles without lipoic acid showed that lipoic acid is not solely responsible for the superior transfection efficiency of the LAMA-mic. More likely, a synergistic effect of the antioxidative lipoic acid and the micellar architecture was identified. Therefore, the incorporation of lipoic acid into the core of hydrophobic-cationic micelles represents a promising tailor-made transfer strategy, which can potentially be beneficial for other difficult to transfect cell types.
尽管在基因传递的研究领域已经取得了实质性的进展，但仍存在一些挑战，例如，仍有一些细胞类型，如原代细胞和悬浮细胞（免疫细胞），已知难以转染。阳离子聚合物由于能够结合、浓缩和掩蔽遗传物质，易于规模化，并且在组成上高度可变，因此受到越来越多的关注。此外，它们可以与具有所需生物和化学性质的进一步单体结合，例如抗氧化、pH 和氧化还原响应或生物相容性特征。通过引入疏水性单体，特别是作为嵌段共聚物，可以形成阳离子胶束，从而提高在具有挑战性的细胞中进行转染的机会。在这项研究中，抗氧化生物分子硫辛酸，也可用作交联剂，被掺入两亲嵌段共聚物聚{[2-（二甲氨基）乙基甲基丙烯酸酯]-b-[n-（丁基甲基丙烯酸酯）-共-（硫辛酸甲基丙烯酸酯）]}（P（DMAEMA-b-[nBMA-co-LAMA]））的疏水性嵌段中，通过 RAFT 聚合合成，并组装成胶束（LAMA-胶束）。研究了这些胶束与 pDNA 的结合、细胞毒性机制以及在 K-562 和 HEK293T 细胞中的转染效率，前者代表了一种难以转染的悬浮白血病细胞系。在应用浓度下，LAMA-胶束表现出低细胞毒性，但在 HEK293T 细胞和特别是 K-562 细胞中转染效率更高。对转染机制的深入研究表明，K-562 细胞中的转染效率不仅取决于特定的致癌融合基因 BCR-ABL。它独立于聚合物-pDNA 复合物的细胞摄取，但与 LAMA-胶束的内体逃逸相关。将 LAMA-胶束与结构相似的不含硫辛酸的胶束的转染效率进行比较表明，硫辛酸并不是 LAMA-胶束优越转染效率的唯一原因。更可能的是，抗氧化的硫辛酸和胶束结构的协同作用被确定。因此，将硫辛酸掺入疏水性阳离子胶束的核心代表了一种有前途的定制转移策略，可能对其他难以转染的细胞类型有益。
