University Medical Center Groningen, University of Groningen, Department of Cell Biology/Membrane Cell Biology, A. Deusinglaan 1, Groningen, The Netherlands.
J Control Release. 2011 Nov 30;156(1):76-84. doi: 10.1016/j.jconrel.2011.07.015. Epub 2011 Jul 20.
Cellular entry of nanoparticles for drug- and gene delivery relies on various endocytic pathways, including clathrin- and caveolae-mediated endocytosis. To improve delivery, i.e., the therapeutic and/or cell biological impact, current efforts are aimed at avoiding processing of the carriers along the degradative clathrin-mediated pathway towards lysosomes, and promoting that along the caveolae-mediated pathway. Here, we demonstrate the effective internalization of branched polyethylenimine polymers (BPEI), complexed with nucleic acids, by HeLa cells along both pathways. However, transfection efficiency or nuclear ODN delivery primarily occurs via the caveolae-mediated pathway, along which delivery into lysosomes is avoided. Interestingly, inhibition of intracellular protein kinase A (PKA) activity modulates the intracellular trafficking of both poly- and lipoplexes along the clathrin-mediated pathway by impeding trafficking into the late endosomal/lysosomal compartments, thus avoiding degradation. In case of BPEI polyplexes this promotes their transfection efficiency by 2-3 fold. Evidence excludes early endosomes as a major site for BPEI-mediated release/delivery. Rather, we identify a novel compartment, tentatively characterized as a transferrin(-)/rab9(-)/LAMP1(-) compartment, to which cargo within the clathrin-mediated pathway of endocytosis is rerouted upon inhibition of PKA, and which may act as an alternative and effective site of cargo release in gene delivery. Our findings offer new opportunities for improving gene delivery by non-viral based nanoparticles.
纳米颗粒的细胞内进入依赖于各种内吞途径，包括网格蛋白和小窝介导的内吞作用。为了提高递送效率，即治疗和/或细胞生物学的影响，目前的努力旨在避免载体沿着降解的网格蛋白介导途径向溶酶体进行处理，并促进载体沿着小窝介导途径进行处理。在这里，我们证明了带支链的聚乙烯亚胺聚合物（BPEI）与核酸复合物可以通过 HeLa 细胞沿着这两种途径有效地被内化。然而，转染效率或核 ODN 递呈主要通过小窝介导的途径发生，通过该途径可以避免递呈到溶酶体中。有趣的是，抑制细胞内蛋白激酶 A（PKA）的活性通过阻碍进入晚期内体/溶酶体区室的运输，从而避免降解，从而调节这两种聚电解质和脂质体复合物沿着网格蛋白介导的途径的细胞内运输。对于 BPEI 多聚物，这将其转染效率提高了 2-3 倍。有证据表明早期内体不是 BPEI 介导的释放/递呈的主要部位。相反，我们确定了一个新的隔室，暂定为转铁蛋白（-）/rab9（-）/LAMP1（-）隔室，该隔室可作为网格蛋白介导的内吞作用中货物再途径的替代和有效释放部位，在基因递送中。我们的发现为基于非病毒的纳米颗粒改善基因递送提供了新的机会。
