Merkel Olivia M, Germershaus Oliver, Wada Carol K, Tarcha Peter J, Merdan Thomas, Kissel Thomas
Department of Pharmaceutics and Biopharmacy, Philipps-Universitat Marburg, Ketzerbach 63, 35032 Marburg, Germany.
Bioconjug Chem. 2009 Jun;20(6):1270-80. doi: 10.1021/bc9001695.
This study describes the synthesis and characterization of five conjugates of poly(ethylene glycol) modified polyethylenimine (PEG-PEIs) coupled in two different synthesis routes to a nonpeptidic pentacyclic RDG-mimetic for integrin receptor-targeted gene delivery. Synthesis of this panel of different conjugates allowed for systematic analysis of structure-activity relationships. Conjugates were therefore characterized regarding molecular composition, DNA condensation, size, and zeta potential of self-assembled polyplexes. In vitro characterization included investigation of blood compatibility, binding affinity to receptor-positive and receptor-negative cells measured by flow cytometry, cellular uptake quantified by scintillation counting, and efficiency and specificity of transfection assayed by reporter gene expression. In a first synthetic approach, low molecular weight PEI (LMW-PEI) was PEGylated using a heterobifunctional PEG linker and coupling of the RGD-mimetic was achieved at the distal end of PEG chains. In a second synthesis route, the RGD-mimetic was directly coupled to AB-block-copolymers of PEI (25 kDa) and PEG (30 kDa). Interactions of RGD-PEG-LMW-PEI conjugates with DNA were strongly impaired, whereas PEG-PEI-RGD conjugates were more promising candidates due to their physicochemical properties and higher receptor specificity. The binding, uptake, and transfection efficiency in receptor-positive cells was strongly increased upon conjugation of the RGD-mimetic to AB-block-copolymers of PEG-PEI and depended on the degree of peptide substitution. The conjugates of PEG-PEI AB-block-copolymers with low ligand density of the RGD-mimetic appear to be promising candidates for in vivo cancer gene therapy.
本研究描述了通过两种不同合成路线将聚乙二醇修饰的聚乙烯亚胺（PEG-PEIs）的五种共轭物与一种非肽五环RDG模拟物偶联，用于整合素受体靶向基因递送的合成与表征。合成这组不同的共轭物有助于系统分析构效关系。因此，对共轭物进行了分子组成、DNA凝聚、自组装多聚体的大小和zeta电位等方面的表征。体外表征包括血液相容性研究、通过流式细胞术测量与受体阳性和受体阴性细胞的结合亲和力、通过闪烁计数定量细胞摄取，以及通过报告基因表达测定转染效率和特异性。在第一种合成方法中，使用异双功能PEG接头对低分子量PEI（LMW-PEI）进行聚乙二醇化，并在PEG链的远端实现RDG模拟物的偶联。在第二种合成路线中，将RDG模拟物直接偶联到PEI（25 kDa）和PEG（30 kDa）的AB嵌段共聚物上。RGD-PEG-LMW-PEI共轭物与DNA的相互作用受到强烈损害，而PEG-PEI-RGD共轭物因其物理化学性质和更高的受体特异性而更具潜力。将RDG模拟物与PEG-PEI的AB嵌段共聚物偶联后，受体阳性细胞中的结合、摄取和转染效率显著提高，且取决于肽取代程度。具有低RGD模拟物配体密度的PEG-PEI AB嵌段共聚物共轭物似乎是体内癌症基因治疗的有潜力的候选物。
