Polycationic Macrocyclic Scaffolds as Potential Non-Viral Vectors of DNA: A Multidisciplinary Study

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• 作者：Ana L. Barr谩n-Berd贸n ; Bel茅n Y茅lamos ; Luis Garc铆a-R铆o ; 脪scar Dom猫nech ; Emilio Aicart ; Elena Junquera
• 刊名：ACS Applied Materials & Interfaces
• 出版年：2015
• 出版时间：July 8, 2015
• 年：2015
• 卷：7
• 期：26
• 页码：14404-14414
• 全文大小：585K
• ISSN：1944-8252

文摘

The potential of lipoplexes constituted by the DNA pEGFP-C3 (encoding green fluorescent protein), polycationic calixarene-based macrocyclic vector (CxCL) with a lipidic matrix (herein named TMAC4), and zwitterionic lipid 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) as nontoxic DNA vectors has been analyzed from both biophysical and biochemical perspectives. For that purpose, several experimental methods, such as zeta potential (PALS methodology), agarose gel electrophoresis, small-angle X-ray scattering (SAXS), transmission electronic cryo-microscopy (cryo-TEM), atomic force microscopy (AFM), fluorescence microscopy, and cytotoxicity assays have been used. The electrochemical study shows that TMAC4 has 100% of its nominal charge available, whereas pDNA presents an effective negative charge that is only 10% that of its nominal one. PALS studies indicate the presence of three populations of nanoaggregates in TMAC4/DOPE lipid mixtures, with sizes of approximately 100, 17, and 6 nm, compatible with liposomes, oblate micelles, and spherical micelles, respectively, the first two also being detected by cryo-TEM. However, in the presence of pDNA, this mixture is organized in L_伪 multilamellar structures at all compositions. In fact, cryo-TEM micrographs show two types of multilamellar aggregation patterns: cluster-type at low and moderate CxCL molar fractions in the TMAC4/DOPE lipid mixture (伪 = 0.2 and 0.5), and fingerprint-type (FP), which are only present at low CxCL molar fraction (伪 = 0.2). This structural scenario has also been observed in SAXS diffractograms, including the coexistence of two different phases when DOPE dominates in the mixture. AFM experiments at 伪 = 0.2 provide evidence that pDNA makes the lipid bilayer more deformable, thus promoting a potential enhancement in the capability of penetrating the cells. In fact, the best transfection perfomances of these TMAC4/DOPE-pDNA lipoplexes have been obtained at low CxCL molar fractions (伪 = 0.2) and a moderate-to-high effective charge ratio (蟻_eff = 20). Presumably, the coexistence of two lamellar phases is responsible for the better TE performance at low 伪.