Effects of a Delocalizable Cation on the Headgroup of Gemini Lipids on the Lipoplex-Type Nanoaggregates Directly Formed from Plasmid DNA

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• 作者：Santosh K. Misra ; M贸nica Mu帽oz-脷beda ; Sougata Datta ; Ana L. Barr谩n-Berd贸n ; Clara Aicart-Ramos ; Pablo Castro-Hartmann ; Paturu Kondaiah ; Elena Junquera ; Santanu Bhattacharya ; Emilio Aicart
• 刊名：Biomacromolecules
• 出版年：2013
• 出版时间：November 11, 2013
• 年：2013
• 卷：14
• 期：11
• 页码：3951-3963
• 全文大小：786K
• 年卷期：v.14,no.11(November 11, 2013)
• ISSN：1526-4602

文摘

Lipoplex-type nanoaggregates prepared from pEGFP-C3 plasmid DNA (pDNA) and mixed liposomes, with a gemini cationic lipid (CL) [1,2-bis(hexadecyl imidazolium) alkanes], referred as (C₁₆Im)₂C_n (where C_n is the alkane spacer length, n = 2, 3, 5, or 12, between the imidazolium heads) and DOPE zwitterionic lipid, have been analyzed by zeta potential, gel electrophoresis, SAXS, cryo-TEM, fluorescence anisotropy, transfection efficiency, fluorescence confocal microscopy, and cell viability/cytotoxicity experiments to establish a structure鈥揵iological activity relationship. The study, carried out at several mixed liposome compositions, 伪, and effective charge ratios, 蟻_eff, of the lipoplex, demonstrates that the transfection of pDNA using CLs initially requires the determination of the effective charge of both. The electrochemical study confirms that CLs with a delocalizable positive charge in their headgroups yield an effective positive charge that is 90% of their expected nominal one, while pDNA is compacted yielding an effective negative charge which is only 10鈥?5% than that of the linear DNA. SAXS diffractograms show that lipoplexes formed by CLs with shorter spacer (n = 2, 3, or 5) present three lamellar structures, two of them in coexistence, while those formed by CL with longest spacer (n = 12) present two additional inverted hexagonal structures. Cryo-TEM micrographs show nanoaggregates with two multilamellar structures, a cluster-type (at low 伪 value) and a fingerprint-type, that coexist with the cluster-type at moderate 伪 composition. The optimized transfection efficiency (TE) of pDNA, in HEK293T, HeLa, and H1299 cells was higher using lipoplexes containing gemini CLs with shorter spacers at low 伪 value. Each lipid formulation did not show any significant levels of toxicity, the reported lipoplexes being adequate DNA vectors for gene therapy and considerably better than both Lipofectamine 2000 and CLs of the 1,2-bis(hexadecyl ammnoniun) alkane series, recently reported.