Research Article: Synthesis and evaluation of L-arabinose-based cationic glycolipids as effective vectors for pDNA and siRNA in vitro

Date Published: July 3, 2017

Publisher: Public Library of Science

Author(s): Bo Li, Wanrong Guo, Fan Zhang, Meiyan Liu, Shang Wang, Zhonghua Liu, Shuanglin Xiang, Youlin Zeng, Valentin Ceña.


Glycolipids might become a new type of promising non-viral gene delivery systems because of their low cytotoxicity, structural diversity, controllable aqua- and lipo-solubility, appropriate density and distribution of positive charges, high transfer efficiency and potential targeting function. In this study, four kinds of L-arabinose-based cationic glycolipids (Ara-DiC12MA, Ara-DiC14MA, Ara-DiC16MA and Ara-DiC18MA) containing quaternary ammonium as hydrophilic headgroup and two alkane chains as hydrophobic domain were synthesized and characterized. They were observed to have strong affinities for plasmid DNA (pDNA) and siRNA, the pDNA can be completely condensed at N/P ratio less than 2, and the siRNA can be completely retarded at N/P ratio less than 3. The dynamic light scattering (DLS) experiment and atomic force microscopy (AFM) experiment demonstrated that cationic lipids and their lipoplexes possessed suitable particle sizes with near-spherical shape and proper ζ-potentials for cell transfection. The Ara-DiC16MA liposome was found to have good transfection efficacy in HEK293, PC-3 and Mat cells compared with other three kinds of liposomes, and also maintain low cytotoxicity and better uptake capability in vitro. Furthermore, the gene silencing assay showed that Ara-DiC14MA and Ara-DiC16MA liposomes have demonstrated effective delivery and higher gene knockdown activity (>80%) in the above mentioned cells than Lipofectamine 2000. These results indicated Ara-DiC16MA can be developed for efficient and low toxic gene delivery.

Partial Text

The gene therapy, using various foreign nucleic acids, containing plasmid DNA (pDNA), small interfering RNAs (siRNAs) and oligonucleotides[1–5] to down or unregulated the expression of defective genes, is a promising strategy for treatment of various inherited or acquired diseases, such as AIDS[6, 7], immunodeficiency[8, 9], degenerative disorders[10] and cancer[11, 12]. Gene therapy has been attracted great attention in the field of biomedical sciences and benefited to develop the human health level over the past few decades. A success of gene therapy is required to design and develop a high efficient gene delivery systems to overcome the obstacles in different stages [13, 14]. In the past decades, a lot of viral and non-viral vector systems were designed and developed to delivery foreign nucleic acids [15–17]. Viral vectors including retrovirus vectors, lentivirus vectors, adenovirus vectors and adeno-associated virus vectors [18], are superior in efficient delivery of genetic material into cells in vivo, and were widely employed in early clinical trials [19, 20]. However, some side effects including host immune and inflammatory reactions, oncogenic transformation by unpredicted, potential to form replication-competent virions and pathogenic risks of the viral vectors severely limited their applications [21–26]. Recently, non-viral delivery systems were keenly explored to avoid the potential problems meet in viral vectors [27]. However, several hurdles and major trafficking barriers, such as effective cellular uptake capability [28], DNA protection and escaped from acidic vesicles (for example, endosomes and lysosomes) [29, 30], nuclear transport targetability to specific cell types and infection of non-dividing cells, need to be solved during constructing an efficient non-viral delivery systems [31, 32].

In summary, as candidates for non-viral gene vectors, a new kind of L-arabinose based cationic lipids bearing different hydrophobic alkane chain were successfully synthesized. Gel electrophoresis assay, AFM images and dynamic light scattering experiments demonstrate that all the liposomes could efficiently bind and compact pDNA and siRNA into nanoparticles with proper size and zeta potential. The gene transfection study showed that lipid Ara-DiC16MA has maximum transfection efficacy in HEK293, PC-3 and Mat cells compared with other three kinds of liposomes, which maintains low cytotoxicity and better cellular uptake capability in vitro. The luciferase knockdown experiments of Ara-DiC16MA and Ara-DiC14MA liposomes demonstrated the higher RNAi activity for siRNA delivery than Lipofectamine 2000 at high N/P ratio. We believed that L-arabinose-based cationic liposomes with differences hydrophobic alkane chain may be a key factor to ensure highly efficient transfection in vitro. Additionally, these works strengthen the validity of the carbohydrates based cationic lipids as effective non-viral gene vectors for gene delivery.




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