Abstract
A histidine-based gemini cationic lipid, which had already demonstrated its efficiency as a plasmid DNA (pDNA) nanocarrier, has been used in this work to transfect a small interfering RNA (siRNA) into cancer cells. In combination with the helper lipid monoolein glycerol (MOG), the cationic lipid was used as an antiGFP-siRNA nanovector in a multidisciplinary study. Initially, a biophysical characterization by zeta potential (ζ) and agarose gel electrophoresis experiments was performed to determine the lipid effective charge and confirm siRNA compaction. The lipoplexes formed were arranged in Lα lamellar lyotropic liquid crystal phases with a cluster-type morphology, as cryo-transmission electron microscopy (cryo-TEM) and small-angle X-ray scattering (SAXS) studies revealed. Additionally, in vitro experiments confirmed the high gene knockdown efficiency of the lipid-based nanovehicle as detected by flow cytometry (FC) and epifluorescence microscopy, even better than that of Lipofectamine2000*, the transfecting reagent commonly used as a positive control. Cytotoxicity assays indicated that the nanovector is non-toxic to cells. Finally, using nano-liquid chromatography tandem mass spectrometry (nanoLC-MS/MS), apolipoprotein A-I and A-II followed by serum albumin were identified as the proteins with higher affinity for the surface of the lipoplexes. This fact could be beyond the remarkable silencing activity of the histidine-based lipid nanocarrier herein presented.
Highlights
The use of nucleic acids as therapeutic agents offers a wide range of possibilities with regard to the treatment of diseases at the molecular genetic level [1,2]
The gemini cationic lipid with functionalized histidine groups in its structure, C3(C16His)2, in combination with the neutral helper lipid monoolein glycerol (MOG) (α = 0.2), has demonstrated in this work its potential to compact, protect and transfect small interfering RNA (siRNA) in two GFP over-expressing cancer cell lines (HeLa-GFP and T731-GFP), provoking in turn the GFP knockdown with efficiency and cell-safety. This affirmation is based on the biophysical study presented that has combined both physicochemical and biochemical experiments to understand the interactions between siRNA and the lipids; the structural patterns of the resulting lipoplexes; and their capacity to cross the cellular membrane, deliver the nucleic acid in the cellular cytoplasm, and knockdown the GFP expression
The histidine-based gemini cationic lipids (GCL) provided the necessary positive and delocalized charge to compact the anionic siRNA molecules by means of a strong electrostatic interaction with an important entropic component associated with the release of Na+ counterions to the bulk
Summary
The use of nucleic acids as therapeutic agents offers a wide range of possibilities with regard to the treatment of diseases at the molecular genetic level [1,2]. The discovery of interference RNA (RNAi) and the development of siRNA molecules have allowed the possibility of controlling a specific and unique mechanism of action in the regulation of genes [3,4] This has been translated into better results in terms of selectivity and efficiency compared to other therapeutic agents used in gene therapy, such as pDNA and oligonucleotides [5,6]. The siRNA molecules can block specific regions in the messenger RNA (mRNA) sequence through the formation of a RNA-induced silencing complex (RISC), suppressing the synthesis of the target pathogenic protein This powerful and selective method for gene silencing can be limited or may even not take place if the siRNA molecules do not reach the cell cytoplasm. The degradation by nucleases present in the bloodstream and their inefficiency to cross the negatively charged cellular membrane are some of the limitations that make the vectorization of nucleic acids necessary
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