Colloids and Surfaces A: Physicochemical and Engineering Aspects
Original Research ArticleEvaluation of siRNA and cationic liposomes complexes as a model for in vitro siRNA delivery to cancer cells
Graphical abstract
Introduction
Over the past decades, several comprehensive studies have led to the discovery of efficient treatment or prevention methods against genetic diseases. For this purpose, gene therapy, which is the introduction of suitable genetic material into cells to correct abnormal genes, appeared as a critical approach [1]. However, the success of gene therapy relies on safe and competent delivery of genetic material into target cells, which, in most cases, is challenging to achieve. Thus far, several genetic materials such as plasmid DNA (pDNA) [2], anti-sense oligodeoxynucleotides (AS-ODNs) [3], and microRNAs [4] have been employed for gene therapy. Furthermore, since the discovery of RNA interference (RNAi) as one of the major breakthroughs in medicine [5], much attention has been given to small interfering RNA (siRNA), which is a double-stranded RNA molecule that is 21–23 nucleotides long and shows a silencing effect on genes via RNAi [6]. Therefore, the delivery of siRNA into cells in order to silence them shows great potential for gene therapy.
Numerous viral and non-viral vectors have been employed in clinical trials of gene therapy. Nevertheless, ∼70% of these trials were performed with viral vectors such as retroviral [7], lentiviral [8], adeno-associated viral [9], and adenoviral vectors [10]. Despite all of the advanced studies performed with viral vectors, they present many limitations such as immunogenicity, toxicity, low gene loading, complexity of vector design, and even carcinogenesis [11,12]. The use of non-viral vectors exhibits great potential to overcome these limitations, particularly given safety considerations. At this point, cationic liposomes (CLs), spherical assemblies of lipids that consist of polar head groups and hydrophobic tails, are important candidates for siRNA delivery due to electrostatic interactions [13] and similarities with the cell membrane.
Besides the study of liposomes’ composition and potential as therapeutics, novel production methods have also been explored. Several techniques have been employed in order to produce liposomes such as reversed phase evaporation [14], thin-film hydration [15], and the solvent injection technique [16]. However, conventional methods usually result in uncontrollable and heterogeneous liposome production, which usually complicates in vivo use [17]. Hence, advanced methods have been developed for more suitable liposome production. At this point, microfluidics, which is the engineering of fluids at micron scales, appears as an emerging technology [18]. Compared to conventional methods, microfluidics exhibits economic and technical advantages such as low reagent use, low-cost design, high control, reduced reaction time, minimization of the effect of mass and heat transfer, low energy consumption, real time data acquisition, and easy automation [19]. Different investigations have been previously performed to evaluate the complexation efficiency between CLs and DNA [20]. Recently, our research group demonstrated the efficient microfluidic complexation of CLs using egg phosphatidylcholine (EPC), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) lipids with pDNA. The study revealed the viability of this system for efficient pDNA delivery [21,22]. Hence, the evaluation of this carrier system for siRNA delivery is also essential for studies in gene therapy.
The electrostatic interaction between siRNA and CLs determines the physico-chemical properties of lipoplexes (siRNA/CL), and is directly related to in vitro and in vivo applications. Living organisms consist of several biological and chemical barriers that complicate the efficient delivery of genetic materials to the treatment site [23]. Therefore, physico-chemical characterization of lipoplexes is critical in order to increase success rates of in vivo siRNA delivery. In this context, one of the important parameters to evaluate is the molar charge ratio (R+/-) between siRNA and CLs as well as its effect on physico-chemical parameters. However, variable lipid composition or surface modifications of CLs that produce target-specific lipoplexes could alter the R+/- value. Hence, this value should be supported with additional tests such as gel retardation, fluorescence assays, or morphological analyses using Cryo-TEM.
Although there are several important studies investigating physico-chemical properties of liposomes complexed with oligonucleotides, pDNA, and polynucleotides [24,25], it is known that lipid composition can interfere in the final lipoplex aggregation [26]. In this context, there is no systematic study reported in the literature that evaluates the lipoplexes of siRNA and CLs using EPC/DOTAP/DOPE phospholipids. Therefore, new investigations are necessary to obtain this critical information. In the present study, siRNA molecules were complexed using a conventional bulk mixing technique with CLs that were produced via a hydrodynamic flow-focusing microfluidic device. Structural stability between the lipid composition of EPC/DOTAP/DOPE and DNA was previously studied [27]. In the present study, the same lipid composition was evaluated for cationic liposome production and the capacity to form lipoplexes with siRNA at different molar charge ratios. Finally, the lipoplexes formed at the optimum molar charge ratio were tested to determine in vitro transfection efficiency, targeted gene silencing, and cytotoxicity in human epithelial carcinoma (HeLa) cells.
Section snippets
Materials
The lipids egg phosphatidylcholine (EPC), 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were purchased from Lipoid (Ludwigshafen, Germany) and used with no further purification. Ethanol used to dissolve lipids was obtained from Labsynth (São Paulo, Brazil). The Sylgard® 184 Silicone Elastomer Kit for fabrication of microfluidic devices was purchased from Dow Corning (Auburn, MI, United States). Deionized water was obtained from
Results and discussion
In our previous studies, the production of cationic liposomes (CLs) using an EPC/DOTAP/DOPE lipid formulation was investigated in order to evaluate the capacity of CLs as a vector system for plasmid DNA delivery [21,22,31]. In the present work, the complexation between EPC/DOTAP/DOPE-based liposomes with siRNA is characterized. We evaluated the physico-chemical properties of lipoplexes as a function of the molar charge ratio (R+/-) and subsequently studied the in vitro silencing as well as
Conclusion
We have studied the physico-chemical properties of siRNA lipoplexes built with different molar charge ratios between siRNA and microfluidics-produced cationic liposomes composed of EPC/DOTAP/DOPE lipids. The zeta potential profile of lipoplexes obtained in this study, representing the constant change in surface charge of the lipoplexes from negative to positive values, was found to be similar to that of previous studies performed with pDNA. The lipoplexes formulated with siRNA showed an average
Conflict of interest
Authors declare that they have no conflict of interest.
Acknowledgments
I. Eş gratefully acknowledges the financial support of the São Paulo Research Foundation (FAPESP) (Grant # 2015/14468-0). M. T. Ok acknowledges the financial support of the MIT International Science and Technology Initiatives Program (MISTI-Brazil).
The authors also thank the Microfabrication Laboratory and the Electron Microscopy Laboratory of Brazilian Nanotechnology National Laboratory (LNNano) at The Brazilian Center for Research in Energy and Materials (CNPEM).
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I. Eş and M. T. Ok contributed equally in this work.