Original Research Article
Evaluation of siRNA and cationic liposomes complexes as a model for in vitro siRNA delivery to cancer cells

https://doi.org/10.1016/j.colsurfa.2018.06.073Get rights and content

Abstract

Controlled release of genetic material such as small interfering RNA (siRNA) using lipid-based non-viral vectors has gained substantial importance in gene therapy applications. Therefore, the interaction between siRNA and these vectors must be well understood. This study aims to investigate the effect of different molar charge ratios (R+/-) between positive charges from microfluidics-produced cationic liposomes (CL) (egg phosphatidylcholine, 1,2-dioleoyl-3-trimethylammonium-propane and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) and negative charges from siRNA and on physico-chemical and morphological properties of the lipoplexes (CL/siRNA) as well as their in vitro luciferase silencing effect in HeLa cells. R+/- 3.27 was found to be the optimum point for complexation. This finding was confirmed by gel retardation and siRNA accessibility assays. According to Cryo-TEM analysis, the lipoplexes had multi-lamellarity. In vitro transfection efficiency of lipoplexes in HeLa cells was tested at three different siRNA concentrations (10, 25, and 35 nM). Significant knockdown of luciferase by siRNA lipoplexes was observed based on reduced luciferase activity of transfected HeLa cells. Our findings were comparable with the silencing effect of siRNA complexed with Lipofectamine®. No cytotoxicity of lipoplexes was detected at the tested concentrations. This study was essential for further complexation studies which will be performed using microfluidic systems to formulate next-generation lipid-based controlled release systems.

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).

References (55)

  • Y. Zhang et al.

    DC-Chol/DOPE cationic liposomes: a comparative study of the influence factors on plasmid pDNA and siRNA gene delivery

    Int. J. Pharm.

    (2010)
  • M. Ramezani et al.

    The influence of size, lipid composition and bilayer fluidity of cationic liposomes on the transfection efficiency of nanolipoplexes

    Colloids Surf. B Biointerfaces

    (2009)
  • C. Foged et al.

    Liposomes for phospholipase A2 triggered siRNA release: preparation and in vitro test

    Int. J. Pharm.

    (2007)
  • S.A. Ásgeirsdóttir et al.

    Targeted transfection increases siRNA uptake and gene silencing of primary endothelial cells in vitro - a quantitative study

    J. Control. Release

    (2010)
  • J. Li et al.

    Biodegradable calcium phosphate nanoparticle with lipid coating for systemic siRNA delivery

    J. Control. Release

    (2010)
  • R.S. Chang et al.

    Cationic drug-derived nanoparticles for multifunctional delivery of anticancer siRNA

    Biomaterials

    (2011)
  • S. Weisman et al.

    Nanostructure of cationic lipid-oligonucleotide complexes

    Biophys. J.

    (2004)
  • J. Kuntsche et al.

    Cryogenic transmission electron microscopy (cryo-TEM) for studying the morphology of colloidal drug delivery systems

    Int. J. Pharm.

    (2011)
  • B. Geusens et al.

    Ultradeformable cationic liposomes for delivery of small interfering RNA (siRNA) into human primary melanocytes

    J. Control. Release

    (2009)
  • D. Bedi et al.

    Delivery of siRNA into breast cancer cells via phage fusion protein-targeted liposomes

    Nanomed. Nanotechnol. Biol. Med.

    (2011)
  • S.L. Ginn et al.

    Gene therapy clinical trials worldwide to 2012 – an update

    J. Gene Med.

    (2013)
  • F. Cao et al.

    Phosphorothioate‑modified antisense oligonucleotides against human telomerase reverse transcriptase sensitize cancer cells to radiotherapy

    Mol. Med. Rep.

    (2017)
  • A. Fire et al.

    Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans

    Nature

    (1998)
  • K.A. Whitehead et al.

    Knocking down barriers: advances in siRNA delivery

    Nat. Rev. Drug Discov.

    (2009)
  • F. Touzot et al.

    Faster T-cell development following gene therapy compared to haplo-identical hematopoietic stem cell transplantation in the treatment of SCID-X1

    Blood

    (2015)
  • S. Hacein-Bey Abina et al.

    Editorial -outcomes following Gene therapy in patients with severe wiskott-aldrich syndrome

    JAMA

    (2015)
  • A.C. Nathwani et al.

    Long-term safety and efficacy of factor IX Gene therapy in hemophilia B

    N. Engl. J. Med.

    (2014)
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    I. Eş and M. T. Ok contributed equally in this work.

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