Interaction of DNA with likely-charged lipid monolayers: An experimental study

https://doi.org/10.1016/j.colsurfb.2019.02.058Get rights and content

Highlights

  • Langmuir monolayer reveal complexation of DOPE/DOPG and DNA.

  • Interaction occurs in the presence and absence of electrolyte and appears non-specific.

  • Surface potential data reveals specificity of the interaction.

  • Normal dipole moment per molecule determines the microstructure of the monolayer.

Abstract

Anionic lipids are increasingly being used in lipoplexes for synthetic gene vectors as an alternative to cationic lipids. This is primarily due to their lower toxicity, which makes them biocompatible and adaptable to be tissue specific. However, anionic lipoplexes require the presence of multivalent cations to promote the electrostatic attraction between DNA and anionic lipid mono- and bilayers. In this work we provide for the first time experimental results of the adsorption of linear DNA onto anionic/zwitterionic lipid monolayers without any addition of cations. This is demonstrated experimentally by means of Langmuir monolayers of DOPE/DOPG (1:1) lipids spread on a water subphase that contains calf thymus DNA. The adsorption of DNA onto anionic/zwitterionic lipid monolayers is discussed in terms of the surface pressure-molecular area isotherms recorded in the absence and in the presence of different electrolytes. Measurements of the surface potential provide additional evidence of the different interaction of DNA anionic/zwitterionic lipid monolayers depending on the presence and nature of electrolyte. These experimental results are further analysed in terms of the overall dipole moment normal to the monolayers providing new insight into the behaviour of anionic lipoplexes and the role of zwitterionic lipids.

Introduction

Lipoplexes (liposomes-DNA) are the most used non-viral vectors in transfection due to their capability to protect genes and introduce them into the target cell [[1], [2], [3]]. Conventionally, mixtures of cationic and zwitterionic lipids are used to spontaneously complexate with DNA [[4], [5], [6], [7], [8], [9], [10], [11], [12], [13]]. However, cationic liposomes induce a large number of adverse effects to DNA delivery such as the lack of efficiency in the transfection, instability of storage, and potential toxicity of the cationic lipid in a cellular environment [[14], [15], [16], [17], [18], [19], [20], [21]]. In contrast, self-assembled DNA delivery systems composed of DNA and anionic/zwitterionic lipids are considered to be promising tools for gene therapy due to their lower cytotoxicity [20,[22], [23], [24], [25], [26], [27], [28], [29]]. Furthermore, naturally occurring mixtures of zwitterionic and anionic phospholipids, which are used to prepare anionic lipoplexes, do not react with the serum proteins, thus enhancing transfection efficiency. Unfortunately, while the complexation of cationic lipids and DNA, based on electrostatic attraction, has been well studied, the molecular mechanisms underlying the formation of anionic lipoplexes still remain unclear [30,31]. In general, the interactions and mechanisms that govern the adsorption of polyelectrolytes (such as DNA) onto like-charged surfaces are a current theme that has been scarcely studied [[30], [31], [32], [33], [34], [35], [36], [37], [38], [39]]. In the case of the adsorption of DNA onto likely-charged lipid surfaces, the presence of multivalent cations is usually required to facilitate the attraction between DNA and the likely-charged surface through direct electrostatic bridging interactions. For instance, Liang et al investigated the structure and interactions of anionic lipoplexes in the presence of different divalent metal cations such as Mg2+, Ca2+, Co2+, Cd2+, Mn2+ and Zn2+ by performing SAXS experiments and confocal microscopy [24]. In this sense, we have compared recently the most relevant formulations for anionic lipoplexes reported in the literature (see Table 1 of the review [30]). The information extracted from this table indicates two important features: on the one hand, the most popular lipids used in anionic lipoplexes are DOPE (1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine) and DOPG (dioleoylphosphatidylglycerol) as zwitterionic and anionic lipids, respectively. On the other hand, divalent cations (especially calcium) are the most employed ions to mediate the adsorption of DNA onto lipid layers. These lipids are known to occur endogenously in vivo and complexate with DNA molecules using Ca2+, being the resulting ternary complex capable of transfecting mammalian cells [23].

In general, the transfection efficiency of anionic lipoplexes depends on the optimal formulation of the complexes, which in turn is a function of the type, concentration and structure of the ternary system lipid-cation-DNA, as well as the binding of lipid-cation, cation-DNA and DNA-lipid. Under this scenario, Langmuir monolayers are a versatile and well established characterisation method to elucidate structural and mechanical properties of DNA interacting with different systems to: octadecylamine monolayers [40], zwitterionic lipid monolayers [41], cationic lipid monolayers [42,43] and anionic lipid monolayers [30]. Namely, this methodology was used by Frantescu et al to characterise the adsorption of DNA onto plasma cell membranes for DNA electrotransfer purposes. More recently, Langmuir monolayers have been also used to investigate the complexation of negatively charged mixed phospholipid monolayers composed of DPPC (dipalmitoylphosphatidylcholine) and DPPS (dipalmitoylphosphatidylserine) with DNA, mediated by Ca2+ [34,44].

Accordingly, in this work we use Langmuir monolayers to investigate the interaction of linear DNA with the most accepted phospholipid mixture found in anionic lipoplexes (DOPE/DOPG) in the absence and presence of electrolyte. Moreover, we have studied the effect of different monovalent and divalent cations in order to evaluate the ionic specificity of the resulting interaction. Hence, we measure the surface pressure (π) – molecular area (A) isotherms of lipids and DNA spread on different subphases. In parallel to the π, we record the surface potential (ΔV) upon compression of the monolayer. From these experiments, we can calculate the dipole moment normal to the interface (μ). This provides a detailed analysis and discussion of the nature of the interaction between DNA and anionic/zwitterionic lipids relevant to the behaviour of anionic lipoplexes.

Section snippets

Materials

The lipids used in this work are: zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) and anionic dioleoylphosphatidylglycerol (DOPG), purchased from Avanti Polar Lipids (> 99% purity) and used as supplied. Solutions of DOPE/DOPG 1:1 (mol/mol) mixtures were prepared by dissolving the phospholipids in chloroform at ambient conditions. These spreading solvents were of HPLC grade (Scharlau) and used as received. Double stranded DNA from calf thymus was purchased from Sigma (>98%

Results and discussion

Although mixtures of DOPE and DOPG lipids are the most popular formulation used in anionic lipoplexes, we have not found explicit reported monolayers of this mixture in the literature. Accordingly, Fig. 1 shows the π-A isotherms of DOPE/DOPG 1:1 spread on pure water and aqueous DNA subphases. The shape of the isotherm recorded on pure water reveals that the lipid mixture remains in a 2D gaseous state until 90 ± 2 Å2/molec where it enters a LE state which endures until collapse at π = 47 ± 1

Conclusions

In this work, we have measured the π-A isotherms of DOPE/DOPG 1:1 monolayers on different subphases to study the interaction of anionic/zwitterionic lipid with DNA. Although it is commonly believed that multivalent cations are required to bring together DNA and likely charged lipids, the π-A isotherms presented here clearly reveal, for the first time, the complexation of DOPE/DOPG monolayers and DNA in the absence of added cations. This outstanding finding is also confirmed by recording the ΔV-A

Acknowledgments

The authors thank the Spanish “Ministerio de Economía y Competitividad (MINECO), Plan Nacional de Investigación, Desarrollo e Innovación Tecnológica (I+D + i)” (Projects FIS2016-80087-C2-1-P, FIS2016-80087-C2-2-P, RYC-2012-10556, MAT2015-63644-C2-1-R), Consejería de Innovación de la Junta de Andalucía (P09-FQM-4698,SOMM17/6105/UGR and PI12.2956), CEI-BioTic (Project BS28-2015) and the European Regional Development Fund (ERDF).

References (61)

  • A. Frantescu et al.

    Interfacial ternary complex DNA/Ca/lipids at anionic vesicle surfaces

    Bioelectrochemistry

    (2006)
  • M. Kapoor et al.

    Physicochemical characterization of anionic lipid-based ternary siRNA complexes

    Biochim. Biophys. Acta – Biomembr.

    (2012)
  • J. Faraudo et al.

    Competing forces in the interaction of polyelectrolytes with charged interfaces

    Curr. Opin. Colloid Interface Sci.

    (2013)
  • R.S. Dias et al.

    Polyelectrolyte condensation in bulk, at surfaces, and under confinement

    Adv. Colloid Interface Sci.

    (2010)
  • D. Mirska et al.

    Biophysical and biochemical properties of a binary lipid mixture for DNA transfection

    Colloids Surf. B Biointerfaces

    (2005)
  • H. Möhwald

    Phospholipid monolayers

    Struct. Dyn. Membr. From Cells to Vesicles

    (1995)
  • A. Michanek et al.

    RNA and DNA interactions with zwitterionic and charged lipid membranes - a DSC and QCM-D study

    Biochim. Biophys. Acta.

    (2010)
  • A. Martín-Molina et al.

    Effect of calcium and magnesium on phosphatidylserine membranes: experiments and all-atomic simulations

    Biophys. J.

    (2012)
  • D.D. Lasic

    Liposomes in Gene Delivery

    (1997)
  • S. Simões et al.

    Cationic liposomes for gene delivery

    Expert Opin. Drug Deliv.

    (2005)
  • A. Rodríguez-Pulido et al.

    A theoretical and experimental approach to the compaction process of DNA by dioctadecyldimethylammonium bromide/zwitterionic mixed liposomes

    J. Phys. Chem. B

    (2009)
  • M. Muñoz-Úbeda et al.

    Effect of lipid composition on the structure and theoretical phase diagrams of DC-Chol/DOPE-DNA lipoplexes

    Biomacromolecules

    (2010)
  • M. Muñoz-Úbeda et al.

    Gene vectors based on DOEPC/DOPE mixed cationic liposomes: a physicochemical study

    Soft Matter.

    (2011)
  • A.L. Barrán-Berdón et al.

    Ribbon-type and cluster-type lipoplexes constituted by a chiral lysine based cationic gemini lipid and plasmid DNA

    Soft Matter.

    (2012)
  • D. Paiva et al.

    The effect of a fluorinated cholesterol derivative on the stability and physical properties of cationic DNA vectors

    Soft Matter.

    (2013)
  • K. Lappalainen et al.

    Comparison of cell-proliferation and toxicity assays using 2 cationic liposomes

    Pharm. Res.

    (1994)
  • Y.N.S et al.

    Cationic lipid-mediated gene delivery to the airways

  • W. Guo et al.

    Efficient Gene delivery using anionic liposome-complexed polyplexes (LPDII)

    Biosci. Rep.

    (2000)
  • S. Dokka et al.

    Oxygen radical-mediated pulmonary toxicity induced by some cationic liposomes

    Pharm. Res.

    (2000)
  • S.D. Patil et al.

    Anionic liposomal delivery system for DNA transfection

    AAPS J.

    (2004)
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