Interaction of DNA with likely-charged lipid monolayers: An experimental study
Graphical abstract
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)
- et al.
Self-organization of nucleic acids in lipid constructs
Curr. Opin. Colloid Interface Sci.
(2016) - et al.
Principles of electrostatic interactions and self-assembly in lipid/peptide/DNA systems: applications to gene delivery
Adv. Colloid Interface Sci.
(2014) - et al.
Cationic liposomes and nucleic acids
Curr. Opin. Colloid Interface Sci.
(2001) Structures of lipid–DNA complexes: supramolecular assembly and gene delivery
Curr. Opin. Struct. Biol.
(2001)- et al.
Comparison of different commercially available cationic liposome–DNA lipoplexes: parameters influencing toxicity and transfection efficiency
Colloids Surf. B Biointerfaces
(2009) - et al.
Transfection of plasmid DNA by nanocarriers containing a gemini cationic lipid with an aromatic spacer or its monomeric counterpart
Colloids Surf. B Biointerfaces
(2018) - et al.
Toxicity and immunomodulatory activity of liposomal vectors formulated with cationic lipids toward immune effector cells
Biochim. Biophys. Acta-Biomembr.
(1997) - et al.
Major limitations in the use of cationic liposomes for DNA delivery
Int. J. Pharm.
(1998) - et al.
Toxicity of cationic lipids and cationic polymers in gene delivery
J. Control. Release.
(2006) - et al.
Biophysical characterization of anionic lipoplexes
Biochim. Biophys. Acta – Biomembr.
(2005)
Interfacial ternary complex DNA/Ca/lipids at anionic vesicle surfaces
Bioelectrochemistry
Physicochemical characterization of anionic lipid-based ternary siRNA complexes
Biochim. Biophys. Acta – Biomembr.
Competing forces in the interaction of polyelectrolytes with charged interfaces
Curr. Opin. Colloid Interface Sci.
Polyelectrolyte condensation in bulk, at surfaces, and under confinement
Adv. Colloid Interface Sci.
Biophysical and biochemical properties of a binary lipid mixture for DNA transfection
Colloids Surf. B Biointerfaces
Phospholipid monolayers
Struct. Dyn. Membr. From Cells to Vesicles
RNA and DNA interactions with zwitterionic and charged lipid membranes - a DSC and QCM-D study
Biochim. Biophys. Acta.
Effect of calcium and magnesium on phosphatidylserine membranes: experiments and all-atomic simulations
Biophys. J.
Liposomes in Gene Delivery
Cationic liposomes for gene delivery
Expert Opin. Drug Deliv.
A theoretical and experimental approach to the compaction process of DNA by dioctadecyldimethylammonium bromide/zwitterionic mixed liposomes
J. Phys. Chem. B
Effect of lipid composition on the structure and theoretical phase diagrams of DC-Chol/DOPE-DNA lipoplexes
Biomacromolecules
Gene vectors based on DOEPC/DOPE mixed cationic liposomes: a physicochemical study
Soft Matter.
Ribbon-type and cluster-type lipoplexes constituted by a chiral lysine based cationic gemini lipid and plasmid DNA
Soft Matter.
The effect of a fluorinated cholesterol derivative on the stability and physical properties of cationic DNA vectors
Soft Matter.
Comparison of cell-proliferation and toxicity assays using 2 cationic liposomes
Pharm. Res.
Cationic lipid-mediated gene delivery to the airways
Efficient Gene delivery using anionic liposome-complexed polyplexes (LPDII)
Biosci. Rep.
Oxygen radical-mediated pulmonary toxicity induced by some cationic liposomes
Pharm. Res.
Anionic liposomal delivery system for DNA transfection
AAPS J.
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