Anionic Nucleotide−Lipids for In Vitro DNA Transfection

Anionic Nucleotide-Lipids for In Vitro DNA Transfection

Salim Khiati,†,‡ Nathalie Pierre,†,‡ Soahary Andriamanarivo,†,‡ Mark W. Grinstaff,| Nessim Arazam,§

Frederic Nallet,§ Laurence Navailles,§ and Philippe Barthelemy*,†,‡

INSERM U869, Bordeaux, F-33076, France, Universite Victor Segalen Bordeaux 2, Bordeaux, F-33076, France, UniversiteBordeaux-1 CNRS, Centre de Recherche Paul Pascal, 115 avenue A. Schweitzer, F-33600 Pessac, France, and Departments ofBiomedical Engineering, Metcalf Center for Science and Engineering, Boston University, Boston, Massachusetts 02215.Received April 10, 2009; Revised Manuscript Received July 20, 2009

A family of new anionic nucleotide based lipids featuring thymidine-3′-monophosphate as nucleotide and 1,2-diacyl-sn-glycerol as lipid moiety for in Vitro delivery of nucleic acids is described. The nucleotide lipids wereprepared in three steps starting from 1,2-diacyl-sn-glycerols and 2′-deoxythymidine-3′-phosphoramidite. Gelelectrophoresis experiments show that nucleotide-based lipid-DNA complexes are observed at Ca2+ concentrationhigher than 1 mM. The transfection experiments carried out on mammalian Hek cell lines clearly demonstratethat the nucleotide moiety enhances the transfection efficacy of the natural anionic DPPA and DPPG lipids.SAXS studies indicate that the enhancement in transfection for nucleotide-based lipid formulations compared tothose of the abasic natural derivative (DPPA) is likely due to the presence of the 2D columnar inverted hexagonalphase (HII) with a unit cell parameter a ) 69.1 Å in the nucleotide lipid formulations. The cytotoxicity studiesof lipoplexes, evaluated against Hek cells using an MTS assay, revealed that palmitoyl nucleotide derivativecomplexes were not toxic even after 4 h of incubation, thus indicating that the anionic nucleotide lipids presentedin this work offer an alternative to cationic transfection reagents.

INTRODUCTION

Therapies ( using natural or synthetic nucleic acids haveemerged as promising strategies for developing cures againstinherited and acquired disorders or diseases, includingcancer (1, 2). One of the significant advantages of nucleicacid based drugs over low molecular weight pharmaceuticaldrugs is their selectivity for their biological target and theirspecificity of ( action (3). To be effective, nucleic acid basedtherapies require the use of vector platforms for deliveringvarious genetic materials into cells. The earliest syntheticDNA delivery systems, first introduced in the 1980s (4, 5),were based on cationic lipids. Today, cationic lipids are stillimportant transfecting tools for research, likely because ofthe researchers’ ability to control their chemical and physicalproperties (6). However, most of these cationic systemsexhibit specific problems outlined by Burgess et al. (7),including cytotoxicity, inactivation in the presence of serum,and inefficiency in ViVo, that scientists are attempting toaddress (8-12). To overcome these limitations, alternativecationic synthetic vectors ( are currently under investigationby a number of research groups (13-16). In ( recent years,there has also been intense research activity in the develop-ment of various innovative lipid systems, including pH-sensitive fusiogenic polymer-modifed liposomes (17), pH-sensitive pegylated lipoplexes (18), nucleolipoplexes (19-21),imidazole (22) and charge reversal lipids (23, 24), lipopoly-thiourea (25), lipophilic peptides (26), zwitterionic lipids (27),and glycosylated lipids (28). Additionally, amphiphilespossessing a nucleoside as a lipid headgroup and lipophilicalkyl chains have been identified as promising tools for gene

delivery into cells (19-21) (28-30) and for self-assemblingnucleic acid-nucleolipid supramolecular systems (31-40).

Examples of anionic amphiphiles for gene delivery are rare.Recently, an anionic amphiphile (1,2-dioleoyl-sn-glyerol-3-[phospho-rac-(1-glycerol] sodium salt), zwitterionic amphiphile(DOPE), and Ca2+ system have been reported that exhibitedtransfection efficiencies similar to the cationic lipid lipo-fectamine (7) (41). To our knowledge, no reports of anionicnucleotide based amphiphiles for gene transfection have beenreported.

Within this context, we hypothesized that an anionic am-phiphile possessing a nucleotide polar head for DNA-lipidassociation would enable formation of lipoplexes in the presenceof Ca2+ and afford in Vitro tranfection. For this purpose, wesynthesized a series of anionic nucleotide-lipids with a thy-midine nucleotide polar head and various 1,2-diacyl-sn-glycerolof C12, C14, and C16 chain length as hydrophobic species. Thepotential of these novel anionic nucleotide based lipids for DNAdelivery and expression was investigated. Herein, we report thesynthesis, physicochemical studies, cytotoxicity, and in Vitrotransfection results of these new anionic nucleotide based lipids(Scheme 1).

EXPERIMENTAL PROCEDURES

Materials. General Experiments and Analytical Condi-tions. Unless noted otherwise, all starting materials and solventswere obtained from Aldrich, Bachem, Genzyme, and GlenResearch and were used without further purification. Allcompounds were characterized using standard analytical andspectroscopic techniques such as 1H, 13C, and 31P spectroscopy(apparatus Bruker Avance DPX-300, 1H at 300.13 MHz, 13C at75.46 MHz, and 31P at 121.49 MHz) and mass spectrometry(Instrument JEOL SX 102, NBA matrix). The NMR chemicalshifts are reported in ppm relative to tetramethylsilane usingthe deuterium signal of the solvent (CDCl3) as a heteronuclearreference for 1H. The 1H NMR coupling constants, Js, arereported in Hz. TEM microscopy experiments were performed

* Corresponding author. [email protected].† INSERM U869.‡ Universite Victor Segalen Bordeaux 2.| Boston University.§ Universite Bordeaux-1 CNRS.

Bioconjugate Chem. 2009, 20, 1765–1772 1765

10.1021/bc900163s CCC: $40.75 2009 American Chemical SocietyPublished on Web 08/27/2009

with a Hitachi H 7650 (negative staining with uranyl acetate1% in water, nickel carbon coated grids). Fluorescence micros-copy experiments were performed on a Zeiss axiovert 200. Silicagel 60 (particle size: 45 µm) was used for flash chromatography(Biotage). Thin layer chromatograms were performed withaluminum plates coated with silica gel 60 F254 (Merck). Therevealing solution for TLC is an anisaldehyde solution (85 mLethanol, 10 mL acetic acid, 5 mL sulfuric acid, and 0.5 mLp-anisaldehyde).

Synthesis. Thymidine 3′-(1,2-Dilauroyl-sn-glycero-3-phos-phate), diC12-3′-dT, Compound 1. 5′-O-(4,4′-Dimethoxytrityl)-2′-deoxythymidine-3′-[(2-cyano-ethyl)-N,N-diisopropyl)] phos-phoramidite (0.500 g, 1 equiv, 0.67 mmol), 1,2-dilauroyl-sn-glycerol (0.398 g, 1.3 equiv, 0.87 mmol/dissolved in 3 mL ofTHF) and a tetrazole solution in acetonitrile (0.45 M, 2 mL,1,3 equiv, 0,87 mmol) were dissolved in 3 mL of dry acetonitrileunder argon. The reaction mixture was stirred for 7 h at roomtemperature followed by oxidation with 40 mL of a solution ofI2 (0.02 M in THF/Pyr/H2O). After 12 h at room temperature,the solvent was evaporated under high vacuum to yieldintermediate products. The contents of the reaction flask weredissolved in 10 mL of methylene chloride and then washed firstwith 3 × 10 mL of HCl (0.1 N) and second with 3 × 10 mL ofa saturated solution of Na2S2O3. Product 1 (160 mg) was isolatedafter purification on silica gel (DCM/MeOH/TEA from 98:2:1to 50:49:1). Yield: 33% Rf ) 0.5 (DCM/MeOH 7:3).

1H NMR (300 MHz, CDCl3): δ in ppm 0.83 (t, 6H, J ) 6.8Hz, 2CH3), 1.21 (m, 32H, 16CH2), 1.54 (m, 4H, 2CH2), 1.86(s, 3H, Me base), 2.25 (m, 4H, 2CH2), 3.04 (t, 2H), 3.7-4.4(m, 7H, 2CH2(glycerol), H4′, H5′), 5.28 (m, 1H, CH glycerol),6.21 (t, 1H, J ) 6.5 Hz, H1′), 7.61 (s,1H, H base).

13C NMR (75 MHz, CDCl3): δ in ppm 12.5 (CH3 base), 14.0(CH3 chain), 19.6 (CH2), 19.7 (CH2), 22.6 (CH2), 24.8 (CH2),29.0-29.5 (CH2), 31.9 (CH2), 33.9 (CH2), 34.1 (CH2), 61.5(CH2), 61.7 (CH2), 62.5 (CH2), 62.6 (CH2), 66.1 (CH2), 66.2(CH2), 69.1 (CH), 78.8 (CH), 85.5 (CH), 86.1 (CH), 110.7 (Cbase), 136.2 (CH base), 150.5 (CdO base), 164.0 (CdO base),172.9 (CdO chain), 173.3 (CdO chain).

31P NMR (121 MHz, CDCl3): δ 2.1 ppm. High-resolutionESI MS- [M-H]-, theorical m/z ) 759.4197, observed m/z )759.4182.

Thymidine 3′-(1,2-Dimyristoyl-sn-glycero-3-phosphate) diC14-3′-dT, Compound 2. A procedure similar to that used for

compound 1 was followed here. 5′-Dimethoxytrityl-2′-deox-ythymidine, 3′-[(2-cyanoethyl)-N,N-diisopropyl)]-phosphoramid-ite (500 mg, 1 equiv, 0.67 mmol), 1,2-dimyristoyl-sn-glycerol(447 mg, 1.3 equiv, 0.87 mmol), and a tetrazole solution 0.45M in acetonitrile (2 mL, 0.87 mmol) were dissolved in dryacetonitrile under argon. Product 2 (160 mg) was isolated afterpurification on silicagel (DCM/MeOH/TEA from 98:2:1 to 50:49:1). Yield: 17% Rf ) 0.6 (DCM/MeOH 7:3).

1H NMR (300 MHz, CDCl3): δ in ppm 0.86 (t, 6H, J ) 6.9Hz, 2CH3), 1.24 (m, 40H, 16CH2), 1.57 (m, 4H, 2CH2), 1.90(s, 3H, Me base), 2.28 (m, 4H, 2CH2), 2.98 (t, 2H), 3.7-4.4(m, 7H, 2CH2(glycerol), H4′, H5′), 5.22 (m, 1H, CH glycerol),6.22 (t, 1H, J ) 6.5 Hz, H1′), 7.61 (s,1H, H base).

13C NMR (75 MHz, CDCl3): δ in ppm 12.5 (CH3 base), 14.0(CH3 chain), 22.6 (CH2), 24.8 (CH2), 29.1-29.6 (CH2), 31.8(CH2), 34.0 (CH2), 34.2 (CH2), 39.0 (CH2), 39.1 (CH2), 61.8(CH2), 62.5 (CH2), 63.4 (CH2), 63.5 (CH2), 70.1 (CH), 74.3(CH), 84.6 (CH), 85.8 (CH), 110.8 (C base), 136.2 (CH base),150.4 (CdO base), 164.0 (CdO base), 173.0 (CdO chain),173,4 (CdO chain).

31P NMR (121 MHz, CDCl3): δ 2.5 ppm.High-resolution ESI MS- [M-H]-, theorical m/z ) 815.4823,

observed m/z ) 815.4819.

Thymidine-3′-(1,2-dipalmitoyl-sn-glycero-3-phosphate) diC16-3′-dT, Compound 3. A procedure similar to that used forcompound 1 was followed here. 5′-Dimethoxytrityl-2′-deox-ythymidine, 3′-[(2-cyanoethyl)-N,N-diisopropyl)]-phosphoramid-ite (1 g, 1 equiv, 1.34 mmol), 1,2-dipalmitoyl-sn-glycerol (992mg, 1.3 equiv, 1,75 mmol), and a tetrazole solution 0.45 M inacetonitrile (4 mL, 1.75 mmol) were dissolved in dry acetonitrileunder argon. Product 3 (552 mg) was isolated after purificationon silica gel (DCM/MeOH/TEA from 98:2:1 to 50:49:1). Yield:47% Rf ) 0.3 (DCM/MeOH 8:2).

1H NMR (300 MHz, CDCl3): δ in ppm 0.88 (t, 6H, J ) 6.9Hz, 2CH3), 1.25 (m, 48H, 16CH2), 1.42 (dd, 4H, J1 ) 8.4 Hz,J2 ) 15.6 Hz, 2CH2), 1.90 (s, 3H, Me base), 2.33 (m, 4H, 2CH2),2.83 (t, 2H J ) 5.6 Hz, H2′), 3.84 (m, 1H, H3′), 4.09-4.35(m, 7H, 2CH2 (glycerol), H4′, H5′), 5.27 (s, 1H, CH glycerol),6.21 (t, 1H, J ) 6.7 Hz, H1′), 7.54 (s,1H, H base).

13C NMR (75 MHz, CDCl3): δ in ppm 12.4 (CH3 base), 14.1(CH3 chain), 19.6 (CH2), 19.7 (CH2), 22.6 (CH2), 24.8 (CH2),29.1-29.6 (CH2), 31.9 (CH2), 33.9 (CH2), 34.1 (CH2), 61.5(CH2), 61.7 (CH2), 62.5 (CH2), 62.6 (CH2), 66.1 (CH2), 66.2(CH2), 69.1 (CH), 78.8 (CH), 85.5 (CH), 86.1 (CH), 111.3 (Cbase), 136.8 (CH base), 150.5 (CdO base), 164.1 (CdO base),173.0 (CdO chain), 173.5 (CdO chain).

31P NMR (121 MHz, CDCl3): δ 2.1 ppm.ESI MS- [M-H]-, theorical m/z ) 871.5449, observed m/z

) 871.5473.

Preparation of Liposomes. Liposomes were prepared frombinary mixtures of the negatively charged lipid nucleolipids (NL)and neutral lipid DOPE. Stock solutions of DOPE and (NL)(10 mg/mL in chloroform) were mixed at the desired ratio andplaced in glass tubes. The mixture was dried under dry N2 andthen desiccated under vacuum overnight. Milli-Q Water wasadded to the dried lipids to obtain liposome solutions (1 mg/mL). This was followed by five thaw-sonication cycles usinga water bath at 55 °C and an ultrasonic bath. The resultantsolution was extruded at room temperature through a 50 nmpore-size nucleopore polycarbonate filter.

Preparation of Plasmid DNA. Plasmid containing a GFPreporter gene under the cytomegalovirus promoter, pE-GFP, waspropagated in transformed Escherichia coli cells. E. coli cellswere grown in standard luria Bertani medium at pH 7.0. Cellswere harvested by centrifugation, and the plasmid DNA was

Scheme 1. Chemical Structure of an Anionic Nucleotide-LipidUsed in This Study, the Thymidine 3′-(1,2-dipalmitoyl-sn-glycero-3-phosphate) (di c16dT) and Non-Nucleotide-Lipids DPPA andDPPG

1766 Bioconjugate Chem., Vol. 20, No. 9, 2009 Khiati et al.

extracted and purified using a Qiagen Plasmid Maxi Kit (Qiagen,Santa Clarita, CA) as per the manufacturer’s recommendedprotocols.

Preparation of Anionic Lipoplexes. DNA (250 µg/mL),liposomes (1 mg/mL), and divalent salt (CaCl2 2H2O, 200 mM)samples were mixed to yield anionic lipoplexes at differentglobal salt concentrations. The DNA to lipid charge stoichi-ometry (D/L ratio) was defined as the total charge of DNAdivided by the total charge of anionic nucleolipides.

Particle Size Determination. Anionic liposomes and li-poplexes were prepared as described above. Particle size wasdetermined using Zetasizer 3000 HAS Malvern. All measure-ments were conducted at 25 °C.

Electrophoresis Studies. Electrophoresis studies were con-ducted in 0.8% agarose gels containing ethidium bromide in0.5 Tris-borate without EDTA buffer. Anionic lipoplexes wereprepared as described above. For this purpose, 20 µL of eachsample was mixed with 4 µL loading buffer (glycerol 30% (v/v), bromophenol blue 0.25% (w/v), and xylene cyanol 0.25 (w/v)) and subjected to agarose gel electrophoresis for 35 min at100 V. The electrophoresis gel was visualized and digitallyphotographed using a G.BOX camera.

Transmission Electronic Microscopy (TEM). Anionic lipo-somes and lipoplexes were prepared as described above andwere visualized by negative staining microscopy. Ten microlitersof liposomes (1 mg/mL) was transferred to a carbon-coated gridfor 10 min. The sample was then dried and stained with 1%(W/W) of uranyl acetate for 30 s. The specimens were observedwith a Hitachi H 7650 electron microscope.

Transfection Assays. Cell lines (Hek 293 human embryokidney) were cultured in Dulbecco’s Modified Eagle’s Medium(D-MEM, Invitrogen) and supplemented with 10% fetal bovineserum (FBS, invitrogen) and 1% not essence AA and 1%L-glutamine at 37 °C in a 5% CO2 atmosphere. Cells were splitevery 3-4 days to maintain monolayer coverage.

The day before transfection, cells were seeded in 24 wellplates (50 000 cells/well). On the day of transfection, themedium containing serum was removed from the well platesand replaced with 200 µL of transfection medium without serum.

Lipoplexes were prepared by adding 4 µL of plasmid (250ng/µL) in 50 µL of medium without serum and 42 µL of anionicliposome dispersions (1 mg/mL). Anionic lipoplex formationwas achieved by the addition of various amounts of a calciumchloride solution (200 mM). These complexes were left at roomtemperature for 15 min before being added to the cells. In thecase of fluorescence microscopy samples, a similar procedurewas followed. In that case, an oligonucleotide fluorescein(random sequence, 18mers) was mixed with the DNA fractionand added to the sample. 50 µL of medium without serum, 42µL of liposomes (1 mg/mL), and 3 µL DNA (250 µg/mL) + 1µL oligonucleotide-fluorescein (250 µg/mL) were mixed. Thecomplex formation was achieved by adding Ca2+ (final con-centration 15 mM).

The cells were incubated with lipoplexes in a media withoutserum for 2 or 4 h to permit transient transfection. Afterward,the complex-containing suspension was removed and replacedby 200 µL of the complete growth medium. GFP expressionwas analyzed after 48 h and measured with flow cytometry.Lipofectamine transfections were performed, as per the manu-facturer’s instructions, using 2 µL of lipofectamine and anidentical amount of plasmid 4 µL (250 ng/µL).

Flow Cytometry Studies. Cells were assayed for GFP expres-sion 48 h after being exposed to the transfection agents. Cellsin the well plates were rinsed with PBS, treated using 100 µLtrypsine EDTA, and incubated for 5 min (37 °C) in 5% CO2

incubator. The cell suspension was then diluted to 200 µL usingPBS, pH 7,4. The cells were then assayed for expression of

GFP. GFP expression was analyzed using a FACS Canto duallaser flow cytometry.

Toxicity Assay. The toxicity of the thymidine-3′-(1,2-diacyl-sn-glycero-3-phosphate) (compounds 1, 2, 3) for the cells wasdetermined 48 h after exposure to the transfection agents usingthe MTS tetrazolium. Cells in the well plates were treated using50 µL of MTS tetrazolium and incubated for 2-4 h (37 °C) ina 5% CO2 incubator. Once coloring developed, the absorbanceof 200 µL of each sample was analyzed at 490 nm.

X-ray Diffraction. SAXS measurements were carried out onthe Bruker Nanostar apparatus at the Centre de Recherche PaulPascal (Pessac, France). The wavelength of the incident beamwas λ ) 1.54 Å, and the sample-to-detector distance was 0.25 m(q range investigated from 0.05 Å-1 to 0.6 Å-1). The diffractionpatterns were recorded by a 2D multiwire gas-filled detector.Anionic membranes formulated with the negatively chargednucleolipids (NL) and the neutral lipid (DOPE) were mixed withdeionized water to obtain solutions with a concentration of 10mg/mL. A plasmid-DNA solution in water was prepared (300mg/mL) and added to the membrane solution in order to getthe molar ratio NL/DNA ) 2. The complex formation wasachieved by adding divalent salt (CaCl2 2H2O, 200 mM) witha final concentration [Ca2+] ) 15 mM. The suspension was heldin a 1.5 mm size glass capillary and then centrifuged for a fewminutes in order to locally concentrate the complex on thebottom of the capillary. The capillary was sealed and insertedin a sample holder at room temperature (25 °C). The collected2D diffraction spectra were angularly integrated to get a 1Dintensity vs q pattern.

RESULTS AND DISCUSSION

In this study, we hypothesized that anionic lipid-nucleic acidcomplexes could take advantage of lipid polar head groupsfeaturing nonionic interactions such as hydrogen-bonding andbase π-π stacking. These additional interactions would likelyinfluence the lipid-DNA associations and enhance the trans-fection efficacy of the natural anionic lipids previously reported(7). To demonstrate this hypothesis, the following constitutingnatural building blocks were selected to prepare the anionicnucleotide based lipids: thymidine (nucleotide polar head) and1,2-diacyl-sn-glycerol (lauroyl, myristoyl, and palmitoyl acylchains). Both moieties, the diacyl-sn-glycerol motif and thethymidine as a nucleotide polar head, were selected on the basisof their natural occurrence in biological systems. In thiscontribution, we report the synthesis of a family of thymidine-3′-(1,2-diacyl-sn-glycero-3-phosphate), the formation of li-poplexes between DNA and nucleotide based lipids in thepresence of Ca2+, SAXS studies, cytotoxicity, and successfulin Vitro gene delivery.

Synthesis. In order to evaluate the impact of the lipid structureon the transfection properties, a series of nucleotide derivativescomposed of thymidine 3′-phosphates bearing natural 1,2-diacyl-sn-glycerol as hydrophobic building blocks was prepared.Although, to the best of our knowledge, nucleoside-5′-mono-phosphate lipids structures have been reported by Baglioni, Berti,and co-workers (31, 32), no double-chain nucleoside-3′-mono-phosphate amphiphile has been described so far. The double-chain nucleoside-3′-monophosphate lipids were synthesized byusing a recently published (37) synthetic approach for single-chain nucleolipids (Scheme 2). Coupling reactions between 5′-DMT protected commercial phosphoramidite (5′-dimethoxytrityl-2′-deoxythymidine,3′-[(2-cyanoethyl)-N,N-diisopropyl)]-phosphoramidite) and lipophilic 1,2-diacyl-sn-glycerol wereundertaken in acidic conditions (tetrazole) similar to those foundin oligonucleotide synthesis. The resulting phosphite intermedi-ates were then oxidized with iodine in a THF/pyridine/watermixture at room temperature under argon to provide the

Anionic Nucleotide-Lipids for DNA Transfection Bioconjugate Chem., Vol. 20, No. 9, 2009 1767

cyanoethyl-protected phosphates. Removal of the DMT groupled to the production of compounds 1, 2, and 3, which werefully characterized by 1H, 13C, 31P NMR, and HRMS. Thecyanoethyl protecting group was removed via an eliminationreaction under basic conditions (TEA). Importantly, the syntheticstrategy developed allowed easy access to the expected thymi-dine-3′-(1,2-diacyl-sn-glycero-3-phosphate) in four steps fromthe commercially available starting materials.

Complexation Studies. Next, to determine whether or notthe nucleotide based lipids 1, 2, and 3 complex nucleic acids,we performed a standard electrophoresis gel shift assay withplasmid-DNA for the assessment of DNA complexation. Sincethe lipid polar head bears an anionic charge, just as was thecase for the previous anionic delivery vectors (7), the formationof lipid-DNA complexes was evaluated in the presence ofdifferent concentrations of divalent calcium. Standard agarosegel electrophoresis revealed two bands for naked DNA (super-coil, nonsupercoil forms, high and low mobility, respectively)(Figure 1A, lane 2). As a control, the addition of Ca2+ to theplasmid DNA samples in the absence of anionic liposomes didnot affect the mobility of the plasmid DNA (Figure 1A, lanes3-5). On the contrary, the presence of anionic liposomes andCa2+ dramatically affected plasmid-DNA migration (Figure1B). On the basis of the hypothesis that the nucleolipid/DNAstability depends on calcium concentration, we also evaluatedthe threshold Ca2+ concentration for complexation. Interestingly,we found that nucleolipid-DNA complexes are observed abovea concentration of 1 mM (Figure 1B, lane 7), which is higherthan the intracellular calcium concentration ([Ca2+] 1 µM)indicating that the lipoplexes can release the DNA inside thecells.Next,weinvestigatedtheoptimumratioofnucleolipid-DOPE.As shown in Figure 2, based upon the lack of free DNA, anoptimal ratio of 10/90 (diC16T/DOPE) was determined.

Nucleolipid-Ca2+-DNA Lipoplexes. The hydrated mixturesof DOPE/diC16-3′-dT in aqueous solution were investigatedwithout plasmid-DNA by transmission electronic microscopy(TEM) and zetasizer (3000 HAS Malvern instrument). As wasexpected, the extruded mixtures were found to self-assemble atroom temperature into liposome-like structures in aqueoussolutions. Under these conditions, at room temperature, theDOPE/diC16-3′-dT were in a “fluid” state. As seen in TEMimages (Figure 3), in the absence of both nucleic acids and Ca2+

and after extrusion with a 50 nm filter, different sizes ofliposomes ranging from 40 to 80 nm were observed. These sizesare consistent with the average sizes of the same samplesmeasured by dynamic light scattering (hydrodynamic diameter:84 nm polydispersity index 0.38). Interestingly, in the presenceof DNA and Ca2+, the mean aggregates size was increased from84 to 128 nm, indicating the formation of larger aggregates.Dried samples of these aggregates investigated by TEM revealedobjects possessing a nonvesicular morphology (data not shown).It is noteworthy that no modification of size was noticed forthe samples containing liposomes in the presence of DNAwithout Ca2+.

It should also be noted that the presence of anionic nucleo-lipids in DOPE/diC16-3′-dT mixtures enhanced the hydrationphenomenon during the preparation of liposomes, which resultedin much more homogeneous colloidal suspensions comparedto DOPE/DPPA samples. This unexpected hydration behavioris likely due to the hydrophilic nucleotide polar head insertedin the molecular structure.

SAXS Studies. Next, to further characterize the molecularorganization within the DOPE-NL-Ca2+-DNA lipoplexes,small-angle X-ray scattering (SAXS) experiments were per-formed. Figure 4 shows the diffraction patterns of theDOPE-DPPA samples with (curve 2) and without (curve 1)

Scheme 2. Synthetic Route to Anionic Nucleotide-Lipidsa

a (a) 0.45 M tetrazole in acetonitrile RT 7 h; (b) 0.02 M I2 pyidine/H2O/THF RT 12 h; (c) 0.1 N HCl.

Figure 1. Effect of Ca2+ on the electrophoretic mobility of (A) Ca2+-DNA complexes and (B) anionic lipoplexes (anionic liposome-Ca2+-DNA)diC16T/DOPE (mol ratio 10/90). (A) The lanes represent control DNA ladder (1); plasmid DNA (2); and Ca2+-DNA complexes at Ca2+ concentrationsof 10 mM (3), 30 mM (4), and 50 mM (5). (B) The lanes represent control DNA ladder (1); plasmid DNA (2); plasmid DNA and empty anionicliposomes (3); anionic lipoplexes at Ca2+ concentrations of 1, 50, and 100 µM, and 1, 5, 10, 15, 30, and 60 mM (7-12).


the plasmid DNA. For the system without plasmid DNA, twodistinct sets of peaks are identified. A 2D hexagonal lattice (q0)with a unit cell spacing of a ) 72.6 Å can be indexed. Thispattern corresponds to the well-known 2D columnar invertedhexagonal phase (HII). The second set of Bragg peaks can beattributed to a lamellar structure with a stacking period d ) 54Å. Bragg peaks up to the fifth and third order are observed for

the hexagonal and lamellar phases, respectively. The lamellarstructure unambiguously corresponds to the lamellar phaseobserved (data not shown) for the pure DPPA system. On theother hand, the 2D hexagonal lattice seems to be very similarto that noticed for the pure DOPE system (42). These resultssuggest a very weak miscibility of the DPPA lipid in the DOPEmembrane. The sample with plasmid DNA exhibits a morecomplicated pattern (empty circles) with a three-phase coexist-ence domain. We can identify the two phases previouslyobserved for the free plasmid DNA system with a 2D hexagonallattice (q0) a ) 71.8 Å and with a lamellar structure (q′0) witha stacking period d ) 54 Å. Interestingly, in the presence ofplasmid DNA, a new lamellar phase (q′′0) can be indexed witha lamellar periodicity d ) 48 Å. This new lamellar phase canbe identified as lamellar complex made from DNA Ca2+ andDOPE (data not shown).

Figure 5 shows the diffraction patterns of the DOPE-diC16-3′-dT samples with (curve 2) and without (curve 1) the plasmidDNA. For the system without plasmid DNA, two distinct setsof peaks are identified which can be indexed on a 2D hexagonallattice (q0 and q′0) with different unit cell spacing, namely, a )77 Å and a′ ) 59 Å. The sample with plasmid DNA also revealstwo sets of peaks. The first set of peaks (q0) corresponds to aHII phase with a unit cell spacing with a ) 69.1 Å. The secondset of peaks (q′0) can be indexed for a lamellar phase with astacking period d ) 48 Å. This lamellar structure is similar tothat observed in the DOPE-DPPA-plasmid DNA sample. The

Figure 2. Electrophoretic mobility of different formulations and Ca2+ concentrations. The lanes represent control DNA ladder (1); plasmid DNAwith different concentrations of Ca2+ (2-4); lipoplexes (anionic nucleotide lipid-Ca2+-DNA) diC16T/DOPE mol ratios of 10/90 (5,6), 25/75(7,8), and 50/50 (9,10) with [Ca2+] ) 15 and 30 mM, respectively.

Figure 3. Transmission electron microscopic (TEM) images of anionicDOPE/diC16-3′-dT liposomes (mol ratio of 10/90) in water with uranylacetate (1%) as negative stainer. Scale bars, 200 nm.

Figure 4. SAXS patterns of DOPE-DPPA-Ca2+ system withoutplasmid DNA (curve 1) and with plasmid DNA (curve 2).

Figure 5. SAXS patterns of DOPE-diC16-3′-dT-Ca2+ system withoutplasmid DNA (curve 1) and with plasmid DNA (curve 2).


differences for the structural parameters observed for the sampleswith and without plasmid DNA strongly suggest the presenceof DNA inside the two phases HII and LR and a good miscibilityof DOPE with the nucleotide lipid (diC16-3′-dT). These resultsprove that the structure of the various complexes is formulation-dependent. In particular, the presence of the nucleotide lipidenhances the existence of a 2D inverse hexagonal phase (a )69.1 Å) which does not occur in the nucleotide free lipid system(DOPE-DPPA-plasmid-DNA-Ca2+), while the lamellarphase with a stacking period d ) 48 Å is present in bothsystems.

Cell Proliferation. The effect on the proliferation of mam-malian cell lines of the nucleolipid-Ca2+-DNA lipoplexes wascompared to a commercially available transfectant, lipofectamineand Ca2+-DNA binary mixtures. The in Vitro lipoplex cytotox-icity was evaluated against Hek cells using an MTS assay. Theproliferation of cells is expressed as percentages of nontreatedcells. As shown in Figure 6, after 2 h of incubation, theproliferation of Hek cells was not affected in the presence ofmyristoyl (DOPE/diC14-3′-dT-Ca2+-DNA) or palmitoyl deriva-tives (DOPE/diC16-3′-dT-Ca2+-DNA), since the cellular growthresponses were essentially higher or the same as the negativecontrols (nontreated Hek cells). Note, furthermore, that notoxicity was observed even after 4 h of incubation for thepalmitoyl derivative. In contrast, Hek cell viability decreasedin the presence of lipofectamine, DOPE/DPPG-Ca2+-DNA,and Ca2+-DNA mixtures for both 2 and 4 h of incubation. Thecytotoxic effect was much stronger for ternary mixtures featuringnon-nucleotide lipid (DOPE/DPPA-Ca2+-DNA) than for di-palmitoyl nucleolipid (diC16-3′-dT), because the cell viabilitywas reduced by 30% and 40% for 2 and 4 h, respectively. Asimilar cytotoxicity effect was observed with DOPE/diC12-3′-dT-Ca2+-DNA samples, indicating that an optimal chain lengthof C16 is required to avoid toxicity. Toxic effects of dilauroylderivatives were previously reported for cationic lipids bearing1,3-diamino-2-propanol backbone (43).

Fluorescence Microscopy Studies. We next used fluores-cence microscopy to determine whether the DOPE/diC16-3′-dT-Ca2+-DNA complexes penetrated the cultured eukaryotic cells.For this purpose, DOPE/diC16-3′-dT liposomes and a mixtureof nucleic acids containing plasmid DNA (0.75 µg) andoligonucleotide-fluorescein (0.25 µg) were mixed. The complexformation was realized by adding Ca2+ (final concentration 15mM). Hek cells were incubated later with the fluorescein-labeledcomplexes for 2 h at 37 °C. As shown in Figure 7 (left), thefluorescein-labeled complexes were internalized at 37 °C,indicating that anionic lipoplexes can transfer DNA into cells.

It is important to note that Hek cells incubated in the presenceof anionic lipoplexessprepared without fluorescein-labeledoligonucleotidess(DOPE/diC16-3′-dT-Ca2+-DNA), where theDNA is an expression vector (pEGFP) encoding GFP, confirmthe release and the expression of the anionic lipoplexes fromthe GFP-plasmid DNA. (Figure 7 right).

Transfection. To investigate the efficiency of the anionicnucleotide based lipids to deliver DNA into cells, we performeda transient transfection assay of mammalian cell lines (Hek 293human embryo kidney). The transfected DNA is an expressionvector (pEGFP) encoding GFP, which enables the detection offluorescence by FACS. Different formulations of anionicnucleolipids were mixed with 1 µg of pEGFP, and thenlipoplexes were incubated with Hek cells. Single-cell suspen-sions were analyzed by FACS Canto dual laser flow cytometry.The GFP negative and positive gates were defined using thecontrol experiment carried out in the absence of lipoplex. Ineach experiment, 50 000 cells were sorted, and the results areshown in Figure 8. Anionic lipoplexes are efficient for trans-fection at a concentration of liposomes (42 µg, DOPE/diCn-3′-dT-Ca2+-DNA) per well. As control experiments, the transfec-tion capability of lipofectamine-DNA and calcium-DNAmixtures were evaluated. As expected for this cell line,lipofectamine appeared to be the most efficient transfectingreagent, whereas calcium salts alone remained less effective intransfecting EGFP. Likewise, with only 18% and 24% oftransfection after 2 h of incubation, the DPPA-Ca2+-DNA andDPPG-Ca2+-DNA lipoplexes, respectively, exhibited weaktransfecting activity. On the contrary, the nucleolipids exhibitedhigher transfecting efficacies. With a level of transfection higher

Figure 6. Cytotoxicities of anionic lipoplexes composed of nucleolipids(diC12,14,16T), DPPA or DPPG and DOPE (10:90 mol ratio)-Ca2+ (15mM)-DNA compared to lipofectamine and DNA with 15 mM Ca2+

and untreated cells.

Figure 7. Fluorescence microscopy images of anionic lipoplexes ([Ca2+]) 15 mM) carrying DNA fluorescein labeled (left) and transfected Hekcells (GFP expression) (right).

Figure 8. Transfection efficiencies of anionic lipoplexes composed ofnucleolipids/DOPE (10:90 mol ratio) (diC12,14,16T), DPPA/DOPE (10:90 mol ratio)-Ca2+(15 mM)-DNA), DPPG/DOPE (10:90 molratio)-Ca2+(15 mM)-DNA), compared with lipofectamine and DNAwith 15 mM Ca2+.


than 40%, the DOPE/diC16-3′-dT-Ca2+-DNA lipoplex shows thebest efficacy among the anionic systems. Note that this levelwas increased to 55% after incubating the Hek cells in thepresence of this complex for 4 h (data not shown). Anexplanation for the lower activity of dilauroyl derivatives couldbe found in the early work of Minsky and co-workers (44). Theyhave suggested that fatty acid chains of 12 carbons are not longenough to create the hydrophobic environment required forefficient DNA compaction. Therefore, it appears that the anionicnucleotide lipids are more efficient compared to bothDPPA-Ca2+-DNA and Ca2+-DNA transfecting systems,demonstrating that the nucleotide moiety influences thelipid-DNA associations and enhances the transfection efficacyof the natural anionic DPPA lipid.

CONCLUSION

In this study, we synthesized a new series of nucleotide-basedlipids featuring thymidine-3′-monophosphate as nucleotide and1,2-diacyl-sn-glycerol as hydrophobic moieties for in Vitrodelivery of nucleic acids. The amphiphilic structures wereprepared in three steps from commercially available startingmaterials: 1,2-diacyl-sn-glycerols and 2′-deoxythymidine-3′phosphoramidite. Gel electrophoresis experiments indicate thatnucleotide-based lipid-DNA complexes are observed above aconcentration of 1 mM, which is higher than the intracellularcalcium concentration ([Ca2+] 1 µM) indicating that DNA canbe released inside the cells. Electronic microscopy and dynamiclight scattering investigations reveal that extrusion of DOPE-diC16-3′-dT mixtures provide homogeneous liposomes, whereaslarger aggregates are observed in the presence of DNA and Ca2+

confirming the formation of complexes.SAXS studies indicate that the structure of the complexes is

strongly dependent on the formulation. Different multiphasesystems can be obtained for the DOPE-diC16-3′-dT andDOPE-DPPA samples with and without plasmid DNA. Thetransfection experiments carried out on mammalian Hek celllines clearly demonstrate that the nucleotide moiety enhancesthe transfection efficacy of the natural anionic DPPA and DPPGlipids. The SAXS studies also indicate that the enhancement intransfection is likely due to the presence of the 2D columnarinverted hexagonal phase (HII) with a unit cell parameter a )69.1 Å, which does not occur in the nucleotide free lipid system(DOPE-DPPA). These results confirm the interest of usinganionic species (45) such as nucleotide lipids in formulationsto optimize transfection efficacy.

The issue of transfection reagent cytotoxicity is particularlyimportant in protocols that attempt to modulate gene functionin Vitro an/or in ViVo. As nontoxic and efficient transfectionreagents, the anionic nucleotide lipids presented in this workoffer an alternative to cationic species, which can be, in someinstances, especially toxic (46). These investigations furthersupport the need for the synthesis and evaluation of nucleotide-based lipids as nucleotide acids delivery vehicles. In fine,nucleotide-based lipids further extend the possibilities ofmodulating the interactions between nucleic acids and lipids.Depending upon the nature of both partners, nucleotide lipidsmay offer new alternatives to lipids, and could be exploited inthe case of small therapeutic oligonucleotides, for example.

ACKNOWLEDGMENT

This research was supported by the Region d’Aquitaine. P.B.acknowledges financial support from the Army Research Office.The authors thank the SERCOMI Centre for technical assistanceduring TEM observations and G. Sigaud for physicochemicalexperiments.

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Anionic Nucleotide−Lipids for In Vitro DNA Transfection