In Vitro Cationic Lipid-Mediated Gene Delivery with FluorinatedGlycerophosphoethanolamine Helper Lipids
Jerome Gaucheron,† Caroline Boulanger,† Catherine Santaella,†,| Nicolas Sbirrazzuoli,‡Otmane Boussif,§,⊥ and Pierre Vierling*,†
Laboratoire de Chimie Bioorganique, UMR 6001 CNRS, Laboratoire de Thermodynamique Experimentale,Universite de Nice-Sophia Antipolis, 06108 Nice Cedex 2, France, and Transgene, SA, 11, rue de Molsheim67082 Strasbourg Cedex, France. Received March 19, 2001; Revised Manuscript Received July 3, 2001
There is a need for the development of nonviral gene transfer systems with improved and originalproperties. “Fluorinated” lipoplexes are such candidates, as supported by the remarkably higher invitro and in vivo transfection potency found for such fluorinated lipoplexes as compared withconventional ones or even with PEI-based polyplexes (Boussif, O., Gaucheron, J., Boulanger, C.,Santaella, C., Kolbe, H. V. J., Vierling, P. (2001) Enhanced in vitro and in vivo cationic lipid-mediatedgene delivery with a fluorinated glycerophosphoethanolamine helper lipid. J. Gene Med. 3, 109-114).Here, we describe the synthesis of fluorinated glycerophosphoethanolamines (F-PEs), close analoguesof dioleoylphosphatidylethanolamine (DOPE), and report on their lipid helper properties vs that ofDOPE, as in vitro gene transfer components of fluorinated lipoplexes based on pcTG90, DOGS(Transfectam), or DOTAP. To evaluate the contribution of the F-PEs to in vitro lipoplex-mediatedgene transfer, we examined the effect of including the F-PEs in lipoplexes formulated with thesecationic lipids (CL) for various CL:DOPE:F-PE molar ratios [1:(1 - x):x with x ) 0, 0.5 and 1; 1:(2 -y):y with y ) 0, 1, 1.5, and 2], and various N/P ratios (from 10 to 0.8, N ) number of CL amines, P )number of DNA phosphates). Irrespective of the F-PE chemical structure, of the colipid F-PE:DOPEcomposition, and of the N/P ratio, comparable transfection levels to those of their respective controlDOPE lipoplexes were most frequently obtained when using one of the F-PEs as colipid of DOGS,pcTG90, or DOTAP in place of part of or of all DOPE. However, a large proportion of DOGS-basedlipoplexes were found to display a higher transfection efficiency when formulated with the F-PEsrather than with DOPE alone while the opposite tendency was evidenced for the DOTAP-basedlipoplexes. The present work indicates that “fluorinated” lipoplexes formulated with fluorinated helperlipids and conventional cationic lipids are very attractive candidates for gene delivery. It confirmsfurther that lipophobicity and restricted miscibility of the lipoplex lipids with the endogenous lipidsdoes not preclude efficient gene transfer and expression. Their transfection potency is ratherattributable to their unique lipophobic and hydrophobic character (resulting from the formulation ofDNA with fluorinated lipids), thus preventing to some extent DNA from interactions with lipophilicand hydrophilic biocompounds, and from degradation.
INTRODUCTION
Gene (or DNA) transfer systems into cells have becomepowerful tools for gene and cellular therapy that are verypromising novel forms of molecular medicine. Successfulgene therapy depends on the efficient delivery of geneticmaterial to cells and its effective expression within thesecells. Although at present, the in vivo expression levelsof synthetic nonviral gene transfer vectors based on(poly)cationic lipids, liposomes, and polymers (e.g., lipo-plexes and polyplexes, respectively (1)) are lower thanfor viral vectors and gene expression is transient, these
systems are likely to present several advantages includ-ing low-cost and large-scale production, safety, lowerimmunogenicity, and capacity to deliver large genefragments. Such systems have therefore gained wideacceptance over the past decade for in vitro and/or in vivogene delivery (2-5). However, there is still a need forthe development of gene transfer systems with improvedand original properties.
Our approach with the development of “fluorinated”lipoplexes based either on highly fluorinated liposper-mines (6, 7) or on a fluorinated glycerophosphoethanol-amine helper lipid (e.g., [F8E11][C16]OPE in Scheme 1which is an analogue of dioleoylphosphatidylethanol-amine, DOPE) (8) proved to some extent successful asthese systems showed a higher in vitro and in vivotransfection potential than conventional lipoplexes (6-8) or PEI polyplexes (8). More particularly, the use of[F8E11][C16]OPE (which on its own is inactive in pro-moting transfection) as colipid of the cationic lipopolyaminepcTG90 (see structure in Scheme 1) increased the in vitroand in vivo gene transfer capability of pcTG90 to a largerextent than DOPE (8). Although the exact mechanismwhich governs lipoplex-mediated gene delivery and ex-
* To whom correspondence should be addressed. E-mail:[email protected]. Phone: 33 (0)4 92 07 61 43. Fax: 33 (0)4 9207 61 51.
† Laboratoire de Chimie Bioorganique.‡ Laboratoire de Thermodynamique Experimentale.§ Transgene.| Present address: CEA Cadarache, DSV:DEVM, Laboratoire
d′Ecologie Microbienne de la Rhizosphere, UMR CNRS-CEA163, 13108 Saint Paul lez Durance, France.
⊥ Present address: Gencell, a division of Aventis Pharma,Process Development, Non Viral Formulation, 13, Quai JulesGuesde, 94400 Vitry/Seine, France.
pression is unclear, several reasons may account for thegene expression improvement resulting from the use of[F8E11][C16]OPE in place of DOPE. This improvementcould be due to the larger ability of the fluorinated colipidto preserve the integrity of complexed DNA in a biologicalenvironment. Indeed, owing to its increased hydrophobicand lipophobic character, this colipid is expected toprevent the fluorinated lipoplexes it forms with cationiclipids from interactions with lipophilic and hydrophilicbiocompounds, and from degradation to a larger extentthan conventional helper lipids, as shown for fluorinatedlipoplexes formed from fluorinated lipospermines ascompared with conventional ones (7). This improvementcould also be related to the larger propensity of thefluorinated colipid to promote fusion with and destabi-lization of the endosome membrane allowing more ef-ficient DNA release in the cytosol, thus preventinginternalized DNA from endosome and lysosome degrada-tion to a larger extent than when DOPE is used as helperlipid (9-12). This fluorinated PE displays also a morepronounced cone-shape geometry than DOPE as a con-sequence of the larger size of its fluorinated chain whencompared with hydrocarbon ones (13). One thereforeexpects a greater tendency for [F8E11][C16]OPE topromote a lamellar to inverted hexagonal HII phasetransition (13) and, thus, a greater effectiveness indisrupting membranes than DOPE (the role in mem-brane fusion of DOPE was indeed attributed to itscapability to initiate such a phase transition (11, 12)).
The goals of the present study was aimed at extendingthe library of fluorinated glycerophosphoethanolamines(F-PEs), and at examining (i) their lipid helper effect onlipofection and (ii) the cationic lipid specificity of theirtransfection enhancement potential, as compared withthat of DOPE. The molecular structure of this secondgeneration of F-PEs (listed in Scheme 1) follows amodular design (one or two fluorinated chains, saturatedor unsaturated chains, ester or ether bond) aimed at theestablishment of structure/properties (transfection helper
potential) relationships. We report here on their synthe-sis and on their helper lipid properties as in vitro genetransfer components of pcTG90, DOGS (Transfectam),and DOTAP-based lipoplexes (see structures in Scheme1), as compared with DOPE. These (poly)cationic lipidswere selected for their well-documented high transfectionefficiency which is significantly improved with DOPE (forpcTG90, see (8, 14, 15), for DOGS, see (16) and forDOTAP, see (17, 18)). They were also chosen for theirspecific structural and geometrical features (linear orbranched polyamino or single quaternary ammoniumpolar head) which might have quite different effects onthe phases these cationic lipids associated to the F-PEsare susceptible to form. The thermotropic phase behaviorof the F-PEs in water was also investigated by differentialscanning calorimetry (DSC) in order to highlight its roleon lipoplex formulation and lipofection (10, 12). In ourinitial study (8), the optimized conditions for the in vitroand in vivo transfection tests, which led us to show asignificantly higher transfection helper potential of[F8E11][C16]OPE as compared with that of DOPE, werecorresponding to pcTG90-based lipoplexes formulated inHEPES and in 5% glucose, respectively. However, invitro, when the lipoplexes were formulated in 5% glucose,both colipids displayed a comparable transfection helperpotential. In anticipation of in vivo testing, lipofectionwith the F-PEs as colipids was investigated here only forlipoplexes formulated in 5% glucose.
EXPERIMENTAL PROCEDURES
General Experimental and Analytical Condi-tions. Most of the reactions were performed in anhydroussolvents under dry and oxygen-free nitrogen. Anhydroussolvents were prepared by standard methods. The puri-fications by column chromatography were carried outusing silica gel 60 (Merck, 70-230 mesh) and chloroform(CHCl3), dichloromethane (CH2Cl2), methanol (MeOH),or mixtures thereof as indicated. Unless noted otherwise,the ratios describing the composition of solvent mixturesrepresent relative volume. Advancing of the reaction wasfollowed by thin-layer chromatography (TLC) on silicaplates F254 (Merck). The following developing systemswere used: UV light, KMnO4, H2SO4/EtOH, Dragendorffreagent (Sigma), ninhydrin reagent (Sigma).
The perfluoroalkyl iodides were purified over neutralalumina before use. DF4C11OPE, DOGS (or Trans-fectam), and DOTAP were prepared as described in refs19, 6, and 17, respectively. The lipopolyamine pcTG90was from Transgene (Strasbourg, France) (20). DOPEwas purchased from Sigma. All other organic chemicalswere from Aldrich, Fluka, or Novabiochem.
1H, 13C, 31P{1H}, and 19F NMR spectra were recordedon a Bruker AC-200 at 200, 50.3, 81, and 188.3 MHz,respectively. Chemical shifts were measured relative toCHCl3 (δ 7.27 ppm) or CH3OD (δ 3.35 ppm) for 1H, toCDCl3 (δ 76.9 ppm) for 13C and expressed indirectly inrelation to TMS, to external ref 75% H3PO4 for 31P, andto CCl3F as internal reference for 19F. The followingabbreviations are used to describe the signal multiplici-ties: s (singlet), d (doublet), t (triplet), q (quadruplet),and m (multiplet). Chemical shifts are expressed in ppmand listed as follow: shift in ppm (multiplicity, coupling,integration and attribution). Elemental analyses werecarried out by the Service Central de Microanalyses duCNRS.
Synthesis of the Glycerol Derivatives 2a-c. rac-2,3-Di-[11-(F-butyl)undecanoyl)]glycerol, 2a. The syn-thesis of 2a from rac-1-O-benzylglycerol was performedaccording to published procedures (21).
rac-3-[11-(F-Octyl)undec-10-enyloxy]-2-(hexadecyl-oxy)propan-1-ol, 2b. The syntheses of derivatives 4 to6 from 1,3-O-benzylideneglycerol, 3, were performedaccording to published procedures (19).
rac-3-[11-(F-Octyl)undec-10-enyloxy]-2-(hexa-decyloxy)propan-1-ol, 2b. A suspension of FeCl3 and4.4 g of 6 in 15 mL CH2Cl2 was stirred for 90 min at roomtemperature. The organic phase was then washed, driedover Na2SO4 and evaporated, and the residue was puri-fied by silica gel chromatography (CH2Cl2:MeOH 100:0
rac-3-[11-(F-Octyl)undecyloxy]-2-(hexadecyloxy)-propan-1-ol, 2c. The simultaneous benzyl deprotectionand CdC hydrogenation of 4 (1.8 g;1.8 mmol) in 15 mLof EtOH and 0.2 mL of AcOH was performed at roomtemperature under 40 atm H2 pressure with Pd/C (10%)as catalyst. Usual workup and silica gel chromatography(CH2Cl2) afforded 1.0 g (60%) of 2c as a white powder(60%). TLC (CH2Cl2; H2SO4): Rf ) 0.55. 1H NMR (CDCl3):δ 0.88 (t, 3H, 3J ) 6.5 Hz, CH3); 1.15-1.48 (broad s, 40H,(CH2)7CH2CH2O, CH3(CH2)13); 1.48-1.72 (m, 4H, CH2-CH2O, CF2CH2CH2); 2.06 (t, 3J ) 7.5 Hz, 3JHF ) 19.0 Hz,2H, CF2CH2); 2.22-2.34 (broad s, 1H, CH2OH); 3.38-3.83 (m, 9H, CH2O, CHO). 13C NMR (CDCl3): δ 14.0(CH3); 20.6 (t, 3JCF ) 4 Hz, CF2CH2CH2); 22.6 (CH3CH2);26.1 (CH2CH2CH2O); 29.2, 29.3, 29.6, 30.0 (CH2CH2O,CH3(CH2)2(CH2)10, CF2(CH2)2(CH2)6); 30.9 (t, 2JCF ) 22Hz, CF2CH2); 31.8 (CH3CH2CH2); 63.1 (CH2OH); 70.4,70.9, 71.8 (CH2O); 78.3 (CHO). 19F NMR (CDCl3): δ -79.6(3F, CF3); -113.3 (2F, CF2CH2); -120.7 (6F, (CF2)3CF2-CH2); -121.6 (2F, CF3(CF2)2CF2); -122.4 (2F, CF3-CF2CF2); -125.0 (2F, CF3CF2).
Synthesis of the F-PE Derivatives. The phos-phorylation of the glycerol derivatives 2a-c and thedeprotection step of the BOC-protected intermediates7a-c with TFA were performed according to the proce-dures described for the synthesis of DF4C11OPE (19).The F-PE derivatives were isolated as TFA salts.
Differential Scanning Calorimetry (DSC). ForDSC measurements in the +4 to 95 °C temperaturerange, the samples were prepared by weighing thepowdered phospholipid (∼10 mg) into pierced aluminumpans and adding a weighed amount of deionized waterto obtain a water concentration of 60% (w/w). DSCmeasurements were carried out at a rate of 10 °C/minwith a Mettler-Toledo DSC 821e apparatus which waspreviously calibrated using indium, zinc, and lead stand-ards. The samples were sealed and heated in a staticatmosphere. Transition enthalpies and temperatureswere determined after several heating/cooling cycles. Thereported values of the Tc represent the temperature atmaximum excess heat capacity.
Preparation of Complexes Composed of theCationic Lipids, the Helper Lipids and PlasmidpTG11033. The plasmid pTG11033 was produced byTransgene. The endotoxin content of the plasmid prepa-ration was checked using a Limulus Amebocyte Lysat kit(Biogenic, Maurin, France). This value was below 1endotoxin unit/mg of plasmid, hence below the 5 e.u./mgof DNA recommended for in vivo protocols. The quantitiesof compounds used were calculated according to thedesired DNA concentration of 0.1 mg/mL, the N/P ratio,the molar weight, and the number of positive charges inthe selected cationic lipid (CL). The N/P ratio of 10, forexample, corresponds to the molar amount of CL neces-sary to have a ratio of 10 amino group nitrogens (for 1mol of CL) per one phosphate in the DNA (330 Da meanMw), as described elsewhere (1, 22, 27). The DNA/CL:colipid(s) complex is formulated by adding a desiredvolume of the liposomal CL:colipid(s) preparation at aCL concentration of 10 mg/mL in 5% glucose to thedesired volume of DNA solution to reach a DNA concen-tration of 0.1 mg/mL. Thus for the preparation of theDNA/DOGS:DOPE (1:1 mol) complex at N/P ratio 5 and0.1 mg/mL DNA, the desired volume of DOPE (10 mg/mL chloroform solution) to get mol:mol DOGS:DOPE was
added to 50 µL of DOGS solution (10 mg/mL in EtOH),and then the mixture was transferred to a borosilicateglass tube (16 × 100 mm). The solvent was evaporatedin Rotavap evaporation system (45 °C, 30 pm, 0.2 bar,40 min). 50 µL of 5% glucose were added to the filmobtained. The preparation was vortexed for 2 h and thensonicated for 5-10 min to yield a liposomal preparationof 50-100 nm mean size as measured by photon correla-tion spectroscopy (see below). Then, 47.9 µL of thispreparation was added to 952.1 µL of DNA solution [(100µL DNA (1 mg/mL) diluted with 852.1 µL of 5% glucose)].This preparation was vortexed for 10 s and was usedwithin 1 h for the particle size measurements and the invitro transfection experiments.
Measurement of the Size of the Lipoplexes. Thesample was diluted with 5% glucose in the measurementtube and homogenized, and the average sizes weremeasured by photon correlation spectroscopy using aCoulter N4Plus particle size analyzer, as described in ref5. The formulations and analyses were reproduced twice.
Agarose Gel Electrophoresis. Each sample wasanalyzed and plasmid integrity in each sample wasconfirmed by electrophoresis after decomplexing thelipoplex with sodium dodecyl sulfate, following the pro-cedures described in ref 5.
In Vitro Transfection of A549 Cells. Twenty-fourhours before transfection, A549 cells (epithelial cellsderived from human pulmonary carcinoma) were grownin Dulbeco-modified Eagle culture medium (DMEM)(GIBCO-BRL, Life Technologies, Cergy Pontoise, France),containing 10% fetal calf serum, FCS (SIGMA, SaintQuentin Fallavier, France), in 96-well plates (2 × 104 cellsper well), in a wet (37 °C) and 5% CO2/95% air atmo-sphere. Volume of DNA/CL:colipid(s) (1:1 mol) or (1:2 mol)lipoplex (5 and/or 1 µL) was diluted to 100 µL in DMEMsupplemented with 10% FCS in order to obtain variousamounts of DNA (0.5 and/or 0.1 µg, respectively) in thepreparation. The culture medium was removed andreplaced by 100 µL of DMEM supplemented with 10%FCS and containing the desired amount of DNA. After 4and 24 h, 50 µL and 100 µL of DMEM supplemented with30 and 10% FCS, respectively, were added. Forty-eighthours after transfection, the culture medium was dis-carded, and the cells were washed twice with 100 µL ofPBS and then lysed with 50 µL of lysis buffer (Promega,Charbonnieres, France). The lysates were frozen at -40°C awaiting analysis of luciferase activity. These meas-urements were done for 10 s on 10 µL of the lysis mixturein a Berthold LB96P luminometer in dynamic mode,using the “Luciferase” determination system (Promega)in 96-well plates. The total protein concentration per wellwas determined using conventional techniques (BCA test,Pierce, Montlucon, France). For cells grown in theabsence of lipoplexes, a well contains around 30 to 50 µgof proteins. The percentage of cell viability of the lipo-plexes was calculated as the ratio of the total proteinamount per well of the transfected cells relative to thatmeasured for untreated cells × 100%. The given means( SEM were calculated from four independent experi-ments.
Statistical Analyses. Statistical analyses were per-formed using a Student t test. The difference betweentwo means was considered as statistically significantwhen p is e 0.05.
RESULTS AND DISCUSSION
Synthesis. The synthesis of the fluorocarbonDF4C11PE, [F8E11][C16]OPE, and [F8C11][C16]OPE
phosphoethanolamines from their respective glycerolprecursors 2a-c was performed according to well docu-mented procedures that have been published for thepreparation of DF4C11OPE (19). These procedures, il-lustrated in Scheme 2 part C, involved phosphorylationof these 2a-c synthons with POCl3, condensation withN-BOC-ethanolamine, hydrolysis, and then BOC-depro-tection using trifluoroacetic acid (TFA), affording theexpected F-PEs as TFA salts (15-50% yields from 2a-c).
The ester-linked glycerol derivative 2a was obtainedin two steps from benzylglycerol which included conven-tional acylation and benzyl deprotection by hydrogenoly-sis (Scheme 2, part A). The more demanding preparationof the mixed fluorocarbon/hydrocarbon 1,2-di-ether linkedglycerol derivatives 1b,c (Scheme 2, part B) was bestperformed from 1,3-O-benzylideneglycerol 3 using astrategy developed for close analogues (19). In this cyclic-strained protected glycerol, the secondary hydroxyl func-tion has been shown to be more accessible and easier toalkylate than in acyclic protected analogues (23). Alkyl-ation of 3 performed under phase catalysis conditionsusing hexadecyl bromide, followed by the reductivecleavage of the 1,3-benzylidene-protecting group withBH3/THF (23) afforded derivative 4 (70% yield). Subse-quent alkylation of the hydroxyl thus liberated in 4 using11-undecenyl bromide yielded the benzyl-protected de-rivative 5 (70% yield). The addition of the linear per-fluorooctyliodide (C8F17I) on the terminal CH2dCH-double bond of 5 was performed using the modifiedmethod of Burton and Kehoe (24): this two-step one-potreaction led to the perfluorooctyl unsaturated derivative6, in 75% yield. According to 19F NMR which showedmore particularly the presence of two resonances for theCF2 in R to the double bond, the isolated compoundconsisted in a mixture of E- and Z-isomers. The mostabundant one was the E-isomer (at least 85%). Theseproducts were also contaminated by a compound (lessthan 5%) which possesses a CF2-CH2-CHdCH- se-quence, as a result of a â,γ-HI elimination during theperfluoroalkylation step (19, 24). This was evident fromthe presence of, respectively, a doublet of triplets (3JHH) 7 Hz, 3JHF ) 18 Hz) at 2.7 ppm, a triplet (2JCF ) 23Hz) at 34.7 ppm for the CH2 group and a CF2 resonanceat -115.8 ppm, in the 1H, 13C, and 19F NMR spectra of6.
Selective benzyl-deprotection was performed by actionof excess FeCl3 in anhydrous CH2Cl2 followed by hydroly-sis (25). This procedure afforded the mixed fluorocarbon/hydrocarbon unsaturated 1,2-glycerol derivative 2b in75% yield. The saturated and benzyl-deprotected ana-logue 2c was obtained in nearly 60% yield by simulta-neous benzyl-deprotection and double-bond hydro-genation using high-pressure hydrogenolysis con-ditions (40 atm H2 in the presence of 10% Pd/C). Undermilder hydrogenolysis conditions (1 atm H2), double-bondhydrogenation but only partial debenzylation was ob-served.
Thermotropic Phase Behavior. The thermotropicphase behavior of samples consisting of hydrated powdersof the F-PEs was investigated by DSC. Table 1 collectsthe thermodynamic parameters (Tc, gel (crystalline) toliquid-crystalline main phase transition and/or lamellarto inverted hexagonal phase transition, see hereafter, andtheir associated ∆H and ∆S) determined on these dis-persions by DSC. The phase behavior of hydratedDF4C11OPE has been investigated in detail by X-raydiffraction, DSC, and optical microscopy with polarizedlight (26). This latter study, performed as a function of
temperature, showed that DF4C11OPE, in excess water,forms a hexagonal phase above 28 °C, (i.e., below butclose to the phase transition temperature measured byDSC). It showed also that the unique and broad phasetransition detected by DSC consisted in a superpositionof transitions, which included the lamellar gel to liquid-crystalline phase transition followed, almost simulta-neously, by a transition to a hexagonal phase. Owing totheir very close molecular structures, one can thereforeassume that the unique and broad phase transitiondetected by DSC for the other fluorinated PEs is alsomost likely a transition to a hexagonal phase. Thepresence of a HII phase at a temperature above the phasetransition temperature detected by DSC was furtherattested by optical microscopy with polarized light.
Lipoplex Formation and Characterization. Thecapability of the various F-PEs as colipid of cationiclipids, CL () DOGS, pcTG90 or DOTAP) to form lipo-plexes was analyzed for various CL:DOPE:F-PE molarratios [1:(1 - x):x with x ) 0.5, 0.75, or 1, and 1:(2 - y):ywith y ) 1, 1.5, or 2], and for various N/P ratios (10, 5,2.5, 1.25, and 0.8), as compared to CL:DOPE (1:1) or (1:2
mol) control lipoplexes. These studies were performedwith pTG11033 plasmid, also used for the in vitro trans-fection assays (see next section). The procedure appliedfor the lipoplex preparation relies on the dilution of aliposomal solution obtained from CL and the colipid(s)in 5% glucose with the DNA solution in 5% glucose, usingN/P ratios of 10, 5, 2.5, 1.25, and 0.8 (N ) number oflipid cations; P ) number of DNA phosphates (22, 27)).These formulations were analyzed by agarose gel elec-trophoresis which showed the absence of , free .plasmid for N/P ratios from 10 to 1.25, DNA being totallyprotected from ethidium bromide interaction. By contrast,the plasmid was accessible to ethidium bromide in thecase of N/P 0.8 formulations. These formulations werealso analyzed by light scattering spectroscopy (LSS) forsize determination (which is an important parameter tocontrol when in vivo uses are contemplated or when theirin vitro transfection efficiency is evaluated (16, 22)). Forthe CL:colipid(s) (1:1 mol) formulations, and irrespec-tive of the nature of CL and colipid(s) and of colipidcomposition, one observed most often lipoplexes of meansize in the 50-180 nm range for an excess of cationiccharges (N/P g 2.5) and in the 150-400 nm range forN/P ) 0.8 while precipitates were systematically detectedfor N/P ) 1.25 (data not shown). Concerning the pcTG90:colipid(s) (1:2 mol) formulations, a very similar behaviorwas observed, i.e., lipoplexes of mean size in the 50-150nm range for N/P g 2.5, in the 150-300 nm range forN/P ) 0.8, and precipitates for N/P ) 1.25 except whenco-formulated with DF4C11OPE or DF4C11PE (data notshown). This latter result is noteworthy as most oftenprecipitates for a N/P ratio of 1.25 (which corresponds toalmost neutraly charged lipoplexes) were reported in theliterature (4, 5, 28). Concerning the N/P 0.8, 2.5, 5, and10 formulations, our results are also close to those
Table 1. Thermodynamic Parameters of the Lamellar toInverted Hexagonal HII Phase Transition for theFluorinated Glycerophosphoethanolamines (F-PEs). DSCExperiments Were Performed in Deionized Water (60%w:w)
reported for other lipoplexes, i.e., stable dispersions for“negatively” (N/P ) 0.8) and positively charged lipoplexes(N/P g 2.5) (4, 5, 28).
As compared with DOPE alone, no impact of the F-PE’snature or of their molecular structure on lipoplex sizecould be evidenced. Our results indicate that the fluori-nated PEs are as effective colipids as DOPE in terms ofcompacting DNA with DOGS, pcTG90 or DOTAP intosmall-sized particles. These data suggest further that thelamellar to hexagonal phase transition temperature ofthe F-PEs, and the expected lower miscibility betweenthe fluorinated colipids and the cationic lipids as com-pared with DOPE (fluorinated amphiphiles are usuallynot miscible with hydrocarbon ones (29)) are not deter-minant for the DNA compaction process when usingcationic lipids in combination with such colipids.
In Vitro Transfection. To evaluate the contributionof the various F-PEs to in vitro lipoplex-mediated genetransfer, we examined the transfection potential of theabove-described lipoplexes formulated with these F-PEs(alone or in combination with DOPE) as colipids ofpcTG90, DOGS, or DOTAP. Both CL:colipid(s) (1:1 mol)and (1:2 mol) formulations were investigated as thepcTG90:colipid(s) (1:1 mol) and (1:2 mol) lipoplexesproved to be more efficient in vitro and in vivo transfec-tion assays, respectively (8, 14, 15). Lipofection wasassayed in vitro on lung epithelial A549 cells, fromhuman pulmonary carcinoma. These assays were per-formed using the luciferase reporter plasmid pTG11033(pCMV-intronHMG-luciferase-SV40pA, 9572 bps) in thepresence of 10% fetal calf serum for 48 h. All the lipoplexformulations described in the precedent section were
Figure 1. A: Luciferase expression (bars) and cell viability (points) in A549 cells of the lipoplexes prepared in 5% glucose and madeof CL() pcTG90, DOGS, or DOTAP):DOPE:[F8E11][C16]OPE (1:1 - x:x with x ) 0.5, 0.75, or 1) and plasmid pTG11033 (DNA) forvarious N/P ratios, as compared to control DOPE lipoplexes (x ) 0). The given means ( SEM were calculated from four independentexperiments. B: Transfection efficiency of the [F8E11][C16]OPE-based lipoplexes vs that of their corresponding DOPE controllipoplexes. The luciferase level ratio LLR is the ratio of luciferase amount measured for the fluorinated formulation vs that measuredfor its corresponding DOPE control. The efficiency is significantly higher if LLR is g 5, significantly lower if LLR e 0.2, or comparableif 0.2 < LLR < 5. When outside this range, p < 0.01 were carried out in a pairwise comparison. The values given as statisticscorrespond to the number of formulations that satisfy the given LLR condition vs the total number of formulations investigated, andto the respective percentages. *The luciferase expression levels (in the 2 × 103 to 7 × 103 fg luciferase per mg protein range)corresponding to the F-PE and control N/P 0.8 pcTG90-based formulations are not shown.
tested, except those that precipitated. The transfectionefficiency of the lipoplexes, expressed in femtograms (fg)of luciferase/mg of protein, was evaluated for a DNAamount of 0.5 µg/well: in our transfection protocol, aplateau of luciferase expression was generally obtainedfor such a DNA amount. For comparison, naked DNA andthe control lipoplexes based on DOPE as sole colipid werealso tested. Cells treated with naked DNA under equiva-lent conditions showed expression levels of about 102-3
fg of luciferase/mg of protein. The cell viability of thelipoplexes was also checked by determining the totalprotein amount per well of the transfected cells relativeto that measured for untreated cells (for which the totalprotein amount per well is in a 30-50 µg/well range).
The transfection and cell viability results for the lipo-plexes based on‚[F8E11][C16]OPE, [F8C11][C16]OPE,DF4C11OPE or DF4C11PE are illustrated in Figures 1A
to 4A for the CL:colipid(s) (1:1 mol) formulations and inFigure 5A for the pcTG90:colipid(s) (1:2 mol) ones. Theseresults indicate that the “fluorinated” lipoplexes formedfrom any of these F-PEs (used as sole colipid or incombination with DOPE) and any of the cationic lipids(pcTG90, DOGS, or DOTAP) do transfect cells to a largerextent than naked DNA.
Concerning the CL:colipid(s) (1:1 mol) formulations(Figures 1A to 4A), the highest luciferase expressionlevels (LEL) were most frequently observed for N/P ratiosof 2.5, 5, and 10 in the case of pcTG90, of 2.5 and 5 inthe case of DOGS (LEL g 106 fg per mg protein), and of0.8, 2.5, and 5 in the case of DOTAP (LEL g 105 fg permg protein). A much lower plasmid expression wasevidenced for the N/P 0.8 lipoplexes formulated withpcTG90 (2 × 103 < LEL < 7 × 103 fg per mg protein,data not shown) or DOGS (104 < LEL e 106 fg per mg
Figure 2. A: Luciferase expression (bars) and cell viability (points) in A549 cells of the lipoplexes prepared in 5% glucose and madeof CL() pcTG90, DOGS, or DOTAP):DOPE:[F8C11][C16]OPE (1:1 - x:x with x ) 0.5, 0.75, or 1) and plasmid pTG11033 (DNA) forvarious N/P ratios, as compared to control DOPE lipoplexes (x ) 0). The given means ( SEM were calculated from four independentexperiments. B: Transfection efficiency of the [F8E11][C16]OPE-based lipoplexes vs that of their corresponding DOPE controllipoplexes. *The luciferase expression levels (in the 2 × 103 to 7 × 103 fg luciferase per mg protein range) corresponding to the F-PEand control N/P 0.8 pcTG90-based formulations are not shown. For more details concerning LLR, and the statistics, see caption ofFigure 1. When LLR is outside the 0.2-5 range, p < 0.01 in a pairwise comparison.
protein). Concerning the pcTG90:colipid(s) (1:2 mol)formulations (Figure 5A), the highest LEL (g 105 fg permg protein) were observed for N/P 5 and 2.5 while forN/P 1.25 and 0.8, LELs remained below 104 fg per mgprotein (results not shown).
The lower transfection efficiency of the N/P 0.8 and1.25 lipoplexes is most probably related to a lower cellularuptake of these “anionic” and “neutral” formulations ascompared with that of the more cationic lipoplexes owingto expected lower lipoplex-cell electrostatic interactions.The mechanism of cellular endocytic uptake of thelipoplexes is indeed mainly governed by electrostaticinteractions between the DNA complexes and the anionicproteoglycans expressed at the surface of these adherentcells (30, 31), this uptake increasing further with raisingthe N/P ratio of the lipoplexes (32). However, lowerluciferase expression was evidenced for some of the N/P
10 lipoplexes as compared with that of their correspond-ing N/P 5 and 2.5 ones (and even of the N/P 0.8 ones inthe case of DOTAP). Concomitantly, cell viability of theN/P 10 lipoplexes (and more particularly for pcTG90 orDOGS) was improved, indicating that cytotoxicity in-duced by these lipoplexes was not responsible for thelower gene expression. That the transfection efficiencyof the lipoplexes does not necessarily increase with a N/Pratio increase was recently shown to be related tosignificant differences in intracellular distribution andtrafficking (probably release from the endosomes orlysosomes) of the lipoplexes between these N/P ratios(32).
As illustrated in Figure 6, it appears, for N/P g 2.5and irrespective of the fluorinated colipid and of theF-PE:DOPE molar ratio, that most of the “fluorinated”pcTG90- and DOGS-based formulations (90 and 79% of
Figure 3. A: Luciferase expression (bars) and cell viability (points) in A549 cells of the lipoplexes prepared in 5% glucose and madeof CL() pcTG90, DOGS, or DOTAP):DOPE:DF4C11OPE (1:1 - x:x with x ) 0.5, 0.75, or 1) and plasmid pTG11033 (DNA) for variousN/P ratios, as compared to control DOPE lipoplexes (x ) 0). The given means ( SEM were calculated from four independentexperiments. B: Transfection efficiency of the DF4C11OPE-based lipoplexes vs that of their corresponding DOPE control lipoplexes.*The luciferase expression levels (in the 2 × 103 to 7 × 103 fg luciferase per mg protein range) corresponding to the F-PE and controlN/P 0.8 pcTG90-based formulations are not shown. For more details concerning LLR, and the statistics, see caption of Figure 1.When LLR is outside the 0.2-5 range, p < 0.01 in a pairwise comparison.
48 formulations, respectively) were at least 10-fold moreefficient (p < 0.01) for transfecting A549 cells than theircorresponding DOTAP lipoplexes. In a pairwise compari-son, two formulations were considered to have compa-rable transfection efficiency when the LLR ratio of theirrespective LEL was within the 0.2-5 range, and ofsignificantly different efficiency if this ratio is outside thisrange, providing its associated p value is e0.05. Oneshould further underscore the higher transfection poten-tial of the pcTG90-based lipoplexes when compared withthe DOGS ones: if 60% of the 48 pcTG90 formulationsinvestigated were found to lead to comparable LELs tothose of their corresponding DOGS complexes, 35% of the48 pcTG90 formulations led however to LELs that wereat least 5-fold higher (p < 0.05). The transfection efficacysequence evidenced for the F-PE-based lipoplexes (i.e.,pcTG90 g DOGS > DOTAP) for N/P g 2.5 is further
identical to that observed for the lipoplexes based onDOPE as sole colipid. The higher in vitro transfectionefficiency of the pcTG90 and DOGS lipopolyamines ascompared with that of DOTAP can be attributed to theirendosomolytic activity related to the presence of proton-able amine functions of low pKa (“proton sponge” effect)(2, 14, 15). However, for the “negatively charged” lipo-plexes (N/P 0.8), the opposite tendency was found for boththe F-PEs and DOPE, as shown in Figure 6. Theirtransfection efficiency was indeed seen to decrease alongthe sequence DOTAP > DOGS > pcTG90. Concerningthe fluorinated lipoplexes, 63 and 100% of the 16 DOTAP-based formulations were significantly more efficient thantheir corresponding DOGS- (at least 10-fold, p < 0.01)or pcTG90-based lipoplexes (at least 10- to 100-fold, p <0.01), respectively, and 69% of the 16 DOGS-basedlipoplexes were significantly more efficient (at least 10-
Figure 4. A: Luciferase expression (bars) and cell viability (points) in A549 cells of the lipoplexes prepared in 5% glucose andmade of CL () pcTG90, DOGS, or DOTAP):DOPE:DF4C11PE(1:1 - x:x with x ) 0.5, 0.75, or 1) and plasmid pTG11033 (DNA) forvarious N/P ratios, as compared to control DOPE lipoplexes (x ) 0). The given means ( SEM were calculated from four independentexperiments. B: Transfection efficiency of the DF4C11PE-based lipoplexes vs that of their corresponding DOPE control lipoplexes.*The luciferase expression levels (in the 2.103 to 7.103 fg luciferase per mg protein range) corresponding to the F-PE and control N/P0.8 pcTG90-based formulations are not shown. For more details concerning LLR, and the statistics, see caption of Figure 1. WhenLLR is outside the 0.2-5 range, p < 0.01 in a pair-wise comparison.
fold, p < 0.01) than the pcTG90-based ones. As for thecationic lipids, the different tendency observed for theirN/P 0.8 lipoplexes as compared with that found for theirmore cationic (N/P g 2.5) ones is most likely attributableto significant differences in the mechanism of cellularuptake and of intracellular traffic of the lipoplexesbetween these cationic lipids and N/P values.
Irrespective of the nature of the F-PE and of the DOPE:F-PE molar ratio, increasing the colipid content from 50to 66.7% mol (1:1 to 1:2 formulations) led, as shown inFigure 7, most often for the N/P 5 lipoplexes to asignificant decrease in transfection (67% of the 12 F-PEformulations) while comparable and higher LELs wereobserved for 67 and 33% of the 12 N/P 2.5 F-PE formula-tions, respectively. These differences can tentatively beattributed to differences in intracellular distribution and
trafficking of the lipoplexes between these N/P andpcTG90:colipid molar ratios (32). One can further observethat, whatever the N/P ratio, [F8C11][C16]OPE consti-tuted the sole colipid (among the F-PEs and DOPE) forwhich transfection was not affected by or increased withthis CL:colipid molar ratio increase.
The main objectives of this study were aimed atexamining the lipid helper effect on lipofection of theF-PEs as compared with that of DOPE and at determin-ing whether their lipid helper potential was cationic lipidspecific or not. Our results indicate that the F-PEsdisplay such a lipid helper effect that is most oftencomparable with that of DOPE. However, their helpereffect shows some dependency on the cationic lipid. Thisis strongly supported by the statistics deduced frompanels B of Figures 1 to 4 and of Figure 5 for the (1:1)
Figure 5. A: Luciferase expression (bars) and cell viability (points) in A549 cells of the lipoplexes prepared in 5% glucose andmade of pcTG90:DOPE:F-PE (1:2 - y:y with y ) 1, 1.5, or 2) and plasmid pTG11033 (DNA) for N/P 2.5 and 5, as compared to controlDOPE lipoplexes (y ) 0). The given means(SEM were calculated from four independent experiments. B: Transfection efficiency ofthe F-PE-based lipoplexes vs that of their corresponding DOPE control lipoplexes. For more details concerning LLR, and the statistics,see caption of Figure 1. When LLR is outside the 0.2-5 range, p < 0.01 in a pair-wise comparison.
and (1:2 mol) formulations, respectively. These panelsdisplay the luciferase level ratios (LLR) measured for theformulations based on‚[F8E11][C16]OPE, [F8C11][C16]-OPE, DF4C11OPE, or DF4C11PE, respectively, vs thatmeasured for their corresponding control lipoplexes(which contain DOPE as sole colipid). The “helper” effectrelated to the presence of F-PE (in place of part of or ofall DOPE) is significantly higher or lower to that of DOPEif this ratio is higher or lower than 5 or 0.2, respectively,providing its associated p value is e 0.05, and this effectis comparable with that of DOPE if this ratio is withinthe 0.2-5 range. Irrespective of the F-PE chemicalstructure, of the colipid mixture composition, and of theN/P ratio, we found indeed that, when using one of theF-PEs as colipid in place of part of or of all DOPE,comparable transfection levels were most frequentlymeasured for the fluorinated lipoplexes and their respec-tive control DOPE lipoplexes. This is the case for 75-100% of the 12 pcTG90-based (1:1 mol) formulationsinvestigated for each F-PE, for 83% of the 24 pcTG90-based (1:2 mol) formulations, and for 75-50% of the 12DOGS-based formulations investigated for each F-PE.However, a significant proportion of fluorinated DOGS-based lipoplexes (8-33% of the 12 formulations investi-gated for each F-PE) were found to display a highertransfection efficiency than their DOPE control. Bycontrast for the DOTAP-based lipoplexes, the oppositetendency was evidenced: if statistically comparable
transfection levels were obtained for 75-25% of the 12formulations investigated for each F-PE, the complemen-tary 25-75% ones displayed significantly lower trans-fection levels than their respective DOPE control lipoplex-es.
The dependence of the lipid helper potential of theF-PEs on the cationic lipid can be tentatively explainedby considering the geometrical complementarity betweenthe cationic lipids and the colipids, which might have aneffect on the packing within the lamellar phases formedbetween each of the CLs and each of the F-PEs or DOPE,and, consequently, on the physicochemical and biologicalproperties of the DNA lipoplexes they form, and ontransfection. Of the three cationic lipids investigated, itis DOGS with its branched polar head which displaysthe most-pronounced “direct” cone-shaped geometry (pureDOGS was shown to form mainly direct micelles whendispersed in water (33)). The F-PEs displaying a more-pronounced “inverted” cone-shaped geometry than DOPE,one therefore expects a higher degree of geometricalcomplementarity between the F-PEs and DOGS, whichshould result in more tightly packed (or more stable)lamellar phases than between DOPE and DOGS or thanbetween any of the F-PEs and any of the two othercationic lipids. Consequently, one expects that, amongthe different combinations, more stable lipoplexes areformed from the F-PEs and DOGS, and thus a morepronounced helper effect for the F-PEs when used with
Figure 6. Dependence of the F-PE lipid helper effect with the cationic lipid. The luciferase level ratios, LLR, correspond to theratios of luciferase amount measured for the formulations based on [F8E11][C16]OPE (A), [F8C11][C16]OPE (B), DF4C11OPE (C),or DF4C11PE (D) and (i) on pcTG90 vs that measured for their corresponding DOGS (upper section) or DOTAP (middle section)lipoplexes, or (ii) on DOGS vs that measured for their corresponding DOTAP (lower section) lipoplexes. For more details concerningLLR, and the statistics, see caption of Figure 1. When LLR is outside the 0.2-5 range, p < 0.01 in a pair-wise comparison.
DOGS. Conversely, DOTAP which, of the three cationiclipids, displays a geometry that is the closest to acylinder, forms lamellar phases which when combinedwith the F-PEs are expected to be substantially lesstightly packed (or more destabilized) than those formedbetween DOTAP and DOPE. Owing to the lower degreeof geometrical complementarity between DOTAP and theF-PEs, one expects a helper effect of the F-PEs that isbelow that of DOPE, as observed.
Where the helper effect on transfection resulting fromthe composition of the colipid mixture (DOPE:F-PE molarratio) is concerned, it seems most preferable to use aDOPE:F-PE (1:3 mol) colipid combination than F-PEalone or than a F-PE:DOPE (1:1 mol) mixture, whateverthe cationic lipid and the F-PE. If a preference for aspecific DOPE:F-PE combination could not be evidencedamong the few (eleven) CL:colipid(s) (1:1 mol) formula-tions that led to significantly higher transfection levelsthan their respective DOPE controls (see Figure 1B to4B), this is however not the case for the 36 formulationsthat displayed a significantly lower transfection levelthan the DOPE controls. Indeed, of these 36 less efficientformulations, only 7 (19%) concerned the DOPE:F-PE (1:3mol) colipid combination while 12 (33%) and 17 (48%)concerned lipoplexes based on the (1:1 mol) colipid mix-ture and on F-PE as sole colipid, respectively, indicating
that transfection was likely optimal for a DOPE:F-PE (1:3mol) colipid combination.
The aim of this study was also to highlight the impactof the F-PE structural elements (one fluorinated tail onone or on both hydrophobic chains, saturated or unsatu-rated hydrophobic chains, ester or ether-linkage) on theirhelper potential. Concerning the effect of the presenceof a fluorinated tail on one or on both hydrophobic chains(as in [F8E11][C16]OPE or DF4C11OPE, respectively),a statistical analysis performed on the 33 [F8E11][C16]-OPE-based formulations as compared with their respec-tive DF4C11OPE ones did not allow to distinguishbetween these two F-PEs (Figure 8, panel A). Thisanalysis demonstrated comparable, higher and lowertransfection levels for 65, 15, and 20% of the formula-tions, respectively. Although of very different structures,these two F-PEs contain almost the same number offluorine atoms (17 vs 18). Both compounds displayfurthermore a phase transition temperature which isbelow or close to the incubation temperature. One expectstherefore a similar hydrophobic and lipophobic character,
Figure 7. Effect of increasing the colipid content on luciferaseexpression in A549 cells of the pcTG90:colipid(s) lipoplexes (from1:1 to 1:2). The luciferase level ratio LLR is the ratio of luciferaseamount measured for the 1:2 mol formulation vs that measuredfor its corresponding 1:1 mol formulation. For more detailsconcerning LLR, and the statistics, see caption for Figure 1.When LLR is outside the 0.2-5 range, p < 0.01 in a pair-wisecomparison.
Figure 8. Panel A: Effect of the presence of a fluorinated tailon one or on both hydrophobic chains on lipofection. LLRrepresents the ratio of luciferase amount measured for the[F8E11][C16]OPE-based formulation vs that measured for itscorresponding DF4C11OPE-based one. Panel B: Effect of thepresence of a double bond in the hydrophobic chain on lipofec-tion. LLR represents the ratio of luciferase amount measuredfor the unsaturated [F8E11][C16]OPE-based formulation vs thatmeasured for its corresponding saturated [F8C11][C16]OPE-based one. Panel C: Impact of the chemical connexion (ester vsether) on lipofection. LLR represents the ratio of luciferaseamount measured for the ester DF4C11PE-based formulationvs that measured for its corresponding ether DF4C11OPE-basedone. For more details concerning LLR, and the statistics, seecaption of Figure 1. When LLR is outside the 0.2-5 range, p <0.01 in a pair-wise comparison.
and comparable interactions with the cationic lipids,serum proteins, and cellular constituents that have noconsequences on transfection.
The presence of a double bond in the hydrophobicchain, although not determinant, was found to favortransfection, as supported by the statistical analysisperformed on the 33 formulations containing [F8E11]-[C16]OPE as compared with their respective [F8C11]-[C16]OPE ones. This analysis (panel B of Figure 8)showed that, irrespective of the cationic lipid, the com-position of the colipid mixture, the CL:colipid(s) molarratio, and the N/P ratio, higher and comparable trans-fection levels were measured for 21 and 70% of theseformulations, respectively. This result is in line withliterature which showed that optimal transfection activitywas preferably achieved when using cationic lipids and/or colipids containing hydrophobic chains that organizeas “fluid” lamellar phases (e.g., lipids with olefinic chainssuch as [F8E11][C16]OPE which displays a phase transi-tion temperature of 38 °C) rather than “gel“ lamellar ones(e.g. lipids with long saturated chains such as [F8C11]-[C16]OPE which displays a phase transition temperatureof 45 °C) (3, 10, 11, 34-37).
Finally, a comparable helper potential was evidencedfor the ester DF4C11PE and ether DF4C11OPE: indeed85% of the 33 formulations containing DF4C11PE dis-played similar transfection levels than their correspond-ing ones based on DF4C11OPE (panel C of Figure 8). Thisresult is not surprising as both colipids possess a veryclose thermotropic phase behavior and is in line with datafrom literature concerning DOTAP and its ether analogueDOTMA (17). One should underscore that, under theconditions of transfection used, the chemical nature ofthe linkage had no impact on lipoplex cell viability.
CONCLUSION
The present work indicates that “fluorinated” lipo-plexes formulated with fluorinated helper lipids andconventional cationic lipids are definitely very attractivecandidates for gene delivery, highlighting the diversityof lipids leading to efficient transfection. The remarkablein vitro and in vivo transfection potency found for suchfluorinated lipoplexes (8) and that reported for fluori-nated lipoplexes formulated with fluorinated liposper-mines (6, 7) confirms further that lipophobicity andrestricted miscibility of the lipoplex lipids with theendogenous lipids does not preclude efficient gene trans-fer and expression. Their transfection potency is ratherattributable to their unique lipophobic and hydrophobiccharacter (resulting from the formulation of DNA withfluorinated lipids), thus preventing DNA to some extentfrom interactions with lipophilic and hydrophilic biocom-pounds, and from degradation (7). Further experimentsaimed at evaluating the in vivo helper transfectionpotential of the fluorinated PEs are currently underway.
ACKNOWLEDGMENT
We wish to thank Drs. H.V.J. Kolbe and O. Meyer(Transgene) for their interest in this project. Manythanks to Dr. B. Cavallini (Transgene) for supplying theplasmid.
Supporting Information Available: Two tables listingthe mean sizes of the CL() pcTG90, DOGS, DOTAP:DOPE:F-PE (1:1 - x:x mol) and pcT9O:DOPE:F-PE (1:2- y:y mol) lipoplexes formed with plasmid pTG11033,respectively, as determined by light scattering spectros-
copy. This material is available free of charge via theInternet at http://pubs.acs.org.
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