Lipoplex formulation of superior efficacy exhibits high surface activity and fusogenicity, and...

a 1768 (2007) 375ndash386wwwelseviercomlocatebbamem

Biochimica et Biophysica Act

Lipoplex formulation of superior efficacy exhibits high surface activityand fusogenicity and readily releases DNA

Rumiana Koynova a Yury S Tarahovsky ab Li Wang a Robert C MacDonald a

a Department of Biochemistry Molecular Biology and Cell Biology Northwestern University 2205 Tech Drive Evanston IL 60208 USAb Institute of Theoretical and Experimental Biophysics Pushchino 142290 Russian Federation Russia

Received 20 July 2006 received in revised form 20 September 2006 accepted 26 October 2006Available online 1 November 2006

Abstract

Lipoplexes containing a mixture of cationic phospholipids dioleoylethylphosphatidylcholine (EDOPC) and dilauroylethylphosphatidylcholine(EDLPC) are known to be far more efficient agents in transfection of cultured primary endothelial cells than are lipoplexes containing either lipidalone The large magnitude of the synergy permits comparison of the physical and physico-chemical properties of lipoplexes that have verydifferent transfection efficiencies but minor chemical differences Here we report that the superior transfection efficiency of the EDLPCEDOPClipoplexes correlates with higher surface activity higher affinity to interact and mix with negatively charged membrane-mimicking liposomes andwith considerably more efficient DNA release relative to the EDOPC lipoplexes Observations on cultured cells agree with the results obtainedwith model systems confocal microscopy of transfected human umbilical artery endothelial cells (HUAEC) demonstrated more extensive DNArelease into the cytoplasm and nucleoplasm for the EDLPCEDOPC lipoplexes than for EDOPC lipoplexes electron microscopy of cells fixed andembedded directly on the culture dish revealed contact of EDLPCEDOPC lipoplexes with various cellular membranes including those of theendoplasmic reticulum mitochondria and nucleus The sequence of events outlining efficient lipofection is discussed based on the presented datacopy 2006 Elsevier BV All rights reserved

Keywords Cationic lipid Lipofection Membrane fusion Surface tension DNA unbinding Gene therapy

1 Introduction

Synthetic cationic lipids have been the subject of intenseexamination in the areas of cell transfection and gene therapyduring the past two decades They are now considered themost promising non-viral gene carriers A critical obstacle forclinical application of lipid-mediated DNA delivery (lipofec-tion) is its unsatisfactorily low efficiency It is believed thatabout 106 copies of plasmid must be delivered to the cellnucleus to achieve expression sufficient for a clinical effecteven though in a typical transfection experiment with cationicliposomes only 102ndash104 copies enter the nucleus and areexpressed [1] Progress in enhancing the lipofection efficacyis impeded because its mechanism is still largely unknownOne of the major obstacles to efficient transfection is the lowlevel of DNA release from the complexes with cationic lipids

Corresponding author Tel +1 847 491 2871 fax +1 847 467 1380E-mail address r-tenchovanorthwesternedu (R Koynova)

0005-2736$ - see front matter copy 2006 Elsevier BV All rights reserveddoi101016jbbamem200610016

(lipoplexes) Cationic lipidndashDNA interactions are strong [2]and the only possibility for release of DNA under cellularconditions appears to be by neutralization of the cationic lipidcharge with cellular anionic lipids as first demonstrated inmodel experiments [3ndash7] A similar process of lipid exchangebetween lipoplexes and cytoplasmic membranes resulting inDNA release into the cytoplasm was later observed incultured cells too [8ndash10]

Dioleoylethylphosphatidylcholine (EDOPC) the cationictriester of DOPC prepared by phosphate ethylation was oneof the first cationic phospholipids applied to transfection It wasrecently found that mixing EDOPC with its shorter-chainanalog dilauroylethylphosphatidylcholine (EDLPC) dramati-cally increased transfection efficacy [11] The largest improve-ment in transfection efficiency (up to 30-fold) was found with a64 (ww) mixture of EDLPCEDOPC when human umbilicalartery endothelial cells (HUAEC) were transfected This findingwas particularly significant given that primary cells ofendothelial tissues have been especially difficult to transfect

376 R Koynova et al Biochimica et Biophysica Acta 1768 (2007) 375ndash386

[12] In an effort to shed light on the mechanism of lipid-mediated DNA delivery we examined the physical andphysico-chemical properties of the EDLPCEDOPC lipoplexesAs described here we found that their superior transfectionefficiency correlates with higher surface activity higher affinityto interact and mix with negatively charged liposomes and withconsiderably faster DNA release relative to the EDOPClipoplexes1 All of these physical characteristics seem importantin the general lipofection pathway Experiments with modelsystems were complemented with observations on transfectedHUAEC cells Confocal microscopy of transfected HUAECcells demonstrated more extensive DNA release into thecytoplasm and nucleoplasm for the EDLPCEDOPC lipoplexesthan for EDOPC lipoplexes close contacts of EDLPCEDOPClipoplexes with various cellular membranes including those ofthe endoplasmic reticulum mitochondria and nucleus wererevealed by electron microscopy

2 Materials and methods

21 Lipids

The triflate salts of 12-dioleoyl-sn-glycero-3-ethylphosphocholine(EDOPC) and 12-dilauroyl-sn-glycero-3-ethylphosphocholine (EDLPC)were synthesized as previously described [14] or these lipids were purchasedas the chloride salt from Avanti Polar Lipids (Alabaster AL) Dioleoylpho-sphatidylcholine (DOPC) dioleoylphosphatidylethanolamine (DOPE) dio-leoylphosphatidylglycerol (DOPG) dioleoylphosphatidylserine (DOPS)cholesterol (Chol) cardiolipin (CL) (heart Na-salt) phosphatidylinositol(PI) (bovine liver Na-salt) and dioleoylphosphatidylethanolamine withcovalently attached rhodamine (Rh-DOPE) all from Avanti Polar Lipidswere used without further purification The phospholipids migrated as a singlespot by thin-layer chromatography BODIPY FL C12-HPC a fluorescentderivative of PC that has spectral characteristics similar to fluorescein waspurchased from Molecular Probes (Eugene OR) Lipids were stored atminus20 degC in chloroform

22 Liposome and lipoplex preparation

Aliquots of lipids EDOPC or an EDLPCEDOPC mixture were placed inborosilicate glass tubes freed of chloroform with an argon stream and keptunder high vacuum for at least 1 h per mg lipid to remove any solventresidues Subsequently samples were hydrated with phosphate-buffered saline(PBS) pH 74 and vortexed for 5 min For preparation of lipoplexes thecationic liposomes were added to plasmid DNA pCMVSport-β-Gal DNA(from Clontech Palo Alto CA propagated and purified by Bayou BiolabsHarahan LA) at the desired ratio as indicated in the text (Assuming anaverage nucleotide mol wt 330 the lipidDNA weight ratios corresponding toisoelectric lipoplexes are 291 for EDOPC 261 for EDLPCEDOPC 64 ww and 241 for EDLPC all lipoplex compositions used in this study werewith some excess of cationic lipid) For most of the experiments the mostefficient lipoplex composition EDLPCEDOPC 64 (ww) [11] was comparedto the EDOPC lipoplexes in some experiments other EDLPCEDOPC ratioswere also used as indicated In some experiments we used DNA covalentlylabeled with fluorescein-5-isothiocyanate (FITC) using the Label IT labelingkit (Mirus Madison WI) according to the protocol supplied by themanufacturer DNA so labeled was purified on Microspin columns includedin the kit or by ethanol precipitation according to the suggested protocol Thenumber of labels per DNA molecule was measured as suggested by Mirus(www genetransfercom FAQ Q26)

1 Lipoplexes of pure EDLPC exhibited enhanced toxicity the viability ofEDLPC-treated cells was only 45 [13]

23 X-ray diffraction

Samples were prepared by adding pre-formed cationic liposomes (5 wtdispersions) to the DNA aqueous solution as previously described [15]Samples were filled into glass capillaries (d=15 mm) (Charles Super CoNatick MA) flame-sealed and equilibrated for 2ndash3 days at room temperaturebefore measurements Small-angle X-ray diffraction (SAXD) measurementswere performed at 37 degC at Argonne National Laboratory Advanced PhotonSource BioCAT (beamline 18-ID) or DND-CAT (beamline 5-IDD) using12 keV X-rays Exposure times were typically sim05ndash1 s Some samples withlonger exposure time were checked by thin layer chromatography after theexperiments Products of lipid degradation were not detected in these samplesand radiation damage of the lipids was not evident from their X-ray patterns

24 Measurement of surface activity at the surface of lipid dispersions

Lipid dispersions (1 mgmL lipid concentration) were prepared in 015 Msodium chloride solution Subsequently appropriate dilutions were made formeasurements Surface tension was measured via the detachment variation ofthe Wilhelmy method [1617] As a Wilhelmy surface we used a roughened05 mm platinum wire Teflon wells d=08 cm contained 75 μL and weremounted on a platform that underwent 2 mm vertical oscillations 4 timesminThe Wilhelmy wire was attached to the underside sensor connection of a CahnRTL electrobalance Maximum excursions of the recorder pen whichcorresponded to the surface tension at zero contact angle and zero buoyancyof the wire were recorded The instrument was calibrated with clean andaspirated water and initial values were set at 715ndash72 mNm The time course ofchange in surface tension of the airndashwater interface of the cationic lipiddispersions was followed using two different techniques (i) by filling the wellwith 50 μgml lipid dispersion and cleaning the surface by aspirationimmediately before the measurement was started [18] (ii) by spreading ofdispersion containing 5 μg cationic lipid over aqueous subphase in the wellimmediately prior to taking measurements [19] The two techniques gavevirtually identical results with respect to the apparent equilibrium surface tensionreached after sim30ndash60 min but because of its greater consistency the secondtechnique was found more appropriate for following the initial kinetics of thesurface tension change

25 Measurement of the kinetics of DNA release from lipoplexes byusing flow fluorometry

This technology allows determination of the relative lipid content perparticle and the lipidDNA composition of individual particles in the lipoplexdispersion (except for very small vesicles and bare plasmid DNA which are notdetectable on our instrument) [2021] A conventional flow cytometerFACSCalibur (Becton and Dickinson) was used A laser beam is focused on avery small portion of a dilute flowing stream and the fluorescence (at up to 3emission wavelengths) as well as light scattering are measured for each particlepassing through the beam It is distinctively useful because it allows access tomeaningful information on the composition of a heterogeneous populationAlthough not previously used for such purposes it was found extremely wellsuited to analyzing highly heterogeneous lipoplex ensembles Cationic lipid waslabeled with 25 wt of the fluorescent label BODIPY FL C12-HPC DNAsamples were labeled with the high-affinity fluorescent dye ethidiumhomodimer-2 (Ethd-2) at 60 bpdye [20] both labels were purchased fromMolecular Probes (Eugene OR) Negatively charged liposomes mimicking atypical membrane lipid composition (MM=DOPCDOPEDOPSChol45202015 ww) were prepared from unlabeled lipids Particles were detectedat the FL1 channel (515ndash545 nm spectral window) by their BODIPY-PCemission (λem=513 nm) The FL3 channel (spectral window gt650 nm)provides information about the amount of DNA in the particle since Ethd-2strongly emits into this channel (λem=624 nm) The FL3FL1 ratio wascalibrated with lipoplexes that had known DNAlipid ratios of less than one aspreviously described [20] To test for DNA release negatively chargedliposomes (unlabeled) were added at 11 lipid weight ratio to the lipoplexesand data were collected as a function of time Typically 10000 particles wereclassified as to lipid content and composition (time for data collection wasusually lt1 min) and the data presented as a 3-D plot with relative lipid content

377R Koynova et al Biochimica et Biophysica Acta 1768 (2007) 375ndash386

per particle on the x axis the charge ratio of DNA to lipid in the particle (DNAlipid stoichiometry) on the y axis and the relative number of particles on the Zaxis (see Results Fig 5B) The short time needed for data collection makes itpossible to study the formation and disintegration of lipoplexes over time

26 Lipid mixing assay

Lipid mixing was monitored with an assay based on fluorescence resonanceenergy transfer (FRET) between two lipid probes as described earlier [22]Lipoplexes containing 1 wt 11prime-dioctadecyl-333prime3prime-tetramethylindocarbo-cyanine perchlorate (DiI) and 1 wt 33prime-dioctadecyloxacarbocyanineperchlorate (DiO) were prepared according to the procedure described aboveNegatively charged liposomes contained 80 mol DOPC and 20 mol of oneof the negatively charged membrane lipids namely PS PI or CL Liposomeswere prepared as described above For some experiments the liposomedispersions were extruded using an Avanti Mini-Extruder (Avanti Polar LipidsAlabaster AL) equipped with a 01 μm polycarbonate membrane to preparelarge unilamellar vesicles Lipoplexes ware prepared according to standardprotocol (see previous paragraph) and analyzed with an Alphascan fluorometer(Photon Technology International Princeton NJ) the final lipid concentrationwas 20 μgml The lipoplexes were treated with unlabeled negatively chargedliposomes with constant stirring Wavelengths were 489 nm for excitation and506 nm for emission The last step of an experiment was to measure thefluorescence in the presence of 02 wt Triton X100 For calibration of thefluorescence scale the initial residual fluorescence intensity before the additionof anionic lipid was set to zero and the intensity at infinite probe dilutionobtained by lysis of the lipoplexes with Triton X-100 was set to 100 Toestimate the influence of charged lipids on the fluorescence of the probes (frompossible effects of surface charge on extinction coefficients and quantum yield)we tested a sequence of cationicanionic lipid mixtures representing the expectedcompositions of liposomes that would be obtained at different extents of lipidmixing The fluorescence intensity as a function of the percentage of lipidmixing was essentially linear (scatter was approx plusmn10) thus providing asimple and unambiguous relationship between the parameters measured and theamount of lipid mixing [142324]

27 Confocal microscopy

The procedure of lipoplex preparation was similar to that presented aboveexcept that in some samples DNAwas covalently labeled with fluorescein usingthe Label-IT kit as described above The protocol used was similar to that usedfor cell transfection [11] and included manipulation of cells under sterileconditions at 37 degC Cells were treated with lipoplexes containing FITC-labeledDNA and unlabeled lipids and ndash in separate experiments ndash with unlabeled DNAand Rh-DOPE-labeled lipids 50 μl of lipoplex suspension was added to cellscultured in 200 μl EBM-2-MV (with 5 serum) Cells were treated for 2 h afterwhich they were washed with HBSS and incubated for 24 h in EBM-2-MV (with5 serum) at 37 degC followed by 1 wt glutaraldehyde fixation andmicroscopy For fluorescein-labeled DNA we used 488 nm laser excitationand collected emission at 500ndash540 nm For rhodamine labeled lipid we used546 nm laser excitation and collected emission at 560ndash600 nm In allexperiments the pinhole was 10 and 15ndash30 Z-sections were taken Images wereanalyzed with Leica and Volocity (Improvision Coventry England) software

28 Electron microscopy

HUAEC were grown to 80 confluence on Permanox plastic slides (Lab-Tek distributed by Electron Microscopy Sciences Hatfield PA) After standardtreatment of cells with lipoplex solution as described in the previous paragraphthe slides with attached cells were treated with 1 vol glutaraldehyde for60 min at intervals of 1 h 3 h 8 h and 24 h following transfection Then theslides were covered with 1 agar for protection and the cells were postfixedwith 1 wt osmium tetroxide overnight at 4 degC and then with 1 wt tannin for3 h at 4 degC All fixative solutions were prepared in 01 M cacodylate buffer (pH74) After dehydration in an acetone series the slides were covered withPELCO Eponate 12 (distributed by Ted Pella Redding CA) After resinpolymerization the embedded cells were removed from the surface of Permanoxslides and re-embedded in Eponate blocks Sections of 50ndash70 nm thickness were

cut in the direction perpendicular to the cell layer on a MT6000-XL microtome(RMC Tucson AZ) Sections were stained with uranyl acetate and lead citrateand observed with a JEM-100CX (JEOL Peabody MA) electron microscope Asimilar procedure was used for lipoplexes

29 Cell viability assay

Cell viability was assayed 24 h after transfection with the MTT method [25]adapted for a microplate reader as follows Briefly 5 mgml MTT (3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium bromide) solution was added tocells in 96-well plates at 15 μl per well and the plate was incubated at 37 degC for4 h Then 100 μl acid-isopropanol (004 M HCl in isopropanol) was added toeach well and mixed thoroughly to dissolve the dark blue crystals The plateswere read on Spectra MAX PLUS microplate spectrophotometer (MolecularDevices Sunnyvale CA) with a test wavelength of 570 nm and a referencewavelength of 630 nm The viability of untreated cells was set as 100

3 Results

31 Structure of lipoplexes

SAXD revealed that the EDOPC and EDLPCEDOPClipoplexes at different compositions are arranged in lamellararrays as shown by the sets of sharp reflections in thediffraction patterns (Fig 1A B) The same is true for the purelipid samplesmdashthey also arrange into lamellar arrays with sim1ndash15 nm smaller repeat period than that of the lipoplexes sim5 nmfor EDOPC and sim42 nm for EDLPC (not illustrated but seeeg [2226]) The difference in the lamellar spacing induced bythe presence of DNA is consistent with reports on lipoplexes ofother EPCs [1527ndash29] the presence of the DNA strandsbetween the lipid bilayers has been verified by the electrondensity profiles of the lipoplexes [22] In addition to the sharplamellar reflections a low-intensity diffuse peak was alsopresent in the lipoplex diffraction patterns Its spacing was 33ndash34 nm for the EDOPC and EDLPCEDOPC (64) lipoplexes at41 lipidDNA (ww) ratio (Fig 1A) Such peaks have beeninterpreted as reflecting the in-plane packing of the DNAstrands intercalated between the lipid lamellae [273031] Thearrangement of the DNA strands between the lipid bilayers hasbeen found sensitive to the lipidDNA stoichiometry of thelipoplex preparations [2632] and is consistent with anexpanding one-dimensional lattice of DNA chains thus theDNA chains confined between bilayers form a 2D smecticphase [2731] The DNA interstrand repeat distance within theEDLPCEDOPC 64 (ww) lipoplexes increased from 34 nm to44 nm when the lipidDNAweight ratio was increased from 41to 61 (Fig 1A B) The lamellar repeat period of the lipoplexesmonotonously decreased with increasing EDLPC fraction (Fig1C) In spite of the pronounced difference in the chain lengths ofthe two cationic lipids the SAXD patterns revealed noindication of phase separation The DNA interstrand distancealso decreased with increasing the EDLPC fraction (Fig 1Cinset)

Thin-section electron microscopy (Fig 2) also revealed amultilamellar structure of both EDOPC lipoplexes andEDLPCEDOPC 64 (ww) lipoplexes In transverse sectionsthe lipoplexes were typically seen to be composed ofperiodically arranged bilayers in concentric circles or in

Fig 1 Small-angle X-ray diffraction profiles of (A) EDOPC and EDLPCEDOPC (64) lipoplexes at 41 lipidDNAweight ratio (arrow points to the peaks originatingfrom the DNAndashDNA in-plane correlation) (B) EDLPCEDOPC lipoplexes at different lipid composition and 61 lipidDNAweight ratio in order to magnify the DNAdiffraction peaks the sharp lipid lamellar reflections are truncated Diffraction data were collected for 1 sec at 37 degC (C) Lipid lamellar repeat distance and DNAspacing (inset) of EDLPCEDOPC lipoplexes at different compositions


uniform spirals (Fig 2) As demonstrated previously thesepatterns represent bilayers alternating with single layers ofparallel DNA molecules [2231] Our dynamic light scatteringexperiments showed that the mean diameter of the cationicliposomes was about 550 nm for EDOPC and about 400 nmfor EDLPCEDOPC 64 (ww) In addition to the demonstra-tion of similar structure and morphology with methods justdescribed fluorescence measurements on lipoplexes treatedwith increasing concentrations of NaCl [2] also revealed

Fig 2 Thin-section electron microscopy of (A) EDOPC lipoplexes and (B)EDLPCEDOPC lipoplexes For both images bar is 01 μm

virtually equivalent DNA-cationic lipid binding energies forthe EDOPC and EDLPCEDOPC 64 lipoplexes (Koynovaunpublished data)

32 Surface activity of the cationic lipid dispersions

The rate of transfer of lipid molecules from the bulk to theairndashwater interface (dynamic surface activity [17]) was assessedby monitoring the changes in the surface tension of theliposome suspension with time [16] The surface tension ofEDLPCEDOPC suspensions of different compositions dis-persed in 015 M NaCl is presented as a function of time in Fig3 As judged from the initial rate of the surface tension decreasethe rate of transfer of lipid molecules to the airndashwater interfacewas greater for dispersions in which EDLPC was the dominantlipid The apparent equilibrium surface tension (sim40 dyncmfor EDOPC vs ltasymp30 dyncm for EDLPCEDOPC 64) as wellas the equilibration time (sim45 min for EDOPC vs sim25 min forEDLPCEDOPC 64) were also considerably lower for themixtures that contained more EDLPC than EDOPC (Fig 3inset)

33 Lipid mixing between lipoplexes and negatively chargedliposomes

In the lipid mixing experiments we used as a targetliposomes containing 80 mol DOPC and 20 mol negativelycharged membrane lipid (PS PI or CL) correspondingapproximately to the amount of anionic lipids in cellularmembranes [3334] The initial rates of lipid mixing with allthree anionic membrane lipids tested were considerably higherfor the EDLPCEDOPC 64 lipoplexes than for the EDOPClipoplexes and in terms of the type of negatively charged lipidinitial rates were in the sequence CLgtPSgtPI for the extrudedunilamellar anionic liposomes (Fig 4) This trend was similar tothat observed with multilamellar negatively charged liposomes

Fig 3 Time course of the change in surface tension of surfaces of dispersions of EDLPCEDOPC at different lipid ratios recorded immediately after spreading adispersion containing a total of 5 μg cationic lipid over the aqueous subphase Inset Equilibration time (upper panel) and apparent equilibrium surface tension (lowerpanel) at different EDLPCEDOPC ratios In the lower panel data obtained both by suspension spreading and by surface aspiration (see Materials and methods) areincluded


(not illustrated) as well as with the trend previously observedfor EDOPC lipoplexes [35]

34 Kinetics of DNA release from lipoplexes induced bynegatively charged membrane lipids

We applied flow-fluorometry [20] to examine as a functionof time the release of DNA from EDOPC and EDLPCEDOPC lipoplexes induced by addition of negatively chargedliposomes that had a composition mimicking natural mem-branes MM=DOPCDOPEDOPSChol 45202015 (ww)[36] The results are presented as plots of the DNAcationiclipid stoichiometry (as a charge ratio) vs the relative cationiclipid content of the individual particles (see Fig 5B) atdifferent times after the addition of the negatively chargedliposomes (Fig 5A) The lipoplexes were prepared at close toisoelectric conditions (DNAlipid sim11 charge ratio) andequilibrated for 30 min before the addition of the negativelycharged membrane lipids measurements were initiatedimmediately upon addition of the negatively charged lipo-somes As seen from the leftmost plots in Fig 5A the EDLPCEDOPC 64 lipoplexes are more homogeneous with respect tolipidDNA composition and have higher lipid content than theEDOPC lipoplexes The latter observation correlates with ourdynamic light scattering experiments the latter indicated that

although EDLPCEDOPC 64 liposomes are somewhat smallerthan EDOPC liposomes (400 nm vs 550 nm) the mixed lipidlipoplexes grew about 4x larger than the EDOPC lipoplexes(not illustrated) Shortly after addition of the negativelycharged liposomes to the EDOPC lipoplexes particles ofintermediate DNAcationic lipid stoichiometries could bedetected but even after 60 min the original 11 lipoplexesstrongly dominated the distribution only after gt2 h was aconsiderable decrease of the DNAcationic lipid stoichiometry(ie DNA release) observed In the case of EDLPCEDOPC64 lipoplexes changes in composition occurred within 5 minafter addition of the negatively charged liposomes the DNAcationic lipid stoichiometry was shifted to lower values andafter 45ndash60 min a considerable portion of the DNA appears tohave been released from the lipoplexes Similar experimentswere also carried out on EDLPCEDOPC 28 and 46 (ww)lipoplexes (not illustrated) The flow-fluorometry data wereused to assess the kinetics of the DNA release from thelipoplexes of different compositions The data calculated asthe portion of the initially contained DNA that was retained inthe lipoplexes after the addition of the negatively chargedliposomes are plotted as a function of time in Fig 5C TheEDLPCEDOPC 64 lipoplex formulation which is the mostactive transfection agent [11] exhibited much faster DNArelease than did lipoplexes of other compositions

Fig 4 FRET assay for mixing between EDOPC lipoplexes (1 2 and 3) orEDLPCEDOPC 64 lipoplexes (1a 2a and 3a) and DOPC unilamellarliposomes containing 20 mol CL (1 and 1a) PS (2 and 2a) or PI (3 and 3a)



The distribution of fluorescent DNA and lipid (Fig 6) wasexamined in HUAEC cells fixed with glutaraldehyde 24 h aftertreatment with lipoplexes Lipid was rendered fluorescent byinclusion of (Rh-DOPE red) and DNA was covalently labeled(FITC-DNA green) In cells treated with EDOPC lipoplexesboth DNA and lipid were localized in the perinuclearendosomes (Fig 6A C) In contrast the distribution offluorescent compounds in cells treated with EDLPCEDOPClipoplexes was quite different (Fig 6B D) clearly showing thatDNA and lipid had both spread into the cytoplasm In additionDNA fluorescence was also detectable in the nucleoplasmIncidentally the latter observation is noteworthy since it is notoften that plasmid fluorescence in the nuclei has been observedSomewhat surprisingly some Rh-DOPE labeled lipid was alsopresent in the nucleoplasm 3-D reconstitution of fluorescenceprofiles revealed presence of Rh-DOPE fluorescent spots on thebottom of the nucleus some fibrillar structures crossing thenucleus were also visible (Tarahovsky et al unpublished data)We were unable to attribute the localization of fluorescent lipidto any of the nuclear regions

36 Electron microscopy of cells

For this study a technique for transverse thin-sectioning ofcell monolayers was developed in which cells were fixed andembedded directly on the plastic growth surface The procedure

excluded enzymatic treatment or centrifugation of cells whichcould lead to changes of the initial shape and possibleredistribution of cytoplasmic components All cells wereobserved to have maintained their native spindle-like shape intransverse sections In the micrograph shown in Fig 7A thebottom of the picture corresponds to the attachment site of thecultured cell to the plastic surface although the plastic itself isnot present having been removed before sectioning Electronmicroscopy of cells treated with lipoplexes revealed easily-recognizable endosomal compartments in the cytoplasm con-taining multilamellar lipoplexes similar to that describedpreviously by others [37ndash39]

EDOPC lipoplexes retained their multilamellar structure forlonger than did lipoplexes prepared from the EDLPCEDOPC64 mixtures In some cases the multilamellar structure of theEDOPC lipoplexes could be seen as long as 24 h aftertransfection (not shown) The lipoplexes containing theEDLPCEDOPC 64 mixture lost their multilamellar structureand after a few hours of residence within cells took on theappearance of vesicles with only a few layers of membranesEndosomes containing EDLPCEDOPC 64 lipoplexes werecommonly seen interacting intimately with various cytoplasmicmembranes (it should be recognized that we cannot distinguishbetween an endosome that completely encapsulates a lipoplexand one that has fused with the outer layers of a lipoplex suchthat its membrane has acquired cationic lipids) In particularthere were numerous examples of endosomelipoplex contactswith mitochondria endoplasmic reticulum (Fig 7A) and nuclei(Fig 7B)

4 Discussion

Understanding the mechanism of gene delivery by cationicliposomes is of utmost importance for effective gene therapyA number of physical and physico-chemical factors havebeen suggested as lipofection modulators but the specificroute of DNA delivery by cationic lipid vectors is still mostlyunknown and their efficiency of delivery is unsatisfactorilylow for many applications To date the primary approach toimproving transfection properties of cationic lipids hasinvolved synthesizing new kinds of cationic amphiphiles orincluding non-cationic helper lipids in lipoplex formulationsAnother strategy more recent and particularly effective is tocombine two cationic lipid derivatives having the same headgroup but different hydrocarbon chains Such combinationsoften synergistically enhance transfection and allow optimiz-ing activity by merely varying the ratio of the twocomponents For example some compositions of the cationiclipid binary mixture EDLPCEDOPC transfected DNA intocells over 30 times more efficiently than either compoundseparately [11] Because of the magnitude of this synergyand the fact that it involved homologs of the same mole-cules this system appeared appropriate for analyzing theorigin of the activity difference between them by comparingthe physical and physico-chemical properties of lipoplexeswith very different transfection efficiencies but minor chemicaldifferences

Fig 5 (A) Plots of DNAlipid stoichiometry (charge ratio) vs cationic lipid content showing the time-course of DNA unbinding from EDOPC (upper panel) orEDLPCEDOPC 64 (lower panel) lipoplexes after addition of a negatively charged model membrane (MM)mixture (MM=DOPCDOPEDOPSChol 45202015 ww) The stoichiometry (y) axis represents the ratio of DNA to lipid charges in the particle Lipoplexes contained a nearly isoelectric ratio of cationic lipid and DNAlabeled with 25 BODIPY-FL and DNA labeled with the high affinity label ethidium homodimer-2 (EthD-2) at 60 bpdye The panels show the stoichiometry vslipid content distributions after different times of incubation at room temperature as indicated Lipoplexes were initially prepared at near the isoelectric lipidDNAratio negatively charged liposomes were added at a 11 weight ratio to the cationic lipid Data on each panel were collected on 10000 particles within 1 min (B)Explanation of the plots (C) kinetics of DNA release (plotted as the portion of the initial DNA retained at different time points) after addition of negatively chargedmembrane-mimicking liposomes for different EDLPCEDOPC compositions


In an earlier publication we described a correlation betweenthe delivery efficiency of these DNA carriers and themesomorphic phases they form after interaction with anionicmembrane lipids Specifically formulations that are particularlyeffective DNA carriers form phases of highest negativeinterfacial curvature when mixed with negatively chargedmembrane lipids whereas less effective formulations formphases of lower curvature under the same conditions [40] In thepresent study we examined further physical characteristics thatmight account for the transfection efficiency of superior

lipoplexes namely their structure surface activity propensityto admixfuse with negatively charged membrane lipids andability to eventually release DNA Experiments with modelsystems were complemented with observations on transfectedHUAEC cells

EDOPC and EDLPCEDOPC lipoplexes are structurallysimilar to each other and to the majority of lipoplexes[27303141] consisting of lamellar lipid arrays with inter-calated DNA threads (Fig 1) The density of DNA packing ishigher (lower interstrand distance) in lipoplexes in which

Fig 6 Confocal microscopy of HUAEC cells fixed 24 h after treatment with lipoplexes that contained rhodamine-labeled lipid (A and B) and fluorescein-labeledDNA (C and D) Cells were treated with EDOPC lipoplexes (A and C) and EDLPCEDOPC lipoplexes (B and D) EDOPC lipoplexes (A C) remained in compactperinuclear endosomes while EDLPCEDOPC 64 lipoplexes (B D) interacted with cellular membranes and released DNA into both cytoplasm and nucleus


EDLPC predominates (Fig 1C inset) as expected from thepresumptive lower area per lipid molecule of the shorter-chaincompound [42]

The lipid concentration of the dispersions used in the X-raydiffraction experiments (sim50 mM) are certainly considerablyhigher than bulk concentrations in the cell however they maybe similar to the local concentrations in the cell (ie atmembranendashmembrane contacts) In any case their relevance tophysiological concentrations with respect to phase andstructural data obtained has been repeatedly checked by controlexperiments at low concentrations (Koynova unpublished databut also see eg the inset of Fig 2A in ref[31])

In order for DNA to be released from lipoplexes and enter thecell nucleus where it is transcribed the cationic lipidelectrostatic charge must be neutralized The unbinding ofDNA from lipoplexes has been identified as one of the criticalsteps along the transfection route Although there may be otherpossibilities according to current understanding it involvesneutralization of cationic lipid by cellular anionic lipids Indeedaddition of negatively charged liposomes to lipoplexes results in

dissociation of DNA from the lipid [34223543] Neutraliza-tion of cationic lipid carriers by anionic membrane lipidsrequired for DNA release presupposes lipid transfer betweencationic lipoplexes and negatively charged membranes mostlikely by fusion of cell membranes with lipoplexes Mixing oflipoplex lipids with cellular lipids was observed a number ofyears ago and interpreted as fusion [8] Precisely how thelipoplex bilayers fuse with cell membranes is unclear [35] butthere is no question that cationic and anionic membranes arecapable of both fusion and hemifusion and do so extremelyrapidly even at relatively low anionic charge densities [44]Because escape of lipoplexes from endosomes prior to theirentry into lysosomes is essential for efficient transgeneexpression fusion of lipoplexes with endosomal membranesshould facilitate DNA release from endosomes into thecytoplasm and thus promote DNA expression To quantifythis fusion process (strictly lipid mixing is what is measuredand indeed lipid mixing is what is required for neutralizationof the lipoplex lipid) we used a FRET assay involving a pairof fluorescent lipid dyes It revealed that EDLPCEDOPC

Fig 7 Electron micrographs of HUAEC cells treated with EDLPCEDOPC 64 lipoplexes and fixed 8 h later Panel A shows a typical transverse section of a treatedcell Lipoplexes perhaps with attached remnants of endosomal membrane (ES) were seen in contact with mitochondria (MC) and endoplasmic reticulum (ER) Inpanel B multilamellar lipoplexes are seen in contact with the nucleus (Nuc) For all images the bar corresponds to 05 μm


lipoplexes fuse faster and more extensively with negativelycharged liposomes (Fig 4) than do EDOPC lipoplexesPreviously it was reported that EDLPCEDOPC lipoplexesexhibit higher fusion capacity with liposomes containing theanionic DOPG [11] Here we report that the superiorfusogenicity of the EDLPCEDOPC mixed lipoplexes is alsomanifested with other anionic membrane lipid classesmdashCL PSand PI Even though fusion of lipoplexes with membranes doesnot necessarily result in release of DNA from the lipoplexes itis nevertheless likely to be beneficial for lipoplex escape fromendosomes That is membrane fusion is probably an essentialstep in transfection but DNA release from the lipoplex may bethe rate-limiting step Indeed a lack of close correlationbetween transfection activity and lipoplexndashmembrane lipidmixing has been reported before [45ndash49] Our recent experi-ments also showed that facile fusion with negatively chargedliposomes characterizes a variety of cationic lipid mixtures(relative to single-component cationic liposomes) regardless oftheir efficacy as transfection agents [13] This phenomenon isperhaps related to nonideal mixing domain formation andorhigher probability for packing defects in the cationic lipidmixtures relative to single component bilayers (packing defectsshould facilitate the fusion of bilayers because bilayer fusion atsome stage in the process must involve merging of thehydrophobic cores of the two participating membranes [50])There is no doubt that two cationic lipids have more degrees offreedom in lipoplexndashmembrane interactions than does a singlelipid Increased degrees of freedom in turn allow for largervariation of membrane curvature which is likely to beimportant in membrane fusion The bilayer bending constantis known to be lowered in lipid mixtures as a result of the factthat a mixed bilayer may assume different compositions in thetwo opposing monolayers the magnitude of the bendingconstant reduction increases with increasing difference between

the two amphiphiles with respect to charge head group sizeand chain length [51] Thus mixed bilayers especially thoseinvolving species of considerable difference in the chain lengthwould exhibit higher tendency to curve and eventually fuse Inany case high fusogenicity is likely to be an attribute of eventhough not a guarantee for superior transfection activity It isclear that extrapolating results from fusion experiments on lipidmodel systems to natural membranes that contain proteins andmuch more complex mixture of lipids requires care hence weemphasize that our lipid mixing (FRET) experiments involvedoppositely charged lipid aggregates (as do the lipoplexndashmembrane interactions) in which fusion is activated byelectrostatic interactions Moreover observations through themicroscope of giant vesicles and fluorescent lipoplexes verifythat true fusion of the lipoplexes with negatively charged bilayermembranes does occur [4452ndash54]

As mentioned above lipid mixing in fact is what wasmeasured by the FRET experiments In principle monomerlipid exchange between aggregates could produce generallysimilar FRET results The presumptive higher CMC of theshorter-chain EDLPC may significantly accelerate the lipidmolecular transfer (the term ldquoCMCrdquo is used here to indicate theminimal lipid concentration required for monomeric lipids toform a supramolecular assembly regardless of its geometry)Indeed a difference of even two methylene groups in each chainresults in a sim30-fold change of the CMC of phosphatidylcho-lines [55] In the case of EDLPC and EDOPC the difference issix CH2 groups so a much higher monomer concentration isexpected in the dispersions with dominant EDLPC content Thehigher concentration of monomers was indeed found tosignificantly facilitate molecular transfer between aggregateseven when the aggregates are all positively charged [56]

The dynamic surface activity of the cationic lipid mixtureswith dominating EDLPC content is also considerably higher


than that of the EDOPC dispersion (Fig 3) The different ratesof monolayer formation at the airwater interface of dispersionsof lipid vesicles may stem from a variety of different causesBoth individual molecules and aggregates may contribute tomonolayer formation In the former case of course the higherCMC of EDLPC might be responsible for acceleratedmonolayer formation Diffusion of whole aggregates to themonolayer with subsequent disruption and rearrangement toform an extended planar monolayer may certainly alsocontribute to the monolayer formation but since the liposomesize is not significantly different for different lipid composi-tions this mechanism could hardly account for the pronounceddifference in the surface activity of the EDOPC and EDLPCEDOPC aggregates In fact with respect to its surface activitythe pure EDLPC dispersions are even superior to the EDLPCEDOPC 64 mixture of maximum transfection activity (Fig 3)The reason for the lower efficiency of the EDLPC lipoplexes isprobably their toxicity the viability of EDLPC-treated cells wasonly 45 and such cells are likely too compromised to expresshigh levels of beta-galactosidase [11] so it may be that thehigher solubility and higher CMC of EDLPC accounts for or atleast contributes to its toxicity when used in the absence ofEDOPC

With respect to the relationship of the processes of molecularlipid exchange and lipoplexndashmembrane fusion to the lipid-mediated DNA delivery we emphasize that they should not beconsidered independent processes in a system involvingpositive and negative lipid aggregates In fact initial molecularexchange between the cationic lipid aggregates and thenegatively charged membranes would create cationic anioniclipid mixtures and such mixtures are known for their strongdisposition to form nonlamellar phases [355758] Thus sincethe propensity for formation of curved nonlamellar phases isthought to play a critical role in fusion of lipid aggregates[5960] molecular lipid exchange could be expected to triggerfusion between cationic and anionic lipid aggregates

DNA release data revealed a correlation between the extentof transfection by different lipoplex formulations and the extentof DNA release that was induced by treating those lipoplexformulations with membrane-mimicking negatively chargedliposomes the highly active transfection agent EDLPCEDOPC64 exhibited considerably faster DNA release relative to theless effective EDOPC (Fig 5A C) after interaction with thenegatively charged membrane lipids The more complete DNArelease from the EDLPCEDOPC 64 lipoplexes is a likelyresult of the propensity of that cationic lipid mixture to formphases of the highest negative interfacial curvature when mixedwith negatively charged membrane lipids [40] Indeed DNArelease has been shown to unambiguously correlate with theinterfacial curvature of the phases that develop when cationiclipids of the lipoplexes interact with the negatively chargedcellular lipids [61]

We have made some effort to relate our experiments onmodel systems to the behavior of the same lipoplex formula-tions in transfected cultured cells It is commonly thought thatduring cell transfection with cationic liposomes lipoplexesenter the cytoplasm by endocytosis and that the DNA is

subsequently released into the cytoplasm and migrates to thenucleus [81037386263] Indeed thin section electronmicroscopy (Fig 7) revealed the presence of lipoplexes insideendosomes as has been previously demonstrated [37ndash39]Furthermore the present data revealed instances of closecontacts of lipoplexes with various cellular membranesincluding those of the mitochondria endoplasmic reticulumand nucleus Membrane contacts were more frequentlyobserved in cells treated with lipoplexes containing themedium-chain cationic lipid (EDLPCEDOPC 64 lipoplexes)than in cells treated with lipoplexes consisting only of the longerchain component (EDOPC) Confocal microscopy also con-firmed a considerable preference of EDLPCEDOPC 64lipoplexes to exchange lipid with cytoplasmic membranes andto release DNA into the cytoplasm and the nucleus (Fig 6)

Lipoplexes with mean diameters of about 400ndash500 nm andlamellar repeat distances ofsim5ndash6 nm as observed in the presentstudy would contain some sim20 membrane bilayers It is thusobvious that lipid exchange between a multilamellar lipoplexand surrounding single bilayer of endosomal membrane is notsufficient to neutralize the cationic charge necessary for DNAunbinding and release from lipoplexes and much moreextensive intermembrane interactions are required for morecomplete DNA release The contacts visualized by electronmicroscopy between the superior EDLPCEDOPC 64 lipo-plexes and various cellular membranes may therefore supportthe concept of gradual lipoplex peeling and DNA release

The lipid composition of different organelles may influencelipoplex lipid exchange and fusion with different cellularmembranes and hence also the distribution of released DNAinside of cell The facile fusion of lipoplexes with CL-containing liposomes (Fig 4) suggests the possibility ofinteraction and fusion of lipoplexes with mitochondrialmembranes and indeed contacts of EDLPCEDOPC 64lipoplexes with mitochondrial membranes were observed byelectron microscopy (Fig 7A) Although the extent to which CLis found on the external mitochondrial membrane remains to besettled [64] the high content of this anionic lipid inmitochondria as a whole has been clearly established [65]

In summary (i) the EDLPCEDOPC 64 mixture which hadsuperior transfection efficiency was found to exhibit consider-ably higher surface activity than EDOPC a characteristic likelyto favor cationic lipid transfer to (negatively charged)membranes Mixtures of anionic and cationic lipids are thuslikely to be generated in cells treated with these lipoplexes suchmixtures are known for their strong disposition to formnonlamellar phases [575861] which propensity is thought toplay a critical role in the fusion ability of lipid aggregates[5960] indeed actual fusion of lipoplexes with anionicliposomes has been demonstrated [54] Furthermore theEDLPCEDOPC 64 mixture exhibits considerably higherability to mix with negatively charged liposomes as demon-strated by FRET Lipoplexndashmembrane mixing contributes tothe charge neutralization of cationic lipids by cellular anioniclipids which is required for DNA unbinding [3414]

(ii) DNA discharge measured subsequent to treatinglipoplexes with negatively charged membrane-mimicking


liposomes was considerably faster for the EDLPCEDOPC 64mixture than for EDOPC lipoplexes An unambiguous correla-tion between DNA release and the phases formed by themixtures of the cellular lipids with the cationic lipids of thelipoplexes has been reported earliermdashspecifically the magni-tude of the negative interfacial curvature of the phase assumedby the cationicndashcellular lipid mixture correlated with release ofDNA [61] The EDLPCEDOPC 64 described here conforms tothat correlation in that it also developed highly curvednonbilayer phases when mixed with negatively chargedmembrane lipids [40]

(iii) thin-section electron microscopy of treated cellsrevealed contacts between EDLPCEDOPC 64 lipoplexes andvarious cellular membranes including those of the endoplasmicreticulum mitochondria and nucleus Because of the multi-layered structure of the lipoplexes multiple encounters betweenlipoplexes and various cellular membranes are expected to beneeded for efficient release of lipoplex DNA

Acknowledgments

NIH grant GM52329 provided primary funding for thisresearch and NIH grant GM57305 provided some importantadditional support We thank Robert A Lamb (NorthwesternUniversity) for access to the flow cytometer instrumentWilliam Russin (Northwestern University) for technical sup-port in confocal microscopy and Harsh Parikh and YaekoHiyama (Northwestern University) for synthesis of cationiclipids BioCAT is a NIH-supported Research Center throughGrant RR08630 Use of the Advanced Photon Source wassupported by the US Department of Energy Basic EnergySciences Office of Energy Research under Contract No W-31-102-Eng-38 DND-CAT is supported by EI DuPont deNemours and Co The Dow Chemical Company NSF GrantDMR-9304725 and the State of Illinois through the Depart-ment of Commerce and the Board of Higher Education GrantIBHE HECA NWU 96 Use of APS was supported by the USDOE Basic Energy Sciences Office of Energy ResearchContract No W-31-102-Eng-38

References

[1] R Tachibana H Harashima N Ide S Ukitsu Y Ohta N Suzuki HKikuchi Y Shinohara H Kiwada Quantitative analysis of correlationbetween number of nuclear plasmids and gene expression activity aftertransfection with cationic liposomes Pharm Res 19 (2002) 377ndash381

[2] E Pozharski RC MacDonald Lipoplex thermodynamics determinationof DNA-cationic lipoid interaction energies Biophys J 85 (2003)3969ndash3978

[3] YH Xu FC Szoka Mechanism of DNA release from cationic liposomeDNA complexes used in cell transfection Biochemistry 35 (1996)5616ndash5623

[4] O Zelphati FC Szoka Mechanism of oligonucleotide release fromcationic liposomes Proc Natl Acad Sci U S A 93 (1996) 11493ndash11498

[5] FC Szoka YH Xu O Zelphati How are nucleic acids released in cellsfrom cationic lipidndashnucleic acid complexes Adv Drug Delivery Rev 24(1997) 291

[6] S Bhattacharya SS Mandal Evidence of interlipidic ion-pairing inanion-induced DNA release from cationic amphiphilendashDNA complexes

mechanistic implications in transfection Biochemistry 37 (1998)7764ndash7777

[7] P Harvie FMP Wong MB Bally Characterization of lipid DNAinteractions I Destabilization of bound lipids and DNA dissociationBiophys J 75 (1998) 1040ndash1051

[8] I Wrobel D Collins Fusion of cationic liposomes with mammalian-cellsoccurs after endocytosis Biochim Biophys Acta Biomembr 1235 (1995)296ndash304

[9] A Noguchi T Furuno C Kawaura M Nakanishi Membrane fusionplays an important role in gene transfection mediated by cationicliposomes FEBS Lett 433 (1998) 169ndash173

[10] M Nakanishi A Noguchi Confocal and probe microscopy to study genetransfection mediated by cationic liposomes with a cationic cholesterolderivative Adv Drug Delivery Rev 52 (2001) 197ndash207

[11] L Wang RC MacDonald New strategy for transfection mixtures ofmedium-chain and long-chain cationic lipids synergistically enhancetransfection Gene Ther 11 (2004) 1358ndash1362

[12] M Teifel LT Heine S Milbredt P Friedl Optimization of transfectionof human endothelial cells Endothelium (New York) 5 (1997) 21ndash35

[13] L Wang R Koynova H Parikh RC MacDonald Transfection Activityof Binary Mixtures of Cationic O-Substituted PhosphatidylcholineDerivatives The Hydrophobic Core Strongly Modulates Their PhysicalProperties and DNA Delivery Efficacy Biophys J 91 (2006) 3692ndash3706

[14] RC MacDonald VA Rakhmanova KL Choi HS Rosenzweig MKLahiri O-Ethylphosphatidylcholine a metabolizable cationic phospholi-pid which is a serum-compatible DNA transfection agent J Pharm Sci 88(1999) 896ndash904

[15] R Koynova RC MacDonald Mixtures of cationic lipid o-ethylpho-sphatidylcholine with membrane lipids and DNA phase diagramsBiophys J 85 (2003) 2449ndash2465

[16] RC MacDonald SA Simon Lipid monolayer states and theirrelationships to bilayers Proc Natl Acad Sci U S A 84 (1987)4089ndash4093

[17] RC MacDonald A Gorbonos MM Mornsen HL Brockman Surfaceproperties of dioleoyl-sn-glycerol-3-ethylphosphocholine a cationicphosphatidylcholine transfection agent alone and in combination withlipids or DNA Langmuir 22 (2006) 2770ndash2779

[18] RZ Qiu RC MacDonald A metastable state of high surface-activityproduced by sonication of phospholipids Biochim Biophys ActaBiomembr 1191 (1994) 343ndash353

[19] A Jordanova Z Lalchev B Tenchov Formation of monolayers andbilayer foam films from lamellar inverted hexagonal and cubic lipidphases Eur Biophys J 31 (2003) 626ndash632

[20] E Pozharski RC MacDonald Analysis of the structure and compositionof individual lipoplex particles by flow fluorometry Anal Biochem 341(2005) 230ndash240

[21] E Pozharski RC MacDonald Single lipoplex study of cationic lipidndashDNA self-assembled complex flow cytometric approach Biophys J 80(2001) 427A

[22] RC MacDonald GW Ashley MM Shida VA Rakhmanova YSTarahovsky DP Pantazatos MT Kennedy EV Pozharski KA BakerRD Jones HS Rosenzweig KL Choi RZ Qiu TJ McIntoshPhysical and biological properties of cationic triesters of phosphatidylcho-line Biophys J 77 (1999) 2612ndash2629

[23] G Ouedraogo P Morliere C Maziere JC Maziere R Santus Alterationof the endocytotic pathway by photosensitization with fluoroquinolonesPhotochem Photobiol 72 (2000) 458ndash463

[24] M Traore RJ Sun S Fawzi-Grancher D Dumas X Qing R Santus JFStoltz S Muller Kinetics of the endocytotic pathway of low densitylipoprotein (LDL) in human endothelial cells line under shear stress an invitro confocal microscopy study Clin Hemorheol Microcirc 33 (2005)243ndash251

[25] T Mosmann Rapid colorimetric assay for cellular growth and survivalmdashApplication to proliferation and cyto-toxicity assays J Immunol Methods65 (1983) 55ndash63

[26] R Koynova RC MacDonald Cationic o-ethylphosphatidylcholines andtheir lipoplexes phase behavior aspects structural organization andmorphology Biochim Biophys Acta Biomembr 1613 (2003) 39ndash48


[27] R Koynova RC MacDonald Columnar DNA superlattices in lamellar o-ethylphosphatidylcholine lipoplexes mechanism of the gelndashliquid crystal-line lipid phase transition Nano Lett 4 (2004) 1475ndash1479

[28] R Koynova L Wang RC MacDonald An intracellular lamellarndashnonlamellar phase transition rationalizes the superior performance of somecationic lipid transfection agents Proc Natl Acad Sci U S A 103(2006) 14373ndash14378

[29] VA Rakhmanova TJ McIntosh RC MacDonald Effects of dioleoyl-phosphatidylethanolamine on the activity and structure of o-alkylphosphatidylcholinendashDNA transfection complexes Cell Mol Biol Lett5 (2000) 51ndash65

[30] DD Lasic H Strey MCA Stuart R Podgornik PM Frederik Thestructure of DNAndashliposome complexes J Am Chem Soc 119 (1997)832ndash833

[31] JO Radler I Koltover T Salditt CR Safinya Structure of DNAndashcationic liposome complexes DNA intercalation in multilamellarmembranes in distinct interhelical packing regimes Science 275 (1997)810ndash814

[32] I Koltover T Salditt CR Safinya Phase diagram stability andovercharging of lamellar cationic lipidndashDNA self-assembled complexesBiophys J 77 (1999) 915ndash924

[33] G Daum Lipids of mitochondria Biochim Biophys Acta 822 (1985)1ndash42

[34] AN Siakotos G Rouser S Fleische Isolation of highly purified humanand bovine brain endothelial cells and nuclei and their phospholipidcomposition Lipids 4 (1969) 234ndash239

[35] YS Tarahovsky R Koynova RC MacDonald DNA release fromlipoplexes by anionic lipids correlation with lipid mesomorphisminterfacial curvature and membrane fusion Biophys J 87 (2004)1054ndash1064

[36] RB Gennis Biomembranes Molecular Structure and Function Springer-Verlag New York 1989

[37] DS Friend D Papahadjopoulos RJ Debs Endocytosis and intracellularprocessing accompanying transfection mediated by cationic liposomesBiochim Biophys Acta Biomembr 1278 (1996) 41ndash50

[38] J Zabner AJ Fasbender T Moninger KA Poellinger MJ WelshCellular and molecular barriers to gene-transfer by a cationic lipid J BiolChem 270 (1995) 18997ndash19007

[39] XH Zhou L Huang DNA transfection mediated by cationic liposomescontaining lipopolylysinemdashCharacterization and mechanism of actionBiochim Biophys Acta Biomembr 1189 (1994) 195ndash203

[40] R Koynova L Wang Y Tarahovsky RC MacDonald Lipid phasecontrol of DNA delivery Bioconjugate Chem 16 (2005) 1335ndash1339

[41] T Boukhnikachvili O Aguerre-Chariol M Airiau S Lesieur MOllivon J Vacus Structure of in-serum transfecting DNAndashcationic lipidcomplexes FEBS Lett 409 (1997) 188ndash194

[42] D Marsh Handbook of Lipid Bilayers CRC Press 1990[43] GW Ashley MM Shida R Qiu MK Lahiri PC Levisay RD Jones

KA Baker RC MacDonald Phosphatidylcholinium compounds a newclass of cationic phospholipids with transfection activin and unusualphysical properties (Abstract) Biophys J 70 (1996) 88A

[44] DP Pantazatos RC MacDonald Directly observed membrane fusionbetween oppositely charged phospholipid bilayers J Membr Biol 170(1999) 27ndash38

[45] T Stegmann JY Legendre Gene transfer mediated by cationic lipids lackof a correlation between lipid mixing and transfection1 Biochim BiophysActa Biomembr 1325 (1997) 71ndash79

[46] B Mui QF Ahkong L Chow MJ Hope Membrane perturbation andthe mechanism of lipid-mediated transfer of DNA into cells BiochimBiophys Acta Biomembr 1467 (2000) 281ndash292

[47] P Pires S Simoes S Nir R Gaspar N Duzgunes MCP de Lima

Interaction of cationic liposomes and their DNA complexes withmonocytic leukemia cells Biochim Biophys Acta Biomembr 1418(1999) 71ndash84

[48] R Leventis JR Silvius Interactions of mammalian-cells with lipiddispersions containing novel metabolizable cationic amphiphiles Bio-chim Biophys Acta 1023 (1990) 124ndash132


[50] D Papahadjopoulos S Nir N Duzgunes Molecular mechanisms ofcalcium-induced membrane-fusion J Bioenerg Biomembr 22 (1990)157ndash179

[51] M Bergstrom Thermodynamics of unilamellar vesicles influence ofmixing on the curvature free energy of a vesicle bilayer J Colloid InterfaceSci 240 (2001) 294ndash306

[52] GH Lei RC MacDonald Lipid bilayer vesicle fusion intermediatescaptured by high-speed microfluorescence spectroscopy Biophys J 85(2003) 1585ndash1599

[53] DP Pantazatos SP Pantazatos RC MacDonald Bilayer mixingfusion and lysis following the interaction of populations of cationic andanionic phospholipid bilayer vesicles J Membr Biol 194 (2003)129ndash139

[54] SP Pantazatos RC MacDonald Real-time observation of lipoplexformation and interaction with anionic bilayer vesicles J Membr Biol191 (2003) 99ndash112

[55] R Smith C Tanford Critical micelle concentration of L-alpha-dipalmitoylphosphatidylcholine in water and watermethanol solutionsJ Mol Biol 67 (1972) 75ndash83

[56] R Koynova RC MacDonald Lipid transfer between cationic vesiclesand lipidndashDNA lipoplexes Effect of Serum Biochim Biophys ActaBiomembr 1714 (2005) 63ndash70

[57] YS Tarahovsky AL Arsenault RC MacDonald TJ McIntosh RMEpand Electrostatic control of phospholipid polymorphism Biophys J 79(2000) 3193ndash3200

[58] RNAH Lewis RN McElhaney Surface charge markedly attenuates thenonlamellar phase-forming propensities of lipid bilayer membranescalorimetric and P-31-nuclear magnetic resonance studies of mixtures ofcationic anionic and zwitterionic lipids Biophys J 79 (2000) 1455ndash1464

[59] H Ellens DP Siegel D Alford PL Yeagle L Boni LJ Lis PJ QuinnJ Bentz Membrane-fusion and inverted phases Biochemistry 28 (1989)3692ndash3703

[60] DP Siegel RM Epand The mechanism of lamellar-to-invertedhexagonal phase transitions in phosphatidylethanolamine implicationsfor membrane fusion mechanisms Biophys J 73 (1997) 3089ndash3111

[61] Y Tarahovsky R Koynova RC MacDonald Efficiency of anionic lipidsto release DNA from lipoplexes correlates with the interfacial curvature ofthe mesomorphic phases of cationicanionic lipid mixtures Biophys J 86(2004) 37A

[62] MB Bally P Harvie FMP Wong S Kong EK Wasan DL ReimerBiological barriers to cellular delivery of lipid-based DNA carriers AdvDrug Delivery Rev 38 (1999) 291ndash315

[63] D Lechardeur KJ Sohn M Haardt PB Joshi M Monck RWGraham B Beatty J Squire H OBrodovich GL Lukacs Metabolicinstability of plasmid DNA in the cytosol a potential barrier to genetransfer Gene Ther 6 (1999) 482ndash497

[64] R Hovius J Thijssen P Vanderlinden K Nicolay B DekruijffPhospholipid asymmetry of the outer-membrane of rat-liver mitochon-driamdashEvidence for the presence of cardiolipin on the outside of the outer-membrane FEBS Lett 330 (1993) 71ndash76

[65] WC Mcmurray RMC Dawson Phospholipid exchange reactions withinliver cell Biochem J 112 (1969) 91ndash108


[12] In an effort to shed light on the mechanism of lipid-mediated DNA delivery we examined the physical andphysico-chemical properties of the EDLPCEDOPC lipoplexesAs described here we found that their superior transfectionefficiency correlates with higher surface activity higher affinityto interact and mix with negatively charged liposomes and withconsiderably faster DNA release relative to the EDOPClipoplexes1 All of these physical characteristics seem importantin the general lipofection pathway Experiments with modelsystems were complemented with observations on transfectedHUAEC cells Confocal microscopy of transfected HUAECcells demonstrated more extensive DNA release into thecytoplasm and nucleoplasm for the EDLPCEDOPC lipoplexesthan for EDOPC lipoplexes close contacts of EDLPCEDOPClipoplexes with various cellular membranes including those ofthe endoplasmic reticulum mitochondria and nucleus wererevealed by electron microscopy

2 Materials and methods

21 Lipids

The triflate salts of 12-dioleoyl-sn-glycero-3-ethylphosphocholine(EDOPC) and 12-dilauroyl-sn-glycero-3-ethylphosphocholine (EDLPC)were synthesized as previously described [14] or these lipids were purchasedas the chloride salt from Avanti Polar Lipids (Alabaster AL) Dioleoylpho-sphatidylcholine (DOPC) dioleoylphosphatidylethanolamine (DOPE) dio-leoylphosphatidylglycerol (DOPG) dioleoylphosphatidylserine (DOPS)cholesterol (Chol) cardiolipin (CL) (heart Na-salt) phosphatidylinositol(PI) (bovine liver Na-salt) and dioleoylphosphatidylethanolamine withcovalently attached rhodamine (Rh-DOPE) all from Avanti Polar Lipidswere used without further purification The phospholipids migrated as a singlespot by thin-layer chromatography BODIPY FL C12-HPC a fluorescentderivative of PC that has spectral characteristics similar to fluorescein waspurchased from Molecular Probes (Eugene OR) Lipids were stored atminus20 degC in chloroform

22 Liposome and lipoplex preparation

Aliquots of lipids EDOPC or an EDLPCEDOPC mixture were placed inborosilicate glass tubes freed of chloroform with an argon stream and keptunder high vacuum for at least 1 h per mg lipid to remove any solventresidues Subsequently samples were hydrated with phosphate-buffered saline(PBS) pH 74 and vortexed for 5 min For preparation of lipoplexes thecationic liposomes were added to plasmid DNA pCMVSport-β-Gal DNA(from Clontech Palo Alto CA propagated and purified by Bayou BiolabsHarahan LA) at the desired ratio as indicated in the text (Assuming anaverage nucleotide mol wt 330 the lipidDNA weight ratios corresponding toisoelectric lipoplexes are 291 for EDOPC 261 for EDLPCEDOPC 64 ww and 241 for EDLPC all lipoplex compositions used in this study werewith some excess of cationic lipid) For most of the experiments the mostefficient lipoplex composition EDLPCEDOPC 64 (ww) [11] was comparedto the EDOPC lipoplexes in some experiments other EDLPCEDOPC ratioswere also used as indicated In some experiments we used DNA covalentlylabeled with fluorescein-5-isothiocyanate (FITC) using the Label IT labelingkit (Mirus Madison WI) according to the protocol supplied by themanufacturer DNA so labeled was purified on Microspin columns includedin the kit or by ethanol precipitation according to the suggested protocol Thenumber of labels per DNA molecule was measured as suggested by Mirus(www genetransfercom FAQ Q26)

1 Lipoplexes of pure EDLPC exhibited enhanced toxicity the viability ofEDLPC-treated cells was only 45 [13]

23 X-ray diffraction

Samples were prepared by adding pre-formed cationic liposomes (5 wtdispersions) to the DNA aqueous solution as previously described [15]Samples were filled into glass capillaries (d=15 mm) (Charles Super CoNatick MA) flame-sealed and equilibrated for 2ndash3 days at room temperaturebefore measurements Small-angle X-ray diffraction (SAXD) measurementswere performed at 37 degC at Argonne National Laboratory Advanced PhotonSource BioCAT (beamline 18-ID) or DND-CAT (beamline 5-IDD) using12 keV X-rays Exposure times were typically sim05ndash1 s Some samples withlonger exposure time were checked by thin layer chromatography after theexperiments Products of lipid degradation were not detected in these samplesand radiation damage of the lipids was not evident from their X-ray patterns

24 Measurement of surface activity at the surface of lipid dispersions

Lipid dispersions (1 mgmL lipid concentration) were prepared in 015 Msodium chloride solution Subsequently appropriate dilutions were made formeasurements Surface tension was measured via the detachment variation ofthe Wilhelmy method [1617] As a Wilhelmy surface we used a roughened05 mm platinum wire Teflon wells d=08 cm contained 75 μL and weremounted on a platform that underwent 2 mm vertical oscillations 4 timesminThe Wilhelmy wire was attached to the underside sensor connection of a CahnRTL electrobalance Maximum excursions of the recorder pen whichcorresponded to the surface tension at zero contact angle and zero buoyancyof the wire were recorded The instrument was calibrated with clean andaspirated water and initial values were set at 715ndash72 mNm The time course ofchange in surface tension of the airndashwater interface of the cationic lipiddispersions was followed using two different techniques (i) by filling the wellwith 50 μgml lipid dispersion and cleaning the surface by aspirationimmediately before the measurement was started [18] (ii) by spreading ofdispersion containing 5 μg cationic lipid over aqueous subphase in the wellimmediately prior to taking measurements [19] The two techniques gavevirtually identical results with respect to the apparent equilibrium surface tensionreached after sim30ndash60 min but because of its greater consistency the secondtechnique was found more appropriate for following the initial kinetics of thesurface tension change

25 Measurement of the kinetics of DNA release from lipoplexes byusing flow fluorometry

This technology allows determination of the relative lipid content perparticle and the lipidDNA composition of individual particles in the lipoplexdispersion (except for very small vesicles and bare plasmid DNA which are notdetectable on our instrument) [2021] A conventional flow cytometerFACSCalibur (Becton and Dickinson) was used A laser beam is focused on avery small portion of a dilute flowing stream and the fluorescence (at up to 3emission wavelengths) as well as light scattering are measured for each particlepassing through the beam It is distinctively useful because it allows access tomeaningful information on the composition of a heterogeneous populationAlthough not previously used for such purposes it was found extremely wellsuited to analyzing highly heterogeneous lipoplex ensembles Cationic lipid waslabeled with 25 wt of the fluorescent label BODIPY FL C12-HPC DNAsamples were labeled with the high-affinity fluorescent dye ethidiumhomodimer-2 (Ethd-2) at 60 bpdye [20] both labels were purchased fromMolecular Probes (Eugene OR) Negatively charged liposomes mimicking atypical membrane lipid composition (MM=DOPCDOPEDOPSChol45202015 ww) were prepared from unlabeled lipids Particles were detectedat the FL1 channel (515ndash545 nm spectral window) by their BODIPY-PCemission (λem=513 nm) The FL3 channel (spectral window gt650 nm)provides information about the amount of DNA in the particle since Ethd-2strongly emits into this channel (λem=624 nm) The FL3FL1 ratio wascalibrated with lipoplexes that had known DNAlipid ratios of less than one aspreviously described [20] To test for DNA release negatively chargedliposomes (unlabeled) were added at 11 lipid weight ratio to the lipoplexesand data were collected as a function of time Typically 10000 particles wereclassified as to lipid content and composition (time for data collection wasusually lt1 min) and the data presented as a 3-D plot with relative lipid content












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Lipoplex formulation of superior efficacy exhibits high surface activity and fusogenicity, and...

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