Proc. Natl. Acad. Sci. USAVol. 93, pp. 11493-11498, October 1996Biochemistry

Mechanism of oligonucleotide release from cationic liposomes(gene therapy/delivery mechanism/cellular/endocytosis/lipid asymmetry)

OLIVIER ZELPHATI AND FRANcis C. SzoKA, JR.*Department of Pharmacy and Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, CA 94143-0446

Communicated by Robert Langer, Massachusetts Institute of Technology, Cambridge, MA, July 23, 1996 (received for review March 14, 1996)

ABSTRACT We propose a mechanism for oligonucleotide(ODN) release from cationic lipid complexes in cells thataccounts for various observations on cationic lipid-nucleicacid-cell interactions. Fluorescent confocal microscopy ofcells treated with rhodamine-labeled cationic liposome/fluorescein-labeled ODN (F-ODN) complexes show the F-ODN separates from the lipid after internalization and entersthe nucleus leaving the fluorescent lipid in cytoplasmic struc-tures. ODN displacement from the complex was studied byfluorescent resonance energy transfer. Anionic liposome com-positions (e.g., phosphatidylserine) that mimic the cytoplas-mic facing monolayer of the cell membrane released ODNfrom the complex at about a 1:1 (-/+) charge ratio. Releasewas independent of ionic strength and pH. Physical separationofthe F-ODN from monovalent and multivalent cationic lipidswas confirmed by gel electrophoresis. Fluid but not solidphase anionic liposomes are required, whereas the physicalstate of the cationic lipids does not effect the release. Watersoluble molecules with a high negative linear charge density,dextran sulfate, or heparin also release ODN. However, ATP,spermidine, spermine, tRNA, DNA, polyglutamic acid, polyl-ysine, bovine serum albumin, or histone did not release ODN,even at 100-fold charge excess (-/+). Based upon theseresults, we propose that the complex, after internalization byendocytosis, induces flip-flop of anionic lipids from thecytoplasmic facing monolayer. Anionic lipids laterally diffuseinto the complex and form a charged neutralized ion-pair withthe cationic lipids. This leads to displacement of the ODNfrom the cationic lipid and its release into the cytoplasm.

Cationic lipids in the form of liposomes or micelles are anefficient carrier for oligonucleotide (ODN) delivery into cellsin culture (1-4). The cationic liposomes form a polyelectrolytecomplex with the ODN, protect them from nuclease degra-dation, enhance their cellular uptake, and improve ODNpotency (1-4). Since Bennett's (2) earlier observation thatcationic lipids alter intracellular localization of ODN, little hasbeen published on the mechanism. This process is, neverthe-less, critical for the delivery of ODN and polynucleotides(DNA) since it seems evident that these macromolecules haveto dissociate from complexes to function. Moreover, theunderstanding of the process of intracellular release shouldhave implications for improvements of ODN and polynucle-otide cationic carriers and for in vivo therapeutic trials. In thisstudy, we use fluorescent confocal microscopy to show thatfluorescein-labeled ODN (F-ODN) separate from the rho-damine-labeled lipid [1,2-dioleoyl-sn-glycero-3-phosphati-dylethanolamine-N-(lissamine rhodamine B sulfonyl); N-Rh-PE] and enters the nucleus leaving the fluorescent lipid inpunctate cytoplasmic structures. We then show that anioniclipids cause a rapid release of ODN from the complex. Basedon these results we propose a mechanism to account for ODNdelivery by cationic lipid complexes.

EXPERIMENTAL PROCEDURESReagents. All lipids were purchased from Avanti Polar

Lipids. Dioctadecylamidoglycylspermine (DOGS) was synthe-sized as described (5). Salmon sperm DNA, tRNA werepurchased from GIBCO/BRL. Poly-L-glutamic acid, ATP,spermine, spermidine, histone type II-A, dextran sulfate,POIY-L-lysine (Mr 25,000), bovine serum albumin, and choles-terol (Chol) were obtained from Sigma. Heparin is fromElkins-Sinn (Cherry Hill, NJ). SYBR Green I was purchasedfrom Molecular Probes.

Oligonucleotides. Phosphorothioate ODN were generouslyprovided by G. Zon (Lynx Therapeutics, Hayward, CA). Twophosphorothioate ODN were used in these studies. An anti-sense anti-rev 28-mer (5'-TCGTCGCTGTCTCCGCTTCT-TCCTGCCA-3') and an anti-murine ,3-actin 25-mer (5'-TCTGGGTCATCTTTTCACGGTTGGC-3') labeled at the 5'end with fluorescein or rhodamine-labeled ODN(Rh-ODN).They were synthesized and purified by a method previouslyreported (6, 7).

Preparation of Liposomes. Liposomes [1,2-dioleoyl-3-trimethylammonium-propane (DOTAP); 1,2-dimyristoyl-3-trimethylammonium-propane (DMTAP); DOGS/1,2-dio-leoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), 1:1(molar ratio of components); phosphatidylserine (PS)/DOPE/ 1,2-dioleoyl-sn -glycero-3-phosphatidylcholine(DOPC), 1:2:1; phosphatidyl-DL-glycerol (PG)/DOPE/DOPC, 1:2:1; phosphatidylinositol (PI)/DOPE/DOPC, 1:2:1;phosphatidic acid (PA)/DOPE/DOPC, 1:2:1; DOPE/DOPC,2:1; DOPC/Chol, 2:1; and L-a-dipalmitoylphosphatidylglyc-erol (DPPG)/1,2-dimyristoyl-sn-glycero-3-phosphatidyleth-anolamine (DMPE)/L-a-dipalmitoyl lecithin (DPPC), 1:2:1]were prepared as described (8). Large unilamellar vesicleswere prepared as described (9). For fluorescently labeledliposomes, 1 mol percent of N-Rh-PE and N-4-nitrobenzo-2-oxa-1,3-diazole phosphatidylethanolamine (NBD-PE) wereadded to cationic lipids. The final concentration of lipids was20 mM, except for DOTAP and DOGS, where the concen-tration was 5 mM.

Fluorescence Resonance Energy Transfer (FRET). Fluores-cence measurements were performed as described (7). For thecharacterization of FRET, unlabeled or fluorescent ODNwere used at 0.5 gg/ml in 2 ml of Tris HCl buffer (30 mMTris HCl, pH 7.5) and cationic lipids were mixed with ODN at

Abbreviations: ODN, oligonucleotide(s); F-ODN, fluorescein-labeledODN; DOTAP, 1,2-dioleoyl-3-trimethylammonium-propane;DMTAP, 1,2-diimnyristoyl-3-trimethylammonium-propane; DOGS, di-octadecylamidoglycylspermine; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine; PS, phosphatidylserine; PG, phosphatidyl-DL-glycerol; PA, phosphatidic acid; PI, phosphatidylinositol; DPPG, L-a-dipalmitoylphosphatidylglycerol; DPPC, L-a-dipalmitoyl lecithin;DMPE, 1,2-dimyristoyl-sn-glycero-3-phosphatidylethanolamine; N-Rh-PE, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine-N-(lissamine rhodamine B sulfonyl); NBD-PE, N-4-nitrobenzo-2-oxa-1,3-diazole phosphatidylethanolamine; FRET, fluorescence reso-nance energy transfer; Chol, cholesterol.*To whom reprint requests should be addressed. e-mail: szoka@cgl.ucsf.edu.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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1Ox (+/-) charge ratio relative to positive charge present oncationic lipids per negative charge present on ODN. FRET wasalso studied in potassium acetate buffer (130 mMC2H302K/10 mM MgCl2/10 mM Hepes, pH 5, 6, or 7.1) andNaCl buffer (150 mM NaCl/2 mM MgCl2/2 mM CaCl2/10mM Tris, pH 7.4). Emission spectra were recorded between500 and 600 nm with excitation at 470 nm. Fluoresceinfluorescence quenching (Q) was calculated as follows: Q = { 1- [F(donor+acceptor)/F(donor alone)]} X 100, where F = fluores-cence level at 520 nm. The rhodamine fluorescence increasevalue (I) was computed as I = {F'(donor+acceptor)/[F'(donor alone)+ F'(acceptor alone)]} x 100, where F' = fluorescence level at 588nm. The fluorescein fluorescence dequenching (dQ) was cal-culated as dQ = Ql(complexes)- Q2(complexes + liposomesor compounds), where Qi represents 100% of quenching.Complexes prepared with DOTAP/N-Rh-PE, DOTAP/NBD-PE, or DOGS/DOPE/N-Rh-PE were made at roomtemperature and those prepared from F-ODN/DMTAP/N-Rh-PE were formed at 37°C. Then, the compounds or lipo-somes were added at different charge ratio and emissionspectra were recorded at various temperatures as above. Forkinetic studies, the different liposome formulations wereadded to complexes and the emission at 520 nm was monitoredas a function of time.

Gel Electrophoresis Experiments. The preparation of theODN/DOTAP or DOGS complexes was carried out in 96-wellplates as follows: 0.75 ,ug of ODN were added to increasingamounts of cationic lipids to result in charge ratio + /- (range,0.625- to 20-fold). The mixture was allowed to stand at roomtemperature for 30 min, then 10 ,ul of30% glycerol were addedand the mixture was loaded on a 20% nondenaturing poly-acrylamide gel. For the ODN release experiments, the variouscompounds or liposomes to be examined were added to thecomplexes and after 15 min, the mixture was loaded on a 20%polyacrylamide gel and a potential of 150 V applied for 4 h.ODN were detected by staining the gel with SYBR Green I.

Confocal Microscopy Study. CV-1 cells (monkey kidneyfibroblast) were grown on coverslips in DME-H21 supple-mented with 10% fetal bovine serum, 2 mM nonessentialamino acids, 10 mM Hepes, and antibiotics at 37°C in 5%CO2/95% air. Then, CV-1 cells were incubated with com-plexes, prepared at 1Ox (+/-) charge ratio at 37°C for 3 h inserum-free medium. The concentrations of ODN and lipidswere 150 nM and 38 ,uM, respectively. After incubation, CV-1cells were washed in PBS and coverslips were mounted onhanging drop slides (Fisher Scientific). Cells were directlyobserved with a MRC-600 (Bio-Rad) confocal laser scanningimaging system equipped with an upright microscope and anx60 oil immersion objective (Nikon).

RESULTSIntracellular Distribution of Fluorescently Labeled ODN

and Cationic Liposomes. To investigate the time course ofdistribution of the ODN and lipid in cells, the complex wasprepared from rhodamine-labeled liposomes (DOTAP/N-Rh-PE) and F-ODN, which are ideal for FRET studies (vide infra).ODN nuclear accumulation has been shown to begin at =30min after addition of the complexes to the cells (4). By 3 hpostincubation the ODN (green color) are localized predom-inantly in the nucleus, whereas fluorescent lipids (red color)were distributed overwhelmingly in the cytoplasm (Fig. 1). Insome places, the colocalization ofODN and lipids are detected(yellow color, Fig. 1) corresponding to complexes at the cellsurface, remaining in intracellular vesicles or associated withthe coverslip. Thus, lipids were physically separated from ODNin cells and ODN have been shown to be stable in cells for thisperiod of time (7).

Interaction of Cationic DOTAP Liposomes with ODN.FRET was used to demonstrate the formation and dissociation

FIG. 1. Intracellular distribution of fluorescent-lipids and F-ODN.F-ODNwere associated with DOTAP/N-Rh-PE at a 10:1 charge ratio.CV-1 cells were incubated in serum-free medium with complexes for3 h at 370C and imaged using a confocal microscope.

of complexes between ODN and cationic liposomes. Thedonor (F-ODN) alone or incubated with unlabeled acceptor(DOTAP liposomes) as well as the acceptor (DOTAP/N-Rh-PE) alone or incubated with unlabeled donor (ODN), show nosignificant alterations in their emission wavelengths (data notshown). In contrast, when the donor (F-ODN) and acceptor(DOTAP/N-Rh-PE) were mixed together, the fluoresceinemission was specifically quenched to Q = 93% (Fig. 2A) andthe rhodamine emission was specifically increased to I = 166%.Similar results were also obtained with the multivalent DOGSlipids (data not shown). This demonstrates that ODN arecomplexed to the cationic liposomes since the ODN attachedfluorophore and lipid associated fluorophore are close enoughto undergo energy transfer.We have also used gel retardation experiments to examine

the interaction between ODN and cationic DOTAP or DOGSlipids. Complexes were prepared at different charge ratios(+/-) DOGS or DOTAP/ODN and separated on 20%PAGE. When complexes were formed with an excess ofpositive charge (.2.5), the ODN did not enter the gel and noband was visible where ODN alone migrated (data not shown).

Effects of Various Liposomes on the Stability of ODN/DOTAP Complexes. The physical separation of the ODN andcationic liposomes in cells raises the question about how theydissociate. Therefore we examined which types of moleculesfound in cells bring about the release of ODN from cationiclipids. Anionic or neutral liposomes were added to the pre-formed complexes (F-ODN/DOTAP/N-Rh-PE) and theemission spectra recorded. All negatively charged liposomesexamined (PS/DOPE/DOPC, PG/DOPE/DOPC, PI/DOPE/DOPC, and PA/DOPE/DOPC) reversed the FRETwhen used with -2-fold excess of negative charge over positivecharge of cationic lipids (Fig. 2A and Table 1). The fluores-cence intensity returned to approximately the levels observedwith F-ODN alone. In contrast, with neutral liposomes(DOPE/DOPC and DOPC/Chol), no dequenching of fluo-rescein fluorescence was observed, indicating that the ODNremain associated with the cationic liposome (Fig. 2A andTable 1). This is also observed when ODN is complexed withDOGS lipids (Table 1). The displacement of ODN fromcomplexes is dependent on the ratio- of negative chargespresent on liposomes to positive charges present in complexes

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Table 1. Biomolecule effects on FRET of F-ODN/cationic lipids

Fluorescein dequenching, %

DOTAP DOGSt

Charge Tris KoAc NaCl NaClratio* (RT)f (370C)§ (370C)§ (370C)

Complex - 0 0 0 0PS/PE/PC 2x 84.9 84.2 74.5 84.3PG/PE/PC 2x 90.8 104.6 84.6 72.6PI/PE/PC 2x 83.5 96.8 47.1 NDPA/PE/PC 4x 49.5 11.9 42.8 NDPE/PC NAI 7.5 0.2 0.6 6.8PC/Chol NA 3.9 0.1 0.4 NDDextran sulfate ix 96.5 48 55 97.2Heparin 15 units 91.1 23.3 55.5 NDPolyglutamic acid 10OX 3.8 -2.5 -1.6 NDBSA NAII -3.2 1.1 2.4 NDATP 1oox -0.5 -0.4 -3.3 -2.9tRNA 1oox -1.5 -0.3 -1.1 NDDNA 1oox -2.9 -0.5 -1.4 4.3Spermine 1oox -0.1 0.4 2.9 1.8Spermidine 1oox -1.7 0.05 2.7 NDHistones 100x 2.5 -0.8 1.3 -1.3Polylysine 100x 0.5 0.1 0.4 ND

ND, not done; NA, not applicable.*Charge of compounds/positive charge of cationic lipids.tSingle experiment.tMean of three experiments and SD . 5%.§Mean of two experiments and range < 5%.lEquivalent amount of lipid used as with anionic lipids.IBSA, 6 mg/ml.

(Fig. 2B). The anionic lipid formulations PG/DOPE/DOPC,PS/DOPE/DOPC, and PI/DOPE/DOPC were most effi-cient. The release of ODN from the complexes was a rapidprocess, since after 100 sec fluorescein fluorescence increasedto a plateau regardless of the composition of the negativelycharged liposomes (Fig. 2C). With ODN/DOGS the releasewas 2-3 times slower (data not shown). Thus, displacement ofODN from complexes by anionic phospholipids can occurwithin the time scale of an endocytotic event.

In the case of the anionic lipids, the ionic conditions (ionicstrength or ion type) had no influence on the dequenchingexcept for PA/DOPE/DOPC liposomes which were less effi-cient in K acetate buffer (Table 1). The importance of pH wasalso investigated by using DOTAP/NBD-PE liposomes andrhodamine-labeled ODN to avoid the problem of fluoresceinquenching at acidic pH (Table 2). Both anionic liposomestested were able to reverse the FRET at pH 5 or 6, whereasneutral liposomes did not reverse the FRET (Table 2). Theseionic strength conditions mimic the extracellular (NaCl-calcium buffer, pH 7.4), intracellular (K acetate-magnesiumbuffer, pH 7.1) and endosomal (K acetate, pH 5.0 and 6.0)ionic and pH environments.The effects of the anionic liposome size on the ODN release

from DOTAP have also been studied. Small unilamellarvesicles (50-60 nm) containing either 15 or 25% of PG or PSwere able to reverse the FRET whereas large unilamellarvesicles (300-400 nm) have shown significantly less ability toinduce the ODN release from complexes (Fig. 2D).

Release of ODN from the complexes was confirmed byelectrophoresis of the complex on a polyacrylamide gel. ODN/DOTAP complexes with a charge ratio of 10 (+/-) analyzedby PAGE was retained and no band was observed where freeODN migrated (Fig. 3A, lanes 1 and 2). Addition of anionic

to preformed complexes with 1x (-/+) and the fluorescence de-quenching was measured at 180 sec as described. Release agreed to±5% in duplicate samples.

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Table 2. Effects of pH on the FRET of Rh-ODN/DOTAP/NBD-PE

NBD dequenching, %

Charge K acetate K acetateratio* buffer, pH 6t buffer, pH 5t

Complex - 0 0PS/PE/PC 2x 92.8 65.6PG/PE/PC 2x 98 50.1PE/PC NA§ -2.1 0.2PC/Chol NA§ -6.2 -0.4Dextran sulfate 1x 33.9 34.8Heparin 15 units 16.3 13.2Polyglutamic acid 1oox 5.1 -0.8BSA NA$ -3.8 -2.5ATP 1oox -7.3 -1.7tRNA 1oox -1.2 -2DNA 1oox -8.3 -0.5Spermine 1oox -6.8 -1.9Spermidine loox -17 -0.5Histones 1oox -8.3 -0.8Polylysine 1oox -5.4 -0.65

NA, not applicable.*Charge of compounds/positive charge of cationic lipids.tSingle experiment.tMean of two experiments and range < 5%.§Equivalent amount of lipid used as with anionic lipids.IBSA, 6 mg/ml.

liposomes to complexes causes the ODN band to reappear(Fig. 3A lanes 3, 4, 7, and 8) whereas with neutral liposomes,ODN are still retained at the point of application (Fig. 3A,lanes 5 and 6). Physical separation of ODN from ODN/DOGScomplexes was also induced by anionic liposomes (data notshown). The gel separation directly confirms the FRET studiesthat anionic liposomes displace ODN from cationic lipidcomplexes. Thus, negatively charged phospholipids, present inthe membrane of the endosome, are capable of releasing ODNfrom the complex.

Effects of Negatively Charged Molecules on the Stability ofODN/DOTAP Complexes. We have also studied the displace-ment of ODN from complexes after addition of differentcellular or synthetic negatively charged molecules. The FRETexperiments revealed that molecules found in high concentra-tions in cells, such as ATP, DNA, and tRNA as well as asynthetic polymer, polyglutamic acid did not release ODNfrom the complex under any conditions examined (Tables 1and 2). BSA, a major serum protein, can interact with ODN/DOTAP complexes and prevent their cellular uptake (unpub-lished observations) but did not induce release of the ODN(Table 1). The addition of an excess of unlabeled ODN (up to100-fold) also has no effect on the complexes (data not shown).The sulfated anionic polymers, dextran sulfate and heparin

displaced ODN from the complex. This displacement bydextran sulfate and heparin was charge ratio-dependent (- /+charge), time-dependent (data not shown), and also saltconcentration and pH dependent (Tables 1 and 2). Theefficiency of both compounds to reverse the FRET weresignificantly reduced in the high salt buffers (Table 1). Atacidic pH, heparin was much less effective at displacing ODNfrom cationic lipids than at neutral pH (Table 2). The kineticsof displacement by the sulfated polymers were as rapid asobserved with the negatively charged liposomes (data notshown).The displacement results were confirmed by the gel retar-

dation assay. After addition of dextran sulfate and heparin tocomplexes, the ODN migrates on the gel where expected (Fig.3B, lanes 3 and 5). In contrast, with ATP, salmon sperm DNA,tRNA, and polyglutamic acid, the ODN remained complexedto cationic lipids (Fig. 3A, lanes 9 and 10; Fig. 3B, lanes 7 and

A1 2 3 4 5 6 7 8 9 10

B 1 2 3 4 5 6 7 8 9 10

FIG. 3. (A) Release of ODN from ODN/DOTAP complexes byanionic liposomes. Various compounds were mixed (with excess ofnegative charge relative to positive charge ofDOTAP) with complexesprepared at lOX (+/-) charge ratio for 15 min and mixtures were runin a 20% nondenaturing polyacrylamide gel. Lanes: 1, ODN alone; 2,1OX ODN/DOTAP; 3, 2x PS/PE/PC; 4, 2x PG/PE/PC; 5, 2xPC/Chol; 6, 2x PE/PC; 7, 2x PI/PE/PC; 8, 4X PA/PE/PC; 9, 10OXpolyglutamic acid; and 10, 10OX ATP. (B) Effects of various cationicand anionic molecules on ODN/DOTAP complex stability. ODN/DOTAP at 10x (+/-) charge ratio were mixed with different anionicor cationic molecules and run as inA. Anionic or cationic compoundswere mixed with complexes with excess of charge relative of thesemolecules to positive charge of DOTAP. Lanes: 1, ODN alone; 2, 1OXODN/DOTAP; 3, lx dextran sulfate; 4, 5Ox histones; 5, 15 unitsheparin; 6, 10OX spermine; 7, 25x salmon sperm DNA; 8, 100Xspermidine; 9, 50x polylysine; and 10, 25X tRNA.

10). Components of both salmon sperm DNA and tRNA enterthe gel where various size fragments are detected, but no bandcorresponding to the ODN is observed.

Effects of Positively Charged Compounds on the Stability ofODN/DOTAP Complexes. The other possibility to induceseparation is that cellular cationic molecules bind to the ODNand displace the cationic lipids. Therefore, we examined ifpositively charged molecules could displace ODN from thecomplex. Histones, spermine, spermidine, and polylysine werenot able to separate the ODN from the cationic lipids underany conditions examined (Tables 1 and 2). With the gel assay,none of the positively charged compounds resulted in ODNmigrating into gel (Fig. 3B, lanes 4, 6, 8, and 9) when added tothe complex. Moreover, the addition of unlabeled DOTAPliposomes also did not modify the fluorescence quenching(data not shown). This implies that once the complex is formed

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Table 3. Effects of the bilayer physical state on the ODN release from cationic lipids

Fluorescein dequenching, %

Charge F-ODN/DOTAP/N-Rh-PEt F-ODN/DMTAP/N-Rh-PEtratio* RT 370C 450C 10°C 370C

PS/PE/PC 2x 79.6 (f/f) 88.8 (f/f) 97.2 (f/f) 58.3 (s/f) 61.1 (f/f)PG/PE/PC 2x 104 (f/f) 106 (f/f) 101 (f/f) 46.5 (s/f) 61 (f/f)DPPG/DPPC/DMPE 2x 4.6 (f/s) 16.6 (f/s) 79 (f/f) 6.1 (s/s) 14.1 (f/s)PE/PC NA§ -1.1 (f/f) 0.3 (f/f) 0.2 (f/f) -0.7 (s/f) -0.8 (f/f)Dextran sulfate 1x 44.4 51.6 55.6 39.7 76.2ATP 1oox -0.7 0.1 -0.7 -0.6 -0.8Histones 1oox -0.5 -0.8 -0.5 -0.8 -3.6

Letters in parentheses are as follows: f, fluid; s, solid. The ratios they show are for: (state of cationic bilayer/state of anionicbilayer). NA, not applicable; RT, room temperature.*Charge of compounds/positive charge of cationic lipids.tMean of two experiments and range s 5%.tSingle experiment.§Equivalent amount of lipid used as with anionic lipids.

with excess positive charge, the surface potential impedes theclose approach of other cationic liposomes.

Effects of Bilayer Physical State of Liposomes on theMechanism of Release. To better understand the ODN releasemechanism from cationic lipids, we have studied the influenceof the physical state of the bilayer (solid gel state versus fluidliquid crystal state) on this process. First, we have examinedthe influence of the physical state of the cationic bilayer onODN release by using DMTAP which has a phase transitiontemperature between 20 and 24°C. In this case, ODN weredisplaced from both solid (10°C) as well as fluid state DMTAPlipids by PS/DOPE/DOPC and PG/DOPE/DOPC liposomesand dextran sulfate (Table 3). Here again, other compoundsexamined did not release ODN. Thus, the physical state of thecationic bilayer does not significantly influence release.

Second, the importance of anionic bilayer fluidity wasstudied by using DPPG/DMPE/DPPC liposomes which havea phase transition temperature around 42°C. Below the phasetransition of the DPPG/DMPE/DPPC liposomes, at roomtermperature or 37°C, no dequenching was observed when thecationic bilayer (DOTAP, Tm < 4°C) of the complex was in afluid state. At 37°C, neutral lipids, ATP, and histones still didnot reverse FRET (Table 3). PS/DOPE/DOPC and PG/DOPE/DOPC liposomes, in fluid state at these temperatures,and dextran sulfate efficiently reversed FRET (Table 3). Incontrast, at 45°C the DPPG/DMPE/DPPC liposomes, now inthe fluid state, reversed FRET (Table 3). No significantdifferences were seen for other compounds or liposomes at thistemperature (Table 3). Thus, the release requires a fluidanionic bilayer.

DISCUSSIONIt is now accepted that cationic lipids deliver nucleic acids intothe cell predominately via an endocytotic pathway (4, 8, 10, 11)rather than after fusion with the plasma membrane as previ-ously suggested (2, 12). How the nucleic acid is released fromthe cationic lipid complex once in the endosome has not beenunderstood. In this work we show using double-label tech-niques that fluorescent lipids associated with the complex arefound in punctate cytoplasmic structures whereas the deliv-ered fluorescent ODN are found in the nucleus as previouslydescribed for the individual components (2, 13). We go on todemonstrate that anionic liposomes are potent agents forreleasing ODN from monovalent and multivalent cationic lipidcomplexes whereas ionic water soluble molecules that arecommon intracellular constituents are not. Based upon thesedata, we propose the scheme illustrated in Fig. 4 to explain howODN and other nucleic acids are released from the complexinto the cytoplasm of the cell. First the cell surface-associated

complex is internalized via an endosome (step 1). The complexinitiates a destabilization of the endosomal membrane thatresults in a flip-flop of anionic lipids (step 2) that are pre-dominately located on the cytoplasmic face of the membrane.The anionic lipids laterally diffuse into the complex and forma charged neutralized ion-pair with the cationic lipid (step 3).This displaces the ODN from the complex and the ODN candiffuse into the cytoplasm (step 4, Fig. 4).

This anionic lipid driven uncoating of the complex is con-sistent with the lipid asymmetry of the plasma and endosomalmembranes (14) as well as all of the known observationsconcerning cationic lipid mediated nucleic acid delivery in-

FIG. 4. Schematic representation of the ODN/cationic lipid com-plex uptake pathway and the mechanism of ODN endosomal release.See text for description.

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cluding cell biological data that shows nucleic acid accessibilityto the cellular translation and transcription machinery. Thus,RNA delivered as a complex is accessible to the ribosome fortranslation (15), a T7 promoter on the delivered plasmid isaccessible to cytoplasmic T7 polymerase (16, 17) and ODN andplasmid DNA are functional upon delivery (1, 2, 4, 8, 13); allwhich suggest the nucleic acid has been separated from thecationic lipid during delivery. Moreover, when plasmid DNA/cationic lipid complexes were directly injected into nucleus,thus bypassing the endocytosis steps, little reporter geneexpression was observed, suggesting that in this instance,where interactions with anionic lipid containing membraneswas bypassed, DNA did not dissociate from the complex (11).The model is also consistent with electron microscopy obser-vations of complexes in cells that show electron dense struc-tures appearing in disrupted endosomes close to the plasmamembrane (10) as well the fluorescence microscopy observa-tions on ODN delivery (2, 4, 13).The model does not attempt to explain the initial membrane

destabilization but is firmly based upon the biophysical prop-erties of membrane fusion since conditions that promote closeapposition of the lipid bilayers that precedes fusion lead tolipid mixing (18, 19). Moreover, anionic lipids and not zwit-terionic lipids dominate the lipid mixing process in an asym-metric fusion system (18). The importance of anionic lipidlateral diffusion in the release is supported by the requirementfor a fluid anionic bilayer to release ODN from the complex(Table 3). Perturbation of the membrane during fusion ordisruption by peptides results in lipid flip-flop (9, 14) and thisphenomenon would supply the anionic lipids from the cyto-plasmic monolayer required to neutralize the cationic lipids inthe complex. Indeed, under certain conditions polycationsinduce the migration of anionic phospholipids from the innerto outer membrane of the bilayer (20). Finally cationic lipo-somes have been shown to fuse with endocytotic vesicles (21,22). The fusion process once started would be autocatalyticsince the appearance of additional anionic lipids at the site ofinteraction would augment the adhesion of the cationic com-plex to the endosomal membrane.The mechanism illustrated in Fig. 4 can explain all of the

known facts about nucleic acid delivery by cationic liposomecomplexes however it is unclear why endocytosis appears to berequired for destabilization and/or fusion since the composi-tion of the outer surface of the plasma membrane and the innersurface of the endosomal vesicles should be similar (14). First,it has been clearly demonstrated that the major route of nucleicacid/cationic lipid complexes delivery is by endocytosis (4, 10,11). Moreover, cationic liposomes have been shown to fusewith membranes only after endocytosis (21). A possible ex-planation is that internalization of the complexes into acompartment with a high radius of curvature may promoteclose contact between the two surfaces and provide a physicaltrigger for membrane destabilization as suggested by theefficiency of small anionic vesicles to displace the ODN incomparison to large vesicles. Alternatively compartmentaliza-tion may be the key; when the ODN is released from thecationic lipid in the endosome there might be either a me-chanical or osmotic stress generated that ruptures the endo-somal bilayer and releases the ODN into the cytoplasm.Release of the ODN from complexes on the surface might notgenerate the necessary stress on the membrane needed torupture it. Finally the absence of clathrin and the accessoryproteins during the release process might make the uncoatedvesicles more prone to rupture. This aspect of the mechanismrequires further work.An intriguing observation is that in addition to the anionic

liposomes, both dextran sulfate and heparin can release ODNfrom the complex but the nucleic acids (ODN, tRNA, DNA)as well as other water soluble charged molecules cannot. This

is most likely due to the greater negative linear charge densityof these molecules (two negative charges per carbohydrateversus one per nucleic acid) (23) compared with the nucleicacids. The fact that the higher linear charge can release ODNindicates that hydrophobic contributions from the lipid tostabilize the complex can be overcome. In the presence of thepotassium acetate buffer (pH 7.1, 6.0, or 5.0) where theircharges are partially shielded, dextran sulfate and heparin areless effective at releasing ODN from the complex whereasanionic liposomes retain much of their release activity. Theeffectiveness of heparin at releasing the ODN from thecomplex in the extracellular mimetic buffer (NaCl, pH 7.4)raises the question if heparin or other glycosaminoglycansfound in extracellular regions of the body (23) might interferewith ODN delivery in vivo by bringing about release of nucleicacids before the complex can reach the target cell? This is apoint for future investigations.

In summary, the biophysical release experiments shownherein, support a model for how anionic lipids mediate ODNrelease from cationic liposome complexes in cells (Fig. 4). Thismodel should provide a useful starting point for the rationaleimprovement of this important nucleic acid delivery system.

We thank Yuhong Xu, Dan Chin, and Christian Plank for helpfulcomments and insights on this problem. We appreciate the encour-agement of Wayne Hendren. This work was partially supported byNational Institutes of Health Grants GM30163 and DK46052 (F.C.S.),Glaxo Inc. (O.Z.) and Association pour la Recherche Contre le Cancer(O.Z.).

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