encapsulation of plasmid dna in stabilized plasmid – lipid particles composed of different...

Encapsulation of Plasmid DNA in Stabilized Plasmid- Lipitd Particles Composed of Different Cationic Lipid Concentration for Optimal Transfection Activity E.G. SARAVOLAC”,‘, 0. LUDKOVSKI”, R. SKIRROW”, M. OSSANLOUd, Y.P. ZHANG”, C. GIESBRECHT”, J. THOMPSON”, S. THOMAS”, H. STARK”, P.R. CULLIS”*” and I’. SCHERRER”.b,” c In previous work (Wheeler et al. (1999) Gelle Therappl, 6, 271--281) we have shown that plasmid DNA can be entrapped in “stabilized plasmid&lipid particles” (SPLP) using low levels (5 10 mol%) of cationic lipid, the fusogenic lipid dioleoylphosphatidylethanola- mine (DOPE), and a polyethyleneglycol (PEG) coating for stabilization. The PEG moieties arc attached to a ceramide anchor containing an arachidoyl acyl group (PEG- Cer&,,). However, these SPLP exhibit low transfection potencies i/z vitro as compared to plasmid/cationic lipid complexes formed with liposomes composed ofcationic and neutral lipid at a 1 : I lipid ratio. The objective of this study was to construct SPLPs with increased cationic lipid contents that result in maximum transfection levels. A phosphate buffer detergent dialysis technique is dcscribcd resulting in formation of SPLP containing 7F42.5mol% DODAC with reproducible encapsulation efficiency of up to 80%. An octanoyl acyl group was used as anchor for the PEG moiety (PEG-CerC8) permilting a quick exchange out ofthe SPLP to further optimize the it? vitro and i/z viva transfection. We have demonstrated that this technique can be used to encapsulate either linearized DNA or supercoiled plasmids ranging from 3-20 kb. The SPLP formed could be isolated from empty vesicles by sucrose density gradient centrifugation, and exhibited a narrow size distribution of approximately 75 i 6 nm as determined by cryo-electron microscopy. The high p&mid-to-lipid ratio observed corresponded to one plasmid per particle. The SPLP consist of a lipid bilayer surrounding the plasmid DNA as visualized by cryo-electron microscopy. SPLP containing a range of DODAC concentrations were tested for in vitro and ill v&o transfection. IIZ l&o, in COS-7 cells transfection reached a maximum after 48 h. The transfection efficiency increased when the DODAC concentration in the SPLP was decreased from 42.5 to 24mol% DODAC. Decreasing the cationic lipid concentration improved lransfection in part due to decreased toxicity. In viva studies using an intraperitoneal B I6 tmllor model and intraperitoncal administration of SPLP showed ‘r Corresponding author. Dcpartmcnt of Biochemistry and Molecular Biology, Liposome Research IJnit. University of British Columbia, 2 14.6 Health Sciences Mall, Vancouver, B.C. V6T 123: Canada. E-mail: schen~eri~~intcl-chg.ul~~.~~. Current address: Johnson and Johnson Research Pty. Ltd., G.P.O. 3331, Sydney, 2001 NSW, Australia. 423

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Encapsulation of Plasmid DNA in Stabilized Plasmid-Lipitd Particles Composed of Different Cationic Lipid

Concentration for Optimal Transfection Activity


P.R. CULLIS”*” and I’. SCHERRER”.b,”


In previous work (Wheeler et al. (1999) Gelle Therappl, 6, 271--281) we have shown thatplasmid DNA can be entrapped in “stabilized plasmid&lipid particles” (SPLP) using lowlevels (5 10 mol%) of cationic lipid, the fusogenic lipid dioleoylphosphatidylethanola-mine (DOPE), and a polyethyleneglycol (PEG) coating for stabilization. The PEGmoieties arc attached to a ceramide anchor containing an arachidoyl acyl group (PEG-Cer&,,). However, these SPLP exhibit low transfection potencies i/z vitro as compared toplasmid/cationic lipid complexes formed with liposomes composed ofcationic and neutrallipid at a 1 : I lipid ratio. The objective of this study was to construct SPLPs with increasedcationic lipid contents that result in maximum transfection levels. A phosphate bufferdetergent dialysis technique is dcscribcd resulting in formation of SPLP containing7F42.5mol% DODAC with reproducible encapsulation efficiency of up to 80%. Anoctanoyl acyl group was used as anchor for the PEG moiety (PEG-CerC8) permilting aquick exchange out ofthe SPLP to further optimize the it? vitro and i/z viva transfection. Wehave demonstrated that this technique can be used to encapsulate either linearized DNA orsupercoiled plasmids ranging from 3-20 kb. The SPLP formed could be isolated fromempty vesicles by sucrose density gradient centrifugation, and exhibited a narrow sizedistribution of approximately 75 i 6 nm as determined by cryo-electron microscopy. Thehigh p&mid-to-lipid ratio observed corresponded to one plasmid per particle. The SPLPconsist of a lipid bilayer surrounding the plasmid DNA as visualized by cryo-electronmicroscopy. SPLP containing a range of DODAC concentrations were tested for in vitroand ill v&o transfection. IIZ l&o, in COS-7 cells transfection reached a maximum after 48 h.The transfection efficiency increased when the DODAC concentration in the SPLP wasdecreased from 42.5 to 24mol% DODAC. Decreasing the cationic lipid concentrationimproved lransfection in part due to decreased toxicity. In viva studies using anintraperitoneal B I6 tmllor model and intraperitoncal administration of SPLP showed

‘r Corresponding author. Dcpartmcnt of Biochemistry and Molecular Biology, Liposome Research IJnit. University of BritishColumbia, 2 14.6 Health Sciences Mall, Vancouver, B.C. V6T 123: Canada. E-mail: schen~eri~~intcl-chg.ul~~.~~.

’ Current address: Johnson and Johnson Research Pty. Ltd., G.P.O. 3331, Sydney, 2001 NSW, Australia.


424 E.G. SARAVOLAC et al.

maximum transfection activity for SPLP containing 24mol% DODAC. Geneexpression observed in tumor cells was increased by approximately one magnitude ascompared to cationic lipid/DNA complexes. The SPLP were stable and upon storage at4°C no significant change in the transfection activity was observed over a one-yearperiod. Thus this phosphate buffer detergent dialysis technique can be used to generateSPLP formulations containing a wide range of cationic lipid concentrations todetermine optimal SPLP composition for high transfection activity and low toxicity.

Keywords: Non-viral gene delivery, Cationic lipid, Cancer gene therapy,P&mid encapsulation, Liposomes

INTRODUCTION (Fraley et al., 1979; 1980; Lurquin, 1979; Wang andHuang, 1987).

Plasmid delivery has primarily relied upon two The focus in our lab has been on the developmentapproaches; virus based gene delivery and non-viral of small (approximately 1OOnm diameter), well-gene delivery. Recombinant viral delivery systems defined lipid-based plasmid carrier systems, wherewhile specific are rapidly cleared from the circula- the plasmid is fully encapsulated within a lipidtion limiting delivery to first pass organs such as the envelope. Previous studies have shown that plasmidliver (Huard et al., 1995; Worgall et al., 1997). Non- DNA can be encapsulated with high efficiencyviral delivery systems have included liposomes, (> 50%) by a detergent dialysis procedure usingcationic lipid-DNA complexes or lipoplexes, cat- low cationic lipid concentration (Wheeler et al.,ionic polymers and cation coated nanoparticles 1999). The encapsulation efficiency was a sensitive(reviews see Ledley, 1995; Felgner, 1997; Lasic, function of the cationic lipid concentration used.1997; Zabner, 1997; Sorgi and Huang, 1997; Chonn The particles formed, of approximately 70 nm diam-and Cullis, 1998; Maurer et al., 1999; Pollard et al., eter, contain one plasmid per particle and are1998). The highly charged lipoplexes and polymers stabilized with a polyethylene glycol (PEG) coating.are quite efficient in gene delivery to cells in vitro The plasmid in these stabilized plasmid-lipid(Gao and Huang, 1991; Felgner et al., 1994; Zabner particles (SPLP) is fully protected from serum nu-et al., 1995; Hofland et al., 1996). However, these cleases and the particles are stable during circula-charged and often large systems are generally tion in the blood stream in vivo (Monck et al., 1999).cleared rapidly following iv. injection limiting the It was shown that the transfection potency of SPLPpotential transfection sites to first-pass organs, such is dependent on the type of ceramide used as anchoras lung liver and spleen (Thierry et al., 1995; Hong for the PEG polymer. The higher transfectionet al., 1997; Hofland et al., 1997; Templeton et al., potency observed with ceramide groups containing1997; Huang and Li, 1997; Liu et al., 1997). The shorter acyl groups was attributed to the fasterDNA is not completely sequestered by these carrier dissociation rate of PEG-Cer with shorter acylsystems, and it is susceptible to degradation by groups, thereby destabilizing the particle and im-nucleases (Wheeler et al., 1999). High concentra- proving cell interaction and uptake into target cells.tions of cationic lipids are known to cause toxicity The inhibitory effect of PEG coating for associationin vivo and in vitro (Harrison et al., 1995, Li and and fusion are well known (Holland et al., 1996a,b).Huang, 1997). Furthermore, these complexes often Initial studies have shown that the transfectionexhibit limited stability, have a tendency for activity of SPLP could be increased significantlyaggregation and exhibit transfection potencies that by increasing the cationic lipid content in the SPLPcan be sample and time dependent. Methods for (Zhang et al., 1999). The formulation methodpassive encapsulation of plasmid into liposomes are described resulted in efficient encapsulation ofhighly inefficient and result in low DNA/lipid ratios DNA into well-defined SPLP containing up to



25 mol% cationic lipid. Citrate was used as counter-ion to efficiently shield the positive charge on thelipid intermediate structures, such as cylindricalmicelles and lamellar sheets formed during dialysisto levels compatible with good entrapment. Citrateconcentrations below the optimal range resulted inaggregate formation while at concentrations abovethe optimal range, interaction of the plasmid withlipid structures was inhibited and little or no encap-sulation occurred. However this protocol was notsuccessful for cationic lipid concentrations higherthan 25-30 mol%.

The focus of this work was to develop a formula-tion protocol that permits efficient encapsulation ofplasmid in SPLP containing 6-50mol% cationiclipid and to determine the optimal cationic lipidconcentration in SPLP for transfection in vitro andin vivo. Formation of SPLP with equimolar con-centrations of cationic and neutral lipids (DODAC/DOPE) were of particular interest, permitting adirect comparison with plasmid/cationic lipid com-plexes of identical lipid composition. In this workit is shown that SPLP containing a range of 6-42.5 mol% cationic lipid can be formed by varyingthe phosphate concentration in the dialysis mediumand that the plasmid DNA is encapsulated within alipid bilayer. Optimal transfection potencies in vitroand in vivo are obtained with SPLP containingapproximately 25 mol% cationic lipid. In a regionalperitoneal tumor model, these optimized SPLPexhibit an increased tumor transfection activity ofabout one magnitude, as compared to the transfec-tion observed with plasmid-cationic lipid com-plexes (lipoplexes). SPLP can be formed withplasmid ranging from 3000 to 50,000 bp in size.



N,N-dioleyl-N,N-dimethylammonium chloride(DODAC) was kindly provided by Dr. Steve Ansell(INEX Pharmaceuticals Corp., Burnaby, BC,Canada) and 1-0-[2’-(w-methoxy-polyethylenegly-c o l ) succinoyll-2-N-octoylsphingosine (PEG-C@

was synthesized as described in Webb et al. (1998)and generously provided by Dr. Zhao Wang (INEXPharmaceuticals Corp.) . Dioleylphosphatidyl-ethanolamine (DOPE) was obtained from North-ern Lipids (Vancouver, BC, Canada). Plasmidsp BS KS+ (2.964 kb), pINEX TK02 (4.262 kb)pGFPemd-c[R] ( 4 . 3 5 6 k b ) , pINEX T K 0 5(5.046 kb), pINEX LO18 (pCMVLuc; 5.650 kb),pINEX DO01 (5.963kb), pINEX PLO1 (8.151),pINEX PO05 (11.125 kb), pINEX BOO1 (15.600 kb)were supplied by the Core Support Group (INEXPharmaceuticals). X-phage DNA (48.502 kb) wassupplied by New England Biolabs (Mississauga,Ont., Canada). Monobasic potassium phosphate(ACS), bovine serum albumin, octyl-P-D-gluco-pyranoside (OGP), Triton X-100TM and DEAE-Sepharose CL-6B were obtained from Sigma (St.Louis, MO, USA). Dialysis membrane (Spetra/Por 2; Spectrum Medical Instruments), HEPES(BDH Analar), dibasic potassium phosphate (BDHAnalar), s u c r o s e ( B D H Analar) and NaCl(BDH ACS) were obtained from VWR Scientific(Edmonton, AB, Canada). The B16/FlO mousemelanoma cell line was obtained from the FrederickCancer Research Center (Frederick, MD, USA).C57/BL67 mice were obtained from HarlanSprague-Dawley Inc (Chicago, IL, USA). The careand handling of the mice followed guidelines set outby the Canadian Council on Animal Care.

Encapsulation of Plasmid DNA

Lipid dispersions were prepared from stock solu-tions of DODAC, DOPE and PEG-CerCs inethanol (typically 10 mg/ml each). Appropriateamounts of lipid were transferred into a glass testtube and the solvent removed under a stream of N2gas followed by storage under vacuum for 3-5 h.The dried lipid film (10 mg lipid per 1 ml formula-tion) was dissolved by gently mixing in a 1 ml solu-tion containing 100 mM octylglucoside (stocksolution 1 M OGP), 200-400 ug/ml plasmid (stocksolution 1 mg DNA/ml in 10 mM Tris-HCl pH 8.0;1 mM EDTA) in the appropriate dialysis buffer. The:lipid/plasmid/detergent suspension was incubated

426 E.G. SARAVOLAC et al.

at RT for at least 30min to dissolve the lipidscompletely. Following incubation the mixture wasdialyzed for 2 days against 100-400 volumes of theappropriate ionic strength dialysis buffer (sodiumphosphate pH 7.4 * NaCl; filtered through 0.2 urnfilter) with 2-3 changes of buffer. After dialysis theSPLP suspension was analyzed for particle sizeand encapsulation efficiency. Non-encapsulatedplasmid was removed by anion exchange chroma-tography on a DEAE Sepharose CL-6B columnequilibrated in dialysis buffer. Where appropriate,empty liposomes were removed by sucrose densitygradient centrifugation as described previously(Wheeler et al., 1999; Zhang et al., 1999) with thefollowing modification. Formulations containinggreater than 30 mol% DODAC were isolated on a1% : 2.5% : 5% sucrose gradient and formulationscontaining 30 mol% and less DODAC were isolatedon a 2.5% : 5% : 10% sucrose gradient. The gradi-ents were centrifuged at 160,000x g for 12- 18 h.The SPLP with > 30 mol% DODAC were buoyanton the 2.5-5% sucrose interface and SPLP with30mol% or less DODAC were buoyant on the 5-10% sucrose interface. The SPLP fraction wasremoved, dialyzed against HBS (20mM HEPES in150 mM NaCl, pH 7.4) and where requiredconcentrated using Aquacide II (Calbiochem,San Diego, CA, USA) to 250-500 ug plasmid/mI.

Evaluation of Plasmid Encapsulation Efficiency

The extent of the plasmid encapsulation in the SPLPwas evaluated by measuring the accessibility of thedsDNA interchelating dye PicoGreenTM (Molec-ular Probes, Eugene, OR, USA) by the followingmethod. Aliquots of the formulation taken directlyfrom detergent dialysis were diluted 1 : 400 in dial-ysis buffer or HEPES buffered saline (pH 7.2)and 2 ~1 of Picogreen reagent was added to 1 .O mlof the diluted samples. The fluorescence was mea-sured at an excitation wavelength of 495nm andemission wavelength at 525 nm using an SLM-Aminco fluorometer (Rochester, NY, USA) both inthe absence (I) and presence (lo) of 10 yl 10% (v/v)

Triton X- 100. The percent plasmid encapsulationwas calculated as E (%) = (Z. - Z)/Z, x 100. Itshould be noted that this assay measures the extentthat the lipid-plasmid association interferes withpicogreen binding to the plasmid DNA. Associationmay be due to encapsulation within lipid vesicles aswell as binding between lipid membrane surfacesdue to aggregation (Zhang et al., 1999). The pres-ence of aggregation was determined separatelyfor each preparation by QELS particle size analysis(below). To determine the total plasmid DNAconcentration in the formulations PicoGreen fluo-rescence was measured in presence of 0.1% TritonX-100 and compared with plasmid DNA at stan-dard concentrations.

Particle Size Determination

Particle sizes were determined by quasi-elastic lightscattering (QELS) using a Nicomp submicronparticle seizer (Model 340, Nicomp particle sizingsystems, Santa Barbara, CA, USA) operated in thevolume-weighted vesicle mode.

Lipid Analysis by HPLC

Lipids were quantified using a Beckman Gold seriesHPLC pump and autosampler system, fitted witha Beckman Ultrasphere Cyano 5 urn column (0.2 xI5 cm) (Beckman Instruments, Fullerton, CA,USA) and the samples were detected on an AlltechModel 500 ELSD - evaporative light scatteringdetector (Alltech Associates Inc., Deerfield, IL,USA). The lipid-containing samples (200 ~1) wereextracted with 1 ml each of HBS/methanol (2: 1)and CHCls. The extract was vortexed for 1 min andthe CHCls layer was separated by centrifugation at2500x g and loaded onto an autosampler forchromatography. The samples were eluted with agradient from 100% solvent A to 90% solvent Bover a time period of 8 min at 0.3 ml/min (SolventA: 99.95% Chloroform, 0.05% Trifluroaceticacid; Solvent B: 90% isopropanol, 9.95 % water,0.05% TFA).


Freeze-Fracture Electron Microscopy

Particle sizes were also determined by freeze-fracture electron microscopy provided by -Dr. KimWong (Department of Biochemistry, University ofBritish Columbia). Freeze-fracture as performedon a Balzers freeze-etching system, BAF 400D(Balzers, Balzers, Liechtenstein). Samples werecryofixed in the presence of 25% glycerol byplunging them into liquid Freon 22. The fracturedsurface was shadowed unidirectionally with plati-num/carbon (45”) and coated with carbon (90”)immediately after fracturing. Replicas were ana-lyzed using a JEOL Model JEM 1200 EX micro-scope (Soquelec, Montreal, QC, Canada).

Cryo-Electron Microscopy

A drop of buffer containing SPLP was applied toa standard electron microscopy grid with a per-forated carbon film. Excess liquid was removedby blotting, leaving a thin layer of the suspensioncovering the holes of the carbon film. The grid wasrapidly frozen in liquid ethane, resulting in vesiclesembedded in a thin film of amorphous ice. Images ofthe vesicles in the ice were obtained under cryogenicconditions at a magnification of 66,000 and adefocus of - 1.5 microns using a Gatan cryo-holderin a Philips CM200 FEG electron microscope.

In Vitro Transfection Assay

COS-7 cells were seeded at a concentration of4.0 x lo4 cells per well in a 24 well dish in a total of2.0 ml media one day prior to transfection. Cells inlogarithmic growth phase were used to seed thedishes. At the time of transfection, cells were lessthan 20% confluent. Formulated plasmid DNA(both SPLP and complexed) was added to the cellsat concentrations ranging from 0.1 to 1 .O ug DNAand remained in the culture medium throughoutthe experiment. At the completion of the incuba-tion period, media was removed from each well andcells were washed twice with PBS prior to storage at-70°C. On the day of analysis, plates were brought

to room temperature and 200 ul cell lysis buffer(0.1% Triton X-100, 250 mM NaH2P04, pH 7.4)was added to each well. Plates were shaken on arotary platform for 10 min at 100 rpm. The expres-sion of the luciferase reporter gene was assayed asdescribed below.

Intraperitoneal Tumor Transfection

B16/FIO tumor cells (1 x IO5 cells in 200~1 PBS)were injected into the peritoneal cavity of femaleC57/BL6 mice. Seven days after tumor seeding,3Opg of formulated plasmid DNA was injectedintraperitoneally (i.p.) into the tumor bearing micein a total volume of 500~1 PBS. Animals weresacrificed 6 or 24 h after transfection. Organs andtumors were collected, weighed, snap frozen inliquid nitrogen and stored at -70°C until analysisfor luciferase gene expression. Frozen samples werebrought to room temperature and transferred intoindividual Fast-Prep tubes pre-loaded with a smallbead. A second bead was added to each tube priorto the addition of 0.5- l.Oml of cell culture lysisreagent (Promega) supplemented with 1 .O mg/mlBSA. Samples were homogenized using the Fast-Prep FP120 (Savant Instruments, Farmingdale,NY, USA) for 10s at a speed setting of 5. Homo-genized samples were then incubated at roomtemperature for 15 min, subjected to centrifugationfor 2 min at 14,000x g to remove debris and theclear lysate was assayed for luciferase activity.

Luciferase Assay

Luciferase assays on both cultured cells or tissuehomogenates were performed using Promega lucif-erase assay substrates (Promega E 1501) accordingto the procedure described by the manufacturer. A20 ul aliquot of cell lysate (or tissue homogenate)was assayed for luciferase activity using a DynexTechnologies ML3000 microplate luminometer(Dynex Technologies, Chantilly, VA, USA). Lumi-nescence readings were calibrated according to astandard curve obtained using a Photinus pyvalisluciferase standard.

428 E.G. SARAVOLAC et al.

Calcein Cell Viability Assay 80

The viability of in vitro cultured cells after incuba-tion with SPLP was determined using a calcein assaytechnique. The media in the wells containing float-ing cells was collected into individual 6 ml tubes. Theadherent cells remaining in the wells were trypsi-nized, harvested in PBS and added to the floatingcells. The pooled cells were centrifuged at 1000 x gfor 10 min and the cell pellet was dissolved in 0.1 mlPBS. A 0.1 ml aliquot of the cell suspension wastransferred to a 96 well plate to perform the viabilityassay. Calcein AM was added in 0.1 ml PBS at afinal concentration of 2 PM per well. After 30 minfluorescence (Ex 485 nm; Em 520 nm) was measuredon a BioLumin (Molecular Dynamics) fluorometer.The measured calcein fluorescence was propor-tional to the cell viability.

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FIGURE 1 Encapsulation of plasmid in SPLP with differentconcentrations of DODAC. SPLP formulations composedof DODAC/DOPE/PEG-CerCs (42.5 - x : 42.5 +x: 15; mol :mol : mol) and pCMVLuc (pINEX L018) (200 ug/ml plasmid;Smg/ml lipid) were prepared with DODAC concentrationvarying between 38 and 42mol% in detergent and dialyzedagainst 150mM NaCl in 150mM sodium phosphate bufferpH 7.4 as described in Materials and Methods. Encapsulationefficiency was determined by PicoGreen assay as outlined inMaterials and Methods. Percent encapsulation is plotted as afunction of DODAC concentration used for SPLP formation.

Encapsulation of Plasmid in SPLPContaining Equimolar Concentrations ofCationic Lipid and DOPE

Previous work showed that citrate can be used tomodulate the ionic interaction between plasmid andlipid intermediate structures, that contain up to25 mol% cationic lipid, during detergent dialysisresulting in efficient encapsulation of plasmid inlipid particles (Zhang et al., 1999). However, athigher cationic lipid concentrations the ionic inter-action was difficult to control and excess aggrega-tion and precipitation was typically observed. Herewe evaluated the shielding effect of phosphateduring dialysis of a near equimolar cationic andneutral lipid mixture of DODAC and DOPE.

Plasmid encapsulation was studied as a functionof DODAC concentration using 150 mM NaCl in150 mM sodium phosphate buffer pH 7.4 as dialysisbuffer (Fig. 1). The PEG concentration was keptconstant at 15% and DOPE concentration variedas required to compensate for changes in DODACconcentration. The extent of plasmid encapsula-tion was estimated by determining the relative

accessibility of the DNA interchelating fluorescentdye PicoGreen to the plasmid, in presence andabsence of Triton X-100 as described in Materialsand Methods. The particle size distribution wasused as an additional criterion for SPLP formationand was required to be approximately 100 nm.Formation of large plasmid-lipid aggregates canlead to an overestimation of the encapsulation bythe PicoGreen assay. The results in Fig. 1 demon-strate the sensitivity of the encapsulation efficiencyto small changes in the cationic lipid concentrationat a given ionic strength. The salt concentration of150 mM NaCl in 150 mM sodium phosphate bufferpH 7.4 was optimal for SPLP formation with42.5 mol% DODAC. A decrease in DODAC con-centration of 2mol% resulted in an up to 40% de-crease in the encapsulation efficiency. At DODACconcentrations higher than 42.5 mol% formation oflarge polydisperse aggregates were detected underthese conditions. In the absence of PEG-Cer, whole-sale aggregation was observed (data not shown).

38 39 40 41 42 43

Mel Percent DODAC


The salt concentration optimal for SPLP forma-tion with equimolar concentration of DODAC andDOPE often required slight adjustments in the NaClconcentration for different plasmid preparations.Without adjustments the encapsulation efficiencycan vary from 40-60% between plasmid batches.

Effect of Lipid and Plasmid Concentration onEncapsulation Efficiency

The effect of the total lipid and plasmid concentra-tion on the encapsulation efficiency was evaluatedfor SPLP with equimolar concentrations ofDODAC and DOPE. A series of SPLP formulationswere prepared where either the plasmid (0.1~ 1 mg/ml) or lipid (l-10 mg/ml) concentrations werevaried in the preparations. Under identical ionicstrength buffer conditions and 200 ug/ml plasmid,increasing lipid concentration resulted in anincrease in the encapsulation efficiency (Fig. 2(a)).Note, that where encapsulation efficiency exceeded70% a significant amount of aggregation (diam-eter > 150 nm) was often observed in these prepara-tions both visually and as measured by particle sizeanalysis, leading to an over-estimate of the encap-sulation by the PicoGreen assay as pointed outearlier. At a constant lipid concentration of 5 mg/ml, increasing plasmid concentration resulted in adecrease of the plasmid encapsulation efficiency(Fig. 2(b)). These results suggest that the plasmidscompete for the cationic lipid intermediate struc-tures bearing a distinct charge density duringdialysis and a larger proportion of plasmid wasencapsulated when the total lipid concentration wasincreased.

Conditions for SPLP Formation with DifferentCationic Lipid Concentrations

A series of studies were performed to determinethe optimal buffer ionic strength in the dialysisbuffer for efficient encapsulation of plasmid(pINEXLO18) in SPLP using a range of cationiclipid concentrations. Replicate SPLP formulationswere prepared containing 30-42.5 mol% DODAC

(a) 100 ,

0 2 4 6 8 10

mglml Total Lipid

@I 80 ,

- I

0 200 400 600 800 1000

pg Plasmid DNA

FIGURE 2 Effect of total lipid and plasmid concentra-tion on encapsulation efficiency. Formulations composed ofDODAC/DOPE/PEG-CerCs (42.5 : 42.5 : 15; mol : mol : mol)and pCMVLuc were dialyzed against 150 mM sodium phos-phate buffer and 150mM NaCl. In (a), the plasmid concen-tration was maintained at 200ug/ml and the total lipidconcentration was varied between 1 and lOmg/ml. In (b), thetotal lipid concentration was maintained at 5mg/ml while theplasmid concentration was varied between 100 and lOOOug/ml. Encapsulation efficiencies of over 70% were typicallyaccompanied with significant particle aggregation as measuredby QELS. Percent plasmid encapsulation was plotted as a func-tion of total lipid concentration (a) and plasmid concentration(b) used in the formulation.

and these were dialyzed against a range of salt con-centrations in the dialysis buffer (Fig. 3). The ionicstrength in the dialysis buffer was titrated byvarying the NaCl concentration from 0 to 200 mMin 150mM sodium phosphate buffer pH 7.4.

430 E.G. SARAVOLAC et al.

0 40 80 120 160 200

NaCl Concentration (mM)

FIGURE 3 Encapsulation efficiency in SPLP containing dif-ferent DODAC concentrations as a function of the salt concen-tration in the dialysis buffer. Formulations containing 30, 34,38 and 42.5 mol% DODAC (DODAC : DOPE : PEG-cerCs;30:55:15; 34:51:15; 38:47:15and42.5:42.5:15;mol:mol:mol) were prepared. Replicate formulations were dialyzedagainst 150mM sodium phosphate buffer containing O-200mM NaCl. Each of the SPLP formulations was preparedwith lOmg/ml total lipid and 400yg/ml plasmid (pCMVLuc,pINEX LO18). Percent encapsulation for SPLP with 30 (m),34 (a), 38 (A) and 42.5mol% (v). DODAC is plotted as afunction of salt concentration in the dialysis buffer.

When the salt concentration in the dialysis bufferwas too high, the association between the cationiclipid and the DNA was inhibited and little encapsu-lation was observed. Encapsulation efficiency in-creased at each DODAC concentration as the ionicstrength of the dialysis buffer was decreased. Aggre-gation was often observed by QELS in prepara-tions where the encapsulation exceeded 60-70%.To obtain efficient encapsulation (> 50%) withDODAC concentrations of 30mol% and lower,the sodium phosphate concentration had to be fur-ther decreased from 150mM to as low as 100 mMsodium phosphate (data not shown). By usingencapsulation efficiency of > 50% and particle sizeof approximately 100 nm in diameter as criteria forSPLP formation, the optimal salt concentrationrequired in the dialysis buffer was determined forDODAC concentrations from 7 to 42.5 mol% andsummarized in Table I.

Following dialysis, the non-encapsulated plas-mid was removed by DEAE Sepharose CL-6B

TABLE I Optimal phosphate buffer concentration for plasmidencapsulation

SPLP DODAC Optimal detergent dialysisconcentration (mol%) buffer concentration

7 150mM NaCl20 90 mM Na2HP0424 110 mM NazHP0430 140 mM Na2HP0434 150 mM Na2HP04 + 50 mM NaCl38 150 mM Na2HP04 + 90 mM NaCl42.5 150 mM Na2HP04 + 140 mM NaCl

TABLE II Diameter and lipid/DNA ratios of purified SPLPformulations


QELS diameter Lipid/DNA ratio

(nmY (ug plasmid/umol lipid)

7 90.0 15.720 64.0 65.524 77.2 13.530 89.3 81.942.5 93.8 59.7

aQELS measured in particle size mode.

chromatography and empty lipid vesicles wereseparated from loaded ones by sucrose density gra-dient centrifugation as previously described (Zhanget al., 1999). Traces of aggregates formed duringdialysis were also removed during gradient isola-tion, resulting in a very narrow particle size dis-tribution. The lipid/DNA ratio was determined foreach SPLP formulation by quantitative HPLC andPicoGreen assay and is presented in Table II. Aconstant lipid to DNA ratio ranging from 60 to82 ug plasmid/umol lipids was observed for isolatedSPLP with pCMVLuc over the whole range ofDODAC concentrations. This ratio is consistentwith that previously reported for SPLP with lowerDODAC concentrations (Wheeler et al., 1999;Zhang et al., 1999). The lipid composition of theisolated SPLP was determined by quantitativeHPLC. It was found to be similar to the initial lipidcomposition used with may be a slight enrichmentin the DODAC content of approximately 2%(data not shown).


Effect of Plasmid Size on EncapsulationEfficiency and SPLP Size

The influence of plasmid size on SPLP formationwas evaluated for formulations containing 7 and24 mol% DODAC respectively. The plasmids usedranged from 2.9 to 15.6 kb and also included aX-phage DNA of 48.5 kb. The formulations withdifferent size plasmids (10 mg/ml lipid; 200 ug/mlplasmid) were dialyzed against identical salt condi-tions. An apparent direct relationship between theencapsulation efficiency and log of the plasmid sizewas observed for both 7 and 24mol% DODACcontaining SPLP formulation (Fig. 4(a)). Encapsu-lation efficiency decreased with increasing plasmidsize. A similar effect was observed for the encapsula-tion efficiency of linearized and supercoiled plasmid(data not shown). In this case the encapsulation ofthe linearized plasmid (which has a larger diameterin solution) was at least 10% less efficient than forthe supercoiled form (p < 0.1). SPLPs containing7 mol% DODAC and different size plasmids wereisolated by DEAE-chromatography and densitygradient centrifugation. The particle size of theisolated SPLPs was analyzed by quasi-elastic lightscattering operated in the volume weighting vesicleand particle mode and was plotted against plasmidsize (Fig. 4(b)). The SPLP size determined in bothvesicle and particle mode, increased with increasingplasmid size. This suggests that the decreased encap-sulation efficiency observed with increasing plasmidsize may reflect a limit to the size of the lipid sheetrequired to encapsulate the plasmid.

Characterization of SPLP byElectron Microscopy

The SPLP structure was analyzed by cryo-electronmicroscopy and compared to large unilamellarvesicles (LUV) of the same lipid compositionprepared by extrusion of a hydrated lipid mixturethrough 100 nm pore size filters (Hope et al., 1985).The electron micrograph of SPLP shows a lipidbilayer surrounding an electron dense internalstructure, which is consistent with plasmid DNA

I ,






1 2 5 10 20 50

Plasmid size (kb)


160 - -m-Ves ic le Size

g 140- -*- Particle Size

I2 4 6 8 10 12

Plasmid Size (kb)

FIGURE 4 Effect of plasmid size on encapsulation effi-ciency and SPLP size. SPLP formulations were prepared witha series of plasmids ranging in size from 2.9 to 15.6 kb (seeMethods) as well as 50 kb X-phage DNA. The SPLP formula-tions were prepared at two DODAC concentrations, 7 and24 mol% (DODAC : DOPE : PEG-cerCs; 7 : 78 : 15 and 24 : 61:15; mol: mol :mol) and were dialyzed against 1lOmM sodiumphosphate buffer, pH 7.2 and 20mM HEPES buffered salinepH 7.2 respectively. All formulations were prepared with10 mg/ml total lipid and 200 ug/ml plasmid. No significantaggregation was observed in any preparation including thosewith encapsulation efficiency > 60%. Encapsulation efficiencywas measured directly after dialysis and plotted as a functionof plasmid size for SPLP containing 7 (m) and 24mol%DODAC (0) in (a). Following removal of non-encapsulatedplasmid by DEAE chromatography and separation of emptyvesicles by sucrose gradient centrifugation, the size of SPLPcontaining 7mol% DODAC and different size plasmid (2.9-15.6 kb) was analyzed by quasi-elastic light scattering usingboth “solid particle” (0) and “vesicle” (H) algorithms in aNICOMP particle size analyzer and plotted as a function ofplasmid size (b). Error bars indicate the standard deviationcalculated by the NICOMP program.

432 E.G. SARAVOLAC et al.

FIGURE 5 Cryo-electron micrographs of SPLP. LUV (A) were prepared by hydration and extrusion through polycarbonatefilters with 100 nm pore size. The lipid concentration of the formulations was adjusted to about 2mg/ml. Isolated SPLP (B) wereprepared as described in Fig. 4(b) with pCMVLuc. The bar in panel (B) indicates 50nm. Details of sample preparation and cryo-electron microscopy are given in Materials and Methods.

encapsulated in a lipid vesicle (Fig. 5(B)). This inter-nal structure in SPLP is not seen in the LUV pre-pared by extrusion (Fig. 5(A)). The SPLPs exhibita homogeneous morphology and size (75 & 6 nmdiameter). This narrow size distribution of the SPLPwas also seen by quasi-elastic light scattering(90 * 16 nm with a narrow Gaussian distribution(x2=0.2) for a 5.65 kb plasmid) and by freeze-fracture electron microscopy (data not shown).SPLP formed with the same size plasmid exhibitan overall similar size distribution regardless of theDODAC concentration used (Table II).

Effect of Cationic Lipid Concentration on theTransfection Potency of SPLP In Vitro

The transfection potency of isolated SPLP contain-ing a range of different DODAC concentrations andluciferase as reporter gene, was investigated in vitrousing COS-7 cells. Measurements of luciferaseexpression in COS-7 cells over a 72 h time coursefollowing transfection with 0.5 ug plasmid in SPLP

containing 42.5,38 and 34 mol% DODAC revealedthat maximal expression occurred after 48 h ofcontinuous incubation with the SPLP formulation(Fig. 6(a)). Decreasing the DODAC concentrationin SPLP from 42.5 to 34mol% increased the extentof luciferase expression approximately 5 fold after48 h of transfection. A dose titration (0.05-l ugplasmid/well) with SPLP containing 20-42.5 mol%DODAC revealed that the transfection potency forSPLP with 20&30mol% DODAC is consistentlyhigher than for SPLP with 42.5 mol% DODAC foreach dose (Fig. 6(b)). The luciferase activities de-tected in cells treated with SPLP were comparable tothose obtained for plasmid lipid complexes formedwith pCMVLuc and DOPE/DOCAC (1: 1; mol :mol) liposomes at a charge ratio of 1.5 : 1 (+ : -which was optimal for this cell line). Cell viabilitymeasured with a calcein hydrolysis assay, clearlyincreased with decreasing cationic lipid content inSPLP. Based on the transfection levels and cellviability SPLP containing 20-25 mol% DODACwere considered optimal for transfection in vitro.


J 0.0 I 124 48 72

Time (hr)

gE 80000

sa 80000.c

5 40000J

.g 20000

3 0

DODAC 20 % 24 % 30 % 42.5 %

FIGURE 6 Effect of DODAC content in SPLP on the trans-fection activity in vitro. Plasmid (pCMVLuc) was encapsulatedin SPLP containing different DODAC concentrations(DODAC : DOPE : PEG-cerCs; x : 85 - x : 15; mol : mol : mol)using dialysis conditions as described in Fig. 3 and Table II.SPLP were isolated by sucrose gradient centrifugation. COS-7cells were plated at 40,000 cells/well in a 24 well plate andincubated for 24 h. For the time course shown in (a) themedium was replaced after 24 h and SPLP containing 0.5 ug ofpINEXL018 added and incubated for 24, 48 and 72 h. Afterincubation was complete the cells were assayed for luciferaseexpression as described in Methods and the luciferase activityplotted as a function of time for SPLP containing 42.5 (m), 38(0) and 34 (&mol% DODAC. For the dose response shownin (b), COS-7 cells, were incubated as described above with 20,24, 30 and 42.5 mol% DODAC SPLP at 0.05, 0.1, 0.5 and 1 ugpCMVLuc/well. The cells were incubated for 24 h and wereassayed for luciferase to quantify transfection and calcein hy-drolysis to estimate cell viability as described in Methods.

SPLP are Stable During Storage

An isolated SPLP preparation containing 24 mol%DODAC was filter-sterilized (0.2um filter) andstored at 4°C for one year. At 3, 6 and 12 monthsaliquots of the formulation were removed fromstorage and assayed for luciferase transfection in adose-response study and compared to the initial

activity. In addition the particle size was character-ized by quasi-elastic light scattering. Throughoutthe storage period the formulation retained bothits transfection activity and small particle size(data not shown).

Effect of Cationic Lipid Concentration on theTransfection Potency of SPLP In Viva

The transfection activity of SPLP formulationscontaining DODAC ranging from 7 to 42.5 mol%were compared in an intraperitoneal B16 tumormodel. B 16 cells were seeded in the peritoneal cavityof C57BL/6 mice. After 7 days the tumors were onaverage approximately 200mg in size and SPLPpreparations containing pCMVLuc were adminis-tered intraperitoneally at a dose of 3Oug plasmidper mouse. The tumors were collected after 24 hand assayed for luciferase activity. Equal plasmiddoses of both, sucrose gradient isolated (excessempty lipid vesicles removed) and non-isolatedformulations were administered to determine if thepresence of empty liposomes would affect the levelof transfection in vivo. The luciferase expressiondetected in tumors treated with non-isolated SPLPcontaining 42.5 mol% DODAC was similar to theexpression following transfection with complexesformed with DODAC/DOPE (1 : 1; mol : mol) lipo-somes and pCMVLuc (3Oug plasmid/mouse) of100&2OOpg/g tumor (data not shown). The com-plexes were formed at a 3 : 1 cationic lipid-to-DNAcharge ratio, which was optimal for transfection inthis tumor model. The highest luciferase expressionwas observed in tumors treated with isolated SPLPcontaining 24 and 30 mol% DODAC (Fig. 7). Theluciferase activity detected was approximately 10fold higher than that measured in tumors treatedwith cationic lipid/DNA complexes. Only very lowlevels of transfection were observed with SPLP con-taining 7mol% DODAC. The presence of excessempty lipid vesicles reduced the transfection pot-ency of SPLP approximately 4 fold, independent ofDODAC concentration in the SPLP. Thus thehighest transfection potency was obtained with iso-lated SPLP containing approximately 25 mol%



2 2500sGJ 2000z3t


5 1000

gJ 500

0 IE.G. SARAVOLAC et al.

FIGURE 7 Transfection of intraperitoneal B16 tumorsusing isolated and non-isolated SPLP. Groups of female C57mice were injected with 100,000 B16 tumor cells 7 days priorto administration of SPLP. Both isolated and non-isolatedSPLP prepared with 7, 20, 24, 30 and 42.5 mol% DODAC(DODAC : DOPE : PEG-cerCs; x : 85 - x : 15; mol : mol : mol)containing 30 pg pINEXL018 were injected into the peritonealcavity of tumor-bearing mice. The tumors were removed 24 hpost-injection and were assayed for luciferase transfection asdescribed in Methods.

DODAC in both cells in vitro and in a regionaltumor model in vivo.


This study demonstrates that SPLP can be formedwith a wide range of cationic lipid concentrations(6-42.5%) by adjusting the salt concentration in aphosphate buffer system used in the detergent dial-ysis process. The formulation process describedeven permits formation of SPLP with equimolarconcentration of cationic lipid and neutral lipidwhich is similar to the lipid composition typicallyemployed to form the plasmid/cationic lipid com-plexes (lipoplexes). The transfection potency ofSPLP formed with equimolar concentrations ofcationic and neutral lipids as determined in cellsin vitro and in a regional tumor model in vivo wascomparable to the one obtained with the plasmid-cationic lipid complexes formed with liposomescomposed of DODAC : DOPE (50 : 50, mol : mol).

But most important, reduction of the cationic lipidcontent in the SPLP to 24mol% resulted in anapproximate 10 fold increase in the transfectionactivity in tumors in vivo.

The SPLPs with the different DODAC concen-trations all exhibit a homogeneous well-definedstructure with plasmid DNA encapsulated withina lipid bilayer envelope. The cryo-electron micros-copy data represent the first demonstration of aplasmid entrapped in a small well-defined lipidvesicle of approximately 75 nm diameter. This struc-ture is clearly distinct from any structures describedfor plasmid/cationic lipid complexes. The completeencapsulation of the plasmid is consistent with theserum stability, protection from DNase and stabil-ity of SPLP in the blood stream (Wheeler et al.,1999; Monck et al., 1999). In contrast to the SPLP,plasmid/cationic lipid complexes are generally muchlarger than 200 nm in diameter and do not providefull protection of the associated plasmid.

The high plasmid-to-lipid ratio of approximately70 ug DNA/urn01 lipid in the isolated SPLP contain-ing pCMV Luc was independent of the DODACconcentration used and corresponds to one plasmidper SPLP. The plasmid-to-particle ratio was cal-culated assuming a lipid molecular area of 0.67 nm2(King et al., 1985) and an average nucleotidemolecular weight of 330. For a SPLP with a diam-eter of 75nm (average size determined by cryo-electron microscopy) and a 5.650 kbp plasmid(pCMV Luc) the plasmid-to-lipid ratio of 70 ugDNA/urn01 lipid corresponds to a plasmid-to-particle ratio of 0.99.

The SPLP can readily be sterilized by passingthrough a 0.22 micron filter and are highly stableduring extended storage. No significant changes intheir size, plasmid encapsulation and transfectionpotency was observed during storage at 4°C over aone-year period. Complexes are usually so unstablethat they have to be formed immediately prior toadministration and their size distribution makesfilter-sterilization impossible.

Entrapment of plasmids of up to 20 kb in 80-100 nm diameter vesicles represents a difficult pack-ing problem. For example, electron micrographs of


a supercoiled 4.4 kb plasmid revealed an extended. length of about 520nm and on average (in two

dimensions) a diameter in the range of 350nm(Lewis et al., 1985). This would suggest an averagediameter of > 1 urn for a 20 kb plasmid. The inverserelationship between encapsulation efficiency andplasmid size observed and the apparent limit forencapsulation of plasmids of 20 kb is therefore notsurprising. The detergent dialysis process withcationic lipids described here must involve partialcondensation of the plasmid to permit encapsula-tion in < 100 nm vesicles. The exact mechanism forplasmid encapsulation is not understood in detail.In an earlier study it was shown that the plasmidencapsulation efficiency was a sensitive function ofthe cationic lipid content and maximum entrapmentwas obtained with 6 mol% DODAC (Wheeler et al.,

1999). It was proposed that plasmid interacts withmacromolecular lipid structures such as cylindricalmicelles and lamellar sheets formed as intermediatestructures during detergent dialysis. The resultsshown here indicate that the positive charge onthese intermediate lipid structures can be shieldedwith phosphate. At phosphate concentrationsabove the optimum range the positive charge onthe lipid structures formed during dialysis is shieldedto the extent that interaction with the plasmid isinhibited, resulting in little or no plasmid encapsula-tion. At the critical optimal phosphate concentra-tion the shielded charge on the lipid structures aresufficient to bind plasmid and encapsulation pro-ceeds as outlined previously (Wheeler et al., 1999).On the other hand, at phosphate concentrationsbelow the optimum range the positive charge on thelipid structures is not shielded sufficiently resultingin strong plasmid lipid interaction with aggregateformation. Adjustments to the NaCl concentrationcan be used in combination with phosphate to fine-tune the system.

The SPLP are stabilized by a PEG coating. ThePEG-ceramide lipids have a dual function by firstregulating the degree of DNA interaction with cat-ionic lipid during SPLP formation and secondlyby providing programmable fusion (Holland et al.,

1996a,b) and circulation lifetimes in vivo (Lasic et al.,

1991; Moncket al., 1999). The PEG-stabilized SPLPexhibit an extended shelve-life of at least one yearwithout a detectable change in particle size and lossof transfection activity. However, the presence ofa PEG coating can inhibit interaction and fusionbetween lipid vesicles (Holland et al., 1996a,b) andthe poor transfection observed with SPLP contain-ing PEG-Cer&a in vitro was attributed to theinability of PEG to dissociate. SPLP containingPEG-CerCi4 with a faster dissociation rate (tip =1.2 h) compared to PEG-CerC2e (t1,2 2 13 days)were shown to have significantly improved trans-fection properties. The SPLP used in the presentstudy contained PEG-CerCs as the stabilizingagent with a very fast dissociation rate (tip 51.2 min) to maximize the transfection and to permitevaluation of the effect of the cationic lipid contentin the SPLP on the transfection potency in vitro andin the regional in vivo model, independent of PEGexchange. It should be noted that these systemswould not be suitable for intravenous delivery fortargeting disease sites such as distal tumors, sincethey will be highly unstable following interactionwith biological fluids.

The most important finding in this study is theimportance of the cationic lipid concentration inSPLP for efficient transfection in vitro and in vivo.

The highest transfection activity was observed withSPLP containing approximately 25 mol% DODACand the expression levels observed in tumors wereabout one magnitude higher than obtained withcorresponding plasmid DNA-cationic lipid com-p lexes . The SPLPs conta in ing 20-30mol%DODAC prepared by the citrate dialysis method(Zhang et al., 1999) exhibit similar transfectionpotencies as SPLP with equivalent DODAC con-centrations made by the phosphate dialysis method(1.2 f 0.6 and 1.7 f 0.8 pg luciferase/g tumor forSPLP with 24mol% DODAC formed by citrateand phosphate dialysis, respectively). There was noapparent toxicity associated with SPLP containing24mol% DODAC as determined by aspartateaminotransferase levels in the serum 24 h followingi.p. injection. The higher concentration of cationiclipid in SPLP may result in enhanced association

436 E.G. SARAVOLAC et al.

with and uptake into cells. In this regard cellulardelivery of plasmid with SPLP containing 6 mol%DODAC and stabilized with PEG-CerCs was lessthan 3% of that observed with DODAC/DOPE(1 : 1) complexes (Mok et al., 1999). In addition it ispossible that it enhances interaction with the endo-somal membrane facilitating intracellular deliveryof the encapsulated plasmid. Excess lipid did notincrease the transfection activity of SPLP in viva incontrast to plasmid/lipid complexes (Song and Liu,1998). The increase in transfection activity observedfor SPLP containing higher cationic lipid concen-trations and the reduced activity by excess emptyvesicles suggest that cellular uptake of SPLP doesrepresent a limiting step for efficient plasmiddelivery. It is important to note that in this studyonly three lipid components DODAC, DOPE andPEG-CerCs have been used. Using a broader rangeof lipids together with other components, such asconjugated targeting ligands, that increase cellularinteraction and uptake, it may be possible toincrease the transfection potential of SPLP evenfurther. The formulation protocol described hereprovides a flexible system to assess different com-ponents for the further development of SPLP typegene delivery systems.

In summary, the results presented in this studyshow that SPLP can be constructed with a widerange of cationic lipid concentrations by includingphosphate in the dialysis medium. The detergentdialysis protocol permits construction of highlyflexible SPLP systems for non-viral gene delivery.SPLP with 24mol% DODAC gives rise to tumortransfection that is superior to plasmid/cationiclipid complexes. Furthermore, these SPLP can bestored over extended period of time without any lossof activity. The well-defined structure sets SPLPclearly apart from lipoplexes and other cationic lipidor cationic polymer complexes.


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