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Preparation and phase behaviour of surface-active pharmaceuticals:self-assembly of DNA and surfactants with membranes.Differential adiabatic scanning microcalorimetric study

Erhan Sleymanoglu a,b,*a Biophysics Section, Department of Physical Chemistry of Drugs, Faculty of Pharmacy, J.A. Comenius University, Odbojarov 10,

83 232 Bratislava, The Slovak Republicb

Department of Biophysics, The Slovak Academy of Sciences, Institute of Experimental Physics, Koice, The Slovak Republic

Received 18 March 2005; accepted 3 May 2005

Available online 14 July 2005

Abstract

Some energetics issues relevant to preparation and surface characterization of zwitterionic phospholipidDNA self-assemblies, as alter-native models of the currently used problematic lipoplexes are presented. Nucleic acid compaction capacities of Mg2+ and N-alkyl-N,N,N-trimetylammonium ions (C

nTMA, n = 12) were compared, with regard to surface interaction with unilamellar vesicles. Differential adiabatic

scanning microcalorimetric measurements of synthetic phosphatidylcholine liposomes and calf thymus DNA and their ternary complexeswith Mg2+ and C12TMA, were employed for deduction of the thermodynamic model describing their structural transitions. Small monodis-

perce and highly stable complexes are established after precompaction of DNA with detergent, followed by addition of liposomes. In contrast,divalent metal cation-mediated aggregation of vesicles either leads to heterogeneous multilamellar DNAlipid arrangements, or to DNA-induced bilayer destabilization and lipid fusion. Possible dependence of the cellular internalization and gene transfection efficiency on thestructure and physicochemical properties of DNAMg2+liposomes or DNAcationic surfactantliposome systems is emphasized by propos-ing the structure of their molecular self-organizations with further implications in gene transfer research. 2005 Elsevier SAS. All rights reserved.

Keywords: Zwitterionic liposomesDNA self-organization; Surface active pharmaceuticals; Differential adiabatic scanning microcalorimetry; Phase behavior;Non-viral gene delivery

1. Introduction

The molecular associations in mixed solutions of DNAwith oppositely charged cosolutes-metal cations, cationicamphiphiles and macromolecules, have attracted researchinterest and efforts not only in terms of their physicochemi-cal [13] and pharmaceutical relevance [46], but also due totheir potential for applications in separation, purification andtransfection of DNA [7,8].

Gene transfection is commonly used in biotechnology andhas received considerable attention in biomedicine for curinggenetic diseases [9,10]. Therapeutic gene transfer is achievedby employing viral [11] or non-viral [1214], synthetic[1517] or physical methods [18]. Research on human genetherapy faces certain hurdles due to the lack of suitable deliv-ery tools for therapeutic nucleic acid transfer to target cells.Having considered the risky viral based delivery systems andproblematic physical methods, nowadays much research effortis devoted to the design of artificial non-viral vehicles formedby association of nucleicacids with lipids (lipoplexes) or poly-mers (polyplexes).Whatever the approach is, in both designs,the objective is to achieve a stable packaging of genes to betransfected, to increase transgene expression and to improvetheir bioavailability, while decreasing their cytotoxicity. In

* Corresponding author. G..E.F., The Central Laboratory, Departmentof Pharmaceutical Chemistry, Gazi Mahallesi, Polatli Caddesi, No: 115/5,

Yenimahalle, 06560 Ankara, Turkey. Tel.: +90 312 211 1947; fax: +90 312223 5018. http://erhan.boom.ru.E-mail address: [email protected] (E. Sleymanoglu).

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0014-827X/$ - see front matter 2005 Elsevier SAS. All rights reserved.doi:10.1016/j.farmac.2005.05.010

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this respect, the desired gene packaging becomes a physicalpharmaceutics problem, requiring contributions from physi-cochemically oriented groups.

There is an urgent need for optimized gene transfection

methods capable of protecting the DNA from degradationvia its route to gene expression [9,10]. Among these, lipid-based delivery method, has gained preference(http://www.wiley.co.uk/genetherapy/clinical/) inthelightofthe possibilities for performing simpler quality controls, aswell as easier satisfaction of pharmaceutical requirements.Designing suitable lipoplex formulations requires systematiccharacterization of the DNAlipid complexes as gene pack-aging systems for transfection [19]. From lipid-based experi-mental designs, various monolayer (LangmuirBlodgettfilms)[20], black lipid membranes (BLMs) [21] and liposomal sys-tems [10,13,14,2225] are studied concerning their ability to

bind nucleic acids. Using a broad range of spectroscopic,microscopic, thermodynamic, hydrodynamic, and other rel-evant techniques [26], a useful laboratory data have been col-lected, in search for a details regarding biophysical and col-loidal factors determining the stability of various DNAlipidself-assemblies. Since mainly electrostatic and hydrophobicinteractions govern the formation of these self-assemblies,most of the designs are concerned with colloidal or interfa-cial electrified surface forces. Thus, mixed systems betweenlipids and surfactants can be employed as packaging agentsfor DNA delivery to the affected cells.

Before switching to real in vitro transfection experimentswith numerous gene reporter molecules, it is crucial first to

achieve a stable DNAlipid formulation with controllableproperties. Measurable parameters of potential interest arephase behavior, morphology and structural characterizationof DNA compaction with various condensing agents, suchas, surfactants, charged and neutral polymers, metal ions, aswell as mixtures of cationic andanionic macromolecules, andthermodynamically stable lipid vesicles. Thus, the moleculardetails of cationic lipid binding to DNA polyanion will beclarified. However, despite the collected research data, cur-rently neither the energetics of these associations nor struc-ture of the resultant complex is well understood.

Following the interesting recent results of using cationic,

small membrane-permeant molecules [27], aswellasourowndata on Mg2+ as bridging DNA with liposomes [28] we havefocused on designs involving such agents as rapidly movingthrough model cellular and nuclear membranes. These couldthen bind nuclear DNA with high affinity. In the light of thelatter proposal, we hypothesized that such molecules couldaffect the topology of bound DNA after associating with it soas to enable penetration of model cell membranes by thisbound nucleic acid. Usually, thepositive charge of these smallmolecules would complex with the negatively charged phos-phate groups of DNA, forming a hydrophobic ion pair. Thelatter would then alter the solution properties of the com-plexed DNA by reducing its polarity, thus giving rise tomembrane-permeant agents to trigger DNA transport throughsimulated cell membranes.

To explore new mechanisms for overcoming physicalmembrane barriers to the intracellular delivery of therapeuticnucleic acids (both DNA and RNA) it is worth studying thedesign of non-viral delivery tools that could form thermody-

namically stable colloidal complexes with DNA, thus obvi-ating the need for chemical conjugation of DNA to variousligands. Interestingly, these small cationic molecules wouldbe able to form membrane-permeant ionpairs with the nucleicacid. In this respect, we have already reported our results withdivalent metal cations, capable of complexing liposomes withDNA, followed by attachment to model cellmembrane, desta-bilizing andinternalizing through it, as an alternative lipoplexentry route to target cell via problematic receptor-mediatedendocytic route. In our opinion, such membrane fluidityeffects of various small cations deserve to be studied withrespect to gene delivery vesicle designs, which potentiallycould enhance their transfectionefficiencies, as suggested pre-viously [28]. Hence, it is interesting to compare our previousresults on divalent metal cations with other promising mol-ecules, such as cationic surfactants for instance, which arewell-known gene transfection enhancers [9]. Experimentaldesigns of this sort focus on features such as influence ofvesicle size and dose, surface charge and properties, and sta-bility characteristics, which are to a major extent, thermody-namically governed processes. Unfortunately, little has beendone so far to relate these physicochemical considerations toin vivo behavior of these dispersions.

To understand the energetics of this important system, thethermodynamics of lipid and amphiphile-like ligand binding

to DNA was studied and some preliminary results are pre-sented here. Both naturally occurring lipids and amphiphileresembling model compounds were used, trying to comparethe phase behavior of various ligands and their binding modesand their effect on the energetics of DNAlipid complex for-mation. Assuming that alkylammonium ion andrelated formsform a relevant structure, their phase paramaters were com-pared with 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine(DPPC) concerning affinity for DNA binding, trying to findmore clues from the correlation between DNA compactionand transfection efficiency obtained from previous studies ofDNA associations with lipid dispersions and polycations with

different chain length.Deducing from both theoretical and experimental studieswill improve the current knowledge concerning molecularinteractions and general biophysical chemistryof these prom-ising formulations in terms of designing improved gene deliv-ery systems.

2. Experimental

2.1. Materials

DPPC, SSC (1.5 104 mol/l Na-citrate, 1.5 103 mol/lNaCl, pH 7.2) reagents were purchasedfrom Sigma (St. Louis,MO, USA) and used without further purification. All other

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reagents were of analytical grade. Alkylamine aqueous solu-tions were stored in tightly sealed containers, to prevent theirreactions with atmospheric CO2, as suggested [29]. Lipid andC12TMA solutions were mixed in polyethylene vials in

desired ratios. Following solvent evaporation under nitrogengas flow, the samples were evacuated at room temperaturefor couple of hours. Cationic detergent was dissolved indouble distilled water and was added to the dry lipid, prior tomeasurement.

2.2. Methods

2.2.1. Preparation of polynucleotide solutions

and concentration determinations

Calf thymus DNA with MW of 8.6 MDa (= 13 kb), 42%GC, Tm = 87 C, ~20 A260 units per mg DNA was used. Poli-nucleotide concentrations and molar ratios are based on theaverage nucleotide molecular weight of 308 [28].

2.2.2. Preparation of liposomes

Chromatographic tests for purity of the lipids were notper-formed, however, the purity of the lipid preparation wasassured from the half-widths of their main phase transitions.1.2 mM lipid in standard SSC buffer, pH 7.2 was used in allexperiments and was stored at 4 C.

The formation of a thin layer of lipids of a 15 ml round-bottomed flask was achieved by a hand-shaking and hydra-tion in particular buffer at around 70 C. Vortexing of thelipid with the desired aqueous solution above the gel-to-

liquid crystalline phase transition of the lipid (Tm) for around30 min resulted in multilamellar vesicles.

Unilamellar vesicles (ULV) were obtained by extrusion ofmultilamellar vesicle (MLV) suspension through two stackedpolycarbonate filters (Nucleopore, Inc.) of 100 nm pore sizeat around 60 C. Repeated extrusion (10 times) through theextruder (LipexBiomembranes, Inc.,Vancouver, BC, Canada)created homogeneous vesicle suspension. This allowed thepreparation of vesicles with a mean diameter of 90 nm and atrap volume in the range of 1.52.0 l/mol.

2.2.3. Preparation of liposomenucleic acid mixtures

Nucleic acidlipid mixtures were prepared 1 h beforemicrocalorimetric measurements by vigorousmixing of eitherphosphatidylcholine MLV or ULV dispersions and solvent,varying nucleic acid concentration and keeping DPPC con-centration fixed. Control experiments of DNAlipids in theabsence of detergent or divalent cations, were performed inparallel. Phospholipid concentration employed was0.3 mg/ml.

The lipid samples were hydrated, as described above toformfirst MLV. The preparationof phosphatidylcholine ULVcalf thymus DNA complexes, was the same as in the case ofMLVs, i.e. by mixing DNA solution with aqueous DPPC dis-persion in the presence of Mg2+.

The DNA concentration used throughout all experimentswas 1.8 mM based on the above-mentioned assumption. A

freezethaw protocol was followed to ensure equal distribu-tion of solutes between lamellae and adequate hydration ofthe lipids.

Comparison with the case of liposomal preparations with-

out employing freezethaw procedure showed no differencein terms of homogeneity of the suspension. This was done byplacing the sample in a cryo-tube and freezing it, in liquidnitrogen for around 30 s. The cryo-tube was subsequentlyremoved and was plunged into warm water (~60 C). Whenthe sample was thawed, the whole cycle of freezethawingwas repeated six times.

2.2.4. Differential scanning calorimetry

Calorimetric measurements were performed using Privalovtype high sensitivity differential adiabatic scanning micro-calorimeter DASM-4 (Biopribor, Pushchino, Russian Federa-

tion) with sensitivity higher than 4 10

6

cal/K and a noiselevel less than 5 107 W. Heating runs were performed witha scan rate of 0.5 K/min. The temperature at the maximum ofthe excess heat capacity curve was taken as the transition tem-perature Tm and the transition width DT1/2 was determined atthe transition half-height. The calorimetric enthalpyDHcal ofthe transition was determined as the area under the excessheat capacity curve [28].

3. Results and discussion

The presentedworkdescribes preliminary microcalorimet-

ric measurements on designing and exploiting of surfaceactive molecular assemblies of DNA, liposomes andN-alkyl-

N,N,N-trimetylammonium ions (CnTMA, n = 12) and Mg2+,

as a model supramolecular pharmaceutical formulation com-pacted for therapeutic gene transfection, or alternatively foruse in DNA chromatography. Following the relevant pro-posal to mimic the way of action of much superior viruses, ascompared to non-viral DNA carriers, for improvement of thetransfection efficiencies of therapeutic gene deliveryvehicles,our efforts concentrated on designing such tools, acting viamembrane destabilization and fusion pathway instead ofreceptor-mediated route [30]. The employment of more natu-

rally encountered surfaces is emphasized, as described in ourrecent study [28]. The advantages of using liposomes as com-pared to other lipid-containing drug carrier systems are wellestablished [31]. Having considered the cytotoxicity prob-lems of the currently employed cationic lipids, neutral phos-pholipid vesicles deserve to be studied in more detailas prom-isingalternatives. Our current researchfocuses on a nanoscalecomplex formations between zwitterionic liposomes andDNA, mediated by various inorganic cations, acting as con-densing agents. Our motivation for such an experimentaldesign has come from recent reports on positive effects ofdivalent cations, such as Ca2+ and Mg2+ on transfection effi-ciency [32,33].

The phase behavior of complex formed between neutrallipid and calf thymus DNA in the presence of Mg2+ is pre-

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sented. N-alkyl-N,N,N-trimetylammonium ions were chosenas an amphiphilic system, whose surface properties are to becompared with Mg2+ with regard to ternary complex forma-tion with liposomes and ability to compact DNA. Mg 2+ is

tested as a compaction agent in the presence of phospholipidvesicle suspensions, in the light of its role in numerous cel-lular events. Its high intracellular concentrations, as well asits well-known property of phosphate group transfer [34]makes it preferred natural divalent inorganic cation in com-parison with commonly used synthetic polymers, which offersystem versatility and large selection of polymer species, butare not encountered in biointerfaces. Ion transport, forinstance, takes place by metal binding to cell membranes.Neutral phospholipids are interesting researchsubjectnot onlyin terms of the abovementioned preference of liposome genedeliverydesigners,but also concerning fundamental cell biol-ogy events. Since phosphatidylcholine moeity is a major con-stituent of the total phospholipid bilayer content, it is usefulto study its interactions with various metals. Hence, the sug-gested phosphatidylcholineMg2+ binary mixture would thengive further data on biological implications of metal ion con-trol of cell membrane fluidity. N-alkyl-N,N,N-trimetyl-ammonium ions (C12TMA) ions were selected due to theirinteresting but yet insufficiently studied effects as membranedestabilizing and lipid penetrating agent. Provided its inter-actions with model membranes are characterized suffi-ciently, a synthesis of new relevant quaternary bisammonium

compounds and further studies on their effects on cell sur-faces with biomedical profits will be stimulated [35].

Fig. 1 depicts DSC heating scans of DPPC vesicles andtheir ternary complexes with calf thymus DNA in the pres-

ence of either Mg2+

or C12TMA. The first curve (1) is a cali-bration mark starting with a typical DPPC multilayer phasetransitions, with a pre-transition temperature peak at around36 C with DHcal of 3.9 kJ/mol and the gel-to-liquid crystaltemperature at 41.9 C (Table 1). The determined values arein a good agreement with those reported previously in thelipid database (LIPIDAT): http://lipidat.chemistry.ohio-state.edu. The signal after the lipid phase peaks is a base linewith calibration mark (50 W, DT= 4), obtained during thefilling of both calorimetric cells with solvent.The next curvesshow the change in phase behavior of extruded unilamellarlipids upon their reaction with DNA in the presence of Mg2+

(2) and (3) in various ratios or C12

TMA (4) and (5), respec-tively. Compared withpureDPPC, triple complexesof DPPCULV with DNA and Mg2+ in equimolar ratios possess broaderlipid peak with decreased maximum. The pre-transition dis-appears (Fig. 1 (2, 3) and Table 1). Similar thermogram isobtained for this ternary complex in ratio of 1:3:1, however,with a shift of the second lipidDNA phase to lower tempera-ture at around 70 C. The last endotherm belonging to freenucleic acid melting depicts a difference with that reportedfor plasmids, which show peaks at 60 C attributed to linearand open-circle plasmid DNA. Another peak of such plasmidDNA phase is usually larger, seen at 80 C and correspondsto supercoiled form of the plasmid [36]. This DSC scan of

ternary complexation was preceded first by DPPCULV peakat 41.9 C, by a second peak belonging to DPPCDNAequimolar binary complex at 51.3 C and a third minor peakof unbound DNA appearing at ~60 C. This is a resolutionprofile of the thermograms (2) and (3), which results onlyafter addition of Mg2+.

The observed peak distribution indicates that the fractionof liposome-free DNA is less encountered than the boundDNA in the lipoplex. The liposomeDNA association resultsin the decrease of the DCp. Interestingly, no any DSC signalwas detected for Mg2+DPPC mixture (data not shown),which was observed previously turbidimetrically [28]. Addi-

tion of C12TMA to DPPCULVDNA mixture results inbroadening of the thermograms leading to difficulties of esti-mations of its onset point, peak top, and hence the endother-mic effect. For these reasons, the quantitative evaluation ofthe surfactant effects should be done with precautions andare, therefore, not represented in Table 1. On the other hand,their properties reported here with respect to DNAliposome

Fig. 1. Thermotropic phase behavior of calf thymus DNA in complex withlipid in the presence of C12TMA and Mg

2+, respectively. (1) DPPCMLV;(2) equimolar mixture of DPPCULV with DNA: (3) DPPCULVDNAcomplex by Mg2+ in 1:3:1 ratio; (4) DPPCULVDNA complex in the pre-senceofC12TMA; (5)DPPCULVDNAmixture in thepresence of C12TMAafter heating of the sample; (6) DNAMg2+ binary mixture.

Table 1Thermodynamics of DPPCvesicle binding to calf thymus DNA in the presence of Mg2+ a

DNAlipid mixtures Tp Tm DT1/2 DHcal DHvH RDPPCMLV 36.05 41.9 0.6 31.9 5501 172DPPCULVMg2+DNA (1:1:1 ratio) 41.7 2.35 10.8 1402 129

DPPCULVMg

2+

DNA (1:3:1 ratio) 41.75 1.65 9.7 1998 206a Represents a mean of six independent DSC measurements. Each measurement was performed as described in Sections 2.1 and 2.2. r is estimated as:r= DH

/DHcal.

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condensation is compared with that of Mg2+. This is donesolely for testing the electrostatics and hydrophobicity rea-soning with regard to the thermodynamics of phospholipidbinding to DNA followed by its compaction. Moreover, DSC

is a very sensitive way of studying certain changes in bilayerpacking, with the presence of units interfering with chainpacking in the bilayer causing a decrease in the temperatureof the main transition [37]. The incorporation of a substancein a liposome bilayer has a more profound effect on the lipidphase transitions peaks. The shift or disappearance of pre-transition or main phase transition can thus be used as aninformative indicator of the existence and level of includedmaterials in the leaflets. Thus, the entrapment of moleculesin liposomes can be quantified by determining the change intemperature of the onset of the transition, as well as by mea-suring of the peak temperature as shifts in the transition tem-

perature, rather than presenting them as abstract values(Table 1).The DSC scan (4) ofFig. 2, represents a thermogram of a

complex formed between DPPC and DNA in the presence ofC12TMAions. It is interesting to study assemblies of this type,since surfactants are used forextraction of proteins from phos-pholipid membranes [38], which could also appear as nucleicacidprotein complexes, specifically or non-specificallyattached to cell membrane fractions. These interactions canbe approached by thermodynamic measurements. It is worthinvestigating their binding characteristics, as drugs usuallyform supramolecular complexes with proteins, membranephospholipids and nucleic acids (DNA and RNA), and their

subsequent release depends on the strength of binding and onthe reversibility of this interaction. The evaluation of physi-cochemical stability of the samples with respect to tempera-ture variations is crucial due to their thermodynamic profilesin terms of manufacturing issues, as recommended also forother relevant multiple emulsions [39]. Moreover, since theenthalpy of binding is a temperature dependent event [29],the effect of heating of the sample is emphasized (Fig. 1 (4)

vs. (5). The single melting peak corresponding to the ternarycomplex formed between DPPCC12TMADNA appears atapproximately 33 C. The melting behavior of the controlsample DNAC12TMA binary mixture (Fig. 2 (1)) is com-

posed of two separate phases. The first situated at 47 Cbelongs to DNA phase, the separate meltingbehavior of whichis shown with two different concentrations on Fig. 2 (2) and(3), respectively. The second phase is a structural transitionseen after 85 C and continues until 100 C, corresponding tocomplex of DNA bound to C12TMA. This type of meltingbehavior indicates the occurrence of aggregation reactionbetween the latter molecules. While, large enthalpy varia-tions with temperature are compensated by the hydrophobiccomponent of entropy, it is possible to estimate electrostaticand hydrophobic contributions to the enthalpy at various tem-peratures by applying additivity, as recently shown for alky-lammonium binding to DNA by isothermal titration calorim-etry [29]. Fig. 2 (2) is a DNA sample, prepared as describedin Sections 2.1 and 2.2, while (3) is the same DNA sample indoubled concentration. These two thermograms show a typi-cal melting behavior of DNA and its synthetic models, whichin calorimetric determinations appear as a single peak. Thishighly cooperative processrepresents unwinding of the doublestranded structure into two polynucleotide strands, which foldinto separate chaotic globules. Theequalityof theheat capaci-ties of the native and denatured DNA states is a typical fea-ture of this process, which makes quantitative determinationof thermal effects of melting, as well as free energy DG ofstabilization of native DNA structure easier. Thiskind of struc-

tural transitions of DNA (Fig. 2 (2) and (3) are different fromthose of lipid phase behavior (Fig. 1 (1)). Lipids undergoanother type of structural transitions, or gel-to-liquid crystal-line transitions, which is also their main phase transition. Inthis respect, these are different from the abovementionedevents in that in phospholipids the degree of cooperativity oftheir main phase transitions entirely depends not on the innermolecular but on interactions between molecules. For suchsystems, the effectiveVant Hoff enthalpy is greater thancalo-rimetrically determined. In general, the degree of cooperat-ivity of the is given as: DHvH = NDH, where Nis the numberof molecules in the cooperativity unit. Testing this kind of

dependence on DNA concentration is crucial, as also shownby another relevant thermodynamic study [29]. In agreementwith this reminding and with the mathematical model pre-sented in the latter study, the prediction is that the position ofthe binding peak would be shifted upon changing the DNAconcentration. Therefore, it is important to perform micro-calorimetric scans at several nucleic acid concentrations atpreviously prepared DNAligand ratios. This is also seen forDPPCMg2+DNA ternary complex in equimolar and1:3:1 ratios, respectively (Fig. 1. (2) and (3)). DNA sampleexaminedin two different concentrations already shows simi-lar melting behavior, but with various peaks (Fig. 2 (2) and(3)), which supports the expectation of dependence of theresults on nucleic acid concentration used. The lower DNAconcentration in lipoplexes results in a little bit prolonged

Fig. 2.

Phase behavior of thecontrolsamples used: (1)DNAC12TMA binarymixture; (2) DNA in concentration used as in Sections 2.1 and 2.2; (3) DNAin doubled concentration; (4) DPPCULVC12TMA binary mixture.


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second peak, as compared with that of higher DNA amount(Fig. 1 (2) vs. (3)), as also indicated by the experimentalenthalpies. However, to relate this effect to transfection effi-ciency of these lipoplexes still remains to be elucidated. Inter-

estingly, this effect is less seen in the case of C12TMADNAbinary mixtures, where the most reproducible results wereobtained for their ratio of 11.8. In this case, the effect ofheating and aggregation is more profound (Fig. 1 (4) and (5)and Fig. 2 (1)). Such dependence of the electrostatic compo-nent of binding Gibbs free energy and entropy on reactantconcentration suggests the existence of an aggregation reac-tion, which was also observed in DNAlipid complexes[28,29].

The Mg2+-ions at the equimolar amounts with DNAincreases the Tm value by 33.7 C, due to Mg

2+-inducedduplex stabilization. ULVs treated with the same Mg2+ con-

centration did not produce such a shift, which is normallydetected spectroscopically [28]. Mg2+ induces the formationof substantial amount of circular DNA, suggesting that Mg2+

cations stabilize the interaction of polynucleotide cohesiveends, the effect being dependent on the concentration ofMgCl2 and possibly being a sequence-specific event [40]. Theformed circular molecules are stabilized by Mg2+, butare notcovalently closed.Although, Mg2+ stabilizes end-to-end inter-actions, it is likely that a dynamic equilibrium exist betweenlinear and circular fragments.

C12TMA ions cause a decrease in lipid ordering by exert-ing a perturbation effect on the lipid bilayer structure. Trim-ethylammonium ions possess an intermediary position among

CnTMA homologues, in terms of lipid ordering, as demon-strated by recent ESR studies [41]. This is characteristicbipha-sic behavior of these alkylammonium series, similar to otheramphiphilic compounds, suggesting that the interaction ofC

nTMA molecules with lipid membranes has important bio-

logical implications. Moreover, other relevant ions, such asbis-quaternary ammonium ions (bis-A2+), e.g. alkane-bis-alkylammonium ion, which is a divalent organic ion with twopositively charged sites connected by a spacer chain and hav-ing also six side chains, has attracted research interest notonly due to its biological properties as ion channel blocker ofacetylcholine receptors, but also as a model compound for

studying the cation effects on hydration of hydrophobic cat-ions. In this respect, it is argued that if its hydrophobicitycould be controlled precisely, it could have had a more sig-nificant value in a two-phase system, such as a membrane,indicating the importance of evaluating various alkylammo-nium species with different structures concerning this fea-ture. C12TMA ions possess a hydrophilic positively chargedammonium group and a hydrophobic alkyl chain of 12 CH2groups. Due to this amphiphilic character these ions partitionbetween aqueous and lipid phases. However, the observedcut-off effect of the whole series of alkylammonium ions indecreasing lipid order cannot be the result only of partitionequilibrium between these two phases. This is also a concen-tration dependent effect possibly caused by lateral bilayerexpansion due to positioning of such amphiphilic ions in the

region of the lipid leaflet, exerting an additional effect ofamphiphile partitioning between aqueous and lipid phases[41]. Hydrophobically driven insertion of C12TMA ions intothe phosphatidylcholine lipid layer brings about attractive

electrostatic forces for DNA polyanion. In this respect,C12TMA ions affect the phospholipid vesicle in a similar wayofMg2+ adsorption onto their surface (Fig.3) creating a posi-tive liposomal interface, which can also be detected by theirelectrophoretic mobility measurements [42].

The structure of the interacting biopolymers in lipoplexformulations is a matter of controversy. Research evidenceindicates that both DNA [43] and lipids [44] affects each oth-ers structural transitions during complex formation. Most ofthe interpretations for this self-assembly in terms of well-established polyelectrolyte theories for interactions betweenoppositely charged macromolecules are an oversimplified

view of the real structures.It is likely that structures similar to cationic lipidDNAcomplexes are formed [35,45]. In ouropinion, kinetic vs. ther-modynamic stability features govern this self-association inmore complex way. Within this respect, the model considersthe lipidDNA structures as overlaying layers of DNAadsorbed onto lipid bilayers, after charge neutralization [28].The process is governed by adsorbed cations (Me2+) on thesurface of the zwitterionic lipids. The presence of such mul-tilayers of alternating lipidDNA assemblies is due to forma-tion of condensed DNA as parallel arrangements between thelipid bilayers [46]. This is expected, due to the existence of3-D correlation forces between the DNA-covered lipid lay-

ers, following DNA-driven formation of multilamellar lipo-somes from ULVs. Based on our own polyelectrolyte data, aswell as on relevant measurements reported in the literature,Fig. 3 represents a proposal of the possible structures ofC12TMADNAliposomes and DNAMg

2+-neutral lipo-somes ternary complexes.

Fig. 3a suggests the possible structure of DNAC12TMAliposome complexes. Under the employed conditions, the ini-tially relaxed DNA in solution is complexed by surfactantmolecules, which adsorb on the nucleic acid surface forminga, micelle-like domains. C12TMA molecules also partition inlipid bilayer forming a swollen mixed bilayers. The latter can

also lead to subsequent humpbacked vesicles with surfactantat regions of high curvature.Afterwards, theDNA in unfoldedform is apparently adsorbed on the surface of surfactantDPPC vesicles, as also suggested by a fluorescent study onneutral lipids, employing cationic surfactant [47]. Since sur-factant molecules become incorporated into the liposomebilayer due to the partition equilibrium between bilayer andaqueous phase, normally thebinding of C12TMADNA com-plexes to the vesicle surface through hydrophobic forcesresults in opening of the micelle-like domains and partition-ing of C12TMA ions in the lipid bilayer. Hence, employmentof cationic surfactant tend to form a fully relaxed DNA, whichis bound with significant stability to plasma membranes,resulting in a difficult to internalize in a cell structure byendocytosis. In contrast, unilamellar phosphatidylcholine


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clearance rate than negatively and positively charged MLVs.ULVs are characterizedwith longer residence timethanMLVs[9].

The unavoidable hurdle until now has been the final fate

of liposome, which is engulfed by macrophages of the RES,via the lysosomal participation. Such fusion with lysosomesleads to destruction of liposomes through the action of phos-pholipases with subsequent release of vesicles content.Hence, efficient strategies to overcome opsonization of lipidvesicles have been designed, thus developing the concept ofliposome targeting to specific target cells and tissues.

Initially, conjugation of various polymers and other mac-romolecules for this purpose was highly cell type-dependentand ended with discouraging results. Therefore, besides therequired knowledge on the influence of in vivo environmenton both vesicle leakage and clearance rates, searching for

improvement of the specificity of cell and tissue recognitionbecomes essential. The objective is to deduce alternative routeof successful gene delivery avoiding the receptor-mediatedintake. With this respect, we have worked on electrostatic andhydrophobic control of membranedestabilization. Despite thefact that often the fluidity of the phospholipid surface isdescribed as disordered phase, a certain level of interfacialorganization or lateral heterogeneity is present due to multi-lamellar nature of the vesicles with high degree of cooperat-ivity. Hence, knowledge of its surface structure is crucial fordesigning efficient lipid-based genedelivery systems and sug-gesting new clues on their interactions with cellular surfaces,as well as internalization mechanisms of the entrapped thera-

peutic DNA through membranes. Although increased sur-face rigidity prolongs the circulatory half-time of lipidvehicles, the commonly employed phospholipids in drugdelivery are at physiological temperature entirely or partly inthe fluid state [52]. For proper understanding of the relation-ships between surface heterogeneity and lipid-carrier func-tion it is important to distinguish between various levels oflipid lateral organization differing in their lifetimes and sizes.Lipid heterogeneity is a consequenceof solidliquid or liq-uidliquid phase separation, as well as by thermal densityfluctuations leading to the appearance of short lived gel-likemicrodomains in a fluid (liquid-crystalline) environment

(Fig. 3). Their appearance implies function of low-orderedboundary regions, which in spite of their short lifetime andsmall size serve as sites of ionpenetration andenzymatic reac-tions. They also may serve as nucleation centers precedinggross lipid phase separation induced by cations and arebelieved to play a role in fusion of the lipid carriers with aplasma membrane of living cells [52].

Numerous suggestions have been proposed for overcom-ing the membraneous barriers, which significantly inhibit theentry of therapeutic DNA to the nucleus. Certain cationicamphiphiles, such as those presented, able of free partition-ing through cell membranes and rapidly localizing to specificnuclear chromosomal regions are being studied for their car-rier potential [53]. The proposal that if such nucleic acid bind-ing agents express their membrane transport properties cre-

ating a more hydrophobic DNA, then they could facilitatesurpassing the membrane barriers that currently limit DNAdelivery into the nucleus by non-viral vectors, remains to betested.

There are several potential ways, though which metal ionscan increase gene transfection efficiency. Thus, their abilityto partition rapidly through cell membranes entering thenucleus may confer novel intracellular trafficking pathwayson complexed DNA. Fig. 4 shows the simplified possibilityof the proposed DNA-mediated liposome fusion with targetcell membranes. The suggested structure of the entrappedDNA [28] is based on Fig. 3b, originally described byKuvitchkin and Suchomudrenko (1987) [54]. The modeldescribes theaggregation of several vesicles resulting in fusionof ULVs, induced by polynucleotide chain unwinding. Thus,the desired highly fusogenic vesicle with higher curvature is

formed. Mg

2+

used, bridge the DNA polyanion to chargereversed liposomes accelerating further their membrane desta-bilizing properties.

We hypothesized that based on these features of Mg2+ as asurface active compound, similarly to cationic peptides, aninorganic cation-mediated cell membrane destabilizationcould occur, governed by electrostaticandhydrophobic forces.As shown on Fig. 4, Mg2+ bring the neutral lipid vesicleswith the encapsulated nucleic acid in close proximity withnegatively charged target cell surface. When optimal combi-nation of surface factors needed for bilayer destabilization is

Fig.4. Proposedmechanism of Mg2+-inducedcellular internalization of neu-tral liposomeDNA formulation.


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reached, coupled with suitable amphiphilicity, the DNA-mediated fusion between zwitterionic liposomes and cellmembrane occurs. This proposal does not, however, rule outthe possibility of existence of another cellular internalization

mechanism of neutral liposomeDNA complexes. Neverthe-less, the described ability of free metal cations for both DNAand lipid binding, partitioning and permeating through cellmembranes entering the nucleus [31,32], coupled with accel-erated nuclear delivery of DNA, supported by helix-bindingmolecules [27], is consistent with the hypothesis that diva-lent metal cations can confer membrane-permeant propertieson complexed DNA. The exact mechanism of nucleic acid-exerted phospholipid fusion in the presence of metal cationsis unknown. One possibility could be, that similarly to cat-ionic peptides [30], the electrostatic screening of the hydra-tion shell of inorganic cations compensates for the inter-vesicle repulsive forces. Once the opposing membranes arein close contact zone, the hydrophobic and electrostatic fea-tures of metal cation form a complex between liposome andcell phospholipids followed by lipid mixing (Fig. 4).Ourpro-posal is further supportedby recent results of Sato et al. (2003)[55] on Mg2+-induced DNA attachment to phospholipids. Inaddition, closely relevant work of describes DNAMg2+

nanoscale mixtures as potential non-viral vector for genedelivery [56].

Besides their membrane permeating properties, Mg2+-ions employed could also increase the transfection efficiencyin other respects. Nuclear stability and topology of genomicDNA seems to be onecellular mechanism forcontrollinggene

transcription [27]. Metal cations may target complexed DNAvia nuclear localization signals (NLS) to transcriptionallyactive chromatin regions.Thus, genecarrier-mediated nucleartargeting could representa way of enhancing thegene expres-sionlevels. This suggestion is in agreement withrecent reportsonCa2+ andMg2+-induced increaseof expression of the trans-fected genes [31,32]. Metal cations can govern the conforma-tion of the transfected DNA, showing the role of nucleic acidtopology control in gene transfection. Employment of natu-rally occurring divalent metal cations as oppose to other syn-thetic and potentially mutagenic molecules [27], deserves tobe studied in more detail.

4. Conclusions

Neutral liposomeMg2+DNA formulations described hereare highly dynamic structures, frequently changing their trans-fection characteristics, depending on particular laboratorypro-tocol. On the other hand, these ternary complexes appear tobe a reproducible lipoplex design, but their serum stabilityprofiles are not yet well defined. While promising results arereported recently on their use [57], more immunological stud-ies are needed for their in vivo evaluation. Nevertheless, theconcept of metal-based pharmaceuticals [58], undertakenhere, will open new insights for studying whether thesesupramolecular complexes follow the similar principles of

binding to cellular receptors and will further define the issueof nucleic acid receptors on cell surfaces.

Acknowledgments

I thank Professor R.I. Zhdanov (V.N. Orekhovich Instituteof Biomedical Chemistry, Russian Academy of Medical Sci-ences) for introducing me to the topic of nucleic acidmembrane interactions and for his help as my postgraduatesupervisor. The hospitality of Professor P. Blgavy (J.A.Comenius University-Bratislava) and Dr. J. Bagelova (Insti-tute of Experimental Physics, The Slovak Academy ofSciences-Koice) is greatly acknowledged.

References

[1] R. Chang, Physical Chemistry for the Chemical and Biological Sci-ences, Chapter 16, Intermolecular Forces, University Science Books,Sausalito, California, 2000 (pp. 669700).

[2] D. Leckband, J. Israelachvili, Intermolecular forces in biology, Q.Rev. Biophys. 34 (2001) 105267.

[3] J.B.F.N. Engberts, M.J. Blandamer, Understanding organic reactionsin water: from hydrophobic encounters to surfactant aggregates,Chem. Commun. (2001) 17011708.

[4] A.T. Florence, D. Attwood, Physicochemical Principles of Pharmacy,third ed, Palgrave, 1998.

[5] E.H. Kerns, L. Di, Physicochemical profiling: overview of thescreens, Drug Discovery Today, Technologies 1 4 (2004) 343348.

[6] P.J. Crowley, L.G. Martini, Formulation design: new drugs from old,

Drug Discovery Today, Therapeutic Strategies 1 4 (2004) 537542.[7] A. Simmonds, M. Cunningham, The preparation of proteinnucleic

acid conjugates, in: M. Aslam, A. Dent (Eds.), Bioconjugation: Pro-tein Coupling Techniques for the Biomedical Sciences, MacmillanReference Ltd, 1998, pp. 483503.

[8] P.-G. de Gennes, Problems of DNA entry into a cell, Physica A(Amsterdam) 274 (1999) 17.

[9] A.J. Kirby, P. Camilleri, J.B.F.N. Engberts, M.C. Feiters,R.J.M. Nolte, O. Sderman, M. Bergsma, P.C. Bell, M.L. Fielden,P. Garca Rodrguez, A. Gudat, C. Kremer, C. McGregor, G. Perrin,M.C.P. Ronsin, van Eijk, Gemini surfactants: new synthetic vectorsfor gene transfection, Angew. Chem. Int. Ed. Engl. 42 (2003) 14481457.

[10] N.S. Templeton, Gene and Cell Therapy-Therapeutic Mechanismsand Strategies, second ed, Marcel Dekker, 2003.

[11] N.A. Kootstra, I.M. Verma, Gene therapy with viral vectors, Annu.Rev. Pharmacol. Toxicol. 43 (2003) 413439.

[12] R. Dass, Cytotoxicity issues pertinent to lipoplex-mediated genetherapy in-vivo, J. Pharm. Pharmacol. 54 (2002) 593601.

[13] A.D. Miller, The problem with cationic liposome/micelle-based non-viral vector systems for gene therapy, Curr. Med. Chem. 10 (2003)11951211.

[14] A.D. Miller, Nonviral liposomes, Methods Mol. Med. 90 (2004)107138.

[15] A.V. Kabanov, V.A. Kabanov, DNA complexes with polycations forthe delivery of genetic material into cells, Bioconjug. Chem. 6 (1)(1995) 720 (JanFeb).

[16] C.L. Gebhart, A.V. Kabanov, Evaluation of polyplexes as gene trans-fer agents, J. Contr. Releas. 73 (2001) 401416.

[17] K.B. Anwer, G. Rhee, S.K. Mendiratta, Recent progress in polymericgene delivery systems, Crit. Rev. Ther. Drug Carrier Syst. 20 (4)(2003) 249293.


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