Evidence for the role of hydrophobic forces on the interactions of nucleotide-monophosphates with cationic liposomes

Journal of Colloid and Interface Science xxx (2013) xxx–xxx

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Journal of Colloid and Interface Science

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Evidence for the role of hydrophobic forces on the interactionsof nucleotide-monophosphates with cationic liposomes

0021-9797/$ - see front matter � 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.jcis.2013.08.013

Abbreviations: DOTAP, 1,2-Dioleoyl-3-Trimethylammonium-Propane; DOPE,1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine; DLS, dynamic light scattering;NMP, nucleotide-monophosphates; AMP, adenosine 50-monophosphate adenosine50-monophosphate disodium salt; GMP, guanosine 50-monophosphate; TMP,thimydine 50-monophosphate; UMP, uridine 50-monophosphate.⇑ Corresponding author. Address: Dipartimento Agricoltura, Ambiente Alimenti

(DIAAA) and Consorzio Interuniversitario per lo Sviluppo dei Sistemi a GrandeInterfase (CSGI), Università del Molise, via De Sanctis, I-86100 Campobasso, Italy.Fax: +39 0874404652.

E-mail address: [email protected] (F. Lopez).

Please cite this article in press as: F. Cuomo et al., J. Colloid Interface Sci. (2013), http://dx.doi.org/10.1016/j.jcis.2013.08.013

Francesca Cuomo a, Monica Mosca a, Sergio Murgia b, Pasquale Avino c, Andrea Ceglie a, Francesco Lopez a,⇑a Dipartimento di Agricoltura, Ambiente Alimenti (DIAAA) and Consorzio Interuniversitario per lo Sviluppo dei Sistemi a Grande Interfase (CSGI), Università degli studi del Molise,I-86100 Campobasso, Italyb Dipartimento di Scienze Chimiche e Geologiche, Università di Cagliari, CNBS e CSGI, s.s. 554 bivio Sestu, I-09042 Monserrato (CA), Italyc DIPIA–INAIL ex-ISPESL, via Urbana 167, 00184 Rome, Italy

a r t i c l e i n f o a b s t r a c t

Article history:Received 11 July 2013Accepted 2 August 2013Available online xxxx

Keywords:Nucleotidesf-PotentialBindingLiposomesMolecular interactions

In this work, the interaction of nucleotide-monophosphates (NMPs) with unilamellar liposomes made of1,2-Dioleoyl-3-Trimethylammonium-Propane (DOTAP) and 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanol-amine (DOPE) was investigated. Here, we demonstrate how adsorption is affected by the type of nucle-otide-monophosphate. Dynamic light scattering (DLS) results revealed, for each NMP, that adistinguishable concentration exists at which a significant growth of the aggregates occurs. Adenosine50-monophosphate (AMP) and guanosine 50-monophosphate (GMP) have shown a higher propensity toinduce liposome aggregation process and in particular GMP appears to be the most effective. From f-potential experiments we found that liposomes loaded with purine based nucleotides (AMP and GMP)are able to decrease the f-potential values to a greater extent in comparison with the pyrimidine basednucleotides thimydine 50-monophosphate (TMP) and uridine 50-monophosphate (UMP). Moreover, acareful analysis of nucleotide-liposome interactions revealed that nucleotides have different capacityto induce the formation of nucleotide-liposome complexes, and purine based nucleotides have higheraffinities with lipid membranes. On the whole, the data emphasize that the mechanisms driving theinteractions between liposomes and NMPs are also influenced by the existence of hydrophobic forces.

� 2013 Elsevier Inc. All rights reserved.

1. Introduction

Liposomes are colloidal particles built up of lipid molecules thatself-assemble in aqueous media into spherical, self-closed struc-tures [1]. Cationic and neutral lipids are typically used for genedelivery and extensively used as in vitro DNA transfection vectors[2,3]. The liposome-based method represents the most studiedtechnology in the area of non-viral vectors [4,5], and a large bodyof data has been already collected in the field of drug deliverymaterials based on cationic liposomes [6–8]. Cationic liposomes in-deed have shown to be able to complex and condense DNA and are

widely used for understanding interactions occurring betweenmolecules and biological membranes [9–13].

The role of the positively charged head group is well recognizedas crucial for binding and complexation of the anionic phosphategroups of the nucleic acid [14], nevertheless, beside the electro-static attraction, the occurrence of hydrophobic forces on the for-mation of oligo/polynucleotide-based lipoplexes was recentlyemphasized [15].

The presence of a net positive or negative charge on the lipo-some surface ensures the formulation stability, a characteristicthat is gradually lost when the surface charge gets close to the neu-trality thus promoting aggregation and fusion phenomena. Accord-ing to the DLVO theory, the stability is governed by the balance oftwo independent types of forces ruling the interaction betweensimilar colloidal particles immersed in aqueous solutions: attrac-tive van der Waals forces and repulsive electrostatic forces. Thistheory analyzes the effects of the van der Waals attraction andthe electrostatic repulsion due to the double layer of counterions[16]. More recently, for the role played by the charge density ahuge amount of new experimental data is available [17,18].

Despite the enormous quantity of information concerning theforces taking part in the interactions between several molecules

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and lipid bilayers this issue still presents the need of fundamentalstudies for a deeper understanding [19,20].

This kind of investigation can be tackled at a minor level ofcomplexity by means of nucleic acid monomers: as a matter of fact,it is expected that the interactions occurring between a cationicliposomes and negatively charged nucleotide-monophosphates(NMPs) should lead to the formation of lipid-mononucleotide com-plexes. The phenomenology of anion–cationic liposome interactionis relatively intricate, depending on a number of parameters suchas the charge density on the particle surface, the ion nature andthe physical–chemical properties of the medium [21]. Kikuchiet al. demonstrated the importance of the hydrophobic forces inthe insertion process of the DNA momomer 20-deoxyadenosine50-monophosphate in cationic liposomes made up of dioctadecyl-dimethylammonium bromide (DODAB) [22]. Later on, the abilityof the nucleotide-monophosphate AMP to induce the aggregationof DODAB vesicles was revealed [23]. Recently, the existence ofother sort of interactions between the lipids and the mononucleo-tides has been demonstrated in a study where the nucleotideswere entrapped inside monoolein-based liquid crystals. It was ex-plained that a recognition process occurs through of the OH groupsof the mono-olein and that, after aging, the mononucleotidesunderwent hydrolysis at the sugar-phosphate ester bond [24].The evidence of interactions with non charged surfaces is a mean-ingful aspect for our investigation because it is a proof of additionalnon-electrostatic interactions taking place at lipid interfaces.

Furthermore the importance of the interactions occurring be-tween the cationic interfaces and nucleotide-monophosphates(NMPs) was recently reported by us through the use of the hexade-cyltrimethylammonium bromide aqueous micellar solution[25,26]. With these studies we demonstrated that although similarin chemical structure, nucleotide-monophosphates influenced thefluorescence of pyrene, secluded in the micelle, in completely dif-ferent ways. The results suggested different roles of the nitroge-nous rings on the response of the pyrene fluorescence tonucleotides loading. The negatively charged phosphate groupdrives the adsorption of NMPs to the micellar surface and leadsto the saturation of the photophysical effects while the specificmoieties of the nucleobases (in the peculiar environment of micel-lar surface) dictate the phenomenology of the fluorescent responseas a consequence of the different behavior toward the cationicsurface.

Given that the net electrical charge of aggregates is a parameterthat strongly affects their physico-chemical behavior, the use ofthe f-potential parameter, defined as the net charge of surfacesincluding bounded ions, could add important information to thisissue [18]. f-Potential has been used to measure the electric chargeon the liposome surfaces, a parameter that affects the stability ofthe formulation [27].

Although it is well known that charge interaction plays a veryimportant role in the complex formation of liposomes and nucleo-tides, the utilization of the f-potential technique for these purposesis still relatively uncharted [28]. For the reasons mentioned, astudy on the affinity of mononucleotides for the liposome wallscould be useful for the analysis of the compaction behavior of poly-nucleotides and eventually for the control of the electrostatic inter-actions between these lipids and DNA.

The goal of the present study was to elucidate the interaction ofnucleotide-monophosphates to lipid membranes. For this purposewe have focused on the association behavior of liposomes andNMPs. Two purine based monophosphates AMP and GMP andtwo pyrimidine based monophosphates TMP and UMP have beenselected. Here, we show how f-potential can be used to studythe binding of nucleotide-monophosphates (NMPs) to a cationic li-pid bilayer. The evolution of size and surface charge of the com-plexes formed between the nucleic acid monomers and the

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cationic liposomes were monitored as the anionic molecules wereadded to a fixed concentration of liposome suspension. A directevidence for the interaction of NMPs was evaluated by the changesin the size value. The binding analysis was done by determiningthe changes of the f-potential surface complex during the titrationprocedure.

2. Materials and methods

2.1. Materials

AMP (P99%, disodium salt), GMP (P99%, disodium salt), UMP(>99%, disodium salt), TMP (P99%, disodium salt) and HEPES, werefrom Sigma. The cationic 1,2-Dioleoyl-3-Trimethylammonium-Propane (DOTAP), the neutral 1,2-Dioleoyl-sn-Glycero-3-Phospho-ethanolamine (DOPE) were purchased from Avanti Polar Lipids,Inc. Alabaster, AL. The water used was from a Milli-Q filtration sys-tem (Millipore).

2.2. Sample preparation

Large unilamellar liposomes were prepared by reverse phaseevaporation, according to the method described by Szoka and Pap-ahadjopoulos [29]. DOTAP and DOPE were mixed (2:1 M ratio) in20 mM HEPES pH 7.4, 50 mM NaCl to achieve a final lipid concen-tration of 15 mg/mL. Liposomes were then extruded through100 nm polycarbonate membranes to obtain small unilamellar lip-osomes [30]. NMP-liposomes complexes were prepared in 5 mMHEPES adding several amounts of stock solution of NMPs to a con-stant volume of monodispersed liposome solution (final concentra-tion 0.75 mg/mL). The final NaCl concentration was 0.25 mM.

2.3. f-Potential and DLS

The measurements of size and f-potential were performedusing a Zetasizer ZS Nano (Malvern, Malvern, UK). For the sizedetermination the light scattering was detected at an angle of90� with a laser He–Ne operating at the wavelength of 633 nm.The working temperature was kept constant at 25 �C with a peltierelement integrated in the apparatus. DLS autocorrelation functionsof the scattered light intensity were carried out with DTS 5.0 soft-ware provided by the manufacturer, which allowed the measure-ment of the distribution of the scattered intensity versus thehydrodynamic diameters. For the measurement of f-potential theelectrophoretic mobility of the aggregates was determined by laserDoppler velocimetry. The samples were placed in dedicated dis-posable capillary cells. The cells were calibrated before each setof measurements with a latex standard solution (�50 mV ± 5 mV).The f-potential values were calculated by the Smoluchowskiapproximation of Henry’s equation [31]. Samples of liposomes con-taining variable NMPs concentrations were injected in dedicateddisposable capillary cells.

2.4. Binding analysis

The binding constants K of NMPs molecules to liposomal bilayerwere determined by the f-potential measurements. The changes inf-potential of the solution containing NMPs at the concentrationranging from 0.25 to 9 mM and liposomes at constant lipid concen-tration of 10 mM were followed in HEPES at pH 7.4. K was then cal-culated according to Langmuir–Freundlich isotherm equation [32].The fraction of coverage of a surface by NMPs can be described byratio: a = A/Amax where A is the amount of adsorbant at a given con-centration in the bulk solution and Amax is the maximum amountof adsorption. This ratio can be expressed as a function of the

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F. Cuomo et al. / Journal of Colloid and Interface Science xxx (2013) xxx–xxx 3

adsorbant concentration [A] in the solution in contact with themembrane by a Langmuir–Freundlich isotherm as:

a ¼ KAð Þn

1þ KAð Þnð1Þ

where K is the Langmuir adsorption constant and increases with anincrease in the binding energy of adsorption and n is the heteroge-neity parameter. The value of a can be determined by the changes inthe f-potential of the liposomes due to the presence of nucleotides[33]:

DfDfmax

¼ K � NMPð Þn

1þ K � NMPð Þnð2Þ

3. Results and discussion

The first parameter considered in this investigation is the vari-ation of dimensions of the anion-liposome complexes induced bythe presence of the nucleotide-monophosphates. Fig. 1 shows thesize evolution and the polydispersity index (PDI) as a function ofthe mononucleotide concentrations increase. The polydispersityindex represents the width of the distribution, measured as the ra-tio between the first and the second cumulants. PDI values close tozero indicate that the sample is monodisperse. When aggregationoccurs the polydispersity index tends to one. The changes causedby the two purine monophosphates (AMP and GMP) and the twopyrimidine monophosphates (TMP and UMP) on the liposomes

Fig. 1. Diameter size (full circles) and PDI values (empty circles) of DOTAP/DOPEliposomes as a function of NMPs concentration. From the upper to the lower panelthe data and the chemical structures of AMP (red), GMP (blue), UMP (green) andTMP (black) are reported. (For interpretation of the references to color in this figurelegend, the reader is referred to the web version of this article.)

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surface, followed by means of DLS, gave rise to noticeable differ-ences in the concentration of nucleotide inducing the aggregation.The graphs reported in Fig. 1 show that no significant size increaseof the aggregates was detected upon the addition of UMP and TMPup to the concentration of 3 mM and 4 mM respectively. Thedimensions were, indeed, stable and approximately close to110 nm for concentrations below the above mentioned values.On the contrary upon the addition of the purine based nucleotidesAMP and GMP, an early constant size rising process takes place(from 100 nm to 200 nm) even in the very low ion concentrationrange (0.125–1 mM).

All the studied mononucleotide–liposome complexes allow theidentification of a distinguishable concentration which corre-sponds to the amount of anions that leads to a significant growthof the aggregate dimensions. The importance of this characteristicpoint was also highlighted from the PDI data. As shown in Fig. 1 forthe pyrimidine based nucleotide the PDI values remain constantabove, 0.1, indicating a small heterogeneity of particle size distri-butions and started to get higher at a defined amount of NMPsadded. On the contrary for AMP and GMP the increase of PDI beginsimmediately at low ion concentrations.

In general the dramatic increase of the liposome dimension inthe presence of anions is determined by self aggregation processof the colloidal particle and it is determined by the liposomes/ioncharge ratio [34]. This means that the aggregation can occur whenelectrostatic repulsions between particles are reduced throughbinding of counterions to the charged lipid surface [35].

To facilitate the following discussion the values of the amountof nucleotides matching the intense growth of the aggregatedimensions were calculated as lipid/NMP ratio. The concentrationvalues of NMP were selected from the size data as the value corre-sponding to a significant size increase (at least twice of the startingsize value) and the ratio values obtained are reported in the secondcolumn of Table 1.

From the data analysis made by keeping constant the lipid con-centration (10 mM), the following sequence can be written:GMP(6.6) > AMP(5) > UMP(2.8) > TMP(2.2). The data presented sofar demonstrate that both AMP and GMP have higher tendencyto induce the aggregation process of the resulting complexes com-pared to UMP and TMP, and GMP is the most effective among thenucleotides considered, in promoting the aggregation. These re-sults suggest that, besides the charged phosphate groups, thereare other interactions involved in the formation of complexes be-tween liposomes and mobile ions. Therefore relevant insights intothe nature of NMP-liposomes interactions must be gained by anelectrokinetical method.

In Fig. 2A f-potential profiles of the cationic liposomes systemsin the presence of the NMPs at different concentrations are shown.By mixing the negatively charged mononucleotides molecules andthe positively charged liposomes a reduction of the f-potential val-ues can be detected. From this graph two different behaviors canbe observed: on the one hand, the liposomes loaded with purinebased nucleotides promote a decrease of the f-potential at lowconcentration. A decrease of f-potential was detected from 58 to

Table 1Lipid/NMP molar ratio values calculated from size data. The parameters K (Langmuiradsorption constant), n (heterogeneity) and R (correlation coefficient) are obtained byfitting the f-potential values to Eq. (2).

Lipid/NMP molar ratio K (mM�1) n R

AMP 5 2.84 (±0.24) 1.56 (±0.18) 0.992GMP 6.6 2.84 (±0.19) 1.74 (±0.18) 0.994TMP 2.2 1.60 (±0.05) 1.74 (±0.08) 0.998UMP 2.8 1.15 (±0.08) 1.18 (±0.11) 0.995

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Fig. 2. (A) Changes of f-potential upon the addition of NMPs to cationic liposomes. (B) Comparisons of the f-potential distribution of DOTAP/DOPE liposomes suspension(black) and liposomes in the presence of 1 mM NMP. AMP (red), GMP (blue), UMP (green) and TMP (black). (For interpretation of the references to color in this figure legend,the reader is referred to the web version of this article.)

Fig. 3. Changes of the Df/Dfmax ratio of DOTAP/DOPE liposomes due to thepresence of NMPs. Fittings are obtained utilizing Eq. (2) (see Section 2.4). AMP (red),GMP (blue), UMP (green) and TMP (black). (For interpretation of the references tocolor in this figure legend, the reader is referred to the web version of this article.)

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28 mV with the addition of 1.25 mM GMP and 1.75 mM AMPrespectively. On the other hand, the pyrimidine monophosphatedid not induce the same f-potential change even at twice the pur-ine concentrations. Indeed the f-potential was 30 mV in the pres-ence of TMP 3 mM and 34 mV after the addition of UMP 3 mM.In Fig. 2B these results were underpinned by the comparison be-tween the f-potential distribution of liposomes with no nucleotideadded (f-potential = 58 mV) and in the presence of 1 mM of all thenucleotides. In every case the nucleotide concentration of 1 mM in-duces a decrease of the net charge which is lower in presence ofAMP and GMP (34 and 32.5 mV respectively) and higher in pres-ence of pyrimidines (38 mV for TMP and 45 mV for UMP). As amatter of fact these results account for the adsorption of the NMPsdriven by the phosphate group on the positively charged liposomesurfaces. The evidence of the important role of the phosphatecharge was previously reported by means of an interaction studybetween unit of nucleic acids (AMP, ADP, and ATP) and DODABvesicles [23].

Nevertheless if we take into account only the electrostatic inter-action one might expect that the coverage profiles should be inde-pendent from the type of nucleotides. This is confirmed neither bythe DLS nor by the f-potential data presented in this work. To bet-ter elucidate this point, in Fig. 3 the previous data are displayed interms of relative changes of the f-potential (Df/Dfmax) as a func-tion of nucleotides concentration. The Dfmax values were chosenconsidering the maximum f-potential amplitude obtained fromall the analyzed systems (see Section 2.4). The curves presentedin Fig. 3 are obtained by the non-linear regression fitting of theexperimental data to Eq. (2). The calculated fitting parametersare gathered in Table 1 and confirmed the differences betweenthe K parameter of the different type of NMPs assayed. Further-more, in all the cases the values of the n parameter were very closeeach other. Taking in mind that K is the Langmuir adsorptionconstant which expresses the binding energy of adsorption, the

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different values of the K show that purine based nucleotides mol-ecules have higher affinity to the liposomes surfaces.

An intriguing result that permits to get more specific informa-tion from size measurements is presented in Fig. 4. The rationalefor the experiment illustrated in this last figure is to demonstratethat when additional interactions, beyond the electrostatic forces,are established between the cationic liposomes and the anionicmononucleotides, the size of the aggregates is barely brought backthrough the restoration of the charge balance.

In row 1 the size distributions of liposomes in the presence ofeach of the mononucleotides under investigation are reported ata defined lipid/NMP molar ratio appropriately chosen. The row 2

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Fig. 4. Size distribution for each NMP at different lipid/NMP ratio. Sample prepared at once (rows 1–2) and sample obtained after the molar ratio restoration through theaddition of a further volume of liposome to the complex reported in row 2 (row 3). AMP (red), GMP (blue), UMP (green) and TMP (black). (For interpretation of the referencesto color in this figure legend, the reader is referred to the web version of this article.)

F. Cuomo et al. / Journal of Colloid and Interface Science xxx (2013) xxx–xxx 5

illustrates the size distributions of liposomes in the presence of aconcentration of NMPs twice the concentration reported in row 1where the dimension of the aggregate started to increase. In row3 are displayed the size distributions observed after the additionof a proper volume of liposome suspension to the latter samples(reported in row 2). The amount of liposome added is able to re-store the molar ratio at the values reported in row 1. Essentially,this procedure was employed to determine if the formation of acomplex between the liposome and the mononucleotides was areversible process in a defined range of the lipid/NMP ratios. Ifthe hypothesis was correct, the reinstatement of the lipid/NMPmolar ratio would bring back the size distribution depicted inrow 1. As reported in figure the complexes formed between thepurine monophosphates AMP and GMP are not reversible. Effec-tively, their size distributions at lipid/NMP molar ratio of 5 forAMP and of 6.6 for GMP, after the molar ratio reinstatement at10 for AMP and 13.2 for GMP do not give back the same dimen-sions as shown in row 1.

The opposite situation is observed when the identical test iscarried out on the pyrimidine mononucelotides. In these cases, infact, the addition of liposomes that restores the lipid/NMP molarratio from 2 to 4 for TMP and from 2.5 to 5 for UMP is effectivein giving back almost the same size distribution as in row 1.Remarkably, this experiment demonstrates the reversibility ofassociation only in the presence of pyrimidine confirming thehypothesis of higher levels of hydrophobic attraction in the pres-ence of purine-based mononucleotides.

The observations reported so far are converging in assigning animportant role to the nitrogenous ring of the mononucleotides inthe interactions between NMPs and liposomes. Bearing in mindthat the occurrence of different forces has been demonstrated totake part in the nucleic acid–cationic liposome interactions, likethe existence of hydrophobic attractions between cationic lipids

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and DNA in the DNA condensation process [36,37], it is reasonableto assume that our results derive from additional forces besides theelectrostatic ones. Among the possible forces the occurrence of rec-ognition process, like that revealed in the presence of monoolein[24], can be kept off because neither DOTAP nor DOPE have moie-ties available for this kind of interaction; moreover also the hydro-lysis of the sugar-phosphate ester bond is ruled out because it tooklonger time to happen compared to the time window of our exper-iments. Furthermore, the behavior of the Adenosine monophos-phate as an hydrophobic anion was demonstrated by means ofcircular dichroism spectroscopy studies underlining the insertionof the Adenosine nitrogenous ring into cationic bilayers of DODAB[38]. The authors reported that the anti conformation of the mono-nucleotide was forced to become syn when interacting with thecationic bilayer.

On the basis of the agreement between the presented data andthe above cited references we corroborate the hypothesis of thecrucial role of the hydrophobic interactions among the nitrogenousmoieties of the mononucleotides and the cationic liposomes. Inparticular we have demonstrated that for the pyrimidine basedmonomers TMP and UMP the hydrophobic propensity to interactwith lipid membrane is to a lesser extent compared with the pur-ine nucleotides AMP and GMP. Additionally we show that GMP isthe nucleotide with the highest affinity for the cationic bilayer.

Indeed, according to the importance ascribed to the purine ringin the insertion process of the adenosine monophosphates in cat-ionic interfaces, we confirm with this study that the feature of thisextra interaction bond is attributable partly to the nucleotide baseinvolved in hydrophobic interactions [22]. The peculiar character ofthe guanosine nucleotides has also been noticed in a recent paperby Caseli et al. [39] who demonstrated that oligonucleotide se-quences consisting of 12 guanosine (G12) and of 5 adenosine with7 guanosine (A5G7) functionalized with poly(butadiene) differently

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6 F. Cuomo et al. / Journal of Colloid and Interface Science xxx (2013) xxx–xxx

behaved with the lipid DPPC, used as a membrane model. In partic-ular they studied the Langmuir Blodgett film formed in the pres-ence of the lipids and the functionalized oligonucleotides, findingout a considerably stronger effect with the G12 sequence that re-duced the elasticity of the DPPC monolayer. Moreover it was re-ported recently that the guanosine monophosphate andadenosine monophosphate are less prone to electrochemical oxida-tion when involved in the formation of complexes with liposomes[40].

4. Conclusions

The present study provides evidence for supplementary forcesin the interactions of NMPs with lipid membranes based on DLSand f-potential data. The importance of AMP phosphate groupsto the surface properties of liposomes has been suggested before[23]. Moreover the presence of hydrophobic interaction was previ-ously reported only for the purine based monomer 20-deoxyaden-osine 50-monophosphate [22]. Here we have extended theinvestigation to other mononucleotides.

From f-potential experiments we established that liposomesloaded with purine based nucleotides (AMP and GMP) exhibit ahydrophobic contribution to a greater extent in comparison withmonophosphates containing the pyrimidine ring (TMP and UMP).

By carrying out DLS experiments we demonstrated that all thestudied NMP-liposomes complexes show a distinguishable concen-tration that triggers the aggregation phenomena. In particular wereported that AMP and GMP have a higher propensity to inducethe aggregation of liposomes and that GMP is the most effective.This evidence agrees with a recent study performed on the interac-tion between oligonucleotides and cationic liposomes [15]. In sum-mary, while supporting the widely accepted idea of a prominentrole of electrostatic forces in nucleotide-liposome interaction, ourstudy highlights the role played by hydrophobic forces in thephases of the DNA compaction process with lipid bilayers. We be-lieve that these findings together with further studies will greatlycontribute to the understanding of the preliminary steps on themechanisms underlying the interaction between DNA and cationicliposomes.

Acknowledgment

This work was financially supported by MIUR (PRIN 20102010BJ23MN), and CSGI.

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