Keywords: beta-lactoglobulin, protein aggregation, metal ions, light scattering, FTIR spectroscopy

In this work, we report a study on the effects of zinc and copper ions on the heat-induced aggregation of β-Lactoglobulin (BLG). Kinetics investigations on aggregates growth by light scattering measurements and on secondary structure changes by FT-IR absorption measurements show the different role played by two metals during the whole process. In particular, the presence of zinc in solution promotes the formation of aggregates of BLG at a lower temperature than the copper. Then, fixed the temperature, formation of a large amount of aggregates, with a large dimension, is observed for the Zn-BLG in shorter time; on the contrary, the presence of the copper in solution does not affect the aggregation process but the secondary structure changes and the formation of different stronger intermolecular H-bonds, which probably lead to build a network of bonds that takes towards gelation. Our studies show as time evolution of aggregation process of BLG is dramatically affected by the presence of metal ions in solution and structural protein modifications are induced by different divalent metal ions.

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M A N U S C R I P T

Introduction

In the last years, it has been observed and studied the aggregation process induced by metal ions, for biomedical and biotechnological purposes. The first of these two aspects is related to studies on several protein systems, most of them on beta amyloid peptide, involved in protein deposition diseases. The idea arises from the finding that increased metal concentrations (mainly copper, iron and zinc) were present in the brains of Alzheimer's disease patients both in the amyloid plaques and in the cortical tissue [START_REF] Lovell | Copper, iron and zinc in Alzheimer's disease senile plaques[END_REF][START_REF] Religa | Elevated cortical zinc in Alzheimer disease[END_REF][START_REF] Stellato | Metal binding in amyloid β-peptides shows intra-and inter-peptide coordination modes[END_REF]. Cu 2+ and Zn 2+ metals ions have a different physiological role and it has been observed that, in vitro, both promote (more zinc than copper) aggregation in amyloid fibrils and/or in amorphous aggregates [START_REF] Wang | Effect of Zinc, Copper and Calcium on the Structure and Stability of Serum Amyloid A[END_REF][START_REF] Tjernberg | A Molecular Model of Alzheimer Amyloid β-Peptide Fibril Formation[END_REF][START_REF] Danielsson | High-resolution NMR studies of the zinc-binding site of the Alzheimer's amyloid b-peptide[END_REF][START_REF] Bush | Rapid induction of Alzheimer A beta amyloid formation by zinc[END_REF][START_REF] Raman | Metal ion-dependent effects of clioquinol on the fibril growth of an amyloid {beta} peptide[END_REF][START_REF] Huang | Zinc-induced Alzheimer's Abeta1-40 aggregation is mediated by conformational factors[END_REF][START_REF] Comai | Cu2+ and Zn2+ effects on betaamyloid aggregation and structural conformation[END_REF][START_REF] Suzuki | Inhibiyory Effect of Copper(II) on Zinc(II)-induced Aggregation of Amyloid b-Peptide[END_REF]. Then, as a function of parameters like solvent's pH and metal concentration, these two ions have shown different behaviors; in particular, under physiological conditions, the amyloid peptide has a higher propensity to bind zinc, whereas, under mildly acidic conditions, as in physiological acidosis following an inflammatory process, copper is preferentially bound; in other words, zinc can protect against copper toxicity, which is induced by acidosis [START_REF] Atwood | Dramatic aggregation of Alzheimer aβ by Cu(II) is induced by conditions representing physiological acidosis[END_REF][START_REF] Miura | Metal Binding Modes of Alzheimer's Amyloid β-Peptide in Insoluble Aggregates and Soluble Complexes[END_REF]. In addition, studies with different concentrations of copper and zinc have shown that these metals prevent fibril formation of amyloid peptide by promoting the formation of no fibril aggregates [START_REF] Raman | Metal ion-dependent effects of clioquinol on the fibril growth of an amyloid {beta} peptide[END_REF][START_REF] Yoshiike | New insights on how metals disrupt amyloid beta-aggregation and their effects on amyloid-beta cytotoxicity[END_REF][START_REF] Brown | Selective aggregation of endogenous beta-amyloid peptide and soluble amyloid precursor protein in cerebrospinal fluid by zinc[END_REF][START_REF] House | Aluminium, iron, zinc and copper influence the in vitro formation of amyloid fibrils of Aβ42 in a manner which may have consequences for metal chelation therapy in Alzheimer's disease[END_REF], like oligomeric aggregates that are more neurotoxic than the same fibrils [START_REF] Garai | Selective destabilization of solubile β oligomers by divalent metal ions[END_REF][START_REF] Cleary | Natural oligomers of the amyloid-β protein specifically disrupt cognitive function[END_REF][START_REF] Westerman | The relationship between Aβ and memory in the Tg2576 mouse model of Alzheimer's disease[END_REF][START_REF] Selkoe | Cell biology of protein misfolding: the examples of Alzheimer's and Parkinson's diseases[END_REF][START_REF] Hoshi | Sato Spherical aggregates of β-amyloid (amylospheroid) show high neurotoxicity and activate tau protein kinase I ⁄ glycogen synthase kinase-3beta[END_REF][START_REF] Lesne´ | A specific amyloid-beta protein assembly in the brain impairs memory[END_REF][START_REF] Walsh | The role of cell-derived oligomers of Abeta in Alzheimer's disease and avenues for therapeutic intervention[END_REF][START_REF] Carrotta | Toxicity of recombinant {beta}-amyloid prefibrillar oligomers on the morphogenesis of the sea urchin Paracentrotus lividus[END_REF]. Finally, other results obtained by structural studies, have shown that this peptide has two metal binding sites with higher and lower affinity: copper in high affinity site is coordinated in a planar configuration with three histidines and terminus or Tyr10 and this binding site is able to bind also a zinc ion [START_REF] Hoshi | Sato Spherical aggregates of β-amyloid (amylospheroid) show high neurotoxicity and activate tau protein kinase I ⁄ glycogen synthase kinase-3beta[END_REF][START_REF] Lesne´ | A specific amyloid-beta protein assembly in the brain impairs memory[END_REF][START_REF] Dong | Metal binding and oxidation of amyloid-beta within isolated senile plaque cores: Raman microscopic evidence[END_REF]. The four aminoacids proposed for zinc binding site are Tyr10, Glu11, Arg5 or the N-terminus [START_REF] Danielsson | High-resolution NMR studies of the zinc-binding site of the Alzheimer's amyloid b-peptide[END_REF][START_REF] Mekmouche | Characterization of the ZnII binding to the peptide amyloidβ1-16 linked to Alzheimer's disease[END_REF][START_REF] Zirah | Structural changes of region 1-16 of the Alzheimer disease amyloid beta-peptide upon zinc binding and in vitro aging[END_REF][START_REF] Zirah | Zinc binding agonist effect on the recognition of the β-amyloid (4-10) epitope by antibeta-amyloid antibodies[END_REF][START_REF] Morante | Inter-and Intra-octarepeat Cu(II) Site Geometries in the Prion Protein: implications in Cu(II) binding cooperativity and Cu(II)-mediated assemblies[END_REF]. It has been made hypothesis about the presence for both metals in the second low affinity binding site but it is still studying [START_REF] Syme | Copper binding to the amyloid-β (Ab) peptide associated with Alzheimer's disease: folding, coordination geometry, pH dependence, stoichiometry, and affinity of Aβ-(1-28): insights from a range of complementary spectroscopic techniques[END_REF][START_REF] Garzon-Rodriguez | Binding of Zn(II), Cu(II), and Fe(II) ions to Alzheimer's A-beta peptide studied by fluorescence[END_REF][START_REF] Bush | The galvanization of beta-amyloid in Alzheimer's disease[END_REF]. More recently, it has also been reported that alterations in the disposition of zinc ions may be important in the initiation and development of amyotrophic lateral sclerosis. In particular, alterations in the expression of metallothioneins, which regulate cellular levels of zinc, have been reported in mutant superoxide

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dismutase SOD1 mice: deletion of metallothioneins in these animals accelerates disease progression [START_REF] Smith | Role of zinc in ALS[END_REF].

The second purpose of the scientific interest, in studying aggregation protein process, drops in the field of biotechnologies as food technology. In fact, most processes in manufacturing of foods are based on thermal treatments. To know the structures of the proteins during and after these treatments is extremely important also for allergenic problems. Regarding dairy production, milk proteins are much studied because some of them are major constituent of fouling deposits in the dairy industry [START_REF] Sakiyama | Protease susceptibility of betalactoglobulin adsorbed on stainless steel surface as evidence of contribution of its specific segment to adsorption[END_REF]. For example, casein, that is the most predominant phosphoprotein found in milk and cheese as a suspension of particles called casein micelles, binds zinc atoms [START_REF] Singh | Binding of zinc to bovine and human milk proteins[END_REF], while βlactoglobulin (BLG) chelates calcium and precipitates when heated and as a function of pH [START_REF] O'connell | Effect of beta-lactoglobulin and precipitation of calcium phosphate on the thermal coagulation of milk[END_REF].

Moreover, in mixtures of whey proteins, fibril-like aggregates have been found [START_REF] Bolder | Fibril assemblies in aqueous whey protein mixtures[END_REF][START_REF] Veerman | Mesostructure of fibrillar bovine serum albumin gels[END_REF] as in production of gels or aggregates in presence of calcium or zinc, respectively [START_REF] Ipsen | Molecular self-assembly of partially hydrolysed alpha-lactalbumin resulting in strong gels with a novel microstructure[END_REF][START_REF] Simons | Role of calcium as trigger in thermal beta-lactoglobulin aggregation[END_REF][START_REF] Saeseaw | Observation of salt-induced beta-lactoglobulin aggregation using sedimentation field-flow fractionation[END_REF]. In fact, metal ions are also able to induce cold gelation and the gel texture formation depends on different parameters as pH conditions and protein and/or iron concentration [START_REF] Bouhallab | Copper-catalyzed formation of disulfide-linked dimer of bovine β-lactoglobulin[END_REF][START_REF] Harvey | A Tryptophan Rotamer Located in a Polar Enviroment Probes pH-Dependent Conformational Changes in Bovine b-Lactoglobulin A[END_REF]. Therefore, protein aggregation is important in food processing and it is relevant to know how aggregation of food proteins occurs and if it is a source of amyloid fibrils [START_REF] Remondetto | Molecular Mechanisms of Fe 2+ -Induced β-Lactoglobulin Cold Gelation[END_REF].

We have already studied thermal induced aggregation of several proteins and we have shown that the whole process is accompanied by significant changes in the conformation and structure of the native molecule [START_REF] Pearce | Formation of amyloid-like fibrils by Ovalbumin and related proteins under conditions relevant to food processing[END_REF][START_REF] Militello | Conformational changes involved in thermal aggregation processes of Bovine Serum Albumin[END_REF][START_REF] Militello | Aggregation kinetics of bovine serum albumin studied by FTIR spectroscopy and light scattering[END_REF][START_REF] Vetri | Thermal induced conformational changes involved in the aggregation pathways of beta-lactoglobulin[END_REF][START_REF] Vetri | Thermal aggregation of bovine serum albumin at different pH: comparison with human serum albumin[END_REF]. Here, we will show, applying the same approach, the study of the aggregation of BLG in presence of copper and zinc in solution. We have selected the BLG because of its double interest above mentioned, as a model beta-protein in the aggregation process and as a thermal marker in the industrial processes involved in preparation of milk [START_REF] Vetri | Amyloid fibrils formation and amorphous aggregation in Concanavalin A[END_REF]. In general, BLG is the major whey protein of ruminant species and is present in the milk of many, but not all, other species. The protein family to which BLG belongs, the lipocalins [START_REF] Chen | Beta-lactoglobulin is a thermal marker in processed milk as studied by electrophoresis and circular dichroic spectra[END_REF], presents a widely diverse series of functions, most of which involve some ligand-binding function; it has been speculated that this binding-function must be the physiological reason for the significant quantities of BLG found

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in milk. This globular protein consists of 162 aminoacids [START_REF] Cleary | Natural oligomers of the amyloid-β protein specifically disrupt cognitive function[END_REF]4 kDa) and is a dimer at neutral pH; it has a core structural pattern formed by one α-helix and eight strands of antiparallel β-sheets and its tertiary structure is stabilized from two disulfide bonds. Moreover, there is also one free SH group (pKa = 8), corresponding to Cys-121, buried in the hydrophobic core of the protein [START_REF] Kontopidis | β-Lactoglobulin: Binding Properties,Structure, and Function[END_REF][START_REF] Relkin | Reversibility of heat-induced conformational changes and surface exposed hydrophobic clusters of β-lactoglobulin: their role in heat-induced sol-gel state transition[END_REF][START_REF] Papiz | The structure of β-lactoglobulin and its similarity to plasma retinol-binding protein[END_REF][START_REF] Burova | Thermodynamic stability of porcine β-lactoglobulin. A structural relevance[END_REF][START_REF] Uhrinova | Structural changes accompanying pH-induced dissociation of the beta-Lactoglobulin dimer[END_REF]. It is a protein extremely sensitive to thermal denaturation [START_REF] Cairoli | Reversible and irreversible modifications of β-lactoglobulin upon exposure to heat[END_REF] and has a mild antioxidant nature due to the free thiol group [START_REF] Monaci | Effect of Heat Treatment on the Detection of Intact Bovine β-Lactoglobulins by LC Mass Spectrometry[END_REF]. In fact, it is already reported that, when heated, globular proteins tend to aggregate, thanks to the contribution of intermolecular disulfide bonds [START_REF] Bush | Rapid induction of Alzheimer A beta amyloid formation by zinc[END_REF][START_REF] Dong | Metal binding and oxidation of amyloid-beta within isolated senile plaque cores: Raman microscopic evidence[END_REF][START_REF] Zirah | Structural changes of region 1-16 of the Alzheimer disease amyloid beta-peptide upon zinc binding and in vitro aging[END_REF][START_REF] Zirah | Zinc binding agonist effect on the recognition of the β-amyloid (4-10) epitope by antibeta-amyloid antibodies[END_REF] and hydrophobic interactions [START_REF] Militello | Aggregation kinetics of bovine serum albumin studied by FTIR spectroscopy and light scattering[END_REF]. In particular, when the temperature increases, monomerization and partial unfolding of the protein are induced with consequent exposure of SH group and formation of an intermolecular bond with a free cysteine of a close molecule. The disulfide exchange can also be promoted in smaller way when the pH value decreases and when the BLG is partly denatured [START_REF] Bouhallab | Copper-catalyzed formation of disulfide-linked dimer of bovine β-lactoglobulin[END_REF][START_REF] Harvey | A Tryptophan Rotamer Located in a Polar Enviroment Probes pH-Dependent Conformational Changes in Bovine b-Lactoglobulin A[END_REF].

To get more insight into the role of the metal ions on the aggregation, here we report a study on the effects of Cu 2+ and Zn 2+ on the heat-induced aggregation of BLG, at pH 7 and at temperature of 60 °C. We monitor the time evolution of the mean dimension of the aggregates by dynamic light scattering measurements; while, structural and conformational changes at secondary and tertiary level respectively, are followed by time evolution of Amide I' and Amide II bands profiles, monitored by infrared absorption spectroscopy. Amide I' is a probe of protein structural organization in terms of α-helix, random coil and β-sheet contents and gives information on intermolecular aggregation through the appearance of two shoulders attributed to β-sheet formation [START_REF] Remondetto | Molecular Mechanisms of Fe 2+ -Induced β-Lactoglobulin Cold Gelation[END_REF][START_REF] Allain | Relationships between conformation of β-lactoglobulin in solution and gel states as revealed by attenuated total reflection Fourier transform infrared spectroscopy[END_REF][START_REF] Dong | Intermolecular β-Sheet Results from Trifluoroethanol-Induced Nonnative a-Helical Structure in β-Sheet Predominant Proteins: Infrared and Circular Dichroism Spectroscopic Study[END_REF][START_REF] Paik | Copper(II)-induced self-oligomerization of α-synuclein[END_REF][START_REF] Renard | Structural investigation of βlactoglobulin gelation in ethanol/water solutions[END_REF]. Amide II is a probe of partial opening of the tertiary structure, which allows the H-D exchange of those hydrogens entrapped in the core of protein before opening [START_REF] Militello | Aggregation kinetics of bovine serum albumin studied by FTIR spectroscopy and light scattering[END_REF]. The temperature selected for all the measurements was 60 °C. D 2 O solutions were used to avoid the IR spectral overlaps between Amide I band and strong ν 2 absorption band of water in the region 1650 cm -1 .

Materials and methods

Samples preparation

IR measurements

IR spectra were measured by using Bruker Vertex 70 spectrophotometer, equipped with a MIR light globar source (i.e. an U-shaped silicon carbide piece), with spectral resolution of 2 cm -1 . Each spectrum was averaged over 100 scans. All samples were placed between two CaF 2 windows, with a 0.05 mm Teflon spacer. The absorption spectrum of the empty beam line was subtracted from the spectrum of each sample. The resultant spectra were smoothed with a 13-point Savitzky and Golay function. In order to identify the time variations of each overlapping component under the broad amides, differential spectra were obtained by subtracting to the spectrum at a generic time t, the spectrum at t 0 (where t 0 was 7 minutes, for reaching thermal equilibrium).

Dynamic Light Scattering measurements

Zetasizer Nano-S90 (Malvern Instruments) Dynamic Light Scattering Instrument was used. It was equipped with a light source from a He-Ne laser (λ = 633 nm) and a scattering angle of 90°.

The sample compartment was completely self enclosed and the temperature was automatically controlled. Temperature scanning was realized by increasing the temperature of 1 °C every 5 minutes and by starting measurement after an equilibration time of 3 minutes. The normalization of

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the scattered intensity was done for each measurement. For polydisperse samples, the correlation function can be written as:

( ) ( ) [ ] 2 1 τ τ i Bg A G + =
where A and B are the baseline and intercept of the correlation function and g i (τ) is the sum of all the exponential decays exp-(2Γτ), with Γ = Dq 2 and where D is translational diffusion coefficient and q = (4πn/λ 0 )sinθ/2 (with n = refractive index of dispersant, λ o = wavelength of the laser, θ = scattering angle). Two approaches are utilized to obtain size information from the correlation function: a) fitting a single exponential to the correlation function to obtain the mean size (zaverage diameter); b) fitting a multiple exponential to the correlation function to obtain the distribution of particle sizes (such as CONTIN). The size distribution obtained is a plot of the relative intensity of light scattered by particles in various size classes and is therefore known as an intensity size distribution. Through Mie theory with the use of the input parameter of sample refractive index, it is possible to convert the intensity distribution to volume and number distributions [START_REF] Mie | Beitrage zur optik truber medien, speziell kolloidaler metallosungen[END_REF][START_REF] Bohren | Absorption and Scattering of Light by Small Particles[END_REF].

Results and discussion

**٭ FIGURE 1 *** Fig. 1 shows the temperature scanning of the scattered light for BLG native and in presence of metal ions (BLG, Cu-BLG and Zn-BLG). As can be seen, only in the Zn-BLG sample, it is evident a rapid increase of intensity, indicating an aggregation process that starts at about 48 °C. For the other two samples, BLG and Cu-BLG, nothing occurs until 60 °C. In view of the fact that for Zn-BLG over 60 °C the aggregation kinetic would be too fast, we have selected this temperature value to follow the aggregation kinetics for all the samples.

**٭ FIGURE 2 ***

To monitor the extent of the aggregation of all the samples we have performed scattering measurements as a function of time, as reported in Fig. 2. In particular, figure 2a reports the time

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evolution of the total scattered intensity for all the three samples. It is relevant to note that, when the aggregation of Zn-BLG is finished, for the other two samples it is not still begun. In the Zn-BLG sample, better showed in the inset, after 10 minutes the total scattered intensity quickly increases for aggregation and after 70 minutes as much quickly decreases for precipitation. The total scattered intensities for BLG and Cu-BLG show behaviors very similar with a light increase for Cu-BLG with respect to that one of BLG, after 400 minutes. We think that this increased behavior, shown by Cu-BLG, with respect to BLG, is due to different conformational changes induced by the metal presence, or to the presence of the clusters of dense liquid (several hundred nanometers in size)

which are metastable with respect to the protein solutions and the which formation takes place within a few seconds after sample preparation [START_REF] Pan | Metastable Mesoscopic Clusters in Solutions of Sickle-Cell Hemoglobin[END_REF].

To evaluate the dimensions of the aggregates during the kinetics and to be sure that we are monitoring self-aggregation of the protein, we present, in Fig. 2b, the kinetics of the diameter mean dimension of molecular species which are present in the samples. It is evident how BLG and Cu-BLG, in these experimental conditions, follow similar evolution in the heat-induced aggregation, producing little species of about 200 nm of diameter (as better seen in the inset of the Fig. 2b), while Zn-BLG rapidly produces aggregates of about 4500/5000 nanometers of diameter. The discontinuity, shown by native BLG after 180 minutes, is not real but has to be attributed to the instrumental analysis and it is due to a superimposition of the correlation functions.

The analysis of these data, already discussed in paragraph 2. it results difficult to find a schematic model that explains the evolution of a so complex process.

To monitor changes in secondary (Amide I') and tertiary (Amide II) structures of protein with and without metal ions, we have performed kinetics of infrared absorption measurement for all the three samples.

We remind that Amide I is an infrared band attributed to an out of phase combination of C = O and C-N stretching of amide groups and that, in D 2 O, it is called Amide I' for the shift towards 1650 cm -1 . Generally, Amide I' band has a composite profile, consisting of several spectral components related to different secondary structures [START_REF] Militello | Aggregation kinetics of bovine serum albumin studied by FTIR spectroscopy and light scattering[END_REF].

**٭ FIGURE 5 *** Fig. 5 shows the differential absorption spectra of the Amide I' band for all the three samples and highlights the time evolution of the main spectral components of the band. The spectral region between 1630 and 1650 cm -1 is attributed to α-helix, disordered random coil and intramolecular native β-sheet and, as it is evident, in all the samples decreases as a function of time (as indicated by arrows). It is noteworthy that the negative spectral component at 1640 cm -1 in the Cu-BLG sample is shifted towards 1635 cm -1 , usually assigned to native β-sheets structures and not

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present in the other two samples. This shift indicates that a possible coordination of the copper, during the aggregation process, can produce different species, as already seen through light scattering. In BLG and Zn-BLG it is evident only a decrease without any shift and in Zn-BLG the component at 1650 cm -1 is present as a shoulder. The latter, attributed to α-helix and disordered structures, is probably less intense because of the specific coordination properties of zinc. Another negative contribution to the Amide I' band is present, at about 1690 cm -1 and assigned to native β-turn structures. We have already performed kinetic measurements of circular dichroism in the far-UV region with native BLG in similar conditions (buffer phosphate, lower concentration and 80 °C) and they showed that, already in the first minutes, native beta structures change in favor of other different type of beta structures [START_REF] Vetri | Thermal induced conformational changes involved in the aggregation pathways of beta-lactoglobulin[END_REF]. Besides, all the samples present a conversion from αhelix and intramolecular β-native towards β-aggregated structures. In particular, the bands due to intermolecular aggregation are at around 1623 and 1685 cm -1 and are assigned to vibrations of strongly bound intermolecular β-strands and of anti-parallel β-sheet, respectively. In all the samples, these bands appear increased as a function of time, giving us the information that aggregation occurs, as seen also by light scattering. Then, the presence of copper determines an increase of the signal in the regions at about 1595 cm -1 and at about 1660 cm -1 with consequent lost of the isosbestic points, well defined in the native BLG sample. The spectral component at about 1590 cm -1 is attributed to asymmetric stretching of CO 2 -, when coordinated with Cu 2+ [START_REF] Mesu | New Insights into the Coordination Chemistry and Molecular Structure of Copper(II) Histidine Complexes in Aqueous Solutions[END_REF].

A study realized on beta amyloid peptide has shown that zinc ion can bind one or two histidine, making itself available for further interaction; this may induce the formation of ordered aggregates, according to a model in which the metal plays the role of a bridge between the imidazole rings of two histidine residues possibly belonging to different peptides [START_REF] Stellato | Metal binding in amyloid β-peptides shows intra-and inter-peptide coordination modes[END_REF]. By the same study results that copper is tightly bound to three histidines of the same protein molecule in a closed structure, which ''protects'' the metal against any further interaction. More in general, the influence of metals in the higher or lower stabilization of native structure, after its coordination with histidines, is reported also for several native metallo-proteins. For example, in native enzyme SOD (superoxide

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dismutase), that has the active site with the two metals, copper and zinc, the FTIR spectroscopy has revealed the influence of metals on the secondary structure of protein matrix [START_REF] Sun | Secondary Strucutre Dependent on Metal Ions of Copper, Zinc Superoxide Dismutase Investigated by Fourier Transform IR Spetroscopy[END_REF]. From a comparison with our results, it is reasonable to think to a similar influence also in our samples.

**٭ FIGURE 6 *** In Fig. 6, we have put together the Amide I' bands of all the three samples, at two different times, to better put in evidence the spectral differences shown in Fig. 5. As can be seen, the profiles of the differential spectra at the initial time are very similar for all the samples; after 5.5 hours, BLG and Zn-BLG show similar behaviors, indeed Cu-BLG shows changes principally in the two regions, at 1660-1680 and 1614 cm -1 . This suggest that Cu 2+ influences the time evolution of the secondary structure of the native protein more than Zn 2+ , which instead has a major influence on the time evolution of the aggregates building.

**٭ FIGURE 7 ***

In Fig. 7 are reported the kinetics of the differential absorption spectra in the Amide II and II' regions. Amide II band is attributed to an out-phase combination of in plane C-N stretching and N-H bending of Amide group. Time evolution of these bands gives us information on the protein partial unfolding with consequent H-D exchange between protein and solvent. The principal negative band at 1540 cm -1 and the positive band at 1436 cm -1 , that are probes of H-D exchange, are present in all the samples. Once again, it results evident that the partial opening of the protein at initial times of the aggregation is necessary [START_REF] Militello | Aggregation kinetics of bovine serum albumin studied by FTIR spectroscopy and light scattering[END_REF]. Then, there is a band at about 1442 cm -1 present only in Cu-BLG, which is probably attributed to the presence and coordination of copper with protein.

**٭ FIGURE 8 *** Summarizing the spectral details, in Fig. 8 we report the time evolution of the component of Amide I' band at 1623 cm -1 for the three samples (8a) and at 1614 cm -1 only for Cu-BLG (8b) and of the component of Amide II' at 1436 cm -1 for all investigated samples (8c). For the three samples, we observe the formation of β-aggregated structures through the increase of the spectral component

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT at 1623 cm -1 .
It is noteworthy to underline that for Cu-BLG the different behavior of the spectral component at 1623 cm -1 is due to the competitive appearance of the spectral component at 1614 cm -1 , after about 45 minutes, thus confirmed in Fig. 8b by the presence of two different trends. This last spectral component is attributed to the formation of very strong intermolecular H-bonds [START_REF] Remondetto | Molecular Mechanisms of Fe 2+ -Induced β-Lactoglobulin Cold Gelation[END_REF][START_REF] Allain | Relationships between conformation of β-lactoglobulin in solution and gel states as revealed by attenuated total reflection Fourier transform infrared spectroscopy[END_REF][START_REF] Jackson | Halogenated alcohols as solvents for protein: FTIR spectroscopic studies[END_REF] and we suggest that the specific presence of copper may promote, in a very short time, the formation of heat-induced network ready to form macromolecules and/or gels, as reported for many globular protein, such as BLG [START_REF] Mulvihill | Gelation characteristics of whey proteins and β-Lactoglobulin[END_REF][START_REF] Ziegler | Gelation of proteins[END_REF]. As it is shown in Fig. 8c, it is evident that the presence of metals in solution affects in a different way the rate and the time evolution of the protein partial unfolding. Moreover, we outline that for Zn-BLG the partial unfolding of the protein seems to stop after 120 minutes, time in which the bigger aggregates are formed, as already reported in figure 2b. Indeed, for BLG and Cu-BLG the contemporary processes of opening and aggregation, at this temperature, are longer, being less pronounced in Cu-BLG, as already seen in the inset of figure 2b. These behaviors confirm us the existence of contemporary steps like conformational opening due to tertiary structure changes, like structural changes due to secondary structures changes, induced also by the coordination with metal ions, and like intermolecular interactions that can select to go towards aggregation and/or gelation process. The Cu-BLG conformation, in our conditions, has some structural changes, due also to the specific coordination with the ion, and some intermolecular interactions that drive the aggregates towards the formation of the gel texture.

Conclusion

The results, shown and discussed in this paper, highlight that metal ions affect the tertiary and secondary structure changes and the formation of supramolecular aggregates in BLG. We outline that Cu 2+ and Zn 2+ play a different role in conditioning the time evolution of the heatinduced aggregation process. In particular, Cu 2+ influences mainly the time evolution of the protein secondary structure and of its initial conformational changes, inducing the formation of stronger Hlinked bonds but not influencing the trend of aggregates formation. Instead, Zn 2+ presents similar

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changes of the conformational and structural changes with respect to the BLG without metal ions, but dramatically influences the formation of big aggregates in a shorter time.

Summarizing the details, conformational and structural changes are the dominant initial steps that drive the aggregation process. In particular, the initial partial opening of the tertiary structures (monitored by Amide II region) and the structural changes between native secondary structures and β-aggregates (monitored by Amide I') occur in all the three samples, BLG, Cu-BLG and Zn-BLG. Following aggregation kinetics, a common trend comes out: at fixed temperature, as time increases, gradually β-aggregates structures increase and ordered α-helix and native β-structures decrease. The presence of metal ions in solution influences these structural changes, due to different coordination of the two metal ions here studied. The presence of zinc ion induces the formation of big size (until to 5000 nm of diameter) aggregates in larger amount, in very short time and in a lower temperature. The presence of copper ion, indeed, destabilizes also some native β-structures not evidenced neither in BLG nor in Zn-BLG (as showed by the shift at 1635 cm -1 ); in fact, copper ion has an own coordination that involves different conformational changes during the aggregation process (as showed by bands at 1660, 1590 and 1445 cm -1 present only in this sample).

The presence of β-aggregates of small dimensions (monitored by band at 1623 and 1685 cm -1 ) together with copper ion seems to favor the formation of very strong intermolecular H-bonds (monitored by band at 1614 cm -1 ) not present in the other samples. This behavior, as reported in literature, is attributed to the initial formation of a network of bonds that can take towards gelation.

As it is known, for example cold gelation consists in a pre-heating of the sample and its subsequent cooling that takes to the gel formation. Our data suggest that, during the heating, pre-aggregates of Cu-BLG can select an alternative patch than that one of aggregation: the patch of the formation of a network of H-bonds that takes towards the gelation.

It will be very interesting to continue to investigate when and why Cu-BLG prefers to go towards the gelation rather than towards formation of larger aggregates and how this depends by

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Figure 3

Navarra et al.

3 ,

 3 permits us to have the distribution of the diameters values with respect to particles numbers into solution and with respect to scattered intensity, at different time instants. **٭ FIGURE 3 *** In Fig. 3 we select the initial times (continuous line), considered the same zero time for all samples, and the final times (dashed line), different as a function of sample, being 11 hours for BLG and Cu-BLG (figs. 3a and 3b) and 50 minutes for Zn-BLG (fig. 3c). We observe that the results obtained for Cu-BLG and BLG are similar, i.e. both the samples produce aggregates of same value of size, A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT about 200 nm. On the contrary, Zn-BLG produces a biggest final size (up to 5000 nm) and a large amount of the aggregates in a shortest time, coherently with the data shown in Fig. 2. **٭ FIGURE 4 *** Analogously, we also show in Fig. 4 the distribution of the diameters values based on the intensity of the scattered light. Here, we report three times, those already shown in previous figure and an intermediate time (short dashed line) with respect to the complete time evolution for all samples. Once again the BLG and Cu-BLG are similar, while Zn-BLG has an aggregation kinetic very different. It is noteworthy that, for Cu-BLG and BLG in the intermediate time (figure 4a and 4b), the species are differently distributed with a main size around to 200 nm. This could explain the different increase of the scattered intensity shown by Cu-BLG in figure 2 with respect to the native protein as due to the presence of different species created by the presence of copper. For this reason,

A

  C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT external solvent, conditions and also by the type of protein. This is an open road to explore and to use in various applied fields.

Figure 1 :

 1 Figure 1: Normalized scattered intensity as a function of temperature for (□) BLG, (○) Cu-BLG

Figure 2 :

 2 Figure 2: (a) Time evolution of the normalized scattered intensity and (b) of the average diameter

Figure 3 :

 3 Figure 3: Size distribution of aggregates dimension based on the numbers of particles present into

Figure 4 :

 4 Figure 4: Size distribution of aggregates dimension based on the intensity of light scattered, at T =

Figure 5 :

 5 Figure 5: Infrared absorption differential spectra in the Amide I' region, defined as ΔA(t) = A(t) -

Figure 6 :

 6 Figure 6: FTIR differential spectra for BLG (continuous line), Cu-BLG (long dashed line), and Zn-

Figure 7 :

 7 Figure 7: Time evolution of infrared absorption differential spectra in the Amide II and Amide II'

Figure 8 :

 8 Figure 8: Time evolution of the differential absorption intensity of Amide I' components: (a) at 1623 cm -1 for (□) BLG, (○) Cu-BLG, (∆) Zn-BLG and (b) at 1614 cm -1 for only Cu-BLG; (c) time evolution of the differential absorption intensity of Amide II' component at 1436 cm -1 . Error bar

Figure 4

 4 Figure 4Navarra et al.

  Figure 5Navarra et al.


Laura Ciani

Dr Sandra Ristori
email: ristori@unifi.it

Claudia Bonechi

Claudio Rossi

Giacomo Martini

Effect of the preparation procedure on the structural properties of oligonucleotide/cationic liposome complexes (lipoplexes) studied by electron spin resonance and zeta potential

Keywords: Lipoplexes, ESR, Zeta Potential, NMR, Cryo-TEM

come

Effect of the preparation procedure on the structural properties of oligonucleotide/cationic liposome complexes (lipoplexes) studied by electron spin resonance and zeta potential

Introduction

Cationic liposomes were first introduced as DNA transfection agents by Felgner et al. thirty years ago [START_REF] Felgner | Lipofection: a highly efficient, lipidmediated DNA-transfection procedure[END_REF] and they are still extensively studied as a valid alternative to viral vectors in gene therapy.

Though generally less efficient than viral-based delivery agents, the complexes formed between liposomes and DNA (lipoplexes) exhibit a number of attractive properties, such as low toxicity, lack of immunogenicity and highly tunable physico-chemical properties (composition, size and surface charge). In this context, the studies of DNA-liposome interactions represent important issues, since knowledge of the structural changes and of the energy involved in lipoplex formation is a pre-requisite for the optimisation of transfection protocols.

Many papers have appeared on this subject [START_REF] Safinya | Structures of lipid-DNA complexes: supramolecular assembly and gene delivery[END_REF][START_REF] Niidome | Gene therapy progress and prospects: Nonviral vectors[END_REF][START_REF] Caracciolo | Multicomponent cationic lipid-DNA complex formation: role of lipid mixing[END_REF][START_REF] Laporte | Design of modular non-viral gene therapy vectors[END_REF][START_REF] Audouy | In vivo characteristics of cationic liposomes as delivery vectors for gene therapy[END_REF][START_REF] Braun | The structure of DNA within cationic lipid/DNA complexes[END_REF][START_REF] Felgner | Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations[END_REF][START_REF] Miller | Cationic liposomes for gene therapy[END_REF][START_REF] Zuidam | Characterization of DNA-lipid complexes commonly used for gene delivery[END_REF][START_REF] Ciani | DOTAP/DOPE and DC-Chol/DOPE lipoplexes for gene delivery: Zeta potential measurements and Electron Spin Resonance spectra[END_REF][START_REF] Bumcrot | RNAi therapeutics: a potential new class of pharmaceutical drugs[END_REF]. For obvious reasons, particular attention has been dedicated to lipoplexes built up with medium/large linear DNA fragments or plasmids (30-200 kbp). In the majority of cases it has been observed that the preparation procedure induces large variations in the lipoplex features [START_REF] Safinya | Structures of lipid-DNA complexes: supramolecular assembly and gene delivery[END_REF][START_REF] Caracciolo | Multicomponent cationic lipid-DNA complex formation: role of lipid mixing[END_REF][START_REF] Meidan | Interaction of oligonucleotides with cationic lipids: the relationship between electrostatics, hydration and state of aggregation[END_REF][START_REF] Meidan | Oligonucleotides lipoplexes: the influence of oligonucleotide composition in complexation[END_REF]. In this paper we turned our attention to lipoplexes formed with a short oligonucleotide (a 20 mer, dsAT) and DOTAP/DOPE (1,2dioleoyl-3-trimethylammonium-propane and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, respectively), which were prepared by two different procedures.

The first procedure was simply based on progressive addition of dsAT to preformed lipoplexes in order to change dsAT concentration up to the desired dsAT content. In the second procedure, each lipoplex was separately prepared, by addition of the components in the appropriated ratio. Therefore, in this latter case the interaction occurred between DNA and unmodified liposomes in each sample, whereas in the case of titration DNA interacted with a preformed lipoplex.

The oligonucleotide used in this work was chosen as a representative example of short nucleic acid. Indeed, the importance of short nuclei acid fragments in gene therapy is demonstrated by outstanding examples such as antisense DNA and small interference RNA (siRNA), recently come into focus as potent inhibitor of disease promoters [START_REF] Peer | Selective gene silencing in activated leukocytes by targeting siRNAs to the integrin lymphocyte function-associated antigen-1[END_REF][START_REF] Liu | The influence of polymeric properties on chitosan/siRNA nanoparticle formulation and gene silencing[END_REF]. Here, we present the results The ESR spectra of spin probes inserted in molecular assemblies are sensitive to changes in their shape, local polarity, and mobility of neighbor molecules [START_REF] Freed | EPR in the 21st century[END_REF][START_REF] Martini | ESR and Scattering studies of perfuorinated surfactants in ordered and disordered systems[END_REF][START_REF] Morandi | Association of sugar-based carboranes with cationic liposomes: an electron spin resonance and light scattering study[END_REF][START_REF] Salvati | Physicochemical characterization and transfection efficacy of cationic liposomes containing the pEGFP plasmid[END_REF][START_REF] Cassera | Differential effects of cholesterol on acyl chain order in erythrocyte membranes as a function of depth from the surface. An electron spin resonance (EPR) spin label study[END_REF][START_REF] Perissi | Electron Spin Resonance and Differential Scanning Calorimetry as combined tools for the study of liposomes in the presence of long chain nitroxides[END_REF][START_REF] Swamy | Coexisting domains in the plasma membranes of live cells characterized by spinlabel ESR spectroscopy[END_REF][START_REF] Šentjurc | Transport of liposome-entrapped molecules into the skin as studied by electron paramagnetic resonance imaging methods[END_REF][START_REF] Stösser | Pharmaceutical aspects of ESR investigation on drug delivery systems, tissues and living systems[END_REF][START_REF] Lurie | Monitoring drug delivery processes by EPR and related techniques. Principles and applications[END_REF][START_REF] Bartucci | Time-Resolved Electron Spin Resonance studies of spin-labelled lipids in membranes[END_REF][START_REF] Bartucci | Chain dynamics in the lowtemperature phases of lipid membranes by Electron Spin-Echo Spectroscopy[END_REF][START_REF] Bartucci | Electron Paramagnetic Resonance investigation of biological systems[END_REF][START_REF] Benatti | Structural and thermal characterization of dioctadecyldimethylammonium bromide dispersions by spin labels[END_REF][START_REF] Caldararu | Structural aspects in self-assembled systems of polyethylene surfactants, as studied by the spin probe technique source[END_REF][START_REF] Erilov | Water concentration profiles in membranes measured by ESEEM of spin-labelled lipids[END_REF][START_REF] Korstanje | Dynamics in lipid vesicles and liposomes studied with ESR: effects of hydration, curvature and unsaturation[END_REF][START_REF] Kristl | Interactions of solid lipid nanoparticles with model membranes and leukocytes studied by ESR[END_REF][START_REF] Marsh | Polarity and permeation profiles in the lipid membranes[END_REF][START_REF] Marsh | High Field Electron Spin Resonance of spin labels in membranes[END_REF].

A C C E P T E

Thus, any change in the ESR line shape observed by passing from free liposomes to lipoplexes reported on the interaction between nucleic material and liposome bilayers at the molecular level. Moreover, the mobility of the spin labels in fluid lipid membranes was close to the optimal range of motional sensitivity in ESR of nitroxides. Zeta Potential (henceforth indicated as ζ) directly gave the surface charge, i.e. a key parameter to study the interaction with cells membranes, which in most cases bear a negative charge. When necessary, Nuclear Magnetic Resonance spectroscopy (NMR) and electron microscopy at low temperature (Cryo-TEM) measurements were carried out on selected samples and added to clarify particular points of the discussion.

The data presented here gave a picture of the formation mechanism in both procedures. The local structure and ordering of lipid molecules in the bilayer were not drastically modified upon interaction with the negatively charged dsAT, although strong binding of dsAT to the liposome surface was found to take place.

Materials and Methods

Materials.

DOTAP (1, purity >99%), and DOPE (2, purity >99%) were purchased from Avanti Polar Lipids, Inc., Alabaster, AL, and used without further purification.

-

O O N + O Cl - O H 3 N + O O P O O O O O O 1 2
The 20-mer oligonucleotides polyA and polyT were obtained from QIAGEN Operon, Alameda, CA. The annealing temperature of the two nucleotides was 312.9 K, as indicated by the manufacturer. The double strand polyA-polyT (henceforth called dsAT) was obtained by mixing equal amounts of polyA and polyT solutions at the same concentration and then annealing in a vial at room temperature for 24 h. All samples were stored at 253 K. The dsAT concentration was determined by measuring the absorbance at 260 nm, taking into account that Abs 260 = 1 for a double strand oligonucleotides corresponded to 50 µg/ml, that is 1.5x10 -4 M.

The cationic spin probe 4-(dimethyldodecylammonio)-2,2,6,6-tetramethyl-1-piperidinyloxy (iodide salt, CAT12) was purchased from Molecular Probe, Inc., Eugene, OR, and used without further purification:

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 6
Phosphate Buffer Saline (PBS) was purchased from Sigma as tablets.

After thawing, the pH value was tested and set to 7.4 with 0. In both procedures stock ethanol solutions (1.1x10 -3 M) of CAT-12 probe were added to preformed liposomes. In practice, the required amount of probe solution was let to evaporate in the bottom of a vial and, after complete ethanol removal, liposomes were added. The system was then equilibrated at room

A C C E P T E D M A N U S C R I P T

temperature for 1 h, prior to ESR spectra recording, as described in ref. [START_REF] Perissi | Electron Spin Resonance and Differential Scanning Calorimetry as combined tools for the study of liposomes in the presence of long chain nitroxides[END_REF]. In order to avoid inhomogeneous line broadening and marked changes in the properties of the liposome bilayer, the ESR probe content was never higher than 1% of total lipids.

The composition r of lipoplexes is expressed as the ratio between the nominal negative charges of phosphate groups in dsAT and the nominal positive charges of DOTAP, i.e.:

[phosphate groups of DNA] r =

[DOTAP molecules in liposomes]

In samples prepared for ESR measurements the charges of CAT-12 were not included in the calculation of r, since their concentration was less than 1%, as mentioned above.

Pure liposomes (r = 0) and samples with r up to 1.5 were investigated. All samples were prepared with the same lipoplex concentration usually tested on cell cultures [START_REF] Salvati | Physicochemical characterization and transfection efficacy of cationic liposomes containing the pEGFP plasmid[END_REF].

Methods.

Electron Spin Resonance Spectroscopy. ESR spectra were obtained with a Bruker ESR spectrometer model 200D, working in the continuous wave mode at X-band (~9.5 GHz). Samples were inserted in the typical rectangular cavity.

Data acquisition and handling were carried out with the ESR software commercialized by STELAR (Meda, Italy). Temperature was controlled with the Bruker VT 3000 apparatus (accuracy ±0.5 °C).

ESR line shapes were analyzed and computed with the procedure given by Freed and coworkers, as described in refs. [START_REF] Budil | Nonlinear-least-square analysis of slow-motion EPR spectra in one and two dimensions using a modified Levenberg-Marquardt algorithm[END_REF][START_REF] Schneider | Spin labeling. Theory and applications[END_REF]. A double-site simulation was used for all the systems investigated.

Zeta potential runs. Measurements of Zeta potential were performed with a Coulter DELSA 440 SX (Coulter Corporation, Miami, FL, USA). Home made

A C C E P T E D M A N U S C R I P T
hemispherical electrodes, covered by a thin gold layer, were used as measure cell. This allowed to reduce the oxidation that partially affects the silver electrodes currently used in this kind of instruments. Runs were performed as detailed in ref. [START_REF] Ciani | DOTAP/DOPE and DC-Chol/DOPE lipoplexes for gene delivery: Zeta potential measurements and Electron Spin Resonance spectra[END_REF]. Size were also calculated in the same experiments according to the procedure described by Langley [START_REF] Langley | Developments in electrophoretic laser light scattering and some of biochemical applications[END_REF].

Nuclear All spectra were processed using the Bruker Software XW INNMR, version 2.5. Bidimensional spectra were analyzed with the SPARKY software [START_REF] Goddard | Francisco UoCaS[END_REF].

Cryogenic Transmission Electron Microscopy (Cryo-TEM). A Philips CM

120 bio TWIN cryo electron microscope equipped with a post-column energy filter and an Oxford CT3500 cryo-holder and its working station was used as a courtesy of the Department of Physical Chemistry, University of Lund, Sweden.

Samples for cryo-TEM analysis were prepared at room temperature and treated as described in previous papers [START_REF] Rossi | Combined cryogenic transmission electron microscopy and electron spin resonance studies of egg phosphatidylcholine liposomes loaded with a carboranyl compound intended for Boron Neutron Capture Therapy[END_REF][START_REF] Rossi | Aggregated structures in dilute aqueous dispersion of a fluorinated/hydrogenated surfactant system. A Cryo-Transmission-Electron-Microscopy study[END_REF].

Results and discussion

Amphiphilic probes, bearing the paramagnetic unit along the alkyl chain, The best-fit simulation of the ESR spectrum with the NLSL software [START_REF] Freed | EPR in the 21st century[END_REF][START_REF] Budil | Nonlinear-least-square analysis of slow-motion EPR spectra in one and two dimensions using a modified Levenberg-Marquardt algorithm[END_REF][START_REF] Schneider | Spin labeling. Theory and applications[END_REF] shown in Figure 1 was obtained with the magnetic and motional parameters reported in table 1.

The same g values were used for both components. The variation due to the different probe environment in these parameters was within the error range that could be appreciated with the NLSL simulation method.

Both species underwent isotropic motion, but, as expected, the 14 Nhyperfine coupling constant decreased in a more hydrophobic environment: <A N > =1.68 ± 0.01 mT for free CAT-12 and <A N >=1.61 ± 0.01 mT for the inserted radicals.

Figure 2 shows the experimental (black line) and simulated (red line) 298 K ESR spectra of CAT-12 in dsAT/DOTAP/DOPE lipoplexes at increasing r, as prepared by the two methods described in the experimental section. The fitting of ESR absorptions showed that all spectra were the sum of two contributions.

These were simulated using the same best-fit parameters of Table 1. It is important to note that the slow motion ESR component had the same line shape in all the spectra collected in this work. Therefore, there was no difference in the 5c and5d, respectively). Peak assignment for pure DOTAP/DOPE liposomes is reported in a previous paper [START_REF] Martini | Boronphenylalanine insertion in cationic liposomes for Boron Neutron Capture Therapy[END_REF].

The proton signals of phospholipids polar heads at 3.3 ppm, as well as those of alkyl chains at 1.4 and 0.9 ppm in the lipoplex spectra were broadened to a greater extent than the corresponding peaks in dsAT free-liposomes (Figure 5c). This was not unexpected because proton signals of most aggregated systems are broadened beyond detection. This occurs because of longer diffusion times and because the molecular order in the bilayer prevented complete averaging out of dipolar interactions. In the present system line broadening was not severe, and most of the relevant signals were distinct and clearly detectable. The observed broadening was therefore due to the interactions with dsAT. Since it was not possible to detect preferential broadening of the polar heads or of the alkyl chain signals, this effect was attributed to growth of the overall aggregates. The practical disappearance of the dsAT peaks in the r = 0.25 lipoplex (Figure 5a) suggested that in this system the oligonucleotide was completely attached to the liposomes surface. This did not contrast with the above presented interpretation of ESR spectra, since the paramagnetic probe, which reported on its specific behavior in the bilayer environment, was not present in the samples prepared for NMR experiments.

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 12

When in the presence of a higher amount of dsAT (r = 0.75, ), the dsAT peaks were again detectable (Figure 5b). In order to have comparable results, the r = 0.75 lipoplexes were prepared at the same dsAT concentration as in the sample in Figure 5a. This allowed to avoid artifacts due to different dsAT content, which also reduced the intensity of the NMR signals. In all cases, the ratio between the intensity of free dsAT and the liposome peaks remained constant, and only the absolute intensity varied. These observation agreed with the information obtained from Cryo-TEM data on the morphology of the dsAT-containing liposomes in the same conditions where NMR spectra were registered, that is far from the neutrality (Figure 6). As it is known [START_REF] Simberg | Phase behavior, DNA ordering, and size instability of cationic lipoplexes. Relevance to optimal transfection activity[END_REF] Cryo-TEM micrographs of plain liposomes deposited on a positive grid indicate the coexistence of unilamellar, bilamellar and invaginated vesicles. After addition of dsAT a drastic change in the vesicle morphology occurred. Specifically, a wealth of large, mainly multilamellar, complexes were observed both in excess of lipids or in excess of dsAT as it is shown in the Figures 6A and6B, where micrographs of samples with r = 0.25 and r = 0.75 are reported. Liposomes and broken liposomal membranes coexisted with novel assemblies of dense multilamellar structures [START_REF] Simberg | Phase behavior, DNA ordering, and size instability of cationic lipoplexes. Relevance to optimal transfection activity[END_REF][START_REF] Alfredsson | Cryo-TEM studies of DNA and DNA-lipid structures[END_REF][START_REF] Weisman | Nanostructure of Cationic Lipid-Oligonucleotide Complexes[END_REF][START_REF] Huebner | Lipid-DNA complex formation: reorganization and rupture of lipid vesicles in the presence of DNA as observed by cryoelectron microscopy[END_REF]. The observed vesicle fusion, twisting and wrapping on one another was due to the presence of short nuclei acid molecules, that induced strong deformation of the original liposomes. This findings were in good agreement with the external model proposed by Felgner and Ringold [START_REF] Felgner | Cationic liposome-mediated transfection[END_REF] and with ZP and ESR results reported in this work.

The fact that dsAT NMR peaks were detectable in the r = 0.75 sample meant that a fraction of the oligonucleotide did not appreciably interact with the large, slowly tumbling, aggregates. The NOESY spectra recorded for free dsAT and for the r = 0.75 lipoplex, could be exactly superimposed and showed crosspeaks with the same sign of diagonal peaks. It is known that these relative signs depend on the rotational correlation time τ c of the molecule embedded in the aggregates [START_REF] Levitt | Basic of Nuclear Magnetic Resonance[END_REF]. In particular, if the rotational correlation time of the molecules is long (i.e. τ c > 3x10 -10 s), the diagonal and cross-peaks have same sign. This meant that dsAT molecules whose peaks were recovered in the NMR spectrum Concerning the second point, ESR measurements did not detect any disruption of the bilayer into small lamellar fragments. This finding has also proved by NMR analysis and by Cryo-TEM micrographs of selected samples.

Cryo-TEM showed that fusion among different vesicles took place.

A C C E P T E D M A N U S C R I P T

4 relative

 4 to molecular properties obtained by Electron Spin Resonance (ESR) spectroscopy of a cationic spin probe inserted into dsAT/DOTAP/DOPE lipoplexes as compared with the results relative to surface charge (i.e. a bulk property) obtained with Zeta Potential (ZP) measurements in the same systems.

  Lipoplexes were prepared according to two different procedures: a) Samples were prepared, as in a typical titration experiments, by progressively adding the desired amount of a 5 mM (in nucleotides) dsAT stock solution to a lipoplex solution. The starting lipid concentration 1.4x10 -3 M). We simply called titration this preparation procedure. In the case of ESR experiments, 20 µ l of lipoplex suspension were collected for the analysis after each addition of dsAT. b) In a second preparation procedure, each sample was obtained by adding dsAT to a fresh liposome solution. This procedure is indicated in the following as single (point to point) preparation.

  have been extensively employed to obtain information on the dynamics and ordering at the molecular level within aggregates such as micelles and natural or synthetic lipid membranes[START_REF] Martini | ESR and Scattering studies of perfuorinated surfactants in ordered and disordered systems[END_REF][START_REF] Morandi | Association of sugar-based carboranes with cationic liposomes: an electron spin resonance and light scattering study[END_REF][START_REF] Cassera | Differential effects of cholesterol on acyl chain order in erythrocyte membranes as a function of depth from the surface. An electron spin resonance (EPR) spin label study[END_REF][START_REF] Perissi | Electron Spin Resonance and Differential Scanning Calorimetry as combined tools for the study of liposomes in the presence of long chain nitroxides[END_REF][START_REF] Swamy | Coexisting domains in the plasma membranes of live cells characterized by spinlabel ESR spectroscopy[END_REF][START_REF] Šentjurc | Transport of liposome-entrapped molecules into the skin as studied by electron paramagnetic resonance imaging methods[END_REF][START_REF] Stösser | Pharmaceutical aspects of ESR investigation on drug delivery systems, tissues and living systems[END_REF][START_REF] Bartucci | Electron Paramagnetic Resonance investigation of biological systems[END_REF][START_REF] Benatti | Structural and thermal characterization of dioctadecyldimethylammonium bromide dispersions by spin labels[END_REF][START_REF] Caldararu | Structural aspects in self-assembled systems of polyethylene surfactants, as studied by the spin probe technique source[END_REF][START_REF] Erilov | Water concentration profiles in membranes measured by ESEEM of spin-labelled lipids[END_REF][START_REF] Korstanje | Dynamics in lipid vesicles and liposomes studied with ESR: effects of hydration, curvature and unsaturation[END_REF][START_REF] Kristl | Interactions of solid lipid nanoparticles with model membranes and leukocytes studied by ESR[END_REF][START_REF] Marsh | Polarity and permeation profiles in the lipid membranes[END_REF][START_REF] Marsh | High Field Electron Spin Resonance of spin labels in membranes[END_REF][START_REF] Ottaviani | Micellar solutions of sulfate surfactants studied by electron spin resonance of nitroxide radicals: 1. Use of neutral and positively charged spin probes[END_REF][START_REF] Baglioni | Micellar solutions of sulfate surfactants studied by Electron Spin Resonance. Part 2. Use of C8-, C12-, and C16-derivatives of piperidinyl-1-oxy[END_REF][START_REF] Ottaviani | Aggregational process of positively charged surfactants CTAC and CAT16 in the presence of starburst dendrimers: an Electron Paramagnetic Resonance spectroscopy study[END_REF][START_REF] Ottaviani | Interaction between starburst dendrimers and SDS micelles studied by continuous-wave and pulsed Electron Spin Resonances[END_REF][START_REF] Schlick | Spatially resolved magnetic resonance: methods, materials, medicine, biology, rheology, hardware[END_REF][START_REF] Schlick | Advanced ESR methods in polymer research[END_REF]. Here, we used CAT-12, i.e. a positively charged amphiphilic nitroxide, with the aim of: i) mimicking the behavior of the cationic component in the bilayer, and ii) obtaining information onA C C E P T E D M A N U S C R I P Tthe liposome outer surface, where interactions with DNA occur. The probe insertion in the bilayer was demonstrated by the change observed in the ESR spectrum, as reported for similar systems described in the literature[START_REF] Ottaviani | Interactions between starburst dendrimers and mixed DMPC/DMPA-Na vesicles studied by the spin label and the spin probe techniques, supported by Transmission Electron Microscopy[END_REF].

Figure 1

 1 Figure1shows the ESR spectrum of CAT-12 (1% of lipid concentration) incorporated into DOTAP/DOPE membranes. Two components were identified, which accounted for the presence of fast and slowly moving species. This finding is quite usual in similar systems. The first absorption corresponded to spin probe molecules almost free to move in the buffer solution (fast component). CAT-12 in pure PBS solution gave exactly the same spectrum. The second absorption was due to nitroxides inserted into the polar head region of the bilayer (slow component). Different CAT-12/lipid ratios were tested in order to minimize the presence of spin probes free in solution. However, in all the investigated systems a spin probe partition between fast and slow moving regions was found.

10 local

 10 lipid arrangement, at least in the region close to the polar heads were the CAT-12 probes are localized.The relative contributions of the two spectral components clearly depended on the r parameter. The slow motion contribution (as % intensity of the total spectrum) is reported in Figure3for both methods of preparation. Surprisingly enough, the behavior of the slow-motion component intensity as a function of r was almost the same for the two preparation procedures. In particular, after an initial decrease, the slow-motion component passed thorough a minimum at r values which were different for samples prepared by titration and by point to point preparation. Beyond the minimum, the intensity of the slowmotion component recovered a high intensity value, which was only slightly dependent on the preparation procedure. This trend agreed with the ζ behavior shown in Figure4, in which the evolution of ζ for the DOTAP/DOPE liposomes-dsAT system, as a function of charge ratio is reported. The inflections points of ζ for the two methods of lipoplex preparation almost exactly corresponded to the minimum points of the slow moving species in Figure3. Complexes with charge ratio well beyond the isoelectric point prepared by both procedures had the same ζ within the limits of experimental error (≈-40 mV). This was a further proof that the local lipid arrangement did not change with the preparation procedure. This finding was in contrast with the behavior of lipoplexes of large DNA[START_REF] Safinya | Structures of lipid-DNA complexes: supramolecular assembly and gene delivery[END_REF][START_REF] Caracciolo | Multicomponent cationic lipid-DNA complex formation: role of lipid mixing[END_REF][START_REF] Safinya | DNA at membrane surfaces: An experimental overview[END_REF][START_REF] Bordi | Time evolution of the formation of different size cationic liposome-polyelectrolyte complexes[END_REF][START_REF] Martini | Boronphenylalanine insertion in cationic liposomes for Boron Neutron Capture Therapy[END_REF] and suggested that short nucleic acid fragments follow a simpler mechanism of complexation.As a possible explanation for the ESR intensity recover after the ζ inversion points, we suggest that addition of low amount of the negative dsAT to liposome dispersion in the proximity of this point, that is almost at the neutrality of the aggregated surface charge, to which the positive CAT-12 probe was loosely bound, favored the formation of CAT-12-dsAT ion-pairs, almost free to move in solution. The nature of this adduct remained ill defined; anyway the reorientational mobility of the >NO unit maintained the same physical features that it had in the dsAT-free paramagnet. Only when the dsAT concentration increased up to negative surface potential, CAT-12 was progressively embeddedA C C E P T E D M A N U S C R I P Tdown to the lipoplex double layer with resultant decrease of its mobility. This way, the negatively charged lipoplexes were not able to expel the spin probe, and the total amount of the slow motional component recovered the high percentage held of pure liposomes.Further proofs of the fact that the interactions between dsAT and lipid bilayer not drastically modified the structure and the order of liposome bilayer came from NMR of selected samples. Figures5a and 5bshow the 1 H-NMR spectrum of the DOTAP/DOPE lipoplexes at dsAT concentrations below and above the change of the surface charge, i.e. at r = 0.25 (spectrum a) and 0.75 (spectrum b). The NMR spectra of the two reference systems, i.e. pure liposomes and dsAT in PBS solution, are also reported for comparison (Figures

  point were in slow motion conditions alike free dsAT, and showed the same dipolar coupling of this latter. This is a valuable piece of information, which is not easily attainable in solution, especially for large DNA fragments.ConclusionsIn this paper ESR and ζ measurements were performed on DOTAP/DOPE/dsAT lipoplexes prepared by two different procedures. This allowed to demonstrate the following four main points: a) the interactions between dsAT and cationic lipids were strong and occurred at the liposome surface; b) the general shape and physicochemical properties of the lipoplexes did not change when short fragments of nucleic acid were introduced into the lipid bilayer of cationic liposomes before the charge neutralization point; c) the bilayer structure of the liposomal carrier in the lipoplexes was substantially preserved at all charge ratio; d) the physical status of the lipoplexes at electrical charge far from the neutrality did not depend on the preparation method.

Figure 2 .Figure 3 .Figure. 4 .

 234 Figure 2. Experimental (black line) and simulated (red line) ESR spectra at 298 K of CAT-12 inserted into dsAT/DOTAP/DOPE lipoplexes prepared by titration (left) and by point-to-point (right) at different r ratios.

Figure 5 . a) 1 HFigure 6 .

 516 Figure 5. a) 1 H NMR proton spectrum of dsAT complexed with DOTAP/DOPE liposomes (r = 0.25, dsAT concentration = 9.45x10 -3 M); b) dsAT complexed with DOTAP/DOPE liposomes (r = 0.75, dsAT concentration = 9.45x10 -3 M); c) pure DOTAP/DOPE liposomes (lipid concentration = 1.4 10 -2 M); d) uncomplexed dsAT (9.45x10 -3 M). The dissolving medium was 0.1 M PBS solution for all samples and spectra were recorded at 298 K.

  Magnetic Resonance Spectroscopy. Mono and bidimensional (NOESY) NMR experiments were performed with a Bruker DRX-600 Avance spectrometer operating at 600.13 MHz for 1 H. A reverse triple resonance ( 1 H,

13

C, BB) with xyz gradients for two-dimensional experiments was used as probe head. NOESY spectra were acquired with 2048 complex points for 256 experiments with a recycle delay of 5 s and a TPPI phase cycle.

Acknowledgements

Thanks are due to Università di Firenze, to Ministero della Università e della Ricerca, and to Consorzio per le Superfici e per le Grandi Interfasi (CSGI) for financial support. Thanks are also due to dr. Anna Salvati, Department of