Microstructural characterization of lysophosphatidylcholine micellar aggregates: The structural basis for their use as biomembrane mimics

Journal of Colloid and Interface Science 336 (2009) 827–833

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Journal of Colloid and Interface Science www.elsevier.com/locate/jcis

Microstructural characterization of lysophosphatidylcholine micellar aggregates: The structural basis for their use as biomembrane mimics Giuseppe Vitiello, Donato Ciccarelli, Ornella Ortona, Gerardino D’Errico * Chemistry Department of Naples University ‘‘Federico II”, Via Cintia, Complesso di Montesantangelo, I-80126 Napoli, Italy CSGI (Consorzio per lo Sviluppo dei Sistemi a Grande Interfase), Firenze, Italy

a r t i c l e

i n f o

Article history: Received 27 February 2009 Accepted 3 April 2009 Available online 12 April 2009 Keywords: Lysophosphatidylcholine (lysoPC) Critical micelle concentration (CMC) Nuclear magnetic resonance (NMR) Electron paramagnetic resonance (EPR)

a b s t r a c t Lysophosphatidylcholines are widely used as biomembrane mimics. In order to furnish a structural basis for this application, in this work the self-aggregation behaviour of n-acyl-lysophosphatidylcholines (CnlysoPC, n = 6,8,10,12), in aqueous solution has been investigated by the PGSTE-NMR and spin probing EPR techniques at 25 °C. The experimental data show that CnlysoPCs behave as zwitterionic surfactants, and permit evaluation of the influence of the acyl chain length on the phospholipid micellization. For all the CnlysoPCs considered, the phospholipid intradiffusion coefficient trend shows a slope change corresponding to the critical micelle concentration (CMC). In the micellar composition range, solubilized tetramethylsilane (TMS) molecules were used to determine the micelle intradiffusion coefficient, from which the aggregate radii and the aggregation numbers were obtained. The solvent intradiffusion coefficient in the CnlysoPC aqueous mixtures has been also measured. The results show that the CnlysoPC micelles present a thick external layer constituted by strongly hydrated glycerophosphocholine groups. The ability of this layer to embed either anionic or cationic guest molecules has been studied by EPR spectroscopy, employing 3-carboxy-PROXYL in its deprotonated form (CP) or TEMPO-choline (TC) as spin probes. In all the considered systems, the nitrogen isotropic hyperfine coupling constant of the spin probe, AN, decreases and the correlation time, sC, increases with increasing phospholipid molality. The results show that CnlysoPC micelles can establish a variety of interaction with different guests. In fact, CP anions interact with the CnlysoPC choline groups adsorbing on the micelle surface, while TC cations interacting with the CnlysoPC phosphate groups are embedded in thick external layer of the micelles. Ó 2009 Elsevier Inc. All rights reserved.

1. Introduction Phospholipids are major components of biological membranes, and for this reason their physicochemical properties have been extensively studied [1]. Generally, phospholipids contain a fatty acid diglyceride, a phosphate group, and an organic hydrophilic moiety such as ethanolamine, serine or choline. Due to their amphipathic character, phospholipids have a great tendency to aggregate, usually forming membrane-like bilayer structures which exhibit complex lyotropic and thermotropic polymorphism. These aggregates have been largely used as cellular membrane models to investigate the membrane interaction with other molecules like peptides or proteins involved in biologically relevant processes [2]. However, the use of bilayers as membrane models presents some technical disadvantages. Dispersion of bilayer aggregates in aqueous medium are opalescent, thus making difficult their use in spectrophotometry, spectrofluorimetry, circular dichroism, and all the other methods in which light scattering is * Corresponding author. Address: Chemistry Department of Naples University ‘‘Federico II”, Via Cintia, Complesso di Montesantangelo, I-80126 Napoli, Italy. Fax: +39 081 674090. E-mail address: [email protected] (G. D’Errico). 0021-9797/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2009.04.008

a problem [3]. Moreover, nuclear relaxation times in bilayer structures are usually very low, so that their use in solution NMR analysis is hampered [4]. Lysophospholipids are amphiphilic single-chain molecules and constitute an important subclass of phospholipids that exhibit unique physical and biological properties not found in their parent dichained phospholipids [5]. Lysophospholipids make up less than 5 mol% of the total phospholipids in a normal cell and higher lysophospholipid concentrations are associated with certain disease states, cell fusion and cell lysis, since they can disrupt the structure of biological membranes. The most important class of lysophospholipids is represented by lysophosphatidylcholines (lysoPC), which are products of phospholipase A2 catabolism of diacylphosphatidylcholine [6]. LysoPC molecules have important biological functions. For example, they regulate a broad range of cell processes [7,8] and inhibit membrane fusion [9,10]. Their ability to micellize is also important in relation to cellular metabolism. In fact, the enzymatic activity of phospholipases toward micellar lipids was apparently much greater than their enzymatic activity toward monomeric lipids [11]. Due to their relatively large hydrophilic group in relation to the hydrocarbon chain, lysophospholipids are more soluble in aqueous

828

G. Vitiello et al. / Journal of Colloid and Interface Science 336 (2009) 827–833 H O

O N

OH

O

O

P

C

O

O

n-3 (a)

O N

O N

C O

(b)

N

O

Na

Cl

+

(c)

Phosphocholine (C10lysoPC), and 1-Lauroyl-2-Hydroxy-sn-Glycero-3-Phosphocholine (C12lysoPC), with a purity > 99%, were obtained from Avanti Polar Lipids (Birmingham, AL, USA). The molecular weights of C6lysoPC, C8lysoPC, C10lysoPC, and C12lysoPC were assumed 355.37, 383.42, 411.47, and 439.53, respectively. The solvent used for PGSTE-NMR measurements was D2O obtained from Sigma (purity > 99.96%) while double-distilled water was the solvent used for EPR measurements. All solutions were prepared by weight. Solubilized tetramethylsilane (TMS), used to measure the micelles’ intradiffusion coefficients, was a Sigma product with purity > 99.9%. The spin probes 4-(N,N-dimethyl-N-(2-hydroxyethyl))ammonium-2,2,6,6-tetramethylpiperidine-1-oxyl chloride (TEMPO-choline) and 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (3-carboxy-PROXYL) were obtained from Molecular Probes (purity > 99%) and Sigma–Aldrich (purity > 98%), respectively. All the experimental data are available by request to the authors.

OH

Fig. 1. Molecular structures of (a) CnlysoPC, (b) deprotonated 3-carboxy-PROXYL (CP), (c) TEMPO-choline (TC).

medium with respect to their di-chained analogues, and they can aggregate into micelles above a critical micelle concentration (CMC) [12,13]. These micellar aggregates can self-organise, with increasing phospholipid concentration, to form a cubic phase [13,14]. Micelles have been largely used as mimics of biological membranes in all those techniques in which bilayers cannot be used. However, phospholipid self-organization in micelles can be very different with respect to that in bilayer structures, and this can strongly affect the interaction with peptides and proteins. In literature, physicochemical and micro-structural characterization of lysophospholipid micelles is not exhaustive. In the present work, we report the results of the physicochemical characterization of the self-aggregation properties of lysoPC’s with different acyl chain lengths (CnlysoPC, n = 6, 8, 10, 12 see Fig. 1 for the molecular structure). A combined experimental strategy, including Pulsed field Gradient STimulated Echo (PGSTE)-NMR and spin probing electron paramagnetic resonance (EPR), has been adopted. Indeed, spectroscopic techniques have been shown to be suitable for microscopic characterization of domains generated by surfactant aggregation as well as for investigation on intermolecular interactions. PGSTE-NMR provides accurate intradiffusion coefficients for the random thermal motion of surfactants in systems of uniform chemical composition, allowing to put in evidence the formation of micelles, and to estimate their dimension [15,16]. Moreover, the water mobility can be followed, furnishing information on the solute hydration [16,17]. The EPR spectroscopy, via the introduction in the system of paramagnetic molecular probes [18], is particularly suitable for the characterization of intermolecular interactions in micellar systems, taking advantage by the direct relation existent between the spectra of spin probes and the micro-environment in which they are embedded [19,20]. The focus of our research is on two main aspects: the influence of the acyl chain length on the CnlysoPC self-aggregation process, in terms of CMC and aggregation number, and the peculiarities of hydrophilic external layers of CnlysoPC micelles in interacting with charged guest molecules. 2. Experimental section 2.1. Materials The phospholipids 1-Hexanoyl-2-Hydroxy-sn-Glycero-3-Phosphocholine (C6lysoPC), 1-Octanoyl-2-Hydroxy-sn-Glycero-3-Phos1-Decanoyl-2-Hydroxy-sn-Glycero-3phocholine (C8lysoPC),

2.2. Intradiffusion measurements Experiments for determination of intradiffusion coefficients were performed on a Bruker 600 DRX spectrometer equipped with a cryo probe. For each water–CnlysoPC system, PGSTE-NMR measurements allowed to determine separately the intradiffusion coefficients of the phospholipid, DP, and water, DW, as a function of the concentration. 1H NMR experiments for diffusion measurements were performed at 25 °C using a stimulated echo sequence with bipolar gradient pulses and one spoil gradient. The sample temperature was controlled within 0.1 °C during measurements by passage of controlled-temperature air through the sample holder. For a system of monodisperse diffusing particles, the PGSTENMR echo signal, I, is given by [21,22]:

IðkÞ ¼

   I0 2s T M þ þ kD exp  2 T2 T1

ð1Þ

where k = c2g2d2 (D  d/3). c is the magnetogyric ratio of the proton, g is the strength of the magnetic field gradient pulses, d is their duration, and D is the distance between the leading edges of the gradient pulses. In this work, echo delays were kept constant so that the relaxation effect would not be accounted for; D was set to 100 ms and the pulsed gradients, with a duration of 2–8 ms depending on the sample, were incremented from 3% to 96% of the maximum strength in 32 equally spaced steps. I0 is the equilibrium magnetization, s and TM are the separations of the rf-pulses of the stimulated spin–echo sequence. T2 and T1 are the transverse and the longitudinal relaxation time, respectively. Eq. (1) can be linearised by reporting ln(I/I0) as a function of k. CnlysoPC intradiffusion coefficient, DP, was determined by following the signal of the CH3 groups’ protons adjacent to the nitrogen atom of the choline moiety (chemical shift = 3.2 ppm). Water intradiffusion coefficient, DW, was determined by following the signal at 4.8 ppm, due to the HDO impurity of the deuterated solvent. The experimental errors on the intradiffusion coefficients were generally less than 2%. 2.3. EPR spectroscopy EPR spectra were obtained employing a Bruker ELEXYS e500 Xband spectrometer equipped with a TE001 cavity. The instrument parameters were as follows: modulation amplitude 0.16 G to avoid signal over modulation, time constant 1.28 ms, receiver gain 60 dB, microwave power 2 mW (20 dB) to prevent saturation effects. We have used two different spin probes containing a cyclic nitroxide: 3-carboxy-PROXYL in its deprotonated form (CP) and TEMPOcholine (TC), see Fig. 1 for their molecular structure.

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DBðmI Þ ¼ A þ BmI þ Cm2I

ð2Þ

where mI is the nitrogen nuclear spin quantum number. As described in a previous work, [19] the values of A, B, and C were determined by means of a least-squares fitting routine of experimental spectra using Eq. (2). In turn, their values allow calculation of AN and sC. The precision on sC values is about 5%, while the experimental error on AN values is estimated to be about 0.01 G.

DP x1010/m2s-1

.0 4.0

4.0 4.

A

3.0 3.

3.0 3.

2.0 .0

2.0 2.

.0 1.0

1.0 1.

0.0 .0 0.0

0.4 0.

m

0.8 0.

DP x1010/m2s-1

1.2 1.

0.0 0. 0.0

0.2

2.0 2.

2.0 2.

1.0 1.

1.0 1.

0.02 0.

m

3.0 3.

C

0. 0.0 0.00

B

C 6 lisoPC

3.0 3.

0.04 0.

0.06 0.

0.4

0.6 0.

0.8

0.06 0.0

0.08 0.0

C 8 lisoPC

D

0. 0.0 0.00 0.0

0.02 0.

0.04

mC12 lisoPC

mC10 lisoPC

DP x1010 /m2s-1

Fig. 2. () Phospholipid intradiffusion coefficients, DP, and (j) TMS intradiffusion 1 ), in coefficients, DM P , as a function of the phospholipid molality (in mol kg CnlysoPC aqueous solutions at 25 °C. (A) C6lysoPC; (B) C8lysoPC; (C) C10lysoPC; (D) C12lysoPC.

4.0 4.

4.0 4.

3.0 3.

3.0 3.

2.0 2.

2.0 2. 1.0 1.

1.0 1. 0.0 0.

A 0

2

0.0 0. 4

6

B 0

10

3.0

3.0 3.

2.0 2.

2.0 2.

1.0

1.0 1.

0.0 0

C 100 10

200 20

300

400 40

500

20

30

1/m /mC 8 lisoPC

1/mC 6 liso soPC PC

DP x1010 /m2s-1

Stock aqueous solutions of CP and TC (1.0  104 mol kg1) were prepared by weight and used as solvent for preparing the samples at various surfactant molalities. The spin probe molality was chosen in such a way that only one spin probe molecule can interact per micellar aggregate, thus avoiding spin-exchange between different spin probe molecules. The pH of CP solution was adjusted to 7.0 by adding small quantities of concentrated aqueous NaOH and it was measured by a Radiometer pH-meter, model PHM240, equipped with a double-junction reference electrode. At pH = 7 the carboxylic group of CP (pKa = 4.0) [23] is almost completely deprotonated, and CP is responsible for the EPR signal. The EPR spectra were performed at 25 °C at CnlysoPC concentrations below, near and above the critical micelle concentration. All sample solutions were put in capillary quartz tubes (0.5 mm i.d.). Since we checked, by prolonged bubbling the solutions with pure nitrogen, that the presence of oxygen in solutions slightly modified the EPR linewidth, we degassed the solutions before insertion into the capillaries. For all the considered samples, the EPR spectrum shows the typical triplet of narrow lines due to hyperfine coupling of the unpaired electron with the 14N nucleus. The narrow line width indicates a fast isotropic tumbling of the spin-probe. In these conditions, as detailed in Section 3, information about the local physicochemical properties of the solubilization site can be obtained by the analysis of the nitrogen isotropic hyperfine coupling constant (AN) and the correlation time (sC) of the nitroxides, determined by the fitting procedure described below. AN depends on the polarity of the medium in which the nitroxide is embedded; in particular, its value increases with both the solvent polarity and Hbonding ability [20,23]. At the same time, sC clearly shows changes in the probe rotational mobility, as determined by the microenvironment viscosity and/or by specific interactions [19,20]. According to the classical theory of motional narrowing of EPR lines [24] the nuclear spin state dependence of the width of the hyperfine line of a nitroxide, DB, is described by the formula:

0.0 0.

D 0

500

000 100

150 500

2000

1/mC12 llisoP 1/ soPC

1/mC10 lilisoPC

Fig. 3. Phospholipid intradiffusion coefficients, DP, as a function of the inverse phospholipid molality (in mol1 kg), in CnlysoPC aqueous solutions at 25 °C. (A) C6lysoPC; (B) C8lysoPC; (C) C10lysoPC; (D) C12lysoPC.

3. Results 3.1. Phospholipid intradiffusion data The experimental intradiffusion coefficients of CnlysoPC in aqueous mixtures are shown, as a function of the phospholipid molality, in Fig. 2A–D. In all cases, DP shows a sigmoidal trend which presents a change of slope at the CMC. An alternative evaluation of the CMC is obtained if the intradiffusion coefficient is reported as a function of the reciprocal molality, as shown in Fig. 3A– D. In this case two straight lines, intersecting at the CMC, are obtained. The measured CMC values are collected in Table 1. They are in good agreement with literature values reported in the same table [25]. The trend of log10 (CMC) vs the number of carbon atoms in the phospholipid acyl chain, n, is well described by the following linear relation:

log10 ðCMCÞ ¼ A þ Bn

ð3Þ

with A = 2.56 ± 0.07 and B = 0.465 ± 0.008. In the case of nonionic surfactants, the plot of log10 (CMC) vs n usually presents a linear trend with slope 0.5 [26], while the slope is 0.3 for ionic surfactants

[27]. Zwitterionic surfactants present a slope value intermediate between these limits, depending on the surfactant headgroup structure [28]. Thus, from this viewpoint, the CnlysoPC behaviour seems to be similar to that of nonionic surfactants. In the premicellar composition range, the DP data may be fitted as a function of phospholipid molality:

DP ¼ DP ð1 þ amÞ

ð4Þ DP

The fitting parameters, and a, are reported in Table 1. The DP values linearly scale with the hydrophobic chain length ðDP =1010 m2 s1 ¼ ð5:3  0:02Þ  ð0:27  0:02Þ  nÞ with a slope which is similar to that found for nonionic ethoxylated surfactants [29]. The absolute a values increase with n, indicating that hydrophobic interactions establish among the phospholipid monomers. Above the CMC the phospholipids start to self-aggregate, so that monomers and micelles coexist. The exchange between the two populations, i.e. between free and micellized phospholipid molecules, is much faster than the diffusion observation time, and consequently the experimental phospholipid intradiffusion coefficient

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G. Vitiello et al. / Journal of Colloid and Interface Science 336 (2009) 827–833

Table 1 Critical micelle concentration and limiting intradiffusion coefficients for the CnlysoPC aqueous mixtures, as evaluated by PGSTE-NMR spectroscopy.

C6lysoPC C8lysoPC C10lysoPC C12lysoPC

CMCa (mol kg1)

DP  1010 ðm2 s1 Þ

a (mol 1 kg)

DM;CMC  1011 ðm2 s1 Þ P

b (mol1 kg)

r  109 (m)

l  109 (m)

0.6 0.7 0.007 0.001

– 0.057 0.0057 0.0006

3.82 ± 0.03 3.24 ± 0.03 2.60 ± 0.02 2.20 ± 0.02

0.57 ± 0.02 1.23 ± 0.18 6.9 ± 1.7 18 ± 7

13.9 ± 1.6 11.2 ± 0.6 9.33 ± 0.03 7.56 ± 0.06

0.97 ± 0.14 0.84 ± 0.04 0.94 ± 0.12 0.98 ± 0.06

1.4 1.8 2.2 2.6

0.65 0.91 1.16 1.41

Data from Ref. [25].

DP ¼ pF DFP þ ð1  pF ÞDM P ¼

mF F ð1  mF Þ M DP D þ m m P

ð5Þ

where pF is the fraction of amphiphile in the monomeric state and mF is the free monomer molality which, in the phase separation model [31], can be assumed to be equal to the CMC. We experimentally estimated DM P by the addition of tetramethylsilane (TMS) to the system. In fact, for a compound which is entirely confined into the micelles and has a negligible solubility in the intermicellar solution, which is an aqueous solution of lipid monomers, the observed intradiffusion coefficient will be the same as the intradiffusion coefficient of the micelles [32]. To be sure that the TMS insertion does not change the shape and dimension of micelles, two measurements were performed for each solution, before and after the TMS addition, checking that the phospholipid intradiffusion coefficients were the same. Inspection of Fig. 2A–D shows that DM P ! DP as the phospholipid molality increases and the monomeric contribution becomes negligible, see Eq. (5). Furthermore, DM P decreases with increasing phospholipid concentration. This evidence could be due to the combination of two effects: intermicellar interactions and change of micelle size. Since the CMC can be considered as the infinite dilution for micelles, the concentration dependence of DM P can be appropriately expanded as a function of (m  CMC) [16], M;CMC DM ½1 þ bðm  CMCÞ P ¼ DP

ð6Þ

The fitting parameters of Eq. (6) are also collected in Table 1. The micelle intradiffusion coefficient extrapolated at the CMC, , can be related to the hydrodynamic size of the aggregates DM;CMC P by using the Stokes–Einstein equation to calculate the apparent radius, r:

r¼

kB T 6pgDM;CMC P

ð7Þ

where g is the viscosity of the aqueous medium. The r values obtained through Eq. (7), collected in Table 1, are compatible with a nearly spherical shape. It is possible to compare these values with those of the extended hydrophobic chain of the phospholipid, l, computed according to the Tanford relation [33]. In doing that, the carbonyl group has been considered as part of the hydrophilic headgroup. Inspection of Table 1 shows that, for all considered phospholipids, l is lower than r, indicating that the CnlysoPC micelle structure present a thick hydrophilic layer (r  l 1  109 m).

2.0

DW x10 9 /m2s-1

is a mean value between that of free monomers, DFP , and that of the micellized molecules, DM P [30]. Thus:

2.0

A

1.5

B

1.5

1.0

1.0

0.5

0.5

0.0 0.0 0.

0.4

0.8 0.

1.2

0.0 0.0

0.2 0.

1.90 1.

1.90

C

1.85

1.85

1.80 1.

1.80

1.75 1. 0.00 0.

0.02 0.

0.4 0.

0.6 0.

0.8

0.06 0.0

0.08 0.

mC 8 lisoP soPC

mC 6 lilisoPC

DW x10 9 /m2s-1

a

CMC (mol kg1)

0.04 0.

0.06 0.

1.75 0.00 0.0

D

0.02 0.0

0.04 0.0

mC

mC10 lilisoPC

12

lisoPC li

Fig. 4. Solvent intradiffusion coefficients, DW, as a function of the phospholipid molality (in mol kg1), in CnlysoPC aqueous solutions at 25 °C. (A) C6lysoPC; (B) C8lysoPC; (C) C10lysoPC; (D) C12lysoPC.

H;m DW ¼ pFW DFW þ pH;m W DW ¼

nFW F nH;m DW þ W DH;m nW nW W

ð8Þ

In the molality scale, nW = 55.5, while the moles of water molecules hydrating phospholipid monomers equal to the phospholipid molality multiplied by the number of water molecules hydrating m each phospholipid molecule (nH;m W ¼ m  h ). In the hypothesis that F DW is the self-diffusion coefficient of neat water, DW , and hydrating water molecules diffuse together with the hydrated phospholipid ðDH;m W ¼ DP Þ, fitting of Eq. (8) to the premicellar DW data allows estimating hm. The values, collected in Table 2, are of the same order of those obtained for other surfactants [17,34]. Intradiffusion coefficients allow to estimate the total number of water molecules whose motion is slowered down by the interaction with a solute. Particularly, in the case of amphiphilic molecules, such as monomeric CnlysoPC, this approach includes, besides the water molecules hydrating the polar headgroup, those forming the cage around the hydrophobic moieties of the solute. This justifies the evidence that hm values increase with n, see Table 2. Above the CMC, the smaller reduction of DW with increasing phospholipid concentration indicates that the number of water

3.2. Solvent intradiffusion data The water intradiffusion coefficient, DW, in water–CnlysoPC mixtures is shown in Fig. 4A–D. In all cases, it decreases with an increase of the phospholipid concentration, showing a slope change at the CMC. In the premicellar region, the decreasing DW trend can be attributed to the fact that some water molecules enter the monomer hydration shell, thus becoming slower with respect to the free ones. Quantitatively, it can be written that:

Table 2 Phospholipid hydration and aggregation numbers for the CnlysoPC aqueous mixtures, as evaluated by PGSTE-NMR spectroscopy.

C6lysoPC C8lysoPC C10lysoPC C12lysoPC

hm

hM

N

73 ± 2 98 ± 6 160 ± 30 209 ± 40

28 ± 4 25 ± 1 22 ± 4 20 ± 2

10 ± 2 19 ± 4 34 ± 4 55 ± 7

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G. Vitiello et al. / Journal of Colloid and Interface Science 336 (2009) 827–833

molecules hydrating the micellized phospholipid is lower than that of water molecules hydrating monomers. Indeed, upon the CnlysoPC self-aggregation, the water molecules involved in the hydrophobic interaction with the acyl chain are released as free molecules in the aqueous bulk. Quantitatively, in the micellar composition range the water intradiffusion coefficient, DW, is an average value between the contributions of free water molecules and molecules hydrating the phospholipid monomers and micelles: H;m H;M H;M DW ¼ pFW DFW þ pH;m W DW þ pW DW

nFW F nH;m nH;M W DW þ W DH;m DH;M W þ nW nW nW W

ð9Þ

The diffusivity of water molecules hydrating micellized phospholipid can be assumed to be equal to that of the micellar aggregates, which is experimentally determined by following the M H;M TMS signal (DH;M W ¼ DP Þ. nW are the moles of water hydrating micellized CnlysoPC, and can be computed as the hydration number of aggregated phospholipid multiplied by its molality ðnH;M W ¼ M ðm  CMCÞ  h Þ. Above the CMC, the micelles exert an obstruction effect on the free water molecules as well as on the phospholipid monomers, and consequently on the water molecules involved in their hydration. Because of the obstruction, DFW is lower than the is lower than the self-diffusion coefficient of neat water, and DH;m W DP value observed in premicellar composition range. In the literature, expressions have been given to account for this effect. Assuming a spherical shape for the micelles under consideration, the following relation holds [35]:

DFW ¼ DW

1 1 þ UM

ð10Þ

where UM is the volume fraction of the obstructing particles, i.e. the micelles, which can be estimated, through an iterative procedure [36], from the radius of the micellar aggregates and their concentration. Fitting of Eq. (9) to the experimental DW data, in the micellar concentration range, allows to obtain the hM values summarized in Table 2. The average hM values for CnlysoPC is 24 ± 3. This indicates that the thick layer formed by phospholipid headgroups in the micelles is strongly hydrated. Indeed, perusal of the table reveals that hM slightly decrease with increasing the phospholipid acyl length. This is probably related to the reduction of the micelle curvature, which brings the headgroups close to each other. The r and hM values can be used to compute the aggregation number of the micelles, N. In fact, assuming a spherical shape, N can be computed by the ratio between the experimental and calculated hydrodynamic volumes, according to the relation:

N¼

4 3

pr 3 M

þ h VW

where V M P is the molecular volume of the micellized phospholipid [37] and VW is the molecular solvent volume. The N values obtained through Eq. (11), collected in Table 2, are of the same order of magnitude of those obtained for ionic surfactants [15,16], generally lower than those expected for nonionic ones [38].

16.25

16.15

12

16.15

12

16.05

8

16.05

8

15.95 0.0

A

0.4

0.8

4 1.2

15.95 0.0

B

0.2

mC 6 lisoPC 16.26

0.6

4 0.8

10

16.26

16.24

8

16.24

8

16.22

6

16.22

6

16.20 0.00

0.05

0.10

mC10 lisoPC

0.15

4 0.20

16.20 0.00

10

D

0.04

0.08

τC x 1012/s

AN /G

0.4

mC8 lisoPC

C

3.3. EPR data In previous works, we have shown that EPR is a suitable technique to study the ability of ionic surfactants to interact with charged nitroxides, used as spin probes [19,20]. Here, we investigate how the zwitterionic CnlysoPC headgroup interacts with both cationic (TC) and anionic (CP) radicals. Much attention has to be paid in deducing microstructural information from spin probes spectroscopic parameters. However, the dependence of the EPR parameters of these radicals on the environment polarity, and/or

16

16

16.25

ð11Þ

τC x 1012/s

VM P

AN /G

¼

on the formation of H-bonds with water molecules has been deeply investigated, in previous study, by combining EPR and ab initio computational results. This makes us more confident in the reliability of our deductions [20,23]. For both the spin probes, in all the systems considered in the present work, the EPR spectrum shows the typical lineshape of fast motion, without superimposition of different signals and/or sudden increase of the line width. This evidence indicates that the chemical exchange rate of the spin label between aqueous bulk and micelles, mCE, is very high with respect to the separation between the EPR lines of the spin probes in both environments, Dx. In this case the experimental EPR spectrum is a weighted average of the signals due to interacting and noninteracting radicals, and can be adequately treated as due to a spin probe presenting a single isotropic motion [39]. In contrast, for amphiphilic spin labels inserted in the micellar aggregates, at least two kind of motional processes have to be considered: local fast molecular reorientations occurring in the motional narrowing regime and aggregate tumbling and lateral diffusion occurring in the intermediate motional regime [40]. The trend of the nitrogen coupling constant of CP and TC, AN, and correlation time, sC, as a function of the phospholipid molality, m, are shown in Fig. 5A–D and Fig. 6A–D, respectively. In all cases, AN decreases and sC increases with increasing m, indicating that the spin probes experience a progressively less polar environment and that their rotational motion is slackened. However, perusal of the figures reveals that the extent of these variations depend on the considered spin probe and CnlysoPC. Note that, in order to make the comparison easier, the figures relative to the same phospholipid (e.g. Figs. 5A and 6A for C6lysoPC) present the same scale on each axis, even if on different absolute values. Indeed, the EPR parameters also depend on the peculiar molecular structure of each spin probe, thus preventing a direct comparison between their absolute values. C6lysoPC establishes comparable interactions with both TC and CP, as evidenced by the similar magnitude of the AN and sC variations. In the case of C8lysoPC, while the AN variation is still comparable, sC increases much more markedly for TC than for CP, indicating that the cationic probe experiences a more viscous microenvironment than the anionic one. For C10lysoPC and C12lysoPC, the CP EPR parameters show very slight changes, while for the TC ones the variation is more evident. Particularly, the sC increase becomes progressively higher with increasing the phospholipid acyl chain. These evidences can be interpreted in terms of the structure of the micellar aggregates formed by the various CnlysoPC. With increasing the phospholipid acyl chain, the curvature

4 0.12

mC12 lisoPC

Fig. 5. () Nitrogen hyperfine coupling constant, AN, and (j) correlation time, sC, of CP as a function of the phospholipid molality (in mol kg1), in CnlysoPC aqueous solutions at 25 °C. (A) C6lysoPC; (B) C8lysoPC; (C) C10lysoPC; (D) C12lysoPC.

832

G. Vitiello et al. / Journal of Colloid and Interface Science 336 (2009) 827–833 17

16.8

16.7 16.

13

16.7

13

16.6 16.

9

16.6

9

A

16.5 16. 0.0 0.

0.4 0.

0.8

mC 6 lisoPC

16.78 16.7

5 1.2

16.5 0.0

16.78

16.76 16.7

9

16.76

16.74 16.7

7

16.74

AN /G

16.72 16.7 0.00 0.0

0.05 0.0

0.10

m

0.15 0.1

C10 lisoPC

5 0.20 0.

16.72 0.00

0.2

0.4

mC8 lilisoPC

0.6

11

D

9 7

0.04

0.08

mC12 lisoP soPC

5 0.12

of micelle surface decreases, and consequently the hydrophilic headgroups come in closer contact. This could also be the reason of the slight decrease of hM observed in Table 2. In the case of CP ions, which interact with the terminal choline group, these changes result in a ‘‘squeezing” of the spin probes from the micelle interior. In other words, CP ions are preferably adsorbed on the micellar surface, with the nitroxide protruding in the aqueous medium, as put in evidence by the limited AN and sC variations. In contrast TC ions, which interact with the inner phosphate group, are embedded in the hydrophilic external layer of the micelles, which becomes more compact and viscous with increasing the phospholipid acyl chain, thus explaining the sC increase. Another consideration has to be done by comparing the trends of the EPR parameters of the cationic probe TC: in the case of C6lysoPC and C8lysoPC, only a slight slope change is detectable in correspondence of the CMC. In the case of C10lysoPC and C12lysoPC, they show an almost abrupt slope change. This evidence confirms that micellization is not a first order transition, and becomes progressively sharper with increasing the phospholipid acyl chain [15,16]. Indeed, for C10lysoPC and C12lysoPC, above the CMC, the TC EPR parameters show a typical saturation trend. Because of the low CMC values, spin probes interaction with the monomers can be neglected, so that in the micellar concentration range, the evaluated AN are mean values between the nitrogen coupling constant of TC localized in the aqueous bulk, AW N and that of TC interacting with the micelles, AM N [39]:

nW nM TC W AN þ TC AM nTC nTC N

ð12Þ

M where nW TC and nTC represent the moles of TC in the aqueous and in the micellar medium, respectively; nTC = nTCW + nTCM is the total number of TC moles in the system. Rizzi et al. defined the micelle–water distribution coefficient of a spin probe as [41]:

Kd ¼

M nM TC =n W W nTC =n

AM N ðGÞ

Kd

12 sM (s) C  10

16.63 ± 0.02 16.65 ± 0.02

150 ± 11 250 ± 30

10.9 ± 0.2 15.4 ± 0.3

4. Discussion

Fig. 6. () Nitrogen hyperfine coupling constant, AN, and (j)correlation time, sC, of TC as a function of the phospholipid molality (in mol kg1), in CnlysoPC aqueous solutions at 25 °C. (A) C6lysoPC; (B) C8lysoPC; (C) C10lysoPC; (D) C12lysoPC.

AN ¼

Table 3 Isotropic hyperfine constant and correlation time of TC in CnlysoPC micellar environment.

C10lysoPC C12lysoPC

5 0.8

τ C x 1012 /s

11

C

17

B

τ C x 1012 /s

AN /G

16.8 16.

ð13Þ

where nW is number of water moles in the medium and nM is the number of moles of micellized phospholipid (in our case, nW = 55.5 and nM = m  CMC). By fitting Eqs. (12) and (13) to the experimental data we obtained the Kd and AM N values that are collected in Table 3. The AM N value is the same for both C10lysoPC and C12lysoPC, while the Kd value only slightly increases. Concerning sC, in the lack of a quantitative theory, its value in micellar environment can be estimated as the intercept of the plot of sC as a function of 1/m. The sM C value is significantly higher for C12lysoPC than for C10lysoPC, see Table 3.

Our investigation has focused on the behaviour of aqueous mixtures of monoacylglycerophosphatidylcholines, CnlysoPC, with a number of carbon atoms in the saturated acyl chain ranging between 6 and 12. Through PGSTE-NMR measurements, both the phospholipid and solvent intradiffusion coefficients were determined. Analysis of these data reveals that, in aqueous solution, CnlysoPCs behave as typical surfactants. They self-aggregate forming micelles and this process is well described by the phase separation model [31]. Micellization is driven by the hydrophobic interaction between the CnlysoPC acyl chains, and consequently, the CMC decreases with increasing n. Interestingly, we found that this decreasing trend presents a slope similar to that reported for non-ionic surfactants. The intradiffusion coefficients of micellar aggregates permitted to determine their hydrodynamic radii, which were found to be compatible with a spherical geometry. The phospholipid aggregation number increases with n. Analysis of the solvent intradiffusion coefficients clearly showed the reduction in the number of hydrating water molecules when a phospholipid molecule passes from the monomeric to the micellized form. The release of hydration water molecules upon phospholipid self-aggregation is the fundamental driving force of the micellization process. Nevertheless, a strong hydration of CnlysoPC headgroups remains also in the micelles. Comparison between the micellar aggregate radius and the length of the extended acyl chain shows that CnlysoPC micelles present a thick hydrophilic external domain, constituted by hydrated glycerophosphatidylcholine groups. In this respect, it is interesting to highlight that the average hM value for CnlysoPC (24 ± 3) is much higher than the value reported for synthetic phosphocholines (hM 15, Ref. [42]), in whose molecular structure an aliphatic alkyl chain is directly bonded, through an ether oxygen, to the phosphate group. This difference is due to the hydration sphere of hydroxyl group in the position 2 of glycerol, and to that of the carboxylic group binding the glycerol and the acyl chain. In this context, it is interesting to observe that the high number of hydration molecules, which further magnifies the steric repulsion among the bulky CnlysoPC headgroups, is likely to be the reason of the low aggregation number of this phospholipids. To test the ability of CnlysoPC headgroup to electrostatically interact with charged guests, we used the spin probing EPR approach. The results show that CnlysoPC micelles, like other zwitterionic ones [43], can interact with both anionic and cationic guests. Anionic molecules preferably adsorb on the micellar surface, while cationic ones can be embedded in the hydrophilic external layer. 5. Conclusions This paper has reported the results of a micro-structural characterization of the micellar aggregates formed by lysophosphatidylcholines, which are zwitterionic phospholipids presenting a single acyl chain. Particularly, the dependence of the aggregation behaviour on the length of this chain has been investigated. Our experimental results have shown that lysophosphatidylcholines behave as zwitterionic surfactants, forming micellar aggre-

G. Vitiello et al. / Journal of Colloid and Interface Science 336 (2009) 827–833

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