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NONLINEAR OPTICAL SPECTROSCOPY OF POPC LIPOSOMES WITH CHOLESTEROL.

Byline: Soheil Sharifi

ABSTRACT: The surface potential and charged density of 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine liposomes with cholesterol is study by using the second harmonic generation. The GouyChapman model is applied to data's to find surface potential of samples. The result shows the surface potential aren't depends to the type of salts but change with the concentration and valence of the electrolyte in solution. The surface potential is changing from 40 to 15 mV with increase of salt concentration from 0 to 5 mM. The charge density was found to be 0.0136 Charge/A 2 which is consistent with the area of a lipid headgroup. Moreover the surface potential of POPC/cholesterol is increasing with increase of cholesterol.

Keywords: Nonlinear Optic Spectroscopy Liposome Nano-structure Second harmonic generation.

Corresponding Author: soheil.sharifi@gmail.com ssharifi@ferdowsi.um.ac.ir

INTRODUCTION:The signal of second harmonic generation (SHG) comes from interaction between light and polarized water close to the lipid of liposomal surfaces. The molecule of water is polarized by the liposomal structure. The SHG is good technique to investigating the electrostatic properties of liposome surface because the SHG methods have interaction with water around of liposome and not exactly with structure of it[1-4]. In general the size of liposomal structure is change from 5 nm to 1 m. So this technique is useful for study the surface potential and charge density of nanostructure in this range of size[56].

Where E(r) is the electric field at a distance r from the charged microparticle surface. The value of (3) is not depend to the distance but E(r)dr is depend to the r. The E(r)dr is the electrostatic potential at the charged surface. It is well known that the SHG field (ESHG) is depend to the nonlinear polarization and it has two part namely (2) and (3) contributions [78].

That S is the surface potential. With change the electrolyte concentration of the aqueous phase the molecules polarized is changing and it can investigate with SHG. The surface potential on the solutions is described with Gouy-Chapman model[9-11]:Equation

That the Boltzmann constant is shows with k the surface potential (FS) and temperature (T) the valence of the electrolyte (Z) the surface charge density(s) dielectric constant (e) and the electrolyte concentration(c) in the solution. The second harmonic field can be expressed as follows:[13-15]Equation

L and M are constant parameters and c is salt concentration that surface potential change with it. It is to be founded that the electrolyte concentration and the valence of the electrolyte can change the second harmonic. Equation 4 can be substituted into eq. 7 leading to the following expression:Equation

where E0 is a constant in the experiment.

In this work we studied surface potential and charge density of the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomal structure with and without cholesterol by using SHG. The surface potential change by adding NaCl MgSO4 Fe2(SO4)3 to the liposomal structure. The structure of POPC liposomal was studied before and this phospholipid shows double layer liposomes[17-20].

EXPERIMENTS:

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was purchased from Avanti polar lipid company and cholesterol (Chol) and chloroform and all type of salts was purchased from Sigma-Aldrich Company Mainz Germany and methanol from Chemical Labs Company Iran. Liposomes containing POPC and cholesterol were prepared using lipid film method. Briefly lipid films consisting of POPC and cholesterol (2:1 M ratio) were prepared in a glass vial by evaporating the chloroform: methanol (2:1 v/v) solution. Traces of organic solvent were removed by keeping the film overnight. The lipid film was then hydrated by adding required amount of distilled water at 55 0C. The resulting multilamellar vesicles (MLVs) were sonicated in a bath-type sonicator (Decon England) to form SUVs (small unilamellar vesicles).

The composition of the liposomal suspension with different present of lipid and cholesterol to water with and without salts is given by the parameter concentration. The experimental solutions were prepared by adding water to the solution. The POPC concentration in the liposomes after mixing with various solutions of electrolytes was kept constant the mass of POPC to the total mass was 0.2.

The salts of Iron(III) sulfate sodium chloride and magnesium sulfate were obtained from Aldrich and put in an oven at 80oC overnight before the preparation of the solutions. All the solutions were prepared in double-distilled water.

The experiments were performed using a pulsed an argon ion laser pumped Ti: Sapphire oscillator. After passing through a half-wave plate and a polarizer the light is gently focused a cylindrical glass sample cell. The scattered light was then collimated with a lens polarization selected and spectrally dispersed on to into the monechromator. The angle of acceptance was at 90o. A photomultiplier tube is used to record the signal from monechromator.

In the experiment the total intensity at frequency 2 is measured that contains SHG signal from the liposome surface and hyper-Rayleigh scattering from aqueous phase. The hyper-Rayleigh scattering originates from orientation

The sizes and polydispersity of the liposome's were estimated from photon correlation spectroscopy experiment we try to keep constant size of liposome by using sonicator. We used dynamic light scattering (DLS) for find the size of liposomal structure. The DLS measurements were performed on filtered samples using Malvern photon correlation spectroscopy instrument at Ferdowsi University of Mashhad. The light source is a He-Ne laser operating at a wavelength of 632.8 nm with vertically polarized light.

3. RESULTS AND DISCUSSION

The Size behavior of liposomal suspension with Salts and Cholesterol was probed with Dynamic light scattering methods. Figure 2 show the correlation function as function delay time for POPC liposomal suspension for pure liposome (down tri-angel) and the liposome with higher concentration of NaCl (up-triangle) MgSO4 (Cross) Fe2(SO4)3(Cubic) and Cholesterol (star) at room temperature. The correlation function for all samples showed a single exponential decay at all concentrations. All the correlation functions in this work were fitted by a single stretchedexponentialfunction.

Boltzmann constant T the absolute temperature and the liquid viscosity. The results of the fitting data's shows the collective diffusion of liposomal suspension is 1.61A-10-11 (m2/S) that hydrodynamic radius RH from stokes law become 150nm.

.The second harmonic field ESHG from the charged surface of POPC liposomes was measured as a function of electrolyte concentration of Fe2(SO4)3(star) NaCl (cubic) and MgSO4(circles) shown in Figure 1. The experimental data was fitted to eq 8 using P Q and s as fitting parameters shown as a solid line in Figure 3. The results of the fitting are summarized in Table 1. The results shows the values of P Q and s obtained from the independent measurements using three electrolytes Fe2(SO4)3 NaCl and MgSO4. The ratio of BkT/Ze of NaCl to that of Fe2(SO4)3 is 3 and NaCl to that of MgSO4 is 2. That is consistent with the prediction of the Gouy-Chapman theory because the valence (Z) of MgSO4 is twice that of NaCl. The charge density obtained from the fitting is about 0.0136 charge/A2. For study the effect of the cholesterol on POPC liposomal structure we used different concentration of cholesterol on liposomal

The results of the fitting with Gouy-Chapman theory are summarized in table 2 and it is showing with increase of cholesterol the charge density increase from 0.0135 to 0.0153 Charge/A 2 by adding cholesterol from 0 to 10 percent. The parameter of P and Q is constant with increase of cholesterol.

Table 1. Fitting Parameters for the Curves of ESHG versus c of

POPC liposomal structure as function of salt concentration using

###the Gouy-Chapman Model.###

###Salt added to###NaCl###MgSO4###Fe2(SO4)3###

###liposome###

###(Charge/A 2)###0.0135###0.0136###0.0136###

###(QKT)/(Ze)###1.6###0.8###0.5###

###P(arb.Unit)###15.2###15.3###15.4

Table 2. Fitting Parameters for the Curves of ESHG versus c of POPC/cholesterol with different cholesterol in lipid as function of salt concentration using the Gouy-Chapman Model.

Cholesterol added to###0%###5%###10%

liposome

(Charge/A 2)###0.0135 0.014 0.0153

(QKT)/(Ze)###1.6###1.4###1.2

P(arb.Unit)###15.2###15.3###15.5

From eq.4 and the fitting parameters in the table 1 we obtained surface potential of POPC liposomal structure as function of different salt concentrations. The fig.5 shows the surface potentials decrease with increase of salt concentration but don't change with type of salt.

The fig.6 is the surface potentials as function of NaCl

Concentration for POPC/cholesterol at three different cholesterol concentrations. This result was obtained from eq.4 and table 2. Our results are show with increase cholesterol to the liposomal structure the surface potential is increasing.

CONCLUSIONS:

We have demonstrated that the outer surface potential and surface charge density of the POPC liposomes with and without cholesterol can be obtained by the SHG technique. The experimental data show agreement with the GouyChapman model. The surface potential measured is in the range of 15-40 mV depending on the valence and concentration of the electrolyte of pure POPC liposomes. The charge density was found to be 0.0136 Charge/A 2 which is consistent with the area of a lipid headgroup. Moreover the surface potential and charge density of POPC/cholesterol is increasing with increase of cholesterol.

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