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Influence of iron oxide nanoparticles on bending elasticity and bilayer fluidity of phosphotidylcholine liposomal membranes
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Influence of iron oxide nanoparticles on bending elasticity and bilayer fluidity of phosphotidylcholine liposomal membranes Poornima Budime Santhosh a , Sophia Ivanova Kiryakova b , Julia Lyubomirova Genova b , Nataˇsa Poklar Ulrih a,c,∗ a
Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia Institute of Solid State Physics, Bulgarian Academy of Sciences, 72, TzarigradskoChaussee Blvd., 1784 Sofia, Bulgaria c Centre of Excellence for Integrated Approaches in Chemistry and Biology of Proteins (CipKeBiP), Jamova 39, 1000 Ljubljana, Slovenia b
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
• Liposomes loaded with bare and silica coated iron oxide particles were prepared. • Silica coated particles have stronger effect on elasticity and membrane fluidity. • Silica coating provide high negative surface potential to the nanoparticles. • Silica coated nanoparticles destabilize the membrane leading to altered properties.
a r t i c l e
i n f o
Article history: Received 30 October 2013 Received in revised form 10 February 2014 Accepted 11 February 2014 Available online xxx Keywords: Fluidity Iron oxide nanoparticles Anisotropy Bending elasticity Liposomes
a b s t r a c t Iron oxide nanoparticles with improved surface characteristics have tremendous applications in various biomedical fields such as magnetic resonance imaging, hyperthermia, immunoassay and targeted drug delivery. The aim of this work was to study the influence of iron oxide (␥-Fe2 O3 ) nanoparticles on the bilayer fluidity and bending elasticity of zwitterionic phosphatidylcholine liposomal membranes. Small unilamellar vesicles prepared with l-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine lipids were encapsulated with uncoated iron oxide nanoparticles and silica coated iron oxide nanoparticles to study their effect on bilayer fluidity. Anisotropy measurements using the fluorescent probes 1,6-diphenyl-1,3,5hexatriene and N,N,N-trimethyl-4-(6-phenyl-1,3,5-hexatrien-1-yl) phenylammonium p-toluensufonate did not show a significant difference in the lipid ordering and bilayer fluidity. Thermally induced shape fluctuations of the giant quasi-spherical lipid vesicles were used to study the influence of both types of iron oxide nanoparticles on the bending elasticity modulus kc of the lipid membrane. The results showed that in the case of uncoated iron oxide (in the studies concentration) the obtained value for the bending elasticity modulus does not differ in the frames of the experimental error from that of pure phospholipid membrane. In the case of silica coated iron oxide nanoparticles the bending elasticity modulus of the membrane decreased by 25%. © 2014 Published by Elsevier B.V.
1. Introduction ∗ Corresponding author at: University of Ljubljana, Department of Food Science and Technology, Biotechnical Faculty, Jamnikarjeva 101, 1000 Ljubljana, Slovenia. Tel.: +386 1 3230780; fax: +386 1 2566296. E-mail address: [email protected] (N.P. Ulrih).
Liposomes represent a simple and convenient model system to study the membrane properties. Liposomes encapsulating the magnetic nanoparticles (NPs) termed as magnetic liposomes act as a promising tool in targeted drug delivery and contrast agents
http://dx.doi.org/10.1016/j.colsurfa.2014.02.035 0927-7757/© 2014 Published by Elsevier B.V.
Please cite this article in press as: P.B. Santhosh, et al., Influence of iron oxide nanoparticles on bending elasticity and bilayer fluidity of phosphotidylcholine liposomal membranes, Colloids Surf. A: Physicochem. Eng. Aspects (2014), http://dx.doi.org/10.1016/j.colsurfa.2014.02.035
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in magnetic resonance imaging [1]. Encapsulation of these magnetic NPs in the phospholipid vesicles has several advantages over the free NPs such as improved stability, reduced NPs toxicity, controlled drug release and the possibility of attachment of specific ligands on the liposomal surface for effective targeting [2,3]. Iron oxide (Fe2 O3 ) NPs are amongst the one of the widely used magnetic NPs in biomedical fields due to their non-toxicity, biocompatibility and unique magnetic properties which enables them to be directed to the target site by the manipulation of an external magnetic field [4–6]. They have shown a great potential in numerous biological and clinical applications including imaging techniques [7], tissue engineering [8], magnetic labelling [9], cell isolation [10], hyperthermia [11], and controlled drug release [12]. As in vivo and in vitro the applications of the magnetic liposomes are tremendously increasing in the clinical arena, it becomes important to validate the effect of iron oxide NPs on the physical properties of the liposomes such as bilayer fluidity, permeability, elasticity and stability. The NPs are usually coated with a variety of biocompatible materials such as polyethylene glycol, silica and dextran [13,14]. Coating enhances the stability of the NPs and improves the binding of various proteins, peptides or specific ligands to the NPs surface to increase their targeting efficiency [15,16]. Depending on the thickness of the coating material, the size of the NPs also increases which may alter their characteristics. For instance, small Fe2 O3 NPs in the size range of 20–100 nm possess a single domain and exhibit superparamagnetic properties, i.e. they behave like ferromagnets under the application of magnetic field and show paramagnetic properties when the field is turned off [17,18]. In contrast, the bulk iron oxide materials possess multiple domains and retain magnetic properties even after the field is turned off. Hence to compare if the coated NPs has some influence on altering the physical properties of liposomes, we have used uncoated Fe2 O3 NPs and silica coated Fe2 O3 NPs in our work. Silica is one of the ideal coating materials for Fe2 O3 NPs as it enhances the stability, compatibility as well as reactivity with other coupling agents making them suitable for various pharmaceutical applications [19–22]. The mechanical properties of lipid bilayer are tightly connected to the problem of cell stability, resistivity and functioning, which drives the strong interest towards investigating these properties. Fluidity represents the viscosity of the lipids in the synthetic phospholipid bilayer or in the cell membrane. It is affected by several factors like temperature, osmotic pressure, composition and the length of fatty acids present in the membrane [23]. The focus of our work is to study the influence of plain Fe2 O3 NPs and silica coated Fe2 O3 NPs on membrane fluidity and bending elasticity of zwitterionic 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) (Fig. 1a) liposomal membranes. Small unilamellar vesicles (SUVs) encapsulated with the both the types of Fe2 O3 NPs were used to study the alterations in the membrane fluidity using the fluorescent probes 1,6-diphenyl-1,3,5-hexatriene (DPH) (Fig. 1b) and N, N, N-trimethyl-4-(6-phenyl-1,3,5-hexatrien-1-yl) phenylammonium p-toluensulfonate (TMA-DPH) (Fig. 1c). Thermally induced shape fluctuations of the giant quasi-spherical lipid vesicles encapsulated with the Fe2 O3 NPs were used to study their influence on the bending elasticity modulus kc of the lipid membrane.
2. Materials and methods The SOPC lipid was purchased from Avanti Polar Lipids Inc. (USA). DPH, TMA-DPH and HEPES [4-(2-hydroxyethyl)-1piperazineethanesulfonic acid] salt were purchased from SigmaAldrich Chemie GmbH (Steinheim, Germany). The nanoparticles were obtained from Joseph Stefan Institute, Ljubljana, Slovenia.
Fig. 1. Structural formulas of (a) 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), (b) 1,6-diphenyl-1,3,5-hexatriene (DPH) and (c) (TMA-DPH).
2.1. Small unilamellar vesicles preparation Small unilamellar vesicles (SUVs) were prepared by the thin film method using the rotary evaporator. The SOPC lipid (2 mg) was dissolved in 1 ml of chloroform in a round bottomed flask and argon gas was allowed to pass through the lipid–chloroform mixture to avoid the oxidation of lipids. The organic solvent was removed from the flask using a rotary evaporator (Buchirotavapor R-114; BuchiLabortechnik, Flawil, Switzerland) operating under a very low pressure (1.7 kPa), until a thin lipid film was produced inside the bottom of the flask. The dried lipid film was then hydrated with 1 ml of distilled water at pH 7.0. To facilitate the hydration process in the formation of liposomes, a small amount of 2 mm-diameter glass beads were added to the lipid suspension in the flask and vortexed vigorously for 10 min to form multilamellar vesicles (MLVs). In order to encapsulate the NPs in liposomes, a thin lipid film was prepared in a similar way and hydrated with 1 ml of aqueous NPs suspension containing 0.5 mg of Fe2 O3 and vortexed to obtain MLVs. The MLVs were then transformed into SUVs by sonication for 30 min total time with 10 s on–off cycles at 40% amplitude using a Vibracell Ultrasonic Disintegrator VCX 750 (Sonics and Materials, Newtown, USA). The non-entrapped NPs were separated from the sample by size exclusion chromatography (SEC) using sepharose gel (CL-4B column, 2.5 × 1.5 cm) and eluted with 10 mM HEPES at pH 7.0. The separated liposomes were collected by a Retriever 500 fraction collector tube. To avoid the possibility of aggregated NPs with larger size to be co-eluted with the SUVs during SEC method, the free NPs were treated by the same procedure used for liposome preparation without adding lipids and analyzed by SEC. 2.2. Preparation of giant unilamellar vesicles Giant unilamellar vesicles (GUVs) were prepared using a modified electroformation method. The SOPC was dissolved in chloroform at 1 mg/ml. The electroformation cell that was used for the preparation of the GUVs consisted of two glass slides, coated with transparent conductor indium tin oxide (thickness, 100 ± 20 nm, resistively of 100 /square) acting as electrodes and a PDMS (Polydimethylsiloxane) spacer, preliminary treated to ensure no emission of impurities in the cell. A few small drops of the lipid solutions were pipetted on the glass surface of the electroformation cell and kept under vacuum for at least 30 min. After the complete evaporation of the solvent, the formation cell was filled with double-distilled water with iron oxide nanoparticles in concentration 0.01 mg/ml. A low frequency (10 Hz) sinusoidal alternative voltage (1.5 V) was applied to the conductive glasses
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overnight. This procedure led to the formation of vesicles appropriate for our experiment—i.e. fluctuating vesicles with a diameter of the order of 20–40 m with no visible defects.
The hydrodynamic size and polydispersity index of the Fe2 O3 NPs and the liposomes were measured using a dynamic light scattering (DLS) instrument, (ANALYSETTE 12 DynaSizer, Fritsch, Germany). The zeta potential of the NPs and the liposomes diluted with 10 mM HEPES buffer at pH 7.0 was analyzed using the ZetaPALS (Brookhaven Instruments Corporation, New York). The NPs were visualized using a field-emission transmission electron microscopy (TEM) (JEOL 2010F) combined with an energy-dispersive X ray spectroscopy (EDXS) microanalysis system operated at an acceleration voltage of 200 kV. For particle visualization, a drop of the NPs suspension was deposited on a copper-grid and air dried before TEM analysis. The silica layer on the NPs was characterized by high-resolution electron microscopy (HREM). The proton conductivity of aqueous NPs suspension was analyzed using an impedance spectrometer (Novocontrol Technologies, Germany). Gold blocking electrodes were used with a frequency range of 0.1 Hz to 1 MHz and the temperature range between 0 and 150 ◦ C. 2.4. Fluorescence anisotropy measurements The effect of uncoated and silica coated Fe2 O3 NPs on the packing order of lipids in the SOPC SUVs was investigated using the fluorimetric method. Temperature dependent fluorescence anisotropy measurements of DPH and TMA-DPH in SOPC liposomes were performed in 10-mm-path-length cuvettes using a Cary Eclipse fluorescence spectrophotometer (Varian; Mulgrave, Australia). In the cuvettes, 10 L of DPH or TMA-DPH was added to 2.5 ml 100 M solutions of SUVs to obtain a final concentration of 0.5 M DPH and 1.0 M TMA-DPH. The anisotropy values of SUVs were measured within the temperature range of 15 ◦ C to 50 ◦ C, by increasing the temperature by 5 ◦ C for every measurement, with a time interval of 7 min with constant mixing. Varian autopolarizers with slit widths with a nominal band-pass of 5 nm were used for both excitation and emission. DPH and TMA-DPH fluorescence anisotropy values were measured at the excitation wavelength of 358 nm and emission wavelength of 410 nm. The anisotropy r values were calculated as shown in Eq. (1) using the built-in software of the instrument: r =
I|| − GI⊥ I|| + 2GI⊥
Applying Eq. (2) the lipid-order parameter S was calculated from the aniostropy values using the following expression [24]:
S=
2.3. Characterization of materials
(1)
where, I|| and I⊥ are the emission intensities with polarizers parallel and perpendicular to the direction of the polarized exciting light, respectively. The values of the G-factor (ratio of the sensitivities of the detection system for vertically [IHV] and horizontally [IHH] polarized light) were determined separately for each sample.
3
2 1/2
1 − 2 r/r0 + 5 r/r0
2 r/r0
− 1 + r/r0 (2)
where r0 is the fluorescence anisotropy of DPH and TMA-DPH in the absence of any rotational motion of the probe. The theoretical value of r0 for DPH is 0.4, while experimental values of r0 lie between 0.362 and 0.394 [24]. In our calculation, the value of r0 was 0.370 for DPH and TMA-DPH in SOPC. 2.5. Thermally induced shape fluctuation method The elastic properties of biomembranes are one of the physical factors ensuring the proper functioning of living matter. This is the reason for the growing interest in the investigation of these properties and their dependence on the presence of various admixtures in the membrane. The analysis of thermally induced shape fluctuations of giant liposomes (vesicles) is a classical method for the investigation of the elastic properties of lipid membranes. It is based on the fact that under the Brownian motion of water molecules, bombarding the lipid membrane the vesicle constantly changes its shape (the vesicle membrane exhibit thermal shape fluctuations). Such fluctuations of the biological or model membranes are a part of the phenomena, describing the deviation of some physical properties from their equilibrium state. The method was proposed by Helfrich [25] and developed in detail by Faucon and Mitov [26,27]. A typical experiment for analyzing the shape fluctuations of nearly spherical lipid vesicles consists in the acquisition of many images of the equatorial cross section of the fluctuating vesicle (see Fig. 2), taken at equal time intervals. The equatorial cross section of a fluctuating nearly spherical lipid vesicle is recorded using a CCD camera for a given period of time (approximately 5–10 min). The obtained sequence of images is analyzed and two mechanical characteristics of the vesicular membrane (bending elasticity modulus and membrane tension) are calculated. The analysis of thermally induced shape fluctuations of giant vesicles was used to study the influence of iron oxide nanoparticles with and without silica coating on the bending elasticity of the membrane of giant SOPC vesicles. GUVs (diameter 20–40 m) without any visible defects were chosen for the bending elasticity measurements. The samples containing fluctuating giant vesicles were observed under phase contrast microscopy (Axiovert 100, Zeiss, Germany, oil immersion objective Ph3 100× magnification). The equipment was improved using stroboscopic illumination [28], based on a flashing xenon lamp (L6604; Hamamatsu, Japan) with a damping vibration system, using short light pulses (less than 3–4 s long for full width at half maximum) and high input energy (2 J). During the experiment every second an image of the equatorial cross section of the
Fig. 2. Three images of the equatorial cross section of a fluctuating vesicle under phase contrast microscopy.
Please cite this article in press as: P.B. Santhosh, et al., Influence of iron oxide nanoparticles on bending elasticity and bilayer fluidity of phosphotidylcholine liposomal membranes, Colloids Surf. A: Physicochem. Eng. Aspects (2014), http://dx.doi.org/10.1016/j.colsurfa.2014.02.035
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Fig. 3. (A) TEM image of silica coated Fe2 O3 NPs (B) High resolution electron microscopy image of the Fe2 O3 NPs coated with 1 nm-thick silica (adapted from Ref. [33]).
vesicle was acquired and recorded, until the total number of images reached a given preliminary number (about 400). All of the experiments were performed in double distilled water environment. Further details on the contour determination, mean squared amplitudes calculation and fitting procedure to determine the bending elastic modulus, kc , and the dimensionless membrane tension , ¯ can be found in the article of Genova [29]. An algorithm for digitalization and processing of image sequences of fluctuating vesicles with a detailed procedure for obtaining the mechanical constants of the vesicular membrane, applying strict objective criteria for qualification of the vesicle as a whole as well as for acceptance or rejection of a given contour of the sequence of recorded images [28] was used for all the experimental data presented in the work. The white noise contribution to the amplitudes of thermal shape fluctuations [30] was evaluated and taken into account in the reported values for the bending elasticity modulus. 3. Results and discussion 3.1. Characterization of iron oxide nanoparticles and liposomes The size of the uncoated Fe2 O3 NPs was found to be 13.7 ± 2.9 nm by TEM analysis. Fig. 3 depicts the TEM image of Fe2 O3 -Si NPs and HREM image of the homogenous silica coating surrounding the NPs with a thickness around 1 nm. DLS results revealed a mean hydrodynamic size of 30 nm ± 1.5 nm for the Fe2 O3 NPs with a polydispersity index (PDI) of 0.44 ± 0.02 (n = 3). The size was found to be 22 nm ± 0.5 nm for Fe2 O3 -Si NPs with a PDI of 0.14 ± 0.01 (n = 3). The density of the iron oxide NPs in aqueous suspension at 25 ◦ C was found to be 1 mg/ml ± 0.01 mg/ml and the pH of both the NPs suspensions was found to be around 8. The zeta potential values of the Fe2 O3 NPs and Fe2 O3 -Si NPs were found to be −6 mV ± 0.004 mV and −28 mV ± 0.007 mV, respectively. The electrical conductivity of the bare and silica coated Fe2 O3 NPs were measured as a function of frequency (0.1 Hz to 1 MHz) and temperature (0–150 ◦ C) to investigate the effect of silica coating (data not shown). The AC activation energy was found to be 0.046 eV for the bare Fe2 O3 NPs and 0.049 eV for Fe2 O3 -Si NPs. The temperature dependent DC resistivity measurements revealed the DC activation energy for the uncoated Fe2 O3 NPs and Fe2 O3 -Si NPs to be 0.24 eV and 0.43 eV, respectively. The AC and DC activation energies of Fe2 O3 NPs are in agreement with the values reported earlier [31,32]. DLS results revealed a mean hydrodynamic size of the control SUVs and NPs loaded SUVs in the range between 70 to 100 nm (PDI = 0.15 ± 0.002) (n = 3). The zeta potential values of the control liposomes, SUVs encapsulated with Fe2 O3 NPs and SUVs encapsulated with Fe2 O3 -Si NPs were found to be −4.13 mV ± 0.009,
−4.75 mV ± 0.007 and −24.14 mV ± 0.015, respectively. Due to the considerable size difference between the NPs (∼20 nm) and SUVs (∼100 nm), we have used SEC using Sepharose CL-4B columns with smaller pore sizes to separate the unbound NPs from the liposomes. SEC is an efficient method to separate the molecules based on their size and widely used to free the encapsulated liposomes from the surrounding particles [33–35]. DLS results revealed a mean hydrodynamic radius of the control SUVs and NPs loaded SUVs in the range between 100 to 110 nm (PDI = 0.15 ± 0.02) (n = 3). The zeta potential values of control liposomes, SUVs encapsulated with Fe2 O3 NPs and Fe2 O3 -Si NPs were found to be −4.13 mV ± 0.009, −4.75 mV ± 0.007 and −24.14 mV ± 0.015, respectively. 3.2. Membrane fluidity The alterations in the membrane fluidity of SOPC SUVs induced by the NPs due their interaction with the phospholipids in the membrane were studied using the fluorescent probes DPH and its cationic derivative TMA DPH. Both the probes are widely used to study the membrane dynamics and architecture in the real cells as well as in artificial lipid vesicles [36]. They are cylindrically shaped molecules and non-fluorescent in water but show intense fluorescence signals after intercalation into the membrane. DPH, being a hydrophobic probe, localizes in the apolar tail region of the membrane and the hydrophilic probe TMA-DPH incorporates near the phospholipid heads at the water membrane interface [37]. Hence these probes are ideal to study the alterations in the membrane viscosity in both the head and tail region of the membrane phospholipids. The anisotropy values of DPH and TMA-DPH rely on the packing order of lipid chains in the membrane. The anisotropy values are directly proportional to the order parameter of the membrane lipids and inversely proportional to the membrane fluidity [38]. Hence from the obtained anisotropy values the lipid order parameter S can be calculated using the formula shown in Eq. (2) and the alterations in the membrane fluidity can be determined accordingly (Fig. 4). The initial order parameter values using DPH for control SUVs devoid of NPs at 15 ◦ C (Fig. 4a) was 0.48 ± 0.01, while for SUVs encapsulated with Fe2 O3 and Fe2 O3 -Si NPs were 0.46 ± 0.03 and 0.43 ± 0.05, respectively. The order parameter values were presented as mean ± SD of five measurements. The error value represents the standard errors of mean of different samples during the measurement. As the temperature was increased, the order parameter values gradually decreased (Fig. 4a) and the final “S” values for control SUVs and SUVs encapsulated with Fe2 O3 and Fe2 O3 -Si NPs at 50 ◦ C were 0.20 ± 0.01, 0.18 ± 0.04 and 0.16 ± 0.01, respectively. The order parameter values for control, SUVs with Fe2 O3 and Fe2 O3 -Si NPs at 15 ◦ C using TMA-DPH (Fig. 4b) were
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Fig. 4. Lipid order parameter of SOPC SUVs determined by (a) DPH anisotropy measurements and (b) TMA-DPH anisotropy measurements ( Control SUVs without NPs; SUVs encapsulated with Fe2 O3 NPs; SUVs encapsulated with Fe2 O3 -Si NPs).
0.68 ± 0.01, 0.67 ± 0.02 and 0.65 ± 0.01, respectively, and at 50 ◦ C the “S” values decreased to 0.59 ± 0.01, 0.58 ± 0.01 and 0.56 ± 0.02 correspondingly. In both the cases, the difference in the “S” values for control vesicles and SUVs with Fe2 O3 NPs was almost negligible whereas a little difference in the order parameter values was observed between the control and SUVs with Fe2 O3 -Si NPs. The difference in the order parameter values caused by the coated and uncoated Fe2 O3 NPs could be interpreted on the basis of their size and the zeta potential values. Silica coating provides more stability, reduces the NPs aggregation leading to smaller size when compared to the plain Fe2 O3 NPs without coating. Since the size of the Fe2 O3 -Si NPs are smaller, they could be easily entrapped in the vesicles and have more probability to interact with the membrane phospholipids thereby altering the lipid ordering and membrane fluidity. Rapid interaction of NPs with the liposomal bilayer may cause membrane disruption which is an important factor leading to altered membrane properties [39]. Recent studies have shown that the interaction of the encapsulated NPs with the liposomal bilayer leads to alteration in the phase behaviour of lipids by reducing the phase transition temperature and increasing the membrane fluidity [40–42]. 3.3. Membrane bending elasticity The analysis of thermally induced shape fluctuations of giant vesicles was used to determine the bending elasticity modulus of giant unilamellar SOPC vesicles in presence of uncoated and silica coated iron oxide (Fe2 O3 ) nanoparticles in concentration 0.01 mg/ml in double distilled water. The experimental data obtained for the bending elasticity modulus kc for SOPC lipid membranes in pure water environment and in presence of 0.01 mg/ml of both types of nanoparticles is presented on Table 1. The values for the bending elasticity modulus of the lipid membrane in all three cases (pure water environment, suspension of coated and uncoated iron oxide nanoparticles) were calculated as the weighted average value of 6–12 giant vesicles, passing all the quality requirements. The obtained experimental results for the bending elasticity modulus show that in the frames of the experimental error the presence of uncoated iron oxide nanoparticles in the studied concentration (0.01 mg/ml in the water suspension) do not influence the elastic properties of SOPC membrane. The presence of silica coated iron oxide nanoparticle in the same concentration (0.01 mg/ml in the water solution) reduces the value of the bending elasticity modulus by about 25%.
Table 1 Bending elasticity modulus kc of SOPC lipid membrane containing coated and uncoated iron oxide nanoparticles. The first column shows the type of the aqueous suspension of the membrane, the second column shows the mean weighted value of the bending elasticity modulus and the third column shows the number of vesicles over which the mean is calculated. Type of aqueous solution
Weighted mean value of the bending elasticity modulus kc (±standard deviation)
Number of vesicles
Uncoated iron oxide NPs −0.01 mg/ml in double distilled water Silica coated iron oxide NPs −0.01 mg/ml in double distilled water Pure water
(1.96 ± 0.10) × 10−19 J
12
(1.45 ± 0.10) × 10−19 J
9
(1.88 ± 0.17) × 10−19 J
6
4. Conclusions The influence of the encapsulated Fe2 O3 NPs on the bilayer fluidity and bending elasticity of the phosphatidylcholineliposomal membranes has been reported. To investigate the effect of coating, uncoated and silica coated Fe2 O3 NPs were used in the experiments. The anisotropy measurements using the fluorescent probes DPH and TMA-DPH revealed that the silica coated Fe2 O3 NPs has a little influence on the order parameter values, whereas the effect was negligible in the case of uncoated Fe2 O3 NPs. A similar result was obtained with the bending elasticity modulus measurements. The Fe2 O3 -Si NPs reduced the bending elasticity modulus values by 25% in comparison with the pure SOPC liposomal membranes while the uncoated Fe2 O3 NPs did not produce a considerable difference. These results imply that significant care has to be taken about the characteristics of the coating material while using the NPs for biomedical applications. Further research on the effect of different coating materials including silica is underway to gain more knowledge and understanding about these materials in membrane interactions.
Acknowledgements This study was supported by Research Program P4-0121, Internal Grant VK-02-13 of the Institute of Solid State Physics, Bulgarian Academy of Sciences, and by Grant 11013-9/2012-6 from the Slovene Human Resources Development and Scholarship Fund.
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Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa
Influence of iron oxide nanoparticles on bending elasticity and bilayer fluidity of phosphotidylcholine liposomal membranes Poornima Budime Santhosh a , Sophia Ivanova Kiryakova b , Julia Lyubomirova Genova b , Nataˇsa Poklar Ulrih a,c,∗ a
Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia Institute of Solid State Physics, Bulgarian Academy of Sciences, 72, TzarigradskoChaussee Blvd., 1784 Sofia, Bulgaria c Centre of Excellence for Integrated Approaches in Chemistry and Biology of Proteins (CipKeBiP), Jamova 39, 1000 Ljubljana, Slovenia b
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
• Liposomes loaded with bare and silica coated iron oxide particles were prepared. • Silica coated particles have stronger effect on elasticity and membrane fluidity. • Silica coating provide high negative surface potential to the nanoparticles. • Silica coated nanoparticles destabilize the membrane leading to altered properties.
a r t i c l e
i n f o
Article history: Received 30 October 2013 Received in revised form 10 February 2014 Accepted 11 February 2014 Available online xxx Keywords: Fluidity Iron oxide nanoparticles Anisotropy Bending elasticity Liposomes
a b s t r a c t Iron oxide nanoparticles with improved surface characteristics have tremendous applications in various biomedical fields such as magnetic resonance imaging, hyperthermia, immunoassay and targeted drug delivery. The aim of this work was to study the influence of iron oxide (␥-Fe2 O3 ) nanoparticles on the bilayer fluidity and bending elasticity of zwitterionic phosphatidylcholine liposomal membranes. Small unilamellar vesicles prepared with l-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine lipids were encapsulated with uncoated iron oxide nanoparticles and silica coated iron oxide nanoparticles to study their effect on bilayer fluidity. Anisotropy measurements using the fluorescent probes 1,6-diphenyl-1,3,5hexatriene and N,N,N-trimethyl-4-(6-phenyl-1,3,5-hexatrien-1-yl) phenylammonium p-toluensufonate did not show a significant difference in the lipid ordering and bilayer fluidity. Thermally induced shape fluctuations of the giant quasi-spherical lipid vesicles were used to study the influence of both types of iron oxide nanoparticles on the bending elasticity modulus kc of the lipid membrane. The results showed that in the case of uncoated iron oxide (in the studies concentration) the obtained value for the bending elasticity modulus does not differ in the frames of the experimental error from that of pure phospholipid membrane. In the case of silica coated iron oxide nanoparticles the bending elasticity modulus of the membrane decreased by 25%. © 2014 Published by Elsevier B.V.
1. Introduction ∗ Corresponding author at: University of Ljubljana, Department of Food Science and Technology, Biotechnical Faculty, Jamnikarjeva 101, 1000 Ljubljana, Slovenia. Tel.: +386 1 3230780; fax: +386 1 2566296. E-mail address: [email protected] (N.P. Ulrih).
Liposomes represent a simple and convenient model system to study the membrane properties. Liposomes encapsulating the magnetic nanoparticles (NPs) termed as magnetic liposomes act as a promising tool in targeted drug delivery and contrast agents
http://dx.doi.org/10.1016/j.colsurfa.2014.02.035 0927-7757/© 2014 Published by Elsevier B.V.
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in magnetic resonance imaging [1]. Encapsulation of these magnetic NPs in the phospholipid vesicles has several advantages over the free NPs such as improved stability, reduced NPs toxicity, controlled drug release and the possibility of attachment of specific ligands on the liposomal surface for effective targeting [2,3]. Iron oxide (Fe2 O3 ) NPs are amongst the one of the widely used magnetic NPs in biomedical fields due to their non-toxicity, biocompatibility and unique magnetic properties which enables them to be directed to the target site by the manipulation of an external magnetic field [4–6]. They have shown a great potential in numerous biological and clinical applications including imaging techniques [7], tissue engineering [8], magnetic labelling [9], cell isolation [10], hyperthermia [11], and controlled drug release [12]. As in vivo and in vitro the applications of the magnetic liposomes are tremendously increasing in the clinical arena, it becomes important to validate the effect of iron oxide NPs on the physical properties of the liposomes such as bilayer fluidity, permeability, elasticity and stability. The NPs are usually coated with a variety of biocompatible materials such as polyethylene glycol, silica and dextran [13,14]. Coating enhances the stability of the NPs and improves the binding of various proteins, peptides or specific ligands to the NPs surface to increase their targeting efficiency [15,16]. Depending on the thickness of the coating material, the size of the NPs also increases which may alter their characteristics. For instance, small Fe2 O3 NPs in the size range of 20–100 nm possess a single domain and exhibit superparamagnetic properties, i.e. they behave like ferromagnets under the application of magnetic field and show paramagnetic properties when the field is turned off [17,18]. In contrast, the bulk iron oxide materials possess multiple domains and retain magnetic properties even after the field is turned off. Hence to compare if the coated NPs has some influence on altering the physical properties of liposomes, we have used uncoated Fe2 O3 NPs and silica coated Fe2 O3 NPs in our work. Silica is one of the ideal coating materials for Fe2 O3 NPs as it enhances the stability, compatibility as well as reactivity with other coupling agents making them suitable for various pharmaceutical applications [19–22]. The mechanical properties of lipid bilayer are tightly connected to the problem of cell stability, resistivity and functioning, which drives the strong interest towards investigating these properties. Fluidity represents the viscosity of the lipids in the synthetic phospholipid bilayer or in the cell membrane. It is affected by several factors like temperature, osmotic pressure, composition and the length of fatty acids present in the membrane [23]. The focus of our work is to study the influence of plain Fe2 O3 NPs and silica coated Fe2 O3 NPs on membrane fluidity and bending elasticity of zwitterionic 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) (Fig. 1a) liposomal membranes. Small unilamellar vesicles (SUVs) encapsulated with the both the types of Fe2 O3 NPs were used to study the alterations in the membrane fluidity using the fluorescent probes 1,6-diphenyl-1,3,5-hexatriene (DPH) (Fig. 1b) and N, N, N-trimethyl-4-(6-phenyl-1,3,5-hexatrien-1-yl) phenylammonium p-toluensulfonate (TMA-DPH) (Fig. 1c). Thermally induced shape fluctuations of the giant quasi-spherical lipid vesicles encapsulated with the Fe2 O3 NPs were used to study their influence on the bending elasticity modulus kc of the lipid membrane.
2. Materials and methods The SOPC lipid was purchased from Avanti Polar Lipids Inc. (USA). DPH, TMA-DPH and HEPES [4-(2-hydroxyethyl)-1piperazineethanesulfonic acid] salt were purchased from SigmaAldrich Chemie GmbH (Steinheim, Germany). The nanoparticles were obtained from Joseph Stefan Institute, Ljubljana, Slovenia.
Fig. 1. Structural formulas of (a) 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), (b) 1,6-diphenyl-1,3,5-hexatriene (DPH) and (c) (TMA-DPH).
2.1. Small unilamellar vesicles preparation Small unilamellar vesicles (SUVs) were prepared by the thin film method using the rotary evaporator. The SOPC lipid (2 mg) was dissolved in 1 ml of chloroform in a round bottomed flask and argon gas was allowed to pass through the lipid–chloroform mixture to avoid the oxidation of lipids. The organic solvent was removed from the flask using a rotary evaporator (Buchirotavapor R-114; BuchiLabortechnik, Flawil, Switzerland) operating under a very low pressure (1.7 kPa), until a thin lipid film was produced inside the bottom of the flask. The dried lipid film was then hydrated with 1 ml of distilled water at pH 7.0. To facilitate the hydration process in the formation of liposomes, a small amount of 2 mm-diameter glass beads were added to the lipid suspension in the flask and vortexed vigorously for 10 min to form multilamellar vesicles (MLVs). In order to encapsulate the NPs in liposomes, a thin lipid film was prepared in a similar way and hydrated with 1 ml of aqueous NPs suspension containing 0.5 mg of Fe2 O3 and vortexed to obtain MLVs. The MLVs were then transformed into SUVs by sonication for 30 min total time with 10 s on–off cycles at 40% amplitude using a Vibracell Ultrasonic Disintegrator VCX 750 (Sonics and Materials, Newtown, USA). The non-entrapped NPs were separated from the sample by size exclusion chromatography (SEC) using sepharose gel (CL-4B column, 2.5 × 1.5 cm) and eluted with 10 mM HEPES at pH 7.0. The separated liposomes were collected by a Retriever 500 fraction collector tube. To avoid the possibility of aggregated NPs with larger size to be co-eluted with the SUVs during SEC method, the free NPs were treated by the same procedure used for liposome preparation without adding lipids and analyzed by SEC. 2.2. Preparation of giant unilamellar vesicles Giant unilamellar vesicles (GUVs) were prepared using a modified electroformation method. The SOPC was dissolved in chloroform at 1 mg/ml. The electroformation cell that was used for the preparation of the GUVs consisted of two glass slides, coated with transparent conductor indium tin oxide (thickness, 100 ± 20 nm, resistively of 100 /square) acting as electrodes and a PDMS (Polydimethylsiloxane) spacer, preliminary treated to ensure no emission of impurities in the cell. A few small drops of the lipid solutions were pipetted on the glass surface of the electroformation cell and kept under vacuum for at least 30 min. After the complete evaporation of the solvent, the formation cell was filled with double-distilled water with iron oxide nanoparticles in concentration 0.01 mg/ml. A low frequency (10 Hz) sinusoidal alternative voltage (1.5 V) was applied to the conductive glasses
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overnight. This procedure led to the formation of vesicles appropriate for our experiment—i.e. fluctuating vesicles with a diameter of the order of 20–40 m with no visible defects.
The hydrodynamic size and polydispersity index of the Fe2 O3 NPs and the liposomes were measured using a dynamic light scattering (DLS) instrument, (ANALYSETTE 12 DynaSizer, Fritsch, Germany). The zeta potential of the NPs and the liposomes diluted with 10 mM HEPES buffer at pH 7.0 was analyzed using the ZetaPALS (Brookhaven Instruments Corporation, New York). The NPs were visualized using a field-emission transmission electron microscopy (TEM) (JEOL 2010F) combined with an energy-dispersive X ray spectroscopy (EDXS) microanalysis system operated at an acceleration voltage of 200 kV. For particle visualization, a drop of the NPs suspension was deposited on a copper-grid and air dried before TEM analysis. The silica layer on the NPs was characterized by high-resolution electron microscopy (HREM). The proton conductivity of aqueous NPs suspension was analyzed using an impedance spectrometer (Novocontrol Technologies, Germany). Gold blocking electrodes were used with a frequency range of 0.1 Hz to 1 MHz and the temperature range between 0 and 150 ◦ C. 2.4. Fluorescence anisotropy measurements The effect of uncoated and silica coated Fe2 O3 NPs on the packing order of lipids in the SOPC SUVs was investigated using the fluorimetric method. Temperature dependent fluorescence anisotropy measurements of DPH and TMA-DPH in SOPC liposomes were performed in 10-mm-path-length cuvettes using a Cary Eclipse fluorescence spectrophotometer (Varian; Mulgrave, Australia). In the cuvettes, 10 L of DPH or TMA-DPH was added to 2.5 ml 100 M solutions of SUVs to obtain a final concentration of 0.5 M DPH and 1.0 M TMA-DPH. The anisotropy values of SUVs were measured within the temperature range of 15 ◦ C to 50 ◦ C, by increasing the temperature by 5 ◦ C for every measurement, with a time interval of 7 min with constant mixing. Varian autopolarizers with slit widths with a nominal band-pass of 5 nm were used for both excitation and emission. DPH and TMA-DPH fluorescence anisotropy values were measured at the excitation wavelength of 358 nm and emission wavelength of 410 nm. The anisotropy r values were calculated as shown in Eq. (1) using the built-in software of the instrument: r =
I|| − GI⊥ I|| + 2GI⊥
Applying Eq. (2) the lipid-order parameter S was calculated from the aniostropy values using the following expression [24]:
S=
2.3. Characterization of materials
(1)
where, I|| and I⊥ are the emission intensities with polarizers parallel and perpendicular to the direction of the polarized exciting light, respectively. The values of the G-factor (ratio of the sensitivities of the detection system for vertically [IHV] and horizontally [IHH] polarized light) were determined separately for each sample.
3
2 1/2
1 − 2 r/r0 + 5 r/r0
2 r/r0
− 1 + r/r0 (2)
where r0 is the fluorescence anisotropy of DPH and TMA-DPH in the absence of any rotational motion of the probe. The theoretical value of r0 for DPH is 0.4, while experimental values of r0 lie between 0.362 and 0.394 [24]. In our calculation, the value of r0 was 0.370 for DPH and TMA-DPH in SOPC. 2.5. Thermally induced shape fluctuation method The elastic properties of biomembranes are one of the physical factors ensuring the proper functioning of living matter. This is the reason for the growing interest in the investigation of these properties and their dependence on the presence of various admixtures in the membrane. The analysis of thermally induced shape fluctuations of giant liposomes (vesicles) is a classical method for the investigation of the elastic properties of lipid membranes. It is based on the fact that under the Brownian motion of water molecules, bombarding the lipid membrane the vesicle constantly changes its shape (the vesicle membrane exhibit thermal shape fluctuations). Such fluctuations of the biological or model membranes are a part of the phenomena, describing the deviation of some physical properties from their equilibrium state. The method was proposed by Helfrich [25] and developed in detail by Faucon and Mitov [26,27]. A typical experiment for analyzing the shape fluctuations of nearly spherical lipid vesicles consists in the acquisition of many images of the equatorial cross section of the fluctuating vesicle (see Fig. 2), taken at equal time intervals. The equatorial cross section of a fluctuating nearly spherical lipid vesicle is recorded using a CCD camera for a given period of time (approximately 5–10 min). The obtained sequence of images is analyzed and two mechanical characteristics of the vesicular membrane (bending elasticity modulus and membrane tension) are calculated. The analysis of thermally induced shape fluctuations of giant vesicles was used to study the influence of iron oxide nanoparticles with and without silica coating on the bending elasticity of the membrane of giant SOPC vesicles. GUVs (diameter 20–40 m) without any visible defects were chosen for the bending elasticity measurements. The samples containing fluctuating giant vesicles were observed under phase contrast microscopy (Axiovert 100, Zeiss, Germany, oil immersion objective Ph3 100× magnification). The equipment was improved using stroboscopic illumination [28], based on a flashing xenon lamp (L6604; Hamamatsu, Japan) with a damping vibration system, using short light pulses (less than 3–4 s long for full width at half maximum) and high input energy (2 J). During the experiment every second an image of the equatorial cross section of the
Fig. 2. Three images of the equatorial cross section of a fluctuating vesicle under phase contrast microscopy.
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Fig. 3. (A) TEM image of silica coated Fe2 O3 NPs (B) High resolution electron microscopy image of the Fe2 O3 NPs coated with 1 nm-thick silica (adapted from Ref. [33]).
vesicle was acquired and recorded, until the total number of images reached a given preliminary number (about 400). All of the experiments were performed in double distilled water environment. Further details on the contour determination, mean squared amplitudes calculation and fitting procedure to determine the bending elastic modulus, kc , and the dimensionless membrane tension , ¯ can be found in the article of Genova [29]. An algorithm for digitalization and processing of image sequences of fluctuating vesicles with a detailed procedure for obtaining the mechanical constants of the vesicular membrane, applying strict objective criteria for qualification of the vesicle as a whole as well as for acceptance or rejection of a given contour of the sequence of recorded images [28] was used for all the experimental data presented in the work. The white noise contribution to the amplitudes of thermal shape fluctuations [30] was evaluated and taken into account in the reported values for the bending elasticity modulus. 3. Results and discussion 3.1. Characterization of iron oxide nanoparticles and liposomes The size of the uncoated Fe2 O3 NPs was found to be 13.7 ± 2.9 nm by TEM analysis. Fig. 3 depicts the TEM image of Fe2 O3 -Si NPs and HREM image of the homogenous silica coating surrounding the NPs with a thickness around 1 nm. DLS results revealed a mean hydrodynamic size of 30 nm ± 1.5 nm for the Fe2 O3 NPs with a polydispersity index (PDI) of 0.44 ± 0.02 (n = 3). The size was found to be 22 nm ± 0.5 nm for Fe2 O3 -Si NPs with a PDI of 0.14 ± 0.01 (n = 3). The density of the iron oxide NPs in aqueous suspension at 25 ◦ C was found to be 1 mg/ml ± 0.01 mg/ml and the pH of both the NPs suspensions was found to be around 8. The zeta potential values of the Fe2 O3 NPs and Fe2 O3 -Si NPs were found to be −6 mV ± 0.004 mV and −28 mV ± 0.007 mV, respectively. The electrical conductivity of the bare and silica coated Fe2 O3 NPs were measured as a function of frequency (0.1 Hz to 1 MHz) and temperature (0–150 ◦ C) to investigate the effect of silica coating (data not shown). The AC activation energy was found to be 0.046 eV for the bare Fe2 O3 NPs and 0.049 eV for Fe2 O3 -Si NPs. The temperature dependent DC resistivity measurements revealed the DC activation energy for the uncoated Fe2 O3 NPs and Fe2 O3 -Si NPs to be 0.24 eV and 0.43 eV, respectively. The AC and DC activation energies of Fe2 O3 NPs are in agreement with the values reported earlier [31,32]. DLS results revealed a mean hydrodynamic size of the control SUVs and NPs loaded SUVs in the range between 70 to 100 nm (PDI = 0.15 ± 0.002) (n = 3). The zeta potential values of the control liposomes, SUVs encapsulated with Fe2 O3 NPs and SUVs encapsulated with Fe2 O3 -Si NPs were found to be −4.13 mV ± 0.009,
−4.75 mV ± 0.007 and −24.14 mV ± 0.015, respectively. Due to the considerable size difference between the NPs (∼20 nm) and SUVs (∼100 nm), we have used SEC using Sepharose CL-4B columns with smaller pore sizes to separate the unbound NPs from the liposomes. SEC is an efficient method to separate the molecules based on their size and widely used to free the encapsulated liposomes from the surrounding particles [33–35]. DLS results revealed a mean hydrodynamic radius of the control SUVs and NPs loaded SUVs in the range between 100 to 110 nm (PDI = 0.15 ± 0.02) (n = 3). The zeta potential values of control liposomes, SUVs encapsulated with Fe2 O3 NPs and Fe2 O3 -Si NPs were found to be −4.13 mV ± 0.009, −4.75 mV ± 0.007 and −24.14 mV ± 0.015, respectively. 3.2. Membrane fluidity The alterations in the membrane fluidity of SOPC SUVs induced by the NPs due their interaction with the phospholipids in the membrane were studied using the fluorescent probes DPH and its cationic derivative TMA DPH. Both the probes are widely used to study the membrane dynamics and architecture in the real cells as well as in artificial lipid vesicles [36]. They are cylindrically shaped molecules and non-fluorescent in water but show intense fluorescence signals after intercalation into the membrane. DPH, being a hydrophobic probe, localizes in the apolar tail region of the membrane and the hydrophilic probe TMA-DPH incorporates near the phospholipid heads at the water membrane interface [37]. Hence these probes are ideal to study the alterations in the membrane viscosity in both the head and tail region of the membrane phospholipids. The anisotropy values of DPH and TMA-DPH rely on the packing order of lipid chains in the membrane. The anisotropy values are directly proportional to the order parameter of the membrane lipids and inversely proportional to the membrane fluidity [38]. Hence from the obtained anisotropy values the lipid order parameter S can be calculated using the formula shown in Eq. (2) and the alterations in the membrane fluidity can be determined accordingly (Fig. 4). The initial order parameter values using DPH for control SUVs devoid of NPs at 15 ◦ C (Fig. 4a) was 0.48 ± 0.01, while for SUVs encapsulated with Fe2 O3 and Fe2 O3 -Si NPs were 0.46 ± 0.03 and 0.43 ± 0.05, respectively. The order parameter values were presented as mean ± SD of five measurements. The error value represents the standard errors of mean of different samples during the measurement. As the temperature was increased, the order parameter values gradually decreased (Fig. 4a) and the final “S” values for control SUVs and SUVs encapsulated with Fe2 O3 and Fe2 O3 -Si NPs at 50 ◦ C were 0.20 ± 0.01, 0.18 ± 0.04 and 0.16 ± 0.01, respectively. The order parameter values for control, SUVs with Fe2 O3 and Fe2 O3 -Si NPs at 15 ◦ C using TMA-DPH (Fig. 4b) were
Please cite this article in press as: P.B. Santhosh, et al., Influence of iron oxide nanoparticles on bending elasticity and bilayer fluidity of phosphotidylcholine liposomal membranes, Colloids Surf. A: Physicochem. Eng. Aspects (2014), http://dx.doi.org/10.1016/j.colsurfa.2014.02.035
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Fig. 4. Lipid order parameter of SOPC SUVs determined by (a) DPH anisotropy measurements and (b) TMA-DPH anisotropy measurements ( Control SUVs without NPs; SUVs encapsulated with Fe2 O3 NPs; SUVs encapsulated with Fe2 O3 -Si NPs).
0.68 ± 0.01, 0.67 ± 0.02 and 0.65 ± 0.01, respectively, and at 50 ◦ C the “S” values decreased to 0.59 ± 0.01, 0.58 ± 0.01 and 0.56 ± 0.02 correspondingly. In both the cases, the difference in the “S” values for control vesicles and SUVs with Fe2 O3 NPs was almost negligible whereas a little difference in the order parameter values was observed between the control and SUVs with Fe2 O3 -Si NPs. The difference in the order parameter values caused by the coated and uncoated Fe2 O3 NPs could be interpreted on the basis of their size and the zeta potential values. Silica coating provides more stability, reduces the NPs aggregation leading to smaller size when compared to the plain Fe2 O3 NPs without coating. Since the size of the Fe2 O3 -Si NPs are smaller, they could be easily entrapped in the vesicles and have more probability to interact with the membrane phospholipids thereby altering the lipid ordering and membrane fluidity. Rapid interaction of NPs with the liposomal bilayer may cause membrane disruption which is an important factor leading to altered membrane properties [39]. Recent studies have shown that the interaction of the encapsulated NPs with the liposomal bilayer leads to alteration in the phase behaviour of lipids by reducing the phase transition temperature and increasing the membrane fluidity [40–42]. 3.3. Membrane bending elasticity The analysis of thermally induced shape fluctuations of giant vesicles was used to determine the bending elasticity modulus of giant unilamellar SOPC vesicles in presence of uncoated and silica coated iron oxide (Fe2 O3 ) nanoparticles in concentration 0.01 mg/ml in double distilled water. The experimental data obtained for the bending elasticity modulus kc for SOPC lipid membranes in pure water environment and in presence of 0.01 mg/ml of both types of nanoparticles is presented on Table 1. The values for the bending elasticity modulus of the lipid membrane in all three cases (pure water environment, suspension of coated and uncoated iron oxide nanoparticles) were calculated as the weighted average value of 6–12 giant vesicles, passing all the quality requirements. The obtained experimental results for the bending elasticity modulus show that in the frames of the experimental error the presence of uncoated iron oxide nanoparticles in the studied concentration (0.01 mg/ml in the water suspension) do not influence the elastic properties of SOPC membrane. The presence of silica coated iron oxide nanoparticle in the same concentration (0.01 mg/ml in the water solution) reduces the value of the bending elasticity modulus by about 25%.
Table 1 Bending elasticity modulus kc of SOPC lipid membrane containing coated and uncoated iron oxide nanoparticles. The first column shows the type of the aqueous suspension of the membrane, the second column shows the mean weighted value of the bending elasticity modulus and the third column shows the number of vesicles over which the mean is calculated. Type of aqueous solution
Weighted mean value of the bending elasticity modulus kc (±standard deviation)
Number of vesicles
Uncoated iron oxide NPs −0.01 mg/ml in double distilled water Silica coated iron oxide NPs −0.01 mg/ml in double distilled water Pure water
(1.96 ± 0.10) × 10−19 J
12
(1.45 ± 0.10) × 10−19 J
9
(1.88 ± 0.17) × 10−19 J
6
4. Conclusions The influence of the encapsulated Fe2 O3 NPs on the bilayer fluidity and bending elasticity of the phosphatidylcholineliposomal membranes has been reported. To investigate the effect of coating, uncoated and silica coated Fe2 O3 NPs were used in the experiments. The anisotropy measurements using the fluorescent probes DPH and TMA-DPH revealed that the silica coated Fe2 O3 NPs has a little influence on the order parameter values, whereas the effect was negligible in the case of uncoated Fe2 O3 NPs. A similar result was obtained with the bending elasticity modulus measurements. The Fe2 O3 -Si NPs reduced the bending elasticity modulus values by 25% in comparison with the pure SOPC liposomal membranes while the uncoated Fe2 O3 NPs did not produce a considerable difference. These results imply that significant care has to be taken about the characteristics of the coating material while using the NPs for biomedical applications. Further research on the effect of different coating materials including silica is underway to gain more knowledge and understanding about these materials in membrane interactions.
Acknowledgements This study was supported by Research Program P4-0121, Internal Grant VK-02-13 of the Institute of Solid State Physics, Bulgarian Academy of Sciences, and by Grant 11013-9/2012-6 from the Slovene Human Resources Development and Scholarship Fund.
Please cite this article in press as: P.B. Santhosh, et al., Influence of iron oxide nanoparticles on bending elasticity and bilayer fluidity of phosphotidylcholine liposomal membranes, Colloids Surf. A: Physicochem. Eng. Aspects (2014), http://dx.doi.org/10.1016/j.colsurfa.2014.02.035
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Please cite this article in press as: P.B. Santhosh, et al., Influence of iron oxide nanoparticles on bending elasticity and bilayer fluidity of phosphotidylcholine liposomal membranes, Colloids Surf. A: Physicochem. Eng. Aspects (2014), http://dx.doi.org/10.1016/j.colsurfa.2014.02.035