Preparation of Complexes of Liposomes with Gold Nanoparticles

C H A P T E R

S E V E N

Preparation of Complexes of Liposomes with Gold Nanoparticles Chie Kojima,* Yusuke Hirano,† and Kenji Kono† Contents 132 134 134 136 137 137

1. 2. 3. 4. 5. 6. 7.

Introduction Preparation of Complexes of EYPC Liposomes with Au NPs Time-Dependent SPR of the Complexes TEM Analysis of the Complexes DLS Analysis of the Complexes Calcein Release from the Complexes Estimation of Numbers of the Au NP and the Liposome in the Complexes 8. Optimization of Lipid Components of the Complexes 9. Concluding Remarks Acknowledgment References

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Abstract Liposomes have been widely used as drug carriers. Visible liposomes have recently become more attractive as drug carriers in personalized medicine. Gold nanoparticles (Au NPs) have unique size- and shape-dependent properties based on their surface plasmon resonance. They can be visualized by computed tomography (CT) and laser optoacoustic imaging. In addition, their photothermogenic properties are useful for photothermal therapy and photoresponsive drug release from liposomes. Therefore, complexation of liposomes with Au NPs is of considerable interest. There are three types of complex: Liposomes containing Au NPs in the inner phase, liposomes with Au NPs at the lipid membrane, and liposomes modified with Au NPs on the surface. This chapter focuses on the preparation and characterization of the third type of complex that is prepared by direct mixing of a Au NP dispersion with a liposome suspension.

* {

Nanoscience and Nanotechnology Research Center, Research Institutes for the Twenty First Century, Osaka Prefecture University, Osaka, Japan Graduate School of Engineering, Osaka Prefecture University, Osaka, Japan

Methods in Enzymology, Volume 464 ISSN 0076-6879, DOI: 10.1016/S0076-6879(09)64007-6

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2009 Elsevier Inc. All rights reserved.

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1. Introduction Drug delivery systems (DDS) are attractive for chemotherapy, because they reduce severe side effects and facilitate effective drug action. Drug carriers are essential for the success of DDS. There are many types of drug carriers available, such as micelles, polymers, virus particles, proteins, and liposomes. Liposomes have classically been used as drug carriers and some, such as Doxil, have already gained Food and Drug Administration (FDA) approval (Immordino et al., 2006). Liposomes are vesicle structures composed of phospholipids with a hydrophobic tail and a hydrophilic head. Due to their amphiphatic character, liposomes can encapsulate water-soluble drug molecules in the inner phase and lipid-soluble ones in the hydrophobic membrane. Improvements to the next generation of liposome drug carriers include controllable release and visibility of the liposome, obtained by addition of functional molecules. Functional molecules that have been added to liposomes include thermosensitive, pH-sensitive, and visible molecules (Al-Jamal and Kostarelos, 2007; Chilkoti et al., 2002; Immordino et al., 2006; Kono, 2001; Kono and Arshady, 2006). Gold nanoparticles (Au NPs) could also be of use as functional molecules because of their interesting shape- and size-dependent physical and chemical properties (Burda et al., 2005; Daniel and Astruc, 2004). Due to their surface plasmon resonance (SPR), Au NPs strongly absorb visible light (Link and El-Sayed, 1999). In addition, they convert this light energy to heat energy (Link and El-Sayed, 2000). Consequently, Au NPs have been considered for photothermal therapy, imaging, and photosensitive drug release (Govorov and Richardson, 2007; Jain et al., 2007; Kim et al., 2007; Pissuwan et al., 2006). Complexes of liposomes with Au NPs are attractive because they can act as both stimuli-responsive and visible drug carriers (Hong et al., 1983; Kojima et al., 2008; Li et al., 2004; Paasonen et al., 2007; Volodkin et al., 2009; Wu et al., 2008). These photochemical properties are only expressed by Au NPs ranging from approximately 2 to 100 nm in diameter (Burda et al., 2005; Daniel and Astruc, 2004). When Au NPs aggregate, they lose their unique photochemical properties. Therefore, the preparation of the complexes also has to be performed without the Au NPs aggregating. Three types of Au NP–liposome complexes exist (Fig. 7.1). The first of these contains Au NPs in the inner phase of the liposome. This type of complex can be prepared by reducing Au ions in the presence of a reductant (Wu et al., 2008), and has been used to investigate in vivo liposome distribution (Hong et al., 1983). In DDS applications, this reduction may be detrimental to drug activity. In the second type of complex, Au NPs are present in the lipid membrane (Paasonen et al., 2007; Park et al., 2006).

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A

B

C

Figure 7.1 A schematic image of the complexes formed between liposomes and Au NPs. (A) A liposome encapsulating Au NPs, (B) a liposome loaded with Au NPs in the membrane, and (C) a liposome modified with Au NPs at the surface (from Kojima et al., 2008).

However, as the thickness of the lipid bilayer is only about 4 nm, a limited number of Au NPs can be incorporated using this strategy. The third complex type is a liposome modified with Au NPs on its surface. This type of complex is simply prepared by mixing an Au NP dispersion with a liposome suspension (Kojima et al., 2008; Volodkin et al., 2009). In this chapter, the third preparation method is described in detail (Kojima et al., 2008), and the influence of lipid components on the liposome are described. The lipids include: egg yolk phosphatidylcholine (EYPC), distearyldimethylammonium bromide (DDAB), distearoyl-sn-glycero-3phosphoethanolamine-N-[methyl(poly(ethylene glycol))] (PEG-PE), and stearylmercaptan (C18-SH). The complexes are characterized by a variety of techniques. UV–Vis spectrometry is used to investigate SPR and its influence on the stability of Au NPs. The size and morphology of the complexes are analyzed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The release of encapsulated molecules from the liposome is examined by using the fluorescent dye calcein.

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2. Preparation of Complexes of EYPC Liposomes with Au NPs Au NPs with a diameter of 13 nm are prepared by reducing Au ions with citric acid, according to a previous report (Grabar et al., 1995). A 5-min reflux is carried out with 50 ml of 1 mM HAuCl4 (Wako Pure Chemical Industries Ltd.). Five milliliters of 38.8 mM sodium citrate (Kishida Chemical) are added with vigorous stirring. The solution changes color from yellow to dark wine red. After cooling, an Au ion dispersion with a concentration of 0.91 mM is obtained by filtration. EYPC can be obtained from a variety of sources (Avanti Polar Lipids, Sigma, etc.). For our studies, it was kindly provided by NOF Corp. (Tokyo, Japan). A chloroform solution of EYPC (10 mg/ml, 500 ml) is evaporated to remove the solvent. The obtained thin lipid membrane (5.0 mg, 6.25 mmol) is further dried under vacuum for at least 2 h, and then dispersed in 0.5 ml of phosphate-buffered saline (PBS; 20 mM Na2HPO3–NaH2PO3, 150 mM NaCl, pH 7) with sonication in a bath type sonicator for 3 min. The liposome suspension is freeze-thawed four times. The obtained liposome suspension is extruded through a polycarbonate membrane with a pore diameter of 200 nm (Kojima et al., 2008; Kono et al., 1999). Lipid concentrations are estimated by Phospholipids C-Test Wako (Wako Pure Chemical Industries Ltd.) according to the manufacturer’s instructions. Sample, blank, and standard solutions are each mixed with the color reagent, which contains phospholipase D, choline oxidase, peroxidase, 4-aminoantipyrine, 3,5-dimethoxy-N-ethyl-N-(2-hydroxy-3-sulfopropyl)aniline sodium, and ascorbic oxidase. After reaction at 37
C for 5 min, the absorbance at 600 nm is measured to estimate the EYPC concentration. The concentration-estimated liposome suspension in PBS is diluted up to 950 ml by addition of distilled water and 10 times concentrated PBS (10 PBS) in an appropriate ratio. Fifty microliters of Au NP dispersion ([Au] ¼ 0.91 mM ) is added to the liposome suspension and vortexed. The final solution is 46 mM of gold ions in 1 PBS. For use in TEM analysis, complexes with an EYPC/Au ratio of 10/1 are prepared by vortex mixing 500 ml of the Au NP dispersion ([Au] ¼ 0.91 mM) with 500 ml of the liposome suspension, diluted with distilled water and 10 PBS. The final solution is 4.6  102 mM of gold ions in 1 PBS.

3. Time-Dependent SPR of the Complexes One of characteristic properties of Au NPs is surface plasmon absorption around 523 nm. To investigate this in each of the complex dispersions, the UV–Vis absorption spectra ranging from 400 to 800 nm are measured

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using a Jasco Model V-560 spectrophotometer ( Jasco Inc., Japan) at 25
C (Haba et al., 2007). As a control, the spectrum of a dispersion solution without liposomes is also measured. The SPR absorption of Au NPs in the absence of liposomes almost disappeared under physiological conditions (Fig. 7.2A). This is due to aggregation resulting from shielding of electrostatic repulsion (Burda et al., 2005; Daniel and Astruc, 2004). In contrast, the SPR absorption in spectra of Au NPs in the presence of EYPC liposomes was retained; this was dependent on the EYPC/Au ratio (Fig. 7.2). At ratios of 1/10 and 1/1, the SPR signal decreased (rapidly and slowly, respectively), while at a ratio of 10/1, the signal remained after 12 h. This indicated that EYPC liposomes contributed to stable dispersion of Au NPs under isotonic conditions, preventing their aggregation.

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Figure 7.2 Time-dependent UV–Vis spectra of the complex of EYPC liposomes (200 nm in diameter) with Au NPs at different ratios in PBS. The time-dependent UV–Vis spectra of Au NPs are only shown as a control (A). The mole ratio of EYPC to Au is 1/10 (B), 1/1 (C), and 10/1 (D). [Au] ¼ 46 mM (from Kojima et al., 2008).

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4. TEM Analysis of the Complexes TEM analysis is performed as follows (Hayashi et al., 1998; Kojima et al., 2008). Collodion-coated grids are coated with a carbon thin film, using an ion sputtering device (E-1030, Hitachi High-Technologies Corp., Japan). After incubation for 1 day in a desiccator, a small drop of sample is placed on the grid for 5 min and then the excess drawn off with filter paper. The Au concentration of the complex at an EYPC/Au ratio of 0/10 and 1/10 was 46 mM. At a ratio of 10/1, the Au concentration of the complex is increased up to 4.6  102 mM, because it is difficult to identify the liposome under this diluted condition. A drop of 2% (w/v) phosphotungstic acid (pH 7) is applied to the grid, drawn off with filter paper, and the stained sample is allowed to dry. The grid is viewed under an electron microscope at 200 kV ( JEOL Ltd., JEM-2000FEX II). Figure 7.3 shows the TEM images of Au NPs in the absence and presence of EYPC liposomes. Large aggregates were observed in TEM

Figure 7.3 TEM images of the complexes of EYPC liposomes with Au NPs at the EYPC/Au ratio of 0/10 (A), 1/10 (B), and 10/1 (C) in PBS after the 6 h incubation. Arrows indicate the Au NPs. Bar ¼ 100 nm (from Kojima et al., 2008).

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images of both the sample comprising only Au NPs and the complex at an EYPC/Au ratio of 1/10. In contrast, no Au NPs aggregates were observed for the complex at an EYPC/Au ratio of 10/1. These findings are consistent with the SPR analysis results. In the TEM image of the complex at an EYPC/Au ratio of 10/1, many Au NPs were observed at the boundary surface within the liposomal assembly. This indicates that the Au NPs complexed with the liposomes.

5. DLS Analysis of the Complexes DLS analysis is performed in the vesicle mode of Nicomp ZLS380 (Nicomp) at room temperature using EYPC suspension (2.5 ml, approximately 0.01 mM) (Kojima et al., 2008). This is performed in both the absence and presence of the Au NP at an EYPC/Au ratio of 10/1. The size of liposome was unchanged before and after the addition of Au NPs (Fig. 7.4), suggesting that Au NPs can complex with liposomes without the aggregation. This finding is not consistent with our TEM results, in which liposomal assembly was observed. It is possible that the liposomal aggregation was an artifact of TEM sample preparation.

6. Calcein Release from the Complexes The collapse behavior of liposomes is analyzed by adding Au NPs to calcein-loaded liposomes. Although the fluorescence of the calcein encapsulated in the liposomes is essentially quenched, an intense florescence is observed after its release from the liposome. The loaded and unloaded percent of calcein is determined from the fluorescence at the initial step and the fluorescence after adding detergent to collapse the liposome, respectively. Calcein release measurements are performed according to the method previously reported (Kono et al., 1994), with some modification (Kojima et al., 2008). Fluorescence intensity is largely influenced by the photochemical properties of the Au NP, so the concentration of Au NPs was decreased to the minimum detection level of calcein. A chloroform solution of EYPC (10 mg/ml, 500 ml) is evaporated to remove the solvent. The obtained thin lipid membrane (5.0 mg, 6.25 mmol) is further dried under vacuum for at least 2 h, and then dispersed in 0.5 ml of 63 mM calcein aqueous solution (pH 7.4) with sonication for 3 min. The liposome suspensions are freeze-thawed four times. The obtained liposome suspension is extruded through a polycarbonate membrane with a pore diameter of 100 nm. Free calcein is removed by gel permeation chromatography using a

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A

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Figure 7.4 Intensity-weighted distributions of the intact liposomes (A) and the complexes of liposomes with Au NPs at the EYPC/Au ratio of 10/1 (B) in PBS after the 6 h-incubation by DLS (from Kojima et al., 2008).

Sepharose 4B column and PBS (10 mM Na2HPO3–NaH2PO3, 150 mM NaCl, pH 7.4). The lipid concentrations are estimated by Phospholipids C-Test Wako (Wako Pure Chemical Industries Ltd.) according to the manufacturer’s instructions, as described earlier. An aliquot of the calceinloaded liposome dispersion is added to 3 ml of PBS containing 0.5 mM ethylenediaminetetraacetic acid (EDTA, Kishida Chemical); the final lipid concentration is 0.2 mM. The fluorescence intensity of the solution is monitored using a spectrofluorometer ( Jasco Inc., FP-6500) at excitation and monitoring wavelengths of 480 and 515 nm, respectively. Measurements are taken before and after the addition of Au NPs to the liposome

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suspension at an EYPC/Au ratio of 1/10. The amount of calcein remaining in the liposome is estimated from Eq. (7.1). 0

Ft  Ft Fluorescence intensity ð%Þ ¼ 0  100 F0  F0

ð7:1Þ

at t and 0 h after the addition of the Au where Ft and F0 refer to the fluorescence 0 0 NP dispersion or PBS, and Ft and F0 refer to the fluorescence of these samples with the addition of 10% Triton X-100 solution (Kishida Chemical, final concentration 0.03%) at t and 0 h. The fluorescence intensity (%) of calcein for the intact liposome decreased after the 24-h incubation (Fig. 7.5). This suggests that the calcein absorbed on the liposome membrane might be released. The fluorescence intensity of calcein after the addition of Au NPs at an EYPC/Au ratio of 1/10 was the same as that in the intact liposome, suggesting that adding Au NPs did not promote calcein release. This implies that the liposomal membrane remained intact after interaction with Au NPs.

7. Estimation of Numbers of the Au NP and the Liposome in the Complexes

Fluorescence intensity (%)

As described earlier, complexed Au NPs at an EYPC/Au ratio of 10/1 were stably dispersed. However, complexes at ratios of 1/1 and 1/10 were not. The particle numbers for Au NPs and liposomes in the complexes can be calculated. A liposome of 200 nm in diameter was determined to contain 3.5  105 EYPC molecules, from Eq. (7.2).

100 80 60 40 20 0

0

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Figure 7.5 Time-dependent fluorescence intensity (%) of calcein in the liposomes with (open symbols) and without (closed symbols) of Au NPs is shown (from Kojima et al., 2008).

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4pr 2  2 ð7:2Þ A where N, r, and A refer to the number of lipids, the radius of the liposomes, and the section area of the EYPC head group (0.717 nm2), respectively (Lasic, 1993). Whereas an Au NP of 13 nm in a diameter is composed of 6.8  104 gold atoms, as determined from Eq. (7.3).    3 2 D U¼ ð7:3Þ p 3 a N¼

where U, D, and a refer to the number of Au atoms, the diameter of the Au NPs, and the edge length of a unit cell (0.40786 nm), respectively (Chithrani et al., 2006). From these calculations, the respective numbers of Au NPs and EYPC liposome in the complex at an EYPC/Au ratio of 10/1 are estimated to be 7.8  1011 and 4.0  1011, under our conditions with 45.5 nmol Au ion and 455 nmol EYPC lipid. Therefore, an EYPC liposome might interact with approximately one Au NP. Overall, these results show that the complexes at an EYPC/Au of 10/1 had a particle ratio of approximately one-to-one and were stably dispersed without disturbing the liposome structure. The enhancement of affinity between the liposome and the Au NPs is of significance for the application.

8. Optimization of Lipid Components of the Complexes As the affinity of liposome to Au NPs should be affected by the lipid component of the liposome, complexes are prepared using both cationic and PEG-modified liposomes. It is expected that cationic lipids and PEG will interact with anionic Au NPs more efficiently. These liposomes are prepared using DDAB (Tokyo Chemical Industry Co. Ltd., Tokyo, Japan) and PEG-PE (Avanti Polar Lipids Inc., Alabaster, AL), in addition to EYPC (Fig. 7.6B and C). DDAB-containing liposomes (10 mol% DDAB (0.625 mmol) and 90 mol% EYPC (5.625 mmol)) are prepared according to the same preparation method of the EYPC liposome before the sonication. The liposomes are incubated at 4
C for 1 day, followed by sonication for 10 min. The size distribution of this liposome preparation is determined to be approximately 200 nm by DLS. A PEG-PE-containing liposome with a diameter of 200 nm and composition of 5 mol% PEG-PE (0.31 mmol) and 95 mol% EYPC (5.94 mmol) is also prepared, according to the same procedure as the EYPC liposomes. The concentration of these lipids is

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O-

+

O

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O

−

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(CH3)3N−CH2CH2−O−P−O−CH2

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O− −

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−

Br−

−

B

O

O

=

O

−

=

(CH2−CH2−O)n C−NH−CH2CH2−O−P−O−CH2 =

CH3O

CH−O−C−(CH2)16CH3 =

CH2−O−C−(CH2)16CH3 O

Figure 7.6 Structures of EYPC (A), DDAB (B), and PEG-PE (C).

estimated from the measured EYPC concentration, considering the in feed ratios. The DDAB- and PEG-bearing liposomes also inhibited the decrease of the SPR signal of the Au NPs, with a dependence on the ratio of lipid to Au (Fig. 7.7). The stability of the SPR signal for these liposomes was similar to the EYPC liposomes. This suggests that complex formation was not improved by DDAB and PEG-PE. Complex formation using liposomes with alkanethiol and different molecular weight preparations of PEG-PE may also be investigated. Liposomes are prepared using C18-SH (Tokyo Chemical Industry Co. Ltd.) and PEG2000-PE or PEG5000-PE (NOF Corp.). PEG-PE- and C18-SHcontaining liposomes with diameters of 100 nm and composition of 5 mol% PEG2000-PE or PEG5000-PE (0.31 mmol), 15 mol% of C18-SH (0.93 mmol), and 80 mol% of EYPC (5.00 mmol) is prepared, according to the same procedure as the EYPC liposome minus the reduction. Before the addition of Au NPs, the liposomes are reduced by reacting with dithiothreitol (DTT; Wako Pure Chemical Industries Ltd.) for 1 h, followed by dialysis using degassed PBS (10 mM Na2HPO3–NaH2PO3, 150 mM NaCl, pH 7.4). The PEG2000- and PEG5000-bearing liposome complexes containing C18-SH inhibited efficiently the decrease of the Au NP SPR signal in the time-dependent SPR absorption spectra, even at a lipid/Au ratio of 1/10 (Fig. 7.8). The PEG5000-PE-bearing liposome was the most stably dispersed complex, suggesting that both the alkanethiol bound to the surface of the Au NPs and PEG play a role in good dispersal of the complex.

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Figure 7.7 Time-dependent UV–Vis spectra of the complex of (A, C, E) DDAB- or (B, D, F) PEG-bearing liposomes with Au NPs at different ratios. The mole ratio of lipid to Au is (A, B) 10/1, (C, D) 1/1, and (E, F) 1/10. [Au] ¼ 46 mM.

9. Concluding Remarks We have described the preparation of various complexes of dispersions of liposomes and Au NPs. Liposomes improve the stability of Au NP dispersions under isotonic conditions. The complexes are formed without disturbing the liposome structure. In addition, complex formation is enhanced by using PEG- and thiol-modified liposomes. It is known that

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A

0.2 PEG-2000 0.15

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Abs

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0.05 Liposome only 0 400

500

600 700 Wavelength (nm)

800

Figure 7.8 Time-dependent UV–Vis spectra of the complex of (A) PEG-2000- or (B) PEG-5000-bearing liposomes containing alkanethiol with Au NPs at the lipid/Au of 1/10. [Au] ¼ 46 mM.

PEG-bearing liposomes are used widely as a drug carrier due to the biocompatibility and the prolonged blood circulation of the liposomes (Greenwald et al., 2000). These types of complexes have potential diagnostic and therapeutic applications in nanomedicine.

ACKNOWLEDGMENT This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT).

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