[methods in enzymology] liposomes, part f volume 464 || preparation of complexes of liposomes with...

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Preparation of Complexes of

Liposomes with Gold Nanoparticles

Chie Kojima,* Yusuke Hirano,† and Kenji Kono†

Contents

1. In

in

076

sciea Prate

troduction

Enzymology, Volume 464 # 200

-6879, DOI: 10.1016/S0076-6879(09)64007-6 All r

nce and Nanotechnology Research Center, Research Institutes for the Twenty Firefecture University, Osaka, JapanSchool of Engineering, Osaka Prefecture University, Osaka, Japan

1

9 Elsevie

ights rese

st Centur

32

2. P
reparation of Complexes of EYPC Liposomes with Au NPs 1 34
3. T
ime-Dependent SPR of the Complexes 1 34
4. T
EM Analysis of the Complexes 1 36
5. D
LS Analysis of the Complexes 1 37
6. C
alcein Release from the Complexes 1 37
7. E
stimation of Numbers of the Au NP and the Liposome
in the Complexes
139
8. O
ptimization of Lipid Components of the Complexes 1 40
9. C
oncluding Remarks 1 42
Ackn
owledgment 1 43
Refe
rences 1 44
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 photother-

mogenic 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 contain-

ing 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.

r Inc.

rved.

y,

131

132 Chie Kojima et al.

1. Introduction

Drug delivery systems (DDS) are attractive for chemotherapy, becausethey reduce severe side effects and facilitate effective drug action. Drugcarriers are essential for the success of DDS. There are many types of drugcarriers available, such as micelles, polymers, virus particles, proteins, andliposomes. 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 structurescomposed of phospholipids with a hydrophobic tail and a hydrophilichead. Due to their amphiphatic character, liposomes can encapsulatewater-soluble drug molecules in the inner phase and lipid-soluble ones inthe hydrophobic membrane.

Improvements to the next generation of liposome drug carriers includecontrollable release and visibility of the liposome, obtained by addition offunctional molecules. Functional molecules that have been added toliposomes 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 interestingshape- 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 andEl-Sayed, 2000). Consequently, Au NPs have been considered forphotothermal therapy, imaging, and photosensitive drug release (Govorovand Richardson, 2007; Jain et al., 2007; Kim et al., 2007; Pissuwan et al.,2006). Complexes of liposomes with Au NPs are attractive because they canact 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 expressedby Au NPs ranging from approximately 2 to 100 nm in diameter (Burdaet al., 2005; Daniel and Astruc, 2004). When Au NPs aggregate, they losetheir unique photochemical properties. Therefore, the preparation of thecomplexes also has to be performed without the Au NPs aggregating.

Three types of Au NP–liposome complexes exist (Fig. 7.1). The first ofthese contains Au NPs in the inner phase of the liposome. This type ofcomplex 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 distri-bution (Hong et al., 1983). In DDS applications, this reduction may bedetrimental to drug activity. In the second type of complex, Au NPs arepresent in the lipid membrane (Paasonen et al., 2007; Park et al., 2006).

A B

C

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

Complex of Liposomes with Gold Nanoparticles 133

However, as the thickness of the lipid bilayer is only about 4 nm, alimited 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 dispersionwith a liposome suspension (Kojima et al., 2008; Volodkin et al., 2009).In this chapter, the third preparation method is described in detail (Kojimaet al., 2008), and the influence of lipid components on the liposome aredescribed. The lipids include: egg yolk phosphatidylcholine (EYPC),distearyldimethylammonium bromide (DDAB), distearoyl-sn-glycero-3-phosphoethanolamine-N-[methyl(poly(ethylene glycol))] (PEG-PE), andstearylmercaptan (C18-SH).

The complexes are characterized by a variety of techniques. UV–Visspectrometry is used to investigate SPR and its influence on the stability ofAu NPs. The size and morphology of the complexes are analyzed bydynamic light scattering (DLS) and transmission electron microscopy(TEM). The release of encapsulated molecules from the liposome isexamined by using the fluorescent dye calcein.


2. Preparation of Complexes of EYPC Liposomes

with Au NPs

Au NPs with a diameter of 13 nm are prepared by reducing Au ionswith 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 PureChemical Industries Ltd.). Five milliliters of 38.8 mM sodium citrate(Kishida Chemical) are added with vigorous stirring. The solution changescolor from yellow to dark wine red. After cooling, an Au ion dispersionwith 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 NOFCorp. (Tokyo,Japan). A chloroform solution of EYPC (10 mg/ml, 500 ml) is evaporated toremove 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 ofphosphate-buffered saline (PBS; 20 mM Na2HPO3–NaH2PO3, 150 mMNaCl, pH 7) with sonication in a bath type sonicator for 3 min. Theliposome suspension is freeze-thawed four times. The obtained liposomesuspension is extruded through a polycarbonate membrane with apore 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’sinstructions. Sample, blank, and standard solutions are each mixed with thecolor 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, theabsorbance at 600 nm is measured to estimate the EYPC concentration.

The concentration-estimated liposome suspension in PBS is diluted upto 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 areprepared by vortexmixing 500 ml of the AuNP dispersion ([Au] ¼ 0.91mM)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 absorp-tion 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


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 solutionwithout liposomes is also measured. The SPR absorption of Au NPs in theabsence of liposomes almost disappeared under physiological conditions(Fig. 7.2A). This is due to aggregation resulting from shielding of electro-static repulsion (Burda et al., 2005; Daniel and Astruc, 2004). In contrast,the SPR absorption in spectra of Au NPs in the presence of EYPC lipo-somes 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. Thisindicated that EYPC liposomes contributed to stable dispersion of Au NPsunder isotonic conditions, preventing their aggregation.

A

Abs

0.2

0.1

0

0 h

Wavelength (nm)400 500 600 700 800

C D

Abs

EYPC/Au = 10/1

Wavelength (nm)4000

0.1

0.2

0.3

0.4

500 600 700 800

B

1 h→12 h

4 h

Au NP only

8 h12 h

Liposome only

Abs

0.2

0.1

0

Wavelength (nm)400 500 600 700

EYPC/Au = 1/10

800

1 h

4 h8 h12 h

Liposome only

1 h

Abs

0.2

0.1

0

Wavelength (nm)400 500 600 700 800

EYPC/Au = 1/1

Liposome only

4 h8 h12 h

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-dependentUV–Vis spectra of Au NPs are only shown as a control (A). The mole ratio of EYPC toAu is 1/10 (B), 1/1 (C), and 10/1 (D). [Au] ¼ 46 mM (from Kojima et al., 2008).


4. TEM Analysis of the Complexes

TEM analysis is performed as follows (Hayashi et al., 1998; Kojimaet 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 isplaced 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 and1/10 was 46 mM. At a ratio of 10/1, the Au concentration of the complexis increased up to 4.6 � 102 mM, because it is difficult to identify theliposome under this diluted condition. A drop of 2% (w/v) phosphotungsticacid (pH 7) is applied to the grid, drawn off with filter paper, and the stainedsample is allowed to dry. The grid is viewed under an electron microscopeat 200 kV ( JEOL Ltd., JEM-2000FEX II).

Figure 7.3 shows the TEM images of Au NPs in the absence andpresence of EYPC liposomes. Large aggregates were observed in TEM

Figure 7.3 TEM images of the complexes of EYPC liposomes with Au NPs at theEYPC/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).


images of both the sample comprising only Au NPs and the complex at anEYPC/Au ratio of 1/10. In contrast, no Au NPs aggregates were observedfor the complex at an EYPC/Au ratio of 10/1. These findings are consistentwith the SPR analysis results. In the TEM image of the complex at anEYPC/Au ratio of 10/1, many Au NPs were observed at the boundarysurface within the liposomal assembly. This indicates that the Au NPscomplexed 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, approxi-mately 0.01 mM) (Kojima et al., 2008). This is performed in both theabsence and presence of the Au NP at an EYPC/Au ratio of 10/1. Thesize of liposome was unchanged before and after the addition of Au NPs(Fig. 7.4), suggesting that Au NPs can complex with liposomes withoutthe aggregation. This finding is not consistent with our TEM results, inwhich liposomal assembly was observed. It is possible that the liposomalaggregation was an artifact of TEM sample preparation.

6. Calcein Release from the Complexes

The collapse behavior of liposomes is analyzed by adding Au NPs tocalcein-loaded liposomes. Although the fluorescence of the calceinencapsulated in the liposomes is essentially quenched, an intense florescenceis observed after its release from the liposome. The loaded and unloadedpercent of calcein is determined from the fluorescence at the initialstep and the fluorescence after adding detergent to collapse the liposome,respectively.

Calcein release measurements are performed according to the methodpreviously reported (Kono et al., 1994), with some modification (Kojimaet al., 2008). Fluorescence intensity is largely influenced by the photo-chemical properties of the Au NP, so the concentration of Au NPs wasdecreased to the minimum detection level of calcein. A chloroform solutionof EYPC (10 mg/ml, 500 ml) is evaporated to remove the solvent. Theobtained thin lipid membrane (5.0 mg, 6.25 mmol) is further dried undervacuum for at least 2 h, and then dispersed in 0.5 ml of 63 mM calceinaqueous solution (pH 7.4) with sonication for 3 min. The liposome suspen-sions are freeze-thawed four times. The obtained liposome suspensionis extruded through a polycarbonate membrane with a pore diameter of100 nm. Free calcein is removed by gel permeation chromatography using a

1.2B

1

0.8

0.6

Inte

nsity

(a.u

.)

0.4

0.2

010 26 66 169 433 1110 2848 7305

Diameter (nm)

1.2A

1

0.8

0.6

Inte

nsity

(a.u

.)

0.4

0.2

010 26 66 169 433 1110 2848 7305

Diameter (nm)

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


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


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

Fluorescence intensity ð%Þ ¼ F0t � Ft

F00 � F0

� 100 ð7:1Þ

whereFt andF0 refer to the fluorescence at t and 0 h after the addition of theAuNP dispersion or PBS, and F

0t and F

00 refer to the fluorescence of these samples

with the addition of 10% Triton X-100 solution (Kishida Chemical, finalconcentration 0.03%) at t and 0 h. The fluorescence intensity (%) of calceinfor the intact liposome decreased after the 24-h incubation (Fig. 7.5). Thissuggests that the calcein absorbed on the liposome membrane might bereleased. The fluorescence intensity of calcein after the addition of Au NPs atan 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 impliesthat the liposomal membrane remained intact after interaction with Au NPs.

7. Estimation of Numbers of the Au NP

and the Liposome in the Complexes

As described earlier, complexed Au NPs at an EYPC/Au ratio of 10/1were stably dispersed. However, complexes at ratios of 1/1 and 1/10 werenot. The particle numbers for Au NPs and liposomes in the complexes canbe calculated. A liposome of 200 nm in diameter was determined to contain3.5 � 105 EYPC molecules, from Eq. (7.2).

0

Flu

ores

cenc

e in

tens

ity

(%)

0

20

40

60

80

100

5 10 15

Incubation time (h)

20 25

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


N ¼ 4pr2 � 2

Að7:2Þ

whereN, 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 of6.8 � 104 gold atoms, as determined from Eq. (7.3).

U ¼ 2

3

� �p

D

a

� �3

ð7:3Þ

where U,D, and a refer to the number of Au atoms, the diameter of the AuNPs, and the edge length of a unit cell (0.40786 nm), respectively(Chithrani et al., 2006). From these calculations, the respective numbersof Au NPs and EYPC liposome in the complex at an EYPC/Au ratio of10/1 are estimated to be 7.8 � 1011 and 4.0 � 1011, under our conditionswith 45.5 nmol Au ion and 455 nmol EYPC lipid. Therefore, an EYPCliposome might interact with approximately one Au NP.

Overall, these results show that the complexes at an EYPC/Au of 10/1had a particle ratio of approximately one-to-one and were stably dispersedwithout disturbing the liposome structure. The enhancement of affinitybetween 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 lipidcomponent of the liposome, complexes are prepared using both cationicand PEG-modified liposomes. It is expected that cationic lipids and PEGwill interact with anionic Au NPs more efficiently. These liposomes areprepared 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 accordingto the same preparation method of the EYPC liposome before the sonica-tion. The liposomes are incubated at 4 �C for 1 day, followed by sonicationfor 10 min. The size distribution of this liposome preparation is determinedto be approximately 200 nm by DLS. A PEG-PE-containing liposome witha diameter of 200 nm and composition of 5 mol% PEG-PE (0.31 mmol) and95 mol% EYPC (5.94 mmol) is also prepared, according to the sameprocedure as the EYPC liposomes. The concentration of these lipids is

EYPC

DDAB

PEG-PE

CH3O (CH2−CH2−O)nC−NH−CH2CH2−O−P−O−CH2

O O

O

CH−O−C−(CH2)16CH3

CH2−O−C−(CH2)16CH3

O−

O

O

O-

O CH−O−C−R

CH2−O−C−R

O

Br−CH3 (CH2)17CH3

(CH2)17CH3CH3

N+

+(CH3)3N−CH2CH2−O−P−O−CH2

A

B

C

−

−

−

−

−

−−−−

= ==

== ==

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


estimated from the measured EYPC concentration, considering the in feedratios. The DDAB- and PEG-bearing liposomes also inhibited the decreaseof the SPR signal of the Au NPs, with a dependence on the ratio of lipid toAu (Fig. 7.7). The stability of the SPR signal for these liposomes was similarto the EYPC liposomes. This suggests that complex formation was notimproved by DDAB and PEG-PE.

Complex formation using liposomes with alkanethiol and differentmolecular weight preparations of PEG-PE may also be investigated. Lipo-somes are prepared using C18-SH (Tokyo Chemical Industry Co. Ltd.) andPEG2000-PE or PEG5000-PE (NOF Corp.). PEG-PE- and C18-SH-containing 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 tothe same procedure as the EYPC liposome minus the reduction. Before theaddition of Au NPs, the liposomes are reduced by reacting with dithio-threitol (DTT; Wako Pure Chemical Industries Ltd.) for 1 h, followed bydialysis using degassed PBS (10 mM Na2HPO3–NaH2PO3, 150 mMNaCl,pH 7.4). The PEG2000- and PEG5000-bearing liposome complexes con-taining C18-SH inhibited efficiently the decrease of the Au NP SPR signalin the time-dependent SPR absorption spectra, even at a lipid/Au ratio of1/10 (Fig. 7.8). The PEG5000-PE-bearing liposome was the most stablydispersed complex, suggesting that both the alkanethiol bound to thesurface of the Au NPs and PEG play a role in good dispersal of the complex.

A0.4

0.3

0.2

0.1

0.0

Liposome only

1h→12h 1h→12h

Lipid/Au=10/1

Abs

Wavelength (nm)400 500 600 700 800

C0.2

0.15

0.1

0.05

0.0

Liposome only

Lipid/Au=1/1

Abs

Wavelength (nm)400 500 600 700 800

E0.2

0.15

0.1

0.05

0.0

Liposome only

Lipid/Au=1/10

Abs

Wavelength (nm)400 500 600 700 800

F0.2

0.15

0.1

0.05

0Liposome only

Lipid/Au=1/10

Abs

Wavelength (nm)400 500 600 700 800

D0.2

0.15

0.1

0.05

0

Liposome only

8h12h

4h1h

Lipid/Au=1/1

Abs

Wavelength (nm)400 500 600 700 800

B0.4

0.3

0.2

0.1

0

Liposome only

Lipid/Au=10/1

Abs

Wavelength (nm)400 500 600 700 800

8h12h

4h1h

8h

12h

4h

1h

8h

12h

4h

1h

Figure 7.7 Time-dependent UV–Vis spectra of the complex of (A, C, E) DDAB- or(B, D, F) PEG-bearing liposomes with AuNPs at different ratios. The mole ratio of lipidto 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 dispersionsof liposomes and Au NPs. Liposomes improve the stability of Au NPdispersions under isotonic conditions. The complexes are formed withoutdisturbing the liposome structure. In addition, complex formation isenhanced by using PEG- and thiol-modified liposomes. It is known that

Liposome only

0

0.05

0.1

0.15

0.2

400 500 600 700 800Wavelength (nm)

Abs

PEG-2000

PEG-5000

A

0

0.05

0.1

0.15

0.2

400 500 600 700 800Wavelength (nm)

Abs

B

Liposome only

8h

12h

4h

1h

8h12h

4h

1h

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 of1/10. [Au] ¼ 46 mM.


PEG-bearing liposomes are used widely as a drug carrier due to the bio-compatibility and the prolonged blood circulation of the liposomes(Greenwald et al., 2000). These types of complexes have potential diagnos-tic 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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[methods in enzymology] liposomes, part f volume 464 || preparation of complexes of liposomes with...

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