IPTC workshop in ChinaIPTC workshop in China

Mahn Won Kim(1), Joon Heon Kim(1,2)

(1)Dept. of Physics, KAIST, (2)APRI, GIST

Adsorption and

Transport of a

Small Molecule on a

Liposome

May 18, 2008

IntroductionIntroduction

Cell Membrane (Molecular Cell Biology, H.Lodish et al.)

Cellular membrane : the boundary of the cell( lipid + protein + carbohydrate )

hydrophobic

hydrophilic

carbon

Hydrogen

Oxygen

Phosphorous

Molecular transport across cell membraneMolecular transport across cell membrane

Non-specific transport of organic cations with hydrophobicity across lipid bilayers

Simple model system

(http://www.bio.psu.edu/Courses/)

A spherical, self-closed structures composed of curved lipid bilayers which entrap part of the solvent into their interior.

endocytosis

(McGraw-Hill Companies, inc.)

Liposome

~140 nm

MaterialsMaterials

Dioleoyl-phosphatidylglycerol (DOPG): Tm = -18 ℃

Malachite Green (MG)

~1 nm

Anionic Lipid (pKa~2)

Cationic Dye (pKa~7)

Distearoyl-phosphatidylglycerol (DSPG): Tm = 54.4 ℃

Technique : Second Harmonic GenerationTechnique : Second Harmonic Generation

)()()()()()(

)()()()(

)3()2()1(

)3()2()1(

tEtEtEtEtEtE

tPtPtPtP

NonlinearMaterial

)2()2( ..

sN

SHG is forbidden in centro-symmetric media in the electric

dipole approximation. At the interface, symmetry is broken.

Intrinsically interface specific !

Where Ns is the surface density, β is the 2nd order

hyperpolarizability, and < > is the orientation-

average.

Technique : Second Harmonic GenerationTechnique : Second Harmonic Generation

I2= (E2)2 N2 Canceled out

E E20

D~λ

E E2=0

D <<

E E2=0

SHG from dye molecules adsorbed on the surface of

microstructures in the centrosymmetric bulk medium

Technique : Second Harmonic GenerationTechnique : Second Harmonic Generation

D~λ

a<<λ

An interface-specific technique for the centrosymmetric media

E2(t) [ No(t) - Ni(t) ]

No(t) : number of MG on the outer surface of liposome bilayer

Ni(t) : number of MG on the inner surface of liposome bilayer

Ref : K.B.Eisenthal et al. Chem. Phys. Lett. 292 (1998) 345

Experimental SetupExperimental Setup

PMT Monoch-romator

Filter

Filter

Sample

PolarizerLens

Lens

Ti:SapphireLaser

Mirror

Beam dump

Ti:Sapphire Laser producing 82 MHz repetition rate, ~100 fs pulses at 840 nm with an energy of about 8nJ

MG solution in 1cm rectangular cell

Inject liposome solutionSyringe and rectangular cell

was temperature-controlled

Transport of dye molecules across liposome bilayersTransport of dye molecules across liposome bilayers

)()())(

()( 22 tntnI

ItItE inout

input

bgsig

-200 0 200 400 600 800 1000 1200 1400 1600 1800 2000

0

1

2

3

4

5

6

7

t [sec]

E2

w [

a.u

.]

Fitted by single exponential decay

y = yo + A

1 exp (-t/

1)

y0 1.0 A1 5.1 t1 302

1/τ1 : a measure of how fast the transport is.

≡ k (the transport rate)

Initial adsorption on the outer layer

nout(i)

mixing

Transport of dye from outer layer to inner layer

nout(t)-nin(t)

Typical SHG data

Transport across the fluid phase of liposome (DOPG)Transport across the fluid phase of liposome (DOPG)

0 200 400 600 800 1000 1200 1400

0

2

4

6

8

10

12

14

E2

w [

a.u

.]time [sec]

MG 1.2uM MG 1.8uM MG 2.4uM MG 3.0uM MG 3.6uM MG 4.2uM MG 4.8uM MG 5.4uM MG 6.0uM

(DOPG 20 uM at 20 ℃)

0 600 1200 1800 24000

1

2

3

4

5

6

7

E2

w [

a.u

.]

time [sec]

T=10 oC

T=15 oC

T=20 oC

T=25 oC

T=30 oC

(DOPG 20 uM, MG 2.4 uM)

MG concentration dependence

Temperature dependence

The temperature and the adsorption of dyes can change

the physical property of lipid bilayer.

Transport rate (temperature, MG concentration)Transport rate (temperature, MG concentration)

0 1 2 3 4 5 6

0.04979

0.13534

0.36788

1

2.71828

7.38906

20.08554

10

0 k

[s

ec

-1]

MG conc [uM]

T = 10 C T = 15 C T = 20 C T = 25 C T = 30 C

]exp[)(1

01

DCBTkk

ko(T) : Transport rate in the limit of CD→0

This is related with the property of lipid bilayer undisturbed by the adsorption of dye.

B=0.56±0.07 [uM-1]

280 285 290 295 300 305-8.5

-8.0

-7.5

-7.0

-6.5

-6.0

-5.5

ln k

0 (se

c-1)

Temperature (K)

omvmof vTTv

vTD

vTv

vTD

v

vTDTD

)](exp[exp)(

)(exp)(exp)()( *

0*

0*

0

Free volume theory for molecular transportFree volume theory for molecular transport

0.5 < < 1 : overlapping constant

v* : the cross-sectional volume of solute

vf : the average free volume of solvent molecule

vm : the average volume at Tm

vo : the close-packing volume of solvent molecule

v : the thermal volume expansion coefficient

Tm : the gel-fluid phase transition temperature

In the lipid bilayer, free surface area can be used instead of free volume.

ommfo aTTa

aT

a

aTDTDTk

)](exp[expexp)()()( *2/1*

0

In the fluid-phase, when solvent molecules fluctuate,

Quite well fitted by free surface area theory

Transport rate across undisturbed lipid bilayerTransport rate across undisturbed lipid bilayer

44)]255(005.0exp[54

139exp045.0

)](exp[exp)(

2/1

*2/10

TT

aTTa

aATTk

omm

If we use

Tm = 255 K : gel-fluid phase transition temperature

ao : the close-packing area in crystal phase 44 Å2

510-3 K-1 : the thermal area expansion coeff.

a* : the cross-sectional area of dye 145 Å2

Then, by fitting

am 54.0 Å2 : lipid area in fluid phase at Tm

0.96 : the overlapping constant (0.5 < < 1)

ko : Transport rate in the limit of CD→0

280 285 290 295 300 305-8.5

-8.0

-7.5

-7.0

-6.5

-6.0

-5.5

ln k

0 (s

ec

-1)

Temperature (K)

Free surface area theory for molecular transportFree surface area theory for molecular transport

In the gel-phase,

Lipid molecules cannot freely move.

The free surface area cannot fluctuate very much.

The probability of finding the free surface area larger than a* is almost

zero.

In the fluid-phase, when lipid molecules fluctuate,

No transport !

ommf aTTa

aTD

a

aTDTD

)](exp[exp)(exp)()( *

0*

0

Liposome made by lipids of different structuresLiposome made by lipids of different structures

1,2-Distearoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (Sodium Salt) (DSPG)

1,2-Dioleoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (Sodium Salt) (DOPG)

Gel-fluid phase transition temperature = -18 ℃

Gel-fluid phase transition temperature = 54.4 ℃

Dependence of SH field on the phase of lipid bilayerDependence of SH field on the phase of lipid bilayer

DOPG (fluid phase at room T) : adsorption + transport

DSPG (gel phase at room T) : adsorption

-200 0 200 400 600 800 1000

0

2

4

6

8

10

12

14

16

DSPG DOPG

E2w

[a

.u.]

time [sec]

If increasing temperature to above phase transition,

What happen ?

Transport of MG across DSPG liposome bilayerTransport of MG across DSPG liposome bilayer

-1000 0 1000 2000 3000 4000 5000 6000 7000 800016

20

24

28

32

36

40

44

48

52

Te

mp

era

ture

[C

]

time [sec]

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

E2w

[a

.u.]

T = 47C T = 47~48~49C T = 47~49C

Transport occurs at near 48℃

Change temperature of premixed solutions equilibrated for more than 3 hrs.

Lower than the previously known phase transition temperature (54.4 ℃) of DSPG.

Real phase transition ?

Phase transition temperature of DSPG bilayerPhase transition temperature of DSPG bilayer

Differential Scanning Calorimetry (DSC) data

Transition temperature is shifted by adsorption of MG on lipid bilayer. (peak at 49.8C and onset at 48.7C)

Transport of MG occurs only at the fluid phase of lipid bilayer.

40 45 50 55 60

Liposome Liposome+MG

54.3 oC

Temperature (oC)

En

do

ther

mic

Hea

t F

low

49.8 oC

J. H. Kim et al. Eur. Phys. J. E 23 (2007) 313

MG transport from inside to outside of liposomeMG transport from inside to outside of liposome

To observe the inside-to-outside transport of dyes, we should make :

the number of dyes on inner layer > the number of dyes on outer layer

To reduce the number of dyes on the outer layer, we need absorbers of

bulk dyes outside liposomes, which shouldn’t contribute to SHG signal.

E E2=0

t <<

D~λ

+ + + + - - - - -

Clay : disk-shaped montmorillonite (diameter~ 500nm, thickness~ 10nm)

Experimental schemesExperimental schemes

DSPG liposome

+

MG

→ Inject clay 0.2 ml at 20 ℃

A1

A2

Mix at 20 ℃

Total 2.0 ml

→ Inject clay 0.2 ml at 20 ℃

MG only R3

20℃→ 50℃→ 20℃ (Transport)

20℃→ 50℃ (Reverse Transport)

Inject clay at 20℃

ResultResult

Inject clayInject clay

Inject clay

0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 600015

20

25

30

35

40

45

50

55

Tem

per

atu

re (

oC

)

time (sec)

0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 60000.0

0.5

1.0

1.5

2.0

timeA1_signal timeA2_signal timeR3_signal

I 2w (

a.u

.)

Transport across the liposome bilayerTransport across the liposome bilayer

0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 600015

20

25

30

35

40

45

50

55

Te

mp

era

ture

(oC

)

time (sec)

0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 60000.0

0.5

1.0

1.5

2.0

timeA1_signal timeA2_signal timeR3_signal

I 2w (

a.u

.)

A

B

0 100 200 300 400 5000.0

0.5

1.0

1.5

2.0

timeA1_signal timeA2_signal timeR3_signal

I 2w (

a.u

.)

A

Outside-to-inside transport

Inject clay

Inject clay

B

Inside-to-outside transport

4800 4900 5000 5100 5200 53000.0

0.5

1.0

1.5

2.0

timeA2_signal

time (sec)

I 2w (

a.u

.)

Desorption from the outer surfaceDesorption from the outer surface

0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 600015

20

25

30

35

40

45

50

55

Te

mp

era

ture

(oC

)

time (sec)

0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 60000.0

0.5

1.0

1.5

2.0

timeA1_signal timeA2_signal timeR3_signal

I 2w (

a.u

.)

A

B

A

B

0 100 200 300 400 500

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

= 33 sec

Experimental Data (A1) Fitted line

time (sec)

E2w (

a.u

.)

1000 2000 3000 4000 50000.0

0.5

1.0

1.5

2.0

2.5

3.0

= 958 sec

Experimental Data (A2) Fitted line

time (sec)

E2w (

a.u

.)

ConclusionConclusion

SHG is an efficient technique to investigate the

transport of dye molecules across liposome

bilayers.

The transport rate of dyes in the fluid phase of

liposome bilayer increases as temperature

increases, and this behavior could be explained by

a free surface area theory.

The transport of dyes can be dramatically

facilitated by the phase transition of the liposome

bilayers from gel to fluid phase. The transition

temperature is affected by the adsorption of dyes.

The equilibrium position of adsorbed MG can be

changed depending on the phase of the lipid

bilayer.