1. IPTC 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

2. Introduction 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

3. Molecular 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

4. Materials Dioleoyl-phosphatidylglycerol (DOPG): T m = -18 ℃ Malachite Green (MG) ~1 nm Anionic Lipid (pKa~2) Cationic Dye (pKa~7) Distearoyl-phosphatidylglycerol (DSPG): T m = 54.4 ℃

5. Technique : Second Harmonic Generation    Nonlinear Material SHG is forbidden in centro-symmetric media in the electric dipole approximation. At the interface, symmetry is broken. Intrinsically interface specific ! Where N s is the surface density, β is the 2 nd order hyperpolarizability, and is the orientation-average.

6. Technique : Second Harmonic Generation I 2  = (E 2  ) 2  N 2 Canceled out EE E20E20 D~λ EE E 2  =0 D << EE E 2  =0 SHG from dye molecules adsorbed on the surface of microstructures in the centrosymmetric bulk medium

7. Technique : Second Harmonic Generation D~λ a<<λ An interface-specific technique for the centrosymmetric media E 2  (t)  [ N o (t) - N i (t) ] N o (t) : number of MG on the outer surface of liposome bilayer N i (t) : number of MG on the inner surface of liposome bilayer Ref : K.B.Eisenthal et al. Chem. Phys. Lett. 292 (1998) 345

8. Experimental Setup 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 solution Syringe and rectangular cell was temperature-controlled

9. Transport of dye molecules across liposome bilayers 1/τ 1 : a measure of how fast the transport is. ≡ k (the transport rate) Initial adsorption on the outer layer n out (i) mixing Transport of dye from outer layer to inner layer n out (t)-n in (t) Typical SHG data

10. Transport across the fluid phase of liposome (DOPG) (DOPG 20 uM at 20 ℃ ) (DOPG 20 uM, MG 2.4 uM) MG concentration dependenceTemperature dependence The temperature and the adsorption of dyes can change the physical property of lipid bilayer.

11. Transport rate (temperature, MG concentration) k o (T) : Transport rate in the limit of C D → 0 This is related with the property of lipid bilayer undisturbed by the adsorption of dye. B=0.56±0.07 [uM -1 ]

12. Free volume theory for molecular transport 0.5 <  < 1 : overlapping constant v * : the cross-sectional volume of solute v f : the average free volume of solvent molecule v m : the average volume at T m v o : the close-packing volume of solvent molecule  v : the thermal volume expansion coefficient T m : the gel-fluid phase transition temperature In the lipid bilayer, free surface area can be used instead of free volume. In the fluid-phase, when solvent molecules fluctuate,

13. Quite well fitted by free surface area theory Transport rate across undisturbed lipid bilayer If we use T m = 255 K : gel-fluid phase transition temperature a o : the close-packing area in crystal phase  44 Å 2   5  10 -3 K -1 : the thermal area expansion coeff. a* : the cross-sectional area of dye  145 Å 2 Then, by fitting a m  54.0 Å 2 : lipid area in fluid phase at T m   0.96 : the overlapping constant (0.5 <  < 1) k o : Transport rate in the limit of C D → 0

14. Free 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 !

15. Liposome 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 ℃

16. Dependence of SH field on the phase of lipid bilayer DOPG (fluid phase at room T) : adsorption + transport DSPG (gel phase at room T) : adsorption If increasing temperature to above phase transition, What happen ?

17. Transport of MG across DSPG liposome bilayer 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 ?

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

19. MG 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E E 2  =0 t << D~λ + + - - - - - Clay : disk-shaped montmorillonite(diameter~ 500nm, thickness~ 10nm)

20. Experimental 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 ℃

21. Result Inject clay

22. Transport across the liposome bilayer A B A Outside-to-inside transport Inject clay B Inside-to-outside transport

23. Desorption from the outer surface A B A B

24. Conclusion 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.