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IPTC workshop in China

IPTC workshop in China. Adsorption and Transport of a Small Molecule on a Liposome. Mahn Won Kim (1) , Joon Heon Kim (1,2 ) (1) Dept. of Physics, KAIST, (2) APRI, GIST. May 18, 2008. Cell Membrane (Molecular Cell Biology, H.Lodish et al.). hydrophilic. Phosphorous. Oxygen. Hydrogen.

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IPTC workshop in China

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  1. IPTC workshop in China Adsorption and Transport of a Small Molecule on a Liposome Mahn Won Kim(1), Joon Heon Kim(1,2) (1)Dept. of Physics, KAIST, (2)APRI, GIST May 18, 2008

  2. Cell Membrane (Molecular Cell Biology, H.Lodish et al.) hydrophilic Phosphorous Oxygen Hydrogen hydrophobic carbon Introduction Cellular membrane : the boundary of the cell ( lipid + protein + carbohydrate )

  3. Liposome endocytosis ~140 nm Molecular transport across cell membrane (McGraw-Hill Companies, inc.) 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. Non-specific transport of organic cations with hydrophobicity across lipid bilayers

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

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

  6. E E20 E E2=0 E E2=0 l D << D~λ Technique : Second Harmonic Generation SHG from dye molecules adsorbed on the surface of microstructures in the centrosymmetric bulk medium I2= (E2)2  N2 Canceled out

  7. a<<λ D~λ Technique : Second Harmonic Generation Aninterface-specifictechnique for the centrosymmetric media No(t) : number of MG on the outer surface of liposome bilayer Ni(t) : number of MG on the inner surface of liposome bilayer E2(t)  [ No(t) - Ni(t) ] Ref : K.B.Eisenthal et al. Chem. Phys. Lett. 292 (1998) 345

  8. Experimental Setup Inject liposome solution Syringe and rectangular cell was temperature-controlled MG solution in 1cm rectangular cell Ti:Sapphire Laser producing 82 MHz repetition rate, ~100 fs pulses at 840 nm with an energy of about 8nJ

  9. Transport of dye molecules across liposome bilayers Typical SHG data Initial adsorption on the outer layer nout(i) Transport of dye from outer layer to inner layer nout(t)-nin(t) mixing 1/τ1 : a measure of how fast the transport is. ≡ k (the transport rate)

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

  11. Transport rate (temperature, MG concentration) B=0.56±0.07 [uM-1] 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.

  12. Free volume theory for molecular transport In the fluid-phase, when solvent molecules fluctuate, • 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.

  13. Transport rate across undisturbed lipid bilayer ko : Transport rate in the limit of CD→0 • If we use • Tm = 255 K : gel-fluid phase transition temperature • ao : the close-packing area in crystal phase  44 Å2 •  510-3 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) Quite well fitted by free surface area theory

  14. Free surface area theory for molecular transport In the fluid-phase, when lipidmolecules fluctuate, 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. No transport !

  15. Liposome made by lipids of different structures 1,2-Dioleoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (Sodium Salt) (DOPG) Gel-fluid phase transition temperature = -18 ℃ 1,2-Distearoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (Sodium Salt) (DSPG) Gel-fluid phase transition temperature = 54.4 ℃

  16. If increasing temperature to above phase transition, What happen ? 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

  17. Transport of MG across DSPG liposome bilayer Change temperature of premixed solutions equilibrated for more than 3 hrs. Transport occurs at near 48℃ 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. t << l - - - - - E E2=0 + + + + D~λ 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. Clay : disk-shaped montmorillonite (diameter~ 500nm, thickness~ 10nm)

  20. 20℃→ 50℃→ 20℃ (Transport) 20℃→ 50℃ (Reverse Transport) Inject clay at 20℃ Experimental schemes Mix at 20 ℃ DSPG liposome + MG A1 → Inject clay 0.2 ml at 20 ℃ Total 2.0 ml A2 MG only → Inject clay 0.2 ml at 20 ℃ R3

  21. Result Inject clay Inject clay Inject clay

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

  23. A A B B Desorption from the outer surface

  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.

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