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Возможности нейтронного и синхротронного излучения для исследования структуры липидных мембран

Возможности нейтронного и синхротронного излучения для исследования структуры липидных мембран. М.А. Киселев Лаборатория нейтронной физики Объединенный институт ядерных исследований , Дубна E-mail: kiselev@nf.jinr.ru.

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Возможности нейтронного и синхротронного излучения для исследования структуры липидных мембран

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  1. Возможности нейтронного и синхротронного излучения для исследования структуры липидных мембран М.А. Киселев Лаборатория нейтронной физики Объединенный институт ядерных исследований, Дубна E-mail: kiselev@nf.jinr.ru Совещание«Современные ядерно-физические методы исследования в физике конденсированных сред», Минск, БГУ, 22-23 апреля, 2013

  2. Phospholipids and ceramides

  3. Phospholipids – major component of cell membranes.Ceramides – major components of stratum corneum lipid matrix.Phospholipids and ceramides – important materials for drug and cosmetic industry.

  4. Objects and methods Multilamelar vesicles (Liposomes) Small-angle and wide-angle powder X-ray diffraction at synchrotron source Unilamellar vesicles Neutron small-angle scattering Oriented multilamellar planar system on quartz substrate Neutron diffraction

  5. Modern methods for the characterization of the lipid nanosystems : • Method of lamellar and lateral synchrotron X-ray diffraction in real time • Method of neutron diffraction for study of model stratum corneum membranes • Method of separated form factors for characterization of vesicular nanodrugs • Method of contrast variation of X-ray scattering by disaccharides.

  6. Real-time X-ray diffraction at cooling DPPC/water system at synchrotron. d=5.9Å Ice crystal Free water Diffusion crystallization Cooling with the rate of 1oC/min, acquisition - 1min/spectrum

  7. Low-temperature phase transition in DPPC/DMSO/water system at Xdmso=0.05. Synchrotron. New phase Ice Cooling with the rate of 1oC/min, acquisition - 1min/spectrum

  8. Drug penetration pathways across the skin. Down hair follicle Down sweat gland Across SC Intercellular route

  9. Fourier profiles atRH=60%, T=32oC DMPC Repeat distance54.750.03 Å Membrane thickness 45.80.1 Å Intermembrane space 9.00.1 Å Model SC membranes Repeat distance 45.63±0.04Å

  10. Nanostructure of fully hydrated model SC membrane ceramide 6/cholesterol/palmitic acid/cholesterol sulfate =55/25/15/5 M. A. Kiselev, N. Yu. Ryabova, A. M. Balagurov, S. Dante, T. Hauss, J. Zbytovska, S. Wartewig, R. H. H. Neubert. New insights into structure and hydration of stratum corneum lipid model membrane by neutron diffraction. European Biophysics J. 34 (2005) 1030–1040.

  11. Hyposesis of super-strong intermembrane interaction created by ceramide 6 in fully extended conformatiion (armature reinforcement). Bricks and mortar model of SC Fully extended conformation of ceramide Hair-pin conformation of ceramide M.A. Kiselev. Conformation of ceramide 6 molecules and chain–flip transitions in the lipid matrix of the outermost layerof mammalian skin, the stratum corneum. Crystallography Reports, 2007, Vol. 52, No. 3, pp. 525–528

  12. Real- time neutron diffraction at DN-2, IBR-2.Time-of-flight. Dehydration 360min Hydration 480min DPPC membrane hydration-dehydration in real time at T=20oC

  13. Nanostructure alteration during hydration-dehydration as result of fourier-synthesis in real-time. T=20oC. Repeat distance d Water layer dw dB Membrane thickness dB dc d Thickness of the hydrocarbon chains region dc Н.Ю. Рябова, М.А. Киселев, А.И. Бескровный, А.М. Балагуров. Исследование структуры многослойных липидных мембран методом дифракции нейтронов в реальном времени. Физика твердого тела, 52 № 5 (2010) 984-991.

  14. Chamber with excess of water • New possibilities to study the influence of the skin penetration enhancer via neutron diffraction at IBR-2 reactor.

  15. Unilamellar vesicles from phospholipid molecules R=250Å n2 ·1014 vesicles/cm3 for 1% DMPC (w/w) 6500 DMPC molecules in one layer for 30oC From point of physics it is nanospheres with liquid crystal surface Neutron small-angle scattering in D2O X-ray small-angle scattering in sucrose solutions M.A. Kiselev, P. Lesieur, A.M. Kisselev, D. Lombardo, M. Killany, S. Lesieur. Sucrose solutions as prospective medium to study the vesicle structure: SAXS and SANS study. J. Alloys and Compounds 328 (2001) 71-76.

  16. SANS pattern at 30 °C of unilamellar DMPC vesicles prepared by extrusion through 500 Å pores at DMPC concentration of 1% (w/w). qR qm

  17. SAXS patterns at 10 °C (circles) and 30 °C (open circles) of unilamellar DMPC vesicles in 40% aqueous trehalose solution, DMPC concentration 3%(w/w)

  18. Method of Separated Form Factors (SFF) c(x)=(x) D2O is contrast, the difference between scattering length densities of the bilayer (x) and the heavy water D2O. For the case of (x)=Const . M.A. Kiselev, P. Lesieur, A.M. Kisselev, D. Lombardo, V.L. Aksenov. Model of separated form factors for unilamellar vesicles. J. Applied Physics A 74 (2002) S1654-S1656

  19. Results for liquid phase at 30oC, HH approximation of (x) Multilamellar (small curved) vesicles d = 44.2 Å A=59.6 Å2 J.Nagle, S. Tristram-Nagle. Structure of lipid bilayers. BBA 1469 (2000) 159-195 М.А. Kiselev, E.V. Zemlyanaya, V.K. Aswal, R.H.H. Neubert. What can we learn about the lipid vesicle structure from the small angle neutron scattering experiment? European Biophysics J. 35 (2006) 477-493.

  20. Practical applications Nanoparticles based on the phospholipids Phosphogliv – 20-50 нм ? ? ? 80% products in the bionanotechnology are drug delivery systems !!! Turnover - 6 billion USD/year

  21. Phospholipid transport nanosystems (FTNS) SAXS patterns. DMPC vesicles in 40% sucrose buffer – red line (LURE, France). 25% solution of FTNS in water – blue line (DIKSI, Sibirea-2, Moscow). Black line - 1/q2law.

  22. Conclusions.Possibilities to study in future: • Drug diffusion through model SC membranes. • Nanostructure of the magnetovesicles. • Elastic properties of the transdermal drug delivery vesicles and magnetovesicles. • Effectiveness of the cryoprotectors applied for bacteria storage.

  23. Participants from Russia • А.М. Balagurov JINR Russua • V.L. Aksenov JINR Russia • E.V. Zemlyanaya JINR Russia • E.V. Ermakova JINR Russia • N.Y. Ryabova JINR Russia • A.Y. Gruzinov KI Russia • A.B. Zabelin KI Russia • O. M. Ipatova IBMC Russia

  24. Foreign participants • P. Lesieur LURE, France • M. Ollivon University Paris-sud, France • R. Neubert MLU Germany • J. Zbytovska MLU Germany • D. Kessner MLU Germany • A. Schroter MLU Germany • M. Janich MLU Germany • T. Gutberlet HMI Germany • Th. Hauss GCB Germany • S. Dante GCB Germany • V.K. Aswal PSI Switzerland

  25. Thank you for the attention

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