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Возможности нейтронного и синхротронного излучения для исследования структуры липидных мембран. М.А. Киселев Лаборатория нейтронной физики Объединенный институт ядерных исследований , Дубна E-mail: [email protected]. - PowerPoint PPT Presentation
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Возможности нейтронного и синхротронного излучения для
исследования структуры липидных мембран
М.А. Киселев
Лаборатория нейтронной физикиОбъединенный институт ядерных исследований, Дубна
E-mail: [email protected]
Совещание«Современные ядерно-физические методы исследования в физике конденсированных сред», Минск, БГУ, 22-23 апреля, 2013
Phospholipids and ceramides
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Phospholipids – major component of cell membranes.Ceramides – major components of stratum corneum lipid matrix.
Phospholipids and ceramides – important materials for drug and cosmetic industry.
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Objects and methods
Unilamellar vesicles
Multilamelar vesicles (Liposomes)
Oriented multilamellar planar systemon quartz substrate
Neutron small-angle scattering
Small-angle and wide-angle powder X-ray diffraction at synchrotron source
Neutron diffraction
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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.
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Real-time X-ray diffraction at cooling DPPC/water system at synchrotron.
Ice crystalFree waterDiffusion
Cooling with the rate of 1oC/min, acquisition - 1min/spectrum
d=5.9Å
c' LL crystallization
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Low-temperature phase transition in DPPC/DMSO/water system at Xdmso=0.05. Synchrotron.
Ice
New phase
Cooling with the rate of 1oC/min, acquisition - 1min/spectrum
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Drug penetration pathways across the skin.
Down hair follicle
Across SC Down sweat gland Intercellular route
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Fourier profiles at RH=60%, T=32oC
-20 -10 0 10 20-15 -5 5 15 25-25
x, Å
- 1
0
1
2
3
sx)
, a.u
.
CH3 groupsCH2 chains
polar head groups
cholesterol
-20 0 20-10 10 30-30
x , Å
- 2
- 1
0
1
2
3
sx
), a
.u.
polar head
C H 2 chainsC H 3 groups
Repeat distance 45.63±0.04Å
Repeat distance 54.750.03 ÅMembrane thickness 45.80.1 ÅIntermembrane space 9.00.1 Å
Model SC membranes
DMPC
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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.
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Hyposesis of super-strong intermembrane interaction created by ceramide 6 in fully
extended conformatiion (armature reinforcement).
Fully extended conformation of ceramide
Hair-pin conformation of ceramide
Bricks and mortar model of SC
M.A. Kiselev. Conformation of ceramide 6 molecules and chain–flip transitions in the lipid matrix of the outermost layer of mammalian skin, the stratum corneum. Crystallography Reports, 2007, Vol. 52, No. 3, pp. 525–528
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Real- time neutron diffraction at DN-2, IBR-2.
Time-of-flight.
DPPC membrane hydration-dehydration in real time at T=20oC
Hydration 480min
Dehydration 360min
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Nanostructure alteration during hydration-dehydration as result of fourier-synthesis in real-time. T=20oC.
Water layer dw
Membrane thickness dB
Repeatdistance d
Thickness of the hydrocarbon chains region dc
d
dB
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.
Chamber with excess of water
• New possibilities to study the influence of the skin penetration enhancer via neutron diffraction at IBR-2 reactor.
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0,00 0,05 0,10 0,15 0,20 0,250
1
2
3
4
5
6
Inte
nsity
, a.u
.
q, Å-1
black t=0 min dryred t=9 min in DMSOblue t=18 min two phasemagenta t=27 min one phase
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Unilamellar vesicles from phospholipid molecules
Neutron small-angle scattering in D
2O
X-ray small-angle scattering in sucrose solutions
From point of physics it is nanospheres with liquid crystal surface
R=250Ån2 ·1014 vesicles/cm3 for 1% DMPC
(w/w)6500 DMPC molecules in one layer for
30oC
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.
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SANS pattern at 30 °C of unilamellar DMPC vesicles prepared by extrusion through 500 Å pores at DMPC
concentration of 1% (w/w).
0 .0 1 0 0 .1 0 00 .0 2 0 0 .0 5 0 0 .2 0 00 .0 0 90 .0 0 4
q , Å -1
1 E -0 0 4
1 E -0 0 3
1 E -0 0 2
1 E -0 0 1
1 E + 0 0 0
1 E + 0 0 1
1 E + 0 0 2
1 E + 0 0 3d
/d,
cm
-1
qR
qm
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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)
0 .1 0 0 0 .2 0 0 0 .3 0 0 0 .4 0 0
q , Å -1
1 E -0 0 1
1 E + 0 0 0
1 E + 0 0 1
1 E + 0 0 2
1 E + 0 0 3
1 E + 0 0 4
1 E + 0 0 5
Inte
nsit
y, a
.u.
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Method of Separated Form Factors (SFF)
IBqSqAnqd
d
mon
)()()( 2
IBqSdqFRqFnqd
dbs
mon
)(),(),()(
F q RR
qRSin qRs ( , ) ( )
4
2 2
.
2
2
2),(
qdSin
qdqFb
For the case of (x)=Const
c(x)=(x) D2O is contrast, the difference between scattering length densities of the bilayer (x) and the heavy water D2O.
dxqxxxqRqR
R
dxqxxqRqR
RqA
d
d c
d
d cves
)sin()()cos(4
)cos()()sin(4)(
2/
2/
2/
2/
2
22/
2/
)()(),(
d
cb
d
dxqxCosxdqF
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
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0,01 0,1
0,01
0,1
1
10
100
1000 1000Å
500Å
d/d,
cm
-1
q, Å-1
Model DF, Å <R>, Å , % d, Å D, Å Nw A, Å2 IB, cm-1
SFF 500 275.6±0.5 27 47.8±0.2 20.5±0.3 11.90.3 61.00.4
0.007
Full 500 275.7±0.4 27 47.8±0.2 20.5±0.3 11.90.3 61.00.4
0.007
SFF 1000 500* 48 45.5±0.6 20.8±0.4 10.8±0.4 62.6±1.0 0.007
Results for liquid phase at 30oC, HH approximation of (x)
Multilamellar (small curved) vesicles
d = 44.2 ÅA=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.
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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
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Phospholipid transport nanosystems (FTNS)
0,01 0,1 1
0,1
1
10
100
1000
10000
1/q2
25% PTNS in waterInte
nsi
ty, a
.u.
Q, Å-1
DMPC vesicles
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/q2 law.
water75%
phospholipid5%
maltose20%
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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.
• 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
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Participants from Russia
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
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Thank you for the attention
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