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Molecular secrets ofmembrane barrier modulators
Jamshed AnwarInstitute of Pharmaceutical Innovation
University of Bradford
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AcknowledgementsAcknowledgements
Rebecca Notman, KCL (now Warwick)
Andrey Gurtovenko, IPIWim Briels, Twente, The NetherlandsWouter den Otter, Twente, The Netherlands
,
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Molecular secrets of
membrane barrier modulators
Molecular secrets of
membrane barrier modulators Significance
Drug & gene delivery; Membrane-lipid therapy;Cryoprotection
Molecular dynamics simulation
Interaction of small molecules with DPPC bilayersOleic acid, DMSO, Ethanol, Urea
Interaction of DMSO with ceramide bilayers
Free energies of pore formation
Concluding remarks
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Ceramide (~50%) (9 types in skin)
Cholesterol (~25%)
Free fatty acids (~10-20%)
OH
O
NH
OH
OH
O
O
NH
OH
OH
O
Ceramide 2
Skin lipidsSkin lipids
CHOL
CER 1
CER 2, 3, 4 and 6
CER 5
Bouwstra et al. (2000) Acta. Derm. Venereol. Supp 208, 23
Sandwich model
OH
CholesterolO
Stearic Acid
O
O
O
O
OPO
N+
O
O
DPPC
Extracted skin lipid behaviouris qualitatively similar to
dipalmitoylphosphatidylcholine(DPPC)
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Proposed Mechanisms of Action
Interact with the skin lipids
Increase the permeability of membranes to drug molecules
Mechanism of action is not fully understood at the molecular level
Common Penetration Enhancers
Chemical penetration enhancersChemical penetration enhancers
Dimethyl-sulphoxide
(DMSO)
Azone
OleicAcid
Ethanol
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Molecular simulationMolecular simulationReductionism
Nothing exists except atoms and
empty space; all else is opinionDemocritus 430-380 BC
Molecular level understanding
ReductionismNothing exists except atoms and
empty space; all else is opinionDemocritus 430-380 BC
Molecular level understanding
Why simulate?Cannot do experiments that we would like to do(Molecular level resolution)Predict behaviour
Why simulate?Cannot do experiments that we would like to do(Molecular level resolution)Predict behaviour
Molecular simulationsAtomistic; Input is atom-atom interactions onlyNo fudge factors
Molecular simulationsAtomistic; Input is atom-atom interactions onlyNo fudge factors
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Interaction potential (molecular mechanics)Interaction potential (molecular mechanics)
+
+
=
10ns)
Accuracy of interaction potential
Periodic boundaries &minimum image convention
NVE, NVT, NPT & NTEmploy extended Lagrangian
e.g. L(r,p,H,s)
1. Initial configuration
2. Equilibration3. Production (averages)
NPT GNPT G
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Molecular dynamics simulation ofdipalmitoylphosphatidylcholine (DPPC)Molecular dynamics simulation ofdipalmitoylphosphatidylcholine (DPPC)
256 DPPC
1 ns
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How faithful are molecular simulation?Comparison with experimentHow faithful are molecular simulation?Comparison with experiment
Electron density profile
Bilayer repeat LzVolume per lipid VLArea per lipid AL
Conformation of molecules
Order parameter for alkyl chain
Diffusion constants
Bending modulus
Area compressibility modulus
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Interaction of oleic acid with DPPC bilayerInteraction of oleic acid with DPPC bilayer
Oleic acid easily accommodated by the DPPC lipids;
No phase separation Notman, Noro & Anwar (2007) J Phys Chem B
OleicAcid
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Interaction of DMSO with DPPC bilayersInteraction of DMSO with DPPC bilayers
350K
Notman et al. (2007) J Amer Chem SocGurtovenko & Anwar(2007a) J Phys Chem B
Pore formation Bilayer disintegration
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DMSO makes DPPC membrane floppyDMSO makes DPPC membrane floppy
Area compressibilitymodulus K
A
33020 mN/m pure DDPC9010 mN/m 20% DMSO
Surface tension v. area per lipid
Circles: without DMSO; Squares: 20% DMSO
5.6x10-21 J pure DPPC1.1x10-21 J for 20% DMSO
Notman et al. (2007) J Amer Chem Soc
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DMSO-induced water pore formationDMSO-induced water pore formation
Pore formation timescale
Dependent on [DMSO]
Typically > 5 -10 ns
Pores stable for
up o ns
Explains for the first timepermeability enhancementof hydrophillic molecules
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Permeability enhancement in action:Ion leakage across DMSO-induced water poresPermeability enhancement in action:Ion leakage across DMSO-induced water pores
Gurtovenko & Anwar (2007b), J Phys Chem B
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DMSO induces translocation (flip-flops) of lipidsDMSO induces translocation (flip-flops) of lipids
Gurtovenko & Anwar (2008) Langmuir
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Rationalization of DMSOs pharmacological effectsRationalization of DMSOs pharmacological effects
Insights from simulations Makes membrane more floppy Induces water pores; enhances permability of ions and water Destroys bilayer structure at high [DMSO] Induces flip-flops (induces programmed cell death?)
Permeability enhancement Cryoprotection
Floppier membrane enable better accommodation of mechanicalstress during ice formation & melting; Water pores control osmoticimbalance
Local analgesicDisrupts action potential by allowing ion leakage in nerves
Cell fusagen in cell biologyReduction in compressibility modulus and bending rigidity + poreformation
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Effect of ethanol on lipid bilayers:Low concentrations (up to 10 mol %)Effect of ethanol on lipid bilayers:Low concentrations (up to 10 mol %)
No ethanol 5 mol % ethanol
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Effect of ethanol on lipid bilayers:Interdigitation of lipid hydrocarbon chainsEffect of ethanol on lipid bilayers:Interdigitation of lipid hydrocarbon chains
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Effect of ethanol on lipid bilayers:High concentrations (~15 mol%)Effect of ethanol on lipid bilayers:High concentrations (~15 mol%)
Micelle-like structuresin the bilayer interior
Gurtovenko & Anwar (2009) J. Phys. Chem. B
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Effect of urea on lipid bilayersEffect of urea on lipid bilayers
No urea 2M of urea
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Structure of ceramide bilayer as a function ofDMSO concentration in the solventStructure of ceramide bilayer as a function ofDMSO concentration in the solvent
Gel phase Liquid crystalline phase
0 10 20 4030 50 60
323K
Notman et al (2007) Biophys. J
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Ceramide bilayers containing DMSOCeramide bilayers containing DMSO
Notman et al (2007) Biophys. J
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Ceramide bilayers containing DMSOCeramide bilayers containing DMSO
=
%60mJ/m190
%10@mJ/m3700
%0mJ/m7900
2
2
2
AK
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Ceramide/fatty acid/cholesterol bilayer (1:1:1)Ceramide/fatty acid/cholesterol bilayer (1:1:1)
Water
Cholesterol
Fatty acid
Ceramide 2
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Free energy of pore formationFree energy of pore formation
R
PoresCell death; Genes/drug delivery (eg electroporation);Lipid asymmetry, Flip-flops & signalling
SL rrF 22 =
Where S is the surface tension (surface energy)L is the line tension (edge energy)
2-d nucleation
Assumes surface lost due to lipids going into bilayer!
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Free energy of pore formationin ceramide bilayersFree energy of pore formationin ceramide bilayers
L=42 pJ/m
L= 6 pJ/m
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Induced pore in ceramide bilayercontaining 60 mol% DMSOInduced pore in ceramide bilayercontaining 60 mol% DMSO
side view
top view
side view
=
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Induced pore in ceramide bilayersInduced pore in ceramide bilayers
side view
top view
top view
side view
=
hydrophobic porebut .
where is the water?
Notman et al (2008) Biophys. J
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Schematic structures of poresSchematic structures of pores
Hydrophilic pore Hydrophobic pore Vapour pore
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Should hydrophobic pores contain water?Should hydrophobic pores contain water?
( ) wlRhRF /edge 2=
2R
h
nm2= hRwv
tvtweq
( ) vwvl RRhRF /2
/edge 22 +=
nm2/
//
= hR
vw
vlwleq
Notman et al (2008) Biophys. J
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Concluding remarksConcluding remarks
DPPC bilayers Oleic acid: accommodated within bilayer without much
perturbation
DMSO induces water pores and makes membrane floppy;induces flip-flops rationalization of DMSOs pharmacological activity
Ethanol: high concentrations cause internalisation of H2O and-
that may serve as delivery packages Urea: replaces H2O at the interface; significance??
Ceramide bilayers DMSO induces gel fluid phase transition; Explains DMSOs
role as a penetration enhancer in transdermal drug delivery;Significance of vapour pores in skin.
Future Impedance measurements & AFM; transport through bilayers In-silico screening of molecules that enhance &/or retard
permeability
