New insights into the stratum corneum lipid membrane organisation [Elektronische Ressource] : an X-ray and neutron scattering study / von Jarmila Zbytovská

Jz

Sujets

• Biologie

Informations

• Publié par: martin-luther-universitat_halle-wittenberg
• Publié le: 01 janvier 2006
• Nombre de lectures: 12
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

NEW INSIGHTS INTO THE STRATUM CORNEUM LIPID
MEMBRANE ORGANISATION
AN X-RAY AND NEUTRON SCATTERING STUDY



Dissertation

zur Erlangung des akademischen Grades
Doctor rerum naturalium (Dr. rer. nat.)
vorgelegt der

Mathematisch-Naturwissenschaftlich-Technischen Fakultät
(mathematisch-naturwissenschaftlicher Bereich)
der Martin-Luther-Universität Halle-Wittenberg

von Frau Mgr. Jarmila Zbytovská
geboren am 17. 9. 1976 in Prag (Tschechische Republik)



Gutachter:
1. Prof. Dr. Dr. Reinhard Neubert
2. Prof. Dr. Siegfried Wartewig
3. Doc. RNDr. Pavel Doležal CSc.

Halle (Saale), den 20. 10. 2006
urn:nbn:de:gbv:3-000012474
[http://nbn-resolving.de/urn/resolver.pl?urn=nbn%3Ade%3Agbv%3A3-000012474]CONTENTS
CONTENTS
LIST OF ABBREVIATIONS IV
LIST OF SYMBOLS V
1 INTRODUCTION 1
2 CURRENT KNOWLEDGE STATUS OF THE STRATUM CORNEUM
STRUCTURE AND TRANSDERMAL PERMEATION ENHANCEMENT 4
2.1 The organization of the mammalian skin 4
2.2 The stratum corneum 5
2.2.1 TheoriginoftheSClipids 5
2.2.2 ThelipidcompositionwithintheSC 6
2.2.3 TheorganizationoftheSClipidmatrix 8
2.3 Drug penetration routes through the skin 10
2.4 Modes of actions of permeation enhancers 10
3 BASIC PRINCIPLES OF EXPERIMENTAL TECHNIQUES EMPLOYED 12
3.1 Differential scanning calorimetry 12
3.2 Infrared and Raman spectroscopy 13
3.2.1 Conformationallysensitivebandsofhydrocarbonchains 13
3.3 Scattering techniques 15
3.3.1 X#raydiffractiononlipids 16
3.3.2 Smallangleneutronscattering 18
3.3.3 SmallangleneutronscatteringonULVs 20
3.3.4 Neutrondiffractiononorientedmultilamellarsamples 22
3.3.5 Particlesizeanalysisviadynamiclightscattering 24
4 INFLUENCE OF PHYTOSPHINGOSINE-TYPE CERAMIDES ON THE
STRUCTURE OF DMPC MEMBRANE 26
4.1 Introduction 27
4.2 Material and Methods 28
4.2.1 Materials 28
4.2.2 Samplepreparation 28
4.2.3 Differentialscanningcalorimetry 29
4.2.4 SmallangleX#raydiffraction 29
4.2.5 Highperformancethinlayerchromatography 29
4.2.6 Smallangleneutronscattering 30
4.2.7 Dynamiclightscattering 30
i CONTENTS
4.3 Results 31
4.3.1 CharacterizationofMLVsbyDSC 31
4.3.2 CharacterizationofMLVsbysmallangleX#raydiffraction 33
4.3.3 CharacterizationofULVs 37
4.4 Discussion 41
4.5 Conclusions 45
5 INFLUENCE OF CHOLESTEROL ON THE STRUCTURE OF STRATUM
CORNEUM LIPID MODEL MEMBRANE 46
5.1 Introduction 47
5.2 Material and Methods 48
5.2.1 Material 48
5.2.2 Vesiclepreparation 49
5.2.3 Vesiclecharacterization 49
5.2.4 SmallangleX#raydiffraction 50
5.2.5 Smallangleneutronscattering 51
5.2.6 Molecularmodelling 51
5.3 Results 52
5.3.1 SmallangleX#raydiffractiononMLVs 52
5.3.2 CharacterizationofULVs 58
5.3.3 SmallangleneutronscatteringfromULVs 59
5.4 Discussion 61
5.5 Conclusions 65
6 THERMOTROPIC PHASE BEHAVIOUR OF A SC LIPID MODEL SYSTEM IN
THE VARIATION OF CHOLESTEROL CONCENTRATION 66
6.1 Introduction 66
6.2 Methods 66
6.2.1 Materialandsamplepreparation 66
6.2.2 Differentialscanningcalorimetry 66
6.2.3 SmallangleX#raydiffraction 67
6.3 Results 68
6.3.1 Differentialscanningcalorimetry 68
6.3.2 SmallangleX#raydiffraction 70
6.4 Discussion 72
6.5 Conclusion 74
7 NEUTRON DIFFRACTION ON THE SC LIPID MODEL SYSTEM 75
i i CONTENTS
7.1 Introduction 75
7.2 Methods 75
7.2.1 Material 75
7.2.2 Samplepreparation 75
7.2.3 Neutrondiffractionmeasurements 76
7.3 Results 77
7.3.1 Neutrondiffractionmeasurementsfromthemixtureswithvarious
CHOLconcentrations 77
7.3.2 NeutronscatteringlengthdensityprofilesoftheSClipidmodel
membrane 79
7.4 Discussion 84
7.5 Conclusions 85
8 INFLUENCE OF PERMEATION MODULATORS ON THE BEHAVIOUR OF A SC
LIPID MODEL MIXTURE 87
8.1 Introduction 87
8.2 Methods 88
8.2.1 Material 88
8.2.2 Samplepreparation 89
8.2.3 SAXDmeasurements 89
8.3 Results and discussion 89
8.3.1 InfluenceofureaontheSClipidsystem 89
8.3.2 InfluenceofoleicacidontheSClipidsystem 91
8.3.3 Influenceof12G12ontheSClipidsystem 93
8.4 Conclusions 95
9 SUMMARY AND PERSPECTIVES 97
10 ZUSAMMENFASSUNG UND AUSBLICK 99
REFERENCES 103
AppendixA
AppendixB
AppendixC
iii LIST OF ABBREVIATIONS
List of Abbreviations

AMD automatedmultipledevelopment
CCD charge#coupleddevice
Cer[AP] ceramide[AP]
Cer[NP] ceramide[NP]
CS cholesterolsulphate
CHOL cholesterol
DLS dynamiclightscattering
DMPC dimyristoylphosphatidylcholine
DSC differentialscanningcalorimetry
HPTLC highperformancethinlayerchromatography
IR infrared
KHGs keratohyalingranules
L#phase longphase
L liquidcrystallinephaseα
L gelphaseβ
L ´ ripplephaseβ
LBs lamellarbodies
LPP longperiodicityphase
MLVs multilamellarvesicles
NIBS non#invasiveback#scatter
NIR nearinfrared
NMR nuclearmagneticresonance
OA oleicacid
PA palmiticacid
RXLI recessiveX#linkedichthyosis
S#phase shortphase
SANS smallangleneutronscattering
SAXD smallangleX#raydiffraction
SC stratumcorneum
SPP shortperiodicityphase
ULVs unilamellarvesicles
UV ultraviolet
VIS visible
WAXD wideangleX#raydiffraction

i v LIST OF SYMBOLS
List of Symbols

2A membraneareapermolecule [Å ]
c lightvelocity [invacuum
8 #12.998*10 ms ]
D lamellarrepeatdistance [Å]
2 #1diffusioncoefficient [m .s ]
d membranethicknessparameterderivedfromMSFF [Å]
d membranethicknessparameterderivedfromKP#plot [Å]g
d distance between two parallel planes characterized by Miller [Å]hkl
indiceshkl
d membranethickness [Å]m
E energy [J]
F structurefactor [a.u.]h
enthalpy [J.mol]H
h diffractionorder;reflexion
#34 Planck’sconstant [6.626176*10
Js]
I intensity [a.u.]
wavelength [Å]or[nm]
m weight [g]
#1M molecularweight [g.mol ]w
23N Avogadro’snumber [6.022*10 ]A
n refractiveindex
η viscosity [Pa.s]
#1ν wavenumber [cm ]
#1q scatteringvector [Å ]
R vesicleradius [Å]
R radiusofgyration [Å]g
R hydrodynamicvesicle radius [Å]h
ρ electron/neutronlengthdensity [cm]
#1Lρ averageexcessscattering#lengthdensityperunitmass [cm.g ]m
T temperature [°C]
t time [s]
θ scatteringangle [°]
3V volume [cm ]or[l]
3V solventaccessiblevolume [Å ]SA

TherestofsymbolsareexplainedinrelevantChapters.
v
lD1. INTRODUCTION
1 Introduction
Theprimaryfunctionofthemammalianskinistheprotectionagainstchemical,pathogen
and UV radiation. It must provide a mechanically strong structure that resists physical
stress.Theskinplaysalsoamajorroleinthermoregulationandwaterbalanceofthebody.
Thefunctionsof skinas sensory,endocrineandimmuneorganshouldbementionedas
well[1,2,3,4].Theskinprotectionfunctionisensuredbyitsuniquebarrierproperties.First
in the 1940s, it was postulated that the outermost layer of the human skin, the stratum
corneum(SC)isresponsiblefortheskinbarrierfunction[5].
TheSCconsistsofdeadcells,thecorneocytes,whicharefilledwithproteinkeratin.The
corneocytes are embedded in a lipid matrix with a unique composition. The SC lacks
phospholipids but it is enriched in ceramides, free fatty acids, cholesterol and its
derivatives. The SC lipids are organized in lipid membranes arranged into a lamellar
structure. Due to the special physicochemical properties of ceramides, these membranes
areextremelyrigidand,therefore,verypoorlypermeable[6].
The necessary impermeability of the human skin represents, however, a very strong
limitationforthesystemictransdermaldrugdelivery.Thisadministrationrouteoffersmany
advantages, namely the avoidance of the first pass metabolism in the liver, reduced side
effects,ortheopportunitytodeliverthedrugcontinuously.Inordertoincreaseabsorption
ofadrugthroughtheskin,areversibledecreaseinitsbarrierfunctionisneeded.
Several physical (sonophoresis, iontophoresis, electro#osmosis, electroporation and
temperature) and chemical or formulation methods have been described, which have
successfullyincreasedthedrugdeliveryacrossorintotheskin.Recently,theapplicationof
permeationenhancershasbeenmostcommonlyusedtoovercometheSClipidbarrier[7].
The most of the non#irritating permeation enhancers are of amphiphilic character. They
canincorporateintotheSClipidmembranesinordertochangethemembranestructure.
Thus, the membrane becomes more fluid and permeable for a drug. This effect was
describedforanumberofsubstances,butonlylittlehasbeenknownaboutthemolecular
background of the permeation enhancers’ mode of action so far. In contrast, only few
1 1. INTRODUCTION
enhancerssuchasureaweredescribedtointeractwiththepolarheadgroupsoftheSC
lipids[8].
Sometopicallyadministratedsubstances(e.g.repellents,sunscreens)shouldreachonlythe
superiorskinlayers,sothatthesystemicabsorptionconnectedwithundesiredsideeffects
is minimized. Here, the so#called transdermal permeation retardants (reducers) can be
applied[9].
Furthermore,damagedskinbarrierduetoadiseaseortraumatendstoabnormalfunction
and increased permeability of the skin. A numbe