die haut als barriere für nanopartikel - das nanoderm projekt
TRANSCRIPT
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Die Haut als Barriere für
NanopartikelDas NANODERM Projekt
Tilman ButzUniversität Leipzig
Nanotechnologie,ihre Produkte und Risiken für den Verbraucher
Expertengespräch im Bundesinstitut für RisikobewertungBerlin, 28. März 2006
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The NANODERM Consortium
Country P.No. Participant Short Name Skills Resp Involved inD 1a Butz Uni Leipzig Ion Microscopy, Radiotracers 1 1 2 3 5 8
Sub Pallon Lund Ion Microscopy
D 1b Sticherling Uni Leipzig Biopsies, tissue 6 2 5 6 8Sub van Vaeck Antwerpen LMMS, S-SIMS
F 2 Surlève-Baz. UBX1/FDRC Electron Microscopy 8 2 4 8Sub UBX I/II
H 3 Hunyadi UD-MHSC Biopsies, cellsSub Kiss Debrecen WP 7 / Microprobe
P 4 Pinhero ITN Biopsies, tissue 2 2 3 6 8
Pl 5 Z. Stachura IFJ Cyclotron, Ion MicroscopySub Budzanowska Krakow Biopsies
Pl 6 J. Stachura Uni Krakow Electron Microscopy 4 4 8
F 7 Moretto UBX1/CENBG Ion Microscopy 3 3 8
7 2 3 6 7 8
5 1 2 3 5 8
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Methods 1
• Tape stripping; quantitat ive
This is a “ horizontal” technique
advantages:
easy and cheap
disadvantages:
does not yield quantitat ive penetrat ion prof iles due to furrow s and hair follicles
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Methods 2
• Confocal Laser Scanning Microscopy
This can be a “ horizontal” technique on explants
and a “ vert ical” technique on cross-sections
advantages:
yields “ 3D” -info w ith “ thick” sect ions and thus
reduces preparation art ifacts
disadvantages:
needs f luorophors; bleaching problems
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Methods 3
• HRTEM
a “ vert ical” method using ultra-thin cross-sections
advantages:
visualize individual part icles
get chemical composit ion of individual part icles
disadvantages:
limited f ield of view (representative ?)
many preparation steps (art ifacts ?)
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Methods 4
Ion beam techniques: PIXE, RBS, STIM: “ vert ical techniques”
PIXE: part icle induced X-ray emission yields elemental maps
RBS: Rutherford backscattering spectrometry and
STIM: scanning transmission ion microscopy yield density
advantages:
large f ield of view
easy preparation
check for preparation art ifacts
disadvantages:
cannot visualize individual part icles
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Methods 5
• Radio-labelling w ith 48V (positron emitter, T1/2 = 16d)
This is a “ vert ical” method using thin cross-sections andnuclear microemulsions
advantages:
ultra-sensit ive
large f ield of view
relat ively easy preparat ion
see individual positron tracks
disadvantages:
cannot visualize individual part icles
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Materials Nanoparticles :
Thioveil , P25, Eusolex T-2000
primary particle size: about 20 nm, coated (?)
Formulations:
various formulations / gels, various commercial sunscreens
Skin:
porcine skin, mouse pads, human skin transplanted to SCID-mice, healthy human skin biopsies (age, sex, caucasian, coloured) and explants, psoriatic skin
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Unprotected skin
Action of Nanoparticles
Reflection and absorptionof UV-radiation
Sun screen withnanoparticles
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Pig Skin Biopsies
areas of biopsy:inner parts of thehind legs
0.3 – 2.750.25 – 2.258, 24, 48Application Times(hours)
•native•water rinsed•ethanol cleaned•tape stripped (10×)
nativeethanol cleanedPretreatment
inner parts of hind legsinner parts ofhind legs
neck & backAreas ofBiopsies
3rd2nd1stCampaign
punch cylinders:5 mm
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TEM of ashed Eucerin Micropigment Lotion 25
Wagner, Uni Leipzig
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Crystalline TiO2 coated with amorphous silica layer
From:
Future Technologies Vol.54
Industrial application of nanomaterials –chances and risks
W. Luther (ed.)
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HRTEM 1
Cross-section through stratum corneum
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HRTEM 2
Nanoparticles between sheets of stratum corneum
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HRTEM 3
Nanoparticles between sheets of stratum corneumdisjunctum
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PIXE on Porcine Skin Cross-Section
red: Pblue: Sgreen: Ti
Clear delineation of strata w ithout staining:• Stratum corneum: sulphur rich• Stratum spinosum: phosphorous rich
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Fast transporter: liposome formulation
Fast transporters do not transport nanoparticles
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Human Skin on SCID-Mouse
C aK
C l
ST iC aK
C l
ST i
Fev 429 – 431 64 µµµµm
Fev 428 – 430 66 µµµµm TiTi
Significant Ti-level instratum corneum compactumand stratum granulosum
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5) M37d1 map (400 µm××××200 µm, 0.40 µC)
In the marked region the Ti MDL is 3ppmThe Ti concentration in this region is below the MDL.
P S Cl K Ca Ti
0,32% 0,74% 0,43% 0,30% 678ppm
optical image
PIXE maps
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Preparation artifacts
Dark spots in optical image are due to detached
horny layer part icles
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Are Microlesions important ?STIM and PIXE images
Anthelios XL 60 F 24h/occlusive
Penetrat ion through stratum corneum can be associated w ith microlesions
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Hair Follicle
400 µm x 400 µm
Penetration into vital t issue?red: P green: Sblue: Ti
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Autoradiography using microemulsions and 48-V labelled TiO2
Hair follicleSkin furrow
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NIH 3T3 internalize Nanoparticles
C
Ti-Wash
15 µg/cm2 TiO2(9 nm, Anatase, uncoated)
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in-vitro Response to TiO2 Nanoparticles
--��������
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Diff. markers/cell adhesionmolecules ����
Proliferation
(����)No����NoNoApoptosis
(����)±����±±Viable cellnumber
fastfastYesNoNoInternalization
NoNo����NoNo[Ca2+]i Change
Dendritic cellsMelanocytesFibroblastsSebocytesKeratinocytes
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Summary
• normally, penetrat ion is restricted to the stratum corneumdisjunctum; sometimes Ti found in s.c. compactum
• occasionally, Ti is detected in the stratum granulosum• very seldom, Ti is detected in the stratum spinosum• in most cases, Ti spots in the dermis are ident if ied as
contaminat ions• fast t ransporters do not transport nanopart icles• no signif icant dif ferences w ere observed for dif ferent
formulat ions nor for dif ferent exposure t imes (no kinet ics !)• to our surprise, the part icle shape had no inf luence• it appears that TiO2 part icles are mechanically rubbed into
the horny layer / hair follicles / furrow s w ithout dif fusivetransport
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Questions to be discussed
• Which tests should be conducted establishing exposurepotent ial through skin barriers?My personal opinion: - never trust a single technique- thus far there is very limited exposure to vital t issue, but
there are open quest ions: part icles in the 1-2 nm rangemight behave like small macromolecules and penetrate;transglandular pathw ayclearence from follicles (+ glands?)
• When is dermal hazard test ing necessary?My personal opinion:- w henever mechanical act ion / microlesions can occur- w henever NPs are t ransporters for toxic substances