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Skinstructureandfunction

Leiden/Amsterdam Center for Drug Research

Integrated Research between


divisions

Education system
No pharmacy students,

Pharmacology, Analytical
Chemistry, Biopharmacy,
Toxicology, Drug Delivery
Technology, etc

Joke A. Bouwstra

DivisionofDrugDeliveryTechnology
Leiden/AmsterdamCenterforDrugResearch(LACDR)

Proteins, vaccination
Skin Research

Science oriented:
Master Degree in BioPharmaceutical Sciences

100 students per year, after


master degree, mostly Ph.D

DDT research lines


Skin Research

Skin barrier and


Lipid organisation
Reconstructed skin

Delivery of
drugs

By pass the skin barrier:


microneedles

Skin research

Biologics formulation

Lipid organization
Skin models
Delivery methods
Diseased skin
Transport mechanisms
Vaccine delivery

Protein stability
Structural characterization
Vaccine delivery
Safety (immunogenicity)
Cell culture models

Overviewofpresentation

Themanyfunctionsoftheskin
Step 2

Step 1
Skin
function

Skin morphology

Step 4
Heterogeneity
of skin

Conventional
-establishes, controls & transmit contacts with
external world
-Protects from the environment
-Maintain temperature, electrolyte and fluid
balance

Step 3
Skin renewal

New
-It is an integral component of immune
and nervous system

Conventionalfunctionoftheskin

Conventionalfunctionoftheskin

Barrier function
Protects body from dessication: water and nutrients
are kept in (physical barrier function)

Barrier function
Protects body from dessication: water and nutrients
are kept in (physical barrier function)

Unwanted substances: bacteria, toxic agents


are kept out (physical barrier function)

Unwanted substances: bacteria, toxic agents


are kept out (physical barrier function)

Protection against the sun (biological barrier


Melanocytes, production of melanin, act as UV filter)

Protection against the sun (biological barrier


melanocytes)

virus/bacteria

Trans epidermal water loss


(TEWL)

Temperature control
Sweat glands& ateria

melanocytes

Surveillance and action


-Immune (langerhans cells, dendritic cells
-repair (wound healing, etc), continuous turnover

Newfunction:immunologicalresponses

Overviewofpresentation

Upon receiving signal from the outside


Step 2

Step 1

Keratinocytes:
release cytokines:
TNF-a, IL-1b:

stratum
corneum

Skin
function

viable
epidermis

Skin morphology

Migration of LCs

Int DC

dermis
LC
IL-12

Lymph node
Int DC
Ueno et al, Immun. Reviews (2007) 219, 118-142

Skinmorphology:overview
hairfollicles

Epidermis
(150 m)
Dermis
(2000 m)

SkinMorphology:somenumbers
Sebaceous glands

Stratum corneum
(15 m)
Systemic
circulation
Nerves (Sensory
Sweat glands

Subcutaneous
fat tissue
Corneocytes
embedded
in lipid domains

16% of body mass: interface to outside


Total surface around 1.8 m2
Epidermis
thickness stratum corneum: 15-20 m
thickness viable epidermis: 100-150 m
cell tissue
Dermis
thickness is 1-2 mm
nerves, vascular bed
cells, connective tissue, collagen
source of most appendages
Hypodermis
thickness 1-2 mm
fat cells

SkinMorphology:routesofpenetrationafterapplying
aproduct
E p id e rm a l
ro u te
In te rce llu la r

SkinMorphology:skinsurface
Important to know: skin has not a flat surface. Large undulations
are present. Rinkels go as deep as the dermis

H a ir

T ra n s ce llu la r

A ppendageal
S tra tu m
ro u te
C o rn e u m

S tra tu m
C o rn e u m

SEM image

E p id e rm is

D e rm is
S e b a ce o u s
g la n d

B lo o d ve s se l

H a ir
fo llic le

Sw eat
gla n d

Desquamation:
Shedding of the cells

Majorcelltypesintheskin

Epidermalrenewing:continuousturnover
Desquamation:
Shedding of cells

Epidermis
Keratinocytes
Langerhans cells
Merkle cells
melanocytes

Keratinocytes

Dermis
Fibroblasts
Dendritic cells
in collagen
matrix

Stratum granulosum
Differentiation
starts

Subcutaneous
fat tissue
Fat cells

Stratum corneum
(15 mm)

Stratum corneum
(15 m)

Corneocytes
embedded
in lipid domains

Stem cells:
Continuous cell
Division: proliferation

Stratum spinosum
Stratum basale

Acloserlookatstratumcorneum
C
C

Penetration
pathway

corneocyte

Stratum granulosum

corneocyte

Epidermis

Stratum spinosum
Stratum basale

Dermis

Blood vessels

HEstaininghumanskin

Stratum granulosum
Stratum spinosum
Stratum basale

Dermis

Lipid
organisation
Penetration
pathway

Epidermis

Blood vessels

Crosssection:cryoscanningelectronmicroscopy
Hydrated stratum corneum:
Individual cells with keratin
filaments

stratum corneum

Stratum corneum

viable epidermis
Viable epidermis
dermis
Upper part of dermis

Finalproductofdifferentiation:stratumcorneum

Desmosomelayeredstructures

12-18 dead cell layers:


Corneocytes: hydrophilic: water, natural moisturising factor, keratin filaments

Lipids: ceramides, cholesterol, fatty acids

15 um

desmosomes
Cornified envelope: thick protein layer:
loricrin, involucrin
(Ca2+ dependent enzymes)

Appendages:Hairfollicle

Importantforskinbarrierfunction

Hair follicle cross section


stratum corneum

gap

epidermis
outer root sheet

Lipid
monolayer
bound to
envelope

Lamellae: lipids
form crystalline
phases

NMF,water, keratin filaments

sebaceous gland
inner root sheet:
Henles layer
Huxleys layer
cuticle

Hair shaft:
cuticle
cortex
medulla

growth cycle
bulge

hair bulb

Cornified envelope
Loricrin, involucrin,etc

Cornified envelope: Almost


impermeable for drugs

Crystalline lipid lamellae and lipids bound to envelope

anagen (growth)

catagen (transition) telogen (rest)

Overviewofpresentation
Step 1
Skin
function

Step 2
Skin morphology

When are these components of


Stratum corneum synthesized during
The differentiation process?

Step 3
Skin renewal

First proteins and then lipids

Renewalofskin

Desmosome break down

Proteinsynthesis

Enzymes affected by
water and pH

Filaggrin break down


Natural Moistursing
Factor (NMF)

A change in
cell morphology
and cell function
There is a constant
renewal of
the epidermis

Stratum corneum

Stratum granulosum
Stratum spinosum
Stratum basale:
Cell division

Differentiation

Differentiation

Desquamation process

Profilaggrin
filaggrin

Desmosome break down


Enzymes affected by
water and pH

Desquamation process
Stratum corneum

Keratohyalin
Granulae:

Stratum granulosum

Cytokeratines

Stratum spinosum

Desmosomes

Stratum basale:
Cell division

Differentiation

Natural Moistursing
Factor (NMF)

Profilaggrin
filaggrin

Cytokeratines

Stratum spinosum

Filaggrin break down

Differentiation

Natural Moistursing
Factor (NMF)

Profilaggrin
filaggrin
Keratohyalin
Granulae:
Involucrin, loricrin,
keratin1,10,profilaggrin

Cytokeratines
keratin 1 and 10

Desmosomes

Natural Moistursing
Factor (NMF)

Stratum corneum

Stratum granulosum

Proteinsynthesis

Filaggrin break down

Desquamation process

Keratohyalin
Granulae:

Desmosomes
Keratin 5, 14

Proteinsynthesis

Differentiation

Filaggrin break down

Desmosome break down


Enzymes affected by
water and pH

Stratum granulosum
Stratum spinosum
Stratum basale:
Cell division

Stratum spinosum

Natural Moistursing
Factor (NMF)

Stratum corneum

Stratum corneum

keratines

Filaggrin break down

Desquamation process

Desquamation process

Stratum granulosum

Proteinsynthesis

Enzymes affected by
water and pH

Desmosome break down


Enzymes affected by
water and pH

Keratohyalin
Granulae:

Desmosomes

Stratum basale:
Cell division

Desmosome break down

Profilaggrin
filaggrin

keratin 1 and 10

Differentiation

Proteinsynthesis

Profilaggrin
filaggrin
Keratohyalin
Granulae:
Involucrin, loricrin,
keratin1,10,profilaggrin

Cytokeratines
keratin 1 and 10

Desmosomes

Stratum basale:
Cell division

Desmosome break down


Enzymes affected by
water and pH

Desquamation process
Stratum corneum

Stratum granulosum
Stratum spinosum
Stratum basale:
Cell division

Proteinsynthesis

Desmosome break down

Filaggrin break down

Natural Moistursing
Factor (NMF)

Desquamation process
Stratum corneum

Profilaggrin
filaggrin
Keratohyalin
Granulae
Involucrin, loricrin,
keratin1,10,profilaggrin

Cytokeratines

Stratum granulosum
Stratum spinosum

keratin 1 and 10

Desmosomes

Stratum basale:
Cell division

Stratumcorneummorphology

Lipid
monolayer
bound to
envelope

Filaggrin break down

Differentiation

Differentiation

Natural Moistursing
Factor (NMF)

Proteinsynthesis

Enzymes affected by
water and pH

Profilaggrin
filaggrin
Keratohyalin
Granulae:
Involucrin, loricrin,
keratin1,10,profilaggrin

Cytokeratines
keratin 1 and 10

Desmosomes

Cornifiedenvelope

lamellae

NMF,water, keratin filaments


envelope
Crystalline lipid lamellae and lipids bound to envelope

Desmosome break down


Enzymes affected by
water and pH

Desquamation process
Stratum corneum

Stratum granulosum
Stratum spinosum
Stratum basale:
Cell division

Stratumcorneummorphology

Lipidcompositioninstratumcorneum
Ceramides (CER)

12 classes of CER

Cholesterol (CHOL)

Lipid
monolayer
bound to
envelope

lamellae
Free fatty acids (FFA)
Cholesterol sulfate
NMF,water, keratin filaments

Calcium

envelope

Minor
amounts

In viable cells phospholipids always play a major in


cell membranes. Therefore a major change in lipid
composition between viable cells and stratum corneum

Crystalline lipid lamellae and lipids bound to envelope

Ceramidesinstratumcorneum

Saturated C22-24
chain length

Synthesisoflipidsinepidermis

EOS/CER1

HN

OH
OH
O

EOP/CER9

HN

OH
OH

OH
O

O
O

EOH/CER4

HN

OH
OH

OH

EOdS

HN

OH
OH

OH

Amide-linked non-hydroxy fatty acids

Amide-linked a-hydroxy fatty acids


OH

NS/CER2

HN

OH
OH

AS/CER5

NP/CER3

OH
OH

AP/CER6

NH/CER8

OH
OH

AH/CER7

OH

HN

OH
OH

HN

OH
OH
OH
OH
O

HN

OH
OH

OH
OH

ND

OH
OH

Stratum granulosum

OH

HN

HN

Stratum corneum

OH

O
HN

Desquamation process

acylceramides
Differentiation

AD

HN

OH
OH

Synthesis of Ceramides:
Glucosyl ceramides and
sphingomyelin

Stratum spinosum

Stratum basale:
Cell division

Synthesisoflipidsintheepidermis

Synthesisoflipidsintheepidermis

Stratum corneum
Lamellar bodies:
Storage glucosylceramides
and sphingomyelin,
phospholipids
Synthesis of Ceramides:
Glucosyl ceramides and
sphingomyelin

Stratum granulosum

Desquamation process

Differentiation

Differentiation

Desquamation process
Glucosylceramides
Ceramides
sphingomyelinCeramides
phospholipidsFatty acids

Stratum corneum

Lamellar bodies:
Storage glucosylceramides
and sphingomyelin,
phospholipids

Stratum granulosum

Synthesis of Ceramides:
Glucosyl ceramides and
sphingomyelin

Stratum spinosum

Stratum spinosum

Stratum basale:
Cell division

Formationofceramides:moredetails

Stratum basale:
Cell division

Synthesisoflipidsintheepidermis
O

1. Glucosylceramides

HN

O-glucose
OH

Desquamation process

Ceramides

O
HN

Deficiency:Gaucher disease
Holleran et al, J. Clin Invest. 81;1338 (1991)

OH
OH

2. Sphingomyeline

HN

O-P-choline
OH

O
HN

Glucosylceramides
Ceramides
sphingomyelinCeramides
phospholipidsFatty acids

Stratum corneum

Lamellar bodies:
Storage glucosylceramides
and sphingomyelin,
phospholipids

Stratum granulosum

Synthesis of Ceramides:
Glucosyl ceramides and
sphingomyelin

sphingomyelinase
Ceramide

Differentiation

glucoceribrosidase

OH
OH

Stratum spinosum

Stratum basale:
Cell division

Formationofphospholipids

Lipidsessentialforbarrierfunction
Step 2

Stratum basale

Stratum granulosum

Stratum corneum

Step 3

Step 1
Step 4

End product: fatty acids and glycerol

Phospholipids
Sphingolipids
Free sterols
Lampe et al, J. lipid Res. 24:131-140 (1983)

Free fatty acids


Triglycerides
sterol-wax esters
hydrocarbons

Lipidcomposition:viableepidermisversuslamellar
bodies

Conclusion
Increase in fatty acids, cholesterol and sphingolipids
(ceramides+glucosylceramides) in stratum corneum
compared to viable epidermis

viable epidermis

lamellar bodies

Lamellar bodies extruded in intercellular regions


between stratum granulosum and stratum corneum

Question
Lipid content lamellar bodies versus whole viable epidermis?

Fatty acids
Cholesterol
Ceramides

Glucosylceramides
Phospholipids

Glucosylceramides preferably stored in lamellar bodies


K.C. Madison et al, J. Invest Derm. Proceedings 3:80-86 (1996)

Formationoflipidlamellae

Fusionoflamellarbodies:formationoflamellae

Differentiation

Stratum corneum-stratum granulosum interface


Formation of lipid lamellae
Located between the cells
Glucosylceramides
Ceramides
sphingomyelinCeramides
phospholipidsFatty acids
Lamellar bodies:
Storage glucosylceramides
and sphingomyelin,
phospholipids
Synthesis of Ceramides:
Glucosyl ceramides and
sphingomyelin

Desquamation process

corneocyte
Stratum corneum
Ceramides
cholesterol
fatty acids

Stratum granulosum
Stratum spinosum

High enzymatic
activity
glucosylceramides
cholesterol
Phospholipids

Stratum basale:
Cell division

Lamellarbodyextrusioprocessinreality

fusion

Lamellar
body

Stratumcorneumboundlipids
LBE=
lamellar body
extrusion
SG= Stratum
Granulosum

Lipid
monolayer
bound to
envelope

LB=Lamellar
Body

lamellae

NMF,water, keratin filaments


envelope
Which lipids are bound to the cornified envelope?

Fusionoflamellae:boundlipidsimportanttoinitiate
thisprocess

Bound lipids synthesis

w-hydroxy acylceramide

Bound lipids

O
=

= =

O
HN

cornified envelope

OH

Ceramide EOS

OH

corneocyte

precursor: acylglucosylceramide EOS


O
=

= =

Ceramides
cholesterol
fatty acids

O
HN

O-glucosyl
OH

hydrolysis
glucose+acyl group

hydrolysis of glucose
group
HN

High enzymatic
activity

O
=

= =

HO

fusion

O
HN

OH
OH

OH
OH

bound to corneocyte envelope

Lamellar
body

Phospholipids
glucosylceramides
cholesterol

Notonlykeratinocytesinepidermis
Stratum corneum
Stratum granulosum
Stratum spinosum
Stratum basale

Langerhans cells
melanocytes

Dermis

Stratum Corneum
Stratum Granulosum
Stratum Spinosum
Stratum Basale

Epidermis

Corneocyte:
soft keratin and
water

Penetration
pathway

FFA
CER
CHOL

Lipid regions

Blood vessels
Langerhans cells: dendritic cells,
Important for protection
and immunisation
Melanocytes: important for melanin
production, UV protection

Cornified envelope and


bound lipids

Overviewofpresentation

Models

Rabbit

Mouse

Rat

Guinea pig

Skin morphology

Step 4
Heterogeneity
of skin

Mini-pig

Step 2

Step 1
Skin
function

Human

Step 3
Skin renewal

Skinregions

Summary

Hand palm: thick stratum corneum (> 60 mm)


Skin region behind the ear: very thin

Skin large and complex organ

Epidermis of old people (around 70 m) thin compared to


epidermis of young (150-200 m)

Body interface with environment


Dynamic organ: able to self repair

Perfusion in different skin regions also different

Forearm dorsal
Shoulder
Buttock

stratum corneum (m)


18.2
11.0
14.9

J. Sandby-Moeller et al, Acta Derm Veverol 2003; 83: 410-413

total epidermis (m)


74.9
81.3
96.5

Maybe not complete perfect barrier: still chances


To perform percutaneous penetration

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