U N I V E R S I T Ä T S M E D I Z I N B E R L I N, Center of Experimental and Applied Cutaneous Physiology, Department of Dermatology

Investigation of the penetration into the skin in vivo/ex vivo

using confocal Raman microscopy

Maxim E. Darvin

Saul Mujica Ascencio

Chun-Sik Choe

Jürgen Lademann

Cosmetic formulations and medical ointments are widely used

in the wellness, cosmetics and in the medicine for topical application

Epidermis

Langerhans cell

Merkel cell

Melanocyte

Corneocytes

Keratinocytes

Transcellular penetration

Follicular penetration

Stratumcorneum

Epidermis

Hair shaft

Intercellular penetration

Penetration pathways

Transcellular penetration

Follicular penetration

Stratumcorneum

Epidermis

Hair shaft

Intercellular penetration

Penetration pathways

Raman microscope „River Diagnostics, Model 3510“(excitation 785 nm)

Darvin et al. J Biomed. Opt. 18(6), 061230, 2013

Raman microscope „Horiba LabRam HR Evolution“(excitation 337 nm, 473 nm, 633 nm)

How the penetration into the skincould be evaluated:

- non-restricted multiple least square fit (NMF) method

- analyses of the lipid to keratin ratio in high wavenumber region usingperpendicular drop down cut-off procedure

- Gaussian-function-based deconvolution method in high wavenumberregion

- tracking specific peak in the fingerprint region complemented withmultivariate statistical analysis

Non-restricted multiple least square fit (NMF) method in the fingeprint region

- Raman spectra of the mammalian stratum corneum are fittedto the spectra of pure stratum corneum compounds, such as cholesterol, ceramide 3, keratin, urea, water, etc.

- The fitting algorithm is a non-restricted multiple least square fit, whichdetermine the coefficients of skin components and investigated oilbased on minimal residual signal.

- The coefficient of investigated oil is recalculated into the contents in thedepth profiles, which are normalized for keratin distribution in the SC.

Non-restricted multiple least square fit (NMF) methodin the fingeprint region

Caspers et al. J Invest. Dermatol. 116(3), 434-442, 2001

Skin Tools 2.0 provided by River Diagnostics

Analyses of the lipid to keratin ratio in the high wavenumber region using perpendicular drop down

cut-off procedure

Stamatas et al. J Dermatol. Sci. 50, 135-142, 2008

Lipids: 2820 – 2900 cm-1

Keratin: 2910 – 2965 cm-1

Gaussian-function based deconvolution procedurein the high wavenumber region

Lipids: 2820 – 2900 cm-1

Keratin: 2910 – 2965 cm-1

Petrolatum-treated skin deconvolution

2600 2800 3000 3200 3400 3600 3800 4000

0

200

400

600

800

1000

Intact human skin (depth 4 µm)

Petrolatum-treated human skin (depth 4 µm)

Difference

Ram

an in

tens

ity, a

.u.

Wavenumber, cm-1

2850

28802935

Choe et al. J Dermatol. Sci. 79, 171-178, 2015

Gaussian-function based deconvolution procedurein the high wavenumber region

2850 2880 2935 2980

Lipid / Keratin = (AUC2850 + AUC2880) / (AUC2935 + AUC2980)

Lipids: 2820 – 2900 cm-1

Keratin: 2910 – 2965 cm-1

Choe et al. Laser Phys, 24, 105601, 2014

Gaussian-function based deconvolution procedurein the high wavenumber region

Choe et al. Laser Phys, 24, 105601, 2014Choe et al. Skin Pharmacol Physiol, accepted, 2015

Human skin spectrum Human skin and petrolatum spectra

Human skin spectrum deconvolution Petrolatum deconvolution

Gaussian-function based deconvolution procedure

Apre-treated skin = C1 Aintact skin + C2 Aoil

Aintact skin = a1f1 + a2f2 + a3f3 + a4f4

Aoil = b1g1 + b2g2 + b3g3 + b4g4 + b5g5

where ai and bj are coefficients averaged from the intact skin and oil spectra,fi and gj are the subsequent Gaussian functions

Residual between Apre-treated skin – (C1 Aintact skin + C2 Aoil) should be minimized for determination of C1 and C2

C2/C1 is the criteria for presence of oil in the skin

Choe et al. Laser Phys, 24, 105601, 2014

Tracking specific peak in the fingerprint region

400 600 800 1000 1200 1400 1600 18000

1000

2000

3000

4000

Ram

an in

tens

ity, a

.u.

Wavenumber, cm-1

Almond oil Jojoba oil

400 600 800 1000 1200 1400 1600 18000

1000

2000

3000

4000

Ram

an in

tens

ity, a

.u.

Wavenumber, cm-1

Paraffin Petrolatum

Tracking specific peak in the fingerprint region

400 600 800 1000 1200 1400 1600 18000

1000

2000

3000

4000 Jojoba oil

Petrolatum

Stratum corneum (human skin in vivo)

Ram

an in

tens

ity, a

.u.

Wavenumber, cm-1

Tracking specific peak in the fingerprint region

400 600 800 1000 1200 1400 1600 18000

1000

2000

3000

4000 Jojoba oil

Petrolatum

Stratum corneum (human skin in vivo)

Ram

an in

tens

ity, a

.u.

Wavenumber, cm-1

Only vegetable oils could be measured

tracking the carbonyl peak

400 600 800 1000 1200 1400 1600 18000

1000

2000

3000

4000

5000

6000

7000R

aman

inte

nsity

, a.u

.

Wavenumber, cm-1

Depth -2 µm

Depth 0 µm

Depth 2 µm

Depth 4 µm

Depth 6 µm

Depth 8 µm

Depth 10 µm

Depth 12 µm

Depth 14 µm

Example of tracking of carbonyl peak (C=O, 1740 cm-1 ) of jojoba oil pre-treated human skin

Raman spectra of phospholipid contained TMX-202 and Imiquimod-based formulation (“IBF”) foams (excitation at 473 nm)

400 600 800 1000 1200 1400 1600 1800

0

200

400

600

800

1000

1200R

aman

inte

nsity

, a.u

.

Raman shift, cm-1

TMX-202 "IBF"~ 690 cm-1

400 600 800 1000 1200 1400 1600 1800

0

500

1000

1500R

aman

inte

nsity

, a.u

.

Raman shift, cm-1

Raman spectra of porcine skin at depth of 2 µm (fat solid line), caffeine (solid line) and propylene glycol (dotted line)

Skin

Propyleneglycol

Caffeine

555 cm-1 840 cm-1

Pectral range selection

400 600 800 1000 1200 1400 1600 1800

1210

86420

-2

-4

Raman shift, cm-1

Pen

etra

tion

dept

h pr

ofile

, m

Treated porcine skin

Caffeine 526-600 cm-1

Propylene glycol 810-880 cm-1

Phenylalanine/Urea 998-1008 cm-1

Skin surface evaluation

998 1000 1002 1004 1006 10080

50

100

150

200

250

Raman Shift, cm-1

Inte

nsity

, a.u

.

-5 0 5 10 15 20 25 300

200

400

600

800

1000

1200

Skin depth, m

AU

C (9

98-1

008

cm-1

), a.

u.

Half value between the minimun and maximum AUC

intensities

Multivariate statistical methods

1. The principal component analysis (PCA) was employed to reduce the noise and highlight the Raman peaks of every spectrum.

2. The linear discriminant analysis (LDA) was employed to classify the groups of untreated and pre-treated skin samples.

3. The T-test (p<0.05) was employed after PCA and LDA to decide whether or not the cream components (caffeine and PG) have been detected.

Spectra reconstruction with PCA

- The noise of the spectra can be reduced

- The classification is related to the similitudes and differences between groups of data

PC1 PC2 PC3 PC4 PC5 PCN

Jolliffe. Principal Components analysis, Springer, Second Edition, 2002Bro et al. Anal. Methods 6, 2812, 2014

Spectra reconstruction with PCA

810 820 830 840 850 860 870 8800

100

200

300

400

Raman Shift, cm-1

Inte

nsity

, a.u

.

810 820 830 840 850 860 870 8800

100

200

300

400

Raman shift, cm-1In

tens

ity, a

.u.

Before PCA After PCA

USTS

USTS

This is a method employed to find tendenciesand classify groups of data

Group 1(5 spectra)

Group 2(5 spectra)

0PC1

PC

20

PCA scores

2 μm 10 μm 16 μm

530 540 550 560 570 580 590 6000

100

200

300

400

500

600

Raman shift, cm-1

Inte

nsity

, a.u

.

USTS

530 540 550 560 570 580 590 6000

100

200

300

400

500

600

Raman shift, cm-1

Inte

nsity

, a.u

.

USTS

530 540 550 560 570 580 590 6000

100

200

300

400

500

600

Raman shift, cm-1

Inte

nsitu

, a.u

.

USTS

-1000 -500 0 500 1000 1500-200

-100

0

100

200

PC1

PC

2

-1000 -500 0 500 1000 1500-200

-100

0

100

200

PC1

PC

2

-1000 -500 0 500 1000 1500-200

-100

0

100

200

PC1

PC

2

USTS

USTS

USTS

PCA scores

It is a method employed to maximized the difference between two groups of data

Group 1(5 spectra)

Group 2(5 spectra)

Number of spectra

LD1 0

LDA scores

1 2 3 4 5 6 7 8 9 10 1112 13 14

-200

-100

0

100

200

Number of measurements

LD1

1 2 3 4 5 6 7 8 9 10 1112 13 14

-200

-100

0

100

200

Number of measurements

LD1

1 2 3 4 5 6 7 8 9 10 1112 13 14

-200

-100

0

100

200

Number of measurements

LD1

USTS

USTS

USTS

2 μm 10 μm 16 μm

LDA scores

2

-200

-100

0

100

200

1

Treated skin

Untreatedskin

2

Untreatedskin

-200

-100

0

100

200

1

Treated skin

2

-200

-100

0

100

200

1

Untreatedskin

Treated skin

2 μm 10 μm 16 μm

p < 0.05

T-test

Substance Range(cm-1)

‘’Horiba’’penetration depth

(μm)

‘’RiverDiagnostics’’

penetration depth(μm)

Caffeine 526-600 12.3±3.0 13.0±1.4

Propylene glycol 810-890 20.7±3.0 22.0±0.1

Results: the caffeine and propylene glycol penetration depths determined by the specific peak tracking method using

multivariate statistical analysis

Oil penetration profiles measured using different methods

0 5 10 15 200

500

1000

1500

2000

2500

Oil

conc

entra

tion,

a.u

.

Penetration depth, µm

Almond oil Jojoba oil Paraffin oil Petrolatum

0 5 10 15 20 25 30

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5 Almond oil Jojoba oil Paraffin oil Petrolatum

Oil

conc

entra

tion,

a.u

.

Penetration depth, µm0 2 4 6 8 10 12 14

0,0

0,5

1,0

1,5

2,0

2,5

3,0 Almond oil Jojoba oil Paraffin oil Petrolatum

Oil

conc

entra

tion,

a.u

.

Penetration depth, µm

0 2 4 6 8 100

2

4

6

8

10

Oil

conc

entra

tion,

a.u

.

Penetration depth, µm

Almond oil Jojoba oil

1740 cm-1 peak tracking method NMF method

Lipid to keratin ratio analyses method Deconvolution method

Results: the oil penetration depths determined by the four different methods

Method 1: Tracking specific peak in the fingerprint region method;Method 2: Non-restricted multiple least square fit (NMF) in the fingerprint region method;Method 3: Method based on analyses of the lipid to keratin ratio in the high wavenumber

region using perpendicular drop down cut-off procedure;Method 4: Method based on analyses of the lipid to keratin ration in the high wavenumber

region using deconvolution procedure.n/a – not applicable

Conclusions

1. Confocal Raman microscopy is well suited to analyze the penetration of substances into the skin in vivo and ex vivo.

2. The specific peak tracking method complemented with the multivariate statistical analysis and Gaussian-function-based deconvolution method aremost sensitive in comparison to other methods for penetration studies.

3. Oils (almond oil, jojoba oil, paraffin oil and petrolatum) and caffeine do not penetrate deeply into the skin saturating only the superficial SC´s layer (~ 10 µm), while propylene glycol and phospholipid containing formulations are ableto reach deep located areas of the stratum spinosum.

Thank you for your attention

Study protocol

Confocal Raman microscopy (River Diagnostics, Model 3510)excitation at 785 nm – fingerprint regionexcitation at 671 nm – high wavenumber region

Topical application

2 mg/cm2 oil homogeneously applied on the skinPenetration time – 1 Hour

In vivo measurements

4 different oils (almond oil, jojoba oil, paraffin oil, petrolatum),6 volunteers, 10-12 CRM measurements for each oil

Measurement of water profile in the skin using „Skin Tools 2.0“ software

Waternormalized = Water / Keratin = AUC3350-3550 / AUC2910-2966

0 5 10 15 20 25 30-2

-1

0

1

2

3

4

Gra

dien

t

Depth, µm

Stratum corneum thickness (gradient 0.5)

0,5

Stratum corneum thickness (intact human skin)

-5 0 5 10 15 20 25 30

35

40

45

50

55

60

65

Wat

er m

ass,

%

Depth, µm

Intact skin