Vibrational Microspectroscopy of Skin: Applications to Pharmacology and Biochemistry. Rich Mendelsohn Rutgers University

Transcription

1 Vibrational Microspectroscopy of Skin: Applications to Pharmacology and Biochemistry Rich Mendelsohn Rutgers University Scientific Students/Associates R. Mendelsohn Dr. G. (Jin) Zhang* C. R. Flach Dr. Andrew Chan Phil Cummins (Estee Lauder) Dr. Chunhong Xiao D. J. Moore (ISP) Collaborators B. Michniak (Rutgers) K. Sloan (Florida-Gainesville) Support: NIH GM Industrial: Estee Lauder, ISP

2 I. Introduction II. Applications a. Spatially resolved transdermal drug delivery Pro 5-FU 5-FU b. Wound healing (preliminary)

3 Methods IR Imaging microscope Spectrometer IR microscopic imaging Stage controller Detector Joystick Radiation The radiation path is orange. Once imaged into sample, the collected radiation passes through a 4x magnifier and then to the detector. 4X magnifier Upper & lower Cassegrains

4 Linear array (16 x 1) detector How are 2D IR Images Generated? Rapid scan linear detector can acquire data continuously. 1) Collect a block of 16 pixels, then shift the stage left. 2) Collect the next block, etc. 3) At the end of a row, return to start the next row. 4) Stage movement occurs only at the ends of the interferometer scan Note: we acquire a full IR spectrum from each pixel Advantages: 1)Rapid scan allows enormous amount of data to be accumulated quickly. 2) Any image with a rectangular geometry may be defined.

5 Raman Microscopy Kaiser Raman microprobe: 1) Excitation wavelength 785 nm minimizes fluorescence. 2) Configured with high-precision mapping stages and confocal optics for both surface and confocal studies 3) Capable of analyzing diffraction-limited areas (typically 1-2 µ m). (~ 3 µm axial resolution in the confocal experiment.) 4) Minimal sample preparation Fiber optic 785 nm in Laser 785 nm Monochromator: notch filter, transmission grating CCD detector Mapping stage Fiber Optic Raman out

6 Schematic representation of IR imaging: Data acquisition and processing Sample interferograms Mirror retardation (cm) FFT and apodization Sample single beam spectra Wavenumber (cm -1 ) Divide Transmittance Spectra Lots of Spectra 15, routine!! 12,, data pts. every ½ hr Wavenumber (cm -1 ) -log 1 Absorbance Spectra Images Wavenumber (cm -1 ) Histograms Spectra Multivariate Analysis Mirror retardation (cm) FFT and apodization Reference pixel interferogram Wavenumber (cm -1 Adapted from: E. N. Lewis, A. M. Gorbach, C. Marcott and I.W. Levin, App. Spec. 5, 263 (1996)

7 Factor Analysis: 1. Too many individual spectra to look at (1,/image in the IR, 1-5/image in the Raman). Need multivariate method. 2. Start with Principal Component Analysis. Each component is ranked according to the % of variance it explains. (If all spectra are the same, then why are we doing imaging?!) But PCA loadings do not resemble real spectra. 3. Factor Analysis seeks transformations from the abstract PCA loadings to the true underlying to the true underlying factors based on spectra at particular locations. 4. Factors Loadings are assumed to represent spectra from the location at which they are determined. Scores indicate the relative contributions from various factors from particular locations.

8 Basic Skin Structure Stratum corneum (sc): primary function: main permeability barrier; maintenance of water homeostasis two major components: terminally differentiated, anucleated corneocytes (keratin), lamellar lipid network thickness:1-2 microns Living Epidermis: primary function: generate the SC principal cell: keratinocyte differentiates in as it migrates to SC thickness: 5-15 microns Dermis: tough connective tissue specialized structures collagen: 75% of dry weight thickness:.6-3 mm from D. M. Pillsbury, W. B. Shelley and A. M. Kligman, "Dermatology", W, B. Saunders Co., Philadelphia, 1956.

9 Vibrational imaging spatial resolution relative to skin region dimensions Confocal Raman Image plane Confocal Raman Z line IR pixel ~Raman laser spot SC 2 microns epidermis Note - IR resolution ~ 2 pixels. Raman resolution ~ laser spot size + leakage

10 Sampling Protocols Intact Skin IR imaging 5 µm thick section cut perpendicular to skin surface (frozen sections) X epidermis dermis Raman microscopic imaging Sample hydration maintained Confocal sampling thick sections parallel to surface sealed in a brass block (SC up) 785 nm in Raman out 1 X objective (oil immersion) 785 nm in Raman out Skin sample Cover glass Z Brass block X Z Well milled into the block Images acquired along X, Z plane Measure along Z-line or along X-Z planes Skin: Yucatan hairless pig, human, or artificial

11 IR Microscopy of Hair in Dermis C-H stretching region factors 5 Factor analysis of hair folicle in dermis f1 f2 f3 f4 f5 5 (f1, hair cuticle (?)) microns microns Factor loading (f3, dermis) L P L P P,L Wavenumber/cm -1 micrometers 1 15 micrometers micrometers 5 F4? 5 (f2, cortex) micrometers 5 micrometers 1 15 micrometers 1 15 micrometers micrometers micrometers micrometers (f5, epidermis)

12 IR imaging Permeation of DPPC-d62 (MLVs) into pigskin IR microscopy 875 x 72 microns Xiao, C. et al., 25, Journal of Investigative Dermatology ( (25)) Liposome CD stretch IR Absorbance (Progres Wavenumber 31 IR Absorbance (Progressively offset) Wavenumber

13 Permeation of Phospholipids into Pigskin Lateral Raman Microscopy (1 µm under the surface) P-d 31 OPC(disordered) DPPC-d 62 (ordered) CD 2 stretching peak area 1e+5 8e+4 6e+4 4e+4 2e Phe P-d 31 OPC Microns from skin surface CD 2 stretching peak area 1e+5 8e+4 6e+4 4e+4 2e Phe DPPC-d Microns from skin surface The disordered lipid permeates much more efficiently J. of Investigative Dermatology, 124, (25)

14 Characterization of Skin Microanatomy by Raman Microscopy Confocal Raman image plane (XZ) was acquired by continually measuring confocal lines in the intact skin. Factor score images X SC Z Factor loadin Factor loadings Raman shift (cm -1 ) Lipid inclusion Nuclei Epidermis SC Lipid Nuclei Epidermis Dermis Dermis Factor analysis: identification of spectral regions + features microns microns

15 Identification of components from factors Lipid inclusion e.g. 65, 7 = cholesterol 113, 159 C-C stretch = ordered lipid Cells identified by DNA bands Labeled bands: Cytosine(785) and from phosphodiester(186) links Raman counts 65 cm -1 7 cm nuclei epidermis cytosine band from DNA Factor loading 158 cm cm cm -1 Raman shift /cm -1 stratum corneum nuclei epidermis ordered lipid 285 cm cm -1 cytosine band from DNA phosphodiester band from DNA Factor loading Raman shift Factor loading Raman shift Raman shift (cm -1 )

16 Applications of Raman microscopic imaging to transdermal drug delivery

17 Imaging the transformation of prodrug drug in skin by confocal Raman microscopy 1) Physicochemical properties of the parent drug are modified to improve solubility in skin for topical delivery. 2) Stability of the parent drug is improved, avoiding degradation by the environment. 3) Prodrug is chemically or enzymatically converted to drug within skin, at locations inaccessible to topically applied drug.

18 5-fluorouracil (5FU) delivery 5-Fluorouracil is used topically for basal cell carcinoma and psoriasis and requires light curettage for the former application, and under occlusion for the latter. hydrolysis 1-ethyloxylcarbonyl-5-fluorouracil (prodrug) 5-fluorouracil (drug) Thanks to Prof. Ken. Sloan, University of Florida Zhang et al. J of Invest. Derm. 127,125 (27)

19 Control studies for In situ prodrug to drug interconversion (prodrug and drug dissolved in water at 65 C) 9 Low frequency Raman spectra of drug, prodrug P,D 8 7 D P D D(637) P P (865) 6 D Raman Counts 5 prodrug in water drug in water Raman shift (cm -1 )

20 Prodrug and drug spectra at different depths under Skin surface Application of prodrug Application of drug C P+D 1 P P D D D 5um 2um Raman counts Raman shift(cm -1 ) 5 25um Raman shift (cm -1 ) um Esterase activity cleaves prodrug to drug permits drug permeation

21 Prodrug,drug distributions in skin as f(t) Ratio (prodrug or drug) / Phe 34 C Prodrug Drug 22 C

22 Lipid structural transitions in native human SC Rocking modes. Take home message I - you can do semiserious spectroscopy on intact tissues (App. Spec. 6, , (26)) Thermal transition: Human SC orthorhombic - to -hexagonal Orthorhombic phase marker Basis of method: The 729 cm -1 component of the rocking mode doublet reflects orthorhombic phase formation. Absorbance T Orthorhombic low T Hexagonal high T Frequency (cm -1 ) 715

23 Solid 5 -FU (crystals) may form in skin after prodrug application 768 cm -1 2 lines of evidence for solid 5FU in skin: 3 25 Raman Intensity FU -solution 5FU -solid Untreated skin Treated skin 996 cm -1 Phe 1. Band near 768 cm -1 in treated skin is same as in solid 5FU, but shifted from 5FU solution. 2. Shoulder at 996 cm -1 in treated skin is same as in solid 5FU. Raman Intensity Expanded scale cm -1 Crystalline 5 -FU Phe (skin proteins) Raman shift (cm -1 ) Raman shift (Wavenumber)

24 Advantages of this approach for transdermal drug delivery 1) Direct detection of prodrug and drug species 2) Spatial distribution of each species 3) Detection of phase transitions in exogenous species 4) Correlation of interconversion with skin molecular regions/properties 5) Kinetics of permeation Drawbacks of this approach 1) Not suitable for low concentrations 2) Relatively long collection times limit in vivo applications.

25 Healing of skin wounds Spatial and temporal sequence of events: 1) Fibrin clot. 2) Clot is infiltrated by inflammatory cells, fibroblasts, granulation tissue 3) Epidermis is reconstituted from wound edges and cut remnants of hair follicles 4) Leading front edge of keratinocytes cut through the clot and over the healthy dermis. 5) Granulation tissue contracts when myofibroblasts tug on one another and the surrounding collagen matrix. Science 276 pp75-81 (1997) Molecular biology does the genotypes, but perhaps we help with the phenotypesconcentrations of proteins and their molecular structures/orientations.

26 Raman and IR imaging of wound healing Wound formed by punch biopsy We examine the residual tissue once the biopsy is taken Wounded area X 1 8 Z 6 micrometers Unwounded Confocal Image plane Wounded micrometers Each pixel represents one spectrum Raman counts Raman shift/ cm Factor analysis performed in different spectral regions 5

27 A Confocal Raman microscopy Punch biopsy: 2mm diameter A B non-wounded Factor loading 5 Factor 1 Factor 2 Factor 1 Factor 2 Factor 3 Factor Raman shift/cm -1 Factor 3 Factor 4 wounded micrometers Left: C-H stretch region Right: Collagen marker region Blue trace disordered lipid (tummy tuck) Red trace Protein, wound collagen Green trace keratin from nonwounded Black trace ordered lipid SC (non-wound) Factor loading 1 5 B Factor 1 Factor 2 Factor 1 Factor 2 Factor Raman shift/cm -1 Factor 3 Collagen from wound Collagen-another type? from wound - Keratin (non-wounded) micrometers

28 IR Orientation study Amide I/Amide II ratio provides an index of collagen orientation (transition moments are ~ perpendicular to each other) Absorbance Polarized FTIR spectra of collagen extracted from highly oriented region 18 I Amide modes II o polarization 9 o polarization Wavenumber/cm -1 Absorbance Polarized FTIR spectra of collagen extracted from poorly oriented region I Amide modes II o polarization 9 o polarization Wavenumber /cm -1

29 IR Orientation study Stronger orientation parallel to the surface of the skin can be observed in the non-wounded region Wounded area contains larger and more randomly orientated collagen region Non-wounded area (dermis) Wounded area 3.5 Amide I/II ratio o * micrometers

30 Closing comments: 1) The molecular structure information inherent in IR and Raman is unique. 2) The analytical methods are so powerful that a wide variety of of biochemical and pharmacological problems may be readily addressed in addition to identification of pathological states.