Tape Stripping & Stratum Corneum

A. VYASA MURTHY
II/II Masters in pharmacy Research Guide Prof. K.V. Ramana Murthy Andhra University

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Introduction uses of tape stripping stratum corneum and barrier functions tape stripping procedure barrier disruption methods types of tapes mechanical removal of corneocytes video disruption of corneocytes on the removed tape strips analytical methods used in tape stripping tape stripping and topical vaccination un answered questions of tape stripping conclusion.
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Introduction:
Tape stripping is an useful method for removing the stratum corneum(SC) and obtaining more information about function of the SC as a main barrier. An adhesive tape is pressed onto the test site and is subsequently abruptly detached. The number of tape strips need to remove SC varies with age , sex , ethnicity. Proposed by FDA as part of a standard method to evaluate bioequivalence of topical dermatological dosage forms.
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Uses of tape stripping:

To measure the SC thickness & mass. Investigate the percutanious penetration & disposition of topically applied drug in vivo. To disrupt the skin barrier function. Collect the SC lipids samples. To detect the proteolytic activity associated with the SC. Quantitatively estimate esterase activity in the SC Quantitative and minimal invasive essay for the detection of metal on and in the skin. Used to disrupt the skin before percutanious peptide and DNA immunization. Investigate fungal and skin infections.
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Stratum corneum & its functions:

SC is a stratified squamous epithelial lining on the body surface that plays an important barrier function and a reservoir for topically applied substances. SC consist corneocytes embedded in lipid-rich intercellular spaces filled with free fatty acids, cholesterol ceramides(1-9),cholesterysulfate,sterol,triglycerides,squalene,nalkanes &phospholipids. These lipids exist as a continuo's lipid phase;occupuying about 20% of the SC volume, arranged in multiple lamellar structures. Rod & cylindrical shape of ceramides makes them suitable for the formation of high ordered gel phase which is fluidized by CHOL which evolves as a competent barrier.

TAPE STRIPPING PROCEDURE(mechanical removal of corneocytes)

Source:.E J of Pharmaceutics and Biopharmaceutics, Volume 72, Issue 2, June 2009, Pages 317 323

Barrier disruption differs on the disruption methods:

Tape stripping method: moderate erythema &glistering surface. The fastest barrier recovery was observed using tape stripping. Acetone treated skin: no erythema & slight superficial dryness. Chloroform-methanol treated skin : deep erythema & edema.
Acetone: No significant difference in SC capacitance between body sites. Scaring or infections Tape stripping: Produced significant difference Strong barrier damage was observed No adverse effects.

Tape stripping depends on: Size of the corneocytes ,age , season , the number of cell layers & corneocytes ,thickness of SC , composition and amount of lipids vary depending on the anatomical site , TEWL pH are affected by the season ,the race , the skin type , the volunteer. the force of removal from the skin. the duration of pressure onto the skin. topically applied substances.
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Type of tapes used:

Commercially 3 adhesive tapes are used : 1) D-square 2) Transpore 3) Micropore No significant difference was found in the kinetic parameters. The mean TEWL was increased with D-square & Transpore as they reach the deeper layers. FDA guidelines: 10 tape strips In vivo experiments resulted 18-20 and for some 40 strips.

Mechanical removal of corneocytes

Source:(http://www.rvc.ac.uk/review/Dermatology/Tests/Stripping.htm)

Distribution of corneocytes on the removed tape strips.

Source : J. Lademann et al. / European Journal of Pharmaceutics and BiopharmaceuticsVolume 72, Issue 2, June 2009, Pages 317323

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 Adhesive films are applied and removed successively from the same treated skin area. The adhesive tape should be pressed onto the skin using a roller to stretch the skin surface. This procedure will avoid the influence of furrows and wrinkles under the tape stripping procedure as described in slide 6. The removed tape strips contain amounts of stratum corneum and amounts of topically applied substances. The typical distribution of the corneocytes on the tape strips removed from different depths of the stratum corneum is shown in the previous slide. The first tape strips contain almost a complete cell layer of corneocytes. With increasing the tape stripping number, the corneocytes and corneocyte aggregates become less and less.
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Percutanious absorption mainly depends on:

1)Permeability coefficient affected by drug polarity, molecular size, vehicle in drug applied ,skin barrier. 2)Occlusive or non-occlusive 3)Skin integrity 4)Intracellular lipid domain of SC
To overcome the barrier function and also to allow the water soluble conferences physical disruption of the corneal layer is the best way. 1)Ultrasound 2)low & high voltage electric pulsing 3)tape stripping 4)Chemical enhancers. SC is removed by serial adhesive TS and consequently percutanious absorption and penetration was significantly increased in stripped skin by this the drug concentration and concentration profile across the SC can be measured.

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In Vivo Drug Penetration Studies in Barrier-Perturbed Skin

Benfeldt et al.

Morgan et al.
Liu et al.

Micro dialysis experiment of TS SALICYLICACID was highly increased in TS. HUMANS( 157 times fold increase)RATS(170 times fold increase) Micro dialysis experiment of TS Pencyclovir in rats(1300 times inc), acyclovir(440 times increase
TS increased the penetration of biological macro molecules such as peptide & DNA INTO viable skin.

Ionetophoresis + TS does not increased the penetration with acetaminophen.

Takigwal et al. Regnier et al. Yu et al.

Ons and DNA do not penetrate SC but removal of SC by TS led to extensive penetration.
TS increased Ons concentration 1 or 2 orders of magnitude Abrasion of the skin prior to DNA application could improve cutaneous gene transfer and expression.
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Tape stripping vs. Analytical methods:

These methods are widely used in determination of drug concentration with in skin: 1) Skin extraction measurement 2) Horizontal stripping & sectioning 3) Quantitative autography 4) Mass spectrophotometry 5) Optical micrography 6) Spectroscopic techniques Methods used to quantify the extracted drug: 1) HPLC 2) spectroscopy 3) scintillating counting Methods with improved sensitivity: 1) ATR-FTIR 2) Fluorescence spectroscopy 3) Confocal microscopy 4) Mass spectrometry 5) Photo thermal spectroscopy
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Technique

Penetrants Detected Any

Measuring Depth All strata

Speed & cost

Complementary Strategies Separation of skin tissue Qualitative autoradiography

Skin extraction

Inexpensive, rapid

Horizontal sectioning

any

All strata

Inexpensive, rapid

Separation of follicles Use of follicle-free skin none

Tape stripping Quantitative fluorescent microscopy Tape stripping 15

Quantitative autoradiograph
ATR-FTIR spectroscopy fluorescence spectroscopy Spectroscopy Microscopy

Radiolabel led only

IR-absorbing only Self-fluorescent only UV/Visible Common or laser

All strata
SC All strata

Expensive, slow
Expensive, Very rapid Medium, Rapid

SC SC

Inexpensive, fast Tape stripping Medium Rapid

Tape stripping versus topical vaccination:

Skin is an active immune surveillance site
It is rich in potent antigenpresenting cells Like LANGERHANS CELLS & DENDRITIC CELLS

there was a significant difference between the results of topical application with and without stripping which was produced in the next slides.

LC plays a key role in the immune response to antigen materials

Skin access makes it is an

Easy target for vaccination

Thus , skin is an alternative target for vaccination topically So focus on topical vaccination for the Induction of ANTIGEN SPECIFIC IMMUNE RESPONSE

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Tape stripping skins behavior

1)Disruption of the epidermal barrier (stripping) enhances the in-vitro T-cell mediated immune responses Production of IL -1,IL-1,IL-8,IL-10,TNF-,INF- 2)TS also increases co-stimulatory molecule expression CD86,CD54,CD40,MHC-II & the antigen capacity of epidermal DCs Also facilitates generation of TH-1 immune responses and stimulates LCs migration to cutaneous lymph nodes. 3)Topical application of tumor associated peptide onto the SC barrier disrupted by tape stripping in mice induces protective anti tumor response in vivo and in vitro. Induction of cytotoxic T lymphocytes on tape stripped ear lobes of mice by application of CTL epitope peptide onto the SC shown a good diffusion (96 micrograms in 24 hours ). However , CTL induction was virtually absent with non stripped mouse skin.

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Tape stripping skins behavior

Kohlen et al found that TAPE STRIPPING significantly enhances antigen specific antibody and CTL responses Stripping resulted in prolonged CTL responses at least 2 months after single immunization. Tape stripping increased the humoral and cellular immune responses of topical DNA antigens ,topical vaccination induced strong antibody responses and elicited substantial CTL responses. These results suggest that TS can be widely used in inducing immune responses with topical vaccination in vivo. Protective affect of topical vaccination was confirmed by watabe et al and seo et al. Efficacy of a topical DNA vaccine that expressed the matrix gene of the influenza virus using a mouse model. Plasmid DNA applied topically on stripped skin on 0,7,14days65% of mice survived when they were topically immunized with plasmid DNA that expressed the matrix gene . Whereas , when applied on in-activated skin only 18% mice were protected and all mice were dead in 7 days after virus inoculation. These results suggest that the topical administration of DNA vaccine induces a protective immunity against influenza challenges.
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Conclusion:
Tape stripping data have been used to estimate dermal absorption, permeability coefficients and partition coefficients, SC mass, barrier function, drug reservoir from in vivo dermal exposures, and even to explain the SC physiology. TS has also been proposed as a method for evaluating the bioequivalence of topical dermatological dosage forms. DPK characterization of the penetration of active drugs in human volunteers has been suggested to be able to replace comparative clinical trials as means of documenting bioequivalence. It is suggested that DPK assessment of drug concentrations in the SC is comparable to blood/urine measurements of systemically administered drugs, where the concentration of a drug in the SC is expected to relate to its concentrations in viable tissue. Short contact DPK experiments can be used to obtain diffusion and partitioning parameters that may subsequently be able to predict drug penetration into the SC following longer application periods.
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Unanswered questions of tape stripping:

1.Intra subject variation. 2.The types and sizes of tapes utilized equally affect the method and the pressure applied to the strip prior to stripping. 3.Unsuitable for volatile chemicals. 4.FDA guidelines describe serial TS to determine the amount of drug with in the skin the first strip is discarded & the extracted drug is expressed as mass per unit area it is impossible to express the amount of drug substance per unit mass area of SC and to determine the proportion of the SC that has been sampled by the method.
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References:
1. 2. Marzulli and Maibachs dermatotoxicology / editors, Hongbo Zhai, Howard I. Maibach, and Klaus-Peter Wilhelm. -- 7th ed. J. J. Escobar Chavez et al, The Tape-Stripping Technique as a Method for Drug Quantification in Skin , J Pharm Pharmaceut Sci (www. cspsCanada.org) 11 (1): 104-130, 2008. J.Lademann et al The tape stripping procedure evaluation of some critical parameters,. European Journal of Pharmaceutics and Biopharmaceutics, Volume 72, Issue 2, June 2009, Pages 317323. University of London(the royal veterinary college 2000 eMedia Unit RVC) (http://www.rvc.ac.uk/review/Dermatology/Tests/Stripping.htm) C. Pailler-Mattei Interpretation of the human skin biotribological behavior after tape stripping J. R. Soc. Interface doi:10.1098/rsif.2010.0672 Published online.

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Thank you

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