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Optimization and validation of freeze-drying for light and electron microscopic autoradiography of percutaneous steroid transport
journal of ELSEVIER
Journal of Controlled Release 42 (1996) 1-13
controlled release
Optimization and validation of freeze-drying for light and electron microscopic autoradiography of percutaneous steroid transport Jan A.M. Neelissen a, Frans H.N. de Haan b, Ad H.G.J. Schrijvers c, Hans E. Junginger ~, Harry E. Bodd6 a'* "LeidenlArasterdam Center for Drug Research, Pharmaceutical Technology, P.O. Box 9502, 2300 RA Leiden, The Netherlands bOrganon International, Oss, The Netherlands CDepartment of Electron Microscopy, State University of Leiden, Leiden, The Netherlands
Received 23 March 1995; revised 4 December 1995; accepted 8 December 1995
Abstract A freeze-drying/autoradiography procedure to visualize steroid transport across human stratum corneum was optimized and validated. A finite dose of [3H]estradiol (3H-E2) was applied to human abdominal skin, i.e. isolated stratum corneum and dermatomed epidermal sheets, in an in vitro permeation set-up. The visualization procedure included cryofixation, freeze-drying, osmium tetroxide (OsO4) vapor fixation, Spurr resin embedding and autoradiography. The different steps of the sample preparation were carefully examined for morphological quality, extraction and diffusion of 3H-E2 from the skin. It appeared that dermatomed skin was, for several reasons, more suitable to process than isolated stratum corneum. While cross-sections of dermatomed skin displayed a well-preserved stratum corneum, the viable epidermis suffered from decreased structural preservation due to cryofixation artifacts. The apparent extraction of 3H-E2 during the infiltration with Spurt resin was due to donor phase residing at the skin surface. In the autoradiographs, there was no indication that 3H-E2 had diffused out of the skin. Silver grains were mainly localized over the superficial corneocytes. Only a few grains were present over the deeper layers of the stratum corneum, both over the inter- and intracellular domains. An equation was deduced to estimate the exposure time of autoradiographs from skin samples, with the advantage that no time was wasted with empirical range finding tests. In the equation a distribution factor was introduced which can be used to quantify the localization of the silver grains over the different tissue structures. In conclusion, it can be stated that the visualization method is suitable to study the distribution of 3H-E2 in human stratum corneum following topical application. Keywords: Percutaneous drug delivery; Human skin; Estradiol; Freeze-drying; Autoradiography
1. Introduction T r a n s d e r m a l drug d e l i v e r y s h o w s advantages o v e r the peroral route for a n u m b e r o f drugs, like
Corresponding author, Tel: +31 71 5274350; fax: +31 71 5274277; e-mail: [email protected].
clonidine, estradiol, fentanyl, nicotine, nitroglycerin and s c o p o l a m i n e [1]. It offers controlled continuous d e l i v e r y o v e r an e x t e n d e d duration and thus maintaining constant therapeutic drug concentrations. O n e o f the limitations is that percutaneous penetration is often too low for a therapeutic effect. H o w e v e r , the properties o f the b i o l o g i c a l p e r m e a b i l i t y barrier, i.e. stratum c o r n e u m and its intercellular lipids, can be
0168-3659/96/$15.0o © 1996 Elsevier Science Ireland Ltd. All rights reserved PII S01 68-3659(96)01324-7
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J.A.M. Neelissen et al. / Journal of Controlled Release 42 (1996) 1-13
modified by using chemical penetration enhancers or iontophoresis. Many questions remain to be answered concerning the mode of action of enhancers and iontophoretic devices, e.g. where and how they act in the stratum corneum. One strategy to study the effect of penetration enhancement is visualization of the distribution of a drug in the stratum corneum after passive permeation compared with an enhanced permeation. This approach requires a visualization method that is generally applicable for highly diffusible drugs, comprising a non-extractive skin sample preparation and a visualization of the drug molecules at both the light and electron microscopical level. The latter is considered necessary to be able to discriminate between the inter- and intracellular domains of the stratum corneum. Several procedures have been used to visualize percutaneous penetration of model compounds and drugs, mainly at the light microscopical level [2-6]. At the electron microscopical level, in situ precipitation [7-9] and electron microscopic autoradiography after cryofixation, freeze-drying, vapor fixation and resin embedding [10,11] have been used. Both methods seem to fulfil the requirements of a non-extractive sample preparation and the possibility to visualize penetrating molecules. In situ precipitation, however, is limited in its use. Only compounds which form an electron-dense precipitate after exposure to a vapor can be used, e.g. osmiophilic agents like n-butanol (exposed to OsO 4 vapor) [9], or heavy metals like mercuric chloride (exposed to ammonium sulfide vapor resulting in a mercuric sulfide precipitate) [7,8]. The other method, also referred to as the method of Stirling and Kinter [12], comprising cryofixation, freeze-drying, OsO 4 vapor fixation, epoxy resin embedding and autoradiography, is generally applicable. It requires a 3H or 14C labeling of the diffusing compound, e.g. [3H]hydrocortisone [ 10], [3H]water, [14C]ethanol and [3H]cholesterol [11]. This paper describes experiments to optimize and validate the method of Stirling and Kinter for the localization of topical applied 3H-E2 in human skin after in vitro permeation. Additionally, an equation is introduced to estimate autoradiographic exposure time.
2. Materials and methods
2.1. Materials
Trypsin (Bovine pancreas type III) and trypsin inhibitor (soybean type II-S) were purchased from Sigma (St. Louis, MO); [2,4,6,7-3H]estradiol (SA 14.6 GBq/mg) from Amersham (Buckinghamshire, UK); Freon 22 from Hoek Loos (Schiedam, The Netherlands); osmium tetroxide from Agar Scientific Ltd. (Stansted, UK); Emulsifier-Safe T M and OptiFluor® O from Packard (Meriden, CT); Spurr resin from Bio-Rad/Polaron (Cambridge, MA, USA); 4% collodion in diethyl ether from Merck (Darmstadt, Germany); Ilford L4 from Ilford Limited (Mobberley, Cheshire, UK). All other chemicals used were of analytical grade. All solutions were prepared with purified water (Milli-Q UF Plus Water System, Millipore, Etten-Leur, The Netherlands). Phosphatebuffered saline (PBS) had the following composition: sodium chloride (NaC1) 8 g/l, potassium chloride (KC1) 0.19 g/l, disodium phosphate (Na2HPO4.12H20) 2.86 g/l, potassium hydrogen phosphate (KH2PO4) 0.20 g/l, pH was set to 7.4 with 0.1 N sodium hydroxide (NaOH). Fresh human abdominal skin was obtained from female donors after cosmetic surgery. Two different types of skin samples were compared:
Dermatomed epidermal sheets. After prior removal of the subcutaneous fat, the flesh skin was dermatomed at 200 /zm (Padgett Electro-Dermatome model B, Kansas City, MO), stored overnight (at 4°C in a petfi dish on filter paper soaked with PBS) and used for permeation experiments the next day. 2. Isolated stratum corneum was obtained from freshly dermatomed skin by incubation overnight on 0.1% trypsin/PBS (w/v) soaked filter paper at 37°C. The stratum corneum was separated from the remaining epidermis, rinsed (at room temperature) with 1% trypsin inhibitor/PBS (w/v), and then rinsed twice by floating on water. The stratum corneum sheets were dried in a desiccator over silica gel, and subsequently stored in a closed container filled with dry nitrogen gas.
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Before use, disks were punched (16 mm diameter) and rehydrated for 24 h over 27% sodium bromide (NaBr)/H20 (w/v), resulting in ~17.5% (w/w) water content of the stratum corneum [13].
2.2. Permeation experiments Franz-type permeation cells with a permeation area of 0.79 cm 2 and a receiver chamber volume of 4.4 ml were kept at 32°C. In experiments with isolated stratum corneum, the stratum corneum (basal side) was supported with a dialysis membrane (Diachema MW-cutoff 5000, Dianorm, Mfinchen, Germany) at the receiver side, in which the receiver chamber was half-filled with 27% NaBr/HzO (w/v). Permeation time was 24 h. In experiments with dermatomed skin, the dermal side was in direct contact with stationary degassed PBS in the receiver chamber. Permeation time was 24 h. In both series of experiments, the donor solution, 25 /xl 1% estradiol (E2)/absolute ethanol (w/v) supplemented with 3.7 × 10 4 Bq 3H-E2 (molecular ratio 3H-E2:E2 ~1:100 000), was evenly distributed over the outer surface of the skin with a syringe. Experiments were non-occlusive, allowing the ethanol to evaporate, resulting in the deposition of a thin layer of E2 crystals on the skin surface. This surface deposit was left untouched throughout the permeation experiment. 3H-E2 permeation experiments were performed in triplicate. Two control permeation experiments were performed with dermatomed skin as described above, with the following modifications: in the first control the donor solution only contained non-radioactive E2. In the second control the experiment was terminated immediately after the ethanol had evaporated from the skin surface, followed by immediate processing as described below, without prior removal of the donor phase. This control should provide an answer if 3H-E2 residing at the skin surface diffuses into the skin during Spurr infiltration.
2.3. Sample preparation 2.3.1. Cryofixation The permeation experiments were terminated by removing the skin from the permeation cell and by
3
immediate removal of the donor phase from the skin surface with one ethanol wipe. The effectiveness of this removal was checked by cleaning one of the skin samples with three ethanol wipes. The skin samples were cut into small pieces (1-1.5 × 2 - 3 mm) with a razor blade and picked up with forceps and immediately plunged into solid/liquid Freon 22. A few non-radioactive pieces of skin were frozen in liquid propane (-180°C) using a plunge cooling device (KF80, Reichert-Jung, Vienna, Austria). Frozen pieces were stored in liquid nitrogen. The PBS acceptor phases were dissolved in Emulsifier-Safe and counted (Packard Tri-Carb 4640 Liquid Scintillation Counter, Tilburg, The Netherlands), to verify 3H-E2 transport across the dermatomed skin. The surface-related activity (defined as the total radioactivity deposited on and taken up in the skin, per unit exposed surface area) of the skin after permeation was determined as follows (disintegrations/min/mm2): first, the surface areas of 3 skin pieces per cell were estimated with the aid of a microscope equipped with an eyepiece micrometer scale. Then, each sample was dissolved in 2 N NaOH at 100°C for 30 min. After chemiluminescence was reduced with HC1, the sample was dissolved in Emulsifier-Safe for liquid scintillation counting.
2.3.2. Freeze-drying Frozen skin pieces from each permeation experiment were transferred to a liquid nitrogen cooled 4-compartment copper sample holder, which was then positioned on a liquid nitrogen precooled stage ( < - 1 8 0 ° C ) in a modified evaporator (Polaron, model 6000, Cambridge, MA) equipped for high vacuum freeze-drying at -80°C. After a high vacuum was established ( < 1 0 - 4 Pa), a liquid nitrogen cooled cold trap was placed over the copper sample holder (distance 1 mm), and the sample holder was heated to - 8 0 ° C (Eurotherm Temperature Controller Type 017, Zoeterwoude, The Netherlands). After freeze-drying (___16 h) overnight, the sample holder was heated slowly (10°C/15 min) to 0°C, and allowed to reach room temperature until the next morning. Then the sample holder was closed with the aid of a manipulator holding a lid. After breaking the vacuum of the freeze-dryer, the sample holder
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J.A.M. Neelissen et al. I Journal of Controlled Release 42 (1996) 1-13
with the pieces of skin still at vacuum, was transported to a desiccator. 2.3.3. Osmium tetroxide vapor fixation
The specially designed desiccator [10,14] was evacuated (about 1 Pa), after which the lid from the sample holder was lifted with a manipulator. OsO 4 vapor was admitted to the working chamber by opening a side port containing 0.1 g or 0.5 g solid OsO 4. Fixation was terminated after 1 or 3 h by pumping away the OsO 4 vapor for 30 min. Next, the working chamber was flushed with dried nitrogen gas (30 min), evacuated overnight, flushed again with dried nitrogen gas (30 min) and again evacuated. 2.3.4. Spurr resin infiltration and embedding
One ml degassed Spurr resin without accelerator was applied to the skin pieces with dried nitrogen gas flushing over the sample holder. The vacuum was restored for an infiltration period of 30 min. Following two more infiltration steps with complete Spurr resin (2 × 30 min) at ambient conditions, the skin pieces were transferred to a prefilled embedding mold for polymerization at 60°C for 48 h. All the Spurr resin used for infiltration was dissolved in Opti-Fluor O and counted to determine any loss of 3H-E2 from the skin pieces. 2.3.5. Sectioning
During both trimming of the blocks as well as sectioning, the skin tended to split at the stratum corneum-Spurr resin interface, thereby destroying stratum corneum ultrastructure. To tackle this problem, attention was paid to the following steps: (1) freshness of the Spurr resin, (2) the trimming procedure, and (3) positioning of the sample block during sectioning. 2.4. Autoradiography
Both light and electron microscopic autoradiography (LM and EM-ARG) were performed according to the 'fiat substrate' procedure described by Salpeter [15]. Briefly, semi-thin (1 /xm) or ultrathin (100 nm) sections were cut perpendicularly to the skin surface with a diamond knife, placed on slides (for EM-ARG collodion coated slides) and dried
under ambient conditions. The water in the knifeboat was checked regularly for radioactivity by liquid scintillation counting (Emulsifier-Safe). A thin carbon layer was evaporated on the surface of the sections to prevent interactions with the photographic emulsion (positive chemography and extraction of 3H-E2). Slides were dipped in a solution of Ilford LA in water (LM-ARG 1:1 v/v, EM-ARG 1:2.5 v/v) at 32°C and withdrawn at a speed of 84 m m / m i n [16]. After vertical drying at ambient conditions for 1 h, the slides were placed in black slide boxes containing silica gel and stored at 4°C. For exposure times see the next section. LM slides were developed at 20°C in Kodak D l 9 b (2 min), EM slides at 20°C in a fine grain developer (GEA; 5 min gold latensification, 5 min elon ascorbic acid) [15,17]. Development was stopped by dipping the slides in water (15 s), 1% acetic acid (30 s) and water (15 s), followed by fixation (2 min) and rinsing in water (3 × 10 min). The non-hardening fixer was made fresh and contained 20% sodium thiosulphate (Na2S203.5H20) and 2.5% potassium metabisulfite (K2S205) in water [15]. LM autoradiographs were stained with toluidine blue and studied with a Leitz Orthoplan microscope (equipped with a Leitz NPL Fluotar 100/1.32 Oel Phaco 2 RK objective, Wetzlar, Germany) in reflection contrast mode (RCM) [18]. The ultrathin sections with the accompanying films were floated on water, covered with copper grids, picked up with parafilm, and examined with a JEOL JEM-100S transmission electron microscope at an accelerating voltage of 80 kV. 2.4.1. Estimation o f autoradiography exposure time
Textbooks [19,20] or articles [21] on autoradiography often lack instructions on the amount of radioactivity a tissue sample should contain for autoradiography and its determination. Also, it would be convenient to know in advance how long sections should be exposed to an emulsion to have the desired density of silver grains. Salpeter [15] described two approaches to estimate the exposure time of electron microscopic autoradiographs: an empirical and a mathematical one. For the empirical approach one has to perform the full autoradiography procedure at the LM level. When the same conditions (i.e. emulsion type and developer) are used as for EM-ARG, the experimen-
J.A.M. Neelissen et al. / Journal o f Controlled Release 42 (1996) 1 - 1 3
tally determined exposure time for LM autoradiographs can be used to estimate the exposure time for EM autoradiographs. It is essential to know how many disintegrations from a 3H-isotope in a 1 /xm section (used for L M - A R G ) are absorbed relative to 100 nm sections (used for EM-ARG). The relative grain yield for 1 /xm sections compared to 100 nm sections is 4 [15]. This means that a 100 nm section requires a 4 times longer exposure to get the same grain yield as in the 1 /xm section. The mathematical approach, based on a calculation of the exposure time, has the advantage that one has an indication if samples contain enough radioactivity before one starts the autoradiography procedure. Then, the exposure time can be determined with the following equation: n
Eq. (4) can be made more accurate, if the percentage activity loss a that might occur during sample preparation is included: n × d,k tca l :
as ×
and
as m
(2)
For a skin sample with thickness d~k, p can be written as: rn p -- dsk
(3)
Inserting Eq. (2) and Eq. (3) in Eq. (1) we obtain: n × d~k tca t =
a~ ×
1440
×
dse × s ×
o-
(4)
× 1440 × dse × s × Or
6.94 × 105 × d s k
where n is the desired grain density in the autoradiograph in grains/ram 2, a m the mass related activity in disintegrations/min/g tissue, 1440 the conversion from min to days, p the density of the tissue in g / r a m 3, ds~ the section thickness in mm, s the sensitivity of the emulsion-developer combination in grains/disintegration, o- the relative sensitivity factor to compensate for 3H-isotope absorption in the sections and teaI the calculated exposure time in days. Instead of mass related activity, which is defined as total radioactivity content in/on a tissue slab per unit weight (a m in disintegrations/min/g), the surface related activity (a~ in " dlsmtegratlons/mln/mm . . . . a) was determined (see Section 2.3.1 on cryofixation). For skin with a surface related weight of m g / r a m 2, a m is written as: am --
1-
For a recommended minimal yield for Ilford L4 emulsion of 105 grains/mm 2 [15], section thicknesses of 0.001 m m (LM) and 0.0001 mm (EM), an Ilford 1A-developer sensitivity of - 1/4 (I grain/4 disintegrations) [15] and a relative sensitivity factor of 0.4 (LM) and 1 (EM) [15], Eq. (5) becomes:
tLM=
a m X 1440 X p X d,e X s X o-
(1+0)
(5)
(1)
tca l =
5
tE~ =
as ×
( 1 - - 1@0)
2.78 X 10 6 × d,k a as X ( 1 - - ] - ~ )
(6a)
(6b)
with tLM and tEM the estimated exposure times for LM and EM autoradiographs respectively. Note that these equations assume that the silver grains are homogeneously distributed over the tissue with a grain density n. Because this is usually not the case, it would be more appropriate to estimate the autoradiography exposure time for the region of interest, in our case the stratum corneum: tcal
t,c-
f
(7)
with tsc the calculated autoradiography exposure time that was corrected for local grain densities over the stratum c o m e u m by means of a distribution factor f Because the stratum corneum is considered the rate limiting barrier in E2 permeation, 3H-E2 is expected to accumulate in the stratum corneum. Therefore, the desired silver grain density n will be reached much sooner over the stratum corneum than over other parts of the section (tsc < teal). Thus the distribution factor is expected to be larger than one ( f > 1). Eq. (7) can only be used if the distribution factor is known, e.g. by previous autoradiography experiments or by layerwise retention measurements
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J.A.M. Neelissen et al. / Journal of Controlled Release 42 (1996) 1-13
in the skin as described by Seta et al. [30]. However, as long as the distribution factor is unknown, the calculated t from Eqs. (6a) and Eqs. (6b) effectively mark the upper limit of exposure time, which corresponds to homogeneously distributed 3H-E2 in the skin.
3. Results and discussion For several reasons, isolated stratum corneum was difficult to process. Firstly, when the sample-holder was positioned on the precooled stage in the modified evaporator, the boiling liquid nitrogen often ejected several stratum corneum pieces from the sample-holder. Secondly, during Spurr resin exchange, the skin pieces often fell apart in several fragments. Thirdly, it was very difficult to cut ultrathin sections from the resin blocks. Because of this, the following applies to dermatomed skin samples only.
3.1. Sample preparation 3.1.1. Cryofixation Although manual plunging of small skin pieces in solid/liquid Freon 22 may be slower and less reproducible then a specially designed plunge cooling device, the ultrastructure of the stratum corneum from dermatomed skin was satisfactory. Cryofixation in propane using a plunge cooling device is supposed to provide better tissue ultrastructure because smaller ice crystals are formed. This is due to a higher cooling rate of the sample caused by a high velocity plunge into a coolant with better cooling properties [22]. The fact that the stratum corneum ultrastructure did not improve with this cryofixation procedure is probably due to the relatively low water content of the corneocytes. 3.1.2. Freeze-drying Many home built freeze-drying devices can be found in the literature [10,12,14,25-28]. Freeze-dry apparatuses commercially available, included the Coulter-Terracio (Ladd Research Industries, Burlington, VT) as well as a recently (by Linner et al. [28]) developed Molecular Distillation Dryer (LifeCell Corporation, The Woodlands, TX). The features of
our newly designed freeze-drying apparatus, a modified Polaron 6000 evaporator, are an accurate thermal control of the samples, a high vacuum, a short distance between samples and condenser, and a great temperature difference between the samples and the condenser surface. Total liquid nitrogen consumption for freeze-drying was about 60 1. Some researchers performed the freeze-drying, OsO 4 vapor fixation and epoxy resin infiltration step in the same apparatus [26,28]. Others used a separate chamber for OsO 4 fixation and epoxy resin infiltration [14,25]. We used a specially designed desiccator [10,14], which necessitated a vacuum tight transfer of the sample holder between the freeze-dryer and the desiccator. Besides the variety in freeze-drying apparatuses, there is also an enormous diversity in freeze-drying protocols. The procedure described in this paper was conformed with the conditions posed in Ingram and Ingrain [29].
3.1.3. Osmium tetroxide vapor fixation With regard to the structural preservation of the skin, it made no difference if OsO 4 vapor fixation was performed with 0.1 or 0.5 g OsO 4 crystals during 1 or 3 h. 3.1.4. Epoxy resin infiltration During OsO 4 fixation, the skin pieces had developed a greyish appearance; upon epoxy resin infiltration, they immediately turned completely black, while a black haze was formed around the skin pieces. A test with blank skin pieces without E2 also displayed this phenomenon, especially after 0.5 g OsO 4 vapor fixation. Therefore, the haze is most likely caused by excess osmium deposited on the skin surface that reacted with the resin. However, a part may originate from a reaction product between OsO 4 and either E2 residing at the skin surface or a skin component. 3.1.5. Sectioning Sectioning was quite cumbersome due to the splitting of the sections. Apparently, no cross-links were formed at the stratum corneum-epoxy resin interface, which might be due to the presence of excess OsO 4 on the skin surface. The best sectioning properties were achieved when four requirements
J.A.M. Neelissen et al. / Journal of Controlled Release 42 (1996) 1-13
were met: (1) instead of premixed Spurr resin stored at -20°C, freshly prepared Spurr resin had to be used; (2) extreme care had to be taken not to put too much force on the stratum corneum-resin interface while trimming the blocks; (3) avoid trimming close to the stratum corneum; and (4) the stratum corneum had to be cut facing the knife at an angle of - 4 5 degrees.
3.1.6. Light and electron microscopy Both semi-thin and ultrathin sections of dermatomed skin revealed good preservation of stratum corneum morphology (Fig. 1 and Fig. 2). Distinct inter- and intracellular domains were visible. In the electron micrographs the stratum corneum appeared electron lucent and the narrow electron-dense areas represented the intercellular lipid domains (Fig. 2). The continuity of this intercellular domain, suggesting little or no loss of intercellular lipids, is evident. Often, the outer 1-3 corneocytes had detached from the rest of the stratum corneum (Fig. 1 and Fig. 2). Desmosomes were present at the basal part of the stratum corneum. The ultrastructure of the viable epidermis varied. In most cases the cells were severely disrupted, but this was expected for any region beyond maximal vitrification depth of 10-20 /zm [23]. However, sometimes a thin layer of stratum granulosum cells was preserved well (Fig. 2). 3.2. Autoradiography 3.2.1. Mathematical estimation of autoradiography exposure time Table 1 summarizes the results of the radioactivity measurements of dermatomed skin pieces after removal of the donor phase by either 1 or 3 ethanol wipes. The difference in efficiency of the removal of the donor phase from the skin surface resulted in a difference in the surface related activity of a factor of 3.4. Table 1 also compiles the results of the radioactivity loss from dermatomed skin pieces during infiltration with Spurr resin. There was no radioactivity detected in the water of the knife-boat after sectioning. The amount of radioactivity found in the Spurr resin was highly dependent on the efficacy of surface wiping. After one ethanol wipe, the subsequent loss of activity from dermatomed skin during
7
infiltration was 79.9%. However, when skin samples were wiped 3 times with ethanol, the total surface specific activity was reduced to 30% of the initially determined activity, and the radioactivity loss during Spurr infiltration was reduced to 5.7% (Table 1). The maximum LM and EM autoradiograph exposure times for dermatomed skin with a thickness dsk of 0.2 mm were calculated with Eqs. (6a) and Eqs. (6b) and expressed in Table 1. The exposure times for skin samples cleaned with either 1 or 3 ethanol wipes indicate that a thorough removal of the residual donor phase from the skin surface (3 wipes) leads to a more precise estimation of the autoradiography exposure time. Autoradiographs of skin pieces immediately processed after the application of the 3H-E2 donor phase were negative. Hence 3H-E2 was not transported from the skin surface into the skin during infiltration. Therefore, a complete removal of the donor phase is not at all costs necessary. The question that remains to be solved is how much 3H-E2 was actually extracted from the skin during infiltration and polymerization. Although this uncertainty still remains a weak point of this technique, it does not harm the results of these experiments. 3.2.2. LM and EM autoradiography After the permeation experiments, radioactivity was found in the acceptor phase, confirming that 3H-E2 was transported across the skin. Autoradiographs of non-radioactive skin pieces were negative, except for very few background grains. There was no indication for positive chemography. Fig. 3 shows a bright-field (a) and a reflection contrast picture (b) of an LM autoradiograph after 43 days of exposure ( - 1 / 2 × tLM, Table 1). Silver grains were mainly located in detached corneocytes. Few grains were present in the intact stratum corneum and viable epidermis. The EM autoradiograph in Fig. 4 was developed after 320 days of exposure (close to the calculated tEM, Table 1), to ensure a higher grain density compared to Fig. 2. Note that the contrast of the section was greatly reduced as compared to Fig. 2. Also small cracks appeared in the intercellular domain. Because both phenomena were never observed in 'non-autoradiographed' sections, they must be an artifact of the autoradiographical procedure. The silver deposits were often located in clusters. It is assumed that clustered silver de-
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J.A.M. Neelissen et al. / Journal of Controlled Release 42 (1996) 1-13
A
Fig. 1. Morphology of dermatomed human skin after freeze-drying. Detached comeocytes (dc), stratum comeum (sc), stratum granulosum (sg) and stratum spinosum (ss) are visible. (A) Bright-field photomicrograph, and (B) reflection contrast photomicrograph of the same section (1 ,am), which was stained with toluidine blue.
posits are d e r i v e d f r o m several latent i m a g e s in a single e x p o s e d silver b r o m i d e crystal and therefore they are considered as one silver grain [16,24].
There was no difference in the E M autoradiographs b e t w e e n skin samples c l e a n e d with 1 or 3 ethanol wipes. In either case, m o s t of the silver
J.A.M. Neelissen et al. / Journal of Controlled Release 42 (1996) 1-13
9
Fig. 2. Ultrastructure of human dermatomed skin after freeze-drying. Stratum corneum (sc) and stratum granulosum (sg) were well preserved. Corneocytes are electron lucent, intercellular lipid domains are electron-dense.
Table 1 Surface related activity, loss of radioactivity and mathematical estimation of autoradiography exposure time for dermatomed human skin pieces (permeation time 24 h) Number of ethanol wipes to remove donor from skinsurface
1 3
Surface related skin activity (a s in disintegrations/min/mm2)
5765 _+ 2118(3) 1704 -4- 168(3)
Radioactivity loss during infiltration with Spurr resin a (%)
Calculated autoradiography exposure time (days) b
1st
2nd
3rd
Total
tLM
tEM
52.3 3.2
22.8 1.8
4.8 0.7
79.9 5.7
120 _+ 44 87 _+ 9
480 _+ 178 346 + 35
"3 × 30 min infiltration with Spurr resin, first under vacuum (no accelerator in Spurr resin), second and third (complete resin) at ambient conditions. btLU and tEM were calculated with Eqs. (6a) and Eqs. (6b).
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J.A.M. Neelissen et al. / Journal of Controlled Release 42 (1996) 1-13
Fig. 3. Light microscopic autoradiograph of stratum corneum and viable epidermis showing that 24 h after topical application of 3H-E2, silver grains were mainly located over the detached corneocytes. (A) Bright-field photomicrograph, and (B) reflection contrast photomicrograph of the same section (1 /zm), which was only faintly stained with toluidine blue, in order not to conceal the silver grains in the bright-field photomicrograph, llford L4 emulsion, 43 days of exposure, Kodak D19b development.
g r a i n s w e r e l o c a t e d o v e r the d e t a c h e d c o r n e o c y t e s (+_2 X 106 g r a i n s / m m 2 ) . T h e s t r a t u m c o r n e u m c o n tained fewer silver grains which were located over
the c o r n e o c y t e s a n d the i n t e r c e l l u l a r lipid d o m a i n (Fig. 4). T h e s i l v e r g r a i n d e n s i t y in the s t r a t u m c o r n e u m was, o n the a v e r a g e , 3 X 10 5 g r a i n s / m m 2.
J.A.M. Neelissen et al. / Journal of Controlled Release 42 (1996) 1-13
11
Fig. 4. Electron microscopic autoradiograph of the stratum corneum showing that 24 h after topical application of 3H-E2, silver grains were located both over the intra- and intercellular domains. Ilford L4 emulsion, 320 days of exposure, gold-elon ascorbic acid development.
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This results in distribution factors for 3H-E2 of f = 22 for detached corneocytes and f = 3.2 for stratum corneum (calculated with Eq. (5) and Eq. (7) for t = 320 days, a s = 1704 disintegrations/min/ 2 m m , c~ = 5.7%). Note that neither a change in the ratio 3H-E2:E2 nor a change in the autoradiography exposure time will effect the distribution factor f (once it is determined). Hence, the f values for detached corneocytes and stratum corneum can be used for future permeation experiments, provided that the same experimental set-up is used. Theoretically, it would be possible to determine the distribution factor separately for corneocytes and the intercellular domains. Due to the limited resolution in EM-ARG, however, this would require extensive statistical quantification of EM autoradiographs [20]. Changes in the distribution of silver grains in the stratum corneum and thus changes in the distribution factor can be used to illustrate effects of for instance:
(a) stage of the transport event, such as lag phase, early and late steady state, elimination phase after removal of the donor phase, (b) donor type, such as finite dose, infinite aqueous dose, patch, (c) type of drug, e.g. lipophilic, hydrophilic, (d) penetration enhancers, such as solubilizers and fluidizers, etc., (e) local drug accumulation in stratum corneum substructures which may be relevant for drugs that need to act in the stratum corneum, such as antimycotics, (f) route of penetration, although it will be quite difficult to translate EM autoradiograph pictures to permeation kinetics. A more promising approach would be a combination of several EM autoradiograph snapshots in time coupled to the actual flux kinetics.
4. Conclusion A visualization method, generally applicable to diffusible substances, was successfully adjusted for the intraepidermal drug localization following a topical applied finite dose of 3H-E2 to human skin in vitro. The equations developed to estimate light and
electron microscopic autoradiography exposure times, can be used to determine distribution factors for E2 in different parts of the skin.
Acknowledgments This work has been financially supported by Schwarz Pharma AG, Monheim, Germany. The author would like to thank G6 van Veen, for adapting the evaporator for freeze-drying; Jos Onderwater, for teaching the fundamentals of autoradiography; Frans Prins, for providing the reflection contrast microscope; and Ger Renes, for placing the electron microscope at my disposal.
Appendix 1 The equation to calculate LM and EM autoradiograph exposure in days: n X dsk tcaI = as
X
1-
× 1440 X
dse X s X o"
[grains x mm -2] X [mm] [disintegrations x min -1 x mm -2] x [ - ] x [1440] x [rnm] × [grains X disintegrations- ~] x [ - ]
1
1
[min- 1] X [1440] ¢ = ~ ¢ : ¢ , [ d a y ]
References [1] B. Berner and V.A. John, Pharmacokinetic characterisation of transdermal delivery systems. Clin. Pharmacokinet. 26 (1994) 121-134. [2] H.J. Bidmon, J.D. Pitts, H.F. Solomon, J.V. Bondi and W.E. Stumpf, Estradiol distribution and penetration in rat skin after topical application, studied by high resolution autoradiography. Histochemistry 95 (1990) 43-54. [3] C. CuUanderand R.H. Guy, Routes of delivery: case studies. 6. Transdermal delivery of pepfides and proteins. Adv. Drug Deliv. Rev. 8 (1992) 291-329. [4] J.N. Mansbridge and A.M. Knapp, Penetration of Lucifer Yellow into human skin: a lateral diffusion channel in the stratum corneum. J. Histochem. Cytochem. 41 (1993) 909914.
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[5] T. Rutherford and J.G. Black, The use of autoradiography to study the localization of germicides in skin. Br. J. Dermatol. 81 (Suppl 4) (1969) 75-87. [6] B.V. Zelei, C.J. Walker, G.A. Sawada, T.T. Kawabe, K.A. Knight, A.E. Buhl, G.A. Johnson and A.R. Diani, Immunohistochemical and autoradiographic findings suggest that minoxidil is not localized in specific cells of vibrissa, pelage, or scalp follicles. Cell Tissue Res. 262 (1990) 407-413. [7] H.E. Bodd~, I. van den Brink, H.K. Koerten and F.H.N. de Haan, Visualization of in vitro percutaneous penetration of mercuric chloride; transport through intercellular space versus cellular uptake through desmosomes. J. Control. Release 15 (1991) 227-236. [8] N.A. Monteiro-Reviere, A.O. Inman and J.E. Reviere, Identification of the pathway of iontophoretic drug delivery - light and ultrastructural studies using mercuric chloride in pigs. Pharm. Res. 11 (1994) 251-256. [9] M.K. Nemanic and P.M. Elias, In situ precipitation: a novel cytochemical technique for visualization of permeability pathways in mammalian stratum corneum. J. Histochem. Cytochem. 28 (1980) 573-578. [10] F.H.N. de Haan, H.E. Boddr, W.C. de Bruin, L.A. Ginsel and H.E. Junginger, Visualizing drug transport across stratum corneum: cryotechniques, vapour fixation, autoradiography. Int. J. Pharm. 56 (1989) 75-86. [11] C.A. Squier and C.A. Lesch, Penetration pathways of different compounds through epidermis and oral epithelia. J. Oral Pathol. 17 (1988) 512-516. [12] C.E. Stirling and W.B. Kinter, High-resolution radioantography of galactose-3H accumulation in rings of hamster intestine. J. Cell Biol. 35 (196"7) 585-604. [13] H.L.G.M. Tiemessen, H.E. Bodd6 and H.E. Junginger, A silicone membrane sandwich method to measure drug transport through isolated human stratum corneum having a fixed water content. Int. J. Pharm. 56 (1989) 87-94. [14] P.M. Frederik and D. Klepper, The possibility of electron microscopic autoradiography of steroids after freeze drying of unfixed testes. J. Microsc. 106 (1976) 209-219. [15] M.M. Salpeter, High resolution autoradiography, in: Techniques in Cellular Physiology, Elsevier, Shannon, Ireland, 1981, pp. P106/1-45. [ 16] G.F.J.M. Vrensen, Some new aspects of efficiency of electron microscopic autoradiography with tritium. J. Histochem. Cytochem. 18 (1970) 278-290. [17] E. Wisse and A.D. Tates, A gold latensification-elon ascorbic acid developer for Ilford L4 emulsion, in: D.S. Bocciarelli (Ed.) Electron microscopy, Vol. II, Tipografia Poliglotta Vaticana, Rome, 1968, pp. 465-466.
13
[18] I. Cornelese-ten Velde, J. Bonnet, H.J. Tanke and J.S. Ploem, Reflection contrast microscopy performed on epi-illumination microscope stands; comparison of reflection contrastand epi-polarization microscopy. J. Microsc. 159 (1990) 1-13. [19] A.W. Rogers, Techniques of Autoradiography, Elsevier, Amsterdam, 1967. [20] J.R.J. Baker, Autoradiography: a comprehensive overview, Microscopy Handbooks 18, Oxford University Press, New York, 1989. [21] B.M. Kopriwa, A reliable standardized method for ultrastructural electron microscopic autoradiography. Histochemie 37 (1973) 1-17. [22] K.P. Ryan, Cryofixation of tissues for electron microscopy: a review of plunge cooling methods. Scan. Microsc. 6 (1992) 715-743. [23] H.Y. Elder, C.C. Gray, A.G. Jardine, J.N. Chapman and W.H. Biddlecombe, Optimum conditions for cryoquenching of small tissue blocks in liquid coolants. J. Microsc. 126 (1982) 45-61. [24] B.M. Kopriwa, A comparison of various procedures for fine grain development in electron microscopic radioautography. Histochemistry 44 (1975) 201-224. [25] H.D. Coulter and L. Terracio, Preparation of biological tissue for electron microscopy by freeze-drying. Anat. Rec. 187 (1977) 477-493. [26] L. Edelmann, Freeze-dried embedded specimens for biological microanalysis. Scan. Electron Microsc. IV (1986) 1337-1356. [27] S. Hisano, T. Adachi, M. Maegawa and S. Daikoku, Some improvement in tissue preparation and colloidal-gold immunolabeling for electron microscopy. Am. J. Anat. 175 (1986) 245-266. [28] J.G. Linner, S.A. Livesey, D.S. Harrison and A,L. Steiner, A new technique for removal of amorphous phase tissue water without ice crystal damage; a preparative method for ultrastructural analysis and immunoelectron microscopy. J. Histochem. Cytochem. 34 (1986) 1123-1135. [29] F.D. Ingram and M.J. Ingram, Freeze-dried, plastic-embedded tissue preparation: a review. Scan. Electron Microsc. IV (1980) 147-160. [30] Y. Seta, A.H. Ghanem, W.I. Higuchi, S. Borsadia, C.R. Behl and A.W. Malick, Physical model approach to understanding finite dose transport and uptake of hydrocortisone in hairless guinea-pig skin, Int. J. Pharm. 81 (1992) 89-99.
Journal of Controlled Release 42 (1996) 1-13
controlled release
Optimization and validation of freeze-drying for light and electron microscopic autoradiography of percutaneous steroid transport Jan A.M. Neelissen a, Frans H.N. de Haan b, Ad H.G.J. Schrijvers c, Hans E. Junginger ~, Harry E. Bodd6 a'* "LeidenlArasterdam Center for Drug Research, Pharmaceutical Technology, P.O. Box 9502, 2300 RA Leiden, The Netherlands bOrganon International, Oss, The Netherlands CDepartment of Electron Microscopy, State University of Leiden, Leiden, The Netherlands
Received 23 March 1995; revised 4 December 1995; accepted 8 December 1995
Abstract A freeze-drying/autoradiography procedure to visualize steroid transport across human stratum corneum was optimized and validated. A finite dose of [3H]estradiol (3H-E2) was applied to human abdominal skin, i.e. isolated stratum corneum and dermatomed epidermal sheets, in an in vitro permeation set-up. The visualization procedure included cryofixation, freeze-drying, osmium tetroxide (OsO4) vapor fixation, Spurr resin embedding and autoradiography. The different steps of the sample preparation were carefully examined for morphological quality, extraction and diffusion of 3H-E2 from the skin. It appeared that dermatomed skin was, for several reasons, more suitable to process than isolated stratum corneum. While cross-sections of dermatomed skin displayed a well-preserved stratum corneum, the viable epidermis suffered from decreased structural preservation due to cryofixation artifacts. The apparent extraction of 3H-E2 during the infiltration with Spurt resin was due to donor phase residing at the skin surface. In the autoradiographs, there was no indication that 3H-E2 had diffused out of the skin. Silver grains were mainly localized over the superficial corneocytes. Only a few grains were present over the deeper layers of the stratum corneum, both over the inter- and intracellular domains. An equation was deduced to estimate the exposure time of autoradiographs from skin samples, with the advantage that no time was wasted with empirical range finding tests. In the equation a distribution factor was introduced which can be used to quantify the localization of the silver grains over the different tissue structures. In conclusion, it can be stated that the visualization method is suitable to study the distribution of 3H-E2 in human stratum corneum following topical application. Keywords: Percutaneous drug delivery; Human skin; Estradiol; Freeze-drying; Autoradiography
1. Introduction T r a n s d e r m a l drug d e l i v e r y s h o w s advantages o v e r the peroral route for a n u m b e r o f drugs, like
Corresponding author, Tel: +31 71 5274350; fax: +31 71 5274277; e-mail: [email protected].
clonidine, estradiol, fentanyl, nicotine, nitroglycerin and s c o p o l a m i n e [1]. It offers controlled continuous d e l i v e r y o v e r an e x t e n d e d duration and thus maintaining constant therapeutic drug concentrations. O n e o f the limitations is that percutaneous penetration is often too low for a therapeutic effect. H o w e v e r , the properties o f the b i o l o g i c a l p e r m e a b i l i t y barrier, i.e. stratum c o r n e u m and its intercellular lipids, can be
0168-3659/96/$15.0o © 1996 Elsevier Science Ireland Ltd. All rights reserved PII S01 68-3659(96)01324-7
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J.A.M. Neelissen et al. / Journal of Controlled Release 42 (1996) 1-13
modified by using chemical penetration enhancers or iontophoresis. Many questions remain to be answered concerning the mode of action of enhancers and iontophoretic devices, e.g. where and how they act in the stratum corneum. One strategy to study the effect of penetration enhancement is visualization of the distribution of a drug in the stratum corneum after passive permeation compared with an enhanced permeation. This approach requires a visualization method that is generally applicable for highly diffusible drugs, comprising a non-extractive skin sample preparation and a visualization of the drug molecules at both the light and electron microscopical level. The latter is considered necessary to be able to discriminate between the inter- and intracellular domains of the stratum corneum. Several procedures have been used to visualize percutaneous penetration of model compounds and drugs, mainly at the light microscopical level [2-6]. At the electron microscopical level, in situ precipitation [7-9] and electron microscopic autoradiography after cryofixation, freeze-drying, vapor fixation and resin embedding [10,11] have been used. Both methods seem to fulfil the requirements of a non-extractive sample preparation and the possibility to visualize penetrating molecules. In situ precipitation, however, is limited in its use. Only compounds which form an electron-dense precipitate after exposure to a vapor can be used, e.g. osmiophilic agents like n-butanol (exposed to OsO 4 vapor) [9], or heavy metals like mercuric chloride (exposed to ammonium sulfide vapor resulting in a mercuric sulfide precipitate) [7,8]. The other method, also referred to as the method of Stirling and Kinter [12], comprising cryofixation, freeze-drying, OsO 4 vapor fixation, epoxy resin embedding and autoradiography, is generally applicable. It requires a 3H or 14C labeling of the diffusing compound, e.g. [3H]hydrocortisone [ 10], [3H]water, [14C]ethanol and [3H]cholesterol [11]. This paper describes experiments to optimize and validate the method of Stirling and Kinter for the localization of topical applied 3H-E2 in human skin after in vitro permeation. Additionally, an equation is introduced to estimate autoradiographic exposure time.
2. Materials and methods
2.1. Materials
Trypsin (Bovine pancreas type III) and trypsin inhibitor (soybean type II-S) were purchased from Sigma (St. Louis, MO); [2,4,6,7-3H]estradiol (SA 14.6 GBq/mg) from Amersham (Buckinghamshire, UK); Freon 22 from Hoek Loos (Schiedam, The Netherlands); osmium tetroxide from Agar Scientific Ltd. (Stansted, UK); Emulsifier-Safe T M and OptiFluor® O from Packard (Meriden, CT); Spurr resin from Bio-Rad/Polaron (Cambridge, MA, USA); 4% collodion in diethyl ether from Merck (Darmstadt, Germany); Ilford L4 from Ilford Limited (Mobberley, Cheshire, UK). All other chemicals used were of analytical grade. All solutions were prepared with purified water (Milli-Q UF Plus Water System, Millipore, Etten-Leur, The Netherlands). Phosphatebuffered saline (PBS) had the following composition: sodium chloride (NaC1) 8 g/l, potassium chloride (KC1) 0.19 g/l, disodium phosphate (Na2HPO4.12H20) 2.86 g/l, potassium hydrogen phosphate (KH2PO4) 0.20 g/l, pH was set to 7.4 with 0.1 N sodium hydroxide (NaOH). Fresh human abdominal skin was obtained from female donors after cosmetic surgery. Two different types of skin samples were compared:
Dermatomed epidermal sheets. After prior removal of the subcutaneous fat, the flesh skin was dermatomed at 200 /zm (Padgett Electro-Dermatome model B, Kansas City, MO), stored overnight (at 4°C in a petfi dish on filter paper soaked with PBS) and used for permeation experiments the next day. 2. Isolated stratum corneum was obtained from freshly dermatomed skin by incubation overnight on 0.1% trypsin/PBS (w/v) soaked filter paper at 37°C. The stratum corneum was separated from the remaining epidermis, rinsed (at room temperature) with 1% trypsin inhibitor/PBS (w/v), and then rinsed twice by floating on water. The stratum corneum sheets were dried in a desiccator over silica gel, and subsequently stored in a closed container filled with dry nitrogen gas.
J.A.M. Neelissen et al. / Journal of Controlled Release 42 (1996) 1 - 1 3
Before use, disks were punched (16 mm diameter) and rehydrated for 24 h over 27% sodium bromide (NaBr)/H20 (w/v), resulting in ~17.5% (w/w) water content of the stratum corneum [13].
2.2. Permeation experiments Franz-type permeation cells with a permeation area of 0.79 cm 2 and a receiver chamber volume of 4.4 ml were kept at 32°C. In experiments with isolated stratum corneum, the stratum corneum (basal side) was supported with a dialysis membrane (Diachema MW-cutoff 5000, Dianorm, Mfinchen, Germany) at the receiver side, in which the receiver chamber was half-filled with 27% NaBr/HzO (w/v). Permeation time was 24 h. In experiments with dermatomed skin, the dermal side was in direct contact with stationary degassed PBS in the receiver chamber. Permeation time was 24 h. In both series of experiments, the donor solution, 25 /xl 1% estradiol (E2)/absolute ethanol (w/v) supplemented with 3.7 × 10 4 Bq 3H-E2 (molecular ratio 3H-E2:E2 ~1:100 000), was evenly distributed over the outer surface of the skin with a syringe. Experiments were non-occlusive, allowing the ethanol to evaporate, resulting in the deposition of a thin layer of E2 crystals on the skin surface. This surface deposit was left untouched throughout the permeation experiment. 3H-E2 permeation experiments were performed in triplicate. Two control permeation experiments were performed with dermatomed skin as described above, with the following modifications: in the first control the donor solution only contained non-radioactive E2. In the second control the experiment was terminated immediately after the ethanol had evaporated from the skin surface, followed by immediate processing as described below, without prior removal of the donor phase. This control should provide an answer if 3H-E2 residing at the skin surface diffuses into the skin during Spurr infiltration.
2.3. Sample preparation 2.3.1. Cryofixation The permeation experiments were terminated by removing the skin from the permeation cell and by
3
immediate removal of the donor phase from the skin surface with one ethanol wipe. The effectiveness of this removal was checked by cleaning one of the skin samples with three ethanol wipes. The skin samples were cut into small pieces (1-1.5 × 2 - 3 mm) with a razor blade and picked up with forceps and immediately plunged into solid/liquid Freon 22. A few non-radioactive pieces of skin were frozen in liquid propane (-180°C) using a plunge cooling device (KF80, Reichert-Jung, Vienna, Austria). Frozen pieces were stored in liquid nitrogen. The PBS acceptor phases were dissolved in Emulsifier-Safe and counted (Packard Tri-Carb 4640 Liquid Scintillation Counter, Tilburg, The Netherlands), to verify 3H-E2 transport across the dermatomed skin. The surface-related activity (defined as the total radioactivity deposited on and taken up in the skin, per unit exposed surface area) of the skin after permeation was determined as follows (disintegrations/min/mm2): first, the surface areas of 3 skin pieces per cell were estimated with the aid of a microscope equipped with an eyepiece micrometer scale. Then, each sample was dissolved in 2 N NaOH at 100°C for 30 min. After chemiluminescence was reduced with HC1, the sample was dissolved in Emulsifier-Safe for liquid scintillation counting.
2.3.2. Freeze-drying Frozen skin pieces from each permeation experiment were transferred to a liquid nitrogen cooled 4-compartment copper sample holder, which was then positioned on a liquid nitrogen precooled stage ( < - 1 8 0 ° C ) in a modified evaporator (Polaron, model 6000, Cambridge, MA) equipped for high vacuum freeze-drying at -80°C. After a high vacuum was established ( < 1 0 - 4 Pa), a liquid nitrogen cooled cold trap was placed over the copper sample holder (distance 1 mm), and the sample holder was heated to - 8 0 ° C (Eurotherm Temperature Controller Type 017, Zoeterwoude, The Netherlands). After freeze-drying (___16 h) overnight, the sample holder was heated slowly (10°C/15 min) to 0°C, and allowed to reach room temperature until the next morning. Then the sample holder was closed with the aid of a manipulator holding a lid. After breaking the vacuum of the freeze-dryer, the sample holder
4
J.A.M. Neelissen et al. I Journal of Controlled Release 42 (1996) 1-13
with the pieces of skin still at vacuum, was transported to a desiccator. 2.3.3. Osmium tetroxide vapor fixation
The specially designed desiccator [10,14] was evacuated (about 1 Pa), after which the lid from the sample holder was lifted with a manipulator. OsO 4 vapor was admitted to the working chamber by opening a side port containing 0.1 g or 0.5 g solid OsO 4. Fixation was terminated after 1 or 3 h by pumping away the OsO 4 vapor for 30 min. Next, the working chamber was flushed with dried nitrogen gas (30 min), evacuated overnight, flushed again with dried nitrogen gas (30 min) and again evacuated. 2.3.4. Spurr resin infiltration and embedding
One ml degassed Spurr resin without accelerator was applied to the skin pieces with dried nitrogen gas flushing over the sample holder. The vacuum was restored for an infiltration period of 30 min. Following two more infiltration steps with complete Spurr resin (2 × 30 min) at ambient conditions, the skin pieces were transferred to a prefilled embedding mold for polymerization at 60°C for 48 h. All the Spurr resin used for infiltration was dissolved in Opti-Fluor O and counted to determine any loss of 3H-E2 from the skin pieces. 2.3.5. Sectioning
During both trimming of the blocks as well as sectioning, the skin tended to split at the stratum corneum-Spurr resin interface, thereby destroying stratum corneum ultrastructure. To tackle this problem, attention was paid to the following steps: (1) freshness of the Spurr resin, (2) the trimming procedure, and (3) positioning of the sample block during sectioning. 2.4. Autoradiography
Both light and electron microscopic autoradiography (LM and EM-ARG) were performed according to the 'fiat substrate' procedure described by Salpeter [15]. Briefly, semi-thin (1 /xm) or ultrathin (100 nm) sections were cut perpendicularly to the skin surface with a diamond knife, placed on slides (for EM-ARG collodion coated slides) and dried
under ambient conditions. The water in the knifeboat was checked regularly for radioactivity by liquid scintillation counting (Emulsifier-Safe). A thin carbon layer was evaporated on the surface of the sections to prevent interactions with the photographic emulsion (positive chemography and extraction of 3H-E2). Slides were dipped in a solution of Ilford LA in water (LM-ARG 1:1 v/v, EM-ARG 1:2.5 v/v) at 32°C and withdrawn at a speed of 84 m m / m i n [16]. After vertical drying at ambient conditions for 1 h, the slides were placed in black slide boxes containing silica gel and stored at 4°C. For exposure times see the next section. LM slides were developed at 20°C in Kodak D l 9 b (2 min), EM slides at 20°C in a fine grain developer (GEA; 5 min gold latensification, 5 min elon ascorbic acid) [15,17]. Development was stopped by dipping the slides in water (15 s), 1% acetic acid (30 s) and water (15 s), followed by fixation (2 min) and rinsing in water (3 × 10 min). The non-hardening fixer was made fresh and contained 20% sodium thiosulphate (Na2S203.5H20) and 2.5% potassium metabisulfite (K2S205) in water [15]. LM autoradiographs were stained with toluidine blue and studied with a Leitz Orthoplan microscope (equipped with a Leitz NPL Fluotar 100/1.32 Oel Phaco 2 RK objective, Wetzlar, Germany) in reflection contrast mode (RCM) [18]. The ultrathin sections with the accompanying films were floated on water, covered with copper grids, picked up with parafilm, and examined with a JEOL JEM-100S transmission electron microscope at an accelerating voltage of 80 kV. 2.4.1. Estimation o f autoradiography exposure time
Textbooks [19,20] or articles [21] on autoradiography often lack instructions on the amount of radioactivity a tissue sample should contain for autoradiography and its determination. Also, it would be convenient to know in advance how long sections should be exposed to an emulsion to have the desired density of silver grains. Salpeter [15] described two approaches to estimate the exposure time of electron microscopic autoradiographs: an empirical and a mathematical one. For the empirical approach one has to perform the full autoradiography procedure at the LM level. When the same conditions (i.e. emulsion type and developer) are used as for EM-ARG, the experimen-
J.A.M. Neelissen et al. / Journal o f Controlled Release 42 (1996) 1 - 1 3
tally determined exposure time for LM autoradiographs can be used to estimate the exposure time for EM autoradiographs. It is essential to know how many disintegrations from a 3H-isotope in a 1 /xm section (used for L M - A R G ) are absorbed relative to 100 nm sections (used for EM-ARG). The relative grain yield for 1 /xm sections compared to 100 nm sections is 4 [15]. This means that a 100 nm section requires a 4 times longer exposure to get the same grain yield as in the 1 /xm section. The mathematical approach, based on a calculation of the exposure time, has the advantage that one has an indication if samples contain enough radioactivity before one starts the autoradiography procedure. Then, the exposure time can be determined with the following equation: n
Eq. (4) can be made more accurate, if the percentage activity loss a that might occur during sample preparation is included: n × d,k tca l :
as ×
and
as m
(2)
For a skin sample with thickness d~k, p can be written as: rn p -- dsk
(3)
Inserting Eq. (2) and Eq. (3) in Eq. (1) we obtain: n × d~k tca t =
a~ ×
1440
×
dse × s ×
o-
(4)
× 1440 × dse × s × Or
6.94 × 105 × d s k
where n is the desired grain density in the autoradiograph in grains/ram 2, a m the mass related activity in disintegrations/min/g tissue, 1440 the conversion from min to days, p the density of the tissue in g / r a m 3, ds~ the section thickness in mm, s the sensitivity of the emulsion-developer combination in grains/disintegration, o- the relative sensitivity factor to compensate for 3H-isotope absorption in the sections and teaI the calculated exposure time in days. Instead of mass related activity, which is defined as total radioactivity content in/on a tissue slab per unit weight (a m in disintegrations/min/g), the surface related activity (a~ in " dlsmtegratlons/mln/mm . . . . a) was determined (see Section 2.3.1 on cryofixation). For skin with a surface related weight of m g / r a m 2, a m is written as: am --
1-
For a recommended minimal yield for Ilford L4 emulsion of 105 grains/mm 2 [15], section thicknesses of 0.001 m m (LM) and 0.0001 mm (EM), an Ilford 1A-developer sensitivity of - 1/4 (I grain/4 disintegrations) [15] and a relative sensitivity factor of 0.4 (LM) and 1 (EM) [15], Eq. (5) becomes:
tLM=
a m X 1440 X p X d,e X s X o-
(1+0)
(5)
(1)
tca l =
5
tE~ =
as ×
( 1 - - 1@0)
2.78 X 10 6 × d,k a as X ( 1 - - ] - ~ )
(6a)
(6b)
with tLM and tEM the estimated exposure times for LM and EM autoradiographs respectively. Note that these equations assume that the silver grains are homogeneously distributed over the tissue with a grain density n. Because this is usually not the case, it would be more appropriate to estimate the autoradiography exposure time for the region of interest, in our case the stratum corneum: tcal
t,c-
f
(7)
with tsc the calculated autoradiography exposure time that was corrected for local grain densities over the stratum c o m e u m by means of a distribution factor f Because the stratum corneum is considered the rate limiting barrier in E2 permeation, 3H-E2 is expected to accumulate in the stratum corneum. Therefore, the desired silver grain density n will be reached much sooner over the stratum corneum than over other parts of the section (tsc < teal). Thus the distribution factor is expected to be larger than one ( f > 1). Eq. (7) can only be used if the distribution factor is known, e.g. by previous autoradiography experiments or by layerwise retention measurements
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J.A.M. Neelissen et al. / Journal of Controlled Release 42 (1996) 1-13
in the skin as described by Seta et al. [30]. However, as long as the distribution factor is unknown, the calculated t from Eqs. (6a) and Eqs. (6b) effectively mark the upper limit of exposure time, which corresponds to homogeneously distributed 3H-E2 in the skin.
3. Results and discussion For several reasons, isolated stratum corneum was difficult to process. Firstly, when the sample-holder was positioned on the precooled stage in the modified evaporator, the boiling liquid nitrogen often ejected several stratum corneum pieces from the sample-holder. Secondly, during Spurr resin exchange, the skin pieces often fell apart in several fragments. Thirdly, it was very difficult to cut ultrathin sections from the resin blocks. Because of this, the following applies to dermatomed skin samples only.
3.1. Sample preparation 3.1.1. Cryofixation Although manual plunging of small skin pieces in solid/liquid Freon 22 may be slower and less reproducible then a specially designed plunge cooling device, the ultrastructure of the stratum corneum from dermatomed skin was satisfactory. Cryofixation in propane using a plunge cooling device is supposed to provide better tissue ultrastructure because smaller ice crystals are formed. This is due to a higher cooling rate of the sample caused by a high velocity plunge into a coolant with better cooling properties [22]. The fact that the stratum corneum ultrastructure did not improve with this cryofixation procedure is probably due to the relatively low water content of the corneocytes. 3.1.2. Freeze-drying Many home built freeze-drying devices can be found in the literature [10,12,14,25-28]. Freeze-dry apparatuses commercially available, included the Coulter-Terracio (Ladd Research Industries, Burlington, VT) as well as a recently (by Linner et al. [28]) developed Molecular Distillation Dryer (LifeCell Corporation, The Woodlands, TX). The features of
our newly designed freeze-drying apparatus, a modified Polaron 6000 evaporator, are an accurate thermal control of the samples, a high vacuum, a short distance between samples and condenser, and a great temperature difference between the samples and the condenser surface. Total liquid nitrogen consumption for freeze-drying was about 60 1. Some researchers performed the freeze-drying, OsO 4 vapor fixation and epoxy resin infiltration step in the same apparatus [26,28]. Others used a separate chamber for OsO 4 fixation and epoxy resin infiltration [14,25]. We used a specially designed desiccator [10,14], which necessitated a vacuum tight transfer of the sample holder between the freeze-dryer and the desiccator. Besides the variety in freeze-drying apparatuses, there is also an enormous diversity in freeze-drying protocols. The procedure described in this paper was conformed with the conditions posed in Ingram and Ingrain [29].
3.1.3. Osmium tetroxide vapor fixation With regard to the structural preservation of the skin, it made no difference if OsO 4 vapor fixation was performed with 0.1 or 0.5 g OsO 4 crystals during 1 or 3 h. 3.1.4. Epoxy resin infiltration During OsO 4 fixation, the skin pieces had developed a greyish appearance; upon epoxy resin infiltration, they immediately turned completely black, while a black haze was formed around the skin pieces. A test with blank skin pieces without E2 also displayed this phenomenon, especially after 0.5 g OsO 4 vapor fixation. Therefore, the haze is most likely caused by excess osmium deposited on the skin surface that reacted with the resin. However, a part may originate from a reaction product between OsO 4 and either E2 residing at the skin surface or a skin component. 3.1.5. Sectioning Sectioning was quite cumbersome due to the splitting of the sections. Apparently, no cross-links were formed at the stratum corneum-epoxy resin interface, which might be due to the presence of excess OsO 4 on the skin surface. The best sectioning properties were achieved when four requirements
J.A.M. Neelissen et al. / Journal of Controlled Release 42 (1996) 1-13
were met: (1) instead of premixed Spurr resin stored at -20°C, freshly prepared Spurr resin had to be used; (2) extreme care had to be taken not to put too much force on the stratum corneum-resin interface while trimming the blocks; (3) avoid trimming close to the stratum corneum; and (4) the stratum corneum had to be cut facing the knife at an angle of - 4 5 degrees.
3.1.6. Light and electron microscopy Both semi-thin and ultrathin sections of dermatomed skin revealed good preservation of stratum corneum morphology (Fig. 1 and Fig. 2). Distinct inter- and intracellular domains were visible. In the electron micrographs the stratum corneum appeared electron lucent and the narrow electron-dense areas represented the intercellular lipid domains (Fig. 2). The continuity of this intercellular domain, suggesting little or no loss of intercellular lipids, is evident. Often, the outer 1-3 corneocytes had detached from the rest of the stratum corneum (Fig. 1 and Fig. 2). Desmosomes were present at the basal part of the stratum corneum. The ultrastructure of the viable epidermis varied. In most cases the cells were severely disrupted, but this was expected for any region beyond maximal vitrification depth of 10-20 /zm [23]. However, sometimes a thin layer of stratum granulosum cells was preserved well (Fig. 2). 3.2. Autoradiography 3.2.1. Mathematical estimation of autoradiography exposure time Table 1 summarizes the results of the radioactivity measurements of dermatomed skin pieces after removal of the donor phase by either 1 or 3 ethanol wipes. The difference in efficiency of the removal of the donor phase from the skin surface resulted in a difference in the surface related activity of a factor of 3.4. Table 1 also compiles the results of the radioactivity loss from dermatomed skin pieces during infiltration with Spurr resin. There was no radioactivity detected in the water of the knife-boat after sectioning. The amount of radioactivity found in the Spurr resin was highly dependent on the efficacy of surface wiping. After one ethanol wipe, the subsequent loss of activity from dermatomed skin during
7
infiltration was 79.9%. However, when skin samples were wiped 3 times with ethanol, the total surface specific activity was reduced to 30% of the initially determined activity, and the radioactivity loss during Spurr infiltration was reduced to 5.7% (Table 1). The maximum LM and EM autoradiograph exposure times for dermatomed skin with a thickness dsk of 0.2 mm were calculated with Eqs. (6a) and Eqs. (6b) and expressed in Table 1. The exposure times for skin samples cleaned with either 1 or 3 ethanol wipes indicate that a thorough removal of the residual donor phase from the skin surface (3 wipes) leads to a more precise estimation of the autoradiography exposure time. Autoradiographs of skin pieces immediately processed after the application of the 3H-E2 donor phase were negative. Hence 3H-E2 was not transported from the skin surface into the skin during infiltration. Therefore, a complete removal of the donor phase is not at all costs necessary. The question that remains to be solved is how much 3H-E2 was actually extracted from the skin during infiltration and polymerization. Although this uncertainty still remains a weak point of this technique, it does not harm the results of these experiments. 3.2.2. LM and EM autoradiography After the permeation experiments, radioactivity was found in the acceptor phase, confirming that 3H-E2 was transported across the skin. Autoradiographs of non-radioactive skin pieces were negative, except for very few background grains. There was no indication for positive chemography. Fig. 3 shows a bright-field (a) and a reflection contrast picture (b) of an LM autoradiograph after 43 days of exposure ( - 1 / 2 × tLM, Table 1). Silver grains were mainly located in detached corneocytes. Few grains were present in the intact stratum corneum and viable epidermis. The EM autoradiograph in Fig. 4 was developed after 320 days of exposure (close to the calculated tEM, Table 1), to ensure a higher grain density compared to Fig. 2. Note that the contrast of the section was greatly reduced as compared to Fig. 2. Also small cracks appeared in the intercellular domain. Because both phenomena were never observed in 'non-autoradiographed' sections, they must be an artifact of the autoradiographical procedure. The silver deposits were often located in clusters. It is assumed that clustered silver de-
8
J.A.M. Neelissen et al. / Journal of Controlled Release 42 (1996) 1-13
A
Fig. 1. Morphology of dermatomed human skin after freeze-drying. Detached comeocytes (dc), stratum comeum (sc), stratum granulosum (sg) and stratum spinosum (ss) are visible. (A) Bright-field photomicrograph, and (B) reflection contrast photomicrograph of the same section (1 ,am), which was stained with toluidine blue.
posits are d e r i v e d f r o m several latent i m a g e s in a single e x p o s e d silver b r o m i d e crystal and therefore they are considered as one silver grain [16,24].
There was no difference in the E M autoradiographs b e t w e e n skin samples c l e a n e d with 1 or 3 ethanol wipes. In either case, m o s t of the silver
J.A.M. Neelissen et al. / Journal of Controlled Release 42 (1996) 1-13
9
Fig. 2. Ultrastructure of human dermatomed skin after freeze-drying. Stratum corneum (sc) and stratum granulosum (sg) were well preserved. Corneocytes are electron lucent, intercellular lipid domains are electron-dense.
Table 1 Surface related activity, loss of radioactivity and mathematical estimation of autoradiography exposure time for dermatomed human skin pieces (permeation time 24 h) Number of ethanol wipes to remove donor from skinsurface
1 3
Surface related skin activity (a s in disintegrations/min/mm2)
5765 _+ 2118(3) 1704 -4- 168(3)
Radioactivity loss during infiltration with Spurr resin a (%)
Calculated autoradiography exposure time (days) b
1st
2nd
3rd
Total
tLM
tEM
52.3 3.2
22.8 1.8
4.8 0.7
79.9 5.7
120 _+ 44 87 _+ 9
480 _+ 178 346 + 35
"3 × 30 min infiltration with Spurr resin, first under vacuum (no accelerator in Spurr resin), second and third (complete resin) at ambient conditions. btLU and tEM were calculated with Eqs. (6a) and Eqs. (6b).
10
J.A.M. Neelissen et al. / Journal of Controlled Release 42 (1996) 1-13
Fig. 3. Light microscopic autoradiograph of stratum corneum and viable epidermis showing that 24 h after topical application of 3H-E2, silver grains were mainly located over the detached corneocytes. (A) Bright-field photomicrograph, and (B) reflection contrast photomicrograph of the same section (1 /zm), which was only faintly stained with toluidine blue, in order not to conceal the silver grains in the bright-field photomicrograph, llford L4 emulsion, 43 days of exposure, Kodak D19b development.
g r a i n s w e r e l o c a t e d o v e r the d e t a c h e d c o r n e o c y t e s (+_2 X 106 g r a i n s / m m 2 ) . T h e s t r a t u m c o r n e u m c o n tained fewer silver grains which were located over
the c o r n e o c y t e s a n d the i n t e r c e l l u l a r lipid d o m a i n (Fig. 4). T h e s i l v e r g r a i n d e n s i t y in the s t r a t u m c o r n e u m was, o n the a v e r a g e , 3 X 10 5 g r a i n s / m m 2.
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Fig. 4. Electron microscopic autoradiograph of the stratum corneum showing that 24 h after topical application of 3H-E2, silver grains were located both over the intra- and intercellular domains. Ilford L4 emulsion, 320 days of exposure, gold-elon ascorbic acid development.
12
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This results in distribution factors for 3H-E2 of f = 22 for detached corneocytes and f = 3.2 for stratum corneum (calculated with Eq. (5) and Eq. (7) for t = 320 days, a s = 1704 disintegrations/min/ 2 m m , c~ = 5.7%). Note that neither a change in the ratio 3H-E2:E2 nor a change in the autoradiography exposure time will effect the distribution factor f (once it is determined). Hence, the f values for detached corneocytes and stratum corneum can be used for future permeation experiments, provided that the same experimental set-up is used. Theoretically, it would be possible to determine the distribution factor separately for corneocytes and the intercellular domains. Due to the limited resolution in EM-ARG, however, this would require extensive statistical quantification of EM autoradiographs [20]. Changes in the distribution of silver grains in the stratum corneum and thus changes in the distribution factor can be used to illustrate effects of for instance:
(a) stage of the transport event, such as lag phase, early and late steady state, elimination phase after removal of the donor phase, (b) donor type, such as finite dose, infinite aqueous dose, patch, (c) type of drug, e.g. lipophilic, hydrophilic, (d) penetration enhancers, such as solubilizers and fluidizers, etc., (e) local drug accumulation in stratum corneum substructures which may be relevant for drugs that need to act in the stratum corneum, such as antimycotics, (f) route of penetration, although it will be quite difficult to translate EM autoradiograph pictures to permeation kinetics. A more promising approach would be a combination of several EM autoradiograph snapshots in time coupled to the actual flux kinetics.
4. Conclusion A visualization method, generally applicable to diffusible substances, was successfully adjusted for the intraepidermal drug localization following a topical applied finite dose of 3H-E2 to human skin in vitro. The equations developed to estimate light and
electron microscopic autoradiography exposure times, can be used to determine distribution factors for E2 in different parts of the skin.
Acknowledgments This work has been financially supported by Schwarz Pharma AG, Monheim, Germany. The author would like to thank G6 van Veen, for adapting the evaporator for freeze-drying; Jos Onderwater, for teaching the fundamentals of autoradiography; Frans Prins, for providing the reflection contrast microscope; and Ger Renes, for placing the electron microscope at my disposal.
Appendix 1 The equation to calculate LM and EM autoradiograph exposure in days: n X dsk tcaI = as
X
1-
× 1440 X
dse X s X o"
[grains x mm -2] X [mm] [disintegrations x min -1 x mm -2] x [ - ] x [1440] x [rnm] × [grains X disintegrations- ~] x [ - ]
1
1
[min- 1] X [1440] ¢ = ~ ¢ : ¢ , [ d a y ]
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