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The Effect of Various In Vitro Conditions on the Permeability Characteristics of the Buccal Mucosa
The Effect of Various In Vitro Conditions on the Permeability Characteristics of the Buccal Mucosa JOSEPH A. NICOLAZZO, BARRY L. REED, BARRIE C. FINNIN Department of Pharmaceutics, Monash University, 381 Royal Parade, Parkville, Victoria, Australia 3052
Received 26 February 2003; revised 6 June 2003; accepted 9 June 2003
ABSTRACT: The effect of various in vitro conditions on the permeability characteristics of the buccal mucosa was assessed using caffeine (CAF) and estradiol (E2) as model hydrophilic and lipophilic markers, respectively. The permeation of CAF and E2 through porcine buccal mucosa was determined in modified Ussing chambers at 378C over 4 h. Comparative permeation studies were performed through full thickness and epithelial tissues, fresh and frozen tissues, and intact and intentionally damaged tissues. Tissue integrity was monitored by the absorption of the normally impermeable fluorescein isothiocyanate (FITC)-labeled dextran 20 kDa (FD20) and tissue viability was assessed using an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) biochemical assay and histological evaluation. Compared to full thickness buccal tissue, permeability through the buccal epithelium was 1.8-fold greater for CAF and 16.7-fold greater for E2. Although the fluxes of the model compounds were no different in fresh and frozen buccal epithelium, histological evaluation demonstrated signs of cellular death in frozen tissue. FD20 permeated damaged tissue, and while this was associated with an increase in CAF transport, no significant change in E2 transport was observed. The tissue appeared to remain viable for up to 12 h postmortem using the MTT viability assay, and this was supported by histological evaluation. ß 2003 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 92:2399–2410, 2003
Keywords: buccal mucosa; permeability; caffeine; estradiol; paracellular; transcellular; tissue integrity; tissue viability
INTRODUCTION Delivery of drugs via the buccal mucosa has several advantages over the traditional oral route that can be exploited.1–3 Like the skin, however, the buccal mucosa acts as a physiological barrier to prevent the absorption of exogenous chemicals and xenobiotics. To most effectively overcome the barrier properties of the mucosa, it is important to be aware of the structure of the tissue and essential to understand the processes involved in drug transport through the tissue.
Correspondence to: Barrie C. Finnin (Telephone: þ61 3 9903 9520; Fax: þ61 3 9903 9583; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 92, 2399–2410 (2003) ß 2003 Wiley-Liss, Inc. and the American Pharmacists Association
Like all stratified squamous epithelia, the buccal mucosa consists of differentiating layers of epithelial cells surrounded by an intercellular lipid matrix. Compared to the lipophilic epithelial cell membranes, the intercellular matrix is relatively hydrophilic, although it has some lipophilic components that are secreted by intracellular membrane coating granules.4 The buccal mucosa therefore consists of hydrophilic and lipophilic regions, and consequently, it has been suggested that the mucosa provides two routes for drug transport—paracellular (between the cells) and transcellular (across the cells).5 Hydrophilic compounds predominantly permeate via the paracellular route due to its relatively hydrophilic nature, whereas lipophilic compounds would permeate via the transcellular route.5,6 An alternative representation of transport routes in the buccal mucosa
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has been suggested, which involves a polar and nonpolar route.7 The nonpolar route involves partitioning of lipophilic molecules into the plasma membrane lipid bilayer or into lipid of the intercellular matrix, and the polar route involves the passage of small hydrophilic molecules through aqueous pores in the plasma membrane of individual cells, or ionic channels in the intercellular spaces.7 Although the polar route can be likened to the paracellular route and the nonpolar route to the transcellular route, this alternative representation does not limit drug transport to only intercellular or intracellular processes. The purpose of this study was to assess the in vitro permeability characteristics of the buccal mucosa using caffeine (CAF) as a model hydrophilic permeant and estradiol (E2) as a model lipophilic permeant. Due to their different physicochemical characteristics, these compounds are expected to be transported across the buccal mucosa via different routes. Therefore, the permeability characteristics of the buccal mucosa under different conditions (full thickness and epithelial tissue, fresh and frozen tissue, intact and damaged tissue) can be assessed in a general manner, because the model permeants have different physicochemical properties, and therefore, different routes of transport. Due to the limited availability of human buccal tissue, porcine buccal mucosa was used for in vitro permeability experiments. Porcine buccal mucosa (a nonkeratinized epithelium) has been suggested as a suitable model membrane because its morphology and permeability are similar to that of human buccal mucosa.8 Integrity markers are often used in in vitro permeability experiments to ensure that the model membrane is intact, and that the observed permeability profiles of model compounds are not a result of compromised tissue integrity. An important component of this study, therefore, was to assess the potential of the high molecular weight fluorescein isothiocyanate (FITC)-labeled dextran 20 kDa (FD20) as a marker of tissue integrity. FD20 was chosen as a marker of tissue integrity because a previous study has revealed that passage of porcine buccal mucosa by FITC-dextrans is restricted to a molecular weight lower than 20 kDa.9 Therefore, appearance of FD20 in the receptor chamber of the in vitro model would be indicative of compromised tissue integrity. When assessing the permeability of compounds through the skin, the issue of viability is often ignored, because the rate-limiting barrier is attributed to the stratum corneum. However, when
assessing the in vitro permeability characteristics of the buccal mucosa, tissue viability may be an important factor to consider. The permeability barrier of the buccal mucosa has been attributed to the top third of the epithelium,10,11 which, unlike the stratum corneum, contains cells with a variety of functional organelles.12 Therefore, it seems appropriate that the viability of excised buccal tissue be assessed, as this may affect the observed permeability results. Consequently, the viability of porcine buccal tissue was determined using histological evaluation and an MTT (3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) biochemical assay that has previously been used in assessing the viability of excised buccal mucosa and cornea.13,14
MATERIALS AND METHODS Materials CAF was obtained from Hunan Pharmaceutical Factory (Hunan, China), and E2 was obtained from AKZO Nobel (Oss, The Netherlands). MTT and FD20 were purchased from Sigma Chemical Co. (St. Louis, MO) and dimethylsulfoxide (DMSO) was purchased from Merck Pty. Limited (Victoria, Australia). Sodium dodecyl sulfate (SDS) was obtained from Boehringer-Mannheim (Germany). Acetonitrile and methanol (Mallinckrodt, KY) were of HPLC grade. Krebs bicarbonate Ringer (KBR) buffer was prepared with 115.5 mM NaCl, 4.2 mM KCl, 21.9 mM NaHCO3, 12.2 mM glucose, 4.0 mM HEPES, 1.2 mM MgSO4 7H2O, 2.5 mM CaCl2 2H2O, and 1.6 mM NaH2PO4 2H2O, and adjusted to pH 7.5 with carbogen (95% O2 þ 5% CO2) bubbling. Water was obtained from a Milli-Q water purification system (Millipore, Milford, MA), and all other chemicals were of analytical grade and were used as received. Tissue Preparation Buccal tissue from domestic pigs (Sus scrofa domestica) was obtained from a local abattoir immediately after slaughter, and was transported in ice-cold KBR. Due to abattoir postmortem processing, pigs underwent a 4-min heat-treatment at 608C, before the buccal tissue could be removed. Therefore, the transport of CAF through this heat-treated mucosa was compared with CAF transport through mucosa obtained from pigs that
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did not undergo postmortem heat-treatment. Additionally, heat-treated mucosa was assessed with light microscopy. For full thickness mucosal studies, buccal mucosa with underlying connective tissue and fat was removed, pinned epithelial side down, and most of the connective tissue and fat removed with surgical scissors. For epithelial studies, the buccal epithelium was carefully separated from the underlying tissues using forceps and surgical scissors. To determine whether the presence of connective tissue alters the overall sensitivity of the tissue to chemical penetration enhancers, both full thickness and epithelial tissues were pretreated with SDS. SDS has been shown to enhance the buccal permeability of compounds,15,16 and therefore, would be expected to affect the permeability in this in vitro model. Consequently, the mucosal surface of buccal tissue was exposed to SDS 1% in KBR for 2 h, CAF transport was monitored over 4 h as detailed below in permeation studies, and the integrity of the tissue was also assessed, as described below in integrity studies. To determine the effect of tissue storage on permeability, buccal epithelium was excised, wrapped in aluminium foil, and stored at 208C in a standard freezer for at least 1 month. On the day of the permeation experiments, the tissue was removed from the freezer, placed in a Petri dish containing KBR for approximately 30 min at room temperature, and then used in permeation experiments. To compare the permeability characteristics of intact and damaged tissue, buccal epithelium was intentionally damaged by placing holes (approximately 5–6 per exposed area) in the epithelium using a Terumo1 25-G needle (Terumo Medical Corporation, Elkton, MD). Light Microscopy To ensure the epithelium had separated from the underlying connective tissue and to assess heat-treated mucosa, epithelial and full thickness tissues were examined with light microscopy. Immediately following excision and/or separation, tissue was placed in 10% buffered formalin. Tissue samples were then dehydrated with increasing concentrations of alcohol, placed in xylene, and embedded in paraffin. Paraffin preparations were then cut to 6-mm thickness using a microtome and stained with hematoxylin and eosin. The prepared slides were examined under a Wild M20 light microscope (Gais, Switzerland). A Spot Jr.
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enhanced digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI) was used to capture images, which were processed by Spot software (Version 2.2, Diagnostic Instruments, Inc., Sterling Heights, MI). Permeation Studies In vitro permeation studies were conducted in modified Ussing chambers (diffusional area 0.64 cm2) maintained at 378C and supplied with carbogen. Porcine buccal mucosa was mounted between the donor and receptor chambers, which were filled with 1.5-mL KBR and allowed to equilibrate for 30 min. For CAF permeation experiments, 100 mL of donor chamber KBR was removed after the equilibration period and replaced with 100 mL of CAF formulation to give a donor concentration of 20 mg/mL (except for full thickness studies, where the donor concentration was 50 mg/mL). For E2 permeation experiments, both donor and receptor chambers were emptied after equilibration and replaced with either E2 saturated suspension (donor) or KBR (receptor). E2 was presented as a saturated suspension due to its poor aqueous solubility (determined to be 2.5 mg/mL in KBR). Samples (100 mL for CAF and 150 mL for E2) were removed from the receptor chamber at periodic intervals over 4 h and replaced with fresh KBR. The results were adjusted to allow for this dilution effect. Samples were assayed for CAF or E2 with HPLC. The cumulative amount of drug permeated was plotted versus time and the steady state flux (Jss) was calculated using eq. 1: Jss ¼
DM A Dt
ð1Þ
where DM is the amount of drug transported across the membrane during time Dt, and A is the diffusional area. In some experiments, the cumulative amount absorbed at 4 h (Q4h) was used for comparison. All experiments were conducted using the buccal mucosa of at least two animals with four to eight replicates and were commenced within 2 h of slaughter. All text results are expressed as mean SD and graphical results are expressed as mean SEM. Integrity Studies FD20 was added to the donor chamber after permeability measurements (donor concentration of approximately 10 mg/mL) and its appearance in
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the receptor chamber was determined after 1 h by fluorescence spectroscopy. The cumulative amount of FD20 absorbed over 1 h was compared in intact and intentionally damaged tissue, as well as in fresh and frozen tissue to determine the effect of freezing on tissue integrity. Analytical Method Concentrations of CAF and E2 were determined using HPLC with a Waters C18 Symmetry1 column (3.9 150 mm) (Waters, Milford, MA). The HPLC system consisted of a Waters 510 HPLC pump, Waters 717 autosampler and Waters 486 tunable absorbance detector. Peak heights were plotted and integrated on a Shimadzu C-R3A Chromatopac integrator (Shimadzu Corporation, Kyoto, Japan). The chromatographic conditions for CAF and E2 are detailed in Table 1. Assay precision (CV%) and accuracy were determined by replicate analyses (n ¼ 6) at the lowest, middle, and highest concentrations validated. These precision and accuracy values are also detailed in Table 1. Concentrations of FD20 were determined using a LS-3 fluorescence spectrometer (Perkin-Elmer Ltd, Norwalk, CT) at an excitation wavelength of 498 nm and emission wavelength of 520 nm. The limit of quantitation for FD20 was 0.01 mg/mL, and precision (CV%) and accuracy values ranged from 0.60 to 11.01% and 98.69 to 100.76%, respectively. Viability Study The MTT assay has been used for quantitative colorimetric measurements of mammalian cell survival and proliferation.17 The original assay
Table 1.
procedure has been modified to assess the viability of tissue specimens rather than cell lines.13,14 MTT is converted in viable cells to formazan (a dark purple water insoluble compound) by enzymes in active mitochondria collectively known as tetrazolium reductase (TR), and the amount of formazan generated is directly proportional to the number of living cells.17 Consequently, the results from the viability assay have been expressed as a TR index (amount of formazan absorbance per mg of tissue). Freshly obtained porcine buccal epithelium was mounted in modified Ussing chambers as in the permeation experiments and at specific time points up to 24 h, the tissue was removed and subjected to the MTT viability assay. MTT was dissolved (2 mg/mL) in freshly prepared phosphate-buffered saline (PBS) and filtered through a Millex1-HA 0.45 mm filter unit (Millipore, Bedford, MA) to remove any undissolved crystals. Biopsy samples (4 mm) of each tissue were obtained at relevant time points using a disposable biopsy punch (Stiefel Laboratories, Germany). The first biopsy sample was taken at 1 h postmortem (quickest feasible time), and this time point was used as the initial viability reading to which all subsequent values were compared. Each biopsy sample was weighed and placed into individual wells of a six-well tissue culture plate (NunclonTM Multidish, Nalge Nunc International, Rochester, NY). Two milliliters of MTT solution was added to each well, and the plate was placed on a rotating platform (100 rpm) at 378C for 2 h. After 2 h, the MTT solution was removed and the tissue was rinsed twice with 1 mL of PBS for 1 min and then minced with surgical scissors. To extract the water-insoluble formazan, 4 mL of DMSO was
Chromatographic Conditions Employed for the Analysis of CAF and E2 Drug
Condition
CAF
E2
Mobile phase Flow rate Injection volume UV detection
Methanol:0.057% H3PO4 (22:78) 1 mL/min 20 mL 273 nm
Assay range Precision (CV%)a Accuracya
0.05–10 mg/mL 0.42–6.35% 100.30–102.59%
Acetonitrile:H2O (40:60) 1 mL/min 100 mL 221 nm (epithelial studies) 277 nm (full thickness studies) 0.02–2 mg/mL 1.02–8.70% 97.89–106.25%
a Precision and accuracy values were determined by replicate analyses (n ¼ 6) at the lowest, middle and highest concentrations validated.
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added to each well and rotated (100 rpm) for 80 min at 378C. The absorbance of formazan was measured at 540 nm (with DMSO as a blank) in a Fluostar Optima microplate reader (BMG Labtechnologies, Offenburg, Germany). The absorbance per mg of tissue was determined and expressed as the TR index. To ensure the assay discriminated between live and dead tissue, tissue was treated for 6 h in DMSO and subjected to the MTT assay. Additionally, the assay was performed in the absence of tissue to ensure no interference and in frozen tissue to assess the effect of freezing on viability. Fresh and frozen tissue, and tissue maintained in the Ussing chambers (for the same length of time as in the MTT viability assay) were also evaluated using light microscopy.
RESULTS Tissue Preparation In initial experiments, the permeation of CAF was compared through buccal mucosa from heattreated and untreated pigs to ensure heat treatment did not affect the barrier properties of the buccal mucosa. The % of applied dose of CAF absorbed over 4 h through heat-treated and untreated porcine buccal mucosa was 10.47 0.60% and 9.00 1.99%, respectively. Additionally, light microscopy revealed no obvious effects on mucosal histology (Figure 1a). Figure 1 also contains the photomicrographs of the buccal epithelium following surgical detachment from the underlying connective tissue. Light microscopy revealed that there was a clear separation of the epithelium from the connective tissue (which appeared intact in full thickness tissue) and that the lowermost layer contained cells that were cuboidal, indicative of basal cells. The undulated appearance of this lower layer was indicative of the junction between the epithelium and lamina propria.18 Permeation Studies The permeability profiles of CAF and E2 through full thickness and epithelial tissues are shown in Figure 2. The cumulative amount of CAF absorbed over 4 h through epithelium was 1.8-fold greater than through full thickness mucosa, with Q4h values of 17.41 1.77 and 9.46 1.63 mg/cm2, respectively ( p < 0.001). When comparing the flux values for each tissue, Jss was 4.68 0.49 mg/cm2/h
Figure 1. Light microscopic evaluation of (a) heattreated porcine full thickness buccal mucosa and (b) heat-treated porcine buccal epithelium after separation from underlying connective tissue (magnification 100). Figure (c) is a high magnification (200) micrograph of the detached epithelium demonstrating the cuboidal shape of the lowest layer of cells and the undulating nature of the basal area.
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Figure 2. Permeation of (a) CAF and (b) E2 through porcine full thickness buccal mucosa (*) and epithelium (*). Data are presented as mean SEM (n ¼ 6).
for epithelium and 3.16 0.26 mg/cm2/h for full thickness tissue ( p < 0.001). A significant ( p < 0.001) difference in lag time was also observed for CAF transport through full thickness tissue (lag time of 1.11 0.24 h) compared with epithelial transport (lag time of 0.41 0.04 h). With E2 transport studies, the difference in full thickness and epithelial permeation was more marked with Q4h values of 0.05 0.12 mg/cm2 (full thickness) and 0.80 0.20 mg/cm2 (epithelium), a 16.7-fold difference. A steady state profile was not achieved for E2 through full thickness mucosa and so steady-state rates could not be compared. The results obtained were very reproducible as intraanimal variation ranged from 6.6–20.2% for CAF permeation and 13.5–20.8% for E2 permeation, and no significant differences were observed with t-tests comparing interanimal data ( p ¼ 0.901 for CAF and p ¼ 0.354 for E2). As demonstrated in Figure 3a, pretreating epithelial tissue with SDS 1% increased CAF flux
Figure 3. Permeability of CAF through (a) porcine buccal epithelium and (b) porcine full thickness buccal mucosa with (*) and without (*) a 2 h pretreatment with SDS 1% w/v in KBR. Data are presented as mean SEM (n ¼ 4–6). *Indicates CAF flux with SDS pretreatment is significantly greater than with no pretreatment (p < 0.001).
from 1.34 0.26 mg/cm2/h to 2.37 0.41 mg/cm2/h (p < 0.001). However, the permeability of CAF was not significantly improved when full thickness tissue was pretreated with SDS. This indicates that the enhancing effect of SDS was negated by the presence of connective tissue in full thickness specimens. Based on these results and the fact that in vivo, blood vessels are directly beneath the epithelium, all further studies were performed using epithelial tissue. The integrity of the polar route of permeation was not affected following SDS pretreatment, as FD20 permeation was well below the threshold indicative of such damage (as discussed in tissue integrity). The Q4h and steady state flux values (Jss) for CAF and E2 through fresh and frozen porcine buccal epithelium are listed in Table 2. Storage of
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Table 2. Q4h and Flux Values for CAF and E2 through Fresh and Frozen Buccal Epithelium Compound Condition Flux (mg/cm2/h) Q4h (mg/cm2) CAF E2
Fresh Frozen Fresh Frozen
1.75 0.25 1.66 0.11 0.41 0.09 0.52 0.06
6.29 0.10 6.13 0.71 1.36 0.36 1.55 0.21
Data are presented as mean SD (n ¼ 4–6).
porcine buccal epithelium at 208C for at least 1 month did not affect the permeation of either model compound. Tissue Integrity In Figure 4, the cumulative amount of FD20 permeating the buccal epithelium over 1 h is compared with the cumulative amount of CAF permeated over 4 h in intact fresh and frozen tissue, and intentionally damaged tissue. Figure 5 shows a comparison between FD20 permeation and E2 transport in the similarly treated tissues. Although there was an increase in CAF permeation associated with increased FD20 appearance in the receptor chamber, a similar result was not observed with E2 permeation. To further investigate the validity of FD20 as an integrity marker, the error associated with CAF permeation data (Q4h) was compared with and without consideration of FD20 permeation. Because FD20 was not a valid marker for the route of permeation taken by E2, a similar data analysis for E2 error is not presented. Figure 6 demonstrates
Figure 4. Comparison of FD20 (gray bars) and CAF (black bars) permeation in intact (fresh and frozen) and intentionally damaged tissue. Data are presented as mean SEM (n ¼ 4–8).
Figure 5. Comparison of FD20 (gray bars) and E2 (black bars) permeation in intact (fresh and frozen) and intentionally damaged tissue. Data are presented as mean SEM (n ¼ 4–7).
that removal of data obtained from tissues through which FD20 permeated resulted in an overall reduction in error. However, when FD20 appearance in the receptor chamber was below 0.58%, the error in CAF permeation did not improve. This indicates that FD20 only acts as a marker of the paracellular route of transport when its permeation exceeds 0.58%. However, this threshold will be reported as 0.6% because such precision (to two decimal places) cannot be guaranteed. Tissue Viability Although the TR index of porcine buccal epithelium appeared to decrease over the first 12 h, there was no significant differences in TR index
Figure 6. Precision (CV%) of CAF permeation data as a function of FD20 permeated (n ¼ 72–84).
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between the first reading (1 h) and any of the readings up to 12 h (Figure 7). However, 24 h postmortem the viability of the tissue had declined significantly when compared to the 1-h postmortem sample (p < 0.05). This indicates that some level of tissue viability can be maintained for up to 12 h in the modified Ussing chambers. The results were reproducible in the two different animals used in this study. As a limit for viability, Imbert et al. used a 50% decrease in TR index from control,13 and as a result, buccal samples were considered viable for up to 20 h postmortem when maintained in KBR. Using this criterion, the buccal epithelium in the present study was viable for up to 12 h, because TR indices were maintained above 0.0059 (50% of control) up to this time point. However, tissue that had been maintained in the Ussing chambers for 24 h and tissue exposed to DMSO for 6 h produced TR indices below 0.0059, values that were significantly different from the 1-h postmortem sample (p < 0.05). Additionally, there was no significant difference in the TR indices produced by fresh (1 h) and frozen tissue. Light micrographs of tissue used in viability studies (Figure 8) demonstrate that the buccal epithelium appeared to have maintained integrity 9 h postmortem and no different to fresh tissue. In both tissues, cells were intact and signs of cell death (cytoplasmic pallor or vacuolization) were not visible. In contrast, light microscopy demon-
Figure 7. TR indices of porcine buccal epithelium maintained in modified Ussing chambers for up to 24 h, exposed to DMSO for 6 h or frozen at 208C for at least 1 month. Data are presented as mean SEM (n ¼ 4). *Indicates that TR index is significantly different (p < 0.05) from TR index of 1 h postmortem sample using Kruskal-Wallis one-way analysis of variance on ranks with Dunnett’s multiple comparison procedure.
Figure 8. Light microscopy of (a) fresh porcine buccal epithelium 1 h postmortem, (b) porcine buccal epithelium 9 h postmortem (maintained in Ussing chambers), and (c) frozen porcine buccal epithelium demonstrating cellular vacuolization and nuclear displacement. All magnification is 200.
strated that there were some morphological changes as a result of tissue storage. Most notably, vacuoles were clearly visible in frozen tissue that may have been a result of ice crystallization.
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DISCUSSION In vitro permeability studies are a useful tool for assessing the potential of a localized anatomical site as a route for drug delivery. It is thus necessary to consider the in vitro conditions and minimize, where possible, artefacts that may not be representative of the in vivo situation. The results from the permeability comparison of epithelial and full thickness tissues demonstrated that the presence of connective tissue significantly reduced CAF and E2 permeation. When a compound traverses a thicker membrane, an increase in lag time is expected. As demonstrated in Figure 2, not only was there an increase in lag time for CAF and E2 transport through full thickness tissue, but also a significant reduction in steady state flux. de Vries et al. also observed this effect with acebutolol and propranolol permeability through porcine buccal mucosa.19 This indicates that connective tissue alters the diffusion characteristics of CAF and E2 in addition to increasing the diffusional pathlength. The difference between full thickness and epithelial permeability was far greater for E2 (16.7-fold) than for CAF (1.8-fold). The greater resistance to permeability observed in full thickness tissue may have been a result of the more hydrophilic nature of the connective tissue, which acts as a greater barrier for lipophilic compounds than for hydrophilic compounds.19 This phenomenon has also been observed in skin penetration studies.20,21 As CAF is a hydrophilic molecule, the presence of connective tissue was not expected to alter permeability; however, these results demonstrate that such assumptions do not always apply. Additionally, the presence of connective tissue in full thickness tissues masked the enhancing effect of SDS, an agent known to enhance oral mucosal permeability.15,16 Because the barrier of the buccal mucosa resides in the upper regions of the epithelium,10,11 and the presence of connective tissue may mask the effects of chemical penetration enhancers, it was concluded that epithelial tissue would serve as a more appropriate model for assessing the in vitro buccal permeability profiles of CAF and E2. Microscopic evaluation of full thickness and epithelial tissues demonstrated a clean removal of epithelial cells from the connective tissue. The epithelium was not strongly attached to the connective tissue making epithelial separation uncomplicated. This may have been a result of the 4-min heat treatment (608C) pigs underwent
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postmortem. Excised buccal mucosa is commonly treated at 60 or 808C to separate epithelium from connective tissue,22–24 so it is expected that the heat treatment pigs underwent postmortem may have created some epithelial separation. To ensure heat treatment did not affect the permeability properties of the buccal mucosa, the permeability of CAF through heat-treated and untreated porcine buccal mucosa was assessed and no differences were observed. Additionally, the heat-treated mucosa was evaluated using light microscopy and no obvious change to tissue morphology was observed. It is assumed that the cells in the lowest layers of the detached epithelium were basal keratinocytes, as indicated by their cuboidal appearance.25 The presence of an undulating surface further supported that these cells would have been in contact with the basement membrane before separation. To accurately identify these lower cells as basal keratinocytes, in situ hybridization and immunohistochemistry could be used to determine the expression of differentiation-specific keratins.26 However, based on light microscopic evaluation and considering that the barrier to drug permeability has been attributed to the upper onethird to one-quarter of the epithelium,10,11 such measures were deemed unnecessary. The effect of tissue storage (208C for up to 1 month) on CAF and E2 permeation was also studied in these experiments. Freezing did not affect the flux or the cumulative amount of CAF or E2 permeating the buccal mucosa, as has been reported with other compounds of similar molecular weight and greater.27,28 In addition, freezing did not affect the integrity of the tissue, as assessed by FD20 permeation (Figures 4 and 5). To assess tissue integrity, FD20 was added to the donor chamber following the completion of CAF and E2 flux measurements. Determining tissue integrity after permeability experiments minimizes any potential interaction between drug and FD20 or interference of FD20 with drug HPLC assays. This method of assessing tissue integrity following permeability experiments has been used for assessing cultured human nasal epithelial integrity with sodium fluorescein29 and Caco-2 membrane integrity with lucifer yellow.30 Studies investigating the permeability of FITC-dextrans have revealed that passage of such hydrophilic compounds through porcine buccal epithelium appears to be through the paracellular route, and is restricted to permeants with a molecular weight less than 20 kDa.9,31,32 Although FD4 and FD10
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permeated porcine buccal mucosa, FD20 and FD40 were too large to permeate the tissue. For this reason, FD20 was chosen as a model nonabsorbable marker. Figure 4 demonstrates that FD20 permeation increased through damaged tissue, which was accompanied by an increase in CAF permeation. In contrast, although there was a large increase in FD20 transport through damaged tissue, there was no corresponding increase in E2 transport (Figure 5). This may be explained by the different pathways that CAF and E2 may follow in permeating the epithelium. CAF may preferentially permeate the buccal epithelium via the polar (or paracellular) route due to its hydrophilic nature. Conversely, E2 may permeate via the nonpolar (or transcellular) route due to its lipophilic character and possibly undergo significant binding and storage within the membrane, as has been observed with other lipophilic compounds.33 By intentionally making holes in the tissue, CAF permeation would be increased because the holes can be likened to an additional polar route. However, the inclusion of additional polar routes (needle holes) did not affect E2 transport, as the polar route would not be its predominant route of transport. This suggests that FD20 is only a valid integrity marker of the polar route, and does not provide information on the integrity of the nonpolar route. The purpose of an integrity marker is to ensure the barrier properties of the tissue are maintained. When the barrier properties of the tissue are compromised, an increase in drug permeability is expected, and the subsequent error associated with a set of drug permeability data would be larger. By excluding drug permeation data on the basis of undue passage of FD20, there should be an overall reduction in variability or error. This is more clearly demonstrated in Figure 6 with CAF permeability error and FD20 permeation. Because FD20 is a large nonabsorbable compound, any degree of absorption should be associated with a large amount of CAF absorption, and consequently, great variability in CAF permeability. However, the variability in CAF permeation data only increased when FD20 appearance in the receptor chamber exceeded 0.6%. This indicates that FD20 only acts as an integrity marker of the polar route above 0.6% and when its appearance in the receptor chamber is below 0.6%, it can be assumed that the polar route integrity has been maintained. However, there can be a partial loss of tissue integrity in the absence of FD20 absorption,
because FD20 is only used to detect gross changes in tissue structure. The barrier properties of the buccal mucosa reside in the top third of the epithelium where the superficial cells, unlike in the stratum corneum, have a variety of functional cell organelles.10–12 Therefore, the viability of the buccal tissue was assessed, so as to gain a clearer understanding on the relationship between viability and permeability. Various methods have been used to assess the viability of excised tissues used in in vitro permeability experiments. It has been reported that the most meaningful method to assess tissue viability is the actual permeability experiment itself, and if the drug permeability does not change during the time course of the experiment, then the tissue is considered viable.34,35 However, tissue death may occur before a permeability experiment begins (especially when tissue transport is required) and so the steady-state profile achieved in the in vitro experiment may be different from that observed in viable tissue. For this reason, great attention was given to assessing the viability of the excised porcine buccal mucosa used in these studies. Histological evaluation of tissue demonstrated that the buccal epithelium appeared viable up to 9 h postmortem, and this was supported by the MTT biochemical assay, where viability was maintained for up to 12 h. Imbert and Cullander have demonstrated that porcine buccal mucosa can be maintained viable for up to 20 h.13 However, in their studies, the buccal tissue was excised and placed in KBR within 5 min of death, and the first MTT incubation occurred within 30 min of death. In the present experiments, buccal tissue was excised and placed in KBR after 20 min of death (due to abattoir postmortem processing) and the first MTT incubation occurred 1 h postmortem. The lag time in immersing the tissue in KBR may have contributed to a more rapid rate of cell death, and this may explain the difference in results. The viability of buccal mucosa was not affected by tissue storage, as assessed by the MTT assay. It, therefore, appears that the activity of tetrazolium reductase is not affected by the freezing process. Enzymes are generally resistant to freezing,36 and so measurement of enzyme function should not be considered as the only method to assess tissue viability. This was demonstrated with the inconsistency between the results from the MTT assay and histological evaluation of frozen tissue. Although the TR index of frozen tissue was not different from fresh tissue, histological evaluation
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demonstrated significant morphological changes in frozen tissue. In frozen specimens, there was a greater degree of cytoplasmic pallor and vacuolization than in fresh tissue. Additionally, some cells in frozen specimens appeared quite swollen with the nucleus compressed to one side of the cell. Overall, freezing appeared to affect structural components of the tissue, but had no effect on biochemical activity. This suggests that the MTT assay should not be used as a sole indicator of tissue viability of frozen specimens, and that a viability assessment should only be made in conjunction with histological consideration. The results from this investigation indicate that viability may not be essential for permeability, because fresh and frozen tissue had similar barrier properties. For the purposes of assessing drug permeability in vitro, however, it is more appropriate to use fresh tissue to avoid artefacts resulting from tissue storage.
CONCLUSIONS The buccal permeability of CAF and E2 was assessed under different in vitro conditions, and the two model compounds demonstrated quite marked differences in their permeability characteristics, as a result of their different physicochemical properties, and therefore, different diffusion pathways. Compared to full thickness buccal tissue, buccal epithelium appeared to be a better model for assessing CAF and E2 permeability, particularly as it is more representative of in vivo conditions. Neither postmortem heat treatment nor freezing of the buccal mucosa affected the barrier properties of the tissue. Although the permeation of FD20 in this in vitro model may be monitored to ensure the integrity of the polar route has not been compromised, it does not provide information on the integrity of the nonpolar route of transport. The MTT biochemical assay can be used to assess the viability of porcine buccal mucosa; however, it should be used in combination with light microscopy to ensure the structural characteristics of the tissue are maintained under different in vitro conditions.
ACKNOWLEDGMENTS The authors would like to thank Mr Ian Boundy (Department of Anatomy and Cell Biology, Monash University, Australia) for his assistance
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in staining and processing of tissue samples for light microscopic studies. Professor Michael Aldred (Dorevitch Pathology, Mayne Health, Australia) is also acknowledged for his advice and assistance in evaluating the tissue samples histologically. The supply of heat-treated porcine buccal tissue from Perfect Pork (Laverton, Australia) and untreated porcine buccal tissue from Meat Research and Training Centre (Victorian Institute of Animal Science, Werribee, Australia) is greatly appreciated. Additionally, Acrux Ltd. (Melbourne, Australia) is acknowledged for financial support.
REFERENCES 1. Rathbone MJ, Hadgraft J. 1991. Absorption of drugs from the human oral cavity. Int J Pharm 74:9–24. 2. de Vries ME, Bodde´ HE, Verhoef JC, Junginger HE. 1991. Developments in buccal drug delivery. Crit Rev Ther Drug Carrier Syst 8:271–303. 3. Shojaei AH, Chang RK, Guo X, Burnside BA, Couch RA. 2001. Systemic drug delivery via the buccal mucosal route. Pharm Tech 25:70–81. 4. Squier CA. 1977. Membrane coating granules in nonkeratinizing oral epithelium. J Ultrastruct Res 60:212–220. 5. Zhang H, Robinson JR. 1996. Routes of drug transport across oral mucosa. In: Rathbone MJ, editor. Oral mucosal drug delivery. New York: Marcel Dekker Inc., pp 51–63. 6. Deneer VHM, Drese GB, Roemele´ PEH, Verhoef JC, Lie-A-Huen L, Kingma JH, Brouwers JRBJ, Junginger HE. 2002. Buccal transport of flecainide and sotalol: Effect of a bile salt and ionization state. Int J Pharm 241:127–134. 7. Gandhi RB, Robinson JR. 1994. Oral cavity as a site for bioadhesive drug delivery. Adv Drug Del Rev 13:43–74. 8. Lesch CA, Squier CA, Cruchley A, Williams DM, Speight P. 1989. The permeability of human oral mucosa and skin to water. J Dent Res 68:1345– 1349. 9. Hoogstraate AJ, Cullander C, Nagelkerke JF, Senel S, Verhoef JC, Junginger HE, Bodde´ HE. 1994. Diffusion rates and transport pathways of fluorescein isothiocyanate (FITC)-labeled model compounds through buccal epithelium. Pharm Res 11:83–89. 10. Squier CA. 1973. The permeability of keratinized and nonkeratinized oral epithelium to horseradish peroxidase. J Ultrastruct Res 43:160–177. 11. Squier CA, Rooney L. 1976. The permeability of keratinized and nonkeratinized oral epithelium to lanthanum in vivo. J Ultrastruct Res 54:286–295.
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12. Wertz PW, Squier CA. 1991. Cellular and molecular basis of barrier function in oral epithelium. Crit Rev Ther Drug Carrier Syst 8:237–269. 13. Imbert D, Cullander C. 1999. Buccal mucosa in vitro experiments I. Confocal imaging of vital staining and MTT assays for the determination of tissue viability. J Controlled Release 58:39–50. 14. Imbert D, Cullander C. 1997. Assessment of cornea viability by confocal laser scanning microscopy and MTT assay. Cornea 16:666–674. 15. Gandhi R, Robinson J. 1992. Mechanisms of penetration enhancement for transbuccal delivery of salicylic acid. Int J Pharm 85:129–140. 16. Healy CM, Cruchley AT, Thornhill MH, Williams DM. 2000. The effect of sodium lauryl sulphate, triclosan and zinc on the permeability of normal oral mucosa. Oral Dis 6:118–123. 17. Mosmann T. 1983. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63. 18. Hoogstraate AJ, Bodde´ HE. 1993. Methods for assessing the buccal mucosa as a route of drug delivery. Adv Drug Del Rev 12:99–125. 19. de Vries ME, Bodde´ HE, Verhoef JC, Ponec M, Crane WIHM, Junginger HE. 1991. Localization of the permeability barrier inside porcine buccal mucosa: A combined in vitro study of drug permeability, electrical resistance and tissue morphology. Int J Pharm 76:25–35. 20. Bronaugh RL, Stewart RF. 1984. Methods for in vitro percutaneous absorption studies III: Hydrophobic compounds. J Pharm Sci 73:1255–1258. 21. Bronaugh RL, Stewart RF. 1986. Methods for in vitro percutaneous absorption studies VI: Preparation of the barrier layer. J Pharm Sci 75:487–491. 22. Squier CA, Cox P, Wertz PW. 1991. Lipid content and water permeability of skin and oral mucosa. J Invest Dermatol 96:123–126. 23. Squier CA, Cox PS, Wertz PW, Downing DT. 1986. The lipid composition of porcine epidermis and oral epithelium. Arch Oral Biol 31:741–747. 24. Law S, Wertz PW, Swartzendruber DC, Squier CA. 1995. Regional variation in content, composition and organization of porcine epithelial barrier lipids revealed by thin-layer chromatography and transmission electron microscopy. Arch Oral Biol 40: 1085–1091. 25. Squier CA, Wertz PW. 1996. Structure and function of the oral mucosa and implications for drug de-
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livery. In: Rathbone MJ, editor. Oral mucosal drug delivery. New York: Marcel Dekker Inc., pp 1–26. Su L, Lane EB, Morgan PR. 1996. Keratin 14 and 19 expression in normal, dysplastic and malignant oral epithelia–A study using in situ hybridization and immunohistochemistry. J Oral Pathol Med 25: 293–301. van der Bijl P, van Eyk AD, Thompson IO, Stander IA. 1998. Diffusion rates of vasopressin through human vaginal and buccal mucosa. Eur J Oral Sci 106:958–962. van der Bijl P, van Eyk AD, Thompson IOC. 1998. Effect of freezing on the permeability of human buccal and vaginal mucosa. S Afr J Sci 94:499– 502. Agu RU, Dang HV, Jorissen M, Willems T, Kinget R, Verbeke N. 2002. Nasal absorption enhancement strategies for therapeutic peptides: An in vitro study using cultured human nasal epithelium. Int J Pharm 237:179–191. Aungst BJ, Nguyen NH, Bulgarelli JP, Oates-Lenz K. 2000. The influence of donor and reservoir additives on Caco-2 permeability and secretory transport of HIV protease inhibitors and other lipophilic compounds. Pharm Res 17:1175–1180. Hoogstraate AJ, Senel S, Cullander C, Verhoef J, Junginger HE, Bodde´ HE. 1996. Effects of bile salts on transport rates and routes of FITC-labelled compounds across porcine buccal epithelium in vitro. J Controlled Release 40:211–221. Junginger HE, Hoogstraate JA, Verhoef JC. 1999. Recent advances in buccal drug delivery and absorption—In vitro and in vivo studies. J Controlled Release 62:149–159. Schu¨rmann W, Turner P. 1978. Membrane model of the human oral mucosa as derived from buccal absorption performance and physicochemical properties of the beta-blocking drugs atenolol and propranolol. J Pharm Pharmacol 30:137–147. Zhang H, Robinson JR. 1996. In vitro methods for measuring permeability of the oral mucosa. In: Rathbone MJ, editor. Oral mucosal drug delivery. New York: Marcel Dekker Inc., pp 85–100. Shojaei AH. 1998. Buccal mucosa as a route for systemic drug delivery: A review. J Pharm Pharmaceut Sci 1:15–30. Mazur P. 1984. Freezing of living cells: mechanisms and implications. Am J Physiol 247:C125– C142.
Received 26 February 2003; revised 6 June 2003; accepted 9 June 2003
ABSTRACT: The effect of various in vitro conditions on the permeability characteristics of the buccal mucosa was assessed using caffeine (CAF) and estradiol (E2) as model hydrophilic and lipophilic markers, respectively. The permeation of CAF and E2 through porcine buccal mucosa was determined in modified Ussing chambers at 378C over 4 h. Comparative permeation studies were performed through full thickness and epithelial tissues, fresh and frozen tissues, and intact and intentionally damaged tissues. Tissue integrity was monitored by the absorption of the normally impermeable fluorescein isothiocyanate (FITC)-labeled dextran 20 kDa (FD20) and tissue viability was assessed using an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) biochemical assay and histological evaluation. Compared to full thickness buccal tissue, permeability through the buccal epithelium was 1.8-fold greater for CAF and 16.7-fold greater for E2. Although the fluxes of the model compounds were no different in fresh and frozen buccal epithelium, histological evaluation demonstrated signs of cellular death in frozen tissue. FD20 permeated damaged tissue, and while this was associated with an increase in CAF transport, no significant change in E2 transport was observed. The tissue appeared to remain viable for up to 12 h postmortem using the MTT viability assay, and this was supported by histological evaluation. ß 2003 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 92:2399–2410, 2003
Keywords: buccal mucosa; permeability; caffeine; estradiol; paracellular; transcellular; tissue integrity; tissue viability
INTRODUCTION Delivery of drugs via the buccal mucosa has several advantages over the traditional oral route that can be exploited.1–3 Like the skin, however, the buccal mucosa acts as a physiological barrier to prevent the absorption of exogenous chemicals and xenobiotics. To most effectively overcome the barrier properties of the mucosa, it is important to be aware of the structure of the tissue and essential to understand the processes involved in drug transport through the tissue.
Correspondence to: Barrie C. Finnin (Telephone: þ61 3 9903 9520; Fax: þ61 3 9903 9583; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 92, 2399–2410 (2003) ß 2003 Wiley-Liss, Inc. and the American Pharmacists Association
Like all stratified squamous epithelia, the buccal mucosa consists of differentiating layers of epithelial cells surrounded by an intercellular lipid matrix. Compared to the lipophilic epithelial cell membranes, the intercellular matrix is relatively hydrophilic, although it has some lipophilic components that are secreted by intracellular membrane coating granules.4 The buccal mucosa therefore consists of hydrophilic and lipophilic regions, and consequently, it has been suggested that the mucosa provides two routes for drug transport—paracellular (between the cells) and transcellular (across the cells).5 Hydrophilic compounds predominantly permeate via the paracellular route due to its relatively hydrophilic nature, whereas lipophilic compounds would permeate via the transcellular route.5,6 An alternative representation of transport routes in the buccal mucosa
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has been suggested, which involves a polar and nonpolar route.7 The nonpolar route involves partitioning of lipophilic molecules into the plasma membrane lipid bilayer or into lipid of the intercellular matrix, and the polar route involves the passage of small hydrophilic molecules through aqueous pores in the plasma membrane of individual cells, or ionic channels in the intercellular spaces.7 Although the polar route can be likened to the paracellular route and the nonpolar route to the transcellular route, this alternative representation does not limit drug transport to only intercellular or intracellular processes. The purpose of this study was to assess the in vitro permeability characteristics of the buccal mucosa using caffeine (CAF) as a model hydrophilic permeant and estradiol (E2) as a model lipophilic permeant. Due to their different physicochemical characteristics, these compounds are expected to be transported across the buccal mucosa via different routes. Therefore, the permeability characteristics of the buccal mucosa under different conditions (full thickness and epithelial tissue, fresh and frozen tissue, intact and damaged tissue) can be assessed in a general manner, because the model permeants have different physicochemical properties, and therefore, different routes of transport. Due to the limited availability of human buccal tissue, porcine buccal mucosa was used for in vitro permeability experiments. Porcine buccal mucosa (a nonkeratinized epithelium) has been suggested as a suitable model membrane because its morphology and permeability are similar to that of human buccal mucosa.8 Integrity markers are often used in in vitro permeability experiments to ensure that the model membrane is intact, and that the observed permeability profiles of model compounds are not a result of compromised tissue integrity. An important component of this study, therefore, was to assess the potential of the high molecular weight fluorescein isothiocyanate (FITC)-labeled dextran 20 kDa (FD20) as a marker of tissue integrity. FD20 was chosen as a marker of tissue integrity because a previous study has revealed that passage of porcine buccal mucosa by FITC-dextrans is restricted to a molecular weight lower than 20 kDa.9 Therefore, appearance of FD20 in the receptor chamber of the in vitro model would be indicative of compromised tissue integrity. When assessing the permeability of compounds through the skin, the issue of viability is often ignored, because the rate-limiting barrier is attributed to the stratum corneum. However, when
assessing the in vitro permeability characteristics of the buccal mucosa, tissue viability may be an important factor to consider. The permeability barrier of the buccal mucosa has been attributed to the top third of the epithelium,10,11 which, unlike the stratum corneum, contains cells with a variety of functional organelles.12 Therefore, it seems appropriate that the viability of excised buccal tissue be assessed, as this may affect the observed permeability results. Consequently, the viability of porcine buccal tissue was determined using histological evaluation and an MTT (3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) biochemical assay that has previously been used in assessing the viability of excised buccal mucosa and cornea.13,14
MATERIALS AND METHODS Materials CAF was obtained from Hunan Pharmaceutical Factory (Hunan, China), and E2 was obtained from AKZO Nobel (Oss, The Netherlands). MTT and FD20 were purchased from Sigma Chemical Co. (St. Louis, MO) and dimethylsulfoxide (DMSO) was purchased from Merck Pty. Limited (Victoria, Australia). Sodium dodecyl sulfate (SDS) was obtained from Boehringer-Mannheim (Germany). Acetonitrile and methanol (Mallinckrodt, KY) were of HPLC grade. Krebs bicarbonate Ringer (KBR) buffer was prepared with 115.5 mM NaCl, 4.2 mM KCl, 21.9 mM NaHCO3, 12.2 mM glucose, 4.0 mM HEPES, 1.2 mM MgSO4 7H2O, 2.5 mM CaCl2 2H2O, and 1.6 mM NaH2PO4 2H2O, and adjusted to pH 7.5 with carbogen (95% O2 þ 5% CO2) bubbling. Water was obtained from a Milli-Q water purification system (Millipore, Milford, MA), and all other chemicals were of analytical grade and were used as received. Tissue Preparation Buccal tissue from domestic pigs (Sus scrofa domestica) was obtained from a local abattoir immediately after slaughter, and was transported in ice-cold KBR. Due to abattoir postmortem processing, pigs underwent a 4-min heat-treatment at 608C, before the buccal tissue could be removed. Therefore, the transport of CAF through this heat-treated mucosa was compared with CAF transport through mucosa obtained from pigs that
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did not undergo postmortem heat-treatment. Additionally, heat-treated mucosa was assessed with light microscopy. For full thickness mucosal studies, buccal mucosa with underlying connective tissue and fat was removed, pinned epithelial side down, and most of the connective tissue and fat removed with surgical scissors. For epithelial studies, the buccal epithelium was carefully separated from the underlying tissues using forceps and surgical scissors. To determine whether the presence of connective tissue alters the overall sensitivity of the tissue to chemical penetration enhancers, both full thickness and epithelial tissues were pretreated with SDS. SDS has been shown to enhance the buccal permeability of compounds,15,16 and therefore, would be expected to affect the permeability in this in vitro model. Consequently, the mucosal surface of buccal tissue was exposed to SDS 1% in KBR for 2 h, CAF transport was monitored over 4 h as detailed below in permeation studies, and the integrity of the tissue was also assessed, as described below in integrity studies. To determine the effect of tissue storage on permeability, buccal epithelium was excised, wrapped in aluminium foil, and stored at 208C in a standard freezer for at least 1 month. On the day of the permeation experiments, the tissue was removed from the freezer, placed in a Petri dish containing KBR for approximately 30 min at room temperature, and then used in permeation experiments. To compare the permeability characteristics of intact and damaged tissue, buccal epithelium was intentionally damaged by placing holes (approximately 5–6 per exposed area) in the epithelium using a Terumo1 25-G needle (Terumo Medical Corporation, Elkton, MD). Light Microscopy To ensure the epithelium had separated from the underlying connective tissue and to assess heat-treated mucosa, epithelial and full thickness tissues were examined with light microscopy. Immediately following excision and/or separation, tissue was placed in 10% buffered formalin. Tissue samples were then dehydrated with increasing concentrations of alcohol, placed in xylene, and embedded in paraffin. Paraffin preparations were then cut to 6-mm thickness using a microtome and stained with hematoxylin and eosin. The prepared slides were examined under a Wild M20 light microscope (Gais, Switzerland). A Spot Jr.
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enhanced digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI) was used to capture images, which were processed by Spot software (Version 2.2, Diagnostic Instruments, Inc., Sterling Heights, MI). Permeation Studies In vitro permeation studies were conducted in modified Ussing chambers (diffusional area 0.64 cm2) maintained at 378C and supplied with carbogen. Porcine buccal mucosa was mounted between the donor and receptor chambers, which were filled with 1.5-mL KBR and allowed to equilibrate for 30 min. For CAF permeation experiments, 100 mL of donor chamber KBR was removed after the equilibration period and replaced with 100 mL of CAF formulation to give a donor concentration of 20 mg/mL (except for full thickness studies, where the donor concentration was 50 mg/mL). For E2 permeation experiments, both donor and receptor chambers were emptied after equilibration and replaced with either E2 saturated suspension (donor) or KBR (receptor). E2 was presented as a saturated suspension due to its poor aqueous solubility (determined to be 2.5 mg/mL in KBR). Samples (100 mL for CAF and 150 mL for E2) were removed from the receptor chamber at periodic intervals over 4 h and replaced with fresh KBR. The results were adjusted to allow for this dilution effect. Samples were assayed for CAF or E2 with HPLC. The cumulative amount of drug permeated was plotted versus time and the steady state flux (Jss) was calculated using eq. 1: Jss ¼
DM A Dt
ð1Þ
where DM is the amount of drug transported across the membrane during time Dt, and A is the diffusional area. In some experiments, the cumulative amount absorbed at 4 h (Q4h) was used for comparison. All experiments were conducted using the buccal mucosa of at least two animals with four to eight replicates and were commenced within 2 h of slaughter. All text results are expressed as mean SD and graphical results are expressed as mean SEM. Integrity Studies FD20 was added to the donor chamber after permeability measurements (donor concentration of approximately 10 mg/mL) and its appearance in
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the receptor chamber was determined after 1 h by fluorescence spectroscopy. The cumulative amount of FD20 absorbed over 1 h was compared in intact and intentionally damaged tissue, as well as in fresh and frozen tissue to determine the effect of freezing on tissue integrity. Analytical Method Concentrations of CAF and E2 were determined using HPLC with a Waters C18 Symmetry1 column (3.9 150 mm) (Waters, Milford, MA). The HPLC system consisted of a Waters 510 HPLC pump, Waters 717 autosampler and Waters 486 tunable absorbance detector. Peak heights were plotted and integrated on a Shimadzu C-R3A Chromatopac integrator (Shimadzu Corporation, Kyoto, Japan). The chromatographic conditions for CAF and E2 are detailed in Table 1. Assay precision (CV%) and accuracy were determined by replicate analyses (n ¼ 6) at the lowest, middle, and highest concentrations validated. These precision and accuracy values are also detailed in Table 1. Concentrations of FD20 were determined using a LS-3 fluorescence spectrometer (Perkin-Elmer Ltd, Norwalk, CT) at an excitation wavelength of 498 nm and emission wavelength of 520 nm. The limit of quantitation for FD20 was 0.01 mg/mL, and precision (CV%) and accuracy values ranged from 0.60 to 11.01% and 98.69 to 100.76%, respectively. Viability Study The MTT assay has been used for quantitative colorimetric measurements of mammalian cell survival and proliferation.17 The original assay
Table 1.
procedure has been modified to assess the viability of tissue specimens rather than cell lines.13,14 MTT is converted in viable cells to formazan (a dark purple water insoluble compound) by enzymes in active mitochondria collectively known as tetrazolium reductase (TR), and the amount of formazan generated is directly proportional to the number of living cells.17 Consequently, the results from the viability assay have been expressed as a TR index (amount of formazan absorbance per mg of tissue). Freshly obtained porcine buccal epithelium was mounted in modified Ussing chambers as in the permeation experiments and at specific time points up to 24 h, the tissue was removed and subjected to the MTT viability assay. MTT was dissolved (2 mg/mL) in freshly prepared phosphate-buffered saline (PBS) and filtered through a Millex1-HA 0.45 mm filter unit (Millipore, Bedford, MA) to remove any undissolved crystals. Biopsy samples (4 mm) of each tissue were obtained at relevant time points using a disposable biopsy punch (Stiefel Laboratories, Germany). The first biopsy sample was taken at 1 h postmortem (quickest feasible time), and this time point was used as the initial viability reading to which all subsequent values were compared. Each biopsy sample was weighed and placed into individual wells of a six-well tissue culture plate (NunclonTM Multidish, Nalge Nunc International, Rochester, NY). Two milliliters of MTT solution was added to each well, and the plate was placed on a rotating platform (100 rpm) at 378C for 2 h. After 2 h, the MTT solution was removed and the tissue was rinsed twice with 1 mL of PBS for 1 min and then minced with surgical scissors. To extract the water-insoluble formazan, 4 mL of DMSO was
Chromatographic Conditions Employed for the Analysis of CAF and E2 Drug
Condition
CAF
E2
Mobile phase Flow rate Injection volume UV detection
Methanol:0.057% H3PO4 (22:78) 1 mL/min 20 mL 273 nm
Assay range Precision (CV%)a Accuracya
0.05–10 mg/mL 0.42–6.35% 100.30–102.59%
Acetonitrile:H2O (40:60) 1 mL/min 100 mL 221 nm (epithelial studies) 277 nm (full thickness studies) 0.02–2 mg/mL 1.02–8.70% 97.89–106.25%
a Precision and accuracy values were determined by replicate analyses (n ¼ 6) at the lowest, middle and highest concentrations validated.
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added to each well and rotated (100 rpm) for 80 min at 378C. The absorbance of formazan was measured at 540 nm (with DMSO as a blank) in a Fluostar Optima microplate reader (BMG Labtechnologies, Offenburg, Germany). The absorbance per mg of tissue was determined and expressed as the TR index. To ensure the assay discriminated between live and dead tissue, tissue was treated for 6 h in DMSO and subjected to the MTT assay. Additionally, the assay was performed in the absence of tissue to ensure no interference and in frozen tissue to assess the effect of freezing on viability. Fresh and frozen tissue, and tissue maintained in the Ussing chambers (for the same length of time as in the MTT viability assay) were also evaluated using light microscopy.
RESULTS Tissue Preparation In initial experiments, the permeation of CAF was compared through buccal mucosa from heattreated and untreated pigs to ensure heat treatment did not affect the barrier properties of the buccal mucosa. The % of applied dose of CAF absorbed over 4 h through heat-treated and untreated porcine buccal mucosa was 10.47 0.60% and 9.00 1.99%, respectively. Additionally, light microscopy revealed no obvious effects on mucosal histology (Figure 1a). Figure 1 also contains the photomicrographs of the buccal epithelium following surgical detachment from the underlying connective tissue. Light microscopy revealed that there was a clear separation of the epithelium from the connective tissue (which appeared intact in full thickness tissue) and that the lowermost layer contained cells that were cuboidal, indicative of basal cells. The undulated appearance of this lower layer was indicative of the junction between the epithelium and lamina propria.18 Permeation Studies The permeability profiles of CAF and E2 through full thickness and epithelial tissues are shown in Figure 2. The cumulative amount of CAF absorbed over 4 h through epithelium was 1.8-fold greater than through full thickness mucosa, with Q4h values of 17.41 1.77 and 9.46 1.63 mg/cm2, respectively ( p < 0.001). When comparing the flux values for each tissue, Jss was 4.68 0.49 mg/cm2/h
Figure 1. Light microscopic evaluation of (a) heattreated porcine full thickness buccal mucosa and (b) heat-treated porcine buccal epithelium after separation from underlying connective tissue (magnification 100). Figure (c) is a high magnification (200) micrograph of the detached epithelium demonstrating the cuboidal shape of the lowest layer of cells and the undulating nature of the basal area.
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Figure 2. Permeation of (a) CAF and (b) E2 through porcine full thickness buccal mucosa (*) and epithelium (*). Data are presented as mean SEM (n ¼ 6).
for epithelium and 3.16 0.26 mg/cm2/h for full thickness tissue ( p < 0.001). A significant ( p < 0.001) difference in lag time was also observed for CAF transport through full thickness tissue (lag time of 1.11 0.24 h) compared with epithelial transport (lag time of 0.41 0.04 h). With E2 transport studies, the difference in full thickness and epithelial permeation was more marked with Q4h values of 0.05 0.12 mg/cm2 (full thickness) and 0.80 0.20 mg/cm2 (epithelium), a 16.7-fold difference. A steady state profile was not achieved for E2 through full thickness mucosa and so steady-state rates could not be compared. The results obtained were very reproducible as intraanimal variation ranged from 6.6–20.2% for CAF permeation and 13.5–20.8% for E2 permeation, and no significant differences were observed with t-tests comparing interanimal data ( p ¼ 0.901 for CAF and p ¼ 0.354 for E2). As demonstrated in Figure 3a, pretreating epithelial tissue with SDS 1% increased CAF flux
Figure 3. Permeability of CAF through (a) porcine buccal epithelium and (b) porcine full thickness buccal mucosa with (*) and without (*) a 2 h pretreatment with SDS 1% w/v in KBR. Data are presented as mean SEM (n ¼ 4–6). *Indicates CAF flux with SDS pretreatment is significantly greater than with no pretreatment (p < 0.001).
from 1.34 0.26 mg/cm2/h to 2.37 0.41 mg/cm2/h (p < 0.001). However, the permeability of CAF was not significantly improved when full thickness tissue was pretreated with SDS. This indicates that the enhancing effect of SDS was negated by the presence of connective tissue in full thickness specimens. Based on these results and the fact that in vivo, blood vessels are directly beneath the epithelium, all further studies were performed using epithelial tissue. The integrity of the polar route of permeation was not affected following SDS pretreatment, as FD20 permeation was well below the threshold indicative of such damage (as discussed in tissue integrity). The Q4h and steady state flux values (Jss) for CAF and E2 through fresh and frozen porcine buccal epithelium are listed in Table 2. Storage of
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Table 2. Q4h and Flux Values for CAF and E2 through Fresh and Frozen Buccal Epithelium Compound Condition Flux (mg/cm2/h) Q4h (mg/cm2) CAF E2
Fresh Frozen Fresh Frozen
1.75 0.25 1.66 0.11 0.41 0.09 0.52 0.06
6.29 0.10 6.13 0.71 1.36 0.36 1.55 0.21
Data are presented as mean SD (n ¼ 4–6).
porcine buccal epithelium at 208C for at least 1 month did not affect the permeation of either model compound. Tissue Integrity In Figure 4, the cumulative amount of FD20 permeating the buccal epithelium over 1 h is compared with the cumulative amount of CAF permeated over 4 h in intact fresh and frozen tissue, and intentionally damaged tissue. Figure 5 shows a comparison between FD20 permeation and E2 transport in the similarly treated tissues. Although there was an increase in CAF permeation associated with increased FD20 appearance in the receptor chamber, a similar result was not observed with E2 permeation. To further investigate the validity of FD20 as an integrity marker, the error associated with CAF permeation data (Q4h) was compared with and without consideration of FD20 permeation. Because FD20 was not a valid marker for the route of permeation taken by E2, a similar data analysis for E2 error is not presented. Figure 6 demonstrates
Figure 4. Comparison of FD20 (gray bars) and CAF (black bars) permeation in intact (fresh and frozen) and intentionally damaged tissue. Data are presented as mean SEM (n ¼ 4–8).
Figure 5. Comparison of FD20 (gray bars) and E2 (black bars) permeation in intact (fresh and frozen) and intentionally damaged tissue. Data are presented as mean SEM (n ¼ 4–7).
that removal of data obtained from tissues through which FD20 permeated resulted in an overall reduction in error. However, when FD20 appearance in the receptor chamber was below 0.58%, the error in CAF permeation did not improve. This indicates that FD20 only acts as a marker of the paracellular route of transport when its permeation exceeds 0.58%. However, this threshold will be reported as 0.6% because such precision (to two decimal places) cannot be guaranteed. Tissue Viability Although the TR index of porcine buccal epithelium appeared to decrease over the first 12 h, there was no significant differences in TR index
Figure 6. Precision (CV%) of CAF permeation data as a function of FD20 permeated (n ¼ 72–84).
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between the first reading (1 h) and any of the readings up to 12 h (Figure 7). However, 24 h postmortem the viability of the tissue had declined significantly when compared to the 1-h postmortem sample (p < 0.05). This indicates that some level of tissue viability can be maintained for up to 12 h in the modified Ussing chambers. The results were reproducible in the two different animals used in this study. As a limit for viability, Imbert et al. used a 50% decrease in TR index from control,13 and as a result, buccal samples were considered viable for up to 20 h postmortem when maintained in KBR. Using this criterion, the buccal epithelium in the present study was viable for up to 12 h, because TR indices were maintained above 0.0059 (50% of control) up to this time point. However, tissue that had been maintained in the Ussing chambers for 24 h and tissue exposed to DMSO for 6 h produced TR indices below 0.0059, values that were significantly different from the 1-h postmortem sample (p < 0.05). Additionally, there was no significant difference in the TR indices produced by fresh (1 h) and frozen tissue. Light micrographs of tissue used in viability studies (Figure 8) demonstrate that the buccal epithelium appeared to have maintained integrity 9 h postmortem and no different to fresh tissue. In both tissues, cells were intact and signs of cell death (cytoplasmic pallor or vacuolization) were not visible. In contrast, light microscopy demon-
Figure 7. TR indices of porcine buccal epithelium maintained in modified Ussing chambers for up to 24 h, exposed to DMSO for 6 h or frozen at 208C for at least 1 month. Data are presented as mean SEM (n ¼ 4). *Indicates that TR index is significantly different (p < 0.05) from TR index of 1 h postmortem sample using Kruskal-Wallis one-way analysis of variance on ranks with Dunnett’s multiple comparison procedure.
Figure 8. Light microscopy of (a) fresh porcine buccal epithelium 1 h postmortem, (b) porcine buccal epithelium 9 h postmortem (maintained in Ussing chambers), and (c) frozen porcine buccal epithelium demonstrating cellular vacuolization and nuclear displacement. All magnification is 200.
strated that there were some morphological changes as a result of tissue storage. Most notably, vacuoles were clearly visible in frozen tissue that may have been a result of ice crystallization.
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DISCUSSION In vitro permeability studies are a useful tool for assessing the potential of a localized anatomical site as a route for drug delivery. It is thus necessary to consider the in vitro conditions and minimize, where possible, artefacts that may not be representative of the in vivo situation. The results from the permeability comparison of epithelial and full thickness tissues demonstrated that the presence of connective tissue significantly reduced CAF and E2 permeation. When a compound traverses a thicker membrane, an increase in lag time is expected. As demonstrated in Figure 2, not only was there an increase in lag time for CAF and E2 transport through full thickness tissue, but also a significant reduction in steady state flux. de Vries et al. also observed this effect with acebutolol and propranolol permeability through porcine buccal mucosa.19 This indicates that connective tissue alters the diffusion characteristics of CAF and E2 in addition to increasing the diffusional pathlength. The difference between full thickness and epithelial permeability was far greater for E2 (16.7-fold) than for CAF (1.8-fold). The greater resistance to permeability observed in full thickness tissue may have been a result of the more hydrophilic nature of the connective tissue, which acts as a greater barrier for lipophilic compounds than for hydrophilic compounds.19 This phenomenon has also been observed in skin penetration studies.20,21 As CAF is a hydrophilic molecule, the presence of connective tissue was not expected to alter permeability; however, these results demonstrate that such assumptions do not always apply. Additionally, the presence of connective tissue in full thickness tissues masked the enhancing effect of SDS, an agent known to enhance oral mucosal permeability.15,16 Because the barrier of the buccal mucosa resides in the upper regions of the epithelium,10,11 and the presence of connective tissue may mask the effects of chemical penetration enhancers, it was concluded that epithelial tissue would serve as a more appropriate model for assessing the in vitro buccal permeability profiles of CAF and E2. Microscopic evaluation of full thickness and epithelial tissues demonstrated a clean removal of epithelial cells from the connective tissue. The epithelium was not strongly attached to the connective tissue making epithelial separation uncomplicated. This may have been a result of the 4-min heat treatment (608C) pigs underwent
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postmortem. Excised buccal mucosa is commonly treated at 60 or 808C to separate epithelium from connective tissue,22–24 so it is expected that the heat treatment pigs underwent postmortem may have created some epithelial separation. To ensure heat treatment did not affect the permeability properties of the buccal mucosa, the permeability of CAF through heat-treated and untreated porcine buccal mucosa was assessed and no differences were observed. Additionally, the heat-treated mucosa was evaluated using light microscopy and no obvious change to tissue morphology was observed. It is assumed that the cells in the lowest layers of the detached epithelium were basal keratinocytes, as indicated by their cuboidal appearance.25 The presence of an undulating surface further supported that these cells would have been in contact with the basement membrane before separation. To accurately identify these lower cells as basal keratinocytes, in situ hybridization and immunohistochemistry could be used to determine the expression of differentiation-specific keratins.26 However, based on light microscopic evaluation and considering that the barrier to drug permeability has been attributed to the upper onethird to one-quarter of the epithelium,10,11 such measures were deemed unnecessary. The effect of tissue storage (208C for up to 1 month) on CAF and E2 permeation was also studied in these experiments. Freezing did not affect the flux or the cumulative amount of CAF or E2 permeating the buccal mucosa, as has been reported with other compounds of similar molecular weight and greater.27,28 In addition, freezing did not affect the integrity of the tissue, as assessed by FD20 permeation (Figures 4 and 5). To assess tissue integrity, FD20 was added to the donor chamber following the completion of CAF and E2 flux measurements. Determining tissue integrity after permeability experiments minimizes any potential interaction between drug and FD20 or interference of FD20 with drug HPLC assays. This method of assessing tissue integrity following permeability experiments has been used for assessing cultured human nasal epithelial integrity with sodium fluorescein29 and Caco-2 membrane integrity with lucifer yellow.30 Studies investigating the permeability of FITC-dextrans have revealed that passage of such hydrophilic compounds through porcine buccal epithelium appears to be through the paracellular route, and is restricted to permeants with a molecular weight less than 20 kDa.9,31,32 Although FD4 and FD10
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permeated porcine buccal mucosa, FD20 and FD40 were too large to permeate the tissue. For this reason, FD20 was chosen as a model nonabsorbable marker. Figure 4 demonstrates that FD20 permeation increased through damaged tissue, which was accompanied by an increase in CAF permeation. In contrast, although there was a large increase in FD20 transport through damaged tissue, there was no corresponding increase in E2 transport (Figure 5). This may be explained by the different pathways that CAF and E2 may follow in permeating the epithelium. CAF may preferentially permeate the buccal epithelium via the polar (or paracellular) route due to its hydrophilic nature. Conversely, E2 may permeate via the nonpolar (or transcellular) route due to its lipophilic character and possibly undergo significant binding and storage within the membrane, as has been observed with other lipophilic compounds.33 By intentionally making holes in the tissue, CAF permeation would be increased because the holes can be likened to an additional polar route. However, the inclusion of additional polar routes (needle holes) did not affect E2 transport, as the polar route would not be its predominant route of transport. This suggests that FD20 is only a valid integrity marker of the polar route, and does not provide information on the integrity of the nonpolar route. The purpose of an integrity marker is to ensure the barrier properties of the tissue are maintained. When the barrier properties of the tissue are compromised, an increase in drug permeability is expected, and the subsequent error associated with a set of drug permeability data would be larger. By excluding drug permeation data on the basis of undue passage of FD20, there should be an overall reduction in variability or error. This is more clearly demonstrated in Figure 6 with CAF permeability error and FD20 permeation. Because FD20 is a large nonabsorbable compound, any degree of absorption should be associated with a large amount of CAF absorption, and consequently, great variability in CAF permeability. However, the variability in CAF permeation data only increased when FD20 appearance in the receptor chamber exceeded 0.6%. This indicates that FD20 only acts as an integrity marker of the polar route above 0.6% and when its appearance in the receptor chamber is below 0.6%, it can be assumed that the polar route integrity has been maintained. However, there can be a partial loss of tissue integrity in the absence of FD20 absorption,
because FD20 is only used to detect gross changes in tissue structure. The barrier properties of the buccal mucosa reside in the top third of the epithelium where the superficial cells, unlike in the stratum corneum, have a variety of functional cell organelles.10–12 Therefore, the viability of the buccal tissue was assessed, so as to gain a clearer understanding on the relationship between viability and permeability. Various methods have been used to assess the viability of excised tissues used in in vitro permeability experiments. It has been reported that the most meaningful method to assess tissue viability is the actual permeability experiment itself, and if the drug permeability does not change during the time course of the experiment, then the tissue is considered viable.34,35 However, tissue death may occur before a permeability experiment begins (especially when tissue transport is required) and so the steady-state profile achieved in the in vitro experiment may be different from that observed in viable tissue. For this reason, great attention was given to assessing the viability of the excised porcine buccal mucosa used in these studies. Histological evaluation of tissue demonstrated that the buccal epithelium appeared viable up to 9 h postmortem, and this was supported by the MTT biochemical assay, where viability was maintained for up to 12 h. Imbert and Cullander have demonstrated that porcine buccal mucosa can be maintained viable for up to 20 h.13 However, in their studies, the buccal tissue was excised and placed in KBR within 5 min of death, and the first MTT incubation occurred within 30 min of death. In the present experiments, buccal tissue was excised and placed in KBR after 20 min of death (due to abattoir postmortem processing) and the first MTT incubation occurred 1 h postmortem. The lag time in immersing the tissue in KBR may have contributed to a more rapid rate of cell death, and this may explain the difference in results. The viability of buccal mucosa was not affected by tissue storage, as assessed by the MTT assay. It, therefore, appears that the activity of tetrazolium reductase is not affected by the freezing process. Enzymes are generally resistant to freezing,36 and so measurement of enzyme function should not be considered as the only method to assess tissue viability. This was demonstrated with the inconsistency between the results from the MTT assay and histological evaluation of frozen tissue. Although the TR index of frozen tissue was not different from fresh tissue, histological evaluation
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demonstrated significant morphological changes in frozen tissue. In frozen specimens, there was a greater degree of cytoplasmic pallor and vacuolization than in fresh tissue. Additionally, some cells in frozen specimens appeared quite swollen with the nucleus compressed to one side of the cell. Overall, freezing appeared to affect structural components of the tissue, but had no effect on biochemical activity. This suggests that the MTT assay should not be used as a sole indicator of tissue viability of frozen specimens, and that a viability assessment should only be made in conjunction with histological consideration. The results from this investigation indicate that viability may not be essential for permeability, because fresh and frozen tissue had similar barrier properties. For the purposes of assessing drug permeability in vitro, however, it is more appropriate to use fresh tissue to avoid artefacts resulting from tissue storage.
CONCLUSIONS The buccal permeability of CAF and E2 was assessed under different in vitro conditions, and the two model compounds demonstrated quite marked differences in their permeability characteristics, as a result of their different physicochemical properties, and therefore, different diffusion pathways. Compared to full thickness buccal tissue, buccal epithelium appeared to be a better model for assessing CAF and E2 permeability, particularly as it is more representative of in vivo conditions. Neither postmortem heat treatment nor freezing of the buccal mucosa affected the barrier properties of the tissue. Although the permeation of FD20 in this in vitro model may be monitored to ensure the integrity of the polar route has not been compromised, it does not provide information on the integrity of the nonpolar route of transport. The MTT biochemical assay can be used to assess the viability of porcine buccal mucosa; however, it should be used in combination with light microscopy to ensure the structural characteristics of the tissue are maintained under different in vitro conditions.
ACKNOWLEDGMENTS The authors would like to thank Mr Ian Boundy (Department of Anatomy and Cell Biology, Monash University, Australia) for his assistance
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in staining and processing of tissue samples for light microscopic studies. Professor Michael Aldred (Dorevitch Pathology, Mayne Health, Australia) is also acknowledged for his advice and assistance in evaluating the tissue samples histologically. The supply of heat-treated porcine buccal tissue from Perfect Pork (Laverton, Australia) and untreated porcine buccal tissue from Meat Research and Training Centre (Victorian Institute of Animal Science, Werribee, Australia) is greatly appreciated. Additionally, Acrux Ltd. (Melbourne, Australia) is acknowledged for financial support.
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