Confocal Laser Scanning Microscopic Visualization of the Transport of Dextrans After Nasal Administration to Rats: Effects of Absorption Enhancers

Purpose. To visualize the transport pathway(s) of high molecular weight model compounds across rat nasal epithelium in vivousing confocal laser scanning microscopy. Furthermore, the influence of nasal absorption enhancers (randomly methylated -cyclodextrin and sodium taurodihydrofusidate) on this transport was studied. Methods. Fluorescein isothiocyanate (FITC)-labelled dextrans with a molecular weight of 3,000 or 10,000 Da were administered intranasally to rats. Fifteen minutes after administration the tissue was fixed with Bouin. The nasal septum was surgically removed and stained with Evans Blue protein stain or DiIC18(5) lipid stain prior to visualization with the confocal laser scanning microscope. Results. Transport of FITC-dextran 3,000 across nasal epithelium occurred via the paracellular pathway. Endocytosis of FITC-dextran 3,000 was also shown. In the presence of randomly methylated -cyclodextrin 2% (w/v) similar transport pathways for FITC-dextran 3,000 were observed. With sodium taurodihydrofusidate 1% (w/v) the transport route was also paracellular with endocytosis, but cells were swollen and mucus was extruded into the nasal cavity. For FITC-dextran 10,000 hardly any transport was observed without enhancer, or after co-administration with randomly methylated -cyclodextrin 2% (w/v). Co-administration with sodium taurodihydrofusidate 1% (w/v) resulted in paracellular transport of FITC-dextran 10,000, but morphological changes, i.e. swelling of cells and mucus extrusion, were observed. Conclusions. Confocal laser scanning microscopy is a suitable approach to visualize the transport pathways of high molecular weight hydrophilic compounds across nasal epithelium, and to study the effects of absorption enhancers on drug transport and cell morphology.     

Diffusion Rates and Transport Pathways of Fluorescein Isothiocyanate (FITC)-Labeled Model Compounds Through Buccal Epithelium

The aim of this study was to characterize transport of FITC-labeled dextrans of different molecular weights as model compounds for peptides and proteins through buccal mucosa. The penetration of these dextrans through porcine buccal mucosa (a nonkeratinized epithelium, comparable to human buccal mucosa) was investigated by measuring transbuccal fluxes and by analyzing the distribution of the fluorescent probe in the epithelium, using confocal laser scanning microscopy for visualizing permeation pathways. The results revealed that passage of porcine buccal epithelium by hydrophilic compounds such as the FITC-dextrans is restricted to permeants with a molecular weight lower than 20 kDa. The permeabilities of buccal mucosa for the 4- and 10-kDa FITC-dextran (of the order of 10^–8 cm/sec) were not significantly different from each other or from the much smaller compound FITC. The confocal images of the distribution pattern of FITC-dextrans showed that the paracellular route is the major pathway through buccal epithelium.     

A Cross-Section Device To Improve Visualization of Fluorescent Probe Penetration into the Skin by Confocal Laser Scanning Microscopy

Pharmaceutical Research -     

Iontophoresis of Poly-L-lysines: The Role of Molecular Weight?

Purpose. (1) To determine the extent of iontophoretic transport as a function of molecular weight (MW) of the penetrant; and (2) to visually and quantitatively characterize the iontophoretic transport pathways (follicular (F) versus nonfollicular (NF)) of the fluorescently-labeled poly-L-lysines employed. Methods. A series of fluorescently-labeled poly-L-lysines (FITC-PLLs) [4 KDa, 7 KDa and 26 KDa] were used to study the extent and distribution of iontophoretic skin penetration as a function of MW using laser scanning confocal microscopy (LSCM). Results. It was found that, relative to the passive controls, and under the electrical conditions considered, iontophoresis greatly enhanced the penetration of the 4 KDa analog, slightly elevated the delivery of the 7 KDa FITC-PLL, but had no effect on the transport of the larger 26 KDa FITC-PLL. Quantitative analyses of LSCM images revealed that iontophoresis increased transport via F pathways only slightly more than that through NF pathways for the 4 KDa and 7 KDa FITC-PLL molecules. Conclusions. It is visually apparent that the iontophoretic transport pathways taken are importantly determined by the physicochemical properties (including size and charge) of the penetrant. The results presented here demonstrate an inverse dependence of iontophoretic delivery upon the MW of the penetrant.     

Buccal mucosa in vitro experiments. I. Confocal imaging of vital staining and MTT assays for the determination of tissue viability.

Delivery of drugs through the skin and the buccal mucosa has been considered as an alternative to per oral dosing for those substances that are degraded in the gastro-intestinal tract, or are subject to first-pass metabolism in the liver. In the buccal mucosa, contrary to skin, the diffusion barriers are located within living cell layers, hence the physiological state of the tissue is likely to significantly affect in vitro diffusion profiles. In this study, we were interested in assessing the viability of excised buccal mucosa and determining the limits of tissue usage under common in vitro experimental conditions. Using confocal laser scanning microscopy (CLSM), we have shown that optical sectioning of samples exposed to calcein AM and ethidium homodimer-1 (used as 'live' and 'dead' cell probes respectively) can be employed to accurately and reliably determine the viability of buccal mucosa biopsies. The results of the CLSM assay were remarkably consistent with that of an MTT assay. In both studies, viability in PBS at 34 degrees C was lost after about 8 h post-mortem, whereas it could be sustained for up to 24 h in KBR.     

Rapid detection of spatial pattern by Fourier analysis

Fast Fourier transform analysis can be used to accurately and rapidly detect pattern in large two-dimensional arrangements of points, such as the locations of cells in culture or plants in a unit square. We present here a sample study of pattern in spatial random point processes. This is evidently the first time that Fourier transform-based cross-correlation techniques have been applied to the analysis of point processes of biological origin. Radial profiles of the power spectra and autocorrelation estimates revealed a nearly constant interpore distance of 0.49 +/- 0.04 mm in the locations of eccrine gland pores on the surface of human skin. Additionally, gland-free areas may exist near hair follicles.     

Sites of iontophoretic current flow into the skin: identification and characterization with the vibrating probe electrode

The routes taken by charged substances (e.g., peptides) through the skin during iontophoretic drug delivery are not well characterized. We have used a vibrating probe electrode to reproducibly identify and vectorize site-specific (spatial resolution = 20 microns) ionic flows as they were occurring in hairless mouse skin clamped at clinically relevant current densities. These iontophoretic currents were primarily appendageal, and certain appendages (e.g., small hairs) appeared to carry most of the current. This finding may have important ramifications with respect to irritation, allergic reaction, and electrical current damage in iontophoretic drug delivery. The size and direction of the current vectors could change under certain conditions (e.g., in an unbuffered preparation, where pH changes occurred during the experiment). The vibrating probe can operate in (and is not adversely affected by) the ranges of pH, tonicity, and current required for the study of iontophoretic currents.     

Effects of bile salts on transport rates and routes of FITC-labelled compounds across porcine buccal epithelium in vitro

In this study the penetration enhancing effect of bile salts on the transport of hydrophilic macromolecular compounds across porcine buccal mucosa was investigated in-vitro. Coadministration of 100 mM of the trihydroxy bile salts sodium glycocholate (GC) and sodium taurocholate (TC) and the dihydroxy bile salts sodium glycodeoxycholate (GDC) and sodium taurodeoxycholate (TDC) increased the in-vitro transport of fluorescein isothiocyanate (FITC) by a factor of a hundred or more, without a significant difference between the four bile salts. The concentration dependence of the enhancing effect of GDC was studied using FITC-labelled dextrans of increasing molecular weight as permeants (FD4, MW 4400; FD10, MW 9400; FD20, MW 19 600). The maximal enhancement was observed when GDC was coadministered in a concentration of 10 mM, resulting in an enhancement ratio of about 2000 for FD4. Using confocal laser scanning microscopy the effects of bile salts on the penetration pathways of hydrophilic compounds were investigated. The uniform distribution of FITC throughout the epithelium was changed by coadministration of 100 mM of bile salt to an increased amount of the fluorescent probe present in the intercellular domains. The intercellular distribution of both FD4 and FD10 was not changed by a low, but effective, concentration of GDC (2 mM, enhancement ratio of 72 for FD4). Increasing the concentration of GDC to 10 and 100 mM resulted in uptake of the fluorescent probe in the epithelial cells. From these results we conclude that the di- and trihydroxy bile salts studied increase the transport of hydrophilic compounds across buccal epithelium in vitro, below 10 mM by increasing the intercellular transport and at 10 mM and higher concentrations by opening up a transcellular route.     

A New System for In Vitro Studies of Iontophoresis

This report describes a new iontophoretic diffusion cell that allows both electrodes to be applied to the same side of the same piece of skin. The cell permits a better approximation of the in vivo situation than do conventional side-by-side cells. The unique construction of the cell allows nonliquid material to be applied to the skin surface and makes it possible to investigate horizontal transport paths. Preliminary results utilizing the cell are described. Iontophoretic enhancement of morphine and clonidine delivery across full-thickness hairless mouse skin has been achieved. The importance of pH control in these experiments is apparent. Further experiments with morphine indicate that, for this drug at least, iontophoretically driven lateral transport within the skin is unimportant. Because the cell design allows significant parallels to the use of iontophoresis in vivo, we suggest that it will prove to be a useful tool in the determination of fundamental structure/transport relationships under the influence of an externally applied current.     

Blockage of skin invasion by schistosome cercariae by serine protease inhibitors.

Invasion of skin by schistosome cercariae is facilitated by a serine protease secreted from the acetabular cells of cercariae in response to skin lipid. Specific inhibitors of the protease, when applied to human skin in formulations designed to retain the inhibitor on and in the upper stratum corneum layers, block cercarial invasion of human skin. Both peptide-based, irreversible inhibitors and non-peptide, reversible inhibitors block cercarial invasion when applied in a propylene glycol:isopropyl alcohol (3:1) formulation in vitro. Arrest of cercarial invasion could be achieved even after immersion of treated skin in water for 2 hr. Peptide-based irreversible inhibitors in the presence of three different Topicare Delivery Compounds optimized arrest of cercarial invasion. The three Topicare Delivery Compounds applied alone prevented 80-100% of cercarial invasion. With inclusion of the inhibitor, there was 97-100% inhibition in vitro. The optimal formulation with inhibitor was then applied to the tails of BALB/c mice, and the mice were exposed to 120 cercariae by tail immersion. With the carrier lotion alone, there was a 50% reduction in worm burden and a 70% reduction in egg burden. When inhibitor was included, an 80% reduction in worm burden and a 92% reduction in egg burden was observed.