Lack of Effect of Ammonium Glycyrrhizinate on the Morphology of Ovine Nasal Mucosa in Vitro

Glycyrrhetinic acid derivatives are reported to be nasal absorption promoters (1). Effects of ammonium glycyrrhizinate (AMGZ) on the in vitro morphology of ovine nasal mucosa were therefore examined by light and electron microscopy. Nasal mucosa was stripped from the submucosa and mounted in Ussing chambers. Exposure of the apical surface to 2% ammonium glycyrrhizinate (24 mM) for 90 min caused no histopathological changes to the nasal epithelium. Epithelial integrity remained intact as evidenced by the continued presence of morphologically intact junctional complexes. No sloughing of the epithelial layer from the basement membrane was observed, and cilia and microvilli were not affected by treatment with AMGZ. The results indicate that short-term exposure in vitro to ammonium glycyrrhizinate caused no overt morphological damage to ovine nasal mucosa.     

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.     

Optimization of Spray-Dried and -Congealed Lipid Micropellets and Characterization of Their Surface Morphology by Scanning Electron Microscopy

Lipid micropellets prepared from glycerides and phospholipids may be a physiological drug carrier system for improving the intestinal absorption of lipophilic drugs. They can be prepared by spray-drying and spray-congealing processes. In this study, formulation and optimization parameters of spray-dried and -congealed lipid pellets in the micro- and nanometer size were investigated. The rapid evaporation of solvents from the droplets, which in turn causes a rapid solidification, influenced the crystalline structures of spray-dried glycerides. Moreover, solvents, the chain length, and the type of lipids and drugs used in the formulations of spray-dried micropellets affected the surface morphology of the micropellets. In contrast to the variations of the surface structure of spray-dried micropellets, formulated spray-congealed micropellets possessed smooth surface properties. The surface morphology and microstructure of both types of micropellets were characterized by SEM.     

Effects of Absorption Enhancers on Rat Nasal Epithelium in Vivo: Release of Marker Compounds in the Nasal Cavity

Purpose. The assessment of the effects of nasal absorption enhancers on the rat nasal epithelium and membrane permeability in vivo after a single nasal dose of the enhancers. Methods. The release of marker compounds (protein, cholesterol and acid phosphatase) from the nasal epithelium was measured using a lavage technique. The nasal membrane permeability was determined after intravenous administration of a systemic tracer (FITC-albumin). Results. The effects of the absorption enhancers could be classified into four categories. The first consisted of HPCD (5%), DMCD (2%) and RAMEB (2%) and was not different from the control (physiological saline). For the second category, DMCD (5%), effects were significantly higher than for the control. The third category, SGC (1%), was more active than DMCD (5%) but less active than the last group. The fourth, most membrane damaging, category consisted of STDHF (1%), laureth-9 (1%) and LPC (1%). Administration of these three enhancers also resulted in release of acid phosphatase, indicating that severe membrane damage occurred. The release of cholesterol from nasal epithelium was largely dependent on the cholesterol solubilisation of the absorption enhancers. The amount of cholesterol released by laureth-9 and LPC was the largest. Conclusions. The results of this in vivo study are in agreement (i.e. similarity in rank order) with morphological and ciliotoxicity studies of nasal absorption enhancers, demonstrating that this in vivo model is a valuable tool to classify nasal absorption enhancers according to their effects on the rat nasal epithelium.     

Formation of Multilayers in the Caco-2 Cell Culture Model: A Confocal Laser Scanning Microscopy Study

Purpose. To introduce confocal laser scanning microscopy (CLSM)combined with digital image restoration to characterise Caco-2 cellsunder different culture conditions, and thus to define additional validcriteria for the optimisation of culture models. Methods. Growth curves were established and transepithelial electricalresistance (TEER) measured for cells grown in EMEM or DMEMmedium on Cyclopore membranes. Cytoskeleton, cell nuclei and tightjunctions (TJ) were investigated by CLSM. Results. Cultures reached a plateau of 4.5 × 10⁵ cells/cm² after 10 days. At the same time TEER reached 750 cm². An irregular,fairly complete network of TJ was present at confluence (2 d).Between 15 and 30 days a regular TJ network was established. Cellsformed mixed mono- and multilayers under most conditions with twoexceptions: flat monolayers were observed on polycarbonate filterswith EMEM and with the Biocoat intestinal epithelium differentiationenvironment system. In multilayers TJ were found in the upper aswell as in the lower cell layers although the regular vertical polaritywas disturbed. Conclusions. CLSM represents an important tool to investigate thecytoarchitecture of Caco-2 cells. 3D-analysis of confocal data givesimportant clues on the characteristics of cell layers and thus helps tovalidate optimisation strategies.     

Development of Octreotide-Loaded Chitosan and Heparin Nanoparticles: Evaluation of Surface Modification Effect on Physicochemical Properties and Macrophage Uptake

Octreotide (OCT) is a therapeutic peptide which is administered for the treatment of acromegaly. The purpose of this study was to design a new polyethylene glycol (PEG)–conjugated nanoparticle (PEG-NP) to overcome the short half-life and poor stability of OCT. The developed PEG-NPs were compared with non-PEGylated NPs with respect to their size, morphological characteristics, loading efficiency, release profile, and macrophage uptake. The OCT-loaded NPs and PEG-NPs were prepared by ionic complexion of chitosan (Cs) with either heparin (Hp) or PEGylated heparin (PEG-Hp). The chemical structure of PEG-Hp was confirmed by IR and proton nuclear magnetic resonance. Morphological analyses by scanning electron microscopy showed that NPs and PEG-NPs have a uniform shape. Dynamic laser scattering measurements indicated that hydrodynamic diameter of NPs and PEG-NPs were 222.5 ± 10.0 nm and 334.9 ± 6.7 nm, respectively. NPs and PEG-NPs had a positive zeta potential of about 32.5 ± 1.1 mv and 20.6 ± 2.4 mv, respectively. Entrapment efficiency was 61.4 ± 1.0% and 55.7 ± 2.4% for NPs and PEG-NPs, respectively. Compared with the NPs, the PEG-NPs exhibited a slower release profile. Subsequently, fluorescein isothiocyanate–labeled chitosanCs was synthesized and used to evaluate the stealth characteristic of PEG-NPs. In vitro macrophage uptake of fluorescently labeled NPs was measured by flow cytometry.