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The aim of our study was to develop an apparatus assessing in vitro permeation through Caco-2 monolayers of oral solid dosage forms as a possible tool to forecast in vivo performance. Therefore, flow through dissolution and permeation modules were connected by means of a stream splitter. Permeation was measured in a specially designed cell, dissolution took place in the apparatus 4, USP. In order to test the apparatus for its reproducibility and conclusiveness, different tablet strengths and varying release profiles of propranolol HCl tablets were produced and evaluated. It was shown that for both tablet species, immediate and extended release, the apparatus was able to measure permeation through Caco-2 monolayer as well as dissolution simultaneously with high precision and reproducibility. The permeated amount of the three immediate release tablets with increasing dosage strength showed linear dependency on the dosage strength. Furthermore, the effect of retarded release on permeation could be detected and conclusive data for dissolution and permeation were obtained. In summary, connecting cell culture based permeability assessment with compendial flow through dissolution equipment led to promising results and poses the base for more advanced studies for detecting influences of dosage forms on permeation process.

Film forming polymeric solutions may present an alternative to the common transdermal dosage forms such as patches or gels. To evaluate the potential of these systems for transdermal drug delivery the permeation of ethinylestradiol from four formulations with different polymers was tested across heat separated human epidermis. The formulation with the best results was then modified by incorporating chemical enhancers to further increase the efficiency of the delivery system. Finally, drug delivery from the developed film forming systems was compared to a commercially available transdermal patch in vitro as well as in vivo in pigs. Among the tested preparations the formulation with polyurethane-14-AMP-acrylates copolymer (DynamX) showed the highest ethinylestradiol permeation. The drug transport was further increased with the incorporation of oleic acid as penetration enhancer, especially when used in combination with propylene glycol. The enhancing effect of oleic acid/propylene glycol was concentration-dependent and increased disproportionately with rising enhancer content. The film forming solution showed a higher ethinylestradiol permeation through heat separated human epidermis than the commercial EVRA patch in vitro and achieved measurable plasma concentrations of ethinylestradiol in vivo in pigs. These promising results encourage the further development of film forming polymeric solutions as novel transdermal dosage form.

Purpose: The aim of this study was to elucidate the expression pattern of transport proteins relevant to drug absorption in human cornea and to assess the human corneal epithelial cell line, HCE-T, regarding its use as an in vitro model for drug-absorption studies., Methods: Human corneal tissue and HCE-T cells were examined for the expression of P-glycoprotein (P-gp/MDR1), multidrug resistance-associated protein 1 (MRP1), multidrug resistance-associated protein 2 (MRP2), lung resistance-related protein (LRP), and breast cancer-resistance protein (BCRP), using reverse transcriptase-polymerase chain reaction and immunofluorescence microscopy. Moreover, transporter activity was measured by bi-directional flux studies across excised human cornea and HCE-T cell layers using a P-gp/MDR1 substrate, rhodamine 123 (Rh123)., Results: Transport studies of Rh123 revealed no significant differences in fluxes in the apical-to-basolateral and basolateral-to-apical directions across excised human corneas or HCE-T cell layers, suggesting the absence or insignificant, if any, participation of P-gp/MDR1 to Rh123 fluxes. Of all the transporter proteins under investigation, only LRP was found in human cornea. By contrast, a signal for LRP was not found in HCE-T, but the expression of MRP1, MRP2, and BCRP could be confirmed. Of note is the lack of P-gp/MDR1 expression in any of the specimens we examined., Conclusions: Only a limited array of ABC-transporters is functionally expressed in human cornea. The expression pattern of HCE-T cells appears to be widely different from that of the native corneal tissue. Hence, the in vitro model of human cornea, HCE-T, should be used with much caution when predicting transport rates across the human corneal epithelial barrier in vivo.

The objective of this work was to compare the barrier function of the small diameter reconstructed human epidermis model Episkin (d=12 mm) to human skin in vitro. For that purpose a modification for the Franz diffusion cell (d=15mm) had to be developed so as to allow direct comparison with the following human skin preparations: Full thickness skin (FTS), split thickness skin (STS), heat-separated epidermis (HSE), and trypsin isolated stratum corneum (TISC). Among the tested preparations, HSE appeared to be the most preferable due to its clear morphological structure and ease of preparation. The lipid profile of HSE and Episkin was analyzed and showed significant differences in terms of cholesterol, ceramides and triglycerides contents, whereas cholesterol esters and fatty acids were not different. Permeation data with HSE and Episkin were then gathered using caffeine and testosterone. Both test compounds permeated much faster through Episkin than through HSE. Moreover, opposed to Episkin, HSE differentiated between the two test compounds. In spite of the remarkable progress in developing RHEs in the past years at this time Episkin can obviously not yet fully replace human skin for in vitro permeability experiments.

The distribution of the P-glycoprotein (P-gp/MDR1) efflux transporter at mucosal barriers has defined it as a functionally important element in limiting drug absorption into the systemic circulation. However, little is known about the distribution and functionality of P-gp/MDR1 in the human lung. Here, the presence of P-gp/MDR1 was investigated immunohistochemically in distal human lung tissue and at mRNA and protein levels in human alveolar epithelial cells (hAEpC) in primary culture. We studied the presence and activity of P-gp/MDR1 in hAEpC monolayers by Western blotting, by immunofluorescence microscopy and by conducting bi-directional transport studies employing a P-gp substrate (rhodamine 123) with and without a P-gp inhibitor (verapamil). The flux of fluorescein sodium was also examined as a paracellular transport marker. Alveolar tissue specimens showed P-gp localised at the luminal membranes of type I pneumocytes. Reverse transcription-polymerase chain reaction revealed the presence of mRNA encoding for P-gp/MDR1 in freshly isolated (i.e. type II) hAEpC and in monolayers of hAEpC cultured for 8 days (i.e. type I-like morphology). At the protein level, P-gp could be detected in hAEpC monolayers after 8 days in culture but not in freshly isolated type II pneumocytes. The flux of rhodamine 123 across hAEpC monolayers on day 8 in culture exhibited net secretion, which disappeared in the presence of verapamil. Fluorescein sodium fluxes showed no distinct directionality. Our findings indicate that P-gp is functionally active in the human alveolar airspace and that hAEpC monolayers might provide a suitable in vitro model for studying P-gp function mechanistically in the distal human lung.

Aim of this study was to automate sampling and quantification of the previously described apparatus for combined determination of dissolution and permeation through Caco-2 monolayer by means of sequential injection analysis (SIA). Native fluorescence of propranolol HCl in Krebs-Ringer buffer (KRB) was used for quantification. Sampling was done at three different locations within the apparatus at a high sampling frequency (approximately 60 h(-1)). Injection volume delivered to the fluorescence detector was 50 microL for permeation monitoring and 25 microL for dissolution monitoring. Linear regression for 50 microL injection yielded a detection limit calculated as 0.04 microg mL(-1) of propranolol HCl in KRB (R(2)>0.999). However, linearity for dissolution monitoring was not given for the complete range of concentrations and first order polynomial calibration was established (R(2)>0.9999). To conclude, the SIA system was able to monitor simultaneously dissolution and permeation of the immediate release propranolol HCl tablets and the authors succeeded in automating the apparatus for combined measurement of dissolution and permeation. In addition, the obtained data was consistent with data obtained by manual sampling followed by HPLC analysis.

Film forming polymeric solutions as a novel approach for skin drug delivery were developed and characterized concerning their mechanical properties and water vapor permeability. They were developed by varying type and content of the film forming polymer as well as nature and content of the plasticizer. The resulting formulations were evaluated according to five criteria: drying time, cosmetic attractiveness, outward stickiness, integrity on skin (after 18 h) and viscosity. Among the 14 tested polymers 10 film formers yielded formulations with a positive evaluation in all five test criteria. Selected formulations were then investigated for tensile strength and elongation at break in vitro and for water vapor permeability in vitro (WVP) and in vivo (TEWL). Their mechanical properties determined in vitro were found to be not predictive for the flexibility and abrasion resistance observed on living skin. Similar to this, the results derived from the WVP and the TEWL methods were not in accordance with each other. Obviously, the investigated in vitro methods do not characterize the properties of the thin films on living skin satisfactorily. Nevertheless, the identified film forming solutions are a promising approach and will provide the basis for the further development of this novel dosage form.

High-resolution atomic force microscopy (AFM) allows a new insight into the surface of mammalian cells. Using the human red blood cell (RBC) as a model, we have demonstrated an important correlation between the conformation of membrane proteins measured from the external face of the cell and the cell shape.

Most human tumours over-express receptors for growth factors and peptide hormones, which are being increasingly studied as a means to selectively deliver cytotoxic agents. An example being the transferrin receptor (TfR, CD71). Here, we studied expression levels and location of TfR in different lung epithelial cell types (i.e., bronchial and alveolar epithelial cells) by flow-cytometry and confocal laser scanning microscopy (CLSM). Furthermore, we assessed uptake levels and cytotoxicity of transferrin (Tf)-conjugated liposomes in vitro. TfR was found to be expressed at a significantly higher level in bronchial epithelial cells compared with their alveolar counterparts. Cells of cancerous origin (i.e., A549 cell line) showed a higher TfR expression level than healthy alveolar epithelial type II cells in primary culture. CLSM revealed TfR to be located primarily at the basolateral aspect of cells, with the exception of cells undergoing mitotic proliferation, which also showed TfR at their apical membranes, due to their loss of cell polarity. Higher expression levels of TfR correlated well with enhanced uptake of Tf-liposomes and increased levels of cytotoxicity. Liposome uptake was temperature-dependent and inhibitable by excess free Tf. Tf-conjugated liposomes appear as good candidates for an approach to deliver cytostatic drugs to sites of lung cancer by inhalation.

The aim of this study was to add knowledge to the existing theories of mucoadhesion and to review mucoadhesive polymers based on their ability to form non-covalent bonds with mucus glycoprotein. Resonant mirror biosensor was used to study the candidate mucoadhesive polymers hydroxypropyl methylcellulose, carboxymethylcellulose, Carbopol, hyaluronate, alginate and chitosan. Bovine submaxillary mucin was chosen as substrate, representing the major glycosylated protein in mucus. For comparison, non-glycosylated bovine serum albumin was used as an alternative substrate. The results of this study reveal that there is a clear correlation between the ionization state of the polymer, which is dependent on the pH of the surrounding environment, and its binding behavior. Ionizable polymers need to be in their unionized state to be able to form non-covalent bonds with mucus glycoprotein. Acidic polymers display binding behavior only at pH around or lower than their corresponding pK(a) values and basic polymers vice versa. Chitosan was found to be the most mucoadhesive polymer. Unionizable polymers like hydroxypropyl methylcellulose did not display any affinity for mucus glycoprotein. Unionized amino- and carboxyl groups on polymers were found to be important structural feature of polymer for the formation of weak chemical bonds to mucus glycoproteins.

Multiphoton microscopy (MPM) of a dually fluorescence-labeled model system in excised human skin is employed for high-resolution three-dimensional (3D) visualization in order to study the release, accumulation, and penetration properties of drugs released from nanoscale carrier particles in dermal administration. Polymer particles were covalently labeled with fluorescein, whereas Texas Red as a drug-model was dissolved in the particles to be released to the formulation matrix. Single nanoparticles on skin could easily be localized and imaged with diffraction-limited resolution. The temporal evolution of the fluorescent drug-model concentration in various skin compartments over more than 5 hours was investigated by multiphoton spectral imaging of the same area of the specimen. The 3D penetration profile of the drug model in correlation with skin morphology and particle localization information is obtained by multiple laser line excitation experiments. MPM combined with spectral imaging was found to allow noninvasive long-term studies of particle-borne drug-model penetration into skin with subcellular resolution. By dual color labeling, a clear discrimination between particle-bound and released drug model was possible. The introduced technique was shown to be a powerful tool in revealing the dermal penetration properties and pathways of drugs and nanoscale drug vehicles on microscopic level.

Rat and pig animal skin has been the most common replacement material for human skin for use in in vitro permeability experiments. Unfortunately, the permeability barrier of skin from laboratory animals is known to be relatively weak, due to significant follicular transport. Pig skin has been shown to be a suitable model for human skin. Unfortunately, it cannot be gathered from the regular slaughtering process, which makes it unsuitable for permeation experiments. We therefore studied the suitability of bovine udder skin, an untreated waste material of the butchering process, as a possible replacement material for use in in vitro permeability tests. We investigated the barrier strength of bovine udder skin against four different substances, and its histology and lipid profile, in comparison with pig skin and heat separated human epidermis. Pig and human skin were found to be equally permeable, whilst bovine udder skin seemed to exhibit a weaker, but less variable, barrier against caffeine, benzoic acid, testosterone, and flufenamic acid. The skin of all three species contained variable contents of the major lipid classes: cholesterol, ceramides, cholesterol ester, fatty acids and triglycerides. Morphological differences mainly comprised variations in the density of hair follicles. Based on these results, the amount of free fatty acids and triglycerides and the density of hair follicles seem to be important factors in the differences between the skin barriers in the three species.

Purpose: The purpose of this study was to establish a primary culture of porcine lung epithelial cells as an alternative to the currently existing cell cultures from other species, such as e.g., rat or human. Primary porcine lung epithelial cells were isolated, cultivated and analyzed at distinct time points after isolation., Materials and Methods: The main part of the work focused on the morphology of the cells and the detection of alveolar epithelial cell markers by using electron microscopy, immunofluorescence microscopy and immunoblotting. Regarding a later use for in vitro pulmonary drug absorption studies the barrier properties of the cell monolayer were evaluated by monitoring bioelectrical parameters and by marker transport., Results: Epithelial cells isolated from porcine lung grew to confluent monolayers with typical intercellular junctions within a few days. Maximum transepithelial resistance of about 2,000 Omega cm2 was achieved and demonstrated the formation of a tight epithelial barrier. Permeability data of sodium fluorescein recommended a minimal transepithelial resistance of 600 Omega cm2 for transport studies. The cell population changed from a heterogeneous morphology and marker distribution (caveolin-1, pro-SP-C, surface sugars) towards a monolayer consisting of two cell types resembling type I and type II pneumocytes., Conclusions: The porcine alveolar epithelial primary cell culture holds promise for drug transport studies, because it shares major hallmarks of the mammalian alveolar epithelium and it is easily available and scaled up for drug screening.

Various polycationic vehicles have been developed to facilitate the transfer of foreign DNA into mammalian cells. Structure-activity studies suggested that biophysical properties, such as size, charge, and morphology of the resulting DNA complexes determine transfection efficiency within one class of vector. To investigate the general validity of these criteria, we studied the efficacy of a variety of DNA delivery vehicles including liposomes (DOTAP, SAINT2) with and without helper lipid (DOPE), the polymer polyethyleneimine (PEI), and cationic nanoparticles (Si26H, PLGA/chitosan) in a comparative manner. Sizes of the DNA complexes varied between 100 and 500 nm for PEI polyplexes and DOTAP/DOPE lipoplexes, respectively. The zeta potential was positive for PEI, Si26H, and DOTAP based complexes, while it was neutral for SAINT2-DNA complexes and negative for PLGA/chitosan-DNA complexes. The latter finding was elucidated by AFM, showing a layer of DNA adsorbed onto the nanoparticles. Transfection activity was negligible for PLGA/chitosan nanospheres, moderate for Si26H nanospheres and high for all other complexes, PEI being the most active carrier. The liposomal preparations were of low (DOTAP) or moderate (SAINT2) stability in serum, resulting in a pronounced reduction of gene expression, which was partially restored by the addition of chloroquine. In conclusion, transfection efficiency (i) seems to require a positive or neutral zeta potential, (ii) is depending on size, e.g., is higher for smaller particles, and (iii) requires a vector that is stable in serum.

Nanoparticles have recently been demonstrated in a rat model to be a promising tool for targeting inflamed areas of the intestinal mucosa in inflammatory bowel diseases whilst concentrating anti-inflammatory drugs at their site of action. Still, however, this novel concept has to be proven in vivo in humans. As a first step biodegradable and biocompatible fluorescent nanoparticles were prepared and characterized to serve as markers for successful inflammation targeting in future clinical trials. To achieve stable fluorescence labelling, fluoresceinamine was covalently bound to poly(L-lactide-co-glycolide) (PLGA) as described by Horisawa et al. The modification rate of carboxyl-end groups of the PLGA chains determined by 1H NMR was 65%. From this modified polymer, nanoparticles (FA-PLGA nanoparticles) of approximately 270 nm size were prepared via nanoprecipitation. Apart from an initial burst effect, most of the label (> 88%) appeared to be strongly bound and was leaked only slowly from the particles. In contrast, we found an immediate leakage of encapsulated sodium fluorescein with nanoparticles prepared by a double emulsion method. In degradation experiments we studied and visualized the changes in morphology and elastic properties of the FA-PLGA nanoparticles within 15 weeks using atomic force microscopy. When FA-PLGA nanoparticles were applied on an in vitro model of the intestinal mucosa (Caco-2 cell culture), only minor amounts of their fluorescent degradation products (approximately 0.02% after 6 h) were transported. In a cytotoxicity study with Caco-2 cells, FA-PLGA nanoparticles yielded an IC50 value as for plain PLGA nanoparticles. In conclusion, the polymer modification method allows to prepare fluorescently labelled nanoparticles from a well-known biodegradable pharmaceutical polymer with sufficient stability to be monitored over a period of several days. Some initial leakage of fluorescence label appears to be unavoidable but negligible with respect to potential absorption and cytotoxicity when applied in vivo.

Oral inhalation of anticancer drugs or drug delivery system is a novel therapeutic approach in the treatment of lung cancer and requires formulations which are sufficiently stabile during nebulisation and subsequent interaction with the surfactant lining of the lungs. In this study, we assessed the stability of plain and PEGylated transferrin-conjugated liposomes after nebulisation using two different nebulisers (i.e., air-jet and ultrasonic type). Furthermore, the integrity of the liposomal membranes was assessed after incubation in commercial lung surfactant solutions (Alveofact). All liposomal formulations showed no significant changes in their size after nebulisation, independent of the type of nebuliser or the liposomal formulation, respectively. However, PEGylation was of advantage when it came to interactions between liposomes and the surfactant lining of the lungs. PEGylated liposomes were significantly more stable and retained > 80% of their drug load over 48 h, which is more than sufficient time for the drug carriers to be taken up by transferrin receptor over-expressing cancer cells in the lung. In conclusion, PEGylated and plain Tf-conjugated liposomes are stable enough to undergo nebulisation in the course of an inhalational therapy, but PEG-stabilisation results in a higher degree of membrane integrity in lung surfactant.

Nanoparticles (NP) are employed in various therapeutic approaches for innovative drug delivery strategies. Among them, there is drug delivery to the brain and sustained release forms for intravenous drug delivery. In order to optimize drug carriers and to elucidate involved mechanisms such as bioadhesion and cellular uptake, NP were surface modified and analyzed for their interaction with human endothelial cells in cell culture. Fluorescently labeled NP of different diameters (50 to 1000 nm) were surface modified either by simple adsorption of chitosan or by covalent binding to the lectin ulex europaeus agglutinin and thereafter applied to human endothelial cells for different incubation periods. After incubation with NP the binding of NP was quantified directly by the fluorescence emission signals from the cell layers. In order to visualize the binding behaviour, NP were localized three-dimensionally in the cell layer by confocal laser scanning microscopy. Cell binding experiments in phosphate buffer were observed to be particle size dependent with the 50 nm NP showing the highest binding percentage over all experiments. Binding decreased with increasing particle diameter and shorter incubation interval. The adhesion was further enhanced by NP surface modifications in the order blank < chitosan < lectin. The presence of plasma proteins enhanced the adhesiveness of chitosan coated NP, while the binding of lectin coated NP was inhibited. Experiments at 4 degrees C indicated the involvement of an active process in the binding of NP to endothelial cells.

In this study, we investigated bi-directional fluxes (i.e., in absorptive and secretive directions) of human serum proteins [albumin (HSA), transferrin (TF), and immunoglobulin G (IgG)] and peptides/proteins of potential therapeutic relevance [insulin (INS), glucagon-like peptide-1 (GLP-1), growth hormone (GH), and parathyroid hormone (PTH)] across tight monolayers of human alveolar epithelial cells (hAEpC) in primary culture. Apparent permeability coefficients (P(app); x10(-7)cm/s, mean+/-S.D.) for GLP-1 (6.13+/-0.87 (absorptive) versus 1.91+/-0.51 (secretive)), HSA (2.45+/-1.02 versus 0.21+/-0.31), TF (0.88+/-0.15 versus 0.30+/-0.03), and IgG (0.36+/-0.22 versus 0.15+/-0.16) were all strongly direction-dependent, i.e., net absorptive, while PTH (2.20+/-0.30 versus 1.80+/-0.77), GH (8.33+/-1.24 versus 9.02+/-3.43), and INS (0.77+/-0.15 versus 0.72+/-0.36) showed no directionality. Trichloroacetic acid precipitation analysis of tested molecules collected from donor and receiver fluids exhibited very little degradation. This is the first study on permeability data for a range of peptides and proteins across an in vitro model of the human alveolar epithelial barrier. These data indicate that there is no apparent size-dependent transport conforming to passive restricted diffusion for the tested substances across human alveolar barrier, in part confirming net absorptive transcytosis. The obtained data differ significantly from previously published reports utilising monolayers from different species. It can be concluded that the use of homologous tissue should be preferred to avoid species differences.

Exposure to chemicals absorbed by the skin can threaten human health. In order to standardise the predictive testing of percutaneous absorption for regulatory purposes, the OECD adopted guideline 428, which describes methods for assessing absorption by using human and animal skin. In this study, a protocol based on the OECD principles was developed and prevalidated by using reconstructed human epidermis (RHE). The permeation of the OECD standard compounds, caffeine and testosterone, through commercially available RHE models was compared to that of human epidermis and animal skin. In comparison to human epidermis, the permeation of the chemicals was overestimated when using RHE. The following ranking of the permeation coefficients for testosterone was obtained: SkinEthic > EpiDerm, EPISKIN > human epidermis, bovine udder skin, pig skin. The ranking for caffeine was: SkinEthic, EPISKIN > bovine udder skin, EpiDerm, pig skin, human epidermis. The inter-laboratory and intra-laboratory reproducibility was good. Long and variable lag times, which are a matter of concern when using human and pig skin, did not occur with RHE. Due to the successful transfer of the protocol, it is now in the validation process.

Purpose: To characterize the telomerase inhibitor and G-quadruplex stabilizing substance 9-[4-(N,N-dimethylamino)phenylamino]-3,6-bis (3-pyrrolodino-propionamido) acridine x 3HCl (BRACO19) in terms of biopharmaceutical properties such as solubility, protein binding, interaction with membrane lipids, cytotoxicity, and permeability across pulmonary epithelial cells., Methods: Protein binding and interaction with membrane lipids were investigated by two high-performance liquid chromatography methods with immobilized human serum albumin and immobilized phosphatidylcholine, respectively. Cytotoxicity (methyl-thiazolyl-tetrazolium assay) and transport studies were performed with the bronchial cell lines 16HBE14o- and Calu-3, primary human alveolar epithelial cells, and the intestinal cell line Caco-2. Transport experiments were also done in the presence of cyclosporin A (10 microM) and tetraethylammonium chloride (5 mM) and at low temperature (4 degrees C)., Results: BRACO19 has good solubility of at least 2 mg/mL in water and in physiological buffers of pH 7.4 and below. Protein binding to human serum albumin was 38%. No interaction with membrane lipids could be found. Cytotoxicity in 16HBE14o-, Calu-3, and human alveolar epithelial cells was in the range of IC50 = 3.5 to 13.5 microM. Caco-2 cells were not affected at concentrations up to 50 microM. No transport of BRACO19 was detected across either cell monolayer in absorptive direction. In secretory direction, permeability was very low, with P (app) values in the range of 0.25 x 10(-7) to 0.98 x 10(-7) cm/s for all epithelial cell cultures tested. The transport was not influenced by cyclosporin A or tetraethylammonium chloride or at 4 degrees C, indicating that no efflux/influx systems or active transport are involved., Conclusions: From these results, we conclude that the very poor permeability of BRACO19 is its main biopharmaceutical limitation. Further applications will require a suitable formulation to warrant adequate delivery across cellular barriers.

The suitability of transepidermal water loss (TEWL) measurements in vitro as a barrier integrity test for human heat separated epidermis (HSE) was investigated. A model system consisting of a Teflon membrane mounted in Franz diffusion cells (FDC) filled with phosphate buffer saline (PBS) was set up. The membrane was used intact and punctured with a needle (up to five holes). After each puncturing the TEWL was measured. Only the TEWL of intact and punctured membrane differed significantly regardless of the number of holes. From three donors intact human HSE and punctured HSE were compared and no significant difference of the TEWL was found. Permeation experiments with flufenamic acid (FFA) showed a significantly higher diffusion rate through punctured HSE. TEWL and drug permeation were compared for skin stripped three, seven and 15 times prior to heat separation to an intact control group. Only the TEWL values of intact HSE and HSE stripped 15 times differed significantly. However, seven and 15 times stripping resulted in significantly higher diffusion rate. In conclusion, TEWL measurements can detect severe damage of the stratum corneum (SC) but not small changes, which nevertheless may already influence drug diffusion. Therefore, TEWL measurements appears to be of limited use as a barrier integrity test for human HSE in in vitro test systems.

D-alpha-tocopheryl poly(ethylene glycol) 1000 succinate (TPGS 1000) is a widely used form of vitamin E. TPGS 1000 is comprised of a hydrophilic polar (water-soluble) head and a lipophilic (water-insoluble) alkyl tail. TPGS 1000 has been used as a solubilizer, an emulsifier and as a vehicle for lipid-based drug delivery formulations. Most recently, TPGS 1000 has been recognized as an effective oral absorption enhancer. An enhancing effect is consistent with a surfactant-induced inhibition of P-glycoprotein (P-gp), and perhaps other drug transporter proteins; however, the exact inhibition mechanism(s) remain unclear. Therefore, in an attempt to generate additional knowledge, we have synthesized and tested various TPGS analogs containing different PEG chain length (TPGS 200/238/400/600/1000/2000/3400/3500/4000/6000). These results demonstrate a relationship between TPGS PEG chain length and influence on rhodamine 123 (RHO) transport in Caco-2 monolayers, a relationship which may be illustrated using a Weibull distribution.

The CFBE41o- cell line was generated by transformation of cystic fibrosis (CF) tracheo-bronchial cells with SV40 and has been reported to be homozygous for the DeltaF508 mutation. A systematic characterisation of these cells, which however, is a pre-requisite for their use as an in vitro model, has not been undertaken so far. Here, we report an assessment of optimal culture conditions, the expression pattern of drug-transport-related proteins and the stability/presence of the CF transmembrane conductance regulator (CFTR) mutation in the gene and gene product over multiple passages. The CFBE41o- cell line was also compared with a wild-type airway epithelial cell line, 16HBE14o-, which served as model for bronchial epithelial cells in situ. The CFBE41o- cell line retains at least some aspects of human CF bronchial epithelial cells, such as the ability to form electrically tight cell layers with functional cell-cell contacts, when grown under immersed (but not air-interfaced) culture conditions. The cell line is homozygous for DeltaF508-CFTR over multiple passages in culture and expresses a number of proteins relevant for pulmonary drug absorption (e.g. P-gp, LRP and caveolin-1). Hence, the CFBE41o- cell line should be useful for studies of CF gene transfer or alternative treatment with small drug molecules and for the gathering of further information about the disease at the cellular level, without the need for primary culture.

The organic anion transporting protein 1B3 (OATP1B3), formerly termed OATP8, is responsible for uptake and subsequent elimination of multiple amphipathic drugs by the liver. In silico methods for the prediction of transport rates for drugs and drug-like molecules might provide an important tool in drug development. Most prediction methods however require a large training set of in vitro experimental data in order to yield reliable results. To obtain these data, we have developed a fluorescence-based assay that allows screening a relatively high number of substances for their transporter affinity. HEK293 cells overexpressing OATP1B3 (HEK-OATP8) [Y. Cui, J. Konig, D. Keppler, Vectorial transport by double-transfected cells expressing the human uptake transporter SLC21A8 and the apical export pump ABCC2, Mol. Pharmacol. 60 (2001) 934-943.] were tested for transport of Fluo-3. Fluo-3 uptake could be seen in a concentration-dependent manner. Uptake can be inhibited completely by the addition of the known OATP1B3-inhibitor rifampicin proving that Fluo-3 is transported by OATP1B3. To verify the suitability of the system to identify modulators of OATP1B3, we tested known substrates for competitively inhibiting the Fluo-3 transport by giving them simultaneously with a 2muM Fluo-3-solution to the cells. The transport of Fluo-3 was decreased by all test substrates in a concentration dependent manner.

The effect of the inclusion of flufenamic acid in poly(lactide-co-glycolide) nanoparticles on the transport of flufenamic acid into excised human skin was investigated. Penetration and permeation data were acquired using two different in vitro test systems: the Saarbrucken penetration model, where the skin acts as its own receptor medium, and the Franz diffusion cell, where the receptor medium is a buffer solution. For the stratum corneum, no differences were found between nanoencapsulated and free drug. Drug accumulation in the deeper skin layers and drug transport across human epidermis were slightly delayed for the nanoencapsulated drug compared to the free drug after shorter incubation times (<12 h). In contrast, after longer incubation times (>12 h), the nanoencapsulated drug showed a statistically significantly enhanced transport and accumulation (p < 0.05). Additionally, nanoencapsulated flufenamic acid was visualized by multiphoton fluorescence microscopy. Particles were found homogeneously distributed on the skin surface and within the dermatoglyphs, but no nanoparticles were detected within or between the corneocytes., (Copyright (c) 2006 S. Karger AG, Basel.)

Telomerase inhibition represents a promising approach to anticancer treatment. In order to clarify the therapeutic potential of telomerase inhibitors we examined different substances (small molecule compounds BIBR1532 and BRACO19, as well as hTR antisense oligonucleotides 2'-O-methyl RNA and PNA) in A-549, MCF-7, and Calu-3 cell lines in a cell-free TRAP assay. We demonstrated that each of the tested agents inhibited telomerase in all used cell lines and that the antisense oligonucleotides represent the most potent inhibitors. Interestingly, upon evaluating the specificity of telomerase inhibitors we found out that not all agents acted specifically against telomerase. We observed that BRACO19 and PNA had an inhibitory effect also on PCR amplification of the TSR8 oligonucleotide which is provided in the TRAP(EZE) kit as a PCR control. By modifying the experimental protocol and using a different reverse primer we were able to enhance PNA selectivity, although the PCR inhibition of the TSR8 control template by BRACO19 could not be prevented. We propose an explanation for the lack of target specificity and suggest caution when testing putative telomerase inhibitors, as it appears that some of those substances may not affect specifically telomerase or telomeric G-rich sequences and thus can lead to the misinterpretation of experimental results.

The purpose of this research was to prepare poly-(epsilon-caprolactone) (PCL) particles by an emulsion-diffusion-evaporation method using a blend of poly-(vinyl alcohol) and chitosan derivatives as stabilizers. The chitosan derivatives used were chitosan hydrochloride and trimethyl chitosans (TMC) with varying degrees of quaternization. Particle characteristics-size, zeta potential, surface morphology, cytotoxicity, and transfection efficiency-were investigated. The developed method yields PCL nanoparticles in the size range of 250 to 300 nm with a positive surface charge (2.5 to 6.8 mV). The cytotoxicity was found to be moderate and virtually independent of the stabilizers' concentration with the exception of the highly quaternized TMC (degree of substitution 66%) being significantly more toxic. In immobilization experiments with gel electrophoresis, it could be shown that these cationic nanoparticles (NP) form stable complexes with DNA at a NP:DNA ratio of 3:1. These nanoplexes showed a significantly higher transfection efficiency on COS-1 cells than naked DNA.

The commercially available reconstructed human epidermis models EpiSkin, SkinEthic and EpiDerm demonstrate reasonable similarities to the native human tissue in terms of morphology, lipid composition and biochemical markers. These models have been identified as useful tools for the testing of phototoxicity, corrosivity and irritancy, and test protocols have been developed for such applications. For acceptance of these tests by the authorities, prevalidation or validation studies are currently in progress. Furthermore, first results also indicate their suitability for transport experiments of drugs and other xenobiotics across skin. Still, however, the barrier function of these reconstructed human epidermis models appears to be much less developed compared to native skin. Further adaptation of the models to the human epidermis, especially concerning the barrier function, therefore remains an important challenge in this area of research.

Site-specific delivery of drugs and therapeutics can significantly reduce drug toxicity and increase the therapeutic effect. Transferrin (Tf) is one suitable ligand to be conjugated to drug delivery systems to achieve site-specific targeting, due to its specific binding to transferrin receptors (TfR), expressed on several cell types of therapeutic interest. TfRs have been reported to be highly expressed on the surfaces of tumour cells and the well-characterised and efficient mechanism of internalisation of Tf has been exploited for the delivery of anticancer drugs, proteins, and therapeutic genes into primarily proliferating malignant cells. Liposomes are effective vehicles for drugs, genes and vaccines and can be easily modified with proteins, antibodies, and other appropriate ligands, resulting in attractive formulations for targeted drug delivery. In this study, we used atomic force microscopy (AFM) and transmission electron microscopy (TEM) to confirm the conjugation of Tf to liposomes by three different coupling methods. In addition, the conventional assays for quantification of protein amount (BCA) and phospholipid content (according to Steward) were performed. AFM and TEM were able to display Tf-molecules on the liposomal surfaces and can be routinely used to obtain additional visual information on the protein-drug carrier conjugation in a fast and reliable manner.

The respiratory tract holds promise as an alternative site of drug delivery due to fast absorption and rapid onset of drug action, with avoidance of hepatic and intestinal first-pass metabolism as an additional benefit compared to oral drug delivery. At present, the pharmaceutical industry increasingly relies on appropriate in vitro models for the faster evaluation of drug absorption and metabolism as an alternative to animal testing. This article reviews the various existing cell culture systems that may be applied as in vitro models of the human air-blood barrier, for instance, in order to enable the screening of large numbers of new drug candidates at low cost with high reliability and within a short time span. Apart from such screening, cell culture-based in vitro systems may also contribute to improve our understanding of the mechanisms of drug transport across such epithelial tissues, and the mechanisms of action how advanced drug carriers, such as nanoparticles or liposomes, can help to overcome these barriers. After all, the increasing use and acceptance of such in vitro models may lead to a significant acceleration of the drug development process by facilitating the progress into clinical studies and product registration.

Objective: The aim of this study was to determine the tissue distribution and epithelial penetration of moxaverine-hydrochloride (MOX) in the rabbit eye., Methods: For systemic application, a radioactively labeled MOX solution was injected into the ear vein of Dutch-belted pigmented male rabbits. For topical dosing, an identical solution was administered. At predetermined time points, rabbits were sacrificed, the eyes dissected, and the amount of MOX in the ocular tissues measured. To examine the MOX permeability across the corneal epithelium, transport studies using rabbit corneal epithelial cell culture were conducted and the respective apparent permeability coefficient in absorptive (a to b) or secretive (b to a) direction was calculated., Results: Topical delivery resulted in high concentrations of MOX in the cornea and conjunctiva, although other tissues of the anterior part yielded lower MOX concentrations. In the tissues of the posterior part, high amounts were detected in the retina. Plasma levels were low. The apparent permeability coefficient across corneal epithelial cell layers was in the range of 10(5) cm/s, exhibiting no apparent directionality., Conclusion: A topical dosing of MOX to posterior regions of the eye seems feasible. MOX levels in the posterior part of the eye were remarkably high, without causing stringent plasma levels. The high apparent permeability coefficient of MOX across the corneal epithelial cell layers might be caused by the lipophilic nature of the drug and was in the range of other compounds with comparable physicochemical properties.

Laser interference lithography (LIL) is a straightforward technique to prepare linear micropatterns for regulating cellular adhesion behaviors on polymer substratum. This process is based on selective laser ablation directly duplicating the interference patterns of two or more coherent laser beams onto the polymer surface. Micropatterns prepared by LIL on poly(ethylene terephthalate) and Thermanox were characterized using atomic force microscopy (AFM) and white light interferometer while the chemical surface modification induced by laser was analyzed by X-ray photoelectron spectroscopy (XPS). The AFM photographs show that the micropatterns are well-defined and of great consistency. Polymer properties and laser parameters related to LIL as well as laser ablation mechanisms are discussed in this technical note.

Freshly isolated rat type II pneumocytes, when grown on permeable tissue culture-treated polycarbonate filters, form confluent alveolar epithelial cell monolayers (RAECM). Cells in RAECM undergo transdifferentiation, exhibiting over time morphological and phenotypic characteristics of type I pneumocytes in vivo. We recently reported that transforming growth factor-beta(1) (TGF-beta(1)) decreases overall monolayer resistance (R(te)) and stimulates short-circuit current in a dose-dependent manner. In this study, we investigated the effects of TGF-beta(1) (50 pM) or 10% newborn bovine serum (NBS) on modulation of paracellular passive ion conductance and its contribution to total passive ion conductance across RAECM. On days 5-7 in culture, tight-junctional resistance (R(tj), kOmegacm(2)) of RAECM, cultured in minimally defined serum-free medium (MDSF) with or without TGF-beta(1) or NBS, was estimated from the relationship between observed transmonolayer voltage and resistance after addition of gramicidin D to apical potassium isethionate Ringer solution under open-circuit conditions. NaCl Ringer solution bathed the basolateral side throughout the experimental period. Results showed that transmonolayer conductance (1/R(te)) and tight-junctional conductance (1/R(tj)) are 0.59 and 0.14 mS/cm(2) for control monolayers in MDSF, 1.59 and 0.38 mS/cm(2) for monolayers exposed to TGF-beta(1), and 0.38 and 0.18 mS/cm(2) for monolayers grown in the presence of NBS. The contributions to total transepithelial ion conductance by the paracellular pathway are estimated to be 23, 23, and 47% for control, TGF-beta(1)-exposed, and newborn bovine serum (NBS)-treated RAECM, respectively.

Background: Macrophages play an important role in ischemia-reperfusion injury of various organs. Liposome-encapsulated dichloromethylene diphosphonate (clodronate-liposome) depletes local macrophages in vivo. However, the effect of this approach on alveolar macrophages in pulmonary ischemia-reperfusion injury has not yet been evaluated., Methods: Clodronate-liposomes in Hanks' balanced salt solution (HBSS) or HBSS alone were given intratracheally to anesthetized male Lewis rats in the clodronate or the control group (n = 6/each group). After 3 days, we subjected the lungs to ischemia (37 degrees C, 60 minutes) and reperfusion (60 minutes) in an isolated blood-perfused rat lung model. Analysis during reperfusion included gas exchange, hemodynamics, and airway mechanics. At the end of reperfusion, we determined leukocyte recruitment and macrophage inflammatory protein-2 (MIP-2) in bronchoalveolar lavage fluid., Results: In the clodronate group, 4 experiments had to be terminated within 10 minutes of reperfusion because of severe lung injury, whereas all lungs of the controls could be studied during the 60-minute reperfusion period (p < 0.05). Clodronate significantly decreased dynamic airway compliance (p < 0.05) and increased airway resistance. Besides a tendency toward greater pulmonary vascular resistance, this was associated with recruitment of polymorphonuclear neutrophils (p < 0.05) and increased MIP-2 concentrations in the bronchoalveolar lavage fluid (p < 0.05)., Conclusions: Intratracheal administration of liposome-encapsulated clodronate does not benefit, but aggravates, warm ischemia-reperfusion injury of the lung, increasing MIP-2-associated alveolar neutrophil recruitment and airway mechanical dysfunction.

Poly(lactide-co-glycolide) (PLGA), a biocompatible and biodegradable polyester co-polymer of PLA and PGA, has been recognized for its ability to deliver genes. However, gene delivery by PLGA nanoparticles is limited by their negative charge and their poor transport through mucosal barriers. In this study, PLGA nanoparticles were surface modified with cationic chitosan in an effort to improve their gene delivery capability. PLGA nanoparticles were synthesized by emulsion-diffusion-evaporation technique using PVA-chitosan (PLGA1) or PVA-chitosan-PEG (PLGA2) blend as stabilizers. This method is reproducible and produces nanoparticles with hydrodynamic diameter <200 nm. The nanoparticles were characterized by zetasizer, photon correlation spectroscopy and atomic force microscopy. A549 epithelial cells were transfected in vitro with PLGA particles complexed with a reporter plasmid encoding green fluorescent protein. PLGA particles transferred EGFP gene, but were less efficient than the lipofectamine control. The nanoparticles were also tested for their ability to transport across the nasal mucosa in vivo in mice. The results show that both PLGA1 and PLGA2 facilitate gene delivery and expression in vivo with increased efficiency and without causing inflammation, as measured by IL-6. Together, these results indicate that chitosan-modified PLGA nanoparticles have greater potential as gene carriers.

Background: The functional response of isolated alveolar epithelial cells (AECs) to ischemia/reperfusion injury (I/R) is incompletely understood. Using a cell culture model, we investigated the tolerance of human type II alveolar cells (ATII) to hypoxia and subsequent reoxygenation., Methods: Cell cultures of A549 cells (human lung adenocarcinoma) and primary ATII were incubated in 95% N(2)/5% CO(2) saturated medium at 37 degrees C for 48 hours or 72 hours. The hypoxic medium was subsequently exchanged to normoxic medium at 37 degrees C. Lactate dehydrogenase (LDH) release and mitochondrial viability, as assessed by WST-1 metabolism, were determined during both hypoxia and reoxygenation. A549 cells and ATII maintained under normoxic conditions served as controls., Results: Before reoxygenation, after 48 or 72 hours of hypoxia, WST-1 metabolism in A549 cells was significantly reduced (p < 0.05), but LDH release remained low in both cell types. Reoxygenation after 48 h of hypoxia was associated with recovery of WST-1 metabolism and an only minimal increase in LDH release. Reoxygenation after 72 hours of hypoxia, in contrast, induced marked injury in both A549 cells and primary ATII as indicated by significantly reduced WST-1 metabolism and a dramatic increase of LDH release compared with normoxic controls (p < 0.05)., Conclusions: Viability of alveolar cell lines and primary ATII is maintained during hypoxia for up to 72 hours. Reoxygenation after 72 hours of hypoxia results in rapid development of injury and cell death in both cell types.

The potential of cationic SiO2 nanoparticles was investigated for in vivo gene transfer in this study. Cationic SiO2 nanoparticles with surface modification were generated using amino-hexyl-amino-propyltri-methoxysilane (AHAPS). The zeta potential of the nanoparticles at pH = 7.4 varied from -31.4 mV (unmodified particles; 10 nm) to +9.6 mV (modified by AHAPS). Complete immobilization of DNA at the nanoparticle surface was achieved at a particle ratio of 80 (w/w nanoparticle/DNA ratio). The surface modified nanoparticle had a size of 42 nm with a distribution from 10-100 nm. The ability of these particles to transfect pCMVbeta reporter gene was tested in Cos-1 cells, and optimum results were obtained in the presence of FCS and chloroquine at a particle ratio of 80. These nanoparticles were tested for their ability to transfer genes in vivo in the mouse lung, and a two-times increase in the expression levels was found with silica particles in comparison to EGFP alone. Very low or no cell toxicity was observed, suggesting silica nanoparticles as potential alternatives for gene transfection.

Cationic solid lipid nanoparticles (SLN) for gene transfer are formulated using the same cationic lipids as for liposomal transfection agents. To investigate the differences and similarities in structure and performance between SLN and liposomes, a SLN preparation (S1), its counterpart formulation without matrix lipid (L1), a commercially available liposomal preparation (DLTR)--all based on the cationic lipid DOTAP--and a liposomal formulation that additionally contained the helper lipid dioleoylphosphatidylethanolamine (DOPE) (Escort) were compared. Photon correlation spectroscopy (PCS) showed that the SLN were smaller in diameter than the corresponding liposomes (88 vs. 148 nm) and atomic force microscopy (AFM) supported the expected structural differences. Desoxy ribonuclein acid (DNA) binding differed only marginally. Surprisingly, reporter gene expression was comparable between all DOTAP based formulations (S1, L1, DLTR), surpassed only by the DOPE containing liposomes (Escort). In conclusion, cationic lipid composition seems to be more dominant for in vitro transfection performance than the kind of colloidal structure it is arranged in. Hence, cationic SLN extend the range of highly potent non-viral transfection agents by one with favourable and distinct technological properties. Further SLN optimisation should be facilitated by the accumulated knowledge about cationic lipids in liposomal formulations., (Copyright 2004 Elsevier B.V.)

Nanoparticles formulated from biodegradable polymers such as poly(lactic acid) (PLA) and poly(lactide-co-glycolide) (PLGA) are being extensively investigated as non-viral gene delivery systems due to their controlled release characteristics and biocompatibility. PLGA nanoparticles for DNA delivery are mainly formulated by an emulsion-solvent evaporation technique using PVA as a stabilizer generating negatively charged particles and heterogeneous size distribution. The objective of the present study was to formulate cationically modified PLGA nanoparticles with defined size and shape that can efficiently bind DNA. An Emulsion-diffusion-evaporation technique to make cationic nanospheres composed of biodegradable and biocompatible co-polyester PLGA has been developed. PVA-chitosan blend was used to stabilize the PLGA nanospheres. The nanospheres were characterized by atomic force microscopy (AFM), photon-correlation spectroscopy (PCS), and Fourier transform infrared spectroscopy (FTIR). Zeta potential and gel electrophoresis studies were also performed to understand the surface properties of nanospheres and their ability to condense negatively charged DNA. The designed nanospheres have a zeta potential of 10mV at pH 7.4 and size under 200nm. From the gel electrophoresis studies we found that the charge on the nanospheres is sufficient to efficiently bind the negatively charged DNA electrostatically. These cationic PLGA nanospheres could serve as potential alternatives of the existing negatively charged nanoparticles.

The purpose of this paper is to review the history of using lectins to target and deliver drugs to their site of action. The hour of birth of "lectinology" may be defined as the description of the agglutinating properties of ricin, by Herrmann Stillmark in 1888, however, the modern era of lectinology began almost 100 years later in 1972 with the purification of different plant lectins by Sharon and Lis. The idea to use lectins for drug delivery came in 1988 from Woodley and Naisbett, who proposed the use of tomato lectin (TL) to target the luminal surface of the small intestine. Besides the targeting to specific cells, the lectin-sugar interaction can also been used to trigger vesicular transport into or across epithelial cells. The concept of bioadhesion via lectins may be applied not only for the GI tract but also for other biological barriers like the nasal mucosa, the lung, the buccal cavity, the eye and the blood-brain barrier.

In the last few years numerous experimental studies have shed light onto the details of the lectin-carbohydrate interaction. X-ray crystallography and NMR spectroscopy have been used to elucidate the structures of lectins, sugars, and their complexes. In addition, an increasing number of experimental methods has been employed to determine the thermodynamic and kinetic parameters of the binding process. Based on this experimental data, computational methods have been developed to model and predict these interactions. A plethora of techniques from Molecular Modeling and Computational Chemistry have been applied to the problem and current models achieve high-quality predictions. These successes are based on both new theoretical approaches and reliable experimental data. The aim of the present article is to outline the most relevant computational and experimental methods applied in the field of lectin-carbohydrate interaction and to give an overview of the current state of the art in the modeling of these interactions with a focus on plant lectins.

This investigation is focused on the enhancement of in vitro transfection activity by optimizing cationic lipid and matrix lipid composition of solid lipid nanoparticles (SLN). For this purpose SLN were formulated by using two different matrix lipids and six different cationic detergents. These 12 formulations were tested for physical parameters such as particle size, zeta potential and DNA-binding capacity, and also for their biological properties such as cytotoxicity and in vitro transfection efficiency. The SLN were produced by hot high-pressure homogenization, all formulations were physically stable and showed a highly positive surface charge (+34 to +45 mV). In vitro cytotoxicity measurements on COS-1 cells revealed that cytotoxicity is strongly dependent on the cationic lipid used. SLN made from one-tailed cationic detergents were highly cytotoxic. In contrast the two-tailed cationic lipids were all well tolerated. Transfection activity seems to be determined by both the cationic lipid and the matrix lipid used. Here, the combination of cetylpalmitate and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride led to significantly higher transfection efficiencies than in all other tested combinations. These results indicate that well tolerated and highly efficient in vitro transfection could be achieved with SLN whenever selecting good combinations of two-tailed cationic lipids and matrix lipids.

Core shell silica particles with a hydrodynamic diameter of 28nm, an IEP of 7.1 and a zeta potential of +35mV at pH 4.0 were synthesised. The role of freeze-drying for the conservation of zwitterionic nanoparticles and the usefulness of different lyoprotective agents (LPA) for the minimisation of particle aggregation were studied. The activity of the nanoparticles was measured as DNA-binding capacity and transfection efficiency in Cos-1 cells before and after lyophilisation. It was found that massive aggregation occurred in the absence of LPA. Of the various LPAs screened in the present investigations, trehalose and glycerol were found to be well suited for conservation of cationically modified silica nanoparticles with simultaneous preservation of their DNA-binding and transfection activity in Cos-1 cells.

Laser-patterning by interference is a method to introduce micropatterns on the surface of TXL and TXB, which were shown to have an effect on the L929 growth. In this experiment, we have produced collagen-coated and laser-patterned TXL and TXB with different dimensions; the groove width of the line patterns varied approximately from 1.2 microm to 9.7 microm, ridge depth varied from 0.4 microm to 1.3 microm, and the groove depth varied between 0.4 microm and 1.3 microm. Therefore, a homogeneous smooth surface was achieved, and that L929 growth was only affected by the different dimensions of the line patterns. All the laser-patterned TXL and TXB have shown inducing different degrees of directional growth of L929 that the cells grew in the direction aligning the microgrooves. However, the different widths of the microgrooves were demonstrated to play an important role in determining cell morphology and growth orientation. For example, cells were elongated when they grew on the narrower widths, which were 1.26 microm, 1.91 microm, and 5.04 microm while cells tended to be triangular when grew on wider width about 9.76 microm. In addition, L929 might grow only on the top of the laser-patterns attaching the ridges when the groove widths were narrow, but might grow into the microgrooves when the width went beyond 5.04 microm.

Purpose: The ability to optimize new formulations for pulmonary delivery has been limited by inadequate in vitro models used to mimic conditions particles encounter in the lungs. The aim is to develop a physiologically-relevant model of the pulmonary epithelial barrier that would allow for quantitative characterization of therapeutic aerosols in vitro., Methods: Calu-3 human bronchial epithelial cells were cultured on permeable filter inserts under air-interfaced culture (AIC) and liquid-covered culture (LCC) conditions. Calu-3 cells grown under both conditions formed tight monolayers and appeared physiologically similar by SEM and immunocytochemical staining against cell junctional proteins and prosurfactant protein-C., Results: Aerosolized large porous particles (LPP) deposited homogeneously and reproducibly on the cell surface and caused no apparent damage to cell monolayers by SEM and light microscopy. However, monolayers initially grown under LCC conditions showed a significant decrease in barrier properties within the first 90 min after impingement with microparticles, as determined by transepithelial electrical resistance (TEER) measurements and fluorescein-sodium transport. Conversely, AIC grown monolayers showed no significant change in barrier properties within the first 90 min following particle application. A dense mucus coating was found on AIC grown Calu-3 monolayers, but not on LCC grown monolayers, which may protect the cell surface during particle impinging., Conclusion: This in vitro model, based on AIC grown Calu-3 cells, should allow a more relevant and quantitative characterization of therapeutic aerosol particles intended for delivery to the tracheobronchial region of the lung or to the nasal passages. Such characterization is likely to be particularly important with therapeutic aerosol particles designed to provide sustained drug release in the lung.