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Claus-Michael Lehr

Publications and source records attributed to Claus-Michael Lehr.

At least 37 records · Page 2Linked to original sources
PubMed2005

Laser interference lithography as a new and efficient technique for micropatterning of biopolymer surface.

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.

Biocompatible Materials↗
PubMed2005

Assessing transferrin modification of liposomes by atomic force microscopy and transmission electron microscopy.

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.

Drug Delivery Systems↗
PubMed2005

Depletion of alveolar macrophages by clodronate-liposomes aggravates ischemia-reperfusion injury of the lung.

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.

Animals↗
PubMed2005

Optimization of the TRAP assay to evaluate specificity of telomerase inhibitors.

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.

Acridines↗
PubMed2005

Tissue distribution of moxaverine-hydrochloride in the rabbit eye and plasma.

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.

Animals↗
PubMed2004

Estimation of paracellular conductance of primary rat alveolar epithelial cell monolayers.

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.

Animals↗
PubMed2004

Transfection with different colloidal systems: comparison of solid lipid nanoparticles and liposomes.

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.

Animals↗
PubMed2004

Computational modeling of the sugar-lectin interaction.

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.

Carbohydrates↗
PubMed2004

Lectin-mediated drug targeting: history and applications.

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.

Drug Delivery Systems↗
PubMed2004

Effect of cationic lipid and matrix lipid composition on solid lipid nanoparticle-mediated gene transfer.

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.

Animals↗
PubMed2003

16HBE14o- human bronchial epithelial cell layers express P-glycoprotein, lung resistance-related protein, and caveolin-1.

PURPOSE: To study the expression of P-glycoprotein (P-gp), lung resistance-related protein (LRP), and caveolin-1 (cav-1) in the human bronchial epithelial cell line 16HBE14o-. METHODS: The presence of P-gp, LRP, and cav-1 in 16HBE14o- cell layers was evaluated using immunocytochemical staining and visualization with confocal laser scanning microscopy (CLSM). Functionality of P-gp was determined by bidirectional transport of rhodamine-123 with and without a P-gp inhibitor, verapamil. Caveolae were visualized using transmission electron microscopy (TEM). Flux of fluorescein-Na was also studied as a paracellular transport marker. RESULTS: Immunocytochemical staining showed expression of P-gp localized at the apical membrane of 16HBE14o- cell layers. The flux of rhodamine 123 across cell layers exhibited a greater Papp value for the secretory (i.e., basolateral-to-apical) direction. This asymmetry disappeared in the presence of verapamil. CLSM provided evidence for the expression of LRP and cav-1. TEM further showed typically shaped caveolae at the apical and basolateral membranes. CONCLUSION: Cell layers of 16HBE14o- express drug transport systems that are also present in the human bronchus in vivo, indicating that the 16HBE14o- cell line may be a suitable candidate for an in vitro model for mechanistic studies of drug transport processes involved in the smaller airways.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2003

Large porous particle impingement on lung epithelial cell monolayers--toward improved particle characterization in the lung.

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.

Aerosols↗
PubMed2003

Laser ablation patterning by interference induces directional cell growth.

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.

Animals↗
PubMed2003

Chemical crosslinking of urokinase to pulmonary surfactant protein B for targeting alveolar fibrin.

Intraalveolar fibrin formation is a consistent finding in acute inflammatory and chronic interstitial lung disease. Polymerization of fibrin in the presence of pulmonary surfactant results in far-reaching incorporation of the hydrophobic surfactant compounds into the growing fibrin matrix, with loss of surface activity, altered fibrin structure and reduced susceptibility of the clot to fibrinolysis. For specific targeting of such alveolar fibrin, we designed a hybrid molecule consisting of the catalytic domain of urokinase (B-chain) and the hydrophobic surfactant protein B (SP-B), termed SPUC. The urokinase B-chain, obtained by limited reduction of human two-chain-urokinase (u-PA) and subsequent affinity purification, was chemically coupled to SP-B in a semi-organic solvent system using a hetero-bifunctional crosslinker. Purification of the chimeric proteins included reversed phase and cation exchange chromatography. SDS-PAGE and Western Blotting with immunostaining were employed for biochemical characterization of the conjugate. Chromogenic substrate assays, (125)I-based fibrin plate assays, active site titration and surface tension measurements (pulsating bubble surfactometer) were performed to analyze the specific fibrinolytic activity of the conjugate and its surface activity. SPUC was found i) to be assembled stoichiometrically in a 1: 1 fashion (SP-B: u-PA), ii) to fully retain the biophysical activity as compared to native SP-B and iii) to also retain the fibrinolytic activity. SPUC was 2-3 fold more effective in lysis of surfactant containing clots and 5-fold more resistant against plasminogen activator 1 (PAI-1) as compared to the native u-PA. We conclude that urokinase and SP-B can be chemically crosslinked, thereby yielding a fibrinolytic enzyme suitable for targeting alveolar fibrin.

Chromogenic Compounds↗
PubMed2002

Differentiation of human alveolar epithelial cells in primary culture: morphological characterization and synthesis of caveolin-1 and surfactant protein-C.

Human alveolar type II cells were isolated from lung tissue and cultured for several days. The morphology of cells was investigated at different time points postseeding and the synthesis of alveolar cell-type specific proteins was analyzed using different methods. The rationale of the study was to characterize a primary cell culture of human alveolar cells for the development of an in vitro model studying pulmonary drug delivery. In vitro test systems based on human cells are attracting increasing interest as important alternatives to animal-derived models because possible interspecies differences in alveolar cell biology and transport mechanisms cannot be excluded. In our study, both morphological characterization and marker protein synthesis of human alveolar cells in culture indicate the differentiation of isolated alveolar type II cells into epithelial monolayers consisting of alveolar type I-like and alveolar type II-like cells, which corresponds to the composition of the alveolar epithelium of the donor tissue. By using flow cytometry, immunofluorescence, immunoblotting and reverse transcriptase polymerase chain reaction (RT-PCR), we observed a shift in the synthesis of important marker proteins. Early cultures were characterized by low caveolin-1 and high Sp-C levels. In comparison, the protein biosynthesis of alveolar cells switched with time of culture to high caveolin-1 and low Sp-C levels. Based on the similarity between human alveolar epithelium and the development of our primary alveolar cell culture, we suggest that the culture may serve as a suitable model to study epithelial transport or cell biological processes in human alveolar cells.

Antibodies↗
PubMed2002

Influence of apical fluid volume on the development of functional intercellular junctions in the human epithelial cell line 16HBE14o-: implications for the use of this cell line as an in vitro model for bronchial drug absorption studies.

Air-interfaced culture (AIC) versus liquid-covered culture (LCC) conditions are known to have different effects on the differentiated phenotype of several cell types, including lung epithelial cells. We report the influence of culture conditions such as apical medium volume on the development of intercellular junctions in the human epithelial cell line 16HBE14o-. Immunofluorescence staining of the tight-junctional protein, ZO-1, has revealed its presence in cells grown in both AIC and LCC. However, only LCC-grown cells exhibit protein ZO-1 localized as a zonula-occludens-like regular belt connecting neighboring cells. The presence of typical tight junctions has been confirmed by electron microscopy. Immunostaining for occludin, claudin-1, connexin43, and E-cadherin has demonstrated intercellular junction structures only in the cells in LCC. These morphological findings have been paralleled by higher transepithelial electrical resistance values and similar fluxes of the hydrophilic permeability marker, fluorescein-Na, under LCC compared with AIC conditions. We conclude that the formation of functional 16HBE14o- cell layers requires the presence of an apical fluid volume, in contrast to other culture conditions for airway epithelial cells.

Actins↗