Biomedical subjects

Thomas Kissel

Publications and source records attributed to Thomas Kissel.

At least 37 records · Page 2Linked to original sources

PubMed2004

Biodegradable polymers and their potential use in parenteral veterinary drug delivery systems.

Biodegradable polymers have been extensively studied for numerous drug delivery systems for human health purposes. The ever-increasing value of animals to human society allows the application of pharmaceutical developments in the veterinary field from those developed in human medicine. Although many similarities between the human and animal health industries exist there are also notable differences. This paper provides an insight into the animal health market with regard to the challenges and special considerations associated with veterinary drug delivery. It also gives an overview of biodegradable polymers that are used or have been tested in the veterinary field. The purpose of this paper is to highlight some recent developments in this area and to investigate the directions in which veterinary pharmaceutics is heading. In particular, examples of existing biodegradable veterinary drug delivery systems are presented together with applications including intravaginal devices, injectables and implantable systems.

Animals↗

PubMed2004

Stabilization of oligonucleotide-polyethylenimine complexes by freeze-drying: physicochemical and biological characterization.

In the present study the lyophilization of oligodeoxynucleotide-polyethylenimine (ODN-PEI) complexes was investigated regarding the maintenance of physicochemical properties and influence on biological activity. To achieve this, we used PEI of different molecular weights, in the range of 800-0.8 kDa, as complexing agents for unmodified ODN and ribozymes. The hydrodynamic diameter was measured by photon correlation spectroscopy (PCS) and the zeta potential was determined using laser Doppler anemometry (LDA) of ODN complexes with PEI derivatives of different molecular weights both before and after lyophilization. Atomic force microscopy (AFM) was used to visualize freshly prepared, stored and lyophilized complexes in solution. The biological activity of the ODN, as well as of plasmid DNA, in lyophilized PEI complexes was examined and compared to freshly prepared complexes using standard transfection assays. All PEI derivatives formed very small complexes with ODN displaying hydrodynamic diameters ranging from 15 to 30 nm. Marginal changes in size after lyophilization were observed for ODN-PEI complexes. In contrast, plasmid complexed with PEI was found to aggregate. In either cases minimal or no influence of the added amount of lyoprotectant was observed. The shape of the very small and highly condensed ODN complexes was not altered by lyophilization as seen in the AFM images. The transfection efficiency of lyophilized ribozyme-PEI complexes relative to freshly prepared complexes was approximately 100%, whereas a decrease was seen for lyophilized plasmid-PEI complexes. An additive of the lyoprotectants trehalose, mannitol or sucrose preserved biological activity. This study demonstrates the particular suitability of ODN-PEI complexes to be formulated as lyophilized systems with no loss in physical stability or biological activity.

3T3 Cells↗

PubMed2004

In vitro and in vivo correlation of buserelin release from biodegradable implants using statistical moment analysis.

Here we investigated the possibility to develop different levels of correlation between in vitro drug release profiles and in vivo pharmacokinetic parameters for three Buserelin implant formulations. The in vitro and in vivo data were analyzed using model-independent and model-dependent methods. Since diffusion, dissolution and erosion effects influence drug release in most cases a simple kinetic model is unlikely to explain the overall in vivo release behavior. Thus the in vitro drug release curves were analyzed according to the theoretical models of Higuchi and Korsmeyer-Peppas. For the formulation with predominant diffusion controlled release level A IVIVC could be established (R2=0.986). Independent on drug release mechanism, a level B correlation between the mean in vitro dissolution time (MDT) and mean in vivo residence time (MRT) was obtained with a correlation coefficient of 0.983. Finally, level C correlation were observed when single in vitro parameters, e.g. T50% (time required to release 50% of drug in vitro) where compared with single in vivo parameters like AUC. This study suggests that a level B correlation could be achieved even when drug release occurs by a combination of diffusion and erosion processes. More sophisticated in vitro models mimicking drug release under in vivo conditions are clearly desirable for parenteral depot formulations.

Absorbable Implants↗

PubMed2004

Variation of peptide transporter (PepT1 and HPT1) expression in Caco-2 cells as a function of cell origin.

Caco-2 cell cultures are a widely used in vitro model for the small intestinal drug transport, although large differences have been reported for actively transported substrates from different laboratories. Therefore, we compared three different Caco-2 clones: (1) from the American Culture Tissue Collection (ATCC), (2) from the German Cancer Research Center (DKFZ) in Heidelberg, and (3) from the University Hospital in Marburg in different passage numbers regarding their morphology, multilayers, and tight junction formation, as well as expression of the peptide transporters, HPT1 and PepT1. We determined tight junction formation by measurement of the transepithelial electrical resistance, multilayer formation by confocal laser scanning microscopy, the expression of PepT1 and HPT1 by RT-PCR, indirect immunofluorescence and the permeability of the PepT1 substrate, cephradine. Morphology and TEER-values varied strongly between the different clones. The expression of PepT1 and HPT1 increased in the following order: HD > ATCC > MR. Indirect immunofluorescence revealed a heterogeneous distribution of the transporters in ATCC-cells, whereas it was homogeneous in HD-cells. Only a very weak expression was found in MR-cells. While in ATCC-cells expression of transporters decreased with increasing passage number, it increased in HD-cells. Expression levels were congruent with the transport of cephradine. Expression of PepT1 and HPT1 was strongly affected by the culture conditions. Under identical culture conditions, Heidelberg (HD) Caco-2 cells seemed to be an appropriate in vitro cell culture model for the transport of actively transported drugs, because interpassage changes are low and the transporter distribution was homogeneous throughout the monolayer.

Caco-2 Cells↗

PubMed2004

Low molecular weight chitosan nanoparticles as new carriers for nasal vaccine delivery in mice.

High molecular weight (Mw) chitosan (CS) solutions have already been proposed as vehicles for nasal immunization. The aim of the present work was to investigate the potential utility of low Mw CS in the form of nanoparticles as new long-term nasal vaccine delivery vehicles. For this purpose, CS of low Mws (23 and 38 kDa) was obtained previously by a depolymerization process of the commercially available CS (70 kDa). Tetanus toxoid (TT), used as a model antigen, was entrapped within CS nanoparticles by an ionic cross-linking technique. TT-loaded nanoparticles were first characterized for their size, electrical charge, loading efficiency and in vitro release of antigenically active toxoid. The nanoparticles were then administered intranasally to conscious mice in order to study their feasibility as vaccine carriers. CS nanoparticles were also labeled with FITC-BSA and their interaction with the rat nasal mucosa examined by confocal laser scanning microcopy (CLSM). Irrespective of the CS Mw, the nanoparticles were in the 350 nm size range, and exhibited a positive electrical charge (+40 mV) and associated TT quite efficiently (loading efficiency: 50-60%). In vitro release studies showed an initial burst followed by an extended release of antigenically active toxoid. Following intranasal administration, TT-loaded nanoparticles elicited an increasing and long-lasting humoral immune response (IgG concentrations) as compared to the fluid vaccine. Similarly, the mucosal response (IgA levels) at 6 months post-administration of TT-loaded CS nanoparticles was significantly higher than that obtained for the fluid vaccine. The CLSM images indicated that CS nanoparticles can cross the nasal epithelia and, hence, transport the associated antigen. Interestingly, the ability of these nanoparticles to provide improved access to the associated antigen to the immune system was not significantly affected by the CS Mw. Indeed, high and long-lasting responses could be obtained using low Mw CS molecules. Furthermore, the response was not influenced by the CS dose (70-200 microg), achieving a significant response for a very low CS dose. In conclusion, nanoparticles made of low Mw CS are promising carriers for nasal vaccine delivery.

Administration, Intranasal↗

PubMed2004

Efficiency of polyethylenimines and polyethylenimine-graft-poly (ethylene glycol) block copolymers to protect oligonucleotides against enzymatic degradation.

Enzymatic instability of oligonucleotides (ON) is one of the major drawbacks of this new class of therapeutic agents. The development of safe, efficient delivery systems capable of stabilizing and protecting these molecules within the formulation, as well as during application, is a challenge in modern gene therapy. In the present study, polyethylenimine (PEI) of different molecular weights and PEGylated PEI block copolymers (PEI-g-PEG) were investigated with regard to their protective properties when complexes with chemically unmodified DNA (d-ON) and RNA (r-ON) oligonucleotides. PEI/ON complexes were incubated with different amounts of serum or nucleases. The influence of pH on the stability was studied and the integrity of the ON was determined by gel electrophoresis. The amount of stable ON within the gels was quantified via densitometric analysis. PEI homopolymers ranging from 800 to 2 kDa protected both types of ON very efficiently, whereas PEI 0.8 kDa demonstrated a slight decrease in protection. The PEGylated PEI derivatives generally protected ON as efficiently as the PEI homopolymers. In particular, the PEI-g-PEG derivative containing 100 PEG chains of 550 Da yielded the highest protection efficiency for both d-ON and r-ON. In general, the highest protection could be achieved at pH 6.7. The ratio of polymer and ON (N/P ratio) also had a great impact on ON stability with higher N/P ratios achieving a better protection. In conclusion, PEIs showed advantageous protective properties for ON. The results of this study offer indications for a rational design of PEI derivatives for the protection and the delivery of ON.

Enzyme Stability↗

PubMed2004

Effects of different application parameters on penetration characteristics and arterial vessel wall integrity after local nanoparticle delivery using a porous balloon catheter.

Catheter-based local delivery of drug loaded nanoparticles agents offers a potential therapeutic approach to reducing restenosis. However, high delivery pressures and large volumes of infusates may cause severe vascular damage and increase intimal thickening. Therefore, we investigated the penetration pattern and vessel wall integrity of fluorescence-labelled nanoparticles (217 nm in diameter) into the non-atherosclerotic aorta abdominalis of New Zealand white rabbits in dependence of the volume (2.5 and 5 ml) and concentration (0.5 and 1 mg/ml) of the nanoparticle suspension, as well as the infusion pressure (2 and 4 atm) using a channelled balloon catheter (SCIMED REMEDY model RC 20/2.5). The location and penetration characteristics of nanoparticles in the arterial vessel wall were visualized using confocal laser scanning microscopy and transmission electron microscopy (TEM). Catheter design and infusion pressure form a radial particle stream through intima and media into the adventitial layer of the aorta abdominalis. Infusion pressures of 4 atm in combination with high particle concentrations lead to effective nanoparticle delivery without severe vessel wall disruptions. Endothelium of the treated vessel segments was slightly affected during catheter insertion showing partly denudation of the innermost cell layer. TEM micrographs underlines transport functional properties of the vasa vasorum inside the vessel wall. Consequently, local delivery efficiency of nanoparticulate carriers is critically affected by infusion pressure, and concentration of carrier suspensions. These factors need to be taken into consideration for the design of in vivo experiments.

Animals↗

PubMed2004

Design of amine-modified graft polyesters for effective gene delivery using DNA-loaded nanoparticles.

PURPOSE: The purpose of this study was the design of a polymeric platform for effective gene delivery using DNA-loaded nanoparticles. METHODS: The polymers were synthesized by carbonyldiimidazole (CDI)-mediated coupling of diamines diethylaminopropylamine (DEAPA), dimethylaminopropylamine (DMAPA) or diethylaminoethylamine (DEAEA) to poly(vinyl alcohol) (PVA) with subsequent grafting of D,L-lactide and glycolide (1:1) in the stoichiometric ratios of 1:10 and 1:20 (free hydroxyl groups/monomer units). The polymers were characterized by 1H-NMR, gel permeation chromatography-multiple-angle laser-light-scattering, and differential scanning calorimetry. DNA-loaded nanoparticles prepared by a modified solvent displacement method were characterized with regard to their zeta (zeta)-potential and size. The transfection efficiency was assessed with the plasmid DNA pCMV-luc in L929 mouse fibroblasts. RESULTS: The polymers were composed of highly branched, biodegradable cationic polyesters exhibiting amphiphilic properties. The amine modification enhanced the rapid polymer degradation and resulted in the interaction with DNA during particle preparation. The nanoparticles exhibited positive zeta-potentials up to +42 mV and high transfection efficiencies, comparable to polyethylenimine (PEI) 25 kDa/DNA complexes at a nitrogen to phosphate ratio of 5. CONCLUSIONS: The polymers combined amine-functions and short poly(D,L-lactic-co-glycolic acid) (PLGA) chains resulting in water-insoluble polymers capable of forming biodegradable DNA nanoparticles through coulombic interactions and polyester precipitation in aqueous medium. The high transfection efficiency was based on fast polymer degradation and the conservation of DNA bioactivity.

Animals↗

PubMed2004

Effect of poly(ethylene imine) molecular weight and pegylation on organ distribution and pharmacokinetics of polyplexes with oligodeoxynucleotides in mice.

The in vivo body distribution and the pharmacokinetics of a 20mer double-stranded nuclear factor kappaB decoy oligodeoxynucleotide (ODN) complexed with 25-kDa poly(ethylene imine) (PEI), low molecular weight 2.7-kDa PEI, and PEGylated PEI [bPEI(25k)-glPEG(550)(50)] after intravenous injection were studied in BALB/c mice using a double-labeling technique to follow simultaneously the distribution of both complex components. The polymers were radioactively labeled with (125)I by Bolton-Hunter reagent and the decoys with [gamma-(32)P]ATP by an enzymatic 5'-end-labeling technique. After i.v. bolus injections into the jugular vein, organ samples were taken after 15 min, 2 h and 12 h. For pharmacokinetic studies blood and plasma samples were collected from 20 s up to 2 h. Uncomplexed decoy was found to be degraded already after 15 min and was rapidly eliminated renally into urine. Complexation with the homopolymers increased the organ levels and circulation time of ODN after 15 min, with similar organ distribution profiles for (125)I and (32)P. In contrast to the behavior of free ODN, the complexes were mainly distributed into liver and spleen. Whereas the organ concentrations of (125)I remained high over 12 h, the (32)P values of ODN decreased in a time-dependent manner, likely due to separation of the complexes and degradation of the DNA. Although PEGylated PEI demonstrated a slower (125)I-uptake into the RES organs compared with 25-kDa PEI due to the shielding effect of PEG [poly(ethylene glycol)], it was not able to better stabilize the complexes in the circulation or protect DNA from degradation.

Adenosine Triphosphate↗

PubMed2003

Hydrolytic degradation of poly(lactide-co-glycolide) films: effect of oligomers on degradation rate and crystallinity.

Oligomers are thought to accelerate the hydrolytic degradation of devices prepared from poly(lactide-co-glycolide), PLGA, due to their increased number of carboxylic end groups. To experimentally verify this hypothesis, two D,L-lactic acid oligomers having molecular weights close to their critical limit of solubility were synthesized and incorporated into PLGA films in three concentrations (0, 10, and 30% w/w). All films were translucent, rather flexible and initially amorphous. With increasing oligomer concentration the glass transition temperature (T(g)) and the molecular weight of films decreased prior to erosion. The degradation studies show that initial mass loss and water absorption are increased in oligomer-containing films as a function of average molecular weight and oligomer concentration. However, the incorporation of oligomers does not accelerate the degradation of films. By contrast, oligomer-containing films show extended lag phase until onset of polymer erosion. This was shown to be related to crystallization. Moreover, it was found that crystallization occurs earlier in oligomer-containing films and that the degree of crystallization is related to the average molecular weight of the oligomer. These findings bring new insight into the role of oligomers in the degradation process and can be used to explain why erosion in massive polymer devices occurs from the center to the surface.

Biotransformation↗

PubMed2003

Low-molecular-weight polyethylenimine as a non-viral vector for DNA delivery: comparison of physicochemical properties, transfection efficiency and in vivo distribution with high-molecular-weight polyethylenimine.

Low-molecular-weight polyethylenimine (LMW-PEI) was synthesized by the acid-catalyzed, ring-opening polymerization of aziridine and compared with commercially available high-molecular-weight PEI (HMW-PEI) of 25 kDa. Molecular weights were determined by size-exclusion chromatography in combination with multi-angle laser light scattering. The weight average molecular weight (M(w)) of synthesized LMW-PEI was determined as 5.4+/-0.5 kDa, whereas commercial HMW-PEI showed a M(w) of 48+/-2 kDa. DNA polyplexes of LMW-PEI and HMW-PEI were characterized with regard to DNA condensation (ethidium bromide fluorescence quenching), size (photon correlation spectroscopy) and surface charge (laser Doppler anemometry). Compared with HMW-PEI, DNA condensation of LMW-PEI was slightly impaired at lower N/P ratios. Complexes with plasmid DNA at a N/P ratio of 6.7 showed significantly increased hydrodynamic diameters (590+/-140 vs. 160+/-10 nm), while the zeta-potential measurements were similar (23+/-2 vs. 30+/-3 mV). The cytotoxicity of LMW-PEI in L929 fibroblasts was reduced by more than one order of magnitude compared with HMW-PEI, as shown by MTT assay. LMW-PEI exhibited increased transfection efficiency in six different cell lines. Reporter gene expression was found to be increased by a factor of 2.1-110. The pharmacokinetics and biodistribution of 125I-PEI in mice were similar for both molecular weights with an AUC of ca. 330+/-100% ID/ml min. Approximately half of the injected dose accumulated in the liver. LMW-PEI proved to be an efficient gene delivery system in a broad range of cell lines. Due to differences in polyplex structure, as well as its relatively low cytotoxicity, which makes the application of high N/P ratios possible, LMW-PEI appears to possess advantageous qualities with regard to transfection efficiency over PEI of higher molecular weight.

Action Potentials↗

PubMed2003

Galactose-PEI-DNA complexes for targeted gene delivery: degree of substitution affects complex size and transfection efficiency.

Complexes of galactosylated polyethylenimines (gal-PEI) with DNA have been proposed for gene delivery to hepatocytes. We synthesized gal-PEI with a broad range of degrees of substitution (DS) ranging from 3.5 to 31% of all PEI amino groups by reductive amination to determine physico-chemical and biological properties with respect to the DS. Gel retardation assay for herring testes DNA-polymer polyplexes showed that increasing DS compromised DNA complexation and especially condensation. Using photon correlation spectroscopy, gal-PEI complexes formed with plasmid DNA were found to increase in size with increasing galactosylation (156+/-7 nm for 0%, 486+/-76 nm for 3.5%, 467+/-86 nm for 9.7% and 652+/-123 nm for 31% DS). Zeta potentials decreased in inverse proportion to DS (0%: 30+/-3 mV, 3.5%: 22+/-2 mV, 9.7%: 15+/-1 mV, 31%: -26+/-3.5 mV) suggesting a shielding effect by carbohydrate coupling. Cytotoxicity of gal-PEI was found to decrease with increasing galactosylation (MTT and LDH assay), no toxicity was detectable for polyplexes with plasmid DNA (LDH assay). The transfection efficiency of a reporter gene complexed with gal-PEI in a hepatocyte cell culture model (HepG2) expressing the asialoglycoprotein receptor was slightly but not significantly increased for galactosylated PEIs at a nitrogen to phosphate (N/P) ratio of 2 and strongly reduced at higher N/P ratios, compatible with only a minor targeting efficiency, strongly affected by DS. In NIH-3T3 mouse fibroblasts, increasing the DS led to a decreased transfection efficiency for all N/P ratios. Our study highlights the necessity of careful optimization of polyplex composition for active gene targeting.

3T3 Cells↗

PubMed2003

Nebulization of biodegradable nanoparticles: impact of nebulizer technology and nanoparticle characteristics on aerosol features.

Nanoparticles may be effective drug delivery systems for use in various pulmonary therapeutic schemes. This study investigated the effect of nebulization technology and nanoparticle characteristics on the features of aerosol generation. Suspensions of biodegradable nanoparticles consisting of commercially available poly(lactide-co-glycolide) and novel comb polymers were nebulized with a jet, ultrasonic, and piezo-electric crystal nebulizer. The effects of the nanoparticle suspensions on the aerosol droplet size, as well as the nanoparticle size before and after nebulization, were characterized via laser diffraction. While the individual nanoparticle suspensions showed no clinically relevant influence on aerosol droplet size, as compared to control experiments, an enhanced nanoparticle aggregation within the droplets was observed. This aggregation was further characterized by fluorescence and scanning electron microscopy. Dependency of aggregation on nebulizer technology and nanoparticle characteristics was noted. Nanoparticles exhibiting the highest surface hydrophobicity were particularly susceptible to aggregation when nebulized with a jet nebulizer. Aggregation was reduced with nanoparticles exhibiting a more hydrophilic surface or when using ultrasonic nebulizers. We conclude that the biodegradable nanoparticles contained in the suspensions did not affect the aerosol droplet size in a clinically relevant manner; however, both the nanoparticle characteristics and the technique of aerosol generation influence nanoparticle aggregation occurring during aerosolization.

Aerosols↗

PubMed2003

Integrin targeting using RGD-PEI conjugates for in vitro gene transfer.

BACKGROUND: Targeting to integrin receptor alpha(nu)beta(3) by RGD peptides seems to be a promising approach for gene delivery to proliferating endothelial cells of tumor metastases. PEGylation of cationic polymers offers a reduction of non-specific binding to cell surfaces. However, little knowledge exists on the influence of charge shielding by PEGylation on targeted gene delivery. Therefore, a variety of RGD peptide-polyethylenimine (PEI) conjugates with different degrees of substitution, with or without poly(ethylene glycol) (PEG) spacer, were synthesized. Influence of degree of substitution and PEG spacer on physicochemical properties as well as on integrin targeting of DNA/polymer complexes was evaluated. METHODS: The tetrapeptide RGDC was coupled to PEI with or without a PEG spacer. Complex formation with DNA was monitored by ethidium bromide (EtBr) fluorescence quenching. Hydrodynamic diameters of complexes and zeta-potential were assessed using a Zetasizer. Fluorescence correlation spectroscopy (FCS) was used to determine peptide binding to living cells. Transfection efficiency was evaluated employing a luciferase reporter gene. Binding of complexes to Mewo cells was monitored by flow cytometry. RESULTS: Polyplexes of RGD-PEI or RGD-PEG-PEI and DNA showed reduced quenching of EtBr fluorescence compared with PEI. All RGD conjugates formed small polyplexes (approximately 100 nm in diameter at a nitrogen/phosphate (N/P) ratio of 6.7). At N/P = 6.7, the zeta-potentials of RGD-PEI complexes were similar to PEI complexes (25-30 mV), while RGD-PEG-PEI formed neutral complexes. FCS showed saturable binding of RGD peptide to Mewo human melanoma cells and only low binding to A549 human lung carcinoma cells. A degree of substitution of 4.6% with SPDP as coupling reagent yielded a conjugate showing 50 times higher luciferase expression in Mewo cells than unmodified PEI at low N/P ratios around 3.3, while a degree of substitution of 1.6% only led to a moderately increased transfection efficiency. Flow cytometry experiments suggest that this effect is partly caused by increased attachment of complexes to cell surfaces. No improvement in transfection efficiency was found in alpha(nu)beta(3)-negative A549 cells. RGD-PEG-PEI complexes showed reasonable transfection efficiencies at high N/P ratios; however, no targeting effect could be found. CONCLUSIONS: Coupling of the tetrapeptide RGDC without a PEG spacer improved transfection efficiency of PEI in integrin-expressing Mewo cells by 1-2 orders of magnitude, especially at low N/P ratios. The use of a PEG spacer seems to impair targeting, possibly by not only shielding PEI, but also the RGD ligand.

Animals↗

PubMed2003

In vitro cytotoxicity testing of polycations: influence of polymer structure on cell viability and hemolysis.

A comparative in vitro cytotoxicity study with different water-soluble, cationic macromolecules which have been described as gene delivery systems was performed. Cytotoxicity in L929 mouse fibroblasts was monitored using the MTT assay and the release of the cytosolic enzyme lactate dehydrogenase (LDH). Microscopic observations were carried out as indicators for cell viability. Furthermore, hemolysis of erythrocytes was quantified spectrophotometrically. To determine the nature of cell death induced by the polycations, the nuclear morphology after DAPI staining and the inhibition of the toxic effects by the caspase inhibitor zVAD.fmk were investigated. All assays yielded comparable results and allowed the following ranking of the polymers with regard to cytotoxicity: Poly(ethylenimine)=poly(L-lysine)>poly(diallyl-dimethyl-ammonium chloride)>diethylaminoethyl-dextran>poly(vinyl pyridinium bromide)>Starburst dendrimer>cationized albumin>native albumin. The magnitude of the cytotoxic effects of all polymers were found to be time- and concentration dependent. The molecular weight as well as the cationic charge density of the polycations were confirmed as key parameters for the interaction with the cell membranes and consequently, the cell damage. Evaluating the nature of cell death induced by poly(ethylenimine), we did not detect any indication for apoptosis suggesting that the polymer induced a necrotic cell reaction. Cell nuclei retained their size, chromatin was homogenously distributed and cell membranes lost their integrity very rapidly at an early stage. Furthermore, the broad spectrum caspase inhibitor zVAD.fmk did not inhibit poly(ethylenimine)-induced cell damage. Insights into the structure-toxicity relationship are necessary to optimize the cytotoxicity and biocompatibility of non-viral gene delivery systems.

Animals↗

PubMed2003

Characterization of a homologous series of D,L-lactic acid oligomers; a mechanistic study on the degradation kinetics in vitro.

A series of low molecular weight polymers of D,L-lactic acid has been synthesized. The oligomers were characterized with respect to molecular weight, glass transition temperature and solubility. The number average molecular weight of the oligomers ranged from 290 to 1320Da. Oligomers with an M(n)<800Da were soluble in buffer at pH 7.4 but insoluble in water and acidic medium. Kinetic studies were performed at pH 1.5, 4.5 and 7.4 using an accelerated in vitro monomer release test. The hydrolytic rate was dependent on molecular weight of oligomer, temperature and pH of the media, with the lowest rate found around pH 4.5. The activation energy was dependent on molecular weight and ranged from 47 to 67kJmol(-1). Random ester cleavage was identified as mechanism of hydrolysis in basic media whereas in acidic media chain-end cleavage ("unzipping") was the mode of action.

Biocompatible Materials↗

PubMed2003

Do cell culture conditions influence the carrier-mediated transport of peptides in Caco-2 cell monolayers?

Despite the fact that different laboratories have reported large differences in permeability for actively transported substrates, Caco-2 cell monolayers are widely used as in vitro model to study small intestinal drug transport. Therefore, we investigated the effect of cell culture conditions, such as time in culture, membrane support, seeding density and supplements to the medium, on the morphology, the formation of tight junctions, as well as the expression of two peptide transporters (PepT1, HPT1) and the efflux pump, P-glycoprotein (Pgp), in Caco-2 cell monolayers. Tight junction formation was assessed by transepithelial electrical resistance measurements; multi-cell layer formation by confocal laser scanning microscopy, the expression of transporters by RT-PCR and the permeability of the PepT1 substrate, cephradine. Both morphology and the expression of carrier-mediated transporters, varied strongly as a function of culture conditions. An increase of differentiation, as documented by tight, homogeneous cell monolayer formation displaying a strong expression of all carrier-mediated transporters, was found up to 3 weeks post seeding. One week later, multi-layer structures were observed and the expression of Pgp decreased. Polyester and polyethylene terephthalate membrane supports decreased the paracellular transport rates substantially, while collagen-coating of PC inserts showed no influence on the morphology and even increased carrier-mediated transporter expression. An average seeding density of 6x10(4) cells/cm(2) seemed to be most favorable, since lower seeding densities led to thin monolayers with altered tight junctions and higher seeding densities to the formation of multilayers. In summary, the expression of carrier-mediated transporters was strongly affected by the culture conditions. The full differentiation was reached after 21 days on collagen-coated polycarbonate inserts at an initial seeding density of 6x10(4) cells/cm(2).

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2002

Distribution and quantification of polyethylenimine oligodeoxynucleotide complexes in human skin after iontophoretic delivery using confocal scanning laser microscopy.

Iontophoresis may be a potentially useful technique for the delivery of oligonucleotides into the skin. To enhance intracellular uptake during iontophoresis, we investigated the dermal delivery of oligodeoxynucleotides (ODN) as a polyelectrolyte complex with polyethylenimine (PEI). Perpendicular cross-sectioning was performed to visualize and quantify the penetration properties of double labeled PEI/ODN complexes across full thickness human skin. Due to the net positive charge of the complexes, anodal iontophoresis was expected to enhance skin delivery by electrorepulsion compared to passive diffusion. Confocal laser scanning microscopy demonstrated that non-complexed ODN could penetrate the skin after 1 h of cathodal iontophoresis but not by passive diffusion or anodal iontophoresis. However, extensive degradation occurred as documented by a dramatic decrease of fluorescence intensity within viable skin tissue after 10 h. Anodal iontophoresis of the complexes led to a deep penetration of both the TAMRA-labeled ODN and the Oregon Green-labeled PEI. A constant increase in fluorescence indicated a protective effect of the polymer against nuclease degradation. Co-localization of red and green fluorescence was noted within numerous nuclei of epidermal keratinocytes. In contrast, passive diffusion of the complexes did not lead to successful uptake into keratinocytes and was limited to the stratum corneum. Complexation of ODN by PEI, therefore, seems to be a promising method to enhance both the transport of charged complexes into the skin and to facilitate intracellular uptake, which may potentially be useful for the local treatment of skin diseases using ODN.

Drug Carriers↗