Biomedical subjects

Jörg Kreuter

Publications and source records attributed to Jörg Kreuter.

At least 19 recordsLinked to original sources

PubMed2006

Nanoparticles--a historical perspective.

The historical development of nanoparticles starting with Paul Ehrlich and then first attempts by Ursula Scheffel and colleagues and the extensive work by the group of Professor Peter Speiser at the ETH Zürich in the late 1960s and early 1970s are described from a personal point of view. Special attention is given to the years between 1970 and the early 1980s. Further developments resulting from this work are also followed, and focus is placed on especially interesting improvements such as nanoparticles for the delivery of drugs across the blood-brain barrier (BBB) and PEGylated nanoparticles with a prolonged blood circulation time.

Drug Delivery Systems↗

PubMed2006

Influence of surfactants, polymer and doxorubicin loading on the anti-tumour effect of poly(butyl cyanoacrylate) nanoparticles in a rat glioma model.

Poly(n-butyl cyanoacrylate) nanoparticles coated with polysorbate-80 can enable the transport of bound drugs across the blood-brain barrier (BBB) after i.v. injection. In the present study the influence of different formulation parameters on the anti-tumoural effects of doxorubicin nanoparticles against glioblastoma 101/8 was investigated. The manufacturing parameters of poly(alkyl cyanoacrylate) doxorubicin-loaded nanoparticles were optimized concerning drug loading. The nanoparticles were coated with different surfactants and injected intravenously on days 2, 5 and 8 after intra-cranial implantation of glioblastoma 101/8 to rats. The survival times of all doxorubicin containing preparations, including a doxorubicin solution, increased the survival times significantly compared to untreated tumour-bearing rats. The most pronounced increase in survival was obtained with the poly(n-butyl cyanoacrylate) doxorubicin-loaded nanoparticles coated with polysorbate 80 and 35% of these animals survived for over 180 days (termination of the experiments). The other nanoparticle preparations yielded lower survival times. Poly(n-butyl cyanoacrylate) doxorubicin-loaded nanoparticles coated with polysorbate 80-coated proved to be very efficient against glioblastoma 101/8. The data suggest that the interaction of nanoparticles with the blood after injection as well as the enhanced permeability and retention effect (EPR effect) contributed differently to the anti-tumoural efficacy depending on nanoparticle formulation and surface properties.

Animals↗

PubMed2006

Biodistribution of polysorbate 80-coated doxorubicin-loaded [14C]-poly(butyl cyanoacrylate) nanoparticles after intravenous administration to glioblastoma-bearing rats.

It was recently shown that doxorubicin (DOX) bound to polysorbate-coated nanoparticles (NP) crossed the intact blood-brain barrier (BBB), and thus reached therapeutic concentrations in the brain. Here, we investigated the biodistribution in the brain and in the body of poly(butyl-2-cyano[3-(14)C]acrylate) NP ([(14)C]-PBCA NP), polysorbate 80 (PS 80)-coated [(14)C]-PBCA NP, DOX-loaded [(14)C]-PBCA NP in glioblastoma 101/8-bearing rats after i.v. injection. The biodistribution profiles and brain concentrations of radiolabeled NP were determined by radioactivity counting after i.v. administration in rats. Changes in BBB permeability after tumour inoculation were assessed by i.v. injection of Evans Blue solution. The accumulation of NP in the tumour site and in the contralateral hemisphere in glioblastoma bearing-rats probably was augmented by the enhanced permeability and retention effect (EPR effect) that may have been becoming instrumental due to the impaired BBB on the NP delivery into the brain. The uptake of the NP by the organs of the reticuloendothelial system (RES) was reduced after PS 80-coating, but the addition of DOX increased again the concentration of NP in the RES.

Animals↗

PubMed2006

Cytotoxicity of doxorubicin bound to poly(butyl cyanoacrylate) nanoparticles in rat glioma cell lines using different assays.

The cytotoxicity of doxorubicin bound to poly(butyl cyanoacrylate) nanoparticles (Dox-PBCA-NP) was investigated in the rat glioma cell lines GS-9L, F-98 and RG-2. MTT and LDH assays were used as cytotoxic assays. In general, the cytotoxicity of nanoparticle-bound doxorubicin (Dox) was enhanced compared to the free drug in solution. However, responses of the cell lines towards the drug effects were different. In the case of free Dox in solution, this difference correlated with different intracellular concentrations of Dox, which in turn, depended on the level of P-glycoprotein (P-gp) expression in these cell lines. Accordingly, the 9L gliosarcoma (GS-9L) cells, which appeared to be most resistant towards Dox, were characterized by the highest P-gp expression.Additionally, the influence of surfactants on the cytotoxic effect was investigated at different Dox concentrations. It was shown that the presence of polysorbate 80 (Tween 80) in the nanoparticle formulation significantly enhanced the cytotoxicity, whereas poloxamer 188 (Pluronic F68) and poloxamine 908 (Tetronic 908) had a negligible influence.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2005

Glycerol-l-lactide coating polymer leads to delay in bone ingrowth in hydroxyapatite implants.

Glycerol-l-lactide as coating polymer for the delivery of basic fibroblast growth factor (bFGF) from hydroxyapatite (HA) ceramic implants was shown to lead to significant delay in bone ingrowth into the implants compared to implants without the coating polymer. The purpose of this work was to study bone ingrowth in HA ceramic implants with and without the coating polymer but without growth factors to enable differentiation between a locking effect of the pores by the polymer and the fact of inactivation of the growth factors by the polymer, which could both be possible for the delay. A defect was created in the subchondral region of both femurs in 24 miniature-pigs and was either filled by the HA implants with or without the coating polymer. Histomorphometry showed a significant delay in bone ingrowth in the polymer coated implants both after 6 and 12 weeks. Detailed histology revealed that the HA pores were completely "locked" by the polymer leading to complete loss of the osteoconductive properties of the HA. Also electron microscopy showed filling of the HA pores by the polymer. Therefore, it can be concluded that glycerol-l-lactide should not be used to coat HA ceramic implants due to significant delay in bone ingrowth.

Animals↗

PubMed2005

Oligonucleotide-protamine-albumin nanoparticles: Protamine sulfate causes drastic size reduction.

Nanoparticles prepared by self-assembly from oligonucleotides (ONs), protamine free base, and human serum albumin ("ternary proticles") are spheres of diameters around 200 nm. Substitution of the protamine free base by protamine sulfate leads to proticles of only around 40 nm in diameter with otherwise unchanged properties. The availability of drug delivery systems of very similar composition but grossly different size may be advantageous when dealing with cells which show size-dependent particle uptake. These nanoparticles are promising candidates for ON delivery to cells because of the following reasons: (1) They are stable for several hours in solutions of up to physiological ionic strength; (2) they are efficiently taken up by cells; (3) after cellular uptake, they easily release the ONs even when these are present as phosphorothioates.

Animals↗

PubMed2005

Molecular weights of poly(butyl cyanoacrylate) nanoparticles determined by mass spectrometry and size exclusion chromatography.

Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and size exclusion chromatography (SEC) were employed to elucidate the chemical composition, mean number average molecular weight (M(n)), mean weight average molecular weight (M(w)), and polydispersity (PD) of poly(butyl cyanoacrylate) (PBCA) manufactured by emulsion polymerisation. Both methods gave similar results for M(n), but substantial differences were observed for M(w) and PD, with MALDI producing consistently lower values which could not be improved by off-line coupling of SEC and MALDI. MALDI gave a more detailed view on the chemical composition of the cyanoacrylate and revealed the presence of two additional polymer series with different end groups besides the expected PBCA series, which showed different retention in SEC. Their formation is explained by the secession/addition of formaldehyde from/to the regular polymer via (reverse) Knoevenagel reaction. In additional experiments, the influence of different pH on PBCA-NP during polymerisation was examined by comparison of polymerisation yield and particle diameter to their chemical composition as revealed by the MALDI spectra. The most uniform nanoparticles, with the highest polymerisation yield, narrowest particle size, and mass distribution were produced at pH 1.

Chromatography, Gel↗

PubMed2005

Selective targeting of antibody-conjugated nanoparticles to leukemic cells and primary T-lymphocytes.

In the present study, surface-modified nanoparticles based on biodegradable material were used for antibody coupling in order to get a selective drug carrier systems. Gelatin nanoparticles were prepared by a desolvation process. Sulfhydryl groups were introduced which enabled the linkage of NeutrAvidin (NAv). Antibodies specific for the CD3 antigen on lymphocytic cells were conjugated to the nanoparticles surface. The binding of biotinylated anti-CD3 antibody was achieved by NAv-biotin-complex formation. Cellular binding and uptake were determined by flow cytometry and confocal laser scanning microscopy (CLSM). Cell-type-specific targeting of anti-CD3-conjugated nanoparticles into CD3-positive human T-cell leukemia cells and primary T-lymphocytes could be shown. Celluar uptake and effective internalization of antibody-conjugated nanoparticles into CD3 expressing cells were demonstrated. Uptake rates of about 84% into T-cell leukemia cells were observed. To confirm selectivity of T-cell targeting, competition experiments were carried out adding excessive free anti-CD3 prior to nanoparticle incubation leading to significantly reduced cellular uptake of antibody-conjugated nanoparticles. Further analysis on the mechanism of uptake confirmed a receptor-mediated endocytotic process. Protein-based nanoparticles conjugated with an antibody against a specific cellular antigen hold promise as selective drug delivery systems for specific cell types.

Antibodies↗

PubMed2005

Degradation of microvascular brain endothelial cell beta-catenin after co-culture with activated neutrophils from patients undergoing cardiac surgery with prolonged cardiopulmonary bypass.

The adhesion of highly activated neutrophils to cerebral microvascular endothelial cells (MVECs) may contribute to disruption and hyperpermeability of the blood-brain barrier (BBB) after cardiac surgery with prolonged cardiopulmonary bypass (CPB). A correlation between CPB duration and neutrophil-mediated BBB damage has not been investigated on the cellular level yet. Therefore, we studied the effects of neutrophils from cardiac surgery patients with CPB time <80 min (group I; n=8) and >80 min (group II; n=8) on the integrity of cultured porcine MVEC. Ex vivo, neutrophils of group II but not of group I significantly degraded the zonula adherens molecule beta-catenin whereas VE-cadherin and occludin were not modified. The transendothelial electric resistance as a measure for the integrity of the endothelial monolayers was reduced over time in both groups. In conclusion, prolonged CPB time entails neutrophil-mediated decrease in MVEC beta-catenin expression, and thus may be an important trigger for BBB disruption.

Animals↗

PubMed2005

Oligonucleotide-protamine-albumin nanoparticles: preparation, physical properties, and intracellular distribution.

Oligodesoxynucleotides (ODNs) or the corresponding phosphorothioates (PTOs) spontaneously form spherical nanoparticles ("proticles") with protamine in aqueous solutions. The proticles can cross cellular membranes and release the ODNs within the cells. Thus, they represent a potential drug delivery system. The major disadvantages of this system are a lack of stability in salt solutions and its inability to also release PTOs. The present study shows, using PTOs and protamine free base, that these shortcomings can be eliminated by the addition of human serum albumin (HSA) as a third component to the starting mixture. The "ternary" proticles thus obtained contain maximally a few percent of the HSA that was originally present. Nevertheless, they differ from the previously studied "binary" proticles: (1) They are stable in salt solutions for at least several hours. (2) They show a high cellular uptake into murine fibroblasts, and they readily release the PTOs after uptake. The ternary proticles therefore represent a considerable improvement over binary proticles for use as drug delivery systems.

Albumins↗

PubMed2005

Preparation and characterisation of antibody modified gelatin nanoparticles as drug carrier system for uptake in lymphocytes.

Established methods of protein chemistry can be used for the effective attachment of drug targeting ligands to the surface of protein-based nanoparticles. In the present work gelatin nanoparticles were used for the attachment of biotinylated anti-CD3 antibodies by avidin-biotin-complex formation. These antibody modified nanoparticles represent a promising carrier system for the specific drug targeting to T-lymphocytes. The objective of this work was the comprehensive quantification of every chemical reaction step during the preparation procedure of these cell specific nanoparticles. Gelatin nanoparticles were formed by a two-step desolvation process. After the first desolvation step the remaining sediment and the supernatant were analysed for molecular weight distribution by size exclusion chromatography (SEC). Nanoparticles then were formed using the high molecular gelatin fraction and subsequently were stabilised by glutaraldehyde crosslinking. A part of the detectable amino groups on the particle surface was reacted with 2-iminothiolane in order to introduce reactive sulfhydryl groups. The thiolated nanoparticles were coupled to NeutrAvidin (NAv) which previously was activated with the heterobifunctional crosslinker sulfo-MBS. All these reaction steps were quantified by photometry or gravimetry. The functionality of NAv after covalent conjugation was confirmed by a biotin-4-fluorescein assay. The NAv-modified nanoparticles then were used for the binding of biotinylated anti-CD3 antibodies by avidin-biotin-complex formation. A highly effective attachment of the ligand was ascertained by different, indirect methods: immunoblotting and fluorimetry. Therefore, a well-defined nanoparticle system with drug targeting ligand modification was established that holds promise for further effective preclinical testing.

Antibodies↗

PubMed2005

Cationic lipid-protamine-DNA (LPD) complexes for delivery of antisense c-myc oligonucleotides.

In the present study, cationic lipid-peptide-DNA-complexes (LPDs) consisting of AH-Chol-liposomes and protamine-phosphodiester-oligonucleotide-particles (proticles) were introduced as carriers for antisense therapy. The LPDs were physically characterized, and a possible mechanism for adsorption of oligonucleotides (ODNs) was suggested. An increase in stability of ODNs against DNase I and serum nuclease digestion by these carriers was demonstrated. The hydrodynamic diameter increased after incubation with FCS which could be attributed to a protein coating of the particle surface. However, in cell culture medium lower particle sizes of the complexes occurred. In an antisense c-myc in vitro model, the effect of LPDs was tested using U937 cells. The C-MYC level was reduced after treatment of these antisense ODN carrier complexes. Furthermore, no changes in target mRNA concentration of the treated cells was found by reverse transcription and competitive multiplex-PCR.

Culture Media↗

PubMed2005

Body distribution of polysorbate-80 and doxorubicin-loaded [14C]poly(butyl cyanoacrylate) nanoparticles after i.v. administration in rats.

Previously it was shown that poly(butyl cyanoacrylate) (PBCA) nanoparticles coated with polysorbate 80 are able to cross the blood-brain barrier (BBB) after i.v. administration. The objective of the present study was to investigate the influence of polysorbate 80 and doxorubicin-loading on the body distribution in rats. The biodistribution profile and brain concentration of (14)C-radiolabeled PBCA nanoparticles, polysorbate 80 coated (14)C-PBCA nanoparticles, and doxorubicin-loaded (14)C-PBCA nanoparticles were determined by radioactivity counting after i.v. administration in rats. The (14)C-PBCA nanoparticles showed a significant accumulation in the organs of the reticuloendothelial system (RES). Polysorbate 80 coating of the (14)C-PBCA nanoparticles decreased this accumulation to about 40% after 1 h post injection. The brain concentration was increased about 2-fold after polysorbate 80-coating at this time point. The presence of doxorubicin in this preparation, however, decreased the brain concentration to levels similar to uncoated particles, probably caused by the positive charge of this compound. After longer time periods after injection the differences between the three preparations decreased.

Animals↗

PubMed2004

Pharmacological activity of DTPA linked to protein-based drug carrier systems.

The chelating agent diethylenetriaminepentaacetic acid (DTPA) inhibits human cytomegalovirus replication. Since chelating agents are known to exhibit anti-cancer effects, DTPA-induced cytotoxicity was evaluated in breast cancer cells (MCF-7) and neuroblastoma cells (UKF-NB-3). DTPA inhibited cancer cell growth in threefold lower concentrations compared to human foreskin fibroblasts (HFF). Antiviral and anti-cancer activity of chelating agents is caused by intracellular complexation of metal ions. DTPA, an extracellular chelator, was covalently coupled to human serum albumin (HSA) molecules, HSA nanoparticles (HSA-NP), gelatin type B (GelB) molecules, and GelB nanoparticles (GelB-NP) to increase cellular uptake. Coupling of DTPA to drug carrier systems increased its cytotoxic and antiviral activity by 5- to 8-fold. Confocal laser scanning microscope examination revealed uptake of DTPA-HSA-NP in UKF-NB-3 cells and HFF. Therefore, coupling of DTPA to protein-based drug carrier systems increases its antiviral and anti-cancer activity probably by mediating cellular uptake.

Antineoplastic Agents↗

PubMed2004

Effect of glycerol-L-lactide coating polymer on bone ingrowth of bFGF-coated hydroxyapatite implants.

Basic fibroblast growth factor (bFGF)-coated hydroxyapatite (HA) cylinders showed good bony incorporation in a previously conducted animal study. However, some cylinders exhibited focal inhomogeneous bone ingrowth. The purpose of the current study was to test whether glycerol-L-lactide polymer coating could improve release properties and bone incorporation of bFGF-coated HA implants. bFGF-coated HA cylinders with or without coating polymer were investigated for in vitro release of bFGF by an immuno-ligand-assay and also for bone ingrowth in miniature pigs after 42 and 84 days. Release from bFGF polymer composites was lower for the first 3 days compared to the other group but was more homogenous and detectable amounts were still found after 20 days. There was significant delay in bone ingrowth of the polymer implants in which even after 84 days bone ingrowth was not completed, whereas in the other group incorporation after 42 days occurred. Detailed histology revealed filling of the HA pores with the polymer, making ingrowth of the surrounding host bone impossible. Only after 84 days starting resorption of the polymer accompanied by bone ingrowth was found. The current study showed that glycerol-L-lactide is not suitable for coating of HA implants due to polymer induced "locking" of HA pores.

Animals↗

PubMed2004

Tumour cell delivery of antisense oligonuclceotides by human serum albumin nanoparticles.

Nanoparticles consisting of human serum albumin (HSA) and containing different antisense oligonucleotides (ASO) were prepared by desolvation. The preparation process was optimised regarding the amount of desolvating agent, stabilisation conditions as well as nanoparticle purification. The glutaraldehyde crosslinking procedure of the particle matrix was identified as a crucial parameter for biodegradability and drug release of the nanoparticles. The influence of chain length and backbone modification of ASOs on the drug loading efficiency was investigated. The loading increased with longer chain length and employment of a phosphorothioate backbone. The resulting nanoparticles were tested in cell cultures for cytotoxicity and cellular uptake. In different tumour cell lines no cytotoxic effect was observed up to nanoparticle concentrations of 5000 microg/ml. All cell lines showed a significant cellular uptake of HSA nanoparticles. The entrapment of a fluorescent labelled oligonucleotide within the particle matrix was used for the detection of the intracellular drug release of the carrier systems. Confocal laser scanning microscopy revealed that nanoparticles crosslinked with low amounts of glutaraldehyde, rapidly degraded intracellularly, leading to a significant accumulation of the ASO in cytosolic compartments of the tumour cells.

Cell Division↗

PubMed2004

Intracellular tracking of protamine/antisense oligonucleotide nanoparticles and their inhibitory effect on HIV-1 transactivation.

Membrane transport of antisense oligonucleotides (ODN) is an inefficient process which requires special carriers for their intracellular delivery. We have developed a delivery system for AS-ODN and their phosphorothioate analogues (AS-PTO) directed against human immunodeficiency virus type 1 (HIV-1) tat mRNA for efficient transfection of HIV-1 target cells. Protamine was used to complex AS-ODN and AS-PTO to form nanoparticles with diameters of about 180 nm and surface charges in the range of -18 to +30 mV. Cellular uptake of these nanoparticles was significantly enhanced compared to naked oligonucleotides. A double labeling technique with fluorescently tagged protamine and AS-ODN was used to follow the intracellular fate of the nanoparticles. Protamine/AS-ODN nanoparticles showed release of the antisense compound leading to specific inhibition of tat mediated HIV-1 transactivation. In contrast, protamine/AS-PTO complexes were stable over 72 h, and failed to release AS-PTO. These results demonstrate that protamine/AS-ODN nanoparticles are useful for future therapeutical application to inhibit viral gene expression.

Cell Survival↗

PubMed2004

Chemotherapy of glioblastoma in rats using doxorubicin-loaded nanoparticles.

Glioblastomas belong to the most aggressive human cancers with short survival times. Due to the blood-brain barrier, they are mostly inaccessible to traditional chemotherapy. We have recently shown that doxorubicin bound to polysorbate-coated nanoparticles crossed the intact blood-brain barrier, thus reaching therapeutic concentrations in the brain. Here, we investigated the therapeutic potential of this formulation of doxorubicin in vivo using an animal model created by implantation of 101/8 glioblastoma tumor in rat brains. Groups of 5-8 glioblastoma-bearing rats (total n = 151) were subjected to 3 cycles of 1.5-2.5 mg/kg body weight of doxorubicin in different formulations, including doxorubicin bound to polysorbate-coated nanoparticles. The animals were analyzed for survival (% median increase of survival time, Kaplan-Meier). Preliminary histology including immunocytochemistry (glial fibrillary acidic protein, ezrin, proliferation and apoptosis) was also performed. Rats treated with doxorubicin bound to polysorbate-coated nanoparticles had significantly higher survival times compared with all other groups. Over 20% of the animals in this group showed a long-term remission. Preliminary histology confirmed lower tumor sizes and lower values for proliferation and apoptosis in this group. All groups of animals treated with polysorbate-containing formulations also had a slight inflammatory reaction to the tumor. There was no indication of neurotoxicity. Additionally, binding to nanoparticles may reduce the systemic toxicity of doxorubicin. This study showed that therapy with doxorubicin bound to nanoparticles offers a therapeutic potential for the treatment of human glioblastoma.

Animals↗