Biomedical subjects

R H Müller

Publications and source records attributed to R H Müller.

At least 91 records · Page 5Linked to original sources

PubMed1998

Analysis of plasma protein adsorption on polymeric nanoparticles with different surface characteristics.

Plasma protein adsorption patterns on colloidal drug carriers acquired after i.v. administration depend on their surface characteristics and are regarded as key factors for their in vivo organ distribution. Polymeric latex particles with strongly differing surface properties were synthesized as models for colloidal drug carriers for tissue-specific drug targeting via the intravenous route. Physicochemical characterization was performed for size, surface charge density, zeta potential, and surface hydrophobicity. The interactions with human plasma proteins were studied by way of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Considerable differences in protein adsorption on the latex particles were detected with regard to the total amount of surface-bound protein on the various particle types as well as specific proteins adsorbed, for example, fibrinogen, albumin, and a recently identified plasma glycoprotein. Possible correlations between protein adsorption patterns and the physicochemical characteristics and topography of the polymeric surfaces are shown and discussed. Knowledge about protein-nanoparticle interactions can be utilized for the rational design of colloidal drug carriers and also may be useful for optimizing implants and medical devices.

Amino Acid Sequence↗

PubMed1998

Degradation of various chlorophenols under alkaline conditions by gram-negative bacteria closely related to Ochrobactrum anthropi.

From concrete debris of a demolished herbicide production plant several Gram-negative bacterial strains were isolated, which exhibit metabolic capabilities for the degradation of 2,4-dichlorophenol (DCP)l), 4-chloro-2-methylphenol (MCP) and 4-chlorophenol (4-CP), while 2-chlorophenol (2-CP) was degraded at a slower rate. Degradative activity was inducible and was impeded by adding of 100 mg/l of chloramphenicol to growing cultures. The strains displayed alkaliphilic properties with optimum DCP/MCP degradation at pH values around 8.5-9.5; activity was observed up to pH values of 11. Degradation was most likely complete according to chlorine balances; formation of intermediary products was observed with MCP some time. Specific activity of up to 380 mumol/h.g dry mass was found within the concentration range of 10-20 mg/l DCP; higher concentrations retarded the activity with complete inhibition at 200-400 mg/l. Some of the strains carry plasmids whose presence was not unambiguously correlated to the degradative properties. Ribotyping revealed a high degree of relationship between the strains. Preliminary taxonomic investigations showed close relationship to Ochrobactrum anthropi.

Anti-Bacterial Agents↗

PubMed1998

Plasma protein adsorption patterns on emulsions for parenteral administration: establishment of a protocol for two-dimensional polyacrylamide electrophoresis.

The two-dimensional polyacrylamide electrophoresis (2-D PAGE) of the plasma protein adsorption pattern previously established for polymeric nanoparticles was modified and transferred to oil in water emulsions for intravenous administration. The emulsions were incubated with citrated plasma, and separation from excess plasma was performed by centrifugation under optimized conditions: 15000 g and three washing steps with 0.05 M phosphate buffer, pH 7.4. With this sample preparation, coalescence of droplets could be avoided and an unchanged surface area maintained, in addition the phosphate buffer minimized artificial IgG adsorption. Critical factors affecting sensitivity were contamination of the sample by oil residues and the use of thiourea in the immobilized pH gradients. Changes in the protein adsorption pattern caused by altered surface properties of the emulsion (i.e. adsorbed Poloxamer 407) were detectable when applying the optimized protocol. Knowledge of the protein adsorption patterns and their correlation to in vivo behavior opens the perspective for the development of intravenous emulsions for controlled drug delivery.

Absorption↗

PubMed1998

Surface-modified amikacin-liposomes: organ distribution and interaction with plasma proteins.

Amikacin-loaded liposomes were produced and surface-modified by adsorption of PEG 4000, Tween 80, poloxamer 407 and gelatin. The organ distribution was studied in mice by analysing the amikacin content in liver, spleen, lung, kidneys and serum. Highest serum levels were obtained with the PEG- and Tween 80 modified liposomes (at 2 hours p.inj.). Modification of the liposomes with gelatin as opsonization promoting agent distinctly increased the amikacin concentration in the liver from 36 to 66 mg/kg. Highest spleen concentrations were observed with non-modified and poloxamer 407 liposomes (242 mg/kg and 248 mg/kg, respectively). The data suggest that modification by a simple adsorption process is sufficient to effectively alter the organ distribution. The liposomes differing in organ distribution exhibited also different plasma protein adsorption patterns, up to 115 spots were detected by 2-D PAGE. Hydrophilic albumin was present in a conc. of appr. 80% on liposomes modified with ethoxylated compounds. On the gelatin liposomes, 14% of alpha-2-Macroglobulin were adsorbed which is a protein typically found on particles rapidly cleared by the RES. IgM, Apo A-I, Apo C-II and alpha-1-Antitrypsin were other detected proteins.

Amikacin↗

PubMed1998

The influence of the sample preparation on plasma protein adsorption patterns on polysaccharide-stabilized iron oxide particles and N-terminal microsequencing of unknown proteins.

The in vivo organ distribution of i.v. injected drug carriers is strongly influenced by the adsorption of plasma proteins after i.v. injection, e.g. uptake by the mononuclear phagocytic system (MPS). 2-D PAGE could be established to analyze plasma protein adsorption patterns on polysaccharide-stabilized aqueous iron oxide dispersions used as contrast agents in Magnetic Resonance Imaging (MRI). After incubation in human plasma, centrifugation, a washing procedure and a solubilization step were carried out to obtain the proteins adsorbed onto these ultrasmall particles (65 nm in diameter). Patterns of adsorbed proteins were analyzed in dependence on the washing medium used, i.e. highly purified water, phosphate buffered saline and Krebs buffer pH 7.4. Conductivity and composition of the washing medium influenced the adsorption of IgG onto the particles, but had little effect on the other proteins present. IgG was strongly reduced when using the relatively high conductive buffers. The more stabilizing polysaccharide was desorbed the larger was the total amount of adsorbed proteins. Appearance of two unknown chains of spots in the range of appr. 92 kDa, accounting for appr. 10% and 2% of the overall detected protein amount, was observed only when using Krebs buffer during the washing process. Performing N-terminal microsequencing one unknown chain of spots could be identified as a dimer of fibrinogen gamma chains.

Adsorption↗

PubMed1998

Etherolytic cleavage of 4-(2,4-dichlorophenoxy)butyric acid and 4-(4-chloro-2-methylphenoxy)butyric acid by species of Rhodococcus and Aureobacterium isolated from an alkaline environment.

Bacterial strains were isolated from the concrete rubble of a demolished herbicide production plant. The predominant feature of these strains was the etherolytic cleavage of 4-(2,4-dichlorophenoxy)butyric acid (DCPB)1) and 4-(4-chloro-2-methylphenoxy)butyric acid (MCPB) while liberating 2,4-dichlorophenol (DCP) and 4-chloro-2-methylphenol (MCP) respectively. Some of the isolates were identified by 16S rDNA sequence analysis and shown to belong to the genera Aureobacterium sp. (strain K2-17) and Rhodococcus (Rh. erythropolis K2-12). The other strains isolated clustered into these two groups according to fatty acid analysis. Etherolytic cleavage proceeded under neutral to alkaline conditions with an optimum at around pH 8.5. With Aureobacterium sp. No. K2-17, the degradation rate was zero at a pH of 6 but as much as 60% of the maximum activity was observed at pH 10.5. With Rh. erythropolis K2-12, by contrast, pronounced activity was detected at pH 6.5 while degradation was no longer observed at pH 10.5. The maximum rates of cleavage were about 1 mmol DCPB/h.g dry mass with Aureobacterium sp. No. K2-17 and about 0.6 mmol DCPB/h.g dry mass with Rh. erythropolis K2-12. DCPB and MCPB were utilized to the same extent. Substrate cleavage and product formation (DCP) proceeded at almost equal rates with Aureobacterium sp. No. K2-17 and Rh. erythropolis K2-12, which indicates that this compound was not further metabolized. Only phenoxybutyric acid compounds served as substrates; phenoxyacetic acid and phenoxypropionic acid derivatives were not utilized by these strains.

2-Methyl-4-chlorophenoxyacetic Acid↗

PubMed1997

Identification of plasma proteins facilitated by enrichment on particulate surfaces: analysis by two-dimensional electrophoresis and N-terminal microsequencing.

Plasma protein adsorption on intravenously injectable drug carriers is regarded as an important factor for the fate of the particles in the body after their administration. Therefore, the plasma protein adsorption patterns on a number of different carrier systems were analyzed in vitro employing two-dimensional electrophoresis (2-DE). The particulate systems presented in this study were polystyrene (PS) model particles, PS nanoparticles surface-modified by adsorption of a surfactant, a commercial fat emulsion, and magnetic iron oxide particles used as contrast agents in magnetic resonance imaging. Most of the spots in the plasma protein adsorption patterns could be identified by matching the resulting 2-DE gels with a reference map of human plasma proteins. Several other proteins that indicated preferentially adsorbed proteins on the surface of the particles investigated have either not been identified on the reference map, or their identity was found to be ambiguous. The relevant proteins are all present in plasma in low abundance. Since these proteins were strongly enriched on the surface of the particles, the resulting spots on the 2-DE gels were successfully identified by N-terminal microsequencing. With this approach, two chains of spots, designated PLS:6 and PLS:8, were determined on a plasma reference map: inter-alpha-trypsin inhibitor family heavy chain-related protein (also named PK-120) and a dimer of fibrinogen gamma, respectively. Plasma gelsolin is presented in a 2-DE adsorption pattern of PS model particles. One of the main proteins adsorbed by droplets of a commercial fat emulsion was identified as apoliprotein H. Moreover, the positions of apolipoproteins apoC-II and apoC-III were also verified on the 2-DE protein map of human plasma. Thus, protein adsorption experiments of the kind presented in this study are increasing our insight into human plasma proteins.

Amino Acid Sequence↗

PubMed1997

Influence of fluorescent labelling of polystyrene particles on phagocytic uptake, surface hydrophobicity, and plasma protein adsorption.

PURPOSE: To investigate the influence of fluorescent labelling of polystyrene particles on phagocytic uptake, surface hydrophobicity and protein adsorption. METHODS: Phagocytic uptake was analysed using chemiluminescence. Hydrophobicity was quantified by adsorption measurements of a hydrophobic dye. Protein adsorption was evaluated by two-dimensional electrophoresis. RESULTS: Commercially available fluorescently labelled particles showed marked differences when compared to unlabelled particles: phagocytic uptake and surface hydrophobicity of labelled particles were diminished. Also the plasma protein adsorption pattern was found to be different from the unlabelled particles: for example, the amount of fibrinogen adsorbed was strongly reduced on the labelled particles. On the other hand, some unknown proteins could be detected on the fluorescently marked particles. In contrast, plain polystyrene particles and labelled ones could be successfully synthesised by Paulke which did not show any considerable differences in phagocytic uptake, surface hydrophobicity and protein adsorption. Polysorbate 20 added as stabilizer to particle suspensions led to completely different behaviour of the particles: the particles showed altered protein adsorption patterns, dominated by immunoglobulins and especially by apolipoproteins. Furthermore, these particles were not phagocytized at all. CONCLUSIONS: Surface hydrophobicity and phagocytic uptake in vitro as well as the interactions with plasma proteins of commercially available polystyrene particles were strongly affected by fluorescent labelling. Particles synthesised by Paulke remained unchanged after labelling. The results show the importance of thorough surface characterization for using particles in test systems in vitro and in vivo.

Adsorption↗

PubMed1997

Cytotoxicity of solid lipid nanoparticles as a function of the lipid matrix and the surfactant.

PURPOSE: Assessment of the in vitro cytotoxicity of solid lipid nanoparticles (SLNs) as a function of lipid matrix (Dynasan 114, Compritol ATO 888), and stabilizing surfactant (poloxamers, Tween 80, soya lecithin, and sodium dodecyl sulphate). Comparison with other colloidal carriers should determine their potential use in the clinic. METHODS: SLNs were produced by high pressure homogenisation. Cytotoxicity was assessed by measuring the viability of HL60 cells and human granulocytes after incubation with SLNs. Particle internalisation was quantified by chemiluminescence measurements. RESULTS: The nature of the lipid had no effect on viability; distinct differences were found for the surfactants. Binding to the SLN surface reduced markedly the cytotoxic effect of the surfactants, e.g., up to a factor of 65 for poloxamer 184. The permanent HL60 cell line-differentiated from cells with granulocyte characteristics by retinoic acid treatment-yielded results identical to freshly isolated human granulocytes. In general, the SLNs showed a lower cytotoxicity compared to polyalkylcyanoacrylate and polylactic/glycolic acid (PLA/ GA) nanoparticles. CONCLUSIONS: Because the results are identical when using human granulocytes, differentiated HL60 cells can be used as an easily accessible in vitro test system for i.v. injectable SLN formulations. The SLNs appear suitable as a drug carrier system for potential intravenous use due to their very low cytotoxicity in vitro.

Cell Survival↗

PubMed1997

Determination of plasma protein adsorption on magnetic iron oxides: sample preparation.

PURPOSE: The purpose of this study was to investigate the influence of the sample preparation on the plasma protein adsorption pattern of polysaccharide-stabilized iron oxide particles by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). METHODS: The iron oxide particles were incubated in vitro in human plasma for five minutes. Thereafter, four different methods for particle recovery, including adsorbed proteins from surplus plasma, were investigated: centrifugation, magnetic separation, gel filtration and membrane-based static microfiltration. Adsorbed proteins were desorbed from the particle surfaces by surfactants and analyzed by 2-D PAGE, as described elsewhere (1,2). RESULTS: All the techniques investigated were able to separate small-size iron oxides (approx. 110 nm) and adsorbed proteins from excess plasma. The gels obtained by the different separation procedures displayed almost identical adsorption patterns. Major proteins identified were: fibrinogen, IgG, albumin and an unclassified protein of about 70 kDa with a pI value of 6.5-7.5. CONCLUSIONS: Centrifugation was regarded as the most suitable separation method due to its speed and ease of use. In contrast to gel filtration, any washing media can be used. The magnetic separation process is restricted to particles with high inducible magnetic saturation, in particular, to iron oxides with overall sizes > 50 nm.

Adsorption↗

PubMed1997

The influence of the sample preparation on plasma protein adsorption patterns on polysaccharide-stabilized iron oxide particles and N-terminal microsequencing of unknown proteins.

Adsorption↗

PubMed1996

Fat emulsions based on structured lipids (1,3-specific triglycerides): an investigation of the in vitro interaction with plasma proteins.

Structured lipids (1,3-specific triglycerides) are new chemical entities made by enzymatic transesterification of the fatty acids in the 1,3-positions of the triglyceride. The purpose of this study was to investigate the in vitro interaction of fat emulsions based on either structured lipids or vegetable oils with human plasma proteins employing two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The structured lipids are triglycerides of the SLS and MLM types, where S is short-chain fatty acids (C4), M is medium-chain fatty acids (C8-10) and L is long-chain fatty acids (C16-18). The vegetable oil-based fat emulsions were the commercially available product, Intralipid, and a soybean oil (LLL) emulsion made de novo identically as the emulsions containing structured lipids. The SLS emulsion was found to adsorb a different protein pattern than the MLM and LLL emulsions. The protein pattern of the SLS emulsion was similar to the protein pattern of Intralipid. These findings might explain the in vivo difference in elimination found in another study, where the emulsion based on structured lipids with short-chain fatty acids in the 1,3-positions was removed more slowly from the general blood circulation compared to emulsions based on lipids with long-chain fatty acids in the 1,3-positions (LLL).

Journal Article↗

PubMed1996

Phagocytic uptake and cytotoxicity of solid lipid nanoparticles (SLN) sterically stabilized with poloxamine 908 and poloxamer 407.

Solid lipid nanoparticles (SLN) as alternative intravenous colloidal drug carriers were produced by high pressure homogenisation of melted lipids (glycerolbehenate, cetylpalmitate). Their surface was modified by using hydrophilic poloxamine 908 and poloxamer 407 blockcopolymers in order to reduce the phagocytic uptake by the reticuloendothelial system (RES) after i. v. injection. The phagocytosis reducing effect of the polymers was investigated in vitro in cultures of human granulocytes, uptake was quantified by chemiluminescence. Modification of the SLN with poloxamine 908 and poloxamer 407 reduced the phagocytic uptake to appr. 8-15% compared to the phagocytosis of hydrophobic polystyrene particles. The modified SLN proved more efficient in avoiding phagocytic uptake than polystyrene particles surface-modified with these blockcopolymers (48% and 38%, respectively). Viability determinations revealed the SLN to be 10 fold less cytotoxic than polylactide nanoparticles and 100 fold less than butylcyanoacrylate particles.

Drug Carriers↗

PubMed1995

[Not Available].

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Dentistry↗

PubMed1993

Colloidal carriers for intravenous drug targeting: plasma protein adsorption patterns on surface-modified latex particles evaluated by two-dimensional polyacrylamide gel electrophoresis.

Targeting to specific sites of the body via colloidal carriers is sought in order to reduce drug side effects. The adsorption of plasma proteins on intravenously injected particles is regarded as the key factor in explaining their organ distribution: total bound protein, or, more likely, the presence of specific proteins and their conformation, are expected to influence macrophage uptake. Polystyrene beads, 60 nm in diameter, were used as model carriers; their surface was differentially modified by adsorption of increasingly hydrophilic block copolymers, poloxamers 184, 188 and 407. After incubation in plasma, the patterns of protein adsorption onto coated beads were analyzed by high-resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The behavior of some representative proteins was monitored, including albumin, fibrinogen, IgG, factor B and the apolipoproteins, A-I, A-IV, C-III, E and J. The more hydrophobic the particles, the larger the total amount of bound protein. However, this correlation was not valid for all of the analyzed protein species, which proves that it is insufficient to look only at physicochemical data to predict organ distribution. On the contrary, it is essential to use 2-D PAGE to establish the correlation between adsorbed proteins and carrier behavior in vivo.

Adsorption↗

PubMed1993

Fat emulsions for parenteral nutrition II: Characterisation and physical long-term stability of Lipofundin MCT LCT.

Lipofundin MCT LCT emulsions for parenteral nutrition were characterized in terms of particle size (bulk population), presence of large particles ( 1 mum) and droplet charge (zeta potential as measure of electrostatic stabilization). Photon Correlation Spectroscopy (PCS), microscopy and Laser Diffractometry (LD) were employed as sizing techniques and compared to each other. Zeta potentials were determined by Laser Doppler Anemometry (LDA). The characterization data showed a good batch-to-batch reproducibility. The physical stability of Lipofundin MCT LCT was monitored over a period of 2 years. The mean diameter of the bulk droplet population did not increase. No formation of larger droplets was found by microscopy and LD. A negligible coalescence could only be detected by the very sensitive PCS measurements (two time window analysis). The zeta potential stayed above -45 mV providing sufficient electrostatic stabilization during the whole storage period.

Journal Article↗

PubMed1992

Particle size, surface hydrophobicity and interaction with serum of parenteral fat emulsions and model drug carriers as parameters related to RES uptake.

Fat emulsions for parenteral nutrition, stabilized by egg lecithin, were characterized in terms of parameters relevant to uptake by the reticuloendothelial system (RES), e.g. size distribution, surface hydrophobicity and adsorption of serum components as a measure of the degree of opsonization. Adsorption of serum components was quantified by zeta potential measurement. Fat emulsions for nutrition were compared with emulsions used for drug delivery and model drug carries for intravenous injection. The emulsions for drug delivery were stabilized by the blockcopolymers Poloxamer 188 and 407 (Pluronic F68 and F127) and Poloxamine 908. Model drug carriers were hydrophobic and hydrophilic polystyrene latex particles. Hydrophilic particles were prepared by adsorption of Poloxamine 908 (coating) onto the particle surface. The hydrophobicity and serum protein adsorption decreased from hydrophobic latex particles to egg lecithin emulsions and blockcopolymer emulsions and particles. The data correlated with that in the literature concerning liver uptake in vivo showing complete RES clearance of hydrophobic latex particles, reduced uptake of egg lecithin emulsions and avoidance of RES uptake by Poloxamine 908 coated particles.

Journal Article↗

PubMed1992

Fat emulsions for parenteral nutrition. I: Evaluation of microscopic and laser light scattering methods for the determination of the physical stability.

Droplet size distribution was studied as a measure of the physical stability of fat emulsions. Test emulsions with increasing fractions of larger particles were prepared and investigated by conventional light microscopy and laser light scattering techniques (Photon Correlation Spectroscopy (PCS), Laser Diffractometer). Light microscopy proved to be most sensitive for detecting a few single large particles. All the techniques placed the test emulsions in the same order of increasing content of larger droplets. The application of a two time window analysis for PCS markedly improved the ability to differentiate between emulsions which were very similar in size distribution. In contrast to the applied semi-quantitative microscopic method, the laser techniques characterise the emulsion in absolute figures. Additional zeta potential measurements by Laser Doppler Anemometry (LDA) can quantify reduced electrostatic repulsion as a possible cause for increased formation of larger droplets.

Journal Article↗