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Biomedical subjects

M Lück

Publications and source records attributed to M Lück.

At least 19 recordsLinked to original sources

Partial solubility parameters of poly(D,L-lactide-co-glycolide).

During production of microparticles by the polymer incompatibility method a polymer solution is demixed. Therefore, investigations into solubility are often carried out when the suitability of a polymer is examined. Solubility parameters can be used to quantify the solubility. For polylactide and polyglycolide as commonly employed copolymers for microparticles the solubility parameters have rarely been documented. This study aimed to determine solubility parameters and partial solubility parameters for different proportions of lactide to glycolide for poly(D,L-lactide-co-glycolide) (PLGA). The employed methods were compared and solubility maps established. Finally the accuracy of the results was discussed for different polymer batches which were used for production of microparticles. Although the turbidity titration method was found to be the most precise, it was not possible to sufficiently explain the differences between three polymer batches during microparticle production.

Chemistry, Pharmaceutical↗

Polymer erosion in PLGA microparticles produced by phase separation method.

This article deals with polymer erosion in biodegradable microparticles produced using the phase separation method. Poly(lactic-co-glycolic acid) copolymers with different compositions and molecular weights were employed. The microparticles were stored in phosphate buffer for 6 months. The molecular weight of the polymers was determined by size exclusion chromatography, and the weight loss was monitored gravimetrically. No weight loss was measured in the first weeks, although the molecular weight decreased significantly already from the start. After a certain storage period which was found to be specific for the type of polymer, the weight of the microparticles decreased rapidly. The start of this weight loss occurred when the molecular weight of the polymer in the degrading microparticles reached a threshold of approximately 15,000. This critical molecular weight was found to be identical for all investigated polymers, i.e. it was independent of the initial molecular weight of the polymer and of the lactic-glycolic ratio.

Chromatography, Gel↗

'Stealth' corona-core nanoparticles surface modified by polyethylene glycol (PEG): influences of the corona (PEG chain length and surface density) and of the core composition on phagocytic uptake and plasma protein adsorption.

Nanoparticles possessing poly(ethylene glycol) (PEG) chains on their surface have been described as blood persistent drug delivery system with potential applications for intravenous drug administration. Considering the importance of protein interactions with injected colloidal dug carriers with regard to their in vivo fate, we analysed plasma protein adsorption onto biodegradable PEG-coated poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA) and poly(varepsilon-caprolactone) (PCL) nanoparticles employing two-dimensional gel electrophoresis (2-D PAGE). A series of corona/core nanoparticles of sizes 160-270 nm were prepared from diblock PEG-PLA, PEG-PLGA and PEG-PCL and from PEG-PLA:PLA blends. The PEG Mw was varied from 2000-20000 g/mole and the particles were prepared using different PEG contents. It was thus possible to study the influence of the PEG corona thickness and density, as well as the influence of the nature of the core (PLA, PLGA or PCL), on the competitive plasma protein adsorption, zeta potential and particle uptake by polymorphonuclear (PMN) cells. 2-D PAGE studies showed that plasma protein adsorption on PEG-coated PLA nanospheres strongly depends on the PEG molecular weight (Mw) (i.e. PEG chain length at the particle surface) as well as on the PEG content in the particles (i.e. PEG chain density at the surface of the particles). Whatever the thickness or the density of the corona, the qualitative composition of the plasma protein adsorption patterns was very similar, showing that adsorption was governed by interaction with a PLA surface protected more or less by PEG chains. The main spots on the gels were albumin, fibrinogen, IgG, Ig light chains, and the apolipoproteins apoA-I and apoE. For particles made of PEG-PLA45K with different PEG Mw, a maximal reduction in protein adsorption was found for a PEG Mw of 5000 g/mole. For nanospheres differing in their PEG content from 0.5 to 20 wt %, a PEG content between 2 and 5 wt % was determined as a threshold value for optimal protein resistance. When increasing the PEG content in the nanoparticles above 5 wt % no further reduction in protein adsorption was achieved. Phagocytosis by PMN studied using chemiluminescence and zeta potential data agreed well with these findings: the same PEG surface density threshold was found to ensure simultaneously efficient steric stabilization and to avoid the uptake by PMN cells. Supposing all the PEG chains migrate to the surface, this would correspond to a distance of about 1.5 nm between two terminally attached PEG chains in the covering 'brush'. Particles from PEG5K-PLA45K, PEG5K-PLGA45K and PEG5K-PCL45K copolymers enabled to study the influence of the core on plasma protein adsorption, all other parameters (corona thickness and density) being kept constant. Adsorption patterns were in good qualitative agreement with each other. Only a few protein species were exclusively present just on one type of nanoparticle. However, the extent of proteins adsorbed differed in a large extent from one particle to another. In vivo studies could help elucidating the role of the type and amount of proteins adsorbed on the fate of the nanoparticles after intraveinous administration, as a function of the nature of their core. These results could be useful in the design of long circulating intravenously injectable biodegradable drug carriers endowed with protein resistant properties and low phagocytic uptake.

Journal Article↗

Complement activation by model drug carriers for intravenous application: determination by two-dimensional electrophoresis.

The interactions of intravenously injected drug carriers with blood proteins are considered as an important factor for the fate of the particles after their administration. Protein adsorption on latex particles applied as model for intravenous drug carriers was analysed using two-dimensional electrophoresis (2-DE). The particles were incubated in citrated plasma, serum and heat-inactivated serum, respectively. Incubation in the various media resulted in clear differences in the protein adsorption patterns. Two characteristic protein spots were determined to be enriched on the 2-DE gels only after incubation of the particles in serum. Employing N-terminal microsequencing these protein spots were identified to be fragments of the complement protein C3. Enrichment of these particular spots was most likely a result of complement activation by the particles. Mechanism of C3 binding to the particle surface and subsequent inactivation by cleavage are discussed in order to explain the results. It could be demonstrated that 2-DE analysis provides the possibility to distinguish between adsorption and covalent attachment of C3 to particulate surfaces. The findings indicate that complement activation was caused by covalent binding of the C3 component C3b to the particles' surface. The influence of the incubation medium on the in vitro protein adsorption of particulate drug carriers has to be considered when a correlation between the protein adsorption pattern and the in vivo behaviour of the particles is approached.

Adsorption↗

Molecular heterogeneity of free PSA in sera of patients with benign and malignant prostate tumors.

OBJECTIVE: To analyze free prostate-specific antigen (f-PSA) in sera from patients with prostate cancer (PCa) and benign prostatic hyperplasia (BPH), and to detect possible differences in subtypes as potential diagnostic parameters. MATERIALS AND METHODS: PSA was purified from sera by an immunoaffinity procedure developed on the basis of oriented antibody immobilization, and subjected to size exclusion chromatography (SEC), Western blotting, and N-terminal amino acid sequencing. RESULTS: The novel procedure allowed the purification of PSA with high yield from sera containing PSA <10 ng/ml. SEC under nonreducing conditions as well as Western blots demonstrated the presence of several molecular forms of f-PSA. Three of the smaller polypeptides exhibited the N-terminal sequence of PSA while one represented the C-terminal fragment Lys(146)-Pro(237). Shortening of some polypeptides by the N-terminal amino acid Ile(1) suggestive of aminopeptidase action was also observed. No propeptide sequence could be detected, and none of the bands from patient sera reacted with antibodies raised against propeptide antigens. BPH sera expressed higher proportions of smaller PSA fragments per unit p33, and contained significant amounts of fragments <14,000 which appeared to be very low or absent from most PCa sera. CONCLUSIONS: f-PSA as obtained from BPH and PCa sera represents a heterogeneous fraction. The major component (p33) is not in the nicked form and does not contain proPSA. Diagnostic potential could arise from the quantitative differences of the smaller PSA derivatives seen between PCa and BPH sera.

Amino Acid Sequence↗

Plasma protein adsorption on biodegradable microspheres consisting of poly(D,L-lactide-co-glycolide), poly(L-lactide) or ABA triblock copolymers containing poly(oxyethylene). Influence of production method and polymer composition.

Biodegradable particulate systems have been considered as parenteral drug delivery systems. The adsorption of plasma proteins on micro- and nanoparticles is determined by the surface properties and may, in turn, strongly influence the biocompatibility and biodistribution of both carriers. In the present study the influence of the polymer composition and the production method of microspheres on the in vitro plasma protein adsorption were investigated using two-dimensional electrophoresis (2-DE). Microparticles were prepared from poly(l-lactide) (l-PLA), poly(d,l-lactide-co-glycolide) (PLGA), and ABA triblock copolymers containing hydrophilic poly(oxyethylene) (B-blocks) domains connected to hydrophobic polyesters (A-blocks). Two different microencapsulation methods were employed, namely the w/o/w emulsion solvent evaporation method and the spray-drying technique. It could be demonstrated that the polymer composition and, especially, the encapsulation technique, influenced the interactions with plasma proteins significantly. For example, the percentages of several apolipoproteins in the plasma protein adsorption patterns of spray-dried PLGA- and l-PLA-particles were distinctly higher when compared to the adsorption patterns of the particles produced by the w/o/w-technique. Some adsorbed proteins were found to be characteristic or even specific for particles produced by the same method or consisting of identical polymers. Polyvinyl alcohol used as stabilizer in the w/o/w-technique may decisively influence the surface properties relevant for protein adsorption. The plasma protein adsorption on particles composed of ABA copolymers was drastically reduced when compared to microspheres made from pure polyesters. The adsorption patterns of ABA-particles were dominated by albumin. The plasma protein adsorption patterns detected on the different microspheres are likely to affect their in vivo performance as parenteral drug delivery systems.

Adsorption↗

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↗

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↗

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↗

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↗

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↗

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↗

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↗

Spreading of trypsinized cells: cytoskeletal dynamics and energy requirements.

The spreading of trypsinized XTH-2 cells (a line derived from Xenopus laevis tadpole heart endothelia) on glass was investigated. Three phases can be distinguished: (1) blebbing of rounded cells, first attachment to a solid substratum and formation of a broad smooth contact area; (2) organization of a peripheral zone of actin fibrils and reinforcement of the basal cytoplasm by a stress fibre-like pattern; (3) extension of lamellae. The first phase seems to be independent of a supply of metabolic energy, while the others clearly depend on it. This is concluded from the close relationship between cellular projection area and energization of mitochondria as revealed by (a) the fluorescence intensity of cells vitally stained with the mitochondria-specific fluorochrome DASPMI (2-4-(dimethylamino)-styryl-1-methylpyridinium-iodine); (b) the degree of spreading in the presence of inhibitors of respiration; (c) effective amelioration of spreading (phases (2) and (3] under conditions of high ATP content. In phase (2) the extension of the central part of the cells becomes stabilized, the cell body settles on the basal cytoplasmic layer and further expansion of the projection area is achieved by lamella formation (phase (3]; motile and stabile regions of the cells become separated. This sequence of events is interpreted as a self-organizing process based on the development of internal hydraulic pressure, actin polymerization and contraction of the newly developed actomyosin network. During trypsinization, depolymerization of actin does not occur but rather on addition of Ca2(+)-containing media. Cellular ATP content drops as well on trypsinization, as on addition of Ca2+. Manganese promotes spreading by decreasing F-actin disassembly and maintaining a high level of cytosolic ATP, most probably because it is not accepted by the calcium pumps. Regarding the association of glycolytic enzymes with F-actin and their influence on actin assembly, lactate dehydrogenase has been inhibited with oxamic acid. This treatment improves the correlation between F-actin content and the degree of spreading; however, the total amount of F-actin remains smaller and the cells spread more.

Actins↗