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Hepatic uptake of polystyrene microspheres in rats: effect of particle size on intrahepatic distribution.

The in vivo disposition of polystyrene microsphere (MS) with the particle size of 50 nm (MS-50) or 500 nm (MS-500) was characterized after intravenous administration to rats. A rapid elimination from systemic circulation was observed for both MSs. Tissue distribution of MS-50 and MS-500 at 1 h after intravenous injection indicated that both MSs were exclusively distributed to liver and that small but significant amounts of MS-50 and MS-500 were also distributed to lung and spleen, respectively. To investigate the intrahepatic distribution of MS, liver was separated into liver parenchymal cells (PC) and non-parenchymal cells (NPC) at 1 or 6 h after intravenous administration. The contribution of each cell fraction was dependent on both the size of MS and the time after administration. Furthermore, by separating the NPC into endothelial cells and Kupffer cells using a centrifugal elutriation method, their contribution was also evaluated. For both MSs, Kupffer cells were recognized to be mostly responsible for the hepatic uptake, although a significant amount of MS-50 (about 28% of total uptake) was taken up by PC. On the other hand, there was little contribution of PC (about 5%) to the hepatic uptake of MS-500. The endothelial cells were contributed larger to the uptake of MS-500 (about 24%) than that of MS-50 (13%).

Animals↗

Mechanisms of hepatic disposition of polystyrene microspheres in rats: effects of serum depend on the sizes of microspheres.

To study the mechanisms of the hepatic disposition of polystyrene microspheres (MS), effects of serum on their hepatic disposition characteristics were investigated for MSs with particle sizes of 50 nm (MS-50) and 500 nm (MS-500) by isolated liver perfusion experiments. It was revealed that serum in the perfusate inhibited and promoted the hepatic disposition of MS-50 and MS-500 at 37 degrees C, respectively. However, pre-heating at 56 degrees C or pre-treatment with anti-C3 antibody of serum reduced the promotive effect of serum on the hepatic uptake of MS-500, suggesting that the complement system should be involved as opsonins for the hepatic uptake of MS-500. Hepatic disposition of both MSs at 4 degrees C was reduced by the addition of serum into the perfusate, which could be ascribed to the reduction of the surface hydrophobicity of MSs due to the adsorption of serum proteins onto the surface of MSs and to resultant decrease in non-specific disposition to the liver. From these results, serum was found to function both as the opsonin to enhance the hepatic uptake of MSs and as the inhibitor by reducing non-specific interaction between MSs and the plasma membrane. Whether serum promotes or inhibits the hepatic disposition of MSs would be dependent on the particle sizes of MSs.

Animals↗

Design and construction of novel molecular conjugates for signal amplification (II): use of multivalent polystyrene microparticles and lysine peptide chains to generate immunoglobulin-horseradish peroxidase conjugates.

Spherical polystyrene microparticles expressing a large number of highly reactive functional groups were chemically engineered to generate antibody-enzyme conjugates as novel signal amplification systems. Chemically modified goat anti-human IgG and horseradish peroxidase (HRP) were combined in a 1:5 ratio and attached to 0.44 microm streptavidin microparticles or N-succinimidyl-S-acetylthioacetate (SATA)-activated 0.29 microm amino microparticles with highly reactive free sulfhydryl groups on their surface. The numbers of HRP molecules/microparticle were further increased by coupling HRP to primary amines on N-terminal biotinylated or bromoacetylated polypeptides containing 20 lysine residues prior to conjugation with streptavidin or sulfhydryl groups-containing microparticles. The antibody-poly-HRP immunoconjugates contained an estimated number of 10(5)HRP/streptavidin microparticle and 10(6)HRP/amino microparticle, respectively. These microparticle immunoconjugates efficiently bound to plasma anti-HIV-1 antibodies that had been captured by HIV antigens on 5 microm carboxyl magnetic microparticles and, upon reaction with orthophenyldiamine substrate, produced a detection signal with 5-8 times more sensitivity as compared to conventional HRP-conjugated goat anti-human IgG. The signal amplification technique by microparticle immunoconjugates may provide potentially novel tools for the development of highly sensitive diagnostic systems.

HIV Antibodies↗

Polystyrene beads coated with antibodies directed to HLA class I intracytoplasmic domain: the use in quantitative measurement of peptide-HLA class I binding by flow cytometry.

Protein-reactive, conformation-independent anti-peptide antibodies were raised in rabbits against a C-terminal sequence SDSAQGSDVSLA, common to most HLA-A and -B locus products. Antibodies were coupled to 4.5-microm polystyrene beads through the Fc portion by the use of protein A. The antibody-coupled beads showed a high capacity to bind HLA-A and -B proteins as well as their alpha chains by the intracytoplasmic domain, keeping the extracellular domains solvent exposed. The density of HLA class I proteins bound on the beads was approximately the same as that on cultured B cells. The antibody beads made it possible to quantitate peptide-HLA class I binding, i.e., in vitro HLA class I assembly by flow cytometry. The assembly rate determined by the provisionally called flow cytometric HLA class I assay was 15%-19% for the reassembly of dissociated HLA class I proteins with the released selfpeptides. With single synthetic peptides, the highest rate so far obtained was 6.5%. The assay specificity and reproducibility were satisfactory.

Animals↗

Intestinal necrosis associated with postoperative orally administered sodium polystyrene sulfonate in sorbitol.

We estimated the incidence of intestinal necrosis in 752 hospitalized patients who had received sodium polystyrene sulfonate (SPS). Of these 752 patients, 117 were exposed within 1 week of surgery. Two cases of intestinal necrosis were discovered, both in patients who had received orally administered SPS in sorbitol within 1 week of surgery. Based on these two cases, the postoperative incidence of intestinal necrosis associated with SPS was 1.8%. For comparative purposes, we identified 862 patients who had undergone hemodialysis, renal transplantation, or cardiac transplantation, but did not receive SPS. No cases of idiopathic intestinal necrosis were found in this second group (P = 0.014). These data suggest that SPS in sorbitol-associated intestinal complications may be a relatively common occurrence in postoperatively exposed patients.

Administration, Oral↗

Endocrine-disrupting effects of styrene oligomers that migrated from polystyrene containers into food.

The endocrine-disrupting effects of styrene dimers (SD: NSD-01, -08 and -09) and styrene trimers (ST: NST -01, -03 and -12), which migrated from polystyrene (PS) containers into instant food, were investigated together with styrene monomer (SM) using in vitro and in vivo assays. In the estrogen (ER) and androgen receptor (AR) binding assay, SM, SD and ST showed no binding activity at concentration of 10(-10)-10(-5) mol/l. In order to evaluate the estrogenic activity in vivo, the uterotrophic assay was conducted. When prepubertal and ovariectomized adult rats were dosed with SM, SD and ST for 3 days by subcutaneous injection, these compounds did not induce significant increase in uterine weight. Additionally, to evaluate anti-androgen activity in vivo, the Hershberger assay for anti-androgenic activity in the presence of testosterone treatment was conducted. When castrated, testosterone-treated immature male rats were dosed SM, SD and ST for 7 days by oral gavage, these compounds did not induce a decrease in the seminal vesicle, ventral prostate and levator ani plus bulbocavernosus muscle weights. To evaluate the effects on hormones other than sex hormones, the thyroid hormone receptor (TR) binding assay and rat serum prolactin (PRL) was conducted. In the TR binding assay, SM, SD and ST showed no binding activity at a concentration of 10(-5) mol/l. When ovariectomized rats were dosed with SM, SD and ST for 3 days by sc injection, the results showed there was no change in rat serum PRL. From the above these results, we concluded that SM, SD and ST exhibit no apparent estrogenic, androgenic, anti-androgenic and thyroid activity.

Animals↗

Evaluation of styrene oligomers eluted from polystyrene for estrogenicity in estrogen receptor binding assay, reporter gene assay, and uterotrophic assay.

Styrene dimers (SDs) and styrene trimers (STs) eluted a little from polystyrene have been suspected of having estrogenic activity in the Wingspread Declaration [Our Stolen Futures, 1996] despite the lack of scientific analysis. Therefore, we have studied and reported styrene oligomers to have no endocrine disrupting effects [J. Food Hygienic Soc. Japan 40 (1999) 36; 41 (2000) 109; Yuki Goseikagaka Kyokaishi 57 (1999) 58; Bunseki Kagaku 49 (2000) 493, 857; Food Chem. Toxicol. 39 (2001) 1233; 40 (2002) 129]. However, Ohyama et al. reported that certain styrene oligomers have estrogenic effects in E-SCREEN and estrogen receptor (ER) binding assay [Environ. Health Perspect. 109 (2001) 699]. Recently, several assay systems have been developed, and a few of them can show false positive reactions at the high concentrations to which test compounds are precipitated [J. Health Sci. 48 (2002) 83]. In order to assess the estrogenic effect of SDs and STs in more detail, we examined the accuracy of the binding assay system and tested SDs and STs by three types of ER binding assay. In one ER binding assay, the same method that Ohyama et al. performed, SDs and STs showed a little estrogenic activity at high concentration; they did not dissolve, but this assay system tended to detect false positive effects at high concentration. In contrast, in the other assay systems, SDs and STs did not show any binding affinity to ER. In addition, luciferase reporter gene assay in HeLa cells transfected with ER expression plasmid and reporter plasmid, as a newly developed standard assay, and immature rat uterotrophic assay were conducted. In these tests, styrene oligomers showed no estrogenic activity.

Animals↗

Measurement of styrene oxide in polystyrenes, estimation of migration to foods, and reaction kinetics and products in food simulants.

The concentration of styrene-7,8-oxide has been measured in nine base resins and 16 samples of polystyrene articles intended for food contact. The epoxide was not detected in the resins (limit of detection 0.5 mg/kg) but was found in 11 of the 16 packaging samples at up to 2.9 mg/kg. Assuming that the propensity of styrene oxide to migrate is the same as styrene monomer, and using existing survey data for styrene monomer in packaging and foods, the migration levels expected for styrene oxide were calculated. Estimates were from 0.002 to 0.15 microgram/kg styrene oxide in foods. The stability of styrene oxide in the four standard EU food simulants was studied at 40, 100, 150 and 175 degrees C, to establish the transformation products to be expected following migration testing. The half-life at 40 degrees C in distilled water, 15% aqueous ethanol, 3% aqueous acetic acid and olive oil was 15, 23, < 1, > 2000 hr, respectively. The principal product was the diol from hydrolysis of the epoxide group. Ring opening in aqueous ethanol simulant gave the diol and also the glycol monoethyl ether. It is concluded that this instability of styrene oxide will reduce concentrations in foods, from an already low migration level to even lower levels with the formation of hydrolysis products that are less toxic than the parent epoxide.

Carcinogens↗

Biliary excretion of polystyrene microspheres depends on the type of receptor-mediated uptake in rat liver.

Hepatic uptake and biliary excretion of fluorescein isothiocyanate-labeled polystyrene microspheres with a particle size of 50 nm (MS-50) were studied in rats. Liver perfusion studies revealed that not only apo-E-mediated but also asialoglycoprotein receptor-mediated uptake is involved in the mechanism of the serum protein-dependent uptake of MS-50 in the liver. The uptake of MS-50 mediated by apo-E contributes more to the total uptake of MS-50 by the hepatocytes than that via asialoglycoprotein receptor in the presence of serum in the perfusate. Furthermore, it was found that MS-50 is substantially excreted into the bile by transcytosis. The extent of exocytosis of MS-50 taken up by the hepatocytes was much higher after MS-50 was endocytosed via asialoglycoprotein receptor than after taken up via the process mediated by apo-E. On the basis of these results, a possible regulation of the intracellular sorting of ligands, depending on the receptor-mediated uptake mechanism, was inferred.

Animals↗

Interaction of polystyrene microspheres with liver cells: roles of membrane receptors and serum proteins.

Our previous studies have demonstrated that serum would play an important role in the hepatic disposition of polystyrene microspheres (MS) and that complement C3 should be involved as the serum opsonin. In this study, we tried to identify the entity of other serum opsonins and dysopsonin for the hepatic uptake of MSs with particle sizes of 50 nm (MS-50) and 500 nm (MS-500) by isolated liver perfusion studies using a recirculation procedure in rats. Pretreatment of the liver by trypsin significantly suppressed the serum-dependent hepatic uptake of both MSs, suggesting that some protein components on the cell surface should be necessary for the serum-dependent phagocytosis of MSs. Pretreatment of the serum by the anti-fibronectin antibody resulted in a significant reduction in the hepatic disposition of MS-500 (49% of control), suggesting that fibronectin should also work as the opsonin for the hepatic uptake of MS-500. The hepatic disposition of both MSs in the presence of serum was inhibited by the addition of N-acetylgalactosamine into the perfusate, suggesting the possible involvement of lectin in the serum-dependent hepatic uptake of MSs. Furthermore, a more intensive hepatic disposition of MSs was observed in the presence of plasma compared with that in the presence of serum in the perfusate, suggesting the possible involvement of blood coagulation factors, such as fibrinogen, as the opsonin in the hepatic disposition of MSs.

Animals↗

Affinity chromatography of thrombin on modified polystyrene resins.

Insoluble polystyrenes substituted with sulphonate and L-arginyl methyl ester (PAOM) present substituents mimicking the reactive binding site of antithrombin III. These materials have a specific affinity for thrombin. The binding of the enzyme is reversible and the eluted thrombin remains active. Consequently, these resins can be used as stationary phases in affinity liquid chromatography in order to purify thrombin with a high biological activity. The influence of different characteristics of such polymers (substitution ratio, average particle size, affinity constant, synthesis conditions) on the purification performance is studied. Human prothrombin complex concentrate is activated and applied onto the gel. A purified human thrombin of high specific activity is separated with a high recovery of biological activity of the enzyme.

Chromatography, Affinity↗

Separation of peptides on a polystyrene resin column.

A high-performance liquid chromatographic column of porous spherical polystyrene--divinylbenzene copolymer (Hamilton PRP-1) was found to be useful for reversed-phase resolution of a wide range of peptides. Recoveries and resolution were comparable with those from more widely used alkyl silica-based columns. Tests involving more than 40 peptides, ranging from 2 to 34 residues, and 3 proteins, indicated wide applicability of this column. The retention times of peptides of known composition are predicted. The stability of the resin at high pH permits protocols of separation involving successive chromatograms at widely different pH values, and offers a second dimension to the resolving power of a single column.

Amino Acids↗

Analytical derivatizations of volatile and hydrophilic carbonyls from aqueous matrix onto a solid phase of a polystyrene-divinylbenzene macroreticular resin.

Extraction and derivatization of carbonyls to benzyloximes, pentafluorobenzyloximes or 2,4-dinitrophenylhydrazones is simplified and reaction times are substantially reduced by simultaneous sorption and derivatization from aqueous solution onto a solid phase. In this reaction a macroreticular polystyrene-divinylbenzene resin acts as a sorbent and catalyst to allow simultaneous extraction and derivatization of hydrophilic and lipophilic aldehydes and ketones from simple as well as complex matrices including plasma. Conversion to the 2,4-dinitrophenylhydrazones or pentafluorobenzyloximation at ambient temperature requires 10 and 20 min, respectively. These reaction conditions correspond to at least a 6-fold reduction in reaction times for derivatization of the reactive aldelhydes and a 36-72-fold reduction for preparation of derivatives for the slower reacting ketones.

Aldehydes↗

Studies on in vitro release behaviour of indomethacin-loaded polystyrene microparticles.

Indomethacin-loaded polystyrene microparticles were prepared by emulsion-solvent evaporation method from an aqueous system. The effect of different parameters like concentration of aqueous phase emulsion stabilizer, volume of the organic disperse phase and initial drug loading on drug content and release of drug were investigated. Keeping the drug-polymer ratio constant, variation in the concentration of emulsion stabilizer and volume of the organic disperse phase did not produce any significant change either in the actual drug content or in the drug release. The initial drug loading, however, greatly influenced the drug release which, as revealed by different analyses, was due to the presence of drug in different physical forms in the microparticles. Physical characterization using thin layer chromatography and infrared spectroscopy apparently revealed the absence of drug degradation and sizeable interaction between the drug and the polymer. Regardless of lack of interaction, thermodynamic properties such as solubility of the drug in the polymer and fraction of the drug present in crystalline form were determined by using differential scanning calorimetry and was further substantiated with scanning electron micrography and X-ray diffraction analysis.

Anti-Inflammatory Agents, Non-Steroidal↗

Adsorption of amphipathic dendrons on polystyrene nanoparticles.

Adsorption of dendrons onto nanoparticles may provide new model structures which may be useful in drug and gene delivery. Tritiated amphipathic dendrons having three lipidic (C(14)) chains coupled to branched (dendritic) lysine head groups with 8, 16 or 32 free terminal amino groups have been synthesised by solid phase peptide techniques. The interaction between these tritiated dendrons and 200 nm polystyrene latex nanoparticles was investigated in phosphate buffered saline. The amount of dendron adsorbed increased with increasing concentration of dendrons and then decreased. Maximum adsorption of dendrons per gram of nanoparticles was found to be between 8.2 and 84 x 10(-6)M, the amounts adsorbed being inversely proportional to the number of amino groups present in the molecule. The number of dendron molecules adsorbed per nanoparticle was found to be between 430 and 4421. The degree of adsorption was found to be slightly altered by the temperature.

Adsorption↗

Sandwich enzyme immunoassay of human chorionic gonadotropin using polystyrene beads as solid support.

A solid-phase sandwich enzyme immunoassay for human chorionic gonadotropin (hCG) employing monoclonal antibodies directed against beta- and alpha-subunits is described. Polystyrene beads used as solid support were coated with monoclonal anti-beta-hCG antibody. Monoclonal anti-alpha-hCG antibody was labelled with the enzyme, horseradish peroxidase. The assay could be performed by a "one-step" or "two-step" procedure. The sensitivity of the two-step procedure was 7.8 ng hCG/ml with a linear range up to 500 ng hCG/ml. The one-step procedure had a measurement range between 15.6 and 125 ng hCG/ml. Within the limits of measuring ranges, the two procedures had similar accuracy, the correlation coefficient being 0.987. Twenty-one urine samples from pregnant women were analysed by the two-step procedure and also by radioimmunoassay. The results were comparable, with r = 0.956.

Antibodies, Monoclonal↗

Hydrolysis of plasmid DNA catalyzed by Co(III) complex of cyclen attached to polystyrene.

Reactivity of the Co(III) complex of cyclen (CoCyc) in the hydrolytic cleavage of supercoiled pUC18 DNA leading to the formation of the corresponding open circular form was enhanced by >200 times upon attachment of CoCyc to cross-linked polystyrenes. Thus, half-lives as short as 40 min were achieved by the resin-based CoCyc in cleavage of the supercoiled DNA at 4 degrees C.

Cobalt↗

Hydrolysis of linear DNA duplex catalyzed by Co(III) complex of cyclen attached to polystyrene.

To design artificial restriction enzymes, synthetic catalytic centers that effectively hydrolyze linear double-stranded polydeoxyribonucleotides are needed. The Co(III) complex of cyclen (CoCyc) attached to polystyrene derivatives hydrolyzes linearized pUC18 DNA with half-lives as short as 30 min at 25 degrees C. The catalytic activity of CoCyc is enhanced by >150 times on attachment to the resin.

Catalysis↗