Ingvar Sjöholm

Determination of the degree of derivatization of acryloylated polysaccharides by Fourier transform proton NMR spectroscopy

Dextran, glycogen, hydroxyethyl starch, and maltodextrin were derivatized with acrylic acid glycidyl ester at alkaline pH. The degree of derivatization was determined by water-elimination Fourier transform nuclear magnetic resonance (NMR) and compared with a bromination method. The signals from the anomeric protons of the glucose residues were used as an internal standard and the degree of derivatization was obtained from the relation between the integrated signals from the acrylic and anomeric protons. The NMR technique is shown to be more precise and convenient for the determination of acryloyl groups than the bromination method used.

Effect of opsonins on the macrophage uptake of polyacrylstarch microparticles

The macrophage uptake of polyacrylstarch and polyacrylamide microparticles was investigated in vitro. Polyacrylstarch microparticles were rapidly phagocytosed by macrophage monolayers. The uptake was dependent on opsonization and could be partly inhibited with a monoclonal antibody against a macrophage-specific receptor for complement. Moreover, the polyacrylstarch microparticles activated complement of the alternative pathway. The uptake of polyacrylamide microparticles by the macrophages was 50–100 times lower and not dependent on opsonins. The polyacrylamide microparticles did not activate complement of the alternative pathway and the uptake was not inhibited by monoclonal antibodies. These results may explain the different vascular half-lives of the polyacrylstarch microparticles (t12 < 5 min) and polyacrylamide microparticles (t12 = 60 min).

Receptor-mediated uptake of starch and mannan microparticles by macrophages: relative contribution of receptors for complement, immunoglobulins and carbohydrates

The contribution of different macrophage receptors to the uptake of polyacryl starch and polyacryl mannan microparticles by macrophages was studied. Both types of microparticle were taken up to a larger and similar extent when they had been pre-incubated with serum, indicating the importance of serum factors in the uptake process. These results were supported by organ distribution studies in mice, showing that the two microparticles were rapidly targeted to the liver and spleen after i.v. injection. The carbohydrate-specific mannose/fucose receptor was found to contribute significantly to the uptake of non-opsonized but not opsonized mannan microparticles. The two different microparticles were found to activate the alternative complement pathway and to adsorb immunoglobulin G, human serum albumin and fibronectin to their surface. Inhibition experiments provided evidence for the involvement of a complement receptor (CR3) and an Fc-receptor (FCR) for IgG in the uptake of opsonized microparticles.

Biodegradable microspheres. VIII. Killing of Leishmania donovani in cultured macrophages by microparticle-bound primaquine

Primaquine covalently bound to polyacryl starch microparticles has been shown to kill Leishmania donovani in cultured mouse peritoneal macrophages. The drug was derivatized with a tetrapeptide spacer and the derivative (Ala-Leu-Ala-Leu-PQ) coupled to the microparticles. This drug-carrier complex did not kill free promastigotes in suspension, but was effective against amastigotes in cultured mouse peritoneal macrophages. These results show that lysosomal processing of the drug-carrier complex is necessary in order to liberate the pharmacologically active drug. Also, the possible role of reactive oxygen intermediates for the anti-leishmanial effect was studied.

Acrylic microspheres in vivo. X. elimination of circulating cells by active targeting using specific monoclonal antibodies bound to microparticles

The elimination from the blood of 51Cr-labelled mouse erythrocytes modified with trinitrophenyl (TNP) groups was followed in mice. After 24 hours, when a stable concentration of the labelled erythrocytes has been attained, monoclonal anti-TNP-antibodies were given intravenously, either in free, soluble form, or bound to microparticles containing immobilized protein A. The anti-TNP-antibodies induced a rapid elimination of the TNP- and 51Cr-labelled erythrocytes. Over the 8-hours time period studied, the elimination rate was significantly faster when the antibodies were administered bound to the particles. After the elimination of the target cells, the radioactivity was found in the liver, spleen and bone marrow. These results and relevant control experiments indicate that a solid carrier 1. can be directed to a specific target cell with a specific antibody and 2. can induce a rapid elimination of the target cell from the circulation.

Inflammatory response to polyacryl starch microparticles, role of arachidonic acid metabolites

The effect of polyacryl starch microparticles on arachidonic acid metabolism in macrophage cultures and in mice was studied. When macrophages were incubated with the microparticles, significant amounts of arachidonic acid metabolites were released. Two of the major metabolites were identified and found to be prostaglandin E2 and leukotriene C4. Intravenous administration in mice of slowly degradable polyacryl starch microparticles, having a mean diameter of 1 μm, resulted in hepatomegaly and occasionally, inflammatory granulomas in the liver. Rapidly degradable microparticles did not give the same response. When the mice were treated with inhibitors of arachidonic acid metabolism, i.e. indomethacin or timegadine, the microparticle-induced hepatomegaly was partly inhibited. The microparticle-mediated effects were compared with those obtained with a well-characterized macrophageactivating/inflammatory agent, 1,3-β-glucan particles.

Comparative conformational analysis of human somatotropin and biosynthetic methionyl human somatotropin by spectroscopic titrations

Abstract Recombinant DNA techniques have been used to prepare human growth hormone (Met-HGH). It differs from the hormone obtained from human pituitaries (HGH) in that it has an additional amino acid (methionine) in the N-terminal position. The proteins from the two sources were subjected to spectroscopic studies in order to detect any conformational differences resulting from the extra methionine residue. Regardless of origin they can be separated into a main component (b-component) and a component with higher anodic mobility (c-component) by polyacrylamide gel electrophoresis. In each case the b-component can be transformed into the c-component by heat treatment at 50°C with a reaction rate constant of about 1 × 10 −6 s −1 . The components have been studied with spectrophotometric tryptophan fluorescence and spectropolarimetric methods by alkaline titrations (up to about pH 12). No differences could be detected between the HGH- and Met-HGH-forms. During the spectrophotometric titration all the tyrosines were titrated in all components with the same average pK a . The tryptophan fluorescence was quenched with increasing pH in the same way in all the components. The decreasing fluorescence was attributed to the ionization of a tyrosine residue with pK a 10.2. Also in CD titrations no differences between HGH and Met-HGH were detected. However the sensitivity of the technique applied was shown to be high since a small difference in ellipticity between the respective b- and c-components was found indicating a small change in the environment of the tryptophan.