Adhesion of platelets to microcapsules and the role of complement.

Rabbit platelets rapidly adhered to carboxylated polyamide microcapsules through the adsorbed layer of plasma components. These components were found to be heat-labile proteins, which exist in fresh serum, and to demand calcium ions to function. These findings ascribed the components to the complement system. In fact, a good correlation was obtained between platelet adhesion to, and complement fixation by, the microcapsules. Moreover, the activation of the complement system by the microcapsules was assumed to proceed via the classical pathway. It was concluded that adhesion of platelets to the microcapsules is brought about by immune adherence.

Preparation of poly(D,L-lactide) and copoly(lactide-glycolide) microspheres of uniform size.

In an attempt to prepare monodisperse poly(D,L-lactide) and copoly(lactide-glycolide) microspheres, a novel emulsification technique (membrane emulsification) was employed and the preparation conditions which might affect the monodispersity were evaluated. With this technique nearly monodisperse poly(D,L-lactide) and copoly(lactide-glycolide) microspheres were successfully prepared and their sizes were controllable only by making use of microporous glass membranes of different pore sizes. However, in the present system of emulsion (methylene chloride/water) the surfactant used was limited to ionic ones and the amount of polymers available for the formation of microspheres was inevitably too small in concentration to entrap sufficient amounts of drug. As for the drug release, the effect of particle size was not appreciable but the method of solvent removal gave a great influence; the solvent extraction method showed a more drug-sustaining effect than did the solvent evaporation method. The present results suggest the possibility of making drug-loaded and biodegradable monodisperse microspheres.

Platelet adhesion onto sulfonated artificial red blood cells.

Rabbit hemolysate-loaded microcapsules having sulfonic acid groups on their surface were prepared and rabbit platelet adhesion onto the sulfonated surface was examined kinetically. Rabbit platelets adhered more markedly onto the surface of a higher charge density than that of a lower one, indicating a trend similar to that found in the case of the surface having carboxyl groups. Moreover, the platelets were found to be stickier in the buffer solution than in the plasma, though the dependence of platelet adhesion in the buffer solution on the magnitude of surface charge of the sulfonated artificial red blood cells was still recognized.

An approach to prepare microparticles of uniform size.

A novel emulsification method which employs a glass membrane of controlled pore size was adopted to prepare various types of microparticles. The microparticles thus obtained were uniform in size and the procedure proved applicable to a wide variety of microencapsulation processes. Furthermore, the particle size could be controlled by selecting preparative conditions.

Platelet adhesion onto microcapsule membranes with different degrees of sulfonation.

Microcapsules having different numbers of sulfonic acid groups on their surfaces were prepared by the interfacial polycondensation method. Platelets adhered to these capsules, and the rate and degree of platelet adhesion were found to be remarkable on those microcapsules that had high surface charge compared to those with low surface charge. This trend was strengthened by the coating of the capsules with plasma, while the electrophoretic mobilities of the plasma-coated capsules showed a considerable reduction and no appreciable difference between the respective mobilities could be observed. This trend suggest that the adsorption of plasma components on microcapsules, and not their surface negative charge, affected platelet adhesiveness directly. In connection with the glycosyl transferase hypothesis, the adsorption pattern and its effect on platelet adhesion of albumin, gamma-globulin, and fibrinogen were examined. The protein adsorption pattern varied depending on both the type of protein and the magnitude of negative charge on the microcapsule surface, but its effect on platelet adhesion was not fully consistent with the glycosyl transferase hypothesis. It was concluded from the findings that the surface negative charge of the capsules strongly affected the composition, molecular orientation, and/or configuration of the adsorbed plasma components, which probably induced the differences in platelet adhesiveness depending on the magnitude of surface negative charge of the microcapsules.

Possible application of polyamine graft copolymer to targeting drug delivery.

Polystyrene microspheres were coated with polyamine graft copolymer and mixed with rat lymphocyte suspension. In the mixture of T and B cells, the microspheres formed large aggregates, while they were fairly well dispersed in the T cell suspension. This result was understood to be the result of preferential adsorption of B cells to the microspheres, indicating it would be possible to deliver drugs to B cells alone with these copolymer-coated microspheres.

Preparation of heat responding artificial cells.

Enzyme-loaded microcapsules were prepared and treated with dimyristoylphosphatidylcholine molecules so as to control the permeation of substrates through the microcapsule membranes. The activities of enclosed enzymes increased with increase in ambient temperature, but gave an abrupt change at around the phase transition temperature of the lipid. The experimental results indicate the possibility of making artificial cells which respond to the temperature change of the surrounding medium.

A study on heat-resistance of microencapsulated glucose oxidase.

Polyurea microcapsules containing glucose oxidase were prepared and their thermodurability was examined. Microencapsulated glucose oxidase was found to be more stable to heat than the enzyme in free solution. This stability was enhanced with an increase in the amount of enzyme entrapped in the microcapsules.

Preparation of polyamide microcapsules having narrow size distributions.

A novel emulsification technique which employs microporous glass membranes was adopted to prepare polyamide microcapsules of narrow size distribution. This technique proved to be simple and quite efficient for getting fairly uniform sizes of dispersed liquid droplets. Polyamide microcapsules prepared by using this technique were found not completely monodisperse, but far more satisfactory than those by any conventional mechanical methods in view of their narrow size distribution. This method also seems promising to get various sizes of the capsules by selecting the pore size of the glass membranes.

Phagocytosis by guinea pig polymorphonuclear leukocytes of liposomes stabilized with polysaccharides.

Hemolysate-loaded liposomes were prepared with their surface coated by polysaccharides, carboxymethylchitin, carboxymethylcellulose and carboxymethylchitosan. Phagocytosis by guinea pig polymorphonuclear leukocytes of these coated liposomes was measured in view of electrostatic interactions between the cells and the liposomes. This view revealed to hold at least in quite an early stage of phagocytosis, but some specific interactions were assumed to exist between the cells and the liposomes. N-acetyl groups of the chitin molecules were suggested to play a role in the complex processes of phagocytosis.

Phagocytosis of monosaccharide-binding latex particles by guinea-pig polymorphonuclear leucocytes.

Phagocytosis of monosaccharide-binding latex particles by guinea-pig polymorphonuclear leucocytes (PMNs) was investigated. When neuraminic acid or glucuronic acid was chemically bound to the surface of latex particles the degree of phagocytosis was somewhat lower than that for bare, glucose-, mannose- or rhamnose-binding latex particles. These experimental findings were interpreted in terms of the kind of monosaccharide and the surface potential and surface hydrophobicity of latex particles. As a result, it was concluded that the inhibitory action of neuraminic acid or glucuronic acid on phagocytosis arises from an increase in the surface hydrophilicity of latex particles brought about by the hydroxyl groups and negative charges on the chemically bound neuraminic acid or glucuronic acid molecules. Physically adsorbed glucuronic acid molecules were also found to decrease the degree of phagocytosis of latex particles by PMNs, though not so remarkable as the chemically bound monosaccharide molecules.

Effect of particle size on phagocytosis of latex particles by guinea-pig polymorphonuclear leucocytes.

The effect of the size of foreign particles on phagocytosis by guinea-pig polymorphonuclear leucocytes (PMNs) was investigated using latex particles having approximately the same surface potentials but different sizes. Two types of latexes with different negative surface potentials, those having amino groups (Ami-latexes) and those having carboxylic acid groups (Ca-latexes), were used in this experiment. Since Ca-latexes had different surface potentials for different particle sizes, the carboxylic acid groups were modified by the carbodiimide method to make the surface potentials almost equal. Phagocytosis was estimated by the rate of oxygen consumption. It was found that the phagocytosis reached its maximum when the latex particle size was about 0.5 microns. This finding was plausibly explained by considering the two probabilities that a latex particle comes into contact with a PMN surpassing a potential barrier and the contact takes place at certain receptor areas favourable to phagocytosis on the PMN surface.

An interpretation of cell separation mechanism on polyamine graft copolymers.

Interaction between polyamide microcapsules having different balances of negative and positive charges on their surface, that is, different isoelectric points and copolymers with different numbers of polyamine macromer of a definite chain length grafted on poly(2-hydroxyethyl methacrylate) backbone was studied by measuring the amount adhered of the microcapsules onto the surface of copolymer-coated glass beads at different pH values. Maximum microcapsule adhesion was observed for a proper combination of microcapsule and copolymer to suggest that separation of a specified cell population from others on the surface of copolymer-coated glass beads can be explained in terms of preferential adhesion through electrostatic interaction of the specified cells with the copolymer.

Adsorption of negatively charged microcapsules to a poly(2-hydroxyethylmethacrylate)/polyamine graft co-polymer surface.

The electrokinetic properties of artificial particles--microcapsules--were compared with those of the rat lymphocyte sub-populations, T and B cells, and similar results were obtained. The affinity of the microcapsules to the polyamine graft co-polymer, which was claimed to be capable of separating the cells efficiently, was measured and it revealed that not the magnitude of the surface negative charge and the kind of dissociable groups, but the balance of the anionic and cationic charges on the microcapsule surface played a key role in their adsorption to the co-polymer.