California Institute of Technology

The United States of America As Represented by the Administrator of the

University of Southern California

Alan Rembaum

Shiao-Ping S Yen

William J Dreyer

Robert S Molday

Willi Volksen

Amitava Gupta

Elias Klein

Manchium Chang

Richard C Yen

Michael Steven Colvin

Harry B Gray

Roger M Williams

Gil F Richards

Won-Kyu Rhim

Zoltan A Tokes

Michael T Hyson

Robert F Landel

Shlomo Margel

Kathryn E Rogers

Vaclav Hadek

Robert B Somoano

Carl J Wallace

Very small, individual polymeric microspheres with very precise size and a wide variation in monomer type and properties are produced by deploying a precisely formed liquid monomer droplet, suitably an acrylic compound such as hydroxyethyl methacrylate into a containerless environment. The droplet which assumes a spheroid shape is subjected to polymerizing radiation such as ultraviolet or gamma radiation as it travels through the environment. Polymeric microspheres having precise diameters varying no more than plus or minus 5 percent from an average size are recovered. Many types of fillers including magnetic fillers may be dispersed in the liquid droplet.

Production of monodisperse, polymeric microspheres

Metal oxide containing polymers and particularly styrene, acrylic or protein polymers containing fine, magnetic iron oxide particles are formed by combining a NO.sub.2 -substituted polymer with an acid such as hydrochloric acid in the presence of metal, particularly iron particles. The iron is oxidized to fine, black Fe.sub.3 O.sub.4 particles which deposit selectively on the polymer particles. Nitrated polymers are formed by reacting functionally substituted, nitrated organic compounds such as trinitrobenzene sulfonate or dinitrofluoro benzene with a functionally coreactive polymer such as an amine modified acrylic polymer or a protein. Other transition metals such as cobalt can also be incorporated into polymers using this method.

Magnetic particles

Microspheres of acrolein homopolymers and copolymer with hydrophillic comonomers such as methacrylic acid and/or hydroxyethylmethacrylate are prepared by cobalt gamma irradiation of dilute aqueous solutions of the monomers in presence of suspending agents, especially alkyl sulfates such as sodium dodecyl sulfate. Amine or hydroxyl modification is achieved by forming adducts with diamines or alkanol amines. Carboxyl modification is effected by oxidation with peroxides. Pharmaceuticals or other aldehyde reactive materials can be coupled to the microspheres. The microspheres directly form antibody adducts without agglomeration.

Polyacrolein microspheres

Substrates, particularly inert synthetic organic resin beads (10) or sheet (12) such as polystyrene are coated with a covalently bound layer (24) of polyacrolein by irradiation a solution (14) of acrolein or other aldehyde with high intensity radiation. Individual microspheres (22) are formed which attach to the surface to form the aldehyde containing layer (24). The aldehyde groups can be converted to other functional groups by reaction with materials such as hydroxylamine. Adducts of proteins such as antibodies or enzymes can be formed by direct reaction with the surface aldehyde groups.

Hybrid microspheres

Anthracycline cancer drugs are covalently coupled to polymeric particles having a diameter of greater than about 0.5 microns to produce pharmaceutical preparations having enhanced cytostatic activity. The coupling of drugs to polymeric carriers shows increased activity against cancer cells, allows the bound drug to retain its cytostatic activity after repeated uses and shows an increase in activity against drug resistant cells.

Drug delivery by polymeric carriers

A synthetic organic resin is coated with a continuous layer of contiguous, tangential, individual microspheres having a uniform diameter preferably between 100 Angstroms and 2000 Angstroms. The microspheres are an addition polymerized polymer of an unsaturated aldehyde containing 4 to 20 carbon atoms and are covalently bonded to the substrate by means of high energy radiation grafting. The microspheres contain reactive aldehyde groups and can form conjugates with proteins such as enzymes or other aldehyde reactive materials.

Microsphere coated substrate containing reactive aldehyde groups

The surface (20) of a moderate band gap semiconductor (12) such as p-type molybdenum sulfide is modified to contain an adherent film (24) of charge mediating ionene polymer containing an electroactive unit such as bipyridinium. Electron transport between the electrode (12) and the mediator film (24) is favorable and photocorrosion and recombination processes are suppressed. Incorporation of particles (26) of catalyst such as platinum within the film (24) provides a reduction in overvoltage. The polymer film is readily deposited on the electrode surface and can be rendered stable by ionic or addition cross-linking. Catalyst can be predispersed in the polymer film or a salt can be impregnated into the film and reduced therein.

Photoelectrochemical electrodes

Microspheres of acrolein homopolymers and co-polymer with hydrophillic comonomers such as methacrylic acid and/or hydroxyethylmethacrylate are prepared by cobalt gamma irradiation of dilute aqueous solutions of the monomers in presence of suspending agents, especially alkyl sulfates such as sodium dodecyl sulfate. Amine or hydroxyl modification is achieved by forming adducts with diamines or alkanol amines. Carboxyl modification is effected by oxidation with peroxides. Pharmaceuticals or other aldehyde reactive materials can be coupled to the microspheres. The microspheres directly form antibody adducts without agglomeration.

Polyglutaraldehyde (PGL) is polymerized in aqueous base or in aqueous highly polar solvent basic media to prepare powders, castable films or coatings for substrates such as amine substituted microbeads. PGL microspheres can be prepared by suspension polymerization in presence of a surfactant or by precipitating PGL from solution containing surfactant. Magnetic PGL microspheres are formed by suspension polymerization in the presence of magnetic particles such as iron oxide. Polyglutaraldehyde can be converted to a fluorescent polymer by reaction with m-aminophenol or other reagent. Proteins can be readily covalently bound to the polyglutaraldehyde.

Polyglutaraldehyde synthesis and protein bonding substrates

Highly fluorescent, stable and biocompatible microspheres are obtained by copolymerizing an acrylic monomer containing a covalent bonding group such as hydroxyl, amine or carboxyl, for example, hydroxyethylmethacrylate, with an addition polymerizable fluorescent comonomer such as dansyl allyl amine. A lectin or antibody is bound to the covalent site to provide cell specificity. When the microspheres are added to a cell suspension the marked microspheres will specifically label a cell membrane by binding to a specific receptor site thereon. The labeled membrane can then be detected by fluorescence of the fluorescent monomer.

Protein specific fluorescent microspheres for labelling a protein

Functional magnetic particles are formed by dissolving a mucopolysaccharide such as chitosan in acidified aqueous solution containing a mixture of ferrous chloride and ferric chloride. As the pH of the solution is raised magnetite is formed in situ in the solution by raising the pH. The dissolved chitosan is a polyelectrolyte and forms micelles surrounding the granules at pH of 8-9. The chitosan precipitates on the granules to form microspheres containing the magnetic granules. On addition of the microspheres to waste aqueous streams containing dissolved ions, the hydroxyl and amine functionality of the chitosan forms chelates binding heavy metal cations such as lead, copper, and mercury and the chelates in turn bind anions such as nitrate, fluoride, phosphate and borate.

Functional magnetic microspheres

Suspension polymerization of aqueous glutaraldehyde in basic conditions in presence of surfactants such as isobutoxy acrylamide copolymers and/or cross-linking agents yield cross-linked, water insoluble glutaraldehyde polymer microspheres ranging in size from 200 A to 10.mu.. Addition of fluorochromes, e.g., FITC, during polymerization yields microspheres of high fluorescent intensity and addition of a suspension of metal containing particles such as Fe.sub.3 O.sub.4 during polymerization yields magnetic microspheres. Immunopolyglutaraldehyde microspheres can be obtained by interacting the polyglutaraldehyde microspheres with antibodies. The immunomicrospheres were used to label and separate cell subpopulations. The labeling specificity is considerably improved by increasing the amount of isobutoxy polyacrylamide incorporated in the microspheres.

Polyglutaraldehyde microspheres

Microspheres are produced by cobalt gamma radiation initiated polymerization of a dilute aqueous vinyl pyridine solution. Addition of cross-linking agent provides higher surface area beads. Addition of monomers such as hydroxyethylmethacrylate acrylamide or methacrylamide increases hydrophilic properties and surface area of the beads. High surface area catalytic supports are formed in the presence of controlled pore glass substrate.

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Alan Rembaum