CA1242671A - Hybrid microspheres - Google Patents

Abstract

HYBRID MICROSPHERES

ABSTRACT

Substrates, particularly inert synthetic organic resin heads (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 alde-hyde with high intensity radiation. Individual micro-spheres (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.

Description

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DESCRIPTION

HY~RID MICROSPHERES

Technical Field The present invention relates to the synthesis of polyacrolein coated substrate, functional derivatives thereof, fluorescent and magnetic variations thereof, protein conjugates thereof and to the uSe of the coated substrate in biological and chemicai research ~.a analysis, as separatior, media for proteins or metals or as a substrate support for meta~ c~talysts c:r en~ymes.

Back~round of the Prior Art The isol~tion and characterization of cell membranes and their components is essential for an understanding of the role in which surface membranes play in regulating a wide variety of biological and immtmological activities. The present techniques used for this purpose are not quite satisfactory.
Knowledge of the nature, number and distribution of specific receptors on cell surfaces is of central importance for an understanding of the molecular basis underlying such biological phenomena as cell-cell recognition in development, cell co~nunication and regulation by hormones and c~emical transmitters, and differences in normal and tumor cell surfaces. In previous studies, the localization of antigens and

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carbohydrate residues on the surface of cells, notably red blood cells ~nd lymphocytes, ha~ been determined by bonding antibodies or lectins to such molecules as ferritin, hemocyanin or peroxidase which have served as markers for transmission electron microscopy.
With advances in high resolution ficanning electron microscopy ~SEM), howe~er, the topographic~l distribution o~ molecular receptors on the surfaces of cell and tissue specimens can be readily determined by similar histochemical techniques using newly developed markers resolvable by SEM.
Recently, commercially available polystyrene latex particles have been utilized as immunologic markers for use in the SEM technique. The surface of such polystyrene particles is hydrophohic and hence certain types of macromolecules such as antihodies are absorbed on the surface under carefully controlled conditions. However, such particles stick non-specifically to many surfaces and molecules and this seriously :20 limits their broad application.
The preparation of small, stable spherical Poly-Hema particles which are biocompatible, i.e., do not interact non-specifically with cells or other biological components and which contain functional groups to which specific proteins and other biochemical molecules can be covalently bonded is disclosed inU.S.
Patent No. 3,957,741.
Smaller, more evenly shaped acrylic microspheres are disclosed inU-S- Patent 4,138,383. Microspheres having a density differing from tl~at of cell membranes are disclosed in U.S. Patent 4,035,316 and fluorescent-acrylic copolymer microspheres are disclosed ln U.S.
Patent 4,326,008.
The hydroxyl groups can be activated by cyanogen bromide for covalent bonding of proteins and other chemicals containing amino groups to the polymeric ~, j z~

bead. Methacrylic acid residues which impart a negative charge onto the particles are likely to prevent non-~pecific binding to cell surface~ and to provide carboxyl groups to which a variety of biochemic~l molecules can be covalently ~onded using the c~rb~di-imide method, The derivatlzation procedure is unneces~arily complex and requires an additional step to prepare the bead surface for covalently ~inding to proteins such as a~tibodie6, lectins and the like or other molecules such a~ DNA, hormones and the like. Therefore, the method of deri~at~zatlon of acrylic microbead~ i5 tedious and availability is limited. Monomeric glutaraldehyde has been used as a biochemical reagent to co~alently bond proteins such as immunoglobulins to ferritin polymeric microspheres and other small particles which were then utilized to map receptors on cell memhranes. However, the reaction mechanism of proteins with glutaraldehyde is difficult to ascertain since its structure is still not clear and it has been reported to be in equilibrium with cyclic and hydrated forms. The reaction is difficult to carry out and frequently gives unsatisfactory results.

U.S. Patents 4,267,234 and 4,267,235 disclose direct bonding of protein to polyglutaraldehyde or copolymers thereof by solution polymerization in aqueous basic medium. In contrast to monomeric glutaraldehyde, the polymers contain conjugated aldehyde groupq. This imparts stability to the Schiff's bases formed after reaction with proteins ana, further, since the hydro-philic polyglutaraldehyde has relatively long chains extending from the ~urface into the surrounding aqueou~ medium, the heterogenous reaction with protein is facilitated, `
Polyglutaraldehyde (PGL) microspheres can be directly prepared by su~pension polymerizatlon with 2~;~7~L
stirring in presence of surfactant or by precipitation from solution containing surEactant. Magnetic, high density or electron dense microspheres can be prepared by coating metal particles or by suspension polymerization oE glutaraldeyde in presence of a suspension of finely divided metal or metal oxide, whereby the metal particles are bound to the surface of the microspheres or are embedded within the microspheres.
The presence of the metals render the microspheres election dense for analysis and in the case of magnetic metals, the microspheres are magnetically attractable for separating cells or por-tions of cells. It has been determined that the PGL microspheres exhibit some degree of non-specific binding to cells. Moreover, though some cross-linking occurs during the homopolymerization of glutaraldehyde, the polymer can be dissolved in highly polar solvents.
A process for polymerizing unsaturated aldehydes such as acrolein is disclosed in U.S. Patent No. 3,105,801 (Bell et al). The process comprises adding a small amount of acid or an acid-acting material to an aqueous solution containing acrolein or other unsaturated aldehyde and exposing the acid medium to high energy ionizing radiation to form high molecular weight polymer in the form of light powders having non-uniform shapes and sizes. The polymers are not utilized as such but are dissolved in aqueous sulfur dioxide solution to form water soluble derivatives which are used as coatings or sizings for paper, cloth, fibers and the like. Bell et al also discusses the copolymerization of acrolein with a wide variety of ethylenically unsaturated monomers such as ethylene diamine, pyridine or acrylic acids or esters, vinyl halides, etc. in amounts from 0.1 to 60%, preEerably from 1%
to 25% by weight of the monomer mixture.
The monomer mixture can contain other agents such as stabilizing, suspending and emulsifying agents. Radiation accelerators such as halides or metal salts may be added to the reaction mixture.
Though the polyacroleins prepared by Bell et al have a high degree of available aldehyde function, there was no recognition of the use of such material as a biological agent.
Furthermore, the presence of _5_ ~z~67~

extraneous ingredients interferes with the purity of the polymer and it would not be suitable as a biochemical protein bonding agent. Speclf~c modification of the material by copolymerization with certain comonomer~
5 designed to impart further properties such a~ non-specific binding and modification to add other functional groups for introduction of dyes, protein~ or other materials which would improve the flexibility of use of the material is disclosed in U.S. Patent 4,413,070.

Description of the Invention It has now been discovered in accordance with the 15 invention that aldehyde substituted microspheres can be formed in situ and grafted onto diverse, inert substrates such as polymeric films, rods, tubes, particles or spheres to ~orm a hybrid coated material. The hybrid material efficiently binds aldehyde reactive organic 20 molecules and proteins. The method converts inert materials into functionally reactive, direct protein bonding materials. The surface area of the substrate is increased due to the coating of covalently bonded submicron sized microsphere~. The coated article such 25 as a sheet or a continuous band of microsphere coated resin or elastomer provides an efficient system for contacting fluids containing the substance to be separated or boundO
The size and functionality of the microspheres can 30 be controlled by selection of polymerization conditions and selection of functionally substituted copolymerizable monomers. The functional nature of the microspheres can be modified by post-polymerization coupling reactions.
The hybrid product i~ produced in accordance with 35 the invention by forming a dllute solution of addition ~4Z~i7~
polymerizable unsaturated aldehyde, placing the inert su~strate in the ~olution and applying high energy radiation to the ~olution. The radiation lnitiates polymexization of the aldehyde which forms microspheres S which attach to the ~ubstrate. Ro~ette appearinq products are formed when spherical polymeric substrates are utilized. Polymerization in the pre~ence of ~urfac-tants results in formation and attachment to th~ surface of more uniformly shaped and smaller microspheres.
Adding a ~olvent to the polymerization that is a non-solvent for the microspheres but i~ A ~olvent for the surace of the ~ubstrate causes ~welling o the surface and a firmer at~achment of the microsphere layer. The layer o microspheres nppears to b~ tran~parent. ~owever, the layer can be rendered fluore~cent by interpolymerization with additional polymerizable or aldehyde reactive monomers. Electron dense or magnetizable metal3 can be incorporated into the microspheres during polymerization.
The invention provides a method of immobilizing high concentrations of enzymes, protein~, hormones, viruses, cells and varied other organic and biological molecules on the ~urface of inert commercial plastics.
The coated substrate6 can be uaed in separation techniques, clinical diagnostic tests, battery separators and as biological and chemical catalyst support~.
The microsphere coated products of the invention exhibit little or no aygregation duxing or after deriva-tization reaction to introduce large amounts of antibodies or other proteins, fluorochromes, etc. The microsphere film is insoluble, has functlonal groups dlrectly reactive with protein, whlch can bind specifically to receptor sltes on cells and the indivldual microsphcres can readlly be preparcd ln size~ from 100 Angs roms to 2,000 Angatrom6, or up to 10 microns or larger lf de~ired.
The derivatization procedure is simplified.
Hydroxyl modified microspheres can be used as a purifica-tion medium to remove metals from solution by chelation and can also bind to catalytic metals as a substrate or support. The microspheres of the invention provide a ~Z4Z6'7~

reliable, simple method to label cells for researeh, analysis or diagnosi 9 .
The micro~phere coated products of the invention ean also be utiliæed as substrates to bind pharmaeeutieals S eontaining functional groups reaetive with aldehyde or with the hydrophilliC hydroxyl, carboxyl or amine Rubstituent or with the funetional group ~ of the adduet.
The mierosphere-pharmaceutieal adduet is less likely to migrate and should reduee side effect~. Furthermore, antibodies can be attached to the microsphere eoated produet so that it migrates to specifie eells having eorresponding antigen receptor sites. Magnetieally attractable micro~phere coated products can be accumulated at a speciflc location in a subject by application of a magnetic field to that location.
These and many other features and attendant advantages of the inventiOn will become apparent as the invention becomes better understood by referenee to the following detailed deseription when considered in conjunction with the accompanying d~awings.

Brief Description of the Dxawings Figure l is a schematic view of the apparatus for forming a microsphere coated product of the invention;
Figure 2 i~ a cross-sectional view of a microsphere coated bead in accordance with the invention; and Figure 3 is a slde view of a mlero~phere coated sheet.

Description of the In~ention Referring now to Figureq 1 to 3, the mierosphere eoated artieles are formed by disposing a sub~trate sueh as a plastie sphere 10 or plastie ~heet 12 in a solution 14 of unsaturated aldehyde eontained in a reaction tank 16. Radiation souree 18 powered by power supply 20 iq turned on and microspheres 22 are formed in solution, migrate to the surface of spheres 10 and sheet 12 and are grafted thereto to form a continuous coating or layer 24 of microspheres 22 which are tangentially-12~

positioned with respect to the substrate. The layer of thebeads need comprise only a continuous monolayer o~
mlcrospheres in order to provide the de~lred functionality.
The layer i5 applied in a very even, uniform manner and generally is present in an amount from 100 Angstroms to 1,000 mlcron~, generally from 500 Angstroms to 100 microns in thickness. The microspheres are produced by addition polymerization of a liquid polymerization system optionally including a dispersion of metal particle~. More uniformly sized and shaped beads are formed in very dilute aqueous monomer mixtures of no more than20~ by weight, preferably 1 tolO~ by weight of dissolved monomers. Surfactants may be present to aid in the di~per~ion of the metal particles and form smaller microspheres.
The polymorization proceeds with or without _ stirring with ~pplication of high energy radiation capablc of genernting free radicals in the aqueous system. The radiatlon source i~ suitably a cobalt 60 gamma sour~e or cesium source and doses of 0.05 to 2.0 megarad~ are sufflcient for polymerizatlon. The reaction is preferably conducted under oxygen excluding condition, generally by applying vacuum to the reaction vessel or by displaclng oxygen gas from the system with an inert gas such as nitrogen.
The additi~n of 0.05 to 5%, by weight, of a stabilizing agent to the aqueous polymerization system bsfore polymerization is found to prevent agglomeration of individual microspheres before they attach to the surface of the substrate. The stabilizing agent is suitably a non-ionlc material such as an aqueou~ soluble polymer such as a polyalkylene oxide polyether or non-ionic surfactants. Representative non-ionic suxfactants are Tweens which are polyoxyethylene derivatives of fatty acid partial esters of sorbitol, Triton Xl or dextrans. The polyethers generally have a molecular weight from 10,000 to 10,000,000, preferably 400,000 * Trade Mark ~. .

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. 9 to 6,000,000 and are poLymers of ethylene oxide, propylene oxide or thelr mixture~. Polyethylene oxides (PEO) and Triton X are preferred, non ionic agent~.
5maller microspheres (50 to 200 Ang~troms in diameter) are formed in solutions containing small amounts, typically from 10 to 150 millimoles, of an anionic ~uractant ~uch as an alkali metal C8 to C20 alkyl sulfate ~urfactaJIt such as sodium lauryl sulfate ~SLSj or ~odiwn dodecyl sulfate (SDS).
The ethylenically unsaturated aldchydes should comprise at least J0~ by weight of the monomer mixture preferably from 20~ to 90% by weight thereof. The aldehyd~s pref~rably have the ethylenia group in alpha-beta po~itlon rel~tive to the aldehyde group and can be selected from those aldehydes containing up to 20 carbon atoms s~lch as acrolein, methacroleill, alpha ethyl acrolein, alpha-butylacrolein, alpha-chloroacrolein, beta-phenylacrolein, alpha-cyclohexyl acrolein and alpha-decylacrolein.
Preferred aldehydes contain 4 to 10 carbon atoms and - 20 especially acrol~in and Cl to C8 aryl alkyl and cycloalkyl substituted derivative~ thereof.
Presence of mono-un3aturated covalent-bonding monomers containing functional hydrophilic groups such as amine, carboxyl or hydroxyl provides non-specific binding and provides further functional ~roups for introduction of proteins, dyes and the like. The comonomers are freely water soluble and can comprise from 10 to 50~ of the monomer mixture. These monomers are suitably selected from amino, carboxyl or hydroxyl substituted acrylic monomers. Exemplary monomers are acrylamide (~M), metha~rylamide (MAM), acrylic acid, methacrylic acid (MA), dimethylaminomethacrylate or hydroxyl lower alkyl or amino-lower-a:Lkyl-acrylates such as those of the formula:

~ 2 ~ 2 where Rl is hydrogen or lower alkyl of 1-8 carbon atoms, R2 i~ alkylene of 1-12 carbon atoms, and Z 18 ~ OH or R3 - N - R4 where R3 or X4 are individually ~elected from H, lower ~lkyl or low~r ~lkoxy of l-B carbon Atoms.
2-hydroxyethyl methacrylnte (HEMA), 3-hydroxypropyl 5 me~hacrylat~ and 2-~minoethyl methacrylat~ are readily available commercially. Poroslty and hydroph~liclty increase wlth incre~slng concentration of monomer.
~ hough apparently the radlation providea cros~-linking of the polymer, further cros~-linking can be provided by 10 inclusion o~ polyunsaturAted compounds which are generally present in O~e monomer mixture in an amount from 0.1-20 by weight, generally 6-12% by weight and are ~uitably a compatible diene or triene polyvinyl compound capable of addition polymerization with the covalent bonding 15 monomer such as ethylene glycol dimethacrylate, tri-methylol-propane-trlmethacrylate, N,N'-methylene-bis-acrylamide ~BAM~, hexahydro-1,3,5-triacryloyl-~-triazine or divinyl benzene.
For small particle size and additional reduction in 20 non-specific binding and agglomeration the monomer mixture prefersbly contains a monomer capable of impartin~
negative charge such as methacrylic acid (MA). The mixture may contain 0-40~ suitably 10 to 30~ of sparingly water fioluble monomers having hydrophobic characteristics 25 since this is found to result in freely-suspended, indi-vidual, small microspheres. The cross-linking agent is sometimes sparingly water soluble. Hydrophobic charac-teristics can also be provided with monomers such as lower alkyl acrylates suitably methyl methacrylate or ethyl 30 methacrylate or a vinyl pyridine. Vinyl pyridines suitahle for use in the invention are 2-vinyl pyridine, 4-vinyl pyridine and 2-methyl-5-vinyl pyridine.
Small microspheres containing electron-dense metals provide higher spatial resolution of cell surface features.
35 Immunomicrosphere~ containing electron-dense metals provide more stable labels than gold particles with physically ab-sorbed anti~odies that are presently used for c~ll labeling.
~he metal containing n~crosphere~ can be ~ynthesized by, '' 11- .~2~i7~
in situ, polymerization of the microsph~re~ in presence of ~ suspension of ~nely-divided metal part~cles or compounds of the metal, preferably a colloidal disper~ion of the metal. The metal is incorporated ~nto the micro-5 sphere in an effective amount of from 0.5~ to 40~ byweight, generally from S~ to 25~ by weight.
~ he m~tal or.metal compound part~cles are preferably fine, evenly sized material~.having a un~for~ di~meter smaller tha~ the resultant microsphere diameter, typically 10 below lOOOA, generally from 25A to SOOA. The metals.are preferably the electron-dense heavy metals having a high atomic n~mber above 50, prefera~ly above 75 such as Pb, ~i, Cot Pt, Au, Fe. The metal may be magnetically attractable such a~ Fe, Ni, Co or alloys thereof or an 15 inorganic magnetic compound such as a metal oxide.
The magnetic mater~al i~ preferably ~ m~gnetic iron o~ide of the formula Fe304. Some hard, ceramic type fcrrites, such as lithium ferrites can also be used.
The metal or c~mpound can be made into a readily disper-~0 sible form by suspension in water containiny a surfactantsuch as polyethylene imine.
~Ofit polymerization reaction with specific fluorocrome reagents that are not in themselves fluorescent, results in a fluorescent microsphere by fo~min~ fluorescent chromo-25 phores attached to the polymer. Anthrone reacts with acrolein units to form ~ benzanthrone fluoroge~ and m-aminophenol reacts with the acrolein ~tructure to form the fluorogen, 7-hydroxyquinoline. ~minofluorescein also reacts with aldehyde yroups to form fluorescent microspheres.
The microspheres can also be rendered fluorescent during polymerization by the covalent coupling of aldehyde or hydroxyl reactive fluorescent chromophores such as aminofluo-rescein, 9-amino acridine, propidium bromide or fluorescein isothiocyanate (FITC) to the hydroxyl or aldehyde groups pre-sent on the microspheres. Highly fluorescent microspheres can also be prepared by suspension polymerization in presence of fluorochromes containing unsaturated groups -- .

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eapable of r~tion with aerolein.
A;nother manner of introducing functionality other than aldehyde onto the m~cro~phere layer i by adduct reaction of the microsphere~ with compou~d~ of ~he fo~mula:
~M - R - Z
where Rl i8 hydrogen or a hydrocarbon group which may be aliphatie or aromatie preferably aryl sueh as phenyl or alkyl of 1 to 10 earbon atoms, R is a divalent hydroearbon group ~uch as alkylene of 1 to 20 earbon atoms and Z is a funetional group ~uch as amine or hydroxyl or R~ ean be hydroxyl. Representative eompounds are hydro~ylamine or ethylene diamine~ The microsphere layer ean be modified to introduee earboxyl groups by oxidation with an agent such as hydrogen peroxide.
The suhstrate can be of diverse physieal or ehemi-cal nature. The substrate must be capable of withstandingthe high energy radiation without deterioration. Though the microsphere layer would probably form on metal, ceramic or glass or plant ma-terial substrates such as wood, the pre-ferred substrat~s are synthetie organie resin materials 20 capable of developing eovalent bonds during high energy radiation.
The resin can be hydrocarbon such as polyethylene polypropylene, pGlystyrene or polyester, polyamide, dextran aerylie polymers SllCh as polyacrylamide, polyaerylate, etc.
25 Though functional groups reactive with aldehyde are not necessary, they would contribute to forming a further means of bonding the microsphere layer to the substrate. The substrate should be at least ten times larger than the mierosphere and can be in the form of a sheet, rod, tuhe, 30 sp~ere, hollow sphere, irregular particle, etc.
Examples oE practice follow-~ea~ents: Methacrylic aeid (MA), 2-hydroxyethyl metha-erylate ~EMA), acrolein, ethylene diamine were fractionally distilled. Polyethylene oxide (PEO, Mw 100,000) N,N'-.

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methylene-bi3~acr~1amicle (~AM), hydroxylamine hydrochloride, 1,6--hexane diamine, l.l.ysine, 1-ethyl-3-~3-dimethyl amino propyl) carbodiimide and sodium dodecyl ~ulfate (SDS) were used as received.

Procedure- Acrolein or monomer mixtures consisting of ~ENA and acrolein or H~MA, BAM, MA and acxolein formed homogeneous ~olutions in distilled water containing PEO or SDS. The polymer substrates were added to the 301ution. After dearat.ion with nitrogen the mixtures were irradiated in CO gamma source at room temperature ~dose rate 0.12 Mrjhour) for various periods. The reaction product was purifi~d and kept in distilled water.

Methods: The aldehyde content was determined from the percent nitrogen of the oxime prepared by the reaction of an aqueous suspeIIsic)n with hydroxylamine hydruchloride [P.J. Bochert Kunstoffe 51 (3) 137 (1961)]. IR spectra were obtained with a Fourrier transform IR (fts-15C, Houston Instruments) spectrophotometer.

Example 1: Polypropylene sheets were placed in a 10 (v/v) acrolein solution in water and irradiated for 0,20,40,60 and 120 minutes. The irradiated sheets were examinea by SEM. There was no coating on the non-irradiated sheet. However, the irradiated sheet showed progressively more attachment of microspheres with irradiation time. Control sheets irradiated in water alone do not exhibit any microspheres on their surface.

Example 2- Sheets of polymethylmethacrylate were run according to the conditions of Example 1. ~he results were similar except that the at-tachment o~ microspheres was visible without microscope.

Exam~le 3: Polypropylene sheets were placed in aqueous solutions each containing 0.4~ by weight PEO and containing 12~67~

respectively; 10%, 8%, 5%, 2~ and 0~ acrolein. Each solution was exposed to irradiation for 90 minutes and each sheet was stained with Geimsa stain and 9-aminoacridine fluorescent dye. The non-irradiated (10% acrolein) sheet and the 0% acrolein sheet did not react with the stain. However, all other sheets exhibited fluorescence.

Example 4: Example 3 was repeated with polystyrene sheats. The sheets were treated overnight with 9-aminoacridine. Again the non-irradiated (10% acrolein) and 0~ acrolein sheets did not exhibit fluore~cence.
~owever, some fluore~ce.nt microsphere~ were visible on the surface without aid of microscope and under 1000 X
magnification~ an even coating of fluorescent micro-spheres was observed.

Example 5: Polypropylene sheets were immersed in anaqueous solutiorI containing 5% acrolein and containing 0%, 0.5~, 2~, an~ 5~ of SDS. Each solution and control sheet in a 0~ acrol~in solution were irradiated for 90 minutes. These sheet~ and a control sheet dipped into the 5% acrolein solution were stained with 9-aminoacridine.
The irradiated shee~ did not fluoresce. The sheet from the solution not containing SDS exhibited fluorescent beads. The sheets treated with SDS solutio~R were fluorescent but indiviclual bead~ could not be observed.

Example 6: Polyst~rene sheets were subjectecl to the condition~ of Exarnple 5 with simllar res~ults.

Exam~le 7: One ml of Styrene-divinyl benzene (S/DVB) beads ~8 micron3); su~ipen~ion (0.14g/ml) containing 0.4~ PE0 and 5~ acrolein, w~re irràdiated in a cobalt gamma source for 0, 21), 40, 60 and 90 minutes. Poly-acrolein (PA) microspIIeres formed in th~! suspen~lion and a~tached to the surface of the heads to form hybrid ~15- ~24~67~

beads. tPA-PS) The PA microspheres were ~eparated from the hybrid beads by ~edimentation; washing once with PE0 and 3X with water.
When the recovered beads were reacted with 1 M hydrox-ylamine hydrochloride for 4 hour~, the non-irradiated beads remained white while the 20-90 minute irradiate beads t~rned yellow indicating shift baae for~ation.
When the recovered beads from the 0 and 90 minute irradiated beads were subjected to reaction with 9-aminoacridine, the non-irradiated beads are ~on-fluorescent while the radiated beads were fluorescent. The same results were observed on treatment with 0.05 ml of 20 mg/ml suqpension of fluorescent goat antirabbit antlbodies (GAR). When the 90 minute irradiated, rosette-shaped hybrid spheres were reacted with ~heep red bloodcells (~C) sensitized with RAS Ig; an adduct was formed.

Example 8: The PS~DVB bead suspension of Example 7 - containing 2 mg/ml of SDS was irradiated for 3 hours and then washed in water. SEM photographs showed small PA microspheres on irradiated PS bead urface and no PA on the non-irradlated control.

Example 9: Sephadex*6-10 (dextran) beads were irradiated for 2 hours in 5% acrolein solution conta~nlng 0.4~
PE0. The irradiated bead~ and non-irradiated control were reacted with 0,05 ml of a 20 mg/ml fluorescent goat antirabbit - Ig for 2 hours. Irradiated heads exhibited fluorescent coating whlch can not be wa~hed off with pH 2.2, 0.2 M glycine wash. The control was non-fluorescent.

Example 10: Biorad ~iogel G-30*(polyacrylamide) beads were treated according to the procedure of Example 9 with the same result~.

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Exam~le 11~ -acrylamide-acrylamide - hydroxyethyl-methacrylate beads (42 mg) were irradiat~d for one hour ~n 20~ acrolein aqueous solution containing 0.4~ PE0.
The non-irradiated control did not form any coating.
The rosette-shaped hybrld beads (6.5 mg) reaulting from the irr~dia~ion procedure were coated w~th GAR
(1.7 mg) and the Ig-bead adduct was added to 10~/ml of sheep RBC ~ensitized with RAS Ig.

A new convenient immunoreagent in form of acrolein hybrid microspheres was synthesized in a variety of sizes. High intensity of fluorescence can be imparted to the microspheres during or after polymerization.
The aldehyde functional groups permit covalent bonding with antibodies, enzymes and other proteins in a single step. There~ore thi6 immunore~gent elimlnates the previously u~ed intermediate steps in which the cyanogen bromide and carbodilmide reaction was u~ed. The high specificity of thc microsphere~, ~t least as far as hurnan rhc is concerncd is a1GO a desirable property. A minor synthetic modific~tion yields ~luorescent, magnetic microsphere~ for a large number of potentlal applications.
The polyacrolein hybrid microspheres of this invention contain more aldehyde groups than the comparable glutaraldchyde copolymer microspheres.
The use of magnetic particles has created a great deal of interest in biochemical research ~nd clinical medicine whenthey are used as supports for immobilized enzymes.
Their easy retrieval from liquors cont~ining colloids and undissolved ~olids should be of practical value.
The separation of proteins and chemical compounds by affinity chromatography can be simplified by elimination of tedious centrifugation procedures and column chroma-tography steps. Magnetic particles have also recently been tested in radioimmunoassay techniques in hyperthermia treatment of cancer and used by being gllided to a vascular malformation such as cerebral aneurism with the -17- ~4Z~

intent to seal the defect by inducing thrombosis.
Other propo~ed applications have been as tracers of blood flow or vehicles for drug delivery. The first succe~sful application of magnetic immunomicrosphere~ to the ~eparation of B and T cells ha~ been demonstrated.
There is lit~le doubt that physical sorting of cell sub-populations has become a necessity. Many ~eparation methods, while useful are limited by the re~tricted set of parameters upon which separation c~n be based and by the f~ct that they are batch techniques.
New flow cytometers and sorters permit quantitative multiparameter meaeur0ments and sorting bnsed on these measurem~nts, but ~r~ limited as to the number of cells that can be ~epurated in a given tlme. Magnetic cell sorters have the potential of cell separation in a continuous proce~s. ~.vidence obtained uxing model cell systems indicates that magnetic immunomicxospheres of desirable si~es can be conjugated with proteins in a simple and convenient manner, therefore offer a potential for large scale immunological cell sorting as well as other applications.
It is to be understood that only preferred embod-iments of the invention have been described and that numerous substitutions, modifications and alterations are permissible without departing from the spirit and scope of the invention as defined in the following claims.

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Claims (17)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A coated article comprising an organic resin substrate, said resin being capable of developing covalent bonds during high energy radiation and being selected from the group consisting of a synthetic resin and a dextran resin;
a continuous layer of contiguous, tangentially-positioned, individual microspheres having a uniform diameter between 100 Angstroms and 2000 Angstroms bound to the surface of the substrate by covalent bonds formed between the resin and the microspheres by means of high energy radiation grafting of the microspheres to the surface of the resin substrate, said microspheres consisting essentially of the addition polymerized polymer of an unsaturated aldehyde containing 4 to 20 carbon atoms.

2. A coated article according to claim 1 in which the resin is selected from polyethylene, polypropylene, polystyrene, or acrylic polymers.

3. A coated article according to claim 1 in which the polymer further consists essentially of at least 20% of an addition polymerizable comonomer having a hydrophilic substituent selected from hydroxyl, amino or carboxyl.

4. A coated article according to claim 1 in which the substrate is at least 10 times thicker than the layer of microspheres.

5. A coated article according to claim 4, in which the thickness of the layer is from 100 Angstroms to 1000 microns.

6. An article according to claim 4 in which the substrate is in the form of spheres, particles, sheets, rods or tubes.

7. An article according to claim 6 in which the aldehyde is selected from acrolein and C1 to C8 alkyl, aryl and cycloalkyl derivatives thereof.

8. A composition comprising an adduct of the article of claim 1 with a material selected from aldehyde reactive proteins, pharmaceuticals and fluorescent chromophores.

9. A composition according to claim 8 in which the aldehyde is acrolein.

10. A method of forming a coated article comprising immersing a substrate of an organic resin capable of developing covalent bonds during high energy radiation in an aqueous solution containing a surfactant and up to 20% by weight of an unsaturated aldehyde containing 4 to 20 carbon atoms, said resin being selected from the group consisting of a synthetic resin and a dextran resin, and irradiating the solution with high energy radiation to provide a continuous layer of contiguous, tangentially positioned, individual microspheres having a uniform diameter between 100 and 2000 Angstroms bound to the surface of the substrate by covalent bonds.

11. A method according to claim 10 wherein the solution further contains from 1 to 50% by weight of a water soluble solvent for the surface of the substrate which is a non-solvent for the coating.

12. A method according to claim 11, wherein the solvent is selected from dimethyl sulfoxide, tetrahydrofuran and dioxane.

13. A method according to claim 10, 11 or 12, wherein the synthetic organic resin is selected from polyethylene, polypropylene, polystyrene, or acrylic polymers.

14. A method according to claim 10, wherein the aldehyde is selected from acrolein and C1 to C8 aryl, alkyl and cycloalkyl derivatives thereof.

15. A method according to claim 10, wherein the aldehyde is acrolein.

16. A method according to claim 10 in which the surfactant is selected from polyalkylene oxide liquid polymers and an alkali metal alkyl sulfate containing 8 to 20 carbon atoms.

17. A method according to claim 16 in which the surfactant is selected from sodium lauryl sulfate and sodium dodecyl sulfate.