CA1218185A - Acrolein microspheres - Google Patents

Abstract

ACROLEIN MICROSPHERES

ABSTRACT

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.

Description

DESCRIPTION
_ ACROLEIN MICRO SPHERES

Technical Field The present invention relates to the synthesis of polyacrolein micro spheres, functional derivatives thereof, fluorescent and magnetic variations thereof, protein conjugates thereof and to the use of the conjugates in biological and chemical research and - 15 testing.

Background of the Prior Art The isolation 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 immunological 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 call-cell recognition in development, cell communication and regulation by hormones and chemical transmitters, and differences in normal and tumor cell surfaces. In previous studies, the localization of antigens and s carbohydrate residues on the surface of cells, notably red blood cells and lymphocytes, has been determined my bonding antibodies or pectins to such molecules as fourteen, hemocyanin or peroxides which have served S as markers for transmission electron microscopy.
With advances in high resolution scanning electron microscopy them), however, the topographical distribution of molecular receptors on the surfaces of cell and tissue specimens can be readily determined by similar histochemical techniques using newly developed markers resolvable by SEMI
Recently, commercially available polystyrene latex particles haze been utilized as immunologic markers for use in the SUM technique. The surface of such polystyrene particles is hydrophobic and hence certain types of macromolecules such as antibodies are absorbed on the surface under carefully controlled conditions, However, such particles stick non-specifically to many surfaces and molecules and this seriously limits their broad application.
he preparation of small, stable spherical Palm 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 in US.
Patent No, 3,957,741.
Smaller more evenly shaped acrylic micro spheres are discos d in US Patent 4,138,383. Micro spheres having a density differing from that of cell membranes are disclosed in US Patent 4,035,316 and fluorescent-acrylic copolymer m~crospheres are disclosed invoice-Patent No. 4,326,00~
The hydroxyl groups can be activated by cyanogen bromide for covalent bonding of proteins and other chemicals containing amino groups to the polymeric bead. Methacrylic acid residues which impart a negative charge onto the particles are likely to prevent non-specific binding to cell surfaces and to provide carboxyl groups to which a variety of biochemical molecules can be covalently bonded using the carbide-imide method.
The derivatization procedure is unnecessarily complex and requires an additional step to prepare the bead surface for covalently binding to proteins such as antibodies, pectins and the like or other molecules such as DNA, hormones and the like. Therefore, the method of deri~atization of acrylic micro beads is tedious and availability is limited. Monomeric glutaraldehyde has been used as a biochemical reagent to covalently bond proteins such as immunoglobulins to fourteen polymeric micro spheres and other small particles which were then utilized to map receptors on cell membranes. However, the reaction mechanism of proteins with glutaraldehyde is difficult to ascertain since its structure is still not Lear 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.
Direct protein bonding polyglutaraldehyde or copolymers therefore disclosed in U-S
Pat nuts 4,267,234 and 4,267,235, prepared by solution polymerization in aqueous basic medium. In contrast to monomeric glutaraldehyde, the polymers contain conjugated alluded groups. This 3Q imparts stability to the Showoffs bases formed after reaction with proteins and, further, since thy hydra-Philip polyglutaraldehyde has relatively long chains extending from the surface into the surrounding aqueous medium, the hetero~enous reaction with protein is facilitated.
Polyglutaraldehyde (PAL) micro spheres can be directly prepared by suspension polymerization with stirring in Rosen ox surf~tant or by precipitation run solution eontainin~ surfactant. Magnetic, huh density or electron dense microphones can be prepared by coating metal particle or by suspension polymeric ration of glutaraldehyde in Presence of suspension of finely divided metal or metal oxide. It has been determined that top PAL micro spheres exhibit some degree of nonspecific binding to cells. Moreover, though some cross-linking occurs during the homopoly-merizatiOn of glutaraldeh~de, the polymer can redissolved in highly polar solvents A process for polymerizing unsaturated aldehydes such as acrolein is disclosed in US. Patent No. 3,105,801. The prowesses comprises adding a small amount of acid or an acid-acting material to an aqueous solution containing acrolein or other unsaturated alluded and exposing the acidic medium to high energy ionizing radiation to form high molecular weight polymer in the for of light powders having nc)n-ulliform shapes and sizes.
The polymers were not utilize as such but are dissolved in aqueous alkaline sulfur dioxide solution to form water soluble derivatives which are used as coatings or sizings for paper, cloth, fibers and the like. Bell et at also discusses the copolymerization of acrolein with a wide variety of ethylenically unsaturated monomers such as ethylene Dianne, pardon or acrylic acids or esters, vinyl halides, etc. in amounts from 0.1 to 60%, preferably from t to 25~ by weight of thy monomer mixture.
The monomer mixture can contain other agents such as stabilizing, suspending as emulsifying agents. Radiation accelerators such as halides or metal salts may be added to the reaction mixture.
Though the polyacroleins prepared by Bell et at have a high degree of available aloud function, there was no recognition of the use of such material as a biological reagent. Furthermore, the presence of extraneous ingredients interferes with the purity of the polymer and it would not be suitable a a biochemical protein boning agent. furthermore, specific modification of the material by copolymeri~ation with certain comonomers designed to impart further properties such as non-specific binding and modification to add other functional groups for introduction of dyes, proteins or other materials would improve the flexibility of use of the material.

Description of the Invention Novel acrolein inter polymer micro spheres and functional, modified reaction products and protein adduces thereof, are produced in accordance with the invention. The size and properties of the micro spheres can be controlled by selection of polymerization conditions and especially by selection of comonomers. The micro-spheres of the invention exhibit exceptional stability and can be derivatized by reaction with amine or with proteins without aggregation -The non-aggregatin~ micro spheres are produced in accordance with this invention by the high-energy initiated inter polymerization of an unsaturated alluded such us acrolein and at least 20% by weight of at least one addition copolym~rizable comonomer having a hydra-Philip functional substituent selected from hydroxyl, amino or carboxyl.
Another manner of introducing functionality other than alluded onto the micro spheres is by adduce reaction of the micro spheres with compounds of the formula: 1 N - R - Z
where I is hydrogen or a hydrocarbon group which may be aliphatic or aromatic preferably aureole such as phenol or alkyd of 1 to 10 carbon atoms, R is a I

diva lent h~c',~ocar~on group such as alkaline of 1 to 20 carbon Amy and % is a functional group such as amine or hydroxyl or RZ can be hydro~yl, Representative come pounds are hy~rox~lamine or ethylene Damon. The micro-spheres can be modified to introduce carboxyl groups by oxidation with an event such as hydrogen peroxide.
The micro spheres of the invention exhibit little or no aggregation during or after derivatization reaction to introduce large amounts of antibodies or other proteins, fluorochromes, etc. The micro spheres ore insoluble, have functional groups directly reactive with protein, are easily dispersed and inn specifically to receptor sites and can be readily prepared in sizes from 100 Angstroms to 2,000 Angstroms, or up to 10 microns or larger if desired.
The ~erivatizatioll procedure is simplified. The hydroxyl modified micro spheres can be used to chelates metals as a purification media or as a support for a catalyst. The micr~s?hPres can be formed into a strong transparent film by drying on a surface or can be forum-lathed to contain metals which can be utilized to form election dense magnetic non-a~gregating particles or magnetic coatings or films. The micro spheres ox the invention provide 2 reliable, simple method to label cells for research or analysis.
The micro spheres of the invention can also be utilized as a substrate to bind pharmaceuticals containing functional groups reactive with alluded, the hydrophillic hydroxyl, carboxyl or mine substituent or the functional group æ of the adduce. The microsphere-pharmaceutical adduce is less likely to migrate end Gould reduce side effects Furthermore, antibodies Jan he attached to the micro sphere so that it migrates to specific cells having corresponding antigen receptor sites. Magnetic micro spheres can be accumlllated at a specific location in a subject by application of a magnetic phyla Jo that location.

I

Russ and many other features and attendant advantage of the invention will become apparent a the invention becomes wetter understood by reference to the following detailed description when considered in conjunction with the accompanying drawings.

Grief Description of the Drawings Figure 1 is a series of graphs showing the effect of addition of comonomers on the size of acrolein copolymer micro spheres.
Figure 2 is a series of schematic reactions of po~yacxolein micro spheres and various modifying and adducing reagents;
Figure 3 is a pair of curves illustrating the alluded and carboxyl content of oxidized Acrolein-Methacrylic Acid copolymer micro spheres; and Figure 4 is a pair of curves demonstrating the kinetics of reaction of polyacrolein micro spheres with an antibody.

I

Detailed Dozier n of the Invention __. ____ Initiation of copolymerization by high energy radiation in absence of chemical initiators or acid materials provides a purer and more evenly sized and shaped micro sphere. The micro spheres are produced by addition polymeriz~lon of a liquid polymerization system optionally including a dispersion of the metal particles in a monomer mixture containing a covalently bondable unsaturated monomer. More uniformly sized and shaped beads are formed in very dilute aqueous monomer mixtures of no more than I by weight, preferably 1 to 4% by weight of dissolved monomers. Surfactants may be present to aid in the dispersion of the metal particles and in suspending the mjcrospherss.
The polymerization proceeds with or without stirring with application of high energy radiation capable of generating free radicals in the aqueous system. The radiation source is suitably a cobalt 60 gamma source or sesame source and doses of 0.05 to 2.0 megarads are sufficient for polymerization. It is believed that polymer chains grow from the surface of metallic particles. The reaction is preferably conducted under oxygen excluding condition, generally by applying vacuum to the reaction vessel or by displacing oxygen gas from the system with an inert gas such as nitrogen. After polymerization has proceeded to completion, the reaction mixture is made neutral by aiding acid or base, passed through mixed ion exchange resins to remove emulsifiers or any free metal particles. Further purification is achieved by centrifugatio}l on a sucrose gradient The addition of 0.05 to 5%, by weight, of a stabilizing agent to the aqueous polymerization system before polymerization is found to further reduce agleam-oration. The stabilizing agent is suitably an aqueous soluble polymer such as a polyalkylene oxide polyether s I

or non-ionic surfac~ants such as Tweet which art polyp oxyethylene derivatives of fatty acid partial esters of sorbitol, Tryout X, or dextr~ns. The polyether~ generally have a molecular weight from 10, 000 to 10, 000, 000, pro-fireball ~00,000 to 6,000,000 and are polymers of ethylene oxide, propylene oxide or their mixtures. Polyethylene oxides (PRO) and Briton X art preferrer.
The smaller micro spheres (50 to 200 Angstroms in diameter) are formed in solutions containing small amount, typically from 10 to 150 millimoles, of an alkali metal I to C20 alkyd sulfate surfactant such as sodium laurel sulfate SLUICE) or sodium dodecyl sulfate (SDS).
The ethylenically unsaturated aldehydes should comprise at least 10~ by weight of the monomer mixture preferably prom 20~ to 90~ by weight thereof. The aldehydes preferably have the ethylenic group in alpha-beta position relative to the alluded group and can be selected from those aldehydes containing up to 20 carbon atoms such as acrolein, methacrolein, alpha ethyl acrolein, alpha-butylacrolein, alpha-chloroacrolein, beta-phenylacrolein, alpha-cyclohexyl acrolein end alpha-decylacrolein.
Preferred aldehydes contain 4 to 10 carbon atoms and especially acrolein and Of to C8 aureole alkyd and cycloalkyl substituted derivatives thereof.
The mono-unsaturated covalent-bondiny monomers are freely waxer soluble and should comprise from 10 to So of the monomer mixture These monomers are suitably selected from amino, carboxyl or hydroxyl 30 substituted acrylic monomers. Exemplary monomers are acrylamide JAM), methacrylamide (MUM), acrylic acid, methacrylic acid (PA), dimethylaminomethacrylate or hydroxyl-lower alkyd or amino-lower-a~kyl~acrylates such as those of the formula:

R2_~ ;

* Trade Mark S

where I -s hydrogen or lower alkyd of 1-8 carbon atoms, R2 is alkaline of 1-12 tarpon atoms and is - OH or R3-- N R4 where R3 or R4 are individually selected from H, lower alkyd or lower alkoxy of 1-8 carbon atoms.

2-hydroxyethyl methacrylate (HEM), 3-hydroxypropyl methacrylate and 2-aminoethyl methacrylate are readily available commercially. Porosity and hydrophilicity increase with increasing concentration of monomer.
Inclusion of polyunsaturated compounds provides 10 cross-linked beads. The polyunsaturated compounds are generally present in the monomer mixture in an amount from 0.1-20~ by weight, generally 6-12~ by weight and are suitably a compatible dine or triune polyvinyl come pound capable of addition polymerization with the covalent 15 bonding monomer such as ethylene glycol dimethacrylate, trimethy]ol-propane-trimethacrylate, N,N'-methylene-bis-acrylamide (BUM), hexahydro-1,3,5-triacryloyl-s-triazine or divinely Bunsen.
Fox small particle size and additional reduction in non-specific binding and agglomeration the monomer mixture preferably contains a monomer capable of imparting negative charge such as methacrylic acid I The mixture may contain 0-40% suitably 10 to 30% of sparingly water soluble monomers having hydrophobic characteristics since this is found to result in freely-suspended, India visual, small beads. The cross-linking agent is sometimes sparingly water soluble. hydrophobic characteristics can also be provided with monomers such as lower alkyd acrylates suitably methyl methacrylate or ethyl Matthew-Creole ox a vinyl pyricline. Vinyl ~yridines suitable for use in the invention are 2-vinyl pardon, 4-vinyl pardon and 2-methyl-5-vinyl pardon.
Small micro spheres of the order of 10~ to 500 Angstroms containing electron dense metals provide higher spatial resolution of it'll surface features. Immunomi-crucifiers containing electron-dense metals provide more stable ibis Han kiwi particles with physically absorbed antibodies what are presently used for call labeling.
The metal containing micrGspheres can be synthesized by, in situ, polymerization of the micro spheres in presence of a suspension of finely-divided metal particles or compounds of the metal, preferably a colloidal dispersion of the metal. The metal is incorporated into the micro-sphere in on effective amount of from 0.5~ to 40% by weight, generally from 5% to 25~ by weight.
The metal or metal compound particles are preferably wine, evenly sized materials having a uniform diameter smaller than the resultant micro sphere diameter, typically o o o below Lowe, generally from AYE to AYE. The metals are preferably the electron-dense heavy metals having a 15 high atomic nwnber above 50, preferably above 75 such as Pub, Nix Co, Pi, A, Fe. The metal may by magnetically attractable such as Fe, Nix Co or alloys thereof or an inorganic magnetic compound such as a metal oxide.
The magnetic material it preferably a magnetic iron 20 oxide of the formula Foe. Some hard, ceramic type ferrite, such as lithium ferrite can also be used.
The metal or compound can be made into a readily dispel-sidle form by suspension in water containing a surfactant such as polyethylene mine.
Post polymerization reaction with specific fluorocrome reagents that are not in themselves fluorescent, results in a fluorescent micro sphere by forming fluorescent chrome-phones attached to the polymer. throne reacts with acrolein units to form a benzanthrone fluorogen and 30 m-aminophenol reacts with the acrolein structure to form the fluorogen, 7-hydroxyquinoline. Aminofluorescein also reacts with alluded groups to Crimea fluorescent micro spheres The micro spheres can also ye rendered fluorescent during polymerization in presence of fluorochrome compounds 35 containing alluded or hydroxyl reactive groups such as aminofluorescein, 9-amino acrîdine, propidium bromide or -12~ I

fluoresce in isothiocyanate (FIT). Highly fluorescent micro spheres can also be prepared by suspension polymer-ization in presence of fluorochromes containing unsaturated groups capable of reaction with acrolein. Examples of practice hollow:

Reagents: Methacrylic acid (MA), 2-hydroxyethyl Matthew-cruelty (HEM), acrolein, ethylene Damon were fractionally distilled. Polyethylene oxide (PRO, My 100000) NUN'-methylene~bis-acrylamide (BUM), hydroxylamine hydrochloride, 1,6-hexane Damon, Lawson l-ethyl-3-~3-dimethyl amino propel) carbodiimide were used as received.

- Synthesis: Acrolein or monomer mixtures consisting of HEM and acrolein or HEM, BUM, MA and acrolein formed homogeneous solutions in distilled water containing 0.4% PRO or 64 my of SDS. After duration with nitrogen the mixtures were irradiated in CO gamma source at room temperature (dose rate 0.12 Mr/hour) for 4 hours. The reaction product was purified by three centrifugations and kept in distilled water.

Methods: The alluded content was determined from the -percent nitrogen of the oxide prepared by the reaction of an aqueous suspension with hydroxylami~e hydrochloride PI Bochert Kunstoffe 51 (3) 137 (1961)l. IT spectra were obtained with a Furrier transform IT ~fts-15C
Houston Instruments) spectrophotometer.

Example 1: Pure acrolein I v/v) in water containing PRO produced colloidal particles (approximately 1,000 Angstroms in diameter) after cobalt gamma irradiation.
Repeat of the procedure substituting 64 my SDS for PRO
resulted in 170 Angstrom micro spheres in higher yield.

Example 2: Acrolein - HEM copol~mer micro spheres of eight different HEM contents were prepared by cobalt I
gamma irradiation of a I TV monomer solution in water containing 0~4% Pi. The diameter of the resulting mlcrosphereS decreased with increasing acrolein content as shown in Figure 1. Over the riddle of the concentration range studied, monomer ratios had little effect on Bite;
permitting the preparation of micro spheres of similar size but different degrees of hydrophobicity.
When the acrolein homopol~ner micro sphere suspension was evaporated to dryness, a brittle film was formed.
However, evaporation of the HEM copolymer ~35 mow percent HOWE micro sphere suspension to dryness results in a strong flexible film.

. : Seven of the copolymers were reacted with hydroxyl~nine chloride to form hydroxyl functional micro spheres. The aloud content was analyzed by this procedure as shown in Table I.

Al r-It Lo us on I o c o I
o Jo c 4-X L o . cry C, o --9 C ID O
E x X .
O C:
mu . o o 0` Lo us Lo Z LO_ q` 0 ._ I o - .
I> Lo L
I .
E O

O o 0 O Jo CJ~_C o V
¢ TV E
o Jo ¢ _ - o I

a --_ o x E L O
V C
D E En o c o I-o ) Q E , _ c c us c-c:
v c o _ Lo c . O a _ U O O Clue O
a o .. Jo ,_ Q I Jo V

The acrolein homopolymer (100~ acrolein~ was found to contain approximately So% of the expected alluded groups. The presence of alluded groups was further confirmed by IT spectra analysis which showed a high intensity peak at 1725 Cal Adduces and reaction products are depicted in Figure 2.

to 4: The hydroxylamine modified copolymer micro spheres containing 35% mow HEM were impregnated with an aqueous solution of copper salt. The copper ions reacted with the micro spheres to form metal chelates adduces.

Example 5: Cross-linked micro spheres containing acid functions were produced by adding MA and BUM to the HEM - Acrolein monomer mixture The porosity of the micro sphere was significantly increased as evidenced by swelling (uptake of liquid). However, the size of the cross-linked micro spheres closely approximated that of the HEM - ACROI.EIN micro spheres of Example 2 as shown in Figure 1. By addition of increasing amounts of BUM to acrolein the hydrophilicity of acrolein spheres could be progressively increased.

Example 6: One of the ~AM-MA-HEMA-Acrolein copolymers was reacted with various Damon to form amine modified adduces. The results are shown in Table 2.

TV
, Jo , -;
it CAL ;:~ O O
o V
. X X I
CO_ Us ,_ E
L
I
I
I co æ
IL _ _ O O
, S X X Jo O Nut Z I
cry Us C ..
__~ ¢
CO
I
,, I
Jo z a) ,_ 1-- Of I- -, Jo cry. ox _. Jo Us TV
LO L awl I' O I ED
I O o o O
I L
o Us o o x x~c pa O C L
C z E lo D ,-at o O I I ED Ox _. _. Us O O O O
Jo .
a E x X x Us Lo ) I-- Z 0. 0 I ,_ O
Z C
a a 'I: O
r ,_ ._ O O
;~: O
V I
O OC~J L Jo Jo I) I) O

r3 X
' X ._ Go C q) E a C J
a _ _ I
I: a .
._ c~J~ I
_ J

it was found that at Hayakawa pi the number of free amino groups was comparable to the number of alluded groups found by hydroxyla~ine analysis. This reaction allows the efficient conversion of alluded functions to amine functions, removed from the surface of the spheres by a two to six carbon spacer arm.
The monomer mixture utilized in the experiment in Table 2 was modified by maintaining the ratio of HEM, MA and BUM constant while adding increasing amounts of acxolein. As shown in Table 3 which follows, the alluded content increased with increasing acrolein content proving that acrolein was being incorporated into the copolymer.

Table 3 No. ALDEHYDE_GROUPS~mg MOLE % ACROLEIN%N X lo-18 35.9 ~.26 1.35 58.5 4.32 1.93 75~8 6.57 guy 95.3 12.16 5.~3 Example 7: The copolymer of Example 6 was reacted with an adduce of fluoresce in isothiocyanate FIT
and 1,6-diamino-n-hexane which resulted in microspher~s of high fluorescent intensity.

En ale I: on allele nine adduce of FIT was prepared.
.__ Addition ox 0.1~ by wright of the adduce to the polyp merization system of Example 6 resulted in on addition inter polymerized fluorescent copolymer micro sphere, Eerily 9: An adduce ox 1,6-diaminohexane (DAY) and _._ FIT was prepared. Addition of 0.1~ of thy adduce to the polymerization system of Example 6 resulted in intro-diction of fluorescent chromophore by condensation with alluded groups to the addition polymerized copolymer.

Example 10: Dispersible iron oxide was prepared by dissolving 10 g of ferrous chloride and 13.5 g of ferris .
chloride in 210 cc of I w/v polyethylene mine (WOW. 1300) aqueous solution. 50% Noah was added to pi 7. The reaction mixture was relaxed for 3 hours, dialyzed extensively against water and separated helically three times from non-magnetic particles. The magnetic polyethylene imine-ixon oxide particles were redispersed in water and then sonicated with a clinical sonicator for 10 minute. Magnetic particles having a diameter of 300 Angstroms with amine groups on the surface were formed. When 1% of the polyethylene imine-iron oxide it added to the solution of monomers of Example 6 and subjected to gamma irradiation micro spheres containing a dispersion of magnetic iron particles is produced Example if: The Acrolein methacrylic acid copolymer micro spheres were oxidized to convert the alluded groups to carboxyl adding 30.5 ml Eye and I ml of H202 (30%) to 14.$ ml of an acrolein micro sphere suspension (33 mg/ml~. The suspension was stirred by a magnetic stirrer for 20 hours. The micro spheres were then washed three times and then centrifuged for 15 minutes in water.
The results are Shari in Figure 3.

Exempt _ : m-J of SDS powder was added to 10 cc of 5 (v/v) ARC solution and irradiated for 4 hours with a cobalt gamma source at 0.12 Mr/h and centrifuged 3 times.
The particles were not visible. Their size as determined under an electron microscope was about 300-500 Angstroms.
When the SDS content was increased to 50 my, the particles were too small to be centrifuged. After dialyzing for several days their size as determined by SUM was about 170 to 340 Angstroms.
The marking of cell surface receptors by means of fluorescent, non-fluorescent or magnetic fluorescent PAL
micro spheres was found to be simple and efficient as evidenced by numerous tests using fixed human or animal antibody labeled cells.
The reactivity is similar to polyglutaraldehyde micro spheres. However, no significant aggregation was observed during reactions with amine, dominoes or proteins under a variety of experimental conditions.
The micro spheres are preferably very small in size from 100 Angstroms to 100 microns, generally from 500 Angstroms to 10 microns so that specific receptor sites on a cell surface can be tagged.

Example 13: To 2.5 ml of a water suspension of acrolein micro spheres (total 15 my) was added 0.5 of a 2 my 1 ml solution of 125 Iodine labeled goat immunoglobulin G (spew. activity 1 x 105 cpm/mc3) in PBS. The mixture was rotated for 3 his. and 400 micro liters allocates were taken at 0,30,60,120 and 180 minutes. Alcott were immediately added to 400 ml of a 1% (w/v) solution of egg albumin in PBS and centrifuged at 15,000 x g for 4 min., resuspended and washed once in PBS as above.
The acrolein micro spheres exhibited direct binding of about 7-9% by weight of antibody whereas a control HEMA-BAM micro sphere was able to bind less than 1% by By weight of the micro sphere. Results are illustrated in Figure 4.

Binding of Methotrexate to Polyacrolein Micro spheres .
I. Preparation of Micro spheres [10%] - Total monomer concentration 90~ Acrolein 10~ Methacrylic Acid in 25 ml 0.4~ PRO 100,000 MY
pi 2.8 Degas with Nitrogen Co Gamma Radiation oh Dose - 0.12 Mocker.
Wash 3X
Resuspend 3b ml H20 lo Cone: 27.5 mg/ml Yield: 46.13~
II. Reaction of Micro spheres with 1-6 Diaminohexane 50 my of micro spheres 0.6 ml DAY t80% aqueous solution) Repeat 4 hr. with shaking at room temperature Wash 3X
Resuspend in 10 ml H20 III~ Reaction of Micro spheres with Carbodiimide Add 20 my of carbodiimide to 50 my of DO micro spheres I Sonic ate 10 minutes Adjust pi to 6 w/ No I PI
Add 10 my methotrexate in 2 ml M20 Check to be sure pi is still 6,0 Sonic ate 2 minutes Shake overnight at room temperature Spin down 3X
Take spectrum of first superannuate. Spectrum indicates that more than 90~ of methotrexate adduced with the micro spheres.

-21~ 5 A new convenient immunoreagent in form of acrolein Copolymer micro spheres was synthesized in a variety of sizes and with a relatively narrow size distribution.
High intensity of fluorescence can be imparted to the micro spheres during or after polymerization. The alluded functional groups permit covalent bonding with antibodies, enzymes and other proteins in a single step. Therefore this immunoreagent eliminates the previously used inter-mediate steps in which the cyanogen bromide and carbide-imide reaction was used. The high specificity of themicrospheres, at least as far as human rbc is concerned is also a desirable property, A minor synthetic modify-cation yields fluorescent, magnetic micro spheres for a large number of potential applications. The polyacrolein copolymer micro spheres of this invention contain approxi-mutely twice as many alluded groups as the comparable glutaraldehyde copolymer micro spheres.
The use of magnetic particles has created a great deal of interest in biochemical research and clinical 20 medicine when used as supports for immobilized enzymes.
Their easy retrieval from liquors containing colludes and undissolved solids should he of practical value.
The separation of proteins and chemical compounds by affinity chromatography can be simplified by elimination 25 of tedious centrifugation procedures and column chrome-tography steps. Magnetic particles have also recently been tested in radio immunoassay techniques in hyperthenmia treatment of cancer r in guidance of magnetic particles to a vascular malformation such as cerebral aneurysm with the 30 intent to seal the defect by inducing thrombosis Other proposed applications have been as tracers of blood flow or vehicles for drug delivery The first successful application of magnetic immunomicrospheres to the separation of B and T cells has been demonstrated.
35 There is little doubt that physical sorting of cell sub-populations has become a necessity. Many separation I

methods Wylie useful are limited by the restricted set of parameters upon which separation can be based and by the fact that they are batch techniques.
New flow cytometers and sorters permit quantitative multi parameter measurements and sorting based on these measurements, but are limited as far as the number of cells that earl be separated in a given time. Magnetic cell sorters have the potential of cell separation in a continuous process Evidence obtained using model cell I systems indicates that magnetic immunomicrospheres of desirably sizes 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 embody mints 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.

Claims (16)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A composition comprising an aqueous suspension of small, polymeric microspheres having a diameter from 500 Angstroms to 10 microns comprising the addition interpolymer-izate of a monomer mixture consisting essentially of:
20% to 90% by weight of an unsaturated aldehyde selected from acrolein and C1 to C8 aryl, alkyl or cycloa]kyl derivatives thereof;
10% to 50% by weight of at least one addition copolymer-izable mono-unsaturated, freely water-soluble acrylic comonomer substituted with a hydrophilic substituent selected from amino, carboxyl or hydroxyl; and 0.1 to 20% by weight of an addition copolymerizable, polyunsaturated cross-linking agent.

2. A composition according to claim 1 in which the diameter of the microspheres is from 100 Angstroms to 2000 Angstroms.

3. A composition according to claim 1 in which the aldehyde is sected from acrolein, methacrolein, alphaethyl acrolein, alpha-butylacrolein, alpha-chloroacrolein, betaphenyl-acrolein, alpha-cyclohexylacrolein and alpha-decylacrolein.

4. A composition according to claim 1 in which the aldehyde is acrolein.

5. A composition according to claim 1 in which the comonomer is selected from acrylamide, methacrylamide, acrylic acid, methacrylic acid, dimethylaminomethyacrylate or compounds of the formula:

-IMAGE-wherein R1 is hydrogen or alkyl of 1-8 carbon atoms, R2 is alkylene of 1-12 carbon atoms and Z is - OH or -IMAGE-where R3 or R4 are individually selected from H, lower alkyl or lower alkoxy of 1-8 carbon atoms.

6. A composition according to claim 5 in which the comonomer comprises hydroxyethyl methacrylate.

7. A composition according to claim 5 in which the comonomer imparts a negative charge to the microsphere.

8. A composition according to claim 7 in which said comonomer is methacrylic acid.

9. A composition according to claim 1 in which the cross-linking agent is present in an amount from 6 to 2% by weight and is selected from a polyvinyl diene or triene capable of addition polymerization with the covalent bonding monomer.

10. A composition according to claim 1 in which the cross-linking agent is bis-acrylamide.

11. A composition according to claim 1 in which the monomer mixture further includes 10% to 30% by weight of sparingly water soluble monomers selected from lower alkyl acrylates or vinyl pyridines.

12. A composition according to claim 1 further in-cluding 0.05 to 5% by weight of a copolymerizable fluorescent chromophore monomer.

13. A composition according to claim 12 in which the fluorescent monomer contains functional groups reactive with aldehyde.

14. A composition according to claim 13 in which the fluorescent monomer contains addition polymerizable unsat-urated groups.

15. A composition according to claim 1 in which said microspheres contain a disperslon of metal particles.

16. A composition according to claim 15 in which the metals are magnetizable.