Production of monodisperse, polymeric microspheres

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.

TECHNICAL FIELD 
The present invention relates to a process for the production of polymeric 
particles and, more particularly, this invention relates to a process for 
the production of evenly-sized, magnetic or non-magnetic, microspheres by 
the polymerization of falling or suspended uniformly-sized and shaped 
droplets in a containerless environment. 
BACKGROUND OF THE INVENTION 
There are extensive biological, medical and industrial uses for small 
polymeric particles having uniform size and even more extensive uses for 
magnetic polymeric microspheres. Small polymeric microspheres, especially 
those containing covalent binding functional groups, are finding 
increasing uses in separation processes such as affinity chromatography, 
in labelling and sorting of biological cells, in diagnostic testing and in 
clinical treatment. Metal and metal oxide containing microspheres, 
particularly those containing magnetically susceptible materials, find use 
in catalysis, and electron microscopy. Uniformly-sized particles can be 
utilized to calibrate instruments or filters and the like. 
Magnetic particles also find use in biology as substrates or carriers for 
enzymes or proteins and in cell biology as substrates derivatized with 
ligands capable of labelling specific cells. The labelled cells can then 
be separated from a mixture containing both labelled and unlabelled cells 
or from mixtures of labelled cells with other proteinaceous material. 
Magnetic microspheres can also be utilized to deliver a pharmaceutical to 
a specified location or organ in an animal or person. 
Microspheres containing magnetic oxides and/or electron dense metals such 
as iron can also be useful in cell identification by electron microscopy. 
Rembaum, et al (Science, 208: 364, 368, [1980]) disclose identification of 
malignant cells in mixture with normal cells by this technique. U.S. Pat. 
No. 4,169,804 discloses use of magnetic-ligand particles for measurement 
of hormones and vitamins. Magnetic microspheres labelled with specific 
antibodies have also been utilized to specifically bind to malignant cells 
in the treatment of leukemia. 
Magnetic polymeric microspheres have been used, for example, to remove 
cancerous cells from bone marrow as a treatment for neuroblastoma. The 
antibody-containing microspheres were mixed with both normal and cancerous 
bone marrow cells. The antibodies attached the microspheres only to the 
cancerous cells. The microsphere-cell conjugates were then removed from 
the solution by being attracted to a strong magnet placed adjacent the 
wall of the container. The cleaned marrow cells were then reimplanted in 
the patient. To date, 55 patients, many of them children, have been 
treated by this technique with promising results (Treleaves et al, Lancet, 
pp. 70-73, Jan. 14, 1984). Molday, et al., (Nature, 268: 437-438 [1974]) 
and U.S. Pat. Nos. 4,157,323; 4,177,253; and 4,267,235 also disclose use 
of magnetic microspheres in the labelling and separation of specific 
animal cells. 
DESCRIPTION OF THE PRIOR ART 
Magnetic polymers have been formed by dispersing the magnetic powders in 
preformed polymers. This technique is limited to soluble or meltable 
polymers and requires separate post-polymerization apparatus and 
processing and adds an additional energy cost to the product. Magnetic 
polymeric materials are generally produced by suspending magnetic 
particles in the liquid phase of the polymerizable formulation and 
polymerizing the monomers in presence of the magnetic particles to form 
polymeric microspheres. Polymerization can be by addition or condensation 
and can be conducted in bulk, emulsion, suspension or solution. Many of 
the magnetic particles are not incorporated into the resulting polymer and 
the size of the polymer particles must necessarily be larger than the 
magnetic particles. It is difficult to maintain a uniform suspension of 
the magnetic particles. The polymer particles are not evenly sized and do 
not contain a uniform amount of magnetic particles. The excess magnetic 
particles must be recovered from the polymerization formulation in 
post-polymerization processing steps. 
U.S. Pat. No. 4,339,337 discloses the preparation of magnetic beads by 
dispersing a magnetic filler in a solution of polymer dissolved in a 
polymerizable vinyl aromatic compound and polymerizing the compound. In 
U.S. Pat. No. 4,358,388, the magnetic filler is dispersed in an organic 
phase containing dissolved initiator and vinyl aromatic monomer. The 
organic phase is emulsified and polymerized to form a latex. 
Magnetic polyglutaraldehyde microspheres are prepared by polymerization of 
glutaraldehyde in presence of magnetic particles (U.S. Pat. Nos. 4,267,234 
and 4,267,235) and magnetic polyacrolein microspheres are also prepared by 
in-situ polymerization of acrolein in presence of magnetic particles (U.S. 
Pat. No. 4,438,239). 
U.S. Pat. No. 4,234,496 discloses the formation of magnetic polyvinyl 
pyridine beads by complexing the amine group with metal salts and reducing 
the complex to form finely divided free metal or metal oxides. This 
technique is limited to complexing with certain acids and the glass 
transition temperature of polyvinyl pyridine is low. 
Porous polystyrene particles containing magnetic iron oxide have been 
prepared by impregnating porous nitrated polystyrene particles with an 
iron salt such as ferrous chloride, ferric chloride or their mixture. 
Magnetic polymer particles having much higher magnetic strength are 
produced by the method described in copending application, Ser. No. 
786,649, filed Oct. 11, 1985. The method utilizes a NO.sub.2 containing 
polymer substrate. Magnetic oxide is introduced into the polymer substrate 
by reacting metal and the polymer in the presence of mineral acid. Very 
fine, black, magnetic oxide is deposited on the surface of the polymer. 
Uniformly sized, small microspheres of the order of 100 Angstroms to 10 
microns in diameter are preferred as carriers for biological substances 
such as antigens or antibodies. Uniformly-sized, small microspheres 
provide monodispersity and result in less non-specific binding to the 
surface of the cells or to the surface of containers. 
Most of the prior methods are incapable of producing uniformly sized or 
shaped spherical particles. The particles are somewhat ovoid in shape and 
are produced in a range of sizes. The magnetic oxide content also varies 
considerably. In some methods the magnetic oxide is present only on the 
surface of the polymeric particles. 
A few types of monodisperse, polymeric particles can be produced by current 
techniques. The particles that are available are very expensive. Some very 
uniform particles produced in space by Vanderhoff are being sold by the 
U.S. Bureau of Standards for $500,000 per gram. Monodisperse polymer 
particles can not be produced from most types of monomers by the methods 
presently utilized. 
Only a complicated process, stepwise seed growth emulsion polymerization, 
produces large, polymer microspheres of nearly uniform size above 2 
microns. This method is very lengthy, leaves unwanted impurities in the 
final product and can only be used with a few materials or monomers--all 
of which are hydrophobic. The microspheres must be washed and freeze-dried 
to obtain a dry product. The microspheres can be coated with magnetite to 
make them magnetic. However, the magnetite forms a loose surface coating 
which interferes with the attachment of antibodies. The magnetite contents 
per microsphere is limited to about 30% since the magnetite is present as 
a surface coating. The densities of polymeric particles produced in the 
presence of fillers such as dense metals or metal oxides such as ferrite 
is very limited. As the concentration of metal or metal oxide filler 
increases the polymeric particles fall out of the emulsion suspension and 
clump together. Hydrophilic, monodisperse particles can not be produced by 
current methods and metal containing hydrophilic, monodisperse particles 
have never been produced by any method. 
STATEMENT OF THE INVENTION 
Very small, individual polymeric microspheres with very precise size and a 
wide variety of properties can be produced in accordance with the present 
invention. Very pure, monodisperse particles can be produced from a wide 
variety of monomers, including hydrophilic monomers, as well as many 
substances which can be sprayed in a liquid form, such as polymers, 
proteins, waxes, starches and even glasses and metals. The particles can 
be produced in a wide range of particle sizes, densities and morphologies. 
Many types of fillers can be incorporated into the particles, e.g., 
magnetic fillers such as magnetite. The fillers are distributed in the 
volume of the particles rather than on the surface as provided by some of 
the prior methods of producing magnetic, polymeric particles. The 
microsphere particles can contain covalent functional groups on the 
surface capable of further reaction with and attachment to other materials 
such as fluorescent dyes, antibodies or other proteins. Macroreticular 
particles can also be made using the present invention simply by 
incorporation of a non-reactive diluent with the monomers. 
The microspheres are produced in accordance with the invention in a simple, 
one-step process. A uniformly-sized droplet of polymerizable liquid is 
formed in an injector device. The droplet is injected into a containerless 
environment and assumes a spheroid shape as it falls or travels through 
the environment. The spheroid droplet is subjected to polymerizing 
inducing radiation as it falls through the environment. Polymeric 
microspheres, having precise size range with diameters varying no more 
than plus or minus 5 percent, usually plus or minus 1 percent from an 
average size, are recovered. 
The method is applicable to any monomer that can be provided in liquid 
form. The monomer can be hydrophobic or hydrophilic. The bulk monomer can 
be a liquid at ambient temperature or can be dissolved in solvent. The 
monomer can also be a solid which is heated before and after being fed to 
the injector in order to convert the solid material to a liquid. Fillers 
can be predispersed in the liquid monomer to form a uniform dispersion 
before the liquid is formed into droplets. The process of the invention 
can be conducted without solvent, catalyst, suspending agents, emulsifiers 
or other reactants providing a very pure particle directly and avoiding 
costly post-polymerization purification techniques. Purity is further 
enhanced by the containerless environment in which the particle is exposed 
during polymerization to a gaseous, vacuum or near vacuum atmosphere 
containing very few molecules. The very pure environment prevents 
contamination by extraneous impurities that could be present in a liquid 
polymerization media or impurities provided by the container itself that 
can be carried into solution. The use of radiation induced polymerization 
also eliminates the introduction of impurities provided by residues of 
catalysts, initiators or suspending agents utilized in emulsion 
polymerization. 
Fluid dynamic forces cause the liquid droplet to assume a spherical shape. 
Subjecting the liquid sphere to polymerizing radiation while spherical 
results in the freezing of the object in a spherical shape as it is 
converted to a solid. In contrast, the forces in a stirred, liquid 
emulsion tend to produce egg-shaped polymeric beads. Radiation-induced 
polymerization rapidly converts the liquid droplets into a solid sphere 
with the expenditure of little energy. The dry product is produced in a 
form ready for use. 
The evenness of the size of the microspheres is due, in large part, to the 
injection of evenly sized drops into the polymerizing environment. A wide 
variety of sizes, including large sizes, can be produced since larger 
drops of higher density can readily be levitated while being polymerized. 
The levitation technique is not sensitive to the ionic or surface 
characteristics of the droplet and hydrophilic or hydrophobic monomers can 
readily be polymerized greatly increasing the range and type of materials 
available in monodisperse form. The internal dispersion of the magnetic or 
other filler reserves the functional sites on the surface. The surface is 
in a more biocompatible form and in a form more available for attachment 
to proteins, dyes or other subtrates. 
The monodisperse polymeric microspheres produced in the method of the 
invention have many uses. The microspheres can be used in polymeric, 
magnetic separation of cancer cells or in labelling and visualization of 
cellular structures. The particles can be used for column packing material 
for liquid chromatography as well as for affinity chromatography. The 
uniformity of size and shape are major factors in the column efficiency 
obtained. The microsphere particles have high surface area and can be made 
porous, like sponges, a form in which they are useful as catalysts or 
catalyst supports The pure hydrophilic materials have a low, non-specific 
absorption of hydrophilic materials such as proteins. Their large, uniform 
size coupled with mechanical strength allows high flow rates and high 
pressures to be used without breaking the particles. Materials stable in 
strong acids and bases can be made, allowing their use in a wide range of 
conditions. Also, if a catalyst were carried on the surface of a magnetic 
particle, the catalyst particle could be magnetically recovered after the 
reaction was completed. Further, by adding various metals, such as 
platinum, the particles themselves could be rendered catalytic. The 
magnetic particles are electron dense and therefore could be used to 
visualize biological or other structures in an electron microscope without 
the necessity to coat the particle with gold. Instruments, filters and the 
like can be calibrated using the very uniform, particles produced in the 
invention. New forms of paint or metal coatings for data storage can be 
fabricated using microsphere particles produced by the invention. The 
microspheres will also find use in diagnosis, therapy and drug targeting. 
These and many other features and attendant advantages of the invention 
will become apparent as the invention becomes better understood by 
reference to the following detailed description when considered in 
conjunction with the accompanying drawings.

DETAILED DESCRIPTION OF THE INVENTION 
The process of the invention involves the steps of (1) liquid droplet 
formation, (2) spheroidization of the droplet and (3) conversion of the 
droplet to a polymerized solid. Steps (2) and (3) are conducted in a 
containerless environment. Containerless environment of the invention 
means a process in which the droplet does not contact the walls of the 
container. Referring now to FIG. 1, a polymerizable liquid is fed from 
supply reservoir 10 to the droplet generator 12. A droplet 14 is deployed 
from outlet 16 of a nozzle 18 into a column 20 of gaseous environment 
contained within a container 22. As the droplet moves through the column 
20, it is formed into a sphere 24 by fluid dynamic forces. 
The droplet solidifies in the adjacent solidifying zone 26 and is collected 
in collector 28. The zone 26 can contain a jacket 30 of cryogenic liquid 
or it can contain a source of polymerizing radiation. If the droplet is 
frozen into spherical shape it can later be polymerized by applying 
radiation to the spherical particle. 
The container is of a length sufficient to complete the solidification 
process. In the case of slowly polymerizing liquid droplets, completion of 
polymerization may require thirty minutes or more. This would require a 
very long drop chamber. It is therefore preferred to levitate the droplet 
during polymerization. Levitation may be provided by acoustic, aerodynamic 
or electrostatic forces. Electrostatic levitation is preferred since 
acoustic or aerodynamic forces are more likely to distort the shape of the 
liquid droplet. The levitation column can be horizontal or vertical. 
Horizontal columns can be slightly tilted so that the droplets and 
hardened particles are pulled toward the outlet of the device by the 
action of gravity. 
Drop generators capable of developing evenly-sized drops are readily 
available commercially. One type of drop generator is based on Rayleigh 
instability of a charged fluid. A hollow needle is filled with liquid so 
that a partial drop protrudes from the end of the needle. Sufficient 
charge applied to the drop causes the drop to deform into a Taylor cone 
and to be ejected from the cone. Very small charged droplets of equal size 
are ejected from the apex of the cone. Control of the size of the tube, 
the character of the liquid and the amount of charge determines the drop 
size. 
A piezoelectric drop generator could be used similar to those used in cell 
sorters and ink jet printers. A piezoelectric crystal connected to the 
back of a small fluid filled cavity having a small opening in front is 
energized with a voltage pulse. The piezoelectric crystal expands, 
reducing the cavity volume. A small, constant-size droplet is ejected with 
each pulse of the crystal. Drops can be made in a size ranging from 1000 
Angstroms up to 100 microns. 
A system for continuously producing uniform microspheres is illustrated in 
FIGS. 2 and 3. The system includes a drop generator 31, a quadropole 
electrodynamic levitator 32 and a radiation source 34. Suitable drop 
generators are piezoelectric injectors or electrostatic drop ejectors. The 
drop generator 31 illustrated is similar to the injector used in ink jet 
printers or cell sorters. A cavity 36 has a piezoelectric crystal 38 
mounted on the rear wall 39 and a small outlet 40 in the front wall 41. 
When the crystal 38 is pulsed by the power supply 42, the crystal expands, 
reducing the volume of the cavity 36 and ejecting a small, constant-size 
droplet 44 of polymerizable liquid through the nozzle 45 with each pulse. 
Drops can readily be made in a size range from 1000 Angstroms to 100 
microns. The drop can be charged by applying a voltage to the tip of the 
nozzle 45 by means of a lead 47 connected to the high voltage terminal of 
a power supply 49. The droplet 44 is deployed into the inlet 48 of the 
central column 50 of the levitator 32. The drop assumes a spherical shape 
due to inherent fluid dynamic forces. The droplet 44 moves horizontally 
along the column by tilting the column toward outlet 52. 
The spherical droplets 44 are deployed into the central column of the 
levitator 32 and become trapped in the field of the levitator. The 
levitator 32 can take the form of four opposed electrodes 60, 62, 64, 66. 
An A.C. field is applied to the side electrodes 60, 62 from an A.C. source 
68 while a D.C. field is applied to the top and bottom electrodes 64, 66 
from a source 70. The varying electric field produces a centering force 
toward the longitudinal axis while the D.C. field serves to cancel the 
effect of gravity. The D.C. field would not be needed in a microgravity 
environment such as space. Since the forces that can be imparted to the 
particles by electrostatic levitators is limited, the zero-gravity of 
space will permit the positioning and control of large numbers of 
particles of high densities. This may provide an effective mass production 
environment. 
Many drops can be held at one time in a row along the axis of the 
levitator. Liquid drops of up to 1 mm can be supported in 1 g gravity for 
several hours. Therefore, the smaller polymer particles can easily be 
held. The tubes of the levitator need not be continuous but can be 
arranged in segments, each controlled separately. This allows the drops to 
be moved along the levitator in steps, or moved to a different level and 
turned around corners and the like which will facilitate large volume 
production of the microspheres. 
Once held in the levitator, the droplets will be polymerized by applying a 
beam of radiation such as ultraviolet energy from a radiation source 34 
such as a high pressure mercury lamp or a UV laser. After polymerization, 
the charged drops will be released from the levitator and collected as a 
dry product in a container 76 by attraction by a grounded plate, an 
electromagnet or permanent magnet placed on the wall of the container 76. 
The entire system can readily be automated to allow continuous production 
of the microspheres using the levitation system much like a factory 
conveyor belt. The environment of space also provides a vacuum environment 
for the column. Inert atmospheres such as vacuum, nitrogen or argon can be 
provided on land based systems by enclosing the column from the injector 
inlet to the particle outlet. The like-charged beads repel each other 
maintaining separation along the axis of the column. 
The liquid droplets may be neat, i.e., pure monomer, or may contain 
vaporizable solvent or diluent such as water or organic solvent usually 
from 0.1 to 30 percent by weight of solvent or diluent. The monomer is 
polymerizable by the radiation applied to the column, either directly or 
indirectly by means of a photoinitiator that is activated by the radiation 
to generate a polymerizing species such as a free radical. Suitable U.V. 
photoinitiators such as a benzoin alkyl ether may be present in an amount 
of 0.1 to 10 percent. The polymerization reaction occurs at higher rates 
as the amount of photoinitiator is increased. 
The droplets may also contain a dispersion of small filler particles such 
as 0.1 to 60 percent by weight of dense metals or metal oxides. 
Fluorescent and nonfluorescent dye may also be incorporated with the 
mixture to prepare colored particles. 
The magnetic particles may be blended into the polymerizable liquid from a 
suspension of the magnetic particles in water or organic liquid. Magnetite 
suspended in an aqueous liquid containing a surfactant suspending agent is 
commercially available. Aqueous suspensions of magnetite without 
surfactants can be made. Other fillers that can be utilized are colloidal 
iron, cobalt or nickel which are all strongly magnetic. High intensity 
magnetic fields can be obtained by dispersing samarium-cobalt or 
neodymium-cobalt magnetic materials in the polymerizable liquid. 
Unsaturated compounds, particularly acrylic monomers, polymerize by 
addition polymerization when subjected to thermal, U.V., gamma or other 
actinic radiation. Representative hydrophobic acrylic monomers are the 
acrylate esters such as compounds of the formula: 
##STR1## 
where R.sup.1 is hydrogen or lower alkyl of 1-8 carbon atoms, R.sup.2 is 
alkylene of 1 to 12 carbon atoms and X is a hydrophobic group such as 
lower alkyl or alkoxy of 1 to 8 carbon atoms. Representative acrylate 
esters are methyl methacrylate, methyl acrylate, ethyl methacrylate or 
propyl methacrylate. 
Hydrophilic and functional microspheres provide biocompatible substrates 
having surface sites available for covalent bonding. Hydrophilic surface 
also reduces the non-specific binding of protein on their surface which 
can cause the denaturing of protein and/or cross reactions. These monomers 
may be mono-unsaturated compounds containing a functional group such as 
aldehyde substituted acrylic monomers. Representative monomers are 
acrolein, acrylamide, methacrylamide, acrylic acid, methacrylic acid, 
dimethylamino-methacrylate or hydroxy-lower alkyl or amino-lower alkyl 
acrylates of the formula: 
##STR2## 
where R.sup.1 is hydrogen or lower alkyl of 1-8 carbon atoms, R.sup.2 is 
alkylene of 1-12 carbon atoms, and Z is --OH or R.sup.3 or R.sup.4 are 
individually selected from H, lower alkyl, or lower alkoxy of 1-8 carbon 
atoms. 2-hydroxyethyl methacrylate (HEMA), 3-hydroxypropyl methacrylate 
and 2-aminoethyl methacrylate are readily available commercially. Porosity 
and hydrophilicity increase with increasing concentration of monomer. 
Inclusion of polyunsaturated compounds also provides cross-linked beads 
which are less likely to agglomerate. The polyunsaturated compounds are 
generally present in the monomer mixture in an amount from 0.1 to 20 
percent by weight, generally 6 to 12 percent by weight and are suitably a 
compatible diene or triene polyvinyl compound capable of addition 
polymerization with the covalent bonding monomer such as ethylene glycol 
dimethacrylate, trimethylol-propane-trimethacrylate, 
N,N'-methylene-bis-acrylamide (BAM), 
hexahydro-1,3,5-triacryloyl-s-triazene or divinyl benzene. 
The monomer mixture may contain a large pecentage, suitably from 40 to 70 
percent of sparingly water-soluble monomers having hydrophobic 
characteristics. The crosslinking agent is sometimes sparingly water 
soluble. Hydrophobic characteristics can also be provided with monomers 
such as lower alkyl acrylates, suitably methyl methacrylate or ethyl 
methacrylate or styrene, or a vinyl pyridine. Vinyl pyridines suitable for 
use in the invention are 2-vinyl pyridine, 4-vinyl pyridine and 
2-methyl-5-vinyl pyridine. 
The metal or metal compound particles are preferably fine, evenly-sized 
materials having a uniform diameter smaller than the resultant microsphere 
diameter, typically below 1000 Angstroms. The metals are preferably the 
electron dense heavy metals having a high atomic number above 50, 
preferably above 75 such as Pb, Co, Pt, Au, Fe. The metal may be 
magnetically attractable such as Fe, Ni, Co or alloys thereof or an 
inorganic magnetic compound such as a metal oxide. The magnetic material 
is preferably a magnetic iron oxide of the formula Fe.sub.3 O.sub.4. Some 
hard ceramic-type ferrites, such as lithium ferrites can also be used. 
EXAMPLES OF PRACTICE FOLLOW 
Microspheres were produced in the system of FIGS. 2 and 3. A liquid monomer 
composition was placed in the cavity of the injector. The levitator was 
operated with a A.C. field of some 4000 volts on the two side electrodes 
and a D.C. field of 500 volts applied to the top and bottom electrodes. 
The charged nozzle sprayer crystal was pulsed to produce droplets from 
1000 Angstroms to 100 microns. In each case, the levitated droplets were 
irradiated with a UV light source for 20 minutes and the particles 
collected on a grounded plate. Sperical, polymeric, polyHEMA microspheres 
were collected. 
EXAMPLE 1 
The following composition was placed in the cavity of the injector: 
______________________________________ 
Material Percent 
______________________________________ 
Trimethylopropane triacrylate (TMPTA) 
5% 
IRGACURE* 184 5% 
Water 10% 
HEMA 80% 
______________________________________ 
* = 1hydroxycyclohexyl phenyl ketone 
Evenly sized, spheroid particles were produced. 
EXAMPLE 2 
______________________________________ 
Material Percent 
______________________________________ 
TMPTA 5% 
IRGACURE 5% 
HEMA 90% 
______________________________________ 
Evenly sized, spheroid particles were produced. 
EXAMPLE 3 
30 percent by weight of fine iron particles was added to the composition of 
FIG. 2 which was coated onto a slide as a layer. The layer polymerized 
when subjected to U.V. radiation. 
It is to be realized that only preferred embodiments of the invention have 
been illustrated and that numerous substitutions, modifications and 
alterations are all permissible without departing from the spirit and 
scope of the invention as defined in the following claims.