Note: Descriptions are shown in the official language in which they were submitted.
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
--3--
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
-4
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
--5--
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.
I -14-
Al r-
0 0
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
-15-
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.
--16 5
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
1-1 E
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
-17-
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.
--19--
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
-20-
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.
