Note: Descriptions are shown in the official language in which they were submitted.
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A l ~; l H~KED POLYMER MACROMOLECULE-EXCLUDING SURFACE,
ITS MODE OF SYNTHESIS AND USE
FIELD OF THE INVENTION
This invention relates to a biomolecule-excl~]tling polymer surface for use as a biocompatible
5 material or for size exclusion chlollla~ography.
BACK~ROUND OF THE INVENTION
Surface and interfacial chemistry concerns the processes that occur at the boundary between
gas- liquid, liquid-liquid, liquid-solid, or gas-solid interfaces. The chemistry and physics at
surfaces and interfaces govern a wide variety of technologically significant processes,
10 incl~1(1ing biocompatible materials, where schemes to reduce adhesion of biomolecules such as
protein and calcium depositions, while enhancing tissue integration, are critical to the
implantation of prosthetic devices. Likewise, this area of chemistry underlies the separation of
molecules using chromatographic techniques.
Chromatography entails a separation method whereby individual chemical compounds which
15 were originally present in a mixture are resolved from each other by the selective process of
distribution between two heterogeneous phases. The distribution of chemical species to be
separated occurs in a dynamic process between the mobile phase and the stationary phase.
The stationary phase is a dispersed medium, which usually has a relatively large surface area,
through which the mobile phase is allowed to flow. The chemical nature of the stationary
20 phase exercises the primary control over the separation process. The greater the affinity of a
particular chemical compound for the stationary medium, the longer it will be retained in the
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system. In other terms, the adsorptive effect of the chromatographic medium for di~el en~
solutes determines their rates of migration through the medium. Exclusion of a compound
will result in a rapid passage through the chromatography medium.
The phenomenon of adsorption, which is a basic thermodynamic property of interfaces,
5 resulting from a discontinuity in intermolecular or interatomic forces, is important in nearly all
industrial processes and products. Not only is adsorption the basic phenomenon of
chromatographic separations, but is a key process that underlies the use of soaps, wetting
agents, lubricants and surface treatments.
One area for which exclusion phenomena plays a foundational role is gel permeation
10 cl~oma~ography, wherein the size separation of macromolecules has become a standard
method for the separation of biopolymers, in particular of proteins and nucleic acid sequences.
Gel exclusion chromatography is associated with the equilibrium behavior of macromolecules
interacting with the gel material, that is, with the partition of a macromolecule between the
stationary and mobile phases (Giddings et al., 1968). Hence, the migration rate of a particular
15 species down a colurnn is directly related to its partition coefficient between the gel and the
surrounding medium. Thermodynamic theories relevant to exclusion chromatography
therefore center on calculation of this partition coefficient.
Gel permeation chromatography requires support materials possessing a hydrophilic surface
and which have if possible no unspecific adsorption behavior. To avoid the strongly
20 unspecified adsorption behavior which occurs when underivatized porous silica gels are used,
US 5,035,803 proposes that the surfaces ofthe pores in the silica gel be occupied by water-
soluble vinyl polymers. The grafting process used in US 5,035,803 provides polymers which
are randomly connected to the base support at any point of the polymer chain.
It is generally believed that the pore width of the chromatographic support material has to be
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matched to the respective separation problem. The processes which are used for setting the
pore width of separation materials frequently require a great deal of effort. For this purpose,
either the degree of cross-linking in the polymerization is adjusted or the pores of silica gel are
widened by post-~le~ elll steps.
It has been found, however, that wide-pored support materials whose pore width is so great
that no separation or only insufficient separation of substances is now possible on the basis of
gel permeation chronlatography give excellent separation results if linear polymers of water-
soluble vinyl monomers are grafted on to the aliphatic hydroxyl groups of these supports. In
these support materials, one terminal monomer unit is in each case covalently bonded to the
base support.
Another gel permeation material, described in WO 94/26379, combines the ideas of grafted
polymers with wide-pore support materials. This material allows substances in a mixture to be
separated on support material comprising linear polymers of water-soluble vinyl monomers
which are grafted onto aliphatic hydroxyl groups of the base support and which are covalently
bonded by a terminal monomer unit to the base support. The separation method of this
invention uses a wide-pored matrix whose pore space is completely acces~ihle to the analyte.
In the chromatographic separation method using this support material, the diffusion of
macromolecules is as strongly influenced by the linear polymers grafted onto the base support
as is similarly known from separations in the gel permeation chromatography of the prior art.
These supports and their methods demonstrate the general theory regarding gel exclusion
chromatography for which, in most calculations of the partition coefficient, the stationary
phase is considered to be C'porous''. The ratio of the equilibrium concentration of the
distributing species inside the gel to that in the bathing medium is the required quantity. It is
generally calculated by ~suming pores of various shapes or size distributions to characterize
the gel (Porath, 1963; Laurent and Kill~n-ler, 1964). The geometric limitations suffered by
the distributing species attempting to occupy the pores produce a geometry-dependent
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reduction in concentration inside the gel that defines the partition coefficient (Porath, 1963;
Laurent and K;1lAn(1Çr~ 1964; Giddings et al., 1968). The results are qualitatively in agreement
with the observation that larger molecules are excluded more than are smaller molecules, but
the molecular property of the distributed material which should correlate best with
5 chromatographic behavior is less clear (Giddings et al., 1968).
United States Patent No. 5585236 describes the separation of nucleic acids on nonporous
polymer beads having an average diameter of about 1 -100 microns, and which are suitable for
chromatographic separation of mixtures of nucleic acids when the polymer beads are alkylated
with alkyl chains having at least three carbon atoms. This procedure is based upon adsorption
10 chromatography, for which an elution profile will be generated wherein the smaller molecules
elute first and the larger molecules elute last. The separation is accomplished within a
gradient that causes the small fragments to elute in front of the larger ones.
It is important to note that this type of adsorption chromatography elution profile is opposite
to that obtained for gel exclusion chromatography. In the latter method, the larger molecules
15 are excluded from the surface and thus pass over the surface, eluting before the smaller
molecules which travel travel the additional distance created by pores or some matrix-like
material such as polyacrylamide gel.
Another area where adsorption of biomolecules on a synthetic surface is of prime importance
is the field of biomaterials science. In fact, the implantation of such biomaterial articles as
20 substitute blood vessels, synthetic and intraocular lenses, electrodes, catheters and the like in
and onto the body is a rapidly developing area of medicine. A pl i,lla~y impediment to the
long-term use of such biomaterial implantables as synthetic vascular grafts has been the lack of
sAti~fActory graft surfaces. The uncoated surfaces of synthetic blood vessels made from
plastics, for example, often stim~llAte rapid thrombogenic action. Various plasma proteins play
25 a role in initi~ting platelet and fibrin deposition on plastic surfaces. These actions lead to
vascular constriction to hinder blood flow, and the inflAmmAtory reaction that follows can lead
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to the loss of function of the synthetic implantable.
It is widely accepted that the biocompatibility of materials depends largely on their surface
properties and the reactions which occur when the material comes in contact with the
biological milieu. These reactions are understood to varying degrees. The most intense effort
has been expended in studying biomaterial/blood interactions (Brash, J. L. and Horbett, T. A.
(Eds), (1987) Proteins at Interfaces: Physicochemical and Biochemical Studies, American
Chemical Society, Washington, 1) but many other areas have received attention, including
restorative dental treatment (Glantz, P.-O. J., Attstrom, R.W., Meyer, A. E. and Baier, R. E.
(1991) In: Interfacial Phenomena in Biological Systems, M. Bender (Ed), Marcel Dekker,
Inc., New York, 77), soft tissue implants (Gristina, A. G., Myrvik, Q. N., Naylor, P.T. and
Meandor, T. L. (1991) In: Interfacial Phenomena in Biological Systems, M. Bender (Ed),
Marcel Dekker, Inc., New York, 105) and tissue culture cell compatibility (Crooks, C. A.,
Douglas, J. A., Broughton, R. L. and Sefton, M. V. (1990) J. Biomed. Mat. Res. 24:1241).
Protein adsorption from plasma or Iymph is a plhllaly event and most investigators in the field
believe that the subsequent fate of foreign material follows directly from the nature of this
adsorption. For instance, whole blood rapidly clots upon exposure to most non- biological
interfaces due to the surface activation of Factor XlI (Ratnoff, O. D. (1971) In: Thrombosis
and Bleeding Disorders: Theory and Methods, N. U. Bang, F. K. Beller, E. Deutsch and E. F.
Mammen (Eds), Academic Press, New York, 214). Platelet adhesion seems to correlate with
the degree of fibrinogen adsorption to many materials (Lindon, J. N., McM~n~m~, G.,
Kushner, L., Kloczewiak, M., Hawiger, J., Merrill, E. W. and S~l~m~n, E. W. (1987) In:
Proteins at Interfaces: Physicochemical and Biochemical Studies, J. L. Brash and T. A.
Horbett, (Eds), American Chemical Society, Washington, 507; Chaikof, E. L., Merrill, E. W.,
Coleman, J. E., Ramber, K., Connolly, R. J. and Callow, A. D., (1990) A. I. Ch. E. J.
36:994.). The complement system is activated by contact of blood with many types of
hemodialysis membranes and oxygenators (Mollnes, T. E., Videm, V., Riesenfeld, J., Garred,
P., Svennevig, J. L., Fosse, E., Hogasen, K. and Harboe, M. (1991) Clin. Exp. Tmmllnol. 86,
Suppl. 1:21).
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Since these reactions are all manifestations of bioincolllpa~ibility whose common thread is
plasma protein interaction with the surface concerned, a natural approach to improving
compatibility is to attempt to control the amount of relevant surface-associated protein.
Insoluble or cross-linked polymers are the most prevalent type of biomaterial and a very wide
variety of types have been tested for biocompatibility. However, the materials currently in use
were not specifically designed for this purpose, rather they were tested because they were
available for other reasons (Ratner, B. D. (1993) J. Biomed. Materials Res. 27:837). As a
result, a need remains for truly biocompatible materials, particularly where blood contacting
applications are concerned.
The surface properties of materials polymerized in bulk are difficult to control due to the
mobility of surface chains and the tendency of the surface material to adapt to the milieu in
which it is located. The surface concentration of component parts of the polymers may not
represent the bulk proportions and in extreme cases, such as with some polyetherurethanes
(Lelah, M. D. and Cooper, S. L. (1986) Polyurethanes in Medicine, CRC Press, Boca Raton,
FL.) local phase separation can occur.
One approach to providing a biocompatible surface with respect to protein adsorption and
platelet adhesion has been to incorporate neutral polymers such as poly(ethylene glycol)
(PEG; also known as poly(ethylene oxide) (Mori, Y., Nagaoka, S., Takiuchi, H., Kikuchi, T.,
Noguchi, N., Tanzawa, H. and Noishiki, Y., (1982) Trans. Am. Soc. Artif. Intern. Organs
28:459) or polyacrylamide (Fujimoto, K. et al. (1993) Biomaterials 14:442) into surface
regions of solid polymers or hydrogels (Drumheller, P. D. and Hubbel, J. A. (1995) J.
Biomed. Mater. Res. 29:207).
PEG may be incorporated into the polymer as a block, cross-linker or macromonomer
(Drumheller, P. D. and Hubbel, J. A. (1995) J. Biomed. Mater. Res. 29:207; Brash, J. L. and
Uniyal, S. (1979) J. Poymer Sci., Polymer Symp. 66:377; Sa Da Costa, V., Brier-Russell, D.,
S~ n, E. W. and Merrill, E. W. (1981) J. Coll. Interface Sci. 80:445; Takahara, A.,
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Tashita, J., Kajiyama, T., Takayanagi, M. and MacKnight, W. J. (1985) Poymer 26:987) or
grafted by reaction of gas phase monomers or oligomers with a substrate in a plasma
discharge (D'Agostino, R. (Ed), Plasma Deposition, Treatment and Etching of Polymers,
l~cademic Press, San Diego, (1990); Lopez, G. P. et al. (1992) J. Biomed. Mater. Res.
26:415). In the above cases it is difficult to measure concentrations, molecular weights and
dispositions of the hydrated components at the surface although x-ray photoelectron
spectroscopy (XPS) gives the elemental composition of the dry surface (Briggs, D. and Seah,
M. P., Practical Surface Analysis by Auger and X-Ray Photoelectron Spectroscopy, Wiley,
N.Y. (1983))
It seems that PEG is somewhat incompatible with a wide variety of proteins so at sufficiently
high PEG concentrations protein is excluded from the polymer coil. In the majority of cases
reported (reviewed in Harris, J. M. (Ed), Biotechl~ical and Biomedical Applications of
Poly(ethylene glycol) Chemistry, Plenum Press, New York, (1992)) both the amount of
protein adsorbed and the adhesion of platelets in vitro has been reduced very significantly.
Increasing PEG densities and molecular weights (up to 2,000) favour this reduction
(Golander, C. and Kiss, E. (1987) J. Coll. Interface Sci. 121:240; Gambotz, W. R. (1988)
Ph.D. Thesis, Centre for Bioengineering, University of W~hin~on; B~ ulll, K.,
Holmberg, K., Safranj, A., Hoffman, A.S., Edgell, M. J., Kozlowski, A., Hovanes, B. A. and
Harris, J. M. (1992) J. Biomed. Mat. Res. 26: 779). Protein adsorption is never ~limin~te~,
however; 5 - 10% or more ofthe control value remains (Golander, C. and Kiss, E. (1987) J.
Coll. Interface Sci. 121:240; Gambotz, W. R. (1988) Ph.D. Thesis, Centre forBioengineering,
University of Washington; Belg~lolll, K., Holmberg, K., Safranj, A., Hoffman, A.S., Edgell,
M. J., Kozlowski, A., Hovanes, B. A. and Harris, J. M. (1992) J. Biomed. Mat. Res. 26: 779;
Llanos, F. R. and Sefton, M. V. (1993) J. Biomed. Mater. Res. 27:1383).
Direct chemical grafting of pl ~rol llled PEG to activated surfaces has been employed
successfully in several studies (Harris, J. M. (Ed), Biotechnical and Biomedical Applications
of Poly(ethylene glycol) Chemistry, Plenum Press, New York, (1992); Golander, C. and Kiss,
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E. (1987) J. Coll. Interface Sci. 121:240; Belg~llolll, K., Holmberg, K., Safranj, A., Hoffman,
A.S., Edgell, M. J., Kozlowski, A., Hovanes, B. A. and Harris, J. M. (1992) J. Biomed. Mat.
Res. 26: 779; Tseng, Y.-C. and Park, K. ~1992) J. Biomed. Mater. Res. 26:373). Uni~lllli~y
of coverage (detected by XPS) can be a problem however (Harris, J. M. (Ed), Biotechnical
5 and Biomedical Applications of Poly(ethylene glycol) Chemistry, Plenum Press, New York,
(1992)) and high density layers, in which the average chain separation is much less than the
radius of gyration in solution, are not generally achieved. Presumably this is because in good
solvents bound chains progressively exclude mobile chains as the layer density builds, reducing
the probability of reaction between mobile chains and unreacted surface sites to negligible
10 levels.
SUMMARY OF THE INVENTION
It is an object ofthis invention to provide a synthetic surface that excludes and/or ~in;~ es
the adsorption of proteins and other macromolecules. The approach taken should work for a
wide variety of macromolecules, not just proteins; e.g.: polysaccharides, nucleic acids,
15 lipoproteins, synthetic polymers, etc. This surface has use as a size exclusion medium for
chromatography and as a biocolllpalible material, because of the exclusion qualities of the
relatively high polymer chain density.
It is another object of this invention to provide a method of synthesis for a high density
tethered polymer surface comprising polymerizing vinyl monomers from an initial high surface
20 concentration of initi~ting groups. The method of synthesizing this surface involves initi~ting
polymerization of the surface polymer chains from the surface groups using CeIV or a metal
carbonyl, which optimizes the density of chains tethered to the surface
DESCRIPTION OF THE FIGURES
Figure 1: Conductometric titration of functional surface groups
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DESCRIPTION OF THE FIGURES
Figure 1: Conductometric titration of functional surface groups
Figure 2: Proton NMR spectrum of latex A3
Figure 3: Conductometric titration for aldehyde content determination
5 Figure 4: Size distribution of Batch #14 seed
Figure 5: Size distribution of Batch #14G1
Figure 6: Size distribution of Batch #14G12
Figure 7: Size distribution of Batch #14G1233
Figure 8: Size exclusion of non-porous beads with grafted neutral polymer chains10 Figure 9: An example of seed production and growing
Figure 10: An example of Acrolein derivatization
Figure 11: An example of grafting using CelV on aldehyde group initiation
Table 1: Seeded Polymerization experiments
Table 2: Recipes for copolymerization styrene/acrolein on the latex
15 Table 3: Characterization of seed latexes
Table 4: First-stage growing latexes
Table 5: Second-stage growing latexes
Table 6: Third-stage growing latexes
Table 7: Surface charge density ofthe final latexes
20 Table 8: Aldehyde content of the derivatized latexes
Table 9: Complete example of parameter set.
Table 10: Optimum range of parameters
Table 11: Aldehyde derivatization
DETAILED DESCRIPTION OF THE INVENTION
25 Definitions
A "biomaterial" may be defined as a material that is substantially insoluble in body fluids and
that is designed and constructed to be placed in or onto the body or to contact fluid of the
- CA 02202424 1997-04-11
body. Vascular grafts and contact lenses are examples of biomaterials.
As used herein, the solid surface of a biomaterial is characterized as "biocompatible" if it is
9a
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capable of functioning or existing in contact with biological fluid and/or tissue of a living
organism with a net beneficial effect on the living organism. Long term biocompatibility is
desired for the purpose of red~lçing disturbance of the host organism.
Size exclusion cl)l o~ ography is a chromatographic process in which the compounds in a
5 mixture are separated on the basis of their hydrated size in solution, the larger molecules
passing through the chromatography medium more rapidly than the smaller molecules, due to
size-dependent exclusion from the stationary phase. Media which produce size exclusion
cl~rollla~ography exclude larger molecules to a greater degree than smaller molecules.
Grafting or surface-initi~ted polymerization is initiation of polymerization from a chemical
10 group associated with the surface. The reaction adds monomers pl t;reren~ially to the polymer
chain attached to the surface as opposed to producing or adding to chains in solution that are
not covalently attached to the surface. Two methods of initiation that produce such reactions
are CeIV initiation and initiation by metal carbonyls in conjunction with UV radiation or heat.
A macromolecule is any oligomeric or polymeric material con1~inin~ more than approxilllately
15 ten monomers, or any non-polymeric species of molecular weight greater than appl o~illlately
500 g/mole. Examples are polypeptides, proteins, nucleic acids, polysaccharides, lipoproteins
and synthetic polymers.
A) The Core Particle or Solid Surface
If the particle core or solid surface to be grafted is polymeric, it consists of precipitated or
20 covalently cross-linked polymer that is insoluble in the solvent in which it is immersed and so
produces a solid particle or other form of solid surface that does not contain pores. Examples
are polystyrene, which is a precipitated polymer subst~nti~lly insoluble in water or divinyl
benzene-styrene copolymer, which forms covalently cross-linked particles that are
substantially insoluble in all solvents that do not break covalent bonds. Many other polymeric
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particles and surfaces are known to those skilled in the art, including newly developed
materials such as urea-melamine beads. In general, all types of polymers which may be
produced in monodisperse beads in the size range between 0.1 to 50 microns may be used for
both particles or solid surfaces.
5 If the particle or surface is not polymeric, it may be a solid, nonporous particle that has on its
surface chemical groups that can be used for surface initiation or that may be chemically
modified to provide such chemical groups. Alternately, it may act as a substrate for the
adsorption of a polymer shell from which surface initiation may be performed. An example
would be silica particles or glass surfaces that can be reacted with silane reagents to provide
10 hydloxylated or aldehyde-co~ -g surface groups. Alternately, hydrophobic silane reagents
may be applied and a copolymer shell of styrene and acrolein adsorbed to the surface. In all
these cases surface initi~ted polymerization could subsequently be carried out by, for instance,
CeIV initiation. There is a large range of solid, non-porous particles and surfaces that could
be used for this purpose, as is well known to those skilled in the art.
15 B) The Polymer Shell: The polymer shell is typically a copolymer of (a) one monomer that is
soluble in or adsorbs to the core particle or the solid polymer whose surface is to be grafted;
~b) a monomer co~ g aldehyde, aliphatic hydroxyl ~or groups that can be readily
converted to aliphatic hydroxyl groups, such as epoxides) or sulfhydryl groups from which
grafting reactions may be initi~te~ In general, any chemical group carrying a hydrogen atom
20 which may be oxidized can form a potential starting point for this process of surface
polymerization. An example is a copolymer of styrene and acrolein that associates with the
surface of polystyrene core latex particles or the surface of bulk polystyrene because of the
solubility of styrene in polystyrene, and which provides aldehyde functions from which to
initiate surface polymerization of vinyl monomers by CeIV initiation.
25 C) The Tethered Polymers: The tethered polymer is a substantially linear polymer formed by
the polymerization of one or more types of vinyl monomer via surface-initi~ted
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polymerization, typically by initi~ting polymerization with metal carbonyls and W radiation
or heat or with CeIV from aliphatic hydroxyl, aldehyde or sulfhydryl groups. For use in
aqueous solutions, the vinyl monomers should be water soluble. For the synthesis of surfaces
for size exclusion chromatography, the tethered polymers should be electrically neutral.
5 D) Modes of Synt~esis: If the core particle or solid surface is a solid polymer latex, either
emulsion polymerization or surfactant-free polymerization may be used for its synthesis. The
shell copolymer typically would be added by solution polymerization of a copolymer, one
monomer of which was dissolved in the latex or adsorbed to its surface, usually using the
same initiator as was used to synthesize the core latex. However, oil soluble initiators could
10 also be used to advantage in certain systems. Those skilled in the art will recognize a number
of well known ways by which to synthesize both the core and shell polymers.
The tethers are synthesized by use of a surface-initi~ted polymerization reaction, such as
initiation with CeIV from aliphatic hydroxyl, aldehyde or sul~ydryl groups, or via metal
carbonyl chemistry in combination with W radiation or heat, lltili7ing water soluble vinyl
15 monomers. Those skilled in the art will know a large number of suitable monomers, including
(meth)acrylic acid derivatives such as acrylamide or methacrylamide, also 2,3-dihydroxypropyl
methacrylate or N-(2-methoxyethyl) acrylamide or N-(2,3-dihydroxypropyl) acrylamide.
Vinylated heterocyclic compounds may also be used to advantage, such as 1-vinylimidazole,
N-vinylpyrrolidone, 2-vinylpyridine, 4-vinylpyridine and 4-vinylpyrrolidone-N-oxide.
20 Macromers such as poly(ethylene glycol) methacrylates may also be used, which will produce
tethers with a comb structure.
Moreover, a process recently developed for grafting polyacrylamide on polyethylene foils
could be used. In this process, radicals are created on the foil using Co(60) radiation and the
foil is immersed in the monomer solution.
12
_ CA 02202424 1997-04-11
E) Other Applica~ions or Uses for Such a Molecule-Excluding Surface: As well as use as for
size exclusion chromatography and in biomaterials, tethered polymer surfaces can be used to
control the adsorption of molecules responsible for adhesion of microorg~ni.~ms to surfaces so
will be useful in producing anti-fouling surfaces. Moreover, depending on the type of polymer
5 grafted onto a surface (ie. "compatible or incompatible" graftings), the polymer surface
coating may be used to render surfaces sticky (ie. gluing together, or repulsing each other).
EXAMPLE I: SYNTHESIS OF A POLYMER SURFACE
Materials
10 All the distilled water used was further purified using a Milli-Q Plus water purification system.
Styrene was Aldrich reagent grade material. It was purified by vacuum distillation at 40~C in
an atmosphere of argon. Purified styrene was stored under argon at -70~C. Acrolein was
Aldrich reagent grade material. It was purified by distillation at 56~C in an atmosphere of
argon. Purified acrolein was stored protected from light, under argon at -70~C. 2,2'-Azobis
15 (2-amidinopropane) dihydrochloride (ABA.2HCl) was supplied by Wako Co. and was used
without further purification. The commercial name for the initiator is V-50 and the formula is
the following:
CH3 CH3
Cl- (+H N ~C--C--N = N--C--C~; NH I ) Cl
C~3 C~3
Sodium chloride Fisher reagent grade was used without further purification.
Preparation of seed lattices
20 The apparatus consisted of a four-necked flask (1 dm3 capacity), equipped with overhead
stirrer, condenser, side-armed addition funnel, argon inlet with stopcock (argon outlet through
the top of condenser). Argon flow was controlled by a needle valve and stirring rate by a
tachometer. The flask was m~int~ined at a constant temperature, by immersion to the neck, in
a thermostated water bath.
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The following quantities of material were used:
0.72 dm3 water
0.872 g NaCl (2.07*10-2 moles/l)
0.54 g ABA.2HCl (2.76*10-3 moles/l)
34.71 g styrene (0.44 moles/l)
0.62 dm3 water and the required amount of sodium chloride were initially placed in the flask,
which was then evacu~ted eight times and flushed with argon under stirring (350 rpm).
The temperature of the bath was then increased to 70 ~ C, under stirring and slow argon flow
initi~ted (1 bubble/s). Styrene was added under argon protection and then the funnel
co~ il-g initiator dissolved in 0.1 dm3 water, previously deg~sed and flushed with argon.
Af[er five minutes, initiator solution was released into the reaction mixture.
The reaction was allowed to proceed for 24 hours, at 70 ~ C, under argon flow, at 350 rpm
stirring rate. The reaction mixture was then cooled at room temperature and filtered through
glass wool to remove big aggregates. The product was cleaned by dialysis against distilled
water for one week, in a 10 1 tank, ch~nging the water every day. Further cleaning was done
by centrifugation and washing at 2500 rpm. The latex suspension was then weighted and the
solid content was determined by freeze drying. The yield was calcul~te-17 relative to the total
amount of styrene introduced.
All the latex suspensions, after cleaning, were stored at 4 ~C, in polypropylene tubes, until
future use.
Seeded polymerizations
A number of expelh~.enls were conducted in order to optimize the conditions for obtaining
reasonably monodisperse polystyrene beads, with a size in the range 1-3 ~lm, using ABA.2HCl
14
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as initiator.
A reaction vessel (as described in Section 6.1.1), having a capacity of either 0.25 1 or 0.5 1
was used. The seed latex was first weighed and put in the flask and the desired concentration
of solids was adjusted using distilled water. Sodium chloride was used in some experiments to
5 achieve a desired ionic strength. The apparatus was ev~cu~ted eight times and flushed with
argon under stirring at 350 rpm, then the temperature was increased to the desired value.
Styrene was added under argon protection. The seed latex was swollen under the same
stirring regime and argon flow (1 bubble/s).
Initiator was then added and the reaction continued for the prescribed time. The product was
10 treated exactly as described above. In some reactions, because the product was aggregated, it
was sonicated for 30 min. before filtering through the glass wool. To prevent further
aggregation, the sonicating bath was cooled with ice.
Grown latexes were characterized with respect to size distribution and solid content. The yield
was calculated for the growth reactions.
15 Detailed recipes for these steps are written in TABLE 1.
In all experiments except for lOG12 (100 g/l), the concentration of styrene relative to total
aqueous phase was 80 g/l.
Surface aldehyde derivatization
The work described in the literature involving polystyrene latex with aldehyde groups on the
20 surface refers to much smaller beads, produced directly by polymerization of a mixture of the
two monomers utili~ing potassium persulphate as initiator. We developed a procedure for
derivatization of previously grown beads. A mixture of styrene and acrolein was used to
CA 02202424 1997-04-11
produce a shell around the polystyrene core.
The monomer ratio was calculated according to the copolymerization curve (Polymer
Handbook, J. Brandrup and E.H. Immergut, eds, Section II: 110, InterScience Publications,
N.Y., 1966) to give azeotropic conditions (ie., composition ofthe feed equal to the
5 composition ofthe reslllting copolymer). The experimental setup was the same as above. The
procedure was as follows:
Seed latex was charged in the flask, which was then ev~c~l~ted and flushed with argon eight
times. The temperature was raised to 50 ~C, under gentle argon flow and stirring at 350 rpm.
Styrene was added and the seed allowed to swell for 15 min., then acrolein dissolved in 10 ml
10 water was put into the reaction vessel, followed by initiator solution (in 10 ml water, washed
in with 10 ml more). The reaction was continued at 50 ~C, under argon flow (one bubble/s)
and with stirring at 350 rpm for 6 h. (See Table 2)
Analytical methods
The size distribution was determined from sc~nning Electron Microscopy images of the latex
15 (one drop was dried on carbon plate, then covered with gold). An image analysis program was
used to measure the diameters of at least 100 beads on several pictures taken at di~l~lll spots
on the plate.
Surface Charge Density
The surface functional groups on the beads are initiator residues only, because the method
20 used to synthesize them was emulsifier-free. They consist of amidine groups, positively
charged. The suspension stability of the latex results from the presence of these groups on the
surface of the microspheres, hence the surface charge density (i.e. concentration of surface
functional groups) is an important characteristic of the product.
16
CA 02202424 1997-04-11
A conductometric titration technique was used to determine surface charge density of the
latexes used for aldehyde derivatization. The method is described in the literature. The only
modification was that the cleaning step to remove detergent, involving ion exchange resins
was omitted, because the beads were surfactant-free. The samples (already dialysed, as
5 described above) were further prepared for titration only by washing once in water
(centrifugation at 1500 rpm, removal of supernatant and replacement with fresh distilled
water). The solid content was determined by freeze drying and weighing the initial suspension
and solid residue. At least 0.5 g of solid latex was then suspended in 10 ml of water, purged
with argon for 5 minutes and placed in the conductometric cell under slow argon flow. A
10 conductivity meter was used to monitor con~-1ct~nce, while 0.01 M titrant (either HCI or
NaOH) was added to the sample, using a precision pump, under vigorous stirring. Titrations
were pelrolllled at constant flow rate (0.0204 mVmin.) with time monitoring. For each
sample, the equivalence point was found twice, once from direct titration using HCI and the
second time from backwards titration using NaOH. The results are reported as the average for
15 two samples.
Both solutions used for titration were standardized by potentiometric titration. NaOH was
first used to titrate a standard solution of potassium biphtalate, then HCI was used to titrate
the NaOH solution of known concentration.
Aldehyde content analysis
20 Nuclear Magnetic Resonance
For the latexes with high aldehyde surface concentration, proton Nuclear Magnetic Resonance
(NMR) spectroscopy was used to determine the aldehyde content. The samples for NMR
were prepared as follows:
The latex suspension co~ g approximately 30 mg solids was freeze dried for 24 hours,
CA 02202424 1997-04-11
then the solid residue dissolved in 1 ml deuterated tetrahydrofuran. Traces of water were
removed from the solution by keeping it in contact with molecular sieves overnight. The
sample was then 11 ~nsrel 1 ed into an NMR tube, previously flushed with argon.
A Brucker 400 Mhz spectrometer was used to record the spectrum.
5 Conductometric Titration
For latexes with low surface aldehyde concentration, NMR was not sensitive enough to
provide a reliable assay. Tn~te~-1, a method from the literature for dete~ inillg aldehyde
content based on conductometric titration was used. This involves reacting the aldehyde
groups with hydroxylamine hydrochloride:
10 NH20H.HCl + R -HC=O HCl + R- CH=N-OH + H20 + HCl
The hydrochloric acid resulting is titrated conductometrically with sodium hydroxide.
The è~)elilllental procedure is described below:
A 0. lM solution of hydroxylamine hydrochloride was prepared in a volumetric flask (M.W.=
69.5; 0.695g/lOOmT ). An exact volume of this solution was added to a concentrated
15 suspension of latex of known solid content. The mixture was left to react overnight while
tumbling in a rotating rack at room temperature. The suspension was then filtered through a
membrane filter (0.22 ~lm pore size), and diluted to 10 mL, then ~ srelled to the
conductometric cell. The titration proceeded under argon. Sodium hydroxide solution,
previously standardized, was added from a glass syringe using a precision pump. During the
20 titration, the time and the conductance were monitored, at constant flow rate and the time
converted to volume. The equivalence point was read on the conductance versus titrant
volume plot. The result reported is the average of two titrations.
18
CA 02202424 1997-04-11
EXAMPLE 2: PRODUCTION OF SEED LATEX
Grafting on latex A8 is accomplished as follows. The reactor is a three-necked vial (capacity
50 ml), equipped with argon inlet and outlet (with stopcocks).
Latex suspension (2 g solids suspended in 9 ml water) and MEA monomer (0.92 g) are
5 introduced in the reactor, which is then deg~sed two times and flushed with argon. The
initiator solution (0.31 g cerium (IV) ammonium nitrate is dissolved in 2 ml of 10 mM nitric
acid solution) is added from a syringe, which is previously degassed. The content of the vial is
mixed by hand, then stirred for one hour at 40 ~C. The reaction is then continued for 64
hours at room temperature.
The product is suspended in 250 ml water, filtered on Millipore ~ ne (1.25,~m), washed
on the filter with 25 ml 0.1 M sodium sulfite solution and then with 50 ml 0.03 M EDTA
(ethylene~ inetetraacetic acid trisodium salt hydrate) solution and rewashed with water.
The product is resuspended in 25 ml water and stored in polypropylene tubes at 4 ~C until
further use.
EXAMPLE 3: CI~ARACTERIZATION OF TEE LATEX
Typical values for the characteristics of the seed latex produced by the described technique are
given in Table 3. Typical values for the conditions that lead to successful first, second and
third growth stages are summarized in Table 9. In this Table, latexes 12G1 and 14G1 have
gone through one growth stage, latexes 12G12 and 14G12 have completed the second growth
stage and 12G1233 and 14G1233 have gone through the third growth stage. For each the
important parameters describing the synthetic conditions that gave stable, uniform latex
plepa.~lions are listed. The optimum range these synthesis parameters could take and still
19
CA 02202424 1997-04-11
produce acceptable products are summarized in Table 10. Table 11 presents two examples of
synthesis of the shell copolymer added to the two parents described in Table 9 where the
critical parameters are listed that again produced stable products. The optimum range for the
shell synthesis parameters are as follows:
1. Acrolein to seed ratio: 1x10-3 to 2x10-3 moles per gram latex
2. Initiator concentration: 4.23x10-4 to 4.4x10-3 moles dm~3
3. Charge density of seed: 154 A2/group to 314 A2/group
The physical properties of typical latexes at each stage of the growth process are given in
Figures 4-7 (size distribution) and Table 7 (surface charge density) and Table 8 (aldehyde
content by two methods).
A grafted layer of N-(2- methoxyethyl) acrylamide (MEA) was added to the core/shell latex
A8, as described above. The amount of monomer grafted was determined from analysis of the
amount left in solution following polymerization. The method was based on the behaviour of
the monomer on HPLC on RP Select B (Merck), 10 m diameter beads, 4.5 cm x 1.0 cmdiam. column, 3ml/min flow rate, in a gradient of 0.01 M tetrafluoroacetic acid (TFA) and
50% acetonitrile in 0.01M TFA. The monomer concentration was determined by the optical
density (OD) at 240 nm. The column was calibrated with pure monomer of known
concentration. The amount grafted was found to be 3.56 x 10 -3 mole/g latex.
The grafted beads were packed into a column 0.46 cm diam x 7.5 cm and a series of proteins
of known molecular weight, dissolved in a buffer of 10 mM phosphate, 300 mM NaCI, pH
7.2, at a concentration of approximately 0.5 mg/ml were chromatographed. The flow rate
was 0.01 ml/min and the OD ofthe eluent stream was monitored at 280 nm. The results are
shown in Figure 8 in which it is seen that good separation on the basis of molecular weight is
obtained.
CA 02202424 1997-04-11
Hence, size exclusion chromatography of proteins is demonstrated on solid polystyrene beads
carrying neutral polymer tethers.
21
ra~e ~: ~ee~ed po~mer~on ~x~erimen~
Batch # ~/. solid~ A.B.A~ 2HCI ~a~l Swelling Re~c~ion Temper~ture Yield
molesn mole~ me t~me ~C %
hours hours
g&l 1 2.2EXP-03 0 24 22 5~ 5
gGlI 1.5 2.2EXP-03 0 2~ 22 50 ~4 5
9GI 11 3. 15 2.2EXP-û3 0 24 22 SO 4U D
9G1112 3 2.2BEXP-~3 2.4EXP-02 I5 I2 50 nega~ive r
gGI 1122 4 2.2EXP-Q3 0 1~ 12 SO llegative r
lOGl 3.17 2.2E~-03 0 27 12 50 nega~e
lO&~ I l.S 6.21E~-04 ~.6EXP-&3 2~ '~2 50 negatrve r
lOG1~ 1 2.4 2.2E~-03 0 3 12 ~0 nega~e
lOGIl 12 2.87 1.47EXP-03 .2EXP-02 0 24 50 3~
llG1 2.9S 2 2EXP-03 ~ 0 12 50 40
11GI1 ~ XP-03 0 0 lO SO nega~ve
1 lG1 I 1 2 1.4EXP-03 0 0 1 1 ~0 ueg~ive
1 lGI2 3.33 1.1EXP-03 2.5EXP-03 0 12 50 nega~e
able 1 cor.t ' d
Ba~S# %so~d~ A~B.A~2E~a Na{~ Swening Reac~on T~nnp~ ~eure Yi~d
mol~s~ moles~ t~ne ~me 'C %
hour~ bour~
l lG12Z 3.33 1.1E~P-03 0 0 9 50 1011&1223 3.17 6.1~E~CP-04 0 0 12 S0 13.~
I lG12234 J. 15 2.56E~CP-04 0 0 }2 50 nega~ve
12G1 3.1S 2 2E~nP-~3 0 0 12 ~0 ~212G12 3.~3 ~. lE~DP-C3 0 0 12 50 6512G123 3.4 ~.16E~{P-04 0 0 12 ~0 ne~ve D
12&1233 3.4 ~.2E~DP-~4 0 0 12 S0 57 r
12&122 3.33 l.I~F.~nP-03 0 0 12 S0 37.5 r
13G] 3.I~ 2.2E~P-03 0 0 12 S0 6g13G12 3.4 1.06E~YP-03 0 0 12 50 22 r~
13G123 ,.4 8.5E~P-04 0 0 12 iO neganve
13GI22 3.3~ 1.08E~P-03 0 0 12 S0 ~ega~ve
14G1 3.15 4.4E~DP-Q3 0 0 12 50 7114GI 1 ~ E~aP-03 0 0 12 45 negat~e
14Gl l l 1.~ 8.4E~DP-0~ 0 0 l2 4~ negatn~e
14G12 3.15 2.7E~P-03 0 0 12 S0 71.g
~a~l e ' con~ ' d
B~l~cb # ~/o so~ds A,B.~. 2~CI N~ wel~i~g Reac~i~n TempeFature Yield
molesJI mole~ ne t~ne ~C %
hours hours
l4~12~ 3.15 4.4EXP-û3 0 0 I2 50 66
14G123 3.4 417E~P-03 0 0 12 40 nega~e
14G12~3 3.4 4.4EXP-03 0 0 12 50 ~6.5
14Gl223 3.15 4 ~EXP-a3 0 0 12 50 ~8.4
CA 02202424 1997-04-11
T~le 2: Recipe~for copoly~ner~zl7~ion s~rene/ncrolein on ~he lat~x
Batch "Pdrent" Solid Acrolein Styrene ~A.2~C~
# conc., ~/O moleslg moles/~ moles/l
latex latex
Al 12G1233 3.4 lE~P-02 1.4EXP-02 3.1EXP-04
A2 12G1233 3.33 2EXP-03 2.8EXP-03 4.23EXP-04
A3 1~G1233 3 33 1EXP-03 1.4EXP-03 4.23EXP-04
A4 12G1233 3.33 2EXP-04 2 8EXP-04 4.23E~-04
A5 12G1233 3.33 4EXP-04 5.7EXP-04 4.23EXP-04
A6 14G 1233 3.33 IEXP~03 1 .4E~XP-03 4.4E~P-03
A7 14G1233 3.33 lEXP-03 lEXP-03 9.22E~'-04
. .
- AX 14G1233 3.33 2EXP-03 2EXP-03 9.22EXI'-04
. .
Ag 14G1233 3.33 lE~P-03 1.4EXP-03 4.23EXP-04
A10 14G1223 3.33 2~XP-03 2E~P-03 9.22EXP-04
CA 02202424 1997-04-11
~able 3: ~karacteriz~tJon vf'seed Intexes
Seed # l~le~n di~meter St~ndnrd Yield
~m devi~tion
0.833 0.04764 70%
0.737 ().03775 71.2%
1 1 0.730 0.03777 70%
12 0.814 0.04863 53.5~/
.. . . .
1 3 1 .020 0 05095 62%
14 0.770 0 0489 67%
~le 4: Firs~-s~e grow~g ~f~s
Batch # r~ r~c ~ ~ N, r, rO" Standard
llm ~m dm~3s~l dm~3 dm-2 ~m deviation
9GI 0.4l5 0.83 2 12EXP~6 3 18E~13 l.32EXP08 bin~odal NA
9G11 0.415 0.725 2.12EXP16 4.77EXP13 1.~8EXP08 bimodal NA
9G] 1 I Q 4~ 5 0.566 2.I2EXP16 10EXP13 4.15EXP08 0 55 0.09
10G~ 0.365 0.497 2.12EXP16 14.8EXP13 5.40EXP08 0.~ NA
1OGI 1 0.365 0.683 0.60EXP16 7.01EXP13 2.~6EXP08 bim~al NA D
~IG1 0.365 0.509 2.12EXP16 I3 8E~13 5.04EXP0~ 0.565 0.04 r
I IG1~ 0.365 0.5?9 1 .06~XP16 g.35~13 3.41EXP08 bimodal NA r
111 0.365 O.S79 1.35~ 9.35EXP13 3.41EXP08 birnoda~ ~A
12G~ 0.41 0.56 2.12EXP16 10.4EXP13 4.26EXP08 0.61 0.05
13G1 0.~ 0.68 2.12EX~16 5.73EXP13 2.86EXP08 0.75 0.0?
14G1 0.385 O.S25 4.24EXPI6 12.6EXP13 4.8~:XP08 O.S5 0.07
14&11 0.385 0.673 3.29EXP16 5.98EXPI3 2.30EXP08 creamed NA
14G~11 Q.385 0.~73 3.g5EX~16 5.98EXP13 2.30EXP08 ~reamed NA
~able 5: .CeconA~ e growing In~P.~
Batch# rS r~k ~ N, N, r, r~ Stand~rd
llm ~m dm~3~ d~-3 dm-2 ~nt deviation
9Gl 112 0 ~5 O.g21 2.2~XP16 4.1EXP13 2.25E~8 bimod~l NA
9~11 I22 0.55 0.746 2.12EXP16 5.47EXP13 3.01EXP08 bimodal NA
lOG12 0.7 0.98~ 1.42EXP16 l.gEXP13 ~.33EXP08 ~i~nodal NA
11G12 0.61 0.8l7 1.06EXP16 - 3.34~XP13 2.Q4EXP08 aggregate~ NA
1 lGl22 0.61 0.817 1 .06EXP1~ 3.34EXP13 2.04EXP08 O.f9 0 07 0
12Gl2 0.613 0.821 1.06~X~16 3.29~XP13 2.02EXP08 0.82 0.14 r
12G122 0.613 0.821 1.11EXP16 3.29EXP13 2.02EXP08 : ag~ee~te~l NA
13G12 0.755 1.0 1.02EXP16 1.8EXP13 1.36E~08 0.88 - 0.15
1 3G 122 0.75~ 1 01 1 .04EXPl 6 1 .76EX~13 1 .33E~0~ 0.~8~unstable) 0.1 5
14&12 0.~5 0.75 2.OEXP16 4.3EXP13 ~.3~;~08 ~.gO 0 21
14&122 0.55 0.75 4.24EX~16 4.3~i:XP13 2.3OEXP08 ~.g6 0.24
7~ab~e 6: rkird-s~age growing ~ s
Batch # r, r~k P ~, N, r5 r~p Stand~rd
-3-1 -3 -2 deviatinn
m ~m dm s dm dm ~m
~IG1223 - 0.79 1.07 5.g2E~15 1.46EXP13 1.15EXP08 1.22 ~.16
12G123 0.82 1.09 5.94EXP1~ 1.4E~13 1.15EXP08 ~.04 0.22
12G1233 0.82 1 09 5.94EXP~5 1.4EXP13 l.l~EXP08 1.15 0.35
13G123 0.88 1.17 8.19EXP15 1.13EXP13 O.99EXP08 creamed NA
14G123 0.90 1.2 8.54EXP15 1.04EXP13 0.93~:XP08 creamed NA O
14G1233 O.gl 1.2 42.4EXPt5 1.04~XP13 O.9SEXP08 1.33 0.31 r
14G1223 0.87 1.18 42.4EXP15 1.11E;XP13 0.97EX~08 1 38 0.32
o
r
CA 02202424 1997-04-11
rnble7: Surf~ce ~harge densi~ oftl~eJ~nallatexes
~atch ~ r Eq/g SAlg ~renlgroup
cnl mole~lg c~2/g ~ /group
12G1233 1.15EXP-04 . 1~31SEXP-06 2.484EXPo4 313.6
14G1233 1.35EXI'-04 2.2~3EXP-06 2 116EXP04 153.9
14G1223 1.38EXP~04 l~ssoExp-o6 2.070EXP04 181~
CA 02202424 1997-04-11
T~ble 8: Aldehyde con~ent ~f t~e der~ zed In~exes
B~tcb # Ald/g ~ Idl~ titr~¢ion Observ~tions
moleslg m~les/~
A I NA NA creamed
~2 2 67EXP-04 4.57E~ 05 bi~nodal
~3 2.94E~-04 3.7EXP-05 some ag~regAtes
A4 l~o peak N~ aggreBflted
AS l'lo peak NA ag~regated
A6 5.~5EXP-OS 4. lEXP~Ob good
A7 ~o peak ~ gnod
A8 8 5EXI~-OS l.OlEXP-OS good
A9 NA 4. IE~-06 good
AlU NA '7.07EXP-06 good
Table 9
~ Seed Gro~h
Batch ~ rempe~h~ r~ s~ % ~lids ~ ~s.~2Ra ~, p
dm2 dm~s~'
i2G1 50 12 80 3.15 2.2~10-' 4.261~l0~ Z.12xiO'~
14GI S0 ' 12 ~0 3.15 4.4xl03 4.85xl0~ 424X10~6 ~-
12G12 S0 12 80 3.33 1.1 xiO~ 2.02~cl0~ I.06~c10~6 ~o
14G12 j~ ]2 8C 3.15 ~.7xl~ ~36 x103 2.6xl0~6
12G1233 S0 12 80 3.4 6.2x10~ I.ISx10~ 5.94~0~5
14G1233 50 12 80 3.4 4.4xlir~ s.sxia~ 4.24~0
CA 02202424 1997-04-11
T~ble ~V
O~tt~u~ ~n~ otn~r~ cter~
P N"r~
dm~ 6 ~
- P~stst~ga g~ 2.12xlOI~to 4.26x~0~ Z.86xlO~toS.~4xlOt
So~oud ~t~g~ growt}l lxlOI~ t~ 4.3x.~01a 1.36xla~t~ ~.36x~0
Third Ra~e ~row~ 5 ~4xlOI~ t~ 4. 24xlO1~ 9,~x~ 07 to 1. l ~xlO~
CA 02202424 1997-04-11
'rnble 11
hy ~ de~Y~t~a~on
(~t S0~~, time fo~ ~on: 6 hour~)
. . .
tch # ~P~rent" Styrenc Acrol~in % 60~ds ~B.A.2~Cl
Inol~gr~soed mol~ran: ~eod molcsdn~3
A~ 12C~123 ~ 2,8xl0~ 2xlO ~ 3 .33 4.~3xlO~
~8 14G1~33 ~x10~ 2xlO~ 3.33 9.22xtO 4
