CA2202424A1 - A tethered polymer macromolecule-excluding surface, its mode of synthesis and use - Google Patents
A tethered polymer macromolecule-excluding surface, its mode of synthesis and useInfo
- Publication number
- CA2202424A1 CA2202424A1 CA 2202424 CA2202424A CA2202424A1 CA 2202424 A1 CA2202424 A1 CA 2202424A1 CA 2202424 CA2202424 CA 2202424 CA 2202424 A CA2202424 A CA 2202424A CA 2202424 A1 CA2202424 A1 CA 2202424A1
- Authority
- CA
- Canada
- Prior art keywords
- polymer
- groups
- aldehyde
- shell
- chains
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
- A61L33/0076—Chemical modification of the substrate
- A61L33/0088—Chemical modification of the substrate by grafting of a monomer onto the substrate
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/291—Gel sorbents
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
- B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
- B01J20/3268—Macromolecular compounds
- B01J20/3276—Copolymers
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
- B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
- B01J20/3268—Macromolecular compounds
- B01J20/3278—Polymers being grafted on the carrier
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/34—Size selective separation, e.g. size exclusion chromatography, gel filtration, permeation
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2220/00—Aspects relating to sorbent materials
- B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
- B01J2220/54—Sorbents specially adapted for analytical or investigative chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
Landscapes
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Health & Medical Sciences (AREA)
- Hematology (AREA)
- Surgery (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Dispersion Chemistry (AREA)
- Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
- Graft Or Block Polymers (AREA)
Abstract
The present invention relates to a high density tethered polymer surface material comprising two regions: (1) a polymer shell characterized by an optimal surface concentration of aldehyde, hydroxyl or sulfhydryl groups; and (2) polymer chains tethered to the shell via the surface groups. The chains function to exclude biomolecules and polymers from approaching the polymer core, thereby minimizing adsorption of such molecules to the surface material.
The method of synthesizing this surface involves initiating polymerization of the surface polymer chains from the surface groups using CeIV, which optimizes the density of chains tethered to the surface. The surface concentration of aldehyde or hydroxyl groups determines the chain density, and the composition of the copolymer constituting the shell determines the surface concentration of the aldehyde or hydroxyl groups. If the polymer shell is coated onto a core particle, this surface has use as a size exclusion medium for chromatography. When coated onto other structures, this surface has application as a biocompatible material, because of the resulting exclusion qualities of the relatively high polymer chain density.
The method of synthesizing this surface involves initiating polymerization of the surface polymer chains from the surface groups using CeIV, which optimizes the density of chains tethered to the surface. The surface concentration of aldehyde or hydroxyl groups determines the chain density, and the composition of the copolymer constituting the shell determines the surface concentration of the aldehyde or hydroxyl groups. If the polymer shell is coated onto a core particle, this surface has use as a size exclusion medium for chromatography. When coated onto other structures, this surface has application as a biocompatible material, because of the resulting exclusion qualities of the relatively high polymer chain density.
Description
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 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 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 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 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).
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., 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, 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 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 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 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 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 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.
_ 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:
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.
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 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 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.
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, 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.
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 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
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 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 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 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 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).
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., 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, 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 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 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 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 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 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.
_ 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:
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.
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 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 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.
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, 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.
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 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.
Hence, size exclusion chromatography of proteins is demonstrated on solid polystyrene beads carrying neutral polymer tethers.
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 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 ~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 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~
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 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~
'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
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.
Hence, size exclusion chromatography of proteins is demonstrated on solid polystyrene beads carrying neutral polymer tethers.
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 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 ~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 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~
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 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~
'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
Claims
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2202424 CA2202424A1 (en) | 1997-04-11 | 1997-04-11 | A tethered polymer macromolecule-excluding surface, its mode of synthesis and use |
| PCT/IB1998/000563 WO1998046351A1 (en) | 1997-04-11 | 1998-04-13 | A tethered polymer macromolecule-excluding surface, its mode of synthesis and use |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2202424 CA2202424A1 (en) | 1997-04-11 | 1997-04-11 | A tethered polymer macromolecule-excluding surface, its mode of synthesis and use |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2202424A1 true CA2202424A1 (en) | 1998-10-11 |
Family
ID=4160390
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2202424 Abandoned CA2202424A1 (en) | 1997-04-11 | 1997-04-11 | A tethered polymer macromolecule-excluding surface, its mode of synthesis and use |
Country Status (2)
| Country | Link |
|---|---|
| CA (1) | CA2202424A1 (en) |
| WO (1) | WO1998046351A1 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ATE484541T1 (en) | 1997-04-21 | 2010-10-15 | California Inst Of Techn | MULTIFUNCTIONAL POLYMER COATING |
| WO2000065352A1 (en) * | 1999-04-28 | 2000-11-02 | Eidgenossisch Technische Hochschule Zurich | Polyionic coatings in analytic and sensor devices |
| US20040171808A1 (en) * | 2001-05-30 | 2004-09-02 | Kazunori Kataoka | Method of bonding substance to be incorporated into polymer terminal |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2534486B1 (en) * | 1982-10-15 | 1987-11-20 | Commissariat Energie Atomique | SURFACE GRAFT PARTICULATE SUPPORT, ITS PREPARATION METHOD AND ADSORBENTS FOR AFFINITY CHROMATOGRAPHY INCORPORATING THIS SUPPORT, AS WELL AS THEIR USE, PARTICULARLY IN BIOLOGY |
| DE3811042A1 (en) * | 1988-03-31 | 1989-10-19 | Merck Patent Gmbh | ION EXCHANGER |
-
1997
- 1997-04-11 CA CA 2202424 patent/CA2202424A1/en not_active Abandoned
-
1998
- 1998-04-13 WO PCT/IB1998/000563 patent/WO1998046351A1/en active Application Filing
Also Published As
| Publication number | Publication date |
|---|---|
| WO1998046351A1 (en) | 1998-10-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6073957B2 (en) | Solid support with grafted chain | |
| CA2074730C (en) | Immobilized polyethylene oxide star molecules for bioapplications | |
| Kayaman et al. | Structure and protein separation efficiency of poly (N‐isopropylacrylamide) gels: Effect of synthesis conditions | |
| US20150018440A1 (en) | Temperature-responsive polymer particles in protein separation applications | |
| Maechling‐Strasser et al. | Preadsorption of polymers on glass and silica to reduce fibrinogen adsorption | |
| JPH021747A (en) | Macro-porous polymer film and its preparation | |
| AU778045B2 (en) | Process for making fluorinated polymer adsorbent particles | |
| Li et al. | Characterization of poly (allylamine) as a polymeric ligand for ion-exchange protein chromatography | |
| JPH0949830A (en) | Stimulus responding type separating material and separating and refining method | |
| EP1436056A2 (en) | Macroporous gel, its preparation and its use | |
| JP2003524680A (en) | Affinity control type material using stimulus-responsive polymer and separation / purification method using the material | |
| CA2202424A1 (en) | A tethered polymer macromolecule-excluding surface, its mode of synthesis and use | |
| JPH07318551A (en) | Chromatography and filler therefor | |
| JP2002030125A (en) | New hydrophilized aromatic polymer | |
| US8940394B2 (en) | Protein imprinting by means of alginate-based polymers | |
| EP0382215A1 (en) | Porous adsorbent for beta-2-Microglobulin | |
| JP3480033B2 (en) | Phosphorylcholine group or its derivative group-containing copolymer, its production method and antithrombotic material | |
| Yavuz et al. | Molecular recognition based iron removal from human plasma with imprinted membranes | |
| Ayhan | Model protein BSA adsorption and covalent coupling onto methyl methacrylate based latex particles with different surface properties | |
| JP3471081B2 (en) | Heparinized material and method for producing the same | |
| JPH0435742A (en) | Adsorbent for beta2-microglobulin | |
| Coad | Evaluation of entropic interaction chromatography media | |
| Kim | Synthesis and engineering of polymeric latex particles for medical applications | |
| JPH09313906A (en) | Reverse phase separating membrane for separating protein | |
| JPS62151752A (en) | Carrier for chromatography and its production |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Dead |