Process for functionalizing a biocompatible polymeric bead, the functionalized beads, and the beads produced thereby

Abstract

The invention involves functionalizing polymeric beads, such as DVB beads, to add an epoxide or aldehyde group. The resulting beads are useful in various applications.

Claims

1. A process for making a polymeric bead having an epoxide group with a biological molecule attached thereto on its surface, said polymeric beads comprising pores having an average diameter of less than 200 Angstroms, comprising simultaneously contacting a polymeric bead which comprises monomeric divinylbenzene and has pores having an average diameter of less than 200 Angstroms with acetic acid and hydrogen peroxide to form an epoxide group on the surface of said polymeric beads, and attaching a biological molecule to said epoxide group.

2. The process of claim 1, further comprising reacting said epoxide group under hydrolysis and oxidation conditions, to form an aldehyde.

3. The process of claim 1, wherein said polymeric beads have a size distribution of 50-150 m diameter.

4. The process of claim 1, further comprising steam stripping said polymeric beads to remove any unreacted divinylbenzene therefrom.

Description

BRIEF DESCRIPTION OF THE FIGURE

(1) FIG. 1 shows the activity of beads prepared according to the invention, with and without a bioactive molecule attached thereto via a functional group attached to the polymeric beads, in accordance with the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(2) Peracetic acid is a strong oxidizing agent, and the reaction of alkene groups with it is also well known. The acid, however, is unstable, and is a severe irritant to the skin, eyes, and respiratory system. Hence, a process which permits the artisan to exploit the oxidative properties of the acid by generating it in situ when needed, is desirable.

(3) The invention involves the in situ generation of peracetic acid in combination with polymeric beads, which are preferably porous and biocompatible, and which present free (unreacted) vinyl groups on their surface. The result is an activated polymeric bead, which presents highly reactive epoxides on its surface. In some embodiments, these epoxides can be reacted to form aldehydes, which are also reactive.

(4) The following examples are intended to be illustrative and not limiting.

Example 1

(5) This example describes the synthesis of dry polymer beads, having a size distribution of 50-150 m diameter.

(6) The beads were prepared using well known suspension polymerization methods. To elaborate, a solution of polyvinyl alcohol in deionized water was used as a dispersant, divinylbenzene (DVB) was the monomer, and toluene as the porogen. Operation of the suspension polymerization method resulted in production of polymer beads, having a size distribution of 50-150 m diameter. The beads were epoxidated using a solution of hydrogen peroxide (30% in water), and a 99% solution of acetic acid.

(7) The reaction of hydrogen peroxide and acetic acid leads to the formation of peroxyacetic acid, as an epoxidizing agent.

Example 2

(8) Polyvinyl alcohol (3.0 g) was dissolved in 500 ml deionized water, to form an aqueous phase. An organic phase was formed by combining 160 g of a DVB solution (63% pure DVB isomer content), 240 g of 99% toluene, and 98% benzoyl peroxide as the radical polymerization initiator. A 1 liter jacketed reactor, equipped with a mechanical agitator, was charged with the aqueous phase referred to supra, and heated to 80 C. at 300 rpm. The organic phase was added and the reaction was allowed to run for 16 hours, at 80 C. After the end of the reaction, the beads produced were washed, three times, in 500 ml deionized water, and steam stripped for 8 hours to remove any residual starting reagents. Beads were sieved, and dried at 90 C., for 24 hours. The yield was 42 g/105 ml dry beads, having diameters over a range of 50-150 m.

Example 3

(9) In this Example, 41 g of the polymer produced in Example 1, supra, was charged into a 1 liter glass reactor equipped with a TEFLON coated agitator. A mixture of 200 ml acetic acid, and 75 ml of a 30% aqueous solution of H.sub.2O.sub.2 were added. The mixture was heated to 50 C., at 100 rpm, and the reaction was allowed to run for 24 hours. The product was yellowish, polymer beads. These were washed in aliquots of deionized water until the pH of the effluent was about 5.5. The washed beads were dried at 80 C., until the weight of the product remained constant. The yield of dry beads was about 43.5 g.

Example 4

(10) This Example describes the attachment of lactate dehydrogenase to the polymer beads of the invention.

(11) A sample (2 g) of the beads synthesized in Examples 1 and 3 was wetted in 70% isopropanol and then washed, four times, with 50 ml of distilled deionized water. LDH was dissolved in a coupling buffer (0.1M Na.sub.2CO.sub.3, pH 9.0) and then reacted with the epoxy functionalized beads described supra, for 16 hours at room temperature, with end over end agitation. Any unreacted epoxy groups in the now functionalized beads were blocked with 1M ethanolamine, at pH 8, for 4 hours at room temperature, also with end over end rocking. The functionalized beads were washed, four times, alternating a wash with 0.1 M acetate buffer containing 0.5 M NaCl (pH 4), with a wash with 0.1 M Tris-HCl buffer containing 0.5 M NaCl, at pH 8.0. Following the four washes, beads were suspended in phosphate saline buffer, and stored at 4 C. until used.

(12) FIG. 1 shows the results of comparative tests to determine LDH activity, using a standard, commercially available LDH activity assay. The samples were conjugated beads, in accordance with this example, and unconjugated beads prepared via Example 1 or 3. Attachment of LDH to the epoxy activated beads shows that the beads can be used in the context of attaching any biological molecule of interest thereto. Polymeric beads having biological materials immobilized therein are well known in the art and their uses need not be elaborated herein.

(13) Once the porous, polymeric beads are activated to present epoxide group on their surface, they can be treated to convert the epoxy group to an aldehyde group. Various methods for accomplishing this are available including, but not being limited to, hydrolysis of epoxide groups, promoted by either acid or base to form benzylic 1,2 diol. The benzylic 1,2 diol is then oxidized directly, at room temperature, with a strong oxidizing agent, such as NaIO.sub.4. The result is benzaldehyde functionality.

(14) The polymeric beads used in the instant invention may have a biocompatible and hemocompatible exterior surface coatings but this is not absolutely necessary. Certain of these coatings are covalently bound to the polymeric beads by free-radical grafting. The free-radical grafting may occur, for example, during the transformation of the monomer droplets into polymeric beads. The dispersant which coats and stabilizes the monomer droplets becomes covalently bound to the droplet surface as the monomers within the droplets polymerize and are converted into polymers. Biocompatible and hemocompatible exterior surface coatings can be covalently grafted onto the preformed polymer beads if the dispersant used in the suspension polymerization is not one that imparts biocompatibility or hemocompatibility. Grafting of biocompatible and hemocompatible coatings onto preformed polymer beads is carried out by activating free-radical initiators in the presence of either the monomers or low molecular weight oligomers of the polymers that impart biocompatibility or hemocompatibility to the surface coating.

(15) A biocompatible material is defined as any natural or synthetic substance/combination of substances (other than drugs) which may be employed for any length of time as a whole or part of a system, to treat, augment, or replace any tissue, organ or function of the body. The polymeric beads of the present invention are preferably non-toxic.

(16) In some embodiments, the polymer has a preferential pore structure such that pore size is below 200 thus allowing efficient steam cleaning to reduce residual organics while still minimizing nonspecific binding of proteins.

(17) Some preferred polymers are coated polymers comprising at least one crosslinking agent and at least one dispersing agent. Suitable dispersing agents include hydroxyethyl cellulose, hydroxypopyl cellulose, poly(hydroxyethyl methacrylate), poly(hydroxyethyl acrylate), poly(hydroxypropyl methacrylate), poly(hydroxypropyl acrylate), poly(dimethylaminoethyl methacrylate), poly(dimethylaminoethyl acrylate), poly(diethylamimoethyl methacrylate), poly(diethylaminoethyl acrylate), poly(vinyl alcohol), poly(N-vinylpyrrolidinone), salts of poly(methacrylic acid), and salts of poly(acrylic acid) and mixtures thereof.

(18) Suitable crosslinking agents include divinylbenzene, trivinylbenzene, divinylnaphthalene, trivinylcyclohexane, divinylsulfone and mixtures thereof. Preferably, the polymer is developed simultaneously with the formation of the coating, such that the dispersing agent gets chemically bound to the surface of the polymer.

(19) Preferred polymers include those derived from one or more monomers selected from divnylbenzene and ethylvinylbezene, styrene and mixtures thereof.

(20) Some preferred polymers are polysaccharide based polymers. Suitable polymers include cross-linked dextran gels such as Sephadex.

(21) Certain preferred polymers are porous highly crosslinked styrene or divinylbenzene copolymer. Other of these polymers are a hypercrosslinked polystyrene produced from crosslinked styrene copolymers by an extensive chloromethylation and a subsequent post-crosslinking by treating with a Friedel-Crafts catalyst in a swollen state.

(22) Some polymers useful in the practice of the invention are hydrophilic self wetting polymers that can be utilized as dry powder containing hydrophilic functional groups such as, amines, hydroxyl, sulfonate, and carboxyl groups.

(23) Certain polymers useful in the invention are polymers prepared from the polymerizable monomers of styrene, divinylbenzene, ethylvinylbenzene, and the acrylate and methacrylate monomers such as those listed below by manufacturer. Rohm and Haas Company, (now part of Dow Chemical Company): (i) macroporous polymeric sorbents such as Amberlite XAD-1, Amberlite XAD-2, Amberlite XAD-4, Amberlite XAD-7, Amberlite XAD-7HP, Amberlite XAD-8, Amberlite XAD-16, Amberlite XAD-16 HP, Amberlite XAD-18, Amberlite XAD-200, Amberlite XAD-1180, Amberlite XAD-2000, Amberlite XAD-2005, Amberlite XAD-2010, Amberlite XAD-761, and Amberlite XE-305, and chromatographic grade sorbents such as Amberchrom CO 71,s,m,c, Amberchrom CG 161,s,m,c, Amberchrom CG 300,s,m,c, and Amberchrom CG 1000,s,m,c. Dow Chemical Company: Dowex Optipore L-493, Dowex Optipore V-493, Dowex Optipore V-502, Dowex Optipore L-285, Dowex Optipore L-323, and Dowex Optipore V-503. Lanxess (formerly Bayer and Sybron): Lewatit VPOC 1064 MD PH, Lewatit VPOC 1163, Lewatit OC EP 63, Lewatit S 6328A, Lewatit OC 1066, and Lewatit 60/150 MIBK. Mitsubishi Chemical Corporation: Diaion HP 10, Diaion HP 20, Diaion HP 21, Diaion HP 30, Diaion HP 40, Diaion HP 50, Diaion SP70, Diaion SP 205, Diaion SP 206, Diaion SP 207, Diaion SP 700, Diaion SP 800, Diaion SP 825, Diaion SP 850, Diaion SP 875, Diaion HP 1MG, Diaion HP 2MG, Diaion CHP 55A, Diaion CHP 55Y, Diaion CHP 20A, Diaion CHP 20Y, Diaion CHP 2MGY, Diaion CHP 20P, Diaion HP 20SS, Diaion SP 20SS, and Diaion SP 207SS. Purolite Company: Purosorb AP 250 and Purosorb AP 400.

(24) Other features of the invention will be clear to the skilled artisan and need not be reiterated here.

(25) The terms and expression which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expression of excluding any equivalents of the features shown and described or portions thereof, it being recognized that various modifications are possible within the scope of the invention.