WO1993014127A1

WO1993014127A1 - Method and composition for heparin binding using polyaminocations covalently immobilised on polymeric surfaces with polyethylene oxide spacers

Info

Publication number: WO1993014127A1
Application number: PCT/US1993/000678
Authority: WO
Inventors: Syed Fazal Mohammad; Xing Hang Ma; Sung Wan Kim
Original assignee: University Of Utah
Priority date: 1992-01-21
Filing date: 1993-01-19
Publication date: 1993-07-22
Also published as: AU3592693A

Abstract

Compositions having formula (I) where Substrate represents any suitable biopolymer to which polycations may be covalently bonded through a spacer grouping; -OC(O)-PEO-C(O)- is a polyethylene oxide polymer having a molecular weight of between about 600 and 8000; and -NH-PAC represents any suitable polyaminocation which contains a positive charge at a neutral pH and effectively removes heparin from blood with little or no loss of plasma proteins or other blood components. The use of polyallylamine or poly-L-lysine as the polyaminocation is most preferable with polyallylamine being the polycation determined to be best suited for purposes of this invention. The polycationic ligand, when covalently bound to a suitable polymeric substrate via a polyethylene oxide (PEO) spacer, provides a solution like polycationic interfacial environment. The solution like conditions are due to the increased mobility of the polycation ligand as a result of the PEO spacer. The flexibility of the polycation attributed to the presence of the spacer at the interface of the polymer minimizes the steric inaccessibility of the heparin thereby improving the binding characteristics. The substrate upon which the spacer molecule is bonded can be any substance that has free hydroxy groups to which the PEO diacid intermediate can be esterified and include cellulose or cellulose acetate.

Description

METHOD AND COMPOSITION FOR HEPARIN BINDING USING POLYAMINOCATIONS COVALENTLY IMMOBILIZED ON POLYMERIC SURFACES WITH POLYETHYLENE OXIDE SPACERS

Background of the Invention

This invention relates to compositions and methods for the removal of heparin from blood to minimize the risk of hemorrhagic complications. More particularly, this invention relates to compositions consisting of a polymeric substrate which has been modified to contain polycation ligands having primary amino groups which are covalently bonded to the substrate through a polyethyleneoxide type spacer and to methods of removing heparin from blood or other solutions by contacting heparinized solution with said compositions.

Heparin is a powerful anticoagulant which is often administered during surgical or other procedures to prevent the formation of clots.

The anticoagulant properties of heparin have been demonstrated to be associated with heparin binding to Antithrombin III (AT III). AT III is a plasma glycoprotein with molecular weight approximately 58,000. AT III binds with thrombin very tightly at a 1:1 stoichiometric ratio, which blocks the active site on thrombin and prevents it from interacting with fibrinogen. However, the inhibition rate of thrombin with AT III is low in absence of heparin. Heparin dramatically accelerates the rate of thrombin inactivation up to 2000-fold. Clinically used heparin can be separated into two distinct fractions according to their affinity for AT III. Approximately 33% of heparin has a high affinity for AT III, which has potent anticoagulant activity (up to 90% of the activity of the unfractionated heparin). A low-affinity heparin binds to the same site on AT III, but with approximately 1000 times lower affinity.

Although anticoagulation is the major pharmacological activity, heparin has many other functions. Heparin inhibits the proliferation of vascular smooth muscle cells and renal mesengial cells, suppresses the delayed-type hypersensitivity, and inhibits angiogenesis. Other pharmacological functions of heparin include antithrombotic effect, antibacterial, antivirus, and antitumor angiogenesis, particularly in combination with cortisone. Although it has been clinically observed that heparin may induce thrombocytopenia, in vitro studies have shown that normal heparin enhances the release of platelets. Moreover, various heparin-binding growth factors can be purified with heparin affinity chromatography.

Heparin has been extensively used in many clinical applications, including cardiac surgery, peripheral vascular surgery, dialysis, autotransfusion, transplantation, the treatment of pulmonary embolism, disseminated intravascular coagulation, and venous thrombosis. The dosage is dependent on the type of application. Heparin has also been used as a prophylactic agent against deep vein thrombosis. Heparin is also of value in the treatment of thromboembolic disorders, such as pulmonary embolism and arterial thrombosis.

As a polyanion, many properties and applications of heparin are associated with electrostatic interactions. Binding of negatively charged heparin onto polymeric surfaces has been applied in the biomedical field in two major ways.

First, heparin is immobilized onto polymers to form nonthrombogenic surfaces to achieve a localized anticoagulant effect at the polymer blood interface. As a well known anticoagulant, heparin has been fixed onto polymers with positive charges by forming a stable complex or by being covalently bound thereto via a spacer grouping. In the case of charged polymers, the immobilized heparin on the surface may then be released into blood by ion exchange, subsequently, the released free heparin interacts with AT III and thus exerts its anticoagulant effect. In the case of being covalently bound to a polymer surface, heparin increases the blood compatibility of the polymer for long-term biomedical applications, particularly when bound through an appropriate spacer grouping.

Polymers bearing positive charges have been used to anchor heparin onto surfaces and have shown good long term blood compatibility. For example, charged polymers such as poly-4-vinylpyridine, Fourt et al., Adv. Chem . Ser. 87, 187 (1968); poly(amidoamine) grafted polyurethane (PUPA), Azzuuoli et al., Biomaterials 8, 61 (1987); and polyvinylchloride grafted with both polyethyleneglycol monomethacrylate and quarternized dimethylaminoethyl methacrylate (Anthron), Nagaoka et al., J. Biomater. Appl . 4 , 3 (1989), have been reported to form a stable complex with heparin.

Polyurethane surfaces modified by polyethylene oxide (PEO) and then reacted with heparin have displayed enhanced blood compatibility due to the synergistic effects of PEO and heparin. See Han et al., Evaluation of Blood Compatibility of PEO Grafted and Heparin Immobilized Polyurethanes, J. Biomed . Matr. Res . : Appl . Biomat . 23(A2), 211-228 (1989); Han et al., Preparation and Surface Characterization of PEO-grafted and Heparin- immobilized Polyurethanes, J . Biomed . Matr . Res . : Appl . Biomat . 23 (Al), 87-104 (1989); and Park et al., J. Biomed . Matr . Res . , 22, 977-992 (1988). The heparin in these applications enhances the blood compatibility of the polymer and is not released into the blood. This use extends only to localized surface effects and does not provide anticoagulant properties to blood in the surgical field of use.

The second means where binding of negatively charged heparin onto polymeric surfaces has been applied in the biomedical field in the area of heparin neutralization where heparin is removed from blood onto the polymer surface by binding.

The administration of inadequate doses of heparin can be dangerous since less than desired concentrations of heparin in blood increases the likelihood of unwanted coagulation after surgery or when blood comes into contact with biopolymeric surfaces thus forming unwanted blood clots in the arteries and veins. It is well documented that most synthetic polymers activate blood coagulation when contact is made between blood and the polymer surface. Therefore, administered heparin is necessary to minimize clotting. This heparin may be present in the blood at relatively high concentrations during contact with polymeric surfaces such as during extracorporeal circulation to prevent clotting in the extracorporeal circuit. Excessive heparin in blood needs to be neutralized at the end of extracorporeal circulation due to the risk of hemorrhagic complications. Usually this is achieved by administration of protamine, a cationic protein, Anido et al., Am . J. Clin . Pathol . 76, 410 (1981). However, protamine also has undesirable side effects, including the risk of anaphylactic reactions in some patients. To circumvent this problem, protamine immobilized cellulose hollow fiber, Kim et al., Trans . Am. Soc . Artif. Intern.

Organs, 35, 644 (1989), and protamine grafted glycidyl methacrylate gel-cellulose, Hou et al., Artif . Organs,

14, 436 (1990), have been developed.

Excessive heparin in the blood can be attracted and removed by electrostatic interactions with polycationic surfaces. Cationic polyelectrolyte surfaces proposed for binding and removal of heparin, include the use of triethylaminoethyl cellulose powder (Heparsorb, Organon Teknika, Durham, NC) and poly-L-lysine bound agarose (Sepharose 4B), Mohammad et al., Thromb . Res . , 20, 599 (1980)

Heparin ionically binds with the polycationic surfaces after exposure to blood and thus is removed from the blood. However, prior art methods have demonstrated the degree of heparin removal that is desired is not sufficient or takes too long to accomplish. One of the reasons for polycationic surfaces not being as effective as might be desired has to do with variables affecting heparin binding onto polycationic surfaces. Already mentioned is the fact that electrostatic attractions exist between the positively charged polycations and the negatively charged heparin. However, steric accessibility of heparin onto polycationic interfaces is also an important consideration. When a polycation is covalently immobilized onto a polymer surface, the polycation is relatively rigid and not laterally movable due to the intramolecular and intermolecular charge repulsions. The unfavorable steric effect restricts heparin binding onto the polycationic interfaces, particularly in the lower regions of the interfacial polycations. Therefore, heparin binding onto polycationic surfaces may be controlled by the balance between the electrostatic attraction and steric accessibility factors.

It would therefore be desirable to increase the heparin binding efficiency on polycation-immobilized surfaces by minimizing the effect of steric inaccessibility.

Objects and Brief Summary of the Invention

It is an object of this invention to provide a means of removing heparin from blood by means of polycationic attraction and binding. It is a further object of this invention to provide means of controlling the amount of heparin in the bloodstream.

An additional object of this invention is the formation of a polymeric substrate containing polycationic ligands having primary amino groups and preferably those selected from the group consisting of polylysine (PLL), polyallylamine (PALA) or polyvinylamine (PVA) bonded through a polyethylene oxide (PEO) spacer grouping to a biocompatible polymeric substrate.

Another object of this invention is to provide a method for the binding and/or removal of heparin in blood by contacting the blood with a polymeric substrate containing polycationic ligands selected from the group consisting of polylysine (PLL), polyallylamine (PALA) or polyvinylamine (PVA) bonded through a polyethylene oxide (PEO) spacer grouping to a biocompatible polymeric substrate.

An additional object to provide a method of removing heparin from blood with a minimum loss of the plasma proteins and other components of blood.

It has now been found that compositions consisting of a polymeric substrate which has been modified to contain polycations such as polyallylamine (PALA), poly- L-lysine (PLL) or polyvinylamine (PVA) covalently bonded to the substrate through a polyethyleneoxide type spacer effectively removes heparin from blood with little or no loss of plasma proteins or other blood components. The use of PALA or PLL is most preferable with PALA being the polycation determined to be best suited for purposes of this invention.

The polycationic ligand, when covalently bound to a suitable polymeric substrate via a polyethylene oxide (PEO) spacer, provides a solution like polycationic interfacial environment. The solution like conditions are due to the increased mobility of the polycation ligand as a result of the PEO spacer. The flexibility of the polycation attributed to the presence of the spacer at the interface of the polymer minimizes the steric inaccessibility of the heparin thereby improving the binding characteristics.

While any suitable polycation ligand rich in NH₂ groups can theoretically be utilized to bind heparin, it has been found preferable to use either polyallylamine (PALA) or poly-L-lysine (PLL) as the ligand. Also, to maximize the interaction between the PEO spacer and the polymer, it has been found preferable to utilize a polymer substrate having free hydroxy groups such as cellulose or cellulose diacetate. The PEO molecule may be derivatized by functional groups, as will be discussed more in detail below, to maximize the ability of the PEO to covalently link the ligand with the polymeric substrate.

Heparin is removed from blood or other fluids by bringing the fluids in contact with the polymeric substrate material containing immobilized polycations with PEO spacers where heparin is adsorbed by the material. Heparin can be released from the immobilized polycations by treatment with a basic solution, thereby allowing the material to be reused when practical.

The substrate containing the immobilized polycationic ligands connected via the PEO spacers must present a large surface area to the blood or other fluid in order to maximize the opportunity for the heparin to come into contact with the positively charged ligands. As such, the substrates can be in the form of films, beads, honeycombs, coated surfaces, strands, filaments, and the like.

The rate of heparin binding is somewhat dependant on controlling parameters such as blood flow rates, concentration of heparin in blood and exposure time of heparinized blood to the binding substrate. The binding of heparin onto the polycationic surfaces grafted to the polymer surface via PEO spacers of molecular weight of between about 600 and 8,000 seems to depend on the balance of several factors such as the size of the polycation ligand, the molecular weight of the PEO spacer, the mobility of the PEO and the chain length of the PEO-polycation at the interface.

PALA having molecular weights ranging from about 8,500 to about 65,000 is suitable for use in the present invention. As used herein, PALA is categorized as PALA(L) for a low molecular weight PALA having a molecular weight of between about 8,500 and 11,000 and PALA(H) for a high molecular weight PALA having a molecular weight range of about 50,000 to 65,000.

When using PALA(L) as the ligand, the maximum heparin binding was observed in the region of PEO molecular weight of between about 2000 and 4000. When using PALA(H) heparin binding did not show clear dependence on the weight of the PEO spacer due to the relatively large size of the PALA(H) ligand.

It further appears that PALA(L) interface with the PEO spacer presents a more solution-like surface than does PALA(H).

As it relates to plasma protein adsorption, it was observed that adsorption reduced with the increase in the chain length of the PEO spacer suggesting that long chain PEO spacers will minimize protein adsorption from blood and thereby improve the biocompatibility of polycationic surfaces. A composition consisting of low molecular weight PALA (M.W. 8500) immobilized onto a biopolymer surface with a PEO spacer (M.W. 3400) is believed to provide optimal heparin binding capacity with decreased protein adsorption.

The heparin removal from polycationic containing substrates is primarily due to the charge interaction between the polyanionic heparin and the polycations. No hemolysis or clotting is observed in circulating blood, suggesting the such a removal system will not cause adverse effects on blood. The system is also effective in removing heparin from aqueous solutions other than blood.

DETAILED DESCRIPTION OF THE INVENTION

The following terminology and/or formulas will be used throughout the description when referring to substrates modified by having a polycation covalently grafted thereon.

By substrate is meant any suitable biopolymer having hydroxy groups to which the polycation may be covalently bonded, either directly or through a PEO type spacer. The substrate may be represented by the following formula:

(Formula l)

where z can be any number, where the number of OH groups is only representative of their presence and where the polymer backbone is representative of any polymer unit. In other words, Formula 1 is meant to depict any suitable biopolymer to which polycations may be covalently bonded unless a particular polymer is specifically designated. Biomedical polymers include, but are not limited to, such hydroxy containing materials as cellulose and derivatives such as cellulose diacetate (CA), poly (ethylene-vinyl alcohol) copolymers

(PEVAL), agarose and the like. Further, one skilled in the art can also utilize other biomedical polymers such as polycarbonates, silicones, acetals, polyesters, polytetrafluroethylene, polyurethane, pyrolytic carbon, nylon or other polyamides, polyacrylonitrile, polyethersulfone and the like by devising reaction sequences and linkages which provide for covalent attachment of polycations to these surfaces.

By polycation is meant a polymeric molecule having a repeating segment containing primary amino groups on the side chain and which is positively charged at neutral pH. These may be generically referred to as polyaminocations (PAC). As stated, polyallylamine (PALA), poly-L-lysine (PLL)) and polyvinylamine (PVA) are preferred with PALA and PLL being most preferred.

PLL is a polypeptide of the formula:

(Formula 2)

which has repeating units containing primary amino groups of the formula:

wherein n is an integer of between about 200 and 240 and which becomes cationic when the e-amino gruups on the side chain become fully protonated. Its ionization depends upon the pH of the medium in which it is contained but is known to form electrostatic interactions with heparin at a neutral pH. The positive charges of PLL per molecule can be adjusted by selecting different molecular PLL weights. The molecular weight range of PLL will generally vary between about 40,000 and 50,000.

PALA is a recently commercialized polymer with primary amino groups as pendant side chains having the formula:

(Formula 3)

which has repeating units containing primary amino groups of the formula:

wherein m is an integer of between about 78 to 500, having a molecular weight of between about 8,500 and 65,000 and is prepared from polymerization of a monoallylammonium salt initiated by an azo compound. PALA is obtained commercially as a HCl salt wherein the HCl is removed by increasing the pH with NaOH. PALA has a higher positive charge density than PLL.

The terms poly(ethylene glycol) (PEG) and poly (ethylene oxide) (PEO) are used interchangeably and identify a neutral polyether of the formula:

HO-(CH₂CH₂O)_n-H

where n is an integer of between about 13 and 180. Throughout this disclosure the term PEO will be used. PEO is extremely water soluble. Low molecular weight PEO, (less than 600) is a viscous fluid at room temperature. As the molecular weight increases, PEO becomes a waxy solid and morphology changes from amorphous to crystalline. PEO-water interaction is found to increase sharply with increasing molecular weight until a weight of about 3400 is reached and then remains constant within the 3400-20000 molecular weight range. When used as a spacer grouping it has been found that the chain mobility of PEO increases with increasing molecular weight up to about 4,000.

PLL and PALA Immobilized Directly on Polymeric surfaces

For comparison purposes PALA and PLL were both immobilized on PEVAL copolymer covalently using cyanogen bromide as a coupling agent. Cellulose diacetate and cellulose hollow fibers could also be used because each of these materials have hydroxyl groups available after proper treatment which can be activated by BrCN. Therefore, the amino groups of PALA and PLL can be coupled directly with the hydroxyl groups on these polymeric supports.

This may be exemplified by the reaction of Formula 1 with BrCN to form a reactive intermediate imidocarbonate represented by Formula 4 as follows:

O

(Formula 4)

The imidocarbonate of Formula 4 is then reacted with either PLL or PALA in an aqueous solution to covalently link a primary amino group from a pendant PLL or PALA chain with the polymer surface as follows in

Formulas 5 and 6 respectively:

/

When the PLL or PALA polycation is covalently immobilized on the polymer surface, it is relatively rigid and not laterally movable due to the intramolecular and intermolecular charge repulsions. The relatively low binding of heparin on polycations rigidly bound may be ascribed to the steric inaccessibility of heparin into the root part of the relatively rigid polycationic interface. Therefore, heparin may not bind efficiently on polycationic directly immobilized on to polymeric surfaces.

When PALA or PLL are coupled directly onto a poly(ethylene-vinyl alcohol) (PEVAL) surface using BrCN as a coupling reagent under exactly the same experimental conditions, the PALA immobilized surface (Formula 6) demonstrates a heparin binding capacity which is about 1.3 times that of PLL (Formula 5) which is believed to be due to the higher charge density of PALA per molecule when compared with PLL. Heparin binding of PALA was 0.71 ± 0.08μg/cm² and of PLL was 0.52 ± 0.19 μg/cm².

Detailed Description Preferred Embodiments of the Invention

As referenced above, the primary factors affecting heparin binding onto polycationic surfaces are electrostatic attractions between the positively charged surfaces and the negatively charged heparin, and steric accessibility of heparin into polycationic interfaces.

When a polycation is covalently immobilized directly onto a polymer surface, the polycation is relatively rigid and not laterally movable due to the intramolecular and intermolecular charge repulsions.

Such unfavorable steric effect restricts heparin binding onto the polycationic interfaces, especially in the lower or 'root' regions of the interfacial polycations.

Therefore, heparin binding onto polycationic surfaces may be enhanced by the balancing of the electrostatic binding and steric accessibility factors. It has been found that the heparin binding efficiency on polycation- immobilized surfaces is increased if the effect of the steric inaccessibility is minimized and a solution like polycationic interfacial environment is provided.

Increasing mobility of the polycation on the surface achieves a solution like interface. Polyethylene glycol (also referred to herein as polyethylene oxide "PEO") has high hydrophilicity and dynamic mobility. Considering these properties PEO is an excellent spacer between the surface and a polycation. The PEO provides a larger intermolecular gap between the polycations at the interface of the polymer surface and therefore minimizes the steric inaccessibility. Therefore, the binding properties of heparin are increased when PEO spacer molecules are inserted between the surface and the polycations.

The heparin removal system is formed by reacting a polymeric surface containing free hydroxy groups, preferably with a PEO molecule which has been derivatized to form a diacid thereby creating an ester linkage between the hydroxy groups of the surface and the carboxylic acid group of the diacid. While the following methods are not considered to be exhaustive, PEO may be derivatized to form diacids by : 1) the reaction of polyethylene oxide with succinic anhydride as shown in Formula 7; 2) the reaction of an alkoxide of polyethylene oxide with 2-halo-alkyl acetate as shown in Formula 8; and 3) the reaction of polyethylene oxide with an alkylene diacid such as azelaic acid as shown in Formula 9. HO-(-CH₂CH₂O-)-_nH + - O -

n=50-100

Formula 7

HO-(-CH₂CH₂O-)-_n-OH + Na/naphthalene + Br-(CH₂ )-_aCO₂Et

O O

( ₂)_a-O-( _{2 2} )_n ( ₂)_a

1

Formula 8

O O O O O O

-(- O)_{n b} O )_b-CO-( _{2 2}O)_n ( ₂)_b-

Formula 9

Depending upon the method of synthesis the diacids may be generically represented by a single formula as seen in Formula 10 as follows:

O O O O

)_d-( _y-O O _y- _d-CO

Formula 10

where d is an integer of 1 to 10, y is an integer of 0 or 1 and n is an integer of between about 10 to 200. The diacid PEO intermediate as represented by Formula 10 is first grafted onto the surface of a polymer, preferably one containing OH groups (Formula 1), using an agent such as 1,3-dicyclohexylcarcodiimide (DCC) to esterify the carboxylic acid groups of the diacid derivatized PEO with the hydroxyl group of the polymer surface according to following sequence:

(Formula 1)

(Formula 10) x Λ J

(Formula 11)

The esterified PEO grafted onto the polymer surface, as represented by Formula 11, then has polycations, such as PLL or PALA, immobilized to the polymer by reacting the -COOH at the opposing end of the PEO with the polycation using an agent such as (3- dimethlyamino-propyl)carbodimide (EDC). Preferably the amount of polycation in the reaction solution will be in excess resulting in only one amino group of PLL or PALA per molecule reacting with each COOH group on the surface. This reaction sequence is represented as follows resulting in PLL or PALA immobilized compositions represents by Formulas 12 and 13 as follows:

(Formula 11)

(Formula 2)

(Formula 12)

(Formula 11)

(Formula 3)

₂

m=78-500

The final product formed contains polylysine (PLL) or polyallylamine (PAPA) separated from the surface by a spacer molecule of PEO. However, any polycation having free amino groups which can be covalently bonded to the PEO spacer may be a suitable agent and is within the scope of this invention. Therefore, the compositions of the invention may be broadly defined by the following formula 14:

(Formula 14)

where PAC stands for any suitable polyaminocation and preferably one selected from the group consisting of polyallyl amine (PALA), poly-L-lysine (PLL) or polyvinlyamine. Again, it is to be emphasized that the material upon which the spacer molecule is bonded can be any substance that has free hydroxy groups to which the PEO diacid intermediate can be esterified, for example, cellulose, cellulose acetate or a poly(ethylene vinyl alcohol) copolymer.

More particularly, the formula representing the compositions of this invention may be further simplified for purposes of claiming. The structures of the PEO spacer, and the PALA and PLL ligands are given in detail above. All have certain repeating units within their respective molecular structures and hence, with the formulas established, it is the molecular weights of these units which determines their functionality in the binding of heparin. Therefore the following formula will be referred to in the claims:

Substrate-OC-PEO-CNH-PAC where Substrate is the same as Formula 1, -OC(O)-PEO- C(O)- has the same meaning as the PEO diacid of Formula 10 and -NH-PAC is the polyaminocation ligand and is preferably the PLL of Formula 2 or the PALA of Formula 3. Obviously, from this simplified definition any of the above formulas and/or structures can be readily determined by knowledge of the molecular weights of the PEO spacer or polycation ingredients and the identity of the specific substrate.

The following examples show a preferred means in which polycations are immobilized onto a polymeric surface with PEO spacers using a reaction sequence to covalently link the amino groups of polycations with carboxyl groups of derivatized PEO derivatives.

Example 1

Synthesis of PEO Dicarboxylic Acids

This examples describes the derivatization of PEO with succinic anhydride to prepare PEO derivatives having carboxyl end groups according to the reaction sequence shown in Formula 7. The hydroxyl end groups of PEO (having molecular weights of 600, 2000, 3400, and 8000) were reacted with succinic anhydride in dioxane using 4-Dimethylaminopyridine (DMAP) and triethylamine (TEA) as catalysts according to the above reaction sequence. Succinic anhydride (20 mmol), 4- Dimethylaminopyridine (10 mmol) and triethylamine (TEA) (10 mmol) were dissolved in 20 ml of dioxane and added to a solution of 5 mmol of polyethylene oxide (PEO) (10 mmmol of OH groups) in 50 ml of dioxane. The mixture was stirred for 12 hr at 60° C. Most of the dioxane was then removed by a rotavapor. The residue was dissolved by dichloromethane and precipitated by ether. The products were dissolved and precipitated twice from dichloromethane/ether and then vacuum dried.

The terminal carboxylic acid groups of polyethylene oxide (PEO) diacids (M. W. 600-8000) was determined by non-aqueous titration. PEO diacids (5 x 10^-4 mol) were dissolved in dioxane and titrated by sodium methoxide (0.1 N) in methanol-benzene (2.8) using 0.2% thymol blue as an indicator. The pure polyethylene oxide (PEO) in dioxane was used as a blank. The amount of sodium methoxide consumed by PEO diacid gave the information on the amount of carboxylic acid groups at two ends of polyethylene oxide (PEO).

Example 2

This example describes the preparation of a cellulose acetate substrate to which is attached the PEO-diacid spacer prepared according to Example 1.

Preparation of Cellulose Acetate Film

Cellulose acetate was dissolved in acetone to make a 10% solution. A casting knife was used to make a film with 0.8 mm thickness on a glass plate and allowed to air dry. The dried cellulose film was peeled off by rinsing the plate with distilled water.

Reaction of PEO Diacid with Cellulose Acetate Film

The cellulose acetate (CA) film (1000 cm²) was immersed in dichloromethane:toluene solvent (5:5) overnight, then 9x10^-4 mol of PEO diacid of Example 1 and 9X10^-1 mol of 4-dimethylaminopyridine (DMAP) were added into the solution and stirred at 4°C for one hour. A 5 ml aliquot of 9X10^-4 mole of DCC in dichloromethane:toluene (5:5) was added dropwise to the reaction mixture and stirred for an additional 1 hour at 4°C. The whole solution was moved to 22 °C and allowed to stand for 48 hr. After the reaction, the CA-PEO films thus formed were washed with toluene and vacuum dried at 40°C for 72 hours to remove residual toluene. The resulting product was characterized by IR spectroscopy and differential scanning calorimetry and is representative of that shown in Formula 11 where n is 87. Reaction of PEO Diacid with CA Solution

Cellulose acetate particles were dissolved in acetone and reacted with polyethylene glycol diacid (M. W. 600) in CH₂Cl₂ using the same procedures as described above. After the reaction, the solution was filtered to remove the precipitate (side product N, N'-dicyclohexyl- carbodimide). The filtrated solution was then precipitated in ether and vacuum dried. The solid residue was washed with distilled water to remove the excess PEO diacid. The resulting product was characterized by IR spectroscopy and differential scanning calorimetry and is representative of that shown in Formula 11 where n is 87. Determination of PEO on CA Surfaces

The amount of PEO coupled onto CA film surfaces was determined by acid-base back titration of its free end of carboxylic acid. The coupled film was thoroughly washed with distilled water and then immersed in distilled water (10 ml). Sodium hydroxide (0.01 N) was added to adjust the pH above 7.0. The solution and film were titrated potentiometrically with 0.01 N hydrochloric acid and the pH was monitored by a Corning pH Meter. CA film without PEO was used as blank. The amount of HCl consumed by PEO grafted film between the second equivalent point subtracted from the amount of HCl used by the blank at the same pH gave the amount of carboxylic acid groups, hence the PEO content on CA film. The results are shown in the table in following Example 3.

Example 3

This example describes the reaction of polyallyl amine with the CA-PEO film formed in Example 2.

Reaction of PALA with CA-PEO Film

CA-PEO-COOH film prepared as described above (1000 cm²) was immersed in water. PALA (2 x10^-4 mol) was added into the film suspension and stirred at 4°C for 1 hr. EDC (2.5x10^-4 mol) in 10 ml of distilled water was added dropwise to the solution and the pH was adjusted to 5. The solution was then stirred for an additional 1 hr. at 4°C. The whole solution was moved to 22°C and allowed to stand for 24 hr. Two additional volumes of EDC were added to the solution at 3 and 6 hr during the reaction. After the reaction was completed, the CA-PEO-PALA films were washed with distilled water. The CA film contained immobilized PALA connected to the film surface via PEO spacers as shown in Formula 13. Both low molecular weight PALA(L) and high molecular weight PALA(H) were immobilized with varying PEO molecular weight spacers.

The content of amino groups from PALA immobilized on the surface was determined by acid-base potentiometric back titration and also by spectroscopy analysis. At pH < 7, all the -NH₂ groups are changed to - NH₃ ⁺ which can be potentiometrically titrated by NaOH. The difference in the amounts of NaOH used between the CA-PEO-PALA and a blank CA gives the amounts of the amino groups on the surface. The results, measured in surface concentrations of 10^-8 mole/cm², are shown in the following table:

^* **

The amount of amino groups on PALA(H) immobilized surfaces is about 2-3 times higher than that on PALA(L) surfaces. A similar procedure to that described above can be used to immobilize PLL onto a CA-PEO film to prepare a product as shown in Formula 12.

Example 4

Heparin Binding in Aqueous Solution

To evaluate the PEO spacer effects on heparin binding efficiency, CA-PEO-PALA films (240 cm²) were placed in 10 ml of 40μg/ml heparin solution (0.1 N Sodium carbonate buffer solution or saline) with shaking for 1 hr. The difference of heparin amount before and after binding gave the net amount heparin bound on the surfaces. CA-glutaric acid-PALA films was also evaluated as a control and compared with CA-PEG-PALA surfaces. The glutaric acid spacer did not provide sufficient linkage space to have any affect on the binding capacity of the PALA.

The amount of heparin on PALA(L) (molecular weight 8,500) immobilized with PEO spacers of varying molecular weight is shown in the following table with a concentration of immobilized PALA on the cellulose acetate surface being given in concentrations of 10^-10 mole/cm²:

The maximum binding of heparin occurred on the PALA(L)-immobilized surfaces with PEO(2K) or PEO (3.4K) spacers. The heparin binding capacities (2.35 ± 0.03 μg/cm² and 2.34±0.01 μg/cm² for PEO(2K) or PEO (3.4 K), respectively) at the maximum binding region were increased 3.5 fold as compared with the PALA(L) surface without PEO spacer (0.68 ± 0.09). To further investigate the PEO effects on the heparin binding, the plot of mole ratio of heparin bound (based on M. W. 5000) to PALA(L) on the surface versus the molecular weight of PEO spacers was drawn. It was found that the heparin binding capacity depended on the chain length of PEO. The maximum synergistic effect for heparin binding on lower molecular weight of PALA surfaces was obtained with the PEO spacers having M.W. 2000 and M.W.3400. The curve (not shown herein) appeared as a reverse bell shape along the X axis: low heparin binding efficiency with the control or PEO(0.6K) with significantly increased efficiency with PEO(2K) and PEO (3.4K), and then a decrease in binding efficiency with PE0(8K). When the molecular weight of PEO was greater than 6000, the segmental motions of PEO was found to be independent of molecular weight of PEO. Other supportive evidence is that the cooperativity of water-PEO interactions increased with the increase in the molecular weight of PEO until a molecular weight of about 3400 was reached.

The amount of heparin on PALA(H) (molecular weight 50,000) immobilized with PEO spacers of varying molecular weight is shown in the following table with a concentration of immobilized PALA on the cellulose acetate surface being given in concentrations of 10^-11 mole/cm²:

For higher M.W. PALA (PALA(H), 50,000-immobilized surfaces, the effects of PEO on heparin binding differed from PALA(L) -immobilized surfaces. Heparin binding efficiency of PALA(H) surfaces with PEO(0.6K) spacer (0.87 ±0.09 μg/cm²,) increased 1.8-fold when compared to PALA(H) surface without PEO spacer. Nevertheless, increase in molecular weight of PEO spacers did not affect heparin binding on PALA(H) surfaces. The maximum heparin binding region was not observed in the plot of the mole ratio of heparin bound to PALA(H) surface against the molecular weight of PEO spacers.

When the PEO synergistic effects on heparin interaction with PALA(H) and PALA(L)-immobilized surfaces were compared, it was found that the PALA(L) was more sensitive to the length or M.W. of PEO spacers than the PALA(H) surfaces. The possible explanation is that for PALA(H)-immobilized surfaces, PEO only slightly enhanced the mobility of PALA(H) due to its relatively large molecular size. However, for PALA(L)-immobilized surfaces, PEO not only enhanced the mobility, but also significantly increased the relative chain length of the dynamic motion part at the PEO-PALA(L) interface. Consequently, the PEO-PALA(L) interface was more solution like and less steric restriction than the PEO- PALA(H) interface. For the PALA(L)-immobilized surfaces, when glutaric acid was used to link PALA(L) with the CA film, no spacer effect was considered due to its short chain. Therefore, heparin binding efficiency was low due to the rigid PALA(L) at the interface. With PEO(0.6K) as a spacer, despite the increased mobility of PALA(L) at the interface, the short chain length of PALA(L)-PEO (0.6K) at the interface could not enhance heparin binding. This minimum chain length requirement at the interface may be ascribed to the relatively large size of the heparin macromolecule. When PEO M.W. was increased to 2000 or 3400, the mobility of PALA(L) at interfaces was further increased, as well as the whole chain length of PALA-PEO was elongated. As the result of both enhancement effects, the heparin binding efficiency increased dramatically. If the chain length of PEO further increased to molecular weight 8000, however, the mobility of PEO did not increase because of the reasons already mentioned above. The positive charges of PALA(L) may also be partially masked by the long chain PEO which may possibly entangle with or bend down to PALA(L).

For the high M.W. PALA-immobilized surfaces, however, PEO(0.6K) increased slightly the mobility of

PALA(H) at the interface as compared with the PALA(H) immobilized surface without PEO spacer. At the same time, the chain length of the PALA(H)- PEO(0.6K) at the interface was already long enough. Therefore, the heparin binding efficiency somewhat increased. Even if the chain length of PEO was further increased (PEO(2K),

PEO3.4K) and PEO(8K)), however, the mobility of PALA(H) may not further increase due to the molecular size of PALA(H). Consequently, heparin binding efficiency remained almost the same as that of PEO(0.6K). Compared to the PALA(L), the heparin binding did not drop down in the PEO(8K) region for PALA(H)-immobilized surface, which may be credited to the long chain of the high molecular weight PALA. It may make PEO(8K) difficult to mask the positive charges of high molecular weight of

PALA. These results suggest that not only does the PEO mobility improve heparin binding on PALA surface, but also the PALA chain length at the interface may also play a role in heparin binding efficiency.

Example 5

Heparin Binding in Blood

CA-PEO (2K) -PALA(L) film was immersed in 1 ml of the heparinized bovine plasma or whole blood (40μg heparin/ml blood, 2% of ³⁵S-labeled heparin). After incubation for 1 hr, the film was rinsed with distilled water and 1 ml of NaOH solution was added. After 5 min shaking, CytoScint cocktail (15ml) was added. The solution was mixed well on a vortex shaker and the radioactivity was counted.

Heparin binding was also evaluated in serum. CA- PEO (2K)-PALA(L) film was immersed in 1:10 diluted serum solution containing heparin (40μg heparin/ml serum solution). After incubation for 1 hr., 0.5ml of solution was taken out for Azure II colorimetric assay.

The amount of heparin bound on CA-PEO(2K)-PALA(L), CA-PEO(2K)-PALA(H), CA-glutaric acid-PALA(L), and CA- glutaric acid-PALA(H) membranes in bovine serum was compared with the corresponding heparin binding amount in aqueous solution. The comparison did not show significant difference between these two media. The amount of heparin binding in bovine plasma or whole blood was determined by counting the radioactivity of ³⁵S-heparin on the film. The amount of heparin bound on the CA-PEO(2K)-PAL-(L) film from plasma or whole blood was compared with the corresponding heparin bound amount in PBS using tracer ³⁵S-heparin, no significant differences in heparin binding amount among the three media were observed. The data shows blood components do not affect heparin binding on CA-PEO-PALA surfaces.

Example 6

Protein Adsorption

Iodine-125 labeled bovine albumin was used as a model protein to study protein adsorption on CA-PEO-PALA surfaces using low molecular weight PALA and PEO spacers varying in molecular weight of from 600 (0.6K) to 8,000 (8K). The concentration of albumin in serum was determined by the bromcresol green method. Different CA-PEO-PALA films (8 cm²) (CA-glutaric acid-PALA(L) (control), CA-PEO(0.6K)-PALA(L), CA-PEO(2K)-PALA(L), CA- PEO(3.4K)-PALA(L), CA-PEO(8K)-PALA(L)) were immersed in the serum solutions (1 ml) which had 50% of normal serum. The film contained solutions were shaken for 1 hr and then rinsed with distilled water and saline until no radioactivity could be detected in the wash. The films were transferred to vials and the radioactivity on the film was counted by Minaxi Gamma Counter (auto-gamma 5000 series, Packard Instrument Company, Downers Grove, IL). The amount of albumin adsorbed (μg/cm²) of the film surfaces is given in the following table:

It is readily apparent that the minimum albumin adsorption was found on the PALA surfaces with PEO(3.4K) and PEO(8K) spacers. There was no significant difference in albumin adsorption between PEO(3.4K) and

PEO(8K) spacers.

Although the above examples were carried out using PALA as the polycation, PLL could have been utilized with comparable results.

Both PALA and PLL are structurally similar in having primary amino groups on the side chain and both being positively charged at neutral pH. PALA has a higher charge density per molecule than PLL due to the smaller repeat unit. Empirical results show that both PALA and PLL have similar heparin binding capacities.

Comparison of average pKa of PALA and PLL indicates that the basic strength of PALA is slightly lower than that of PLL but the difference was not significant. The repulsive interactions of the intramolecular amino groups on the PALA chain is greater than that of PLL which may be attributed to the higher charge density per molecule of PALA than PLL. The greater intramolecular force between functional amino groups on PALA also translates into the fact that changes of pKa value of PALA is more sensitive than to the changes of pH than with PLL. Another similarity with PALA and PLL is that the degree of ionization is similar at about neutral pH. The pKa of PLL is lightly higher than that of PALA inferring that he interaction between PLL and polyanions such as heparin may be somewhat stronger than that of PALA. However, the higher positive charge density of PALA per molecule when compared with PLL suggests that PALA may actually bind more heparin than PLL.

In view of the above, the criteria for selecting a polycation as a heparin binding reagent are (a) capability of binding heparin, (b) functional groups which can be covalently grafted onto polymeric surfaces and (c) commercial availability at a reasonable price. Any polycation having primary amino groups is considered to be a candidate for use in this invention due to the likelihood of various chemical reactions with the free amino groups. Inclusive of polycations which are currently commercially available and have free amino groups are PLL, PALA and PVA. The high price of PLL limits its practical usage. Furthermore, the terminal

COOH group on this amino acid makes it more difficult to covalently bond to the PEO spacer by linking the amino groups on the PLL chain with the COOH groups of the derivatized PEO. This creates the possibility of self- cyclization between the pendant amino groups and the carboxyl end group of the same PLL molecule. Neither PALA nor polyvinylamine have this terminal COOH problem. However, that does not remove the possibility of utilizing PLL in the present invention. The difficulties encountered merely make the PALA the preferred polycation to use because it does not possess carboxyl groups. The PVA molecule has not been utilized in tests but is also believed to be functional in the present invention.

In view of the above, it was deemed that PALA would be the most practical and best polycation embodiment of the invention to illustrate. Therefore, while the invention has been described and illustrated with reference to certain preferred embodiments thereof, those skilled in the art will appreciate that various modifications, changes, omissions, and substitutions can be made without departing from the spirit of the invention. It is intended, therefore, that the invention be limited only by the scope of the following claims.

To simplify claim terminology certain shortened definitions will be utilized in view of the detailed description given above. The substrate of Formula 1 will simply be referred to as "Substrate".

Claims

1. A composition having the formula:

O O

Substrate-OC-PEO-CNH-PAC

where Substrate represents any suitable biopolymer to which polycations may be covalently bonded through a spacer grouping; -OC(O)-PEO-C(O)- is a polyethylene oxide polymer having a molecular weight of between about 600 and 8000; and -NH-PAC represents any suitable polyaminocation which contains a positive charge at a neutral pH.

2. A composition according to Claim 1 wherein - NHPAC is a member selected from the group consisting of polyallylamine (PALA), poly-L-lysine (PLL) and polyvinylamine (PVA).

3. A composition according to Claim 2 wherein - NHPAC is a member selected from the group consisting of PALA and PLL.

4. A composition according to Claim 3 wherein - NHPAC is PLL.

5. A composition according to Claim 3 wherein - NHPAC is PALA having a molecular weight of between about 8,500 and 65,000.

6. A composition according to Claim 5 wherein the PALA is PALA(L) having a molecular weight of between about 8,500 and 11,000.

7. A composition according to Claim 6 wherein the polyethylene oxide polymer has a molecular weight of about 2000 to 3400.

8. A composition according to Claim 7 wherein PALA has a molecular weight of about 8,500.

9. A composition according to Claim 5 wherein PALA is PALA(H) having a molecular weight of between about

50,000 and 65,000.

10. A composition according to Claim 1 wherein Substrate is a member selected from the group consisting of cellulose, cellulose diacetate, poly(ethylene-vinyl alcohol) and agarose.

11. A method for removing heparin from an aqueous solution containing the same which comprising (a) providing a composition of the formula:

O O

II

Substrate-OC-PEO-CNH-PAC where Substrate represents any suitable biopolymer to which polycations may be covalently bonded through a spacer grouping; -OC(O)-PEO-C(O)- is a polyethylene oxide polymer having a molecular weight of between about 600 and 8000; and -NH-PAC represents any suitable polyaminocation which contains a positive charge at a neutral pH; (b) bringing said solution containing heparin into contact with said composition at a neutral pH causing heparin to bind to said polyaminocation and (c) removing said solution depleted of heparin from contact with said composition.

12. A method according to Claim 11 wherein -NHPAC is a member selected from the group consisting of polyallylamine (PALA), poly-L-lysine (PLL) and polyvinylamine (PVA).

13. A method according to Claim 12 wherein -NHPAC is a member selected from the group consisting of PALA and PLL.

14. A method according to Claim 13 wherein -NHPAC is PLL.

15. A method according to Claim 13 wherein -NHPAC is PALA having a molecular weight of between about 8,500 and 65,000.

16. A method according to Claim 5 wherein the PALA is PALA(L) having a molecular weight of between about

8,500 and 11,000.

17. A method according to Claim 16 wherein the polyethylene oxide polymer has a molecular weight of about 2000 to 3500.

18. A method according to Claim 17 wherein PALA has a molecular weight of about 8,500.

19. A method according to Claim 15 wherein PALA is PALA(H) having a molecular weight of between about 50,000 and 65,000.

20. A composition according to Claim 11 wherein Substrate is a member selected from the group consisting of cellulose, cellulose diacetate, poly(ethylene-vinyl alcohol) and agarose.