WO1996008211A2 - Plasma grafting methods and compounds - Google Patents

Abstract

This invention relates to plasma grafting methods and compounds. More particularly, the present invention relates to a novel plasma grafting process compound for producing a surface suitable for immobilizing bioactive substances.

Description

DESCRIPTION

Plasma Grafting Methods and Compounds

Background and Introduction to the Invention

Publications and other references referred to herein are incorporated herein by reference and are numerically referenced in the following text and respectively grouped in the appended Bibliography which immediately precedes the claims.

Modification of surfaces has been of interest in industrial and medical applications as well as academic interest for many years. These applications include immobilization of proteins (1), application in solid-phase immunoassay (2), control of electroosmosis (3), affinity chromatography (4), solid phase peptide synthesis (5) and modification of the interface between an urethane potting resin and a bundle of siloxane coated fiber (6) or for solid phase catalysts (7).

Over the years, a large number of medical devices have been developed which contact blood. The degree of blood contact varies with the device and its use in the body. For instance, catheters may briefly contact the blood, while implants, such as heart valves and vascular grafts, may contact blood for a number of years. Regardless of the device, contact of blood with foreign materials initiates the process of thrombosis, often followed by formation of thromboemboli.

Adsorption of proteins is normally one of the first events to occur when blood contacts a foreign surface. The composition and conformation of adsorbed proteins influence subsequent cellular responses such as platelet adhesion, aggregation, secretion, complement activation, and ultimately, the cross-linked fibrin formation and thrombus formation.

The initial protein layer at the blood-material interface is subject to denaturation, replacement, and further reaction with blood components. During this phase of protein adsorption, adsorbed fibrinogen is converted to fibrin. Fibrin formation is accompanied by the adherence of platelets and possibly other cellular componenets. The platelets become activated and release the contents of their granules. This activates other platelets, thereby resulting in platelet aggregation.

A thrombus eventually forms from entrapment of erythrocytes (red blood cells) and other blood constituents in the growing fibrin network. Thrombus growth can eventually lead to partial or even total blockage of the device unless the thrombus is sheared off or otherwise released from the foreign surface as an embolus. Unfortunately, such emboli can be as dangerous as blockage of the device because emboli can travel through the blood-stream, lodge in vital organs, and cause infarction of tissues. Infarction of the heart, lungs, or brain, for example, can be fatal. Therefore, the degree to which foreign material used in biomedical implants inhibits thrombus formation, embolization, and protein denaturation determines its usefulness as a biomaterial.

In the past, the thrombogenicity of biomedical implants has been treated by the administration of systemic anticoagulants, e.g., heparin and warfarin. However, long-term systemic anticoagulation therapy is disadvantageous due to the risk of hazardous side effects. Moreover, overdose of anticoagulants may cause lethal side reactions, such as visceral or cerebral bleeding. For these reasons, there have been extensive efforts to develop materials which can be used in biomedical devices or implants which can contact blood with minimal or no systemic anticoagulation therapy being necessary to avoid thrombus formation.

Production of a nonthrombogenic blood-contacting surface through immobilization of biologically active molecules is one method which has been attempted to avoid thrombus formation. Such bioactive molecules include anticoagulants, such as heparin. Heparin is a highly sulfonated mucopolysaccharide containing a number of charged functional groups. The heparin/antithrombin III complex formed inactivates Factor Xa. Factor Xa is responsible for the conversion of prothrombin to thrombin which mediates the conversion of fibrinogen to fibrin (8).

Surface modification of polymeric materials offers the advantage of optimizing the chemical nature of the blood/polymer interface while allowing a choice of the substrate to be based upon the necessary properties of the blood-contacting device. The methods used to immobilize bioactive molecules onto blood-contacting surfaces fall into four general groups: physical adsorption, physical entrapment, electrostatic attraction, and covalent binding.

Surfaces incorporating bioactive molecules by physical adsorption or entrapment beneath the blood-contacting surface exhibit a significant degree of thromboresistance. However, depletion of the bioactive molecules by leaching into the blood environment causes the surface to rapidly lose its thrombo-resistant character. Entrained molecules diffuse to the surface which, along with physically adsorbed bioactives, are then "leached" from the surface into the blood plasma by mechanical and chemical mechanisms. Similarly, electrostatically or ionically bound molecules are subject to partitioning and ion exchange between the blood-contacting surface and the electrolyte-rich plasma resulting in depletion.

Covalently bound bioactive molecules resist depletion sufficiently to offer a potentially "long term" thromboresistant effect. Most prior attempts to covalently bind heparin to a blood-contacting surface have reportedly resulted in severely decreased activity of the bound heparin. For example, heparin coupled to a blood-contacting surface through one of its carboxyl groups using a carbodiimide method has been reported to lose up to 90% of its activity (9). Others have reported "covalent" attachment of heparin, but actually describe heparin covalently bound to a tether molecule which is ionically bound to the substrate (10).

Additional problems are encountered in situations in which the blood-contacting surface must be gas permeable. For example, siloxane polymers are of particular interest in blood gas exchange devices due to their inherent thromboresistant properties and gas permeability (11). Siloxane polymers, however, are relatively inert and thus relatively resistant to modification of their surfaces except for the use of ionizing radiation or free-radical initiators in the presence of a vinylic monomer (11-17). However, th use of plasma discharge processes provide one technique for altering the surface chemistry of silicones (18) and other polymeric materials (19-26). The introduction of functional groups, such as amine or hydroxyl groups, onto a materials surface may provide sites for subsequent covalent immobilization of graft polymers, tethers and/or biomolecules (27-28). Use of chemical methods for surface modification of siloxanes and silicones can be problematic (29-31).

Essentially three different processes can be accomplished in a plasma environment: coating, grafting and etching. These three processes are depicted schematically in Figure 1. Coating (Fig. 1a) refers to the ability to lay down on a surface by a plasma polymerization process a film of polymerized monomer. The coating process described in co-assigned, co-pending U.S. Application filed November 12, 1993, entitled "Hydrocyclosiloxane Membrane Prepared by Plasma Polymerization Process," is one example of a coating process. In that process, a siloxane film is deposited on a surface. This particular siloxane, 1,3,5,7-tetramethylhydrocyclosiloxane, produces a membrane film that is flexible but relatively resistant to abrasion unlike the monomer tetramethyldisiloxane. (32) Other monomers used in coating processes include octamethylcyclotetrasiloxane (33), ethylene (34), fluorocarbons (35) and others. For all practical purposes coating is defined as application of a film or layer of deposited polymerized monomer forming a "skin" or membrane over the substrate. In applications where for example gas transfer is a desired property, a "skin" of polymer may disrupt or abolish desirable gas transfer properties.

Etching (Fig. 1b) is a process of cleaning a surface which is analogous to "surface ablation" and is commonly used for metals (36). Typically, in a plasma discharge system a common monomer used for "etching" is argon gas. Plasma derived from argon is high energy and has the ability to remove molecular species from the surface exposing substrate that was located under the surface prior to treatment. This process is particularly useful for the removal of organic contaminates.

Grafting (Fig. 1c) refers to the ability to attach by end-point attachment individual molecules of a monomer used in a plasma process. End point attachment is important because the unattached end of the molecule can be used to conduct further chemical modification. For example with a tether molecule, one end is attached to the surface leaving the other end of the tether extending away from the surface. The extended end can be chemically modified to attach a biomolecule.

During a plasma process such as grafting, a plasma glow zone is created by passing monomer molecules through a high frequency (13.6 MHz radio frequency power supply is used in this invention) electric field at pressures lower than atmospheric pressure. Electrons under the influence of the electric field will interact with the monomer molecules and gain kinetic energy. A low temperature plasma will form when the energetic electrons collide with the monomer molecules. A plasma is composed of electrons, free radicals and molecules in the excited state. The general process of creating a low temperature plasma is known to those in the art (37-39) and accordingly, a detailed discussion of the theory behind plasma processes is not provided here.

Plasma grafting is a process of passing a monomer species through a plasma glow zone which generates free radicals. At the same time the substrate either sits in the glow zone (batch type reactor) or is continously feed

(continous type reactor) through the plasma glow zone.

During the time that the substrate resides in the plasma glow zone, the substrate is bombarded by electrons and ions. When the electrons become energetic enough, chemical bonds are broken in the top layers of the substrate becoming chemically active due to radical anions or cations.

Because of the low pressure environment of the plasma system, the free radicals generated have longer lifetimes than free radicals generated in the atmosphere. The majority of free radicals generated in the glow zone will recombine with each other forming low molecular weight oligomers. Only a fraction of free radicals will interact with the chemically activated substrate surface. For polymeric materials, covalent bonds are formed between the monomer molecules and the polymeric substrate through free radical mechanisms.

By controlling the monomer flow rate, system pressure, residence times of monomer and substrate in the plasma glow zone, the surface of the substrate is grafted with monomer molecules which has a desired functional group for further chemical manipulation by wet chemistry processes.

Thus, it will be appreciated that there is a need for a plasma grafting process which achieves end point attachment of the grafting reagent to the surface while maintaining the integrity of the nucleophilic or other desired functional group. This nucleophilic group can then be used as a handle to conduct further chemical modifications. It will also be appreciated that there is a need for a plasma process which is capable of introducing masked or protected nucleophilic groups without damaging pH sensitive substrates such as siloxanes. In addition, this process will be useful for any application in which one desires to correct any type of interfacial problems between two materials. The grafting of amino groups to one or both of the substrates to be bonded can and will lead to increased strength of the bond.

Summary of the Invention

The present invention is directed to methods for producing a surface suitable for immobilizing a biomolecule onto the surface. The methods are particularly useful for those surfaces used in constructing biomedical devices and implants. A variety of bioactive molecules, for example, those which counteract specific blood-foreign material incompatibility reactions, may be immobilized onto the surface. The present invention pertains to the use of N-protected unsaturated or cyclic amines (see Figure 2) to plasma graft amine groups onto a surface. Increasing interest in the synthesis and use of N-silylated amines have recently been reported. (40-42) An example of a desired substrate, a siloxane surface, grafting would rendering the surface able to bond to a bioactive molecule or tether or a tether/biomolecule combination of molecules. Siloxane is a preferred substrate because the substrate itself is relatively thromboresistant. Moreover, siloxane is gas permeable, which is important in many applications, for example, in gas exchange devices. Nevertheless, it will be appreciated that other substrates are within the scope of the present invention. It will also be appreciated that methods and grafted surfaces involving biomolecules other than those which counteract blood-foreign material incompatibility are within the scope of the present invention. Plasma processes on various substrates can be characterized using the surface analysis methods known as ESCA43 (electron spectroscopy for chemical analysis) and TIRF44 (Total internal reflection fluorescence) for primary amine containing surfaces. For example, TMCTS siloxane surfaces can initially be analyzed looking at specifically the elements of silicone, carbon, nitrogen and oxygen. The atomic percentages of silicone, carbon, nitrogen and oxygen are typically 33%, 33%, 0% and 33%. After grafting using N-trimethylsilylallylamine (TMSAA) and treating the 1,3,5,7-tetramethylhydrocyclosiloxane

(TMCTS) surface with water, the surface atomic percentage for nitrogen is typically in the 4-8% range. This means that 4-8 % nitrogen has been introduced on the surface. It can not be stated that all of this nitrogen will be useful for chemical modification which would involve only primary and secondary amines as nucleophiles capable of reacting. TIRF spectroscopy using the fluoroprobe fluorescamine (45), generates a fluorescent adduct only upon reaction with a primary amino groups. Accordingly, TIRF can be used to determine if primary amino groups are present on a grafted surface.

The present invention pertains to the use of N-protected unsaturated or cyclic amines to plasma graft amine groups onto a surface, for example, a siloxane surface, thus rendering the surface capable of bonding to a tether or bioactive molecule. To overcome the inertness of the surface while retaining desirable properties such as gas transfer, amine groups are introduced onto the surface using a plasma grafting process. In a plasma grafting process a reactive molecule is introduced onto the substrate surface.

It is believed that the unsaturated or cyclic amine-containing compound is 'activated' by the electrode of the plasma appartaus resulting in generation of a radical or similar reactive intermediate by homolytic or heterolytic cleavage of the carbon-carbon double bond, carbon-carbon triple bond or strained carbocyclic ring system. The reactive intermediate then undergoes silicon-hydrogen and/or carbon-hydrogen insertion on a siloxane substrate surface. In turn, the siloxane surface may also be 'activated' by the electrode possibly generating radicals on the surface. Other polymeric materials and other substrates may be treated by only slight modifications of existing methods. By this method, as will be later discussed in detail, the amine-containing compound is attached to the surface.

Alternatively, as described in the commonly assigned and concurrently filed U.S. Application, entitled "Electrophilic polyethylene oxides for the modification of polysaccharides, polypeptides (proteins) and surfaces", after the amine groups are plasma grafted onto the surface, the surface may be reacted with epoxide-, isocyanate-terminated or other electrophilic-terminated poly-(ethylene) oxide (hereinafter referred to as "PEO") tether molecules. After the reaction occurs, one end of the PEO molecule is covalently bound to the surface, while the other end of the PEO molecule retains an intact electrophilic group (epoxide, isocyanate or others) for further attachment of a biomolecule in the next processing step. Polyethylene oxide is presently preferred because it exhibits low protein adsorption. (46) Other molecules may be used as "spacer or tether chains," as well. For example, these can include peptides, polypeptides, long chain alkanes, proteins, polysaccharides, saccharides, fatty acids, poly(amino acids), poly(vinyl alcohols), poly (vinyl pyrrolidinones), polyphosphazenes, poly(acrylic acid) and other biologically derived polymers.

The intact electrophilic group on the PEO that is surface bound may react with the nucleophilic groups (hydroxyl, amine, sulfhydryl) present in biomolecules and pharmaceutical agents. Thus, various bioactive molecules may be covalently bonded to one end of the PEO molecule in the same way that the other end of the PEO molecule is covalently bonded to the siloxane blood-contacting surface.

In this embodiment, because a bioactive molecule is spaced away from the surface at one end of a PEO chain, the bioactive molecule may possess an activity approaching the activity of the bioactive molecule in solution. Because of the mobility of the bioactive molecules near the surface, the effectiveness of the bioactive molecule may be substantially greater than the same bioactive molecule bound directly to the surface or by a very short tether molecule. (47) For these reasons, this embodiment may be preferred.

Bioactive molecules which may be immobilized on a surface include: heparin, urokinase, plasmin, and tissue plasminogen activator (TPA), streptokinase, albumin, IgG, hirudin, other proteins, modified prostaglandins and other pharmaceuticals. Heparin inhibits the blood incompatibility reaction which normally causes clotting and thromboemboli formation. Heparin acts by interacting with antithrombin III, Factor Xa and thrombin to inhibit the conversion of fibrinogen to fibrin. Urokinase, plasmin, and TPA are serine proteases which lyse protein deposits and networks formed during blood incompatibility reactions that lead to thrombosis.

The U.S. Patent application Serial No. 08/152,176 in the names of Chen-Ze Hu, E. Kurt Dolence, Shigemasa Osaki, and Clifton G. Sanders and entitled "Hydrocyclosiloxane membrane prepared by plasma polymerization process" and the commonly assigned and concurrently filed patent application in the names of E. Kurt Dolence, Chen-Ze Hu, Ray Tsang, Clifton G. Sanders and Shigemasa Osaki and entitled "Electrophilic polyethylene oxides for the modification of polysaccharides, polypeptides (proteins) and surfaces" the disclosures of which are incorporated by this reference, more fully describe aspects related to the present invention. Brief Description of the Figures

Figure 1 depicts plasma processes: coating, etching and grafting

Figure 2 depicts monomer candidates for plasma grafting agents as the mono-N-trimethylsilyl protected analogs

Figure 3 depicts modified electrodes configuration of the Plasma Science 0500 plasma system

Figure 4 depicts engineering STAR-PL plasma system Figures 5A to 5D depict ESCA spectra of two minute ammonia grafting of tetramethylhydrocyclosiloxane (TMCTS) coated KPF-190 fiber

Figures 6A to 6D depict ESCA spectra of two minute ammonia grafting of tetramethylhydrocyclosiloxane (TMCTS) coated KPF-190 fiber following water treatment

Figures 7A to 7D depict ESCA spectra of two minute allylamine grafting of a tetramethyldisiloxane (TMDS) surface following a dichloromethane wash

Figures 8A to 8D depict ESCA spectra of two minute allylamine grafting of a tetramethyldisiloxane (TMDS) surface following water wash

Figure 9 depicts a reaction scheme and reagent stochiometry used in the synthesis of N-trimethylsilylallylamine

Figures 10A to 10C depict ESCA spectra of tetramethylhydrocyclosiloxane (TMCTS) plasma coated KPF-190 polypropylene fiber

Figures 11A to 11D depict ESCA spectra of TMCTS coated KPF-190 fiber grafted with N-trimethylsilylallylamine using the STAR-PL plasma grafting system

Figures 12A to 12D depict ESCA spectra of TMCTS coated KPF-190 fiber grafted with N-trimethylsilylallylamine using the STAR-PL plasma grafting system followed by water treatment

Figures 13A to 13D depict ESCA spectra of TMCTS coated KPF-190 fiber grafted with N-trimethylsilylallylamine using the Plasma Science 0500 plasma grafting system Figures 14A to 14D depict ESCA spectra of TMCTS coated KPF-190 fiber grafted with N-trimethylsilylallylamine using the Plasma Science 0500 plasma grafting system followed by water treatment

Figures 15A to 15D depict ESCA spectra of TMCTS coated quartz slide grafted fifty seconds with N-trimethylsilylallylamine using the Plasma Science 0500 plasma grafting system

Figure 16 depicts surface fluorescence of a fluorescamine treated tetramethylhydrocyclosiloxane coated quartz slide grafted for 50 seconds using

N-trimethylsilylallylamine (TMSAA) versus a non-grafted siloxane control

Figure 17 depicts ESCA spectra of polystyrene spin-coated quartz slide

Figures 18A to 18D depict ESCA spectra of polystyrene spin-coated quartz slide grafted fifty seconds with N-trimethylsilylallylamine using the Plasma Science 0500 plasma grafting system

Figure 19 depicts surface fluorescence of a fluorescamine treated polystyrene spin-coated quartz slide grafted for 50 seconds using N-trimethylsilylallylamine (TMSAA) versus a non-grafted polystyrene control

Figures 20A to 20C depict Carbon-13 nuclear magnetic resonance spectrum of STAR-PL deposited oligomer

Figures 21A to 21G depict Carbon-13 nuclear magnetic resonance spectrum of STAR-PL deposited oligomer-95% carbon-13 HPEO reaction product

Detailed Description of the Invention

Applicants have discovered that, surprisingly, use of an N-protected unsaturated or cyclic amine allows the achievement of desirable end-point attachment. As described previously, carbon end-point attachment offers the advantage of preserving the nucleophilic group intact for further chemical elaboration. In addition, use of N-protected amines as plasma grafting agents with siloxane substrates, protects these normally pH sensitive surfaces from extensive damage due to the basicity of amines. This observation has allowed the application of the N-trimethylsilylallylamine (TMSAA) grafting process to siloxane coated hollow fiber in a gas transfer application without critical loss of gas permeability as compared with an allylamine or ammonia grafting processes.

Applicants have also discovered optimal conditions for maximizing the amount of primary and possibly secondary amines produced during plasma grafting. This is important because only primary and secondary amines are useful for covalent coupling appropriate electrophilic agents such as mixed carbonates, active esters, isocyanates, isothiocyanates, epoxides and other electrophiles. By "optimal conditions" is meant those conditions which maximize water stability (i.e., minimize the amount of nitrogen lost upon water exposure as monitored by ESCA analysis); those conditions which maximize nitrogen available to react with a tether or spacer (i.e., maximize available primary and secondary amino groups); those conditions which minimize gas permeability degradation as a result of grafting.

The optimal conditions chosen are a function of several variables. The most important is the type of plasma reactor apparatus (plate reactor, tube type reactor, or other configurations) and its construction geometry. Two types of plasma reactors are utilized in this invention.

A. Plasma Science 0500 ("PL-500") System

The internal electrode R.F. plasma reactor is a plate type reactor depicted in Figure 3. This system is a modified Plasma Science 0500 plasma system (PL-0500) that is a capacitively coupled with an internal electrode radio frequency system (Plasma Science, Inc, 272 Harbor Boulevard, Belmont, CA 94002). The reactor consists of a hot electrode 1 and the ground electrode 2 with an electrode size of 14.75 inches by 17 inches. Both electrodes are located inside of the vacuum system and are located 5 inches apart. The radio frequency (R.F.) system, which includes the 13.6 MHz power generator 7 , matching network 6 and R.F. cable 5, are connected to the hot electrode 1 through the R.F. cable 5 and the electrical ground 2 through the R.F. cable 5 to the power generator 7 and to the earth ground 12. A fixed amount of monomer is fed continuously from the monomer reservoir 4, through the mass flow controller 3, into the plasma chamber 13 and pumped out of the plasma chamber 13 through the output port 8. The unused monomer is condensed in the liquid nitrogen cold trap 11.

The PL-0500 is a batch type plasma reactor. The substrate to be plasma grafted is placed in the space located between the hot and ground electrodes. The plasma chamber 13 is pumped down by opening the gate valve 9 . The gate valve 9 is located in between the pump system and the plasma chamber 13. After opening the gate valve 9 , the pressure of the plasma chamber 13 will drop to the millitorr range and reaches a steady state equilibrum pressure within a few minutes. After the plasma chamber pressure has reached steady state, the mass flow controller is turned on and the monomer is fed from the monomer reservoir 4 into the plasma chamber 13. The throttle valve 10 is then turned on to regulate and maintain a constant pressure in the plasma chamber 13. After the plasma chamber 13 has reached steady state, the plasma glow zone is activated in the space located between the hot electrode 1 and the ground electrode 2 by introducing the R.F. power into the hot electrode 1 . The plasma glow zone can be viewed through the view port 4.

When the grafting process is completed, the R.F. power is turned off and the grafted substrate is allowed to remain inside of the system for two minutes, a post monomer quench, to eliminate radical species that have activated the substrate surface. After this two minute post monomer quench period, the mass flow controller 3 and the throttle valve 10 are closed. The residual monomer is continuously pumped out of the plasma chamber 13 through the gate valve 9 . The gate valve 9 is then closed and the plasma chamber 13 is vented to the atmosphere by opening the vent valve 14. The plasma grafted substrate can then be removed from the plasma chamber. B. STAR-PL System

The STAR plasma coating system (STAR-PL) is an external electrode radio frequency plasma system that has been developed in-house and is depicted in Figure 4. This system is capacitively coupled and is designed for the continuous plasma grafting of substrates such as fibers and catheter tubing. The STAR-PL system continuously grafts substrate by drawing the substrate through the plasma glow zone. The plasma glow zone is controlled by parameters such as radio frequency (R.F.) power, monomer flow rate, system pressure and the substrates dwell time in the plasma glow zone.

The STAR-PL systems consists of two chambers and a plasma glow zone. Chamber .14. contains a supply spool 10, pulleys 9 and clutch 21. Chamber 15 contains take-up spool 11, pulleys 9 and a coating speed control pulley 16.

Grafting using the STAR-PL system is accomplished as follows. The fiber or catheter tubing 5 is wound onto the supply spool 10. The fiber or catheter tubing is tied with 15 turns of extra guide thread. A loop is formed between the supply spool 10, the chamber 14, the plasma glow zone 6, the chamber 15, pulleys 9 and the take-up spool 11 with the aid of the guide thread. The fiber or catheter will pass through the Pyrex^® glass tube 7 three times before winding up on the take-up spool 11.

After the system has been pumped down, the monomer is fed form the monomer reservoir 12 into the system through the mass flow controller 13, the chamber 14, the plasma glow zone 6, the chamber 15 and is continuously pumped out through the outlet port 17. The unreacted monomer is condensed out at the liquid nitrogen trap system 20. The system pressure is controlled by the throttle valve 18. The plasma glow zone consists of a Pyrex^® glass tube 7 with the R.F. electrode 4 attached to the exterior glass surface and the ground electrode 22 attached to the R.F. shielding box 8. The R.F. shielding box 8 is attached to both chambers 14 and 15. The plasma glow zone 6 is activated inside of the Pyrex^® glass tube 7 .

The R.F. system, which supplies the power to maintain the plasma glow zone, includes the R.F. power supply 1 , the R.F. cable 2 , the R.F. matching network 3, the R.F. electrodes 4, and the ground electrode 22. The electrodes are made of 1 inch wide copper tape.

When the substrate moves through the plasma glow zone 6, the proper substrate tension is maintained by the clutch 21 and the speed of the substrate is measured by the speed control pulley 16. The plasma grafted substrate is continuously wound onto the take-up spool 11. After the plasma grafting is completed, the R.F. power supply 1 , mass flow controller 13 and the throttle valve 18 are closed. The system is vented to the atmosphere and the plasma grafted substrate spools can be removed from the system.

C. Preferred N-Protected Unsaturated or Cyclic Amines

Preferred N-protected unsaturated and cyclic amines include compounds selected from

wherein R₁ is -CH₂- or -CH(CH₃)-, R₂ is hydrogen, lower alkyl, tri-loweralkylsilyl or lower alkylsilane and R₃ is tri-lower alkylsilyl or a lower alkylsilane group.

More preferably the N-protected amine is a mono- or bis- N-triloweralkylsilylallylamine. Preferred lower alkyl groups include methyl and ethyl. Other protecting groups may be used which are removable under mild, non- oxidizing conditions by a chemical means and include, e.g., acetyl, trifluoroacetyl and others known in the art. Appropriate N-protecting groups include groups which would give sufficient vaporpressure to be put in a gas phase for plasma grafting.

As described herein, we have found N-trimethylsilyl protected unsaturated and cyclic amines to be particularly preferred, more particularly mono and bis brimethylsilylallyl amines.

We have found by use of tri-lower alkylsilylallyl- amines such as those described herein in the grafting process provide advantages over coatings obtained using either ammonia or allylamine. Use of ammonia and allylamine for grafting has resulted in greatly diminished gas permeability of the resulting membrane; in addition although those procedures resulted in introduction of nitrogen into the membrane, those nitrogens were unstable under aqueous conditions.

D. Substrates

In addition, the substrate can play an important part in the conditions that can be utilized. Many substrates are very sensitive to temperature and UV radiation and are easily damaged. For these two important reasons, one can not make generalizations which would make it easy to predict results of a grafting process without considerable experimentation. In the case of siloxane membranes, the variables involved in plasma grafting are geometry dependent based on the reactor design. However, the values of the variables are not unique, simply the correct combination of variables must be found. Typically, this is determined using a response-surface approach.

Introduction of Amine Groups by Plasma Grafting - Optimization of grafting conditions and monomer choice

The methods of the present invention involve introducing amine groups on a surface by plasma grafting using a gas of an N-protected unsaturated or cyclic amine. In one plasma grafting process, microporous hollow fibers coated with a plasma-polymerized tetramethylhydrosiloxane are used as the substrate. These fibers are subjected to additional plasma exposure in the presence of gas of an N-protected unsaturated amine, N-trimethylsilylallylamine (TMSAA).

In order to demonstrate the important alteration of the plasma grafting results by using N-protected amines, the following examples will include the use of ammonia and allylamine. Analysis by electron spectroscopy for chemical analysis (ESCA) 48-49 is used to identify and quantitate surface nitrogen relative to the other elements observed. Because ESCA analyzes only up to the top 100 angstroms of a surface, analysis of bulk structure below the sampling depth is not possible with ESCA. In addition, the atomic percent reported by ESCA is for the entire volume analyzed (i.e., the top 50-100 angstroms). Thus, 3% nitrogen does not correspond with 3% of the surface atoms being nitrogen. This is because the nitrogen atoms would be found only on the surface and atoms (i.e., carbon/silicone) from below the surface are also detected. Nevertheless, ESCA analysis of percent nitrogen provides a valuable approximation for the number of amino groups on the surface.

Grafting using N-protected unsaturated amines results in amine incorporation into or onto the siloxane polymer. ESCA and fourier transform infrared spectroscopy analysis of the resulting surface demonstrates the existence of Si-H bonds, C-N bonds, Si-O bonds, and Si-CH₃ groups. While the exact surface structure resulting from these reaction processes is not known, the use of TIRF and the fluoroprobe fluorescamine allows one to probe for the presence of free primary amine groups.

The use of N-protected unsaturated amines in plasma grafting to attach functional amine groups to the siloxane substrate, as discussed below, is an important improvement within the scope of the present invention. Several advantages have been found in employing N-protected unsaturated amines in a plasma grafting process.

Experiments have shown that when using the N-trimethylsilylallylamine grafting process, more amine groups are made available for covalent binding, these amino groups are hydrolytically stable and gas permeability of the substrate is retained to a greater extent than surface grafted with ammonia or allylamine.

In the present application, reaction of a nucleophilic group (amino) covalently bound to the surface with electrophilic tether followed by an appropriate biomolecule produces a bioconjugate in which a strong covalent bond is formed. The type of bond formed in this process is a function of the type of starting electrophilic group present on the PEO molecule. In the case of an amino group reacting with PEO bis-glycidyl ether, an alpha-amino alcohol is formed. Reaction of an amino nucleophile with a PEG mixed carbonate produces an urethane or carbamate covalent bond. Several examples of an electrophilic PEO reactions with biomolecules are present in the literature (50-51). To assist in understanding the present invention, the following examples are included which describe the results of a series of experiments. The following examples relating to this invention should not, of course, be construed as specifically limiting the invention and such variations of the invention, now known or later developed, which would be within the purview of one skilled in the art are considered to fall within the scope of the present invention as hereinafter claimed. Examples

Example 1: Ammonia Plasma Treatment of a

Tetramethyldisiloxane Coated Silicon Chip

Initial attempts to use ammonia as a reagent for the introduction of amine groups capable of reacting with a PEO "tether" were conducted on a plasma deposited membrane of tetramethyldisiloxane on a silicon wafer chip. (52) The resulting membrane was characterized using ESCA. During ammonia plasma treatment, power was varied from 40 to 180 watts, times from one to 15 minutes with a ammonia mass flow of 190 μmoles per second and throttle pressure of 180 millitorr. Similar results were obtained for all conditions attempted. A tetramethyldisiloxane surface was treated with ammonia plasma for a period of 15 minutes

(180 watts). The spectrum was obtained immediately after plasma treatment. An identical chip was treated with ammonia as described above except that the chip was treated with distilled deionized water prior to obtaining the ESCA spectrum. Table 1 summarizes the atomic percentages of the chips.

Inspection of this data reveals that upon exposure to water, the surface content of nitrogen groups drops drastically indicating that the nitrogen groups appear to be hydrolytically unstable. In all likelihood, this is a result of nitrogen-silicon bond formation during plasma treatment which are hydrolytically unstable. It has also been reported (53) that ammonia plasma does not produce primary amines efficiently on polymeric substrates.

In any event, these studies demonstrate that ammonia is not a viable candidate for plasma treatment of siloxanes due to the instability of the nitrogen groups upon exposure to water. Further evidence supporting nitrogen-silicon bonding arises upon treatment of ammonia treated siloxane with polyoxyethylene bis-glycidyl ether in water. Inspection of the resulting surface by ESCA indicates that a large amount of PEO appears to be simply adhered to the surface . The amount of PEO present is much too large considering the small amount of nitrogen present that could participate in covalent bonding forming an alpha-amino alcohol. Apparently, upon hydrolysis of a nitrogen-silicon bond, a silicone-hydroxyl bond is formed releasing ammonia. This silicon-hydroxyl moiety could interact with PEO via hydrogen bonding leading to adhered, adsorbed PEO without covalent bonding occuring.

Example 2: Ammonia Plasma Treatment of Tetramethylhydro- cyclotetrasiloxane Coated KPF-190 Fiber

Attempts to treat tetramethyltetrahydrocyclosiloxane (TMCTS) coated KPF-190 polypropylene fiber with ammonia plasma using the Plasma Science 0500 plate type reactor also gave poor results as depicted in Table 2 and Figures 5A to 5D and 6A to 6D.

a - Gas to gas permeability: Oxygen/Carbon dioxide - 2.08/12.66

b - Gas to gas permeability: Oxygen/Carbon dioxide - 0.51/1.19 c - Gas to gas permeability: Oxygen/Carbon dioxide - Unmeasureable

Conditions for this ammonia plasma treatment were pressure=180 mtorr, power=180 watts, ammonia flow=190 micromoles per second with the times of 2,5,10 and 15 minutes. In each case, following water treatment greater than 40% of the initially grafted nitrogen washed away. Most detrimental is the fact that ammonia plasma totally destroyed the gas permeation ability of the fiber. In any event, unacceptable loss of both gas permeability and nitrogen make ammonia treatment unacceptable.

Example 3: Allylamine Plasma Treatment of Tetramethyldisiloxane Coated Silicon Wafer Chips

It may be theorized that allylamine (NH₂CH₂CH=CH₂) offers an "advantage" over ammonia in the grafting process based on an analysis of the bond energies (54) of all bonds in the molecules allylamine and ammonia. Indeed, allylamine (NH₂CH₂CH=CH₂) has been used previously (55-61) in plasma applications to deposit "nitrogen" groups on surfaces.

Attempts to use allylamine as a grafting agent are depicted in Table 3 and Figures 7A to 7D and 8A to 8D. Conditions for this grafting attempt included power=20 watts, and pressure=542 mtorr with times of 2, 4, 6, 8 and 10 minutes.

In addition, two chips were treated at the same time using the Plasma Science 0500 plate reactor. One chip was washed with distilled dichloromethane in an attempt to remove any organic soluble material deposited by the plasma treatment. The second chip was washed with distilled deionized water. Each chip then had an ESCA spectrum taken and atomic percentages determined as summarized in Table 3. It is clear from the data that following water exposure from 38 to 56% of the nitrogen depositied was removed by a water wash, again suggesting that possibly nitrogen-silicon bonding is occuring which subsequently hydrolyzes upon water exposure. In the case of a gas transfer application, creation of a film or gross modification of the siloxane chemical structure will inhibit efficient gas transfer and can radically lead to changes in the thromboresistance of the surface. Any film formation has the potential of delamination and will enter general blood circulation in the case of a blood contacting device application.

Example 4: N-Trimethylsilyl Allylamine Synthesis

A common theme in organic synthesis is to protect amino groups with a protecting group such that other chemical manipulation can be made on a molecule without interference from the amino nucleophile. After such modifications the protecting group is removed from the amino group. In an attempt to modify the plasma grafting ability of allylamine, the amino nitrogen was protected as the N-trimethylsilyl derivative. Choice of an N-trimethylsilyl group arose from literature (62) use of N-trimethylsilylallylamine use in hydrosilylation of silicones containing silicon-hydrogen functional groups. N-Trimethylsilylallylamine was synthesized based on the procedure described in the literature63 and is described in detail below. Other N-trimethylsilylated amines may be synthesized from the parent amine by modification of the below described procedure followed by distillation.

Procedure: Synthesis of N-trimethylsilylallylamine (TMSAA)

The synthetic procedure for the synthesis of N-trimethylsilylallylamine is described below. Reagent stoichiometry is described as follows:

Distill 600 mL total volume of allylamine from calcium hydride (reflux for 30 minutes before distilling) while under a nitrogen environment. Assemble the reaction apparatus. Be sure that the nitrogen flow is started such that the bubbler is slowly bubbling. Add to the reaction flask by removing one of the 24/40 glass stoppers the following:

1. 600 mL of allylamine distilled from calcium hydride

2. 844 mL of hexamethyldisilazane (HMDS)

3. 0.220 grams of ammonium sulfate

Double check to ensure that the reaction mixture is under a slight positive pressure of nitrogen.

Initiate refluxing of the mixture by setting the potentiostat at a setting of 140/50% setting. Reflux the mixture for 60 hours under a nitrogen environment. At this time the mixture should be a clear solution.

Quickly remove, using a disposable pasteur pipette, a small sample of the mixture and obtain a proton and carbon NMR spectrum in deuterated chloroform. Note that nearly all allylamine should be gone with the major product being TMSAA. In addition, some of the disilylated product and hexamethyldisilazane will be present. The spectrum should confirm this. Allow the reaction mixture to cool down while under a positive pressure of nitrogen. Carefully and quickly remove the reaction flask and attach it to the distillation apparatus. Immediately place the apparatus under a positive pressue of nitrogen. Turn on the potentiostat setting for the heating mantle to 140/42%. Call the local National Weather service office and get the atmospheric pressure reading for the day. Record the value.

Slowly allow the still to heat up. Turn the distillation reciever to the 250 mL flask for fraction one. This fraction boiling point collection point is ambient temperature to 106 degrees centigrade. This fraction will consist of allylamine and TMSAA in approximately a 1:1 ratio. Save this fraction for further resynthesis by reworking.

Once the boiling point has reached 106 degrees centigrade turn the distllation reciever to the 1000 mL flask and begin collection of the fraction two. Continue collecting distillate until the boiling point has reached 109 degrees centigrade. Once the boiling point has reached 109 degrees discontinue distillation.

Tare a 1.0 liter glass amber bottle with label. Quickly pour into the glass bottle the fraction 2 material with a boiling point of 106-109 degrees centigrade. Determine the amount in grams of product obtained.

Obtain both a proton, carbon-13 and silicon-29 nuclear magnetic resonance spectrum with deuterated chloroform without tetramethylsilane. The concentration for the test sample is 0.05 mL of TMSAA in 0.55 mL of deuterated chloroform. The internal reference for proton NMR is the residual non-deuterated chloroform signal located at 7.26 ppm. The internal reference for carbon NMR is the deuterated chloroform signal triplet signal located at 77.0 ppm. The internal reference for silicon NMR is tetramethylsilane located at 0.00 ppm. NMR data is as follows: ¹H NMR (CDCl₃) δ 7.26 (singlet, internal reference CHCl₃) 5.95-5.78 (multiplet, 1 H, olefinic proton), 5.14-4.90 (multiplet, 2 H, terminal olefinic protons), 3.36-3.26 (multiplet, 2 H, allylic protons), 0.70-0.20 (broad singlet, 1 H, NH proton), 0.03 (singlet, 9 H, (CH₃)₃Si); ¹³C NMR (CDCl₃) δ 140.8 (olefinic carbon), 112.8 (olefinic carbon), 77.0 (triplet center signal, internal reference CDCl₃), 44.5 (allylic carbon), 2.4 (trace 1,1,1,3,3,3-hexamethyldisilazane impurity, this signal must not be larger than the 44.5 ppm signal), 0.1 (methyl carbon from the trimethylsilyl group); ²⁹Si NMR (CDCl₃) δ 4.23 (N-trimethylsilylallylamine), 2.44 (trace 1,1,1,3,3,3-hexamethyldisilazane impurity), 0.00 (internal reference tetramethylsilane).

Calculate the percentage of allylamine present in the TMSAA using the quality assurance inspection document located in document control. This value should well less that the specification limit of less than 5%. If it is NOT up to specification, redistill the material as described above.

In addition, N-trimethylsilylallylamine may be purchased from HULS America, Inc., Piscataway, NJ.; however, quality must be monitored carefully. Synthesized N-trimethylsilylallylamine used in the plasma grafting process may contain up to 15% 1,1,1,3,3,3- hexamethyldisilazane impurity without affecting the quality of grafting results. In fact, attempts to utilize 1,1,1,3,3,3-hexamethyldisilazane as a grafting agent did not result in the implantation of nitrogen on the surface.

Example 5 : N-Trimethylsilyl Allylamine Plasma Treatment of Tetramethylhydrocyclotetrasiloxane KPF-190 Fibers

Table 4 and Figures 10A to 14D describe plasma grafting using N-trimethylsilylallylamine on tetramethyl- hydrocyclotetrasiloxane KPF-190 polypropylene fiber. In contrast to surfaces grafted with non-silylated amino groups, use of N-trimethylsilylallylamine afforded quite different results than examples 1 to 3. Table 4 includes data utilizing both the Plasma Science 0500 and STAR plasma units which demostrates that the conditions used to obtain similar results from the different geometrically configured machines can vary. Conditions used in grafting is configuration dependent. The ESCA data reveals that following water treatment the amount of nitrogen lost to hydrolysis is at most 11%. This is in contrast to examples 1, 2 and 3 utilizing ammonia and allylamine which afforded nitrogen lose values following water treatment of 40 to 70%. Although the gas to gas permeability of N-trimethylsilylallylamine grafting of tetramethyltetrahydrocyclosiloxane has decreases by 50%, the ratio of carbon dioxide (CO₂) : oxygen (O₂) permeation has remained approximately 6:1 as in the starting TMCTS only coated fiber. Most importantly, the fact that the nitrogen introduced by grafting appears to be stable to aqueous conditions. This will prove to be very important in providing a coating which has a minimized potential for large scale leaching of a coating.

a Flow rate = 17.2 μmoles/second, pressure = 20 mtorr, power = 80 watts, time = 2.5 minutes

b Flow rate = 20.7 μmoles/second, pressure = 80 mtorr, power = 25 watts, fiber speed = 10.9 cm/second

c Gas/Gas permeation rate oxygen: carbon dioxide - 2.08 (±0.1)/12.66 (±0.3)

d Gas/Gas permeation rate oxygen:carbon dioxide - 1.01 (±0.0)/5.7 (±0.1)

e Gas/Gas permeation rate oxygen:carbon dioxide - 1.1 8 (±0.1)/6.1 (±0.6) Example 6: N-Trimethylsilyl Allylamine Plasma Treatment of Tetramethylhydrocyclosiloxane Coated Quartz Glass

Slides

Although grafting using N-trimethylsilylallylamine produces nitrogen on a siloxane surface as monitored by ESCA, the true identity as whether these nitrogen groups are primary, secondary, tertiary or quaternary remains an unknown. For attachment of an electrophilic tether such as a mixed carbonate polyethylene oxide, only primary and secondary amino groups will be useful nucleophiles. In order to probe for the presence of primary amino groups, the fluoroprobe fluorescamine (45) and the method of total internal reflectance fluorescence spectroscopy (44) (TIRF) were used. The probe, fluorescamine, will produce surface fluorescence only upon reaction with primary amino groups. Other oxidation states of amino groups do not form fluorescent products. In order to conduct surface fluorescence measurements, a series of quartz slides were coated with TMCTS followed by grafting using N-trimethylsilylallylamine. Grafting conditions and reactor used were the Plasma Sciences 0500 reactor using the conditions: power = 19 watts, flow = 4.6 μmoles per second, pressure = 56 mtorr and variable times of 10, 30, 50, 70 and 90 seconds. ESCA spectra for these chips grafted fifty seconds are depicted in Figures 15A to 15D.

Atomic percentages are listed in Table 5. For the time values of 10 to 90 seconds little difference exists in the percentage of nitrogen introduced ranging from 9 to 10.5 atomic percentage. Preliminary qualitative TIRF data for the 50 second sample is depicted in Figure 16 clearly demonstrates the presence of surface fluorescence indicating that primary amino groups are present on the surface as compared to an ungrafted control. ESCA data for the 50 second sample used before and after water exposure are summarized in Table 5.

Example 7: N-Trimethylsilyl Allylamine Plasma Treatment of Polystyrene (MW 250.000) Film Cast on a Quartz Slide-50 Second Grafting Exposure

In order to determine whether N-trimethylsilylallylamine grafting will anchor primary amino groups on the surface of other polymeric material other than siloxane, grafting was conducted on polystyrene. A polystyrene film was spin-cast on a quartz TIRF slides using a 3% solution of polystyrene (molecular weight 250,000) in toluene. The spin cast film/slide was then inspected by ESCA as depicted in Figures 17A to 17D and summarized in Table 6. The polystyrene slide was then subjected to the following grafting conditions using the Plasma Science 0500 reactor and inspected by ESCA to determine atomic percentages as depicted in Figures 18A to 18D and Table 6. Interestingly, following grafting, the grafted surface reveals not only the incorporation of nitrogen but also oxygen. The presence of oxygen must arise due to incorporation during the grafting process (due to imperfect seals leading to oxygen leaking into the system) or upon shut-down of the system and exposure to the atmosphere. Apparently, oxygen is being trapped by the radicals formed during the grafting process or generated on the surface. Prior to conducting grafting on a non-oxygen containing substrate such as polystyrene, oxygen incorporation was not noticed due to the substrates high oxygen content. However, the presence of oxygen is not detrimental to the ability of the amino groups to react with fluorescamine . Qualitative TIRF results for a polystyrene - TMSAA grafted surface are depicted in Figure 19 indicating that reactive primary amino groups are present on the surface of the grafted polystyrene. In addition, preliminary TIRF data indicates that N-trimethylsilylallylamine grafting appears to work on polycarbonate, polyurethanes such as Tecoflexr and other polymeric substrates.

Example 8: Oligomer Deposit Characterization

In all plasma polymerization processes, downstream toward the vacuum port, will be depositied low molecular weight oligomeric material arising from gas phase reaction of ionized monomer with itself . Since grafting on a surface can not be identified and structurally characterized on a surface by classical methods such as nuclear magnetic resonance spectroscopy, the depositied oligomeric polymer should in some degree reflect the structural identification of the grafted species on the surface. In order to learn more about the structural features of the grafting species, the deposited oligomer was investigated by spectroscopic methods and the chemistry of the oligimer probed.

The oligomer deposited in the downstream vacuum portion of the tube of the STAR system is typically a gummy, amber colored resin. The Fourier transform infrared spectrum reveals that the oligomer is an alkane (2954 cm^-1) which contains amino and or hydroxyl groups (3279 cm^-1) in addition to a possible silyl ether being present (1251 cm^-1). More detailed structural information on the oligomer can be obtained using nuclear magnetic resonance spectroscopy. This oligomer is soluble in organic solvents such as chloroform and alcohols such as methanol, however it takes considerable time for dissolution to occur. The proton NMR spectra, reveals that no structural features relating to olefinic or double bond character remain in the oligomer as is present in N-trimethylsilylallylamine. The broad character of the alkane protons located between 0.5 to 4.0 parts per million demonstrates that this oligomer is highly crosslinked. This diversity of alkane backbone is demonstrated in the carbon-13 NMR spectra (Figures 20A to 20C) since all of the alkane carbons appear as a very broad small hump ranging from 10 to 65 parts per million. However, the silicon-29 NMR spectrum (64) provides confirmation of information provided by ESCA. Table 7 lists the silicon chemical shift values for for a variety of silanes including N-trimethylsilylallylamine. Clearly, from the silicon-29 NMR spectrum, it can be observed that the STAR oligimer contains three magnetically distinct silicon atoms. Most importantly is the signals located between -20 and -23 ppm which suggest that the silicon is bound with oxygen. This confirms the incorporation of oxygen into the grafting as was discussed in Example 8 involving polystyrene. The broad nature of the oligomer silicon signals suggest that the silyl groups are involved in the crosslinking of the deposited polymer.

Finally, Table 7 lists the results of combustion analysis for the oligomeric material with the balance of atoms being silicon and oxygen that can not be measured by combustion analysis. This data confirms that based on two independent analysis runs that the process deposits material from run to run that are very similar from an elemental composition point of view. Table 7A - Silicon-29 NMR chemical shift values (relative to TMS at 0.00 ppm) and carbon-hydrogennitrogen combustion analysis data for STAR oligomer

1. N-trimethylsilylallylamine: 4.23 ppm

2. Bis-(N-trimethylsilyl)allylamine: 6.52 ppm

3. 1,1,1,3,3,3-hexamethyldisilazane: 2.46 ppm

4. Tetramethyltetrahydrocyclosiloxane :

-32.11, -32.37, -32.70 ppm

5. Tetramethyldisiloxane: -4.65 ppm

6. STAR oligomer: range 5-10 ppm with main signal at 7.15 ppm, broad signal from 0-5 ppm (small), -20 to -23 ppm small broad signal with spikes at -20.99 and -21.67 ppm

Example 9: Oligomer Reaction With Carbon-13 Enriched Polyoxyethylene Bis-(N-Hydroxybenzotriazolyl) Carbonate - Confirmation of Urethane Bond Formation

The purpose of grafting a surface is to implant an amino group or other nucleophile such that an electrophilic tether can be attached for the purpose of binding a biomolecule or other compounds. In order to confirm the reaction of polyoxyethylene bis-(N-hydroxybenzotriazolyl) carbonate (HPEO) with the amino groups in the STAR oligomer, the residue was reacted with 95% carbon-13 enriched HPEO in dichloromethane. Following an aqueous workup, the product obtained was characterized by both infrared spectroscopy and nuclear magnetic resonance spectroscopy. Figures 21A to 21G depict the carbon-13 spectral data for the product confirming urethane bond formation. The infrared spectrum reveals little since the carbon-13 enrichment results in an isotopic shift of the urethane carbonyl. Reaction of natural abundance carbon-13 HPEO reveals the presence of an urethane carbonyl in the 1715-1720 cm^-1 range. However, the proton and carbon NMR confirmed clearly the presence of urethane bonding. The proton NMR spectra reveals a multiplet centered at 4.21 ppm which is characteristic of the methylene protons on the PEG backbone that are located alpha to the urethane oxygen. The carbon-13 NMR (Figures 21A to 21G) reveals carbonyl carbon signals located at 156.4 and 155.0 ppm confirming the formation of an urethane bond. Model reactions for urethane bond formation show that typically urethane bonds show up in the 155-157 ppm region. The silicon-29 NMR, shows no real change in the types of silicon present in the product .

In conclusion, from the foregoing, it will be appreciated that the parameters associated with N-trimethylsilylallylamine grafting are highly interdependent and dependent upon the specific plasma chamber. One skilled in the art would appreciate that the parameters must be modified and the correct combination determined experimentally when using different geometry plasma chambers/glow zones.

The applicants determined optimal conditions for N-trimethylsilylallylamine (TMSAA) plasma grafting as described above. By "optimal conditions" is meant to be conditions which produce "nitrogen" on the treated surface having the following characteristics: (1) stability in water, i.e., to minimize the amount of nitrogen lost on exposure to water; (2) capability of covalent binding to electrophilic PEO molecules, i.e., maximize primary and secondary amines; (3) maintenance of gas permeability of substrate; i.e., minimize gas permeability degradation which may result from the plasma grafting process; and (4) reproducibility of atomic percent nitrogen as monitored by ESCA analysis. Bibliography

1. Bayer, E.; Rapp, W. "Polystyrene-immobilized PEG Chains: Dynamics and application in peptide synthesis, immunology, and chromatography" in Poly (ethylene glycol) Chemistry: Biotechnical and Biomedical Applications, editor: J. Milton Harris, Chapter 20, pages 325-342 (1992)

2. Holmberg. K.; Bergstrom, K.; Stark, M.;

"Immobilization of proteins via PEG chains" in Poly (ethylene glycol) Chemistry: Biotechnical and Biomedical Applications, editor: J. Milton Harris, Chapter 19, pages 303-324 (1992)

3. Poly (ethylene glycol) Chemistry: Biotechnical and Biomedical Applications, editor: J. Milton Harris, Chapter 1, page10 (1992)

4. Bayer, E.; Rapp, W. "Polystyrene-immobilized PEG Chains: Dynamics and application in peptide synthesis, immunology, and chromatography" in Poly (ethylene glycol) Chemistry: Biotechnical and Biomedical Applications, editor: J. Milton Harris, Chapter 20, pages 325-342 (1992)

5. TentaGel resins, manufactured by Peptide International, Inc., Louisville, KY: A polystyrene resin modified with poly (ethylene) glycol 6. E. Kurt Dolence, Chen-Ze Hu, Shigemasa Osaki, unpublished results. Siloxane coated fiber grafted with TMSAA prior to potting using a polyurethane based potting resin demonstrated much improved bonding when compared to non-grafted siloxane coated fiber. 7. Manecke, G.; Storck, W.; "Polymeric Catalysts"; Angew.

Chem. Int. Ed. Engl. 17, 657-670 (1978) 8. Heparin: Chemical and biological properties and clinical applications, edited by D. A. Lane and U. Lindahl, CRC Press, Inc., Boca Raton, FL USA, 1989. 9. Ebert, CD., "Evaluation of Diaminoalkane Mediated Immobilized Heparin," PhD. dissertation, Dept . of Pharmaceutics, Univ. of Utah (1981)

10. O.Larm, R. Larsson and P. Olsson; "A New non-thrombogenic surface prepared by selective covalent binding of Heparin via a modified Reducing Terminal Residue" Biomat., Med. Dev., Art. Org., 11(2&3), 161-173 (1983)

11. Warrick, E. L.; Pierce, O. R.; Polmanteer, K. E.;

Saam, J. C.; "Silicone elastomer developments 1967-1977," Rubber Chemistry and Technology, 52, 437-525 (1979)

12. Kammermeyer, K.; "Silicone rubber as a selective barrier - gas and vapor transfer"; Indust. Eng. Chem. 49, 1685-1686 (1957) 13. Chawla, A.S. "Use of plasma polymerization for preparing silicone-coated membranes for possible use in blood oxygenators"; Art. Organs 3, 92-96 (1979)

14. Kopecek, J.; Ulbrich, K. "Biodegradation of Biomedical Polymers" Prog. Polym. Sci. 9, 1 (1983) 15. Chawla, A.S. "Evaluation of plasma polymerized hexamethylcyclotrisiloxane biomaterials towards adhesion of canine platelets and leucocytes"; Biomaterials 2, 83 (1981)

16. Chawla, A.S. "Preparation of silicone coated biomaterials using plasma polymerization and their preliminary evaulations" ; Trans. Am. Soc. Artif. Int. Organs. 25, 287 (1979)

17. M. Yamamoto, J. Sakata, M. Hirai "Plasma Polymerized Membranes and Gas Permeability"; J. Applied Polym. Sci. 29, 2981-2987 (1984)

18. Urban, M. W.; Stewart, M. T.; "DMA and ATR FT-IR studies of gas plasma modified silicone elastomer surfaces"; J. Applied Polym. Sci. 39, 265-283 (1990) 19. Silicone: Hollahan, J.; Carlson, S. "Hydroxylation of polymethylsiloxane surfaces by oxidizing plasmas"; J. Applied Poly. Sci., 14, 2499-2508 (1970)

20. Polypropylene hollow fibers: Denes, F. ; Percec, V.;

Totolin, M. ; Kennedy, J.P. "Cationic grafting from plasma modified polymer surfaces 2. Preliminary investigations with polypropylene fibers"; Polymer Bulletin 2, 499-504 (1980)

21. Polyethylene : Guthrie, J.T. ; Kotov, S. ; "The radiation induced graft copolymerization of methacrylic acid with polyethylene. Ion-exchage properties of the copolymeric composites"; J. Applied Polym. Sci. 37, 39-54 (1989)

22. Polypropylene: Yokoyama, Y. ; Tanioka, A.;

Miyasaka, K. "Preparation of a single bipolar membrane by plasma-induced graft polymerization"; J. Membrane Sci. 43, 165-175 (1989)

23. Poly (tetrafluoroethylene): Hegazy, E.A.; Taher, N.H.;

Kamal, H. "Preparation and properties of cationic membranes obtained by radiation grafting of methacrylic acid onto PTFE films " J. Applied Polym. Sci. 38, 1229-1242 (1989) 24. Polyethylene hollow fibers: Saaito, K. ; Kaga, T.; Yamagishi, H.; Furusaki, S. "Phosphorylated hollow fibers synthesized by radiation grafting and cross-linking" J. Membrane Sci. 43, 131-141 (1989) 25. Polystyrene: Strobel, M.; Thomas, P.A.; Lyons, C.S.

"Plasma fluorination of polystyrene" J. Polym. Sci.: Part A Polymer Chemistry 25, 3343-3348 (1987)

26. Acrylates: Epaillard, F.; Brosse, J.C.; Legeay, G.

"Plasma-induced polymerization" J. Applied Polym. Sci. 38, 887-898 (1989).

27. Gombotz. W. R.; Hoffman, A. S.; "Functionalization of polymeric films by plasma polymerization of allyl alcohol and allylamine"; J. Appl . Polym. Sci., Polym. Symp. Ed. 42, 255 (1988)

28. Griesser, H. J.; Chatelier, R. C.; "Surface characterization of plasma polymers from amine, amide and alcohol monomers"; J. Appl. Polym. Sci.: Appl. Polym. Symposium, 46, 361-384 (1990) 29. Balykova, T.N.; Rode, V.V. "Progress in the study of the degradation of stabilization of siloxane polymers."; Russ. Chem. Rev. 38, 306-317 (1969)

30. Ratner, B.D., Hoffman, A.S. "Radiation Grafted Hydrogels on Silicone Rubber as New Materials", Polym. Sci. Technol. 7, 159-171 (1975).

31. Merker, R.L.; Elyash, L.J.; Mayhew, S.H.; Wang, J.Y.C.

"The Heparinization of Silicone Rubber Using Aminoorganosilane Coupling Agents" in U.S. Dept. of Health, Education and Welfare Contract PH 43-66-977, report 3, October 31, 1969. 32. Nomura, H.; U.S. Patent No. 4,824,444; "Gas permselective composite membrane prepared by plasma polymerization coating techniques", assignee: Applied Membrane Technology, Inc. 33. Chawla, A. S.; Sipehia, R. "Characterization of plasma polymerized silicone coatings useful as biomaterials", J. Biomed. Materials Res. 18, 537 (1984)

34. Chang, F. Y.; Shen, M.; Bell, A. T. "Effects of electric discharge surface treatment on the diffusion characteristics of polymers" J. Appl. Polym. Sci. 17, 2915 (1973)

35. Colter, K. D.; Bell, A. T.; Shen, M. "Control of the pilocarpine release rate through hydrogels by plasma treatment" Biomatls., Med. Dev., Art. Org. 5(1), 1 (1977)

36. O'Kane, D. F.; Mittal, K. L.; "Plasma cleaning of metal surfaces", J. Vac. Sci. Technol. 11, 567-569

(1974)

37. Yasuda, H.; "Plasma Polymerization"; Academic Press, 1985

38. Chapman, B. N.; "Glow Discharge Processes: Sputtering and Plasma Etching", Wiley, New York (1980)

39. Biederman, H.; "Plasma Polymerization Processes", Elsevier Scientific Publishers , Amsterdam and New York, 1992

40. Murai, T.; Yamamoto, M.; Kondo, S.; Kato, S. "Silver Iodide mediated amination reaction of allylic chlorides with lithium bis (trimethylsilyl)amide: A new synthetic method to N,N-disilylallylamines via lithium amide argentates" J. Org. Chem. 58,

7440-7445 (1993)

41. Corriu, R. J. P.; Geng, B.; Moreau, J. J. E. "An efficient synthesis of substituted (Z)-allylamines and 7-membered nitrogen heterocycles from

(Z)-3-(tributylstannyl)allylamines" J. Org. Chem. 58, 1443-1'448 (1993)

42. Grignon-Dubois, M.; Fialeix, M.; Laguerre, M.; Leger, J.; Gauffre, J. "An organosilicon route to the B-norbenzomorphan skeleton" J. Org. Chem. 58,

1926-1931 (1993)

43. Dilks, A. "X-Ray photoelectron spectroscopy for the investigation of polymeric materials", in Electron Spectroscopy: Theory, Techniques and Applications 4, 277-359 (1981)

44. Hlady, V.; Van Wagenen, R.A.; Andrade, J.D. "Total internal reflection intrinsic fluorescence (TIRIF) spectroscopy applied to protein adsorption" in Surfaces and Interfacial Aspects of Biomedical Polymers volume 2: Protein Adsorption, Ch. 2, 81-117

45. Stocks, S.J.; Jones, A.J.M.; Ramey, C.W.; Brooks, D.E.

"A fluoremetric assay of the degree of modification of protein primary amines with polyethylene glycol" Anal. Biochem. 154, 232-234 (1986) 46. Gombotz, W.R.; Guanghui, W.; Horbett, T.A.; Hoffman, A.S. "Protein adsorption to and elution from polyether surfaces" in Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical Applications, editor: J. Milton Harris, Chapter 16, 247-261 (1992) 47. Park, K. D.; Kim, W. G.; Jacobs, H.; Okano, T.; Kim, S. W. "Blood compatibility of SPUU-PEO-heparin graft copolymers", J. Biomed. Mat. Res. 26, 739-756 (1992)

48. Everhart, D. S.; Reilley, C. N.; "The effects of functional group mobility on quantitative ESCA of plasma modified polymer surfaces", Surface and Interface Analysis, 3, 126 (1981)

49. Dilks, A. "X-Ray photoelectron spectroscopy for the investigation of polymeric materials", in Electron Spectroscopy: Theory, Techniques and Applications 4, 277 (1981)

50. Furukawa, S.; Katayama, N.; Hzuka, T.; Urabe, I.;

Okada, H. "Preparation of polyethylene glycol-bound NAD and its application in a model enzyme reactor" FEBS LETTERS 121, 239-242 (1980)

51. Suzuki, T.; Kanbara, N.; Tomono, T.; Hayashi, N.;

Shinohara, I. "Physicochemical and biological properties of poly(ethylene glycol)-coupled immunoglobulin G" Biochimica et Biophysica Acta 788, 248-255 (1984)

52. Hu, C. Z.; Dolence, E. K.; Osaki, S.; unpublished results.

53. Nakayama, Y.; Takahagi, T.; Soeda, F.; Hatada, K.;

Nagaoka, S.; Suzuki, J.; Ishitari, A. " XPS analysis of ammonia plasma treated polystyrene films utilizing gas phase chemical modification" J. Polym. Sci.: Polym. Chem. Ed. 26, 559-572 (1988).

54. "The Chemist's Companion: A Handbook of Practical Data, Techniques, and References", edited by A.J. Gordon and P.A. Ford and published by John Wiley and Sons, New York.

55. Gombotz, W.R., Guanghui, W. ; Hoffman, A.S.

Immobilization of poly (ethylene oxide) on poly (ethylene terephthalate) using a plasma polymerization process" J. Appl. Polym. Sci. 37: 91-107 (1989)

56. Krishnamurthy, V.; Kamel, I.L.; Wei, Y. "Analysis of plasma polymerization of allylamine by FTIR" J. Poly.

Sci.: Polym. Chem. Ed. 27, 1211-1224 (1989)

57. Gombotz, W. R. ; Hoffman, A. S.; "Functionalization of polymeric films by plasma polymerization of allyl alcohol and allylamine"; J. Appl. Polym. Sci., Polym. Symp. Ed. 42, 255 (1988)

58. Hollahan, J.R.; Wydeven, T. "Synthesis of reverse osmosis membranes by plasma polymerization of allylamine" Science 179, 500 (1973)

59. Yasuda, H.; Bumgarner, M.O.; Marsh, H.C.; Morosoff, N.

"Plasma polymerization of some organic compounds and properties of the polymers" J. Polym. Sci.: Polym. Chem. Ed. 14, 195-224 (1976)

60. Peric, D.; Bell, A.T.; Shen, M. "Reverse osmosis characteristics of composite membranes prepared by plasma polymerization of allylamine. Effects of deposition conditions" J. Appl. Polym. Sci. 21, 2661-2673 (1977)

61. Sakata, J.; Wada, M. "Preparation of ion-exchange membranes by plasma polymerization. 1." J. Appl. Polym. Sci. 35, 875-884 (1988) 62. Speier, J.L.; Zimmerman, R.; Webster, J. "The addition of silicon hydrides to olefinic double bonds. Part 1. The use of phenylsilane, diphenylsilane, phenylmethylsilane, amylsilaneand tribromosilane" J. Am. Chem. Soc. 78, 2279-2281 (1956)

63. Saam, J.C.; Speier, J.L. "Preparation of 3-triethoxysilylpropylamine and 1,3-bis(3-aminopropyl) tetramethyldisiloxane" J. Org. Chem. 24, 119 (1959)

64. Taylor, R.B.; Parbhoo, B.; Fillmore, D.M. "Nuclear magnetic resonance spectroscopy" in The Analytical Chemistry of Silicones, edited by A. L. Smith, Wiley and Sons, 371-414 (1991)

Claims

Claims

1. A method for producing a grafted nucleophile on a polymeric surface suitable for attachment of a bioactive molecule comprising the steps of:

(a) introducing a gas of an N-protected unsaturated or cyclic amine within a plasma chamber usable for plasma grafting;

(b) exposing said gas to a radio frequency (13.6 MHz), microwave or other energy sources of sufficient power to create a plasma; and

(c) exposing a substrate to said plasma for sufficient time to activate the surface and graft said N-protected unsaturated or cyclic amine molecules onto the activated sites of said surface in such a way that the resulting amine groups are available for attachment to an appropriate tether

2. The method of claim 1 wherein said N-protected unsaturated amine is a compound of the formula:

CH₂=CH-R₁-N(R₂XR₃) wherein R₁ is -CH₂- or -CH(CH₃)-, R₂ is hydrogen, lower alkyl, tri-loweralkylsilyl or lower alkylsilane and R₃ is tri-lower alkylsilyl or a lower alkylsilane group.

3. The method of claim 1 wherein said N-protected unsaturated amine is a compound of the formula:

HC≡C—R₁-N(R₂XR₃)

wherein R₁ is -CH₂- or -CH(CH₃)-, R₂ is hydrogen, lower alkyl, tri-loweralkylsilyl or lower alkylsilane and R₃ is tri-lower alkylsilyl or a lower alkylsilane group.

4. The method of claim 1 wherein said N-protected cyclic amine is a compound of the formula:

wherein R₁ is -CH₂-, -CH(CH₃)-; R₂ is hydrogen, lower alkyl or lower alkylsilane and R₃ is tri-lower alkylsilyl or a lower alkylsilane group and n = 2 (cyclopropyl) or n = 3 (cyclobutyl).

5. A method according to claim 1 wherein said unsaturated amine is a mono or bis N-tri- loweralkylsilylallylamine.

6. The method of claim 5 wherein said N-protected unsaturated amine is selected from ^

and a combination thereof.

7. The method of claim 5 wherein said N-protected unsaturated amine is N-trimethylsilylallylamine:

8. The method of claim 5 wherein said N-protected unsaturated amine is bis (N-trimethylsilyl)allylamine:

9. A method according to claim 6 wherein said polymeric surface is a membrane formed by the plasma polymerization of a hydrocyclosiloxane monomer of the general formula:

where R an aliphatic group having 1 to about 5 carbon atoms and n is an integer from 2 to about 10.

10. A method of claim 9, wherein said hydrocyclosiloxane monomer is selected from the group consisting of 1,3,5,7,tetramethylhydrocyclotetrasiloxane, 1,3,5,7,9-pentamethylhydrocyclopentasiloxane, 1,3,5,7,9,11-hexamethylhydrocyclohexasiloxane, and a mixture of 1,3,5,7,9-pentamethylcyclopentasiloxane and 1,3,5,6,9,11-hexamethylcyclohexasiloxane monomers.

11. A grafted nucleophile on a polymeric surface prepared according to the method of claim 1.

12. A grafted nucleophile on a polymeric surface prepared according to the method of claim 2.

13. A grafted nucleophile on a polymeric surface prepared according to the method of claim 8.

14. An amine grafted polymeric membrane having amino groups which was prepared by reacting a N-protected unsaturated or cyclic amine within a plasma chamber usable for plasma grafting with a membrane formed from the plasma polymerization of hydrocyclosiloxane monomer of the general formula:

wherein R an aliphatic group having 1 to about 5 carbon atoms and n is an integer from 2 to about 10, under reaction conditions wherein said N-protected amine reacts with activated sites of said membrane to graft said N- protected amine molecules on to the activated sites on the membrane.

15. A membrane according to claim 14 wherein said hydrocyclosiloxane monomer is selected from the group consisting of 1,3,5,7,tetramethylhydrocyclotetrasiloxane, 1,3,5,7,9-pentamethylhydrocyclopentasiloxane, 1,3,5,7,9,11-hexamethylhydrocyclohexasiloxane, and a mixture of 1,3,5,7,9-pentamethylcyclopentasiloxane and 1,3,5,6,9,11-hexamethylcyclohexasiloxane monomers.

16. A membrane according to claim 14 wherein said N- protected amine is a mono or bis N-tri- loweralkylsilylallylamine.

17. A membrane according to claim 15 wherein said N- protected amine is a mono or bis N-tri- loweralkylsilylallylamine.

18. A membrane according to claim 15 wherein is selected from

a combination thereof.

19. A membrane according to claim 18 wherein said N- protected unsaturated amine is N- trimethylsilylallyalamine:

20. A membrane according to claim 18 wherein said N- protected unsaturated amine is bis(N- trimethylsilyl)allylamine: