FIELD OF INVENTION

The invention relates to a polymer network surface having a plurality of molecular imprints from components in blood and the ability to bind said components present in blood. Examples of components include exogenous components as well as endogenous such as endogenous

coagulation cascade inhibitors or glycosaminoglycan. The invention also relates to implants or extracorporeal devices comprising said polymer network surfaces or a method producing them.

BACKGROUND OF INVENTION

Control of the interaction of blood with foreign materials is the central issue in the attenuation of patient response to implantation or to a process, e.g. dialysis or a cardiac-pulmonary bypass, which involves direct contact between the defence systems of the blood and structures placed in an internal human biological environment. The initial event that takes place when blood comes into contact with a foreign object involves rapid

adsorption of proteins to the material surface.

This, in turn, triggers platelet adhesion, activation of the intrinsic coagulation pathway and complement activation. The ability to design a material that does not present a threat to the host, and thus possesses good biocompatibility, is important on account of the need for devices for interaction with blood in clinical medicine.

The primary function of the complement system is to engender the host with innate immunity to defend against foreign cells and organisms. It consists of -30 plasma and cellular proteins (receptors and regulators).

Complement is activated when host recognition molecules encounter non-self structures, e.g. microbial carbohydrates or artificial (bio)materials. This leads to activation of the central component, C3, by proteolytic enzyme complexes, convertases, into the anaphylatoxin C3a which, together with C5a, activates and recruits phagocytes, and C3b which binds to target surfaces and promotes phagocytosis. Further activation leads to the formation of the membrane attack complex (MAC) that may cause cell lysis. The complement system also has cross-activating or cross-inhibiting activities affecting parts of the other cascade systems in blood such as the coagulation, and fibrinolytic and kinin systems. In vivo, the system is under delicate regulation at multiple steps in order to protect autologous cells against damage caused by complement activation. The majority of all known inhibitors, both cell bound and in the fluid phase, regulate the activity of the convertases.

The main event in the coagulation cascade is the enzymatic activation of prothrombin to thrombin by factor Xa in complex with factor Va. Thrombin is a potent activator of platelets and cleaves fibrinogen to fibrin. The main coagulation inhibitor is the serine protease inhibitor antithrombin (AT) which inactivates most of the enzymes in the cascade, the major targets being thrombin and factor Xa. The activity of AT is greatly enhanced by the presence of the glycosaminoglycans heparin or heparan sulfate.

Nature employs a large variety of proteoglycans, which comprise linear polysaccharides consisting of repeating disaccharide units known as glycosaminoglycans (GAGs), e.g. chondroitin sulfate/dermatan sufhate, and heparin/ heparin sulphate, which are linked to a core protein. The number and length of the GAG chains in a proteoglycan varies extensively, as do their degree and pattern of sulfonation. As a result, a single proteoglycan fraction derived from cell surfaces may contain many thousands of different

components and their characterisation in terms of structure and biological function is a major challenge.

Heparin coated surfaces have been shown to suppress coagulation through reduced adsorption of several plasma proteins, including Factor XII and fibrinogen, that normally lead to thrombogenicity. However, the complexity of the coagulation cascade, and the diversity of roles that heparin itself plays in the body, mean that surfaces containing exogenous heparin, might have unexpected biological effects in vivo. Consequently, surfaces with 'heparin-like' functionality, or that can recruit heparin or other sulphated carbohydrates are of considerable interest. It was envisaged that an alternative strategy for controlling the activating properties of a surface would be to create a material with a sufficiently moderate affinity for a regulatory target molecule such as heparin or another sulfonated carbohydrate such as chondroitin sulfate. However, the use of heparin requires that there is a continuous access to blood, which makes it impossible to be used in the future. Accordingly the life span is also limiting and therefore in principle, surfaces capable of recruiting a patient's endogenous glycosaminoglycans or administered heparin would provide a self-regulating biomaterial.

One strategy for developing such materials is molecular imprinting, a means to produce synthetic polymers with antibody-like recognition behaviour. The technique utilises a molecular template to direct the three dimensional arrangement of polymerisable structural building blocks (monomers) through complex formation. The strength and fidelity of the interactions of the template with the various monomers and solvent employed are of paramount importance for the fabrication of sites with affinity for the template. Stabilisation of the complexes through polymerisation and subsequent removal of the template reveals a surface capable of selectively rebinding the template molecular structures. However, there are a number of problems associated with using the technique together with

biomacromolecular templates due to the difficulties associated with the release of large template structures from a three dimensional polymer matrix and the necessity for working in aqueous media.

SUMMARY OF THE INVENTION

The invention relates to a polymer network surface having a plurality of molecular imprints from components in blood and having the ability to bind said components in the blood, such as endogenous and/or exogenous components.

In a first aspect the invention relates to a polymer network surface having a plurality of molecular imprints from components present in the blood and the ability to bind said components present in the blood comprising crosslinking agents and monomers in the ratio from 60-90: 10-40 wherein at least one of the monomers or crosslinking agents comprises at least one functional group selected from the group consisting of hydroxyl, carboxyl, acrylate, carbonyl, anhydride, imide, amine, amide, piperidine, piperazine or pyridine.

Examples include endogenous coagulation cascade inhibitor or glycosaminoglycan molecular imprints. By such an invention it is for the first time possible to create molecular imprints from large complexes, complexes that has the size of the examples mentioned herein or about the same size or even larger, without release of the large template structures as well as being able to work in aqueous media. By the development of the new method it was for the first time possible to create the molecular imprints which will be useful in a huge number of applications within the medical device area in which blood is treated or for some reason involved in the therapy. The invention relates to all kinds of products that come into contact with blood from a mammal, wherein there is a need of providing a system which will not activate the complement system of the mammal and thereby providing systems which eliminates specific problems connected to the activation of the complement system.

In a second aspect the invention relates to a coating comprising a polymer network surface having a plurality of molecular imprints from components present in the blood and the ability to bind said components present in the blood comprising crosslinking agents and monomers in the ratio from 60-90: 10-40 wherein at least one of the monomers or crosslinking agents comprises at least one functional group selected from the group consisting of hydroxyl, carboxyl, acrylate, carbonyl, anhydride, imide, amine, amide, piperidine, piperazine or pyridine.

In a third and fourth aspect the invention relates to an implant or extracorporeal medical device comprising the polymer network surface as defined above, wherein said implant or device may be used in any connection with blood, wherein there is a need of reducing the possibility that the patient will be exposed to thrombogenicity

In a fifth aspect the invention relates to the use of a self-regulating polymer network surface for implantation and/or extracorporeal blood contact.

In a sixth aspect the invention relates to a method of producing a polymer network surface as described above.

By the new invention it will for the first time be possible to put on the market new products which could be used when there is a need of reducing and/or eliminating the possibility of thrombogenicity and/or complement activation.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig 1 shows schematic of the imprinting process. In (i) silica glass slides are reacted with 3-aminopropyltrimethoxysilane (APTMS) and glutardialdehyde in the presence of heparin to generate the template surface. A polymerisable layer is formed on a second slide by treatment with 3- methacryloyloxpropyl-triethoxysilane. Polymerisation of a solution

containing methacrylic acid (MAA) and diacryloylpiperidine (DAP) is carried out between the glass slides held at a defined distance (140 μιη) by the use of spacers to generate an imprinted surface (ii). Non-imprinted (reference) surfaces are prepared using MAA and DAP but with a non-templated surface. Separation of the upper and lower slides and extensive washing exposes the imprinted surface (iii, AFM shown in inset) with a 'memory' for heparin. Exposure of imprinted surfaces to blood leads to lower complement activation than occurs for the reference surfaces.

Fig 2 shows decrease in platelet counts and detection of complement activation products C3a and sC5b-9 complexes after contact between whole blood and imprint and control surfaces. The tested surfaces consisted of glass, heparin imprinted surfaces before (MIP) or after (MIP + Hep) saturation with soluble heparin, and reference polymers before (Ref) or after (Ref + Hep) saturation with heparin. Panel A: Platelet loss after incubation of blood without additions for 1 hr in the blood chamber model. Complement activation was monitored as the generation of C3a (Panel B) and sC5b-9 complexes (Panel C). Data are presented as means ± SEM from five experiments, each performed in duplicate, using blood from four different donors.

Fig 3 shows atomic force microscopy of imprinted (a) and non- imprinted (b) surfaces. Topographic imaging was performed in air and aqueous buffer using a closed wet cell. Contact mode imaging utilised an applied load and scan rate limited to ca. 1 nN and 3 Hz, respectively, to minimize compression and lateral damage to polymer grafts and underlying surfaces. Surface roughness data (c) shows significantly higher roughness (t- test, unequal variances) between the imprinted and control surfaces. Adhesion forces of Si ₃ N ₄ tip-to-surface for both imprinted and non-imprinted surfaces were significantly lower (p < 0.001) than for glass and mica, but the

differences of adhesion force between imprinted and non-imprinted surfaces did not differ significantly (p > 0.05).

DETAILED DESCRIPTION OF THE INVENTION

In the context of the present application and invention the following definitions apply:

The term "polymer network" is intended to mean a network of polymers comprising a polymer polymerized from monomers cross-linked with cross-linker in the presence of the target analyte, the polymer having a capacity for selectively binding the target analyte or a target analyte analogue.

The term "target analyte" is intended to mean any component of the blood, such as those defined herein.

The term "molecular imprint" is intended to mean the construction of ligand selective recognition sites in synthetic polymers where a template (atom, ion, molecule, complex or a molecular, ionic or macromolecular assembly, including micro-organisms) is employed in order to facilitate recognition site fomiation during the covalent assembly of the bulk phase by a polymerization or polycondensation process, with subsequent removal of some or all of the template being necessary for recognition to occur in the spaces vacated by the templating species'. The term "self-regulating" is intended to mean that the polymer network adsorbs an endogenous or administered substance, which enhances the biocompatibility of the material Description

The invention relates to a polymer network surface having the ability to bind a plurality of blood components such as endogenous and/or exogenous components present in the blood.

In one embodiment the invention relates to a polymer network surface having a plurality of molecular imprints from components present in the blood and the ability to bind said components present in the blood comprising crosslinking agents and monomers in the ratio from 60-90:10-40 wherein at least one of the monomers and/or crosslinking agents comprise at least one functional group selected from the group consisting of hydroxyl, carboxyl, acrylate, carbonyl, anhydride, imide, amine, amide, piperidine, piperazine or pyridine. The monomers may be the same or a mixture of monomers and the crosslinking agents may be the same or a mixture. The invented polymer network enables the possibility for the components to bind to said polymer network surface. The molecular imprints being exposed from one surface of the polymer network. Examples include endogenous

coagulation cascade inhibitors or glycosaminoglycans. Thereby it is for the first time possible to us a polymeric network to bind components from the blood of a human being that is to be treated for any kind of disorder or disease. Thereby, complications such as immunological reactions against foreign components are reduced and or eliminated. Additionally, there is no need to separate tissue obtained from other humans from blood. The risk of transferring diseases from one human to another via blood is also removed, moreover, the costs related to transplantations are reduced.

The polymer network surface may attract components present within the blood of a mammal such as a human being. This includes both components produced by the mammal as well as components which are injected or introduced into the mammal such as to treat a disorder or disease. Examples of components include proteins, peptides, heparin, chondroitin sulphate, dermatan sulphate, heparin sulphate or heparin. One specific example is heparin. The polymer network surface may as well attract/be able to bind more than one and the same components.

The monomer and/or the crosslinking crosslinking agent comprise at least one functional group selected from the group consisting of hydroxyl, carboxyl, acrylate, carbonyl, anhydride, imide, amine, amide, piperidine, piperazine or pyridine. For example may the functional group be piperazine or carboxyl or the functional groups may be piperazine and carboxyl.

The ratio between said crosslinking agents and monomers may be 60-90: 10-40, such as 65-85: 15-35, 70-85: 15-30, 75-85:15-25, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 80:20.

The polymer network surfaces may have any kind of suitable thickness and one example is a thickness of about 130-150 μιη, such as 140 μπι.

The polymer network surfaces comprise at least a monomer and a crosslinking agent and there are a huge amount of different ones available on the market well-known for a person skilled in the art. Examples of

crosslinking agents includes DAP, DVB and EGDMA and examples of monomers includes MAA, MA, AAm, IPaam, HEMA, AMPSA, ethylene and methylene bisacrylamide.

One examples being wherein said polymer network surface comprises the crosslinking agent diacryloylpiperazine and the monomer methacrylic acid. The solvent may be water.

Accordingly, the invention relates to a coating comprising the polymer network surface as defined above.

Additionally the invention relates to an implant comprising the polymer network surface as defined above. Examples includes stents, cardiac valves, needles, syringes, equipment for dialysis or any equipment which concerns extracorporeal devices which are used for handling blood from a mammal such as a human being, dog, camel, horse, cow etc.

Accordingly the invention relates to an extracorporeal medical device comprising the polymer network surface as defined above. Examples include all equipment that are used in contact with blood such as during dialysis, i.e., tubes and bags. The invention also relates to implants as well as implant devices and controlled release devices.

The invention also relates to the use of a self-regulating polymer network surface for implantation and/or extracorporeal blood contact.

In a final aspect the invention relates to a method of producing a polymer network as defined above comprising the steps of:

i) providing a first and a second surface provided on two separate plates;

ii) arranging on the first surface a template coating comprising one or more blood components or an analogues thereof, wherein the components are substantially arranged at the surface of the coating; iii) providing a solution comprising one or more crosslinking agents and monomers in the ratio from 60-90: 10-40; iv) attaching the second surface with a functional group reactive with one of the monomers and/or the crosslinking agents during polymerisation;

vi) arranging the solution between the first and the second surface which are separated from each other with a distance;

vii) polymerising the solution; and

viii) removing one of the two plates leaving an imprinted polymer surface on the second surface.

The polymerisation used in the method may be with radical or ionic polymerisation. The monomers may be one type of monomers or a mixture of different ones and the crosslinking agent may be one type or a mixture of crosslinking agents.

The ratio between the crosslinking agents and monomers agent may be 60-90: 10-40, such as 65-85: 15-35, 70-85: 15-30, 75-85:15-25, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 80:20.

The functional groups on the monomer and/or the crosslinking agent may be as defined above for the polymer network surface.

The functional groups on the second surface may be vinyl, acrylate, nitrile or peroxide, such as acrylate.

Additionally the invented polymer network may contain components which are released from the polymer network at controlled conditions, such as different pharmaceutical compositions, active

components, as well as antimicrobial agents etc. One example is heparin and analogues to heparin.

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those skilled in the art that the disclosed techniques that follows represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, a person skilled in the art, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and the scope of the invention. EXAMPLES

EXAMPLE 1

Different combinations of monomers: crosslinkers were evaluated in a primary test, wherein it was found that DAP in combination with MAA had increased properties compared to other combinations (see table 1 and 2 below).

Table 1 : Composition of the synthesized polymers, with regard to monomers, crosslinkers, monomer: crosslinker ratio and solvent. To select polymers for further studies the synthesised materials were screened based on their complement activating ability in hirudin plasma (generation of C3a). The results (n=4) are shown as: + 0-50% increase in C3a generation compared to control, ++ 50-150% increase compared to control and +++ >150 %

compared to control.

Polymer composition C3a total (60:40)* C3a total (80:20)* C3a total (90:10)*

DAP + water:

MAA ++ +++ (P1) ++4

MA n.d +++ n.d.

AAm n.d +++ n.d

IPAAm n.d +++ n.d

HEMA n.d +++ n.d

AMPS A n.d +++ n.d

DVB + EtOH:

MAA ** +++ + (P2) ++

MA n.d + n.d

AAm n.d ++ n.d

IPAAm n.d + n.d

HEMA n.d + n.d

Styrene n.d + n.d

4-VP n.d + n.d

AMPSA n.d *** n.d

EGDMA + EtOH: MAA ** + + (P3) +

MA ++ + ***

AAm n.d ++ n.d

IPAAm ** ++ ++ (P4) ++

HEMA ** ++ ++ (P6) ++

Styrene ** +++ + (P5) ++

4-VP n.d ++ n.d

AMPSA n.d *** n.d V, V-diacryloylpiperazine (DAP); divinylbenzene (DVB); ethylene glycol dimethacrylate (EGDMA);

methacrylic acid (MAA), V-isopropyl acrylamide (IPAAm), 2-hydroxyethyl methacrylate (HEMA), styrene, methallyl alcohol (MA), acrylamide (AAm), 4-vinylpyridine (4-VP) or 2-acrylamido-2-methylpropane sulfonic acid (AMPSA).

n.d.: Not determined

* Ratio crosslinker: monomer

** Polymers in the ratio 80:20 were selected for further studies.

*** Not fully polymerized.

Table 2. Polymer compositions, physical data for synthesized polymers, and contact angles obtained from the captive bubble measurements in water. Data for the contact angle measurement is expressed as mean ± SEM, n≥l 1.

PI P2 P3 P4 P5 P6

MAA (mmol) 1.159 1.318 0.910

IPAAm (mmol) 0.883

Styrene (mmol) 0.892

HEMA (mmol) 0.867

DAP (mmol) 4.635

DVB (mmol) 5.273

EGDMA (mmol) 3.640 3.532 3.567 3.467

AIBN (mmol) 0.108 0.082 0.079 0.080 0.078

APS ^a (μΙ_) 133

TEMED ^b (μΙ_) 13

Water (μ¾ 1330

Ethanol (μΕ) 1200 1200 1200 1200 1200

% C found 58.20 85.63 61.47 62.17 65.60 61.10

% H found 7.40 8.00 7.40 7.40 7.37 7.40

% N found 10.83 <0.3 <0.3 0.80 <0.3 <0.3

BET surface area (m ² /g) 188 161 203 130 25 105

Average pore diameter 133.5 27.6 84.4 32.1 101.6 116.6

(A)

Swelling degree 1.78 ± 1.15 ± 1.09 ± 1.19 ± 1.19 ± 1.18 ±

(mL/mL) 0.03 0.05 0.01 0.01 0.01 0.01

Specific swelling 6.38 3.07 5.57 5.81 5.41 5.46

(mL/g)

Contact angle (°) 19.9 ± 19.8 ± 20.0 ± 19.7 ± 18.6 ± 19.4 ±

0.6 0.4 0.4 0.8 0.5 0.5

Functional groups -COOH -COOH -COOH -NH- -OH

CH ₂ - CH=CH-

(CH ₃ ) ₂ ^a 33 % APS solution, ^b 30% TEMED solution.

_V,V-diacryloylpiperazine (DAP); divinylbenzene (DVB); ethylene glycol dimethacrylate (EGDMA); methacrylic acid (MAA), V-isopropyl acrylamide (IPAAm), 2-hydroxyethyl methacrylate (HEMA).

EXAMPLE 2

Preparation of template surfaces Glass microscope slides (26 x 76 mm) were immersed overnight in solutions of H ₂ S0 ₄ (aq, 0.1 M), then rinsed sequentially with water and acetone before treatment (45 min, 37 °C) with a solution of (3-aminopropyl)- trimethoxysilane (APTMS, Fluka, >97%) in acetone (5%, v/v). The slides were rinsed with acetone and then water before treatment (1 h, 22 °C) with an aqueous solution of heparin (Bioiberica, Barcelona, Spain, mean M _w 12 kDa, 110 IU/mg, 7.5%, w/v) and aqueous glutardialdehyde (GA, Merck, 3%, v/v, pH 5.2 adjusted with H ₂ S0 ₄ ). After incubation, the slides were rinsed with water, dried and stored at 8 °C until further use.

Stability of heparin bound to template surface:

Quantification of heparin bound to template surface

The surface concentration of heparin was determined by Corline Systems AB, Uppsala, Sweden using toluidine blue staining. The AT binding capacity of the immobilised heparin, with a Corline™ Heparin Surface (CHS) as reference, was determined by a colorimetric assay based on the fact that AT in complex with heparin inhibits coagulation factor FXa, performed as described Kodama et al.. ²⁰ Briefly, the surfaces were incubated with AT, then washed and incubated with FXa (both from Enzyme Research Laboratory Inc, South Bend, IN, USA). The remaining FXa in solution was determined using a chromogenic substrate specific for FXa, thus giving an indirect estimate of the amount of bound AT.

Stability of immobilized heparin surfaces

Template surfaces and CHS references (coated on glass) were incubated with citrate plasma in the chamber model described below. After incubation, detection was carried out by incubating aliquots of plasma with AT. Thereafter, FXa was added and the amount of active FXa {i.e. which had not been inhibited by the complexes formed between AT and the heparin which had detached from the surfaces) was determined as described below.

Blood and plasma-based models

Heparin treatment of equipment and preparation of blood

All equipment used in contact with blood (the slide chamber, tubes, pipette tips, etc.) were furnished with a CHS according to the

manufacturer's recommendations. The surface concentration of heparin was 0.5 μg/cm ² , with an AT binding capacity of 2-4 pmol/cm ² (Andersson, J. et al. J. Biomed. Mater. Res. 6 Ά, 458-66 (2003)). Whole blood (25 mL) from healthy volunteers who had received no medication for 10 days was collected in 50 mL Falcon ^® tubes (Becton Dickinson, San Jose, CA, USA) without the addition of soluble heparin, or other anticoagulants, and was used within 5 min in the slide chamber model described below.

Test surface (slide) chambers

The effect on coagulation and complement activation (below) as well as the leakage of heparin from different surfaces (above) was determined using a slide chamber model which consists of a polymethylmethacrylate (PMMA) microscope slide-sized two-well tray (well- volume 1.65 mL) each of which were filled with 1.3 mL of blood (activation studies) or citrate plasma (heparin release) (Hong, J. et al. Thromb. Haemost. 82, 58-64 (1999)). Thereafter, the test surfaces (template, reference and imprinted polymers, CHS or glass) were attached as a lid with clips, creating two circular chambers. The device was rotated for 60 min vertically at 22 rpm in a 37 °C water bath. Reference glass surfaces were cleaned in 5% (w/v) ammonium persulfate at 60°C for 60 min and rinsed in MilliQ water prior to use. Synthesis of heparin imprinted polymer surfaces

Microscope slides were cleaned (5 min, 80 °C) in a series of washing solutions containing 4% NH ₃ (aq), 5% ¾(¾ (aq), 4.5% HC1 (aq) and 3.8% H2O2 (aq), then carefully rinsed with water, acetone, dry tetrahydrofuran and dry toluene and were immediately transferred into a solution comprised of (3-methacryloxypropyl)-trimethoxysilane (3mL, Sigma), triethylamine (0.3 mL) and in dry toluene (150 mL). After 24 h at 22 °C the surfaces were washed with dry toluene, dry tetrahydrofuran and dry acetone, and dried.

For a batch of 5 polymer films, diacryloylpiperazine (288 mg, Fluka, >99%) was dissolved in water (400 μL) by sonication, and 4.6 μL methacrylic acid (MAA, Merck) was added. The solution was sparged with nitrogen, 40 μΤ 33% (w/v) ammonium persulfate (Sigma) and TEMED (4 μΕ, 30%), v/v) was added and 120 μΕ of the solution was spread between the two surfaces. The monomerized and template surfaces were separated using spacers (140 μιη) cut from teflon membrane filters (Pall Corp., MI, USA), figure 1. After polymerisation (16 h), which was initiated by heat the surfaces were carefully separated after treatment with an aqueous ammonia solution (5%), w/v), followed by sequential washes with the separation solution and then with 4M Guanidine HC1. The resultant polymers were inspected for completeness of surface coverage, absence of scratches and air bubbles using a light microscope. Reference polymers were synthesised as described above, but instead of a heparinised glass template, a clean glass surface was used. The polymer films were stored in 20% ethanol at 8 °C for up to 6 months. Heparin adsorption/ Resorption studies

Polymer surfaces were equilibrated in 20 mM phosphate buffer, pH 7.5 (30 min) before PMMA masks (26 x 76 x 10 mm containing 8 wells with i.d. 6 mm) were mounted over the polymer surfaces. The wells were incubated with ³ H-heparin (American Radiolabeled Chemicals Inc, St Louis, MO, USA; 0.4 mCi/mg, Mw 6-20 kDa) solutions (100 μΐ,, 1 h, 22 °C) in the concentration range 0.75 to 75 μg/mL diluted in phosphate buffer. Aliquots (50 μΤ) were removed and mixed with scintillation cocktail (Beckman Ready Safe™, Sweden, 2 mL) and counted in a liquid scintillation counter (Beckman LS 6000, Sweden). The values were normalised to avoid day-to-day

variability and the difference between reference and imprinted polymers was determined. Seven individual experiments were performed (n=3 to 12 wells).

In separate experiments, polymer surfaces (imprinted and reference) were incubated for 45 min at 22°C with heparin (Bioiberica, 8 mg/mL in 0.9% NaCl) and washed 15 min with 0.9% NaCl (aq). The degree of desorption of heparin from these surfaces after incubation in plasma was studied as described above for immobilized heparin surfaces.

Interaction of whole blood and surfaces

Incubation of surfaces with blood

Imprint, reference and glass surfaces, incubated with heparin as above, or with 0.9% NaCl only, and heparin surfaces (template and CHS coated onto glass) were exposed to whole blood (without added

anticoagulants) in the chamber model as described above. After incubation, EDTA was added to a final concentration of 10 mM, to terminate further activation of complement and coagulation. The experiment was performed five times (four different donors) with duplicate analyses with similar results.

Platelet consumption

The platelets remaining in the blood samples were counted using a Coulter A ^C T diff™ haematology analyser (Coulter Corporation, Miami, FL, USA). Due to large intra-individual differences in platelet count data are expressed as percentage of initial values. The blood was centrifuged (10 min, 2200 x g, 4 °C) within 30 min. and the plasma stored at -70 °C. Complement activation

Complement activation was monitored as the generation of C3a and sC5b-9 complexes (the soluble form of MAC) in the plasma by enzyme immuno-assays (EIAs) as described previously (Mollnes, T. E., Lea, T., Froland, S. S., Harboe, M. Scand. J. Immunol. 22, 197-202 (1985)). In the C3a assay the monoclonal antibody 4SD17.3 was used for capture and biotinylated polyclonal rabbit anti-C3a and HRP-conjugated streptavidin (Amersham, Little Chalfort, UK) for detection. In the sC5b-9 assay, anti neoC9 monoclonal antibody aEl 1 (Diatec AS, Oslo, Norway) was used for capture and polyclonal rabbit anti-C5 antibodies and HRP-conjugated anti- rabbit immunoglobulin (Dako A/S, Glostrup, Denmark) for detection.

Zymosan-activated serum served as standard and values are given as ng/mL (C3a) or arbitrary units/ml (AU/mL; sC5b-9). Physical characterisation of polymer surfaces

Surface free energies of the polymer surfaces were determined by contact angle goniometry performed with a Kriiss G10 goniometer (Kruss Optronic, Hamburg, Germany) fitted with an enclosed thermostated cell and interfaced to image-capture software (Drop Shape Analysis 1.00.14.0, Kriiss Optronic, Hamburg, Germany). For advancing (0A) contact angle

experiments, measurements of air bubbles (2-8 μΕ) using the captive bubble technique were recorded at 20 °C using either doubly distilled water, diiodomethane (Fluka, >98%) or ethylene glycol (Sigma, >99%). Surfaces were equilibrated (2 x 50 min) in the solvent before contact angle experiment was performed and measurements were carried out with 4-9 droplets on a minimum of two independently prepared surfaces to obtain an average contact value.

Images of reference and imprinted polymers were acquired using an atomic force microscopy (AFM, TopoMetrix TMX2000 Discoverer

Scanning Probe Microscope, ThermoMicroscopes, UK) with a 70 x 70 x 12 μιη tripod piezoelectric scanner. Topography measurements were conducted using "V" shaped silicon nitride cantilevers bearing an integrated standard profile tip (length 200 μιη, nominal spring constant (K) 0.032 N m ^"1 ; Part. No. 1520-00, ThermoMicroscopes, Santa Clara, CA, USA). Topographic imaging was performed in air and aqueous buffer using a closed wet cell, modified to allow variable temperature adjustment. Contact mode imaging utilised an applied load and scan rate limited to ca. 1 nN and 3 Hz, respectively, to minimize compression and lateral damage to polymer grafts and underlying surfaces. Statistical treatments

Statistical significance was calculated with Student's i-test using GraphPad

Prism ^® 3.02 (GraphPad Software Inc., San Diego, CA, USA). Data are

presented as mean values ± SEM.

Table 3 - XPS analysis - elemental composition of polymers

Hydro Nitroge

Carbon -gen n Oxygen Sulfur Total

Expected analysis

(based on

DAP/MAA ratios) 61.3 7.2 13.8 17.6 100

Found (Non- 69.7 ± 20.5 ± 0.10 ±

imprinted) 0.3 N.D 9.0 ± 0.1 0.1 0.1 99.2

68.4 ± 22.1 ± 0.18 ±

Found (Imprint) 0.2 N.D 8.7 ± 0.1 0.1 0.2 99.2

#) Performed at Fraunhofer Institute for Interfacial Engineering and Biotechnology

Table 4 Advancing contact angles of water, diiodomethane (DIM) and ethylene glycol (EG) on polymer surfaces and surface free energies of these systems.

Polymer Contact angle, 0 ^a (°) Surface energy (mJ

¾0 DIM EG 7s ⁺ s

Ref 36 ± 52 ± 35 ± 33 ± 0.1 ± 54 ± 39 ±

3 2 2 1 0 3 1

MIP 34 ± 61 ± 42 ± 28 ± 0.1± 63± 34±

5 2 2 1 0 4 1

#) Each contact angle value is the mean of at least four drops on the minimum of two independently prepared polymer samples. ##) Differences between ys ^LW , 7s ^" and ys ^tot were all statistically significant (p < 0.05) as determined by t-Test (two-sample assuming unequal variances).

When introducing elements of the present invention or the

20 preferred embodiments (s) thereof, the articles "a", "an", "the" and "said" are intended to mean that there are one or more of the elements. The terms "comprising", "including" and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.

Results and Discussion

Full blood compatibility requires that the surface in contact activates neither coagulation nor complement cascades. While heparinization, which is always optimized for minimizing coagulation rather than

complement activation, ²¹ has been used for decades to produce surfaces with minimal activating character, alternatives not relying on the use of substances of biological origin are most desirable. We envisaged that a surface capable of recruiting exogenic heparin from a patients' own blood (at most 3 IU/mL)( Reber, D. et al. Heart Surg. Forum 11, E276-E280 (2008)) during an extracorporal procedure such as during coronary-pulmonary bypass surgery should have the possibility to be self-regulating thus diminishing the risk of thrombosis.

Molecular imprinting was perceived as a means to produce surfaces with such self regulatory function. However, to achieve imprinted polymer surfaces capable of recognizing heparin in its biologically active conformation necessitates a polymerization system amenable to use in aqueous media and capable of supporting monomer-template interactions while not compromising the three dimensional structure of the

macromolecular template. Polymers comprised of diacryloyl piperazine and methacrylic acid have previously been demonstrated to be suitable for imprinting in aqueous media (Piletsky, S. A., Andersson, H. S., Nicholls I. A. Macromolecules 32, 633-636 (1999)). However, the physical size of a macromolecular structure such as heparin prohibits its use in conjunction with established imprinting strategies (bulk polymerization) on account of the difficulties associated with the removal of the template from the polymer. To avoid this problem, and in order to have a material to which blood proteins have access suggested a surface imprinting strategy, Figure 1. Whitcombe and colleagues have described the preparation of quasi-2-dimensional surface imprints (a textured 2-D polymer surface) of inorganic materials (D'Souza, S. M. et al. Directed nucleation of calcite at a crystal-imprinted polymer surface. Nature 398, 312-316 (1999)), and a related approach has been adopted by the groups of Shie et al. and Hoshiri et al, J. Am. Chem. Soc. 130, 15242 (2008) for proteins and peptides. Preparation of template surfaces & Quantification of heparin bound to template surface

Template surfaces were prepared in a two-step process whereby- glass surfaces were derivatized with (3-aminopropyl)trimethoxysilane, then treated with a solution containing (3%, w/v) glutardialdehyde and heparin (7.5%, w/v). The surface concentration of heparin on the template surface was shown by toluidine staining to be 0.14 ± 0.01 g/cm 2 (12 ± 1 pmol/cm 2 ). The theoretical surface loading (close packing) of heparin (mean Mw 12 kDa) was estimated to 7 pmol/cm based on the heparin structure determined from NMR studies by Mulloy et al, Biochem. J. 293, 849-58 (1993) and the assumptions that all heparin molecules lie flat on the surface and that charge repulsion does not influence packing density. These data indicate a higher packing density which suggests that portions of the heparin structure are free in solution, i.e. that on average only a portion of each heparin molecule is physically bound to the derivatized surface, a process driven by repulsive coulombic interactions.

The conformation of immobilized heparin is critical for the recognition of the active pentasaccharide by AT. The quality of the template surface was therefore determined by the efficiency of which AT bound to the surface bound heparin. The AT binding capacity of the template surface was determined to be 4.4 ± 0.2 pmol/cm compared to for the CHS reference 2.8 ±

2

0.3 pmol/cm .

Stability of heparin bound to template surface:

The stability of the immobilised heparin was demonstrated by determining the presence of active heparin- AT complex, after incubation of the template surface with citrated plasma. The amount of heparin released into the plasma from a surface area of 2.8 cm ² was determined to be 0.017 ± 0.003 IU/mL (8.8 ± 1.5 pmol/cm ). This value, close to the detection limit of the assay, indicates that the template surface is as stable as the reference CHS, i.e. with no leakage of heparin into the blood. 25

Synthesis of heparin imprinted polymer surfaces

Heparin imprinted and reference polymers were synthesized according to the strategy summarized in Figure 1. The surfaces onto which the polymers were to be attached were first derivatized with (3- methacryloxypropyl)-trimethoxysilane. Solutions containing the functional monomer, methacrylic acid, i.e. the cross-linking agent, diacryloylpiperazine, were polymerized, between the template and (3-methacryloxypropyl)- trimethoxysilane derivatized surfaces to yield thin films of 140 μιη thickness. The two surfaces were subsequently separated revealing a thin film.

Reference polymers were prepared identically, though using a freshly cleaned glass surface as template. Although readily obtained, the yield of surfaces of suitable quality (size) for use in studies varied between the heparin imprinted (40%) and glass reference systems (75%). The polymers were brittle in the dry state which necessitated their storage in aqueous solution. X-ray

photoelectron spectroscopy studies (XPS, Table 3) confirmed the successful elution of template from the surface, as reflected in an S-content of <0.1 pM/mm ² .

Polymer heparin binding

Polymer binding characteristics were studied using a radioligand binding assay with H-heparin (Mw 6-20 kDa) in the concentration range from 0.75 to 75 μg/mL. The imprinted polymer showed an higher affinity for heparin relative to the reference polymer over a narrow concentration interval. The difference between reference and imprinted polymer was statistically significant at a heparin concentration of 7.5 μg/ml (i-test with unequal variances, p=0.009), where the reference surface bound 317 ± 19 fmol/mm ² and the selective binding was determined to 23 ± 12 fmol/mm .

Release of heparin from heparin incubated imprinted and control polymer surfaces

The polymer surfaces were tested for their ability to release heparin into plasma after saturation with heparin. Following incubation with 8 mg/mL, -1000-fold more than used for the radioligand binding studies, the plasma was analysed for heparin content as above. This experiment showed that the amount of heparin released from the imprinted polymer, 0.036 ± 0.008 IU/mL (18 ± 4 pmol/cm ), was lower than for the reference polymer, 0.060 ± 0.020 IU/mL (31 ± 10 pmol/cm ² ), indicating that heparin is bound more strongly to the imprinted surface than to the reference. This

concentration can be compared to the patient blood concentration normally employed during bypass surgery (up to 3 IU/mL). ²³ Moreover, since the amount of heparin released from the surfaces is very low, the effect seen on C3a and sC5b-9 measurements (see later) can not be a result of heparin leaking since no effect on complement activation is seen at concentrations of heparin below 0.5 IU/mL. ²⁶ This is further supported by the results of the XPS studies described above. Surface contact induced platelet consumption

Imprinted and control polymer surfaces as well as unmodified glass surfaces, after saturation with soluble heparin followed by incubation with 0.9 % NaCl or incubated with NaCl only, were tested in the blood chamber model described above. The platelet loss was significantly lower for all polymer surfaces compared to the unmodified glass (Figure 2A). There were no differences between heparin imprinted or control polymer surfaces (61 ± 12% and 57 ±13%, respectively). Preincubation of the heparin imprinted surface with soluble heparin did not affect the decline in platelet counts (44 ± 11%). In contrast, there was a tendency, although not statistically significant, to lower platelet consumption in the blood which had been in contact with control polymers that had been preincubated with soluble heparin (30 ± 9%). This apparent effect can be accredited to the greater amount of heparin released (see above) from the reference polymer surface compared to the heparin imprinted surface. All platelets were consumed after contact with glass, regardless of whether or not the glass had been preincubated with soluble heparin.

Complement activation upon contact between imprint surfaces and whole blood

The activation of complement by glass is significant, and provides a meaningful reference for comparison with treated glass surfaces. The diacryloylpiperazine-methacrylic acid co-polymer used in this study was found to be as potent as unmodified glass with respect to complement activation. This high complement activating capacity was efficiently overcome by the heparin imprinting process (Figure 2 B, C). Neither the levels of generated C3a nor of sC5b-9 complexes were affected by

preincubation with soluble heparin. In absolute numbers, 680 ± 115 ng/mL of C3a was generated in blood after contact with imprinted surfaces without heparin contact, and 660 ± 125 ng/mL with heparin imprints that have been preincubated with heparin (Figure 2B). The corresponding values for reference polymers are 1350 ± 190 ng/mL, and 1480 ± 260 ng/mL,

respectively. Levels of 1200 ± 260 ng/mL were detected in samples which had been in contact with glass, and no effect was seen upon preincubation with heparin (data not shown). Similar observations but with even greater differences between heparin imprinted and reference polymer surfaces were seen when the end product of the complement cascade, sC5b-9, was measured (Figure 2C). The heparin imprinted surfaces induced the formation of 77 ± 19 AU of sC5b-9 per mL before and 89 ± 24 AU/mL after heparin saturation. Corresponding values for the polymer reference were 188 ± 26 AU/mL and 211 ± 34 AU/mL, respectively. The values detected after glass contact were 190 ± 58 AU/mL and did not change when the glass had been preincubated with soluble heparin (data not shown). Importantly, despite the larger surface area of the imprinted polymers, as observed in AFM studies (Figure 2A & 2B), the inherent activation of the entire complement cascade by the MIP was significantly lower than for the corresponding reference polymers, as reflected by the observed levels of sC5b-9. Collectively, these data provide strong support for the presence of heparin selective sites in the molecularly

imprinted polymer film. These results suggest the use of molecularly imprinted polymer films as potential autoregulatory surface coatings for materials to be placed in contact with blood.

Unravelling the mechanisms underlying the unique behaviour of the imprinted polymer films when in contact with blood is challenging. As heparin is not an endogenous component of blood an important question arises, namely, to what component(s) present in blood can we attribute the observed differences in the hemocompatibility of the two surfaces? The main proteoglycan present in all blood cells consists of the protein serglycine, linked to multiple chondroitin sulfate chains, The concentration of chondroitin sulfate in plasma has recently been demonstrated to increase by 4 g/mL upon platelet activation. 28 We hypothesise that the interaction of the polymers with this complex endogenous proteo-glycosoaminoglycan could provide the basis for the differences in degree of complement activation of the two surfaces. Studies using pure chondroitin sulfate (the glycosoaminoglycan subunit) revealed no conclusive evidence of a role for this structure, which suggested that the intact glycosoaminoglycan structure with the capacity for multiple simultaneous interactions with surface is necessary.

To summarize the results thus far presented, the studies using whole blood demonstrate that the influence of the heparin template on the polymer resulted in a surface capable of attenuating complement activation, as determined through studying two separate points in this cascade system. Furthermore, as pre-incubation of surfaces with soluble heparin did not influence complement activation, it must be concluded that the specific characteristics of the heparin molecularly imprinted polymer surface are directly responsible for the biological activity of the surface. Importantly, the diacryloylpiperazine-methacrylic acid co-polymer has an inherently low thrombogenicity, which when coupled with the relatively low complement activating character induced by the molecular imprinting process renders a material with enhanced hemocompatibility. Moreover, we propose a role for the proteo-glycosoaminoglycan serglycine in the observed compatibility of the molecularly imprinted surfaces with the complement cascade.

Physical characterisation of the polymer

In view of the known activation of the coagulation cascade via contact of blood with synthetic surfaces, surfaces were investigated using Atomic Force Microscopy (AFM) in order to establish if there were any marked differences in topography and/or roughness of imprinted surfaces compared to controls, Figure 3. AFM imaging of the polymer films was conducted in water using contact mode. There were no significant differences in the overall topographies at the micron scale and both polymer films displayed features corresponding to interconnected agglomerates (-50-75 nm in diameter), spaced evenly across the surface. These agglomerates were indicative of the macroporous polymer structure formed by cross-linking in a porogenic solvent. Separation of the polymer aggregates was <10 nm. Further analysis indicated a slightly greater overall roughness in the case of the heparin imprinted polymer surface, than in the case of the reference polymer, with a difference in height over the areas studied of ± lnm for the imprinted surfaces than for the control polymer. Generally speaking, complement activation is proportional to blood accessible surface area. In this case, the imprinted polymer surface was less complement activating than the reference, despite a greater apparent surface area. Together with the binding and platelet and complement activation studies, this data provides further support for the role of the heparin template induced influence on polymer structure on the behaviour of the imprinted polymer when in contact with blood.

In an attempt to determine the influence of the effect of the template surface on the corresponding polymer at a higher level of chemical detail, the surface free energies of reference and imprinted materials was determined by contact angle goniometry using the captive bubble technique. The contact angles for water, diiodomethane and ethylene glycol as well as the surface free energy components were determined, Table 4. The advancing water contact angle, 0 _W , for the reference and imprinted surfaces were determined to be 35.7 ± 0.6 and 34.4 ± 0.9 degrees, respectively, which indicated that both surfaces were similar with respect to hydrophilic character. The hydrophilic nature of the material itself may explain the strong

complement activation seen when presented to blood in the absence of a heparin surface imprint.

Examination of the Lewis base and Lifschitz van der Waals components revealed noticeable differences between reference and imprinted polymers, suggesting a higher electron pair density at the imprinted surface. This may be explained by an enrichment of methacrylic acid residues

(reported as carboxylates during contact angle measurements at pH 7) ¹ at the imprinted surface through prior binding to polar and hydrogen bonding sites on the heparin chain during the templated polymerisation. Similarly, the localised higher polarity of the imprinted surface was confirmed through the reduced van der Waals' component compared to the non-imprinted. The low magnitude of the Lewis acid components for both surfaces suggested that residual non-ionised carboxylic acid functionality contributed little to the measured total surface energy during contact angle analysis. The total surface free energy of the polymer films was evaluated to be 39 mJ/m for the reference and 34 mJ/m for the imprinted surface which is similar to those obtained for other polyacrylamides. The indication of functionalities complementary to the template, and especially their surface enrichment, provides further support for the presence of sites with selectivity for an active form of heparin or similar sulphated polysaccharides like serglycine on the surface of the imprinted polymers.

¹ The methacrylic acid residues were likely to have been non-ionised during the initial imprinting, but would have been converted to carboxylates during contact angle studies in distilled water: the Lewis base content thus reports the relative surface content of these residues.