TECHNICAL FIELD

The present invention relates to solid substrates, the surfaces of which have been modified to introduce re¬ active groups of a hydrophilic nature thereon. The inven¬ tion also covers a method for preparing such solid sub¬ strates.

BACKGROUND OF THE INVENTION

In medicinal technology, such as technology involvin medicinal equipment such as devices for implantation or apparatus exposed to living tissue, it is often desirable to make the exposed surfaces compatible with the environ¬ ment. This is often done by immobilizing biologically active compounds onto the exposed surfaces. This is usually made in two steps, viz. a first step of activatin the surface in question and a second step of coupling the biologically active compound to the activated surface.

In the first step residing in activation which fre¬ quently takes place by the treatment using a polymeric compound, the introduced reactive and functional groups should be attached to the substrate by strong binding thereto. In the second step residing in the coupling the binding shall also be as strong as possible. However, the biological activity of the immobilized substance must not be impaired.

As examples of techniques for the immobilization of biologically active compound onto the surface of for ex¬ ample hospital equipment are the immobilization of gly- coseaminoglycans (GAGs) on intraocular eye lenses, certai wound dressings, orthopedic implants etc. Such immobili¬ zation of GAGs is usually performed in two steps, namely pretreatment of the surface to make it more reactive and/or hydrophilic, and immobilization of the molecule by ionic or covalent binding. In such pretreatment procedure

a reagent or primer containing reactive amino functions i adhered to the surface. This reagent can be further sta¬ bilized by the addition of a crosslinking agent, usually by a functional organic substance.

BACKGROUND ART

The primers and crosslinkers hitherto used are usual ly prepared from materials of a non-biological origin and are of a non-biodegradable type. Examples of reagents are polyethylen imine and tridodecylmethylammonium chloride. Further details on this immobilization techniques are found in Hoffman J., Larm 0. and Sholander S., A new meth od for covalent coupling of heparin and other glycosamino glycans to substances containing primary amino groups, Carbohydrate Research (1983) 117, 328; Larm 0., Larsson R and Olsson P., A new non-thrombogenic surface prepared by selective covalent binding of heparin via a modified reducing terminal residue, Biomaterials, Medical Devices and Artificial Organs (1983) 11, 161. The present invention has for a main object to pro¬ vide new techniques for modifying surfaces to enable the introduction of reactive groups of a hydrophilic nature o such surfaces.

Another object of the invention is to provide new techniques for performing such modification using substan ces that are of a biological origin and also are of a bio degradable type.

Yet another object of the invention is to introduce reactive amino and/or hydroxyl groups suitable for cova- lent binding of biologically active substances to the sur faces involved.

Still another object of the invention is to provide new techniques enabling modification of surfaces to make said surfaces more hydrophilic by the introduction of functional groups that can be used for covalent coupling of biologically active substances to such surfaces.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides a solid substrate, the surface of which has been modified to in¬ troduce reactive groups of a hydrophilic nature thereon. The modification of the surface is provided by a primer comprising a first polysaccharide containing as reactive groups amino and hydroxyl groups. Thus, it has been sur¬ prisingly found that such first polysaccharide can be ef¬ fectively attached to the surface of solid substrates, an a particularly preferred embodiment of the invention is constituted by said first polysaccharide being chitosan.

Chitosan consists of 1,4-β-bound D-glucosamine units The polysaccharide is linear and the qualities differ wit regard to the degree of N-acylation. In nature all amino groups are acetylated and the polysaccharide is then ter¬ med chitin. It is mainly obtained from shells of crabs an shrimps.

It is preferred that chitosan has a degree of N-aσy- lation of at most about 90%. A preferred degree of N-acy- lation is at most about 50% and preferably less than abou 25%.

According to a preferred aspect of the invention sai first polysaccharide has been stabilized by crosslinking using a periodateoxidated second polysaccharide having vi cinal hydroxyl groups or amino and hydroxyl groups in a vicinal position, said polysaccharide having been subjec¬ ted to a periodate oxidation to form at least one pair of dialdehyde functions.

It is preferred that said second polysaccharide used for stabilization by crosslinking is selected from poly- saccharides the biodegradation products of which are non- toxic, such as D-glucose amine and D-glucose. It is par¬ ticularly preferred that said second polysaccharide is selected from the group comprising chitosan, amylose and glycosaminoglycans.

The substrates having modified surfaces in accordanc with the present invention are usually of a hydrophobic o inert nature, and can be selected from the group compri¬ sing polyolefins, polyurethanes, polyvinyl chloride, poly styren, silicone and polytetrafluoroethylene or from the group comprising medicinally acceptable metals and glass. Among the polymeric materials polyolefins are preferred, such as polyethylene or polypropylene.

The invention also provides for a process for the preparation of a solid substrate, the surface of which ha been modified to introduce reactive groups of a hydrophi¬ lic nature thereon. Said process involves the following steps: a) providing a substrate, the surface of which is to be modified; b) preparing a solution of said first polysaccharide; c) coating said surface with the solution resulting from step b); and d) providing precipitation of said first polysaccha- ride on said surface resulting in modification of its pro perties.

In such process it is preferred to use as said first polysaccharide a chitosan.

According to a preferred embodiment of the process o the invention the solution prepared in step b) above is supplemented with a second polysaccharide having vicinal hydroxyl groups or amino and hydroxyl groups in a vicinal position, and said second polysaccharide has been subjec¬ ted to a periodate oxidation to form at least one pair of aldehyde functions. The function of said second poly¬ saccharide is to stabilize said first polysaccharide by crosslinking.

It is preferred that said second polysaccharide is selected from polysaccharides the biodegradation products of which are non-toxic, such as D-glucosamine and D- glucose. Among particularly preferred polysaccharides con¬ stituting said second polysaccharide are chitosan, amylos

and glycoseaminoglycans.

The present invention will now be further illustrate by non-limiting examples with reference to the appended drawings, wherein: Figures 1 and 2 illustrate by chemical formulae a covalent coupling of carbonic anhydrase onto a surface containing primary amino groups; and

Figure 3 is a schematic representation of conceivabl coupling reactions to obtain covalent coupling of a biolo gically active substance to a modified substrate surface.

Illustration of reactions resulting in covalent binding

Figure 3 illustrates a number of reactions that can be used in association with the present invention in orde to covalently bind biologically active substances to sub¬ strates containing amino and/or hydroxyl groups. The il¬ lustrated reactions are to be construed as examples only and are not intended to restrict the scope of the inven¬ tion. In Figure 3 illustrating covalent coupling to amino or hydroxyl groups, EDC is a water-soluble carbodiimide, and Z equals 0 or NR, R, R' and R' ' are organic groups, optionally immobilized.

In regard to substrates constituted by polymers of a hydrophobic nature, such as polyethylene and polypropy- lene, it is preferred to etch the surface before applying the first polysaccharide. Such etching can be performed using an oxidizing agent in acid solution, such as potas¬ sium permanganate in sulfuric acid. Such etching improves adherence of the first polysaccharide, whether stabilized by crosslinking or not.

In the non-limiting examples below percentages and parts refer to weight unless otherwise indicated.

EXAMPLE 1

Etching of polyethylene surfaces

Polyethylene film or tubing is incubated for 2 min, at room temperature with a solution of 2% potassium per- manganate (KMnO. ) (w/v) in concentrated sulfuric acid H ₂ S0. and carefully rinsed with distilled water.

EXAMPLE 2

Periodate oxidation of chitosan In a typical example when about 10% of the mono- saccharide residues are oxidized the polysaccharide chitosan containing 15% N-acetyl groups is dissolved in water (100 ml) and sodium periodate (0.5 g) were added.

The solution is kept in the dark at room temperature for 24 hours. The reaction mixture is then dialysed against distilled water and freeze dried to give 4.1 g chitosan containing dialdehyde functions.

EXAMPLE 3 Example 2 is repeated using amylose instead of chito¬ san. Similar results are obtained.

EXAMPLE 4

Example 2 is repeated using hyaluronic acid instead of chitosan. Similar results are obtained.

EXAMPLE 5

Amination with chitosan and crosslinking

The surface resulting from Example 1 is treated at room temperature with a solution of chitosan containing 15% N-acetyl groups (0.25% w/v) in water, together with a crosslinking agent (0.015% w/v) prepared as described in Example 2 above. The surface is carefully rinsed with ethanol (80%) and then stabilized by reaction for 2 hours at 50°C with sodium cyanoborohydride (0.00025% w/v in 0.15 M NaCl, pH 3.9). The surface is rinsed with water and treated with a solution of dextran sulphate (Pharmacia AB,

Uppsala Sweden) 0.1 g/L in 0.15 M NaCl, pH 3.0 for 10 min at 55°C. After rinsing with large volumes of distilled water, the surface is treated with a solution of 0.25% chitosan in aqueous solution at pH 9.0, 10 min at room temperature and washed as above. The presence of amino - groups is verified with an indicator (ponceau S, Sigma).

EXAMPLE 6

Example 5 is repeated but using the crosslinking agent of Example 3.

EXAMPLE 7

Example 5 is repeated but using the crosslinking agent of Example 4.

EXAMPLE 8

Amination with chitosan only.

The surface resulting from Example 1 is treated at room temperature with a solution of chitosan containing 15% N-acetyl groups (0.25% w/v) in water.

EXAMPLE 9

Covalent coupling of biologically active substance to modified surface Polyethylene beads are etched as in Example 1 above and aminated with crosslinking as in Example 5 above. The aminated beads are further activated by treatment with a solution of borate buffer (50 ml, pH 9.0), ethanol (10 ml and chloroacetaldehyde dimethylacetal (1 ml). To the clea solution another 40 ml of borate buffer are added and the suspension is stirred overnight at room temperature. The granulate is hydrolyzed in aqueous HC1 (100 ml, 0.05 M) for 25 minutes at 70°C. The reactions involved so far are illustrated in appended Figure 1. After washing with large volumes of water carbonic anhydrase (CA) originating from bovine erythrocytes (Sigma) is coupled by reductive amination. The granulate

is stirred in an aqueous solution (200 ml, pH 6.1) con¬ taining CA (128 mg) and NaBHgCN (40 mg) at room tempera¬ ture for 24 hours. This reaction is illustrated in appen¬ ded Figure 2. After washing with water and drying the coupling yield is measured colorimetrically with respect to the ability for the immobilized enzyme to hydrolyse

2 p-Nitrofenylacetate. The coupling yield is 5 μg/cm .

EXAMPLE 10

Carbodiimide coupling of hyaluronic acid.

Polyethylene film is etched as in Example 1 and ami¬ nated as in Example 5 and treated with a solution of hya¬ luronic acid (Pharmacia, 0.195 mg in 100 mL water). A so- lution of l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, Merck, 0.5 g in 1.0 mL water) is adde gradually during 0.5 h. After adjustment of pH to 4.75 (0.1 M HC1) the reaction is allowed to proceed over night.

The films are carefully rinsed with large volumes of ultr

2 pure water and dried. The coupling yield is 1.8 μg/cm as determined by FTIR.

EXAMPLE 11

Immobilisation of carbonic anhydrase (CA) by carbodiimide coupling

Polyethylene beads (3 mm i diameter, 40 ml) are etched as in Example 1 and aminated as in Example 5. The beads are suspended in an aqueous solution of CA (100 ml, pH 5.5 adjusted with M HC1). EDC (2 g in 5 mL water, see Example 8) is added gradually to the stirred suspension. The pH-value is maintained at 5.5 for 24 h at room tempe¬ rature. The beads are washed with large volumes of NaCl (5

L) and analysed as in Example 9. The coupling yield is 2

EXAMPLE 12

Carbodiimide coupling of heparansulphate

Polyethylene is etched as in Example 1 above and treated with heparansulphate (35 mg in 50 mL of water). The coupling procedure is performed as described in

Example 10 with the modification that 0.2 g EDC in 1.0 mL of water is used in the coupling procedure. The coupling yield is determined semiquantatively with toluidine blue which gives a lilac colour and quantatively with FTIR (1.6

2 μg/cm ).