GLUTATHIONE MODIFIED SURFACES

Abstract: The invention relates to a novel thiol modified substrate or article with a noble metal or polymer surface on to which glutathione has been immobilized, said novel product possessing an outstanding combination of biological activities making it possible for e.g., blood contact uses, such as an implant. The product is preferably prepared by chemisorption of the glutathione from an aqueous solution thereof.

Description: GLUTATHIONE MODIFIED SURFACES Technical field The present invention is within the field of sub¬ strates having at least a surface layer modified by an immobilized thiol compound. More specifically it relates to the immobilization of surface layers by a specific novel thiol compound which has unexpectedly been shown to possess outstanding biological activities as compared to previously known immobilized thiol compounds. The inven- tlon also relates to some specific favourable uses of said substrate, said uses being made possible by the new acti¬ vities found. Background of the invention Gold surfaces modified by immobilized thiol compounds are previously known per se. Generally such modified sur¬ faces have appeared to be of interest as model systems for the study of protein-surface interactions. It has also been found by the inventors that the thiol-gold immobili- zation technique is a reliable technique for studies of coagulation and complement activation under well control¬ led conditions. Thus, the surface modification technique offers possibilites to separate the influence of different functionalities from those of physical factors, such as surface mobility (tail group) and morphology (surface roughness). As an example of prior art within this field refe¬ rence can be made to P. Tengvall, M. Lestelius, I. undstrϋm and B. Liedberg "Plasma Protein and Antisera Interactions with L-cysteine and 3-Mercaptopropionic Acid Monolayers on Gold Surfaces", Langmuir, 8, 1236-1238 (1992). In this recent study it was shown that short and fairly simple thiols, like MPA (HSCH 2 CH 2 COOH) and L-cys (HSCH 2 CH(NH 3 + )C0 2 " ), immobilized onto gold, bound diffe- rent antibodies after incubation in human plasma. Both surfaces are hydrophilic but with different net charges and chemical compositions. Polymer surfaces modified with immobilized molecules are of course also previously known. General description of the invention According to the present invention it has unexpec¬ tedly appeared that a specific thiol that is more complex as to chemical structure than those fairly simple thiols which have hitherto been investigated for protein-surface interactions, viz. glutathione, shows outstanding proper- ties when immobilized onto a noble metal surface. More specifically it has been found that said immobilized mole¬ cule is essentially completely inactive both with referen¬ ce to coagulation activity and with reference to comple¬ ment activity. Thus, although some molecules have pre- viously been found to possess a low coagulation activity, they have in fact not been useful for medical purposes in practice due to a pronounced complement activity, i.e. they activate the complement system of the body and pro- voce the cell injurious effects thereof. That is, gene- rally they cause inflammatory reactions. In other words the new immobilized system according to the present invention shows a unique combination of non-adverse activities which makes it possible for a large number of medical applications, especially where blood contact is involved, particularly for short time blood contacts, such as up to about 2 hours. For instance this means that the new system should be a competitor to the well known technique of heparinizing surfaces to be used for different medical purposes. The fact that it may well be a strong competitor to heparin, especially at short time contacts, is not only because of its outstanding bio¬ logical activities but also a result of the fact that the method of immobilizing glutathione onto a noble metal sur¬ face is extremely simple and reliable, as will be discus- sed more below. Thus, a first object of the invention is to provide a new modified substrate possessing a unique combination of biological activities which makes it possible for a large number of valuable medical applications. Another object of the invention is to provide a modi- fled substrate that can be manufactured or prepared in an extremely simple and reliable way. Still another object of the invention is to provide a modified substrate carrying an immobilized thiol compound that is non-synthetical, i.e. completely native to the hu¬ man or animal body, as said substance per se is a substan¬ ce found in blood and human and animal tissues. One additional object of the invention is to provide a modified substrate, the other part of which, i.e. in ad- dition to the thiol ingredient, is completely inactive or inert to its environment as it is of a noble metal. Still another object of the invention is to provide a very simple and reliable process for the preparation of the above-mentioned substrate. Other objects of the invention include the use of the modified substrate referred to in various medical articles and assays for different medical or diagnostic purposes. Furthermore, the immobilized new thiol compound should work similarly also on a polymer surface, which means that one additional object of the invention is to provide a modified substrate having at least a surface layer of a polymer, to which said thiol has been immobi¬ lized. Such a polymer article should be especially well suited as a disposable article. Other objects of the invention should be obvious to a person skilled in the art when reading the present de¬ scription or claims. More specifically, according to a first aspect of the invention, there is provided a substrate of the type ha- ving at least a surface layer modified by an immobilized thiol compound, the characteristic feature of said sub¬ strate being that said surface layer is a noble metal or polymer layer modified by immoblized glutathione. In connection with the invention the term noble metal is utilized in its common sense, i.e. generally a metal or alloy that is relatively superior in resistance to corro- sion or oxidation. Generally it can also be said that the term is an equivalent to precious metal. As such noble metals have previously been utilized to immobilize thiol compounds reference can also be made to the prior art concerning details of said metals. Prefer- ably, however, the noble metal is selected from the group consisting of gold, silver and platinum, most preferably gold. The term polymer generally relates to rather inert polymers, especially such which have been modified accor- ding to previously known techniques. Preferably said poly¬ mer is selected from the group consisting of polyethylene, polypropylene, polystyrene, polyurethane or terephtalates. The term substrate is used in a broad sense in con¬ nection with the invention. Thus, generally it means any part, substance, element, etc., which lies beneath and supports another, i.e. in this case it supports the gluta¬ thione. In other words the substrate may well be a part of a larger article, e.g. a medical article, or represent such an article per se. A person skilled in the art should have no problem in making a proper choice of said substra¬ te, neither as to configuration or shape, nor as to the nature thereof. In general the nature of the substrate is selected according to known principles, as this part of the invention also represents prior knowledge. Preferably, however, the substrate base material is selected from the group consisting of silicon, glass, polymer and metal. Theoretically the substrate might be of said noble metal in its entirety, but for obvious cost reasons this is not the case for larger articles. On the contrary integral articles made completely from the polymer referred to are possible for cost ri * :i3ors. Furthermore, it has been found that the invention works extremely well with a rather thin surface layer of said noble metal or polymer, preferably within the range of 50-500 nm. According to an especially preferable embo- diment of the invention the thickness of said surface layer is 100-300 nm, such as approximately 200 nm. As concerns the technique of accomplishing the noble metal surface layer a preferable embodiment of the inven¬ tion means that said noble metal layer has been sputter- deposited or physically vaporized onto the substrate re¬ ferred to. Such sputtering technique is well known and has been utilized per se in connection with immobilizing thiol compounds onto gold surfaces, and reference is therefore made to prior literature as to the preparation of said noble metal layers. How to provide a surface layer of the polymer should be well-known to a person skilled in the art and need not be described here. With reference to the thickness of the Immobilized glutathion layer it has been found in accordance with the present invention that a preferable range thereof is 5-25 A, preferably 5-13 A, e.g. about 10 A. Extremely good re¬ sults have been obtained when using a mono-layer of gluta¬ thione immobilized on said surface layer. According to still another preferable embodiment of the invention the substrate is a substrate or which the glutathione has been immobilized on to the surface layer by chemisorption from an aqueous solution thereof, which technique is especially preferable for said noble metal layer. Preferably the concentration of said aqueous solu¬ tion of glutathione is within the range of 0.1-10 mM. Otherwise the technique of immobilizing the glutathione molecule is similar to the techniques already used for immobilizing other thiol compounds onto gold surfaces. Therefore, further details concerning this part of the invention as well as the immobilization onto polymer sur¬ faces can be found in relevant literature. In addition thereto further details about the immobilization will of course be found in the more detailed description of the invention below. According to a second aspect of the invention there is also provided a process for the preparation of the sub¬ strate defined above. Said process, which is very simple and reliable and which may even be used by the final uti¬ lizer of the substrate, is characterized by starting from a substrate having a noble metal or polymer surface or alternatively sputter-depositing on a substrate a noble metal surface and modifying said surface with glutathione by chemisorption from an aqueous solution of said gluta¬ thione so as to immobilize the same thereupon. As referred to above the technique of sputter-depo- siting the noble metal onto a substrate is well known and such principles may be used here. Although further details can be found in the literature it may perhaps be added that to improve the adherence of the noble metal layer to the substrate a very thin layer of an adherence-promoting metal, such as chromium, could be used. Furthermore, the surface is preferably cleaned, for instance in distilled water, ammonium hydroxide and/or hydrogen peroxide, before immobilizing the glutathione. In addition thereto the sur¬ face is also rinsed, for instance in distilled water and stored therein until the chemisorption takes place. The chemisorption of the glutathione can also be per¬ formed according to known principles. For instance this means that it may well be performed at room temperature or at slightly elevated temperatures, which Is of course ex- tremely advantageous. After the chemisorption step the glutathione layer is preferably treated by sonication, which sonication is suitably performed after having rinsed the glutathione layer in water, such as distilled water. According to still another aspect of the invention there is also provided an article which comprises a sub¬ strate as defined above or as manufactured in the above- described way, for use in medical or diagnostic treatment of the human or animal body. In this context the term me¬ dical is to be understood in a very broad sense, i.e. the article referred to need not primarily be used for a the- rapeutic purpose but may well be utilized for applications like implants, surgical instruments and sensors. However, as referred to above, the article is useful for almost any application where coagulation and compelement activities are to be avoided, such as in all contacts with blood. For instance this means that the article is well suited for cell culture uses. However, other medical uses should be obvious to a person skilled in the art and need not be enumerated here. Thus, according to an additional aspect of the inven- tlon there is also provided a use of the substrate as de¬ fined above or as manufactured above, for the manufacture of any article to be used in blood contacting medical or diagnostic treatments or surgical treatment of the human or animal body. As mentioned above, this may for instance be a use of the substrate for the manufacture of an im¬ plant or a surgical instrument article. Examples The Invention will now be further described more speci ically by means of working examples where the im¬ mobilized glutathione according to the invention is pre¬ pared and compared to the previously known immobilized molecules 3-mercaptopropionic acid (MPA) and L-cysteine (L-cys). Before presenting the examples, however, the Figures referred to therein should be briefly commented on. Thus, In the accompanying figures the following is shown: Fig. 1 Suggested molecular structures of 3-mercapto- propionic acid, L-cysteine and glutathione respectively, at physiological pH (7.4) Fig. 2 FT-IRAS spectra of a) 3-mercaptopropionic acid, b) L-cysteine and c) glutathione monolayers on gold, formed at spontaneous pH (3.3, 5.3 and 3.4, respec¬ tively). Fig. 3 Kalllkrein formation and indirect quantification of F XII and F XIIa on surfaces and in solutions. The sodium glass surface was used as a highly activa¬ ting reference. Fig. 4 Anti-C3c depositions (15 min incubations) after in¬ cubation in human serum for 10 min, under room con¬ ditions. Fig. 5 Total plasma protein and antlsera adsorptions onto gold, gold-MPA, gold-L-cys and gold-GSH surfaces at room conditions. The surfaces were incubated in 10% citrated human plasma for 10 min and then in anti- sera for X5 min (for details, see Materials & Methods). In a) the horizontal dashed line indica¬ tes the over-all pasma level and all samples to the right of the vertical dashed line have been treated with amplification antlsera. In b) all samples to the right of the dashed line have been treated with amplification antlsera. Material and Methods Gold films, about 200 nm thick each, were sputter- deposited on glass substrates precoated with a thin layer of chromium - 1 nm. Scanning force microscopy (contact mode, 400 nm scanside) indicated that the surfaces were flat with a surface roughness * 6 nm (see Table 1). The gold surfaces were cleaned in five parts (v/v) distilled water, one part ammonium hydroxide (25%) and one part of hydrogen peroxide (30%) for 10 min at 80°C. After the cleaning procedure, the hydrophilic surfaces were rinsed and stored in distilled water for no more than 15 min before chemisorption of the thiol. Monolayers of glutathione (GSH), L-cysteine (L- cys) and 3-mercaptopropionic acid (MPA) were formed in aqueous solutions of 1 mM L-cys (pH - 5.3 ± 0.3) (from Sigma), 1 mM MPA (pH - 3.3 ± 0.3) (from Fluka) for 15 min, or in 2 mM GSH (pH - 3.4 ± 0.1) (from Sigma) for 1 hour, at room temperature (RT). After chemical modification the surfaces were rinsed in distilled water and finally soni¬ cated for 3 min before contact angle and ellipsometric measurements were made (for surface characterization, see Table 1). The molecular structure of the formed monolayers were analysed using Fourier Transform Infrared Reflection- Absorption Spectroscopy (FT-IRAS) in a dry state under mild vacuum. The analyses were performed using a BRUKER IFS 113 v Fourier transform spectrometer, equipped with a DTGS detector and a GIR (Grazing angle Infrared Reflec¬ tion) accessory aligned at 83°. All spectra were obtained at a spectral resolution of 4 cm by averaging 500 inter- ferograms. Prior to plasma incubations, one of six identically prepared surfaces was rinsed in distilled water, blown dry in nitrogen, and the complex refractive index of the sur¬ face was calculated from ellipsometric measurements in ten different points. The sample-surfaces were incubated for 10 min in 10% citrated human plasma in 0.01 M Phosphate Buffered Saline (PBS, 82 volume % 0.01 M Na 2 HP0 4 , 18 volume % 0.01 M KH 2 P0 4 and pH setting to 7.4 by NaOH/HCl), at RT. After rinsing in buffer they were immersed in antlsera at 1/20 or 1/50 dilutions for 15 min. Some samples were then transferred to the amplification antlsera (1/50 dilution, 15 min). After rinsing in distilled water and drying in nitrogen the amount of organic material was determined by elllpsometry at five points on each sample. Five samples for each antlsera were analysed. The equivalent organic layer thickness was calculated from the ellipsometric data using an isotropic three-phase model and a refractive index n - 1.465 for the organic layer. The maximum error of the calculated organic layer thicknesses was low, ± 0.3 nm. The error of the calculated thickness was determined using the formula for pooled standard deviations, the standard deviation was then multiplied with values from the Student's t-table (95%-level of confidence). The error bars in figures 4-5 thus represent the calculated error (from standard deviation) plus the estimated error for the ellipsometer (± 0.3 nm). The thicknesses of the L-cys, MPA and GSH layers were calculated from the changes in the op¬ tical properties of the gold surface after the chemical modification. The ellipsometer used was a Rudolph Research AutoEl III, equipped with a He-Ne laser working at 632.8 nm. Citrated plasma and serum were prepared from blood from apparently healthy donors and stored at -80°C until use. The IgG fraction-antisera used were Swine anti-Albu¬ min (SwaAlb), Swine anti-Immunoglobulin G (SwalgG) and Swine anti-Complement 3c (SwaC3c) from Orion Diagnostica, Finland; Rabbit anti-Fibrinogen (RbaFG), Rabbit anti- Fibronectin (RbaFN), Rabbit anti-c Macroglobulin (Rbac_ 2 M) and Rabbit anti-Complement 3c (RbaC3c) from Dakopatts a/s, Denmark; Goat andi-High Molecular Weight Kininogen (GtaHMWK), Goat anti-Factor XII (GtaFXII), Goat anti- Factor VIII (GtaFVIII), Goat anti-AntiThrombin III (GtaAThXII), Goat anti-Lipoprotein (GtaLp) and Goat anti- Prekallikrein (GtaPK) were all from Nordic Immunochemical Laboratories, the Netherlands. Human Serum C-Reactive Protein (CRP) Calibrator and Goat a CRP were from Dakopatts a/s, Denmark. The surface concentrations of low concentration plasma proteins (HMWK, LP, FXII, FVIII, ATh III and PK) were amplified with secondary antibodies: Rabbit anti-Swine Immunoglobulins (RbaSw) and Rabbit anti- Goat Immunoglobulins (RbaGt), both from Dakopatts a/s. Control tests showed only a small crossreactivity with plasma proteins, and the amplification antisera had low affinity for surfaces incubated in human plasma only. A small amount of human IgG was added to diluted amplifica¬ tion antisera to further suppress the crossreactivity, as recommended by the suppliers. Subsequently, the results should therefore be compared only for each protein and do not allow quantitative comparisons between different pro¬ teins on a specific surface. Complement factor deposition on solid surfaces can be detected using elllpsometry and antisera techniques. Here, detection of complement-activation, as Indicated by anti- C3c binding, was performed in the following manner. Sur¬ faces were incubated in 10% serum diluted in HANK's buffer (solution A:30 g NaCl, 4 g KC1, 2 g MgS0 4 * 7 H 2 0, 0.94 g CaCl 2 '2H-,0 and 500 ml distilled water; solution B: 0.6 g Na 2 HP0., 0.6 g KH 2 P0 4 , 10 g dextrose and 500 ml distilled water, A and B were mixed and the pH set with NaOH/HCl) at pH 7.4 for 10 min, rinsed in buffer followed by distilled water and blown drv. The adsorbed layer thickness was mea¬ sured in five points. The surfaces were again rinsed in buffer and incubated in an anti-C3c solution diluted 1/50 in HANK's, for ten minutes. Finally, the surfaces were re¬ moved and rinsed with buffer and distilled water, blown dry and the organic layer thickness was once more deter¬ mined in five points. Parallel to this, a control set of surfaces was incubated in heat deactivated 10% serum (56°C for 30 min before use) and antisera as above. The diffe¬ rence in the amount of deposited anti-C3c for the two series was Interpreted as a measure of the relative degree of complement activation. Surface bound kalllkrein and kalllkrein in solution were determined using soda watch-glass surfaces covered with (200 nm thick) gold films. The gold films were made by thermal evaporation on top of a thin layer (4 nm) of chromium. Pure glass, known to be a rapid contact active- tor, served as the reference material. The glass, gold and thiolated gold watch-glasses were cleaned like the other gold substrates and incubated in 10% citrated human plasma in PBS, pH 7.4, for 10 min. The surfaces were then rinsed 3 times in 0.05 M Trls buffer (25 ml Tris-(hydroxymethyl)- amino-methane, 42 ml 0.1 M HC1 and 100 ml distilled water) plus 0.15 M NaCl, at pH 7.5. The kalllkrein assays (de- scribed below) were made using normal citrated plasma, citrated factor XII deficient (F XII d f ) plasma from Sigma as the extra prekallikrein source, and Cephotest (cephalin, phosphatidylethanol-amine) from Nyco ed AB, Lidingβ, Sweden, was used to activate F XII. The kalli- krein-specific H-D-Pro-Phe-Arg-pNA'2HCl peptide S-2302 was from Chromogenlx AB, Mβlndal, Sweden. The principle of the test is kalllkrein cleavage of the S-2302 substrate. The end product (pNA) has a high absorbance at 405 nm and is quantified by sepctrophotometry. Peptide-pNA (S-2302) kallikreln - Peptide-OH+pNA (yellow, 405 nm). The kalllkrein assays were performed as follows: The surfaces were rinsed thoroughly with Trls buffer and incubated in 400 μl, 10% (PBS, pH 7.4) plasma for ten min. The rinsed surfaces were then tested for (the boxes indi¬ cate additions into the watch glasses after plasma treat¬ ment) total amount of surface bound Factor XII. 200 μl Cephottst 1 12210μl 20 μl Tris buffer 10 min 100 μl S-2302 tOmln cciittήric acid 10 μl FXπ da tpl_.mι (to stop reaction) aciivurri Factor XTI fr YH^ on the surface 220 μl Trls buffer 120μl 10 min 100 μl 30 μl FXπ dd pl_-ma S-2302 10 min citric acid V-lli-rt-in on _ι_ 5_*Hace 200 μl S-2302- 10 rain 200 l citric acid The plasma used in the incubations was tested for _____________ 10 ale 100 μl S-2302 lO mia 120μl citric a d 20μl plasma previously incubated on the surface J min 200μl tric add 200μl S-23C The assay solutions were transferred into a 1 ml polystyrene disposable cuvette, the volumes were doubled with Tris buffer, and the absorbance was measured at 405 nm. The errors presented in Figure 3 are maximum errors. The number of tests for each component varied from three to six for the various surface modifications. Results and Discussion Suggested molecular structures All three modified surfaces were hydrophilic with water contact angles smaller than 15". The MPA and L-cys are likely to give weakly negatively charged and zwitte- rionic surfaces, respectively, at physiological pH. Glu¬ tathione presents one negatively charged and one zwitte- rionic end, separated by a polypeptide-like backbone as shown in Figure 1. Surface characterization with FT-IRAS The reflection absorption (R-A) spectra of MPA, L-cys and GSH are shown in Fig. 2. There exist unfortunately no complete assignments of the spectra of these molecules in the literature. However, the peak positions of the strong¬ est bands and their tentative assignments are summarized in Table 2. All three R-A spectra lack the characteristic peak of the SH group at 2550 cm (spectral region not shown in Fig. 2), confirming that the binding to the gold surface occurs via the thiol group. The peak positions and general appearance of the R-A spectra for the L-cys and MPA monolayers correspond well with prior art. Looking at the spectrum of GSH, the most intensive peaks are those originating from the amide groups. The so- called Amide I peak (C-0 stretching) occurs at 1663 cm " and the Amide II and III peaks (C-N-H stretching and ben¬ ding) occur at 1549 and 1242 cm " , respectively. For the carboxyl groups (COOH) of GSH, the C-0 stretching mode at 1728 cm " , reveals the acid form. It should be emphasized, however, that all three monolayers investigated in this study are prepared at a significantly lower pH than em¬ ployed in the plasma protein adsorption experiments. The occurrence of charged and dipolar species (C0 2 and NH ) at physiological pH is therefore expected to be more frequent as indicated in Fig. 1. Kalllkrein assays In normal coagulation activated blood factor XII ft (or the Hageman factor) cleaves prekallikreln to form kalli- krein, which in turn activates high molecular weight kininogen (HMWK) and inactivate F XII, thus resulting in the intrinsic coagulation cascade. The relative amounts of total surface bound F XII, surface bound activated F XII (F XII.), surface bound kalllkrein, plasma F XII., and plasma kalllkrein for the various surfaces are shown in Fig. 3. The glass and MPA surfaces bound comparatively large amounts of F XI and F XII. and released kalllkrein into solution. L-cys and GSH showed low or undetectable kalllkrein formation in all the assays (Fig. 3), indicating a low surface F XII activity. This was expected for L-cys since contact activation of coagulation is known to preferen¬ tially take place on negatively charged surfaces and L-cys is suggested to be non-charged (zwitterionic) at pH 7 (Fig. 1). The low kalllkrein formation on GSH is, however, not yet understood, since significant amounts of C00- groups are expected to be present at pH 7. Gold exhibited large and significant amounts of sur¬ face bound F XII and kalllkrein, respectively, although the plasma levels appeared to be low. The low release of kalllkrein from the gold surface indicates a low F XII. formation in plasma. This in turn suggests that gold binds and activates F XII, but the F XIIa. and kalllkrein turn- over is low. The results may be due to plasma proteins being tightly bound to the surface. Control experiments for unspecific kalllkrein forma¬ tion with pure gold substrates showed that i) no kalllkrein was formed in any assay when using F χII dβf plasma instead of normal. This shows that F XII was a prerequisite for kalllkrein formation ii) kalllkrein was formed by using cephalin in the plasma after Incubation on λu, implying that the plasma was not depleted of F XII or kalllkrein. ill) a layer of low but still significant thickness of the S-2302 reagent was adsorbed, on top of the plasma protein layer on gold, as measured by elllpsometry. This demonstrates that the reagent was also surface active, iv) experiments performed with 1 or 20 min plasma incubations gave similar results. This indicates that the incubation time-chosen for the present set of experiments was not the cause for the obtained results. Another set of control experiments using glass sub¬ strates incubated in prekallikrein deficient plasma gave negative results for kalllkrein in plasma and positive when tested for surface bound F XII. Thus again, F XII was necessary for the colouring to occur. In summary, the con¬ trol tests showed that the pNA formation was correlated to both the presence of F XII and prekallikrein on surfaces or in plasma. Deposition of anti-C3c Elllpsometry was used to detect the increase in thickness of the organic layer after serum and subsequent anti-C3c incubations. The results are shown in Figure 4. L-cysteine surfaces incubated in normal serum showed a small thickening of the organic layer after incubation in the antibody solution. The results were similar when cit¬ rated plasma was used instead of serum. Antisera binding In Figures 5a and 5b the amounts (equivalent thick¬ nesses) of deposited organic material on the surfaces after plasma and antisera Incubations are summarized. In this context it should be noted that the observed amount of organic material in each case is the amount which re¬ mains after the rinsing and drying steps. Pure gold deposited significant quantities of a-IgG, a-FG, a-ATh III, a-PK and a-HMWK (Fig. 5a). The deposition of a-HMWK and a-PK suggests that the surface may be coagu¬ lation active, although no a-F XII bound onto the surface. The kalllkrein assays, however, revealed the presence of FXII on the surface (see Fig. 3) and that only low amounts of kalllkrein were released into the solution. The MPA surfaces bound relatively large-amounts of a- HMWK (- -^plification antisera) and significant quantities of a-F XII, a-ATh III and a-PK (Fig. 5b). The low overall a-FG binding onto the--investigated hydrophilic thiol modi¬ fied surfaces and the large a-HMWK binding onto the (nega- tively charged).;MPA surface (-C00-), are not surprising. The anti-HMWK followed an expected binding pattern of HMWK, i.e. the histidlne rich positively charged portion of HMWK may be deposited onto the negative MPA surface, perhaps physisorbed via electrostatic interaction. The kalllkrein assays (Fig. 3) also showed elevated F XII. and kalllkrein formation on and outside the MPA surfaces. The combined antisera binding and kalllkrein assay results suggest that MPA is clot promoting, when immobilized onto gold. The L-cys surfaces bound significant amounts of a- IgG, a-ATh III, a-LP and a-PK and relatively low amounts of a-FG and a-HMWK after the plasma incubations. The sig¬ nificant a-IgG deposition (see Fig. 5b) could be related to the increased a-C3c binding after exposure to human serum (Fig. 4). The glutathione surfaces did not deposit detectable amounts of any of the antisera tested for. The thickness of the total adsorbed protein layer after plasma incuba¬ tions and rinsings was less than 1 nm. This suggests that GSH, when chemisorbed onto gold, presents a surface with advantageous properties for blood contact applications. Another intriguing observation in Figures 4 and 5, was the large difference in the adsorbed amounts of plasma and serum protein onto the modified surfaces. This diffe- rence was particularly large for GSH. Control tests with heparinized plasma in PBS buffer and citrated plasma in citrate buffer, showed no elevated depositions of organic material on GSH. Thus, in summary it can especially be seen (Fig. 5a- b) that pure gold (Au) and MPA bind large amounts of a- HMWK, indicating contact activation of coagulation. L- cysteine shows increased binding of a-IgG, a-ATh III and a-Lipoprotein. This combined with the results of Figure 4 (showing increased a-C3c binding onto serum incubated L- cysteine surfaces) indicate that L-cysteine imparts some activation to the complement system but not the coagula¬ tion. On the contrary, glutathione does not activate any of these systems. Finally, preliminary results from vital microscopy experiments on rats confirm that GSH-surfaces have a pro¬ nounced low activation of the inflammatory system. Thus, it has been observed that platelets from body fluids were associated to the GSH-surfaces but were not activated during the test period. Table 1. Physical surface characterization Au Au+MPA Au+L ys Au+GSH Static water contact angle <20 14±3 5±3 7±2 (degrees) Refractive index at (0.16±Q.02) (0.18±0.02) (0.18±Q.02) (0.19±0.04) λ=632.8 nm *i(3.63±0.02) +i(3.5 ±0.02) +i(3.5 ±0.02) +i(3.51±0.03) Layer thickness (nm) 0.6 *0.2 0.6 ±0.2 1.3 ±0.2 Surface roughness (nm) <6 <6 <6 <7 (rms-valuc) The errors given as m ucimurn errors Table 2. FT-IRAS cha actcrization of MPA, L-cys and GSH on gold. Peak positions are given in vavenumbers (cπr*). MPA L-cys GSH Assignment 1720 1714 1647 1514 1408 1421 1396 1329 1242 Amide EH 1223 1144 1053 974 Q.Ho_ (faeing) v_/__ : syznmetric asynτ αsαic siretching mode δj ts : syπxmetric asy : netric def oπn__ion mode δaς : scissoring mode opb : out-of-plane bend

Claims: CLAIMS 1. A substrate having at least a surface layer modi¬ fied by an immobilized thiol compound, characterized in that said surface layer is a noble metal or polymer layer modified by immobilized glutathione. 2. A substrate according to claim 1, characterized in that the noble metal is selected from the group consisting of gold, silver and platinum. 3. A substrate according to claim 2, characterized in that the noble metal is gold. 4. A substrate according to claim 1, characterized in that the polymer is selected from the group consisting of a polyethylene, polypropylene, polystyrene, polyurethane or terephtalate polymer. 5. A substrate according to any one of the preceding claims, characterized in that said surface layer is a 50 - 500 nm thick layer on--another substrate, preferably selec¬ ted from the group consisting of silicon, glass, polymer and metal. 6. A substrate according to claim 5, characterized in that said surface layer is 100-300 nm thick. 7. A substrate according to any one of claims 5 and 6, characterized in that said noble metal layer has been sputter- or vapourdeposited on said substrate. 8. A substrate according to any one of the preceding claims, characterized In that the thickness of the immobi¬ lized glutathione layer is within the range of 5-25 A, preferably 5-13 A. 9. A substrate according to any one of the preceding claims, characterized in that said glutathione is present as a monolayer immobilized on said surface layer. 10. A substrate according to any one of the preceding claims, characterized in that the glutathione has been immobilized on said surface layer by chemisorption from an aqueous solution thereof. 11. A substrate according to claim 10, characterized in that the concentration of said aqueous solution of glutathione is 0.1-10 mM. 12. A process for the preparation of a substrate having at least a surface layer modified by an immobilized glutathione layer as defined in any one of claims 1-11, characterized by starting from a substrate having a noble metal or polymer surface or alternatively sputter-deposi¬ ting on a substrate a noble metal surface and modifying said surface layer with glutathione by chemisorption from an aqueous solution of said glutathione so as to immobi¬ lize the same thereupon. 13. A process according to claim 12, characterized by sonication of the chemisorbed glutathione layer, preferab- ly after rinsing thereof in water. 14. An article comprising a substrate as defined in any one of claims 1-11 or manufactured as defined in any one of claims 12-13, for use in medical or diagnostic treatment of the human or animal body, especially for blood contact treatments thereof. 15. An article according to claim 14, characterized in that it is selected from the group consisting of im¬ plants, surgical instruments and sensors. 16. An article according to claim 14, characterized in that it is intended for cell culture uses. 17. Use of a substrate as defined in any one of claims 1-11 or manufactured as defined in any one of claims 12-13 for the manufacture of an article to be used in blood contacting medical or diagnostic treatments or surgical treatment of the human or animal body. 18. Use according to claim 17 for the manufacture of an implant or surgical instrument article.

Inventors: LESTELIUS MAGNUS, LUNDSTROEM INGEMAR, NYGREN HAAKAN, TENGVALL PENTTI, LESTELIUS, MAGNUS, LUNDSTROEM, INGEMAR, NYGREN, HAAKAN, TENGVALL, PENTTI
Applicants: FORSKARPATENT I LINKOEPING AB, LESTELIUS MAGNUS, LUNDSTROEM INGEMAR, NYGREN HAAKAN, TENGVALL PENTTI, LESTELIUS, MAGNUS, LUNDSTROEM, INGEMAR, NYGREN, HAAKAN, TENGVALL, PENTTI
Application year: 1995
Main classification: G01N33
IPC codes: A61L33, G01N33