EP3038665A1

EP3038665A1 - Method for improving the biocompatibility of a surface

Info

Publication number: EP3038665A1
Application number: EP14758341.3A
Authority: EP
Inventors: Andreas Bollmann; Klaus LÜCKE; Fritz Scholz; Katja VAHL; Robert SMAIL; Ulrich Hasse; Heike Kahlert
Original assignee: Gilupi GmbH
Current assignee: Gilupi GmbH
Priority date: 2013-08-27
Filing date: 2014-08-27
Publication date: 2016-07-06
Also published as: CN105744963A; MX2016002584A; WO2015028503A1; US20160208389A1; RU2016109458A; AU2014314261A1; JP2016529015A; DE102013217085A1

Abstract

The present invention relates to a method for improving the biocompatibility of a surface (1), in particular the surface of a solid, and to a device (3), for example an implant, sensor or cell culture vessel, which is brought into contact with biological systems via a biocompatible surface. In order to improve the biocompatibility of surfaces, in particular with respect to cell cultures and tissues, in a simple way and for a large number of different surfaces, provision is made, according to the invention, for the surface (1) to be brought into contact with reactive radicals (2). The device (3) according to the invention has a biocompatible surface (1) that has been treated by the method according to the invention.

Description

Method for improving the biocompatibility of a surface

The present invention relates to a method for improving the biocompatibility of a surface, in particular a solid surface.

The invention further relates to a device, for example an implant, a sensor or a cell culture vessel, which is brought into contact with biological systems, with a biocompatible surface.

Materials that come into contact with biological systems must have high biocompatibility, i. (I) the materials must not have any deleterious effect on the biological system; and (ii) the biological environment must not undergo changes in material, e.g. Cause corrosion, biodegradation, etc.

To increase the biocompatibility of materials, there are a variety of mechanical, chemical and physical methods for modifying surfaces. By mechanical modification (eg, by polishing or grinding), certain surface topographies or roughnesses are to be obtained, impurities of the surface to be removed, and the adhesion properties for subsequent attachment of molecules to be improved (I. Milinkovic, R. Rudolf, KT Raic, Z. Aleksic V. Lazic, A. Todorovic, D. Stamenkovic, Materiali in tehnologije / Materials and Technology 46 (2012) 251-256).

Surfaces can be chemically modified by direct reactions with specific reagents, by covalent attachment of molecules to the surface, by plasma-based techniques, such as. Plasma assisted etching, deposition or polymerization, and plasma immersion ion implantation (P.K. Chu, J.Y. Chen, L.P. Wang, N. Huang, Mater, Sci. Eng., R 36 (2002) 143-206).

Liu, Chu, and Ding review various possibilities of surface modification of titanium and titanium alloys for biomedical applications (X. Liu, PK. Chu, C. Ding, Mater. Sei. Eng., R 47 (2004) 49-121). Chemically, titanium surfaces can be treated with acids or alkalis. The chemical modifications also include sol-gel coatings, anodic oxidation, chemical vapor deposition, and biochemical modifications. Physically, titanium surfaces can be modified by thermal spraying (eg, flame spraying or plasma spraying), by physical vapor deposition, or by ion implantation and deposition. Electrochemically, the biocompatibility of titanium surfaces with the help of anodic oxidation and increased by electrophoretic or cathodic deposition of hydroxyapatite (K.-H. Kim, N. Ramaswamy, Dent. Mater. J. 28 (2009) 20-36).

Various physical and chemical methods are also described for the modification of polymer surfaces (F. Abbasi, H. Mirzadeh, A.-A. Katbab, Polym. Int 50 (2001) 1279-1287). The most common physical methods for surface modification of silicone polymers are plasma and laser treatments as well as corona discharges. Chemically, the surfaces of silicone polymers can be modified by etching, oxidation, hydrolysis, functionalization and surface grafting.

In all these methods, the properties of surfaces can be changed to increase biocompatibility in various respects.

The present invention has for its object to provide a method for the treatment of surfaces, which allows to improve the biocompatibility of the surface, in particular to cell cultures and tissues, in a simple manner, and that for a variety of different surfaces, in particular solid surfaces, can be used ,

The above object is achieved according to the invention by treating the surface with at least one species of reactive radical. The device mentioned at the beginning solves this problem by treating its biocompatible surface with a method of the present invention.

According to the invention, it has surprisingly been found that a surface treatment with at least one species of reactive radicals detoxifies the surface and thus improves the biocompatibility of the surface. In contrast to the existing methods, the surface is detoxified by the reactive radicals, which increases their biocompatibility with biological systems without, for example, applying additional layers to the surface.

Another advantage is that the radicals can be generated in very different ways and thus the method can be adapted to a wide variety of material requirements. If the biocompatibility of, for example, heat-sensitive surfaces is to be improved, the radicals can be produced at room temperature, for example by the Fenton reaction. If the surface is to be treated as free of chemicals as possible, eg photolysis or radiolysis can be used to generate radicals. A "reactive radical" is an atom or molecule with at least one unpaired electron that is reactive, and reactive radicals usually react very quickly, often in less than a second. "At least one species of reactive radical" includes both versions where the surface is treated with only one type of radical (radical atom, radical ion, radical molecule, or radial molecular ion) as well as those in which different types of radicals come into contact with the surface. An "improvement in biocompatibility" in the context of the present invention is manifested by a detoxification of the surface, ie the surface treated according to the invention is less cytotoxic, that is less damaging to the cell and / or tissue, compared to an untreated surface which is not reactive with free radicals The improved biocompatibility can be determined by a cytotoxicity test in which the untreated surface and once the reactive radical treated surface is contacted with a cell culture and then the cell vitality in the solution is determined the cell vitality can be increased by at least 10%, preferably by at least 25%, and more preferably by 50-100%.

The solution according to the invention can be further improved by various configurations which are advantageous in each case and can be combined with one another as desired. These embodiments and the advantages associated with them will be discussed below.

According to one embodiment of the method, the reactive radicals can deactivate active sites that cause biological reactions and have cell and / or tissue damage to the surface. Thus, the cytotoxic reactions triggering active sites on the surface are specifically and specifically deactivated by the treatment with reactive radicals. This is surprising and unexpected because one would expect that reactive radicals on surfaces trigger chemical reactions that generate active sites and thereby act as cytotoxic agents. An active site that causes cytotoxic reactions is an atom or substance on the cell surface that is cell and / or tissue damaging. By means of reactive radicals, these active sites can be specifically and specifically deactivated, for example, by converting them into non-cytotoxic substances or by leaching them out of the cell surface, for example by reactive cleavage.

In another embodiment, the reactive radicals may comprise at least one species of oxygen radicals, nitrogen radicals, carbon radicals, sulfur radicals, and / or a species of halogen radicals. The reactive oxygen radicals include all radicals in which the at least one unpaired electron is attached to an oxygen radical. substance atom sits. Examples of oxygen radicals are hyperoxide anions, hydroxyl radicals, hydroperoxyl radicals, peroxyl radicals or alkoxyl radicals. Examples of nitrogen radicals are nitrogen monoxide and nitrogen. Carbon radicals include, for example, triplet carbene and alkyl radicals, and sulfur radicals include, for example, thiyl radicals. Halogenated radicals include, among others, chlorine radicals and bromine radicals.

According to another embodiment, reactive radicals can be generated by cleavage of a radical initiator. A radical starter is a molecule that can be converted into at least one reactive radical. For example, the chlorine-chlorine bond in molecular chlorine (Cl ₂ ) or the bromine-bromine bond in molecular bromine (Br ₂ ) can be cleaved by exposure to light and the molecular radical initiators are thereby converted into reactive radicals.

In one embodiment, the surface may be contacted with the radical initiator, which is typically stable as opposed to reactive radicals, and the radical initiator subsequently converted to the reactive radical in situ. This ensures that the entire surface is treated evenly.

The free radical initiator can be converted into the reactive radical by photolysis, radiolysis, thermolysis, by plasma, and / or by a chemical, for example electrochemical, and / or a biochemical, for example an enzymatic reaction. Radical production can thus be adapted in different ways and to the properties of the surface to be treated, for example non-thermally by light, for example UV radiation, or the use of X-ray and other ionizing radiation. A chemical reaction, for example in the form of a chemical or electrochemical Fenton reaction, in which hydrogen peroxide is removed by reaction with Fe (II) ions or with other transition metal ions, e.g. Cu (II), Ti (III), Cr (II) or Co (II) is decomposed in an acidic medium to form the highly reactive hydroxyl radical is also possible at room temperature.

According to another embodiment of the method according to the invention, the reactive radical may be a hydroxyl radical. Hydroxyl radicals can easily from biologically harmless substances, eg. As water, are generated. Hydroxyl radicals can be formed in particular: a) in a Fenton reaction; b) by photolysis of a peroxide; c) by radiolysis of water or other oxygenate radiolysable to hydroxyl radicals; or d) by a plasma reaction of an oxygen compound which can be converted by means of plasma treatment into hydroxyl radicals, preferably water or a peroxide.

The surface whose biocompatibility is improved by means of the method according to the invention can comprise, for example, a noble metal, a noble metal compound or a polymer, or a polymer. Precious metals, such as gold, are often used as electrodes in biosensors and as implant material. Implants and cell culture vessels are often made of polymers which, although in a biological environment do not cause any material change, such as corrosion, but which have cell and / or tissue damage to biological systems, thus can be improved by the method according to the invention in their biocompatibility.

According to a further embodiment, the surface may belong to an implant, a sensor or a cell culture vessel. One advantage is that the implant, the sensor or the culture vessel can first be prepared and then treated according to the invention. The method of the invention is universal, i. can be used for every kind of surface and every surface type, because for a given surface type or a given surface type with particularly suitable reactive radicals can be provided by different methods adapted to the material requirements.

Also contemplated in accordance with the present invention is a device that is contacted with biological systems, such as an implant, a sensor, or a cell culture vessel having a biocompatible surface treated according to any of the above methods. The device is characterized by a surface with improved biocompatibility, which is easily demonstrated by comparison of a surface before treatment with reactive radicals and a surface treated with reactive radicals, the latter has a significantly higher cell vitality, if with a Cell culture is brought into contact. Another feature of the device according to the invention is that the active sites that trigger biological reactions and cell and / or tissue damage, deliberately deactivated, ie converted into biologically inactive molecules or, z. B. in the case of biologically active gold ions, detached from the surface. In the following the invention will be explained in more detail by means of exemplary embodiments with reference to the drawings and concrete experiments. The feature combinations exemplified in the embodiments can be supplemented in accordance with the above statements in accordance with the necessary for a particular application properties of the device according to the invention or the method according to the invention by further features. Also, also in accordance with the above statements, individual features may be omitted in the described embodiments, if the effect of this feature in a specific application does not matter.

In the drawings, the same reference numerals are used for elements of the same function and / or the same structure.

Show it:

1 shows a schematic representation of the method according to the invention for improving the biocompatibility of a surface according to a first embodiment;

2 shows a schematic illustration of a method for improving the biocompatibility of a surface according to a second embodiment;

3 shows a graph relating to the dependence of cell vitality on the amount of gold detached from a gold surface;

4: AFM images and cross-sectional analyzes of (a) a mechanically polished gold surface before implantation, (b) a mechanically polished gold surface after implantation, (c) a "Fenton-polished" gold surface before implantation, and (d) a "Fenton" polished "gold surface after implantation in the peritoneal cavity of mice.

In the following, a first embodiment of a method according to the invention for improving the biocompatibility of a surface 1, in the schematic representation of FIG. 1 a solid surface, will be explained with reference to the schematic illustration of FIG. The surface 1 is brought into contact with reactive radicals 2. The radical may have a number of n unpaired electrons (represented by a ·). If the radical contains two unpaired electrons, one speaks of a diradical, with three unpaired electrons of a triradical, etc. The surface 1 may be the surface of a device 3, for example an implant, a sensor or a cell culture vessel, whose biocompatibility is to be improved.

The reactive radicals 2 cause active sites 4, which cause biological reactions and have cell and / or tissue damage, to be deactivated from the surface 1. The active center 4 is schematically indicated in the figures as a circle with a star located therein, wherein the star symbolizes the cytotoxic effect, ie the cell or tissue damaging property of the active center 4.

As shown on the right side in FIG. 1, the reactive radical 2 deactivates the active center 4 of the surface 1. The deactivation can be done, for example, by the active center 4 being split off from the surface and being dissolved out at the surface, as shown on the right side of FIG. 1 above. The deactivation may also be such that the active center 4 is converted by the reactive radical 2 so that it no longer has a cell- or tissue-damaging effect, which is symbolized in FIG. 1, bottom right, by the star indicating the cytotoxic effect no longer exists.

2, a further embodiment of the method according to the invention is shown schematically. In the embodiment of FIG. 2, reactive radicals 2 are generated by cleaving a radical initiator 5. In contrast to a reactive radical 2, the radical starter 5 is stable, ie less reactive and more durable. In the method of the embodiment shown in FIG. 2, the radical starter 5 is first brought into contact with the surface 1 of the device 3. Subsequently, the radical starter 5 is reacted in situ, ie in place in the reactive radical 2. To implement the radical starter 5 is converted by a cleavage agent 6 in the reactive radical 2.

The cleavage agent 6 may be both a chemical substance or an enzyme, as well as radiation such as UV, X-ray or ionizing radiation, as well as the change of a parameter, for example the temperature or the pressure, which the cleavage of the radical initiator 5 in the reactive Radical 2 causes. Thus, depending on the nature and nature of the surface 1, a cleavage agent 6 and thus a conversion method of the radical initiator 5 can be selected which does not modify the properties of the surface 1, with the exception of biocompatibility, which is improved according to the invention. For example, by means of a photolysis (light irradiation) or radiolysis (ionizing radiation), the biocompatibility of the surface 1 can be improved without an increase in temperature. This is particularly advantageous for thermosensitive surfaces. After the radical starter 5 has been converted into the reactive radical 2 by means of the cleavage agent 6 (right-hand side of FIG. 2), the process according to the invention of the second embodiment proceeds analogously to the process shown in FIG. 1, by reactive radicals 2 rendering the biocompatibility of the surface 1 improved by active centers 4 of the surface 1 are specifically deactivated.

In the following, the teaching according to the invention is explained on the basis of concrete test results.

1. Reduction of the cytotoxicity of gold layers

The cell vitality of electrodeposited gold layers on stainless steel wires was investigated after gamma sterilization. Untreated and oxygen radical-treated gold layers were subjected to a cytotoxicity test with human adult skin fibroblasts (NHDF cells). For this eluates were prepared from the wires and their influence on the cell vitality of the NHDF cells was examined by means of a colorimetric assay (TTC assay) (for detailed description see: N. Saucedo-Zeni et al., Int. J. Oncol. 41 (2012 ) 1241-1250).

The radicals were generated using Fenton solutions and UV photolysis of hydrogen peroxide. The following composition of the Fenton solution was used: c ₍ NH ₄ ) ₂ Fe (so ₄ ) ₂ .6 (H ₂ O) = 0.01 ίποΐ · L ¹ , c _NaiEDJA = 0.01 ίποΐ L ¹ , c _{acetate buffer} 0.1 mol · L ¹ and ^C H ₂ O ₂ ⁼ 0.1 mol - L ¹ . The total treatment time was 120 minutes, replacing the "old" Fenton solution with a fresh Fenton solution every 5 minutes.

For the generation of radicals by means of UV photolysis of H ₂ 0 ₂ a '705 UV Digester "(Metrohm, Switzerland) was used. It has been shown that a 30-minute treatment of the gold layer using a 0.3% H ₂ 0 ₂ solution is sufficient to completely detoxify the gold layers.

While cell vitality in untreated gold layers was only between 20 and 60%, cell vitality in gold layers after the above reactive oxygen radical treatment was virtually 100%; the gold layers were completely detoxified by the radical treatment.

Furthermore, the Fenton solutions used were examined for their gold content by means of ICP-AES with an "Optima 2100 DV ICP Optical Emission Spectrometer" (PerkinElmer, USA). It was found that the greater the amount of gold removed, the higher the cell vitality is (see FIG. 3).

Larsen, K. Kolind, DS Pedersen, P. Doering, M., Pedersen, G. Danscher, M. Penkowa, M. Stoltenberg, Histochem., Cell, are known to have gold ions eluting from gold implants Biol., 130 (2008) 681-692, G. Danscher, A. Larsen, Histochem, Cell, Biol., 133 (2010) 367-373). In the case of the organisms used for the cytotoxicity tests (human dermal fibroblasts) they are obviously toxic. From Fig. 3 it can thus be seen that the released gold atoms are active centers of the surface which trigger biological reactions. The detachment of these active sites detoxifies the surface.

2. Implantation of gold plates into the peritoneal cavity of mice

First, six gold sheets (size: 15 mm 5 mm 0.05 mm) were mechanically polished with alumina powder. Three of the mechanically polished gold sheets were then treated with oxygen radicals generated by Fenton solutions. For this purpose, the gold plates were placed in a freshly prepared solution consisting of (NH ₄ ) ₂ Fe (SO ₄ ) ₂ - 6 (H ₂ O) (c _{Fe 2+} = 1 - 1 0 ³ mol L ¹ , Merck), Na ₂ EDTA - 2H ₂ O

(c _EDTA = 1 - 0 ³ mol L ^"1 ; Merck) and acetate buffer (c _{CH; jCOOH} = c _{cH COO} _ = 1 - 1 0 ² mol L ^{" 1} , pH =

4.7; Merck) The Fenton reaction was started by the addition of H ₂ O ₂ (Merck) and the gold sheets were exposed to this solution for 5 minutes. This procedure was repeated 12 times so that the total treatment time was 120 minutes. In the Fenton solution c _HO and c _{pe2 + were} always in the ratio 10: 1.

Both the mechanically polished and the Fenton-treated gold sheets were AFM images taken (see Fig. 4a and 4c) and determines the surface roughness factors (see Table 1). The AFM measurements were made using a "NanoScope I" (Digital Instruments, USA) in contact mode.

The gold sheets were implanted in the peritoneal cavity of mice (one gold sheet per mouse). After 14 days, the gold sheets were removed from the mice and AFM images of the gold surfaces were taken again (see FIGS. 4b and 4d) and the roughness factors determined (see Table 1). The AFM images and roughness factors make it clear that the gold surfaces treated only by mechanical polishing are smoothed in the peritoneal cavity of the mice, ie biologically active, ie cell-damaging, gold is detached from the implants. On the other hand, the mechanically polished and subsequently radical-treated gold surfaces show no change in the roughness of the surface because the active sites are deactivated when reactive radicals are treated. Therefore, no gold was detached from the gold surfaces in the peritoneal cavity. This proves that implants have a higher biocompatibility through pre-treatment with radicals (not to be attacked). Table 1: Roughness factors of differently treated gold surfaces

Roughness factor [nm]

Mechanical Before implantation 14.7 ± 2.1

polished post-implantation 10.2 ± 0.5

"Fenton pre-implantation 12.6 ± 1.0

polished "post-implantation 13.4 ± 1.3

Reference surface

reactive radical

Device (for example, implant, sensor or cell culture vessel) active center

radical initiator

cleavage agent

Claims

claims

Method for improving the biocompatibility of a surface (1), in particular a solid-state surface, wherein the surface (1) is brought into contact with at least one species of reactive radical (2).

A method according to claim 1, wherein the reactive radicals (2) deactivate active sites (4) which cause biological reactions and cause cell or tissue damage to the surface (1).

The method of claim 1 or 2, wherein the reactive radicals (2) comprise at least one oxygen radical, a nitrogen radical, a carbon radical, a sulfur radical and / or a halogen radical.

Method according to one of claims 1 to 3, wherein the reactive radicals (2) are generated by cleaving a radical initiator (5).

The method of claim 4, wherein the radical initiator (5) is contacted with the surface (1) and reacted in situ into reactive radicals (2).

The method of claim 4 or 5, wherein the radical starter (5) by photolysis, radioysis, thermolysis, by plasma, by a chemical and / or biological reaction in reactive radicals (2) is reacted.

A process according to any one of claims 1 to 6, wherein the reactive radical (2) is a hydroxyl radical which is formed:

a. in a Fenton reaction;

b. by photolysis of a peroxide;

c. by radiolysis of water or another oxygen radical radiolysable oxygen compound; or

d. by a plasma reaction of an oxygen compound which can be converted by means of plasma treatment into hydroxyl radicals, preferably of water or of a peroxide.

A method according to any one of claims 1 to 7, wherein the surface (1) comprises a noble metal, a noble metal compound or alloy, or a polymer. Method according to one of claims 1 to 8, wherein the surface (1) belongs to an implant, a sensor or a cell culture vessel.

0. Device (3), for example an implant, sensor or cell culture vessel, which is brought into contact with biological systems with a biocompatible surface (1) which has been treated according to the method of any one of claims 1 to 9.