DE19960660A1 - Generation of reactive groups on poly(tetrafluoroethylene) by treatment with hydrogen peroxide and sulfuric acid, are useful for subsequent attachment of cells to vascular prostheses - Google Patents

Abstract

Generation of reactive surface groups (A) on poly(tetrafluoroethylene) (PTFE) by wet chemical methods, comprises reaction with a mixture of hydrogen peroxide and sulfuric acid. Independent claims are also included for: (a) coupling of cyanuric chloride to (A) to allow covalent binding of additional functional molecules; (b) covalent binding of protein, peptide or derived mimetics to the surface of (a); (c) other chemical and physical methods for introducing reactive surface groups on to PTFE that can be combined with methods (a) and (b); (d) coupling a crosslinker, reactive with hydroxy, amino or thiol groups, to the modified PTFE surface, provided this reaction can be combined with method (b); (e) loading implants made in (b) with autologous cells; (f) conditioning materials of (e) by culturing under flow conditions with shearing forces comparable with those encountered in vivo; (g) introduction of an intermediate layer (IL) during modification or loading of the implant material; and (h) IL that provides controlled release of a growth factor or pharmaceutical or provides for proteolytic cleavage by cell-specific enzymes.

Description

The invention relates to a novel, multi-stage method for subsequent Surface modification and functionalization more commercially available Implant materials based on PTFE to increase the biological Long-term tolerance and stability. In particular, the invention relates to Modification of vascular implants with defined adhesion factors for permanent Colonization with autologous endothelial cells.

STATE OF THE ART

In biotechnology, biomedicine have a variety of materials different material properties found entrance. Especially in the modern Medical devices will use these properties as permanent or temporary implant materials applied. The properties of biological inert to be completely biocompatible. Examples of used here Materials are hydrocarbon polymers, fluorocarbon polymers, semiconductors, ceramics and Metals. Of these materials, the compact polytetrafluoroethylene and the porous polytetrafluorethylene (PTFE) for the medical application of Importance. The PTFE is a hydrophobic fluorocarbon polymer with a large Resistance to biochemical and chemical influences. It is also inert to a variety of solvents. There are many different manufacturing processes for PTFE and a wealth of them commercial forms. Both the properties of the surface, Hydrophilicity and hydrophobicity, mechanical resilience and surface morphology (Pores, strands) can be changed within defined limits. In the foreground this Modifications are the possibilities of grafting other more hydrophilic ones Polymers (patent US 4661383) or polymers with free reactive groups (patent US 5914182) or the pressing of metals such as aluminum (patent US 5118524) into the PTFE polymer matrix.

Building on these composite materials, bioactive species can now be Crosslinking agents are fixed on the surface. The chemisorption or  Physisorption of bioactive species such as enzymes, organic catalysts, ribozymes, Organometallic compounds, proteins, glycoproteins, peptides, polyamino acids, Nucleic acids, antibodies, carbohydrates, cytokines, lipids and components of the extracellular matrix provides the basis for a biosymphatization, increasing the Biocompatibility of such materials in the organism. By modifying the Surface are the energetic properties of the surface like free energy and changes the wettability and / or the surface morphology.

Due to its inert surface properties, PTFE is difficult to access for direct modification. The number of reactive groups on the surface of PTFE materials depends on the manufacture and the base material used. Free hydroxyl groups are available on the surface in less than 1%. For a direct modification of PTFE surfaces, the number of reactive groups has to be increased by physical and chemical methods. Various methods (patents US 5369012, US 4946903) were developed. Combined chemical and physical methods have so far been used to generate hydroxyl groups and amino groups. The polymer surface was exposed to differently concentrated gas mixtures of ammonia, water vapor, H ₂ , various organic nitrites and nitrates or alcohols. These substances were connected by glow discharge, laser radiation or other forms of hard radiation as an energy source. A further variant of the generation of reactive groups could be achieved by reducing the surface with various mixtures of water and hydrogen under analogous experimental conditions with the production of unsaturated hydrocarbons on the surface. The modification was carried out directly on the surface of the material up to a layer depth of 10 nm (patent US 4946903). Percentage changes in the proportion of hydroxyl or nitrogen groups between 3.5% and 33% depending on the test conditions. The interfacial energy of the surfaces obtained increased significantly, the critical angle with water decreased noticeably. The hydrophilized surface can now be used for the physisorption or chemisorption of the above-mentioned bioactive substances. Depending on the method used, the bulk material is influenced to a greater or lesser extent.

The activated PTFE surface with reactive groups is now a covalent one Binding of functional molecules as described in 1. accessible. Previous Technologies are largely based on an exclusively adsorptive, due to the hydrophilic properties of the surface based attachment of the Functional molecules on the polymer surface. The addition of Composite polymers (grafting) as hydrophilic intermediate layers between the inert Polymer material and the functional molecules are primarily made by adsorption and subsequent coupling by high-energy radiation. These are expanded Work through the possibility of using positively charged polymers and monomeric molecules such as aminosilanes, chitosans, lysine, polylysine and the like. a. These can be linked to the surface using the above methods and the Functional molecules both covalently and via electrostatic interaction be connected (patent US 5512474).

The prosthetic replacement of blood vessels represents one of the important principles of surgery cardiovascular surgery. The use of large-lumen arterial vessels is included good long-term results possible, with smaller arterial vessels or in the venous System, however, the current limits of prosthetic vascular replacement show up. It can accumulate in these situations of relatively low blood flow by Platelets and local activation of blood clotting to excess Formation of wall adherent thrombus with thrombotic early closure of the Perform vascular prosthesis. Research results of arteriosclerosis of native vessels such as the observation of the behavior of prosthetic vessels also leaves the local Activation of coagulation due to the lack of a complete endothelial cell population as central mechanism appear. Therefore, it should be possible for a complete Colonization of the vascular prosthesis with autologous endothelial cells the occurrence of the above Complications can be kept to a minimum. The principle of the settlement of Vascular prostheses with endothelial cells are already largely established experimentally, and it initial clinical experience is also available. Up to 70% of the endoluminal Surface with endothelial cells is possible within hours to days. Clinically 70% colonization of the endoluminal surface is sufficient despite the use of However, growth factors do not rule out. Lack of quality and density of settlement obviously the reason for the fact that in previous applications of  endothelial cell populated vascular prostheses did not significantly improve the results could be achieved. The expression and maintenance of the endothelium-specific Properties play a vital role in cell anchoring on the Vascular prostheses. The cell anchoring on the vascular prostheses is improving expect, given the specific properties of the extracellular matrix of the Blood vessels transferred to the surface of the artificial vascular prosthesis.  

OBJECT OF THE INVENTION

The subject of the protection is a new, multi-stage process for the subsequent surface modification and functionalization of commercially available PTFE materials. The materials that can be considered here are both solid, mechanically resilient PTFE block composites, as well as flexible foils and spun fabrics with a wide variety of external appearances. The basic properties of these substances are high surface inertness, the basic molecular composition of the base polymer according to C _n F _m at n = 2 and m = 1-4 and a contact angle with water of greater than 100 °. The base polymers can also have a different microscopic surface morphology, such as smooth, porous, voided surface, etc.

The new method consists of five partial steps that build on one another. The The first step is the creation of reactive surface groupings on the Polymer surface through oxidation. The method here was wet chemical Oxidation of the surface caused by different concentrations (from 1 vol% Hydrogen peroxide up to 70 vol%) Hydrogen peroxide / sulfuric acid mixtures reached could be used. By varying the reaction temperature and the Incubation time can be the number of reactive hydroxyl groups obtained on the surface be varied. The oxidation takes place primarily on the surface of the polymer up to a depth of approx. 20 nm. With modifications of the polymers in the areas of sufficient activation does not change the surface morphology. This could be done with the help of atomic force microscopy as well as electron microscopy be occupied. Evidence of the formation of free hydroxyl groups was made with the help FT-IR spectroscopy. In addition to the unchanged basebands of PTFE new bands of different hydroxyl groups were formed Intensity. The contact angle with water decreased depending on the Reaction parameters to values between 88 ° and 115 ° (pure PTFE 125 °).

The activation strategies currently used for the GCE surface are based on an oxidative change in a first step. The oxidation can take place both wet-chemically with sulfuric acid / hydrogen peroxide / 1, 7, 8 /, potassium dichromate / 7 / and cerium ^IV / perchloric acid / 7 / or electrochemically by anodic oxidation / 9, 11 /. Different oxygen compounds such as aliphatic and phenolic hydroxyl groups, quinoid structures, aldehyde and carboxyl groups are obtained on the surface / 9, 10 /.

The second step is the chemical activation of the generated reactive Hydroxyl groups protected by small reactive molecules. Among little ones reactive molecules are understood to be compounds based on their chemical Structure linking other dissolved substances with polymer surfaces make possible. These reactive molecules (hereinafter referred to as reactants) can remain on the surface after the coupling reaction, but also afterwards be desorbed again. Are reactants in the sense of the method presented Molecules that connect to hydroxyl groups still have the ability possess to react with free hydroxyl, amino and thiol groups. As reactants find carbodiimide / 2 /, thionyl chloride / 4, 5 /, various silanes / 3 /, cyanogen bromide / 8 /, Cyanuric chloride / 6 / use.

The surface morphology of the PTFE polymers is not changed. The activation of the Surface can be done in different solvents. Be typical Chloroform, methanol, ethanol, dioxane, acetone or their mixtures with various Catalysts such as n, n-diisopropylethylamine or dimethylfluoropyriden u. a., used. The reaction can be monitored and characterized using FT-IR spectroscopy. The example is the coupling of cyanuric chloride to the PTFE surface a new band was observed in FT-IR. At the same time, the contact angle increases against water from approx. 90 ° to 110 °. The polymer activated in this way can be stored. At Using the given method, the reactivity remains unchanged for 6 months receive.

In a third sub-step, selected functional molecules are activated on the so activated PTFE surface coupled, which allows targeted attachment and spread of cells convey a defined type and origin. This is done through an affine and specific Interaction of these molecules with the physiological adhesion molecules of the cells reached. Candidates here are: components of the extracellular matrix such as collagens, fibronectin, laminin, vitronectin; linear derived from these molecules and cyclic peptide sequences (RGD sequences), receptor proteins from Microorganisms such as Invasin A from Yersinia spec. or Picornavirus VP1-3 as well as from these derived (fusion) proteins, peptides and mimetic structures. The The desired cellular receptors are in particular integrins (beta-1 and beta 3).  

In the application example presented, a fusion protein from the Maltose-binding protein (MBP) and the C-terminal extracellular domain of Invasin A used by Yersinia enterocolitica as a cell-binding functional molecule.

An optional fourth sub-step can be introduced at this point in the manufacturing process to insert an intermediate layer of monomeric or polymeric substances, which the activity of the covalently fixed functional molecules in the sense of the binding of Cells do not interfere with their adhesion proteins. They are not in the third step used linker molecules, which in the second step on the surface of the Implant material have been applied. Alternatively there is an absorptive Connection of the intermediate layer in question. The aim of this intermediate layer is a selective one Support of the cell adhesion to be achieved by the functional molecules Stimulation of growth and division of the desired cell type or inhibition and Killing unwanted cell types. This can be due to the release of Growth factors or pharmaceuticals (e.g. anticoagulants or toxins) from the Intermediate layer can be achieved, which can be controlled if necessary. A Control of the release of the effectors from the intermediate layer can be ensured by incorporating the effector molecules into them as fusion proteins (F-Linker-E) be, the fusion portion is used for fixation on the implant material and the Linker contains specific protease sequences, which are the degradation by proteases of the respective cell type are accessible.

In the final fifth sub-step of the invention, the modified Implant materials undergo a final colonization with cells, of which a long-term stabilization of the maximum for the respective application Implanates and its functionality can be expected. This is preferred Colonization was carried out ex vivo and with autologous, d. H. from the recipient of the Cells obtained from implant performed. After the settlement, the applied cell layer conditioned. To do this, it becomes the after implantation expected stress factors and stimuli such as shear stress, pulsation, growth factors, Hormones or metabolic products. Through this settlement, one Masking of the implant material can be achieved, which is accepted by the body will, no immunological reactions and activation of coagulation and Complements cascades.  

The combination of chemical modifications with selection of the appropriate ones Functional molecules with the permanent, sufficiently resistant to shear forces Colonization with specific cells can be described as "biosympathization" and represents the essential novelty of this invention.  

APPLICATION EXAMPLE

PTFE implant material for the prosthetic replacement of small-lumen blood vessels (diameter) (origin) was oxidized on the luminal surface by incubation for 15 minutes with a mixture of 30% hydrogen peroxide and 96% sulfuric acid. The decrease in surface hydrophobicity was demonstrated by measuring the wetting angle (water). This decreased from the original 125 ° to approx. 90 °. An analysis of the IR spectrum showed new bands between 1000 and 1400 cm ^{-1 which are} characteristic of unmodified PTFE and which are characteristic of hydroxyl groups.

The implant material activated in this way was linked to the OH-reactive crosslinker cyanuric chloride. After neutralization of the oxidized surface, this could be achieved by incubation in a saturated solution of cyanuric chloride in chloroform for 30 minutes. The successful covalent modification with cyanuric chloride was verified by means of IR spectroscopy. The spectrum of the surface of the material treated in this way showed additional bands characteristic of C = N bonds at 2200 to 2400 cm ^-1 . The wetting angle increased from 90 ° to 110 ° after the reaction with cyanuric chloride. The structure and strength of the implant material were not adversely affected by the described procedure.

Following the oxidation and reaction with the cross-linker cyanuric chloride, the proteins MD1, MD2, Min3 and MBP were covalently coupled to the implant material by reacting one of its amino groups with one of the two still available and amino-reactive C-Cl functions (Protein concentration 100nM, 16 h). The proteins MD1, MD2 and Min3 represent fusion proteins from the maltose-binding protein (Swiss-Prot: P02928) at the N-terminus and sequences of the extracellular domain of Invasin A from Yersinia enterocolitica (Swiss Prot: P19196) of different lengths at the C-terminus The primary structures are shown in Fig. 1. All proteins were expressed from the corresponding plasmids based on the pMal system in E. coli and purified by maltose affinity chromatography and ion exchange chromatography. The activity of MD1, MD2 and Min3 in terms of mediating cell adhesion has been shown in independent studies. Inhibition studies with beta-1 integrin-specific antibodies and the RGD peptides GRGDS and GRGDTP also demonstrated that the observed adhesion is mediated by specific interactions between the fusion proteins and cellular beta-1 integrins. The successful coupling was successfully demonstrated in all cases by means of an enzyme-linked immunoassay based on an anti-MBP antibody. An examination of the surface of the modified implant material with the aid of atomic force microscopy showed typical, 5-10 nm large spherical structures ( Fig. 2). These corresponded in size to the proteins used and were missing on surfaces that were not reacted with the protein (s).

Then attempts to colonize unmodified implant material, oxidized implant material, oxidized and reacted with cyanuric chloride Implant material as well as either the control protein MBP or the active Fusion proteins Min3, MD1 and MD2 covalently coated implant material carried out. Since the implants are vascular prostheses, Colonization experiments selected primary human endothelial cells. These were previously by enzymatic digestion (0.1% type I collagenase) from a saphenous vein magna isolated. The cell culture medium used for the colonization contained in addition to the Standard components an optimized composition of different Growth factors.

Figures 3A to 3D show the result of a 24-hour colonization of unmodified implant material (A), oxidized implant material (B) and with the control protein MBP or Min3-modified implant material (C, D). It can clearly be seen that on samples A to C only a small part of the surface could be populated with cells, while with the Min3 coating (D) a very uniform and complete colonization was achieved. The identity of the endothelial cells was additionally demonstrated by staining intracellular, von Willebrand factor positive storage granules. Perfusion tests also showed sufficient stability of the formed cell layer against shear forces.

/ 1 / Heiduschka, P., Dittrich, J., Electrochimica Acta, 37, (1992), 2573-2580
/ 2 / Bourdillon, C., Bourgeois, JP, J. Am. Chem. Soc., 102, (1980), 4231
/ 3 / Armstrong, FA, Browen, KJ, J. Electroanal. Chem., 219, (1987), 319
/ 4 / von der Linden, WE, von Dieker, JW, Anal. Chim. Acta, 119, (1980), 1
/ 5 / Kamau, GN, Anal. Chim. Acta, 207, (1988), 1
/ 6 / Ianniello, RM, Yacynych, AM, Anal. Chem., 53, (1981), 2090-2095
/ 7 / Kepley, LJ, Bard, AJ, Anal. Chem., 60, (1988), 1459-1467
/ 8 / P. Heiduschka, dissertation, Halle University of Education, (1990)
/ 9 / Engstrom, RC, Strasser, VA, Anal. Chem., 56, (1984), 136
/ 10 / Evans, JF, Kuwana, T., Henne, MT, Royer, GP, J. Elektroanal. Chem., 80, (1977), 409
/ 11 / Blaedel, WJ, Jenkins, RA, Anal. Chem., 46, (1974), 1952

Claims (14)

1. The wet chemical generation of reactive surface groups on PTFE materials by reaction with mixtures of hydrogen peroxide and sulfuric acid. 2. Other chemical and physical methods for introducing reactive surface groups on PTFE materials such as OH, NH ₂ , COOH or CHO, provided that these are combined with the following patent claims. 3. The coupling of cyanuric chloride to according to claims 1 and 2 of the Surface of PTFE materials generated reactive groups for the purpose of covalent attachment of other functional molecules. 4. The coupling of other cross linkers to according to claims 1 and 2 of the Surface of PTFE materials created reactive groups, which continue to Have the ability to react with free hydroxyl, amino or thiol groups, if this reaction is combined with those of the following claims. 5. The covalent attachment of proteins, peptides or derived mimetics Ligands for cellular adhesion molecules to one activated according to claim 3 or 4 PTFE surface. 6. Use of proteins according to claim 5, characterized in that these Sequences of the extracellular domain of the Yasinia invasin A protein enterocolitica included. 7. Use of proteins according to claim 5, characterized in that these Represent antibodies or antibody fragments with specificity for integrins. 8. The colonization of implant materials according to claim 5 to 7 with autologous cells for the purpose of masking the material and increasing the biological Long-term functionality and stability.   9. The colonization of small-lumen (1-10 mm diameter) vascular prostheses on PTFE Basis according to claims 5 to 7 with autologous endothelial cells. 10. The use of endothelial cells characterized by in vitro differentiation Hematopoietic stem cells (hemangioblasts) were obtained according to claim 9. 11. The conditioning of populated vascular prostheses according to claim 9 and 10 by Cultivation under flow conditions with shear forces according to the Conditions under physiological conditions at the location of the planned Forces that occur during implantation. 12. The introduction of an intermediate layer in the modification and settlement of Implant materials according to claims 1 to 11. 13. An intermediate layer according to claim 12, from which growth factors or pharmaceuticals released in a controlled manner. 14. An intermediate layer according to claim 13, wherein the release by the proteolytic cleavage is achieved by cell-specific enzymes.