Patent Grant
8168423
This invention relates generally to the generation of nitric oxide in situ and to nitric oxide sensors, and more particularly to biocompatible materials having surfaces that are capable of generation of nitric oxide in situ when contacted with nitrite, nitrate or nitrosothiols in blood, and to a reproducible nitric oxide sensor.
Although medical devices such as extracorporeal circuits and hemodialysis tubes are widely used in clinical settings, the polymers typically used to fabricate such devices (PVC, polyurethane, silicone rubber, etc.) are still subject to platelet aggregation and adhesion onto the surface of these materials. Thus, patients are often given anti-clotting agents (i.e., heparin) in order to reduce thrombosis on the surface of these devices. Similarly, implanted devices made of stainless steel or other alloys, or even carbon, can cause thrombus formation when in direct contact with blood. There is, therefore, a need for materials that more closely simulate the antithrombogenic properties of the endothelial cells that line blood vessels in order to obviate the need to administer anticoagulants.
Nitric oxide (NO) is an important intracellular and intercellular messenger molecule that plays an important physiological role in anti-platelet aggregation and anti-platelet activation, vascular relaxation, neurotransmission, and immune response. It has been proposed that synthetic materials that release low levels of NO would, therefore, more closely simulate the natural activity of endothelial cells, and therefore, would have improved biocompatibility.
Several classes of NO-releasing materials are currently under investigation worldwide. These include NO donors (i.e., diazeniumdiolates, nitrosothiols) that may be relatively complicated to synthesize and may in some instances require stringent storage conditions.
Currently, NO generation is determined by water uptake (such as in the case of diazeniumdiolates) or the intensity of light (as with iron nitrosyls). However, blood already contains a host of species that are derived from, or are physiologically-generated in vivo that may be reduced to NO. These species include nitrites, nitrates, and a host of nitrosothiols (e.g., nitrosoglutathione, nitroso albumin, etc.). The presence of these species raises the possibility of recycling these species back to nitric oxide.
S-nitrosothiols (RSNO) are believed to take part in the storage and transportation of NO in biological systems. While RSNOs are generally stable in physiological conditions, they may, in some instances, be sensitive to photolytic breakdown and catalytic decomposition in the presence of transition metals to produce the free thiol and NO. Modification of the membrane design of NO sensors may be used to fabricate a nitrosothiol sensor and thus detect the presence of nitrosothiols in fluids.
A device for detecting nitrosothiol content in a solution includes at least two electrodes disposed in a housing, wherein one of the at least two electrodes is a working electrode having a platinized tip and the other of the at least two electrodes is a counter electrode. A filter membrane is disposed at an end of the housing and is configured to come in contact with the solution. The filter membrane and at least a portion of the working electrode have a material coated thereon. The material includes a polymer and a source of copper dispersed within the material. The material and the platinized tip are configured to come into contact with the solution containing nitrosothiols to convert the nitrosothiols to nitric oxide in order to detect the nitrosothiol content.
Features and advantages of embodiment(s) of the present disclosure will become apparent by reference to the following detailed description and drawings, in which:
An embodiment of the present disclosure provides a novel, reproducible nitric oxide sensor for the detection of the S-nitrosothiols. In an embodiment, the sensor is coated with a material having a catalytic or consumptive/reactive copper ion complex therein. The sensor may advantageously be used to take numerous measurements of the nitrosothiol content in a sample over several days.
A further embodiment of the present disclosure provides a novel approach for enhancing the biocompatibility of materials of the type suitable for implantation in a human or animal body and/or for prolonged contact with the body or blood. In accordance with a broad aspect of the disclosure, materials have been developed to have reactive agents that are capable of generating, at the materials' surface/blood interface, physiologically significant amounts of NO when in contact with blood. A reactive agent, having nitrite reductase and/or nitrite reductase-like activity, nitrate reductase activity, or nitrosothiol reductase activity, may be immobilized, adsorbed, adhered, or otherwise made available at a surface of the material. Still further, the reactive agent may be covalently attached to a polymer backbone within the material.
As used herein, the term “material,” when referring to the material that is provided with the reactive agents, may be any material that may be suitable for contact with the body and/or body fluids, particularly blood, of a living being, e.g., is physiologically acceptable and non-toxic. In some embodiments, the material should be suitable for long-term contact, or in-dwelling uses. Non-limitative examples of such materials include polymers, metals and alloys thereof, and carbon (graphite).
Many polymeric materials are suitable for the practice of the disclosure, and the following illustrative list of polymers that have been used for biomedical applications is not intended to be limiting in any manner. Examples include synthetic polymers such as polyurethanes, polydimethylsiloxane, ethylene vinyl acetate, nylons, polyacrylics, polymethyl methacrylate, polyamides, polycarbonates, polyesters, polyethylenes, polypropylenes, polystyrenes, poly(vinyl chloride) (PVC), polytetrafluoroethylene (PTFE) and cellulose acetate.
In an embodiment, a material includes a hydrophobic polymer, such as poly(vinyl chloride), polyurethane, and silicone rubber, and a layer of a reactive agent having nitrite reductase activity, nitrite reductase-like activity, nitrate reductase activity or nitrosothiol reductase activity attached to a surface of the hydrophobic polymer. The attachment may be by adsorption, covalent bonding, and the like. In an embodiment, the polymer may include lipophilic salts of nitrite/nitrate or nitrosothiols within its matrix to create a reservoir of nitrite/nitrate or nitrosothiol that can continuously leak to the catalytic surface.
In embodiments where the material is a polymer, the NO-releasing polymer may be formed, cast, or otherwise shaped to form a monolithic device, such as implantable devices such as drug depot or in-dwelling devices, catheters, extracorporeal tubing sets (including kidney dialysis or open-heart surgery heart-lung machines), and/or the like. Other examples of such devices include, but are not limited to medical devices such as arterial stents, guide wires, bone anchors, bone screws, protective platings, hip implants, joint implants, spine appliances, electrical leads, biosensors, vascular grafts, heart valves, extracorporeal circuits, and probes. In an alternate embodiment, the material may also be applied as a film on another substrate that may be a polymer, or another surface, such as the surface of a metal device. Suitable metals for such devices include, but are not limited to stainless steel, nickel, titanium, aluminum, copper, gold, silver, platinum and/or combinations thereof.
Further, the material may be a metal substrate. In an embodiment, the metal material may have a reactive agent covalently attached to its surface. It is to be understood that in this embodiment, and other embodiments discussed herein, the reactive agent may be a biomimetic catalytic or consumptive/reactive agent. As used herein the term “biomimetic agent” refers to a species possessing nitrite reductase-like activity, or the ability to reduce nitrosothiols which converts endogenous or exogenous nitrite, nitrate and/or nitrosothiols to NO when in contact with blood.
Illustratively, the biomimetic catalytic agent may be a metal ion ligand complex wherein the metal ion is capable of reducing one or more of nitrate, nitrite, nitrosothiols, and other blood species to nitric oxide. In an embodiment, the metal ion ligand complex is a Cu(II) complex. Neutral carrier type ligands that have high metal binding affinity, particularly for copper, and, preferably, planar square-type geometry that provides a minimum amount of steric hindrance to the approach of the electron source (e.g., ascorbate or NADH) to the center metal of the complex so that the copper ion can easily be reduced from Cu(II) to Cu(I), are suitable for the practice of the disclosure. Non-limitative examples include nitrogen or sulfur donating compounds, such as Nx-donor macrocyclic ligands (x=2, 4, 5, 6, 8) such as cyclen, cyclam and their derivatives, and crown ethers and Sx-donor macrocyle-type ligands (x=2, 4, 5, 6, 8).
Attachment of the metal ion ligand to the metal surface may be accomplished by any suitable means known to a person of ordinary skill in the art. One such technique involves silanizing the surface of the metal to provide reactive sites to bind the ligand.
In specific illustrative embodiments, the biomimetic catalytic agent is a Cu(II) metal ion ligand complex selected from the group consisting of dibenzo[e,k]-2,3,8,9-tetraphenyl-1,4,7,10-tetraaza-cyclododeca-1,3,7,9-tetraene; dibenzo[e,k]-2,3,8,9-tetramethyl-1,4,7,10-tetraaza-cyclododeca-1,3,7,9-tetraene; dibenzo[e,k]-2,3,8,9-tetraethyl-1,4,7,10-tetraaza-cyclododeca-1,3,7,9-tetraene; and dibenzo[e,k]-2,3,8,9-tetraphenyl-1,4,7,10-tetraaza-cyclodeca-1,3,7,9-tetraene.
In other embodiments, the reactive agents are biocatalysts, such as enzymes, having nitrate reductase activity, nitrite reductase activity, nitrite reductase-like activity, and/or nitrosothiol reductase activity. Illustratively, nitrite reductases, nitrate reductases, enzymes having nitrosothiol reducing ability, xanthine oxidase, and/or combinations thereof may be selected. Due to the ease of procuring xanthine oxidase commercially (e.g., Sigma, St. Louis, Mo.), xanthine oxidase may be used in an embodiment. Other potentially useful immobilized biocatalysts may include nitrite reductases and nitrate reductases from plants or bacteria.
Still further, the reactive agents may be metal complexes (hydrogels) that are covalently attached to the polymer backbone of the material.
In an alternate embodiment, the reactive agent may be a source of metals and/or metal ions that either converts the nitrates, nitrites and/or nitrosothiols, or generates a species capable of converting nitrates, nitrites and/or nitrosothiols to NO. In one embodiment, copper, copper (I) or copper (II) containing materials may be exposed to endogenous or exogenous sources of nitrates, nitrites, or nitrosothiols with appropriate reducing agents to generate the active metal (I) species for the generation of NO at the surface of the material. It is to be understood that the sources of nitrates, nitrites, nitrosothiols and reducing agents may be from the blood, the polymer matrix, the metal substrate, and/or may be injected intravenously. The metal/metal ion source degrades (via corrosion) or leaches onto the surface of the material to yield the required and/or desired catalytic/reactive species that is capable of converting the nitrates, nitrites and/or nitrosothiols to NO. Other non-limitative examples of metal/metal ion sources include calcium, magnesium, cobalt, manganese, iron, molybdenum, tungsten, vanadium, aluminum, chromium, zinc, nickel, platinum, tin, ions thereof, and/or mixtures thereof. Some non-limitative examples of a metal ion source are copper (II) phosphate and various copper salts.
In certain embodiments, an exogenous source of nitrites, nitrates or nitrosothiols is provided in the material to form a reservoir of nitrites, nitrates or nitrosothiols that can continuously leak to the surface of the material. In these embodiments, the exogenous source (a non-limitative example of which includes lipophilic salts of nitrites, nitrates or nitrosothiols) is dispersed within the material. In some embodiments, the material containing the exogenous source of nitrites, nitrates or nitrosothiols is applied to a device as a coating. Some non-limitative examples of nitrites, nitrates or nitrosothiols, include, without limitation, quaternary ammonium salts, such as tridodecylmethylammonium nitrite (TDMA+NO2−/NO3−); trimethyl phenyl ammonium; dimethyl dioctadecyl ammonium; etc. In addition to quaternary ammonium salts, quaternary phosphonium salts or quaternary arsonium salts may be used in the practice of embodiments of the disclosure.
Methods of making the disclosure include swelling a polymer, such as a polyvinyl chloride (PVC) or silicone, or a polymer-coated device in the presence of an organic solvent containing an appropriate copper (I) or copper (II) containing moiety or the nitrite/nitrate salt to form a copper(I), copper (II) or a nitrite/nitrate salt-containing polymer. The copper(I), copper(II) or nitrite/nitrate salt-containing polymer may then be coated with a layer of immobilized enzyme, illustratively a nitrite reductase enzyme, such as xanthine oxidase. Many techniques are available for immobilizing enzymes. For example, see, Hasselberger, “Uses of Enzymes and Immobilized Enzymes, Nellson-Hall,” Chicago (1978) or Guilbault, “Analytical Uses of Immobilized Enzymes,” Marcel Dekker, New York (1984). Alternately, the copper(I), copper(II) or nitrite/nitrate salt-containing polymer-coated device may then be dried to return the device to its functional form.
In another embodiment of the inventive method, the biomimetic generation of NO may be further achieved by immobilizing metal-ion ligand complexes, on the surface of the material, or by dispersing these ligands within the material, which, as previously discussed, may be a polymer. In some embodiments, additional lipophilic nitrite/nitrate salts, or nitrosothiols, are added to an underlying polymer matrix material or provided as a coating on the material, or as an additional layer.
In other embodiments, the reactive agent may be added during the processing stage when the desired end product (e.g. device) is molded or cast from the native polymer material. In still other embodiments, the surface of the device that will be exposed to blood, for example, the outside surface of a catheter or the inner surface of tubing of the type used in extracorporeal circuits, or the surface of metal stents, may be coated, either by dip-coating or by another method with the material containing the reactive agent capable of reducing nitrate, nitrite or nitrosothiols to NO.
In an alternate embodiment, the reactive agents (including, but not limited to metal sources, metal ion sources, biocatalysts or biomimetic catalytic or consumptive/reactive agents, and/or the like) may also be covalently tethered to the surface of the material. It is to be understood that covalent macromolecules having a copper containing moiety may be covalently attached to a polymer backbone. One non-limitative example includes a copper cylan complex bound to a polyhydroxyethylmethyloxylate matrix. It is to be understood that such copper complexes may be attached to other polymers, including, but not limited to at least one of polyurethanes and poly(vinyl chlorides).
Preliminary feasibility studies have been carried out to demonstrate the basic concept of this disclosure. Using xanthine oxidase as a model enzyme for nitrite reductase activity. PVC polymer films were doped with TDMA+NO2− and then coated with a layer of immobilized XO.
Illustratively, the PVC polymeric film, or membrane, was prepared by a cocktail solution casting method as described, for example, in Mathison et al., Anal. Chem., Vol. 71, pages 4614-4621 (1999) or any of the patents referenced herein. The cocktail solution was prepared by dissolving the appropriate amounts of membrane components (polymer, plasticizers and, in some cases, an ion-exchanger) into a solvent, illustratively tetrahydrofuran (THF). The membranes were cast in a mold to a final thickness of about 150 μm.
The polymer film was then coated with immobilized XO prepared by crosslinking XO with bovine serum albumin (BSA) in the presence of glutaraldehyde. The cross-linked product forms a hydrogel that is dip-coated on the PVC polymer substrate.
An electrochemical sensor was used to probe the surface concentrations of NO generated when the coated film were placed into a buffered solution containing NADH at physiological pH. Significant levels of NO were generated at the surface of the film under these conditions. The generation of NO near the surface of the polymer film continued for several hours as the nitrite in the film was exchanged for anions in the buffer phase.
In this particular embodiment, the electrochemical NO sensor used was similar in style to conventional Clark type oxygen sensor. Glass coated Platinum (Pt) wire served as the anode and Ag/AgCl wire (0.25 mm dia.) was used as the cathode. The internal filling solution was composed of 0.3 mM HCl and 30 mM NaCl, pH 3.5. An outer gas permeable membrane (Goretex, polytetrafluoroethylene with 50% porosity and 0.2 μm pore size) was placed between the internal filling solution and sample solution. Amperometric NO measurements were performed by applying a potential of 0.9 V.
In another illustrative embodiment, biomimetic catalysts and/or reactants, such as Cu(II)-ligand complexes, for example, dibenzo[e,k]-2,3,8,9-tetraphenyl-1,4,7,10-tetraaza-cyclododeca-1,3,7,9-tetraene, were either incorporated in or tethered to a polymer or other material surface, such as a metal. Examples of this embodiment are shown in
Three films having the following formulation were prepared in accordance with the method set forth above: 66.7 wt % PVC polymer (132 mg); 33.3 wt % plasticizer, illustratively nitrophenyloctyl ether (NPOE; 66 mg), and Cu(II) ligand complex, CuLxC12 (2 mg), Lx being ligands, L1-L3 as shown on
Referring back to
The polymeric film compositions used in this experiment are as follows:
Film 1:
66 mg PVC; 132 mg NPOE; 4 mg Cu(II) complex; and 20 mg ion pair, or TDMA+NO2−
Film 2:
100 mg PVC; 100 mg NPOE, 4 mg Cu(II) complex; and 20 mg ion pair
Film 3:
132 mg PVC; 66 mg NPOE; 4 mg Cu(II) complex; and 20 mg ion pair
These results show generation of NO by the polymer film that is particularly good for the highly plasticized embodiments.
Referring now to
In an embodiment, the housing 118 is configured to hold the glass capillary 116, the working electrode 110, and the counter electrode 114. It is to be understood that the housing 118 is also configured to have a space 120 therein that may be filled with an internal filling solution (not shown). Still further, the housing 118 has a membrane 122 covering opposed open ends 126, 127. It is to be understood that any suitable material may be used for the housing 118. In an embodiment, the housing 118 is formed from polypropylene.
The membrane 122 is configured to receive the working electrode 110, the counter electrode 114, and the glass capillary 116, as all three elements 110, 114, and 116 extend and/or protrude from upper end 126 of the housing 118. At lower end 127, membrane 122, and filter membrane 123 (and subsequently working electrode 110) come in contact with external solutions/samples 130 (shown in
It is to be understood that any suitable electrode materials may be used for the working electrode 110 and the counter electrode 114 as long as one acts as an anode and the other acts as a cathode. In an embodiment, the working electrode 110 is a platinum wire and the counter electrode 114 is a silver wire. The counter electrode 114 is in close proximity to, but spaced from the membrane 122.
In an embodiment, the glass capillary 116 is centered within the housing 118 and has a portion protruding through the membrane 122 and out the upper end 126 of the housing 118.
The working electrode 110 is positioned such that the glass capillary 116 substantially surrounds the working electrode 110. It is to be understood that the portion of the working electrode 110 that extends from the end 126 of the housing 118 extends further than that of the glass capillary 116, therefore a portion of the working electrode is not surrounded by the glass capillary 116.
The working electrode 110 (prior to being inserted in the glass capillary 116) may also be substantially coated with material 124 that advantageously assists in obtaining numerous nitrosothiol measurements. It is to be understood that the polished and platinized tip 112 of the working electrode 110 may not be coated with the material 124, while the rest of the working electrode 110 may be coated. The material 124 includes a polymer having a source of copper, which acts as a catalyst or a reducing agent, therein. It is to be understood that any suitable polymer may be selected. Examples of suitable polymers include, but are not limited to hydrophilic polyurethanes, PTFE, poly(vinyl chloride), silicon rubbers and/or mixtures thereof. In an embodiment, a hydrophilic polyurethane is selected due in part to its high water-uptake capability.
It is to be further understood that the source of copper within the material 124 may be an inorganic copper source (such as copper particles and copper compounds/salts (a non-limitative example of which is copper (II) phosphate)) and/or a copper complex (including those illustrated in
A non-limitative example of the material 124 may be prepared by adding 2 mg of DTTCT (or 2 mg of copper particles) and about 20 mg of polyurethane to about 1 mL of tetrahydrofuran (THF).
Another device 100′ for detecting nitrosothiol content in a solution is shown in
For illustrative purposes,
Upon contacting the nitrosothiol sensor device 100 with the sample containing RSNO, the nitrosothiol sensor device 100 detects the released NO, which then allows the user to calculate the amount of RSNO in the sample.
Specifically referring to
Referring now to
The nitrosothiol sensor device 100 of the present disclosure may measure the RSNO concentration in a sample, where the RSNO concentration ranges between about sub-μM and about 40 μM. Further, the nitrosothiol sensor device 100 was found to have a detection limit of about 0.2 μM for SNAP and CysNO and about 0.5 μM for GSNO and SNAC.
It is to be understood that the nitrosothiol sensor device's 100 relatively long-term sensitivity may decrease continuously, due in part to the leaching of the copper source (complexes, compounds, ions) from the material 124 to a solution phase. Further, it is contemplated that the sensor device's 100 sensitivity may also be dependant on the amount of reducing agent in the solution/sample, the type of RSNO species being detected and/or the amount of EDTA present, if any. Still further, it is to be understood that the number of uses of the sensor device 100 may also impact the sensitivity because the stirring condition may facilitate leaching of the catalytic active copper species.
Further, it is contemplated that the nitrosothiol sensor device 100 of the present disclosure may advantageously remain unclogged due in part to the existence of the copper source in the material 124. It is believed, without being bound to any theory, that the existence of the copper source may also advantageously prolong the sensitivity of the nitrosothiol sensor device 100 such that reading may be reproduced over time (generally a few days).
The present disclosure offers many advantages, including, but not limited to the following. An important advantage of the nitrosothiol sensor device 100 over previous sensors for measuring nitrosothiols is that the readings are reproducible for relatively long term use. An important advantage of embodiments of the method for generating NO locally at the surface of polymers or other materials is the potential simplicity of simply dip-coating the material with a material containing a reactive agent. The material may have a single reactive agent or mixture of reductase activities. It may be a biological protein (enzyme), a source of metal/metal ions, or a metal ion ligand complex that mimics the enzyme function. Even in those embodiments where added TDMA+NO2−/NO3− or some other nitrite, nitrate, or nitrosothiol, such as nitroso cysteine, is required and/or desired within the material, the stability of such a species is likely to far exceed the stability of diazeniumdiolates and other NO donors used to date.
In a clinical situation, it should be noted that, even if the amount of reducing equivalent species in the blood were to vary from test subject to test subject, it is possible to add reducing equivalents of an alternate electron donor to the blood, illustratively in the form of ascorbic acid, by administering low doses of Vitamin C to the patient. This may aid in ensuring the presence of adequate levels of reducing equivalents.
While several embodiments of the disclosure have been described in detail, it will be apparent to those skilled in the art that the disclosed embodiments may be modified. Therefore, the foregoing description is to be considered exemplary rather than limiting.
This application is a divisional of U.S. patent application Ser. No. 11/944,034 filed Nov. 21, 2007 now U.S. Pat. No. 8,034,384, which itself is a continuation of U.S. patent application Ser. No. 10/794,878 filed Mar. 5, 2004, now U.S. Pat. No. 7,335,383, which itself is a continuation-in-part of U.S. Ser. No. 10/052,239 filed on Jan. 16, 2002, now U.S. Pat. No. 7,128,904, which itself claims the benefit of U.S. Provisional Patent application Ser. No. 60/262,014 filed on Jan. 16, 2001. Each of the above-identified applications is incorporated by reference herein in its entirety.
This invention was made in the course of research partially supported by a grant from the National Institutes of Health, Grant Number GM 56991. The U.S. government has certain rights in the invention.
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| Number | Date | Country | |
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| Child | 12061271 | US |
| Number | Date | Country | |
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| Child | 11944034 | US |
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| Parent | 10052239 | Jan 2002 | US |
| Child | 10794878 | US |
