Title:
METHOD FOR MAKING NONTHROMBOGENIC SURFACES
United States Patent 3634123
Abstract:
The specification discloses a method for reducing thrombosis of blood induced by contact with a foreign surface by treating the surface with a cationic surface-active agent, and a conventional anticoagulant, such as heparin.
Inventors:
Eriksson, Jan Christer (Bromma, SW)
Lagergren, Hans Ragnar (Stockholm, SW)
Johansson, Anders Lennart (Enebyberg, SW)
Gillberg, Elsa Gunilla (Bromma, SW)
Lagergren, Hans Ragnar (Stockholm, SW)
Johansson, Anders Lennart (Enebyberg, SW)
Gillberg, Elsa Gunilla (Bromma, SW)
Application Number:
04/738826
Publication Date:
01/11/1972
Filing Date:
06/21/1968
View Patent Images:
Export Citation:
Primary Class:
Other Classes:
428/522, 428/523, 514/56, 424/447, 427/343
International Classes:
C12M1/00; C12Q1/56; A61K17/18
Field of Search:
117/138.8B,138.8E,138.8UA,62.1,118,47A 424/28,183 128/DIG.22 3/1,DIG.2
Other References:
gott et al., "Heparin Bonding on Colloidal Graphite Surfaces" Science, V. 132 Dec. 1963, pp. 1297-1298.
Primary Examiner:
Martin, William D.
Assistant Examiner:
Cohen D.
Parent Case Data:
This application is a continuation-in-part of our U.S. application Ser. No. 510,355, filed Nov. 29, 1965, now abandoned.
Claims:
We claim
1. A method for retarding coagulation of blood in contact with a plastic surface, which comprises treating such plastic surface with a cationic surface-active agent in an aqueous medium at a temperature sufficiently elevated so as to cause absorption of the cationic agent onto the surface, and treating such plastic surface with heparin.
2. The method of claim 1 wherein the aqueous solution of cationic surface-active agent is degassed before treating said plastic surface.
3. The method of claim 1 wherein the plastic surface is a polyolefin surface.
4. The method of claim 1 wherein the plastic surface is a polyvinyl chloride surface.
5. The method of claim 1 wherein the plastic surface is a silicone resin surface.
6. The method of claim 1 wherein the cationic surface-active agent is a member of the group consisting of primary, secondary and tertiary amines and salts thereof and quaternary ammonium compounds.
7. The method of claim 1 wherein the cationic surface-active agent has an alkyl chain of at least four carbon atoms.
8. The method of claim 1 wherein the plastic surface is first treated with the cationic surface-active agent and is thereafter treated with the heparin.
9. The method of claim 1 wherein said plastic is a thermoplastic.
10. The method of claim 1 wherein said surface-active agent has an alkyl chain of about 12 to 18 carbon atoms.
11. The method of claim 1 wherein the plastic surface is treated with an aqueous solution containing the surface-active agent in a concentration of at least about 10 -4 moles per liter and subsequently treated with an aqueous solution containing the heparin.
12. The method of claim 1 wherein the plastic surface is treated with an aqueous dispersion or emulsion of a complex compound formed by reacting a cationic surface-active agent with heparin.
13. A plastic article suitable for use in contact with blood without significant coagulation, said article having a plastic surface which is to contact the blood, said surface having been treated with a cationic surface-active agent and heparin by the method of claim 1.
14. A plastic article as described in claim 13 wherein the surface-active agent has an alkyl chain of at least four carbon atoms.
15. An article as described in claim 13 wherein said plastic is a thermoplastic.
16. A plastic article as described in claim 13 wherein the surface-active agent has an alkyl chain of about 12 to about 18 carbon atoms.
17. A plastic article as described in claim 13 wherein said plastic is a polyolefin.
18. An article as described in claim 13 wherein said plastic is a polyvinyl chloride.
19. An article as described in claim 13 wherein said plastic is a silicone resin.
20. An article as described in claim 13 wherein said surface-active agent is a member of the group consisting of primary, secondary and tertiary amines and salts thereof and quaternary ammonium compounds.
1. A method for retarding coagulation of blood in contact with a plastic surface, which comprises treating such plastic surface with a cationic surface-active agent in an aqueous medium at a temperature sufficiently elevated so as to cause absorption of the cationic agent onto the surface, and treating such plastic surface with heparin.
2. The method of claim 1 wherein the aqueous solution of cationic surface-active agent is degassed before treating said plastic surface.
3. The method of claim 1 wherein the plastic surface is a polyolefin surface.
4. The method of claim 1 wherein the plastic surface is a polyvinyl chloride surface.
5. The method of claim 1 wherein the plastic surface is a silicone resin surface.
6. The method of claim 1 wherein the cationic surface-active agent is a member of the group consisting of primary, secondary and tertiary amines and salts thereof and quaternary ammonium compounds.
7. The method of claim 1 wherein the cationic surface-active agent has an alkyl chain of at least four carbon atoms.
8. The method of claim 1 wherein the plastic surface is first treated with the cationic surface-active agent and is thereafter treated with the heparin.
9. The method of claim 1 wherein said plastic is a thermoplastic.
10. The method of claim 1 wherein said surface-active agent has an alkyl chain of about 12 to 18 carbon atoms.
11. The method of claim 1 wherein the plastic surface is treated with an aqueous solution containing the surface-active agent in a concentration of at least about 10 -4 moles per liter and subsequently treated with an aqueous solution containing the heparin.
12. The method of claim 1 wherein the plastic surface is treated with an aqueous dispersion or emulsion of a complex compound formed by reacting a cationic surface-active agent with heparin.
13. A plastic article suitable for use in contact with blood without significant coagulation, said article having a plastic surface which is to contact the blood, said surface having been treated with a cationic surface-active agent and heparin by the method of claim 1.
14. A plastic article as described in claim 13 wherein the surface-active agent has an alkyl chain of at least four carbon atoms.
15. An article as described in claim 13 wherein said plastic is a thermoplastic.
16. A plastic article as described in claim 13 wherein the surface-active agent has an alkyl chain of about 12 to about 18 carbon atoms.
17. A plastic article as described in claim 13 wherein said plastic is a polyolefin.
18. An article as described in claim 13 wherein said plastic is a polyvinyl chloride.
19. An article as described in claim 13 wherein said plastic is a silicone resin.
20. An article as described in claim 13 wherein said surface-active agent is a member of the group consisting of primary, secondary and tertiary amines and salts thereof and quaternary ammonium compounds.
Description:
The instant invention is directed to a method for reducing the tendency of blood to coagulate when brought into contact with foreign surfaces and to a method for treating a foreign surface to render it nonthrombogenic.
The tendency of blood to coagulate when brought into contact with a foreign surface causes considerable difficulty in many fields of medicine and medical technology. In surgical operations involving the use of an extra corporeal circulatory system, e.g., a heart-lung machine or an artificial kidney, it is usually necessary to employ an anticoagulant to prevent thrombosis. Similarly, in cardiovascular surgery it is often desirable to use artificial blood vessels and valves made of synthetic material. At present this technique is possible to a limited extent only, since available materials usually induce thrombosis after having been incorporated into the body. Therefore, although artificial heart valves having satisfactory mechanical and hydromechanical properties are available, their use is limited because of the danger of thrombosis.
When blood is stored, sodium citrate is often added to retard coagulation. The citrate, however, may produce undesirable physiological effects during blood transfusions and often interferes with blood analyses. It would be advantageous if such difficulties could be avoided by storing blood in such a way as to prevent contact between the blood and surfaces which have a tendency to promote coagulation. The term "foreign surfaces," as used herein, refers to surfaces of a material, other than body tissue, which normally induces coagulation or thrombosis of blood.
Accordingly, the principal object of this invention is to provide surfaces which do not promote coagulation.
Another object of the invention is to provide a means for preventing or diminishing blood coagulation induced by contact with foreign surfaces.
A further object of the invention is to provide a method for treating a surface to reduce the incidence of blood coagulation seen in the absence of conventional anticoagulants.
A still further object of the invention is to provide a method for binding a conventional anticoagulant to a foreign surface, thereby providing a nonthrombogenic surface.
Another object of the invention is to provide instruments, conduits, containers, membranes, and the like, suitable for use in contact with blood.
These and other related objects are achieved by a method which comprises treating a surface with a surface-active agent at an elevated temperature whereby the hydrocarbon portion of the surface-active agent is bound to the surface.
In a particular embodiment of the invention after treating the surface with the cationic surface-active agent, the treated surface is further treated with or exposed to a conventional anticoagulant, such as heparin and other sulfuric acid esters of mucopolysaccarides.
It has been found that a paraffin surface which has been treated with a cationic surface-active agent having an excess of positive hydrophilic groups is characterized by a prolonged coagulation time in vitro. It has also been discovered that the coagulation time can be further increased by subsequent treatment of the surface with an anticoagulant, such as heparin.
The most important natural anticoagulant, identified as heparin, occurs in the body as its sodium salt. The active component of heparin is a negative ion having a large number of sulfate and sulfonate groups. It has now been found possible to bind heparin, by chemisorption, to a surface which bears positive hydrophilic groups. Similarly, an anticoagulant having positively charged active groups can be fixed to a surface having negatively charged groups, e.g., by means of an anionic surface-active agent.
Surface-active agents, e.g., anionic and cationic surfactants, can be fixed to a surface by contacting the surface with the surface-active agent at an elevated temperature. The elevated temperature is believed to increase the permeability of the molecular structure of the plastic. The increased permeability allows the hydrophobic end of the molecule, e.g., the alkyl chain of the surface-active molecule, to become primarily fixed to the plastic surface. Subsequent reduction of the permeability of the plastic surface, by lowering the temperature, causes the primarily fixed molecules of the surface-active agent to become permanently bound to the plastic surface. The surface-active agent thus fixed to the plastic surface cannot be dislodged by normal mechanical methods or by washing the surface with a solvent at temperatures below the fixing temperature. It is possible, however, to remove the surface-active agent by bringing the plastic surface into contact with a solvent or the surface-active agent at a temperature which is approximately equal to or higher than the temperature utilized for fixing the surface, active materials to the surface.
The method of this invention is applicable to the treatment of a wide variety of materials including glass and metal surfaces. Glass and metal articles are first coated with suitable plastics before treatment. The preferred materials are plastics, including polyethylene, polypropylene, polyvinyl chloride, polystyrene, polytetrafluorethylene, and the like. It is possible to utilize this invention in the treatment of articles comprising inert polyolefins and also of the general class of thermoplastics. Elastomers and cellulose derivatives can also be rendered nonthrombogenic by this invention.
In order to form a firm bond between the surface and the surface-active agent it is preferred to use a surface-active agent containing an alkyl chain of at least four carbon atoms. Surface-active agents having a long hydrocarbon chain are preferred, particularly those containing a hydrocarbon chain of from about 12 to about 18 carbon atoms.
Suitable cationic surface-active agents can be of a varied nature and include primary, secondary, tertiary amines, and their salts, as well as quaternary ammonium compounds, pyridinium, and guanidium salts which have at least one alkyl group with a chain length longer than two carbon atoms and preferably longer than about eight carbon atoms. In the secondary, tertiary, and quaternary ammonium compounds, respectively, the nitrogen atom can bear one, two and three hydrocarbon atoms, such as a lower alkyl group, e.g., methyl, ethyl, or propyl; a benzyl, or an alkylol group, or the nitrogen atom can bear one or two hydrocarbon groups having an arbitrary chain length.
Another class of suitable cationic surface-active agents are the alkyl ammonium salts of the formula:
XNH 3 --(CH 2) y NH 3 X
wherein X is a halogen and y is a number of at least about 4 and preferably from about 8 to 18 .
Since primary amines provide stronger heparin complexes than other ammonium salts of equivalent chain length, it is preferred to use primary amines as the surface-active agents in the treatment of chemically inert plastics.
The primary amines are generally employed in the salt form, i.e., as a salt made by adding HC1, HBr or HI. Water solutions of salts of primary amines are preferred since the amine molecules absorbed onto the plastic surface from these solutions are to a large extent ionized whereas this is not normally the case for solutions formed by dissolving amine in e.g., organic solvents. The Krafft point is the temperature at which the solubility of the surface-active agent suddenly increases and provides homogeneous solutions. Absorbed amine molecules are more difficult to rinse off. Accordingly, if water or an aqueous solution is used for rinsing the plastic articles after treatment with the surface-active agent, the temperature of the water should preferably be above the Krafft point of the surface-active agent.
When using an anticoagulant having positively charged reactive groups, e.g., protamine hydrochloride, the surface-active agent should be anionic. For instance, a surface can be treated with sodium cetyl sulfate and subsequently treated with protamine hydrochloride.
In the practice of this invention, the surface-active agent can be used in the form of an aqueous solution. For example, an article having a plastic surface and the aqueous solution of the surface-active agent are heated, if necessary in an autoclave, to an elevated temperature near or above the softening temperature of the plastic. Generally, when treating polyolefins, the temperature should preferably be at least about 80°C. The concentration of the surface-active agent in the aqueous solution in equilibrium with the surface-active agent absorbed onto the surface of the plastic article should preferably be higher than about one-third the so-called critical micelle concentration which is characteristic of the surface-active agent. This assures that a monolayer of high concentration of the surface-active agent is provided on the surface. It is usually preferred to start with an aqueous solution containing from about 10 -4 to about 10 -4 moles of surface-active agent per liter of solution. The aqueous solution of the surface-active material should preferably be degassed at a temperature of about 100°C. In order to remove oxygen and other gases which might impair absorption. Degassing is particularly important when treatment is conducted in an autoclave, as otherwise an undesired oxidation of a polymer surface might occur at this high temperature.
Water solutions of primary amines preferably have a pH above about 4 at about 55°C. when the concentration is 0.005 moles per liter. Suitable solutions can be prepared by dissolving a sufficient amount of an amine salt in water or by neutralizing the free amine, e.g., in an aqueous solution with an aqueous acid. If it is desired to transform the entire amount of free amine to the salt form, precautions should be taken to avoid use of excessive amounts of acid, which, if present during treatment of the surface, may interfere with absorption of the surface-active molecule. Suitable solutions can be prepared by dissolving the amine, preferably a primary amine, in a small volume of ethanol, and adding the resulting solution, at room temperature with vigorous stirring to an aqueous solution containing an equivalent amount of acid. Stirring is continued for about 10 minutes at room temperature to disperse the entire amount of amine in the liquid and neutralize it with the acid. The temperature is then raised to a temperature above the Krafft point of the actual ammonium salt, at which point heating may be stopped. However, it is preferred to heat the solution to about 90°C., so that dissolved gases, e.g., oxygen and carbon dioxide, are removed.
In some cases, washing of the plastic surface with an organic solvent facilitates fixing of the surface-active agent to the surface, probably due to cleaning or swelling of the plastic surface as a result of contact with the organic solvent. If an organic solvent of hydrophobic character is employed, it is preferred to rinse the washed plastic surface with a hydrophilic organic solvent, e.g., acetone, before treatment of the plastic surface with the aqueous solution of the surface-active agent.
It is also possible to treat a plastic surface with a liquid which is not a true solution of the surface-active agent but rather a dispersion of the surface-active material, i.e., an emulsion or suspension. The surface-active agent may be dispersed in water or an organic liquid, preferably a lower alcohol. The plastic surface is treated with the dispersion of the surface-active agent at an elevated temperature for a time sufficient to insure fixing of the desired concentration of surface-active molecules on the surface.
When the surface has been exposed to the solution or dispersion at an elevated temperature for a long enough time to insure that a proper amount of surface-active material is provided, the temperature is reduced to ensure firm bonding of the surface-active molecules to the surface. Generally, the temperature should be reduced by at least about 20° C., and it is preferred to reduce the temperature to a level of from about 40° C. to about 50° C. The reduction in temperature may be accomplished while the plastic article is immersed in the dispersion or solution or the article may be removed from the liquid and allowed to cool in air.
It is also possible to treat a plastic surface by wetting the surface with a solution or dispersion of a surface-active agent and subsequently heating the air or an inert gas to the elevated temperature necessary to fix the surface-active molecules to the plastic surface. The heating first evaporates the solvent, leaving a thin film of the surface-active molecules on the surface of the articles. Subsequent heating produces an increased mobility of the chains of the plastic, allowing the alkyl chain of the dry surface-active agent to penetrate into the plastic.
A suitable nonthrombogenic surface can be provided by treating the plastic surface, with a surface-active agent anticoagulant complex prepared by reacting a surface-active agent, preferably a cationic agent, with an anticoagulant, to form a complex compound. The treatment of the surface is conducted at elevated temperatures, as discussed herein. Formation of the complex does not significantly impair the thrombosis-retarding capacity of the anticoagulant. A blood anticoagulant, such as heparin, is a polyelectrolyte having a large number or ionic groups to which the surface-active molecules can be attached. The ionic groups of heparin occur in a regular pattern and it is a theory of ours that the positions of the ionic groups of the surface-active molecules should be consistent with the regular pattern of the ionic groups of the heparin molecule if a maximum amount of heparin is to be bound to a surface having a given number of surface-active molecules.
This consistency may not always be obtained if the surface is first treated with the surface-active agent alone, as these molecules may become fixed to the surface in an irregular pattern, especially at a low surface concentration of the surface-active agent. By reacting the anticoagulant with the surface-active agent in an aqueous solution to form an emulgated or dispersed complex and fixing the complex to the surface, this irregularity can be diminished. For example, heparin can be reacted with cetyltrimethylammonium bromide and a plastic article can be treated with an aqueous dispersion of the resulting complex compound. Binding of the complex compound to the plastic is effected by penetration of the plastic surface by the hydrocarbon chains of the cetyltrimethylammonium portion of the molecule.
Since certain plastics of an extremely inert nature, e.g., polytetrafluoroethylene, require a treatment temperature which is above the decomposition point of heparin, particularly when operating in an acidic solution, it is frequently desirable to treat the article with a solution of a chemical compound having an alkyl chain and an ionic group in its molecule, such as cetyl amine hydrochloride, at a high temperature, and subsequently contact the treated surface at a lower temperature with a dispersion of a surface-active agent anticoagulant compound, as described above. In this manner, binding of the complex compound to the surface is effected by means of an ionic bond between the cetyl amine hydrochloride and the heparin.
It is desired that the concentration of heparin on the surface of the plastic article shall be at least 0.1 International Unit (IU) per square centimeter. Consequently, the concentration of heparin in the aqueous dispersion or emulsion of a cation-heparin complex compound should preferably be so high that said surface concentration is obtained. The ratio of cationic surface agent to heparin should preferably be from about 10 -7 to 10 -8 mole of surface-active agent per International Unit of heparin. Suitable cationic surface-active agents include monoamines having a long hydrocarbon chain, in acid solution, alkyl pyridinium chloride and alkyl trimethyl ammonium halides, e.g., chloride bromide or iodide, containing a long chain alkyl radical.
The data in table I, below, illustrates the significance of the length of the hydrocarbon chain.
In these experiments plates of polypropylene with a surface area of 5 square centimeters were treated with aqueous solutions of various surface-active agents at elevated temperatures, as shown. After cooling the plates were washed four times with 0.9 percent sodium chloride solution at 40° C. Each wash had a duration of 10 minutes. The plates where then treated, at 70° C., with an aqueous solution containing 0.9 percent sodium chloride and 2.5 IU heparin per milliliter. The heparin has been labeled with the radioactive isotope tritium. The plates were then washed at 40° C. with 200 milliliters of 0.9 percent sodium chloride solution for longer periods, and subsequently with distilled water; the plates used in experiments 6-9 were additionally washed with 200 millimeters of citrate blood at 40° C.
In the experiments 1-5 the surface-active agent consisted of an alkyl-ammonium salt of the type
where R is a hydrocarbon chain having at least 12 carbon atoms, as shown, X is a negative monovalent ion, e.g., chlorine or bromine, and R 1 , R 2 , and R 3 can be the same or different members of the group consisting of hydrogen, and lower alkyl.
In experiments 6-8 the surface-active agent consisted of an alkyl ammonium salt of the type R--NH 3 Cl, where R is a hydrocarbon chain with not more than six carbon atoms, and in experiment 9, the surface-active agent was comprised of an alkyl ammonium salt of the formula:
ClNH 3 --(CH 2) 12 --NH 3 Cl
The results are given in table 1, which states the type of radicals, the treatment time, the treatment temperature, the concentration of the surface-active agent in the solution, and the results of measurement of radioactivity as the number of decays observed per minute. The radioactivity values represent a measure of the quantity of heparin bound to the plastic surface.
The activity of the heparin differs in experiments 1-5 from the activity of the heparin in experiments 6-9. Therefore, these two series of experiments are not directly comparable. ##SPC1##
The experiments 1-5 indicate that a free hydrocarbon chain with 16-18 carbon atoms gives approximately the same degree of fixing between the surface-active agent and the plastic surface, irrespective of what other radicals are bound to the amine group. Reduction of the number of carbon atoms to 12 produces a significant decrease in the degree of fixing between the surface-active agent and the plastic surface. Experiments 6-8 indicate that the degree of fixing between the surface-active agent and the plastic surface is largely dependent on the length of the hydrocarbon chain. Experiment 9 shows that a hydrocarbon chain with ammonium groups at both ends permits fixing, although to a lower degree than with a free hydrocarbon chain.
To illustrate that absorption of the surface-active agent on a plastic surface is relatively independent of the nature of the solvent, the following experiments were made.
Plates of polypropylene with a surface of 5 square centimeters were treated with a solution of C 14 labeled cetyltrimethylammonium bromide (CTAB) at an elevated temperature. The solvents used were water, ethanol, and cyclohexane containing 0.5 percent by weight of water. After cooling, the plates were washed four times with an isosmotic salt solution and then with distilled water. To determine the amount of surface-active agent primarily absorbed on the surface, the number of decays per minute were determined. The plates were than repeatedly washed at 40° C. with 200 milliliters of 0.9 percent sodium chloride solution for 1 hour until the measured radioactivity of the plates remained constant, i.e., until loosely adsorbed surface-active agent molecules had been removed.
Plates identically treated as above, but with nonlabeled CTAB, were treated with an aqueous solution containing 0.9 percent sodium chloride and 2.5 IU of tritium-labeled heparin per milliliter at 70° C. for 1 hour. The plates were then washed at 40° C. with 200 milliliters of an isosmotic salt solution for longer periods until a stable radioactive level was obtained. The results are given in table 2 which shows the type of solvent used, the surface-active agent concentration, treatment time and temperature, and the stable levels of heparin activity which are a measure of the quantity of heparin united to the plastic surface. ------------------------------------------------------------ --------------- TABLE 2
Radioactiv- ity of Hep- CTAB--conc. Time Temp. arin Decays Solvent mol/l. Hr. ° C. per Min. ____________________________________________________________ ______________ H 2 O 3 . 10 - 5 2 130 8,000 ethanol 1 . 10 - 3 2 60 9,560 cyclohexane 1 . 10 - 4 1 80 12,600 ____________________________________________________________ ______________
Table 3 shows some examples of suitable plastics and suitable treatment time and temperature for such plastics. In these tests plastic plates were treated with an aqueous solution of cetyltrimethylammonium bromide. The concentration of the solution was 3 . 10 - 5 mol/1., except for the first two plastics mentioned in the table for which a concentration of 7 . 10 - 5 mol/1. was used. In general, a somewhat higher concentration is to be preferred, viz, about 10 - 4 to about 10 - 2 mol/1. The cetyltrimethylammonium bromide was labeled with radioactive isotope C 14, which made it possible to determine the quantity of surface-active agents fastened to the surface. Any quantity of surface-active agent loosely adhering to the surface was first removed by thorough washing. Table 3 shows the result of the radioactivity measurement, as number of decays per minute. The results obtained for one plastic are not directly comparable to that of another plastic due to differences in surface area. ------------------------------------------------------------ --------------- TABLE
3 Time Temp. Radio- Plastics Hr. ° C. activity ____________________________________________________________ ______________ copolymer of styrene and propylene 2 130 1,000 copolymer of styrene and acrylic nitrile 2 120 3,000 polyethylene 2 98 48,500 polypropylene 2 140 42,800 polystyrene 2 100 3,200 polyvinylchloride 1 100 220,000 polytetrafluorethylene 1 180 3,000 polyamide (Nylon 66) 2 170 83,000 polyamide (Nylon 6) 2 l30 270,000 acetalcopolymerisate 2 150 8,000 polycarbonate 2 130 17,700 cellulose acetate 1 100 24,100 nitrile rubber 2 100 360,700 silicon rubber 2 160 7,900 ____________________________________________________________ ______________
metal articles can be provided with a coating of a plastic, and such plastic surface subsequently treated to make it coagulation retarding.
The following examples further illustrate the invention.
EXAMPLE 1
Small tubes of polyethylene are treated with an aqueous solution of cetyltrimethylammonium bromide having a concentration of 0.0005 mole per liter, for 24 hours at 100° C. The tubes are allowed to cool down at 70° C. in the solution. They are now rinsed five times with an isosmotic solution of sodium chloride and 10 times with distilled water. They are now allowed to stand for 1 hour in an isosmotic sodium chloride solution containing 100 IU of heparin per milliliter. The tubes are now rinsed one time with an aqueous solution of cetyltrimethylammonium bromide having a concentration of 0.0002 mole per liter and 10 times with distilled water. In the tubes, thus treated, blood begins to coagulate after an average of 80 minutes. After having been transferred from said tubes into a glass container, the blood coagulates after 5- 20 minutes.
EXAMPLE 2
Small tubes of polypropylene are treated with an acidified aqueous solution of cetylamine, having a concentration of 0.1 mole per liter, for 24 hours at 150° C. The tubes are rinsed five times with distilled water and are treated with a solution of heparin, having a concentration of 200 IU per milliliter for 15 minutes. The treated tubes are rinsed five times with an isosmotic solution of sodium chloride. Blood in contact with the tubes, thus treated, does not coagulate after 1 hour.
EXAMPLE 3
Two milliliters of an aqueous solution of heparin having a concentration of 5,000 IU per milliliter are added, with stirring, to 1 liter of an aqueous solution of cetylpyridinium bromide having a concentration of 0.001 mole per liter. A polyethylene surface having a size of 20 square decimeters is treated in the emulsion thus prepared for 24 hours at 83° C. The surface is now repeatedly rinsed with an isosmotic solution of sodium chloride. Blood is brought into contact with the surface thus treated in an experiment in vitro. No coagulation can be found after 2 hours. After having been in contact with said surface and having been transferred to a container of a material, such as glass, which promotes coagulation, the blood will coagulate after 10- 15 minutes.
EXAMPLE 4
Two milliliters of an aqueous solution of heparin containing 5,000 IU per milliliter are added, while stirring, to 1 liter of an aqueous solution of cetyltrimethylammonium bromide having a concentration of 0.001 mole per liter. A polypropylene surface having a size of 17 square decimeters is treated in the emulsion thus prepared for 2 hours at 165° C. The surface is now repeatedly rinsed with an isosmotic solution of sodium chloride. Blood is brought into contact with the surface thus treated in an experiment in vitro. No coagulation can be found after 2 hours. After having been in contact with said surface and having been transferred to a container of a material, such as glass, which promotes coagulation, the blood will coagulate after 10- 15 minutes.
EXAMPLE 5
Small tubes of polyethylene are treated for 36 hours at 93° C. with an emulsion prepared by mixing 500 milliliters of a heparin solution containing 40.000 IU of heparin and 500 milliliters of an aqueous solution of cetyltrimethylammonium bromide having a concentration of 0.002 mole per liter. The tubes are allowed to cool down in the emulsion to 45° C., and are subsequently rinsed one time with an aqueous solution of cetylpyridinium bromide having a concentration of 0.1 mole per liter and 10 times with distilled water. Blood is stored in the tubes thus treated. No coagulation can be found after 2 hours. After having been transferred from tubes thus treated into a glass container, the blood will coagulate in the normal time.
EXAMPLE 6
Small tubes of polypropylene are treated with an emulsion as described in example 5, except that the temperature is 150° C. After cooling the tubes are rinsed 10 times with distilled water and one time with an isosmotic solution of sodium chloride. No coagulation can be found in blood having been stored for 2 hours in tubes thus treated. After having been transferred to a glass container the blood will coagulate in the normal time.
EXAMPLE 7
Five hundred milliliters of an aqueous solution of cetyltrimethylammonium bromide having a concentration of 0.002 mole per liter are mixed with 500 milliliters of a solution of heparin containing 40,000 IU of heparin. Small containers of polystyrene are treated with the emulsion thus prepared for 2 hours at 80° C. The containers are allowed to cool down in the emulsion, and are now rinsed 10 times with distilled water and two times with an isosmotic solution of sodium chloride. When stored in such containers blood will begin to coagulate after approximately 80 minutes.
EXAMPLE 8
An emulsion is prepared as described in example 7. Tubes of polyvinyl chloride are treated in the emulsion for 15 minutes at 120° C., and are subsequently rinsed 10 times with distilled water and two times with an isosmotic solution of sodium chloride. When stored in such tubes blood will begin to coagulate after approximately 60 minutes.
EXAMPLE 9
Small tubes of polyethylene are treated with an acidified aqueous solution of octadecylamine (0.005 mole) for 17 hours at 98° C. The tubes are allowed to cool down to 50° C. in the solution of the surface-active agent. They are now rinsed four times, 10 minutes at a time, with 50° C. isosmotic solution of sodium chloride. The tubes are then treated for 4 hours at 70° C. with an isosmotic sodium chloride solution containing 2.5 IU of heparin per milliliter and then rinsed for 1 hour at 40° C. with an isosmotic salt solution. Blood stored in these tubes shows no signs of coagulation after 3 hours storage and gives, when transferred to glass tubes, normal coagulation times.
EXAMPLE 10
Small tubes of polypropylene are treated with a 0.005-molar cetylammonium chloride aqueous solution for 15 hours at 130° C. After cooling in the solution the tubes are rinsed four times, 10 minutes at a time, with an isosmotic salt solution at 40° C. The tubes are then treated for 2 hours at 70° C. with an isosmotic salt solution containing a 2 IU of heparin per milliliter. The tubes are then treated for 1 hour at 40° C. with an aqueous solution containing a cetylammonium chloride (0.01 weight percent) and sodium chloride (0.9 weight percent). The tubes are then rinsed five times with a 40° C. isosmotic salt solution. Blood is stored in these tubes for 2 hours. There is no sign of coagulation. The tubes are now carefully rinsed 10 times with an isosmotic salt solution. Fresh blood is then again brought into contact with the surface. After 2 hours storage without coagulation this blood is poured out and the tubes are again rinsed 10 times with an isosmotic salt solution and more blood is brought into contact with the treated tubes. After a 2-hour storage period the blood in these tubes is transferred, without coagulation, to untreated glass tubes. A normal coagulation time is now obtained.
EXAMPLE 11
Tubes of polycarbonate were treated for 3 hours at about 112° C. in a water solution of octadecylamine. The amine concentration was 0.002-molar and the amine was neutralized with hydrochloric acid to a pH of 7. The amine solution was poured off at 60° C. and the tubes were immediately rinsed four times with 60° C. isotonic salt solution, once with 60° C. 0.01-molar hydrochloric acid, and five times with 60° C. isotonic salt solution. The tubes were treated for 2 hours at 60° C. with an isotonic salt solution containing 2.5 IU per mole of heparin and afterwards the tubes were rinsed 10 times with 40° C. isotonic salt solution. The tubes were then filled with blood. No coagulation was observed after 3 hours storage at 37° C.
EXAMPLE 12
Cetylamine (0.0005 mole) was added at room temperature to 1 liter of water containing 0.0005 mole of hydrochloric acid. After 10 minutes of vigorous stirring, the solution was heated. The pH of the solution at 55° C. was equal to 4.4. When the temperature has increased to 90° C., polypropylene tubes were placed in the solution. The heating was now continued in an autoclave. At 100° C. the autoclave was degassed and was then kept at 145° C. for 17 hours. The autoclave was allowed to cool to 60° C., and the tubes were immediately rinsed carefully 10 times with 50° C. isotonic salt solution. The tubes were now treated for 4 hours at 70° C. with an isotonic salt solution containing 2.5 IU per milliliter of heparin. The tubes were rinsed 10 times with a 40° C., 10 percent sodium chloride solution. The tubes were now filled with blood. No coagulation was observed after 3 hours storage at 37° C.
EXAMPLE 13
Plates of silicone resin were treated for 14 hours at 160° C. with a 0.005-molar cetylamine hydrochloride aqueous solution. The solution was allowed to cool to 60° C., and the plates were immediately rinsed three times at 50° C. for 10 minutes with an isotonic salt solution and then 10 times with a 40° C. isotonic salt solution. The plates were divided into two groups and now treated with heparin solutions in two different ways:
a. One group of plates was treated for 4 hours at 70° C. with an isotonic salt solution containing 2.5 IU per milliliter heparin which had been labeled with tritium.
b. The other group of plates was treated for 4 hours at 70° C. in a dispersion of complex compound of heparin-cetylammonium chloride. The complex compound was produced by reacting an aqueous solution containing 12 IU per milliliter of heparin with a 0.0003-molar cetylamine-hydrochloride aqueous resulting in a heparin having three-fourths of its negative groups bound as a complex compound. The heparin was labeled with tritium.
Both groups of plates were now rinsed four times for one-half hour at 40° C. with an isotonic salt solution, for 1 hour at 40° C. with citrate blood, and 10 times for 5 minutes at 40° C. with isotonic salt solutions. Radioactive measurements showed that in both groups the heparin molecules had been firmly adsorbed to the surface of the plates.
EXAMPLE 14
Woven articles of fibers of polytetrafluorethylene were treated in a 0.005-molar cetylamine hydrochloride aqueous solution either for 4 hours at 230° or for 18 hours at 195° C. The amine solution in both series was allowed to cool to 60° C. The articles were now rinsed 20 times with 50° C. isotonic salt solution. The articles of both series were treated with heparin solutions as described in paragraphs (a) and (b) of example 13, and were subsequently rinsed as described in said example. In all articles thus produced the heparin was firmly adsorbed to the surface.
EXAMPLE 15
An artificial heart valve consisted of parts of tantalum meta, woven pieces of polytetrafluoroethylene and a ball made from a silicone resin. The parts were treated in the following way in an aqueous 0.005 molar cetylamine hydrochloride solution.
The silicone resin ball was treated in a solution at 160° C. for 14 hours. The tantalum and the polytetrafluoroethylene parts were treated in the solution at 195° C. for 14 hours. In both cases, the amine solution was allowed to cool to 60° C., and the parts were carefully rinsed 15 times with an isotonic salt solution at 50° C. The parts were now assembled to form two artificial valves. One of these valves was now treated for 4 hours at 70° C. in an isotonic salt solution containing 2.5 IU per milliliter of heparin. The other valve was treated in an aqueous dispersion of a complex compound of heparin and cetylammonium chloride containing 12 IU per milliliter of heparin and 3 . 10 - 7 moles per milliliter of cetylammonium chloride. The values were now carefully rinsed 10 times with an isotonic salt solution at 40° C. Experiments in vivo revealed that both valves had a nonthrombogenic surface.
EXAMPLE 16
Tubes of polypropylene were treated for 17 hours at 130° C. in 0.5-molar cetyltrimethylammonium bromide aqueous solution, which had been degassed at 100° C. The solution was poured off at 40° C. and the tubes were then rinsed six times with a 40° C. isotonic salt solution. Each rinsing had a duration of 10 minutes.
The tubes were then divided into four groups. One group of tubes was heparinized for 2 hours at 40° C. with an isotonic salt solution containing 2.5 IU per milliliter of heparin.
A second group of tubes was heparinized for 2 hours at 70° C. with an isotonic salt solution containing 10 IU per milliliter of heparin. A third group of the tubes was heparinized for 2 hours at 70° C. with an isotonic salt solution containing 100 IU per milliliter of heparin. A fourth group of the tubes was heparinized for 2 hours at 20° C. with an isotonic salt solution containing 100 IU or milliliter of heparin. All tubes were rinsed 20 times with 40° C. isotonic salt solution after the heparin treatment. At a following in vitro test none of the series produced any signs of coagulation in human blood having been stored for 2 hours in the tubes.
EXAMPLE 17
Polyethylene catheters (length 60- 80 centimeters, diameter 2.5 millimeter) were under sterile conditions inserted via a vein in the backbone of a dog up into Vena Cava Inferior to a level with the heart. Eight catheters had been treated in distilled water for 17 hours at 98° C. (so-called nontreated catheters) and eight catheters had been treated 17 hours at 98° C. in a water solution containing heparin-CTAB-complex produced by reacting 40 IU of heparin per milliliter with 10 116 6 moles of CTAB per milliliter, i.e., three-fourths of the heparin negative groups are neutralized. After a 1-month insertion the dogs were killed and post mortemed. The post mortem findings in dogs with nontreated catheters showed several thrombi adherent to the wall of Vena Cava or a totally occluded vessel and several big pulmonary emboli, while the dogs in which the treated catheters had been inserted, had no pulmonary emboli and, in general, the Vena Cava Inferior was free from thrombus formation except for some cases where one small thrombus was observed.
The tendency of blood to coagulate when brought into contact with a foreign surface causes considerable difficulty in many fields of medicine and medical technology. In surgical operations involving the use of an extra corporeal circulatory system, e.g., a heart-lung machine or an artificial kidney, it is usually necessary to employ an anticoagulant to prevent thrombosis. Similarly, in cardiovascular surgery it is often desirable to use artificial blood vessels and valves made of synthetic material. At present this technique is possible to a limited extent only, since available materials usually induce thrombosis after having been incorporated into the body. Therefore, although artificial heart valves having satisfactory mechanical and hydromechanical properties are available, their use is limited because of the danger of thrombosis.
When blood is stored, sodium citrate is often added to retard coagulation. The citrate, however, may produce undesirable physiological effects during blood transfusions and often interferes with blood analyses. It would be advantageous if such difficulties could be avoided by storing blood in such a way as to prevent contact between the blood and surfaces which have a tendency to promote coagulation. The term "foreign surfaces," as used herein, refers to surfaces of a material, other than body tissue, which normally induces coagulation or thrombosis of blood.
Accordingly, the principal object of this invention is to provide surfaces which do not promote coagulation.
Another object of the invention is to provide a means for preventing or diminishing blood coagulation induced by contact with foreign surfaces.
A further object of the invention is to provide a method for treating a surface to reduce the incidence of blood coagulation seen in the absence of conventional anticoagulants.
A still further object of the invention is to provide a method for binding a conventional anticoagulant to a foreign surface, thereby providing a nonthrombogenic surface.
Another object of the invention is to provide instruments, conduits, containers, membranes, and the like, suitable for use in contact with blood.
These and other related objects are achieved by a method which comprises treating a surface with a surface-active agent at an elevated temperature whereby the hydrocarbon portion of the surface-active agent is bound to the surface.
In a particular embodiment of the invention after treating the surface with the cationic surface-active agent, the treated surface is further treated with or exposed to a conventional anticoagulant, such as heparin and other sulfuric acid esters of mucopolysaccarides.
It has been found that a paraffin surface which has been treated with a cationic surface-active agent having an excess of positive hydrophilic groups is characterized by a prolonged coagulation time in vitro. It has also been discovered that the coagulation time can be further increased by subsequent treatment of the surface with an anticoagulant, such as heparin.
The most important natural anticoagulant, identified as heparin, occurs in the body as its sodium salt. The active component of heparin is a negative ion having a large number of sulfate and sulfonate groups. It has now been found possible to bind heparin, by chemisorption, to a surface which bears positive hydrophilic groups. Similarly, an anticoagulant having positively charged active groups can be fixed to a surface having negatively charged groups, e.g., by means of an anionic surface-active agent.
Surface-active agents, e.g., anionic and cationic surfactants, can be fixed to a surface by contacting the surface with the surface-active agent at an elevated temperature. The elevated temperature is believed to increase the permeability of the molecular structure of the plastic. The increased permeability allows the hydrophobic end of the molecule, e.g., the alkyl chain of the surface-active molecule, to become primarily fixed to the plastic surface. Subsequent reduction of the permeability of the plastic surface, by lowering the temperature, causes the primarily fixed molecules of the surface-active agent to become permanently bound to the plastic surface. The surface-active agent thus fixed to the plastic surface cannot be dislodged by normal mechanical methods or by washing the surface with a solvent at temperatures below the fixing temperature. It is possible, however, to remove the surface-active agent by bringing the plastic surface into contact with a solvent or the surface-active agent at a temperature which is approximately equal to or higher than the temperature utilized for fixing the surface, active materials to the surface.
The method of this invention is applicable to the treatment of a wide variety of materials including glass and metal surfaces. Glass and metal articles are first coated with suitable plastics before treatment. The preferred materials are plastics, including polyethylene, polypropylene, polyvinyl chloride, polystyrene, polytetrafluorethylene, and the like. It is possible to utilize this invention in the treatment of articles comprising inert polyolefins and also of the general class of thermoplastics. Elastomers and cellulose derivatives can also be rendered nonthrombogenic by this invention.
In order to form a firm bond between the surface and the surface-active agent it is preferred to use a surface-active agent containing an alkyl chain of at least four carbon atoms. Surface-active agents having a long hydrocarbon chain are preferred, particularly those containing a hydrocarbon chain of from about 12 to about 18 carbon atoms.
Suitable cationic surface-active agents can be of a varied nature and include primary, secondary, tertiary amines, and their salts, as well as quaternary ammonium compounds, pyridinium, and guanidium salts which have at least one alkyl group with a chain length longer than two carbon atoms and preferably longer than about eight carbon atoms. In the secondary, tertiary, and quaternary ammonium compounds, respectively, the nitrogen atom can bear one, two and three hydrocarbon atoms, such as a lower alkyl group, e.g., methyl, ethyl, or propyl; a benzyl, or an alkylol group, or the nitrogen atom can bear one or two hydrocarbon groups having an arbitrary chain length.
Another class of suitable cationic surface-active agents are the alkyl ammonium salts of the formula:
XNH 3 --(CH 2) y NH 3 X
wherein X is a halogen and y is a number of at least about 4 and preferably from about 8 to 18 .
Since primary amines provide stronger heparin complexes than other ammonium salts of equivalent chain length, it is preferred to use primary amines as the surface-active agents in the treatment of chemically inert plastics.
The primary amines are generally employed in the salt form, i.e., as a salt made by adding HC1, HBr or HI. Water solutions of salts of primary amines are preferred since the amine molecules absorbed onto the plastic surface from these solutions are to a large extent ionized whereas this is not normally the case for solutions formed by dissolving amine in e.g., organic solvents. The Krafft point is the temperature at which the solubility of the surface-active agent suddenly increases and provides homogeneous solutions. Absorbed amine molecules are more difficult to rinse off. Accordingly, if water or an aqueous solution is used for rinsing the plastic articles after treatment with the surface-active agent, the temperature of the water should preferably be above the Krafft point of the surface-active agent.
When using an anticoagulant having positively charged reactive groups, e.g., protamine hydrochloride, the surface-active agent should be anionic. For instance, a surface can be treated with sodium cetyl sulfate and subsequently treated with protamine hydrochloride.
In the practice of this invention, the surface-active agent can be used in the form of an aqueous solution. For example, an article having a plastic surface and the aqueous solution of the surface-active agent are heated, if necessary in an autoclave, to an elevated temperature near or above the softening temperature of the plastic. Generally, when treating polyolefins, the temperature should preferably be at least about 80°C. The concentration of the surface-active agent in the aqueous solution in equilibrium with the surface-active agent absorbed onto the surface of the plastic article should preferably be higher than about one-third the so-called critical micelle concentration which is characteristic of the surface-active agent. This assures that a monolayer of high concentration of the surface-active agent is provided on the surface. It is usually preferred to start with an aqueous solution containing from about 10 -4 to about 10 -4 moles of surface-active agent per liter of solution. The aqueous solution of the surface-active material should preferably be degassed at a temperature of about 100°C. In order to remove oxygen and other gases which might impair absorption. Degassing is particularly important when treatment is conducted in an autoclave, as otherwise an undesired oxidation of a polymer surface might occur at this high temperature.
Water solutions of primary amines preferably have a pH above about 4 at about 55°C. when the concentration is 0.005 moles per liter. Suitable solutions can be prepared by dissolving a sufficient amount of an amine salt in water or by neutralizing the free amine, e.g., in an aqueous solution with an aqueous acid. If it is desired to transform the entire amount of free amine to the salt form, precautions should be taken to avoid use of excessive amounts of acid, which, if present during treatment of the surface, may interfere with absorption of the surface-active molecule. Suitable solutions can be prepared by dissolving the amine, preferably a primary amine, in a small volume of ethanol, and adding the resulting solution, at room temperature with vigorous stirring to an aqueous solution containing an equivalent amount of acid. Stirring is continued for about 10 minutes at room temperature to disperse the entire amount of amine in the liquid and neutralize it with the acid. The temperature is then raised to a temperature above the Krafft point of the actual ammonium salt, at which point heating may be stopped. However, it is preferred to heat the solution to about 90°C., so that dissolved gases, e.g., oxygen and carbon dioxide, are removed.
In some cases, washing of the plastic surface with an organic solvent facilitates fixing of the surface-active agent to the surface, probably due to cleaning or swelling of the plastic surface as a result of contact with the organic solvent. If an organic solvent of hydrophobic character is employed, it is preferred to rinse the washed plastic surface with a hydrophilic organic solvent, e.g., acetone, before treatment of the plastic surface with the aqueous solution of the surface-active agent.
It is also possible to treat a plastic surface with a liquid which is not a true solution of the surface-active agent but rather a dispersion of the surface-active material, i.e., an emulsion or suspension. The surface-active agent may be dispersed in water or an organic liquid, preferably a lower alcohol. The plastic surface is treated with the dispersion of the surface-active agent at an elevated temperature for a time sufficient to insure fixing of the desired concentration of surface-active molecules on the surface.
When the surface has been exposed to the solution or dispersion at an elevated temperature for a long enough time to insure that a proper amount of surface-active material is provided, the temperature is reduced to ensure firm bonding of the surface-active molecules to the surface. Generally, the temperature should be reduced by at least about 20° C., and it is preferred to reduce the temperature to a level of from about 40° C. to about 50° C. The reduction in temperature may be accomplished while the plastic article is immersed in the dispersion or solution or the article may be removed from the liquid and allowed to cool in air.
It is also possible to treat a plastic surface by wetting the surface with a solution or dispersion of a surface-active agent and subsequently heating the air or an inert gas to the elevated temperature necessary to fix the surface-active molecules to the plastic surface. The heating first evaporates the solvent, leaving a thin film of the surface-active molecules on the surface of the articles. Subsequent heating produces an increased mobility of the chains of the plastic, allowing the alkyl chain of the dry surface-active agent to penetrate into the plastic.
A suitable nonthrombogenic surface can be provided by treating the plastic surface, with a surface-active agent anticoagulant complex prepared by reacting a surface-active agent, preferably a cationic agent, with an anticoagulant, to form a complex compound. The treatment of the surface is conducted at elevated temperatures, as discussed herein. Formation of the complex does not significantly impair the thrombosis-retarding capacity of the anticoagulant. A blood anticoagulant, such as heparin, is a polyelectrolyte having a large number or ionic groups to which the surface-active molecules can be attached. The ionic groups of heparin occur in a regular pattern and it is a theory of ours that the positions of the ionic groups of the surface-active molecules should be consistent with the regular pattern of the ionic groups of the heparin molecule if a maximum amount of heparin is to be bound to a surface having a given number of surface-active molecules.
This consistency may not always be obtained if the surface is first treated with the surface-active agent alone, as these molecules may become fixed to the surface in an irregular pattern, especially at a low surface concentration of the surface-active agent. By reacting the anticoagulant with the surface-active agent in an aqueous solution to form an emulgated or dispersed complex and fixing the complex to the surface, this irregularity can be diminished. For example, heparin can be reacted with cetyltrimethylammonium bromide and a plastic article can be treated with an aqueous dispersion of the resulting complex compound. Binding of the complex compound to the plastic is effected by penetration of the plastic surface by the hydrocarbon chains of the cetyltrimethylammonium portion of the molecule.
Since certain plastics of an extremely inert nature, e.g., polytetrafluoroethylene, require a treatment temperature which is above the decomposition point of heparin, particularly when operating in an acidic solution, it is frequently desirable to treat the article with a solution of a chemical compound having an alkyl chain and an ionic group in its molecule, such as cetyl amine hydrochloride, at a high temperature, and subsequently contact the treated surface at a lower temperature with a dispersion of a surface-active agent anticoagulant compound, as described above. In this manner, binding of the complex compound to the surface is effected by means of an ionic bond between the cetyl amine hydrochloride and the heparin.
It is desired that the concentration of heparin on the surface of the plastic article shall be at least 0.1 International Unit (IU) per square centimeter. Consequently, the concentration of heparin in the aqueous dispersion or emulsion of a cation-heparin complex compound should preferably be so high that said surface concentration is obtained. The ratio of cationic surface agent to heparin should preferably be from about 10 -7 to 10 -8 mole of surface-active agent per International Unit of heparin. Suitable cationic surface-active agents include monoamines having a long hydrocarbon chain, in acid solution, alkyl pyridinium chloride and alkyl trimethyl ammonium halides, e.g., chloride bromide or iodide, containing a long chain alkyl radical.
The data in table I, below, illustrates the significance of the length of the hydrocarbon chain.
In these experiments plates of polypropylene with a surface area of 5 square centimeters were treated with aqueous solutions of various surface-active agents at elevated temperatures, as shown. After cooling the plates were washed four times with 0.9 percent sodium chloride solution at 40° C. Each wash had a duration of 10 minutes. The plates where then treated, at 70° C., with an aqueous solution containing 0.9 percent sodium chloride and 2.5 IU heparin per milliliter. The heparin has been labeled with the radioactive isotope tritium. The plates were then washed at 40° C. with 200 milliliters of 0.9 percent sodium chloride solution for longer periods, and subsequently with distilled water; the plates used in experiments 6-9 were additionally washed with 200 millimeters of citrate blood at 40° C.
In the experiments 1-5 the surface-active agent consisted of an alkyl-ammonium salt of the type
where R is a hydrocarbon chain having at least 12 carbon atoms, as shown, X is a negative monovalent ion, e.g., chlorine or bromine, and R 1 , R 2 , and R 3 can be the same or different members of the group consisting of hydrogen, and lower alkyl.
In experiments 6-8 the surface-active agent consisted of an alkyl ammonium salt of the type R--NH 3 Cl, where R is a hydrocarbon chain with not more than six carbon atoms, and in experiment 9, the surface-active agent was comprised of an alkyl ammonium salt of the formula:
ClNH 3 --(CH 2) 12 --NH 3 Cl
The results are given in table 1, which states the type of radicals, the treatment time, the treatment temperature, the concentration of the surface-active agent in the solution, and the results of measurement of radioactivity as the number of decays observed per minute. The radioactivity values represent a measure of the quantity of heparin bound to the plastic surface.
The activity of the heparin differs in experiments 1-5 from the activity of the heparin in experiments 6-9. Therefore, these two series of experiments are not directly comparable. ##SPC1##
The experiments 1-5 indicate that a free hydrocarbon chain with 16-18 carbon atoms gives approximately the same degree of fixing between the surface-active agent and the plastic surface, irrespective of what other radicals are bound to the amine group. Reduction of the number of carbon atoms to 12 produces a significant decrease in the degree of fixing between the surface-active agent and the plastic surface. Experiments 6-8 indicate that the degree of fixing between the surface-active agent and the plastic surface is largely dependent on the length of the hydrocarbon chain. Experiment 9 shows that a hydrocarbon chain with ammonium groups at both ends permits fixing, although to a lower degree than with a free hydrocarbon chain.
To illustrate that absorption of the surface-active agent on a plastic surface is relatively independent of the nature of the solvent, the following experiments were made.
Plates of polypropylene with a surface of 5 square centimeters were treated with a solution of C 14 labeled cetyltrimethylammonium bromide (CTAB) at an elevated temperature. The solvents used were water, ethanol, and cyclohexane containing 0.5 percent by weight of water. After cooling, the plates were washed four times with an isosmotic salt solution and then with distilled water. To determine the amount of surface-active agent primarily absorbed on the surface, the number of decays per minute were determined. The plates were than repeatedly washed at 40° C. with 200 milliliters of 0.9 percent sodium chloride solution for 1 hour until the measured radioactivity of the plates remained constant, i.e., until loosely adsorbed surface-active agent molecules had been removed.
Plates identically treated as above, but with nonlabeled CTAB, were treated with an aqueous solution containing 0.9 percent sodium chloride and 2.5 IU of tritium-labeled heparin per milliliter at 70° C. for 1 hour. The plates were then washed at 40° C. with 200 milliliters of an isosmotic salt solution for longer periods until a stable radioactive level was obtained. The results are given in table 2 which shows the type of solvent used, the surface-active agent concentration, treatment time and temperature, and the stable levels of heparin activity which are a measure of the quantity of heparin united to the plastic surface. ------------------------------------------------------------ --------------- TABLE 2
Radioactiv- ity of Hep- CTAB--conc. Time Temp. arin Decays Solvent mol/l. Hr. ° C. per Min. ____________________________________________________________ ______________ H 2 O 3 . 10 - 5 2 130 8,000 ethanol 1 . 10 - 3 2 60 9,560 cyclohexane 1 . 10 - 4 1 80 12,600 ____________________________________________________________ ______________
Table 3 shows some examples of suitable plastics and suitable treatment time and temperature for such plastics. In these tests plastic plates were treated with an aqueous solution of cetyltrimethylammonium bromide. The concentration of the solution was 3 . 10 - 5 mol/1., except for the first two plastics mentioned in the table for which a concentration of 7 . 10 - 5 mol/1. was used. In general, a somewhat higher concentration is to be preferred, viz, about 10 - 4 to about 10 - 2 mol/1. The cetyltrimethylammonium bromide was labeled with radioactive isotope C 14, which made it possible to determine the quantity of surface-active agents fastened to the surface. Any quantity of surface-active agent loosely adhering to the surface was first removed by thorough washing. Table 3 shows the result of the radioactivity measurement, as number of decays per minute. The results obtained for one plastic are not directly comparable to that of another plastic due to differences in surface area. ------------------------------------------------------------ --------------- TABLE
3 Time Temp. Radio- Plastics Hr. ° C. activity ____________________________________________________________ ______________ copolymer of styrene and propylene 2 130 1,000 copolymer of styrene and acrylic nitrile 2 120 3,000 polyethylene 2 98 48,500 polypropylene 2 140 42,800 polystyrene 2 100 3,200 polyvinylchloride 1 100 220,000 polytetrafluorethylene 1 180 3,000 polyamide (Nylon 66) 2 170 83,000 polyamide (Nylon 6) 2 l30 270,000 acetalcopolymerisate 2 150 8,000 polycarbonate 2 130 17,700 cellulose acetate 1 100 24,100 nitrile rubber 2 100 360,700 silicon rubber 2 160 7,900 ____________________________________________________________ ______________
metal articles can be provided with a coating of a plastic, and such plastic surface subsequently treated to make it coagulation retarding.
The following examples further illustrate the invention.
EXAMPLE 1
Small tubes of polyethylene are treated with an aqueous solution of cetyltrimethylammonium bromide having a concentration of 0.0005 mole per liter, for 24 hours at 100° C. The tubes are allowed to cool down at 70° C. in the solution. They are now rinsed five times with an isosmotic solution of sodium chloride and 10 times with distilled water. They are now allowed to stand for 1 hour in an isosmotic sodium chloride solution containing 100 IU of heparin per milliliter. The tubes are now rinsed one time with an aqueous solution of cetyltrimethylammonium bromide having a concentration of 0.0002 mole per liter and 10 times with distilled water. In the tubes, thus treated, blood begins to coagulate after an average of 80 minutes. After having been transferred from said tubes into a glass container, the blood coagulates after 5- 20 minutes.
EXAMPLE 2
Small tubes of polypropylene are treated with an acidified aqueous solution of cetylamine, having a concentration of 0.1 mole per liter, for 24 hours at 150° C. The tubes are rinsed five times with distilled water and are treated with a solution of heparin, having a concentration of 200 IU per milliliter for 15 minutes. The treated tubes are rinsed five times with an isosmotic solution of sodium chloride. Blood in contact with the tubes, thus treated, does not coagulate after 1 hour.
EXAMPLE 3
Two milliliters of an aqueous solution of heparin having a concentration of 5,000 IU per milliliter are added, with stirring, to 1 liter of an aqueous solution of cetylpyridinium bromide having a concentration of 0.001 mole per liter. A polyethylene surface having a size of 20 square decimeters is treated in the emulsion thus prepared for 24 hours at 83° C. The surface is now repeatedly rinsed with an isosmotic solution of sodium chloride. Blood is brought into contact with the surface thus treated in an experiment in vitro. No coagulation can be found after 2 hours. After having been in contact with said surface and having been transferred to a container of a material, such as glass, which promotes coagulation, the blood will coagulate after 10- 15 minutes.
EXAMPLE 4
Two milliliters of an aqueous solution of heparin containing 5,000 IU per milliliter are added, while stirring, to 1 liter of an aqueous solution of cetyltrimethylammonium bromide having a concentration of 0.001 mole per liter. A polypropylene surface having a size of 17 square decimeters is treated in the emulsion thus prepared for 2 hours at 165° C. The surface is now repeatedly rinsed with an isosmotic solution of sodium chloride. Blood is brought into contact with the surface thus treated in an experiment in vitro. No coagulation can be found after 2 hours. After having been in contact with said surface and having been transferred to a container of a material, such as glass, which promotes coagulation, the blood will coagulate after 10- 15 minutes.
EXAMPLE 5
Small tubes of polyethylene are treated for 36 hours at 93° C. with an emulsion prepared by mixing 500 milliliters of a heparin solution containing 40.000 IU of heparin and 500 milliliters of an aqueous solution of cetyltrimethylammonium bromide having a concentration of 0.002 mole per liter. The tubes are allowed to cool down in the emulsion to 45° C., and are subsequently rinsed one time with an aqueous solution of cetylpyridinium bromide having a concentration of 0.1 mole per liter and 10 times with distilled water. Blood is stored in the tubes thus treated. No coagulation can be found after 2 hours. After having been transferred from tubes thus treated into a glass container, the blood will coagulate in the normal time.
EXAMPLE 6
Small tubes of polypropylene are treated with an emulsion as described in example 5, except that the temperature is 150° C. After cooling the tubes are rinsed 10 times with distilled water and one time with an isosmotic solution of sodium chloride. No coagulation can be found in blood having been stored for 2 hours in tubes thus treated. After having been transferred to a glass container the blood will coagulate in the normal time.
EXAMPLE 7
Five hundred milliliters of an aqueous solution of cetyltrimethylammonium bromide having a concentration of 0.002 mole per liter are mixed with 500 milliliters of a solution of heparin containing 40,000 IU of heparin. Small containers of polystyrene are treated with the emulsion thus prepared for 2 hours at 80° C. The containers are allowed to cool down in the emulsion, and are now rinsed 10 times with distilled water and two times with an isosmotic solution of sodium chloride. When stored in such containers blood will begin to coagulate after approximately 80 minutes.
EXAMPLE 8
An emulsion is prepared as described in example 7. Tubes of polyvinyl chloride are treated in the emulsion for 15 minutes at 120° C., and are subsequently rinsed 10 times with distilled water and two times with an isosmotic solution of sodium chloride. When stored in such tubes blood will begin to coagulate after approximately 60 minutes.
EXAMPLE 9
Small tubes of polyethylene are treated with an acidified aqueous solution of octadecylamine (0.005 mole) for 17 hours at 98° C. The tubes are allowed to cool down to 50° C. in the solution of the surface-active agent. They are now rinsed four times, 10 minutes at a time, with 50° C. isosmotic solution of sodium chloride. The tubes are then treated for 4 hours at 70° C. with an isosmotic sodium chloride solution containing 2.5 IU of heparin per milliliter and then rinsed for 1 hour at 40° C. with an isosmotic salt solution. Blood stored in these tubes shows no signs of coagulation after 3 hours storage and gives, when transferred to glass tubes, normal coagulation times.
EXAMPLE 10
Small tubes of polypropylene are treated with a 0.005-molar cetylammonium chloride aqueous solution for 15 hours at 130° C. After cooling in the solution the tubes are rinsed four times, 10 minutes at a time, with an isosmotic salt solution at 40° C. The tubes are then treated for 2 hours at 70° C. with an isosmotic salt solution containing a 2 IU of heparin per milliliter. The tubes are then treated for 1 hour at 40° C. with an aqueous solution containing a cetylammonium chloride (0.01 weight percent) and sodium chloride (0.9 weight percent). The tubes are then rinsed five times with a 40° C. isosmotic salt solution. Blood is stored in these tubes for 2 hours. There is no sign of coagulation. The tubes are now carefully rinsed 10 times with an isosmotic salt solution. Fresh blood is then again brought into contact with the surface. After 2 hours storage without coagulation this blood is poured out and the tubes are again rinsed 10 times with an isosmotic salt solution and more blood is brought into contact with the treated tubes. After a 2-hour storage period the blood in these tubes is transferred, without coagulation, to untreated glass tubes. A normal coagulation time is now obtained.
EXAMPLE 11
Tubes of polycarbonate were treated for 3 hours at about 112° C. in a water solution of octadecylamine. The amine concentration was 0.002-molar and the amine was neutralized with hydrochloric acid to a pH of 7. The amine solution was poured off at 60° C. and the tubes were immediately rinsed four times with 60° C. isotonic salt solution, once with 60° C. 0.01-molar hydrochloric acid, and five times with 60° C. isotonic salt solution. The tubes were treated for 2 hours at 60° C. with an isotonic salt solution containing 2.5 IU per mole of heparin and afterwards the tubes were rinsed 10 times with 40° C. isotonic salt solution. The tubes were then filled with blood. No coagulation was observed after 3 hours storage at 37° C.
EXAMPLE 12
Cetylamine (0.0005 mole) was added at room temperature to 1 liter of water containing 0.0005 mole of hydrochloric acid. After 10 minutes of vigorous stirring, the solution was heated. The pH of the solution at 55° C. was equal to 4.4. When the temperature has increased to 90° C., polypropylene tubes were placed in the solution. The heating was now continued in an autoclave. At 100° C. the autoclave was degassed and was then kept at 145° C. for 17 hours. The autoclave was allowed to cool to 60° C., and the tubes were immediately rinsed carefully 10 times with 50° C. isotonic salt solution. The tubes were now treated for 4 hours at 70° C. with an isotonic salt solution containing 2.5 IU per milliliter of heparin. The tubes were rinsed 10 times with a 40° C., 10 percent sodium chloride solution. The tubes were now filled with blood. No coagulation was observed after 3 hours storage at 37° C.
EXAMPLE 13
Plates of silicone resin were treated for 14 hours at 160° C. with a 0.005-molar cetylamine hydrochloride aqueous solution. The solution was allowed to cool to 60° C., and the plates were immediately rinsed three times at 50° C. for 10 minutes with an isotonic salt solution and then 10 times with a 40° C. isotonic salt solution. The plates were divided into two groups and now treated with heparin solutions in two different ways:
a. One group of plates was treated for 4 hours at 70° C. with an isotonic salt solution containing 2.5 IU per milliliter heparin which had been labeled with tritium.
b. The other group of plates was treated for 4 hours at 70° C. in a dispersion of complex compound of heparin-cetylammonium chloride. The complex compound was produced by reacting an aqueous solution containing 12 IU per milliliter of heparin with a 0.0003-molar cetylamine-hydrochloride aqueous resulting in a heparin having three-fourths of its negative groups bound as a complex compound. The heparin was labeled with tritium.
Both groups of plates were now rinsed four times for one-half hour at 40° C. with an isotonic salt solution, for 1 hour at 40° C. with citrate blood, and 10 times for 5 minutes at 40° C. with isotonic salt solutions. Radioactive measurements showed that in both groups the heparin molecules had been firmly adsorbed to the surface of the plates.
EXAMPLE 14
Woven articles of fibers of polytetrafluorethylene were treated in a 0.005-molar cetylamine hydrochloride aqueous solution either for 4 hours at 230° or for 18 hours at 195° C. The amine solution in both series was allowed to cool to 60° C. The articles were now rinsed 20 times with 50° C. isotonic salt solution. The articles of both series were treated with heparin solutions as described in paragraphs (a) and (b) of example 13, and were subsequently rinsed as described in said example. In all articles thus produced the heparin was firmly adsorbed to the surface.
EXAMPLE 15
An artificial heart valve consisted of parts of tantalum meta, woven pieces of polytetrafluoroethylene and a ball made from a silicone resin. The parts were treated in the following way in an aqueous 0.005 molar cetylamine hydrochloride solution.
The silicone resin ball was treated in a solution at 160° C. for 14 hours. The tantalum and the polytetrafluoroethylene parts were treated in the solution at 195° C. for 14 hours. In both cases, the amine solution was allowed to cool to 60° C., and the parts were carefully rinsed 15 times with an isotonic salt solution at 50° C. The parts were now assembled to form two artificial valves. One of these valves was now treated for 4 hours at 70° C. in an isotonic salt solution containing 2.5 IU per milliliter of heparin. The other valve was treated in an aqueous dispersion of a complex compound of heparin and cetylammonium chloride containing 12 IU per milliliter of heparin and 3 . 10 - 7 moles per milliliter of cetylammonium chloride. The values were now carefully rinsed 10 times with an isotonic salt solution at 40° C. Experiments in vivo revealed that both valves had a nonthrombogenic surface.
EXAMPLE 16
Tubes of polypropylene were treated for 17 hours at 130° C. in 0.5-molar cetyltrimethylammonium bromide aqueous solution, which had been degassed at 100° C. The solution was poured off at 40° C. and the tubes were then rinsed six times with a 40° C. isotonic salt solution. Each rinsing had a duration of 10 minutes.
The tubes were then divided into four groups. One group of tubes was heparinized for 2 hours at 40° C. with an isotonic salt solution containing 2.5 IU per milliliter of heparin.
A second group of tubes was heparinized for 2 hours at 70° C. with an isotonic salt solution containing 10 IU per milliliter of heparin. A third group of the tubes was heparinized for 2 hours at 70° C. with an isotonic salt solution containing 100 IU per milliliter of heparin. A fourth group of the tubes was heparinized for 2 hours at 20° C. with an isotonic salt solution containing 100 IU or milliliter of heparin. All tubes were rinsed 20 times with 40° C. isotonic salt solution after the heparin treatment. At a following in vitro test none of the series produced any signs of coagulation in human blood having been stored for 2 hours in the tubes.
EXAMPLE 17
Polyethylene catheters (length 60- 80 centimeters, diameter 2.5 millimeter) were under sterile conditions inserted via a vein in the backbone of a dog up into Vena Cava Inferior to a level with the heart. Eight catheters had been treated in distilled water for 17 hours at 98° C. (so-called nontreated catheters) and eight catheters had been treated 17 hours at 98° C. in a water solution containing heparin-CTAB-complex produced by reacting 40 IU of heparin per milliliter with 10 116 6 moles of CTAB per milliliter, i.e., three-fourths of the heparin negative groups are neutralized. After a 1-month insertion the dogs were killed and post mortemed. The post mortem findings in dogs with nontreated catheters showed several thrombi adherent to the wall of Vena Cava or a totally occluded vessel and several big pulmonary emboli, while the dogs in which the treated catheters had been inserted, had no pulmonary emboli and, in general, the Vena Cava Inferior was free from thrombus formation except for some cases where one small thrombus was observed.