Reduced and controlled surface binding of biologically active molecules

Articles of manufacture which are adapted for use in contact with one or more biologically active agents are coated with a glassy carbohydrate film. The glassy film provides a reduced surface energy coating which exhibits a reduced degree of binding with biologically active agents. Methods for applying the glassy carbohydrate film are disclosed wherein the glassy film is adsorbed directly onto the article surface. The coated articles are for use both in vitro and in vivo where contact with biologically active agents is expected.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates generally to articles which are designed to 
be in contact with biologically active agents. Such articles include 
implant devices and other structures which are designed to be utilized in 
vivo. Such articles also include containers, supports, and transport 
systems wherein biologically active agents are in continual contact with 
the surfaces of the article. More particularly, the present invention 
relates to reducing and thereby controlling the degree to which 
biologically active agents bind to the surfaces of such articles. 
2. Description of Related Art 
Most biologically active agents interact with other molecules present on 
either surfaces or membranes. In fact, the effectiveness of many 
biological systems is dependent on the presence of certain intrinsic 
binding properties between biologically active agents and biological 
surfaces. For example, biological surfaces, such as endothelial linings or 
receptor-embedded cell membranes, incorporate high affinity (energy) 
binding properties to achieve optimal biological function. Although the 
binding properties of biologically active agents is essential for proper 
biological function, there are many situations where binding of these 
biologically active agents to non-biological surfaces presents a problem. 
For example, the coagulation protein factor XII is a biologically active 
agent which binds to healthy vascular endothelial cells. Protein factor 
XII plays an important role in the naturally occurring coagulation 
process. However, when protein factor XII binds to the surface of an 
implanted biomaterial, the result may be a thrombotic or thromboembolic 
complication of the prosthetic device. 
Other situations where reduced surface binding of biologically active 
agents would be desirable include vessels used to transport biologically 
active agents. In these situations, binding of the agent to the wall of 
the transport container results in reduced yield of the transported 
product. In addition, reduced binding would be desirable in a vascular 
prosthesis where interactions of biologically active agents can promote 
complications and reduce the medical utility of the device. For example, 
it would be desirable to reduce surface binding of biologically active 
agents to hip prostheses where the binding of such agents can result in 
denaturization of the agents and the initiation of an inflammatory 
reaction clinically associated with pain and reduced utility of the 
device. 
Another situation where reduced and thereby controlled surface binding of 
biologically active agents would be desirable includes the fabrication of 
biological opto-electronic devices. These devices would provide electronic 
output from electron transporting biologically active molecules responding 
to photoelectric, thermal, or other environmental stimulus. To fabricate 
these devices, only limited numbers of biologically active molecules would 
be deposited ideally on a solid support. Moreover, the reduced and thereby 
controlled binding of the biologically active molecules would ideally not 
result in conformational denaturation of the molecules. 
The non-biological materials which are commonly used in the manufacture of 
biomedical and food service devices include polymers, ceramics and metals, 
most of which have high surface energies. These high surface energies 
result frequently in increased binding of biologically active molecules in 
situations, such as those described above, where such binding is 
undesirable. Accordingly, it would be desirable to provide a treatment for 
the surfaces of such non-biological materials which would effectively 
reduce the surface energy and thereby decrease undesirable binding of 
biologically active agents thereto. 
Over the years, various materials have been developed for use as surface 
modifying agents which reduce the binding of biologically active agents to 
their surfaces. Examples include polymers, such as silicone, polystyrene, 
polyethylene and polytetrafluoroethylene. All of these materials have low 
surface energies. Accordingly, the binding affinities between these 
materials and biologically active agents is reduced. These materials are 
generally used in bulk form, i.e., the entire device is made from the 
materials. 
More recently, different alcohol based compounds have been either 
physically adsorbed or chemically bonded to the surface of non-biological 
materials to reduce the subsequent surface binding of biologically active 
agents. Among the more commonly used are polyethylene glycol and sodium 
heparin. While affording improved resistance to absorption of proteins and 
other biologically active agents, these two exemplary materials are each 
subject to their own specific problems. For example, non-biological 
surfaces, such as immunoaffinity chromatography columns and 
electrophoretic capillaries, have been coated with polyethylene glycol. 
Although such coatings have reduced binding of biologically active agents, 
the nephrotoxic effects of polyethylene glycol are well documented. 
Further, binding of polyethylene glycol to the non-biological surface is 
possible only through various forms of covalent chemistry. 
Sodium heparin is a well-recognized anti-coagulation factor whose use 
entails correlative physiological effects. Most often, sodium heparin is 
covalently bound directly to the non-biological surface or indirectly 
through various carbon chain extenders. In addition, sodium heparin has 
been physically absorbed onto the non-biological surface. Other surface 
modification techniques have involved the coating of electrophoretic 
capillaries with phosphate moieties and conventional silanes and 
polyacrylimides. 
Other attempts at reducing the surface activity of non-biological materials 
have involved the covalent bonding of maltose to silica substrates wherein 
an additional silicone-based intermediate moiety 
(3-aminopropyltriethoxysilane) is covalently bound to both the 
fused-silica capillary walls and the disaccharide. In another procedure, 
cellulose has been absorbed onto non-biological surfaces. Specifically, 
methylcellulose has been used to coat the inside of quartz electrophoresis 
tubes to reduce or eliminate electroendosmosis. The protocol used in 
applying the methylcellulose coating involves three steps. First, the 
electrophoresis tube is washed with detergent. The possibility of 
detergent residues present on the quartz surface is not desirable since it 
may block carbohydrate adsorption. The second step involves addition of 
formaldehyde and formic acid to the methylcellulose solution to catalyze 
the cross-linking of the carbohydrate molecules which are present in the 
coating. Finally, the quartz tube is heated between applications of the 
methylcellulose. 
There presently is a need to provide a simple, quick, and efficient 
technique for reducing the surface energy of articles which are designed 
for use in contact with biologically active agents. The technique should 
be capable of reducing surface energy levels sufficiently to reduce and 
thereby control the binding of biologically active agents to the article's 
surface. 
SUMMARY OF THE INVENTION 
In accordance with the present invention, a method is provided for reducing 
the surface energy of materials which are used in articles that are 
designed for contact with biologically active agents. The present 
invention involves coating the surface of the article with a relatively 
low energy glassy carbohydrate film. The carbohydrate film has a surface 
energy which is well below the surface energy of many non-biological 
materials, such as metals, ceramics, and certain polymers. The glassy 
carbohydrate film provides a sufficient reduction in surface energy to 
reduce the binding energy between the surface and biologically active 
agents. 
As a feature of the present invention, the glassy carbohydrate film is 
simply applied to the article surface by adsorption. An essential aspect 
of the present invention is that the article surface must be substantially 
free of contaminating material. It was discovered that glassy carbohydrate 
films adsorb readily to article surfaces provided that the surfaces are 
contaminant free. The simplicity of adsorbing glassy carbohydrate films 
onto clean article surfaces makes the invention well suited for use in a 
wide variety of situations where it is desired to reduce the surface 
energy of a particular device or article of manufacture. 
As a further feature of the present invention, carbohydrate films which are 
especially amenable to reducing surface energy were found to include 
cellobiose, trehalose, isomaltose, nystose, sucrose and related 
oligosaccharides. In addition to basic sugars, allosteric effectors may 
also be used alone or in combination with the basic sugars to provide an 
effective glassy carbohydrate film which provides substantial reductions 
in surface energy. 
The above-discussed and many other features and attendant advantages of the 
present invention will become better understood by reference to the 
following detailed description when taken in conjunction with the 
accompanying drawing.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The present invention has wide application to articles of manufacture which 
are used in contact with one or more biologically active agents. The 
articles may be designed for in vivo or in vitro use. Examples of articles 
designed for in vivo use which may be treated in accordance with the 
present invention include implant devices, such as a cardiac pacemaker, 
electrode, central nervous system fluid shunt, and infusion pump. Other 
articles which are designed for in vivo use which are amenable to 
treatment in accordance with the present invention include percutaneous 
electrodes and transcortical percutaneous orthopedic pins. Articles which 
are designed for in vitro use which may be treated in accordance with the 
present invention include containers for biologically active agents, 
transport devices and virtually any article or device which is designed to 
be in continual contact with solutions that contain biologically active 
agents. Examples are intravenous fluid solution bags, hypodermic syringes 
and needles, food processing conduits, pesticide applicators, and cans of 
motor oil. 
The articles which may be treated in accordance with the present invention 
are made from metal, metal alloys, ceramics and polymers. Specific 
examples of metals and metal alloys include stainless steel, gold, silver, 
aluminum, silicon and titanium. Specific examples of ceramic materials 
include glass (sodium borosilicate and other types), aluminum oxide, 
silicon oxide, zirconium oxide, silicon nitride, and diamond. Polymer 
materials include polystyrene, polyethylene, polyacrylate, 
polymethylmethacrylate, polycarbonate, polyvinylchloride, polyurethane and 
silicone. 
In accordance with the present invention, the surface of the article is 
coated with a glassy carbohydrate film. The glassy films are preferably 
made from sugars selected from the group of basic sugars, such as 
cellobiose, trehalose, isomaltose, nystose, and related oligosaccharides. 
In addition, the glassy film may be made from allosteric effectors such as 
pyridoxal-5-phosphate, or 2,3 phosphoglycerate. If desired, the glassy 
film may be made from a combination of basic sugars and one or more 
allosteric effectors. 
In accordance with the present invention, it is essential that the surface 
of the article be free of contaminants prior to application of the glassy 
carbohydrate film. Any of the conventional techniques commonly used to 
provide ultra cleaning of surfaces may be used. These techniques include 
acid washing, washing with super critical fluids, or heating. Combinations 
of these methods, along with more sophisticated techniques such as plasma 
glow discharge cleaning, may be used. The particle cleaning technique used 
is not particularly important. What is important is that the surface to be 
coated be substantially contaminant free. 
The coating of the clean article surface is accomplished by simple 
adsorption of the glassy film onto the ultra clean surface. As will be 
realized, it is necessary that the surface must remain clean until the 
carbohydrate film is applied. Ultra clean, high energy surfaces are very 
reactive and will bind with a wide variety of materials other than 
carbohydrates. Accordingly, it is necessary that the cleaned surface be 
maintained in a contaminant free environment until the glassy film is 
applied. 
Any number of techniques may be utilized for applying the glassy film to 
the article surface. A convenient method involves simply immersing the 
article into a concentrated solution of the carbohydrate. Other techniques 
may be used, provided that they are capable of applying a uniform coating 
of glassy carbohydrate. The film thickness is not particularly important, 
so long as the underlying high energy surface is substantially covered. 
Film thicknesses on the order of less than 1 nanometer to 1 micron are 
suitable. The glassy film may also be applied as a pattern on the surface 
of the support material. Support material surfaces with patterns of glassy 
films thereon would be useful in more complex systems such as 
bio/opto-electric devices. Patterns of glassy films can be created using 
photoetching or other chemical/masking operations which are routinely used 
to create integrated circuits. 
The present invention is particularly well suited for treating articles and 
devices which are used in vivo to reduce binding of biologically active 
agents within the mammalian body. However, the present invention may be 
used to coat any article wherein it is desired to reduce the binding 
energy between the article surface and biologically active agents. For 
example, various applications include the coating of articles such as 
bottles for the transportation of pharmacologic agents, tubing and bags 
containing pharmacologic agents for administration, implantable medical 
devices, tubing used to conduct biological fluids (e.g., extracorporeal 
hemodialysis and extracorporeal blood oxygenation). Also, articles such as 
primary stainless steel used in the food industry may be coated in 
accordance with the present invention. For example, conduits and tubing 
used to transport various prepared foods from preparation vats to the 
canning or bottling assembly line may be coated in accordance with the 
present invention to reduce binding of biologically active agents. 
Supports used to anchor biologically active molecules, such as support 
particles and beads, may also be coated. 
The present invention is especially well suited for large scale operations 
where the simplicity of reducing surface activity by coating with glassy 
carbohydrate films is desirable. Further, the inexpensive nature of the 
carbohydrate coating process and the abundance of surface modifying 
carbohydrates makes the present invention especially well suited for 
commercial use. Further, the resulting glassy carbohydrate surface is a 
highly biocompatible surface which is glassy, water-like and relatively 
low in surface binding energy. 
An example of an exemplary embodiment of the present invention wherein 
glass storage vessels are coated with a cellobiose coating is as follows: 
Glass vials (4.0 ml.) were sonicated in 10 N hydrochloric acid for 20 
minutes and rinsed liberally in high performance liquid chromatography 
(HPLC) grade water. The vials were then baked at 210.degree. C. in a 
glassware oven for at least 18 hours before being cooled to 25.degree. C. 
in a laminar flow hood under nitrogen gas. Half of the vials were then 
incubated with a 500 mM cellobiose solution overnight at 5.degree. C. 
After incubation, both coated and non-coated vials were washed with 
sterile HPLC grade water three times. The vials were then allowed to dry 
in a laminar flow hood before insulin solutions were added. 
To demonstrate the reduced surface binding of biologically active molecules 
to he cellobiose treated glass surface, the loss of insulin from solution 
was measured over time. Clean, heat treated, vials (both cellobiose coated 
and non-coated) were incubated with Novalin R recombinant insulin over a 
24 hour time frame. A concentration of 10 units/ml of a pH 6.1 phosphate 
buffered saline solution was employed because of the good DEAE column 
sensitivity by HPLC. Unadsorbed insulin concentration was calculated from 
the integration of a 280 mn absorbing peak with an average retention time 
of three minutes. The mobile phase was a 20 mM acetic acid buffer (pH 4.5) 
with a linear 0-800 mM NaCl gradient over a 30 minutes interval at a flow 
rate of 1.0 ml/minute through a Waters R DEAE 5PW column. Determinations 
were taken in triplicate and averaged at times zero. 2, 4, 7, 18 and 27 
hours. The Drawing is a graph of the adsorption isotherms for recombinant 
insulin which shows that from an initial concentration of 10 units/ml, 
only 60% was recoverable after 6 hours in the untreated glass vials while 
approximately 90% was recoverable after 6 hours in the cellobiose treated 
vial. The percent recovery was stable for the subsequent 27 hours. 
Having thus described exemplary embodiments of the present invention, it 
should be noted by those skilled in the art that the within disclosures 
are exemplary only, and that various other alternatives, adaptations, and 
modifications may be made within the scope of the present invention. 
Accordingly, the present invention is not limited to the specific 
embodiments as illustrated herein, but is only limited by the following 
claims.