Biomolecule attachment to hydrophobic surfaces

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BACKGROUND OF THE INVENTION

Biomolecules--that is, molecules of compounds which engage in a biological activity or are effective in modulating a biological activity--commonly are used in solution or are adsorbed or otherwise attached to solid supporting surfaces such as glass beads. U.S. Pat. No. 3,959,078 refers to the attachment of enzymes to solid surfaces.

Biomolecules can alter the solid or semi-solid surfaces to which they are attached Heparin, for example, can be attached to polyethylene surfaces of a blood bag to provide the surface with anticoagulant properties. Ebert et al.; The Anticoagulant Activity of Derivatized and Immobilized Heparins, in Biomaterials: Interfacial Phenomena and applications, Cooper et al., eds., Am. Chem. Soc. 1982, pp.161-176.

Several procedures currently exist for immobilizing synthetic and naturally produced molecules onto solid or semi-solid substrates. The chemistries typically utilized are either highly substrate dependent or result in a significantly reduced activity of the immobilized species. An example of such chemistries include copolymer grafting procedures Larsson, P. H. Johansson, S. G.; Hult, A.; and Gothe, S.: Covalent Binding of Proteins to Grafted Plastic Surfaces Suitable for Immunoassays; J. Immuno. Methods, 98, 1987, p-129-135.

The tertiary and quaternary structures of such biomolecules as proteins and polysaccharides have historically been viewed as being "static" in nature. This static view of biomolecular function recently has given way to understanding the dynamic motions of intramolecular structures as a basis for function. Jarplus, M.; McCammon, J. A.: The Dynamics of Proteins, Scientific American, April, 1986, p. 42-51. An implication of this understanding is that for optimal activity, proteins and other biomolecules should be immobilized by methods that distort neither the conformation nor the molecular motions of the biomolecules.

In addition to the loss of activity, the conformational distortion that may occur upon immobilization of a biomolecule can give rise to undesired biological responses, especially on implant and medical device surfaces. For instance, increased thrombogenicity or induction of foreign body reactions and rejection following implantation have been reported. When certain macromolecules (e.g. proteins and polysaccharides) encounter previously untreated polymers or other medical device materials, they may adsorb onto these surfaces and undergo alterations in both conformation and activity. Foreign body reactions to soft tissue implants and the thrombogenicity of most polymers involve a cellular level response in the host after a layer of protein has adsorbed onto the device surface. The so called adverse host response may be attributed to the altered macromolecule structure producing an abnormal function when immobilized on the device's surface.

Various plasma proteins undergo slow conformational changes resulting in the loss of secondary structures, such as altered helix or beta-sheet. The denaturation of surface immobilized proteins through these type of changes may render them antigenic. Peters, J. H. and Goetzl, E.: Recovery of Greater Antigenic Reactivity in Conformationally Altered Albumin, J. Biological Chem., 224, 1969, p. 2068; Stern, I. J., et al.: Immunogenic Effects of Materials on Plasma Proteins, Conf. Proc. Artificial Heart Program, National Heart Institute, 1969, p. 259. The immobilization of some plasma proteins which may produce an altered thrombogenic response. Brash, J. L.; Protein Interactions with Artificial Surfaces, Interaction of the Blood with Natural and Artificial Surfaces, ed. Salzman, E. W., 1981 by Marcel Dekker, Inc., p.39-44.

By spacing biomolecules away from a support, it would be expected that somewhat improved biomolecule activity should be observed. Initial studies indicating the utility of spacer arms have been conducted with heparin. Ebert et al. supra, reported that the bioactivity of heparin could be correlated, to a limited degree, with the length of a spacer holding heparin molecules apart from a supporting surface. Activated partial thromboplastin time was assayed with bovine plasma as an indicator of heparin activity. Heparin was immobilized with hydrophobic aliphatic spacers of varying lengths and produced heparin activity that increased with spacer length. However because of the hydrophobic nature of alkane spacers, the use of longer chains under aqueous physiological conditions would be expected to result in a coiling or doubling back of the spacer molecules, the spacers thus losing their ability to space biomolecules from a solid or semi-solid surface.

SUMMARY OF THE INVENTION

Certain of the practical problems associated with attachment of a biomolecule onto a surface can be avoided by employing a long chain chemical spacer which has two reactive groups separated by a chain length of at least 25 Angstroms when extended. The length of the spacer between the reactive groups is sufficient to space the biomolecule group beyond any substantial adverse interactions associated with the surface to which the spacer is attached. The spacers of the invention generally comply with the following requirements:

1. The spacer may be covalently attached to a support surface and to a biomolecule, and the covalent attachments desirably occur under different predetermined conditions.

2. The reaction conditions for coupling the spacer to a biomolecule are sufficiently gentle as to avoid damaging the biomolecule, and adverse interactions between the biomolecule and the spacer are minimized.

3. Preferably, the spacer contains repeating units such that its length may be adjusted as desired to optimize the specific activities of a given biomolecule.

4. There are no significant bulk physical changes to the surface to which the biomolecule is attached.

The process of the invention involves the treatment of a surface by the immobilization thereon of a biomolecule through the use of a heterobifunctional spacer having two reactive groups thereon, the chemical chain providing a spacing between the groups of at least 25 Angstroms, measured along an extended length of the chain. As used herein, "extended" chain length refers to the straight line distance between two positions along a chemical chain when the chain is stretched to its maximum length consonant with maintaining proper bond angles. For example, propane, CH.sub.3 --CH.sub.2 --CH.sub.3, exhibits an "extended" chain length between terminal carbon atoms of about 2.5 A (observing a bond angle of 109.5.degree.), whereas the sum of the (carbon-carbon) bond distances along the molecule is about 3.06 A. The distances measured along the spacer chains described herein are all "extended" chain lengths.

The spacers desirably are totally self-contained in that no pretreatment of a target surface to generate bonding groups is required to effect immobilization. An important aspect of the invention is the discovery that adverse substrate/biomolecule interactions can be predictably reduced.

In one embodiment, the invention comprises a long chain spacer for tethering a biomolecule to a surface while avoiding substantial deleterious effect upon the biomolecule by the presence of the surface, the spacer comprising a chemical backbone having two reactive groups attached thereto and separated by a backbone extended chain length of not less than about 25 Angstroms, one such reactive group being capable of forming a covalent bond to a sur