Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
2001-03-21
2004-02-03
Killos, Paul J. (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C560S205000, C562S598000
Reexamination Certificate
active
06686144
ABSTRACT:
BACKGROUND
1. Technical Field
The present invention relates to the adsorption of polyampholytes to charged surfaces to form matrices. More particularly, surface charged density of a synthetic material is manipulated to effect assembly and matrix formation polyampholytic molecules.
2. Description of Related Art
During tissue development, repair and adaptation, the behavior of cells is largely controlled by interactions with their extracellular matrix (ECM). While the local cell population synthesizes the bulk of the extracellular matrix material, the ECM, correspondingly, influences cell phenotypic expression by providing attachment sites which modulate cell morphology and intracellular signal transduction events. The result of this pas-de-deux is a tissue with the requisite structural and functional capabilities. While the dramatic influences of subtle changes in matrix conformation are well demonstrated in Weaver, et al.,
J. Cell Biol.
137, 231-245 (1997), an understanding of the processes regulating the deposition and remodeling of the ECM forms the basis of much of the current research in developmental biology. Cells can play a critical role in the organization of the ECM, but physico-chemical dynamics carry the dominant responsibility for the nucleation, initiation and formation of the matrix.
The prototypical example of ECM organization remains that of collagen. The self-assembly of solubilized collagen to form gels was demonstrated close to 50 years ago Gross, et al.,
Proc. Soc. Exp. Biol. Med.
80, 462 (1952), Gross, et al.,
J. Biol. Chem.
233, 355-360 (1958). The triple helical collagen molecule spontaneously assembles under appropriate pH and ion conditions to form fibrils 20 nm to 100 nm or greater in diameter, and subsequently into fibril bundles as large as several hundred micrometers in diameter, Wood, et al.,
Biochem. J.
75, 588 (1960), Birk, et al., in
The Cell Biology of the Extracellular Matrix
, E. D. Hay, Ed. (Academic Press, New York, 1991) pp 221. The local cell population also plays an important role in this organizational process, both by secreting cross-linking enzymes, and by mechanically organizing the collagen fibers through attachment and contraction, Bell, et al.,
Proc. Natl. Acad. Sci. USA
76(3), 1274-1278 (1979), Kleinman, et al.,
Analytical Biochemistry
94, 308-312 (1979).
The organization of other major ECM molecules such as fibronectin, tenascin, and titin, remains far less well understood. Fibronectin, for example, is known to assemble into fibrils 100-1000 nm in diameter, but will not do so spontaneously in solution, generally requiring the presence of cells or cell-surface-like structures, Hynes,
Proc. Natl. Acad. Sci. USA
96(6), 2588-2590, (1999). Fibronectin is an adhesive protein, acting as the primary intermediate between cells and the collagen matrix for many cell types. It is a large (M
w
=450-500 kDa) glycoprotein, consisting of two nearly identical covalently linked subunits, each composed of three types of repeating modules. This complex forms a globular tertiary structure in solution, but an elastic, extended structure when formed into fibrils, Christopher, et al.,
J. of Cell Science
110, 569-581, (1997); Krammer, et al.,
Proc. Natl. Acad. Sci. USA
96(4), 1351-1356, (1999). However, the forces that provide the extension of the globular molecule into a fibrillar structure in the presence of cells remain undefined. Recent work suggests that plasma membrane lipid domain expansion could provide this necessary force for fibril assembly though the applicability of this proposed mechanism to the physiologic condition remains unclear, Pankov, et al.,
Mol. Biol. Cell
10, 4A-4A, (1999), Baneyx, et al.,
Proc. Natl. Acad. Sci. USA
96(22), 12518-12523, (1999).
Under in vivo conditions, fibronectin is a highly charged macromolecule with a net negative charge of approximately 47 (pI=5.6-6). While this molecule appears to have a cylindrical shape in solution, the contour of this molecule in the unfolded state has been visualized by electron microscopy, and these studies indicate a globular strand-like molecule approximately 2 nm in diameter and 140 nm in length, Petersen, et al., (1989)
Fibronectin
, Academic Press, NY. pp. 1-25. These dimensions would suggest that the molecule presents an average surface charge density of approximately 0.025 C/m
2
. The inherent difficulty in understanding fibronectin adsorption and fibrillogenesis is the identification of the process by which these net negatively charged molecules not only adsorb onto negatively charged substrates with charge densities of 0.1 C/m
2
or more, but undergo multilayer adsorption. Moreover, given that the Debye screening length at physiologic salt concentrations is on the order of only 10 angstroms, it is important to address how substrate surface charge density can influence the formation of fibrillar structures as large as one micrometer in diameter.
The adsorption of charged polymers and polyelectrolytes onto both uniformly and non-uniformly charged surfaces, has been analyzed both experimentally and theoretically, Fleer, et al., (1993),
Polymers at Interfaces
; Chapman and Hall: London, Dobrynin, et al., (1999), Phys. Rev. Ltrs. 84, 3101-3104, Ellis, et al., (2000),
J. Chem. Phys.
112:8723-8729, Sens, P. et al., (2000), Phys. Rev. Ltrs. 84, 4862-4865. However, while proteins commonly support a net charge (typically negative), they are polyampholytic in character, that is, after dissociation in a physiologic medium proteins support both positive and negative charge domains. Though the theory of polyampholyte adsorption remains relatively undeveloped, there have been substantial experimental efforts in this area due to the relevance of protein adsorption processes in technologies such as photography, Vaynberg, et al., (1998),
Colloid Interface Sci.
205:131-140. These studies have shown that adsorption can occur even when both the protein and surface have the same net (negative) charge, Kamiyama, et al., (1992),
Macromolecules
25:5081-5088, Neyret, et al., (1995),
J. Colloid Sci.
176:86-94. Theoretical studies of single chain polyampholyte adsorption suggest that such adsorption can occur due to the polarization of the polymer chains in the electric field created by the charged surface, Dobrynin, et al., (1997), Macromolecules 30:4332-4341, Netz, et al., (1998), Macromolecules 31, 5123-5141. Recently, these theories have been extended to address multilayer adsorption of polyampholytes, Dobrynin, et al., (1999),
Macromolecules
32:5689-5700.
Notwithstanding the above, it is widely believed that assembled forms of fibronectin, unlike fibrillar collagen molecules which can be made to assemble into fibrils in a test tube, will assemble into filaments only on the surface of certain cells, suggesting that additional proteins are needed for filament formation. See, e.g., Alberts et al., Molecular Biology of the Cell, 3
rd
Ed., Garland Publishing, (1994) pg. 987.
The ECM serves as an important influence in the normal processes of growth, repair, and adaptation as well as in the development of disease states and cell transformation. An understanding of how environmental agents can influence ECM formation would be extremely beneficial in the efforts to identify harmful or beneficial environmental agents. The ability to create ECM-like structures in environments free from cells and undesirable proteins and/or peptides would provide a tremendous tool in the study of ECMs and the interactions of various substances and stimuli with ECMs.
SUMMARY
A method for causing aggregation of a polyampholyte selected from the group consisting of fibronectin molecules and aggrecan molecules is provided which includes subjecting the polyampholyte molecules to a charge density of greater than about 0.01 C/m
2
generated by a non-living system to cause aggregation of the polyampholyte molecules.
Also provided is a composition including a synthetic surface having a charge density greater than about 0.01 C/m
2
in contact with a polyampholyte selected from the grou
McLeod Kenneth J.
Pernodet Nadine
Rafailovich Miriam
Dilworth & Barrese LLP.
Killos Paul J.
The Research Foundation of the State University of New York
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