Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
2001-08-30
2003-04-22
Ketter, James (Department: 1636)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C424S409000, C424S489000, C435S177000, C435S182000, C435S395000, C530S383000, C530S402000
Reexamination Certificate
active
06552172
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to fibrin nanoparticles, methods for preparing fibrin particles of various sizes, including fibrin nanoparticles and fibrin microbeads, and uses thereof.
BACKGROUND OF THE INVENTION
Fibrinogen is the main structural protein of the clot and has been developed in concentrated form as a fibrin glue or sealant. Fibrinogen's structure and the biochemical mechanisms of clot formation as well as fibrin interactions with platelets have been elucidated and are well documented. Beyond hemostasis, fibrin also serves as an interim matrix to recruit cells needed for the regeneration of granulation tissue. Different cell types are drawn into the clot from the tissues surrounding the wound. Ultimately, the cells drawn into the clot secrete new collagen and other extracellular matrix (ECM) molecules.
Fibrinogen is not immunogenic within the same species, as attested by the use of pooled fibrin glue for clinical applications (Radosevich M., Goubran H. A. & Burnouf T., Fibrin sealant: Scientific rationale, production methods, properties and current clinical use.
Vox Sang
., 72: 133-143, 1997; Marx G. Kinetic and mechanical parameters of fibrin glue. Symposium on Surgical Tissue Adhesives: Atlanta Ga. In: Current Trends in Surgical Tissue Adhesives, Sierra D. & Saltz R. (Eds) (1996)). In addition to fibrinogen's hemostatic activity, fibrin(ogen) (which is known in the art as a reference to fibrin and/or fibrinogen) has been shown to elicit cell attachment (haptotaxis) and migratory (chemotaxtic) responses with different cell types including mouse and human fibroblasts (MF & HF), bovine aortic endothelial (BAEC) and smooth muscle cells (SMC) (Gorodetsky, R., et al.
J. Lab. Clin. Med
., 131: 269-280, 1998).
Fibrinogen is heat sensitive and becomes denatured above 50° C. By contrast, endogenous factor XIII is less heat sensitive and can induce cross-links even at higher temperatures. Based upon these properties of fibrinogen and factor XIII, the inventors devised a patented process for preparing fibrin microbeads (“FMB”) (see, U.S. Pat. No. 6,150,505, issued Nov. 21, 2000). The process involved reacting fibrinogen and thrombin, in heated vegetable oil (70-80° C.) in the presence of endogenous factor XIII. The crude FMB isolated from the above procedure were filtered through fine mesh filter and sedimented in 95% ethanol. Microscopic evaluation indicated that the FMB were 50-200 &mgr;m in diameter (Gorodetsky, et al.,
J. Invest. Dermatol
., 112(6): 866-872, June 1999). Tests with sucrose solutions indicated that the FMB have a density 1.32+0.1 gm/ml.
The FMB offered a significant improvement over fibrin microbeads of the prior art. Specifically, since factor XIII was used as the cross linking agent rather than exogenous chemical cross linking agents, such as glutaraldehyde (which was typically used in the preparation of fibrin beads (see, Ho, et al.,
Drug Dev. and Ind. Pharm
. 20(4):535-546, 1994)), the FMB retained the haptotactic and chemotaxtic properties of fibrin(ogen) for a variety of cell types. It is believed that the use of exogenous chemical cross linking agents, such as glutaraldehyde, not only destroys certain biologically active sites, thereby interfering with the binding of the microbeads to cells, but also may render the microbeads immunogenic.
In addition, it is believed that the combination of heat and factor XIII in the preparative process of the FMB allowed a significantly greater degree of cross-linking of the fibrin(ogen) than would otherwise occur in physiologic fibrin, or fibrin-based drug delivery systems such as described in Senderoff, et al., (
J. Parenteral Sci
. &
Tech
., 45(1): 2-6, 1991). This renders the FMB stable for prolonged periods in aqueous solution, a property that is particularly desirable for use as vehicles for culturing cells, as well as for other uses.
SUMMARY OF THE INVENTION
The present invention is directed to fibrin nanoparticles (FNP) having a mean diameter of 200-2000 nm. The FNP of the present invention, like the FMB of U.S. Pat. No. 6,150,505, do not contain any exogenous cross-linking agents such as glutaraldehyde that can damage certain biologically active sites and affect the rate of their enzymatic degradation, and therefore FNP are reactive with various types of cells. Rather, the FNP of the present invention are prepared by reacting fibrinogen, thrombin and Factor XIII under heat, where the Factor XIII acts as the cross-linking agent. As a result of the preparation process, the FNP of the present invention contain extensive cross-linking of fibrin(ogen), which renders the fibrin particles stable for prolonged periods in aqueous solution.
However, it has been unexpectedly found that the FNP of the present invention, unlike the larger FMB, are taken up into the cytoplasm of cells. Therefore, the FNP of the present invention offers an innovative and unique solution for selectively delivering agents into cells, including agents that are not normally taken up by cells. Since the FNP of the present invention are also non-toxic, minimally non-immunogenic and biodegradable, FNP is a significant advancement in the field of drug delivery.
In addition to FNP, the present invention also provides a method for preparing FNP comprising the steps of: (i) mixing an aqueous solution comprising fibrinogen, thrombin and Factor XIII in an oil emulsion at a temperature of 50-80° C., without the addition of an exogenous chemical cross-linking agent, until fibrin microbeads are formed in the mixture; (ii) homogenizing the mixture to form FNP having a mean diameter of 200-2000 nm in the mixture; and (iii) isolating FNP having a mean diameter of 200-2000 nm from the mixture.
In addition, the present invention provides another method for preparing fibrin particles of any size. This method is comprised of the steps of: (i) mixing an aqueous solution comprising fibrinogen, thrombin and Factor XIII, without the addition of an exogenous chemical cross-linking agent, in a manner to obtain a fibrin clot; (ii) incubating the fibrin clot at ambient temperature for a period of time sufficient to obtain a cross-linked fibrin clot; (iii) mixing the cross-linked fibrin clot in oil at a temperature of 50-80° C. to obtain dehydrated fibrin pieces; (iv) homogenizing or grinding the fibrin pieces to obtain fibrin particles of the desired size; and (v) isolating the fibrin particles of the desired size. This method may be used to prepare particles of any size, including FNP and FMB.
The present invention also provides another method for preparing fibrin particles of any size. This method comprises the steps of: (i) mixing an aqueous solution comprising fibrinogen, thrombin and Factor XIII, without the addition of an exogenous chemical cross-linking agent, in a manner to obtain a fibrin clot; (ii) incubating the fibrin clot at ambient temperature for a period of time sufficient to obtain a cross-linked fibrin clot; (iii) heating the cross-linked fibrin clot at a temperature of 50-80° C. to obtain fibrin pieces; (iv) homogenizing the fibrin pieces to obtain fibrin particles of the desired size; and (v) isolating the fibrin particles of the desired size. This method also may be used to prepare FNP, FMB and fibrin particles of other sizes.
The present invention provides yet another method for preparing fibrin particles of any size, that comprising the steps of: (i) introducing an aqueous solution comprising fibrinogen, thrombin and Factor XIII, without the addition of an exogenous chemical cross-linking agent, into a spray dryer; (ii) spray drying the mixture to obtain spray dried fibrin particles; (iii) heating the spray dried particles at a temperature of 50-80° C. to dehydrate or remove water; and (iv) isolating fibrin particles of the desired size. Here again, this method may be used to prepare fibrin particles of various sizes, including FNP and FMB.
Still further, the present invention provides a composition comprising fibrin nanoparticles and an agent, wherein the agent is admixed with the fibrin nanoparticles.
In add
Gorodetsky Raphael
Marx Gerard
Amster Rothstein & Ebenstein
Habto Biotech, Inc.
Ketter James
Sullivan D
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