Growth factor modified protein matrices for tissue engineering
View Patent ↗Proteins are incorporated into protein or polysaccharide matrices for use in tissue repair, regeneration and/or remodeling and/or drug delivery. The proteins can be incorporated so that they are released by degradation of the matrix, by enzymatic action and/or diffusion. As demonstrated by the examples, one method is to bind heparin to the matrix by either covalent or non-covalent methods, to form a heparin-matrix. The heparin then non-covalently binds heparin-binding growth factors to the protein matrix. Alternatively, a fusion protein can be constructed which contains a crosslinking region such as a factor XIIIa substrate and the native protein sequence. Incorporation of degradable linkages between the matrix and the bioactive factors can be particularly useful when long-term drug delivery is desired, for example in the case of nerve regeneration, where it is desirable to vary the rate of drug release spatially as a function of regeneration, e.g. rapidly near the living tissue interface and more slowly farther into the injury zone. Additional benefits include the lower total drug dose within the delivery system, and spatial regulation of release which permits a greater percentage of the drug to be released at the time of greatest cellular activity.
1. A fusion protein, comprising:
(i) a first protein domain;
(ii) a second protein domain; and
(iii) an enzymatic or hydrolytic cleavage site between the first and the second domains;
wherein the first domain is a growth factor selected from the group consisting of the platelet derived growth factor superfamily and the transforming growth factor beta (TGFβ) superfamily;
wherein the second domain is a crosslinking Factor XIIIa substrate domain;
wherein the enzymatic cleavage site is selected from the group consisting of proteolytic substrates and polysaccharide substrates, and
wherein the hydrolytic cleavage site comprises a substrate with a linkage which undergoes hydrolysis by an acid or a base catalyzed reaction.
2. The fusion protein of claim 1 wherein the growth factor is selected from the group consisting of platelet derived growth factor (PDGF), TGFβ, bone morphogenic protein (BMP), vascular epithelial growth factor (VEGF), and insulin-like growth factor (IGF).
3. The fusion protein of claim 2 wherein the growth factor is selected from the group consisting of platelet derived growth factor-aloha-beta (PDGF-ABA), TGFβ1, TGFβ3, BMP2, BMP 7, VEGF 121, and IGF 1.
4. The fusion protein of claim 1 , wherein the Factor XIIIa substrate domain comprises SEQ ID NO:20.
5. The fusion protein of claim 1 wherein the cleavage site is an enzymatic cleavage site.
6. The fusion protein of claim 1 , wherein the cleavage site is an enzymatic cleavage site, which is cleaved by an enzyme selected from the group consisting of plasmin and matrix metalloproteinase.
7. A kit comprising:
(A) a fusion protein, comprising:
(i) a first protein domain;
(ii) a second protein domain; and
(iii) an enzymatic or hydrolytic cleavage site between the first and the second protein domains;
wherein the first domain is a growth factor selected from the group consisting of the platelet derived growth factor superfamily and the transforming growth factor beta (TGFβ) superfamily;
wherein the second domain is a Factor XIIIa crosslinking substrate domain;
wherein the enzymatic cleavage site is selected from the group consisting of proteolytic substrates and polysaccharide substrates; and
wherein the hydrolytic cleavage site comprises a substrate with a linkage which undergoes hydrolysis by an acid or a base-catalyzed reaction,
(B) fibrinogen,
(C) thrombin, and
(D) a calcium source.
8. The kit of claim 7 wherein the kit further comprises a Factor XIIIa.
9. The kit of claim 7 wherein the growth factor is selected from the group consisting of PDGF, vascular epithelial growth factor (VEGF), TGFβ, bone morphogenic protein (BMP), and insulin-like growth factor (IGF).
10. The kit of claim 9 wherein the growth factor is selected from the group consisting of platelet derived growth factor-alpha-beta (PDGF-AB), TGFβ1, TGFβ3, BMP2, BMP 7, VEGF 121 and IGF 1.
11. The kit of claim 7 wherein the Factor XIIIa substrate domain comprises SEQ ID NO:20.
12. The kit of claim 7 wherein the fusion protein comprises an enzymatic cleavage site.
13. The kit of claim 7 , wherein the enzymatic cleavage site is cleaved by an enzyme selected from the group consisting of plasmin and matrix metalloproteinase.