Controlled release of FGF‐2 using fragmin/protamine microparticles and effect on neovascularization

Abstract

Water-insoluble fragmin/protamine microparticles of about 0.5-1 mum in diameter were prepared by simple mixing of low-molecular-weight heparin (fragmin) with protamine. We investigated the capability of these microparticles to immobilize fibroblast growth factor (FGF)-2, to protect FGF-2 against degradation, to enhance FGF-2 activity, and to facilitate controlled release of FGF-2. FGF-2 bound to the fragmin/protamine microparticles with high affinity (Kd = 2.08 x 10(-9) M) and the half-life of FGF-2-activity was prolonged substantially through binding of FGF-2 to the microparticles, by protection of FGF-2 from inactivation by heat and proteolysis. After subcutaneous injection into the back of mice, the fragmin/protamine microparticles underwent biodegradation and disappeared in about 2 weeks. A similar injection of FGF-2-containing microparticles resulted in significant neovascularization and fibrous tissue formation near the injection site after 1 week. These results indicate that controlled release of biologically active FGF-2 occurs through both slow diffusion and biodegradation of the microparticles, with subsequent induction of neovascularization. (c) 2008 Wiley Periodicals, Inc. J Biomed Mater Res, 2009.

Preparation and characterization of low-molecular-weight heparin/protamine nanoparticles (LMW-H/P NPs) as FGF-2 carrier

“…19 FGF-2-containing LMW-H/P MPs showed substantial induction of vascularization and fibrous tissue formation due to the gradual release of FGF-2 molecules from LMW-H/P MPs. 19 However, LMW-H/P MPs are unstable; the particles irreversibly fuse and become larger (10 µm in diameter) during long-term storage (1 week or longer). Furthermore, after LMW-H/P MPs are freeze-dried, cotton-like materials are produced, and these do not dissolve in water.…”

“…19 Briefly, 0.3 mL of protamine solution (10 mg/mL; Mochida Pharmaceutical Co., Ltd., Tokyo, Japan) was drop by drop added to 0.7 mL of LMW-H solution (Fragmin ® , 6.4 mg/mL; Kissei Pharmaceutical Co., Ltd., Nagano, Japan) with vortexing for approximately 2 min. To maximize the production of microparticles, LMW-H and protamine were mixed at a ratio at 7:3 (vol:vol).…”

“…Heparin protects FGF-2 from inactivating treatments by heat and proteolysis. [19][20][21] To determine whether LMW-H/P NPs with dextran could protect FGF-2 against heat inactivation, FGF-2 (10 µg/mL) was heated in the presence of LMW-H/P NPs (3.14 mg/mL). As shown in Figure 6, heat exposure at over 51°C in the absence of LMW-H with Concentration of FGF-2 (ng/mL) Figure 5 Protective effects of LMW-h/P NP on FgF-2 activity.…”

“…Despite the difficulty of size distribution control of LMW-H/P MPs, it should be noted that any problems did not exist in the repeatability of the experiments using LMW-H/P MPs studied to date. 19 On the other hand, in 20-fold and more diluted systems, only nanoscale particles were formed. And SEM of 10-fold diluted system showed that the products were a mixture of aggregations (MPs) and NPs.…”

“…17,18 We also prepared water-insoluble microparticles (1 µm in diameter) by mixing LMW-H (6.4 mg/mL) and protamine (10 mg/mL) at a ratio of 7:3 (vol:vol), and investigated the capacity of the resulting injectable LMW-H/protamine microparticles (LMW-H/P MPs) to protect the activity of FGF-2. 19 However, after the LMW-H/P MPs were freeze-dried, a cotton-like amorphous material was generated, and this was not dissolved with water. In this study, we used diluted LMW-H (0.32 mg/ml) as an anion molecule and the diluted protamine as a cation molecule to synthesize LMW-H/protamine nanoparticles (LMW-H/P NPs; about 120 nm in diameter).…”