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

Abstract

Abstract:We produced low-molecular-weight heparin/protamine nanoparticles (LMW-H/P NPs) as a carrier for heparin-binding growth factors, such as fibroblast growth factor-2 (FGF-2). A mixture of low-molecular-weight heparin (MW: about 5000 Da, 6.4 mg/mL) and protamine (MW: about 3000 Da, 10 mg/mL) at a ratio of 7:3 (vol:vol) yields a dispersion of microparticles (1-6 µm in diameter). In this study, diluted low-molecular-weight heparin solution in saline (0.32 mg/mL) mixed with diluted protamine (0.5 mg/mL) at a ratio at 7:3 (vol:vol) resulted in soluble nanoparticles (112.5 ± 46.1 nm in diameter). The generated NPs could be then stabilized by adding 2 mg/mL dextran (MW: 178-217 kDa) and remained soluble after lyophilization of dialyzed LMW-H/P NP solution. We then evaluated the capacity of LMW-H/P NPs to protect activity of FGF-2. Interaction between FGF-2 and LMW-H/P NPs substantially prolonged the biological half-life of FGF-2. Furthermore, FGF-2 molecules were protected from inactivation by heat and proteolysis in the presence of LMW-H/P NPs.

Improved angiogenesis and healing in crush syndrome by fibroblast growth factor-2–containing low-molecular-weight heparin (Fragmin)/protamine nanoparticles

“…The F/P NPs were prepared, as described previously [7,23]. In brief, 0.3 mL of protamine solution (10 mg/mL; Mochida Pharmaceutical Co, Tokyo, Japan) was added dropwise to 0.7 mL of Fragmin solution (6.4 mg/mL; Kissei Pharmaceutical Co, Tokyo, Japan).…”

“…The final concentrations of FGF-2 and F/P NPs were estimated at 25 mg/mL and 3.75 mg/mL, respectively. Under these conditions, approximately 75% of FGF-2 molecules were immobilized on the F/P NPs because approximately 5 mg of FGF-2 could be immobilized onto 1 mg of F/P NPs [7,8].…”

“…We have reported Fragmin/protamine nanoparticles (F/P NPs: 112.5 AE 46.1 nm in diameter), which we originally prepared as polyelectrolyte complexes [7,8]. The F/P NPs are specifically bound to various heparinbinding GFs such as FGF-2 [7e9], hepatocyte GFs [10], and other GFs in platelet-rich plasma [11,12].…”

“…The F/P NPs are able to protect these GFs from heat and proteolytic inactivation and enhance their biological activities. Because the diameter of fibroblast GF-2econtaining low-molecular-weight heparin (Fragmin)/protamine nanoparticles (FGF-2 þ F/P NPs) is estimated to be 200 AE 20 nm depending on the concentration of FGF-2 (data not published yet), they can be easily injected [7]. Furthermore, GFs þ F/P NPs substantially induce vascularization and fibrous tissue formation because of stabilizing, activating, and gradually releasing GFs molecules from GFs þ F/P NPs [7e10].…”

Enhanced regenerative healing efficacy of a highly skin-permeable growth factor nanocomplex in a full-thickness excisional mouse wound model

“…38 In addition, the LMWP-conjugated growth factor nanocomplex resulted in significantly accelerated wound closure compared with the LMWP-conjugated growth factor combination -it may be that the LMWP-conjugated growth factors were protected from rapid elimination by proteolytic inactivation, and that binding to their receptors was facilitated. 39 Whereas, the native growth factors also formed nanocomplexes after combining with LMWH, the native growth factor complexes failed to exhibit wound-healing efficacy. This might have been caused by the relatively weak interaction between the growth factors and LMWH, and the poor skin permeability of the native growth factors and their complex.…”