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

Mori Y, Nakamura S, Kishimoto S, Kawakami M, Suzuki S, Matsui T, Ishihara M - Int J Nanomedicine (2010)

Bottom Line: 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 microm 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).Interaction between FGF-2 and LMW-H/P NPs substantially prolonged the biological half-life of FGF-2.

View Article: PubMed Central - PubMed

Affiliation: Research Institute, National Defense Medical College, Tokorozawa, Saitama, Japan.

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 microm 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.

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Protective effects of LMW-H/P NPs on bioactivity of trypsin-treated FGF-2. Stock solutions (10 μg/mL FGF-2 with 3.14 mg/mL LMW-H/P NPs with 20 mg/mL dextran (•), 1.6 mg/mL LMW-H with 20 mg/mL dextran (Δ), 20 mg/mL dextran alone (□) or control (non) (○)) were treated with trypsin at 37°C for the indicated periods of time. After trypsinization, proteolysis was stopped by adding FBS, and inactivated FGF-2 in the stock solutions was diluted to 10 ng/mL with culture medium. HMVECs were cultured for 3 days using one of the prepared media, and data represent means ± SD of quadruplicate determinations.Abbreviations: FGF-2, fibroblast growth factor-2; HMVECs, human dermal micro-vascular endothelial cells; LMW-H, low-molecular-weight heparin; LMW-H/P, low-molecular-weight heparin/protamine; NP(s), nanoparticle(s); SD, standard deviation.
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f7-ijn-5-147: Protective effects of LMW-H/P NPs on bioactivity of trypsin-treated FGF-2. Stock solutions (10 μg/mL FGF-2 with 3.14 mg/mL LMW-H/P NPs with 20 mg/mL dextran (•), 1.6 mg/mL LMW-H with 20 mg/mL dextran (Δ), 20 mg/mL dextran alone (□) or control (non) (○)) were treated with trypsin at 37°C for the indicated periods of time. After trypsinization, proteolysis was stopped by adding FBS, and inactivated FGF-2 in the stock solutions was diluted to 10 ng/mL with culture medium. HMVECs were cultured for 3 days using one of the prepared media, and data represent means ± SD of quadruplicate determinations.Abbreviations: FGF-2, fibroblast growth factor-2; HMVECs, human dermal micro-vascular endothelial cells; LMW-H, low-molecular-weight heparin; LMW-H/P, low-molecular-weight heparin/protamine; NP(s), nanoparticle(s); SD, standard deviation.

Mentions: LMW-H with dextran, dextran, and LMW-H/P NPs with dextran were able to protect inactivation of FGF-2 after trypsin treatment, while activity was lost in controls (Figure 7). In the presence of trypsin, the biological half-life of 10 ng/mL FGF-2 with LMW-H with dextran, dextran and LMW-H/P NPs with dextran was about >120 min, 80 min, and >120 min, respectively (with control (non) = 20 min). Thus, LMW-H/P NPs protects FGF-2 from inactivating treatments, similar to LMW-H/P MPs.19


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

Mori Y, Nakamura S, Kishimoto S, Kawakami M, Suzuki S, Matsui T, Ishihara M - Int J Nanomedicine (2010)

Protective effects of LMW-H/P NPs on bioactivity of trypsin-treated FGF-2. Stock solutions (10 μg/mL FGF-2 with 3.14 mg/mL LMW-H/P NPs with 20 mg/mL dextran (•), 1.6 mg/mL LMW-H with 20 mg/mL dextran (Δ), 20 mg/mL dextran alone (□) or control (non) (○)) were treated with trypsin at 37°C for the indicated periods of time. After trypsinization, proteolysis was stopped by adding FBS, and inactivated FGF-2 in the stock solutions was diluted to 10 ng/mL with culture medium. HMVECs were cultured for 3 days using one of the prepared media, and data represent means ± SD of quadruplicate determinations.Abbreviations: FGF-2, fibroblast growth factor-2; HMVECs, human dermal micro-vascular endothelial cells; LMW-H, low-molecular-weight heparin; LMW-H/P, low-molecular-weight heparin/protamine; NP(s), nanoparticle(s); SD, standard deviation.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2865009&req=5

f7-ijn-5-147: Protective effects of LMW-H/P NPs on bioactivity of trypsin-treated FGF-2. Stock solutions (10 μg/mL FGF-2 with 3.14 mg/mL LMW-H/P NPs with 20 mg/mL dextran (•), 1.6 mg/mL LMW-H with 20 mg/mL dextran (Δ), 20 mg/mL dextran alone (□) or control (non) (○)) were treated with trypsin at 37°C for the indicated periods of time. After trypsinization, proteolysis was stopped by adding FBS, and inactivated FGF-2 in the stock solutions was diluted to 10 ng/mL with culture medium. HMVECs were cultured for 3 days using one of the prepared media, and data represent means ± SD of quadruplicate determinations.Abbreviations: FGF-2, fibroblast growth factor-2; HMVECs, human dermal micro-vascular endothelial cells; LMW-H, low-molecular-weight heparin; LMW-H/P, low-molecular-weight heparin/protamine; NP(s), nanoparticle(s); SD, standard deviation.
Mentions: LMW-H with dextran, dextran, and LMW-H/P NPs with dextran were able to protect inactivation of FGF-2 after trypsin treatment, while activity was lost in controls (Figure 7). In the presence of trypsin, the biological half-life of 10 ng/mL FGF-2 with LMW-H with dextran, dextran and LMW-H/P NPs with dextran was about >120 min, 80 min, and >120 min, respectively (with control (non) = 20 min). Thus, LMW-H/P NPs protects FGF-2 from inactivating treatments, similar to LMW-H/P MPs.19

Bottom Line: 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 microm 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).Interaction between FGF-2 and LMW-H/P NPs substantially prolonged the biological half-life of FGF-2.

View Article: PubMed Central - PubMed

Affiliation: Research Institute, National Defense Medical College, Tokorozawa, Saitama, Japan.

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 microm 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.

Show MeSH
Related in: MedlinePlus