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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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Related in: MedlinePlus

Illustration of LMW-H/P MP and LMW-H/P NP generation by mixing protamine to LMW-H at various ratios.Abbreviations: LMW-H, low-molecular-weight heparin; LMW-H/P, low-molecular-weight heparin/protamine; MP, microparticle; NP, nanoparticle.
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f3-ijn-5-147: Illustration of LMW-H/P MP and LMW-H/P NP generation by mixing protamine to LMW-H at various ratios.Abbreviations: LMW-H, low-molecular-weight heparin; LMW-H/P, low-molecular-weight heparin/protamine; MP, microparticle; NP, nanoparticle.

Mentions: When nondiluted protamine (10 mg/ml) was drop by drop added to nondiluted LMW-H (6.4 mg/mL) until 0.4 of the volume fraction, a gradual increase in turbidity was observed (Figure 1). Figure 2 shows that the high turbidity was due to the generated LMW-H/P MPs (2.93 ± 1.11 μm in diameter). Turbidity was measured by reading OD at 630 nm using a Mini Plate Reader (Nunc InterMed., Tokyo, Japan). When the volume fraction of protamine was low (0.10–0.15), the turbidity was also low. However, electron microscopic observation confirmed the generation of LMW-H/P NPs (about 100 nm in diameter) and LMW-H/P MPs (about 1 μm in diameter) (data not shown). Furthermore, when the volume fraction of protamine was high (>0.5), insoluble oily precipitates were generated, and turbidity of the supernatant was almost zero (Figure 1). An illustration of the generation of micro/nanoparticles by mixing protamine (10 mg/mL) and LMW-H (6.4 mg/mL) at various volume ratios is shown in Figure 3. In contrast, when nondiluted LMW-H was drop by drop added to nondiluted protamine, insoluble oily precipitates were immediately generated. The reaction was irreversible, and the generation of micro/nanoparticles was not observed.


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)

Illustration of LMW-H/P MP and LMW-H/P NP generation by mixing protamine to LMW-H at various ratios.Abbreviations: LMW-H, low-molecular-weight heparin; LMW-H/P, low-molecular-weight heparin/protamine; MP, microparticle; NP, nanoparticle.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2865009&req=5

f3-ijn-5-147: Illustration of LMW-H/P MP and LMW-H/P NP generation by mixing protamine to LMW-H at various ratios.Abbreviations: LMW-H, low-molecular-weight heparin; LMW-H/P, low-molecular-weight heparin/protamine; MP, microparticle; NP, nanoparticle.
Mentions: When nondiluted protamine (10 mg/ml) was drop by drop added to nondiluted LMW-H (6.4 mg/mL) until 0.4 of the volume fraction, a gradual increase in turbidity was observed (Figure 1). Figure 2 shows that the high turbidity was due to the generated LMW-H/P MPs (2.93 ± 1.11 μm in diameter). Turbidity was measured by reading OD at 630 nm using a Mini Plate Reader (Nunc InterMed., Tokyo, Japan). When the volume fraction of protamine was low (0.10–0.15), the turbidity was also low. However, electron microscopic observation confirmed the generation of LMW-H/P NPs (about 100 nm in diameter) and LMW-H/P MPs (about 1 μm in diameter) (data not shown). Furthermore, when the volume fraction of protamine was high (>0.5), insoluble oily precipitates were generated, and turbidity of the supernatant was almost zero (Figure 1). An illustration of the generation of micro/nanoparticles by mixing protamine (10 mg/mL) and LMW-H (6.4 mg/mL) at various volume ratios is shown in Figure 3. In contrast, when nondiluted LMW-H was drop by drop added to nondiluted protamine, insoluble oily precipitates were immediately generated. The reaction was irreversible, and the generation of micro/nanoparticles was not observed.

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