Figures & data
Figure 1 Changes in turbidity by mixing protamine to LMW-H at various ratios. When protamine (10 mg/mL) was added dropwise to LMW-H (6.4 mg/mL) until 0.4 of the volume fraction, gradual increases in turbidity were observed. When the volume fraction of protamine was high (>0.5), turbidity was very low due to generated insoluble oily precipitates.
Abbreviations: LMW-H, low-molecular-weight heparin.
Figure 2 LMW-H/P MPs produced by mixing protamine to LMW-H. LMW-H (6.4 mg/mL) and protamine (10 mg/mL) were mixed to produce LMW-H/P MPs at a ratio of 7:3 (vol:vol). Produced microparticles were aggregates composed of many nanoparticles and particle size was 2.93 ± 1.11 μm.
Abbreviations: LMW-H, low-molecular-weight heparin; LMW-H/P, low-molecular-weight heparin/protamine; MPs, microparticles.
Figure 3 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.
Figure 4 LMW-H/P NPs produced by mixing diluted protamine and LMW-H. Equally diluted aqueous LMW-H solution and protamine solution were mixed at a ratio of 7:3 (vol:vol) to produce LMW-H/P NPs. Produced nanoparticles were PECs and nanoparticle size was 84.6 ± 26.8 and 95.0 ± 27.0 nm after mixing 100- and 50-fold diluted protamine and LMW-H, respectively. When mixing 20-fold diluted protamine and LMW-H solutions, LMW-H/P NPs were generated and the size was 112.5 ± 46.1 nm.
Abbreviations: LMW-H, low-molecular-weight heparin; LMW-H/P, low-molecular-weight heparin/protamine; NPs, nanoparticles; PECs, polyelectrolytes.
Figure 5 Protective effects of LMW-H/P NP on FGF-2 activity. 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 incubated at 37°C for 0, 1, 3, and 7 days. FGF-2 in the stock solution was diluted to the indicated concentrations 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.
Figure 6 Protective effects of LMW-H/P NPs on bioactivity of heat-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 incubated at 37, 44, 51, 58, 65 and 72°C for 30 min. FGF-2 in the stock solution was diluted to the indicated concentrations 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.
Figure 7 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.
