Sustained-release G-CSF microspheres using a novel solid-in-oil-in-oil-in-water emulsion method

Figures & data

Figure 1 Schematic drawing of microsphere preparation.

Abbreviations: PLGA, poly(lactic-co-glycolic acid); MS, microspheres.

Figure 2 (A–D) Scanning electron micrographs of different samples. (A) Dextran nanoparticles; (B) dextran nanoparticles recovered from the prepared dextran nanoparticle–loaded microspheres using S/O/O/W; (C) control microspheres (prepared microspheres using W/O/W); (D) prepared dextran nanoparticle–loaded microspheres using S/O/O/W.

Abbreviations: S/O/O/W, solid-in-oil-in-oil-in-water; W/O/W, water-in-oil-in-water.

Figure 3 Monomer content of recovery G-CSF from different samples during preparation processes by SEC-HPLC (n = 5).

Notes: Samples: original G-CSF (G-CSF:dextran = 1.4:5.0 ± 0.3 mg) solution; G-CSF from dextran aqueous phase/polyethyene glycol aqueous phase emulation; G-CSF from dextran nanoparticles; G-CSF from prepared microspheres using the S/O/O/W method (PLGA 50/50 2A: 100.0 ± 0.5 mg PLGA, dextran nanoparticles [G-CSF:dextran, 1:4] = 10.0 ± 0.3 mg); G-CSF from control microspheres (prepared microspheres using the W/O/W method) (PLGA 50/50 2A: 100.0 ± 0.5 mg, G-CSF solution [G-CSF:dextran = 1:4] = 10.0 ± 0.3 mg). *P > 0.05; **P < 0.05.

Abbreviations: G-CSF, granulocyte colony–stimulating factor; SEC-HPLC, size-exclusion chromatography–high-pressure liquid chromatography; PLGA, polylactic-co-glycolic acid; S/O/O/W, solid-in-oil-in-oil-in-water; W/O/W, water-in-oil-in-water.

Figure 4 Bioactivity from different samples during preparation processes (n = 5).

Notes: Samples: original G-CSF (G-CSF:dextran = 1.4:5.0 ± 0.3 mg) solution; G-CSF from dextran aqueous phase/polyethylene glycol aqueous phase emulation; G-CSF from dextran nanoparticles; G-CSF from prepared microspheres using the S/O/O/W method (PLGA 50/50 2A: 100.0 ± 0.5 mg PLGA, dextran nanoparticles [G-CSF:dextran, 1:4] = 10.0 ± 0.3 mg); G-CSF from control microspheres (prepared microspheres using the W/O/W method) (PLGA 50/50 2A: 100.0 ± 0.5 mg, G-CSF solution [G-CSF:dextran = 1:4] = 10.0 ± 0.3 mg). *P > 0.05; **P < 0.05.

Abbreviations: G-CSF, granulocyte colony–stimulating factor; PLGA, polylactic-co-glycolic acid; S/O/O/W, solid-in-oil-in-oil-in-water; W/O/W, water-in-oil-in-water.

Figure 5 In vitro release profiles of microspheres (n = 5, P < 0.05).

Notes: ⋄: Prepared microspheres using the S/O/O/W method (PLGA 50/50 2A: 100.0 ± 0.5 mg PLGA, dextran nanoparticles (G-CSF:dextran, 1:4) = 10.0 ± 0.3 mg); ♦: control microspheres (prepared microspheres using W/O/W method) (PLGA 50/50 2A: 100.0 ± 0.5 mg, G-CSF solution [G-CSF:dextran = 1:4] = 10.0 ± 0.3 mg).

Abbreviations: G-CSF, granulocyte colony–stimulating factor; PLGA, polylactic-co-glycolic acid; S/O/O/W, solid-in-oil-in-oil-in-water; W/O/W, water-in-oil-in-water.

Figure 6 In vitro release relative bioactivity of microspheres (n = 5, P < 0.05).

Notes: ⋄: Prepared microspheres using the S/O/O/W method (PLGA 50/50 2A: 100.0 ± 0.5 mg PLGA, dextran nanoparticles [G-CSF:dextran, 1:4] = 10.0 ± 0.3 mg); ♦: control microspheres (prepared microspheres using the W/O/W method) (PLGA 50/50 2A: 100.0 ± 0.5 mg, G-CSF solution [G-CSF:dextran = 1:4] = 10.0 ± 0.3 mg).

Abbreviations: G-CSF, granulocyte colony–stimulating factor; PLGA, polylactic-co- glycolic acid; S/O/O/W, solid-in-oil-in-oil-in-water; W/O/W, water-in-oil-in-water.

Figure 7 In vivo release plasma G-CSF level of PLGA microspheres (n = 5, P < 0.05).

Notes: ▲: Original G-CSF (G-CSF:dextran = 1.4:5.0 ± 0.3 mg) solution; ⋄: G-CSF from prepared microspheres using the S/O/O/W method (PLGA 50/50 2A: 100.0 ± 0.5 mg PLGA, dextran nanoparticles [G-CSF:dextran, 1:4] = 10.0 ± 0.3 mg); ♦: G-CSF from control microspheres (prepared microspheres using the W/O/W method) (PLGA 50/50 2A: 100.0 ± 0.5 mg, G-CSF solution [G-CSF:dextran = 1:4] = 10.0 ± 0.3 mg).

Abbreviations: G-CSF, granulocyte colony–stimulating factor; PLGA, polylactic-co-glycolic acid; S/O/O/W, solid-in-oil-in-oil-in-water; W/O/W, water-in-oil-in-water.

Figure 8 Increasing ratio of in vivo neutrophil levels (n = 5, P < 0.05).

Notes: ▲: Original G-CSF (G-CSF:dextran = 1.4:5.0 ± 0.3 mg) solution; ⋄: G-CSF from prepared microspheres using the S/O/O/W method (PLGA 50/50 2A: 100.0 ± 0.5 mg PLGA, dextran nanoparticles [G-CSF:dextran, 1:4] = 10.0 ± 0.3 mg); ♦: G-CSF from control microspheres (prepared microspheres using the W/O/W method) (PLGA 50/50 2A: 100.0 ± 0.5 mg, G-CSF solution [G-CSF:dextran = 1:4] = 10.0 ± 0.3 mg).

Abbreviations: G-CSF, granulocyte colony–stimulating factor; PLGA, polylactic-co-glycolic acid; S/O/O/W, solid-in-oil-in-oil-in-water; W/O/W, water-in-oil-in-water.

Figure 9 (A and B) The released mechanism diagram from the PLGA microsphere. (A) Mechanism diagram stability of G-CSF; (B) Released mechanism diagram of G-CSF.

Abbreviations: G-CSF, granulocyte colony–stimulating factor; PLGA, polylactic-co-glycolic acid.