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International Journal of Nanomedicine Volume 8, 2013 - Issue
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Original Research

Novel preparation method for sustained-release PLGA microspheres using water-in-oil-in-hydrophilic-oil-in-water emulsion

Xiaoyun Hong1 Department of Neurology, Xinhua Hospital affiliated to Shanghai JiaoTong University, School of Medicine, Shanghai, People’s Republic of China;2 School of Pharmacy, Shanghai JiaoTong University, Shanghai, People’s Republic of China
,
Liangming Wei3 Key Laboratory for Thin Film and Microfabrication Technology, Ministry of Education, Research Institute of Micro/Nanometer Science and Technology, Shanghai JiaoTong University, Shanghai, People’s Republic of China
,
Liuqing Ma2 School of Pharmacy, Shanghai JiaoTong University, Shanghai, People’s Republic of China
,
Yinghui Chen4 Department of Neurology, Jinshan Hospital, Fudan University, Shanghai, People’s Republic of ChinaCorrespondence[email protected]
,
Zhenguo Liu1 Department of Neurology, Xinhua Hospital affiliated to Shanghai JiaoTong University, School of Medicine, Shanghai, People’s Republic of ChinaCorrespondence[email protected]
&
Weien Yuan2 School of Pharmacy, Shanghai JiaoTong University, Shanghai, People’s Republic of China
Pages 2433-2441 | Published online: 08 Jul 2013

Figures & data

Figure 1 Optical microscopic images of free solidified microspheres. (A) PLGA (3A50/50), (B) PLGA (3A50/50)/LPLA = 40/80, (C) PLGA (3A50/50)/HPLA = 40/60, (D) PLGA (3A50/50)/HPLA = 40/80, (E) PLGA (2A50/50)/HPLA = 40/80, and (F) PLGA (2A50/50)/LPLA = 40/80. W1, 5% w/w BSA and dextran (BSA to dextran ratio 6 mg:6 mg) water cosolution; Oh, PG/G = 4 (5.5 mL containing 0.5 mL of 1% PVA and 5% NaCl); W2, 5% NaCl 1000 mL; oil phase 10%, 1200 mg of dichloromethane solvent. (G) PLGA (3A50/50)/LPLA = 40/80 and (H) PLGA (3A50/50)/HPLA = 40/80. W1, 5% w/w BSA and dextran (BSA to dextran ratio 6 mg:6 mg) water solution; Oh, DEG/G = 4 (5.5 mL containing 0.5 mL of 1% PVA and 5% NaCl); W2, 5% NaCl 1000 mL; oil phase 10%, 1200 mg of dichloromethane solvent.

Abbreviations: BSA, bovine serum albumin; PLGA, poly(lactic-co-glycolic acid); LPLA, low viscosity polylactide (molecular weight 60,000); HPLA, high viscosity polylactide (molecular weight 83,000); PVA, poly(vinyl alcohol); PG/G, 1, 2-propylene glycol/glycerol; Oh, hydrophilic oil; DEG/G, ethylene glycol/glycerol; NaCl, sodium chloride; W1, 1% PVA and 5% NaCl water solution; W2, 5% NaCl water solution; w/w, trehalose 1%

Figure 1 Optical microscopic images of free solidified microspheres. (A) PLGA (3A50/50), (B) PLGA (3A50/50)/LPLA = 40/80, (C) PLGA (3A50/50)/HPLA = 40/60, (D) PLGA (3A50/50)/HPLA = 40/80, (E) PLGA (2A50/50)/HPLA = 40/80, and (F) PLGA (2A50/50)/LPLA = 40/80. W1, 5% w/w BSA and dextran (BSA to dextran ratio 6 mg:6 mg) water cosolution; Oh, PG/G = 4 (5.5 mL containing 0.5 mL of 1% PVA and 5% NaCl); W2, 5% NaCl 1000 mL; oil phase 10%, 1200 mg of dichloromethane solvent. (G) PLGA (3A50/50)/LPLA = 40/80 and (H) PLGA (3A50/50)/HPLA = 40/80. W1, 5% w/w BSA and dextran (BSA to dextran ratio 6 mg:6 mg) water solution; Oh, DEG/G = 4 (5.5 mL containing 0.5 mL of 1% PVA and 5% NaCl); W2, 5% NaCl 1000 mL; oil phase 10%, 1200 mg of dichloromethane solvent.Abbreviations: BSA, bovine serum albumin; PLGA, poly(lactic-co-glycolic acid); LPLA, low viscosity polylactide (molecular weight 60,000); HPLA, high viscosity polylactide (molecular weight 83,000); PVA, poly(vinyl alcohol); PG/G, 1, 2-propylene glycol/glycerol; Oh, hydrophilic oil; DEG/G, ethylene glycol/glycerol; NaCl, sodium chloride; W1, 1% PVA and 5% NaCl water solution; W2, 5% NaCl water solution; w/w, trehalose 1%

Figure 2 Optical microscopic images of solidified microspheres. W1, 5% w/w BSA and dextran (BSA to dextran ratio 6 mg:6 mg) water solution; Oh, DEG/G = 4 (5.5 mL containing 0.5 mL of 1% PVA and 5% NaCl); W2, 5% NaCl 1000 mL; oil phase, 10% of 1200 mg dichloromethane solvent. (A) PLGA (3A50/50), (B) PLGA (3A50/50)/LPLA = 40/80, (C) PLGA (3A50/50)/HPLA = 40/60, (D) PLGA (3A50/50)/HPLA = 40/80; (E) PLGA (2A50/50)/HPLA = 40/80, and (F) PLGA (2A50/50)/LPLA = 40/80. W1, 5% w/w BSA and dextran (BSA to dextran ratio 6 mg:6 mg) water cosolution; Oh, PG/G = 4 (5.5 mL containing 0.5 mL of 1% PVA and 5% NaCl); W2, 5% NaCl 1000 mL; oil phase 10%, 1200 mg of dichloromethane solvent. (G) PLGA (3A50/50)/LPLA = 40/80 and (H) PLGA (3A50/50)/HPLA = 40/80.

Abbreviations: BSA, bovine serum albumin; PLGA, poly(lactic-co-glycolic acid); LPLA, low viscosity polylactide (molecular weight 60,000); HPLA, high viscosity polylactide (molecular weight 83,000); PVA, poly(vinyl alcohol); PG/G, 1, 2-propylene glycol/glycerol; Oh, hydrophilic oil; DEG/G, ethylene glycol/glycerol; NaCl, sodium chloride; W1, 1% PVA and 5% NaCl water solution; W2, 5% NaCl water solution; w/w, trehalose 1%.

Figure 2 Optical microscopic images of solidified microspheres. W1, 5% w/w BSA and dextran (BSA to dextran ratio 6 mg:6 mg) water solution; Oh, DEG/G = 4 (5.5 mL containing 0.5 mL of 1% PVA and 5% NaCl); W2, 5% NaCl 1000 mL; oil phase, 10% of 1200 mg dichloromethane solvent. (A) PLGA (3A50/50), (B) PLGA (3A50/50)/LPLA = 40/80, (C) PLGA (3A50/50)/HPLA = 40/60, (D) PLGA (3A50/50)/HPLA = 40/80; (E) PLGA (2A50/50)/HPLA = 40/80, and (F) PLGA (2A50/50)/LPLA = 40/80. W1, 5% w/w BSA and dextran (BSA to dextran ratio 6 mg:6 mg) water cosolution; Oh, PG/G = 4 (5.5 mL containing 0.5 mL of 1% PVA and 5% NaCl); W2, 5% NaCl 1000 mL; oil phase 10%, 1200 mg of dichloromethane solvent. (G) PLGA (3A50/50)/LPLA = 40/80 and (H) PLGA (3A50/50)/HPLA = 40/80.Abbreviations: BSA, bovine serum albumin; PLGA, poly(lactic-co-glycolic acid); LPLA, low viscosity polylactide (molecular weight 60,000); HPLA, high viscosity polylactide (molecular weight 83,000); PVA, poly(vinyl alcohol); PG/G, 1, 2-propylene glycol/glycerol; Oh, hydrophilic oil; DEG/G, ethylene glycol/glycerol; NaCl, sodium chloride; W1, 1% PVA and 5% NaCl water solution; W2, 5% NaCl water solution; w/w, trehalose 1%.

Figure 3 Scanning electron micrographs of microspheres prepared using PLGA (3A50/50) (A), and PLGA (3A50/50)/HPLA (B). W1, 5% w/w BSA and dextran (BSA to dextran ratio 6 mg:6 mg) water solution; Oh, DEG/G = 4 (5.5 mL containing 0.5 mL of 1% PVA and 5% NaCl); W2, 5% NaCl 1000 mL; oil phase 10%, PLGA (3A50/50)/HPLA = 40/80, 1200 mg of dichloromethane solvent.

Abbreviations: BSA, bovine serum albumin; PLGA, poly(lactic-co-glycolic acid); HPLA, high viscosity polylactide (molecular weight 83,000); PVA, poly(vinyl alcohol).

Figure 3 Scanning electron micrographs of microspheres prepared using PLGA (3A50/50) (A), and PLGA (3A50/50)/HPLA (B). W1, 5% w/w BSA and dextran (BSA to dextran ratio 6 mg:6 mg) water solution; Oh, DEG/G = 4 (5.5 mL containing 0.5 mL of 1% PVA and 5% NaCl); W2, 5% NaCl 1000 mL; oil phase 10%, PLGA (3A50/50)/HPLA = 40/80, 1200 mg of dichloromethane solvent.Abbreviations: BSA, bovine serum albumin; PLGA, poly(lactic-co-glycolic acid); HPLA, high viscosity polylactide (molecular weight 83,000); PVA, poly(vinyl alcohol).

Figure 4 Optical microscopic images of protein dextran microparticles recovered from microspheres. (A) W1, 5% w/w BSA and dextran (BSA to dextran ratio 6 mg:6 mg) water solution; Oh, DEG/G = 4 (5.5 mL containing 0.5 mL of 1% PVA and 5% NaCl); W2, 5% NaCl 1000 mL; oil phase 10%, PLGA (3A50/50)/LPLA = 40/80; 1200 mg of dichloromethane solvent; (B) W1, 5% w/w BSA and dextran (BSA to dextran ratio 6 mg:6 mg) water solution; Oh, PG/G = 4 (5.5 mL containing 0.5 mL of 1% PVA and 5% NaCl); W2, 5% NaCl 1000 mL; oil phase, 10% PLGA (3A50/50)/LPLA = 40/80, 1200 mg of dichloromethane solvent.

Abbreviations: BSA, bovine serum albumin; PLGA, poly(lactic-co-glycolic acid); LPLA, low viscosity polylactide (molecular weight 60,000); PG/G, 1, 2-propylene glycol/glycerol; Oh, hydrophilic oil; DEG/G, ethylene glycol/glycerol; NaCl, sodium chloride; W1, 1% PVA and 5% NaCl water solution; W2, 5% NaCl water solution; w/w, trehalose 1%.

Figure 4 Optical microscopic images of protein dextran microparticles recovered from microspheres. (A) W1, 5% w/w BSA and dextran (BSA to dextran ratio 6 mg:6 mg) water solution; Oh, DEG/G = 4 (5.5 mL containing 0.5 mL of 1% PVA and 5% NaCl); W2, 5% NaCl 1000 mL; oil phase 10%, PLGA (3A50/50)/LPLA = 40/80; 1200 mg of dichloromethane solvent; (B) W1, 5% w/w BSA and dextran (BSA to dextran ratio 6 mg:6 mg) water solution; Oh, PG/G = 4 (5.5 mL containing 0.5 mL of 1% PVA and 5% NaCl); W2, 5% NaCl 1000 mL; oil phase, 10% PLGA (3A50/50)/LPLA = 40/80, 1200 mg of dichloromethane solvent.Abbreviations: BSA, bovine serum albumin; PLGA, poly(lactic-co-glycolic acid); LPLA, low viscosity polylactide (molecular weight 60,000); PG/G, 1, 2-propylene glycol/glycerol; Oh, hydrophilic oil; DEG/G, ethylene glycol/glycerol; NaCl, sodium chloride; W1, 1% PVA and 5% NaCl water solution; W2, 5% NaCl water solution; w/w, trehalose 1%.

Figure 5 Scanning electron micrographs of protein dextran recovered from microspheres. (A) W1, 5% w/w BSA and dextran (BSA to dextran ratio 6 mg:6 mg) water solution; Oh, DEG/G = 4 (5.5 mL containing 0.5 mL of 1% PVA and 5% NaCl); W2, 5% NaCl 1000 mL; oil phase 10%; PLGA (3A50/50)/LPLA = 40/80, 1200 mg of dichloromethane solvent. (B) W1, 5% w/w BSA and dextran (BSA to dextran ratio 6 mg:6 mg) water solution; Oh, PG/G = 4 (5.5 mL containing 0.5 mL of 1% PVA and 5% NaCl); W2, 5% NaCl 1000 mL, oil phase 10%; PLGA (3A50/50)/LPLA = 40/80, 1200 mg of dichloromethane solvent.

Abbreviations: BSA, bovine serum albumin; PLGA, poly(lactic-co-glycolic acid); LPLA, low viscosity polylactide (molecular weight 60,000); PVA, poly(vinyl alcohol); PG/G, 1, 2-propylene glycol/glycerol; Oh, hydrophilic oil; DEG/G, ethylene glycol/glycerol; NaCl, sodium chloride; W1, 1% PVA and 5% NaCl water solution; W2, 5% NaCl water solution; w/w, trehalose 1%.

Figure 5 Scanning electron micrographs of protein dextran recovered from microspheres. (A) W1, 5% w/w BSA and dextran (BSA to dextran ratio 6 mg:6 mg) water solution; Oh, DEG/G = 4 (5.5 mL containing 0.5 mL of 1% PVA and 5% NaCl); W2, 5% NaCl 1000 mL; oil phase 10%; PLGA (3A50/50)/LPLA = 40/80, 1200 mg of dichloromethane solvent. (B) W1, 5% w/w BSA and dextran (BSA to dextran ratio 6 mg:6 mg) water solution; Oh, PG/G = 4 (5.5 mL containing 0.5 mL of 1% PVA and 5% NaCl); W2, 5% NaCl 1000 mL, oil phase 10%; PLGA (3A50/50)/LPLA = 40/80, 1200 mg of dichloromethane solvent.Abbreviations: BSA, bovine serum albumin; PLGA, poly(lactic-co-glycolic acid); LPLA, low viscosity polylactide (molecular weight 60,000); PVA, poly(vinyl alcohol); PG/G, 1, 2-propylene glycol/glycerol; Oh, hydrophilic oil; DEG/G, ethylene glycol/glycerol; NaCl, sodium chloride; W1, 1% PVA and 5% NaCl water solution; W2, 5% NaCl water solution; w/w, trehalose 1%.

Figure 6 Distribution of microspheres. W1, 5% w/w BSA and dextran (BSA to dextran ratio 6 mg:6 mg) water solution; Oh, PG/G = 4 (5.5 mL containing 0.5 mL of 1% PVA and 5% NaCl); W2, 5% NaCl 1000 mL, oil phase 10%; PLGA (3A50/50)/LPLA = 40/80, 1200 mg of dichloromethane solvent.

Abbreviations: BSA, bovine serum albumin; PLGA, poly(lactic-co-glycolic acid); LPLA, low viscosity polylactide (molecular weight 60,000); PVA, poly(vinyl alcohol); PG/G, 1, 2-propylene glycol/glycerol; Oh, hydrophilic oil; NaCl, sodium chloride; W1, 1% PVA and 5% NaCl water solution; W2, 5% NaCl water solution; w/w, trehalose 1%; dV/dD, d volume/d density; SD, standard deviation.

Figure 6 Distribution of microspheres. W1, 5% w/w BSA and dextran (BSA to dextran ratio 6 mg:6 mg) water solution; Oh, PG/G = 4 (5.5 mL containing 0.5 mL of 1% PVA and 5% NaCl); W2, 5% NaCl 1000 mL, oil phase 10%; PLGA (3A50/50)/LPLA = 40/80, 1200 mg of dichloromethane solvent.Abbreviations: BSA, bovine serum albumin; PLGA, poly(lactic-co-glycolic acid); LPLA, low viscosity polylactide (molecular weight 60,000); PVA, poly(vinyl alcohol); PG/G, 1, 2-propylene glycol/glycerol; Oh, hydrophilic oil; NaCl, sodium chloride; W1, 1% PVA and 5% NaCl water solution; W2, 5% NaCl water solution; w/w, trehalose 1%; dV/dD, d volume/d density; SD, standard deviation.

Table 1 Effect of PLGA/PLA ratio on loading efficiency and encapsulation efficiency (n = 3)

Table 2 Effect of PLGA to PLA ratio on loading and encapsulation efficiency (n = 3)

Table 3 Effect of PLGA to PLA ratio on loading and encapsulation efficiency (n = 3)

Table 4 Effect of different Oh on loading efficiency and encapsulation efficiency (n = 3)

Table 5 Amount of myoglobin and BSA protein aggregated before and after each formulation step (n = 5)

Figure 7 Comparison of recovery rates for protein activity from microspheres prepared using the different methods (n = 3).

Abbreviation: β-gal, β-galactosidase; w/o/w, water-in-oil-in-water.

Figure 7 Comparison of recovery rates for protein activity from microspheres prepared using the different methods (n = 3).Abbreviation: β-gal, β-galactosidase; w/o/w, water-in-oil-in-water.

Figure 8 Effect of ratio of PLGA/PLA on release in vitro (n = 3). W1, 5% w/w BSA and dextran (BSA to dextran ratio 6 mg:6 mg) water solution; Oh, DEG/G = 4 (5.5 mL containing 0.5 mL of 1% PVA and 5% NaCl); W2, 5% NaCl 1000 mL, oil phase 10%, 1200 mg of dichloromethane solvent. (A) PLGA (3A50/50), (B) PLGA (3A50/50)/LPLA = 40/80, and (C) PLGA (3A50/50)/HPLA = 40/80.

Abbreviations: BSA, bovine serum albumin; PLGA, poly(lactic-co-glycolic acid); LPLA, low viscosity polylactide (molecular weight 60,000); HPLA, high viscosity polylactide (molecular weight 83,000); PVA, poly(vinyl alcohol); PG/G, 1, 2-propylene glycol/glycerol; Oh, hydrophilic oil; DEG/G, ethylene glycol/glycerol; NaCl, sodium chloride; W1, 1% PVA and 5% NaCl water solution; W2, 5% NaCl water solution; w/w, trehalose 1%; PLA, polylactide.

Figure 8 Effect of ratio of PLGA/PLA on release in vitro (n = 3). W1, 5% w/w BSA and dextran (BSA to dextran ratio 6 mg:6 mg) water solution; Oh, DEG/G = 4 (5.5 mL containing 0.5 mL of 1% PVA and 5% NaCl); W2, 5% NaCl 1000 mL, oil phase 10%, 1200 mg of dichloromethane solvent. (A) PLGA (3A50/50), (B) PLGA (3A50/50)/LPLA = 40/80, and (C) PLGA (3A50/50)/HPLA = 40/80.Abbreviations: BSA, bovine serum albumin; PLGA, poly(lactic-co-glycolic acid); LPLA, low viscosity polylactide (molecular weight 60,000); HPLA, high viscosity polylactide (molecular weight 83,000); PVA, poly(vinyl alcohol); PG/G, 1, 2-propylene glycol/glycerol; Oh, hydrophilic oil; DEG/G, ethylene glycol/glycerol; NaCl, sodium chloride; W1, 1% PVA and 5% NaCl water solution; W2, 5% NaCl water solution; w/w, trehalose 1%; PLA, polylactide.

Figure 9 Effect of different materials on release in vitro. W1, 5% w/w BSA and dextran (BSA to dextran ratio 6 mg:6 mg) water solution; Oh, DEG/G = 4 (5.5 mL containing 0.5 mL of 1% PVA and 5% NaCl); W2, 5% NaCl 1000 mL; oil phase 10%, 1200 mg of dichloromethane solvent; formulation I, PLGA (2A50/50)/LPLA = 40/80; formulation II, PLGA (2A50/50)/HPLA = 40/80; formulation III, PLGA (3A50/50)/LPLA = 40/80; formulation IV, PLGA (3A50/50)/HPLA = 40/80.

Abbreviations: BSA, bovine serum albumin; PLGA, poly(lactic-co-glycolic acid); LPLA, low viscosity polylactide (molecular weight 60,000); HPLA, high viscosity polylactide (molecular weight 83,000); PVA, poly(vinyl alcohol); Oh, hydrophilic oil; DEG/G, ethylene glycol/glycerol; NaCl, sodium chloride; W1, 1% PVA and 5% NaCl water solution; W2, 5% NaCl water solution; w/w, trehalose 1%.

Figure 9 Effect of different materials on release in vitro. W1, 5% w/w BSA and dextran (BSA to dextran ratio 6 mg:6 mg) water solution; Oh, DEG/G = 4 (5.5 mL containing 0.5 mL of 1% PVA and 5% NaCl); W2, 5% NaCl 1000 mL; oil phase 10%, 1200 mg of dichloromethane solvent; formulation I, PLGA (2A50/50)/LPLA = 40/80; formulation II, PLGA (2A50/50)/HPLA = 40/80; formulation III, PLGA (3A50/50)/LPLA = 40/80; formulation IV, PLGA (3A50/50)/HPLA = 40/80.Abbreviations: BSA, bovine serum albumin; PLGA, poly(lactic-co-glycolic acid); LPLA, low viscosity polylactide (molecular weight 60,000); HPLA, high viscosity polylactide (molecular weight 83,000); PVA, poly(vinyl alcohol); Oh, hydrophilic oil; DEG/G, ethylene glycol/glycerol; NaCl, sodium chloride; W1, 1% PVA and 5% NaCl water solution; W2, 5% NaCl water solution; w/w, trehalose 1%.

Figure 10 Effect of PLGA/PLA ratio on release in vitro. W1, 5% w/w BSA and dextran (BSA to dextran 6 mg:6 mg) water solution; Oh, DEG/G = 4 (5.5 mL containing 0.5 mL of 1% PVA and 5% NaCl); W2, 5% NaCl 1000 mL; oil phase 10%, 1200 mg of dichloromethane solvent; formulation I, PLGA (3A50/50)/HPLA = 40/60; formulation II, PLGA (3A50/50)/HPLA = 40/80.

Abbreviations: BSA, bovine serum albumin; PLGA, poly(lactic-co-glycolic acid); HPLA, high viscosity polylactide (molecular weight 83,000); PVA, poly(vinyl alcohol); Oh, hydrophilic oil; DEG/G, ethylene glycol/glycerol; NaCl, sodium chloride; W1, 1% PVA and 5% NaCl water solution; W2, 5% NaCl water solution; w/w, trehalose 1%; PLA, polylactide.

Figure 10 Effect of PLGA/PLA ratio on release in vitro. W1, 5% w/w BSA and dextran (BSA to dextran 6 mg:6 mg) water solution; Oh, DEG/G = 4 (5.5 mL containing 0.5 mL of 1% PVA and 5% NaCl); W2, 5% NaCl 1000 mL; oil phase 10%, 1200 mg of dichloromethane solvent; formulation I, PLGA (3A50/50)/HPLA = 40/60; formulation II, PLGA (3A50/50)/HPLA = 40/80.Abbreviations: BSA, bovine serum albumin; PLGA, poly(lactic-co-glycolic acid); HPLA, high viscosity polylactide (molecular weight 83,000); PVA, poly(vinyl alcohol); Oh, hydrophilic oil; DEG/G, ethylene glycol/glycerol; NaCl, sodium chloride; W1, 1% PVA and 5% NaCl water solution; W2, 5% NaCl water solution; w/w, trehalose 1%; PLA, polylactide.

Figure 11 Effect of different Oh on release in vitro (n = 3). W1, 5% w/w BSA and dextran (BSA to dextran ratio 6 mg:6 mg) water solution; Oh, PG/G = 4 (5.5 mL containing 0.5 mL of 1% PVA and 5% NaCl); W2, 5% NaCl 1000 mL; oil phase 10%, 1200 mg of dichloromethane solvent; formulation A, PLGA (3A50/50)/LPLA = 40/80; formulation B, PLGA (3A50/50)/HPLA = 40/80.

Abbreviations: BSA, bovine serum albumin; PLGA, poly(lactic-co-glycolic acid); HPLA, high viscosity polylactide (molecular weight 83,000); PVA, poly(vinyl alcohol); PG/G, 1, 2-propylene glycol/glycerol; Oh, hydrophilic oil; NaCl, sodium chloride; W1, 1% PVA and 5% NaCl water solution; W2, 5% NaCl water solution; w/w, trehalose 1%; LPLA, low viscosity polylactide (molecular weight 60,000).

Figure 11 Effect of different Oh on release in vitro (n = 3). W1, 5% w/w BSA and dextran (BSA to dextran ratio 6 mg:6 mg) water solution; Oh, PG/G = 4 (5.5 mL containing 0.5 mL of 1% PVA and 5% NaCl); W2, 5% NaCl 1000 mL; oil phase 10%, 1200 mg of dichloromethane solvent; formulation A, PLGA (3A50/50)/LPLA = 40/80; formulation B, PLGA (3A50/50)/HPLA = 40/80.Abbreviations: BSA, bovine serum albumin; PLGA, poly(lactic-co-glycolic acid); HPLA, high viscosity polylactide (molecular weight 83,000); PVA, poly(vinyl alcohol); PG/G, 1, 2-propylene glycol/glycerol; Oh, hydrophilic oil; NaCl, sodium chloride; W1, 1% PVA and 5% NaCl water solution; W2, 5% NaCl water solution; w/w, trehalose 1%; LPLA, low viscosity polylactide (molecular weight 60,000).

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