Acidic microclimate pH distribution in PLGA microspheres monitored by confocal laser scanning microscopy

Abstract

Purpose: The acidic microclimate pH (micropH) distribution inside poly(lactic-co-glycolic acid) (PLGA) microspheres was monitored quantitatively as a function of several formulation variables.

Methods: A ratiometric method by confocal laser scanning microscopy with Lysosensor yellow/blue dextran was adapted from those previously reported, and micropH distribution kinetics inside microspheres was examined during incubation under physiologic conditions for 4 weeks. Effects of PLGA molecular weight (MW) and lactic/glycolic acid ratio, microspheres size and preparation method, and polymer blending with poly(ethylene glycol) (PEG) were evaluated.

Results: micropH kinetics was accurately sensed over a broadly acidic range (2.8 < micropH < 5.8) and was more acidic and variable inside PLGA with lower MW and lactic/glycolic acid ratio. Lower micropH was found in larger microspheres of lower MW polymers, but size effects for lactic-rich polymers were insignificant during 4 weeks. Microspheres prepared by the oil-in-oil emulsion method were less acidic than those prepared by double emulsion, and blending PLGA 50/50 with 20% PEG increased micropH significantly (micropH > 5 throughout incubation).

Conclusions: Coupling this method with that previously developed (SNARF-1 dextran for micropH 5.8-8.0) should provide microclimate pH mapping over the entire useful pH range (2.8-8.0) for optimization of PLGA delivery of pH-sensitive bioactive substances.

Figure 1

The pH sensitivity and concentration-independence of the standard curve of dextran conjugated Lysosensor yellow/blue^®. The third-order polynomial curve fitting the data was Y = −0.15x³ + 1.84x² −6.51x + 7.26, where Y = I_450nm/I_520nm; x = pH, r² = 0.98.

Figure 2

Typical processed confocal images of microspheres (size 45 – 65 μm) prepared by w/o/w during incubation at 37°C for 4 weeks for PLGA 50/50 (i.v. = 0.2 dl/g) (A), 50/50 (i.v. = 0.6 dl/g) (B), 85/15 (i.v. = 0.6 dl/g) (C), and 100/0 (i.v. = 0.6 dl/g) (D). Microspheres were incubated for 1 (A1 – D1), 7 (A2 – D2), 14 (A3 – D3), 21 (A4 – D4), and 28 (A5 – D5) days.

Figure 3

Typical processed confocal images of w/o/w microspheres (size 90 – 125 μm) during incubation at 37°C for 4 weeks for PLGA 50/50 (i.v. = 0.2 dl/g) (A), 50/50 (i.v. = 0.6 dl/g) (B), 85/15 (i.v. = 0.6 dl/g) (C), and 100/0 (i.v. = 0.6 dl/g) (D). Microspheres were incubated for 1 (A1 – D1), 7 (A2 – D2), 14 (A3 – D3), 21 (A4 – D4), and 28 (A5 – D5) days.

Figure 4

Effect of polymer MW on μpH distribution kinetics inside w/o/w PLGA 50/50 microspheres (n = 20) during incubation for 4 weeks in PBST at 37°C for PLGA with i.v. = 0.2 dl/g (A) and 0.6 dl/g (B). Microsphere size was 45 – 65 μm (A1, B1) and 90 – 125 μm (A2, B2).

Figure 5

Effect of polymer composition (lactic/glycolic acid ratio) on μpH distribution kinetics inside w/o/w PLGA microspheres (n = 20) during incubation at 37°C in PBST for PLGA 50/50 (A), 85/15 (B) and 100/0 (C). Microsphere size was 45 – 65 μm (A1 – C1) and 90 – 125 μm (A2 – C2) and PLGA i.v. was 0.6 dl/g

Figure 6

Typical processed confocal images of PLGA 50/50 (i.v. = 0.6 dl/g) microspheres (size 45 – 65 μm) prepared by w/o/w (A) and o/o (B) methods during incubation at 37°C for 4 weeks. Microspheres were incubated for 1 (A1, B1), 7 (A2, B2), 14 (A3, B3), 21 (A4, B4), and 28 (A5, B5) days.

Figure 7

μpH distribution kinetics inside PLGA 50/50 (i.v. = 0.6 dl/g) o/o microspheres during incubation in PBST at 37°C.

Figure 8

Typical processed confocal images of o/o microspheres prepared with polymer blends of PLGA 50/50 and 20 % PEG (size 45 – 65 μm) during incubation at 37°C for 4 weeks for PLGA 50/50 with i.v. = 0.2 dl/g (A) and 0.6 dl/g (B). Microspheres were incubated for 1 (A1, B1), 14 (A2, B2), 21 (A3, B3), and 28 (A4, B4) days.

Figure 9

Effect of PEG blending on μpH distribution kinetics inside PLGA o/o microspheres during incubation for 4 weeks in PBST at 37°C for PLGA 50/50 with i.v. = 0.2 dl/g (A) and 0.6 dl/g (B). PEG was blended at 20% w/w

Figure 10

Effect of PLGA MW and composition (w/o/w) (A) and microsphere preparation method with or without addition of PEG (o/o) (B) on dye release kinetics during incubation for 4 weeks in PBST at 37°C. Dye loading efficiency was 0.40, 0.41, 0.38, 0.36% for PLGA 50/50 (i.v. = 0.2 dl/g), 50/50 (i.v. = 0.6 dl/g), 85/15 (i.v. = 0.6 dl/g), and 100/0 (i.v. = 0.6 dl/g) (A), and 0.92 and 0.88% with and without PEG blending (B), respectively. All microspheres were 45 – 65 μm.