Protein Loading into Spongelike PLGA Microspheres

Abstract

A self-healing microencapsulation process involves mixing preformed porous microspheres in an aqueous solution containing the desired protein and converting them into closed-pore microspheres. Spongelike poly-d,l-lactide-co-glycolide (PLGA) microspheres are expected to be advantageous to protein loading through self-healing. This study aimed to identify and assess relevant critical parameters, using lysozyme as a model protein. Several parameters governed lysozyme loading. The pore characteristics (open-pore, closed-pore, and porosity) of the preformed microspheres substantially affected lysozyme loading efficiency. The type of surfactant present in the aqueous medium also influenced lysozyme loading efficiency. For instance, cetyltrimethylammonium bromide showing a superior wetting functionality increased the extent of lysozyme loading more than twice as compared to Tween 80. Dried preformed microspheres were commonly used before, but our study found that wet microspheres obtained at the end of the microsphere manufacturing process displayed significant advantages in lysozyme loading. Not only could an incubation time for hydrating the microspheres be shortened dramatically, but also a much more considerable amount of lysozyme was encapsulated. Interestingly, the degree of microsphere hydration determined the microstructure and morphology of closed-pore microspheres after self-healing. Understanding these critical process parameters would help tailor protein loading into spongelike PLGA microspheres in a bespoke manner.

Keywords: closed-pore; microencapsulation; open-pore; poly-d,l-lactide-co-glycolide; porous microspheres; protein.

Figure 1

The schematic representation of various experimental settings for lysozyme loading into porous poly-d,l-lactide-co-glycolide (PLGA) microspheres. RT stands for room temperature.

Figure 2

A SEM image showing (a) the surface and (b) internal morphology of a typical spongelike PLGA microsphere (bar size = 10 µm). As a part of the microsphere surface is peeled off, its inside is exposed without damaging its internal structure. The surface and inside of the microsphere have numerous pores that are inter-connected to have an open pore structure.

Figure 3

The pore size distributions of spongelike PLGA microspheres prepared using (a) 3 mL or (b) 7 mL of the ammonia solution. The microsphere porosity is determined by the amount of the ammonia solution used to trigger ammonolysis of isopropyl formate, the dispersed solvent of PLGA.

Figure 4

Lysozyme loading into wet PLGA microspheres prepared using 7 mL of the ammonia solution. They were incubated in aqueous 25 mg/mL to 200 mg/mL lysozyme solutions containing 0.2% Tween 80 for the specified periods of time. Lysozyme contents in the microspheres were then determined.

Figure 5

Lysozyme loading into dried microspheres prepared using 7 mL of the ammonia solution. They were dispersed in a 100 mg/mL lysozyme solution containing 0.2% Tween 80 at RT. At specified time intervals, lysozyme contents in the microspheres were measured.

Figure 6

Sedimentation rates of dried microspheres prepared using 7 mL of the ammonia solution. They were dispersed in 0.2% Tween 80 solution at RT, and the amounts of their sediments were determined over time.

Figure 7

Effect of surfactant type upon lysozyme loading in dried microspheres prepared using 7 mL of the ammonia solution. In terms of lysozyme loading efficiency, CTAB shows a superior functionality than Tween 80.

Figure 8

Comparison of lysozyme payloads in porous microspheres before and after pore closure. Wet microspheres prepared using 3 mL and 7 mL of the ammonia solution were equilibrated at RT for 2 h in (a) 50 mg/mL and (b) 100 mg/mL lysozyme solutions containing 0.2% Tween 80, which was further subjected to self-healing. Lysozyme contents in the microspheres were determined before and after self-healing.

Figure 9

Effect of surfactant type upon lysozyme payloads in PLGA microspheres before and after pore closure. Dried microspheres prepared using 3 mL of the ammonia solution were first incubated in a 100 mg/mL lysozyme solution containing 0.2% either Tween 80 or CTAB (RT/2 h), and self-sealing (41 °C/2 h) was carried out. Lysozyme contents in microspheres before and after self-healing were determined.

Figure 10

SEM images of the internal morphology of closed-pore PLGA microspheres prepared using 7 mL of the ammonia solution. Dried microspheres were first equilibrated in an aqueous solution containing 0.2% either (a) Tween 80 or (b) CTAB, and were then subjected to self-healing.

Figure 11

SEM images of the internal morphology of various PLGA microspheres (a) before and (b) after self-healing (bar size = 10 µm). Wet microspheres prepared using 3 mL and 7 mL of the ammonia solution were first incubated in 0.2% Tween 80 solution at RT for 2 h and were followed by self-healing.

Figure 12

SEM images of the internal morphology of dried PLGA microspheres after self-healing (bar size = 10 µm). Dried microspheres prepared using 3 mL and 7 mL of the ammonia solution were first incubated at RT for (a) 2 h and (b) 24 h and were followed by self-healing.

Figure 13

Confocal microscopic images showing the distributions of (a) nile red, (b) FITC-dextran, and (c) nile red and FITC-dextran within PLGA microspheres.