Controlled Release of Lysozyme from Double-Walled Poly(Lactide-Co-Glycolide) (PLGA) Microspheres

Abstract

Double-walled microspheres based on poly(lactide-co-glycolide) (PLGA) are potential delivery systems for reducing a very high initial burst release of encapsulated protein and peptide drugs. In this study, double-walled microspheres made of glucose core, hydroxyl-terminated poly(lactide-co-glycolide) (Glu-PLGA), and carboxyl-terminated PLGA were fabricated using a modified water-in-oil-in-oil-in-water (w1/o/o/w2) emulsion solvent evaporation technique for the controlled release of a model protein, lysozyme. Microspheres size, morphology, encapsulation efficiency, lysozyme in vitro release profiles, bioactivity, and structural integrity, were evaluated. Scanning electron microscopy (SEM) images revealed that double-walled microspheres comprising of Glu-PLGA and PLGA with a mass ratio of 1:1 have a spherical shape and smooth surfaces. A statistically significant increase in the encapsulation efficiency (82.52% ± 3.28%) was achieved when 1% (w/v) polyvinyl alcohol (PVA) and 2.5% (w/v) trehalose were incorporated in the internal and external aqueous phase, respectively, during emulsification. Double-walled microspheres prepared together with excipients (PVA and trehalose) showed a better control release of lysozyme. The released lysozyme was fully bioactive, and its structural integrity was slightly affected during microspheres fabrication and in vitro release studies. Therefore, double-walled microspheres made of Glu-PLGA and PLGA together with excipients (PVA and trehalose) provide a controlled and sustained release for lysozyme.

Keywords: controlled release; double-walled microspheres; drug delivery; poly(lactide-co-glycolide); therapeutic proteins.

Figure 1

Scanning electron microscope (SEM) images of lysozyme loaded double-walled microspheres prepared in different formulations. (a) LF1; (b) LF2; (c) LF3; and, (d) LF4.

Figure 2

Scanning electron microscope (SEM) images of lysozyme loaded single-walled microspheres prepared in different formulations. (a) LF5; (b) LF6; (c) LF7; (d) LF8.

Figure 3

In vitro release profiles of lysozyme for (a) 24 h double-walled; (b) 24 h single-walled; and (c) 70 days double-walled and single-walled microspheres prepared in different formulations. The data are presented as mean ± SD (n = 3).

Figure 4

Field-emission scanning electron microscope (FE-SEM) images of lysozyme loaded double-walled microspheres prepared with 2.5% trehalose (w/v) in internal aqueous phase at different resolution: (a) 2000× mag; (b) 6000× mag and double-walled microspheres prepared with 1% PVA (w/v) in internal aqueous phase; and, 2.5% trehalose (w/v) in external aqueous phase at different resolution: (c) 2000× mag; (d) 6000× mag.

Figure 5

In vitro release profiles of lysozyme from double-walled microspheres prepared together with excipients and without excipients in the internal and external aqueous phase during preparation. The data are presented as mean ± SD (n = 3).

Figure 6

Polyacrylamide Gel Electrophoresis (SDS-PAGE) of lysozyme extracted and released from double-walled microspheres. Lane 1: standard molecular weight marker (M_w range: 3.5–260 kDa); Lane 2: fresh lysozyme standard; Lane 3: lysozyme extracted from microspheres prepared without excipients; Lane 4: day 1 released lysozyme from microspheres prepared without excipients; Lanes 5–6: day 1 and day 42 released lysozyme from microspheres prepared with 1% PVA (w/v) in internal aqueous phase and 2.5% trehalose (w/v) in external aqueous phase, respectively.