An Approach to the Design of a Particulate System for Oral Protein Delivery .II. Preparation and Stability Study of rhGH-Loaded Microspheres in Simulated Gastrointestinal Fluids

Abstract

The delivery of therapeutic proteins has gained momentum with development of biotechnology. However, large molecular weight, hydrophilic nature and susceptibility to harsh environment of gastrointestinal tract (GIT) resulted in low absorption. The main objective of this work was the design of a particulate system for oral delivery of recombinant human growth hormone (rhGH) on the basis of particle uptake mechanism in GIT. Biodegradable protein-loaded microspheres were prepared using Resomers (RG207, RG756 and RG505) by double emulsion methods. Aqueous solution of protein and freshly prepared rhGH-zinc complex were used for loading process. Various analytical methods, including fluorescence spectroscopy, SDS-PAGE electrophoresis and reversed-phase chromatography, were set up for the quantification and qualification of rhGH before and after the formulation and fabrication procedures. At the optimum conditions, microspheres were mostly below 10 μm with relatively high protein loading (> 50%). Obtained data showed that the stability of protein did not change during the formulation and microencapsulation processes. Results also showed that the encapsulation process in the presence of zinc caused no detectable change in the protein chemical stability. In-vitro stability study of microspheres in different simulated GI media indicated that the entrapped protein was physically stable. Less than 20% of rhGH was released from the microspheres incubated in both simulated stomach and intestine fluids for 3 and 6 h, respectively.

Keywords: Biodegradable polymer; Microencapsulation; Microparticle; Oral protein delivery; Resomer®.

Figure 1

Scanning electron micrograph of microspheres prepared. a) formulation G, b) formulation H, c) formulation I

Figure 2

SDS-PAGE results of rhGH encapsulated in various microspheres. Lane 1: Standard protein markers, Lane 2: Standard rhGH, Lane 3: Standard rhGH complexed with zinc (1:100 molar ratio), Lanes 4-7: F, G, H and I microspheres, respectively, fabricated by method II, Lane 8: E microspheres fabricated by method I

Figure 3

Reversed-phase HPLC results of (a) standard solution of rhGH, and (b) rhGH extracted from G microspheres (peak I: native rhGH, peak II: Physiogel^®).

Figure 4

Scanning electron micrographs of G microspheres after incubation at 37°C in SGF-a for 6 h.

Figure 5

Scanning electron micrographs of G microspheres after incubation at 37°C in SIF-a for 12 h.

Figure 6

Scanning electron micrographs of G microspheres after incubation for 12 h at 37°C in FaSSIF.

Figure 7

Scanning electron micrographs of G microspheres after incubation for 12 h at 37°C in FaSSIF.

Figure 8

Scanning electron micrograph of G microspheres after incubation for 3 h at 37°C in SGF-a followed by a 6-hr incubation at 37°C in SIF-a