Preparation and characterization of silk fibroin as a biomaterial with potential for drug delivery

Abstract

Background: Degummed silk fibroin from Bombyx mori (silkworm) has potential carrier capabilities for drug delivery in humans; however, the processing methods have yet to be comparatively analyzed to determine the differential effects on the silk protein properties, including crystalline structure and activity.

Methods: In this study, we treated degummed silk with four kinds of calcium-alcohol solutions, and performed secondary structure measurements and enzyme activity test to distinguish the differences between the regenerated fibroins and degummed silk fibroin.

Results: Gel electrophoresis analysis revealed that Ca(NO3)2-methanol, Ca(NO3)2-ethanol, or CaCl2-methanol treatments produced more lower molecular weights of silk fibroin than CaCl2-ethanol. X-ray diffraction and Fourier-transform infrared spectroscopy showed that CaCl2-ethanol produced a crystalline structure with more silk I (α-form, type II β-turn), while the other treatments produced more silk II (β-form, anti-parallel β-pleated sheet). Solid-State 13C cross polarization and magic angle spinning-nuclear magnetic resonance measurements suggested that regenerated fibroins from CaCl2-ethanol were nearly identical to degummed silk fibroin, while the other treatments produced fibroins with significantly different chemical shifts. Finally, enzyme activity test indicated that silk fibroins from CaCl2-ethanol had higher activity when linked to a known chemotherapeutic drug, L-asparaginase, than the fibroins from other treatments.

Conclusions: Collectively, these results suggest that the CaCl2-ethanol processing method produces silk fibroin with biomaterial properties that are appropriate for drug delivery.

Figure 1

SEM photographs of B. mori silk fibroin prepared with various solutions. (A) Degummed silk fibroin. (B) Silk fibroin prepared from Ca(NO₃)₂·4H₂O-methanol solution. (C) Silk fibroin prepared from Ca(NO₃)₂·4H₂O-ethanol solution. (D) Silk fibroin prepared from CaCl₂-methanol-H₂O solution. (E) Silk fibroin prepared from CaCl₂-ethanol-H₂O solution.

Figure 2

SDS-PAGE analysis of B. mori silk fibroins prepared with various solutions. Silk fibroins were prepared from four different calcium-alcohol solutions (as described below) then dissolved in hot water. The range of molecular weight of the proteins produced by each solution was determined by SDS-PAGE with 12% acrylamide gel and 4% condensing gel, which was stained with 0.25% Coomassie Brilliant Blue R-250. Lanes: M, marker. (A) silk fibroin prepared from Ca(NO₃)₂·4H₂O-methanol solution. (B) silk fibroin prepared from Ca(NO₃)₂·4H₂O-ethanol solution. (C) silk fibroin prepared from CaCl₂-methanol-H₂O solution. (D) silk fibroin prepared from CaCl₂-ethanol-H₂O solution. (E) Degummed silk fibroin.

Figure 3

FTIR transmittance spectra of B. mori silk fibroins prepared with various solutions. (A) Degummed silk fibroin. (B) Silk fibroin prepared from Ca(NO₃)₂·4H₂O-methanol solution. (C) Silk fibroin prepared from Ca(NO₃)₂·4H₂O-ethanol solution. (D) Silk fibroin prepared from CaCl₂-methanol-H₂O solution. (E) Silk fibroin prepared from CaCl₂-ethanol-H₂O solution.

Figure 4

Wide-angle X-ray diffraction patterns of B. mori silk fibroins prepared with various solutions. (A) Degummed silk fibroin. (B) Silk fibroin prepared from Ca(NO₃)₂·4H₂O-methanol solution. (C) Silk fibroin prepared from Ca(NO₃)₂·4H₂O-ethanol solution. (D) Silk fibroin prepared from CaCl₂-methanol-H₂O solution. (E) Silk fibroin prepared from CaCl₂-ethanol-H₂O solution.

Figure 5

Solid-state¹³C CP/MAS-NMR spectra of B. mori silk fibroins prepared with various solutions. (A) Degummed silk fibroin. (B) Silk fibroin prepared from Ca(NO₃)₂·4H₂O-methanol solution. (C) Silk fibroin prepared from Ca(NO₃)₂·4H₂O-ethanol solution. (D) Silk fibroin prepared from CaCl₂-methanol-H₂O solution. (E) Silk fibroin prepared from CaCl₂-ethanol-H₂O solution.

Figure 6

Enzyme activity tests for silk fibroins prepared with various solutions. L-ASNase (A) and glucose oxidase (B) were separately immobilized to degummed silk fibroin or regenerated fibroins prepared from four different calcium-alcohol solutions. The activities of these enzymes attached to the fibroins were calculated as a change in optical density at 450 or 460 nm measured on a microplate reader, accordingly. Results are presented as mean ± SD (n = 3 assays of triplicate samples). P < 0.05 was considered to be statistically significant.