Functionalization of silk with actinomycins from Streptomyces anulatu s BV365 for biomedical applications

Abstract

Silk, traditionally acclaimed as the "queen of fiber," has been widely used thanks to its brilliant performance such as gentleness, smoothness and comfortableness. Owing to its mechanical characteristics and biocompatibility silk has a definitive role in biomedical applications, both as fibroin and fabric. In this work, the simultaneous dyeing and functionalization of silk fabric with pigments from Streptomyces anulatus BV365 were investigated. This strain produced high amounts of orange extracellular pigments on mannitol-soy flour agar, identified as actinomycin D, C2 and C3. The application of purified actinomycins in the dyeing of multifiber fabric was assessed. Actinomycins exhibited a high affinity towards protein fibers (silk and wool), but washing durability was maintained only with silk. Acidic condition (pH5) and high temperature (65°C) facilitated the silk dyeing. The morphologies and chemical components of the treated silk fabrics were analyzed using scanning electron microscopy and Fourier transform infrared spectroscopy. The results showed the pigments bind to the silk through interaction with the carbonyl group in silk fibroin rendering the functionalized, yet surface that does not cause skin irritation. The treated silk exhibited a remarkable antibacterial effect, while the biocompatibility test performed with 3D-reconstructed human epidermis model indicated safe biological properties, paving the way for future application of this material in medicine.

Keywords: Streptomyces; actinomycins; anticancer; antimicrobial; biocompatibility; functional biomaterials; nonactin; silk.

FIGURE 1

The appearance of pigmented S. anulatus BV365 grown on MSF (A, B) or TSB (C, D) solid media for 5 days at 30°C (A). Scanning electron micrograph shows the spore morphology (E). Stereo microscope images, ×6.3 magnifications (System SZX10, Olympus), Field emission scanning electron microscopy (FESEM, Mira3 Tescan).

FIGURE 2

HR-LCMS/MS identification of the actinomycins in fraction F3. Different amino acid substitutions leading to various actinomycin structures can be observed with valine (red) or isoleucine (blue).

FIGURE 3

Photograph of the actinomycins fraction F3 in a solvent system consisting of acetone:water (10:90) and photographs of samples of multifiber fabric dyed at 65°C, taken before and after washing.

FIGURE 4

Dyeing of multifiber fabric, results obtained for silk part at different pH and temperature using 1% o.w.f. in the dyebath (A); Absorbance of the dyebath after dyeing of silk on multifiber fabric (left) and K/S values of silk part dyed at 25, 45°C and 65°C and at pH 3 and pH 5 of the dyebath (B).

FIGURE 5

Photographs of silk after dyeing for different durations with actinomycins fraction F3 (A). Absorbance of the dyebath and corresponding color strength of silk fabric dyed during different times (B) and color difference ΔE* between samples dyed during various times and sample dyed for 10 min (C).

FIGURE 6

Stereo light microscope images of untreated sample of silk (left) and dyed silk (right) at 6,3× magnification (A); SEM images of untreated sample of silk at different magnifications (top) and dyed silk at different magnifications (bottom) (B). ATR-FTIR spectra of S. anulatus BV365 pigment actinomycins (top) and silk fabric before and after dyeing with actinomycins containing F3 (bottom) (C).

FIGURE 7

Macroscopic appearance of Labskin1.1 model (A). Cell viability was calculated as % of MTT reduction to formazan compared to negative control treated with PBS (B). Quantification of release of proinflammatory cytokine IL-1α after treatment (C). Results are averages of n = 3, error bars represent SD and values were compared to the PBS-treated control using a t-test; (*p ≤ 0.01, **p ≤ 0.01) (* = p < 0.01; ** = p < 0.001).