The silk of gorse spider mite Tetranychus lintearius represents a novel natural source of nanoparticles and biomaterials

Abstract

Spider mites constitute an assemblage of well-known pests in agriculture, but are less known for their ability to spin silk of nanoscale diameters and high Young's moduli. Here, we characterize silk of the gorse spider mite Tetranychus lintearius, which produces copious amounts of silk with nano-dimensions. We determined biophysical characteristics of the silk fibres and manufactured nanoparticles and biofilm derived from native silk. We determined silk structure using attenuated total reflectance Fourier transform infrared spectroscopy, and characterized silk nanoparticles using field emission scanning electron microscopy. Comparative studies using T. lintearius and silkworm silk nanoparticles and biofilm demonstrated that spider mite silk supports mammalian cell growth in vitro and that fluorescently labelled nanoparticles can enter cell cytoplasm. The potential for cytocompatibility demonstrated by this study, together with the prospect of recombinant silk production, opens a new avenue for biomedical application of this little-known silk.

Figure 1

T. lintearius silk. (A) Gorse plant covered with thick “film” of T. lintearius silk; (B) adult T. lintearius; (C) native silk taken directly from the plant and visualised by SEM; (D) low density T. lintearius silk visualised by SEM; (E) measurements of individual silk fibers (SEM); (F) individual silk fiber (arrowhead) deposited on a silicon slide used for AFM measurements. Scale bar: b—100 µm, c, d and f—10 µm, e—2 µm.

Figure 2

T. lintearius individual silk fiber thickness and Young’s modulus compared with silk of T. urticae. (A) Silk fiber thickness of T. urticae and T. lintearius measured by AFM; (B) Young’s modulus of individual silk fiber of T. urticae and T. lintearius measured by AFM. Data shown are mean ± SEM (n = 7).

Figure 3

SDS-PAGE gel of regenerated silk fibroin from B. mori (L1) and silk from T. lintearius (L2, L3, L4). Arrowheads mark highly stained bands at 15 kDa, 40 kDa and 85 kDa and asterisks indicate moderately stained bands at 35 kDa, 50 kDa, 140 kDa and ≈ 200 kDa.

Figure 4

Amide region (I–IV) of the spectra of: (A) silk from T. lintearius; (B) degummed fibroin from B. mori, and (C) and (D) the nanoparticles obtained from them, respectively.

Figure 5

Secondary structures calculated from analysis of the IR spectra covering the amide I region (1735–1580 cm^–1) of freeze dried silk from T. lintearius, degummed silk fibroin from B. mori, and the nanoparticles obtained from them. Data are expressed as mean ± SD (n = 3). Groups denoted by different letters are statistically different (p < 0.05).

Figure 6

Focused Ion Beam milling combined with Scanning Electron Microscopy (FIB-SEM) of T. lintearius nanoparticles at different concentrations. (A) 10X dilution of T. lintearius nanoparticles showing dense conglomerates of particles. (B) 100X dilution shows spontaneous formation of silk fibers (arrowheads). (C) At 1000X dilution individual nanoparticles show diameter of ca. 20 nm (arrowheads) (D) Silicone wafer with no sample loaded (control). Scale bar: a—200 nm, b–d—100 nm.

Figure 7

Proliferation of the L929 fibroblast cell line growing on the T. lintearius silk (Tl-S) and B. mori silk fibroin (Bm-SF) films at 48 h, 5 days and 7 days after seeding. The control corresponds to cells growing on the nude culture plates. Data are expressed as the average values of relative fluorescence units (RFU) (570–610 nm) ± SD (n = 5) of the PrestoBlue test. (*) represents significant statistical differences relative to group control.

Figure 8

Cell proliferation of the L929 cell line for aqueous dispersions of Tl-SN and Bm-SFN at different concentrations, based on the PrestoBlue assay. Data are expressed as mean ± SD (n = 4). (*represents significant statistical differences between different nanoparticles concentration data and controls (100% proliferation), while significant statistical differences among different studied silk types are indicated by the (•) symbol.

Figure 9

Confocal laser scanning microscopy of HDF cell line after 24 h of exposure to FITC-labelled silk nanoparticles (green). (A) Control cells grown without NP exposure; (B) HDF + Tl-SN; (C) HDF + Bm-SFN. Nuclei were stained with DAPI (blue) and cytoplasmic actin filaments with Atto Rho6G phalloidin (red). Scale bar: 50 µm.

Figure 10

Detection and quantification by flow cytometry of HDF and HepG2 cellular uptake of FITC-labelled nanoparticles of T. lintearius silk (Tl-SN) and B. mori silk fibroin (Bm-SFN). Flow cytometry histograms of cell count versus FITC intensity of the studied FITC-labeled NP added to (A) HDF and (B) HepG2 cells after 24 h of cell exposure.