Tracking Bacterial Nanocellulose in Animal Tissues by Fluorescence Microscopy

Abstract

The potential of nanomaterials in food technology is nowadays well-established. However, their commercial use requires a careful risk assessment, in particular concerning the fate of nanomaterials in the human body. Bacterial nanocellulose (BNC), a nanofibrillar polysaccharide, has been used as a food product for many years in Asia. However, given its nano-character, several toxicological studies must be performed, according to the European Food Safety Agency's guidance. Those should especially answer the question of whether nanoparticulate cellulose is absorbed in the gastrointestinal tract. This raises the need to develop a screening technique capable of detecting isolated nanosized particles in biological tissues. Herein, the potential of a cellulose-binding module fused to a green fluorescent protein (GFP-CBM) to detect single bacterial cellulose nanocrystals (BCNC) obtained by acid hydrolysis was assessed. Adsorption studies were performed to characterize the interaction of GFP-CBM with BNC and BCNC. Correlative electron light microscopy was used to demonstrate that isolated BCNC may be detected by fluorescence microscopy. The uptake of BCNC by macrophages was also assessed. Finally, an exploratory 21-day repeated-dose study was performed, wherein Wistar rats were fed daily with BNC. The presence of BNC or BCNC throughout the GIT was observed only in the intestinal lumen, suggesting that cellulose particles were not absorbed. While a more comprehensive toxicological study is necessary, these results strengthen the idea that BNC can be considered a safe food additive.

Keywords: absorption; bacterial cellulose nanocrystals; bacterial nanocellulose; cellulose binding module; fluorescence microscopy; food additive; gastrointestinal tract.

Figure 1

FTIR spectra of BNC and BCNC.

Figure 2

(A,B) TEM micrographs of the BCNC prepared by the acid hydrolysis of BNC and (C) the corresponding particle-size distributions. Scale bars: (A) 500 nm and (B) 200 nm.

Figure 3

Adsorption isotherm of GFP–CBM onto: (A) BNC and (B) BCNC, after 2 h of incubation.

Figure 4

FM images of GFP–CBM (0.05 mg/mL) bound onto (A) BNC and (B) BCNC after 2 h of incubation. BNC and BCNC stock solutions were both at 0.25 mg/mL. Scale bar: 10 µm.

Figure 5

Microscopic images of GFP–CBM:BCNC (0.002 mg/mL) mixed with 2% gelatin (1:1) (A,B) and of GFP–CBM:BCNC aqueous suspension (C). (A) FM image. (B,C) TEM images. Scale bars: (A) 20 µm and (B,C) 1 µm.

Figure 6

CLEM images of BCNC labeled with GFP–CBM. Small green dots in FM correspond to single BCNC in TEM (highlighted in the red boxes). The solid part of the TEM grid had some autofluorescence (yellow arrow); however, it did not interfere with the visualization as the GFP–CBM:BCNC-gelatin mixture was fixed in the transparent network mesh. Scale bars: (A–C) 10 µm for FM, (A,C) 500 nm and (B) 1 µm for TEM.

Figure 7

Metabolic viability of (A) L929 and (B) BMMΦ cells when cultured with increasing doses of BCNC for 24 h, as assessed by the Resazurin assay. PBS was used as the control. Data is expressed as a percentage relative to the control and is the mean ± SD of three independent experiments. All treatment conditions were compared with the control using Dunnett’s multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 compared to control (non-treated cells).

Figure 8

Cellular uptake of BCNC by phagocytic cells. FM images of macrophages exposed to 0.001 mg/mL BCNC for 2, 4, 6 and 24 h. DAPI (blue), Pha-red (red) and GFP–CBM (green) were used for nuclei, actin cytoskeleton and BCNC visualization, respectively. Scale bar: 50 µm.

Figure 9

FM images of a male rat histological sample after a 21-day repeated-dose study. Four GIT regions (duodenum, jejunum, ilium and colon) were stained for nuclei (blue), actin cytoskeleton (red) and cellulose (green). Control (no gavage) and BNC (oral gavage) animals. Cellulose fibers were detected at the intestinal lumen or trapped between the villi (pointed out by the yellow arrow and the respective amplified image in the yellow box). Scale bar: 200 µm.