Stability of plant virus-based nanocarriers in gastrointestinal fluids

Abstract

Cowpea mosaic virus (CPMV) is a plant virus which is being extensively investigated as a drug delivery and vaccine nanocarrier for parenteral administration. However, to date little is known about the suitability of plant-based nanocarriers for oral delivery. In this study, the colloidal (i.e. aggregation), physical (i.e. denaturation) and chemical (i.e. digestion of the polypeptides) stability of CPMV and its empty virus-like particles (eVLPs) in conditions resembling the gastrointestinal fluids were evaluated. The nanoparticles were incubated in various simulated gastric and intestinal fluids and in pig gastric and intestinal fluids. CPMV and eVLPs had similar stabilities. In simulated gastric media, they were stable at pH ≥ 2.5. At lower pH destabilisation of the particle structure occurred, which, in turn, rendered the polypeptides extremely sensitive to pepsin digestion. However, both CPMV and eVLPs were stable in simulated intestinal fluids, in pig gastric fluids and in pig intestinal fluids. Thus CPMV, despite being a protein-based nanoparticle, was much more resistant to the harsh GI conditions than soluble proteins. Remarkably, both CPMV and eVLPs incubated in pig gastric and intestinal fluids were not subject to protein adsorption, with no formation of a detectable protein corona. The lack of a protein corona on CPMV and eVLP surfaces in GI fluids would imply that, if orally administered, these nanoparticles could maintain their native surface characteristics; thus, their biological interactions would remain predictable and unchanged. In summary, CPMV and eVLPs can be considered promising nanocarriers for applications requiring oral delivery, given their chemical, physical and colloidal stability and lack of protein adsorption from the environment in most of the tested conditions.

Fig. 1. CPMV physical stability in pH 1.2. A: schematic diagram of the stability studies: the sucrose gradient ultracentrifugation was used as a means to assess the VNPs’ integrity. CPMV was incubated for two hours in either PBS (control) or pH 1.2, followed by density gradient ultracentrifugation with the sucrose dissolved in either PBS or pH 1.2, respectively. A third sample was exposed to pH 1.2, the pH neutralised after two hours and the density gradient ultracentrifugation performed in PBS. B: SDS-PAGE (top) and UV absorbance at 280 nm (bottom) of the sucrose gradient fractions, after 2 hours incubation of CPMV in PBS (control), pH 1.2 and pH 1.2 followed by pH neutralisation. S, indicates the supernatant; 10, 20, 30, 40, 50 and 60 indicate the percentages of sucrose of the collected fractions; Precip.↓ indicates the precipitate formed after the ultracentrifugation. C: TEM images of CPMV after 2 hours incubation in PBS (control), pH 1.2 and pH 1.2 followed by pH neutralisation. D: DLS of CPMV after 2 hours incubation in PBS (control), pH 1.2 and pH 1.2 followed by pH neutralisation.

Fig. 2. Physical stability of CPMV in simulated gastric fluids at different pH (without pepsin). A: Coomassie-stained (top) and ethidium bromide-stained (bottom) native agarose gel of CPMV after 2 hours incubation in simulated gastric fluids at different pH and in PBS (control). B: Intensity-based particle size distribution of CPMV after 2 hours incubation in simulated gastric fluids at different pH (I) and at the different pH, but followed by pH neutralisation (II), as measured by DLS; CPMV was incubated in PBS for positive control.

Fig. 3. Chemical and physical stability of CPMV in simulated gastric fluids with pepsin. A and B: SDS-PAGE of CPMV incubated for 2 hours in SGF containing different concentrations of pepsin at pH 1.2 (A) or at pH 3 (B). CPMV (in water) and 3.2% pepsin were used as positive and negative controls, respectively. BSA was used as control to verify the enzymatic activity. C: Coomassie-stained (top) and ethidium bromide-stained (bottom) native agarose gel of CPMV after 2 hours incubation in SGF at different pH and containing fixed concentration of pepsin [0.32% (w/v)]. CPMV (in PBS) and SGF were used as positive and negative controls, respectively.

Fig. 4. Chemical and physical stability of CPMV upon exposure to pig gastric fluids. A: SDS-PAGE of CPMV incubated in PGF for 2 hours. CPMV (in water) and PGF were used as positive and negative controls, respectively. BSA (green arrow) was used as control to verify the enzymatic activity. BSA and CPMV samples were also added to pre-boiled, i.e. inactivated, PGF for comparison purposes. L and S protein are indicated by red arrows. B: Schematic diagram of the sucrose gradient ultracentrifugation performed to separate CPMV from PGF. C: UV absorbance (mean ± SD, n = 3) at 280 nm of the sucrose gradient fractions of PGF, or of CPMV incubated for 2 hours in either PBS or PGF. D: SDS-PAGE of the same sucrose gradient fractions analysed in C; S, indicates the supernatant; 10, 20, 30, 40, 50 and 60 indicate the percentages of sucrose of the collected fractions. E and F: fractions of the gradient containing CPMV were pooled together, dialysed and concentrated and then analysed by SDS-PAGE (E left), native agarose gel (E right) and TEM (F).

Fig. 5. Chemical and physical stability of CPMV upon exposure to pig intestinal fluids. A: SDS-PAGE of CPMV incubated in PGF for 4 hours. CPMV (in water) and PIF were used as positive and negative controls, respectively. BSA was used as control to verify the enzymatic activity. BSA and CPMV samples were also added to pre-boiled, i.e. inactivated, PIF for comparison purposes. S protein is indicated by a red arrow. B: UV absorbance (mean ± SD, n = 3) at 280 nm of the sucrose gradient fractions of PIF or of CPMV incubated for 4 hours in PBS or PIF. C: SDS-PAGE of the same sucrose gradient fractions analysed in B; S, indicates the supernatant; 10, 20, 30, 40, 50 and 60 indicate the percentages of sucrose of the collected fractions. D and E: fractions of the gradient containing CPMV were pooled together, dialysed and concentrated and then analysed by SDS-PAGE (D left), native agarose gel (D right) and TEM (E).

Fig. 6. Physical stability of eVLPs in simulated gastric fluids at different pH (without pepsin). A: SDS-PAGE of sucrose gradient fractions, after 2 hours incubation of eVLPs in PBS (control), pH 1.2 and pH 1.2 followed by pH neutralisation. S, indicates the supernatant; 10, 20, 30, 40, 50 and 60 indicate the percentages of sucrose of the collected fractions; Prec.↓ indicates the precipitate formed after the ultracentrifugation. B: TEM images of eVLPs after 2 hours incubation in PBS (control), pH 1.2 and pH 1.2 followed by pH neutralisation; (scale bar = 100 nm). C: Coomassie-stained (top) and ethidium bromide-stained (bottom) native agarose gel of eVLPs after 2 hours incubation in simulated gastric fluids at different pH and in PBS (control). CPMV was also used as control.