Protection and Delivery of Anthelmintic Protein Cry5B to Nematodes Using Mesoporous Silicon Particles

Abstract

The ability of nano- and microparticles of partially oxidized mesoporous silicon (pSi) to sequester, protect, and deliver the anthelmintic pore-forming protein Cry5B to nematodes is assessed in vitro and in vivo. Thermally oxidized pSi particles are stable under gastric conditions and show relatively low toxicity to nematodes. Fluorescence images of rhodamine-labeled pSi particles within the nematodes Caenorhabditis elegans and Ancylostoma ceylanicum show that ingestion is dependent on particle size: particles of a 0.4 ± 0.2 μm size are noticeably ingested by both species within 2 h of introduction in vitro, whereas 5 ±2 μm particles are excluded from C. elegans but enter the pharynx region of A. ceylanicum after 24 h. The anthelmintic protein Cry5B, a pore-forming crystal (Cry) protein derived from Bacillus thuringiensis, is incorporated into the pSi particles by aqueous infiltration. Feeding of Cry5B-loaded pSi particles to C. elegans leads to significant intoxication of the nematode. Protein-loaded particles of size 0.4 μm display the highest level of in vitro toxicity toward C. elegans on a drug-mass basis. The porous nanostructure protects Cry5B from hydrolytic and enzymatic (pepsin) degradation in simulated gastric fluid (pH 1.2) for time periods up to 2 h. In vivo experiments with hookworm-infected hamsters show no significant reduction in worm burden with the Cry5B-loaded particles, which is attributed to slow release of the protein from the particles and/or short residence time of the particles in the duodenum of the animal.

Recombinant Paraprobiotics as a New Paradigm for Treating Gastrointestinal Nematode Parasites of Humans

“…Cry5B in IBaCC showed a dose-dependent inhibition of motility similar to previously published studies with purified Cry5B (30). We had previously confirmed uptake of 0.4 µm particles by adult hookworms (56). To confirm uptake of IBaCC by hookworms, we labeled IBaCC with rhodamine, which predominantly labels full length Cry5B.…”

Bacillus thuringiensis Cry5B protein as a new pan-hookworm cure

“…Since >99.9% of Cry5B is rapidly degraded in stomach acid ( Hu et al, 2010a ), we hypothesized that protection from stomach acid should improve efficacy. Previous attempts to protect Cry5B from stomach acid using nanoparticles failed, likely due to slow release kinetics ( Wu et al, 2015 ). To circumvent the challenges with formulation strategies, we tested whether direct inhibition of stomach acid proteolysis would improve Cry5B efficacy.…”

The adsorption features between insecticidal crystal protein and nano-Mg(OH) 2

“…The adsorption kinetics study indicated that the adsorption of protein was gradually increased, then approached the adsorption equilibrium after around 2 h. This means that the adsorption could be regulated in a short time in the practical application of nano-Mg(OH) 2 as an adsorbent. Previous study also loaded the crystal protein anthelmintic Cry5B with partially oxidized mesoporous silicon (pSi), and the loading time was 16 h [ 17 ]. In our system, the adsorption of Cry11Aa by nano-Mg(OH) 2 was a rapid process, and the adsorption capacity could achieve equilibrium in the first 2 h. This excellent adsorption ability might be attributed to the large adsorption affinity of nano-Mg(OH) 2 and the relatively high BET areas.…”