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

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 <i>in vitro</i> and <i>in vivo</i>. 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 <i>in vitro</i>, 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 <i>in vitro</i> 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. <i>In vivo</i> 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

Dose response of tribendimidine against hookworm <i>A</i>. <i>ceylanicum</i> infections in hamsters.

<p>(A), (C), and (E): Effects of tribendimidine at indicated doses on intestinal hookworm burdens in hookworm-infected hamsters. For (A), (C), and (E) and in similar figures below, the hookworm burdens in each hamster are indicated with a separate symbol, long horizontal bars represent mean hookworm burdens per group; small bars indicate standard error. (B), (D), and (F): Effects of tribendimidine at indicated doses on parasite egg production (fecal egg counts) in hookworm-infected hamsters. For (B), (D), and (F) and in similar figures below, shown are the average eggs per gram of feces in each group on day 4 post-treatment. The fecal egg counts in each animal are indicated with a separate symbol. Long horizontal bars represent mean eggs per gram of feces (EPG) per group; small bars indicate standard error. Panel (A) & (B); (C) & (D) and (E) & (F) came from three independent <i>in vivo</i> experiments. TrBD = tribendimidine.</p

The 1:1 Cry5B:tribendimidine combination at a higher dose eliminates hookworm infections in hamsters.

<p>Effects of treatment of higher dose Cry5B alone or higher dose tribendimidine alone or higher dose Cry5B plus higher dose tribendimidine in combination on (A) intestinal hookworm burdens and (B) fecal egg counts in hookworm-infected hamsters. None of the individual drugs eradicated the hookworm infections in hamsters, but the combination of both drugs did. TrBD = tribendimidine.</p

<i>In vivo</i> data associated with experimental results in Fig 6.

<p><i>In vivo</i> data associated with experimental results in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006506#pntd.0006506.g006" target="_blank">Fig 6</a>.</p

Cry5B and tribendimidine in combination are synergistic <i>in vivo</i> against hookworm infections in hamsters.

<p>Effects of individual treatment of Cry5B alone or tribendimidine alone and combination treatment of Cry5B plus tribendimidine at indicated doses on (A) intestinal hookworm burdens and (B) fecal egg counts in hookworm-infected hamsters. Brackets indicate statistical comparisons between groups, with p values shown. Data come from the combination of two independent <i>in vivo</i> experiments. TrBD = tribendimidine.</p

Dose response of pyrantel against hookworm <i>A</i>. <i>ceylanicum</i> infections in hamsters.

<p>Effects of pyrantel at indicated doses on (A) intestinal hookworm burdens and (B) fecal egg counts in hookworm-infected hamsters. PYR = pyrantel.</p

The 3:1 Cry5B:pyrantel combination at a higher dose eliminates hookworm infections in hamsters.

<p>Effects of treatment of higher dose Cry5B alone or higher dose pyrantel alone or higher dose Cry5B plus higher dose pyrantel in combination on (A) intestinal hookworm burdens and (B) fecal egg counts in hookworm-infected hamsters. None of the drugs individually eradicated the hookworm infections in hamsters but the combination of both drugs did. PYR = pyrantel.</p

<i>In vivo</i> data associated with experimental results in Fig 3.

<p><i>In vivo</i> data associated with experimental results in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006506#pntd.0006506.g003" target="_blank">Fig 3</a>.</p

Pyrantel-resistant cyathostomins are hypersusceptible to Cry5B.

<p>Dose-dependent developmental inhibition of a non-anthelmintic resistant population of cyathostomins (Barn 10; [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006506#pntd.0006506.ref062" target="_blank">62</a>]) and of an anthelmintic-resistant population of cyathostomins (pyrantel-resistant Population S or PYR_R) to (A) pyrantel and (B) purified Cry5B. Each data point represents the % of larvae that matured to the infectious L3 stage (L3i). Error bars indicate standard error from three independent replicates. Susep = Barn 10, pyrantel susceptible; PYR_R = pyrantel-resistant population.</p

Cry5B and pyrantel in combination are synergistic <i>in vivo</i> against hookworm infections in hamsters.

<p>Effects of individual treatment of Cry5B alone or pyrantel alone and combination treatment of Cry5B plus pyrantel at indicated doses on (A) intestinal hookworm burdens and (B) fecal egg counts in hookworm-infected hamsters. Black arrow indicates the effect of the combination of Cry5B and pyrantel at ratio of 3:1 (1 mg/kg Cry5B + 0.33 mg/kg pyrantel), showing greater than expected impact based on individual doses or next highest combination dose. PYR = pyrantel.</p