Bioavailability of CVTMGMV and free CV in C. elegans was investigated in liquid culture. C. elegans nematode motility was classified as either totally immobilized, impaired motility, or completely mobilized nematodes. To illustrate the data that was collected, a series of snap shots of C. elegans incubated with no treatment, 10 μM CV, and 10 μM CVTMGMV was taken every second for 60 seconds. The corresponding videos can be found in the supporting information. Figure 3.5 A-C illustrates the nematodes observed after 3 h of incubation. Five nematodes were selected in each treatment regime and pseudo-colored to illustrate their motility. Untreated C. elegans showed no impaired motility . For example, the nematode colored in pink moves across the frame within the 40 sec interval, while other nematodes disappear from or appear in the frame during that time interval. Although the motility of these nematodes is evident, most nematodes do not travel far but rather move within a restricted area,blueberry packaging such as the nematode colored in yellow. C. elegans treated with 10 μM of CV or 10 μM of CVTMGMV behaved differently and showed severe motility impairment .
All pseudo-colored nematodes in Figure 3.5 B+C were paralyzed or dead and did not move. However, this is not true for all nematodes, as a population of nematodes showed little to no motility impairment when treated with CV or CVTMGMV. From the imaging data there were no apparent differences between the two treatment groups, free drug vs. CVTMGMV .To quantitatively analyze the motility effects of CV on C. elegans, nematodes were treated with various concentrations of free CV, CVTMGMV, or TMGMV for 24 h at 22°C. At specific time points nematodes were observed under a white light microscope and the percent of affected nematodes and was quantified as a function of time. The effective concentration , defined as the concentration of CV at which half of the maximum immobilization of C. elegans was reached, was determined for free CV and CVTMGMV . Sixty percent of nematodes treated with 100 μM CV were paralyzed/dead within 1 h and no further improvements were observed within 24 h . When treated with 10 μM or 1 μM of CV, only ~30% or ~15% of nematodes were paralyzed/dead within the first hour, respectively. In those cases, maximum efficacy was observed after 6 h of incubation, when ~50% and ~25% of nematodes were affected. In both treatment regimes, a decrease in efficacy was observed after 6 h of incubation – this phenomenon may be explained because remaining unaffected population of nematodes continued to progress through their life cycle; consequently eggs were laid and nematodes hatched, which led to an overall increase in population and a decrease in percent of nematodes affected by the treatment. Furthermore, it is possible that at low doses of CV, nematodes are able to recover and slowly become mobile again.
At doses of CV lower than 1 μM, there was no significant effect on nematode motility compared to the untreated population. The EC50, defined as the concentration of CV at which half of the maximum immobilization of C. elegans was reached, was quantified at various time points and was determined to be 3.7 μM. CVTMGMV showed a similar trend to free CV , and, as expected, TMGMV alone did not show any nematicide properties When treated with 100 μM of CVTMGMV, ~40% of nematodes were paralyzed/dead within the first hour, and maximum efficacy was reached in the first 3 h. Therefore the efficacy of 100 μM of CV and CVTMGMV is identical after 3 h of incubation. However, when the concentration of CVTMGMV was dropped to 10 μM, the maximum efficacy was ~30% and was reached after ~8 h of incubation. Interestingly, CV release from CVTMGMV in nematode media conditions revealed a half-life of 8 h , thus supporting the idea that CV was released from TMGMV and made available to treat the nematode infestation. All studied concentrations of CVTMGMV lower than 10 μM led to no significant treatment of the nematode infestation compared to the untreated population. The calculated EC50 of CVTMGMV is 13.8 μM, which is approximately 4 times greater than the EC50 of free CV. While reduced efficacy was observed in the petri dish experiments, I envision that CVTMGMV will outperform free CV in the field based on the enhanced drug delivery aspect . Next, I set out to understand the bio-distribution of CV in the nematodes. I prepared fluorescently labeled TMGMV and analyzed whether TMGMV would interact with or be ingested by C. elegans.
Briefly, diazonium coupling and click chemistry was used to conjugate a Cy5 dye to TYR side chains on TMGMV, as structural studies indicated that TYR2 is solvent-exposed . I conjugated ~160 dyes per full length TMGMV, or about 7.5% of CPs were modified with Cy5 . We have previously demonstrated that a minimum conjugation of Cy5 to ~8% of TMV coat proteins is sufficient to yield maximum fluorescence intensity,237 thus the prepared samples were thought to be sufficient for imaging experiments. Fluorescent TYR-Cy5TMGMV was incubated with C. elegans nematodes for 3 h at 22°C and subsequently analyzed by fluorescent microscopy . Results indicate that nematodes ingest the proteinaceous TMGMV carrier and that while TMGMV distributes throughout the entire nematode body, the majority of TMGMV accumulates in the gastrointestinal tract. A soil mobility test was designed to establish the leaching of CVTMGMV and free CV in soil. Briefly, top soil was packed in a plastic column up to a height of 4 cm and saturated with deionized water. CVTMGMV or free CV was applied atop the soil columns, followed by DI water. Fractions were collected from the soil column, purified, and analyzed by UV/visible spectroscopy for the presence of TMGMV and CV. The λ260 and λ280 wavelengths were monitored to quantify the amount of TMGMV that leached through the soil. A background 260:280 absorbance was observed in a CV soil leaching column, which most likely corresponds to the absorbance of organic matter present in top soil . CVTMGMV showed enhanced mobility over free CV in the soil column,blueberry packaging box eluting from the column at high concentrations in the 5th to 15th elution fractions . In stark contrast, the efflux of CV from the soil column was delayed until the 25th to 50th elution fractions at a concentration 3.6 times lower than CVTMGMV . CV is hydrophobic and has a strong binding affinity to soil particles rendering the drug mostly immobile in soil, which explains the delayed efflux and lower concentrations eluted. Taken together, the data demonstrates the potential of TMGMV as a drug carrier to enable penetration of CV or other nematicides through soil to reach nematodes feeding on the roots of plants. In this study, I have demonstrated the potential of tobacco mild green mosaic virus as a carrier for anthelmintic drugs, such as crystal violet , to treat plants infected with parasitic nematodes. After careful analysis of the TMGMV structure, I identified solvent exposed TYR2 on the exterior surface enabling chemical modification. I also identified solvent exposed carboxylates, GLU145 and ASP66 on the exterior surface and GLU95 and GLU106 on the interior surface, and established the chemical addressability of these residues. I also showed the potential for electrostatic encapsulation of positively charged guest molecules in TMGMV. Further studies are needed to identify which of the identified GLU and ASP residues are chemically reactive. Electrostatic drug loading using crystal violet was achieved, yielding TMGMV carriers loaded with ~1500 CV per CVTMGMV nanocarrier.
Treatment efficacy, while lower compared to free drug, was demonstrated using liquid C. elegans nematode cultures . Diffusion experiments revealed significantly increased soil mobility of CVTMGMV vs. free CV; the latter was unable to sufficiently diffuse and disperse through soil. Overall CVTMGMV demonstrates efficacy and superior soil motility, and as such makes a promising platform technology as a drug carrier targeting agricultural application. Pesticides are needed to protect our crops and thus maximize crop yields.However, the efficacy of chemical pesticides is limited by their instability and strong binding to organic matter in soil, which can render them inactive or prevent their accumulation at the root level, where many pests reside.Large doses are applied to compensate, resulting in the accumulation of pesticide residues in soil, water and agricultural products.Long-term exposure to these chemicals is a risk to human health and threatens the biodiversity of an already fragile ecosystem.Advances in nanotechnology have led to the development of more effective drug delivery and medical imaging methods , and the same innovations are now being applied to smart agrochemical delivery systems, known as nanopesticides.These involve the use of nanomaterials for the adsorption, encapsulation or conjugation of pesticides, improving the biodegradability, stability, permeability and dispersion of the active pesticide ingredient. Nanopesticides have a much greater surface area than conventional pesticides, increasing their potential for interaction with target pests at lower doses. The encapsulation of pesticides within nanoparticles also prevents premature degradation and the risk of direct human exposure to the active ingredient. There is also evidence that nanopesticides and conventional pesticides differ in their environmental behaviour, so it is necessary to understand the fate of nanopesticides in detail in order to ensure they comply with regulatory guidelines and legislation.Most of the nanopesticides investigated thus far are based on synthetic or natural polymers, metallic compounds or liposomes, which tend to persist in the environment.As a biodegradable alternative, nanopesticides can be developed from plant viruses.One example, already EPA-approved, is the application of Tobacco mild green mosaic virus as the herbicide Solvinix, which is produced by BioProdex for deployment against invasive tropical soda apple weed in the state of Florida.The safety profile and possible risks of TMGMV have been reported.TMGMV cannot self-disseminate and is not transmitted by vectors such as insects, seeds, or pollen. Mechanical transmission through insects or contact between plants is thus the only route of transmission. Only plants of the Solanaceae are susceptible to TMGMV infections. Therefore, TMGMV offers a good safety profile for crops that are not part of the Solanaceae. Nonetheless, TMGMV as well as other plant virus-based systems could be inactivated through ultraviolet radiation for safe use on any crop.To investigate the potential of plant virus nanoparticles and virus-like particles as nanopesticides in more detail, I compared the behaviour of three viruses and two synthetic particle formulations in soil column experiments and computational models, as a way to gauge their ability to deliver pesticides to the rhizosphere and thus prevent infestation by root pests . I tested two VNPs, based on the rod-like TMGMV and the icosahedral Cowpea mosaic virus , and a virus-like particle based on Physalis mosaic virus . These were compared to mesoporous silica nanoparticles and a poly formulation , which have already been developed as synthetic nanopesticides.TMGMV was oβtained from Bioprodex, DegraFluorex Fluorescent PLGA nanoparticles were purchased from Phosphorex, and MSNPs functionalized with propylcarboxylic acids were obtained from Sigma-Aldrich. I resuspended 3 mg ml-1 of PLGA and 1 mg ml-1 of MSNP in distilled water and sonicated them using a Branson 2800 device for 10 min to obtain homogeneous solutions. CPMV was propagated in Burpee black-eyed pea plants and purified as previously described.PhMV VLPs were prepared in ClearColi BL21 cells as previously described.USDA Permits were obtained for any work with plant viruses. TMGMV comprises 2,130 identical coat proteins arranged helically around a single-stranded RNA genome, forming a hollow rigid rod measuring 300 × 18 nm with a 4-nm internal channel.The external surface features two solvent-exposed tyrosine side chains , which can be functionalized using diazonium coupling reactions. I used sulfo-Cy5-azide to modify these Tyr residues as previously described.10 Briefly, I mixed 25 μl 0.68 M 3-ethynylaniline with 75 µl 3 M sodium nitrite in 400 μl 0.3 M p-toluenesulfonic acid monohydrate for 1 h on ice. I then added 15 equivalents of the resulting diazonium salt to 2 mg ml-1 TMGMV in 10 mM borate buffer for 30 min on ice. The particles were centrifuged at 112,000 g for 1 h on a 30% sucrose cushion to separate the TMGMV-alkyne particles from the excess DS. TMGMV-alkyne was resuspended in 10 mM KP buffer overnight before adding sulfo-Cy5-azide via a Cu-catalysed alkyne-azide cyclo-addition reaction. I added two equivalents of Cy5 per coat protein to 2 mg ml-1 TMGMV-alkyne in the presence of 2 mM aminoguanidine, 2 mM L-ascorbic acid sodium salt and 1 mM copper sulfate in 10 mM KP buffer on ice for 30 min.