The particles were again centrifuged at 112,000 g for 1 h on a 30% sucrose cushion to remove excess Cy5, and resuspended in 10 mM KP buffer overnight. Further purification to remove aggregates involved centrifugation at 16,000 g for 10 min. TMGMV-Cy5 was eluted using PD Minitrap G-25 desalting columns to remove free Cy5 dye. CPMV comprises 180 coat proteins and displays a total of 300 surface-exposed lysine side chain.PhMV also comprises 180 identical coat proteins, but each displays four surface-exposed lysine side chains making 720 in total.CPMV and PhMV were labelled with sulfo-Cy5-NHS using NHS-activated esters targeting the surface lysine residues. The reactions were carried out with a 1200-fold or 900-fold molar excess of sulfo-Cy5-NHS in 10 mM KP buffer at room temperature overnight, with agitation. Alkynes were conjugated to carboxylate groups on the MSNP surface using 1.5 mM propargylamine per gram of MSNP and 2.5 mM EDC in 10 mM HEPES buffer . The reaction was allowed to proceed for 24 h at room temperature followed by an alkyne-azide click reaction induced by adding 250 nmoles of sulfo-Cy5-azide per gram of MSNP. The components were incubated at 4°C with gentle agitation for 30 min using 1 mg ml-1 MSNP in 10 mM KP buffer in the presence of 1 mM CuSO4, 2 mM aminoguanidine and 2 mM ascorbate . MSNPs were purified by centrifugation at 7,000 g for 10 min and buffer exchange at least five times.Garden Magic Top Soil was packed at a density of 0.32 g cm-3 into a cylindrical column and saturated with deionized water to remove air pockets.
This was the maximum density achievable under our experimental conditions,blueberry packaging containers but the density of soil in real environments can be higher due to compaction effects with depth and over time. I injected a bolus containing 1 mg of each formulation with and without conjugated or infused dye molecules at the top of the soil column and saturated the column with water at a constant flow rate of 1.5 cm3 min-1 in 10 mM KP buffer The eluent was collected at the base of the column in 500-μl fractions. Up to 200 fractions were collected in each trial . The elution fractions containing TMGMV, PhMV or CPMV were analysed by SDSPAGE to determine the mass of nanoparticles recovered in each elution fraction. CPMV was analysed on 4−12% NuPage pre-cast gels in 1× MOPS buffer. TMGMV and PhMV were analysed on 4−12% NuPage polyacrylamide SDS gels cast according to the Surecast Handcast protocol . I mixed 23 μl of each elution fraction with 7 μl 5x SDS loading buffer and separated the samples for 1 h at 200 V and 120 mA with SeeBlue Plus2 ladder size and three standards containing known amounts of nanoparticles for comparison. The gels were then incubated in 20% methanol and 10% acetic acid in water 30 min before staining with Coomassie Brilliant Blue for an additional 30 min. The gels were imaged using the AlphaImager HP system under white light and the FluorChem R system under MultiFluor red light. The elution fractions containing PLGA and MSNP were imaged as 20-μl droplets on Parafilm on the FluorChem R imaging system under MultiFluor red light in the presence of the nanoparticle standards described above. All nanoparticles were imaged in triplicate and the images were analysed using ImageJ.
The area under the curve of the standards was used to create a linear standard curve relating the AUC of the elution samples to the total mass of nanoparticles present in the corresponding elution fraction. Finally, fractions that appeared to contain no nanoparticles were centrifuged at 160,000 g for 3 h and the pellet was resuspended in 1 mL 10 mM KP buffer for SDSPAGE analysis to determine the recovered mass of nanoparticles.To establish the soil transport behaviour of each formulation, I conducted mobility studies using a cylindrical column . As a reference, I ran 500 μg of free Cy5 through a soil column with a smaller diameter of 10 mm. Cy5 was unable to penetrate further than 4 cm through the soil because it bound strongly to the soil particles . About 40% of the mass of injected Cy5 was recovered from a column with a soil depth of 2 cm. These results are comparable to data reported for abamectin, fenamiphos and oxamyl, as well as other pesticides.No matter which nanoparticle type was used as a carrier, the mobility of Cy5 within the column was significantly enhanced . The best-performing carrier was TMGMV, which penetrated to a soil depth of 30 cm regardless of whether the cargo was conjugated or encapsulated . The spatiotemporal distribution of the nanoparticles and the Cy5 cargo was very similar, indicating that in each formulation the carrier and cargo were co-eluted . The quantity of encapsulated Cy5 that co-eluted with its carrier decreased with soil depth, indicating that a portion of the cargo was released over time. To determine the quantity of particles loss from the soil transport experiment, I pooled all elution samples that showed no evidence of nanoparticles in SDS-PAGE analysis and collected any trace amounts of the virus. I found that the residual mass of nanoparticles accounted for only ~2.5% of the overall mass of particles injected . TEM imaging of the eluted particles revealed that they remained intact .
In terms of soil transport behaviours, TMGMV and CPMV were able to penetrate through 30 cm of soil, whereas PhMV, MSNP and PLGA only penetrated 4, 12, and 8 cm of soil, respectively. The mobility of the carriers in soil can therefore be ranked from highest to lowest as follows: TMGMV >> CPMV >>> MSNP > PLGA > PhMV. These data suggest that the PhMV, MSNP and PLGA formulations are not suitable for pesticide delivery deep into the soil, to target the rhizosphere, but may be suitable for the delivery of pesticides that must remain close to the surface, such as herbicides. In the latter context, PhMV demonstrated the greatest pesticide delivery capability within the first 4 cm of soil .The particle size may influence the mobility of the carriers, but there was no particular trend within the size range I tested. For example, the 250-nm MSNP particles penetrated further than the 65-nm PLGA formulation, which in turn penetrated further than the 31-nm PhMV particles, but the 31-nm CPMV particles were much more mobile than all of the above. This is interesting given that CPMV and PhMV are similar in size and geometry, so the remarkable difference in mobility must reflect their surface chemistries. Both CPMV and PhMV are proteinaceous,blueberry packing boxes but the distinct amino acid sequences of their coat proteins ensure that CPMV carries a negative surface charge whereas PhMV is positive . Furthermore, the rod-like TMGMV particles were the most mobile of all, suggesting that the elongated shape may facilitate their transport through the soil. In the field of nanomedicine, elongated nanoparticles are better at margination and transport through membranes than spherical particles, which improves their tumour homing and penetration characteristics.A high aspect ratio therefore appears to be a generally favourable property that facilitates movement between obstacles by influencing particle behaviour in flowing liquids. I therefore speculate that the field of nanopesticide delivery should further focus on designing nanoparticles with high aspect ratio in addition to the traditional spherical counterparts. Particles with overall neutral to negative surface charge should also be favoured over positively charged nanoparticles to prevent early binding to soil matter. The concentration of Cy5 as a function of soil depth was higher when the dye was conjugated to the particles rather than encapsulated . This reflects the slower release of the conjugated dye from the carrier, allowing it to be carried further, whereas the encapsulated dye leaks more readily from the carrier and once released is rendered less mobile by its affinity for soil particles. Interestingly, Cy5 was released rapidly from the TMGMV*Cy5 and MSNP*Cy5formulations which suggests that the electrostatic forces between Cy5 and the carboxylate residues of the TMGMV interior and the MSNP mesopores are not strong enough to overcome the attraction between Cy5 and the soil. Therefore, for field applications, the conjugated formulation appears superior to the encapsulated formulation. Both TMGMV and CPMV were able to deliver Cy5 deep in the soil, but TMGMV-Cy5 showed by far the better performance.
In Chapter II, I have demonstrated that nematodes ingest nematicide-loaded TMGMV particles, which resulted in the death of 60% of the nematode population in liquid cultures within 24 h.To increase the efficacy, future TMGMV formulations should include cleavable linkers to promote the slow and controlled release of the pesticide at the root level. But in order to translate such pesticide formulations from the bench to the field, it is first necessary to establish the dose required to eradicate rhizosphere-dwelling pests. I therefore developed a mathematical model and validated it using our experimental data as discussed below. A model column of length L [cm] and constant cross-sectional area A [cm2 ] was filled with a mixture of stationary soil particles and fluid . The input to this model was a known mass nanoparticles, with or without pesticide, introduced over a short period of time to the soil surface. The outputs were the concentrations of the nanoparticle ΩNP [mg cm-3 ], the nanoparticlepesticide formulation CNPS [mg cm-3 ], and free pesticide CP [mg cm-3 ] at the base of the soil column as a function of time for a specific depth of soil. Following the injection, fluid flow was established at top the column at a rate Q [cm3 min-1 ]. Nanoparticles were subsequently transported through the void volume fraction [dimensionless] of the saturated soil column, with an adsorption surface per soil particle volume [cm-1 ]. The soil particle density within the column was assumed to be uniform. The rates of nanoparticle degradation and pesticide deactivation were assumed to be negligible during the experiment, as confirmed empirically . Nanoparticle binding to soil particles was modelled as a first-order irreversible reaction with rate constant kNPS [cm min- 1 ] dependent on the nanoparticle size, aspect ratio and surface chemistry. The pesticide release rate was modelled as a first-order irreversible reaction with rate constant kPF [min-1 ]. While simple, using a first-order release mechanism led to comparative errors ranging from 10-4 to 10-9 between the empirical data and the model output, which are sufficiently small to be acceptable differences . The resulting free pesticide may bind to soil particles through a firstorder irreversible reaction with rate constant kPS [cm min-1 ]. The interaction mechanisms are summarized in Supplementary Figure 8 and the corresponding partial differential equations are shown in the methods section. These equations were made dimensionless and solved using MATLAB . The system contained five unknowns: the dispersion constants of the nanoparticle DNP and pesticide DP, and the rate constants of nanoparticle absorption to soil kNPS, pesticide absorption to soil kPS, and pesticide release from nanoparticles in fluid kPF. These values were obtained by comparing the model output to the empirical data and minimizing the error in MATLAB. This computational model is therefore semiempirical in nature. . The resulting model outputs closely matched the empirical data , although the values of DNP and kNPS differed slightly for each depth due to experimental error caused by the need to use a new soil column in each test. Although the bulk density of the soil was kept constant across all experiments, the soil particle distribution and the soil packing may have differed from column to column. To compensate for these variables, the average values of DNP and kNPS at different depths were computed to model the average nanoparticle soil transport profile . The nanoparticle dispersion DNP and rate of absorption to soil kNPS determine the ability of a nanoparticle to carry pesticide deep in the soil. With greater mechanical dispersion, the nanoparticles become more widely distributed at a given soil depth over time. Therefore, mechanical dispersion greatly influences the concentration of nanoparticles at any given soil depth and time. The average DNP of each nanoparticle can be ranked from highest to lowest as follows: TMGMV > CPMV > MSNP > PhMV > PLGA. As the absorption to the soil becomes stronger, the nanoparticles become less mobile. The average rate constant of nanoparticle absorption to soil kNPS can also be ranked from highest to lowest as follows: MSNP >>> PLGA ≈ PhMV >> TMGMV > CPMV. The model confirms the superior mobility of TMGMV and its suitability to deliver pesticides to the rhizosphere.