They demonstrated that abamectin encapsulated in RCNMV had increased stability and superior mobility in soil compared to free abamectin, which resulted in enhanced bio-availability and treatment efficacy in tomato seedlings. As expected, no viral infection in the tomato seedlings was observed, as tomato is not a RCNMV host species. In the present work, I propose the use of tobacco mild green mosaic virus , also known as the U2 strain of tobacco mosaic virus , as a carrier to deliver nematicides. TMGMV self-assembles into a 300 by 18 nm rod-shaped virus with a 4 nm wide hollow channel. The high aspect ratio soft-matter nanotube may provide a promising alternative over the spherical platform technologies, i.e. most synthetic nanoparticles as well as RCNMV. The nanomedicine field has demonstrated that carrier shape impacts the in vivo fates with elongated materials conferring advantages with enhanced margination, diffusion, and penetration through tissue.Whether high aspect ratio materials perform better in soil remains to be seen. Another advantage of the TMGMV platform is the high surface area , 3.9 times larger than RCNMV , which may allow for higher payload delivery. TMGMV is already EPA-approved: Solvinix, a formulation of TMGMV mass-produced by BioProdex, is commercially available as an herbicide in the state of Florida for the treatment of the invasive weed tropical soda apple.TMGMV is not transmitted by insects, pollen, or other vectors; it is not seed borne and cannot self-disseminate. While TMGMV is capable of infecting solanaceous plants , TMGMV is unable to penetrate and infect healthy plants in the absence of a lesion wound.
Furthermore, Solvinix was tested on 435 plants representing 311 species,blueberry box among which only 8% of plants were killed.It is therefore safe to conclude that TMGMV can be applied in the field with little to no risk to the environment or the crop itself. Although there are a few species of plants to which TMGMV is lethal,it is important to remember that the remaining 3000 species infected by parasitic nematodes are not susceptible to TMGMV. While its structure is known to atomic resolution ,the chemistry of TMGMV has not yet been established. Making use of the structural information and the well established chemistries for tobacco mosaic virus , I developed bio-conjugation techniques and non-covalent drug loading strategies for TMGMV. As a proof of concept, I used the anthelmintic drug Crystal Violet in our studies.The supernatant was subsequently analyzed by UV/visible spectroscopy .TMGMV is the U2 strain of tobacco mosaic virus ; the latter has been extensively studied in plant pathology and structural biology since the 1900s and more recently in nanomedicine, biotechnology and energy research.173 Therefore, the surface chemistry of TMV is well understood. Here I set out to establish the chemistry of TMGMV. The amino acid sequences of the coat proteins of TMV and TMGMV present 72% homology; also the structural overlay of a single CP of TMV and TMGMV reveals a high degree of structural similarity, only 14% of the amino acids do not overlap in the crystal structures . This is also reflected when comparing the assembled nucleoprotein complexes of TMV and TMGMV . Just like TMV, TMGMV forms a cylindrical structure measuring 300 by 18 nm with a 4 nm-wide hollow interior channel. The TMGMV particles consist of 2130 identical copies of CP units arranged helically around a single-stranded RNA genome .
Analysis of the structure reveals the amino acid profile on the exterior and interior surface: because LYS, CYS, TYR, ASP, and GLU are often targeted for bio-conjugation or electrostatic drug loading, I analyzed the TMGMV structure for presence of these residues. While solvent-exposed LYS and CYS side chains were not identified in TMGMV , several TYR, ASP and GLU residues were found to be solvent-exposed on the exterior/interior TMGMV surfaces. Structural data indicate TYR2 to be exposed on the exterior surface – this is different from the structure of TMV, for which both TYR2 and to a greater extent TYR139 are solvent-exposed on the exterior surface . The TYR2 side chain of TMGMV could provide a potential target for bio-conjugation, e.g. the introduction of a fluorescent label for imaging and tracking studies as described below. Further, I identified ASP66 and GLU95, 106, and 145 to be solvent exposed, with GLU145 and ASP66 located on the exterior surface and GLU95 and GLU106 on the interior surface . This is similar to the structure of TMV, for which GLU145, ASP64 and ASP66 are solvent-exposed on the exterior surface while GLU97 and GLU106 are solvent-exposed on the interior surface . However, it should be noted that previous research identified GLU97 and GLU106 to be the only carboxylates in TMV that are reactive toward carboxylate-specific chemistries; GLU145 and ASP64 and 66 were not found to be reactive.The presence of GLU/ASP residues in TMGMV would allow for functionalization through bio-conjugate chemistry or electrostatic loading of positively charged guest molecules, as we previously described in the case of TMV.Lastly, I analyzed the surface charge of TMGMV and determined that the inner and outer surfaces carry a net negative coulombic charge with the interior being more electronegative than the exterior . Together these data indicate solvent-exposed TYR side chains on the exterior surface of TMGMV and addressable carboxylates – possibly on the exterior and interior surfaces. With the structural information in hand, I set out to develop TMGMV as a carrier for nematicide delivery.
Specifically, I chose to work with crystal violet as a proof-of-concept, because this therapeutic compound is fluorescent and thus streamlines the analysis. The positively charged CV was loaded into TMGMV making use of electrostatic interactions and concepts that were previously developed to load positively charged platinum drug candidates and porphyrin derivatives into TMVThe reaction mix of 6000:1 CV:TMGMV resulted in the highest loading efficiency while still yielding dispersed TMGMV particles: 68% of the CPs were modified with a CV molecule. Assuming a full length TMGMV particle , each TMGMV would carry ~ 1500 drug molecules. This formulation was subsequently used for all following studies. When compared with TMV-drug formulations, the TMGMV formulation yielded comparable results: I previously reported the loading of 2,000 phenanthriplatin per TMV206 and 900 copies of a porphyrin derivative ZnPr per TMV.235 In those cases, the loading procedure was similar, in which a positively-charged guest molecule was loaded via electrostatic interaction with TMV’s interior carboxylates. As in the case of TMV, the interior channel of TMGMV is lined with a dense layer of carboxylates – this, in combination with the more electronegative interior surface,blueberry package may suggest that drug loading occurs on the inside channel. However further studies would be needed to rule out drug association with the exterior surface in both the cases of TMV and TMGMV. To compare the drug loading efficiency of the rod-shaped TMGMV system to the icosahedral RCNMV-based nematicide carrier, the number of drug molecules was normalized to the molecular weight of the nanocarrier yielding ~3.6 × 10-5 CV per dalton of TMGMV protein, while only ~1.8 × 10-5 abamectin molecules were loaded per dalton of RCNMVprotein. In other words, when normalized per molecular weight, twice as much drug molecule can be loaded per TMGMV than compared to RCNMV. The structural integrity of non-modified TMGMV and CVTMGMV were assessed by size exclusion chromatography and transmission electron microscopy . SEC measurements revealed no significant difference comparing native TMGMV and CVTMGMV; both particles showed the same elution profile . Further, TEM imaging of TMGMV and CVTMGMV revealed rod-shaped samples with no apparent differences when comparing TMGMV and CVTMGMV ; TEM imaging indicates that the average length of TMGMV and CVTMGMV is comparable measuring 146 ± 97 nm and 136 ± 76 nm, respectively .It is possible that this is an artifact from the TEM grid preparation, i.e. the particles may break during the drying process. However, it is important to note that there are no apparent differences comparing the TMGMV and CVTMGMV, indicating that the nucleoprotein complex withstands the loading and purification process. To gain insights into whether CV- loading into TMGMV is indeed via electrostatic interactions with GLU and/or ASP residues, chemically modified TMGMV in which the carboxylates were neutralized was prepared. To do so, EDC coupling was used to introduce alkyne ligands at the carboxylates, for subsequent addition of biotin labels using Cu-catalyzed alkyneazide cycloaddition . The protocols are detailed in the methods and were adapted from previous methods established for bioconjugation to TMV.Biotinylation was confirmed by western blot , yet quantitative data could not be obtained. To quantify the degree of labeling, the fluorescent Cy5 dye was conjugated to GLU/ASPTMGMV, yielding ~275 dyes per full length TMGMV, or about 13% of CPs were modified with Cy5 . Biotinylated and alkyne-labeled TMGMV were then used in CV-loading experiments, and I observed a 40% decrease in CV loading when using alkyne-labeled TMGMV compared to unmodified GLU/ASPTMGMV . Severe aggregation was observed when biotinylated TMGMV was used in CV loading experiments .
This phenomenon may be explained as follows: if the chemistry of TMGMV and TMV is matched, then biotins will be displayed along the interior channel, preventing the positively charged guest molecules to be loaded and protected inside the TMGMV channel – instead CV may cross link the particles through interactions with the while less negative, also negatively-charged exterior surface. Because the data indicate that CV-loading is mediated through the solvent-exposed GLU/ASP acids, and in light of the TMGMV structure and its similarities to the known biochemistry to TMV,I therefore expect that interior loading of CV is achieved by this method. Next, I evaluated the release profile of CV from the TMGMV nanocarrier. The release rate of CV from CVTMGMV is expected to be proportional to the pH of the bathing conditions as well as temperature. Based on thermodynamics, the rate of diffusion should increase with temperature. Furthermore, as pH decreases, a larger number of carboxylate groups become protonated and carry a net neutral charge that can no longer interact with the positively charge CV and consequently, free CV should diffuse away from TMGMV. Therefore the release rate of CV should be higher at lower pH and higher temperatures. To test this experimentally, 1 mg of a 1 mg.mL-1 solution of CVTMGMV was prepared as described above and dialyzed against various buffers for 72 h . I tested the release profile at room temperature and 4°C to evaluate two extreme upper soil thermal conditions.Sodium acetate and PBS buffer solutions were chosen to mimic the acidic and neutral soil environments respectively. Diffusion of CV from CVTMGMV was also evaluated in KP buffer, which was used during loading and storing conditions of the sample. Free CV, in a concentration matched to the concentration and number loaded into TMGMV, was also dialyzed in KP buffer at 4°C as a positive control. As expected, increased release rates of CV from CVTMGMV were observed at low pH and high temperature . Approximately half of the free CV was dialyzed within 1.6 h and complete release was observed in less than 18 hrs, while delayed release profiles were observed for the CVTMGMV nanoparticle formulations. For CVTMGMV, 50% of CV was released only after 5 h in acidic conditions at room temperature , with complete release achieved after about 24 hrs. These conditions most realistically mimic the soil environment. In stark contrast, release in storing conditions was significantly slower, with 50% of CV released within 13 hrs, and complete release was not observed within 72 h. This is promising for application of these nanoparticles, however it would be advised to prepare fresh formulations before application in the field. We have previously reported similar results with the release of the cancer drug phenanthriplatin from TMV;half of the encapsulated chemotherapeutic was released after 1 h at pH 5 and 24 h at pH 7.4. On the other hand, encapsulated porphyrin derivatives loaded in TMV were found to be stably encapsulated for at least one month when stored at 4°C and pH 7.I hypothesize that the increase in stability of the porphyrin drug was due to its higher electropositivity: the compound used carries 3 positive charges. In contrast, phenanthriplatin and CV carry 2 and 1 positive charges, respectively. Compared to the previously reported RCNMV carrier, the release rate of CV from TMGMV is slightly faster than that of abamectin from RCNMV in acidic soil conditions. 50% of abamectin was released within 8 and 7 h at pH 5.2 and 7.4 respectively .