PLANT VIRUS-LIKE NANOPESTICIDES FOR PRECISION FARMING

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: EXTENDED
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1022093
• Grant No.: 2020-67021-31255
• Project No.: CALW-2019-05732
• Proposal No.: 2019-05732
• Program Code: A1511
• Project Start Date: Jun 1, 2020
• Project End Date: May 31, 2025
• Grant Year: 2020

Project Director
Steinmetz, N.

Recipient Organization
UNIV OF CALIFORNIA-SAN DIEGO
9500 GILMAN DRIVE
LA JOLLA,CA 92093

Performing Department
NanoEngineering

Non Technical Summary
Pesticides are extensively used for food production in field. But pesticides and methods used to apply them are inefficient. Pesticides accumulate in the environment, on crops, and in drinking water. Pesticides are toxic to the environment and human health. It important to develop better ways to apply pesticides. A nanotechnology is proposed to package and safely deliver pesticides. This targeted delivery is expected to reduce the amount of pesticide needed. Pests will be treated more effectively, and less amount of pesticides will accumulate in the environment. Nanotechnology has already made an impact medicine. We seek to make an impact on agriculture using these tiny yet powerful nanoparticles. A nanoparticle is 2500 times smaller than the width of a human hair. A key innovative aspect of this approach is that we use the nanoparticles from a harmless plant virus. Plant viruses naturally occur in soil and plants. Plant viruses can be engineered to customize size and materials properties. Under this proposal we will use such plant viruses to answer how size and materials properties affects its interactions with soil and plants. Understanding these fundamental questions is expected to make an impact on next-generation pesticides. Findings from this research will help guide the design of more effective and safer pesticides. The overarching goal is to improve food quality and safety.Plant viruses can be engineered to customize size and materials properties. Under this proposal we will use such plant viruses to answer how size and materials properties affects its interactions with soil and plants. Understanding these fundamental questions is expected to make an impact on next-generation pesticides. Findings from this research will help guide the design of more effective and safer pesticides. The overarching goal is to improve food quality and safety.

Animal Health Component

0%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	0110	2020	100%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
0110 - Soil;

Field Of Science
2020 - Engineering;

Keywords

bio/nanotechnology

nanopesticide

nematodes

precision farming

soil mobility

Goals / Objectives
Major GoalsThe extensive use of inefficient agricultural pesticides leads to the accumulation of chemical residues on crops, in soil, as well as in drinking and groundwater, causing severe harm to ecosystems and endangering human health. TTobacco mild green mosaic virus (TMGMV) as a nanopesticide platform. Biological pesticides based on TMGMV have the potential to make a strong impact on food security by improving quality and yields (i.e. more effective crop treatment), while protecting the environment and agricultural ecosystems. The use of TMGMV rather than synthetic nanoparticles or the more commonly explored spherical nanoparticles is expected to offer unique advantages. In nanomedicine, carrier shape influences in vivo fate, with elongated materials conferring advantages such as enhanced margination, diffusion, and tissue penetration. We hypothesize that the high aspect ratio of TMGMV will improve soil mobility and we will systemically investigate the effect of carrier size and shape.We will fulfill the following objectives: Objective 1. Study the effect of nanocarrier size, shape, and surface chemistry on soil mobility. Native TMGMV forms a nucleoprotein assembly measuring 300x18 nm. Using a combination of plant molecular farming, protein self-assembly, and thermal treatment, we will formulate TMGMV nanocarriers varying in size (50-300 nm), shape (sphere vs rod), and surface chemistry (zwitterionic vs passivated) and will test their mobility in soil. Objective 2. Develop nanopesticides for safe field use and broad applications. TMGMV has a narrow host range but it does infect several solanaceous species among others. We will determine whether UV treatment can render the formulation inactive and safe without affecting its nanocarrier properties. In parallel, we will develop a genome-free VLP carrier based on TMGMV.Objective 3. Optimize the nanocarrier to achieve efficient loading and controlled release. Most pesticides (e.g. avermectin) are hydrophobic, which limits their mobility in soil, restricts their potential for bioconjugation and inhibits cargo delivery. We will develop orthogonal strategies that allow bioconjugation to the pesticide carrier in aqueous solutions using non-ionic surfactants (poloxamers).Objective 4. Experimental treatment of nematode-infested plants with candidate nanopesticides. Nanopesticide candidates will be tested against free nematicides using tomato as a model crop. We hypothesize that the biopesticide will be more effective at lower doses than the free drug. Leaching behavior will be studied to determine the environmental impact of nanopesticide vs conventional pesticide exposure.

Project Methods
We propose plant virus-based pesticide nanocarriers as an economically and environmentally viable alternative to synthetic nanoparticles. The application of plant virus-based nanocarriers to crops is a novel research direction and the methods developed under this award will yield novel nanopesticide formulations making use of the plant virus tobacco mild green mosaic virus (TMGMV). We will develop a library of nanomaterials derived from TMGMV to study structure-function relationships in terms of nanocarrier size, shape, and surface chemistry and the impact on soil mobility and ultimately pesticide delivery and efficacy. Size and shape of TMGMV will be tailored through self-assembly and heating protocols; surface chemistry will be altered through bioconjugation methods.Soil mobility will be assessed as a function of nanocarrier size, shape, and surface chemistry.For safe use for field applications we will develop inactivation protocols to render TMGMV non-infectious toward its host plant; we will also use self-assembly techniques to produce genome-free TMGMV variants.Bioconjugate and formulation chemistries will be developed to produce pesticide-laden TMGMV; such formulations will be assessed for efficacy and pesticide delivery using liquid nematode cultures and nematode-infested plants. Pesticide leaching in the environment will be studied.

Progress 06/01/23 to 05/31/24

Outputs
Target Audience:Plant scientists, chemists, engineering with an interest in pesticide delivery, nematodes, nanotechnology, plant virology. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Post-doctoral, graduate and undergraduate students are being trained in nanotechnology, applied virology, and precision farming. I train students according to the NPA guidelines and focus on the six classifiedcompetencies:#1. Discipline-specific conceptual knowledge,#2. Research skill development,#3. Communication skills,#4. Professionalism,#5. Leadership and management skills, and#6. Responsible conduct of research. To monitor research and professional development progress we use an Individual Development Plan that is reviewed annually. How have the results been disseminated to communities of interest?Invited Presentations National Institute for Quantitative Health Science and Engineering, Michigan State University, Nov 29th 2023 (online) UC San Diego, Biological Science Department, Oct 25th 2023 IMDD Summit, UC San Diego, 5/12/2023 International RWTH Aachen University, Institute for Center for Biohybrid Medical Systems, Dec 13th 2023 RWTH Aachen University, Bio 7, Molecular Biotechnology, Dec 14th 2023 CIC NanoGune San Sebastián, Spain, November 15th 2023 Sogang University, South Korea, September 5th 2023 University of Twente, Netherlands, July 4th 2023 Fraunhofer IME, Aachen, June 22, 2023 Invited Lectures at International and National Conferences: Green-bio Industry, Pohang, Korea, September 7-8th 2023 What do you plan to do during the next reporting period to accomplish the goals?Under the approved NCE, we will complete Objective4. We are preparing ivermectin-laden TMGMV and test efficacy againstnematode-infected tomato plants.We hypothesize that the biopesticide will be more effective at lower doses than the free drug. Leaching behavior will be studied to determine the environmental impact of nanopesticide vs conventional pesticide exposure.

Impacts
What was accomplished under these goals? Objective 1. Study the effect of nanocarrier size, shape, and surface chemistry on soil mobility. Objective 1 was completed and results were discussed in previous reports. Objective 2. Develop nanopesticides for safe field use and broad applications. Objective 2 was completed and results were discussed in previous reports. Objective 3. Optimize the nanocarrier to achieve efficient loading and controlled release. We focused on chemical optimization to enable nematicide loading into or onto the protein-based carriers. First, refined the chemical bioconjugation strategies of TMGMV - this was discussed previously and published: I. Gonzalez-Gamboa, A.A. Caparco, J.M. McCaskill, N.F. Steinmetz, Bioconjugation Strategies for Tobacco Mild Green Mosaic Virus, Chembiochem 23(18) (2022) e202200323.. This deeper understanding of the chemical properties of TMGMV further enables its functionalization and use as a multifunctional nanocarrier platform for applications in precision farming (and medicine). Second, because the conjugation of hydrophobic pesticides (e.g., IVM) to hydrophilic plant virus-based nanocarriers resulted in extensive aggregation, we developed a pre-incubation and coating strategy using non-ionic surfactants, specifically Pluronic F127. This polymer coat strategy enabled the stable formulation of IVN-TMGMV - also this work was published and previously reported;M.D. Shin, J.D. Hochberg, J.K. Pokorski, N.F. Steinmetz, Bioconjugation of Active Ingredients to Plant Viral Nanoparticles Is Enhanced by Preincubation with a Pluronic F127 Polymer Scaffold, ACS Appl Mater Interfaces 13(50) (2021) 59618-59632. Third - the focus of PR4 - as a new direction and a departure from multi-step bioconjugation reactions (which are resource-intensive and often produce low yields), we explored non-covalent encapsulation techniques. Not only are these techniques simpler - avoiding multistep purification processes and expensive linkers - and able to achieve higher yields, the regulatory steps also should be streamlined because the active ingredient is not modified. In one application, we made use of the modular nature of TMGMV and carefully adjusted the bathing conditions to allow the structures to "breathe" and open pockets between coat proteins. This carefully controlled swelling enabled the inter-coat protein loading of several pesticides, including fluopyram, clothianidin, rifampicin, and IVN into the macromolecular TMGMV structure with up to ~1000 cargo molecules per TMGMV particle (this work is now accepted for publication in Scientific Reports). In another approach, we made use of the thermal shape-switching properties of high-aspect-ratio virus nanoparticles to form SNPs. We found that shape transformation occurs while heating well above the melting temperature of the nucleoprotein assembly, causing unfolding and reassembly into ordered aggregates, which assume a spherical shape. We investigated a phase space for TMGMV rod-to-SNP transition as a function of TMGMV concentration, heating time, and temperature. The size of the SNPs is a function of the temperature/heating time as well as concentration, with higher TMGMV starting concentrations leading to larger SNPs. A set of SNPs was prepared for soil mobility analysis. We found that SNPs have good soil mobility regardless of their size. Furthermore, we demonstrated that IVN can be loaded into SNPs during this thermal reshaping process.The one-step synthesis of IVN-laden SNPs was highly versatile and reproducible, with up to ~50 IVN molecules per coat protein for SNPs measuring 100-200 nm (i.e., 1×106 IVN molecules per SNP, or 60% IVN by mass). The soil mobility was maintained for the IVN-laden SNPs and most importantly, we confirmed that the cargo and SNP carrier co-eluted from the soil columns, and this held true for all sizes tested [3]. These data indicate that the non-covalent loading of cargo into SNPs facilitates soil transport. This contrasts with the TMGMV rods, where non-covalent loading strategies based on electrostatic binding resulted in stable particles in the test tube, but the cargo was stripped from the particles when added to soil. Finally, we confirmed the superior efficacy of IVN-SNPs vs free IVN against C. elegans. To mimic in-field application, the formulations were added to soil columns and C. elegans was exposed to the eluted fractions. Only IVN-SNPs significantly reduced the number of nematodes and their surface mobility, whereas soluble IVN had no effect, probably because it was lost in the soil. From these data, we conclude that the entrapment of agrochemical cargo molecules in SNPs promotes soil mobility, resulting in the effective treatment of nematodes. This work is now published:A.A. Caparco, I. Gonzalez-Gamboa, S.S. Hays, J.K. Pokorski, N.F. Steinmetz, Delivery of Nematicides Using TMGMV-Derived Spherical Nanoparticles, Nano Lett 23(12) (2023) 5785-5793.

Publications

• Type: Journal Articles Status: Published Year Published: 2023 Citation: Venkateswaran U.P.#*, Caparco A.A.#*, Gonzalez-Gamboa I.*, Caballero R.M.*, Schuphan J., Steinmetz N.F. (2023) Plant Viral Nanocarrier Soil Mobility as a Function of Soil Type and Nanoparticle Properties. ACS Agricultural Science & Technology. as-2023-00074n (acsagscitech.3c00074) in press. #authors contributed equally.
• Type: Book Chapters Status: Accepted Year Published: 2023 Citation: Ma Y.*, Steinmetz N.F. (2023) Potato Virus X Inactivation and Characterization. Methods Mol Biol. 2023;2671:257-271. doi: 10.1007/978-1-0716-3222-2_15. PMID: 37308650
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Caparco A.A.*, Gonz�lez-Gamboa I.*, Hays S.S., Pokorski J., Steinmetz N.F. (2023) Delivery of Nematicides Using TMGMV-Derived Spherical Nanoparticles. Nano Letters https://doi.org/10.1021/acs.nanolett.3c01684.

Progress 06/01/22 to 05/31/23

Outputs
Target Audience:Plant scientists, chemists, engineering with an interest in pesticide delivery, nematodes, nanotechnology, plant virology. Changes/Problems:We have made consirable progress under the proposed objectives with no changes in scope but there were delays due to delayed recruitment of a post-doc. We are in the process of hiring a post-doc to increase personnel effort. There were delays in hiring and we expect that we need to submit an NCE at the end of the grant term (05/31/2024) to complete all aims as proposed. What opportunities for training and professional development has the project provided?Post-doctoral, graduate and undergraduate students are being trained in nanotechnology, applied virology, and precision farming. I train students according to the NPA guidelines and focus on the six classifiedcompetencies:#1. Discipline-specific conceptual knowledge,#2. Research skill development,#3. Communication skills,#4. Professionalism,#5. Leadership and management skills, and#6. Responsible conduct of research. To monitor research and professional development progress we use an Individual Development Plan that is reviewed annually. How have the results been disseminated to communities of interest?Through manuscripts and presentations: Manuscripts See under Products Presentations? School of Pharmacy Seminar, University of Utah, Utah, February 9, 2023 Department of Biological Engineering, MIT, Boston, MA, October 27th 2022 Uniklinikum RWTH Aachen University, June 14th 2022 10-year Anniversary Biointerfaces Institute Research Symposium, Michigan University, Nov 10th 2022 (Distinguished Lectureship) GRC Drug Carriers in Medicine and Biology. NanoEngineering gone viral: plant virus based therapeutics. Mount Snow, West Dover, VT. July 21-August 5th 2022. First International Blackforest Virology Symposium - in memory of Holger Jeske, NanoEngineering gone viral: plant virus based therapeutics Fritz Lauterbad, Germany, June 23-25th 2022 GRC Bioinspired Materials: NanoEngineering gone viral: plant virus based therapeutics, June 5-10th 2022, Les Diablerets, Switzerland. International Congress of Nemotology (ICN2022): NanoEngineering gone viral: plant virus nanotechnologies for precision farming, May 1-6th 2022, Juan-Les-Pins, Antibes, France. International Society for Plant Molecular Farming (ISPMF), Plant viruses against cancer. Rome, Italy, 09/26-28 2022 Gordon research conference (GRC) of Physical Virology, Lucca (Barga), LU, Italy, January 22--27/2023. González-Gamboa I., McCaskill J., Caparco A.A., Fuenlabrada P., Jin Z., Jokerst J.V., Steinmetz N.F. Inter Coat Protein Molecule Loading into TMGMV. Poster presentation. Gordon research conference (GRC) of Physical Virology, Lucca (Barga), LU, Italy, January 22--27/2023. Caparco A.A., Gonzalez-Gamboa I., Steinmetz N.F. Thermal transition of TMGMV to spherical nanoparticles enables encapsulation of hydrophobic cargo. Poster presentation González-Gamboa I., McCaskill J., Caparco A.A., Fuenlabrada P., Jin Z., Jokerst J.V., Steinmetz N.F. Inter Coat Protein Molecule Loading into TMGMV. Poster presentation and Oral presentation. Gordon research seminar (GRS) of Physical Virology, Lucca (Barga), LU, Italy, January 21--22/2023. Caparco A.A., Gonzalez-Gamboa I., Steinmetz N.F. Thermal transition of TMGMV to spherical nanoparticles enables encapsulation of hydrophobic cargo. Poster presentation 2022 ACS Spring, March 20-24 2022, San Diego, CA.González-Gamboa I., Caparco A., McCaskill J.,Steinmetz N.F.Tobacco mild green mosaic virus as a multifunctional platform for efficient pesticide delivery. Oral presentation. ACS Spring 2022, March 20-24 2022, San Diego, CA. Caparco, A.A., Gonzalez Gamboa, I.,Steinmetz N.F.Thermal transformation of rod-shaped viruses into spherical nanoparticles for precision agriculture and drug delivery.Poster presentation. What do you plan to do during the next reporting period to accomplish the goals?In the context of the 4 objectives, we will continue to establish the chemistry for optimized pesticide loading (Objectives 2-3) and systematically investigate soil diffusion properties and pesticide delivery nematodes (Objective 1). As the project matures a focus will lie on the applications with nematodes - initially in the petri dish in media and soil cultures and then in field (Objevtive 4).

Impacts
What was accomplished under these goals? Objective 1. Study the effect of nanocarrier size, shape, and surface chemistry on soil mobility.Native TMGMV forms a nucleoprotein assembly measuring 300x18 nm. Using a combination of plant molecular farming, protein self-assembly, and thermal treatment, we will formulate TMGMV nanocarriers varying in size (50-300 nm), shape (spherevsrod), and surface chemistry (zwitterionicvspassivated) and will test their mobility in soil. We have optimized the synthesis of spherical nanoparticles of TMGMV and defined the space-phase diagram to control the shape-switching yielding nano- to micronscale spherical formulations of TMGMV; we also established the encapsulation of agriculturally relevant cargo, namely ivermectin. We are finalizing studies to demonstrate efficacy of such SNPs against nematodes and also detail the soil mobility properties. This work will be written up for publication. SNP soil mobility is compared also to the mobility of TMGMV rods. PEGylated and native TMGMV as well pesticide-laden TMGMV were analyzed systemically using Magic Topsoil Veggie, Potting, and 50/50 soils provided by SoCal Mulch (Menifee, CA, USA) as testbeds. In addition to the TMGMV nanoparticles, we also considered filamentous plant viruses and spherical formulations with varying surface charge. We note significant differences in soil mobility in the various testbeds and this is attributed to differing chemical composition of the soils. Importantly rod and spherical formulations of TMGMV outperform any other system tested. This work will be written up for publication. Objective 3. Optimize the nanocarrier to achieve efficient loading and controlled release.Most pesticides (e.g.avermectin) are hydrophobic, which limits their mobility in soil, restricts their potential for bioconjugation and inhibits cargo delivery. We will develop orthogonal strategies that allow bioconjugation to the pesticide carrier in aqueous solutions using non-ionic surfactants (poloxamers). We completed to probe the bioconjugate chemistry space of TMGMV and reported the results in González-Gamboa I.*#; Caparco A.A.*#;McCaskill J.M.*;Steinmetz N.F.(2022) Bioconjugation strategies for Tobacco mild green mosaic virus. ChemBioChemhttp://dx.doi.org/10.1002/cbic.202200323 In brief, we focused on the establishment and refinement of chemical bioconjugation strategies to load molecules into or onto TMGMV for targeted delivery. The chemical addressability of TMGMV was established using fluorescence and biotin labeling, proteomic analysis, electron microscopy, and a structural model. The yield of modification and distribution of modified amino acid residues for three types of bioconjugations, targeting the N-terminus, tyrosine and glutamic acid residues using a combination of NHS, diazonium, EDC and click chemistry was determined, and the results were corroborated with analysis of the available TMGMV structure. A combination of NHS, EDC, and diazo coupling reactions in combination with click chemistry were used to modify theN-terminus, glutamic/aspartic acid residues, and tyrosines in TMGMV. We report loading with over 600 moieties per TMGMV via diazo-coupling, which is a >3-fold increase compared to previous studies - this was achieved by careful optimization of the reaction conditions. We also report that cargo can be loaded to the solvent-exposedN-terminus and carboxylates on the exterior/interior surfaces. Mass spectrometry revealed the most reactive sites to be Y12 and Y72, both tyrosine side chains are located on the exterior surface. For the carboxylates, interior E106 (66.53 %) was the most reactive for EDC-propargylamine coupled reactions, with the exterior E145 accounting for >15 % reactivity, overturning previous assumptions that only interior glutamic acid residues are accessible. A deeper understanding of the chemical properties of TMGMV further enables its functionalization and use as a multifunctional nanocarrier platform for applications in medicine and precision farming. In ongoing work, we also turned toward making use of the dynamic nature of TMGMV to non-covalently trap pesticide at the coat protein interface. Data indicate that both pH and solvent-assisted loading is feasible - however with great variation comparing various target pesticides. We will continue to optimize and refine these methods.

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: 236. Gonz�lez-Gamboa I.*#; Caparco A.A.*#; McCaskill J.M.*; Steinmetz N.F. (2022) Bioconjugation strategies for Tobacco mild green mosaic virus. ChemBioChem http://dx.doi.org/10.1002/cbic.202200323. #co-first authors.
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Monroy-Borrego A.G.*, Steinmetz, N.F. (2022) Three methods for inoculation of viral vectors into plants. Front. Plant Sci. - Plant Biotechnology, DOI: 10.3389/fpls.2022.963756.
• Type: Book Chapters Status: Accepted Year Published: 2023 Citation: Charudattan, R., Boyetchko, S.M., Rosskopf, E.N., Williams, K.T., Borrego, A.M.,. Steinmetz, N.F. 202x. Ecologically based weed management: bioherbicides, nanotechnology, heat, and microbially-mediated soil disinfestation. In N.E. Korres, E. Travlos and T.K Gitsopoulos (Eds.) "Ecologically-based weed management: Concepts, challenges and limitations. ", Wiley Pub. (in press).
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Bioconjugation of Active Ingredients to Plant Viral Nanoparticles Is Enhanced by Preincubation with a Pluronic F127 Polymer Scaffold. ACS Applied Materials & Interfaces, https://doi.org/10.1021/acsami.1c13183
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Chung Y.H.*, Church D., Koellhoffer E.C.*, Osota E.*, Shukla S.*, Rybicki E.P., Pokorski J.K., Steinmetz N.F. (2021) Integrating plant molecular farming and materials research for next-generation vaccines. Nature Reviews Materials. https://doi.org/10.1038/ s41578-021-00399-5
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Ma Y.*, Commandeur U., Steinmetz N.F. (2021) Three Alternative Treatment Protocols for the Efficient Inactivation of Potato Virus X. ACS Appl. Bio Mater. https://doi.org/10.1021/acsabm.1c00838
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Chariou, P.L., Ma, Y., Hensley, M., Rosskopf, E.N., Hong, J.C., Charudattan, R., Steinmetz, N.F. (2021) Inactivated Plant Viruses as an Agrochemical Delivery Platform; ACS Agricultural Science & Technology; https://doi.org/10.1021/acsagscitech.1c00083

Progress 06/01/21 to 05/31/22

Outputs
Target Audience:Target audiences: Plant scientists, chemists, engineering with an interest in pesticide delivery, nematodes, nanotechnology, plant virology. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A post-doc, graduate and master student are being trained in nanotechnology, applied virology, and precision farming. I train students according to the NPA guidelines and focus on the six classifiedcompetencies:#1. Discipline-specific conceptual knowledge,#2. Research skill development,#3. Communication skills,#4. Professionalism,#5. Leadership and management skills, and#6. Responsible conduct of research. To monitor research and professional development progress we use an Individual Development Plan that is reviewed annually. How have the results been disseminated to communities of interest?Through manuscripts and presentations: Manuscripts ShinM.D.*, HochbergJ.D., Pokorski J.K.,Steinmetz N.F.(2021) Bioconjugation of Active Ingredients to Plant Viral Nanoparticles IsEnhanced by Preincubation with a Pluronic F127 Polymer Scaffold. ACS Applied Materials & Interfaces,https://doi.org/10.1021/acsami.1c13183 Chung Y.H.*, Church D., Koellhoffer E.C.*, Osota E.*, Shukla S.*, Rybicki E.P., Pokorski J.K., Steinmetz N.F. (2021) Integrating plant molecular farming and materials research for next-generation vaccines. Nature Reviews Materials. https://doi.org/10.1038/ s41578-021-00399-5 Ma Y.*, Commandeur U., Steinmetz N.F. (2021) Three Alternative Treatment Protocols for the Efficient Inactivation of Potato Virus X. ACS Appl. Bio Mater. https://doi.org/10.1021/acsabm.1c00838 Chariou, P.L., Ma, Y., Hensley, M., Rosskopf, E.N., Hong, J.C., Charudattan, R., Steinmetz, N.F. (2021) Inactivated Plant Viruses as an Agrochemical Delivery Platform; ACS Agricultural Science & Technology; https://doi.org/10.1021/acsagscitech.1c00083 Presentations 2. REU program at UC San Diego, Department of Bioengineering, July 26th 2021 (online) 3. 3M, Nanotechnology seminar, June 28th 2021 (online) 4. UC San Diego Jacob's School of Engineering Research Expo, May 19th 2021 (online) 5. University of Notre Dame, Chemical and Biomolecular Engineering, May 18th 2021 (online) 6. UC Berkeley, Department of Bioengineering, March 3rd 2021 (online) 7. CaliBaja Webinars for college students for the US and Mexico, UC San Diego, Feb 24 2021 (online) 93. Centre for Plant Biotechnology and Genomics CBGP (UPM-INIA), November 19th 2021 (online) 94. Department of Molecular and Cell Biology, University of Cape Town, September 22nd 2021 (online) 95. King Abdullah University of Science and Technology (KAUST), Department of Bioengineering, Feb 9th 2021 (online) 113. Virtual Conference- Materials in the Anthropocene (CVMA), NanoEngineering gone #viral: Plant virus-based therapeutics. October 27-29th 2021 (online). (Keynote Lecture) 115. Swiss NanoConvention 2021 online, Plenary Lecture, June 24-25th 2021 (online) (Plenary Lecture) 127. Pacifichem 2021; Symposium 332: Chemistry and applications of protein and virus-based nanotechnologies, Nanoengineering gone viral: plant virus-based therapeutics and pesticides, Dec 20th 2021 (online) 128. NSF Grantees Conference; Session: Nanomanufacturing in Agriculture and Biomedicine, NanoEngineering gone #viral Dec 7-8th 2021 (online) 129. BASF CARA Research Agreements Fall Review: Going Viral: Tailoring soil mobility of agrochemicals with virus-like particles, November 15-17th 2021 (online) 131. 2021 AFRI nanotechnology annual grantees' conference: Plant virus-like nanopesticides for precision farming. October 6-7 2021 (online). 132. Plant Health 2021: Session Fighting Virus with Virus: NanoEngineering gone viral: plant virus-based COVID-19 vaccine candidates. August 2-6th 2021 (online) What do you plan to do during the next reporting period to accomplish the goals?We will focus our efforts on Objectives 1 and 3 and will refine the nanoparticle design (size and shape and surface chemistry) as well as to improve loading for agrochemical compounds. The goals are to assay soil mobility and efficacy against nematodes.

Impacts
What was accomplished under these goals? Objective 1. Study the effect of nanocarrier size, shape, and surface chemistry on soil mobility.Native TMGMV forms a nucleoprotein assembly measuring 300x18 nm. Using a combination of plant molecular farming, protein self-assembly, and thermal treatment, we will formulate TMGMV nanocarriers varying in size (50-300 nm), shape (spherevsrod), and surface chemistry (zwitterionicvspassivated) and will test their mobility in soil. We have begun to establish methods for SNP formulations to probe the structure-function relationship of nanoparticle geometry and soil diffusion: The SNPsare thermally transformed virus nanoparticles whose size can be tuned by adjusting the initial concentration of virus in solution. Previous work by us and others has shown SNPs can be formed using tobacco mosaic virus (TMV), which has very high sequence homology with TMGMV and is also a rod-shaped virus. These SNPs have also been shown to be reactive and capable of loading cargo via bioconjugation reactions. Building off this work, we hypothesized that TMGMV should undergo a similar thermal transformation to SNPs that would be dependent on the heating temperature, heating time, and initial concentration of virus. We developed a phase space for the formation of SNPs using SEM and TEM and found that TMGMV SNPs did form in conditions where temperature was higher than 96 °C for at least 30 seconds. In preliminary studies, we also tested whether TMGMV could be used to load cargo via entrapment or encapsulation. Encapsulation is desirable as it is low-cost and less time-intensive than covalent attachment strategies, which is desirable for the agricultural applications we have in mind for this material. We quantified the degree of encapsulation for several SNP formulations and found nearly 100% of Cyanine 5 in solution could be entrapped in some cases. In ongoing and upcoming work, we will assess the soil mobility of these SNP materials and compare with TMGMV rods. Objective 2. Develop nanopesticides for safe field use and broad applications.TMGMV has a narrow host range but it does infect several solanaceous species among others. We will determine whether UV treatment can render the formulation inactive and safe without affecting its nanocarrier properties. In parallel, we will develop a genome-free VLP carrier based on TMGMV. During the previous reporting period, we described out efforts and results to develop non-infectious TMGMV. We successfully established 3 methods of viral inactivation: chemical inactivation by βPL or formalin treatment as well as by radiation using UV light. We report the successful inactivation of TMGMV using 10 J cm−2 of ultraviolet light, 1.5 M βPL, or 1 M formalin; the lack of infectivity was confirmed using Nicotiana tabacum Tennessee 86, N. tabacum Samsun nn, and tropical soda apple (Solanum viarum). This is now published: Chariou, P.L., Ma, Y., Hensley, M., Rosskopf, E.N., Hong, J.C., Charudattan, R., Steinmetz, N.F. (2021) Inactivated Plant Viruses as an Agrochemical Delivery Platform; ACS Agricultural Science & Technology; https://doi.org/10.1021/acsagscitech.1c00083 Moreover we sought to test the robustness of these methods and applied the approaches to another plant virus nanoparticle, namely the potato virus X. We report experiments showing that PVX can be completely inactivated by exposure to UV irradiation (0.5 J cm−2) or chemical treatment (1 mM β-propiolactone or 10 mM formalin) without interfering with the chemical addressability of lysine or cysteine residues, which are typically used as conjugation handles for virus nanoparticle functionalization. Also this work was published: Ma Y.*, Commandeur U.,Steinmetz N.F.(2021) Three Alternative Treatment Protocols for the Efficient Inactivation of Potato Virus X. ACS Appl. Bio Mater. https://doi.org/10.1021/acsabm.1c00838 Objective 3. Optimize the nanocarrier to achieve efficient loading and controlled release.Most pesticides (e.g.avermectin) are hydrophobic, which limits their mobility in soil, restricts their potential for bioconjugation and inhibits cargo delivery. We will develop orthogonal strategies that allow bioconjugation to the pesticide carrier in aqueous solutions using non-ionic surfactants (poloxamers). Proteinaceous nanoparticles, such as TMGMV and other plant viruses as well as protein cages, can be used to deliver large payloads of active ingredients, which is advantageous in medicine and agriculture. However, the conjugation of hydrophobic ligands to hydrophilic nanocarriers such as plant viral nanoparticles (plant VNPs) can result in aggregation by reducing overall solubility. Given the benefits of hydrophilic nanocarrier platforms for targeted delivery and multivalent ligand display, coupled with the versatility of hydrophobic drugs, contrast agents, and peptides, this is an issue that must be addressed to realize their full potential. Here, we report two preincubation strategies that use a Pluronic F127 polymer scaffold to prevent the aggregation of conjugated plant VNPs: a plant VNP− polymer precoat (COAT) and an active ingredient formulation combined with a plant VNP−polymer precoat (FORMCOAT). The broad applications of these modified conjugation strategies were highlighted by testing their compatibility with three types of bioconjugation chemistry: N-hydroxysuccinimide ester−amine coupling, maleimide−thiol coupling, and copper(I)-catalyzed azide−alkyne cycloaddition (click chemistry). The COAT and FORMCOAT strategies promoted efficient bioconjugation and prevented the aggregation that accompanies conventional bioconjugation methods, thus improving the stability, homogeneity, and translational potential of plant VNP conjugates in medicine and agriculture. The chemistries were applied to ivermectin conjugation and solubility of the pesticide-loaded nanoparticle were greatly increased which also allowed increase in a.i. loading. This work was published: ShinM.D.*, HochbergJ.D., Pokorski J.K.,Steinmetz N.F.(2021) Bioconjugation of Active Ingredients to Plant Viral Nanoparticles IsEnhanced by Preincubation with a Pluronic F127 Polymer Scaffold. ACS Applied Materials & Interfaces,https://doi.org/10.1021/acsami.1c13183 In ongoing work, we are also further refining the chemical bioconjugation methods to load agrochemicals into or onto TMGMV for targeted delivery to plant pests. We were able to improve AI loading by 3-fold from previously published data and report bioconjugation to the N-terminus for the first time in this virus. Also, the specific modified residues (and their location) are elucidated in this work using a proteomic approach - this is ongoing work. These results will further extend the understanding of the chemical addressability of TMGMV and enable development of novel multifunctional platforms for pesticide delivery and precision farming.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2021 Citation: Chariou, P.L., Ma, Y., Hensley, M., Rosskopf, E.N., Hong, J.C., Charudattan, R., Steinmetz, N.F. (2021) Inactivated Plant Viruses as an Agrochemical Delivery Platform; ACS Agricultural Science & Technology; https://doi.org/10.1021/acsagscitech.1c00083
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Shin M.D.*, Hochberg J.D., Pokorski J.K., Steinmetz N.F. (2021) Bioconjugation of Active Ingredients to Plant Viral Nanoparticles Is Enhanced by Preincubation with a Pluronic F127 Polymer Scaffold. ACS Applied Materials & Interfaces, https://doi.org/10.1021/acsami.1c13183
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Chung Y.H.*, Church D., Koellhoffer E.C.*, Osota E.*, Shukla S.*, Rybicki E.P., Pokorski J.K., Steinmetz N.F. (2021) Integrating plant molecular farming and materials research for next-generation vaccines. Nature Reviews Materials. https://doi.org/10.1038/ s41578-021-00399-5
• Type: Journal Articles Status: Accepted Year Published: 2021 Citation: Ma Y.*, Commandeur U., Steinmetz N.F. (2021) Three Alternative Treatment Protocols for the Efficient Inactivation of Potato Virus X. ACS Appl. Bio Mater. https://doi.org/10.1021/acsabm.1c00838

Progress 06/01/20 to 05/31/21

Outputs
Target Audience:Under this award we will study nanotechnology approaches to package and safely deliver pesticides to treat crops. The research described is of fundemantal and applied nature and the target audience are scientists and engineers in the space of agricultural nanotechnology. In the long term the data gained and products developed under this award will be of interest to industries developing next-generations pesticides. Changes/Problems:The COVID-19 pandemic delayed the start of the project as personnel hires were delayed. However personnel has now been hired and we are on track with the objectives. What opportunities for training and professional development has the project provided?A graduate student and post-doc are trained under this award. The trainees will be training in nanoscience and nanotechnology approaches applied to pesticide delivery. To provide professional development, the trainess are trained along the guidelines and established core competencies described by the National Postdoctoral Association (NPA). "Competency" has been defined as "an acquired personal skill that is demonstrated in [one's] ability to provide a consistently adequate or high level of performance in a specific job function." These competencies are meant to serve primarily as: i) a basis for self-evaluation by the student and ii) a basis for developing training opportunities that can be evaluated by mentors. To develop these competencies, traineesl meet with their mentor, Dr. Steinmetz, for biweekly small group meetings and 1-1 meetings. The six core competencies are: 1. Discipline-specific conceptual knowledge, 2. Research skill development, 3. Communication skills, 4. Professionalism, 5. Leadership and management skills, and 6. Responsible conduct of research. Further, Dr. Steinmetz uses a formal Individual Development Plan (IDP) training, this is a living document to help trainees in their self-assessment of his strengths and weaknesses, thus allowing identifying areas that his mentor needs to pay attention to and provide guidance to turn potential weaknesses into strengths. In this IDP document trainees will also list short/long-term goals and milestones to be accomplished associated with this research award and their long-term career goals. This formal form of training has been valued highly by the National Postdoctoral Association and is expected to focus trainee and trainer toward achieving the short-term and long-term goals. How have the results been disseminated to communities of interest?A manuscript describing the results described above has been submitted and is in review with the Journal of Agriculture and Food Chemistry. A review article is under consideration with Nature Reviews Materials; the work is partially supported by this award as the review article is focused on "Integrating molecular farming and materials research for manufacture of next-generation vaccines" What do you plan to do during the next reporting period to accomplish the goals?We have begun to work on the following objectives: Objective 1. Study the effect of nanocarrier size, shape, and surface chemistry on soil mobility. Native TMGMV forms a nucleoprotein assembly measuring 300x18 nm. Using a combination of plant molecular farming, protein self-assembly, and thermal treatment, we will formulate TMGMV nanocarriers varying in size (50-300 nm), shape (sphere vs rod), and surface chemistry (zwitterionic vs passivated) and will test their mobility in soil. Objective 3. Optimize the nanocarrier to achieve efficient loading and controlled release. Most pesticides (e.g. avermectin) are hydrophobic, which limits their mobility in soil, restricts their potential for bioconjugation and inhibits cargo delivery. We will develop orthogonal strategies that allow bioconjugation to the pesticide carrier in aqueous solutions using non-ionic surfactants (poloxamers).

Impacts
What was accomplished under these goals? We focused on Objective 2. Develop nanopesticides for safe field use and broad applications. Nanoparticle-based pesticide delivery systems have emerged to decrease the environmental and health impact of pesticides while increasing their efficacy. The majority of nanopesticides in the development pipeline are synthetic materials and these present their own set of environmental risks. As an alternative, we proposed the development of naturally occurring nanomaterials, namely plant viruses such as tobacco mild green mosaic virus (TMGMV) for the delivery of pesticides. We and others have previously shown that plant virus-based nanoparticles have favorable soil mobility properties and thus could offer new avenues to deliver pesticides to target root-feeding pests. However, it is imperative to develop methods for safe deployment of plant viruses in the environment. Therefore, to avoid plant infection, we investigated viral inactivation methods to render TMGMV non-infectious toward plants. TMGMV was treated with UV light, β-propiolactone (βPL) or formalin; dose-escalation studies were performed, and infectivity or lack thereof was assessed using three plant host species: Nicotiana tabacum Tennessee 86 (Tn86), N. tabacum Samsun nn, and tropical soda apple (TSA) (Solanum viarum). We report the successful inactivation of TMGMV using 10 J cm-2 of UV light, 1.5 M βPL, or 1 M formalin, laying the groundwork for the development of eco-friendly, non-infectious viral pesticide nanocarriers that could be applied on crops.

Publications