Progress 09/01/19 to 08/31/20
Outputs
Target Audience:Individuals, groups, and market segments involved in nanotechnology, pesticide delivery, and innovative agricultural technologies. Researchers, scientific professionals, agricultural technolgists, and administrators. Pesticide companies, pesticide developers, and technology investors. Teachers, students, and trainees. Changes/Problems:In April 2020, our ability to make progress on the project came to an unexpected halt due to the coronavirus nCoV-19 outbreak. We hope to make further, swift progress as soon as the statewide and institutional restrictions are lifted at the Principal Investigators/Project Directors' respective locations in California and Florida. What opportunities for training and professional development has the project provided?A graduate student, partially funded on this project is completing his training and expects to graduate. How have the results been disseminated to communities of interest?Through scientific journal articles. What do you plan to do during the next reporting period to accomplish the goals?Complete the remaining tasks to address the stated objectives, prepare material for publication in scientific journals, present the results in appropriate scientific venues, and file for patents, as indicated.
Impacts
What was accomplished under these goals?
Objective 1. To develop and optimize TMGMV with nematicide payloads and assess the efficacy of TMGMV-delivered nematicide (TDN) vs free nematicide against nematodes in vitro. Nematicide-loaded TMGMV will be prepared and optimized. Loading and release will be studied; nematicide release will be tailored making use of counterions and covalent chemistries. The effective concentration (EC50) of encapsulated and free nematicides to treat C. elegans will be compared in liquid cultures. In parallel, chosen nematicide-loaded TMGMV will be tested in vitro for activity and effective concentration against Meloidogyne incognita. We have synthesized various avermectin-derivatives with ligation handles for coupling to the TMGMV nanocarrier as shown in Figure 1. Synthesis of azido-avermectin. Ethyl 5-bromovalerate (0.100 g, 0.478 mmol, 1 eq) was reacted with sodium azide (NaN3) (155 mg, 2.391 mmol, 5 eq) overnight in dimethylformamide (DMF) (~10 mL). After reaction, crude product was washed with H2O three times to remove excess NaN3. Organic layer was dried with sodium sulfate (Na2SO4) and reduced in vacuo to give ethyl 5-pentanoate (81.9% recovery, 67 mg). Ethyl 5-pentanoate (67 mg, 0.392 mmol, 1 eq) was reacted with KOH (33 mg, 0.589 mmol, 1.5 eq) in a 1:1 mixture of MeOH (~5 mL) and H2O (~5 mL) overnight. After reaction, crude product was diluted with H2O and acidified with 1 M HCl. Aqueous layer was extracted three times with ethyl acetate. The organic layer was dried with Na2SO4 and reduced in vacuo to give 5-azidopentanoic acid. (63.6% recovery, 35 mg). 5-azidopentanoic acid (12 mg, 0.086 mmol, 1.5 eq) and abamectin (50 mg, 0.057 mmol, 1 eq) were reacted via a Steglich Esterification consisting of N, N' dicyclohexylcarbodiimide (DCC) (18 mg, 0.086 mmol, 1.5 eq) and catalytic 4-dimethylaminopyridine (DMAP) (1 mg, 5 mol %) in dichloromethane (DCM) (~5 mL) overnight. Reaction began on ice and proceeded as ice melted. Following the reaction, the crude product was placed in the freezer for 30 minutes and gravity filtered to remove the precipitated Dicyclohexylurea (DCU) byproduct. The remaining product was then extracted with H2O to remove excess DMAP. The product was then filtered via silica column chromatography with a mobile phase of 4% MeOH in DCM. Both single and double conjugated azido-avermectin was isolated and reduced in vacuo. Azido-avermectin was dissolved in tetrahydrofuran (THF) prior to use. Synthesis of azido-PEG-avermectin. N3-PEG500-COOH (39 mg, 0.086 mmol, 1.5 eq) and abamectin (50 mg, 0.057 mmol, 1 eq) were reacted via a Steglich Esterification consisting of DCC (18 mg, 0.086 mmol, 1.5 eq) and catalytic DMAP (1 mg, 5 mol %) in DCM (~5 mL) overnight. Reaction began on ice and proceeded as ice melted. Following the reaction, the crude product was placed in the freezer for 30 minutes and gravity filtered to remove the precipitated DCU byproduct. The remaining product was extracted with H2O to remove excess DMAP. The product was then filtered via silica column chromatography with a mobile phase of 4% MeOH in DCM. Azido-PEG500-avermectin was isolated and reduced in vacuo. Azido-PEG-avermectin was dissolved in THF prior to use. Synthesis of azido-peptide-avermectin. 6-maleimidohexanoic acid (49 mg, 0.109 mmol, 1.1 eq) was conjugated to avermectin (13 mg, 0.099 mmol, 1 eq) via a Steglich Esterification consisting of DCC (21 mg, 0.099 mmol, 1 eq) and catalytic DMAP (1 mg, 0.010 mmol, 0.1 eq) in DCM. Following the reaction, the crude product was placed in the freezer for 30 minutes and gravity filtered to remove the precipitated DCU byproduct. The remaining product was then washed three times with sodium bicarbonate and once with H2O to remove excess DMAP and 6-maleimidohexanoic acid. The product was then purified via silica column chromatography with a mobile phase of 1/1:Hexane/Ethyl Acetate to further remove the 6-maleimidohexanoic acid. Single conjugated maleimide-avermectin was isolated and reduced in vacuo. (Recovery: 54.7 mg, 55%). Azido-peptide (50 mg, 0.047mmol, 1 eq) and maleimide-avermectin (15 mg, 0.052mmol, 1.1 eq) was combined with trifluoroacetic acid (TFA) (5mL) in THF and H2O (5mL) overnight. The product was then filtered via silica column chromatography with a mobile phase of 4% MeOH in DCM. Azido-peptide-avermectin was isolated and reduced in vacuo. Azido-peptide-avermectin was dissolved in THF prior to use. Figure 1 Avermectin Synthesis. A) azido-avermectin, B) azido-PEG-avermectin, C) azido-Peptide-avermectin. Conjugation of azide-modified avermectin to TMGMV. We then tested the solubility of azido-avermectin (Figure 1A) and found that it remained insoluble in water, ethanol, methanol, dimethyl sulfoxide (DMSO), and DMF, but soluble in THF. This was expected since the linker is an azide-coupled carbon rich chain, which therefore does not increase hydrophilicity. This high hydrophobicity prevented the hydrolysis of the ester bond between avermectin and the linker, which resulted in a severe loss of potency against liquid cultures of C. elegans. While 0.39 μM avermectin immobilized 50% of the nematode population within 48 h, 60 μM azido-avermectin were required to reach the same result. We therefore tested whether the conjugation of azido-avermectin to the alkyne modified TMGMV would increase its solubility and therefore its bioavailability to nematodes in an aqueous solution. TMGMV was conjugated to 5 eq of azido-avermectin using our established click chemistry protocol. To quantify the avermectin loading efficiency, TMGMV-avermectin was reacted with 2 eq of sulfo-Cy5-azide for 30 min on ice followed by purification and characterization by UV-spectroscopy. Results indicated that ~100 avermectin were successfully conjugated per TMGMV particle. Size exclusion chromatography revealed no signs of particle breakage but indicated particle aggregates. Since avermectin is hydrophobic and the conjugation is done in an aqueous solution, it was not surprising that a portion of the TMGMV-avermectin product aggregated. With a level of loading efficiency of ~100 avermectin per TMGMV, we were unable to execute a nematode killing assay, which would have required 39.4 mg of TMGMV-avermectin per 1 mL of nematode culture to reach the maximum concentration of 100 μM avermectin. So, to date we successfully derivatives of avermectin and begun bioconjugation to the TMGMV carrier. Loading efficiencies to date are inefficient and our goal is to improve the chemistry through inclusion of water-soluble linkers such as PEG or peptides (as outlined in Figure 1B+C) to yield more stable constructs that are higher yielding. The compounds have been synthesized and conjugation to TMGMV are the next steps. Objective 2. Systems modeling of TMGMV soil diffusion behavior and nematicide release profile in soil. A computational model will be developed to predict the diffusion profile of the TDN in soil. The model will be validated with soil leaching experiments considering soil grain size, pH, temperature, and concentration of organic matter. These studies will provide insight on the dose regimen required to treat various types of crops in different soils. The model was developed while the SBIR grant was under review and the model was published in Chariou P.L. et al. 2019, Nature Nanotechnology, cited under Publications. Objective 3. Experimental treatments of tomato infested with Meloidogyne incognita, root knot nematode, with TDN and free nematicide. The treatment efficacy of TDN vs free nematicides will be assessed using tomato plants in pots using field-soil conditions. Deriving from Objective 2 data, TDN formulations that have been optimized to diffuse and release its cargo to the right soil depth will be tested. We hypothesize that TDN will be more efficacious and deliver lower doses than free nematicide. Pending.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2020
Citation:
Chariou P.L., Dogan A.B., Welsh A.G., Saidel G.M., Baskaran H., Steinmetz N.F. (2019) Soil mobility of synthetic and virus-based model nanopesticides. Nature Nanotechnology, 14, 712-718. PMID: 31110265, PMCID: pending (NIHMS 1066976).
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Progress 09/01/19 to 04/30/20
Outputs
Target Audience:Individuals, groups, and market segments involved in nanotechnology, pesticide delivery, and innovative agricultural technologies. Researchers, scientific professionals, agricultural technolgists, and administrators. Pesticide companies, pesticide developers, and technology investors. Teachers, students, and trainees. Changes/Problems:In April 2020, our ability to make progress on the project came to an unexpected halt due to the coronavirus nCoV-19 outbreak. We hope to make further, swift progress as soon as the statewide and institutional restrictions are lifted at the Principal Investigators/Project Directors' respective locations in California and Florida. What opportunities for training and professional development has the project provided?A graduate student, partially funded on this project is completing his training and expects to graduate. How have the results been disseminated to communities of interest?Through scientific journal articles. What do you plan to do during the next reporting period to accomplish the goals?Complete the remaining tasks to address the stated objectives, prepare material for publication in scientific journals, present the results in appropriate scientific venues, and file for patents, as indicated.
Impacts
What was accomplished under these goals?
Objective 1. To develop and optimize TMGMV with nematicide payloads and assess the efficacy of TMGMV-delivered nematicide (TDN) vs free nematicide against nematodes in vitro. Nematicide-loaded TMGMV will be prepared and optimized. Loading and release will be studied; nematicide release will be tailored making use of counterions and covalent chemistries. The effective concentration (EC50) of encapsulated and free nematicides to treat C. elegans will be compared in liquid cultures. In parallel, chosen nematicide-loaded TMGMV will be tested in vitro for activity and effective concentration against Meloidogyne incognita. We have synthesized various avermectin-derivatives with ligation handles for coupling to the TMGMV nanocarrier as shown in Figure 1. Synthesis of azido-avermectin. Ethyl 5-bromovalerate (0.100 g, 0.478 mmol, 1 eq) was reacted with sodium azide (NaN3) (155 mg, 2.391 mmol, 5 eq) overnight in dimethylformamide (DMF) (~10 mL). After reaction, crude product was washed with H2O three times to remove excess NaN3. Organic layer was dried with sodium sulfate (Na2SO4) and reduced in vacuo to give ethyl 5-pentanoate (81.9% recovery, 67 mg). Ethyl 5-pentanoate (67 mg, 0.392 mmol, 1 eq) was reacted with KOH (33 mg, 0.589 mmol, 1.5 eq) in a 1:1 mixture of MeOH (~5 mL) and H2O (~5 mL) overnight. After reaction, crude product was diluted with H2O and acidified with 1 M HCl. Aqueous layer was extracted three times with ethyl acetate. The organic layer was dried with Na2SO4 and reduced in vacuo to give 5-azidopentanoic acid. (63.6% recovery, 35 mg). 5-azidopentanoic acid (12 mg, 0.086 mmol, 1.5 eq) and abamectin (50 mg, 0.057 mmol, 1 eq) were reacted via a Steglich Esterification consisting of N, N' dicyclohexylcarbodiimide (DCC) (18 mg, 0.086 mmol, 1.5 eq) and catalytic 4-dimethylaminopyridine (DMAP) (1 mg, 5 mol %) in dichloromethane (DCM) (~5 mL) overnight. Reaction began on ice and proceeded as ice melted. Following the reaction, the crude product was placed in the freezer for 30 minutes and gravity filtered to remove the precipitated Dicyclohexylurea (DCU) byproduct. The remaining product was then extracted with H2O to remove excess DMAP. The product was then filtered via silica column chromatography with a mobile phase of 4% MeOH in DCM. Both single and double conjugated azido-avermectin was isolated and reduced in vacuo. Azido-avermectin was dissolved in tetrahydrofuran (THF) prior to use. Synthesis of azido-PEG-avermectin. N3-PEG500-COOH (39 mg, 0.086 mmol, 1.5 eq) and abamectin (50 mg, 0.057 mmol, 1 eq) were reacted via a Steglich Esterification consisting of DCC (18 mg, 0.086 mmol, 1.5 eq) and catalytic DMAP (1 mg, 5 mol %) in DCM (~5 mL) overnight. Reaction began on ice and proceeded as ice melted. Following the reaction, the crude product was placed in the freezer for 30 minutes and gravity filtered to remove the precipitated DCU byproduct. The remaining product was extracted with H2O to remove excess DMAP. The product was then filtered via silica column chromatography with a mobile phase of 4% MeOH in DCM. Azido-PEG500-avermectin was isolated and reduced in vacuo. Azido-PEG-avermectin was dissolved in THF prior to use. Synthesis of azido-peptide-avermectin. 6-maleimidohexanoic acid (49 mg, 0.109 mmol, 1.1 eq) was conjugated to avermectin (13 mg, 0.099 mmol, 1 eq) via a Steglich Esterification consisting of DCC (21 mg, 0.099 mmol, 1 eq) and catalytic DMAP (1 mg, 0.010 mmol, 0.1 eq) in DCM. Following the reaction, the crude product was placed in the freezer for 30 minutes and gravity filtered to remove the precipitated DCU byproduct. The remaining product was then washed three times with sodium bicarbonate and once with H2O to remove excess DMAP and 6-maleimidohexanoic acid. The product was then purified via silica column chromatography with a mobile phase of 1/1:Hexane/Ethyl Acetate to further remove the 6-maleimidohexanoic acid. Single conjugated maleimide-avermectin was isolated and reduced in vacuo. (Recovery: 54.7 mg, 55%). Azido-peptide (50 mg, 0.047mmol, 1 eq) and maleimide-avermectin (15 mg, 0.052mmol, 1.1 eq) was combined with trifluoroacetic acid (TFA) (5mL) in THF and H2O (5mL) overnight. The product was then filtered via silica column chromatography with a mobile phase of 4% MeOH in DCM. Azido-peptide-avermectin was isolated and reduced in vacuo. Azido-peptide-avermectin was dissolved in THF prior to use. Figure 1 Avermectin Synthesis. A) azido-avermectin, B) azido-PEG-avermectin, C) azido-Peptide-avermectin. Conjugation of azide-modified avermectin to TMGMV. We then tested the solubility of azido-avermectin (Figure 1A) and found that it remained insoluble in water, ethanol, methanol, dimethyl sulfoxide (DMSO), and DMF, but soluble in THF. This was expected since the linker is an azide-coupled carbon rich chain, which therefore does not increase hydrophilicity. This high hydrophobicity prevented the hydrolysis of the ester bond between avermectin and the linker, which resulted in a severe loss of potency against liquid cultures of C. elegans. While 0.39 μM avermectin immobilized 50% of the nematode population within 48 h, 60 μM azido-avermectin were required to reach the same result. We therefore tested whether the conjugation of azido-avermectin to the alkyne modified TMGMV would increase its solubility and therefore its bioavailability to nematodes in an aqueous solution. TMGMV was conjugated to 5 eq of azido-avermectin using our established click chemistry protocol. To quantify the avermectin loading efficiency, TMGMV-avermectin was reacted with 2 eq of sulfo-Cy5-azide for 30 min on ice followed by purification and characterization by UV-spectroscopy. Results indicated that ~100 avermectin were successfully conjugated per TMGMV particle. Size exclusion chromatography revealed no signs of particle breakage but indicated particle aggregates. Since avermectin is hydrophobic and the conjugation is done in an aqueous solution, it was not surprising that a portion of the TMGMV-avermectin product aggregated. With a level of loading efficiency of ~100 avermectin per TMGMV, we were unable to execute a nematode killing assay, which would have required 39.4 mg of TMGMV-avermectin per 1 mL of nematode culture to reach the maximum concentration of 100 μM avermectin. So, to date we successfully derivatives of avermectin and begun bioconjugation to the TMGMV carrier. Loading efficiencies to date are inefficient and our goal is to improve the chemistry through inclusion of water-soluble linkers such as PEG or peptides (as outlined in Figure 1B+C) to yield more stable constructs that are higher yielding. The compounds have been synthesized and conjugation to TMGMV are the next steps. Objective 2. Systems modeling of TMGMV soil diffusion behavior and nematicide release profile in soil. A computational model will be developed to predict the diffusion profile of the TDN in soil. The model will be validated with soil leaching experiments considering soil grain size, pH, temperature, and concentration of organic matter. These studies will provide insight on the dose regimen required to treat various types of crops in different soils. The model was developed while the SBIR grant was under review and the model was published in Chariou P.L. et al. 2019, Nature Nanotechnology, cited under Publications. Objective 3. Experimental treatments of tomato infested with Meloidogyne incognita, root knot nematode, with TDN and free nematicide. The treatment efficacy of TDN vs free nematicides will be assessed using tomato plants in pots using field-soil conditions. Deriving from Objective 2 data, TDN formulations that have been optimized to diffuse and release its cargo to the right soil depth will be tested. We hypothesize that TDN will be more efficacious and deliver lower doses than free nematicide. Pending.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2020
Citation:
Chariou P.L., Dogan A.B., Welsh A.G., Saidel G.M., Baskaran H., Steinmetz N.F. (2019) Soil mobility of synthetic and virus-based model nanopesticides. Nature Nanotechnology, 14, 712-718. PMID: 31110265, PMCID: pending (NIHMS 1066976).
|