[go: up one dir, main page]

WO2022125520A1 - Microparticules de membrane cellulaire d'insectes pour la détoxification d'insectes pollinisateurs - Google Patents

Microparticules de membrane cellulaire d'insectes pour la détoxification d'insectes pollinisateurs Download PDF

Info

Publication number
WO2022125520A1
WO2022125520A1 PCT/US2021/062163 US2021062163W WO2022125520A1 WO 2022125520 A1 WO2022125520 A1 WO 2022125520A1 US 2021062163 W US2021062163 W US 2021062163W WO 2022125520 A1 WO2022125520 A1 WO 2022125520A1
Authority
WO
WIPO (PCT)
Prior art keywords
oil
insect
cell membrane
composition
microparticles
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2021/062163
Other languages
English (en)
Inventor
James Webb
Jing Chen
Minglin Ma
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cornell University
Original Assignee
Cornell University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cornell University filed Critical Cornell University
Publication of WO2022125520A1 publication Critical patent/WO2022125520A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
    • A01N25/32—Ingredients for reducing the noxious effect of the active substances to organisms other than pests, e.g. toxicity reducing compositions, self-destructing compositions
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/10—Animals; Substances produced thereby or obtained therefrom
    • A01N63/14—Insects

Definitions

  • the present invention generally relates to methods for detoxifying or preventing toxification of insect pollinators, such as bees.
  • the present invention more particularly relates to detoxifying or preventing toxification of insect pollinators by feeding the insect pollinators a composition that removes and/or degrades a toxifying compound, such as a pesticide toxic to the insect pollinators.
  • insecticides and other agrochemicals
  • Insecticides can have lethal and sub-lethal effects on pollinators, both at an individual and colony level, often through the impairment of vital neuronal pathways (J. Yao et al., Journal of Economic Entomology, 111, 4, August 2018, 1517-1525).
  • Other agrochemicals, such as fungicides can cause synergistic effects with other toxins by destroying beneficial gut bacteria, which are essential for defending against viruses, parasites and insecticides (A.
  • Organophosphate (OP) pesticides are heavily relied upon in agricultural production to prevent crop loss due to numerous types of insects.
  • OPs have a market of over $7 billion and account for more than a third of insecticide sales worldwide, and often lead to pollinator exposures and exhibit high toxicity towards honey bees and bumble bees (S. R. Rissato et al., Food Chem., 101, 1719-1726, 2007).
  • OP insecticides influence insect cholinergic neural signaling through inhibition of carboxyl ester hydrolases, particularly acetylcholinesterase (AChE) which breaks down acetylcholine.
  • AChE acetylcholinesterase
  • organophosphate pesticides include malathion, parathion, methyl parathion, chlorpyrifos, diazinon, dichlorvos, phosmet, fenitrothion, tetrachlorvinphos, azamethiphos, azinphos-methyl, azinphos-ethyl, and terbufos.
  • Malathion and parathion are the two of the most widely applied OPs in commercially pollinated crops. Malaoxon, malathion’s metabolite, is 1000-fold stronger at inhibiting AChE than malathion (O. P. Rodriguez et al., Bull. Environ. Contam.
  • insect pollinators such as bees (e.g., honey bees and bumble bees), as discussed above.
  • insect pollinators are critical for agriculture and farming, the use of such pesticides can result in a critical decline in pollination, which represents a threat to global food production and ecological balance.
  • the present invention provides a downstream solution to the persistent and pernicious problem of inadvertent pesticide toxification of insect pollinators, particularly bees.
  • a method is herein described for detoxifying insect pollinators that have ingested one or more pesticides.
  • the method more particularly involves feeding a detoxifying formulation to a community of insect pollinators, wherein the detoxifying formulation includes microparticles containing: i) an oil phase and (ii) insect cell membrane material.
  • the detoxifying formulation further includes: (iii) a surface active agent.
  • the oil phase may be or include a plant oil, such as one or more of coconut oil, olive oil, almond oil, avocado oil, com oil, cottonseed oil, flax seed oil, sesame seed oil, walnut oil, soybean oil, safflower oil, sunflower oil, palm oil, grape seed oil, lemon oil, and orange oil.
  • the oil phase occupies a core portion of the microparticle and the insect cell membrane material encapsulates the oil phase.
  • the insect cell membrane material can be derived from any insect.
  • the insect cell membrane material is derived from an insect in the superorder Polyneoptera (e.g., mantids or termites) or an insect in the order Orthoptera, (e.g., crickets, grasshoppers, and katydids).
  • Polyneoptera e.g., mantids or termites
  • Orthoptera e.g., crickets, grasshoppers, and katydids
  • the microparticles are provided to the insect pollinators in the form of a suspension of the microparticles in an aqueous medium, typically with an insect pollinator attractant included in the aqueous medium.
  • the method is typically practiced by placing the detoxifying aqueous suspension in a location accessible to the insect pollinators to permit the insect pollinators to ingest the detoxifying aqueous suspension.
  • FIG. 1 depicts a cross-section of the microsponge outer layer presenting the potential interactions of the different major pesticide groups; neonicotinoids, organophosphates, carbamates and pyrethroids with the membrane surface proteins nAChR, AChE and sodium-gated ion channels. Pyrethroids are shown being absorbed into the coconut oil core phase following interaction with the sodium-gated ion channel.
  • FIG. 2 is a schematic depicting microsponge fabrication, consumption and their subsequent passage through the bee digestive tract.
  • Microsponges are extracted into the midgut from the crop stomach where they are grouped with pollen grains. Pollen grains release contaminants during digestion which are specifically sequestered by the consumed microsponges, before the microsponges are excreted as feces.
  • FIGS. 3A-3C Fluorescent microscope images of the microsponge structure.
  • FIG. 3A is an image of a cell membrane stained using FITC-phalloidin.
  • FIG. 3B is an image of oil microspheres stained using Nile red.
  • FIG. 3C is an image overlay of FIGS. 3A and 3B.
  • FIGS. 4A-4E In vitro characterization of microsponge detoxification capacity. HPLC determination of paraoxon removal (FIG. 4A), malathion removal (FIG. 4B) and imidacloprid removal (FIG. 4C) by microsponges fabricated using differing membrane concentrations.
  • FIG. 4D shows the results of an assay assessing the OPT-catalyzed production of nitrophenol from paraoxon hydrolysis to confirm HPLC findings of paraoxon removal using differing microsponge concentrations.
  • FIG. 4E shows the results of an HPLC determination of paraoxon removal using differing microsponge concentrations.
  • FIGS. 5A-5B In vivo assessment of bumblebees.
  • FIG. 5A graph showing survival rates of bumblebees fed paraoxon-contaminated pollen balls (10 pg g -1 pollen) over 6 days and either microsponge treatments or plain sucrose. Survival was monitored over 120 hours to infer in vivo microsponge efficacy.
  • FIG. 5B depicts an exemplary apparatus for determining mortality following contaminated pollen ball consumption against microsponge treatment in syrup.
  • FIG. 6 Fluorescent microscope imaging of the nanosponge structure. Left panel: cell membrane stained using FITC-phalloidin. Middle panel: Oil microspheres stained using Nile red. Right panel: Overlay of left and middle panels. [0018] FIG. 7. Characterization of nanosponge using scanning electron microscopy (SEM) imaging. Image of nanosponge with successfully coated cell membrane (left panel) and image of nanosponge without membrane coating (right panel).
  • SEM scanning electron microscopy
  • FIG. 8 In vitro characterization of cell membrane (left panel) and oil concentration (right panel) influence on detoxification capacity. Paraoxon remaining following mixing with nanosponges was characterized via spectrophotometric analysis at 405 nm of paraoxon hydrolysis using phosphotriesterase.
  • FIG. 9 In vitro characterization of cell membrane coated and non-coated nanosponge detoxification capacity of a library of 11 pesticides; malathion, coumaphos, imidacloprid, clothianidin, acetamiprid, aldicarb, carbofuran, carbaryl, mancozeb, captan and propiconazole. Pesticide concentration was measured by HPLC analysis.
  • the present disclosure is directed to a composition for detoxifying insect pollinators that have ingested or otherwise internalized one or more pesticide compounds or substances.
  • compositions for detoxifying insect pollinators include detoxifying compositions, microparticles, microsponges, and nanosponges.
  • the term “pesticide,” as used herein, broadly includes any substance applied onto plants to improve the quality, growth, or product yield of the plants.
  • the pesticide generally possesses one or more properties of controlling or regulating agricultural or horticultural pests, wherein the pests may be crop-damaging insects, animals, fungi, or undesired plant life (e.g., invasive species or weeds).
  • pesticides are used for controlling or killing crop-damaging insects, agricultural pesticides are generally not intended for controlling or killing insect pollinators.
  • the pesticide may be, more specifically, an insecticide, herbicide, fungicide, or nematicide.
  • pesticides include neonicotinoids, pyrethroids, carbamates, organophosphates, organochlorides, butenolides, ryanoids, diamides, dinitrophenols, fluorine-containing insecticides, formamidines, insect growth regulators, isoxazoline- containing insecticides, macrocyclic lactones, nereistoxin and analogues thereof, oxadiazine-containing insecticides, oxadiazolone-containing insecticides, phthalimidecontaining insecticides, pyrazole-containing insecticides, pyrimidinamine insecticides, pyrrole-containing insecticides, quaternary ammonium insecticides, sulfoximines, tetramic acid insecticides, thiazole-containing insecticides, thiazolidine insecticides, and thioureacontaining insecticides.
  • one or more pesticides ingested in the insect pollinator are organophosphate pesticides.
  • organophosphate pesticides include malathion, parathion, methyl parathion, chlorpyrifos, diazinon, dichlorvos, phosmet, fenitrothion, tetrachlorvinphos, azamethiphos, azinphos-methyl, azinphos-ethyl, and terbufos.
  • a first component (component i) of the detoxifying composition is an oil phase.
  • the oil phase occupies a core portion of the microparticle.
  • the oil phase contains at least one type of oil or a mixture of at least two types of oils.
  • the oil may be any type of oil, provided that the oil is non-toxic to insect pollinators or other life forms.
  • the oil is a natural oil, but a non-toxic synthetic oil may also be used.
  • the oil phase may contain, for example, a mono-, di-, or tri-glyceride.
  • the oil phase is at least or more than 50 wt%, 60 wt%, 70 wt%, 80 wt%, or 90 wt% saturated, or the oil phase contains a level of saturation within a range bounded by any two of the foregoing values.
  • the oil phase is or contains a plant oil.
  • plant oils include coconut oil, olive oil, almond oil, avocado oil, com oil, cottonseed oil, flax seed oil, sesame seed oil, walnut oil, soybean oil, safflower oil, sunflower oil, palm oil, grape seed oil, lemon oil, and orange oil.
  • a second component (component ii) of the detoxifying composition is insect cell membrane material.
  • the insect cell membrane material can be derived from any insect.
  • the insect cell membrane material is derived from an insect in the superorder Polyneoptera (e.g., mantids or termites) or an insect in the order Orthoptera, (e.g., crickets, grasshoppers, and katydids).
  • the insect cell membrane material is at least partially purified by being substantially devoid of connective tissue material.
  • the insect cell membrane material generally possesses phospholipids and possibly other types of compounds known to form lipid bilayers and micellular structures.
  • the phospholipids and possibly other types of compounds found in the insect cell membrane material have the ability to encapsulate oil droplets and stabilize them as aqueous dispersions.
  • the oil phase occupies a core portion of the microparticle and the insect cell membrane material encapsulates the oil phase.
  • the insect cell membrane material comprises one or more membrane proteins including, but not limited to integral membrane proteins, transmembrane proteins, surface displayed membrane proteins, and other membrane proteins.
  • the membrane protein may include ion channel proteins, receptor proteins, and pore proteins.
  • the insect cell membrane material comprises one or more membrane proteins distinct from membrane proteins found in red blood cells.
  • the insect cell membrane material comprises one or more membrane proteins distinct from membrane proteins found in mammals.
  • the insect cell membrane material comprises insect-type nicotinic acetylcholine receptors (nAChRs), which are targets for insect-specific neonicotinoid insecticides.
  • nAChRs insect-type nicotinic acetylcholine receptors
  • the insect cell membrane material comprises one or more of insect-type nicotinic acetylcholine receptors (nAChRs), AChE (acetylcholinesterase receptor membrane protein) and sodium-gated ion channels.
  • insect cell membrane material comprises membrane proteins capable of selectively binding pesticides, including but not limited to neonicotinoids, organophosphates, carbamates and pyrethroids.
  • the detoxifying composition includes a surface active agent.
  • the surface active agent should be non-toxic to insect pollinators.
  • the surface active agent may be any substance known in the art to have a surface active property, i.e., surfactant ability, including any of the non-toxic surfactants known in the art.
  • the surface active agent may be, for example, a natural or synthetic polymer.
  • the surface active agent is a natural-based surfactant, such as a polypeptide (e.g., protein) or polysaccharide (sugar or carbohydrate).
  • polypeptide surface active agents include gelatin, collagen, fibrin, polylysine, and polyaspartate.
  • polysaccharide surface active agents include dextran, dextrose, starch, maltodextrin, chitosan, pectin, agarose, hemicellulose (e.g., xylan), alginate, carrageenan, guar gum, xanthan gum, locust bean gum, and cellulose gum.
  • the surface active agent may alternatively be amphiphilic by containing one or more hydrophilic portions and one or more hydrophobic sections.
  • amphiphilic surface active agents include sodium lauryl sulfate, alkylbenzene sulfonates, and lignin sulfonates.
  • synthetic polymers include polyvinyl alcohol, polyvinyl acetate, and polysorbate-type nonionic surfactants (e.g., polysorbate 80).
  • the surface active agent may alternatively be a non-ionic surfactant, which typically contains at least one polyalkylene oxide (hydrophilic) portion attached to a hydrophobic hydrocarbon portion.
  • the polyalkylene oxide (PAO) portion is typically polyethylene oxide (PEO), although polypropylene oxide (PPO), and poly butylene oxide (PBO) may also serve as the PAO.
  • the PAO typically includes at least or greater than 5, 10, 15, 20, 30, 40, or 50 alkylene oxide units.
  • the non-ionic surfactant may alternatively or in addition include one or more hydroxy (OH) or cyclic ether (e.g., tetrahydrofuran) groups per molecule.
  • the hydrocarbon portion is generally constructed solely of carbon and hydrogen atoms, except that one or more fluorine atoms may or may not be present.
  • the hydrocarbon portion may be or include one or more alkyl groups, alkenyl groups, cycloalkyl groups, and aromatic groups (e.g., phenyl).
  • the non-ionic surfactant includes a hydrocarbon group corresponding to a linear or branched hexyl, heptyl, octyl, nonyl, decyl, undecyl, or dodecyl group.
  • non-ionic surfactants include: (i) Triton® X-100 and Igepal® surfactants, which contain a (l,l,3,3-tetramethylbutyl)phenyl portion; (ii) polysorbate (Tween®) surfactants, such as polysorbate 80, which contain a polyethoxylated sorbitan moiety attached (typically via an ester bond) to a hydrocarbon group, such as an undecyl group; (iii) non-ionic triblock copolymers, also known as poloxamers, such as Pluronic® surfactants, which typically contain alternating PEO and PPO units, such as PEO-PPO-PEO and PPO-PEO-PPO surfactants; and (iv) Brij® surfactants, which contain a PEO portion attached to an alkyl portion (typically 12-20 carbon atoms).
  • Triton® X-100 and Igepal® surfactants which contain a (l,l,3,3
  • the above described components (i)-(ii) or (i)-(iii) are included as components of microparticles.
  • the microparticles are composed of at least or solely components (i)-(ii) or (i)-(iii).
  • the insect cell membrane material is dispersed throughout the oil, while in other embodiments, the insect cell membrane material forms an encapsulating coating around a micron-sized droplet of the oil, or the insect cell membrane material may be dispersed throughout the oil and also form an encapsulating coating around a micron-sized droplet of the oil.
  • the surface active agent may be in the oil core, insect cell membrane material shell, or both.
  • the insect cell membrane material has the ability to stabilize the oil core and maintain its micron size when the microparticles are dispersed in aqueous medium.
  • the microparticles may or may not include one or more additional components.
  • the microparticles further include an insect pollinator attractant admixed with components (i)-(ii) or (i)-(iii).
  • the microparticles further include pollen admixed with components (i)-(ii) or (i)-(iii).
  • the microparticles further include one or more nutrients for insect pollinators.
  • the one or more nutrients may be, for example, one or more carbohydrates (e.g., sugar or nectar), amino acids, vitamins, minerals, or lipids (e.g., fatty acids or sterols).
  • the microparticles typically have a size of at least 0.1 microns and up to 200 microns.
  • the size of the microparticles is generally substantially equivalent to the size of the oil core, except that the thickness of the insect cell membrane material, if surrounding the oil core, will increase the size of the microparticle.
  • the microparticles have a size of precisely, about, at least, up to, or less than 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 30, 40, 50, 100, 150, or 200 microns, or a size within a range bounded by any two of the foregoing values (e.g., 0.1-200 microns, 0.1-150 microns, 0.1-100 microns, 0.1-50 microns, 1-200 microns, 1-150 microns, 1-100 microns, 1-50 microns, 10-200 microns, 10- 150 microns, 10-100 microns, or 10-50 microns). In some embodiments, any range of microparticle sizes derivable from the above values may be excluded.
  • the microparticles may also possess an outer surface porosity, with the pores typically being nanosized, such as 1-500 nm or 1-100 nm in size.
  • the pores correspond to interstitial spaces within or between portions or segments of the insect cell membrane material.
  • the pores have a size of precisely, about, at least, greater than, up to, or less than, for example, 1, 2, 5, 10, 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 nm, or a pore size within a range bounded by any two of the foregoing values.
  • the present disclosure is directed to a detoxifying aqueous suspension containing any of the detoxifying microparticles described above suspended in an aqueous medium.
  • the aqueous medium may have an acidic, neutral, or alkaline pH.
  • the aqueous medium has an alkaline pH, such as a pH of at least or greater than 7, 7.5, 8, 8.5, 9, 9.5, or 10, or a pH within a range bounded by any two of the foregoing values.
  • the detoxifying aqueous suspension typically contains an insect pollinator attractant in the aqueous medium, the detoxifying microparticles, or both.
  • the insect pollinator attractant may be or include, for example, sucrose, a plant extract, fruit extract, or a pheromone.
  • the attractant may be present in an amount of, for example, 1-5 g/mL in the aqueous medium. However, in some embodiments, an attractant is not included.
  • the aqueous medium includes a surface active agent to help stabilize the suspension.
  • the aqueous medium may also include one or more auxiliary agents, such as, for example, a buffer, anti-bacterial agent, or nutrient appropriate for insect pollinators.
  • the suspended microparticles are mixed with pollen to form a macroscopic pollen ball, which is then administered to the insect pollinators in the same manner described above, such as in the form of an aqueous suspension.
  • the present disclosure is directed to a method for using the detoxifying composition to protect insect pollinators from the harmful effects of pesticides.
  • the insect pollinators are more specifically protected from the harmful effects of organophosphate pesticides.
  • the insect pollinators typically belong to the order Hymenoptera, such as bees (e.g., honey bees or bumble bees) or wasps.
  • the detoxifying composition in the form of microparticles or suspension thereof, as described above, is placed in a location accessible to the insect pollinators to permit the insect pollinators to ingest the detoxifying composition.
  • the pesticide Upon ingestion, the pesticide is absorbed from the gut of the insect pollinator into the oil phase of the detoxifying microparticles.
  • the method results in at least or above 50%, 60%, 70%, 80%, or 90% survival of the insect pollinators compared to insect pollinators administered the aqueous medium without the detoxifying microparticles.
  • the microparticles are provided to the insect pollinators in the form of a suspension of the microparticles in an aqueous medium, as described above, typically with an insect pollinator attractant included in the aqueous medium.
  • the attractant may be present in the aqueous medium in an amount of, for example, 1-5 g/mL in the external aqueous medium.
  • the insect pollinator attractant may be or include, for example, sucrose, a plant extract, fruit extract, or a pheromone.
  • the method is typically practiced by placing the detoxifying aqueous suspension in a location accessible to the insect pollinators to permit the insect pollinators to ingest the detoxifying aqueous suspension.
  • the microparticles are spray dried with a protective coating to result in a further stabilization of the encapsulated oil core.
  • the spray dried microparticles are further capable of retaining the integrity of the original microparticles when suspended in an aqueous medium.
  • the spray dried coating preferably has the property of at least partially dissolving or degrading when in the digestive tract of the insect pollinator.
  • the spray dry coating is disposed as a layer covering the insect cell membrane material surrounding each oil phase core.
  • the spray dry coating is any of the well known compositions used for this purpose in the pharmaceutical industry.
  • the spray dry coating is crosslinked for additional stability.
  • the spray dry coating material may be, for example, a polysaccharide (e.g., chitosan or maltodextrin) or a protein (e.g., gelatin) or combination thereof, or any of the compositions provided above for the surface active agent.
  • the spray dry coating is insoluble at neutral pH and/or at least partially soluble at acid pH.
  • the resulting spray dried microparticles necessarily have a particle size larger than the original microparticles.
  • the particle size of the spray dried particles may be at least 0.1 microns and up to 1000 microns.
  • the spray dried microparticles have a size of precisely, about, at least, or greater than 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 30, 40, 50, 100, 150, 200, 500, or 1000 microns, or a size within a range bounded by any two of the foregoing values (e.g., 0.1-1000 microns, 0.1-500 microns, 0.1- 200 microns, 0.1-100 microns, 1-1000 microns, 1-500 microns, 1-200 microns, 1-100 microns, 10-1000 microns, 10-500 microns, 10-200 microns, 10-100 microns, 100-1000 microns, or 100-500 microns.
  • cricket Acheta domesticus
  • coconut oil microcores that can be suspended in sucrose to perform broad-spectrum pesticide detoxification in bees. These materials were selected based on several design considerations. Crickets present a scalable source of insect cells as crickets are produced in high volumes from insect farms for food production. Further, the derived cell membranes from insect cells express insect-type nicotinic acetylcholine receptors (nAChRs), which are targets for insect-specific neonicotinoid insecticides.
  • nAChRs insect-type nicotinic acetylcholine receptors
  • microsponge core because of its natural origins and high saturated fat content which results in strong emulsion stability.
  • the bimodal microsponge design combines the specific binding capacity of a cricket cell membrane, with the non-specific absorption of coconut oil.
  • the microsponges can advantageously be suspended in sucrose to form an emulsion with the addition of the non-toxic surfactant Tween 80.
  • FIG. 1 depicts a cross-section of an exemplary microsponge outer layer showing the potential interactions of different pesticide groups (neonicotinoids, organophosphates, carbamates, and pyrethroids) with the membrane surface proteins nAChR, AChE and sodium-gated ion channels.
  • pesticide groups neonicotinoids, organophosphates, carbamates, and pyrethroids
  • nAChR membrane surface proteins
  • AChE sodium-gated ion channels
  • FIG. 2 is a schematic depicting fabrication of an exemplary microsponge, followed by consumption and their subsequent passage through the insect pollinator digestive tract.
  • Microsponges are extracted into the midgut from the crop stomach where they are grouped with pollen grains. Pollen grains release contaminants (e.g., pesticides) during digestion, but such contaminants are sequestered by the consumed microsponges before the microsponges are excreted as feces.
  • contaminants e.g., pesticides
  • Microsponge fabrication House crickets (Acheta domesticus)' were euthanized via freezing and subsequently homogenized in a 50 ml centrifuge tube containing a metal ball bearing via vigorous shaking for 10 seconds. The product was filtered and collected in an ice bath through a 200 pm sieve followed by a 100 pm cell filter to remove connective tissues. The filtrate was centrifugated at 3,200rpm for 5 minutes and the supernatant discarded.
  • the pellet was resuspended with lysis buffer (25 mM HEPES, 100 mM NaCl, 1 mM EDTA, 10% (v/v) glycerol, 1% (v/v) Triton X-100) and incubated for 10 minutes on ice.
  • the solution was centrifugated again at 3,200 rpm for 5 minutes and the was pellet washed with 0.9% (w/v) saline. This step was repeated two further times to leave a purified solution of cell membrane in saline. Protein concentration was then determined using a BCA protein assay kit.
  • a volume of Tween 80 was added to the solution to form a final concentration of 2% (v/v) once diluted with sucrose.
  • a volume of liquified coconut oil was subsequently added to form a volume ratio of 2:1 cell membrane:sucrose. The mixture was blended vigorously for 20 seconds and diluted with sucrose to form 0.5 mg/mL coconut oil in solution.
  • Microsponge visualization Microsponges were prepared as previously mentioned with the exception of staining purified cell membranes with FITC-phalloidin for 24 hours and staining liquid coconut oil with Nile red (Ipg/mL) dissolved inside. Stained microsponges were then visualized under CLSM.
  • Microsponges were prepared with varying concentrations of cell membrane protein (0, 0.1, 0.4, 1.6, 2 mg/mL), 2 mL of 0.5 mg/mL microsponges were mixed with 2 mL of the following insecticides; 0.5mM paraoxon, 0.44mM malathion, 0.5mM imidacloprid, and left for 10 minutes at room temperature. Samples were centrifugated and the aqueous solution was collected for HPLC analysis.
  • Microsponges were prepared as usual with the exception of mixing varying oil concentrations: 0.21, 0.31, 0.47 and 0.71 g/mL. 2 mL of microsponges were mixed with 2 mL 0.5mM paraoxon and left for 10 minutes at room temperature. Samples were centrifugated and the aqueous phase was collected for HPLC analysis. In addition, the aqueous phase was mixed with 0.5 mg/mL phosphotriesterase and immediately read under a spectrophotometric plate reader at 405 nm to quantify nitrophenol production. Absorbance readings were analyzed against a standard curve readings from known paraoxon concentrations.
  • the culture was subsequently centrifuged (10 minutes, 4,000 rpm), the supernatant was discarded, and the pellet consisting of cells was resuspended in 40 mL resuspension buffer (3.15g Tris-HCl, 29.22g NaCl, 56g glycerol, 44 pL CoCh (IM), 144 mg imidazole, IL H2O).
  • the suspension was sonicated (65% amplitude, 5 s on, 25 s off, 20 minutes total) in an ice bath.
  • the suspension then underwent further centrifugation (1.5 hours, 13,000 rpm) and the supernatant containing crude OPT was collected.
  • Crude OPT was purified using a HIS-select, NTA-nickel bead affinity column. The column was successively equilibrated using an equilibration buffer (20 rnM phosphate buffer, 300 mM NaCl, 10 rnM imidazole), before protein was fed through the column and washed with further equilibration buffer. Captured OPT was then eluted with elution buffer (20 mM phosphate buffer, 300 mM NaCl, 250 mM imidazole). The eluted protein was concentrated using Amicon Ultra 15 mL 3 kDa-membrane tubes and subsequently washed with saline three times. OPT concentration was determined using a Bicinchoninic acid assay (BCA) assay kit. Confirmation of OPT production was carried out via SDS-PAGE.
  • BCA Bicinchoninic acid assay
  • Pollen balls were prepared by mixing 5 mL of 20 pg/mL paraoxon, with 10g of high desert bee pollen granules. The mixture was shaken until a homogeneous slurry was formed, then left at room temperature to allow full absorption of paraoxon. The contaminated pollen was then crushed in a pestle and mortar. The mixture containing pollen and sucrose was rolled by hand into equally sized 3 g pollen balls. Treatments were prepared by diluting microsponges sucrose (1 g/mL) to 0.5 mg/mL oil concentrations.
  • Cricket cell membranes were isolated first by homogenizing frozen crickets in saline solution and filtering the mixture through a 100 pm filter to remove unwanted connective tissues. The subsequent solution was centrifugated, the supernatant containing unwanted cell debris discarded and the pellet resuspended with a cell lysis solution (10 mM Tris-HCl, pH 7.5, 1% Triton-X, 150mM NaCl). The solution then underwent three further centrifugation steps followed each time by washing with saline to purify a solution of cell membranes. The microsponge fabrication process was optimized by trialing varying cell membrane concentrations.
  • a Bicinchoininic acid assay BCA assay was performed on the cell membrane solution to determine protein concentration and used this quantification as a proxy to set the membrane concentrations.
  • Cell membranes were then mixed with a volume of Tween 80 that formed a final surfactant concentration of 2% following microsponge suspension in sucrose.
  • Liquid coconut oil was added to the mixture at varying oil concentrations: 0.21, 0.31, 0.47 and 0.71 g/mL and blended vigorously using a vortex for three minutes.
  • the resultant emulsion was diluted in 100% sucrose to 4% (v/v) microsponges, a concentration based on the volumes of coconut oil and cell membrane.
  • the cell membrane acts as a further agent to stabilize the sucrose and oil phases.
  • the emulsion suspension stability remained stable for at least two weeks following dilution in sucrose.
  • FIGS. 4A-4E show in vitro characterization of microsponge detoxification capacity.
  • HPLC was used to determine level of paraoxon removal (FIG. 4A), malathion removal (FIG. 4B) and imidacloprid removal (FIG. 4C) by microsponges fabricated using differing membrane concentrations.
  • FIG. 4D shows the results of an assay assessing the OPT-catalyzed production of nitrophenol from paraoxon hydrolysis to confirm HPLC findings of paraoxon removal using differing microsponge concentrations.
  • FIG. 4E shows the results of an HPLC determination of paraoxon removal using differing microsponge concentrations. Removal of 0.44 mM malathion increased two-fold by increasing membrane concentrations from 0 to 2 mg/mL. Removal of 0.4 mM paraoxon increased 4-fold by increasing oil concentrations from 0.21 to 0.71 mg/mL.
  • FIGS. 3A-3C are fluorescent microscope images of the microsponge structure.
  • FIG. 3A is an image of a cell membrane stained using FITC-phalloidin.
  • FIG. 3B is an image of oil microspheres stained using Nile red.
  • FIG. 3C is an image overlay of FIGS. 3A and 3B.
  • FIGS. 5A-5B show in vivo assessment of bumblebees.
  • FIG. 5A shows survival rates of groups fed paraoxon-contaminated pollen balls and either microsponge treatments or plain sucrose. Survival was monitored over 120 hours to infer in vivo microsponge efficacy.
  • FIG. 5B depicts an exemplary apparatus for determining mortality following contaminated pollen ball consumption against microsponge treatment in syrup.
  • microsponges retain detoxification functionality following consumption and that the microsponges divert sequestered toxins away from their intended targets.
  • the emulsion was then combined in a crosslinking bath of 100 ml Sodium Tripolyphosphate (TPP) 3% (w/w) and stirred for 10 minutes. Under continuous stirring, the emulsion was then fed into an Ollital SS-2000 spray dryer at an inlet temperature of 180°C, peristaltic pump set to 30, needle spray set to 4.0 s and fan set to 100. The subsequent powder product was then collected and stored.
  • TPP Sodium Tripolyphosphate
  • Nanosponge visualization Nanosponges were prepared as previously described, with the exception of Cell membranes were stained with FITC-phalloidin for 24 hours, olive oil was stained with Nile red (Ipg/mL). Nanosponges were then prepared as previously described without spray drying. Stained nanosponges were then visualized under CLSM.
  • Nanosponges were prepared without spray drying with varying concentrations of cell membrane protein (0, 0.1, 0.4, 1.6, 2 mg/mL). 2 mL of 0.5 mg/mL nanosponges were mixed with 2 ml of the following insecticides; 0.5 mM paraoxon, 0.44 mM malathion, 0.5 mM imidacloprid, and left for 10 minutes at room temperature. Samples were centrifuged and the aqueous solution was collected for HPLC analysis.
  • Nanosponges were prepared without spray drying and with the exception of mixing varying oil concentrations: 0.21, 0.31, 0.47 and 0.71 g/mL. 2 mL of nanosponges were mixed with 2 mL 0.5 mM paraoxon and left for 10 minutes at room temperature. Samples were centrifuged and the aqueous phase was mixed with 0.5 mg/mL phosphotriesterase and immediately read under a spectrophotometric plate reader at 405 nm to quantify nitrophenol production. Absorbance readings were analyzed against standard curve readings from known paraoxon concentrations.
  • the culture was subsequently centrifuged (10 minutes, 4,000 rpm), the supernatant was discarded, and the pellet consisting of cells was resuspended in 40 mL resuspension buffer (3.15 g Tris-HCl, 29.22 g NaCl, 56 g glycerol, 44 pL CoCh (IM), 144 mg imidazole, IL H2O).
  • the suspension was sonicated (65% amplitude, 5 s on, 25 s off, 20 minutes total) in an ice bath.
  • the suspension then underwent further centrifugation (1.5 hours, 13,000 rpm) and the supernatant containing crude OPT was collected.
  • Crude OPT was purified using a HIS-select, NTA-nickel bead affinity column. The column was successively equilibrated using an equilibration buffer (20 mM phosphate buffer, 300 mM NaCl, 10 mM imidazole), before protein was fed through the column and washed with further equilibration buffer. Captured OPT was then eluted with elution buffer (20 mM phosphate buffer, 300 mM NaCl, 250 mM imidazole). The eluted protein was concentrated using Amicon Ultra 15 mL 3 kDa-membrane tubes and subsequently washed with saline three times. OPT concentration was determined using a Bicinchoninic acid assay (BCA) assay kit. Confirmation of OPT production was carried out via SDS-PAGE.
  • BCA Bicinchoninic acid assay
  • Nanosponges were prepared without spray drying. 2 mL of 0.5 mg/mL nanosponges were mixed with 2 ml of the following pesticides at 0.5mM: malathion, coumaphos, imidacloprid, clothianidin, acetamiprid, aldicarb, carbofuran, carbaryl, mancozeb, captan, and propiconazole, and left for 10 minutes at room temperature. Samples were centrifuged and the aqueous solution was collected for HPLC analysis.
  • Pollen balls were prepared by mixing 5 mL of 20 pg/mL paraoxon, with 10 g of high desert bee pollen granules. The mixture was shaken until a homogeneous slurry was formed, then left at room temperature to allow full absorption of paraoxon. The contaminated pollen was then crushed in a pestle and mortar. The mixture containing pollen and sucrose was rolled by hand into equally sized 3 g pollen balls. Treatments were prepared by diluting nanosponges (1 g/mL) to 0.5 mg/mL oil concentrations.
  • Groups of 10 bumblebees (Bombus impatiens) were placed in microcolony rearing cages and treated with a 10 pg/g contaminated pollen ball, and 5 mL of either plain sucrose or a nanosponge solution in a centrifugal tube with a small aperture for feeding. Microcolonies were monitored every 12 hours for mortalities until 6 days had elapsed.
  • This work is based on an adaptation of the nanosponge fabrication process using cricket (Acheta domesticus) cell membranes and olive oil nanocores, spray dried using a crosslinked chitosan-gelatin polymer shell, to perform broad-spectrum pesticide detoxification in bees. These materials were selected based on several design considerations. Crickets present a scalable source of insect cells, as crickets are produced at volume for food production. Further, the derived cell membranes express insect-type nicotinic acetylcholine receptors (nAChRs) which are targets for insect- specific neonicotinoid insecticides.
  • nAChRs insect-type nicotinic acetylcholine receptors
  • pyrethroids and organophosphates can also be diverted through their respective targets found in the cell membrane: sodium-gated ion channels and acetylcholinesterase.
  • oils are adept in capturing major pesticide groups such as organophosphates, carbamates, and pyrethroids, which display extreme lipophilicity; and eonicotinoids display moderate lipophilicity.
  • Chitosan and gelatin are biocompatible polymers that can be used in combination during spray drying to encapsulate oils. Both polymers are insoluble in neutral water at room temperature, yet will readily dissolve around pH 4.8. It was herein hypothesized that spray drying using these wall materials would form oil-encapsulated nanoparticles, capable of retaining integrity in a solid state, when suspended in water at room temperature. When the nanoparticles reached the digestive tract of a bee ( ⁇ pH 4.8), the chitosan-gelatin shell would begin to partially dissolve.
  • Gelatin is an appropriate polymer for spray drying because in addition to its aforementioned properties, it serves as both an emulsifying and film forming agent.
  • Chitosan has been widely used as a vehicle for loading drugs or biologies due to its cationic charge. It was herein further hypothesized that the chitosan component of the shell is able to form in complex with the anionic plasma cell membrane, forming a functional shell layer, capable of identifying pesticides.
  • Olive oil was utilized as the nanosponge core because of its high biocompatibility and reasonably strong Hansen solubility parameter.
  • the bimodal nanosponge design combines the specific binding capacity of a cricket cell membrane, with the non-specific absorption of olive oil.
  • FIG. 9 shows in vitro characterization of cell membrane coated and non-coated nanosponge detoxification capacity of a library of 11 pesticides; malathion, coumaphos, imidacloprid, clothianidin, acetamiprid, aldicarb, carbofuran, carbaryl, mancozeb, captan and propiconazole. Pesticide concentration was measured by HPLC analysis.
  • nanosponges were subsequently removed from the suspension and the mass of remaining pesticides was detected to determine the percentage captured. It was found that the nanosponges were able to successfully remove over 50% of the mass of 7 pesticides. In all cases, membrane coated nanosponges removed a greater mass of pesticides relative to noncoated oil nanodroplets, with 6 out of 11 examples exhibiting a statistically significant difference. At least one pesticide from each of the pesticide groups tested showed a significantly improved capture efficiency using membrane coated nanoparticles relative to uncoated nanoparticles. This indicates the membrane was able to support the varying mechanisms of action of the pesticide groups tested.
  • nanosponges were effective in capturing paraoxon within the gut of the bumblebee before paraoxon was able to interact with the brain or nervous system. In this way, the nanosponges provided a successful and safe diversion for the neurotoxin.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Dentistry (AREA)
  • Plant Pathology (AREA)
  • Pest Control & Pesticides (AREA)
  • Wood Science & Technology (AREA)
  • Agronomy & Crop Science (AREA)
  • Environmental Sciences (AREA)
  • Toxicology (AREA)
  • Insects & Arthropods (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Virology (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

L'invention concerne une composition pour détoxifier des insectes pollinisateurs d'un ou de plusieurs pesticides, la composition contenant des microparticules comprenant : (I) une phase huileuse et (ii) un matériau de membrane cellulaire d'insecte et, éventuellement, (iii) un agent tensioactif. Une suspension aqueuse comprenant les microparticules détoxifiantes décrites ci-dessus dans un milieu aqueux, qui peut également contenir un attractif pour les insectes pollinisateurs, est également divulguée. L'invention concerne également un procédé de détoxification d'insectes pollinisateurs d'un ou de plusieurs pesticides, le procédé comprenant le placement de la suspension aqueuse détoxifiante dans un emplacement accessible aux insectes pollinisateurs pour permettre aux insectes pollinisateurs d'ingérer la suspension aqueuse détoxifiante, la suspension aqueuse détoxifiante comprenant des microparticules, telles que décrites ci-dessus, en suspension dans un milieu aqueux, comprenant typiquement aussi un attractif pour insectes pollinisateurs dans le milieu aqueux.
PCT/US2021/062163 2020-12-08 2021-12-07 Microparticules de membrane cellulaire d'insectes pour la détoxification d'insectes pollinisateurs Ceased WO2022125520A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202063122698P 2020-12-08 2020-12-08
US63/122,698 2020-12-08

Publications (1)

Publication Number Publication Date
WO2022125520A1 true WO2022125520A1 (fr) 2022-06-16

Family

ID=81974807

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2021/062163 Ceased WO2022125520A1 (fr) 2020-12-08 2021-12-07 Microparticules de membrane cellulaire d'insectes pour la détoxification d'insectes pollinisateurs

Country Status (1)

Country Link
WO (1) WO2022125520A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009011808A1 (fr) * 2007-07-13 2009-01-22 President And Fellows Of Harvard College Sélection basée sur des gouttelettes
WO2014012099A1 (fr) * 2012-07-13 2014-01-16 Tufts University Encapsulation de fragrance et/ou d'arômes dans des biomatières de fibroïne
US20150217024A1 (en) * 2012-08-08 2015-08-06 Nanyang Technological University Methods of manufacturing hydrogel microparticles having living cells, and compositions for manufacturing a scaffold for tissue engineering

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009011808A1 (fr) * 2007-07-13 2009-01-22 President And Fellows Of Harvard College Sélection basée sur des gouttelettes
WO2014012099A1 (fr) * 2012-07-13 2014-01-16 Tufts University Encapsulation de fragrance et/ou d'arômes dans des biomatières de fibroïne
US20150217024A1 (en) * 2012-08-08 2015-08-06 Nanyang Technological University Methods of manufacturing hydrogel microparticles having living cells, and compositions for manufacturing a scaffold for tissue engineering
US9974886B2 (en) * 2012-08-08 2018-05-22 Nanyang Technological University Methods of manufacturing hydrogel microparticles having living cells, and compositions for manufacturing a scaffold for tissue engineering

Similar Documents

Publication Publication Date Title
Abdollahdokht et al. Conventional agrochemicals towards nano-biopesticides: an overview on recent advances
Oliveira et al. Nanopesticide based on botanical insecticide pyrethrum and its potential effects on honeybees
Wang et al. Enhanced antibacterial activity of eugenol-entrapped casein nanoparticles amended with lysozyme against gram-positive pathogens
Ale et al. Nanotechnology-based pesticides: Environmental fate and ecotoxicity
EP3849308B1 (fr) Composition de spores et champignons entomopathogènes, et son utilisation
JP2021528484A (ja) 病原体防除組成物及びその使用
Pinto et al. Micro-and nanocarriers for encapsulation of biological plant protection agents: A systematic literature review
JP2018503694A (ja) 高効力活性薬剤の封入
US20210307320A1 (en) Microbial microcapsule compositions, methods and related methods
Ahmed et al. Effect of Lambda-Cyahalothrin as nanopesticide on cotton leafworm, Spodoptera littoralis (Boisd.)
Jampílek et al. Bioactivity of nanoformulated synthetic and natural insecticides and their impact on environment
Graily-Moradi et al. Nanoinsecticides: preparation, application, and mode of action
de Assis et al. Therapeutic potential of Melaleuca alternifolia essential oil in new drug delivery systems
Paiva Filho et al. Design of chitosan-alginate core-shell nanoparticules loaded with anacardic acid and cardol for drug delivery
Cortesi et al. Natural antimicrobials in spray-dried microparticles based on cellulose derivatives as potential eco-compatible agrochemicals
Habood et al. Nanochitosan to control the red palm weevil Rhynchophorus ferrugineus (Olivier)(Coleoptera: Curculionidae)
WO2022125520A1 (fr) Microparticules de membrane cellulaire d'insectes pour la détoxification d'insectes pollinisateurs
CN103975919A (zh) 一种爪哇棒孢霉菌分生孢子微胶囊的制备方法
Mishra et al. Bio-based nanoemulsions: An eco-safe approach towards the eco-toxicity problem
Adetuyi et al. Nanobioinsecticide and Nanoemulsions: Recent Advances
Gamal Eldin et al. Efficacy of thymol nanoemulsion against Varroa destructor mites infesting Apis mellifera colonies under the stress of abiotic factors in Egypt
Hosny et al. Nanocarrier-Based Formulation and Encapsulation of Imidacloprid: Design and Toxicological Assessment on Spodoptera littoralis Larvae Using Chitosan-Dextran Sulfate
FR2999096A1 (fr) Capsule a membrane etanche aux liquides et permeable aux gaz, procede de fabrication et utilisation pour l'elevage d'arthropodes in vitro
US6248321B1 (en) Encapsulation of microparticles in teardrop shaped polymer capsules of cellular size
US11607424B2 (en) Carbon-rich micro-particles for protecting bees and overcoming colony collapse disorder

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21904230

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21904230

Country of ref document: EP

Kind code of ref document: A1