WO2022125520A1 - Insect cell membrane microparticles for detoxification of insect pollinators - Google Patents

Abstract

A composition for detoxifying insect pollinators of one or more pesticides, the composition containing microparticles comprising: (i) an oil phase and (ii) insect cell membrane material, and optionally (iii) a surface active agent. Also disclosed herein is an aqueous suspension comprising the above-described detoxifying microparticles in an aqueous medium, which may also contain an insect pollinator attractant. Also described herein is a method for detoxifying insect pollinators of one or more pesticides, the method comprising placing the detoxifying aqueous suspension in a location accessible to the insect pollinators to permit the insect pollinators to ingest the detoxifying aqueous suspension, wherein the detoxifying aqueous suspension comprises microparticles, as described above, suspended in an aqueous medium, typically also including an insect pollinator attractant in the aqueous medium.

Description

INSECT CELL MEMBRANE MICROPARTICLES FOR DETOXIFICATION OF INSECT POLLINATORS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority from U.S. Provisional Application No. 63/122,698, filed on December 8, 2020, which is herein incorporated by reference in its entirety.

GOVERNMENT SUPPORT

[0002] This invention was made with government support under grant number 2017-18-107 awarded by the National Institute of Food and Agriculture, US Department of Agriculture, Hatch. The government has certain rights in the invention.

FIELD OF THE INVENTION

[0003] 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.

BACKGROUND OF THE INVENTION

[0004] One-third of U.S. crops are dependent on managed and native bees for sustained production, yield, and quality. Pollinators contribute $24 billion to the U.S. economy, of which honeybees are responsible for over $15 billion (The White House Archives, 2014, Fact Sheet: The Economic Challenge Posed By Declining Pollinator Populations, Washington D.C.: Office of the Press Secretary). However, bee populations are rapidly declining (Kulhanek, K., et al., Journal Of Apicultural Research, 56(4), 328-340, 2017. https://doi.org/10.1080/00218839.2017.1344496). Beekeepers lose on average one-third of colonies each winter and more than 700 U.S. native bee species are now at risk of extinction (The Center for Biological Diversity, 2017. Pollinators In Peril. Portland Oregon).

Between 2013-2019, the U.S. beekeeping industry spent $2 billion, a third of its income, on replacing 10 million hives (Amadeo, K. (2019). Bee Colony Collapse Disorder and Its Impact on the Economy. The Balance). The loss of managed colonies has caused a rise in pollination fees for many crop farmers. Growers are currently facing diminished crop yields as a result of poor pollination from weakened colonies.

[0005] The application of insecticides, and other agrochemicals is considered to be a major cause of managed and native bee population loss (D. Goulson et al., Science, 347, 10, 2015). 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. Iverson et al., Apidologie, 50, 733-744, 2019). In the case of social bees, chemicals can be transported back into the hive within pollen and nectar and accumulate within wax, developing brood and other bee castes (S. McArt et al., Sci. Rep., 7, 46554, 2017). Research has shown over the course of a year up to 93 different foreign compounds accumulated within a colony and that the number of pesticides was a strong predictor of colony death (K. Traynor et al., Sci. Rep., 6, 33207, 2016).

[0006] Organophosphate (OP) pesticides, in particular, 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. OPs inactivate AChE through irreversible covalent inhibition, causing a build-up of acetylcholine and overstimulation of nicotinic and muscarinic receptors (J. V. Peter et al., Indian J. Crit. Care Med., 18, 735, 2014).

[0007] Some examples of 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. Toxicol., 58, 171-176, 1997). Malathion and parathion exhibit oral LDso’s of 0.38 and 0.175 pg/bee respectively (C. D. S. Tomlin, The Insecticide Manual: A World Compendium, British Crop Production Council, ISBN 9781901396188, 2009).

[0008] Although these pesticides are useful in mitigating the damage caused by agricultural pests, they unfortunately also have an adverse effect on insect pollinators, such as bees (e.g., honey bees and bumble bees), as discussed above. As 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.

SUMMARY OF THE INVENTION

[0009] The present invention provides a downstream solution to the persistent and pernicious problem of inadvertent pesticide toxification of insect pollinators, particularly bees. To achieve the solution, 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. In some embodiments, the detoxifying formulation further includes: (iii) a surface active agent.

[0010] 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. In some embodiments, 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. In particular embodiments, 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).

[0011] Typically, 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. BRIEF DESCRIPTION OF THE FIGURES

[0012] 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.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] 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).

[0019] 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.

[0020] 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.

DETAIEED DESCRIPTION OF THE INVENTION

[0021] In one aspect, 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. Some examples of 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). As noted earlier above, although 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.

[0022] Some examples of 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. In particular embodiments, one or more pesticides ingested in the insect pollinator are organophosphate pesticides. Some examples of organophosphate pesticides include malathion, parathion, methyl parathion, chlorpyrifos, diazinon, dichlorvos, phosmet, fenitrothion, tetrachlorvinphos, azamethiphos, azinphos-methyl, azinphos-ethyl, and terbufos.

[0023] A first component (component i) of the detoxifying composition is an oil phase. In some embodiments, 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. Typically, 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. In some embodiments, 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. In particular embodiments, the oil phase is or contains a plant oil. Some examples of 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.

[0024] 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. In particular embodiments, 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). Typically, 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. In some embodiments, for each microparticle, the oil phase occupies a core portion of the microparticle and the insect cell membrane material encapsulates the oil phase.

[0025] In some embodiments, 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. In some embodiments, the insect cell membrane material comprises one or more membrane proteins distinct from membrane proteins found in red blood cells. In some embodiments, the insect cell membrane material comprises one or more membrane proteins distinct from membrane proteins found in mammals. In some embodiments, the insect cell membrane material comprises insect-type nicotinic acetylcholine receptors (nAChRs), which are targets for insect-specific neonicotinoid insecticides. In some embodiments, 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. In some embodiments, the insect cell membrane material comprises membrane proteins capable of selectively binding pesticides, including but not limited to neonicotinoids, organophosphates, carbamates and pyrethroids.

[0026] In some embodiments, 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. In some embodiments, the surface active agent is a natural-based surfactant, such as a polypeptide (e.g., protein) or polysaccharide (sugar or carbohydrate). Some examples of polypeptide surface active agents include gelatin, collagen, fibrin, polylysine, and polyaspartate. Some examples of 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. Some examples of amphiphilic surface active agents include sodium lauryl sulfate, alkylbenzene sulfonates, and lignin sulfonates. Some examples of synthetic polymers include polyvinyl alcohol, polyvinyl acetate, and polysorbate-type nonionic surfactants (e.g., polysorbate 80).

[0027] 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. As part of the hydrophilic portion, 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). In some embodiments, the non-ionic surfactant includes a hydrocarbon group corresponding to a linear or branched hexyl, heptyl, octyl, nonyl, decyl, undecyl, or dodecyl group. Some examples of 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).

[0028] The above described components (i)-(ii) or (i)-(iii) are included as components of microparticles. The end result is that the microparticles are composed of at least or solely components (i)-(ii) or (i)-(iii). In some embodiments, 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.

[0029] The microparticles may or may not include one or more additional components. In one embodiment, the microparticles further include an insect pollinator attractant admixed with components (i)-(ii) or (i)-(iii). In another embodiment, the microparticles further include pollen admixed with components (i)-(ii) or (i)-(iii). In another embodiment, 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).

[0030] 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. In different embodiments, 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.

[0031] 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. Typically, the pores correspond to interstitial spaces within or between portions or segments of the insect cell membrane material. In different embodiments, 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.

[0032] In another aspect, 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. In particular embodiments, 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. At least when being used to administer to insect pollinators, 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. In some embodiments, 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. In some embodiments, 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.

[0033] In another aspect, the present disclosure is directed to a method for using the detoxifying composition to protect insect pollinators from the harmful effects of pesticides. In some embodiments, 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. In the method, 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. Upon ingestion, the pesticide is absorbed from the gut of the insect pollinator into the oil phase of the detoxifying microparticles. In some embodiments, 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.

[0034] Typically, 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.

[0035] In some embodiments, 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. However, the spray dried coating preferably has the property of at least partially dissolving or degrading when in the digestive tract of the insect pollinator. In the case where the insect cell membrane material encapsulates the oil phase in each microparticle, the spray dry coating is disposed as a layer covering the insect cell membrane material surrounding each oil phase core. In some embodiments, the spray dry coating is any of the well known compositions used for this purpose in the pharmaceutical industry. In some embodiments, 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. In some embodiments, the spray dry coating is insoluble at neutral pH and/or at least partially soluble at acid pH.

[0036] After spray drying, 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. In different embodiments, 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. In some embodiments, any range of spray dried microparticle sizes derivable from the above values may be excluded.

[0037] Examples have been set forth below for the purpose of illustration and to describe the best mode of the invention at the present time. However, the scope of this invention is not to be in any way limited by the examples set forth herein.

Examples

[0038] Overview

[0039] The following experiments describe a nanosponge fabrication process using cricket (Acheta domesticus) cell membranes and 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. Other more generalist pesticides, such as pyrethroids and organophosphates, can also be diverted through their respective targets found in the cell membrane: sodium- gated ion channels and acetylcholinesterase. In addition, oils are adept in capturing major pesticide groups such as organophosphates, carbamates, and pyrethroids, which display extreme lipophilicity; neonicotinoids display moderate lipophilicity. Coconut oil was utilized as the 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. Herein is described the successful preparation of microsponges from cricket cell-membranes and coconut oil. The microsponges can advantageously be suspended in sucrose to form an emulsion with the addition of the non-toxic surfactant Tween 80.

[0040] 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. Notably, pyrethroids are shown being absorbed into the coconut oil core phase following interaction with the sodium-gated ion channel.

[0041] 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.

[0042] Methods

[0043] 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. Having diluted the solution to a desired protein concentration, in a vortex blender, 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.

[0044] 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.

[0045] In vitro performance using differing cell membrane concentrations. 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.

[0046] In vitro performance using differing oil concentrations. 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.

[0047] Phosphotriesterase synthesis. Ampicillin, chloramphenicol, and IPTG solutions were sterilized before use. E. coli containing pQE30-PTE was cultured using Miller Grade LB broth with 100 pg/mL ampicillin and 25 pg/mL chloramphenicol under 37°C. Once cultures in 5,000 mL flasks attained an OD of 0.4, 500 pL CoCh (IM) and at OD 0.8-1.0, 500 pL IPTG (200 mg/mL) was supplemented per liter of culture. The flask was left to culture for three further hours. 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.

[0048] Mortality testing. 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. 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 microsponge solution in a centrifugal tube with a small aperture for feeding. Microcolonies were monitored every 12 hours for mortalities until 6 days had elapsed.

[0049] Results and Discussion

[0050] 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. Therefore, 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.

[0051] The detoxification capacity of the emulsions improved with increasing cell membrane and oil concentrations. 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.

[0052] To visualize the successful formation of micro-scale cores encapsulated in membranes, membrane solutions were stained using FITC-phalloidin and coconut oil using Nile red dye before preparing microsponges as previously mentioned. Confocal laser scanning microscopy (CLSM) imaging displayed a core-shell structure to confirm the formation of microscale oil droplets surrounded by a layer of cell membrane (FIGS. 3A- 3C). 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.

[0053] Preliminary tests were conducted to determine in vivo microsponge efficacy by assessing mortality in treated and untreated groups of bumbles bees (Bonibus impatiens). 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. As shown, groups of bees treated with pollen balls containing 15 pg/g paraoxon displayed improved mortality when treated with 4% (v/v) of microsponges in 100% sucrose. Groups without microsponge treatment experienced 100% mortality after 5 days, whereas microsponge treated groups experienced only 73% mortality. This data demonstrates that the microsponges retain detoxification functionality following consumption and that the microsponges divert sequestered toxins away from their intended targets.

[0054] Methods for Spray Drying Experiments

[0055] Preparation of cell membrane isolate. House crickets (Acheta domesticns) and a working buffer (50mM TRIS at pH 7.4 containing 150mM NaCl and 2.55mg/ml Trehalose), 1:2 cricket to buffer mass ratio, were homogenized in a Ninja BN701 food blender at low speed for 30 seconds. The homogenate was filtered in three stages, first through a 1/16 inch sieve, followed by a 160 pm filter bag and finally a 75 pm filter bag. The filtrate was then fed once through a M- 11 OP Microfludics™ Microfluidizer to lyse cells. The solution was then centrifuged at 2500g for 20 minutes, before the supernatant was collected as a cell membrane isolate. Protein concentration was then determined via BCA protein assay kit. Membrane protein was then diluted to desired concentrations using the working buffer.

[0056] Preparation of spray-dried pH responsive, cell-membrane coated nanosponges. A 0.5% (w/w) chitosan solution was prepared in 0.05M acetic acid, before the solution pH was adjusted to 7.4 using NaOH. A 3% (w/w) gelatin solution was prepared using DI water heated to 50°C. Then at room temperature, 9 ml cell membrane isolate, 100 ml gelatin solution, 400 ml chitosan solution, 5 ml olive oil and 3 ml Polysorbate 80 were combined, before mechanically emulsifying using a Huxi JRJ-300SH. 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.

[0057] 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.

[0058] In vitro performance using differing cell membrane concentrations. 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. 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.

[0059] In vitro performance using differing oil concentrations. 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.

[0060] Phosphotriesterase synthesis. Ampicillin, chloramphenicol, and IPTG solutions were sterilized before use. E. coli containing pQE30-PTE was cultured using Miller Grade LB broth with 100 pg/mL ampicillin and 25 pg/mL chloramphenicol under 37°C. Once cultures in 5,000 mL flasks attained an OD of 0.4, 500 pL CoCh (IM) and at OD 0.8-1.0, 500 pL IPTG (200 mg/mL) was supplemented per liter of culture. The flask was left to culture for three further hours. 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.

[0061] In vitro performance using a library of pesticides. 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.

[0062] Mortality testing. 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.

[0063] Spray Dried Formulations

[0064] 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. Other more generalist pesticides, such as pyrethroids and organophosphates, can also be diverted through their respective targets found in the cell membrane: sodium-gated ion channels and acetylcholinesterase. In addition, oils are adept in capturing major pesticide groups such as organophosphates, carbamates, and pyrethroids, which display extreme lipophilicity; and eonicotinoids display moderate lipophilicity.

[0065] Finally, a picketing emulsion process was employed using chitosan and gelatin, followed by spray drying, to improve the stability and versatility of the design as a solid powder. 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. This would reveal the oil-membrane core-shell complex whilst offering ‘nano-buffering’ protection of cell membrane proteins in the gastric environment. 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.

[0066] 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.

[0067] Characterization of spray dried nanosponges. The experimentation depicted in this manuscript has used the nanosponge fabrication protocol without the spray drying wall step using chitosan and gelatin. To visualize the successful coating of cell membranes on nanosize oil droplets, each of the cell membrane and oil components was stained with FITC- phalloidin and Nile Red, respectively. Fluorescent imaging presented the successful fabrication of a homogenous dispersion of oil droplets with reasonable size distribution (FIG. 6). The nano-sized droplets displayed colocalization of the oil and cell membrane fluorescence. Successful coating was confirmed by scanning electron microscopy (FIG. 7). In samples fabricated with cell membranes, a core-shell structure could be visualized, consistent with the encapsulation of an oil droplet within a thin membrane layer, whereas nanosponges fabricated without cell membranes visually lacked this outer layer component.

[0068] In vitro capture of paraoxon. To first optimize oil and cell membrane concentration for optimal capture of pesticides, paraoxon was used as a model pesticide as its concentration in a solution can easily be measured by quantifying nitrophenol, a byproduct of paraoxon hydrolysis, using the enzyme phosphotriesterase. Having mixed solutions of nanosponges with increasing cell membrane and increasing oil concentrations, a significant positive correlation was observed between increased paraoxon removal and increased concentrations of both design components (FIG. 8). From this, cell membrane concentrations at a protein concentration of 2 mg/ml and 0.71 g/ml concentration of oil were used in further investigations. This also indicates that both the oil and cell membrane components of the design each contribute to the capture of paraoxon, as a reduction in the concentration of either of these components would reduce the efficiency of capture.

[0069] The pesticide capture of a larger library of pesticides was subsequently investigated in vitro using an HPLC analysis. The aim was to determine whether the design was able to capture pesticides that are less lipophilic than the organophosphate paraoxon. A further aim was to gauge the efficiency of pesticide capture for pesticides that interact with cells via differing modes of action. 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.

[0070] Upon incubating various 0.5 mM solutions of pesticides with nanosponges, 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. All organophosphate and fungicide pesticides assessed had data points of over 50% pesticide mass removed, as well as two out of three carbamate pesticides. All neonicotinoids tested, and the carbamate aldicarb, had relatively lower capture efficiencies, with the majority of data points lying under 50% removal. Generally speaking, pesticides with a higher octanol-water partition coefficient, (high values indicate an affinity for diffusing into a hydrophobic phase), were captured more efficiently by the nanosponge design relative to pesticides with coefficient low values.

[0071] In vivo performance. To investigate the nanosponge’s potential to prevent mortality in pollinators, groups of 40 bumblebees (B. impatiens) were simultaneously fed an acutely lethal dose of paraoxon in pollen balls (lOpg g^-1 pollen), as well as suspension of nanosponges in sucrose. The negative control group received a sucrose solution without nanosponges. Bumblebees in the negative control group promptly died off, less than half of bumblebees remained alive after two days. All bees had died within 5 days. In the nanosponge fed group however, 80% of bees remained alive after 6 days, despite continuous paraoxon exposure. This data suggested that the 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.

[0072] While there have been shown and described what are at present considered the preferred embodiments of the invention, those skilled in the art may make various changes and modifications which remain within the scope of the invention defined by the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A composition comprising an aqueous suspension of microparticles wherein each microparticle comprises:

(i) an oil phase; and

(ii) insect cell membrane material; wherein the microparticles are suspended in an aqueous medium.

2. The composition of claim 1 , wherein component (i) occupies a core portion of each microparticle and component (ii) encapsulates component (i) in each microparticle.

3. The composition according to any one of claims 1-2, wherein said oil phase comprises a mono-, di-, or tri-glyceride.

4. The composition according to any one of claims 1-3, wherein said oil phase is at least 50 wt% saturated.

5. The composition according to any one of claims 1-4, wherein said oil phase comprises a plant oil.

6. The composition of claim 5, wherein said plant oil is selected from the group consisting 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.

7. The composition according to any one of claims 1-6, wherein said insect cell membrane material is derived from an insect in the order Orthoptera.

8. The composition according to any one of claims 1-7, wherein said insect cell membrane material is cricket cell membrane.

9. The composition according to any one of claims 1-8, wherein the insect cell membrane material comprises one or more membrane proteins.

10. The composition of claim 9, wherein the one or more membrane proteins comprise insect- type nicotinic acetylcholine receptors (nAChRs).

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11. The composition according to any one of claims 1-10, further comprising: (hi) a surface active agent.

12. The composition of claim 11, wherein said surface active agent is a non-ionic surfactant.

13. The composition of claim 11, wherein said surface active agent is a polysorbate-type non-ionic surfactant.

14. The composition of claim 13, wherein said surface active agent is polysorbate 80.

15. The composition of claim 11, wherein said surface active agent is a non-ionic surfactant containing at least one polyalkylene oxide group.

16. The composition according to any one of claims 1-15, wherein said microparticles have a size in a range of 0.1-100 microns.

17. The composition according to any one of claims 1-15, wherein said microparticles have a size in a range of 1-100 microns.

18. The composition according to any one of claims 1-17, wherein said aqueous medium contains an insect pollinator attractant.

19. The composition of claim 18, wherein said insect pollinator attractant is sucrose.

20. The composition of claim 19, wherein said sucrose is present in a concentration of 1-5 g/mL in said aqueous medium.

21. The composition according to any one of claims 1-18, wherein each microparticle is encapsulated with a spray dry coating.

22. The composition of claim 21, wherein the spray dry coating comprises chitosan and gelatin.

23. A method of detoxifying insect pollinators from one or more pesticides, the method comprising placing a detoxifying aqueous suspension in a location accessible to the insect pollinators to permit the insect pollinators to ingest the detoxifying aqueous suspension, wherein said detoxifying aqueous suspension comprises microparticles suspended in an aqueous medium containing an insect pollinator attractant, wherein said microparticles contain (i) an oil phase; and (ii) insect cell membrane material.

24. The method of claim 23, wherein component (i) occupies a core portion of each microparticle and component (ii) encapsulates component (i) in each microparticle.

25. The method according to any one of claims 23-24, wherein said pesticide is selected from the group consisting of 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, phthalimide-containing insecticides, pyrazole- containing insecticides, pyrimidinamine insecticides, pyrrole-containing insecticides, quaternary ammonium insecticides, sulfoximines, tetramic acid insecticides, thiazole- containing insecticides, thiazolidine insecticides, and thiourea-containing insecticides.

26. The method according to any one of claims 23-25, wherein said insect pollinators comprise the order Hymenoptera.

27. The method of claim 26, wherein said insect pollinators are bees.

28. The method according to any one of claims 23-27, wherein said insect pollinator attractant is sucrose.

29. The method of claim 28, wherein said sucrose is present in a concentration of 1-5 g/mL in said external aqueous medium.

30. The method according to any one of claims 23-29, wherein said method results in at least 50% survival of the insect pollinators compared to insect pollinators administered said aqueous medium without said microparticles.

31. The method according to any one of claims 23-30, wherein said oil phase comprises a mono-, di-, or tri-glyceride.

32. The method according to any one of claims 23-31, wherein said oil phase has an iodine value of no more than 100.

33. The method according to any one of claims 23-32, wherein said oil phase comprises a plant oil.

34. The method according to any one of claims 23-33, wherein said plant oil is selected from the group consisting 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.

35. The method according to any one of claims 23-34, wherein said insect cell membrane material is derived from an insect in the order Orthoptera.

36. The method according to any one of claims 23-35, wherein said insect cell membrane is cricket cell membrane.

37. The method according to any one of claims 23-36, wherein the insect cell membrane material comprises one or more membrane proteins.

38. The method of claim 37, wherein the one or more membrane proteins comprise insect- type nicotinic acetylcholine receptors (nAChRs).

39. The method according to any one of claims 23-38, further comprising: (iii) a surface active agent.

40. The method of claim 39, wherein said surface active agent is a non-ionic surfactant.

41. The method of claim 39, wherein said surface active agent is a polysorbate-type non-ionic surfactant.

42. The method of claim 41, wherein said surface active agent is polysorbate 80.

43. The method of claim 39, wherein said surface active agent is a non-ionic surfactant containing at least one polyalkylene oxide group.

44. The method according to any one of claims 23-43, wherein said microparticles have a size in a range of 0.1-100 microns.

45. The method according to any one of claims 23-43, wherein said microparticles have a size in a range of 1-100 microns.

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46. The method according to any one of claims 23-45, wherein each microparticle is encapsulated with a spray dry coating.

47. The composition of claim 46, wherein the spray dry coating comprises chitosan and gelatin.

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