Patent Application
20190098895
The present disclosure relates to agriculture and specifically to formulations for delivery of agriculturally active agents.
References considered to be relevant as background to the presently disclosed subject matter are listed below:
Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter.
In agriculture delivery of active agents is at times hampered by the presence of the cuticle layer that prevents the agent from penetration into the plant's vascular system and delivery of same to the various plant parts.
U.S. Pat. No. 4,394,149 describes liposomes encapsulating biologically active material. The liposomes are formed by mixing lipid or organic solvent with aqueous solution of the biologically active material, emulsifying the mixture, removing the solvent and suspending the gel in water.
U.S. Pat. No. 5,958,463 describes a method for preparing boron containing liposomes for agricultural use. The method involves mixing lecithin with organic solvent in specific proportions. After allowing the mixture to settle the top layer is saved while the bottom layer is discarded. Next the active agent is added to form a concentrate. When the concentrate is added to water the vesicle is formed.
U.S. Pat. No. 6,165,500 describes the preparation of liposomes that comprise a lipid and surfactant (referred to as transfersomes) for transporting medical agent through membranes. The transfersomes were shown to penetrate into the surface of leaves which resulted in a slightly reddish appurtenance at the surface of the leaves.
Finally, International patent application publication No. WO 13/192190 describes liposomal formulation for agricultural use, that comprise as active ingredient pesticides, nematicides, or herbicides. The formulation is applied to the soil or to the plant media.
The present disclosure provides, in accordance with a first of its aspects, a formulation comprising (i) liposomes comprising a lipid membrane and an intraliposomal aqueous core, wherein the liposome has a diameter in the range of between 100 nm to 300 nm; and the lipid membrane comprises at least one liposome forming phospholipid; and (ii) an agriculturally acceptable carrier.
In some embodiments, the present disclosure provides a formulation comprising:
In accordance with a second aspect, the present disclosure provides a method of treatment, comprising applying the formulation disclosed herein. In some embodiments, the method is for treating a plant and comprises applying to a surface of a plant part a formulation as disclosed herein.
Also disclosed herein is the use of a formulation as disclosed herein for agriculture.
In accordance with a third aspect, the present disclosure provides a kit comprising (a) an agriculturally acceptable carrier; (b) liposomes or liposome forming lipids as defined herein; and (c) instructions for use of the carrier and liposomes for treating a plant.
In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:
The present disclosure is based on the development of a liposomal formulation, applied onto leaves of a plant, that was effective in distributing an agent encapsulated within the intraliposomal core of the liposome into various plant parts, including the apical shoot, stem and roots.
Thus, the present disclosure provides, in accordance with its first aspect, a formulation comprising (i) liposomes comprising a lipid membrane and an intraliposomal aqueous core, wherein the liposome has a diameter in the range of between 100 nm to 300 nm; and the lipid membrane comprises at least one liposome forming phospholipid; and (ii) an agriculturally acceptable carrier.
In accordance with some embodiments, the present disclosure provides, a liposomal formulation comprising within a carrier suitable for agricultural use, liposomes comprising a lipid membrane and an intraliposomal aqueous core, wherein
Liposomes are sealed sacs in the micron and submicron range dispersed in an aqueous environment in which one or more bilayers (lamellae) separate the external aqueous phase from the internal aqueous phase. The bilayer is composed of amphiphiles, the latter having defined polar and apolar regions. When amphiphiles are present in an aqueous phase, they self-aggregate such that their hydrophilic moiety faces the aqueous phase, while their hydrophobic domain is “protected” from the aqueous phase.
As a prerequisite in order to form liposomes, amphiphiles must be organized in a lamellar phase. However, the formation of lamellar phases is not sufficient to lead to liposome formation [Seddon, J. M., Biochemistry, 29(34):7997-8002, (1990)]. Liposome formation also requires the ability of the lamellae to close up on themselves to form vesicles.
Various approaches were proposed to classify amphiphiles into sub-groups. One approach is based on geometric and energetic parameters of amphiphiles. According to this approach proposed by Israelachvili and co-workers [Israelachvili, J, Physical principles of membrane organization, Q. Rev Biophys, 13(2):121-200 (1980), Lichtenberg and Barenholz, In Methods of Biochemical Analysis, D. Glick (Ed), 33:337-462, 1988; Kumar, Biophys J., 88:444-448, (1991)] amphiphiles are defined by a packing parameter (PP), which is the ratio between the cross sectional areas of the hydrophobic and hydrophilic regions.
In the context of the present disclosure, the liposomes are used as a carrier for agriculturally beneficial agents and to this end, various types of liposomes can be used. Specifically, yet without being limited thereto, the liposomes of the disclosed formulation can be any one or combination of vesicles selected from the group consisting of small unilamellar vesicles (SUV), large unilamellar vesicles (LUV), multilamellar vesicles (MLV), multivesicular vesicles (MVV), large multivesicular vesicles (LMVV, also referred to, at times, by the term giant multivesicular vesicles, “GMV”), oligolamellar vesicles (OLV), and others.
In some embodiments, the liposomes are large unilameller vesicles (LUV).
It has been found by the inventors that there is a significance, for the purpose of, inter alia, liposome penetration and/or distribution within the plant, to use a liposomal population having an average diameter in the range of 100 to 300 nm. In some embodiments, the liposomes have an average diameter in the range of 100 nm to 250 nm, at times, in the range of 110 nm to 230 nm, at times, in the range of 120 nm to 220 nm, at times, at least 100 nm but no more than 200 nm.
The lipid membrane comprises two or more phospholipids, at least one of which is a liposome forming lipid. It is noted in this connection that the amount of phospholipids in the liposome can be determined as organic phosphorous by the modified Bartlett method [Shmeeda H, Even-Chen S, Honen R, Cohen R, Weintraub C, Barenholz Y. 2003. Enzymatic assays for quality control and pharmacokinetics of liposome formulations: comparison with nonenzymatic conventional methodologies. Methods Enzymol 367:272-92].
As used herein, the “liposome forming lipids” are firstly phospholipids which when dispersed in aqueous media by itself at a temperature above their solid ordered to liquid disordered phase transition temperature (Tm, the temperature in which the maximal change in heat capacity occurs during the phase transition) will form stable liposomes.
In some embodiments, the phospholipids are selected from glycerophospholipids and sphingomyelins. The glycerophospholipids have a glycerol backbone wherein at least one, preferably two, of the hydroxyl groups at the head group is substituted by one or two hydrocarbon tails (chains), typically, an acyl, alkyl or alkenyl tails, and the third hydroxyl group is substituted by a phosphate (phosphatidic acid) or a phospho-ester such as phosphocholine group (as exemplified in phosphatidylcholine), being the polar head group of the glycerophospholipid or combination of any of the above, and/or derivatives of same and may contain a chemically reactive group (such as an amine, acid, ester, aldehyde or alcohol). The sphingomyelins consists of a ceramide (N-acyl sphingosine) unit having a phosphocholine moiety attached to position 1 as the polar head group. The term “sphingomyelin” or “SPM” as used herein denotes any N-acetyl sphingosine conjugated to a phosphocholine group, the later forming the polar head group of the sphingomyelin (N-acyl sphingosyl phospholcholines). The acyl chain bound to the primary amino group of the sphingosine (to form the ceramide) may be saturated or unsaturated, branched or unbranded.
In some embodiments, at least one of the liposome forming lipid is a phospholipid having one or two C14 to C24 hydrocarbon tails, typically, acyl, alkyl or alkenyl chain) and have varying degrees of unsaturation, from being fully saturated to being fully, partially or non-hydrogenated lipids (the level of saturation may affect rigidity of the liposome thus formed (typically liposomes formed from lipids with saturated chains are more rigid than liposomes formed from lipids of same chain length in which there are un-saturated chains, especially having cis double bonds).
Further, the lipid membrane may be of natural source (e.g. naturally occurring phospholipids), semi-synthetic or fully synthetic lipid, as well as electrically neutral, negatively or positively charged.
Examples of liposome forming glycerophospholipids include, without being limited thereto, phosphatidylglycerols (PG) including dimyristoyl phosphatidylglycerol (DMPG); phosphatidylcholine (PC), including egg yolk phosphatidylcholine, soybean PC, sunflower PC, rapeseed PC, hill PC, canola PC, flax seed lecithin, wheat lecithin, dimyristoyl phosphatidylcholine (DMPC, Tm 24° C.), 1-palmitoyl-2-oleoylphosphatidyl choline (POPC), hydrogenated soy phosphatidylcholine (HSPC, Tm 65° C.), distearoylphosphatidylcholine (DSPC, Tm 55° C.); di-lauroyl-sn-glycero-2phosphocholine (DLPC); 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, Tm 41° C.); 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine; 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC); 1,2-dihenarachidoyl-sn-glycero-3-phosphocholine; 1,2-dibehenoyl-sn-glycero-3-phosphocholine 1,2-ditricosanoyl-sn-glycero-3-phosphocholine 1,2-dilignoceroyl-sn-glycero-3-phosphocholine; 1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine; 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC); 1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (SPPC); 1,2-di-oleoyl-sn-glycero-3-phosphocholine (DOPC Tm −17° C.); phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE).
In some embodiments, the at least one liposome forming lipid has a choline head group.
In some embodiments, the at least one liposome forming lipid is a phosphatidylcholine (PC) carrying one or two saturated or unsaturated C14 to C24 hydrocarbon tails, or at times or two saturated or unsaturated C14 to C20 hydrocarbon tails, or at times or two saturated or unsaturated C16 to C20 hydrocarbon tails, or at times one or two saturated or unsaturated C16 or C18 hydrocarbon tails.
In some embodiments, the lipid membrane comprises a combination of PC's carrying at least one hydrocarbon tail selected from the group consisting of C16:0, C18:0, C18:1, C18:2, and C18:3.
In some embodiments, the lipid membrane comprises at least one unsaturated C16 or C18 PC.
In some embodiments, the lipid membrane comprises a mole ratio between saturated and non-saturated liposome forming lipids of between 10%:90% to 90%:10%, at times, a mole ratio of between 20%:80% to 80%:20%, at times, a mole ratio between 30%:70% to 70%:30%, at times, a mole ratio between 20%:80% to 50%:50%, at times, a mole ratio of between 20:80 to 40%:60%.
In some embodiments, at least one of said two or more phospholipids comprise at least one unsaturated hydrocarbon tail, namely, at least one double bond in the hydrocarbon chain. At times, the unsaturated hydrocarbon chain can comprise two, three, four or more double bonds.
In some embodiments, at least one of said two or more phospholipids is a lipid comprising a polar head group.
In the context of the present disclosure, when referring to a polar head group it is to be understood as one encompassing an alcohol moiety.
In some embodiments, the polar head group is one comprising a serine moiety.
In some embodiments, the polar head group is one comprising a choline moiety.
In some embodiments, the polar head group is one comprising ethanolamine.
In some embodiments, the polar head group is one comprising glycerol.
In some embodiments, at least one of said two or more phospholipids comprise a polar inositol head group. In some embodiments, the phospholipid comprising an inositol head group is selected from the group consisting of phospatidylinositol (PI), PI(4)P, PI(3)P, PI(3,4,5)P3, PI(4,5)P2, PI(3,5)P2, PI(3,4)P2.
In some embodiments, at least one of said two or more phospholipids has an acidic head group.
In some embodiments, when referring to an “acidic head group” it is to be understood as encompassing a moiety selected from the group consisting of glycerol, hydroxyl, carboxyl, amine, and phosphoric group.
Examples of the acidic phospholipids include natural or synthetic lipid selected from phosphatidylglycerols (PGs) such as dilauroylphosphatidylglycerol (DLPG) dimyristoylphosphatidylglycerol (DMPG) dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG) dioleoylphosphatidylglycerol (DOPG), egg yolk phosphatidylglycerol (egg yolk PG), hydrogenated egg yolk phosphatidylglycerol; phosphatidylinositols (PIs) such as phosphatidylinositol, dimyristoylphosphatidylinositol, dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol (DSPI), dioleoylphosphatidylinositol (DOPI), soybean phosphatidylinositol (soybean PI), hydrogenated soybean phosphatidylinositol, phosphoinositides, sphingomyelin and phosphatidic acid. Each of these acidic phospholipids can be used alone or in combination of two or more.
In some embodiments, the acidic head group comprises a phosphatidic acid. In some embodiments, the liposomes in the disclosed formulation comprise within the lipid membrane at least one non-liposome forming lipid.
When referring to a non-liposome forming lipid it is to be understood as referring to a lipid that does not spontaneously form into a vesicle when brought into an aqueous medium.
There are various types of lipids that do not spontaneously vesiculate and yet are used or can be incorporated into vesicles. These include, for example, sterols, saponins, sphingolipids, e.g. sphingomyelin, lipoproteins, e.g. PEG-DSPE, etc.
Such additional lipids can be used to affect any one of the stability, surface charge and membrane fluidity, as well as assist in the loading of active agents into the liposomes.
In some embodiments, the non-liposome forming lipid is a sterol.
A non-limiting list of sterols that can be part of the lipid membrane of the liposomes includes β-sitosterol, β-sitostanol, stigmasterol, stigmastanol, campesterol, campestanol, ergosterol, avenasterol, brassicasterol, fucosterol, cholesterol (CHOL), cholesteryl hemisuccinate, and cholesteryl sulfate.
In some embodiments, the sterol is a plant derived sterol, namely, a phytosterol. In accordance with this embodiment, the sterol is selected from the group consisting of β-sitosterol, β-sitostanol, stigmasterol, stigmastanol, campesterol, campestanol, ergosterol, avenasterol, brassicasterol and any combination of two or more of these sterols.
In some further embodiments, the lipid membrane comprises one or more phytosterols selected from the group consisting of β-sitosterol, stigmasterol, and ergosterol.
Another non-liposome forming lipid that can form part of the lipid membrane is a hydrophobic aglycone or a saponin. Structurally, saponins contain a hydrophobic aglycone and a hydrophilic glycoside (sugar) head group.
Saponins can be used as an alternative to sterols or they can be used in combination with sterols. In some embodiments, the lipid membrane comprises a combination of at least one sterol and at least one saponin.
When the lipid membrane comprise a combination of one or more sterol and one or more saponins, the mole % ratio between the phospholipids in the lipid membrane and the sterols and saponins in the lipid membrane is between 20 mol %:80 mol % to 80 mol %:20 mol %. At times, the mole ratio between the phospholipids and the sterols and saponins is between 40 mol %:60 mol % to 60 mol %:40 mol %. At times, the mole ratio between the phospholipids and the sterols and saponins is between 50 mol %:50 mol % to 80 mol %:20 mol %.
In some embodiments, the saponin is selected from the group consisting of dammaranes, tirucallanes, lupanes, hopanes, oleananes, taraxasteranes, ursanes, cycloartanes, lanostanes, cucurbitanes, solanine, solanidine, tomatine, chaconine, tomatidine and steroids, with or without a linked sugar moiety.
In some embodiments, the saponin is selected from the group consisting of solanine, solanidine, tomatine, chaconine, tomatidine and any combination of same.
The ratio between the liposome forming lipid and the non-liposome forming lipid can vary depending on the desired effect of the non-liposome forming lipids on the membrane. At times, the lipid membrane comprises a liposome forming lipid to non-liposome forming lipid mole ratio of between 20%:80% to 80%:20%, at times, a mole ratio of between 30%:70% to 70%:30%, at times, a mole ratio of between 40%:60% to 60%:40%.
In some embodiments, the lipid membrane comprises at least one cationic lipid (monocationic or polycationic lipids). Cationic lipids typically consist of a lipophilic moiety, such as a sterol or the same glycerol backbone to which two acyl or two alkyl, or one acyl and one alkyl chain contribute the hydrophobic region of the amphipathic molecule, to form a lipid having an overall net positive charge.
Monocationic lipids may include, for example, 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP) 1,2-dioleyloxy-3-(trimethylamino) propane (DOTAP); N-[1-(2,3,-ditetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE); N-[1-(2,3,-dioleyloxy)propyl]-N,N-dimethyl-N-hydroxy ethyl-ammonium bromide (DORIE); N-[1-(2,3-dioleyloxy) propyl]-N,N,N-trimethylammonium chloride (DOTMA); 3β-[N—(N′,N′-dimethylaminoethane) carbamoly] cholesterol hydrochloride (DC-Chol); and dimethyl-dioctadecylammonium (bromide salt, DDAB), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MLV), 1,2-di-O-octadecenyl-3-trimethylammonium propane (chloride salt) (DOTMA), Ethyl PC, 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ)
Polycationic lipids, due to their large polycationic head group may, at times, be considered as non-liposome forming lipids. Such lipids, when mixed with other lipids such as sterols and saponins together with liposome forming phospholipids at suitable mole ratio will be incorporated into the lipid membrane of the liposomes. The polycationic lipids include a similar lipophilic moiety as with the mono cationic lipids, to which polycationic head groups are covalently attached such as the polyalkyamines spermine or spermidine. The polycationic lipids include, without being limited thereto, N-[2-[[2,5-bis[3-aminopropyl)amino]-1-oxopentyl]amino]ethyl]-N,N-dimethyl-2,3-bis[(1-oxo-9-octadecenyl)oxy]-1-propanaminium (DOSPA), and ceramide carbamoyl spermine (CCS). The cationic lipids may form part of a derivatized phospholipids such as the neutral lipid dioleoylphosphatidyl ethanolamine (DOPE) derivatized with polylysine to form a cationic lipopolymer.
The liposomes may further comprise lipopolymers. The term “lipopolymer” is used herein to denote a lipid substance modified by inclusion in its polar head group a hydrophilic polymer. The polymer head group of a lipopolymer is typically water-soluble. Typically, the hydrophilic polymer has a molecular weight equal or above 750 Da. There are numerous polymers which may be attached to lipids to form such lipopolymers, such as, without being limited thereto, polyethylene glycol (PEG), polysialic acid, polylactic (also termed polylactide), polyglycolic acid (also termed polyglycolide), apolylactic-polyglycolic acid, polyvinyl alcohol, polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline, polyhydroxyethyloxazoline, polyhydroxypropyloxazoline, polyaspartamide, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, polyvinylmethylether, polyhydroxyethyl acrylate, derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose. The polymers may be employed as homopolymers or as block or random copolymers. The lipids derivatized into lipopolymers may be neutral, negatively charged, as well as positively charged.
The most commonly used and commercially available lipids derivatized into lipopolymers are those based on phosphatidyl ethanolamine (PE), usually, distearoylphosphatidylethanolamine (DSPE).
One particular family of lipopolymers that can be employed according to the present disclosure are the monomethylated PEG attached to DSPE (with different lengths of PEG chains, in which the PEG polymer is linked to the lipid via a carbamate linkage resulting in a negatively charged lipopolymer, or the neutral methyl polyethyleneglycol distearoylglycerol (mPEG-DSG) and the neutral methyl poly ethyleneglycoloxy carbonyl-3-amino-1,2-propanediol distearoylester (mPEG-DS) [Garbuzenko O. et al., Langmuir. 21:2560-2568 (2005)]. Another lipopolymer is the phosphatidic acid PEG (PA-PEG).
The PEG moiety can have a molecular weight of the head group from about 750 Da to about 20,000 Da, at times, from about 750 Da to about 12,000 Da and typically between about 1,000 Da to about 5,000 Da. While the lipids modified into lipopolymers may be neutral, negatively charged, as well positively charged, i.e. there is not restriction to a specific (or no) charge. For example, the neutral distearoyl glycerol and the negatively charged distearoyl phosphatidylethanolamine, both covalently attached to methoxy poly(ethylene glycol) (mPEG or PEG) of Mw 750, 2000, 5000, or 12000 [Priev A, et al. Langmuir 18, 612-617 (2002); Garbuzenko O., Chem Phys Lipids 135, 117-129 (2005); M. C. Woodle and D D Lasic Biochim. Biohys. Acta, 113, 171-199. 1992].
The most commonly used and commercially available lipids derivatized into lipopolymers are those based on phosphatidyl ethanolamine (PE), usually, distearylphosphatidylethanolamine (DSPE). A specific family of lipopolymers employed by the invention include methoxy PEG-DSPE (with different lengths of PEG chains) in which the PEG polymer is linked to the DSPE primary amino group via a carbamate linkage. The PEG moiety preferably has a molecular weight of the head group is from about 750 Da to about 20,000 Da. More preferably, the molecular weight is from about 750 Da to about 12,000 Da and most preferably between about 1,000 Da to about 5,000 Da. One specific PEG-DSPE that can be employed herein is that wherein PEG has a molecular weight of 2000 Da, designated herein 2000PEG-DSPE or 2kPEG-DSPE [M. C. Woodle and D D Lasic Biochim. Biohys. Acta, 113, 171-199. 1992].
In some embodiments, the liposomes contain up to 5 mole % lipopolymer. In some embodiments, the liposome is either lipopolymer free or contains between 0.1 mole % to 5 mole %, at times, between 0.5 mole % to 4 mole %, at times between 1 mole % to 3 mole %.
In some embodiments, the liposome contains at least one anti-oxidant. In some embodiments, the anti-oxidant is within the lipid membrane.
In some embodiments, the anti-oxidant is selected from the group consisting of α-tocopherol, tocotrienols, tocopherol succinate, tocopherol acetate, ascorbyl palmitate, coenzyme Q10 (ubiquinone), vitamin A, bioflavonoids, carotenoids, sodium escorbate, glutathione.
In some embodiments, the anti-oxidant is α-tocopherol.
In some embodiments, the liposome comprise a targeting moiety exposed at the external surface of the liposomes. In some embodiments, the targeting moiety is a low molecular weight compound, a protein, a peptide or a glycoprotein linked embedded to at least the outer surface of the liposomes.
In some embodiments, the liposomes can comprise, embedded in the lipid membrane, a protein that can facilitate specific plant organ targeting or penetration.
In some embodiments, the liposome can comprise other hydrophobic and/or other lipids or combination of lipids such as glycosphingolipids (i.e., gangliosides), and phosphatidyl ethanolamines (PE). Such groups would typically have a functional group extending from the liposome membrane, the exposed groups may then be used, for example, as a targeting moiety. Examples of such exposed groups may include, without being limited thereto, sugars (glycolipid), polymers (lipopolymer), proteins (lipoprotein).
In some embodiments, the lipid membrane comprises lipids that are essentially all from natural source. In some embodiments, the lipid membrane comprises lipids that are all from plant source.
When referring to a lipid or lipids from plant source it is to be understood that the lipid(s) is isolated from a plant or from a part of a plant (e.g. the seeds) such that when applied onto a plant as part of the formulation disclosed herein, no plant hyper sensitive response is launched.
In some embodiments, natural/plant derived lipids can be obtained from vegetable sources like, e.g., seed oil (from soybeans, rape (canola), wheat germ, sunflower, flax, cotton, corn, coconut, arachis, sesame), pulp oil (palm, olive, avocado pulp), desert shrub, tobacco, bean, and carrot. These raw materials are world-wide produced at very large scale. Natural phospholipids may be further converted to saturated phospholipids by means of hydrogenation or further treated with enzymes to, e.g., remove partially fatty acids (e.g. using phospholipase A2) or to convert a polar head group (e.g. using phospholipase D). The saturated phospholipids are considered as natural phospholipids because the resulting saturated lipids are also occurring in nature (i.e., natural identical).
In accordance with some embodiments, the lipid membrane comprises plant derived phospholipids comprising lecithin or portion thereof. Lecithin is described in the United States Pharmacopoeia (USP) as a complex mixture of acetone-insoluble phosphatides, which consists chiefly of PC, PE, phosphatidylserine, and phosphatidylinositol, combined with various amounts of other substances such as triglycerides, fatty acids, and carbohydrates, as separated from the crude vegetable oil source.
In some embodiments, the lipid membrane comprises lipids and phospholipids derived from lecithin. When referring to “lipids and phospholipids derived from lecithin” it is to be understood as a lipid combination comprising at least two phospholipids, at least one of which is a PC, and at least one of which (which is or is not a PC) is characterized by one or more of the following features: (a) it has an unsaturated lipid tail; (b) it comprises a polar head group; (c) it comprises an acidic head group.
The plant lipids can be incorporated into the liposomes in their natural form (as they appear in nature) or they can be subjected to chemical modifications, such as, without being limited thereto, hydrogenation and oxidation.
In some embodiments, the liposome within the formulation disclosed herein comprise a lipid membrane composed of a combination of two or more liposome forming lipids, and the lipid membrane further comprises (i) at least one unsaturated lipid (that can be one of the liposome forming lipids or a non-liposome forming lipid) specifically unsaturated PC, (ii) PI; and (iii) sterol.
In accordance with some embodiments, the lipid membrane comprises at least a combination of (i) phospholipids that comprise PC, PI and PA; (ii) one or more sterols; and (iii) on or more saponins.
In some embodiments, the liposomes carry one or more (the same or different) agriculturally active agents or ingredients. In some embodiments, the one or more active agents are encapsulated within the intraliposomal internal aqueous core. In yet some other embodiments, the active agent is embedded in the lipid bilayer membrane.
Methods for loading of active agents within liposomes (either into the aqueous, intraliposomal core or within the lipid bilayer membrane) are well known in the art. The selection of a preferred loading technique, may depend, inter alia, on the type of active agent to be loaded, e.g. hydrophilic, hydrophobic, amphipathic, low molecular weight, macromolecule etc.
When referring to an agriculturally active agent, it is to be understood as any agent that provides a beneficial agricultural effect. The active agent may be a low molecular weight compound or a macromolecule, e.g. polymer.
The active agent may be classified according to its effect on the plant.
The active agent may be, but not limited to, pesticides, fertilizers, bio stimulants, and/or plant nutrients.
In some embodiments, the active agent is a pesticide. In the context of the present disclosure, when referring to a pesticide, it is to be understood as encompassing any substance used for destroying organisms harmful to cultivated plants.
In some embodiments, the pesticide is any member of the group consisting insecticides, herbicides, rodenticides, bacteriocides, fungicides and nematocides.
In some embodiments, the pesticide is a herbicide. Non limiting examples of herbicides include: Glufosinate, Propaquizafop, Metamitron, Metazachlor, Pendimethalin, Flufenacet, Diflufenican, Clomazone, Nicosulfuron, Mesotrione, Pinoxaden, Sulcotrione, Prosulfocarb, Sulfentrazone, Bifenox, Quinmerac, Triallate, Terbuthylazine, Atrazine, Oxyfluorfen, Diuron, Trifluralin, Chlorotoluron.
For example, and without being limited thereto, the herbicide can be used against weeds known to damage plants. For example, and without being limited thereto, the weeds can be any member of the following group of families: Gramineae, Umbelliferae, Papilionaceae, Cruciferae, Malvaceae, Eufhorbiaceae, Compositae, Chenopodiaceae, Fumariaceae, Charyophyllaceae, Primulaceae, Geraniaceae, Polygonaceae, Juncaceae, Cyperaceae, Aizoaceae, Asteraceae, Convolvulaceae, Cucurbitaceae, Euphorbiaceae, Polygonaceae, Portulaceae, Solanaceae, Rosaceae, Simaroubaceae, Lardizabalaceae, Liliaceae, Amaranthaceae, Vitaceae, Fabaceae, Primulaceae, Apocynaceae, Araliaceae, Caryophyllaceae, Asclepiadaceae, Celastraceae, Papaveraceae, Onagraceae, Ranunculaceae, Lamiaceae, Commelinaceae, Scrophulariaceae, Dipsacaceae, Boraginaceae, Equisetaceae, Geraniaceae, Rubiaceae, Cannabaceae, Hyperiacaceae, Balsaminaceae, Lobeliaceae, Caprifoliaceae, Nyctaginaceae, Oxalidaceae, Vitaceae, Urticaceae, Polypodiaceae, Anacardiaceae, Smilacaceae, Araceae, Campanulaceae, Typhaceae, Valerianaceae, Verbenaceae, Violaceae. For example, and without being limited thereto, the weeds can be any member of the group consisting of Lolium Rigidum, Amaramthus palmeri, Abutilon theopratsi, Sorghum halepense, Conyza Canadensis, Setaria verticillata, Capsella pastoris, and Cyperus rotundus. Additional weeds include, for example, Mimosapigra, salvinia, hyptis, senna, noogoora, burr, Jatropha gossypifolia, Parkinsonia aculeate, Chromolaena odorata, Cryptoslegia grandiflora, Anndropogon gayanus.
In some embodiment, the pesticide is a fungicide. Non limiting examples of fungicides include: azoxystrobin, mancozeb, prothioconazole, folpet, tebuconazole, difenoconazole, captan, bupirimate, fosetyl-A1.
In some embodiment, the pesticide is an insecticide. Non limiting examples of insecticides include Imidacloprid, Acetamiprid, Indoxacarb, Pymetrozine, Novaluron, Bifenthrin, Beta-Cyfluthrin, Spinosad, Acephate, Tau-Fluvalinate.
In some embodiments, the pesticide is a bacteriocide.
In some embodiments, the active agent is a plant nutrient. When referring to a plant nutrient it is to be understood as encompassing any substance that has a beneficial effect on the growth of the plant, substances necessary for plant growth and metabolism and completion of life cycle.
In some embodiments the plant nutrient is selected from the group consisting of macro-nutrients (N, P, K), secondary nutrients (S, Si, Ca, Mg) and micronutrients (Fe, B, Cl, Mo, Co, Cu, Zn, Ni, Al).
In some embodiments the plant nutrient is a plant hormone (phytohormone) or its metabolite or precursor, and plant growth regulators. In some embodiments, the phytohormone is selected from abscisic acid hormone, auxins, cytokinins, gibberellins, ethylene, brassinosteroids (polyhydroxysteroids), salicylic acid, jasmonates, plant peptide hormones, polyamines, nitric oxide, strigolactones, karrikins, and triacontanols.
The liposomes are carried by an agriculturally acceptable carrier.
In some embodiments, the “agriculturally acceptable carrier” is a carrier that is non-phytotoxic.
In some embodiments, the “agriculturally acceptable carrier” is a carrier that can be phytotoxic.
In some embodiments, the agriculturally acceptable carrier is selected from a polar organic solvent, water, water dispersible particulate matter (e.g. granules, capsules, beads, pellets, tablets, etc.).
In some embodiments, the agriculturally acceptable carrier is inert, i.e. while it may facilitate in the delivery of the liposomes to the plant (e.g. in penetration), it does not abrogate the integrity of the liposomes and/or the activity of any active agent carried by the formulation, and preferably within the liposomes.
The selection of the carrier may depend on the manner of bringing the formulation into contact with the plant, as further discussed below.
The present disclosure also provides a method for treating in the field of agriculture. In some embodiments, the treatment can be of a plant or plant part.
In some other embodiments, the treatment can be via application of the formulation to the soil, or to a plant growth medium (e.g. hydroponic growing medium).
In some embodiments, the method is for treating a plant and the method comprises applying to the surface of the plant the formulation disclosed herein.
In the context of the present disclosure “treating” denotes an effect on the plant that can be for reducing, inhibiting or eliminating a plant pathological condition, be it one caused by a pathogen or by an environmental condition (physiological factors); or for preventing from the pathological condition from developing. Thus, in the context of the present disclosure, treatment encompasses treatment for curing from a pathological condition, as well as protective treatment.
In some embodiments, the treatment is for pest control.
In some embodiments, the treatment is for improving crop.
In some embodiments, the treatment is for imparting the plant with a desired trait. In the context of the present disclosure, a plant trait can include, without being limited thereto, abiotic or biotic stress tolerance, drought tolerance, high harvest yield, high biomass and/or vigor, high seed yield/quality, increased crop/flower per plant, growth rate, fruit quality (fruits color break, firmness, shine, etc) etc.
The liposomal formulation disclosed herein is to be applied to the plant by direct contact with the plant or part thereof. Direct contact requires that intact liposomes within the formulation are in contacted with the plant or plant part.
In the context of the present disclosure, a “plant part” denotes any one or combination of the meristems, leaves, root, stem, shoot, flower, fruit, tuber, seed, onion, petriole, bud, tendril, trunk, bulb, rhizome and stolon.
It has been found, as also exhibited in the following non-limiting examples, that when applied onto the plant's leaves, the formulation effectively penetrates and is distributed from the leaves to the plant parts, including the apical shoot. Thus, in some embodiments, the formulation is applied onto at least a portion of the plant's foliage.
Thus, in the context of the present disclosure, the liposomes effectively penetrate into the plant, and are distributed throughout portions of the plant.
Specifically, in the context of the present disclosure, when referring to “penetration” it is understood to encompass the translocation of intact liposomes from the external surface of the plant part that has been brought into contact with the formulation, into the plant, via the plant cuticles, epidermis or hypodermis.
Penetration into a plant part can be determined by measuring the amount of a detectable liposome component, e.g. a marked membrane lipid or other membrane component or a marked encapsulated agent, after the plant part has been thoroughly rinsed. The mark can be by the use of a fluorescent dye.
Further, in the context of the present disclosure, when referring to “distribution” it is to be understood to encompass the translocation of at least the active agent from the plant part onto which the formulation has been applied, to at least one other plant part.
In some embodiments, distribution is of intact liposomes.
Distribution of the active agent and/or intact liposomes carrying the active agent can be determined, for example, by thoroughly rinsing or entirely removing the plant part onto which the formulation has been applied and measuring the amount of a detectable liposome component, e.g. a marked membrane lipid or other membrane component or a marked encapsulated agent. Also in this case, the mark can be by the use of a fluorescent dye. Distribution can be to any plant part, such as apical shoot, leaves, stem and roots.
In some embodiment, distribution is at least to the apical shoot.
Delivery of the formulation to the plant's part can be by any one or combination of spraying the plant, smearing the formulation onto the plant part, submerging the plant part within the formulation, fumigation, applying ultrasonic droplets, dusting with the formulation.
In some embodiments, the formulation is applied by spraying. To this end, and in accordance with some embodiments, the formulation is in a form of an aqueous formulation in which the liposomes are suspended or dispersed. The aqueous formulation may also contain, suspended therein, particulate matter (e.g. bead, capsules, etc.) carrying the liposomes.
In some embodiments, the liposomal formulation is applied onto the plant part by smearing.
In some embodiments, the liposomal formulation is applied onto the plant part by submerging the plant part, e.g. leaves, into the liquid formulation.
In some other embodiments, the formulation is applied onto the plant's seeds. According to this embodiment, and without being limited thereto, the seeds can be sprayed with the formulation and/or be submerged within or drenched by the formulation.
In some embodiments, the formulation is applied to the plant leaves. This may be achieved by any of the above listed delivery techniques, i.e. smearing, spreading, spraying, immersing, fumigating, applying US droplets, dusting. In some embodiments, the leaves are sprayed with the formulation.
In some other embodiments, the formulation is applied onto the plant's roots. According to this embodiment and without being limited thereto, direct contact of the roots with the formulation can be achieved by the use of hydroponic systems with the formulation being dispersed or suspended in the plant reservoir within the hydroponic tank or tray.
In some embodiments, the liposomes are delivered to the plant through irrigation.
The formulation disclosed herein is applied onto the plant or plant part in an amount, and at a schedule that is effective to treat the plant. The amount and schedule can be easily determined by those versed in the art and will depend, inter alia, on the type of the plant, the pathology, whether it is protective or curative treatment, the environmental conditions etc.
Also disclosed herein is the use of the formulation for agricultural applications, e.g. in accordance with the method disclosed herein.
In addition, disclosed herein is a kit comprising (a) an agriculturally acceptable carrier; (b) liposomes or liposome forming lipids as defined herein; and (c) instructions for use of the carrier and liposomes for treating a plant.
In some embodiments, the liposomes within the kit are in dry form, e.g. lyophilized and the instructions comprise, steps for rehydrating the liposomes together with the carrier.
In some embodiments, the kit comprises separately, the lipid membrane components, i.e. the liposome forming lipids and other lipid components as defined herein with the active agent being within the agriculturally acceptable carrier, such that when mixed together, liposomes encapsulating the active agent are formed.
As used herein, the forms “a”, “an” and “the” include singular as well as plural references unless the context clearly dictates otherwise. For example, the term “a liposome” includes one or more liposomes.
Further, as used herein, the term “comprising” is intended to mean that the formulation includes the recited liposome and carrier but not excluding other elements, such as a surfactant or other components that may be part of the liposome or part of the carrier. The term “consisting essentially of” is used to define formulations which include the recited elements but exclude other elements that may have an essential significance on the formulation. “Consisting of” shall thus mean excluding more than trace elements of other elements. Embodiments defined by each of these transition terms are within the scope of this invention.
Further, all numerical values, e.g. when referring the amounts or ranges of the elements constituting the formulation are approximations which are varied (+) or (−) by up to 20%, at times by up to 10% of from the stated values. It is to be understood, even if not always explicitly stated that all numerical designations are preceded by the term “about”.
The invention will now be exemplified in the following description of experiments that were carried out in accordance with the invention. It is to be understood that these examples are intended to be in the nature of illustration rather than of limitation. Obviously, many modifications and variations of these examples are possible in light of the above teaching. It is therefore, to be understood that within the scope of the appended claims, the invention may be practiced otherwise, in a myriad of possible ways, than as specifically described hereinbelow.
Materials:
L-α-Phosphatidylcholine, hydrogenated from soy bean (HSPC) was obtained from Avanti Lipids, Inc.
Soy lecithin was obtained from various commercial suppliers
Cholesterol was obtained from Sigma Aldrich.
1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (PEG-DSPE 2000) as an ammonium salt was obtained from Lipoid.
Calcium Acetate (CaAc), Fluorescein, EuCl3, Mannitol, MES, Cellulase R10, 0.66-0.8% Macerozyme R10, and 0.1% BSA were obtained from Sigma Aldrich.
Sequesterene 138 (iron chelate microelement nutrient) was obtained from 15 Syngenta.
MgSO4 was obtained from Merck.
All other reagents and solvents were obtained from known vendors.
Methods:
Preparation of Liposomes
Extraction of Lipids from Soy Lecithin for Liposomes B
Lipids are extracted from soy lecithin by dissolving the lecithin in EtOH and heating to 60° C. for approximately an hour. The upper liquid phase is collected and the EtOH evaporated. The lipid mixture powder obtained comprises phosphatidylcholine (PC)—35-50%; phosphatidylinositol (PI)—10-20%; phosphatidic acid (PA)—3-6%; phytosterols and saponins—25-30%. The chain types in the mixture comprise 16:0—20-25%; 18:0—10-15%; 18:1—15-22%; 18:2—35-40%; 18:3—10-15%.
Lipids extracted from lecithin by this method were used for preparation of Liposomes B, for the experiments presented in
Liposome Production by Ethanol Injection with Passive Loading
An organic phase is prepared by dissolving a selected lipid formulation in a water-miscible organic solvent such as EtOH (10% volume or less from the final volume of a combined organic and inorganic emulsion) at a temperature above the gel-to-liquid crystalline phase transition temperature (Tm) of the dominant lipid (e.g., 65° C. for HSPC) to obtain a concentration of 50 mM.
An aqueous phase is prepared separately by dissolving the compound (agent) to be encapsulated in water at 65° C.
The organic and aqueous phases are then merged by injecting the organic phase into the inorganic phase, preferably in a rapid and consistent motion, to thereby obtain a cloudy solution (an emulsion), which is vortexed for a few seconds. Vesicles encapsulating the compound of interest are spontaneously formed, and are thereafter downsized by stepwise extrusion through 400, 100, 80 nm membranes (5 repetitions for each membrane). The extruded solution is then subjected to dialysis (at e.g., 12-14 kD cutoff) at room temperature.
This method was used for preparation of Liposomes A, B for the experiments presented in
Liposomes A, B were prepared according to the passive loading procedure described hereinabove, using an aqueous phase prepared by dissolving Diethylenetriaminepentaacetic acid gadolinium(III) dihydrogen salt hydrate in aqua solution, to achieve a final concentration of 100 Mm Gd. The obtained concentration of Gd in the liposomes was approximately 2 mM.
Liposomes A were prepared according to the passive loading procedure described hereinabove, using an aqueous phase prepared by dissolving EuCl3 in a pre-prepared 5% DEX solution, to achieve a final concentration of 50 mg/ml. The obtained concentration of EuCl3 in the liposomes was approximately 2 mg/ml
Liposomes A were prepared according to the passive loading procedure described hereinabove, using an aqueous phase prepared by dissolving 20% w/w MgSO4.7H2O in a pre-prepared 5% DEX solution, to achieve a final MgSO4 concentration of about 2% wt. in the liposomes, which is in line with the commonly-used amounts of this fertilizer in traditional plant fertilizing.
Liposomes A were prepared according to the passive loading procedure described hereinabove, using an aqueous phase prepared by dissolving 16-17% w/w Sequestrene™ 138 (chelated iron) in a pre-prepared 5% DEX solution, to achieve a final Sequesterene-derived iron concentration of about 0.1% wt. in the liposomes, which is in line with the commonly-used amounts of this fertilizer in traditional plant fertilizing.
Liposomes encapsulating non-chelated iron were prepared in a similar manner. The aqueous phase was prepared by mixing a 10 grams/liter solution of AAS-grade Fe-standard in a pre-prepared 5% DEX solution, to achieve a final Iron concentration of about 0.1% wt. in the liposomes.
Fluorescein-encapsulating Liposomes A for the experiment presented in
Liposomes encapsulating CaAc-encapsulating are first prepared by ethanol injection and passive loading, as described above, wherein the concentration of the lipid formation was 50 mM and the aqueous (inorganic) phase was prepared separately by dissolving CaAc in water at 65° C., at a concentration of 100 mM. Following successive encapsulation of CaAc and dialysis (performed in order to dismiss excess non-encapsulated CaAc), CaAc-containing liposomes are introduced into a 2 mg/ml 5 Fluorescein-containing (free acid, from Sigma) 5% D+-Glucose (Dextrose, DEX) solution in a 1:2 ratio (such that liposomes are diluted 1:2 within the solution, and the final concentration of Fluorescein post-mixing is 1 mg/ml), at 55° C., and the obtained mixture is subjected to constant magnetic stirring for 60 minutes. The Fluorescent compound is then mobilized by its concentration gradient through the 10 temperature-disturbed liposomal membranes, where conjugation with Ca occurs causing the newly formed salt to precipitate inside the particle. The obtained liposome solution is thereafter left to cool to room temperature and further dialysis is performed in order to remove non-precipitated/non-encapsulated dye. The obtained concentration of fluorescein in the liposomes was approximately 0.6 mg/ml
Liposomes were measured using Dynamic Light Scattering (DLS) instrument (http://www.malvern.com/en/products/technolog/dynamic-light-scattering/) for of particle size characterization and size distribution. Samples were diluted 1:100 with the buffer in which the liposomes were prepared.
Zeta potential is a measure of the magnitude of the electrostatic or charge repulsion/attraction between particles, and is one of the fundamental parameters known to affect stability. Zeta potential for liposomes solution was measured using DLS instrument. Samples were diluted in ratio of 1:100 with the buffer which the liposomes were prepared in.
To quantify pyranine entrapment, liposomes encapsulating pyranine were decomposed with 0.1% triton and pyranine was determined using Tekan, Multimode Microplate Reader with fluorescence in excitation wavelength 413 nm and emission wavelength 510 nm.
To quantify fluorescein entrapment, liposomes encapsulating fluorescein were decomposed with 0.1% triton and fluorescein was determined using Tekan, Multimode microplate reader with absorbance in wavelength of 525 nm.
To quantify metal entrapment, liposomes encapsulating metals were dissolved in 1% HNO3 at a ratio of 1:100, vortexed and filtered through a 0.45 m syringe filter. Metals were determined using Inductively Coupled Plasma Optical Emission Spectroscopy (ICP OES). Wavelengths used were 342 nm for Gd, 397 nm for Eu, 280 nm for Mg and 259 nm for Fe.
To quantify glufosinate entrapment liposomes were decomposed using Bligh and Dayer method, Glufosinate being dissolved in the upper phase. Glufosinate was determined using High Performance Liquid Chromatography with Diode-Array Detection (HPLC-DAD) according to the method described in Changa et. al, Journal of the Chinese Chemical Society, 2005, 52, 785-792. Briefly, 9-fluorenylmethyl chloroformate (FMOC-Cl) was used for pre-column derivatization of the non-absorbing glufosinate. The samples were separated by HPLC-DAD at 12 min with 25 mM borate buffer at pH 9, followed by determination with a UV detector at 260 nm.
Liposomes were applied to plants using three different application methods
At the determined time following liposomal application, plants were sliced into different organs, washed and counterstained using propidium iodide solution 75 mM. Samples were examined in the Confocal Zeiss LSM 510 META.
Cherry tomato (Shiren variety) seeds of uniform genetics were germinated and grown in a designated nursery in soil (experiments presented in
Gd-encapsulating Liposomes A, B (concentration of 2 mg/ml) were prepared as described above and 0.1 ml was applied to a single leaf by smearing as described above.
72 hours after application, the leaf on which the formulation was applied was thoroughly rinsed for 45 seconds, under running water. Plants were thereafter cut and divided to leaflet samples, petiole samples, stem sample and root sample, samples were dehydrated in oven (2 hours at 105° C.) and the dry weight recorded. The dried samples were placed in ceramic bowls and fully digested by cremation for 3-5 hours at 550° C. Ash residues were dissolved in 1% nitric-acid and collected to tubes, at a final volume of 10 ml for each sample. Samples were filtered through 0.45 μm syringe filter and analyzed using Inductively Coupled Plasma Optical Emission Spectroscopy (ICP OES) with a wavelength of 342 nm.
Penetration was calculated as the % of Gd applied that was found in the entire plant, including the leaf on which the formulation was applied.
Total distribution is the amount of Gd (microgram Gd/gram fresh tissue) found in the entire plant excluding the leaf on which the formulation was applied. Distribution to different organs is the amount of Gd (microgram Gd/gram fresh tissue) found in each organ separately.
The objective was to determine movement of liposome in woody plants (mature vine in vineyard), as well as to evaluate the movements from the younger to older plant parts (from top of the branch toward the base).
Eu-encapsulating Liposomes A (concentration of 50 mg/ml) were prepared as described above.
The liposomes were applied to the youngest leaf of a mature vine branch by the leaflet submerge method, as described above.
After application (72 hours), sample vine leaves were collected at distances of 5 cm from the application point downward toward the branch base, a distance of 60 cm in total. Samples were dehydrated in oven (2 hours at 105° C.) and the dry weight recorded. The dried samples were placed in ceramic bowls and fully digested by cremation for 3-5 hours at 550′C. Ash residues were dissolved in 1% nitric-acid and collected to tubes, at a final volume of 10 ml for each sample. Samples were filtered through 0.45 μm syringe filter and analyzed using Inductively Coupled Plasma Optical Emission Spectroscopy (ICP OES) with a wavelength of 280 nm.
Liposomes A encapsulating MgSO4, Fe and Sequestrene™ were prepared as described above.
Hoagland solution formulation was prepared according to Epstein, E. Mineral Nutrition of Plants: Principles and Perspectives. John Wiley & Sons, Inc. 1972, pp. 412, with some modification, using the ingredients listed in Table 2 below.
Cherry tomato (Sheran variety) seeds of uniform genetics were germinated and grown in designated nursery for about 3 weeks (e.g., until generation of 4th leaf), stripped from soil and washed thoroughly with DDW. Each plant's exposed root system was immersed in 250 ml of full 0.5 Hoagland solution, as described above, and air-pumped constantly for 7 days under steady ambient temperature, humidity and CO2 levels (data recorded by Rotronic CL11 sensor). After adaptation of the plants to full Hoagland media, plants were taken out of the beakers (excluding Group 3), washed thoroughly with DDW and transferred to a premixed microelement-deficient Hoagland solution (Mg-deficient for the experiments presented in
Plants were visually examined daily for signs of nutrient deficiency.
Once deficiency was identified, commercial and liposomal formulations were applied by smearing on a single leaf:
Ten days after application, the plants were examined for signs of deficiency-interveinal chlorosis and epinasty of older leaves, and overall growth and appearance were compared with untreated controls.
Assessment of Glufosinate Activity
Liposomes B encapsulating glufosinate were prepared as described above.
Cotton plant seeds of uniform genetics were germinated and grown in designated nursery for 6 weeks (2-3 leaves).
Either commercial formulation of glufosinate or glufosinate-encapsulating Liposome B were applied by spraying as described above.
Glufosinate activity was assessed phenotypically (signs of chlorosis and wilting, necrosis, plant death) on days 22 and 35 after treatment.
In the following examples, Gd was used either in free form or encapsulated in Liposome A or Liposome B, with variations in liposome composition, as specifically indicated.
For the purpose of quantifying penetration and distribution of Gd into various organs of the tomato plant, liposome formulation was smeared on a single leaf of 4-8 weeks old tomato plants, at a physiological age of 4-7 true leaves. Unless otherwise stated, 72 hours after application, the plants were dissected and cremated. Concentration of Gd in different plant organs was detected by ICP-OES. The plant organs examined were: apical shoot, leaf above loading point, leaf below loading point, roots and stem. Overall, the entire plant was cremated.
Effect of liposome Size
Comparison Between Effect of Free Gd in 0.1% Triton Vs. Liposomal Gd on Penetration and Distribution
However,
For the purpose of determining the effect of cholesterol concentration on penetration and distribution, the following liposome compositions were compared:
For the purpose of determining the effect of PEG-DSPE on penetration and distribution of Liposomes A, PEG-DSPE in the indicated amounts were added, as indicated in Table 3 below:
For the purpose of determining the effect of chain length on penetration and distribution, phospholipids with different chain lengths were used to prepare Liposome A (each variant of Liposome A comprised a single type of chain):
The lipid mixture consisted of one of the phospholipids listed above (60 mol %), cholesterol (38 mol %) and PEG-DSPE (2 mol %).
Effect of Positively Charred Livid. DOTAP
In the following, the effect of N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium) (DOTAP) incorporated into liposome A or Liposome B, on the distribution was determined. The liposomal composition is presented in Tables 4A-4B:
When incorporated into Liposome B, an even stronger positive effect was observed, where distribution even to the root was significantly high, as shown in
Notably, Liposome B, being made of soy lecithin has a negative charge. The addition of 15% DOTAP to Liposome B has changed the zeta potential positively by 10 mv, but the zeta of the liposomes was nevertheless negative—−20 mv.
In the following, the effect of α-tocopherol incorporated into Liposome A or Liposome B, on the distribution was determined. The liposomal compositions are presented in Tables 5A-5B:
The effect of α-tocopherol is shown in
As shown in
In the following examples, Liposome A encapsulating Fluorescein (fluorescent marker) was applied using leaflet-submerging method on tomato plants. Liposome presence in the roots was detected using confocal microscopy 24-96 hours after application.
Similar results were obtained when pyranine was encapsulated within Liposome B (phospholipids 51%, phytosterol 47%, PEG-DSPE 2%, where presence of the fluorescent marker in the roots, 72 hours post application was observed, as shown in
In a similar experiment, the presence of Fluorescein encapsulated in Liposome A in adjacent leaves was observed after 24 hr, 48 hr and 72 hr, as shown in
The following experiment demonstrated the translocation of Europium in mature woody plants. To this end, a single vine leaf was submerged in a solution containing Europium encapsulated in Liposomes A.
As shown in
Three plants of Eleusine indica were grown in the same pot. Fourteen days after germination, each plant was subjected to a different herbicidal treatments by smearing a single leaf of each plant. Specifically, the following groups were examined:
Left Plant: Plant treated with a commercial Glufosinate herbicide at the recommended dose (6 mg gluofosinate/ml);
Center Plant: Plant treated with Liposome B encapsulating Glufosinate at 65% of its recommended dose (3.9 mg glufosinate/ml).
Right Plant: non-treated plant.
In a similar experiment, a comparison was made between commercial and liposomal formulation of Glufosinate at 1/16 its recommended dose applied on the entire foliage of the plant by sparying.
Specifically, liposomal glufosinate was prepared, with a final glufosinate concentration in the liposomes of 0.35 mg/ml. Three treatment groups (in three replicas) were examined as follows:
Plant A: 1 ml per plant of commercial Glufosinate at a dose of 0.375 mg/ml.
Plant B: 1 ml per plant of Liposome B encapsulating Glufosinate at a dose of 0.35 mg/ml
Plant C: Untreated control (UTC)
The same trend continues 35 days after application. Plants treated with commercial formulation (left) continued to grow, while plants applied with liposome B (right) died as shown in
Tomato plants were grown under Mg-deficient conditions (interveinal chlorosis and leaf epinasty were observed). Two treatments were applied on the topmost leaf of the Mg-deficient tomato plant: Commercial unformulated MgSO4 and Liposome A containing MgSO4. Basipetal movement of Mg was evaluated through deficiency correction of lower leaves (3rd and 4th leaves) 10 days after application.
As shown in
This observation supports the finding that liposomal formulation improved basipetal movement of Mg.
In a further experiment, Liposome A containing MgSO4 and Liposome A containing Sequestrene (Fe-chelate) were applied on the topmost leaflet of Mg and Fe deficient tomato plants. Also in this case, basipetal movement was evaluated through deficiency (interveinal chlorosis, leaf epinasty) correction of lower leaves (3rd and 4th leaves) 10 days after application.
As shown in
The same effect is observed in
Finally, acripetal movement of Fe applied on the lowest leaf by smearing was observed when Fe-deficient tomato plants were treated as follows:
The applied concentration was 50% of the recommended Fe rate for tomatoes. Acripetal movement was evaluated through deficiency correction of other parts of the foliage 10 days after application.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IL2017/050290 | 3/8/2017 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62305629 | Mar 2016 | US |
