Continuous production of fertilizer-loaded liposomes

Abstract

A composition for nutrient delivery to plant roots, the composition comprising liposomes, wherein each liposome comprises a lipid bilaycr; and an aqueous core encapsulated within the lipid bilaycr, wherein the aqueous core includes at least one active agricultural compound that includes a plant nutrient.

Description

[0001] CONTINUOUS PRODUCTION OF FERTILIZER-LOADED LIPOSOMES

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 634,628, filed April 16, 2024, which is incorporated herein by reference in its entirety.

[0003] ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

[0004] This invention was made with government support under grant number 2133423 awarded by the National Science Foundation. The government has certain rights in the invention.

[0005] BACKGROUND

[0006] Current fertilization practice results in less than 50% of the nutrient applied to a location making it to the crop. This can lead to significant waste and environmental burden. Conservative estimates can be as high as 6.8 million tons of nitrogen fertilizer wasted annually (based on 2012 production values). Embedded resources (i.e., energy, water, and materials used to produce fertilizer) associated with this nitrogen waste include more than 332 million gallons of wasted fuel. Further, it is estimated that 360,000 tons of phosphorus, another major component of fertilizer, are lost annually from U.S. cropland. Rather than being produced via chemical processes, phosphorus is mined and therefore introduces wasted embedded resources associated with mining and refinement processes. This inefficient use of vital resources is a major contributor to severe nitrogen and phosphorus cycle imbalances, and is highlighted as such in the National Academy’s Grand Challenges of the 21st century (NAE Grand Challenges for Engineering. Manage the Nitrogen Cycle, 2016).

[0007] Upon fertilizer application, nitrogen and phosphorus participate in various biological and chemical fixation processes, as well as physicochemical interactions with the soil environment, that can significantly reduce a plant’s ability to take up these important nutrients. Physicochemical interactions may influence nutrient mobilization in the soil and / or result in volatilization due to changes in charge and physical state. Nitrogen is a primary nutrient that is essential to plant function, and accordingly is a principal component of most fertilizers. However, fertilizer use significantly disrupts the nitrogen cycle, currently accounting for more than half of nitrogen fixation that is attributed to human activity. The impact to environmental and human health associated with nitrogen fertilization cascades across the entire life cycle, including energy and resource intensive processes during production, risks associated with fertilizer storage, release of nitrous oxide (N2O, a potent greenhouse gas) and eutrophication caused by nutrient leaching.

[0008] Phosphorus is another primary nutrient that is important for crop production and therefore, also is a principal component of fertilizer. Management of the phosphorus cycle is highlighted as an important challenge of the 21st century that must be addressed to ensure future security of food and water resources. Fertilizer use significantly disrupts the phosphorus cycle, and accounts for more than become a global concern. It is a finite resource and the geographical location of the largest reserves are often difficult or dangerous to reach and / or work in. In addition to potentially having reached global ‘peak phosphorus’ , there are significant environmental and human health concerns associated with phosphorus. These include energy and resource intensive upstream processes during phosphorus extraction and refinement, and eutrophication caused by nutrient leaching. Improved efficiency of phosphorus use in crop production has been identified as one of two primary mechanisms to ameliorate the current issues of phosphorus cycle sustainability.

[0009] SUMMARY

[0010] In one aspect, disclosed herein is a composition for nutrient delivery to plant roots, the composition comprising liposomes, wherein each liposome comprises: a lipid bilayer; and an aqueous core encapsulated within the lipid bilayer, wherein the aqueous core includes at least one active agricultural compound that includes a plant nutrient.

[0011] Another aspect disclosed herein is a liposome comprising: a lipid bilayer comprising soybean lecithin and cholesterol; and an aqueous core encapsulated within the lipid bilayer, wherein the aqueous core includes at least one dissolved active agricultural compound.

[0012] A further aspect disclosed herein is a method comprising: applying to soil a composition comprising liposomes, wherein each liposome comprises a lipid bilayer, and an aqueous core encapsulated within the lipid bilayer, wherein the aqueous core includes at least one active agricultural compound that contains a plant nutrient that is delivered to a plant root when the composition is applied to the soil.

[0013] An additional aspect disclosed herein is a method for delivering an agricultural active compound to plant roots comprising: applying to soil a liposome, the liposome comprising: a lipid bilayer comprising soybean lecithin and cholesterol; and an aqueous core encapsulated within the lipid bilayer, wherein the aqueous core includes at least one dissolved active agricultural compound, thereby releasing the active agricultural compound into the soil for delivery to plant roots.

[0014] A further aspect disclosed herein is a method comprising: introducing a lipid-containing water miscible organic solvent stream into a hydrodynamic fluid focusing chip; introducing two lateral streams of an aqueous phase that includes an active agricultural compound into the hydrodynamic fluid focusing chip such that the solvent stream is sheathed by the aqueous phase streams; and forming a liposome that includes a core comprising the active agricultural compound and a lipid bilayer encapsulating the core.

[0015] The foregoing will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

[0016] BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1 is a schematic representation of a liposome structure as disclosed herein.

[0018] FIG. 2 is a schematic representation of a liposome production setup and the liposome formation during self-assembly process in a hydrodynamic fluid focusing (HFF) chip.

[0019] FIG. 3 is a graph showing nitrate (NO3- ) concentration measured in prepared liposomes at different Flow Rate Ratios (FRR), and Total Flow Rates (TFR).

[0020] FIG. 4 is a graph showing liposome size (as hydrodynamic diameter) at different soybean lecithin / cholesterol (SoyL / Chol) molar ratios immediately after production (red; hatched) and after two- week storage at 4°C (grey).

[0021] FIGS. 5A and 5B are images showing the effect of NO3 concentration (noted above each image) dissolved in PBS (FIG. 5A) and MilliQ water (FIG. 5B) on salt precipitation in a HFF chip.

[0022] FIGS. 6 A and 6B are graphs showing the hydrodynamic diameter (FIG. 6A) and PDI (FIG. 6B) of various liposomal carriers after production in HFF chip and after 2-week storage at 4 °C. Blank carrier in PBS (Bink liposome - PBS), nitrate-containing liposomes in PBS (N liposome - PBS), and nitrate-containing liposomes in MilliQ water (N liposome - MilliQ).

[0023] FIGS. 7A and 7B show that producing the liposomes with filtered deionized water increased the NO3 concentration in the liposomes at least twice compared to liposomes formed in PBS.

[0024] FIG. 8 is a graph demonstrating the results of study regarding several processing parameters.

[0025] FIGS. 9A-9C. (FIG. 9A) Breakthrough curves of bromide tracer (mg Br L-1) over pore volumes in saturated soil columns (flow rate: 5 mL minute1) in Chatham soil (top) and sand (bottom) for free bromide (red) and liposomes (blue). Shaded region indicates range across three replicates. (FIG. 9B) Comparison of mean residence time (T, pore volumes) (top) and bromide retention (%) (bottom) for free bromide (red) and liposomes (blue) in Chatham soil and sand. Asterisks indicate a statistically significant difference in the means (p < 0.0005) by unpaired t test. (FIG. 9C) The bromide extracted in increments of 2.5 cm of depth from one each of the saturated Chatham (green) and sand (purple) soil replicate columns. Shaded regions indicate range across technical replicates of extraction and measurement.

[0026] FIGS. 10A-10B. (FIG. 10A) Breakthrough curves of bromide tracer (mg Br L1) over 20 days in unsaturated soil columns (flow rate: 12.5 mm day1) in Chatham soil (top) and sand (bottom) for free bromide (red) and liposomes (blue). Shaded region indicates range across three replicates. (FIG. 10B) Comparison of mean residence time (T, days) and bromide retention (%) for free bromide (red) and liposomes (blue). Asterisks indicate a statistically significant difference in the means (p < 0.005) by unpaired t test.

[0027] FIG. 11. Batch adsorption isotherms for free bromide (top) and liposomal encapsulated bromide (bottom), with Chatham soil (green) and sand (purple), showing the adsorption capacity at equilibrium (qeq) for bromide concentrations in the solution at equilibrium (ceq). Error bars display ± one standard deviation across three replicates. Hill Isotherm fits with 95% confidence intervals (dotted lines) are shown for the liposome encapsulated bromide data.

[0028] FIG. 12. Cumulative release profiles of nitrate from liposomes with varying soybean lecithimcholesterol molar ratios (100:0, 87.5:12.5, 62.5:37.5) over 10 days.

[0029] DETAILED DESCRIPTION

[0030] I. Terminology

[0031] The following explanations of terms and abbreviations are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. As used herein, “comprising” means “including” and the singular forms “a” or “an” or “the” include plural references unless the context clearly dictates otherwise. The term “or” refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise.

[0032] Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples arc illustrative only and not intended to be limiting. Other features of the disclosure are apparent from the following detailed description and the claims.

[0033] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, percentages, temperatures, times, and so forth, as used in the specification or claims are to be understood as being modified by the term “about.” Accordingly, unless otherwise indicated, implicitly or explicitly, the numerical parameters set forth are approximations that may depend on the desired properties sought and / or limits of detection under standard test condi tions / methods. When directly and explicitly distinguishing embodiments from discussed prior art, the embodiment numbers are not approximates unless the word “about” is recited.

[0034] “Agricultural active compound” refers to a biologically active compound that is used in agriculture and targeted to plants to affect their biology. An agricultural active compound agent may have activity as an herbicide, pesticide, fertilizer, or a combination thereof. In certain embodiments, an agricultural active compound may be an organic or inorganic compound that acts as a plant nutrient. In some embodiments, the agricultural active compound is not water. “Rhizosphere” refers to a narrow region of soil adjacent to a plant’ s roots that is directly influenced by secretions from roots and associated soil microorganisms.

[0035] II. Overview

[0036] Given the necessity of nitrogen and phosphorus for food production, the increased demand on the food supply chain, and the increasing constraints on resources, there is an opportunity to advance the sustainability of current fertilization practices through improved design of nutrient (i.e. agricultural active compound) delivery systems. Improving the nutrient use efficiency (NUE) can result in significant savings in the amount of fertilizer used, which, in turn, can provide significant economic and environmental benefits. NUE is defined as the total amount of nutrients taken up by plants divided by the total amount of nutrients applied to the soil. A relatively modest 2% increase in NUE can result in savings of half a million tons of nitrogen and over 7,000 tons of phosphorus in the U.S. alone, equivalent to several $100 million savings and a significant reduction in the amount of nutrients released from the soil to surrounding bodies of water.

[0037] Disclosed herein are agricultural active compound-loaded liposomes. The liposomes are produced via microfluidic hydrodynamic fluid focusing methods. The formulation methodology disclosed herein enables sustainable production of agricultural active compound-loaded liposomes. This technology offers a route to deliver an agricultural active compound(s) to crop roots more efficiently by potentially reducing the amount of an agricultural active compound(s) that is added to farm fields and / or increasing the percentage of an agricultural active compound(s) that is absorbed by the plant. For example, nitrogen-loaded liposomes can address the global challenge of rebalancing the nitrogen cycle such as immense environmental and human health costs associated with excess nitrogen use, including eutrophication in lakes and coastal waters, associated cyanotoxins produced by algal blooms, greenhouse gas emissions from production of nitrogen-based fertilizer, on-farm greenhouse gas emissions from nitrogen processes, and contaminated drinking water sources.

[0038] Drug-loaded liposomes typically need a very small amount of drug to be encapsulated in the liposomes to produce therapeutic effect. However, to satisfy the needs of a plant for fertilizer, the encapsulated fertilizer should be higher in the range of several orders of magnitude compared to drug- loaded liposomes. The microfluidic production methods disclosed herein include several parameters such as lipid concentration, Flow Rate Ratio (FRR), and Total Flow Rate (TFR). The importance of these sets of parameters is that the cargo that is being encapsulated here (e.g., potassium nitrate) is a salt and the ionic components of it are orders of magnitude smaller than typical cargos (drugs, proteins, macromolecules, etc.) that have been encapsulated in liposomes. As a result, the design space for agricultural active compound-loaded liposomes is very different compared to other liposomes.

[0039] In another aspect of the methods, a buffer in the aqueous phase of the microfluidic process is not used. The typical buffer solution in the aqueous phase is replaced with ultrapure water (e.g., using MilliQ® system) as the aqueous phase carrier. The ultrapure water (MilliQ water hereafter) is deionized (i.e. charged molecules and ions are removed by filtration) and the ultrapure water is also RNase-free water that has low organic content.

[0040] III. Liposomes and Methods of Making

[0041] Liposomes are vesicles composed of one or more lipid bilayer shells, encapsulating a small volume core of aqueous solution. The lipid molecules form a generally spherical liposome that encapsulates the core aqueous solution, and the aqueous solution includes the agricultural active agent (e.g., nitrate (NO3)) derived from the agricultural active compound. An example is shown in FIG. 1. In certain embodiments, the liposome has one lipid bilayer shell. In certain embodiments, the liposome has multiple lipid bilayer shells.

[0042] Illustrative lipids for the lipid bilayer shell include soybean lecithin, cholesterol, and mixtures thereof. The total lipid concentration may range from 10 to 300 mg / ml.

[0043] In one example, the lipid bilayer includes soybean lecithin and cholesterol. In one example, the lipid bilayer includes soybean lecithin and cholesterol, and no other lipids. In another example, the soybean lecithin is the only lipid present in the lipid bilayer. The relative amounts of soybean lecithin to cholesterol may range from 100:0 to 1:99 molar ratio %. In certain embodiments, the molar ratio of soybean lecithin to cholesterol is 66:33 or higher molar ratio%. In certain embodiments, the molar ratio of soybean lecithin to cholesterol is at 90: 10 or higher molar ratio%.

[0044] The agricultural active compound may be a phosphorus nutrient, a nitrogen nutrient, a potassium nutrient, a secondary nutrient (e.g., sulfur, such as from a sulfate salt such as ammonium sulfate, potassium sulfate, manganese sulfate, or copper sulfate), a micronutrient (e.g., boron, such as from boric acid; copper, such as from a copper salt, such as copper sulfate or cupric chloride; or manganese, such as from manganese sulfate), or a mixture thereof. In some embodiments, the agricultural active compound is urea, ammonium nitrate, potassium nitrate, calcium nitrate, sodium nitrate, mono-ammonium phosphate, di-ammonium phosphate, calcium dihydrogen phosphate, potassium chloride, ammonium sulfate, potassium sulfate, or a mixture thereof. In certain embodiments, the amount of the agricultural active compound in the liposome is 25 to 1,800, more particularly 500 to 1,800, mg / mL.

[0045] In one example, the liposome formulation includes soybean lecithin (SoyL) and cholesterol (Choi) as the lipids in the lipid bilayer shell with potassium nitrate (KNO3) as the source of nitrogen encapsulated within the lipid bilayer shell.

[0046] In another aspect, the liposome delivery platform may have enhanced pH sensitivity. The enhanced pH sensitivity enables localized release of the agricultural active compound cargo at the plant root. There is a localized change in pH at the rhizosphere compared with the bulk soil, which can be up to 2.5 pH units. For example, the pH sensitivity may be enhanced by including at least one pH-sensitizing agent in the liposome delivery platform so the liposome carrier is pH sensitive. Illustrative pH-sensitizing agents include sunflower lecithin, a mixture of cholesteryl hemisuccinate (CHEMS) and dimethyldioctadecylammonium bromide (DDAB), a mixture of cholesteryl hemisuccinate (CHEMS) and N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), a mixture of cholesteryl hemisuccinate (CHEMS) and l,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), or a combination thereof.

[0047] Illustrative enhanced pH-sensitivity formulations for including in the lipid bilayer include: Sunflower Lecithin / Cholesterol / polyoxyethylene sorbitan monooleate (i.e., Tween 80);

[0048] Sunflower Lecithin / Soybean Lecithin / Cholesterol / polyoxyethylene sorbitan monooleate (i.e., Tween 80);

[0049] Soybean Lecithin I cholesteryl hemisuccinate (CHEMS) / dimethyldioctadecylammonium bromide (DDAB);

[0050] Soybean Lecithin / N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC) / CHEMS; or l,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) / CHEMS / Soybean Lecithin.

[0051] The delivery formulations disclosed herein are liposome systems prepared using a microfluidic hydrodynamic fluid focusing method. Microfluidics refers to manipulation of small amounts of fluids using channels with tens to hundreds of micrometers in dimensions. It offers rapid and adjustable mixing, homogeneous reaction environments, and a high-throughput experimental platform. The most common microfluidic method for production of liposomes is when a stream of lipid-containing water miscible organic solvent (referred to as the “solvent phase”) is sheathed by two lateral streams of aqueous phase (see FIG. 2). The water and lipid molecules diffuse into the solvent phase and aqueous phase, respectively, reducing the alcohol concentration to below the solubility limit of the lipids. This initiates the self-assembly of lipid molecules which eventually conforms to a spherical shape that entraps a small volume of aqueous phase.

[0052] In certain examples, the total flow rate (TFR) for making the liposomes, which is the sum of the aqueous flow rate and solvent phase flow rates, is 0.1 to 1.5 mL / min, or 0.1 to 0.5 mL / min.

[0053] In certain examples, the flow rate ratio (FRR) for making the liposomes, which is the ratio of the aqueous flow rate to solvent phase flow rate, is 1 to 20, or 1 to 2.

[0054] The solvent phase (solution of lipid) can be made by dissolving the lipid dissolution in a water miscible organic solvent. Illustrative solvents include isopropyl alcohol (IP A), methanol, ethanol, chloroform, or a mixture thereof.

[0055] The aqueous phase (solution of agricultural active compound) can be made by dissolution of the agricultural active compound in phosphate -buffered saline (PBS) or water. In certain examples, the water is filtered, ultrapure deionized water (MilliQ water) with resistivity of 18.2 MQ*cm and total organic carbon below 5 ppb.

[0056] In certain examples, the liposome particles have a hydrodynamic radius of 50 nm to 1 pm, more particularly 600 nm to 1 pm. In certain examples, the liposomes have a polydispersity index (PDI) of 0.1 to 0.5, more particularly 0.3 to 0.5.

[0057] The product has liposome particles homogeneously dispersed in the aqueous phase.

[0058] IV. Applications

[0059] Disclosed herein are delivery formulations that target an agricultural active compound, such as a nutrient, to plants, especially the rhizosphere and plant root cells, thereby reducing, or substantially preventing, leaching of the compound into the surrounding soil. In certain examples, a difference in pH triggers release of the nutrient from the liposome.

[0060] Embodiments of the disclosed delivery formulations may be prepared as a liquid for transportation and / or application to the soil. The platform may be applied to the soil by any suitable technique, including, but not limited to, fertigation, spraying, or drip-administration. The platform may be administered via an irrigation system. The components of the platform may be provided separately to a location and mixed on site before administration. Alternatively, the platform may be prepared off site and transported to the administration site.

[0061] The disclosed platform also may be useful for hydroponic plant production. Such applications may significantly reduce or substantially eliminate concerns relating to selecting stabilizers, or lipids, based on the pH of the environment.

[0062] V. Examples

[0063] A HFF microfluidic chip was utilized to produce liposomes with SoyL / Chol molar ratio of %66:%33 at 65 mg / ml lipid concentration at FRR= 2,5,10 and TFR=300, 600, 1000 pL / min. Results indicate that the encapsulated NOa concentration is dependent on the TFR, FRR, and lipid concentration (FIG. 3).

[0064] SoyL-Chol liposomes of different molar ratios were produced, and the liposome stability was assessed after two weeks of storage at 4°C by comparing the liposome hydrodynamic diameter (determined by dynamic light scattering, FIG. 4). Results indicate that liposome carriers are stable even without addition of Choi (100:0 in FIG. 4). This is also important for cost minimization of the formulation since Choi contributes 60% of the cost of the lipid formulation while only constitutes 33% of the molar ratio.

[0065] The microfluidic system parameters, including TFR, FRR, and lipid concentration, influence liposome characteristics like size and polydispersity index (PDI). However, their impact on liposome encapsulation remains unstudied. The most straightforward way to increase the amount of encapsulated NO3 concentration in the liposomes is to increase the input NO3 concentration in the aqueous solution. However, too high of concentration has led to undesired precipitation in the chip, causing microchannel clogging. The precipitation may be from the combined KNO3 and ionic concentration of the phosphate buffer (PBS) being beyond the solubility limit of the alcohol solvent phase. To address this, PBS was replaced with DI water, resulting in a significant reduction in chip precipitation without affecting liposome stability (FIG. 5).

[0066] Dynamic light scattering (DLS) was used to measure the hydrodynamic diameter and polydispersity indexes (PDI) of the produced liposomal carriers, as an indication of their stability over time. The hydrodynamic diameter does not significantly change over 14 days post-production at 4’C (FIG. 6), showing the stability of liposomes even when produced in MilliQ water.

[0067] Producing the liposomes with filtered deionized water increased the NO3 concentration in the liposomes at least twice compared to liposomes formed in PBS the production parameters were the same for both cases TFR= 750 ijL / min, FRR=10, and at SoyL / Chol molar ratio of %66:%33 at 13 mg / ml lipid concentration (FIG. 7).

[0068] Considering TFR, FRR, and lipid concentrations as processing parameters influencing encapsulated NO3 concentration in liposomes, a 3D design space was utilized. Parameters were randomly selected, liposomes prepared, nitrate concentration measured, and parameters adjusted accordingly. Positive changes were pursued if nitrate concentration increased, and adjustments were made in reverse or explored other parameters if it decreased. FIG. 8 demonstrates results of this study confirming the effect of TFR, FRR, and lipid concentration on the encapsulated NO3 concentration in liposomes (in MilliQ water).

[0069] Liposomes were prepared specifically to encapsulate a cargo of sodium bromide. Bromide (Br ) is commonly used as a conservative tracer for tracking the movement of water in soils. Because it is anionic and largely chemically inert under most environmental conditions, like those typical of agricultural soils, it has limited interactions or transformations in soil water. While bromide is not a nutrient that is delivered to crops, it allowed the fate and transport of liposomes without the influence of sorption or biological transformations of the tracer load itself to be examined. Thus, any reduction in bromide leachate concentrations or increase in mean residence time observed in soil column studies is engendered by the liposomes through the physical, chemical, or biological interactions with the soil.

[0070] Liposomes were produced via continuous flow processing using a 3-input hydrodynamic fluid focusing chip (Dolomite, Royston, UK). Mixing of the liposomal components (i.e., lipids in an organic phase and ionic cargo in an aqueous phase) at the junction of the chip causes self-assembly of lipid molecules which eventually conform to a spherical shape that entraps and encapsulates the cargo.

[0071] Saturated soil column experiments were conducted using liposome encapsulated bromide and a free bromide control in Chatham soil and sand. Breakthrough curves display the concentration of tracer that leaches out of the column over time (FIGS. 9A-9C). The shift in the breakthrough curves to the right observed for the liposome encapsulated bromide for both the Chatham soil and the sand treatments suggests slower transport than when applied as free bromide (FIG. 9A). Bromide residence time, T, was significantly longer when encapsulated in liposomes than compared to the free bromide control in both Chatham soil (67 ± 8% increase, p < 0.0005 by t-test) and sand (43 ± 4% increase, p < 0.0005 by t test). Bromide retention in both soils was found to be negligible in free bromide control experiments (9.8% and 3.4% in Chatham and sand, respectively) (FIG. 9B). Bromide retention significantly increased when encapsulated in liposomes, with 55 ± 5% retention in Chatham soil (p < 0.0005 by t test) and 72 ± 9% retention in sand (p < 0.0005 by t test), demonstrating that liposomes reduce transport and leaching of the encapsulated load. Analysis of the 2.5 cm soil increments extracted from a representative column provided insight into the depth of penetration (or retention) of the bromide. The majority of liposome encapsulated bromide was retained in the top 10 cm of the column soil for both Chatham soil (82 + 1%) and sand (100%) (FIG. 9C). Together, these results indicate that when in saturated conditions, liposomal encapsulation of an ionic load (here, bromide) effectively reduces the rate of transport, increases retention in the soil, and reduces losses to leachate across soil types. In agricultural settings where surface soils can become saturated due to irrigation or heavy precipitation, this suggests that liposomal encapsulation would help slow the transport of ionic loads through the soil profile.

[0072] Unsaturated conditions were also tested to further assess the effect of liposome encapsulation on transport (FIG. 10A). In addition, these experiments were carried out over 20 days, a much longer duration than the saturated experiments (2.5 hours). For the unsaturated column experiments there was no statistically significant difference in the residence time (T) between encapsulated and free bromide for either soil type (FIG. 10B), meaning encapsulation did not reduce the rate of transport of bromide when unsaturated conditions were simulated. The free bromide control was similarly retained in both soil types (22 ± 7% in Chatham and 23 ± 7% in sand). This non-conservative behavior, where bromide persists in soil under unsaturated conditions, may result from biogenic immobilization via enzymatic bromination of organic compounds, as noted in the literature. Preferential flow, where percolating water moves in non-uniform paths and bypasses some pore spaces, could also leave behind pockets of free bromide in less traveled flow paths, leading to its eventual retention, rather than it being flushed from the system. In contrast to retention time, liposomal encapsulation fostered a significant increase in the mass of bromide retained over the free bromide for Chatham soil following the 20 day experiment (53 ± 2% retention, p < 0.005 by t test). This increase in retention demonstrates that liposomes have promise to reduce the quantity of leached ionic agrochemical loads in the vadose zone of agricultural soils. No significant difference was found between the retention of free bromide and liposomal encapsulated bromide in unsaturated sand.

[0073] The liposomes interact with the surface of soil particles as they travel with water through the soil pore space, and the result of these interactions could further influence liposome residence time and retention. To characterize these liposome-soil interactions, batch adsorption experiments were performed to produce adsorption isotherms of adsorption capacity (qeq) as a function of bromide concentration (ceq) (FIG. 11). It is important to note that the solid suspension was separated via gravity settling at the completion of the experiment. As a result, it is not feasible to distinguish how much bromide settled out of solution due to complexation with soil versus homo-aggregation of liposomes. Thus, the adsorption capacity data presented reflects the combined effect of liposomal adsorption to soil and homo-aggregation of liposomes on bromide removal.

[0074] There was no adsorption of free bromide to either soil (FIG. 11, top), which was expected, given it is an anion and soil particles have a negative surface charge. Therefore, any observed adsorption in the liposome experiments must emerge as a result of bromide being encapsulated in the liposomes, either through homo-aggregation or soil complexation. In both the Chatham soil and sand, the adsorption capacity of the bromide encapsulated liposomes increases with increased bromide concentration (FIG. 11, bottom). Both the Chatham soil and sand follow an S-type trend, where there is limited adsorption at low concentrations of the adsorbate, but a rapid increase in adsorption at higher concentrations of the adsorbate. The observed trend suggests a cooperative adsorption mechanism, in which the initial binding of molecules to a sorbent surface enhances the subsequent adsorption of additional molecules. The larger n in the Chatham soil suggests that cooperative adsorption is greater than in the sand. Translating this observation to the liposomes, either as individual units or as aggregates ranging in size, it suggests that they adsorb to the soil surface through electrostatic interactions, or through additional attachment processes, such as Van der Waals attraction after overcoming repulsive forces from the electric double layer. Initial interactions with soil particles can foster increased adsorption through subsequent aggregation. Cooperative adsorption becomes more favorable at higher liposome concentrations due to a shift in equilibrium that favors liposome aggregation. This observed difference in behavior between the Chatham soil and sand could be due to differences in the soil pore water chemistry (i.e., ionic strength, pH, presence of organic molecules, microbial activity). The high degree of liposome aggregation upon dilution in Chatham pore water further supports the hypothesis that cooperative adsorption is a major mechanism of interaction. In addition, maximum adsorption capacity (qmax) was determined from the Hill Isotherm fits, with the Chatham soil and sand having qmax values of 222 and 137 mg Br kg1soil, respectively. The higher adsorption capacity in the Chatham soil is hypothesized to be due to the higher soil particle surface area found in silty clay loam soil relative to sand.

[0075] The adsorption interactions and complexation between soil and liposomes, and perhaps liposomes with themselves, could contribute to the retarded transport of liposomes observed in the saturated column experiment and the retention of bromide found in all liposome column experiments. These interactions between liposomes and soil particles could be further leveraged for desired transport outcomes. For example, liposomes could be further engineered to include specific lipid terminal chemistry, to achieve preferred charge interactions, or selective ligands, to bind to specific epitopes found in soil.

[0076] The findings demonstrate that liposome encapsulation reduces leaching of the conservative tracer, bromide, across two diverse soil types in saturated conditions. Increased retention was also observed in a natural agricultural soil (Chatham soil) in unsaturated conditions. Further investigation found that the increase in retention of the encapsulated load is likely caused by a combination of factors, including adsorption, size exclusion, and microbial immobilization. Together, these results indicate that liposomes could act as effective carriers of ionic loads (e.g., ionic nutrients) in soil while reducing losses to leaching. The increase in soil retention enabled by liposome encapsulation could prolong the availability of nutrients in the root zone, allowing for greater opportunity for crop assimilation and thus, increased nutrient use efficiency.

[0077] Nitrate-loaded liposomes were synthesized using a hydrodynamic fluid focusing microfluidic chip, employing soybean lecithin and cholesterol as the lipid components. The lipids were dissolved in isopropyl alcohol (IP A) and introduced as the core stream, sheathed by an aqueous solution of potassium nitrate (KNO3) at a total flow rate (TFR) of 1200 (tL / min and a flow rate ratio (FRR) of 5. Three different molar ratios of Soybean Lecithin to Cholesterol (100:0, 87.5:12.5, and 62.5:37.5) were evaluated to investigate the effect of cholesterol content on nitrate release kinetics.

[0078] Release studies were performed using a dynamic dialysis method. Ten milliliters of the liposomal suspension (donor compartment) were placed inside dialysis bags and immersed in 500 mL of ultrapure water (sink compartment). At specified time intervals, 0.2 mL samples were collected from the donor compartment, and the overall nitrate concentrations were quantified using a vanadium chloride (VC13) reduction assay.

[0079] As shown in FIG. 12, the release profiles revealed a distinct influence of cholesterol content on the release behavior. Liposomes without cholesterol (100:0, red circles) exhibited rapid release, reaching over 90% within the first 4 days. Incorporation of cholesterol significantly slowed nitrate release, with the 87.5:12.5 (green squares) and 62.5:37.5 (blue downward triangles) formulations showing more gradual release kinetics over the 10-day period. An additional sample containing 180 ppm free KNO3(purple diamonds) served as a control, displaying immediate and complete diffusion of nitrate within the first few hours.

[0080] These results indicate that cholesterol plays a stabilizing role in the lipid bilayer, reducing permeability and thereby extending the release duration of encapsulated nitrate. This tunability makes liposomal nitrate delivery a promising platform for controlled nutrient release applications.

[0081] In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention.

Claims

What is claimed is:

1. A composition for nutrient delivery to plant roots, the composition comprising liposomes, wherein each liposome comprises: a lipid bilayer; and an aqueous core encapsulated within the lipid bilayer, wherein the aqueous core includes at least one active agricultural compound that includes a plant nutrient.

2. The composition of claim 1, wherein the lipid bilayer comprises soybean lecithin, cholesterol, or a mixture thereof.

3. The composition of claim 1 or 2, wherein the total lipid concentration is from 10 to 300 mg / ml.

4. The composition of any one of claims 1 to 3, wherein the agricultural active compound is a phosphorus nutrient, a nitrogen nutrient, a potassium nutrient, a sulfate salt, a boron source, a copper source, or a manganese source.

5. The composition of any one of claims 1 to 3, wherein the agricultural active compound is urea, ammonium nitrate, potassium nitrate, calcium nitrate, sodium nitrate, monoammonium phosphate, di-ammonium phosphate, calcium dihydrogen phosphate, potassium chloride, ammonium sulfate, or potassium sulfate.

6. The composition of any one of claims 1 to 3, wherein the agricultural active compound is potassium nitrate.

7. The composition of any one of claims 1 to 6, wherein the amount of the agricultural active compound in the liposome is 25 to 1,800 mg / L.

8. A liposome comprising: a lipid bilayer comprising soybean lecithin and cholesterol; and an aqueous core encapsulated within the lipid bilayer, wherein the aqueous core includes at least one dissolved active agricultural compound.

9. The liposome of claim 8, wherein the amount of soybean lecithin to cholesterol is 66:30 or higher molar ratio %.

10. The liposome of claim 8 or 9, wherein the agricultural active compound is a phosphorus nutrient, a nitrogen nutrient, a potassium nutrient, a sulfate salt, a boron source, a copper source, or a manganese source.

11. The composition of any one of claims 8 to 10, wherein the agricultural active compound is urea, ammonium nitrate, potassium nitrate, calcium nitrate, sodium nitrate, monoammonium phosphate, di-ammonium phosphate, calcium dihydrogen phosphate, potassium chloride, ammonium sulfate, or potassium sulfate.

12. The composition of claim 8 or 9, wherein the agricultural active compound is potassium nitrate.

13. The composition of any one of claims 8 to 12, wherein the amount of the agricultural active compound in the liposome is 25 to 1,800 mg / L.

14. A method comprising: applying to soil a composition comprising liposomes, wherein each liposome comprises a lipid bilayer, and an aqueous core encapsulated within the lipid bilayer, wherein the aqueous core includes at least one active agricultural compound that contains a plant nutrient that is delivered to a plant root when the composition is applied to the soil.

15. The method of claim 14, wherein the lipid bilayer comprises soybean lecithin, cholesterol, or a mixture thereof.

16. The method of claim 14 or 15, wherein the total lipid concentration is from 10 to 300 mg / ml.

17. The method of any one of claims 14 to 16, wherein the agricultural active compound is a phosphorus nutrient, a nitrogen nutrient, a potassium nutrient, a sulfate salt, a boron source, a copper source, or a manganese source.

18. The method of any one of claims 14 to 16, wherein the agricultural active compound is urea, ammonium nitrate, potassium nitrate, calcium nitrate, sodium nitrate, mono-ammonium phosphate, di-ammonium phosphate, calcium dihydrogen phosphate, potassium chloride, ammonium sulfate, or potassium sulfate.

19. The method of any one of claims 14 to 16, wherein the agricultural active compound is potassium nitrate.

20. The method of any one of claims 14 to 20, wherein the amount of the agricultural active compound in the liposome is 25 to 1,800 mg / L.

21. A method for delivering an agricultural active compound to plant roots comprising: applying to soil a liposome, the liposome comprising: a lipid bilayer comprising soybean lecithin and cholesterol; and an aqueous core encapsulated within the lipid bilayer, wherein the aqueous core includes at least one dissolved active agricultural compound, thereby releasing the active agricultural compound into the soil for delivery to plant roots.

22. The method of claim 21, wherein the agricultural active compound is a phosphorus nutrient, a nitrogen nutrient, a potassium nutrient, a sulfate salt, a boron source, a copper source, or a manganese source.

23. The method of claim 21 or 22, wherein the agricultural active compound is urea, ammonium nitrate, potassium nitrate, calcium nitrate, sodium nitrate, mono-ammonium phosphate, diammonium phosphate, calcium dihydrogen phosphate, potassium chloride, ammonium sulfate, or potassium sulfate.

24. The method of claim 21 or 22, wherein the agricultural active compound is potassium nitrate.

25. The method of any one of claims 21 to 24, wherein the amount of the agricultural active compound in the liposome is 25 to 1,800 mg / L.

26. A method comprising: introducing a lipid-containing water miscible organic solvent stream into a hydrodynamic fluid focusing chip; introducing two lateral streams of an aqueous phase that includes an active agricultural compound into the hydrodynamic fluid focusing chip such that the solvent stream is sheathed by the aqueous phase streams; and forming a liposome that includes a core comprising the active agricultural compound and a lipid bilayer encapsulating the core.

27. The method of claim 26, the organic solvent stream has an organic solvent stream flow rate and the aqueous phase stream has an aqueous phase stream flow rate, wherein the sum of the aqueous phase stream flow rate and an organic solvent stream flow rates, is 0.1 to 1.5 mL / min.

28. The method of claim 26, the organic solvent stream has an organic solvent stream flow rate and the aqueous phase stream has an aqueous phase stream flow rate, wherein the ratio of the aqueous phase stream flow rate to organic solvent stream flow rate is 1 to 20.

29. The method of claim 27, wherein the ratio of the aqueous phase stream flow rate to organic solvent stream flow rate is 1 to 20.

30. The method of any one of claims 26 to 29, wherein the lipid comprises soybean lecithin, cholesterol, or a mixture thereof, and the wherein the agricultural active compound is a phosphorus nutrient, a nitrogen nutrient, a potassium nutrient, a sulfate salt, a boron source, a copper source, or a manganese source.

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