Compositions and methods for seed nanopriming

Abstract

Nanoparticulate compositions and methods of use thereof, are disclosed. The nanoparticulate compositions include: (a) nanoparticles comprising a homogenous network of plant secondary metabolites optionally metals bound to the plant secondary metabolites, preferably a metal-phenolic network comprising one or more metals and one or more phenolic compounds, the network having dispersed therein one or more plant treatment agents, or (b) nanoparticles comprising a core and a shell, wherein the shell comprises one or more plant secondary metabolites and metal, preferably a metal-phenolic network comprising one or more metals and one or more phenolic compounds in the form of a metal phenolic network, wherein the core comprises hydrophobic molecules (a) in a membrane encapsulating one or more plant treatment agents and / or (b) having dispersed therein one or more plant treatment agents, and optionally wherein the nanoparticulate composition comprises a surfactant at the interface between the core and the shell.

Description

[0001]KAUST 2024-082-02 PCT COMPOSITIONS AND METHODS FOR SEED NANOPRIMING CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 696,679 filed September 19, 2024, which is herein incorporated by reference in its entirety. FIELD OF THE INVENTION The disclosed invention is generally in the field of seed nanopriming and specifically in the area of controllable release of plant growth regulators for seed nanopriming. BACKGROUND OF THE INVENTION The global agriculture and food security sector faces pressing challenges, including low crop yields, compromised soil health, and inefficient use of agrochemicals.1Salinity, a significant stressor in arid regions, particularly impacts wheat production. To ensure food security by 2050, it is imperative to increase wheat yields.2Bio-stimulants like plant growth regulators (PGRs) can enhance plant resilience to abiotic stresses. However, their low water solubility, instability in environmental conditions, and potential phytotoxicity at high concentrations limit their effectiveness.3Furthermore, they must be used at appropriate levels to induce the desired effects because, at high concentrations, instead of functioning as hormones or regulators, they can become phytotoxic, hinder plant growth, or act as herbicides.4,5To overcome these challenges, innovative solutions are urgently needed. Nanotechnology offers promising approaches, as demonstrated by the European Commission.6Nano- enabled technologies, particularly those with surface functionalization, can integrate multiple functions into a single platform. These multicomponent systems enhance physicochemical properties, such as stability, solubility, and controlled release.7-12Despite their potential, the application of these systems in agriculture is still limited,13primarily due to challenges in bulk-scale production and the lack of reactive groups on nanoparticle surfaces. These limitations can complicate synthesis and potentially affect stability.14One such application is seed nanopriming, an eco-friendly technique that prepares seeds to withstand environmental stresses15,16. Unlike traditional coating methods that remain on the seed surface and directly interact with the environment, nanopriming delivers essential nutrients and plant growth regulators into the seed, enhancing its ability to cope with abiotic stress conditions.17-21Thus, enhancing seed vigor, promoting early germination, and improving overall crop production. In this regard, careful design of the nanopriming platform can be a promising solution for crop resilience in arid environments through controlled PGR delivery. Tannin-based bio-stimulants have shown potential in increasing root growth and enhancing plant resistance to salt stress due to their antioxidant properties.22However, environmental stresses such as high salinity and reactive oxygen species (ROS) can pose significant threats to plants, 145765042.1 KAUST 2024-082-02 PCT including DNA damage and programmed cell death.23,24Additionally, excess sodium in saline soils can limit the uptake of essential micronutrients like iron (Fe), an essential micronutrient for plants, and other inorganic minerals by inducing hyperosmotic stress in the plant as well as oxidative stress.25Indole-3-acetic acid (IAA), the most common naturally occurring PGR, can be combined with essential micronutrients like Fe and tannins to help plants manage these stresses. Fe supports chlorophyll synthesis, while tannins and IAA regulate stress responses by managing root growth and nutrient uptake.26,27Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application. BRIEF SUMMARY OF THE INVENTION The accompanying drawings illustrate several embodiments of the disclosed method and compositions and together with the description, serve to explain the principles of the disclosed method and compositions. Nanoparticulate compositions and methods of use thereof, are disclosed. The nanoparticulate compositions include: (a) nanoparticles comprising a homogenous network of plant secondary metabolites optionally metals bound to the plant secondary metabolites, preferably a metal-phenolic network comprising one or more metals and one or more phenolic compounds, the network having dispersed therein one or more plant treatment agents, or (b) nanoparticles comprising a core and a shell, wherein the shell comprises one or more plant secondary metabolites and metal, preferably a metal-phenolic network comprising one or more metals and one or more phenolic compounds in the form of a metal phenolic network, wherein the core comprises hydrophobic molecules (a) in a membrane encapsulating one or more plant treatment agents and / or (b) having dispersed therein one or more plant treatment agents, and optionally wherein the nanoparticulate composition comprises a surfactant at the interface between the core and the shell. In some forms, the plant secondary metabolites comprise tannins, phenolic acids, alkaloids, flavonoids, terpenoids, glycosides, glucosinolates, cucurbitacins, or a combination thereof. In some forms the one or more phenolic compounds are caffeic acids, gallic acids, quercetins, resveratrols, vanillic acids, tannic acids, or a combination thereof. In some forms, the plant treatment agents include biostimulants, nutrients, pesticides, biopesticides, plant growth regulators, and / or vitamins. 245765042.1 KAUST 2024-082-02 PCT Exemplary plant growth regulators include, but are not limited to indole-3-acetic acids (IAA), gibberellic acids, abscisic acids, jasmonic acids, kinetins, zaxinones, lactones, humic acids, cytokinins, micronutrients, or a combination thereof. The disclosed compositions include metals bound to the plant secondary metabolites; the metals include, but are not limited to iron, magnesium, zinc, manganese, copper, nickel, molybdenum, in elemental and / or ionic forms. In some forms, the membrane comprises a monolayer or a bilayer, optionally, a surfactant is at an interface between the shell and the core. In some forms the core is a solid core comprising the hydrophobic molecules and / or the one or more plant treatment agents dispersed therein. Methods for treating a plant, plant seed or plant part, includes administering the disclosed nanoparticulate to a plant, plant seed or plant part, for an effective amount of time prior to planting. Exemplary plant seeds include, but are not limited to a wheat seed, tomato seed, capsicum seed, and pearl millet seed In some forms the nanoparticulate composition localizes in the plant seed after step (i). In some forms the nanoparticulate composition improves one or more growth metrics selected from the group consisting of seed germination rate, see germination percentage, of the plant seed in the range of 1% to 20%. In some forms wherein the nanoparticulate composition improves growth enhancement of the plant seed in the range of 1% and 70. In some forms the nanoparticulate composition increases shoot and root length in comparison to a plant seed that has not been administered the composition. In some forms the nanoparticulate composition increases fresh biomass in comparison to a plant seed that has not been administered the composition. In some forms the nanoparticulate composition improves uptake of Mg2+, Zn2+, in comparison to a plant seed that has not been administered the composition. In some forms nanoparticulate composition maintains a K+ / Na+ratio, in comparison to a plant seed that has not been administered the composition. BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings illustrate several embodiments of the disclosed method and compositions and together with the description, serve to explain the principles of the disclosed method and compositions. 345765042.1 KAUST 2024-082-02 PCT FIG.1A-1E. Characterization of SoLIN and Phelm nano assembly. (FIG.1A-1B) Transmission electron microscopy (TEM) image of SoLIN and Phelm. (FIG.1C-1D) Particle size distribution of SoLIN and Phelm. (FIG.1E) ζ-potential of the unloaded SLC, SoLIN, and Phelm (FIG.1F) SoLIN and Phelm as a final product. FIG.2A-2D. IAA release in different conditions. (FIG.2A) Cumulative IAA release from Phelm under varying pH conditions. (FIG.2B) Comparative Phelm and SoLIN release profiles under physiological conditions (PBS, pH 7.4, 37°C). The position of release at time 6h was added to compare with the release in other buffers at the same time point. (FIG.2C-2D) IAA release from Phelm and SoLIN in a simulated salinity stress environment (100 mM NaCl) and other saline buffers. FIG.3A-3D. Fe3+release and kinetics. (FIG.3A) Cumulative release of Fe3+from Phelm in water, hydrochloric acid, and sodium chloride, (FIG.3B) zero-order, (FIG.3C) first-order, and (FIG.3D) Korsmeyer-Peppas kinetic release models. FIG.4A-4B. Impact of nano-priming on wheat seedlings under simulated salinity environment. (FIG.4A) Spider charts of seed germination rate, seed germination percentage, yield enhancement percentage, root and shoot length, auxin concentration, and fresh biomass measurements. (FIG.4B) Auxin quantification in wheat roots. Asterisks denote significance (one- way ANOVA, ns: non-significant, *p <0.05, **p<0.005). Data are shown as ±STDEV (n=4, each with 20 seeds). FIG.5A-5B shows plant growth metrics of plants irrigated with freshwater (FIG.5B), and (FIG.5B) 100 mM NaCl. Asterisks denote significance (one-way ANOVA, ns: non-significant, (*p<0.05, **p<0.005, ***p<0.0005) Data are shown as ±STDEV (n=4, each with 20 plants). FIG. 6. Colloidal stability of SoLIN and Phelm. Data represent mean ± SD (n=3). Statistical analysis was performed using a t-test. The asterisk denotes significance (*p <0.05, ns= non-significant). FIG.7A-7D. Zero-order, first-order, and Korsmeyer-Peppas kinetic release models of IAA from (FIG.7A) Phelm at different pH values, (FIG.7B) Phelm and SoLIN under physiological conditions (PBS, pH 7.4 at 37°C), (FIG.7C) Phelm and (FIG.7D) SoLIN under salt stress stimulated conditions (100 mM NaCl) and other saline buffers. FIG.8A-8C. Three-dimensional (3D) plots illustrating the temporal changes in UV-vis absorption spectra of (FIG.8A) encapsulated and (FIG.8B) non-encapsulated IAA during thermal treatment at 45°C in a simulated soil environment. (FIG.8C) Normalized concentration plot depicts the degradation of IAA over time. 445765042.1 KAUST 2024-082-02 PCT FIG.9A-9D. (FIG.9A) IAA release from Phelm in a simulated soil environment. (FIG. 9B) Linear fitting of zero-order, (FIG.9C) first-order, and (FIG.9D) Korsmeyer-Peppas kinetic release model. FIG.10A-10B. Seed germination rate of wheat seedlings under (FIG.10A) salt stress simulated conditions, and (FIG.10B) freshwater. FIG.11A-11E. Impact of nano-priming on (FIG.11A) germination rate, (FIG.11B) germination percentage, (FIG.11C-11D) root and shoot length, FIG.11E) fresh biomass measurements of wheat seedlings under simulated salinity conditions. Asterisks denote significance (one-way ANOVA, ns: non-significant, *p <0.05, **p<0.01, ***p<0.001, ****p<0.0001). Data are shown as ±STDEV (n=4, each with 20 seeds). FIG.12A-12E. Impact of nano-priming on (FIG.12A) seed germination rate, (FIG.12B) seed germination percentage, (FIG.12C-D) root and shoot length, (FIG.12E) fresh biomass measurements of wheat seedlings under normal conditions. Asterisks denote significance (one-way ANOVA, ns: non-significant, *p<0.05, ***p<0.0005) Data are shown as ±STDEV (n=4, each with 20 seeds). FIG.13. Comparison of the H2O2scavenging capacity of Phelm at different concentrations after one hour with respect to different precursors and the blank control. FIG.14. Synthetic route of metal–phenolic nanocarriers (MPNs). Empty nanocarriers (CAFZin) were synthesized via coordination-driven self-assembly of caffeic acid and ZnSO₄·7H₂O. Kinetin was encapsulated under the same conditions to produce CAFZin-K nanocarriers. FIG.15 A-15C. Physicochemical characterization of MPNs. (FIG.15A) FTIR spectra showing representative bands of CAFZin-K in comparison with non-encapsulated Kn, caffeic acid, and CAFZin. (FIG.15B) XPS broad scan (O 1s, N 1s, C 1s, and Zn 2p) of Caffeic acid, Kinetin, and CAFZin-K. (FIG.15C) UV-vis spectra of CAFZin-K compared with non-encapsulated Kinetin (Kn), caffeic acid, and CAFZin. FIG.16A-16E. Release and stability profile of kinetin from CAFZin-K metal–phenolic nanocarriers. (FIG.16A) Cumulative release of kinetin from CAFZin-K at different pH conditions (pH 3, 5, and 9). (FIG.16B) Kinetin release under saline stress conditions, including 15% and 30% seawater, and 100 and 150 mM NaCl solutions. (FIG.16C) Release profile of kinetin in a soil model system simulating environmental conditions. (FIG.16D) Stability comparison of CAFZin-K and free kinetin following exposure to UV irradiation (~365 nm) for 24 hours. (FIG.16E) Thermal stability of CAFZin-K and free kinetin after incubation at 45^°C for 24 hours. 545765042.1 KAUST 2024-082-02 PCT FIG.17A-17B. Hydrogen peroxide (H₂O₂) scavenging activity of CAFZin-K. (FIG.17A) H₂O₂ scavenging percentage of CAFZin-K at various concentrations, compared with corresponding controls at the same concentrations. (FIG.17B) Overall scavenging activity of CAFZin-K compared to non-encapsulated kinetin and individual precursors. A control containing only H₂O₂ was used as a reference. All samples were incubated for 24 hours, and H₂O₂ levels were quantified by UV absorbance at 230^nm. Experiments were performed in triplicate (n = 3). FIG.18A-18B. Impact of seed nanopriming on tomato plant growth under normal and saline conditions. Growth parameters of tomato plants derived from primed seeds and irrigated with (FIG.18A) tap water and (FIG.18B) 15% seawater. Plants were treated with control (non-primed), kinetin, CAFZin, and CAFZin-K formulations. Root length, fresh biomass, and dry biomass were measured at week 4, and analyzed using one-way ANOVA. All experiments were performed in triplicate (n = 3), and data are presented as mean ± standard deviation (SD). Asterisks indicate statistical significance: ns = not significant, *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001. FIG.19A-19B. Elemental distribution in tomato plant tissues. (FIG.19A) Essential micronutrients zinc (Zn) and magnesium (Mg), and (FIG.19B) Sodium (Na) and potassium (K) distribution in tomato stems, leaves, and roots after four weeks of irrigation with tap water (TW) or 15% seawater (SW). FIG.20A-20B. Kinetin retention in primed seeds. (FIG.20A) Comparative Zn content in different seed types primed with CAFZin-K. (FIG.20B) Quantification of kinetin retained in tomato seeds after priming with either CAFZin-K or non-encapsulated Kinetin. FIG.21. Size and surface charge characterization of metal–phenolic nanocarriers. Dynamic Light Scattering (DLS) was used to determine the hydrodynamic diameter of CAFZin and CAFZin- K nanocarriers. Zeta potential measurements were performed for free Kinetin, CAFZin, and CAFZin-K to evaluate surface charge variations and confirm successful Kinetin encapsulation. FIG.22. Calibration curve of Kinetin. A standard calibration curve was generated by measuring the absorbance of Kinetin at 270 nm using UV–vis spectroscopy. This curve was used to calculate the encapsulation efficiency and loading capacity of CAFZin-K nanocarriers. FIG.23. Release Kinetics at different pH. Kinetin release profiles at different pH values were fitted to zero-order, first-order, and Korsmeyer–Peppas kinetic models to investigate the release mechanism from CAFZin-K nanocarriers. The results indicate a complex release behavior governed by both diffusion of Kinetin and relaxation of the metal–phenolic network structure. Figure 24. Release Kinetics in salt media. Kinetin release profiles in different salt media, values were fitted to zero-order, first-order, and Korsmeyer–Peppas kinetic models to investigate 645765042.1 KAUST 2024-082-02 PCT the release mechanism from CAFZin-K nanocarriers. The results indicate a complex release behavior governed by both diffusion of Kinetin and relaxation of the metal–phenolic network structure. FIG.25. Release kinetics in a soil model. Kinetin release profiles were evaluated in a simulated soil environment and fitted to zero-order, first-order, and Korsmeyer–Peppas kinetic models to elucidate the release mechanism. The data indicate a Fickian diffusion-controlled process, where the release rate is proportional to the remaining amount of Kinetin within the nanoparticle matrix. FIG.26. Biocompatibility assessment of nanoparticle components and formulations. Biocompatibility of caffeic acid, zinc sulfate heptahydrate (ZnSO₄·7H₂O), Kinetin, CAFZin, and CAFZin-K nanocarriers evaluated on HepG2 (left) and NIH-3T3 (right) cell lines after 24^h of exposure. Cell viability was measured using the CCK-8 assay to assess potential cytotoxic effects. FIG.27A-27B. Impact of nanopriming on tomato seeds. Growth parameters of tomato plants derived from primed seeds and irrigated with (FIG.27A) tap water and (FIG.27B) 15% seawater during early germination. Seeds were treated with control (non-primed), kinetin, CAFZin, and CAFZin-K formulations. Measurements were analyzed using one-way ANOVA. All experiments were performed in triplicate (n = 3), and data are presented as mean ± standard deviation (SD). Asterisks indicate statistical significance: ns = not significant, *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001. FIG.28A-28D. Impact of nanopriming on tomato plant growth under normal and saline conditions. Plants were treated with control (non-primed), Kinetin, CAFZin, and CAFZin-K formulations. Plant height was recorded at weeks 1, 2, and 4 (FIG.28A-28B), and analyzed using two-way ANOVA to evaluate the effect of treatment and time. All experiments were performed in triplicate (n = 3), and data are presented as mean ± standard deviation (SD). Asterisks indicate statistical significance: ns = not significant, *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001. Representative pictures of tomato plants and roots at different stages of the treatment with (FIG. 28C) tap water and (FIG.28D) 15% seawater. DETAILED DESCRIPTION OF THE INVENTION I. Definitions Use of the term "about" is intended to describe values either above or below the stated value in a range of approx. + / - 10%; in other embodiments the values may range in value either above or below the stated value in a range of approx. + / - 5%; in other embodiments the values may range in value either above or below the stated value in a range of approx. + / - 2%; in other embodiments the 745765042.1 KAUST 2024-082-02 PCT values may range in value either above or below the stated value in a range of approx. + / - 1%. The preceding ranges are intended to be made clear by context, and no further limitation is implied. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. The term “plant substrate” refers to a substrate commonly used for growing plants, including plant seeds, plant roots and plant seedlings. Non-limiting examples of such plant substrates include, but are not limited to soil, peat, compost, vermiculite, perlite, sand, clay, and combinations thereof. The term "culture" describes a population of microorganisms cultivated or grown in a nutrient medium. Also as used herein the phrases "fresh nutrient medium", "suitable nutrient medium" and the like mean an aqueous solution or suspension of nutrients which are necessary and sufficient to support the growth of the disclosed microorganisms. By the term “effective amount” of a composition as provided herein is meant a nontoxic but sufficient amount of the composition to provide the desired result. As will be pointed out below, the exact amount required will vary from plant to plant, depending on the species and the severity of the disease that is being treated, the mode of administration, and the like. Thus, it is not possible to specify an exact “effective amount.” However, an appropriate effective amount can be determined by one of ordinary skill in the art using only routine experimentation. The term “plant substrate” refers to a substrate commonly used for growing plants, including plant seeds, plant roots and plant seedlings. Non-limiting examples of such plant substrates include, but are not limited to soil, peat, compost, vermiculite, perlite, sand, clay, and combinations thereof. The term “plant” is used in it broadest sense. It includes, but is not limited to, any species of woody, ornamental or decorative crop or cereal, and fruit or vegetable plant. It also refers to a plurality of plant cells that are largely differentiated into a structure that is present at any stage of a plant’s development. Such structures include, but are not limited to, a fruit, shoot, stem, leaf, flower petal, etc. The term “plant cell” refers to a structural and physiological unit of a plant, comprising a protoplast and a cell wall. The plant cell may be in form of an isolated single cell or a cultured cell, 845765042.1 KAUST 2024-082-02 PCT or as a part of higher organized unit such as, for example, plant tissue, a plant organ, or a whole plant. The term “plant cell culture” refers to cultures of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes and embryos at various stages of development. The term “plant material” refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant. The term “plant organ” refers to a distinct and visibly structured and differentiated part of a plant such as a root, stem, leaf, flower bud, or embryo. As used herein, “plant part” or “part of a plant” can include, but is not limited to cuttings, cells, protoplasts, cell tissue cultures, callus (calli), cell clumps, embryos, stamens, pollen, anthers, pistils, ovules, flowers, seed, petals, leaves, stems, and roots. The term “plant tissue” includes differentiated and undifferentiated tissues of plants including those present in roots, shoots, leaves, pollen, seeds and tumors, as well as cells in culture (e.g., single cells, protoplasts, embryos, callus, etc.). Plant tissue may be in planta, in organ culture, tissue culture, or cell culture. The term “plant part” as used herein refers to a plant structure, a plant organ, or a plant tissue. “Seed germination” refers to growth of an embryonic plant contained within a seed resulting in the formation and emergence of a seedling. II. Nanoparticulate Composition The nanoparticulate compositions disclosed herein are metal phenolic nanocarriers. Metal phenolic nanocarriers (MPNs) feature a structure of interconnected metal ions and phenolic ligands. These components form a three-dimensional network through coordination bonds and other interactions, creating stable nanocarriers that are roughly spherical in shape. The specific metal ion and phenolic compound determine the nanocarrier's size, charge, and functionality. Depending on the materials and conditions, MPNs can form either homogeneous networks or core- shell structures where the metal-phenolic network forms a coating. In some forms, the disclosed nanoparticulate composition contains homogenous network. In these forms, the metal phenolic nanocarriers (MPNs) as disclosed herein feature a structure of interconnected metal ions and phenolic ligands. These components form a three-dimensional network through coordination bonds and other interactions, creating stable nanocarriers that are roughly spherical in shape. These nanocarriers are formed directly through the controlled self- 945765042.1 KAUST 2024-082-02 PCT assembly of metal and polyphenol components, without the need for a template. The forms, metals and phenolic network as described below, which can be in the form of a homogenous network as described herein, or in the form of shell, as disclosed for nanocarriers with a core-shell structure. In some form, the disclosed nanoparticulate composition contains a core and a shell, wherein the shell contains one or more metals and one or more phenolic compounds and the nanoparticles are in the form of a metal phenolic network. In this structure, a pre-existing nanoparticle forms the core, and a layer of MPN is coated around it to provide a shell. In some embodiments, the core is in the form of a solid lipid matrix, containing one or more surfactants and one or more lipids; The disclosed nanoparticulate compositions contains one or more plant treatment agents, in its core. In some forms the one or more plant treatment agents contains one or more hydrogen- bonding functional groups, and the one or more plant treatment agents are incorporated into the matrix of the nanoparticle, for example, solid lipid matrix through hydrogen bonding between at least one of the one or more hydrogen-bonding functional groups and the solid lipid matrix. In some instances, the nanoparticulate composition enhances the stability, solubility, sustained release, and / or bioavailability of the one or more plant treatment agents in a plant system, especially under stress conditions such as high salinity, for example 100 mM NaCl. In some instances, co-release of the one or more phenolic compounds and the one or more metals via nanoparticulate composition provides abiotic stress resilience in plant systems, for example resilience against high salinity conditions (e.g.100 mM NaCl). By shielding active compounds from premature degradation or environmental loss, these carriers enable controlled and sustained release. Upon application, the nanoparticulate composition localizes into the plant system and undergoes gradual disassembly, often triggered by enzymatic or pH-responsive mechanisms, allowing localized, time-regulated cargo release. The disclosed nanoparticulate compositions demonstrate higher uptake and bioavailability of encapsulated one or more plant treatment agents in comparison to conventional formulations. In some forms, the nanoparticulate compositions include: (a) nanoparticles comprising a homogenous network of plant secondary metabolites optionally metals bound to the plant secondary metabolites, preferably a metal-phenolic network comprising one or more metals and one or more phenolic compounds, the network having dispersed therein one or more plant treatment agents, or (b) nanoparticles comprising a core and a shell, wherein the shell comprises one or more plant secondary metabolites and metal, preferably a metal-phenolic network comprising one or more 1045765042.1 KAUST 2024-082-02 PCT metals and one or more phenolic compounds in the form of a metal phenolic network, wherein the core comprises hydrophobic molecules (a) in a membrane encapsulating one or more plant treatment agents and / or (b) having dispersed therein one or more plant treatment agents, and optionally wherein the nanoparticulate composition comprises a surfactant at the interface between the core and the shell. In some forms, the plant secondary metabolites comprise tannins, phenolic acids, alkaloids, flavonoids, terpenoids, glycosides, glucosinolates, cucurbitacins, or a combination thereof. In some forms, the one or more phenolic compounds include, but are not limited to caffeic acids, gallic acids, quercetins, resveratrols, vanillic acids, tannic acids, or a combination thereof. In some forms the one one or more plant treatment agents include, but are not limited to biostimulants, nutrients, pesticides, biopesticides, plant growth regulators, and / or vitamins. In some forms the plant growth regulators include, but are not limited to indole-3-acetic acids (IAA), gibberellic acids, abscisic acids, jasmonic acids, kinetins, zaxinones, lactones, humic acids, cytokinins, micronutrients, or a combination thereof. The nanoparticulate composition include comprising metals bound to the plant secondary metabolites. The method can be iron, magnesium, zinc, manganese, copper, nickel, molybdenum, in elemental and / or ionic forms. In some forms the hydrophobic molecules include lipids, hydrophobic polymers, or a combination thereof, optionally wherein the hydrophobic polymers comprise polyesters, polyanhydrides, poly(p-dioxanone)s, polycarbonates, or a combination thereof, such as poly(α- hydroxy acid)s, poly(lactone)s, or combination thereof, such as poly(lactic acid)s, poly(glycolic acid)s, poly(lactic acid-co-glycolic acid)s, poly(caprolactone)s, poly(pentadecalactone)s, poly(hydroxybutyrate-co-hydroxyvalerate)s, poly(hydroxybutyrate)s, or a combination thereof. Useful lipids include waxes, triglycerides, partial glycerides, phospholipids, fatty acids, glycolipids, or a combination thereof. Exemplary waxes includenatural waxes (such as carnauba wax, shea butter, beeswax, candelilla wax, rice bran wax, jojoba wax, and bayberry wax), vegetable oils ( (such as castor oil, palm oil, or soybean oil), synthetic waxes (such as paraffin wax or microcrystalline wax), synthetic esters (such as cetyl palmitate or stearyl stearate), or a combination thereof. Exemplary triglycerides include natural or semi-synthetic triglycerides (such as glyceryl tristearate, glyceryl tripalmitate, glyceryl trimyristate, glyceryl trilaurate, glyceryl triricinoleate palm oil triglycerides, coconut oil triglycerides, soybean oil triglycerides, or a combination thereof. The partial glycerides include, but are not limited to, monoglycerides (such as Glyceryl monostearate (GMS), glyceryl monopalmitate, glyceryl monolaurate, or glyceryl monooleate), 1145765042.1 KAUST 2024-082-02 PCT diglycerides (such as glyceryl distearate, glyceryl dipalmitate, glyceryl distearate or oleate mixtures), or a combination thereof. The fatty acids include, but are not limited to, saturated fatty acids: (such as stearic acid, palmitic acid, myristic acid, lauric acid, or behenic acid), unsaturated fatty acids (such as oleic acid, linoleic acid, linolenic acid, or ricinoleic acid), hydroxylated or functionalized fatty acids, or a combination thereof. Useful phospholipids include, but are not limited to, soy lecithin, egg lecithin, hydrogenated soy phosphatidylcholine (HSPC), hydrogenated egg phosphatidylcholine (HEPC), distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dibehenoylphosphatidylcholine, hydrogenated phosphatidylethanolamine, hydrogenated phosphatidylglycerol, hydrogenated phosphatidylinositol, or a combination thereof. In some forms the membrane comprises a monolayer or a bilayer. In some forms, the nanoparticles include surfactant at an interface between the shell and the core. The surfactant can be nonionic surfactants, anionic surfactants, cationic surfactants, amphiphiles, or a combination thereof. In some forms, the core is a solid core comprising the hydrophobic molecules and / or the one or more plant treatment agents dispersed therein. A. Shell 1. Form The shell of the nanoparticle composition described herein is in the form of a metal phenolic network. The metal phenolic network is formed via the coordination between the one or more metals and the one or more phenolic compounds. The one or more phenolic compounds are capable of chelating the one or more metals to form a cross-linked network structure. The network may be further formed through additional interactions such as hydrogen bonding, π–π stacking, or hydrophobic association. The structure is generally porous and permeable to small molecules, and its physical and chemical properties can be tuned by selection of the one or more metals, the one or more phenolic compounds, and the assembly conditions. The metal phenolic network of the shell provides a biodegradable and biocompatible platform for protection and release of the one or more plant treatment agents 2. Metal The metal–phenolic network is formed using one or more metals capable of coordinating with the one or more phenolic compounds to form stable complexes. In some embodiments, the one or more metals are divalent, trivalent, and tetravalent metal ions, for example iron, aluminum, chromium, gallium, zinc, copper, cobalt, nickel, manganese, magnesium, calcium, titanium, 1245765042.1 KAUST 2024-082-02 PCT zirconium, hafnium, molybdenum, gold, silver, and platinum. Preferably, the one or more metals are magnesium, zinc, manganese, copper, nickel, molybdenum, or iron. In some instances, the one or more metals, such as zinc, serve as a source of micronutrients and are delivered to a plant system as the nanoparticulate composition undergoes gradual disassembly to promote development, yield, stress tolerance, and / or nutrient uptake. 3. Phenolic Compound The metal-phenolic network is formed using one or more phenolic compounds capable of coordinating with the one or more metals to form stable complexes. Suitable phenolic compounds include naturally occurring or synthetic phenolic compounds. In some instances, the one or more phenolic compounds are phenols and catechols (such as phenol, catechol, resorcinol, and hydroquinone), gallic acid derivatives (such as gallic acid, methyl gallate, and propyl gallate), tannins (such as tannic acid, ellagic acid, and pyrogallol), flavonoids (such ascatechin, epicatechin, epigallocatechin gallate (EGCG), quercetin, rutin, and myricetin, phenolic acids (such as caffeic acid, chlorogenic acid, ferulic acid, sinapic acid, coumaric acid, and vanillic acid), or other polyphenols (such as dopamine, norepinephrine, l-DOPA, lignin-derived phenolics). Preferably, the one or more phenolic compounds are caffeic acid, gallic acid, quercetin, resveratrol, vanillic acid, or tannic acid. In some instances, the one or more phenolic compounds, such as caffeic acid, serve as a source of micronutrients and are delivered to a plant system as the nanoparticulate composition undergoes gradual disassembly to promote development, yield, stress tolerance, and / or nutrient uptake. B. Core 1. Form (i). Core In some instances, the core is in the form of a solid lipid matrix, formed from (and therefore containing) one or more surfactants and one or more lipids. In some forms the one or more plant treatment agents contain one or more hydrogen-bonding functional groups. In some forms the one or more plant treatment agents are incorporated into the solid lipid matrix through hydrogen bonding between at least one of the one or more hydrogen-bonding functional groups and the solid lipid matrix. The solid lipid matrix core is functionalized on the outer surface with the metal- phenolic network formed by the complexation of the one or more phenolic compounds and the one or more metals. 1345765042.1 KAUST 2024-082-02 PCT (a). Surfactants The solid lipid matrix is made using (and therefore, contains) one or more surfactants to stabilize the one or more lipids by reducing interfacial tension between the lipid phase and the surrounding medium, promoting uniform particle size distribution, preventing aggregation, and enhancing the dispersibility and stability of the matrix. In some instances, the one or more surfactants are nonionic surfactants, anionic surfactants, cationic surfactants, or amphiphiles. In some instances, the nonionic surfactants are polysorbate derivatives (such as TWEEN 20, TWEEN 40®, TWEEN 60®, and TWEEN 80®), polyethylene glycol (PEG) derivatives (such as PEG-40 stearate or PEG-60 hydrogenated castor oil), poloxamer derivatives (PLURONIC F68®, PLURONIC F127®, and PLURONIC P85®), polyvinyl alcohol (PVA) derivatives, sorbitan ester derivatives (SPAN 20®, SPAN 40®, SPAN 60®, SPAN 80®), or polyoxyethylene ethers. In some instances, the anionic surfactants are sodium dodecyl sulfate (SDS), sodium cholate, or sodium deoxycholate. In some instances, the amphiphiles are bile salts, phospholipids, or lecithin. In some instances, the cationic surfactants are cetyltrimethylammonium bromide (CTAB) or stearylamine. (b). Lipids The solid lipid matrix contains one or more lipids to provide a solid or semi-solid hydrophobic environment capable of encapsulating the one or more plant treatment agents, stabilizing the core structure, influencing the release of encapsulated the one or more plant treatment agents, and contributing to the overall mechanical and thermal stability of the matrix. In some instances, the one or more lipids are triglycerides, partial glycerides, phospholipids, fatty acids or waxes. In some instances, the waxes are natural waxes (such as shea butter, beeswax, carnauba wax, candelilla wax, rice bran wax, jojoba wax, and bayberry wax), vegetable oils (such as castor oil, palm oil, or soybean oil), synthetic waxes (such as paraffin wax or microcrystalline wax) and / or synthetic esters (such as cetyl palmitate or stearyl stearate). Preferably, the wax is cetyl palmitate, paraffin wax, carnauba wax, beeswax, shea butter, or candelilla wax. In some instances, the triglycerides are natural or semi-synthetic triglycerides (such as glyceryl tristearate, glyceryl tripalmitate, glyceryl trimyristate, glyceryl trilaurate, glyceryl triricinoleate palm oil triglycerides, coconut oil triglycerides, or soybean oil triglycerides. In some instances, the partial glycerides are monoglycerides (such as Glyceryl monostearate (GMS), glyceryl monopalmitate, glyceryl monolaurate, or glyceryl monooleate) and / or diglycerides (such as lyceryl distearate, glyceryl dipalmitate, glyceryl distearate or oleate mixtures) 1445765042.1 KAUST 2024-082-02 PCT In some instances, the fatty acids are saturated fatty acids: (such as stearic acid, palmitic acid, myristic acid, lauric acid, or behenic acid), unsaturated fatty acids (such as oleic acid, linoleic acid, linolenic acid, or ricinoleic acid), or hydroxylated or functionalized fatty acids. Preferably, the fatty acids are stearic acid, palmitic acid, behenic acid, and / or lauric acid. In some instances, the phospholipid is soy lecithin, egg lecithin, hydrogenated soy phosphatidylcholine (HSPC), hydrogenated egg phosphatidylcholine (HEPC), distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dibehenoylphosphatidylcholine, hydrogenated phosphatidylethanolamine, hydrogenated phosphatidylglycerol, and / or hydrogenated phosphatidylinositol. (c). Solid Lipid Matrix Properties In instances wherein the core is a solid lipid matrix, the nanoparticulate composition has less crystalline arrangement in comparison to solid lipid matrix without a metal phenolic network shell. In these instances, the nanoparticulate composition has a lower enthalpy of fusion in comparison to a crystalline phase of the one or more lipids used in the nanoparticulate composition alone. This difference is attributed to the solid lipid core crystalline phase being less organized than the crystalline phase of the pure one or more lipids, which requires more energy to overcome cohesion forces in the crystal lattice. In instances wherein the core is a solid lipid matrix, the nanoparticulate composition has no change in particle size after 1 month of storage at 4oC. Without being bound, less ordered crystallinity leads to higher physical stability and prevents early release of the loaded one or more plant treatment agents from the nanoparticulate composition during storage. In instances wherein the core is a solid lipid matrix, the nanoparticulate composition releases more of the one or more plant treatment agents in comparison to the solid lipid matrix without a metal phenolic network shell. Without being bound by theory, it is hypothesized that the hydrophilic groups of the solid lipid core, remain on the surface of the solid lipid core, and the interaction between the metal-phenolic shell and the exposed hydrophilic groups makes the solid lipid core a little labile, which leads to more release of the one or more plant treatment agents from the core. In instances wherein the core is a solid lipid matrix, the nanoparticulate composition releases more of the one or more plant treatment agents under salinity stress conditions (e.g.100 mM NaCl) in comparison to the solid lipid matrix without a metal phenolic network shell. • In these instances, the nanoparticulate composition releases the one or more plant treatment agents in 1545765042.1 KAUST 2024-082-02 PCT the range of 70% and 90%, in the range of 75% and 90%, in the range of 80% and 90%, in the range of 82% and 88%, or in the range of 84% and 88% in 100 mM NaCl. Without being bound to theory, it is hypothesized that higher ionic strength (such as at least 100 mM NaCl or 2 M NaCl), sodium ions shield the one or more phenolic compounds, allowing them to extend away from the one or more metals rather than forming a closed complex and increasing solubility. This ‘loosening up’ of the coating around solid lipid matrix core increases the release of the one or more plant treatment agents. In instances wherein the core is a solid lipid matrix, the nanoparticulate composition release the one or more plant treatment agents faster as the pH values lower. In these instances, the nanoparticulate composition releases the one or more plant treatment agents in the range of 90% and 100%, in the range of 92% and 100%, in the range of 94% and 100%, in the range 96% and 100%, or in the range of 98% and 100% at a pH of 3. In these instances, the nanoparticulate composition releases the one or more plant treatment agents in the range of 70% and 90%, in the range of 72% and 90%, in the range of 74% and 90%, in the range of 76% and 90%, in the range of 78% and 90%, in the range of 78% and 88%, in the range of 78% and 86%, in the range of 78% and 84%, or in the range of 78% and 82% at a pH of 5. In these instances, the nanoparticulate composition releases the one or more plant treatment agents in the range of 50% and 70%, in the range of 52% and 70%, in the range of 54% and 70%, in the range of 56% and 70%, in the range of 58% and 70%, in the range of 58% and 68%, in the range of 58% and 66%, in the range of 58% and 66%, in the range of 58% and 64%, in the range of 58% and 62% at a pH of 7.5. Without being bound to theory, the change in structure from bis- and tris- to mono- complexes triggered by the protonation of the one or more phenolic compounds, for example the catechol groups in tannic acid, results in the partial disassembly of the shells cross-links. In instances wherein the core is a solid lipid matrix, the core has a particle size of at least 100 nm, such as in the range of 100 and 200 nm, in the range of 100 and 180 nm, in the range of 100 and 160 nm, in the range of 100 and 140 nm, in the range of 100 and 130 nm, in the range of 110 and 130 nm, or in the range of 120 and 130 nm. In these instances, the nanoparticulate composition has a particle size of at least 200 nm, such as in the range of 250 and 400 nm, in the range of 250 and 380 nm, in the range of 250 and 360 nm, in the range of 250 and 340 nm, in the range of 250 and 320 nm, in the range of 250 and 300 nm, in the range of 260 and 300 nm, in the range of 270 and 300 nm, or in the range of 270 and 290 nm. 1645765042.1 KAUST 2024-082-02 PCT In instances wherein the core is a solid lipid matrix, the nanoparticulate composition has a zeta potential of at least -40 mV, such as in the range of -20 and -40 mV, in the range of -20 and -38 mV, in the range of -20 and -36 mV, in the range of -20 and -34 mV, in the range of -20 and -32 mV, in the range of -22 and -32 mV, in the range of -24 and -32 mV, in the range of -26 and -32 mV, in the range of -28 and -32 mV or in the range of -29 and -31 mV. In instances wherein the core is a solid lipid matrix, the nanoparticulate composition has a loading capacity of at least 3%, such as in the range of 3% and 20%, in the range of 5% and 20%, in the range of 7% and 20%, in the range of 10% and 20%, in the range of 10% and 18%, in the range of 10% and 16%, in the range of 12% and 16%, or in the range of 12% and 14%. In instances wherein the core is a solid lipid matrix, the nanoparticulate composition has an encapsulation efficiency of at least 75%, such as in the range of 75% and 100%, in the range of 77% and 100%, in the range of 79% and 100%, in the range of 81% and 100%, in the range of 83% and 100%, in the range of 83% and 98%, in the range of 83% and 96%, in the range of 83% and 94%, in the range of 83% and 92%, in the range of 83% and 90%, in the range of 83% and 88%, or in the range of 85% and 88%. In instances wherein the core is a solid lipid matrix, the nanoparticulate composition releases the one or more plant treatment agents in the range of 20% and 50%, in the range of 20% and 45%, in the range of 20% and 40%, in the range of 20% and 35%, in the range of 25% and 35%, in the range of 27% and 35%, in the range of 27% and 33%, or in the range of 29% and 33%, in PBS In instances wherein the core is a solid lipid matrix, the nanoparticulate composition has a hydrogen peroxide scavenging activity of at least 60%, such as in the range of 60% and 85%, in the range of 62% and 85%, in the range of 64% and 85%, in the range of 66% and 85%, in the range of 68% and 85%, in the range of 68% and 82%, in the range of 68% and 80%, in the range of 68% and 78%, in the range of 68% and 76%, or in the range of 68% and 74%. 2. Plant Treatment Agents The nanoparticulate composition disclosed herein contains one or more plant treatment agents encapsulated by the metal-phenolic network shell. The one or more plant treatment agents are biostimulants, nutrients, pesticides, biopesticides, plant growth regulators, and / or vitamins. In some instances, the biostimulants are microbial biostimulants, plant-derived biostimulants, inorganic / chemical biostimulants, and / or physical biostimulants. In some instances, the nutrients are primary macronutrients (such as nitrogen, phosphorus, potassium), secondary macronutrients (such as calcium, magnesium, or sulfur), micronutrients 1745765042.1 KAUST 2024-082-02 PCT (such as iron, manganese, zinc, copper, boron molybdenum, chlorine, or nickel), silicon, cobalt, and / or selenium. In some instances, the pesticides are insecticides (such as organophosphates: malathion, chlorpyrifos, carbamates: carbaryl, methomyl, pyrethroids: permethrin, cypermethrin, neonicotinoids: imidacloprid, thiamethoxam, Bacillus thuringiensis toxins, or Spinosad), herbicides (such as glyphosate, 2,4-Dichlorophenoxyacetic acid, atrazine, paraquat, or dicamba), fungicides (such as azoles: tebuconazole, propiconazole, strobilurins: azoxystrobin, kresoxim-methyl, copper- based fungicides: copper hydroxide, copper oxychloride, or sulfur-based compounds), rodenticides (such as bromadiolone, warfarin, or zinc phosphide) and / or Nematicides (such as fosthiazate, oxamyl, or fluensulfone). In some instances, the biopesticides are microbial, plant-derived, or biochemical agents bacteria such as Bacillus thuringiensis, Bacillus subtilis, and Pseudomonas fluorescens; fungi (such as Beauveria bassiana, Metarhizium anisopliae, and Trichoderma spp.), viruses (such as baculoviruses including Nucleopolyhedrovirus) entomopathogenic nematodes (such as Steinernema and Heterorhabditis), plant-derived compounds (such as neem-based products like azadirachtin, pyrethrum, rotenone, and essential oils including thyme, clove, and peppermint) and / or biochemical agents (such as insect growth regulators, like methoprene and pyriproxyfen, pheromones and plant- incorporated protectants). In some instances, the vitamins are fat-soluble vitamins in plants (such as vitamin A: beta- carotene, alpha-carotene, and lycopene; vitamin E: tocopherols and tocotrienols; or Vitamin K: phylloquinone), and / or water-soluble vitamins (such as vitamin C: ascorbic acid; and B-complex vitamins: thiamine, riboflavin, niacin, pantothenic acid, pyridoxine, pyridoxal, pyridoxamine, biotin, and folate In some instances, the plant growth regulators are auxins (such as indole-3-acetic acid, indole-3-butyric acid, naphthaleneacetic acid, or 2,4-Dichlorophenoxyacetic acid), gibberellins (such as gibberellic acid) cytokinins (such as kinetin, zeatin, 6-benzylaminopurine, or thidiazuron), abscisic acid, ethylene, jasmonates (such as jasmonic acid and methyl jasmonate), strigolactones, brassinosteroids, zaxinones, and / or lactones. Preferably, the one or more plant treatment agents are indole-3-acetic acid (IAA), gibberellic acid, abscisic acid, jasmonic acid, kinetin, zaxinones, and / or lactones. (i). Hydrogen Bonding Groups In instances wherein the core is a solid lipid matrix, the one or more plant treatment agents contain one or more hydrogen functional groups for the one or more plant treatment agents to 1845765042.1 KAUST 2024-082-02 PCT incorporate into the solid lipid matrix through hydrogen bonding between at least one of the one or more hydrogen-bonding functional groups and the solid lipid matrix. In these instances, the hydrogen bonding functional groups are hydroxyl groups, amino groups, amide groups, carboxyl groups, carbonyl groups, ester groups, ether groups, thiol groups, thioether groups, nitrile groups, nitro groups, sulfoxide groups, sulfone groups, phosphate groups, phosphonate groups and.or halogens C. Nanoparticulate Composition Properties The nanoparticulate compositions disclosed herein are uniformly dispersed spherical nanoparticles with an amorphous structure that provides solubility and stability for the one or more plant treatment agents. Encapsulation within the metal-phenolic network provides protection for the one or more plant treatment agents against degradation. In some instances, the nanoparticulate composition disclosed herein provide antioxidant protection, especially under stress conditions such as high salinity (at least 100 mM NaCl), to improve plant resilience. Without being bound to theory, the antioxidant protections is provided by the one or more metals (such as zinc) and the one or more phenolic compounds (such as caffeic acid). In some instances, the hydrogen peroxide scavenging activity of the nanoparticulate composition is greater compared to the scavenging activity of the one or more phenolic compounds, the one or more metals, or the one or more plant treatment agents alone. In some instances, the nanoparticulate composition has a hydrogen peroxide scavenging activity of at least 10%, such as in the range of 10% and 50%, in a range of 15% and 50%, in a range of 20% and 50%, in a range of 25% and 50%, in a range of 30% and 50%, in a range of 35% and 50%, or in a range of 35% and 45%. In some instances, administration of the nanoparticulate composition improves proximity and / or uptake of the one or more plant treatment agents in a plant system in comparison to administration the one or more plant treatment agents alone. In some embodiments, the nanoparticulate composition is administered to prime a plant seed, contributing to early-stage physiological preparedness under stress conditions, such as high salinity. In some instances ,the nanoparticulate composition provides support for root activity and transport processes of one or more plant treatment agents. In some instances, the nanoparticulate composition promotes ion absorption and supports ion homeostasis, even under stressed conditions (for example high salinity conditions). For example, the nanoparticulate composition maintains a 1945765042.1 KAUST 2024-082-02 PCT healthy K⁺ / Na⁺ ratio as a key marker of salt tolerance by simultaneously limiting Na⁺ accumulation and sustaining K⁺ availability. The nanoparticulate composition disclosed herein exhibits pH responsive release of the one or more plant treatment agents. Without being bound to theory, at alkaline pH, deprotonation of the one or more phenolic compounds promotes strong coordination with metal ions, resulting in a more rigid and stable structure that hinders release. In contrast, acidic conditions lead to protonation of the one or more phenolic compounds, weakening metal–ligand coordination and triggering disassembly of the network, thereby facilitating release of the one or more plant treatment agents. In some instances, the nanoparticulate composition releases the one or more plant treatment agents in a range of 45% and 60%, in a range of 47% and 60%, in a range of 49% and 60%, in a range of 49% and 58%, in range of 49% and 56%, or in a range of 49% and 54% at a pH of 3. In some instances, the nanoparticulate composition releases the one or more plant treatment agents in a range of 30% and 45%, in a range of 32% and 45%, in a range of 34% and 45%, in a range of 36% and 45%, in a range of 36% and 43%, or in a range of 36% and 41%, at a pH of 5. In some instances, the nanoparticulate composition releases the one or more plant treatment agents in a range of 10% and 30%, in a range of 12% and 30%, in a range of 14% and 30%, in a range of 16% and 30%, in a range of 18% and 30%, in range of 20% and 30%, in range of 20% and 28%, or in range of 20% and 26%, at a pH of 7.5. In some instances, the presence of multivalent ions (such as Mg²⁺ and Ca²⁺), can disrupt or reorganize the coordination matrix, enabling both diffusion and matrix relaxation. In some instances, the presence of simpler ionic composition (such as only NaCl) solutions leads to more uniform, diffusion-dominated release. In some instances, the nanoparticulate composition has a diffusion-controlled process of release for the one or more plant treatment agents. For example, release from a nanoparticulate composition encapsulating kinetin with a metal phenolic network containing caffeic acid and zinc followed a Fickian diffusion mechanism, characteristic of primarily diffusion-controlled processes In some instances, the nanoparticulate composition has particle size of at least 80 nm, such as between 80 and 200 nm, between 80 and 180 nm, between 80 and 160 nm, between 80 and 140 nm, between 90 and 140 nm, between 100 and 140 nm, between 110 and 140 nm, or between 110 and 130 nm. In some instances, the nanoparticulate composition has a zeta potential of at least -20 mV, such as between -1 and -20 mV, between -1 and -18 mV, between -1 and -16 mV, between -1 and - 2045765042.1 KAUST 2024-082-02 PCT 14 mV, between -1 and -12 mV, between -1 and -10 mV, between -3 and -10 mV, between -5 and - 10 mV, or between -6 and -9 mV. In some instances, the nanoparticulate composition has a loading capacity of at least 1% or at least 5%, such as in the range of 1% and 20%, in the range of 3% and 20%, in the range of 5% and 20%, in the range of 5% and 18%, in the range of 5% and 16%, or in the range of 8% and 16%. In some instances, the nanoparticulate composition has an encapsulation efficiency of at least 80% or at least 90%, such as in the range of 80% and 100%, in the range of 83% and 100%, in the range of 86% and 100%, in the range of 88% and 100%, in the range of 90% and 100%, in the range of 92% and 100%, in the range of 94% and 100%, in the range of 96% and 100%, or in the range of 98% and 100%. D. Formulations The nanoparticulate composition disclosed herein may be used in unmodified form or formulated into formulations in combination with one or more formulation excipients, in a suitable carrier. The disclosed nanoparticulate composition are included in the formulation in an effective amount to improve agricultural crop yield and quality in a plant. The formulations can be in various forms, as described below. Formulation excipient are generally materials that can be used to deliver active ingredients, such as the nanoparticulate compositions described herein, to a plant, a plant part(e.g., a seed or seedling), plant cells or tissue, or a growing site of a plant (e.g., soil or growth medium), without having an adverse effect on plant growth, growth medium structure, soil drainage, or the like. Generally, the nanoparticulate compositions in a formulation is in an effective amount to promote plant growth, and / or inhibit growth of parasitic weed. The formulations may contain from about 0.1% to about 95% of the nanoparticulate compositions by weight, such as between about 5% and about 95%, between about 0.1% and about 90% by weight, between about 1% and about 80% by weight, between about 1% and about 60% by weight, between about 1% and about 50% by weight, between about 1% and about 40% by weight, between about 1% and about 30% by weight, between about 1% and about 20% by weight, or between about 1% and about 10% by weight. In embodiments where the formulation is a liquid, the nanoparticulate compositions can have a concentration between about 0.1 µM and about 10 M, between about 1 µM and about 1 M, between about 1 µM and about 100 mM, or between about 1 µM and about 10 mM, such as up to about 1 M, up to about 500 mM, up to about 100 mM, at least 1 mM, at least 10 mM, or at least 50 mM. 2145765042.1 KAUST 2024-082-02 PCT In embodiments where the formulation is diluted prior to use, after dilution, the nanoparticulate compositions can have a concentration between about 0.1 µM and about 1 mM, between about 0.1 µM and about 10 µM, or between about 1 µM and about 10 µM, such as about 2.5 µM or about 5 µM. The specific amount of the nanoparticulate compositions in the formulation depends on the formulation form, application equipment, and nature of the plants to be treated. 1. Formulation Excipients and Carriers Suitable formulation excipients are known in the art and are described below. The carrier can be any carrier, including, but not limited to, solid carriers and liquid carriers. Optionally, more than one suitable formulation excipient may be formulated together and / or mixed separately. Formulating formulations containing one or more the nanoparticulate compositions disclosed herein, are known in the art. (i). Solid Carriers Suitable solid carriers include, but are not limited to, plant powders (e.g., soybean flour, tobacco flour, wheat flour, wood flour, walnut shell four, cotton seed hulls, and the like), mineral powders (e.g., clays such as attapulgite clay, kaolin clay, Fubasami clay, pyrophyllite clay, bentonite and acid clay, talcs such as talc powder and agalmatolite powder, silicas such as diatomaceous earth and mica powder, pumice, fuller's earth, diatomaxeous earth, and the like), synthetic hydrated silicon oxide, alumina, talc, kieselguhr, chalk, titanium dioxide, ceramic, other inorganic minerals (e.g., sericite, quartz, sulfur, active carbon, calcium carbonate, hydrated silica, lime, and the like), lignin, and a combination thereof. (ii). Liquid Carriers Suitable liquid carriers include, but are not limited to, water, alcohols (e.g., methyl alcohol, ethyl alcohol, n-propyl alcohol, isopropyl alcohol, butyl alcohol, hexyl alcohol, benzyl alcohol, ethylene glycol, propylene glycol, phenoxyethanol), ketones (e.g., acetone, methyl ethyl ketone, methyl isobutyl ketone, cyclohexanone), ethers (e.g., diisopropyl ether, 1,4-dioxane, tetrahydrofuran, ethylene glycol monomethyl ether, ethylene glycol dimethyl ether, diethylene glycol monomethyl ether, propylene glycol monomethyl ether, dipropylene glycol monomethyl ether, 3-methoxy-3-methyl-1-butanol), aliphatic hydrocarbons (e.g., hexane, cyclohexane, kerosene, lamp oil, fuel oil, machine oil and so on), aromatic hydrocarbons (e.g., toluene, xylene, ethylbenzene, dodecylbenzene, phenylxylylethane, solvent naphtha, methylnaphthalene), halogenated hydrocarbons (e.g., dichloromethane, trichloroethane, chloroform, carbon 2245765042.1 KAUST 2024-082-02 PCT tetrachloride), acid amides (e.g., N,N-dimethylformamide, N,N-dimethylacetamide, N- methylpyrrolidone, N-octylpyrrolidone), esters (e.g., butyl lactate, ethyl acetate, butyl acetate, isopropyl myristate, ethyl oleate, diisopropyl adipate, diisobutyl adipate, propylene glycol monomethyl ether acetate, fatty acid glycerin ester, Y-butyrolactone), nitriles (e.g., acetonitrile, isobutyronitrile, propionitrile), carbonates (e.g., propylene carbonate and so on), and vegetable oils (e.g., soybean oil, olive oil, linseed oil, coconut oil, palm oil, peanut oil, malt oil, almond oil, sesame oil, mineral oil, rosmarinic oil, geranium oil, rapeseed oil, cotton seed oil, corn oil, safflower oil, orange oil). Water is generally the carrier of choice for the dilution of concentrates. In some embodiments, the liquid carrier is water, acetone, cyclohexanone, or a combination thereof. (iii). Surface-Active Agents Surface-active agents may be mixed with any solid and liquid carriers described above to form the formulations. Optionally, one or more surface-active agents are included in formulations designed to be diluted with a carrier, such as water, before application. Surface-active agents can be anionic, cationic, non-ionic or polymeric. Optionally, surface-active agents are employed as emulsifying agents, wetting agents, and / or suspending agents. Suitable surface-active agents include, but are not limited to, salts of alkyl sulfates, such as Atlas G-1086 (product name, manufactured by Croda industrial chemicals), diethanolammonium lauryl sulphate; alkylarylsulfonate salts, such as calcium dodecylbenzenesulfonate; alkylphenol- alkylene oxide addition products, such as nonylpheno l-C. sub.18 ethoxylate; alcohol-alkylene oxide addition products, such as tridecyl alcoho l-C. sub.16 ethoxylate; soaps, such as sodium stearate; alkylnaphthalenesulfonate salts, such as sodium dibutylnaphthalenesulfonate; dialkyl esters of sulfosuccinate salts, such as sodium di(2-ethylhexyl) sulfosuccinate; sorbitol esters, such as sorbitol oleate; quaternary amines, such as lauryl trimethylammonium chloride; polyethylene glycol esters of fatty acids, such as polyethylene glycol stearate; block copolymers of ethylene oxide and propylene oxide; and salts of mono and dialkyl phosphate esters. (iv). Other Formulation Excipients Other excipients commonly utilized in agricultural compositions can be included in the disclosed formulations and they include, but are not limited to, crystallisation inhibitors, viscosity modifiers, suspending agents, spray droplet modifiers, pigments, antioxidants, foaming agents, light-blocking agents, compatibilizing agents, antifoam agents, sequestering agents, neutralising 2345765042.1 KAUST 2024-082-02 PCT agents and buffers, corrosion inhibitors, dyes, odorants, spreading agents, penetration aids, micronutrients, emollients, lubricants, sticking agents, and the like. Casein, gelatin, saccharides (e.g., starch, Xanthan gum, gum arabic, cellulose derivatives, and alginic acid), lignin derivatives, bentonite, synthetic water-soluble polymers (e.g., polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylic acids), PAP (acidic isopropyl phosphate), BHT (2,6-di-tert-butyl-4-methylphenol), and BHA (mixture of 2-tert-butyl-4-methoxyphenol and 3-tert-butyl-4-methoxyphenol) can also be used. Optionally, the formulations can include one or more fertilizers, for example, liquid fertilizers or solid, particulate fertiliser carriers such as ammonium sulfate, ammonium phosphate, ammonium nitrate, urea, and ammonium chloride. Optionally, the formulation can include one or more pesticides, such as such as insecticides, nematicides, fungicides or herbicides, or additional plant growth regulators. Alternatively, the fertilizers, pesticides, and / or additional plant growth regulators can be separate from the formulation and applied concurrently or sequentially with the formulation to a plant or growing site of a plant. 2. Forms of Formulation The formulations can be in various physical forms, for example, dusting powders, aerosols, gels, wettable powders, water-dispersible granules, water-dispersible tablets, effervescent compressed tablets, emulsifiable concentrates, microemulsifiable concentrates, oil-in-water emulsions, oil flowables, aqueous dispersions, oil dispersions, suspoemulsions capsule suspensions, emulsifiable granules, soluble liquids, water-soluble concentrates (with water or a water miscible organic solvent as carrier), or impregnated polymer films. The above described formulations can be applied directly or diluted prior to use. Diluted formulation can be prepared, for example, with water. (i). Exemplary Formulations (a). Wettable Powders Wettable powders are generally in the form of finely divided particles which disperse readily in water or other liquid carriers. The particles contain the nanoparticulate compositions retained in a solid matrix. Suitable solid matrices include, but are not limited to, fuller's earth, kaolin clays, silicas and other readily wet organic or inorganic solids. Wettable powders normally contain a small amount of surface-active agents as emulsifying agents, wetting agents, and / or suspending agents. 2445765042.1 KAUST 2024-082-02 PCT (b). Emulsifiable Concentrates Emulsifiable concentrates are homogeneous liquid compositions dispersible in water or other liquid and may consist entirely of the nanoparticulate compositions with a liquid or solid emulsifying agent, or may also contain a liquid carrier, such as xylene, heavy aromatic naphthas, isophorone and other non-volatile organic solvents. Emulsifiable concentrates are typically dispersed in water or other liquid carriers as described above. (c). Granular Formulations Granular formulations include both extrudates and relatively coarse particles. Granular formulations are generally applied without dilution to a plant or growing site of a plant. Typical carriers for granular formulations include, but are not limited to, fertilizer, sand, fuller's earth, attapulgite clay, bentonite clays, montmorillonite clay, vermiculite, perlite, calcium carbonate, brick, pumice, pyrophyllite, kaolin, dolomite, plaster, wood flour, ground com cobs, ground peanut hulls, sugars, sodium chloride, sodium sulphate, sodium silicate, sodium borate, magnesia, mica, iron oxide, zinc oxide, titanium oxide, antimony oxide, cryolite, gypsum, diatomaceous earth, calcium sulphate and other organic or inorganic materials which absorb or can be coated with the nanoparticulate compositions. Granular formulations normally contain one or more surface-active agents such as heavy aromatic naphthas, kerosene and other petroleum fractions, or vegetable oils; and / or stickers such as dextrins, glue, or synthetic resins. The granular substrate material can be a solid carriers described above and / or a fertilizer, such as urea / formaldehyde fertilisers, ammonium, liquid nitrogen, urea, potassium chloride, ammonium compounds, phosphorus compounds, sulphur, similar plant nutrients and micro nutrients and mixtures or combinations thereof. The nanoparticulate compositions may be homogeneously distributed throughout the granule or may be spray impregnated or absorbed onto the granule substrate after the granules are formed. (d). Encapsulated Granules Encapsulated granules are generally porous granules with porous membranes sealing the granule pore openings, retaining the nanoparticulate compositions in liquid form inside the granule pores. Granules typically have a diameter ranging from about 1 mm to about 1 cm, such as from about 1 mm to about 50 mm, from about 1 mm to about 20 mm, from about 1 mm to about 10 mm, from about 1 mm to about 2 mm. 2545765042.1 KAUST 2024-082-02 PCT Granules are formed by extrusion, agglomeration or prilling, or are naturally occurring. Examples of naturally occurring granule materials include, but are not limited to, vermiculite, sintered clay, kaolin, attapulgite clay, sawdust, and granular carbon. Exemplary materials for porous membranes include, but are not limited to, natural and synthetic rubbers, cellulosic materials, styrene-butadiene copolymers, polyacrylonitriles, polyacrylates, polyesters, polyamides, polyureas, polyurethanes, and starch xanthates. (e). Dusts Dusts are free-flowing admixtures of the nanoparticulate compositions with finely divided solids, such as talc, clays, flours and other organic and inorganic solids. The finely divided solids act as dispersants and carriers. (f). Microcapsules Microcapsules are typically droplets or granules where the nanoparticulate compositions enclosed in an inert porous shell, which allows escape of the enclosed nanoparticulate compositions to the surroundings at controlled rates. (g). Encapsulate Droplets Encapsulated droplets are typically about 1 to 50 microns in diameter. The enclosed liquid may include a liquid carrier as described above in addition to the nanoparticulate compositions. (ii). Other Formulations Other useful formulations include simple solutions of the nanoparticulate compositions in a solvent in which it is completely soluble at the desired concentration, such as acetone, cyclohexanone, alkylated naphthalenes, xylene, and other organic solvents. Pressurised sprayers, wherein the nanoparticulate compositions are dispersed in finely- divided form as a result of vaporisation of a low boiling dispersant solvent carrier, may also be used. Method for making compositions of: (a) nanoparticles comprising homogenous metal- phenolic network comprising one or more metals and one or more phenolic compounds or (b) nanoparticles comprising a core and a shell wherein the shell comprises a metal-phenolic network comprising one or more metals and one or more phenolic compounds in the form of a metal phenolic network; wherein the nanoparticulate composition comprises one or more plant treatment agents are disclosed in Examples 1 and 2, the methods of making incorporated herein by reference. Synthetic route of metal–phenolic nanocarriers (MPNs) is shown in Fig.14, exemplified therein using CAFZin. Empty nanocarriers (CAFZin) were synthesized via coordination-driven 2645765042.1 KAUST 2024-082-02 PCT self-assembly of caffeic acid and ZnSO₄·7H₂O. A plant treatment agent, Kinetin, is encapsulated under the same conditions to produce CAFZin-K nanocarriers. For example, a method of making a nanoparticulate composition containing a core and a shell, wherein the core is in the form of a solid lipid matrix, containing one or more surfactants and one or more lipids, the method involving; (i) emulsification of one or more surfactants and one or more lipids in a suitable solvent with one or more plant treatment agents to form a core, and (ii) assembly and crosslinking of one or more phenolic compounds and one or more metals on an outer surface of the core to form a shell. The disclosed core-shell nanoparticulate composition can be fabricated via a two-step process: 1) plant treatment agent (for example, IAA) loading in SLC via o / w emulsification (SoLIN) and 2) TA-Fe3+interfacial self-assembly on SoLIN liquid-liquid interface. Emulsification of the carnauba wax mixture in water in the presence of Tween 20 ® generated SLCs to which tannic acid was self-assembled and cross-linked by Fe3+to form a film on the emulsion phase and ultimately produce Phelm. IV. Methods of Using The disclosed methods in some forms does not involve coating the seed surface to counteract NaCl ions directly. Instead, the method employs nanopriming to deliver essential nutrients and plant growth regulators into the seed (or in some forms a plant part), enhancing its ability to cope with abiotic stress such as saline conditions. Seed priming is a pre-sowing treatment that causes a physiological change in the seed that permits it to germinate more rapidly. Priming is the process of pre-treating seeds before planting, using traditional methods such as pre-soaking and coating. Nano-priming uses nanoparticles (NPs) with a size of less than 100 nm. In some forms, "priming" using the disclosed compositions results in the development of stress tolerance such as high salt stress (100 mM NaCl) plants germinated from the primed seeds. Generally, a method for promoting plant growth using the disclosed compositions includes (i) applying one or more formulations described above to a plant seed, a plant, plant part, plant cell, plant tissue or growing site of plant. Preferably the composition is applied to a plant seed. Step (i) may be repeated, (e.g., more than once). Optionally, step (i) may be repeated twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, etc. Optionally, step (i) may be repeatedly applied to different objects of a plant and / or growing site of the plant at each time. For example, the one or more formulations may be applied to a seed of a plant, seedling of the plant, the soil before, during, or after planting of the plant, and / or to the root of the plant. 2745765042.1 KAUST 2024-082-02 PCT Methods for treating a plant, plant seed or plant part, includes administering the disclosed nanoparticulate to a plant, plant seed or plant part, for an effective amount of time prior to planting. Exemplary plant seeds include, but are not limited to a wheat seed, tomato seed, capsicum seed, and pearl millet seed In some forms the nanoparticulate composition localizes in the plant seed after step (i). In some forms the nanoparticulate composition improves one or more growth metrics selected from the group consisting of seed germination rate, see germination percentage, of the plant seed in the range of 1% to 20%, in the range of 2% to 20%, in the range of 4% to 20%, in the range of 6% to 20%, in the range of 8% to 20%, in the range of 8% to 18%, in the range of 8% to 16%, in the range of 8% to 14%, in the range of 8% to 12%, in the range of 8% to 11%, or in the range of 9% to 11%. In some forms wherein the nanoparticulate composition improves growth enhancement of the plant seed in the range of 1% and 70%, in the range of 5% and 70%, in the range of 10% and 70%, in the range of 15% and 70%, in the range of 20% and 70%, in the range of 25% and 70%, in the range of 30% and 70%, in the range of 35% and 70%, in the range of 40% and 70%, in the range of 45% and 70%, in the range of 50% and 70%, in the range of 55% and 70%, in the range of 55% and 65%, in the range of 55% and 60%, or in the range of 57% and 60%. In some forms the nanoparticulate composition increases shoot and root length in comparison to a plant seed that has not been administered the composition. In some forms the nanoparticulate composition increases fresh biomass in comparison to a plant seed that has not been administered the composition. In some forms the nanoparticulate composition improves uptake of Mg2+, Zn2+, in comparison to a plant seed that has not been administered the composition. In some forms nanoparticulate composition maintains a K+ / Na+ratio, in comparison to a plant seed that has not been administered the composition. A. Plants 1. Plant Types In some embodiments, the plant (or plant part, cells or tissues thereof) to be treated can be a cereal, grain, or vegetable plant. Exemplary plants include, but are not limited to, corn, rice, maize, wheat, barley, rye, oat, sorghum, pearl millet, millet, cotton, bean, soybean, pea, peanut, buckwheat, beet, rapeseed, sunflower, sugarcane, tobacco, solanaceous vegetables (eggplant, tomato, pimento, pepper, potato, etc.), hemp, clover, melon, legume, cucurbitaceous vegetables (cucumber, pumpkin, zucchini, water melon, melon, squash, etc.), cruciferous vegetables (Japanese radish, white turnip, 2845765042.1 KAUST 2024-082-02 PCT horseradish, kohlrabi, Chinese cabbage, cabbage, leaf mustard, broccoli, cauliflower, etc.), asteraceous vegetables (burdock, crown daisy, artichoke, lettuce, etc.), liliaceous vegetables (green onion, onion, garlic, and asparagus), ammiaceous vegetables (carrot, parsley, celery, parsnip, etc.), chenopodiaceous vegetables (spinach, Swiss chard, etc.), lamiaceous vegetables (Perilla frutescens, mint, basil, etc.), strawberry, sweet potato, dioscorea japonica, colocasia, etc. In some preferred embodiments, the plant is a rice plant. The plant may be wild-type or mutant. In some embodiments, the plant is a wild-type plant, such as a wild-type rice plant. The one or more formulations may be applied to a plant, plant part, or growing site of plant by any method known in the art, including both foliar and non-foliar application. Exemplary application method to a plant or plant part include, but are not limited to, spraying, drenching, dripping on, or dusting the plant or plant part, coating a seed, and / or applying as a cream or paste or as a vapor. For example, application methods for a foliage of plants may be applying to surfaces of plants, such as foliage spraying and trunk spraying. In some embodiments, the method of application can be absorbing to plants transplantation such as soaking entire plant or roots. In some embodiments, a formulation formulated with a solid carrier may be adhered to the roots. Methods for coating a seed is known in the art, for example, US 2007 / 0105721 by Flematti, et al. Exemplary application method to soil include, but are not limited to, spraying onto the soil, drenching the soil, dripping onto the soil, dusting the soil, and / or soil incorporation. Examples of places where the one or more formulation can be applied include, but are not limited to, planting hole, furrow, around a planting hole, around a furrow, entire surface of cultivation lands, the parts between the soil and the plant, area between roots, area beneath the trunk, main furrow, growing soil, seedling raising box, seedling raising tray and seedbed. Examples of the treating period include before seeding, at the time of seeding, immediately after seeding, raising period, before settled planting, at the time of settled planting, and growing period after settled planting. Exemplary application method to a growing medium, such as a culture solution, include, but are not limited to, mixing the compounds or formulations into the growing medium. Optionally, the method can include a step of applying one or more agriculturally beneficial agents prior to, during, or after step (i). Agriculturally beneficial agents generally refer to any agent 2945765042.1 KAUST 2024-082-02 PCT or combination of agents capable of causing or providing a beneficial and / or useful effect in agriculture. 2. Growing Sites The growing site of plant can be soil or a growing medium. In some embodiments, the growing site of plant is soil. The soil may be soil before, during, or after planting the plant. In some embodiments, the growing site of plant is a growing medium. Exemplary growing medium include, but are not limited to, water, culture solution, urethane, and rock wool. Culture solution generally refers to a water solution containing nutrient components required for plant growth. The nutrient components and their relative concentrations can be easily adjusted to a proper concentration for different plants and application, which are known in the art. In some embodiments, the one or more agriculturally beneficial agents are applied prior to applying the one or more formulations of the compounds or salts thereof. In some embodiments, the one or more agriculturally beneficial agents are applied during (e.g., simultaneous or substantially simultaneous with) applying the one or more formulations of the compounds or salts thereof. In some embodiments, the one or more agriculturally beneficial agents are applied after applying the one or more formulations of the compounds or salts thereof. Optionally, the agriculturally beneficial agents may be part of a formulation described above, or independently from the one or more formulations. The agriculturally beneficial agents can be fertilizers, micronutrients, microorganisms, or a combination thereof. Fertilizers are generally known to the person skilled in the art, e.g., see Ullmann’s Encyclopedia of Industrial Chemistry, 5thedition, Vol. A 10, Verlagsgesellschaft, Weinheim, 1987. Exemplary fertilizers that can be used in the disclosed methods are generally organic and inorganic nitrogen-containing compounds, such as ureas, urea / formaldehyde condensates, amino acids, ammonium salts and ammonium nitrates, potassium salts (e.g., chlorides sulphates, nitrates), salts of phosphoric acid, salts of phosphorous acid (e.g., potassium salts and ammonium salts), and a combination thereof. The growing medium may be applied, for example, to soak a plant or plant part, such as seeds or seedling for their germination or rotting, or to soak roots of plants or spraying it to roots to culture the plants. 3045765042.1 KAUST 2024-082-02 PCT The disclosed compositions and methods can also involve other active ingredients, which in some embodiments form all or part of at least one layer of a seed coating composition, include but are not limited to herbicides, plant growth regulators, crop desiccants, fungicides, insecticides, insect repellants, and combinations thereof. Suitable pesticides include, for example, triazine herbicides; sulfonylurea herbicides; uracils; urea herbicides; acetanilide herbicides; and organophosphonate herbicides such as glyphosate salts and esters. Suitable fungicides include, for example, nitrilo oxime fungicides; imidazole fungicides; triazole fungicides; sulfenamide fungicides; dithio-carbamate fungicides; chloronated aromatic; and dichloro aniline fungicides. Suitable insecticides, include, for example, carbamate insecticides; organo thiophosphate insecticides; and perchlorinated organic insecticides such as methoxychlor. Suitable miticides include, for example, propynyl sulfite; triazapentadiene miticides; chlorinated aromatic miticides such as tetradifan; and dinitrophenol miticides such as binapacryl. Other active ingredients can include adjuvants, and surfactants, and fertilizers. The methods, compounds, and compositions herein described are further illustrated in the following examples, which are provided by way of illustration and are not intended to be limiting. It will be appreciated that variations in proportions and alternatives in elements of the components shown will be apparent to those skilled in the art and are within the scope of disclosed forms. All parts or amounts, unless otherwise specified, are by weight. EXAMPLES Example 1: Smart Nano-Priming of Wheat Seeds by a Core / Shell Metal Coordinated Assembly for Elevated Germination at High Salinity Materials and Methods All chemicals used in this work were of analytical reagent grade from commercial sources and used without further purification. Carnauba wax and phosphate-buffered saline (PBS) were obtained from Thermo Scientific (Waltham, MA, USA); Tannic acid, sodium chloride (NaCl), hydrogen peroxide (H2O2), Tween 20®, glacial acetic acid (CH3COOH), 4-(2- Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), Tris-Buffered Saline (TBS), Sodiumphosphate dibasic heptahydrate (Na2HPO4 · 7H2O), Sodium phosphate monobasic monohydrate(NaH2PO4 · H2O), and 70% perchloric acid (HClO4) were purchased from Sigma-Aldrich(Darmstadt, Germany). Iron chloride hexahydrate (FeCl3.6H2O) was purchased from PanReac Quimica (Barcelona, Spain). MilliQ water was used in the experiments unless otherwise stated. 3145765042.1 KAUST 2024-082-02 PCT Experimental design The experimental design was a completely randomized block design using the Taguchi method. Four continuous factors were considered as inputs for the fabrication of SoLIN: surfactant concentration, lipid amount, homogenization time, and cargo amount. Encapsulation efficiency (EE) and loading capacity (LC) percentages were chosen as outputs. Similarly, five continuous factors were considered as inputs for the fabrication of Phelm: SoLIN volume, phenol volume, metal volume, phenol concentration, and metal concentration, whereas particle size was chosen as the output. Thus, optimization for SoLIN and Phelm formulations was performed accordingly. Fabrication of SLC and indole 3-acetic acid encapsulation (SoLIN) SLCs were fabricated by using the hot homogenization method. In brief, carnauba wax (400 mg) was melted above the glass transition temperature (90°C) for 10 min; simultaneously, a surfactant solution of Tween 20® (1.5 % w / v) was stirred for 20 min at 90°C. Afterward, the surfactant solution was poured into the melted carnauba wax and homogenized three times using Ultraturrax® at 10,000 rpm for 1 minute with 5 min rest intervals. Encapsulation of IAA in solid lipid nanoparticles was performed as follows: IAA (2 mg) was added to the lipid phase consisting of molten carnauba wax (400 mg, 90°C) and mixed with a surfactant solution in water of Tween 20® (1.5% w / v). The mixture was homogenized thrice using an Ultraturrax® at 10,000 rpm for 1 min with 5-min rest intervals. The temperature was kept constant throughout the entire experiment. The samples were then cooled to room temperature to enable SLC formation and stored at 4°C for further analysis. Fabrication of the Phelm platform Fabrication of the Phelm platform was performed by the interfacial self-assembly of tannic acid and Fe3+onto the SoLIN surface. First, an aliquot of SoLIN (1.3 mL) was mixed with tannic acid (925 µL, 19 mM) and vortexed for 30 s at a reduced speed. Subsequently, FeCl3.6H2O (684 µL, 34 mM) was added to the mixture and vortexed for 30 s. The obtained solution was adjusted to pH 8 using Tris-HCl buffer (300 µL, 1M) and purified with a PD-10 column to remove excess starting material. Dynamic Light Scattering (DLS) The size, ζ-potential, and polydispersity index (PdI) of Phelm and SoLIN were analyzed by dynamic light scattering (DLS) on a Zetasizer Nano (Malvern Instruments). To obtain the particle size and PdI, 30 µL of Phelm and SoLIN were dispersed in 1 mL of MilliQ® water, followed by a brief sonication for 1 min at room temperature. After sonication, the samples were placed on a glass cuvette and scanned at 25°C and 60-s intervals in triplicate. 3245765042.1 KAUST 2024-082-02 PCT For measurements of the ζ-potential, the samples were diluted in MilliQ® water solution using the above-mentioned conditions and transferred to a Universal Dip Cell (ZEN 1002). Entrapment efficiency (EE) and loading capacity (LC) percentages The encapsulation efficiency (EE%) and loading capacity (LC%) were determined using a UV-Vis Spectrophotometer (UV-2600, Shimadzu Europe) at 280 nm absorbance. A calibration curve of IAA was prepared at different concentrations (0.002, 0.004, 0.006, 0.008, and 0.01 mg / mL) as follows: IAA (1 mg) was dispersed in water (1 mL), and the obtained mixture was adjusted to pH 13 by adding a NaOH solution (300 µL, 1 N). The EE and LC percentages were obtained using the following equations:^^% = ^^^^ ^^ ^^^^^^ ^^^^^^^^^^ ^^^^ ^^ ^^^ ^ 100 Equation (1)^^% = ^^^^ ^^ ^^^^^^ ^^^^^^^ ^^ ^^^^^ ^ 100 Equation (2)Where ^^^^^^ !^"" #$ ^%%= weight of initial IAA used in the formulation (mg),&^"" #$ #^'(' ^%% = weight of IAA loaded in SoLIN (mg), &^"" #$ )#^^* = total weight ofthe complex (mg). Indole 3-acetic acid release SoLIN and Phelm (1 mL) were dispersed in PBS solution (1 mL, pH 7.4). The solutions were collected in Eppendorf ® tubes and shaken (Eppendorf™ Thermomixer) for 20 min at 36°C and 400 rpm. Upon completion, the samples were centrifuged at 20,000 rcf for 20 min at 4°C. The recovered supernatants were analyzed and replaced with the same amount of fresh PBS. To measure the concentration of IAA released from SoLIN, a NaOH solution (300 µL, 1 N) was added to each supernatant solution at different time intervals to facilitate IAA solubility in the media. These solutions were analyzed by UV-vis spectroscopy (UV-2600, Shimadzu Europe) in triplicate at 280 nm wavelength. Briefly, aliquots (1 mL) were taken from the supernatant at 0, 1, 2, 3, 24, 48, 72, 96, 120, 144,168,192, and 216 h. The percentage cumulative release (% R) at each time point was calculated using the following equations:%!#+^^^ = (^#^-(^^.^^^#^ #$ ^%% )^^ "+0(.^^^^^^ (^1^^ ) ^ 2# +!((!^) Equation (3)Equation (4) zero order, first order, and Korsmeyer-Peppas. The corresponding equations are:<(.# #.'(.: >^ = >^ + @^. ^ Equation (5)B^."^ #.'(.: ^>^ = ^>^ − @D. ^33Equation (6) >^ = Equation (7) KAUST 2024-082-02 PCTWhere >^ is initial release capacity; >^ is the release capacity at time t; @^, @D, @; are the releaseconstants of each model; ^ is time, and ^ is the diffusional exponent or release exponent. Indole 3-Acetic Acid Release in simulated salinity conditions SoLIN and Phelm (1 mL) were dispersed in NaCl (1 mL, 100 mM) and shaken (Eppendorf™ Thermomixer) for 20 min at 36°C and 400 rpm. After the incubation, the samples were centrifuged at 20,000 rcf for 10 min and the supernatants were collected for further analysis. The selected time points for incubation were 0, 1, 2, 3, 4, 5, and 6 h. During the entire experiment, the volume remained constant by adding fresh NaCl (1 mL) after each centrifugation or any of the buffers that were chosen as controls: Hydroxyethyl piperazine-1-ethanesulfonic acid (HEPES), Tris Buffer Saline (TBS), and Phosphate Buffer (PB). Thermal stability of Phelm and IAA kinetics release in a simulated soil environment The thermal stability of both Phelm and IAA was studied in a simulated soil environment. The soil model was prepared through a mixture of amino acids as follows: Aspartic acid (5mM), Glutamine (5mM), Histidine (5mM), PBS (1x), Asparagine (5mM), Glutamic acid (5mM), Alanine (5mM), HEPES (10mM) all in 5mL of PBS and 1 drop of ethanol. Encapsulated and non-encapsulated IAA were added to the simulated soil environment at a final concentration of 45 µM. The samples were vortexed for 10 seconds to ensure thorough mixing and then incubated at 45°C for 24 hours. Aliquots of 500 µL were taken at different time intervals 0h, 1h, 6h, 12h, and 24h. The concentration of IAA in the samples was calculated by using UV-vis spectrophotometry at 280 nm wavelength. The release experiment was performed by adding 1 mg of Phelm in 1 mL of the amino acid mixture used as a soil model and incubated at 45°C under continuous shaking (600 rpm). The selected point times for incubation were 0, 1, 2, 4, 24, 48, 72, 96, 120, 144 and 168 h. After the incubation, the samples were centrifuged at 20,000 rcf for 10 min and the supernatants were collected for further analysis. During the experiment, the volume remained constant by adding fresh amino acid media (1 mL) after each centrifugation. The kinetics release was fitted with three different models using the equations (5-7). Fe3+release One milliliter of Phelm was dispersed in 1 mL of H₂O, HCl (10 µM), and NaCl (100 mM) solutions, then shaken using an Eppendorf™ Thermomixer for 20 minutes at 36°C and 400 rpm. 3445765042.1 KAUST 2024-082-02 PCT Following incubation, the samples were centrifuged at 20,000 rcf for 10 minutes, and the supernatants were collected for subsequent analysis. Incubation time points were selected at 0, 1, 2, 3, 4, 5, and 6 h. Throughout the experiment, the volume was maintained by adding 1 mL of fresh media after each centrifugation. A standard calibration curve for Fe³⁺ determination was prepared using aqueous FeCl₃ solutions with concentrations ranging from 50 to 500 µM. The cumulative release data was also fitted with the three kinetic models presented in equations 5-7. Powder X-Ray Diffraction (PXRD) Powder X-ray diffraction (PXRD) analysis was performed using an X-ray diffractometer (D2 PHASER XE-T, Bruker®). The reported patterns correspond to SoLIN and Phelm structures. Thermogravimetric Analysis (TGA) Thermal stability was measured using a TGA 5500 (TA ® instruments), and sample containers (Platinum HT pans) were heated in N2 flow up to 1000°C with a heating rate of 10 °C / min). Differential scanning calorimetric (DSC) Differential scanning calorimetry (DSC) of SoLIN and Phelm nanoparticles was performed using a Discovery DSC (TA ® instruments), operating at a 20 °C / min heating rate from 30°C to 420 °C under nitrogen. The enthalpy of the transition (∆I) and the crystallinity index percentage (CI %) were calculated using the following equations: °P∆I = ^J^^ 4^^^J ^K^ ^^^^^^^1 ;^^L (MNQ R)Equation (8) n (9) Where, ∆I^[^^^ℎ^ 0E #$ ^.^^"^^^#^ #$ )^*∆I^X = ^^^ℎ^ 0E #$ ^.^^"^^^#^ #$ ]+ ^ ^0^'Scanning Electron Microscopy (SEM) SEM imaging was performed on a Quattro (ThermoFisher) instrument operated at 3.0 kV. The samples were prepared by dropping diluted nanoparticles onto a silicon wafer and kept in a vacuum desiccator overnight to allow drying. Furthermore, the samples were coated with an iridium layer of 1.0 nm thickness. 3545765042.1 KAUST 2024-082-02 PCT Scanning electron microscopy / energy dispersive spectroscopy (SEM-EDS) Phelm nanoparticles were analyzed by scanning electron microscopy (SEM; Zeiss Merlin Microscope) operating at 20 KV, 2nA, and equipped with an 80 mm2Oxford detector for EDS analysis. The Mapping data acquisition and processing was realized using the Aztec Oxford software. Fe, C, and O distributions in the Phelm area were detected using an acceleration potential of 20 kV at a working distance of 10 mm and a spot size of 2 µm. Scanning transmission electron microscopy (STEM) The structure and the elementary composition of the sample were examined using a Cs probe-corrected microscope from Thermo Fisher Scientific by operating it at the accelerating voltage of 300 kV and with a beam current of 0.1 nA. Darkfield imaging was performed by scanning transmission electron microscope (STEM) coupled to a high-angle annular dark-field (HAADF) detector. Furthermore, an X-ray energy dispersive spectrometer (SuperXG2) was also utilized in conjunction with DF-STEM imaging to acquire STEM-EDS spectrum-imaging data sets. Transmission Electron Microscopy (TEM) TEM imaging was performed on an EI-Tecnai Twin instrument operated at 120 kV in the bright-field mode to evaluate the morphology of the synthesized SoLIN and Phelm. The samples were prepared by dropping SoLIN and Phelm (5 µL ) nanoparticle solutions onto 200 square mesh carbon-coated copper grids, followed by the removal of excess water using a piece of filter paper and allowed vacuum drying overnight. Fourier transform infrared (FTIR) The chemical composition and identification of functional groups were characterized by using Fourier transform infrared spectroscopy with attenuated total reflection mode (Smart iTR, Thermo Scientific™), reported in transmittance units for the as-synthesized Phelm and compared with the SoLIN and IAA spectra. Hydrogen Peroxide Scavenging Activity The H2O2 scavenging assay of Phelm, SoLIN, SLC, IAA, and TA-Fe3+was performed by preparing a stock solution of H2O2(15 mM) in PBS. The solutions were then prepared at different concentrations (0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, and 0.8 mg / mL), and each solution (1 mL) was added to the H2O2solution (1 mL). Subsequently, the absorbance of the samples containing the above-mentioned formulations at different concentrations and H2O2 were measured at 240 nm after 1 hour for each concentration. The H2O2 solution in PBS was used as a control. The scavenging activity percentage was calculated using the following equation: 3645765042.1 KAUST 2024-082-02 PCTI_`_ = ^a6^U^a ^ 100% Equation (10)of the control sample, and %^is the absorbance of the SoLIN, SLC, IAA or TA-Fe3+. Preparation of the priming solutions The as-synthesized Phelm and SoLIN were used as seed priming solutions (100 mL, 2 µM). In addition, an IAA solution (100 mL, 2 µM) and MilliQ® water (Mock) were designated as positive and negative controls respectively. Seed priming method Healthy seeds were selected and weighed from the same seed lot and used in all the experiments. For the seed priming method, seeds were soaked either in water, IAA, SoLIN, or Phelm priming solutions (100 mL) for 24 h in the dark at room temperature. Upon priming completion, seeds were surface-dried on a paper towel to eliminate excess moisture. Wheat plate germination assay and growth metrics. Wheat seedlings primed with chemicals or water were grown on filter paper in Petri plates sealed with parafilm. For each treatment, four biological replicates, each with 20 seeds, were distributed uniformly on each plate and treated with either a NaCl solution (10 mL, 100 mM) or MilliQ® water (10 mL) at room temperature. Seedlings were placed in a chamber adjusted to 25°C and collected 72 h after germination. Root and shoot, lengths were measured using a ruler and gently wiped to remove excess water before recording the fresh-weight biomass. The seed germination rate (b3) and seed germination percentage (bF%) were calculated based on the following equations:b3 = ^4^c^J ^^ 1^J^^^^^^^ ^^^^^^^1^^4^c^J ^^ 1^J^^^^^^^ ^^^^^^^1^ d^e^ ^^ ^^J^^ :^4^^ + ⋯ d^e^ ^^ ^^^^^ :^4^^Equation (11) Equation (12) Wheat seedlings primed with chemicals or water were grown on seed trays and transferred to pots (6 x 5.5 x 4.5 cm) under a semi-controlled environment adjusted to 26ºC day / night temperature, under 12h-light photoperiod with 60% humidity for nursery and no fertilizer or nutrient solution was added. Irrigation was done daily either with 10 mL of freshwater or 100 mM NaCl solution. Plant height, root length, and fresh biomass were measured after 10 days of growth. Indole 3-acetic acid quantification Roots of seven-day-old wheat seedlings were frozen and homogenized manually in a chilled mortar using liquid nitrogen until obtaining a fine powder. Subsequently, a methanol solution (5 3745765042.1 KAUST 2024-082-02 PCT mL, 80 %) was added to the ground mixture and sonicated for 30 min in the dark. The homogenate was then centrifuged at 10,000 g for 20 min at 4°C. The supernatants were reduced in volume by drying under a fume hood. Reverse-phase liquid chromatography (RPLC) was performed to elute IAA. The column was packed to a length of approximately 16 cm using C18 (Agela Technologies, USA), and the column width was approximately 1.5 cm. A small cotton plug was kept at the top to maintain the column intact. The supernatants were directly poured above the cotton plug and eluted with acetic acid (2 %) in methanol until complete recovery of IAA. The collected fractions were analyzed using UV-vis spectrophotometry at 280 nm, and the IAA concentrations were calculated using a calibration curve. A qualitative analysis was conducted on the collected fractions to detect IAA using Salkowski’s reagent. The reagent was prepared by mixing (0.5 M) ferric chloride (1mL) solution with a perchloric acid solution (49 mL, 35 %,) and vortexed for 30 seconds. Subsequently, (1 mL) of the collected fractions described above was mixed with 1 mL of Salkowski reagent and incubated at 30°C in the dark for 30 min. The presence of IAA was detected by measuring the pink color development after 30 min. During the test, a pure IAA solution (1mM) and a blank sample containing (1 mL, 2 %) acetic acid solution were used as controls. Confocal microscopy A qualitative analysis of Phelm was performed by using confocal microscopy. The preparation of Phelm followed the outlined procedure in the fabrication section with slight adjustments. Specifically, 1.3 mL of SoLIN-loaded Nile Red dye (0.304 mM) was combined with 925 µL of TA (19 mM) and 925 µL of FITC green dye (0.304 mM), followed by vortexing for 30 seconds. Subsequently, 684 µL of FeCl3 (34 mM) was added. The pH was carefully adjusted to 8 using 300 µL of Tris-HCl buffer (1 M), and the mixture was subjected to three washes with water to eliminate any excess starting materials. Phelm internalization study in wheat seeds was performed as follows: seeds were incubated in either water or Phelm solution overnight. Treated seeds were washed three times with water. The seeds were crossed-sectioned into thin layers horizontally and vertically, placed on glass slides, and covered with a glass coverslip. The images for both studies were taken using a laser scanning confocal microscope (Leica SP8) following a standard protocol. The Rhodamine signal was detected using the Rhd filter with Ex.650 / Em.668nm. Statistical Analysis A one-way ANOVA test was used to calculate the significant difference (p-value). Asterisks denote significance (ns: non-significant, *p <0.05, ***p<0.0005, ****p<0.0001). Software GraphPad 3845765042.1 KAUST 2024-082-02 PCT Prism 8 software was used for statistical analysis. Data are shown as ± STDEV (n=4, each with 20 seeds) for, fresh biomass analysis and germination rate of wheat seedlings treated with water, SoLIN, IAA, and Phelm. Results and Discussion Preparation and characterization of the SoLIN and Phelm nanoparticles A set of experiments was conducted as depicted in (Tables 1 and 2) by using Taguchi’s experimental design to optimize the fabrication procedure. This method uses orthogonal arrays to reduce the number of experiments required when investigating several factors.28Thus, SoLIN and Phelm nanoparticles were prepared via two key steps. (1) SLC formation and encapsulation of IAA using hot homogenization until a viscous solution was formed, and the solution was allowed to cool down at room temperature. (2) Metal phenolic network formation at the liquid-liquid interface was achieved by adding tannic acid to the SoLIN solution followed by the dropwise addition of iron chloride after gentle vortexing for 30 s. The resulting solution was adjusted to pH 8 to obtain a tris- type complexation. Table 1. Taguchi’s experimental design for the fabrication of SoLIN. 3945765042.1 KAUST 2024-082-02 PCT Table 2. Taguchi’s experimental design for the fabrication of Phelm. . average was 7.3 nm with a PdI of 0.25 ± 0.02 (data not shown). Phelm nanoparticles exhibited a particle size of 282.2 ± 3.1 nm (data not shown) and a PdI value of 0.47 ± 0.17. The transmission electron microscopy (TEM) images provided detailed insights into the morphology of the nanoparticles under investigation. The analysis revealed the spherical shape and the monodisperse distribution of SoLIN. Notably, a well-defined surfactant layer surrounding the crystalline lipid core was evidenced by variations in intensity and the presence of fringes within the core region (Figure 1A). This observation is attributed to the higher density of carboxylic groups within the lipid composition compared to the surfactant constituents, thereby resulting in pronounced intensity contrasts.29,30In contrast; Phelm nanoparticles displayed a different arrangement, characterized by a core-shell architecture where the core fringes were not prominently displayed (Figure 1B). Similar findings were reported for the characterization of chitosan-hyaluronan solid lipid nanoparticles.31The size obtained from TEM was smaller than the one obtained in DLS (Figure 1C-1D), as 40 45765042.1 KAUST 2024-082-02 PCT expected since it measures the actual physical diameter of the particles without considering the surrounding medium or hydration effects. Zeta potential measurements were conducted in unloaded SLC (13.1 ± 3.5 mV), SoLIN (-26.3 ± 4.7 mV), and Phelm (-30.1 ± 6.9 mV). IAA was found to be incorporated into the SLC matrix because of the -COOH and -NH groups present in IAA, as may be seen by the variation in charge from positive to negative in unloaded SLC and loaded SoLIN (Figure 1e). During the optimization process, the as-synthesized SoLIN and Phelm nanoparticles were characterized by SEM. As depicted by the images, both systems exhibited a spherical morphology (data not shown). FTIR analysis (data not shown) of IAA displayed a strong band at 3381 cm-1attributed to the –OH or –NH group, 1688 cm-1carbonyl group (C = O) stretching, and 1456 cm-1corresponding to the C=C bond in the IAA molecule.32Furthermore, the spectra of SoLIN and Phelm possessed characteristic bands at 2915 and 2846 cm-1attributed to stretching vibrations of (–CH3 and –CH2). Additionally, the vibration at 1734 cm-1is representative of the ester group also present in the carnauba wax.33It can be observed that the –NH band of IAA is concealed after incorporation either in SoLIN or Phelm matrix, this can be associated with a hydrogen bond formation between –OH and –NH groups with the carnauba wax’ ester / alcohol / carboxylic group, thus, allowing the incorporation of IAA into the SLC. The PXRD analysis confirmed the presence of two large peaks (2θ = 21.5°) and (2θ = 23.8°) characteristic of carnauba wax,34as observed in the SoLIN diffractogram (data not shown). The changes observed in the Phelm diffractogram indicate a decrease in the crystalline arrangement,35although the corresponding peaks of carnauba wax are noticeable; the presence of a more amorphous phase was evident. After encapsulation, we noticed that the characteristic peaks of IAA were not evident. This could be due to several factors. One possibility is that IAA is dispersed at the molecular level in the Phelm matrix, preventing the formation of detectable IAA crystals post-encapsulation.36This suggests that carnauba wax is present in larger quantities compared to IAA. Consequently, we hypothesize that the low concentrations of IAA might result in undetectable peaks in the XRD spectra. These results align with those of the DSC analysis (data not shown), which measures the enthalpy of fusion (ΔH) and material crystallinity by comparing it to 100% crystalline or amorphous samples. Thermal behavior varies among pure lipid, SoLIN, and Phelm. According to the literature, the ΔH of carnauba wax is 168.3 J / g,37which differs from that obtained for SoLIN (ΔH = 126.4 J / g) and Phelm (16.3 J / g). This difference is attributed to the SLC crystalline phase being less organized than the crystalline phase of the pure wax, which requires more energy to overcome cohesion forces in the crystal lattice.38Moreover, the CI percentages were found to be 37.5% for SoLIN and 4.83% for Phelm. The degree of organization is lower for Phelm because of 4145765042.1 KAUST 2024-082-02 PCT the MPS formation in the outer layer of SoLIN. According to previous literature reports, less ordered crystallinity could lead to higher physical stability and prevent the early outflow of loaded molecules from particles during storage time.39This idea was supported by comparing Phelm colloidal stability to SoLIN, and finding no significant change in particle size at time zero and after 1 month of storage at 4°C compared with SoLIN (Figure 6). Thermogravimetric analysis (data not shown) is consistent with similar reports in the literature. The IAA thermal decomposition was̴ 210 °C40(95.09% weight loss), SoLIN degradation was̴ 389 °C (51.39% weight loss) corresponding to carnauba wax,41and Phelm exhibited five degradation zones and weight losses: 167°C (4.3%) due to dehydration, 281 °C (16.55%), and 433 °C (34.1%) decomposition of outer gallic acid constituents, 547 °C (6.95%) gallic acid in the inner layer, and 735°C (8.93%) carbon- oxygen functional groups in the glucose central ring of tannic acid.42The UV-vis spectra of SLC (unloaded), MPS (TA-Fe complexation), SoLIN, and IAA were analyzed and compared with those of Phelm (data not shown). IAA exhibited characteristic peaks at a strong maximum at 220 nm and another peak at 280 nm.43,44The unloaded SLC absorption bands at 217 and 312 nm are representative of the carnauba wax.34,41SoLIN bands displayed a new peak at 280 nm corresponding to IAA. Phelm exhibited four main absorption peaks the ones at 200 nm corresponding to TA45and that was also present in the spectra of MPS, the peak at 280 was associated with the presence of IAA, whereas the ones at 253 and 353 correspond to SLC. These results are consistent with those available in the literature46–48and suggest the successful incorporation of IAA into Phelm nanoparticles. The successful formation of MPS was indicated by the color change in the solution from white to purple (Figure 1F). The encapsulation efficiency and loading capacity were calculated to be 86.9% ± 0.29 and 13.1% ± 0.23 based on equations 1 and 2, as described in the methodology section. SEM-EDS and STEM images (data not shown) shows the elemental mapping and distribution for carbon, iron, and oxygen present in the Phelm sample. Confocal imaging was performed to visualize Phelm inner core and outer shell after encapsulating Red Nile dye in the lipid part of the system and FITC in the outer metal-phenolic layer (data not shown). Indole 3-acetic acid (IAA) release from Phelm: A range of factors, such as solubility, pH sensitivity, ionic strength, pairing, and buffering capacity can influence the release profile of agricultural molecules. In our research, we examined the behavior of the nanopriming system in terms of PGR release and diffusion in various media, including salt buffers and a soil mimic model. This evaluation is intended to understand the kinetics release and how these substances diffuse under different conditions. This does not imply that the 4245765042.1 KAUST 2024-082-02 PCT nanopriming material is applied directly to the environment in which the plant grows. Rather, the focus is on how the priming process prepares the seed internally to better handle the stresses imposed by high salinity during subsequent growth stages. To investigate the factors influencing IAA release from Phelm, we initially assessed its pH responsiveness (Figure 2A). We found that lower pH values led to faster release, likely due to structural changes in the outer layer. This is attributed to the protonation of catechol groups in TA55,56, resulting in partial disassembly of bis- and tris- cross-links and the formation of mono-complexes. The release kinetics and mechanism of IAA were investigated under these conditions and analyzed using three different kinetic models: zero-order, first-order, and Korsmeyer-Peppas. Each model was applied to different stages of the release process (Figure 7A). The model fit was evaluated using the coefficient of determination (R²) values, with higher R² indicating a better model fit. The R² values and other model parameters derived from each release stage are presented in (Table 3). It was found that the linear correlation coefficient (R² =0.99) of the Korsmeyer-Peppas model was the highest for the first stage of IAA from Phelm, and it follows a Quasi-Fickian diffusion mechanism (n ≤ 0.45). Table 3. Kinetic parameters for Indole-3-Acetic Acid (IAA) release from Phelm under varying pH conditions. spectrophotometry at 280 nm under physiological conditions (PBS, pH 7.4 at 37°C) and compared the release profile between Phelm and SoLIN at different time points. Phelm released 38.17% IAA after 24 h, compared with 34.98% in SoLIN at the same time (Figure 2B). The kinetics release of Phelm indicated the highest linear correlation coefficient (R² =0.99) for the Korsmeyer-Peppas model and was governed by a Quasi-Fickian diffusion mechanism (n≤ 0.45) whereas SoLIN followed a first-order kinetics (R² =0.99) (Figure 7B, Table 4). SoLIN was composed of carnauba 4345765042.1 KAUST 2024-082-02 PCT wax and Tween 20®. Carnauba wax contains a significant amount of ω-hydroxycarboxylic acids and fatty alcohols, whereas Tween 20® has multiple polyethylene glycol (PEG) chains. Because of the hydrophilicity of the carboxylic, alcohol, and PEG groups, they are expected to remain on the surface of SoLIN. It is hypothesized that the interaction between the metal phenolic complex and the exposed hydrophilic groups of the SoLIN surface makes the solid core a little labile, which leads to more release of IAA from the inner core. Table 4. Kinetic parameters for Indole-3-Acetic Acid (IAA) release from Phelm and 296 SoLIN at physiological conditions. To determine if similar behavior occurs in other saline conditions, we compared IAA release in NaCl, which simulates the salinity stress environment, and other saline buffers, including HEPES, PB, and TBS. The results indicated that IAA release was highest in NaCl, followed by TBS, PB, and HEPES. In NaCl, Phelm released a significantly higher amount of IAA (91.09%), compared to SoLIN (53.97%) after 6 h (Figure C-2D). Kinetic modeling using the Korsmeyer- Peppas equation revealed that the release of IAA from Phelm and SoLIN in NaCl-containing buffer was governed by non-Fickian diffusion mechanisms, classified as Super Case II transport (n>1) and anomalous diffusion (n>0.5). In contrast, IAA release in the other buffer solutions followed a quasi- Fickian diffusion mechanism (Figure 7C and 7D, Table 5 and 6). It has been reported that at a higher ionic strength (2 M NaCl), sodium ions shield the galloyl groups of tannic acid, allowing them to extend away from the Fe3+center rather than forming a very closed complex and increasing 44 45765042.1 KAUST 2024-082-02 PCT solubility.49This ‘loosening up’ of the coating around SoLIN and the consequent effect on SoLIN influenced the very high release of IAA. Table 5. Kinetics parameters for Indole-3-Acetic Acid (IAA) release from Phelm in 299 simulated salt stress conditions and other saline buffers. Table 6. Kinetic parameters for Indole-3-Acetic Acid (IAA) release from SoLIN in 302 simulated salt stress conditions and other saline buffers. In addition to ionic strength, this behavior could be attributed to other factors, for example, IAA has limited solubility in water but can dissolve more readily in solutions with certain salts due to the formation of ion pairs. In a NaCl solution, the presence of sodium and chloride ions may increase the solubility of IAA through ion pairing, potentially leading to higher cargo release compared to the other buffer solutions.50Moreover, individual buffer constituents can also interact with our system and influence the release of IAA. On the other hand, buffer solutions are designed to resist pH changes, which can be advantageous for maintaining consistent cargo release kinetics. In contrast, the NaCl solution may lack buffering capacity, leading to pH changes that could influence IAA solubility and release.51,52Thus, while buffer solutions can maintain a constant pH, the pH of a NaCl solution may vary depending on its initial pH and any changes induced by dissolution or reaction of components. If the pH of the NaCl solution is closer to the acidic range favored by IAA, it could enhance its solubility and release compared to other buffers.5345 45765042.1 KAUST 2024-082-02 PCT Evaluation of Phelm's performance in a simulated soil environment To assess the thermal stability and release kinetics of encapsulated and non-encapsulated indole-3-acetic acid (IAA), both formulations were subjected to a 24-hour incubation at 45°C in a previously described soil-mimicking model54. The concentration of IAA was monitored over time using UV-vis spectrophotometry. Results revealed that both encapsulated and non-encapsulated IAA underwent thermal degradation. However, encapsulated IAA demonstrated significantly higher retention, retaining 52.06% of its initial concentration after 24 hours compared to only 19.3% for non-encapsulated IAA (Figure 8A-8C). This suggests that the encapsulation matrix effectively protects IAA from thermal degradation, making it more suitable for applications in thermally challenging environments. Furthermore, the release kinetics of encapsulated IAA in Phelm were investigated in the soil-mimicking model over an 8-day period. The release kinetics of Phelm exhibited the highest linear correlation coefficient (R² = 0.99) when modeled using zero-order kinetics, indicating a constant release rate throughout the experimental period (Figure 9A-9D, Table 7). Table 7. Kinetics parameters for Indole-3-Acetic Acid (IAA) release from Phelm in a simulated soil environment. * The Korsmeyer-Peppas model is valid for the first 60% of the compound released. To evaluate the controlled release of Fe3+from the Phelm system, we conducted release studies in deionized water, 10 µM HCl (pH 5), and 100 mM NaCl (pH 7). After 6 hours, it was observed that the rate of Fe3+release was faster in the NaCl solution compared to both, H2O and HCl (Figure 3A-3D). This suggests that the presence of chloride ions (Cl-) in the NaCl solution, in combination with its higher pH, facilitated the release of Fe3+ions. Kinetic modeling using the Korsmeyer-Peppas equation indicated a quasi-Fickian diffusion mechanism for Fe3+(n ≤ 0.5) release under all conditions, as evidenced by the highest correlation coefficients (Table 8). 46 45765042.1 KAUST 2024-082-02 PCT Table 8. Kinetic parameters of Fe3+ release from Phelm under varying conditions was assess wheat seeds in the presence of Phelm tagged with a fluorescent dye (Rhodamine B) in distilled water for 24 and 48 h. Before characterization by confocal microscopy, the wheat seeds were washed three times with distilled water to remove any excess material on the seed surface. The seeds were crossed-sectioned into thin layers horizontally and vertically, placed on glass slides, and covered with a glass coverslip. Nanoparticles were found in the inner tegmen, cotyledon, and radicle (data not shown). In the control samples that were merely treated with water, no fluorescence was observed. These findings confirmed the internalization of Phelm within wheat seedlings after priming. Effects of seed nano-priming in wheat under normal and simulated salinity stress environment Seed priming is a hydration method that activates metabolic processes before radicular protrusion in germination, thereby enhancing germination, abiotic stress tolerance, and plant yields.15Salinity affects germination by reducing water uptake and disrupting nutrient mobility. Salt-inhibited root growth reduces auxin accumulation, independent of auxin biosynthesis. IAA regulates lateral root formation through complex crosstalk with abscisic acid, displaying induction under mild salt stress (NaCl ≤50 mM) but inhibition under high-salinity conditions.57,58Nevertheless, the exogenous application of free PGRs faces challenges such as dosage determination, concentration, and application method influence, which are expensive at the field scale.59For example, in Iris hexagona, exposure of leaves and seeds to high salinity stress (100 mM NaCl) led to a significant drop in IAA levels. Specifically, IAA levels in young leaves decreased by 65%, resulting in a higher number of aborted seeds and reduced aboveground biomass.60In this sense, we hypothesized that seed priming with a controlled-release IAA delivery system would equip seeds with the necessary tools for successful establishment in the field, enhancing germination rates, seedling vigor, and overall plant performance. We conducted a plate germination 47 45765042.1 KAUST 2024-082-02 PCT essay to investigate the potential of IAA-encapsulated within Phelm nanoparticles to enhance wheat seedling growth under both normal and saline conditions. Wheat seeds were subjected to four treatments: a control (Mock), IAA, SoLIN, and Phelm nano-priming. Germination tests were carried out under both normal conditions (MilliQ® water) and simulated salinity stress (100 mM NaCl). The impact of these treatments on seedling growth parameters was evaluated. Overall, we can observe that seeds treated with Phelm displayed better performance in growth metrics and auxin le compared to the control groups (Figure 4A, 4B). Under a simulated salinity stress environment, seeds treated with Phelm after 72 hours exhibited a higher number of germinated seeds per plate (Figure 10A and 10B). The total germination percentage differed significantly between treatments Phelm vs. Mock and non- encapsulated IAA. Treatment type exerted a significant influence on root length, germination rate, and fresh biomass. However, no significant differences (ns) were observed in shoot length among the treatments. Notably, germination activity significantly decreased in wheat seeds primed with non-encapsulated IAA compared to other treatments, accompanied by significantly different germination rates (Figure 11A-11E). Conversely, seedlings primed with Phelm and treated with freshwater, showed significant improvements in root length and fresh biomass, with no significant differences in shoot length among all treatments (Figure 12A-12E). Following the petri dish experiment, we planted wheat seeds in soil pots to assess the effects of various treatments on plant growth. These treatments included a control group primed with water and three groups primed with either IAA, SoLIN, or Phelm. We cultivated the plants in both normal and saline conditions to evaluate how each treatment influenced their growth. Our findings revealed that plants treated with Phelm exhibited significantly greater fresh biomass and root length compared to all control groups. However, plant height was significantly taller only when compared to the Mock and IAA groups, and there was no significant difference in height between Phelm-treated plants and those treated with SoLIN (data not shown, Figure 5A). Under high salinity conditions, plants treated with Phelm exhibited significantly greater fresh biomass compared to all control groups. Additionally, these plants displayed significantly taller heights and longer roots when compared to Mock and IAA- treated plants (data not shown, Figure 5B). This can be associated with several reasons. First, salinity can affect seed germination by reducing the amount of growth stimulants such as gibberellic acid, then, increasing the amount of abscisic acid, and altering the seed membrane permeability and water activity.17This is in agreement with previous studies that associate salinity stress to the detriment of regenerative growth and seed development.44,45For instance, suppressed seedling growth in maize after the 4845765042.1 KAUST 2024-082-02 PCT exogenous application of IAA has been reported.61Moreover, the poor capability of seeds to uptake IAA62coupled with the associated drawbacks of this plant hormone, such as low solubility in aqueous media, decomposition under light irradiation, oxidation, and heating,63may affect the effectiveness of IAA during seed priming. In this regard, the encapsulation of IAA facilitated seed germination even under high-salinity conditions by conferring a protective effect. On the other hand, salt stress exerts osmotic and ionic impacts that produce excessive reactive oxygen species (ROS) and result in oxidative damage, thus damaging proteins, lipids, nucleic acids, and the cellular structure.17Plants with high antioxidant activities can scavenge and detoxify ROS, thus increasing their salt tolerance.64Moreover, it has been reported that cereals may be able to tolerate stress better when ROS detoxifying systems are strengthened with IAA.65Under salt stress, Na+accumulation promotes ROS production, which negatively modulates root growth by regulating auxin polar transport in roots.66We hypothesized that the outer Phelm layer may facilitate seed germination due to the antioxidant capacity of phenolic groups. Thus, we evaluated the H2O2scavenging capacity of Phelm at different concentrations for 1 hour according to a reported protocol67and compared it to five control groups. Phelm demonstrated the highest antioxidant activity at the highest concentration, exhibiting a scavenging percentage of approximately 72%. This was followed by the TA-Fe3+complex, which exhibited a scavenging percentage of approximately 64% (Table 9, Figure 13). These results suggest that the antioxidant properties of Phelm are primarily attributed to its outer shell. Table 9. Comparative evaluation of the hydrogen peroxide (H2O2) scavenging capacity of Phelm at its highest concentration after a 1-hour incubation period, relative to other precursors and a blank control. Subsequently, we conducted a qualitative essay to screen the presence of IAA in wheat roots by extraction and purification. The resulting products were combined with a Salkowski reagent, which produced a pink color upon reaction with IAA and reduction of Fe3+.68,57(data not shown) 4945765042.1 KAUST 2024-082-02 PCT To further detect and quantify IAA, the purified products were analyzed using UV-vis spectrophotometry (data not shown), and the concentrations were calculated using a calibration plot. The quantification of IAA levels in wheat roots (Figure 4B) indicates a significant elevation in auxin levels in seedlings treated with Phelm compared to other groups. This observed increase in auxin levels is noteworthy due to its potential positive impacts. For instance, with more developed roots, seedlings are better equipped to absorb water and nutrients from the soil; thus, improving their chances of stress survival and growth in the field and reducing the time it takes for them to reach maturity.69Ultimately, healthier and more vigorous seedlings with higher levels of auxin are likely to result in higher yields at harvest time. Based on these results, we conclude that the developed delivery system serves as a synergistic platform that effectively protects and disperses IAA. Additionally, it incorporates iron as an essential micronutrient and leverages phenol antioxidant activity, collectively enhancing plant growth parameters. Conclusion The current study introduces Phelm as an innovative, cost-effective, and scalable technology for encapsulating and releasing the plant growth regulators, exemplified herein using indole-3-acetic acid (IAA) in wheat seedlings. The platform's core component, SoLIN, demonstrated high encapsulation efficiency (86.9%) for poorly water-soluble compounds like IAA. The outer shell provided stability over time during storage, pH responsiveness, antioxidant capacity, and micronutrient delivery. When applied as a nano-priming solution, Phelm significantly enhanced seedling development in wheat, boosting growth metrics by 9.8% under normal conditions and 58.6% under simulated abiotic stress compared to control groups. These findings underscore the efficacy of bio-stimulants delivered through appropriate vehicles, even under challenging conditions such as salt stress. The Phelm system offers the advantage of a pH-responsive mechanism coupled with synergistic priming. This unique approach not only supplies seeds with essential iron micronutrients crucial for plant growth but also delivers IAA simultaneously. 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Int. J. Mol. Sci.2022, 23 (23). (66) Fu, et al. Front. Plant Sci.2019, 10 (April). (67) Wei, et al. Adv Funct Mater 2020, 30 (49), 2002234. (68) Goswami, et al. J Microbiol Methods 2015, 110, 7–14. (69) Kazan, et al. Ann. Bot.2013, 112 (9), 1655–1665. Example 2: Abiotic Stress Triggered Nanocarriers for Climate- Resilient Agriculture Materials and Methods All chemicals used in this study were of analytical reagent grade and obtained from commercial suppliers without further purification. Caffeic acid (C9H8O4), Kinetin (C10H9N5O), zinc sulfate heptahydrate (ZnSO4·7H₂O), sodium hydroxide (NaOH), sodium chloride (NaCl), and hydrogen peroxide (H2O2) were purchased from Sigma-Aldrich (Darmstadt, Germany). Rhodamine B was sourced from Alfa Aesar (Massachusetts, United States). Milli-Q water was used for all experiments unless otherwise specified. Seawater utilized in the experiments was provided by the KAUST Water Desalination Center. Tomato seeds (Arkansas traveler) were obtained from the Center of Desert Agriculture at KAUST. Fabrication of CAFZin and CAFZin-K nanocarriers CAFZin nanocarriers were synthesized by dissolving 5 mg of caffeic acid in 2 mL of 0.1 M NaOH (pH 12), followed by brief sonication. Separately, 65 mg of ZnSO₄·7H₂O was dissolved in 1 mL of deionized water. The ZnSO₄ solution was then added dropwise to the caffeic acid solution under continuous stirring. The reaction mixture was stirred for 30 minutes at room temperature. The pH was monitored and adjusted to 8 by the gradual addition of 0.1 M NaOH. The resulting precipitate was washed twice with deionized water and once with acetone. CAFZin-K nanocarriers 5245765042.1 KAUST 2024-082-02 PCT were prepared following a similar procedure with slight modifications. Initially, 2 mg of kinetin was dissolved in 2 mL of 0.1 M NaOH (pH 12) and briefly sonicated. Subsequently, 5 mg of caffeic acid was added to the solution. In a separate vial, 65 mg of ZnSO₄·7H₂O was dissolved in 1 mL of deionized water and then added dropwise to the kinetin-caffeic acid mixture under stirring. The reaction was allowed to proceed for 30 minutes at room temperature. The pH was adjusted to 8 using 1 mL of 0.1 M NaOH added dropwise. The nanocarriers were collected by centrifugation (7000 rcf, 30 min), washed twice with deionized water and once with acetone, and subsequently dried at 45°C under dynamic vacuum in a glass oven for 24 hours. The dried samples were used for characterization purposes. Dynamic Light Scattering (DLS) The hydrodynamic size, ζ-potential, and polydispersity index (PdI) of CAFZin and CAFZin- K nanocarriers were determined using a Zetasizer Nano (Malvern Instruments). For size and PdI measurements, 30 µL of each nanoparticle dispersion was diluted in 1 mL of Milli-Q® water and sonicated for 5 minutes at room temperature. The samples were then transferred to a glass cuvette and analyzed at 25°C, with measurements recorded in triplicate at 60-second intervals. For ζ- potential measurements, the samples were prepared under the same conditions, diluted in Milli-Q® water, and transferred to a Universal Dip Cell (ZEN 1002) for analysis. Entrapment Efficiency and Loading Capacity Percentages The encapsulation efficiency (EE%) and loading capacity (LC%) of the nanocarriers were determined using a UV-Vis spectrophotometer (UV-2600, Shimadzu Europe) at an absorbance wavelength of 270 nm. A calibration curve for kinetin was generated using standard solutions at concentrations of 0.002, 0.004, 0.006, 0.008, and 0.01 mg / mL. A kinetin stock solution (1 mg / mL) was prepared in 1N NaOH for calibration.^^% = ^^^^ ^^ ^^^^^^ g^^^^^^^^^^^^^ ^^^^ ^^ g^^^^^^ ^ 100^^% = ^^^^ ^^ ^^^^^^ g^^^^^^^^^^ ^^ Z^hi^^6g ^ 100Where ^^^^^^ !^"" #$ @^^(^^^ = weight of initial Kinetin used in the formulation (mg),&^"" #$ #^'(' @^^(^^^ = weight of Kinetin loaded in CAFZin-K (mg), &^"" #$ ^%B<^^ −@ = total weight of the complex (mg).Kinetin Release Studies The release kinetics of kinetin from CAFZin-K nanocarriers were evaluated under various conditions and modeled using three different mathematical approaches: zero-order, first-order, and 5345765042.1 KAUST 2024-082-02 PCT Korsmeyer-Peppas models. The cumulative release data were fitted to the corresponding equations to assess the release mechanism.<(.# #.'(.: >^ = >^ + @^. ^B^."^ #.'(.: ^>^ = ^>^ − @D. ^@#."!(E(. F(00^": >^ = @;. ^^Effect of pH on Kinetin Release To investigate the impact of pH, CAFZin-K (1 mg) was dispersed in 0.5 mL of methanol solutions adjusted to pH 3, 5, or 9. The samples were transferred to Eppendorf tubes and incubated in an Eppendorf Thermomixer at 36 °C with constant shaking at 400 rpm for 30 minutes. Following incubation, the suspensions were centrifuged at 17,000 rcf for 10 minutes, and the supernatants were collected for analysis. At specific time intervals (0, 1, 2, 3, 4, 5, and 6 hours), an equal volume of fresh methanol solution was added to maintain constant volume conditions. Kinetin concentrations in the collected supernatants were quantified using a UV-Vis spectrophotometer (UV-2600, Shimadzu Europe) at 270 nm, based on a standard calibration curve prepared for kinetin in methanol. All measurements were performed in triplicate. The percentage of cumulative release (%R) at each time point was determined using the following equations. %!#+^^^ = ^#^-(^^.^^^#^ #$ @^^(^^^ ^^ "+0(.^^^^^^ (!j !^) ^ 2# +!((!^) Effect of Salinity on Kinetin Release To examine kinetin release under varying salinity conditions, CAFZin-K (1 mg) was dispersed in 0.5 mL of different salt solutions, including 15% seawater, 30% seawater, 100 mM NaCl, and 150 mM NaCl. The samples were incubated under the same conditions as described for the pH study (36 °C, 400 rpm, 30 minutes). After incubation, the samples were centrifuged at 17,000 rcf for 10 minutes, and the supernatants were collected. At predetermined time points (0, 1, 2, 3, 4, 5, and 6 hours); fresh saline solutions of the corresponding concentration were added to replace the withdrawn supernatant, maintaining a constant volume. Kinetin concentrations were quantified using UV-Vis spectrophotometry at 270 nm, following a standard calibration curve. All measurements were conducted in triplicate, and the cumulative release (%R) was calculated accordingly. 5445765042.1 KAUST 2024-082-02 PCT Kinetin Release in a Simulated Soil Environment Kinetin release from CAFZin-K was further assessed in a simulated soil environment, mimicking natural soil composition through an amino acid-based medium. The soil model was prepared by dissolving aspartic acid (5 mM), glutamine (5 mM), histidine (5 mM), asparagine (5 mM), glutamic acid (5 mM), alanine (5 mM), phosphate-buffered saline (PBS, 1×), and HEPES (10 mM) in 5 mL of PBS. CAFZin-K (1 mg) was dispersed in 1 mL of this simulated soil medium. The sample was vortexed for 10 seconds to ensure homogeneity, followed by incubation at 45 °C for 24 hours. At predefined time points (0, 1, 2, 4, 6, and 24 hours), the samples were centrifuged at 17,000 rcf for 10 minutes, and the supernatants were collected for analysis. To maintain constant volume conditions, 1 mL of fresh amino acid medium was added after each centrifugation step. Kinetin concentrations in the collected supernatants were determined using UV-Vis spectrophotometry at 270 nm. All experiments were conducted in triplicate. Photostability assessment The stability of CAFZin-K and free kinetin under UV radiation was assessed by preparing solutions at three different concentrations (10, 25, and 50 µM) in 1N NaOH. The samples were exposed to UV light (~365 nm) for varying durations: 0, 0.5, 1, 2, 4, and 24 hours. At each time point, 500 µL aliquots were collected and analyzed using UV-Vis spectrophotometry (UV-2600, Shimadzu Europe) to quantify the remaining Kinetin. A calibration curve was constructed to determine the kinetin concentration at the initial time point (0 h) and after UV exposure. Additionally, the amount of non-encapsulated kinetin was quantified to evaluate potential losses due to UV degradation. All experiments were performed in triplicate to ensure reproducibility. Thermal stability The thermal stability of CAFZin-K and free kinetin was evaluated at 45°C, a temperature representative of field conditions in the Kingdom of Saudi Arabia. Solutions were prepared at three different concentrations (10, 25, and 50 µM) in 1N NaOH and incubated at 45°C for various durations (0, 0.5, 1, 2, 4, and 24 hours). At each time point, 500 µL aliquots were collected and analyzed using UV-Vis spectrophotometry (UV-2600, Shimadzu Europe) to quantify the remaining kinetin. A calibration curve was established to determine kinetin concentrations at the initial time point (0 h) and after thermal exposure. Additionally, the concentration of non-encapsulated kinetin was quantified to assess potential degradation losses. All experiments were conducted in triplicate to ensure accuracy and reproducibility. 5545765042.1 KAUST 2024-082-02 PCT High-Resolution Transmission Electron Microscopy (HRTEM) High-resolution transmission electron microscopy (HRTEM) was performed using an FEI Titan ST operating at 300 kV to examine the morphology of the synthesized nanocarriers. Sample preparation involved extruding CAFZin-K nanocarriers through a 1.0-µm membrane, followed by depositing 5 µL of the recovered solution onto 200-square-mesh carbon-coated copper grids. Excess solvent was removed using filter paper, and the grids were left to dry under vacuum overnight before imaging. Fourier Transform Infrared (FTIR) The chemical composition and functional groups of the synthesized CAFZin-K nanocarriers were analyzed using Fourier transform infrared (FTIR) spectroscopy in attenuated total reflection (ATR) mode (Smart iTR, Thermo Scientific™). Spectra were recorded in transmittance mode over a wavenumber range of ~4000–700 cm⁻¹ to identify characteristic functional groups. The FTIR spectra of CAFZin-K were compared with those of its individual components, including CAFZin, free kinetin, and caffeic acid, to confirm successful encapsulation and interactions between the constituents. Hydrogen Peroxide Scavenging Activity The H2O2 scavenging assay of CAFZin, CAFZin-K, kinetin, and caffeic acid was conducted using a hydrogen peroxide (H2O2) stock solution (15 mM) prepared in phosphate-buffered saline (PBS). Test solutions were prepared at varying concentrations (5 µM, 10 µM, 15 µM, and 30 µM), and 500 µL of each solution was mixed with 1 mL of the H2O2solution. The absorbance of the reaction mixtures was measured at 230 nm after 24 hours of incubation. The H2O2 solution in PBS served as the control. The scavenging activity percentage was calculated using the following equation: H2O2 =^a6^U^a^ 100 % where Ac represents the absorbance of the control sample (H₂O₂ in PBS), and As corresponds to the absorbance of the sample containing CAFZin, CAFZin-K, kinetin, or caffeic acid. Powder X-Ray Diffraction (XRD) Powder X-ray diffraction (PXRD) analysis was performed using an X-ray diffractometer (D8 Advance A25, Bruker®). Data were collected over a 2θ range of 4° to 80° with a step size of 0.02°. The recorded diffraction patterns correspond to CAFZin-K and CAFZin, along with the physical mixture of precursors and kinetin, which were used as controls. 5645765042.1 KAUST 2024-082-02 PCT Confocal Microscopy A qualitative analysis of CAFZin-K (loaded with Rhodamine B instead of kinetin) was conducted using confocal microscopy. The preparation of CAFZin-K followed the procedure outlined in the fabrication section, with slight modifications. Specifically, caffeic acid was dissolved in 0.1 M NaOH to a final concentration of 14 mM (2 mL), while ZnSO₄·7H₂O was prepared in water at a 0.226 M concentration (1 mL). A Rhodamine B solution (0.84 mM, 2 mL) was also prepared. To synthesize CAFZin-K (Rhodamine B loaded), the caffeic acid solution was first mixed with the Rhodamine B solution and vortexed for 30 seconds. ZnSO₄ was then added dropwise, and the reaction pH was adjusted to 8. The mixture was stirred for 30 minutes at room temperature, followed by centrifugation (20,000 rcf, 10 minutes). The resulting product was washed three times with water to remove excess Rhodamine B and unreacted precursors. For the internalization study in tomato seeds, seeds were incubated in either free Rhodamine B (non- encapsulated) or Rhodamine B-loaded CAFZin solution for 6 hours at a 1:1 weight-to-volume ratio. After incubation, the seeds were washed three times with Milli-Q® water to remove the excess dye, cross-sectioned horizontally and vertically into thin layers, and mounted on glass slides with a coverslip.3D images were taken using a Leica SP8 Laser Confocal Microscope (CLSM). The uptake was detected using Rhod channel (Ex.545 nm), and a Z-stack of the samples was generated. Preparation of the priming solutions Seed priming solutions were prepared using the as-synthesized CAFZin-K, CAFZin, and Kinetin at a final concentration of 10 µM in a 1:1 weight-to-volume ratio. Non-primed seeds served as the control. Seed Priming Method Healthy tomato seeds from the same lot were weighed and soaked in CAFZin-K, CAFZin, or Kinetin solutions for 6 hours in the dark at room temperature. After priming, the seeds were surface-dried on paper towels to remove excess moisture. Tomato Plate Germination Assay and Growth Metrics Primed and non-primed tomato seeds were placed on filter paper in Petri dishes and sealed with parafilm. Each treatment included three biological replicates of 20 seeds per plate, irrigated with either 10 mL of 15% seawater or Milli-Q® water at room temperature. Plates were kept in a controlled chamber (20.5°C, 63% humidity), and seedlings were collected 7 days post-germination. Root and shoot lengths were measured using a ruler, and fresh biomass was recorded after gently removing excess moisture. Germination rate (GR) and germination percentage (GP% ) were calculated as follows: 5745765042.1 KAUST 2024-082-02 PCTb3 = ^4^c^J ^^ 1^J^^^^^^^ ^^^^^^^1^d^e^ ^^ ^^J^^ : + ⋯^4^c^J ^^ 1^J^^^^^^^ ^^^^^^^1^ ^4^^ d^e^ ^^ ^^^^^ :^4^^^^ ^^^^^ ^^^^^ inch deep) filled with potting soil and maintained in a provisional growth station (23°C, 40% humidity, 15 h light / 9 h dark). During this growth stage (approximately 1 week), plants were irrigated only with distilled water. At the three-leaf stage, three healthy plants per treatment (CAFZin-K, CAFZin, Kinetin, and control) were transplanted into pots (6 × 5.5 × 4.5 cm) and grown under semi-controlled conditions (26°C, 60% humidity, 12 h light / 12 h dark). No fertilizers or nutrient solutions were added. Plants were irrigated daily with either 10 mL of freshwater or a 15% seawater solution. After 4 weeks, plant height, root length, and fresh biomass were recorded. Mass Spectrometry CAFZin-K (1 mg) was dispersed in 1 mL of methanol (0.1 M, pH 5.5) and stirred for 1 hour. The dispersion was then centrifuged at 20,000 rcf for 10 minutes, and the supernatant was collected for mass spectrometry (MS) analysis in positive reflecting mode. For kinetin, the same procedure was followed using methanol without pH modification. CAFZin was prepared following the same protocol as CAFZin-K and analyzed in negative reflecting mode. All MS analyses were performed using a QTOF mass spectrometer (MaXis QTOF, Bruker, MA, USA). Ultrawave Digestion Each sample was weighed into acid-washed PTFE digestion tubes, and 5 mL of concentrated nitric acid (HNO₃) was added. Samples were placed in the ultrawave digestion unit and subjected to controlled microwave-assisted digestion following the manufacturer's recommended settings. Upon completion of the digestion cycle, indicated by the instrument's green status signal, samples were removed, cooled, quantitatively transferred, and diluted to a final volume of 50 mL with Milli-Q water before ICP-OES analysis. Elemental analysis in plant tissues and seeds (ICP-OES) Samples underwent microwave-assisted acid digestion using an ultrawave Single Reaction Chamber (SRC) Microwave Digestion System (Milestone Inc., Italy). Elemental analysis was performed using an inductively coupled plasma optical emission spectrometer (ICP-OES, Agilent 5900, Agilent Technologies, USA) equipped with an axial-view plasma torch. Calibration curves for Zn, K, Mg, and Na were established using freshly prepared multi- element standard solutions at concentrations ranging from 0.1 to 100 ppm. A stock standard 5845765042.1 KAUST 2024-082-02 PCT solution (100 ppm) was prepared by diluting commercially available certified standards (1000 ppm) in 1% HNO₃. Subsequent working standards (0.1, 1, 10, and 100 ppm) were obtained via serial dilution. Quality control (QC) standards at 100 ppm and 0.1 ppm were prepared similarly to validate analytical accuracy. Calibration curves employed quadratic regression fits, demonstrating excellent linearity with correlation coefficients (R²) greater than 0.999. Cell viability studies The biocompatibility of the formulations (CA, ZnSO4.7H2O, Kinetin, CAFZin, and CAFZin-K) was tested using the CCK-8 assay according to the manufacturer’s protocol. Briefly, HepG2 cells and NIH-3T3 cells were seeded into 96-well plates 10x103cells / well in 100 µl DMEM culture medium with 10% FBS and 1% Penicillin-Streptomycin, and incubated in a 5% CO2 incubator at 37°C overnight. After that, the cells were incubated with 100 µl DMEM medium containing different concentrations of formulations, ranging from 0.01 to 5 µg / mL, except for Kinetin, the range was 0.001 to 0.5 µg / mL. Cells without any treatment were used as a control, and all the conditions were done in triplicate. After 24 h, the media was discarded from the plate and cells were washed with PBS. Then, 10 µl of CCK-8 and 90 µl of fresh DMEM media were added to each well and incubated for 4 h. The absorbance was measured at OD = 570 nm and 605 nm using a microplate spectrophotometer (BioRad-xMark Microplate Absorbance Spectrophotometer). The ratio of the absorbance at 570 / 605 nm was calculated, and the average absorbance value of wells without cells was considered blank and subtracted from all wells to cancel the background noise. The cells without any treatment were used as a control; their mean value was considered 100% viable, and the viability of the other treated cells was calculated based on that, using the equation: ^( " 2^^]^ ^^E% =^#..(-^(' !(^^ ^]"#.]^^-( #$ ^.(^^(' -( " ^#..(-^(' !(^^ ^]"#.]^^-( #$ -#^^.# ^ 100X-ray photoelectron spectroscopy (XPS) X-ray photoelectron spectroscopy (XPS) analyses were performed on a Kratos Amicus system to determine the elemental composition. The binding energy (BE) scale was referenced to the C 1s peak of aromatic carbon at 284.5 eV. The measurement depth was 5–6 nm, and the analytical area ranged from 0.3 to 0.7 mm². All samples were washed several times with water and dried overnight under dynamic vacuum at 45 °C. Kinetin Extraction and Purification Tomato seeds were primed either with non-encapsulated Kinetin or CAFZin-K solutions. Post-primed seeds were frozen in liquid nitrogen, crushed, and incubated overnight in modified 5945765042.1 KAUST 2024-082-02 PCT Bieleski’s solvent: methanol (MeOH): formic acid (HCOOH): water (H2O), 15:1:4 ratio at –20^°C to facilitate Kinetin release from internal seed tissues. The mixture was centrifuged, and the supernatant was collected for further purification. The supernatant containing the extracted Kinetin was purified using a Sephadex G-25 column pre-equilibrated with 1 M ammonium bicarbonate (NH4HCO3) buffer at pH 7.2–7.5. Elution was performed in two sequential steps: initially with 1 M NH4HCO3, followed by 80% methanol. The collected fractions were subsequently dried under reduced pressure using a rotary evaporator to partially remove residual solvents. Liquid Chromatography with tandem mass spectrometry LC-MS-MS Quantification was carried out on an LC-MS / MS system (Thermo Altis Plus, MA, USA) with an electrospray ionization (ESI⁺) source. Samples were separated on a C18 reverse-phase column (2.1 × 50 mm, 1.7 µm) using a gradient of (A) 0.1% acetic acid in water and (B) 0.1% acetic acid in acetonitrile at 0.3 mL / min. Statistical Analysis All statistical analyses were performed using GraphPad Prism 8. Data are presented as mean ± standard deviation (SD) from three independent replicates (n = 3). For germination assays, each replicate was one plate containing 20 seeds. A one-way ANOVA was used to determine statistically significant differences between treatment groups. Where appropriate, post hoc comparisons were performed, and significance was reported as follows: ns (not significant), *p < 0.05, **p < 0.005, and ***p < 0.0005. For plant growth metrics in potting soil, one-way ANOVA was also employed, with significance levels indicated as: ns (not significant), *p < 0.05, **p < 0.01, ***p < 0.0005, and ****p < 0.0001. A two- way ANOVA was applied to evaluate plant height over time, with significance levels consistent with the aforementioned scale. A two-tailed unpaired t-test was used to assess differences in Kinetin concentration between free and encapsulated kinetin (****p<0.001). The analyzed parameters included shoot length, root length, germination percentage, and fresh biomass of tomato seedlings, comparing non-primed controls to seeds primed with CAFZin-K, CAFZin, or Kinetin, and irrigated with either fresh water or 15% seawater. Results and discussion Synthesis and characterization of CAFZin-K CAFZin-K nanocarriers were synthesized through a simple mixing process involving Kn, caffeic acid, and zinc sulfate under basic conditions, as described in the Materials and Methods section. The addition of zinc sulfate caused the reaction mixture to turn green, indicating successful complexation between the metal ion and the organic linker. 6045765042.1 KAUST 2024-082-02 PCT Following the reaction at room temperature, the pH was adjusted to 8, and the nanocarriers were isolated, washed, and dried prior to characterization (Figure 14). A control formulation lacking Kn, referred to as CAFZin, was also prepared for comparison. Dynamic light scattering (DLS) analysis of CAFZin-K revealed a uniform particle size of 122.67 ± 6.94 nm and a zeta potential of -7.8 ± 0.36 mV, in contrast to the +13.36 ± 0.58 mV measured for CAFZin, suggesting successful incorporation of Kn (Figure 21). High-resolution transmission electron microscopy (HRTEM) confirmed the formation of uniformly dispersed spherical nanocarriers, with sizes consistent with DLS measurements (Figure 15A). Fourier-transform infrared spectroscopy (FTIR) (Figure 15B) showed characteristic peaks of caffeic acid at 3396 and 3211 cm⁻¹ (hydroxyl groups), and a broad band from 1535 to 1650 cm⁻¹ associated with C=C stretching. The peak at 1442 cm⁻¹ corresponded to the -OH bending of the carboxylic group.34In both CAFZin and CAFZin-K, new bands in the 1420–1492 cm⁻¹ range were attributed to metal–carboxylate bond formation.35While both samples exhibited similar IR patterns, a distinct peak at 1118 cm⁻¹ in CAFZin-K indicated C—O stretching of the furan ring, confirming the presence of Kn in the nanoparticle matrix.36,37X-ray photoelectron spectroscopy (XPS) further supported that the coordination of Zn²⁺ with caffeic acid (through phenolic –OH and carboxylic –COOH groups) significantly reduces the local electron density (Figure 15B, Figure 15A and data not shown). Upon encapsulation of Kn, additional hydrogen bonding or coordination interactions may form, further withdrawing electron density from aromatic C, C–N, and C=N groups. These factors lead to a net decrease in electron density on carbon atoms, thus, higher C 1s binding energy.38The observed increase in O 1s binding energy is attributed to the coordination of Zn²⁺ with oxygen atoms from the carboxyl and hydroxyl groups of caffeic acid, which alters their electronic environment and leads to higher binding energy values. These oxygen atoms directly coordinate with Zn²⁺, donating their lone pairs, resulting in a significant reduction in electron density.39Additionally, possible hydrogen bonding further reduces the local electron density.40UV–vis analysis (Figure 15C) showed spectral shifts and reduced intensity near ~270 nm and ~380 nm in both CAFZin and CAFZin-K compared to pristine caffeic acid, indicating metal–phenolic coordination.41–43Although no significant spectral differences were observed between CAFZin and CAFZin-K, the appearance of a representative Kn band around 270–280 nm in CAFZin-K confirmed its incorporation.44Encapsulation efficiency and loading capacity were calculated as 98.8 ± 4.45% and 12.5 ± 3.81%, respectively, using a standard plot at different concentrations of Kn (Figure 16) and the equations provided in the Materials and Methods section. These values indicate high loading performance, as nanoparticle systems containing more than 10% active compound by weight are generally considered efficient carriers.45Moreover, 6145765042.1 KAUST 2024-082-02 PCT mass spectrometry analysis was performed to further confirm the presence of Kn in the CAFZin-K nanocarriers. Mass spectra were obtained using QTOF-MS to confirm the presence of Kinetin and caffeic acid in the nanoparticle formulations. CAFZin-K and free Kinetin were analyzed in positive ion reflecting mode, both showing a characteristic signal at m / z 216.09 corresponding to Kinetin. CAFZin was analyzed in negative ion mode, exhibiting a representative signal at m / z 178.93, confirming the presence of caffeic acid. (data not shown). X-ray diffraction (XRD) patterns revealed predominantly amorphous structures in both CAFZin and CAFZin-K, indicating that Kn loading did not alter the overall structural phase.46,47Physical mixtures and individual precursors were also analyzed as controls. PXRD patterns were recorded for the physical mixtures of nanoparticle precursors (ZnSO4, caffeic acid, and Kinetin) at two ratios: (i) 100:100:12.5, reflecting the Kinetin loading capacity within CAFZin- K, and (ii) 1:1:1 for equimolar comparison. Raw individual precursors were included as controls (left). PXRD patterns of CAFZin and CAFZin-K nanocarriers (right) revealed a semi-amorphous structure in both cases, indicating that Kinetin encapsulation did not induce significant structural changes in the nanoparticle matrix. (data not shown). Release Kinetics and stability of CAFZin-K Understanding the release kinetics of Kn from CAFZin-K nanocarriers is essential to evaluate their potential as responsive and sustained release systems for agricultural applications. The release profile governs not only the timing and extent of bioavailability but also how effectively the system performs under diverse field conditions. Soil temperature and moisture, along with environmental factors such as pH fluctuations, ionic strength, and the complexity of soil matrices, can all influence the stability of agrochemicals. Encapsulation within protective carriers like metal–polyphenol networks (MPNs) offer a promising strategy to enhance the stability and efficiency of Kn by modulating its release across these variable conditions. The pH-responsive release of Kn from CAFZin-K nanocarriers was evaluated at pH 3, 5, and 9 using UV–vis spectrophotometry, monitoring the characteristic absorption at 280 nm in methanol. As anticipated, the release rate was significantly higher under acidic conditions. After 6 hours, only 23.9% of Kn was released at pH 9, compared to 38.5% and 52.7% at pH 5 and pH 3, respectively (Figure 16A). This behavior is attributed to the pH-dependent coordination dynamics of MPNs. At alkaline pH, deprotonation of the polyphenol hydroxyl groups promotes strong coordination with metal ions, resulting in a more rigid and stable structure that hinders release. In contrast, acidic conditions lead to protonation of hydroxyl groups, weakening metal–ligand coordination and triggering disassembly of the network, thereby facilitating Kn release.48The release kinetics and mechanism 6245765042.1 KAUST 2024-082-02 PCT of Kn were investigated under these conditions and analyzed using three different kinetic models: zero-order, first-order, and Korsmeyer−Peppas. Each model was applied to the release plots. The model fit was evaluated using the coefficient of determination (R2) values, with higher R2 values indicating a better model fit. The R2 values and other model parameters derived from each release stage are presented in Figure 23, Table 10. It was found that the linear correlation coefficient (R2 = 0.99) of the Korsmeyer−Peppas model was the highest for the first stage of Kn from CAFZin-K, and it follows a non-Fickian diffusion mechanism (0.5 < n < 1). Table 10. pH release kinetics. Release conditions pH 3 pH 5 pH 9 Zero-order @^3_8.59 6.57 4.01 0.92 0.96 0.94 First order @D_ 0.12 0.08 0.04 3 0.96 0.98 0.96 Korsmeyer Peppas @l19.85 11 7.84 ^ _ 0.57 0.72 0.65 3 0.99 0.99 0.99 To assess the effect of ionic strength and salt composition on Kn release, UV–vis spectrophotometry at 280 nm was used in different saline environments. After 6 hours, the cumulative release followed the trend: 30% seawater < 150 mM NaCl < 15% seawater < 100 mM NaCl (Figure 16B). This variation likely stems from differences in ionic strength and ion composition affecting the stability of the metal–phenolic network. While 100 mM NaCl promoted the highest release and followed a Fickian diffusion mechanism (n < 0.5), 15% seawater (~76 mM NaCl) showed non-Fickian release behavior (0.5 < n < 1), despite its lower salt concentration (Figure 24, Table 11). This may be due to the presence of multivalent ions in seawater (e.g., Mg²⁺, Ca²⁺), which can disrupt or reorganize the coordination matrix, facilitating both diffusion and matrix relaxation.49,50In contrast, the simpler ionic composition of NaCl solutions leads to more uniform, diffusion-dominated release.51Table 11. Release in different salt media. Release conditions 15% Seawater 30% Seawater 100 mM NaCl 150 mM NaCl Zero-order @ 1.57 0.98 1.82 1.28 3^_ 0.96 0.97 0.87 0.95 First order @ 3D_ 0.02 0.01 0.02 0.01 0.96 0.97 0.88 0.95 Korsmeyer Peppas@l2.49 1.73 5.46 2.61 ^ _ 0.84 0.70 0.45 0.63 3 0.99 0.99 0.99 0.99 6345765042.1 KAUST 2024-082-02 PCT Soil model release kinetics To evaluate this, the thermal stability of CAFZin-K was compared with non-encapsulated Kn by simulating soil-like environments. Given the complexity of soil, where organic matter interacts with microbial activity and environmental variables (e.g., pH, temperature, CO₂), amino acid (AA) media was used to mimic soil organic content and incubated samples at 45 °C for 24 h. Additionally, PBS and HEPES buffers were used to assess stability under physiological conditions (Figure 16C). Kn release under these conditions was analyzed using zero-order, first-order, and Korsmeyer–Peppas models. The Korsmeyer–Peppas model showed the best fit (R² = 0.99), indicating that release from CAFZin-K followed a Fickian diffusion mechanism (n < 0.5), characteristic of primarily diffusion-controlled processes (Figure 25, Table 12). Table 12. Release in soil model. Release conditionsSoil model 1st stage Soil model 2nd stage Zero-order @ 3.624 0.041 3^_ 0.865 0.957 First order @D3_0.071 0.010 0.995 0.871 Korsmeyer Peppas @ 6.455 ^l_ 0.103 3 0.997 UV and thermal treatment The stability of CAFZin-K and free Kn under UV radiation and thermal stress was evaluated to assess the protective effect of MPN encapsulation. Kn solutions at concentrations of 10, 25, and 50 μM were exposed to UV light (~365 nm) and incubated at 45°C, conditions chosen to mimic environmental stressors typical of field settings such as those in the Kingdom of Saudi Arabia. For both treatments, samples were collected at 0, 0.5, 1, 2, 4, and 24 hours and analyzed by UV–vis spectrophotometry to quantify the remaining Kn. A progressive decrease in absorbance was observed for non-encapsulated Kn across all concentrations and time points, indicating significant degradation under both UV and thermal conditions. In contrast, CAFZin-K samples showed markedly reduced loss of absorbance, confirming that encapsulation within the MPN framework provides enhanced protection against degradation (Figure 16D-E, data now shown). Thermal stability of Kinetin and CAFZin-K. Stability comparison of free Kinetin and Kinetin encapsulated within CAFZin-K nanocarriers after incubation at 45^°C for 24^h. UV–vis spectroscopy was used to monitor changes in absorbance intensity at various concentrations, revealing improved thermal stability upon encapsulation. Comparative analysis of the stability of free Kinetin and Kinetin 6445765042.1 KAUST 2024-082-02 PCT encapsulated in CAFZin-K nanocarriers following UV irradiation (~365^nm) for 24^h. UV–vis spectroscopy was employed to track changes in absorbance intensity at various concentrations, demonstrating enhanced photostability of Kinetin upon encapsulation. Biocompatibility of CAFZin-K Prompted by the promising effect of CAFZin-K on plant germination and growth, its biocompatibility was evaluated on mammalian cell lines due to the possible presence of Zn traces in the fruits before human consumption. The biocompatibility of Caffeic Acid was measured, ZnSO4.7H2O, Kn, CAFZin, and CAFZin-K on liver (HepG2) cells and fibroblasts (NIH-3T3) cells after 24 h measured by CCK-8 (Figure 26). The concentrations of Kn (cargo) were 10-fold lower due to the lower amount compared to the carrier, based on the encapsulation capacity. It was found that CAFZin-K was biocompatible up to 5 μg / mL, and the presence of Kn helped in decreasing the cytotoxic effects of the network since it is known to decrease cellular stress.52Functional antioxidant properties Under abiotic stress conditions, plants generate elevated levels of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), which can cause oxidative damage to cellular components, impair growth, and reduce nutrient uptake.53Thus, materials capable of scavenging ROS may provide additional protective benefits when applied in agricultural settings. The ROS- scavenging capacity of CAFZin-K was evaluated using H2O2 as a model oxidant. Compared to control groups, CAFZin-K demonstrated significantly higher scavenging activity, reaching approximately 40% at a Kn-equivalent concentration of 10 μM after 24 hours (Figure 17). This enhanced activity may be attributed to the synergistic contribution of both the phenolic framework and zinc ions within the metal–phenolic network, which are known to exhibit antioxidant properties.33,54These findings suggest that, beyond controlled hormone release, CAFZin-K may also confer antioxidant protection under stress conditions, potentially contributing to improved plant resilience. CAFZin-Assisted Seed Priming and Cargo Localization Rhodamine B (RhB) was used as a fluorescent model to assess the potential of CAFZin as a seed nanopriming carrier. Z-stack confocal microscopy revealed that free RhB primarily localized at the seed surface, indicating minimal penetration. In contrast, seeds treated with RhB-loaded CAFZin showed fluorescence beyond the outer layers, suggesting enhanced association with internal tissues (data not shown). This supports the role of CAFZin in improving cargo proximity or uptake during priming, potentially contributing to early-stage physiological preparedness under stress conditions. 6545765042.1 KAUST 2024-082-02 PCT Impact of CAFZin-K on tomato growth metrics Seed priming is a pre-sowing technique that partially hydrates seeds without triggering radicle emergence, enhancing germination rate, uniformity, and stress tolerance. This increased resilience is thought to result from early activation of germination-related processes (e.g., respiration, gene transcription) and the induction of mild stress responses, such as LEA protein accumulation. Together, these effects establish a “priming memory” that improves seed performance under subsequent stress.55In the present work, the growth parameters were evaluated following the nanopriming of tomato seeds during early germination stages, with subsequent irrigation using either tap water or 15% seawater for one week. Physiological traits such as shoot length, root length, fresh biomass, germination rate per plate, and total germination percentage were recorded across all treatment groups. Overall, seeds treated with CAFZin-K demonstrated statistically significant improvements in shoot and root length compared to control groups under tap water conditions (Figure 27A) (***p < 0.0005) and under 15% seawater (*p < 0.05) (Figure 27B). After one week of germination, the seedlings were transplanted into potting soil and allowed to acclimate for an additional week. Following this period, plants were irrigated daily with either tap water (Figure 18A) or 15% seawater (Figure 18B) for one month. Plant height was recorded at weeks 1, 2, and 4 (Figure 28A-28D). Across all developmental stages, plants primed with CAFZin- K consistently outperformed the control groups, with the benefits of Kn encapsulation being particularly pronounced under seawater irrigation. By the fourth week, additional physiological parameters such as root length, fresh biomass, and dry biomass were assessed, and plant tissues (stem, root, and leaf) were collected for elemental analysis. Plants treated with CAFZin-K exhibited superior growth compared to control treatments, including those primed with non-encapsulated Kn. These findings confirm that encapsulation provides a protective effect, enhancing the release and bioavailability of Kn, especially under stress conditions. Building on this concept, these results show that the efficacy of Kn, a plant growth regulator known to alleviate salinity stress,56,57can be further enhanced through nanopriming. Co-release with phenolic compounds and essential nutrients via nanocarriers improves molecular stability and triggers stronger physiological responses, highlighting the potential of multifunctional nanopriming strategies to boost abiotic stress resilience in crops.58–60CAFZin-K bioavailability and nutrient distribution in plant tissues. Ensuring balanced uptake of essential ions like magnesium (Mg²⁺) and zinc (Zn²⁺) is helpful for plant growth and stress tolerance, particularly under salinity, where ionic toxicity disrupts nutrient homeostasis.61,62ICP-OES was used to examine nutrient distribution in plant tissues among 6645765042.1 KAUST 2024-082-02 PCT different groups irrigated with fresh and saltwater, and mainly to assess the impact of priming with CAFZin-K and non-encapsulated Kn and their influence on elemental uptake under tap and seawater irrigation. Empty nanocarriers (CAFZin) and non-primed seeds were also used as controls. Under tap water, both Mg²⁺ and Zn²⁺ levels were highest in CAFZin-K treated plants across all parts (Figure 19A), suggesting enhanced availability and transport driven by the combination of the nanocarrier and Kn, improved zinc release and movement through the plant. While salinity typically suppresses micronutrient uptake, the advantage of CAFZin-K persisted under seawater irrigation, maintained elevated Zn²⁺ levels, and fully restored Mg²⁺ uptake, particularly in roots. This likely reflects the role of Kn in supporting root activity and transport processes.63These insights highlight the synergistic effect, which is the ability of CAFZin-K to both promote and protect ion absorption, supporting ion homeostasis even under stressed conditions.64Sodium (Na+) and potassium (K+) uptake patterns further indicate the impact of the treatments on ionic balance under both normal and saline conditions.65 In tap water, sodium levels remained low overall but were slightly elevated in CAFZin and CAFZin-K treatments, likely due to baseline uptake.66(Figure 19B) Under seawater irrigation, sodium increased across all groups, with Kn-treated plants showing the highest accumulation in roots. In contrast, CAFZin-K effectively reduces sodium, especially in roots and leaves, indicating better control than free Kn over salt stress. For potassium, CAFZin-K again reached the highest levels across stem, leaf, and root tissues under tap water irrigation. In addition, CAFZin-K preserved root potassium uptake under seawater conditions, despite the expected drop caused by sodium interference. These patterns highlight the role of CAFZin-K in maintaining a healthy K⁺ / Na⁺ ratio as a key marker of salt tolerance, by simultaneously limiting Na⁺ accumulation and sustaining K⁺ availability where it is most needed. Nano-Enabled Release at the Seed Interface Comparative Zinc Uptake Across Seed Species Nanoparticle release systems, including metal-phenolic carriers, have been reported to significantly enhance the stability, solubility, and sustained release of biostimulants in plant systems.67By shielding active compounds from premature degradation or environmental loss, these carriers facilitate controlled and sustained release.16Once localized into the seed through nanopriming, they undergo gradual disassembly, often triggered by enzymatic or pH-responsive mechanisms, allowing localized, time-regulated cargo release.68 This approach has consistently demonstrated higher uptake and bioavailability of encapsulated compounds than conventional formulations.696745765042.1 KAUST 2024-082-02 PCT To assess the release efficiency of zinc from the CAFZin-K metal–phenolic nanocarriers, inductively coupled plasma optical emission spectroscopy (ICP-OES) was performed on three crop species: tomato, capsicum, and pearl millet. These measurements aimed to explore whether seed traits influence zinc uptake following nano-priming. The results revealed high zinc retention in tomato (~70%) and capsicum (~80%) seeds, while pearl millet showed significantly lower uptake (~40%) (Figure 20A). This differential uptake is likely attributable to physical and structural differences in seed coats.70Tomato and capsicum seeds, which typically have thinner or more permeable seed coats, may facilitate greater nanoparticle availability. In contrast, pearl millet seeds, known for their harder and more impermeable seed coats, may pose barriers to nanoparticle entry. These findings are consistent with prior studies highlighting the role of seed structural properties in regulating nanoparticle absorption.71–73While physical limitations reduced zinc uptake in pearl millet, measurable zinc levels in all three species confirm the capacity of CAFZin-K to deliver micronutrients across diverse seed types. This underscores its potential for seed-level biofortification strategies, especially in species with favorable seed permeability traits. Kinetin Bioavailability in Tomato Seeds To evaluate the effectiveness of Kn release via nanoencapsulation, a sensitive and selective LC-MS / MS assay was developed to quantify Kn content at the seed interface following priming. This analysis focused exclusively on tomato seeds, the primary crop of this study. Kn content was significantly higher in seeds treated with CAFZin-K encapsulated Kn (1.537 ± 0.022 μg / mL) compared to those treated with free Kn (0.414 ± 0.021 μg / mL), representing a 3.7-fold increase (****p < 0.0001) (Figure 20B, data now shown). These results confirm that nanoencapsulation using CAFZin-K enhances Kn stability and bioavailability, likely through protection from degradation and controlled release mechanisms triggered within the seed microenvironment, hence, maintaining a higher effective dose within the seed tissues compared to non-encapsulated treatment. Conclusions Overall, these findings demonstrate that the encapsulation of biostimulants, such as phytohormones, holds significant potential for advancing climate-resilient agriculture. Encapsulation provides essential protection against environmental factors such as light and high temperatures, which is particularly advantageous during the storage and transport of agrochemicals. Moreover, it addresses common challenges related to the poor solubility of certain compounds, enhancing their effectiveness during seed priming. Delivering encapsulated phytohormones at the seed level improves their bioavailability within seed tissues, allowing plants to withstand environmental stresses from the earliest stages of 6845765042.1 KAUST 2024-082-02 PCT development. Furthermore, integrating naturally occurring plant-derived molecules and essential nutrients into the nanocarrier system promotes synergistic effects that support both plant growth and nutrition, reducing the need for multiple external inputs, as evidenced in these experimental trials. Such systems can be tailored for specific agricultural applications, including seed priming, hydroponics, soil amendment, and foliar fertilization, offering a path toward more sustainable and precision-based crop management strategies. It is understood that the disclosed method and compositions are not limited to the particular methodology, protocols, and reagents described as these can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the method and compositions described herein. Such equivalents are intended to be encompassed by the following claims. References (1) Laxman, et al. Abiotic Stress Management for Resilient Agriculture 2017 Springer Singapore: Singapore, pp 399–412. (2) Rai, et al. Sustainable Agriculture in the Era of Climate Change 2020 Springer International Publishing: Cham, pp 143–168. (3) Chi, et al. Bioself-Assembled Crystals in Plants Promote Photosynthesis and Salt Stress Resistance. ACS Nano 2021, 15 (3), 5165–5177. (4) Zhang, et al. ACS Sustain. Chem. Eng.2023, 11 (8), 3346–3358. (5) Wu, et al. 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Claims

KAUST 2024-082-02 PCT We claim:

1. A nanoparticulate composition comprising: (a) nanoparticles comprising a homogenous network of plant secondary metabolites optionally metals bound to the plant secondary metabolites, preferably a metal-phenolic network comprising one or more metals and one or more phenolic compounds, the network having dispersed therein one or more plant treatment agents, or (b) nanoparticles comprising a core and a shell, wherein the shell comprises one or more plant secondary metabolites and metal, preferably a metal-phenolic network comprising one or more metals and one or more phenolic compounds in the form of a metal phenolic network, wherein the core comprises hydrophobic molecules (a) in a membrane encapsulating one or more plant treatment agents and / or (b) having dispersed therein one or more plant treatment agents, and optionally wherein the nanoparticulate composition comprises a surfactant at the interface between the core and the shell.

2. The nanoparticulate composition of claim 1, wherein the plant secondary metabolites comprise tannins, phenolic acids, alkaloids, flavonoids, terpenoids, glycosides, glucosinolates, cucurbitacins, or a combination thereof.

3. The nanoparticulate composition of claim 1 or 2, wherein the plant secondary metabolites comprise tannins comprising phenolic structures.

4. The nanoparticulate composition of claim 1 to 2, wherein the one or more phenolic compounds are caffeic acids, gallic acids, quercetins, resveratrols, vanillic acids, tannic acids, or a combination thereof.

5. The nanoparticulate composition of any one of claims 1 to 4, wherein the one or more plant treatment agents comprise biostimulants, nutrients, pesticides, biopesticides, plant growth regulators, and / or vitamins.

6. The nanoparticulate composition of claim 5, wherein the plant growth regulators comprise indole-3-acetic acids (IAA), gibberellic acids, abscisic acids, jasmonic acids, kinetins, zaxinones, lactones, humic acids, cytokinins, micronutrients, or a combination thereof.

7. The nanoparticulate composition of any one of claims 1 to 6, comprising metals bound to the plant secondary metabolites.

8. The nanoparticulate composition of claim 7, wherein the metals comprise the iron magnesium, zinc, manganese, copper, nickel, molybdenum, in elemental and / or ionic forms.

9. The nanoparticulate composition of any one of claims 1 to 8, wherein the hydrophobic molecules comprise lipids, hydrophobic polymers, or a combination thereof, optionally wherein the 7245765042.1KAUST 2024-082-02 PCT hydrophobic polymers comprise polyesters, polyanhydrides, poly(p-dioxanone)s, polycarbonates, or a combination thereof, such as poly(α-hydroxy acid)s, poly(lactone)s, or combination thereof, such as poly(lactic acid)s, poly(glycolic acid)s, poly(lactic acid-co-glycolic acid)s, poly(caprolactone)s, poly(pentadecalactone)s, poly(hydroxybutyrate-co-hydroxyvalerate)s, poly(hydroxybutyrate)s, or a combination thereof.

10. The nanoparticulate composition of any one of claims 1 to 9, wherein the hydrophobic molecules comprise lipids.

11. The nanoparticulate composition of claim 10, wherein lipids comprise waxes, triglycerides, partial glycerides, phospholipids, fatty acids, glycolipids, or a combination thereof.

12. The nanoparticulate composition of claim 11, wherein the waxes comprise natural waxes (such as carnauba wax, shea butter, beeswax, candelilla wax, rice bran wax, jojoba wax, and bayberry wax), vegetable oils ( (such as castor oil, palm oil, or soybean oil), synthetic waxes (such as paraffin wax or microcrystalline wax), synthetic esters (such as cetyl palmitate or stearyl stearate), or a combination thereof.

13. The nanoparticulate composition of claim 11, wherein the triglycerides comprise natural or semi-synthetic triglycerides (such as glyceryl tristearate, glyceryl tripalmitate, glyceryl trimyristate, glyceryl trilaurate, glyceryl triricinoleate palm oil triglycerides, coconut oil triglycerides, soybean oil triglycerides, or a combination thereof.

14. The nanoparticulate composition of claim 11, wherein the partial glycerides comprise monoglycerides (such as Glyceryl monostearate (GMS), glyceryl monopalmitate, glyceryl monolaurate, or glyceryl monooleate), diglycerides (such as glyceryl distearate, glyceryl dipalmitate, glyceryl distearate or oleate mixtures), or a combination thereof.

15. The nanoparticulate composition of claim 11, wherein the fatty acids comprise saturated fatty acids: (such as stearic acid, palmitic acid, myristic acid, lauric acid, or behenic acid), unsaturated fatty acids (such as oleic acid, linoleic acid, linolenic acid, or ricinoleic acid), hydroxylated or functionalized fatty acids, or a combination thereof.

16. The nanoparticulate composition of claim 11, wherein the phospholipids comprise soy lecithin, egg lecithin, hydrogenated soy phosphatidylcholine (HSPC), hydrogenated egg phosphatidylcholine (HEPC), distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dibehenoylphosphatidylcholine, hydrogenated phosphatidylethanolamine, hydrogenated phosphatidylglycerol, hydrogenated phosphatidylinositol, or a combination thereof. 7345765042.1KAUST 2024-082-02 PCT 17. The nanoparticulate composition of claim 11, wherein the glycolipids comprise cerebrosides, galactocerebrosides, glucocerebrosides, sulfatide, gangliosides (e.g., ganglioside GM1, ganglioside GM2, etc.), globoside, lactosylceramide, or a combination thereof.

18. The nanoparticulate composition of any one of claims 1 to 17, wherein the membrane comprises a monolayer or a bilayer.

19. The nanoparticulate composition of any one of claims 1 to 18, comprising a surfactant at an interface between the shell and the core.

20. The nanoparticulate composition of any one of claims 1 to 19, wherein the surfactant comprises nonionic surfactants, anionic surfactants, cationic surfactants, amphiphiles, or a combination thereof.

21. The nanoparticulate composition of claim 20, wherein the nonionic surfactants comprise polysorbate derivatives (such as TWEEN 20®, TWEEN 40®, TWEEN 60®, and TWEEN 80®), polyethylene glycol (PEG) derivatives (such as PEG-40 stearate or PEG-60 hydrogenated castor oil), poloxamer derivatives (PLURONIC F68®, PLURONIC F127®, and PLURONIC P85®), polyvinyl alcohol (PVA) derivatives, sorbitan ester derivatives (SPAN 20®, SPAN 40®, SPAN 60®, SPAN 80®), polyoxyethylene ethers, or a combination thereof.

22. The nanoparticulate composition of claim 20, wherein: (a) the anionic surfactants comprise sodium dodecyl sulfate (SDS), sodium cholate, and sodium deoxycholate; (b) the amphiphiles comprise bile salts, phospholipids, or lecithin; and / or (c) the cationic surfactants comprise cetyltrimethylammonium bromide (CTAB) or stearylamine.

23. The nanoparticulate composition of any one of claims 1 to 22, wherein the core is a solid core comprising the hydrophobic molecules and / or the one or more plant treatment agents dispersed therein.

24. The nanoparticulate composition of any one of claims 1 to 23, wherein the nanoparticles have particle size of at least 80 nm, such as between 80 and 200 nm, between 80 and 180 nm, between 80 and 160 nm, between 80 and 140 nm, between 90 and 140 nm, between 100 and 140 nm, between 110 and 140 nm, or between 110 and 130 nm.

25. The nanoparticulate composition of any one of claims 1 to 24, wherein the nanoparticles have a zeta potential of at least -20 mV, such as between -1 and -20 mV, between -1 and -18 mV, between -1 and -16 mV, between -1 and -14 mV, between -1 and -12 mV, between -1 and -10 mV, between -3 and -10 mV, between -5 and -10 mV, or between -6 and -9 mV.

26. The nanoparticulate composition of any one of claims 1 to 25, wherein the nanoparticles have a loading capacity of the one or more one or more plant treatment agents of at least 1% w / w or 7445765042.1KAUST 2024-082-02 PCT at least 5% w / w, such as in the range of 1% w / w and 20% w / w, in the range of 3% w / w and 20% w / w, in the range of 5% w / w and 20% w / w, in the range of 5% w / w and 18% w / w, in the range of 5% w / w and 16% w / w, or in the range of 8% w / w and 16% w / w.

27. The nanoparticle composition of any one of claims 1 to 26, wherein nanoparticles have an encapsulation efficiency of at least about 80% or at least about 90%, such as about 80% to about %, about 83% to about 100%, about 86% and to about 100%, in the range of about 88% and to about 100%, about 90% to about 100%, about 92% to about 100%, about 94% to about100%, about 96% to about 100%, or about 98% to about 100%.

28. The nanoparticulate composition of any one of claims 1 to 27, wherein the nanoparticulate composition releases about 70% to about 90%, about 75% to about 90%, about 80% to about 90%, about 82% to about 88%, or about 84% to about 88% of the one or more plant treatment agents in 100 mM NaCl.

29. A method for treating a plant, plant seed or plant part, the method comprising: administering the composition of any one of claims 1-28 to the plant, plant seed or plant part.

30. The method of claim 29, comprising administering the composition to plant seed for an effective amount of time prior to planting.

31. The method of claim 29 or 30, wherein the plant seed is a wheat seed, tomato seed, capsicum seed, and pearl millet seed 32. The method of any one of claims 29 to 31, wherein the nanoparticulate composition localizes in the plant seed after step (i).

33. The method of any one of claims 29 to 32, wherein the nanoparticulate composition improves one or more growth metrics selected from the group consisting of seed germination rate, see germination percentage, of the plant seed in the range of 1% to 20%, in the range of 2% to 20%, in the range of 4% to 20%, in the range of 6% to 20%, in the range of 8% to 20%, in the range of 8% to 18%, in the range of 8% to 16%, in the range of 8% to 14%, in the range of 8% to 12%, in the range of 8% to 11%, or in the range of 9% to 11%.

34. The method of any one of claims 29 to 33, wherein the nanoparticulate composition improves growth enhancement of the plant seed in the range of 1% and 70%, in the range of 5% and 70%, in the range of 10% and 70%, in the range of 15% and 70%, in the range of 20% and 70%, in the range of 25% and 70%, in the range of 30% and 70%, in the range of 35% and 70%, in the range of 40% and 70%, in the range of 45% and 70%, in the range of 50% and 70%, in the 7545765042.1KAUST 2024-082-02 PCT range of 55% and 70%, in the range of 55% and 65%, in the range of 55% and 60%, or in the range of 57% and 60%.

35. The method of any one of claims 29 to 34, wherein the nanoparticulate composition increases shoot and root length in comparison to a plant seed that has not been administered the composition of any one of claims 1 to 28.

36. The method of any one of claims 29 to 35, wherein the nanoparticulate composition increases fresh biomass in comparison to a plant seed that has not been administered the composition of any one of claims 1 to 28.

37. The method of any one of claims 29 to 36, wherein the nanoparticulate composition improves uptake of Mg2+, Zn2+, in comparison to a plant seed that has not been administered the composition of any one of claims 1 to 28.

38. The method of any one of claims 29 to 37, wherein the nanoparticulate composition maintains a K+ / Na+ratio, in comparison to a plant seed that has not been administered the composition of any one of claims 1 to 28.

39. The method of any one of claims 28 to 38, wherein the nanoparticulate composition improves bioavailability of the one or more plant treatment agents in comparison to a plant seed that has not been administered the composition of any one of claims 1 to 28. 7645765042.1

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