Nanoparticles, their preparation and use

Abstract

Hydrophilic nanoparticles are disclosed comprising CuS and a hydrophobic surface. Also disclosed are amphiphilic polymeric nanoparticles useful in transporting the CuS or other hydrophobic payloads, and composites containing the polymeric nanoparticles and the hydrophobic CuS payloads. This system can be used, for example, to modulate plant growth and development, resulting in improved yields, crop quality, and shelf life.

Description

NANOPARTICLES, THEIR PREPARATION AND USECristina M. Sabliov, Carlos E. Astete, Alvaro Gustavo Garcia,Fannyuy V. Kewir, Jeffrey A. Davis, Maria da Graga HenriquesVicenteFile No. Sabliov AG-2024-002-02

[0001] This invention was made with United States Government support under 20236701840722 awarded by the National Institute of Food and Agriculture, and 1632854 awarded by the National Science Foundation. The United States Government has certain rights in the invention.

[0002] The benefit of the 14 June 2024 filing date of United States provisional patent application serial number 63 / 660,125; and the benefit of the 15 August 2023 filing date of United States provisional patent application serial number 63 / 532,694; are claimed under 35 U.S.C. § 119(e) in the United States, and are claimed under applicable treaties and conventions in all countries.TECHNICAL FIELD

[0003] This invention pertains to hydrophobic CuS nanoparticles; their preparation and use; and amphiphilic polymeric nanoparticles useful in conjunction with CuS nanoparticles or other payloads.BACKGROUND ART

[0004] While the use of plastics (polymers) in agriculture has increased productivity in many ways, it has also increased waste generation and farmland contamination. Agricultural plastic waste raises concerns about microplastic contamination of soil ecosystems and possible uptake by plants, spreading plastics within the food chain. More sustainable alternatives are needed to maintain and enhance agricultural productivity while minimizing plastic waste. Biopolymers, bio-friendly materials with controlled degradation profiles, have emerged as a promising alternative.

[0005] Natural biopolymers, such as lignin (LN), have vast potential for use in synthesized, lignin-modified polymers. LN is one of the most abundant aromatic substances in nature. LN is generally obtained as a byproduct of the pulp and paper industry. An abundance of hydroxyl groups in aromatic and aryl chains allows functionalization of LN, for example covalent attachment to other polymers. Modifying LN, a hydrophilic polymer, with hydrophobic polymers by grafting can impart amphiphilicity to the resulting lignin-grafted polymer, allowing the polymer to self-assemble into nanostructures without the need for surfactants, and allowing the controlled release of payloads such as drugs or agrochemicals from the nanostructures. Lignin-modified polymers find potential uses in various fields, including biomedicine, packaging, and agriculture.

[0006] Mendez, O. E et al. Lignin Nanoparticles as Delivery Systems to Facilitate Translocation of Methoxyfenozide in Soybean (Glycine max). J. Agric. Food Res. (2022) 7:100259. doi.org / 10.1016 / j.jafr.2O21.100259 reported the use of lignin-based nanoparticles (LNPs) as a delivery system to control the release and translocation of methoxyfenozide(MFZ), a non-systemic insecticide, in soybean plants. LNPs were synthesized from lignin-graft-poly(lactic-co-glycolic) acid (PLGA) and loaded with 2.7 % MFZ (w / w), presenting a size of 113.8 ± 3.5 nm, PDI of 0.313 ± 0.029, and zeta potential of -53.3 ± 6.9 mV. The LNPs facilitated the controlled release and translocation of MFZ through soybean tissues. Although LN-modified PLGA nanoparticles comprise a promising delivery system, the cost of the PLGA component is relatively high.

[0007] Poly(£-caprolactone) (PCL), an FDA-approved polymer with hydrophobic, biodegradable, and non-toxic properties, exhibits a slow degradation rate compared with other biodegradable polymers, allowing the controlled release of entrapped compounds for extended periods.

[0008] Delivery of agrochemicals by current practices is inefficient. Although millions of tons of agrochemicals are used around the world annually, it has been estimated that only 1-20% of these compounds actually reach their intended targets. Active ingredients can be lost, for example, through volatilization, fixation, or leaching in the soil.

[0009] Copper-based agrochemicals, such as copper sulfide, CuS, are widely used in agriculture for their protective effects on plant health. CuS exhibits antifungal and bactericidal properties, and it is also a nutrient source for plants.

[0010] Current agricultural practices can lead to frequent use of and long-term exposure to copper-based agrochemicals. However, long-term exposure to and frequent use of Cu-based agrochemicals create potential risks to the environment and the soil microbiome. A Cu concentration above about 200 mg / kg in the soil is considered to present a risk ofpollution for ground water supplies, and a potential risk of toxicity towards non-target microorganisms.

[0011] There is an unfilled need for better targeting and controlled delivery of CuS in agricultural applications.

[0012] In nanoform, CuS is more effective than the bulk material in agricultural applications. Foliar application of CuS NPs to rice plants has been reported to reduce the severity of bakanae disease by 32%, while applying a bulk Cu-based pesticide only reduced disease incidence by 12%. Additionally, CuS NPs enhances plant defenses by increasing salicylic acid and jasmonic acid production.

[0013] J. Almarashi etal., “Exploring Nano-sulfide Enhancements on the Optical, Structural and Thermal Properties of Polymeric Nanocomposites,” J. Inorg. Org. Polymer (2020) 30:3230-3240 described the incorporation of CuS into bulk-scale polymeric matrices. Nanocomposite films of polyvinyl alcohol / polyethylene glycol (PVA / PGE) were prepared by casting, with the incorporation of 10-12 nm CuS particles at concentrations between 1% to 5% w / w.

[0014] M. Du et al., “CuS nanoparticle-based microcapsules for solar- induced phase-change energy storage,” ACS Appl. Nano Mater. (2022) 5:13009-13017 describes entrapping CuS NPs about ~10 nm in a dodecanol tetradecyl ester / melamine formaldehyde nanostructure.

[0015] Nanotechnology for agriculture is a tool that might be used for more efficient agrochemical applications and reduced environmental pollution. One example is a nanoparticle delivery system to deliverbioactive payloads, specifically polymer-based nanoparticulate systems.

[0016] A. Pereira et al., “Chitosan nanoparticles as carrier systems for the plant growth hormone gibberellic acid” (2017) Colloids and Surfaces B 150:141-152 reported the synthesis of a chitosan-based nanocarrier for gibberellic acid, GA3, with a 90% encapsulation efficiency, and its use in sustained release when applied to Solanum lycopersicum and Phaseolus vulgaris. See also A. Pereira et al., “Polymeric nanoparticles as an alternative for application of gibberellic acid in sustainable agriculture” (2019) Scientific Reports 9:7135.

[0017] Plant growth regulators (PGRs) are hormones or other molecules with broad applications in agriculture and horticulture. PGRs alter plant hormonal homeostasis and signaling, and modulate plant growth and development. Selective application of PGRs can improve yields, crop quality, and shelf life. An example of a PGR is gibberellic acid (GA3), a naturally occurring plant hormone found in young leaves, shoots, and flowers. GA3 helps improve plant growth through its influence on shoot elongation, breaking seed dormancy, inducing organ differentiation, and increasing fruit production. The phytochrome GA3 is one of the 136 known gibberellic acids (having 19 or 20 carbon atoms). GA3 can be derived from fermentation products of the fungus Gibberrella fujikuroi. GA3 (C19H22O6) is a tetracyclic diterpenoid having two hydroxyl groups, a lactone ring, and a carboxyl group.DISCLOSURE OF THE INVENTION

[0018] We have discovered new hydrophilic nanoparticles comprising CuS and a hydrophobic surface. We have also discovered amphiphilic polymeric nanoparticles useful in transporting the CuS orother hydrophobic payloads, and composites containing the polymeric nanoparticles and the hydrophobic CuS payloads.

[0019] The invention allows improved control of agrochemical release and reduced environmental contamination. The delivery system is synthesized from renewable polymers. MFZ, an insecticide used to control lepidopteran larvae, was used as a model agrochemical in a prototype demonstration of the novel system. To our knowledge, there are no prior reports of LN-grafted PCL (LN-g-PCL) nanoparticles, nor their use in agrochemical or drug delivery systems.

[0020] We have employed two grafting methods, a ring-opening polymerization (ROP)(LN-g-PCLp) and an acylation reaction (LN-g-PCLa), to create polymers capable of self-assembling into nanoparticles having different properties, without requiring surfactants. Payload release profiles can be tailored by using different grafting methods, and by manipulating the PCL chain length grafted to LN.

[0021] To the inventors’ knowledge, there are no previous reports describing compositions comprising hydrophobic CuS nanoparticles, or concerning CuS nanoparticles that are themselves embedded within coreshell polymeric nanoparticles. We have prepared biodegradable, ligninbased delivery systems with controlled properties for delivering CuS nanoparticles efficiently. The hydrophobicity of the surface-modified CuS NPs facilitates their entrapment in the hydrophobic core of the LN-polymer core-shell structures. Tuning the NP properties can help control the release profile by varying the type of lignin, the lignin-polymer grafting ratio, and the method of making the LN-polymer nanoparticles.

[0022] In one embodiment we prepared a GAs-grafted lignin-based nanoparticle to deliver nanoscale CuS, a plant micronutrient complex and antifungal compound. The GA3 acts as a signaling agent in the translocation of the nano-delivered agrochemicals, improving their targeting.MODES FOR CARRYING OUT THE INVENTIONLN-g-PCL Polymers and Nanoparticles

[0023] Materials. PCL (Mn= 45,000 g mol'1), £-ca pro lactone (CL, >99%), and stannous 2-ethyl hexanoate (Sn(Oct)2), purity 92.5-100.0%), cyclohexanol (purity 98.5%), and 2-chloro-4,4,5,5-tetramethyl-1 ,3,2— dioxaphospholane (TMDP, purity 95%) were acquired from Sigma-Aldrich (St. Louis, MO). Alkaline LN was acquired from TCI Inc. (Portland, OR). Methanol (purity >99%), dichloromethane (DCM, purity >99.9% extra dry), oxalyl chloride (purity >99%), dimethylformamide (DMF, purity >99.5%), dimethyl sulfoxide (DMSO, purity >99.7%), ethyl ether (purity >99%), toluene (purity >99.5%, extra dry), and trehalose (purity >99%) were acquired from Fisher Scientific (Pittsburgh, PA).

[0024] Synthesis of LN-g-PCL polymers, Synthesis 1 : "Grafting From." LN-g-PCLp polymers were synthesized by ring-opening polymerization (ROP) of the CL monomer, as shown in Scheme 1. Alkali lignin was dried in a vacuum oven before the synthesis. The synthesis was conducted under anhydrous bulk conditions without any solvent, under argon flux. Briefly, 2 g of alkali lignin was placed in a 250 ml 3-neck round-bottom flask, followed by adding CL at a given ratio. Different CL / LNratios were used (w / w) = 2, 6, and 10 in the grafting reactions. Three different degrees of polymerization (DPs) were thus obtained (22, 57, 101 by1H NMR), with correspondingly different PCL chain lengths.

[0025] The mixture remained under consistent magnetic agitation at ambient temperature until the LN was completely dissolved. Then the mixture was heated and stirred in an oil bath at 130°C. Subsequently, 0.2% (v / v according to CL volume) of Sn(Oct)2 (tin(ll) 2-ethylhexanoate) was added as a catalyst. The grafting reaction proceeded for 24 hours with constant stirring at 200 rpm under argon flow to maintain anhydrous conditions. When the reaction was complete, the solution was precipitated dropwise into 200 mL cold methanol. The supernatant was discharged, and the precipitate was washed five times with cold methanol.

[0026] Next the resulting grafted polymer was diluted in 200 mL DCM, and washed five times with distilled water to remove unreacted LN. Then the polymer was concentrated in a Rotavapor R-300 (Buchi Corporation, New Castle, DE). The resulting solids were frozen at -80 °C for 24 h and lyophilized (FreeZone Plus 2.5; Labconco, Kansas City, MO) to remove water and solvents from the polymer. Finally, the LN-g-PCLp polymer was stored in a desiccator at room temperature until used.LN s-ca pro lactone LN-g-PCLpScheme 1. Synthesis of LN-g-PCLp by ROP.

[0027] Synthesis of LN-g-PCL polymers, Synthesis 2: "Grafting To." LN-g-PCLa polymers were synthesized by an acylation reaction, as depicted in Scheme 2. Briefly, 4 g of PCL (103 DP by1H NMR) was dissolved in 50 mL of DCM at room temperature in a three-neck roundbottom flask under anhydrous conditions with argon flux. Oxalyl chloride (110 pL) was added dropwise with a glass syringe, and 4 mL of DMF were added as a catalyst. The mixture reacted at room temperature at 300 rpm for four hours.

[0028] DCM was evaporated in a Buchi R-300 rotavapor (Buchi Corporation, New Castle, DE) to obtain a viscous solution of PCL-CI. For the coupling reaction 4 g PCL-CI was dissolved in 50 mL of DCM, which was then added dropwise to 2 g of alkali lignin dissolved in 50 mL of DMSO. Then the mixture was left to react for 24 hours under argon flow. At the end of the reaction period, ethyl ether (200 mL) was added to the mixture to precipitate the LN-g-PCLa polymer.

[0029] Precipitation, washing, and lyophilization were carried out, following essentially the same methodology as described above for “Synthesis 1 ,” and the resulting polymer was called LN-g-PCLa.Scheme 2. Synthesis of LN-g-PCLa by acylation. R are the randomly distributed monolignols of lignin (e.g., p-coumaryl, sinapyl, and coniferyl alcohol).

[0030] Characterization of LN-g-PCL polymers. Fourier transform infrared (FTIR) spectroscopic analysis was used to confirm the attachment of LN to PCL using a Bruker Tensor 27 (Bruker 500, Billerica, MA). Dried samples were analyzed with 32 scans between 400 to 600 cm'1, at a resolution of 4 cm'1. Changes in hydroxyl and carbonyl groups were analyzed to evaluate interactions between the polymers.

[0031] Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker 400 (Billerica, MA) at 400 Hz in deuterochloroform (CDCh).1H NMR spectra allowed estimating the length of the PCL arms that had been grafted onto LN, by calculating the ratio of the areas of the signal peaksat 4.03 ppm (repeating -CH2O-) and at 3.65 ppm (terminal -CH2OH).

[0032] Quantitative31P NMR was used to determine the hydroxyl content of lignin. Briefly, 20 mg dried LN were dissolved in a mixture of 0.5 mL pyridine / deuterated chloroform (1.6:1 , v / v). Cyclohexanol (100 pL / mL) was used as an internal standard, and TMDP (0.1 mL) was added to phosphorylate the hydroxyl groups.31P NMR spectra were measured 15 minutes after the addition of TMDP. The spectra were acquired with an inverse-gated decoupling pulse and a 12 s relaxation delay, with 256 scans. The chemical shift was calibrated by the peak of the product of TMDP plus water at 132.2. The hydroxyl content of lignin and LN-g-PCL polymers was determined based on the concentration of cyclohexanol in the internal standard. Monomer conversion was calculated using the integrated peaks of the1H NMR spectra as follows:Monomer Conversion (%) — 100 (1 )' 'Polymerization yield was calculated as: 100 (2)

[0033] where WCL is the initial weight of CL and WPCL is the final weight of PCL in the grafted polymer.

[0034] Differential scanning calorimetry (DSC) was performed using a TA Q100 DSC (TA Instruments, New Castle, DE). The crystallization temperature (Tc) and melting (Tm) temperature were determined by standard methods. About 2-4 mg of pure LN, PCL, or LN-g-PCL polymer was sealed in an aluminum pan, followed by a heating and cooling cyclefrom 0 to 120°C at 10 °C / min under a nitrogen atmosphere.

[0035] The crystallinity of the samples was calculated as:

[0036] where AHf refers to the melting enthalpy (J g'1) obtained from the fusion peak of DSC, and AHf* = 136.1 J g'1is the heat of fusion for 100% crystalline PCL.

[0037] Thermogravimetric analyses (TGA) were used to evaluate the thermal stability of pure LN, PCL, and LN-g-PCL polymer samples on a TA TGA550 (TA instruments). About 3-5 mg of the samples were placed in an aluminum pan, and were then heated to 600°C at a rate of 50°C min'1under nitrogen flow.

[0038] Gel Permeation Chromatography (GPC) was used to determine the weight-average molecular weight (Mw) and the number-average molecular weight (Mn) of PCL and LN-g-PCL polymers. The polydispersity index (PDI) is defined as the ratio of Mw to Mn, and represents a measure of the distribution breadth of molecular weights. LN-g-PCLpor PCL polymer, 5 mg, were dissolved overnight in 1 mL of THF, and then filtered with a 0.45 pm PTFE syringe filter. The Mw and Mn analyses for LN-g- PCLp polymers were performed on newly-synthesized polymers with DPs of 20, 53, and 99. Separations were carried out with an EcoSEC Elite GPC System, equipped with two TSK Gel columns from Tosoh: 4.6 mm x 15 cm x 3 pm (Super HZ2400, Super HZ4000), and a guard column (Super HZ-L 3 pm x 4.6 mm x 2 cm) (Tosoh Bioscience LLC, King of Prussia, PA). Two detectors were used: a EcoSEC differential refractive indexdetector, and a Lens3 multiangle light scattering detector (Tosoh) equipped with a 505 nm Diode Laser at 20 mW. 20 pL of sample in THF, stabilized with 250 ppm BHT, were injected at 0.3 mL I min. The EcoSEC was calibrated with polystyrene standards or Multiangle Light Scattering. Polymer mass was determined using the specific refractive index increment (dn / dc) of 0.071 mL / g for all samples containing PCL; dn / dc denotes the change in the refractive index n of a solution with respect to changes in the solute concentration c.

[0039] LN-g-PCL NP synthesis. LNPs were synthesized from LN-g- PCL polymers by the oil in water (O / W) emulsion evaporation technique, which produced particles having a core-shell structure with a hydrophilic LN outer shell layer, and a hydrophobic PCL core. Briefly, lignin-g-PCL (400 mg) was dissolved in DCM (20 mL). Deionized water (120 mL) was used as the aqueous phase, into which the organic solution was gently introduced as droplets. Subsequently, microfluidization was carried out in four cycles utilizing an M-110P microfluidizer (Microfluidics, Newton, MA, USA). The organic solvent was then evaporated with a Rotavapor R-300 (Buchi Corporation, New Castle, DE). After adding trehalose (800 mg) as a cryoprotectant, the solution was frozen at -80°C for 24 hours, followed by lyophilization with a FreeZone Plus 2.5 freeze dryer (Labconco, Kansas City, MO). Payloads (e.g., agrochemicals) were incorporated by essentially the same method, with the payload (e.g., methoxyfenozide, 40 mg) being added in the organic phase.

[0040] Nanoparticle characterization. Size distribution, polydispersity, and zeta potential of the fresh LN-g-PCL nanoparticles were determined by Dynamic Light Scattering (DLS) using a Malvern Zetasizer ZS (Malvern, Panalytical Westborough, MA) at 25°C. The nanoparticles wereresuspended in water at a concentration of 0.2-0.5 mg / mL and placed in a cuvette for characterization.

[0041] Morphology and aggregation of the resuspended nanoparticles were analyzed by Transmission electron microscopy (TEM) using a JEM- 1400 TEM (Jeol USA.inc Peabody, MA). LN-g-PCL nanoparticles were resuspended in water, placed on a copper grid with a contrast agent (uranyl acetate 1 %), and dried at room temperature. Then the grid was placed in the TEM chamber for analysis.

[0042] Resuspended freeze-dried LN-g-PCL nanoparticles were used for evaluating loading capacity (LC) and entrapment efficiency (EE). 20 mg of nanoparticle powder were suspended in 2 ml of water. Subsequently, 100 pL of the mixture were diluted in 500 pL acetonitrile. The total payload content of the solution was quantified by high- performance liquid chromatography (HPLC) at an absorption wavelength of 254 nm, using 20 pL of sample. The Agilent HPLC system was equipped with an LC-20AT pump and SPD-20A PDA detector, SIL-20AC interfaced with the LC- solution software system (Agilent, USA). An elution gradient was used with water as solvent A and acetonitrile as solvent B. The initial condition was set to 30% of solvent B for 10 min. Subsequently, a solvent gradient was set to 90% of solvent B within 10 min, holding for 5 min. A flow rate of 1 mL min'1was used with the Zorbax Agilent C18 column (150 mm x 4.6 mm i.d. x 5 pm particle size). The calculations of LC and EE for the MFZ nanoparticles were determined as: 100 (4)

[0043] The number of particles from the LN-g-PCL copolymers was measured by nanoparticle tracking analysis (NTA) using a ZetaView® Quart PMX-420 (Particle Metrix GmbH, Meerbusch, Germany). Resuspended LN-g-PCL nanoparticles were tracked using a laser (A = 488 nm) and scattered light.

[0044] Payload release. The release of payload (MFZ in prototype experiments) was analyzed by dialysis, and MFZ concentrations were measured by high-performance liquid chromatography (HPLC). Briefly, LNPs were dispersed in water at a concentration of 10 mg / mL, placed in a dialysis bag (MWCO of 12-14 kDa, Fisherbrand, USA), and immersed in 1 L of PBS solution at 23°C or 30°C. 300 pL aliquots were taken at 0, 1 , 2, 3, 6, 12, 24, 48, 72, 96, 144, and 192 h, and the PBS was replaced every 24 h. MFZ was extracted from the nanoparticles by adding 100 pL of sample to 500 pL of acetonitrile. For each aliquot, agrochemical concentrations were quantified by HPLC. For the cumulative release analysis, the MFZ amount at time zero was treated as the initial 100% level for each respective NP system, and the subsequent release was reported relative to the initial amount.

[0045] Statistical analysis. Experimental data were collected in triplicate (n=3) and analyzed using R Studio for Windows v2023.06.0+421 (RStudio Inc., Boston, Ma, USA). Statistical significance was determined as < 0.05 using one-way ANOVA to compare statistical differences between treatments; and using two-way ANOVA to determined significant differences between treatments, or within a treatment though the releasetimeframe. Tukey HSD was performed to test differences among sample means ( < 0.05). Data are reported as mean ± standard deviation.Results and discussion

[0046] We used two approaches for modifying LN to synthesize LN- g-PCL copolymers: a "grafting from" ring-opening polymerization, and a "grafting to" acylation reaction. We developed nanodelivery systems for the controlled release of payloads such as MFZ. Selecting among the different polymeric structures formed by the different grafting methods, as well as controlling the DP with the ROP method, allowed the particles to be tuned and facilitated the controlled release of the MFZ payload.

[0047] Polymer characterization. Inter- and intramolecular interactions between LN and PCL were observed in the FT-IR spectra , confirming covalent PCL attachment to LN. Functional groups such as hydroxyl (OH, 3000-3700 cm'1), methylene (CH2, 2945-2864 cm'1), carbonyl (C=O, -1721 cm'1), and aromatic ring (1600 cm'1) were observed. The intensity of the OH peak decreased for the LN-g-PCL copolymer after grafting as compared to unmodified LN, indicating that some of the OH from the LN had reacted to form ester linkages. The strong C=O absorption peak for both PCL and LN-g-PCL copolymers suggested the formation of intermolecular hydrogen bonds between carboxylic groups in PCL. Additionally, the presence of an aromatic ring, a characteristic of LN, confirmed the incorporation of LN into the LN-g-PCL copolymer.

[0048] The chemical structure of LN-g-PCL was analyzed by1H-NMR and compared to PCL. Several PCL peaks were seen in the grafted polymer: 4.03 ppm (d, repeating -CH2O-), 3.65 ppm (d', terminal -CH2OH), 2.29 ppm (a, -COCH2-), 1.63 ppm (b, -CH2-), and 1.38 ppm (c, -CH2-).

[0049] The number of repeated units of CL grafted to LN, the “DP,” was calculated from the integrated ratio of the NMR peaks for different methylene subtypes: repeating -CH2O- and terminal -CH2OH. The DP for the ROP-synthesized copolymers ranged from 22 to 101 . By contrast, the LN-g-PCLa copolymers, synthesized through acylation, had no detectable terminal methylene peak, indicating essentially complete modification of the end groups of the PCL chain. Additionally, monomer conversion analysis revealed a high monomer conversion during bulk polymerization of LN and CL.

[0050] We examined changes in the LN hydroxyl groups by31P NMR following modification with PCL, specifically the Aliphatic OH (145.0 ppm to 149.0 ppm), phenolic OH (140.4 ppm to 144.4 ppm), and carboxylic OH (134 ppm to 136 ppm) groups. Quantitative analysis showed an average reduction for all copolymers of 85.9 ± 6.4% for the aliphatic OH groups, and 78.3 ± 22.9% for the phenolic OH groups after the modification reaction. Increasing the ratio of CL to LN caused a greater reduction in total OH. This trend is presumably related to the miscibility of LN in CL during the bulk reaction, such that a higher volume of CL increases the amount of dissolved LN, and increases the availability of OH for the ROP reaction. A similar effect was observed in the acylation reaction, where the reduced miscibility of LN and modified PCL in the DCM:DMSO solution decreased the availability of OH from lignin, resulting in a higher OH content at the end of the reaction.

[0051] Notably, the LN-g-PCLa copolymers synthesized by acylation lacked aliphatic and carboxylic OH peaks from PCL, indicating essentiallycomplete reaction of the PCL hydroxyl groups.

[0052] Although the precise quantification of aliphatic and carboxylic OH was obstructed by the OH peaks generated by the ROP reaction itself, our results confirmed that the polymerization of CL also started at the carboxylic OH of LN, leading to a PCL arm terminating with a carboxylic OH group. Overall, the grafting modifications resulted in different PCL chain attachments to LN, showing that the LN-g-PCL properties can be controlled by proper choice of the grafting method.

[0053] DSC was used to characterize the thermal properties of the LN- g-PCL samples, alkaline LN, and PCL. Over the temperature range from 0°C to 120°C, no glass transition temperature (Tg) was observed for either the LN-g-PCL polymer samples or for alkaline LN. However, melting and crystallization temperatures were observed for both the grafted polymers and the PCL. The average melting point temperature (Tm) for all LN-g- PCL polymers was 55.2 ± 1 .8°C, similar to the Tm of PCL (57.4 ± 1 .3°C). There was a trend, in which the Tm of the copolymers approached the T m of PCL as the length of the PCL arms increased.Table 1. Summary of certain properties of LN-g-PCL copolymers.Yto / \a OH Mn Mwp- |CMonomer Polymerization(mmol / g)b(Da)c(Da)cConversion YieldLN - 3.94LN-g-PCLp 22DP 59.4±3.3 0.75 7199 7522 1.04 96% 91.1%LN-g-PCLp 57DP 53.0±2.6 0.39 17777 20812 1.17 98% 66.7%LN-g-PCLp 101DP 49.0±2.2 0.17 21943 31817 1.45 99% 89.5%LN-g-PCLa 103DP 31.0±1.2 1.13PCL 48.9±2.1 0.10 30195 36225 1.2a: Xc determined by DCS.b: OH quantification obtained by31P NMR.c: Data obtained by GPC.d: Data obtained by 1 H NMR.

[0054] Crystallinity, as measured by DSC, decreased slightly with an increase in the DP of the PCL attached to the LN. When the DP of the PCL arm was short (22 DP), the LN-g-PCLpcopolymer synthesized by ROP exhibited high crystallinity (59.0%). With a larger DP (101 DP), the crystallinity decreased to 49.0%. The lowest crystallinity seen (31.0%) was for the LN-g-PCLacopolymer prepared by acylation.

[0055] These observations showed that both the grafting method (ROP or acylation) and the DP of the LN-g-PCLp polymers influenced copolymer crystallinity. Additionally, the acylation reaction resulted in LN attaching to both ends of the PCL, leading to more rigid and compact chains, with reduced crystallinity.

[0056] This behavior can be attributed both to the nature of LN, which is a nonuniform and amorphous polymer, impeding the alignment of the PCL arms; and also to the relaxation dynamics of the grafted copolymers. Shorter PCL chains exhibit a diffusive motion, supporting freer movement and crystal formation. Conversely, longer PCL chains tend to have a more subdiffusive motion, in which movement of the polymer chains tends to bemore restricted and slower due to interaction between PCL chains, resulting in lower crystallization.

[0057] TGA was used to investigate how the grafting method and DP influenced thermal stability. Typically, pure PCL undergoes a two-stage degradation process: Hydrolytic Degradation. In the first stage, PCL undergoes hydrolytic degradation, in which water molecules attack the ester bonds in the polymer backbone. Water, CO2, and carboxylic acid groups are released by pyrolysis, characterized by chain breakage and depolymerization. Thermal and Oxidative Degradation (Pyrolysis). The second stage is a pyrolysis step, involving thermal and oxidative degradation.

[0058] Our observations found that the LN-g-PCL polymers were more thermally stable than neat PCL. The enhanced thermal stability was attributed to the decomposition of LN aromatic compounds at temperatures above 600°C.

[0059] The observed total weight losses for PCL and LN at 400°C were 93.6% and 32.2%, respectively, while that for the LN-g-PCLpcopolymer with 101 DP was 87.1%. Other LN-g-PCL polymers with different DP showed similar total weight percent loss, regardless of the DP. By contrast, polymers grafted by acylation showed the lowest percentage weight loss by TGA. Among all LN-g-PCL copolymers, the maximum rate of degradation temperature (MRDT) was similar (339.9 ± 1.3°C), except for LN-g-PCL 101 DP, which had a slightly higher MRDT of 344.4 °C - still lower than that of PCL (372.1 °C).

[0060] Conversely, the DP influenced the initiation temperatures for thermal degradation. The initial decomposition temperature decreased from 341 °C to 328°C when the DP decreased from 101 to 22. The highest and lowest onset temperatures were found for PCL (371.6°C) and LN (318.5°C), respectively. These results suggest that grafting PCL onto LN enhances thermal stability and resistance, while the grafting method does not significantly alter the degradation pattern of the copolymers.

[0061] Nanoparticle characterization. In the emulsion evaporation technique for nanoparticle synthesis, the solvation of LN-g-PCL decreases as the DCM evaporates. Consequently, introducing the LN-g- PCL / DCM solution into water leads to the aggregation of polymer molecules and the formation of nanoparticles. The hydrophobic PCL segments tend to avoid contact with the aqueous environment and aggregate to form the core of the nanoparticles, facilitating the encapsulation of hydrophobic substances in the hydrophobic core. Conversely, the hydrophilic LN segment, orients towards the outer part of the nanoparticle, towards the aqueous environment. The LN shell enhances the stability of the NPs in aqueous media.

[0062] Dynamic light scattering (DLS) was used to determine the mean size, distribution, and zeta potential of LNPs synthesized from the four polymers. See Table 2. We observed that the grafting method affected the particle size, with LNPs from polymers synthesized by acylation being smaller. The higher DP of polymers synthesized by ROP led to a larger particles. This behavior is consistent with the notion that polymers with shorter hydrophobic sections should tend to produce smaller micelles.Table 2. Properties of LN-g-PCL copolymers nanoparticles. EE = Encapsulation efficiency. LC = Loading capacity.NumberSize (nm) PDI Zeta (mV) EE(%) LC(%) ofParticlesLN-g-PCLp 22DP 184.6±3.5b0.045±0.01 -57.0±1.8 77.8±1.5a7.8±0.1a2.70E+10LN-g-PCLp 57DP 269.6±0.4a0.099±0.02 -58.8±1.0 79.5±1.5a8.0±0.1a1.81 E+10LN-g-PCLp 101DP 245.0±1.5a0.056±0.02 -58.3±1.2 78.9±1.2a7.9±0.1a1.85E+10LN-g-PCLa 103 DP 80.21±0.2c0.050±0.02 -62.3±6.6 57.6±1.8b5.8±0.2b2.73E+10

[0063] The LNP mean size was also influenced by the type of copolymer used. Despite a general tendency for NPs made with amphiphilic copolymers to have smaller sizes and narrower size distributions, the LN-g-PCLpNPs had a larger mean size - and considerably larger when the DP was increased. The increase in LNP mean size might be attributed to two factors: (1 ) limited solubility of LN-g- PCLp polymers with higher DP during nanoparticle synthesis, promoting agglomeration of polymer chains, and (2) unrestricted arrangement of PCL chains grafted to LN by ROP, creating a larger hydrophobic core within the LNPs. Additionally, the distinct molecular structures of the LN- g-PCLa (103 DP) and LN-g-PCLp (101 DP), even at similar DP levels, can influence LNP size. In LN-g-PCLa, some PCL is attached at both ends, resulting in a more rigid but shorter PCL chain structure. Conversely, in LN-g-PCLp, PCL is grafted from one side, leading to a longer chain for the same DP. While an increase in DP from 22 to 57 indeed led to an increased NP size, no significant additional size differences were observed when DP increased further from 57 to 101. This observation might be attributed to changes in the Mw of the polymers, and possible entanglement between PCL chains at higher DP. The increase of DP of PCL in the LN-g-PCLp polymers allows greater interaction between PCLchains, resulting in a more entangled core without significant change in the spatial dimensions of the hydrophobic core and the NP. These differences in structure were evident in the crystallinity analysis shown in Table 1 , and suggest that a higher DP could slow the diffusion of a hydrophobic payload molecule through a more compact PCL matrix.

[0064] Additionally, nanoparticle tracking analysis suggested a trend in which lower NP size correlated with a greater number of NPs in aqueous solution, in which the LNPs were more-or-less monodispersed, and in which the LNPs had a consistent negative zeta potential from -62.3 ± 6.6 mV to -57.0 ± 1.8 mV when suspended in water. This outcome is attributed in large part to the influence of various functional groups, such as phenolic and carboxylic OH, and possibly other anionic moieties in the LN outer shell. These anionic groups can ionize in an aqueous environment.

[0065] This interpretation is supported by the31P NMR data, which showed a reduction in but not a complete conversion of the aliphatic and phenolic groups in the modified LN. There was an observed “remnant” of 15.5 ± 10.8 % of OH groups on average for all LN-g-PCL copolymers. The presence of unmodified OH groups could contribute to a negative zeta potential. For the LN-g-PCLaparticles, the absence of a carboxylic OH peak in the31P NMR analysis suggested a nearly complete modification of the PCL end groups. However, the LN component of LN-g-PCLa still retained some carboxylic and phenolic OH groups after grafting, contributing to the negative Z potential (Table 1 ). These observations confirmed the influence of lignin’s functional groups on the negative Z potential of the nanoparticles.

[0066] The morphology of the LN-g-PCL nanoparticles was investigated through TEM. The LN-g-PCL NPs displayed regular, spherical shapes, with diameters primarily in the 80-270 nm range. The TEM images showed distinct dark and clear regions, with the darker portions attributed to the LN block, and the clear areas to the hydrophobic PCL blocks. This core-shell morphology indicates the formation of micelle-like nanoparticles in aqueous solution. The TEM imaging was performed after drying and resuspension of the particles. Post-drying DLS analysis of particles revealed higher PDI values, consistent with the TEM observations.

[0067] Payload loading and entrapment efficiency. Table 2 includes data on loading capacity (LC) and entrapment efficiency (EE) for LNPs with a prototype payload, namely the agrochemical MFZ. LC and EE were not significantly affected by the DP of polymers made by ROP. However, lower LC and EE were seen for NPs synthesized from LN-g-PCLa, made by the acylation grafting reaction. The reduced LC and EE for the LN-g- PCLa NPs might be attributed to the smaller size of the nanoparticles, and the structure of the amphiphilic polymer. For LN-g-PCLa, PCL is attached at both ends, leading to a more compact and entangled PCL chain structure, and hence a smaller nanoparticle core with lower EE. On the other hand, in LN-g-PCLp, PCL is grafted from one side only, resulting in a longer chain for the same DP. The longer PCL chain structure resulted in a larger core size with correspondingly higher EE. In summary, the observations showed that the grafting method and the NP size both affect the NP entrapment properties.

[0068] Payload release. The payload release profile from LN-g-PCL NPs was investigated, to see the effects of variations in the DP, graftingmethod, and temperature (at 23 °C and 30 °C). Among NPs made by the ROP grafting process, the LN-g-PCLp22 DP, having the shortest PCL chains attached to LN, released 31.7 % of MFZ in the first 24 hours, and behaved similarly to LNPs from the LN-g-PCLp 57 DP. By contrast, LN- g-PCLp 101 DP NPs showed a slightly lower cumulative release of 29.2% of MFZ over the same time span. These results showed that DP had a small, but measurable effect on cumulative release of the payload. The grafting method also influenced the release of MFZ from the LNPs. LN-g- PCLa NPs, containing PCL chains with DP of 103 or smaller, showed the lowest cumulative release of MFZ of all LNPs tested in these experiments.

[0069] The slower release from LN-g-PCLarelative to LN-g-PCLp resulted from the different grafting methods. The smaller and more compact PCL core matrix in LN-g-PCLa NPs presented more restricted PCL chains and slower diffusive motion from relaxation of the grafted chains. The more densely packed and entangled PCL core may hinder diffusion of the payload (MFZ) though the polymeric matrix, resulting in a higher effective binding affinity between MFZ and the PCL core.

[0070] Changes in the DP had a smaller effect on release of the hydrophobic agrochemical MFZ. The release kinetics followed two different phases as a function of time. No significant differences were seen in the cumulative release of MFZ for different DPs over the initial hours, perhaps because the initial release was that of more weakly-bound MFZ molecules located near the shell were initially released. However, following the initial 24 hours, the strong interactions of PCL with MFZ in the core led to a more gradual, DP-dependent release. An increase in the DP of PCL grafted to LN molecules may increase the likelihood of entanglement between PCL chains during NP formation, influencing thestructure and stability of the resulting micelles. Increased entanglement within the core decreases the accessibility of water molecules to the core, and decreases diffusion of MFZ through the PCL matrix, resulting in slower release rates with higher DP for PCL in the LN-g-PCL NPs. These observations are consistent with the observation that NP size was relatively independent of DP, at least between 55 and 101 DP.

[0071] Temperature influenced the release behavior, with a faster release at higher temperatures as expected. No significant differences were seen between the release profile of LN-g-PCLpNPs with 22 DP (100%) and 57 DP (97.7%) over 196 h at 30 °C, while LN-g-PCLp 101 DP NPs showed a more delayed release (86.3%). All LN-g-PCLp NPs had higher cumulative release at 30°C than at 23°C over 196 h. By contrast, the LN-g-PCLa NPs had a lower cumulative release (37.3%) of all four polymeric NPs at 30°C, but that release was not significantly altered by lowering the temperature to 23°C (36.8%).CuS Nanoparticles, and LN-g-PLGA Nanoparticles entrapping CuSNanoparticles

[0072] Copper is commonly used as a contact fungicide. An efficient means to distribute Cu systemically within a plant could improve the ability of Cu to enhance natural plant defenses. Combining the contact mode of action of Cu-based compounds with systemic deployment of Cu using LNPs could be a particularly efficient strategy to combat fungal / bacterial infections.Materials

[0073] PLGA was acquired from Sigma-Aldrich (St. Louis, MO). LN was acquired from TCI Inc. (Portland, OR). Methanol (purity >99%),dichloromethane (DCM) (purity >99.9% extra dry), oxalyl chloride (purity >99%), DMF (purity >99.5%), dimethyl sulfoxide (DMSO, purity >99.7%), ethyl ether (purity >99%), toluene (purity >99.5%, extra dry), and trehalose (purity >99%) were acquired from Fisher Scientific (Pittsburgh, PA).Synthesis of hydrophobic CuS nanoparticles

[0074] CuS NPs were synthesized by reaction with sodium citrate, in a modification of the method of Zhou, M., et al. (2010). A Chelator-Free Multifunctional [64Cu]CuS Nanoparticle Platform for Simultaneous Micro- PET / CT Imaging and Photothermal Ablation Therapy. Journal of the American Chemical Society, 132(43), 15351-15358. doi.org / 10.1021 / ja106855m. Two solutions were prepared: solution A was made by combining 10 mM copper (II) chloride (CUCI2) with 5 mM sodium citrate with vigorous mixing in deionized (DI) water; Solution B was a 10 mM solution of sodium sulfide (Na2S) in DI water. The synthesis reaction involved mixing 25 mL of Solution A with 25 mL of Solution B under constant stirring for 1 hour at room temperature. A change from pale-blue to dark-brown was observed after the addition of Na2S. Subsequently, the reaction mixture was transferred to a hot oil bath at 90 °C for either 15 or 30 minutes, followed by cooling to room temperature.

[0075] For surface modification, a 5 mmol solution of 1 -octadecanethiol (ODT) in dimethylformamide (DMF) was prepared and sonicated until the ODT had fully dissolved. Then 50 mL of the ODT solution were added to the previously-described reaction mixture of CuS, and allowed to react overnight with constant magnetic stirring. The next day, the solution was washed multiple times with 150 mL of hexane until the modified CuS NPshad been extracted essentially completely. Hexane was then evaporated using a Rotavapor R-300 (Buchi Corporation, New Castle, DE). The resulting CuS-ODT NPs were resuspended in ethyl acetate and centrifuged (Beckman Coulter Avanti JXN-26, 7000 rpm, rotor JA-30.50Ti, Beckman Coulter, Brea, CA) for 5 minutes several times to remove excess ODT. The resulting pellet (CuS NPs) was collected and dried in a vacuum oven for 24 h. The CuS NPs were stored in a desiccator at room temperature until used.Characterization of CuS-ODT NPs

[0076] Transmission electron microscopy (TEM) was used to examine the morphology of CuS NPs synthesized during a 15 min reaction (CuSismin), during a 30 min reaction (CuSaomin), and after modification with ODT (CuSismin-ODT and CuSaomin-ODT, respectively). TEM was performed with a JEM-1400 instrument (JEOL, Japan), following established protocols from the literature. Elemental analysis was conducted with a JEM 661 0LV Scanning Electron Microscope (SEM)(JEOL, Japan) equipped with EDS.

[0077] X-ray photoelectron spectroscopy (XPS) was used to analyze the chemical composition and oxidation states of CuSismin and CuSaominwith a Scienta Omicron ESCA 2SR X-ray Photoelectron Spectroscope (Scienta Omicron GmbH, Taunusstein, Germany).

[0078] Structural analysis of CuS NPs was performed by X-ray diffraction using a PANalytical Empyrean X-Ray Diffractometer with Cu Ka radiation (A = 0.15406 nm) with a 20 range of 20°- 80°.

[0079] Average crystallite sizes were obtained by Scherrer’s Equation: (eq 6)where A is the X-ray wavelength, K is a dimensionless geometric factor with the value 0.9, 0 is Bragg’s angle, and p is full width at half maximum (FWHM). FTIR spectroscopy analysis was used to assess the surface modification of CuS after the addition of ODT using a Bruker Tensor 27 instrument (Bruker 500, Billerica, MA) in the range 400 to 4000 cm'1.Synthesis of (AZS)LN-PLGA polymers

[0080] (A / S)LN-PLGA polymers were synthesized via an acylation reaction, following a modification of the method of Astete, C. E., et al. (2020). Lignin-Graft-Poly(lactic- co -glycolic) Acid Biopolymers for Polymeric Nanoparticle Synthesis. ACS Omega, 5(17), 9892-9902. doi.org / 10.1021 / acsomega.0c00168. First, 4 g of PLGA were dissolved in 100 mL of DCM at room temperature overnight in a three-neck roundbottom flask under anhydrous conditions with argon flow. Oxalyl chloride (110 pL, catalyst) was added with a glass syringe, along with 4 mL of DMF. The mixture then at room temperature, with constant stirring at 300 rpm for 5 hours.

[0081] DCM was then removed using a Rotavapor Buchi R-300 (Buchi Corporation, New Castle, DE) to yield a viscous PLGA-CI solution. The coupling reaction involved dissolving PLGA-CI in 50 mL of DCM, whichwas then added dropwise to 4 g of (A / S) LN dissolved in 50 mL of DMSO at a LN-PLGA ratio of 1 :1 (w / w). A 2:1 (w / w) ratio composition was also prepared by the same method, changing the initial PLGA amount to 2 g. In either case, the mixture was allowed to react for 48 hours under argon flow with constant magnetic stirring at room temperature. Ethyl ether (200 mL) was then added to precipitate the LN-PLGA polymer.

[0082] Subsequently, the grafted polymer was diluted in 200 mL of DCM overnight, and washed with distilled water to remove unreacted LN. The polymer was further extracted by adding 50 mL of DCM to the distilled water until essentially complete extraction had been achieved. The polymer solution was concentrated with a Rotavapor R-300 (Buchi Corporation, New Castle, DE). The resulting solids were frozen at -80°C for 24 hours and then lyophilized (FreeZone Plus 2.5; Labconco, Kansas City, MO) to remove residual water and solvents. The LN-PLGA polymer was then stored at -20 °C until used.

[0083] The chemical structure of the (A / S)LN-PLGA was assessed by1H-NMR using a Bruker 400 MHz system (Billerica, MA) at 400 MHz in CDCh, and by FT-IR analysis using a Bruker Tensor 27 instrument (Billerica, MA).Polymeric nanoparticle synthesis and characterization

[0084] A / SLN-PLGA core-shell NPs were synthesized using the oil in water (O / W) emulsion evaporation technique as generally described in Astete et al. 2020. The (A / S)LN-PLGA polymers self-assemble into micelles with a hydrophilic LN outer layer and a hydrophobic PLGA core. Briefly, (A / S)LN-PLGA (200 mg) was dissolved in DCM (10 mL). To loadCuS NPs into the LN-PLGA NPs, 20 mg of CuS-ODT was added to the DCM solution, and this organic-phase solution was added dropwise into the aqueous phase (60 mL deionized water). Subsequently, the mixture was microfluidized in four cycles with an M-110P microfluidizer (Microfluidics, Newton, MA, USA).

[0085] The organic solvent was evaporated using a Rotavapor R-300 (Buchi Corporation, New Castle, DE), trehalose (200 mg) was added as a cryoprotectant, and the solution was then frozen at -80 °C for 24 hours before lyophilization with a FreeZone Plus 2.5 freeze dryer (Labconco, Kansas City, MO).

[0086] Particle size, zeta potential, and number of particles were evaluated using nanoparticle tracking analysis (NTA) with a ZetaView Quart PMX-420 instrument (Particle Metrix GmbH, Meerbusch, Germany. The LNPs were resuspended in water at 0.5 mg / mL. A volume of 1 mL of a diluted sample (1 :1000) was analyzed using a laser at A = 480 nm. The morphology of LNPs was examined using TEM with a JEM-1400 instrument.CuS release from LNPs

[0087] We used the dialysis bag method to measure Cu ion and S ion release from (A / S)LN-PLGA NPs (LNPs loaded with either CuSismin or CuSaomin), at either of two temperatures (25 and 35°C). CuS-loaded LNPs were resuspended in deionized water (DI) at a concentration of 15 mg / mL to a total volume of 10 mL and placed inside a dialysis bag having a Mw cutoff of 12-14 kDa (Fisherbrand, USA). The dialysis bag was immersed in 500 mL of DI held at either 25°C or 35°C. Aliquots of 10 mL were takenfrom outside the dialysis bag at 0, 1 , 3, 6, 12, 24, 48, 144, and 240 h, and replaced with an equal volume of DI with each sample taken. Cu ions were extracted from samples by acidification with nitric acid (HNO3) to a final concentration of 2%. The concentrations of Cu and S ions were quantified with inductively-coupled plasma (ICP) mass spectrometry (MS), and also with ICP optical emission spectroscopy (OES). The cumulative release of ions is reported as a percentage relative to the amount at time zero.Statistical Analyses

[0088] Each release experiment was replicated three times (n=3). Statistical analyses of the data were performed with RStudio for Windows v2023.06.0 +421 (RStudio Inc., Boston, MA). Statistical differences were found using one-way ANOVA with a significance level (a) of 0.05. Data are reported as mean ± standard deviation.Results and discussionSynthesis and characterization of CuS NPs (CuS, CuS-ODT).

[0089] The morphology and size of CuS nanoparticles were characterized by TEM and SEM. By increasing the duration of the CuS synthesis reaction, the size of the particles increased from 8.8 ± 1.3 nm (at 15 min, CuSismin) to 14.7 ± 3.3 nm (at 30 min, CuSaomin). As one hypothesis, this increase in particle size with increasing time may be related to Ostwald ripening, a process in which smaller nanoparticles disperse and redeposit onto larger particles.

[0090] The chemical composition of CuS nanoparticles was analyzed by energy dispersive spectroscopy (EDS). Table 3 shows elemental analysis for CuS nanoparticles with different reaction synthesis times, 15 and 30 min. At both time points, the three elements representing the greatest weight percentage in the CuS NPs were C, S, and Cu. The Cu : S stoichiometric ratio was 1 .8:1 of Cu:S for both CuSismin and CuSaomin. This ratio suggests the formation of CU9S5, also known as digenite, at both reaction times. Evidently, increasing the reaction time from 15 to 30 min did not greatly affect the chemical composition of the product.

[0091] CuS NPs were modified with ODT, an 18-carbon primary thiol (R- SH). It is hypothesized that ODT forms an S-Cu bond that modifies the surface of the CuS NPs. Elemental analysis following the ODT modification showed a decrease in the Cu:S ratio to 0.80 ± 0.21 (Table 3), suggesting that the CuS surface had been modified by formation of Cu-S bonds between Cu and S from ODT. Additionally, the combined weight percentages of elemental Cu and S in the CuS-ODT powder decreased from 74.17 ± 1.46 % to 24.86 ± 2.24 % for CuSismin, and from 77.74 ± 3.68 % to 35.29 ± 8.21 % for CuSaomin. The decrease in elemental Cu and S levels from the native CuS NPs presumably follows from the increase in C levels, resulting from the 18-C chain of ODT.Table 3. Elemental composition of the CuS NPs synthesized at 15(CuSismin) and 30 min (CuSaomin), before and after ODT surface modification.

[0092] XPS analysis of the CuSismin and CuSaomin NPs helped confirm the chemical composition and oxidation states of CuSismin and CuSaomin. The Cu 2p spectra had prominent peaks at 932 and 951.9 eV, and a shoulder peak at 933 eV, corresponding to the +1 and +2 oxidation states of Cu, respectively. (Other studies have reported copper sulfides having more Cu+1than Cu+2). Additionally, satellite peaks at -945 and 965 eV from Cu+2were present in the sample synthesized at 30 min, indicating that the proportion of Cu+2increased with increasing reaction time, possibly due to oxidation of copper sulfide to copper sulfate. This change in oxidation state was also indicated by an increase in the strength of apeak at 933 eV as reaction time increased from 15 minutes to 30 minutes. The S 2p spectra showed the presence of disulfide (S2)2' at 162.5 eV for 2p3 / 2 and 163 eV for 2pi / 2, and monosulfide (S2') at 161 .2 eV for 2p3 / 2 and 162 eV for 2pi / 2. A peak at -169 eV indicated the possible oxidation of S'2to S+2to form SO4. The sulfide oxidation could result from greater exposure to oxygen and water vapor during the longer reaction time.

[0093] XRD patterns from CuSismin and CuSsomin were consistent with published standards for the crystal structure of covellite (CuS) AMCSD No. 0010980, and digenite (Cui.sS) AMCSD No. 0006939, suggesting that the samples comprised a mixture of CuS and Cui.sS. These results support the XPS finding that a mixture of valences is present in the CuS NPs.

[0094] The diffraction peaks for the CuSismin and CuSsomin samples were similar, with predominant 20 peaks around 27.8°, 29.4°, 31.9°, 33.0°, 48.1 °, 52.9°, and 59.6°, corresponding to the (101 ), (102), (103), (006),(110), (108), and (116) crystal planes of the CuS standard. Likewise, diffraction peaks at 27.7°, 32.0°, 46.0°, 54.5°, and 67.1 ° correspond to the(111 ), (200), (220), (311 ), and (400) crystal planes of Cui.sS. The average crystallite sizes obtained by the Scherrer Equation were estimated to be 5.2 ± 1 .3 nm for CuSismin and 5.5 ± 2.0 nm for CuSsomin.

[0095] The surface modification of CuS upon the addition of ODT was analyzed by FTIR. Symmetric and asymmetric methylene (CH2) vibrations were present at 2848 and 2915 cm'1, respectively. The presence of both symmetric and asymmetric CH2 peaks suggested that the alkyl chains had predominantly adopted an all-trans conformation. This alkyl chain configuration suggests that the molecules form acrystalline structure over the CuS NP surface. Lower-intensity symmetric and asymmetric methyl (CH3) peaks were seen at 2872 and 2953 cm'1, respectively. CH2 bending peaks were visible at 1471 and 1461 cm'1, and peaks corresponding to C-S bonds were seen at 729 and 717 cm'1. Note that vibration signals from CuS are not visible in a conventional IR spectrum, because CuS itself is IR-inactive. Moreover, the absence of a band at 2577 cm'1, which would be a typical vibration for S-H in ODT, indicated the successful formation of S-Cu bonds. Overall, the presence of the alkyl chains and C-S bonds confirmed the surface modification of the CuS NPs.LN-PLGA polymer characterization

[0096] FTIR spectra indicated the presence of LN and PLGA in the copolymers. Several functional groups typical of LN were present in all (A / S)LN-PLGA copolymers, including hydroxyl groups (OH, 3000-3700 cm'1), aromatic rings (symmetric a-carbonyl, 1590 cm'1; asymmetric a-carbonyl, 1508 cm'1), and primary and secondary alcohols (1030 cm'1). Similarly, we observed the signatures of functional groups characteristic of PLGA, such as carbonyl (C=O, 1721 cm'1), methylene (CH2, 2900-2800 cm'1), and C-CO-O (symmetric and asymmetric, 1300 and 1150 cm-1). The presence of these functional groups in the copolymers confirmed the successful grafting of PLGA to (A / S)LN.

[0097] 1H NMR spectra of the A / SLN-PLGA copolymers allowed further elucidation of their structures. Peaks characteristic of lactic acid were observed in the NMR spectra, such as methyl (-CH3, 1.5 ppm) and methine (-CH, 5.2 ppm). Likewise, the characteristic methylene group (CH2, 4.8 ppm) peak from glycolic units is present, and the methoxy group(-OCH3, 3.6 ppm) from lignin was seen in all copolymers. These results are consistent with the FTIR results, supporting the conclusion that PLGA had successfully grafted onto LN.Lignin-PLGA Nanoparticle Characterization

[0098] The A / SLN-PLGA NPs had a hydrophilic LN outer shell layer, and an inner hydrophobic PLGA core. Using DLS analysis to evaluate the mean size of ALN-PLGAi:iNPs following the entrapment of CuS, we observed a substantial size increase: from 93.71 ± 0.5 nm for “empty” NPs, to 176.9 ± 1 .3 nm for ALN-PLGAi:i(CuSismin-ODT).

[0099] The (A)LN-PLGA 1 :1 and 2:1 w / w loaded with CuSis-ODT presented an average size of 132.1 ± 0.5 and 149.3 ± 0.3 nm, with a zeta potential of -41.53 ± 1.01 and -46.23 ± 1.05 mV, respectively, similar to values for particles loaded with CuSao-ODT. On the other hand, lignosulfonate formed smaller NPs. For example, NPs made with (S)LN- PLGA 1 :1 and 2:1 w / w loaded with CuSao-ODT presented an average size of 130.3 ± 0.7 and 127.2 ± 0.7 nm, respectively; with zeta potentials of -45.96 ± 0.92 and -48.23 ± 0.76, respectively.Table 4. Size, # NP / ml, and zeta potential of (AZS)LN-PLGA NPs loaded with CuS-ODT. n=3

[0100] TEM images confirmed the core-shell NP structure. The micelle-like NPs presented a dark outside layer by TEM, attributed to (A / S)LN, and a clear core, corresponding to PLGA. Measurements of size by TEM for the (A / S)LNPs ranged from 101 to 154 nm, with no significant differences seen for different LN-PLGA ratios. By contrast, earlier studies had found that increasing the LN ratio in LN-based polymers generally resulted in smaller NP sizes. Our observations thus suggest that the CuS-ODT affected the self-assembly process, perhaps by interfering with interfacial tension.

[0101] TEM analysis also confirmed the entrapment of CuS NPs inside the core-shell structure made from LN-PLGA. TEM images of ALN-PLGA1 :1 NPs containing CuSismin-ODT showed the CuS NPs as black dots dispersed inside the larger, generally spherical, polymeric nanoparticles. TEM analysis indicated that the (A / S)LN-PLGA NPs had successfully entrapped CuS within the polymeric nanostructure.

[0102] The elemental composition of (A / S)LN-PLGA NPs was confirmed by EDS analysis (Table 5). Elemental analysis showed a stoichiometric Cu:S ratio of 0.59 ± 0.30 for ALN-PLGA; and 0.25 ± 0.06 for SLN-PLGA; with both values being substantially smaller than the 1.78 ± 0.06 ratio for unmodified CuS NPs. The lower copper levels were attributed to the addition of S into the system during the modification of the CuS NPs, as well as from the LN structure after entrapment into LNPs. After modification with ODT, the Cu:S ratio decreased to 0.80 ± 0.21 due to the introduction of thiol sulfur atoms from ODT. The further lowering of the Cu:S ratio after entrapment into the LNPs was attributed to the sulfur atoms from lignin, and especially from lignosulfonate. The sulfite pulping process results in a higher S content in SLN than in ALN.

[0103] The elemental weight percentage of sulfur for LNPs loaded with CuS-ODT was 2.37 ± 0.50% for ALN-PLGA delivery systems, and was 2.82 ± 0.52% for SLN-PLGA delivery systems. These results are generally consistent with the EDS analysis of CuS-ODT, considering the ratio between polymer and CuS during synthesis, giving concentrations close to the theoretical figures of 2.29% and 3.21% for CuSismin-ODT and for CuSaomin-ODT, respectively.Table 5. Elemental-composition of the (AZS)LN-PLGA NPs loaded with CuS. n=3CuS release study (n=2)

[0104] Cu and S ions could be successfully released from the novel composition as needed. We tested Cu and S ion release over seven days from several systems, including ALN-PLGAi:iloaded CuSaomin-ODT, empty ALN-PLGAi:i, CuSaomin-ODT, and un-modified CuS NPs; n=2. The fresh CuS NPs showed the highest release of S at 0.77 ppm and Cu at 0.24 ppm after 1 day, increasing to 1.59 and 1.47 ppm over 7 days, respectively. By contrast, only 0.016 ppm of Cu ions were released from ALN-PLGAi:i NPs over 7 days, while the other systems released no measurable Cu during the 7-day observation period.

[0105] The Cu ions were released from CuS NPs embedded well within the cores of the PLGA nanoparticles, gradually diffusing outside the coreshell structure in the presence of water. The slower release of Cu ions from the ALN-PLGA NP systems was attributed to the hydrophobic PLGA core, the electrostatic interaction of the positively charged Cu with the negatively charged lignin surface, and the chelating properties of the thiol functional groups in ODT.

[0106] The observed release rates from the nanoparticles were relatively slow and gradual; a slow release rate is desirable for many applications. The ALN-PLGA NPs delivery systems slowed the dissolution and release of Cu, and illustrate the potential of the novel delivery systems in the controlled release of Cu and S as micronutrients, which can potentially enhance plant disease resistance and overall health by slowly release nutrients used in plant defenses.

[0107] In summary, in this set of experiments CuS NPs were synthesized by the citrate reaction for two different reaction times, 15 min and 30 min. The particle size increased with increasing reaction time, from 8.8 ± 1 .3 nm to 14.7 ± 3.3 nm. Fresh CuS NPs were surface-modified with an 18-carbon thiol (ODT), and the resulting hydrophobic CuS NPs were then entrapped in LNPs. Elemental analysis showed changes in the stoichiometric Cu:S ratio from 1.8:1 in fresh CuS NPs to 0.8:1 after surface modification, due to the introduction of an 18-carbon chain, and a further lowering to 0.59 ± 0.30 and 0.25 ± 0.06 after entrapment in ALN-PLGA and SLN-PLGA core-shell NPs, respectively. The CuS-loaded NPs measured 122-162 nm in diameter, depending on the type of LN, the LN LGA ratio, and the CuS entrapped. Release of Cu and S into aqueous solution was controlled over a duration of at least 168 h. Theseresults demonstrated the ability of the LN-based NPs to control the release of modified CuS NPs into aqueous solution.Hydrophobic CuS nanoparticles: entrapment in and release from lignin-PCL nanoparticlesMaterials

[0108] PCL was acquired from Sigma-Aldrich (St. Louis, MO). LN was acquired from TCI Inc. (Portland, OR). Methanol (purity >99%), dichloromethane (DCM) (purity >99.9% extra dry), oxalyl chloride (purity >99%), DMF (purity >99.5%), dimethyl sulfoxide (DMSO, purity >99.7%), ethyl ether (purity >99%), toluene (purity >99.5%, extra dry), and trehalose (purity >99%) were acquired from Fisher Scientific (Pittsburgh, PA).Synthesis of hydrophobic CuS nanoparticles

[0109] CuS NPs were synthesized by reaction with sodium citrate, generally following the method of Zhou et al., 2010. Two solutions were prepared for the synthesis: Solution A was made by combining 10 mM copper chloride (CuCh) with 5 mM sodium citrate through vigorous mixing in deionized (DI) water; solution B was prepared as a 10 mM solution of sodium sulfide (Na2S) in DI water. The synthesis reaction involved mixing 25 mL of Solution A with 25 mL of Solution B under constant stirring for 1 hour at room temperature. A change from pale-blue to dark-brown was observed following the addition of Na2S. Subsequently, the reaction mixture was transferred to a hot oil bath at 90°C for either 15 or 30 minutes, followed by cooling to room temperature.

[0110] For surface modification, a 5 mM solution of 1 -octadecanethiol (ODT) was prepared in dimethylformamide (DMF), with sonication until fully dissolved. Separately, 50 mL ODT solution was added to the previously-described CuS reaction mixture, and allowed to react overnight under constant magnetic stirring. The following day the solution was washed multiple times with 150 mL of hexane until the modified CuS NPs had been essentially completely extracted. Hexane was then evaporated with a Rotavapor R-300 (Buchi Corporation, New Castle, DE). The resulting CuS-ODT NPs were resuspended in ethyl acetate and centrifuged (Beckman Coulter Avanti JXN-26, 7000 rpm, rotor JA-30.50Ti, Beckman Coulter, Brea, CA) for 5 minutes several times to remove excess ODT. The pellet (CuS NPs) was collected and dried in a vacuum oven for 24 h. The resulting CuS NPs were stored in a desiccator at room temperature until used.Synthesis of ALN-PLC

[0111] LN-PCL polymers were synthesized generally following the method of Garcia, A., et al. Modulation of Methoxyfenozide Release from Lignin Nanoparticles Made of Lignin Grafted with PCL by ROP and Acylation Grafting Methods. Langmuir (2024) 40(10), 5433-5443. https: / / doi.org / 10.1021 / acs.langmuir.3c03965. Briefly, 2 g of alkali LN was placed in a three-neck round-bottom flask (250 ml), followed by the addition of £-ca pro lactone (CL) at specific CL : LN ratios (w : w). A CL : LN ratio of 3:1 was used in many of these experiments, which was a ratio that led to a DP of 36 (as determined by1H NMR).

[0112] The reaction was held in an oil bath at 130°C. Following the addition of 0.2% Sn(Oct)2 catalyst (v / v based on CL volume), the reaction proceeded for 24 hours with constant stirring. Next 200 mL cold methanolwere added to remove monomers and unreacted PCL. The resulting grafted polymer was washed five times with 200 mL DCM and distilled water to remove unreacted LN. Subsequently, the polymer was concentrated, frozen, and lyophilized (FreeZone Plus 2.5) to remove residual water and solvents. Finally, the LN-PCL polymer was stored in a desiccator at room temperature until used.Polymeric nanoparticle synthesis and characterization

[0113] ALN-PCL core-shell NPs were synthesized following generally the same methodology as previously described for the (A / S)LN- PLGA NPs. The ALN-PCL NPs were then loaded with CuS-ODTaomin.

[0114] The LN-PCL nanoparticles were resuspended in water at 0.2 - 0.5 mg / mL and placed in a cuvette for characterization. The size distribution, polydispersity, and zeta potential of the LN-PCL nanoparticles were determined by dynamic light scattering (DLS) with a Malvern Zeta Sizer ZS (Malvern, Panalytical, Westborough, MA) at 25°C. Elemental analysis was conducted with a JEM 6610LV Scanning Electron Microscope (SEM) (JEOL, Japan) equipped with EDS. Results are shown in Table 6.Table 6. Size, PDI, and zeta potential of ALN-PLGA NPs loaded withCuS-ODT. n=3Materials

[0115] Alkaline lignin; 2,4-dimethoxy benzyl alcohol; 1 -ethyl-3-(3- dimethyl aminopropyl)-carbodiimide hydrochloride (EDCI); dimethyl amino pyridine (DMAP); and zinc chloride were purchased from TCI (Portland, OR). Hydrochloric acid (HCI) was supplied by VWR International (West Chester, PA). Poly [lactic-co-glycolic acid] (PLGA); 2- chloro-4,4,5,5-tetramethyl-1 ,3,2-dioxaphospholane (TMDP); and gibberellic acid were purchased from Sigma Aldrich (St. Louis, MO). Dimethylformamide (DMF), pyridine, chloroform-d, chromium (III) 2,4- pentanedionate (Cr(acac)3), cyclohexanol, dichloromethane (DCM), and sodium chloride were purchased from Thermo Fischer Scientific (Waltham, MA).MethodsGibberellin A3 methyl ester

[0116] Gibberellin A3 methyl ester was synthesized as follows: 0.75 g GA3 was dissolved in dichloromethane (25 mL). EDCI-HCL (625 mg, 3.27 mmol) was slowly added, followed by the addition of the acyl transfer agent and catalyst DMAP (1.25 g, 6.54 mmol) at 0°C. The carboxyl activating agent (EDCI-HCI) and GA3 were reacted in a 1 .5:1 (EDCI: GA3- COOH) molar ratio. The DMAP : EDCI molar ratio was 2 : 1. After stirring at room temperature for 90 min, excess methanol (0.5 mL, 12.36 mmol) was added dropwise at 0°C. The mixture was stirred at room temperature for 12 h, after which the reaction mixture was washed with dilute hydrochloric acid (1 M, 10 mL) and extracted with dichloromethane (3 x 50 mL). The collected organic phase was washed twice with D.L water with added DCM to ensure maximum extraction. Afterward, the solventwas evaporated under reduced pressure using a Rotavapor Buchi R-300 (Buchi New Castle, DE, USA), and the resulting concentrate was dried in a vacuum oven (Fisher Scientific, Haverhill, MA, USA) for 24 h.Polymer synthesis

[0117] The LN-PLGA graft polymer was synthesized in an acylation reaction. Briefly, for lignin-PLGA (1 :1 w / w), 1.2 g of PLGA was dissolved overnight in 35 mL of anhydrous dichloromethane (DCM) in a 3-neck round-bottom flask at room temperature under argon flux. After PLGA had completely dissolved, 31.76 pL of oxalyl chloride was added dropwise with a glass syringe, followed by 3 mL of dimethylformamide (DMF) catalyst. The mixture was stirred continuously at 200 rpm and room temperature for 5 hours. The mixture was then added dropwise into the lignin previously dissolved in 25 mL of DMSO in a 3-neck round-bottom flask, under argon gas. The mixture was left at room temperature with stirring at 250 rpm for 48 hours. Afterward, the polymer was precipitated by washing with 40 mL ethyl ether 5 times, and kept overnight in 60 mL DCM (not anhydrous). The suspended polymer was then washed with water to remove unreacted lignin, and the polymer was collected from the bottom phase. The DCM was evaporated with a Rotavapor R-300 (Buchi Corporation, New Castle, DE, USA), and the residual polymer solution was frozen at -80°C, and then freeze-dried (FreeZone Plus 2.5; Labconco, Kansas City, MO, USA) under high vacuum at 300 mbar for 4 days at 30°C.

[0118] Essentially the same two-step acylation reaction was used to link the newly-synthesized polymer to the previously esterified GA3 in a 1 :2 molar ratio. The first step involved activating the OH group of the GA3methyl ester using oxalyl chloride (Reaction 1 ). The second step was the reaction of the acyl chloride from reaction 1 with LN-PLGA (Reaction 2).R OH + CICOCOCI -> R'COCl + CO2+ HCl + COCl2Reaction 1R'COCl + lignin_PLGA -> R'COO_lignin_PLGA Reaction 2(wherein R’ represents gibberellin A3 methyl ester)

[0119] The resulting GA3-LN-PLGA polymer was stored at -20°C until used.CuS and GA3-LN-PLGA nanoparticle synthesis

[0120] The copper sulfide (CuS) nanoparticles were synthesized by the citrate method. Initially, two solutions were prepared, with solution A comprising 10 mmol copper chloride (CUCI2) and 5 mmol sodium citrate, and solution B comprising 10 mmol sodium sulfide. 40 mL aliquots of each of solutions A and B were mixed together (1 :1 v / v), and the mixture was stirred continuously at 300 rpm and room temperature for one hour. The mixture was then placed in a hot oil bath at 90°C for 15 or 30 minutes, and then cooled to room temperature. The resulting CuS nanoparticles were extracted by washing the solution with 150 mL hexane multiple times until extraction was essentially complete. The hexane was removed with a Rotavapor R-300 (Buchi Corporation, New Castle, DE, USA). The solids were then resuspended in ethyl acetate, and allowed to precipitate by gravity overnight to remove impurities.

[0121] The GA3-LN-PLGA NPs were synthesized by an emulsion evaporation technique. Briefly, at room temperature an organic phase was prepared by dissolving 125 mg of LN-PLGA biopolymer in 5 mL of DCM.After complete dissolution, the organic phase was added dropwise into the aqueous phase (70 mL of DI water) with stirring for 15 minutes. The resulting emulsion was homogenized at 30,000 psi with a microfluidizer (Microfluidics, MA, USA) four times. After emulsification, the DCM solvent was evaporated over 2 hours in a Rotavapor R-300 (Buchi Corporation, New Castle, DE, USA). 135 mg of Trehalose were then added into the resulting nanoparticle suspension (at a 1 :1.1 mass ratio) as a cryoprotectant before freezing and lyophilizing the NPs (FreeZone Plus 2.5, Labconco, Kansas City, MO, USA). The dried NPs were stored at -20°C until used.

[0122] The entrapped-CuS GA3-LN-PLGA nanoparticles were synthesized following the same protocol, except that the CuS nanoparticles were added to the organic phase before emulsification.Polymer and nanoparticle characterization

[0123] Gibberellic acid and synthesized polymers dissolved in DMSO were characterized by1HNMR (Bruker 500, Billerica, MA) at 400 Hz. FT-IR spectra were obtained with a spectrometer (Bruker Alpha P, Germany) over the range 4000-400 cm'1.

[0124] 31P NMR was used to quantify OH groups in the LN-PLGA. Briefly, 25 mg of vacuum oven-dried polymer was dissolved in 0.5 mL pyridine / chloroform (1.6:1 v / v) plus 0.5 mL anhydrous DMF (to enhance solubility). After complete dissolution, 0.1 mL of an internal standard solution was added, and the mixture was phosphitylated with 2-chloro- 4,4,5,5-tetramethyl-1 ,3,2-dioxaphospholane (TMDP) before takingspectral readings, using a minimum 128 scans.

[0125] Scanning electron microscope (SEM) images of synthesized polymers were taken with a JSM-6700F (JEOL Ltd, Tokyo, Japan) after coating the polymer with a thin layer of platinum with a sputter coater. Transmission electron microscopy (TEM) images of nanoparticles were acquired with a JEOL JEM 1400 (Jeol USA Inc., MA, USA). Before TEM imaging, the NPs were suspended in DI water at a concentration of 4 mg / mL. A droplet of the sample was mixed with the contrast agent uracil acetate (2%) and placed on a copper grid. Excess was removed with filter paper (through capillary action), and the remaining solution was left to dry for 3 min at room temperature before the grid was placed on the holder.

[0126] The size and the zeta potential of the nanoparticles were obtained with a PMX-430 ZetaView QUATT Laser analyzer (Particle Matrix, Ammersee, Germany). The PDI was obtained with a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK).Results and discussionGA3 esterification

[0127] The carboxy group of GA3 was protected as a methyl ester by reacting the GA3 with methanol to inhibit unwanted side reactions. The COOH proton, which absorbs at 12.59 ppm in1H NMR, disappeared following esterification; and a new peak appeared at 3.67 ppm, characteristic of methyl esters. The C-3 and C-13 OH protons, which absorb at 5.59 ppm and 4.85 ppm for pure GA3, were still seen in the1H NMR spectrum for COOH-protected GA3.

[0128] Protection of the carboxyl group by esterification was further confirmed by FTIR. In pure GA3 the C=O from the carboxylic group appeared as a very strong band at 1738 cm'1, which split into two bands (1768 cm'1and 1729 cm'1) in the COOH-protected GA3. Thus the COOH group was successfully protected at a yield of 66.5%, without altering the C-3 (and C-13) OH groups that are later used for attaching the LN-PLGA polymer.

[0129] For subsequent deprotection of the COOH group, active hydrolysis of GA esters by carboxylesterases occurs in plant cells. Hence, within the plant, in situ re-conjugation of the gibberellin A3 methyl ester is expected to occur after the nanocarrier system has been introduced into a plant, which should eliminate the need for deprotection before the compositions are used in the field.Polymer synthesis and characterization

[0130] The successful grafting of PLGA onto lignin was confirmed by FTIR. The FTIR spectrum displayed bands corresponding to the expected product. The strong vibration of the carbonyl group in PLGA appeared at 1748 cm'1. Vibrations corresponding to the aromatic rings of lignin were observed at 1515-1595 cm'1for the lignin-grafted PLGA polymer (LN- PLGA). The degree of attachment of GA3 to LN-PLGA was based on phosphorus NMR (31P NMR). We thereby estimated totals for the different types of OH groups (aliphatic, phenolic, and carboxylic) in the LN-PLGA (0.4459 mmol / g). The available OH groups were used to estimate the amount of GA3 methyl ester (mole ratio 1 to 2) used in the coupling reaction.

[0131] The FTIR spectrum of GA3-LN-PLGA was insufficient to confirm successful linking of the methyl ester onto the LN-PLGA polymer, probably due to the small total amount of GA3 incorporated.1H NMR was therefore used for this purpose instead. The newly-formed polymer showed characteristic GA3 methyl ester peaks in the NMR spectrum. A peak at 1 .06 ppm corresponded to the hydrogen of the C-18 methyl group of GA3, while a peak at 3.67 ppm corresponded to the methyl ester peak previously formed during the COOH protection step.Nanoparticle synthesis and nano CuS entrapment

[0132] Emulsion evaporation was used to synthesize three sets of nanoparticles: (1 ) NPs were synthesized from the LN-PLGA polymer only; (2) NPs were made from the polymer after grafting with the GA3 methyl ester; and (3) NPs were made from the GAs-grafted polymer, with entrapped nanoscale CuS. The entrapped CuS particles could be seen as small dots spread throughout the polymer nanoparticle, both entrapped and unentrapped. The GA3-LN-PLGA NPs showed a distinct core-shell demarcation, with a core diameter of approximately 60 nm and a shell thickness of approximately 10 nm. The CuS-entrapped GA3-LN-PLGA NPs showed a similar morphology, except for the appearance in the core of dents or what appeared to be patches, in addition to the dot-like structures attributed to the CuS NPs.

[0133] Nanoparticle characterization is summarized in Table 7. The LN-PLGA NPs were in the range of 50 nm, with a PDI of 0.155. After grafting with GA3, the NP size increased by approximately 25 nm; and it increased by about 15 nm when CuS was entrapped.Table 7: DLS characterization of nanoparticles synthesized from 1 :1 w / w LN:PLGA polymerNP type Size PDI ZP pH(nm) (mV)LN-PLGA 52.6 0.155 -62.7 5.5Freshly prepared NPs GA3-LN-PLGA 79.9 0.231 -62.7 5.5CUS+GA3-LN-PLGA 95.8 0.245 -66.2 5.0LN-PLGA 57.3 0.214 -53.7 7.8Resuspended NPs (dried with trehalose) GA3-LN-PLGA 428.2 0.556 -66.1 8.0CUS+GA3-LN-PLGA 96.7 0.268 -60.5 7.4Resuspended NPs (dried LN-PLGA 67.9 0.273 -53.2 8.3 without trehalose) GA3-LN-PLGA 9380.3 0.980 -63.6 8.3CUS+GA3-LN-PLGA 1179.7 0.848 -44.8 8.4

[0134] To confirm that the CuS nanoparticles had localized, both empty and CuS-loaded nanoparticles were embedded in epoxy resin, sectioned, and imaged with high-resolution TEM. The images confirmed the presence of CuS nanoparticles within the hydrophobic core of the polymer nanoparticles.

[0135] To assess the impact of the LN:PLGA ratio on the nanoparticles, nanoparticles were synthesized with varying LN:PLGA polymer ratios, and were characterized before and after freeze-drying. Results are shown in Table 8. The particles were generally small in size, -55-60 nm, at these LN:PLGA ratios; and in general the particles will be larger in size with a higher ratio of PLGA. NPs with entrapped CuS were larger, with diameters~ 65 nm, and those containing GA were slightly larger still, measuring ~ 80 nm. All nanoparticles were larger after freeze drying, but with the addition of trehalose, the resuspended particles measured <100 nm for all formulations.Table 8: Characterization of nanoparticles synthesized from various LN:PLGA ratiosBefore / after Replicate Zetafreeze Type of system and potential drying LN:PLGA ratio (w / w) Size (nm) PDI (mV)1 57.47 0.103 -55.13Before Empty 2:1 LN:PLGA 2 55.06 0.159 -52.173 56.70 0.142 -48.12Avg. 56.41 0.1351 60.30 0.292 -54.98After Empty 2:1 LN:PLGA 2 57.37 0.207 -52.741 57.54 0.175 -55.87Before Empty 1:1 LN:PLGA 2 60.35 0.247 -58.353 58.51 0.194 -51.21Aw, 58.80 0,205 -55.141 59.07 0.154 -62.97After Empty 1:1 LN:PLGA 2 60.69 0.213 -62.273 59.88 0.224 -73.531 65.52 0.210 -58.54Before CuS+l:l LN:PLGA 2 64.64 0.228 -57.773 64.80 0.271 -66.061 95.64 0.230 -65.41After CuS+l:l LN:PLGA 2 94.31 0.244 -67.653 94.97 0.240 -64.411 81.46 0.097 -46.92Before C11S+GA3-LN-PLGA 2 82.64 0.125 -51.213 78.02 0.163 -53.16DEFINITIONS / GENERAL DISCUSSION

[0136] In discussing the present compounds, compositions, and methods, it is to be understood that they are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.

[0137] While aspects of the present invention may be described or claimed in a particular statutory class, such as the statutory class of compositions of matter, this is for convenience only and one of skill in the art will understand that each aspect of the present invention can be described and claimed in any statutory class. Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0138] As used in the specification and the claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearlydictates otherwise; and vice versa. Thus, for example, reference to “a functional group,” “an alkyl,” or “a residue” includes mixtures of two or more such functional groups, alkyls, or residues, and the like; and vice versa. Further, a “functional group” or a “group” may consist of just one atom, or it may contain several atoms.

[0139] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed, and vice versa. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11 , 12, 13, and 14 are also disclosed.

[0140] References in the specification and claims to parts by weight (or mass) of a particular element or component in a composition denotes the weight (or mass) relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weight of component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardlessof whether additional components are contained in the composition.

[0141] A weight (or mass) percent (wt. %) of a component, unless specifically stated to the contrary, is based on the total weight (or mass) of the formulation or composition in which the component is included.

[0142] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance or component may or may not occur or be included, and that the description includes both instances where said event or circumstance or component occurs or is included and instances where it does not.

[0143] As used herein, the term “prevent” or “preventing” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed.

[0144] As used herein, the term “derivative” refers to a compound having a structure derived from the structure of a parent compound (e.g., a compound disclosed herein) and whose structure is sufficiently similar to those disclosed herein that, based upon that similarity, would be expected by one skilled in the art to exhibit the same or similar activities and utilities as the claimed compounds, or to induce, as a precursor, the same or similar activities and utilities as the claimed compounds. Exemplary derivatives include salts, esters, amides, salts of esters or amides, and N- oxides of a parent compound.

[0145] In one aspect, the disclosed compounds comprise the products of the synthetic methods described herein. In a further aspect, the disclosed compounds comprise a compound produced by a synthetic method described herein. In a still further aspect, the invention comprisesa composition comprising an effective amount of the product of the disclosed methods and an acceptable carrier. In a still further aspect, the invention comprises a method for manufacturing a composition comprising combining at least one compound of any of disclosed compounds or at least one product of the disclosed methods with an acceptable carrier or diluent.

[0146] As used herein, the terms “effective amount” and “amount effective” refer to an amount that is sufficient to achieve the desired result or to have an effect on an undesired condition. As used in the specification and claims, an “effective amount” of a composition refers to a quantity of the composition sufficient to be effective to prevent, inhibit, slow the progression, or treat the symptoms of a disease. Depending on context, these measurements and determinations may be statistical in nature, or they may be made in the professional judgment and assessment of an experienced individual.

[0147] The complete disclosures of all references cited in this application are hereby incorporated by reference. Also incorporated by reference are the complete disclosures of the two priority applications, United States provisional application serial number 63 / 660,125, filed 14 June 2024; and United States provisional patent application serial number 63 / 532,694, filed 15 August 2023. In the event of an otherwise irreconcilable conflict, however, the present specification shall control.

[0148] Note that in the interest of brevity, in some instances data and observations are summarized in the present specification that are presented in greater detail in one or both of the two priority applications, where the more detailed data may be viewed for reference; and the complete disclosures of which are hereby incorporated by reference.

[0149] PARTIAL LIST OF ABBREVIATIONSC=O, carbonyl group; CDCI3, deuterated chloroform; CH2, methylene; CL, £-caprolactone; DCM, dichloromethane; DLS, dynamic light scattering; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; DP, degree of polymerization; DSC, differential scanning calorimetry; EE, entrapment efficiency; FDA, Food and Drug Administration; FTIR, Fourier transform infrared spectroscopy; HPLC, high-performance liquid chromatography; IRT, Interborough Rapid Transit; LC, loading capacity; LN, lignin; LN-g-PCL, lignin-grafted-poly(£-caprolactone); LN-g-PCLa, LN- g-PCL by acylation reaction; LN-g-PCLp, LN-g-PCL by ROP; LN-g-PLGA, lignin-grafted-poly(lactic-co-glycolic) acid; LNPs, lignin nanoparticles; MFZ, methoxyfenozide; MRDT, maximum rate of degradation temperature; NMR, nuclear magnetic resonance; NPs, nanoparticles; OH, hydroxyl group; PCL, poly(£-caprolactone); PLGA, poly(lactic-co-glycolic) acid; ROP, ring-opening polymerization; Sn(Oct)2, stannous 2-ethyl hexanoate; Tc, crystallization temperature; TEM, transmission electron microscopy; Tg, glass transition temperature; TGA, thermogravimetric analyses; Tm, melting temperature; TMDP, 2-chloro-4, 4,5,5- tetramethyl-1 ,3,2-dioxaphospholane; Xc(%), crystallinity.

Claims

What is claimed:

1. Nanoparticles comprising a copper sulfide interior bonded to an aliphatic thiol exterior, wherein said nanoparticles have a mean diameter from 5 to 50 nanometers, and wherein said nanoparticles are hydrophobic.

2. The nanoparticles of Claim 1 , wherein said thiol comprises 1 -octadecanethiol.

3. A method for making the nanoparticles of Claim 1 , said method comprising reacting a solution of said thiol with copper sulfide nanoparticles, and evaporating solvent to leave said solid-phase nanoparticles.

4. Core-shell nanoparticles comprising a lignin-graft-polyester; wherein said core predominantly comprises polyester; and wherein said shell predominantly comprises lignin; provided that said polyester is not lignin- graft-poly(lactic-co-glycolic) acid.

5. The nanoparticles of Claim 4, wherein said polyester comprises lignin-graft-poly(£-caprolactone).

6. The nanoparticles of Claim 4, wherein said lignin comprises an alkali lignin or a lignosulfonate.

7. A method for making the nanoparticles of Claim 4, said method comprising forming an aqueous emulsion of said lignin-graft-polyester without added surfactant, and evaporating water from the emulsion to leave solid-phase nanoparticles.

8. A composition comprising an admixture of the nanoparticles of Claim 4 and a payload molecule; wherein said payload molecule associates primarily with said core if said payload molecule is hydrophobic; wherein said payload molecule associates primarily with said shell if said payload molecule is hydrophilic; and wherein said payload molecule associates with both said core and said shell if said payload molecule is amphiphilic.

9. A method for administering a payload molecule to a plant, said method comprising administering to the plant the composition of Claim 8; wherein the payload molecule is taken up by the plant’s cells in substantially greater amounts than would be taken up from an equimolar amount of the payload molecule alone, otherwise administered to the plant in the same manner, but in the absence of the nanoparticles.

10. The composition of Claim 8, wherein the payload molecule is a copper sulfide.

11. The composition of Claim 10, wherein the payload molecules are contained within second nanoparticles that comprise a copper sulfide interior bonded to a thiol outer layer, wherein said second nanoparticles have a mean diameter from 5 to 50 nanometers.

12. The composition of Claim 8, additionally comprising a plant growth regulator covalently bonded to said lignin-graft-polyester.

13. The composition of Claim 12, wherein said plant growth regulator comprises gibberellic acid.

14. A method for administering copper sulfide to a plant, said method comprising administering to the plant the composition of Claim 11 ; wherein the copper sulfide is taken up by the plant’s cells in substantially greater amounts than would be taken up from an equimolar amount of the copper sulfide alone, otherwise administered to the plant in the same manner, but in the absence of the nanoparticles.

15. A method for administering a payload to a plant, said method comprising administering to the plant the composition of Claim 12; wherein the payload is taken up by the plant’s cells in substantially greater amounts than would be taken up from an equimolar amount of the payload alone, otherwise administered to the plant in the same manner, but in the absence of the nanoparticles.

16. The method of Claim 15, wherein said payload comprises a copper sulfide, and wherein said plant growth regulator comprises gibberellic acid.

17. The nanoparticles of Claim 4, wherein the mass ratio of said lignin to said polyester is from 1 : 0.25 to 1 : 6.

18. The nanoparticles of Claim 4, wherein the lignin comprises an alkaline lignin.

19. The nanoparticles of Claim 4, wherein the lignin comprises a lignosulfonate.

20. The nanoparticles of Claim 4, wherein said nanoparticles have a mean diameter from 50 to 300 nanometers.

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