Recipient Organization
LOUISIANA STATE UNIVERSITY
202 HIMES HALL
BATON ROUGE,LA 70803-0100
Performing Department
Biological & Agricultural Engineering
Non Technical Summary
Our goal is to develop lignin- based biomaterials to be used in agriculture. The biodegradable films developed herein are envisioned to replace synthetic plastic mulch films used to cover the soil and increase the productivity of a crop by avoiding the loss of water, reducing soil erosion, and preventing growth of weeds near the crop. The ability of the amphiphilic film and lignin nanoparticles to co-deliver agrochemicals to the plants can prove beneficial. A lower dose of agrochemicals (pesticides or fertilizer) will be necessary by creating a more focused delivery to thesite of action and avoiding leaching and loss due to water washing. Biodegradable methods of delivery will reduce the number of applications necessary by controlling the release profile of nutrients and pesticides over time, decreasing the environmental impact of the agrochemical in addition to replacing currently used, petroleum-based, nondegradable films.
Animal Health Component
80%
Research Effort Categories
Basic
10%
Applied
80%
Developmental
10%
Goals / Objectives
The goal of the project is to develop biodegradable materials based on lignin in the form of films to aid crop productivity by preventing soil erosion, retaining soil moisture, and preventing weed growth, and nanoparticles designed for delivery of nutrients and agrochemicals. Amphiphilic polymers will be made from lignin (LGN) grafted with PLGA (LGN-g-PLGA) and the graft polymers will be used to develop biodegradable films and nanoparticles of controlled properties for agricultural applications. The following objectives are proposed:Objective 1. Chemically modify lignin to form novel amphiphilic polymer: Form amphiphilic lignin-based biopolymer by grafting a hydrophobic biodegradable synthetic polyester on a hydrophilic natural polymer (lignin) from different sources. PLGA will be covalently linked to lignin at different LGN:PLGA ratios in a two-step acylation reaction to form the LGN-g-PLGA amphiphilic polymer. LGN-g-PLGA will be characterized by H-NMR and FTIR.Objective 2. Form biodegradable lignin films and nanoparticles of controlled properties: Synthesize amphiphilic, biodegradable, and multifunctional film (LGN FMs) and core-shell lignin nanoparticle (LGN NPs) for agrochemical delivery. LGN NPs of controlled physical characteristics will be synthesized by an emulsion evaporation method and will be characterized by measuring the morphology, size, size distribution, zeta potential, and hydrophobicity using DLS, TEM, spectrophotometry, and water contact angle. LGN FMs will be made by a solution casting method (with and without plasticizers). Mechanical properties, thermal properties, surface properties, hydrophobicity, moisture absorption, and UV absorbance of the films will be measured.Objective 3.Evaluate films and NPs as potential biodegradable mulch films or film coatings, and environmentally responsive agrochemical delivery systems:Develop prototypes of films and nanoparticles deemed most suitable for application in agriculture based on their properties. Assess enzymatic and hydrolytic degradation of the biomaterials at temperatures and pHs relevant to agricultural applications, using HPLC and spectrophotometric methods.Load films and nanoparticles with agrochemicals (methoxyfenozide, adiacyl hydrazine hydrophobic insecticide and zeatin, a cytokinin) and measure time-release as a function of temperature and pH by HPLC.
Project Methods
Objective 1. Chemically modify lignin to form novel amphiphilic polymerSynthesis of ALGN-g-PLGA biopolymer: The coupling of lignin to PLGA will be based on acylation reaction at two different LGN:PLGA ratios 1:2 and 1:4 (w/w) (Scheme 1). Briefly, for the 1:4 LGN:PLGA w/w, an amount of 2 g of PLGA will be dissolved in 30 ml of dichloromethane (DCM) at room temperature in a 3 neck round bottom flask. A nitrogen flow is connected to a bubbler bottle with 1 M NaOH to neutralize HCl produced during the reaction. After complete dissolution of PLGA at room temperature, 5 eq. of oxalyl chloride will be added dropwise with a glass syringe. The reaction will be performed at room temperature with mild stirring for 4 hours and the solution will be concentrated with a rotavapor Buchi R-300 (Buchi Corporation, New Castle, DE). Next, the polymer will be precipitated with addition of 150 ml of ethyl ether, and the white precipitate will be washed at least three times to remove impurities. The solids will be dried overnight under high vacuum. Next, the second reaction will be performed with dissolution of 1 g of dry PLGA-Cl in 20 ml of DMSO, which it is added dropwise to 500 mg of LGN dissolved in 20 ml of DMSO. The reaction will be performed overnight at room temperature and nitrogen flow. The LGN-g-PLGA polymer will be precipitated with addition of 200 ml of ethyl ether. The precipitated polymer suspended in 20 ml of DCM will be washed with water to remove unreacted lignin obtaining a clear supernatant. Finally, the DCM will be evaporated with a rotavapor Buchi R-300, and the polymer will be dried under high vacuum for 3 days at 30°C. The LGN-g-PLGA polymer will be stored at 2-4°C for further characterization and use in nanoparticle and film formation.Biopolymer characterization: The LGN-g-PLGA conjugates will be characterized by 1H-NMR, (Bruker 500 MHz system) at 500 MHz in dimethyl sulfoxide (DMSO) and FT-IR Bruker Tensor 27 (Bruker, Billerica, MA).Objective 2. Form biodegradable nanoparticles and lignin films of controlled propertiesSynthesis of LGN-g-PLGA nanoparticles with entrapped agrochemicals: The polymeric nanoparticles will be synthesized by the emulsion evaporation technique with important modifications. No surfactants will be added in the aqueous phase and no purification steps will be needed. Briefly, around 500 mg of PLGA-lignin will be dissolved in 8 ml of ethyl acetate at room temperature under strong stirring. Around 50 mg of methoxyfenozide will be added and mixed for 15 minutes for complete dissolution. Next, the organic phase will be added to the aqueous phase (80 ml of DI water). After 10 minutes of mixing, the suspension will be homogenized with a microfluidizer (Microfluidics Corp.) at 30,000 psi four times at 4°C. Also, sonication can be used for small volumes. Next, the organic solvent will be evaporated in a Buchi rotavapor R-300 at 32°C under vacuum for at least 45 min. Finally, trehalose will be added (1 to 1 mass ratio) as cryoprotectant, and the samples will be placed in a freeze drier Labconco FreeZone 2.5 (Labconco Corporation, Kansas City, MO) for 2 days at -80°C to remove water. Finally, the LGN-g-PLGA NPs will be stored at -20°C before characterization.Nanoparticle characterization: Size, polydispersity and zeta potential of the nanoparticles will be measured by dynamic light scattering (DLS) by using a Malvern zetasizer ZS (Malvern Panalytical, Westborough, MA). Briefly, 1 ml of the resuspended polymeric nanoparticles sample in low resistivity water at concentrations of 0.2-0.4 mg/ml will be placed in a cuvette or zeta potential cell and analyzed at 25°C. The nanoparticle morphology will be analyzed by Transmission Electron Microscopy (TEM) Jeol JEM-1400. One droplet of the resuspended polymeric nanoparticles in high resistivity water is placed over a carbon copper grid with a contrast agent, the excess sample removed, and the sample dried for 10 minutes before placing the grid on the TEM chamber.Film formation: Film formation will be accomplished by the solution casting technique with modifications.36 The LGN-g-PLGA will be dissolved in DMSO at a concentration of (2.0%) (w/v) at 60°C. The solution will be mildly stirred for 3 h and sonicated 20min. After solubilization, the plasticizer will be added at the concentrations (0%), (5%), (10%) and (20%) and stirred for 24h. The solution will be poured in polyester plates and stored under vacuum at 0.5 atm for an additional 24 h. Finally, the resulting film will be stored in a desiccator at room temperature for 7 days to remove the remaining solvent.Film Characterization: Film thickness will be measured using a micrometer (Mitutoyo Digimatic Micrometer 293 MDC-MX Lite). Five measurements will be taken at different places and the mean will be calculated. The hydrophobicity of the film on both sides (lignin-hydrophilic and PLGA-hydrophobic) will be measured by water contact angle measurement by the wetting process. Thermal properties of the films will be measured using DSC, TGA, and TMA. Scanning electron microscopy (SEM) will be used to visualize the morphology and texture of the films with different LGN:PLGA ratios and types of lignin. The mechanical properties of the film will be measured to observe the effects of LGN:PLGA ratios and the addition of plasticizes on the tensile strength, elongation at break, penetration, and Young's modulus. A texture analyzer will be used to analyze the samples following the standard method ASTM D882-12. The test will be repeated five times with film strips previously prepared and conditioned at 75% relative humidity by placing the samples in a desiccator with a saturated saline solution of NaCL for 7 days [94].37 Moisture absorption of film will be measured by standard methods.38 The UV absorbance of the film will be measured by Ultraviolet-visible spectroscopy using a transmittance wavelength of 200-800nm using a cleaned quartz slide as a reference.26Objective 3. Evaluate films and nanoparticles as potential biodegradable mulch films or film coatings, and environmentally responsive agrochemical delivery systemsThose films and nanoparticles deemed most suitable for application in agriculture based on physical-chemical properties measured under Objective 2 will be prototyped. The degradation and release kinetics will be measured from prototypes exposed to environmental conditions specific to agriculture applications.Agrochemical loading and release: Films and nanoparticles will be loaded with agrochemicals, methoxyfenozide, a diacyl hydrazine hydrophobic insecticide, and zeatin, a cytokinin. Time-release will be measured as a function of temperature (25, 35, 45°C) and pH (5 and 7). It is estimated that by tuning the Tg and hydrophobicity of the films and nanoparticles, their release response can be calibrated to respond to environmental stimuli (temperature, water availability, pH changes). The paddle over disk method will be used to measure release kinetics of the film.39 Agrochemical release from the NPs will be assessed by the dialysis method. High performance liquid chromatography analysis of the samples will be performed to measure released agrochemical content.40,41The materials will be optimized by comparing them against commercially available materials commonly used in similar applications.Nanoparticle and film degradation: Enzymatic and hydrolytic degradation of the nanoparticles and films will be measured as a function of temperature and pH, using spectrophotometric methods. A slightly modified method by Xie et al.42 will be utilized to depolymerize LGN-g-PLGA NPs and FMs. LGN-PLGA degradation will be induced by the enzyme laccase and quantified by the Prussian Blue Assay,42 and the hydrolytic degradation of the PLGA moities will be evaluated by GPC and mass lost over time.43