(182ax) From Medicine to Agriculture: Microfluidic-Produced N-Loaded Liposomes as an Enhanced-Efficiency Fertilizer

Nitrogen-based fertilizers have been central to modern agriculture, driving crop productivity and supporting global food security. Nearly half of the world’s population depends on the widespread use of inorganic nitrogen (N) fertilizers [1]. However, this achievement comes at a significant environmental cost. On average, only about 50% of applied nitrogen is taken up by crops, while the remainder is lost to the environment through leaching, volatilization, and runoff [2]. These inefficiencies contribute to water pollution, greenhouse gas emissions, and ecosystem degradation. Excess nitrogen is increasingly recognized as a major threat to aquatic ecosystems and public health [3].
To address these challenges, we draw inspiration from nanomedicine, where nanoscale carriers deliver therapeutic agents with spatial and temporal precision. Translating these principles to agriculture offers a promising strategy to improve nitrogen use efficiency (NUE) by enhancing the precision and responsiveness of fertilizer delivery. Compared to conventional controlled-release and slow-release fertilizers, nanocarrier-based systems may enable more targeted nutrient availability, stronger interaction with plant roots, and responsiveness to environmental cues such as pH and moisture. However, broad adoption in agriculture remains limited by issues of scalability, cost, potential ecotoxicity, and a lack of standardized manufacturing platforms [4].Liposomes—vesicles composed of lipid bilayers—have been widely used in biomedicine for controlled delivery of both hydrophilic and hydrophobic molecules. They are biodegradable, biocompatible, and capable of stimuli-responsive release triggered by factors such as pH, temperature, or light [5], [6]. These features make them attractive candidates for nutrient delivery. Yet their application in agriculture has been constrained by scale and cost [7]. While biomedical systems operate at small volumes with high cost tolerance, agricultural deployment requires cost-effective production at kilogram-to-ton scales. Recent reductions in lipid cost [8], along with advances in continuous microfluidic manufacturing [9], have improved the scalability, reproducibility, and efficiency of liposome production, making them increasingly viable for agricultural applications. In this study, we demonstrate that liposomal formulations encapsulating ionic cargoes can effectively delay nutrient transport through soil, reduce leaching, and enhance nutrient retention. We present a novel platform that adapts clinically validated liposomal encapsulation technologies for delivering potassium nitrate (KNO₃) as a nitrogen fertilizer in soil systems. Using a continuous microfluidic process, we generated KNO₃-loaded liposomes and systematically examined how fabrication parameters influence their physicochemical properties, including size distribution, encapsulation efficiency, and stability. To evaluate agricultural performance, we conducted a controlled plant growth experiment comparing liposomal KNO₃, free KNO₃, and water-only treatments. Results show that liposomal KNO₃ improves soil nitrate retention, modulates microbial respiration, and—importantly—causes no detrimental effects on grass growth, highlighting its safety and suitability for field application. This work represents the first successful adaptation of liposomal encapsulation systems from medicine to agriculture for soil-applied fertilizer delivery. By integrating scalable microfluidic manufacturing with environmentally responsive nutrient carriers, this interdisciplinary approach offers a promising path toward next-generation fertilizers that are both efficient and sustainable.

Methods

Liposome Preparation

Nitrate-loaded liposomes were synthesized using a microfluidic hydrodynamic flow-focusing chip to enable controlled and continuous production. The lipid phase, composed of soybean lecithin and cholesterol dissolved in isopropanol (IPA), was introduced through the central inlet, while the aqueous phase containing potassium nitrate (KNO₃) was pumped through the flanking side inlets. Total flow rates (TFRs) were set at 1200, 2400, and 3600 µL/min, with flow rate ratios (FRRs) between aqueous and organic streams adjusted to 5, 10, 15, and 20. Inside the chip, the three solutions converge at the junction (Fig. 1 inset, optical micrograph), where the aqueous streams sheath the lipid stream. This configuration induces rapid mixing and drives the spontaneous self-assembly of lipid molecules into spherical liposomal nanoparticles, encapsulating nitrate ions from the aqueous phase (see schematic, Fig. 1). The microfluidic format enables reproducible liposome formation with tunable
size and encapsulation efficiency.

Following production, liposome suspensions were immediately dialyzed against ultrapure water to remove residual solvent and unencapsulated KNO₃. Dialysis was carried out over 3 days at 4°C using Spectra/Por® dialysis membranes (MWCO: 12–14 kDa; Fisher Scientific, USA). The dialysate was refreshed twice daily to maintain a strong concentration gradient.

Physicochemical Characterization

The resulting liposomes were characterized for hydrodynamic diameter and zeta potential using dynamic light scattering (DLS) on a Zetasizer Nano instrument. Measurements were performed in triplicate for each formulation to assess size uniformity, stability, and surface charge.

NO₃ content

Liposome suspensions were diluted to 2% (v/v) and lysed with 1% (v/v) Triton X-100 to release encapsulated nitrate. Nitrate-N content was quantified using a colorimetric microwell assay adapted from Doane & Horwáth [10], in which VCl₃ reduces nitrate to nitrite under acidic conditions. The resulting nitrite reacts with sulfanilamide and NEDD to form a pink azo dye, measured at 540 nm absorbance.

Soil Column Transport Study

To evaluate the transport behavior of encapsulated solutes, saturated soil column experiments were conducted using silty clay soil. Liposome-encapsulated bromide and free bromide (as KBr) were applied to separate columns under constant flow. Effluent samples were collected over time and analyzed for bromide content using a colorimetric assay adapted from [11]. Breakthrough curves were compared to assess mobility and retention differences between free and encapsulated forms.

Plant Growth and Treatment Experiment

A greenhouse experiment was conducted using Tall Fescue (Festuca arundinacea) grown in pots filled with silty clay soil. Plants were grown for six weeks under controlled environmental conditions. Treatment groups included: water-only control, free KNO₃, and liposome-encapsulated KNO₃. Liposomal nitrate was applied at equivalent nitrogen levels to the free nitrate group. Plant height was measured weekly, and dry biomass was recorded at harvest after oven-drying samples at 60°C for 72 hours.

References

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