Constructing co-assembly derived smart lipid nanoparticles for enhanced antimicrobial potency with plant growth-promoting potential

Abstract

Lipid nanoparticles (LNP) are widely used in biomedical applications due to their biocompatibility and safety, yet their translation to plant systems remains limited. Here, we establish a unified strategy integrating antibacterial compound discovery, plant-adapted nanocarrier design, and mechanistic elucidation to combat bacterial plant diseases. A series of thymol-derived quaternary ammonium compounds were synthesized, among which C2 was identified as the lead candidate with potent antibacterial activity (EC₅₀ = 0.78 µg/mL). Upon co-assembly into lipid nanoparticles, C2@LNP exhibited pH-responsive release under infection-associated acidic conditions and achieved a high drug-loading efficiency of 82.41%. This plant-tailored LNP system enhances cuticle penetration and foliar retention, as demonstrated by systematic analyses of leaf deposition, droplet behavior, and microscopic adhesion. Consequently, C2@LNP enabled improved disease control at reduced dosages, with protective and curative efficacies 1.94-fold and 1.56-fold higher than those of free C2, respectively. Interestingly, certain lipid components of the nanocarrier, particularly HSPC, exhibited a preliminary plant growth–promoting potential. Mechanistic studies indicated that the enhanced antibacterial performance arises from suppression of quorum sensing, membrane disruption, and induction of apoptosis-like cell death. Overall, this work establishes a sustainable pesticide delivery paradigm for environmentally friendly crop protection.

Graphical abstract

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12951-026-04885-2.

Introduction

Traditional pesticide formulations are increasingly inadequate in addressing critical challenges such as off-target deposition, rapid resistance development, and ecological toxicity. These limitations are further exacerbated by their intrinsic instability, including high susceptibility to photodegradation and microbial decomposition, underscoring the urgent need for advanced delivery platforms [1, 2]. Among emerging alternatives, lipid-based nanocarriers—particularly lipid nanoparticles (LNP, a term uniformly used herein to denote the liposomal vesicle systems constructed in this study)—have attracted growing attention due to their intrinsic biocompatibility, biodegradability, low toxicity, and dynamic structural tunability [3]. The remarkable success of LNP as precision delivery systems in biomedical applications has naturally stimulated interest in their agricultural translation [4–6]. However, this translation is far from straightforward [7]. Fundamental physiological and structural differences between mammalian and plant systems—including the presence of a waxy cuticle, stomatal barriers, and distinct immune responses—constitute major obstacles that limit the direct deployment of biomedical LNP technologies in plants [8–13]. Consequently, effective agricultural application of LNP requires plant-oriented structural re-engineering rather than simple technological transfer, enabling robust foliar adhesion, efficient cuticle penetration, and biocompatibility within plant tissues. Addressing these plant-specific constraints is therefore essential for achieving both specificity and efficacy in next-generation pesticide delivery systems.

The limited efficacy of conventional antibiotics against plant pathogenic bacteria is largely attributed to the formidable outer membrane barrier and the pervasive formation of biofilms. A representative and economically devastating example is Xanthomonas oryzae pv. oryzae (Xoo, PXO^99A), a Gram-negative pathogen that primarily infects gramineous crops, particularly rice [14, 15]. During infection, Xoo secretes extracellular enzymes and virulence-associated proteins that degrade leaf tissues, giving rise to a localized weakly acidic microenvironment (pH 5.5–6.5) [16]. This infection-induced acidity not only enhances bacterial virulence and complicates chemical intervention but also provides a physiologically relevant trigger for pH-responsive delivery systems. Importantly, this microenvironmental feature creates an opportunity for the on-demand and localized release of antibacterial agents from lipid nanoparticles (LNP) directly at infection sites. However, current control strategies still rely heavily on conventional bactericides, such as bismerthiazol and thiodiazole copper, whose extensive use has accelerated resistance development and progressively diminished field efficacy [17]. More alarmingly, these agents are increasingly associated with ecological toxicity and environmental risk. As a result, the number of effective and sustainable bactericidal options for crop protection is rapidly shrinking [18]. Under these constraints, the integration of mechanistically innovative antibacterial agents with stimuli-responsive delivery platforms has emerged as a critical and timely strategy to enhance disease control, delay resistance evolution, and promote environmentally sustainable and user-friendly plant protection solutions [19, 20].

Quaternary ammonium (QA) compounds, characterized by their permanent positive charges, exert antimicrobial activity primarily through electrostatic disruption of negatively charged bacterial membranes [21]. Owing to this mechanism, QA compounds have been extensively applied as disinfectants in medical and industrial settings, underscoring their intrinsic antimicrobial potency [22, 23]. However, their dose-dependent cytotoxicity has substantially restricted broader agricultural application [24]. Notably, the cationic nature of QA compounds renders them inherently compatible with lipid nanoparticle (LNP) systems. Many lipid components used in LNP formulations are cationic or ionizable, enabling stable nanoparticle assembly through electrostatic and non-covalent interactions with QA molecules [25]. Such integration not only attenuates nonspecific toxicity by modulating charge density and release behavior, but also allows the incorporation of stimuli-responsive lipid components to achieve controlled and targeted antibacterial delivery [26]. Recently, various stimuli-responsive LNP have shown promise in other agricultural sectors: thermoresponsive liposomes improve efficacy by 1.4-fold [27], PEG-modification enhances foliar penetration by 38.7% [28], and PEI-functionalization reduces pesticide requirements by 75% [29]. However, while these precedents show promise in managing fungal pathogens or insects, their application against the unique physiological barriers of plant bacterial infections remains relatively fragmented. Therefore, coupling QA molecules with rationally designed and plant-adapted LNP offers a viable strategy to preserve antimicrobial potency while mitigating off-target effects and reducing effective dosages. To further introduce a mechanistically complementary antibacterial dimension, the natural compound thymol was rationally incorporated into the QA framework, enhancing antimicrobial efficacy through antibiofilm activity and enabling synergistic performance against plant bacterial pathogens [30, 31]. Crucially, unlike persistent aliphatic QAs (e.g., DDAC) that face strict regulatory scrutiny due to environmental accumulation, our QA derivative incorporates an environmentally cleavable ester linkage. This design ensures that it can be hydrolyzed into the safe natural product thymol and fatty acids, thereby providing a viable pathway to overcome current regulatory hurdles in agricultural applications.

Building on the physicochemical properties of liposomal nanomaterials, we designed a novel thymol-derived pyridine derivative (C2) featuring an environmentally cleavable ester linkage to reduce intrinsic toxicity. To date, lipid nanoparticles (LNP) have evolved into promising delivery carriers for crop disease control. Numerous research teams have engineered LNP systems for the encapsulation of fungicides, herbicides, insecticides and bactericides toward agricultural applications, among which LNP-stabilized essential oil emulsions have been customized to suppress bacterial leaf blight [32]. Even so, the majority of existing work prioritizes payload loading efficiency and sustained release profiles, whereas far fewer efforts focus on characterizing the interfacial interactions between nanocarriers and plant tissue surfaces. In addition, prevailing agricultural LNP are largely adapted from nanocarrier architectures originally developed for biomedical or industrial scenarios [33–35]. Due to the absence of surface groups for targeted interaction, common liposomes show weak adhesion to the superhydrophobic and microstructured plant cuticles, which easily leads to droplet bouncing. To explicitly distinguish our work from generic LNP delivery systems, we establish a plant-tailored “co-assembly” paradigm that confers three unique advantages. First, moving beyond traditional passive encapsulation, C2 functions dually as a potent biocidal payload and an integral cationic structural unit, overcoming the common bottleneck of low drug loading while ensuring superior colloidal stability. Second, the system is engineered to optimize the plant–nano interface and support plant vigor; the lipid matrix not only improves foliar wetting and retention on the superhydrophobic cuticle but also independently promotes plant growth, providing an ancillary biocompatibility benefit beyond disease control. Finally, the formulation incorporates pH-responsive lipid architectures (pH 5.0–7.4), enabling on-demand release triggered by the localized acidification from high-density Xoo colonization. Together, by integrating rational molecular design with stimulus-responsive co-assembly, this work elevates C2@LNP from a conventional nanocarrier to a smart, highly biocompatible dynamic defense platform (Scheme 1) [36–38].

Scheme 1.

Schematic diagram of the preparation of C2@LNP via liposome–C2 co-assembly for acid-responsive release and enhanced foliar adhesion, leading to improved disease control and rice growth, along with the dual antibacterial mechanism of C2 against Xoo

Experimental section

Instruments and chemicals

Nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS) data were acquired using a Bruker Biospin-AG-400 spectrometer, JEOL ECX-500 spectrometer, and Thermo Scientific Q Exactive system, respectively. Morphological analysis and energy-dispersive X-ray spectroscopy (EDS) were performed with an FEI Nova NanoSEM 450 scanning electron microscope (SEM) and a HITACHI HT7800 transmission electron microscope (TEM) equipped with an Oxford X-MaxN 80T detector. X-ray photoelectron spectroscopy (XPS) was conducted on a Thermo Scientific ESCALAB Xi+ spectrometer. Fluorescence imaging and apoptosis analysis were carried out on an Olympus BX53 microscope and a BD FACSCalibur flow cytometer, respectively. Optical density (OD) was measured with a Cytation^TM5 multi-mode reader. Dynamic light scattering (DLS) and zeta potential analyses were performed on a Nano Brook Omni analyzer. Drug release kinetics were determined by an Agilent 1260 Infinity II HPLC system. Fourier-transform infrared (FT-IR) spectroscopy and Raman spectroscopy were recorded on a Thermo Nicolet iS50 FT-IR spectrometer and a Horiba HR800 confocal Raman microscope, respectively. UV-Vis absorption spectra were acquired with a Shimadzu UV-2600i spectrophotometer. Lipid nanoparticle (LNP) assembly was conducted using an IKA RV 10 rotary evaporator, and surface wettability was analyzed with a JC-2000D contact angle meter.

Thymol, pyridinium quaternary ammonium derivatives, lipids (HSPC, CHEMS, PE), pH-responsive acids (oleic acid, azelaic acid), and solvents were purchased from Bide Pharmatech and Energy Chemical. Dialysis bags (MWCO 3.5 kDa), phosphate-buffered saline (PBS), and trifluoroacetic acid (TFA) were sourced from Shanghai Yuanye Bio-Technology Co., Ltd. Staining reagents, biochemical assay kits (AO/EB, ROS, amylase/cellulase), and other chemicals were obtained from Shanghai Xinweier Glass Instrument Co., Ltd, Beijing Solarbio Science & Technology Co., Ltd, Tianjin Zhiyuan Chemical Reagent Co., Ltd, and Energy Chemical. All chemicals were of analytical grade.

Synthesis of target compounds

The synthesis of the target compounds (A1–C4) proceeded via a general multi-step protocol. First, 4-hydroxypyridine was alkylated with ethyl 6-bromohexanoate in the presence of KOH in MeCN under reflux to afford intermediate 1 (general route for intermediates 5–8). Subsequent hydrolysis of the ester group in 1 using NaOH in H₂O/MeCN yielded the carboxylic acid intermediate 2 (general route for intermediates 9–12). Intermediate 2 was then coupled with thymol via EDCI/DMAP-mediated esterification to provide intermediate 3 (general route for intermediates 4, 13–16). Finally, a quaternization reaction between intermediate 3 and various substituted benzyl derivatives in MeCN furnished the target pyridinium compounds. All intermediates and final products were purified by standard column chromatography. Detailed synthetic procedures and characterization data (¹H NMR, ¹³C NMR, ¹⁹F NMR, HRMS) for all compounds are provided in the Supporting Information.

Preparation and Characterization of Liposomes (C2@LNP)

Liposomes were formulated using a lipid film hydration method, adapted from established protocols for pH-responsive nanocarriers [11]. Briefly, hydrogenated soy phosphatidylcholine (HSPC, 6 mg), cholesteryl hemisuccinate (CHEMS, 2 mg), a pH-responsive component (2 mg; e.g., oleic acid, azelaic acid), and compound C2 (1 mg) were dissolved in 10 mL ethanol. The solvent was removed by rotary evaporation at 40 °C to form a thin lipid film, which was hydrated with 10 mL ultrapure water at 55 °C for 45 min to obtain C2-loaded liposomes (C2@LNP). Blank LNP were prepared similarly without C2. The hydrodynamic diameter, polydispersity index (PDI), and zeta potential of the liposomes were measured in triplicate at 25 °C using a Nano Brook Omni analyzer. Formulations meeting the criteria of PDI ≤ 0.3 and |zeta potential| ≥ 30 mV were selected for further study to ensure colloidal stability. Encapsulation efficiency (EE) and drug loading (DL) were determined by a dialysis-HPLC method [9]. Purified liposomes were lyophilized, redissolved in methanol, and analyzed by HPLC. EE and DL were calculated as: EE (%) = (Mass of encapsulated drug / Initial drug input) × 100%; DL (%) = (Mass of encapsulated drug / Total mass of lyophilized liposomes) × 100%. UV-Vis absorption spectra of C2@LNP were recorded against a blank LNP reference.

Antibacterial activity assay

The antibacterial activity against Xoo was evaluated using turbidimetric and agar plate assays, following standard methods for bactericide evaluation [23, 29]. For the turbidimetric assay, compound C2 or C2@LNP (in a series of concentrations) was mixed with nutrient broth (NB) and a log-phase Xoo suspension (OD₅₉₅ ≈ 0.6). After incubation at 28 °C with shaking (180 rpm) for 24–48 h, bacterial growth inhibition was assessed by measuring OD₅₉₅. The half-maximal effective concentration (EC₅₀) was calculated via Probit analysis. For the agar plate assay, compound solutions were mixed with melted nutrient agar (NA), poured into plates, and inoculated with a diluted bacterial suspension. After 4 days of incubation at 28 °C, colony formation inhibition was observed.

(XPS, FT-IR, Raman, TEM, EDS)

The molecular interactions, composition, and morphology of the liposomes were characterized using X-ray photoelectron spectroscopy (XPS) [39, 40], Fourier-transform infrared (FT-IR) spectroscopy, Raman spectroscopy [41, 42], transmission electron microscopy (TEM) [12], and energy-dispersive X-ray spectroscopy (EDS) [31], following established analytical protocols for nanocarrier systems. For XPS analysis, lyophilized liposome powder was directly analyzed on a Thermo Scientific ESCALAB Xi+ spectrometer. Survey and high-resolution spectra were acquired, with charge correction referenced to the C 1s peak at 284.8 eV. For FT-IR spectroscopy, samples were prepared as KBr pellets and spectra were recorded in transmission mode (4000 –400 cm^{− 1}). Raman spectra were acquired on a Horiba HR800 confocal microscope using a 532 nm laser. For TEM and EDS analysis, a diluted liposome suspension was deposited onto carbon-coated copper grids, negatively stained with 2% phosphotungstic acid, and imaged using a Hitachi HT7800 TEM at 120 kV. Concurrent EDS analysis was performed on selected regions using an Oxford X-MaxN 80T detector to determine elemental distribution.

pH-Responsive drug release

The pH-dependent release profile of C2 from C2@LNP was investigated using a dialysis method [11]. Liposomes dispersed in phosphate-buffered saline (PBS) at different pH values (7.4, 7.0, 6.0, 5.0) were placed in dialysis bags (MWCO 1 kDa) and immersed in the corresponding release media at 37 °C under gentle agitation. The released drug in the dialysate was collected at predetermined intervals, concentrated, and quantified using an Agilent 1260 Infinity II HPLC system equipped with a UV-Vis detector. Chromatographic separation was achieved on an Agilent ZORBAX Eclipse Plus C18 column (4.6 × 250 mm, 5 μm) maintained at an operating temperature of 25 °C. The mobile phase consisted of acetonitrile (Solvent A) and methanol (Solvent B). Elution was performed under isocratic conditions at a volume ratio of 90:10 (A: B) at a constant flow rate of 1.0 mL/min. The detection wavelength was set at 274 nm. For quantitative analysis, a calibration curve (R² > 0.999) was established using accurately weighed free C2 standards dissolved in the mobile phase across a concentration range of 1 to 100 µg/mL.

Hypersensitivity response (HR) ASSAY

The ability of C2@LNP to induce a hypersensitivity response (HR) in Nicotiana benthamiana was assessed to evaluate its potential to trigger plant defense, adapting methods used for pathogen-plant interaction studies [7]. Leaves of five-leaf stage plants were infiltrated with mixtures containing Xoo suspension and test solutions (C2@LNP, C2, or controls). After 24 h incubation at 28 °C, necrotic lesion areas were measured under natural light, and tissue autofluorescence was examined under UV excitation (365 nm) to assess HR intensity.

In vivo bioactivity against Xoo in rice

The protective and therapeutic efficacy of C2@LNP against Xoo was evaluated in rice using established methodologies described in the literature [19, 26]. For therapeutic tests, rice leaves were inoculated with Xoo (OD₅₉₅ = 0.6) by clipping, then sprayed with C2@LNP, C2, or commercial bactericides (BT: bismerthiazol; TC: thiadiazole copper). For protective tests, leaves were sprayed prior to inoculation. Plants were maintained at 28 °C and 90% relative humidity. Disease severity was assessed 14 days post-inoculation by calculating the disease index (DI) and control efficiency, based on a standardized grading scale for lesion area.

Foliar wettability and retention analysis

The adhesion and retention properties of C2@LNP on rice leaves were evaluated by measuring contact angle, rebound height, surface tension, and sliding angle, using methodologies similar to those for assessing agrochemical formulations [43, 44]. Droplets (5 µL) of C2@LNP, C2, LNP, or water were placed on rice leaf surfaces. Static contact angles were measured with a goniometer. Droplet rebound behavior was recorded with a high-speed camera. Surface tension was determined via the pendant drop method. The sliding angle, defined as the minimum tilt angle causing droplet movement, was measured on pre-treated leaves to compare formulation adhesion.

Molecular dynamics (MD) simulation

MD simulations were performed to investigate the co-assembly mechanism of C2@LNP, following computational approaches used for lipid-based systems [30]. The initial structures of HSPC, CHEMS, PE, and C2 were optimized. A simulation system was built with a molar ratio of HSPC: CHEMS: PE: C2 = 6:2.7:2.7:1 in a water box. Simulations were run for 100 ns using GROMACS-2022.2 under the CHARMM36 force field. Trajectories were analyzed for root-mean-square deviation (RMSD), solvent-accessible surface area (SASA), radius of gyration (Rg), and intermolecular interactions (hydrogen bonds, electrostatic forces).

Antibacterial mechanism studies

A series of assays were conducted to elucidate the antibacterial mechanism of C2 and C2@LNP against Xoo, adapting established protocols [14, 23, 40]. Biofilm Inhibition (Crystal Violet Assay): Xoo was treated with C2 at sub-inhibitory concentrations (0.5×EC₅₀ to 10×EC₅₀) for 72 h. Biofilm biomass was stained with crystal violet, dissolved in ethanol, and quantified by measuring OD₅₇₀. Membrane Integrity Assays: Extracellular conductivity was monitored over time after treating Xoo pellets with C2. Membrane permeability was assessed using the N-phenyl-1-naphthylamine (NPN) uptake assay for outer membrane damage and the o-nitrophenyl-β-D-galactopyranoside (ONPG) hydrolysis assay for inner membrane integrity. Apoptosis and Cell Death Analysis: Xoo cells treated with C2, C2@LNP, or controls were stained with acridine orange/ethidium bromide (AO/EB) and observed under a fluorescence microscope to differentiate live, apoptotic, and dead cells. Apoptosis was further quantified by flow cytometry using Annexin V-FITC/PI staining. Bacterial Motility and Extracellular Enzyme Activity: Swimming motility was assessed on low-agar plates containing C2. Extracellular amylase and cellulase activities were evaluated on specific indicator plates supplemented with C2, with hydrolysis zones visualized after staining. Transcriptomic Analysis: Xoo was treated with C2 (1×EC₅₀ and 5×EC₅₀) for 15 h. Total RNA was extracted, sequenced, and analyzed to identify differentially expressed genes and pathways.

Toxicity and growth promotion evaluation

Rice phytotoxicity and growth promotion were evaluated by foliar-spraying rice plants at the tillering stage with C2@LNP, C2, LNP, and individual lipid components (HSPC, CHEMS, PE), each applied at a concentration consistent with the in vivo antibacterial dosage (100 µg/mL), every 3 days for three consecutive cycles. Leaf yellowing was observed for phytotoxicity. Plant height, leaf length and biomass (fresh/dry weight) were measured 14 days post-treatment to assess growth promotion effects. Zebrafish were exposed to C2, LNP or C2@LNP at various concentrations for 96 h to evaluate acute aquatic toxicity, with mortality and toxic symptoms recorded periodically. The pharmacokinetic and toxicity profiles of compound C2 were predicted using the ADMETlab 2.0 platform to assess potential human health and environmental risks.

Statistical analysis

All in vitro and in planta experiments were performed with at least three independent biological replicates, and each biological replicate included three technical replicates to ensure measurement accuracy. Data are presented as mean ± standard deviation (SD). Statistical significance was determined using one-way analysis of variance (ANOVA) followed by Duncan’s post hoc test (p < 0.05) in SPSS 25.0. Graphs were generated using Origin 2021 and GraphPad Prism. The more detailed experimental procedures can be found in the Supporting Information.

Results and discussion

Synthesis of liposome-mimetic structures and antibacterial evaluation

All compounds were fully characterized by NMR and HRMS (Fig. S1–S121) (Scheme 2). Antibacterial activity against Xoo was evaluated using a turbidimetric assay (Table 1; Tables S1–S2). Among the tested compounds, pronounced structure–activity relationships were observed. Incorporation of the quaternary ammonium framework combined with thymol resulted in a dramatic enhancement of antibacterial potency, as exemplified by A4 (EC₅₀ = 0.84 µg/mL) compared with the non-thymol analogue B1 (EC₅₀ = 57.08 µg/mL), highlighting the critical contribution of thymol-mediated antibiofilm and membrane-disruptive effects. Substitution at the pyridinium nitrogen with a benzyl group proved superior to heteroaryl or extended aromatic substituents, with A4 (0.84 µg/mL) outperforming A1 (2.15 µg/mL), A2 (13.80 µg/mL), and A3 (2.82 µg/mL). Building on our previous research, halogen substitution has been shown to exert an effect on antimicrobial activity. To systematically investigate the impact of halogen atoms on bactericidal efficacy, we synthesized and evaluated compounds A4 and A5. Their antibacterial activities exhibited no significant difference, with EC₅₀ values of 0.81 µg/mL for A4 and 0.84 µg/mL for A5. Furthermore, compound A4 was selected for further investigation owing to its readily available raw materials and straightforward synthetic route. Additional substitution on the benzyl ring generally reduced antibacterial activity, as illustrated by A14 (p-methylbenzyl, 1.79 µg/mL), suggesting that steric or electronic perturbations may compromise optimal membrane interactions. Furthermore, alkyl chain length exerted a decisive influence on activity, with the octyl-substituted derivative C2 exhibiting the highest potency (EC₅₀ = 0.78 µg/mL) among the C-series (C1–C4). Collectively, these results identify C2 as the most balanced and potent candidate for subsequent incorporation into lipid nanoparticle (LNP) delivery systems.

Scheme 2.

Synthetic route of the target compounds

Table 1.

The EC₅₀ values of target compounds against Xoo

Compound	EC50 (µg/mL)	Regression equation	Compound	EC50 (µg/mL)	Regression equation
1	61.95 ± 1.54	y = 1.4167x + 2.4612	B1	57.08 ± 1.84	y = 0.9889x + 3.263
2	67.33 ± 0.84	y = 1.7054x + 1.8822	B2	2.53 ± 0.08	y = 1.7687x + 4.2872
3	2.74 ± 0.16	y = 2.092x + 4.0828	5	67.36 ± 2.28	y = 1.4321x + 2.3815
A1	2.15 ± 0.24	y = 2.6539x + 4.1178	6	65.53 ± 1.65	y = 1.1337x + 2.9407
A2	13.80 ± 0.35	y = 1.996x + 2.7247	7	75.62 ± 1.83	y = 1.081x + 2.9692
A3	2.82 ± 0.17	y = 3.0703x + 3.6175	8	68.45 ± 0.92	y = 1.2727x + 2.6641
A4	0.81 ± 0.04	y = 1.5507x + 5.1393	9	84.15 ± 2.81	y = 1.4063x + 2.2928
A5	0.84 ± 0.06	y = 1.5563x + 5.1151	10	62.74 ± 1.18	y = 1.1544x + 2.9249
A6	0.86 ± 0.05	y = 1.6718x + 5.1073	11	79.65 ± 1.84	y = 1.007x + 3.0855
A7	0.95 ± 0.11	y = 2.2731x + 5.0486	12	64.41 ± 1.45	y = 1.3938x + 2.4787
A8	0.96 ± 0.06	y = 2.5111x + 5.0399	13	4.85 ± 0.18	y = 1.2597x + 4.1361
A9	0.98 ± 0.07	y = 2.3233x + 5.0233	14	2.38 ± 0.24	y = 1.6368x + 4.3822
A10	1.16 ± 0.11	y = 2.9014x + 4.8076	15	3.31 ± 0.11	y = 1.4294x + 4.2578
A11	1.29 ± 0.09	y = 1.9309x + 4.786	16	3.71 ± 0.13	y = 1.4494x + 4.1751
A12	3.56 ± 1.51	y = 1.7572x + 4.0318	C1	0.86 ± 0.25	y = 1.5787x + 5.1008
A13	3.11 ± 0.16	y = 2.2074x + 3.9114	C2	0.78 ± 0.02	y = 1.4702x + 5.1612
A14	1.79 ± 0.21	y = 1.6828x + 4.5764	C3	1.11 ± 0.08	y = 1.306x + 4.9422
A15	1.21 ± 0.19	y = 2.5126x + 4.7883	C4	1.05 ± 0.10	y = 1.6614x + 4.9676
A16	11.29 ± 0.54	y = 2.6498x + 2.2101	BT	36.05 ± 0.47	y = 1.7037x + 2.3475
A17	6.68 ± 0.37	y = 1.9439x + 3.3966	TC	88.79 ± 1.91	y = 0.9972x + 3.0571
4	6.21 ± 0.18	y = 1.4283x + 3.8672

^a)Average of three replicates

Co-assembly and characterization of C2@LNP

To translate the optimized antibacterial molecule C2 into a plant-compatible nanocarrier system, a facile thin-film hydration method was employed to construct lipid nanoparticles (LNP) via co-assembly [38]. Hydrogenated soybean phosphatidylcholine (HSPC), cholesteryl hemisuccinate (CHEMS), functional lipids, and C2 were co-dissolved in ethanol, followed by rotary evaporation and hydration to yield C2-loaded LNP (C2@LNP) (Fig. S122A). Functional lipid components—including oleic acid (OA), lauric acid (LA), stearic acid (SA), azelaic acid (AA), decanoic acid (DA), and phosphatidylethanolamine (PE)—were systematically introduced to modulate membrane properties and pH responsiveness [45, 46]. The pH-responsive behavior of C2@LNP was attributed to the acid-induced structural rearrangement of CHEMS and functional lipids, which facilitates payload release under the weakly acidic microenvironment characteristic of Xoo infection sites [39]. Based on preliminary screening, a mass ratio of HSPC: CHEMS: functional lipids: C2 = 6:2:2:1 was selected as the initial formulation framework. Six pH-responsive LNP variants (OA-, LA-, SA-, AA-, DA-, and PE-based) were subsequently prepared and comprehensively characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential analysis, encapsulation efficiency (EE), drug loading (DL), and UV–vis spectroscopy (Fig. S122B–S122E). Among these formulations, PE-based LNP exhibited the most balanced physicochemical performance, including favorable particle size, surface charge, and loading capacity, and was therefore selected for further compositional optimization. Fine-tuning the relative proportions of PE, CHEMS, and HSPC yielded an optimized formulation with a mass ratio of HSPC: CHEMS: PE: C2 = 6:2.7:2.7:1, corresponding to a hydrodynamic diameter of 611.24 nm, a zeta potential of 65.28 mV, an encapsulation efficiency of 82.41%, and a drug loading of 6.34% (Fig. S123A–S123C). Macroscopically, free C2 appeared as a clear yellow solution, whereas blank LNP formed a non-uniform milky suspension; simple mixing of the two resulted in an inhomogeneous dispersion. In contrast, hydration-driven co-assembly led to a homogeneous dispersion with disappearance of the characteristic yellow color and the emergence of a pronounced Tyndall effect (Fig. 1A and B), indicating successful nanoparticle formation. At the molecular level, XPS analysis revealed a near-complete disappearance of Cl 2p signals and an increase in the N⁺:NH₂ ratio from 1:1 in LNP to 2:1 in C2@LNP, accompanied by a shift in the C = O: C–O: COOH ratio from 3:2:1 to 6:3:1 (Fig. 1C and Fig. S124). Furthermore, elemental mapping by EDS confirmed a bilayer liposomal structure, with Cl⁻ signals derived from C2 localized within the membrane despite its low abundance (Fig. 1D). Collectively, these results provide converging evidence for successful co-assembly and rational optimization of C2@LNP, ensuring that the nanocarrier architecture is directly built upon the molecular efficacy of C2.

Fig. 1.

Co-assembly and Characterization of C2@LNP. A) Schematic illustration of the co-assembly process of the lipid-like compound C2. B) Appearance characteristics and Tyndall effect of the individual components of C2, LNP, unhydrated C2@LNP and hydrated C2@LNP. (C) XPS spectrum of the co-assembled liposome C2@LNP. (D) EDS spectrum of the co-assembled liposome C2@LNP

Co-assembly mechanism and antibacterial evaluation of C2@LNP

To elucidate the molecular basis underlying the co-assembly of C2@LNP, we first performed 100 ns molecular dynamics (MD) simulations (Fig. 2A). The trajectories revealed a pronounced increase in hydrogen-bond density between CHEMS and HSPC (Fig. S125A), accompanied by enhanced electrostatic interactions between C2 and HSPC (Fig. S125B), indicating preferential and cooperative binding during nanoparticle formation. Root mean square deviation (RMSD) and solvent-accessible surface area (SASA) analyses further showed a rapid decrease in system energy within 10–45 ns (Fig. S125C–S125E), ultimately yielding a compact and stable nanostructure (Fig. 2B) [47]. These computational insights were corroborated by spectroscopic analyses. In the infrared (IR) spectrum of C2@LNP, the carbonyl stretching bands originating from HSPC (1733 cm⁻¹), CHEMS (1707 cm⁻¹), and PE (1741 cm⁻¹) converged into a merged peak centered at 1733 cm⁻¹. Concurrently, the phosphate vibrations of HSPC and PE (P = O and P–O at 1251 and 1033 cm⁻¹) shifted to 1178 and 1017 cm⁻¹, respectively (Fig. 2C), reflecting altered intermolecular interactions. Broadening of the N–H stretching band (3298 cm⁻¹), together with red-shifts of C2-associated carbonyl, ether, and pyridinium vibrations and a slight blue-shift of the C = N/C = C band, collectively indicated encapsulation driven by hydrogen bonding and electrostatic coordination. Raman spectroscopy further supported this assembly model, showing reduced P–O–C vibrational intensity and enhanced C–H stretching signals, consistent with tighter alkyl-chain packing within the lipid bilayer (Fig. 2D). Based on these combined results, a plausible co-assembly mechanism is proposed (Fig. 2E). Initially, C2 electrostatically associates with HSPC and PE, followed by hydrogen-bond-mediated coupling of CHEMS with HSPC to form basic structural units. This cooperative interaction promotes inward ordering of hydrophobic alkyl chains while exposing hydrophilic domains to the aqueous environment. Subsequently, the rigid moieties of CHEMS and C2 jointly modulate membrane fluidity and reinforce the structural integrity of the LNP. After confirming successful encapsulation, the antibacterial activity of C2@LNP and its individual components against Xoo was evaluated using a turbidimetric assay, with all formulations normalized to the same C2 content. As summarized in Table 2 and Table S3, C2@LNP exhibited an EC₅₀ of 3.85 µg/mL, representing an apparent reduction in potency relative to free C2 (EC₅₀ = 0.78 µg/mL). This outcome underscores the necessity of considering release kinetics and microenvironment-responsive behavior when evaluating nanoformulated antibacterial agents, thereby motivating further investigation of the pH-triggered release and in situ antibacterial performance of C2@LNP under biologically relevant conditions.

Fig. 2.

The co-assembling mechanism of C2@LNP. (A) Molecular dynamics simulation of the co-assembly process of C2@LNP. (B) Molecular dynamics simulation study on the co-assembly binding mechanism of C2@LNP. (C) FTIR spectra of C2@LNP and its components. (D) Raman spectra of C2@LNP and its components. (E) Illustration of the binding mechanism of the co-assembly of C2@LNP

Table 2.

EC₅₀ values of target compounds against Xoo. ^a)

Compound	EC50 (µg/mL)	Regression equation	Compound	EC50 (µg/mL)	Regression equation
C2	0.78 ± 0.02	y = 1.9632x + 5.2097	PE	239.69 ± 2.48	y = 0.9933x + 2.6363
C2@LNP	3.85 ± 0.15	y = 2.2987x + 3.6545	Thymol	18.75 ± 0.84	y = 2.1369x + 2.2797
LNP	286.83 ± 3.25	y = 0.9545x + 2.6542	BT	35.53 ± 0.47	y = 1.6821x + 2.3917
HSPC	278.27 ± 3.81	y = 0.7112x + 3.2615	TC	87.58 ± 1.91	y = 1.8508x + 1.4050
CHEMS	281.70 ± 3.54	y = 0.952x + 2.6678

^a)Average of three replicates

pH-responsiveness of C2@LNP

To investigate the pH-responsive release behavior of C2@LNP under mildly acidic conditions relevant to bacterial infection, samples were incubated at different pH values. At neutral pH, C2@LNP dispersions remained uniformly white and stable, whereas samples adjusted to pH 6.0 and 5.0 rapidly turned yellow and turbid, indicating acid-triggered disruption of the nanostructure and release of encapsulated C2 (Fig. 3A). Quantitative release profiles further confirmed this pH-dependent behavior (Fig. 3B). C2@LNP exhibited relatively controlled release at neutral pH (7.0 and 7.4), releasing only ~ 55% of C2 over 7 days. In contrast, mildly acidic conditions markedly accelerated release, with the cumulative release at pH 5.0 exceeding that at pH 6.0 by approximately 10% within 24 h. A broad pH range of 5.0–7.4 was systematically evaluated to ensure the wide applicability and robustness of the delivery system under various field conditions. Results indicate that high-density Xoo proliferation leads to a progressive decline in the environmental pH from 7.04 to 5.59 (Fig. S126), providing a clear biological signal for triggered cargo release. In cases of severe late-stage infection, localized acidification can be further exacerbated toward pH 5.0 due to host tissue disintegration, ensuring that LNP maintains a robust triggered-release capacity throughout the entire infection cycle. Considering the acid-producing nature of Xoo, we hypothesized that C2@LNP would undergo microenvironment-triggered structural reorganization upon localization at infection sites, thereby restoring antibacterial activity in situ. To test this hypothesis, growth-curve assays were conducted using C2@LNP at concentrations normalized to C2 content (Fig. 3C). At low doses (0.5×EC₅₀ to 2×EC₅₀), Xoo growth was progressively suppressed in a dose-dependent manner, while exposure to 5×EC₅₀ resulted in pronounced inhibition of bacterial proliferation. To further evaluate antibacterial performance under solid-phase conditions that more closely mimic localized infection environments, an agar-plate assay was performed at 5×EC₅₀ (Fig. 3D). While LNP and individual lipid components (HSPC, CHEMS, and PE) exhibited minimal inhibition (< 10%), C2@LNP achieved an inhibition rate of 85.31%, closely approaching that of free C2 (87.32%). These findings indicate that the weakly acidic microenvironment generated by Xoo on solid media substantially enhances the release of C2 from C2@LNP, enabling its antibacterial efficacy to approximate that of the free compound. The hypersensitive response assay further corroborated effective antibacterial performance under practical illumination conditions (Fig. 3E). Under both natural daylight and 365 nm UV irradiation, C2@LNP and free C2 induced near-complete healing of necrotic lesions, whereas control treatments showed no significant lesion recovery. Finally, transmission electron microscopy (TEM) directly visualized the acid-triggered structural evolution of C2@LNP (Fig. 3F). Intact spherical liposomes with well-defined bilayers were observed at pH 7.4 and 7.0, whereas partial bilayer disruption occurred at pH 6.0 and complete rupture was evident at pH 5.0. Together, these results provide direct evidence that C2@LNP undergoes weak-acid-triggered disassembly, thereby enabling controlled release of C2 under infection-relevant conditions without compromising its intrinsic antibacterial potency.

Fig. 3.

The pH response and antibacterial effect of C2@LNP. (A) Solution states and Tyndall effects of C2@LNP after hydration self-assembly at different pH conditions. C2 + LNP represents the state before hydration and C2@LNP represents the state after hydration self-assembly. (B) Release profile of C2@LNP after 7 days at different pH conditions. (C) Growth curve of C2@LNP against Xoo. (D) Agar plate assay to evaluate the antibacterial activity differences of LNP, C2, and C2@LNP at five times the EC₅₀ concentration of C2. (E) Hypersensitive response assay of C2, LNP, and C2@LNP against Xoo. (F) SEM images of C2@LNP released at different pH conditions. Data in B) and C) are presented as mean ± SD (n = 3 independent biological replicates)

Excellent deposition performance of C2@LNP on rice

To evaluate the plant–nano interface behavior of C2@LNP, its deposition and retention performance on rice leaves were systematically investigated. Rice leaves present a highly hydrophobic and micro-/nanostructured surface, posing a major barrier to effective agrochemical delivery. Contact angle measurements revealed that free C2 exhibited limited wettability on rice leaves (123.4°), comparable to that of deionized water (124.4°) (Fig. 4A). In contrast, C2@LNP showed a markedly reduced contact angle of 67.7°, outperforming both blank LNP (78.6°) and a reported cyclodextrin-based formulation (FT6@β-CD, 92.5°) [40]. Sliding angle analysis further demonstrated enhanced leaf retention, with C2@LNP droplets exhibiting significantly shorter sliding distances than water, free C2, and LNP formulations (Fig. 4B–C). Surface tension measurements provided quantitative insight into this improved interfacial behavior (Fig. 4D). C2@LNP displayed a surface tension of 51.35 mN/m, substantially lower than that of water (73.34 mN/m), free C2 (70.74 mN/m), and LNP (54.47 mN/m), corresponding to reductions of 42.8% and 6.1% relative to water and LNP, respectively. This reduction is attributed to the amphiphilic nature of the C2 pyridinium cation, which preferentially orients at the air–water interface and disrupts the hydrogen-bonding network via charge–dipole interactions, thereby lowering interfacial energy. Consistent with these measurements, spraying experiments on rice leaves revealed that C2@LNP formed smaller, more uniformly distributed droplets with higher surface coverage, whereas water and free C2 produced larger and aggregated droplets (Fig. 4E). High-speed imaging further showed that water and free C2 droplets underwent pronounced rebound upon impact, while LNP-based formulations effectively suppressed splashing (Fig. 4F). Notably, C2@LNP reached a maximum rebound height approximately half that of water at 40 ms and achieved complete leaf retention within 60 ms. Scanning electron microscopy (SEM) provided direct morphological evidence for enhanced foliar adhesion (Fig. 4G). C2@LNP was observed to anchor within the micro-/nanostructured papillae and epicuticular wax layer of rice leaves, whereas free C2 formed large, uneven precipitates due to its limited water compatibility. In contrast, our co-assembled C2@LNP platform showed excellent adaptability to rice leaf surfaces. Lipids with complementary carboxyl and amino groups (CHEMS and PE) interact with the cationic pyridinium moiety of C2, enabling the nanoplatform to form a multivalent interaction network with foliar waxes. This Velcro-like adhesion within leaf micro/nanostructures effectively inhibits droplet bouncing and improves particle deposition, outperforming conventional liposomes. Collectively, these results demonstrate that rational LNP engineering markedly improves wetting, retention, and foliar deposition on rice leaves, thereby establishing a favorable plant–nano interface for efficient agrochemical delivery.

Fig. 4.

The wetting, bouncing, retention, and surface deposition performance of H₂O, C2, LNP and C2@LNP on rice leaves. (A) Static contact angles. (B) High-speed camera images taken from a height of 14.0 cm and inclined at 45°, showing vertical changes corresponding to the initial stage (D₀), the maximum sliding distance stage (D_max), and the final stage of the droplet (Final). (C) The related statistical maximum sliding distance (D_max) of droplets from the above sliding experiment (n = 3, ANOVA; Tukey HSD; p < 0.05). (D) Surface tensions. (E) The practical simulation of spraying on rice leaves. (F) High-speed camera images of bouncing behaviors of their corresponding droplets (diameter: 2.0 mm) from a height of 14.0 cm. (G) SEM images of deposition behaviors on the rice leaf surfaces. Data in C) and D) are presented as mean ± SD (n = 3 independent biological replicates)

In vivo antibacterial assay

To evaluate whether the enhanced pH responsiveness and optimized plant–nano interface of C2@LNP translate into improved disease control under realistic conditions, in vivo antibacterial assays were conducted using a potted rice bacterial leaf blight model [41]. Rice plants were treated at a dosage of 100 µg/mL, and disease severity was assessed following inoculation with Xoo (Fig. 5). In preventive treatments, C2@LNP achieved a protective efficiency of 65.42%, significantly outperforming free C2 (33.65%), BT (26.17%), and TC (16.82%), corresponding to 1.94-, 2.49-, and 3.89-fold improvements, respectively. In curative assays, C2@LNP similarly exhibited superior performance, affording a curative efficiency of 67.82%, exceeding that of free C2 (43.48%), BT (33.04%), and TC (17.39%) by 1.56-, 2.05-, and 3.84-fold, respectively. While the superior in vivo efficacy of C2@LNP over commercial bactericides is evident at this uniform concentration, this comparison should be interpreted within the proper context. Commercial products like BT and TC are industry standards that are typically applied at significantly higher concentrations in field settings to compensate for severe foliar run-off associated with conventional formulations. The experimental results reveal that the ability of C2@LNP to surpass these agents at 100 µg/mL is primarily driven by the explicit contribution of the liposomal delivery system in enhancing interfacial affinity and enabling microenvironment-responsive release. Importantly, the inclusion of free C2 as a control effectively decouples intrinsic molecular toxicity from formulation benefits. Although C2@LNP displayed lower apparent potency in static in vitro assays, its superior in vivo performance highlights the role of the nanocarrier in maximizing drug bioavailability on superhydrophobic rice leaves. Specifically, this markedly enhanced efficacy is rationalized by the synergistic contributions of pH-triggered release and optimized foliar deposition. The pH-triggered disassembly of C2@LNP at infection sites, combined with reduced contact angle and surface tension, ensures superior leaf retention and prolonged pathogen exposure. Compared to previously reported lipid-based nanocarriers or conventional QA formulations that often suffer from premature leakage and poor adhesion [36–38], C2@LNP explicitly overcomes these bottlenecks through interfacial engineering and spatiotemporal responsive release. Thus, rather than claiming absolute molecular superiority, this comparison demonstrates how the rational integration of the liposomal system enables robust disease control at chemical dosages significantly lower than those used in conventional applications. From a translational perspective, the practical viability of this platform is supported by the high synthetic yield of the active ingredient C2 (84.37%) and its potential compatibility with scalable production techniques like ethanol injection. While the initial formulation cost is higher than that of traditional pesticides, the C2@LNP platform offers significant differentiated competitive advantages. Specifically, the drastically reduced field application dosage (100 µg/mL) and superior biological efficacy minimize total chemical input. Furthermore, the potential plant growth–promoting effect provides additional value to growers by enhancing crop yield and quality. These combined benefits effectively offset the formulation costs, establishing a high-value, competitive crop-protection paradigm for large-scale rice cultivation.

Fig. 5.

Curative and protective efficacy of C2, C2@LNP, LNP, BT, and TC (100 µg/mL) against Xoo infection. Significant differences (Tukey’s HSD test, p < 0.05) are indicated by different lowercase letters (a, b, c, d). Data are presented as mean ± SD (n = 3 independent biological replicates)

Safety assessment of C2@LNP in rice and zebrafish, and evaluation of plant biocompatibility

To assess the biosafety of the C2@LNP system and its suitability for practical agricultural application, a series of toxicity and plant growth assays were conducted. Although quaternary ammonium compounds are often associated with dose-dependent toxicity, their integration into lipid nanoparticle (LNP) systems is expected to mitigate nonspecific biological effects. The intrinsic toxicity of C2 was first evaluated using in silico ADMET prediction (Fig. 6A), which suggested a low risk toward representative human cell models and environmental receptors. Phytotoxicity was then examined on rice plants at a concentration of 100 µg/mL (Fig. 6B and Fig. S127). While free C2 did not significantly affect overall plant height, it induced mild leaf-tip yellowing (approximately 5–10 cm), a phenomenon that was more pronounced in plants treated with commercial bactericides BT and TC. Notably, this phytotoxic symptom was almost completely eliminated upon liposomal encapsulation, demonstrating the effective shielding effect of the LNP matrix. Aquatic toxicity was further evaluated using a zebrafish model (Fig. 6C and Fig. S128). At an exposure concentration of 10 µg/mL, free C2 resulted in one observed mortality, whereas no deaths occurred in the C2@LNP-treated group at an equivalent C2 dose, corresponding to 100% survival. Moreover, unlike DDAC, which shows high aquatic toxicity with a 96 h LC₅₀ below 1 mg/L toward zebrafish (Danio rerio) [43, 44, 48], our results confirm the low intrinsic toxicity of C2 (LC₅₀ > 10 µg/mL) and demonstrate that our delivery system further enhances biocompatibility. Beyond acute toxicity, the regulatory viability of quaternary ammonium (QA) compounds strongly depends on their environmental persistence. The favorable ADMET properties of C2 stem from its hydrolyzable ester group, which allows the molecule to be readily degraded by plant esterases and soil microorganisms into non-toxic thymol and fatty acids in agricultural environments, potentially mitigating long-term residue issues associated with persistent QAs such as DDAC. Beyond biosafety assessment, we systematically quantified how lipid compositions regulate rice seedling growth via a 14-day cultivation assay, covering free C2 and fully formulated C2@LNP (Fig. 6D). Among all tested lipids, hydrogenated soybean phosphatidylcholine (HSPC) generated the noticeable growth-stimulating effect: compared with the untreated control, HSPC treatment elevated plant height, leaf length, fresh weight and dry weight by 18.2%, 40.9%, 38.6% and 35.3%, respectively. Blank LNP induced moderate growth promotion, while free C2 showed no detectable growth-boosting activity. The assembled C2@LNP displayed growth-promoting effects equivalent to blank LNP yet slightly inferior to HSPC. Given the limited biological replicates used in this cultivation test, we treat this result only as preliminary evidence of latent growth-promoting potential, rather than a research conclusion. In our follow-up work, we will further dissect the growth-regulating activity of HSPC and explore its broader ecological impacts, including interactions with soil microbiomes, effects on non-target plant species, and environmental degradation profiles.

Fig. 6.

Biological safety and growth-promoting assessment of C2@LNP. A) ADMET toxicity prediction of C2. B) Toxicity of C2@LNP and its components toward rice growth. C) Zebrafish toxicity assays of C2, LNP, and C2@LNP. D) Promoting effects of the components of liposomes on the growth of rice. Data in D) are presented as mean ± SD (n = 3 independent biological replicates)

Transcriptomics analysis

To elucidate the antibacterial mechanism underlying the activity of C2 against Xoo, transcriptomic analysis was performed on the PXO^99A strain following 10 h exposure to low (1×EC₅₀) and high (5×EC₅₀) concentrations of C2. A total of 4,258 and 4,246 genes were detected under low- and high-dose treatments, respectively (Fig. 7A and Fig. S129A), among which 544 and 611 genes were identified as differentially expressed (DEGs) (Fig. 7B and Fig. S129B). KEGG pathway enrichment analysis revealed distinct concentration-dependent regulatory profiles (Fig. 7C and Fig. S129C). At the lower concentration, C2 predominantly downregulated genes associated with two-component signaling systems and biofilm formation (Fig. 7D), pathways that are critical for environmental sensing, quorum regulation, and host colonization. Because two-component systems serve as the primary sensory receptors for quorum sensing autoinducers, their suppression at the transcriptional level provides a direct molecular explanation for the inhibition of bacterial group behaviors. In contrast, high-concentration treatment primarily suppressed genes involved in fructose and mannose metabolism, starch and sucrose metabolism, and the phosphotransferase system (PTS), indicating severe disruption of carbon utilization and membrane-associated transport processes (Fig. 8A). The significant downregulation of these membrane-bound transport proteins (e.g., PTS) aligns closely with physical damage to the lipid bilayer, which compromises the functional integrity of membrane protein complexes. Notably, ribosomal pathway genes were consistently upregulated under both treatment conditions, suggesting a common stress response characterized by elevated protein synthesis in response to membrane perturbation. At the gene level, low-dose C2 treatment resulted in significant downregulation of PXO_RS07330 (TssD, biofilm formation; 2.5-fold) and PXO_RS23615 (alkaline phosphatase D, two-component system; 8-fold) (Fig. 7E–F). In contrast, high-dose exposure caused marked suppression of PXO_RS18335 (TssD, carbohydrate metabolism; 8.67-fold) and PXO_RS18535 (membrane transport protein; 5.22-fold) (Fig. 8B–C). Collectively, these transcriptomic data support a concentration-dependent, multi-level antibacterial mechanism of C2. At lower concentrations, C2 primarily attenuates bacterial virulence by disrupting signal transduction and biofilm formation, thereby impairing colonization and quorum-associated behaviors. At higher concentrations, C2 exerts direct bactericidal effects by collapsing membrane transport and central carbon metabolism, ultimately destabilizing intracellular homeostasis and precipitating the metabolic crisis that triggers apoptosis-like cell death.

Fig. 7.

Transcriptomic analysis and experimental validation of the antibacterial mechanism of Xoo following treatment with C2 at a concentration of 1×EC₅₀. (A) Venn diagram comparing the transcriptomes of the C2-treated group and the control group. (B) Volcano plot depicting differentially expressed genes between the C2-treated and control groups. (C) Bubble plot from KEGG pathway enrichment analysis highlighting significantly enriched pathways in the C2-treated group compared to the control group. (D) Specific upregulated and downregulated pathways identified among the significantly enriched KEGG pathways between the C2-treated and control groups. (E) GSEA enrichment analysis of the biofilm formation pathway, along with the corresponding heatmap of associated genes. (F) GSEA analysis of the two-component system pathway and the corresponding heatmap of related genes. (G) Crystal violet staining assay assessing the effect of C2 on Xoo biofilm formation. (H) Bar chart illustrating the impact of C2 treatment on the swimming motility of Xoo. (I) Bar graph showing the inhibitory effect of C2 on starch enzyme activity in Xoo. (J) Bar chart depicting the inhibition of cellulase activity in Xoo following C2 treatment. K) Bar chart demonstrating the effect of varying C2 concentrations on Xoo motility. L) Bar chart showing suppression of extracellular amylase activity in Xoo upon treatment with C2 at different concentrations. M) Bar chart illustrating inhibition of extracellular cellulase activity in Xoo after exposure to different concentrations of C2. Data in G), H), I), and J) are presented as mean ± SD (n = 3 independent biological replicates)

Fig. 8.

Transcriptomic analysis and experimental validation of the antibacterial mechanism of Xoo (PXO^99A) following treatment with C2 at a concentration of 5×EC₅₀. (A) Specific upregulated and downregulated pathways identified among the significantly enriched KEGG pathways between the C2-treated and control groups. (B) GSEA enrichment analysis of the carbohydrate metabolism pathway, along with the corresponding heatmap of associated genes. (C) GSEA enrichment analysis of the transporter pathway and the corresponding heatmap of related genes. (D) Measurement of extracellular membrane conductivity in Xoo following treatment with varying concentrations of C2. (E) ONPG assay assessing changes in inner membrane permeability of Xoo after exposure to different concentrations of C2. (F) NPN assay evaluating alterations in outer membrane permeability of Xoo upon C2 treatment. (G) AO/EB dual-staining assay of Xoo treated with C2 at the EC₅₀ concentration. (H) Apoptosis analysis of Xoo treated with C2 (1×EC₅₀) for 1 h, 2 h, and 4 h. (I) SEM images of Xoo exposed to C2 at concentrations of 1×EC₅₀ and 5×EC₅₀. (J) Schematic diagram illustrating the proposed antibacterial mechanism of C2 against Xoo. Data in D), E), and F) are presented as mean ± SD (n = 3 independent biological replicates)

Experimental verification of the C2 antibacterial mechanism

To experimentally validate the antibacterial mechanism of C2 predicted by transcriptomic analysis, a series of assays related to quorum sensing, virulence behavior, and membrane integrity were conducted [49]. At sublethal concentrations, C2 markedly impaired virulence-associated phenotypes of Xoo. Crystal violet staining revealed a pronounced inhibition of biofilm formation, with OD₅₇₀ values decreasing from 1.5 in the control group to 0.2 upon C2 treatment (Fig. 7G). Motility assays further demonstrated dose-dependent suppression of bacterial movement, with swimming diameters reduced from 23.9 mm (control) to 15.2 mm and 8.9 mm at 1×EC₅₀ and 2×EC₅₀, respectively (Fig. 7H and K), indicating compromised colonization and dissemination [50]. These phenotypic attenuations perfectly correlate with the transcriptomic downregulation of two-component systems and biofilm formation pathways (e.g., TssD), which orchestrate these quorum-sensing-dependent traits. Consistent with quorum sensing disruption, C2 significantly suppressed extracellular enzyme secretion, as evidenced by reduced hydrolytic zones in amylase and cellulase assays (Fig. 7I–J and L–M) [51]. At higher concentrations, C2 exerted direct bactericidal effects through membrane disruption. Electrolyte leakage measurements revealed a substantial increase in extracellular conductivity to 165.3 µS/cm at 5×EC₅₀, approaching that of the boiled positive control (192.7 µS/cm), indicating severe loss of membrane integrity (Fig. 8D). ONPG and NPN uptake assays further confirmed enhanced permeability of the inner and outer membranes, with 2.0-fold and 14.7-fold increases, respectively, relative to the control (Fig. 8E–F). This physical membrane damage corroborated the transcriptomic finding that membrane-associated transport processes were severely impaired, as the breached lipid bilayer can no longer support transmembrane protein functions. These results are consistent with the proposed electrostatic disruption of lipid packing by quaternary ammonium cations [52]. Cellular fate analysis provided further insight into the downstream consequences of membrane damage. Acridine orange/ethidium bromide (AO/EB) staining revealed a progression from viable cells in the control group to mixed viable and apoptotic populations at 1×EC₅₀, and exclusively non-viable cells at 5×EC₅₀ (Fig. 8G and Fig. S130). Annexin V-FITC/PI flow cytometry confirmed a time-dependent apoptotic response, with the proportion of apoptotic cells increasing from 59.8% after 1 h to 80.0% after 8 h of C2 exposure (Fig. 8H and Fig. S131). The rapid induction of this apoptosis-like state serves as the terminal consequence of the severe metabolic collapse and massive membrane failure identified in the high-dose transcriptomic profile. Scanning electron microscopy further visualized pronounced membrane shrinkage and structural collapse at 5×EC₅₀ (Fig. 8I). Collectively, these results experimentally substantiate the transcriptomic predictions and establish a concentration-dependent antibacterial mechanism of C2. At lower concentrations, C2 attenuates Xoo virulence by disrupting quorum sensing, motility, and extracellular enzyme secretion. At higher concentrations, it compromises membrane integrity, perturbs osmotic balance and metabolism, and induces apoptosis-like cell death. This mechanistic validation completes a unified framework linking rational compound design, lipid nanoparticle engineering, and antibacterial function (Fig. 8J) [53].

Conclusion

Liposomes have been widely validated as safe and effective carriers in biomedical drug delivery, yet their translation into agricultural applications, particularly for the management of recalcitrant plant bacterial diseases, remains largely underexplored. In this work, we present a systematic strategy that integrates antibacterial compound discovery, lipid nanocarrier engineering, and mechanistic elucidation to address rice bacterial blight. Through rational molecular design, we identified a thymol-derived pyridine candidate C2, featuring an environmentally cleavable ester linkage to mitigate intrinsic toxicity. C2 manifests a concentration-dependent dual mechanism, characterized by virulence suppression at low doses and membrane-disruptive bactericidal action at higher concentrations. To explicitly distinguish this work from generic delivery systems, we established a plant-tailored “co-assembly” paradigm. By integrating C2 into a pH-responsive lipid nanoparticle (C2@LNP), we successfully optimized the plant–nano interface, markedly enhancing foliar wetting, retention, and cuticle penetration. This smart platform not only enables on-demand cargo release triggered by Xoo-induced acidification (pH 5.0–7.4) but also achieves substantially enhanced in vivo protective and curative efficacies at reduced dosages. Notably, specific lipid components of the delivery platform, particularly HSPC, exhibited a preliminary plant growth-promoting effect. Overall, this study constructs a plant-compatible LNP system integrating molecular innovation and interface engineering to address the persistent hurdles of low bioavailability and insufficient effective bactericides. Benefiting from the high synthetic yield of compound C2 (84.37%) and its dual antibacterial mechanism involving quorum sensing disruption and the induction of apoptosis-like cell death, the as-constructed LNP features scalable preparation, pH-responsive behavior, and preliminary plant growth-promoting potential, to advance sustainable agricultural management.

Author contributions

Jia-Rui Rao: Conceptualization, Investigation, Formal analysis, Writing – original draft. Lei Liu: Writing – review & editing. Zhi-Jun Luo: Investigation. Pan Xu: Investigation. Chao-Yu Zhao: Investigation. Tai-Hong Zhang: Investigation. Jin-Hong Hu: Investigation. Li-Wei Liu: Investigation. Zhi-Bing Wu: Conceptualization, Project administration, Supervision. Xiang Zhou: Investigation, Supervision, Writing – review & editing. Song Yang: Conceptualization, Supervision, Project administration, Resources, Writing – review & editing. Funding acquisition.

Data availability

Data will be made available on request.

Declarations

Consent for publication

All authors consent for publication.

Competing interests

The authors declare no competing interests.