Water-soluble Fullerene Derivatives Research Articles

(Invited) Highly Water-Soluble Fullerene Derivatives for Durable Polymer Electrolyte Membrane Fuel Cells

Recently, the focus of proton-exchange membrane fuel cell (PEMFC) applications has shifted from fuel cell vehicles (FCVs) to heavy-duty vehicles (HDVs), necessitating enhanced fuel cell durability. The degradation of carbon-supported metal nanoparticle catalysts and electrolyte membranes are the primary factors responsible for fuel cell durability. Particularly in the context of HDVs, augmenting the chemical stability of electrolyte membranes, such as Nafion, stands as a crucial factor in improving fuel cell lifetime. The degradation mechanism of Nafion is believed to involve the cleavage of polymer chains by OH∙ radicals, formed by crossover hydrogen and crossover oxygen in the presence of trace transition metal ions. A common strategy to mitigate Nafion's chemical degradation involves adding radical quenchers like cerium ion (Ce+) to Nafion. However, Ce+ may migrate out of Nafion during fuel cell operation, leading to a decline in its highly efficient radical quenching ability over extended operational periods.In this study, we demonstrate that a fullerene-derived radical quencher with multiple hydroxy groups significantly contributes to enhancing the durability of Nafion. Our approach involves synthesizing fullerene derivatives with various kind of hydroxy and functional groups for remarkable water solubility to realize their homogeneous dispersion within the Nafion membrane. The abundant functional groups also aid in improving proton conductivity. Moreover, the chelating effect of the spherically distributed functional groups immobilizes themselves, preventing any observed leakage from the Nafion membrane. These fullerene-derivative radical quenchers effectively enhance fuel cell durability and hold promise for future applications in HDVs.This study was supported by the New Energy and Industrial Technology Development Organization (NEDO), Japan (JPNP20003).

Metabolite responses of cucumber on copper toxicity in presence of fullerene C60 derivatives

Copper (Cu) toxicity in crops is a result of excessive release of Cu into environment. Little is known about mitigation of Cu toxicity through the application of carbon-based nanomaterials including water-soluble fullerene C60 derivatives. Two derivatives of fullerene were examined: polyhydroxylated C60 (fullerenol) and arginine C60 derivative. In order to study the response of Cu-stressed plants (Cucumis sativus L.) to these nanomaterials, metabolomics analysis by gas chromatography-mass spectrometry (GC-MS) was performed. Excess Cu (15 μM) caused substantial increase in xylem sap Cu, retarded dry biomass and leaf chlorosis of hydroponically grown cucumber. In Cu-stressed leaves, metabolomes was disturbed towards suppression metabolism of nitrogen (N) compounds and activation metabolism of hexoses. Also, upregulation of some metabolites involving in antioxidant defense system, such as ascorbic acid, tocopherol and ferulic acid, was occurred in Cu-stressed leaves. Hydroponically added fullerene adducts decreased the xylem sap Cu and alleviated Cu toxicity with effectiveness has been most pronounced for arginine C60 derivative. Metabolic responses of plants subjected to high Cu with fullerene derivatives were opposite to that observed under Cu alone. Fatty acids up-regulation (linolenic acid) and antioxidant molecules (tocopherol) down-regulation might indicate that arginine C60 adduct can alleviate Cu induced oxidative stress. Although fullerenol slightly improved cucumber growth, its effect on metabolic state of Cu-stressed plants was not statistically significant. We suggest that tested fullerene C60 adducts have a potential to prevent Cu toxicity in plants through a mechanism associated with their capability to restrict xylem transport of Cu from roots to shoot, and to maintain antioxidative properties of plants.

Rewiring Photosynthesis by Water-Soluble Fullerene Derivatives for Solar-Powered Electricity Generation.

Natural photosynthesis holds great potential to generate clean electricity from solar energy. In order to utilize this process for power generation, it is necessary to rewire photosynthetic electron transport chains (PETCs) of living photosynthetic organisms to redirect more electron flux toward an extracellular electrode. In this study, a semi-artificial rewiring strategy, which use a water-soluble fullerene derivative to capture electrons from PETCs and donate them for electrical current generation, is proposed. A positively charged fullerene derivative, functionalized with N,N-dimethyl pyrrolidinium iodide, is found to be efficiently taken up by the cyanobacterium Synechocystis sp. PCC 6803. The distribution of this fullerene derivative near the thylakoid membrane, as well as site-specific inhibitor assays and transient absorption spectroscopy, suggest that it can directly interact with the redox centers in the PETCs, particularly the acceptor side of photosystem I (PSI). The internalized fullerene derivatives facilitate the extraction of photosynthetic electrons and significantly enhance the photocurrent density of Synechocystis by approximately tenfold. This work opens up new possibility for the application of fullerenes as an excellent 3D electron carrier in living biophotovoltaics.

Protective action of water-soluble fullerene adducts on the example of an adduct with l-arginine

We present radioprotective, antiglycating, and photoprotective properties of a water-soluble C60 fullerene derivative with l-arginine (C60-Arg) and composite films based on collagen containing C60-Arg. The synthesis of these materials is described. The identification of the synthesised materials was carried out using modern physicochemical methods of analysis. The physicochemical properties of aqueous solutions of C60-Arg, such as, particle size distribution, zeta potentials, distribution coefficient in the octan-1-ol–water system were measured. The computer simulation of the process of C60-Arg association in aqueous and isotonic solutions was carried out using Molecular Dynamics. Composite films based on collagen containing C60-Arg demonstrate significant improvement in mechanical properties, cell adhesion, and cell proliferation when the nano-modifier is added. This shows high potential for the use of the C60-Arg adduct in biomedicine.

Protective role of fullerenol and arginine C60 fullerene against copper toxicity in cucumber

Copper (Cu), when in excess, is one of the most toxic and hazardous metals to all living organisms, including plants. Engineered nanomaterials have the potential for increasing crop protection. However, the protective role of fullerenes (carbon-based nanoparticles with wide application in various areas) against Cu toxicity in plants is, so far, understudied. The present study investigated whether fullerenes can potentially alleviate Cu toxicity in plants (Cucumis sativus L.). Two water-soluble fullerene C60 derivatives were examined: fullerenol [C60(OH)22–24] and arginine-functionalized fullerene [C60(C6H13N4O2)8H8], under controlled conditions using hydroponics. Plants treated with 15 μM of Cu exhibited typical symptoms of Cu toxicity: impaired growth, leaf chlorosis, reduced photosynthetic activity, nutritional imbalances, and enhanced lipid peroxidation. These symptoms were alleviated in the presence of fullerene derivatives with arginine C60 having the more pronounced effect. Improved cucumber Cu tolerance was attributable to Cu buffering in the root zone (roots and medium), which caused a dramatic decline in Cu transport towards leaves and the elimination of oxidative damage. The Cu removal efficacy of arginine C60 was much greater than that of fullerenol. These fullerenes acted in a dose-dependent manner and removed Cu selectively without significant modification of the bioavailability of other essential nutrients. Treatment with free arginine did not affect Cu immobilization or Cu toxicity. These results suggest that the surface chemistry of the fullerene core is important for the protection of plants under excessive Cu conditions. The information offered a new approach to preparing promising practical materials for alleviating Cu toxicity in plants with potential application in fields.

Facile preparation of fullerenol-based humidity sensor with highly fast response

As a typical representative of water-soluble fullerene derivatives, fullerenols have been widely used in biomedicine, adsorption, and sensor detection. This paper has studied the structure and composition of commercial fullerenols, which indicate abundant hydrophilic groups exist on the surface of carbon cages to promote the adsorption of water molecules. It is found that the humidity sensor coated with fullerenols has a fast response (19s)/recovery time (12s) and low hysteresis (ΔH ≈ 2.68%RH) at the relative humidity level of 11–97%. The change mechanism of the impedance signal is due to the ions’ formation caused by the adsorption of water molecules. Excellent repeatability and stability are still maintained after a long time of exposure in the air, which displays great application potential.

Electrostatic effects on water-soluble fullerene derivatives interaction with cytochrome c and cytochrome c oxidase.

Water-soluble fullerene derivatives are good candidates for various biological applications such as anticancer or antimicrobial therapy, cytoprotection, enzyme inhibition, and many others. Their toxicity, both in tissue culture and in vivo, is a critical characteristic for the development and restriction of these applications. The effects of six water-soluble cationic and anionic polysubstituted fullerene derivatives on cytochrome c oxidase activity in rat brain mitochondria and the possibility of cytochrome c binding were studied. We found that the ability of these fullerene derivatives to bind with cytochrome c oxidase and charged molecules like eosin Y strongly depends on their electrostatic charge. As was shown, the cationic fullerene derivative inhibits cytochrome c oxidase that has the overall negative electrostatic potential completely, unlike anionic derivatives. Thus, it confirms the essential role of electrostatic interactions in the interaction of fullerene derivatives with the active site of enzymes. The results explore how cationic fullerene derivatives play a role in mitochondrial dysfunction, oxidative stress, and cytotoxicity.

Water-Soluble Fullerene C60 Derivatives Are Effective Inhibitors of Influenza Virus Replication

The influenza virus genome features a very high mutation rate leading to the rapid selection of drug-resistant strains. Due to the emergence of drug-resistant strains, there is a need for the further development of new potent antivirals against influenza with a broad activity spectrum. Thus, the search for a novel, effective broad-spectrum antiviral agent is a top priority of medical science and healthcare systems. In this paper, derivatives based on fullerenes with broad virus inhibiting activities in vitro against a panel of influenza viruses were described. The antiviral properties of water-soluble fullerene derivatives were studied. It was demonstrated that the library of compounds based on fullerenes has cytoprotective activity. Maximum virus-inhibiting activity and minimum toxicity were found with compound 2, containing residues of salts of 2-amino-3-cyclopropylpropanoic acid (CC50 > 300 µg/mL, IC50 = 4.73 µg/mL, SI = 64). This study represents the initial stage in a study of fullerenes as anti-influenza drugs. The results of the study lead us conclude that five leading compounds (1-5) have pharmacological prospects.

Investigation of the biological activity of the water-soluble C<sub>60</sub>/poly-<i>N</i>-vinylpyrrolidone complex

Objectives. The study aimed to investigate the biological activity of the C60/poly-N-vinylpyrrolidone (C60/PVP) complex representing a water-soluble fullerene derivative. In vitro and in vivo techniques were used to analyze the effect of the C60/PVP complex on the activity of lactate dehydrogenase (LDH) and evaluate changes in the biochemical parameters of blood serum when per os administered to mice.Methods. In order to determine the activity of a commercial LDH preparation and study the kinetics of this process, the standard Warburg photometric method was used. To assess the effect of polyvinylpyrrolidone (PVP) and the C60/PVP complex on some biochemical parameters in vivo, a study was conducted on two-month-old male white mongrel mice weighing 20 ± 3 g. Determination of biochemical parameters of blood serum was carried out using a semi-automatic biochemical analyzer according to standard methods.Results. The effect of the C60/PVP complex on LDH activity was studied along with changes in the biochemical parameters of mouse blood serum characterizing carbohydrate metabolism. As well as increasing the glucose and pyruvic acid content, the C60/PVP complex was found to reduce lactate content and LDH activity in blood serum along with in vitro LDH activity according to the type of mixed inhibition.Conclusions. The C60/PVP complex and PVP were shown to exhibit biological activity in vitro and in vivo. The C60/PVP complex, representing a mixed-type LDH inhibitor, was shown to inhibit LDH activity, as well as contributing to a decrease in lactate concentration and an increase in the concentration of pyruvic acid and glucose in blood serum when administered per os to mice. The inhibitory effect of PVP on LDH activity was revealed in both in vivo and in vitro investigations. In vivo, PVP contributes to a decrease in the concentration of lactate in the blood. The less pronounced effect of the C60/PVP complex as compared to PVP alone may be due to the fact that C60 molecules are “hidden” in cavities formed in PVP molecules.

Interactions between fullerene derivatives and biological systems

This review highlights the design of water-soluble fullerene derivatives, their cellular trafficking, and their applications in therapeutics and diagnostics towards various cell pathologies.

Dynamic surface properties of carboxyfullerene solutions

Application of conventional methods of dilatational surface rheology in combination with optical techniques and atomic force microscopy allowed determination of the surface properties of a water-soluble fullerene derivative, carboxyfullerene C60(C(COOH)2)3. It has been shown that the surface activity of this derivative is higher than that of other soluble fullerene derivatives, for example, of fullerenols. The dynamic surface elasticity of carboxyfullerene solutions reaches up to values of about 180 mN/m. Brewster angle microscopy indicates a liquid-like nature of carboxyfullerene adsorption layers. The application of atomic force microscopy shows that these adsorption layers consist of numerous interconnected surface aggregates. The size and number of aggregates increase during adsorption. The compression of carboxyfullerene spread layers results in its partial dissolution. At the same time, the compression isotherms indicate a high stability of the layers of carboxyfullerene aggregates.

Effect of Water-Soluble Chlorine-Containing Buckminsterfullerene Derivative on the Metabolism of Reactive Oxygen Species in Human Embryonic Lung Fibroblasts

The development of novel biologically active nanopharmaceuticals is a topical problem of medicine. Water-soluble fullerene derivatives are of particular interest due to their ability to regulate intracellular metabolism of reactive oxygen species (ROS) by direct oxidation or effects on oxidative and signaling enzymes. Here, we studied the effect of a water-soluble chlorine-containing derivative of C60 fullerene on human embryonic lung fibroblasts. MTT tests, intracellular ROS visualization, detection of the ROS-associated gene and protein expression, repair, cell proliferation and cell cycle regulation, and quantitation of oxidative DNA damage were used. Within the first three hours after exposure, antioxidant effects were revealed. Later, oxidative damage appeared. Thus, the studied compound had an ambiguous effect on ROS metabolism associated with a switch in the regulatory effect, which must be taken into account when assessing its biological activity and toxicity.

Влияние водорастворимых производных фуллерена С60 на уровень окислительного стресса и на транскрипционную активность генов, регулирующих про- и антиоксидантную активность эмбриональных фибробластов легких человека, культивируемых in vitro

The paper considers the effect of two water-soluble C60 fullerene derivatives modified with amino acid residues on the level of reactive oxygen species (ROS) in cultured human cells and on the transcriptional activity of genes that regulate pro- and antioxidant activity of cells. C60 fullerene derivatives with attached serine and phenibut amino acid residues were synthesized from C60Cl6 chlorofullerenes. Human embryonic lung fibroblast cultures were used in in vitro experiments. Various concentrations of fullerene derivatives were added to the culture medium, the cells were cultured in the presence of compounds from 1 to 72 hours. The level of ROS was determined using the dye 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), which is oxidized by free radicals to form fluorescent 2,7-dichlorofluorescein (DCF), by fluorescence microscopy, flow cytometry, and using a plate reader. The level of protein expression was determined by flow cytometry using specific antibodies. The level of gene expression was assessed by real-time polymerase chain reaction. It was shown that when the studied compounds are introduced into the cellular environment, they intensively absorb reactive oxygen species due to double conjugated bonds in the framework. At the same time, fullerene derivatives contribute to the development of secondary oxidative stress in cells 24 hours after administration. This effect occurs due to the activation of the NOX4 enzyme. In the cells of human embryonic lung fibroblasts, which were injected with the studied C60 fullerene derivatives, a correlation was found between the average value of the NOX4 enzyme protein and the level of reactive oxygen species. The lack of activation of the antioxidant transcription factor NRF2 upon incubation of human embryonic lung fibroblasts in the presence of C60 fullerene derivatives contributes to the development of secondary oxidative stress in cells.

Self-Organization of Fullerene Derivatives in Solutions and Biological Cells Studied by Pulsed Field Gradient NMR.

Fullerene derivatives are of great interest in various fields of science and technology. Fullerene derivatives are known to have pronounced anticancer and antiviral activity. They have antibacterial properties. Their properties are largely determined by association processes. Understanding the nature and properties of associates in solvents of various types will make it possible to make significant progress in understanding the mechanisms of aggregation of molecules of fullerene derivatives in solutions. Thus, this work, aimed at studying the size and stability of associates, is relevant and promising for further research. The NMR method in a pulsed field gradient was used, which makes it possible to directly study the translational mobility of molecules. The sizes of individual molecules and associates were calculated based on the Stokes-Einstein model. The lifetime of associates was also estimated. The interaction of water-soluble C60 fullerene derivatives with erythrocytes was also evaluated. The values of self-diffusion coefficients and the lifetime of molecules of their compounds in cell membranes are obtained. It is concluded that the molecules of fullerene derivatives are fixed on the cell surface, and their forward movement is controlled by lateral diffusion.

Long-term maintenance of synaptic plasticity by Fullerenol Ameliorates lead-induced-impaired learning and memory in vivo

Fullerenol, a functional and water-soluble fullerene derivative, plays an important role in antioxidant, antitumor and antivirus, implying its enormous potential in biomedical applications. However, the in vivo performance of fullerenol remains largely unclear. We aimed to investigate the effect of fullerenol (i.p., 5 mg/kg) on the impaired hippocampus in a rat model of lead exposure. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF–MS) is a kind of newly developed soft-ionization mass spectrometry technology. In the present study, an innovative strategy for biological distribution analysis using MALDI-TOF–MS confirmed that fullerenol could across the blood–brain barrier and accumulate in the brain. Results from behavioral tests showed that a low dose of fullerenol could improve the impaired learning and memory induced by lead. Furthermore, electrophysiology examinations indicated that this potential repair effect of fullerenol was mainly due to the long-term changes in hippocampal synaptic plasticity, with enhancement lasting for more than 2–3 h. In addition, morphological observations and biochemistry analyses manifested that the long-term change in synaptic efficacy was accompanied by some structural alteration in synaptic connection. Our study demonstrates the therapeutic feature of fullerenol will be beneficial to the discovery and development as a new drug and lays a solid foundation for further biomedical applications of nanomedicines.

(Invited, Digital Presentation) Aggregation Switchable Fullerene-Peptides Conjugates

Intensive research has been applied on chemical functionalization of fullerenes over the past decades, aiming at utilizing their broad availabilities in chemical biology field. Chemical moieties was induced to improve their water solubility and thus enhance their properties or interaction with biomolecules. In our previous study, we reported the synthesis of fullerene-PEG conjugates and demonstrated their ability for photoinduced DNA damage and ROS generation under visible light irradiation.In this work, we report a water-soluble fullerene derivative by constructing a fullerene-peptide conjugates. The aggregation behavior as well as bio properties has been systematically studied for this series of fullerene-peptide conjugates (Figure 1a). By altering the attached peptides, fullerenes could exist as monomer or micelle in aqueous phase (Figure 1b). Figure 1

Novel mixed matrix membranes based on polyelectrolyte complex modified with fullerene derivatives for enhanced pervaporation and nanofiltration

Solution-processable polyelectrolyte complex (PEC) modified with various water-soluble fullerene derivatives (fullerenol, carboxyfullerene, fullerene derivative with L-arginine) were synthesized by using sodium carboxymethyl cellulose (CMC) and poly(diallyldimethylammonium chloride) (PDADMAC) for the creation of novel supported mixed matrix membranes for enhanced pervaporation and nanofiltration. The optimal preparation conditions and membrane composition were found. The structural characteristics and physicochemical properties of PEC-based membranes were analysed by Fourier-transform infrared (FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), scanning electron (SEM) and atomic force (AFM) microscopies, thermogravimetric analysis (TGA), contact angle measurements and swelling experiments. The developed membranes were tested in pervaporation dehydration of isopropanol (12–50 wt% water) and, for the first time, in nanofiltration of heavy metals (model solutions and wastewater from galvanic production). Optimal transport characteristics were possessed by a supported membrane with a selective layer based on PEC-fullerenol (4%) composite: improved permeation flux of 0.28–1.62 kg/(m2h) and 99.99–79.30 wt% water in permeate in pervaporation dehydration of isopropanol (12–50 wt% water) at 22 °C, and 2.5 times improved permeability at a high rejection coefficients in nanofiltration of heavy metals compared to the pristine CMC membrane, which indicated its promise industrial application for water purification.

Water-Soluble, Alanine-Modified Fullerene C60 Promotes the Proliferation and Neuronal Differentiation of Neural Stem Cells.

As carbon-based nanomaterials, water-soluble C60 derivatives have potential applications in various fields of biomedicine. In this study, a water-soluble fullerene C60 derivative bearing alanine residues (Ala-C60) was synthesized. The effects of Ala-C60 on neural stem cells (NSCs) as seed cells were explored. Ala-C60 can promote the proliferation of NSCs, induce NSCs to differentiate into neurons, and inhibit the migration of NSCs. Most importantly, the Ala-C60 can significantly increase the cell viability of NSCs treated with hydrogen peroxide (H2O2). The glutathioneperoxidase (GSH-Px) and superoxide dismutase (SOD) activities and glutathione (GSH) content increased significantly in NSCs treated even by 20 μM Ala-C60. These findings strongly indicate that Ala-C60 has high potential to be applied as a scaffold with NSCs for regeneration in nerve tissue engineering for diseases related to the nervous system.

Endohedral Gd-Containing Fullerenol: Toxicity, Antioxidant Activity, and Regulation of Reactive Oxygen Species in Cellular and Enzymatic Systems.

The Gd-containing metallofullerene derivatives are perspective magnetic resonance imaging contrast agents. We studied the bioeffects of a water-soluble fullerene derivative, gadolinium-endohedral fullerenol, with 40–42 oxygen groups (Gd@Fln). Bioluminescent cellular and enzymatic assays were applied to monitor toxicity and antioxidant activity of Gd@Fln in model solutions; bioluminescence was applied as a signaling physiological parameter. The Gd@Fln inhibited bioluminescence at high concentrations (>2·10−1 gL−1), revealing lower toxicity as compared to the previously studied fullerenols. Efficient activation of bioluminescence (up to almost 100%) and consumption of reactive oxygen species (ROS) in bacterial suspension were observed under low-concentration exposure to Gd@Fln (10−3–2·10−1 gL−1). Antioxidant capability of Gd@Fln was studied under conditions of model oxidative stress (i.e., solutions of model organic and inorganic oxidizers); antioxidant coefficients of Gd@Fln were determined at different concentrations and times of exposure. Contents of ROS were evaluated and correlations with toxicity/antioxidant coefficients were determined. The bioeffects of Gd@Fln were explained by hydrophobic interactions, electron affinity, and disturbing of ROS balance in the bioluminescence systems. The results contribute to understanding the molecular mechanism of “hormetic” cellular responses. Advantages of the bioluminescence assays to compare bioeffects of fullerenols based on their structural characteristics were demonstrated.

The effects of fullerene on photosynthetic apparatus, chloroplast-encoded gene expression, and nitrogen assimilation in Zea mays under cobalt stress.

Carbon nanostructures, such as the water-soluble fullerene (FLN) derivatives, are considered perspective agents for agriculture. FLN can be a novel nano-agent modulating plant response against stress conditions. However, the mechanism underlying the impacts of FLN on plants in agroecosystems remains unclear. Zea mays was exposed to exogenous C60 -FLN applications (FLN1: 100; FLN2: 250; and FLN3: 500 mg L-1 ) with/without cobalt stress (Co, 300 μM) for 3 days (d). In the maize chloroplasts, Co stress disrupted the photosynthetic efficiency and the expression of genes related to the photosystems (psaA and psbA). FLNs effectively improved the efficiency and photochemical reaction of photosystems. Co stress induced the accumulation of reactive oxygen species (ROS) as confirmed by ROS-specific fluorescence in guard cells. Co stress increased only chloroplastic superoxide dismutase (SOD) and peroxidase (POX). Stress triggered oxidative damages in maize chloroplasts, measured as an increase in TBARS content. In Co-stressed seedlings exposed to FLN1 and FLN2 exposures, the hydrogen peroxide (H2 O2 ) was scavenged through the nonenzymes/enzymes-related to the AsA-GSH cycle by preserving ascorbate (AsA) conversion, as well as GSH/GSSG and glutathione (GSH) redox state. Also, the alleviation effect of FLN3 against stress could be attributed to increased glutathione S-transferase (GST) activity and AsA regeneration. FLN applications reversed the inhibitory effects of Co stress on nitrogen assimilation. In maize chloroplasts, FLN increased the activities of nitrate reductase (NR), glutamate dehydrogenase (GDH), nitrite reductase (NiR), and glutamine synthetase (GS), which provided conversion of inorganic nitrogen (N) into organic N. The ammonium (NH4 + ) toxicity was removed via GS and GDH but not glutamate synthase (GOGAT). The increased NAD-GDH (deaminating) and NADH-GDH (aminating) activities indicated that GDH was needed more for NH4 + detoxification. Therefore, FLN exposure to Co-stressed maize plants might play a role in N metabolism regarding the partitioning of N assimilates. Exogenous FLN conceivably removed Co toxicity by improving the expressions of genes related to reaction center proteins of photosystems, increasing the level of enzymes related to the defense system, and improving the N assimilation in maize chloroplasts.