Belén
Parra-Torrejón
a,
Marta
Salvachúa-de la Fuente
a,
Maria J.
Giménez-Bañón
b,
Juan D.
Moreno-Olivares
b,
Diego F.
Paladines-Quezada
b,
Juan A.
Bleda-Sánchez
b,
Rocío
Gil-Muñoz
b,
Gloria B.
Ramírez-Rodríguez
*a and
José M.
Delgado-López
*a
aDepartment of Inorganic Chemistry, Faculty of Science, University of Granada, Granada, Spain. E-mail: gloria@ugr.es; jmdl@ugr.es
bInstituto Murciano de Investigación y Desarrollo Agrario y Medioambiental, Calle Mayor s/n. La Alberca, Murcia, Spain
First published on 15th March 2023
Nanotechnology is emerging as a potential strategy to achieve sustainable agricultural productivity and global food security. Engineered nanoparticles (NPs) are endowed with the ability to deliver active ingredients in a responsive manner, reducing the adverse environmental impacts in comparison to the conventional practices. However, the relationship between NP features (i.e., size, morphology, surface charge, structure, etc.) and functionality has been scarcely studied so far. In this work, two types of calcium phosphate NPs were functionalized with the plant resistance-inductor methyl jasmonate (MeJ), which stimulates the production of secondary plant metabolites (e.g., anthocyanins, stilbenes, and flavonols) in grapes. The properties of the resulting nanomaterials, namely, elongated apatite (Ap-MeJ) and round-shaped amorphous calcium phosphate NPs (ACP-MeJ), were studied in detail. In addition, the loading capacity, release kinetics and elicitor thermal stability of the two nanosystems were compared. The results indicated that the differences in terms of morphology, crystalline structure and surface charge did not affect the release kinetics nor the protective action offered by the NPs. However, ACP-MeJ showed much higher MeJ loading capacity than Ap-MeJ. Both types of nanomaterials exhibited similar performance in field experiments on Monastrell vineyards (Vitis vinifera L.). Foliar application of ACP-MeJ and Ap-MeJ (1 mM MeJ) produced wines with similar contents of anthocyanins and tannins (around 500 mg L−1 and 1100 mg L−1, respectively), but Ap-MeJ treatment required doubling the NP amount due to its lower MeJ adsorption capacity. Both treatments produced wines with higher tannin concentration than wines from non-treated grapes or wine treated with free MeJ at a much higher dosage (10 mM). Results highlight the potential of ACP and Ap NPs as elicitor nanocarriers enabling enhancement of the quality of wine in a more sustainable manner.
Calcium phosphate NPs, mainly nanocrystalline hydroxyapatite (Ap, Ca10(PO4)6(OH)2) and its transient precursor phase, amorphous calcium phosphate (ACP, Ca3(PO4)2), are ideal nanocarriers for the controlled delivery of agrochemicals due to: i) their main composition, which is rich in plant nutrients (Ca and P), ii) biocompatibility and biodegradability, iii) pH-responsive solubility, iv) high surface-to-volume ratio and v) easy surface functionalization.3,4 The vast majority of the studies were focused on the most thermodynamically stable phase, Ap, which is by far the most extended in the literature for medical applications.5 It has been demonstrated the potential of Ap NPs as nanocarriers for the sustainable release of plant macronutrients, mainly phosphorus and urea, the most common source of nitrogen for field crops.3,6–11 Ap NPs have been also functionalized with humic substances providing the synergistic co-release of crop nutrients and stimulants.12 A recent study demonstrated the potential of Ap NPs in promoting disease suppression.13
ACP NPs have been much less explored for environmental applications, most likely due to their transient nature. However, they can be stabilized with the use of organic molecules, such as citrate, which delays their rapid conversion into more stable crystalline calcium phosphate phases.14–16 In a recent study, we demonstrated that ACP has higher loading capacity to host exogenous ions (e.g., nitrate) or molecules (e.g., urea) than Ap. This, along with the higher solubility at neutral or slightly acidic pH, and the more cost-effective production for industrial exploitation in agriculture make ACP an ideal candidate for the controlled delivery of agrochemicals (e.g., fertilizers, pesticides, and elicitors).17 ACP has been also recently used as nanocarriers of methyl jasmonate (MeJ), an elicitor able to stimulate plants to produce secondary metabolites (SMs), which protect plants against biotic and abiotic stresses.18,19 It stimulates the production of low molecular mass SMs with antimicrobial activity, such as anthocyanins, stilbenes, and flavonols, in grapevines.20,21 However, the treatment of free MeJ in fields presents several drawbacks due to its high volatility and its phytotoxicity when used at high doses.22,23 We found that ACP NPs provided a sustainable release and protection against thermal degradation of the elicitor, ensuring activity over longer period of times. In fact, MeJ-doped ACP NPs reduced by 10 times the required dosage of the elicitor while maintaining the quality of the harvested grapes.24,25
However, the specific role of the NP properties in the performance (i.e., loading capacity, release kinetics, protection, etc.) and efficiency in field experiments has not been studied so far. This work is aimed at evaluating how the properties of the NPs, and concretely the structure, morphology and surface charge, affect the efficiency in delivering and protecting the elicitor and the response of the plants with the two types of NPs: amorphous or nanocrystalline calcium phosphate (ACP-MeJ or Ap-MeJ, respectively). The two types of NPs were in-depth characterized to evaluate the impact of MeJ adsorption on the NP features, including loading capacities and release kinetics. Finally, the performance of the nanoassemblies was evaluated by means of field experiments in grapevines. Monastrell grapevines were treated with free MeJ (10 mM), ACP-MeJ and Ap-MeJ (both 1 mM MeJ). The effect of these preharvest treatments on the content of two important components of wines, i.e., anthocyanins and tannins, was evaluated. While the anthocyanins are responsible for the red color and astringency and bitterness,26 tannins play a relevant role in the organoleptic properties including wine mouthfeel, such as bitterness, hardness, dryness, astringency, structure, and body.20,27
Apatite (Ap) NPs were synthesized following a similar protocol consisting of mixing two solutions of equal volume (A) CaCl2 (0.1 M) and Na3(C6H5O7) (0.4 M) and (B) Na2HPO4 (0.12 M) and Na2CO3 (0.1 M). In this case, the NPs were aged in the same solution at 60 °C for 24 hours.14,17 After that, the precipitates were collected and repeatedly washed with ultrapure water by centrifugation (5000 rpm, 15 min, 18 °C).
The loading capacity (Qmax) was calculated by quantifying the MeJ released from 20 mg of ACP-MeJ or Ap-MeJ NPs in 1 mL of ultrapure water. After 72 hours of release, the supernatant was collected by centrifugation (12000 rpm, 15 min, 18 °C) and the amount of MeJ in solution was estimated by UV-vis spectroscopy (λ = 291 nm, Agilent Technologies, Santa Clara, CA, USA).24 After that, the NP pellet was again dispersed in 0.4 mL of water and left for 72 hours to confirm if MeJ has been completely desorbed. We found that after 72 hours of release, MeJ was almost completely desorbed, with the absorbance (λ = 291 nm) of the supernatant of the second cycle being close to cero (Fig. S4, ESI†).
The elemental chemical composition (Ca and P) was evaluated by inductively coupled plasma optical emission spectrometry (ICP-OES, Optima 7300DV from University of Málaga, SCAI). Firstly, 2 mL of ultrapure nitric acid was used to dissolve 20 mg of the powdered sample. Secondly, the mix was diluted up to 100 mL with ultrapure water. Three measurements of Ca and P contents were carried out each in triplicate. The corresponding emission wavelengths were 317.93 nm (Ca) and 213.62 nm (P).
The two types of NPs (ACP and Ap) were then functionalized with MeJ through a previously optimized protocol.24 The TEM micrograph of ACP-MeJ NPs (Fig. 1a) shows round-shaped NPs with diameters falling in the 10–20 nm range, in perfect agreement with the size of pristine ACP NPs synthesized through the same protocol.17 The SAED pattern (inset Fig. 1a) along with the XRD pattern (Fig. S1†) confirmed the amorphous nature of the NPs, before and after MeJ adsorption.
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Fig. 1 TEM images of ACP-MeJ (a) and Ap-MeJ (b). SAED patterns of the respective NPs are shown as an inset. |
On the other hand, the TEM micrograph of Ap-MeJ (Fig. 1b) shows elongated NPs with a length of around 20 nm and a width of around 5 nm (Fig. S2C, Table S1†). The SAED pattern (inset Fig. 1b) displays the typical reflections 002, 112 and 004 of hydroxyapatite at a d-spacing equal to 3.44, 2.75 and 1.72 Å, respectively (ASTM Card file No. 9-432). Rietveld analysis of the XRD patterns of Ap-MeJ (Fig. S2B†) confirmed that anisotropic (elongated) Ap nanocrystals were produced (Table S1†). Neither the morphology nor the size of Ap NPs was affected by MeJ adsorption (Table S1†).14,15,17 Nevertheless, the crystal size slightly increased after the adsorption process, due to the fact that they remained 24 hours in aqueous solution (Fig. S2, Table S1†).
The chemical composition (Ca and P contents) of the NPs was scarcely affected by MeJ adsorption (Table 1). ACP and ACP-MeJ NPs have a Ca/P molar ratio close to 1.5, in agreement with the molecular formula of ACP (Ca3(PO4)2).30 Conversely, Ap and Ap-MeJ presented a higher Ca/P molar ratio, close to 1.67, which fits with the molecular formula of stoichiometric hydroxyapatite (Ca5(PO4)3OH).16 In both cases, neither Ca nor P contents were affected by MeJ adsorption (Table 1).
Sample | Ca (wt%) | P (wt%) | Ca/P | ζ-Potential (mV) |
---|---|---|---|---|
ACP | 14.7 ± 0.1 | 8.1 ± 0.1 | 1.41 ± 0.03 | −10.5 ± 0.1 |
ACP-MeJ | 15.6 ± 1.3 | 8.8 ± 1.3 | 1.49 ± 0.13 | −6.2 ± 1.0 |
Ap | 31.0 ± 0.9 | 14.7 ± 0.2 | 1.62 ± 0.03 | −21.6 ± 0.2 |
Ap-MeJ | 31.9 ± 0.7 | 14.9 ± 0.8 | 1.66 ± 0.05 | −16.4 ± 2.2 |
The successful MeJ adsorption was confirmed by FTIR, X-ray photoelectron spectroscopy (XPS) and ζ-potential. Fig. 2a shows the FTIR spectra of ACP and Ap NPs before and after MeJ adsorption. The Ap and Ap-MeJ spectra show the characteristic phosphate vibrational bands of apatite (ν4PO4 at 561 and 602 cm−1 and ν3PO4 at 1032, 1046 and 1087 cm−1). These phosphate absorption bands are broader for ACP and ACP-MeJ due to the lack of long-range order.14,24 The FTIR spectra before and after MeJ adsorption were similar, confirming that the composition of ACP and Ap NPs was not affected by MeJ adsorption. The unique difference was the presence of a vibrational band at 1740 cm−1 ascribed to carbonyl (ketone) groups of MeJ.31 This band is more intense in the ACP-MeJ spectrum than that for Ap-MeJ, indicating that MeJ is bound to a larger extent on ACP in comparison to Ap. In fact, MeJ quantification by UV-vis spectroscopy revealed a higher MeJ loading capacity for ACP-MeJ (10.6 ± 0.5% wt) than for Ap-MeJ NPs (4.5 ± 1.1% wt) (Fig. 2b).
![]() | ||
Fig. 2 (a) FTIR spectra of Ap, ACP, Ap-MeJ and ACP-MeJ. (b) MeJ loading capacity of ACP-MeJ and Ap-MeJ NPs calculated by UV-vis spectroscopy. |
The XPS spectra of the survey scan of Ap and Ap-MeJ were quite similar (Fig. S3†), displaying the characteristic peaks of apatite: the Ca 1s, Ca 2p and Ca 2s, P 2p and P 2s.32 The high-resolution P 2p and Ca 2p spectral lines in Ap and Ap-MeJ were identical (Fig. S3†). The unique difference between both XPS spectra was found in the C 1s peak, which is shifted to higher energies, due to the bonding of MeJ to the Ap surface (Fig. S3†).
The changes in surface charge of the NPs before and after the functionalization were also evaluated (Table 1). The ζ-potential of ACP and Ap NPs before MeJ adsorption was −10.5 ± 0.1 mV and −21.6 ± 0.2 mV, respectively. The negatively charged carboxyl groups of citrate at the NP surface were the main responsible for the negative values. MeJ functionalization provided NPs with less negative surface charge, i.e. ζ-potential of −6.2 ± 1.0 mV (ACP-MeJ) and −16.4 ± 2.2 mV (Ap-MeJ) (Table 1). To evaluate whether the reduction of MeJ loading of Ap-MeJ was associated with the more negative surface charge of Ap, in comparison to the ACP/ACP-MeJ samples, we modified the surface charge of Ap NPs. To this aim, the Ap NPs were immersed in sodium hydroxide solution to remove the citrate molecules from the NP surface. Ap* NPs with less negative surface charge (−0.7 ± 0.2 mV, Table S2†) had a MeJ loading capacity of 6.9 ± 0.4% wt, slightly higher than that of Ap-MeJ (4.5 ± 1.1% wt). This finding indicated that the surface charge has a very low impact on the loading capacity of calcium phosphate NPs. In contrast, other systems (i.e., polymeric NPs) showed a more prominent effect of the surface charge on the encapsulation efficiency.33 In this study, the highest MeJ content on ACP-MeJ compared to Ap-MeJ and Ap*-MeJ may be associated with the NP structure (amorphous vs. crystalline). ACP NPs have higher capacity to host exogenous ions or molecules (e.g., citrate, urea, nitrate) than the crystalline apatite ones due to their short-range order.17 The greater loading capacity of ACP is also associated with its higher surface reactivity and the presence of a high content of tightly bound water.17,34 To this respect, ACP is more promising as nanocarriers of agrochemicals than Ap, even though the latter has been much more explored in the literature.3,6
We also evaluated the protective effect of the NPs against MeJ thermal degradation. To this aim, we measured the remaining MeJ concentration in drops of Ap-MeJ, ACP-MeJ and free MeJ after 24 hours at 50 °C by UV-vis spectroscopy (Fig. 3b). A solution containing free MeJ mixed with Ap NPs (Ap + MeJ) was also included. We observed no significant differences between the ACP-MeJ and Ap-MeJ samples, remaining in both cases more than 90% of the initial MeJ. This finding indicated that MeJ was protected by both types of NPs, regardless of their crystallinity, morphology or surface charge. On the other hand, free MeJ was almost degraded/evaporated after 24 hours. In the case of Ap + MeJ, only 25% of initial MeJ remained in the drops (Fig. 3b), confirming that our experimental protocol provides composite nanomaterials (ACP-MeJ or Ap-MeJ) with a higher level of performance in comparison to the sum of the parts (Ap + MeJ).
In this work, we compared the composition of anthocyanins and tannins in wines from Monastrell grapes treated during veraison with 10 mM MeJ (positive control), Ap-MeJ (1 mM MeJ), ACP-MeJ (1 mM MeJ) and water as a control. Grapes treated with MeJ resulted in wines with a significant increase of the total anthocyanin concentration with respect to non-treated grapes (Fig. 4a). The anthocyanins are water-soluble pigments responsible for the red color and its tonality variations in grapes and red wines. These compounds also can contribute to the astringency and bitterness of grapes and wines.26 Previous studies in Vitis vinifera L. Monastrell revealed that MeJ treatment (10 mM) increases the anthocyanin content of grapes and wines in two consecutive years (2009 and 2010).20 It has been pointed out that jasmonic acid and MeJ are naturally occurring plant growth regulators that modulate chlorophyll degradation and anthocyanin biosynthesis.20 However, ACP-MeJ and Ap-MeJ treatment did not enhance the anthocyanin content of wines with respect to the control (Fig. 4a).
More interestingly, both nanoparticle-based treatments enhanced the tannin concentration in wines with respect to the control and free MeJ treatments (Fig. 4b), showing statistically significant differences only in Ap-MeJ treatment. Proanthocyanidins, commonly known as tannins, are flavonoid compounds found in grape skin and seeds. They play a relevant role in the organoleptic properties including wine mouthfeel, such as bitterness, hardness, dryness, astringency, structure, and body. Moreover, tannins participate in reactions with wine anthocyanins, favoring wine color stability with time.20,27 It has been shown that premium Monastrell wines with high projected market prices are rich in tannins.37 Gil-Muñoz et al. demonstrated that pre-harvest treatments with MeJ stimulated the production of tannins in both Tempranillo and Monastrell grapes and the corresponding wines, enhancing their quality and health-related properties.20,27 However, they required ten times higher MeJ dosage (10 mM). In this work, we obtained similar results but at much reduced dosages using the NPs.
We can conclude that there were no significant differences between the two nanocarriers in the enhancement of anthocyanin and tannin contents in wine. Nonetheless, a recent study about the effect of the different MeJ treatments on the phenolic composition of the grape during the ripening period revealed a delayed effect of Ap-MeJ treatment on the increase of the anthocyanin content in the grape skin with respect to MeJ and ACP-MeJ treatments.25 This delayed effect was most probably associated with the need for a greater Ap-MeJ uptake, at least twice that of ACP-MeJ, due to the lower MeJ content (Fig. 2b). At the harvest time, the three treatments showed similar improvement of anthocyanin and tannin contents in the grape skin.25 Further experiments dedicated to track the fate of the NPs inside the plants are needed to evaluate the absorption mechanisms (and NP translocation) of the two types of NPs.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d3ce00185g |
This journal is © The Royal Society of Chemistry 2023 |