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Article
Engineering biomimetic calcium phosphate nanoparticles: a green
synthesis of slow-release multinutrient (NPK) nano-fertilizers
Gloria Belén Ramírez-Rodríguez, Gregorio Dal Sasso, Francisco J. Carmona, Cristina Miguel-Rojas,
Alejandro Pérez de Luque, Norberto Masciocchi, Antonietta Guagliardi, and José Manuel Delgado-López
ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00937 • Publication Date (Web): 26 Jan 2020
Downloaded from pubs.acs.org on February 3, 2020

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Page 1 of 35 ACS Applied Bio Materials

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4 Engineering biomimetic calcium phosphate nanoparticles:
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a green synthesis of slow-release multinutrient (NPK) nano-fertilizers
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Gloria B. Ramírez-Rodríguez#,‡, Gregorio Dal Sasso¥, Francisco J. Carmona‡,
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14 Cristina Miguel-Rojas†,‡, Alejandro Pérez-de-Luque†, Norberto Masciocchi‡,
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16 Antonietta Guagliardi¥ and José M. Delgado-López*,#
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19 #Department of Inorganic Chemistry, University of Granada, Faculty of Science, Av. Fuente
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22 Nueva, s/n, 18071 Granada, Spain. E-mail: jmdl@ugr.es
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25 ‡Department of Science and High Technology and To.Sca.Lab. University of Insubria. Via
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27 Valleggio 11, I-22100 Como, Italy.
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30 ¥Institute
31 of Crystallography and To.Sca.Lab. Consiglio Nazionale delle Ricerche (IC-CNR). Via
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33 Valleggio 11, I-22100 Como, Italy.
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36 †IFAPA Alameda del Obispo, Area of Genomic and Biotechnology, Avenida Menéndez Pidal,
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S/N, 14004 Córdoba, Spain.
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45 KEYWORDS: amorphous calcium phosphate, crystallization, nitrate, urea, slow release,
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47 nanofertilizer
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3 ABSTRACT
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6 Biomimetic calcium phosphate nanoparticles (CaP) have been actively used in biomedicine,
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8 due to their high biodegradability and biocompatibility. However, much less progress has been
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10 made regarding its application in precision agriculture, i.e., for the controlled delivery of active
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species to plants. Herein we report a straightforward and green synthetic method to dope CaP
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15 with potassium (K) and nitrogen (N, as nitrate and urea). By modulating the synthetic conditions
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17 in terms of maturation time (at 37 ºC) and doping, we prepared K and N-doped nanoparticles in
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the form of either amorphous calcium phosphate (ACP) or nanocrystalline apatite (Ap) and
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22 studied the impact of the dopants on the ACP-to-Ap transformation pathways. Importantly, we
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24 found out that ACP, isolated at low maturation times, incorporates nitrogen (in the form of
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26 nitrate and urea) to a larger extent than Ap (2.6 vs 1.1 %wt, respectively). Multinutrient
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29 nanofertilizers (so-called nanoU-NPK) with the following composition (wt%) were obtained: Ca
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31 (23.3), P (10.1), K (1.5), NO3 (2.9) and urea (4.8). The nanoU-NPK provides a slow and gradual
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33 release of the most important plant macronutrients (NPK), with N in two chemical forms, and
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different kinetics. The concentration of nutrients supplied by 10 g L-1 of nanoU-NPK to the
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38 media after one week (in mg L-1) was: Ca (27.0), P (6.2), K (41.0), NO3 (134.0) and urea (315.0).
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40 Preliminary tests on durum wheat have shown that the application of nanoU-NPK allows
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reducing the amount of nitrogen supplied to the plants by 40% with respect to a conventional
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45 treatment, without affecting the final kernel weight per plant. The application of these slow-
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47 release NPK nanofertilizers is a promising strategy toward enhancing the efficiency of the
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49 fertilization, complying with the concept of precision agriculture.
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3 1. Introduction
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6 Calcium phosphates (CaP) are the most important inorganic constituents of biological hard
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8 tissues such as bone, dentine and deer antlers.1 The natural formation of these biominerals has
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10 long been used as a source of inspiration for the design of advanced nanomaterials with high(er)
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levels of performance and new functionalities.2 Synthetic nanocrystalline apatite (Ap) and its
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15 ephemeral precursor (i.e., amorphous calcium phosphate, ACP), and prepared in close to
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17 physiological, “biomimetic” conditions, are remarkably biocompatible, non-toxic and
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biodegradable.3 Moreover, they present superior adsorption capacity for organic molecules
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22 (drugs, proteins or other active species),4 and can host a large variety of cationic (Ca, K, Mg, Zn,
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24 etc.) and anionic substitutions5 (mostly carbonate, as occurring in bone6). More importantly, their
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26 pH-responsive solubility allows a controlled and gradual release of the ionic constituents and
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29 dopants upon partial dissolution in aqueous media.7,8 Owing to these peculiar properties, CaP,
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31 under many crystal forms, has been widely used for a broad range of applications,9 including
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33 regenerative medicine and hard tissue repair10,11 and as pH-responsive nanocarriers for the
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controlled delivery of therapeutic molecules, drugs and genes.12–15
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38 Despite all these desirable properties and the fact that they are intrinsically rich in Ca and P
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40 (important plant nutrients), CaP applications in precision agriculture for the controlled delivery
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of (macro)nutrients in plants, are still in their infancy.16–23 Currently, the use of conventional,
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45 highly soluble, fertilizers to supply the chief macronutrients (the N, P, K elements) is one of the
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47 key practices to increase the crop yields and face the expected rise in global food demand.24
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49 However, only a small percentage (for N and K, well below 40%, and, for P, down to 10%)25 of
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52 the spread fertilizers is assimilated by plants, while the rest is eventually washed into water
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54 bodies through leaching and surface run-off, or lost by volatilization. The low nutrient use
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3 efficiency (NUE) of the conventional fertilizers has then become an enormous problem not only
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6 from the economical point of view but also for the compelling environmental aspects derived
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8 therefrom.26 Indeed, although conventional fertilizers have ensured enough food production to
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10 feed world population over the last decades, its intensive usage is nowadays recognized as being
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one of the major anthropogenic factors in the eutrophication and contamination of surface and
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15 groundwater bodies.27
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17 In this work, taking inspiration from bone biomineralization process, we have tuned a simple
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green precipitation method using citrate and carbonate ions28,29 (two important components of
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22 bone nanoapatite4,30,31) to obtain multinutrient (NPK) nano-fertilizers. By finely tuning the
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24 experimental conditions, we have doped calcium phosphate nanoparticles, intrinsically very rich
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26 in Ca and P, with two chemical forms of nitrogen (nitrate and urea) and potassium ions. The
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29 effect of doping species on the mineralization pathway of CaP has been monitored by combining
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31 Synchrotron Wide-Angle X-Ray Total Scattering (WAXTS) experiments with transmission
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33 electron microscopy (TEM) imaging, Fourier transform infrared (FTIR) and Raman
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spectroscopies, inductively coupled plasma optical emission spectrometry (ICP-OES) and
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38 elemental analysis. Finally, the nutrient release kinetics from engineered ACP nanoparticles
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40 doped with potassium, nitrate and urea (nanoU-NPK) and its efficiency as nitrogen supplier on
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durum wheat has been preliminarily evaluated. Thus, we foresee that providing these slow-
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45 release NPK nanofertilizers to plants can be considered as a promising strategy toward
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47 enhancing the efficiency of the fertilization, fulfilling with the requirements of precision
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49 agriculture.
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3 2. Materials and Methods
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2.1. Materials
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9 Calcium nitrate tetrahydrate (CaNO3·4H2O, ≥99.0% pure, Bioxtra), sodium citrate tribasic
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11 dihydrate (Na3(C6H5O7)∙2H2O, ≥99.0% pure, urea (pellets, ≥99.5%, ReagentPlus®), potassium
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13 phosphate dibasic anhydrous (K2HPO4, ≥99.0% pure), sodium carbonate (Na2CO3, ≥99.0% pure,
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16 Bioxtra) and potassium nitrate (KNO3, ≥99.0%) were purchased from Sigma Aldrich. CellTiter
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18 96® AQueous One Solution Cell Proliferation Assay (MTS) was purchased from Promega. All
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20 the solutions were prepared with ultrapure water (0.22 µS, 25 °C, MilliQ©, Millipore).
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25 2.2. Synthesis of pristine and doped nanoparticles
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27 CaP nanoparticles were obtained by a simple batch precipitation method at physiological
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temperature (37 ºC). For the control samples (nano-PK), two solutions: A) CaCl2 (0.2 M) and
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32 Na3Cit (0.2 M) and B) K2HPO4 (0.12 M) and Na2CO3 (0.1 M) were mixed (1:1 v/v, 200 mL
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34 total).28,29 Carbonate and citrate ions, in the form of sodium salts, were added with the aim of
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36 mimicking the composition of bone nanoparticles (in a so-called biomimetic approach).28 The
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39 precipitates were collected at different maturation times (5 minutes and 4, 24 and 48 hours) and
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41 repeatedly washed with ultrapure water by centrifugation (5000 rpm, 15 minutes) and then
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43 freeze-dried overnight under vacuum (Telstar Cryodos Freeze-drier).
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46 In order to obtain nitrate-doped CaP nanoparticles (nano-NPK), we adapted our previous
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48 precipitation method by replacing CaCl2 by Ca(NO3)2 in solution A and adding KNO3 (0.2 M) in
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With the aim of further increasing the nitrogen content, nano-NPK were functionalized with
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55 urea, the most used N-based fertilizer, generating a nanoU-NPK fertilizer. To this aim, 8 g of
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3 urea were added to the Ca(NO3)2 solution. After maturation at 37 ºC (5 minutes and 9.5, 24 and
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6 48 and 100 hours), the nanoparticles were collected, washed and dried as described before. Table
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8 1 summarizes the concentration of the ionic species used to obtain the corresponding
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10 nanoparticles, i.e., nano-PK, nano-NPK and nanoU-NPK.
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15 Table 1. Ionic molar concentrations for each synthesis and the corresponding pH measured just
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17 after mixing both solutions (pH0).
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20 [Ca2+] [Cl-] [NO3-] [Na+] [Cit3-] [K+] [PO42-] [CO32-] [Urea] pH0
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22 nano-PK 0.1 0.2 --- 0.4 0.1 0.12 0.06 0.05 --- 9.06
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24 nano-NPK 0.1 --- 0.3 0.4 0.1 0.22 0.06 0.05 --- 9.00
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26 nanoU-NPK 0.1 --- 0.3 0.4 0.1 0.22 0.06 0.05 0.67 9.00
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30 2.3. Characterization techniques
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Transmission electron microscopy (TEM) images were collected with a LIBRA 120 PLUS
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35 (Carl Zeiss SMT, Centre for Scientific Instrumentation of the University of Granada (CIC-
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37 UGR)), operating at 120 kV. Nano-NPK nanoparticles collected by centrifugation were
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ultrasonically dispersed in ethanol and then few drops of the slurry were deposited on 200 mesh
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42 copper grids covered with thin amorphous carbon films.
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44 Fourier transform infrared (FTIR) spectra of freeze-dried samples were collected on a Bruker
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46 Tensor 27 spectrometer with spectral resolution of 2 cm-1 by accumulating 100 scans in the
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49 4000–400 cm-1 range. 2 mg of sample was mixed with 200 mg of potassium bromide (KBr). The
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51 mixture was pressed at 5 tons into a 12 mm diameter disc using a hydraulic press (Specac). Three
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53 KBr pellets were produced for each sample and a pure KBr disk was used as a blank.
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3 Raman spectra were collected with a LabRAM-HR spectrometer with a backscattering
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6 geometry (Jobin-Yvon, Horiba, Japan). The excitation line was provided by a diode laser
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8 emitting at a wavelength of 532 nm and a Peltier cooled charge-couple device (CCD) (1064 x
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10 256 pixels) was used as detector. The Raman shift of the spectrometer was calibrated, before and
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after each experiment, using the 520.7 cm-1 peak of crystalline silicon as standard. The final
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15 spectrum is the result of averaging three acquisitions of 40 seconds each with a resolution better
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Laboratory X-ray Powder diffraction (PXRD) data were collected on a Bruker AXS D8
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22 Advance diffractometer using Cu Kα radiation (λ = 1.5418 Å), from 5° to 55° (2θ) with a scan
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24 rate of 3 s step-1, step size of 0.02° with a HV generator set at 40 kV and 40 mA. Details of
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26 diffractometer calibration are reported in the supporting information.
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31 2.4. Quantitative Compositional Analysis
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33 The chemical composition (Ca, P and K) was analyzed by inductively coupled plasma optical
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emission spectrometry (ICP-OES, Optima 8300, Perkin-Elmer) from CIC-UGR. 20 mg of the
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38 powdered sample were dissolved in 2 ml of ultrapure nitric acid and then diluted up to 100 mL
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40 with Milli-Q water. Three replicates were prepared and the Ca, P and K content was measured
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three times in each replicate. The emission wavelengths were 317.93 nm (Ca), 213.62 nm (P)
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45 and 766.49 nm (K).
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47 The nitrogen and carbon content was measured by elemental analysis with a Thermo Scientific
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49 Flash 2000 Organic Elemental Analyzer equipped with a microbalance (XP6, Mettler Toledo)
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52 from CIC-UGR.
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3 The quality assurance (QA) and quality control (QC) operations performed to establish the
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6 methods for sample quantifications and to validate the analytical instruments (ICP-OES and
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8 Elemental analyzer) are described in the supporting information (SI).
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2.5. Wide-Angle-X-Ray Total Scattering experiments (WAXTS)
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15 Synchrotron WAXTS data were collected at the X04SA Material Science beamline of the
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17 Swiss Light Source32 in transmission mode, with X-rays energy set at 16 KeV, corresponding to
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an operational wavelength λ = 0.775108 Å. Glass capillaries of 0.5 mm in diameter were loaded
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22 with the powders of nano-PK and nano-NPK (5 min, 4h, 24h, 48h). The beamline is equipped
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24 with a single-photon counting silicon microstrip MYTHEN II detector enabling the fast
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26 collection of X-ray data over the 2°-120° angular range. A data reduction procedure was applied
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29 aiming at properly subtracting scattering contributions arising from the capillary and surrounding
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31 background; the WAXTS signals of the empty capillary and air were independently acquired.
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33 Direct and transmitted beams through the filled capillaries were measured in order to
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experimentally determine the sample linear absorption coefficient and correct the data for this
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38 effect. Details of the data reduction procedure on similar samples can be found in ref.29
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40 The Debye Scattering Equation (DSE) method of analysis33 is particularly suitable to the
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characterization of nanocrystalline materials as both the Bragg and diffuse scattering are
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45 modeled and equally treated; thus providing, simultaneously both structural and morphological
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47 information, at the atomic and nanometer scale, respectively. Recent examples of its successful
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49 usage in characterizing nanoparticles and colloids can be found in topical reviews.34,35 The basics
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52 of the DSE approach used in this work are described in detail in the supporting information (SI).
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3 2.6. Nutrient release in aqueous media
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6 The Ca, K and P release was measured in water at room temperature by ICP-OES. 100 mg of
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8 powdered sample was immersed in 10 mL of ultrapure water. At scheduled times (30 minutes
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10 and 8, 24, 72 and 168 hours), the sample was centrifuged (5000 rpm for 15 min) and the
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supernatant was filtered (0.2 µm). Then, 8 mL of supernatant was treated with 0.3 mL of
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15 ultrapure nitric acid and diluted in Milli-Q water up to 10 mL. The emission wavelengths were
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17 317.93 nm (Ca) and 213.62 nm (P) and 766.49 nm (K). Three samples per group were analyzed
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at each time point.
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22 The total nitrogen release was measured by colorimetric assays. 10 mg of powdered sample
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24 was dispersed in 1 mL of ultrapure water. At scheduled times (5, 15 and 45 minutes and 1, 6, 24,
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26 96 and 168 hours) the sample was centrifuged and the supernatant was used to measure the urea
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29 and nitrate concentration at each time point by UV-Vis spectroscopy. Urea was quantified using
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31 the para-dimethyl-amino-benzaldehyde colorimetric method36 whereas nitrate was quantified by
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33 Griess assay.37 Both analyses were performed in triplicates at each time point.
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38 2.7. Urea release kinetics in simulated solid media: leaching experiments
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Experiments were performed in a vertical column filled with inert soil (a simulated solid medium
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43 consisting of a mixture of vermiculite and sand 1:3, 120 mesh) and a pellet containing 12 mg of
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45 urea (supplied as pure urea or as nanoU-NPK). The pellet was manufactured by pressing the
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powdered sample at 5 tons during 60 second into a 12 mm diameter disk using a hydraulic press.
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50 The column was rinsed with ultrapure water until reaching the soil-holding capacity. Then,
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52 ultrapure water was poured into the column at a constant flow rate of 0.86 mL·h-1 using a
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54 peristaltic pump. At scheduled time, the leachate from the column was collected and filtered
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3 through a 0.2 µm membrane filter. The urea content of the leachate was quantified by UV-Vis
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6 using the para-dimethyl-amino-benzaldehyde colorimetric method.36
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9 2.8. In-vitro cyto-compatibility assays
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12 Human Embryo Kidney cells (HEK293) from CIC-UGR were cultured in standard tissue culture
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14 flask and maintained in Eagle's Minimum Essential Medium (EMEM) with Earle’s Balanced
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Salt Solution (EBSS) supplemented with 2mM Glutamine, 1% Non Essential Amino Acids
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19 (NEAA), 1 mM sodium pyruvate (NaP) and 10% Foetal Bovine Serum (FBS) at 37ºC with 5%
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21 CO2. Cells were detached from culture flasks by trypsinization, centrifuged and resuspended.
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The cells were passed into a 96 flat transparent well (1.0 · 104 cells/well) and were cultured at
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26 37ºC with 5% CO2 during 24 hours. Then, HEK293 cells were incubated with aqueous
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28 suspensions of nanoU-NPK at different concentrations (1, 50, 100 and 200 µg/mL) using as
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30 control cells treated only with PBS. After 48 hours of incubation, cell viability was evaluated by
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33 the MTS colorimetric assay using CellTiter 96® AQueous One Solution Reagent. The MTS
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35 assay is based on the conversion of a tetrazolium salt into a coloured aqueous soluble formazan
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37 product mediated by mitochondrial activity of viable cells. Briefly, 20 µL of AQueous One
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Solution Reagent was added to each well and after 4 h of incubation at 37 ºC, the absorbance at
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42 490 nm was measured with a spectrophotometer (Infinite® 200 PRO NanoQuant). Formazan
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44 concentration, as measured by the absorbance at 490 nm, is proportional to the number of
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metabolically active cells. The relative cell viability (%) corresponds to the absorbance ratio
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49 between the treated and non-treated cells (i.e., [A]sample/[A]control x 100). Each experiment was
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3 2.9. Experiments on durum wheat under controlled conditions
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6 The efficiency of nanoU-NPK nanoparticles as nitrogen nanofertilizer was tested on wheat
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8 under controlled conditions (growth chamber), using the commercial variety of durum wheat
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10 (Triticum durum) Amilcar (Monsanto). One durum wheat seed was sowed in each 113 cm2
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plastic container (plant pots) filled with a non-sterile 1:1 soil/sand mixture and cultivated in a
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15 growth chamber at 22 ± 1 °C, illuminated with simulated sunlight (12:12h light/dark cycle). The
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17 soil was collected from Santaella, Southern Spain (province CO Lat. 37º34´03´´N; Long.
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4º50´48´´O), which has clay loam soil type. Three nitrogen-fertilizing treatments were used in
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22 this study with a completely randomized duplicated block design with 12 plants per block (12
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24 pots x 2 blocks x 3 treatments). These treatments included tap water only, conventional
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26 fertilization with (NH4)2HPO4 (diamoniumm hydrogen phosphate, DAP) and nanoU-NPK. DAP,
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29 which is one of the most common fertilizers used in the field, is particularly suitable as control in
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31 these experiments because it contains high content of P, as occurring with the nanoparticles. The
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33 experimental setup applied an initial amount of conventional fertilizer (36 kg of N ha-1) to all the
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treatments at planting. Tap water was applied as irrigation water when needed, usually three
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38 times a week. During the stem elongation, and just before ear formation, the three groups of
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40 plants were treated as follows: (1) untreated control group, received only water; (2) conventional
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fertilization group, received 150 kg of N ha-1 of granular DAP (thus, with P at a dosage of 170 kg
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45 of P ha-1); (3) nanoU-NPK fertilization group, received 15 kg of N ha-1 of a sprayed aqueous
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47 suspension of nanoU-NPK and 60 kg of N ha-1 of granular DAP (with P at a total dosage of 120
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49 kg of P ha-1). Since it is known that P is not a limiting factor for yield in wheat, the study was
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3 2.10. Statistical Analyses
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6 Statistical comparisons were performed with GraphPad Prism software (version 6.0) using
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8 one-way ANOVA and Bonferroni’s post hoc test. Differences in the obtained numerical results
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10 were considered statistically significant when P-values are less than 0.05 (i.e., P < 0.05).
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16 3. Results and discussion
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18 3.1. Bio-inspired CaP mineralization in the presence of nitrate ions
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20 WAXTS data of powdered nano-PK and nano-NPK, collected at increasing maturation times,
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23 evidence the structural evolution of the nanoparticles upon maturation (Figure 1a,c). At low
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25 maturation times (5 min and 4 hours), only two very broad humps are observed, indicating the
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27 precipitation of ACP both in the presence and absence of nitrate. TEM imaging confirms the
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precipitation of amorphous round-shaped nanoparticles with diameters falling in the 10 - 25 nm
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32 range at the early stages of maturation (Figure 2a). On the other hand, broad Bragg peaks emerge
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34 after 24 hours of maturation in both samples (Figure 1a,c), witnessing the formation of poorly
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36 crystalline apatite nanocrystals. These diffraction data exclude the co-precipitation of highly
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39 crystalline nitrate salts jointly with the nanoparticles.
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33 Figure 1. Synchrotron (λ = 0.775 Å) WAXTS data of nano-PK (a) and nano-NPK (c) samples at
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35 increasing maturation times (5 min, 4 h, 24 h and 48 h, vertically offset for the sake of clarity).
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37 Asterisks indicate the presence of small fraction of potassium chloride salts. Mass fraction (from
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DSE analysis) of amorphous (ACP) and crystalline (Ap) components in nano-PK (b) and nano-
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42 NPK (d) samples at increasing maturation time.
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47 The WAXTS data were analyzed by the DSE method.29 Atomistic models of nanocrystalline
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apatite were generated, in the form of tiny platelets as those of bone,29 and used to calculate the
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52 corresponding simulated X-ray pattern, to be matched to the measured data (as detailed in
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54 Materials and Methods and SE of SI). With this modeling approach, simulations of the
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3 nanoapatite fraction carry both structural (crystal arrangement and vacancies) and
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6 microstructural (size, morphology and lattice strain) information, within a unique atomic-to-
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8 nanoscale model. The best fit of nano-PK sample (maturation time: 48 hours) provided by the
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10 DSE method is shown in Figure S1 (Supporting Information, SI). WAXTS data collected on pure
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ACP samples were used as part of the model and, jointly to the nanoapatite simulation, scaled to
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15 the experimental pattern (Figure S1). The mass-based average thicknesses (T), widths (W) and
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17 lengths (L) of nano-PK and nano-NPK nanocrystals as retrieved through the DSE analysis are
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shown in Table S1. Very small crystals were found after 24 hours of maturation, exhibiting a
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22 platy morphology with thicknesses of around 2 nm, which is in agreement with the value
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24 reported for bone apatite nano-platelets.30 The presence of nitrate does affect neither the size nor
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26 the morphology of the nanocrystals (Table S1).
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45 Figure 2. TEM images of nano-NPK samples at 5 minutes (a) and 24 hours (b) of maturation at
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48 37 ºC. Insets show high magnification of nano-NPK particles (scale bar=50 nm). TEM images of
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50 pristine CaP nanoparticles showed very similar morphologies (data not shown).
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53 In addition, DSE analysis allowed us to quantify the mass fractions of the amorphous (ACP)
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55 and nanocrystalline (Ap) components (Figure 1b,d). The weight percentage of ACP accounts for
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3 ~60% (nano-PK) and ~79% (nano-NPK) after 24 hours and ~51% (nano-PK and nano-NPK)
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6 after 48 hours (Figure 1b,d). TEM micrographs of nano-NPK after 24 hours of maturation
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8 (Figure 2b) show elongated nanoparticles with platy morphology. Since the co-precipitation of
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10 round-shaped purely amorphous nanoparticles was not detected by TEM at these maturation
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times, we can infer that the amorphous and crystalline components are associated resulting in
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15 composite nanoparticles, as it has been previously observed.28,29,38,39 However, a very small
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17 fraction of non-transformed amorphous particles (with round and/or platy morphology)
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remaining after 24 hours (and not detected by TEM) cannot be totally ruled out from our
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22 experimental results. Thus, by combining WAXTS and TEM imaging we can conclude that ACP
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24 precipitates at the early stages and then gradually transforms upon maturation to nanocrystalline
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26 apatite composites, containing an amorphous component. The addition of nitrate in the solution
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29 slightly delays the transformation of the ACP phase, but does neither modify the ACP-to-Ap
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31 conversion pathway nor the morphology/sizes of the nanocrystals.
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51 Figure 3. (a) FTIR spectra of the nano-PK and nano-NPK (maturation time: 24 hours). (b) Time-
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54 dependent evolution of the ν3(NO3) band of nano-NPK samples. (c) Nitrogen content
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56 (determined by elemental analysis) of nano-NPK collected at 5 minutes, 4, 24 and 48 hours. Data
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3 are expressed as mean with the corresponding standard deviation as error bars. Statistically
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6 significant differences between measurements are marked with * (P-value < 0.05).
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9 FTIR spectra of nano-PK and nano-NPK at low maturation times are characteristic of ACP, with
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11 broad phosphate vibrational bands, which become progressively sharper upon maturation (Figure
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13 S2 and Table S2, SI).40 These variations are fully consistent with the initial precipitation of ACP,
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16 which gradually transforms to Ap. FTIR spectra also show additional peaks related to carbonate,
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18 citrate and water (Figures 3a-b and S2 and Table S2, SI).40 The effective doping with nitrate
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20 (Figure 3a-b and S2b) is witnessed by the sharp band appearing at 1385 cm-1, which is assigned
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23 to the anti-symmetric stretching mode (ν3) of NO3. Since the precipitation of non-reacting nitrate
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25 salts was excluded by WAXTS data, we can confirm that nitrate ions are trapped within the
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27 nano-NPK nanoparticles and/or adsorbed at the surface. The intensity of nitrate band and,
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consequently, the amount of nitrate of the nano-NPK, increases with the maturation time up to
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32 24 hours and then abruptly decreases (Figure 3b). This trend in nitrogen content was confirmed
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34 by elemental analysis (Figure 3c). The maximum amount of nitrogen (0.75 wt% N) was indeed
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36 found after 24 hours and fell below 0.20 wt% after 48 hours (Figure 3c). The decrease of
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39 nitrogen content seems to be associated to the ACP transformation. Indeed, the amount of ACP
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41 drops from 79 wt% to 51 wt% in 24h and 48h maturated nano-NPK. To further confirm the
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43 influence of ACP to Ap conversion on nitrogen incorporation, we carried out the synthesis of
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46 nano-NPK at 80 ºC and analyzed the powders at increasing maturations times by WAXTS
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48 (Figure S3a, SI). As expected, raising the temperature resulted in a much faster ACP-to-Ap
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50 transformation: indeed Ap diffraction peaks are well visible after 4h (Figure S3a,b), whereas,
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when the synthesis is carried out at 37 °C, incipient crystallization takes place after 24h (Figure
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55 1c,d). The decrease in nitrogen content occurs concomitantly with the ACP-to-Ap transformation
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3 also in the syntheses at 80°C (Figure S3c-d). In fact, nano-NPK samples synthesized at 80ºC
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6 incorporated nitrogen, in a much lower amount (0.31 wt%, Figure S3d), at 5 minutes of
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8 maturation, i.e. when ACP is the unique phase (Figure S3b). Though, according to WAXTS-DSE
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10 analysis (Figure S3b), some amorphous material is still present after 4 hours, the nitrogen content
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nearly vanishes when the crystallization occurs (from 4 hours onwards, Figure S3d). These
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15 findings highlight the higher adaptability of ACP structure to incorporate nitrate to a higher
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17 extent than (nano)crystalline Ap does. The high(er) loading capacity of ACP is associated to its
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higher surface reactivity, short-range order and to the presence of a high content of tightly bound
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22 water.4,38,41,42
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28 3.2. Increasing the nitrogen content by urea functionalization
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30 Nano-NPK nanoparticles were also precipitated in the presence of urea, (NH2)2CO, the most
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32 used N fertilizer (containing 46 wt% of N), with the aim of increasing the nitrogen content of the
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nanoparticles and providing two nitrogen sources: nitrate and urea, the latter being easily
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37 hydrolyzed to NH3 and CO2 in water. The structural evolution of urea-doped NPK nanoparticles
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39 (nanoU-NPK) upon maturation was studied by Raman Spectroscopy, which allows the ACP-to-
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41 Ap conversion to be monitored by the characteristic blue-shift of the ν1PO4 Raman peak (Figure
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44 4a).38 Moreover, Raman signals of urea and nitrate can be detected at ca. 1000 and 1050 cm-1,
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46 respectively (Figure 4a and Figure S4a, SI). According to the position of the ν1PO4 Raman peak,
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48 ACP is stable until 24 hours of maturation, but for prolonged times, is then transformed to Ap
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51 (inset of Figure 4a). Laboratory XRPD results confirm this evolution, with the appearance of the
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53 Bragg reflection of nanocrystalline apatite after 48 hours (Figure S4b). In addition, combining
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55 Raman and XRPD results, the co-precipitation of other calcium phosphate phases, highly
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3 crystalline salts (including alkali nitrates) or crystalline urea can be excluded. Accompanied by a
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6 blue-shift of ν1PO4 peak upon maturation, a significant decrease in the intensity of nitrate (Figure
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8 S5, SI) and urea Raman peaks (Figure 4b) becomes evident. Elemental analysis confirms that the
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10 total nitrogen content of as synthesized samples abruptly decreased after 48 hours of maturation
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(Figure 4c), when ACP is partially converted. This behavior corroborates the higher loading
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15 capacity of ACP (than Ap) to host exogenous ions (e.g., nitrate) or molecules (e.g., urea).
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Figure 4. (a) Raman Spectra of nanoU-NPK at different maturation times: 5 min and 9.5, 24, 48
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56 and 100 h. The inset displays the shift of the symmetric stretching mode (ν1) of the tetrahedral
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3 PO4 group from 952 cm-1 (ACP) to 959 cm-1 (Ap). (b) Histograms of the normalized Raman peak
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6 intensity of urea (IνCN/Iν1PO4). (c) Total N content determined by elemental analysis. Data are
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8 expressed as mean with the corresponding standard deviation as error bars. Statistically
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10 significant differences between measurements are marked with * (P-value < 0.05) or *** (P-
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value < 0.001).
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18 3.3. Nutrient release kinetics in aqueous media
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20 Calcium phosphate nanoparticles are only partially soluble in water and, therefore, provide a
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slow and gradual release of the ionic constituents (Ca2+ and PO43-).9 ACP is the most soluble
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25 calcium phosphate phase at neutral or slightly acidic pH.9 Thus, the higher solubility and higher
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27 loading capacity make ACP the ideal candidate for the controlled delivery of agrochemicals
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29 (e.g., fertilizers, pesticides, and herbicides) and target-specific biomolecules (e.g., DNA,
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32 proteins, etc).43 The fully amorphous nanoparticles obtained at very short maturation times (5
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34 minutes) and doped with multi-nutrients (potassium, nitrate and urea) were used in the release
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36 tests. The data were compared to those obtained from nano-PK nanoparticles (amorphous
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39 nanoparticles not containing nitrogen), with the aim of evaluating the impact of nitrogen in the
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41 release kinetics (Figure 5a). The composition of both types of nanoparticles is summarized in
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43 Table 2.
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Table 2. Chemical composition (wt%) of nano-PK and nanoU-NPK quantified by ICP-OES(a),
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47 elemental analysis(b) and colorimetric methods(c).
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50 Caa Pa Ka Nb Nitratec Ureac
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Nano-PK 24.9±0.25 11.3±0.13 1.1±0.03 ----- ----- -----
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55 NanoU-NPK 23.3±0.55 10.1±0.09 1.5±0.05 2.8±0.3 2.9±0.4 4.8±0.34
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Dried nanoparticles were immersed in water and the concentrations of the released nutrients
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9 (Ca, P, K and N) were analyzed, typically, over few days. Figure 5a displays the time-dependent
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11 Ca, P and K release from nanoU-NPK and nano-PK nanoparticles; for each nutrient, the
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13 cumulative wt% is normalized to its initial amount, experimentally determined (Table 2). For
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16 both types of nanoparticles, Ca and P follow a gradual and slow release profile, which can be
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18 fitted to first order kinetics with release rates. Both amorphous nanomaterials are non-
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20 stoichiometric with Ca/P ratio (Table 2) significantly higher than 1.5 (i.e., the stoichiometric
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23 Ca/P ratio of pure ACP), due to citrate7 and carbonate incorporation.41 The high Ca/P ratio can
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25 then explain the slightly faster release of Ca than P (k(Ca) = 0.03 h-1 vs k(P) = 0.02 h-1, Table S3,
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27 SI).
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The release of K follows very similar kinetics (k = 0.03 h-1), after a substantial sudden release
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32 (a so called burst effect), likely due to K ions weakly bound at the ACP surface (ca. 20 % wt of
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34 the total K, Figure 5a). Ca, P and most of the K ions are then released by nanoparticle
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36 dissolution, witnessed by similar kinetic constants in both nano-PK and nanoU-NPK (Table S3,
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39 SI), suggesting that N-doping does not significantly influence the dissolution kinetics attributed
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41 to nanoparticle degradation.
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19 Figure 5. (a) Ca, P and K release profiles from amorphous nanoU-NPK (spheres) and
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22 amorphous nano-PK (diamonds) in aqueous media during one week. Dashed lines represent the
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24 theoretical curves estimated according to first order equation (release constants, k, are shown in
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26 Table S3, SI). Data are expressed as mean with the corresponding standard deviation as error
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bars. (b) Total nitrogen release profile from nanoU-NPK (spheres) resulting from the sum of
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31 nitrogen released as nitrate (empty squares) and as urea (empty diamonds). The N release shows
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33 two kinetic regimes: a first burst release (Fitting 1, k = 1.97 h-1, Table S3, SI) followed by a
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smooth(er) release (Fitting 2, k = 0.01 h-1, Table S3, SI).
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40 The delivery of nitrogen follows a typical profile of weakly bonded species at the surface of
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42 the nanoparticles (Figure 5b). Most of the nitrogen (~90 %) was found in solution after few
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45 hours, 80 % in form of urea and 20% in form of nitrate (Figure 5b). The rest of the nitrogen (~10
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47 %) is then gradually and slowly released (Figure 5b) with a rate comparable to that of
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49 nanoparticle dissolution (Table S3). This much slower release of N is likely due to N-species
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either strongly bounded on the surface and/or incorporated into the labile ACP structure, and are
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54 released only when the nanoparticle undergoes partial dissolution.
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3 Summarizing, the nanoU-NPK provide a slow and gradual release of the most important plant
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6 macronutrients (NPK), with N in two chemical forms and different kinetics; this are another
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8 important feature, so far unreported, of our N-bearing nano-fertilizers, which also carry another
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10 important micronutrient (Ca). Eventually, the concentration of nutrients supplied by 10 g L-1 of
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nanoU-NPK nanoparticles to the media after one week was: 27 for Ca, 6.2 for P, 41 for K, 315
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15 urea and 134 for NO3 (values in mg L-1).
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3.4. Urea release kinetics in simulated solid media: leaching experiments
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22 The release of urea from nanoU-NPK was also monitored in a vertical column containing a
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24 simulated solid medium (leaching experiments, inset of Figure 6). Time-dependent eluted urea
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26 concentrations from the nanocomposite were analyzed by UV-Vis spectroscopy and compared to
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29 those eluted from a crystalline urea pellet (Cry-U, commonly used in conventional treatments).
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31 The high solubility of urea in water provides an instantaneous release when applied in Cry-U
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33 form, 90% of N being leached in less than one hour (Figure 6). However, after 1 h, the released
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36 amount was reduced by ∼10 times when urea is incorporated into nanoU-NPK (Figure 6), 83 %
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38 of urea being leached away only after ca. 6 hours. Henceforth, urea, supplied as Cry-U, as in
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40 conventional fertilization treatments, is readily lost, with a kinetic constant (k = 1.99 h-1) five
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times higher than that estimated for nanoU-NPK (k = 0.38 h-1) (Table S4, SI). This release rate is
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45 in agreement with that previously obtained from urea-hydroxyapatite nanohybrids.20,23
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Figure 6. Urea release from pellets of Cry-U (blue) and nanoU-NPK (magenta) embedded in a
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37 simulated solid medium, mimicking an inert soil (a low-diffusive medium) during water
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39 irrigation. Dashed-line curves represent the best fits of the experimental data, obtained by first
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41 order kinetic models (kinetic constants are shown in Table S4, SI). The inset shows a graphical
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44 representation of the column used in the leaching experiments.
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49 3.5. In vitro cyto-compatibility of nanoU-NPK
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52 It is widely accepted that biomimetic calcium phosphate nanoparticles as such have no
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54 inherent toxicity.3 This makes them ideal biomaterials for biomedical applications (e.g., tissue
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3 engineering and controlled delivery of drugs).9,11,12 With the aim of proving that urea and nitrate
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6 incorporation does not induce nanoparticles toxicity, which would be detrimental for food-based
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8 applications, we performed cell viability experiments (MTS assays) using Human Embryonic
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10 Kidney cells (HEK293) (Figure 7). As expected, results indicate excellent cell viability (ca.
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100%) even when high concentrations of nanoU-NPK nanoparticles (200 µg/ml) are used. This
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15 in vitro analysis demonstrates the good cyto-compatibility of our nanomaterials, confirming their
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39 suitability as green agrochemicals for the controlled delivery of nitrogen.
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Figure 7. Viability of Human Embryonic Kidney cells (HEK 293) in contact with nanoU-NPK
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46 at increasing nanoparticle concentrations (1, 50, 100 and 200 µg mL-1). Data are expressed as
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48 mean with the corresponding standard error as error bars. Any significant differences were found
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between measurements.
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6 3.6. Experiments on Durum wheat
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8 Preliminary experiments on durum wheat (triticum durum)44 under controlled conditions
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10 (growth chambers) were also performed with the aim of demonstrating the efficiency of the
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nano-composites for the controlled delivery of nitrogen in plants. During stem elongation, and
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15 just before ear formation, two fertilization treatments were applied: i) conventional treatment at a
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17 N dosage of 150 kg of N ha-1 (rates recommended for Andalusia and the Mediterranean area for
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cultivating wheat45) in the form of granular DAP and ii) treatment with nanoU-NPK at a total N
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22 dosage of 75 kg of N ha-1. The kernel weight, an important yield parameter strongly affected by
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24 the amount of applied nitrogen, was measured once the final plant physiological maturity was
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26 reached for the three treatments (Figure 8). Similar average kernel weights were obtained with
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29 the two treatments, despite the huge reduction of N applied with the nanoparticles (40 %wt in
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31 total). As expected, the untreated plants (receiving only tap water, control) showed a strong
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33 shrinkage of the average kernel weight. Significantly, it is well known that a relevant decrease of
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36
yield would be obtained with conventional fertilizers (e.g., DAP), if the nitrogen rate is reduced
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38 up to 40%.45,46 This increase in efficiency can be due to the controlled release of N (Figure 5 and
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40 6) and/or to a better immobilization within the soil matrix avoiding lixiviation (Figure 6).
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Furthermore, N-doped amorphous nanoparticles could favor the absorption and translocation of
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45 the nutrients through the plant tissues. However, further works are required to gaining a deeper
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47 understanding in the mechanisms underlying nanoparticle uptake and nutrients release inside the
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49 plants.
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28 Figure 8. Average kernel weight per plant. Data are expressed as mean with the corresponding
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31 standard error as error bars. Statistically significant differences between measurements are
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33 marked with * (P-value < 0.05).
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35 4. Conclusions
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Taking inspiration from bone biomineralization, we have synthesized multinutrient nano-
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40 fertilizers through a simple and green synthetic route, potentially scalable for industrial
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42 exploitation. By modulating the synthetic method in terms of maturation time and doping, we
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firstly evaluated the likelihood of N and K doping of CaP nanoparticles in the form of
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47 amorphous (ACP) or nanocrystalline apatite (Ap), as well as the impact of these species on the
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49 mineralization pathways. Then we optimized synthetic conditions in terms of macronutrient
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51 loading (N - both in the form of nitrate and urea - and K) and release kinetics performance. We
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54 obtained biomimetic ACP nanoparticles, intrinsically rich in Ca and P, doped with potassium,
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56 nitrate and urea (so called nanoU-NPK). While the amount of NK dopants is small compared to
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3 the molar content found in soluble conventional fertilizers, this work demonstrates that doped
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6 nanoparticles show a much more controlled release profile. Accordingly, comparative
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8 fertilization tests on wheat plants (triticum durum) in growth chambers showed that comparable
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10 yields of grains are obtained using nanoU-NPK, with the advantage of using a much lower N
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quantity (a reduction of 40% wt) than that supplied by conventional treatments.
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15 Our work evidences the potential benefits of engineering nanoparticles via bioinspired calcium
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17 phosphate crystallization to obtain controlled-release multinutrient nanosystems, promising
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materials for more efficient and sustainable practices in agriculture. Future work will be
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22 addressed to further enhance the encouraging properties in terms of increasing the N loading and
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24 favor a progressive release over a prolonged time (within days, or even weeks).
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36 ASSOCIATED CONTENT
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40 Supporting Information. The following content is supplied as Supporting Information:
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- S1. Analytical methods validation
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45
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47 - S2. Debye Scattering Equation (DSE) approach
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- List of figures (S1-S5).
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54 - List of tables (Table S1-S4).
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AUTHOR INFORMATION
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7 Corresponding Author
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10 * Author to whom correspondence should be addressed (José M. Delgado-López,
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12 jmdl@ugr.es)
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14
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16 Author Contributions
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19 The manuscript was written through contributions of all authors. All authors have
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21
22 given approval to the final version of the manuscript.
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24
25 ACKNOWLEDGMENTS
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29 This work has been performed thanks to the funding by Fondazione CARIPLO (Project No.
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31 2016-0648: Romancing the stone: size-controlled HYdroxyaPATItes for sustainable Agriculture
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33 – HYPATIA) and FEDER/Ministerio de Ciencia, Innovación y Universidades–Agencia Estatal
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36 de Investigación (FEDER/MCIU/AEI) through the projects NanoVIT (RTI-2018-095794-A-
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38 C22) and NanoSmart (RYC-2016-21042). GBRR acknowledges the FEDER/MCIU/AEI for her
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40 postdoctoral contract within the Juan de la Cierva Program (JdC-2017). The courtesy of the
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43 scientific and technical staff at the MS-X04SA beamline of the Swiss Light Synchrotron at the
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45 Paul Scherrer Institut in Villigen, CH, and the Centre for Scientific Instrumentation of University
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47 of Granada (CIC-UGR) is heartily acknowledged.
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53 REFERENCES
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56 (1) Wegst, U. G. K.; Bai, H.; Saiz, E.; Tomsia, A. P.; Ritchie, R. O. Bioinspired Structural
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12 (3) Epple, M. Review of Potential Health Risks Associated with Nanoscopic Calcium
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14 Phosphate. Acta Biomater. 2018, 77, 1–14.
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17 (4) Gómez-Morales, J.; Iafisco, M.; Delgado-López, J. M.; Sarda, S.; Drouet, C. Progress on
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20 the Preparation of Nanocrystalline Apatites and Surface Characterization: Overview of
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22 Fundamental and Applied Aspects. Prog. Cryst. Growth Ch. 2013, 59, 1–46.
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25 (5) Uskoković, V.; Tang, S.; Wu, V. M. On Grounds of the Memory Effect in Amorphous and
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27 Crystalline Apatite: Kinetics of Crystallization and Biological Response. ACS Appl.
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