Unconventional and Sustainable Nanovectors for Phytohormone

Research Article Cite This: ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

pubs.acs.org/journal/ascecg

Unconventional and Sustainable Nanovectors for Phytohormone Delivery: Insights on Olea europaea Ilaria Clemente,† Felicia Menicucci,† Ilaria Colzi,‡ Luca Sbraci,† Carla Benelli,§ Cristiana Giordano,§ Cristina Gonnelli,‡ Sandra Ristori,*,† and Raffaella Petruccelli§ †

Chemistry Department & CSGI, University of Florence, via Della Lastruccia 3, Sesto Fiorentino 50019, Italy Department of Biology, University of Florence, via Micheli 1, Florence 50121, Italy § Trees and Timbers Institute, CNR, via Madonna del Piano 10, Sesto Fiorentino 50019, Italy Downloaded via UNIV OF TASMANIA on October 29, 2018 at 08:42:17 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

ABSTRACT: Nanoscience has allowed outstanding progress in many fields of research. Concerning the transport and delivery of active principles (drugs, contrast agents, sensitizers etc.), the improvements obtained by miniaturized carriers have recently been extended from medicine and pharmacy to agriculture. However, when coming to crop production, issues such as scalability, eco-compatibility, and sustainability represent a veritable challenge, so far only partially addressed. In this study, we propose lipid-based nanoformulations for the administration of root-promoting phytohormones to different cultivars of Olea europaea. In order to maximize the efficiency of these novel carriers, we devised nanovectors made by lipids extracted from olive pomace, a material derived from the plant itself and representing a waste in oil processing. This allowed combining cost-effectiveness and environmentally friendly procedures. The implementation obtained using adjuvants, such as purified natural lipids forming stable and well-defined nanoobjects, was also investigated. Controlled and reproducible formulations were achieved after extensive study of the obtained systems, in particular by physicochemical characterization through advanced methods. Trials in vivo and in vitro showed that rooting was enhanced with respect to conventional treatments, thus indicating that the innovative formulations here fabricated have significant potentiality for the large-scale administration of agrochemicals in the context of sustainable economy. KEYWORDS: Green nanodelivery, Olive pomace, Rooting, Phytohormones, Small angle scattering

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delivering molecules of biomedical interest,9−13 and their precise atomic/molecular engineering has been exploited for imaging purposes, as well as for the treatments of different diseases.14,15 This knowledge can be expanded to design suitable nanovectors able to interact with plant cell membranes. For this purpose, the superior biocompatibility of lipid-based nanocarriers makes them very attractive to devise ad hoc formulations bearing similarities with the plants to be treated. The use of lipid mixtures extracted from target organisms themselves is an innovative procedure in drug delivery that can facilitate incorporation, thus improving the performance of encapsulated drugs. Following the present essential procedures of “green chemistry”, lipids to fabricate nanocarriers can be obtained from microorganisms, animals, or plants. In order to make the process not only eco-friendly, but also helpful for the circular economy, it is desirable to reutilize difficult to eliminate wasteproducts, such as olive mill pomace (OMP), as sources of

INTRODUCTION To feed the ever-growing global population, the scientific community is called to face the problem of increasing agricultural productivity while preserving soil health and crop biodiversity. Over the last decades, extensive use of pesticides and agrochemicals has led to soil degradation, environmental pollution, and resistance to plant pathogens, thus claiming for sustainable and eco-friendly technologies to increase the quantity and quality of agricultural products.1 Among the possible ways suitable to promote greener practices for agricultural production, the fostering of local products and of their cultivation through safer technologies has gained importance and support from regional and international institutions, especially in Europe.1 Recently, new plant biotechnologies at the nanoscale level have proven to be useful tools to manage some challenges of agricultural sciences.2−5 Phytochemical drugs can be encapsulated within inorganic nanoparticles (i.e., obtained from metal or metal oxides) or in soft matter nanocarriers. Both these vectors can allow microtransportation and intracellular delivery of poorly water-soluble molecules.6−8 Lipid-based nanocarriers, such as liposomes and micelles, have been extensively used for © XXXX American Chemical Society

Received: July 19, 2018 Revised: October 10, 2018 Published: October 15, 2018 A

DOI: 10.1021/acssuschemeng.8b03489 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

Research Article

ACS Sustainable Chemistry & Engineering Table 1. Nomenclature and Sample Composition sample acronym E IBA NAA EPE IPE NPE EPC IPC NPC

composition 200 200 200 160 160 160 160 160 160

mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL

pomace pomace pomace pomace pomace pomace pomace pomace pomace

+ IBA 10−2 M + NAA 10−2 M +20 mg/mL DOPE +20 mg/mL DOPE+ IBA 10−2 M +20 mg/mL DOPE+ NAA 10−2 M +20 mg/mL DOPC +20 mg/mL DOPC + IBA 10−2 M + 20 mg/mL DOPC + NAA 10−2 M

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lipids.16 The OMP is the solid waste made up of small pieces of stone and parts of the olive pulp and skin. It is produced in large quantities from the olive oil industry and it is potentially harmful if freely discarded in the environment, because of its low pH, high salinity, and presence of phytotoxic substances.16 Nanotechnologies could open up new opportunities in agriculture, especially for one of its most limiting factors, that is a sustainable vegetative propagation of plants.1,2 In vivo and in vitro vegetative propagation is necessary to produce clones, individuals genetically equal to each other and to the mother plant. Vegetative propagation allows one to obtain healthier crops with desirable agronomical and morphological traits. So far, in the context of plant multiplication, the most widely used root promoting compounds have been auxins (mainly IAA, indole-3-acetic acid, IBA, indole-3-butyric acid, and NAA, 1-naphtaleneacetic acid), a class of molecules that can either be found in nature or synthesized.17 Despite the help provided by phytohormone administration, the rooting process is quite difficult, not only because of the intrinsically different root ability of the cultivated varieties (cultivars), but also of the poor auxin solubility in water media. The latter negatively influences the transport from the site of application to the root initiation, thus reducing auxin availability.18 To overcome this limiting issue, large amounts of growth regulators are used in conventional practices, but still many horticultural and woody species remain recalcitrant to rooting.18 Therefore, studies aimed at developing novel techniques for phytohormone administration must be necessarily directed to increase their plant availability, while decreasing the total amount of auxins employed. In this work, we devised innovative lipid-based nanovectors to abide with the requirements of compatibility and ecosustainability principles, preparing auxin carriers using lipids extracted from OMP. The obtained nanosystems were extensively characterized in terms of structure and surface charge by physicochemical techniques, such as Dynamic Light Scattering, Zeta potential, and Small Angle (X-ray and Neutron) Scattering. We also engineered the new carriers by adding small amounts of purified, yet natural, phospholipids, to improve the structure and shelf-stability of our formulations. Finally, we evaluated the efficacy of our unconventional method of phytohormone administration on the initiation of the rooting process (in vivo and in vitro) in Olea europaea L., an extensively cultivated species typical of the Mediterranean area. Moreover, the preparation protocol was optimized to be easily scalable, in view of establishing a new nanobiotechnology standard, which is a cutting-edge issue19 for applications in agricultural research.

lipid employed

auxin loaded

only pomace lipids only pomace lipids only pomace lipids pomace lipids + DOPE pomace lipids+ DOPE pomace lipids+ DOPE pomace lipids+ DOPC pomace lipids+ DOPC pomace lipids+ DOPC

empty nanovectors IBA NAA empty nanovectors IBA NAA empty nanovectors IBA NAA

MATERIAL AND METHODS

Reagents. 1,2-Dioleyl-sn-glycero-3-phosphocoline (DOPC, 98% purity) and 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE ≥ 97% purity) were purchased from Lipoid GmbH Indole-3-butyric acid (IBA, 99% purity) and 1-naphtaleneacetic acid (NAA, ≥ 95% purity), Agar and all the components used in tissue culture were purchased from Sigma-Aldrich. Preparation of Liposomes Containing Rooting Hormones. Olive pomace from various cultivars was provided by local oil producers, then mixed together to obtain a homogeneous starting material, stored in freezer at −20 °C to prevent oxidation and defrozen right before use. For lipid extraction, aliquots of 200 mg of olive pomace were treated with 1 mL Folch solution (CHCl3/ CH3OH 2:1 v/v) under stirring at room temperature for 24 h. Two types of systems were prepared: (i) samples containing only lipids from pomace, (ii) samples containing olive pomace and pure phospholipids (DOPE or DOPC), added as adjuvant in small amount, i.e., 1:10 w/w with respect to the pomace. Consequently, by taking 50% as average lipid extraction, the final ratio was estimated to be 1:5 purified lipid/natural lipid. After solvent evaporation, a lipid film was obtained to which auxins dissolved in the proper organic solvents were added. Specifically, CHCl3/C3H6O 2:1 v/v solution was used for IBA, while NAA was dissolved in CHCl3. The stock concentration for both auxins was 10−2 M. Again, a dry lipid film was obtained by evaporation under-vacuum. This film was rehydrated with Milli-Q water and equilibrated for 8−12 h. The final suspension was homogenized through extensive vortexing, followed by eight cycles of freeze−thaw (liquid nitrogen/40 °C bath). Subsequent sonication (5 cycles of 3 min each) at high power was used to downsize the lipid vectors. Noteworthily, this protocol allowed to scale preparation up to a total volume of 1.2 L of lipid formulation in each trial. Smaller volumes (1−2 mL) for in vitro administration were prepared by extrusion performed 27 times with 100 nm polycarbonate porous membranes. These samples were filtered using sterile 0.45 μm pore filters and treated with UV light to avoid in vitro explant contamination during the treatments. In Table 1 all sample names, composition, types of lipid used, and encapsulated auxin are summarized. Dynamic Light Scattering, Zeta Potential, Small Angle Scattering, and Mass Spectrometry. Dynamic Light Scattering (DLS) is a powerful tool for assessing the structure of soft matter colloids (micelles, proteins, vesicles) in the length scale of a few nm to hundreds of nm. In this range, DLS can provide the mean size and size distribution of scattering objects for populations spreading over a single or double distribution, together with the corresponding polydispersity parameters. In this work DLS was performed on a Malvern Zetasizer (Nano ZS) equipped with a He−Ne 633 nm, 4 mW laser with backscattering optics and microDoppler effect for Zeta potential measures, after 1:20 dilution of all samples to adjust the optical turbidity. The cumulant expansion was employed to analyze the autocorrelation function of the scattered intensity and to obtain mean size and polydispersity index.20 In this work, the information obtained with DLS were complemented by Zeta potential measurements, which report on the surface charge of colloidal aggregates in solution. Specifically, Zeta B


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ACS Sustainable Chemistry & Engineering Table 2. Rooting Induction Treatments in Experiments 1, 2, and 3 code

treatment (T)

Experiment 1 January (48 h)

Experiment 2 April (72 h)

Experiment 3 July (96 h)

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11

control (C)a SP-IBA (4000 ppm) SP-NAA (2000 ppm) IBA (50 ppm) NAA (50 ppm) IBA (200 ppm) NAA (200 ppm) IPE (200 ppm) NPE (200 ppm) IPC (200 ppm) NPC (200 ppm)

x x x x x − − − − − −

x x x − − x x x x − −

x x x − − x x − − x x

a

Controls in plain water and in nanovectors without hormones, comprehensive of E, EPE, and EPC (Table 1).

potential data were obtained from the electrophoretic mobility employing the Helmholtz−Smoluchowski equation.21 The structural properties of plain and auxin loaded nanovectors at high resolution were then determined by Small Angle X-ray and Neutron Scattering (SAXS and SANS). SAXS experiments were performed at the ID02 beamline of the European Synchrotron Radiation Facility (Grenoble, France). The wavelength of the incoming beam was 1 Å, and the sample detector distances was 1 m, which covered a q range 0.103−6.5 nm−1 (q = (4π/λ) sin θ, where 2θ is the scattering angle). The 2D SAXS patterns initially recorded were normalized to the absolute scale using a standard procedure reported elsewhere.22,23 Samples were loaded on a flow through capillary of 2 mm diameter to ensure accurate background (water) subtraction. Data fitting was performed by using GAP (Global Analysis Program) package,24,25 that allows to reproduce the SAXS intensity diagrams of both quasi-Bragg peaks (arising from oligolamellar structures) and diffuse scattering (originated from monolamellar vesicles). It also provides the electron density profile as the sum of Gaussian distributions corresponding to the hydrophobic tails and the polar heads. The routine used in this work is based on the modified Caillé theory,26,27 which accounts for the bilayer bending rigidity. The main fitting parameters were extracted from the following expression for the scattered intensity:

I(q) =

sample to detector). All samples diluted 1:2 before measurement. The 2D SANS patterns were normalized to the absolute scale and the 1D patterns from the different configurations were concatenated.22 The 1D SANS curves fitting was performed by using SasView package (https://www.sasview.org/.). Mass spectrometry was used to assess the loading ability and possible cargo limits. Specifically, a liquid chromatography-electrospray mass spectrometer (HPLC−ESI MS, Thermo Instruments, CA, U.S.A.) was employed. In order to avoid possible masking effects exerted from the vectors, these latter were disrupted by 1:100 dilution with ethanol prior to column injection. Two different concentrations (5 × 10−3 M and 2 × 10−2 M) of auxin were tested and for all samples independently from the starting concentration of phytohormones, a loading capacity of 200−300 ppm was measured. The loading efficiency expressed as a percentage of the lower starting concentration, i.e., 500 ppm, was around 60%, corresponding to 1 molecule of auxin every 3−5 lipids. This insertion rate was high enough to allow the preparation of reasonable volumes of carriers for in vivo administration. Application of Nanovectors Loaded with Hormones to In Vivo and In Vitro Rooting. In Vivo Study. One-year old olive scions were collected from plants of cultivar (cv) Leccino (easy-toroot), one of the most cultivated cultivars in Italy and cv Leccio del Corno (difficult-to-root) an interesting cultivar for its agronomic and qualitative characteristics.18 After the harvest, scions 3−3.5 mm in diameter were cut to a length of 10−15 cm, with 4−6 nodes and 4 leaves, to obtain semihardwood cuttings. Each treatment consisted of 20 cuttings per experimental unit. After each treatment, the cuttings were transferred in greenhouse benches containing pearlite and kept under mist for 90 days. Swelling of the cutting base, callus presence, rooting percentage, and number and length of roots were analyzed. Eleven treatments were carried out during three independent experiments performed in January, April, and July of 2017 (Table 2). Cutting basal ends of Leccino and Leccio del Corno were dipped in IBA and NAA nanovectors with two different cargo concentrations, 50 ppm (T4, T5) and 200 ppm (T6, T7), and in 200 ppm cargo PE and PC formulations (T8-T11). Cuttings were soaked for 48, 72, and 96 h in different plain pomace, PE and PC series formulations (Table 2, Experiment 1; 2; 3). Respectively two types of controls were chosen, i.e., the “standard procedure” (SP; T2, T3) usually followed for olive propagation, and control (C; T1) in water and without hormones. For the standard procedures, powder IBA was dissolved in hydro-alcoholic solution at 4000 ppm; powder NAA was dissolved in aqueous solution at 2000 ppm; cuttings basal ends were dipped in the solutions for 7 s following the recommendations of Fabbri et al.18 For control cuttings, bases were dipped in nonloaded nanovectors and water for 48, 72, and 96 h, respectively (Table 2). In Vitro Study. Microcuttings (3 nodes length) of “Canino” were used in the experiment. “Canino” is a widespread olive cultivar in central Italy with high and constant productivity, and low susceptibility to the major diseases that affect olive.28 Various in vitro studies on this cultivar have been performed,29 but no

(1 − Ndiff )S(q)P(q) + Ndiff P(q) q2

(1)

where Ndiff is the fraction number of uncorrelated bilayers per scattering domain, S(q) the structure factor which accounts for interparticle interactions, and P(q) the absolute square of the form factor, accounting for intraparticle interactions. Other variables taken into account for the fits were the lamellar repeating distance (d) and the average number of bilayers per scattering domains (Nmean). The electron density profile, was modeled by symmetric Gaussian distributions centered at distance zh from the bilayer middle point. The polar headgroups and the hydrophobic tails standard deviations were σH and σc, respectively. The amplitude of the hydrophobic tail Gaussian relative to the headgroup was ρ′c, and fluctuaction were accounted for by the adimensional Caillè parameter. Regarding the SAXS diagrams of samples where a long-range ordering existed, as indicated by Bragg’s peaks, which could not be attributed to bilayers stacked in a lamellar arrangement, a phenomenological approach was chosen for data fitting. An example of this type, where three Gaussians centered at q values of 0.101, 0.175, and 0.201 A−1 (fwhm of 6.6 × 10−3, 1.0 × 10−2, and 1.2 × 10−2 A−1, respectively) was used to describe the sharp peaks superimposed to a background which was described by a Lorentzian broad distribution. SANS experiments were performed at the PAXY beamline of the Laboratoire Léon Brillouin (Saclay, France). Four instrumental configurations were chosen to cover a large q range, from 2 × 10−3 to 0.4 Å−1, with a good overlap between them (large q: λ = 5 Å; D = 1 m, middle q: λ = 5 Å, D = 3.5m, small q: λ = 8 Å, D = 5m, very small q: l λ = 15 Å, D = 7m, where λ is the wavelength and D the distance C


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ACS Sustainable Chemistry & Engineering information about its rooting ability is reported. Four different treatments were tested (Table 3): (1) IBA 2 mg/mL loaded

complexity expected for these systems. Nevertheless, all samples were stable over time, as checked by visual inspection and by repeated DLS measurements, and the suspensions remained monophasic and homogeneous even after 4−6 months. The main differences were observed as a function of lipid composition. In particular, samples of the PE series (Figure 1B) had smaller average dimensions (200−230 nm), with respect to samples from pomace only or to the PC series (350−400 nm, Figure 1A and C), thus evidencing the ability of DOPE to impart size control and improve monodispersity (Figure 2). All nanovectors prepared with purified phospholipids showed narrower size distribution with respect to pomace lipids (Figure 1). The same trend was found when comparing auxin-loaded systems. Moreover, aggregates containing purified lipids (either of the PE or PC series) did not show any marked size change due to the cargo loading, whereas for samples of the series prepared from pomace lipids only the average dimensions increased upon association with auxins. This confirmed that in the absence of purified phospholipids the size and distribution of nanoaggregates were less controlled and, consequently, guest molecules were able to easily induce variations. Concerning the surface charge, all samples had negative Zeta potential (∼−23b ÷ ∼−13 mV) and conductivity were in the range 0.03−0.08 mS/cm (Table 4). Both these properties explained the observed stability over time. After auxin loading, all nanovectors showed slightly less negative surface charge with respect to the corresponding empty systems, in line with the neutrality of the inserted molecules, expected to dilute the overall surface charge.33 Structural Characterization by SAXS and SANS. The scattering intensity of the formulations made from only pomace showed unstructured SAXS profiles, indicating that very polydisperse aggregates were in solution (Figure 3, green curve). The presence of either DOPE or DOPC, even though added in small percentage, significantly modified the SAXS curves (Figure 3, red curve). In the case of samples from PC series (Figure 3, blue curve), SAXS diagrams were dominated by the large oscillations pattern characteristic of monolamellar vesicles with mean diameter in the range of 100−200 nm and by the typical signature of lipid bilayers, as it can be inferred by the comparison with similar lipid systems.33 However, the presence of a quasi-Bragg correlation peak (Figure 4A) indicated that a fraction (around 12%) of bilamellar aggregates was present, in agreement with the value 2.0 of the Ndiff

Table 3. Treatments on Microcuttings cv Canino code

treatments

1 2 3 4

IBA (nanovectors) E (empty nanovectors) IBA liquid IBA 2 mg/L medium

length liquid treatments (h) 6 6 6

24 24 24

for 6 weeks OM OM OM OM

semisolid semisolid semisolid semisolid

medium medium medium medium

nanovectors, (2) nonloaded nanovectors (E), (3) IBA 2 mg/mL simply dissolved in aqueous solution, and (4) IBA 2 mg/L added in OM (olive medium30 semisolid medium (conventional method). The liquid solutions, used for rooting experiment, were sterilized by filtration (Sartorius 0.45 μ pore filter) and maintained under UV light for 12h. “Canino” microcuttings were soaked in 1 mL of all liquid treatments (1, 2, 3−Table 3) under laminar flux hood in sterile conditions and covered by plastic containers to prevent dehydration. The length of liquid pulse treatment was 6 and 24 h, following the explants were placed on hormone free OM semisolid medium supplemented with 50 mg/L Fe-EDDHA, 36 g/L mannitol, and 3 g/L Gelrite. Microcuttings in conventional method were cultured in semisolid medium for 6 weeks. In the experiment, 3 glass jars (500 mL) with 100 mL of semisolid OM medium containing 6 microcuttings each were employed, for a total of 12 jars and 72 microcuttings. All jars were maintained at 22 °C ± 1 °C, under a 16 h light/8h dark photoperiod at 60 μmol m−2 s−1 photosynthetically active radiation provided by cool-white fluorescent lamps. To favor root emission, the basal area of the microcuttings was kept in the dark by covering the base of the jars.31,32 Rooting percentage of microcuttings was evaluated after 6 weeks.

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RESULTS AND DISCUSSION Characterization by Dynamic Light Scattering and Zeta potential. DLS data showed that aggregates with mean size in the range 170−400 nm were formed both by the natural lipid (pomace series, E, IBA, and NAA) and composite formulations with DOPE (PE series, EPE, IPE, and NPE) or DOPC (PC series, EPC, IPC, and NPC), as shown in Figure 1. The polydispersity index (PDI) of all samples was in the range 0.2−0.4, indicating the presence of scattering objects with wide but controlled size distribution, which resulted from the balance among local spontaneous curvatures, component mixture and the procedure followed for preparation. The observed polydispersity was consistent with the structural

Figure 1. Size distributions obtained from Dynamic Light Scattering intensity, showing comparison among samples with different lipid composition. In each graph samples curves from the same series are reported, i.e. pomace only (1A), PE (1B), and PC (1C), thus assessing differences in size and monodispersity based on the lipids employed in each one. D


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ACS Sustainable Chemistry & Engineering

Figure 2. Dynamic Light Scattering intensity distributions as a function showing comparison among the plots made by taking into account the loaded compound. In the same graph samples from the three series loaded with the same molecule are plotted, namely no compound (2A), IBA (2B), and NAA (2C), to assess the presence (or absence) of trends in size and monodispersity depending on the cargo.

Table 4. Zeta Potential Values for the Three Series of Samplesa only pomace series

ZP (mV)

PE series

ZP (mV)

PC series

ZP (mV)

E IBA NAA

−19.4 −16.3 −16.5

EPE IPE NPE

−22.4 −23.5 −23

EPC IPC NPC

−17.6 −13.4 −13.6

Error value calculated over three different runs was ±0.2 mV.

a

SANS intensity profiles confirmed that the structural characteristics of the investigated systems mainly depended on their lipid composition. Indeed, samples from the PC series (Figure 6, left panel) showed the typical form factor of uncorrelated flat objects, in agreement with the bilayer-based aggregates evidenced by SAXS. On the contrary, the broad peak present in the SANS diagrams of samples from the PE series arose from long-range correlations among the nanostructures in solution (Figure 6, right panel) and was indicative of a more ordered arrangement in solution. The apparent differences in the SAXS and SANS diagrams are due to the well-known different contrast source and mechanism of interaction with matter of X-ray and neutrons, as reported in fundamental texts about Small Angle Scattering.21 Application of Nanovectors Loaded with Hormones to In Vivo and In Vitro Rooting Trials. In Vivo Study. The efficacy of the nanovectors for auxins delivery were tested on olive tree cuttings of the two different cvs easy-to-root “Leccino” and difficult-to-root “Leccio del Corno” in greenhouse under mist. In the first experiment conducted in January 2017, after 90 days from exposure to IBA and NAA loaded nanovectors at 50 ppm concentration for 48 h, for all treatments cv Leccino showed 65% of cuttings with swollen base and callus, while cv Leccio del Corno exhibited only callus in 25% of cuttings. Callus formation can be important in the rooting of olive cuttings since in several cultivars a close correlation was observed between callus development and rooting.18 In any case, the rooting process was observed only in cv Leccino (Figure 7), with a percentage of rooted cuttings in T4 (IBA 50 ppm) of 15% with an average root length of 0.4 cm, (Figure 8A). In T2 (SP-IBA) 10% of rooting with 0.8 cm of roots length was observed. No rooting was observed in all control treatments (Figure 7A). Exposing the cuttings to only pomace (T6, T7) and IPE and NPE (T8, T9) nanovectors loaded with IBA and NAA at 200 ppm for 72 h (Experiment 2, April 2017), the cvs Leccino and Leccio del Corno presented swollen base and callus in 24% and 10% of the cases, respectively. Rooting process was present

Figure 3. SAXS Intensity diagrams (I(q) vs q, curves shifted vertically for clarity) of samples loaded with NAA for each series, showing the different patterns due to the employed lipid.

parameter in Table 5. The other systems with the same lipid composition (BPC, IPC) showed similar patterns. The electron density profile (Figure 4B) was obtained from the best fit parameters listed in Table 5. This pattern shows the amplitude of the Gaussians for both polar heads and hydrocarbon chains as a function of the distance (in Angstrom) from the center of the bilayer, which corresponds to the minimum value. For samples of the PE series, the observed SAXS profile was characteristic of a more ordered structure, i.e., a lyotropic phase, as evidenced by three well-defined Bragg peaks (Figure 5) with maxima in the ratio 1: √3:2, typical of a hexagonal arrangement. This was consistent with the higher local curvature that DOPE is able to impart.35,36 The superposition of the Bragg peaks to a nonflat background stands for the presence of another, coexisting, population whose mean size is in the range of ∼100 Å, as estimated from the shoulder position. E


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Figure 4. SAXS diagram and best fit for sample loaded with NAA of the PC series (A), arrows on the peak corresponding to the first harmonics and the expected second harmonics,34 and the corresponding electron density profile (B), assessing the presence of a bilayer.

In a third experiment (July 2017), after 90 days a high percentage of Leccino and Leccio del Corno cuttings (more than 95%), soaked in nanovectors (T6, T7, T10, T11 treatments), showed signs of basal rot (Figure 8E, red arrow) although many examples of callous formation were observed (Figure 8E and F, green arrows). Nevertheless, in T10 treatment 1 cutting had root emission (Figure 8F black arrow). In T2 and T3 standard treatments, cuttings showed neither basal rot nor rooting, as expected for the brief soaking time of this procedure. The cuttings of all controls (T1) showed no sign of root formation. In olive trees, the success of rooting process can be affected by many factors, included the type of cultivar and the timing of cutting collection.18 The results of a screening on rooting rate of 426 cultivars reported that more than 60% of cultivars present a low rooting ability (0−33%), 20% have a medium rooting ability (33−66%), and only 16% of the total show a rate of rooting higher than 70%.37 The definition of the most suitable season for cutting preparation is drawn on the production cycle of self-rooted plants, which coincides with two annual peak points. The first period coincides with April− June (summer cycle), when vegetative growth is at its peak, and the second one with September−October (autumn cycle), before the physiological activity of the plant decreases owing to low winter temperatures.18 In vivo results confirmed the higher rooting ability of cv Leccino compared to cv Leccio del Corno. Furthermore, this study reported that cv. Leccino cuttings, dipped in IBA nanovectors in January, emitted roots, thus suggesting that the application of loaded nanovectors could increase the period of propagation of the cultivars in the nursery. In the second experiment, carried out in a favorable period for rooting, the aforementioned results were confirmed, and the addition of PE nanocarriers in treatments with both hormones favored the rhizogenic effect and increased the roots number and length. This is in line with the enhanced control imparted by DOPE on nanovectors design, stability, and cargo release, even in the case of NAA, the less bioavailable auxin. Moreover, the data revealed that 96 h dipping, regardless of the treatment applied, is unsuitable for olive rooting. However, even in these unfavorable conditions PC nanovectors induced swollen basal end (Figure 8E), which could prelude to subsequent root emission (Figure 8F). In this case further protocol adjustments need to be explored.

Table 5. SAXS Parameters for Best Fit and Electron Density Profile of the Sample in Figure 4a parameters d (Å) Nmean fluctuation parameter zh (Å)

values

parameters

± ± ± ±

σH (Å) σc (Å) ρ′c Ndiff

63.4 2.00 0.40 17.00

0.2 0.05 0.06 0.37

values 3.28 8.70 −0.880 0.7800

± ± ± ±

0.01 0.32 0.007 0.04

a

See text for detailed parameter description.

Figure 5. SAXS diagram and corresponding best fit for the sample loaded with NAA of the PE series, showing the ratios of the relative positions of the Bragg peaks.

only in Leccino and the maximum rooting percentage was observed in T6 and T8 treatment (Figure 7B) with, respectively, 7 and 25 mean number of fully developed roots, and an average length of 1.8 and 2.2 cm in both treatments (Figure 8B and C). Interestingly, rooting was observed also in NPE (NAA-DOPE) nanovectors (T9), with 10% of rooting, an average of 18 roots, whose length ranged from 1.5 to 1.7 cm (Figure 8D), while the cuttings dipped in NAA standard (T3) or in NAA nanovectors (T7) did not show roots. In T2 treatment, the percentage of rooted cuttings was of 10%, while no rooting was observed in all control treatments. F


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Figure 6. SANS diagrams and corresponding best fits for samples loaded with NAA of the PC series (left panel) and PE series (right panel), respectively.

Figure 7. Rooting percentage of Leccino cv cuttings in January (Experiment 1, A) and in April (Experiment 2, B). Bar diagram for a percentage made Standard Error values.

A previous approach using IBA- and IAA-stabilized silver nanoparticles (AgNPs) in Hibiscus rosa sinensis was reported by Thangavelu et al.38 They observed a higher rooting percentage and root number per cuttings in treatments with AgNPs in ex vitro condition. In our research, similarly, the treatment with hormones encapsulated in nanocarriers enhanced the rooting process, this effect might be due to improved hormone bioavability and easier accumulation, with supposedly a slow release in the basal part of the cutting.39 In Vitro Study. In the evaluation of loaded nanovectors efficacy in “Canino” cultures, after 6 h of pulse liquid treatment and transferring into OM semisolid medium for 6 weeks (Figure 9), the percentage of root development was higher in microcuttings exposed to IBA loaded nanovectors (27%) than in microcuttings treated with liquid IBA administration (18%) (Figure 10). The extension of the length of treatment to 24 h was not effective in increasing the percentage of rooting, reducing it to 16% in the case of IBA loaded nanovectors and to 10% in liquid IBA (Figure 10). Moreover, a prolonged treatment revealed symptoms of suffering in microcuttings. The treatment with nonloaded formulations (E), both at 6 and 24 h, showed some callus formation and no rooting in microcuttings (Figure 11A). Semisolid medium with conventional IBA administration recorded 16% of rooting (Figure 11B), similar to IBA liquid after 6 h, but poor rooting was

produced in this treatment with respect to 6 h IBA loaded nanovectors application (Figure 11C−E). In our conditions in vitro cultures treated with nanovectors showed less contamination compared to nonloaded nanovectors ones. Therefore, 6 h liquid pulse treatment with loaded nanovectors at the concentration of 2 mg/mL was associated with more favorable emission and development of roots. The rooting microcuttings were in healthy conditions, thus further confirming the compatible and nontoxic nature of these treatments. The same condition was recorded also in microcuttings without roots treated with empty nanovectors. Phytotoxic effect is an aspect that can characterize carbon particles used in plant treatments40 and that needs to be taken into account. The lipid-based nanovectors used in our study, being of plant origin, because deriving from olive pomace, did not show any visual sign of toxicity in the microcuttings themselves. In in vitro culture, in most cases, the culture medium is directly supplemented with IBA, before autoclaving, at the rate increasing from 1 to 4 mg/L41 to obtain highest rooted explants and root quality. In our research, the application of 2 mg IBA in conventional method showed lower rooting percentage in respect to other in vitro olive cultivars.41 To improve the rooting capability an increase in IBA concentration would be desirable for each treatment assessed. G


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Figure 8. Experiment 1 - Rooting effect of in vivo cultured cv Leccino cuttings treated with IBA loaded nanovectors 50 ppm (A): callus formation, roots emission, and swelling of the base; Experiment 2Fully developed root in in vivo cultured cv Leccino cuttings treated with IBA 200 ppm loaded nanovectors (B); IPE loaded nanovectors (C) and NPE loaded nanovectors (D); Experiment 3Callus formation in in vivo cultured cv Leccino cuttings treated with NPC loaded nanovectors (E) and IPC loaded nanovectors (F): basal rot (red arrows), callus formation (green arrows), and root emission (black arrow).

Figure 9. In vitro olive microcuttings: A, B during treatment with nanovectors loaded with IBA for 6 h; C microcuttings transferred in OM semisolid medium for 6 weeks.

COOH) was beneficial for plant growth and rooting.44 The possibility that the SWCNT particles establish interactions with the mineral salts, the organic acids, and carbohydrates that compose the medium was supposed. The use of SWCNTs in Ficus carica, on the contrary, have no influence on rooting of in vitro plants.40 Experience of the nanobiotechnology approach using plant rooting hormones stabilized in silver nanoparticles (AgNPs) was reported by Thangavelu et al.;38 in this study the application of AgNPs in Nicotiana tabacum enhanced in vitro root growth respect to plant control. Similar results, with the

However, rooting abilities vary significantly among different olive cultivars.18,41 Regarding the sterilization method of liquid solutions, filtration and UV irradiation procedures have been considered,42 as the elevated temperatures used for autoclaving are not suited for formulation stability of lipid-based nanovectors. The effect of nanoparticles in plants depends and varies according to their composition, size, and physical and chemical properties, all these characteristics can influence absorption, translocation, and interaction with the cells of plant.43 The application in in vitro culture of blackberry (Rubus adenotrichos) of functionalized nanoparticles (SWCNTsH


Research Article


biocompatible methods in next generation agricultural practices, under the auspices of the circular economy.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: sandra.ristori@unifi.it. ORCID

Sandra Ristori: 0000-0003-0708-3956 Notes

The authors declare no competing financial interest.

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Figure 10. Rooting percentage after 6 and 24 h liquid pulse treatments in “Canino” microcuttings Bar diagram for a percentage made Standard Error values.

ACKNOWLEDGMENTS The authors wish to thank Michele Sorini for contributing to the experiments in micropropagation. Thanks are also due to Dr. Giulia Fadda and Dr. Michael Sztucki for their help as local contacts at PAXY and ID02, respectively. The ESRF (Grenoble, France) and to the LLB (Saclay, France) are acknowledged for beam time allocation.

application of lipid-based nanovectors in in vitro olive culture are obtained in our study.

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CONCLUSIONS In this research, we fabricated innovative and sustainable lipid nanovectors, developing a scalable protocol and obtaining stable formulations from olive pomace to encapsulate auxins. The potential action of auxin-loaded nanovectors was tested in olive trees and was found to improve the rooting process both in in vivo and in in vitro trials. Moreover, in in vivo conditions, the administration of the loaded nanocarriers to cuttings decreased the concentration of supplied phytohormones used in standard nursery conditions at least 10-fold. This study can be a significant initial step in the improvement of the rooting process in recalcitrant cultivars and in the application of green nanobiotechnology to Olea europaea. These results open new perspectives for agrochemical delivery by means of advanced

REFERENCES

(1) Kashyap, P. L.; Xiang, X.; Heiden, P. Chitosan Nanoparticle Based Delivery Systems for Sustainable Agriculture. Int. J. Biol. Macromol. 2015, 77, 36−51. (2) Khot, L. R.; Sankaran, S.; Maja, J. M.; Ehsani, R.; Schuster, E. W. Application of Nanomaterials in Agricultural Production and Crop Protection: A Review. Crop Prot. 2012, 35, 64−70. (3) Ranjan, S.; Dasgupta, N.; Lichtfouse, E., Eds. Nanoscience in Food and Agriculture 3. Sustainable Agriculture Reviews; Springer, Vol 23, 2016. (4) Lu, W.; Kelly, A. L.; Miao, S. Improved Bioavailability of Encapsulated Bioactive Nutrients Delivered through MonoglycerideStructured O/W Emulsions. J. Agric. Food Chem. 2017, 65, 3048− 3055.

Figure 11. In vitro olive microcuttings rooting after 6 weeks: A callus formation after treatment with nonloaded nanovectors; B roots development in treatment with IBA added in OM semisolid medium; and C−E after treatment of 6 h with IBA-loaded nanovectors. I


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ACS Sustainable Chemistry & Engineering (5) Zhou, L.; Cai, D.; He, L.; Zhong, N.; Yu, M.; Zhang, X.; Wu, Z. Fabrication of a High-Performance Fertilizer To Control the Loss of Water and Nutrient Using Micro/Nano Networks. ACS Sustainable Chem. Eng. 2015, 3, 645−653. (6) Malmsten, M. Soft Drug Delivery Systems. Soft Matter 2006, 2, 760−769. (7) Angelova, A.; Garamus, V. M.; Angelov, B.; Tian, Z.; Li, Y.; Zou, A. Advances in Structural Design of Lipid-Based Nanoparticle Carriers for Delivery of Macromolecular Drugs, Phytochemicals and Anti-Tumor Agents. Adv. Colloid Interface Sci. 2017, 249, 331−345. (8) Roy, I.; Ohulchanskyy, T. Y.; Pudavar, H. E.; Bergey, E. J.; Oseroff, A. R.; Morgan, J.; Dougherty, T. J.; Prasad, P. N. CeramicBased Nanoparticles Entrapping Water-Insoluble Photosensitizing Anticancer Drugs: A Novel Drug−Carrier System for Photodynamic Therapy. J. Am. Chem. Soc. 2003, 125, 7860−7865. (9) Harris, L.; Batist, G.; Belt, R.; Rovira, D.; Navari, R.; Azarnia, N.; Welles, L.; Winer, E. Liposome-Encapsulated Doxorubicin Compared with Conventional Doxorubicin in a Randomized Multicenter Trial as First-Line Therapy of Metastatic Breast Carcinoma. Cancer 2002, 94, 25−36. (10) Morandi, S.; Ristori, S.; Berti, D.; Panza, L.; Becciolini, A.; Martini, G. Association of Sugar-based Carboranes with Cationic Liposomes: an Electron Spin Resonance and Light Scattering Study. Biochim. Biophys. Acta, Biomembr. 2004, 1664, 53−63. (11) Li, N.; Zhuang, C.; Wang, M.; Sun, X.; Nie, S.; Pan, W. Liposome Coated with Low Molecular Weight Chitosan and its Potential Use in Ocular Drug Delivery. Int. J. Pharm. 2009, 379, 131− 138. (12) Torchilin, V. P. Lipid-Core Micelles for Targeted Drug Delivery. Curr. Drug Delivery 2005, 2, 319−327. (13) Ristori, S.; Ciani, L.; Candiani, G.; Battistini, C.; Frati, A.; Grillo, I.; In, M. Complexing a Small Interfering RNA with Divalent Cationic Surfactant. Soft Matter 2012, 8, 749−756. (14) Yih, T.; Al-Fandi, M. Engineered Nanoparticles as Precise Drug Delivery Systems. J. Cell. Biochem. 2006, 97, 1184−1190. (15) Doane, T. L.; Burda, C. The Unique Role of Nanoparticles in Nanomedicine: Imaging, Drug Delivery and Therapy. Chem. Soc. Rev. 2012, 41, 2885−2911. (16) Gómez-Muñoz, B.; Hatch, D. J.; Bol, R.; García-Ruiz, R. The Compost of Olive Mill Pomace: From a Waste to a Resource Environmental Benefits of Its Application in Olive Oil Groves. In Sustainable Development Authoritative and Leading-Edge Content for Environmental Management; Curkovic, S., Ed.; INTECH: Rijeka, Croatia, 2012; pp 459−484. (17) Saini, S.; Sharma, I.; Kaur, N.; Pati, P. K. Auxin: a Master Regulator in Plant Root Development. Plant Cell Rep. 2013, 32, 741− 757. (18) Fabbri, A.; Bartolini, G.; Lambardi, M.; Kailis, S. G. Olive Propagation Manual; Landlinks Press: Australia, 2004. (19) Abbati de Assis, C.; Greca, L. G.; Ago, M.; Balakshin, M. Y.; Jameel, H.; Gonzalez, R.; Rojas, O. J. Techno-Economic Assessment, Scalability and Applications of Aerosol Lignin Micro- and NanoParticles. ACS Sust. Chem. Eng. 2018, Article ASAP, DOI: 10.1021/ acssuschemeng.8b02151. (20) Koppel, D. E. Analysis of Macromolecular Polydispersity in Intensity Correlation Spectroscopy: the Method of Cumulants. J. Chem. Phys. 1972, 57, 4814−4820. (21) Hunter, R. J. Zeta Potential in Colloid Science; Academic Press: London; 1981. (22) Lindner, P., Zemb, T., Eds. Neutron, X-Rays and Light Scattering Methods Applied to Soft Condensed Matter; North Holland Press: Amsterdam, 2002. (23) Boesecke, P. Reduction of Two-Dimensional Small- and WideAngle X-Ray Scattering Data. J. Appl. Crystallogr. 2007, 40, s423− s427. (24) Pabst, G.; Rappolt, M.; Amenitsch, H.; Laggner, P. Structural Information from Multilamellar Liposomes at Full Hydration: Full qRange Fitting with High Quality X-Ray Data. Phys. Rev. E: Stat. Phys., Plasmas, Fluids, Relat. Interdiscip. Top. 2000, 62, 4000−4009.

(25) Pabst, G.; Koschuch, R.; Pozo-Navas, B.; Rappolt, M.; Lohner, K.; Laggner, P. Structural Analysis of Weakly Ordered Membrane Stacks. J. Appl. Crystallogr. 2003, 36, 1378−1388. (26) Caillé, A. Remarques sur la Diffusion des Rayons X dans les Smectiques. C. R. Acad. Sci. Paris B 1972, 274, 891−893. (27) Zhang, R.; Suter, R. M.; Nagle, J. F. Theory of the Structure Factor of Lipid Bilayers. Phys. Rev. E: Stat. Phys., Plasmas, Fluids, Relat. Interdiscip. Top. 1994, 50, 5047−5060. (28) Bartolini, G. 2008, Oleadatabases http://www.oleadb.it/. (29) Rugini, E.; Cristofori, V.; Silvestri, C. Genetic Improvement of Olive (Olea europaea L.) by Conventional and In Vitro Biotechnology Methods. Biotechnol. Adv. 2016, 34, 687−696. (30) Rugini, E. In Vitro Propagation of Some Olive (Olea europaea sativa L.) Cultivars with Different Root-ability, and Medium Development Using Analytical Data from Developing Shoots and Embryos. Sci. Hortic. 1984, 24, 123−134. (31) Rugini, E.; Bazzoffia, A.; Jacoboni, A. A Simple In Vitro Method to Avoid the Initial Dark Period and to Increase Rooting in Fruit Trees. Acta Hortic. 1988, 227, 438−440. (32) Mencuccini, M. Effect of Medium Darkening on In Vitro Rooting Capability and Rooting Seasonality of Olive (Olea europaea L.) Cultivars. Sci. Hortic. 2003, 97, 129−139. (33) Ristori, S.; Di Cola, E.; Lunghi, C.; Richichi, B.; Nativi, C. Structural Study of Liposomes Loaded with a GM3 Lactone Analogue for the Targeting of Tumor Epitopes. Biochim. Biophys. Acta, Biomembr. 2009, 1788, 2518−2525. (34) Kucerka, N.; Pencer, J.; Sachs, J. N.; Nagle, J. F.; Katsaras, J. Curvature Effect on the Structure of Phospholipid Bilayers. Langmuir 2007, 23, 1292−1299. (35) Siegel, D. P.; Epand, R. M. The Mechanism of Lamellar-toInverted Hexagonal Phase Transitions in Phosphatidylethanolamine: Implications for Membrane Fusion Mechanisms. Biophys. J. 1997, 73, 3089−3111. (36) Marrink, S.-J.; Mark, A. E. Molecular View of Hexagonal Phase Formation in Phospholipid Membranes. Biophys. J. 2004, 87, 3894− 3900. (37) Cimato, A. Olive Nursery Production and Plant Production Techniques; International Olive Council. www.internationaloliveoil.org 2008, 53−55. (38) Thangavelu, R. M.; Gunasekaran, D.; Jesse, M. I.; Su, M. R.; Sundarajan, D.; Krishnan, K. Nanobiotechnology Approach Using Plant Rooting Hormone Synthesized Silver Nanoparticle as “Nanobullets” for the Dynamic Applications in Horticulture−An In Vitro and Ex Vitro Study. Arabian J. Chem. 2018, 11, 48−61. (39) Ram, P.; Vivek, K.; Kumar, S. P. Nanotechnology in Sustainable Agriculture: Present Concerns and Future Aspects. Afr. J. Biotechnol. 2014, 13, 705−713. (40) Flores, D.; Chaves, J.; Chacón, R.; Schmidt, A. A Novel Technique Using SWCNTs to Enhanced Development and Root Growth of Fig Plants (Ficus carica). In Nanotechnology 2013. Biosensors, instruments, medical, environment and energy technical proceedings of the 2013 NSTI Nanotechnology Conference and Expo.; Nano Science and Technology Institute: Boston, 2013, pp 167−170. (41) Lambardi, M.; Rugini, E. Micropropagation of Olive (Olea europaea L.). In Jain, S. M., Ishii, K, Eds.; Micropropagation of Woody Trees and Fruits. Forestry Science; Springer: Dordrecht, 2003; Vol. 75. (42) Toh, M.-R.; Chiu, G. N. C. Liposomes as Sterile Preparations and Limitations of Sterilization Techniques in Liposomal Manufacturing. Asian J. Pharm. Sci. 2013, 8, 88−95. (43) Rico, C.; Majumdar, S.; Duarte-Gardea, M.; Peralta-Videa, J. R.; Gardea-Torresdey, J. L. Interaction of Nanoparticles with Edible Plants and Their Possible Implications in the Food Chain. J. Agric. Food Chem. 2011, 59, 3485−3498. (44) Flores, D.; Chacón, R.; Alvarado, L.; Schmidt, A.; Alvarado, C.; Chaves, J. Effect of Using Two Different Types of Carbon Nanotubes for Blackberry (Rubus adenotrichos) in Vitro Plant Rooting, Growth and Histology. Am. J. Plant Sci. 2014, 5, 3510−3518.

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Unconventional and Sustainable Nanovectors for Phytohormone

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