Synthesis of positively charged hybrid PHMB-stabilized silver nanoparticles: the search for a new type of active substances used in plant protection products

Silver nitrate (Sigma-Aldrich, 99+%), sodium borohydride (Acros Organics, 99%), 20% PHMB hydrochloride aqueous solution with M  =  2200 (Arch Biocidals, 98%), 20% PHMB hydrochloride aqueous solution with M  =  3300 (Shanghai Terppon Chemical, 98%), 20% PHMB hydrochloride aqueous solution with M  =  4400 (Hainan Zhongxin Chemical, 98%), PHMB hydrochloride aqueous solution with M  =  28 500 (Shijiazhuang Lemandou Chemicals, 98%) were used without further purification.

Colloidal solutions of nanocrystalline silver containing 0.5 g l⁻¹ of silver and 0.01–0.4 g l⁻¹ of PHMB were obtained by reduction of silver nitrate with sodium borohydride in the presence of PHMB. 50 ml of a 0.046 M aqueous solution of silver nitrate were added dropwise to 300 ml of a 0.008–0.333% aqueous solution of PHMB (with varying average molecular weight) with continuous stirring. As a result a white suspension was obtained. The suspension was kept for 15 min. Then an aqueous solution of sodium borohydride (150 ml, 0.062 M) was added dropwise with continuous stirring. After a reducing agent was added the mixture was kept for 1 h to interfuse, its color going from dark brown to dark green, depending on the average molecular weight of the stabilizer. For further analysis a part of the resulting colloidal solutions was rinsed with distilled water several times, isolated by centrifugation (25 000 rpm) and air-dried for 24 h at 50 °С.

2.3.1. UV–visible spectrophotometry

2.3.2. Dynamic light scattering

2.3.3. Transmission electron microscopy

2.3.4. X-ray powder diffraction

2.3.5. IR spectroscopy

2.3.6. X-ray photoelectron spectroscopy

2.3.7. XPS analysis

Growth stimulating properties of PHMB-stabilized silver nanoparticles were evaluated during field experiments on potato (Solanum tuberosum L., 1753 cv. Meteor), spring wheat (Triticum L. 1753 cv. Triso) and apple (Malus P. Mill., 1754 cv. Lobo and Vishnevoye) plants [41–43].

2.4.1. Design of the experiment on potato

2.4.2. Design of the experiment on wheat

2.4.3. Design of the experiment on apple

2.5.1. Determining activity of polyphenol oxidase

2.5.2. Determining activity of peroxidase

2.5.3. Determining activity of catalase

Acute and chronic toxicity of the single-time intragastrically administered and long-term enterally administered aqueous dispersion of PHMB stabilized AgNPs was studied on white non-linear mice. All studies were carried out in Scientific Institution Research Center of Federal Medical and Biological Agency of Russia according to 'OECD Guidelines for the Testing of Chemicals, section 4: Health Effects' and 'Guideline for hygienic estimation of novel pesticides, № 4263-87 (Russia).

The content of silver in tissues or organs of plants and laboratory animals was determined by means of inductively coupled plasma—atomic emission spectrometry using a Varian 720-ES ICP-AES spectrometer (Agilent Technologies, USA). Samples of biomaterial were decomposed with a mixture of saturated HCl and HNO₃ 3:1 v/v and then analyzed. All samples of biomaterial were prepared after crop harvesting.

Standard dispersions were calculated for the obtained data (n  =  3 or 4). Analysis of statistically significant deviations of the processed data was carried out by means of the one-way ANOVA test with further application of Tukey test at the significance level 0.05. In some cases the least-significant difference was also provided (multiple t-test without α-correction).

Silver reduction with sodium borohydride is one of the most applicable methods for silver nanoparticles manufacturing both in homogeneous and heterogeneous systems. Sodium borohydride, unlike any of other reducing agent (including citric and ascorbic acids, polyphenols and carbohydrates), possesses high reactivity; it is easier to use compared to the reduction with gaseous hydrogen or physical methods and it is also characterized by comparatively low toxicity. Simplicity of the methods allows to upscale this technique of PHMB-stabilized silver nanoparticles production to the industrial level.

Silver reduction with sodium borohydride proceeds according to the following scheme:

$$\begin{eqnarray}&&\text{2AgN}{{\text{O}}_{\text{3}}}+\text{4NaB}{{\text{H}}_{\text{4}}}+\text{7}{{\text{H}}_{\text{2}}}\text{O}\to \text{2Ag}+\text{N}{{\text{a}}_{\text{2}}}{{\text{B}}_{\text{4}}}{{\text{O}}_{\text{7}}}+\text{2NaN}{{\text{O}}_{\text{3}}}+\text{15}{{\text{H}}_{\text{2}}}\end{eqnarray} \tag{ 1 }$$

Silver reduction with sodium borohydride in the presence of PHMB (figure 1(a)) with a varying degree of polymerization resulted in formation of variously colored sols (figure 1(b)); these sols were stable for over 18 months. The sample names of colloidal solutions of silver nanoparticles stabilized with PHMB with various degrees of concentration and polymerization are listed in table 1.

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In each case during the reduction of silver nitrate the formation of crystalline silver nanoparticles was observed that was verified by the data obtained from RFA and electron diffraction. In figure 2(a) an x-ray photograph of an Ag-PHMB5(20) sample that is typical for all obtained samples can be observed. Silver phase formation (PDF 004-0783) can be observed in the figure 2. It has to be noted that a halo is present at 40° 2θ which is likely to correspond to the amorphous polymer. An electron diffraction pattern of the Ag-PHMB5(20) sample is presented in figure 2(b); it correlates with the data obtained from the x-ray phase analysis. Measuring of the first four rings of the diffraction pattern allowed us to determine the corresponding interplanar spaces d (2.34 Å, 2.01 Å, 1.43 Å, 1.22 Å). Their closeness to those of metallic silver (2.36 Å, 2.04 Å, 1.44 Å, 1.23 Å) indicates that the obtained nanoparticles have the same crystalline structure as bulk silver [53]. It should be noted that a wide ring (d  =  2–2.3 Å) is observed in the diffraction pattern which is likely to correspond to diffraction on amorphous PHMB attached to the particles' surfaces.

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Given that PHMB is a polycationic polymer containing imine nitrogen in its chain it can effectively stabilize silver nanoparticles by formation of a strong coordination bond Ag-N [54].

Presence of PHMB macromolecules on the surface of AgNPs is confirmed by the data obtained from XPS, namely by the presence of bands with a specific structure in C1s and N1s spectra (figure 3). Deconvolution analysis showed two C1s lines at 285 eV (carbon in CH₂-groups) and at 287 eV (carbon in C =N-groups). N1s band contains three lines with energy peaks at 402, 403 and 403.5 eV that corresponds to the three various types of nitrogen compounds in PHMB molecules.

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Coordination of PHMB with AgNPs is indicated by the IR-spectroscopy results for the Ag-PHMB5(15) sample, presented in figure 4. Thus 3294 and 3157 cm⁻¹ stretching vibration bands of N–H groups of PHMB(15) shift to 3073 and 3023 cm⁻¹ correspondingly. Similar shift for the N–H amino groups stretching vibrations is observed in the presence of octadecylamine coordination on the AgNPs surfaces indicating formation of a strong coordination bond [55]. Disappearance of the C=N stretching vibration band with the peak at 1539 cm⁻¹ also indicates the imine groups coordination with the AgNPs. Two narrow bands with peaks at 686 and 753 cm⁻¹ appear in the IR spectrum of Ag-PHMB5(15) compared with the polymer spectrum corresponding to N–H bending. High intensity of the bands may correlate with participation of these groups in hydrogen bonds [40].

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Average size of the AgNPs according to TEM was from 5 to 15 nm (table 1). The mean size and the size distribution of particles depend on the chain length of stabilizing PHMB and its concentration.

Figure 5 (the second line) represents microphotographs of the AgNPs obtained in the presence of 0.1, 0.5 and 4 g l⁻¹ solutions of PHMB(15). At the low PHMB concentration a small amount of polycrystalline aggregates (~ 20–25 nm) is formed along with the major fraction of particles (~ 6 nm). Increase in the PHMB concentration up to 4 g l⁻¹ provides a means to reduce the aggregates fraction as well as the average particle size from 6 to 4 nm. Evidently it happens because the polymer adsorption by the AgNPs blocks both their growth and aggregation.

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Data obtained from the electron microscope studies correspond well with the results obtained from the x-ray phase analysis and presented in figure 6.

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According to TEM (table 1) the variations in the PHMB polymerization degree n from 10 to 20 have almost no effect on the nanoparticles' size, while the increase in the polymerization degree up to n  =  130 results in the sharp rise of the nanoparticles' size in sols from 4–6 nm to 13–14 nm. It has been established that the molecular weight of stabilizing polymers (polyethylene glycol, polyvinylpyrrolidone, polyethyeneimine, natural polymers, chitosan) is a strong influence on formation [28–30], aggregative stability [31, 32], the shape [33, 34] and the size [35–38] of AgNPs. Thus in the case of polyvinylpyrrolidone the particle size enlargement along with the polymer chain elongation is generally associated with the increase of the specific sites of a macromolecule (free amide I groups), on which silver ions are deposited [36, 37]. PHMB also has an ability to coordinate silver ions through the secondary nitrogen [56]. From reduction of these complexes silver atoms may appear which later form clusters and nanoparticles. If we assume that initially a nanoparticle is formed out of atoms of metal coordinated by a single polymer molecule, then a particle size ought to depend linearly on the polymerization degree. However, a twofold increase in n from 10 to 20 results in no significant particle enlargement.

Particle size increase observed along with the enlargement of the stabilizing polymer molecules might be connected to the decrease in the nanoparticle stabilization efficiency because of reduction of the average number of macromolecules per each silver particle. Yet this assumption fails to square with the data as the sizes of the silver particles obtained at the same Ag:PHMB mole ratio for PHMB(15) and PHMB(130) differ twofold (table 1).

TEM data analysis indicates formation of large spherical cavities inside the particles (figure 5, bottom line). Apparently, AgNPs grow on the specific sites of polymer globules which get encapsulated inside the metallic shell and, as a result, large particles of the core–shell type are formed.

Absorption spectrum characteristic for the obtained AgNPs samples is presented in figure 7. A wide band with a peak near 390–405 nm is connected to the plasmon absorption of silver nanoparticles.

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PHMB has proven to be an exceptionally good stabilizer allowing obtaining of highly concentrated dispersions of silver nanoparticles (silver density up to 5 g l⁻¹) dilution resistant at least up to a silver density of 0.02 mg l⁻¹. Nevertheless, PHMB-stabilized dispersions lost their aggregative stability in the presence of a fractional amount of chloride ion (figure 7(a)). With the increase in the ionic strength of the solution the ζ-potential of the nanoparticles falls below the dispersion stability level (±30 mV) leading to aggregation and sedimentation of the sol particles (table 2). Along with the increase in the stabilizing polymer chain length zeta potential of the particles reaches its peak value at n  =  15 (table 2) while their resistance to the ionic strength remains unchanged (figure 7(b)). This phenomenon may be due to the increase in steric hindrances to aggregation because of the polymer molecular chain elongation which adds to the particles' stability when they avoid coagulation even when the ζ-potential is below the threshold level of 30 mV.

According to table 3 silver nanoparticles stabilized with PHMB(15) possess the highest ζ-potential, that is why a sample based on this solution was selected for further study of its growth-stimulating and enzymatic activity because of its resistance to aggregation.

In experiments with potato the pre-planting treatment of tubers (50, 75 and 100 ml metric ton⁻¹) followed by the foliar application (50, 75 and 100 mg ha⁻¹) of Ag-PHMB(15) resulted in a considerable increase in the overall potato productivity and the food and seed grades of the tuber yield (table 3). The maximum additional yield was registered in the cases of pA₂ and pA₃ treatment and amounted to 4.9–5.7 metric ton ha⁻¹ or 16–19 %; the seed potato yield was by 13.2 thousand tubers (4.5%) higher than the baseline value. We observed an increase in the food grade yields after each treatment with Ag-PHMB(15), this effect was dose-dependent and amounted to 68–100% compared to the baseline value (figure 8). After the treatment with various doses of Ag-PHMB(15) the weight of seed tubers remained stable (63–64 g) while the food grade tubers became larger weighing up to 118–126 g compared to 63 g and 82 g respectively.

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Treatment of spring wheat seeds with Ag-PHMB(15) increased their germination energy by 1.7–4.9% over control (table 4) while the field germination rate increased by 9.2%, number of seedlings from the treated seeds was above control values by 11.1–11.4 %, on the average.

Positive effect from treatment with the colloidal solution of Ag-PHMB(15) was registered in the variants where the spring wheat seeds and plants received 60 ml and 80 ml of the solution: the number of fertile stems increased by 18 and 35 pcs m⁻², number of grains per ear increased to 20.6 and 21.1 grains, the weight of 100 grains increased by 0.5–1.1 g, respectively. Treatment with 40 ml produced results equal to those of the control experiment. Significant yield increase in the cases when the spring wheat was treated with 80 ml and 60 ml of Ag-PHMB(15) amounted to 3.9 dt ha⁻¹ and 2.5 dt ha⁻¹, respectively at LSD₀₅  =  2.38 dt ha⁻¹. The minimum dose of Ag-PHMB(15) resulted in no significant increase in the spring wheat yield.

It should be noted that in the case of the plants treated with Ag-PHMB(15) phenological growth stages were hastened by 1–4 days compared to the control (figure 9). Early and vigorous tillering indicates favorable growth conditions and is important for obtaining highly productive plants.

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Study of foliage treatment of apple trees (cv. Lobo and Vishnevoye) with Ag-PHMB(15) has exposed the positive effect of the preparation which is manifested in slowing down the plants' vegetative growth and the improvement of their productivity (table 5). When the dose of the agent was increased from 150 ml ha⁻¹ to 200–250 ml ha⁻¹ the vegetative development indicators improved in both cultivars, at the same time productivity of cultivar Lobo decreased. The highest productivity rates for apple cultivar Vishnevoye were registered in the case of the 200 ml ha⁻¹ treatment dose.

Foliage treatment of apple trees cultivar Lobo with Ag-PHMB(15) in larger doses results in an increase in bud primordia formation by 74.8 %. Foliage treatment of apple plants cultivars Lobo and Vishnevoye with Ag-PHMB(15) lead to almost no significant increase in Vitamin C and sugars content of fruits.

Thus, the PHMB-stabilized silver nanoparticles have a considerable growth-stimulating effect on the studied agricultural plants. This effect can be explained in terms of the biological activity of silver nanoparticles. Silver has the most pronounced effect on physiological mechanisms regulated by the ethylene plant hormone [57]. Often the observed biological influence of low and medium doses of silver ions is explained by the activity of an inhibitor of the ethylene response [58]. For example, silver salts have a stimulating effect on the stem, root and leaves growth, root branching, early flowering, also these compounds prevent leaf abscission and blossom withering. A number of other effects traditionally associated with the inhibition of the ethylene biosynthesis or a decrease in the plant sensitivity to ethylene are observed, in particular the stimulation of the transport of auxins, an increase in production of biogenic polyamines [58], which accelerate cell proliferation.

Such activity of silver can be attributed to the ability of Ag⁺ ions to displace copper ions (I) with the resembling structure from the ethylene receptor ETR1 localized in the cell membrane [59]. Silver ions interfere with the normal activity of the ethylene receptor and prevent it from transmitting signals to the cell [59].

Phytopathogenic microorganisms have a strong impact on biochemical processes in plants. In the course of evolution plants have developed various systems allowing them to resist diseases. Mechanisms of plant immunity vary greatly. Production of reactive oxygen intermediates is one of the most common responses to the phytopathogens' influence [60]. Interaction between a pathogen and a host plant results in a serious alteration in the functional status of the plant that is reflected, first of all, in the activity of enzymes [60].

Peroxidase is one of such enzymes. It is a two-component enzyme, an iron containing protein. The enzyme is localized in the cell wall, mitochondria, chloroplasts, cell nucleus, cytoplasm and other organelles. Peroxidase is highly sensitive to the environmental influence on the plants, moreover, the plant's response to biotic stressors resembles the response to the abiotic ones [61].

Figure 10(a) represents the experimental data on peroxidase activity in potato, wheat and apple. Its activity increased in every case. The highest degree of increase was registered in apple leaves after treatment with Ag. It may be connected to the development of the hypersensitive reaction due to the oxidative stress. This suggestion is supported by the observation of marginal necrosis on apple leaves on a number of occasions (figure 11).

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Along with the aforementioned the peroxidase content can increase in response to the invasion by pathogens and the infection contamination of the plant tissues [61]. Quinones and products of their condensation resulting from oxidation with peroxidase possess fungitoxic and bactericidal properties and serve as a chemical barrier during necrotic surface formation. This biochemical response can be used as an indicator of the plant's resistance because these processes often occur simultaneously. Nevertheless, the increased peroxidase activity does not always correlate with resistance and does not always induce it. Some infections cause reduction in the peroxidase activity.

Figure 10(b) represents the activity of peroxidase in potato infested with late blight and Alternaria blight. The study showed that as a whole, activation dynamics of this enzyme is higher in plants treated with Ag-PHMB(15) than in control plants. Also, the peroxidase activity in the infested plants treated with Ag-PHMB(15) appears to be higher compared to non-infested plants treated with the same dispersion. This observation indicates an increase in one of the main non-specific defense reactions and, consequently, in non-specific resistance of the treated plants.

As in the case of peroxidase, treatment with Ag-PHMB(15) influenced catalase activity, especially in the affected plants. Catalase activity in the leaves increased to some degree (figure 12(a)), particularly in the apple plants, which, similarly to the case with peroxidase, indicates high sensitivity of the plant to the preparation. Catalase activity in the potato leaves slightly decreased under the influence of late blight and Alternaria blight (figure 12(b)).

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Alteration in the polyphenol oxidase activity under the influence of preparations containing silver (figure 13) was not as pronounced as in the case of other enzymes. We observed no significant difference between the variants.

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Bioaccumulation of silver nanoparticles in plant tissues after the spraying treatment of potato, wheat and apple trees with the Ag-PHMB(15) dispersion was studied after harvesting; silver content in the potato tubers, wheat grain, apple fruits, leaves and branches (table 6) was determined. After the spraying treatment silver was usually found to accumulate in the leaves and, less often, in the branches. When the dose of the agent was increased from 150 ml ha⁻¹ to 200–250 ml ha⁻¹ the silver content increased respectively. No significant migration of silver in edible parts of the plants was observed. Silver content in the harvested crops did not exceed 0.02 ppm, which is safe enough for human consumption. For example, the US EPA drinking water alert limits of silver is 0.1 ppm [62].

The Ag-PHMB(15) dispersion was registered as a PPP in Russia and some other countries. As a part of the registration procedure the safety of PHMB-stabilized silver nanoparticles was estimated; Ag-PHMB(15) was characterized by low acute and chronic toxicity when intragastrically administered to laboratory animals (white non-linear mice). The value of DL₅₀ of Ag-PHMB(15) in the case of single-time intragastric administration exceeds 10 ml kg⁻¹ of body weight. According to the classification DL₅₀ the Ag-PHMB(15) dispersion is referred to as a low hazard substance when intragastrically administered to laboratory animals (mice). Also long-term 28 days enteral administration in doses of 2 ml kg⁻¹ of body weight did not lead to a damaging effect on the performance status of the mice. Thus, the Ag-PHMB(15) dispersion may be considered safe enough to be used as a plant protection agent.