RU2347366C2 - Composition for trasporting active substances to wheat roots - Google Patents

Abstract

FIELD: agriculture.

SUBSTANCE: invention contains micro-particles each including matrix and chemical substance distributed thereinto. The matrix corresponds to liposomes or nano-particles of silicon dioxide; also the surface of the matrix contains natural hydrocarbon containing compounds able specifically to bind with root surface. Liposomes are obtained out of egg phosphatidylcholine and their surface contains lipopolysaccharides of the outer membrane of bacteria Azospirillum brasilense Sp245. The surface of the matrix out of silicon dioxide contains polysaccharides of wheat roots Triticum aestivum L of Saratovskaya 29 breed. Micro-particles are of 0.01-0.2 mcm size.

EFFECT: diminishing anthropogenic load to arable land and environment.

3 cl, 6 ex, 6 tbl

Description

The invention relates to the field of ecology, biotechnology and methods applicable in crop production and can be used to deliver various chemical compounds with biological activity to the roots of plants. The proposed composition can be used for targeted delivery of growth stimulants and other active substances to the roots of wheat, as well as for basic research on plants grown in laboratory conditions.

Known nanosystems for the delivery of drugs in the human body. For example, liposomes that carry carbohydrate determinants are used to transport cytotoxic drugs to tumor cells (Vodovozova E.L., Interaction of liposomes that carry carbohydrate determinants with melanoma cells // Biol. Membranes. 2004. V.21. No. 1. S. 53-65). As specific carbohydrate determinants, the residues of trisaccharide A, tetrasaccharide sialyl LewisX, capable of specifically binding to pectins located on the surface of a cancer cell are used. Various vector liposomes loaded with lipid derivatives of the antitumor drug sarcolisin exhibit better therapeutic properties compared to the original sarcolysin.

However, nanosystems with these specific carbohydrate determinants are not applicable in agricultural practice.

Closest to the proposed solution is a composition with a controlled gradual release of the active ingredient for the delivery of an agricultural chemical to plants. The invention describes a composition for delivering an agricultural chemical to plants containing microparticles having an average diameter of 0.2 to 200 microns, each of which includes a polymer matrix and an agricultural chemical distributed in the polymer matrix selected from cyproconazole, epoxyconazole and tebuconazole, the microparticles including from 1 to 50% by weight of an agricultural chemical and from 50 to 99% by weight of said polymer matrix, consisting of a polymer selected from the group consisting of polymethyl methacrylate, poly p of lactic acid, copolymers of lactic and glycolic acids, cellulose acetate butyrate, polystyrene, copolymers of hydroxybutyric acid and hydroxyvaleric acid, copolymers of styrene and maleic anhydride, extraction rosin, acrylic polymer and vinylpyrrolidone and vinyl acetate copolymer and their combination (see. A01N 43/653).

These microsystems do not contain on their surface specific determinants capable of recognizing certain plant crops, i.e. they are not intended for the targeted delivery of active substances. In addition, they include polymers that are poorly degraded in the environment; therefore, the use of such compounds can increase the anthropogenic load on natural ecosystems.

The objective of the invention is to develop a composition for targeted delivery of active substances to the roots of wheat, as well as for basic research on plants grown in laboratory conditions.

The technical result is to reduce the anthropogenic load on agricultural land and the environment.

The problem is achieved in that in the composition for delivering active substances to wheat roots containing microparticles, each of which includes a matrix and a chemical distributed in it, according to the solution, the matrix is liposomes or silicon dioxide nanoparticles, while the matrix surface contains natural carbohydrate-containing compounds capable of specifically binding to the root surface.

Liposomes can be obtained from egg phosphatidylcholine.

The surface of the liposome matrix may contain lipopolysaccharides of the outer membrane of bacteria Azospirillum brasilense Sp245.

The surface of the matrix of silicon dioxide may contain polysaccharides of wheat roots Triticum aestivum L. varieties Saratovskaya 29.

Microparticles can have a size of 0.01-0.20 microns.

In the well-known authors of scientific, technical and patent literature, compositions with a similar set of features were not found. The result, due to the combination of these features, was not achieved in the known solutions. The advantages of the claimed composition is the ability to deliver with its help chemical compounds to the roots of wheat purposefully, with greater drug savings and reduced environmental load.

The method of preparation of the composition is as follows.

If the matrix is liposomes, the composition is prepared as follows. In an aqueous solution of a chemical (active) substance at 50 ° C, a 10% ethanol solution of phosphatidylcholine is added to a final concentration of 1.4 g / l with stirring, which is carried out until the smell of alcohol disappears. The resulting suspension is voiced on an ultrasonic disintegrator 3 times for 30 s at maximum power. Liposomes containing lipopolysaccharides (LPS) of the outer membrane of bacteria Azospirillum brasilense Sp24 are prepared as described above by injecting an ethanolic solution of phosphatidylcholine into aqueous LPS solutions with a concentration of 20-150 mg / L and a chemical. The technique for isolating LPS from bacteria Azospirillum brasilense Sp245 is described in (Fedonenko Yu.P., Egorova I.V., Konnova S.A. et al. Participation of azospirillum lipopolysaccharides in interaction with wheat root surface // Microbiology. - 2001. - Tom 70. - No. 3. - S.384-390.).

If the matrix is silicon dioxide nanoparticles, the composition is prepared as follows. Create in water a concentration of silicon dioxide nanoparticles ("Fluka", diameter 7 nm) 1 g / L. The resulting suspension is also voiced on an ultrasonic disintegrator. To obtain nanoparticles inlaid with polysaccharides, solutions of polysaccharides isolated from wheat roots of Triticum aestivum L. cultivar Saratovskaya 29 are poured into a suspension of particles to a final concentration of 10 ^-3 -10 ^-1 g / l and stirred for about 1 hour. Isolation of polysaccharides of wheat roots carried out by any known method (Isolation and analysis of glycopolymers of plant and bacterial origin: textbook for student biol. faculty / Konnova S.A., Sachkova (Fuchedzhi) O.A., Konnova O.N., Fedonenko Yu .P. - Saratov: Publishing House of SSU, 2005, - 39 pp. For distribution in microch On the basis of a chemical substance, the suspension of nanoparticles is diluted 100-10000 times with an aqueous solution of the substance and stirred for about 1 hour.

Examples of practical implementation of the method

Example 1. Evaluation of the effectiveness of targeted delivery of a chemical to the roots of wheat Triticum aestivum L. variety Saratovskaya 29 using liposomes enriched with LPS bacteria Azospirillum brasilense Sp245.

Methylene blue is used as the delivered chemical. Natural carbohydrate-containing compounds that can specifically bind to the root surface of wheat are bacterial LPS of Azospirillum brasilense Sp245 (Aref'eva O.A., Rogacheva S.M., Kuznetsov P.E. et al. Liposomes in the study of the mechanism of bacterial aggregation and their adsorption on the roots plants // Biological membranes. - 2006. - No. 1. - T.23. - S.14-21).

Preparing a composition with a matrix in the form of liposomes from egg phosphatidylcholine. A 0.1% ethanol solution of egg phosphatidylcholine (Biolek, Ukraine) is added to an aqueous solution containing methylene blue at a concentration of 0.1 g / L to a final concentration of 1.4 g / L with stirring on a magnetic stirrer at 500 rpm. The resulting suspension is voiced on an ultrasonic disintegrator UD-11 ("Elpan", Poland) 3 times for 30 s at maximum power.

Preparing a composition with a matrix in the form of liposomes from egg phosphatidylcholine containing LPS on the surface. In this case, the injection of a 10% ethanol solution of egg phosphatidylcholine is carried out in a solution of lipopolysaccharide with a concentration of 23 and 150 mg / l, containing methylene blue with a concentration of 0.1 g / l.

Low molecular weight compounds, including dye not included in liposomes, are removed by dialysis (pore diameter of the dialysis membrane is 12-14 kDa) against water for 2 days.

The composition of the solutions and suspensions used in the experiments are shown in table 1.

Table 1 Suspension / solution Composition, mg / l Phosphatidylcholine LPS * Methylene blue Methylene blue (control) 0 0 one Liposomes 1400 0 0.7 ± 0.1 Low LPS Liposomes 1400 23 0.7 ± 0.2 High LPS Liposomes 1400 150 0.7 ± 0.1 LPS solution 0 150 0.9 * Concentration before dialysis

Whole roots are separated from wheat seedlings, placed in boxes with test solutions and suspensions. Root incubation is carried out in two ways: a) completely immerse the roots in the incubation medium and incubate for 45 minutes; b) the roots are immersed in the medium so that the places of their sections remain above the surface of the studied solutions and suspensions, and incubated for 24 hours. After incubation, the roots are thoroughly washed with distilled water, triturated in a mortar and extracted with 96% ethanol for 1 hour. In the alcohol extract of the roots, the dye content is determined spectrophotometrically at 632 nm, with an optical layer length l = 1 cm. The concentration of methylene blue is determined by preliminary constructed calibration dependence of optical density on concentration. An ethanol extract of wheat roots serves as a comparison solution for the extracts. The results of the experiments are presented in table 2, which shows that there is an increase in the degree of sorption of methylene blue depending on the concentration of LPS in liposomes more than 2.5 times,

table 2 Suspension / Solution The concentration of methylene blue, ppm (by weight of the root) 24 h incubation Incubation 45 min Methylene blue (control) 20 ± 5 8 ± 4 Liposomes 15 ± 6 8 ± 3 Low LPS Liposomes 20 ± 10 16 ± 6 High LPS Liposomes 54 ± 9 46 ± 5 LPS solution 9 ± 2 12 ± 5

The presence of LPS in liposomes increases not only the degree, but also the rate of sorption of the dye. So, in 45 min, 85 ± 9% of the dye of the maximum possible amount is sorbed from the suspension of liposomes with a high LPS content on the roots. In a suspension of liposomes without LPS, this indicator is 53 ± 20%. It should be noted that the number of adsorbed liposomes with integrated LPS on the roots does not increase after 24 hours of incubation. Thus, delivery efficiency is increased when using LPS.

Example 2. Targeted delivery of indolyl-3-acetic acid to wheat roots.

The composition for the delivery of indolyl-3-acetic acid (IAA) to the roots of plants is obtained as follows. An LPS solution is added to a citrate-phosphate buffer (pH 6.0) containing IAA at a concentration of 1-100 mg / L to a final concentration of 150 mg / L. Then, a 10% ethanol solution of egg phosphatidylcholine is added to the resulting solution to a final concentration of 1.4 g / l with stirring on a magnetic stirrer at 500 rpm. The resulting suspension is voiced on an ultrasonic disintegrator 3 times for 30 s at maximum power. Undispersed phospholipids are removed by centrifugation (K-24 centrifuge, Germany) for 10 minutes at 8000 rpm.

The compositions are introduced into test tubes with a hydroponic complex medium in a ratio of 1:10, sticky wheat seeds of Triticum aestivum L. cultivar Saratovskaya 29 are placed on the surface of the hydroponic solution and grown in a climate chamber (KTLK 1250, Germany) for 2 weeks with the regime: 16- hour light period, 25 ° C. The length of the seedlings is measured. The results of the experiments are presented in table.3.

Table 3 Solution / Suspension The average length of the plant, mm Hydroponic solution (control) 1456 ± 30 IAA solution, 10 mg / l 1620 ± 10 Liposomes with LPS (150 mg / L), and IAA, 10 mg / L 1697 ± 20

From the data presented in Table 3, it can be seen that the greatest growth of plants is observed on a hydroponic medium, when processing liposomes with LPS and IAA, which indicates the effectiveness of these microsystems.

Example 3. Evaluation of the effectiveness of the delivery of IAA liposomes with LPS to coleoptiles of wheat

Prepare liposomes enriched with LPS and loaded IAA, as described in example 2.

Wheat coleoptiles are prepared. To do this, the sticky wheat seeds are laid out on stainless steel lattices in glasses, which are placed in a thermostat at 27 ° C for cultivation. Seedlings when reaching the length of coleoptiles 20-22 mm are removed from the thermostat, cut them off at the base and measured. Coleoptiles of equal length are selected, of which lengths of 5 mm are cut into the machine at a distance of 4 mm from the upper end. A dull glass needle removes the first real sheet from them. After preparation, the segments are placed in cups with the studied solutions and suspensions (Table 4). The glasses are kept in a thermostat at 27 ° C for 20 hours. After incubation, the length of the coleoptile segments is measured with an accuracy of 0.5 mm. By the difference in their lengths in suspensions, the growth effect of the systems used is judged (Table 4).

Table 4 Suspension / solution The growth of coleoptiles, (% of the original length) Water 14 ± 2 LPS solution, 150 mg / l 12 ± 2 IAA solution 1 mg / l 48 ± 1 10 mg / l 49 ± 1 100 mg / l 47 ± 2 Liposomes without LPS with IAA, the initial state of IAA: 1 mg / l 14 ± 1 10 mg / l 14 ± 1 100 mg / l 13 ± 2 Liposomes with LPS (150 mg / l) with IAA; initial IAA condition: 1 mg / l 50 ± 1 10 mg / l 53 ± 2 100 mg / l 50 ± 1

The results presented in Table 4 indicate, firstly, the absence of biological activity in LPS: almost the same increase in coleoptiles in water and a solution of LPS was noted. Secondly, it has been shown that the maximum biological effect of IAA is at a concentration of 1 mg / l, which is also confirmed by literature data (Palamarchuk I.A., Veselova T.D. Tutorial on botanical histochemistry. - Moscow: Moscow. University, 1965. - 93 p.). Liposomes without LPS loaded with IAA do not exhibit biological activity. On the other hand, the use of liposomes, inlaid with LPS and loaded IAA, leads to the same result as the use of solutions of IAA.

It should be noted that the amount of substance in the internal volume of liposomes in suspensions is much less than in the corresponding IAA solutions. Those. to obtain a high biological effect in the case of delivery systems (liposomes), it is necessary to use a significantly smaller amount of biologically active substances.

Example 4. Delivery of fluorescein sodium (FN) liposomes to the roots of wheat.

As a delivered chemical substance, a dye is used - sodium fluorescein (FN). The composition is prepared on the basis of liposomes, as described in the preparation method, by injecting an alcoholic solution of egg phosphatidylcholine into a 10 ^-5 M aqueous solution of FN. Then the mixture is disintegrated with ultrasound 3 times for 30 s at maximum power. Non-dispersed phospholipids are removed by centrifugation (K-24 centrifuge, Germany) for 10 minutes at 8000 rpm. The dye not included in the liposomes is removed for 1 day. dialysis against water.

Whole roots are separated from wheat seedlings, their boxes with solutions and suspensions are placed (Table 5). The roots are immersed in media so that the sections of their slices remain above the surface and incubated for 1 hour. To determine the degree of sorption of FN by roots, they are crushed, extracted with water for 1 hour, centrifuged for 10 minutes at 10,000 rpm. Select 1 ml of the supernatant, mix with 5 ml of a pH 8.25 phosphate buffer and measure the fluorescence intensity at an absorption wavelength of 491 nm, an emission of 512 nm.

Table 5 Solution / Suspension Fluorescence intensity 10 ^-5 M aqueous solution of fn 2.2 Liposomes with fn 1095

From table 5 it is seen that the sorption of FN on wheat roots, after their incubation with emulsion of liposomes, is approximately 500 times higher than during incubation in a dye solution. Since it was previously established (examples 1, 3) that liposomes are practically not adsorbed on the roots of wheat, we associate this result with the incorporation of FN into the lipid membrane of liposomes and the participation of a chemical in a specific process of sorption on the roots. A similar result can be expected if a compound with a structural similarity to FN is used as an active chemical in delivery systems.

Example 5. Targeted delivery of fn nanoparticles of silicon dioxide enriched in plant polysaccharides to the roots of wheat

The composition for the directed transport of FN to wheat roots based on silicon dioxide nanoparticles is prepared as follows.

Plant polysaccharides are isolated from wheat root biomass. A portion of the roots of wheat (10 g) is ground in a mortar. The crushed biomass is transferred to a beaker and, to fix the material, it is poured with a 5-fold volume of 96% hot ethanol for 5 min, after which the fixing solution is drained. Then, an 80% ethanol solution is added to the plant biomass and incubated for 20 minutes in a boiling water bath, periodically stirring the contents with a glass rod. The extract is poured through a filter into a dry glass, the extraction procedure is repeated two more times. All extracts are combined and concentrated by evaporation in a water bath at a temperature of 40-50 ° C to a volume of 20 ml.

To separate polysaccharides, gel filtration was used on a chromatographic column (2 × 50 cm) filled with Sephadex G-75 carrier (Pharmacia, Sweden), and pyridine acetate buffer (0.025 M, pH 4.1) was used as an eluent. Get polysaccharides of high molecular weight (IPN) and low molecular weight (NPS).

A hydrosol of silica nanoparticles is obtained as described in the method for preparing the composition. It is diluted with a 10 ^-5 M solution of FN to a final concentration of particles of 10 ^-2 , 10 ^-3 , 10 ^-4 g / l and solutions of IPN and NPS to their final concentration of 10 ^-1 , 10 ^-2 , 10 ^-3 g / l at stirring on a magnetic stirrer for 1 h. The resulting compositions are used in experiments.

Roots separated from wheat seedlings are incubated in media (Table 6), and the degree of sorption of FN by roots is evaluated, as described in Example 4. The experimental results are presented in Table 6.

From the data of Table 6 it can be seen that the degree of sorption of FN on wheat roots depends both on the concentration of polysaccharides and on the concentration of nanoparticles in the hydrosol. The highest degree of sorption is observed for nanoparticles inlaid with IPN and NPS at a concentration of 10 ^-1 g / l, with their content in a hydrosol of 10 ^-4 g / l. Compared with particles without a polysaccharide coating, it increases by 19.5 times for IPN and 11.3 for NPS. It was noted that the degree of sorption of nanoparticles increases with increasing concentration of polysaccharides. The decrease in the degree of sorption of particles when their concentration in hydrosols is more than 10 ^-4 g / l is apparently associated with the process of aggregation of nanoparticles.

It was shown that nanoparticles without a polysaccharide coating are also able to be sorbed by wheat roots, but less efficiently. Compared with the control, the degree of adsorption of FN in the hydrosols of such particles increases only 2.1–2.8 times. As for solutions of polysaccharides (without nanoparticles), the degree of adsorption of dye from them does not differ significantly from the control. Those. in the delivery of a chemical substance to the roots of plants, the main role is played by silicon dioxide nanoparticles.

Table 6.
The fluorescence intensity of root extracts after their incubation in the hydrosols of silicon dioxide nanoparticles and solutions of various compositions The concentration of polysaccharides, g / l The concentration of nanoparticles, g / l 10 ^-2 10 ^-3 10 ^-4 10 ^-5 0 UPU 10 ^-2 9.1 ± 0.8 23.5 ± 4.3 42.8 ± 6.2 6.5 ± 2.4 3.9 ± 1.1 10 ^-3 6.6 ± 0.4 8.1 ± 0.9 11.1 ± 1.2 3.2 ± 1.9 2.5 ± 0.8 Npc 10 ^-1 5.7 ± 0.6 9.0 ± 0.9 24.9 ± 3.7 5.6 ± 1.3 4.2 ± 1.4 10 ^-2 7.1 ± 1.4 7.7 ± 0.2 18.7 ± 0.9 4.3 ± 1.2 3.5 ± 1.2 10 ^-3 6.5 ± 0.4 6.4 ± 0.5 6.8 ± 0.6 4.6 ± 0.6 3.3 ± 0.6 0 6.2 ± 0.9 4.9 ± 0.5 4.7 ± 0.6 5.0 ± 1.3 2.2 ± 0.2

Thus, the most effective sorption was found for nanoparticles inlaid with IPN at a concentration of 10 ^-2 g / L. This once again confirms that specific determinants on the surface of the matrix contribute to the targeted delivery of microparticles to the roots of plants, in this case wheat.

Example 6. Sizing of microparticles

The liposome size was estimated by the spectrum of the turbidity of the suspensions (Bezrukova A.G., O.A. Rosenberg // Bull. Exp. Biol. Honey. - 1981. - No. 4. - S.506-507.). In the wavelength range of suspensions of 370-500 nm, their optical density values are determined. A linear dependence of IgD on logλ is built, the wave exponent n is found as the tangent of the slope of the line. The wave exponent depends on the particle diameter n (d). According to the tabulated values (Bezrukova, Rosenberg, 1981), the average value of the particle diameter is found. The diameter of the used liposomes is 0.1-0.18 microns.

Claims (3)

1. A composition for delivering active substances to the roots of wheat, containing microparticles, each of which includes a matrix and a chemical distributed in it, characterized in that the matrix is liposomes obtained from egg phosphatidylcholine, while the surface of the matrix contains lipopolysaccharides of the outer membrane of bacteria Azospirillum brasilense Sp245. 2. A composition for delivering active substances to wheat roots, containing microparticles, each of which includes a matrix and a chemical distributed in it, characterized in that the matrix is silicon dioxide nanoparticles, while the surface of the matrix contains polysaccharides of wheat roots Triticum aestivum L. cultivars Saratov 29. 3. The composition according to claims 1 and 2, characterized in that the microparticles have a size of 0.01-0.20 microns.