Polyelectrolyte complex and production thereof

Abstract

The present invention relates to a polyelectrolyte complex, comprising at least one nucleic acid, the polyelectrolyte complex having, in addition to the at least one nucleic acid, at least one ionotropic gel-forming anionic polymer and a cationic polymer, and the polyelectrolyte complex is present in the form of a particle having a size in a range of ≤ 2 µm.

Description

[0001] Polyelectrolyte complex and its preparation

[0002] TECHNICAL FIELD

[0003] The present invention relates to polyelectrolyte complexes of nucleic acids with at least one ionotropic gel-forming anionic polymer and one cationic polymer, which exhibit improved long-term stability and improved efficacy. The present invention further relates to a plant protection agent or a plant strengthening agent comprising such a polyelectrolyte complex. The present invention further relates to a process for producing such a polyelectrolyte complex.

[0004] BACKGROUND

[0005] Biologically active substances based on nucleic acids are usually very fragile and can be rapidly degraded by biotic stress, i.e., enzymes, or abiotic stress, such as heat, UV radiation, or pH influences, rendering them ineffective. For example, dsRNA, as a biological pesticide, must overcome this hurdle to remain functional over the long term and thus be used effectively. Accordingly, the stabilization of nucleic acids, such as single- and double-stranded nucleic acids such as dsRNA, may be necessary to ensure their long-term effectiveness when used as pesticides or plant strengtheners.

[0006] Double-stranded RNA (dsRNA) is considered a promising tool for protecting plants from viruses, insects, fungal pathogens, and oomycetes (Niehl & Heinlein 2019, Liu et al. 2020, Niu et al. 2021). In most eukaryotes, dsRNA triggers RNA interference (RNAi), a gene regulatory pathway that promotes genome stabilization and defense against RNA viruses and viroids (Fire et al. 1998, Fedoroff 2012, Torri et al. 2022). Furthermore, in vertebrates and plants, dsRNA plays a crucial role as a pathogen-associated molecular pattern (PAMP), triggering nonspecific defense responses that mitigate potential viral infections. However, how fungi respond to dsRNA remains largely unexplored.

[0007] In crop protection, the RNAi mechanism can be used for pest and disease control by using either exogenous dsRNA (spray-induced gene silencing, SIGS) or host-expressed dsRNA (host-induced gene silencing, HIGS) to reduce key gene activities of pests and pathogens and thereby reduce their virulence (Cai et al. 2018). In HIGS, plants are transgenically modified to endogenously express specific dsRNA sequences, thus causing gene silencing of the pathogen in question. In contrast, SIGS relies on the exogenous application of dsRNA derived from chemical or biotechnological processes and is usually administered through conventional spray applications.

[0008] SIGS offers distinct advantages over HIGS: It does not require the use of transgenic plants, which addresses public concerns about genetic modification (Tom et al. 2022). Furthermore, SIGS can be targeted, allowing farmers to apply it selectively when certain pathogens pose a threat to crops. However, there are several challenges that complicate the use of dsRNA in agriculture, such as high production and purification costs, and the susceptibility of dsRNA to premature degradation, which occurs both before and after application and during storage. In addition, the cuticle, cell wall, and cell membrane pose major hurdles for the efficient uptake of negatively charged dsRNA. These challenges have been discussed in many studies, which also highlight the size exclusion limits posed by plant barriers (Liu et al. 2020, Torri et al. 2021).2022, Tatematsu et al. 2018, Chen & Hur 2022, Dang et al. 2011, Nicolas & Garre 2016).

[0009] To overcome these challenges, numerous formulations have been developed in the prior art, generally aiming to attenuate the anionic charge of dsRNA or circumvent size exclusion limits. These formulations include various strategies, such as the use of carbon nanoparticles, e.g., E.g., carbon nanodots cationically functionalized with polyethylenimine and subsequently loaded with dsRNA, layered double hydroxide nanosheets also known as bioclays, DNA nanostructures, especially those based on DNA origami, other cationic or amphiphilic oligopeptides, including plant-penetrating peptides, and encapsulation in cationic liposomes (Nicolas & Garre 2016, Cai et al. 2018, Dean et al. 2012, Raman et al. 2017, Zanini et al. 2021, Wang & Dean 2022, Sarkar & Roy-Barman 2021, Delgado-Jarana et al. 2006, John et al. 2016, Marques et al. 2006, Abdellatef et al.2015, Hur 2019, Yin et al. 2016). Despite all these formulation efforts, none of these products has been successfully transferred from laboratory development to practical application.

[0010] It is known from the prior art that nucleic acids are protected by complexation with EDTA, thus inactivating the nucleases. However, this solution should be avoided because EDTA is harmful to humans and aquatic organisms. D. Arruda, I. Gonzalez, S. Finet, L. Cordova, V. Trichet, G. Andrade, C. Hoffmann, P. Bigey, W. Marcedo, A. Conha, A. Souza, v. Escriou, Journal of Colloid and Interface Science, 540, 342-353, 2019, describe siRNA lipoplexes supplemented with sodium alginate. These were prepared by mixing cationic liposomes containing cationic DMAP AP lipid and DOPE with a previously prepared mixture of siRNA and sodium alginate. The produced lipoplexes have a size of 268nm + / - 87nm.

[0011] WO 2022 / 076877 A1 describes a layer arrangement intended to provide a stabilizing layer or a protective layer. This layer arrangement comprises different layers, each of which can be formed from one of alginate and chitosan.

[0012] WO 2012 / 027713 A2 describes that dsRNA can be complexed with alginate as a complexing agent, whereby nanoparticles can be produced.

[0013] US2020 / 0330395A1 generally describes the improvement of the efficiency of RNA interference (RNAi) in insects, as well as the formation of a complex of chitosan (CS), sodium tripolyphosphate (TPP) and dsRNA (CS-TPP-dsRNA).

[0014] US2010 / 0092572A1 describes chitosan-based colloidal particles comprising a ribonucleic acid, a chitosan and a polyanion, as well as compositions of these particles suitable for the delivery of ribonucleic acids into mammalian cells.

[0015] US2008 / 0160096A describes the production of nanoparticles from biopolymers such as polycations and polyanions for encapsulating therapeutic nucleic acids. These nanoparticles are intended to protect the active ingredients and prevent immune reactions in the body, thus enabling targeted delivery of drugs and chemotherapies to protect healthy body parts, as well as reducing dosage. However, the current state of the art still offers potential for improvement, particularly with regard to effective protection of nucleic acids in particles used in crop protection.

[0016] The object of the present invention is to create a measure by which at least one disadvantage of the prior art is at least partially overcome. In particular, the object of the present invention is to develop a measure by means of which effective protection of nucleic acids in particles is possible in the field of plant protection.

[0017] It is therefore the object of the present invention to provide a process for the preparation of polyelectrolyte complexes which offer effective protection of nucleic acids in the field of plant protection.

[0018] These and other objects are achieved by methods and means according to the independent claims of the present invention. The dependent claims relate to specific embodiments.

[0019] SUMMARY OF THE INVENTION

[0020] The present invention provides polyelectrolyte complexes of nucleic acids with at least one ionotropic gel-forming anionic polymer and one cationic polymer, which exhibit improved long-term stability and improved efficacy. The present invention further provides a plant protection agent or a plant strengthening agent comprising such a polyelectrolyte complex. The present invention further provides a process for producing such a polyelectrolyte complex. The invention and the general advantages of its features are explained in more detail below.

[0021] The invention is defined by the features of the appended independent claims. Preferred embodiments of the invention are disclosed in the subclaims, the description, and the figures. Further features described or shown in the subclaims, the description, or the figures may, individually or in any combination, constitute a subject matter of the invention, unless the context clearly indicates otherwise.

[0022] SHORT DESCRIPTION OF THE CHARACTERS

[0023] Fig. 1 shows a schematic diagram illustrating the influence of polymer concentration on the size of the formed particles.

[0024] Fig. 2 shows a schematic diagram illustrating the influence of charge ratios on the size of the formed particles.

[0025] Fig. 3 shows an enlarged section of Figure 2.

[0026] Fig. 4 shows the characterization of chitosan-alginate-dsRNA interpolyelectrolyte complexes. The β-potential of CAR-IPECs prepared with a molar charge ratio (z) of 0.75, 0.9, 1.25, 1.6, and 2 (+ / -) was determined by ELS (n=3). One-way ANOVA F4,10 = 1048.588; p<0.01 and Bonferroni's post-hoc test at p<0.05. Sigmoidal Boltzmann fit (line) with R 2 = 0.996.

[0027] Fig. 5. schematically shows the formulation process of chitosan-alginate-dsRNA IPECs.

[0028] First, dsRNA and alginate are mixed in a 1:1 ratio to form an anionic solution. The anionic solution is added to the chitosan, after which the interpolyelectrolyte complexes (IPECs) form via condensed phase separation. The latter is driven by electrostatic interactions between positively charged amino groups and negatively charged phosphate and carboxyl groups of chitosan, dsRNA, and alginate, respectively. The charge of the IPECs is determined by an excess of positive or negative groups during the condensed phase separation.

[0029] Fig. 6. Shows the effect of dsRNA treatment against Magnaporthe oryzae (Mo) infection in Brachypodium distachyon (Bd): Relative size of the affected area on Bd leaves sprayed with Mo conidia and with naked Pmkl dsRNA (1 ng / pL), chitosan-based Pmkl dsRNA nanoparticles (NPs), or empty NPs. Control plants were sprayed with conidial solution only (CTR). The relative size of the affected area relative to the entire leaf was calculated at 5 dpi using ImageJ.

[0030] Fig. 6A: Simultaneous treatment with Mo conidia and naked or formulated dsRNA or empty NPs.

[0031] Fig. 6B: Sequential treatment with a time interval of one day between dsRNA / NP treatment and subsequent Mo inoculation.

[0032] Fig. 6C: Sequential treatment with a time interval of 7 days between dsRNA / NP treatment and subsequent Mo inoculation. The results of three independent replicates were combined, and the boxplots represent the mean with standard deviation. Statistical significance was determined using the Kruskal-Wallis test (p<0.05), and the asterisks indicate the difference from the control group according to the Dunn test for multiple comparisons. Pairwise comparisons in (C) were performed using the Mann-Whitney test (*: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001).

[0033] Figure 7 shows the spray-mediated control of Magnaporthe oryzae on Brachypodium distachyon leaves infected with Mo conidia 7 days (Fig. 7 AC) or 14 days (Fig. 7 DF) after dsRNA treatment. Plants were treated with empty NPs, naked Phi6 dsRNA, or formulated Phi6 dsRNA (Phi6 dsRNA NP) as a control, or with naked Pmkl dsRNA or nanoparticle-formulated Pmkl dsRNA (Pmkl dsRNA NP). Control plants were sprayed with Mo conidia solution (CTR) only. The results of three independent replicates were combined.

[0034] Fig. 7 A and D: Infection symptoms on Bd leaves. A representative image from three independent experiments is shown.

[0035] Fig. 7 B and E: Quantification of the affected area on Mo-infected Bd plants. The relative size of the affected area relative to the total leaf was quantified using ImageJ at 6 dpi. Boxplots show the mean with standard deviation. Statistical significance was determined using the Kruskal-Wallis test (p < 0.05), and asterisks indicate the difference from the control group according to Dunn's multiple comparison test.

[0036] Fig. 7 C and F: Relative fungal growth determined from harvested leaves by RT-qPCR-based analysis of the expression of the Mo housekeeping gene MoGPD. The Bd housekeeper gene BdUbilO was used for normalization. Bars represent the mean with standard deviation from three independent replicates. Statistical significance was assessed by one-way ANOVA (p<0.05), and asterisks indicate the difference from the control group according to Dunnett's multiple comparison test (*: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001).

[0037] Fig. 8A shows remaining dsRNA ph16in the supernatant of dsRNA formulated in chitosan-alginate-dsRNA interpolyelectrolyte complexes (CAR-IPECs) and unformulated control dsRNA. The dsRNA concentration of the CAR-IPECs and the dsRNA controls (n=3) was determined using a nanophotometer with an extinction coefficient of 46.5 ng / pL A260nm-l. Subsequently, the samples and controls were centrifuged at 21380 g for 1 h at 4°C, and the dsRNA in the supernatant was analyzed using the nanophotometer to determine the concentration of free dsRNA in the samples. Significant differences between the supernatant and the untreated samples were determined according to the paired t-test: t(2)=63.23; p<0.01.

[0038] Fig. 8B shows the comparison of formulation efficiency achieved by different dsRNA formulation methods and the present application (★), ie, CAR-IPECs.

[0039] Fig. 9 shows the protection of dsRNA in chitosan-alginate-dsRNA interpoly electrolyte complexes. Fig. 9 A,B: The results were analyzed by gel electrophoresis at 110 V for 40 min in a 1% high-resolution agarose gel with the relevant dsRNA. phl6 L, M, and S segments were evaluated at 7599 bp, 4063 bp, and 2948 bp, respectively. Ladder (L) (Quick-Load® Purple 1 kb Plus DNA, New England Biolabs). Black bands in the gel wells indicate unreleased dsRNA.

[0040] Fig.9A: 180-minute RNase III treatment of free and formulated CAR phl6 -IPECs (0.1 g / L). In both cases, 1196 ng dsRNA phl6 treated with 0.05 U RNase III at 37°C for 0 and 180 min in the case of free dsRNA and for 0, 60, 120, and 180 min (') in the case of formulated dsRNA. The enzymatic reaction was stopped with 2.5 pmol EDTA.

[0041] Fig. 9B: RNase III treatment and EDTA release of dsRNA ph16, formulated in 0.1 g / L CAR-IPECs. 1196 ng of free or formulated dsRNA phiö were first treated with 0.05 U RNase III at 37°C for 0, 2, 5, 15, 30, and 60 min and then treated with 11 pmol EDTA for 60 min to stop the enzymatic reaction and release the dsRNA from the formulation.

[0042] Fig. 9 CE: Gel electrophoresis was performed in 1% agarose and stained with ethidium bromide for 30 min (50 min in E) at 70 V, whereby the relevant dsRNA™ v L, M, and S segments were located at 7599 bp, 4223 bp, and 3268 bp, respectively. Ladder (L) (MassRuler 1 kb DNA ladder, Thermofisher). All samples except the controls were treated with 11 pmol EDTA for 60 minutes to stop the enzymatic reaction (CD).

[0043] Fig. 9C: RNAse III treatment of free and formulated dsRNA™ v Control (“C”): untreated sample. 1200 ng dsRNA™ v were treated with 0.05 U RNAse III at 37°C for 0, 2 or 20 min.

[0044] Fig. 9D: MNase treatment of free and formulated dsRNA™ v 600 ng of each sample was treated with 3 U or 30 U MNAse at 37°C for 30 min.

[0045] Fig. 9E: Temperature treatment of free and formulated dsRNA™ v Control 1 (CI): untreated sample; Control 2 (C2): sample was incubated with only 11 pmol EDTA for 60 min at 37°C. 700 ng of each sample was temperature-treated for 30 min at 37°C, 45°C, and 60°C.

[0046] Fig. 10A shows representative images of TMV:GFP infection sites in N. benthamiana leaves 4 days post-inoculation (dpi), as seen under UV light. The leaves were inoculated with 20 ng of TMV-GFP virions and water or with 2 pg of free or formulated dsRNA™. v that has undergone a heat treatment. Scale = 1 cm.

[0047] Fig. 10B shows the number of infection sites in inoculated leaves at 4 dpi. Data are expressed as mean ± SEM and follow a normal distribution, as confirmed by the Shapiro-Wilk test (n = 6-7). Asterisks indicate significant differences according to unpaired t-tests. *P < 0.05.

[0048] Figure 11 A shows the comparison of in vitro produced 300 bp dsRNA formulated in chitosan-alginate-dsRNA interpolyelectrolyte complexes (CAR-IPECs) and chitosan-dsRNA (CR-IPECs) with different charge ratios. (1) DNA ladder (Quick-Load® Purple 1 kb Plus DNA, New England Biolabs), (2-6) CAR-IPECs with a charge ratio of (+ / -) 1.25, 1.5, 2, and 3, respectively, (7-11) CR-IPECs with a charge ratio of 1.25, 1.5, 2, and 3, respectively, and (12) unformulated dsRNA. Each pocket was loaded with 300 ng of dsRNA, either formulated in CAR-, CR-IPECs, or unformulated. Gel electrophoresis was performed in a 1% agarose gel with Roti gel staining in TA buffer at 110 V.

[0049] Fig. 11B shows the hydrodynamic diameter and PDI of dsRNA ph16 CAR (n = 5) and CR-IPECs (n = 3) with a loading ratio of 1.25. One-way ANOVA Fl, 6 = 7.447, p < 0.5.

[0050] Figure 12 shows the stability of polyelectrolyte complexes during storage. Chitosan-alginate-dsDNA interpolyelectrolyte complexes (“CAD-IPECs”) were prepared at a concentration of 1.25 (+ / -) and 0.05 g / L and stored for 16 days under sterile conditions at room temperature. 80–100 pL samples were measured in disposable UV microcuvettes (Z-height 8.5 mm, 70–850 pL UV microcuvette, Brand, Germany) by dynamic light scattering (Delsa Nano C, Beckman Coulter, Brea, USA) to determine the mean hydrodynamic diameter. Non-significant (ns) according to one-way ANOVA, n=3.

[0051] DETAILED DESCRIPTION OF THE INVENTION

[0052] Before describing the invention in detail, it should be noted that this invention is not limited to the individual components of the described devices or method steps of the described methods, since these devices and methods may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It is to be noted that where parameter ranges are given that are limited by numerical values, the ranges include these limits. It is further to be understood that the embodiments disclosed herein are not to be construed as individual embodiments that would not be related to one another. Features discussed in connection with one embodiment are also intended to be disclosed in connection with other embodiments shown herein.If, in one case, a particular feature is not disclosed in one embodiment but in another embodiment, this does not necessarily mean to a person skilled in the art that this feature should not be disclosed in the other embodiment. Those skilled in the art will understand that the purpose of this application is to disclose the mentioned feature for the other embodiment as well, but that this has not been done for reasons of clarity and to keep the description manageable.

[0053] Furthermore, the content of the prior art documents referred to herein is incorporated by reference. This applies in particular to prior art documents that disclose standard or routine processes. In this case, the primary purpose of incorporation by reference is to facilitate sufficient disclosure and avoid tedious repetition.

[0054] The present invention relates to a polyelectrolyte complex comprising at least one nucleic acid, wherein the polyelectrolyte complex comprises, in addition to the at least one nucleic acid, at least one ionotropic gel-forming anionic polymer and one cationic polymer, and wherein the polyelectrolyte complex is present as particles having a size in a range of < 2 pm.

[0055] Such a complex offers significant advantages over prior art solutions, particularly when used in a crop protection agent or plant strengthening agent. The present invention thus relates to a polyelectrolyte complex. A polyelectrolyte complex, also referred to as a simplex, is understood, for the purposes of the present invention, to be a complex in which two polymers interact electrostatically with one another due to charge differences. Depending on the excess charge, positive or negative complexes are formed that repel one another and thus neither aggregate nor agglomerate. Due to their charge and size, these charged polyelectrolyte complexes can form stable colloidal suspensions, for example in aqueous solutions.

[0056] Furthermore, it is characteristic of the polyelectrolyte complexes according to the invention that they are formed as particles which have the components, at least the anionic polymer, over their entire extent or across their entire cross-section. This should also include when the components, at least the anionic polymer, are present in variable concentrations or proportions along the diameter. This is, for example, a distinction from lipoplexes known from the prior art, in which alginate is present in the interior of their lipoplexes but not in the outer shell, and is therefore only released when the lipoplex is destroyed. According to the invention, the anionic polymer is present in the entire particle and in particular also on the outer circumference of the particle. Furthermore, liposomes are known for their comparatively short lifespan, so that the complexes according to the invention are more stable.Furthermore, the complexes according to the invention are smaller than lipoplexes, even with comparatively long nucleic acids, so that the complexes according to the invention can be absorbed particularly advantageously by plants.

[0057] The present invention encompasses the polyelectrolyte complex comprising at least one outer layer comprising an ionotropically gel-forming anionic polymer. Thus, a core can be present which has one or more layers on its exterior, of which at least one, preferably the outermost, and particularly preferably all, layers comprise an ionotropically gel-forming anionic polymer. For example, precisely one layer, also referred to as the shell, can be present as a coating.

[0058] The polyelectrolyte complex comprises at least one nucleic acid. As described above, nucleic acids can be advantageously used, for example, as active ingredients in crop protection products and should therefore remain stable for as long a period as possible to ensure their effectiveness. However, biologically active substances based on nucleic acids are usually very fragile and can be quickly degraded by biotic stress, i.e., enzymes, or abiotic stress, such as heat, UV radiation, or pH influences, rendering them ineffective. For example, dsRNA, as a biological crop protection product, must overcome this hurdle to remain functional over the long term and thus to be effectively used. Accordingly, the stabilization of nucleic acids, such as single- and double-stranded nucleic acids, such as dsRNA, may be necessary to ensure their long-term effectiveness when used as crop protection products.

[0059] To achieve this, the polyelectrolyte complexes comprise at least one ionotropically gel-forming anionic polymer. For the purposes of the present invention, an ionotropically gel-forming anionic polymer is understood in particular to mean a polymer with an anionic charge that can form a gel with ionic components, in particular with cationic components. In particular, the anionic polymers present form a gel with a metal ion. Furthermore, for the purposes of the present invention, a gel is defined as a particularly disperse system consisting of at least one solid and one liquid phase, in particular a colloid. The solid phase forms in particular a porous, three-dimensional network whose pores are filled by the liquid.

[0060] In the formulation, the anionic polymer acts as a biodegradable metal ion complexing agent, analogous to complexation with EDTA. This chelates the metal ions from the environment, which are cofactors of nucleases, rendering them nonfunctional and leaving the integrity of the formulated nucleic acids unaffected. One advantage is that this component offers an effective replacement for EDTA, which is often used in the state of the art. The metal ion chelator EDTA is usually used in various forms, e.g., EDTA disodium salt. However, EDTA is harmful to humans and aquatic organisms (GHS: H332, H373, H412), and the German Environment Agency recommends replacing EDTA with substances that are as easily degradable as possible and, above all, have no harmful effects.According to the invention, the existing anionic polymer can be used as a replacement for EDTA and, in a co-formulation with nucleic acids, reliably protects them from enzymatic degradation.

[0061] Preferably, the ionotropic gel-forming anionic polymer can be selected from the group consisting of alginate, furcellaran, guarate, carrageenan, pectinate and, from the polymer derivatives, amidated pectinate, carboxymethylcellulose, carboxyguargum, phosphoguargum, whereby alginate, especially sodium alginate, can be preferred.

[0062] Due to a high proportion of guluronic acid or mannuronic acid in the alginate, stability through nuclease inhibition can be particularly effective in alginate.

[0063] Furthermore, the polyelectrolyte complex of the invention comprises a cationic polymer. Therefore, by co-formulating anionic polymer and nucleic acid(s) with cationic polymers, simplexes (polyelectrolyte complexes) are formed as submicroparticles, so that the nucleic acids remain in close proximity to the anionic polymer and are thus protected with long-term stability. Submicroparticles, as defined by the invention, are those having a size in the range of equal to or less than 2 pm. Furthermore, the cationic polymer masks the charge of the nucleic acid, such as dsRNA. This is advantageous for plant penetration, since negatively charged substances are generally less able to penetrate plant cells.Accordingly, charge masking by the cationic polymer can enable improved penetration of the polyelectrolyte complex into the cells of the plants to be protected, which can further increase the effectiveness of a plant protection product based on the polyelectrolyte complex.

[0064] The present invention is thus based on preferably biodegradable products and is easily combined with known nucleic acid formulation methods. During production, the nucleic acids and the anionic polymer can first be mixed, thus protecting them from nuclease digestion from this point on. It can then be formulated, for example, together with a positively charged polymer, and thus be used in a plant protection product that can be used for various purposes, for example, as an agent for controlling pests or plant-pathogenic fungi and viruses.

[0065] Furthermore, the polyelectrolyte complex is present as particles with a size in the range of < 2 pm, approximately < 1 pm, for example < 500 nm, preferably < 200 nm, particularly preferably < 100 nm. Such a size range enables the particle and thus the nucleic acid to particularly efficiently overcome the size exclusion barrier of the plants to be protected, including the cuticle, the stomata, the cell membrane and the cell wall, and thus the nucleic acid can presumably reach the cells, particularly the cytoplasm, of plants. In the case of dsRNA, it is processed there by the cell's RNA interference machinery into effective siRNA. This siRNA, in turn, can recognize the messenger RNA (mRNA) of pathogens and pests and prevent their protein expression. Through the targeted selection of target (mRNA) sequences, the plant can be protected from specific pathogens and pests.Furthermore, it is possible that the formulation also enables the direct uptake of the nucleic acid into the pest and pathogen, and in particular into the cell interior or cytoplasm. In the case of dsRNA, this can also be processed into active siRNA in pathogens such as plant pathogenic fungi and plant pests, where it can become active. This can enable a highly effective action of the nucleic acid. Where sizes or diameters are mentioned in the context of the invention, this generally refers to the hydrodynamic diameter.

[0066] The nucleic acid can preferably be selected from the group consisting of dsDNA, ssRNA, sRNA, dsRNA, ssDNA, dsDNA, nRNA (poly(1:C)), circular nucleic acids (circ RNA, circ DNA). It has been shown that, in particular, the nucleic acids described above can be effectively complexed by the anionic polymer and, furthermore, form stable particles with the cationic polymer. These particles can also be easily produced in the desired size. Accordingly, the advantages of the present invention can be particularly effective, particularly when using the aforementioned nucleic acids. Thus, an effective and stable plant protection agent can be produced. However, it should be noted that the present invention is not strictly limited to the nucleic acids described above.

[0067] The cationic polymer can be selected from the group consisting of chitosan, poly-lysine, poly-arginine, poly-histidine, polydiallyldimethylammonium chloride, diethylaminoethyl cellulose, diethylaminoethyl dextran, chitosan-

[0068] Hydroxypropyltrimonium chloride, dextran-hydroxypropyltrimonium chloride, polyethyleneimine, and lysine-dextran, with chitosan being preferred. It has also been shown that, particularly when using the aforementioned polymers, such as chitosan in particular, as the cationic polymer, mechanically stable particles can also be produced. Thus, their use can provide high stability both against the degradation of the nucleic acids and against mechanical influences. Furthermore, corresponding biopolymers are biodegradable, so that the use of the polyelectrolyte complexes in this embodiment is particularly sustainable and ecologically advantageous. The ionotropically gel-forming anionic polymer can preferably be present, based on the at least one nucleic acid, in an amount of >0.1 parts by weight to <10 parts by weight, approximately from >0.75 parts by weight to <1.25 parts by weight, preferably from >0.95 parts by weight to <1.05 parts by weight.In this case, an amount of 1 part by weight relative to a reference should mean the same amount by weight and a part by weight of 0.75 should mean a weight amount of approximately 75% by weight relative to the reference, i.e. approximately that of at least one nucleic acid.

[0069] Alternatively or additionally, the cationic polymer may be present, based on the at least one nucleic acid, in an amount of > 0.1 parts by weight to < 10 parts by weight, for example from > 0.75 parts by weight to < 1.25 parts by weight, preferably from > 0.95 parts by weight to < 1.05 parts by weight.

[0070] It has been shown that, particularly in the above-described quantity ranges, stable polyelectrolyte complexes can be formed in which the nucleic acid can be effectively protected.

[0071] It may further be preferred that the polyelectrolyte complex be free of EDTA. As described above, EDTA is sometimes problematic for humans and the environment, and has harmful effects. Accordingly, substituting EDTA with biopolymers, which are preferably biodegradable, is highly advantageous. By using the ionotropic gel-forming anionic polymer, the use of EDTA can be completely eliminated according to the invention.

[0072] For further technical features and advantages of the polyelectrolyte complex, reference is made to the description of the crop protection agent, the plant strengthening agent, the process, the figures, and the description of the figures. Also described is a crop protection agent comprising a polyelectrolyte complex as described above. Furthermore, a plant strengthening agent comprising a polyelectrolyte complex as described above is described.

[0073] It has been shown that the polyelectrolyte complex can be an effective plant protection agent by containing the nucleic acid. In particular, the plant protection agent, which comprises one or more nucleic acids, can be effective against pests as well as other pathogens.

[0074] Because the polyelectrolyte complex contains at least one ionotropic gel-forming anionic polymer in addition to the nucleic acid and is further provided with a cationic polymer, the nucleic acid or crop protection agent can be particularly stable over the long term. In particular, the nucleic acid in the polyelectrolyte complex can exhibit high stability against nucleases, UV radiation, and elevated temperatures or heat.

[0075] A corresponding effect could be demonstrated regardless of whether the polyelectrolyte complexes are used preventively, i.e. in a plant protection product, or when at least partial damage or infestation has already occurred, i.e. in a biological pest control product.

[0076] Furthermore, very good long-term stability has also been demonstrated for a plant tonic intended to generally maintain plant health. In particular, plant tonics are intended to increase plant resistance to external influences. Accordingly, a plant tonic containing the polyelectrolyte complexes described above has also been described. In principle, it has been shown that the plant protection agent (dsRNA) is effective more effectively due to its charge, size, and RNase protection.

[0077] For further technical features and advantages of the plant protection product and the plant strengthening agent, reference is made to the description of the polyelectrolyte complex, the process, the figures and the description of the figures.

[0078] Also described is a method for producing a polyelectrolyte complex, comprising at least the following process steps: a) producing a solution comprising a nucleic acid, at least one ionotropic gel-forming anionic polymer, and a positively charged polymer; b) optionally heating the solution, preferably in a temperature range of > 30°C to < 90°C; c) mixing the solution; and d) incubating the solution.

[0079] The above-described process advantageously makes it possible to produce a polyelectrolyte complex as described above.

[0080] For this purpose, according to process step a), a solution comprising a nucleic acid, alginin, and a positively charged polymer is first generated. The solution can, in principle, comprise any suitable solvent, although water may be particularly preferred.

[0081] In principle, the nucleic acid used can be selected. However, it has been shown that the nucleic acid can preferably be selected from the group consisting of dsDNA, ssRNA, sRNA, dsRNA, ssDNA, dsDNA, poly (1:C)), and circular nucleic acids. The cationic polymer can also be selected in principle. However, it has been shown that the aforementioned advantages can be particularly effective when chitosan is used as the cationic polymer.

[0082] In principle, the order in which the solution or the combination of the individual reactants is generated can be freely selected. However, it has been shown that the order in which the respective components are mixed can influence the size of the polyelectrolyte particles produced. In order to produce small particles with a size in the range of < 2 pm, it can be advantageous to first produce a mixture of nucleic acid and anion in process step a) and then mix this mixture with cationic polymer. Surprisingly, it has been shown that the size of the polyelectrolyte particles can be particularly small, especially in this embodiment.In particular, in this embodiment, it can preferably be made possible for the polyelectrolyte particles produced to have a size in a range of < 2 pm, so that polyelectrolyte particles can be produced as submicroparticles according to the invention.

[0083] The result of the inventive process is therefore that the particles are initially produced in a suitable size. Once they are formed and surrounded by particles of the same charge, the particles will repel each other. This keeps the particles small and maintains this state over an extended period. However, agglomeration can occur over very long periods.

[0084] If the particles are located in a larger area, for example due to agglomeration, the agglomerated particles can be separated again by applying energy, such as ultrasound, and the existing particle agglomerates can be reduced in size. Furthermore, the size of the particles can be controlled, for example, by the concentration of the respective substances used; for example, high concentrations can increase the size of the particles.

[0085] It may further be preferred that the nucleic acid, cationic polymer, and ionotropic gel-forming anionic polymer are each present in the solution produced in process step a) in the same mass fraction or part by weight. This can enable particularly effective long-term stability of the nucleic acid in the polyelectrolyte complex. Furthermore, it can be supported so that the particles have a particularly small particle size in the range of < 2 μm. Such a size of the polyelectrolyte particles can, as already indicated above, enable particularly good bioavailability.

[0086] With regard to the proportion of the cationic polymer and, correspondingly, preferably also of the ionotropic gel-forming anionic polymer and the nucleic acid, it may further be preferred that these are each present in the solution produced in process step a) in a proportion of <0.01 wt.%, preferably <0.05 wt.%. These proportions are present in particular in the starting solution and, accordingly, also in the final solution. This configuration can also help ensure that the formed complex particles have a small size in the range of <200 nm.

[0087] The same applies if a polyelectrolyte complex is produced with a charge ratio in a range of >1.25, purely by way of example to <3. In the context of the present invention, a charge ratio should be understood in particular as meaning that the ratio of the positive charges of the polycation, i.e., for example, the charges of all amine groups of chitosan, to the negative charges of the polyanions, i.e., for example, the charges of the phosphate groups of the nucleic acid, such as in particular dsRNA and the carboxyl groups of alginate, for example, lies within the predetermined range. In principle, all charge ratios >1.25 will produce positive particles. From a certain point, however, even positive polymer, such as chitosan, would presumably be unbound, i.e., not as part of a polyelectrolyte complex.

[0088] Finally, it may be preferable for all substances used in the process, in particular all solvents used in the process, to be nuclease-free. This prevents nuclease attack during the production of the polyelectrolyte particles, in which the nucleic acid is still unprotected. To ensure that the nucleic acids do not come into contact with nucleases and are not degraded prior to the formulation process, nuclease-free solvents, such as DEPC water, are used. Furthermore, all solutions and materials are treated with, among other things, UV-C, autoclaving, or decontamination reagents, such as the agent known as RNase-AWAY, for nucleases.

[0089] After the solution has been produced, the solution is optionally heated according to process step b), preferably to a temperature range of > 30 °C to < 90 °C, such as to 45 °C.

[0090] According to process step c), the solution is then mixed, which can be achieved, for example, by mechanical means such as stirring. In principle, this is also possible using a vortex mixer.

[0091] Finally, to form the polyelectrolyte particles, in particular the polyelectrolyte submicroparticles, the solution is incubated according to process step d). In this process step, the solution can be stored essentially untreated under ideally unchanged parameters, such as temperature, to enable growth of the polyelectrolyte particles.

[0092] The product can then be stored until further use. This can be done, in particular, at temperatures at or below room temperature, i.e., temperatures in a range of > 1 °C to < 25 °C. Further steps are also conceivable, such as increasing the particle concentration through dialysis and adding additional adjuvants to improve spray application.

[0093] EXAMPLES

[0094] While the invention has been shown and described in detail in the drawings and the foregoing description, these illustrations and descriptions are to be considered as illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations of the disclosed embodiments may be understood and practiced by those skilled in the art in practicing the claimed invention, having regard to the drawings, the disclosure, and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. The mere fact that certain means are recited in different dependent claims does not mean that a combination of those means cannot be advantageous. Any reference signs in the claims are not to be understood as limiting the scope of application.

[0095] All amino acid sequences disclosed herein are presented from N-terminus to C-terminus; all nucleic acid sequences disclosed herein are presented 5'->3'.

[0096] The terms "polyelectrolyte complex," "nanoparticle" (NP), and "interpolyelectrolyte complex" (IPEC) are used interchangeably in this application. For example, the terms "dsRNA-NP," "dsRNA-IPEC," and "CAR-IPEC" refer to the same formulation (containing chitosan, alginate, and dsRNA).

[0097] Example 1 - Preparation and characterization of the polyelectrolyte complex In a specific embodiment, the process can proceed as follows.

[0098] I. All polymers are diluted to the same concentration with nuclease-free water. a. 1% (w / w) chitosan is dissolved in 1% (w / w) acetic acid solution and then diluted to 0.005% with water; b. 0.005% (w / w) alginate is dissolved in water; c. dsRNA is diluted to 50 ng / pL with nuclease-free water (this corresponds to 0.005% (w / w);

[0099] II. The anionic solutions are mixed 1 : 1 so that a 0.005 wt.

[0100] % anionic polymer solution is created. a. This consists of 0.0025% alginate and 0.0025% dsRNA to ensure good protection of the nucleic acid;

[0101] III. For optimal mixing and formulation reaction, both solutions are heated to a range of > 40 °C to < 90 °C, for example to 45 °C.

[0102] IV. The cationic (chitosan) solution is added (in heated state, 45°C) in a charge ratio of 1.25 (up to 2.0) + / - to the anionic solution (RNA and alginate).

[0103] V. To ensure optimal mixing, the solution is optionally vortexed, although other mechanical energy input such as stirring is also possible.

[0104] VI. The mixture is incubated for 1 hour at room temperature (23°C in the laboratory) before the solution is subsequently analyzed.

[0105] VII. Storage is then possible at 1° to 25°C until use.

[0106] The polyelectrolyte complexes prepared as described above can be characterized as follows. 1. Characterization of the formulation particles:

[0107] The formulations had a charge ratio of 1.25 and were prepared using 0.005 wt% solutions. Long dsRNA (three segments with 3000 to 8700 base pairs) was formulated into these.

[0108] The formulations produced with this dsRNA, these parameters, and the above preparation method were analyzed for size, size distribution and charge using Dynamic Light Scattering (DLS) and Electrophoretic Light Scattering (ELS)

[0109] (Zeta Potential), whereby the particle sizes were additionally determined by

[0110] Scanning electron microscope. The following parameters were obtained, which are to be considered as examples.

[0111] • hydrodynamic diameter (Z-average): 93.7 nm ± 2.9 nm

[0112] • Volume distribution: 61.8 nm ± 3.4 nm

[0113] • Polydispersity index: 0.214 ± 0.024

[0114] • Charge (zeta potential) ± 27.7 ± 1.4 mV

[0115] The volume distribution is a common measure of particle size known to those skilled in the art, measured using DLS. It represents the average particle size relative to the volume of all particles in the solution.

[0116] The polydispersity index indicates the monodispersity of the particles, essentially whether there are multiple particle populations of different sizes. Generally speaking, the smaller the particles, the more monodisperse they are. This results in a so-called smaller bell curve.

[0117] Regarding charge, this refers to the positively charged particles. This determination is helpful because positively charged particles / molecules are more easily absorbed by plant cells. This improves the uptake of dsRNA, which is inherently negatively charged.

[0118] With regard to the above-mentioned characterizations, the following standards are applicable with regard to the hydrodynamic diameter, the polydispersion index and the zeta potential:

[0119] Z-Average: ISO 13321 and ISO 22412:2017

[0120] Polydispersity Index: 13321 : 1996 E and ISO 22412:2008

[0121] Zeta Potential: ISO 13099 (ISO 13099-3:2014)

[0122] The above-described process allows the polyelectrolyte particles according to the present invention to be produced in a particularly advantageous manner. Accordingly, the process serves to produce nucleic acid-containing polyelectrolyte particles in which the nucleic acid is effectively stabilized and, in particular, protected from nucleases, UV radiation, and heat.

[0123] The invention is explained below by way of example with reference to the attached drawings, wherein the features shown below can represent an aspect of the invention both individually and in combination, and wherein the invention is not limited to the following drawing, the following description and the following embodiment.

[0124] Figure 1 shows the influence of the total polymer concentration, i.e. the concentration of the cationic polymer, for example chitosan, together with the anionic polymer, for example alginate and dsRNA, on the size of the polyelectrolyte complexes produced, where the X-axis describes the polymer concentration of the polymer mixture in wt.% in the mixture produced in process step a) and where the Y-axis shows the average hydrodynamic diameter of the polyelectrolyte complexes formed in nm.

[0125] The experiments with different solution concentrations showed that the smallest particles were obtained at low polymer concentrations such as 0.005 wt% in the solution produced in process step a).

[0126] Figure 2 shows the influence of the charge ratios on the size of the produced polyelectrolyte complexes, where the X-axis shows the charge ratios [+ / -] of the produced polyelectrolyte complex and the Y-axis shows the average hydrodynamic diameter of the formed polyelectrolyte complexes in nm.

[0127] It has been shown that the particles become very large near a charge ratio of 1, which is presumably due to the fact that the particles have little to no surface charge (zeta potential). As a result, the particles no longer repel each other, allowing them to agglomerate and aggregate. Accordingly, Figure 2 shows that particles with a charge ratio > 1 are positively charged, and particles with a charge ratio < 1 are negatively charged. Presumably, there is a transition point (theoretically at exactly 1) where the particles are uncharged, which theoretically should be at a charge ratio of 1.

[0128] Surface charge

[0129] The molar charge ratio, often referred to in the literature as the N / P ratio, is a crucial parameter in the formulation of nanoparticles through electrostatic interaction (Mao et al. 2010). In the present example, particles formed by electrostatic interaction of oppositely charged polyelectrolytes (IPECs) are treated. The molar charge ratio (z) is defined herein as the ratio of the positive and negative charges of the formulation components that can interact with each other. For z > 1, there is an excess of cationic charges, and for z < 1, there is an excess of anionic charges. Therefore, the resulting particles should be positively and negatively charged, respectively. For z ~ 1, cationic and anionic charges are approximately balanced, so that the average charge of the particles in the suspension is approximately neutral.Therefore, at charge ratios close to z = 1, aggregation of particles with low positive or negative charges and agglomeration of neutral particles are to be expected.

[0130] To investigate these hypotheses, CAR-IPECs were formulated with charge ratios between 0.75 and 2.0 (+ / -). The resulting particles were analyzed by DLS and ELS to determine their hydrodynamic size and ^-potential, respectively. The corresponding ELS results are presented in Fig. 4. As shown, ELS experiments with dsRNA confirmed ph16 - formulations at charge ratios above 1, the positive surface charge of the IPECs with C, from +27.7 ± 1.4 mV at z = 1.25, which increased to +35.5 ± 0.4 mV at z = 2.0. The increase of the ÜP° tenzThe potentials of the IPECs correlate well with the charge ratio used in the formulation process. Furthermore, the formulations prepared with z of 0.75 and 0.9 had a negative (^-potential of -31.1 ± 0.98 mV and -23.7 ± 1.4 mV, respectively. It is also plausible that IPECs formed at z < 0.75 are increasingly negatively charged, i.e., follow the same sigmoidal function (sigmoidal fit) observed between z of 0.75 and 2.0 (Fig. 4; Table 2).

[0131] Table 2: Boltzmann fit, performed on ÜP° ten ti a Chitosan-alginate-dsDNA interpolyelectrolyte complexes were prepared with a molar charge ratio (z) of 0.75, 0.9, 1.25, 1.6, and 2 (+ / -) and measured by electrophoretic light scattering (Delsa Nano C, Beckman Coulter, Brea, USA). The underlying zeta potential data were generated by N=3.

[0132] Overall, these results suggest that predictions about the size and charge of IPECs can be made based on the molar charge ratio. Generally, positive particles are developed for foliar application (Rank et al. 2021), however, there is no clear consensus in the literature regarding benchmark potential due to the wide range of different targets (Zhang et al. 2023 and Parkinson et al. 2022). Based on these results, CAR-IPECs were formulated at z = 1.25 (+ / -), at low polymer concentrations < 0.1 g / L, and with an equal amount of alginic acid and dsRNA for subsequent experiments, as these parameters produced cationic IPECs containing the largest amount of alginic acid without significantly changing the charge or size.

[0133] Example 2 - Effect of dsRNA treatment against Magnaporthe oryzae (“Mo”) infection in Brachypodium distachyon (“Bd”) Production of dsRNA

[0134] The dsRNA sequences for the Pmkl gene were synthesized using MEGAscript (ThermoFisher) according to the manufacturer's instructions. The Pmkl PCR templates for in vitro transcription were prepared from cDNA of axenically grown fungi using primers with the T7 sequence.

[0135] Phi6 dsRNA was produced in Pseudomonas syringae LM2691, which stably replicates the phage genome. The produced tri-segmented phi6 dsRNA genome, which contains a kanamycin resistance gene insertion in the largest segment, comprises 2948, 4063, and 7599 bp dsRNA molecules. After culturing the bacteria overnight in liquid culture, the cells were harvested, and the dsRNA was purified by nucleoZol-chloroform extraction, stepwise LiCl precipitation, and ammonium acetate precipitation.

[0136] The aforementioned Pmkl and phi6 genes, their sequences, and the production of corresponding dsRNA are known to those skilled in the art (see, for example, Niehl et al. 2018; the content of this document is incorporated herein in its entirety for the purpose of explanatory power). The L, S, and M segments of the Phi6 genome are also known to those skilled in the art (L: https: / / www.ncbi.nlm.nih.gov / nuccore / 20330564; M: https: / / www.ncbi.nlm.nih.gov / nuccore / 20330569; S: https: / / www.ncbi.nlm.nih.gov / nuccore / 20330558).

[0137] It should also be noted that one skilled in the art is able to select suitable primer combinations (optionally with a probe) to identify and quantify the expression of each of these genes based on the disclosure provided herein in conjunction with their routine knowledge. dsRNA Formulation in NPs pmkl dsRNA (SEQ ID NO: 1) was encapsulated in dsRNA-alginate-chitosan nanoparticles (NPs) according to the method of the present invention (see Example 1). A 50 ng / μl chitosan solution (30 kDa, 90% deacetylation; Glentham Life Sciences Ltd, United Kingdom) dissolved in 0.005% (v / v) acetic acid and a 50 ng / μl anionic polymer solution consisting of an equal concentration of dsRNA and sodium alginate (Algogel 3001, Cargill, USA) were heated to 45°C for 1 minute.Subsequently, the chitosan solution was pipetted into the anionic solution at a volume ratio of 1:1.17 (chitosan solution: anionic solution), achieving a positive-to-negative charge ratio of 1.25:1, i.e., the ratio of the positively charged amino groups of the chitosan to the negatively charged phosphate and carboxyl groups of the dsRNA and alginate, respectively. The resulting mixture was shaken briefly and incubated at RT for 1 h. Samples were taken for particle characterization, and the remaining NPs were stored at 4°C. In addition, alginate-chitosan control NPs were prepared with the same positive-to-negative charge ratio, which is why the volume ratio was adjusted to 1:0.94 (chitosan solution: alginate solution).

[0138] The formulation prolongs the protective effect of dsRNA treatment against Magnaporthe oryzae infections in Brachypodium distachyon.

[0139] The activity of naked Pmkl-dsRNA was compared with formulated Pmkl-dsRNA-NP. Three-week-old Bd plants were first sprayed with a mixture of conidia and 1 ng / μl of dsRNA, dsRNA-NP, or empty NPs. Analysis of the affected leaf area at 5 dpi showed that unformulated and formulated Pmkl-dsRNA protected Bd plants to the same extent, while the empty NP did not (Fig. 6A). Thus, dsRNA is released from NPs and can activate RNAi to the same extent as naked dsRNA. The present inventors then designed an experiment that better reflects the agronomic practice of preventive treatment, where a longer time interval between dsRNA application and inoculation could more accurately demonstrate the benefits of a dsRNA formulation. The plants were sprayed with dsRNA and inoculated with Mo conidia 1 day or 7 days later.No difference was observed between plants treated with naked or formulated Pmkl dsRNA when a one-day gap was chosen between treatment and infection. However, when a 7-day gap was maintained, the Pmkl dsRNA NP showed significantly greater protective efficacy against Mo infection than the naked Pmkl dsRNA (Mann-Whitney test, p<0.0001) (Fig. 6 B and C; Table 1). Table 1: Disease severity in Mo-infected Bd leaves after treatment with naked and formulated dsRNA.

[0140] In summary, the results described above demonstrate that the double-stranded RNA (pmkl) has a significant effect on fungal infection. In contrast, RNA with a different sequence (phiö) showed no effect. The formulation substances chitosan and alginate, which were formulated together as particles (empty NPs), also showed no effect.

[0141] Furthermore, the dsRNA formulated with chitosan and alginate (pmkl-dsRNA-NP) showed a more significant effect than the reformulated agent (pmkl-dsRNA). In other words, the results demonstrate that the dsRNA formulated with chitosan and alginate (pmkl-dsRNA-NP) functions as a pesticide against this fungus and shows improved efficacy compared to the unformulated pmkl-dsRNA.

[0142] Example 3 - In vivo produced dsRNA with chitosan and alginate for improved plant protection against tobacco mosaic virus (TMV)

[0143] Materials and Methods dsRNA production and purification

[0144] TMV is a single-stranded RNA virus (ssRNA). The sequence of the viral genome is fully known to those skilled in the art (https: / / www.ncbi.nlm.nih.gOv / nuccore / NC_001367.l).

[0145] High-quality (fully duplexed) dsRNA was produced in Pseudomonas syringae strain LM2691, in which a lysis-defective modified bacteriophage phiö replicates as a stable episome, expressing the desired dsRNA sequences (see Niehl et al. 2018 and Sun et al. 2004). P. syringae-Z \\\ mn for the production of TMV-specific dsRNA (dsRNA™ v ) and target-unspecific dsRNA ph16 have already been established and stored as a glycerol stock at -80 °C (Niehl et al. 2018, the contents of this document are incorporated herein in their entirety for the purpose of executability).

[0146] For this study, dsRNA ph16 and dsRNA™ vprepared as follows: The dsRNA-producing bacteria were incubated in Luria-Bertani broth containing kanamycin (25 pg / ml) at 28 °C for 18–20 h, followed by centrifugation at 6000 g for 10 min to collect the cells. The cell pellet was dissolved in ultrapure water (Merck, Darmstadt, Germany), and the cell suspension was mixed with an equal volume of NucleoZOL (Macherey-Nagel, Düren, Germany) and 1 / 5 volume of chloroform (VWR, Bratislava, Slovakia). The mixture was shaken vigorously and centrifuged at 8000 g for 15 min. The aqueous phase was collected, and the RNA extraction procedure was repeated two more times. After the final extraction step, the RNA was precipitated with isopropanol (Emsure, Merck, Darmstadt, Germany), washed with 75% ethanol, and dissolved in ultrapure water.The dsRNA was fractionated from the ssRNA using a stepwise LiCl precipitation as described by Levanova and Poranen (2018). The ssRNA was first precipitated from the RNA mixture using 2 M LiCl, followed by dsRNA precipitation using 4 M LiCl (Merck, Darmstadt, Germany). The LiCl fractionation was repeated twice to increase the purity of the dsRNA. The concentration and purity of the dsRNA were first determined using the NanoDrop 2000c UV-Vis spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). The quality of the dsRNA was then checked by agarose gel electrophoresis. The final dsRNA preparation comprised three dsRNA segments: 7599 bp, 4223 bp, and 3268 bp for dsRNA. ph16 and 7599 bp, 4063 bp and 2948 bp for dsRNA™ v The largest segment is the original L segment of Phi6 with a kanamycin resistance gene insert, while the middle (M) and small (S) segments are either originally (for dsRNA ph16) or modified (for dsRNA™ v ) and consist of TM V-specific dsRNA. The dsRNA was stored in aliquots in RNase-free microcentrifuge tubes at -80 °C. For the experiments, the aliquots were thawed on ice. Thawed samples were stored at 4 °C for up to 1 week. In addition, a batch of dsRNA ph16 sonicated to obtain fragmented dsRNA. In this study, dsRNA ph16 full-length or sonicated, dsRNA™ v and sonicated dsDNA from salmon sperm (UltraPure™ Salmon Sperm DNA Solution, Thermo Fisher Scientific, USA) was used for experiments and treated ceteris paribus. The dsRNA ph16 came from a single production and purification batch stored in aliquots at -80 °C.

[0147] Formulation of interpolyelectrolyte complexes (IPECs)

[0148] First, a 1% acetic acid solution was prepared using double-autoclaved MilliQ water. To prepare the chitosan solution, 1 g of chitosan with a molecular weight of 30 kDa and a degree of deacetylation of 90% (Glentham Life Sciences Ltd, United Kingdom) was dissolved in 99 g of the 1% acetic acid solution and stirred overnight to obtain a 1% (w / w) stock solution. Separately, 0.5% sodium alginate (Algogel 3001, Cargill, USA) was dissolved in ddH2O by stirring. Both solutions were autoclaved at 121 °C for 6 minutes to ensure that the biopolymers were not depolymerized and to ensure the sterility and homogeneity of the solutions, which were then sterile filtered using 0.2 pm cellulose acetate syringe filters.

[0149] The dsRNA aliquots were then thawed on ice, and their concentrations were determined using a nanophotometer (NanoPhotometer™ N60, Implen, Germany) with a dsRNA absorption coefficient of 46.5 ng / pL A260nm determined from the literature (see Nwokeoji et al. 2017 and Strezsak et al. 2021). The concentrations were then adjusted by dilution with nuclease-free water. The dsRNA and alginate solutions, both at the same concentration, were then combined in various ratios to form the anionic precursor solutions. Precursor solutions with lower polymer concentrations were prepared by further dilution with nuclease-free water. The formulation procedures were carried out in a dedicated sterile workbench (VWR PCR Workstation, VWR International, USA) sterilized with UV-C radiation.The indoor air was continuously circulated past UV-C lamps, and the surfaces were cleaned with RNase AWAY® (Molecular BioProducts, USA). To ensure homogeneous solutions, the chitosan and the anionic precursor solutions were briefly heated to 45°C in a thermomixer (CellMedia, Gutenborn, Germany). The anionic precursor was then pipetted into the chitosan solution, and the solutions were briefly mixed with a micropipette. The mixture was then shaken at low speed for 5 s, and the IPECs formed by electrostatic interaction of positively charged N and negatively charged C and P groups (Fig. 4A). The formulation was then incubated for 1 h at room temperature or stored at 4°C prior to analysis. CAD and CA IPECs, i.e., with DNA andwith only chitosan and alginate (CA-IPEC), were prepared following the same procedure, adjusting the polymer solutions to maintain the same molar charge ratio. To prepare IPECs with the desired charge ratios (z), molar charge ratio calculations were performed based on the assumed electrostatic interaction of a cationic, positive charge (+), i.e., an amino (N) group of chitosan monomers, with an anionic, negative charge (-), i.e., a carboxyl (C) group of alginic acid monomers, or a phosphate (P) group of nucleic acid nucleotides. Therefore, the molar charge ratio was calculated based on the positively charged N groups to negatively charged C and P groups. For example, a ratio of 1.25 positive to negative charges (+ / -) means B. 1.25 times or a 25% excess of N groups compared to the combined C and P groups.To determine the molar charge ratio, factors such as the degree of deacetylation of chitosan, the dry weight of the polymers, and the molecular weight of the charge-bearing monomers must be considered. The calculations assumed equal amounts of all nucleotides in the dsRNA and dsDNA sequences, and thus equal amounts of guanosine monophosphate, cytidine monophosphate, adenosine monophosphate, and uridine or thymidine monophosphate. The average molar mass of (deoxy)ribonucleic monophosphate was calculated to be 321.5 g mol-1 for dsRNA and 309.0 g / mol for dsDNA (Strezsak et al. 2021). Furthermore, due to the length of the L, M, and S segments of dsRNA, the following calculations were made: ph16 the charge and weight differences of the 5'-triphosphates are neglected. Formulation efficiency

[0150] To determine formulation efficiency (FE), CAR-IPECs were formulated with polymer concentrations of 0.5 g / L and a molar charge ratio of 1.25 (+ / -) to achieve a dsRNA concentration of 125.3 ng / pL. Control samples containing 132 ng / pL of unformulated dsRNA were prepared analogously. The samples were centrifuged at 21380 g for 1 h at 4 °C (MIKRO 200 R, Andreas Hettich, Tuttlingen, Germany), and the supernatant from each sample was collected for subsequent analysis. The dsRNA concentration of all samples was determined using a nanophotometer as above.

[0151] The formulation efficiency (also referred to as encapsulation or entrapment efficiency in the literature) of dsRNA in CAR-IPECs was calculated using the following equation:

[0152] FE % = (input dsRNA-unformulated dsRNA) / input dsRNA *100 %

[0153] In the present experimental setup, the unformulated dsRNA remains in the supernatant, while the formulated dsRNA sediments in the CAR-IPECs, which means that the above equation can also be stated as follows

[0154] FE % = (input dsRNA supernatant dsRNA) / input dsRNA *100 %

[0155] Polyelectrolyte complex characterization

[0156] The hydrodynamic diameter (Dh) of the samples was determined using dynamic light scattering (DLS) with a Beckman Coulter Delsa Nano C instrument with a scattering angle of 165°. Approximately 80–100 pL of each sample was dispensed into disposable UV microcuvettes (Z-height 8.5 mm, 70–850 pL UV microcuvette, Brand, Germany) and placed in the cell counting chamber of the DLS instrument. If necessary, highly concentrated colloidal suspensions were diluted with MilliQ water to ensure accurate measurements. Seventy scans were recorded to calculate the number, volume, and intensity distributions, as well as the Z-mean (or cumulative result) according to ISO 22412:2017, along with the polydispersity index (PDI) of the particles.

[0157] The zeta potentials C of the IPECs were determined using electrophoretic light scattering (ELS) with the same Delsa Nano C instrument. To ensure the absence of air bubbles in the flow cell, 800–1200 pL of the sample volume was injected into the flow cell using sterile syringes (Fisherbrand™, Thermo Fisher Scientific Inc., USA).

[0158] Stability studies

[0159] The stability of free and formulated dsRNA to nucleases and heat stress was assessed either by exposure to RNase III (Shortcut® RNase III; New England Biolabs, Ipswich, USA) or micrococcal nuclease (MNase; Thermo Fisher Scientific, USA), or by incubation at elevated temperatures followed by gel electrophoresis. To achieve detectable band intensity, 0.1 g / L (100 ng / pL) of dsRNA was formulated in 0.1 g / L CAR-IPECs with a charge ratio of 1.25 (+ / -), yielding 26 ng / pL of dsRNA.

[0160] For RNase III, a stability test was performed in which the dsRNA was treated as specified by the manufacturer, but with adjusted volumes and concentrations to meet the assay requirements. Shortcut® Reaction Buffer * 10 was diluted 10-fold and 1 pL was added to the reaction. Subsequently, 46 pL of 0.1 g / L dsRNA, either free or encapsulated, was added. Then, 0.05 U of RNase III diluted in enzyme dilution buffer (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 0.1 mM EDTA in 50% (v / v) glycerol) was added prior to the addition of 0.02 pmol MnCl2. The final volume of the reaction mixture was 50 pL. Depending on the experiment, the samples were incubated for 0–180 min at 37°C. The reaction was then terminated with 2.5 pmol EDTA, and the mixture was incubated for 5 min. To further release the dsRNA from the formulation, the mixture was incubated with 11 pmol EDTA (final concentration 152.8 mM) for 60 min, based on the method described by Lallana et al.(2017) developed heparin-mediated nucleic acid release.

[0161] The samples were prepared at staggered times to ensure the simultaneous release of dsRNA prior to gel electrophoresis. 25 pL of each treated sample was mixed with 2.5 pL of 60% (v / v) glycerol. Gel electrophoresis was performed in high-resolution 1% agarose gels (Carl Roth, Karlsruhe, Germany) stained with ROTI gel stain (2.5 pL gel stain / 50 mL agarose; Carl Roth, Karlsruhe, Germany). Gel electrophoresis was performed at 110 V for approximately 40 minutes, and images were acquired using an ethidium bromide filter (UVP Ethidium Bromide Filter, Analytik Jena™, Germany) with the gels illuminated under UV light at 312 nm. The results are shown in Fig. 9A-C.

[0162] For MNase treatment, the enzyme was first diluted in reaction buffer pH 7.9 (50 mM Tris-HCl and 5 mM CaCl2) to 3 U / pL or 30 U / pL. Subsequently, 24 pL containing 600 ng of free or formulated dsRNA™ v (25 ng / pL stock) were incubated with 3 U or 30 U of MNase for 30 min at 37 °C in a thermal cycler (VeritiPro™ Thermal Cycler, Applied Biosystems). The reaction was stopped, and the formulations were dissolved by adding 11 pmol of EDTA (final concentration of 152.8 mM), followed by incubation for 60 min at 37 °C. The integrity of the dsRNA was assessed by gel electrophoresis in a 1% agarose gel (Euromedex) stained with ethidium bromide for 30 min at 70 V. Before loading the gel, the treated samples (36 pL) were mixed with 3.6 pL of 60% (v / v) glycerol. The results are shown in Fig. 9D.

[0163] The stability of the dsRNA at different temperatures was investigated by incubating 56 pL with 700 ng of free or formulated dsRNA™ v (12.5 ng / pL strain) were incubated at 37°C, 45°C, and 60°C for 30 minutes in a thermal cycler (VeritiPro™ Thermal Cycler, Applied Biosystems). To dissolve the formulations, 11 pmol EDTA (final concentration of 152.8 mM) was added to all samples and then incubated for 60 minutes at 37°C. The integrity of the dsRNA-treated samples was assessed by gel electrophoresis in a 1% agarose gel (Euromedex) stained with ethidium bromide for 50 minutes at 70 V. The results are shown in Fig. 9E. dsRNA treatment of Nicotiana benthantiana against TMV

[0164] The temperature treatment of free or formulated dsRNA was carried out in vitro as described above: Samples of 25 ng / pl (dsRNA™ v) stocks were incubated at either 37 °C or 60 °C for 30 minutes in a thermocycler to obtain heat-treated samples of free CAR-IPEC formulated dsRNA™ v Before application to plants, the efficacy of the treatment was confirmed by visualizing the samples in a 1% agarose gel stained with ethidium bromide. Before loading the gel, 500 ng of the heat-treated dsRNA was incubated with 11 pmol EDTA (final concentration of 152.8 mM) for an additional 60 minutes at 37 °C to dissolve the formulations. Gel electrophoresis was performed for 50 minutes at 70 V. Since EDTA salt can be toxic to plants, the remaining samples were applied directly to the leaves of Nicotiana benthamiana without added EDTA.

[0165] N. benthamiana seedlings were grown in a greenhouse for 4 weeks under 16h / 8h light / dark periods at 22°C / 18°C. Leaves of uniform size were selected, and one leaf per plant was simultaneously treated with either 2 pg of heat-treated free or formulated dsRNA™. v(80 pL of a 25 ng / pL stock) together with 20 ng of GFP-expressing TMV virions (TMV:GFP) (3 pL of a 0.67 ng / pL stock) (Lindbo, 2007). In the control treatment, the volume of dsRNA was replaced with water. The mixture was mechanically inoculated by rubbing the leaves in the presence of Celite. To remove excess Celite, the leaves were washed with distilled water 30 minutes after inoculation, and the plants were then returned to the greenhouse under the same growth conditions. Images were taken 4 days post-inoculation (dpi) under UV light, and the number of TMV:GFP infection sites on the treated leaves was quantified. The results are shown in Fig. 9.

[0166] Statistical analysis

[0167] Statistical analyses were performed using IBM SPSS Statistics V22.0 software (SPSS, Chicago, USA). All values ​​are expressed as mean ± standard deviation (SD), derived from at least 3 or 5 replicates for β-potential and Dh, or PDI, respectively. Normality and homogeneity of variance were tested using the Shapiro-Wilk test and the Levene test, respectively. For data with homogeneous variance, a one-way analysis of variance (ANOVA) was performed, followed by a Bonferroni post hoc test to determine significant differences between the means. In cases where the variance was not homogeneous, Welch's ANOVA was performed, followed by Dunnett's T3 post hoc test. Post hoc tests were used to determine statistically significant differences between the means. The significance level for all tests was set at p < 0.05. The statistical analysis and graphical presentation of the plant experiments (Fig.9) was performed using GraphPad Prism 8 software. The data were normally distributed, as confirmed by the Shapiro-Wilk test. An unpaired t-test was performed to evaluate differences between the free and formulated dsRNA sample treatments. All values ​​are expressed as the mean ± standard error of the mean (SEM) of 6 individual replicates per treatment.

[0168] Results

[0169] Polyelectrolyte complexes: For the use of long dsRNA (L, M, S segments) formulations in chitosan-based polyelectrolyte complexes as crop protection agents, it is necessary to (i) improve the stability of the dsRNA, (ii) potentially facilitate plant uptake, and (iii) release the dsRNA from the formulation at the site of action. To address these challenges, the dsRNA was formulated with chitosan, a sustainably sourced cationic biopolymer that offers additional antimicrobial and plant-strengthening effects (Mao et al. 2010, Wattjes et al. 2020). Therefore, we adapted the formulation methods of nucleic acid with chitosan described by Gurusamy et al. (2020) for long dsRNA to generate positively charged IPECs. For this purpose, alginate was added as a biological chelating agent to increase protection against nucleases (Douglas et al. 2006).Thus, the electrostatic interaction between the differently charged polyelectrolytes chitosan, alginate and dsRNA. ph16 / dsRNA™ v , used to formulate the desired IPECs.

[0170] Formulation efficiency

[0171] The formulation efficiency (FE) of dsRNA in IPEC formulations was investigated, i.e. how much of our active ingredient (dsRNA ph16) was incorporated into the colloidal IPECs during formulation. To determine formulation efficiency, 0.5 g / L CAR-IPECs were formulated with a molar charge ratio of 1.25 (+ / -), containing approximately 125 ng / pL (0.125 g / L) of dsRNA. To ensure the accuracy of the dsRNA concentrations in the CAR-IPECs measured with the nanophotometer, spectral analysis was first used to confirm the absorption peak at 260 nm, which corresponds to the absorption peak of the nucleic acids. In addition, the other formulation polymers, chitosan and alginate, do not exhibit absorption at 260 nm.

[0172] Figure 8A shows that the control unformulated dsRNA remained unchanged in the pre-centrifugation fractions and in the supernatant, confirming that the dsRNA remains in solution at the applied centrifugal force of 21380 g. In contrast, the mean dsRNA concentration in CAR-IPECs decreased significantly in the supernatant. This suggests that the majority of the dsRNA is formulated in the CAR-IPECs. The FE was calculated from the initial concentration of dsRNA in the CAR-IPECs and the remaining dsRNA in the supernatant, i.e., the unbound dsRNA, using the following equation: FE = (125.32 ng / pL - 7.18 ng / pL) / 125.32 ng / pL x 100% = 94.3%.

[0173] The method showed higher FE compared to the other chitosan-based formulations as well as other methods (see Fig. 8 B, Kumar et al. 2016, Dhandapani et al. 2019, Kumar et al. 2016), including lipid nanoparticles, carbon dots, layered double hydroxide nanosheets, and star polycations with 49%, 68.3%, 86.4%, and 63.1%, respectively.

[0174] The addition of alginate also increased the dsRNA binding capacity of CAR-IPECs at lower charge ratios compared to chitosan-dsRNA (CR)-IPECs, although CR-IPECs were smaller than CAR-IPECs (Fig. 11). dsRNA protection in CAR-IPECs

[0175] Alginic acid, known for its ability to chelate metal ions, was incorporated into the IPEC formulations of chitosan and dsRNA to enhance protection against nuclease digestion. This approach is based on the fact that nucleases such as RNase III require divalent metal ions to maintain their catalytic activity or stabilize their dimeric structure (Nicholson 2014).

[0176] Therefore, the inventors of the present application hypothesized that chelation of these divalent metal ions inhibits RNase activity and thereby increases the stability of dsRNA in the formulation. The stability of dsRNA ph16 and dsRNA™ v in IPECs, ie CAR phl6 -IPECs or CAR™ v -IPECs, compared to free dsRNA and digestion by the nucleases RNase III or MNase (Fig. 8A-D). The formulated and unformulated dsRNA ph16was treated with 0.05 U RNase III for up to 3 hours before the reaction was stopped with 2.5 pmol EDTA. This was sufficient to inhibit the enzyme but not to dissolve the IPECs (Fig. 9A). The subsequent gel retardation test is shown in Fig. 9A. The formulated dsRNA remained visible in the gel wells throughout the entire duration of the experiment (Fig. 9A). In contrast, the free dsRNA was only visible before treatment (0'-treatment) and showed the distinct bands of dsRNA. ph16 , i.e., L, M, and S segments at 7599 bp, 4063 bp, and 2948 bp, respectively. After nuclease treatment, only a weak lag signal below 3 kbp remained from the free dsRNA, indicating that the dsRNA was reduced to fragments of various lengths < 3 kbp (Fig. 9A). In addition, the L, M, and S segments of the formulated dsRNA remain ph16not only in the gel wells, but also appear as (weak) bands before and after enzyme treatment (Fig. 9A). The appearance of this (presumably) intact dsRNA phl6 Segments is further evidence of the protection against enzymatic degradation provided by the IPEC formulation. In agreement with previous work, the sample remaining in the gel wells indicates successful formulation of the total dsRNA (Dhandapani et al. 2019, Sun et al. 2022). To force partial release of the dsRNA, the final EDTA concentration was increased from 83 mM to 152 mM in a second experiment (Fig. 9B). The subsequent gel retardation assay demonstrated that free dsRNA ph16was completely degraded within 2 min, as indicated by a dsRNA fragment cloud < 3 kbp and the absence of the distinct bands of the L, M, and S segments (Fig. 9B). In contrast, the corresponding segments of the formulated and then released dsRNA were still detectable even after 60 min of RNase treatment. These results demonstrate effective protection against RNase III digestion of dsRNA incorporated into IPECs. These results are consistent with those of Douglas et al. (2006), who showed comparable protection against nuclease digestion of plasmid DNA loaded onto the surface of alginate-chitosan particles.

[0177] However, the inclusion of alginate in the IPECs of the present invention significantly improved protection against nucleases, consistent with the ability of alginate to inhibit RNase III activity and EDTA. Experiments with dsRNA™ v and CAR™ v-IPECs stored at 4 °C for 14 days showed comparable results (Fig. 9C). Furthermore, DLS measurements of CAD-IPECs showed that the hydrodynamic diameter of the IPECs remained constant over a period of 16 days (Fig. 12).

[0178] Taken together, these results indicate a long shelf life for the formulations. Furthermore, the additional experiments with MNase demonstrate the broad RNase protection of CAR-IPECs (Fig. 9D). Free dsRNA™ v was degraded after 30 minutes of incubation with 3 U and 30 U MNase, while formulated dsRNA™ v released from the formulations after MNase treatment by EDTA still contained the same three segments of the dsRNA™ v These results on formulated dsRNA™ v also show that the protection of dsRNA in CAR-IPECs is independent of the dsRNA sequence.

[0179] To demonstrate the heat stability of the CAR-IPEC formulations, free and formulated dsRNA™ v Treated for 60 minutes at temperatures between 37°C and 60°C (Fig. 9E), i.e., temperatures that can occur under field conditions depending on plant height, climate zone, and season. The free dsRNA™ v showed initial signs of degradation at 37°C with a weakening of the M segment (Fig. 9E). At higher temperatures of 45°C, the M segment was completely degraded and the S segment became weak (Fig. 9E). Finally, after treatment at 60°C, only a weak L segment was visible, while the bands of the formulated dsRNA™ v remained stable after treatment at all temperatures (Fig. 9E). These results indicate that the formulation also stabilizes dsRNA against the degradation processes induced by higher temperatures.

[0180] Fig. 10 shows a statistically significant reduction in the number of infection sites on leaves treated with formulated dsRNA™ v treated, compared to leaves treated with free dsRNA™ v treated after both formulated and free dsRNA™ v previously incubated at 37 °C or 60 °C for 30 min.

[0181] Conclusions

[0182] The examples of the present application demonstrate that a formulation of long double-stranded RNA (dsRNA) using interpolyelectrolyte complexes (IPECs) consisting of the biopolymers chitosan and alginate is advantageous to protect the dsRNA from biotic and abiotic factors.

[0183] The inventive approach aims to increase the stability of dsRNA against environmental nucleases and, secondarily, to reduce the negative charge of the dsRNA, which could promote leaf uptake. The inventive approach is based on submicroscopic particles whose surface charge can be either positive or negative. A positive surface charge is preferred for the application of the inventive formulation as a crop protection agent. Using this approach, it was possible to produce -100 nm particles with adjustable size and charge, achieving a formulation efficiency of 94.3%.

[0184] The experiments on Brachypodium dislachyond^&nz n demonstrate that the dsRNA formulated with chitosan and alginate (pmkl-dsRNA-NP) acts as a plant protection agent against Magnaporthe oryzae, showing improved efficacy compared to the unformulated pmkl-dsRNA. The experiments on Nicotiana benthamiana plants demonstrated that formulated dsRNA provides protection against tobacco mosaic virus. Furthermore, the IPEC formulation protected dsRNA from enzymatic degradation by RNase III and micrococcal nuclease (MNase). Furthermore, excellent protection of the formulated dsRNA against heat degradation was observed.

[0185] Subjects of the invention

[0186] 1. A method for producing a polyelectrolyte complex, comprising at least the process steps: a) producing a solution comprising a nucleic acid, at least one ionotropic gel-forming anionic polymer, and a cationic polymer; b) optionally heating the solution, preferably in a temperature range from > 30 °C to < 90 °C; c) mixing the solution; and d) incubating the solution, wherein the polyelectrolyte complex is produced as particles with a size in a range of < 2 pm.

[0187] 2. The method according to claim 1, characterized in that all substances used in the method are free of nuclease.

[0188] 3. The method according to item 1 or 2, characterized in that the nucleic acid, the cationic polymer, and the at least one ionotropic gel-forming anionic polymer are each present in the same mass fraction in the solution produced in process step a). 4. The method according to any one of claims 1 to 3, characterized in that the cationic polymer is present in the solution produced in process step a) at a concentration of <0.006 wt.%.

[0189] 5. Method according to one of items 1 to 4, characterized in that in method step a) a mixture of nucleic acid and ionotropic gel-forming anionic polymer is first produced and this mixture is then mixed with cationic polymer.

[0190] 6. Process according to one of items 1 to 5, characterized in that a polyelectrolyte complex having a charge ratio in a range of > 1.25 to < 3 is produced.

[0191] 7. Process according to one of items 1 to 6, characterized in that a polyelectrolyte complex with a positive surface charge (zeta potential) is produced.

[0192] 8. Method according to one of items 1 to 7, characterized in that the nucleic acid is selected from the group consisting of dsDNA, ssRNA, sRNA, dsRNA, ssDNA, dsDNA, poly (1:C)), and circular nucleic acids.

[0193] 9. Method according to one of items 1 to 8, characterized in that the nucleic acid is dsRNA.

[0194] 10. The method according to any one of items 1 to 9, characterized in that the ionotropically gel-forming anionic polymer is selected from the group consisting of alginate, furcellaran, guar gum, carrageenan, pectinate, and, from the polymer derivatives, amidated pectinate, carboxymethylcellulose, carboxyguar gum, and phosphoguar gum. 11. The method according to any one of items 1 to 10, characterized in that the ionotropically gel-forming anionic polymer is alginate.

[0195] 12. Method according to one of items 1 to 11, characterized in that the cationic polymer is selected from the group consisting of chitosan, poly-lysine, poly-arginine, poly-histidine, polydiallyldimethylammonium chloride, diethylaminoethyl cellulose, diethylaminoethyl dextran, chitosan hydroxypropyltrimonium chloride, dextran hydroxypropyltrimonium chloride, polyethyleneimine, and lysine dextran.

[0196] 13. Method according to one of items 1 to 12, characterized in that the cationic polymer is chitosan.

[0197] 14. Method according to one of items 1 to 13, characterized in that the nucleic acid is dsRNA, the ionotropic gel-forming anionic polymer is alginate, and the cationic polymer is chitosan.

[0198] 15. Polyelectrolyte complex prepared by the process according to any one of items 1 to 14.

[0199] 16. Polyelectrolyte complex comprising at least one nucleic acid, wherein the polyelectrolyte complex comprises, in addition to the at least one nucleic acid, at least one ionotropic gel-forming anionic polymer and one cationic polymer, and wherein the polyelectrolyte complex is present as particles having a size in a range of < 2 pm.

[0200] 17. The polyelectrolyte complex according to item 16, characterized in that the nucleic acid is selected from the group consisting of dsDNA, ssRNA, sRNA, dsRNA, ssDNA, dsDNA, poly(1:C)), and circular nucleic acids. 18. The polyelectrolyte complex according to either item 16 or 17, characterized in that the nucleic acid is dsRNA.

[0201] 19. Polyelectrolyte complex according to one of items 16 to 18, characterized in that the ionotropic gel-forming anionic polymer is selected from the group consisting of alginate, furcellaran, guaranate, carrageenan, pectinate and, from the polymer derivatives, amidated pectinate, carboxymethylcellulose, carboxyguargum, and phosphoguargum.

[0202] 20. Polyelectrolyte complex according to any one of items 16 to 19, characterized in that the ionotropic gel-forming anionic polymer is alginate.

[0203] 21. Polyelectrolyte complex according to one of items 16 to 20, characterized in that the cationic polymer is selected from the group consisting of chitosan, poly-lysine, poly-arginine, poly-histidine, polydiallyldimethylammonium chloride, diethylaminoethyl cellulose, diethylaminoethyl dextran, chitosan hydroxypropyltrimonium chloride, dextran hydroxypropyltrimonium chloride, polyethyleneimine and lysine dextran.

[0204] 22. Polyelectrolyte complex according to any one of items 16 to 21, characterized in that the cationic polymer is chitosan.

[0205] 23. Polyelectrolyte complex according to one of items 16 to 22, characterized in that the nucleic acid is dsRNA, the ionotropic gel-forming anionic polymer is alginate, and the cationic polymer is chitosan.

[0206] 24. Polyelectrolyte complex according to one of items 16 to 23, characterized in that at least one ionotropically gel-forming anionic polymer is present in an amount of > 0.1 part by weight to < 10 parts by weight, based on the at least one nucleic acid.

[0207] 25. Polyelectrolyte complex according to one of items 16 to 24, characterized in that the cationic polymer, based on the at least one nucleic acid, is present in an amount of > 0.1 part by weight to < 10 parts by weight.

[0208] 26. Polyelectrolyte complex according to one of items 16 to 25, characterized in that the polyelectrolyte complex is free of EDTA.

[0209] 27. Polyelectrolyte complex according to one of items 16 to 26, characterized in that the polyelectrolyte complex has its components throughout its cross-section.

[0210] 28. Polyelectrolyte complex according to one of items 16 to 27, characterized in that the polyelectrolyte complex comprises at least one outer layer comprising an ionotropically gel-forming anionic polymer.

[0211] 29. Polyelectrolyte complex according to one of items 16 to 28, characterized in that the polyelectrolyte complex has a positive surface charge (zeta potential).

[0212] 30. Polyelectrolyte complex according to any one of items 16 to 29, characterized in that the nucleic acid in the polyelectrolyte complex, compared to a free nucleic acid, has a higher stability against at least one selected from the group of nucleases, UV radiation, and elevated temperature or heat. 31. Polyelectrolyte complex according to any one of items 16 to 29, characterized in that the dsRNA is selected from the group consisting of

[0213] • Pmkl -specific dsRNA (SEQ ID No. 1), and

[0214] • TMV-specific dsRNA.

[0215] 32. Plant protection products comprising a polyelectrolyte complex according to any one of items 16 to 31.

[0216] 33. Plant strengthening agent comprising a polyelectrolyte complex according to any one of items 16 to 31.

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[0261] Abkürzungen

[0262] Bd Brachypodium distachyon CAD-IPEC chitosan-alginate-dsDNA interpolyelectrolyte complex CA-IPEC chitosan-alginate interpolyelectrolyte complex CAR-IPEC chitosan-alginate-dsRNA interpolyelectrolyte complex CR-IPEC chitosan-dsRNA interpolyelectrolyte complex Dh hydrodynamic diameter DLS dynamic light scattering Dpt days after treatment ELS electrophoretic light scattering FE formulation efficiency HIGS host-induced gene silencing IPECs interpolyelectrolyte complexes Mo Magnaporthe oryzae NP nanoparticles PAMP pathogen-associated molecular pattern PDI polydispersity index RN Ai RNA interference SIGS spray-induced gene silencing TMV tobacco mosaic virus

[0263] sequences

[0264] Table 3: Plasmids used for the transformation of Pseudomonas syringae

[0265] The following sequences are part of the disclosure of this application. A WIPO ST.26-compliant electronic sequence listing is also provided with this application. In case of discrepancies between the sequences in the table below and the electronic sequence listing, the sequence in this table is considered the correct one.

[0266] Table 4: Sequence list

Claims

Patent claims 1. A method for producing a polyelectrolyte complex, comprising at least the process steps: a) producing a solution comprising a nucleic acid, at least one ionotropic gel-forming anionic polymer, and a cationic polymer; b) optionally heating the solution, preferably in a temperature range from > 30 °C to < 90 °C; c) mixing the solution; and d) incubating the solution, wherein the polyelectrolyte complex is produced as particles with a size in a range of < 2 pm.

2. The method according to claim 1, characterized in that all substances used in the method are free of nuclease.

3. The method according to claim 1 or 2, characterized in that the nucleic acid, the cationic polymer and the at least one ionotropic gel-forming anionic polymer are each present in the same mass fraction in the solution produced in process step a).

4. Process according to one of claims 1 to 3, characterized in that the cationic polymer is present in the solution produced in process step a) in a concentration of < 0.006 wt.%.

5. Method according to one of claims 1 to 4, characterized in that in process step a) first a mixture of nucleic acid and ionotropic gel-forming anionic polymer is produced and this mixture is then mixed with cationic polymer.

6. Process according to one of claims 1 to 5, characterized in that a polyelectrolyte complex having a charge ratio in a range of > 1.25 to < 3 is produced.

7. Process according to one of claims 1 to 6, characterized in that a polyelectrolyte complex with a positive surface charge (zeta potential) is produced.

8. The method according to any one of claims 1 to 7, characterized in that the nucleic acid is selected from the group consisting of dsDNA, ssRNA, sRNA, dsRNA, ssDNA, dsDNA, poly (1:C)), and circular nucleic acids.

9. Method according to one of claims 1 to 8, characterized in that the nucleic acid is dsRNA.

10. The method according to any one of claims 1 to 9, characterized in that the ionotropic gel-forming anionic polymer is selected from the group consisting of alginate, furcellaran, guaranate, carrageenan, pectinate and, from the polymer derivatives, amidated pectinate, carboxymethylcellulose, carboxyguargum, and phosphoguargum.

11. Process according to one of claims 1 to 10, characterized in that the ionotropic gel-forming anionic polymer is alginate.

12. The method according to any one of claims 1 to 11, characterized in that the cationic polymer is selected from the group consisting of chitosan, poly-lysine, poly-arginine, poly-histidine, polydiallyldimethylammonium chloride, diethylaminoethyl cellulose, Diethylaminoethyl dextran, chitosan hydroxypropyltrimonium chloride, dextran hydroxypropyltrimonium chloride, polyethyleneimine, and lysine dextran.

13. Process according to one of claims 1 to 12, characterized in that the cationic polymer is chitosan.

14. The method according to any one of claims 1 to 13, characterized in that the nucleic acid is dsRNA, the ionotropic gel-forming anionic polymer is alginate, and the cationic polymer is chitosan.

15. Polyelectrolyte complex prepared by the process according to any one of claims 1 to 14.

16. Polyelectrolyte complex comprising at least one nucleic acid, wherein the polyelectrolyte complex comprises, in addition to the at least one nucleic acid, at least one ionotropic gel-forming anionic polymer and one cationic polymer, and wherein the polyelectrolyte complex is present as particles having a size in a range of < 2 pm.

17. Polyelectrolyte complex according to claim 16, characterized in that the nucleic acid is selected from the group consisting of dsDNA, ssRNA, sRNA, dsRNA, ssDNA, dsDNA, poly (1:C)), and circular nucleic acids.

18. Polyelectrolyte complex according to one of claims 16 or 17, characterized in that the nucleic acid is dsRNA.

19. Polyelectrolyte complex according to one of claims 16 to 18, characterized in that the ionotropic gel-forming anionic polymer is selected from the group consisting of from alginate, furcellaran, guarate, carrageenan, pectinate and from the polymer derivatives amidated pectinate, carboxymethylcellulose, carboxy-guargum, and phospho-guargum.

20. Polyelectrolyte complex according to one of claims 16 to 19, characterized in that the ionotropic gel-forming anionic polymer is alginate.

21. Polyelectrolyte complex according to one of claims 16 to 20, characterized in that the cationic polymer is selected from the group consisting of chitosan, poly-lysine, poly-arginine, poly-histidine, polydiallyldimethylammonium chloride, diethylaminoethyl cellulose, diethylaminoethyl dextran, chitosan hydroxypropyltrimonium chloride, dextran hydroxypropyltrimonium chloride, polyethyleneimine and lysine dextran.

22. Polyelectrolyte complex according to one of claims 16 to 21, characterized in that the cationic polymer is chitosan.

23. Polyelectrolyte complex according to one of claims 16 to 22, characterized in that the nucleic acid is dsRNA, the ionotropic gel-forming anionic polymer is alginate, and the cationic polymer is chitosan.

24. Polyelectrolyte complex according to one of claims 16 to 23, characterized in that at least one ionotropic gel-forming anionic polymer is present in an amount of > 0.1 part by weight to < 10 parts by weight, based on the at least one nucleic acid.

25. Polyelectrolyte complex according to one of claims 16 to 24, characterized in that the cationic polymer, based on the at least one nucleic acid, is present in an amount of > 0.1 part by weight to < 10 parts by weight.

26. Polyelectrolyte complex according to one of claims 16 to 25, characterized in that the polyelectrolyte complex is free of EDTA.

27. Polyelectrolyte complex according to one of claims 16 to 26, characterized in that the polyelectrolyte complex has its components throughout its cross-section.

28. Polyelectrolyte complex according to one of claims 16 to 27, characterized in that the polyelectrolyte complex comprises at least one outer layer comprising an ionotropic gel-forming anionic polymer.

29. Polyelectrolyte complex according to one of claims 16 to 28, characterized in that the polyelectrolyte complex has a positive surface charge (zeta potential).

30. Polyelectrolyte complex according to one of claims 16 to 29, characterized in that the nucleic acid in the polyelectrolyte complex, compared to a free nucleic acid, has a higher stability against at least one selected from the group of nucleases, UV radiation, and elevated temperature or heat.

31. Polyelectrolyte complex according to one of claims 16 to 29, characterized in that the dsRNA is selected from the group consisting of • Pmkl -specific dsRNA (SEQ ID No. 1), and • TMV-specific dsRNA.

32. A plant protection agent comprising a polyelectrolyte complex according to any one of claims 16 to 31.

33. Plant strengthening agent comprising a polyelectrolyte complex according to any one of claims 16 to 31.

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