Interpolyelectrolyte complexes of in vivo produced dsRNA with chitosan and alginate for enhanced plant protection against tobacco mosaic virus

Benjamin W Moorlach, Ana Rocío Sede, Katharina M Hermann, Alesia A Levanova, Minna M Poranen, Michael Westphal, Martin Wortmann, Elzbieta Stepula, Desiree Jakobs-Schönwandt, Manfred Heinlein, et al.

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To cite this version:

Benjamin W Moorlach, Ana Rocío Sede, Katharina M Hermann, Alesia A Levanova, Minna M Poranen, et al. Interpolyelectrolyte complexes of in vivo produced dsRNA with chitosan and alginate for enhanced plant protection against tobacco mosaic virus. International Journal of Biological Macromolecules, 2025, 306, pp.141579. 10.1016/j.ijbiomac.2025.141579. hal-04975292

HAL Id: hal-04975292

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Submitted on 4 Mar 2025

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# Interpolyelectrolyte complexes of in vivo produced dsRNA with chitosan and alginate for enhanced plant protection against tobacco mosaic virus

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Benjamin W. Moorlach<sup>a,b</sup>, Ana R. Sede<sup>c</sup>, Katharina M. Hermann<sup>a,b</sup>, Alesia A. Levanova<sup>d</sup>, Minna M. Poranen<sup>d</sup>, Michael Westphal<sup>e</sup>, Martin Wortmann<sup>e</sup>, Elzbieta Stepula<sup>a</sup>, Desiree Jakobs-Schönwandt<sup>a,f</sup>, Manfred Heinlein<sup>c,*</sup>, Waldemar Keil<sup>a,*</sup>, Anant V. Patel<sup>a,*</sup>

## ARTICLE INFO

### Keywords:

dsRNA

RNAi

SIGS

Formulation

Alginate

Chitosan

Interpolyelectrolyte complex

## ABSTRACT

We developed a formulation of long double-stranded RNA (dsRNA) using interpolyelectrolyte complexes (IPECs) composed of the biopolymers chitosan and alginate, in order to protect the dsRNA from biotic and abiotic factors. Our primary objectives were to enhance stability of dsRNA against environmental nucleases and, secondarily, to mitigate the negative charge of the dsRNA, which may promote foliar uptake. Our approach relies on submicron particles with adjustable surface charge being either positive or negative. Following this approach, we obtained a high encapsulation efficiency of 94 %. Subsequently, we investigated the influence of the charge ratio and total polymer content on the size, size distribution and ζ-potential of the IPECs. We discovered that formulating at low polymer concentrations ≤0.05 g/L with charge ratios of ≤0.9 (+/-) and ≥ 1.25 (+/-), respectively, produced <100 nm particles. Furthermore, the IPEC formulation protected dsRNA from enzymatic degradation by RNase III and micrococcal nuclease. In addition, we observed outstanding protection of formulated dsRNA from heat degradation. Experiments on Nicotiana benthamiana plants showed that formulated dsRNA offered protection against tobacco mosaic virus. In essence, this formulation demonstrates versatility for the production of IPECs with customizable size, surface charge, and nucleic acid content.

## 1. Introduction

Over the past decades, conventional plant protection agents have garnered substantial attention and raised notable social, political, and health concerns [1]. The increased demand for environmentally friendly plant protection agents reflects a growing awareness and prioritisation of sustainable agricultural practices. Nevertheless, the implementation of these environmentally friendly plant protection methods in current agricultural practices continues to remain elusive. One highly promising candidate for sustainable plant protection is double-stranded RNA (dsRNA). In eukaryotic cells dsRNA serves as a nature-derived and environmentally safe molecule, which triggers RNA interference (RNAi) upon uptake [2,3]. RNAi represents a highly conserved cellular

mechanism in eukaryotic cells, employed for gene regulations and self-protection against pathogens [2,3].

The main biotechnological approaches based on RNAi for plant protection *are i*) host-induced gene silencing (HIGS), and *ii*) spray-induced gene silencing (SIGS) [2]. HIGS involves the transgenic modification of plants to endogenously express specific dsRNA sequences, thereby inducing gene silencing of the targeted pathogen. On the contrary, SIGS relies on the exogenous application of dsRNA derived from chemical or biotechnological processes, typically administered through conventional spray applications [2]. Several commercially available HIGS crops have been introduced in various countries such as Brazil, Canada, Japan, Nigeria, and the USA. Notable examples include transgenic maize exhibiting resistance against corn rootworm, plum trees

<footnote><sup>a</sup> Faculty of Engineering and Mathematics, Fermentation and Formulation of Biologicals and Chemicals, Hochschule Bielefeld, University of Applied Sciences and Arts, Interaktion 1, 33619 Bielefeld, Germany</footnote>

<footnote><sup>b</sup> Department of Technology, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany</footnote>

<footnote><sup>c</sup> Institut de biologie moléculaire des plantes, CNRS, Université de Strasbourg, 67000 Strasbourg, France</footnote>

<footnote><sup>d</sup> Molecular and Integrative Biosciences Research Programme, Faculty of Biological and Environmental Sciences, University of Helsinki, 00014 Helsinki, Finland</footnote>

<footnote><sup>e</sup> Faculty of Physics, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany</footnote>

<footnote><sup>f</sup> Bioengineering and Sustainability, Westphalian University of Applied Sciences, August-Schmidt-Ring 10, 45665 Recklinghausen, Germany</footnote>

<footnote>* Corresponding authors.
E-mail addresses: manfred.heinlein@ibmp-cnrs.unistra.fr (M. Heinlein), waldemar.keil@hsbi.de (W. Keil), anant.patel@hsbi.de (A.V. Patel).</footnote>
engineered for resistance to plum pox virus, and common beans resistant to bean golden mosaic virus [4]. Despite these successes, regulatory concerns have prevented the approval of HIGS products for European Union markets, as discussed by Arpaia, et al. in 2020 [4].

HIGS requires farmers to purchase patented seeds before the planting season and anticipate the specific pathogens that may threaten their crops, leading to potential uncertainties. Moreover, the development of each transgenic plant is accompanied by considerable costs and have the risk of resistance breakdown [5,6]. SIGS presents distinct advantages over HIGS. It does not necessitate the use of transgenic crops, addressing public concerns associated with genetic modifications [7]. Additionally, SIGS offers targeted application, allowing farmers to apply it selectively when specific pathogens pose a threat to the crops.

Overall, SIGS products offer the convenience of application through conventional spray methods, which is important as it aligns well with established practices for chemical pesticides [2,8]. The targeted application of specific dsRNA can be employed on demand, either pre-emptively or during pathogen infestation, in precisely measured quantities tailored to the specific needs of the plants and plant pests [8]. However, several significant challenges hinder the agricultural application of utilising dsRNA. These obstacles include the high costs associated with production and purification, the susceptibility of dsRNA to premature degradation—occurring both before and after application, as well as during storage. Furthermore, dsRNA undergoes rapid degradation in soils, especially at elevated temperatures and pH levels which increase the degradation of RNA molecules present in the environment [9–11]. Moreover, the cuticle, cell wall, and cell membrane present major hurdles for efficient uptake of negatively charged dsRNA. These challenges are discussed in many studies, which also highlights the size exclusion limits imposed by plant barriers [2, 7, 12–15]. To surmount these challenges, numerous formulations have been devised, generally aimed at mitigating the anionic charge of dsRNA or circumventing size exclusion limits. These formulations encompass diverse strategies, including the use of carbon nanoparticles, such as carbon nanodots cationically functionalized with polyethyleneimine and subsequently loaded with dsRNA [16,17]. Other approaches involve layered double hydroxide nanosheets (bioclays), DNA nanostructures (especially those based on DNA origami), cationic or amphiphilic oligopeptides (including plant-penetrating peptides), and encapsulation in cationic liposomes [15,18–27]. Additional formulation strategies involve the direct utilisation of dsRNA production systems, where genetically modified bacteria capable of producing dsRNA are applied directly to the field [4,28–30]. Moreover, insights from pharmaceutical science, particularly in the context of RNAi-based treatments and gene therapy, offer potential formulations applicable to RNAi in agriculture [31–35].

Despite these formulation efforts, none have successfully transitioned from laboratory development to field application. Among the latter, chitosan complexes present a promising approach for dsRNA delivery, as they enhance dsRNA stability and RNAi efficacy, after which they degrade into non-toxic products (Quilez-Molina 2024). For example, in insect feeding assays chitosan-based formulations have demonstrated effectiveness against pests such as *Helicoverpa armigera* [36,37]. In general, chitosan complexes are either formed with a crosslinking agent, such as tripolyphosphate and then loaded with dsRNA in a subsequent step, or the dsRNA functions as a crosslinker during particle formation and is present throughout the particle [38,39]. Chitosan is a nature-derived biopolymer of β(1 → 4)-linked monomers of N-acetyl-D-glucosamine (acetylated or chitin monomer), and D-glucosamine (deacetylated or chitosan monomer), which form a linear polysaccharide [40]. In acidic media, the amine groups of the deacetylated monomers are protonated which gives the chitosan its cationic charge and makes it soluble in water. This enables the formation of complexes when combined with anionic crosslinkers or polymers. Chitosan is antimicrobial, plant strengthening and may trigger unspecific antiviral effects to varying degrees depending on the polymer length, i.e., molecular weight (MW), and degree of deacetylation [40–44].

Desoxyribo- and ribonucleases often require the presence of bivalent cations, such as Ca²⁺ or Mn²⁺, as cofactors in order to function and degrade nucleic acids [45]. Consequently, chelators such as ethylenediaminetetraacetic acid (EDTA), which bind metal ions, inhibit enzymatic degradation of RNA. Therefore, the inclusion of EDTA or other chelators in formulation can protect nucleic acids, as shown by Lorentz, et al. (2006) with EDTA-chitosan complexes loaded with plasmid DNA [46]. For agricultural applications, environmentally friendly alternatives to EDTA are required, such as alginate, which is an anionic polysaccharide derived from brown algae [47]. It is a linear polymer with homopolymeric blocks of (1 → 4)-linked β-D-mannuronate (M) and α-L-guluronate (G) residues covalently linked together. The G-G blocks of alginate can bind metal ions, which causes the formation of alginate hydrogels [47,48]. Alginate-derived products are already established for agricultural applications, e.g., seed and fruit coatings, as absorbent polymer, or to formulate active substances, such as agrochemicals or beneficial bacteria [49]. Numerous studies have shown that alginate can form particles, such as interpolyelectrolyte complexes (IPECs), with chitosan [50,51]. For example, Douglas, et al. (2006) loaded chitosan-alginate (CA-)IPECs with plasmid DNA [52]. This formulation increased the in vitro transfection efficiency in human 293 T cells and provided protection of the DNA against nucleases, which was not observed with plasmid DNA formulated with chitosan particles alone [52]. Additionally, CA-IPEC have proven less toxic than state-of-the-art transfection vectors formulations [53]. Other researcher loaded messenger (m)RNA onto CA-IPECs and showed that and showed 79 % of mRNA was released after 21 days compared to 42 % released from mRNA loaded onto β-glycerophosphate crosslinked chitosan particles. To benefit from enhanced nuclease protection, low toxicity, and improved release of nucleic acids, we chose to formulate dsRNA into chitosan-algiantide-dsRNA IPECs (CAR-IPECs) [54].

In this study, we used intensively purified and formulated dsRNA molecules which were produced in *Pseudomonas* bacteria. The dsRNA synthesis in *Pseudomonas* is based on the exploitation of bacteriophage phi6 polymerase complexes which are responsible for the amplification of the tri-segmented dsRNA genome of the phage (Fig. 1) [55,56]. Stable amplification of heterologous dsRNA sequences is achieved in this system by mimicking the properties of the phage genome. Therefore, the dsRNA produced consists of three dsRNA molecules of approximately 3, 4 and 7 kilobase pairs (kbp) in size. Compared to 21 bp short interfering dsRNA (siRNA), which cause the cleavage of the cognate RNA target sequence at a single unique site, the long dsRNAs produced in this system generate a large pool of unique siRNAs that all act individually in target site cleavage within the same target RNA. This enables greater target sensitivity and reduces the risk of resistance [55]. In the current study, such long dsRNA was formulated into CAR-IPECs) and subjected to a series of measurements to (i) determine the required formulation parameters to produce positively charged CAR-IPECs, and to (ii) substantiate the effect of alginate on the stability of the incorporated dsRNA against biotic stress, such as nucleases, and abiotic stress, e.g., elevated temperature. Finally, we carried out an *in planta* experiment with long, tobacco mosaic virus (TMV)-specific dsRNA produced in our *Pseudomonas* system, formulated into CAR-IPECs, and applied to *Nicotiana benthamiana* plants (Fig. 1). Unlike non-formulated dsRNA, the formulated dsRNA significantly reduced TMV infection even when exposed to 60 °C before application.

## 2. Materials and methods

### 2.1. dsRNA production and purification

High quality (fully duplexed) dsRNA was produced in *Pseudomonas syringae* strain LM2691 in which a lysis-defective modified bacteriophage phi6 replicates as a stable episome while expressing the dsRNA sequences of interest [55,57]. *P. syringae* cell lines for the production of TMV-specific dsRNA (dsRNA<sup>TMV</sup>) and target-unspecific dsRNA<sup>phi6</sup> have
<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Pipeline for dsRNA-based plant protection with the model pathogen tobacco mosaic virus. (1) Sequences of essential genes are selected from the genome of targeted pathogen tobacco mosaic virus, and (2) combined with sequence elements derived from the bacteriophage phi6. (3) The produced chimeric nucleic acid is introduced into Pseudomonas bacterial cell to amplify the target specific dsRNA within the phi6 polymerase complexes. (4) Subsequently, the produced dsRNA is extracted, and purified, and (5) the dsRNA is formulated with the biopolymers chitosan and alginate into chitosan-alginate-dsRNA interpolyelectrolyte complexes (CAR-IPECs). (6) The CAR-IPECs are applied to the crops by conventional spraying to induce RNA interference against the target plant pathogen.</figcaption></figure>

been established previously and kept as glycerol stocks at -80 °C [55]. For this study, dsRNA<sup>phi6</sup> and dsRNA<sup>TMV</sup> were prepared as follows: The dsRNA producing bacteria were incubated in Luria-Bertani broth containing kanamycin (25 µg/mL) at 28 °C for 18-20 h, followed by 10-min centrifugation at 6000g 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 1/5th volume of chloroform (VWR, Bratislava, Slovakia). The mixture was vigorously vortexed and centrifuged at 8000g for 15 min. The aqueous phase was collected, and RNA extraction procedure was repeated two more times. After the final extraction step, RNA was precipitated with isopropanol (Emsure, Merck, Darmstadt, Germany), washed with 75 % ethanol and dissolved in ultrapure water. The dsRNA was fractionated from ssRNA with a stepwise LiCl precipitation, as described by Levanova and Poranen (2018) whereby ssRNA was initially precipitated from the RNA-mixture with 2 M LiCl followed by dsRNA precipitation with 4 M LiCl (Merck, Darmstadt, Germany) [58]. The LiCl fractionation was repeated twice to enhance dsRNA purity. The concentration and purity of dsRNA was initially assessed using UV-Vis spectrophotometer NanoDrop 2000c (Thermo Fisher Scientific, Wilmington, DE, U.S.A.). Subsequently, the quality of dsRNA was verified by agarose gel electrophoresis (Fig. S1). The final dsRNA preparation comprised three dsRNA segments: 7599 bp, 4223 bp, and 2948 bp for dsRNA<sup>phi6</sup>, and 7599 bp, 4063 bp, and 3268 bp for dsRNA<sup>TMV</sup>. The largest segment is the original L segment of Phi6 with a kanamycin resistance gene insert, whereas the medium (M) and small (S) segments are either original (for dsRNA<sup>phi6</sup>) or modified (for dsRNA<sup>TMV</sup>) and consist of TMV-specific dsRNA. The dsRNA was stored in aliquots in RNase-free microcentrifuge tubes at -80 °C. For experiments, aliquots were thawed on ice. Thawed samples were stored at 4 °C for up to 1 week. Additionally, a batch of dsRNA<sup>phi6</sup> was sonicated to obtain fragmented dsRNA (Fig. S2). In this study full-length or sonicated dsRNA<sup>phi6</sup>, dsRNA<sup>TMV</sup>, and sonicated salmon sperm dsDNA (UltraPure™ Salmon Sperm DNA Solution, Thermo Fisher Scientific, USA) were employed for experiments and treated ceteris paribus. The dsRNA<sup>phi6</sup> originated from a single production and purification batch, which had been stored at -80 °C in aliquots.

## 2.2. Formulation of interpolyelectrolyte complexes

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 yield a stock solution of 1 % (w/w). Separately, 0.5 % sodium alginate (Algogel 3001, Cargill, USA) was dissolved by stirring in ddH₂O. Both solutions were autoclaved for 6 min at 121 °C sterilisation phase, to ensure the biopolymers are not depolymerized while ensuring the sterility and homogeneity of the solutions, which were subsequently sterile-filtered using 0.2 µm cellulose acetate syringe filters.

Next, dsRNA aliquots were thawed on ice, and their concentrations were determined using a nanophotometer (NanoPhotometer™ N60, Implen, Germany) with a custom dsRNA absorption coefficient of 46.5 ng/µL A<sub>260nm</sub>, established from literature [59,60]. Subsequently, the concentrations were adjusted by dilution with nuclease-free water. The dsRNA and alginate solutions, both at the same concentration, were then combined at various ratios to form the anionic precursor solutions. Precursor solutions with lower polymer concentrations were prepared by further dilution with nuclease-free water. Formulation procedures were conducted within a dedicated sterile workbench (VWR PCR Workstation, VWR International, USA), sterilised using UV-C radiation. The interior air was continuously circulated past UV-C lamps, and surfaces were cleaned with RNase AWAY® (Molecular BioProducts, USA). To ensure homogenous solutions, the chitosan and anionic precursor solutions were briefly heated to 45 °C in a thermomixer (CellMedia, Gutenborn, Germany) (Fig. 2). Subsequently, the anionic precursor was pipetted into the chitosan solution and the solutions were briefly mixed with micropipette. Then the mixture was agitated by vortexing at low speed for 5 s, and the IPECs formed through electrostatic interaction of positively charged N and negatively charged C and P groups (Fig. 2). Then the formulation was incubated at room temperature for 1 h prior to analysis, or storage at 4 °C. CAD- and CA-IPECs, i.e., with DNA or only containing chitosan and alginate, respectively, were prepared by the same method, with adjustments made to polymer solutions to maintain
<figure><img src="image_6.png" /><figcaption>**Fig. 2.** The formulation process of chitosan-alginate dsRNA IPECs. First dsRNA and alginate are mixed in a 1:1 ratio. The anionic solution is added to the chitosan upon which the interpolyelectrolyte complexes form via a condensed phase separation. The latter is driven by electrostatic interactions of positively charged amine groups with negatively charged phosphate and carboxyl groups from chitosan, dsRNA, and alginate, respectively. The charge of the IPECs is determined by excess of either positive or negative groups during the condensed phase separation.</figcaption></figure>

the same molar charge ratio.

To create IPECs with desired charge ratios (z), calculations of the molar charge ratio were performed based on the assumed electrostatic interaction of one cationic, positive charge (+), i.e., an amine (N) group from chitosan monomers, with one anionic, negative charge (-), i.e., a carboxyl (C) group of alginic acid monomers, or one phosphate (P) group of nucleic acid nucleotides. Therefore, the charge ratio was calculated based on the moles of positively charged N groups to negatively charged C and P groups. For example, a 1.25 positive to negative charge ratio (+/-), has 1.25 times or an excess of 25 % N groups compared to the combined C and P groups. To determine the molar charge ratio, factors including the degree of deacetylation of chitosan, the dry weight of the polymers, and the molecular weight of charge-bearing monomers need to be considered. In our calculation we assumed an equal amount of all nucleotides in the dsRNA and dsDNA sequence and therefore an equal amount of guanosine monophosphate, cytidine monophosphate, adenosine monophosphate, and uridine or thymidine monophosphate, to calculate the average molar mass of (deoxy)ribonucleic monophosphate as 321.5 g/mol for dsRNA and 309.0 g/mol for dsDNA, respectively [60]. Moreover, due to the length of L, M, and S segments of dsRNA<sup>ph16</sup>, the charge and weight differences of 5' triphosphates were disregarded.

## 2.3. Encapsulation efficiency

To determine the encapsulation efficiency (EE), CAR-IPECs were formulated at polymer concentrations of 0.5 g/L and a molar charge ratio of 1.25 (+/-) to reach a dsRNA concentration of 125.3 ng/µL. Control samples with 132 ng/µL unformulated dsRNA were prepared analogously. The samples were centrifuged at 21380g for 1 h at 4 °C (MIKRO 200 R, Andreas Hettich, Tuttlingen, Germany) and the supernatant of each sample was collected for subsequent analysis. The dsRNA concentration of all samples was determined by nanophotometer as described above (section 2.2).

The encapsulation efficiency (EE), also known as entrapment efficiency in the literature, of dsRNA in CAR-IPECs was calculated by the following equation as described by Schober, et al. 2024 [61]:

$$EE\% = (\text{input dsRNA} - \text{unformulated dsRNA})/\text{input dsRNA} \times 100\%$$

In our experimental setup the unformulated dsRNA will remain in the supernatant while the formulated dsRNA will be sedimented in the CAR-IPECs, which means the above equation can also be stated as:

$$EE\% = (\text{input dsRNA} - \text{supernatant dsRNA})/\text{input dsRNA} \times 100\%$$

## 2.4. Interpolyelectrolyte complex characterization

The hydrodynamic diameter Dh of the samples was determined employing dynamic light scattering (DLS) with a Delsa Nano C instrument from Beckman Coulter with a scattering angle of 165°. Approximately 80–100 µL of each sample was added to disposable UV micro

cuvettes (Z-height 8.5 mm, 70–850 µL UV microcuvette, Brand, Germany) and placed into the cell counting chamber of the DLS device. If necessary, high-concentration colloidal suspensions were diluted with MilliQ water to ensure accurate measurements. Seventy scans were recorded to compute the number, volume, and intensity distributions, as well as the Z-average (or cumulant result) according to ISO 22412:2017, along with the polydispersity index (PDI) of the particles. Exact parameters used for measurements can be found in the supporting information (table S1).

Zeta (ζ) potentials 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 measurement flow cell, 800–1200 µL of sample volume were injected into the flow cell using sterile syringes (Fisherbrand™, Thermo Fisher Scientific Inc., USA).

Raman spectra of the formulation and its pure components were recorded using a WITec alpha300 apyron microscope (WITec, Ulm, Germany). The spectra were acquired with a 532 nm laser operating at a power output of 10 mW, an integration time of 3 s, and a 100× objective. Spectral data processing, including spectral truncation, baseline correction, and normalization, was performed with an in-house script written in MATLAB.

Additionally, the particles were imaged in a helium ion microscope Orion Plus (Carl Zeiss, Oberkochen, Germany) applying an acceleration voltage of 28 kV. Scanning with He⁺ ions generates positive surface charging on insulating samples, allowing the use of an electron flood gun during imaging for charge stabilisation. The sample could thus be imaged without the need for a conductive coating, leaving the samples in a more native condition [62].

## 2.5. Stability assays

The stability of free and formulated dsRNA against nucleases and heat stress was evaluated by exposure to either RNase III (ShortCut® RNase III; New England Biolabs, Ipswich, USA), or micrococcal nuclease (MNase; Thermo Fisher Scientific, USA), or by incubation at increased temperatures and subsequent gel electrophoresis. In order to achieve a detectable band intensity, 0.1 g/L (100 ng/µL) dsRNA was formulated in 0.1 g/L CAR-IPECs with a charge ratio of 1.25 (+/-), resulting in 26 ng/ µL dsRNA.

For RNase III, a stability assay was established, in which the dsRNA was treated as specified by the manufacturer, but with adjusted volumes and concentrations to meet the requirements of the assay. ShortCut® Reaction Buffer ×10 was diluted 10-fold and 1 µL added to the reaction. Subsequently, 46 µL of 0.1 g/L dsRNA either free or encapsulated was added. Then, 0.05 U RNase III diluted in enzyme dilution buffer, i.e., 50 mM Tris-HCl (pH 7.5), 100 mM NaCl and 0.1 mM EDTA in 50 % (v/v) glycerol, was added, prior to addition of 0.02 µmol MnCl₂. The final volume of the reaction mix was 50 µL. Depending on the experiment, the samples were incubated for 0–180 min at 37 °C. The reaction was then terminated with 2.5 µmol EDTA and the mixture incubated for 5 min. To additionally release the dsRNA from the formulation, the mixture was
incubated with 11 µmol EDTA (final concentration of 152.8 mM) for 60 min, which was based on the heparin-mediated nucleic acid release developed by Lallana, et al. (2017) [63]. The samples were prepared with staggered timing to ensure simultaneous dsRNA-release prior to gel electrophoresis. From each of the treated samples, 25 µL were mixed with 2.5 µL 60% (v/v) glycerol. Gel retardation assays were performed in 1 % high-resolution agarose gels (Carl Roth, Karlsruhe, Germany) stained with ROTI gel stain (2.5 µL gel stain/50 mL agarose; Carl Roth, Karlsruhe, Germany). Gel electrophoresis was conducted at 110 V for approximately 40 min, and images were captured using an ethidium bromide filter (UVP Ethidium Bromide Filter, Analytik Jena™, Germany) with gels illuminated under UV light at 312 nm.

For MNase treatment, the enzyme was first diluted in reaction buffer pH = 7.9 (50 mM Tris-HCl and 5 mM $CaCl_2$) to 3 U/µL or 30 U/µL. Next, 24 µL containing 600 ng of free or formulated dsRNA<sup>TMV</sup> (25 ng/µL stock) were incubated with 3 U or 30 U of MNase for 30 min at 37 °C in a thermocycler (VeritiPro™ Thermal Cycler, Applied Biosystems). The reaction was stopped and formulations were dissolved by adding 11 µmol EDTA (final concentration of 152.8 mM) followed by an incubation for 60 min at 37 °C. The integrity of the dsRNA was evaluated by gel electrophoresis in 1 % agarose gel (Euromedex) stained with ethidium bromide for 30 min at 70 V. Before gel loading, treated samples (36 µL) were mixed with 3.6 µL 60% (v/v) glycerol.

The stability of the dsRNA against different temperatures was investigated by incubating 56 µL containing 700 ng of free or formulated dsRNA<sup>TMV</sup> (12.5 ng/µL stock) at 37 °C, 45 °C and 60 °C for 30 min in a thermocycler (VeritiPro™ Thermal Cycler, Applied Biosystems). To dissolve the formulations, 11 µmol EDTA (final concentration of 152.8 mM) was added to all the samples, then incubated for 60 min at 37 °C. The integrity of the dsRNA treated samples was evaluated by gel electrophoresis in 1 % agarose gel (Euromedex) stained with ethidium bromide for 50 min at 70 V. Before gel loading, glycerol was added to 6 % (v/v) final concentration.

## 2.6. dsRNA treatment of Nicotiana benthamiana against tobacco mosaic virus

Temperature treatment of free or formulated dsRNA was performed in vitro following the protocol detailed in section 2.5. Briefly, samples from 25 ng/µL (dsRNA<sup>TMV</sup>) stocks were incubated either at 37 °C or 60 °C for 30 min in a thermocycler to produce heat-treated samples of free CAR-IPEC formulated dsRNA<sup>TMV</sup>. Prior to application to plants, we confirmed that the treatment was effective by visualising the samples in a 1 % agarose gel stained with ethidium bromide. Before gel loading, 500 ng of heat treated-dsRNA were incubated with 11 µmol EDTA (final concentration of 152.8 mM) for another 60 min at 37 °C to dissolve the formulations. Gel electrophoresis was conducted for 50 min at 70 V. Since EDTA salt can be toxic to plants, the rest of the samples were applied directly on *Nicotiana benthamiana* leaves without EDTA addition.

N. benthamiana seedlings were grown for 4 weeks in a greenhouse under 16 h/8 h light/dark periods at 22 °C/18 °C. Leaves of uniform size were selected, and one leaf per plant was treated simultaneously with either 2 µg of heat-treated free or formulated dsRNA<sup>TMV</sup> (80 µL of 25 ng/µL stock) along with 20 ng of GFP-expressing TMV (TMV:GFP) virions (3 µL of 0.67 ng/µL stock) [64]. For the control treatment, the volume of dsRNA was replaced by water. The mixture was mechanically inoculated by rubbing the leaves in the presence of celite. In order to remove excess celite, the leaves were washed with distilled water 30 min after inoculation and the plants were then returned to the greenhouse under the same growing conditions. Images were taken at 4 days-post-inoculation (dpi) under UV light and the number of TMV:GFP infection sites on treated leaves were quantified.

## 2.7. Statistical analysis

Statistical analyses were conducted using IBM SPSS Statistics V22.0 software (SPSS, Chicago, USA). All values are presented as mean ± standard deviation (SD) derived from at least 3 or 5 replicates for ζ-potential, and Dh or PDI, respectively. Normality and homogeneity of variance were assessed using Shapiro-Wilk's and Levene's tests, respectively. One-way analysis of variance (ANOVA) was employed for data exhibiting homogeneity of variance, followed by Bonferroni's post-hoc test to determine significant differences between means. In cases where variance was not homogeneous, Welch's ANOVA was performed, followed by Dunnett's T3 post-hoc test. Post-hoc tests were utilised to identify statistically significant differences among means. The significance level for all tests was set at *p* < 0.05.

Statistical analysis and graphical representation for the *in planta* trials (Fig. 8) were performed using GraphPad Prism 8 software. Data followed a normal distribution as confirmed by the Shapiro-Wilk test. To assess the differences between the treatment with free and formulated dsRNA samples, an unpaired *t*-test was conducted. All values are presented as mean ± standard error of the mean (SEM) from 6 individual replicates per treatment.

# 3. Results and discussion

## 3.1. Interpolyelectrolyte complexes

From a broad range of formulated nucleic acids formulated with chitosan complexes for varying applications, we now focus on the formulation of long dsRNA (L, M, S segments) in chitosan-based poly-electrolyte complexes as plant protectants. This necessitates formulations, which (i) improve the stability of dsRNA, (ii) possibly facilitate the uptake into plants and (iii) release the dsRNA from the formulation at the site of action. To achieve these aims, we chose to formulate the dsRNA with chitosan, sustainably derived cationic biopolymer, which offers additional antimicrobial and plant strengthening effects [39,40]. Therefore, we adapted the formulation methods previously published by Gurusamy, et al. (2020) of nucleic acid with chitosan for our long dsRNA to create positively charged IPECs [65]. We decided to improve upon this formulation method by adding alginate as a biological chelator to increase the protection against nucleases inspired by Douglas, et al. 2006 [52]. Therefore, the present study distinctly takes advantage of the electrostatic interaction between the differently charged poly-electrolytes chitosan, alginate and dsRNA<sup>phi6</sup>/ dsRNA<sup>TMV</sup> to formulate the desired IPECs.

## 3.2. Interpolyelectrolyte complex formation

In order to track the formation of IPECs during the formulation of chitosan with alginic acid or nucleic acids, DLS measurements were performed on the pristine polymers, i.e., cationic chitosan and the anionic polyelectrolytes dsRNA, dsDNA, alginic acid, and mixtures thereof. The results of the DLS measurements are shown in Fig. 3a, in which the autocorrelation function $g^2$ is plotted against the delay time. All pristine polymer solutions showcase undefined and noisy autocorrelation curves, indicative for the absence of well-defined structured phase. In contrast, IPECs formed by the electrostatic interaction of chitosan with at least one negatively charged polymer exhibit well defined autocorrelation functions (Fig. 3a). The latter were fitted and subsequently transformed via inverse Laplace transformation, resulting in the Dh distributions, as depicted in Fig. 3b. Clearly, a similar size distribution is found throughout the formulated IPECs, which can be rationalised by a constant charge ratio employed throughout the sample series (ceteris paribus). Surprisingly, the chitosan-alginate IPECs autocorrelation function is shifted towards larger correlation times compared to the other IPECs, which is indicative for larger particles as confirmed by inverse Laplace transformation (Fig. 3b). Therefore, the DLS results
<figure><img src="image_7.png" /><figcaption>**Fig. 3.** Interpolyelectrolyte complex formation. a) Dynamic light scattering autocorrelation function G2 against the delay time. Autocorrelation functions of the pure anionic polymers DNA, dsRNA, and alginic acid, and cationic chitosan, and the correlation function of interpolyelectrolyte complexes (IPECs) formed by the mixture of anionic polymers and chitosan are displayed. b) Inverse Laplace transformation of chitosan-alginate IPECs (CA-IPECs) and chitosan-alginate-dsRNA IPECs correlation functions. c) The hydrodynamic diameter (Dh) of CAD-IPECs formulated at 0.05 g/L polymer and 1.25 (+/-) molar charge ratio was determined by dynamic light scattering (n = 3). Lower case letters indicate significant differences according to one-way ANOVA F<sub>4,10</sub> = 37.481; *p* < 0.001 and Bonferroni's post-hoc-test at *p* < 0.05. d) The ζ-potential of CAD-IPECs (0.05 g/L, 1.25 (+/-) molar charge ratio) which created substituting up to 100% of DNA with alginate, i.e., CA-IPECs was determined by electrophoretic light scattering (n = 3). Welch's ANOVA F<sub>4,4,142</sub> = 5.358, *p* = 0.063 and Dunnet's-T3 post-hoc-test at *p* < 0.05.</figcaption></figure>

indicate that there is no well-defined phase (i.e. particles) in the polymer solutions, however, IPEC formation was confirmed for the combination of cationic chitosan with all anionic polymers and polymer mixtures. These results are supported by Raman spectroscopy data show that all compounds (nucleic acid, chitosan and alginate) are present in the CAD-IPEC formulation (Fig. S3).

As previously stated, the formulation aims to improve the stability of dsRNA by incorporation into submicron chitosan-alginate-dsRNA IPECs with cationic charge. To ensure optimal size and charge of the IPECs, we investigated several formulation parameters, that is, the alginic acid content and ratio of alginic acid to nucleic acid, the polymer concentration, and the molar charge ratio of cationic to anionic monomers. We determined the influence of these parameters on the IPECs hydrodynamic diameter and ζ-potential by DLS and ELS, respectively.

In turn, the main reason for incorporating alginate into our IPECs is to increase the stability against RNases. However, adding alginate to the IPECs shifts the surface charge (ζ-potential) more negative. Therefore, the bottleneck is to employ a reasonable amount of alginate that enables good stability against RNases while still exhibiting a positive surface charge. Thus, we investigated which proportion of nucleic acid can be substituted in the formulations by alginate, without changing the main IPEC particle characteristics, i.e., mean particle diameter and ζ-potential. Other formulation parameters, such as the total polymer concentration and the molar charge ratio (z) remained constant during the experiment at 0.05 g/L and z = 1.25, respectively. This means that the chitosan and anionic polymer mixture volumes were adjusted accordingly at each ratio of alginate and DNA in the anionic polymer solution to maintain the same charge ratio throughout the experiment. The results obtained from DLS and ELS measurements are shown in Fig. 3c-d. Unambiguously, substituting larger amounts of DNA with alginate increased the diameter of the IPECs up to 145.0 ± 11.1 nm, a two-fold increase over the chitosan-DNA IPECs (CD-IPECs) (71.3 ± 10.9 nm)

(Fig. 3c). Furthermore, it can be noted that up to half of the nucleic acid content can be substituted by alginate without changing the IPECs size significantly (Fig. 3c).

The ELS results are presented in Fig. 3d. The surface charge of the IPECs determined by ELS, was 24.1 mV and 29.9 mV, for CD-IPECs and chitosan-alginate (CA)-IPECs, respectively (Fig. 3d). These data reveal slightly lower ζ-potentials at higher DNA contents, and conversely lower alginate concentrations. Taken together, these results indicated that up to half of the nucleic acid content of the IPECs can be substituted by alginate without changing the particle size or surface charge significantly (Fig. 3c-d). Therefore, IPECs were formulated with equal amounts of alginate and dsRNA for all following experiments if not otherwise stated. Compared to DNA, RNA possesses an additional oxygen atom on its ribose sugar site. Moreover, in dsRNA, thymidine monophosphate is replaced by uridine monophosphate. Nonetheless, it is noteworthy that both dsDNA and dsRNA double helices exhibit the same outward-facing charge due to an equal number of phosphate groups, resulting in an equivalent anionic charge. Therefore, results from CAD- and CD-IPECs should be applicable to CAR- and CR-IPECs, respectively.

### 3.3. Encapsulation efficiency

The formulated IPECs should contain the nucleic acid. However, solely by observing the DLS data the presence of nucleic acid in the formulation cannot be verified. Therefore, we investigated the encapsulation efficiency (EE) of dsRNA in IPEC formulations, i.e., how much of our active ingredient (dsRNA<sup>phi6</sup>) was incorporated into the colloidal IPECs during formulation. To determine the encapsulation efficiency, 0.5 g/L CAR-IPECs were formulated at a molar charge ratio of 1.25 (+/-) containing approximately 125 ng/µL (0.125 g/L) dsRNA. To ensure the accuracy of dsRNA concentrations measured within CAR-IPECs by nanophotometer, we first confirmed the absorbance peak at
260 nm, i.e., the absorbance peak of nucleic acids, by spectral analysis (Fig. S4). Additionally, we confirmed that the other formulation poly- mers chitosan and alginate exhibited no absorbance around 260 nm (Fig. S4). In Fig. 4a the control of unformulated dsRNA remained un- changed in the pre-centrifugation and supernatant fractions, confirming that the dsRNA remains in solution at the applied centrifugal force of 21,380 g (Fig. 4a). In contrast, the mean dsRNA concentration in CAR- IPECs dropped significantly in the supernatant (Fig. 4a). This suggests that the majority of dsRNA is formulated in the CAR-IPECs. We calcu- lated the EE from the initial concentration of dsRNA in the CAR-IPECs and the remaining dsRNA in the supernatant, i.e., the unbound dsRNA, with the equation: $EE = (125.32 \text{ ng/µL} - 7.18 \text{ ng/µL})/125.32 \text{ ng/µL} \times 100\% = 94.3\%$. Our method showed a higher EE compared to the other chitosan-based formulations as well as other methods, including lipid nanoparticles, carbon dots, layered double hydroxide nanosheets, and star polycations with 49%, 68.3%, 86.4% and 63.1%, respectively (Fig. 4b) [5,61,66–68]. Additional evidence of a high EE was obtained by subsequent gel retardation assays, in which cationic CAR-IPECs remained in the gel pockets in contrast to control dsRNA, which migrated as expected (Fig. S5 and Fig. S6a). The addition of alginate also increased dsRNA binding capacity of CAR-IPECs at lower charge ratios at lower charge ratios compared to chitosan-dsRNA (CR)- IPECs, although CR-IPECs were smaller than CAR-IPECs (Fig. S6). Lower charge ratios require less chitosan, which may otherwise trigger un- specific effects in plants [43,44]. In our previous work we did not encounter unspecific effects from the chitosan or alginate in CAR-IPEC formulations of in vitro dsRNA targeting *Magnaporthe oryzae* in *Brachypodium distachyon* [69]. Overall, the better binding capacity, high EE and low charge ratios, and hypothesized greater stability against nu- cleases, led to focus on the development of CAR-IPECs.

### 3.4. IPECs surface charge

In order to determine the impact of the polymer concentration dur- ing formulation on the resulting IPEC size, we created CAR-IPECs with 0.035 g/L – 0.5 g/L total polymer concentration, i.e., the combined anionic and cationic polymer concentrations. We observed a correlation between increased polymer concentrations and larger particle di- ameters, as displayed in Fig. 5a. From 0.035 g/L to 0.075 g/L the mean IPEC diameters were between 94.3 ± 5.6 nm and 111.4 ± 4.3 nm,

respectively, however these results were not significantly different (Fig. 5a). At higher polymer concentrations of 0.35 g/L and 0.5 g/L the particles diameter increased up to 167.2 ± 36.3 nm and 280.1 ± 38.7 nm, which is a two- and three-fold increase of their initial particle size, respectively. Overall, a clear correlation emerges between mean diam- eter and the polymer concentration during formulation, which can be described by a linear equation ($R^2 = 0.941$, Fig. 5a). Essentially, these results are consistent with the work of Sreekumar, et al. 2018, which demonstrated that higher chitosan concentrations increased the size of chitosan particles cross-linked with tripolyphosphate (TPP) at 0.25 to 5 g/L chitosan solution [70]. The results presented in our study extend these findings and hint towards a similar trend for IPECs at concentra- tions between 0.035 g/L and 0.5 g/L (Fig. 5a).

The molar charge ratio, often referred to as N/P ratio in literature pertaining to chitosan complexes formed through electrostatic in- teractions with molecules containing phosphate groups, is a crucial parameter in the formulation of nano- and microparticles through electrostatic interaction [39]. Here we discuss particles formed via electrostatic interaction of oppositely charged polyelectrolytes, i.e., IPECs. We defined the molar charge ratio ($z$) as the ratio of positive and negative charges of the formulation components, which can interact with each other. At $z > 1$ there is an excess of cationic charges and at $z < 1$ there is likewise an excess of anionic charges. Therefore, the resulting particles should, respectively, be positively and negatively charged. Furthermore, at $z \approx 1$ there is an approximately equal cationic and anionic charge, and the resulting mean particle charge in the suspension will be close to neutral. Therefore, aggregation of particles with minor positive or negative charges and agglomeration of neutral particles were expected and observed at charge ratios near $z = 1$.

To investigate these hypotheses, CAR-IPECs were formulated at charge ratios between 0.75 and 2.0 (+/-). The resulting particles were analyzed with DLS and ELS to determine their hydrodynamic size and $\zeta$-potential, respectively. The corresponding ELS results are displayed in Fig. 5b. As showcased, ELS experiments with dsRNA<sup>phi6</sup>-formulations at charge ratios above 1 confirmed the positive surface charge of the IPECs with $\zeta$ of +27.7 ± 1.4 mV at $z$ of 1.25 increasing to +35.5 ± 0.4 mV at $z = 2.0$ (Fig. 5b). The increase in $\zeta$-potential of the IPECs correlates well with the employed charge ratio in the formulation process. Moreover, the formulations created with $z$ of 0.75 and 0.9 had a negative $\zeta$-po- tential of -31.1 ± 0.98 mV and -23.7 ± 1.4 mV, respectively (Fig. 5b).

<figure><img src="image_8.png" /><figcaption>**Fig. 4.** a) Remaining dsRNA<sup>phi6</sup> in supernatant of dsRNA formulated in chitosan-alginate-dsRNA interpolyelectrolyte complexes (CAR-IPECs) and unformulated control dsRNA. The dsRNA concentration of the CAR-IPECs and dsRNA controls ($n = 3$) was determined by nanophotometer with the extinction coefficient of 46.5 ng/µL $A_{260nm}^{-1}$. Then the samples and controls were centrifuged at 21380 g for 1 h at 4 °C and the dsRNA in the supernatant was analyzed by nanophotometer to determine the concentration of free dsRNA in the samples. Significant differences between supernatant and untreated samples according to paired t-test ($t(2) = 63.23$; $p < 0.001$). b) Comparison of encapsulation efficiency obtained by different dsRNA formulation methods and the current work (★), i.e., CAR-IPECs.</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>**Fig. 5.** Characterization of chitosan-alginate dsRNA interpolyelectrolyte complexes. a) Hydrodynamic diameter (Dh) determined by DLS of chitosan-alginate-dsRNA<sup>phi6</sup> interpolyelectrolyte complexes (CAR-IPECs) created with a charge ratio of 1.25 and polymer concentrations between 0.035 g/L and 0.5 g/L (n = 5). One-way ANOVA F<sub>5,24</sub> = 40.08; p < 0.001 and Bonferroni's post-hoc-test at p < 0.05. Linear fit (red line) with R<sup>2</sup> = 0.941. b) The ζ-potential of CAR-IPECs created with the molar charge ratio (z) of 0.75, 0.9, 1.25, 1.6, and 2 (+/-) was determined by ELS. (n = 3). One-way ANOVA F<sub>4,10</sub> = 1048.588; p < 0.001 and Bonferroni's post-hoc test at p < 0.05. Sigmoidal Boltzmann fit (red line) with R<sup>2</sup> = 0.996. c) Mean Dh of CAR-IPECs created with a concentration of 0.05 g/L and z between 0.75 and 2.0 (+/-). d) Graph excluding z from 1.0 to 1.1, in which large particles agglomerates form (n = 5). Welch's ANOVA F<sub>5,10.96</sub> = 11.14 and p < 0.001, and Dunnet-T3 post-hoc-test at p < 0.05. Lower case letters indicate statistical differences.</figcaption></figure>

Moreover, it is reasonable that IPECs created at *z* < 0.75 will be increasingly negatively charged, i.e., following the same sigmoidal function (sigmoidal fit) observed between *z* of 0.75 and 2.0 (Fig. 5b; table S2). Nevertheless, further measurement would be required to confirm this; however, these were omitted in this study to preserve dsRNA. Samples at 0.9 < *z* < 1.25 were not assessed with ELS due to the formation of large particles as shown in Fig. 5c, which are unsuitable for the ELS measurement system, as they precipitate during analysis.

Submicron IPECs formed at 0.9 ≥ *z* ≥ 1.25, with hydrodynamic diameters of 88.1–103.4 nm (Fig. 5c-d), whereas, at charge ratios of 1.0 and 1.1 the mean hydrodynamic diameter was 1943.0 nm and 4681.5 nm, respectively (Fig. 5c). It can be concluded that charge ratios of approximately 1 produce neutral particles, which aggregate or agglomerate to larger structures. However, the large particles sediment during the DLS measurement, which means that we cannot accurately quantify the mean size of these large aggregates.

Additionally, the ELS measurements confirmed that the particle charge shifts sigmoidally from negative to positive between *z* of 0.9 and 1.25 (Fig. 5b). Overall, these results suggest that predictions can be made on the size and charge of the IPECs based on the molar charge ratio. However, currently the *z* calculation marginally undercounts the positive charge (overcounts the negative charge), as the particles increased in diameter at *z* = 1.1 (+/-), i.e., an inverted molar charge ratio *z*<sup>-1</sup> (-/+) of 0.91, whereas at *z* = 0.9 (+/-), i.e., *z*<sup>-1</sup> = 1.11 (-/+), submicron particles were produced (Fig. 5d). Note that the exact sequence of base pairs, or simply the guanine-cytosine (GC) content needs to be taken into account to improve the calculation of the average monomer weight of a (desoxy)ribose monophosphate and thereby the calculation of *z*. Despite these shortcomings, the current calculation is accurate for a good estimation and production of submicron particles

with either negative or positive ζ-potentials. In general, positive particles are commonly developed for foliar application [2]. However, there is no clear consensus in literature about a benchmark ζ-potential, due to the wide range of different targets [71,72].

Based on these results, we formulated CAR-IPECs at *z* = 1.25 (+/-), at low polymer concentrations ≤0.1 g/L, and with an equal amount of alginic acid and dsRNA, for all subsequent experiments, as these parameters formed cationic IPECs which contained the greatest amount of alginic acid without significantly altering the charge or size. Based on the current understanding, we expect cationic submicron IPECs to improve the foliar uptake most.

### 3.5. Helium ion microscopy

Fig. 6 depicts HIM images of CAR-IPECs formulated as described above at a concentration of 0.1 g/L at multiple levels of magnification (Fig. 6 a-c). The CAR-IPECs appear as distinct smooth particles. Compared to DLS measurements of the hydrodynamic diameter of the same sample (102 nm), the mean size of the particles in the examined area of the HIM images was almost three times the diameter (295 nm), although many particles of the expected diameter were also present in the sample (Fig. 6 c-d). It is clear from these images that particle streams formed during the drying process of the sample, as areas of higher and lower particle concentrations are visible (Fig. 6 a-b). Therefore, the particles were forced into close proximity, which may have led to aggregation phenomena during the drying process [73]. These results may, however, indicate that complete drying of the formulations should be avoided. This is especially relevant for foliar applications, as similar drying phenomena should be expected on plant leaves, if the CAR-IPECs do not enter the plant vascular system directly from the aqueous
<figure><img src="image_10.png" /><figcaption>**Fig. 6.** Helium ion microscopy (HIM) images of chitosan-alginate-dsRNA<sup>phi6</sup> interpolyelectrolyte complexes (CAR<sub>phi6</sub>-IPECs) created at the molar charge ratio 1.25 (+/-) and a concentration of 0.1 g/L at different magnifications (a-c) and (d) the corresponding diameter histogram for n = 175 particles. (b,c) CAR<sub>phi6</sub>-IPECs are highlighted in orange.</figcaption></figure>

medium of the spray formulation. Therefore, this insight may put greater emphasis on the selection of appropriate adjuvants to achieve uptake into plants before the spray droplets dry on the leaf surface. Future studies of CAR-IPECs should employ surfactants to lower surface tension of spray droplets to enable rapid uptake through stomata and cuticle pathways, as reported by Hu, et al. 2020 [74]. Previous studies have found three uptake pathways for formulated dsRNA which may be exploited, (i) internalization of dsRNA released outside the cell wall, (ii) fusion of lipid-based carriers with the cell membrane, (iii) endocytosis, which can be clathrin-mediated, and which requires subsequent endosomal escape mechanisms [13,75]. Moreover, chitosan-based formulations have already shown to enhance clathrin-mediated endocytosis [65,76].

### 3.6. dsRNA protection in CAR-IPECs

Alginic acid, known for its metal ion chelation capabilities, was incorporated into our IPEC formulations of chitosan and dsRNA to enhance the protection against nuclease digestion. This approach is based on the requirement of nucleases, such as RNase III, for bivalent metal ions to maintain catalytic activity, or stabilise their dimeric structure [77]. Therefore, we hypothesized that the chelation of these bivalent metal ions inhibits the RNase activity, thereby increasing the dsRNA stability in the formulation. We believe this will increase the protection offered by formulation in chitosan-based IPECs shown in previous studies [36,37].

Consequently, we investigated the stability of dsRNA<sup>phi6</sup> and dsRNA<sup>TMV</sup> within IPECs, i.e., CAR<sub>phi6</sub>-IPECs and CAR<sub>TMV</sub>-IPECs, respectively, compared to free dsRNA against digestion by the nucleases

<figure><img src="image_11.png" /><figcaption>**Fig. 7.** Protection of dsRNA in chitosan-alginate-dsRNA interpolyelectrolyte complexes. a-b) Results were assessed by gel electrophoresis at 110 V for 40 min in a 1 % high resolution agarose gel with the relevant dsRNA<sup>phi6</sup> L, M and S segments 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 pockets indicate unreleased dsRNA. a) RNase III treatment of free and formulated CAR<sub>phi6</sub>-IPECs (0.1 g/L). In both cases, 1196 ng of dsRNA<sup>phi6</sup> were treated with 0.05 U RNase III at 37 °C. The enzymatic reaction was stopped with 2.5 µmol EDTA. b) RNase III treatment and EDTA-release of dsRNA<sup>phi6</sup> formulated in a 0.1 g/L CAR-IPECs. 1196 ng of free or formulated dsRNA<sup>phi6</sup> were first treated with 0.05 U RNase III at 37 °C, and subsequently treated with 11 µmol EDTA for 60 min to stop the enzymatic reaction and release the dsRNA from the formulation. c-e) Gel electrophoresis was performed in a 1 % agarose and stained with ethidium bromide for 30 min (50 min in e)) at 70 V with the relevant dsRNA<sup>TMV</sup> L, M and S segments at 7599 bp, 4223 bp, and 3268 bp, respectively. Ladder (L) (MassRuler 1 Kb DNA ladder, Thermofisher). All samples except controls were treated with 11 µmol EDTA for 60 min to stop the enzymatic reaction (c-d). c) RNase III treatment of free and formulated dsRNA<sup>TMV</sup>. Control (C): non-treated sample. 1200 ng of dsRNA<sup>TMV</sup> were treated with 0.05 U RNase III at 37 °C. d) MNase treatment of free and formulated dsRNA<sup>TMV</sup>. 600 ng of each sample were treated with MNase at 37 °C. e) Temperature treatment of free and formulated dsRNA<sup>TMV</sup>. Control 1 (C1): non-treated sample, control 2 (C2): sample only incubated with 11 µmol EDTA for 60 min at 37 °C. 700 ng of each sample were temperature-treated for 30 min. a-e) For treatment time (min) and temperatures see headers.</figcaption></figure>
RNase III or MNase. Therefore, formulated and unformulated dsRNA<sup>phi6</sup> was treated with 0.05 U RNase III for up to 3 h before the reaction was halted with 2.5 µmol of EDTA, which was sufficient to inhibit the enzyme but insufficient to dissolve the IPECs (Fig. 7a). The subsequent gel retardation assay is shown in Fig. 7a. Apparently, the formulated dsRNA remained visible in the gel pockets for the entire duration of the experiment (Fig. 7a).

In contrast, the free dsRNA was only visible before treatment (0' treatment), showing the distinct bands of the dsRNA<sup>phi6</sup>, i.e., L, M, and S segments at 7599 bp, 4063 bp, and 2948 bp, respectively. After nuclease treatment only a weak trailing signal below 3 kbp remains of the free dsRNA, indicating that the dsRNA was reduced to fragments of various lengths <3 kbp (Fig. 7a). Moreover, in addition to staying in the gel pockets the L, M and S segments of formulated dsRNA<sup>phi6</sup> also appear as (weak) bands before and after the enzyme treatment (Fig. 7a). The appearance of these presumably intact dsRNA<sup>phi6</sup> segments is further evidence for the protection against enzymatic degradation offered by the IPEC formulation. In accord with previous works, the sample remaining in the gel pockets indicates the successful formulation of all dsRNA [66,78]. However, the concentration of EDTA employed to terminate the RNase III reaction may have been sufficient to partially release dsRNA from the IPEC formulations by competitively binding with chitosan through electrostatic interaction, allowing dsRNA to migrate freely in the gel retardation assay (Fig. S7). Therefore, we performed additional experiments which confirmed that weak dsRNA bands were only present in gel retardation assays in the presence of sufficient EDTA concentration (Fig. S8). Moreover, we investigated a series of EDTA concentrations to establish the optimal conditions for dsRNA release from IPEC formulations (Fig. S8 and Fig. S9). However, the EDTA concentrations necessary for the partial dsRNA release produced diffuse bands, with a trailing centre, making the comparison of the band intensities challenging (Fig. S9). Furthermore, the band intensity should only be compared between formulated samples, and not with free dsRNA bands, as there is only partial release, as indicated by the remaining black bands in the gel pockets (Fig. S5, and Fig. 7b-e).

In order to force a partial dsRNA release, we performed a second assay by increasing the final EDTA concentration from 83 mM to 152 mM (Fig. 7b). The subsequent gel retardation assay clarified that free dsRNA<sup>phi6</sup> was already fully 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. 7b). In contrast, the respective segments of the formulated and then released dsRNA were still detectable even after 60 min of RNase treatment. These results showcase effective protection against RNase III digestion of dsRNA incorporated within IPECs. The latter results are in line with Douglas, et al. (2006) who showed comparable protection against nuclease digestion of plasmid DNA loaded onto the surface of alginate-chitosan particles [52]. Nevertheless, further investigation is necessary to illuminate the specific mechanism of protection by alginate [36]. The inclusion of alginate into our IPECs has, however, clearly improved the protection against nucleases, which is consistent with the ability of alginate to inhibit RNase III activity as well as EDTA (Fig. S10). We repeated the experiment with similar results with dsRNA<sup>TMV</sup> and CAR<sub>TMV</sub>-IPECs stored for 14 days at 4 °C (Fig. 7c). Moreover, DLS measurements of CAD-IPECs revealed that the hydrodynamic diameter of IPECs remained constant over a period of 16 days (Fig. S11). Taken together these results indicate a long shelf life of the formulations.

Furthermore, we performed additional experiments with MNase to demonstrate the broad RNase protection offered by CAR-IPECs (Fig. 7d). The free dsRNA<sup>TMV</sup> was degraded after 30 min incubation with either 3 U and 30 U MNase, whereas the formulated dsRNA<sup>TMV</sup> released from the formulations by EDTA after MNase treatment still displayed the same three segments of dsRNA<sup>TMV</sup>. These results on formulated dsRNA<sup>TMV</sup> also demonstrated that the protection of dsRNA in the CAR-IPECs is both independent of the nuclease and the dsRNA sequence, i.e. dsRNA<sup>phi6</sup> or dsRNA<sup>TMV</sup> sequence. To demonstrate the heat stability offered by CAR-

IPEC formulations, the free and formulated dsRNA<sup>TMV</sup> was also treated for 60 min with temperatures between 37 °C and 60 °C (Fig. 7e), which are temperatures that can occur in field conditions depending on plant height, climate zone and season. The free dsRNA<sup>TMV</sup> showed first signs of degradation at 37 °C with a weakening of the M-segment (Fig. 7e). At increased temperatures of 45 °C the M-segment was fully degraded and the S-segment became faint (Fig. 7e). Finally, only a weak L-segment was visible after treatment at 60 °C, whereas the formulated dsRNA<sup>TMV</sup> bands remained stable after treatment at each temperature (Fig. 7e). In contrast, the formulated dsRNA remained visible at each treatment temperature, however, the band intensity was diminished after 60 °C. Indicating a similar trend to the free dsRNA<sup>TMV</sup> the M-segment appeared most faint (Fig. 7e). These results indicate that the formulation stabilises the dsRNA against degradation processes induced at higher temperatures.

### 3.7. Application of formulated dsRNA<sup>TMV</sup>

The efficacy of the free dsRNA with sequence homology to TMV (dsRNA<sup>TMV</sup>) on conferring protection against the targeted virus in *N. benthamiana* plants was previously assessed by Niehl, et al. (2018) [55]. Additionally, we confirmed that the dsRNA<sup>phi6</sup>, which contains no targets against TMV, did not have unspecific effects on *N. bethamiana*, as it demonstrated no protection compared to dsRNA<sup>TMV</sup> (Fig. S12). Therefore, any effects observed with dsRNA<sup>TMV</sup> must stem from the TMV target regions on the M and S segments, which are not shared with dsRNA<sup>phi6</sup>.

Since we demonstrated that CAR-IPEC formulations stabilised dsRNA against degradation at 60 °C (Fig. 7e), we now aim to evaluate whether the still “intact” dsRNA<sup>TMV</sup> from the temperature-treated formulation is also still functional and can provide antiviral protection to plants. To this end, temperature treatment of free- and formulated dsRNA<sup>TMV</sup> was repeated as indicated above and the integrity of the dsRNA was again confirmed by agarose gel electrophoresis (Fig. S13). Subsequently, *N. benthamiana* plants (one leaf per plant) were co-treated with a mixture containing water or 2 µg of the treated sample together with 20 ng of TMV:GFP virions. Fig. 8 shows a statistically significant reduction in the number of infection sites on leaves treated with formulated dsRNA<sup>TMV</sup> compared to those treated with free dsRNA<sup>TMV</sup>, after both formulated and free dsRNA<sup>TMV</sup> were previously incubated at 37 °C or 60 °C for 30 min. Notably, no protective effect was observed when free dsRNA<sup>TMV</sup> subjected to 60 °C was applied, which can be attributed to the complete degradation of the dsRNA (Fig. S13). These results clearly demonstrate that the prepared CAR-IPEC formulation protects the dsRNA from heat stress and that the molecules remain functional. The efficacy of CAR-IPEC formulations was also reported for shorter, in vitro produced dsRNA sequences, i.e., 448 bp dsRNA<sup>Pmk1</sup>, which was formulated at 0.05 g/L at z = 1.25 (+/-) and significantly increased the protection against the fungal pathogen *M. oryzae* 14 days after foliar application of CAR-IPECs [69]. This demonstrates the versatility and efficacy of CAR-IPEC formulations to protect various dsRNAs against nuclease and heat degradation and possibly enable the implementation of RNAi based plant protection against viral and fungal pathogens following the approach presented in Fig. 1. The improvement of RNAi observed in this study and previously in *M. oryzae* are likely due to a combination of better dsRNA stability and protection from nucleases as demonstrated in this study, and increased uptake of intact dsRNA. Therefore, we expect CAR-IPECs to improve other SIGS applications, e. g., against insects, where other chitosan-based formulations have already proven effective, as well as in plant genetic engineering which faces analogous uptake and stability challenges to virus protection [15,37].

## 4. Conclusions

The scrutinised IPECs fabrication method is versatile and can easily
<figure><img src="image_12.png" /><figcaption>**Fig. 8.** a) Representative images of TMV:GFP infection sites in N. benthamiana leaves at 4 days-post-inoculation (dpi) as seen under the UV light. Leaves were co-treated with 20 ng of TMV-GFP virions and water or 2 µg of free or formulated dsRNA<sup>TMV</sup> subjected to the temperature treatment. Scale = 1 cm. b) Number (n°) of infection sites in inoculated leaves at 4 dpi. Data are shown as the 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.</figcaption></figure>

be adapted to produce IPECs with desired size, surface charge, and nucleic acid content. We discovered that formulating nucleic acids and polymers at low concentrations ≤0.05 g/L, at charge ratios of z ≤ 0.9 (+/-) and z ≥ 1.25 (+/-) produced submicron particles, with negative and positive ζ-potentials, respectively. The dsRNA remained protected from enzymatic digestion and heat treatment for hours compared to free dsRNA which degraded in minutes. Additionally, we found that the L, M and S segments remained intact in the formulation at 4 °C and room temperature for 14 days and the formulation protected the dsRNA against enzymatic digestion, which may indicate a long shelf life. Furthermore, the charge ratio and the polymer concentration was optimised during the formulation process, i.e., long dsRNA was formulated in chitosan-alginate-dsRNA IPECs with <100 nm hydrodynamic diameter and positive ζ-potential. These properties are expected to improve RNAi, as displayed in the experiments with *N. benthamiana* protected against TMV. Moreover, long dsRNA produced biotechnologically in the phi6 production system can be successfully formulated with biological polymers to produce a formulation suitable for SIGS. The integration of target-specific sequences into this formulation pipeline will likely facilitate the implementation of RNAi based crop protection strategies.

### CRediT authorship contribution statement

**Benjamin W. Moorlach:** Writing – review & editing, Writing – original draft, Visualization, Investigation, Formal analysis. **Ana R. Sede:** Writing – review & editing, Writing – original draft, Visualization, Investigation, Formal analysis. **Katharina M. Hermann:** Formal analysis. **Alesia A. Levanova:** Writing – review & editing, Investigation, Formal analysis. **Minna M. Poranen:** Writing – review & editing, Supervision, Resources, Methodology, Funding acquisition, Conceptualization. **Michael Westphal:** Investigation, Formal analysis. **Martin Wortmann:** Writing – review & editing, Visualization, Investigation, Formal analysis. **Elzbieta Stepula:** Methodology. **Desiree Jakobschönwandt:** Writing – review & editing, Project administration, Methodology, Conceptualization. **Manfred Heinlein:** Writing – review & editing, Supervision, Resources, Funding acquisition, Conceptualization. **Waldemar Keil:** Writing – review & editing, Writing – original draft, Validation, Supervision, Methodology, Data curation. **Anant V. Patel:** Supervision, Resources, Funding acquisition, Conceptualization.

### Declaration of competing interest

The authors declare no conflict of interest.

### Acknowledgments

This work was funded by the Deutsche Forschungsgemeinschaft (DFG project no. 459501999) to AP, the ANR (grant ANR-21-SUSC-0003-01) to MH, and the Ministry of Agriculture and Forestry Finland (grant VN/8755/2021) to MMP as part of the project "BioProtect" carried out under the second call of the ERA-NET Cofund SusCrop, being part of the Joint Programming Initiative on Agriculture, Food Security and Climate Change (FACCE-JPI). SusCrop has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 771134. Funding was also received from the Research Council of Finland (grant 331627) to MMP. The authors thank Lukas E. Hoffmann for help on the molar charge ratio calculations. The facilities and expertise of the HiLIFE Biocomplex unit at the University of Helsinki, members of Instruct-ERIC Centre Finland, FINStruct, and Biocenter Finland are gratefully acknowledged. The authors thank Prof. Dr. Armin Gölzhäuser from Bielefeld University for providing access to the helium ion microscope. The Raman microscope was funded by the Deutsche Forschungsgemeinschaft (DFG) in the scope of "Großgeräteaktion für Hochschulen für Angewandte Wissenschaften (HAW) 2022".

### Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijbiomac.2025.141579.

### Data availability

Data will be made available on request.

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