rus
Issue #5/2023
A.I.Akhmetova, I.V.Yaminsky, I.Yu.Ilyina, Yu.D.Aleksandrov, V.E.Tikhomirova, E.V.Popova
AFM STUDY OF NANOPLATFORMS FOR THE BIOLOGICALLY ACTIVE COMPOUNDS DELIVERY TO PLANTS
AFM STUDY OF NANOPLATFORMS FOR THE BIOLOGICALLY ACTIVE COMPOUNDS DELIVERY TO PLANTS
DOI: https://doi.org/10.22184/1993-8578.2023.16.5.248.255
In this work, nanoplatforms based on calcium phosphate (CaP), chitosan, and gold nanoparticles, as well as nanoemulsions based on nonionic surfactants, are studied for the delivery of biologically active compounds, including double-stranded RNA (dsRNA), into a plant cell. Using atomic force microscopy, the morphological characteristics of the carriers, the nature of the arrangement on graphite and mica substrates, and the geometric dimensions were evaluated to further assess their ability to form complexes with dsRNA. The project is aimed at searching for nanoplatforms of various chemical nature to create composites with biologically active RNA, which ensure the safety of molecules during exogenous application (plant spraying) and increase the efficiency of their delivery to plant leaves to protect plants from pathogens (viruses).
In this work, nanoplatforms based on calcium phosphate (CaP), chitosan, and gold nanoparticles, as well as nanoemulsions based on nonionic surfactants, are studied for the delivery of biologically active compounds, including double-stranded RNA (dsRNA), into a plant cell. Using atomic force microscopy, the morphological characteristics of the carriers, the nature of the arrangement on graphite and mica substrates, and the geometric dimensions were evaluated to further assess their ability to form complexes with dsRNA. The project is aimed at searching for nanoplatforms of various chemical nature to create composites with biologically active RNA, which ensure the safety of molecules during exogenous application (plant spraying) and increase the efficiency of their delivery to plant leaves to protect plants from pathogens (viruses).
INTRODUCTION
Due to climate change, outbreaks of agricultural plant diseases and environmental stresses are likely to have an increasing negative impact on food production. This problem requires new innovative methods development of plant protection against pathogens based on the achievements of molecular, cell biology and biotechnology. Such methods are RNAi technologies based on the mechanism of RNA interference (RNAi), which is the main mechanism of plant defense against viral infections. In viral infections, this mechanism is induced by dsRNA (replicative form of viral RNA) and leads to degradation of complementary viral RNA [1].
The most promising and safe (environmentally friendly) approach seems to be the one where RNAi-based antiviral defense is induced by spraying plants with exogenous dsRNA (or hairpin RNA) complementary to viral RNA, called spray-induced gene silencing (SIGS). [2] Spraying plants with dsRNA has been successfully used for induction of virus resistance in various crops. One of the problems in dsRNA molecules application is their instability in the environment, resulting in short time window of their antiviral activity. Another major challenge is to ensure efficient delivery of the RNA molecules into plant cells. The specific structure of the leaf surface and the plant cell itself make the problem of delivery of biologically active molecules into the plant particularly challenging. A variety of nanoplatforms are used to solve these problems [3]. However, biocompatible, biodegradable nanoplatforms development that provide protection of regulatory RNAs and their effective penetration into plant cells is still an urgent task, and without their solution the wide RNAi technologies application in practice of crop production is impossible.
The nanoplatform sizes is a significant factor limiting penetration of vehicles delivery into plants. Yong et al. (2021) [4] concluded that a size of 50 nm is the reference size for nanomaterials that can freely penetrate tomato pollen cells [5]. Other studies have shown that the nanoparticles size that can freely penetrate plant cells should be less than 20 nm in at least one dimension. The penetration of composites into plant cells is also influenced by nanomaterial characteristics such as, for example, shape and zeta potential. Therefore, a careful study of their morphological, geometric, adsorption and other physical characteristics is required to select a platform to act as a carrier for stabilization, protection and delivery of dsRNA molecules into plant cells.
MATERIALS AND METHODS
CaP-particles: CaP-particles of 80±20 nm and CaP-particles coated with 5kDa chitosan of 160±25 nm were synthesized according to the method [6]. The 1 μl sample of particle suspension was applied to graphite, incubated for 5 min, and blotted with paper. Chitosan particles of 5 kDa chitosan size 110±20 nm, chitosan particles of 72 kDa glycol-chitosan and size 250±10 nm were obtained by ionotropic gelation method [7]. The size data were obtained by dynamic light scattering method.
Gold nanoparticles: the work considered gold nanoparticles in polyethylenimine, concentration – 0.2 mmol, 0.5 μl were applied on mica and on graphite.
Nanoemulsions based on non-ionogenic surfactants: niosomes with chlorhexidine, emulsions with clay particles with modified surface, 0.5 µl applied on mica and on graphite.
The main method of the present study is atomic force microscopy. Scanning was performed in air in contact mode with a CSG10 cantilever on freshly pierced graphite (highly oriented pyrolytic graphite) and mica substrates using a FemtoScan atomic force microscope. The results were processed in FemtoScan Online software, which allows obtaining three-dimensional images, constructing contour lengths, filtering, image processing, and performing the necessary quantitative calculations: area, volume, perimeter, greatest height, and roughness of the object [8, 9].
RESULTS
The morphological characteristics of the carriers, and nature of their arrangement on graphite and mica substrates, and their geometric dimensions were evaluated in order to further assess their ability to form complexes with dsRNA (Fig.1).
A characteristic surface profile was formed for each sample so that in future, when creating a complex for dsRNA delivery, it would be possible to identify the carrier by characteristic patterns of surface morphology.
CaP-PARTICLES, HYBRID PARTICLES BASED ON CALCIUM PHOSPHATE AND CHYTOSANE
Since calcium phosphate is part of a human body, it is a promising material for use in medicine. Due to its biocompatibility, biodegradability, and controlled properties, calcium phosphate can be used in the form of nano- and microparticles as a carrier of drugs [10].
CaP-particles are the good candidates as carriers because they have a high affinity for nucleic acids [11], and due to controlled synthesis it is possible to obtain particles of suitable size for gene delivery inside cells. The particles considered in this work were: CaP particles (Fig.2), as well as hybrid particles, which are CaP particles coated with 5kDa chitosan (Fig.3). We hypothesize that such hybrid particles can combine the promising properties for two materials.
CHITOSAN PARTICLES
Chitosan-based carriers are extremely promising because of its proven biocompatibility, biodegradability, nontoxicity, and adsorption ability. Nanoparticles made of chitosan and its derivatives can be used for nucleic acids delivery [12, 13], the positive surface charge of such particles can provide efficient loading and protection of incorporated drugs from nuclease activity [14]. However, despite the great progress in the use of chitosan in the field of medicine and pharmaceuticals, the chitosan-based delivery systems application in agriculture is still limited [15].
In our study, we examined particles derived from chitosan lactate with an average molecular weight of 5 kDa (Fig.4) and particles from a 72 kDa glycol-chitosan derivative (Fig.5).
GOLDEN NANOPARTICLES
Gold nanoparticles have minimal cytotoxicity [16]. However, the use of this vector in plant cells has been reported relatively rarely. Zhang et al. used different sizes and shapes of gold nanoparticles to introduce DNA-Cy3 into plant cells by injection, spherical particles of 10 nm size, functionalized with small interfering RNAs, were the most effective in delivering miRNAs and induced gene silencing in mature leaves of Nicotiana benthamiana plants [17].
Both small particles around 100 nm and larger particles around 400 nm are present in the sample on mica, with a characteristic height range of 8 to 30 nm (Fig.6). Gold nanoparticles on graphite aggregated.
NANOEMULSIONS BASED ON NON-IONOGENIC SAAs
Aqueous polyethylene glycol solution changes permeability of the cell membrane, which allows foreign genes to easily penetrate into the nucleus [18]. This method is relatively easy to apply on many plant species. According to the measurements results, all tested surfactant samples lay on the graphite surface as a film without characteristic particles, the sample with clay showed particles (Fig.7). The sample with chlorhexidine forms niosomes, but they are destroyed in the air.
CONCLUSIONS
RNAi technologies being developed based on the achievements of molecular, cell biology and biotechnology are new innovative methods of plant protection against pathogens (in particular, viruses). The development of effective composites for exogenous treatment of crops is an urgent task. In this work, 3D images of carriers based on calcium-phosphate, chitosan, gold nanoparticles, as well as nanoemulsions based on non-ionic surfactants were obtained. Such parameters as adsorption on the hydrophobic surface of graphite, tendency to aggregation, their distribution on the surface, and the geometric dimensions of particles were evaluated.
Scanning probe microscopy can be used as a tool for three-dimensional visualization with nanometer precision and for measuring local mechanical and physicochemical properties of nanoplatforms in the composites design with biologically active molecules (various regulatory RNAs and peptides).
ACKNOWLEDGMENTS
The work of A.I.Akhmetova, I.Y.Ilyina, Y.D.Alexandrov, V.E.Tikhomirova, and E.V.Popova was supported by the Russian Science Foundation (Project No. 23-74-30003).
PEER REVIEW INFO
Editorial board thanks the anonymous reviewer(s) for their contribution to the peer review of this work. It is also grateful for their consent to publish papers on the journal’s website and SEL eLibrary eLIBRARY.RU.
Declaration of Competing Interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Due to climate change, outbreaks of agricultural plant diseases and environmental stresses are likely to have an increasing negative impact on food production. This problem requires new innovative methods development of plant protection against pathogens based on the achievements of molecular, cell biology and biotechnology. Such methods are RNAi technologies based on the mechanism of RNA interference (RNAi), which is the main mechanism of plant defense against viral infections. In viral infections, this mechanism is induced by dsRNA (replicative form of viral RNA) and leads to degradation of complementary viral RNA [1].
The most promising and safe (environmentally friendly) approach seems to be the one where RNAi-based antiviral defense is induced by spraying plants with exogenous dsRNA (or hairpin RNA) complementary to viral RNA, called spray-induced gene silencing (SIGS). [2] Spraying plants with dsRNA has been successfully used for induction of virus resistance in various crops. One of the problems in dsRNA molecules application is their instability in the environment, resulting in short time window of their antiviral activity. Another major challenge is to ensure efficient delivery of the RNA molecules into plant cells. The specific structure of the leaf surface and the plant cell itself make the problem of delivery of biologically active molecules into the plant particularly challenging. A variety of nanoplatforms are used to solve these problems [3]. However, biocompatible, biodegradable nanoplatforms development that provide protection of regulatory RNAs and their effective penetration into plant cells is still an urgent task, and without their solution the wide RNAi technologies application in practice of crop production is impossible.
The nanoplatform sizes is a significant factor limiting penetration of vehicles delivery into plants. Yong et al. (2021) [4] concluded that a size of 50 nm is the reference size for nanomaterials that can freely penetrate tomato pollen cells [5]. Other studies have shown that the nanoparticles size that can freely penetrate plant cells should be less than 20 nm in at least one dimension. The penetration of composites into plant cells is also influenced by nanomaterial characteristics such as, for example, shape and zeta potential. Therefore, a careful study of their morphological, geometric, adsorption and other physical characteristics is required to select a platform to act as a carrier for stabilization, protection and delivery of dsRNA molecules into plant cells.
MATERIALS AND METHODS
CaP-particles: CaP-particles of 80±20 nm and CaP-particles coated with 5kDa chitosan of 160±25 nm were synthesized according to the method [6]. The 1 μl sample of particle suspension was applied to graphite, incubated for 5 min, and blotted with paper. Chitosan particles of 5 kDa chitosan size 110±20 nm, chitosan particles of 72 kDa glycol-chitosan and size 250±10 nm were obtained by ionotropic gelation method [7]. The size data were obtained by dynamic light scattering method.
Gold nanoparticles: the work considered gold nanoparticles in polyethylenimine, concentration – 0.2 mmol, 0.5 μl were applied on mica and on graphite.
Nanoemulsions based on non-ionogenic surfactants: niosomes with chlorhexidine, emulsions with clay particles with modified surface, 0.5 µl applied on mica and on graphite.
The main method of the present study is atomic force microscopy. Scanning was performed in air in contact mode with a CSG10 cantilever on freshly pierced graphite (highly oriented pyrolytic graphite) and mica substrates using a FemtoScan atomic force microscope. The results were processed in FemtoScan Online software, which allows obtaining three-dimensional images, constructing contour lengths, filtering, image processing, and performing the necessary quantitative calculations: area, volume, perimeter, greatest height, and roughness of the object [8, 9].
RESULTS
The morphological characteristics of the carriers, and nature of their arrangement on graphite and mica substrates, and their geometric dimensions were evaluated in order to further assess their ability to form complexes with dsRNA (Fig.1).
A characteristic surface profile was formed for each sample so that in future, when creating a complex for dsRNA delivery, it would be possible to identify the carrier by characteristic patterns of surface morphology.
CaP-PARTICLES, HYBRID PARTICLES BASED ON CALCIUM PHOSPHATE AND CHYTOSANE
Since calcium phosphate is part of a human body, it is a promising material for use in medicine. Due to its biocompatibility, biodegradability, and controlled properties, calcium phosphate can be used in the form of nano- and microparticles as a carrier of drugs [10].
CaP-particles are the good candidates as carriers because they have a high affinity for nucleic acids [11], and due to controlled synthesis it is possible to obtain particles of suitable size for gene delivery inside cells. The particles considered in this work were: CaP particles (Fig.2), as well as hybrid particles, which are CaP particles coated with 5kDa chitosan (Fig.3). We hypothesize that such hybrid particles can combine the promising properties for two materials.
CHITOSAN PARTICLES
Chitosan-based carriers are extremely promising because of its proven biocompatibility, biodegradability, nontoxicity, and adsorption ability. Nanoparticles made of chitosan and its derivatives can be used for nucleic acids delivery [12, 13], the positive surface charge of such particles can provide efficient loading and protection of incorporated drugs from nuclease activity [14]. However, despite the great progress in the use of chitosan in the field of medicine and pharmaceuticals, the chitosan-based delivery systems application in agriculture is still limited [15].
In our study, we examined particles derived from chitosan lactate with an average molecular weight of 5 kDa (Fig.4) and particles from a 72 kDa glycol-chitosan derivative (Fig.5).
GOLDEN NANOPARTICLES
Gold nanoparticles have minimal cytotoxicity [16]. However, the use of this vector in plant cells has been reported relatively rarely. Zhang et al. used different sizes and shapes of gold nanoparticles to introduce DNA-Cy3 into plant cells by injection, spherical particles of 10 nm size, functionalized with small interfering RNAs, were the most effective in delivering miRNAs and induced gene silencing in mature leaves of Nicotiana benthamiana plants [17].
Both small particles around 100 nm and larger particles around 400 nm are present in the sample on mica, with a characteristic height range of 8 to 30 nm (Fig.6). Gold nanoparticles on graphite aggregated.
NANOEMULSIONS BASED ON NON-IONOGENIC SAAs
Aqueous polyethylene glycol solution changes permeability of the cell membrane, which allows foreign genes to easily penetrate into the nucleus [18]. This method is relatively easy to apply on many plant species. According to the measurements results, all tested surfactant samples lay on the graphite surface as a film without characteristic particles, the sample with clay showed particles (Fig.7). The sample with chlorhexidine forms niosomes, but they are destroyed in the air.
CONCLUSIONS
RNAi technologies being developed based on the achievements of molecular, cell biology and biotechnology are new innovative methods of plant protection against pathogens (in particular, viruses). The development of effective composites for exogenous treatment of crops is an urgent task. In this work, 3D images of carriers based on calcium-phosphate, chitosan, gold nanoparticles, as well as nanoemulsions based on non-ionic surfactants were obtained. Such parameters as adsorption on the hydrophobic surface of graphite, tendency to aggregation, their distribution on the surface, and the geometric dimensions of particles were evaluated.
Scanning probe microscopy can be used as a tool for three-dimensional visualization with nanometer precision and for measuring local mechanical and physicochemical properties of nanoplatforms in the composites design with biologically active molecules (various regulatory RNAs and peptides).
ACKNOWLEDGMENTS
The work of A.I.Akhmetova, I.Y.Ilyina, Y.D.Alexandrov, V.E.Tikhomirova, and E.V.Popova was supported by the Russian Science Foundation (Project No. 23-74-30003).
PEER REVIEW INFO
Editorial board thanks the anonymous reviewer(s) for their contribution to the peer review of this work. It is also grateful for their consent to publish papers on the journal’s website and SEL eLibrary eLIBRARY.RU.
Declaration of Competing Interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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