1 Chitosan-based delivery systems for plants: A brief overview of recent advances and future directions Muhammad Mujtaba1, Khalid Mahmood Khawar2, Marcela Candido Camara3, Lucas Bragança Carvalho3, Leonardo Fernandes Fraceto3, Rania E. Morsi4,5, Maher Z. Elsaabe7, Murat Kaya6, Jalel Labidi8, Hidayat Ullah9, Depeng Wang10* 1Institute of Biotechnology, Ankara University, Ankara 06110, Turkey 2Ankara University, Faculty of Agriculture, Department of Field Crops, 06100 Ankara, Turkey 3São Paulo State University (UNESP), Institute of Science and Technology of Sorocaba, Department of Environmental Engineering, Sorocaba, Brazil 4Egyptian Petroleum Research Institute, Nasr City, 11727, Cairo, Egypt 5EPRI-Nanotechnology Center, Egyptian Petroleum Research Institute, 11727 Cairo, Egypt 6Department of Biotechnology and Molecular Biology, Faculty of Science and Letters, Aksaray University, 68100 Aksaray, Turkey 7Department of Chemistry, Faculty of Science, Cairo University, 12613 Cairo, Egypt 8Biorefinery Processes Research Group, Department of Chemical and Environmental Engineering, University of the Basque Country (UPV/EHU), Plaza Europa 1, 20018 Donostia- San Sebastian, Spain 9Department of Agriculture, The University of Swabi, Anbar 23561, Swabi, Khyber Pakhtunkhwa, Pakistan 10College of Life Science, Linyi University, Linyi, China *Corresponding author Depeng Wang Address: College of Life Science, Linyi University, Linyi, China E-mail: wyandywang@163.com wangdepeng@lyu.edu.cn Tel: +86-0539-7258717 2 Abstract Chitosan has been termed as the most well-known of these biopolymers, receiving widespread attention from researchers in various fields mainly, food, health, and agriculture. It is a deacetylated derivative of chitin, mainly isolated from waste shells of the phylum Arthropoda after their consumption as food. Chitosan molecules can be easily modified for the adsorption and slow release of plant growth regulators, herbicides, pesticides, and fertilizers, etc. Chitosan as a carrier and control release matrix offers many benefits including; protection of biomolecules from harsh environmental conditions such as pH, light, and temperatures and slow, prolonged release of active ingredients from its matrix consequently protecting the plant's cells from the hazardous effects of burst release. In the current review, we will discuss the recent advances in the area of chitosan application as a control release system. In addition, future recommendations will be made in light of current advancements and major gaps. Keywords: Chitosan nanoparticles; slow delivery; phytohormones; plants 1. Introduction Advancements in the field of nanotechnology open up new doors in terms of applications for various nanomaterials in different fields including plant sciences. The application of different biomaterials as delivery systems showed many advantages like biocompatibility, nontoxicity, an affinity for biomolecules, efficient encapsulation, prolonged release, etc. [1, 2]. Agriculture science is confronted by a number of challenges such as nutrient losses, pathogens, and low crop yield due to water, fertilizer and pesticide mismanagement [3]. To manage such problems, farmers are using intensive amounts of pesticides and other agrochemicals (nitrogenous, phosphoric, potassic fertilizers and organometal) which are ultimately leading to environmental problems such as air pollution, soil toxicity, degradation of agro-ecosystems, residue accumulation and pesticide resistance in insects and pathogen as well as human health problems. In order to overcome the described problems due to the indiscriminate use of synthetic agrochemicals, it is important to develop new strategies for the safer and effective application of these chemicals. It has been shown by many researchers that controlling the release of agrochemicals (fertilizers, pesticides, fungicides, and herbicides) can lead to higher activity and enrich the soil content of the natural biodegradable components [4]. That is how the new smart materials (the nanomaterials) can play a crucial role in enhancing the agricultural process [3, 5-9]. 3 Considering the recent advancements in nanotechnology, encapsulation of ingredients such as pesticides, herbicides and nutrients can be termed as the most favorable solution to the current challenges [6, 10, 11]. Recently the utilization of nano-carriers and nanosensors has drawn the attention of researchers from divergent fields of plant sciences and extensive research efforts are underway to enhance the synthesis and efficiency of biopolymers based on nanocarriers [12, 13]. What characteristic of nanomaterials makes them so effective in improving crop production? Primarily their size. The small molecular size can easily penetrate the plant surface membrane through the cuticles, trichomes, stomata, stigma, hydathodes, wounds and root junctions [14, 15]. For example, controlled release of agro nutrients from nanocarriers enhances the efficacy of the active ingredients, reduces the loss by decreasing the volatilization and prevents contamination risks (environmental and health). The desirable properties of biomaterials based nanocarriers are the reason behind the growing interests in their applications for plants [16]. A wide range of biopolymers including cellulose, chitin, chitosan, collagen, alginate and starch have been actively applied as nanocarriers for the controlled delivery of agrochemicals [12, 17]. The use of cellulose was not as extensive as starch or lignin derivatives [18]. A set of criteria has been defined for a polymer matrix intended to be used as a microencapsulation agent [19]. i. The biopolymer must have a glass transition state, molecular weight and molecular structure suitable for the release (prolonged) of the load. ii. The surface functional groups should not react with the loaded content (agrochemicals etc.) iii. It should be biodegradable and non-toxic to the environment. iv. The polymer should have enough stability during storage and application and should be economical. A large number of studies have been conducted on the encapsulation of antimicrobial agents, pesticides, agrochemicals and agro-nutrients to define the required dosage for specific plants or crops to inhibit microbial growth, diseases or stresses. But, this does not solve the problem completely because the problem is linked to the application of synthetic agrochemicals [20]. Since the last decade, a hunt for an alternative, natural, economical, biodegradable and nontoxic carrier for agro nutrients and agrochemicals has been underway. Chitosan has emerged as a potentially applicable polysaccharide in agriculture, medicine, cosmetic and biomedical industry for different applications [21, 22]. In plants, chitosan is 4 gaining attention as one of the most suitable carrier matrices for agro-nutrients and agrochemicals, owing to its wide range of properties including biocompatibility, biodegradability, high permeability, cost-effectiveness, non-toxicity and excellent film-forming ability [23]. Chitosan and its derivatives, alone or in the composite form, are being used for the controlled release of pesticides, macro and micronutrients, herbicides and plant hormones [20]. An overview of current research shows that the number of articles in this area has been increasing which is a sign of growing interest of researchers in this field. In this review, we will discuss the current status of chitosan application as a nano-carrier in agricultural science. In addition, we will provide a future perspective explaining the gaps in the research with possible solutions moving forward. 2. Overview of chitosan Chitosan is a polycationic polymer isolated after the deacetylation of chitin. Chitin is a structural polymer of many organisms such as crustaceans, insects, mollusks, fungi, and shrimps, etc [24, 25]. The detailed compilation of chitosan sources has been given in Table 1. Chitin a very similar polymer to cellulose, as they function the same; structural integrity (Fig.1). Plants produce cellulose in their cells and chitin has been produced by crustaceans and arthropods in their shells. Up to now, three crystallographic forms of chitin have been reported as alpha (α), beta (β) and gamma (γ) [26]. The α and β are the most commonly existing forms of chitin and can be extracted from a variety of organisms. However, the sources of gamma chitin are scares and hard to find for mass production. For a long time all the literature was relying on a single article for the existence and knowledge of physicochemical properties of gamma chitin [27]. But a very recent report clarifies the big picture and answered a number of questions which were remained unanswered in the previous report [26]. Chemically it is a linear polymer consist of 2-amino-2-deoxy-D-glucose and 2-acetamido-2-deoxy-D-glucose monomers linked through 1–4 bonds. Structurally, α and γ are similar in terms of physicochemical properties compared to the β form of chitin. Compared to chitin chitosan is more functional, thanks to its amino based functional groups stretching along the chain [21]. The molecular weight, biological and physicochemical properties and purity of the isolated chitosan mainly depend upon the source and extraction method [28]. In addition, the protonation intensity of amino groups also plays a vital role in the functionality of chitosan. The major properties of chitosan include nontoxic for humans (LD50=16 g kg-1 in rat) mucoadhesive [29], hemostatic, film-forming [30], excellent adsorption matrix, antiviral, 5 antibacterial and antifungal [31], antioxidative [32]anticholesterolaemic agent [33]. Literature lists multiple references dealing with the properties and wide range applications in the fields of biology, medicine, agriculture, and other vital areas [34]. All the above-mentioned properties of chitosan make it a suitable candidate for myriads of application in different fields including, medicine, cosmetics, and agricultural sciences. Table 1. Aquatic, terrestrial and microorganisms sources of chitin and chitosan (Reprinted from [25]). Aquatic Terrestrial Microorganisms Crustaceans Crab Chionoecetes opilio[35] Podophthalmus vigil[36] Paralithodes amtschaticus[37] Carcinus mediterraneus [38] Arthropods Spiders Geolycosa vultuosa [39] Hogna radiate [39] Nephila edulis [40] Fungi (cell walls) Ascomydes Mycelium[41] Penicillium [42] Yeast (b type)[43] Blastomycota Blastocladiaceae [44] Chytridiomycota Chytridiaceae [24] Protista Brown algae [45] Planta Green algae [45] Scorpions Mesobuthus gibbosus [46] Water lobster Crayfish [47] Insects Ants Prawn Aristens antennatus [48] Beetles Bombyx mori [49] Holotrichia parallela Leptinotarsa decemlineata Cockroaches Krill Daphnia longispina [50] Anax imperator [51] Hydrophilus piceus [51] Notonecta glauca [51] Agabus bipustulatus [51] Asellus aquaticus [51] Mollusca Squid pens Loligo [52] Todarodes pacificus[53] Coelenterata 6 Figure 1. Chemical structure of chitosan; showing all amino groups in blue (Reprinted from with permission from MDPI from Sharif, Mujtaba, Ur Rahman, Shalmani, Ahmad, Anwar, Tianchan and Wang [25]). 3. Chitosan as a matrix for control release After World War II, the agriculture sector had mainly started relying on synthetic pesticides. According to a report, only 0.1 % of the pesticides and other agrochemicals reach the target while the remaining have entered the environment through leaching or evaporation causing contamination for both humans and animals [54]. Control release can be defined as ‘‘the permeation-regulated transfer of an active ingredient from a reservoir to a targeted surface to maintain a predetermined concentration level for a specified period of time’’[55]. A control release matrix can maintain the release of active compounds, in this case, is synthetic pesticides, for a longer time. Additionally an effective release matrix can act as a reservoir for the load and protecting it from degradation mainly due to environmental factors such as UV radiations, water, evaporation, harsh pHs, etc [54]. The main objective of a nanocarrier for plants is the prolonged release of load, enhanced efficacy, and safety of the load and reduction of contamination. The concept of microencapsulation evolved in the late 1980s as an alternative to liposomes for solving the problem of stability as liposomes were unstable in biological solutions [56]. In the following years, researchers focused their attention on exploring and 7 designing different matrices for control release through encapsulation. Chitosan became a dream polymer for encapsulation applications due to its surface amine-based functional groups stretching across the chain. Chitosan can form films, hydrogels, scaffolds, fibers and micro- and nanoparticles thanks to deacetylated surface making easier for myriads of modifications [57, 58]. With the increasing importance of control release in plants, chitosan has been emerged as an ideal polymer due to its desirable physicochemical and biological properties. Since chitosan-based NMs control the release of active ingredients such as pesticides, micronutrients, and plant hormones, they have become rather popular and are being produced in great amounts and marketed around the world. They are now an important part of the defense systems against plant diseases and crop growth promotors [59-65]. Several international organizations; the Codex Alimentarius, a joint body of the UN World Health Organization (WHO) and Food Agriculture Organization (FAO), the European Union (EU) and other organizations have introduced the concept of acceptable maximum residual levels of pesticides (MRLs) in order to confirm the acceptable levels of food quality [4]. Based on its biodegradability, chitosan- based nanomaterials are advantageous over the synthetic agrochemicals, since they are able to escape some stringent regulation of nanomaterials applications [66]. Considering the desired features of chitosan, it can be stated that chitosan-based nanocarriers have a bright future ahead in the field of control delivery in plant sciences. 4. Production of chitosan nanocarriers Chitosan nanomaterials were prepared for the first time in 1994 by emulsification and cross- linking method as drug carriers [67]. Then after, other methods have been developed as ionic gelation [68], reverse micellar method [69], precipitation [70], sieving [71], emulsion droplet coalescence [72] and spray drying [73] most of them have been developed for pharmaceutical and medical fields [66] (Table 2). The production methods mainly depend upon the mode of action needed, for example, the release rate of the active ingredient will depend on the size and shape of the nanoparticles, the thermomechanical behavior and the level of toxicity of the degradable residues [23, 74, 75]. Ionotropic gelation is a simple and mild procedure that uses no chemical cross-linking reducing thus the possible toxic side effects of chemicals or reagents used in the procedure. The emulsion cross-linking method [20, 76] provides high loading capacity and better particle size and release control. Emulsion-droplet coalescence method [72] results in small particle size with high loading capacity. Precipitation method [77] does not involve toxic solvents and leads to better control of particle size and controlled release of the active materials. Reverse micellar method [74] is generally a tedious and laborious process but 8 it has the advantage of producing suitable particle size with a small polydispersity index. The sieving process [71] uses a nano-scaled release device, however, it produces irregular particle sizes. Spray drying method gives a powder formulation with good entrapment efficiency, prolonged active ingredient release however, a lot of parameters can influence the final product particle size [70]. The ionic gelation method which is based on the ionic interactions between the positively charged chitosan and the polycationic negatively charged tripolyphosphate (TPP) is now among the most used method for the preparation of chitosan-based NMs and is recently has been adapted for agricultural applications. Several parameters control the size of the nanoparticles and consequently affects the plant responses towards the nano-chitosan, among these parameters are the degree of deacetylation (DD), molecular weight and concentration of chitosan, chitosan: TPP mass ratio, pH, temperature and rate of mixing of TPP and chitosan [78, 79] in addition to size distribution/polydispersity index (PDI), surface charge (zeta- potential) and functional/encapsulated component [78]. Table 2. Major advances in chitosan nanoparticles production protocols and applications. Matrices Method Encapsulated ingredient Reference carboxymethyl chitosan modified carbon nanoparticles Fast oxidation Carbon nanoparticles [80] Tebuconazole metal- organic framework/chitosan Coating Tebuconazole metal-organic framework [81] Chitosan Emulsion Glyphosate [82] N-deoxycholic acid- O-glycol chitosan Reverse micelles Rotenone [83] N-hexanoyl-O-glycol chitosan Reverse micelles Atrazine [84] Chitosan nanocapsules Ionotropic gelation Achillea millefolium essential oil [85] Chitosan Coating Pesticide/diatomit e/Fe3O4 [86] 9 N,N- dimethylhexadecyl carboxymethyl chitosan Isopropanol agitation Rotenone [83] chitosan Emulsion Imazamox [87] Pectin, chitosan, and sodium tripolyphosphate Ionotropic gelation Paraquat [88] Chitosan nanoparticles Ionic gelation S- nitrosoglutathione [89] Chitosan nanoparticles Ionic gelation Salicylic acid [90] Chitosan nanoparticles Ionic gelation Hexaconazole [91] Chitosan nanoparticles Emulsion crosslinking Cymbopogon martinii Essential Oil [92] chitosan (CS) nanoparticles Nitrogen, phosphorous and potassium [93] chitosan (CS) nanoparticles Spray drying Cu [64] 5. Chitosan as a controlled delivery system 5.1 Delivery of plant growth promoters Phytohormones are molecules responsible for promoting plant growth and development. They are applied to crops to improve production, visual and nutritional aspects, alleviate biotic and abiotic stresses, and increase product shelf life. Although these molecules show the potential to be applied in many crops, few carriers have been explored as a matrix for plant growth promoters. Some examples of plant growth promoters coated in the chitosan matrix are described in Table 3. A carrier system composed of chitosan (CS) and γ-polyglutamic acid (CS/PGA) was developed for gibberellic acid (GA3) delivery [94]. This system presented homogeneous particle size distribution (134 nm) and the GA3 encapsulation efficiency was 61%. In other work [95], the same author's proposed chitosan/alginate (CS/ALG) and 10 chitosan/tripolyphosphate (CS/TPP) nanocarriers for GA3 encapsulation. The GA3 encapsulation efficiency was higher than CS/PGA, reaching 100% CS/ALG and 90% CS/TPP. For the three carriers tested it was possible to observe that the encapsulation of GA3 prevented its degradation and allowed the controlled release of the active materials when compared with the free hormone. Maximum GA3 release was reached after 48h, 17h, and 10h, for CS/PGA, CS/ALG, and CS/TPP, respectively (Figure 2). These authors also performed biological activity assays on beans to evaluate the effects of free and encapsulated GA3 on plant growth. The CS/PGA was more efficient than free GA3 in stimulating seed germination, root and shoot growth, promoting an increase of 15-45% in the shoot length of beans plants. The CS/ALG showed significant effects on beans leaf development and in carotenoids levels compared to free GA3 and CS/TPP nanoparticles. However, no differences were observed in relation to shoot growth between them Figure 2: Biological activities of the alginate (AL)/chitosan (CS)-GA3 and CS/TPP-GA3 nanoparticles towards Phaseolus vulgaris: a) shoot development (cm); b) root development (cm); c) leaf area development (cm2); d) images of the leaves for the treatment at a concentration of 0.012%; e) chlorophyll ‘a’ content (mg mm−2); f) chlorophyll ‘b’ content (mg mm−2); g) total content of carotenoids (mg mm−2). Statistical analysis using one-way ANOVA (Reprinted with permission of Elsevier from Santo Pereira, Silva, Oliveira, Oliveira and Fraceto [95]. 11 Salicylic acid (SA) is another important phytohormone responsible for plant defense response. Chitosan has been used as a matrix for the synthesis of SA nanoparticles to alleviate abiotic stress caused by heavy metals in Isatis cappadocia [96]. The CS/SA nanoparticles application could improve plant growth and phytoremediation efficiency under metal stress by helping plant roots build efficient barriers restricting the entrance of toxic compounds into the xylem, and thus reducing its translocation into shoots [96]. SA microencapsulation using chitosan from crustaceans’ residues was developed by Martin-Saldaña, Chevalier, Iglesias, Colman, Casalongué, Álvarez and Chevalier [97]. The microparticles (1.57 µm to 2.45 µm) were obtained through the gelation method and showed encapsulation efficiency ranging from 59-99%. The lowest efficiency was obtained using the highest concentration of SA, which according to the authors, can be explained by the competitive interaction between TPP and SA to bond with CS. The bioassay on lettuce seedlings showed that low doses of chitosan-SA microparticles can favor the growth of lettuce root and induce defense proteins, and therefore are a promising formulation for activating the defense response of horticultural plants. Nanoparticles with micronutrients like manganese, boron, copper, iron, zinc can be used as plant growth promoters and for soil nutrition [23]. As described by Choudhary and co- workers [98], CS nanoparticles coupled with copper show the ability to improve plant growth even under disease conditions. This is mainly correlated to the strong fungicidal activity of copper and its important role in electron transfer and redox reactions, boosting plant growth. In addition, chitosan is also an inducer of defense response in plants, helping in plant protection. The CS as micro- or nano-carrier for plant growth promoter delivery presents great potential to be used in agriculture, and thus, could be further explored. 5.2 Delivery of fertilizers Soil fertilization is a common practice in agriculture to supply macro and micronutrients to crops, improving their quality. However, when in excess, they result in environmental contamination, increasing the salinity and unbalancing the pH of the soil [99]. Moreover, large amounts of nutrients may be lost prior to absorption by plants. To avoid these problems, chitosan micro- or nanoparticles and hydrogels have been studied as a strategy to enhance the efficiency of fertilizers, reduce soil contamination and promote a sustainable release of these molecules on the soil. Nitrogen, potassium, phosphate, copper, zinc, and vitamins were the focus of many pieces of researches in the last five years (Table 3). Chitosan-urea nanoparticles were successfully synthesized and characterized by Araújo and co-workers [99]. Their work 12 describes a controlled release system for urea delivery using an interaction between humic substances and urea, loaded into chitosan nanoparticles. NPK nano fertilizers were prepared by ionic gelation method using chitosan [100]. The biological activity on coffee seedlings showed a significantly increased in nitrogen and potassium content in the leaves, which the suitable doses of NPK nano fertilizer for improvement of nutrients uptake were of 30 to 40 ppm. The nano fertilizer also enhanced the contents of chlorophyll and carotenoids, and using doses of 30 ppm, the NPK-nanoparticles stimulated the growth of coffee seedlings8. In 2016, Abdel- Aziz and co-workers [101] studied the uptake and translocation of chitosan/NPK nanoparticles in wheat seedlings. They discuss that the nanoparticles entered in the stomata of the leaves and were translocated by the phloem system, which is the main and unique pathway for translocation of nanoparticles and in consequence, are carried out to shoots and roots, improving plant development. Hydrogels films were another strategy adopted for fertilizer delivery. Hydrogel shows the ability to gradually release their load material in the environment through water absorption [102]. Iftime, Ailiesei, Ungureanu and Marin [103] designed a controlled release system for urea by hydrogelation of chitosan with salicylaldehyde. The aim of the authors was to promote soil fertilization and water retention. The water absorbency capacity of the formulation was 68 g/g and water holding capacity in the soil about 154%. The release of urea was carried out in three stages, firstly, releasing 46% in 5h, then a slowed release reaching 75% after 11 days, and lastly, slower continuous release in the next 23 days when almost all the urea was released. In addition, due to the increment of nitrogen in the soil, a growth of over 70% in tomato plants was observed in treated soils, when compared to untreated ones Figure 3. 13 Figure 3: a) X-ray diffractograms of the formulations, b) FTIR spectra of a series of formulations and c) Percentages of urea released during 35 days from the formulations detailed for the first 10 days and first 10 h (Reprinted with permission of Elsevier from Iftime, Ailiesei, Ungureanu and Marin [103]). Biofertilizers based on living microorganisms can be applied to seeds or soil, where bacteria or rhizobacteria colonize the plant rhizosphere and improve its growth. In this context, Perez, Francois, Maroniche, Borrajo, Pereyra and Creus [104] proposed ionically crosslinked beads composed of chitosan and starch to coat Azospirillum brasilense and Pseudomonas fluorescens. The authors produced microbeads around 1.82 mm, which remained relatively stable for the period of one year. This polymeric matrix loaded with plant growth-promoting 14 bacteria was capable to release viable cells in a fast and prolonged manner on the non-sterile soil. Therefore, biofertilization is also a promising alternative practice to contribute with sustainable agriculture and to the recovery of poor-quality soils. 5.3 Delivery of genetic materials The development of nanocarriers to deliver genetic materials, such as plasmid DNA, siRNA, proteins and double-stranded RNA or DNA, into protoplasts or plant cells, opens new opportunities in plant genetic engineering. This pioneer area still must face important challenges, including genetic insertion safety and nano-plant-gene interaction to ensure popular acceptance [105]. Chitosan is a potential carrier to gene delivery and plant transformation due to its capability to complex with negatively charged genetic material via electrostatic interactions, protecting against enzymatic degradation [106, 107]. Kwak, Lew, Sweeney, Koman, Wong, Bohmert-Tatarev, Snell, Seo, Chua and Strano [106] developed a chloroplast- target transgene delivery by the complexation between plasmid-DNA with chitosan-complexed single-walled carbon nanotubes. The system was able to penetrate through the protoplast and chloroplast membranes of the arugula plants showing an estimated efficiency of pDNA delivery of 47%. The complexation with chitosan also protected pDNA from cellular degradation, and it was demonstrated that the nanoparticle-mediated delivery approach could also be applied to other plant species such as watercress, spinach, and tobacco plants. 5.4 Delivery of pesticides The excessive use of pesticides was a traditional way of fighting pests and microbes in a trail to increase crop yields, however, the excess pesticides were a major threat to the humans and animals who consume the agricultural products, in addition to a contamination of the underground water, This was a challenging problem which was a concern of a great number of researchers. How to control and optimize the amount and rate of release of the pesticides in the agricultural industry? Chitosan is a versatile polysaccharide that has enabled the development of intelligent systems for pesticide release in agriculture, favoring the reduction of the consumption of hazardous compounds and reducing environmental and human health impacts. The new work has produced asset release systems responsive to the environmental or intrinsic conditions of its target. Efforts are also made to increase formulation loading and the production of controlled release carriers that promote protection against photosensitive and highly volatile 15 compounds. Relevant work in recent years is shown in Table 3 and some of them are reported in more detail below. Chitosan-Isoleucine-based nanomicellar systems and copolymers were developed to encapsulate the spinosad fungicide, whose results indicated effective protection against photodegradation [108]. The association with nanomicella promoted an active release was faster at pH 6.4 than at pH7, indicating the formulation is pH-responsive, and the activity showed higher activity against fungi treated with the encapsulated formulation compared to unencapsulated spinosad. He and coworkers coated crystals of Bacillus thuringienses with chitosan and observed that chitosan improves the stability and resistance of these crystals to environmental stress and is responsive to alkaline pH, indicating its potential to fight specific insects in their larval stage as it may release the active in the environment. midgut of these pests [109]. Paula, Sombra, de Freitas Cavalcante, Abreu and de Paula [110] synthesized chitosan/cashew tree gum microspheres loaded with Lippia sidoides (LS) essential oil, as a natural insecticide. A slow release of the LS was achieved only after the microspheres have been crosslinked. In this way, the loaded spheres are efficient pest control. Guan, Chi, Yu and Li [111] prepared chitosan/alginate beads loaded with imidacloprid a photodegradable insecticide. The rate of the insecticide release from the beads was much slower than that of the free insecticide (Figure 4). The rate of release was a function of alginate: chitosan ratio. Chitosan derivative containing hydrophilic and hydrophobic groups was prepared and it forms spherical micelles with a size of 167.7–214.0 nm by self-assembly in aqueous solution [112]. Rotenone, a water-insoluble botanical insecticide, was loaded into the micelles solution by reverse micelle method in much higher concentration than in aqueous solution. This assembly provided a successful vehicle for the controlled release of pesticides into the soil. 16 Figure 4: i) Transmission CLSM images of the imidacloprid release process from the Chitosan/Alginate (CHI-ALG)10 microcapsules. (a) Morphologies of imidacloprid microcrystal before dissolution. (b) Images of imidacloprid microcrystal in dissolution. (c) Images of polysaccharide capsules after removal of the crystal cores; ii) SEM images of IMI microcrystals (a) uncoated, (b) coated with 5 layers of CHI/ALG, (c) coated with 10 layers of CHI-ALG; iii) SEM micrographs of photocatalysts: (a): TiO2; (b) SDS/TiO2; (c) Ag/TiO2; (d) SDS/Ag/TiO2; iv) In vitro dissolution profile of coated and uncoated imidacloprid microcrystals; v) The degradation of nano- imidacloprid on photocatalysts (a): catalyst powders under UV light; (b): catalyst powders under natural light (Reprinted with permission of Elsevier from Guan, Chi, Yu and Li [111]). Chitosan coating on mesoporous silica nanoparticles may increase loading in pesticide formulations [113, 114]. A study by Liang and co-authors reached a loading value for prochloraz fungicide molecules of 25.4%. In addition to improving light stability, the study reported that acid and enzymes from infected fruits favor the release of the active ingredient present in the nanoparticles, characterizing them with this double responsiveness character [113]. The effectiveness of this system has been proven by improving antifungal activity against citrus diseases and reducing toxicity, in zebrafish assays, of six times the toxicity about the 17 application of the isolated active. In the case of a synchronous system for encapsulating the emulsion-based azoxystrobin fungicide and modifying the surface of mesoporous silica nanoparticles, loading may be increased from 3.6% to 21% while maintaining pH-responsive release, which is controlled by the carboximethychitosan gatekeeper. The reported system showed a positive effect on late tomato pest, Phytophthora infestans, when compared to the active applied alone in the same dosages [114]. Metal-organic systems can be combined with chitosan to produce fungicidal nanocapsules, such as adsorbed charged tebuconazole and assembled by electrostatic interactions with chitosan and pectin. The study demonstrated that the release is responsive to the action of pectinase in slightly acidic medium, and the fungicide nanocapsules showed microbicidal efficacy and did not affect the growth of Chinese cabbage, providing evidence that they may be safe for application [115]. The association between chitosan and botanical compounds have also been reported in studies [116]. Plants produce compounds as defence mechanisms with the proven activity of these compounds, such as Carvacrol and Linalool which have insecticidal activity and repellents. The problem encountered with the use of botanicals in agricultural formulations is related to photosensitivity and high volatility. A study by Campos and co-workers reports the production of beta-cyclodextrin and chitosan nanoparticles for inclusion compound formation with linalool and carvacrol and suggests an improvement in the life of the compounds. The results indicate that the formulation enables a more sustained release in these actives, which were significantly more effective in terms of acaricidal activities and oviposition against Tetranychus urticae and enabling applications in an environmentally friendly manner by the use of natural compounds. 18 5.5 Delivery of herbicides Herbicides are compounds traditionally used by agriculture to combat unwanted plants, classified as weeds, which cause yield losses. These compounds have their importance due to the growing demand for food, however, they pose risks to several organisms [117]. Technologies to improve the effectiveness of these molecules and reduce potential risks have been developed, such as the association of these compounds with chitosan. Important studies published in recent years (Table 3) demonstrate the potential of chitosan to be used in herbicide formulations. Table 3. Examples of chitosan application as a polymeric matrix for agricultural compounds delivery. Matrix Active ingredient Properties Benefits References Cu-chitosan Copper MD: 361.3 nm ZP: +22.1 mV PDI: 0.20 Antifungal activity, induction of antioxidant and defense enzymes, and significant increasing in plant development [64] Chitosan/TPP (Sodium tripolyphosphate) Salicylic acid MD: 1.57-2.45 um PDI: 0.08-0.22 EE: 59-99% Induction of lettuce plant defense response and growth [97] Chitosan/TPP Salicylic acid - Improve plant defense to overcome metalloid stress caused by arsenic [96] Chitosan/Alginate Gibberellic acid MS: 450 nm ZP: -29 PDI: 0.3 EE: 100% Improve the growth of bean plants and fruit growth of tomato [94, 118] 19 Chitosan/TPP (Sodium tripolyphosphate) MS: 195 nm ZP: +27 mV PDI: 0.3 EE:90% Chitosan/y-polyglutamic acid Gibberellic acid MS: 134 nm ZP -27.9 mV PDI 0.35 EE: 61% Enhanced seed germination, root growth and leaf area, and avoid GA3 degradation [94] Chitosan Nitric oxide MD: 38.81 nm ZP: +17.7 mV PDI: 0.30 EE: 91.07% Alleviate salt stress in maize plants preventing negative effects on plant growth [119] Chitosan/TPP Zinc MD: 250-300 nm ZP: +42.34 mV PDI: 0.28 EE: 10-40% Overcome zinc deficiency in poor soils, prevent nutrient loss and environmental pollution [120] Chitosan/sodium montmorillonite clay KNO3 EE: 40 - 65% Continuous release of potassium for 60 days [102] Chitosan/TPP Urea mixture with peat, humic acid or humin - Controlled release of urea by humic acid substances and by pH [99] Chitosan/ poly- methacrylic acid NPK - Nanoformulation with the lowest concentration of NPK promoted greater wheat development [101] Chitosan/starch Plant growth- promoting bacteria MD: 2.97 nm The hydrogel structure allowed a fast and prolonged release of bacteria in soil, which has strong biotechnological potential as biofertilizer [104] Chitosan/TPP Thiamine MD: 596 nm ZP: +37.7mV PDI <1 Thiamine nanoparticles improved chickpea seed germination and seedling vigor, as well as the content of IAA and defense enzymes [121] 20 EE: 90% Chitosan/TPP KNO3 EE: 92 - 136% The systems showed a swelling-controlled mechanism to release the fertilizer on soil [122] Chitosan/ sodium montmorillonite clay Chitosan/starch/TPP KNO3 MD: 3.0 - 3.7 mm EE: 36-40% The cumulative release of KNO3 achieved 70- 80% after 14 days, which was influenced by the presence of water [123] Chitosan/hydroxypropyl methylcellulose KNO3 - The blend increased about 50 days the residence time of the fertilizer on soil [124] Chitosan/salicylaldehyde Urea - The hydrogel promoted a sustained delivery of urea on soil, reaching maximum release after 35 days. Moreover, the formulation improves the water-holding capacity of the soil and stimulated tomato growth [103] Chitosan/TPP - MD: 259 nm ZP: 40.9 mV PDI: 0.281 Chitosan nanoparticles showed growth effects in wheat seeds and seedlings in low concentrations (5ug/mL) due to better absorption by seed surface and regulation of IAA synthesis [125] Chitosan/TPP NPK MD: 500 nm ZP: 50 mV The nanoparticles showed a sustainable release of NPK during 240h. The application of nano fertilizer in coffee seedlings enhanced NPK levels in the leaves, which improved plant development [100] Chitosan/Polymethacrylic acid NPK MD: 24 - 87 nm ZP: 10 - 85 mV The system was toxic when applied on Pisum sativum in highest doses, inhibiting root growth, lateral root formation and causing cell abnormalities [93] 21 Chitosan/ Polydopamine ZnPN Film thickness: 50-70 nm ZP: -42.7 mV The nanoparticles showed pH-responsive release profile, increased the nutrient utilization efficiency and improved corn seedlings growth [126] Chitosan/Polymethacrylic acid Potassium MD: 368 nm EE: 34.98% The system showed a slow release profile fitting in the criteria for slow-release fertilizer [127] Chitosan/polyacrylic acid Copper MD: 4.8 - 10.7 nm ZP: -22.9 - +26.8 mV The nanoparticles present antifungal and antibacterial activity and improved onion plants growth when treated with a concentration of 75 ppm [128] Chitosan Urea EE: 60 - 96% The hydrogel was prepared using oxidized and grafted chitosan showing a swelling degree of 1500 to 2300%, being pH- dependent. The urea release is favored in alkaline soils [129] Chitosan-complexed single-walled carbon nanotubes Plasmid DNA MD: 104 – 190 nm ZP: 28.1 – 32.3 mV Chloroplast transformation in Eruca sativa, Nasturtium officinale, Nicotiana tabacum, and Spinacia oleracea plants [106] Chitosan Harpinpss protein MD: 133.7 nm ZP: +48.6 mV EE: 89.7 % Tomato plants treated with Harpin nanoparticles showed resistance against fungi disease caused by Rhizoctonia solani [130] Pectin/Chitosan/TTP Paraquat MD: 520 nm ZP: -16 mV PDI: 0.4 EE: 89% Trial with corn and mustard plants demonstrated improved herbicidal activity. The herbicide nanoparticles showed reduced toxicity and mutagenicity to human cells, lower absorption, and deep soil penetration. [88] N-octyl derivates/chitosan Atrazine MD: 41-177 nm EE: > 90% 37.5% reduction in the use of organic solvents in herbicide formulations and promotes the slower release of the active. [131] 22 Smectite/Chitosan/Fe(III) Imazamox - Assays with Brassica oleracea botrytis have shown herbicidal activity similar to the commercial formulation, although the release is slower. However, there was a 15% reduction in total soil leaching losses and a 40% reduction in the maximum concentration in soil leachate. [87] Alginate/Chitosan Imazapic and imazapyr MD: 377 nm ZP: -30 mV EE: 62-71% 1.8×109 particles/mL Increased herbicidal activity tested on Bidens pilosa. Slower release compared to isolated assets and reduced toxicity and genotoxicity. [132] Chitosan/TPP Imazapic and imazapyr MD: 478 nm ZP: +15mV EE: 58-70% 1.2×109 particles/mL Increased herbicidal activity tested on Bidens pilosa. Slower release compared to isolated assets and reduced toxicity and genotoxicity. [132] Chitosan/TPP Paraquat MD: 282 nm ZP: +5 mV EE: 63% The influence of humic substances promoted a reduction in the toxicity of encapsulated Paraquat compared to commercial. [133] Carboxymethyl/chitosan Diuron MD: 250 nm ZP: -30 mV PDI: 0.16 EE: 78% Controlled release and Glutathione-responsive for herbicide release. Efficacy in pre-emergent target species (Echinochloa crusgalli) trials and preservation of non-target species. [134] Carboxymethyl chitosan/2-nitrobenzyl Diuron MD: 140nm ZP:-30mV EE: 91% The micellar system was photoresponsive to herbicide release and kept it encapsulated in the absence of light. [135] Chitosan-Isoleucine/ copolymers Spinosad MD: 454 nm PDI: 0.2 ZP: +16 mV Photodegradation rate reduction, pH- responsive, antifungal activity against pathogenic bacteria found in bananas. [108] 23 Porphyrinic metal- organic/pectin/chitosan Tebuconazole MD: 158 nm ZP: +26 mV EE: 30% The tebuconazole nanocapsules did not affect the growth of Chinese cabbage and had a dual microbicidal effect on plant pathogens, responsive to pH and pectinase action. [115] Mesoporous silica/carboxymethyl chitosan Azoxystrobin MD: 152 nm EE: 71% Loading: 21% It presents pH-responsive release and improved fungicidal activity against late tomato pest, Phytophthora infestans. [136] Mesoporous silica/chitosan Prochloraz MD: 340 nm ZP: +35 mV Loading: 25% Nanoparticles showed good properties responsive to esterase and pH, sustained antifungal efficacy against citrus pathogens and reduction of potential environmental damage. [113] Mesoporous silica/chitosan derivative Pyraclostrobin MD: 299 nm Loading: 40% ZP: 30mV The coating of mesoporous silica nanoparticles increased loading from 26 to 40% and demonstrated the same half-dose fungicidal activity against Phomopsis asparagi. [137] Carboxymethyl chitosan/deoxycholic acid Rotenone spherical micelles MD: 91-140 nm EE: 60-98% Loading: 1-6% Increase in the amount of water-soluble fungicide in the order of 50 times that of the isolated active, resulting in a reduction in the use of organic solvents. [114] Poly(2-dimethylamino- ethyl methacrylate)/chitosan Pyraclostrobin EE: 64% Loading: 19% The microcapsules presented pH release and responsive term. Toxicity reduction in zebrafish assays. [138] Beta- cyclodextrin/chitosan Carvacrol and linalool MD: 175 and 245 nm ZP: +23 and +43 mV PDI: 0.1 and 0.3 Inclusion compounds between beta- cyclodextrin / chitosan essential oils and nanoparticles showed higher acaricidal activity and reduced oviposition against Tetranychus urticae. [116] 24 EE >90% Chitosan/alginate or carboxymethyl cellulose Bacillus thuringiensis Bipyramidal shape MD: 0,6x1,2 µm ~2x107 spore/mL Protection of parasporal crystals from environmental stresses prolonging their activity. Toxins released at pH above 9.0 and larvicidal toxicity equivalent to unencapsulated crystal toxins. [109] Modified clay/chitosan/alginate Acetamiprid D: 1.2-1.7 mm Loading: 4-6% EE: 43-90% Coating the chitosan clay increases loading and encapsulation efficiency for the slower released pesticide. [139] Alginate/chitosan Acetamiprid D: 201 nm PDI: 0.39 ZP: -32 mV EE: 62% The formulation acts by releasing the pesticide in a controlled manner over a wide pH range. [140] Chitosan/TTP hexaconazole D: 100 nm ZP: +35mV EE: 73% Effective in fungal control in alkaline soils, superior to the commercial formulation and less toxic in non-target cell lines. [141] MD: mean diameter; ZP: zeta potential; PDI: polydisperse index; EE: encapsulation efficiency; CE: crosslinking efficiency 25 Paraquat is a potent herbicide but has considerable toxicity and potential for environmental contamination in soils and water. The encapsulation of this compound in pectin/chitosan/TTP nanoparticles demonstrated that herbicidal activity could be improved concomitantly with the reduction of its toxic and mutagenic effects in human cells. Also in this study, it is shown that Pectin/Chitosan/TTP reduces the absorption and deep penetration of the herbicide in the soil, thus reducing the risks of contamination [88]. Grillo, Clemente, de Oliveira, Campos, Chalupe, Jonsson, de Lima, Sanches, Nishisaka and Rosa [133] and co-authors compared paraquat formulations under the influence of humic substances, the results showed that humic substances promoted a reduction in the toxicity of chitosan/TTP-encapsulated paraquat about the commercial formulation, due to changes in the dynamic equilibrium in the medium. Even in formulations where the association with chitosan-containing systems does not promote any increase in herbicidal activity, it can be maintained, and it is worth highlighting features that can be improved in these formulations. The smectite/chitosan/ Fe(III) combination, for example, promoted a slower release for the imazamox compound and maintained the herbicidal activity at commercial formulation levels, but reduced total soil leaching losses by 15% and by 40%. % concentration of leached herbicide, thus contributing to less soil contamination [87]. Studies involving chitosan and herbicidal molecules have also made efforts to demonstrate the advantages of this association for reducing toxicity and genotoxic effects. Alginate/chitosan and chitosan/TPP nanoparticles were employed for the formulation of slow- release nanoherbicides containing imazapic and imazapyr by Maruyama, Guilger, Pascoli, Bileshy-José, Abhilash, Fraceto and De Lima [132]. Herbicidal efficacy trials were tested against the target species Bidens pilosa, presenting higher efficacy than the isolated actives. For nano encapsulated herbicidal actives, cytotoxicity was reduced, less cell damage was identified in genotoxicity assays, and there was less interference in the amount and types of bacteria associated with the nitrogen cycle in the soil compared to the isolated actives [132]. Also, chitosan may be employed for the formulation of nanoherbicides responsive to a given stimulus [134]. Carboxymethyl/chitosan nanoparticles were glutathione-responsive for the controlled release of the diuron herbicide. The study showed that diuron release in vitro was dependent on glutathione concentration. Nanoformulation was effective in the preemergence treatment of target species and did not affect the development of non-target species. The use of carboxymethyl/chitosan micelles was combined with photolabile side groups of 2-nitrobenzyl and showed light irradiation sensitive herbicide release, indicating that reticulated micelles can be used as photo-controlled carrier system [140]. The use of chitosan, therefore, configures a 26 promising alternative to produce intelligent and more efficient herbicidal formulations for use in agriculture. 6. Conclusion and Future prospects Considering all the recent advancements made in the last five years in the application and production of chitosan-based nanocarriers, it can be stated that a major improvement has been made in this area. Yet, there is a lot still to achieve by advancing the production and application techniques of chitosan-based carrier matrices. The application of chitosan-based nanoparticles for control delivery purposes is still under research. Major constraints in transferring the chitosan-based delivery matrices for the control controlled release of pesticides, herbicides and other genetic materials to agriculture include; 1) Economical production techniques of nanocarriers (as the current synthesis procedures require time, money and labor), 2) Application of these nanocarriers for the dose reduction of already applied synthetic agrochemicals, 3) Guided delivery of agrochemicals through modified chitosan nanocarriers (ability to sense the target tissues of cells and release the load at the specific target), 4) An Intensive investigation of chitosan interaction with the plant’s vascular system is required. It can help design the plant-specific or species-specific chitosan-based nanocarriers for the control release application. 5) Delivery of genetic materials through chitosan-based nanocarriers is still an expanding research area. A few number of articles have demonstrated the potential of chitosan nanocarriers for genetic materials. More research is required to fully explore the hidden potential of chitosan for carrying genetic materials in plants. 6) Lack of chitosan based nanocarriers available on the market for use in agriculture. For this reason, more detailed studies of chitosan-based carriers are needed. 7) The use of chitosan as nanocarriers in herbicide formulations has shown through studies that this technology improves the effectiveness of molecules and reduces potential environmental and human health risks. Existing studies with herbicides and chitosan are still considered 27 excipients, however, they direct to the potential application as controlled release systems and responsive to specific stimuli. 7) Advances in technology transfer to large producers and standardization of technology validation protocols are required to encourage the large production of these chitosan-based nano-formulations. 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