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International Journal of Biological Macromolecules 77 (2015) 36–51

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

j ourna l ho me pa g e: www.elsev ier .com/ locate / i jb iomac

eview

hitosan nanoparticle based delivery systems for sustainablegriculture

rem Lal Kashyapa,b,∗, Xu Xiangb, Patricia Heidenb

ICAR-National Bureau of Agriculturally Important Microorganisms (NBAIM), Mau, Uttar Pradesh 275101, IndiaMichigan Technological University (MTU), Houghton, MI 49931, USA

r t i c l e i n f o

rticle history:eceived 18 October 2014eceived in revised form 3 February 2015ccepted 16 February 2015vailable online 5 March 2015

eywords:hitosanncapsulation

a b s t r a c t

Development of technologies that improve food productivity without any adverse impact on the ecosys-tem is the need of hour. In this context, development of controlled delivery systems for slow and sustainedrelease of agrochemicals or genetic materials is crucial. Chitosan has emerged as a valuable carrierfor controlled delivery of agrochemicals and genetic materials because of its proven biocompatibility,biodegradability, non-toxicity, and adsorption abilities. The major advantages of encapsulating agro-chemicals and genetic material in a chitosan matrix include its ability to function as a protective reservoirfor the active ingredients, protecting the ingredients from the surrounding environment while they arein the chitosan domain, and then controlling their release, allowing them to serve as efficient gene deliv-

griculturelant protectionontrolled releaseanoparticles

ery systems for plant transformation or controlled release of pesticides. Despite the great progress inthe use of chitosan in the area of medical and pharmaceutical sciences, there is still a wide knowledgegap regarding the potential application of chitosan for encapsulation of active ingredients in agriculture.Hence, the present article describes the current status of chitosan nanoparticle-based delivery systemsin agriculture, and to highlight challenges that need to be overcome.

© 2015 Elsevier B.V. All rights reserved.

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371.1. Chitosan in crop production and protection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371.2. Chitosan as a promising delivery system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411.3. Strategies for production of chitosan nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411.4. Emulsion cross-linking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411.5. Emulsion-droplet coalescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 441.6. Ionotropic gelation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 441.7. Precipitation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 441.8. Reverse micelles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 441.9. Seiving method. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 441.10. Spray drying . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451.11. Strategies for loading active ingredient into chitosan nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451.12. Release kinetics of active ingredients from chitosan nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

2. Applications of chitosan nanoparticles as a delivery system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462.1. Pesticide delivery for crop protection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
2.2. Fertilizer delivery for balanced and sustained nutrition . . . . . . . . . . .2.3. Herbicide delivery for weed eradication . . . . . . . . . . . . . . . . . . . . . . . . . . .2.4. Micronutrient delivery for crop growth promotion . . . . . . . . . . . . . . . .
∗ Corresponding author at: ICAR-National Bureau of Agriculturally Important MicroorgE-mail address: [email protected] (P.L. Kashyap).

ttp://dx.doi.org/10.1016/j.ijbiomac.2015.02.039141-8130/© 2015 Elsevier B.V. All rights reserved.

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anisms (NBAIM), Mau, Uttar Pradesh 275103.

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P.L. Kashyap et al. / International Journal of Biological Macromolecules 77 (2015) 36–51 37

2.5. Soil health improvement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 472.6. Delivery of genetic material for plant transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

3. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48. . . . . .

1

piamgualfiapuiapstttrnatanieotnpstiapaciia

fncoap[roaacs

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. Introduction

The biggest challenge faced by agricultural researchers is toroduce sufficient quantity and quality of food to feed the ever

ncreasing global population without degrading the soil health andgro-ecosystem. It has been estimated that global food productionust increase by 70–100% by 2050 to meet the demand of the

rowing population explosion [1]. Agricultural production contin-es to be challenged by a large number of insect pests, diseases,nd weeds accounting for 40% losses to the tune of US $2000 bil-ion per year [2]. To manage these losses and enhance productivity,armers are making excessive and indiscriminate use of agrochem-cals which leads to deterioration of soil health, degradation ofgro-ecosystems, residue problems, environmental pollution andesticide resistance in insects and pathogens. Hence, there is anrgent need to change the manner in which we use agrochem-

cals. Changes can include (i) judicious deployment of pesticidend fertilizer, (ii) rapid and precise detection of pathogens andests, as well as pesticides and nutrient levels, and (iii) promotingoil health by agrochemical degradation. In this context, nano-echnology has emerged as a technological advancement that canransform agriculture and allied sectors by providing with novelools for the molecular management of biotic and abiotic stresses,apid disease detection and enhancing the ability of plants to absorbutrients or pesticides [3–5]. Besides this, nanobiotechnology canlso improve our understanding of crop biology and thus can poten-ially enhance crop yields or their nutritional values. Nanosensorsnd nano-based smart delivery systems are some of the nanotech-ology applications that are currently employed in the agricultural

ndustry to aid with combating crop pathogens, minimizing nutri-nt losses in fertilization, improving crop productivity throughptimized water and nutrient management as well as to enhancehe efficiency of pesticides at lower dosage rates [6,7]. Nanotech-ology derived devices are also being explored in the field oflant breeding and genetic transformation [8,9]. Table 1 describesome of the advancements made in the field of agricultural nano-echnology. Among all these advancements, encapsulating activengredients, such as fertilizers, herbicides, fungicides, insecticides,nd micronutrients in controlled release matrices is one of the mostromising and viable options for tackling current challenges in therea of agricultural sustainability and food security in the face oflimate change. It has been shown that encapsulation of activengredients in nanoparticles enhances the efficacy of chemicalngredients, reducing their volatilization, and decreasing toxicitynd environmental contamination [40].

Chitosan has emerged as one of the most promising polymersor the efficient delivery of agrochemicals and micronutrients inanoparticles (Fig. 1; Table 2). The enhanced efficiency and effi-acy of nanoformulations are due to higher surface area, inductionf systemic activity due to smaller particle size and higher mobility,nd lower toxicity due to elimination of organic solvents in com-arison to conventionally used pesticides and their formulations62,63]. Chitosan nanoparticles have been investigated as a car-ier for active ingredient delivery for various applications (Fig. 1)wing to their biocompatibility, biodegradability, high perme-
bility, cost-effectiveness, non-toxicity and excellent film formingbility [64]. Over the past three decades, various procedures likeross-linking, emulsion formation, coacervation, precipitation andelf-assembly, etc. have been employed to synthesize chitosan
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

nanoparticles [65,66]. Chitosan has also known for its broad spec-trum antimicrobial and insecticidal activities [67,68]. Further, it isbiodegradable giving non-toxic residues with its rate of degrada-tion corresponding to molecular mass and degree of deacetylation[69,70]. However, the low solubility of bulk chitosan in aqueousmedia limits its wide spectrum activity as an antimicrobial agent.Therefore, various strategies have been employed to enhance itsantifungal potential [41]. Chitosan is able to chelate various organicand inorganic compounds, making it well-suited for improving thestability, solubility and biocidal activity of chelated fungicides orother pesticides [64]. For example, copper (Cu) compounds are wellknown for their antifungal nature and have been used with chi-tosan for antibacterial and antifungal activities. The majority of theresearch on chitosan nanoparticles in agricultural research studiedtheir biocidal and antagonistic effects on bacteria and fungi, andgave encouraging results [71–73]. Chitosan-based nanocompos-ite films, especially silver-containing ones, showed antimicrobialactivity against several pathogens [74], but some effect was alsoobserved with chitosan films alone [75]. Other studies investigatedthe use of chitosan–PVA hydrogels for antimicrobial and food pack-aging applications [76–78]. The combination of silver nanoparticleswithin a chitosan–PVA polymeric material also emerged as oneof the most promising candidates for new antimicrobial materials[44]. Recently, application of chitosan particles loaded with copperhas been reported in waste water treatment [79,80]. Consideringthe growing interest, and recent advances, in chitosan-based nano-materials in medical and pharmacological applications, the purposeof this article is to review the current and ongoing research anddevelopmental efforts into chitosan nanoparticles as a delivery sys-tem, with particular focus on describing methods that would besuitable for promoting crop productivity.

1.1. Chitosan in crop production and protection

There have been several reports describing the use of chitosanfor biotic and abiotic stress management in agriculture [73,81–85].Table 3 lists some of the applications of chitosan in crop pro-duction and protection. For the first time, Allan and Hadwiger[130] described the application of chitosan as an antimicrobialagent. This has led to the exploitation of its antimicrobial potentialin various sectors of agriculture. Since the 1980s, the study ofchitosan has been shift from a general sewage treatment agentto plant growth regulator, soil conditioner, vegetables and fruitsantistaling agent, and seed coating agent, especially in the cropdisease management. Several studies showed that chitosan is notonly an antimicrobial agent but also an effective elicitor of plantsystemic acquired resistance to pathogens [73,82,84,131]. Thispolymer has been reported to be the enhancer and regulator ofplant growth, development and yield [85,132,133]. Chitosan hasbeen demonstrated to induce plant defences in tomato [87,89],cucumber [97], chilli seeds [102], strawberry fruits [88] and roseshrubs [99]. Chitosan can activate innate immunity by stimulatinghydrogen peroxide (H2O2) production in rice [134,135], inducea defense response by nitric oxide (NO) pathways in tobacco[136,137], promote the development and drought resistance of
coffee [138], support the synthesis of phytoalexin [139], impact thejasmonic acid–ethylene (JA/ET) signaling marker in oilseed rape[140], cause changes in protein phosphorylation [141], activatemitogen-activated protein kinases (MAPKs) [142] and trigger

38 P.L. Kashyap et al. / International Journal of Biological Macromolecules 77 (2015) 36–51

Table 1Major advancements of nanotechnology in agriculture.

Year Advancement/application(s) Institute(s)/company Reference

2003 Soil binder product based, on a nano-siliica component, toprevent soil runoff and allow seeds blended into the product togerminate

US based company (ETC Group, 2004) [10]

2005 Inorganic Zn–Al layered double hydroxide (ZAL) nanocompositebased controlled release of herbicide(2,4-dichlorophenoxyacetate (2,4-D))

Advanced Materials Laboratory, Institute of AdvancedTechnology (ITMA), Malaysia

[11]

2006 Rapid analysis of pirimicarb residues in vegetables usingmolecularly imprinted polymers (methacrylic acid with carboxylfunctional groups) as recognition elements

University of Hong Kong, Hong Kong SAR, China. [12]

2006 Nano-TiO2 on glassy carbon electrode to detect parathion(pesticide) residue in vegetables

Wuhan University, Wuhan, China; Chinese Academy of Sciences,Beijing, China

[13]

2006 Porous hollow silica nanoparticles (PHSNs) for controlleddelivery system for water-soluble pesticide (validamycin)

Beijing University of Chemical Technology, Beijing, China [14]

2006 Filters coated with TiO2 nanoparticles for the photocatalyticdegradation of agrochemicals in contaminated waters

University of Ulster, UK [15]

2007 Pesticide detection with aliposome-based nano-biosensor University of Crete, GR [16]2007 Mesoporus silica nanoparticles transporting DNA to transform

plant cellsIowa State university, US [8]

2008 Primo MAXX® , nano emulsions as plant growth regulator andstress alleviator

Syngenta Crop Protection, Greensboro, NC [17]

2008 Starch nanoparticles conjugated with fluorescent materialtransporting DNA to transform plant cells

Université de Perpignan via Domitia, Perpignan, France; Institutefor Bioengineering of Catalonia, Barcelone, Spain

[18]

2008 Nanofibres from wheat straw and soy hulls forbio-nanocomposite production

Canadian Universities and Ontario Ministry of Agriculture, Foodand Rural Affairs, CA

[19]

2009 PEG coated nanoparticles loaded with garlic essential oil forcontrol of storage pests (Tribolium castaneum)

Huazhong Agricultural University, Wuhan, China [20]

2009 Cadmium telluride quantum dots (CdTe QDs) to detect 2,4-dichlorophenoxyacetic acid (2, 4-D), (herbicide)

Central Food Technological Research Institute, Mysore, India. [21]

2009 Methyl parathion and chlorpyrifos residue detection using nanosize polyaniline matrix with SWCNT, single stranded DNA andenzyme

Institute of Animal Reproduction and Food Research, Tuwima,Poland

[22]

2009 Nano-sensor for early detection of grain spoilage during storage University of Manitoba, Winnipeg [23]2009 Pesticide detection using gold nanoparticles based dipstick

competitive immuno-assayCentral Food Technological Research Institute, Mysore, India. [24]

2009 Fluorescence silica nanoparticles in combination with antibodyto detect Xanthomonas axonopodis pv. Vesicatoria insolanaceaous crops

MingDao University, Taiwan; National Chung-Hsing University,Taiwan

[25]

2010 Soil-enhancer product, based on a nano-clay component, forwater retention and release

Geohumus-Frankfurt, DE [7]

2010 Pesticide detection using nano-Au/nafion composite invegetables (cabbage, spinach, lettuce)

Beijing University of Technology, China [26]

2010 Carbon nanotube (CNT) conjugated with INF24 oligonucleotidesto reduce the bean rust disease severity

Universidad de Chile, Chile [27]

2010 Magnetic carbon coated nanoparticles as smart agrochemicaldelivery system

IFAPA, Centro Alameda del Obispo, Área de Mejora y Biotecnología,Córdoba, Spain; CSIC-Universidad de Zaragoza, Spain; CSIC,Instituto de Agricultura Sostenible, Alameda Córdoba, Spain

[28]

2010 Polyhydroxybutyrate-co-hydroxyvalerate microspheres ascontrolled release herbicide delivery system for atrazine

UNESP—Univ. Estadual Paulista, Brazil; UNICAMP, CidadeUniversitária Zeferino, Brazil; University of Sorocaba, Sorocaba,SP, Brazil

[29]

2010 Pathogen detection (Tilletia indica) using nano-gold basedimmunosensors based on surface plasmon resonance (SPR)

G.B Pant University of Agri. & Tech., Pantnagar, India; NationalPhysical Laboratory, New Delhi, India

[30]

2010 Pathogen (Sclerotinia sclerotiorum) detection based onelectrochemical sensor, using modified gold electrode withcopper nanoparticle to monitor the levels of salicylic acid in oilseeds

Huazhong University of Science and Technology, Hubei China;Chinese Academy of Agricultural Sciences-Key Laboratory forGenetic Improvement of Oil Crops, China

[31]

2011 Amino-functionalized nanocomposite withtetra-ethylene-pent-amine for organochlorine andorganophosphorus pesticides in cabbage

Ningbo Municipal Center for Disease Control and Prevention,Zhejiang, China.

[32]

2011 Optical sensor for the detection of pesticides (Dipel, Siven 85%WP) in water using ZnCdSe Quantum dots films

Universiti Kebangsaan Malaysia, Malaysia [33]

2012 Neem oil (Azadirachta indica) nanoemulsion as larvicidal agent VIT University, India [34]2012 Macronutrient fertilizers coated with zinc oxide nanoparticles University of Adelaide, AU CSIRO Land and Water, AU Kansas

State University, US[35]

2012 Amphotericin B nanodisks (AMB-NDs) for the treatment offungal pathogens in chickpea and wheat plants

Área de Mejora y Biotecnología, Córdoba, Spain [36]

2013 Pheromone nanogel for the efficient management of fruit-fly Indian Institute of Science (IIS), Bangalore, India; NationalBureau of Agriculturally Important Insects (NBAII), India

[37]

2013 1-Naphthylacetic acid silica conjugated nanospheres for controlrelease and as a plant growth regulator

China Agricultural University, China [38]

2014 Nanoformulation based on chitosan/tripolyphosphatenanoparticles loaded with paraquat herbicide for control releaseand eco-friendly weed management

UNESP—Univ. Estadual Paulista, Brazil; UNICAMP, CidadeUniversitária Zeferino, Brazil; University of Sorocaba, Sorocaba,SP, Brazil and Max Rubner Institut, Karlsruhe, Germany

[39]

2014 Poly(epsilon-caprolactone) nanoparticles containing atrazineherbicide as an alternative technique to control weeds andreduce damage to the environment

UNESP—Univ. Estadual Paulista, Brazil; UNICAMP, CidadeUniversitária Zeferino, Brazil;

[7]


Fig. 1. Strategies for the production of chitosan naoparticles and their applications as a delivery system in agriculture.

Table 2Some examples of active ingredients encapsulated in chitosan-based controlled release matrices in agriculture.

Matrices Method Active ingredient Characteristics Reference(s)

Cu-chitosan nanoparticles Ionic gelation CuSO4 Enhanced antifungal activity against Alternariaalternate, Macrophomina phaseolina andRhizoctonia solani

[41]

�-Fe3O4-CS nanocomposite film Cross-linking �-Fe3O4 Heavy metals monitoring with low detection limit [42]Chitosan microspheres Emulsion cross-linking Urea Controlled release of the urea fertilizer [43]Chitosan–PVA hydrogel Cross-linking Silver nanoaprticles Size of 13 nm; exhibits good antibacterial activity [44]Alginate reinforced chitosan and

starch beadsCross-linking Imazaquin (Herbicide) Porous spherical beads of 2.31 mm size; sustained

slow release of active material[45]

Composite gel Cross-linking Atrazine (Herbicide) andimidacloprid

Sustained release of active material in water for572 h for atrazine and 24 h for imidacloprid,respectively

[46]

Chitosan microspheres Emulsion cross-linking Auxins (Agrochemical) Chitosan microspheres extended action of auxinrelease (up to 120 h)

[47]

Chitosan microspheres Cross-linking Paraquat (Herbicide) Sustained release of active material in water for 8 h [48]Chitosan–silver nanoparticles

composite micro-beadsCross-linking Silver nanoparticles Pesticide removal for extended periods [49]

Chitosan-coated NPK compoundfertilizer

Urea, calcium phosphate andpotassium chloride

Size of 78 nm; controlled release of the NPKfertilizer

[50]

Chitosan hydrogels Cross-linking Potassium nitrate (KNO3) andDihydrogen ammoniumphosphate [(NH4)2HPO4]

Hydrogel in the form of circular pads with 2 mm inthickness and 120 mm in diameter; controlledrelease of the potassium fertilizer; enhanced up to25% water retention of the soil

[51]

Chitosan microcapsules Precipitation 3-Hydroxy-5-methylisoxazole(Herbicide)

Size of 5 �m; sustained release of active materialin water for 80–160 h

[52]

Chitosan gel beads (with acetic orpropionic anhydride)

Cross-linking Atrazine (Herbicide) and urea(Fertilizer)

Extended release period of atrazine to 7 months;chitosan-coated urea beads extended action ofurea release (up to 180 h)

[53]

Beauvericin–chitosannanoparticles

Ionic gelation Beauvericin (Pesticidalcyclodepsipeptide)

Improved pesticidal activity against groundnutdefoliator Spodoptera litura

[54]

Alginate–chitosan microcrystals Self-assembly Imidacloprid (Insecticide) A novel photodegradable insecticide; controlledand sustained release of midacloprid; showedtoxicity against Martianus dermestoides adults

[55]

Chitosan nanoparticles + chitosan – Dichlorprop (Herbicide) Enhanced toxicity to fresh water green algae andslow release of Dichlorprop

[56]

Chitosan microspheres Coacervation–cross-linking Brassinosteroids (Hormones) Controlled delivery of brassinosteroids withbiological activity as agrochemicals

[57]

Chitosan – Dichlorprop (Herbicide) Controlled and slow release of dichlorprop [58]Chitosan nanoparticles – Hexavalent chromium (Metal) Effective agent for in situ subsurface environment

remediation[59]

N-(octadecanol-1-glycidylether)-O-sulfate chitosan(NOSCS) micelle

Reverse micelle Rotenone (Insecticide) Useful as a prospective carrier for control releasedagrochemical

[60]

Chitosan – 1-Naphthylacetic acid(Hormone)

Controlled and slow release of 1-Naphthylaceticacid

[61]

–, Not mentioned.


Table 3Principal studies reported in the literature involving chitosan use for plant growth promotion and protection from 1984 to 2015.

Year Plant/crop Effect/impact of chitosan application Reference

1984 Pea Antifungal activity against Fusarium solani due to synthesis and elicitation of pisatin phytoalexin [86]1992 Tomato Enhanced resistance of tomato plants to the crown and root rot pathogen Fusarium oxysporum f. sp.

radicis-lycopersici[87]

1992 Strawberry Antifungal activity against postharvest pathogens [88]1994 Tomato Induction of systemic resistance to Fusarium crown and root rot [89]1998 Celery Reduction in the incidence and severity of Fusarium yellows [90]2001 Pepper Enhanced biomass production and yield by decreasing transpiration and water use by 26–43% [91]2001 Maize Induction in endogenous hormone content, alpha-amylase activity and chlorophyll content in

seedling leaves[92]

2002 Mulberry Enhancement in respiration rate of germination seeds, root vigor, chlorophyll, protein content andperoxidase in seedlings as well as nitrate reductase and amylase activities

[93]

2002 Cucumber, Chilli, pumpkin,and cabbage

Increment in the seed germination rate [94]

2002 Peanut Enhancement in the energy of germination and germination percentage [95]2002 Soybean Enhancement in growth and yield [96]2003 Cucumber Containment of gray mold infection in plants caused by Botyrtis cinerea [97]2003 Potato Enhancement in yield and late blight resistance by Arbuscular mycorrhizal fungi band chitosan sprays [98]2004 Rose shrubs Enhanced resistance against foliar diseases [99]2004 Date palm Antifungal activity against Fusarium oxysporum f. sp. albedinis and elicitor of defence reactions [100]2005 Maize Increased in plant vigor [101]2006 Chilli Enhanced resitance against Colletotrichum sp.; promote of seedling growth [102]2006 Grapevine Induction of plant defence system against gray mold and downy mildew [103]2007 Rice (Oryza sativa) Induction of defence response against Pyricularia grisea [104]2007 Papaya Antifungal activity against anthracnose and improvement in quality retention of papaya during storage [105]2008 Tobacco Elicitation of callose apposition and abscisic acid accumulation in response to Tobacco necrosis virus

attack[106]

2008 Pearl millet Enhanced seed germination and seedling vigor [107]2009 Maize Increased chilling tolerance of maize seedlings and induced higher activities of antioxidative enzymes [108]2010 Pear Elevated defense-related enzymes activity [109]2010 Grape Direct antifungal activity against Botrytis bunch rot and induction of defense-related enzymes

activities[110]

2010 Sweet cherry Maintained quality attributes and extended the postharvest life by inducing defense-related enzymesactivities

[111]

2010 Mango Combined effects of postharvest heat treatment and chitosan coating on quality and antimicrobialproperties of fresh cut mangoes

[112]

2011 Hypericum perforatum Produced xanthone-rich extracts with antifungal activity [113]2011 Tomato Accumulated phosphatidic acid and nitric oxide [114]2011 Apricot Direct inhibition activity against fruit rot [115]2011 Radish Promoted the uptake of nutrients, nitrogen, potassium and phosphorous, decreased cadmium

concentration[116]

2011 Barley Induced stomatal closure [117]2012 Okra Foliar application of chitosan (100 ppm) enhanced growth and fruit yield [118]2012 Sycamore Enhanced the production of H2O2 and nitric oxide [119]2012 Rice Sheath blight induced activity of defense-related enzymes [120]2013 Ajowan (Carum copticum) Improvement in the germination and growth performance of ajowan (Carum copticum) under salt

stress[121]

2013 Safflower and sunflower Elevated activity of antioxidant enzymes [122]2013 Rice Showed positive and promising effects in increasing rice yield and inhibiting brown backed rice

plant-hoppers[123]

2013 Watermelon Direct killing effect and protection from fruit blotch disease [124]2013 Peach Reduced brown rot infection and enhanced antioxidant and defense-related enzymes [125]2013 Pine Up-regulated the expression level of defense-related enzymes and Pitch canker [126]2013 Camellia Accumulated H2O2, defense-related enzymes, and soluble protein and Anthracnose [127]

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2013 Broccoli Antimicrobial coating served as c2014 Tomato Alternatives fungicide for control2015 Wheat Potential application as a plant gr

efense-related gene expression [143]. Even applied on plantsogether with biological control agents, chitosan enhanced thefficacy in the control of pathogens [144,145]. Soil amendmentith chitosan has frequently been shown to control Fusariumilts [90,146,147] and gray molds [97,103] in a number of crops.

t is interesting to note that these studies show chitosan to beungistatic against both biotrophic and necrotrophic pathogens.esides this, another one of the most important bioactivity ofhitosan on plants is stimulation of seed germination in responseo abiotic stress. In peanut, seed coated with chitosan enhance thenergy of germination and germination percentage [95]. Dzung
nd Thang [96] suggested that chitosan could enhance growth andield in soybean. Seed soaked with chitosan increased germinationate, length and weight of hypocotyls and radicle in rapeseed [148].handrkrachang [94] also found that the application of chitosan
for bioactive compounds [128]sarium crown and root rot [129]regulator [85]

could increase the germination rate of cucumber, chilli, pumpkinand cabbage. Manjunatha et al. [107] reported that seed primingwith chitosan enhances seed germination and seedling vigor inpearl millet. Further, it is also noticed that seed priming with acidicchitosan solutions improved the maize vigor [101]. Similarly,rice seedlings treated with chitosan induced defense responsesagainst the rice blast pathogen, Magnaporthe grisea by inducingthe production of the phytoalexins (sakuranetin and monilactoneA) in leaves [104]. Moreover, chitosan also stimulated the growthand yield of rice along with reinforcing the defense response [149].In addition, other studies also supported a role of chitosan in
modulating the plant response to several abiotic stresses includingsalt and water stress [121,138,150]. For instance, Boonlertnirunet al. [151] found that chitosan treatments had a significant effecton the growth or yield of drought-stressed rice plants compared to


Table 4Pros and cons of various strategies used for the synthesis of chitosan encapsulated active compounds.

Strategies Pros Cons Reference(s)

Ionotropic gelation Simple and mild procedure; no chemicalcross-linking; reduce the possible toxic sideeffects of chemicals or reagents used in theprocedure; and better control of degradationkinetics

Release of active ingredient depends onmolecular weight, degree of deacetylation, andconcentration of chitosan

[66,158–162]

Emulsion cross-linking High drug loading efficiency; controlledrelease with improved bioavailability; andeasy to control particle size

Tedious process, uses harsh crosslinkingagents, problem of reactivity of active agentwith cross-linking agent, and challenge ofcomplete removal of unreacted cross-linkingagent

[40,66,159,161,163]

Emulsion-droplet coalescence High loading efficiency and smaller particlesize

Particle size depends on the degree ofdeacetylation of chitosan. The decreaseddegree of deacetylation increases particle sizewhich in turn decreases drug content

[66,164]

Precipitation Efficient control of particle size and drugrelease; and avoids the use of toxic organicsolvents

Partial protection of the loaded active agentfrom nuclease degradation

[40,159]

Reverse micellar method Thermodynamically stable particle size withsuitable polydispersity index; and narrow sizedistribution with smaller particle size

Tedious and laborious process [40,66]

Sieving method Simple procedure and can be easily scaled up Irregular particle shape [165]Spray drying method High drug stability, good entrapment

sParticle size depends on size of nozzle, spray [40,159]

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efficiency, prolonged drug release attributeand useful method to prepare powderformulation

ontrol plants. It is interesting to note that the effect was greatesthen chitosan was applied before the onset of stressful conditions.ittelli et al. [91] also noticed that the water use of pepper plantsreated with chitosan reduced by 26–43%, with no significanthange in biomass production or yield. These findings indicatehat chitosan has potential to be developed as an antitranspirant ingricultural situations where excessive water loss is undesirable.

Recently, chitosan coatings have emerged as an ideal alterna-ive to chemically synthesized pesticides. It has been reported toeduce the growth of decay and induced resistance in the hostissue [152]. Chitosan can also help to protect the safety of edi-le products. The protection of fresh cut broccoli with chitosangainst E. coli and Listeria monocytogenes was assisted with bioac-ive components such as bee pollen and extracts from propolis andomegranate [153]. Chitosan protection by exclusion occurs withoybean seed treatments. In this case the major advantage was pro-ection from insects such as agarotis, ypsilon, soybean pod borer,nd soybean aphids. Additionally, the treatment was also accom-anied by increases in seed germination, plant growth and soybeanield. From the above points, it is clear that the chitosan productsre more effective and can be used in a numbers of ways to reduceisease levels and enhance crop productivity in a eco-friendly andustainable manner.

.2. Chitosan as a promising delivery system

Chitosan is one of the most widely used polymers in the fieldf drug delivery. Its attractiveness relies on its useful structuralnd biological properties [154,155], which include a cationic char-cter, solubility in aqueous acidic media, and biodegradability.hitosan has a low solubility at physiological pH of 7.4 as it is

weak base (pKa 6.2–7). Chitosan is synthesized by removinghe acetate moiety from chitin through amide hydrolysis underlkaline conditions (concentrated NaOH) or through enzymaticydrolysis in the presence of chitin deacetylase [156]. Chitosan’smine groups readily complex with a variety of oppositely charged
olymers such as poly(acrylic acid), sodium salt of poly(acryliccid), carboxymethyl cellulose, xanthan, carrageenan, alginate andectin, etc. [157]. Chitosan also provides considerable flexibilityor development of formulation, as it is available in wide range
flow rate, pressure inlet air temperature; andencapsulation efficiency depends on themolecular weight of chitosan

of molecular weights (500–1400 kDa) and degrees of acetylation.Chitosan’s amine group also readily lends itself to other chem-ical modifications. Chitosan easily absorbs to plant surfaces (e.g.leaf and stems), which helps to prolong the contact time betweenagrochemicals and the target absorptive surface. Chitosan nanopar-ticles are known to facilitate active molecule or compound uptakethrough the cell membrane. The absorption enhancing effect of chi-tosan nanoparticles improves the molecular bioavailability of theactive ingredients contained within the nanoparticles [158]. Takentogether, these advantages indicate that chitosan has a bright futureas a drug delivery system in the field of sustainable agriculture.

1.3. Strategies for production of chitosan nanoparticles

Chitosan nanoparicles can be synthesized by various techniquesviz., emulsion cross-linking, emulsion-droplet coalescence, precip-itation, ionotropic gelation, reverse micelles and sieving throughnano-scaled controlled release devices. A comparison of these tech-niques, their merits and demerits are summarized in Table 4. Theselection of methods for chitosan nanoparticles synthesis dependson requirements such as the particle size and shape, thermal sta-bility, release time of the active ingredients, and residual toxicityof the final product.

1.4. Emulsion cross-linking

Emulsions are a standard process leading to nanoparticulatephases, while cross-linking is a common way to stabilize a par-ticle structure and to manipulate the controlled-release propertiesof that particle. Altering the cross-linking degree of a particlemodifies an agrochemical’s permeability through it. Cross-linkingenhances the mechanical strength of the final particle by introduc-ing a three-dimensional network structure into the nano-emulsion.The process begins when a chitosan solution is emulsified inan oil phase (water-in-oil emulsion). The chitosan phase is firststabilized by a suitable surfactant, and is then reacted with an
appropriate cross linking agent (e.g. formaldehyde, glutaraldehyde,genipin, glyoxal, etc.). This is followed by washing and drying ofthe resulting nanoparticles [159]. This method is schematicallyrepresented in Fig. 2(A) . The particle size is mainly determined


Fig. 2. Schematic representation of various methods for the synthesis of chitosan nanoparticles. (A) Emulsion cross-linking; (B) emulsion-droplet coalescence; (C) ionotropicgelation; (D) precipitation; (E) reverse micelles; (F) sieving; and (G) spray drying. The term ‘drug’ is used to represent an agrochemical compound, micronutrient and geneticmaterial, etc.


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y the size of the emulsion droplet, which in turn is dependent
n the type of surfactant, degree of crosslinking and the stirringpeed [166]. The molecular weight and concentration of chitosanlso affect the preparation and performance of the nanoparti-les [40,167]. The major drawback of this method is that it is
nued ).

somewhat tedious and the use of harsh, and often expensive, cross-
linking agents can induce undesirable chemical reactions with theactive agent. Recently, Fan et al. [47] studied the synthesis andcontrolled release characteristics of auxin-loaded chitosan micro-spheres using a cross-linker. They found that the cumulative release

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f the auxins from the particles reached a maximum (60%) afterbout 120 h. They also observed that maximum encapsulation effi-iency was significantly influenced by the type of cross-linker,ross-linking time and the oil/water phase ratio. Based on theseesults this procedure is suitable to prepare chitosan nanoparticlesor prolonged controlled release of compounds, possibly spanningeeks or months, and do so with greater safety to non-target orga-isms.

.5. Emulsion-droplet coalescence

This method follows the principles of emulsion by cross-linkingut uses precipitation techniques [168,169]. An emulsion is firstrepared by dispersing chitosan solution and liquid paraffin oil. Thective ingredient and a sodium hydroxide solution are combinednd added to the first emulsion to produce additional droplets.igh-speed mixing is then used to generate collisions between

he different droplets, randomly combining them and precipitatingarticles of small size [169]. The particle size depends primarily onhe degree of deacetylation of chitosan. Generally, at lower degreef deacetylation, large size particles with less ability to retain thective ingredients are obtained [170]. The pictorial representationf the method is shown in Fig. 2B. Using this procedure, Toku-istu et al. [164] synthesized gadopentetic acid loaded chitosan

anoparticles (452 nm) with 45% drug loading efficiency. A similarethodology has been adopted by Anto et al. [168] to encapsulate

-fluorouracil. Interestingly, when two emulsions with equal outerhase are mixed together, droplets of each collide randomly andoalesce, resulting in final droplets with uniform content.

.6. Ionotropic gelation

The chitosan nanoparticles produced through this method aretable, non-toxic and organic solvent free [41,48,169–172]. It isery simple, and employs the use of oppositely charged complexespolyanions) to bond to the oppositely charged amino groups of chi-osan (NH3

+). Tripolyphosphate (TPP) is the most commonly usedonic cross-linker, and relies on electrostatic interaction instead ofhemical cross-linking, avoiding the possible toxicity of reagentsnd other adverse reactions. However, the cross-linking is pH-ependent. In this procedure, chitosan is dissolved in a weak acidicedium and added drop wise under constant stirring to an aqueous

olution containing the other reagents (Fig. 2C). Due to the com-lexation between oppositely charged species, chitosan undergoes

onic gelation and the spherical nanoparticles precipitate [173]. Thehitosan/TPP molar ratio largely controls the mean diameter of theanoparticles, which can also affect the drug release characteristics.

nterestingly, the mechanism of nanoparticle formation throughonic gelation is well described by several workers [174,175]. It haseen suggested that all ionic groups of TPP participated in interac-ions with chitosan amine groups. The ion pairs, formed throughhe negatively charged TPP with the protonated amine function-lity of chitosan in ionotropic gelation provided chitosan with anmphoteric character, which enhanced the protein adhesion andubsequently accelerated the attachment of anchorage dependantells. Recently, Koukaras et al. [174] provided insights into thentermolecular interactions responsible for the ionic cross-linkinguring ionotropic gelation by means of all electron density func-ional theory. They reported that the maximum-interaction relativeonfigurations of TPP and chitosan oligomers depended on the pri-ary ionic cross-linking types (H-, M- and T-links). In all three of the

inking types, there is a high degree of correspondence between chi-
osan monomers and TPP polyanions, and thus, these correspondo low � (and �) ratios. As a result, at low � ratios, the high con-entration of TPP permits the formation of dense H-links. At high
(and �) ratios, the dihedral bias deters the formation of parallel

logical Macromolecules 77 (2015) 36–51

CS chains and impels the formation of irregular and smaller sizenanoparticle cores. At even higher � ratios, the very low concen-tration of TPP results in low nanoparticle core densities because ofthe increased distance between successive H-links, ultimately lead-ing to an increased nanoparticle size. Besides this, a recent work onchitosan/TPP nanoparticles has also established that the concentra-tion of acetic acid used to dissolve chitosan and the temperature atwhich the cross-linking process occurs, strongly affect the size dis-tribution of the obtained nanoparticles [176]. Fàbregasa et al. [177]found that the stirring speed during ionic gelation significantlyaffect reaction yield. Therefore, manipulation of this parameter canbe used to give some control over size range that is obtained to favorthe maximum yield of nanoparticles of desired size.

1.7. Precipitation

This method is quite simple. Chitosan nanoparticles are pro-duced by blowing a chitosan solution into an alkaline solution [e.g.NaOH(aq)] or methanol. The blowing is accomplished with a com-pressed air nozzle, thereby forming the coacervate particles. Theseare separated and purified by filtration and followed by washingwith hot and cold water [178]. The method is schematically repre-sented in Fig. 2D. Generally, various parameters viz., compressed airpressure, spray nozzle diameter and chitosan concentration affectsthe particle shape and size. Although this method is simple, cross-linking is required to enhance the particle stability, and even thenparticles have weak mechanical strength and irregular morphol-ogy.

1.8. Reverse micelles

This method uses a thermodynamically stable mixture of water,oil and lipophilic surfactant. Using this method, it is possible toobtain very small polymeric nanoparticles (≤10 nm) with a uniformdistribution compared with other methods. The size, polydisper-sity and thermodynamic stability of the particles are maintainedin a dynamic equilibrium system. Briefly, the method consists ofpreparing a surfactant solution (e.g. sodium bis(ethyl hexyl) sul-fosuccinate or cetyl trimethylammonium bromide) in an organicsolvent (e.g. n-hexane), to which a chitosan solution and the activeingredient are added under constant stirring, forming a transpar-ent mini- or micro-emulsion. Subsequently, a cross-linking agent(e.g. glutaraldehyde) is added and the system is maintained underconstant agitation. The organic solvent is then evaporated, pro-ducing a dry and transparent mass that is dispersed in water. Asalt is then added to this system, which precipitates the surfac-tant. The resulting mixture is centrifuged and the supernatant,containing nanoparticles loaded with the active substance, is col-lected. The nanoparticles are separated by dialysis and lyophilizedto obtain a dry powder [165]. The method is schematically repre-sented in Fig. 2E. Brunel et al. [179] used a reverse micellar methodto prepare chitosan nanoparticles. They emphasized that chitosanof low molecular weight is preferable to achieve better control overparticle size and distribution. This may be due to a reduction inthe viscosity of the internal aqueous phase or entanglement of thepolymer chains during the process. In recent times, this methodhas been use for enzyme immobilization [180] and to encapsulateoligonucleotides [181]. To date, despite some of the advantages, theuse of this method to produce chitosan nanoparticles is limited, dueto the difficulty of isolating the nanoparticles and need to use a largequantity of organic solvent.

1.9. Seiving method

This method involves the cross-linking of an aqueous acidicsolution of chitosan using glutaraldehyde. The cross-linked

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hitosan is then passed through a sieve with suitable mesh sizeo obtain microparticles. The microparticles are then washedith sodium hydroxide (0.1 N) solution to remove the unreacted

lutataraldehyde and dried at 40 ◦C [159]. This method does noteem to be being extensively researched for agricultural use. Theethod is schematically shown in Fig. 2F.

.10. Spray drying

This method is extensively used for the production of matri-es to produce dry powders, granules and pellets from chitosanolutions and suspensions [160]. The technique is quite versatilend can be used for drugs with high or low heat-sensitivity andith high or low water solubility, and hydrophilic or hydropho-

ic polymers [182]. This procedure is inexpensive and employs single step to produce small-sized particles that are typicallyicro-sized, and then these particles are often reformulate into

uspensions, capsules or tablets [183]. This technique uses a chi-osan solution in acetic acid, to which the active ingredient and theross-linking agent (glutaraldehyde or sodium tripolyphosphate)re consecutively added (Fig. 2G). The resulting solution is atom-zed through a hot air stream, causing flash evaporation of theolvent to form the desired particles [184]. The important parame-ers to modulate particle size in this process are the type of needle,ow speed of the compressed air, air temperature and degree ofross-linking [185]. This method can be used to synthesize particlesith or without cross-linking, and has been used to prepare chi-

osan micro-particles for the delivery of cimetidine, famotidine andizatidine [186]. Recently, Tokárová et al. [187] used spray-driedhitosan microcarriers for the delivery of silver nanoparticles.

.11. Strategies for loading active ingredient into chitosananoparticles

Loading active ingredient into nanoparticulate systems can beone at the time of preparation of particles (incorporation) or afterhe formation of particles (incubation). In these systems, the activengredient is physically embedded within the matrix or adsorbedn the surface. Various techniques have been developed to improvehe efficiency of loading the active ingredient, but the efficiencyargely depends on the method of preparation and the physico-hemical properties of the substance. Loading efficiency is generallyaximized when the substance is incorporated during the forma-

ion of particles, while incubations typically give a much loweregree of incorporation. However, the degree of incorporation islso influenced by the specific process parameters such as exactethod of preparation, presence of additives (e.g. cross linking

gent, surfactant stabilizers, etc.), and agitation intensity [159].oth hydrophillic and hydrophobic compounds can be loaded intohitosan-based particulate systems. Water-soluble compounds areixed with chitosan solution to form a homogeneous mixture, and

hen, particles can be produced by any of the methods describedarlier in the section. Water-insoluble compounds that precipitaten the acidic chitosan solutions can be incorporated after particlereparation by soaking the preformed particles with a saturatedolution of the active ingredient. Water-insoluble drugs can alsoe loaded using a multiple emulsion technique. In this method,ompound is dissolved into a suitable solvent and then emulsi-ed in the chitosan solution to form an oil-in-water type emulsion.ometimes, compounds can be dispersed within a chitosan solutiony using a surfactant to form a suspension. The oil in water (o/w)
mulsions or suspensions prepared in this manner can be furthermulsified into liquid paraffin to get oil-water-oil multiple emul-ions. The resulting droplets can be hardened by using a suitableross-linking agent.
logical Macromolecules 77 (2015) 36–51 45

1.12. Release kinetics of active ingredients from chitosannanoparticles

The release of an active ingredient from chitosan-based parti-cles depends upon the morphology, size, density, and extent andrate of cross-linking of the particles, as well as the physicochemicalproperties of the drug. If any adjuvant is used this can also affectthe release rate. Studies showed that under in vitro conditions, therelease of an active ingredient is affected by pH, solvent polarity,and the presence of enzymes in the dissolution media [188,189].Generally, the release of drug from chitosan particles occurs byone, or a combination of three different mechanisms: (i) an osmot-ically driven burst mechanism, (ii) a diffusion mechanism, and (iii)erosion or degradation of the polymer. In agricultural systems therelease mechanisms are by diffusion release and/or degradationrelease. The diffusion release mechanism includes several stepsviz., (i) penetration of water into particulate system, which causesswelling of the matrix; (ii) conversion of a glassy polymer into aplasticized or rubbery swollen matrix, and (iii) diffusion of com-pound from the swollen matrix.

The original active ingredient content contained in chitosan par-ticles is determined in different ways, but the release from thechitosan particles, is typically measured from particles placed inphosphate buffer saline (PSB; pH 7.4) and kept in a thermostaticincubator at 37 ◦C. Specified volumes of the buffered medium areremoved at regular intervals from the sample being analyzed, andthat same amount of fresh buffer is added back into the flask tokeep the total solution volume constant throughout the durationof the study. The aliquot of removed sample is then filtered andthe transparent filtrate is analyzed. The quantity of active ingre-dient in the aliquot is typically determined by spectroscopic orchromatographic methods.

Diffusion release of active ingredient is typical for hydrophilicpolymers that form hydrogels (e.g. polyvinyl alcohol), while dif-fusion and degradation release occurs with chitosan. It is notuncommon to observe an initial “burst” release of active ingre-dient from particles that predominantly release active ingredientby diffusion or degradation. This happens due to the adsorptionof active ingredients onto the surface of the particles. Once thisburst is exhausted, a slow and steady release is observed that accel-erates if and when the particle matrix begins to degrade. Kweonand Kang [190] synthesized chitosan–polyvinylalcohol (PVA) par-ticles to study the compound release mechanism of the activeingredient under various conditions. They calculated the diffusioncontrolled release by analysis of the linear relationship betweenthe amount of active ingredient released and the square root ofthe time. Jamnongkan and Kaewpirom [51] demonstrated potas-sium release kinetics and water retention of controlled-releasefertilizers based on chitosan hydrogels is through a quasi-Fickiandiffusion mechanism. Similarly, Jameela et al. [191] obtained a goodcorrelation fit for the cumulative drug released vs. square root oftime, demonstrating that the drug release from the microspherematrix is diffusion-controlled and obeys the Higuchi equation[190]. It was demonstrated that the rate of release depends uponthe size of microspheres. Orienti et al. [192] studied the correlationbetween matrix erosion and release kinetics of indomethacin-loaded chitosan microspheres. Release kinetics was correlatedwith the concentration of chitosan in the microsphere and pH ofthe release medium. Nam and Park [188] have demonstrated thein vitro release test of drug loaded chitosan microspheres. Agni-hotri and Aminabhavi [165] also analyzed the dynamic swellingdata of chitosan microparticles and concluded that with increase
in cross-linking, swelling of chitosan microparticles decreases.Recently, Khan and Ranjha [193] studied the swelling behavior ofchitosan/poly(vinyl alcohol) hydrogels as a function of pH, poly-meric compositions and degree of cross-linking. They noticed that

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welling increased by increasing poly(vinyl alcohol) contents inhe structure of hydrogels at higher pH. They also observed thathe cross-linking ratio was inversely related with the swellingf hydrogels. Similar results were also described by Martínez-uvalcaba et al. [194], where drug release increased with increasingrug contents in the hydrogels, while release of drug decreaseds the ratio of crosslinking agent increased in the hydrogel struc-ure owing to strong physical entanglements between polymers.t is also important to note that the release rate of drugs fromydrophilic matrices based on chitosan is greatly affected byhanges in pH. The increase in release rates could be due to anssociated increase in the fluid-filled cavities created by dissolu-ion and diffusion of the drug particles near the surface, which inurn results in an increase in the permeability of the drug [195].

. Applications of chitosan nanoparticles as a deliveryystem

.1. Pesticide delivery for crop protection

The difficulties in controlling pests along with concern about thendiscriminate use of pesticides in agriculture have been the subjectf intense debate and discussion. The pressure to devise alternativeethods of pest control, to reduce the dependency on synthetic

esticides and reduce residue problem, is rising steadily. Therere several examples of slow release of encapsulated agrochemi-als by polymeric nanoparticles. For example, Liu et al. [196] usedolyvinylpyridine and polyvinylpyridine-co-styrene nanoparticleso control release of tebuconazole and chlorothalonil fungicidesor solid wood preservation. That method has given near quan-itative incorporation of the active ingredient. A few years laterolymeric nanocapsules were described as vehicles for the pesti-ides ivermectin and acetamiprid [197], while Wang et al. [198]sed nanosized inorganic particles such as TiO2, SiO2, Fe2O3, orl2O3 as pesticide carriers for increased bioactivity and reduc-

ion in residues. Boehm et al. [199] obtained stable polymericanospheres (135 nm) with 3.5% encapsulation rate and despitehe low active ingredient content, this formulation yielded signif-cant improvements in the bioavailability of the insecticide (RPA07382) to plants. These researchers also performed biologicaltudies on cotton plants infested with aphids to estimate theirect contact efficacy of nanosphere formulations on insects. Theanosphere formulations performed better than the classical sus-ension to manage the infestation. It is important to note that theeveloped nanosphere formulations are not better than the clas-ical suspension in terms of speed of action and sustained release.evertheless, nanosphere formulation performed better than thelassical suspension to enhance the systemicity of the active ingre-ient and improve its penetration through the plant. It has beeneported that nanoparticles loaded with essential garlic oil areffective against Tribolium castaneum [20]. It has also been reportedhat aluminosilicate-filled nanotubes stick to plant surfaces whilehe nanoscale aluminosilicate particles leach from the nanotubesnd subsequently stick to the surface hair of insect pests. Thesearticles ultimately enter the body and influence certain physio-

ogical functions [200,201]. Recently, a pesticide company releasedn aqueous dispersion formulated with nano-sized biocide (Ban-er MAXX® from Syngenta) having a broad spectrum systemic

ungicidal action. The active ingredient controls leaf spots, blights,usts and powdery mildew diseases on various horticultural andrnamental crops [17,202]. At present, there are several reports
vailable regarding the production and use of chitosan nanoparti-les as a delivery matrix for the release of pesticides in agriculture.s an example, Paula et al. [203] prepared and characterized micro-pheres composed of chitosan and cashew tree gum, which were

used as carriers of the essential oil of Lippia sidoides, which pos-sesses insecticidal properties. The findings indicated the suitabilityof chitosan for use as matrices to carry bioinsecticides designed tocontrol the proliferation of insect larvae. Similarly, microcapsulesof alginate and chitosan were prepared, characterized, and evalu-ated as a carrier system for imidacloprid [55]. The particles obtainedwere stable and imidacloprid was encapsulated with an efficiencyof around 82%. In release assays, it was shown that the releasetime of the encapsulated insecticide was up to eight times longer,compared to the free insecticide, and that alterations in the con-centrations of alginate and chitosan affected the release profile. Inanother independent study, Quinones et al. [57] described the useof chitosan microspheres to carry synthetic analogs of brassinos-teroids and diosgenin derivatives. The release kinetics assay usingwater revealed that the least efficiently encapsulated steroids werereleased fastest from the particles. These results demonstrate thatmolecular modifications can be used to design effective systems forthe delivery and release of agrochemicals. Besides this, amphiphilicderivative of chitosan, N-(octadecanol-1-glycidyl ether)-O-sulfatechitosan has been evaluated as a carrier for the insecticide rotenone[60]. The polymeric micelles formed were spherical, with a sizerange of between 167 and 204 nm, and the nanoparticles wereformed by self-assembly in aqueous solution. The encapsulation ofrotenone increased its solubility in water 1300-fold, while in vitrorelease assays demonstrated that the nanoparticles provided sus-tained release of the insecticide. The properties of nanomicellesbased on NOSCS enable them to be used as carriers to encapsulateand subsequently release insoluble pesticides employed in agri-culture. Feng and Peng [204] synthesized a new compound basedon chitosan, using carboxymethyl chitosan (CM-C) with ricinoleicacid (RA) for use as a carrier of the biopesticide azadirachtin (AZA).The particles presented good polydispersion, with a size range of200–500 nm, as well as smooth spherical morphology and high zetapotential. The AZA encapsulation efficiency was 56%, and the par-ticles were able to release the pesticide over a period of 11 days.The use of the carrier assisted the solubilization in water of thislipid-soluble pesticide, and could therefore offer advantages in agri-cultural applications.

2.2. Fertilizer delivery for balanced and sustained nutrition

The extent and quality of plant growth is largely dependenton the quantity of fertilizer and water. So, improvement in cropoutcomes requires improved utilization of water resources andfertilizer nutrients. It is estimated that about 40–70% of nitrogen,80–90% of phosphorus, and 50–70% of potassium of the appliedfertilizers is lost to the environment and cannot be absorbed bythe intended plants. This is not only a substantial monetary andresource loss but also results in serious environmental pollution[50,205]. Several recent research studies have been publishedthat describe the use of superabsorbent polymers to enhancegermination and crop growth under arid and desert environments.The results are encouraging, and show that use of such materialscan reduce water consumption in irrigation, and reduce the plantdeath rate [206,207]. An optimized combination of slow releasefertilizers and superabsorbent polymers may not only significantlyimprove plant nutrition and yields, but might be a method tomitigate the stressed environmental impact, reduce water lossesto evaporation, and reduce irrigation frequency [208]. Indeed, thedevelopment of slow release fertilizers from chitosan nano- ormicroparticles is a relatively new concept to reduce fertilizer con-sumption and to minimize environmental pollution. With these
principles in mind, Wu et al. [209] developed a chitosan-coatedNPK compound fertilizer with both controlled-release and water-retention capabilities, by using an inner coating of chitosan, and anouter coating was poly (acrylic acid-co acrylamide) [P(AA-co-AM)],

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hich is a superabsorbent polymer. It was observed that the prod-ct showed a slow controlled release of the nutrients. The nutrientseleased did not exceed 75% on the 30th day. Furthermore, chitosans a readily biodegradable material, while the P(AA-co-AM) canlso be degraded in soil, so neither the matrix polymers nor theiregraded products were harmful to the soil. We believe that suchroducts have a great potential as eco-friendly nano-fertilizers,specially in drought-prone regions with limited water availability.n similar efforts, Corradini et al. [50] explored the possibility oftilizing chitosan nanoparticles for slow release of NPK fertilizer,hile Hussain et al. [43] reported controlled release of urea from

hitosan microspheres. Although preparation of nanoparticles asontrolled release devices may be more costly than simple broadpplication of fertilizer, it is clear that these materials can not onlyynchronize the release of nitrogen, phosphorous and potassiumertilizer for their optimum uptake by crops, but they can alsorevent undesirable nutrient losses to soil, water, and air. This hashe compensatory benefits of requiring less use of fertilizers, asell as undesirable environmental impact. Nevertheless, because

f the “upfront” higher costs, it is clear that if the materials inevelopment are to become commercial successes, they will needo offer better value to growers by reducing the overall cost ofertilizer, and enhance crop productivity.

.3. Herbicide delivery for weed eradication

Every year approximately 10–15% the principal food productions lost due to weeds and other plant competition. In recent decades,here has been an alarming increase in the use of herbicides to man-ge the weeds that are responsible for these losses. Each year 47.5%f the total pesticides that are used have been applied to crops toanage these pests [210]. The heavy use of herbicides has given

ise to serious environmental and public health problems. Problemsrising from the herbicides currently in use are attributed to theirhemical stability, solubility, bioavailability, photodegradation andoil sorption. In addition, transfer of these agents to aquatic sys-ems affects water quality, resulting in adverse impacts to humans,ther biota, and the wider environment. In this sense, controlledelease formulations of herbicides have become a necessity, sincehey often increase herbicide efficacy at reduced doses. Recently,ilva et al. [48] used alginate/chitosan nanoparticles as a carrier sys-em for paraquat application. They demonstrated that associationf paraquat with alginate/chitosan nanoparticles alters the releaserofile of the herbicide, as well as its interaction with the soil, andence this system could be an effective means of reducing negative

mpacts caused by paraquat. Similarly, Grillo and co-workers [39]repared and evaluated chitosan/tripolyphosphate nanoparticless carrier systems to paraquat herbicides. The results showed thathe nanoparticles were able to decrease the herbicide toxicity [39].n another study, Celis et al. [211] used bionanocomposite mate-ial based on chitosan and clay (montmorillonite) as an adsorbentor the herbicide clopyralid present in an aqueous solution or in

mixture of water and soil. The bionanocomposites showed gooderbicide adsorption capacity at pH levels at which the anionic formf the active principle and the cationic form of chitosan predomi-ated. Removal of the herbicide from aqueous solution was moreffective when a higher concentration of chitosan was used in theionanocomposite. At slightly acid pH, the composites effectivelydsorbed clopyralid from soil. The use of this type of formulationould help to limit the mobility of anionic pesticides in the environ-ent, reducing risks of contamination of surface and subterranean
ater bodies. Wen et al. [212] studied the bioavailability of the chi-
al herbicide dichlorprop to the green alga Chlorella pyrenoidosa, inhe absence and presence of chitosan nanoparticles. These observa-ions provided a clear indication that chitosan was able to modify


the enantioselective bioavailability of the herbicide, which couldbe of use in environmental protection applications.

2.4. Micronutrient delivery for crop growth promotion

It is a well known fact that micronutrients like manganese,boron, copper, iron, chlorine, molybdenum, and zinc promote opti-mum plant growth. Steady increases in crop yields following the1960s ‘green revolution’ has progressively depleted the level ofessential micronutrients like zinc, iron and molybdenum in thesoil [213]. Farming practices, such as liming acid soils, contributeto micronutrient deficiencies in crops by decreasing the availabil-ity [213]. Foliar application of micronutrients is now a commonagricultural practice and is shown to enhance its uptake by theleaves [214]. Nanoformulations of micronutrients may be usedto spray crops for enhanced foliar uptake, or nanomaterials withmicronutrients may be used as a soil addition for their slow releaseto promote plant growth and improve soil health [215]. For anexample, Tao et al. [61] synthesized chitosan modified with 1-naphthylacetic acid, which is an important plant growth hormone.The results indicated that the release of the 1-naphthylacetic acidwas strongly dependent on pH and temperature, and could con-tinue for 55 days at pH 12 and 60 ◦C. Despite this dependence, theformulation offers potential for the slow release of plant growthhormones.

2.5. Soil health improvement

The installation of nanosensors in farmers’ fields is being appliedto enable the real time monitoring of soil conditions and the earlydetection of potential problems such as nutrient depletion or insuf-ficient water [216]. In this context, nanosensors can help to extendthe new practices of precision farming by detecting and rectifyingagronomic problems in a very short time span. Nanomaterials, suchas hydrogels and zeolites, were reported to be useful for improvingthe water-holding capacity of soil [217] and to absorb environ-mental contaminants [218]. Recently, efforts have been made todevelop a nanoparticle modified chitosan sensor for the determina-tion of heavy metals [42]. The biosensor is based on the combinationof chitosan cross-linked with glutaraldehyde modified with para-magnetic Fe3O4. The �-Fe3O4/CS nanocomposite film, which can beeasily prepared, exhibits high accumulation ability for the deter-mination and removal of heavy metals (arsenic, lead, and nickel)and ‘reports’ the process by an electrical response. Agnihotri et al.[44] developed a novel antimicrobial chitosan–PVA-based hydro-gel, which can behave both as a nanoreactor and an immobilizingmatrix for silver nanoparticles (AgNPs) with promising antibacte-rial applications.

2.6. Delivery of genetic material for plant transformation

The biggest challenge for gene delivery in agricultural crops isthe plant cell wall. Traditional gene transfer methods in plantssuch as Agrobacterium-mediated gene transfer, electroporation,PEG-mediated gene transfer, particle gun bombardment, etc., arecostly, labor intensive and cause significant perturbation to thegrowth of cells. In addition, these methods have very low effi-ciency (0.01–20% efficiency). Nevertheless that has been relativelysuccessful for genetic transformation of dicots [219]. Hence, thereis interest in utilizing novel delivery systems for the develop-ment of successful transformants. Nanotechnology has shown itsvalue in the genetic modification of plants by introducing new
genes with a corresponding crop improvement. This system hassignificant advantages in comparison to conventional and tradi-tional gene transformation tactics. Firstly, nanoparticle approachesare applicable to both monocot and dicot plants, irrespective of

4 of Bio

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issue or organ type. Secondly, they can be used to overcomeransgenic silencing via regulating the DNA copies combined withanoparticles. Thirdly, nanoparticles can be easily functionalized

or further enhancement of transformation efficiency, if needed.inally, nanoparticle-mediated multigene transformation is possi-le without involving traditional methods that require complexarriers. Overall, these key features make nanoparticles excel-ent gene carriers for the genetic engineering of crops. Zinc oxideanoparticles and carbon nanotubes were both reported to pen-trate tomato (Lycopersicon esculentum) seed tissues and plantoots, indicating that new nutrient delivery systems can be devel-ped by exploiting the nanoscale porous domains of the planturfaces [220]. Gene transfer by bombardment of DNA-absorbedn gold particles has also been successfully harnessed for theevelopment of transgenic plants in a species-independent man-er [221]. Torney et al. [8] demonstrated the delivery of DNA andhemicals through silica nanoparticles internalized in plant cells,ithout any help from specialized equipment. Martin et al. [222]

eported protein and DNA co-delivery to plant cells via the biolisticethod, using mesoporous silica nanoparticles. The potential for

iodegradable chitosan to be used in gene delivery is supported byts ability to protonate in acidic solution and to form a complex

ith DNA through electrostatic interactions [223]. Furthermore,ome reports provide evidence that polymer/DNA complexes areore stable than those involving cationic lipids, and can protectNA from nuclease degradation [224]. Chitosan/DNA nanoparti-les may be readily formed by coacervation between the positivelyharged amine groups on chitosan and negatively charged phos-hate groups on DNA. However, the transfection efficiency ofhitosan is low. The transfection efficiency has been shown toepend on the chitosan molecular weight, degree of deacetyla-ion, pH of the transfecting medium, and cell type [225]. A pHf 6.8–7.0 is critical for transfection [226], and evidence suggestshat DNA complexes formed by shorter and close to monodispersehitosan oligomers (24-mer) have more desirable properties thanltrapure chitosan and are therefore more attractive as gene deliv-ry systems than the conventional high molecular weight chitosans227]. Besides this, the degree of chitosan deacetylation also actss an important factor in chitosan-DNA nanoparticle formulations it affects DNA binding, release and gene transfection efficiencyn vitro and in vivo [228].

RNA interference (RNAi) is a powerful strategy for post trans-riptional gene silencing that can be mediated by delivery of syn-hetic double stranded small interfering RNA (siRNA). This processesults in the degradation of homologous RNA and thereby causesnockdown of the specific target gene [229–231]. This method hasmerged as a recognized strategy to control insect pests that feedpon plants producing double stranded RNA (dsRNA). For effec-ive insect control the production of sufficient dsRNA by transgeniclants as well as their delivery in an effective and non-degradedanner is required, which in turn required continuous feeding of

igh levels of dsRNA, because of dsRNA degradation in the insect gut232,233]. Recently, chitosan has attracted significant attention forse in formulations with small interfering RNA (siRNA). Because ofhe cationic nature, chitosan can make complex with siRNA easilynd forms nanoparticles. Several reports indicate the applicationf chitosan nanoparticle-entrapped siRNA as a carrier for siRNAelivery [234–236]. In one recent study, Zhang et al. [237] havehown that chitosan nanoaprticles successfully delivered dsRNAagainst chitin synthase genes) in stabilized form, to mosquito lar-ae via feeding. Chitosan nanoparticles could prove to be efficientn dsRNA delivery due to their efficient binding with RNA, pro-
ection and the ability to penetrate through the cell membrane.hese results clearly indicate that chitosan nanoparticles basediRNA formulations may contribute to plant pathogen and pestontrol while avoiding the lengthy process of conventional plant

transformations. There are some mixed results with regard to genedelivery via chitosan nanoparticles, but given the potential advan-tages of chitosan nanoparticles to assist in the delivery of geneticmaterial to design new and improved plant genotypes, chitosanwill continue to be an important research topic. And, there is a realpotenital to use DNA-coated chitosan nanoparticles as a nanocar-rier in a gene gun system, for bombardment of plant cells and tis-sues to achieve efficient and targeted gene transfer, in near future.

3. Conclusions

Application of chitosan based nanoparticles in agriculture isstill in a nascent stage. Encouraging and promising results arealready being achieved in delivery of agrochemicals and genesfor plant transformation using chitosan nanoparticles. The useof such nanomaterials for the delivery of pesticides, micronutri-ents and fertilizers is expected to reduce the required dosage forefficacy and ensure a controlled delivery. An important advancewill have been realized once the application of nanoparticles isable to deliver biocontrol materials and control the release at anappropriate rate for effective crop protection and nutrition, anddo so with a reduced environmental hazard. In the context ofhost–pathogen interactions, application of nanoparticle technologyand efficient transportation of substances, such as systemic plantdefence activators (e.g. salicylic acid, jasmonic acid and benzothia-diazole, etc.), to specific target sites provide novel solutions for cropstress alleviation. If it is possible to have a distribution of properlyfunctionalized nanoparticles throughout the plant vascular systemand direct them to targeted sites, then these nanoformulations canbe successfully used to deliver chemicals (fungicides, herbicidesand insecticides, etc.), or other substances (plant hormones, elic-itors and nucleic acids) into localized areas of plant tissues. Thiscould help to decode ever – unveil insight story at physiological,biochemical and genetic levels, ultimately helping to make sus-tainable agriculture a reality. The foundations of new crop healthtechniques have been laid, and research into many of the poten-tial applications is ongoing, especially in the field of crop geneticengineering and production of selective, effective and low doseplant protection products using chitosan-nanoparticle as a prin-ciple. However, issues such as increasing the scale of productionprocesses and lowering costs, as well as toxicological perspectives,still must be addressed to further advance nanotechnology intosustainable agriculture.

While research interest into chitosan nanoparticle based deliv-ery systems is increasing, the current level of knowledge does notallow a fair assessment of the pros and cons that will arise fromthe use of chitosan based nano-pesticides in agriculture. As a pre-requisite for such assessment, a better understanding of the fateand effect of such products after their application is required. Thesuitability of current regulations should also be analyzed so thatrefinements can be implemented, if needed. Another major hurdlein sustainable agriculture is the removal of harmful contaminantsfrom soil. This is another area where it is believed that uniqueproperties of chitosan nanoparticles may prove to be useful, in envi-ronmental detection, sensing and remediation systems. Overall,it can be concluded that chitosan nanoparticles based technologyhave a promising future with value in crop productivity in sustainedand eco-friendly ways.

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