Carbohydrate Polymers 165 (2017) 394–401

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Zinc complexed chitosan/TPP nanoparticles: A promising

micronutrient nanocarrier suited for foliar application
Paresh Deshpande a , Ashwin Dapkekar a , Manoj D. Oak b , Kishore M. Paknikar a,∗ ,
Jyutika M. Rajwade a,∗
a
Nanobioscience Group, Agharkar Research Institute, G. G. Agarkar Road, Pune 411 004, India
b
Genetics and Plant Breeding, Agharkar Research Institute, G. G. Agarkar Road, Pune 411 004, India

a r t i c l e i n f o a b s t r a c t

Article history: Cultivation of cereals in zinc deficient soils leads to declined nutritional quality of grain. Zinc deficiency
Received 26 August 2016 in humans is a consequence of consumption of micronutrient deficient cereals as staple food. To achieve
Received in revised form an increase in zinc density in grain, we evaluated zinc complexed chitosan nanoparticles (Zn-CNP) as a
16 December 2016
potential ‘nanocarrier’ suited for foliar fertilization. Zn-CNP were synthesized using tri-polyphosphate
Accepted 16 February 2017
as a cross-linker. Spherical Zn-CNP (diameter 250–300 nm) were positively charged (zeta potential,
Available online 20 February 2017
+42.34 mV) and contained ∼20 mg Zn/g (w/w). Plant growth in zinc deficient sand media, followed by
foliar application of Zn-CNP (twice-a-week, for 5 weeks) after anthesis resulted in 27 and 42% increase in
Keywords:
Zinc (Zn)
grain zinc content of MACS 3125 and UC1114 (durum wheat cultivars) respectively. Translocation of zinc
Agronomic biofortification ions from foliar applied Zn-CNP into the leaf and seed tissue was demonstrated using zinquin and dithi-
Wheat zone stains, respectively. The study indicates the suitability of chitosan-based nanocarriers in agronomic
Chitosan nanocarriers biofortification.
© 2017 Elsevier Ltd. All rights reserved.

1. Introduction
Today, crop production has achieved “yield sufficiency” in most
of the parts of the world but cereals and grains are deficient in
micronutrients. Micronutrient deficiency is referred to as “hidden
hunger” and is mainly with respect to iron and zinc (Kennedy,
Nantel, & Shetty, 2003). In India, zinc deficiency in agricultural soils
is widespread (∼50%) and is likely to increase to ∼63% by 2025. The
majority of cereals such as wheat and rice are cultivated in zinc-
deficient soils, which ultimately leads to a significant decrease in
grain zinc content. Zinc is an essential micronutrient for both plants
and humans. Zinc acts as a co-factor for an array of enzymes that are
involved in DNA replication, protein and lipid metabolism (Maret,
2013). Zinc plays a major role in growth, development, immunity
and reproductive health. Stunted growth, enhanced susceptibility
to infectious diseases, skin lesions, and diarrhoea (Prasad, 2013)
are reported to be a result of deficiency of this vital micronutrient.
Presently, 26% of the Indian population is at risk of zinc deficiency,
∗ Corresponding authors.
E-mail addresses: kmpaknikar@aripune.org (K.M. Paknikar),
jrajwade@rediffmail.com, jrajwade@aripune.org (J.M. Rajwade).
and it is estimated that a small effort towards enriching cereals with
zinc could save the lives of up to 48,000 children in India annually
(www.zinc.org.in).
Micronutrient deficiency in cereals can be overcome by ‘genetic’
and ‘agronomic’ biofortification (Clemens, 2014). Genetic biofor-
tification includes the use of inherent genetic variations in crop
species along with conventional breeding practices for achieving
desirable traits in the cultivated genotypes. Development of new
genotypes with higher mineral densities has been suggested for
zinc enrichment in wheat (Cakmak, 2008). Genetic biofortification
is time-intensive and requires extensive experimentation before
the mineral-enriched varieties can be cultivated by the farmer. Con-
sidering these aspects, agronomic biofortification is increasingly
becoming popular. Agronomic biofortification can be achieved by
micronutrient application via seed priming, soil and/or foliar fer-
tilization. Fertilization of soils is a simple and efficient strategy
suggested for achieving micronutrient biofortification. However, it
must be borne in mind that soil properties such as high pH, salinity,
and calcareous nature lead to complexation of micronutrients mak-
ing them unavailable to the plant (Alloway, 2009). Enhanced zinc
in wheat has been reported earlier (Zhao, Tian, Cao, Lu, & Lio, 2014)
after foliar application of zinc-containing salts and chelates (e.g.
ZnSO4 , Zn-EDTA, typically @ 0.3–0.5 kg/ha). The application of the

http://dx.doi.org/10.1016/j.carbpol.2017.02.061
0144-8617/© 2017 Elsevier Ltd. All rights reserved.
 P. Deshpande et al. / Carbohydrate Polymers 165 (2017) 394–401
excessive amount of salts (as source of micronutrients) may lead
to environmental hazards such as leaching of micronutrients and
runoff due to rain resulting in contamination of local ground water
bodies. Furthermore, much of the applied fertilizer is wasted since
only a small portion of applied micronutrient is taken up by the
plant. Foliar application may also cause scorching of leaves. There-
fore, ensuring availability of zinc to the plants, avoiding excessive
usage of micronutrients and consequent environmental hazards
remains a challenge.
In the recent years, the potential applications of nanotech-
nology in agriculture are being put forth. It is speculated that
nanoparticles containing micronutrients can be used very effec-
tively to correct micronutrient deficiencies in cereals. (Ghormade,
Deshpande, & Paknikar, 2011; Kashyap, Xiang, & Heiden, 2015).
Nanotechnology could help achieve the desired effects at very
low doses, thus overcoming the adverse effects associated with
excessive fertilizer application. Besides this, the ‘controlled release’
property of nanoparticles can give an edge over conventional appli-
cation methods by providing the ‘right dose’ at the ‘right time’
(Kumar, Bhanjana, Sharma, Sidhu, & Dilbaghi, 2014). A variety of
biopolymers such as, chitosan and alginate which were developed
earlier for medical and pharmacological applications are now being
studied for the delivery of agrochemicals (Wang, Lombi, Zhao, &
Kopittke, 2016). The structure of chitosan is well suited for metal
ion binding via ion exchange, complexation, physical sorption and
by inter- and intra-molecular entrapment (Qi, Xu, Jiang, Hu, & Zou,
2004). Chitosan shows surface adherence (Zeng & Luo, 2012) and
exhibits antibacterial properties. In addition, thiolated derivatives
of chitosan showed enhanced antibacterial activity than pristine
chitosan (Croce, Conti, Maake, & Patzke, 2016). Similary, loading of
metal ions on chitosan nanoparticles lead to increased antibacte-
rial action (Du, Niu, Xu, Xu, & Fan, 2009). Chitosan, in particular,
has been studied as an immune elicitor in plants (Chandra et al.,
2015). However, the use of chitosan nanoparticles as ‘nanocarrier’
for delivery of micronutrients in cereals has not been reported so
far.
Over this background, it was hypothesized that zinc complexed
chitosan can be used to synthesize nanoparticles [Zn-complexed
chitosan/TPP nanoparticles, Zn-CNP] suited for foliar delivery of
zinc thereby achieving agronomic biofortification. The potential of
Zn-CNP as a ‘nanocarrier’ for micronutrient delivery was assessed
under zinc deficient conditions.
2. Materials and methods
Chitosan (poly [ˇ-2-amino-2- deoxy-(1-4)-d glucopyranose])
with a molecular weight of 60,000 Da and a deacetylation degree
of 85% was obtained from India Sea Foods, Cochin, Kerala. Chi-
tosan flakes were dissolved in acetic acid and re-precipitated with
sodium hydroxide (NaOH). Zinc sulfate (ZnSO4. 7H2 O), sodium tri-
polyphosphate (TPP) and acetic acid were purchased from SD Fine
Chemicals, Mumbai, India; dithizone, zinquin were procured from
Sigma, USA.
Durum wheat varieties MACS 3125 (a high yielding variety
developed by our institute) and UC 1114 (a kind gift from Prof.
Jorge Dubcovsky, UC Davis, CA, USA. The variety contains HGPC-B1
gene responsible for high protein content) were used in the study.
2.1. Synthesis of zinc complexed chitosan nanoparticles (Zn-CNP)
Chitosan nanoparticles were synthesized by ionotropic gelation
process (Calvo, Remunan-Lopez, Vila-Jato, & Alonso, 1997). Briefly,
chitosan (0.3 g) was dissolved in 1% v/v acetic acid. The solution
was stirred continuously on magnetic stirrer at ambient tempera-
ture (25 ± 3 ◦ C) and 1 mL of sodium tripolyphosphate (1% TPP, v/v)
395
was added dropwise to 25 mL chitosan solution to form chitosan
nanoparticles. For hardening, stirring was further continued for
20 min. These particles have been referred to as CNP. To prepare
Zn-CNP, zinc sulfate was dissolved in chitosan solution and TPP
was added as described above. The effect of different concentra-
tions of chitosan, TPP, and ZnSO4 on complexation efficiency and
nanoparticles size was studied by varying the concentration of each
reactant while the other two were kept constant. Further, the effect
of pH on zinc content was evaluated, in which pH of chitosan dur-
ing zinc addition was adjusted using 2N NaOH. The zinc content
in Zn-CNP was determined indirectly. Zn-CNP were separated by
centrifugation at 14,000 rpm for 20 min and zinc content in the
supernatant was estimated using an atomic absorption spectropho-
tometer (AAS, AAnalyst 400 Perkin Elmer, USA). The complexation
efficiency
(CE%) was calculated by the following formula
Total zinc added − unbound zinc in supernatant
CE (%) = × 100
Total zinc added
2.2. Physicochemical characterization of Zn-CNP
The analysis of the size (Z-average mean) and zeta potential
of the nanoparticles was performed at a scattering angle of 135◦
at ambient temperature (25 ± 3 ◦ C) using a nanoparticles analyzer
(DelsaNanoTM, Beckman Coulter, USA). For zeta potential mea-
surements, samples were dispersed in quartz-distilled water. For
morphological analysis, nanoparticles suspensions were deposited
on vitreous carbon stubs and dried at ambient temperature. Sam-
ples were coated with gold and observed under scanning electron
microscope (EVO MA15, Zeiss, Germany) operated at 20 kV.
For FT-IR analysis samples (7.0–9.0 mg) were freeze-dried,
mixed with KBr to form a pellet and spectra were recorded in range
400–4000 cm−1 (resolution: 4 cm−1 , 16 scans) using an FT-IR spec-
trometer (Spectrum 2000, Perkin Elmer, USA). For determining the
crystallinity, the samples were freeze dried and ground to form a
powder. X-ray diffraction patterns were recorded using desktop X-
ray diffractometer (Miniflex II, Rigaku, Japan) with Cu K␣ radiation
of 1.54 Å, 40 kV, and current of 40 mA. Samples were scanned from
5◦ to 80◦ at a scan speed of 0.5◦ /min.
2.3. Effect of foliar applied Zn-CNP on zinc content in leaf tissue
and wheat grain
Coarse sand was used instead of soil for the cultivation of wheat.
Sand was pre-treated by soaking in acid (5 N HCl) for 18 h and was
thoroughly washed with distilled water until pH was neutral. PVC
columns (95 cm long with inner diameter 10 cm, bottom supported
by a perforated plastic plate) were filled with dry sand and the
sand column was moistened with distilled water. Wheat, Triticum
durum seeds (two varieties, MACS 3125 and UC1114) were surface
sterilized using 0.5% sodium hypochlorite (NaOCl) for 3 min, rinsed
thoroughly with distilled water. Seeds were placed in a petri dish
in dark for germination. After 48 h, 7–8 germinated seedlings were
transferred to PVC columns filled with sand.
The sand was irrigated daily using distilled water and every
alternate day with 250 mL zinc free Hoagland nutrient solution
(Hoagland & Arnon, 1950) until plant maturity. At flowering stage
(i.e. anthesis, ∼60 days after germination); a suspension of freshly
prepared Zn-CNP (∼25 mL, zinc content ∼20 mg/g) was applied to
the leaves as a foliar spray. The foliar application was done twice a
week and continued for 5-weeks.
During growth of the plants following analyses were performed
which include
(i) Localization of zinc in leaf tissue, (ii) assessment of grain zinc
content and (iii) localization of zinc in grain
396 P. Deshpande et al. / Carbohydrate Polymers 165 (2017) 394–401
To study the uptake of zinc in leaf tissue, control and treated
leaves were collected 2 h after application of CNP and Zn-CNP,
respectively. Leaf surface was washed carefully with deionized
water and transverse sections of leaves were obtained using a sharp
blade. Sections were treated with 25 ␮M zinquin solution prepared
in phosphate buffered saline (PBS, pH 7.4) and incubated in dark for
45 min, washed with PBS and mounted on a glass slide. For imaging,
sections were excited at 405 nm and images were obtained using
an upright fluorescence microscope (magnification 200×, EVOS FL,
Thermo Fisher Scientific, USA) with green filter.
At maturity of the plants, spikes and flag leaves were collected
from plants grown in sand column. Grains from each plant were
harvested and stored separately, while the leaf surface was care-
fully washed using deionized water and the leaves were dried. For
determining the zinc content, a representative aliquot of grains
(0.1 g) was digested in three acid mixture (Nitric acid: Perchloric
acid: Sulfuric acid, 3:2:1) over a hot plate at 250 ◦ C until the sam-
ple was clear. After filtration and suitable dilution using 5% HNO3 ,
grain zinc content was estimated by atomic absorption spectrome-
try. Similar protocol was followed for estimation of leaf Zn content.
Plants of both varieties, which received a foliar application of CNP
served as control. All analyses were performed in triplicates.
The localization of Zn within the seeds was studied by dithi-
zone (DTZ, diphenyl thiocarbazone) staining method described by
Ozturk et al. (2006). Briefly, dry seed were excised longitudinally
and the resulting halves (inner seed portion facing upwards) were
fixed on a glass slide using cyanoacrylate adhesive resin. Fixed
seeds were polished using 300- grit sandpaper followed by a 600-
grit sandpaper to create a uniform topology for visualization post
DTZ staining. After polishing, the seed sections were rinsed with
water and stained with freshly prepared dithizone stain (500 mg/L
w/v in methanol) for 30 min. Finally, sections were rinsed thor-
oughly in water, gently dried with tissue paper and observed under
a reflected light microscope equipped with a high-resolution digital
camera. Grain Zn localization in different parts of grain viz. embryo,
aleurone and endosperm was measured separately using imageJ
software tool (Duarte et al., 2016).
Fig. 1. Characterization of Zn-loaded chitosan nanoparticles. Size distribution of nanoparticles a: CNP, c: Zn-CNP; SEM image of nanoparticles showing spherical morphology,
b: CNP and d: Zn-CNP (scale bar 10 ␮m).
3. Results and discussion
3.1. Synthesis and characterization of zinc loaded chitosan
nanoparticles
Chitosan nanoparticles were formed instantaneously because
of the interaction between the negatively charged groups of TPP
and the positively charged amino groups of chitosan as reported
earlier (Calvo et al., 1997). Mixing of ZnSO4 , in chitosan before addi-
tion of TPP, did not slow down the formation of nanoparticles. The
average hydrodynamic size of CNP and Zn-CNP was 290 ± 35 nm
and 325 ± 40 nm (Fig. 1a, c). The particles exhibited a polydisper-
sity index of 0.19 and 0.28, respectively. The surface charge of CNP
and Zn-CNP was +32.2 mV and +42 mV, respectively.
Scanning electron microscopy revealed that (Fig. 1b, d) both
CNP and Zn-CNP were spherical in shape with average diameter
of 200 nm. During the process of Zn-CNP formation, the structure
of chitosan is altered due to the interaction with zinc. Addition of
TPP further leads to ionic gelation and formation of nanoparticles.
TPP probably induces the formation of secondary structures in the
native chitosan polymer. The particles size data show that there
are no major differences in the particles size of CNP and Zn-CNP.
The small difference in surface charges (c.a. + 10 mV) observed in
both types of nanoparticles indicate that zinc is encapsulated into
the nanoparticles and only very few zinc moieties remain exposed
on the surface of nanoparticles. On basis of the surface charge, the
Zn-CNP nanoparticles show increased stability.
Fig. 2 depicts the FTIR spectra of chitosan, CNP and Zn-CNP. The
spectrum of chitosan showed a band at 3405 cm−1 that could be
attributed to stretching vibrations typical of the NH2 and OH
groups. The small peak at 1660 cm−1 could be assigned to CONH2
group of chitosan while, a band at 1060 cm−1 could be assigned
to the second OH group. On this background, the FTIR spectra
of CNP and Zn-CNP showed new peaks which could be attributed
to chitosan- TPP-Zn interaction. FTIR spectrum of Zn-CNP showed
a shift in peak from 3375 cm−1 to 3437 cm−1 which corresponds
to the combined peaks of the NH2 and OH group stretching
vibrations in chitosan. The peak at 1594 cm−1 in chitosan (which
 P. Deshpande et al. / Carbohydrate Polymers 165 (2017) 394–401
Fig. 2. FT-IR spectra of chitosan, CNP and Zn-CNP.
corresponds to CONH2 group) appeared to shift to 1560 cm−1 and
1554 cm−1 in CNP and Zn-CNP, respectively indicating the interac-
tion between the amino group of chitosan, metal ion (zinc) and
TPP. The peaks obtained were in agreement with an earlier study
(Dudhani and Kosaraju, 2010). The band at 1060 cm−1 in chitosan
showed a significant shift to higher wavenumber from 1072 cm−1
to 1084 cm−1 in CNP and Zn-CNP, respectively which suggested that
the second OH group, was involved in complexation. These results
corroborate the findings of Wang, Du, and Liu (2004). Thus, it is
postulated that enhancement in inter- and intra- molecular inter-
actions in CNP and Zn-CNP occurred due to the ionic interaction
between negatively charged polyphosphoric group and positively
charged ammonium groups of chitosan. The bending vibration
in C H group of acetyl at 1360 cm−1 in CNP showed a shift to
1381 cm−1 in Zn-CNP. The change in vibration frequencies in this
particular region could be specifically attributed to the bonding of
zinc with OH and NH groups present in chitosan.
The chitosan-Zn binding in Zn-CNP can be explained either by
pendant or bridge formation. In pendant formation, the metal ions
bind to the amino group of chitosan whereas during bridge forma-
tion, metal ions bind to two or more amino groups and hydroxyl
groups of one or more chitosan chains forming a bridge (Wang
et al., 2004). In the case of Zn-CNP, the amino group of chitosan is
involved in binding of zinc ions as well as ionic cross-linking with
TPP molecules.
Powder X-ray diffraction patterns of chitosan, CNP and Zn-CNP
are depicted in Fig. 3. Pure chitosan showed two main characteristic
diffraction peaks at 2␪ = 9.5◦ and 20.6◦ which correspond to 020 and
110 crystal lattices. The peaks obtained were in agreement with
an earlier report (Ioelovich, 2014). In the case of CNP and Zn-CNP,
diffraction peaks were obtained at 22◦ and 21.5◦ , respectively. Peak
broadening in CNP and Zn-CNP indicated the amorphous nature of
the nanoparticles formed. During interaction of chitosan with tri-
polyphosphate the compact inter- and intra-chain bonding of the
molecule is lost, which manifests as a decrease in crystallinity. The
loss of crystallinity of chitosan nanoparticles permits interaction
of metal ions (Qi et al., 2004; Qi & Xu, 2004). Our findings on the
decrease in crystallinity are in agreement with the earlier studies
(Govindan, Nivethaa, Saravanan, Narayanan, & Stephen, 2012).
Effects of initial concentration of chitosan, TTP, and ZnSO4
on zinc complexation and size of nanoparticles are presented
in Fig. 4(a–c). The particle size increased linearly from 103 to
465 nm as a function of chitosan concentration in the range of
0.1–0.4 g% (R2 = 0.98). Similarly, zinc loading increased from 17
to 24% indicating a positive corelation with chitosan concentra-
397
Fig. 3. XRD pattern of chitosan, CNP and Zn-CNP.
tion (R2 = 0.77). Similarly, the increase in TPP concentration from
0.5 to 2 g% resulted in increased size of the nanoparticles as well
as zinc complexation efficiency (CE) with correlation values of
0.68 and 0.79, respectively (Fig. 4b). When the ZnSO4 concentra-
tion was increased from 0.5 to 2 mg/mL, it was observed that size
of nanoparticles changed from 300 nm to 550 nm. Furthermore,
ZnSO4 concentration and size of nanoparticles were positively cor-
related (R2 = 0.97). The complexation efficiency increased up to a
concentration of 2 mg/mL ZnSO4 (41%) and beyond 2 mg/mL ZnSO4 ,
CE was significantly reduced (Fig. 4c). In the presence of >2 mg/mL
ZnSO4 , the charges on Zn-chitosan complexes are not balanced by
the negatively charged TPP, leading to the formation of larger par-
ticles. In this experiment, the zinc complexation efficiency showed
high negative correlation (R2 = 0.04). Further, complexation effi-
ciency is reduced probably due to the displacement of zinc ions
from the chitosan polymer chains by the TPP molecules. Similar
results were reported (Luo, Zhang, Cheng, & Wang 2010), where the
increase in selenite concentration was negatively correlated with
chitosan-selenite binding. A recent study revealed the importance
of process parameters such as rate of stirring, rate of TPP addition,
temperature crosslinking, needle diameter etc. in the synthesis of
colloidal chitosan-TPP nanoparticles (Rázga, Vnukova, Némethova,
Mazancova, & Lacík, 2016).
It is reported that smaller nanoparticles have a high uptake
through stomata and can be used for achieving maximum deliv-
ery of the active ingredient to the plant (Fernández & Eichert,
2009). Therefore, selection of the appropriate size of nanoparti-
cles with an effective dose of active ingredient is a crucial factor for
nanocarrier-mediated agrochemical delivery. In the present study,
lower concentration of chitosan (0.1 g%) resulted in particles that
were small (103 nm size) but this was accompanied by reduced
Zn complexation. An increase in the chitosan concentration led
to improvement in zinc complexation but resulted in larger par-
ticles (size, 465 nm). Therefore, the concentration of chitosan was
selected on the basis of zinc complexation efficiency. Based on the
above data, 0.3 g% chitosan, 0.1 g% ZnSO4 and 1 g% TPP were selected
as optimal concentrations that produced nanoparticles with a size
∼325 nm, containing ∼20 mg/g zinc which were used in subsequent
studies.
When the effect of pH on zinc complexation was investigated
(Fig. 4d), it was observed that zinc complexation with chitosan was
influenced by the initial pH of chitosan solution. The increase in
pH from 4.5 to 6.5 had a significant effect on zinc complexation.
At pH 4.5 the zinc content was 20.6 mg/g which rose to 32.6 mg/g
398 P. Deshpande et al. / Carbohydrate Polymers 165 (2017) 394–401
Fig. 4. Effect of initial concentration of a: chitosan, b: TTP and c: ZnSO4 on nanoparticles size and Zn complexation efficiency, d: Effect of pH on Zn content of Zn-CNP (Error
bar shows mean ± SD, n = 3).
at pH 5.5. However, at pH ≥ 6.0, a decrease in zinc content was
observed (21.2 mg/g). It may also be noted that high pH affected the
nanoparticles formation and agglomerated particles were formed.
The above observation could be explained based on de-protonation
of NH2 group and precipitation of chitosan solution as well as
decreased solubility of ZnSO4 at higher pH.
3.2. Assessment of Zn-CNP as a micronutrient nanocarrier
3.2.1. Translocation of foliar applied Zn-CNP in leaf tissue
After foliar application of Zn-CNP on the leaf, the localiza-
tion pattern of zinc was observed microscopically. For this, the
transverse sections (T.S.) of leaf were stained with a fluorophore
viz., zinquin. Zinquin is known to bind Zn2+ and exhibits green
fluorescence under UV excitation. A low-level zinc-dependent flu-
orescence was detected in the T.S. of control plants, specifically
in the lower epidermal region. With Zn-CNP treatment, the fluo-
rescence was mainly localized in the upper and lower epidermal
layers and vascular bundles (Fig. 5a). The increased fluorescence
could be attributed to the close contact of epidermal cells with
outermost cuticular layer which allows the diffusion of ionic zinc
through aqueous pores. As compared to epidermal layers, lower
fluorescence intensity was observed in regions containing vascular
bundles. Overall, fluorescence intensity of Zn-CNP treated leaves
was higher than control samples. Further, fluorescence intensity
was quantified using ImageJ software (Fig. 5b) where, a signifi-
cant increase (P < 0.001) in fluorescence intensity was observed in
treated samples.
Results obtained in the present study were in agreement with a
recent report (Feigl et al., 2015), which demonstrates the accumu-
lation of zinc in different plant parts. The latter was proved using
zinquin dye and the maximum fluorescence intensity was recorded
in meristem and roots of Brassica. It is reported that, once the zinc
is taken by epidermal cells its transport to vascular bundle and fur-
ther transport to sink organs such as grain occurs via the phloem
(Eichert and Goldbach, 2008). The release of active ingredient from
chitosan is based on three mechanisms viz., diffusion, osmotically
driven burst mechanism and erosion or degradation of the poly-
mer (Kashyap et al., 2015). In case of Zn-CNP the release of zinc
from chitosan nanoparticles is mainly due to uptake of nanoparti-
cles through stomata followed by diffusion release involving series
of changes in polymer matrix. Polymer swells upon penetration of
water followed by conversion of polymer to swollen matrix and
diffusion of zinc from the swollen matrix. It is proposed that intra-
cellular spaces inside the cell are slightly acidic in nature which
further helps in release of Zn from the chitosan nanoparticles.
In addition, Zn content of dried flag leaves were measured using
atomic absorption spectrometry. Leaf Zn content was increased in
both (MACS 3125 and UC1114) cultivars as shown in Fig. 5c. Zn
content of Zn-CNP treated samples was found to be 31 ␮g/g and
28 ␮g/g, in MACS 3125 and UC1114 cultivar, respectively while in
untreated control Zn content was 20.2 ␮g/g and 21 ␮g/g, respec-
tively. The increased Zn content in leaf tissue further confirms the
observed significant Zn dependent fluorescence in treated samples.
On the basis of these results, we propose that uptake of zinc from
Zn-CNP takes place via a well-reported stomatal uptake pathway.
To further understand the uptake pathway of Zn-CNP via stomata
a scannig electron microscopy and fluorescence microscopy was
performed. It was observed that post Zn-CNP application to leaves
the particles localize near the stomata (Supplementary Fig. S1a).
The stomatal localization of Zn-CNP was further confirmed using
fluorescence microscopy (Supplementary Fig. S1b). Additionally,
nanoparticles internalization inside the leaves was studied using
confocal laser scanning microscopy (Supplementary Fig. S1c). Con-
focal microscopy revealed the internalization of nanoparticles as
seen by intense green signal of FITC-tagged Zn-CNP.
3.2.2. Agronomic biofortification
The Zn-CNP were applied as a foliar spray post anthesis, and the
growth of plants was monitored up to maturity. It was observed
that zinc content of Zn-CNP treated UC1114 variety was found to
be 27 ␮g/g in comparison with control (19 ␮g/g). Similarly, zinc
content of MACS-3125 variety treated with Zn-CNP was 21 ␮g/g
as compared to control (15 ␮g/g) (Fig. 6). The increase in grain
zinc content was 27% and ∼42% in MACS 3125 and UC1114 vari-
eties respectively, which was statistically significant (P < 0.05). Our
results are comparable to earlier reports explaining the effect of
foliar zinc application on grain zinc enhancement in plants that are
cultivated in zinc-deficient soils (Zhao et al., 2014). In the present
study, carried out in zinc deficient sand, grain zinc enrichment upon
foliar application of Zn-CNP is demonstrated. We also observed the
differences in the zinc content between two durum wheat geno-
 P. Deshpande et al. / Carbohydrate Polymers 165 (2017) 394–401 399

Fig. 5. a: Fluorescence microscopy representative images for Zn localization in leaves using Zinquin dye (scale bar 400 ␮m) b: Fluorescence intensity of Zinquin stain (Error
bar shows mean ± SD, n = 8), ***P < 0.001 (One-way ANOVA, Tuckey’s multiple comparison test), c: leaf Zn content (Error bar shows mean ± SD, n = 3).

types tested. The higher zinc content in UC1114 could be attributed
to the quantitative trait loci (QTL) viz., GPC-B1 responsible for grain
protein and micronutrient enhancement (Kade, Barneix, Olmos, &
Dubcovsky, 2005). For effective grain zinc enhancement, the tim-
ing of foliar application is very crucial. Earlier studies have shown
that zinc application during senescence stages leads to maximum
nutrient transport from mother plant to developing seed (Cakmak,
Pfeiffer, & McClafferty, 2010). In the present study, Zn-CNPs were
applied at post anthesis stages (during grain development), dur-
ing which zinc enrichment was realized. Further, in the present
case, it is noteworthy that despite lower zinc content of Zn-CNP
as compared to conventional micronutrient spray fertilizers, zinc
enrichment of grain was observed.
Use of sand columns instead of soil or hydroponic system to
assess the response of the developed nanocarrier offered sev-
eral advantages. Sand is an inert solid substitute for soil where
controlled nutrient supply can be ensured. Further, it provides
a substratum for penetration of roots thereby ensuring that the
growth of the plant can be followed up to maturity. In addition, it is
known that the effectiveness of a foliar formulation can be masked
by soil environment because the soil itself can act as a source of
‘test’ material. Cultivation of plants in sand media can thus help in
400 P. Deshpande et al. / Carbohydrate Polymers 165 (2017) 394–401
Fig. 6. Grain Zn content in MACS 3125 and UC1114 cultivar (Error bars represents
mean ± SD of 3 independent replicates) *: P < 0.05 (One-way ANOVA, Tuckey’s mul-
tiple comparison test).
overcoming such ‘soil effect’. Although, use of hydroponic systems
to monitor the response of nutrient on plant growth is well known
but, it is mainly employed to study nutrient uptake through roots
and not via leaves. Also, hydroponically grown plants require fre-
quent replenishment of nutrients, and it does not mimic the ideal
field condition for plant growth (Clark, 1982). The sand columns
used in present study thus mimics the zinc deficient environment
and tests the true potential of developed nanocarrier. The results
indicate that Zn-CNP can be used as a micronutrient nanocarrier
to overcome the zinc deficiency in wheat cultivated in areas that
contain calcareous soils with alkaline pH.
3.2.3. Localization of zinc in grains
After establishing the biofortification due to application of Zn-
CNP, it was necessary to study localization of zinc in the grain.
The wheat grain consists of three major parts viz., aleurone layer,
embryo, and endosperm. It is known that during the process of
milling when flour is obtained, most of the aleurone and embryo is
removed. Hence, it is important to achieve maximum zinc loading
in the endosperm region. The DTZ dye was used because it forms
a pink/red colored complex upon reaction with zinc. The colored
complex aids visualization of zinc in grains (Duarte et al., 2016).
The pink color complex was intense in grain samples treated with
Zn-CNP as compared to control samples as shown in Fig. 7a and
b. Embryo and some part of endosperm stained intense pink color
Fig. 7. Grain Zn localization by DTZ staining method a: No treatment (Control), b: Zn-CNP treated grain of MACS 3125 cultivar c: Different parts of grain aleurone, embryo
and endosperm of UC1114 cultivar. d: DTZ Intensity measurement using imageJ software (Error bar shows mean ± SD, n = 8).
in treated samples than control indicating the major Zn enriched
parts of the grain.
We observed variations in the intensity of pink color in different
parts of the grain and therefore the color was quantified using the
imageJ software (NIH, USA). The intensity of staining in embryo,
aleurone, and endosperm regions (Fig. 7c) was measured sepa-
rately through the RGB color space (Red, Green, and Blue). From
Fig. 7d it can be clearly seen that in treated samples, embryo region
of grain had highest zinc content followed by aleurone layer and
endosperm. It is known that the embryo and aleurone layer are rich
in protein and phytate (phosphate reserve) and high concentration
of these components act as a sink for zinc. The endosperm is poor
in phytate and protein and therefore low amount of zinc (Ozturk
et al., 2006) is found in this region. The use of DTZ is advantageous
over the X-ray-based imaging techniques such as ␮-XRF (micro-X-
ray fluorescence) with reference to ease of sample preparation and
observation.
The data indicate that the nanocarrier-mediated delivery of
micronutrient was indeed successful, leading to enrichment of zinc
in grain. Use of such nanocarriers can be a promising short-term
approach for achieving agronomic biofortification.
4. Conclusion
The present study demonstrates that Zn-complexed chitosan
nanoparticles (Zn-CNP) can act as a ‘micronutrient nanocarrier’
suited for foliar application in wheat. The nanoparticles can cer-
tainly be employed for the controlled delivery of agrochemicals
because they are effective at low concentrations. Zn-CNP not only
releases the active ingredient in slow fashion but also prevents
the nutrient loss and possible environmental pollution. The results
thus have the potential to be translated as a successful commercial
technology after suitable field trials.
Conflict of interest
Authors declare no conflict of interest.
Acknowledgments
Authors gratefully thank the Council of Scientific and Industrial
Research (CSIR), University Grant Commission (UGC) and National
 P. Deshpande et al. / Carbohydrate Polymers 165 (2017) 394–401 401

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