Development and evaluation of alginate–chitosan nanocapsules for controlled release of acetamiprid

International Journal of Biological Macromolecules 81 (2015) 631–637

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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Development and evaluation of alginate–chitosan nanocapsules for controlled release of acetamiprid Sandeep Kumar a,∗ , Neetu Chauhan a , Madhuban Gopal b , Rajesh Kumar b , Neeraj Dilbaghi a a b

Department of Bio and Nano Technology, Guru Jambheshwar University of Science & Technology, Hisar, Haryana 125 001, India Division of Agricultural Chemicals, Indian Agricultural Research Institute (IARI) , New Delhi 110 012, India

a r t i c l e

i n f o

Article history: Received 17 May 2015 Received in revised form 23 August 2015 Accepted 25 August 2015 Available online 28 August 2015 Keywords: Nanocapsules Controlled release Chitosan Polyelectrolyte complexation Acetamiprid Pesticide

a b s t r a c t Smart formulations based on nanomaterials have the capability to reduce the consumption of hazardous pesticides and their impact on human health and environment. Nanoformulations of agrochemicals have the potential to improve food productivity without compromising with the ecosystem. In the present work, controlled release nanocapsules containing acetamiprid were prepared by polyelectrolyte complexation of two natural macromolecules, i.e. alginate and chitosan. The size, morphology and chemical interaction studies of the prepared nanocapsules were investigated by Dynamic Light Scattering (DLS), Transmission Electron Microscopy (TEM), and Fourier Transform Infrared Spectroscopy (FTIR). The zetapotential studies revealed stability of the nanocapsules. TEM results show spherical morphology of the nanocapsules. The encapsulation efficiency was found to be 62% as quantified by Ultra High Pressure Liquid Chromatography (UHPLC). Nanocapsules were analysed for controlled release in vitro at three different pH. Maximum release was observed at pH 10 followed by pH 7 and 4, respectively. A non-Fickian release mechanism was found to be followed by the nanoformulation. A controlled release pattern was also found from nanoformulation as compared to commercial formulation in soil. Thus this formulation can reduce the frequency of application of pesticides by controlling the release and will subsequently reduce their side effects. © 2015 Elsevier B.V. All rights reserved.

1. Introduction The production of quality food to feed growing population is the major challenge faced by agricultural scientists. Excessive and repetitive use of pesticides is a matter of major environmental concern as numbers of pesticides are identified as noxious or carcinogenic and can adversely affect human health and ecosystem. Ineluctable use of pesticides in modern agricultural practices has motivated researchers worldwide to focus attention in developing smarter formulations that can minimize use of such hazardous agrochemicals. The utilization of nanoplatforms in diagnostics and medicine under in-vitro conditions has generated interest in agrinanotechnology for site specific and controlled release of various macromolecules enabling efficient use and safer handling of these agrochemicals. Controlled release formulations (CRFs) are emerging continuously to combat the issues associated with pesticides. Controlled release formulations of pesticides are emerging continuously to decrease the consumption and related side effects

∗ Corresponding author. E-mail address: [email protected] (S. Kumar). http://dx.doi.org/10.1016/j.ijbiomac.2015.08.062 0141-8130/© 2015 Elsevier B.V. All rights reserved.

of pesticides. Controlled release formulations are reported to be superior to conventional formulations as they extend activity of pesticide [1], prevent pollution by reducing leaching [2] and volatilization [3], minimize residues on food stuffs, reduce health problems by decreasing dermal and inhalation toxicity [4,5], minimize cost involved with the production of pesticide in bulk and lastly but not the least, CRFs also reduce the harm caused to human health directly involved in handling of pesticides or indirectly through environment. Thus, the challenge of agricultural sustainability and food security can be addressed by encapsulating active ingredients such as herbicides, fungicides, fertilizers, in controlled release matrices, for decreasing toxicity and providing ecoprotection. Nanotechnology based controlled release formulations are finding application in drug delivery [6,7] for improving healthcare and has been investigated extensively by researchers across the globe. A carrier must possess high drug loading and should prevent any premature release before reaching the intended site. The combination of nanotechnology and polymer science has resulted in the development of several novel formulations of existing compounds with improved characteristics. Polymeric nanocapsules have been frequently used as drug-delivery systems [8,9] but only a few reports

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are available on pesticide or herbicide release [10–12]. Nanocapsules are solid hollow particles which are used actively in controlled release formulation as these use little amount of pesticide compared to conventional formulation. Nanocapsules have small size and high surface area which helps them deposit on the plant leaves and thus reduces pesticide waste [10]. In the present study, acetamiprid was chosen as the active ingredient for encapsulation in nanocapsules. Acetamiprid, (E)-N-1-[(6chloro-3-pyridyl)methyl]-N-2-cyano-N1-methylacetamidine is a neonicotinoid insecticide, used on vegetables, fruits and tea crops for control of insects of Hemiptera, mainly aphids, Thyasnoptera and Lepidoptera sps, [13]. The major problem associated with acetamiprid is its high water solubility that allows it to enter the natural water resources and poses threat to environment. Thus its great demand for a variety of crops and environmental concern prompted us to encapsulate it in nanocapsules which can provide a physical barrier as well as control its release. A variety of natural polymers such as sodium alginate, chitosan, gelatin, albumin, etc., are used to develop nanocapsules as these are non-toxic, biodegradable and inexpensive. The alginate–chitosan system has been studied widely for drug delivery [14–24]. Alginate is an anionic biopolymer of ␣-l-guluronic acid and ␤D-mannuronic acid units linked together by 1,4-glycosidic bonds [25]. Nanocapsules formed by an alginate polymer are found to have low stability that results in loss of the encapsulated materials. Cationic polymers such as chitosan have been employed with alginate for overcoming limitations associated with swift release of encapsulated material [26–28]. Chitosan, a naturally occurring linear polysaccharide consisting of copolymers of d-glucosamine and N-acetyl-d-glucosamine units joined through ␤-(1-4)-glycosidic bonds, has emerged as an exciting prospect for efficient delivery of micronutrients and agrochemicals [29]. Chitosan, due to its cationic character forms complex with negatively charged polymers such as alginate, sodium tripolyphosphate (STPP), TPP, xanthan gum, carrageenan, etc., and has been examined thoroughly for active ingredient delivery owing to its cost effectiveness, biodegradability, high permeability and low toxicity. In the present work alginate–chitosan nanocapsules were prepared by two step procedure, first the formation of pregel on addition of calcium chloride to sodium alginate and the second step involved formation of polyelectrolyte complex between carboxyl group of alginate and amine group of chitosan. A pregel nucleus forms upon interaction of alginate and Ca2+ at certain ion concentration on stirring [17]. The addition of chitosan solution into the pregel forms a polyelectrolyte complex which stabilizes the pregel into separate sponge-like nanoparticles [30]. The polyelectrolyte complex protects the encapsulated active ingredient, and limits its release more effectively than matrix formed from alginate or chitosan alone [31]. Thus, the present study was taken up with the aim to develop alginate–chitosan nanocapsules for controlled release of acetamiprid. These kinds of formulations are capable of providing alternative strategies for pest control in agriculture along with reducing dependency on synthetic pesticides and pesticide residue problems. The various process steps have been examined by microscopic and spectroscopic techniques. The release studies were performed at different pH ranges and in soil.

2. Experimental 2.1. Materials Chitosan was purchased from HiMedia Laboratories Pvt. Ltd. (Mumbai, India). Sodium Alginate was purchased from Sisco Research laboratories Pvt. Ltd. (Mumbai, India) Dichloromethane

(DCM), glacial acetic acid and acetone were of analytical reagent quality and purchased from Merck. Methanol and water for HPLC were HPLC grade. Acetamiprid technical (96.5%) was obtained as a gift from Nagarjuna Agrichem chemicals pvt ltd. Ultra high pressure liquid chromatogram (UHPLC) from Thermo Fisher Scientific was used to estimate the amount of active ingredient in nanoformulation. 2.2. Preparation of acetamiprid containing alginate–chitosan nanocapsules Alginate–chitosan nanocapsules were prepared by ionic pregelation and polyelectrolyte complexation method [22]. 0.06% Sodium alginate aqueous solution and 0.05% chitosan solution in 1% acetic acid were prepared and kept overnight. Thereafter, alginate and chitosan solutions were filtered and their pH was adjusted to 4.9 and 4.6, respectively. Acetamiprid (25 mg) was ultrasonicated with 100 mL of alginate solution for 15 min. Then 20 mL of 0.067% calcium chloride solution was dropped slowly to the above solution and stirred continuously for 30 min. Chitosan solution (15 mL) was added dropwise into the above and stirred further for 30 min which resulted in a colloidal suspension (pH 4.7). Nanocapsules were recovered by centrifugation at 14,000 rpm for 30 min at 4 ◦ C. 3. Characterization 3.1. Size estimation and stability study The average particle size of the acetamiprid loaded alginate–chitosan nanocapsules was determined using the Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). Dynamic light scattering (DLS) technique was used to measure the average size of nanocapsule and size distribution (polydispersity index), while zeta potential was studied for determining stability of the prepared nanocapsules which depends on electrophoretic movement of nanocapsules in the solution. 3.2. Transmission electron microscopy (TEM) The morphology of acetamiprid loaded alginate–chitosan nanocapsules was determined by JEOL’s JEM 1011 transmission electron microscope by taking drop of aqueous solution of nanocapsules on carbon coated Cu grid. 3.3. Fourier transform infrared spectroscopy (FTIR) Alginate–chitosan nanocapsules separated from suspensions were lyophilized and their FTIR spectra were obtained using a Bruker’s alpha spectrophotometer. Pellet was made by grinding 1% (w/w) of sample, with respect to potassium bromide (KBr) and compressed into KBr disc under a hydraulic press at 10,000 psi. Samples were scanned over a wavenumber region of 600–4000 cm−1 . The characteristic peaks were recorded for different samples. 3.4. Encapsulation efficiency Acetamiprid content was estimated by UHPLC (Fig. 6). For this, nanoformulation was centrifuged and the pellet obtained was washed twice with distilled water to remove any untrapped pesticide. The pellet was dried and ultrasonicated with methanol, filtered through 0.22 ␮ filters into vials and kept in UHPLC apparatus. The syringe itself takes the sample (1 ␮L) from vials which are sequenced in the software. Methanol and water (50:50, v/v) was used as mobile phase at a flow rate of 0.1 mL/min. The absorbance was taken at 254 nm. The total analysis time for one sample was 10 min with elution of acetamiprid at 4.8 min (retention time).

S. Kumar et al. / International Journal of Biological Macromolecules 81 (2015) 631–637

Encapsulation efficiency of nanocapsules was calculated according to equation (1). Encapsulation efficiency% =

Amount of entrapped acetamiprid Total amount of acetamiprid added × 100

(1)

3.5. Controlled release of pesticide at different pH Three buffer solutions viz. acetate for pH 4, phosphate buffer solutions for pH 7 and 10 were used for release analysis of acetamiprid from nanocapsules. The release of acetamiprid from the nanocapsules and the commercial formulation (20% SP) was determined as per literature [32]. Nanocapsules taken in parchment paper strips in three replicates were added to 25 mL of each buffer in conical flasks and kept in Biological Oxygen Demand (BOD) incubator at 30 ◦ C. At time intervals (from 2 to 60 h), 1 mL solution was removed for acetamiprid determination by UHPLC, and replaced with fresh buffer to keep

633

the volume same as before. The sample was taken in a separatory funnel and 10% NaCl solution (100 mL) was added. The resultant mixture was extracted with dichloromethane (DCM) (30 mL × 3). The solvent DCM was removed in rotary evaporator. The residue obtained was dissolved in 50:50 (v/v) methanol and water mixture and analyzed by UHPLC. The amount of acetamiprid in samples was calculated using equation (2) =

A  m  1 2 A2

×

m1

×P

(2)

Here, A1 = peak area of acetamiprid in sample, A2 = peak area of acetamiprid in technical, m1 = mass, in grams, of the test sample, m2 = mass, in grams, of the technical and P = purity of the technical sample. The acetamiprid technical available was of 96.5% purity. 3.6. Controlled release studies in soil Release studies were done according to the literature method [33]. Soil was collected from the Indian agricultural research

Fig. 1. (a) Size distribution of nanocapsules, (b) zetapotential of nanocapsules.

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institute (IARI), New Delhi (pH 8.3). Nanocapsules were taken in parchment paper strips and placed inside 25 g of soil taken in 50 mL beaker. Water was added to maintain 60% water-holding capacity of the soil. The beakers then covered with parafilm in which holes were made for gas exchange, and kept in a BOD incubator at 30 ◦ C temperature. For regular sampling, 3 beakers (replicates) per treatment were taken time to time (2–60 h). The parchment strips were removed from the soil. Extraction of acetamiprid from soil sample was done according to method found in literature [34]. As acetamiprid is very soluble in acetone, the extraction was done with acetone. The soil samples were suspended in 50 mL acetone, placed at shaker for half an hour, and then filtered. The procedure was repeated with acetone twice. These extracts were combined and concentrated in a rotary evaporator. 150 mL of 10% NaCl aqueous solution was added to the residue left in rotary evaporator. This solution was transferred into a separatory funnel and extracted with dichloromethane (3 × 30 mL). The DCM extract was collected in a round bottomed flask after passing through anhydrous Na2 SO4 and concentrated in a rotary evaporator. The residues were dissolved in 5 mL methanol/water mixture. The amount of acetamiprid was assessed by UHPLC. 3.7. Statistical analysis All the values of controlled release in buffers and soil, done in triplicate were analysed by one way-ANOVA. The value of p < 0.05 indicates statistical significance. 3.8. Release kinetics study The release profile of acetamiprid from alginate–chitosan nanocapsule was investigated from Eq. (3): Mt = kt n M∞

Fig. 2. TEM micrograph of acetamiprid loaded alginate–chitosan nanocapsules (a) at ×80,000, (b) at ×1,20,000 magnification.

(3)

Here Mt is acetamiprid amount released at given time, M∞ is the maximum acetamiprid available for release, t is time of release, k is a kinetic constant which includes characteristics of macromolecular system and active ingredient, and the value of n defines diffusion. The type of mechanism is reflected by the value of n. Sphere shaped systems which have n value less than or equal to 0.43 shows Fickian mechanism or Case I type of delivery, which is diffusion controlled. If the value of n is equal to or greater than 0.85, it is case II type delivery which depends on the relaxation of polymer matrix. The value of n in between 0.43 and 0.85 shows a non-Fickian mechanism, which depends on both diffusion and polymer relaxation simultaneously [35,36]. By plotting graph between ln(Mt /M∞ ) and ln(t), the values of n, k and r2 can be found [35]. 4. Results and discussion The results of study using DLS are shown in Fig. 1(a). The particle size of nanocapsules was 201.5 nm with a polydispersity index (PDI) of 0.390. A PDI value <0.5 is good for colloidal suspension. It portrays that the suspension has homogenous size distribution. Zeta potential value of −32.1 mV confirmed the stability of synthesized nanocapsules (Fig. 1b). The zeta potential value around ±30 mV is assumed to be good for suspension. The negative value of zetapotential shows the presence of more –COO− groups, which is due to the high concentration of alginate than chitosan [20]. The carboxyl groups present on alginate form complexes with amino group of chitosan but due to more concentration of alginate, there remains some free carboxyl groups which renders a negative value of zetapotential. TEM micrograph displayed solid dense nanocapsules linked to one another with their size ranging from 30 to 40 nm. Fig. 2 shows

TEM image at different magnifications, one at ×80,000, and second at ×1,20,000 magnification. The variation in size measured by PSA and TEM is observed because average size of the particles are measured using PSA taking into account possible agglomeration of different size classes. In PSA technique, hydrodynamic layers are formed upon submicron particles which results in increase in size [37]. The peaks in alginate spectra near 1616 and 1418 cm−1 are associated with asymmetric and symmetric stretching vibration of COO− groups respectively of alginate. The formation of alginate–chitosan nanocapsules was confirmed by FTIR spectra as peak was slightly shifted from 1418 to 1417 cm−1 after complex formation between alginate and chitosan [38]. The presence of acetamiprid in nanocapsules was confirmed by the presence of peak at 1633 cm−1 which corresponds to stretching vibration of C N/C N, [39]. The peak around 3400–3500 cm−1 becomes broad due to hydrogen bonding between COOH group of alginate and NH2 of chitosan (Fig. 3). The quantification from UHPLC revealed that alginate–chitosan nanocapsules encapsulated 62% of acetamiprid insecticide (Fig. 4). Acetamiprid has high solubility in polymer solution which allows its easy encapsulation in polymer. There are two peaks in the chromatogram. The peak before acetamiprid is because of the solvent. UHPLC allowed detection of acetamiprid in less time (4.9 min) compared to conventional HPLC with an analysis time of 30 min. Release study of acetamiprid in different buffers presented a controlled pattern with pleatue reaching in 36 h for pH 10 and 48 h for pH 7 and 4 for nanoformulation (Fig. 5a) while it was 12 h (pH 4) and 24 h (pH 7 and 10) for commercial formulation of acetamiprid (Fig. 5b).

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Fig. 3. FTIR spectra of (a) sodium alginate, (b) chitosan, (c) acetamiprid technical, (d) acetamiprid containing alginate–chitosan nanocapsules.

In case of nanoformulation, 50% of the insecticide was released after 24 h at pH 10 and after 36 h at pH 7 and 4 while commercial formulation released approximately half of the pesticide within 6 h at all the three pH. The rate of release from polymer matrix depends on two factors (a) solubility of encapsulated material and (b) polymer characteristics. The rate of release from nanoformulation was almost similar upto 12 h at all the pH ranges. Swelling of polymer matrix results in diffusion of active ingredient due to concentration gradient. The comparatively fast release at pH 10 may be due to the more solubility of acetamiprid at this pH and dissolution of alginate. The retarded release at pH 4 can be accounted for the neutralization of carboxyl groups present on alginate which leads

70.0

ACETAMRID #266 mAU

to less repulsion and more complexation of polymer matrix. More compaction of the polymer matrix results in less swelling and thus less release of pesticide. The results indicated that nanoformulation of acetamiprid took longer time (approximately 24 h) to release the insecticide as compared to its commercial formulation. It was also found that the pesticide degradation after maximum release was less in nanoformulation as compared to commercial formulation which may be due to protection of pesticide in polymer nanocapsules for a longer time. Alginate–chitosan combination has been used to encapsulate Paraquat [particle size 635 nm, release time 8 h, ref. 38], Gatifloxacin [particle size 205–572 nm, release time 24 h, ref. 20] and Chloramphenicol [particle size 265 nm, release time

2 ppm std

UV_VIS_1 WVL:254 nm

1 - Acetamiprid - 4.840

40.0

20.0

min

-10.0 0.0

1.3

2.5

3.8

5.0

6.3

7.5

Fig. 4. UHPLC Chromatogram showing peak of acetamiprid.

8.8

10.0

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(a)

pH 4 pH 7 pH 10

Cumulative release (%)

Cumulative release (%)

100 80 60 40 20 0 0

10

20

30

40

50

Nanoformulation Commercial formulation

100 80 60 40 20 0

60

0

Time (hrs) pH 4 pH 7 pH 10

Cumulative release (%)

100 80 60 40 20 10

20

30

20

30

40

50

60

Time (hrs)

(b)

0

10

40

50

60

Time (hrs) Fig. 5. (a) Cumulative release% of acetamiprid from alginate–chitosan nanocapsules. (b) Cumulative release% from commercial formulation of acetamiprid at different pH.

24 h, ref. 21] whereas acetamiprid has been used as active ingredient in polymers polylactic acid-polycaprolactone [particle size 617–700 ␮m, release time 24 h, ref. 40] and Tapioca starch, urea and sodium borate [particle size 2–20 ␮m, release time dependent on conc of urea, sodium borate and size, ref. 41] for studying release profile. Statistical analysis done on controlled release with ANOVA shows that the results are of statistical significance. The release of acetamiprid followed a non-Fickian behavior at all the pH ranges which is predicted from n values (0.6431–0.6759) [34]. In case of non-Fickian profile, both diffusion rate of pesticide and relaxation rate of polymer matrix are comparable. Two simultaneous processes occur, i.e. swelling of polymer matrix through water movement and diffusion of active ingredient. The k value is used to find out release rate. Higher the value of k, faster will be the release. The value of k was highest for acetamiprid release in buffer of pH 10 (0.8279) followed by pH 7 (0.7883) and pH 4 (0.7296), as shown in Table 1. Table 1 Diffusion exponential (n), Pearson coefficient (r2 ), and diffusion constant (k) for acetamiprid released from alginate–chitosan nanocapsules at different pH. pH

n

r2

k

4 7 10

0.6759 0.6431 0.6619

0.9595 0.9616 0.9536

0.7296 0.7883 0.8279

Fig. 6. Comparison of cumulative release% of acetamiprid from commercial and nano formulation in soil.

Thus, it can be concluded that the diffusion of acetamiprid from nanoformulation, is faster in basic solution (pH 10) than in neutral (pH 7) and acidic media (pH 4), respectively. Thus the relationship between release rate and slope of release behavior supports the assumption (Fig. 5a). Fig. 6 shows the comparison of controlled release of pesticide from commercial and nano formulation in soil. The recovery of acetamiprid from soil by the method adopted for the extraction was 96%. It was found that rate of release from both the formulations was almost similar to that of pH 10 buffer. It may be due to the alkaline nature of the selected soil. Thus acetamiprid reflected non-Fickian profile in soil also which includes swelling of the polymer matrix coupled to release of pesticide by diffusion. Maximum release of 93% was observed from nanoformulation in 36 h while commercial formulation released the maximum amount in 24 h. At higher pH ranges, chitosan becomes insoluble which reduces the active ingredient release. A little degradation of pesticide was found after maximum release which may be due to the microbial attack or environmental conditions in soil. The release from nanoformulation was slow as compared to commercial formulation which is due to the physical barrier provided by nanocapsules.

5. Conclusion The alginate and chitosan macromolecules were used for preparation of controlled release formulation of acetamiprid. The estimation of acetamiprid was done by a highly sensitive instrument UHPLC, which is very useful in the detection of very low amounts of pesticide. The release of pesticide was observed at different pH to find its stability and controlled release profile in different types of soil. Thus, this newer formulation can be used in any type of soil due to the stability of acetamiprid and controlled release profile of nanocapsules at all the pH ranges but will be best for acidic soil as the rate of release is retarded in acidic conditions due to the electrostatic interaction between alginate and chitosan. The nanoformulation was found to be superior to commercial formulation in terms of controlled release. Therefore this formulation can be helpful in the reduction of consumption of pesticide. In this way there are dual benefits for human and environment, first, non-toxic, biodegradable and inexpensive polymer for encapsulation and other is encapsulation of hazardous pesticide.

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