Environmentally benign nanometric neem-laced urea emulsion for controlling mosquito population in environment
Prabhakar Mishra 1, Merlyn Keziah Samuel1, Ruchishya Reddy 1, Brij KishoreTyagi 2,
Amitava Mukherjee 1, Natarajan Chandrasekaran 1*
1.Centre for Nanobiotechnology, VIT University, Vellore-632014, Tamil Nadu, India
2.Department of Zoology & Environment Science, Punjabi University, Patiala, Punjab, India.
Supplementary document
*Correspondence:
Dr. N. Chandrasekaran, PhD
Senior Professor & Director
Centre for Nanobiotechnology
VIT University, Vellore-632014, India
E-mail: ;
Phone: 91 416 2202624
Material and methods
Optimization of No. of cycles in Microfluidization
The optimization of the no. of cycles applied in the microfluidization was determined through an experimented conducted using different ratios of neem oil: surfactant for neem urea nanoemulsion formation. The three different ratios used for the application was 1:1 – 1:3. Each coarse emulsion prepared through usage of different ration of oil:surfactant was subjected to microfluidization for a set of 30 cycles. After each 5th cycle the droplet diameter of the obtained nanoemulsion was analysed using the DLS technique (Nanoparticle analyser, SZ-100, Horiba, Japan). The experiment was carried out in triplicated to obtain accuracy in the results.
Stability studies of NUNE
Thermodynamic stability
To determine the thermodynamic stability of the formulated nanoemulsion it was centrifuged at 10,000 rpm for a duration of 30 min and was observed for the occurrence of any phase separation. Heating–Cooling cycle was commenced through keeping the formulated nanoemulsion at 40º C and 4º C alternatively at each temperature. This cycle was repeated for three times. This process was carried out to check the stability of the formulated at varying storage temperature. Freeze–Thaw stress was performed by placing the nanoemulsion alternatively at -21 ºC and 25 ºC for a duration of 48 h at each of the temperature. The cycle was repeated twice, and the entire experimentation was carried out in triplicates.
Kinetic Stability
The formulated nanoemulsion (NUNE) was tested for its kinetic stability through the measurement of its mean hydrodynamic size at different time intervals. The NUNE was checked for the occurrence of phase separation and creaming.
Biosafety studies against the non-target studies
Antibacterial activity against the beneficial bacterial isolate
Well Diffusion Assay
The well diffusion assay was commenced using the sterilised petri dishes of 10 cm diameter. Autoclaved Muller Hinton agar medium was used for the experimentation. The 1.0x107CFU/mL of bacterial suspension was prepared and overlaid on the surface of the agar plate. The different wells with different concentrations (10- 500 µg/mL) of NUNE, were prepared on the surface of the agar plates already inoculated with bacterial inoculum. The plates were kept for incubation at 37 ºC and were observed after 24 h for the occurrence of a zone of inhibition around the wells. Further, the measurement of the zone of inhibition was carried out. Ampicillin was used in the assay as a standard control drug. Entire experimentation was conducted in triplicates.
Microscopic analysis
Live cell dead cell experimentation
The nanoemulsion toxicity causing alterations in the membrane integrity and mortality of the bacterial cells was studied through the fluorescence microscopic analysis (Leica, DM-2500) as per Dalai et al. (2012). To 400 μL of the bacterial suspension of both control and treatment, 16 μL of Acridine orange (AO) (15 μg mL−1 in PBS) and 16 μL of Ethidium bromide (EtBr) (50 μg mL−1 in PBS) were added. The stained bacterial suspension (both treated and control) was kept for the incubation for 10 min at room temperature and subjected to centrifugation at 5000 rpm for 10 min. The supernatant was discarded to remove the unbound dyes. The resuspension of the cell pellet was carried out in 20 μL of PBS. The experimental setup was performed throughout in the dark condition to avoid the photobleaching of dyes. Fluorescence microscopy was performed using BP 450-490, LP 590 filter; images were captured with a Leica-DFC-295 camera and further processed using Leica-Application Suite 3.8
Scanning Electron Microscopy
The NUNE toxicity towards the morphological alterations in bacterial isolate was studied through the scanning electron microscopy. The samples treated with the NUNE, and control were examined using the scanning electron microscopy (EVO-18, Carl Zeiss, Germany) for alterations in the membrane integrity. The bacterial isolate in the NB (Nutrient Broth) medium was used for the treatment with NUNE. The untreated bacterial samples were used as a control. Both treated and control samples were kept for the incubation of 24 h. Later the samples were rinsed thrice using the phosphate buffer saline. After the proper rising, the samples were fixed using the 2% glutaraldehyde solution. The samples were then dehydrated using the ethanol dilution in water (20%, 40%, 60%, 80%, and 100%) and were allowed to air dry overnight.
Phytotoxicity study of the formulated nanoemulsion
Seed Germination index
Paddy seeds were sterilised by soaking them in the sodium hypochlorite (NaOCl) solution of 2.5% for a duration of 15 minutes as per Lin et al. 1996. After the sterilisation, these seeds were rinsed thoroughly using the Milli-Q water and then soaked in the nanoemulsion solution of different concentrations (2-200 µg/mL) for various time intervals from 24 h – 72 h at a proper laboratory condition (Temperature 27± 2º C, Relative humidity of 65%). The seeds soaked in the Milli-Q water was kept as the control.
A piece of filter paper (90 mm X 10 mm, Whatman no. 1), 5 ml of Milli-Q water or the NUNE were added to the Petri plates. 20 seeds were then moved into each dishes. Later the Petri dishes were kept in the incubator. Following the 7 days of the treatment, the seed germination was recorded through counting the germinated seeds that had the coleoptile of length 2 mm while remaining seeds were considered as the non-germinated seeds.
Determination of Root and Shoot Length
The toxicity of the NUNE was tested on the paddy plant through assessing the length of the root and shoot of the paddy plant as per Rahman et al. (2007). The NUNE toxicity profile on the root and shoot length were carried out by soaking the seeds in the different concentrations (2-200 µg/mL), and the distilled water was used as the control. The seeds of the paddy were soaked in the various solutions of the nanoemulsion, and the Milli-Q water was later were sowed into the soil and left for the germination. The germination rate was checked for the duration of 30 days at an interval of 5 days period. The shoot and tiller length of the paddy plant was measured using the metric scale. The entire experimentation was carried out in triplicates.
Total chlorophyll estimation
The total chlorophyll estimation in the paddy plants was determined as per Arnon et al. (1949). The leaves of the paddy plant were cut into small pieces leaving behind the midrib, were mixed thoroughly and 0.10 g of the leaves were weighed and put into mortar and pestle. 7ml of dimethyl sulphoxide (DMSO) was later added to the finely chopped leaves and were thoroughly homogenised. The homogenised mixture was subsequently kept in incubation (65ºC) for a duration of 24 hours. The extract obtained from the incubation of leaves was then transferred, and the total samples volume was adjusted to 10 ml using the DMSO. After this, the samples were stored at 2-4 ºC and were determined for the chlorophyll content at a wavelength (λmax) of 645 and 663 nm. The entire experimentation was carried out in triplicates. The below-mentioned equation was used to calculate the amount of chlorophyll a (chl a), chlorophyll b (chl b) and total chlorophyll.
Chl a (g l-1) = 0.0127 A663 – 0.00269 A645 (1)
Chl b (g l-1) = 0.0029 A663 – 0.00468 A645 (2)
Total Chl (g l-1) = 0.0202 A663 + 0.00802 A645 (3)
Biochemical analysis
The toxicity of the NUNE towards the biochemical profile of the paddy field was studied through the estimation of the total protein (Bradford 1976) and lipid peroxidation (Detailed description is been provided in the supplementary document). The homogenate was prepared using the root and shoot samples. The samples homogenate was prepared using the root samples in 50µM phosphate buffer (pH 7.2). The extract of the root and shoot samples was then centrifuged at 2000xg for 30 mins. The temperature maintained during the centrifugation was 4º C. Later the supernatant obtained after centrifugation was used for further experimentation.
Biochemical analysis
Total protein estimation
Total protein estimation of the paddy roots and shoots was carried out as described by Bradford 1976. The root and shoot samples (10 µL) of both NUNE treated, and control was mixed with the 200 µL of Bradford reagent. Later the sample mixture was incubated for a duration of 5 min at room temperature. The estimation of the total protein was carried out at a wavelength of 595 nm. The bovine serum albumin was used as n standard in the experimentation. Entire experimentation was conducted in triplicates.
Lipid peroxidation assay
The toxicity of the nanometric emulsion on the paddy biochemical profile was assessed by determining the activity of lipid peroxidation. The lipid peroxidation assay was carried out through measurement of malondialdehyde (MDA) formation using TBA (thiobarbituric acid) method as described by Stewart et al. 1980. Lipid peroxidation was determined by measuring the amount of malondialdehyde (MDA) formation using the method containing 20% (w/v) trichloroacetic acid. The mixture was then incubated at 95°C for 30 min, and then the reaction was stopped by spontaneously placing the sample vials in an ice bath. The cooled mixture was then centrifuged at 10000 g for 10 min, and later the absorbance of the supernatant was read at wavelengths of 532 and 600 nm. The final concentration of the MDA was calculated by subtracting the non-specific binding at 600 nm and then using the molar extinction coefficient of 155 mM-1 cm-1.
Results and discussion
The experimentation was carried out to optimize the no. of cycles applied in the process of microfluidization. The three different ratios exhibited a significant reduction in the droplet diameter from the 1st cycle to 25th cycle. Later the droplet diameter became constant, without any significant reduction in it. Fig. S1 depicts the results of three different nanoemulsion being formulated using a pressure of 20,000 psi for 30 cycles. The three different ratio of nanoemulsion showed the similar trend of droplet diameter reduction, further which the droplet dimeter became constant. In the ratio 1 (Figure S1A), the initial size at 1stcycle (135.2±0.23 nm) significantly reduced to (98.32±0.11 nm) at 25th cycle, which further became insignificant at 30th cycle (97.4±0.65 nm). This trend was observed in remaining nanoemulsion prepared using ratio of 1:2 (Fig. S1B) and ratio 1:3 (S1C). Therefore the final optimized condition for the neem urea nanoemulsion formulation was at 20,000 psi for 25 cycles using microfluidizer, without occurrence of any instability phenomenon.
Fig. S1. Optimization of number of cycles for different ratios of Oil: Surfactant (A) 1:1, (B) 1:2 and (C) 1:3 for neem urea nanoemulsion formulation using microfluidizer
Stability of NUNE
The NUNE which was experimented for the thermodynamic stability through freeze-thaw and centrifugation studies, which was found to be stable without an occurrence of any instability phenomenon such as creaming and phase separation. The kinetic stability was further assessed through determination of the droplet size and was found to be 20.3 ±0.23nm and 19.6±0.61 in Milli-Q and Paddy field water dispersion respectively on the day of initiation of experimentation. The insignificant increase in the droplet size been observed as 24.3±0.88 nm in Milli-Q water and 26.7±0.87 nm in Paddy field water till the day 20, depicted its stability. Further, the droplet size of the formulated nanometric emulsion increased significantly and reached till 260.7±0.29 nm and 272.3±0.80 nm on the 40th day of experimentation for Milli-Q and Paddy field water dispersion; this led to a termination of the study (Table S1).
From the study, it was observed that the formulated nanometric emulsion exhibited the stability till day 20 upon which the droplet size increased significantly and resulted in instability phenomenon. Later the stability of the nanoemulsion was determined in the field conditions. In this experimentation the formulated nanoemulsion was found to be stable for four days, then the droplet size increased significantly making it unstable. The initial droplet size was found to be 20.2± 1.01nm which insignificantly elevated to 24.3± 1.23 nm till day 4. Further commencement of the experiment leads to significant increase in the droplet size of the nanoemulsion dispersion to 263.4 ± 2.23 nm on the 16th day, further which the study was terminated (Fig.S1A). This result of the stability at field scale was further found to be corroborative with the zeta potential analysis, which displayed its remarkable stability for 4 days, further which upon the increase in the droplet size the study was terminated (Fig. S2B). The stability of the nanoemulsion for four days in the field experimentation defines its applicability as a larvicidal agent.
The trend of the nanoparticles towards the instability phenomenon such as aggregation and agglomeration which can be considered in the context of different electrostatic, steric and van der Waals forces between nanometric particles using the DLVO (Derjaguin-Landau-Verwey-Overbeek) theory (Derjaguin and Landau1941; Vassilev et al. 2006). The inflexible distribution of the nanoparticles depicts its stability in the aqueous stability. There is a substantial relation between the physiological characterizations i.e. the size surface and shape features of a nanoparticle aqueous dispersion and its toxicological impact on the host organisms. The mean hydrodynamic size and the surface charge of the nano-dispersions can show the dramatic effect of its uptake by the organisms and their exposure to the nanoparticles. The exposure of the nanoemulsion towards the different physicochemical parameters such as higher temperatures and pH range makes the emulsion system to become instable also leads to the reduction in the azadirachtin content (Kumar et al. 2000)
Fig.S2. The stability of the NUNE in field conditions through estimation of droplet size (14A) and zeta potential (14B). Corresponding error bar represents Standard error of 3 replicates (SE, n =3).
Table S1. Stability of the NUNE in the lab conditions
Days / Droplet size inMilli -Q water (nm) / Droplet size in
Paddy field water (nm)
1 / 20.3 ±0.23 / 19.6±0.61
10 / 22.3±0.67 / 24.5±0.73
20 / 24.3±0.88 / 26.7±0.87
30 / 93.4± 2.23 / 103.6±0.55
40 / 260.7±0.29 / 272.3±0.80
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