Sigma-Aldrich India supplied the halloysite nanotubes (HNTs). Neem oil was obtained using the cold press process. Acephate was obtained from Rallies India Pvt. Ltd. Fresh cotton (Gossypium) and maize (Zea mays) leaves were obtained from the Jalgaon area of Maharashtra, India. Deionized water was utilized in all of the trials. Fourier transform infrared (FT-IR) spectra were recorded using FT-FTIR affinity model no. 1 with 2.0 cm⁻¹ resolution in the 4000 - 400 cm⁻¹ range. Microscopic images of Pickering emulsions were captured using a Bio Mini IS 4381 digital microscopes at 40 X magnification. The contact angle analyzer (DSA 100, Data Physics, OCA-25, Germany) was used to measure the droplet contact angle on the leaf surface. The vacuum freeze drier FDL2-10N -2-60 B was utilized, with the temperature set at −55˚. High Speed homogenizer (Remi RQ-127 A/D) was used for homogenization.

Pickering emulsion is prepared in two steps. It is described in Figure 2. In first step Pickering emulsions were created by homogenizing and sonicating an aqueous solution of neem oils for 20 minutes at 1:9, 2:8, and 3:7 oil/water volume ratios. To stabilize Pickering emulsions, different amounts of HNTs (0, 0.5, 1, 2, and 5 weight percent) were added in it.

A pesticide inserted Pickering emulsions were created by adding 0.5 g of Acephate to previously prepared Pickering emulsions at various concentrations (0, 0.5, 1, 2, and 5 wt%) of HNTs, homogenizing, and sonicating for 20 minutes.

Figure 2. Preparation of Neem oil-based Pickering emulsion.

Microscopic images of Droplets of Various Neem oil-based Pickering emulsion

Droplet size depends upon interfacial tension. Lower the interfacial tension, smaller is the droplet size and vice versa [3]. Here for these emulsions, HNT reduces interfacial tension, which in turn reduces droplet size. Emulsions with smaller droplets are more likely to exhibit kinetic stability. Smaller droplet sizes usually result in greater stability. This is because smaller droplets have a higher surface area to volume ratio, which makes it easier for stabilizing particles to be adsorbed at the interface. Figure 3 represents the microscopic images of Droplets of Various Neem oil-based Pickering emulsion. Halloysite nanotubes adsorb at the oil-water interface in the previous Pickering emulsions, creating a rigid barrier. Compared to surfactants, this barrier inhibits droplet coalescence far more effectively. Neem oil Samples A, B, and C represent Pickering emulsions according to the oil to water ratio. For samples A, B, and C, the corresponding O/W ratios are 2:8, 3:7, and 1:9 Sample A exhibits greater stability and smaller droplets at HNT concentrations of 0.2% and 0.5%. In contrast, Sample B, which contains 0.1% and 0.2% HNT, has more stability and smaller droplets. While, Sample C, which has a 0.2% HNT content, exhibits the smallest droplets and the highest stability. As a result amongst all these samples, the sample containing 0.2% HNT show smallest droplet size and maximum stability. Therefore, the stability of the Pickering emulsion will increase with the HNT content. A Digital Binocular Microscope (Micron optic) was used to study droplet images of these emulsions at magnifications ranging from 4x to 100x. A digital micro imaging camera manufactured by Micaps Ltd. was used to take microscopic pictures.

Figure 3. Microscopic images of Droplets of Various Neem oil -based Pickering emulsion.

Antimicrobial activity of Pickering emulsions prepared by using Neem oil -Methodology

The cultures Staphylococcus aureus,Bacillus cereus, Pseudomonassp., and Escherichia coli were grown in nutrient broth medium. The nutrient broth medium was purchased from “HI Media Laboratories, Mumbai”. The Nutrient broth composition was (g/l) Peptone 10.0 Beef extract 10.0, Sodium chloride 5.0, pH after sterilization 7.3 ± 0.1. It was grown at 37˚C for its accelerated growth. Freshly grown culture was spread on the nutrient agar plate. A well 0.7 mm diameter with sterile borer was prepared in the medium. Each sample having a varied concentration each 100 μl (microliter) was loaded to well in plate. Plates were incubated at 37˚C and observed for growth of that respective microorganism on plate. After incubation, the zone of inhibition was checked around the colony and its diameter were measured.

Controls:

Negative control: Sterile distilled water and blank Pickering emulsion (without neem oil).

Positive control: Streptomycin (10 µg/mL) solution.

The zone of inhibition data obtained from antimicrobial assays were analyzed using one-way analysis of variance (ANOVA) to determine significant differences among treatment concentrations and formulation types. The antimicrobial activity of neem oil-based Pickering emulsions prepared at different oil-to-water (O/W) ratios (2:8, 3:7, and 1:9) was evaluated against Gram-positive and Gram-negative bacterial strains including Staphylococcus aureus, Bacillus cereus, Escherichia coli, and Pseudomonas aeruginosa using the agar well diffusion method. Test samples and its control against different bacteria is demonstrated.

Results:

Sample A (2+8 O/W ratio):Table 1 describes the zone of inhibition, while Figure 4 shows a graphical representation of sample A against various bacterial strains.

Staphylococcus aureus: The zone of inhibition significantly increased from 1.2 cm at 0% concentration to 1.4 cm at both 1% and 2% concentrations. This indicates a positive correlation between concentration and antimicrobial activity.

Bacillus cereus: The inhibition zone increased from 0.9 cm to 1.4 cm at 2%, suggesting moderate activity.

Pseudomonassp.:No significant antimicrobial activity was observed across all concentrations (0%, 0.05%, and 2%), indicating resistance.

Escherichia coli:A notable increase in the zone of inhibition was recorded, from 0.8 cm at 0% to 1.35 cm at 1% and subsequently decreasing to 1.2 cm at 2%.

Table 1. Zone of inhibition for Sample-A.

Figure 4. Graphical representation of zone of inhibition for Sample-A.

Sample B (3+7 O/W ratio):Table 2 describes the zone of inhibition, while Figure 5 depicts a graphical representation of sample B against numerous bacterial strains.

Staphylococcus aureus: A consistent increase in the zone of inhibition was observed, peaking at 1.65 cm at the 5% concentration.

Bacillus cereus:The inhibition remained relatively constant (0.8 cm) across most concentrations, except for a slight increase at the 5% concentration.

Pseudomonassp.:Inhibition was noted only at concentrations of 0%, 0.05%, and 5%, while no activity was observed at 1% and 2%.

Escherichia coli:The highest zone of inhibition (1.5 cm) was recorded at the lowest concentration (0.05%), indicating significant activity compared to other samples.

Table 2. Zone of inhibition for Sample-B.

Figure 5. Graphical representation of zone of inhibition for Sample-B.

Sample C (1+9 O/W ratio):Table 3 describes the zone of inhibition, while Figure 6 depicts a graphical representation of sample C against numerous bacterial strains.

Staphylococcus aureus:The zone of inhibition showed an increasing trend from 1.3 cm at 0% to a peak of 2 cm at the 2% concentration, followed by a decrease at the highest concentration (5%).

Bacillus cereus:Moderate antimicrobial activity was observed, with an inhibition zone of up to 1 cm.

Pseudomonassp.: Similar to sample 8 + 2, there was no significant activity noted except for concentrations of 0%, 1%, and 5%.

Escherichia coli:Moderate activity was observed with a maximum zone of inhibition of approximately 1.2 cm.

Table 3. Zone of inhibition for Sample-C.

Figure 6. Graphical representation of zone of inhibition for Sample-C.

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

Figure 7. Antibacterial activity against various bacterial strain, (a) Test samples for Staphylococcus aureus; (b) Control for Staphylococcus aureus; (c) Test samples for Pseudomonas aeruginosa; (d) Control for Pseudomonas aeruginosa; (e) Test samples for E. coli; (f) Control for E. coli; (g). Test samples for Bacillus cereus; (h) Control for Bacillus cereus.

Conclusion: When compared to control groups: Sample 8 + 2 demonstrated high antimicrobial activity against S. aureusat concentrations of 0.05%, 1%, and 2%. For Bacillus cereus, significant activity was noted only at concentrations of 1% and 2%. Sample 7 + 3 exhibited higher antimicrobial efficacy against S. aureusand E. colispecifically at concentrations of 2% and above. Sample 9 + 1 showed enhanced activity against S. aureusacross multiple concentrations (0.05%, 1%, and up to 5%) compared to control. In contrast, no significant antimicrobial activity was recorded against Pseudomonassp. across all samples, indicating inherent resistance. The findings suggest that samples containing varying concentrations exhibit differing degrees of antimicrobial efficacy against specific bacterial strains. Notably, samples containing higher concentrations demonstrated enhanced inhibitory effects against Staphylococcus aureus and Escherichia coli, while showing limited effectiveness against Pseudomonassp. The results obtained warrant further investigations to explore potential applications of these compounds to be used as antimicrobials and also to design newer molecules with better functionality Figure 7.

Contact Angle of HNTs-AcephateEmulsion on the Leaf Surface—The spreading, wetting, and adhesion behavior of pesticide formulations on plant leaves are all influenced by the contact angle. The contact angle plays a major role in determining the droplet wettability of a leaf surface. The droplet’s wettability can be classified as good (θ < 60˚), moderate (60˚ ≤ θ < 80˚), poor (80˚ ≤ θ < 100˚), or very poor (θ ≥ 100˚). The higher the contact angle of the droplet on the leaf surface, the less wet the liquid will be on that surface. The surface of Maize and cotton leaves were coated with 8 μL of Acephate emulsion containing 0, 0.5, 1, 2, and 5 weight percent HNTs in water in order to assess the wetting properties of the emulsions on the crop leaves. Data Physics, OCA-25, Germany’s DSA 100 contact angle analyzer was used to measure the droplet contact angle’s magnitude on the leaf surface.

On cotton leaves, an emulsion based on neem oil with a ratio of 2 + 8 ML displays the contact angle trend shown below. Its range of 61.82˚ to 69.08˚ indicates a high degree of wettability. The emulsions with 0.2 and 0.5% HNT concentrations had lower contact angles (61.82˚ & 64.76˚, respectively). In a similar vein, an emulsion with a 7 + 3 ML ratio of water to Neem oil exhibits an irregular contact angle trend. The contact angle first decreases and then slightly increases as the emulsifier concentration rises. The contact angles of the emulsions with 0.05 and 0.2% HNT concentrations were lower than those of the others (48.62˚ and 56.56˚, respectively). Conversely, an emulsion with a 9 + 1 ML ratio of water to Neem oil exhibits exceptional wettability on cotton leaves due to a contact angle that falls between 58.82˚ and 50.07˚. The contact angles of the emulsions containing 0.05 and 0.5% HNT concentrations were lower than those of the others (51.06˚ and 50.07˚, respectively). When the contact angle for cotton leaves is compared, the maize leaves perform better. A Pickering emulsion based on neem oil with a proportion of 8 + 2 ML exhibits excellent wettability. As the concentration of emulsifier HNT increases, the contact angle decreases. Its location ranges from 48.09˚ to 29.0˚. The emulsion with the shortest contact angle and the best hydrophilic nature is the 0.5% concentration. The contact angle for Pickering emulsions with an oil to water ratio of 3 + 7 and 1 + 9 falls between 58˚ and 61˚. For both kinds of emulsions, the contact angle shows an irregular trend. However, contact angle on maize leaves shows a modest degree of wettability. The contact angle for different concentrations of neem oil-based emulsion on cotton and maize leaves is displayed in Figures 8-13.

Figure 8. Contact angle of Neem oil-based Pickering emulsion on Maize leaves with the proportion (8 + 2 ML).

Figure 9. Contact angle of Neem oil-based Pickering emulsion on Maize leaves with the proportion (7 + 3 ML).

Figure 10. Contact angle of Neem oil-based Pickering emulsion on Maize leaves with the proportion (9 + 1 ML).

Figure 11. Contact angle of Neem oil-based Pickering emulsion on Cotton leaves with the proportion (8 + 2 ML).

Figure 12. Contact angle of Neem oil-based Pickering emulsion on Cotton leaves with the proportion (7 + 3 ML).

Figure 13. Contact angle of Neem oil-based Pickering emulsion on Cotton leaves with the proportion (9 + 1 ML).

FTIR Analysis

Fourier transform infrared (FTIR) spectroscopy was used to confirm the successful fabrication of the neem oil-based Pickering emulsion stabilized by halloysite nanotubes (HNTs) and loaded with acephate. Figure 14 describes the FTIR Analysis for Neem oil-based Pickering emulsion.

Figure 14. FTIR analysis for Neem oil-based Pickering emulsion.

The FTIR spectrum of HNT exhibited characteristic aluminosilicate bands. The sharp absorption peaks observed at 3695 - 3620 cm⁻¹ correspond to the stretching vibrations of inner surface Al-OH groups, confirming the structural hydroxyl groups of halloysite. A broad band centered around 3450 - 3200 cm⁻¹ is attributed to hydrogen-bonded O-H stretching of adsorbed water molecules [5][24]-[27]. The band near 1630 cm⁻¹ corresponds to H-O-H bending vibrations of interlayer water. A strong and prominent band in the region of 1080 - 1030 cm⁻¹ is assigned to Si-O-Si stretching vibrations, which is the fingerprint region of aluminosilicate frameworks. Additionally, the band at ~910 cm⁻¹ is due to Al-OH bending, while bands below 600 cm⁻¹ correspond to Si-O and Al-O lattice vibrations. These results confirm the structural integrity of the halloysite nanotubes after formulation.

Neem oil showed characteristic lipid absorption bands. The peaks at ~2925 and ~2855 cm⁻¹ correspond to asymmetric and symmetric stretching vibrations of aliphatic -CH₂ groups, confirming the presence of long hydrocarbon chains. The intense band at ~1740 cm⁻¹ is attributed to the C=O stretching vibration of ester groups present in triglycerides. Additional bands at 1465 - 1375 cm⁻¹ arise from CH₂ bending and CH₃ deformation vibrations. The peaks observed in the range of 1240 - 1160 cm⁻¹ correspond to C-O stretching of ester linkages. An increase in the intensity of these characteristic peaks with higher neem concentration confirms the proportional increase of the oil phase in the emulsion system.

The spectrum of acephate displayed distinct absorption bands characteristic of organophosphate compounds. The band observed around 1650 cm⁻¹ is attributed to amide C=O stretching, while peaks in the region of 1250 - 1020 cm⁻¹ correspond to P=O and P-O-C stretching vibrations. The presence of bands near 650 - 560 cm⁻¹ is associated with P-S stretching vibrations of the phosphorothioate group. These peaks confirm the structural identity of acephate.

In the formulated Pickering emulsion system, characteristic peaks corresponding to HNT (Si-O-Si and Al-OH), neem oil (C-H and ester C=O), [28]-[31] and acephate (amide and phosphate groups) were retained, indicating the successful incorporation of all components. Notably, slight broadening of the O-H stretching band and minor shifts in the carbonyl and phosphate regions were observed, suggesting hydrogen bonding interactions between the surface hydroxyl groups of HNT and the functional groups of neem oil and acephate. The strong Si-O band of HNT partially overlaps with the P-O-C stretching of acephate, indicating possible adsorption or confinement within the nanotube lumen or at the oil-water interface.

Importantly, no new absorption bands were detected, indicating the absence of covalent bond formation and confirming that the encapsulation process is primarily physical in nature. The results demonstrate that acephate is successfully loaded within the HNT-stabilized neem oil Pickering emulsion through adsorption and interfacial stabilization mechanisms without altering the chemical structure of the individual components.

Overall, FTIR analysis confirms the successful preparation of a neem oil-based Pickering emulsion stabilized by halloysite nanotubes and effectively loaded with acephate, with evidence of intermolecular interactions contributing to system stability.

The three-dimensional structure of the freeze-dried emulsion droplets was investigated by SEM. Figure 15 is the images for Neem oil Pickering emulsion without emulsifier, it shows that droplets are irregularly shaped and partially merged, which indicates droplet coalescence, and boundaries between droplets are not sharply defined. Surface of droplets appears rough and disrupted, suggesting instability in the emulsion. The Instability of emulsion may be due to absence of emulsifier or may be due to deformation of droplets. While SEM images with presence of emulsifier HNT (Figure 16) shows that particles are spherical, well defined and uniform in size and shape. Which shows that no coalescence or deformation occurs. Emulsifier HNT adsorbs at oil/water interface by capillary forces and covers oil droplets and prevents them from coalescing. The boundary between droplets and surrounding phase appears sooth and well defined. A more stable emulsion with well-maintained droplet integrity and dispersion is depicted in this image.

Figure 15. SEM images with 0% HNT.

Figure 16. SEM images with 0.2 % HNT.

Figure 17. EDS analysis of Neem oil with 0% HNT.

Table 4. Elemental analysis of Neem oil with 0% HNT.

Figure 18. EDS analysis of Neem oil with 0.2% HNT.

Table 5. Elemental analysis of Neem oil with 0.2% HNT.

The EDS data show a clear compositional shift when halloysite nanotubes are used as an emulsifier. The neem-oil emulsion without emulsifier exhibits 32.6 wt% Si and 48.6 wt% C, (Shown in Table 4) whereas the halloysite-stabilized emulsion shows a marked increase in carbon (59.3 wt%) and a concurrent decrease in silicon (24.7 wt%) and oxygen. (Shown in Table 5). The dominance of the Si peak in both spectra indicates the presence of aluminosilicate material, but the reduced relative Si (and O) signal in the halloysite-containing formulation, together with the higher surface C fraction, suggests a thicker organic overlayer or greater surface coverage of oil/organics on halloysite particles in that sample [32]-[34]. In other words, halloysite nanotubes appear to be well incorporated into the organic phase and covered/coated by the neem oil/acephate matrix, attenuating the detectable Si signal from the particle surface. The small change in Al is consistent with a low absolute Al content and is within typical measurement uncertainty for light Al signals in organic matrices. Overall, the EDS images (Figure 17 & Figure 18) and results are consistent with halloysite acting as a Pickering-type stabilizer that is embedded within or coated by the organic phase, which should enhance interfacial stabilization of droplets compared to the formulation without emulsifier [35]-[38].

Insecticidal Activity:

Spodopterafrugiperda is commonly known as the fall armyworm. It is polyphagous lepidopteran pest which has become a significant threat to agricultural industry all over the globe. Its aggressive feeding behavior during larval stage causes extensive foliar damage, whorl tissue destruction, [38]-[41] and compromised reproductive structures. It also affects photosynthetic efficiency and plant Vigor. Spodopterafrugiperda mainly attacks on plants like Maize, Cotton, Sorghum, rice, sugarcane etc. It has an ability to develop resistance to multiple classes of insecticides [42]-[48]. So, it is essential to create alternative, environmentally friendly pest management techniques because of the negative effects linked to S.frugiperda. Neem oil is an organic pesticide. Its Pickering emulsions are created using water and the insecticide acephate; HNTs stabilize these emulsions. The insecticidal activity of neem oil-based Pickering emulsions was evaluated against larvae of Spodopterafrugiperda using a standard leaf-dip method [49]-[53]. Fresh maize leaves were washed with distilled water and air-dried. Leaves were dipped into respective Pickering emulsions containing varying concentrations of neem oil and HNT-acephate formulations for 30 s and air-dried at room temperature. Each treatment consisted of 10 larvae per replicate, with three independent replicates (n = 30 larvae per treatment). Figure 19 illustrates how treated leaves were put in Petri dishes and how the larvae were left to feed for 24 hours in a controlled laboratory setting (27 ± 2˚C; 65 ± 5% relative humidity).

Controls Negative control: Distilled water-treated leaves, Positive control:Commercial acephate formulation (0.1%).

Larvae were considered dead if no movement was observed upon gentle probing. Table 6 and Table7 demonstrates the Mortality (%) of Spodopterafrugiperda larvae after 24 h exposure to Pickering emulsions. Sample B (2:8 O/W ratio) demonstrated significantly higher larval mortality (86.7 ± 3.1ᶜ %) compared to Sample A (68.3 ± 2.5ᵇ %) and Sample C (52.1 ± 3.8ᵃ %). No mortality was observed in the negative control (distilled water-treated leaves), confirming that larval death was attributable to the treatment formulations. The positive control (commercial acephate 0.1%) exhibited 96.2 ± 1.4ᵈ % mortality.

These findings indicate that the 2:8 O/W ratio provides optimal dispersion and bioavailability of active ingredients, leading to enhanced insecticidal efficacy. Larval mortality increased significantly with increasing concentration of halloysite nanotubes (HNTs). Emulsions containing 0.5% HNT exhibited the highest mortality (91.4 ± 2.2ᶜ %), followed by 0.2% HNT (84.6 ± 2.8ᵇ %). Treatments with 0.1% and 0% HNT showed significantly lower mortality (61.5 ± 3.3ᵃ % and 49.8 ± 4.0ᵃ %, respectively) (p < 0.05). This suggests that higher emulsifier concentration enhances formulation stability and improves sustained release of neem oil and acephate.

Table 6. Mortality (%) of Spodopterafrugiperda larvae after 24 h exposure to Pickering emulsions.

Table 7. Effect of HNT concentration on larval mortality Insecticidal Bioassay.

Figure 19. Larval mortality Insecticidal Bioassay.