Introduction

The International Association for the Study of Pain defines pain as “an unpleasant sensory and emotional experience associated with actual or potential tissue damage”1. This definition acknowledges both the physical and emotional components of pain, reflecting its multifaceted nature. The sensation of pain directly impairs quality of life2,3.

Pain can be categorized according to multiple parameters, including duration and pathophysiology. Based on duration, pain is categorized as acute or chronic. Acute pain constitutes a short-term response to tissue injury and generally reduces as the healing process progresses. In contrast, chronic pain is defined as a persistent discomfort for more than 3 months, often in the absence of tissue damage. This condition should exhibit dynamic patterns, alternating between periods of exacerbation and remission2,3,4.

From a pathophysiological perspective, pain is classified into nociceptive, neuropathic or nociplast subtypes3,4,5. Nociceptive pain arises from the activation of nociceptors in response to noxious stimuli. Neuropathic pain results from structural or functional alterations within the central or peripheral nervous system. Nociplastic pain is characterized by altered nociceptive processing without evidence of tissue injury or lesions in the nervous system3,4.

In many cases, pain management requires a multifactorial approach, involving combined administration of drugs, such as: opioids, antidepressants, analgesics and/or local anesthetics (LAs)3,6. However, the diverse etiology of pain and variability in the treatment response are challenges that must be addressed3,7,8. Many individuals continue to suffer from pain even after receiving significant pharmacological interventions9,10. Furthermore, the growing awareness of the risks associated with prolonged opioids use, including dependence and overdose, limit the use of certain drugs for pain treatment, driving the search for safer and more effective alternative methods11.

The human endocannabinoid system (ECS) is a complex regulatory network responsible for maintaining physiological processes, including sleep, pain, appetite, immune functions and nervous system derived pathologies. It is composed of endogenous mediators and cannabinoids receptors – CB1 and CB212. Active compounds from Cannabis, as cannabidiol (CBD), have gained prominence for its potential therapeutic benefits in the nervous system13,14,15,16. This phytocannabinoid exhibits analgesic effects through allosteric modulation of cannabinoids receptors12,16, reducing the release of excitatory neurotransmitters and inhibiting pain signal transmission16. CBD is a lipophilic molecule12,17, which results in low bioavailability in biological tissues18, compromising the effectiveness of pain management19,20,21. To overcome this limitation, strategies such as drug delivery systems have been developed to enhance absorption and therapeutic efficacy18,19,20,22.

Nanocolloidal systems, such as liposomes, nanoemulsions and lipid nanoparticles, allow the encapsulation of therapeutic agents to be sustained released over time17,18,23,24. It has been demonstrated that the drugs loaded by nanocarriers have ability to improve the efficacy, minimizing side effects25,26,27. In this sense, solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) are lipid-based nanocarriers that have been used for multiple applications in drug and gene delivery17,24,28. These systems have been explored in a wide range of therapeutic fields, such as in neurological disorders, cancer treatment, analgesia, inflammation, autoimmune conditions and immunization strategies26,28,29,30,31,32.

SLN and NLC can be used to encapsulated lipophilic compounds with success, given by its lipid matrices stabilized by surfactant. SLN contain a lipid matrix composed exclusively of solid lipids. In contrast, NLC is based on a blend of solid and liquid lipids at room temperature. Resulting in amorphous or partially crystalline matrix with interstitial spaces that enhance the encapsulation of hydrophobic molecules, without its expulsion over time17,24. The advantages of NLC, such as: biocompatibility, long-term stability, bioavailability improvement, make them a highly attractive system24,33.

Lately, NLC have been formulated using natural excipients, as vegetable butters, waxes and oils, forming the bioactive lipid matrices24,31. Such excipients exert dual role – structural and bioactive – contributing to the structural integrity of nanoparticle, while also provides therapeutic properties30,34. Therefore, the incorporation of CBD oil into NLC represents a promisingapproach to enhance its administration by several routes and optimize the analgesic efficacy, being useful for pain management18,20,23.

The aim of this work was to develop NLC formulations composed of palm butter and CBD oil as a nanoherbal strategy for pain management. It was monitored the long-term stability over a year (25 °C) of the systems, in terms of particle size (nm), polydispersity index (PDI) and Zeta potential (mV). The in vivo nanotoxicity and efficacy assays were determined by chicken embryo and Drosophila melanogaster biological alternative models, respectively.

Results

Long-term physicochemical stability

NLC and NLC/CBD formulations were evaluated regarding particle size (nm), PDI and Zeta potential (mV) at predefined time points over 365 days at 25 °C (0, 7, 15, 30, 60, 90, 120, 150, 180 and 365 days). Initially, the average particle size was 383.80 ± 12.80 nm for NLC and 236.3 ± 3.40 nm for NLC/CBD, as shown in Fig. 1. After 365 days, these values were 369.80 ± 0.40 and 235.70 ± 2.00 nm, respectively. No statistically significant intragroup variation (p < 0.05) was observed during the monitoring period.

Fig. 1
Fig. 1The alternative text for this image may have been generated using AI.
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Long-term physicochemical stability of NLC and NLC/CBD formulations in terms of particle size (nm) over 365 days at 25 °C. One-way ANOVA/Tukey post hoc were used to analyze intragroup statistics; p < 0.05, n = 3.

Figure 2 demonstrated that NLC/CBD exhibited PDI ≤ 0.2, indicating homogeneous particle size distribution. Initially, PDI values were 0.221 ± 0.01 for NLC and 0.153 ± 0.01 for NLC/CBD. After 365 days, these values were 0.250 ± 0.01 and 0.148 ± 0.02 for NLC and NLC/CBD, respectively. No statistically significant intragroup differences (p < 0.05) were observed over time.

Fig. 2
Fig. 2The alternative text for this image may have been generated using AI.
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Long-term physicochemical stability of NLC and NLC/CBD formulations in terms of polydispersity index over 365 days at 25 °C. One-way ANOVA/Tukey post hoc were used to analyze intragroup statistics; p < 0.05, n = 3. PDI: polydispersity index.

Figure 3 displayed the Zeta potential (mV) values over a year, exhibiting statistically significant intragroup variations (p < 0.05) for both formulations. After a year, the mean Zeta potential values were − 30.50 ± 1.10 mV for NLC and − 32.30 ± 0.70 mV for NLC/CBD.

Fig. 3
Fig. 3The alternative text for this image may have been generated using AI.
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Long-term physicochemical stability of NLC and NLC/CBD formulations in terms of Zeta potential (mV) over 365 days at 25 °C. One-way ANOVA/Tukey post hoc were used to analyze intragroup statistics; p < 0.05, n = 3.

Morphological analysis using FE-SEM

Field Emission Scanning Electron Microscopy (FE-SEM) analysis was employed to characterize the morphology of the prepared NLC. Figure 4 presented FE-SEM images of the NLC (A) and NLC/CBD (B) formulations, revealing spherical nanoparticles with well-defined contours.

Fig. 4
Fig. 4The alternative text for this image may have been generated using AI.
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Field Emission Scanning Electron Microscopy (FE-SEM) images of NLC (A) and NLC/CBD (B) at 1390× magnification.

In vivo nanotoxicity assay using chicken embryo model

Chicken embryos (CE) were treated with NLC, CBD emulsion as control and NLC/CBD at 6 different concentrations (23.00, 11.50, 5.75, 2.87, 1.43, and 0.72 mg/mL). Figure 5 illustrated the mortality rate (%) of the CE. At the highest concentration tested (23.00 mg/mL), all treatments resulted in embryo mortality.

For NLC, the mortality increased at 11.50 mg/mL, whereas CBD and NLC/CBD maintained comparatively lower mortality rates in the same concentration. Moreover, at lower concentrations (0.72–5.75 mg/mL), all treatments induced low to moderate mortality rates, generally below or around 20%.

Overall, the results indicated a concentration-dependent effect, with acceptable safety observed at lower concentrations and increased embryotoxicity at higher doses. Particularly, NLC/CBD at 2.87 mg/mL demonstrated biocompatibility, with no evident acute toxicity effects in CE.

Fig. 5
Fig. 5The alternative text for this image may have been generated using AI.
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In vivo toxicity study in terms mortality (%) of chicken embryos treated with NLC, cannabidiol emulsion (CBD) and NLC/CBD at different concentrations. CN: treated with 0.85% NaCl (n = 5); NLC: NLC without cannabidiol (n = 27); CBD: cannabidiol oil emulsion (n = 29); NLC/CBD: NLC with cannabidiol oil (n = 30). Chi-square test was used followed by the difference between two proportions test (p < 0.05).

Fig. 6
Fig. 6The alternative text for this image may have been generated using AI.
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Showed the incidence of severe lesions in CE. NLC (control) induced the highest percentage of severe lesions, with range values exceeding 70%. In contrast, CBD resulted in lower lesion severity, with percentages remained below 50%. On the other hand, NLC/CBD exhibited a concentration-dependent increase in severe lesions but showed reduced lesion incidence compared to NLC.

Figure 6: In vivo toxicity study in terms of severe lesions in chicken embryos treated with NLC, cannabidiol emulsion (CBD) and NLC/CBD at different concentrations. CN: treated with 0.85% NaCl (n = 5); NLC: NLC without cannabidiol (n = 27); CBD: cannabidiol oil emulsion (n = 29); NLC/CBD: NLC with cannabidiol oil (n = 30). Chi-square test was used followed by the difference between two proportions test (p < 0.05).

Figs. 7
Figs. 7The alternative text for this image may have been generated using AI.
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Indicated the weight changes in CE and annexes. Compared to the NC, none of the treatments showed statistically significant changes. The nanotoxicity assessment in CE showed that the CBD-based nanoparticles did not interfere with embryo or extraembryonic tissue weight at the tested concentrations.

Figure 7: In vivo toxicity in terms of the changes of weight and annexes of chicken embryo under treatment with NLC, cannabidiol emulsion (CBD) and NLC/CBD at different concentrations. CN: treated with 0.85% NaCl (n = 5); NLC: NLC without cannabidiol (n = 27); CBD: cannabidiol oil emulsion (n = 29); NLC/CBD: NLC with cannabidiol oil (n = 30). One-way ANOVA/Tukey tests were used to analyze the intergroup statistical difference (p < 0.05).

Nociceptive activity assessment

Figure 8 displayed the results of nociception assay on Drosofila melanogaster. The larvae treated with NLC at 2.0 mg demonstrated a prolonged nociception response time (p = 0.0036). Additionally, NLC/CBD containing 1.0 mg of CBD was also able to delay the nociception response time in the larvae (p = 0.0077). These results suggested that both NLC (2.0 mg) and NLC/CBD (1.0 mg) exhibited analgesic activity compared with the NC group. No significant differences were observed between CBD emulsion and NLC/CBD.

Fig. 8
Fig. 8The alternative text for this image may have been generated using AI.
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Nociceptive assay of Drosophila melanogaster larvae treated with NLC, cannabidiol emulsion (CBD) and NLC/CBD at different concentrations (1.0 and 2.0 mg/mL). CN: treated with 0.85% NaCl; NLC: NLC without cannabidiol; CBD: cannabidiol oil emulsion; NLC/CBD: NLC with cannabidiol; (n = 20). Statistical analyses were carried out by ANOVA and Tukey post hoc tests (p < 0.05).

Discussion

The development of more biocompatible and effective pain management therapy is still a challenge. CBD have shown the ability to decrease the painful symptoms in different efficacy model assays14,16,20. In this work, it was proposed a pre-formulation study of NLC loading CBD that demonstrated control quality, safety and efficacy analyzed by different alternative biological models.

The control quality of a pharmaceutical product will determine the shelf life of formulations under specific conditions. In general, the measurements of particle size (nm), PDI and Zeta potential (mV) of nanocolloids may be monitored. These parameters are crucial for understanding the stability, biodistribution and therapeutic efficacy of nanoparticles34,35. Several factors, including surface properties, colloidal stability and composition are critical determinants of biodistribution, safety and therapeutic performance36.

The incorporation of CBD oil in the lipid matrix decreased the nanoparticle size, as expected. The presence of oil reduced the viscosity and the crystallinity of the solid lipid in the matrix, forming internal structural imperfections24. Thus, CBD oil exerted a dual role in the system, acting both as the bioactive compound, responsible for the therapeutic effect, and as a structural excipient that contributed to the particle size reduction, improving the size uniformity34.

Specifically, in the case of nanocolloids, the limit size range considered for parenteral administration is up to 250 nm, given by the biodegradability and hydrophobicity properties34. In here, it was reached suitable particle size even using low surfactant concentration, once it is strongly correlated with the toxicity of nanosystems37. In addition, lipid nanoparticles have high affinity to interact with the lipid bilayer of biological membranes, allowing the permeation of CBD with success. The reports regarding CBD-loaded NLC were effective for brain or local delivery with particles in the range of approximately 100–250 nm17,18,19,38. Furthermore, NLC/CBD showed an homogenous size distribution, even after a year with PDI values close to 0.1, indicating homogeneous dispersion, whereas values higher than 0.2 are related to heterogeneity30,31,39.

Zeta potential is a measure of the electrokinetics potential of nanoparticles and is directly related to colloidal stability34,39,40. Nanoparticles with positive or negative Zeta potential are considered cationic or anionic, respectively39. Although a positive surface charge can enhance cellular uptake, they are often associated with increased cytotoxicity, hemolysis and nonspecific interactions with plasma proteins and DNA. Still, all the cationic surfactants are more toxic than anionic and non-ionic surfactants. For these reasons, the most of lipid nanoparticles are anionic41,42. Fluctuations in Zeta potential values are inherent to the dynamic nature of colloidal systems, reflecting their structural complexity30,31,34. Despite variations in such values observed for NLC and NLC/CBD, no signs of physicochemical instability were detected over a year of storage, indicating effective steric stabilization.

FE-SEM images of NLC and NLC/CBD revealed spherical particles, as expected for this type of system. However, particle sizes observed in FE-SEM micrographs were higher than those measured by DLS. This discrepancy is widely reported in the literature. FE-SEM and DLS have different principles and require distinct sample preparation procedures. DLS measures the hydrodynamic diameter of nanoparticles in dispersion through Brownian motion. In contrast, FE-SEM analysis is performed with dried samples followed by a gold bath that cover all the samples (sputtering process), which increase the nanoparticles sizes and can promote particle aggregation36,39. Therefore, DLS results is used to determine particle size distribution, which is directly correlated with efficacy and biodistribution39,43.

The monitoring of the biophysical properties of the formulations confirmed the long-term physicochemical stability for at least 12 months of storage at 25 °C. These results indicated suitable structural properties for administration via different routes, including parenteral delivery, pending further in vivo safety and pharmacokinetic investigations. Moreover, the scalable production technology employed for NLC/CBD, together with the absence of special requirements for storage, transport and distribution, makes this system particularly promising in terms of economic viability.

Assessing the toxicity of nanoparticles intended for biomedical applications is essential to ensure the safety of the system44. In vivo nanotoxicity assessments utilizing CE model constitute a pivotal phase in evaluating the biocompatibility and safety profile of nanoparticles. This is an accepted model for nanotoxicity assays, due to their ease of handling, rapid development and well-understood physiology44,45.

An important aspect of nanotoxicity assays is the analysis of weight variation in embryos and their annexes, such as the yolk sac and chorioallantoic membrane, which provide quantitative data on the impact of nanoformulations on embryo growth and development. This allows for the identification of potential adverse effects, such as growth retardation or developmental abnormalities34,44. The mortality rate of embryos exposed to nanoformulations provides a direct measure of acute toxicity45.

In this work, the pattern of severe lesion formation was directly correlated with the embryo mortality indexes, reinforcing a clear concentration-dependent toxicological response. At lower concentrations – 1.43 and 2.87 mg/mL – NLC/CBD has induced a low incidence of severe lesions, which is consistent with the reduced mortality observed at these doses. These results indicated that NLC/CBD is well tolerated at lower concentrations, whereas higher doses lead to progressive tissue damage and embryotoxicity, highlighting the importance of dose optimization in future in vivo studies.

Particularly, the dose treatment at 2.87 mg/mL indicated that NLC/CBD induced a relatively low percentage of severe lesions and mortality in CE, suggesting a improve of tolerability associated with the CBD encapsulation. These results indicated that nanoencapsulation of CBD modulated the biological response, reducing lesion severity at lower concentrations34.

The efficacy of nanoparticles was evaluated through a nociception assay in DM larvae. This in vivo model is widely recognized for assessing nociceptive responses, due to its genetic simplicity and capacity for high-throughput experiments. Conducting a nociception assay in DM larvae allows the evaluation of the impact of nanoparticles on pain perception and nociceptive pathways46,47.

Stage III DM larvae has well-characterized nociceptive systems, due to their class IV sensory neurons, which share similarities with those of mammals, facilitating the extrapolation of results to other organisms. The nociception assay in the alternative DM model provides critical information on potential adverse effects of nanoparticles, especially if they are being developed for therapeutic applications in humans46,47.

Notably, it was determined the analgesic effect of DM starting from 1.0 mg/mL of NLC/CBD treatment, as evidenced by the modulation of the larval nociceptive response34. In addition, this effective concentration of NLC/CBD was considered safe in the nanotoxicity assay (up to 2.87 mg/mL). Interestingly, NLC control composed of palm butter, has also demonstrated analgesic activity on DM. In this case, a synergistic effect can be observed and should have more deeply investigated.

There are a few works that have developed nanocarriers as micelles, vesicles, nanoemulsions and lipid nanoparticles for CBD encapsulation, demonstrating its potential analgesic for topical and brain applications. Such results reinforced the advantages of nanostructured-based CBD delivery systems in optimize its therapeutic effect18,19,20,23. This work was pioneer in the evaluation of antinociceptive activity of herbal NLC using alternative biological models. It is worth mentioning that such technology was recently patented by us.

Finally, these results supported the potential of NLC/CBD to modulate nociceptive responses more effectively than non-encapsulated CBD, particularly at lower doses, reinforcing this strategy for pain management. Moreover, further investigation in more complex biological models and clinical assays should be conducted to obtain robust data reinforcing its potential clinical use.

Conclusions

Pain is manifested in different ways impacting the quality of life. The traditional treatments are highly toxic to the cardiovascular and central nervous systems. Lately, cannabidiol (CBD) has attracted attention for its analgesic potential. Therefore, the development of NLC formulations represents a strategy to enhance the bioavailability of CBD, optimizing therapeutic properties for pain management.

In this work, the NLC/CBD demonstrated shelf life of at least a year when stored at 25 °C. Moreover, the safety of the system was confirmed by a nanotoxicity test on chicken embryo model, which showed biocompatibility up to 2.87 mg/mL. Finally, the efficacy assay conducted on Drosophila melanogaster larvae model showed that NLC/CBD at 1.0 mg/mL was able to improve the nociception time of the larvae, suggesting its analgesic potential. These findings selected a safe and efficient NLC/CBD concentration to be further testing in more complex biological models and clinical trials to find a novel system for pain management.

Materials and methods

Preparation of nanostructured lipid carriers

The hot emulsification method was used to prepare the control nanoparticles (NLC) and the CBD oil nanoparticles (NLC/CBD). The lipid phase, consisting of palm butter (5%; w/v) and cannabidiol oil (5%; w/v), was heated in a water bath 10 °C above the melting point of palm butter (~ 47 °C). Simultaneously, the aqueous phase composed of poloxamer 188 (3%; w/v) was heated to the same temperature. Then, it was gradually added to the lipid phase under stirring at 10,000 rpm for 2 min using an Ultra-Turrax® T18 homogenizer. The resulting microemulsion was subsequently subjected to ultrasonication for 10 min. Following this process, the resulting nanoemulsion was cooled in an ice bath to 25 °C to form NLC. All nanoparticles were successfully obtained as solid particles exhibiting a milky, colloidal appearance and were stored at 25 °C until further use31,34.

Physicochemical stability study of the formulations

Particle size (nm), PDI and Zeta potential (mV) were analyzed using dynamic light scattering (DLS) with the LiteSizer 500 (Anton Paar) equipament. To assess the quality control of the NLC and NLC/CBD formulations, these parameters were monitored over 365 days at 25 °C. Statistical analyses were performed using ANOVA and Tukey’s post hoc test to determine significant intragroup differences over time (p < 0.05) (19,23). Origin (USA) software was used for these analyses30.

Nanoparticle morphology

The morphology of the NLC and NLC/CBD nanoparticle samples was evaluated using Field Emission Scanning Electron Microscopy (FE-SEM). A drop of each sample was added to a glass coverslip previously mounted on an aluminum stub. After complete solvent evaporation, the stubs were coated with a conductive layer using sputtering for 120 s at 30 kV. The nanoparticles were then visualized using a Zeiss EVO MA10 SEM with secondary and backscattered electron detectors, operating in high vacuum at 20 kV30,48.

In Vivo nanotoxicity assay on chicken embryo model

The nanotoxicity of the formulations and their respective controls was evaluated through on vivo chicken embryo model (CE), according to the following parameters: mortality (%), change in embryo weight and weight of annexes (g) (yolk, chorioallantoic membrane, and amnion)31,44. NLC/CBD (5%; w/v) was compared with different control solutions : 0.85% saline solution (NC); cannabidiol oil emulsion (5%; w/v) with poloxamer 188 (3%; w/v) (CBD); and NLC control (without cannabidiol) with poloxamer 188 (3%; m/v) and palm butter (5%; m/v).

91 eggs of Gallus gallus eggs (W-36 lineage) were used. Prior to analysis, eggs were submitted to ovoscopy to ensure the embryo viability at 7 days of development31. Eggs were weighed and divided into 4 groups: negative control (NC) (n = 5), treated with saline solution; NLC control (n = 27); CBD (n = 29); and NLC/CBD (n = 30). All treatments were administrated on embryo chorioallantoic membrane (CAM). For NLC, CBD and NLC/CBD the following concentrations were tested: 23, 11.5, 5.75, 2.87, 1.43, and 0.72 mg/mL. Then, the eggs were incubated in automatic incubator (Premium Ecológica®) at 37.5 °C and 55% air humidity for 7 days31.

Embryo mortality was monitored daily to determine viability (%). At days 14 of embryonic development, the eggs were weighed. Then, embryos and their annexes were individually weighed31. Weight changes in embryos and associated tissues were calculated based on the difference between the egg’s weight before and after treatment, according to the following equation:

$${\rm aW = (ce.ysW \times 50) \div ieW}$$
(1)

where: aW: adjusted egg weight for 50 g; ce.ysW: weight of the embryo or annex; ieW: initial egg weight31,44.

One-Way ANOVA/Tukey tests were used to elucidate the intergroup statistical differences, in terms of chicken embryo weight changes (p < 0.05). For the chicken embryo viability test, chi-square test was used followed by the difference between two proportions test (p < 0.05). GraphPad Prism 8 and Origin (USA) software were used31.

Assessment of nociceptive activity on Drosophila melanogaster model

The Drosophila melanogaster (DM) w1118 (white) was acquired from the Bloomington Stock Center at Indiana University, USA. The flies were maintained in vials containing ¼ of Bloomington culture medium (1500 mL water, 27g yeast, 15g soy flour, 109.5g cornmeal, 9g agar, 115.5g glucose syrup, acid and nipagin solution) in a BOD incubator at 25 °C, 70% relative humidity, and a 12:12 h light-dark cycle49. All formulation concentrations were diluted in the medium before introducing the flies.

The analgesic activity of the formulations were evaluated using a nociception test in third-instar DM larvae46,47. 2 concentrations – 1.0 and 2.0 mg/mL) of emulsion, NLC control and NLC/CBD were analyzed. Additionally, a negative control (NC) feeded with Bloomington culture medium with distilled water was evaluated.

Groups of 30 flies (15 females, 15 males) were fed with Bloomington culture medium with NLC formulations or control solutions (i.e. emulsion). The flies were maintained in BOD for 24 h. Then, were removed, anesthetized with ethyl ether and sacrificed. Vials were maintained for 72 h until reaching the third larval stage, which allows nociceptive analysis due to the presence of class IV type II sensory neurons46,47.

At the third-instar, 10 larvae per group were individually placed in Petri dishes for 10 s to explore the environment. Then, a chemical stimulation, with HCl (9%; w/v), was administrated to the posterior of larva and their behavioral responses was recorded by video (30 frames/s). A complete roll of 360° the body axis of DM larvae was considered an aversive behavior. Other responses such as rapid locomotion and partial rotation were excluded47. The experiment was conducted in duplicate and each larva was tested once.

Behavioral responses of DM larvae were recorded following HCl exposure (a pain-inducing agent). The nociceptive time (s) was defined as the time until a pain-like behavior and determined by video recording (30 frames/s)47. ANOVA/Tukey post hoc statistical test were used to analyzed intergroup differences (p<0.05). GraphPad Prism 8.0 (USA) was used for these analyses.