The effects of gold nanoparticles and nano-emulsions synthesized from cannabidiol oil on oxytocin-induced uterine contractions in rats

This study investigates the development of innovative, naturally derived therapeutic agents for managing uterine contractions, which have significant implications for tocolysis and dysmenorrhea treatment. Three nano-formulations were evaluated: chemically synthesized gold nanoparticles (CS-AuNPs), green-synthesized gold nanoparticles using cannabidiol oil (CBDO-AuNPs), and a CBD nano-emulsion (CBDO-NE2). Advanced analysis techniques, including UV-Vis spectrophotometry, SEM-EDX, XRD, FTIR, and DLS, were employed for the characterization. These NPs were mostly spherical, ranging in size from 10 to 213 nm. Using an isolated tissue bath system with rat uteri, the study compared the antispasmodic effects of these nanoformulations with those of CBD on oxytocin-induced contractions. Results demonstrated that CS-AuNPs and CBDO-AuNPs significantly outperformed CBD across all contractile parameters (p < 0.004). Notably, CS-AuNPs exhibited the highest potency due to direct interactions with cell membranes and ion channels, as well as the influence of citrate stabilizers. In contrast, the CBDO-NE2 showed more selective effects, significantly affecting the cumulative area under the curve (AUC, p = 0.001) but not the contraction frequency (BPM, p = 0.222). These findings suggest that the physical and chemical properties of the nano-delivery system influence the therapeutic potential of CBD. The synthesized nano-formulations offer a promising approach to enhancing bioavailability and antispasmodic potency, indicating the need for subsequent in vivo clinical evaluation.

The uterus plays a fundamental function in the life of a woman, facilitating various reproductive processes. Uterine contractions play a vital role in numerous reproductive processes, including sperm and embryo transfer, the menstrual cycle, gestation, and parturition (Myers and Elad, 2017; Yaman et al., 2018). Electrical impulses generated at the cellular level induce spontaneous contractions within the smooth muscle of the uterus (Rosen and Yogev, 2023). Aberrant activity of the uterus may lead to dysmenorrhea and the onset of preterm labor. Although anti-inflammatory pharmacological agents are employed to alleviate dysmenorrhea, which is characterized by painful menstrual cramps, this therapeutic approach is ineffective in approximately 20%–25% of instances. Moreover, chronic administration of these medications has been reported to elicit detrimental effects on renal, hepatic, and gastrointestinal systems (Ameen, 2023). Preterm labor constitutes a significant contributor to both stillbirth and neonatal mortality.

Tocolysis, defined as the inhibition of uterine contractions, possesses the capacity to mitigate the incidence of preterm labor. Systemically administered tocolytic agents, such as indomethacin, carry inherent risks of transplacental passage, potentially causing adverse fetal complications alongside their intended uterine relaxation effects. Currently, the development of biocompatible targeted nano-delivery systems is the focus to overcome these risks (Xin et al., 2024).

In contemporary research, naturally derived nanocarriers have been engineered to treat various diseases, including cancer. The distribution and effectiveness of nanocarriers in the body vary depending on their source, size, shape, surface charge, and surface chemistry (N’Dea et al., 2021). For instance, lipophilic and positively charged nanoparticles (NPs) tend to pass through cell membranes more readily, leading to greater toxicity due to rapid clearance and tissue accumulation. Thus, the characteristics of NPs must be carefully investigated to balance tissue uptake, toxicity, and effect on the treatment of dysmenorrhea and preterm labor (Figueroa Espada et al., 2020). The effects of NPs on muscle contractions have been studied (Vidanapathirana et al., 2018; Yaman et al., 2018; Oueslati and Ben, 2021; Tsymbalyuk et al., 2021). Investigating the distribution of NPs in the tissues of pregnant mice or rats, AuNPs were primarily selected as carrier agents. This selection was based on the fact that AuNPs allow precise surface modification, size control during synthesis, and highly sensitive quantification of gold accumulation in tissues using inductively coupled plasma mass spectrometry (ICP-MS) (N’Dea et al., 2021). Studies have reported size-dependent accumulation of AuNPs in reproductive tissues. A survey by Semmler Behnke et al. showed that anionic AuNPs of 1.4 nm and 18 nm crossed the placenta and accumulated in fetal tissue (Semmler Behnke et al., 2014).

In contrast, the AuNPs larger than 80 nm may be used for drug delivery to maternal tissues (Semmler Behnke et al., 2014). Regarding uterine muscle contraction, Yaman et al. reported that SiO 2 NPs decreased contraction frequency and energy consumption in rats, particularly at higher concentrations (Yaman et al., 2018). Tsymbalyuk et al. found that chronic exposure to ZnO and TiO 2 NPs induced uterine contractions but not spontaneous pacemaker activity (Tsymbalyuk et al., 2021). Vidanapathirana et al. indicated that in the latter stages of pregnancy, citrate-stabilized AgNPs may adversely affect maternal uterine vascular tissue contractility and influence fetal weight, in contrast to the reactions observed after exposure to PVP-stabilized AgNPs (Vidanapathirana et al., 2018). Among metallic NPs, AuNPs are widely used in medical applications as biocompatible materials and drug-delivery platforms, due to their nanodimensions and ability to penetrate cellular structures. Cannabinoids of natural origin, particularly cannabidiol (CBD), possess the capability to induce relaxation in uterine smooth muscle by modulating myometrial contractility. However, the precise receptor levels and ion channel pathways mediating this response remain a subject of debate. CBD exhibits a complex pharmacological profile, interacting not only with the cannabinoid receptors (CB1 and CB2) but also modulating transient receptor potential channels (such as TRPV1), and directly or indirectly influencing voltage-gated calcium channels (Olivas Aguirre et al., 2022). While CB1 receptors are expressed in the non-neuronal tissues including the uterus, the acute antispasmodic effects of CBD might be heavily reliant on the desensitization of TRPV1 channels and the subsequent inhibition of extracellular calcium influx, which is crucial for oxytocin-induced sustained contractions (Lefebvre et al., 2024). CBD oil, extracted from the hemp plant, contains CBD, which is a therapeutic agent used to treat many diseases, such as cancer, diabetes, inflammatory pathological disorders, chronic pain, and neurodegenerative disorders. Despite its therapeutic effects, CBD’s highly hydrophobic nature limits its use by reducing its bioavailability. To address this concern, nano-emulsion (NE) formulations can be used to maximize the surface-to-volume ratio, thereby enhancing tissue permeability and retention (Khorsand et al., 2025), facilitating transport across biological membranes, and improving the bioavailability of lipophilic compounds (Liu et al., 2024).

The principal objective of this study is to synthesize an innovative, naturally derived nanotherapeutic agent for the management of preterm labor (tocolysis) and dysmenorrhea. Therefore, this study is the first to synthesize AuNPs from CBD oil and evaluate the effects of CBDO-NE2 and CBDO-AuNPs on uterine muscle activity. The nanoformulations of CBDO-AuNPs and CBDO-NE2 are expected to improve bioavailability, tissue-targeting specificity, and sustained activity relative to CBD. Additionally, chemically synthesized gold nanoparticles (CS-AuNPs) were used to evaluate the effect of CBD-free nanoparticles on biological activity. Assessing the impacts of these nano-based agents on uterine smooth muscle contractility will enable the development of more effective and targeted antispasmodic therapies.

Chemicals

40% CBD (without THC) oil was obtained from Naturecan, Turkey. Gold (III) chloride trihydrate (HAuCl 4 ·3H 2 O) (purity, >%99.9), trisodium citrate, ethanol, and Tween 20 were purchased from Sigma-Aldrich (USA). D-glucose, MgSO 4, CaCl 2, and KCl were purchased from Merck (Darmstadt, Germany), while NaCl and KH 2 PO 4 were obtained from Isolab Laborgeräte GmbH (Germany). Deionized water was used throughout all experimental procedures.

Chemical synthesis of CS-AuNPs

The synthesis of CS-AuNPs was conducted using the Turkevich method, described by Niyomthai, Supaphol, and Niyomthai (Niyomthai et al., 2022) with some modifications. In this method, citrate was used to reduce Au 3+ ions to Au 0 at high temperatures, acting as both a capping and a stabilizing agent. During the synthesis, a 22-W daylight fluorescent lamp (as a natural light source) was used. For the preparation of CS-AuNPs, the effects of the initial HAuCl 4 ·3H 2 O concentration and reaction temperature were systematically studied (Shah et al., 2024). The synthesis and optimization steps are summarized in the Supplementary Material.

Biosynthesis of CBDO-AuNPs

For the green synthesis of CBDO-AuNPs, 40% CBD oil was used. Initially, a CBD microemulsion was prepared by mixing CBD oil with 0.5% (v/v) ethanol. The prepared CBD microemulsion was subsequently added to 50 mL of a 1 mM HAuCl 4.3H 2 O solution at a 1:10 (v/v) ratio. The resulting mixture was kept under visible light and on a heater for 60 min. The solution color changed from light yellow to gray-lilac, indicating the formation of AuNPs. This AuNP solution was centrifuged at 6500 rpm for 7 min, washed thoroughly with deionized water and ethanol to remove impurities, and the resulting CBDO-AuNPs were dried in the oven at 30 °C.

Preparation of CBDO-NEs

Deionized water, CBD oil, and Tween 20 (as an emulsifier) were used to prepare the CBDO-NEs. The prepared 1% Tween 20 solution (v/v) and CBD oil were mixed at different volumetric percentages (V Tween20 /V oil: 1/1, 1.5/1, 2/1). The emulsifier concentration was planned to be between 0.5% and 2% (v/v), and the sonication time was 500 s. Sonication was applied for the specified time at 98% power setting using the SONICS Vibra-cell brand CV18 model ultrasonic homogenizer (130 W, 20 kHz). To prevent overheating, the samples were kept in an ice/water bath throughout sonication (Kampa et al., 2022; Grifoni et al., 2023). The compositions of the prepared CBDO-NEs are given in Table 1.

Characterization of the CS-AuNPs, CBDO-AuNPs, and CBDO-NE2

Various analytical techniques, such as ultraviolet-visible spectroscopy (UV-vis) (Thermo Scientifc™, United States), scanning electron microscope (SEM) (JEOL-JSM-7001F, Japan) coupled to an energy dispersive X-ray spectroscopy (EDX), Fourier transform infrared (FTIR) spectroscopy (Perkin-Elmer Spectrum Two FT-IR), X-ray powder diffraction (XRD) (Rigaku Smart Lab X-ray diffractometer), were used to confirm the synthesis of the prepared nano-formulations. 2θ angle scanning was performed using the XRD device with a 40 kV voltage and a 30 mA tube current, coupled with a Cu K-beta tube (λ: 1.54 Å). The synthesized nanoformulations were coated with Au-Pt prior to SEM analysis. Average surface charge measurements and size distributions of CBDO-AuNPs and CBDO-NE2 were conducted utilizing Malvern Nano Zetasizer (Malvern Instruments, Serial number: MAL1124417).

Preparation of uterine tissue samples

A total of 40 uterine tissue samples were obtained from 10 female Wistar rats (250–300 g) housed at the Experimental Animal Application and Research Center of Ondokuz Mayıs University. In each group, 10 rats were included (n = 10 rats in total, each contributing one tissue to each treatment group). The animals were maintained under a constant 12-h light-dark cycle and an environmental temperature of 21 °C–23 °C. Food and water were available ad libitum. The animals, all of which had experienced at least one pregnancy, were euthanized under high-dose ketamine (300 mg/kg) and xylazine (30 mg/kg), IP anesthesia.

Uterine tissues excised from each animal were carefully dissected in Krebs solution under laboratory conditions. The samples were prepared as strips measuring 1 cm × 0.5 cm × 0.2 cm. Each tissue strip was meticulously weighed using a precision balance (Shimadzu, Japan), and the recorded weights were documented. The tissue strips were subsequently mounted vertically and affixed to force transducers (Commat, Ankara, Türkiye) under a baseline tension of 2 g. Experimental procedures were conducted in 10 mL tissue baths (Commat, Ankara, Türkiye) under controlled conditions: a gas mixture of 95% O 2 and 5% CO 2, pH 7.4, and 38 °C. The tissue samples were incubated under these conditions for 1 h. The bath solution was refreshed with fresh Krebs solution every 15 min during the incubation.

Measurement of uterine muscle activity

A total of 40 uterine muscle strips were used to evaluate the effects of four different formulations (CBD, CBDO-NE2, CBDO-AuNPs, and CS-AuNPs). Each tissue sample was incubated for 1 h, during which the bath solution was refreshed with fresh Krebs solution every 15 min. Four strips were tested simultaneously in four separate baths. Once the system reached stability, muscle activities were recorded using an isometric transducer, and passive tension was applied to the preparation. During a 20-min observation period, tissue viability was assessed by spontaneous contractions, and recordings were obtained using a data acquisition system (Biopac, United States). Subsequently, to induce uterine contractions, 0.5 nM oxytocin (Sigma-Aldrich, Germany) was added to each bath (Das et al., 2012). This concentration was determined based on preliminary experiments. Following the induction of maximum contractions with oxytocin, the experimental drug applications were administered. Each drug was tested individually on each tissue strip. Once the plateau of maximum oxytocin-induced contractions (0.5 nM) was reached, the baseline contractile state (amplitude, frequency, and cumulative AUC) for each separate tissue sample was normalized to exactly 100%. The inhibitory effects induced by cumulative experimental doses (1 × 10 −9 M to 2 × 10 −4 M) were calculated as a percentage reduction relative to each tissue’s individual oxytocin plateau. This normalization strategy effectively corrected for inherent biological variations between individual tissue strips.

The following concentrations were utilized in the experimental applications:

1 × 10 −9, 1 × 10 −8, 1 × 10 −7, 1 × 10 −6, 1 × 10 −5, 2 × 10 −5, 3 × 10 −5, 1 × 10 −4, and 2 × 10 −4 M. Following each dose application, contractions were recorded for 20 min. The area under the curve (AUC), number of peaks per minute, frequency (BPM), and peak amplitudes (P MAX) were calculated as mean ± standard error of the mean (SEM) from 40 uterine tissue samples obtained from 10 female rats.

Composition of physiological salt solution

According to established protocols, Krebs’ solution was freshly prepared daily (McCafferty et al., 2007). This solution was used both for tissue incubation and transportation. The composition of the Krebs solution was as follows: 118 mM sodium chloride (NaCl), 4.7 mM potassium chloride (KCl), 1.2 mM calcium chloride dihydrate (CaCl 2 ·2H 2 O), 1.2 mM magnesium sulfate heptahydrate (MgSO 4 ·7H 2 O), 25 mM sodium bicarbonate (NaHCO 3), 1.2 mM potassium dihydrogen phosphate (KH 2 PO 4), and 11 mM D-glucose monohydrate (C 6 H 12 O 6 ·H 2 O). The pH of the solution was adjusted to 7.4 by adding 0.1 M hydrochloric acid (HCl). The isolated tissue bath system was maintained at 38 °C and aerated with a gas mixture of 95% O 2 and 5% CO 2.

Statistical analysis

The inhibitory concentrations (IC 50) of CBD, CBDO-NE2, CBDO-AuNP, and CS-AuNP, as well as comparisons of AUC, BPM, and P MAX values, were calculated using nonlinear regression analysis in Prism software (GraphPad, version 10.3.1). Before performing variance analysis, the Shapiro-Wilk test was applied to assess the normality of the data distribution. AUC, BPM, and PMAX values were compared across formulations using a one-way analysis of Variance (ANOVA) followed by Tukey’s post hoc test. Results were reported as mean ± SEM with 95% confidence intervals (CIs). A significance level of p < 0.05 was considered statistically significant, and the F test was used for the analysis.

Characterization of the CS-AuNPs

The color changes that occur during the synthesis of CS-AuNPs are given in Supplementary Figures S1, S2 shows the UV-Vis spectra, SEM images, and particle size distribution of CS-AuNPs synthesized using 0.25 mM HAuCl 4 at two different temperatures (45 °C and 75 °C). According to the UV-vis spectra, the observed peak in the 500–600 nm wavelength range indicates the reduction of Au 3+ to AuNPs, thus synthesizing CS-AuNPs. UV-vis spectra revealed that the synthesis of CS-AuNPs at 75 °C (completed in 30 min) was faster than the synthesis at 45 °C (completed in 40 min). Furthermore, a narrower maximum absorbance peak was observed at 75 °C in the 500–550 nm wavelength range. SEM images and size distribution graphs also confirmed the formation of uniform and smaller particles with an average diameter of 10.09 ± 0.09 nm (Supplementary Figures S2E–H).

In contrast, at a temperature of 45 °C, a broad peak was observed extending over a wider wavelength range (500–600 nm), and the average particle size was determined to be 17.02 ± 0.45 nm, as shown in Supplementary Figures S2A–D. A temperature of 75 °C was selected as optimal for the synthesis of CS-AuNPs, facilitating the production of smaller particles.

The concentration effect of HAuCl 4.3H 2 O on particle size distributions was investigated at different concentrations (0.5 mM and 1 mM) at 75 °C. SEM images and size distributions of CS-AuNPs synthesized using HAuCl 4.3H 2 O at concentrations of 0.5 mM and 1 mM are shown in Supplementary Figure S3.

The yield of the CS-AuNPs synthesis reaction using HAuCl 4.3H 2 O (0.25 mM) was found to be low. Utilizing 1 mM HAuCl 4.3H 2 O solution resulted in CS-AuNPs with diverse geometric structures (spherical, triangular pyramidal, hexagonal, etc.) with an average size of 50.00 ± 0.37 nm. CS-AuNPs prepared using a 0.5 mM HAuCl 4.3H 2 O solution exhibited a more spherical structure with an average size of 17.09 ± 0.16 nm (Supplementary Figures S3A–C). A concentration of 0.5 mM HAuCl 4.3H 2 O was chosen as the optimal concentration.

Characterization of the CBDO-AuNPs

CBD functioned as a reducing agent during the synthesis of CBDO-AuNPs by reducing Au 3+ ions to Au, thus leading to the formation of CBDO-AuNPs. During the synthesis process, a change in the color of the reaction medium (from light yellow to violet-gray) was observed, as shown in Figures 1a–c. The UV-Vis spectrum of the 0.001M CBD oil exhibited characteristic absorption peaks in the UV region between 200 and 300 nm, with no significant absorbance in the visible range (400-800 nm), confirming the optical profile of the pure CBD oil as shown in Figure 1d. The formation of CBDO-AuNPs was confirmed by the presence of a characteristic surface plasmon resonance (SPR) observed at a maximum wavelength λmax ∼530 nm in the UV-Vis spectrum, as seen in Figure 1e (Oueslati and Ben, 2021; Dheyab et al., 2023).

The SEM images, EDX spectrum, particle size distribution, and XRD pattern of CBDO-AuNPs are given in Figure 2. The synthesized CBDO-AuNPs exhibited diverse geometric structures (e.g., spherical, triangular, prismatic, and hexagonal), with spherical and subspherical shapes being predominant (Raguindin and Mercado, 2023). Particle sizes ranged from 20 to 120 nm, with an average particle size of 28.03 ± 0.40 nm. According to EDX analysis, Au peaks were observed in the elemental composition of CBDO-AuNPs. Furthermore, the EDX spectrum showed a strong gold signal absorption at 2.2 kV (Singh and Mijakovic, 2022). The crystal structure of biosynthesized CBDO-AuNPs was elucidated by XRD pattern (Figure 2f). The characteristic Au diffraction peaks observed at 2θ = 38.28°, 44.59°, 64.78°, 77.67, and 81.97°, corresponding to the (111), (200), (220), (311), and (222) planes, respectively. XRD confirmed that the biosynthesized CBD-coated gold nanoparticles possess a face-centered cubic (FCC) crystal structure (Sekar et al., 2022; Hassan et al., 2023).

FTIR absorption spectra of CBD oil and CBDO-AuNPs in the range of (4000–450 cm -1) are shown in Figures 3a,b. A strong and broad band emerged from the FTIR spectra in approximately 3653–3031 cm -1. This band is related to the O-H stretching vibrations in the CBD oil and CBDO-AuNPs structures. It may also originate from the ethyl alcohol molecules present in the sample compositions during the measurement. The bands at approximately 2967 cm -1 and 2884 cm -1 in the spectrum of CBD oil show asymmetric and symmetric C-H stretching vibrations. The peaks at approximately 1653 cm -1 and 1635 cm -1 in the CBD oil and CBDO-AuNPs correspond to the C=O stretching vibration. The peaks at 1378 cm -1 and 1394 cm -1 correspond to C-H bond bending vibrations, while the peaks at 1327 cm -1 and 1337 cm -1 correspond to C-O bending vibrations, and the peaks observed at 1041 cm -1 and 1085 cm -1 correspond to C-O stretching vibrations (Josiah et al., 2021; Barnawi et al., 2022). A decrease in the intensity of the C-O stretching peaks, along with an increase in the intensity of the C=O bond expansion peaks, was observed in the CBD-AuNPs spectrum. This indicates that the -C-OH structures in the CBD structure have been transformed into -C=O structures during the formation of CBDO-AuNPs, and that the CBD component present in the oil structure has contributed to its formation. The hydrodynamic size distribution and polydispersity index (PDI) of the synthesized CBDO-AuNPs are shown in Figures 3a,b. The size of CBDO-AuNPs was measured using dynamic light scattering (DLS). The size distribution and PDI value of the synthesized CBDO-AuNPs are shown in Figure 3c. From the DLS analysis, it was determined that the CBDO-AuNPs have an average size of 213 nm, and the polydispersity index (PDI) is 0.298. A polydispersity index between 0.08 and 0.7 indicates moderate polydispersity. Therefore, a PDI value of 0.298 indicates that CBDO-AuNPs have a moderately polydisperse particle size distribution.

NPs with zeta potential values > +25 mV or < −25 mV are considered quite stable. Lower ZP values can lead to aggregation, coagulation, or flocculation due to van der Waals attraction between particles and show low colloidal stability in NPs. As seen in Figure 3d, CBDO-AuNPs were determined to have a zeta potential of −6.04 (mV) (Hood et al., 2024; Niu et al., 2025).

Characterization of CBDO-NE2

Supplementary Figure S4 illustrates the apparatus used for sonication (an ultrasonic homogenizer) and the resulting CBD oil NEs. The CBDO-NE2 was chosen as the optimal formulation, as it showed no phase separation after 2 weeks and remained stable. SEM and EDS images of the prepared CBDO-NE2 are shown in Figure 4. The size distribution, PDI value, and ZP of the synthesized CBDO-NE2 are shown in Figure 4. The average hydrodynamic diameter of CBDO-NE2 was determined to be 170.5 nm, the PDI value was 0.416, and the ZP was −28 mV. The PDI (0.416) and ZP (−28 mV) measurements proved that CBDO-NE2 exhibited a moderate polydisperse distribution and moderate stability. Blal et al. formulated NEs to optimize a CBD-based NE (using Tween 80 as the surfactant) to enhance oral mucosal absorption. The stabilities of NEs composed of varying concentrations of CBD (400, 800, 2000, and 4000 μg/mL) were assessed by DLS analysis of droplet size distributions. The average droplet sizes of the NEs incorporated cannabis at lower concentrations (400 and 800 μg/mL) were determined to be ∼300 and 600 nm, respectively. The PDI values were also found between 0.2 and 0.4. These findings indicate that the NEs (incorporated cannabis at 400 and 800 μg/mL) exhibited moderate polydispersities and demonstrated stabilities; the formulations with higher concentrations (2000, and 4000 μg/mL) revealed an increase in droplet sizes (up to 1000 and 1250 nm) and PDI values (between 0.7 and 0.9), indicating phase separation, aggregation, and inadequate homogeneous distribution (Blal et al., 2025).

Isolated tissue bath evaluation of the effects of CBD, CBDO-AuNPs, CS-AuNPs, and CBDO-NE2 on uterine contractility

This study evaluated the effects of different CBD formulations (CBDO-NE2, CBDO-AuNPs) and CS-AuNPs on smooth muscle contractions. The obtained IC 50 values showed that the muscle-relaxant effects of these compounds varied significantly across formulations. The effects of CBD-NE2, CBDO-AuNPs, and CS-AuNPs formulations on uterine smooth muscle were assessed using BPM, P MAX, and AUC parameters, and the formulations were compared based on their IC 50 values (Figure 5). The IC 50 value is an important parameter that reflects a compound’s potency in inhibiting biological activity. Lower IC 50 values indicate more substantial biological effects (Table 2).

The CBD group exhibited an IC 50 value of 2.39 for BPM, suggesting its effectiveness in suppressing uterine muscle activity. However, the IC 50 values for P MAX (5.91) and AUC (2.56) were higher than for BPM, indicating that the effect of CBD on maximum contractile force may be limited.

The CBDO-NE2 formulation showed higher IC 50 values for BPM (8.87) and AUC (9.86) than CBD, suggesting reduced acute potency and cumulative effect, respectively, likely due to altered bioavailability from the emulsifier.

In the CBDO-AuNPs group, IC 50 values were 2.23 for BPM, 4.54 for P MAX, and 9.0 for AUC. The high AUC IC 50 value suggests the most extended duration of action.

CS-AuNPs demonstrated the highest inhibitory potency across two major parameters. CS-AuNPs achieved the lowest IC 50 values for both contraction frequency (1.97) and total cumulative activity (2.28). This finding indicates that the NP structure possesses significant antispasmodic properties against uterine contractions, acting as the most potent inhibitor of the BPM and AUC parameters among all tested compounds. However, fluctuations in the P MAX IC 50 value support the assumption that the effect of this formulation may be shorter or more transient.

When comparing CBD with CBDO-NE2, a significant difference was observed for AUC (F = 10.69, p = 0.0011), indicating that the NE formulation substantially enhanced the cumulative effect. While the difference in BPM (p = 0.2221) was not statistically significant, a significant difference was observed in P MAX (p = 0.0098), suggesting that the muscle-relaxing effect of the CBDO-NE2 primarily manifests as a prolonged action over time.

Comparisons between CBD and CBDO-AuNPs demonstrated statistically significant differences in all parameters (BPM: p = 0.0014; P MAX: p = 0.0028; AUC: p < 0.0001). These results indicate that the CBDO-AuNPs markedly enhance both the rapid effects (BPM and P MAX) and the cumulative effect (AUC) of CBD.

Similarly, comparing CBD with CS-AuNPs revealed significant differences across all parameters (BPM: p = 0.0049; P MAX: p = 0.0288; AUC: p < 0.0001). The pronounced difference in AUC for the CS-AuNPs group suggests a longer-lasting effect at the target tissue.

When CBDO-NE2 was compared with CBDO-AuNPs, no significant differences were observed for BPM and P MAX (p = 0.1717 and p = 0.8685, respectively), whereas a significant difference was noted for AUC (p = 0.0370). This suggests that both formulations exhibit comparable immediate relaxation effects but differ in their long-term efficacy. Similarly, comparisons between CBDO-NE2 and CS-AuNPs revealed no significant differences in BPM and P MAX. However, a significant difference in AUC was observed (p = 0.0100), suggesting that the NE provides a more stable yet moderate effect. At the same time, CS-AuNPs produce a more substantial cumulative effect.

No significant differences were detected between CBDO-AuNPs and CS-AuNPs for any parameter (p > 0.5), indicating that both nano-formulations have similar pharmacodynamic profiles. However, the carrier matrices may influence pharmacokinetic properties (Table 3). This study demonstrates that formulating CBD within various pharmaceutical carrier systems yields distinct pharmacological effects. CBDO-AuNPs and CS-AuNPs are particularly notable for rapid onset and strong cumulative effects, whereas the emulsion form (CBDO-NE2) provides a more stable but moderate effect profile. These findings underscore the importance of nano-based agents in enhancing the therapeutic potential of CBD (Supplementary Figures S5–7).

AuNPs are prominent agents in the treatment of uterine smooth muscle disorders, particularly uterine myomas. Their unique properties enable targeted drug delivery, enhancing treatment therapeutic efficacy and making AuNPs a promising alternative to conventional therapies. Therefore, AuNPs are designed to overcome biological barriers that hinder conventional treatments, enabling target-site-specific drug delivery (Van Staden et al., 2024). Studies have shown that AuNPs can induce apoptosis in uterine smooth muscle cells and may be beneficial for reducing tumor size during treatment (Wang et al., 2024). The surface properties of AuNPs significantly affect their cytotoxicity and cellular uptake (Sun et al., 2018). AuNPs have been shown to inhibit the proliferation and migration of vascular smooth muscle cells, suggesting analogous effects in uterine smooth muscle (Lo et al., 2018). Despite the promising applications of AuNPs in treating uterine smooth muscle disorders, concerns regarding their long-term safety and potential toxicity remain. Our findings confirmed that the structural, chemical, and physical properties of the natural-based delivery system have a significant impact on the therapeutic effectiveness. The synthesized AuNPs-based nanoformulations were found to exhibit a dominant, intrinsic antispasmodic activity as therapeutic carrier transporters in uterine tissue. Further research is essential to fully understand their biological interactions and optimize their therapeutic use in gynecological applications.

NPs that enter the human body through various routes may influence ion channels either directly or indirectly. Regarding the direct mechanism, the physicochemical characteristics of NPs—including morphology, dimensions, surface-to-volume ratio, surface chemistry, and surface charge—are suggested to play a significant role. Nanoparticles could theoretically interact with ion channels, potentially blocking them in the cell membrane by creating a physical barrier or altering their kinetics. For instance, in a study by Yin (2019), spermidine-modified AuNPs were prepared, and it was hypothesized that spermidine might direct the AuNPs toward the ion pores of K+ channels. Under that experimental design, the authors proposed that gold-sulfur bonds could form between AuNPs and the sulfur atom in the cysteine ring within the channels, thereby synthesizing an AuNP-spermidine complex capable of blocking K+ channels. While such a molecular interaction represents a plausible theoretical mechanism for explaining the potent antispasmodic activity observed in our ex vivo results, it remains speculative for our current study, as specific patch-clamp electrophysiological or molecular docking analyses were not performed. On the other hand, in the indirect mechanism of action, changes in the structural and elastic properties of the cell membrane, ion release from NPs, and oxidative stress may play a role. Needle-like or sharp-edged NMs can disrupt lipid layers with appropriate flexibility and fluidity, thereby negatively affecting the function of ion channels located between them. Additionally, oxidative stress induced by NMs can lead to damage in ion channels (McCarthy et al., 2011; Yin et al., 2019). Oxytocin-induced uterine contractions primarily rely on G-protein-coupled receptor (OTR) activation, which triggers phospholipase C activation and subsequent calcium mobilization from intracellular stores and extracellular influx via L-type voltage-gated calcium channels. This allows us to assess the receptor-level pathways critically. According to our research, CBD compositions inhibit these contractions. This effect can be explained mechanistically by CBD’s multi-target nature, which prevents the intracellular calcium spike required for myometrial cross-bridge cycling by acting as an antagonist to GPR55, blocking voltage-gated calcium channels, and stimulating/desensitizing TRPV1 channels. However, a crucial question is whether the improved effects are due to intrinsic nanoparticle activity or better CBD distribution. The maximum potency (IC 50 for BPM and AUC) was demonstrated by chemically synthesized blank gold nanoparticles (CS-AuNPs), indicating that the observed tocolytic effects are not exclusively dependent on CBD delivery augmentation. Rather, the gold core itself drives a significant intrinsic antispasmodic effect. By physically adhering to cell membranes, AuNPs can produce a steric barrier that modifies membrane fluidity and interferes with the kinetics of ion channels that are chemically or mechanically gated. Furthermore, the citrate stabilizers on CS-AuNPs may directly interact with membrane proteins or chelate extracellular ions. A pharmacodynamic counterbalance occurs when CBD is green-synthesized onto AuNPs (CBDO-AuNPs): the nanoparticle core retains its channel-blocking properties while delivering CBD to target myometrial receptors, producing a different, longer cumulative profile (AUC) than pure CBD.

The most compelling result was the extremely high potency of the CS-AuNPs, which exhibited the lowest IC 50 values for both contraction frequency (1.97) and cumulative activity (2.28). This places the gold carrier as the most potent inhibitor of BPM and AUC among all tested formulations. The intrinsic biological activity of synthesized CS-AuNPs with a particle size of 17.09 ± 0.16 nm suggests the existence of two predominant mechanisms. Firstly, the low IC 50 indicates that CS-AuNPs may be directly interfering with the influx of extracellular calcium ions or the release of intracellular calcium stores, which are essential for the sustained contraction of smooth muscle cells. Secondly, surface charge and cellular uptake may play a role; the high uptake efficiency required to achieve such low IC 50 values may be linked to the high surface charge of these NPs (Lavoie Cardinal et al., 2016).

The CBDO-AuNPs formulation showed a synergistic effect, reducing the maximum contractile force (P MAX: 4.54) compared to pure CBD (P MAX: 5.91). This improvement supports the hypothesis that the AuNP carrier enhanced CBD delivery to the cellular targets. However, the CBDO-AuNPs exhibited the lowest potency in reducing total cumulative activity (AUC: 9.00). The CBDO-NE2 performed poorly in the ex vivo environment, showing the lowest acute efficacy across BPM and AUC (8.87 and 9.86). This acute reduction in potency compared to CBD (BPM: 2.39) suggests that the emulsifier system, designed to improve solubility and bioavailability, may have actively hindered CBD availability in the isolated tissue bath. In the Krebs solution environment, the CBD may have been trapped within the emulsion droplets, preventing its rapid partition from the droplet interface to the aqueous medium and subsequent cellular membrane interaction (Feng, 2022; Stasiłowicz Krzemień et al., 2023).

CS-AuNPs, CBDO-AuNPs, and CBDO-NE2 were successfully synthesized and comprehensively characterized. CBDO-AuNPs exhibited characteristic SPR absorption, an FCC crystalline structure, and predominantly spherical morphology. Optimizing CS-AuNP synthesis at 75 °C and 0.5 mM HAuCl 4 favored the formation of smaller, more homogeneous particles with a high yield. The CBDO-NE2 formulation exhibited nanoscale droplet dispersion with moderate polydispersity and good colloidal stability, as indicated by particle size, PDI, and zeta potential. Functional evaluations revealed that the effects of CBD pharmaceutical formulations (CBDO-NE2 and CBDO-AuNPs) on smooth muscle contractions varied significantly. The obtained data clearly demonstrate that AuNPs-based systems are significantly superior to NE in enhancing the bioavailability and pharmacological efficacy of CBD. Compared to CBD, the CBDO-AuNPs and CS-AuNPs formulations produced statistically highly significant differences across all parameters, including rapid onset of action (BPM and P MAX) and cumulative activity (AUC) (all p values ≤0.0049). In particular, the CS-AuNPs group exhibited the most substantial antispasmodic potential among the tested formulations, yielding the lowest IC 50 values for BPM (IC 50: 1.97 M) and AUC (IC 50: 2.28 M). The superior activity of CS-AuNPs most likely stems from two main factors: enhanced interaction with the target tissue due to its interaction with ion channels, and the direct antispasmodic or signaling pathway-modulating effects of the AuNP core or its surface stabilizers (citrate). This effect suppresses uterine contractions either additively to CBD’s effect or independently of it. Although CBDO-NE2 did not show a significant improvement over pure CBD for BPM (p = 0.2221) and P MAX (p = 0.0098), it did show a significant increase in the AUC parameter (p = 0.0011), indicating a more stable and prolonged cumulative effect. Furthermore, the lack of a significant difference between CBDO-AuNPs and CS-AuNPs for any parameter (p > 0.5) confirms that both nanoparticle systems share a similarly high pharmacodynamic profile. Consequently, these findings highlight the crucial role of naturally derived nanoparticle formulation strategies in enhancing the therapeutic potential of CBD for the treatment of uterine dysfunction.

These formulations have promising antispasmodic efficacy, but their safety profiles, especially regarding gold nanoparticles (AuNPs) need close toxicological evaluation. It is well known that metallic nanoparticles exhibit cytotoxicity that depens on surface charge and size. Reproductive toxicity is a major concern in maternal-fetal pharmacology. According to earlier research, tiny AuNPs (less than 20 nm) can cross the placental barrier, accumulate in fetal tissues, and possibly cause embryotoxicity and oxidative stress in the placental architecture (Semmler-Behnke et al., 2014). Although green synthesis utilizing CBD oil aims to reduce toxic residues and improve biocompatibility compared to chemical synthesis, the risk of nanoparticle-induced reactive oxygen species (ROS) generation in uterine tissues and subsequent histopathological alterations cannot be overlooked. Therefore, while these nano-formulations effectively suppress hyper-contractility, their clinical transition is strictly bottlenecked by the need for comprehensive reproductive and systemic toxicity profiling (Chang et al., 2021).