Nanosuspensions Loaded with Acetogenins: Physical Stability During In Vitro Digestion, Genotoxicity and Cytotoxicity

TL;DR

Researchers tested nanosuspensions for delivering acetogenins, finding that different stabilizers affect stability and release during digestion.

Contribution

The study compares two nanosuspension formulations for oral delivery of acetogenins, highlighting their stability and bioavailability differences.

Findings

PEGSL-ACG-NSps maintained structural integrity and showed controlled acetogenin release during digestion.

βCDSL-ACG-NSps rapidly released acetogenins and achieved higher bioaccessibility and potential bioavailability.

Neither formulation showed genotoxic or cytotoxic effects in mice at high doses.

Abstract

This study assesses the stability, in vitro bioaccessibility and potential bioavailability, and in vivo genotoxicity and toxicity of polyethylene glycol–soy lecithin (PEGSL-ACG-NSps) or β-cyclodextrin–soy lecithin (βCDSL-ACG-NSps) nanosuspensions (NSps). Both formulations exhibited initial particle sizes below 130 nm and PDI values below 0.3. Under simulated gastrointestinal conditions, PEGSL-ACG-NSps preserved structural integrity, with only a moderate size increase (~239 nm) in the intestinal phase and controlled release of acetogenins (ACGs); in contrast, βCDSL-ACG-NSps destabilized considerably (size > 500 nm) and released ACGs rapidly. Consistently, βCDSL-ACG-NSps achieved higher in vitro bioaccessibility and a potential bioavailability (up to 95% from post-digestion recovery). In contrast, PEGSL-ACG-NSps displayed a more gradual release profile (up to 55%). In vivo toxicity tests in mice showed no significant genotoxic or cytotoxic effects for either formulation, even at high doses. These findings suggest that selecting appropriate food-grade stabilizing polymers is crucial for optimizing NSps for the oral delivery of ACGs as therapeutic agents.

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Full text

1. Introduction

Acetogenins (ACGs) are secondary metabolites produced through the acetate-polyketide pathway and are primarily extracted from plants of the Annonaceae family. These compounds are molecules with 35–37 carbon atoms connected to one, two, or three tetrahydrofuran or tetrahydropyran rings in their central region, along with several oxygenated groups (–OH) and an α-β-unsaturated or saturated γ-lactone [1]. ACGs exhibit a wide range of biological activities, including antibacterial, antifungal, and antitumor effects, which have generated significant interest for their potential therapeutic applications [2,3]. Their primary mechanism involves inhibiting mitochondrial complex I (NADH: ubiquinone oxidoreductase), thereby decreasing ATP production and inducing cell apoptosis [3,4].

However, despite their high therapeutic potential, ACGs are limited by their very lipophilic structure, which reduces their solubility in water, stability under physiological conditions, and oral bioavailability [5,6,7]. In addition to pharmacokinetic challenges, some in vivo studies in rats have shown that certain ACGs can be toxic, especially when administered systemically at high doses and under conditions that promote their accumulation in sensitive tissues. Champy et al. [8] reported that annonacin, a monotetrahydrofuran acetogenin, induced neurotoxicity in rats when administered intravenously at doses of 3.8–7.6 mg/kg/day for 28 days. This exposure resulted in inhibition of mitochondrial complex I in brain tissue, reduced cerebral ATP levels, and pronounced neurodegeneration, characterized by significant losses of dopaminergic, cholinergic, and GABAergic neurons, as well as marked astrocytic and microglial proliferation. Consequently, Chen et al. [9] reported that repeated administration of bullatacin (300 µg/kg) over three weeks caused liver and kidney toxicity by increasing calcium levels, reactive oxygen species production, Bax expression, and the Bax/Bcl-2 ratio in rats. These findings highlight the risks linked to ACGs’ use, particularly when systemic exposure and accumulation in the central nervous system are involved.

To overcome these obstacles and reduce systemic toxicity while maintaining antitumor effectiveness, new and promising drug delivery strategies, such as nanosuspensions (NSps), have been developed. These colloidal systems consist of nanometer-sized (<200 nm) particles of the active ingredient dispersed in water and stabilized by polymeric matrices, thereby improving the dispersibility and bioavailability of hydrophobic drugs [10].

In this context, several studies have demonstrated that using hydrophilic polymers, such as polyethylene glycol (PEG) and modified cyclodextrins (βCD), in combination with soy lecithin (SL) enables the formation of NSps capable of encapsulating ACGs. This improves their pharmacokinetic profile, reduces systemic toxicity, and provides self-assembly properties that are resistant to gastrointestinal conditions [11,12,13,14,15]. A study by Hong et al. [11] revealed that ACG NSps stabilized with hydroxypropyl–beta-cyclodextrin–soy lecithin showed sustained in vitro release, good stability in plasma and simulated gastrointestinal fluid, and significant antitumor efficacy both orally (49.74%) and intravenously (70.31%). Similarly, Hong et al. [12] prepared ACG NSps using mPEG2000–PCL2000 as a stabilizer, achieving particle sizes of 123.2 nm, high ACG loading, efficient in vitro cytotoxicity (4T1, MCF-7, and HeLa lines), good stability in physiological media, sustained release, and improved antitumor efficacy (74.83%) in mice bearing the 4T1 tumor compared to free ACGs (45.53%).

Although NSps show significant potential as delivery systems for ACGs, few studies have simultaneously examined their physicochemical stability in a gastrointestinal environment, bioaccessibility, bioavailability, genotoxicity, and cytotoxicity.

From a therapeutic perspective, ACGs are promising compounds with bioactive potential and health-promoting properties. However, their practical application in functional foods or therapeutic products is limited by poor water solubility, low gastrointestinal tract stability, and safety concerns arising from uncontrolled exposure. In this context, nanosuspension-based delivery systems made with food-grade polymers and surfactants are a viable strategy to enhance the oral delivery, gastrointestinal behavior, and safety of lipophilic food bioactives. Therefore, this study aimed to evaluate two acetogenin-loaded NSps stabilized with polyethylene glycol–soy lecithin (PEGSL-ACG-NSps) and β-cyclodextrin–soy lecithin (βCDSL-ACG-NSps). The focus was on their physical stability during simulated gastrointestinal digestion, in vitro bioaccessibility and potential bioavailability, and in vivo cytotoxicity and genotoxicity. The formulations were developed using food-grade excipients, including soy lecithin, PEG, and β-CD, and tested under gastrointestinal conditions relevant to oral consumption. In this sense, the present study contributes to evaluating whether these NSPs meet the preliminary criteria that justify their future exploration as potential therapeutic agents, while recognizing the need for further studies to confirm their safety and applicability.

2. Materials and Methods

2.1. Materials

The ACGs used in this study were obtained from the endosperm of A. muricata seeds at the Bromatology and Nutrition Laboratory of the National Technological Institute of Mexico in Tepic, following the methodology described by López-Romero et al. [2]. Briefly, ACGs were extracted from the defatted endosperm by thermosonication (50 °C, 50 min, 100% amplitude, and 0.5 s pulse cycles) using an ultrasonic system (UP400S, Hielscher Ultrasonic, Teltow, Germany). The optimized extract was purified by open-column chromatography on silica gel (60 mesh) and then further purified on silica gel (230–400 mesh). TLC was used to analyze the resulting fractions, and ACG-rich fractions were characterized by HPLC-DAD. Also, structural elucidation of the ACGs was performed by HPLC-MS and NMR, confirming purities greater than 95% [16]. The isolated ACGs mainly consisted of pseudoannonacin (60%), annonacin (6%), and other ACGs (34%) such as bullatacin, squamostatin-D, squamocin, isodesacetyluvarcicin, and desacetyluvaricin [2]. Polyethylene glycol 6000 (PEG), (2)-hydroxypropyl-β-cyclodextrin (βCD), soy lecithin (SL), pancreatic lipase, porcine pepsin, gastric mucin, bile salts, and cyclophosphamide (1000 mg/mL) were purchased from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA). Acetonitrile, HPLC-grade water, and all analytical-grade reagents such as sodium hydroxide (NaOH), hydrochloric acid (HCl), sodium chloride (NaCl), ammonium nitrate (NH_4_NO_3_), potassium phosphate (KH_2_PO_4_), potassium chloride (KCl), potassium citrate (C_6_H_5_K_3_O_7_), urea (CO(NH_2_)2), and calcium chloride (CaCl_2_) were purchased from Jalmek Scientific S.A., Guadalajara, Jalisco, Mexico.

2.2. Preparation of Nanosuspensions Loaded with Acetogenins

Nanosuspensions (NSps) loaded with acetogenins (ACG) were prepared following the protocols established by Montalvo-González et al. [17] and López-Romero et al. [18]. In summary, PEG (0.20% w/v) or βCD (16% w/v), dissolved in sterile water (10 mL), was added dropwise to 800 µL of a surfactant solution (10–15 mg/mL SL in ethanol). The mixture was stirred at 500 rpm for 30 min to form amphiphilic complexes (PEGSL and βCDSL). Amphiphilic complexes are self-assembled supramolecular structures formed by the association of hydrophilic and hydrophobic domains in aqueous media [12]. During this process, the hydrophilic portion of PEG or βCDSL interacts with water. In contrast, the hydrophobic portion of SL organizes toward the core of the complex, enabling the subsequent association with ACGs.

Subsequently, the ethanol was evaporated using a rotary evaporator (Yamato RE300, Yamato, Tokyo, Japan). Next, 160 µL of an ACG solution (62.5 mg/mL in ethanol) was added dropwise, and the mixture was stirred at 500 rpm for 30 min. Finally, the ethanol was evaporated, yielding ACG-loaded nanosuspensions (PEGSL-ACG-NSps and βCDSL-ACG-NSps) at a final concentration of 1.024 mg/mL. The resulting systems are obtained as stable nanosuspensions, consisting of colloidal particles in the nanometer range dispersed in an aqueous continuous phase [10]. Particle size depends on the concentration of amphiphilic complexes and ACGs, as well as on the dispersion conditions [19]. The particle size of the nanosuspensions ranged from 113.32 nm for PEGSL-ACG-NSps to 133.77 nm for βCDSL-ACG-NSps, with a polydispersity index (PDI) between 0.19 and 0.20, indicating a homogeneous size distribution and good colloidal stability. Encapsulation efficiencies of 83.33% and 56.09% for PEGSL-ACG-NSps and βCDSL-ACG-NSps, respectively, have been reported in these previous studies [19].

2.3. Simulated In Vitro Gastrointestinal Digestion Study

The simulated in vitro digestion of PEGSL-ACG-NSps and βCDSL-ACG-NSps was performed in three sequential phases (mouth, gastrointestinal, and small intestine) following the methodology described by Brodkorb et al. [20] and Bravo-Alfaro et al. [21], with some modifications.

During the mouth phase, 7 mL of PEGSL-ACG-NSps or βCDSL-ACG-NSps was mixed with 7 mL of simulated salivary fluid (see Table 1) and 0.3 g/L of gastric mucin. The pH was adjusted to 6.8, and the mixture was incubated for 10 min at 37 °C with agitation at 100 rpm.

For the gastrointestinal phase, 14 mL of simulated gastric juice (containing 0.0034 g NaCl, 0.7 mL HCl, and 0.32 g pepsin per 100 mL of distilled water, with a pH of 2.0) was added to the oral bolus. The pH was then adjusted to 2.5 using 1 M NaOH, and the mixture was incubated for 2 h at 37 °C with shaking at 100 rpm.

During the small intestine phase, the pH was adjusted to 7.0 using 1 M NaOH, and then 3.6 mL of bile salt solution (46 mg/mL in phosphate buffer, pH 7.0) and 2.2 mL of pancreatic lipase (15 mg/mL in 100 mM phosphate buffer, pH 7.0) were added. The mixture was incubated for 2 h at 37 °C with shaking at 100 rpm.

The stability analysis was conducted to evaluate the resistance of NSps to simulated human digestion conditions. During each digestion phase, 1 mL aliquots were collected to determine particle size and PDI and then stored at −20 °C until further analysis.

In Vitro Bioaccessibility and Potential Bioavailability of Nanosuspensions During In Vitro Digestion: Analysis by High-Performance Liquid Chromatography (HPLC).

The collected sample (2 mL) at each stage of the digestion assay was mixed with 2 mL of chloroform to extract the ACGs, which are highly soluble in this solvent. The mixture was homogenized at 10,000 rpm for 1 min (Ultraturrax Kaibrite FS-2A, Shanghai, China) to promote effective disruption of the nanosuspensions and facilitate transfer of ACGs into chloroform, followed by centrifugation at 8000 rpm for 10 min at 4 °C. The chloroform was then evaporated to dryness. The residue was reconstituted in 1 mL of HPLC-grade methanol.

The resulting solutions were then filtered through nylon filters (0.22 µm), and 10 µL was injected into an HPLC system (Agilent Technologies 1260 Infinity, Agilent Technologies, Waldbronn, CA, USA) equipped with a diode array detector and a C18 column (5 μm, 4.6 mm × 250 mm; Thermo Scientific, Sunnyvale, CA, USA). Separation was carried out using a gradient elution of acetonitrile (eluent A) and water (eluent B) as follows: 60% A (0–30 min), 70% A (35–40 min), and 60% A (41–60 min), at a flow rate of 0.8 mL/min. Peak areas were recorded at 210 nm and quantified using calibration curves for annonacin (0.093–1.50 mg/mL, R^2^ = 0.9991) and pseudoannonacin (0.13–2.10 mg/mL, R^2^ = 0.9998) [16].

In vitro bioaccessibility and potential bioavailability were calculated according to Zhao et al. [22] using Equations (1) and (2).

$$[eqn]$$ $$[eqn]$$

where the micellar fraction refers to the ACGs solubilized in mixed micelles, which are colloidal structures formed during intestinal digestion through the interaction of bile salts, phospholipids, and lipid digestion products. The digested fraction, in turn, represents the total amount of ACGs recovered after digestion, including both micellized compounds (ACGs incorporated into and solubilized in mixed micelles, making them potentially available for intestinal absorption) and those still associated with the nanoparticles present in the NSps.

2.4. In Vivo Genotoxicity and Cytotoxicity Study

2.4.1. Animal Handling and Care

The male C57BL/6 mice (6–8 weeks old, 20–25 g) used in this study were sourced from the Animal Facility at the University Health Sciences Center, University of Guadalajara. Animals were acclimated under standard housing conditions in polycarbonate cages at 24 ± 2 °C, 50–60% relative humidity, and a 12 h light/dark cycle, with ad libitum access to food and water. The protocol was approved by the Research Ethics Committee of the University of Guadalajara (No. Protocol CI-03523). All procedures adhered to the Guide for the Care and Use of Laboratory Animals and the CONCEA Euthanasia Practice Guidelines.

2.4.2. Formation of Experimental Groups

Animals (36 mice) were randomly assigned to nine experimental groups, with four mice in each group. The number of animals per group was determined by the nature of the in vivo micronucleus assay, which allows the evaluation of a large number of erythrocytes per animal, providing high analytical sensitivity. This experimental design was selected in accordance with internationally accepted guidelines for genotoxicity testing, emphasizing ethical principles and the reduction in animal use (3Rs), while maintaining sufficient power to detect biologically relevant genotoxic effects when appropriate positive controls are included [23].

Group 1 served as the positive control and received cyclophosphamide (10 mg/kg of body weight). Groups 2 and 3 served as negative controls and received the PEGSL or βCDSL complex, respectively. Groups 4, 5, and 6 were treated with PEGSL-ACG-NSps at doses of 400, 800, and 1600 µg/kg of body weight, respectively. Groups 7, 8, and 9 received βCDSL-ACG-NSps at the same doses.

All treatments were administered via orogastric tube once daily for five consecutive days. At the end of the experiment, the animals were euthanized using an overdose of tiletamine–zolazepam anesthesia (Zoletil^®^ 50, Virbac, Carros, France).

2.4.3. Micronucleus Test

Cytotoxic and genotoxic effects were assessed using the micronucleus assay in bone marrow. Before administering the treatment, peripheral blood smears were collected by making a small cut at the tip of the tail at 0, 24, 48, 72, and 96 h. The samples were fixed with absolute ethanol for 10 min, stained with acridine orange, and examined under an epifluorescence microscope (Olympus CX3, Olympus, Tokyo, Japan).

Recent genotoxic damage, induced 24 h after treatment, was assessed by counting micronucleated polychromatic erythrocytes (MNPCE) per 1000 total polychromatic erythrocytes (TPE). Accumulated genotoxicity was measured by the number of micronucleated normochromatic erythrocytes (MNE) per 10,000 total erythrocytes (TE). At the same time, cytotoxicity was evaluated as the reduction in the number of polychromatic erythrocytes (PCE) per 1000 TE [24,25,26].

2.4.4. Ethical Considerations

The study was conducted in accordance with Mexican Official Standards NOM-062-ZOO-1999, NOM-033-SAG/ZOO-2014, and NOM-087-ECOL-SSA1-2002 [27,28,29]. All experimental procedures were reviewed and approved by the appropriate ethics committee and followed NOM-062-ZOO-1999 for laboratory animal care and use, NOM-033-SAG/ZOO-2014 for humane euthanasia, and NOM-087-ECOL-SSA1-2002 for the management of bio-infectious waste.

2.5. Statistical Analysis

The data obtained from the in vitro digestion study were expressed as mean ± standard deviation and analyzed using one-way analysis of variance (ANOVA), followed by Fisher’s least significant difference (LSD) post hoc test for multiple comparisons between formulations and controls. For the in vivo study, data normality (MNE, PCEMN, and PCE) was evaluated using the Kolmogorov–Smirnov test before parametric analysis. Intragroup comparisons were made relative to baseline values, while intergroup comparisons were conducted relative to the positive control (cyclophosphamide, 10 mg/kg) and the negative controls (PEGSL and βCDSL complex). Repeated measures ANOVA followed by Fisher’s LSD test was applied, and differences were considered statistically significant (p < 0.05). All statistical analyses were performed using STATISTICA software (v.10, StatSoft Inc., Tulsa, OK, USA).

3. Results and Discussion

3.1. Analysis of the Stability of Nanosuspensions During the In Vitro Digestion Study

Figure 1 and Figure 2 show the results obtained for particle size and PDI of the PEGSL-ACG-NSps (Figure 1A,B) and βCDSL-ACG-NSps (Figure 2A,B) formulations during each stage of the simulated digestion study.

Initially, PEGSL-ACG-NSPs and βCDSL-ACG-NSPs had average sizes of ~100 nm and ~128 nm, respectively, with PDI values of ~0.22, indicating a homogeneous size distribution. At the start of digestion, in the buccal and gastric phases, particle size remained constant (p > 0.05). Although a moderate increase in PDI (~0.32) was observed for PEGSL-ACG-NSps, the values remained within acceptable ranges for colloidal systems [5].

These results suggest good colloidal stability of NSps under adverse gastrointestinal conditions, such as bile salts, digestive enzymes, or acidic pH, which can alter particle stability, leading to possible aggregation or structural collapse of the systems [21]. Furthermore, Figure 1A shows that the PEGSL-ACG-NSps formulation exhibited a gradual increase in particle size, from 130.8 nm at the beginning of the intestinal phase to a maximum rise of 239.25 nm at the end of digestion. The gradual increase in size in PEGSL-ACG-NSps is due to the hydration of the PEG coating, which forms a flexible steric barrier that protects the particles from digestive conditions, allowing only moderate swelling without compromising their structural integrity. These results indicate a stable system, ideal for controlled release of active compounds [7].

In contrast, βCDSL-ACG-NSps (Figure 2A) showed a dramatic increase at the beginning of this intestinal phase (297.22 nm), reaching 518.23 nm at 120 min. This behavior suggests that βCD does not provide as adequate steric protection against digestive conditions as PEG, potentially leading to destabilization of the colloidal system and premature or uncontrolled release of ACGs. These results suggest that PEG formulations may be suitable and effective for the controlled release of ACGs during gastrointestinal transit.

Particle size is a critical parameter in lipid nanotransport systems, as it directly influences their stability, encapsulation efficiency, release profile, distribution in the body, interaction with mucous membranes, and, especially, cellular uptake. The latter process, essential for in vivo efficacy, occurs mainly through endocytosis mechanisms, such as pinocytosis and phagocytosis. In general, particles smaller than 1 µm are internalized by cells via different pinocytosis pathways (mediated or not by clathrin or caveolin), which depend on the particle’s size and uniformity (PDI). Therefore, characterizing the size and distribution of NSps is essential for predicting their biological behavior and optimizing their therapeutic efficacy [5].

3.2. In Vitro Bioaccessibility and Potential Bioavailability of Nanosuspensions During In Vitro Digestion

The results obtained (Figure 3 and Figure 4) show significant differences (p < 0.05) between the PEGSL-ACG-NSps and βCDSL-ACG-NSps formulations in terms of release, in vitro bioaccessibility, and potential bioavailability during simulated digestion. The PEGSL-ACG-NSps formulation exhibited lower bioaccessibility (25–29%) and gradual potential bioavailability (25–55%), with ACGs released 60 min after the intestinal phase. In contrast, the βCDSL-ACG-NSps formulation showed faster release (p < 0.05), with higher bioaccessibility (15–50%) and high potential bioavailability (35–95%) of ACGs from the first 30 min of the small intestine phase.

In this context, evaluating in vitro bioaccessibility and potential bioavailability is essential for predicting the gastrointestinal behavior of NSps after oral administration. These determinations allow us to estimate how much of the ACGs could be released under physiological digestive conditions and resist degradation caused by gastrointestinal factors (pH, acidity, enzymatic activity, bile salts), and what fraction is actually available for intestinal absorption.

Additionally, HPLC-DAD analysis performed at each stage of the simulated gastrointestinal digestion did not show significant changes in ACGs concentration or chromatographic profiles (Figure 5), suggesting that the acetogenins did not undergo relevant chemical degradation under the evaluated conditions. In addition, determining in vitro potential bioavailability provides critical information for the design of optimal formulations, such as selecting appropriate coatings (e.g., PEG or βCD) and anticipating in vivo behavior, thereby reducing the time and costs associated with animal testing.

These results reflect a controlled release profile for PEGSL-ACG-NSps, due to the protection provided by the flexible steric layer formed by PEG, which acts to protect the particles from digestive conditions and delay the release of ACG, as mentioned by Wiecinski et al. [30], who demonstrated that PEG 5000 improves the structural stability of nanoparticles, slowing their degradation under gastric conditions and improving their biodegradability. Furthermore, Hong et al. [11] and Kesisoglou et al. [31] confirmed that the use of high-molecular-weight PEG improves dispersion and colloidal stability and promotes slower, prolonged release by forming a physical barrier around the nanoparticles, which is key for applications requiring controlled drug release.

In contrast, the βCDSL formulation, with its lower structural protection capacity against gastric conditions, allowed for a more rapid release of the ACGs. This characteristic could favor rapid, albeit less controlled, absorption, which is appropriate in applications requiring immediate therapeutic action, such as skin wounds at risk of infection [32]. Therefore, although the βCDSL formulation may not be ideal for sustained-release therapies, its kinetic profile positions it as a promising alternative for rapid, effective intervention.

In a study conducted by Hong et al. [13], it was reported that NSps loaded with ACGs and stabilized with βCD and soy lecithin, evaluated under controlled laboratory conditions at pH 7.4, exhibited more sustained ACGs release than in the present study. In contrast, the faster release profile reported here may be attributable to several factors, including differences in formulation composition, experimental design, and the gastrointestinal conditions under which the NSps were exposed. These conditions may involve more aggressive physicochemical and enzymatic environments, which can compromise the stability of βCD/ACGs complexes and promote accelerated desorption of the active compounds during the gastric and intestinal phases. Consequently, the results obtained in this study provide data that could be considered for future in vivo conditions.

Taken together, these results demonstrate that the choice of coating has a decisive impact on the stability and release profile of NSps, highlighting the effectiveness of PEG in designing colloidal systems with sustained release and greater gastrointestinal resistance.

3.3. In Vivo Genotoxicity and Cytotoxicity Study

The results obtained from this study demonstrate that neither the amphiphilic complexes nor the PEGSL-ACG-NSps and βCDSL-ACG-NSps at any of the concentrations evaluated caused significant genotoxic effects (Figure 6) in the in vivo model used (p < 0.05).

The frequency of micronucleated polychromatic erythrocytes (MNPCE), a reliable biomarker for assessing chromosomal damage [33], remained constant (3 MNPCE) up to 72 h after administration. However, a slight increase (7 MNPCE) was observed in the group treated with PEGSL-ACG-NSps at 1.6 mg/kg. Nevertheless, these results are lower than those observed with cyclophosphamide (p < 0.05), which showed a gradual increase of 3–23 MNPCE at 72 h, confirming its well-established genotoxic capacity and suggesting ongoing DNA damage or a deterioration of repair mechanisms [26,34].

Similarly, analysis of cumulative genotoxic damage (Figure 7) showed no evidence of progressive accumulation of normochromatic micronuclei (MNE) in groups treated with NSps, even after exposure for up to 96 h. These results indicate that NSps do not cause cumulative genotoxic stress or DNA integrity alterations over the exposure period. In contrast, the group treated with cyclophosphamide (10 mg/kg) showed a significant increase (p < 0.05) in MNE formation, reaching up to 46 MNE at 72 h, which corroborates its cumulative genotoxic effect on DNA.

Finally, Figure 8 shows that the amphiphilic complexes did not cause genetic damage. Figure 8A shows that the number of polychromatic erythrocytes (PCE), an indicator of bone marrow damage, remained constant for PEGSL-ACG-NSps at any of the concentrations evaluated. In contrast, βCDSL-ACG-NSps at 1.6 mg/kg showed a slight increase from 45 PCE at 24 h to 58 PCE at 96 h. Other indicators of cytotoxicity did not accompany this variation and therefore do not suggest a cytotoxic effect under the evaluated conditions. In contrast, cyclophosphamide showed a gradual decrease in the number of PCE (from 53 to 25) from 48 h, suggesting possible mitotic suppression or severe cytotoxic damage to bone marrow cells [34].

In contrast to these findings, the genotoxic potential of ACGs administered in their free form has been reported previously. García-Aguirre et al. [35] evaluated acetogenin isomers isolated from Annona cherimolia in a murine model using male NIH mice, administering doses of 1–5 mg/kg. Their results demonstrated a significant, dose-dependent increase in genotoxicity, with a peak effect at 48 h, comparable to that induced by daunorubicin. This discrepancy underscores the importance of the amphiphilic complexes employed in the present NSps, as these may modulate the biodistribution, cellular interactions, and biological activity of ACGs, thereby reducing their genotoxic potential.

Evaluation of genotoxicity and cytotoxicity is a key aspect of characterizing NSps for potential use in pharmaceuticals, as their nanometric size can induce unwanted interactions with cellular components, including DNA, causing toxic damage in the short or long term. Therefore, it is necessary to evaluate the biological safety of any nanosystem before its application. In this study, the toxicity of NSps was assessed using the micronucleus assay. This sensitive and reliable tool allows the evaluation of DNA damage induced by physical and chemical agents at early stages, by quantifying complete or fragmented chromosomes that are not incorporated into the nucleus during mitosis due to aneugenic or clastogenic effects. This method can be applied to any nucleated cell and requires small sample volumes, making it an ideal approach for toxicological studies [36]. Overall, these findings indicate that both PEGSL-ACG-NSps and βCDSL-ACG-NSps did not induce significant genotoxic or cytotoxic effects in the micronucleus assay at the tested doses following short-term exposure.

Despite the comprehensive characterization performed in this study, some limitations should be considered. The gastrointestinal behavior and potential bioavailability of ACGs were evaluated using an in vitro digestion model, which, although widely accepted, cannot fully replicate the complexity of in vivo digestion and absorption processes. In addition, the in vivo experiments were conducted at short-term exposure levels, which do not allow conclusions regarding chronic toxicity or long-term safety. Furthermore, pharmacokinetic parameters were not evaluated, and no specific biomarkers of oxidative stress were included, limiting the mechanistic interpretation of the observed biological responses. However, based on the results obtained in this first stage, we will conduct the second in vivo study to assess the pharmacokinetic profile, long-term toxicity, and incorporation of relevant biomarkers of oxidative stress.

4. Conclusions

This study demonstrates that both NSps are effective strategies for transporting ACGs without undergoing any changes during in vitro digestion. The βCDSL-ACG-NSps had 95% in vitro potential bioavailability, while PEGSL-ACG-NSps had only 55%. Finally, none of the formulations or their individual components induced detectable genotoxic or cytotoxic effects under the short-term conditions evaluated.