Chemicals

ISL was obtained from Damas-beta (Shenzhen, China). Casein was purchased from Adamas (Shanghai, China). Soybean phospholipid was acquired from Aiweituo Pharmaceutical Technology Co., LTD (Shanghai, China). Cholesterol was obtained from Aladdin Reagent Co., LTD (Shanghai, China). Zein was purchased from Tokyo Chemical Industry (Tokyo, Japan). Lecithin was obtained from Solarbio science and technology company (Beijing, China), hematoxylin-eosin staining solution (Sigma Aldrich, MO, USA), Triton x-100 (Sigma), paraformaldehyde (Sigma). The purity of the reagents above are all 98%. Solvents including dimethyl sulfoxide, ethanol, acetonitrile, and methanol (Sigma Aldrich, MO, USA), were of chromatographic grade. All other experiments were performed with Milli-Q deionized water (EMD Millipore, Billerica, MA, USA).

Animals

Both sexes of Kunming (KM) mice (15–20 g) and SD rats (105–120 g) were provided by SPF Biotechnology Co., Ltd, China. This study strictly adhered to the guidelines set forth in the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals. The research protocol received approval from the Ethics Committee of the Affiliated Hospital of Chengdu University of Traditional Chinese Medicine (approval number: 20220421). The room was maintained at a temperature of 25°C ± 1°C, with a relative humidity of 60%, and a 12-h daylight to darkness ratio. The animals had constant access to sufficient food and water. The animals were divided into six groups: Group 1 served as the control group, Group 2 received free ISL treatment, Group 3 was treated with the excipient (ZLH NPs), and Groups 4–6 were administered with respective ISL@ZLH NPs at doses of 27.5 mg/kg, 55.0 mg/kg, and 110.0 mg/kg body weight. All euthanasia procedures were carried out in accordance with the guidelines and regulations established by the institution’s animal care and use committee or equivalent regulatory body. For euthanasia, CO₂ was used as the anesthetic for euthanasia purposes. The animal was placed into a euthanasia box and CO₂ was infused at a rate of 30% of the box volume per minute until the animal stopped breathing and their pupils dilated. After turning off the CO₂ supply, the animal was observed for an additional 2 min to confirm death. Then their organs (liver, spleen, lung, kidney, prostate, testes, urinary bladder, stomach, duodenum, jejunum, colon, thyroid, adrenals, pancreas, salivary gland, lymph nodes, and thymus) were dissected and measured for histopathological analysis.

Preparation of ISL@ZLH NPs

ISL@ZLH NPs were prepared by the solvent evaporation method, which were formulated with distilled water to the required concentration for the test. Briefly, 30 mg of soybean phospholipid, 6 mg of cholesterol, 1 mg of zein, and 3 mg of ISL were dissolved in 2 mL of 90% ethanol (v/v). The resulting organic phase was slowly added to a 10 mL aqueous phase containing 1 mg of casein, while stirring gently at 500 r/min for 2 h under 40°C to evaporate the ethanol. After stirring, the ISL@ZLH NPs suspensions were centrifuged at 12,000 rpm for 10 min to remove non-encapsulated drugs, and then immediately filtered through a 0.45 μmol/L microporous membrane.

Acute toxicity of ISL@ZLH NPs

The study monitored the toxic effects of ISL@ZLH NPs in mice (15–20 g) and rats (105–120 g) with a sample size of 10 animals per group. The mice received ISL@ZLH NPs at a concentration of 160 mg/kg body weight via oral gavage, while the rats received 110 mg/kg body weight through the same route. Following administration, the animals were closely monitored for any signs of toxicity or adverse effects, including changes in behavior, appearance, and physiological parameters. Observations were made at regular intervals for a specified duration (e.g., from day 1 to day 7) to assess acute toxicity. The number of deaths and any morbidities were recorded, along with any abnormal behavior or clinical signs. The body weight of the animals was measured before the administration of ISL@ZLH NPs and at specified time points during the observation period. Food consumption was also monitored to assess any potential effects on appetite or feeding behavior. At the end of the observation period, surviving animals were euthanized, and a necropsy was performed. Organs such as the liver, kidney, heart, lungs, and spleen were collected and examined for macroscopic abnormalities. The median lethal dose (LD₅₀) values were calculated using the Bliss method (1938) or any other specific method utilized for this calculation.

Subacute toxicity of ISL@ZLH NPs

A total of 120 rats, aged 5–6 weeks, were divided into six groups of 20 animals each, with both sexes used in the study. Ten males and ten females were included in each group. Group 1 served as the control group, Group 2 received free ISL treatment [55 mg/(kg∙day)], Group 3 was treated with the excipient (ZLH NPs), and Groups 4–6 were administered with respective ISL@ZLH NPs at doses of 27.5 mg/kg, 55 mg/kg, and 110 mg/kg body weight. Subacute studies typically involve daily exposure of test subjects to a substance for a period of 30–90 days. These studies aim to identify the toxic effects of a substance that may occur within this relatively short exposure period. Changes in behavior, clinical signs, and body weight were monitored closely. Each rat was housed individually and provided with a rat diet. Before the endpoint of each period, the rats’ bladders were emptied by suprapubic manipulation immediately after saline or drug administration. Then, they were given 2 mL of water by gavage and placed in metabolism cages. After 5 h, urine was collected and a urine glutamic oxaloacetic transaminase (UGOT) assay was performed. At the end of each study, blood samples were taken from the orbital sinus for red blood cell (RBC) and white blood cell (WBC) counts, prothrombin time (PT), hemoglobin (Hb) and hematocrit (HCT) determinations, and blood glucose (GLU), blood urea nitrogen (BUN), and serum glutamic-pyruvic transaminase (SGPT) assays. At necropsy, the thyroid, adrenals, ovaries, uterus, prostate, testes, spleen, heart, kidneys, and liver were weighed, and the ratios of organ weight to body weight were calculated. The methodology involved evaluating the organ coefficients in ISL@ZLH NPs in comparison to free ISL and control-treated SD rats. The study measured the changes in the size and weight of organs in the rats to determine the effectiveness of the ISL@ZLH NPs. The formula used to calculate the organ coefficients was as follows: [organ weight (g) ÷ body weight] × 100%.

Representative tissues from organs including the liver, spleen, lung, kidney, prostate, testes, urinary bladder, stomach, duodenum, jejunum, colon, thyroid, adrenals, pancreas, salivary gland, lymph nodes, and thymus were fixed in formalin for histopathological examination.

Evaluation of toxicity in various organ systems

The evaluation of the animals’ eyes, skin, fur, mucous membranes, respiratory system, circulatory system, autonomic nervous system, and central nervous system was performed according to the previous methods [37–40]. The animals’ eyes were visually inspected for any signs of redness, discharge, swelling, or other abnormalities. Additionally, the anterior and posterior segments of the eye, including the cornea, lens, and retina were also examined. The skin and fur were carefully examined for any signs of irritation, redness, swelling, lesions, or abnormal hair loss. The presence of any abnormalities, such as rashes or wounds, was noted. The mucous membranes, including those of the oral cavity and nasal passages, were observed for any signs of inflammation, ulcers, or discoloration. The animals’ respiratory system was assessed by monitoring their respiratory rate and observing any abnormal breathing patterns. The circulatory system was evaluated by monitoring the animals’ heart rate and pulse. Additionally, the color and temperature of the extremities were assessed. The autonomic nervous system was assessed by observing the animals’ response to various stimuli, such as light or sound. Changes in pupil size, salivation, lacrimation, urination, or defecation were noted. The central nervous system was evaluated through a comprehensive neurological examination, which included assessing the animals’ coordination, balance, reflexes, and overall behavior. Any signs of motor abnormalities, tremors, seizures, or changes in consciousness were recorded.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 9.0 software. The data was presented as means ± standard deviations for 5 animals each. The Dunnett (1964) t-test was used to determine the significance of data variability between the treated and control groups. Statistical significance was considered at P < 0.05.

Preparation and storage of ISL@ZLH NPs

Previously, ISL@ZLH NPs were prepared and characterized in the laboratory. These NPs had a size of approximately 132.06 nm, a polydispersity index of 11.2, and a zeta potential of −37.2 mV [12]. The stability of these nanomaterials was crucial for subsequent experiments. Therefore, ISL@ZLH NPs were stored at 4°C for a period of 14 days. The findings of the present study indicated that ISL@ZLH NPs exhibited consistent particle size, and there were no observable turbidity or impurity peaks. This suggests that the ISL@ZLH NPs were highly stable, even at 14 days of storage at 4°C.

Evaluation of the acute toxicity of ISL@ZLH NPs

In the acute toxicity study, a single oral dose of ISL@ZLH NPs at a high concentration (160 mg/kg for mice and 110 mg/kg for rats) did not result in mortality in both KM mice and SD rats. Normal patterns of behavior were observed in all groups (Table 1). There were no abnormalities in animals’ eyes, skin, fur, mucous membranes, respiratory system, circulatory system, autonomic nervous system, or central nervous system when treated with ISL@ZLH NPs. In addition, no significant difference was detected in body weights of these groups (Figures 1 and 2).

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Figure 1. 

The changes in body weight (g) of KM mice during acute toxicity studies for 7 days. Presented as mean ± standard deviation, with a sample size of 5 mice per group

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Figure 2. 

The changes in body weight (g) of SD rats during acute toxicity studies for 7 days. Presented as mean ± standard deviation, with a sample size of 5 rats per group

Evaluation of the subacute toxicity of ISL@ZLH NPs

During the 30-day subacute toxicity studies (Table 2), the body weight of the SD rats remained consistent across all groups, including control, free ISL, excipient, and ISL@ZLH NPs (27.5 mg/kg, 55.0 mg/kg, 110.0 mg/kg body weight) groups, with no statistically significant differences observed among the groups. In male rats, measurements taken at the beginning of the study and at various intervals (1 day: 126.38 g ± 4.85 g, 10 days: 202.04 g ± 19.82 g, 20 days: 247.66 g ± 20.73 g, and 30 days: 276.52 g ± 17.16 g) showed no significant changes compared to the respective control group. Similarly, in female rats, there were no significant changes in body weight at various intervals (1 day: 113.88 g ± 3.91 g, 10 days: 147.42 g ± 8.51 g, 20 days: 170.98 g ± 13.04 g, and 30 days: 189.08 g ± 17.36 g) compared to the respective control group.

During the 90-day subacute toxicity studies (Table 3), the body weight of the SD rats remained consistent across all groups, including control, free ISL, excipient, and ISL@ZLH NPs groups, with no statistically significant differences observed among the groups. In both male and female rats, measurements taken at the beginning of the study and continued at 10-day intervals until 90 days showed no significant changes compared to the respective control group. The lack of change in body weight suggests that the administration of ISL@ZLH NPs and free ISL and excipient did not have any adverse effects on rats’ growth or metabolism. In the context of an excipient, it refers to the ZLH NPs administered group. This is a positive finding, as changes in body weight could indicate toxicity or other negative effects on rats’ overall health. The stable body weight also suggests that rats’ food intake and activity levels were not affected by the treatment. This supports the overall safety of ISL@ZLH as well as free ISL and excipients.

Evaluation of organ coefficients in ISL@ZLH NPs-treated SD rats

In the study, organ coefficients in SD rats were evaluated at two different time points: 30 days and 90 days. The results are presented in Tables 3 and 4. During the 30-day subacute toxicity studies (Table 4), the organ coefficients of SD rats remained consistent across all groups, including control, free ISL, excipient, and ISL@ZLH NPs (27.5 mg/kg, 55.0 mg/kg, 110.0 mg/kg body weight) groups, with no statistically significant differences observed. In male rats, organ coefficients were calculated on the 30th day and showed no significant changes compared to the respective control group for heart (0.371% ± 0.022%), thyroid (0.012% ± 0.001%), spleen (0.228% ± 0.032%), liver (2.497% ± 0.086%), kidneys (0.546% ± 0.012%), adrenals (0.019% ± 0.001%), prostate (0.124% ± 0.009%), and testes (0.660% ± 0.010%). Similarly, in female rats, there were no significant changes in organ coefficients on the 30th day for heart (0.372% ± 0.043%), thyroid (0.013% ± 0.001%), spleen (0.227% ± 0.027%), liver (2.563% ± 0.106%), kidneys (0.551% ± 0.005%), adrenals (0.019% ± 0.001%), ovaries (0.230% ± 0.016%), and uterus (0.055% ± 0.005%) compared to the respective control group. During the 90-day subacute toxicity studies (Table 5), the organ coefficients of SD rats remained consistent across all groups, indicating that the administration of different doses of ISL@ZLH NPs and free ISL-treated groups did not have any adverse effects on the organs compared to the control group.

Organ coefficients are a measure of the relative weight of an organ compared to the total body weight of an animal. It is an important parameter that is used to evaluate the toxicity of a substance. An increase in organ coefficients indicates that the organ is growing larger than expected, which could be a sign of toxicity. On the other hand, a decrease in organ coefficients could indicate that the organ is shrinking, which could also be a sign of toxicity. The fact that there were no significant differences in organ coefficients between the groups at both time points suggests that the substance did not cause any adverse effects on the organs of the rats. This is an important finding as it indicates that the substance is safe for use in animals.

Evaluation of urine volume and UGOT in ISL@ZLH NPs-treated SD rats

The results of the volume of urine and UGOT did not show any differences over the 30-day and 90-day periods, as presented in Table 6. During the 30-day subacute toxicity studies (Table 6), the urine volume and UGOT of SD rats remained consistent across all groups, including control, free ISL, excipient, and ISL@ZLH NPs (27.5 mg/kg, 55.0 mg/kg, 110.0 mg/kg body weight) groups, with no statistically significant differences observed. In male rats, urine volume was 1.37 mL ± 0.03 mL and UGOT levels were 3.13 mL ± 0.05 mL on the 30th day, and 3.37 mL ± 0.08 mL and 3.21 mL ± 0.06 mL, respectively, on the 90th day. The data indicated that there were no significant changes in the volume of urine or UGOT compared to the respective control group in male rats. In female rats, urine volume was 1.25 mL ± 0.05 mL and UGOT levels were 2.73 mL ± 0.07 mL on the 30th day, and 2.12 mL ± 0.04 mL and 2.90 mL ± 0.06 mL, respectively, on the 90th day (Table 5). The data revealed no significant alterations in the volume of urine or UGOT compared to the corresponding control group in female rats. These findings imply that the experimental treatment did not exert significant effects on these measures in male or female rats, thus indicating no significant detrimental impacts on the rats’ health throughout the study.

Evaluation of hematological parameters in ISL@ZLH NPs-treated SD rats

The results of the hematological parameters did not show any differences over the 30-day and 90-day periods, as presented in Tables 7 and 8. During the 30-day subacute toxicity studies (Table 7), the hematological parameters of SD rats remained consistent across all groups, including control, free ISL, excipient, and ISL@ZLH NPs (27.5 mg/kg, 55.0 mg/kg, 110.0 mg/kg body weight) groups, with no statistically significant differences observed. In male rats, hematological parameters were determined on the 30th day and showed no significant changes in RBC [(7.06 ± 0.07) × 10¹²/L], WBC [(12.29 ± 1.10) ×10⁹/L] Hb (218.49 g/L ± 3.94 g/L), HCT (45.24% ± 0.43%), GLU (9.62 mmol/L ± 0.32 mmol/L), BUN (5.62 mmol/L ± 0.23 mmol/L), SGPT (51.40 U/L ± 2.36 U/L), and PT (12.90 s ± 0.43 s) compared to the respective control group. Similarly, in female rats, there were no significant changes in hematological parameters on the 30th day for RBC [(7.06 ± 0.07) × 10¹²/L], WBC [(12.29 ± 1.10) × 10⁹/L], Hb (218.49 g/L ± 3.94 g/L), HCT (45.24% ± 0.43%), GLU (9.62 mmol/L ± 0.32 mmol/L), BUN (5.62 mmol/L ± 0.23 mmol/L), SGPT (51.40 U/L ± 2.36 U/L), and PT (12.90 s ± 0.43 s) compared to the respective control group.

During the 90-day subacute toxicity studies (Table 8), the hematological parameters of SD rats remained consistent across all groups, indicating that the administration of different doses of ISL@ZLH NPs and free ISL-treated groups did not have any adverse effects on the blood parameters compared to the control group. These findings suggest that the subacute toxicity test over both 30 days and 90 days did not have any significant effects on the hematological parameters in rats. It is important to note that hematological parameters are crucial indicators of the overall health of the animals, and the lack of any significant changes in these parameters indicates that the subacute toxicity test did not have any adverse effects on the health of the rats.

Evaluation of histological observation in ISL@ZLH NPs-treated SD rats

Upon microscopic examination of the lung tissue, it was observed that the cellular structures of the bronchiole, alveoli, alveolar duct, and blood vessels were normal in rats administered ISL@ZLH NPs and ISL with excipient, as compared to the control group. Similarly, the architecture of cardiac muscle cells and connective tissue was found to be normal in the heart tissue of treated rats. The liver tissue of treated rats showed no abnormalities in the cellular structures of hepatocytes, sinusoids, and the central vein, which were comparable to those of the control group. In the kidney, the epithelial lining of glomerular tufts and renal tubules were observed with no abnormalities. Furthermore, there were no abnormalities observed in the acinar and islet cells of the pancreas of rats treated with ISL@ZLH NPs, as compared to rats in the control group. These findings suggest that the administration of ISL@ZLH NPs and ISL with excipient did not result in any significant structural changes in the cellular tissue of the lung, heart, liver, kidney, and pancreas of rats. Furthermore, there were no abnormalities or any significant structural changes in the cellular tissue of the salivary glands, stomach, duodenum, jejunum, colon, urinary bladder, lymph nodes, thymus, spleen, mammary gland, ovaries, uterus, prostate, testicles, thyroid and adrenal glands. Based on the findings, it can be concluded that the treatment of ISL@ZLH NPs did not lead to any significant adverse effects on the morphology of these tissues. This information is supported by the data presented in Figures 3, 4, 5, and 6.

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Figure 3. 

Histology of tissue from the subacute toxicity testing of ISL@ZLH NPs in male rats of 30 days. All histological sections of the heart, liver, spleen, lung, kidney, prostate, testes, urinary bladder, stomach, duodenum, jejunum, colon, thyroid, adrenals, pancreas, salivary glands, lymph nodes, and thymus (hematoxylin and eosin staining, ×10) of rats

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Figure 4. 

Histology of tissue from the subacute toxicity testing of ISL@ZLH NPs in female rats of 30 days. All histological sections of the heart. livers, spleen, lungs, kidneys, ovaries, uterus, urinary bladder, stomach, duodenum, jejunum, colon, thyroid, adrenals, pancreas, salivary glands, lymph nodes, and thymus (hematoxylin and eosin staining, ×10) of rats

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Figure 5. 

Histology of tissue from the subacute toxicity testing of ISL@ZLH NPs in male rats of 90 days. All histological sections of the heart. livers, spleen, lungs, kidneys, prostate, testes, urinary bladder, stomach, duodenum, jejunum, colon, thyroid, adrenals, pancreas, salivary glands, lymph nodes, thymus (hematoxylin and eosin staining, ×10) of rats

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Figure 6. 

Histology of tissue from the subacute toxicity testing of ISL@ZLH NPs in female rats of 90 days. All histological sections of the heart. livers, spleen, lungs, kidneys, ovaries, uterus, urinary bladder, stomach, duodenum, jejunum, colon, thyroid, adrenals, pancreas, salivary glands, lymph nodes, thymus, mammary glands (hematoxylin and eosin staining, ×10) of rats

Study limitations

The findings of this study should be interpreted with caution due to the use of animal models, which may not fully capture the complexity and variability of human biology and disease conditions. Extrapolation of these results to human populations should be done cautiously. Additionally, longer-term studies are necessary to gain a better understanding of the sustained effects and potential long-term safety of ISL@ZLH NPs. Conducting long-term follow-up studies would be valuable to assess any potential delayed or cumulative effects. Furthermore, future studies should explore the optimal dosage range and potential dose-dependent effects of ISL@ZLH NPs. Although this study evaluated the anti-inflammatory effects of ISL@ZLH NPs, it did not measure specific inflammatory markers such as C-reactive protein or interleukins [ILs (IL-1, IL-6)]. Incorporating these measurements in future studies would provide a more comprehensive assessment of the anti-inflammatory activity of ISL@ZLH NPs.

Conclusion

Animal models, including KM mice and SD rats, were utilized to assess the acute and subacute toxicity of ISL@ZLH NPs. The findings demonstrated that ISL@ZLH NPs exhibit safety and non-toxicity in these animal models, thereby indicating their potential for clinical application. The study observed no noteworthy alterations in biochemical, hematological, and histopathological parameters in both species across all tested doses, highlighting the promising potential of ISL@ZLH NPs as a drug delivery system for ISL. This innovative approach could enhance therapeutic efficacy and bioavailability while upholding safety considerations. However, further extensive investigations are warranted to validate these findings and establish the full extent of their clinical utility.