Preparation of SiNPs
SiNPs were purchased from Evonik with a purity of 99.99% and stored in a cool and dry environment to ensure their stability. SiNPs were identified and characterized by various techniques, including X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), and Zeta potential measurement. SiNPs were prepared to the appropriate concentration using 0.5% sodium carboxymethyl cellulose (CMC-Na) solution as a solvent before experimental administration. The solvent was obtained from Sinopharm Chemical Reagent Co., Ltd. (batch number: 20200713) and was preserved at room temperature. Additionally, the anesthetic drug pentobarbital sodium was obtained from Shanghai Yuyan Scientific Instrument Co., Ltd. (Batch number: 202003152) and was properly stored in a safe box at room temperature. Moreover, 0.9% sodium chloride injection (batch number: 2008192711) produced by Cisen Pharmaceutical Co., Ltd. was chosen as the anesthetic solvent. Sodium pentobarbital was dissolved and formulated into a 2% concentration anesthetic, specifically for animal anesthesia.
Animals and Husbandry
In this study, 60 healthy male and 60 healthy female rats were sourced from Shanghai Sippr-BK Laboratory Animals Co., Ltd. All experimental animals were certified as specific-pathogen-free. From the date of purchase, the rats were subjected to a 9-day quarantine observation period. At the beginning of the experiment, the rats were aged 7–9 weeks and weighed 218.7 ~ 267.3 g for males and 181.5 ~ 220.7 g for females. During the experimental period, five rats were housed per cage. The rats were maintained under rigorous environmental conditions, with the temperature stabilized at 21.1–24.1℃, humidity controlled at 53.1–57.1%, more than 15 air changes per hour, and a 12-hour light cycle provided daily. The feed was sourced from Shanghai Shihlin Biotechnology Co., Ltd and was given through rationing and free intake. All drinking water and feed were sterilized by autoclaving. Throughout the animal husbandry and dissection process, the guidelines for the Care and Use of Laboratory Animals by the National Institutes of Health (NIH) were always followed. In addition, this study program was approved by the Biomedical Ethics Committee of the Second Military Medical University. (approval number: IACUC-2019053).
Experimental design
Based on relevant data, the average daily consumption of silica for adults is estimated to be 9.4 mg/kg body weight [20]. Zande et al. conducted a repeated dose toxicity study for 84 days on SD rats with a dose of 1000 mg/kg [21]. The results showed that the 1000 mg/kg dose caused liver fibrosis in SD rats. Therefore, to evaluate the toxicity of SiNPs by long-term gavage on SD rats, three dosage groups were proposed: low, medium, and high, at 125, 250, and 500 mg/kg, respectively. The administration volume for rats was 10 ml/kg body weight, administered once a day. Additionally, a vehicle control group was established, which received an equivalent volume of 0.5% CMC-Na solution orally. A total of 120 SD rats (half male and half female) were assigned to four groups according to body weight using a randomized block group design. The rats were administered the respective solutions by gavage for 94 consecutive days, followed by withdrawal of the drug for a 30-day recovery period. The first administration date was defined as day 0, the dosing period ended on day 94 (d94), and the recovery period ended on day 124 (d124). The study was designed and implemented by the OECD Guidelines 408 [22]. The specific experimental design is shown in Fig. 1.
Clinical observation
From the beginning of the quarantine period, rats were monitored once a day for clinical symptoms and mortality. The frequency of observations was increased to twice a day from the beginning of treatment to the end of the trial. The observations included any abnormalities in the appearance and signs of the rats, their activities, the nature of their excreta, and the local reaction to the administration of the drug.
Body weight and food consumption
The body weight and food consumption of the rats were measured weekly after administration. Additionally, the body weight of the rats was determined at the beginning of the administration and before autopsy. Food consumption was assessed as follows: On the first day, a set quantity of feed (200g) was allocated to each group of both sexes. Subsequently, the residual feed was measured the following day. The difference between the two values was considered as the daily food consumption for the respective groups of animals.
Hematology and serum biochemistry
On days 94 and 124, hematological and serum biochemical examinations were performed on rats scheduled for dissection. Before sampling, the rats were fasted for at least 12 hours while maintaining normal water intake. On the day of sampling, the rats were first anesthetized using pentobarbital sodium, and the required blood samples were gathered from the abdominal aorta. The samples were gathered separately for the following assays: approximately 1.0 mL for hematology assays, approximately 0.9 mL for coagulation time assays, and approximately 3 mL for serum biochemistry assays. Hematological testing samples were treated with EDTA-K2 to prevent coagulation and then subjected to testing. For coagulation time testing, the samples were first anticoagulated with sodium citrate, and the plasma was separated through centrifugation (3000 g for 10 minutes) for testing. For serum biochemical testing, no anticoagulation was required and the serum was separated by centrifugation (3000 g for 10 minutes) for testing. Hematology tests cover a wide range of parameters, including the count of erythrocytes (RBC), the level of hemoglobin (HGB), hematocrit value (HCT), the average hemoglobin content of red blood cells (MCH), the average volume of a red blood cell (MCV), the average concentration of hemoglobin in red blood cells (MCHC), the count of platelets (PLT), the count of leukocytes (WBC), the proportion of lymphocytes (LYMPH), the proportion of reticulocyte count (RETIC), the proportion of neutrophils (NEUT), the proportion of large unstained cells (LUC), the proportion of eosinophils (EOS), the proportion of monocytes (MONO), the proportion of basophils (BASO), and prothrombin time (PT). Meanwhile, serum biochemical tests include total cholesterol (TCH), alanine aminotransferase (ALT), glucose (GLU), total bilirubin (TBIL), aspartate aminotransferase (AST), albumin (ALB), alkaline phosphatase (ALP), triglyceride (TG), creatinine (CREA), total protein (TP), lactate dehydrogenase (LDH), phosphorus (P), calcium (Ca), urea nitrogen (BU), creatine kinase (CK), and electrolytes such as chloride (Cl), potassium (K), and sodium (Na).
Urinalysis
On days 93 and 123, urinalysis was performed on rats. The urinalysis included leukocytes, urobilinogen, occult blood, protein, pH, specific gravity, glucose, bilirubin, nitrite, and ketones.
Pathological examination
On days 94 and 124, all rats scheduled for autopsy were euthanized by bloodletting under pentobarbital sodium anesthesia and underwent a detailed necropsy. The heart, brain, liver, spleen, adrenal glands, thymus, kidneys, testes, epididymis, uterus, and ovaries of the rats from each group were weighed, and the organ-to-body weight coefficient and organ-to-brain weight coefficient were calculated. The specific formulas are as follows. Organ-to-body weight coefficient = organ weight (g) / body weight (g) × 100%; Organ-to-brain weight coefficient = organ weight (g) / brain weight (g) × 100%. An exhaustive histopathological analysis was conducted on all organs and tissues of the animals within the high-dose group and the vehicle control group. The presence of drug-related toxic changes was determined by visual observation, organ weighing, and pathological histologic examination results. After fixation, the tissues underwent fine block trimming and were subjected to routine dehydration and paraffin embedding. Then, these tissues were cut into thin slices of approximately 4–5 µm using a slicer and colored with hematoxylin-eosin (HE) for examination. Finally, an Olympus imaging system was used for image recording. Based on the observed histopathological and morphological features, the characteristics of the lesions, the extent involved, and their severity were analyzed in detail.
Statistical analysis
The experimental data were presented as mean ± standard deviation and statistically analyzed using GraphPad Prism 5.0 software. For between-group comparisons, one-way analysis of variance (ANOVA) was used, and multiple comparisons were performed by Dunnett's test after statistical differences were found. For urine indicators, the Kruskal-Wallis test was chosen for analysis. A P value of less than 0.05 was considered statistically significant.