Cardiotoxicological assessment of crocin-coated silver nanoparticles in an experimental rat model

Abstract

Silver nanoparticles (AgNPs) are widely used for various industrial and biomedical applications; however, their potential cardiotoxicity remains insufficiently understood and requires further investigation. We evaluated the cardiotoxic effects of AgNPs and crocin-coated AgNPs (CR-AgNPs) administered at doses of 20, 40, and 80 µg/kg in male Wistar rats. Forty-two rats were randomly allocated to seven groups as follows: The groups included a control group (normal saline), three AgNPs-treated groups (20, 40, and 80 µg/kg), and three crocin-coated AgNPs-treated groups (20, 40, and 80 µg/kg), all of which received treatment via oral gavage for 30 days. At the end of the experiment, the animals were anesthetized with ketamine and xylazine, and blood samples and cardiac tissues were collected for biochemical and histopathological analyses. Our findings showed that malondialdehyde (MDA) levels were significantly increased in the AgNPs 80 group compared with the control group. In addition, administration of CR-AgNPs at 80 µg/kg significantly reduced MDA levels compared with the AgNPs 80 group. Histopathological examination confirmed the toxic effect of AgNPs at 80 µg/kg, as evidenced by mild interstitial edema and localized separation of myocardial muscle fibers. In conclusion, these findings suggest that CR-AgNPs may represent a safer alternative for pharmaceutical applications by reducing the risk of subacute cardiotoxicity in experimental animals. Further investigations are required to provide additional evidence in this field.

Graphical Abstract

Highlights

• •CR-AgNPs may represent a safe alternative for pharmaceutical applications.
• •CR-AgNPs did not induce cardiotoxicity.
• •The safety of AgNPs is related to their physico-chemical properties.

1. Introduction

Metal nanoparticles have attracted considerable attention worldwide, and their products are used in various pharmaceutical, industrial, and environmental applications [1]. Among the various metal nanoparticles developed through nanotechnology, silver nanoparticles (AgNPs) possess unique physicochemical properties that support their use in biomedical products, including wound dressings, creams, and antiseptic sprays [2]. The increasing integration of nanotechnology with biomedical applications has also contributed to the development of innovative approaches for diagnosis, sensing, and therapeutic applications, highlighting the growing importance of nanomaterials in modern healthcare [3].The widespread use of metal nanoparticles raises concerns regarding their release into and accumulation within environmental systems, which may pose risks to human health [4]. Several studies have reported toxic effects of AgNPs in agricultural systems and animal models [5]. The toxicity of AgNPs is strongly influenced by physicochemical properties such as size, shape, surface charge, aggregation or agglomeration state, crystallinity, surface coating, functionalization, and composition [6]. These properties influence biological interactions, cellular uptake, tissue distribution, and elimination, thereby determining the toxicity of AgNPs within biological systems [7]. However, studies have reported inconsistent findings regarding their toxicity, which may be related to differences in synthesis methods [8]. Conventional physicochemical synthesis methods have several limitations, including particle agglomeration and potential adverse health effects [9]. Green synthesis methods using plants, bacteria, and fungi have gained attention as eco-friendly and sustainable alternatives in recent years [10]. These biological materials act as reducing and stabilizing agents and may help minimize toxicity-related concerns [11]. Among these approaches, the green synthesis of AgNPs using plant extracts or plant-derived flavonoids has been shown to effectively reduce and stabilize silver ions [12]. Saffron, the dried stigma of Crocus sativus, is well known for its potent antioxidant properties [13]. The major bioactive constituents of saffron include crocin, crocetin, picrocrocin, and safranal, which contribute to its free-radical-scavenging activity [14]. More broadly, recent research has highlighted the therapeutic and protective potential of bioactive compounds derived from medicinal plants, supporting continued investigation of plant-derived compounds as potential agents in biomedical applications [15]. Numerous human and animal studies have demonstrated the ability of crocin to reduce oxidative stress and enhance antioxidant status 16, 17, 18. Howere, using of crocin in green-synthesized AgNPs and investigating the toxicological effects of this formulationin animal models that may help clarify how these nanoparticles can be safely utilized in biomedical and pharmaceutical applications has not been evlauated til now [5]. Despite extensive research in this field, several uncertainties remain regarding how the physicochemical properties of AgNPs influence nanotoxicity. For the first time, this study was desgined to disclose the pharmacologically important biosynthesizing of AgNPs coated with crocin and find a rapid and cost-effective of drug delivery formulation. Also, we aimed to determine the safety or toxicity of crocin-coated AgNPs on cardiac tissue of amle rats by evalating cardiac enzyme and oxidative stress indices and also histopathological altartions.

2. Materials and methods

2.1. Chemicals

Silver nitrate (AgNO3) was optained from the Pallava brand (Catalog Number: 0232, made in India). Crocin was obtained from Sigma Aldrich (Catalog Number: 42553–65–1). CK-MB (Catalog Number: CK-MB−917-SP) and LDH (Catalog Number: LDH-H−917-S) kits were obtained from Delta.DP. CO, Iran. GSH (Catalog Number: NS−15087) MDA (Catalog Number: NS−15023), NO (Catalog Number: NS−15043), SOD (Catalog Number: NS−15033) kits were obtained from Navand Salamat co., Iran.

2.2. Ethical approval

This experiment was approved by the Animal Ethics Committee of Birjand University of Medical Sciences, Birjand, Iran, and was conducted in accordance with the ARRIVE guidelines (Ethical Approval No. IR.BUMS.AEC.1404.016).

2.3. Silver nanoparticle synthesis

Crocin-coated AgNPs were synthesized as follows: Ten milliliters of an aqueous crocin solution was added dropwise to 90 mL of a 1 mM AgNO₃ solution.The mixture was then placed on a shaker and incubated at room temperature for 24 h [19]. After 24 h, the color of the solution changed, indicating the formation of nanoparticles.The silver nanoparticles were collected by centrifugation at 10,000 rpm for 10–15 min and washed thoroughly.The supernatant was discarded, and deionized water was added to the pellet, which was then sonicated to ensure proper dispersion.This procedure was repeated three times to ensure complete removal of residual nitrate. The resulting sediment containing silver nanoparticles was maintained at 50–55 °C for 24 h, cooled to room temperature, and stored in the dark at 4 °C.

2.4. Characterization of silver nanoparticles

Characterization of the synthesized nanoparticles is essential for understanding their physicochemical and functional properties. The synthesized nanoparticles were characterized using zeta potential/dynamic light scattering analyzer (PZ/DLS) and transmission electron microscopy (TEM; Zeiss EM10, Carl Zeiss, Germany) at MHAMAX Company, Tehran, Iran.

2.5. Animal model

The rats were obtained from the Experimental Medicine Center of Birjand University of Medical Sciences.Before the start of the study, the animals were acclimatized to the laboratory environment for at least one week.Throughout the experimental period, the rats had free access to standard pellet chow and water and were maintained under controlled conditions of humidity (60 ± 5%), temperature (30 ± 2 °C), and a 12-h light/12-h dark cycle.

2.6. Determining sample size

The animals used in this study were obtained from the Experimental Medicine Center of Birjand University of Medical Sciences.Based on the resource equation approach described in the literature and considering k = 5 experimental groups, the calculated sample size was approximately five animals per group.To account for possible attrition or mortality, six rats were included in each group (2).

2.7. Animal grouping

A total of 42 adult male rats weighing 250–300 g were assigned to seven groups, with six animals in each group according to the simple randomization method.The experimental groups were as follows:Group 1 (control): animals received 0.5 mL of normal saline by oral gavage.Group 2 (AgNP20): animals received silver nanoparticles at a dose of 20 µg/kg by oral gavage. Group 3 (AgNP40): animals received silver nanoparticles at a dose of 40 µg/kg by oral gavage.Group 4 (AgNP80): animals received silver nanoparticles at a dose of 80 µg/kg by oral gavage.Group 5 (CR-AgNP20): animals received crocin-coated silver nanoparticles at a dose of 20 µg/kg by oral gavage.Group 6 (CR-AgNP40): animals received crocin-coated silver nanoparticles at a dose of 40 µg/kg by oral gavage. Group 7 (CR-AgNP80): animals received crocin-coated silver nanoparticles at a dose of 80 µg/kg by oral gavage [20].After 30 days of treatment, all animals were anesthetized with ketamine (50 mg/kg) and xylazine (10 mg/kg).Following tissue collection, a portion of each sample was fixed for histopathological evaluation.The remaining tissue samples were stored at −80°C until analysis of oxidative stress markers. The biochemical and histopathological tests were perfomed by blinded investigators.

2.8. Assessment of Oxidative Stress Markers

2.8.1. Measurement of reduced glutathione

Reduced glutathione (GSH) levels were measured using a commercial assay kit (Kiazist, Iran). According to the manufacturer's protocol, Ellman's reagent (DTNB) reacts with free sulfhydryl (–SH) groups in their reduced form, producing a yellow-colored compound. Thus, only free reduced thiol groups participate in this reaction, whereas oxidized thiols remain unreactive.Reduced thiol groups, including glutathione (GSH), reflect the antioxidant status of the tissue and contribute to antioxidant defense by serving as cofactors for glutathione peroxidase during free-radical neutralization.The absorbance of the DTNB–thiol complex was measured at 405 nm.

2.8.2. Measurement of Superoxide Dismutase

Superoxide dismutase (SOD) is an antioxidant enzyme that plays a key role in protecting cells against oxidative stress by neutralizing superoxide anions (O₂⁻). SOD activity was determined using a commercial assay kit (Kiazist, Iran).

2.8.3. Measurement of Nitric Oxide

Nitric oxide (NO) acts as a cellular signaling molecule in vertebrates, plants, bacteria, and fungi. In vertebrates, NO can be produced by phagocytic cells and may activate downstream inflammatory pathways.Its vasodilatory effect is well established. NO is also considered a free radical that may contribute to oxidative stress under certain conditions.

2.8.4. Measurement of Malondialdehyde

Malondialdehyde (MDA) is a widely used biomarker of lipid peroxidation and oxidative damage to cell membranes.One of the most commonly used methods for measuring MDA is the TBARS assay, in which MDA reacts with thiobarbituric acid to form a colored complex.The intensity of the resulting color is directly proportional to the MDA concentration and is measured spectrophotometrically at approximately 535 nm.

2.8.5. Measurement of serum cardiac enzymes

Serum levels of lactate dehydrogenase (LDH) and creatine kinase MB (CK-MB) were measured using commercial assay kits (PARS AZMON, Iran) according to the manufacturer's instructions.

2.9. Histopathological Assay

Following collection, heart tissue samples were fixed in 10% formalin.The specimens were subsequently dehydrated in graded ethanol solutions and cleared with xylene. The tissues were embedded in paraffin at 60 °C and sectioned into 4–5 μm thick slices.The sections were stained with hematoxylin and eosin (H&E) for histological evaluation.Three sections from each specimen were evaluated independently in a blinded manner by an experienced pathologist.

2.10. Data Analysis

All data were analyzed using GraphPad Prism software (version 10.4.1).Following preliminary evaluation of the data, appropriate statistical tests were selected to assess differences among groups. The normality of data was determined by Kolmogorov–Smirnov (K–S) test.

After determining the normality of our data, one-way analysis of variance (ANOVA) and Tukey post hoc test were used for group comparisons, as appropriate. A P value of less than 0.05 was considered statistically significant. The results are presented in tables and figures.

3. Results

3.1. Findings of nanoparticles charactrization

The DLS findings showed that AgNPs had a hydrodynamic diameter of 28.37 ± 6.57 nm, a PDI of 0.238 and a zeta potential of −5.41 (Fig. 1A) and CR-AgNPs had a size of 36.18 ± 9.46 nm, PDI of 0.298 and a zeta potential of −1.93 (Fig. 1B).

Fig. 1.

DLS analysis showed hydrodynamic diameter of AgNPs (1 A) and CR-AgNPs (1B). Abbrevation: AgNPs: silver nanoparticles, CR-AgNPs: crocin-coated silver nanoparticles.

TEM micrographs indicated the formation of predominantly spherical AgNPs and CR-AgNPs (Figs. 2A and 2B). Analysis of particle size distribution demonestrated average diameters of 8.91 ± 1.59 nm for AgNPs and 11.81 ± 3.08 nm for CR-AgNPs, respectively (Figs. 2A and 2B). The particle sizes were distributed within the range of 6–18 nm, confirming the successful synthesis of nanoparticles smaller than 20 nm by both chemical and green methods. The relatively narrow size distribution suggested a good degree of particle uniformity. The nanoscale size and morphology observed for CR-AgNPs indicate that these nanoparticles may be suitable for further investigation in biomedical and antibacterial applications (Figs. 3A and 3B).

Fig. 2.

TEM analysis of AgNPs (2 A) and CR-AgNPs (2B) showed spherical shape as well as distribution. Abbrevation: AgNPs: silver nanoparticles, CR-AgNPs: crocin-coated silver nanoparticles.

Fig. 3.

TEM images of particle size of AgNPs with average diameters of 8.91 ± 1.59 nm was showen in Fig. 1C. Fig. 1D showed a TEM picture of 11.81 ± 3.08 nm CR-AgNPs. Abbrevation: AgNPs: silver nanoparticles, CR-AgNPs: crocin-coated silver nanoparticles.

3.2. Findings of serum cardiac enzyme function

No significant differences were observed in serum creatine kinase MB (CK-MB) or LDH levels between the treated groups and the control group (Table 1).

Table 1.

The serum levels of CK-MB and LDH of experimental groups.

Groups Parameters	C	AgNPs 20	AgNPs40	AgNPs80	CR-AgNP20	CR-AgNP40	CR-AgNP80
CK-MB (U/L)	221.28 ± 14.21	223.11 ± 17.13	223.43 ± 9.87	235.71 ± 21.27	230.09 ± 11.84	237.18 ± 22.35	230.91 ± 20.11
LDH (U/L)	434.75 ± 6.56	430.70 ± 10.71	425.82 ± 7.27	430.68 ± 9.99	430.00 ± 11.21	430.07 ± 10.25	423.93 ± 8.40

Data are presented as mean ± SD.

C: control, AgNPs 20: silver nanoparticles 20 µg/kg, AgNPs 40: silver nanoparticles 40 µg/kg, AgNPs 80: silver nanoparticles 80 µg/kg, CR-AgNPs 20: crocin coated- silver nanoparticles 20 µg/kg, CR-AgNPs 40: crocin coated- silver nanoparticles 40 µg/kg, CR-AgNPs 80: crocin coated- silver nanoparticles 80 µg/kg groups.

3.3. Findings of oxidative stress indices

Analysis of oxidative stress markers showed that only MDA levels differed significantly between the control group and the AgNPs 80 group (P < 0.001).Analysis of oxidative stress markers showed that only MDA levels differed significantly between the control group and the AgNPs 80 group (P < 0.001). A significant difference in MDA levels was also observed between the CR-AgNPs80 and AgNPs 80 groups (P < 0.001). No significant differences were detected among the remaining groups for MDA or any of the other oxidative stress parameters (Table 2).

Table 2.

Results of oxidative stress indexes in the cardiac tissue samples of experimental groups.

Groups Parameters	C	AgNPs 20	AgNPs40	AgNPs80	CR-AgNP20	CR-AgNP40	CR-AgNP80
SOD (U/mg tissue)	1.163 0.09 ±	1.23 ± 0.17	1.18 ±0.12	1.19 ± 0.07	1.29 ±0.17	1.20 ±0.11	1.30 ±0.19
GSH(µM/mg tissue)	2.76 ± 0.66	2.69 ±0.40	2.77 ±0.15	2.64 ± 0.69	2.88 ±0.39	2.88 ±0.42	3.01 ±0.61
NO (µM/mg tissue)	0.101 ± 0.026	0.104 ±0.027	0.109 ± 0.019	0.107 ±0.012	0.105 ± 0.025	0.093 ±0.018	0.106 ±0.031
MDA (nM/mg tissue)	2.46 ±0.52	2.13 ± 0.67	2.06 0.49 ±	3.93 ±0.79 ***	1.90 ±0.32	1.86 ± 0.28	2.38 ±0.49 ###

Data are presented as mean ± SD.

*Significant difference compared to the control group: ***;P < 0.001.

#Significant difference between AgNPs 80 group versus CR-AgNPs group: ###: P < 0.001.

C: control, AgNPs 20: silver nanoparticles 20 µg/kg, AgNPs 40: silver nanoparticles 40 µg/kg, AgNPs 80: silver nanoparticles 80 µg/kg, CR-AgNPs 20: crocin coated- silver nanoparticles 20 µg/kg, CR-AgNPs 40: crocin coated- silver nanoparticles 40 µg/kg, CR-AgNPs 80: crocin coated- silver nanoparticles 80 µg/kg groups.

3.4. Histopathological findings

Histopathological examination of cardiac tissue (Fig. 4) showed normal myocardial architecture in the control group (A), characterized by regularly arranged cardiac muscle fibers, preserved cross-striations, centrally located nuclei, and no evidence of degeneration, necrosis, inflammatory cell infiltration, edema, or vascular congestion.No remarkable histopathological alterations were observed in the myocardial tissue of the AgNPs 20 group (B) compared with the control group.Likewise, myocardial tissue from the AgNPs 40 group (C) appeared comparable to that of the control group, with no notable pathological changes.In the AgNPs 80 group (D), mild interstitial edema and focal separation of myocardial muscle fibers were observed. However, there was no clear evidence of necrosis, hemorrhage, or inflammatory cell infiltration.No significant histopathological changes were observed in the CR-AgNPs 20 (E), CR-AgNPs 40 (F), or CR-AgNPs 80 (G) groups when compared with the control group. Histopathological scoring of cardiac tissue samples is showed in Table 3.

Fig. 4.

Histopathological findings of cardiac sections of C: control, AgNPs 20: silver nanoparticles 20 µg/kg, AgNPs 40: silver nanoparticles 40 µg/kg, AgNPs 80: silver nanoparticles 80 µg/kg, CR-AgNPs 20: crocin coated- silver nanoparticles 20 µg/kg, CR-AgNPs 40: crocin coated- silver nanoparticles 40 µg/kg, CR-AgNPs 80: crocin coated- silver nanoparticles 80 µg/kg groups,Staining: Hematoxylin and eosin (H&E), Magnification: x400, Scale bar = 100 µm. A: normal normal sturture of cardiac tissue that is characterized by regularly arranged cardiac muscle fibers, preserved cross-striations, centrally located nuclei, and no evidence of degeneration, necrosis, inflammatory cell infiltration, edema, or vascular congestion (control group). B and C: No remarkable histopathological alterations were observed in the myocardial tissue (AgNPs 20 and AgNPs 40 groups) D: Mild interstitial edema and focal separation of myocardial muscle fibers were observed. However, there was no clear evidence of necrosis, hemorrhage, or inflammatory cell infiltration (AgNPs 80 group). E, F and G: No significant histopathological changes (CR-AgNPs 20 (E), CR-AgNPs 40 (F), or CR-AgNPs 80 (G) groups.

Table 3.

Semi-quantities evaluation of cardiac tissue samples.

Groups Pathological changes	C	AgNPs 20	AgNPs40	AgNPs80	CR-AgNP20	CR-AgNP40	CR-AgNP80
Degeneration	1	1	1	2	1	1	1
Necrosis	1	1	1	1	1	1	1
Inflammation	1	1	1	1	1	1	1
Edema	1	1	1	2	1	1	1
Interstitial hemorrhage	1	1	1	1	1	1	1

C: control, AgNPs 20: silver nanoparticles 20 µg/kg, AgNPs 40: silver nanoparticles 40 µg/kg, AgNPs 80: silver nanoparticles 80 µg/kg, CR-AgNPs 20: crocin coated- silver nanoparticles 20 µg/kg, CR-AgNPs 40: crocin coated- silver nanoparticles 40 µg/kg, CR-AgNPs 80: crocin coated- silver nanoparticles 80 µg/kg groups.

4. Discussion

To the best of our knowledge, this is the first experimental study to directly compare the cardiotoxic effects of crocin-coated AgNPs (20, 40, and 80 µg/kg) with those of uncoated AgNPs administered at the same doses in adult male rats. Most studies investigating AgNPs toxicity have focused on bacteria, fish species, cell lines, or isolated tissues, whereas in vivo evidence remains limited. Toxicological studies in mammalian models are essential for understanding how the physicochemical characteristics of nanoparticles influence living systems. Our findings showed no significant differences in serum LDH and creatine kinase MB (CK-MB) levels or in cardiac GSH levels, NO levels, and SOD activity between the treated groups and the control group.Likewise, MDA levels did not differ significantly from those of the control group in any treatment group except the AgNPs 80 group. Furthermore, histological examination of cardiac tissue revealed mild interstitial edema with focal separation of muscle fibers, without evidence of necrosis, hemorrhage, or inflammatory cell infiltration, only in rats receiving AgNPs at 80 µg/kg. In the present study, TEM analysis confirmed the successful synthesis of AgNPs and CRO-AgNPs, which appeared as predominantly spherical and relatively uniform particles.The average particle sizes of AgNPs and CRO-AgNPs were 8.91 and 11.81 nm, respectively.Li et al. (2020) and Hassanen et al. (2019) reported that nanoparticle size is an important determinant of toxicological properties 21, 22.AgNPs with diameters of approximately 10 nm have been reported to exert toxic effects in animal tissues, whereas particles measuring ≥ 40 nm generally show lower toxicity.Consistent with these observations, our findings suggest that administration of AgNPs at 80 µg/kg may induce lipid peroxidation in rat cardiac tissue. Lipid peroxidation is one of the main manifestations of oxidative damage and has been found to play an important role in the toxicity of many xenobiotics.The increase in cardiac MDA levels suggests that AgNP-induced oxidative injury occurred in a dose-dependent manner.The highest MDA levels were observed in animals receiving the highest dose of AgNPs.Similar findings have been reported in previous studies, which linked AgNP-induced cardiac toxicity to enhanced lipid peroxidation.For example, AgNPs (50 nm) were reported to induce cardiotoxicity in a dose-dependent manner by increasing lipid peroxidation and reducing SOD and CAT activities in the cardiac tissue of Catla catla fish [23].Another study showed that oral administration of AgNPs (8–20 nm) for two weeks increased MDA levels and SOD activity while reducing CAT activity in the cardiac tissue of Wistar rats [24].AgNPs exposure has also been reported to induce persistent vasoconstriction and increased cardiac contractility in spontaneously hypertensive rats through oxidative stress-related mechanisms 25, 26. In this study, elevated MDA levels with unchanged antioxidant content typically indicated a mild oxidative stress response that the antioxidant system could counteract, thereby preventing severe toxic effects [27]. These findings suggest that lipid peroxidation is a sensitive index, with changes occurring during the early stage of toxic damge. However, the stability of the antioxidant system indicated the possible role of limited oxidative stress in AgNP-induced toxicity at low doses without eextensive oxidative stress induction [27]. A previous study has indicated that AgNPs can induce an indirect oxidative stress response following activation of inflammation as evidenced by a slight increase in MDA levels [28].

AgNPs exposure appears to affect not only the oxidant–antioxidant balance in cardiac tissue but also cardiac histopathology [29].In our study, H&E staining revealed mild interstitial edema in the AgNPs 80 group, without obvious evidence of necrosis, hemorrhage, or inflammatory cell infiltration.Myocardial edema refers to the excessive accumulation of fluid within the myocardial interstitium or cardiac cells, usually resulting from increased capillary permeability.Myocardial edema may impair cardiomyocyte contractility, leading to systolic and diastolic dysfunction and potentially contributing to inflammation and fibrosis [30]. Previous studies reported greater cardiomyocyte damage, fibrosis, and inflammatory cell infiltration in AgNPs-treated hypertensive mice compared with saline-treated hypertensive controls 25, 31. Our histopathological findings support the presence of mild cardiac tissue injury that may be associated with lipid peroxidation and increased capillary permeability.Moreover, because serum LDH and CK-MB levels remained within the normal range, oral exposure to AgNPs at 80 µg/kg appears to induce only mild histopathological alterations, possibly through lipid peroxidation-related mechanisms.Previous studies have shown that the toxic effects of AgNPs in experimental animals are largely influenced by their size, shape, surface characteristics, dose, and route of administration [32].Therefore, characterization of synthesized nanoparticles, including their shape, size, surface properties, and stability, is important for biomedical applications.Biological synthesis of AgNPs using medicinal plants and their major flavonoids has been reported as a promising strategy for pharmaceutical applications.Green synthesis using plant extracts and flavonoids with strong reducing activity may help protect the nanoparticle surface against oxidation [33]. The broader development of nanotechnology-based biomedical approaches further supports the potential of engineered nanomaterials for diverse healthcare applications, although their safety and biological compatibility require careful evaluation [B].

In the present study, crocin was used as a coating agent for AgNPs.This approach is based on the highly polar nature of crocin, which may contribute to the reduction of metal ions during nanoparticle formation.Several parameters were adjusted to optimize the size of the crocin-coated AgNPs while maintaining a suitable concentration for experimental administration.These parameters included AgNO₃ concentration, pH, temperature, and the volume of the crocin solution.Different synthesis protocols were evaluated to optimize AgNO₃ concentration, temperature, pH, and crocin volume. The final synthesis conditions included an AgNO₃ concentration of 1 mM.These conditions were selected based on nanoparticle stability and particle size, as confirmed by TEM analysis [34].

Our toxicological assessment showed that crocin-coated AgNPs (80 µg/kg) prevented the mild histopathological changes observed in the cardiac tissue of animals treated with uncoated AgNPs at the same dose.The protective effect of crocin coating may be attributed to several mechanisms, including free-radical scavenging, reduction of Ag⁺ release, enhancement of enzymatic and non-enzymatic antioxidant defenses, and inhibition of nanoparticle aggregation 14, 35.The antioxidant properties of crocin may therefore contribute to reducing the adverse biological effects associated with AgNP exposure. Consistent with our findings, several studies have reported that green-synthesized AgNPs and flavonoid-coated AgNPs can reduce or prevent toxic effects associated with AgNPs of different sizes.Tareq and colleagues (2022) reported that AgNPs synthesized using Psidium guajava leaf extract produced less oxidative damage in the brains of rats than conventionally synthesized AgNPs, likely because of the antioxidant activity of plant-derived flavonoids [34].

Green-synthesized AgNPs prepared from combined extracts of P. crispum, Z. mays silk, and A. senegal were reported to reduce cadmium chloride-induced oxidative damage in hepatic, renal, and testicular tissues [36].The antioxidant and free-radical-scavenging activities of these plants have been suggested to contribute to their protective effects against cadmium-induced toxicity.

However, a crocin-only group was not included in this experimental design, and the the antioxidant activity of CR-AgNPs was not direcly evaluated, which limits our interpretation of the pharmacological activity of crocin in the nanoformulation. Although, crocin significantly reduced toxic damage in animals exposed to CR-AgNPs, the exact mechanism underlying this effect remains unclear. Future studies are needed to clarify the the contribution of crocin to diminishing the toxic effect of AgNPs.

5. Conclusion

The results demonstrated that crocin-coated AgNPs administered at doses of 20, 40, and 80 µg/kg did not produce detectable cardiotoxic effects. It was observed that AgNPs at a dose 80 µg/kg induced lipid peroxidation accompanied by edema in the cardiac tissue. However, crocin coated AgNPs at a dose 80 µg/kg inhibited the cardiotoxicity effects induced by AgNPs. These findings highlight the potential of crocin-coated AgNPs lower evidence of cardiotoxicity under the experimental conditions than conventionally synthesized AgNPs, supporting their potential for pharmaceutical applications. Biological synthesis of AgNPs based on the coating with flavonoids not only aligns with environmentally friendly principles but also appears to enhance the biocompatibility of the AgNPs. This successful synthesis approach compared with the chemical methods used most studies, emphasizes the necessity of identifying suitable methods to optimize nanoparticle synthesis while minimizing toxic effects. However, more investigations are needed to elucidate the long-term effects, biodistribution, mechanisms of action, and interactions of crocin-coated AgNPs in in vivo models. Such studies will not only validate the present findings but also support the broader application of crocin-coated AgNPs in medicine and pharmaceutical sciences.

Author’s contributions

Study concept: Tahereh Farkhondeh, Saeed Samargahndian. Performing experimental procedure and laboratory tests: Mohammad Amin Abdolahi Ziaaldini, Tahora Fakhrtaha. Drafting of the manuscript: Tahereh Farkhondeh, Mohammad Amin Abdolahi Ziaaldini, Tahora Fakhrtaha. Critical revision of the manuscript for important intellectual content: Saeed Samargahndian and Tahereh Farkhondeh, Supervision: Tahereh Farkhondeh, Saeed Samargahndian. Approved final version: all authors.

CRediT authorship contribution statement

Saeed Samarghandian: Writing – review & editing, Supervision, Conceptualization. Tahora Fakhrtaha: Writing – original draft, Investigation. Tahereh Farkhondeh: Writing – review & editing, Writing – original draft, Supervision, Conceptualization. Mohammad Amin Abdolahi Ziaaldini: Writing – original draft, Investigation.

Ethical approval

This study was approved by the ethics committee of Birjand University of Medical Sciences, (number: IR.BUMS.REC.1401.016).

Funding

The research presented in this publication received support from the Birjand University of Medical Sciences [Grant ID: 7038], in Birjand, Iran.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.