A Step Forward in the Suitability of Silver Nanoparticle Properties for Nanomedicine Applications

1. Introduction

The growing challenge of treating complex diseases, such as drug-resistant infections and cancer, has intensified the search for new, safer therapeutic strategies. Among the most promising nanomaterials, silver nanoparticles (AgNPs) have gained significant attention due to their broad-spectrum biological activities, including antimicrobial, antiviral, anti-fungal, anti-inflammatory, and anticancer effects [1,2,3,4,5]. As a result, AgNPs are currently among the most widely studied nanomaterials in the biomedical sciences.

However, ongoing concerns regarding their safety have limited the efficient clinical translation of AgNP application. The prevailing paradigm asserts that chemically or physically synthesized AgNPs are inherently toxic, while green-synthesized AgNP formulations, although considered less harmful, exhibit inconsistent chemical composition. Indeed, various studies have reported cytotoxic and genotoxic effects of AgNPs across multiple models, closely related to their physicochemical properties (size, shape, surface coating, dose, and exposure time) [6,7,8,9,10]. Surprisingly, despite extensive studies of AgNPs, only a few works have accurately determined the median lethal dose (LD₅₀) of AgNPs—an essential toxicological benchmark—and even fewer have included complete formulation data such as contents of metallic silver, stabilizer, and solvent. This gap hinders meaningful comparisons between AgNP formulations and weakens the toxicological foundation necessary for clinical development.

To address this issue, we evaluated five well-characterized formulations of Argovit™ AgNPs synthesized by a high-energy electron beam method and stabilized with polyvinylpyrrolidone (PVP) with different molecular masses. All AgNP formulations contain a specific Ag/PVP ratio, with well-characterized concentrations of metallic silver, the stabilizer, and the solvent. In earlier studies, these formulations exhibited high physicochemical stability and scalability and have demonstrated selective anticancer activity, genoprotective effects, and minimal environmental toxicity [11,12,13,14]. Despite all studied formulations exhibiting from good to remarkable results on each evaluated system, differences in PVP used for each formulation show a specific response, e.g., AgNP formulation synthesized with PVP K-30 exhibits the best anticancer effect in colon; AgNP formulation with K-17 results in the best antihyperglycemic effect [15] while using PVP 12.6 KDa provides the best abiotic elicitor to improve the yields of plant-derived bioactive compounds of pharmaceutical interest. Differences in the nature of coating agents play a key role in optimizing AgNP formulations for specific applications.

Therefore, the purpose of this article was to measure the LD₅₀ of these five AgNP formulations in mice using the standardized scientific test method, OECD Guideline 420 (Table 1), and to compare the obtained LD₅₀ values with those published for other AgNP formulations.

Table 1 presents GHS Hazard Categorization for Acute Toxicity by Oral Route according to OECD 420 [16]. The categorization includes the LD50 corresponding to five acute toxicity levels: 1) lethally toxic, 2) toxic, 3) harmful, 4) low hazard, 5) without significant danger, and a not classified level defined as harmless.

2. Materials and Methods

2.1. Silver Nanoparticles

The Scientific and Production Center Vector-Vita Ltd. (Novosibirsk, Russia) kindly donated five formulations of ArgovitTM AgNPs. Each of the five formulations contains approximately 1.2% wt. metallic silver, 18.8% wt. of stabilizing agent (polyvinylpyrrolidone (PVP)), and 80% wt. of distilled water. Each formulation has an overall concentration of 200 mg/mL of AgNPs (considering metallic Ag and PVP) suspended in water. All formulations have been characterized by DLS, UV-Vis, HR-TEM, TGA, and DSC. The summary of the physicochemical properties of each AgNPs formulation is shown in Table 2, adapted from [17].

Characteristics of the PVP stabilizer and electron adsorbed doses used in the preparation of every AgNP formulation are presented in Table 3.

Ag1 and Ag2 samples showed the influence of PVP MW variation at 15 kGy, while Ag3 and Ag4 at 45 kGy. Ag2 and Ag3 revised the absorbed dose variation, with 15 kGy for Ag2 and 45 kGy for Ag3 at constant MW. The influence of the PVP manufacturers (Boai NKY Pharmaceuticals Ltd. and LLC AK Sintvita) was revised on Ag2 and Ag5, respectively, maintaining the same MW and electron-absorbed dose.

2.2. Acute Toxicity Test (OECD Guideline 420)

The GHS approach (Table 1) uses the observation of clear signs of toxicity or death in at least three of the five mice as the test endpoint. The lethal dose for all AgNP formulations was studied on BALB/c mice following the instructions of the OECD Guideline 420 for Acute Oral Toxicity Assay-Fixed Dose Procedure [18]. The experimental protocol (No. 001/2018) was approved by the Ethical Committee of the Autonomous University of Baja California, México. First, an observational study was conducted with each formulation to select the appropriate starting dose. A female mouse was administered a single dose of 4,300 mg/kg of each AgNP formulation and observed for 14 days. Changes in clinical signs, including skin and fur appearance, eyes, membranes, nausea, vomiting, diarrhea, tremors, convulsions, salivation, lethargy, sleep, coma, general behavior, respiratory pattern, and death, were carefully recorded. Since no signs of toxicity were observed during the observational study, the limit test with five healthy nulliparous females was conducted at a starting dose of 4,300 mg/kg of AgNPs. Twenty-five female mice (body weight ~18.6 g) were randomly divided into five groups and kept in polycarbonate cages at 22±2 °C, 60% humidity, and a 12/12-h light-dark cycle. These groups were designated by the AgNP formulation administered: AgNP1, AgNP2, AgNP3, AgNP4, and AgNP5. Mice were given repeated doses of 4,300 mg/kg every two hours until three of the five mice in each group died. During the experiment, the changes in clinical signs were carefully registered. Animals with very low mobility and a decreased respiratory rate, without coma, for up to 30 minutes were classified as agonic. LD₅₀ doses were calculated in mg/kg for AgNPs (metallic silver with stabilizer) and the complete AgNPs formulation (metallic silver with stabilizer and water).

2.3. Histopathology

In addition to the main method used in this work (Acute toxicity test), histopathological studies were also conducted. Since histological studies are time-consuming, the number of animals per group was determined based on LD₅₀ results, survival rate, and clinical signs. The intestine, liver, kidney, lungs, and heart were collected and placed in 10% formaldehyde for histopathological evaluation as recommended by the OECD Guide 420 [18]. Tissues were stained with hematoxylin and eosin (H&E) and examined microscopically (ZEISS Primo Star 1, Jena, Germany) for evidence of cellular inflammation or damage.

2.4. Statistical Analysis

Kaplan-Meier survival curves were constructed to assess survival probability as a function of cumulative dose for each AgNP formulation, using dose (mg/kg of metallic silver) as a proxy for time in classical survival analysis. The Kaplan–Meier estimator is a nonparametric statistic that estimates the survival function S(t), which represents the probability that an individual survives beyond a given dose. It is calculated as:

$$S\left(t\right)=\coprod _{t_i\le }\left(1-{\displaystyle \frac{d_i}{n_i}}\right)$$

where dᵢ is the number of events (deaths) at dose level tᵢ, and nᵢ is the number of subjects at risk before dose tᵢ.

In this context, each increment in the administered dose was treated as a time-like event, and death was considered the event of interest. The survival function was updated at each dose level where at least one death occurred. Right censoring was applied to animals that survived the most significant cumulative dose without dying. The curves were generated using GraphPad Prism version 8.0, and the survival functions for each AgNP group were compared descriptively.

3. Results and Discussion

Table 4 presents the LD₅₀ calculated for two cases: for AgNP (metallic Ag and PVP) and the complete formulation (metallic Ag, PVP, and H₂O). The LD₅₀ (metallic Ag and PVP) range for the five AgNP formulations is 17,204-30,107 mg/kg (Table 4). The administration of AgNP1 exhibits the lowest LD₅₀ value of 17,204 mg/kg, whereas the AgNP4 formulation led to the highest dose (30,107 mg/kg) (Table 4). The LD₅₀ values follow the order: AgNP4 > AgNP5 > AgNP3 = AgNP2 > AgNP1.

3.1. The Kaplan–Meier Survival Curves

Figure 1 shows the Kaplan–Meier survival curves for mice exposed to five different Argovit™ AgNPs formulations. As the number of administered doses increases, distinct differences in survival appear among the groups. AgNP1 produced the fastest decline, reaching the death of three out of five mice at a cumulative dose of 17,204 mg/kg of AgNPs and total mortality by the fifth dose. AgNP2 gradually decreased, with complete mortality after the sixth dose. In contrast, AgNP3, AgNP4, and AgNP5 showed delayed lethality. AgNP4 maintained 80% survival through the seventh dose at 30,100 mg/kg, followed closely by AgNP5, which maintained 100% survival through the fifth dose and 80% through the sixth. These survival trends align with the calculated LD₅₀ values and confirm the lower acute oral toxicity of AgNP4 and AgNP5.

3.2. Frequency and Severity of Clinical Signs

Table 5 illustrates the frequency and severity of clinical signs presented by the AgNP formulations. The most frequently observed symptoms (observed in 15-25 mice across all groups) were: hypothermia (the most severe symptom), lethargy and closed or half-closed eyes (moderate symptoms), and hunched back/ruffled fur (mild symptoms). The least frequently observed (in only 1-2 mice across all groups) were the following moderate symptoms: mustard-colored urine, eye discharge, blepharitis, and hiccup. The remaining symptoms were observed at an average frequency (ranging from 3 to 10 mice across all groups).

The percentages of mild, moderate, and severe clinical signs for each formulation are presented in Table 6. AgNP3, AgNP4, and AgNP5 caused milder clinical signs. AgNP3 was mainly associated with general fatigue and vomiting reflexes, whereas AgNP4 and AgNP5 showed limited systemic symptoms, suggesting lower potential for toxicity. The clinical sign patterns reinforce the survival ranking and LD₅₀ data, positioning AgNP3, AgNP4, and AgNP5 as the least toxic formulations, and highlighting the utility of clinical signs as early indicators of nanoparticle toxicity. The samples can be arranged in the following order: AgNP3 < AgNP5 < AgNP4 < AgNP1 < AgNP2, with AgNP2 showing the highest percentage of severe signs and AgNP3 the lowest.

Figure 2 displays the percentage of mice with severe symptoms relative to the initial number of mice. Severe dehydration has been reported to cause cardiovascular effects that affect thermoregulation, which could explain the hypothermia. Another complication of dehydration is the neurological effects, such as confusion, delirium, and seizures [19], which could explain agony. Thus, high doses of AgNPs can lead to dehydration of the body, which in turn can trigger other severe symptoms, including death. On the other hand, frequent administration of AgNPs could lead to overhydration, resulting in water intoxication and most of the symptoms described in Table 4 and Table 5 [20]. In the case of dyspnoea, it could have been due to pulmonary aspiration, since these mice also exhibited a vomiting reflex. Figure SI-1 presents the same data as Figure 2 for all observed symptoms.

3.3. Histopathological Results

The histopathological changes observed in five organs after administration of silver nanoparticles (AgNPs) are presented in Table 7, which illustrates the percentage of mice with these changes. No specific changes were identified in the kidney after targeted searching. Fewer changes were observed in the heart and lungs. A greater number of changes were observed in the liver and spleen. The percentages of mice with significantly different changes are marked in blue in Table 6. Analysis of these values indicates that the highest percentage of histological change was observed in AgNP3, followed by AgNP4 and AgNP5, with the lowest changes in AgNP1 and AgNP2. Interestingly, two of five mice in the AgNP3 group developed pericardial calcification, and the AgNP3 formulation was the only one to show lymphoid infiltrate in the heart. Hence, the samples can be arranged in the following histological abnormalities order: AgNP1~AgNP2>AgNP5~AgNP4>AgNP3

The photographs in Figure 3 illustrate the representative changes observed in the five organs. In the liver, megahepatocytes and vacuolization were observed (Figure 3a and b, respectively). The pigmented areas in the red pulp, apoptosis in the white pulp, congestion, and extramedullary hematopoiesis (Figure 3c and d) were observed in the spleen. Congestion and hemorrhage were registered in the kidney (Figure 3e and f). Figure 3g and h demonstrate hemorrhage and alveolar macrophages with pigment, respectively, in the lung. In the heart, pigment deposits on the valvular surface and pigment residues in the lumen of the cardiac chambers and pericardial calcification (Figure 3i and j) were registered.

3.4. Performance Order of AgNP Formulations Regarding LD₅₀, Survival Rate, Clinical Signs, and Histopathological Changes

Our results showed that each formulation exhibits distinct performance across LD₅₀, survival rate, clinical signs, and histopathological changes. Table 8 summarizes the identified patterns in AgNP formulations with respect to LD₅₀, survival rate, clinical signs, and histopathological changes.

Data from Table 8 on LD₅₀, survival rate, and clinical signs indicate that AgNP4 and AgNP5 are the least toxic and most promising for completing their preclinical portfolio first. On the other hand, although AgNP1 and AgNP2 formulations exhibited the highest toxic effects among the five formulations assessed in this work, their LD₅₀ values are sufficiently high to warrant consideration for future biomedical applications.

It is interesting to examine the cause of the observation described above. Regarding LD₅₀, survival rate, and clinical signs, the AgNP4 and AgNP5 groups exhibit lower toxicity, while the AgNP1 and AgNP2 groups show more serious damage in the experimental group (Table 8). This pattern is consistent with the recorded histopathological damage. The histological changes in the AgNP1 and AgNP2 groups are minimal compared with those in the other groups because they died more rapidly at a lower dose than those in the AgNP4 and AgNP5 groups (Figure 1). Considering the similarity in physicochemical properties among the five AgNP formulations assessed in this work (Table 3), the main difference that can explain the patterns described in Table 8 is the size of the coating polymer. The longer the polymer, the higher the stability of the AgNP formulation and the lower the toxicity in whole animals.

3.5. Comparison with LD₅₀’s of Other AgNP Formulations

It should be emphasized that toxicologists use the LD₅₀ value as the first step in assessing a substance’s toxicity [21]. LD₅₀ is the basis for planning further assessments, including chronic and sub-chronic toxicity, pharmacodynamic and pharmacokinetic assays, and other studies. The OECD guidelines help classify new substances in the Global Harmonized System (GHS). This system includes five LD₅₀ categories, with substances in Category 1 considered the most toxic and those in Category 5 considered harmless [22] (Table 1). The values of LD₅₀ of the five AgNPs formulations studied (metallic Ag with PVP) are in the range of 17,204-30,107 mg/kg (Table 4). According to the Hazard Characterization presented in Table 1, the values for the AgNPs studied here exceed 3.4-6.0 times the LD₅₀ category 5 (>5,000 mg/kg) (Table 4). In other words, the LD₅₀ of the studied AgNP formulations exceeds the LD₅₀ of the “Harmless” category by 340-600%. What category do the complete AgNP formulations correspond to? The LD₅₀ values for the five AgNP formulations studied, calculated for the complete formulations, range from 86,020 to 150,535 mg/kg (Table 4). These values exceed the LD₅₀ for the “Harmless” category by 17.2-30.1 times, or 1,720-3,010%. It was of great interest to compare the LD₅₀ values of the AgNP formulations studied here with those of other formulations of silver nanoparticles.

Considering that the determination of the LD₅₀ is a key factor in assessing the toxicity of a substance [21], it was surprising to find that we could identify only twelve articles reporting this parameter for AgNP formulations. Table 9 summarizes the experimental details and results obtained from these publications. The concentration of AgNPs was mentioned only in two of these works [17,23], while the other 10 did not specify the concentration of the AgNPs or the content of metallic silver within the AgNP formulation [24,25,26,27,28,29,30,31,32,33]. The AgNP size and stabilizer type were also unspecified in several studies. In 9 previous publications, precise LD₅₀ values were not provided; LD₅₀ was reported as >2,000 or >5,000 mg/kg. This is further confirmation that the LD₅₀ of AgNP formulations has been poorly studied. The estimated Human equivalent dose for the complete formulation studied in this work and in previously published works was also included in Table 9.

So, we could not find any other LD₅₀ results for AgNP formulations beyond those presented in Table 9. Comparing research results under identical conditions is ideal, but when unavailable, data should be compared with the closest available studies by highlighting similarities and differences. We could not compare our results with other AgNP formulations stabilized with PVP, as LD50 values for such formulations have not been reported in the literature. Table 9 shows that we could compare our LD₅₀ results only with those obtained for AgNPs synthesized by green synthesis, as they are the only ones reported in the literature.

Thus, despite the current paradigm that AgNPs are toxic and dangerous for medical use, we found that the most basic toxicological study (LD₅₀) was conducted on only a small number of AgNP formulations. The comparison of the LD₅₀ values for the five Argovit™ AgNP formulations studied in this work, and those reported for other AgNP formulations, is presented in Figure 4. When the concentrations of metallic silver and stabilizing agents of the formulations are indicated in the works, it is possible to calculate the LD₅₀ for comparative purposes: 1) active component (metallic Ag), 2) complete AgNPs (metallic Ag with stabilizer), and 3) complete formulation (metallic Ag + stabilizer + water). Regrettably, the previous studies presented in Figure 4 do not specify which of the three methods was used to report the LD₅₀. Figure 4a shows the comparison for AgNPs, considering the complete formulation (metallic Ag + stabilizer + water).

For five of the twelve AgNP formulations, it was found that the LD₅₀ was more than 2,000 [27,29,31,32,33] and five works found that were greater than 5,000 [23,25,26,28,30] (Figure 4a). In any case, the difference between the LD₅₀ of Argovit™ AgNPs tested in this research and those previously published is notable (Figure 4a). As confirmed via email, some researchers calculate the LD₅₀ using the complete AgNP formulation [23,33]. In this case, the LD₅₀ difference between AgNP4 and the values reported in those works ranged from 30.11-188.17 times (3,011-18,817%) higher. Two other surprising points are presented below. Firstly, four of our five AgNPs formulations have similar LD₅₀ as drinking water (LD₅₀ >90,000 mg/kg) [34] (Figure 4a). Secondly, the LD₅₀ of our five AgNPs formulations is 1.64-3.63 times higher than that of sucrose (LD₅₀ of 32,500 mg/kg) [34] (Figure 4a). Hence, Argovit™ AgNP formulations exhibit lower acute oral toxicity than sucrose, a naturally occurring disaccharide composed of glucose and fructose, the main constituent of white sugar, and one of the least toxic compounds. The LD₅₀ of AgNP4 (calculated for Ag and stabilizer) is 6.02–37.6 times (602-3,760%) higher than those published in previous works (Figure 4b). Figure 4b also shows that the LD₅₀ values for some pharmacological drugs (horizontal-colored lines) are close to those reported for previously published AgNP formulations.

LD₅₀ values for Ag2-Ag5 formulations range from 107,525 to 150,535 mg/kg. As mentioned above, the LD50 of tap water is >90,000 mg/kg [34]. This indicates that the LD50 values reported for Ag2-Ag5 are the same as those for tap water, which is among the least toxic substances [31]. It suggests that, in the case of Ag2-Ag5, it is impossible to distinguish which compound exactly (AgNPs or water) killed the mice. Is it possible that water killed the mice? Is this a reasonable suggestion? Due to the characteristics of AgNPs assessed in this work, it is possible that water intoxication by overhydration could produce death in mice. Also, overhydration may be responsible for the symptoms observed in mice described in Table 4 and Table 5.

Analysis of LD₅₀ and physicochemical properties of five AgNP formulations showed that LD₅₀ rises (Table 3) with an increase in PVP molecular mass (Table 3). The high polymer content stabilizes the formulations, forming a nanogel that prolongs AgNPs’ lifespan and appears to act as an extended-release system [35,36]. However, further work will be necessary to examine more deeply the relationship between physicochemical parameters and LD₅₀. The most important revelation of the present work is that all five Argovit™ AgNP formulations showed significantly higher LD₅₀ values (up to 37.6-fold, or 3,760% higher) than the value corresponding to a “Harmless” level.

Although calculating human LD₅₀ values from mouse LD₅₀ data is not always accurate, this approach remains one of the most effective methods to date for a wide range of chemical compounds [37]. The scaling factor to transform LD₅₀ from a mouse to a human is 0.081 [38]. The LD₅₀ for AgNP4 in humans corresponds to 12,193 mg/Kg or 853.88 g of concentrated AgNP/day/person (average weight of 70 Kg). It is difficult to imagine that a human could withstand such a dose, because concentrated AgNP formulations have a very bitter taste with a strong metallic aftertaste. Figure 5 and Figure 6 illustrate the conversion of the administered volume of the AgNP formulation to a human equivalent.

From the LD₅₀ values reported for rodent species (Table 9), we calculated the corresponding human dose using the previously published scaling factors by Nair and colleagues (2008) [38]. The result of this exercise is shown in Figure 6. LD₅₀ values calculated for humans for five Argovit AgNP formulations range from 6,967.62 to 12,193.34 mg/kg, while the human LD₅₀ values reported in twelve earlier publications do not exceed 405 mg/kg. Hence, based on data from literature and present research, we conclude that the human LD₅₀ of Argovit AgNP formulations is 17-30 times higher than that of other AgNP formulations.

Notably, nine of the twelve AgNP formulations reported by other scientific groups were obtained via green synthesis. Of the remaining two, one was obtained using mild green conditions, and the other does not indicate the method (Table 9). Many studies report that AgNPs synthesized by green synthesis (using microbiological or plant derivatives) are less toxic than AgNPs synthesized by chemical methods [39,40]. Recent advancements in nanobiotechnology have demonstrated the potential of biosynthesized AgNPs as eco-friendly, cost-effective, and biocompatible agents, particularly for cancer treatment [41]. On the other hand, green synthesis has some disadvantages, such as variability in the chemical composition of synthesized AgNPs, primarily due to environmental factors like weather conditions, time constraints, and overall plant production. Additionally, the availability of raw materials in some regions, the need for extensive extraction techniques [42], and the difficulty in monitoring and controlling the biomolecules involved in the synthesis [41].

Five Argovit™ AgNP formulations studied in this work were synthesized by the physicochemical method, which included 1) the reduction of silver ions with high-energy electrons (physical stage) and 2) the stabilization of the formed metallic silver nuclei with organic molecules of polyvinylpyrrolidone (chemical stage). Hence, the results obtained here for Argovit™ AgNPs challenge the paradigm that AgNPs produced via green synthesis are less toxic than those synthesized by chemical or physical methods. Argovit™ AgNP formulations synthesized via physicochemical methods exhibit extremely low toxicity, with in vivo acute oral toxicity at least 6.02–37.6 times lower than that of biogenically synthesized AgNPs.

As shown in Table 6 and Table 7, the histopathological analysis reveals no severe changes. The liver and spleen present the most significant changes (Table 7). We found several publications reporting the acute oral toxicity of AgNPs in Albino rats with total doses of 4,000 and 4,200 mg/kg [43,44], and in Wistar rats with doses of 3,000 and 3,950 mg/kg [45,46]. The AgNP LD₅₀ in our study ranged from 17,204 to 30,107 mg/kg. Hence, applying 6-10 times higher AgNPs dose we did not observe kidney tissue damage (Table 7), while other AgNP formulations (with 6-10 times lower doses) showed swollen epithelium with cytoplasmic vacuolization in renal tubules, thickening of the basement membrane, and destruction of some mitochondrial cristae, podocyte elongation and thinning of their primary and secondary processes, and thickening of the basement membrane of the capillary tufts [44].

Using very high doses in the present work (17,204–30,107 mg/kg of AgNPs), pigmented deposits are observed on valves, pericardial calcification, and scant lymphoid infiltrate, while other AgNP formulations at much lower doses—4,000 mg/kg in albino rats [43] and 3,950 mg/kg in Wistar rats [46] caused the appearance of extravasated red blood cells and congested blood vessels, inflammatory cellular infiltration and vacuolation of cardiac muscle fibers, interruption with wide spaces, deposition of collagen fibers between cardiac muscle fibers [43], as well as fragmentation of sarcoplasm and degenerative changes in myocardial fibers [46]. We used very high doses (17,204–30,107 mg/kg) to observe only hemorrhage and pigment in alveolar macrophages in the lung (Table 7). In contrast, with other AgNP formulations administered at 4,000 mg/kg [43] and 3,950 mg/kg [46], additional findings were reported, including collapse of some alveoli, emphysematous dilatation, thickening of interalveolar septa, deposition of collagen fibers, congested blood vessels, and inflammatory infiltrates [43]. An influx of polymorphonuclear cells, lymphocytes, and macrophages into alveoli and perivascular areas was also described [46].

The liver is the largest solid organ in the body. It removes toxins from the blood and maintains blood sugar levels, among other fundamental physiological functions. Obviously, at such high doses (17,204–30,107 mg/kg), it is hard to expect AgNPs not to affect hepatic tissue. We observed congestion, vacuolization, megahepatocytes, Kupffer cell pigmentation, and, to a lesser extent, cytoplasmic inclusions (Table 5). Notably, Kupffer cell pigmentation may reflect the internalization of AgNPs. On the other hand, AgNPs formulations reported in other publications, administered at 4,200 mg/kg in albino rats [44], more pronounced liver damage was observed, including sinusoidal narrowing, exaggerated hepatocytes with hypertrophic nucleoli, and accumulation of lipid globules in both the nucleoplasm and cytoplasm. Furthermore, electron microscopy revealed that Kupffer cells exhibited a hypertrophied morphology with altered mitochondrial cristae, large lysosomes, and endosomes filled with silver nanoparticles [44].

3.6. The Reasons for the Low Toxicity of Argovit^TM AgNP Formulations

It is worth mentioning that the Argovit™ AgNP formulation (AgNP2) has previously demonstrated low cyto- and genotoxicity in mouse erythrocytes and human lymphocytes, and low hemolysis in human erythrocytes [11,47,48]. Why do ArgovitTM AgNPs studied in the present work exhibit lower toxicity than AgNP formulations studied by other authors? The extremely low toxicity could be related to the high stability conferred by the Ag/PVP [17] which also promotes the formation of nanogels [50,51]. The results of recent studies using HRTEM, DLS, UV-visible spectroscopy, XPS, luminescence, FTIR, viscosity, and electrophoresis indicate that the structure of AgNPs in the Argovit™ formulation family is a novel model comprising PVP nanohydrogels incorporating Ag cores and/or PVP-capped Ag cores [49,50]. It is known that nanogels, through their unique physicochemical properties, such as small size, high surface area, and tunable surface charge, can effectively encapsulate and deliver therapeutic agents to target sites while minimizing off-target effects and toxicity [35,36,51]. Further research is needed to better understand how the unique Argovit™ AgNP structure contributes to their low toxicity.

The evidence for the AgNPs-nanogel structure is provided in [50,51], with increased viscosity and FTIR data (the appearance of new peaks attributed to vibrations of C-N and C-O bands) being the most direct indicators of nanogel formation [50]. We were unable to find any studies in the literature on the LD₅₀ of AgNPs-nanogel formulations. To our knowledge, the present work is the first to study the LD₅₀ of AgNPs-nanogel systems. Currently, the development of AgNPs-nanogels is an emerging field of nanoscience. Only a few studies have focused on the synthesis and characterization of these systems. In [50], data on synthesis, characterization methods, toxicity, biological activity results, certification, and commercialization of AgNPs-nanogels reported in the literature are compared with those of Argovit^TM AgNPs. In terms of these parameters, Argovit AgNPs-nanogels have been studied incomparably more extensively than other AgNPs-nanogel formulations [49]. The extraordinary properties of Argovit^TM AgNPs attract the attention of researchers. In earlier studies, AgNP1-AgNP5 formulations studied here exhibited high physicochemical stability and scalability and have demonstrated selective anticancer activity, genoprotective effects, and minimal environmental toxicity [11,12,13,14]. Despite all studied formulations exhibiting from good to remarkable results on each evaluated system, differences in PVP used for each formulation show a specific response, e.g., AgNP formulation synthesized with PVP K-30 exhibits the best anticancer effect in colon; AgNP formulation with K-17 results in the best antihyperglycemic effect [15], while using PVP 12.6 KDa provides the best abiotic elicitor to improve the yields of plant-derived bioactive compounds of pharmaceutical interest. Differences in coating agents play a key role in optimizing AgNP formulations for specific applications.

Furthermore, the remarkable stability of Argovit™ AgNP formulations is also reflected in their clearance efficiency. Biodistribution and bioaccumulation of AgNP5 have been measured by the radioactive tracer method (RTM) after the intragastric administration of a single 1 mg dose of AgNPs, following the activity of the radioactive isotope ^110mAg in whole blood, liver, spleen, pancreas, gonads, kidneys, lungs, heart, brain, gastrointestinal organs, musculoskeletal frame with hair and skin, and feces. The results shown that more than 98% of the administered silver was eliminated in feces after 24h with minimal percentages in target organs [52]. Similar results were obtained after subchronic administration of this AgNP formulation [53,54].

In addition to very low toxicity, Argovit™ used in this research has demonstrated high potential for several medical uses, e.g., treating foot injuries in diabetic patients [55,56], returning the bacteria’s susceptibility to antibiotics [47], antiparasitic activity [57], as a preventive agent for SARS-CoV-2 infection [58], antiviral activity [59], as an antifungal agent [60]. Regarding cancer treatments, Argovit™ AgNPs have demonstrated that they can induce non-immunogenic tumour cell death [61] decrease the tumor growth rate as well as increase the life expectancy in mice with melanoma [62]. Moreover, [13] found that healthy cells were 16 times more resistant than cancer cells, indicating the selective cytotoxic response of AgNPs.

One of the most significant challenges for medicine is to achieve an effective treatment against cancer without adverse effects, considering that cancer treatments are among the most toxic drugs. Argovit™ formulations are between 107 and 940 times less harmful than the antitumor drug cyclophosphamide, which has an oral LD₅₀ of 160 mg/kg [63] (Figure 4b). A similar pattern is observed when comparing the LD₅₀ of Argovit™ with those of other drugs shown in Figure 4b. For example, Argovit™ AgNP formulations have LD₅₀ values 5.23-9.91 times higher than tetracycline, a widely used broad-spectrum antibiotic; 10.46-19.07 times higher than albendazole, an antiparasitic drug; and 60.44-106.52 times higher than amphotericin B, an antifungal drug.

The low toxicity and broad biological activity of Argovit™ AgNP formulations set them apart from many other AgNP formulations, making them viable candidates for further development across diverse fields, including nanomedicine, environmental science, and food safety. Further in vivo studies and clinical trials will be required to investigate their potential and examine new areas where these nanoparticles could affect public health and industrial applications.

5. Conclusions

This study assesses the acute oral toxicity associated with five well-characterized formulations of Argovit™ AgNPs. The results reveal LD₅₀ values ranging from 86,020 to 150,535 mg/kg for the complete AgNP formulations, placing them above the “harmless” threshold under the OECD classification. The LD₅₀ of Argovit AgNP formulations was found to be 17-30 times higher than the human LD₅₀ reported for other AgNP formulations in the literature.

Survival analysis, clinical observations, and histopathological evaluations collectively confirm the low acute toxicity of these formulations, even at doses substantially higher than those used in previous studies. Notably, no significant tissue damage was observed in critical organs, such as the kidneys and heart, despite high exposure levels. The previous results revealed that more than 98% of the administered silver was eliminated in feces after 24h, with minimal percentages in target organs, which also contributes to the low LD₅₀ of these AgNPs formulations. The study suggests that the low LD₅₀ may be due to the higher stability and the peculiar structure of Argovit AgNP formulations, which, according to recent data, consists of AgNPs incorporated into PVP nano- and microgels. To our knowledge, the present work is the first to study the LD₅₀ of AgNPs-nanogel systems.

These findings challenge the prevailing notion that AgNPs synthesized by physicochemical methods are inherently more toxic than those synthesized by green methods. Argovit™ AgNP formulations are promising candidates characterized by their low toxicity, high physicochemical stability, and robust potential for scalable biomedical applications.