In Vitro Evaluation of DNA Damage Induction by Silver (Ag), Gold (Au), Silica (SiO₂), and Aluminum Oxide (Al₂O₃) Nanoparticles in Human Peripheral Blood Mononuclear Cells

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In the paper under revision, the Authors studied the DNA damaging properties of several nanoparticles (NPs) which are different in material and size. The NPs internalization and induction of reactive oxygen species were also evaluated. In general, this work is interesting and well written. However, the Authors should address these points before publication of the manuscript:

 - I suggest improving the setting of the introduction in order to make the reader better understand how the tested particles were chosen and thus the final purpose of the work. In particular, the authors should better describe the particles characteristics and their applications/limitations. I suggest to add in the manuscript these interesting articles about this topic:  Int. J. Mol. Sci. 2023, 24(6), 5133. DOI: 10.3390/ijms24065133; Nature Reviews Materials volume 5, pages886–909 (2020). DOI:1038/s41578-020-0230-0;  Micro & Nano Letters Volume13, Issue9 (2018). DOI:1049/mnl.2018.5070; Front. Chem. 2021. 9, 736519. DOI: 10.3389/fchem.2021.736519.

 - The size of the nanoparticles seems to be an important parameter related to their toxicity, as the Authors also noted. For this reason, it is important to add a paragraph explaining how the sizes of the different particles tested were chosen.

 - Since even at time zero the hydrodynamic diameter of the particles in the medium is very different to the reported diameter of the particles, it would be advisable to analyse the particles in water and compare their hydrodynamic diameter with that on the datasheet in order to better assess whether the agglomeration is due to the medium or incorrect storage. Could the fact that the particles are in an agglomerated form have given rise to the altered results? Please comment.

 

 

 

Author Response

1. I suggest improving the setting of the introduction in order to make the reader better understand how the tested particles were chosen and thus the final purpose of the work. In particular, the authors should better describe the particles characteristics and their applications/limitations. I suggest to add in the manuscript these interesting articles about this topic:  

Int. J. Mol. Sci. 2023, 24(6), 5133. DOI: 10.3390/ijms24065133; 

Nature Reviews Materials volume 5, pages886–909 (2020). DOI:1038/s41578-020-0230-0;  

Micro & Nano Letters Volume13, Issue9 (2018). DOI:1049/mnl.2018.5070; 

Front. Chem. 2021. 9, 736519. DOI: 10.3389/fchem.2021.736519.

 

Thank you for the reference suggestions. Introduction improved; new references added (underlined):

Silver nanoparticles are widely used in biomedicine due to their antibacterial, antiviral, and antifungal properties against Staphylococcus aureus [9], Candida albicans [10], herpes simplex virus (HSV), and human parainfluenza virus type 3 (HPIV-3) [11]. Currently, silver nanoparticles are included in many products, such as antibacterial dressings, home water treatment systems, cosmetics, and textiles [12]. In recent years, special attention has been paid to silica nanoparticles (SiO₂) because of their wide range of applications in drug delivery, environmental remediation, and advanced catalysis [13], making them one of the most abundant nanoparticles on Earth [14]. Similarly, Al₂O₃ nanoparticles have been successfully used in drug delivery [15] and in ceramics, to enhance the mechano-physical properties of ceramic tiles [16]. The unique chemical, physical, and photonic properties of gold nanoparticles have facilitated their use in medical biophysics [17], molecular imaging [18], and biosensors [19]. Gold nanoparticles tend to accumulate at tumor sites through a process known as the enhanced permeability and retention effect (EPR) [20]. When exposed to radiation, these NPs emit secondary electrons, which induce indirect DNA damage within cancer cells. Therefore, gold nanoparticles can be successfully used as radiosensitizers in cancer therapy [21]. Despite the numerous benefits of NPs in various industries, the exponential growth of nanotechnology has led to increased human exposure to nanomaterials, raising concerns about their safety.

 

2. The size of the nanoparticles seems to be an important parameter related to their toxicity, as the Authors also noted. For this reason, it is important to add a paragraph explaining how the sizes of the different particles tested were chosen.

 

Thank you for your insightful question. Additional information, regarding the question is added to the manuscript:

„The selection of nanoparticles for our study was, primarily based on their commercial availability and the established applications of different nanoparticle sizes. Gold nanoparticles are preferred to be as small as possible in biomedical applications such as imaging, therapy, and diagnostics due to their biocompatibility and unique optical properties [31,32]. Hence, we chose 5 nm nanoparticles for these reasons. To evaluate the impact of size on particle toxicity, we also included 40 nm gold nanoparticles. This larger size will help us understand any size-dependent differences in genotoxicity. A similar rationale was applied to the selection of Al₂O₃ NPs. Larger nanoparticles (50-150 nm) can be used in coatings, electronics, and ceramics [33,34], whereas smaller particles tend to be more reactive and are used in catalysis [35]. We chose both sizes, to encompass many applications in the field. The predominant use of SiO2 nanoparticles is in drug delivery, where smaller sizes are preferred, because of the easier loading and uptake of NPs [36,37]. Finally, for the silver nanoparticles their reactivity and antibacterial properties are most effective at sizes up to 50 nm [38], therefore 35 nm size particles were selected.  Overall, selected nanoparticles possess unique properties, that lead to widespread applications, therefore evaluation of their safety is important.“

 

3. Since even at time zero the hydrodynamic diameter of the particles in the medium is very different to the reported diameter of the particles, it would be advisable to analyse the particles in water and compare their hydrodynamic diameter with that on the datasheet in order to better assess whether the agglomeration is due to the medium or incorrect storage. Could the fact that the particles are in an agglomerated form have given rise to the altered results? Please comment.

 

Thank you for your important observation. First, after purchasing, particles were stored according to manufacturer’s recommendations. Secondly, it is quite common, for the hydrodynamic size of particles to change in cell culture media, due to presence of proteins. Additionally, nanoparticles tend to agglomerate because of their high surface area and strong attractive interactions, making them larger than their primary size. However, it is an interesting point to evaluate particle agglomeration in water, that we surely will explore in the future.

However, in this paper we added the following paragraph discussing possible relation between agglomeration of nanoparticles and their toxicity (lines 345-369):

“Nanoparticle tracking analysis (NTA) revealed that all tested nanoparticles agglomerated in cell culture media, in most cases, resulting in sizes larger than their primary size. Regarding the impact of nanoparticle agglomeration on their toxicity, there are no consensus. Murugadoss et al. [48] investigated the toxicity of small agglomerates (SA) and large agglomerates (LA) of TiO₂ nanoparticles. The study revealed, that in most in vitro analyses, there were no significant differences between SA and LA samples, leading to the conclusion that LA are not less active than SA. Interestingly, notable differences were observed in THP-1 cells, where LA induced more damage than SA. THP-1 cells, being phagocytic, may be more suitable for the uptake of submicron and micron-sized agglomerates, resulting in higher LA uptake and increased cellular damage, compared to SA. In our study, the peripheral blood mononuclear cell layer contains mainly lymphocytes with a small number of monocytes [49], which could explain slightly higher uptake levels of larger agglomerates (SiO₂, Al₂O₃ NPs, etc.), compared to smaller ones (Au NPs). However, it is important to note, that the DNA-damaging potential is influenced not only by the agglomeration or uptake of NPs, but also by the composition of particles and selected cell lines. Magdolenova et al. [50] proposed that larger agglomerates might be less stable, allowing individual NPs to be released from the agglomerate and subsequently taken up by the cells. They also suggested that larger agglomerates precipitate quickly, potentially leading to higher real exposure to NPs, compared to particles dispersed in the cell culture media, thus making them more toxic. Their study showed that large agglomerates induced more DNA damage in all tested cell cultures in vitro, whereas NP suspensions with agglomerates smaller than 200 nm had no genotoxic effects. Overall, there are mixed opinions on whether agglomeration increases the toxicity of nanoparticles. We believe, that while agglomeration can facilitate the uptake of NPs in some cases, toxicity is influenced by multiple factors beyond agglomeration alone.”

Reviewer 2 Report

Comments and Suggestions for Authors

Comments to Author:

Referee Report

Title: In vitro evaluation of DNA damage induction by silver (Ag), 2 gold (Au), silica (SiO2), and aluminum oxide (Al2O3) nanoparticles in human peripheral blood mononuclear cells

Submitted to Current Issues in Molecular Biology

Manuscript Number: cimb-3075755

This study evaluated the in vitro DNA damage induced by silver, gold, silica, and aluminum oxide nanoparticles (NPs) in human blood mononuclear cells. I have the following concerns regarding this work:

1. Introduction: For the application of NPs in medication, please use more updated references such as Siddique et al. (Nanomaterials 2022; 12:2826) and Moore et al. (Nano Ex 2021; 2:022001).
2. Introduction: There are many different types of NPs available on the market. The authors need to justify why they selected these four NPs for the study. Why are these particular NPs significant in causing DNA damage compared to others such as polymer and magnetic NPs?
3. Introduction: An important aspect of NPs application is their irradiation to generate secondary electrons. The authors should explain this process, using references like Jabeen et al. (Nanomaterials 2021; 11:1751) and Santiago et al. (App. Sci. 2023; 13:4916).
4. Section 3.1: Please provide more details about the NTS analysis. Including a reference for the analysis would be beneficial. Additionally, it would be helpful if the authors could provide TEM or SEM images of the NPs.
5. Figures 1 and 2: Ensure consistency in labeling the subfigures, whether using capital or small letters.
6. Section 3.3: The authors should carefully explain the relationship between DNA damage and cytotoxicity, as cell death may not solely result from DNA damage.
7. Discussion of Size Effect: The authors need to explain the size effect of the NPs, as particle size is a critical parameter alongside the material of the particles.

Comments on the Quality of English Language

No comment.

Author Response

1. Introduction: For the application of NPs in medication, please use more updated references such as Siddique et al. (Nanomaterials 2022; 12:2826) and Moore et al. (Nano Ex 2021; 2:022001).

 

Thank you for the reference suggestions. New references added.

 

2. Introduction: There are many different types of NPs available on the market. The authors need to justify why they selected these four NPs for the study. Why are these particular NPs significant in causing DNA damage compared to others such as polymer and magnetic NPs?

 

Thank you for your insightful question. We selected these nanoparticles for our study, because of their diverse applications which makes them highly relevant for assessing their safety.

SiO2 nanoparticles are the most abundant NP on Earth and are widely used in many industries, including food, electronics, and pharmaceuticals. Al₂O₃ NPs is frequently used in ceramics, coatings, and catalysis. Silver nanoparticles are among the most widely used nanoparticles due to their antimicrobial properties, making them prevalent in medical devices, textiles, and consumer products. Finally, gold nanoparticles are extensively used in biomedical applications, including drug delivery, diagnostics, and imaging. Therefore, the extensive presence and use of these nanoparticles make it crucial to understand their potential biological impacts, particularly DNA damage.

While polymer and magnetic nanoparticles also have significant applications, our focus on SiO2, Al2O3, Ag, and Au nanoparticles was driven by their extensive use across diverse fields and the existing literature suggesting their potential impact on cellular and genetic material. Future studies could certainly include polymer and magnetic nanoparticles to provide a more comprehensive understanding of nanoparticle-induced DNA damage across an even broader range of materials.

 

Additional information added to the manuscript:

The selection of nanoparticles for our study was, primarily based on their commercial availability and the established applications of different nanoparticle sizes. Gold nanoparticles are preferred to be as small as possible in biomedical applications such as imaging, therapy, and diagnostics due to their biocompatibility and unique optical properties [31,32]. Hence, we chose 5 nm nanoparticles for these reasons. To evaluate the impact of size on particle toxicity, we also included 40 nm gold nanoparticles. This larger size will help us understand any size-dependent differences in genotoxicity. A similar rationale was applied to the selection of Al₂O₃ NPs. Larger nanoparticles (50-150 nm) can be used in coatings, electronics, and ceramics [33,34], whereas smaller particles tend to be more reactive and are used in catalysis [35]. We chose both sizes, to encompass many applications in the field. The predominant use of SiO2 nanoparticles is in drug delivery, where smaller sizes are preferred, because of the easier loading and uptake of NPs [36,37]. Finally, for the silver nanoparticles their reactivity and antibacterial properties are most effective at sizes up to 50 nm [38], therefore 35 nm size particles were selected.  Overall, selected nanoparticles possess unique properties, that lead to widespread applications, therefore evaluation of their safety is important.

 

3. Introduction: An important aspect of NPs application is their irradiation to generate secondary electrons. The authors should explain this process, using references like Jabeen et al. (Nanomaterials 2021; 11:1751) and Santiago et al. (App. Sci. 2023; 13:4916).

 

Thank you for pointing that out. It is an important aspect, so we added it to our introduction (changes underlined):

The unique chemical, physical, and photonic properties of gold nanoparticles have facilitated their use in medical biophysics [17], molecular imaging [18], and biosensors [19]. Gold nanoparticles tend to accumulate at tumor sites through a process known as the enhanced permeability and retention effect (EPR) [20]. When exposed to radiation, these NPs emit secondary electrons, which induce indirect DNA damage within cancer cells. Therefore, gold nanoparticles can be successfully used as radiosensitizers in cancer therapy [21].

 

4. Section 3.1: Please provide more details about the NTA analysis. Including a reference for the analysis would be beneficial. Additionally, it would be helpful if the authors could provide TEM or SEM images of the NPs.

 

Additional information and additional reference inserted (underlined):

“Hydrodynamic particle size was evaluated by Nanoparticle Tracking Analysis (NTA) (Nanosight LM10, Malvern Panalytical Ltd, Malvern, UK) immediately (0 h), 1 or 3, and 24 hours after sonication. The samples were injected into the chamber with a sterile syringe until the liquid reached the tip of the nozzle. Each measurement was performed at 22 , with a camera level at 10. The Nanosight NTA 3.1 analytical software was employed. The highest peak size (size distribution peak with most particles) and mean particle size distribution was determined by tracking analysis of the particle Brownian motion in solution [31].“

 

SEM or TEM images of NPs were not made, so, we are unable to provide them.

 

5. Figures 1 and 2: Ensure consistency in labeling the subfigures, whether using capital or small letters.

 

Thank you for pointing that out. Labeling is corrected and consistent now.

 

6. Section 3.3: The authors should carefully explain the relationship between DNA damage and cytotoxicity, as cell death may not solely result from DNA damage.

 

In our study, we evaluated the potential of SiO₂, Al₂O₃, Ag, and Au nanoparticles to induce DNA damage and cytotoxicity. It is crucial to note that DNA damage can lead to cytotoxicity, as severe or unrepaired DNA lesions can trigger cell death pathways such as apoptosis or necrosis. However, cytotoxicity can occur independently of DNA damage, as nanoparticles may cause cell death through mechanisms that do not directly involve genetic material, such as disrupting cellular membranes or inducing oxidative stress.

Therefore, while there is a relationship between DNA damage and cytotoxicity, the presence of cytotoxic effects does not necessarily imply that DNA damage is the primary cause. Rather, we are highlighting that high levels of cytotoxicity and cell death can impact the results of DNA damage assessments, potentially leading to false positive results in the comet assay, therefore should be avoided.

 

Changed or edited parts in the manuscript underlined:

“According to Azqueta et al., [36] high levels of cytotoxicity can influence DNA migration in the comet assay. To minimize the risk of false positive results, it is recommended to maintain cell viability above 70-75 %.“

 

7. Discussion of Size Effect: The authors need to explain the size effect of the NPs, as particle size is a critical parameter alongside the material of the particles.

 

Thank you for pointing that out. Additional information added to the discussion part (underlined):

“Our study confirmed the intracellular uptake of Al₂O₃ nanoparticles, with 50 nm size particles being internalized more efficiently than particles with a primary size of 13 nm. It is known, that the NP uptake strongly depends on their size [52]. Several studies indicated, that the optimal size for efficient uptake is approximately 50nm [53–55], however, other properties, including shape, composition, and surface charge of NP has to be taken into consideration, as well [52].”

 

“Similarly, Xia et al. showed that 5 nm AuNPs induced a dose-dependent increase in DNA damage in the HepG2 cell line, while 20 and 50 nm size particles did not [59]. Generally, smaller nanoparticles have enhanced surface area, exposed surface atom ratio and elevated catalytic capabilities, resulting in higher toxicity [60]. It was suggested, that smaller gold nanoparticles release toxic ions and inhibit thioredoxin reductase, damaging mitochondria and inducing secondary DNA damage [61].”

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I am satisfied with the corrections and modifications from the authors as per my comments.

Comments on the Quality of English Language

No comment.