Antibacterial Properties of PMMA Functionalized with CuFe₂O₄/Cu₂O/CuO Nanoparticles

Round 1

Reviewer 1 Report

1. Figure 3A, there seems to be a certain aggregation of nanometers. Does it have an effect on antimicrobial efficiency?

 

2. Figure 7, the bacterial status of the painted plate seems to be different. The bacteria of the mixture is much larger.

 

3. What is the mechanism of antibacterial? Whether Cu ion release will be fast. This point needs to be described by the authors.

 

4. Antibacterial mechanism can be strengthened by citing 10.1016/j.mtbio.2022.100264; 10.1016/j.cej.2022.135691; 10.1002/advs.202106015.

 

5. TGA  results, why is the final remaining temperature of the nano-doped system more than that of the PMMA group and the 0.5% nano group?

 

6. What are the advantages of the designed CuFe2O4/Cu2O/CuO/PMMA in this paper compared to other systems? The comparison and the advantages can be added to the manuscript.

 

7. There are some formatting errors in the article. For example, spelling of references must be checked to meet the journal style. The case of the magazine name should be consistent. Please check carefully and use it properly.

Author Response

1. Figure 3A, there seems to be a certain aggregation of nanometers. Does it have an effect on antimicrobial efficiency?

We added in Result and discussion (Page 5, Line 2, marked yellow):

«One of the main problems in the introduction of nanoparticles into polymeric matrices is their agglomeration [38]. EETW nanoparticles  are capable of forming aggregates and agglomerates. Nanoparticle aggregates are easily destroyed by ultrasonic treatment. To break down agglomerates of assemblied nanoparticles is significantly more difficult. The ability to form agglomerates is determined by the surface energy of the nanoparticles (size factor) and the melting temperature of the metal. Silver nanoparticles form solid agglomerates which is confirmed by the sedimentation analysis data».

(Page 5, Line 3, marked yellow):

«The agglomeration of nanoparticles leads to a decrease in antibacterial activity, as shown in work [39]. However, it was not possible to break down the nanoparticle agglomerates by ultrasonic treatment. A further increase in the dispersion time leads to local heating and sintering of nanoparticle».

2. Figure 7, the bacterial status of the painted plate seems to be different. The bacteria of the mixture is much larger.

The size of bacterial colonies is determined by their number, the fewer bacterial colonies, the larger their size.

3. What is the mechanism of antibacterial? Whether Cu ion release will be fast. This point needs to be described by the authors.

Antibacterial mechanism can be strengthened by citing 10.1016/j.mtbio.2022.100264; 10.1016/j.cej.2022.135691; 10.1002/advs.202106015.

We added in Result and discussion part (Page 9, Line 4, marked yellow):

«As shown authors [49] the modification of ationic polyeth-yleneimine/anionic pectin hydrogel with polydopamine@Cu nanoparticles reduced survival rates for S. aureus from 80.2% to 34.6%, for E.coli - from 71.1% to 22.4%. Antibacterial activity of CuFe2O4/Cu2O/CuO/PMMA composites against E. coli is higher than S. aureus (Fig. 7b). It can be ascribed to the diferences in the membrane structure and composition of these bacteria. E. coli has a complicated cell wall with internal and thin external membranes which composed of peptidoglycan, lipopolysaccharides, lipoproteins, and phospholipids molecules. The S.aureus cell wall has a single thick membrane of peptidoglycan. Terefore, E.coli are more impressible for penetrating antibacterial agents [50]. The antibacterial ef-fect of CuFe2O4/Cu2O/CuO/PMMA composite may be described using three mechanisms: (1) its efective interaction nanoparticle with the bacterial cell wall due to its high sur-face-to-volume ratio, (2) copper ions releasing, which had a destructive effect on the structures of bacteria cells membrane, and (3) their capability to produce reactive oxygen species (ROS). According to [51] the main mechanism of the ferrite nanoparticle action is based on ROS generation. ROS generation causes lipid peroxidation, destruction or modification of proteins, violation of the integrity of the bacterial membrane, destruction of enzymes and RNA damage, leading to cell death.»

5. TGA  results, why is the final remaining temperature of the nano-doped system more than that of the PMMA group and the 0.5% nano group?

We added in Result and discussion (Page 8, Line 1, marked yellow):

TG curves can be represented as a sequence of logistic curves, each describing a different stage of the chemical process. To evaluate TG thermal parameters, fitting model of mass gain as function of temperature can be represented in the form [45]

where M(t) is current value of the sample mass, N is the number of logistic components, i.e. the number of process stages, t is the temperature, M_i – mass change of the sample, b_i –rate parameter of mass change, θ_i - the temperature of the maximum rate of mass change in each stage, and τ_i - the asymmetry parameter of each stage.

Fitted thermal parameters are listed in Table 1.

 

Table 1. Fitted TG thermal parameters

		PMMA	0.5-CuFe2O4/PMMA	2-CuFe2O4/PMMA	5-CuFe2O4/PMMA
I stage	M	5.7	4.96	5.33	10.71
	θ	156	150	154	173
	b	18.2	9.65	16.7	16.2
	τ	1.72	3.58	4.41	5.12
II stage	M	94.10	94.90	88.30	82.07
	θ	377	378	378	378
	b	15.1	17.89	16.48	13.60
	τ	0.11	0.10	0.27	0.49
	R2	0.9999	0.9999	0.9999	0.9999

 

As can be concluded from the data presented, the destruction process of PMMA is two stage process. The effect of the introduced ferrite nanoparticles on the process of PMMA destruction at the first stage is evident only at a ferrite nanoparticles content of more than 2 %, which results from the increase in the temperature of the maximum destruction rate and the increase in the mass fraction of the polymer subjected to destruction. Also, with increasing ferrite nanoparticles content in the composite, the asymmetry parameter slightly increases, which may be related to the deceleration of PMMA nanoparticles at a higher temperature with the formation of volatile products. In the second stage of the PMMA destruction process, the effect of injected ferrite nanoparticles is less noticeable. The temperature of the maximum destruction rate is almost constant, but the mass fraction of the polymer subjected to destruction is lower. The overall rate of the degradation process decreases with increasing ferrite nanoparticles content in the composite, and from the increase in the asymmetry parameter it can be concluded that the process of PMMA destruction with the formation of nonvolatile products (carbonization) dominates over the process of PMMA destruction with the formation of volatile products. Thus, it can be concluded that the first stage of composite destruction is dominated by PMMA destruction with the formation of volatile products, while the second stage of the destruction is dominated by the process with the formation of non-volatile products».

6. What are the advantages of the designed CuFe₂O₄/Cu₂O/CuO/PMMAin this paper compared to other systems? The comparison and the advantages can be added to the manuscript.

 Каковы преимущества разработанных CuFe2O4/Cu2O/CuO/ПММА в этой статье по сравнению с другими системами? Сравнение и преимущества могут быть добавлены в рукопись.

We added in Introduction (Page 2, Line 4, marked yellow):

«Moreover, the complex microstructure of CuFe₂O₄/Cu₂O/CuO nanoparticles has several advantages, such as (1) CuFe₂O₄ electrostatic interaction and complexing with phosphate in the cell wall [34]; (2) copper ions Cu²⁺ released from the samples would change the permeability of bacterial cell wall, prevent the cell from absorbing nutrients, and ultimately affect the cell growth and vitality [35]; (3) iron (III) and copper ions can play a synergistic role in antibacterial activity of nanoparticles [36]».

7. There are some formatting errors in the article. For example, spelling of references must be checked to meet the journal style.The case of the magazine name should be consistent.Please check carefully and use it properly.

We checked article and corrected errors.

.

Reviewer 2 Report

review attached

Comments for author File: Comments.pdf

Author Response

The study of Glazkova et al. aims to develop polymethylmethacrylate (PMMA)-based coatings doped vith oxide nanoparticles for antibacterial protection of surfaces. Nanoparticles of complex phase composition (CuFe2O4/Cu2O/CuO) and semi-conducing interfaces suitable for photoexcitation were obtained by detonation – quite an unusual synthesis technique. They were later incorporated in the PMMA precursors, and the antibacterial activity of the coating assessed using standard procedures. The materials seem to display impressive (if not somewhat overstaded) performance in terms of surface protection. Overall, I found the study to be quite interesting in terms of methodology and materials performance. The rationale for this study is well explained, the protocols correctly described. Materials characterization adequately, although slightly limited. The assessment of the antibacterial efficiency seems correct, but the quantitative figure seems exaggerated, and the way this efficiency has been calculated should be explained. Most important, however, the manuscript is of unequal redacting and editing quality, which makes it hardly fit for publication in its present state. I therefore recommend major corrections in order to improve the overall technical quality, and, perhaps, improve the data analysis Although data seem to have been collected under correct conditions, their interpretation has been limited by lack of quantitative analysis.

1. For example, Rietveld modelling of DRX patterns could have been performed to obtain some indication on the relative proportions of CuFe₂O₄, Cu₂O and CuO phases. This modelling would also have provided insight into the size of diffraction domains, and this information used to discuss the size of particles as observed by TEM and SEM. The SEM-EDX data showing homogeneous composition in terms of Cu and Fe is in apparent contradiction with the DRX observations of distinct phases, some of them containing only Cu. The authors can clarify this apparent contradiction, based on data derived from DRX quantitative analysis, or from the knowledge of the interaction volume of backscattered electrons (the excitation source for the EDX signal).

 

We added table on Figure 3 and proportions of CuFe₂O₄, Cu₂O and CuO phases on Figure 2. We added in Result and discussion part (Page 4, Line 3, marked yellow):

«The Reference Intensity Ratio (RIR) method was used for quantitative analysis by powder diffraction. The mass ratio of copper ferrite phase was 72.4 % wt., copper oxide (I) and (II) – 20,5 % wt. and 7.1 % wt. respectively».

 

2. The antibacterial activity was assessed by the so-called drop contamination method, and the observation of only one to three CFU (Figure 8) lead to the conclusion that the growth of MRSA was inhibited by 99.999 %. I do not deny that impressive antibacterial properties were demonstrated by the doped PMMA, but I question this figure. First, 99.999% relative to what: an ideal culture media for which each bacteria would result in a colony; or PMMA, for which the number of formed colonies is already far lower than the number of CFU in the drop? Second, because a single test per condition has been performed, the stastical uncertzinty on the efficiency cannot be estimated. This uncertainty could be assessed for example by running several distinct Petri dished and performing adequate statistical analysis of the results.

We agree with this comment. We have recalculated the bacterial reduction relative to PMMA. Figure 7 shows only typical images of Petri dishes. For each sample, antibacterial assay was performed with three independent replicates (n = 3). The results of determining the number of surviving clones were added to Table 2.

3. I am also surprised by the inference made in the manuscript that the results from contact angle analysis can be used to directly assessprotein adsorption and bacterial biofilm formation (L. 231). L. 231. To the best of my knowledge, protins are not made of water only, and bacterial biofilms are far more complex than simple water. Therefore, I woluld like the authors how their observations can actually support this conclusion. At the very least, a reference is needed.

We added in Result and discussion (Page 7, Line 3, marked yellow):

«Authors [48] noted hydrophobity to be a dominant factor in influencing adhesion on surfaces. Bacteria with hydrophilic properties (E. coli, MRSA) prefer to adhere to hydrophilic surfaces. For example, S. aureus (include MRSA) has a hydrophilic character, and thus this bacterium favors metal alloys over ultra-high molecular weight polyethylene that present a more hydrophobic surface.»

 

4. The technical quality of the manuscript should be significantly improved prior to resubmission. On the one hand, the figure captions are laconic at best, and sometimes fail to provide some necessary information on the nature of the figure. For example, Figure caption 3 2 does not even indicate that figures Cu, Fe and O correspond to EDX maps of elements in the preparation.

 

Corrected, marked yellow.

 

In figure 4 it is not clear whether the images were obtained from optical or SEM microscope.

 

Corrected, marked yellow.

 

Figure caption for figure 8 is an extreme case – it has been obviously copied and pasted, but not edited to explained the actual content of Figure 8.

 

Corrected, marked yellow.

 

On the other hand, the manuscript is unnecessary redundant and lengthy at places and should be rewritten for clarity and conciseness; For example, the following sentences (L. 229-231): “However, a decrease in contact angle was observed when 10 wt. % of NPs was added in PMMA, and this composite showed the contact angle of 66.5° ± 2.8°. The addition of 10 230 wt. % of NPs decreased the contact angle.” can be simply reduced to : “when 10 wt. % of NPs was added in PMMA, the contact angle for the composite was 66.5° ± 2.8, significantly lower than for pure PMMA.”

 

Corrected, marked yellow.

 

A through proofreading by a native English would also remove obvious mishaps such as the wrong legend of figure 8, or the presence of words written in Cyrillic (L. 222), the meaning of which are quite cryptic.

 

Corrected, marked yellow.

 

Other items to be addressed include the name of bacterial species which must be systematically italicized (e.g. L. 72 and 275);

 

Usually the name of MRSA is not in italics.

 

The correct position of commas and hyphens (e.g. L. 60); and the correct definition of acronyms, such and TGA, SEM, or TEM.

 

Corrected, marked yellow.

 

Finally, for all instruments used in the characterization section, the model name, brand name, and country should be given. For example, LEO EVO 50, Zeiss, Germany).

 

Corrected, marked yellow.

 

Specific comments. L. 17. Please indicate the size (or size range) of individual nanoparticles in the abstract. L. 26. The mechanism given in this sentence has not been demonstrated. I suggest to remove this sentence. L. 41. Bacterial infection or contamination? You cannot infect a non-living material. L. 48 sq: I understand: introduction of antibacterial nanoparticles in the monomer preparation prior to synthesis. L; 51. Move the size value right after “silver nanoparticles”. L. 71. Please correct “CuFe2O4” (remove the extra space). L 97. So obviously it is not a simple hetero-junction, but to heterojunctions in series. Please clarify this point. L. 117 please indicate the amount of NPs added to the PMMA solution. L. 135. It’s Nicolet. L. 159. The phase diagram given in Figure 1 does not contain any field explicitly related to CuFeO2 or CuFe2O4, but “delafossite” and “spinel”. I understand from the manuscript and from additional research that delafossite corresponds to (CuFeO2) and spinel to CuFe2O4, but this has to be explictely stated in the manuscript. Also why writing CuFe2O4 in parenthese right after CuFeO2 , L. 159? This has the implicit meaning that CuFeO2 in the diagram actually corresponds to CuFe2O4, which is confusing. L. 174. Only one diffraction pattern is shown. L. 187 replace “include” with “result from”. L. 197. Where is the subject of the sentence? L. 202. “Uniform distribution” may be mistaken for a uniform distribution in size. Please write that they are uniformly distributed in the matrix 3 L. 209. Obviously it’s figure 5a. L. 219 “contains”… L; 262 Please insert the right figure caption. L. 343. Journal name in italics

 

Corrected, marked yellow.

 

Author Response File: Author Response.docx

Reviewer 3 Report

Line 72 The author should write the species name in italics: Klebsiella pneumoniae and Staphylococcus aureus

Lin 83-84. The author should check the sentence.

Figure 1: The author should either replace the Figure 1 or improve the resolution of the picture.

Figure 5: please mark the peaks.

Line 221. The author should check the sentence. “Тhe hydrophobicity of the composite surfaces plays an important role in term of биообрастания в морской воде.”

Line 231-233: The author should add reference.

Line 256: What is the number of bacteria?

Figure 7 tittle is wrong. “Figure 7. TGA curves of PMMA and CuFe2O4/Cu2O/CuO/PMMA composites”.

What about the comparison of the MIC values for the CuFe2O4/Cu2O/CuO with those values that were assessed for the other synthesized nanoparticle?

It would be beneficial if the authors carefully reviewed the paper for technical mistakes.

Author Response

Line 72 The author should write the species name in italics: Klebsiella pneumoniae and Staphylococcus aureus

Corrected, marked yellow.

Lin 83-84. The author should check the sentence.

Corrected, marked yellow.

Figure 1: The author should either replace the Figure 1 or improve the resolution of the picture.

Figure was improved.

Figure 5: please mark the peaks.

Was added.

Line 221. The author should check the sentence. “Тhe hydrophobicity of the composite surfaces plays an important role in term of биообрастания в морской воде.”

Corrected.

Line 231-233: The author should add reference.

The reference was added.

Line 256: What is the number of bacteria?

Corrected. «The bacteria reduction as a function…»

Figure 7 tittle is wrong. “Figure 7. TGA curves of PMMA and CuFe2O4/Cu2O/CuO/PMMA composites”.

Corrected.

What about the comparison of the MIC values for the CuFe₂O₄/Cu₂O/CuO with those values that were assessed for the other synthesized nanoparticle?

According to [            Gheidari, D.; Mehrdad, M.; Maleki, S.; Hosseinic, S. Synthesis and potent antimicrobial activity of CoFe₂O₄ nanoparticles under visible light. Heliyon, 2020, 6(10), e05058. https://doi.org/10.1016/j.heliyon.2020.e05058] the main mechanism of the ferrite nanoparticle action is based on ROS generation. Copper ions releasing will too small. We think the MIC value will be high without visible light irradiation.

It would be beneficial if the authors carefully reviewed the paper for technical mistakes.

The article was corrected.

 

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

see attached pdf file. 

Comments for author File: Comments.pdf

Author Response

1. First, uncertainties on the antibacterial efficiency are still missing. These uncertainties would be derived from the spread between the experimental values and the calculated curve plotted in figure 7b, using statistic tools that would be available in standard textbooks.

 

Figure 7 was changed.

 

2. Second, some figure captions may still be developed. For example, when several curves are plotted in figure 7, the sample nature and relationship to plot color should be indicated. Figure 6 would also need some development.

Fig. 7 caption was changed, marked yellow.

Fig. 6 caption was edited.

 

3. Third, the manuscript tries to correlate hydrophobicity and antibacterial properties. However, I doubt that the relatively modest variations in hydrophobicity between PMMA and composites makes a real difference in terms of bacteria adhesion. I would note in passing that 66.5 ± 2.8 is not significantly different from 69.7 ± 1.1, since the two values overlap within uncertainty (L. 248). So this is actually a demonstration that hydrophobicity is not an explanation for the difference in antibacterial properties.

We added in Result and discussion (Page 8, line 1, marked yellow):

«As the content of the NPs in the composite increased to 5%, the contact angles of the deionized water increased to 86.33 + 5.75°. This can be attributed to a increase in surface tension by the hydrophobic CuFe₂O₄/Cu₂O/CuO nanoparticles. When 10 wt. % of NPs was added in PMMA, the contact angle for the composite was 66.5° ± 2.8, significantly lower than for pure PMMA. This decrease in the contact angle reflects a reduction in surface hydrophobicity, which could be caused by a reduction in the total surface area of the nanoparticles due to their agglomeration».

4. Fourth, if I understand well, the equation given at L. 268 describes the evolution of the sample mass as a function of temperature, written here as t instead of the standard notation T. in addition, M(T) can simply be defined as the mass at temperature T – the definition as “the current value of the sample mass” is confusing. I don’t believe the term “logistic” is appropriate to describe this equation: in English, logistics refers to “the practical organization that is needed to make a complicated plan successful when a lot of people and equipment are involved” (Oxford Learner’s dictionary).

The variance in the designation of the term "temperature" has been corrected. As for the term "logistic”, “Logistic function”, “Logistic Equation”, “Logistic curve” are common terms widely used, in particular, in the study of the kinetics of heterogeneous chemical reactions involving solids. For example, this report: A. K. Burnham, Use and misuse of logistic equations for modeling chemical kinetics, Journal of Thermal Analysis and Calorimetry 127 (2017) 1107–1116. Synonyms for the term "logistic curve" are often used – “S-shaped curve”, “sigmoid curve”. A logistic curve in its simplest form (τ = 1) is symmetric about the point T = θ, which is on the abscissa axis. In the case of τ ≠ 1, the symmetry of the curve is broken.

At an arbitrary point of the curve, the rate of the chemical process dM/dT is proportional to the tangent of the curve slope at that point. And then it follows that as the value of τ increases, the slope of the curve decreases, which means that the rate of the process decreases, as shown in the figure below.

5. Finally, the issues related to the manuscript redaction and editions have been only partially addressed, and new ones have appeared in the corrections. The assistance and proofreading of a native English would probably be helpful here.

We checked article and corrected errors.

 

Specific comments and corrections.

 

All comments have been taken into account and necessary corrections have been made to the manuscript.

17. 17. Please rewrite : “the coating increases to 5 % wt. % 17 and 10 wt. %, as the size of

particle agglomerates increases to 50 μm and 100 μm respectively”.

60. 60. Please rewrite: “a… class of photocatalytic st ‒ antibacterial agents”.
61. 90. Please rewrite: “…but is complicated by a number…”, and remove “complicates it” t the

end of the sentence.

95. 95. Please rewrite: “gen-containing atmosphere of an iron wire together with copper,” (there

is no zinc in the present study).

104. 104. Please rewrite: “Furthermore,…”
105. 114. Please rewrite: “a gas mixture of 80 vol. % Ar + 20 vol. % O2”
106. 123. Please rewrite: “Then, 0.01 g, 0.04 g, 0.1 g, or 0.2 g of CuFe2O4/Cu2O/CuO powder…”
107. 129. Please rewrite: “The morphology of NPs was characterized using scanning (SEM; LEO

EVO 50, Zeiss, Germany) and transmission electron microscopy (TEM; JEM 2100,

JEOL, Japan).”

151. 151. Please rewrite: “. A volume of 400 μL”

L154. Upon (=during) incubation, or after it?

168. 168. Please rewrite: “When the Fe/Cu atomic ratio equals 50:50,”

 

1. 190: what is the difference between aggregate and agglomerates?

The difference between agglomerates and aggregates lies in the nature of the interaction between particles. There are weak physical interactions between particles in agglomerates, while in aggregates there are strong chemical forces between particles. Particle aggregation is beyond the scope of the present study, so the authors have excluded it from the manuscript. The corresponding paragraph has been corrected.

 

214. 214. Please rewrite: “However, they are strongly agglomerated and the size of NP

agglomerates increases”

239. 239. Please rewrite: “For example, S. aureus (include MRSA) has a hydrophilic character,

which favors adhesion of this bacterium to metal alloys over ultra-high molecular

weight polyethylene…”

250. 250. Please rewrite: “Thus, the active biocidal antifouling composite coatings can control

bacteria adhesion.” (but I actually do not agree with this sentence; please see general

coments above).

277. 277. Please rewrite: “As can be concluded from the data presented, the destruction of PMMA

occurs in two stages.”

283. 283. “, the asymmetry parameter slightly increases, which may be related to the deceleration of PMMA nano-particles at a higher temperature with the formation of volatile products.” It is impossible to understand this sentence; please clarify.

At an arbitrary point of the curve, the rate of the chemical process dM/dT is proportional to the tangent of the curve slope at that point. And then it follows that as the value of τ increases, the slope of the curve decreases, which means that the rate of the process decreases, as shown in the figure below.

 

333. 333. Please rewrite: “(1) effective interaction of the nanoparticle with the bacterial cell wall

due to its high surface-to-volume ratio, (2) release of copper ions”

Figure 3. Do the figures in the inserted table result from (an average of) point analyses, or form an integration of EDX

The figures in the inserted table result from integration of EDX.

Author Response File: Author Response.docx

Reviewer 3 Report

Revisions were made.

Author Response

All comments have been taken into account and necessary corrections have been made to the manuscript.

Author Response File: Author Response.docx