Development of Nanomedicine from Copper Mine Tailing Waste: A Pavement towards Circular Economy with Advanced Redox Nanotechnology
and Samir Kumar Pal 3,*Round 1
Reviewer 1 Report
In this manuscript, the authors have extracted CuO nano granules from copper mine tailings of two places with different geographical locations and functionalized with citrate ligands, and found that the extracted functionalized CuO from copper tailings offers similar property compared to that of the synthesized CuO. Overall, this work is well organized and meaningful, which can be accepted for publication in Catalysts after a minor revision by addressing the following comments.
1 In Page 7, the authors described that “The additional peaks are appearing due to the attribution of the elements like Au, Cr etc. present in the mine samples as revealed from the SEM EDAX analysis (Figure 2).” However, EDAX analysis of extracted and capped CuO from mines should be provided.
2 The authors described that “The UV-Vis spectra of the citrate capped CuO samples (Figure 2a) exhibited a broad absorbance peak at 285 nm characteristic of surface plasmon resonance of the CuO nanparticles (46).”, Figure 2a should be revised to Figure 3a.
3 In Figure 3, the absorbance peak of synthesized citrate-CuO nanohybrids at 200-300 nm is required.
4 The authors described that “The ζ-potential for the Peru and Bhopal samples were obtained to be -11.2mV and -8mV respectively.” The ζ-potential peaks of Peru and Bhopal samples should be added into Figure 2a.
5 Some new and relevant literatures should be cited such as Journal of Materials Chemistry A 10 (2022) 6835-6871; Materials Reports: Energy 2 (2022) 100081; Materials Today Energy 23 (2022) 100896; Materials Reports: Energy 2 (2022) 100092; Materials Today Energy 23 (2022) 100899; Materials Reports: Energy 2 (2022) 100091; Materials Reports: Energy 1 (2021) 100019.
Author Response
Point by point response to the reviewers (Manuscript ID: catalysts-2183620)
The authors would like to thank the learned reviewers for their careful scrutiny as well as valuable comments and suggestions. We have addressed their constructive comments pointwise which have been extremely useful in improving the quality of our revised manuscript.
Reviewer 1
Comments and Suggestions for Authors:
In this manuscript, the authors have extracted CuO nano granules from copper mine tailings of two places with different geographical locations and functionalized with citrate ligands, and found that the extracted functionalized CuO from copper tailings offers similar property compared to that of the synthesized CuO. Overall, this work is well organized and meaningful, which can be accepted for publication in Catalysts after a minor revision by addressing the following comments.
Reply: The authors would like to thank the learned reviewer for the nice briefing and kind appreciation of our work. As per the kind suggestion of the learned reviewer, we have addressed all the points raised by the learned reviewer and incorporated in the revised manuscript. The constructive comments from the reviewer have facilitated the further improvement of the quality of the manuscript.
Query 1. In Page 7, the authors described that “The additional peaks are appearing due to the attribution of the elements like Au, Cr etc. present in the mine samples as revealed from the SEM EDAX analysis (Figure 2).” However, EDAX analysis of extracted and capped CuO from mines should be provided.
Reply 1: The authors appreciate the concern of the learned reviewer. EDAX analysis of extracted and capped CuO from mines are provided in the revised manuscript as per the kind suggestion of the learned reviewer (Figure:2, Page:7-8, Line:274-279; and Page:8, Line: 324-326).
Query 2. The authors described that “The UV-Vis spectra of the citrate capped CuO samples (Figure 2a) exhibited a broad absorbance peak at 285 nm characteristic of surface plasmon resonance of the CuO nanparticles (46).”, Figure 2a should be revised to Figure 3a.
Reply 2: The authors are thankful to the learned reviewer for the critical reading of the manuscript and pointing out this issue. This unintentional mistake has been corrected and Figure 2a is modified to ‘Figure 3a’ in the revised manuscript as per the kind suggestion (Page:9, Line:339-341).
Query 3. In Figure 3, the absorbance peak of synthesized citrate-CuO nanohybrids at 200-300 nm is required.
Reply 3: We would like to thank the learned reviewer for pointing out this issue. The unintentional mistake has been corrected in the revised version of the manuscript. All the absorbance spectra in the panel ‘a’ of Figure 3 (Page:9, Line:358 and Page:4, Line:159-161) are shown in the range 200 – 800nm.
Query 4. The authors described that “The ζ-potential for the Peru and Bhopal samples were obtained to be -11.2mV and -8mV respectively.” The ζ-potential peaks of Peru and Bhopal samples should be added into Figure 2a.
Reply 4: The authors appreciate the concern raised by the learned reviewer. A NanoS Malvern (Zeta-seizer) instrument equipped with a 4 mW He:Ne laser (λ = 632.8 nm) and a thermostat coupled sample chamber was employed for dynamic light scattering (DLS) and ζ potential measurements. For the kind information of the learned reviewer, we would like to point out that the full curve for a sample is only provided by the instrument software while titration experiment is performed as we obtained in case of synthesized pure Citrate capped CuO nanohybrid. However, for the extracted Citrate CuO nanoparticles from Peru and Bhopal mine samples only zeta potential measurements were carried out for which only values in mV unit are provided, no full curves were available from the instrument. We have addressed this issue in line171-174 of page4 and line 316-321 of page 8 of the revised manuscript.
Query 5. Some new and relevant literatures should be cited such as Journal of Materials Chemistry A 10 (2022) 6835-6871; Materials Reports: Energy 2 (2022) 100081; Materials Today Energy 23 (2022) 100896; Materials Reports: Energy 2 (2022) 100092; Materials Today Energy 23 (2022) 100899; Materials Reports: Energy 2 (2022) 100091; Materials Reports: Energy 1 (2021) 100019.
Reply: The authors thank the learned reviewer for the kind suggestion. We have cited all of the references in our revised manuscript with great appreciation (Page:15, Line:520-522, 518-519, 515-517, 513-514; Page:16, Line: 551-553, 548-550).
Please see the attachment.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Authors report the synthesis of CuO nano particles and study their anti microbial activities. The novelty of the work is not clear. As the authors state in the intro, metallic Cu and CuO are already know to exhibit multi-toxicity on multi-drug resistant bacterial species , and the effect might be associated with reactive oxygen species (ROS) production. In the present manuscript authors confirmed that their synthesised CuO nano particles show the same feature and behaviour already reported in the literature. Moreover, some aspects of the characterisation of the material need clarification:
1) In the FTIR spectrum of the synthesized CuO (Fig 1b, red curve) something is off with its baseline.
2) Authors state: "The CuO obtained from Peru and Bhopal mine samples exhibited peaks corresponding to the synthesized CuO NPs." Which peaks are they refering to?
3) In Fig 2a, there is a peak assigned to Si in the EDAX graph. Where does it come from since this is the synthesised sample?
4) Authors state that “the results of DLS corroborates with the size obtained from XRD (34.4nm) and FESEM analysis (38.1nm)”. However, the DLS results are between 78-190nm. Why this difference of >50% can be assumed small enough to affirm that both values from the DLS and XRD/SEM are comparable?
5) Figure 5 and Table 1 need to be explained and contextualised in the paper.
: 6) The mechanism of CuO is shown in the TOC, but not explained in the manuscript. Ideally the mechanism should be detailed in the text, or the TOC should be redrawn and represent the context of the work.
Author Response
Point by point response to the reviewers (Manuscript ID: catalysts-2183620)
The authors would like to thank the learned reviewers for their careful scrutiny as well as valuable comments and suggestions. We have addressed their constructive comments pointwise which have been extremely useful in improving the quality of our revised manuscript.
Reviewer 2
Comments and Suggestions for Authors:
Authors report the synthesis of CuO nano particles and study their anti-microbial activities. The novelty of the work is not clear. As the authors state in the intro, metallic Cu and CuO are already known to exhibit multi-toxicity on multi-drug resistant bacterial species, and the effect might be associated with reactive oxygen species (ROS) production. In the present manuscript authors confirmed that their synthesised CuO nano particles show the same feature and behaviour already reported in the literature. Moreover, some aspects of the characterisation of the material need clarification:
Reply: The authors would like to thank the learned reviewer for carefully reading and scientifically reviewing as well as briefing our work. As correctly pointed out by the learned reviewer, CuO nanoparticles are already known to exhibit multi-toxicity on multi-drug resistant bacterial species. However, it is for the kind information of the learned reviewer, citrate ligand capped CuO nanoparticles of average diameter around 40nm which are suspended in aqueous solution has not been reported earlier. Although CuO is a well-known material, its surface functionalization with a biocompatible citrate ligand and its antibacterial studies for APDT (Antibacterial Photo-dynamic Therapy) applications are sparse in contemporary literature.
The size, morphology and mainly solubility play a significant role on the antibacterial activi-ties of Cu, CuO and Cu2O [G. McDonnell and A. D. Russell, "Antiseptics and disinfectants: activity, action, and resistance," Clinical microbiology reviews, vol. 12, pp. 147-179, 1999. ; H. Pang, F. Gao, and Q. Lu, "Morphology effect on antibacterial activity of cuprous oxide," Chemical Communications, pp. 1076-1078, 2009. ; G. Ren, D. Hu, E. W. Cheng, M. A. Vargas-Reus, P. Reip, and R. P. Allaker, "Characterisation of copper oxide nanoparticles for antimicrobial applications," International journal of antimicrobial agents, vol. 33, pp. 587-590, 2009.]. The major limitation of metallic CuO NPs in the nano-size range is the lack of significant stability in dispersions due to their strong tendencies of aggregation and formation of larger clusters to reduce the energy associated with their high surface area [M. J. Wozniak-Budych, Ł. Przysiecka, B. M. Maciejewska, D. Wieczorek, K. Staszak, M. Jarek, et al., "Facile synthesis of sulfobetaine-stabilized Cu2O nanoparticles and their biomedical potential," ACS Biomaterials Science & Engineering, vol. 3, pp. 3183-3194, 2017. ; J. Zhou, H. Xiang, F. Zabihi, S. Yu, B. Sun, and M. Zhu, "Intriguing anti-superbug Cu2O@ ZrP hybrid nanosheet with enhanced antibacterial performance and weak cytotoxicity," Nano Research, vol. 12, pp. 1453-1460, 2019. ; E. M. Hotze, T. Phenrat, and G. V. Lowry, "Nanoparticle aggregation: challenges to understanding transport and reactivity in the environment," Journal of environmental quality, vol. 39, pp. 1909-1924, 2010.]. The formation of clusters results in sedimentation leading to loss of reactivity and antimicrobial performance in which a nanometric size is essential [E. M. Hotze, T. Phenrat, and G. V. Lowry, "Nanoparticle aggregation: challenges to understanding transport and reactivity in the environment," Journal of environmental quality, vol. 39, pp. 1909-1924, 2010.]. Further surface modifications of CuO NPs by post-functionalization approach not only enhances their colloidal stability but also introduces unique physical and chemical properties including possible enhancement of their antimicrobial activities. Functionalization or capping of an inorganic nanoparticle with an organic ligand like citrate or folate are evidenced to produce nanohybrids that have unique therapeutic potentials [S. Mondal, R. Ghosh, A. Adhikari, U. Pal, D. Mukherjee, P. Biswas, et al., "In vitro and Microbiological Assay of Functionalized Hybrid Nanomaterials to Validate Their Efficacy in Nanotheranostics: A Combined Spectroscopic and Computational Study," ChemMedChem, vol. 16, pp. 3739-3749, 2021.]. We have addressed this issue in the revised manuscript (Page:2, Line:77-88) and all the concerns raised by the learned reviewer point by point in the following section.
Query 1. In the FTIR spectrum of the synthesized CuO (Fig 1b, red curve) something is off with its baseline.
Reply 1: The authors appreciate the concern of the learned reviewer. For the kind note of the learned reviewer, we would like to address that the FTIR spectrum of the synthesized citrate functionalized CuO nanoparticles provided in Figure 1b of the revised manuscript is baseline corrected. The apparent baseline shift in the stacked plot may be due to the excess concentration of the synthesized nanoparticles in comparison with the extracted CuO NPs from the copper mine tailings which has broadened the peak at 3307.4 cm-1. This issue has been addressed in the revised manuscript (Page:8, Line:296-299).
Query 2. Authors state: "The CuO obtained from Peru and Bhopal mine samples exhibited peaks corresponding to the synthesized CuO NPs." Which peaks are they referring to?
Reply 2: The authors appreciate the concern of the learned reviewer. The peaks at 32.5, 35.4, 35.5, 38.7, 38.9,46.2, 48.8, 51.3, 53.4, and 56.7 in 2θ correspond to the different CuO planes, respectively of the synthesized CuO nanoparticles as depicted in Figure 1a (Page:6, Line:246) of the revised manuscript. The peaks at the same 2θ positions for the extracted CuO nanoparticles from both Peru and Bhopal mine samples are referred here which confirm the extraction of CuO from the mine tailings. This issue has been addressed in the revised manuscript (Page:6-7, Line:266-273).
Query 3. In Fig 2a, there is a peak assigned to Si in the EDAX graph. Where does it come from since this is the synthesised sample?
Reply 3: The authors appreciate the concern raised by the learned reviewer. The peak of Si appearing in the EDAX graph of Figure 2a is attributed from the silicon substrate. For EDAX analysis, the test sample was drop casted on Si wafer substrate and the thickness of the drop casted layer may not be uniform throughout the substrate. This issue has been addressed in the revised manuscript (Page:8-9, Line:328-331).
Additionally, the EDAX analysis of extracted and capped CuO NPs from Peru and Bhopal mines are also provided in the revised manuscript along with the synthesized citrate functionalized CuO NPs (Figure 2b and 2c inset, Page:7-8, Line:274-279).
Query 4. Authors state that “the results of DLS corroborates with the size obtained from XRD (34.4nm) and FESEM analysis (38.1nm)”. However, the DLS results are between 78-190nm. Why this difference of >50% can be assumed small enough to affirm that both values from the DLS and XRD/SEM are comparable?
Reply 4: The authors appreciate the concern of the learned reviewer. It has to be noted that the Dynamic Light Scattering (DLS) accounts the hydrodynamic diameter of the functionalized nano-material comprising of core CuO and citrate ligands at the surface along with associated water molecules and some possible aggregation of the functionalized nanoparticles in the aqueous solution. The electron micrograph of the nanoparticles shows the inorganic nanomaterial as a whole containing crystalline and amorphous inorganic core substrate. On the other hand, the size from the XRD accounts only the crystalline materials in the core inorganic particles. Thus, it is obvious that DLS overestimates the size, while XRD reveals the crystalline nanoparticles in the core [Pabisch S, Feichtenschlager B, Kickelbick G, Peterlik H. Effect of interparticle interactions on size determination of zirconia and silica based systems–A comparison of SAXS, DLS, BET, XRD and TEM. Chemical physics letters. 2012 Jan 10;521:91-7.]. This issue is addressed in page:8 in line:304-312 of the revised manuscript.
Query 5. Figure 5 and Table 1 need to be explained and contextualised in the paper.
Reply 5: The authors would like to thank the learned reviewer for pointing out this issue. As per the kind suggestion of the learned reviewer, Figure 5 and Table 1 are explained and contextualised in the revised manuscript. We have used computational biology strategy in order to rationalize the antibiotic resistant bacterial remediation found in our experimental studies. In introduction section (page:3) line no. 113-114, states the objective behind using the computational biology tool, which relies on the existing research works, and related predictive models. The section 2.8 viz. ‘Method of Computational Biology’, under ‘Materials and Methods’, described in detail, the tool used to study computational biology in the revised manuscript (Page:5, Line:237-244).
Figure 5 along with the accompanying descriptive text (Page:13, Line:464-472) in the results and discussion section of the revised manuscript, indicated thoroughly the name of the proteins of S. hominis, which are getting affected by citrate functionalized CuO. Figure 5 was generated using the tool mentioned in the material and method section earlier. Table 1 depicted the activities of the affected proteins and how the proteins help S. hominis in its survival. The extraordinary effect of the synthesized copper nanohybrid may be hypothesized using predictive biological interactions. To compare our findings with the existing research works, and related predictive models, the STITCH database was used. It generated an interaction network (Figure 5) between the citrate capped CuO and its effect on various proteins of S. hominis. To understand how CuO affects the survival of the bacteria, a comprehensive table (Table 1) was populated with the protein names and their respective activities. The table suggests the negative impact of CuO on key-proteins of S. hominis, which help the organism to deal with the environmental stress, energy metabolism, GTP binding, translation regulation etc.
Query 6. The mechanism of CuO is shown in the TOC, but not explained in the manuscript. Ideally the mechanism should be detailed in the text, or the TOC should be redrawn and represent the context of the work.
Reply 6: The authors appreciate the concern of the learned reviewer. The therapeutic action of the CuO NPs against S. hominis bacteria occur primarily via the generation of reactive oxygen species which mainly include formation of hydroxyl radical (HO.*) and superoxide radical (O2.-*). Initially an electron-hole (e-/h+) pairs are formed when the electromagnetic radiation of energy (hu) is either greater than or equal to the bandgap energy (Eg) of the CuO NPs. This phenomenon excites the electron from the valence band (VB) to the conduction band (CB), leaving holes behind in the VB. These photoexcited electrons reduce the surface adsorbed O2 to O2-.*, while the holes oxidize H2O or HO- to OH.*[A. Ajmal, I. Majeed, R. N. Malik, H. Idriss, and M. A. Nadeem, "Principles and mechanisms of photocatalytic dye degradation on TiO 2 based photocatalysts: a comparative overview," Rsc Advances, vol. 4, pp. 37003-37026, 2014.; M. Pirilä, M. Saouabe, S. Ojala, B. Rathnayake, F. Drault, A. Valtanen, et al., "Photocatalytic degradation of organic pollutants in wastewater," Topics in Catalysis, vol. 58, pp. 1085-1099, 2015. ; A. K. Sibhatu, G. K. Weldegebrieal, S. Sagadevan, N. N. Tran, and V. Hessel, "Photocatalytic activity of CuO nanoparticles for organic and inorganic pollutants removal in wastewater remediation," Chemosphere, p. 134623, 2022.] as described in the graphical abstract. These in-situ production of reactive radicals starts off attacking the bacterial population and eliminates them by the production of toxic by-products as a result of ROS mediated damage to the cellular system.
CuO+hu= CuO (e-+h+)
O2+e-=O2-.*
H2O+h+=H++HO.*
In absence of an electromagnetic radiation, the anti-microbial activity may be attributed to the ligand to metal charge transfer (LMCT) of the C-CuO NPs. The LMCT bands originate due to the interaction of the Cu1+/2+ centres in the NP with the surface bound citrate ligands. In the dark condition the origin of the anti-bacterial activity might be due to the conversion of the Cu1+ to Cu2+ states at the centre accompanied by the direct injection of the electrons into the CB of the C-CuO NPs followed by the reduction of the surface adsorbed O2 to O2-*.
Cu1+-O=Cu+2-O + e-
O2+e-=O2-*
The enhanced efficacy of the citrate functionalized CuO NPs in presence of light for more bacterial remediation establishes its credentials for the application of APDT (Antibacterial Photo-dynamic Therapy). The mechanism of action citrate-CuO has been detailed in the text of the revised manuscript (Page:12-13, Line:436-461) as per the kind suggestion of the learned reviewer.
Please see the attachment.
Author Response File: 
Author Response.pdf
Round 2
Reviewer 2 Report
The authors have addressed all the concerns of the reviewers, and the new version of the manuscript has been improved significantly.
