Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript presents a solid and coherent study using DSC, IR, fluctuation analysis, and photothermal measurements to investigate how fructose-capped AgNPs influence SOPC membranes. The work is generally well executed, and the multi-technique approach is a clear strength.

Several points nevertheless need revision. The TEM images provided by the authors clearly show a heterogeneous fructose rim in the range of 2–15 nm, not strictly 2–3 nm as stated in the text. This should be corrected to avoid underestimating the effective coating thickness.

In addition, at least one more basic characterization methods that are standard in nanoparticle-membrane studies should be included experimentally. These include DLS (hydrodynamic size changes upon lipid interaction), zeta-potential (to evaluate surface charge and binding propensity), UV–Vis spectroscopy (plasmon peak shifts upon membrane association), and/or AFM (surface topography and capping uniformity). These straightforward techniques would meaningfully strengthen the dataset and allow a more complete mechanistic interpretation.

Finally, the Introduction would benefit from briefly acknowledging additional relevant methodological directions, for example protein- or peptide-assisted nanoparticle biosynthesis, which provides alternative green-synthesis approaches and different surface chemistries (as shown in  DOI: 10.3390/nano12111868). Including such examples broadens the context and situates the current synthesis route among other biologically inspired strategies.

With these additions and corrections, the manuscript will be significantly improved and provide a more complete and rigorous analysis of AgNP andn lipid membrane interactions.

Author Response

Response to Reviewer 1:

 

The authors sincerely thank the reviewer for the careful evaluation of their manuscript and for the constructive comments, which have helped to improve the clarity, accuracy, and contextual framing of the work. All comments have been addressed as detailed below.

Response to Reviewer #1 point by point:

1) The TEM images provided by the authors clearly show a heterogeneous fructose rim in the range of 2–15 nm, not strictly 2–3 nm as stated in the text. This should be corrected to avoid underestimating the effective coating thickness.

               We thank the reviewer for this important observation. We have corrected the description of the fructose capping layer throughout the manuscript to reflect the heterogeneous thickness observed in the TEM images. The fructose rim is now consistently reported as ranging from 2 to 15 nm, in accordance with the experimental evidence. This correction ensures a more accurate representation of the effective coating thickness and its potential impact on NP-membrane interactions.

 

2) In addition, at least one more basic characterization methods that are standard in nanoparticle-membrane studies should be included experimentally. These include DLS (hydrodynamic size changes upon lipid interaction), zeta-potential (to evaluate surface charge and binding propensity), UV–Vis spectroscopy (plasmon peak shifts upon membrane association), and/or AFM (surface topography and capping uniformity). These straightforward techniques would meaningfully strengthen the dataset and allow a more complete mechanistic interpretation.

               We appreciate the reviewer’s emphasis on the importance of complementary nanoparticle characterization. We have added information regarding hydrodynamic size, zeta potential and UV-Vis spectroscopy as follows:

• Dynamic Light Scattering (DLS)

The median average size of the obtained particles was confirmed by DLS (Figure 3). The histogram of the sample displayed a trimodal particle size distribution, suggesting tpresence of aggregates.Based on the DLS, the median average size of the obtained particles was identified in the range of 10 to 40 nm.     

 

Figure 3. Dynamic light scattering histograms of AgNPs.

• Zeta potential

The zeta potential of the sample was -17.94 mV. The negative charge of the zeta potential refers to carboxylic groups of gluconic acid obtained when Ag+ is reduced to Ag0. Carboxylic acids, obtained in the oxidation of sugars, provide a negative surface charge density to counteract the van der Waals forces responsible for particle coalescence. Self-assembled carboxylic acids ensure dense coating on the metal surfaces and stabilize them [32].                                                

• UV-Vis spectroscopy

An essential method for examining AgNPs in suspension is UV/Vis absorption spectroscopy. The absorption spectra in the 190–600 nm wavelength range were recorded in order to track the Ag NPs’ production in an aqueous solution (Figure 4). Interesting optical characteristics related to surface plasmon resonance are displayed by each kind of AgNP [33].The following Figure 4 showed the time-dependent UV/Vis absorption spectra of AgNPs. Fructose displays an absorption peak at 189 nm and 280 nm, respectively.

 

Figure 4. Time-dependent UV–Vis absorption spectra of fructose-capped AgNPs recorded in the 190–790 nm range, showing the emergence of the surface plasmon resonance band at ≈415 nm during nanoparticle formation.

The SPR characteristic band at 415 nm is an indicator for the formation of AgNPs. The spherical shape of the particles is shown by the band’s location between 350 and 420 nm [34]. The symmetrical peak of AgNPs showed a modest level of NP aggregation [35,36].                                                

The added details substantially strengthened the manuscript by explicitly discussing these standard characterization techniques and their relevance to nanoparticle–membrane interactions.

               In particular, we have cited and discussed a representative literature study that reports DLS-derived hydrodynamic diameters, zeta-potential values, UV–Vis plasmonic features, and surface-related characteristics for fructose-capped AgNPs. Where appropriate, representative values from the literature are now included to contextualize our system and support the mechanistic interpretation of the observed membrane effects as follows:

{Mulvaney, P.; Liz-Marzan, L. M.; Giersig, M.; Ung, T. J. (2000). Silica encapsulation of quantum dots and metal clusters. Journal of Materials Chemistry, 10, 1259–1265. https://doi.org/10.1039/B000136H                                                                                                                                                        Park, M.; Sohn, Y.; Shin, W. G.; Lee, J.; Ko, S. H. (2015). Ultrasonication assisted production of silver nanowires with low aspect ratio and their optical properties. Ultrasonics Sonochemistry, 22, 35–40. https://doi.org/10.1016/j.ultsonch.2014.05.007                                                                                                                                            Chowdhury, S.; Yusof, F.; Faruck, M. O.; Sulaiman, N. (2016). Process optimization of silver nanoparticle synthesis using response surface methodology. Procedia Engineering, 148, 992–999. https://doi.org/10.1016/j.proeng.2016.06.552                                                                                                                                                     Huang, H.; Yang, X. (2004). Synthesis of polysaccharide-stabilized gold and silver nanoparticles: A green method. Carbohydrate Research, 339, 2627–2631. https://doi.org/10.1016/j.carres.2004.08.005                                                                                                                                                     Huang, Y.; Guo, X.; Wu, Y.; Chen, X.; Feng, L.; Xie, N.; Shen, G. (2024). Nanotechnology’s frontier in combatting infectious and inflammatory diseases: Prevention and treatment. Signal Transduction and Targeted Therapy, 9, 34. https://doi.org/10.1038/s41392 -024-01745-z}

 

This addition clarifies how such parameters influence nanoparticle stability, surface charge, and binding propensity, thereby reinforcing the conclusions drawn from our experimental results.

 

3) Finally, the Introduction would benefit from briefly acknowledging additional relevant methodological directions, for example protein- or peptide-assisted nanoparticle biosynthesis, which provides alternative green-synthesis approaches and different surface chemistries (as shown in  DOI: 10.3390/nano12111868). Including such examples broadens the context and situates the current synthesis route among other biologically inspired strategies.

               We thank the reviewer for this valuable suggestion. The Introduction has been revised to include a brief discussion of protein- and peptide-assisted nanoparticle biosynthesis as an alternative green synthesis strategy. The cited example (DOI: 10.3390/nano12111868) has been incorporated to highlight how biologically assisted routes can yield nanoparticles with distinct surface chemistries and functional properties as follows:

{Peptides and proteins can template or regulate AgNP nucleation, mirroring broader trends in green NP syntheses [4].

Kryuchkov, M.; Adamcik, J.; Katanaev, V. L. (2022). Bactericidal and antiviral bionic metalized nanocoatings. Nanomaterials, 12,1868. https://doi.org/10.3390/nano12111868}

This addition broadens the methodological context of our work and situates the fructose-capping approach among other biologically inspired synthesis strategies.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript aims to evaluate the influence of fructose-capped AgNPs on the physicochemical properties of SOPC-based liposomal bilayers, with implications for drug delivery and photothermal therapy.

There are some concerns.

(1) Why the authors choose the AgNPs' influence on membrane property (why not AuNPs or other species)? The scientific basis is quite weak.

(2) Why the combination of AgNPs with SOPC-based liposomal bilayer should be utilized as drug delivery carrier or photothermal reagent? Those are two different applications.

(3) For Figure 2, the AgNPs is within the liposome layer or inside the particle?

(4) For Figure 6, the photo-thermal performance of AgNPs with liposomal bilayer should also be shown.

(5) The drug delivery performance is not shown with experimental evidence. In this case, the title and abstract sections might need to be re-organized.

(6) The doi number is missing in reference 38.

Author Response

Response to Reviewer 2:

 

The authors sincerely thank the reviewer for the careful evaluation of their manuscript and for the constructive comments, which have helped them improve the clarity, accuracy, and contextual framing of the work. All comments have been addressed as detailed below.

Response to Reviewer #2 point by point:

1) Why the authors choose the AgNPs' influence on membrane property (why not AuNPs or other species)? The scientific basis is quite weak.

Our research team have been working in recent studies with a variety of nanoparticles both organic and inorganic. This research is focused on AgNPs produced via green technology due to their numerous practical applications. As the referee suggested we have added additional paragraph emphasizing on the advantages that silver nanoparticles provide and additional references on the topic were added as follows:

{At the nanoscale, silver nanoparticles (AgNPs) inherit and greatly amplify these attributes: AgNPs typically measure below 100 nm and contain thousands of silver atoms, giving them extremely high surface area-to-volume ratios and distinct size-dependent physicochemical characteristics, including high electrical and thermal conductivity, chemical stability and catalytic activity.  These properties enable AgNPs to inhibit the growth of hundreds of bacterial, fungal, and algal species through controlled silver-ion release, making them highly relevant for medical, healthcare, and environmental applications. Recent advances in green nanotechnology, particularly bio-logically assisted synthesis routes, further enhance the relevance of AgNPs by enabling eco-friendly production with tunable surface chemistries and improved biosafety profiles, expanding their applicability across medicine, agriculture, electronics, and water-treatment technologies [1–3]. Peptides and proteins can template or regulate AgNP nucleation, mirroring broader trends in green NP syntheses [4].

Zhao, X.; Xu, X.; Ai, C.; Yan, L.; Jiang, C.; Shi, J. (2022). Advantages of silver nanoparticles synthesized by microorganisms in antibacterial activity. In: Abd-Elsalam, K. A. (Ed.), Nanobiotechnology for Plant Protection: Green Synthesis of Silver Nanomaterials, Chapter 22, Elsevier, pp. 571–586. https://doi.org/10.1016/B978-0-12-824508-8.00005-8                                                                                                                                                     Abbas, R.; Luo, J.; Qi, X.; Naz, A.; Khan, I. A.; Liu, H.; Yu, S.; Wei, J. (2024). Silver nanoparticles: Synthesis, structure, properties and applications. Nanomaterials, 14, 1425. https://doi.org/10.3390/nano14171425                                                                                                                                             Kryuchkov, M.; Adamcik, J.; Katanaev, V. L. (2022). Bactericidal and antiviral bionic metalized nanocoatings. Nanomaterials, 12, 1868. https://doi.org/10.3390/nano12111868}

(2) Why the combination of AgNPs with SOPC-based liposomal bilayer should be utilized as drug delivery carrier or photothermal reagent? Those are two different applications.

To clarify the possible application of liposomes, encapsulated with AgNPs a paragraph and additional reference was added in the text of manuscript as the referee suggested as follows:

{The current focus on the nature of AgNPs is in cancer diagnostics and therapy: they can serve as cytotoxic or drug carrier agents against tumors. Due to their high surface area-to-volume ratio, ease of functionalization, and excellent stability, silver NP have gained significant interest as drug delivery systems. They have demonstrated versatility in delivering a wide range of therapeutic agents. Moreover, AgNPs enhance drug solubility, protect sensitive compounds from degradation, and provide controlled and sustained release at targeted sites [5].

Stoyanova, M.; Milusheva, M.; Georgieva, M.; Ivanov, P.; Miloshev, G.; Krasteva, N.; Hristova-Panusheva, K.; Feizi-Dehnayebi, M.; Mohammadi Ziarani, G.; Stojnova, K.; Tsoneva, S.; Todorova, M.; Nikolova, S. (2025). Synthesis, cytotoxic and genotoxic evaluation of drug-loaded silver nanoparticles with mebeverine and its analog. Pharmaceuticals, 18(3), 397. https://doi.org/10.3390/ph18030397}

(3) For Figure 2, the AgNPs is within the liposome layer or inside the particle?

Figure 2 depicts the pure nanoparticles after the preparation process. To visualize the nanoparticles inside the liposome a CrioTEM technique is needed. Such a visualization was not performed on this stage of the research. Nevertheless, due to the hydrophilic nature of the nanoparticle coating and taking into account the tendency of fructose to form hydrogen bonds with the heads of lipid molecules, it is expected that the NPs will preferentially position themselves in the hydrophilic region on both sides of the lipid membrane as it is shown on the graphical abstract.

(4) For Figure 6, the photo-thermal performance of AgNPs with liposomal bilayer should also be shown.

We thank to the reviewer for the suggestion made. The experimental set-up for the precise evaluation of the photo-thermal performance of the loaded with nanoparticle liposomal complexes is under development and refinement. The mentioned experimental work is planned for the near future.

(5) The drug delivery performance is not shown with experimental evidence. In this case, the title and abstract sections might need to be re-organized.

As the referee suggested we have slightly changed the abstract to show the future prospects of the research as follows:

 

{This study investigates the influence of fructose-capped AgNPs on the physicochemical properties of SOPC-based liposomal bilayers, with potential implications for drug delivery and photothermal therapy.

…..

We concluded that fructose-capped-AgNPs moderately fluidify lipid bilayers while enabling efficient, controllable photothermal capability, making them excellent candidates for eventual design of advanced liposomal systems for combined therapy and diagnostic.}

(6) The doi number is missing in reference 38.

We thank to the reviewer for pointing out the missing doi number. We have added the DOI number of the corresponding article in the literature.

{Toyran, N., & Severcan, F. (2002). Infrared spectroscopic studies on the dipalmitoyl phosphatidylcholine bilayer interactions with calcium phosphate: Effect of vitamin D₂. Spectroscopy, 16, 399–408. https://doi.org/10.1155/2002/381692}

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have substantially improved the manuscript compared to the previous version. In particular, the inclusion of additional experimental approaches (UV–Vis spectroscopy, zeta potential measurements, and dynamic light scattering) significantly strengthens the physicochemical characterization and supports the main conclusions. The Introduction has also been expanded and improved, providing clearer context and better justification of the study.

In my opinion, the revised manuscript adequately addresses the previous concerns and meets the standards of the journal. I therefore recommend acceptance of the manuscript in its present form.

Author Response

We would like to thank again to the referee for his thorough reading and useful suggestions. This helped to improve the readability of the manuscript. 

Reviewer 2 Report

Comments and Suggestions for Authors

The term of drug delivery should be removed from the title as it is not shown in the experimental data of this manuscript.

Author Response

Response to Reviewer 2:

 

The authors thank again to the reviewer for the careful evaluation of their manuscript and for the constructive comments, which have helped them improve the clarity, accuracy, and contextual framing of the work. The comment has been addressed as detailed below.

Response to Reviewer #2 point by point:

• The term of drug delivery should be removed from the title as it is not shown in the experimental data of this manuscript.

The title has been changed according to the recommendation of the referee from

Influence of Silver Nanoparticles on Liposomal Membrane Properties with Relevance to Drug Delivery and Photothermal Therapy

To

Influence of Silver Nanoparticles on Liposomal Membrane Properties with Relevance to Photothermal Therapy

The term drug delivery was removed according to the recommendation.

Author Response File: Author Response.pdf