The study by Glory Kah, Rahul Chandran, and Heidi Abrahamse explores the formulation of silver nanoparticles conjugated with PEG and curcumin as a novel approach for photodynamic therapy targeting lung cancer cells, particularly lung cancer stem cells. This research highlights an intriguing and promising strategy in the evolving field of cancer therapy. However, the manuscript, in its current form, could benefit from further clarification and improvements.

Introduction:
• In the introduction, the authors mention guaranteeing tumor-specific accumulation, which does not appear to be the primary aim of the manuscript and could be confusing for readers.
• Certain phrases in the introduction, such as “functionalizing photosensitizers have been suggested by researchers” or “curcumin can be explored as a photosensitizer,” may be misleading. These statements could give the impression that the concept is entirely novel, even though the authors discuss related previous studies later in the discussion. I recommend rephrasing these parts of the introduction for clarity.
• The manuscript does not explain the use of the plant Bidens pilosa. It would be helpful to clarify why this plant was chosen. While I recognize that the authors have previously published a review on the benefits of biogenic silver nanoparticles, it is important to provide some background on this topic within the manuscript itself, rather than relying on readers’ familiarity with prior publications.

Materials/Methods:
• There is some inconsistency in the level of detail provided across different sections. For instance, while some parts offer background information on the methodology (e.g., zeta potential), others only mention the equipment used (e.g., UV-Vis). It would be beneficial to standardize the level of detail throughout.
• Regarding the statistical analysis, it is unclear whether the authors tested for the normal distribution of data points and if the appropriate statistical methods were subsequently applied. This should be clarified to ensure the validity of the analysis.
• The manuscript does not address the stability of the produced nanoparticles. Were the nanoparticles used immediately after fabrication, or were they stored? If storage occurred, did the authors conduct stability studies, particularly looking at agglomeration and the stability of the conjugates over time?
• Concerning immunofluorescence staining, it is important to confirm whether appropriate controls were performed to rule out nonspecific staining. For example, were secondary antibody-only controls used to exclude the possibility of nonspecific binding?
• Since cancer cell lines are prone to dedifferentiation, affecting the sensitivity in toxicity studies, more information about the age of the cells might be of interest to the reader: which passages were used in the experiments?
• In light of the RSC’s strict data availability guidelines, the current statement—"The authors confirm that the data supporting the results are available in this article. The datasets obtained and analyzed during the current study can be available from the corresponding author upon reasonable request"—is not sufficient for the journal. I recommend that the authors deposit the datasets in one of the recommended repositories or, at least include the raw data points in Figures 6 and 8. Additionally, I suggest uploading higher-quality versions of all figures, particularly Figure 2.
• I also encourage the authors to provide detailed information on the sample size and the number of repetitions for each experiment in the Methods section. The current mention of “n=3” in the figure descriptions (Figures 6 and 8) should be supplemented with this information to ensure transparency and reproducibility.

Results:
• The presentation of the FTIR results is notably more detailed compared to other sections, and it includes passages that verge on discussion rather than a straightforward presentation of the results. It would be beneficial to streamline this section to focus on the factual data, reserving interpretative commentary for the discussion section.
• Some images/figures appear to be stretched (Figure 2 A-D) or compressed in width (Figure 3 A-C). Ensuring consistent aspect ratios would improve their presentation.
• Overall, the figures would benefit from higher resolution and uniform lettering, particularly in font size, to enhance clarity and readability.
• Figure 2: The description for panels (E) and (F) is missing from the figure legend. Please provide this information for completeness.
• Figure 3: I recommend using the same magnification for all HRTEM micrographs. Currently, panel (A) has a scale bar of 200 nm, the scale bar in (B) is illegible, and (C) has a scale bar of 100 nm. Consistent magnification and clearly visible scale bars would aid in better comparison between images.
• In the Results section "3.5 Immunofluorescence," the label should be CD44-FITC, not CD44-PE. Please correct this to reflect the accurate staining used.
• Figure 6: The legends on the right side of the figure are cut off in some places (e.g., ‘alone’ in (F) is missing the ‘-ne’). Adjusting the layout to ensure all text is fully visible would improve the figure’s legibility.
• Figure 8: It is unclear why “experimental groups” is written between the graphs. It would be helpful to clarify which sub-figure this label pertains to or adjust the placement to avoid confusion.

Discussion:
• The authors frequently mention "targeting" lung cancer cells but do not describe an active targeting mechanism. Whether the uptake of nanoparticles by tumor cells is intended to rely on passive uptake or if an active targeting mechanism is being considered should be clarified and discussed in more detail.
• While the authors demonstrate differentiated toxicity in lung cancer cells (A549) and lung cancer stem cells (CD44+/CD133+ A549), they do not address toxicity in non-cancerous lung epithelial cells. This is particularly important given the potential for passive uptake and the absence of a defined targeting mechanism. The implications for healthy tissue should be discussed to provide a more comprehensive assessment of the therapeutic approach.
• Have the authors investigated the uptake of nanoparticles into the cells? It is unclear whether nanoparticles that are not taken up are removed after the incubation period or before irradiation with the light source, as the methods section suggests they are not. Clarification on this point would be beneficial.
• On page 15, when discussing toxicity, the authors state that "the toxicity of the drug was noticed to be dose-independent, as an increased concentration of Cum-PEG-BpAgNPs led to higher toxicity in both the A549 and A549 SC." This should be corrected to "dose-dependent" to accurately describe the observed relationship between concentration and toxicity.

Language:
• The abstract would benefit from minimizing the use of abbreviations, such as MTT, LDH, and ATP, to enhance readability and accessibility for a broader audience.
• The manuscript could significantly improve with more thorough language editing. There are noticeable issues, such as sentences missing verbs (e.g., the last sentence of the abstract), word repetitions ("assay assay" in the abstract, "mean mean" in the description of Figure 8), and improper formatting of numbers (e.g., "104" should be "10^4" and "CO2" should be written with a subscript "2"). Additionally, a large section of the discussion (almost a full column) is repeated on page 15 when discussing the cytotoxicity data.
• In some instances, the authors have omitted references, leaving placeholders like "(refers)" on page 8 and "(references)" on page 16. These need to be addressed to ensure the manuscript is properly cited.
• There are several errors in the numbering of subheadings in the Results section: subheadings 3.1 and 3.5 appear twice, while 3.7 is missing, and the numbering for 3.13 is in italics. Consistent and accurate numbering is essential for clarity.
• Abbreviations are not used consistently throughout the manuscript. For example, "photosensitizer" is introduced as an abbreviation but is sometimes written out later in the text. Similarly, there is inconsistency in abbreviations for time, with variations like minutes/mins and hours/hr/h. Ensuring uniformity in abbreviation usage would improve the manuscript's clarity.
• The last sentence of the introduction—"This work is part of the author’s Thesis to be submitted to the Department of Biomedical Sciences, University of Johannesburg, South Africa"—would be more appropriately placed in the Acknowledgment section.
• The nomenclature of the nanoparticles in the study is unnecessarily confusing. They are referred to by several different names throughout the manuscript, such as:
o "green silver nanoparticles curcumin conjugate" (title)
o "photoactive curcumin nano-silver functionalized polymer" (abstract)
o "biologically AgNPs" (introduction)
o "curcumin-green nanometallic silver conjugate" (introduction)
o "nano-metallic photoactive bioconjugate molecule" (discussion)
o "green synthesized nanosilver" (discussion)
o "anticancer molecule"
This inconsistency in terminology can be confusing for readers. Additionally, the use of "Bp" in the abbreviation of the different nanoparticles is inconsistent, particularly in the discussion and in Figures 4 and 9. Standardizing the terminology and abbreviations throughout the manuscript would greatly enhance readability and comprehension.

The authors have made significant efforts to address the reviewers' suggestions, and their revisions have notably improved the manuscript. The changes enhance the clarity and presentation of the research, making it more suitable for publication in RSC Advances.