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The authors have addressed all the comments accordingly.
[# PeerJ Staff Note - this decision was reviewed and approved by Konstantinos Kormas, a PeerJ Section Editor covering this Section #]
Thank you for revising the manuscript based on the reviewers' comments. However, further refinement is needed, particularly in terms of language and clarity. We recommend reviewing the English for improved readability and clear communication of your findings. Please revise the manuscript according to Reviewer #1 recommendations.
The authors addressed satisfactorily the scientific issues, and minor alterations are necessary, as specified in points 1 to 3 below. However, I wonder why the group has other papers in the field that have an acceptable English language, and in this one, the grammar and scientific style seem neglected. In some instances, the comprehension of the work is hindered by the incorrect usage of terms and violations of basic grammatical rules. I have suggested improvements from item 4 onwards. Note: These are just a few examples of how the text could be enhanced, but all work should undergo professional editing to correct English and scientific style. This measure is essential to take the work to another quality level.
1. Line 54: Remove the term “parasite”. Acanthamoeba is not a stricto sensu parasite. (Suggestion: “Acanthamoeba spp. is a waterborne, opportunistic protozoan commonly that can cause amebic keratitis and granulomatous amebic encephalitis.”)
2. Lines 365-369: The description is incorrect or misinterpreted. Figures 2C and 2D do not indicate that the "survival rate of Vero and HaCaT cells was lower than 80% when treated with those NPs at lower concentrations of 0.008-0.25 mg/mL". In addition, only Kre loaded-PEG-b-PCL showed acceptable cytotoxicity (VERO and HatCaT cell viability >80%) at 0.250 mg/mL, the best MIC for A. triangularis, as shown in Table 2.
3. The authors use the term "normal cells" to refer to the cell lines used in the cytotoxicity assays (e.g., Table 2 title. Figure 2 caption, lines 66, 80, 83, 141, 359, 422, 430, 433, 434, 435, lines 98, 145). I suggest using their specific designations (VERO, HatCaT) or replacing the term "normal" with "mammalian cells" or "mammalian cell lines" if referring to them generically.
4. Lines 56-59: In the abstract, study proposal should be rewritten to be more assertive (Suggestion: “In this study, we characterized niosomes, PEG-b-PCL, and their combination loaded with Knema retusa extract (KRe) and tested the effect of these nanoparticles (NPs) on Acanthamoeba triangularis stages”)
5. Line 60-63: The initial part in the Methods of the Abstract seems to be a study aim. I recommend starting with the description of synthesis and characterization of NPs, which is a substantial part of the study. (Suggestion: “ KRe-loaded PEG-b-PCL, KRe-loaded niosome, and KRe-loaded PEG-b-PCL plus niosome were synthesized and characterized regarding particle size and charge, yield, encapsulation efficiency, and drug loading content. The effect of these KRe-loaded NPs on trophozoite and cystic forms of A. triangularis was assessed through assays of minimal inhibitory concentration (MIC), using trypan blue exclusion to determine the viability.”)
6. Lines 68-80: Results in abstract remains confusing and wordy. It should be rewritten to adopt a scientific, polish English style. Here a suggestion for improvement: “KRe-loaded niosome presented the higher yield (87.93 ± 6.03%) at 286 nm UV-Vis detection and exhibited a larger size (199.3 ± 29.98 nm) and drug loading concentration (DLC; 19.63 ±1.84 %) than KRe-loaded PEG-b-PCL (45.2 ± 10.07 nm and 2.15 ± 0.25%). Encapsulation efficiency (EE; %) of KRe-loaded niosome was 63.67 ± 4.04, significantly lower (than the combination PEG-b-PCL plus niosome (79.67 ± 2.08). However, the particle charge of these NPs was similar (-28.2 ± 3.68 mV and -28.5 ± 4.88, respectively). Additionally, KRe-loaded niosome and KRe-loaded PEG-b-PCL plus niosome showed the lower MIC in 24h (0.25 mg/mL), inhibiting 90-100% of Acanthamoeba trophozoites, an effect that persisted until 72h. KRe-loaded niosome affected the adherent ability around 40-60% at 1/4-1/2 MIC (0.125-0.25 mg/mL) and removal of Acanthamoeba adhesion on the surface by about 90% at 2MIC (0.5 mg/mL). Cell viability of VERO and HaCaT cells treated with 0.125 mg/mL of KRe-loaded niosome and KRe-loaded PEG-b-PCL plus niosome was superior to 80%."
7. Line 338-341: Results from cysts assays should be anticipated in the section (insert after line 323). A suggestion for description improvement of this part, to avoid redundancy: “All the tested KRe-loaded NPs have a weak inhibitory effect (<90%) on cysts independently of the Acanthamoeba species, indicating the MIC values on these resistance forms were higher than 1 mg/mL (Table 2).”
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I have reviewed all the suggestions I provided to the authors and am pleased with their responses. I continue to find the text highly relevant for advancing effective solutions against Acanthamoeba trophozoites and cysts. Thank you
The review process has been completed and the letter includes two thorough reviews from qualified referees at the bottom.
1. Revise the manuscript and ensure that all the concerns raised by the reviewers are addressed with utmost precision. Make sure that all the necessary changes are incorporated in the manuscript before resubmitting it.
2. Work on improving the language of the manuscript to make it more clear and comprehensible to readers. Make sure that your writing style is consistent throughout the manuscript.
Chimplee et al study describes the production and characterization of niosomes and PEG-b-PCL [poly(ethylene glycol)-block-poly(•-caprolactone)] loaded with Knema retusa extract, a native plant from Malaysia, and the effect of these nanoparticles, alone and in combination, on Acanthamoeba trophozoites. The writing needs an overall revision in English language regarding grammar and style. The text sometimes seems redundant and has incorrect grammar constructions (e.g.line 234: The anti-adhesion property of KRe-loaded niosomes found that the nanoparticles could significantly reduce the adhesive ability of trophozoites).
I strongly recommend the assistance of a colleague who is proficient in English or from a professional editing service. The authors provided a good set of references concerning nanoparticles synthesis and characterization. However, literature on Acanthamoeba should be improved to reiterate the significance of the study in the context of anti-amoebic therapy. Raw data on MIC determination is lacking.
The authors described the preparation of the plant extracts, synthesis of nanoparticles, loaded-particles characterization, and assays to determine the effect of loaded-nanoparticles on Acanthamoeba trophozoites´ viability and adhesion. The study idea is original and relies on previous evidence of antibacterial and anti-parasitic effect of Knema. However, the authors insufficiently explored the difficulties in treating Acanthamoeba infections and the need for new therapeutic alternatives.
I have concerns about the anti-amoebic assays performed in triplicate (n=3) and in a single replication. Besides, would the test t Student be appropriated without a normality evaluation? Other point of concern is the lack of discussion on safety issues. Corneal cytotoxicity of available anti-amoebic agents is one of the drawbacks on Acanthamoeba therapy, and authors did not cover in the paper the potential cytotoxicity of these nanoparticles.
Specific issues:
1. Background: The authors justify the study mentioning that their comparative approach is unprecedented (“it is never been known”), which sounds superficial in terms of motivation. I suggest exploring the idea of therapeutic alternatives for Acanthamoeba infections is an issue of health concern.
2. Introduction: Information on Acanthamoeba in the first and the last paragraph should be joined, to avoid the split of the topics. I recommend including more detailed information on the drawbacks of anti-Acanthamoeba therapy, which would better justify the study.
3. Introduction: line 115: Acanthamoeba infections are not highly prevalent!
4. Mat& Met – Line 193: insert the amount of distilled water used for these reagents quantity in PYG preparation. Lines 195-2011: The text is redundant and do not meet an adequate, objective scientific style. The assays related to time-dependent analysis (results from Table 3) are not described. The rationale behind using ½ MIC, ¼ MIC … 1MIC, 2MIC in the adhesion tests is not clear. This is not discussed either.
5. Do the authors think a single assay in triplicate without repetition provides a robust result? Is the chosen statistical method adequate?
6. Raw data from MIC determination must be included.
7. Figure 1 is small, the layout of Fig 1A, B, C, D and E must be improved. Did 1D and IE represent results from DLS? It must be included in legend.
8. The authors barely cover Acanthamoeba in Discussion, the issues related to drawbacks in therapy should be better explored. The need of evaluate cytotoxicity for human cells and the effect of the nanoparticles in resistant cystic stages should also be mentioned. Is there any previous study on safety of Knema extract? If so, the author should discuss them.
9. In conclusion, I believe the study is of interest, but the authors should reformulate the manuscript regarding to language and discuss the Acanthamoeba topic in more depth.
Excellent and important article on the effectiveness of a plant extract through the use of nanoparticles against trophozoites of Acanthamoeba triangularis.
In the Introduction section
1) I suggest adding more references about Acanthamoeba characteristics in addition to the one mentioned (Marciano-Cabral & Cabral, 2003) (lines 78-83).
2) Are there toxicity data for Knema retusa? If so, what would they be? Are there cytotoxicity data for Knema retusa? For which cells would it be toxic? (lines 84-94).
3) In lines 95 -115, the text talks about niosomes and micelles. It would be interesting to add a figure about these nanoparticles. I believe it would be enriching for the paper. Perhaps a Graphical abstract would be necessary.
In the Material and methods section:
Acanthamoeba culture and anti-Acanthamoeba activity (line 187).
1) line 189. Why was only one strain used in the experiment? Why was an ATCC strain of clinical origin not used to compare with A. triangularis? And why only this isolate and not others such as A. polyphaga or A. castellanii?
2) Line 201: in relation to chlorhexidine, what concentration was used? It must be described in the text.
3) Line 203: what percentage of trypan blue was used?
Anti-adhesion activity (line 215)
1) Line 223: what concentration of chlorhexidine was used?
In the Results section:
Effect of KRe-loaded nanoparticles on trophozoite viability (line 263)
1) Line 264: I think table 2 is unnecessary. Some data from table 2 can be inserted into table 3.
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As it is an original and unpublished work:
1) Although the article deals with and focuses on the comparison of which method would be most effective for the anti-adhesion and inhibition of Acanthamoeba triangularis, I would like to know what would be the target of using the niosome loaded with the extract used? Would it be used for keratitis, for example? For formulating eye drops? Do you already have any ideas?
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