DOPC Liposomal Formulation of Antimicrobial Peptide LL17-32 with Reduced Cytotoxicity: A Promising Carrier Against Porphyromonas gingivalis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Comments

1. 
   The emergence of antibiotic resistance in periodontal bacteria is a significant issue in dental health that is addressed in this publication. The study investigates a novel approach to improve peptide stability, lower cytotoxicity, and boost antibacterial efficiency by concentrating on liposomal delivery of the antimicrobial peptide LL17-32. One noteworthy strength is the comparison of neutral dioleoyl-phosphatidylcholine (DOPC) liposomes with negatively charged soybean lecithin (SL) liposomes, which emphasizes how lipid charge affects antibacterial activity. The subject is current and highly pertinent to oral biology and pharmaceutics.
2. The structural characteristics of LL17-32, a fragment generated from LL-37, and earlier data supporting its antibacterial activity provide strong justification for its selection. Comparing LL17-32 in further detail with other synthetic or cathelicidin-derived antimicrobial peptides that have been tested against oral infections would improve the manuscript, nevertheless. A contextualization of this kind would enable the reader to recognize the relative benefits of LL17-32 with regard to stability, selectivity, and potential therapeutic benefits.
3. 3. The manuscript might benefit from more mechanistic confirmation, such as fluorescence microscopy or peptide localization experiments, to ascertain whether the peptide is supplied at the bacterial surface rather than buried within the liposomal bilayer.
4. the methods for Transmission electron microscopy (TEM) imaging, In vitro RTF release study, Bacteria-killing activity against Porphyromonas gingivalis , FITC labelling of Porphyromonas gingivalis, Treatment of LL17-32 loaded liposomes with P. gingivalis, Cell viability assay, Cytotoxicity assay needs references.
5. In vitro release and therapeutic implications
   It is noteworthy that LL17-32 remained unreleased from both liposomal formulations for a period of several hours. Although this stability is beneficial in safeguarding the peptide payload, it also implies that the therapeutic efficacy in vivo may be restricted by the absence of sustained peptide release. The authors should further elaborate on this point and evaluate whether the formulation could be modified to achieve a balance between retention and controlled release, a feature that is frequently desired for clinical applications.

6 Antibacterial testing
It is comprehensible that Porphyromonas gingivalis is the only priority, given its status as a critical periodontal pathogen. Nevertheless, the translational significance of the research would be further elevated if the activity were also evaluated against other representative members of the oral microbiota, such as Streptococcus spp. or Fusobacterium nucleatum. This data would assist in determining whether the approach is extremely pathogen-specific or has a broader utility.

7. Cytotoxicity evaluation
   The decreased cytotoxicity toward gingival keratinocytes is a positive development. Nevertheless, a more comprehensive biosafety profile could be achieved by investigating immune cell viability or hemolysis test.
8. Some methodological details (e.g., TEM imaging difficulties with SL liposomes) could be clarified further for reproducibility.
9. Statistical rigor: Statistical analyses are mentioned but could be described in more depth (e.g., number of replicates, specific tests).
10. Some figures (e.g., TEM images, flow cytometry results) could benefit from improved clarity and annotation to guide interpretation.
11. discussion: Although the discussion is thorough, there are some speculative moments (e.g., peptide inner vs. outer membrane distribution). The authors should be very explicit about the difference between hypotheses and conclusions based on data.
12. conclusion and Future directions: the conclusion should be brief. The conclusion may benefit from a specific list of future actions. These could involve assessing effectiveness in co-cultivation with host cells, testing in biofilm models, or moving on to animal models of periodontitis. These prospective viewpoints might aid in framing the data' translational significance.

 

Author Response

Please see attached file with the reply to the reviewers´ comments. 

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This article is almost identical to the ones published by the authors last year. The authors have even increased the level of journal from the previously published article with the same work only performing the control that should have b een included in the first paper. 

Author Response

Please see attached letter with reply to reviewers.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript entitled “DOPC Liposomal Formulation of Antimicrobial Peptide 2 LL17-32 with Reduced Cytotoxicity: A Promising Carrier against 3 Porphyromonas gingivalis” explored two types of liposomes made from different lipids to deliver the antimicrobial peptide LL17-32 against Porphyromonas gingivalis, a dental pathogen. Both liposomes were stable and had small, round particles. LL17-32 showed high association efficiency with both liposomal membranes, with no detectable LL17-32 in vitro release. In biological assays, peptide-loaded DOPC liposomes exhibited dose-dependent bactericidal activity against P. gingivalis, whereas SL liposomes significantly attenuated the bactericidal effect of LL17-32. Both formulations displayed reduced cytotoxicity toward human gingival keratinocyte (TIGK) cells versus the free peptide. The manuscript is well written; however, there are some issues that should be addressed to improve the quality of the submitted manuscript.

1. The materials section has quite a few grammatical mistakes, like using “was purchased” when it should be “were purchased” for more than one item. Few sentences are often too long and hard to follow, punctuation is sometimes confusing, and different materials are grouped unclearly.
2. In the methods section 2.2.1, the liposome preparation method is unclear because it does not specify whether soybean lecithin (SL) or DOPC was used as the phospholipid in each formulation. It is explained in the result, but it should also be clear in the method part.
3. In section 2.2.4, there seems to be some inconsistency between the terms encapsulation efficiency and association efficiency.
4. In section 2.2.6, the timing is described as up to 4 h/18 h is a bit confusing; it's not clear which time refers to which medium, although this is explained in the result section, but it should also be clarified in the method.
5. In the results and discussion section 3.1, there is an inconsistency in the terminology, as both SL liposome and LS liposomes are used. There should be the use of a single term.
6. Section 3.5 shows a lack of quantitative data; this section mentions a progressive reduction in size and a minimal reduction, but exact values or percentage changes are not provided.
7. Any particular reasons authors would like to mention for the different scale used in TEM image of loaded SL (Figure 5).
8. Did the authors try to characterize the formulations using DSC, TGA, and PXRD to confirm amorphization?

Minor issues

1. The font size of the reference is inconsistent with the main text. Please ensure uniform font size, font style, and space between lines throughout the manuscript for better readability.
2. A few sections of results, such as section 3.5, lack sufficient quantitative data. So, the authors are encouraged to include relevant numerical values throughout the manuscript wherever applicable to enhance clarity.

 

 

Author Response

Please see attached letter with replies to reviewers.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

Jinyang Han et al.'s in their study "DOPC Liposomal Formulation of Antimicrobial Peptide LL17-32 with Reduced Cytotoxicity: A Promising Carrier against Porphyromonas gingivalis" examined the antimicrobial peptide (AMP) LL17-32's ability to bind and be delivered by two liposomal formulations against the dental bacterial pathogen Porphyromonas gingivalis. Liposomes were created and evaluated using hydrodynamic size distribution, ζ-potential, morphology, membrane fluidity, peptide association efficiency, stability, and peptide release in vitro under physiological conditions. They were made of either negatively charged soy lecithin (SL) or neutrally charged dioleoyl-phosphatidylcholine (DOPC) phospholipids.

Both liposomal formulations showed good colloidal stability and spherical morphology with hydrodynamic diameters less than ~170 nm. With no discernible LL17-32 in vitro release, LL17-32 demonstrated a high association efficiency with both liposomal membranes. Peptide-loaded DOPC liposomes demonstrated dose-dependent bactericidal activity against P. gingivalis in biological assays, while SL liposomes markedly reduced the bactericidal effect of LL17-32. Both formulations displayed reduced cytotoxicity toward human gingival keratinocyte (TIGK) cells versus free peptide.

The study, according to the authors, has implications for comprehending the factors taken into account during the early phases of liposome development for the delivery of AMPs in antibacterial applications. There is yet a lot of effort to be done; advancement calls for a better comprehension of the behavior of peptide-associated nanoparticles in intricate physiological settings. Since peptides are extremely dynamic molecules, it is necessary to thoroughly assess their stability and interactions with proteins, lipid membranes, and other biological components under a variety of circumstances.

 

The manuscript has a highly original idea and was well-written. However, before any publication, a few crucial issues must be resolved.

1. The authors discuss their work on "Chitosan-coated liposomal systems for delivery of antibacterial peptide LL17-32 to Porphyromonas gingivalis," which was published in Heliyon 2024, in the paragraph that begins with line 90. Comparing the benefits and drawbacks of the two liposomal formulations in the submitted study with those of chitosan will yield greater results (Heliyon 24).
2. Comparing the findings of this work to those of other nanoparticle systems, such as inorganic/metallic, polymeric, lipid-based, and dendrimer nanostructures, which were published in a review article BME Front. 2025 Mar 4;6:0104 doi: 10.34133/bmef.0104, can also be beneficial.

 

Author Response

Please see attached document with replies to reviewers´comments.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have replied to my comments

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have addressed all the raised queries so the manuscript can be accepted in its present form.