Phosphatidylinositol Stabilizes Fluid-Phase Liposomes Loaded with a Melphalan Lipophilic Prodrug

Round 1

Reviewer 1 Report

In this manuscript entitled “Phosphatidylinositol Stabilizes Fluid-Phase Liposomes Loaded with a Melphalan Lipophilic Prodrug”, the authors describe the interactions of MlphDG-liposomes with plasma albumin using FTIR and the release of MlphDG from the liposomes using AF4. In previous study, the authors confirmed the antitumor effect of MlphDG liposomes in mouse models.  In this work, they explained MlphDG stability and releasability from liposome well, these data support the antitumor effect of MlphDG liposome in mouse  models.  As the authors themselves bring up in line 536, it seems that data on the reconversion of MlphDG prodrug to the parent drug (melphalan) will be needed in the future.

1. Line 268, 369, 480, 484, 487, 494, 496,  figure number in the text are written by a large letter (ex. Figure 2C), but in figure, they are written by a small letter (ex. (c)).
2. In Figure 6, I don’t know where grey line is.
3. Line 453, Please fix typos (routs to routes).
4. Line 460, The section number would be 3.3, not 3.4.
5. Line 507, Please change c, d to bold on “c and d respectively”.

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript entitled "Phosphatidylinositol Stabilizes Fluid-Phase Liposomes Loaded with a Melphalan Lipophilic Prodrug" is well written.
Nevertheless, there are some points that should be adressed before being published in Pharmaceutics.
Line 34: However,in circulation, liposomes like any other nanoparticles are instantly covered with a complex layer of proteins and lipoproteins.
The term nanoparticle should be removed. Rephrase this sentence, please.
Line 113: Liposomes (large unilamellar vesicles) were prepared as described earlier [22, 23]. Please, include reference: J. DRUG DEL. SCI. TECH., 19 (1) 51-59 2009
Line 130: Samples were measured 10 times over 30 s at 20 °C. Average particle size and width of the particle size distribution were determined by cumulant method. Why haven't the authors used 25ºC?
It is necessary to add the standard deviations in the PDI and zeta potential values. In addition, discuss the effect of the incorporation of the different components on the size of the liposomes obtained.
   

 

Author Response

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Author Response File: Author Response.docx

Reviewer 3 Report

Authors proposed a paper entitled “PI stabilizes FP Liposomes loaded with a Melphalan Lipophilic prodrug” for the publication in Pharmaceutics.

 

The author has a good scientific soundness and the English is used in a proper manner.

The major issues are that there is no information about drug encapsulation efficiency into liposomes. This is a very important information that authors need to give the reader. Indeed, if the amount of lost drug is high in terms of percentage, this means that the cost of the process in terms of raw materials is high.

 

Another major issue is the not consistency of the particle size distributions of the liposomes produced in this manner and shown in this paper. I find difficult to believe that the mean size of the produced liposomes is around 110 nm and the standard deviations is only 1-2 nm. Look at PSDs of Figure 8a. How can you say that the standard deviation is 1 or 2 nm ?

 

The third issue is that the used process for the production of liposomes is the conventional thin layer hydration, followed by freeze drying and extrusion. A lot of cycles of post-processing steps are required, but no information is given on the loss of drug and lipids during all these steps. Which is the yield in terms of lipids? If the method is thin layer hydration, large unilamellar vesicles are produced, as stated in line 113. However, post-processing steps are selecting only liposomes of around 100 nm, extracting them from the overall produced population. Therefore, what is the yield in terms of lipids?

 

The fourth issue is that a SEM or TEM image of the produced liposomes is not present. How can I be sure that your liposomes are spherical in shape?

 

I have some other minor issues listed here:

Line 13. Please do not use personal form “we developed…”

Line 35. Explain what precisely happens when liposomes are covered.

Line 36. Nanoparticulate means nanosized?

Line 41. The state of the art about PEGylation methods and liposomes PEGylated could be enlarged by reading this three papers:

Milla, P., Dosio, F., & Cattel, L. (2012). PEGylation of proteins and liposomes: a powerful and flexible strategy to improve the drug delivery. Current drug metabolism, 13(1), 105-119.

Trucillo, P., & Reverchon, E. (2021). Production of PEG-coated liposomes using a continuous supercritical assisted process. The Journal of Supercritical Fluids, 167, 105048.

Naik, S., Patel, D., Surti, N., & Misra, A. (2010). Preparation of PEGylated liposomes of docetaxel using supercritical fluid technology. The Journal of Supercritical Fluids, 54(1), 110-119.

Line 65. A double space is present in this line.

Line 91. As Line 13.

Lines 100-102. These last three lines are for Results sections. In this last part of the introduction, only the aims should be.

Line 133. Why you measured zeta potential only on the selected nanosized liposomes?

Line 285 and 303. These sub-paragraphs numbered as 1 and 2 should be numbered following the other sub-paragraphs numeration.

Line 465. “due to their…” instead of “due their”

Future perspectives section is missing in the conclusions or next to the conclusions section.

Abbreviation List is highly suggested.

Author Response

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Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

Authors provided a new version of the manuscript.

As I said, the manuscript has a quite good scientific soundness.

The paper has slightly improved, but there are still some issues that, in my opinion, need to be addressed.

The authors said that the drug delivery systems are covered by protein and lipoprotein during circulation. however, then, liposomes should fuse with cell membrane. therefore, it is also important to say that liposomes are characterized by an external barrier that is similar to cell barrier. this is why liposomes are biocompatible systems since they can be fused with cells and release their content directly inside. therefore, these proteins covering the drug carriers should be degraded before fusing with target cells or tissues. maybe authors can add this concept.

Table 1. “ml” should be “mL”

I am not at all convinced about your explanation on standard deviations reported on the mean liposome sizes in table 2. I repeat my comment, table 2 should be supported by particle size distribution diagrams.

Lipid yield has not been included in the manuscript text.

I suggest adding a sketch of the process employed in this work.

I suggest using impersonal forms in the manuscript text. Examples are: Line 19. “we explored”. Line 57 “we developed”. Line 85 “we explored”. Line 112. “we employed”. Line 164 “we acquired”. Line 321 “ we performed” Line 411 “we calculated” Line 418 “we did not get” Line 420. “we used” Line 536 “we have no data” Line 558 “we can assume” Line 566 “we have developed”.

Line 568. define the “sufficient amount”

Authors provided this answer, but they did not include it or part of it into the manuscript text: “Response 8: We thank the reviewer for the interesting articles to expand our horizons. While the possibility to anchor PEG-lipid in the bilayer depends on the lipid composition (PEG-lipid leaves fluid-phase bilayers and forms micelles), physical adsorption of PEG on liposome surface is less studied and could indeed improve pharmacokinetics of the formulation. However, to the best of our knowledge, the data on the behavior and stability of such liposomes in physiological media is scarce yet”

Thank you.

Author Response

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Author Response File: Author Response.pdf

Round 3

Reviewer 3 Report

Authors provided a significantly improved version of their paper. After the third revised version, I can say that this paper deserves to be published in the present form.

Best regards