Review Reports - Liposomes as “Trojan Horses” in Cancer Treatment: Design, Development, and Clinical Applications

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have provided a comprehensive evaluation of the physicochemical principles guiding liposome design for anticancer drug delivery. The paper reviews how advancements in lipid composition, particle engineering, PEGylation, and functionalization strategies have enhanced liposome stability, targeting, and controlled release. Overall, it highlights the evolution from conventional to multifunctional liposomal systems and their translational potential in cancer therapeutics.

Comments:

The authors have reported commercialized liposomal formulations in a very comprehensive table; however, they have not cited recent liposomal formulations that are under clinical trials currently. This could be included.
Another important aspect missing in this review is including a discussion about theranostic liposomes for tumor targeting. The authors are advised strongly and encouraged to include this to enhance the quality of the review.
The authors must include a higher resolution figure for Figures 1, 4 and 5 since these figures are blurred.

Author Response

Comments:

The authors have reported commercialized liposomal formulations in a very comprehensive table; however, they have not cited recent liposomal formulations that are under clinical trials currently. This could be included.

We thank the reviewer for the interesting suggestion. Two paragraphs briefly mentioning some ongoing clinical trials were included in lines 721 to 735.

Another important aspect missing in this review is including a discussion about theranostic liposomes for tumor targeting. The authors are advised strongly and encouraged to include this to enhance the quality of the review.

We thank the reviewer for this helpful suggestion. A new subsection has been added discussing theranostic liposomes for tumor targeting (beginning “In parallel, theranostic liposomes have emerged…”). This section highlights recent advances in multifunctional liposomal systems integrating imaging and therapy, including photothermal, MRI-guided, and hybrid nanohybrids, as well as their translational challenges.

The authors must include a higher resolution figure for Figures 1, 4 and 5 since these figures are blurred.

We have replaced al figures with higher resolution versions to ensure that all details are clearly visible.

Reviewer 2 Report

Comments and Suggestions for Authors

This review paper describes the preparation, drug loading and clinical prospects of liposomes, particularly, in anticancer therapy. There are some important issues that should be resolved.

Lines 24-25: “Critical parameters such as…” As the authors later mention the phase transitions of the lipids in the liposomes, such words as “critical parameters” should be avoided in order to not confuse the reader. Otherwise, the reader might be puzzled whether the authors describe the critical parameters of the phase transition.

Lines 46-53: The authors wish to show the history and the context of their review. If so, they should clearly explain, what the novel concepts and findings do they describe, and how their review differs from the numerous recent reviews. Just a very brief search provides the following reviews on liposomes-assisted drug delivery: https://doi.org/10.3390/ph14090835, https://doi.org/10.1208/s12249-021-02179-4, https://doi.org/10.1155/2021/3041969, https://doi.org/10.1021/acsptsci.1c00066, https://doi.org/10.3390/pharmaceutics14102195.

Line 54: “Phosphatidylcholines are the main components of the biological lipidic bilayer” This statement is too broad. There are many different lipid bilayers in biology. Probably, the authors implied lipid bilayers of eukaryotes?

Lines 54-55 and Fig.1: The authors wish to introduce chemical structure to the reader (who might be new to the field). If so, they should make it more systematic.

The possible approach: first, show the structure of the phosphatidic acid and some its derivatives (PC, PE, PS, etc.). Second, make the structures consistent (regarding the dissociation of the phosphate, e.g., in the typical physiological conditions).

Lines 58-59: “cooperative orientation of the dipoles in the bilayer can infer an overall net charge to the membrane” I have to disagree. No matter how you arrange the dipoles, the overall charge will be zero. Maybe, the authors wanted to describe electric potential instead of the charge?

Line 66: “(number of methyl groups)” I must disagree. No matter the length of the straight-chain fatty acid, it has only one methyl group: at the very end of the chain. Maybe, “methylene groups”?

Lines 68-69: The authors can be more specific and describe between what phases the transition occurs (see, e.g., https://doi.org/10.1016/j.molliq.2022.119874).

Fig.1a: Figure does not match the caption. This is not a phosphatidylcholine.

Lines 104-105: “as ethanol evaporates, form stable closed liposomal structures” I doubt that the ethanol might simply evaporate after dilution in water. Please, check this statement.

Lines 263-283: The authors should be more specific and show the schemes of the hydrolysis and oxidation chemical reactions.

Line 312: “tilts the lipidic acyl chains towards the normal bilayer” Please, check and rephrase.

Figure 5. The font size should be larger. The text is barely readable.

The paper would benefit from the added concise conclusion section.

Author Response

This review paper describes the preparation, drug loading and clinical prospects of liposomes, particularly, in anticancer therapy. There are some important issues that should be resolved.

Thanks for pointing out this possible confusion. We can change the paragraph accordantly.

Lines 46-53: The authors wish to show the history and the context of their review. If so, they should clearly explain, what the novel concepts and findings do they describe, and how their review differs from the numerous recent reviews. Just a very brief search provides the following reviews on liposomes-assisted drug delivery: https://doi.org/10.3390/ph14090835, https://doi.org/10.1208/s12249-021-02179-4, https://doi.org/10.1155/2021/3041969, https://doi.org/10.1021/acsptsci.1c00066, https://doi.org/10.3390/pharmaceutics14102195.

We have extended the Abstract including what, we believe, are the unique features of this review and what differ from other review in liposomes.

We thank the reviewer for the observation, and we already made the correction in line 54.

Lines 54-55 and Fig.1: The authors wish to introduce chemical structure to the reader (who might be new to the field). If so, they should make it more systematic.

We thank the reviewer for this valuable suggestion. Following the comment, Figure 1 has been completely revised. We have now included representative examples of the chemical structures of each main phospholipid class (PC, PE, PS, and PG). In addition, for each phospholipid, an example corresponding to those listed in Table 1 has been included. All molecular structures have been carefully checked to ensure consistency under physiological conditions, particularly regarding the phosphate dissociation state.

We thank the reviewer for this observation. We agree that the cooperative orientation of dipoles does not generate a net charge, but rather an electric potential across the membrane. The sentence has been corrected accordingly.

We thank the reviewer for this comment. You are completely right; the correct term is “methylene groups”. The manuscript has been revised accordingly at line 66.

Lines 68-69: The authors can be more specific and describe between what phases the transition occurs (see, e.g., https://doi.org/10.1016/j.molliq.2022.119874).

We appreciate the reviewer suggestion; it certainly helps to improve the standard of the manuscript. In the revised text, we have specified that the transition described corresponds to the passage from the gel phase (L?) to the liquid-crystalline or liquid-disordered phase (L? or L?), in some cases mediated by an intermediate rippled phase (P?’), depending on the lipid composition.

As in other mesoscale phenomena, phase transitions in lipid bilayers can be addressed from a macroscopic or microrheological perspective, as done by Sot et al. (https://doi.org/10.1016/j.molliq.2022.119874), or from a microthermodynamic or statistical-mechanical perspective, as discussed in Domínguez-Arca et al. (https://doi.org/10.1016/j.molliq.2022.120230) In the latter approach, the L?–L? transition is explained in terms of the trans–gauche conformational change of the hydrocarbon chains of the lipids. In the ordered (gel) phase, around 90% of the chain segments adopt the trans conformation, while the gauche conformers account for only 8–9%. In the liquid-crystalline phase, this ratio shifts significantly, with the gauche population reaching about 20%, thereby increasing molecular disorder and system entropy.

These conformational changes, verified by calorimetric measurements, explain the energy involved in the transition and directly affect the microviscosity, permeability, and mechanical properties of the bilayer — key factors determining its behavior in drug delivery processes and its adjuvant role in liposomal systems.

Fig.1a: Figure does not match the caption. This is not a phosphatidylcholine.

Thank you very much for your observation. You are absolutely right. The previous figure did not correspond to a phosphatidylcholine. We have now corrected this mistake and replaced it with a fully revised figure. The updated version includes a representative example of each phospholipid mentioned, ensuring that all structures are accurate and consistent with the figure caption and the text.

Lines 104-105: “as ethanol evaporates, form stable closed liposomal structures” I doubt that the ethanol might simply evaporate after dilution in water. Please, check this statement.

We appreciate the reviewer’s insightful comment. We agree that ethanol does not simply evaporate upon dilution in water, and the previous wording could be misleading. The formation of liposomes in the ethanol injection method is not primarily driven by ethanol evaporation but rather by the abrupt change in solvent polarity upon injection of the ethanolic lipid solution into the aqueous phase.

The rapid dilution of ethanol leads to a local supersaturation of lipids because the ethanolic solvent suddenly loses its capacity to solubilize the phospholipids. This causes the lipids to precipitate and spontaneously self-assemble into bilayer fragments, which subsequently reorganize and close to form stable liposomal vesicles.

Accordingly, we have revised the sentence to accurately reflect this mechanism. We thank the reviewer for this valuable observation, which helped us clarify the mechanistic description of liposome formation in this section.

Lines 263-283: The authors should be more specific and show the schemes of the hydrolysis and oxidation chemical reactions.

Thank you very much for this helpful suggestion. We have expanded the explanation of both lipid hydrolysis and peroxidation to provide greater detail and clarity. In addition, we have included a new illustrative figure showing the schemes of each process, which we believe enhances the understanding of these reactions.

Line 312: “tilts the lipidic acyl chains towards the normal bilayer” Please, check and rephrase.

We have checked the sentence and detected some mistake that makes the phase difficult to understand. We have rephrased the sentence.

Figure 5. The font size should be larger. The text is barely readable.

Thank you very much for your comment. We have modified Figure 5 to increase the font size, ensuring that the text is now clear and easily readable for the reader.

The paper would benefit from the added concise conclusion section.

Thanking the reviewer for his suggestion, which allows us to defend the position of the review, a concise conclusion section has been added to the revised manuscript, summarizing the main contributions of the review as follows:

This review consolidates key physicochemical principles underlying the design and optimization of liposomes for anticancer drug delivery. Cancer was selected as a paradigmatic target, not only due to its clinical relevance but also because it illustrates the full potential of liposomal systems—from chemotherapeutic drug delivery to the transport of biomolecules used in vaccination and immunotherapy. Liposomes are ideal candidates owing to their remarkable biocompatibility, derived from their structural mimicry of biological membranes, and their thermodynamic stability under diverse environmental conditions. Furthermore, their capacity for controlled or thermodynamically triggered release—through surfactant-mediated degradation or membrane poration—makes them versatile carriers for molecules with a broad range of solubilities and therapeutic functions.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have improved the paper, and it is suitable for publication now