Cisplatin-Loaded Thermosensitive Liposomes Functionalized with Hyaluronic Acid: Cytotoxicity and In Vivo Acute Toxicity Evaluation

Round 1

Reviewer 1 Report

Cisplatin is a potent antitumor drug used in first-line chemotherapy against several solid tumors, including breast cancer. However, toxicities and drug resistance limit its clinical application. This study evaluated the cytotoxicity of Cisplatin-loaded thermosensitive liposomes functionalized with hyaluronic acid. The question posed by the authors is new; the methods and results are relevant and accurate. The experimental data indicated that this new formulation is a potential candidate for the intravenous therapy of solid tumors. Therefore, I think that this manuscript is worth publication.

Author Response

We thank the reviewer#1 for the positive analysis of our manuscript.

Reviewer 2 Report

1. Abstract must be revised with the important numerical data.

2. Introduction is not in good shape. It must be updated with delivery systems and drug formulation findings with reported literature.

3. What is the rationale of using this formulation design.

4. Include the HPLC method in manuscript.

5. How the composition of only 2 formulation optimized. 

6. Statistical analysis must be added in Fig 1A.

7. Has the author checked the formulation on normal cells.

8. What may be reason for death of rats in CDDP group.

 

 

Author Response

We thank the reviewer for the insightful comments, after which substantial changes were made to the manuscript. 

1. Abstract must be revised with the important numerical data.

Answer:  Thank you for your consideration. The abstract was rewritten and numerical data were included.

2. Introduction is not in good shape. It must be updated with delivery systems and drug formulation findings with reported literature.

Answer: As suggested by the reviewer, the introduction session was rewritten and new references were added in the revised version.

3. What is the rationale of using this formulation design.

Answer: The thermosensitive formulation developed in this study was designed to allow selective CDDP delivery in the tumor region and decrease the serious side effects provoked by the drug. The rationale behind the formulation design is based on: (i) Thermosensitive liposomes are composed of lipids that undergo the transition from the gel phase to the crystalline liquid phase in response to heat, thus allowing drug release specifically in the heated region; ii) hyperthermia is a method used to treat tumors by raising local or regional temperature through controlled heat sources; iii) hyaluronic acid has been utilized in the active targeting of nanoparticles to tumors due to its ability to bind to CD44 receptors overexpressed in some cancer cells. Thus, the association of these features in one system could be a powerful strategy to overcome the CDDP toxicity that limits the successful treatment of cancer patients as well as to improve cancer therapy. The rational basis of our formulation is discussion through the 2^nd-4^th paragraphs in the introduction section.

4. Include the HPLC method in manuscript.

Answer: The description of the HPLC method was added in the new version of the article, as requested by the reviewer. The following text was added:

“The chromatographic apparatus of the HPLC analysis consisted of a quaternary pump (G1311B), an auto-injector (G1329B), and a diode array detector (DAD) (G4212B) connected to the EzChrom integration program (Agilent Technologies, California USA). Separation was performed using a 250 cm × 25 mm with particle of 5 μm Hypersil C18 column (Agilent Technologies, California USA). The mobile phase composed of methanol and water (65:35 v/v) was filtered and degassed by suction-filtration through a nylon membrane. The flow rate was 1.5 mL min-1 in isocratic flow, and the injection volume was 20 μL. The eluate absorbance was monitored at 254 nm.”

5. How the composition of only 2 formulation optimized. 

Answer: The development and optimization of formulation were the subjects of study of the paper published by our group recently (Gomes IP et al. Thermosensitive liposomes containing cisplatin functionalized by hyaluronic acid: preparation and physicochemical characterization. J Nanopart Res., 2022, 24, 30). In this paper, we evaluated the influence of different ratios of lipid composition as well as hyaluronic acid concentrations on the physicochemical parameters (mean diameter, polydispersity index, and zeta potential) in order to optimize the liposomal composition. Furthermore, we performed a detailed analysis of supramolecular organization by the small-angle X-ray scattering (SAXS) technique. The data allow us to choose the adequate HA-coated TSL-CDDP and non-coated TSL-CDDP formulations used in the present work.

6. Statistical analysis must be added in Fig 1A.

Answer: Thank you for your comment. We have included the statistical analysis in Figure 1A as suggested.

 Has the author checked the formulation on normal cells.

Answer: We thank the reviewer for the comment, and unfortunately, we do not have data from normal cells for our formulation. However, although a selectivity index is one of the parameters to determine the safety of a treatment, other approaches may be used for this end. In our specific case, a detailed in vivo toxicity study was performed which allows us to track potential toxicities in different organs and tissues. As demonstrated by our findings, the TSL-CDDP-HA showed much less toxicity to the mice when compared to the free drug. These findings confirm the safety of the new formulation proposed and, we believe, they overcome the need for an in vitro selectivity evaluation.    

8. What may be reason for death of rats in CDDP group.

Answer: Unfortunately, we were not able to assess the cause of death of this animal since it died at night, and we found it in an advanced state of decomposition. However, we believe that a possible reason for death is due to the nephrotoxicity since the surviving animals of this group showed increased urea levels compared to the saline control group. Previous studies also reported renal toxicity as the cause of animal deaths (Leite EA et al., 2012a; Leite EA et al., 2012b).

1. Leite EA et al., Acute toxicity study of cisplatin loaded long-circulating and pH-sensitive liposomes administered in mice. J Biomed Nanotechnol, 2012a, 8, 229–239.
2. Leite EA et al., Encapsulation of cisplatin in long-circulating and pH-sensitive liposomes improves its antitumor effect and reduces acute toxicity. Int J Nanomedicine 2012b, 7, 5259–5269.

Reviewer 3 Report

remove repetition of abbrevations in the abstract

present results more clearly in abstract

need extensive revision in the introduction section

need to add study rationale by and draw hypothesis with the previous studies

Is reverse phase evaporation method previouslly reported for the preparation of thermosensitive liposomes?

what is the use of finding zeta potential in this study, mention in method section

what is the role of HA in TSL-CDDP-HA, particle size in both cases almost similar but PDI differ.

Author Response

We thank the reviewer for the insightful comments, after which substantial changes were made to the manuscript. 

1. remove repetition of abbrevations in the abstract

Answer: The repetition was removed from the abstract, as suggested by the reviewer.

 

2. present results more clearly in abstract.

Answer: Thank you for your consideration. The abstract was rewritten and numerical data were included.

 

3. need extensive revision in the introduction section

Answer: As suggested by the reviewer, the introduction session was rewritten and new references were added in the revised version.

 

4. need to add study rationale by and draw hypothesis with the previous studies

Answer: The thermosensitive formulation developed in this study was designed to allow selective CDDP delivery in the tumor region and decrease the serious side effects provoked by the drug. The rationale behind the formulation design is based on: (i) Thermosensitive liposomes are composed of lipids that undergo the transition from the gel phase to the crystalline liquid phase in response to heat, thus allowing drug release specifically in the heated region; ii) hyperthermia is a method used to treat tumors by raising the local or regional temperature through controlled heat sources; iii) hyaluronic acid has been utilized in the active targeting of nanoparticles to tumors due to its ability to bind to CD44 receptors overexpressed in some cancer cells. Thus, the association of these features in one system could be a powerful strategy to overcome the CDDP toxicity that limits the successful treatment of cancer patients as well as to improve cancer therapy.  The rational basis of our formulation is discussed in the 2^nd-4^th paragraphs in the introduction section.

 

5. Is reverse phase evaporation method previously reported for the preparation of thermosensitive liposomes?

Answer: Recently, our group published a paper with the same liposomal composition using reverse-phase evaporation as the preparation method (Gomes et al., 2022). Moreover, as can be seen in the literature, other studies used the same methodology to produce thermosensitive liposomes with different lipidic compositions (Li et al., 2020; Yang et al., 2020; Zhu et al., 2014; Zen et al., 2009).

1. Gomes IP et al. Thermosensitive liposomes containing cisplatin functionalized by hyaluronic acid: preparation and physicochemical characterization. J Nanopart Res., 2022, 24(2), 30.
2. Li Yanan et al., Long-Circulating Thermosensitive Liposomes for the Targeted Drug Delivery of Oxaliplatin. Int J Nanomedicine, 2020, 15, 6721-6734.
3. Yang S et al., Thermosensitive Liposomes Encapsulating Anti-Cancer Agent Lomustine, and Contrast Medium Iohexol, for Thermochemotherapy: Preparation, Characterization, and In Vivo Evaluation. J Nanosci Nanotechnol. 2020, 20(10), 6070-6076.
4. Zhu X et al. Thermo-sensitive liposomes loaded with doxorubicin and lysine modified single-walled carbon nanotubes as tumor-targeting drug delivery system. Biomater Appl. 2014, 29(5), 769-779.
5. Zeng Z et al. Preparation and characterization of tegafur magnetic thermosensitive liposomes. Pharm Dev Technol. 2009, 14(4), 350-357.

 

6. what is the use of finding zeta potential in this study, mention in method section

Answer: Zeta potential measurements are presented as a useful and efficient method of polymer adsorption evaluation since a change in the surface charge generally occurs in absorbing polymers' presence (Tinoco et al, 2018; Ramasamy et al., 2014; Zadaka et al., 2010; Rivkin et al., 2010). Thus, in this study, we also evaluated the efficiency of HA coating onto TSL-CDDP by zeta potential analysis and attributed it as an adequate efficiency of the process when the values were close to neutrality. Since uncoated liposome is positively surface charged and free HA presents a negative charge, a neutralization of surface charge evidenced by zeta potential indicates the formation of the TSL-CDDP-HA. As requested by the review, the following sentence was added in the method section:

“Zeta potential measurement was used to evaluate the efficiency of HA coating on TSL-CDDP by electrostatic interaction since a neutralization in the surface charge might indicate the association of HA to the liposome surface.”

1. Tinoco LMS et al., Hyaluronic acid-coated nanoemulsions loaded with a hydrophobic ion pair of all-trans retinoic acid for improving the anticancer activity. J. Pharm. Sci. 2018;54(4):e17361.
2. Ramasamy T et al., Chitosan-based polyelectrolyte complexes as potential nanoparticulate carriers: physicochemical and biological characterization. Pharm Res. 2014;31(5):1302-1314.
3. Zadaka D et al., Applying zeta potential measurements to characterize the adsorption on montmorillonite of organic cations as monomers, micelles, or polymers. J Colloid Interface Sci 2010; 352; 171–177.
4. Rivkin I et al., Paclitaxel-clusters coated with hyaluronan as selective tumor targeted nanovectors. 2010;31(27):7106-7114.

 

7. what is the role of HA in TSL-CDDP-HA, particle size in both cases almost similar but PDI differ.

Answer: Hyaluronic acid is an anionic polymer utilized in the active targeting of nanoparticles to tumors due to its ability to bind to CD44 receptors overexpressed in some cancer cells. In addition, it might contribute to reducing the adsorption of blood proteins, increasing the blood circulation time, as well as act as increases the cell uptake by endocytosis. Therefore, coating TSL with HA could favor the selective targeting to the tumor that could improve the anticancer therapy and reduce toxicity. 

Round 2

Reviewer 2 Report

Accept

Author Response

We thank the reviewer#2 for the positive analysis of our manuscript.