Topical Delivery of Carvedilol Loaded Nano-Transfersomes for Skin Cancer Chemoprevention

Round 1

Reviewer 1 Report

This manuscript by Chen et al describes the incorporation of carvedilol in a transferosome formulation for potential topical delivery. Below are some comments and suggestions for consideartion

1. Do the authors want to suggest incorporating carvedilol loaded transferosomes in a sunscreen type product for prevention of skin cancer?

2. It would be valuable to include a microscopic image of the transferosome microstructure

3. What was the rationale behind stability evaluations conducted at 4C since the real time conditions and product use conditions eventually will be at 25C/30C. Suggestion is to conduct stability at real time conditions.

4. Can the authors justify using Pur-A-Lyzer mini dialysis kit? Was there a membrane selection process involved? The dialysis membrane would be very porous and if the aim is to mimic topical delivery where skin is the target. A release membrane  with optimal hydrophilic and lipophilic properties must be selected.

5. Can the authors elaborate on the procedure of removal of outer surface of skin from ear.

6. In terms of skin penetration and retention, significant difference is not observed between free carvedilol and transferosome loaded carvedilol. Ideally the replicates should be the same for both groupls. Also, how many donors were used? Is the data from 10 skin sections from 1 ear? please clarify

Minor revision- check degree symbol (superscript) throughout the document

Final comment: After reviewing the results presented in the manuscript, the significance of incorporating carvedilol in transferosomes is not apparent since in most of the studies the results were not statistically significant between the free carvedilol and transferosome loaded carvedilol. There were instances were free drug showed better permeation, release, skin retention compared to the transferosome formulation.

Author Response

Response to Reviewer 1 Comments:

Reviewer 1: 

This manuscript by Chen et al describes the incorporation of carvedilol in a transferosome formulation for potential topical delivery. Below are some comments and suggestions for consideration:

1. Do the authors want to suggest incorporating carvedilol loaded transferosomes in a sunscreen type product for prevention of skin cancer?

Yes. Although sunscreens have been recommended as a primary preventive approach for everyone to prevent UV-induced skin damage and cancer, current sunscreen products in the market cannot provide sufficient protection. Therefore, the cases of skin cancer are still rising. The chemicals used in most marketed sunscreens merely absorb or reflect UV radiation but do not have pharmacological properties to prevent oxidative damage and malignant transformation of skin cells. The ultimate goal of this project is to improve the effects of sunscreen. Previous studies showed that carvedilol protected skin damage/cancer beyond acting as a sunscreen (ref#3). Future studies will be directed to the development of sunscreen in the form of gel or cream containing optimized carvedilol transfersome, or incorporating the transfersome into commercial sunscreen products. Further in vivo studies are necessary before we can suggest how carvedilol loaded transfersomes can be used to prevent skin cancer. This statement has been added to the Conclusions (line 511-514).

2. It would be valuable to include a microscopic image of the transferosome microstructure

We were planning to take the TEM images for the transfersomes. However, due to the pandemic, we have not yet obtained the access to the TEM facility. This image analysis has to be conducted in the future.

3. What was the rationale behind stability evaluations conducted at 4C since the real time conditions and product use conditions eventually will be at 25C/30C. Suggestion is to conduct stability at real time conditions.

We haven’t examined the stability of the formulations at room temperature yet, although it is likely they will be stable. Since storage of the current formulation in suspension at room temperature may result in bacteria growth, chemical preservatives are needed. To make the formulation composition simple, the transfersomal formulation was only evaluated for stability at 4°C. Future studies are needed to develop this formulation into a form that can be incorporated into a gel or cream for long term room temperature storage. This statement has been added to the Conclusions (line 510-511).

4. Can the authors justify using Pur-A-Lyzer mini dialysis kit? Was there a membrane selection process involved? The dialysis membrane would be very porous and if the aim is to mimic topical delivery where skin is the target. A release membrane with optimal hydrophilic and lipophilic properties must be selected.

The mini dialysis kit with artificial membrane was used in the in vitro drug release study as the first step to predict the in vivo performance of carvedilol loaded transfersomes. The advantage of the Pur-A-Lyzer kit, which is cell free, are rapid and reproducible.

The molecular weight cut off (MWCO) of the dialysis membrane is 3.5 KDa, which is the smallest one available at Sigma. This information has been added to the “Methods” (line 133-134). By selecting this dialysis membrane, our purpose is to make sure the pore size is small enough so that the nanoparticles cannot pass through, but only solubilized drug can. As expected, the drug release from the transfersome was much slower than the free drug. We totally agree with the reviewer that skin membrane should be the model for the drug release study. Therefore, drug release studies were further conducted on porcine ear skin and artificial 3D human skin cultures.

5. Can the authors elaborate on the procedure of removal of outer surface of skin from ear.

More detailed description was added to the methods (lines 144-145).

6. In terms of skin penetration and retention, significant difference is not observed between free carvedilol and transferosome loaded carvedilol. Ideally the replicates should be the same for both groups. Also, how many donors were used? Is the data from 10 skin sections from 1 ear? please clarify.

Each piece of porcine ear skin from a single donor was used for six Franz diffusion cells. We commonly performed 2~3 replicates for F18 and 3~4 replicates for free drug in one experiment. To obtain the data shown in Fig. 3, more than five independent experiments were performed. For F18, all the data were used, while for free drug dissolved in acetone, the failure rate was high, probably because the acetone evaporated too fast before carrying the drug through the skin (for acetone dissolved drug, some diffusion cells produced very low or no permeation at all; the drug level in receiver compartment was below the detection level of HPLC, which were called “failure” and removed from the data analysis). Therefore, 10 replicates for F18 but only 6 replicates for free drug were used to obtain the data shown in the figure.

Minor revision- check degree symbol (superscript) throughout the document

It has been checked and corrected.

Final comment: After reviewing the results presented in the manuscript, the significance of incorporating carvedilol in transferosomes is not apparent since in most of the studies the results were not statistically significant between the free carvedilol and transferosome loaded carvedilol. There were instances were free drug showed better permeation, release, skin retention compared to the transferosome formulation.

The goal of this project is to develop a topical delivery system for human application. Therefore, carvedilol in the free drug form dissolved in acetone is not ideal vehicle in human product because acetone can dry the skin and break the skin barrier function. In addition, the ideal topical delivery system should be the one to deliver the drug in a slower rate (as can be seen from F18), because systemic absorption needs to be avoided, but local skin retention and local pharmacological activity remain. Therefore, transfersomal formulation such as F18, with slower permeation and slower release, is what we desired as the product of this project. Importantly, in vivo studies confirmed that F18 showed better local efficacy than the free drug (unpublished; studies are ongoing).

Reviewer 2 Report

The manuscript by Chen et al describes the preparation and evaluation of carvedilol loaded transfersome. It is professionally written, well presented and clearly show the potential of their method and efficacy. The authors used in vitro and ex vivo drug release methods as well as cell culture and 3D reconstructed skin equivalent model to validate the results. Impressive work had been made here.

The following are recommended to:

general:  the results and discussion are typically separated in this journal

Major:

1. One of the key markers used in the 3D skin equivalent is safety and toxicity test. Although some data can be obtained by the histology (that are not quantified here), MTT, XTT, LDH (in the media) or other specific methods should be used to evaluate the impact of the system (transfersome), the free- and of carvedilol loaded formula. Without it, one can say that the impressive reduction in the parameters is due to reduce in viable cells.
2. Mass balance – why wasn’t it calculated? Do the authors still have the SC and residual formulation from their experiments?
3. The use 3D skin model is impressive but it is not “human skin” per se, the term used in ex vivo skin models obtained from surgeries. The authors should always write skin equivalent or other common terminology (for instance line 218).

Minor:

4. Line 58: “Carvedilol is a highly lipophilic compound with low molecular weight and a favorable logarithmic partition coefficient, making it suitable for skin-specific delivery”. Please add a reference or an illustration with log p.
5. Line 75-76 please rephrase as there are some liposomes that work fine…
6. Line 129: please add RT time of the HPLC
7. Line 145: no equilibrium with the buffer was performed prior to skin mounting?
8. Sulforhodamine B? isn’t it binds total proteins (live and dead cells)? Why not MTT, cell count?
9. 231 please add the primer sequences
10. 237 histological examination of the 3d model is not typical or easy. Please add the exact procedure: formalin – time duration, dehydration steps and time, h&e ext..
11. As small and large baches of preparation can differ. Please write the volume/gr ex used in line 279 and 287
12. 315 are the particle not stable at RT? This should be addressed in the conclusion
13. 325-326 as also seen in of Figure 3, “the skin permeation still limits immediate release” of the free form. Please rephrase to focus on the compliance
14. 385,386 change symbols

Author Response

Response to Reviewer 2 Comments

Reviewer 2:

The manuscript by Chen et al describes the preparation and evaluation of carvedilol loaded transfersome. It is professionally written, well presented and clearly show the potential of their method and efficacy. The authors used in vitro and ex vivo drug release methods as well as cell culture and 3D reconstructed skin equivalent model to validate the results. Impressive work had been made here.

The following are recommended to:

general: the results and discussion are typically separated in this journal

Based on “Instruction to Authors”, results and discussion could be combined. This way, it is easier to discuss at the same time presenting the results.

Major:

1.One of the key markers used in the 3D skin equivalent is safety and toxicity test. Although some data can be obtained by the histology (that are not quantified here), MTT, XTT, LDH (in the media) or other specific methods should be used to evaluate the impact of the system (transfersome), the free- and of carvedilol loaded formula. Without it, one can say that the impressive reduction in the parameters is due to reduce in viable cells.

The main purpose of using the 3D human skin culture model in this study is to evaluate and compare the drug diffusion/permeation and photoprotective efficacy. Future studies will be designed to evaluate toxicity and safety using the same model. Since in the monolayer cultures of both JB6 and HaCaT cells, the formulation showed little cytotoxicity, the possibility that observed differences on the 3D skin culture resulting from reduced viability is small.

2.Mass balance – why wasn’t it calculated? Do the authors still have the SC and residual formulation from their experiments?

Since the main purpose in the present study is to evaluate and compare permeation and skin retention, the tapes containing the SC layers were discarded.

3.The use 3D skin model is impressive but it is not “human skin” per se, the term used in ex vivo skin models obtained from surgeries. The authors should always write skin equivalent or other common terminology (for instance line 218).

We agree with the reviewer. The changes were made accordingly: “Human reconstituted skin” is used throughout the manuscript.

Minor:

4.Line 58: “Carvedilol is a highly lipophilic compound with low molecular weight and a favorable logarithmic partition coefficient, making it suitable for skin-specific delivery”. Please add a reference or an illustration with log p.

Log P value was added to line 59.

5.Line 75-76 please rephrase as there are some liposomes that work fine…

Changes were made.

6.Line 129: please add RT time of the HPLC

This information has been added into the “Methods” (line 130)

7.Line 145: no equilibrium with the buffer was performed prior to skin mounting?

The skin was equilibrated in the PBS buffer for 30 min before drug loading.

8.Sulforhodamine B? isn’t it binds total proteins (live and dead cells)? Why not MTT, cell count?

The SRB assay has been used in our lab for many years. It produced reproducible cytotoxicity data consistent with MTT, XTT and MTS assays.

9.231 please add the primer sequences

Primer sequences have been added.

10.237 histological examination of the 3d model is not typical or easy. Please add the exact procedure: formalin – time duration, dehydration steps and time, h&e ext..

The detailed method has been added.

11.As small and large baches of preparation can differ. Please write the volume/gr ex used in line 279 and 287

This information has been added into the notes part of Table 1 and Table 2.

12.315 are the particle not stable at RT? This should be addressed in the conclusion

The formulation may be stable at room temperature, but storage of the current formulation at room temperature may perish quickly. To avoid bacteria growth, chemical preservatives are needed. To make the formulation composition simple, the current transfersomal formulation in liquid suspension was only evaluated for stability at 4 degree. Future studies are needed to change the formulation into more stable after incorporation into a gel/cream or sunscreen. This statement has been added to the results and conclusions.

13.325-326 as also seen in of Figure 3, “the skin permeation still limits immediate release” of the free form. Please rephrase to focus on the compliance

Changes were made.

14.385,386 change symbols

Changes were made.

Round 2

Reviewer 1 Report

Based on the response to my comments,  the paper can be accepted.

Author Response

Thanks!

Reviewer 2 Report

The viability issue still remains -  the MOA could be penetration due to hampering the skin barrier and not permeation. The use of skin (ear) and the 3D model w/o examination safety is not obvious.

The authors could:

Add data regarding the histological examination (that had been performed) (no/minor/major structural modifications were observed due to the formulation and the SC was not alter due to treatment or add a statement such as "further studies are required to determine the safety of the proposed formulation, and to exclude skin irritation".

All other comments were addressed. The work is of high interest to the readers

Author Response

We agree with the reviewers and therefore revised the manuscript by adding the statement, “Since drug permeation may result from damaged skin barrier, further studies are needed to determine acute and repeated dose toxicity of F18 and skin irritation on the same model” (line 438-439).