Preparation and Characterization of Patch Loaded with Clarithromycin Nanovesicles for Transdermal Drug Delivery

Round 1

Reviewer 1 Report

Row 31-21: Delete semicolons.

Row 45: Delete semicolon.

Row 62: Delete double bracket.

Rows 116-117: Instead „. Table (1) shows the amounts of CLR, surfactants, Chol, All niosomal dispersions were kept refrigerated at 4 ℃.” You should write: “Prepared niosomal dispersions  were kept refrigerated at 4 ℃. Table (1) shows the amounts of CLR, surfactants, and Chol.”

Row 148: Delete double period.

Row 173: replace “physiological solution” with “normal saline”.

Row 184: Delete brackets.

Figure 1. Should be improved it is not clearly visible. Also the font should be increased to be clearly readable.  Providemore photos in higher resolution of your CLR niosomal paches and provide dimensions of manufactured patches: width, length and thickness on figure

Row 226: Delete extra space:“ in  .“

Section 2.9. Cytotoxicity assay-Why did you choose MCF-7 263 cell line?

Did you performed the skin irritation studies? If not,why?

Figure 2. Improve figure caption, to be readable.

Figure 5. Remove the caption on figure, the resolution of caption is not good and it is already clearly written under the figure that it is a S. aureus. If you decide to keep the caption on the figure, please move it in the corner of the figure, not on the Petri dish.

Row 538: unbold “The”.

Section 5. Conclusions-Should contain conclusions derived from your findings. For example:

1.       CLR-loaded niosomal nanovesicles were successfully prepared and incorporated  into a skin patch for transdermal administration.

2.       The optimal Chol: surfactant ratio for drug relese was found to be….

3.       Encapsulation of CLR into niosomes did not reduce the antibacterial activity

4.       Encapsulation of CLR niosomes into transdermal patch, significantly enhanced the CLR transdermal  delivery.

5.       The shear-thinning characteristic of CLR-loaded niosomal patches was maintained by the incorporation  of niosomes.

 Please take your findings into account and rewrite conclusions.

“Niosomes-based patches that mediate TDD is a potential strategy for avoiding the bitterness of oral liquid dosage form of CLR, lowering GIT-related side effects, and extending CLR's presence in the systemic circulation. Thus, allowing for less frequent dosing. Furthermore, as niosomal patches are simple needle-free alternative systems they enhanced pediatrics’ adherence which may help to reduce antibiotic resistance. In conclusion, niosomes based transdermal patches may be a promising  method for TDD of class II drugs and drugs experiencing GIT side effects.” –Those sentences are generalized and they can fit better in the introduction and not in the conclusion.

 

Author Response

Response to reviewers

We would like to thank the reviewers for the careful and thorough reading of this manuscript and for the thoughtful comments and constructive suggestions, which help to improve the quality of this manuscript. Kindly find our response to reviewers. We have been able to incorporate changes to reflect most of the suggestions provided by the reviewers. We have tracked the changes within the manuscript. If there are any questions or it is not clear, please do let me know.

Here is a point-by-point response to the reviewers’ comments and concerns.

 

Reviewer 1

Row 31-21: Delete semicolons.

Row 45: Delete semicolon.

Row 62: Delete double bracket.

Rows 116-117: Instead „. Table (1) shows the amounts of CLR, surfactants, Chol, All niosomal dispersions were kept refrigerated at 4 ℃.” You should write: “Prepared niosomal dispersions  were kept refrigerated at 4 ℃. Table (1) shows the amounts of CLR, surfactants, and Chol.”

Row 148: Delete double period.

Row 173: replace “physiological solution” with “normal saline”.

Row 184: Delete brackets.

We thank the reviewer for pointing that out. We have modified all of them and the changes visible in the revised manuscript.

Figure 1. Should be improved it is not clearly visible. Also the font should be increased to be clearly readable.  Providemore photos in higher resolution of your CLR niosomal paches and provide dimensions of manufactured patches: width, length and thickness on figure

We thank the reviewer for pointing that out. We have improved Figure 1 and the changes visible in the revised manuscript.

Row 226: Delete extra space:“ in  .“

We thank the reviewer for pointing that out. We have deleted it and the changes visible in the revised manuscript.

 

Section 2.9. Cytotoxicity assay-Why did you choose MCF-7 263 cell line?

We thank the reviewer for pointing that out. The addition of CLR to MCF-7 cells was shown to have cytotoxic effects in breast cancer cell lines as reported by many other studies most particularly when added as a combination treatment with other chemotherapeutic agents (1,2). Here, we aimed to evaluate if this cytotoxicity is seen and enhanced when formulating the drug as nanoparticles. Furthermore, our blank transdermal patch was not toxic to the cells, therefore, testing nano-CLR in a transdermal formulation will not affect the results. This transdermal formulation will be tested in skin cancer cell lines alone and in combination with other drugs (Under study). 

References

1. Clarithromycin effectively enhances doxorubicin-induced cytotoxicity and apoptosis in MCF7 cells through dysregulation of autophagy 
2. Clarithromycin enhances bortezomib-induced cytotoxicity via endoplasmic reticulum stress-mediated CHOP (GADD153) induction and autophagy in breast cancer cells 

Did you performed the skin irritation studies? If not,why?

We thank the reviewer for pointing that out. We have not done irritation test. We are planning to do it in the future with in vivo studies.

 

Figure 2. Improve figure caption, to be readable.

We thank the reviewer for pointing that out. We have improved the caption of Figure 2 and the changes visible in the revised manuscript.

 

Figure 5. Remove the caption on figure, the resolution of caption is not good and it is already clearly written under the figure that it is a S. aureus. If you decide to keep the caption on the figure, please move it in the corner of the figure, not on the Petri dish.

We thank the reviewer for pointing that out. We have deleted the caption on figure 5 and the changes visible in the revised manuscript.

 

Row 538: unbold “The”.

We thank the reviewer for pointing that out. We have unbolded “The” and the changes visible in the revised manuscript.

 

Section 5. Conclusions-Should contain conclusions derived from your findings. For example:

1. CLR-loaded niosomal nanovesicles were successfully prepared and incorporated  into a skin patch for transdermal administration.
2. The optimal Chol: surfactant ratio for drug relese was found to be….
3. Encapsulation of CLR into niosomes did not reduce the antibacterial activity
4. Encapsulation of CLR niosomes into transdermal patch, significantly enhanced the CLR transdermal  delivery.
5. The shear-thinning characteristic of CLR-loaded niosomal patches was maintained by the incorporation  of niosomes.

 Please take your findings into account and rewrite conclusions.

“Niosomes-based patches that mediate TDD is a potential strategy for avoiding the bitterness of oral liquid dosage form of CLR, lowering GIT-related side effects, and extending CLR's presence in the systemic circulation. Thus, allowing for less frequent dosing. Furthermore, as niosomal patches are simple needle-free alternative systems they enhanced pediatrics’ adherence which may help to reduce antibiotic resistance. In conclusion, niosomes based transdermal patches may be a promising method for TDD of class II drugs and drugs experiencing GIT side effects.” –Those sentences are generalized and they can fit better in the introduction and not in the conclusion.

We thank the reviewer for pointing that out. We agree with your comment. We have rewritten the conclusion section and the changes visible in the revised manuscript.

Conclusion (Page 20)

CLR-loaded niosomal nanovesicles were successfully prepared and incorporated into a skin patch for transdermal administration. Interestingly, the encapsulation efficiency of niosomes containing Tween 80 was significantly higher than those prepared using Span 60. The optimal Chol: surfactant ratio for drug release was found to be 0.5:1. In addition, the encapsulation of CLR into the niosomal nanovesicles did not reduce the antibacterial activity of CLR. The shear-thinning behavior of CLR-loaded niosomal patches was maintained by the incorporation of niosomes. The niosomal patch has a significantly higher permeability coefficient of CLR than the conventional patch which markedly enhance the CLR transdermal delivery. Proceeding from the aforementioned findings, it can be concluded that niosomes-based patches that mediate TDD is a potential strategy for avoiding the bitterness of the oral liquid dosage form of CLR, lowering GIT-related side effects,  extending the  presence of CLR in the systemic circulation, and allowing for less frequent dosing. In conclusion, niosomes-based transdermal patches may be a promising method for TDD of class II drugs and drugs experiencing GIT side effects.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

The study conducted by the authors is quite useful and interesting, however it still remains some problems that need to be improved before considering for publication. I suggest the improvement as follow.

1. The author should explain more detail the correlation between physiological condition and used condition in the work (PBS containing 20% isopropanol).

2. In general, the particles size measured by TEM usually smaller when used DLS method (which determine dynamic diameter or in swelling state of particles). However, the results showed in this work does not follow that rule.

3.  Some results should be explained and discussed, for example, F7 and F8 have same composition of niosomes (span 60 and cholesterol), only differ in drug content, but the %EE and ZP values are huge difference; another is effect of cholesterol amount on PS and stability of niosomes. Besides, the profiles of in vitro release also need to discuss, why F9 release 60% drug after 1h compare with only 20% for F1.

4. The authors stated that effective delivery happen when niosomes possess PS smaller 300 nm or best value in range 10-210 nm, but the authors chose F9 for further study.

5. I recommend the author provide images to show flexibility and smoothness of patches, in addition, the morphology of patches should be evaluated by using SEM.

6. The authors should check carefully all error bar in all graphs.

Author Response

Response to reviewers

We would like to thank the reviewers for the careful and thorough reading of this manuscript and for the thoughtful comments and constructive suggestions, which help to improve the quality of this manuscript. Kindly find our response to reviewers. We have been able to incorporate changes to reflect most of the suggestions provided by the reviewers. We have tracked the changes within the manuscript. If there are any questions or it is not clear, please do let me know.

Here is a point-by-point response to the reviewers’ comments and concerns

Reviewer 2

The study conducted by the authors is quite useful and interesting, however it still remains some problems that need to be improved before considering for publication. I suggest the improvement as follow.

1. The author should explain more detail the correlation between physiological condition and used condition in the work (PBS containing 20% isopropanol).

We thank the reviewer for pointing that out. The following paragraph has been added to the revised manuscript and the changes are visible in the revised manuscript.

In vitro (Page 11)

For poorly water-soluble drugs, adding solubilizers such as Tween-80, ethanol, and isopropanol in the release medium is recommended [48-50]. PBS containing 20% isopropanol at pH 7.4 was chosen as the receptor media in both drug release and drug permeation studies to achieve the required sink conditions. This is because the sink condition is attained when the equilibrium solubility of drugs in the dissolving medium is at least three times the volume required for drug saturation [50]. In addition, these tests were carried out using PBS solution containing 20% isopropanol (pH 7.4) at 37 ± 0.5 °C and 600 rpm to simulate physiological condition as previously described in [51]. Additionally, to preserve the sink condition, sampling was done at specific time intervals and after each sampling, a fresh buffer solution was supplied to the release medium [52].

References

48. Bashash, M., M. Varidi, and J. Varshosaz, Sucrose stearate based niosomes as an alternative to ordinary vehicles for efficient curcumin delivery. Journal of Food Measurement and Characterization, 2022. 16(3): p. 2104-2118.
49. Sabry, S., et al., Formulation, characterization, and evaluation of the anti-tumor activity of nanosized galangin loaded niosomes on chemically induced hepatocellular carcinoma in rats. Journal of Drug Delivery Science and Technology, 2021. 61: p. 102163.
50. Zaki, R.M., M.M. Alfadhel, and S.M. Alshahrani, Formulation of Chitosan-Coated Brigatinib Nanospanlastics: Optimization, Characterization, Stability Assessment and In-Vitro Cytotoxicity Activity against H-1975 Cell Lines. 2022. 15(3).
51. Chen, S., et al., Recent advances in non-ionic surfactant vesicles (niosomes): Fabrication, characterization, pharmaceutical and cosmetic applications. Eur J Pharm Biopharm, 2019. 144: p. 18-39.
52. Hnin, H.M., E. Stefánsson, and T. Loftsson, Physicochemical and Stability Evaluation of Topical Niosomal Encapsulating Fosinopril/γ-Cyclodextrin Complex for Ocular Delivery. 2022. 14(6).

 

2. In general, the particles size measured by TEM usually smaller when used DLS method (which determine dynamic diameter or in swelling state of particles). However, the results showed in this work does not follow that rule.

We thank the reviewer for pointing that out. We agreed with reviewer. The following paragraph has been added to the revised manuscript and the changes are visible in the revised manuscript.

Page 9, line 332:  In general, the particles size measured by TEM usually smaller when used DLS method but the particle size measured by TEM could be comparable to DLS method, as described in previous studies [33-36]

References

33. De Silva, L., et al., Characterization, optimization, and in vitro evaluation of Technetium-99m-labeled niosomes. International journal of nanomedicine, 2019. 14: p. 1101.
34. Maurer, V., et al., In-Vitro Application of Magnetic Hybrid Niosomes: Targeted siRNA-Delivery for Enhanced Breast Cancer Therapy. 2021. 13(3).
35. Obeid, M.A., et al., Niosome-encapsulated balanocarpol: compound isolation, characterisation, and cytotoxicity evaluation against human breast and ovarian cancer cell lines. Nanotechnology, 2020. 31(19): p. 195101.
36. Sezgin-Bayindir, Z. and N. Yuksel, Investigation of formulation variables and excipient interaction on the production of niosomes. AAPS PharmSciTech, 2012. 13(3): p. 826-35.

 

 

3. Some results should be explained and discussed, for example, F7 and F8 have same composition of niosomes (span 60 and cholesterol), only differ in drug content, but the %EE and ZP values are huge difference; another is effect of cholesterol amount on PS and stability of niosomes. Besides, the profiles of in vitro release also need to discuss, why F9 release 60% drug after 1h compare with only 20% for F1.

We thank the reviewer for pointing that out. The following paragraph has been added to the revised manuscript and the changes are visible in the revised manuscript.

Page10 / line 356 . F7 and F8 have same composition of niosomes (Span 60 and Chol with a ratio of 1:0.5, but they only differ in drug content which was 200mg and 100mg, respectively. However, the results in Table 4 showed that the increase in the amount of CLR in the hydration media decreases the EE%, where the EE% of F7 and F8 was 12.00 ± 1.00 and 50.00 ± 1.00%, respectively, which was significantly different (p < 0.05). This is because drug entrapment efficiency of the niosomes is affected by drug concentration, where the higher the amount of drug added to niosomes, the lower the drug encapsulation efficiency as described in [42-44]. A high loading concentration of the drug lowers entrapment efficiency because a high concentration of the drug hinders vesicle formation, as was previously de-scribed in [45].

 

F9 , which composed of  a 100 mg Tween 80 and 50 mg Chol, has a considerably (p < 0.05) larger PS compared to F6, composed of 1:1 Chol: Tween 80 ratio. In addition, F7 and F8 have significantly (p < 0.05) larger PS than those of F4. Since these niosomes have lower Chol to surfactant ratios than F4. Our findings may be explained by the fact that niosomes with lower Chol levels have larger particle sizes. Our findings were in line with those of Akbari et al. [46], who observed that the PS of the niosomes significantly dropped from 444.27 ± 4.86 to 383.67 ± 5.03 nm (P= 0.0001) as the ratio of Chol : surfactant in-creased from 0:10 to 5:5. El-far et al. [47] reported that a greater Chol ratio made with Tween 80 significantly reduced the particle size of niosomes. This is because higher Chol levels in niosomes result in a greater bilayer hydrophobicity, which raises surface energy and reduces particle size [38, 48]. Moreover, as the amount of CLR increases from 100 to 200 mg, the vesicular size increases. A study by Miatmoko et al. [42] revealed that the PS of niosomes increased with the addition of ursolic acid, but the encapsulation efficiency de-creased when the amount of ursolic acid was increased.

Regarding in vitro studies, we discussed the results in Page 11/Line 427.

References

38. Waqas, M.K., et al., Development and characterization of niosomal gel of fusidic acid: in-vitro and ex-vivo approaches. Des Monomers Polym, 2022. 25(1): p. 165-174.
39. Miatmoko, A., et al., Characterization and distribution of niosomes containing ursolic acid coated with chitosan layer. Res Pharm Sci, 2021. 16(6): p. 660-673.
40. Joshi, S., et al., Comprehensive Screening of Drug Encapsulation and Co-Encapsulation into Niosomes Produced Using a Microfluidic Device. Processes, 2020. 8(5): p. 535.
41. Tabandeh, H. and S.A. Mortazavi, An Investigation into Some Effective Factors on Encapsulation Efficiency of Alpha-Tocopherol in MLVs and the Release Profile from the Corresponding Liposomal Gel. Iran J Pharm Res, 2013. 12(Suppl): p. 21-30.
42. Bhardwaj, P., et al., Niosomes: A review on niosomal research in the last decade. Journal of Drug Delivery Science and Technology, 2020. 56: p. 101581.
43. Akbari, J., et al., Innovative topical niosomal gel formulation containing diclofenac sodium (niofenac). Journal of Drug Targeting, 2022. 30(1): p. 108-117.
44. El-Far, S.W. and H.A. Abo El-Enin, Targeting Colorectal Cancer Cells with Niosomes Systems Loaded with Two Anticancer Drugs Models; Comparative In Vitro and Anticancer Studies. 2022. 15(7).
45. Bashash, M., M. Varidi, and J. Varshosaz, Sucrose stearate based niosomes as an alternative to ordinary vehicles for efficient curcumin delivery. Journal of Food Measurement and Characterization, 2022. 16(3): p. 2104-2118.

 

4. The authors stated that effective delivery happen when niosomes possess PS smaller 300 nm or best value in range 10-210 nm, but the authors chose F9 for further study.

We thank the reviewer for pointing that out. We wrote that nanovesicles having a diameter of 300 nm or less can deliver their contents into the deeper skin layers to some extent. Particles in 10-210 nm size range, on the other hand, may preferentially penetrate via the transfollicular route [37]. Therefore, niosomes with PS of less than 300 nm are considered to be in the optimum range for nanocarriers for TDD.

37. Danaei, M., et al., Impact of Particle Size and Polydispersity Index on the Clinical Applications of Lipidic Nanocarrier Systems. 2018. 10(2).

 

5. I recommend the author provide images to show flexibility and smoothness of patches, in addition, the morphology of patches should be evaluated by using SEM.

We thank the reviewer for pointing that out. The following figure has been added to the revised manuscript using digital camera. Regarding evaluation of the morphology of patches using SEM, SEM is not available in our facility and it requires more resources, therefore we used digital camera to show the smoothness and surface of patches and the changes are visible in the revised manuscript.

Figure 6. Appearance of matrix type patches containing CLR niosomes (P4) under digital camera

6. The authors should check carefully all error bar in all graphs.

We thank the reviewer for pointing that out. The error bars have been checked in all graphs.

 

 

Author Response File: Author Response.pdf

Reviewer 3 Report

This research article shows transdermal delivery  of clarithromycin niosomes by using transdermal patches. The clarithromycin formulation and patches are well  characterized and evaluated. My comments are as follow

 

#1. Author should remove the word novel from the title because this formulation is not novel.

#2. Research team should highlight the key finding  in the abstract which I can’t  find.

#3.Figure1 quality is so poor. Team should improve the resolution up to 300 dpi for all figure.

#4. There are lots of typos and grammatical errors. Team should go through the English editing from the native speakers.

#5.  In Fig. 6 and 7, there should be single Y axis for all compounds. Moreover, FTIR is not so much suitable for this kind of analysis. We cannot see that much differentiation in the figure between two or more spectra.

#6. Figure 5 data is so much confusing. Author should provide a clear image for better understanding.

#7. What is the reason behind the selection of breast cancer cell line for the transdermal patches for cell toxicity. Why team did not select the normal skin cancer cell line.

Author Response

Response to reviewers

We would like to thank the reviewers for the careful and thorough reading of this manuscript and for the thoughtful comments and constructive suggestions, which help to improve the quality of this manuscript. Kindly find our response to reviewers. We have been able to incorporate changes to reflect most of the suggestions provided by the reviewers. We have tracked the changes within the manuscript. If there are any questions or it is not clear, please do let me know.

Here is a point-by-point response to the reviewers’ comments and concerns

Reviewer 3

This research article shows transdermal delivery of clarithromycin niosomes by using transdermal patches. The clarithromycin formulation and patches are well characterized and evaluated. My comments are as follow

 

#1. Author should remove the word novel from the title because this formulation is not novel.

We thank the reviewer for pointing that out. The title has been modified in the revised draft and the changes are visible in the revised manuscript.

 

#2. Research team should highlight the key finding in the abstract which I can’t find.

We thank the reviewer for pointing that out. We have added the key findings in the abstract section and the changes are visible in the revised manuscript.

Abstract: Clarithromycin (CLR), categorized as a Biopharmaceutical Classification System class II drug, has several gastrointestinal tract side effects and an extremely unpalatable bitter taste. The current study aims to design transdermal patch-embedded CLR niosomes to overcome the aforementioned CLR-related challenges. Various niosomal formulations have been successfully fabricated and characterized for morphology, size, in vitro release, and antimicrobial efficacy. Subsequently, the CLR niosomes were loaded into transdermal patches using solvent casting method. The polydispersity index of niosomes ranged from 0.005 to 0.360, indicating the uniformity of niosomes. The encapsulating efficiency varied between 12 and 86%. The optimal Chol: surfactant ratio for drug release was found to be 0.5:1. In addition, the encapsulation of CLR into niosomal nanovesicles did not reduce the antibacterial activity of CLR. Moreover, a shear-thinning behavior was observed in the niosomal gels before loading them into the niosomal patches. The niosomal patch has a significantly higher permeability coefficient of CLR than the conventional patch. The flux (Jss) of the niosomal patch was significantly higher than the conventional patch by more than 200 times. In conclusion, niosomes based transdermal patches could be a promising strategy for transdermal drug delivery of class II drugs and drugs experiencing GIT side effects.

 

#3.Figure1 quality is so poor. Team should improve the resolution up to 300 dpi for all figure.

We thank the reviewer for pointing that out. We have improved the quality of Figure 1 and the changes are visible in the revised manuscript.

#4. There are lots of typos and grammatical errors. Team should go through the English editing from the native speakers.

We sincerely appreciate the reviewer’s comment. The revised manuscript was edited for proper English language, grammar, punctuation, spelling, and overall style by Grammarly .

 

#5.  In Fig. 6 and 7, there should be single Y axis for all compounds. Moreover, FTIR is not so much suitable for this kind of analysis. We cannot see that much differentiation in the figure between two or more spectra.

We thank the reviewer for pointing that out. We have updated and clarified the figures and merge them in one figure and the changes are visible in the revised manuscript.  

Figure 7. ATR-FTIR spectra of CLR, blank niosomes, niosomes, and niosomal patch (P4).

 

#6. Figure 5 data is so much confusing. Author should provide a clear image for better understanding.

We thank the reviewer for pointing that out. We have modified it and the changes are visible in the revised manuscript.

Figure 5. The zones of inhibition of S. aureus using the niosomal formula (F9). Note: Well No.1: Negative control (50% DMSO: PBS), Well No.2: Positive control (CLR 12 μg /100 μl) Well No.3: Test Item , niosomal formulation (F9) (CLR 12 μg /100 μl).

 

#7. What is the reason behind the selection of breast cancer cell line for the transdermal patches for cell toxicity. Why team did not select the normal skin cancer cell line.

We thank the reviewer for pointing that out. The addition of CLR to MCF-7 cells was shown to have cytotoxic effects in breast cell lines as reported by many other studies most particularly when added as a combination treatment with other chemotherapeutic agents(1,2). Here, we aimed to evaluate if this cytotoxicity is seen and enhanced when formulating the drug as nanoparticles. Furthermore, our blank transdermal patch was not toxic to the cells, therefore, testing nano-CLR in a transdermal formulation will not affect the results. This transdermal formulation will be tested in skin cancer cell lines alone and in combination with other drugs (Under study). 

 References

1. Clarithromycin effectively enhances doxorubicin-induced cytotoxicity and apoptosis in MCF7 cells through dysregulation of autophagy 
2. Clarithromycin enhances bortezomib-induced cytotoxicity via endoplasmic reticulum stress-mediated CHOP (GADD153) induction and autophagy in breast cancer cells 

 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The authors have addressed all my concerns, I recommend for publication after English checking again.

Reviewer 3 Report

I think this manuscript can be published  now.