Itraconazole-Loaded Ufasomes: Evaluation, Characterization, and Anti-Fungal Activity against Candida albicans
, Neveen M. Saleh 3, Rehab N. Shamma 4,* and Noha I. Elsherif 5,*
Round 1
Reviewer 1 Report
Manuscript Number: pharmaceutics-2024034
Title: Itraconazole loaded ufasomes: evaluation, characterization, and anti-fungal activity against Candida albicans
Authors: Sara M. Hashem, Mary K. Gad, Hend M. Anwar, Neveen M. Saleh, Rehab N. Shamma and Noha I. Elsherif
The manuscript “Itraconazole loaded ufasomes: evaluation, characterization, and anti-fungal activity against Candida albicans” describes the design and development of colloidal carriers (ufasomes) based on oleic acid for encapsulation of itraconazole to improve its antifungal activity. The use of various types of biocompatible nanocontainers makes it possible to enhance the biological effect, reduce toxicity, dosages, and increase the stability of drugs. Therefore, this area of nanotechnology is certainly relevant. The topic of the paper and the material presented are in the scope of Pharmaceutics. However, there are some critical points that need to be addressed before the manuscript can be recommended for publication.
Major comments:
1. Please improve the introduction by adding references to papers from the last two years (2021-2022). What is the current status of ufasomes? Overall, the introduction looks like separate blocks that are not related to each other. What other nanocontainers have been used to encapsulate the itraconazole?
2. The novelty of the work is not obvious. An appropriate explanation should be added to the text.
3. Why were the best results obtained in the absence of cholesterol? Justify the choice of cholesterol concentration.
4. In the part “Preparation of ITZ-loaded ufasomes” you mentioned that use the hydration method for its ability to give uniform particles. Please explain the formation of particles with a very high polydispersity index.
5. Supplement the article with in vitro release profiles of the itraconazole from ufasomes, for example, by dialysis.
6. Explain the choice of the itraconazole concentration to be loaded. Give the results of optimizing this value.
7. Explain the mechanism of increasing antifungal activity using ufasome? Give a graphical representation of the process.
8. Are formulations designed for topical or systemic use? Is it possible to predict the stability of developed formulations in a biological environment with changes in pH and ionic strength?
9. The quality of TEM images is not suitable for such a high standard journal.
10. Does the spectrophotometry method really allow you to determine EE (Table 2) with an accuracy of up to 0.001?
11. Add measurement error for all biological experiments.
Minor comments:
1. The list of references is not formatted according to the guidelines of Pharmaceutics. Please correct.
2. The quality of Figure 1 needs to be improved, but at the same time it should be transferred to SI.
3. Table 2 should be improved. For instance, replace «208.1 ± 3.203» with «208 ± 3». Correct all values according to the given example.
4. References 11 and 12 are repeated. Please double check references.
5. Figure 6: replace «itraconazole» with «ITZ».
6. Line 166 «Candida. albicans»: please delete the dot.
Author Response
Reviewer 1 comments:
- Comment 1
Please improve the introduction by adding references to papers from the last two years (2021-2022). What is the current status of ufasomes? Overall, the introduction looks like separate blocks that are not related to each other. What other nanocontainers have been used to encapsulate the itraconazole?
*Reply:
We thank you for this direction, we have updated yje references in the introduction section in the revised manuscipt.
- Comment 2
The novelty of the work is not obvious. An appropriate explanation should be added to the text.
*Reply:
Thank you for this correction, we have updated in the aim to include:
“To the best of our knoweldge, this is the first study evaluating the formulation of itraconazole in unstaurated faaty acid enriches vesicles, ufasomes”
- Comment 3
Why were the best results obtained in the absence of cholesterol? Justify the choice of cholesterol concentration.
*Reply:
Previous studies reported that cholesterol can modulate the fluidity, elasticity, and permeability of the vesicle. This happens by filling the gaps in the vesicles.In our study we tried to optimize our vesicles in absence and presence of different concentrations of cholesterol. The optimization was done using Design® expert software; and the results observed showed that the presence of cholesterol had no significant effect on the %EE (p= 0.8754), increased the PS insignificanlty (p= 0.1095), and had no significant effect on the ZP (p=0.3842).
- Comment 4
In the part “Preparation of ITZ-loaded ufasomes” , you mentioned that use the hydration method for its ability to give uniform particles. Please explain the formation of particles with a very high polydispersity index.
*Reply:
The results of most samples were in the monodispersed phase, and the optimized and selected sample had a PDI value of 0.297. Moreover, this method is known to give mostly uniform, as observed in several previous researches, including those done by Jamal et al. [1] and Waqas et al. [2].
- Comment 5
Supplement the article with in vitro release profiles of the itraconazole from ufasomes, for example, by dialysis.
*Reply:
We are so grateful for such guidance. In our work, we have used the Agar well diffusion method. This method, developed for the first time in 1940, is used for antimicrobial susceptibility testing as a replacement for the in-vitro method.
- Comment 6
Explain the choice of the itraconazole concentration to be loaded. Give the results of optimizing this value.
*Reply:
The rationale behind using these ITZ concentrations lie partially in the trial and error, and in the revious searches that entrapped ITZ, including ITZ loaded PLGA nanoparticles (used 20 mg) [3], ITZ loaded PLGA microspheres (9.1 and 16.7%) [4], and ITZ loaded liposomes (1:10 ITZ to dry contents) [5].
- Comment 7
Explain the mechanism of increasing antifungal activity using ufasome. Give a graphical representation of the process.
Reply:
Thank you. A graphical representation is attached.
- Comment 8
Are formulations designed for topical or systemic use? Is it possible to predict the stability of developed formulations in a biological environment with changes in pH and ionic strength?
*Reply:
We are so grateful for your direction. The formulation is designed for topical use. So, there was no need to investigate the stability of the formulation in a biological environment.
- Comment 9
The quality of TEM images is not suitable for such a high standard journal.
*Reply:
We are so grateful for your direction. Better quality images are attached.
- Comment 10
Does the spectrophotometry method really allow you to determine EE (Table 2) with an accuracy of up to 0.001?
*Reply:
We are grateful for your comment, and as per your suggestion, we will remove all the un-necessary extra digits from those values in table 2. However, please note that the results attached are not the absorbance results, but the %EE ± SD results derived from the equation of the standard calibration curve conducted on the spectrophotometer prior to starting our work.
- Comment 11
Add measurement error for all biological experiments.
*Reply:
We are so grateful for your direction, and the required measurements were added.
- Minor comments:
- The list of references is not formatted according to the guidelines of Pharmaceutics. Please correct.
Reply: The list of references has been updated as instructed.
- The quality of Figure 1 needs to be improved, but at the same time it should be transferred to SI.
Reply: The image quality was updated.
- Table 2 should be improved. For instance, replace «208.1 ± 3.203» with «208 ± 3». Correct all values according to the given example.
Reply: The required correction has been performed in the revised manuscript.
- References 11 and 12 are repeated. Please double check references.
Reply: All references were double checked and updated.
- Figure 6: replace «itraconazole» with «ITZ».
Reply: The required correction has been performed in the revised manuscript.
- Line 166 «Candida. albicans»: please delete the dot.
Reply: The required correction has been performed in the revised manuscript.
Reviewer 2 Report
This is a very interesting study of artificially manufactured itraconazole loaded ufasomes, a promising new drug-delivery system, their properties and their efficacy against Candida albicans infections. Indeed, the authors have labored significantly to manufacture, characterize and investigate the different properties of the different formulations.
Interestingly, these laboratory produced products in this study appear to be significantly smaller than previously reported ones, despite similar lipid characteristics (
The authors present the methods used clearly and in detail and analyze the results of the study clearly and sufficiently.
Although this is clearly an in vitro study, analogous studies may be the forerunner leading eventually to the application of novel technologies in clinical and real life practice.
Author Response
We would like to thank you for granting us the chance to resubmit the enclosed electronic files of our manuscript entitled “Itraconazole Loaded Ufasomes: Evaluation, Characterization, and Anti-fungal Activity Against Candida Albicans.” Authored by: Sara M. Hashem, Mary K. Gad, Hend M. Anwar, Neveen M. Saleh, Rehab N. Shamma and Noha I. Elsherif
We are ensuring that the manuscript represent a great value regarding the efficacy of the Itraconazole-loaded ufasomes against fungal infections. Our formulation represents a new solution to combat fungal resistance. We hope that this work to be considered for publication in PHARMACEUTICS.
Reviewer 3 Report
In this manscript, the inhibitory effect of Ufasomidazole loaded with itraconazole on Candida albicans fungi and its mechanism were studied. I'm not an expert on pharmaceutics, so I only care about antifungal activity. From the analysis of the active part, the manscript has not reached the level of publication.
1. The logic or purpose of this paper is unclear. I don't understand why the author does this experiment. In fact, Ufasolimidazole loaded with itraconazole did not significantly improve the inhibitory effect of candida albicans.
2. Why did the author express some inflammatory factors (IL-1b)? In fact, these have nothing to do with antifungal mechanisms. Also, the primers used are of human origin, not fungal origin.
3. The relevant activity values of the author have not been analyzed by error analysis and significance analysis, so the data are not credible.
4. Many pictures are not clear and the resolution is not enough.
Author Response
Reviewer 3 comments:
- Comment 1
The logic or purpose of this paper is unclear. I don't understand why the author does this experiment. In fact, Ufasolimidazole loaded with itraconazole did not significantly improve the inhibitory effect of candida albicans.
*Reply:
We thank the reviewer for his comment. In our study we aim to improve and restore the activity of itraconazole that was decreased due to the development of antimicrobial resistance that need an urgent action. The discovery of new agents that take a long time and can’t face the rapidly increasing in the resistance, on the other hand, when new agents come to market, pathogens can quickly develop resistance to them. However, an alternative approach to restore activity of antimicrobials can be done by functionalized with oleic acid that that considered as antimicrobial tenements that usually provide a sustained drug release effect, minimize the drug toxicity, and increase overall drug efficacy that was proved in our study by comparing the inhibition zone of control (itraconazole alone =15 mm) and the formula (25 mm).
- Comment 2
Why did the author express some inflammatory factors (IL-1b)? In fact, these have nothing to do with antifungal mechanisms. Also, the primers used are of human origin, not fungal origin.
*Reply:
We appreciate your opinion, in our study we aim to apply the current formula on topical infection with Candida albicans, we studied the effect of fungal infection on human fibroblast cells and compared with formula application by studying the inflammation in presence of Candida albicans and that is why we use the primer for human fibroblast cells.
- Comment 3
The relevant activity values of the author have not been analyzed by error analysis and significance analysis, so the data are not credible.
*Reply:
Thank you for your direction, we have updated our data in the whole manuscript.
- Comment 4
Many pictures are not clear and the resolution is not enough.
*Reply:
The quality of all attached figures were updated.
Reviewer 4 Report
The authors of the manuscript ”Itraconazole Loaded Ufasomes: Evaluation, Characterization, and Anti-fungal Activity Against Candida Albicans” present a paper where ufasomes with encapsulate ITZ were evaluated to improve drug penetration. Authors also reports an in vitro microbiological study for supporting the ITZ-loaded ufasomes. The manuscript is clearly written and the results are of interest but, it contains some flawn to be improved before acceptance.
Points that need to be addressed.
TITLE
"Candida albicans" should be written in italic "Candida albicans".
ABSTRACT
Lines 23 and 25: Please, authors shoul use italic format for scientific names.
INTRODUCTION
Line 23: Reference [1] seems to be a wrong reference, please ckeck and change it.
Line 35: “Candida albicans” should be written in italic format “Candida albicans”. Please, starting from this line, 99% of scientific names are written in wrong format. Please check carefully and correct them!
Line 54: Reference [11] is doubled, in fact #11 and #12 are the same (see reference list). Please, check them and change reference numbers on text.
Line 54: Reference [15] is doubled, in fact #15 and #19 are the same (see reference list). Please, check them and change reference numbers on text.
Line 62: “in vitro” should be written in italic format “in vitro”. Please, check and correct them on text. Same situation of line 35.
Materials and Methods
Line 80: Reference [18] is doubled, in fact #18 and #21 are the same (see reference list). Please, check them and change reference numbers on text.
Line 115: Is there any reason for jumping the reference numbers from 20 to 52? Please, authors should setup the right number sequence.
Line 132 (Table 2): Please, authors must improve the quality of the table 2.
Line 166: “Candida albicans” should be written “C. albicans” in italic format. Please, starting from this line, check carefully the scientific names and correct them in italic format.
RESULT AND DISCUSSION
Figure 1: Please, improve the quality of the figure. It is really difficult read words and numbers. Please delete what is not necessary.
Line 395 - Legend Figure 1: Part of the text is in bold, correct it.
Lines 401-402: Please, use the right format for paragraph titles.
Figure 2: Please, the quality should be improved (maybe a smaller one?) and put a bit more information on legend and picture (arrows, for example).
Line 437: Please, follow indications already seen above, about italic format of scientific names. Please use C. albicans, it was already written many times.
Line 439: Please, correct italic format in all “result and discussion”!
Figure 5: Authors should make a control with oleic acid, basically a third well. The haloes (A and B) seems to have the same dimension. Please, authors should improve the figure.
Figure 6: Authors should explain better what are negative and positive controls. It is not really clear. Did you set up more than 1 assay? So, put SE or SD.
CONCLUSION
Lines 566-568: Please, follow indications already seen above, about italic format of scientific names.
Author Response
Reviewer 4 comments:
The authors of the manuscript ”Itraconazole Loaded Ufasomes: Evaluation, Characterization, and Anti-fungal Activity Against Candida Albicans” present a paper where ufasomes with encapsulate ITZ were evaluated to improve drug penetration. Authors also reports an in vitro microbiological study for supporting the ITZ-loaded ufasomes. The manuscript is clearly written and the results are of interest but, it contains some flawn to be improved before acceptance.
Results and discussion part:
- Figure 5:Authors should make a control with oleic acid, basically a third well. The haloes (A and B) seems to have the same dimension. Please, authors should improve the figure.
- Figure 6:Authors should explain better what are negative and positive controls. It is not really clear. Did you set up more than 1 assay? So, put SE or SD.
*Reply: Thank you sir for your kind words. The requested changes were all done. Also please note that all the requested italic words were done in the original manuscript, however by placing the manuscript in the journal’s format, all of them disappeared.
Conerning figure 5, better quality images were attached for each well individually.
Conerning figure 6, it was updated as requested. The selected positive control was fibroblast cell culture containing Candida, while the negative control was a blank fibroblast cell culture (section 2.6.4.1., lines 245).
Round 2
Reviewer 1 Report
The manuscript can be accepted for publication.
A minor comment. The list of references is not formatted according to the guidelines of Pharmaceutics https://www.mdpi.com/authors/references. Please correct.
Reviewer 3 Report
The manuscript looked fine after revised, so it could be accepted in present form.
Reviewer 4 Report
Authors made the requested changes. The manuscript can be published.
